updates on pdfs

.gitattributes

@@ -1,35 +1,35 @@
-*.7z filter=lfs diff=lfs merge=lfs -text
-*.arrow filter=lfs diff=lfs merge=lfs -text
-*.bin filter=lfs diff=lfs merge=lfs -text
-*.bz2 filter=lfs diff=lfs merge=lfs -text
-*.ckpt filter=lfs diff=lfs merge=lfs -text
-*.ftz filter=lfs diff=lfs merge=lfs -text
-*.gz filter=lfs diff=lfs merge=lfs -text
-*.h5 filter=lfs diff=lfs merge=lfs -text
-*.joblib filter=lfs diff=lfs merge=lfs -text
-*.lfs.* filter=lfs diff=lfs merge=lfs -text
-*.mlmodel filter=lfs diff=lfs merge=lfs -text
-*.model filter=lfs diff=lfs merge=lfs -text
-*.msgpack filter=lfs diff=lfs merge=lfs -text
-*.npy filter=lfs diff=lfs merge=lfs -text
-*.npz filter=lfs diff=lfs merge=lfs -text
-*.onnx filter=lfs diff=lfs merge=lfs -text
-*.ot filter=lfs diff=lfs merge=lfs -text
-*.parquet filter=lfs diff=lfs merge=lfs -text
-*.pb filter=lfs diff=lfs merge=lfs -text
-*.pickle filter=lfs diff=lfs merge=lfs -text
-*.pkl filter=lfs diff=lfs merge=lfs -text
-*.pt filter=lfs diff=lfs merge=lfs -text
-*.pth filter=lfs diff=lfs merge=lfs -text
-*.rar filter=lfs diff=lfs merge=lfs -text
-*.safetensors filter=lfs diff=lfs merge=lfs -text
-saved_model/**/* filter=lfs diff=lfs merge=lfs -text
-*.tar.* filter=lfs diff=lfs merge=lfs -text
-*.tar filter=lfs diff=lfs merge=lfs -text
-*.tflite filter=lfs diff=lfs merge=lfs -text
-*.tgz filter=lfs diff=lfs merge=lfs -text
-*.wasm filter=lfs diff=lfs merge=lfs -text
-*.xz filter=lfs diff=lfs merge=lfs -text
-*.zip filter=lfs diff=lfs merge=lfs -text
-*.zst filter=lfs diff=lfs merge=lfs -text
-*tfevents* filter=lfs diff=lfs merge=lfs -text
+*.7z filter=lfs diff=lfs merge=lfs -text
+*.arrow filter=lfs diff=lfs merge=lfs -text
+*.bin filter=lfs diff=lfs merge=lfs -text
+*.bz2 filter=lfs diff=lfs merge=lfs -text
+*.ckpt filter=lfs diff=lfs merge=lfs -text
+*.ftz filter=lfs diff=lfs merge=lfs -text
+*.gz filter=lfs diff=lfs merge=lfs -text
+*.h5 filter=lfs diff=lfs merge=lfs -text
+*.joblib filter=lfs diff=lfs merge=lfs -text
+*.lfs.* filter=lfs diff=lfs merge=lfs -text
+*.mlmodel filter=lfs diff=lfs merge=lfs -text
+*.model filter=lfs diff=lfs merge=lfs -text
+*.msgpack filter=lfs diff=lfs merge=lfs -text
+*.npy filter=lfs diff=lfs merge=lfs -text
+*.npz filter=lfs diff=lfs merge=lfs -text
+*.onnx filter=lfs diff=lfs merge=lfs -text
+*.ot filter=lfs diff=lfs merge=lfs -text
+*.parquet filter=lfs diff=lfs merge=lfs -text
+*.pb filter=lfs diff=lfs merge=lfs -text
+*.pickle filter=lfs diff=lfs merge=lfs -text
+*.pkl filter=lfs diff=lfs merge=lfs -text
+*.pt filter=lfs diff=lfs merge=lfs -text
+*.pth filter=lfs diff=lfs merge=lfs -text
+*.rar filter=lfs diff=lfs merge=lfs -text
+*.safetensors filter=lfs diff=lfs merge=lfs -text
+saved_model/**/* filter=lfs diff=lfs merge=lfs -text
+*.tar.* filter=lfs diff=lfs merge=lfs -text
+*.tar filter=lfs diff=lfs merge=lfs -text
+*.tflite filter=lfs diff=lfs merge=lfs -text
+*.tgz filter=lfs diff=lfs merge=lfs -text
+*.wasm filter=lfs diff=lfs merge=lfs -text
+*.xz filter=lfs diff=lfs merge=lfs -text
+*.zip filter=lfs diff=lfs merge=lfs -text
+*.zst filter=lfs diff=lfs merge=lfs -text
+*tfevents* filter=lfs diff=lfs merge=lfs -text

app.py

@@ -43,7 +43,7 @@ with gr.Blocks(theme="dark") as demo:
                 with gr.Group():
                     gr.Markdown(f'<center><h2>Or Load Offline</h2></center>')
                     questions = gr.CheckboxGroup(choices = QUESTIONS, value = QUESTIONS, label="Questions (Please don't change this part now)", info="Please select the question you want to ask")
-                    answer_type = gr.Radio(choices = ["ChatGPT_txt", "GPT4_txt"], label="Answer_type", info="Please select the type of answer you want to show")
+                    answer_type = gr.Radio(choices = ["ChatGPT_txt", "GPT4_txt", 'New_GPT_4_pdf'], label="Answer_type", info="Please select the type of answer you want to show")
                     btn_submit_txt_offline = gr.Button(value='Show Answers')
                     # btn_submit_txt.style(full_width=True)
 

backend.py

@@ -351,7 +351,7 @@ class Backend:
             res_list.append(tmp_res_list)
         return res_list
 
-    def process_file_offline(self, questions, answer_type):
+    def process_file_offline(self, questions, answer_type, progress = gr.Progress()):
         # record the questions
         self.questions = questions
 
@@ -360,17 +360,30 @@ class Backend:
             df = pd.read_csv('./offline_results/results_all.csv')
         elif answer_type == 'GPT4_txt':
             df = pd.read_csv('./offline_results/results_all_gpt4.csv')
+        elif answer_type == 'New_GPT_4_pdf':
+            df = pd.read_csv('./offline_results/results_new_pdf.csv')
 
         # make the prompt
         self.res_list = self.phase_df(df)
-
-        txt_root_path = './20230808-AI coding-1st round'
-        self.filename_list = df['fn'].tolist()
+        if answer_type == 'ChatGPT_txt' or answer_type == 'GPT4_txt':
+            txt_root_path = './20230808-AI coding-1st round'
+            self.filename_list = df['fn'].tolist()
+        elif answer_type == 'New_GPT_4_pdf':
+            txt_root_path = './new_pdfs'
+            self.filename_list = df['fn'].tolist()
+            self.filename_list = ['.'.join(f.split('.')[:-1]) + '.txt' for f in self.filename_list]
+        
         self.text_list = []
-        for file in self.filename_list:
-            text_path = os.path.join(txt_root_path, file)
-            with open(text_path, 'r', encoding='utf-8') as f:
-                text = f.read()
+        for file in progress.tqdm(self.filename_list):
+            if file.split('.')[-1] == 'pdf':
+                # convert pdf to txt
+                text = self.phrase_pdf(os.path.join(txt_root_path, file))
+
+            else:    
+                text_path = os.path.join(txt_root_path, file)
+                with open(text_path, 'r', encoding='utf-8') as f:
+                    text = f.read()
+
             self.text_list.append(text)
 
         # Use the first file as default

new_pdfs/10.1002_brb3.2556.txt

@@ -0,0 +1,1045 @@
+Received: 30 October 2021
+
+Revised: 20 January 2022
+
+Accepted: 27 February 2022
+
+DOI: 10.1002/brb3.2556
+
+O R I G I N A L A R T I C L E
+
+miRNA-384-3p alleviates sevoflurane-induced nerve injury by inhibiting Aak1 kinase in neonatal rats
+
+Yuanyuan Chen1
+
+Xuan Gao2
+
+Hao Pei3
+
+1Department of Anesthesiology, Yancheng Maternity and Child Health Care Hospital, Yancheng, Jiangsu, China
+
+Abstract
+
+Objective: Sevoflurane is a common anesthetic and is widely used in pediatric clinical
+
+2Department of Anesthesiology, Shanghai Blue Cross Brain Hospital, Shanghai, China
+
+surgery to induce and maintain anesthesia through inhalation. Increasing studies
+
+3Department of Anesthesiology, Children’s Hospital of Fudan University, Shanghai, China
+
+have revealed that sevoflurane has neurotoxic effects on neurons, apoptosis, and
+
+memory impairment. miR-384 is involved in the process of neurological diseases.
+
+Correspondence Hao Pei, Department of Anesthesiology, Children’s Hospital of Fudan University, No. 399, Wanyuan Road, Minhang District, Shanghai City 201102, China. Email: lebajie_pei@hotmail.com
+
+However, the role of miRNA-384-3p in sevoflurane-induced nerve injury is not clear.
+
+This study focused on exploring the roles and mechanisms of miRNA-384-3p in
+
+sevoflurane-induced nerve injury.
+
+Methods: Seven-day-old rats were exposed to 2.3% sevoflurane to induce nerve injury.
+
+The morphological changes in neurons in the hippocampal CA1 region were detected
+
+Yuanyuan Chen and Xuan Gao contributed equally to this work.
+
+by HE staining and Nissl staining. Neuronal apoptosis was detected by TUNEL and
+
+Western blot assays. Spatial memory and learning ability were detected by the Morris
+
+water maze assay. The target gene of miRNA-384-3p was verified through a luciferase
+
+reporter assay. A rescue experiment was used to confirm the miRNA-384-3p pathway
+
+in sevoflurane-induced nerve injury.
+
+Results: Sevoflurane reduced miRNA-384-3p expression in the rat hippocampus.
+
+miRNA-384-3p alleviated sevoflurane-induced morphological changes in hippocampal
+
+neurons and apoptosis of neurons in the hippocampal CA1 region. Meanwhile, miRNA-
+
+384-3p attenuated the decline in spatial memory and learning ability induced by
+
+sevoflurane. miRNA-384-3p alleviated sevoflurane-induced nerve injury by inhibiting
+
+the expression of adaptor-associated kinase 1 (Aak1).
+
+Conclusion: Our findings revealed the role and mechanism of miRNA-384-3p in
+
+sevoflurane-induced nerve injury, suggesting that miRNA-384-3p could be a novel and
+
+promising strategy for reducing sevoflurane-induced neurotoxicity.
+
+K E Y W O R D S Aak1, miRNA-384-3p, nerve injury, sevoflurane
+
+This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Brain and Behavior published by Wiley Periodicals LLC.
+
+Brain Behav. 2022;12:e2556. https://doi.org/10.1002/brb3.2556
+
+wileyonlinelibrary.com/journal/brb3
+
+1 of 11
+
+21579032, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.2556 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+2 of 11
+
+CHEN ET AL.
+
+were kept on a 12-h light–dark cycle in individually ventilated cages at 21 ± 1◦C with free access to food and water. All animal experi- ments were approved by the Institutional Animal Care and Use Com-
+
+1
+
+INTRODUCTION
+
+Sevoflurane, an inhalation anesthetic, is widely used in clinical surgical
+
+mittees and performed according to the institution’s guidelines and
+
+operations (Egan, 2015; Yi et al., 2015). However, recent studies have
+
+animal research principles.
+
+shown that sevoflurane has neurotoxic effects, increases neuronal
+
+The rats were randomly divided into three groups: control, sevoflu-
+
+apoptosis, and reduces learning and memory ability (H. He et al., 2018;
+
+rane, and miRNA-384-3p agomir injection. Each group consisted of 12
+
+Perez-Zoghbi et al., 2017). The mechanism of sevoflurane-induced neu-
+
+rats. Sevoflurane was used to anesthetize rats as previously described
+
+rotoxicity remains mostly unknown. Hence, it is necessary to explore
+
+(Zhou et al., 2017). Briefly, rats were exposed to 2.3% sevoflurane
+
+the underlying molecular mechanism of sevoflurane-induced neuro-
+
+for 2 h every day for 3 continuous days. The gas flow was 2 L/min,
+
+toxicity to reduce sevoflurane-induced nerve injury.
+
+and the concentration of sevoflurane was measured by a gas monitor
+
+MicroRNAs (miRNAs) are noncoding RNAs, and their expres-
+
+(Detex Ohmeda, CO, USA). The NPS-A3 heating device (Midea Group, Beijiao, China) was used to heat the chamber up to 38◦C. Rats in the sevoflurane group were injected with 2 nmol agomir NC (volume is 2 ��l) into the hippocampus on the left lateral cerebral ventricles
+
+sion is involved in various physiological and pathological processes
+
+(Gjorgjieva et al., 2019; Sun et al., 2018). Some studies have con-
+
+firmed that miRNAs play a vital role in sevoflurane-induced neurotox-
+
+icity. For example, Zhao et al. (2018) found that sevoflurane upregu-
+
+after the first day of exposure to sevoflurane. The miRNA-384-3p
+
+lates miR-34a expression in the hippocampus. miR-34a also promoted
+
+agomir was purchased from RiboBio (Guangzhou, China) and diluted
+
+neuronal apoptosis and memory impairment induced by sevoflurane through the wnt1/β-catenin pathway (Zhao et al., 2018). In neonatal
+
+with Entranster transfection reagent (Engreen Biosystem Co., Beijing,
+
+China). Then, bilateral intrahippocampal administration was per- formed by injection with 2 nmol miRNA-384-3p agomir (volume is 2 μl)
+
+rats, the level of miR-96 is positively correlated with the concentra-
+
+tion of exposed sevoflurane. The increased expression of miR-96 aggra-
+
+into the hippocampus using a stereotaxic apparatus (RWD Life Science,
+
+vates sevoflurane-induced hippocampal neuron apoptosis and cogni-
+
+Shenzhen, China) and a 33-gauge beveled NanoFil needle. On the first
+
+tive function injury (C. Xu et al., 2019). X. He et al. (2007) found that
+
+day of exposure to sevoflurane, the cells were exposed to sevoflurane
+
+miR-384 expression was higher in the hippocampus than in other tis-
+
+for 2 days. Control group rats were exposed to air for 2 h/day and over
+
+sues. In addition, miR-384-5p expression was more than 10 times
+
+3 consecutive days. After being exposed to sevoflurane for 3 days, the
+
+higher than that of miRNA-384-3p in the rat hippocampus (X. He
+
+rats were euthanized, and the hippocampus was collected for further
+
+et al., 2007). Liu et al. found that chronic cerebral ischemia increased
+
+experiments.
+
+miR-384 expression in the hippocampus and hippocampal neurons.
+
+Knockdown of miR-384 inhibits the apoptosis of hippocampal neurons
+
+induced by chronic cerebral ischemia (Liu et al., 2019). Similarly, miR-
+
+2.2
+
+Cell isolation and culture
+
+384-5p promotes neurotoxicity and attenuates learning and memory
+
+in rats (Jiang et al., 2016; Q. Xu et al., 2019). However, whether the
+
+The hippocampus was dissected from neonatal rats (7 days old),
+
+roles of miRNA-384-3p are consistent with those of miR-384-5p in
+
+triturated, and dissociated through trypsin. The dissociated cells were
+
+neurotoxicity remains obscure. Therefore, we focused on exploring the
+
+filtered and centrifuged and then resuspended in Dulbecco’s Modified
+
+effects of miRNA-384-3p on sevoflurane-induced neurotoxicity.
+
+Eagle Medium/F12 medium (DMEM/F12, Thermo-Scientific, MA,
+
+Aak1, adaptor-associated kinase 1, has been reported to be associ-
+
+USA). Then, the cells were seeded onto dishes coated with poly-D-
+
+ated with nervous-related diseases and nerve injury (Shi et al., 2014).
+
+lysine and cultured with DMEM/F12 supplemented with 10% fetal
+
+For instance, Aak1 regulates clathrin-mediated endocytosis, thereby
+
+bovine serum (FBS, Thermo Scientific, MA, USA), 1% glutamine, 4.5 g/L
+
+affecting the cognitive ability of AD mice (Fu et al., 2018). However, the
+
+B27 plus glucose, and 1% penicillin–streptomycin (Sigma–Aldrich, MI, USA). After culturing for 3 days, 5 μg/ml cytosine arabinoside C
+
+role of Aak1 in anesthesia-induced neurotoxicity remains unclear.
+
+The role and mechanism of miRNA-384-3p in sevoflurane-induced
+
+(Sigma–Aldrich, MI, USA) was added to the medium and cultured for 24 h. The neurons were cultured in a humidified incubator at 37◦C and 5% CO2 for 14 days.
+
+neurotoxicity were investigated in this study, and the results confirmed
+
+that miRNA-384-3p attenuated sevoflurane-induced neuronal apop-
+
+tosis and memory disorder by inhibiting the expression of Aak1. Our
+
+findings suggest that miRNA-384-3p may be a promising strategy for
+
+resolving sevoflurane-induced nerve injury during clinical surgery.
+
+2.3
+
+Cell treatment and transfection
+
+2
+
+MATERIALS AND METHODS
+
+Neurons were cultured in a humidified incubator chamber with
+
+a gas mixture of 1% sevoflurane, 94% air and 5% CO2 for 6 h. Sevoflurane was delivered to the chamber at a rate of 10 L/min
+
+Animals and treatment
+
+2.1
+
+through a vaporizer (Datex-Ohmeda, Helsinki, Finland). Control
+
+Seven-day-old Sprague–Dawley rats were used in this study. They were
+
+neurons were cultured in a humidified incubator with 95% air and 5%
+
+obtained from the GemPharmatech Company (Nanjing, China). All rats
+
+CO2.
+
+21579032, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.2556 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+CHEN ET AL.
+
+3 of 11
+
+2.6
+
+Hematoxylin and eosin staining
+
+Hippocampal neurons, including control and sevoflurane-exposed neurons, were seeded into 24-well plates at 104 cells/well. When the
+
+confluence of the cells reached 60%, Lipofectamine 3000 (Thermo Sci-
+
+Rats were euthanized under the anesthesia of pentobarbital sodium
+
+entific, MA, USA) was used for transfection. miRNA-384-3p mimics,
+
+(80 mg/kg), and then the hippocampal tissues were removed. Tissues
+
+NC mimics, pcDNA-Aak1 vector (pc-Aak1), and pcDNA control vec-
+
+were fixed with 4% paraformaldehyde for 24 h and paraffin embed- ded. Sections of 4 μM were cut, and staining was carried out according
+
+tor (pc-NC) were obtained from GeneChem (Shanghai, China). miRNA-
+
+384-3p mimics and NC mimics were transfected into control neu-
+
+to the hematoxylin and eosin (HE) protocol. Neural injury scoring was
+
+rons. NC mimics and pc-NC were transfected into control neurons
+
+performed according to the following standard: no nerve cell death, 0
+
+simultaneously. NC mimics and pc-NC, miRNA-384-3p mimics and pc-
+
+points; scattered single nerve cell death, 1 point; slight nerve cell death,
+
+NC, and miRNA-384-3p mimics and pc-Aak1 were transfected into
+
+2 points; mass nerve cell death, 3 points; and almost complete nerve
+
+sevoflurane-exposed neurons simultaneously. The transfection con-
+
+cell death, 4 points.
+
+centration was 10 nM. After transfection for 48 h, the cells were col-
+
+lected for further experiments.
+
+2.7
+
+Nissl staining
+
+Real-time quantitative polymerase chain
+
+2.4 reaction
+
+Paraffin sections of hippocampal tissues were deparaffinized and stained with cresyl violet solution for 45 min at 37◦C. Next, sections were washed with distilled water and differentiated with gradient con-
+
+Total RNA was isolated from hippocampal tissue or transfected neu-
+
+centration ethanol. The differentiation was stopped when the tissue
+
+rons by using TRIzol reagent (Thermo-Scientific, MA, USA). RNA was
+
+was clear by transferring the sections to distilled water. Then, the sec-
+
+reverse transcribed into cDNA by using the PrimeScript RT reagent kit
+
+tions were dehydrated through a gradient concentration of ethanol and
+
+(Takara, Japan). A SYBR green PCR kit (Vazyme, Nanjing, China) was
+
+covered with neutral resin. Optical microscopy (Nikon, Tokyo, Japan)
+
+used to perform real-time quantitative polymerase chain reaction (RT–
+
+was used to observe the neurons in the hippocampal CA1 regions. The
+
+qPCR). U6 and GAPDH were used to normalize the relative expres-
+
+number of Nissl bodies was analyzed in a double-blinded manner with
+
+sion of miRNA-384-3p and Aak1. The miRNA-384-3p forward primer sequence (5′−3′) was AATTCCTAGAAATTGTT, and the reverse primer sequence (5′−3′) was AGTGCAGGGTCCGAGGTATT. The U6 forward primer sequence (5′−3′) was CTCGCTTCGGCAGCACATATACT, and the reverse primer sequence (5′−3′) was ACGCTTCACGAATTTGCGT- GTC. The Aak1 forward primer sequence (5′−3′) was CGGGTCACTTC- CGGGTTTA, and the reverse primer sequence (5′−3′) was TTCTTCTC- CGGTTTCAGCCC. The GAPDH forward primer sequence (5′−3′) was GAACGGGAAGCTCACTGG, and the reverse primer sequence (5′−3′)
+
+Image-Pro Plus 6.0.
+
+2.8
+
+Cell apoptosis
+
+The cell apoptosis ratio was measured in transfected neurons and hip-
+
+pocampal tissues by using the In Situ Cell Death Detection kit (Roche,
+
+Basel, Switzerland). After staining, the positive neurons were ran-
+
+domly observed by a fluorescence microscope (Nikon, Tokyo, Japan)
+
+was GCCTGCTTCACCACCTTCT.
+
+in five fields. The apoptosis ratio was measured by TUNEL-positive
+
+neurons/DAPI-positive neurons.
+
+2.5
+
+Subcellular fractionation
+
+2.9 Western blot analysis
+
+After hippocampal microdissection, tissues were immediately treated
+
+with freshly prepared ice-cold homogenization buffer (20 mM HEPES,
+
+Protein was extracted from hippocampal tissue or transfected neurons
+
+2 mM EGTA, 0.3 mg/ml dithioerythritol, 0.16 mg/ml phenylmethyl-
+
+using RIPA lysis buffer containing a protease inhibitor (Promega
+
+sulfonyl fluoride, and 0.020 mg/ml aprotinin) and homogenized. The homogenate was centrifuged at 17,000 × g for 5 min to obtain the
+
+Corporation, WI, USA). The protein samples were fractionated by
+
+SDS–PAGE and transferred to a polyvinylidene difluoride membrane
+
+cytoplasmic fraction. The pellet was washed with buffer B (150 mM NaCl; 10 mM HEPES; 1 mM EDTA), centrifuged at 17,000 × g for 1 min at 4 ◦C, resuspended in buffer C (25% v/v glycerol; 20 mM HEPES; 400 mM NaCl; 1.2 mM MgCl2; 0.2 mM EDTA), vortexed for 30 s and incubated on ice for 10 min (five times) to finally centrifuge at 17,000 ×
+
+(PVDF, Millipore, MA, USA). Afterwards, the membranes were incu-
+
+bated with 5% nonfat milk for 2 h at room temperature. Then, the
+
+membranes were incubated with the primary antibody overnight at 4◦C. Then, the membranes were incubated for 2 h with the secondary antibody at room temperature and visualized with a chemilumines-
+
+g for 20 min to obtain the nuclear fraction (Caviedes et al., 2021). RNA
+
+cence kit (Vazyme, Nanjing, China). ImageJ software was used to
+
+expression of GAPDH, U6, miRNA-384-3p, and Aak1 in the nuclear
+
+analyze the protein expression. In this study, antibodies against Bax,
+
+and cytoplasmic fractions was detected by RT–qPCR as mentioned
+
+Bcl-2, cleaved caspase-3, PCNA, and Aak1 were diluted to 1:1000 for
+
+above.
+
+use, cleaved caspase-9 was diluted to 1:200, and Ki-67 was diluted to
+
+21579032, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.2556 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+4 of 11
+
+CHEN ET AL.
+
+1:100. β-actin was used as the internal control, and the antibody was
+
+ANOVA were used to test the mean difference between groups. Statis-
+
+diluted to 1:5000. The goat anti-rabbit HRP antibody was used as a
+
+tical analysis was carried out using GraphPad Prism 7 (GraphPad Inc., San Diego, CA, USA). A p-value < .05 was considered statistically sig-
+
+secondary antibody and diluted to 1:5000 for use. All antibodies were
+
+nificant.
+
+purchased from Abcam (London, England).
+
+2.10
+
+Morris water maze test
+
+3
+
+RESULTS
+
+The Morris water maze (MWM) test was used to evaluate the learning
+
+3.1 miRNA-384-3p in the rat hippocampus
+
+Sevoflurane reduces the expression of
+
+and memory abilities of rats at the age of 2 months. The MWM con- sisted of a pool (100 cm × 100 cm × 60 cm) and a platform (1 cm × 1 cm). The pool was filled with warm water (25◦C) to 1 cm. Rats were ran- domly placed in the pool and allowed to swim to the platform. The time
+
+To detect the effect of sevoflurane on miRNA-384-3p expression, we
+
+collected hippocampal tissues from control and sevoflurane-exposed
+
+that the rats spent swimming to a hidden platform was measured at 90
+
+neonatal rats and detected the expression of miRNA-384-3p through
+
+s, and the rats were allowed to rest on the platform for 20 s. The time
+
+RT–qPCR. The results showed that miRNA-384-3p expression was
+
+was recorded as 90 s if the rats did not find the platform within 90 s, and
+
+decreased in the hippocampal tissues of sevoflurane-exposed rats
+
+the rats were also placed on the platform for 20 s to rest. In the acqui-
+
+compared with control rats (Figure 1a). miRNA-384-3p was primarily
+
+sition phase, five training sessions were conducted every day for 5 con-
+
+located in the cytoplasm in hippocampal tissues (Figure 1b). The results
+
+tinuous days. After the training, probe trials were performed. The time
+
+suggested that miRNA-384-3p was downregulated by sevoflurane in
+
+of plateau quadrant residence and the number of traversing platforms
+
+the rat hippocampus.
+
+were recorded by computerized tracking/analyzing video systems to
+
+suggest the spatial memory and learning ability of the rats.
+
+miRNA-384-3p restores sevoflurane-induced
+
+3.2 morphological changes in neurons in the hippocampal CA1 region
+
+Dual-luciferase reporter assay
+
+2.11
+
+Aak1 wild type (Aak1 WT) containing the miRNA-384-3p binding sites in the 3′UTR of Aak1 was inserted into the firefly luciferase vector.
+
+Sevoflurane-induced neurotoxicity has been reported previously
+
+(Perez-Zoghbi et al., 2017). To confirm the role of miRNA-384-3p
+
+To confirm specific binding, an Aak1 mutant (Aak1 Mut) containing the mutated binding sites of miRNA-384-3p in the Aak1 3′UTR was
+
+in sevoflurane-induced neurotoxicity, miRNA-384-3p agomir was
+
+injected into the rat hippocampus after the first day of sevoflurane
+
+constructed. For the luciferase reporter assay, hippocampal neurons
+
+exposure. We detected morphological changes in neurons through HE
+
+were cultured and plated in 24-well plates. Each well was transfected with 1 μg Aak1 WT vector or Aak1 Mut vector, 1 μg Renilla luciferase
+
+and Nissl staining. The HE results showed that sevoflurane induced
+
+neuronal injury and decreased the number of neurons in the hippocam-
+
+plasmid, and 100 pM miRNA-384-3p mimics or NC mimics by using
+
+pal CA1 regions, and the decreased injury and number of neurons were
+
+Lipofectamine 3000 (Invitrogen, CA, USA). After 48 h of transfection,
+
+attenuated by the miRNA-384-3p agomir (Figure 2a). The Nissl
+
+the dual-luciferase reporter assay system (Promega Corporation, WI,
+
+results showed that Nissl bodies and neurons were decreased in the
+
+USA) was used to measure the firefly and Renilla luciferase activities.
+
+hippocampal CA1 regions of sevoflurane-exposed rats compared
+
+with control rats. The sevoflurane-induced decrease in Nissl bodies
+
+was attenuated by the miRNA-384-3p agomir (Figure 2b). These
+
+2.12
+
+Cell viability assay
+
+results demonstrated that miRNA-384-3p restored the sevoflurane-
+
+induced morphological changes in neurons in the hippocampal CA1
+
+Cell viability was detected by the cell counting kit-8 (CCK8) assay.
+
+regions.
+
+Transfected hippocampal neurons were seeded onto 96-well plates at approximately 103 cells/well (100 μl/well). Then, the neurons were cul- tured for 1 h and mixed with 10 μl CCK8 reagent (Dojindo, Kumamoto,
+
+miRNA-384-3p inhibits sevoflurane-induced 3.3 neuronal apoptosis in the hippocampal CA1 region
+
+Japan) for 2 h. Next, the optical density was measured at 450 nm by uti-
+
+lizing a Bio-EL340 automatic microplate reader (Tek Instruments, Hop-
+
+kinton, USA).
+
+We detected the function of miRNA-384-3p in sevoflurane-induced
+
+neuronal apoptosis through the TUNEL assay and Western blot
+
+2.13
+
+Statistical analysis
+
+assay. The TUNEL assay results demonstrated that the apoptosis
+
+ratio of neurons was increased in the hippocampal CA1 region of
+
+All data are presented as the mean ± standard deviation (SD) of
+
+sevoflurane-treated rats compared with control rats. Overexpression
+
+three independent experiments. Unpaired Student’s t test and one-way
+
+of miRNA-384-3p inhibited the apoptosis induced by sevoflurane
+
+21579032, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.2556 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+CHEN ET AL.
+
+5 of 11
+
+F I G U R E 1
+
+Sevoflurane reduces the expression of miRNA-384-3p in the rat hippocampus. (a) The expression of miRNA-384-3p was detected
+
+by RT–qPCR in the hippocampus of sevoflurane-exposed rats and control rats. (b) Nuclear and cytoplasmic expression of miRNA-384-3p in the hippocampus from control rats was assessed by RT–qPCR. **p < .01, the difference was compared to control rats. The error bars represent the mean ± SD in three independent repetitions
+
+F I G U R E 2 miRNA-384-3p restores sevoflurane-induced morphological changes in neurons in the hippocampal CA1 region. Neonatal rats were exposed to sevoflurane-induced nerve injury and were divided into two groups; one group was injected with miRNA-384-3p agomir into the hippocampus. Normal neonatal rats were used as a negative control. (a) HE staining detected morphological changes in neurons in the hippocampal CA1 region. (b) Nissl staining detected Nissl bodies and neurons in the hippocampal CA1 region. The scale bar is 50 μM. Every experiment had three independent repetitions. ***p < .001, **p < .01 vs. the control group, ##p < .01, #p < .05 vs. the sevoflurane group. The error bars represent the mean ± SD in three independent repetitions
+
+3.4 and learning ability of sevoflurane-treated rats
+
+miRNA-384-3p improves the spatial memory
+
+(Figure 3a). Similar to the TUNEL assay results, Western blot results
+
+showed that the expression of Bax, cleaved-caspase-3, and cleaved-
+
+caspase-9 was increased. Meanwhile, Bcl-2 expression was decreased
+
+in the hippocampal CA1 region of sevoflurane-treated rats compared
+
+Next, we tested the function of miRNA-384-3p in sevoflurane-induced
+
+with control rats. Overexpression of miRNA-384-3p attenuated
+
+changes in spatial memory and learning ability through the MWM
+
+sevoflurane-induced expression changes in these apoptosis-related
+
+test. The results showed that the time of plateau quadrant residence
+
+genes (Figure 3b). These results suggested that miRNA-384-3p inhib-
+
+and the number of traversing platforms were reduced in sevoflurane-
+
+ited sevoflurane-induced neuronal apoptosis in the hippocampal CA1
+
+treated rats compared with control rats, suggesting that sevoflurane
+
+region.
+
+impaired the spatial memory and learning ability of rats (Figure 4a,b).
+
+21579032, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.2556 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+CHEN ET AL.
+
+6 of 11
+
+F I G U R E 3 miRNA-384-3p inhibits sevoflurane-induced neuronal apoptosis in the hippocampal CA1 region. Neonatal rats were exposed to sevoflurane-induced nerve injury and were divided into two groups; one group was injected with miRNA-384-3p agomir into the hippocampus. Normal neonatal rats were used as a negative control. (a) Cell apoptosis was detected by a TUNEL assay in the hippocampal CA1 region. (b) Western blot analysis of the expression of apoptosis-related genes. **p < .01. The difference was compared to control rats. ##p < .01, #p < .05, the difference was compared to sevoflurane-treated rats. The error bars represent the mean ± SD in three independent repetitions
+
+Meanwhile, overexpression of miRNA-384-3p increased the plateau
+
+and miRDB databases to predict the target genes of miRNA-384-3p.
+
+quadrant residence time and the number of traversing platforms in
+
+The predicted results showed that Aak1 was the only target gene of
+
+sevoflurane-treated rats, suggesting that miRNA-384-3p attenuated
+
+miRNA-384-3p in the three databases (Figure 5a). The predicted bind-
+
+sevoflurane-induced injury to spatial memory and learning ability
+
+ing sequence of Aak1 and miRNA-384-3p is shown in Figure 5b. The
+
+(Figure 4a,b). These results demonstrated that miRNA-384-3p had a
+
+luciferase assay was used to confirm the binding site, and the results showed that the luciferase activity was decreased in Aak1 3′UTR
+
+protective effect on spatial memory and learning ability in rats.
+
+WT and miRNA-384-3p mimic cotransfected neurons compared with Aak1 3′UTR WT and NC mimic cotransfected neurons. However, the luciferase activity was not significantly changed in Aak1 3′UTR
+
+3.5
+
+Aak1 is a target gene of miRNA-384-3p
+
+MUT-transfected neurons (Figure 5c). To confirm that miRNA-384-3p
+
+To explore the underlying mechanism of miRNA-384-3p in
+
+regulates Aak1 expression, Western blotting was performed on neu-
+
+sevoflurane-induced nerve injury, we used the TargetScan, miRWalk,
+
+rons transfected with NC mimics or miRNA-384-3p mimics. The results
+
+21579032, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.2556 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+CHEN ET AL.
+
+7 of 11
+
+F I G U R E 4 miRNA-384-3p improves the spatial memory and learning ability of sevoflurane-treated rats. Neonatal rats were exposed to sevoflurane-induced nerve injury and were divided into two groups; one group was injected with miRNA-384-3p agomir into the hippocampus. Normal rats were used as a negative control. When rats were at the age of 2 months, plateau quadrant residence (a) and the number of traversing platforms (b) were detected by the MWM test. *p < .05 vs. the control group, #p < .05 vs. the sevoflurane group. The error bars represent the mean ± SD. Every experiment had three independent repetitions
+
+F I G U R E 5 Aak1 is a target gene of miRNA-384-3p. (a) Prediction of target genes of miRNA-384-3p through the miRDB, miRWalk, and TargetScan databases. (b) The putative target sequence of miRNA-384-3p in the 3′UTR of Aak1 and the mutated sequence. (c) Luciferase assays in neurons transfected with Aak1 WT or Aak1 Mut and NC mimics or miR-384 mimics. (d) Western blot analysis of Aak1 expression in neurons transfected with miRNA-384-3p mimics or NC mimics. (e) RT-qPCR detected Aak1 expression in the hippocampus of sevoflurane-treated rats and control rats. (f) Nuclear and cytoplasmic expression of Aak1 in the hippocampus from normal rats was assessed by RT-qPCR. **p < .01. The difference was compared to control rats or transfected NC mimic neurons. The error bars represent the mean ± SD in three independent repetitions
+
+21579032, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.2556 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+8 of 11
+
+CHEN ET AL.
+
+showed that the expression of Aak1 was decreased in miRNA-384-3p
+
+neurodevelopmental defects (Warner et al., 2018; Zhang et al., 2016).
+
+For instance, ropivacaine exposure induces significant sciatic nerve
+
+mimic-transfected neurons compared with NC mimic-transfected
+
+injury in diabetic rats (Yu et al., 2019). Ketamine, midazolam, or a
+
+neurons (Figure 5d). Additionally, RT-qPCR results showed that Aak1
+
+combination of the two drugs induce apoptotic neurodegeneration in
+
+was upregulated in the hippocampus of sevoflurane-treated rats
+
+the developing mouse brain (Young et al., 2005). Therefore, exploring
+
+compared with control rats (Figure 5e). Moreover, Aak1 was primarily
+
+the methods of reducing the injury induced by anesthesia is impor-
+
+located in the cytoplasmic fraction in the hippocampus of control rats
+
+tant and necessary. Sevoflurane is an anesthetic and contributes to
+
+(Figure 5f). These results demonstrated that Aak1 was a target gene
+
+neurological disorders and neurodegeneration in the development of
+
+of miRNA-384-3p and that its expression was negatively regulated by
+
+miRNA-384-3p.
+
+the brain and affects memory and cognition (O’Farrell et al., 2018;
+
+Zhang et al., 2016). Sevoflurane at subanesthetic concentrations trig-
+
+gers neuronal apoptosis in 7-day-old mouse brains (Zhang et al., 2008).
+
+3.6 neuronal apoptosis and nerve injury through Aak1
+
+miRNA-384-3p alleviates sevoflurane-induced
+
+Sevoflurane exposure repeatedly induces cognition-related biochem-
+
+ical changes in the hippocampus and impairs learning and memory
+
+ability (Guo et al., 2018). Therefore, we established a sevoflurane
+
+To confirm whether miRNA-384-3p plays a role in sevoflurane-induced
+
+neurotoxicity model in neonatal rats through repeated exposure to
+
+nerve injury through Aak1, we transfected miRNA-384-3p mimics and
+
+sevoflurane.
+
+the Aak1 vector into hippocampal neurons simultaneously, and the
+
+Previous studies have reported that neurotoxicity induced by anes-
+
+neurons were cultured with sevoflurane. The RT-qPCR results showed
+
+thesia is regulated by miRNA (Bahmad et al., 2020). For example,
+
+that miRNA-384-3p expression was decreased in sevoflurane-treated
+
+miR-34a expression is upregulated in propofol-treated neurons and
+
+neurons compared to control neurons, and miRNA-384-3p mimics
+
+rats. Meanwhile, inhibition of miR-34a improves propofol-induced
+
+restored the expression of miRNA-384-3p. Meanwhile, the expres-
+
+cognitive dysfunction by suppressing cell apoptosis and recovering
+
+sion of Aak1 was increased in sevoflurane-treated neurons compared
+
+the expression of MAPK/ERK pathway genes (Xin, 2018). miR-124
+
+with control neurons. miRNA-384-3p mimics decreased the expres-
+
+increases ketamine-induced apoptosis in the hippocampal CA1 region
+
+sion of Aak1 in sevoflurane-treated neurons, and the miRNA-384-3p-
+
+and improves the memory performance of mice (H. Xu et al., 2015).
+
+induced decrease in Aak1 was partially restored by transfection with
+
+miR-384 is also involved in the progression of brain development, cog-
+
+the Aak1 vector (Figure 6a). To detect whether Aak1 participated in
+
+nition, and pathophysiology of neurological disorders (Gu et al., 2015).
+
+the regulation of miRNA-384-3p on sevoflurane-induced cell viability,
+
+However, the roles of miRNA-384-3p in anesthesia-induced neurotox-
+
+a CCK8 assay was used. The results showed that miRNA-384-3p atten-
+
+icity remain unclear. Here, we detected the expression of miRNA-384-
+
+uated the inhibitory effect of sevoflurane on cell viability, while the
+
+3p in sevoflurane-exposed rat hippocampi and found that sevoflurane
+
+miRNA-384-3p effect was remarkably undermined after the overex-
+
+decreased the expression of miRNA-384-3p. A miRNA-384-3p agomir
+
+pression of Aak1 (Figure 6b). Western blotting was used to measure
+
+was injected into neonatal rats to overexpress miRNA-384-3p. We fur-
+
+proliferation-related gene expression at the protein level. The results
+
+ther confirmed that miRNA-384-3p improved neuronal morphology,
+
+showed that sevoflurane inhibited the expression of PCNA and Ki-
+
+neuronal apoptosis, and learning and memory ability in sevoflurane-
+
+67, which was partially restored by miRNA-384-3p. Meanwhile, over-
+
+treated rats.
+
+expression of Aak1 attenuated miRNA-384-3p-mediated expression
+
+miRNAs mainly regulate the mRNA degradation or posttranscrip-
+
+changes in PCNA and Ki-67 in sevoflurane-treated neurons (Figure 6c).
+
+tional repression of the targeted gene (Saliminejad et al., 2019). To
+
+The TUNEL assay was used to detect whether Aak1 participated in
+
+explore the mechanism of miRNA-384-3p in sevoflurane-induced
+
+the regulation of miRNA-384-3p on sevoflurane-induced cell apop-
+
+nerve injury, we predicted and confirmed that Aak1 is a target
+
+tosis, and the results showed that overexpression of miRNA-384-3p
+
+gene of miRNA-384-3p. Aak1 plays vital roles in neuropathic pain,
+
+reduced the apoptosis of hippocampal neurons induced by sevoflu-
+
+schizophrenia, Parkinson’s disease and other neuropathic disorders
+
+rane, while the miRNA-384-3p effect was inhibited by increasing the
+
+(Abdel-Magid, 2017). For example, Fu et al. found that Aak1 expression is highest on day 14 and is reduced on day 30 in the Aβ1-42-induced AD model. The expression of Aak1 is negatively correlated with cognitive
+
+expression of Aak1 (Figure 6d). Similar to the results, the Western blot
+
+results demonstrated that sevoflurane-induced changes in apoptosis-
+
+related genes were attenuated by miRNA-384-3p. Meanwhile, Aak1
+
+ability by regulating the process of clathrin-mediated endocytosis (Fu
+
+overexpression restored the miRNA-384-3p-mediated changes in the
+
+et al., 2018). Kostich, Walter et al. discovered that Aak1 knockout mice
+
+expression of these genes in sevoflurane-treated hippocampal neurons
+
+have an antinociceptive phenotype, which may be a novel therapeutic
+
+(Figure 6e). These results demonstrated that miRNA-384-3p allevi-
+
+approach for neuropathic pain by inhibiting Aak1 kinase (Kostich et al.,
+
+ated sevoflurane-induced neuronal apoptosis and nerve injury through
+
+2016). Leger, Helene et al. found that Ndr kinases inhibit the prolifer-
+
+Aak1.
+
+ation of terminally differentiated cells and modulate the function of
+
+interneuron synapses through Aak1 (Leger et al., 2018). However, the
+
+DISCUSSION
+
+4
+
+role of Aak1 in anesthesia-mediated nerve injury remains unknown.
+
+Here, we confirmed that Aak1 expression was negatively regulated
+
+Anesthesia is widely used in modern medicine; however, a multitude
+
+by miRNA-384-3p in hippocampal neurons. Meanwhile, we demon-
+
+of evidence has demonstrated that anesthesia increases the risk of
+
+strated that miRNA-384-3p alleviated sevoflurane-induced neuronal
+
+21579032, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.2556 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+CHEN ET AL.
+
+9 of 11
+
+F I G U R E 6 miRNA-384-3p alleviates sevoflurane-induced neuronal apoptosis and nerve injury through Aak1. Hippocampal neurons were divided into four groups, including NC mimic- and pc-NC-transfected neurons cultured under control conditions, NC mimic- and pc-NC-transfected neurons cultured with 1% sevoflurane, miRNA-384-3p mimic- and pc-NC-transfected neurons cultured with sevoflurane, miRNA-384-3p mimic-, and pc-Aak1-transfected neurons cultured with sevoflurane. (a) RT-qPCR detected miRNA-384-3p and Aak1 levels. (b) CCK8 assay detected cell viability. (c) Western blot analysis of the expression of proliferation-related genes. (d) TUNEL assay detected cell apoptosis. (e) Western blot analysis of apoptosis-related gene levels. **p < .01 vs. the NC mimics + pc-NC group. ###p < .001, ##p < .01 vs. the sevoflurane group. &&&p < .001, &&p < .01, &p < .05 vs. the miR-384-3p mimic + pc-NC + sevoflurane group. The error bars represent the mean ± SD in three independent repetitions
+
+apoptosis and nerve injury by inhibiting the expression of Aak1 via
+
+CONFLICT OF INTEREST
+
+rescue experiments.
+
+The authors declare that they have no conflict of interest.
+
+AUTHOR CONTRIBUTIONS
+
+CONCLUSION
+
+5
+
+Xuan Gao and Hao Pei conceived and designed the study. Xuan Gao and
+
+Yuanyuan Chen performed the literature search and data extraction.
+
+Hao Pei drafted the manuscript. All authors read and approved the final
+
+In neonatal rats, we confirmed the roles and mechanisms of
+
+version of the manuscript.
+
+miRNA-384-3p in sevoflurane-induced nerve injury,
+
+including hip-
+
+pocampal neuron apoptosis and memory impairment. The findings
+
+of our study suggest that miRNA-384-3p could be a promising
+
+DATA AVAILABILITY STATEMENT
+
+strategy for reducing sevoflurane-induced nerve injury in clinical
+
+All data generated or analyzed during this study are included in the
+
+surgery.
+
+article.
+
+21579032, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.2556 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+10 of 11
+
+CHEN ET AL.
+
+PEER REVIEW
+
+Leger, H., Santana, E., Leu, N. A., Smith, E. T., Beltran, W. A., Aguirre, G. D., & Luca, F. C. (2018). Ndr kinases regulate retinal interneuron prolifera- tion and homeostasis. Science Reports, 8, 12544. https://doi.org/10.1038/ s41598-018-30492-9
+
+The peer review history for this article is available at https://publons.
+
+com/publon/10.1002/brb3.2556
+
+Liu, J., An, P., Xue, Y., Che, D., Liu, X., Zheng, J., Liu, Y., Yang, C., Li, Z., & Yu, B. (2019). Mechanism of Snhg8/miR-384/Hoxa13/FAM3A axis regulating neuronal apoptosis in ischemic mice model. Cell Death & Disease, 10, 441. https://doi.org/10.1038/s41419-019-1631-0
+
+ORCID
+
+Hao Pei
+
+https://orcid.org/0000-0002-6777-5463
+
+O’Farrell, R. A., Foley, A. G., Buggy, D. J., & Gallagher, H. C. (2018). Neurotox- icity of inhalation anesthetics in the neonatal rat brain: Effects on behav- ior and neurodegeneration in the piriform cortex. Anesthesiology Research and Practice, 2018, 6376090.
+
+REFERENCES
+
+Abdel-Magid, A. F. (2017). Inhibitors of adaptor-associated kinase 1 (AAK1) may treat neuropathic pain, schizophrenia, Parkinson’s disease, and other disorders. ACS Medicinal Chemistry Letters, 8, 595–597. https://doi. org/10.1021/acsmedchemlett.7b00208
+
+Perez-Zoghbi, J. F., Zhu, W., Grafe, M. R., & Brambrink, A. M. (2017). against sevoflurane-induced neurotoxicity extends to several brain regions in neonatal rats. British Journal of Anaesthesia, 119, 506–516. https://doi.org/10.1093/bja/aex222
+
+Dexmedetomidine-mediated
+
+neuroprotection
+
+Bahmad, H. F., Darwish, B., Dargham, K. B., Machmouchi, R., Dargham, B. B., Osman, M., Khechen, Z. A., El Housheimi, N., Abou-Kheir, W., & Chamaa, F. (2020). Role of microRNAs in anesthesia-induced neurotoxicity in animal models and neuronal cultures: A systematic review. Neurotoxic- ity Research, 37, 479–490. https://doi.org/10.1007/s12640-019-00135- 6
+
+Saliminejad, K., Khorram Khorshid, H. R., Soleymani Fard, S., & Ghaffari, S. H. (2019). An overview of microRNAs: Biology, functions, therapeutics, and analysis methods. Journal of Cellular Physiology, 234, 5451–5465. https: //doi.org/10.1002/jcp.27486
+
+Shi, B., Conner, S. D., & Liu, J. (2014). Dysfunction of endocytic kinase AAK1 in ALS. International Journal of Molecular Sciences, 15, 22918–22932. https://doi.org/10.3390/ijms151222918
+
+Caviedes, A., Maturana, B., Corvalán, K., Engler, A., Gordillo, F., Varas-Godoy, M., Smalla, K. H., Batiz, L. F., Lafourcade, C., Kaehne, T., & Wyneken, U. (2021). eNOS-dependent S-nitrosylation of the NF-κB subunit p65 has neuroprotective effects. Cell Death & Disease, 12, 4. https://doi.org/10. 1038/s41419-020-03338-4
+
+Sun, Z., Shi, K., Yang, S., Liu, J., Zhou, Q., Wang, G., Song, J., Li, Z., Zhang, Z., & Yuan, W. (2018). Effect of exosomal miRNA on cancer biology and clinical applications. Molecular Cancer, 17, 147. https://doi.org/10.1186/ s12943-018-0897-7
+
+Egan, T. D. (2015). Total intravenous anesthesia versus inhalation anesthe- sia: A drug delivery perspective. Journal of Cardiothoracic and Vascular Anesthesia, 29 Suppl (1), S3–S6. https://doi.org/10.1053/j.jvca.2015.01. 024
+
+Warner, D. O., Zaccariello, M. J., Katusic, S. K., Schroeder, D. R., Hanson, A. C., Schulte, P. J., Buenvenida, S. L., Gleich, S. J., Wilder, R. T., Sprung, J., Hu, D., Voigt, R. G., Paule, M. G., Chelonis, J. J., & Flick, R. P. (2018). Neuropsychological and behavioral outcomes after exposure of young children to procedures requiring general anesthesia: The Mayo Anes- thesia Safety in Kids (MASK) Study. Anesthesiology, 129, 89–105. https: //doi.org/10.1097/ALN.0000000000002232
+
+Fu, X., Ke, M., Yu, W., Wang, X., Xiao, Q., Gu, M., & Lu, Y. (2018). Periodic vari- ation of AAK1 in an Abeta1-42-induced mouse model of Alzheimer’s dis- ease. Journal of Molecular Neuroscience, 65, 179–189. https://doi.org/10. 1007/s12031-018-1085-3
+
+Gjorgjieva, M., Sobolewski, C., Dolicka, D., Correia de Sousa, M., & Foti, M. (2019). miRNAs and NAFLD: From pathophysiology to therapy. Gut, 68, 2065–2079. https://doi.org/10.1136/gutjnl-2018-318146
+
+Xin, P., Kuang, H. X., Li, X. L., Wang, Y., Zhang, B. M., Bu, H., Wang, Z. B., Meng, Y. H., Wang, Y. H., & Wang, Q. H. (2018). Proteomics and its application to determine mechanism of action of traditional Chinese medicine. Zhong- guo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chi- nese Materia Medica, 43, 904–912.
+
+Gu, Q. H., Yu, D., Hu, Z., Liu, X., Yang, Y., Luo, Y., Zhu, J., & Li, Z. (2015). miR-26a and miR-384-5p are required for LTP maintenance and spine enlargement. Nature Communication, 6, 6789. https://doi.org/10.1038/ ncomms7789
+
+Xu, C., Niu, J. J., Zhou, J. F., & Wei, Y. S. (2019). MicroRNA-96 is respon- sible for sevoflurane-induced cognitive dysfunction in neonatal rats via inhibiting IGF1R. Brain Research Bulletin, 144, 140–148. https://doi.org/ 10.1016/j.brainresbull.2018.09.001
+
+Guo, S., Liu, L., Wang, C., Jiang, Q., Dong, Y., & Tian, Y. (2018). Repeated expo- sure to sevoflurane impairs the learning and memory of older male rats. Life Sciences, 192, 75–83. https://doi.org/10.1016/j.lfs.2017.11.025 He, H., Liu, W., Zhou, Y., Liu, Y., Weng, P., Li, Y., & Fu, H. (2018). Sevoflurane post-conditioning attenuates traumatic brain injury-induced neuronal apoptosis by promoting autophagy via the PI3K/AKT signaling pathway. Drug Design, Development and Therapy, 12, 629–638. https://doi.org/10. 2147/DDDT.S158313
+
+Xu, H., Zhang, J., Zhou, W., Feng, Y., Teng, S., & Song, X. (2015). The role of miR-124 in modulating hippocampal neurotoxicity induced by ketamine anesthesia. International Journal of Neuroscience, 125, 213–220. https:// doi.org/10.3109/00207454.2014.919915
+
+Xu, Q., Ou, J., Zhang, Q., Tang, R., Wang, J., Hong, Q., Guo, X., Tong, M., Yang, L., & Chi, X., (2019). Effects of aberrant miR-384-5p expression on learn- ing and memory in a rat model of attention deficit hyperactivity disor- der. Frontiers in Neurology, 10, 1414. https://doi.org/10.3389/fneur.2019. 01414
+
+He, X., Zhang, Q., Liu, Y., & Pan, X. (2007). Cloning and identification of novel microRNAs from rat hippocampus. Acta Biochimica et Biophys- ica Sinica (Shanghai), 39, 708–714. https://doi.org/10.1111/j.1745-7270. 2007.00324.x
+
+Yi, W., Zhang, Y., Guo, Y., Li, D., & Li, X. (2015). Elevation of Sestrin-2 expres- sion attenuates sevoflurane induced neurotoxicity. Metabolic Brain Dis- ease, 30, 1161–1166. https://doi.org/10.1007/s11011-015-9673-1 Young, C., Jevtovic-Todorovic, V., Qin, Y. Q., Tenkova, T., Wang, H., Labruyere, J., & Olney, J. W. (2005). Potential of ketamine and midazolam, individ- ually or in combination, to induce apoptotic neurodegeneration in the infant mouse brain. British Journal of Pharmacology, 146, 189–197. https: //doi.org/10.1038/sj.bjp.0706301
+
+Jiang, M., Yun, Q., Shi, F., Niu, G., Gao, Y., Xie, S., & Yu, S. (2016). Down- regulation of miR-384-5p attenuates rotenone-induced neurotoxicity in dopaminergic SH-SY5Y cells through inhibiting endoplasmic reticulum stress. American Journal of Physiology. Cell Physiology, 310, C755–C763. https://doi.org/10.1152/ajpcell.00226.2015
+
+Kostich, W., Hamman, B. D., Li, Y. W., Naidu, S., Dandapani, K., Feng, J., Easton, A., Bourin, C., Baker, K., Allen, J., Savelieva, K., Louis, J. V., Dokania, M., Elavazhagan, S., Vattikundala, P., Sharma, V., Das, M. L., Shankar, G., Kumar, A., . . . Albright, C. F. (2016). Inhibition of AAK1 kinase as a novel therapeutic approach to treat neuropathic pain. Journal of Pharmacology and Experimental Therapeutics, 358, 371–386. https://doi. org/10.1124/jpet.116.235333
+
+Yu, Z. Y., Geng, J., Li, Z. Q., Sun, Y. B., Wang, S. L., Masters, J., Wang, D. X., Guo, X. Y., Li, M., & Ma, D. (2019). Dexmedetomidine enhances ropivacaine- induced sciatic nerve injury in diabetic rats. British Journal of Anaesthesia, 122, 141–149. https://doi.org/10.1016/j.bja.2018.08.022
+
+21579032, 2022, 7, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.2556 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+CHEN ET AL.
+
+11 of 11
+
+Zhang, X., Xue, Z., & Sun, A. (2008). Subclinical concentration of sevoflu- rane potentiates neuronal apoptosis in the developing C57BL/6 mouse brain. Neuroscience Letters, 447, 109–114. https://doi.org/10.1016/j. neulet.2008.09.083
+
+sevoflurane-induced apoptosis in the developing rat brain potentially via the mitochondrial pathway. Molecular Medicine Reports, 15, 2204–2212. https://doi.org/10.3892/mmr.2017.6268
+
+Zhang, X., Zhou, Y., Xu, M., & Chen, G. (2016). Autophagy is involved in the sevoflurane anesthesia-induced cognitive dysfunction of aged rats. Plos One, 11, e0153505. https://doi.org/10.1371/journal.pone.0153505 Zhao, X., Sun, Y., Ding, Y., Zhang, J., & Li, K. (2018). miR-34a inhibitor may effectively protect against sevoflurane-induced hippocampal apopto- sis through the Wnt/beta-catenin pathway by targeting Wnt1. Yonsei Medical Journal, 59, 1205–1213. https://doi.org/10.3349/ymj.2018.59. 10.1205
+
+How to cite this article: Chen, Y., Gao, X., & Pei, H. (2022).
+
+miRNA-384-3p alleviates sevoflurane-induced nerve injury by
+
+inhibiting Aak1 kinase in neonatal rats. Brain and Behavior, 12,
+
+e2556. https://doi.org/10.1002/brb3.2556
+
+Zhou, X., Xian, D., Xia, J., Tang, Y., Li, W., Chen, X., Zhou, Z., Lu, D., & Feng, X. (2017). MicroRNA-34c is regulated by p53 and is involved in

new_pdfs/10.1002_brb3.514.txt

@@ -0,0 +1,671 @@
+Early-life single-episode sevoflurane exposure impairs social behavior and cognition later in life Daisy Lin1,2,*, Jinyang Liu1,*, Lea Kramberg1, Andrea Ruggiero1, James Cottrell1 & Ira S. Kass1,2,3 1Anesthesiology Department, SUNY Downstate Medical Center, Box 6, 450 Clarkson Ave, Brooklyn, New York 11203 2Department of Physiology and Pharmacology, SUNY Downstate Medical Center, 450 Clarkson Ave, Brooklyn, New York 11203 3The Robert F. Furchgott Center for Neural and Behavioral Sciences, Brooklyn, New York 11203
+
+Keywords Cognition and social interaction, postnatal day 7, sevoflurane
+
+Correspondence Daisy Lin, Anesthesiology Department, SUNY Downstate Medical Center, Box 6, 450 Clarkson Ave, Brooklyn, NY 11203. Tel: 718-270-2048, 718-270-1709; Fax: 718- 270-3928; E-mail: daisy.lin@downstate.edu
+
+Funding Information This study was supported by the Anesthesiology Department, Brooklyn Anesthesia Research Division of the University Physicians of Brooklyn, Brooklyn, New York.
+
+Received: 3 September 2015; Revised: 12 May 2016; Accepted: 13 May 2016
+
+Brain and Behavior, 2016; 6(9), e00514, doi: 10.1002/brb3.514
+
+These authors contributed equally to the work.
+
+Abstract
+
+Background: Single-episode anesthetic exposure is the most prevalent surgery- related incidence among young children in the United States. Although numer- ous studies have used animals to model the effects of neonatal anesthetics on behavioral changes later on in life, our understanding of the functional conse- quences to the developing brain in a comprehensive and clinically relevant manner is unclear. Methods: The volatile anesthetic, sevoflurane (sevo) was administered to C57BL6 postnatal day 7 (P7) mice in a 40% oxygen and 60% nitrogen gas mixture. In order to examine the effects of sevo alone on the developing brain in a clinically relevant manner, mice were exposed to an aver- age of 2.38 (cid:1) 0.11% sevo for 2 h. No sevo (control) mice were treated in an identical manner without sevo exposure. Mice were examined for cognition and neuropsychiatric-like behavioral changes at 1–5 months of age. Results: Using the active place avoidance (APA) test and the novel object recognition (NOR) test, we demonstrated that P7 sevo-treated mice showed a deficit in learning and memory both during periadolescence and adulthood. We then employed a battery of neuropsychiatric-like behavioral tests to examine social interaction, communication, and repetitive behavior. Interestingly, compared to the no- sevo–treated group, sevo-treated mice showed significant reductions in the time interacting with a novel mouse (push–crawl and following), time and interac- tion in a chamber with a novel mouse, and time sniffing a novel social odor. Conclusions: Our study established that single-episode, 2-h sevo treatment dur- ing early life impairs cognition later on in life. With this approach, we also observed neuropsychiatric-like behavior changes such as social interaction defi- cits in the sevo-treated mice. This study elucidated the effects of a clinically rel- evant single-episode sevo application, given during the neonatal period, on neurodevelopmental behavioral changes later on in life.
+
+Introduction
+
+General anesthesia has been used in young children dur- ing surgical procedures dating back as early as the 1800s (Costarino and Downes 2005; Mai and Cote 2012). Cur- rently, an annual estimate of over 1 million children under the age of 4 received medical procedure-related anesthesia in the United States (Rabbitts et al. 2010). However, it is only in the last decade that we started to recognize that general anesthesia has deleterious effects on the developing brain (Jevtovic-Todorovic et al. 2003; Fre- driksson et al. 2007; Mellon et al. 2007; Slikker et al. 2007; Loepke and Soriano 2008; DiMaggio et al. 2009,
+
+2011; Wilder et al. 2009; Brambrink et al. 2010; Sun 2010; Stratmann 2011; Amrock et al. 2015). With arising concerns regarding the safe use of general anesthesia in young children, there is an urgent demand to clearly understand the resulting functional changes in the brain in a comprehensive and clinically relevant manner.
+
+This current study investigates the resulting functional changes that have been widely established, such as cogni- tion, as well as those still unknown, such as neuropsychi- atric disorders. Using the mouse as a model system, we examined these functional changes while maintaining a close relevance of our study approach to clinical settings. Our focus is to examine the implications of single-episode
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc. Brain and Behavior, doi: 10.1002/brb3.514 (1 of 14) This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
+
+21579032, 2016, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.514 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+D. Lin et al.
+
+Early-Life Sevoflurane Exposure on Behavior
+
+anesthetic exposure on early brain development because it is the most prevalent surgery-related incidence among children under the age of 4 (Wilder et al. 2009).
+
+et al. 2005; Read and Hammersley 2005; Cath et al. 2008; Champagne and Curley 2009; Roullet et al. 2013; Lee et al. 2015). Exposure to anesthetics during a critical developmental time period is a major environmental insult to the brain. However, the effect of early-life expo- sure to anesthetics on the development of neuropsychi- atric disorders is not clear and therefore is investigated as an area of functional changes in this current study.
+
+Numerous human and animal studies had been con- ducted to examine the association between early-life gen- eral anesthetic exposure and behavioral changes later on in life, with a focus on cognitive function (Jevtovic- Todorovic et al. 2003; DiMaggio et al. 2009; Sprung et al. 2009; Wilder et al. 2009; Flick et al. 2011; Murphy and Baxter 2013; Shen et al. 2013a). A majority of the human studies were conducted with a retrospective population cohort approach, by gathering data from a specific sub- population and identifying incidences of general anes- ages of 3–6. However, thetic the variations in these human study approaches such as dif- ferent assessment tools have led to inconsistent conclu- impairment sions; (DiMaggio et al. 2009, 2011; Sprung et al. 2009; Wilder et al. 2009), while others did not (Bartels et al. 2009; Kalkman et al. 2009; Hansen et al. 2011, 2013). Although prospective human studies have started to emerge in recent years, an agreement in outcome has yet to be established. An interim secondary outcome study from one group did not find increased risk of neurodevelop- mental outcome at 2 years of age (Davidson et al. 2016), while another group found cognitive impairment in chil- dren ages 6–11 years (Stratmann et al. 2014).
+
+We investigated the role of early-life exposure to sevoflurane on cognition and neuropsychiatric-like behav- ioral changes. Sevo is the most commonly used volatile anesthesia for surgical procedures on both children and adults in the United States (Sakai et al. 2005). By expos- ing postnatal day 7 (P7) mice to a single episode of sevo for 2 h, we established that cognitive ability was impaired later on in life. Interestingly, we also observed with three different behavior paradigms that early-life exposure to sevo resulted in social deficits. This study extends our awareness of the insults that single-episode exposure to sevo has on the developing brain, resulting in long-lasting functional changes that we can observe through behavior later on in life.
+
+exposure before
+
+some
+
+groups
+
+reported cognitive
+
+Materials and Methods
+
+Treatment with sevoflurane
+
+C57/BL6 mice were used throughout the study, which was approved by the SUNY Downstate IACUC. A total of nine litters of mice were used to establish the approxi- mate MAC for P7 mice. A separate set of 11 litters of mice were used for treatment without tail clamp and mice from this group were used for behavioral tests later on in life. At P7, all male pups from each litter (ranging from 2 to 6 pups) were randomly assigned to either the sevo or the no sevo (control) group, while the female pups remained with the dam. During a 2-h treatment period, pups from the sevo group were separated from the dam and exposed to sevo in a 40% oxygen (O2) and 60% nitrogen (N2) gas mixture (GTS-WELCO, Newark Distri- bution, Morrisville, PA). These pups were placed on a 37°C heating pad to prevent hypothermia during treat- ment. A pulse oximeter sensor (MSTAT 4 mm, Kent Sci- entific Corporations, Torrington, CN) was placed on one of the hind paws of the pup and measurements for heart rate (HR) and blood oxygen saturation (SpO2) were recorded every 5 min. To establish the approximate MAC of sevo on P7 mice, each treatment of sevo consisted of two mice and tail clamp was done every 10 min. The sevo concentration was adjusted to a higher concentration if both mice moved during tail clamp, adjusted to a lower concentration when neither mouse moved during tail clamp, and no adjustment was made when only one
+
+Animal studies, ranging from rodents to nonhuman primates, have persistently showed an association between early-life general anesthetic exposure and deficits in learn- ing and memory-related behavior. However, anesthetics such as isoflurane (iso), sevoflurane (sevo), or ketamine were generally given in the range of 4–8 h in rodents et al. 2008; (Jevtovic-Todorovic Loepke et al. 2009; Satomoto et al. 2009; Stratmann et al. 2009; Liang et al. 2010; Murphy and Baxter 2013; Shen et al. 2013a; Wang et al. 2013; Lee et al. 2014a), to as long as 5–24 h in monkeys (Zou et al. 2009, 2011; Bram- brink et al. 2010; Paule et al. 2011). As a comparison, children undergoing typically exposed to only 1 MAC (minimum alveolar concentra- tion) of iso or sevo for <1 h (Rabbitts et al. 2010). There- the reported behavior changes as a result of fore, anesthetic in animals have not accurately depicted the effects of anesthetics on the developing brains of young children.
+
+et al. 2003; Sanders
+
+surgeries
+
+are
+
+routine
+
+exposure
+
+The developing brain is vulnerable to a variety of envi- ronmental insults, ranging from deprivation of maternal care to toxin and drug exposure, resulting in increased risk of neuropsychiatric disorders, such as autism spec- trum disorder, depression, anxiety, bipolar disorder, schizophrenia, and obsessive compulsive disorder (Pichot 1986; Ansorge et al. 2004; Batten et al. 2004; Phillips
+
+Brain and Behavior, doi: 10.1002/brb3.514 (2 of 14)
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.
+
+21579032, 2016, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.514 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+D. Lin et al.
+
+Early-Life Sevoflurane Exposure on Behavior
+
+mouse responded to stimuli. This was our approach to determine that with a given sevo concentration, 50% of mice did not respond to stimuli. The sevo concentration was recorded every 5 min since that was the time interval that we used to record peripheral capillary oxygen satura- tion (SpO2) and HR. The pups from the control group were also separated from the dam and exposed only to 40% O2 and 60% N2. At the end of the 2-h treatment the pups were returned to their home cage and reunited with their dams. All pups were then reared and weaned follow- ing standard institution procedures.
+
+explore the open field arena. Locomotion activities such as distance and time in different compartments of the arena were automatically measured using a computerized track- ing apparatus (Versadat, Versamax, Groovy, CA).
+
+Learning and memory-like behavior
+
+Active place avoidance test
+
+The APA test is a hippocampus-dependent spatial mem- ory test. A rotating arena consisting of a circular platform (40 cm diameter) was placed in the center of a dimly lit room. The mouse was trained to avoid a 60° shock zone, which could be defined within a region of the room iden- tified by multiple visual cues (Fenton et al. 1998; Wesier- ska et al. 2005). Two-day trials were performed as described previously (Burghardt et al. 2012). Briefly, the mouse was given 10 min for each trial with at least a 50- min intertrial interval. The locomotion of the mouse was tracked by computer-based software that analyzed images from an overhead camera and delivered shocks appropri- ately (Tracker, Bio-Signal Group Corp., Brooklyn, NY). A brief constant current shock (500 msec, 60 Hz, 0.2 mA) across pairs of rods was delivered to the shock zone upon entrance of the mouse. Track analysis software (Bio-Signal Group Corp.) was used to compute the num- ber of times that the mouse entered the shock zone.
+
+Behavior tests
+
+The mice that were involved in the behavior tests had undergone a 2-h sevo or no sevo treatment at P7 without tail clamping. They were reared and group housed under standard conditions. The sevo-treated mice were marked to distinguish them from the no-sevo–treated mice within a litter. We examined at most one to two litters of mice at a time for each behavior, with at least 1 week of resting time in between different behavioral tests. The behavior tests were given sequentially for the active place avoidance (APA), reciprocal social interaction, and olfaction habitua- tion/dishabituation. After completion of these tests, we then introduced three-chamber interaction, open field, and novel object recognition (NOR). All behavioral apparatus were assembled and remained in their original locations throughout the entire duration of the project. The APA test was done on mice starting at the age of P27. All other behaviors were conducted on mice within the age range of 1.5–5 months old. The following reasons contributed to variation in the number of mice used for some tests. First, we were not able to examine all treated mice on the APA due to irreparable malfunctioning of the APA apparatus. Therefore, we introduced NOR as a second cognition test on mice that had not been used for the APA. Second, some mice were not included in testing if they were not within the age range at the time of the test, specifically the second group of tests such as three-chamber interaction, open field, and NOR. Besides the APA and the open field, all other tests that required manual scoring were first video- taped and then scored by experimenters who were blind to the treatment status of the mice.
+
+foot
+
+Novel object recognition test
+
+This is a two consecutive day test examining learning and memory-like behavior on adult male mice (3–5 months of age; Leger et al. 2013). The test was conducted in a room with dim lighting. Day 1 is considered the familiarization phase. Mice were individually habituated in a standard open field apparatus for 10 min. They were then taken out of the arena briefly and two identical glass bottles filled with pink silica gel were placed in the center of the arena. The glass bottles were positioned 5 inches from each other such that the mouse can travel freely across the center of the arena without obstruction. The mouse was then put back in the arena and allowed 10 min to become familiar with the two identical objects. Day 2 is the test phase. One of the glass bottles is taken out of the arena and replaced with a yellow laboratory tube rack (H 6.5, W 3.5, D 2 inches) as a novel object. The holes on the sides of the tube rack were taped to prevent the mouse from climbing on them during the experiment. The same mouse was placed in the arena for 10 min in an identical manner as day 1 and allowed 10 min of exploration time. The times spent sniff- ing and interacting with (attempting to climb up or jump on or at) the familiar and the novel objects were scored for each mouse.
+
+Locomotion and anxiety-like behavior
+
+Open field test
+
+An open field apparatus was used to assess the general physical and anxiety-like performance of the mice based on their ambulatory locomotion in the arena (Crawley 1985). In a well-lit novel room, each mouse was given 30 min to
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.
+
+Brain and Behavior, doi: 10.1002/brb3.514 (3 of 14)
+
+21579032, 2016, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.514 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+D. Lin et al.
+
+Early-Life Sevoflurane Exposure on Behavior
+
+presentation of odor lasted 2 min. The amount of time that the mouse spent sniffing the cotton swab, including nose poking, chewing, sniffing, and close proximity (2 cm) of the nose to cotton swab was scored (Silverman et al. 2010).
+
+Social interactions
+
+Reciprocal social interaction
+
+The subject mouse (P7 sevo treated or no sevo control) was transferred from his home cage to a new cage with fresh bedding and allowed to habituate to the cage for 10 min. At the end of this 10-min session, a novel male target mouse (that had not undergone treatment during P7) of similar age was introduced into the same cage. The subject and the target mice were allowed to interact for 10 min. The amount of time that the subject mouse spent interacting with the target mouse (push–crawl/fol- lowing behavior), self-grooming, and exploring the arena and the total time the mouse was mobile were scored manually (Silverman et al. 2010).
+
+Statistical analysis
+
+All statistical analysis was done using GraphPad Prism 5.0 (GraphPad, San Diego, CA). Data with one variable such as the open field and the reciprocal social interaction were analyzed by t test. Data with two variables such as the APA, the NOR, the three-chamber interaction, and the olfaction habituation/dishabituation were analyzed by two-way ANOVA, followed by Bonferroni posttests.
+
+Results
+
+Three-chamber interaction
+
+A three-chamber apparatus made of clear plexiglass was used for this study (Nadler et al. 2004). The apparatus is divided into three equally sized compartments (H 9.5, W 8, D 16 inches). First, the subject mouse was habituated for 10 min in the center chamber. Then the doors that give access to the left and right sides of the chamber were opened allowing the subject mouse to freely explore all three chambers for 10 min. During this time, the novel target mouse was habituated under a wire pencil cup on a separate tabletop. After 10 min of three-chamber explo- ration, the doors were closed and the subject mouse was briefly confined in the center chamber. During this time, we set up the three-chamber apparatus such that the novel target mouse was placed on one side of the cham- ber and a novel empty pencil cup on the other side. A weighted plastic cup was placed on the top of each pencil holder to prevent the subject mouse from climbing on the top of it. The doors were then opened to allow the subject mouse to explore the three chambers for 10 min.
+
+Establishing a mouse model of neonatal sevoflurane treatment
+
+In order to understand the effect that neonatal sevo treat- ment alone has on behavioral changes later on in life, we established two different sevo treatment groups. During a 2-h treatment period, we used tail clamp to first establish that the approximate MAC of sevo for P7 mice averaged 3.58 (cid:1) 0.07% (Fig. 1A). Since tail clamp may result in pain and scaring of the tail similar to the act of clinical surgery, we then treated a separate group of mice in a sim- ilar manner but without tail clamp. This second group of mice was treated with a reduced concentration of sevo, which was sufficient to keep the mice immobilized/uncon- scious and this sevo concentration averaged 2.38 (cid:1) 0.11% (Fig. 1B). These mice were subsequently examined for the effect of neonatal sevo treatment alone on behavioral changes later on in life. Mice were monitored closely for their measurements of peripheral capillary oxygen satura- tion (SpO2) and HR during the treatment. An average SpO2 of 97 (cid:1) 0.11% and HR of 427 (cid:1) 2.02 beats per min suggest the mice were in a physiological healthy state, without any signs of hypoxia (Fig. 1C and D).
+
+Communication
+
+Olfaction habituation/dishabituation
+
+The mouse was transferred to a new cage containing a thin layer of fresh bedding and a hole for inserting a cotton tipped swab. After a 10-min habituation period in the new cage, the mouse was presented with nonsocial and social odors. Each odor was presented for three consecutive times; the order of presentation was water, almond extract (1:100, Spice Supreme), orange extract (1:100, McCor- mick), mouse socials 1 and 2. The mouse social odors were taken by wiping in a zigzag pattern across the bottom sur- face of different cages for odors 1 and 2; each cage housed the same sex and strain. Each unfamiliar mice of
+
+Locomotion and anxiety-like behavior
+
+Locomotion and movement of the limbs are critical to all mouse behavior. Therefore, the no–sevo- and sevo-treated mice were examined for their locomotion in the open field apparatus as a general physical assessment (Crawley 1985, 2007; Fig. 2). This brightly lit, novel test environ- ment with an unprotected center is also anxiety provok- ing. The two groups of mice were examined for their exploration in the center versus the total arena as a mea- surement of anxiety-like behavior. We observed no
+
+Brain and Behavior, doi: 10.1002/brb3.514 (4 of 14)
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.
+
+21579032, 2016, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.514 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+D. Lin et al.
+
+Early-Life Sevoflurane Exposure on Behavior
+
+Figure 1. Two approaches of sevo treatment on postnatal day 7 (P7) mice. (A) The approximate minimum alveolar concentration (MAC) of sevo necessary for P7 mice was established by tail clamping every 10 min during treatment. The approximate MAC of sevo was averaged to be 3.58 (cid:1) 0.07% based on the concentration recorded every 5 min (N = 13). (B) On a separate group of mice, less than one MAC (2.38 (cid:1) 0.11%) of sevo was given without tail clamping. This group of mice was then used in all the subsequent behavior paradigms (N = 17). (C and D) Data shown are measurements taken from the group of mice that were exposed to sevo with tail clamp. SpO2 and heart rate were recorded every 5 min on all mice undergoing sevo treatment.
+
+Figure 2. Postnatal day 7 sevo treatment did not have an effect on locomotion and anxiety-like behavior later on in life. No differences were observed between the two groups (no sevo vs. sevo) on different measurements of the open field apparatus such as (A) total distance traveled and the (B) ratio of center/total distance traveled. Unpaired t-test with Welch’s correction was used for statistical calculation (N = 14 for no sevo; N = 13 for sevo).
+
+two groups on locomotion differences between the (Fig. 2A) and anxiety-like behavior (Fig. 2B; unpaired t- test with Welch’s correction). Data suggest that locomo- tion and anxiety-like behaviors are not potential con- founds to subsequent behavioral tests.
+
+five trials during day 1 and this behavior persisted into day 2. A similar observation was not present in the sevo group. Sevo-treated mice showed significantly more entrances into the shock zone over the 2 days, 10-trial period (two-way ANOVA followed by Bonferroni postt- ests, P < 0.01 for treatment, P < 0.001 for trial, P > 0.05 for interaction between treatment and trial, and P < 0.05 for treatment effect within trials, day 1—trials 4 and 5 and day 2—trial 2).
+
+Learning and memory
+
+Active place avoidance
+
+We observed learning and memory impairment as early as periadolescent age (P27 and P28), based on the hip- pocampus-dependent APA test. The mouse learned to avoid a stationary shock zone in a constant rotating arena using the distal room landmarks as cues (Fig. 3). The no- sevo–treated mice learned to avoid the shock zone over
+
+Novel object recognition
+
+We examined cognitive function of adult mice (ages 4– 5 months) by the NOR test. The NOR is a widely used learning and memory task, which offers no external stim- uli or reinforcement (Leger et al. 2013). During day 1—
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.
+
+Brain and Behavior, doi: 10.1002/brb3.514 (5 of 14)
+
+21579032, 2016, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.514 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+D. Lin et al.
+
+Early-Life Sevoflurane Exposure on Behavior
+
+Figure 3. Neonatal sevo treatment impaired learning and memory during periadolescence. (A) Locomotion of a no- sevo- and a sevo-treated mouse are represented by traces on the circular rotating platform. The arrow indicates the direction of platform movement (1 revolution per minute). Entrances into the shock zone (represented by the 60° red zone) are marked as the red dots. (B) At P27 and P28, sevo-treated mice entered the shock zone significantly more compared to the no-sevo–treated group during a total of 10 trials. Two-way ANOVA was used for statistical calculation: F(1, 63) = 1.28, P < 0.01 for treatment; F(9, 63) = 14.6, P < 0.001 for trials; F(9, 63) = 1.8, P > 0.05 for interaction between treatment and trials. Bonferroni posttests showed P < 0.05 for treatment on day 1, trials 4 and 5; day 2, trial 2. Asterisk (*) denotes P < 0.05 for treatment effect within the trials (N = 4 for no sevo, N = 5 for sevo).
+
+familiarization (Fig. 4A), both the no-sevo- and sevo-trea- ted groups spent a similar amount of time exploring the two identical objects, with no differential preference for a specific object (Fig. 4B; two-way ANOVA). However, dur- ing day 2—testing (Fig. 4A), the no sevo group spent sig- this nificantly more time exploring the novel object; difference was not found in the sevo-treated group (Fig. 4C; two-way ANOVA, followed by Bonferroni postt- ests, P < 0.01 for time exploring the objects in the no sevo group). Combining the two sets of learning and memory behavioral tests, data demonstrated for the first time, a 2-h, single-episode neonatal exposure to less than one MAC of sevo impairs learning and memory as early as periadolescence and this persists to adulthood.
+
+direct insight into how two unfamiliar mice interact in a standard new cage.
+
+We scored the four most predominant behaviors of the subject mouse while paired with the target mouse in the novel cage, such as push–crawl/following, arena explo- ration, self-grooming, and time being mobile. Among the four measurements, sevo-treated mice showed a specific deficit compared to no-sevo–treated mice on the amount of time they engage in push–crawling and following the unfamiliar mouse (Fig. 5; unpaired t-test with Welch’s correction, P < 0.05). Data show mice exposed to single- episode sevo treatment on P7 had impaired social interac- tion later on in life.
+
+Three-chamber social interaction
+
+Social interactions
+
+We wanted to further understand whether the two groups of P7-treated mice would differ in a social behavior para- digm that is self-directed, without the physical elicitation from the novel target mouse. To address this question, interaction three-chamber we
+
+Reciprocal social interaction
+
+Mice are social animals that engage in a variety of social interaction behaviors. The reciprocal social interaction paradigm is designed to provide the most detailed and
+
+employed the
+
+social
+
+Brain and Behavior, doi: 10.1002/brb3.514 (6 of 14)
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.
+
+21579032, 2016, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.514 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+D. Lin et al.
+
+Early-Life Sevoflurane Exposure on Behavior
+
+confined under a wire pencil cup on one of the two sides of the three-chamber apparatus (Fig. 6A). Confining the target novel mouse under the wire pencil cup prevented aggressive interaction between the two unfamiliar mice while providing olfactory, visual, auditory, and tactile contact.
+
+In the no sevo group, mice spent significantly more time in the chamber with the confined novel mouse com- pared to the chamber with the novel object (an empty pencil cup; Fig. 6B; two-way ANOVA, followed by Bon- ferroni posttests, P < 0.001). This behavior was not pre- sent in which mice spent similar amount of time in the mouse chamber and the object chamber. Although the subject mouse was always placed in the center chamber at the initiation of the experiment, both no sevo and sevo groups showed the least interest in the center chamber compared to the two side chambers (Fig. 6B; two-way ANOVA, followed by Bonferroni posttests, P < 0.001). While in the specific chambers, we made similar observations on the time that the no sevo versus sevo mouse spent interacting with the novel mouse or the novel object, such as nose poking or sniffing. The mice from the no-sevo–treated group had significantly more interest in interacting with the novel mouse rather than the object, while the sevo-treated two-way group did not show a preference (Fig. 6C; followed by Bonferroni posttests, P < 0.001). ANOVA, Combining data from this experiment and reciprocal social interaction, we demonstrated that early-life sevo treatment impacts social interaction later on in life.
+
+for the sevo-treated group,
+
+Communication
+
+Olfactory
+
+Olfactory cues are considered to be important for rodent choice, mother–infant communication such as mate bonding, aggressive interaction, territory recognition, and social bonding (Harrington 1976; Clutton-Brock 1989; Ferguson et al. 2001; Broad et al. 2006; Stowers et al. 2013). Since the effect of early-life general anesthetic exposure on communication is unclear, we examined this behavior by the olfactory habituation/dishabituation para- digm (Crawley et al. 2007).
+
+Figure 4. Neonatal sevo-treated mice showed impairment in learning and memory during adulthood. (A) A schematic layout of the novel object recognition task that was used to examine the learning and memory behavior in mice. (B) During day 1—familiarization, both no sevo and sevo groups spent similar amounts of time exploring the two identical objects. No preference for a specific object was detected. A statistical analysis, P > 0.05 for two-way ANOVA was used for treatment, object, and interaction between treatment and object. (C) During day 2—testing, while the no-sevo–treated group spent significantly more time exploring the novel object, the sevo-treated group did not show an increased interest. Two-way ANOVA, followed by Bonferroni posttest were used for statistical analysis, F(1, 36) = 3.6, P > 0.05 for treatment; F(1, 36) = 10.4, P < 0.01 for object; F(1, 36) = 3.6, P > 0.05 for interaction between treatment and object. Asterisk (**) denotes P < 0.01 for exploration time between the two different objects in the no sevo group (N = 11 for no sevo; N = 9 for sevo).
+
+Mice were presented with three different nonsocial odors and two different social odors on cotton swabs. Both the sevo- and no-sevo–treated groups were able to habituate to the same odor when it was presented three consecutive times. This indicated by a significant decrease in time spent sniffing the cotton swabs of the same odor from the first to the third presentation (Fig. 7; repeated measure two-way ANOVA, P < 0.0001 for time spent sniffing cotton swabs from the first to the third
+
+is
+
+paradigm (Nadler et al. 2004; Silverman et al. 2010). In this experimental setup, the subject mouse initiated the social approach, while the target novel mouse was
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.
+
+Brain and Behavior, doi: 10.1002/brb3.514 (7 of 14)
+
+21579032, 2016, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.514 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+D. Lin et al.
+
+Early-Life Sevoflurane Exposure on Behavior
+
+Figure 5. Neonatal sevo-treated mice had a deficiency in social interaction behaviors as shown by reciprocal social interaction. (A) The sevo-treated mice showed significantly less push–crawl and following toward a novel mouse of the same sex and similar age compared to the no-sevo– treated mice. (B–D) During the 10 min of the reciprocal social interaction paradigm, the two groups of mice did not display a difference in arena exploration, self- grooming, or total time being mobile in the cage. Unpaired t-test with Welch’s correction was used for statistical calculation. Asterisk (*) denotes P < 0.05 (N = 18 for no sevo; N = 17 for sevo).
+
+(A)
+
+(B)
+
+(C)
+
+Figure 6. Neonatal sevo-treated mice had a deficiency in social interaction behaviors as shown by three-chamber social interaction. (A) A three- chamber apparatus was used to examine social interaction as shown in the photograph. The apparatus is divided into an object chamber (left), a center chamber (center), and a novel mouse chamber (right). The doors on both the left and the right side of the center chamber are opened to allow the subject mouse to travel freely in between all three chambers. (B) The no sevo group spent significantly more time in the novel target mouse chamber compared to the object chamber, while the sevo group showed no such preference. Both groups of mice showed the least preference for the center chamber in which there was no novel target mouse or object present. Two-way ANOVA, followed by Bonferroni posttest were used for statistical analysis, F(1, 81) = 0, P > 0.05 for treatment; F(2, 81) = 46.3, P < 0.0001 for chamber; F(2, 81) = 2.9, P > 0.05 for interaction between chamber and treatment. Asterisk (***) denotes P < 0.001 for time in mouse versus object chamber for the no sevo group; P < 0.001 for time in mouse versus center and object versus center chamber for both the no sevo and the sevo groups. (C) A similar observation was made for the time the mice spent interacting with the novel target mouse or the novel object (sniffing and nose poking). The no-sevo– treated mice spent significantly more time interacting with the novel target mouse than the novel object. Such an observation was not present in the sevo-treated group. Two-way ANOVA, followed by Bonferroni posttest were used for statistical analysis, F(1, 53) = 1.3, P > 0.05 for treatment; F(1, 53) = 14.6, P < 0.001 for subject/object; F(1, 53) = 1.8, P > 0.05 for interaction between treatment and subject/object. Asterisk (***) denotes P < 0.001 for time spent interacting with subject/object for the no sevo group (N = 16 for no sevo; N = 13 for sevo).
+
+Brain and Behavior, doi: 10.1002/brb3.514 (8 of 14)
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.
+
+21579032, 2016, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.514 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+D. Lin et al.
+
+Early-Life Sevoflurane Exposure on Behavior
+
+dishabituation in nonsocial odors, we noticed a difference in their behavior toward the social odors. The sevo-trea- ted mice showed significantly less interest in sniffing com- pared to no-sevo–treated mice when novel mouse 2 odors were presented (repeated measure two-way ANOVA, P = 0.05 for treatment). These data further validate a deficiency in social interaction behavior in P7 sevo-treated mice, an observation that has been recapitulated in two other behavioral paradigms in this current study: recipro- cal social and three-chamber social interaction.
+
+Discussion
+
+In the United States, children under the age of 4 requir- ing single-episode exposure have the highest prevalence compared to two or more exposures (Wilder et al. 2009). Therefore, the effect of early-life single-episode anesthetic exposure on the long- term functional consequence of brain development is par- ticularly important. Our goal for this study is to capture this effect in our mouse-model system using a clinically relevant anesthetic concentration and duration such that we can apply the results to better understand the risks in children. The major findings in this study are that P7 sevo-exposed mice had impairments in cognition and social interaction behaviors later on in life.
+
+surgery-related anesthetic
+
+Figure 7. Both neonatal no-sevo- and sevo-treated mice showed no impairment olfactory habituation/dishabituation. Mice were able to habituate to the same scent when presented three consecutive times. This was shown by a significant decrease in the time spent sniffing from the first to the last presentation of the same scent. Repeated measure two-way ANOVA resulted in F(2, 66) = 40, P < 0.0001 for almond; F(2, 66) = 40, P < 0.0001 for orange; and F(2, 66) = 40, P < 0.0001 for social 1 and F(2, 66) = 5, P < 0.01 for social 2. There was no treatment difference on nonsocial and social 1 odor habituation. These mice were also able to dishabituate from old scents when new scents were presented. This was shown by a significant increase in every transition between the last presentation of the old scent to the first presentation of the new scent. A two-way ANOVA resulted in F(1, 66) = 31, P < 0.0001 for transition from water to almond; F(1, 66) = 20, P < 0.0001 for transition from almond to orange; F(1, 66) = 127, P < 0.0001 for transition from orange to social 1; and F(1, 66) = 14, P < 0.001 for transition from social 1 to social 2. There was no treatment difference on odor dishabituation. However, sevo-treated mice were observed to have a social interaction abnormality in this paradigm. There was a treatment difference on in which a repeated measure two-way social 2 odor habituation, ANOVA resulted in F(1, 66) = 4, P = 0.05 for treatment. Asterisk (*) denotes P = 0.05 for treatment effect on social 2 odors: mouse 2-1, 2-2, and 2-3 (N = 18 for no sevo; N = 17 for sevo).
+
+in
+
+communication
+
+behavior
+
+based
+
+on
+
+F(2,
+
+66) = 30, P < 0.0001 for water;
+
+It has been more than a decade since the first study in rodents demonstrated anesthetic neurotoxicity in the developing brain, resulting in impairment in cognitive function later on in life (Jevtovic-Todorovic et al. 2003). However, the effects of single-episode neonatal anesthetic exposure still face skepticism due to three major issues. First, animal studies lack a close mimic of anesthetic commonly applied to children treatment parameters among the single-episode exposure group. Second, a more comprehensive understanding of the functional conse- quences of single-episode exposure on different behavioral changes with disease implications has not been estab- lished. Third, it is difficult to dissociate general anesthesia from the underlying disease, coexisting conditions, or sur- gical procedures study focuses on addressing these critical questions and they are discussed in detail in the following sections.
+
+presentation of the odors: water, almond, orange, and social 1; repeated measure two-way ANOVA, P < 0.01 for time spent sniffing cotton swabs from the first to the third presentation of the odor, social 2). There was no treatment difference on habituation for nonsocial and social 1 odors. Presentation of new odors elicited increased interest such that both groups of mice were able to dishabituate from the old scent to the new scent. This is indicated by a significant increase in time spent sniffing the cotton swab from the last presentation of the old odor to the first presentation of the new odor (two-way ANOVA, P < 0.01–0.0001 for change of odor: transition from an old to a new odor). While both groups of mice showed similar behavior in olfactory cue habituation/
+
+that
+
+require anesthesia. This
+
+Single-episode anesthetic treatment in neonates
+
+Skepticism toward the effect of neonatal single-episode anesthetic exposure arises mainly due to inconsistency in experimental approaches, which ranges from anesthetic type to treatment duration and concentration. We chose to study only one type of anesthetic, sevoflurane, for sev- eral reasons. Unlike some other animal studies that used
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.
+
+Brain and Behavior, doi: 10.1002/brb3.514 (9 of 14)
+
+21579032, 2016, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.514 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+D. Lin et al.
+
+Early-Life Sevoflurane Exposure on Behavior
+
+different anesthetic cocktails, treatment with sevo alone in this study provides a clear picture of its specific effects on the developing brain. Anesthetic cocktails that others have used to establish mouse models of neonatal anesthetic effects included nitrous oxide, which is often used as one of the induction agents, or ketamine, which is often used as procedural sedation or premedication in emergency rooms, but not as anesthetic maintenance for pediatric ambulatory surgeries (Alderson and Lerman 1994; Mellon et al. 2007; Lee et al. 2013). However, sevo is used for both anesthetic induction and maintenance, therefore it is often the main anesthetic agent during a pediatric surgical procedure (Goa et al. 1999).
+
+The effect of neonatal single-episode anesthetic exposure on different behavioral changes
+
+One of the significances of the current study is that we demonstrated impairment in cognitive behavior by adher- ing our study approach closely to clinical scenarios and is based on two cognitive tasks that involve different brain regions. By the APA test, we demonstrated that P7 sevo- treated mice had impairment in the hippocampus-depen- dent spatial memory as early as periadolescent age (P27– P28; Fig. 3). Previous functional inactivation of the dorsal spatial hippocampus blocked both the acquisition of memories and the retrieval of long-term spatial avoidance memories on the APA (Fenton et al. 1998; Cimadevilla et al. 2000; Kubik and Fenton 2005; Wesierska et al. 2005). Lesions in other brain regions such as the fornix or the anterior thalamus have also resulted in deficits in long-term spatial memory (Aggleton et al. 1996); however, region-specific tasks are needed to suggest their vulnerabil- ity to the effects of sevo during neonatal period. Since sevo targets receptors such as GABAA, glycine, and nicotinic acetylcholine that are ubiquitously expressed throughout the central nervous system (CNS) (Campagna et al. 2003; Rudolph and Antkowiak 2004), we wondered what other brain regions might be involved in neonatal sevo-induced cognitive deficits. To approach this question, we examined the mice on a different cognition task, NOR (Fig. 4). This task was chosen specifically because unlike the APA, which is hippocampus-dependent, this task examines recognition memory formation and is associated with the cortex, espe- cially the medial temporal lobe and the thalamus (Brown and Aggleton 2001; Norman and Eacott 2004; Aggleton et al. 2011; Warburton and Brown 2015). We speculate that neonatal sevo-induced cognitive impairment is associ- ated with these brain regions, but would require future work at the anatomical, morphological, and electrophysi- ology level to support this.
+
+To translate animal data and apply it to understand the clinical implications of general anesthesia, it is critical to adhere to treatment parameters commonly used among young children. Several of the most frequently performed procedures among children under the age of 4 are tonsil- lectomy, circumcision, hernia repair, and myringotomy with ear tube (Rabbitts et al. 2010). Children undergoing these types of surgeries are typically among the single-epi- sode anesthetic exposure group. The average duration of these types of surgeries is under 1 h and general anesthe- sia is most frequently used. However, among P7 rodent studies, exposure to anesthetics ranged from 4 to 6 h et al. 2008; (Jevtovic-Todorovic Loepke et al. 2009; Satomoto et al. 2009; Stratmann et al. 2009; Liang et al. 2010; Lee et al. 2014a,b). Longer expo- sure times ranging from 5 to 24 h were examined in monkeys (Zou et al. 2009, 2011; Brambrink et al. 2010; Paule et al. 2011). This is a 4- to 24-fold increase in treat- ment duration compared to pediatric ambulatory surg- eries and is not a realistic prediction for children among the single-episode exposure group.
+
+et al. 2003; Sanders
+
+Our study approach consisted of 2 h of sevo treatment (Fig. 1). We did not reduce exposure to 1 h, but recog- nize that further reducing the duration would be a closer mimic to the common clinical surgeries among young children. Nevertheless, our approach is already the short- est duration currently used in animal studies, as we are unaware of any animal study with <2 h exposure dura- tion. While others have used a range of 1–4% sevo on P7 rodents (Satomoto et al. 2009; Liang et al. 2010; Feng et al. 2012; Kato et al. 2013; Ramage et al. 2013; Shen et al. 2013b; Amrock et al. 2015), we used an average dosage of 2.38% of sevo. This concentration was less than the MAC for P7 mice, but was sufficient to keep the mice immobilized/unconscious throughout the duration of the treatment (Fig. 1). This further illustrates the deleterious effects of early-life sevo, resulting in behavioral changes later on in life. Future works on fine-tuning procedural approaches in order to establish the best rodent model for translational research are still necessary.
+
+Another significance of the current study is our investi- gation of the effects of neonatal sevo exposure on the development of neuropsychiatric-like behavioral changes. Perturbation of the developing brain due to environmen- tal insults is associated with numerous neurodevelopmen- tal disorders. The exposure of human fetuses or neonatal rodents is paradoxically linked to changes in emotional behavior or the development of depression/anxiety later on in life (Ansorge et al. 2004; Hanley et al. 2013). Alcohol exposure during gestation is associated with a wide range of neurobehavioral disorders, including mood, cognition, and social interaction changes (Streissguth et al. 2004). Aside from toxins and drugs, early-life abuse and neglect has been demonstrated to increase susceptibility to depression, schizophrenia, and
+
+to antidepressants
+
+Brain and Behavior, doi: 10.1002/brb3.514 (10 of 14)
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.
+
+21579032, 2016, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.514 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+D. Lin et al.
+
+Early-Life Sevoflurane Exposure on Behavior
+
+accompany surgery-related anesthesia. Many studies used tail clamping of animals to establish the required MAC of anesthetics. Such approaches mimic clinical surgical pro- cedures and are good animal models used to understand surgical-related anesthesia. However, to understand the effect of anesthesia alone, we need to completely dissoci- ate anesthesia from the act of surgery. Previously, two groups conducted learning and memory behavioral tests on neonatal sevo-treated rats without tail clamping (Shih et al. 2012; Stratmann et al. 2014). However, rats in both groups were treated for 4 h with sevo ranging from 2.1% to 5.3%. Long treatment duration indicates potential resulted in physiologically decreased survival rates from 92% to 67% between the 2 and 4 h (Shih et al. 2012). The present study demon- strated for the first time a single-episode, 2-h treatment by sevo alone during the neonatal period impairs both social interaction and cognition.
+
+anxiety-related disorders (Batten et al. 2004; Phillips et al. 2005; Read and Hammersley 2005; Champagne and Cur- ley 2009). Anesthetic exposure during neonatal surgical treatment is also an environmental insult to the develop- ing brain. Therefore, we used a comprehensive approach by including social interaction (Figs. 5, 6), communica- tion (Fig. 7), and repetitive behavior (Data S1) to better understand the neurodevelopmental changes of these mice. Among these behaviors, social interaction impair- ment associated with neonatal sevo treatment has been reported previously by another group (Satomoto et al. 2009). Using a caged social target in an open field appa- ratus, the group demonstrated that the no-sevo–treated control group interacted significantly more with the social target compared to the sevo-treated group. The novelty of the current study is, not only did we observed a deficit in social behavior in three different paradigms, reciprocal social interaction, and olfactory habituation/dishabituation (Figs. 5–7); our study approach, compared to Satomoto et al., consisted of a threefold reduced treatment duration (2 hr, Lin et al., vs. 6 hr, Satomoto et al.) and a lower sevo dosage (2.38% sevo, Lin et al., vs. 3% sevo, Satomoto et al.).
+
+intolerable
+
+toxicity;
+
+this
+
+interaction, three-chamber social
+
+Concluding Remarks
+
+We took the top-down approach to study the effects of neonatal exposure to sevo by incorporating a battery of behavior paradigms. Our group showed for the first time that a single-episode, 2-h treatment of 2.3% sevo during the neonatal period impairs both social interaction and cognition later on in life. This study provides insight into the effects that clinically relevant neonatal anesthesia expo- sure have on the long-term functional changes in the brain. With this information, we will then be able investigate the associated changes in electrophysiology, morphology, sig- nal pathways, and molecular mechanisms. We are hopeful that our cumulative understanding will result in future therapeutic targets that may reverse the deleterious effects of early-life anesthetic exposure on the developing brain.
+
+Changes in social behavior could arise from an imbal- ance in excitatory/inhibitory neurotransmission, which has been shown in both mouse models of autism (Chao et al. 2010; Auerbach et al. 2011; Peca et al. 2011; Penagarikano et al. 2011; Han et al. 2012) and by optogenetic manipula- tion (Yizhar et al. 2011). It is temping to hypothesize that sevo’s activation of GABAA receptors in the developing brain may contribute to this imbalance. GABA exerts exci- tatory signaling in neurons during early development and then undergoes a switch to inhibition (Ben-Ari et al. 2007). In rodents, the GABA switch extends over the entire second postnatal week and is completed in the third extruder K+/ week. Developmental expression of the Cl cotransporter, KCC2 is pivotal for the change from Cl depolarizing to hyperpolarizing GABAA-mediated action (Ben-Ari et al. 2007). Exposure to sevo at P7 is concurrent with GABA’s excitatory/inhibitory switch and the expres- sion of KCC2. Whether excess activation of the GABAA receptor by sevo during this critical developmental period has an effect on the expression of KCC2, is unknown. Future investigation on sevo’s interference with the nor- mal developmental excitatory/inhibitory switch would provide insight into the mechanisms underlying the observed functional changes of the brain.
+
+(cid:3)
+
+(cid:3)
+
+Acknowledgments
+
+We are thankful for the generous funds to carry out this research that were provided by the Anesthesiology Department through the Brooklyn Anesthesia Research Division of the University Physicians of Brooklyn, Brook- lyn, New York.
+
+Conflict of Interest
+
+None declared.
+
+Dissociating general anesthesia from surgery
+
+References
+
+Aggleton, J. P., P. R. Hunt, S. Nagle, and N. Neave. 1996. The effects of selective lesions within the anterior thalamic nuclei on spatial memory in the rat. Behav. Brain Res. 81:189–198.
+
+Animal models provide an ideal system to dissociate the that underlying diseases
+
+and coexisting
+
+conditions
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.
+
+Brain and Behavior, doi: 10.1002/brb3.514 (11 of 14)
+
+21579032, 2016, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.514 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+D. Lin et al.
+
+Early-Life Sevoflurane Exposure on Behavior
+
+autism-like stereotypies and Rett syndrome phenotypes. Nature 468:263–269.
+
+Aggleton, J. P., J. R. Dumont, and E. C. Warburton. 2011. Unraveling the contributions of the diencephalon to recognition memory: a review. Learn. Mem. 18:384–400. Alderson, P. J., and J. Lerman. 1994. Oral premedication for
+
+Cimadevilla, J. M., A. A. Fenton, and J. Bures. 2000.
+
+Functional inactivation of dorsal hippocampus impairs active place avoidance in rats. Neurosci. Lett. 285:53–56. Clutton-Brock, T. H. 1989. Mammalian mating systems. Proc.
+
+paediatric ambulatory anaesthesia: a comparison of midazolam and ketamine. Can. J. Anaesth. 41:221–226.
+
+R. Soc. Lond. B Biol. Sci. 236:339–372.
+
+Amrock, L. G., M. L. Starner, K. L. Murphy, and
+
+M. G. Baxter. 2015. Long-term effects of single or multiple neonatal sevoflurane exposures on rat hippocampal ultrastructure. Anesthesiology 122:87–95.
+
+Costarino, A. T. Jr, and J. J. Downes. 2005. Pediatric
+
+anesthesia historical perspective. Anesthesiol. Clin. North America 23:573–595, vii.
+
+Ansorge, M. S., M. Zhou, A. Lira, R. Hen, and J. A. Gingrich. 2004. Early-life blockade of the 5-HT transporter alters emotional behavior in adult mice. Science 306:879–881.
+
+Crawley, J. N. 1985. Exploratory behavior models of anxiety in
+
+mice. Neurosci. Biobehav. Rev. 9:37–44.
+
+Crawley, J. N.. 2007. What’s wrong with my mouse?:
+
+Auerbach, B. D., E. K. Osterweil, and M. F. Bear. 2011.
+
+behavioral phenotyping of transgenic and knockout mice. Wiley-Interscience/John Wiley & Sons, Hoboken, NJ. Crawley, J. N., T. Chen, A. Puri, R. Washburn, T. L. Sullivan,
+
+Mutations causing syndromic autism define an axis of synaptic pathophysiology. Nature 480:63–68.
+
+Bartels, M., R. R. Althoff, and D. I. Boomsma. 2009.
+
+J. M. Hill, et al. 2007. Social approach behaviors in oxytocin knockout mice: comparison of two independent lines tested in different laboratory environments. Neuropeptides 41:145–163. Davidson, A. J., N. Disma, J. C. de Graaff, D. E. Withington,
+
+Anesthesia and cognitive performance in children: no evidence for a causal relationship. Twin Res. Hum. Genet. 12:246–253.
+
+Batten, S. V., M. Aslan, P. K. Maciejewski, and C. M. Mazure. 2004. Childhood maltreatment as a risk factor for adult cardiovascular disease and depression. J. Clin. Psychiatry 65:249–254.
+
+L. Dorris, G. Bell, et al. 2016. Neurodevelopmental outcome at 2 years of age after general anaesthesia and awake- regional anaesthesia in infancy (GAS): an international multicentre, randomised controlled trial. Lancet 387:239– 250.
+
+Ben-Ari, Y., J. L. Gaiarsa, R. Tyzio, and R. Khazipov. 2007.
+
+DiMaggio, C., L. S. Sun, A. Kakavouli, M. W. Byrne, and
+
+GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiol. Rev. 87:1215– 1284.
+
+G. Li. 2009. A retrospective cohort study of the association of anesthesia and hernia repair surgery with behavioral and developmental disorders in young children. J. Neurosurg. Anesthesiol. 21:286–291.
+
+Brambrink, A. M., A. S. Evers, M. S. Avidan, N. B. Farber, D.
+
+J. Smith, X. Zhang, et al. 2010. Isoflurane-induced neuroapoptosis in the neonatal rhesus macaque brain. Anesthesiology 112:834–841.
+
+DiMaggio, C., L. S. Sun, and G. Li. 2011. Early childhood exposure to anesthesia and risk of developmental and behavioral disorders in a sibling birth cohort. Anesth. Analg. 113:1143–1151.
+
+Broad, K. D., J. P. Curley, and E. B. Keverne. 2006. Mother- infant bonding and the evolution of mammalian social relationships. Philos. Trans. R. Soc. Lond. B Biol. Sci. 361:2199–2214.
+
+Feng, X., J. J. Liu, X. Zhou, F. H. Song, X. Y. Yang,
+
+X. S. Chen, et al. 2012. Single sevoflurane exposure decreases neuronal nitric oxide synthase levels in the hippocampus of developing rats. Br. J. Anaesth. 109:225–233. Fenton, A. A., M. Wesierska, Y. Kaminsky, and J. Bures. 1998.
+
+Brown, M. W., and J. P. Aggleton. 2001. Recognition memory:
+
+what are the roles of the perirhinal cortex and hippocampus? Nat. Rev. Neurosci. 2:51–61.
+
+Both here and there: simultaneous expression of autonomous spatial memories in rats. Proc. Natl Acad. Sci. USA 95:11493–11498.
+
+Burghardt, N. S., E. H. Park, R. Hen, and A. A. Fenton. 2012.
+
+Adult-born hippocampal neurons promote cognitive flexibility in mice. Hippocampus 22:1795–1808.
+
+Ferguson, J. N., J. M. Aldag, T. R. Insel, and L. J. Young.
+
+Campagna, J. A., K. W. Miller, and S. A. Forman. 2003.
+
+2001. Oxytocin in the medial amygdala is essential for social recognition in the mouse. J. Neurosci. 21:8278–8285. Flick, R. P., S. K. Katusic, R. C. Colligan, R. T. Wilder, R. G. Voigt, M. D. Olson, et al. 2011. Cognitive and behavioral outcomes after early exposure to anesthesia and surgery. Pediatrics 128:e1053–e1061. Fredriksson, A., E. Ponten, T. Gordh, and P. Eriksson. 2007. Neonatal exposure to a combination of N-methyl-D-aspartate and gamma-aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. Anesthesiology 107:427–436.
+
+2001. Oxytocin in the medial amygdala is essential for social recognition in the mouse. J. Neurosci. 21:8278–8285. Flick, R. P., S. K. Katusic, R. C. Colligan, R. T. Wilder, R. G. Voigt, M. D. Olson, et al. 2011. Cognitive and behavioral outcomes after early exposure to anesthesia and surgery. Pediatrics 128:e1053–e1061. Fredriksson, A., E. Ponten, T. Gordh, and P. Eriksson. 2007. Neonatal exposure to a combination of N-methyl-D-aspartate and gamma-aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. Anesthesiology 107:427–436.
+
+Mechanisms of actions of inhaled anesthetics. N. Engl. J. Med. 348:2110–2124.
+
+2001. Oxytocin in the medial amygdala is essential for social recognition in the mouse. J. Neurosci. 21:8278–8285. Flick, R. P., S. K. Katusic, R. C. Colligan, R. T. Wilder, R. G. Voigt, M. D. Olson, et al. 2011. Cognitive and behavioral outcomes after early exposure to anesthesia and surgery. Pediatrics 128:e1053–e1061. Fredriksson, A., E. Ponten, T. Gordh, and P. Eriksson. 2007. Neonatal exposure to a combination of N-methyl-D-aspartate and gamma-aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. Anesthesiology 107:427–436.
+
+Cath, D. C., D. S. van Grootheest, G. Willemsen, P. van Oppen,
+
+and D. I. Boomsma. 2008. Environmental factors in obsessive-compulsive behavior: evidence from discordant and concordant monozygotic twins. Behav. Genet. 38:108–120.
+
+Brain and Behavior, doi: 10.1002/brb3.514 (12 of 14)
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.
+
+21579032, 2016, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.514 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+D. Lin et al.
+
+Early-Life Sevoflurane Exposure on Behavior
+
+Goa, K. L., S. Noble, and C. M. Spencer. 1999. Sevoflurane in paediatric anaesthesia: a review. Paediatr. Drugs 1:127–153. Han, S., C. Tai, R. E. Westenbroek, F. H. Yu, C. S. Cheah, G. B. Potter, et al. 2012. Autistic-like behaviour in Scn1a+/- mice and rescue by enhanced GABA-mediated neurotransmission. Nature 489:385–390.
+
+with infection during pregnancy and risk of autism spectrum disorders. Brain Behav. Immun. 44:100–105.
+
+Leger, M., A. Quiedeville, V. Bouet, B. Haelewyn, M. Boulouard, P. Schumann-Bard, et al. 2013. Object recognition test in mice. Nat. Protoc. 8:2531–2537.
+
+Liang, G., C. Ward, J. Peng, Y. Zhao, B. Huang, and H. Wei. 2010. Isoflurane causes greater neurodegeneration than an equivalent exposure of sevoflurane in the developing brain of neonatal mice. Anesthesiology 112:1325–1334.
+
+Hanley, G. E., U. Brain, and T. F. Oberlander. 2013. Infant developmental outcomes following prenatal exposure to antidepressants, and maternal depressed mood and positive affect. Early Hum. Dev. 89:519–524.
+
+Loepke, A. W., and S. G. Soriano. 2008. An assessment of the effects of general anesthetics on developing brain structure and neurocognitive function. Anesth. Analg. 106:1681–1707.
+
+Hansen, T. G., J. K. Pedersen, S. W. Henneberg, D. A.
+
+Pedersen, J. C. Murray, N. S. Morton, et al. 2011. Academic performance in adolescence after inguinal hernia repair in infancy: a nationwide cohort study. Anesthesiology 114:1076–1085.
+
+Loepke, A. W., G. K. Istaphanous, J. J. 3rd McAuliffe, L.
+
+Miles, E. A. Hughes, J. C. McCann, et al. 2009. The effects of neonatal isoflurane exposure in mice on brain cell viability, adult behavior, learning, and memory. Anesth. Analg. 108:90–104.
+
+Hansen, T. G., J. K. Pedersen, S. W. Henneberg, N. S. Morton,
+
+and K. Christensen. 2013. Educational outcome in adolescence following pyloric stenosis repair before 3 months of age: a nationwide cohort study. Pediatr. Anaesth. 23:883–890.
+
+Mai, C. L., and C. J. Cote. 2012. A history of pediatric
+
+anesthesia: a tale of pioneers and equipment. Paediatr Anaesth. 22:511–520.
+
+Harrington, J. E. 1976. Recognition fo territorial boundaries by olfactory cues in mice (Mus musculus L.). Z. Tierpsychol. 41:295–306.
+
+Mellon, R. D., A. F. Simone, and B. A. Rappaport. 2007. Use
+
+of anesthetic agents in neonates and young children. Anesth. Analg. 104:509–520.
+
+Murphy, K. L., and M. G. Baxter. 2013. Long-term effects of neonatal single or multiple isoflurane exposures on spatial memory in rats. Front. Neurol. 4:87.
+
+Jevtovic-Todorovic, V., R. E. Hartman, Y. Izumi, N. D.
+
+Benshoff, K. Dikranian, C. F. Zorumski, et al. 2003. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J. Neurosci. 23:876–882.
+
+Nadler, J. J., S. S. Moy, G. Dold, D. Trang, N. Simmons, A.
+
+Perez, et al. 2004. Automated apparatus for quantitation of social approach behaviors in mice. Genes Brain Behav. 3:303–314.
+
+Kalkman, C. J., L. Peelen, K. G. Moons, M. Veenhuizen, M.
+
+Bruens, G. Sinnema, et al. 2009. Behavior and development in children and age at the time of first anesthetic exposure. Anesthesiology 110:805–812.
+
+Norman, G., and M. J. Eacott. 2004. Impaired object
+
+recognition with increasing levels of feature ambiguity in rats with perirhinal cortex lesions. Behav. Brain Res. 148:79–91. Paule, M. G., M. Li, R. R. Allen, F. Liu, X. Zou, C. Hotchkiss, et al. 2011. Ketamine anesthesia during the first week of life can cause long-lasting cognitive deficits in rhesus monkeys. Neurotoxicol. Teratol. 33:220–230.
+
+Kato, R., K. Tachibana, N. Nishimoto, T. Hashimoto, Y.
+
+Uchida, R. Ito, et al. 2013. Neonatal exposure to sevoflurane causes significant suppression of hippocampal long-term potentiation in postgrowth rats. Anesth. Analg. 117:1429– 1435.
+
+Kubik, S., and A. A. Fenton. 2005. Behavioral evidence that
+
+Peca, J., C. Feliciano, J. T. Ting, W. Wang, M. F. Wells, T. N. Venkatraman, et al. 2011. Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature 472:437–442.
+
+segregation and representation are dissociable hippocampal functions. J. Neurosci. 25:9205–9212.
+
+Lee, S. Y., S. L. Cheng, S. B. Ng, and S. L. Lim. 2013. Single-
+
+Penagarikano, O., B. S. Abrahams, E. I. Herman, K. D.
+
+breath vital capacity high concentration sevoflurane induction in children: with or without nitrous oxide? Br. J. Anaesth. 110:81–86.
+
+Winden, A. Gdalyahu, H. Dong, et al. 2011. Absence of CNTNAP2 leads to epilepsy, neuronal migration abnormalities, and core autism-related deficits. Cell 147:235–246.
+
+Lee, B. H., J. T. Chan, O. Hazarika, L. Vutskits, and J. W. Sall. 2014a. Early exposure to volatile anesthetics impairs long- term associative learning and recognition memory. PLoS One 9:e105340.
+
+Phillips, N. K., C. L. Hammen, P. A. Brennan, J. M. Najman,
+
+and W. Bor. 2005. Early adversity and the prospective prediction of depressive and anxiety disorders in adolescents. J. Abnorm. Child Psychol. 33:13–24.
+
+Lee, B. H., J. T. Chan, E. Kraeva, K. Peterson, and J. W. Sall. 2014b. Isoflurane exposure in newborn rats induces long- term cognitive dysfunction in males but not females. Neuropharmacology 83:9–17.
+
+Pichot, P. 1986. DSM-III: the 3d edition of the Diagnostic and Statistical Manual of Mental Disorders from the American Psychiatric Association. Rev. Neurol. (Paris) 142:489–499.
+
+Lee, B. K., C. Magnusson, R. M. Gardner, A. Blomstrom, C. J. Newschaffer, I. Burstyn, et al. 2015. Maternal hospitalization
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.
+
+Brain and Behavior, doi: 10.1002/brb3.514 (13 of 14)
+
+21579032, 2016, 9, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/brb3.514 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+D. Lin et al.
+
+Early-Life Sevoflurane Exposure on Behavior
+
+Stratmann, G. 2011. Review article: neurotoxicity of anesthetic drugs in the developing brain. Anesth. Analg. 113:1170– 1179.
+
+Rabbitts, J. A., C. B. Groenewald, J. P. Moriarty, and R. Flick.
+
+2010. Epidemiology of ambulatory anesthesia for children in the United States: 2006 and 1996. Anesth. Analg. 111:1011–1015.
+
+Stratmann, G., J. W. Sall, L. D. May, J. S. Bell, K. R.
+
+Ramage, T. M., F. L. Chang, J. Shih, R. S. Alvi, G. R. Quitoriano, V. Rau, et al. 2013. Distinct long-term neurocognitive outcomes after equipotent sevoflurane or isoflurane anaesthesia in immature rats. Br. J. Anaesth. 110 (Suppl 1):i39–i46.
+
+Magnusson, V. Rau, et al. 2009. Isoflurane differentially affects neurogenesis and long-term neurocognitive function in 60-day-old and 7-day-old rats. Anesthesiology 110:834– 848.
+
+Stratmann, G., J. Lee, J. W. Sall, B. H. Lee, R. S. Alvi, J. Shih, et al. 2014. Effect of general anesthesia in infancy on long- term recognition memory in humans and rats. Neuropsychopharmacology 39:2275–2287.
+
+Read, J., and P. Hammersley. 2005. Child sexual abuse and schizophrenia. Br. J. Psychiatry 186:76; author reply 76.
+
+Roullet, F. I., J. K. Lai, and J. A. Foster. 2013. In utero
+
+exposure to valproic acid and autism–a current review of clinical and animal studies. Neurotoxicol. Teratol. 36:47–56. Rudolph, U., and B. Antkowiak. 2004. Molecular and neuronal
+
+Streissguth, A. P., F. L. Bookstein, H. M. Barr, P. D. Sampson, K. O’Malley, and J. K. Young. 2004. Risk factors for adverse life outcomes in fetal alcohol syndrome and fetal alcohol effects. J. Dev. Behav. Pediatr. 25:228–238.
+
+substrates for general anaesthetics. Nat. Rev. Neurosci. 5:709–720.
+
+Sun, L.. 2010. Early childhood general anaesthesia exposure
+
+Sakai, E. M., L. A. Connolly, and J. A. Klauck. 2005.
+
+and neurocognitive development. Br. J. Anaesth. 105(Suppl 1):i61–i68.
+
+Inhalation anesthesiology and volatile liquid anesthetics: focus on isoflurane, desflurane, and sevoflurane. Pharmacotherapy 25:1773–1788.
+
+Wang, S. Q., F. Fang, Z. G. Xue, J. Cang, and X. G. Zhang. 2013. Neonatal sevoflurane anesthesia induces long-term memory impairment and decreases hippocampal PSD-95 expression without neuronal loss. Eur. Rev. Med. Pharmacol. Sci. 17:941–950.
+
+Sanders, R. D., J. Xu, Y. Shu, A. Fidalgo, D. Ma, and M.
+
+Maze. 2008. General anesthetics induce apoptotic neurodegeneration in the neonatal rat spinal cord. Anesth. Analg. 106:1708–1711.
+
+Satomoto, M., Y. Satoh, K. Terui, H. Miyao, K. Takishima, M. Ito, et al. 2009. Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice. Anesthesiology 110:628–637.
+
+Warburton, E. C., and M. W. Brown. 2015. Neural circuitry
+
+for rat recognition memory. Behav. Brain Res. 285:131–139.
+
+Wesierska, M., C. Dockery, and A. A. Fenton. 2005. Beyond
+
+memory, navigation, and inhibition: behavioral evidence for hippocampus-dependent cognitive coordination in the rat. J. Neurosci. 25:2413–2419.
+
+Shen, X., Y. Dong, Z. Xu, H. Wang, C. Miao, S. G. Soriano,
+
+et al. 2013a. Selective anesthesia-induced neuroinflammation in developing mouse brain and cognitive impairment. Anesthesiology 118:502–515.
+
+Wilder, R. T., R. P. Flick, J. Sprung, S. K. Katusic, W. J. Barbaresi, C. Mickelson, et al. 2009. Early exposure to anesthesia and learning disabilities in a population-based birth cohort. Anesthesiology 110:796–804.
+
+Shen, X., Y. Liu, S. Xu, Q. Zhao, X. Guo, R. Shen, et al.
+
+2013b. Early life exposure to sevoflurane impairs adulthood spatial memory in the rat. Neurotoxicology 39:45–56. Shih, J., L. D. May, H. E. Gonzalez, E. W. Lee, R. S. Alvi, J. W. Sall, et al. 2012. Delayed environmental enrichment reverses sevoflurane-induced memory impairment in rats. Anesthesiology 116:586–602.
+
+Yizhar, O., L. E. Fenno, M. Prigge, F. Schneider, T. J.
+
+Davidson, D. J. O’Shea, et al. 2011. Neocortical excitation/ inhibition balance in information processing and social dysfunction. Nature 477:171–178.
+
+Zou, X., T. A. Patterson, R. L. Divine, N. Sadovova, X. Zhang, J. P. Hanig, et al. 2009. Prolonged exposure to ketamine increases neurodegeneration in the developing monkey brain. Int. J. Dev. Neurosci. 27:727–731.
+
+Silverman, J. L., M. Yang, C. Lord, and J. N. Crawley. 2010. Behavioural phenotyping assays for mouse models of autism. Nat. Rev. Neurosci. 11:490–502.
+
+Slikker, W. Jr, X. Zou, C. E. Hotchkiss, R. L. Divine, N.
+
+Zou, X., F. Liu, X. Zhang, T. A. Patterson, R. Callicott, S. Liu, et al. 2011. Inhalation anesthetic-induced neuronal damage in the developing rhesus monkey. Neurotoxicol. Teratol. 33:592–597.
+
+Sadovova, N. C. Twaddle, et al. 2007. Ketamine-induced neuronal cell death in the perinatal rhesus monkey. Toxicol. Sci. 98:145–158.
+
+Sprung, J., R. P. Flick, R. T. Wilder, S. K. Katusic, T. L. Pike, M. Dingli, et al. 2009. Anesthesia for cesarean delivery and learning disabilities in a population-based birth cohort. Anesthesiology 111:302–310.
+
+Supporting Information
+
+Additional supporting information may be found online in the supporting information tab for this article:
+
+Stowers, L., P. Cameron, and J. A. Keller. 2013. Ominous
+
+odors: olfactory control of instinctive fear and aggression in mice. Curr. Opin. Neurobiol. 23:339–345.
+
+Data S1. Repetitive behavior.
+
+Brain and Behavior, doi: 10.1002/brb3.514 (14 of 14)
+
+ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.

new_pdfs/10.1002_cbin.10349.txt

@@ -0,0 +1,329 @@
+Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10349
+
+RESEARCH ARTICLE
+
+Role of miR-34c in ketamine-induced neurotoxicity in neonatal mice hippocampus
+
+Shu-e Cao*, Jianmin Tian, Shengyang Chen, Xiaoran Zhang and Yongqiang Zhang
+
+Department of Anesthesiology, The First Affiliated Hospital of XinXiang Medical College, WeiHui, HeNan Province 453100, China
+
+Abstract
+
+Ketamine is a commonly used pediatric anesthetic, but it might affect development, or even induce neurotoxicity in the neonatal brain. We have used an in vivo neonatal mouse model to induce ketamine-related neurotoxicity in the hippocampus, and found that miR-34c, a microRNA associated with pathogenesis of Alzheimer’s disease, was significantly upregulated during ketamine-induced hippocampal neurodegeneration. Functional assay of silencing miR-34c demonstrated that downregulation of miR-34c activated PKC-ERK pathway, upregulated anti-apoptotic protein BCL2, and ameliorated ketamine-induced apoptosis in the hippocampus. Cognitive examination with the Morris water maze test showed that ketamine-induced memory impairment was significantly improved by miR-34c downregulation. Thus, miR-34c is important in regulating ketamine-induced neurotoxicity in hippocampus.
+
+Keywords: hippocampus; ketamine; miR-34c; neurotoxicity
+
+Introduction
+
+Ketamine, synthesized in 1962, has been widely used in clinic anesthesia due to its rapid onset and minimal side-effects (Domino, 2010). Pharmacologically, the major mechanism of ketamine working as anesthetic is to inhibit the glutamate neuro-transmission through N-methyl-D-aspartate (NMDA) receptors (Ikonomidou et al., 1999 Olney et al., 1991), and a ketamine overdose could severely affect the development of neonatal brain and induce cortical neurotoxicity in both animals and humans (McGowan and Davis, 2008; Brambrink et al., 2012 Dong and Anand, 2013). In hippocampus, the major component of brain associated with memory and learning, new evidence had revealed through both in vitro and in vivo animal models, that repetitive or high dose adminis- tration of ketamine suppressed neural excitability, induced apoptosis impair learning and memory functions (Huang et al., 2012 Huang et al., 2013). Little is known about the underlying mechanisms or the associated signaling pathways during the process of hippocampal or memory neurodegeneration induced by anesthesia.
+
+significantly
+
+in hippocampus,
+
+thus
+
+MicroRNAs are endogenously expressed noncoding short RNAs regulating gene silencing by suppressing the transla- tion or degrading targeted messenger RNAs (Kim, 2005). They are abundantly expressed in various components in the brain, being involved in modulating embryogenesis, neural development, and maturation (Kosik and Krichevsky, 2005; Darnell et al., 2006 Kosik, 2006; Lau and Hudson, 2010). Among them, microRNA 34 c (miR-34c) is a member of miR-34 family, which includes three homologous miRNAs expressed at two different loci of chromosome (miR-34a, miR-34b, and miR-34c), and are involved in various aspects of neural development or degeneration (Agostini et al., 2011; Casci, 2012; Liu et al., 2012). miR-34c is a newly discovered modulator associated with pathogenesis of neurodegenera- tive disease (Zovoilis et al., 2011). Thus, we have determined whether miR-34c may regulate anesthesia-related neurotox- icity in hippocampus. Ketamine was introduced into an in vivo neonatal mouse model to induce anesthesia-related hippocampal neurotoxicity, and the effect of ketamine- induced neurodegeneration on the expression level of miR- 34c in hippocampus was measured. A lentiviral vector was used to downregulate miR-34c to investigate its functional
+
+(cid:1)Corresponding author: e-mail: yongqiang.zhang@aol.com
+
+164
+
+Cell Biol Int 39 (2015) 164–168 © 2014 International Federation for Cell Biology
+
+Y. Zhang et al.
+
+role in modulating anesthesia-induced neurotoxicity in hippocampus in vivo.
+
+Materials and methods
+
+Animals
+
+C57BL/6 mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). The in vivo induction of ketamine-related hippo- campal neurotoxicity was done at 2 weeks. Quantitative real time PCR of miR-34 family was used at 3 weeks, as was hippocampal injection of lentivirual vector of miR-34c. For analyses of TUNEL staining and Western blotting, 2-month old mice were used. All experimental procedures were reviewed and approved by the Animal Care Committee at the first affiliated Hospital of XinXiang Medical College.
+
+Induction of ketamine-related hippocampal neurotoxicity
+
+The in vivo protocol to induce ketamine-related hippocam- pal neurotoxicity was done as before with slight modifica- tions (Hayashi et al., 2002; Huang et al., 2012, Liu et al., 2012). Young C57BL/6 mice, postnatal 14 days, were intraperitoneally administrated with repeated dosage of 75 mg/kg ketamine per day for six consecutive days (n ¼ 28). Normal saline was injected in the control group of mice (n ¼ 25).
+
+RNA isolation and reverse transcription
+
+Hippocampal RNA was isolated with Trizol reagent (In Vitrogen, Carlsbad, CA, USA). Briefly, mice were anesthe- tized and decapitated. Hippocampal samples were retrieved and homogenized at 1 mL Trizol/0.1 g tissue. The quantity of RNA was assessed by spectrophotometry followed by 1% agarose gel electrophoresis. Total RNA was treated with 10 U of RNase free DNase I, and reverse transcription (RT) was done in a total volume of 20 mL with random hexamer primers using a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). cDNA was stored at (cid:3)20(cid:4)C until further use.
+
+Quantitative RT-PCR
+
+Expression of miR-34a, miR-34b, miR-34c, and house- keeping gene GAPDH were measured by TaqMan micro- RNA RT-PCR on the ABI 7900 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Expression profiles of each gene were quantified using corresponding standard curves. End-point RT-PCR of miR-34a, miR-34b, miR-34c, and GAPDH used 50 ng of total RNA with a mirVana RT-PCR miRNA Detection Kit (Ambion, Austin,
+
+Cell Biol Int 39 (2015) 164–168 © 2014 International Federation for Cell Biology
+
+miR-34c in hippocampus
+
+Texas, USA). PCR products were separated and visualized on a 4% agarose gel. Each sample was run in triplicate and a mean value of each Ct triplicate was used.
+
+Lentivirus production and transduction
+
+To downregulate miR-34c, the coding sequence for a 2’-O- methyl oligonucleotide of miR-34c inhibitor was UCCGU- CACAUCAAUCGACUAACG, and the non-specific control antisense sequence was UACUCUUUCUAGGAGGUU- GUUAUU (Yu et al., 2012). These two sequences were amplified and cloned into pCDH-CMV-MCS-EF1-coGFP for in vivo gene transfer, resulting in a miR-34c inhibitor vector (lenti-miR34c-I) and miR-34c non-specific control vector (lenti-miR34c-C) (System Biosciences, Mountain View, CA, USA). The lentivirual expression vectors and pPACK packaging vector were co-transfected into 293T cells, and viral particles were collected and concentrated to high titer.
+
+Hippocampal injection
+
+One day after the 6-day ketamine treatment, the injections of lent viruses were performed on the right side of the cortex. A tiny hole was drilled above hippocampus and a Hamilton syringe was used to inject 2 mL of lentivirus of miR-34c inhibitor (lenti-miR34c-I, 20 mM, n ¼ 17) or non- specific control (lenti-miR34c-C, 20 mM, n ¼ 14) at the coordinates assessed from bregma and skull surface: lateral þ1.5 mm, and vertical anteroposterior (cid:3)2.0 mm, (cid:3)1.5 mm. After injection, the incision was quickly sealed with dental cement.
+
+Western blotting
+
+Western blotting analysis was conducted at 2 months. Four mice with Lenti-miR34c-I injection and four mice with Lenti-miR34c-C injection were included in this analysis. Forty micrograms of hippocampal protein were collected and separated on an 8% NuPage Gel with MES buffer (Invitrogen, Carlsbad, CA, USA) and transferred to a polyvinylidene difluoride membrane. Primary antibody dilutions included 1:500 BCL2 (Santa Cruz, USA), 1:100 phosphorylated-PKC (p-PKC) (Sant Cruz Biotechnologies, Santa Cruz, CA, USA), 1:100 phosphorylated-ERK (p-ERK) (Sant Cruz Biotechnologies, Santa Cruz, CA, USA), and 1:1,000 b-actin (Cell Signaling, Danvers, MA, USA). Membranes were then incubated in primary antibody in Odyssey Blocking Buffer at 4(cid:4)C for 24 h, followed by three washes in 0.1% PBS-T and 1 h incubation at RT with 1:1,000 secondary antibodies. The films were visualized and quantified on the Odyssey Infrared Imaging Center (Li-Cor, Lincoln, NE, USA).
+
+165
+
+miR-34c in hippocampus
+
+TUNEL staining for hippocampal apoptosis
+
+Hippocampal slices (350 mm) were prepared for terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining to detect the apoptosis, using an In Situ Cell Death Detection Kit according to manufacturer’s protocol (Roche, Branchburg, NJ, USA). Five mice with Lenti-miR34c-I injection and 5 mice with Lenti- miR34c-C injection were included in the analysis. Hippo- campal CA1 region was examined under a fluorescent scope. The apoptotic CA1 neurons were identified based on their size, location and immuno-reaction to TUNEL staining. The average number of the apoptotic neurons per 0.01 mm2 was measured and compared between control hippocampi and hippocampi treated with miR-34c inhibitor.
+
+Morris water maze (MWM) testing
+
+The MWM testing was carried out 1 month after hippocampal transfection of miR-34c knockdown. Eight mice with Lenti-miR34c-I injection and 5 mice with Lenti- miR34c-C injection were included in this analysis. In a large circular tank with a transparent platform (10 cm (cid:5) 10 cm), warm water at 26(cid:4)C was added to submerge the platform 1 cm below the surface. Visual cues of color paints were used to aid mice in locating the platform. The mice were given training sessions four times per day for one week before final testing. In each training session, the mice were put in the maze to locate the platform in 2 min followed by resting on the platform for 30 s. If mice did not locate the platform in 2 min, they were aided with flashing lights to the platform with 30 s of rest on top of the platform. On the final day of examination, the average swimming time and swimming distance were compared between control mice and the mice with miR-34c knockdown.
+
+Statistical analysis
+
+Statistic analysis was conducted with SPSS software (version 11.0). The measured data were presented as mean (cid:6) stan- dard deviations. The statistical differences were measured with a Student’s t-test, and the significance set at P < 0.05.
+
+Results
+
+miR-34c is upregulated in hippocampus by excessive ketamine administration
+
+We examined whether the expression profiles of the miRNAs in the mirR-34 family would be modulated by excessive ketamine application that induces neurodegener- (Hayashi et al., 2002, Huang ation in hippocampus et al., 2012; Liu et al., 2012). After injecting C57BL/6 mice at 2 weeks with 75 mg/kg ketamine for 6 days, they were
+
+166
+
+Y. Zhang et al.
+
+killed on the 7th day. Hippocampal mRNAs of miR-34 family, including miR-34a, miR-34b, and miR-34c, were measured by quantitative real-time PCR (qPCR). miR-34c was particularly upregulated, whereas miR-34a or miR-34b was relatively unchanged (Figure 1).
+
+Knocking down miR-34c ameliorates ketamine-induced hippocampal neurotoxicity
+
+To see if miR-34c has a role in modulating the neurotoxicity in hippocampus induced by excessive administration of keta- mine, we gave repeated daily administration of ketamine through systemic injection in young mice (P14) for six consecutive days. In hippocampus, the neurons in CA1 regions underwent significant apoptotic neurodegeneration, as previously reported (Hayashi et al., 2002, Huang et al., 2012; Liu et al., 2012). However, after injection of lentivirus containing miR-34c inhibitor (lenti-miR34-I) into mouse hippocampus on P21, TUNEL staining on 2-month old mouse hippocampal CA1 region showed that the number of apoptotic neurons was significantly reduced (Figure 2A), being about half of the number of apoptotic neurons in mice injected with control lentivirus (lenti-miR34c-C) (P < 0.05). Thus, inhibition of miR-34c helps ameliorate anesthesia- induced neurotoxicity in the hippocampus.
+
+Knocking down miR-34c upregulates anti-apoptotic pathways during ketamine-induced hippocampal neurotoxicity
+
+After systemic ketamine administration and hippocampal miR-34c lentivirus injection, Western blotting analysis was used on the 2-month old mice (Figure 2B). Anti-apoptotic protein Bcl2 was significantly upregulated by knocking
+
+n o s s e r p x e A N R m 4 3 - R m
+
+i
+
+i
+
+450%
+
+400%
+
+350%
+
+300%
+
+250%
+
+200%
+
+150%
+
+100%
+
+50%
+
+Contrl Ketamine
+
+
+
+0%
+
+miR-34a
+
+miR-34b
+
+miR-34c
+
+Figure 1 Hippocampal miR-34c expression is upregulated by ketamine. Mice were treated with repetitive IP administration of ketamine, or normal saline (control) for 6 days. Expression of miR34a/b/c mRNAs (normalized to GAPDH) in hippocampus were examined by q-PCR. (*, P < 0.05, n ¼ 5).
+
+Cell Biol Int 39 (2015) 164–168 © 2014 International Federation for Cell Biology
+
+Y. Zhang et al.
+
+A
+
+lenti-miR34c-C
+
+lenti-miR34c-I
+
+B
+
+lenti-miR 34c-C
+
+lenti-miR 34c-I
+
+p-PKC
+
+p-ERK
+
+Bcl2
+
+β-action
+
+Figure 2 Knocking down miR-34c reduces apoptosis, upregulated Bcl2 and PKC-ERK signaling pathway after ketamine-induced hippocampal neurotoxicity. (A) TUNEL staining of hippocampal CA1 region in 2-month-old mice induced with ketamine-related neurotoxicity. Hippocampal injection of control vector (lenti-miR34c-C), or vector of miR-34c inhibitor (lenti-miR34c-I) was performed on P21. (B) Western blotting was also used to compare Bcl2 protein, phosphorylated PKC (p- PKC), and phosphorylated ERK (p-ERK), between miR-34c inhibitor treated mice and control mice after ketamine-induced hippocampal neurotoxicity. (Scale bar: 50 mM).
+
+down miR-34c in the hippocampus. It was previously demonstrated that ketamine downregulated PKC pathway in the hippocampus to induce neuronal apoptosis (Huang et al., 2012; Liu et al., 2012). Here, we demonstrate that PKC pathway is indeed activated or strengthened, as more phosphorylated PKC and phosphorylated ERK were induced by genetically knocking down miR-34c after ketamine treatment.
+
+Knocking down miR-34c increases memory performance after ketamine-induced hippocampal neurotoxicity
+
+The question remained as to whether knocking down miR- 34c could rescue the memory loss after ketamine-induced neurotoxicity in hippocampus. Two-month-old mice were examined by the MWM test. For the control mice, lentivirus containing the non-specific control vector was injected in the hippocampus. Mice with hippocampal downregulation of miR-34c following ketamine-induced memory im- pairment, the averaged swimming time and swimming distance were both markedly reduced compared to the mice without hippocampal miR-34c downregulation (Figure 3). Thus, our result suggests that downregulation of miR-34c could increase memory.
+
+Cell Biol Int 39 (2015) 164–168 © 2014 International Federation for Cell Biology
+
+miR-34c in hippocampus
+
+A
+
+80
+
+) c e s ( e m
+
+60
+
+
+
+i t g n m m w S
+
+i
+
+i
+
+40
+
+20
+
+0
+
+lenti-miR34c-C
+
+lenti-miR34c-I
+
+B
+
+)
+
+m c (
+
+e c n a t s d
+
+i
+
+1300
+
+1000
+
+
+
+g n m m w S
+
+i
+
+i
+
+500
+
+lenti-miR34c-C
+
+lenti-miR34c-I
+
+Figure 3 Knocking down miR-34c increases memory performance after ketamine-induced hippocampal neurotoxicity. Mice were initially induced with hippocampal neurotoxicity by repeated administra- tion of ketamine, and then followed by hippocampal injection of control vector (lenti-miR34c-C, left), or miR-34c inhibitor (lenti-miR34c-I, right). Morris water maze was used at 2 months to compare memory performance. Both swimming time (A) and swimming distance (B) were shortened in the mice receiving miR-34c inhibitor after ketamine- induced memory dysfunction in hippocampus. *, P < 0.05 (n ¼ 5).
+
+Discussion
+
+Ketamine is commonly used in pediatric anesthesia, but evidence suggests that excessive or repetitive usage of ketamine hinders or even damages the normal development of neonatal brain in both animal and human. We need to understand the underlying molecular mechanisms of this cortical neurotoxic event, as well as identify therapeutic targets to reduce or inhibit anesthesia-induced neuro- degeneration in the brain.
+
+miR-34c had an important role in anesthesia-induced hippocampal neurodegeneration. Though all three members of the miR-34 family, miR-34a, miR-34b, and miR-34c, are expressed in hippocampus, little is known about their exact roles in modulating hippocampal maturation or develop- ment (Juhila et al., 2011). miR-34c is upregulated in neurodegenerative diseases or under stress condition, and targeted inhibition of miR-34c markedly improved learning capability in mice (Haramati et al., 2011; Zovoilis et al., 2011). After introducing hippocampal neurodegen- in vivo eration in mouse through ketamine induction,
+
+167
+
+miR-34c in hippocampus
+
+inhibition of miR-34c in hippocampus significantly im- proved MWM performance, with shortened swimming time and distance to locate platforms. Thus, along with previous findings, the results point to a critical role of miR-34c in regulating memory function through hippocampus in the brain.
+
+It is noteworthy that anti-apoptotic protein Bcl2 was upregulated by miR-34c being knocked down after keta- mine-induced neurotoxicity in hippocampus. The expres- sion, or induced overexpression of Bcl2 protein by tumor necrosis factor (Tamatani et al., 1999) or estrogen receptors (Zhao et al., 2004), proved protective against neuronal apoptosis in the hippocampus. And miR-34a is inversely associated with Bcl2 expression in cortex in Alzheimer’s disease (Wang et al., 2009), but there has been no report identifying any of the miR-34 family in the regulation of Bcl2 expression in hippocampus. Thus, our finding showing that knocking down miR-34c upregulated Bcl2 expression level in hippocampus after ketamine-induced neurotoxicity is novel, and also indicates that miR-34 family microRNA might be directly involved in the regulation of neuronal apoptosis in the hippocampus. Thus, our data could further our understanding on identifying the underlying mecha- nisms of anesthesia-induce neurotoxicity, as well as developing targeted clinic therapies to treat anesthesia- induced neurotoxicity in neonatal brains.
+
+Acknowledgements and funding
+
+References
+
+Agostini M, Tucci P, Killick R, Candi E, Sayan BS, di val Cervoval PR, et al. (2011) Neuronal differentiation by TAp73 is mediated by microRNA-34a regulation of synaptic protein targets. Proc Natl Acad Sci 108: 21093–8.
+
+Brambrink AM, Orfanakis A, Kirsch JR (2012) Anesthetic
+
+neurotoxicity. Anesthesiol Clin 30: 207–28.
+
+Casci T, (2012) Ageing: MicroRNA tunes ageing pathway in flies.
+
+Nat Rev Genet 13: 222–3.
+
+Darnell DK, Kaur S, Stanislaw S, Konieczka JH, Yatskievych TA, Antin PB (2006) MicroRNA expression during chick embryo development. Dev Dyn 235: 3156–65.
+
+Domino EF (2010) Taming the ketamine tiger. 1965. Anesthesi-
+
+ology 113: 678–84.
+
+Dong C, Anand K (2013) Developmental neurotoxicity of
+
+ketamine in pediatric clinical use. Toxicol Lett.
+
+Haramati S, Navon I, Issler O, Ezra-Nevo G, Gil S, Zwang R, et al. (2011) MicroRNA as repressors of stress-induced anxiety: the case of amygdalar miR-34. J Neurosci 31: 14191–203.
+
+Hayashi H, Dikkes P, Soriano SG (2002) Repeated administration of ketamine may lead to neuronal degeneration in the developing rat brain. Paediatr Anaesth 12: 770–4.
+
+168
+
+Y. Zhang et al.
+
+Huang L, Liu Y, Jin W, Ji X, Dong Z (2012) Ketamine potentiates hippocampal neurodegeneration and persistent learning and memory impairment through the PKCgamma-ERK signaling pathway in the developing brain. Brain Res 1476: 164–71. Huang L, Liu Y, Zhang P, Kang R, Li X, Bo L, et al. (2013) In vitro dose-dependent inhibition of the intracellular spontaneous calcium oscillations in developing hippocampal neurons by ketamine. PLoS One 8: e59804.
+
+Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikranian K, et al. (1999) Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 283: 70–4.
+
+Juhila J, Sipila T, Icay K, Nicorici D, Ellonen P, Kallio A, et al. (2011) MicroRNA expression profiling reveals miRNA families regulating specific biological pathways in mouse frontal cortex and hippocampus. PLoS One 6: e21495.
+
+Kim VN (2005) Small RNAs: classification biogenesis, and
+
+function. Mol Cells 19: 1–15.
+
+Kosik KS (2006) The neuronal microRNA system. Nat Rev
+
+Neurosci 7: 911–20.
+
+Kosik KS, Krichevsky AM (2005) The elegance of the MicroRNAs:
+
+a neuronal perspective. Neuron 47: 779–82.
+
+Lau P, Hudson LD (2010) MicroRNAs in neural cell differentia-
+
+tion. Brain Res 1338: 14–9.
+
+Liu N, Landreh M, Cao K, Abe M, Hendriks G-J, Kennerdell JR, et al. (2012) The microRNA miR-34 modulates ageing and neurodegeneration in Drosophila. Nature 482: 519–23.
+
+McGowan FX, Jr., Davis PJ (2008) Anesthetic-related neurotox- icity in the developing infant: of mice, rats, monkeys and, possibly, humans. Anesth Analg 106: 1599–602.
+
+Olney J, Labruyere J, Wang G, Wozniak D, Price M, Sesma M (1991) NMDA antagonist neurotoxicity: mechanism and prevention. Science (Washington) 254: 1515–8.
+
+Tamatani M, Che YH, Matsuzaki H, Ogawa S, Okado H, Miyake S-i , et al. (1999) Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NFŒ B activation in primary hippo- campal neurons. J Biol Chem 274: 8531–8.
+
+R
+
+Wang X, Liu P, Zhu H, Xu Y, Ma C, Dai X, et al. (2009) MiR-34a, a microRNA up-regulated in a double transgenic mouse model of Alzheimer’s disease, inhibits bcl2 translation. Brain Res Bull 80: 268–73.
+
+Yu F, Jiao Y, Zhu Y, Wang Y, Zhu J, Cui X, et al. (2012) MicroRNA 34c gene down-regulation via DNA methylation promotes self- transition in breast renewal and epithelial-mesenchymal tumor-initiating cells. J Biol Chem 287: 465–73.
+
+Zhao L, Wu T-w, Brinton RD (2004) Estrogen receptor subtypes alpha and beta contribute to neuroprotection and increased Bcl-2 expression in primary hippocampal neurons. Brain Res 1010: 22–34.
+
+Zovoilis A, Agbemenyah HY, Agis-Balboa RC, Stilling RM, Edbauer D, Rao P, et al. (2011) MicroRNA-34c is a novel target to treat dementias. Embo J 30: 4299–308.
+
+Received 25 February 2014; accepted 5 June 2014. Final version published online January 2015.
+
+Cell Biol Int 39 (2015) 164–168 © 2014 International Federation for Cell Biology

new_pdfs/10.1007_s10072-014-1726-4.txt

@@ -0,0 +1,159 @@
+Neurol Sci (2014) 35:1401–1404 DOI 10.1007/s10072-014-1726-4
+
+O R I G I N A L A R T I C L E
+
+Sevoflurane postconditioning provides neuroprotection against brain hypoxia–ischemia in neonatal rats
+
+Xiaoyan Ren • Zhi Wang • Hong Ma • Zhiyi Zuo
+
+Received: 25 February 2014 / Accepted: 15 March 2014 / Published online: 5 April 2014 (cid:2) Springer-Verlag Italia 2014
+
+Abstract Application of volatile anesthetics after brain ischemia provides neuroprotection in adult animals (anes- thetic postconditioning). We tested whether postcondi- tioning with sevoflurane, the most commonly used general anesthetic in pediatric anesthesia, reduced neonatal brain injury in rats. Seven-day-old Sprague–Dawley rats were subjected to brain hypoxia–ischemia (HI). They were postconditioned with sevoflurane in the presence or absence of 5-hydroxydecanoic acid, a mitochondrial KATP channel inhibitor. Sevoflurane postconditioning dose- dependently reduced brain tissue loss observed 7 days after brain HI. This effect was induced by clinically relevant concentrations and abolished by 5-hydroxydecanoic acid. These results suggest that sevoflurane postconditioning protects neonatal brain against brain HI via mitochondrial KATP channels.
+
+Abbreviations 5-HD KATP channels HI
+
+5-Hydroxydecanoic acid ATP sensitive potassium channels Hypoxia–ischemia
+
+Introduction
+
+Neonatal brain injury occurs at about one in every 4,000 live births [1]. Most of them survive to adulthood and can long-term neurological and cognitive have significant impairment, such as cerebral palsy and epilepsy [2–4]. Thus, neonatal brain injury has a major impact on patients, their families and our society. However, there are no effective and practical interventions available for use in clinical practice to reduce neonatal brain injury, indicating an urgent need to identify these interventions.
+
+Keywords Brain hypoxia–ischemia (cid:2) Neonates (cid:2) Postconditioning (cid:2) Sevoflurane
+
+X. Ren (cid:2) H. Ma (&) Department of Anesthesiology, The First Hospital of China Medical University, Shenyang 110001, People’s Republic of China e-mail: mahong5466@yahoo.com
+
+X. Ren (cid:2) Z. Wang (cid:2) Z. Zuo (&) Department of Anesthesiology, University of Virginia Health System, 1 Hospital Drive, PO Box 800710, Charlottesville, VA 22908-0710, USA e-mail: zz3c@virginia.edu
+
+Neonatal brain injury is usually caused by brain hypoxia, ischemia or the combination of hypoxia and ischemia [1]. This situation is often modeled in rodents by inducing brain hypoxia–ischemia (HI) [5]. It has been shown that applica- tion of isoflurane, a volatile anesthetic used clinically, after focal brain ischemia provides neuroprotection in adult rodents [6]. This isoflurane postconditioning effect also protects brain against neonatal brain HI in rats [7]. However, isoflurane is now not commonly used in pediatric anesthesia in the USA. Sevoflurane is the most commonly used general anesthetic in pediatric anesthesia and also more commonly used in adults than isoflurane in current clinical practice in the USA and many other developed countries [8]. However, it is not known yet whether sevoflurane can induce a post- conditioning effect against neonatal brain HI.
+
+Z. Wang Department of Anesthesiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China
+
+Based on the information of isoflurane postconditioning, we hypothesize that sevoflurane postconditioning reduces
+
+123
+
+1402
+
+neonatal brain injury. To test this hypothesis, we applied sevoflurane after brain HI in neonatal rats. Because mito- chondrial ATP-sensitive potassium (KATP) channels may be involved in neuroprotection and cardioprotection induced by volatile anesthetics [6, 9], we also determined whether mitochondrial KATP channels played a role in the sevoflurane postconditioning effects on neonatal rats using 5-hydroxydecanoic acid (5-HD), a specific inhibitor of mitochondrial KATP channels.
+
+Methods
+
+All experimental protocols were approved by the Institu- tional Animal Care and Use Committee of the University of Virginia (Charlottesville, VA). All surgical and experi- mental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publications number 80-23) revised in 2011. Efforts were made to minimize the number of animals used and their suffering.
+
+Neonatal brain hypoxia–ischemia modal
+
+Brain HI was performed in 7-day-old male and female Sprague–Dawley rats as described previously [10, 11]. In brief, neonates were anesthetized by isoflurane and their left common carotid arteries were permanently ligated with a double 7-0 surgical silk. The procedure lasted \5 min. After surgery, neonates were returned to the cages with their mothers for 3 h. The neonates were then placed in a chamber filled with humidified 8 % oxygen–92 % nitrogen for 2 h at 37 (cid:3)C. The oxygen concentration and tempera- ture in the chamber were continuously monitored.
+
+Drug application
+
+The neonates were randomly divided into the following groups: (1) control, (2) brain HI, (3) brain HI and post- conditioning with 1, 2 and 3 % sevoflurane, (4) brain HI and 5-HD treatment (10 mg/kg) and (5) brain HI, 5-HD treatment and postconditioning with 2 % sevoflurane. Sevoflurane postconditioning was performed by exposing neonates to various concentrations of sevoflurane in 30 % O2 for 1 h immediately after brain HI. Neonates of brain HI alone group were placed in a chamber flushed with 30 % O2 for 1 h. The mitochondrial KATP channel inhibitor 5-HD was dissolved in normal saline and administered intraperitoneally just before the start of brain HI. The dose of 5-HD was based on a previous study in which intra- peritoneal injection of 10 mg/kg 5-HD blocked ischemic preconditioning-induced protection [12].
+
+123
+
+Neurol Sci (2014) 35:1401–1404
+
+Fig. 1 Neuroprotective effects of sevoflurane. Seven-day-old rats were subjected to or were not subjected to the brain HI and then postconditioned with various concentrations of sevoflurane. Brain was harvested at 7 days after the brain HI. The results are mean ± SD (n = 8–10). *P \ 0.05 compared with control rats. ^P \ 0.05 com- pared with rats that had brain HI only
+
+Brain injury/tissue loss quantification
+
+After 7 days of the brain HI, rats were sacrificed under deep isoflurane anesthesia and then their brains were har- vested as described previously [11, 13]. The hindbrain was removed from cerebral hemispheres and bilateral hemi- spheres were weighed separately. The weight ratio of left to right hemispheres was calculated.
+
+Statistical analysis
+
+The results are presented as mean ± SD (n C 6). Statisti- cal analysis was performed by one-way analysis of vari- ance followed by the Tukey’s test. A P B 0.05 was considered statistically significant.
+
+Results
+
+The control rats had similar right and left hemisphere weights. The left brain HI significantly reduced left cere- bral hemisphere weight examined 7 days after brain HI (weight to right cerebral hemispheres: 0.987 ± 0.012 of control group vs. 0.740 ± 0.049 of brain HI group, n = 8–10, P \ 0.001), suggesting brain tissue loss in this side of brain. This tissue loss was dose- dependently reduced by sevoflurane postconditioning, an effect that was already significant even after being post- conditioned with 1 % sevoflurane (Fig. 1) (weight ratio of left to right cerebral hemispheres: 0.740 ± 0.049 of brain HI group vs. 0.820 ± 0.051 of brain HI plus 1 % sevo- flurane group, n = 8–10, P = 0.003). The sevoflurane
+
+ratio of
+
+left
+
+Neurol Sci (2014) 35:1401–1404
+
+Fig. 2 Inhibition of sevoflurane postconditioning-induced neuropro- tection by a mitochondrial KATP inhibitor. Seven-day-old rats were subjected to or were not subjected to the brain HI and then postconditioned with 2 % sevoflurane in the presence or absence of 10 mg/kg 5-HD. a Representative brain images. The inserted length marker = 2 mm; b Quantification results. The results are mean ± SD (n = 6–10). *P \ 0.05 compared with control rats, ^P \ 0.05 com- pared with rats that had brain HI only, #P \ 0.05 compared with rats that had brain HI and sevoflurane postconditioning. HD 5-HD, Sevo sevoflurane
+
+postconditioning effects were abolished by 5-HD (weight ratio of left to right cerebral hemispheres: 0.884 ± 0.049 of brain HI plus 2 % sevoflurane group vs. 0.759 ± 0.044 of brain HI plus 2 % sevoflurane plus 5-HD group, n = 8–10, P \ 0.001), although 5-HD alone did not affect the brain tissue loss caused by brain HI (Fig. 2).
+
+Discussion
+
+Our results clearly showed that postconditioning with sevoflurane-induced neuroprotection against neonatal brain HI. These results extend the previous finding of isoflurane postconditioning effects in neonatal rats [7] to sevoflurane,
+
+1403
+
+the most commonly used general anesthetic in pediatric anesthesia. To test the effects of sevoflurane, we used the classical Rice–Vannucci model, a widely used and well- characterized animal model to study neonatal brain injury. This model incorporates both brain ischemia and hypoxia, factors that contribute to neonatal brain injury in humans [1, 5]. We monitored brain tissue loss that signifies brain structure damage as showed in our previous studies [11, 13].
+
+Our results suggest that the sevoflurane postconditioning effects may be mediated by mitochondrial KATP channels because 5-HD, a mitochondrial KATP channel inhibitor, abolished sevoflurane effects and 5-HD alone did not alter brain tissue loss after brain HI. Consistent with this finding, our previous study has implicated the involvement of mitochondrial KATP channels in isoflurane postcondition- ing-induced neuroprotection in adult rats after focal brain ischemia [6]. These channels are inhibited by ATP and activated under energy-depleted conditions. They also can be activated by volatile anesthetics [14]. The opening of these channels produces an outward current. This current can maintain the mitochondrial membrane potential and reduce the opening of mitochondrial permeability transi- tion pore to inhibit cell injury and death [15, 16]. Of note, previous studies have shown that application of sevoflurane before brain ischemia (sevoflurane preconditioning) also provides neuroprotection. This effect may be mediated by inhibition of brain inflammation and activation of gluta- mate transporters in the brain [17–19]. Although these mechanisms play a role in the sevoflurane postcondition- ing-induced neuroprotection and whether there is a rela- tionship between these mechanisms and the mitochondrial KATP channel pathway require further investigation.
+
+We used cerebral weight ratio to quantify the brain tissue loss after brain HI. The method is used by many studies before and the brain injury quantified by this method is highly correlated with that measured by bio- chemical, electrophysiological and morphometric methods [20, 21]. In addition, the cerebral weight ratio method is simple to perform and very objective.
+
+Our finding may have clinical translational implications. Sevoflurane is the most commonly used general anesthetic in pediatric population [8]. It is often used in patients for Cesarean section if general anesthesia is used. One mini- mum alveolar concentration (the concentration in the lungs to prevent movement in 50 % subjects in response to sur- gical stimuli) of sevoflurane is *2 % in human [22] and 2.9 % in rats younger than 30 days [23]. Our results showed that sevoflurane at a concentration as low as 1 % was effective to induce neuroprotection, suggesting that a subclinical concentration for anesthesia can induce the postconditioning effect. In addition, we have demonstrated the postconditioning effect, which may be easy to apply
+
+123
+
+1404
+
+clinically because its application does not require the predication of ischemia occurrence. However, exposure of neonatal mice to 3 % sevoflurane for 6 h can lead to brain cell apoptosis and increase in brain tumor necrosis factor a [24]. Thus, it is judicious to avoid exposure to a high concentration of sevoflurane for a long time so that the potential sevoflurane-induced neurotoxicity will not occur. In summary, we have shown that sevoflurane at clini- cally relevant concentrations can induce a postconditioning effect against neonatal brain injury. This effect may be mediated by mitochondrial KATP channels.
+
+Acknowledgments This study was supported by Grants (R01 GM065211 and R01 GM098308 to Z. Zuo) from the National Insti- tutes of Health, Bethesda, Maryland, by a Grant from the International Anesthesia Research Society (2007 Frontiers in Anesthesia Research Award to Z. Zuo), Cleveland, Ohio, by a Grant-in-Aid from the American Affiliate (10GRNT3900019 to Z. Zuo), Baltimore, Maryland, and the Robert M. Epstein Professorship Endowment, University of Virginia.
+
+Heart
+
+Association
+
+Mid-Atlantic
+
+Conflict of interest None.
+
+References
+
+1. Ferriero DM (2004) Neonatal brain injury. N Engl J Med 351(19):1985–1995
+
+2. Lynch JK, Nelson KB (2001) Epidemiology of perinatal stroke. Curr Opin Pediatr 13:499–505
+
+3. Sran SK, Baumann RJ (1988) Outcome of neonatal strokes. Am J Dis Child 142:1086–1088
+
+4. Sreenan C, Bhargave R, Robertson CM (2000) Cerebral infarc- tion in the term new-born: clinical presentation and long-term outcome. J Pediatr 137:351–355
+
+5. Silbereis JC, Huang EJ, Back SA, Rowitch DH (2010) Towards improved animal models of neonatal white matter injury associ- ated with cerebral palsy. Dis Models Mech 3(11–12):678–688 6. Lee JJ, Li L, Jung H–H, Zuo Z (2008) Postconditioning with isoflurane reduced ischemia-induced brain injury in rats. Anes- thesiology 108:1055–1062
+
+7. Zhou Y, Lekic T, Fathali N, Ostrowski RP, Martin RD, Tang J et al (2010) Isoflurane posttreatment reduces neonatal hypoxic- ischemic brain injury in rats by the sphingosine-1-phosphate/ phosphatidylinositol-3-kinase/Akt 41(7): 1521–1527 pathway. Stroke
+
+8. Fox AJ, Rowbotham DJ (1999) Anaesthesia. BMJ 319(7209): 557–560
+
+9. Obal D, Dettwiler S, Favoccia C, Scharbatke H, Preckel B, Schlack W (2005) The influence of mitochondrial KATP-chan- nels in the cardioprotection of preconditioning and postcondi- tioning by sevoflurane in the rat in vivo. Anesth Analg 101(5):1252–1260
+
+123
+
+Neurol Sci (2014) 35:1401–1404
+
+10. Zhao P, Peng L, Li L, Xu X, Zuo Z (2007) Isoflurane precon- ditioning improves long-term neurologic outcome after hypoxic- rats. Anesthesiology brain ischemic 107(6):963–970 neonatal injury in
+
+11. Wang Z, Zhao H, Peng S, Zuo Z (2013) Intranasal pyrrolidine dithiocarbamate decreases brain inflammatory mediators and provides neuroprotection after brain hypoxia-ischemia in neona- tal rats. Exp Neurol 249:74–82
+
+12. Gozen A, Demiryurek S, Taskin A, Ciralik H, Bilinc H, Kara S et al (2013) Protective activity of ischemic preconditioning on rat testicular ischemia: effects of Y-27632 and 5-hydroxydecanoic acid. J Pediatr Surg 48(7):1565–1572
+
+13. Zhao P, Zuo Z (2004) Isoflurane preconditioning induces neu- roprotection that is inducible nitric oxide synthase-dependent in the neonatal rats. Anesthesiology 101:695–702
+
+14. Jiang MT, Nakae Y, Ljubkovic M, Kwok WM, Stowe DF, Bosnjak ZJ (2007) Isoflurane activates human cardiac mito- chondrial adenosine triphosphate-sensitive K? channels recon- stituted in lipid bilayers. Anesth Analg 105(4):926–932 table of contents
+
+15. Kowaltowski AJ, Seetharaman S, Paucek P, Garlid KD (2001) Bioenergetic consequences of opening the ATP-sensitive K(?) channel of heart mitochondria. Am J Physiol Heart Circ Physiol 280(2):H649–H657
+
+16. Gateau-Roesch O, Argaud L, Ovize M (2006) Mitochondrial permeability transition pore and postconditioning. Cardiovasc Res 70(2):264–273
+
+17. Wang H, Lu S, Yu Q, Liang W, Gao H, Li P et al (2011) Sevoflurane preconditioning confers neuroprotection via anti- inflammatory effects. Front Biosci 3(1):604–615
+
+18. Sanders RD, Manning HJ, Robertson NJ, Ma D, Edwards AD, Hagberg H et al (2010) Preconditioning and postinsult therapies for perinatal hypoxic-ischemic injury at term. Anesthesiology 113(1):233–249
+
+19. Wang C, Lee J, Jung H, Zuo Z (2007) Pretreatment with volatile anesthetics, but not with the nonimmobilizer 1,2-dichlorohexa- fluorocyclobutane, reduced cell injury in rat cerebellar slices after an in vitro simulated ischemia. Brain Res 1152:201–208
+
+20. Andine P, Thordstein M, Kjellmer I, Nordborg C, Thiringer K, Wennberg E et al (1990) Evaluation of brain damage in a rat model of neonatal hypoxic-ischemia. J Neurosci Methods 35(3):253–260
+
+21. McDonald JW, Roeser NF, Silverstein FS, Johnston MV (1989) Quantitative assessment of neuroprotection against NMDA- induced brain injury. Exp Neurol 106(3):289–296
+
+22. Behne M, Wilke HJ, Harder S (1999) Clinical pharmacokinetics of sevoflurane. Clin Pharmacokinet 36(1):13–26
+
+23. Orliaguet G, Vivien B, Langeron O, Bouhemad B, Coriat P, Riou B (2001) Minimum alveolar concentration of volatile anesthetics in rats during postnatal maturation. Anesthesiology 95(3): 734–739
+
+24. Lu Y, Wu X, Dong Y, Xu Z, Zhang Y, Xie Z (2010) Anesthetic sevoflurane causes neurotoxicity differently in neonatal naive and Alzheimer disease transgenic mice. Anesthesiology 112(6): 1404–1416

new_pdfs/10.1007_s12035-017-0730-0.txt

@@ -0,0 +1,237 @@
+Mol Neurobiol (2018) 55:164–172 DOI 10.1007/s12035-017-0730-0
+
+Early Exposure to Ketamine Impairs Axonal Pruning in Developing Mouse Hippocampus
+
+Aleksandar Lj. Obradovic 1 & Navya Atluri 2 & Lorenza Dalla Massara 3 & Azra Oklopcic 4 & Nikola S. Todorovic 5 & Gaurav Katta 6 & Hari P. Osuru 2 & Vesna Jevtovic-Todorovic 2
+
+Published online: 24 August 2017 # Springer Science+Business Media, LLC 2017
+
+Abstract Mounting evidence suggests that prolonged expo- sure to general anesthesia (GA) during brain synaptogenesis damages the immature neurons and results in long-term neurocognitive impairments. Importantly, synaptogenesis re- lies on timely axon pruning to select axons that participate in active neural circuit formation. This process is in part depen- dent on proper homeostasis of neurotrophic factors, in partic- ular brain-derived neurotrophic factor (BDNF). We set out to examine how GA may modulate axon maintenance and prun- ing and focused on the role of BDNF. We exposed post-natal day (PND)7 mice to ketamine using a well-established dosing regimen known to induce significant developmental neurotox- icity. We performed morphometric analyses of the infrapyramidal bundle (IPB) since IPB is known to undergo intense developmental modeling and as such is commonly used as a well-established model of in vivo pruning in rodents. When IPB remodeling was followed from PND10 until PND65, we noted a delay in axonal pruning in ketamine-
+
+treated animals when compared to controls; this impairment coincided with ketamine-induced downregulation in BDNF protein expression and maturation suggesting two conclu- sions: a surge in BDNF protein expression Bsignals^ intense IPB pruning in control animals and ketamine-induced down- regulation of BDNF synthesis and maturation could contrib- ute to impaired IPB pruning. We conclude that the combined effects on BDNF homeostasis and impaired axon pruning may in part explain ketamine-induced impairment of neuronal cir- cuitry formation.
+
+Keywords Infrapyramidal bundle . Synaptogenesis . Brain-derived neurotrophic factor . Mossy fibers . General anesthesia . Immature brain
+
+Introduction
+
+Vesna Jevtovic-Todorovic
+
+vesna.jevtovic-todorovic@ucdenver.edu
+
+1 Department of Neuroscience, Mount Sinai School of Medicine, New
+
+York, NY, USA
+
+2 Department of Anesthesiology, University of Colorado School of Medicine, 12401 East 17th Avenue, Aurora, CO 80045, USA 3 Department of Anesthesiology and Pharmacology, University of
+
+Padua, Padua, Italy
+
+4 Department of Medicine, University of Virginia Health System,
+
+Charlottesville, VA, USA
+
+5 University of Virginia College of Arts and Sciences,
+
+Charlottesville, VA, USA
+
+6 Department of Anesthesiology, University of Michigan, Ann
+
+Arbor, MI, USA
+
+Advances in modern medicine enable us to care for sick and premature children but force us to rely on extensive and fre- quent use of sedatives and general anesthetics during painful interventions. Unfortunately, recent discoveries show that ex- posure to sedation and general anesthesia (GA) during critical stages of brain development (i.e. synaptogenesis) may be damaging to immature neurons by causing extensive apopto- tic cell death ultimately resulting in long-term neurocognitive and behavioral impairments [1–7].
+
+Synaptogenesis involves two equally important regressive events: (1) naturally occurring neuronal death by apoptosis to eliminate neurons that are not appropriately connected with their targets [8, 9] and (2) axon pruning to select axons that participate in active neural circuits [8, 9]. We know that GA exacerbates neuronal apoptosis, thus causing widespread de- letion of developing neurons in vulnerable brain regions. However, it is not yet clear whether or how GA impairs
+
+Mol Neurobiol (2018) 55:164–172
+
+selection of appropriate axons and elimination of redundant axons, two balancing forces necessary for the formation and fine-tuning of neuronal networks. Considering the protracted nature of cognitive and behavioral impairments that appear to worsen over a lifetime [1, 10], we set out to examine whether an early exposure to GA causes long-term impairments of proper axon maintenance and pruning in neurons that survive the initial apoptotic Battack.^
+
+It seems that axon pruning manifests through at least two major phases: one, more robust and related to interplay of major neurotrophic factors and later phase, dominated by lo- cally produced and secreted neuronal growth factors. Hence, to begin to understand the mechanisms of anesthesia-induced modulation of axon maintenance and pruning, we focused on the role of neurotrophic factors. Brain-derived neurotrophic factor (BDNF) was of particular interest for this study because the disturbances in BDNF expression and function have been shown to impair axon pruning. For example, sympathetic neu- ron targets are inappropriately innervated in BDNF(+/−) knock- out mice [11], whereas BDNF deprivation in neuronal cultures impairs axonal growth, causes extensive axonal degeneration and impairs axon competition [11]. Given that GA exposure during critical stages of synaptogenesis causes perturbation in BDNF regulation and impairment of Trk pathway activation [12, 13] while decreasing neuronal activity, we hypothesize that GA impairs the axon selection and pruning, which may explain long-term defects in neuronal networking and may in turn be the culprit for reported functional impairments.
+
+We used an early exposure to ketamine as a well- established model of anesthesia-induced developmental neu- rotoxicity in mice [14] and developmental modeling of infrapyramidal bundle as a well-established model of in vivo pruning [15]. We report that an early exposure to ketamine delays axonal pruning; this impairment coincides with ketamine-induced downregulation in BDNF expression and maturation suggesting that ketamine-induced modulation of BDNF synthesis and maturation could at least in part contrib- ute to the impairment of neuronal circuitry formation.
+
+Materials and Methods
+
+Animals
+
+We used 7-day-old (PND7) CD-1 mice (Harlan Laboratories, Indianapolis, IN) for all experiments. We chose this age be- cause (1) it is when rodents are most vulnerable to GA- induced developmental neurotoxicity [16] and (2) it falls be- fore developmental pruning of the infrapyramidal bundle (IPB) begins [15]. Our ketamine anesthesia protocol was as follows: experimental mouse pups were exposed to 6 h of ketamine anesthesia, and controls were exposed to 6 h of mock anesthesia (vehicle) injected IM. During anesthesia,
+
+pups were carefully monitored. After the administration of anesthesia, mice were reunited with their mothers until sacri- fice (from P8 until P65). The weaning was done at P21 using the standard protocol. At the desired age, mice were divided randomly into two groups: one group for assessing expression of pro- and the mature form of BDNF using the Western blotting technique and the second group for morphometric studies of IPB development. Our randomization process was designed to provide each group with roughly equal represen- tation of pups from each dam.
+
+The experiments were approved by the Animal Use and Care Committees at the University of Colorado, the Office of Laboratory Animal Resources (OLAR), Aurora, Colorado and the Animal Use and Care Committees of the University of Virginia, Charlottesville, Virginia. The experiments were done in accordance with the Public Health Service’s Policy on Humane Care and Use of Laboratory Animals. Efforts were made to minimize the number of animals used while being able to conduct meaningful statistical analyses.
+
+Anesthesia Administration
+
+To achieve general anesthesia state, we used a ketamine pro- tocol known to cause significant developmental neurotoxicity in PND7 mice whereby mouse pups received a total of four doses of ketamine, at 75 mg/kg, IM every 90 min so that the loss of righting reflex and lack of response to tail pinch could be maintained for 6 h [14]. For control animals, saline was administered using the same volume and administration schedule. During entire anesthesia procedure, animals were kept away from their mother and were housed in standard, tightly closed mice cages, with free air flow through air filters. Animals were kept in close proximity to each other in the cages, so they could preserve, even under anesthesia, impor- tant olfactory cues and stimuli, necessary to bust and sustain their metabolic output. During the experiment, we carefully monitored animals and measured environmental temperature in their breeding cages. We established that the ambient tem- perature in the breeding cage was around 37.0 ± 1 °C. Considering that animals at this age are very sensitive to change in body temperature, they were kept under constant ambient temperature maintained with heating pads conve- niently set up around the cages. The ambient temperature was assessed at frequent time intervals using a thermometer.
+
+Western Blot Studies
+
+For BDNF protein quantification, we dissected the hippocampus immediately after the brains were removed from the individual pups using a dissecting scope (10× magnification). Tissue was collected on ice and was snap-frozen in liquid nitrogen immedi- ately. The protein concentration of the lysates was determined with the Total Protein Kit using the Bradford method (Cayman
+
+165
+
+166
+
+Chemical, Ann Arbor, MI). Approximately 10–25 μg of total protein was heat-denatured and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) through 4–20% Tris-glycine polyacrylamide gradient gels (BioRad, Hercules, CA). Separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked at room temperature for 1 h in 3% bovine serum albumin (BSA) followed by incubation at 4 °C overnight with primary antibody, anti-BDNF (1:500, Alomone Labs, Jerusalem, Israel), and anti-β-actin antibody (1:10,000, Sigma- Aldrich, USA) as a loading control.
+
+Membranes were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies—goat anti-rabbit or goat anti-mouse IgG (1:10,000, Santa Cruz, Dallas, TX). Three washes with 0.3% Tween-20 in Tris-buffered saline were performed between all steps. Immunoreactivity was detected using en- hanced chemiluminescence substrate (SuperSignal West Femto, Thermo Scientific, UT). Images were captured using GBOX (Chemi XR5, Syngene, MD), and gels were analyzed densitometrically with the computerized image analysis pro- gram ImageQuant 5.0 (GE Healthcare, Life Sciences, Piscataway, NJ).
+
+Histological Preparation
+
+Mice were deeply anesthetized with 2% isoflurane and imme- diately perfused with 4% paraformaldehyde in 0.1 M phos- phate buffer (at pH 7.4). Brains were extracted and immersed in fresh 4% paraformaldehyde and incubated at 4 °C for ad- ditional 2–3 days before being embedded in agar. Briefly, brain coronal sections (50 μm thickness) were cut using vibratome. Tissue sections were blocked with 1× TBS con- taining 5% normal goat serum, 1% BSA and 0.1% Triton X- 100 for 1 h at room temperature before incubated with primary antibodies against calbindin (anti-calbindin D-28K antibody, 1:1000; Gene Tex, CA, USA) overnight at 4 °C. Free floating sections were then washed three times with TBS and then incubated with corresponding HRP-conjugated secondary an- tibodies (1:200) at room temperature for 2 h. Tissue sections were mounted on glass slides and air-dried. For detection, we used DAB Peroxidase substrate kit (Vector Laboratories) fol- lowing manufacturer’s instructions.
+
+Histological Morphometric Assessment
+
+The morphometric analyses of IBP developmental shortening (from PND10 until PND65 in both control and ketamine- treated mice) were performed using coronal hippocampal slices (50 μm) cut from bregma − 1.34 to − 2.30 mm (as determined using a mouse brain atlas). The images were scanned at 20× magnification using an Aperio Scanscope XT digital slide scanner (Aperio Technologies Inc., Vista,
+
+Mol Neurobiol (2018) 55:164–172
+
+CA) at the University of Virginia, Charlottesville, VA and at the University of Colorado, Aurora, CO. The hippocampal area in digital sections (.svs file) was extracted at 600 μm scale to convert to a .tiff file and was spatially calibrated using 1000 μm2 grid prior to quantifying using Image-Pro Plus 7.0 software (Media Cybernetics, MD). The morphometric ap- proach was used to evaluate so-called Bnormalized length of IPB,^ which takes into consideration individual variability and developmental growth of hippocampus. The IPB length was approximated from the tip of the inferior blade of the dentate granule cell layer (Ba^). The length of CA3 was approximated from the tip of the inferior blade to the apex of the curvature of the CA3 pyramidal cell layer (Bb^). Normalized IPB length was taken as a ratio between Ba^ and Bb.^ The values from serial sections (n = 3–6 serial sections per animal from 6 to 7 animals per age group) were averaged to provide a single data point and were presented as normalized IPB length. The re- sults from different age groups were statistically analyzed by t test and between both groups by two-way ANOVA using GraphPad Prism 5.01 software (GraphPad, CA). The experi- menters were blinded to the experimental condition.
+
+Statistical Analysis
+
+Comparisons among groups were made using one-way and two-way ANOVAs followed by Tukey’s post-hoc test. Using the standard version of GraphPad Prism 5.01 software (Media Cybernetics, Inc., Bethesda, MD), we considered p < 0.05 to be statistically significant. All data are presented as mean ± SD or mean ± SEM. The sample sizes reported throughout the BResults^ section and in the figure legends were based on previous experience.
+
+Results
+
+To begin to understand the effects of general anesthesia on neuronal circuitry remodeling, we focused on well- established IPB model of in vivo axon connectivity. The IPB is formed from the axons of granule cells in the dentate gyrus projecting to pyramidal cells mostly in region CA3 (Fig. 1). During normal development, the IPB undergoes a progressive decrease in size due to tightly controlled axon selection and pruning, a process necessary to assure proper circuitry forma- tion in developing hippocampus [15].
+
+The IPB Fails to Undergo Proper Shortening in Ketamine-Treated Mice
+
+To begin to understand whether an early GA exposure impairs axon pruning, we examined the IPB length in CD-1 mice that were exposed to either saline (vehicle control) or ketamine at post-natal day (PND)7 (Fig. 2a). We chose this age because (1)
+
+Mol Neurobiol (2018) 55:164–172
+
+Fig. 1 Schematic representation of the infrapyramidal bundle (IPB). The IPB is formed from the axons of granule cells in the dentate gyrus projecting to pyramidal cells in region CA3. During normal development, the IPB undergoes progressive decrease in size due to tightly controlled axon selection and pruning
+
+it is when rodents are most vulnerable to GA-induced develop- mental neurotoxicity [16] and (2) it falls immediately before developmental pruning of the IPB begins [15]. As stated earlier, a total of four doses of ketamine, at 75 mg/kg, IM, were admin- istered every 90 min so that the loss of righting reflex and lack of response to tail pinch could be maintained for 6 h. The IPB length was measured at different age points—when IPB length is about maximal (PND10); the IPB length begins to decrease (PNDs 20 and 30); and the IPB length reaches its final length (PNDs 40 and 65) [15]. The morphometric approach used to evaluate so-called Bnormalized length of IPB,^ which takes into consideration individual variability and developmental growth of hippocampus, is shown in Fig. 2b. The IPB length was measured from the tip of the inferior blade of the dentate gran- ule cell layer (Ba^). The length of CA3 was measured from the tip of the inferior blade to the apex of the curvature of the CA3 pyramidal cell layer (Bb^). Normalized IPB length was taken as a ratio between Ba^ and Bb^ [15].
+
+As shown in Fig. 3, control animals underwent substan- tial shortening of the IPB from PND20 until the PND65 compared to PND10 (shortening from 20 to 80%,
+
+Fig. 2 The time line of the experimental design and the IPB morphometry in mice. a Ketamine exposure occurred at post-natal day (PND)7 when synaptogenesis is at the peak in mice. The IPB length was measured at different age points—when IPB length is about maximal (PND10); IPB length begins to decrease (PNDs 20 and 30); and IPB length reaches its final length (PNDs 40 and 65). b The morphometric approach used to evaluate so-called Bnormalized length of IPB,^ which
+
+respectively; see table for pairwise comparison to PND10), whereas ketamine-treated ones maintained the the PND30 IPB length close to PND10 level until (p = 0.572), resulting in a significantly longer IPB throughout the experimental time line [two-way ANOVA main effect on treatment (F (1,56) = 9.247); (**, p < 0.01)] (n = 6–7 pups per data point) suggesting a rightward shift with ketamine treatment (Fig. 3a). Representative microphotographs from the control and ex- perimental animals are depicted in Fig. 3b (black line underlines the IBP length. As stated earlier, we used calbindin staining (with anti-calbindin D-28K antibody) to label mossy fibers in the IPB since it labels long un- myelinated axons [17].
+
+BDNF Protein Expression is Downregulated in Ketamine-Treated Mice
+
+To begin to decipher the mechanism of ketamine-induced delay in the IPB pruning, we assessed BDNF protein ex- pression changes during early stages of brain development.
+
+takes into consideration individual variability and developmental growth of hippocampus. The IPB length was approximated from the tip of the inferior blade of the dentate granule cell layer (Ba^). The length of CA3 was approximated from the tip of the inferior blade to the apex of the curvature of the CA3 pyramidal cell layer (Bb^). Normalized IPB length was taken as a ratio between Ba^ and Bb^
+
+167
+
+168
+
+Fig. 3 Ketamine exposure impairs IPB pruning in young mice. a Control animals underwent significant shortening of the IPB from PND20 until the PND65 compared to PND10, whereas ketamine-treated ones maintained the IPB length at PND10 level until the PND30 (see the table depicting within- the-group comparisons). The IPB remained longer throughout the experimental time line in ketamine-treated animals suggesting a rightward shift with ketamine treatment [two-way ANOVA main effect on treatment (F (1, 56) = 9.247); (**, p < 0.01)] (n = 6–7 pups per data point). b Representative microphotographs from the control and experimental animals are depicted from PND10 to PND65. To label mossy fibers in the IPB, we used calbindin staining (with anti-calbindin D-28K antibody) since it labels long unmyelinated axons (magnification 20×). Note a delay in the IPB shortening in ketamine- treated animals (right panels) when compared to controls (left panels)
+
+We measured protein levels of mature (Fig. 4a; n = 3–4) and pro- (Fig. 4b; n = 3–4) forms of BDNF in saline- and ketamine-treated mice. The treatment was performed at PND7, and the Western blot analyses of hippocampal tis- sue were performed in frequent intervals (until PND30) to capture the BDNF changes during the initial phase of IPB pruning. As shown in Fig. 4a, the mature BDNF levels rise steadily in both control and ketamine-treated mice over the course of first 20 post-natal days, peak around PND22, and slowly decline thereafter in controls (PND22 vs. PND19, ††, p < 0.01 and PND22 vs. PND26, †, p < 0.05).
+
+Mol Neurobiol (2018) 55:164–172
+
+Ketamine-treated animals exhibit a much less robust in- crease in BDNF levels throughout with a lower BDNF level when compared to age-matched controls. Note that there is over a twofold decrease in BDNF expression in the ketamine group on PND22 as compared with age-matched controls (***, p < 0.001). Interestingly, pro-BDNF levels appeared to be somewhat elevated in ketamine-treated an- imals shortly after the treatment (Fig. 4b). However, starting from PND13, there was a precipitous decline in their pro-BDNF levels that was significant on PND22 as compared with age-matched controls (*, p < 0.05).
+
+Mol Neurobiol (2018) 55:164–172
+
+The Impairment of BDNF Homeostasis Corresponds with the Timing of Intense IPB Pruning
+
+To assess how the changes in mature BDNF levels correspond to the time line of the IPB pruning, we superimposed the IPB pruning curve on the mature (Fig. 5a) and pro-BDNF expres- sion curves (Fig. 5b) and discovered that around the time when intense IPB pruning is normally initiated (the time course of normal IPB pruning is superimposed with a blue dashed line), there is a peak in BDNF expression followed by a precipitous decline in control animals suggesting that under normal circumstance, an increase in pro- and mature
+
+ƒFig. 4 Ketamine exposure impairs BDNF homeostasis in young mice. a Mature BDNF protein expression was examined during the early stage of brain development in saline and ketamine-treated mice. The treatment was performed at PND7, and the Western blot analyses of hippocampus were performed in frequent intervals as indicated (until PND30). The mature BDNF levels rise steadily in both control and ketamine-treated mice over the course of first 20 post-natal days, peak around PND22, and slowly decline thereafter in controls (PND22 vs. PND19, ††, p < 0.01 and PND22 vs. PND26, †, p < 0.05). Ketamine-treated animals exhibit much less robust increase in BDNF levels throughout with a lower BDNF level when compared to age-matched controls. Note that there is over a twofold decrease in BDNF expression on PND22 in the ketamine group as compared with age-matched controls (***, p < 0.001) (n = 3–4 animals per data point). b Pro-BDNF protein expression was examined during the early stage of brain development in saline and ketamine-treated mice. The treatment was performed at PND7, and the Western blot analyses of CA3 and dentate gyrus were performed in frequent intervals (until PND30). Pro-mature BDNF levels appeared to be somewhat elevated (although not significantly) in ketamine-treated animals shortly after the treatment. However, starting from PND13, there was a precipitous decline in their pro-BDNF levels found to be significant on PND22 as compared with age-matched controls (*, p < 0.05) (n = 3–4 animas per data point)
+
+BDNF provides a homeostatic Bsignal^ for intense IPB prun- ing (around PND22; marked with shaded rectangles).
+
+Discussion
+
+We show in this study that ketamine exposure during the crit- ical stages of mouse brain development impairs timely IPB pruning. Since both pro- and mature BDNF forms exhibit a significant decline at the time when intense IBP shortening begins (around PND20), we propose that ketamine-induced impairment in developmental axon pruning could be, at least in part, BDNF-dependent. We further hypothesize that distur- bance in axonal selection and pruning may result in faulty formation of functional neuronal networks among the remain- ing (Bnormal^) neurons. This notion remains to be confirmed in future mechanistic and functional studies.
+
+The importance of neuronal activity in regulating develop- mental axon competition is well-established. For example, developing rat sympathetic eye-projecting neurons initially extend axon collaterals to two different eye sections, but then axon elimination occurs, so that any individual neuron ulti- mately only projects to one section [18]. If, however, the ac- tivity is disturbed during this critical time period, axon selec- tion does not occur, thus affecting the innervations of an eye. Although the effect of general anesthesia on neuronal activity in vast brain circuitries is complex and not well understood, it is clear that a substantial decrease in neuronal activity has to occur to induce the state of unconsciousness, amnesia, and insensitivity to pain—three key features of the general anes- thesia state. Hence, GA-induced neuronal inhibition may be the cause of improper axon selection manifested as delayed pruning.
+
+169
+
+170
+
+Fig. 5 The peak of BDNF protein expression coincides with the initiation of intense IPB pruning in young mice. a When the IPB pruning curve (dashed blue line) was superimposed on the mature BDNF protein expression curve in control animals, it showed that a substantial peak in BDNF expression occurs around the time when intense IPB pruning is normally initiated. b When the IPB pruning curve (dashed blue line) was superimposed on the pro-BDNF protein expression curve in control animals, it showed that an increase in pro- BDNF expression occurs around the time when intense IPB pruning is normally initiated
+
+The role of neurotrophic factors in developmental axon competition is becoming more appreciated. We, along with others have previously reported that the exposure to GA causes profound disturbances in homeostasis of the neuro- trophic factor, BDNF. This, in turn, leads to the inhibition of Trk-B-dependent pathways, either directly or indirectly via p75NTR signaling, ultimately resulting in neuronal death [12, 13] since both Trk and p75NTR receptors modulate the activa- tion of major survival pathways for neurons [19, 20]. However, after a decade of intense investigation, we question whether the detrimental effects of GA on BDNF signaling have consequences beyond inducing neuronal death to in- clude compromising the development and function of the re- maining surviving neurons by impairing not only current but also future connections, maintenance of neuronal circuits, and general plasticity of dentate gyrus-CA3 communications. We base this hypothesis on data presented herein which suggests the impairment of proper and timely axon pruning, a crucial process during normal development that allows removal of exuberant or misguided axon branches while maintaining oth- er appropriate connections of the same neuron. If, indeed, GA
+
+Mol Neurobiol (2018) 55:164–172
+
+impairs axon selection during critical stages of synaptogene- sis, this effect may account for long-lasting impairment of neuronal networking and circuitry formation [21].
+
+We find that the time course of this impairment correlates with GA-induced downregulation of BDNF protein expres- sion in developing hippocampus in vivo. Although our work was not focused on studying Trk-mediated signaling, based on presently available knowledge, the basic mechanism suggests that active axons secrete BDNF enabling extensive activation of p75NTR receptors located on a Blosing^ (less active) axon. p75NTR activation inhibits Trk-mediated signaling, which is essential for axon maintenance, thus promoting degeneration and pruning of losing axons [11, 22]. When BDNF levels are downregulated, this timely activation of p75NTR receptors is impaired resulting in faulty axon pruning similar to the one we report herein.
+
+Our findings indicate that GA impairs the normal progres- sive decrease in the IPB of mossy fiber projections (IMF) in the hippocampus, an important event in the formation of proper circuitry between the dentate gyrus and the CA3 region, a neu- ronal circuitry that plays a crucial role in learning and memory [23]. We focused on this well-established IPB model of in vivo axon connectivity because during normal development, the IPB undergoes a progressive decrease in size due to tightly con- trolled axon selection and pruning, a process necessary to assure proper circuitry formation in developing hippocampus [15]. The size of the IPB correlates inversely with performance in a variety of cognitive tasks, i.e. the longer the IPB, the more learning and memory development (in particular spatial learn- ing) [23] are impaired, suggesting that subtle disturbances in hippocampal circuitries could have detrimental long-term ef- fects on cognitive development. Because spatial learning is im- paired in animals exposed to ketamine [24] (intravenous anes- thetics used in pediatric medicine) and because our data suggest that ketamine compromises IPB pruning, we propose a tempo- ral association between ketamine-induced impairment of BDNF homeostasis and disturbances in normal development of IPB. It remains to be determined whether this temporal asso- ciation could explain the impairment in cognitive functioning previously reported. This notion could be considered based on the observation that BDNF is critical for cognitive development [25], modulates the strength of existing synaptic connections, and assists in the formation of new synaptic contacts [26, 27]. In addition, pro- and mature forms of BDNF can induce long-term potentiation and depression [28], known to be impaired after an early exposure to anesthesia [1]. A decrease greater than two- fold in BDNF expression in the ketamine group around the time of intense IPB pruning approaches a reduction in BDNF previ- ously reported to be sufficient to eliminate the competitive ad- vantage of active neurons, thus resulting in impaired pruning [29]. It is noteworthy that somewhat elevated pro-BDNF levels in ketamine-treated animals we report herein could be an at- tempt to compensate for a decrease in mature BDNF.
+
+Mol Neurobiol (2018) 55:164–172
+
+In conclusion, we report that ketamine exposure during critical stages of mammalian brain development leads to an impaired BDNF homeostasis and delayed pruning of axons known to be critically important for proper cognitive develop- ment. We suggest that disturbance in axonal selection and pruning may lead to a faulty formation of functional neuronal networks among the remaining (Bnormal^) neurons thus resulting in an impaired synaptic neurotransmission we and others have previously reported [1, 21]. Further studies of neuronal circuitry formations vis-à-vis the studies of axonal selection and pruning are needed to make final determination.
+
+Acknowledgments This study was supported by the grants R0144517 (NIH/NICHD), R0144517-S (NIH/NICHD), R01 GM118197 (NIH/ NIGMS), R21 HD080281 (NIH/NICHD), John E. Fogarty Award 007423-128322 (NIH), and March of Dimes National Award, USA (to Vesna Jevtovic-Todorovic). Vesna Jevtovic-Todorovic was an Established Investigator of the American Heart Association. We thank Jonathan Park for his assistance with the morphometric analysis of the IPB pruning.
+
+Compliance with Ethical Standards
+
+Conflicts of Interest The authors declare that they have no competing interests.
+
+Human and Animal Rights and Informed Consent The experiments were approved by the Animal Use and Care Committees at the University of Colorado, the Office of Laboratory Animal Resources (OLAR), Aurora, Colorado and the Animal Use and Care Committees of the University of Virginia, Charlottesville, Virginia. The experiments were done in accordance with the Public Health Service’s Policy on Humane Care and Use of Laboratory Animals.
+
+References
+
+1.
+
+Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF (2003) Early exposure to common anesthetic agents causes widespread neurode- generation in the developing rat brain and persistent learning defi- cits. J Neurosci 23(3):876–882
+
+2. Loepke AW, Istaphanous GK, McAuliffe JJ 3rd, Miles L, Hughes EA, McCann JC, Harlow KE, Kurth CD et al (2009) The effects of neonatal isoflurane exposure in mice on brain cell viability, adult behavior, learning, and memory. Anesth Analg 108(1):90–104 3. Paule MG, Li M, Allen RR, Liu F, Zou X, Hotchkiss C, Hanig JP, Patterson TA et al (2011) Ketamine anesthesia during the first week of life can cause long-lasting cognitive deficits in rhesus monkeys. Neurotoxicol Teratol 33(2):220–230
+
+4. Rizzi S, Carter LB, Ori C, Jevtovic-Todorovic V (2008) Clinical anesthesia causes permanent damage to the fetal guinea pig brain. Brain Pathol 18(2):198–210
+
+5. Fredriksson A, Pontén E, Gordh T, Eriksson P (2007) Neonatal exposure to a combination of N-methyl-D-aspartate and gamma- aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. Anesthesiology 107(3):427–436
+
+6. Viberg H, Pontén E, Eriksson P, Gordh T, Fredriksson A (2008) Neonatal ketamine exposure results in changes in biochemical
+
+substrates of neuronal growth and synaptogenesis, and alters adult behavior irreversibly. Toxicology 249(2–3):153–159
+
+7. Kodama M, Satoh Y, Otsubo Y, Araki Y, Yonamine R, Masui K, Kazama T (2011) Neonatal desflurane exposure induces more ro- bust neuroapoptosis than do isoflurane and sevoflurane and impairs working memory. Anesthesiology 115(5):979–991
+
+8. Bishop DL, Misgeld T, Walsh MK, Gan WB, Lichtman JW (2004) Axon branch removal at developing synapses by axosome shed- ding. Neuron 44:651–661
+
+9. Luo L, O’Leary DD (2005) Axon retraction and degeneration in development and disease. Annu Rev Neurosci 28:127–156 10. Wilder RT, Flick RP, Sprung J et al (2009) Early exposure to anes- thesia and learning disabilities in a population-based birth cohort. Anesthesiology 110:796–804
+
+11. Singh KK, Park KJ, Hong EJ, Kramer BM, Greenberg ME, Kaplan DR, Miller FD (2008) Developmental axon pruning mediated by BDNF-p75NTR-dependent axon degeneration. Nat Neurosci 11: 649–658
+
+12. Lu LX, Yon JH, Carter LB, Jevtovic-Todorovic V (2006) General anesthesia activates BDNF-dependent neuroapoptosis in the devel- oping rat brain. Apoptosis 11:1603–1615
+
+13. Pearn ML, Hu Y, Niesman IR, Patel HH, Drummond JC, Roth DM, Akassoglou K, Patel PM et al (2012) Propofol neurotoxicity is mediated by p75 neurotrophin receptor activation. Anesthesiology 116:352–361
+
+14. Young C, Jevtovic-Todorovic V, Qin YQ, Tenkova T, Wang H, Labruyere J, Olney JW (2005) Potential of ketamine and midazo- lam, individually or in combination, to induce apoptotic neurode- generation in the infant mouse brain. Br J Pharmacol 146(2):189– 197
+
+15. Bagri A, Cheng HJ, Yaron A, Pleasure SJ, Tessier-Lavigne M (2003) Stereotyped pruning of long hippocampal axon branches triggered by retraction inducers of the semaphorin family. Cell 113:285–299
+
+16. Yon JH, Daniel-Johnson J, Carter LB, Jevtovic-Todorovic V (2005) Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience 135:815–827
+
+17. Liu XB, Low LK, Jones EG, Cheng HJ (2005) Stereotyped axon pruning via plexin signaling is associated with synaptic complex elimination in the hippocampus. J Neurosci 25:9124–9134 18. Lawrence JM, Black IB, Mytilineou C, Field PM, Raisman G (1979) Decentralization of the superior cervical ganglion in neo- nates impairs the development of the innervations of the iris. A quantitative ultrastructural study. Brain Res 168:13–19
+
+19. Majdan M, Miller FD (1999) Neuronal life and death decisions: functional antagonism between the Trk and p75 neurotrophin re- ceptors. Int J Dev Neurosci 17:153–161
+
+20. Miller FD, Kaplan DR (2001) Neurotrophin signaling pathways regulating neuronal apoptosis. Cell Mol Life Sci 58:1045–1053 21. Mintz CD, Barrett KM, Smith SC, Benson DL, Harrison NL (2013) Anesthetics interfere with axon guidance in developing mouse neo- cortical neurons in vitro via a γ-aminobutyric acid type A receptor mechanism. Anesthesiology 118:825–833
+
+22. Singh KK, Miller FD (2005) Activity regulates positive and nega- tive neurotrophin-derived signals to determine axon competition. Neuron 45:837–845
+
+23. Crusio WE, Schwegler H (2005) Learning spatial orientation tasks in the radial-maze and structural variation in the hippocampus in inbred mice. Behav Brain Funct 1(1):3–11
+
+24. Fredriksson A, Archer T, Alm H, Gordh T, Eriksson P (2004) Neurofunctional deficits and potentiated apoptosis by neonatal NMDA antagonist administration. Behav Brain Res 153:367–376 25. Lu Y, Christian K, Lu B (2008) BDNF: a key regulator for protein synthesis-dependent LTP and long-term memory? Neurobiol Learn Mem 89(3):312–323
+
+171
+
+172
+
+26. Thoenen H (1995) Neurotrophins and neuronal plasticity. Science 270:593–598
+
+27. Lu B, Figurov A (1997) Role of neurotrophins in synapse develop- ment and plasticity. Rev Neurosci 8:1–12
+
+28. Woo NH, Teng HK, Siao CJ, Chiaruttini C, Pang PT, Milner TA, Hempstead BL, Lu B (2005) Activation of p75NTR by proBDNF
+
+Mol Neurobiol (2018) 55:164–172
+
+facilitates hippocampal long-term depression. Nat Neurosci 8: 1069–1077
+
+29. Chen ZY, Ieraci A, Teng H, Dall H, Meng CX, Herrera DG, Nykjaer A, Hempstead BL et al (2005) Sortilin controls intracellu- lar sorting of brain-derived neurotrophic factor to the regulated secretory pathway. J Neurosci 25:6156–6166

new_pdfs/10.1007_s12640-009-9063-8.txt

@@ -0,0 +1,675 @@
+Neurotox Res (2009) 16:140–147 DOI 10.1007/s12640-009-9063-8
+
+Neurodegeneration in Newborn Rats Following Propofol and Sevoflurane Anesthesia
+
+Sven Bercker Æ Bettina Bert Æ Petra Bittigau Æ Ursula Felderhoff-Mu¨ ser Æ Christoph Bu¨ hrer Æ Chrysanthy Ikonomidou Æ Mirjam Weise Æ Udo X. Kaisers Æ Thoralf Kerner
+
+Received: 13 June 2008 / Revised: 21 April 2009 / Accepted: 12 May 2009 / Published online: 27 May 2009 (cid:1) Springer Science+Business Media, LLC 2009
+
+Abstract Propofol and sevoflurane are commonly used drugs in pediatric anesthesia. Exposure of newborn rats to a variety of anesthetics has been shown to induce apoptotic neurodegeneration in the developing brain. Newborn Wistar rats were treated with repeated intraperitoneal injections of propofol or sevoflurane inhalation and compared to controls. Brains were examined histopathologically using
+
+S. Bercker and B. Bert contributed equally to this article.
+
+S. Bercker (&) (cid:1) U. X. Kaisers Department of Anesthesiology and Intensive Care Medicine, University Hospital Leipzig, Leipzig, Germany e-mail: sven.bercker@medizin.uni-leipzig.de
+
+B. Bert Institute of Pharmacology and Toxicology, School of Veterinary Medicine, Freie Universitaet Berlin, Berlin, Germany
+
+the De Olmos cupric silver staining. Additionally, a sum- mation score of the density of apoptotic cells was calculated for every brain. Spatial memory learning was assessed by the Morris Water Maze (MWM) test and the hole board test, performed in 7 weeks old animals who underwent the same anesthetic procedure. Brains of propofol-treated animals showed a significant higher neurodegenerative summation score (24,345) when compared to controls (15,872) and to sevoflurane-treated animals (18,870). Treated animals also demonstrated persistent learning deficits in the hole board test, whereas the MWM test revealed no differences between both groups. Among other substances acting via GABAA agonism and/or NMDA antagonism propofol induced neurodegeneration in newborn rat brains whereas a sevoflurane based anesthesia did not. The significance of these results for clinical anesthesia has not been completely elucidated. Future studies have to focus on the detection of safe anesthetic strategies for the developing brain.
+
+P. Bittigau Children’s Hospital, Campus Virchow-Klinikum, Charite´- Universitaetsmedizin Berlin, Berlin, Germany
+
+Keywords Neurotoxicity (cid:1) Propofol (cid:1) Sevoflurane (cid:1) Newborn (cid:1) Anesthesia (cid:1) Behavior (cid:1) Histology (cid:1) Rodents
+
+U. Felderhoff-Mu¨ser Department of Neonatology, University Hospital of Essen, Essen, Germany
+
+Introduction
+
+C. Bu¨hrer Department of Neonatology, Children’s Hospital, Campus Virchow-Klinikum, Charite´-Universitaetsmedizin Berlin, Berlin, Germany
+
+C. Ikonomidou Department of Neurology, University of Wisconsin, Madison, WI, USA
+
+M. Weise (cid:1) T. Kerner Department of Anesthesiology and Intensive Care Medicine, Asklepios Klinikum Hamburg, Hamburg, Germany
+
+Propofol (6,2 Diisopropylphenol) has been introduced as a in 1977 (Kay and Rolly sedative and anesthetic agent 1977). In pediatric anesthesia it is widely used if intra- venous induction is needed, as a sedative for short interventions, in day case surgery, and in total intravenous anesthesia. Furthermore, propofol is popular due to its short context-sensitive half-life combined with rapid clear-headed reawakening and its antiemetic properties. After the report of 5 children dying from myocardial
+
+123
+
+Neurotox Res (2009) 16:140–147
+
+failure after long-term propofol infusion (Parke et al. 1992) its use in pediatric critical care has been restricted; however, short-term application in children is considered safe. In 1999 the U.S. Federal Drug Administration decreased the approved age for maintenance of anesthesia with propofol to 2 months, whereas in Germany the use of propofol 1% for induction of anesthesia and mainte- nance is approved for children older than 1 month (Motsch and Roggenbach 2004). Despite these restrictions off-label use is common for anesthesia even in preterm infants and neonates.
+
+Due to its favorable properties for inhalational induction of anesthesia, its sweet odor and its rapid onset and offset the fluorinated hydrocarbon sevoflurane (2,2,2-trifluoro-1- [trifluoromethyl]ethyl fluoromethyl ether) is also widely used in current pediatric anesthesia. Similar to most anes- thetics, sevoflurane acts via GABA agonism (Hapfelmeier et al. 2001).
+
+Recently, it was shown that the combined application of GABA-enhancing and NMDA-blocking agents (midazo- lame, nitrous oxide and isoflurane) induced widespread neurodegeneration in the brains of newborn rats (Jevtovic- Todorovic et al. 2003). In addition, this was demonstrated for a variety of other agents with the same mechanisms of action, e.g. barbiturates, benzodiazepines, alcohol and ketamine (Ikonomidou et al. 2001; Bittigau et al. 2002). These findings have been followed by a lively discussion regarding their clinical relevance (Anand and Soriano 2004; Olney et al. 2004).
+
+The presented study was conducted to evaluate the neurotoxic effects of sevoflurane and propofol in newborn rats by analyzing the immediate histological damage and behavioral changes in the adult animals.
+
+Materials and Methods
+
+The experiments were performed according to the guide- lines of the German Animal Protection Law and were approved by the Berlin State authorities.
+
+Wistar rat pups were purchased from the Bundesinstitut fu¨r gesundheitlichen Verbraucherschutz und Veterina¨r- medizin BgVV, Berlin, Germany.
+
+Experimental Protocol
+
+Six-day-old Wistar rats received either intraperitoneal (i.p.) injections of propofol or underwent inhalational anesthesia with sevoflurane and were separated from their mother during the experimental phase. In every litter animals were randomized either for anesthesia or controls.
+
+141
+
+For propofol anesthesia doses of 30 mg/kg body weight were given every 90 min up to a cumulative dose of 90 mg/kg. For gas administration rats were placed into an incubation chamber (Billups Rothenberg Inc., Del Mar, USA), which was connected to an anesthesia system (F.Stephan GmBH, Gackenbach) for 6 h. Carbon dioxide and sevoflurane concentrations were monitored using a gas monitor (Datex Ohmeda, GE Healthcare, Munich, Ger- many). To avoid rebreathing of carbon dioxide, inspired CO2 concentration was continuously monitored and kept below 1 vol% by adjusting the fresh gas flow. Pilot studies established that sevoflurane concentrations between 3 and 5% maintained sufficient depth of anesthesia, as deter- mined by lack of reaction to a painful stimulus. However, it was also observed that skin color changed and respiratory diminished in some animals, suggesting respiratory insuf- ficiency. Accordingly, animals were closely monitored during the experiment and sevoflurane concentration adjusted between 3 and 5 vol% to maintain normal skin color and adequate respiratory efforts.
+
+The animals were observed for another 90 min until they were awake and active to be returned to their mother after the last injection and 6 h of gas application respec- tively. To maintain body temperature and prevent hypo- thermia, animals were placed on a heating device. During anesthesia respiratory frequency and skin color were observed to detect apnea and hypoxia. If bradypnoe occurred, rats received a pain stimulus, if breathing did not restart or resuscitation efforts were necessary rats were excluded from further processing and analysis. Verum and control animals received injections of PA¨ D II cristalloid/ glucose (50 g/l) solution (Fresenius-Kabi, Bad Homburg, Germany) to prevent hypoglycemia and hypovolemia. Control animals were separated from the mother for the same period as the anesthetized animals and received injections of the crystalloid/glucose solution as well. In order to reduce the amount of laboratory animals, the control animals of the propofol group were pooled with control animals from the sevoflurane group. Therefore, sham injections have not been performed. However, all experiments were performed using the same experimental settings and laboratories during the same time.
+
+For perfusion fixation animals were killed with an injection of an overdose of chloral hydrate 24 h after starting anesthesia. A solution of PBS (phosphate buffered saline) mixed with heparine (Thrombophob 25,000, Hep- arin-Natrium; Nordmark Arzneimittel GmbH, Uetersen, Germany) was injected slowly into the heart and ascending aorta in order to wash out the blood from the vessels. Afterwards, rats were perfused with a solution containing paraformaldehyde 4% (Merck, Darmstadt, Germany) with cacodylate buffer for 10 min (De Olmos cupric silver staining).
+
+(Sigma, Deisenhofen, Germany)
+
+123
+
+142
+
+Histology
+
+To visualize degenerating cells, coronal sections (70 lm) of the whole brain were cut on a vibratome and stained with silver nitrate and cupric nitrate (De Olmos and Ingram 1971). This technique stains degenerating cells dying via an apoptotic or non-apoptotic mechanism. Degenerating cells were identified by their distinct dark appearance due to silver impregnation.
+
+Quantification of Damage
+
+Quantification of brain damage was assessed in the frontal, parietal, cingulate, retrosplenial cortex, caudate nucleus (mediodorsal part), thalamus (laterodorsal, mediodorsal, and ventral nuclei), septum, dentate gyrus, hypothalamus, cornu ammonis field CA1, and subiculum in silver stained sections by estimating mean numerical densities (Nv) of degenerating cells (Gundersen et al. 1988). An unbiased counting frame (0.05 mm 9 0.05 mm: dissector height 0.07 mm) and a high aperture objective were used for sampling. The Nv for each brain region was determined with 8–10 dissectors. Regional Nv values from 17 brain regions were summed to give a total score for degenerating neurons for each brain. Figure 1 shows representative sil- ver stained brain regions.
+
+Fig. 1 Light microscopic overviews of silver-stained transverse sections picturing neurodegenerative changes in 6-day-old rats. The images of the rat thalamus show examples 24 h after treatment a after propofol treatment, b sevoflurane treatment, and c for controls. Degenerated neurons are pictured as small dark dots
+
+123
+
+Neurotox Res (2009) 16:140–147
+
+Behavioral Task
+
+treated, n = 7 testing n = 13 propofol For behavioral sevoflurane treated animals and n = 6 controls were anesthetized as described above. Only male pups were used. At the beginning of behavioral testing, animals were 7 weeks old. Animals were group-housed (4–5 animals per cage) under standard laboratory conditions (22 ± 2(cid:2)C room temperature) with an artificial 12 h light-dark cycle (lights on 6.00–18.00 h). Rats had access to food (Altromin 1326, Lage, Germany) and water ad libitum. They were acclimated to the animal unit for at least 2 weeks before testing. One hour prior to testing rats were transferred to a quiet anteroom. All experiments were performed between 8.00 and 13.00 h and only male animals were tested. In order to avoid possible carry-over effects, a pause of 7 days, during which the animals were left undisturbed in their home cages, was introduced between the hole board and water maze task.
+
+Morris Water Maze (MWM) Test
+
+The water maze task was conducted by using the same experimental design as described previously (Bert et al. 2002). A blue circular tank (diameter 200 cm, 60 cm deep) was filled up with water (21 ± 1(cid:2)C) to a height of 42 cm.
+
+Neurotox Res (2009) 16:140–147
+
+The tank was surrounded by several visual cues and was indirectly illuminated (120 lx at the centre of the pool).
+
+For a single adaptation trial (day 0, without platform), the rats were released into the pool for 90 s with no escape platform present. On the following 8 days (day 1–8, place version) a transparent platform (16 9 16 cm) was sub- merged 1.5 cm below the surface in the middle of one of four virtual quadrants which was according to adaptation trial neither preferred nor avoided by the rat. Each day the animals were lowered into the water facing the wall from three different starting points (left, opposite, and right from the platform quadrant). Animals that did not find the escape platform within 90 s were placed onto it by the experi- menter. All rats were allowed to remain on the platform for 30 s for orientation and were afterwards removed to rest for 60 s in a heated cage until the next trial. For each trial the escape latency to reach the platform was measured by a computerized tracking system (TSE VideoMot, Version 1.43, Bad Homburg, Germany). For each animal the three daily trials were averaged. On day 9 the escape platform was removed (spatial probe) and the time spent in each quadrant during a single 90 s trial was registered. On day 10 (cued version) the platform was elevated 1 cm above water level, signaled by a white cylinder (diameter 3 cm and 4 cm high), and moved to the quadrant opposite to the initial quadrant. This test was performed to assess the motivation to escape from the water and sensor-motor integrity. The testing procedure and recorded parameter during the cued version were the same as for the hidden platform version of the task (Morris 1984).
+
+Hole Board Test
+
+The hole board apparatus consisted of a square box (50 9 50 cm) made of grey Perspex with 16 equally spaced holes (diameter 2.5 cm), and was situated in a sound-attenuated chamber. The behavior of rats was monitored by an overhead installed video camera which was linked to a computerized tracking system (TSE Vid- eoMot2, Bad Homburg, Germany). The test was conducted on two consecutive days. On both days rats were placed in the centre of the apparatus and observed for 10 min. The numbers of nose pokes and rearings as well as the distance traveled were recorded. After each animal the box was cleaned with 2-propanol 30%. Habituation to the apparatus was defined as a significant reduction of nose pokes, rearings and locomotor activity from the 1st to the 2nd day (Voits et al. 1995).
+
+Blood Gas Analysis
+
+To exclude severe hypoxia, hypercapnia or lactic acidosis, a blood gas analysis was performed for example in one
+
+143
+
+animal of each group by transcutaneous puncture of the left ventricle. The probe was analyzed by a blood gas analyzer (Radiometer ABL series, Radiometer, Copenhagen, Denmark).
+
+Statistical Analysis
+
+The Kolmogorov Smirnov test was used to test for normal distribution. The results of the sum scores were compared using the Mann–Whitney U test between controls and propofol as well as between controls and sevoflurane. The place version data of the water maze test were analyzed by two-way ANOVAs on repeated measures followed by Holm-Sidak method for post-hoc multiple pair-wise com- parisons. The spatial probe and the cued version data were analyzed by one-way ANOVAs followed by Holm-Sidak post-hoc tests. The data of the hole board test were ana- lyzed by paired t-tests. Differences were considered to be significant if P \ 0.05.
+
+Results
+
+Anesthetic Procedures
+
+All rats subjected to propofol injections or inhalational anesthesia with sevoflurane developed deep anesthesia with no or only minor reaction to pain stimuli. Rats were observed concerning skin color and respiratory frequency. In all but two rats no pathological findings were observed. Of the anesthetized rats, n = 1 in each treatment group had apnea and died during the experiment. Blood gas analyses revealed normal results.
+
+Histology
+
+For silver nitrate and cupric nitrate staining, 26 animals have been processed after propofol anesthesia and 13 ani- mals after sevoflurane anesthesia whereas 18 controls were separated from their mothers as described above. The results of the sum score, the cell counts of every brain region and the differences in whole body weight as well as the absolute brain weight between anesthetized and control animals are summarized in Table 1. There was a significant higher sum score of degenerated neurons in propofol treated animals compared to the control group (P \ 0.001). Main differences in the amount of degenerated cells could be detected in the mediodorsal and laterodorsal part of the thalamus and in the subiculum. There was no significant difference between controls and sevoflurane treated ani- mals (P = 0.944).
+
+123
+
+144
+
+Neurotox Res (2009) 16:140–147
+
+Table 1 Results for controls, propofol and sevoflurane treated animals concerning brain weight (g), difference of body weight before and 24 h after the experiments, and histological damage as sum score and in the several regions
+
+Percentiles
+
+Controls
+
+Propofol
+
+Sevoflurane
+
+25
+
+50
+
+75
+
+25
+
+50
+
+75
+
+25
+
+50
+
+75
+
+Brain weight (g)
+
+0.64
+
+0.69
+
+0.7
+
+0.6
+
+0.64
+
+0.68
+
+0.575
+
+0.6
+
+0.61
+
+Change in body weight (g)
+
+1.35
+
+1.9
+
+2.55
+
+0.4
+
+1.4
+
+1.8
+
+0.6
+
+1.2
+
+1.45
+
+Sum score CFR II
+
+13140 2000
+
+15872 2571
+
+19821 3571
+
+18622.75 2000
+
+24345.5 3714
+
+32677 5571
+
+12765 2571
+
+17410 3000
+
+18537 3928.5
+
+CFR IV
+
+635
+
+952
+
+1270
+
+635
+
+952
+
+1270
+
+635
+
+952
+
+1270
+
+CING II
+
+1143
+
+2143
+
+2571
+
+1429
+
+2571
+
+4143
+
+1785.5
+
+2429
+
+2857
+
+CING IV
+
+1270
+
+1270
+
+1587
+
+1270
+
+1270
+
+1587
+
+1270
+
+1587
+
+1746
+
+caud
+
+228
+
+314
+
+674
+
+200
+
+286
+
+800
+
+328.5
+
+400
+
+700
+
+septum
+
+71
+
+143
+
+286
+
+114
+
+200
+
+371
+
+171
+
+257
+
+314
+
+CPR II
+
+1500
+
+2143
+
+4071
+
+2571
+
+4000
+
+6143
+
+1643
+
+2571
+
+3856.5
+
+CPR IV
+
+793
+
+952
+
+1270
+
+635
+
+635
+
+1270
+
+635
+
+635
+
+1269.5
+
+RSC II
+
+500
+
+1000
+
+1214
+
+571
+
+857
+
+1143
+
+857
+
+1143
+
+2428.5
+
+RSC IV
+
+952
+
+1270
+
+1905
+
+952
+
+952
+
+1587
+
+952
+
+952
+
+1587.5
+
+TH MD
+
+86
+
+171
+
+285
+
+257
+
+343
+
+486
+
+114.5
+
+200
+
+257
+
+TH LD
+
+314
+
+400
+
+643
+
+1057
+
+2371
+
+4171
+
+585.5
+
+771
+
+1814.5
+
+TH V
+
+71
+
+114
+
+328
+
+86
+
+200
+
+314
+
+100
+
+143
+
+228.5
+
+hyth
+
+385
+
+486
+
+743
+
+371
+
+571
+
+1114
+
+285.5
+
+543
+
+700
+
+ca1 dg
+
+286 171
+
+429 229
+
+571 343
+
+286 229
+
+429 286
+
+571 400
+
+214.5 171
+
+286 286
+
+429 485.5
+
+subic
+
+400
+
+686
+
+900
+
+943
+
+1200
+
+1972
+
+486
+
+600
+
+671
+
+CFR frontal cortex, CING cingulated cortex, caud caudate, septum, CPR parietal cortex, RSC retrosplenial cortex, TH thalamus, hyth hypo- thalamus, ca1 ca1 hippocampus, dg dentate gyrus, subic subiculum
+
+MWM Test
+
+Place Version
+
+motor integrity. Also, in the cued version a difference between the propofol or sevoflurane treated animals and controls was not detected.
+
+There was a significant effect of factor day during the place version (P \ 0.001) in all groups. Post-hoc analysis revealed that all groups exhibited decreasing time escaping the water on days 2–8 when compared to day 1. Even though it seems that on day 2 and 4 propofol treated rats were seeking the platform for a longer period, a significant group effect was not observed (F(1, 17) = 2.050; P = 0.170).
+
+Spatial Probe
+
+The good performance during the place version was reflected in the spatial probe. In all three groups, rats preferred the quadrant where the platform was located during the place version to the other three quadrants. Accordingly, the test did not show any difference between controls and anesthetized rats.
+
+Hole Board
+
+Control animals showed a significant reduction of nose pokes behavior (P = 0.030) and a decrease in locomotor activity from day 1 to day 2 (P = 0.012), referring to a non- spatial habituation learning. However, the numbers of rear- ings were not significantly decreased on day 2. In contrast, propofol-treated animals showed no habituation to the hole board. The number of nose pokes and distance traveled remained unchanged, whereas the number of rearings was even increased on day 2 (P = 0.01). In contrast, a significant reduction of nose pokes behavior from day 1 to day 2 could be shown in sevoflurane-treated animals (P \ 0.05).
+
+Discussion
+
+Cued Version
+
+All animals showed a decrease in the escape latencies during the 2nd and 3rd trial indicating no deficient sensory-
+
+In our study a short-term propofol anesthesia of approxi- mately 4.5 h duration induced histological neurodegener- ation in the immature rat brain and led to persistent
+
+123
+
+Neurotox Res (2009) 16:140–147
+
+learning deficits. Anesthesia with a cumulative dose of 90 mg/kg propofol caused significantly higher scores of degenerated neurons compared to controls.
+
+Accordingly, animals showed significant learning defi- cits during behavioral testing after 7 weeks. In contrast, a prolonged exposure to sevoflurane did not lead to mor- phological brain damage or to learning deficits when compared to controls.
+
+A broad variety of psychoactive and sedative substances such as barbiturates, benzodiazepines, antiepileptic drugs (Bittigau et al. 2002), Ketamine (Fredriksson et al. 2007) and anesthetics with a combination of sedatives (Jevtovic- Todorovic et al. 2003) as well as different doses of pro- pofol (Fredriksson et al. 2007; Cattano et al. 2008) have been shown to induce apoptosis in the central nervous system of animals.
+
+Jevtovic-Todorovic et al. demonstrated that anesthesia with a cocktail of midazolam, Isoflurane and nitrous oxide led to similar deficiencies.
+
+In most monitored brain regions propofol-treated ani- mals showed an increase in degenerated cells. The maxi- mum difference was detected in the thalamus. Learning deficits have been shown to be associated with circum- scribed hippocampal damage (Morris et al. 1982). How- ever, in our study neuronal damage was distributed diffusely and the hippocampus was also affected. The location of the maximum effects in the thalamus is in accordance with a recent study of Nikizad et al. (2007) who demonstrated the neurotoxic effects of a 6-h anesthesia with Isoflurane, Midazolam and nitrous oxide.
+
+In the learning tests there were only minor effects of propofol treatment on spatial navigation, as estimated by the MWM test. Although the learning curve during the place version showed a slight difference toward a learning deficit no group effect for the whole investigation period could be shown. Furthermore, results did not differ on day 8. In contrast, propofol-treated animals showed no habit- uation to the hole board. The number of nose pokes and distance traveled remained unchanged. Thus, propofol anesthesia affected the animals’ capability for explorative learning.
+
+It has been criticized that neurotoxic effects of anes- thetics in animal models might be induced by uncontrol- lable the mother, such as deprivation of hypoglycemia or hypoxia (Soriano et al. 2005). In the lit- erature there is evidence for an aggravation of apoptosis by glucose deprivation (Wise-Faberowski al. 2001). Therefore, it might be speculated that isolation from the mother and hypoalimentation alone induced apoptosis of neurons. However, in our experiments there was no sig- nificant difference in the increase of body weight between controls and propofol-treated animals in the 24 h after testing. Additionally, the brain weights did not differ. The
+
+effects
+
+et
+
+145
+
+blood gas analysis for one propofol-treated animal revealed normal results. The death of one animal possibly caused by depth of propofol anesthesia might be seen as an indication for asphyxia in at least some animals. However, a corre- sponding case was also seen in the sevoflurane group and here no difference in neurotoxic effects or any learning deficits could be demonstrated.
+
+A possible limitation might be that sham injections have not been performed in the control and in the sevoflurane group. However, there is no evidence in the literature that intraperitoneal injections alone may lead to a comparable increase in neurodegeneration. Furthermore, it has to be considered that the majority of the studied substances show a dose-dependent toxicity. In our study a distinct dose has been chosen to maintain a predefined depth of anesthesia. Therefore, we cannot exclude that higher doses of sevo- flurane would have had more distinct effects.
+
+As discussed earlier, a variety of anesthetics and other substances obviously may induce impaired brain develop- ment and cognitive or behavioral defects. This is well known for alcohol abuse in pregnancy leading to the fetal Impaired neurological development alcohol syndrome. could also be shown in infants after maternal benzodiaze- pine (Laegreid et al. 1992) or antiepileptic medication (Meador et al. 2009) during pregnancy. Furthermore, the application of Phenobarbital in infants suffering from sei- zures was associated with a significant lower intelligence quotient when compared to placebo (Farwell et al. 1990). In experimental studies an induction of apoptotic neuro- degeneration in newborn rats by high-dose GABAA ago- nists such as diazepam and Phenobarbitone has been described by Bittigau et al. (2003). The exact mechanism by which these classes of medicaments induce neuroa- poptosis remains unclear. It has been hypothesized that depression of neuronal activity during synaptogenesis is a common denominator. Recently, it has been shown that isoflurane-induced apoptosis is dependent on cytosolic calcium levels and therefore disruption of intracellular calcium homeostasis is a potential pathway (Wei et al. 2008; Zhang et al. 2008).
+
+In the data presented here, we could show that the GABA agonist sevoflurane did not lead to increased cell death even though the animals were deeply anesthetized indicating sufficient depression of neuronal activity.
+
+We cannot conclude why sevoflurane in contrast to other tested substances with the same mechanism of action did not lead to neuronal cell damage. However, data concern- ing the influence of inhalational anesthetics on brain development are not consistent. To our knowledge only few recently published studies could show neurotoxic effects of inhalational anesthetics individually and not in combination with other substances (Johnson et al. 2008). Li et al. (2007) demonstrated that exposure to isoflurane
+
+123
+
+146
+
+decreased apoptosis in 21-day-old rats. In the above dis- cussed study of Jevtovic-Todorovic et al. (2003) only a cocktail of three anesthetic substances led to the described effects. We suggest that volatile halogenated hydrocarbons do not have comparable neurotoxic effects as benzodiaze- pines, ketamine, propofol and other substances. It has to be discussed if the prolongation of exposure or an increase of concentrations might lead to more pronounced neurotoxic effects. Additionally, in this study rats were euthanized 24 h after the anesthetic procedure whereas other protocols waited for a shorter period (Jevtovic-Todorovic et al. 2003; Li et al. 2007). We cannot exclude that sevoflurane anes- thesia would have more pronounced effects if this period would have been shorter. The experimental
+
+results concerning sedatives and anesthetics raised serious concerns about the safety of common NMDA antagonists and GABA agonists used in pediatric anesthesia. Consecutively, the transferability of such results to human beings has been discussed contro- versially. The main arguments against transferability were that the reported neurodegeneration may be also caused by ischemia, hypoxia, hypoglycemia or hypothermia due to insufficient monitoring (Anand and Soriano 2004). During our experiments animals were deeply sedated and two animals even died during anesthesia. As we could not demonstrate any increase in scores of degenerated cells and accordingly could not observe any learning deficits in sevoflurane animals when compared to controls, we sug- gest that depth of anesthesia did not influence cell death in the described experimental setting.
+
+In clinical studies, neurotoxic effects of a propofol- based anesthesia causing learning disorders or develop- mental retardation have not been confirmed yet. Currently, neurological sequelae in children submitted to prolonged propofol infusion have been described in case reports (Lanigan et al. 1992; Trotter and Serpell 1992). After surgery was performed in newborn infants for transposition of the great arteries, prolonged intensive care (and hence prolonged exposure to sedatives) is associated with reduced intelligence quotients at 8 years of age (Newburger et al. 2003). But learning deficits after extensive surgery in the newborn are most likely influenced by a variety of reasons and should not be referred to anesthesia alone.
+
+However, the issue of neurotoxicity after administration of GABAA agonists or NMDA antagonists has not been subject to a randomized controlled trial. To date no clinical trial investigated neurological sequelae after application of different anesthetic regimens in the newborn.
+
+Concerns have to be raised that the use of propofol in neonates might induce apoptotic neurodegeneration and may lead to specific behavioral deficits. This has been already shown for other frequently used substances (i.e., benzodiazepines, barbiturates, volatile anesthetics). In the
+
+123
+
+Neurotox Res (2009) 16:140–147
+
+presented experiments, inhalational anesthesia did not have such consequences.
+
+In clinical pediatric anesthesia, propofol is considered to be a safe intravenous agent. Apart from former clinical practice it has been shown extensively that there is unconditional necessity for anesthesia during surgery in the neonatal period. In contrast, apoptotic neurodegeneration has been demonstrated exclusively in animal experiments. As clinical studies are still lacking, future research has to focus on the detection of safe anesthetic strategies and substances.
+
+Acknowledgments This study was funded by institutional resources of the Department of Anesthesiology and Intensive Care Medicine, and of the Children’s Hospital, Campus Virchow Klinikum, Charite´- Universitaetsmedizin in Berlin, Germany.
+
+References
+
+Anand KJ, Soriano SG (2004) Anesthetic agents and the immature therapeutic? Anesthesiology 101:
+
+brain: are these toxic or 527–530
+
+Bert B, Fink H, Huston JP, Voits M (2002) Fischer 344 and Wistar rats differ in anxiety and habituation but not in water maze performance. Neurobiol Learn Mem 78:11–22
+
+Bittigau P, Sifringer M, Genz K, Reith E, Pospischil D, Govindarajalu S, Dzietko M, Pesditschek S, Mai I, Dikranian K, Olney JW, Ikonomidou C (2002) Antiepileptic drugs and apoptotic neuro- degeneration in the developing brain. Proc Natl Acad Sci USA 99:15089–15094
+
+Bittigau P, Sifringer M, Ikonomidou C (2003) Antiepileptic drugs and apoptosis in the developing brain. Ann NY Acad Sci 993: 103–114; discussion 123–104
+
+Cattano D, Young C, Straiko MM, Olney JW (2008) Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain. Anesth Analg 106:1712–1714
+
+De Olmos JS, Ingram WR (1971) An improved cupric-silver method for impregnation of axonal and terminal degeneration. Brain Res 33:523–529
+
+Farwell JR, Lee YJ, Hirtz DG, Sulzbacher SI, Ellenberg JH, Nelson KB (1990) Phenobarbital for febrile seizures—effects on intel- ligence and on seizure recurrence. N Engl J Med 322:364–369 Fredriksson A, Ponten E, Gordh T, Eriksson P (2007) Neonatal exposure to a combination of N-methyl-D-aspartate and gamma- aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. Anesthesiology 107:427–436
+
+Gundersen HJ, Bendtsen TF, Korbo L, Marcussen N, Moller A, Nielsen K, Nyengaard JR, Pakkenberg B, Sorensen FB, Vesterby A et al (1988) Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 96:379–394
+
+Hapfelmeier G, Schneck H, Kochs E (2001) Sevoflurane potentiates through recombinant and blocks GABA-induced currents alpha1beta2gamma2 GABAA receptors: implications for an enhanced GABAergic transmission. Eur J Anaesthesiol 18: 377–383
+
+Ikonomidou C, Bittigau P, Koch C, Genz K, Hoerster F, Felderhoff- Mueser U, Tenkova T, Dikranian K, Olney JW (2001) Neuro- transmitters and apoptosis in the developing brain. Biochem Pharmacol 62:401–405
+
+Neurotox Res (2009) 16:140–147
+
+Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikr- anian K, Zorumski CF, Olney JW, Wozniak DF (2003) Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 23:876–882
+
+Johnson SA, Young C, Olney JW (2008) Isoflurane-induced neuroa- poptosis in the developing brain of nonhypoglycemic mice. J Neurosurg Anesthesiol 20:21–28
+
+Kay B, Rolly G (1977) I.C.I. 35868, a new intravenous induction
+
+agent. Acta Anaesthesiol Belg 28:303–316
+
+Laegreid L, Hagberg G, Lundberg A (1992) Neurodevelopment in late infancy after prenatal exposure to benzodiazepines—a prospective study. Neuropediatrics 23:60–67
+
+Lanigan C, Sury M, Bingham R, Howard R, Mackersie A (1992) Neurological sequelae in children after prolonged propofol infusion. Anaesthesia 47:810–811
+
+Li Y, Liang G, Wang S, Meng Q, Wang Q, Wei H (2007) Effects of fetal exposure to isoflurane on postnatal memory and learning in rats. Neuropharmacology 53:942–950
+
+Meador KJ, Baker GA, Browning N, Clayton-Smith J, Combs- Cantrell DT, Cohen M, Kalayjian LA, Kanner A, Liporace JD, Pennell PB, Privitera M, Loring DW (2009) Cognitive function at 3 years of age after fetal exposure to antiepileptic drugs. N Engl J Med 360:1597–1605
+
+Morris R (1984) Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods 11:47–60 Morris RG, Garrud P, Rawlins JN, O’Keefe J (1982) Place navigation impaired in rats with hippocampal lesions. Nature 297:681–683 infusion syndrome.
+
+Motsch J, Roggenbach J
+
+(2004) Propofol
+
+Anaesthesist 53:1009–1022; quiz 1023–1004
+
+Newburger JW, Wypij D, Bellinger DC, du Plessis AJ, Kuban KC, Rappaport LA, Almirall D, Wessel DL, Jonas RA, Wernovsky G (2003) Length of stay after infant heart surgery is related to cognitive outcome at age 8 years. J Pediatr 143:67–73
+
+147
+
+Nikizad H, Yon JH, Carter LB, Jevtovic-Todorovic V (2007) Early exposure to general anesthesia causes significant neuronal deletion in the developing rat brain. Ann NY Acad Sci 1122: 69–82
+
+Olney JW, Young C, Wozniak DF, Ikonomidou C, Jevtovic-Todorovic V (2004) Anesthesia-induced developmental neuroapoptosis. Does it happen in humans? Anesthesiology 101:273–275 Parke TJ, Stevens JE, Rice AS, Greenaway CL, Bray RJ, Smith PJ, Waldmann CS, Verghese C (1992) Metabolic acidosis and fatal myocardial failure after propofol infusion in children: five case reports. BMJ 305:613–616
+
+Soriano SG, Anand KJ, Rovnaghi CR, Hickey PR (2005) Of mice and men: should we extrapolate rodent experimental data to the care of human neonates? Anesthesiology 102:866–868; author reply 868–869
+
+Trotter C, Serpell MG (1992) Neurological sequelae in children after
+
+prolonged propofol infusion. Anaesthesia 47:340–342
+
+Voits M, Fink H, Gerhardt P, Huston JP (1995) Application of ‘nose- poke habituation’ validation with post-trial diazepam- and cholecystokinin-induced hypo- and hypermnesia. J Neurosci Methods 57:101–105
+
+Wei H, Liang G, Yang H, Wang Q, Hawkins B, Madesh M, Wang S, Eckenhoff RG (2008) The common inhalational anesthetic isoflurane induces apoptosis via activation of inositol 1,4,5- trisphosphate receptors. Anesthesiology 108:251–260
+
+Wise-Faberowski L, Raizada MK, Sumners C (2001) Oxygen and glucose deprivation-induced neuronal apoptosis is attenuated by halothane and isoflurane. Anesth Analg 93:1281–1287
+
+Zhang G, Dong Y, Zhang B, Ichinose F, Wu X, Culley DJ, Crosby G, Tanzi RE, Xie Z (2008) Isoflurane-induced caspase-3 activation is dependent on cytosolic calcium and can be attenuated by memantine. J Neurosci 28:4551–4560
+
+123

new_pdfs/10.1007_s12640-016-9615-7.txt

@@ -0,0 +1,627 @@
+Neurotox Res (2016) 30:185–198 DOI 10.1007/s12640-016-9615-7
+
+O R I G I N A L A R T I C L E
+
+Ketamine Affects the Neurogenesis of the Hippocampal Dentate Gyrus in 7-Day-Old Rats
+
+He Huang1 Dan Wang2
+
+Cun-Ming Liu1 • Yu-Qing Wu2
+
+Jie Sun1
+
+Ting Hao2
+
+Chun-Mei Xu2
+
+
+
+Received: 15 December 2015 / Revised: 22 February 2016 / Accepted: 1 March 2016 / Published online: 10 March 2016 (cid:2) Springer Science+Business Media New York 2016
+
+Abstract Ketamine has been reported to cause neonatal neurotoxicity via a neuronal apoptosis mechanism; how- ever, no in vivo research has reported whether ketamine could affect postnatal neurogenesis in the hippocampal dentate gyrus (DG). A growing number of experiments suggest that postnatal hippocampal neurogenesis is the foundation of maintaining normal hippocampus function into adulthood. Therefore, this study investigated the effect of ketamine on hippocampal neurogenesis. Male Sprague– Dawley rats were divided into two groups: the control group (equal volume of normal saline), and the ketamine- anesthesia group (40 mg/kg ketamine in four injections at 1 h intervals). The S-phase marker 5-bromodeoxyuridine (BrdU) was administered after ketamine exposure to postnatal day 7 (PND-7) rats, and the neurogenesis in the hippocampal DG was assessed using single- or double- immunofluorescence staining. The expression of GFAP in the hippocampal DG was measured by western blot anal- ysis. Spatial reference memory was tested by Morris water maze at 2 months after PND-7 rats exposed to ketamine treatment. The present results showed that neonatal keta- mine exposure significantly inhibited neural stem cell (NSC) proliferation, decreased astrocytic differentiation, and markedly enhanced neuronal differentiation. The dis- ruptive effect of ketamine on the proliferation and differ- entiation of NSCs lasted at least 1 week and disappeared the by 2 weeks after ketamine exposure. Moreover,
+
+migration of newborn neurons in the granule cell layer and the growth of astrocytes in the hippocampal DG were inhibited by ketamine on PND-37 and PND-44. Finally, ketamine caused a deficit in hippocampal-dependent spatial reference memory tasks at 2 months old. Our results sug- gested that ketamine may interfere with hippocampal neurogenesis and long-term neurocognitive function in PND-7 rats. These findings may provide a new perspective to explain the adult neurocognitive dysfunction induced by neonatal ketamine exposure.
+
+Keywords Ketamine (cid:2) Neonatal (cid:2) Neurogenesis (cid:2) Hippocampal dentate gyrus (cid:2) Morris water maze test
+
+Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, is widely used in anesthesia, analgesia, and sedation during the neonatal period (Asadi et al. 2013; Guerra et al. 2011). However, ketamine has been reported short-term and long-term neurotoxicities, to induce including neuronal apoptosis and neurocognitive dysfunc- tion in the adult stage (Ikonomidou et al. 1999; Liu et al. 2011; Paule et al. 2011; Zou et al. 2009; Pfenninger et al. 2002; Wilder et al. 2009). As a commonly used anesthetic the safety of during pediatric anesthesia and sedation, ketamine has been the subject of concern for anesthesiol- ogists and the public.
+
+& Yu-Qing Wu
+
+xymzyqwu@126.com
+
+1 Department of Anesthesiology, The First Affiliated Hospital
+
+of Nanjing Medical University, Nanjing, China
+
+2
+
+Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, China
+
+The causal link between neuronal death in the hip- pocampus of the developing brain and neurocognitive dysfunction in the adult stage has not been investigated in detail. Curiously, a previous study showed that 4 h of hypercapnia exposure caused a similar degree of hip- pocampal neuronal death as 4 h of isoflurane in PND-7 rats, but only 4 h of isoflurane caused a long-term neu- rocognitive deficit (Stratmann et al. 2009). This raises
+
+123
+
+186
+
+suspicion regarding whether ketamine-induced hippocam- pal neuronal death in PND-7 rats can fully account for the neurocognitive dysfunction observed in the adult stage. Hence, it is worthwhile to study whether there is any other mechanism to explain the cognitive deficit in the adult stage after neonatal ketamine exposure other than anes- thesia-induced neuronal death.
+
+The developing central nervous system (CNS) has a critical period called brain growth spurt (BGS), which lasts from the end of pregnancy to the first 2–3 weeks after birth in rodents; in humans, the corresponding period begins in the last trimester of pregnancy and continues until 2 years after birth (Byrnes et al. 2001). During this period, the brain exhibits a high degree of plasticity, and substantial neuro- genesis occurs rapidly and lays the foundation for the normal structure and function of the brain. The hippocampal dentate gyrus (DG) is one of only two restricted regions other than the subventricular zone (SVZ) where neurogenesis occurs during development and continues, at a slower rate, into adulthood (Lledo et al. 2006; Mongiat and Schinder 2011; Luskin 1993; Vadodaria and Jessberger 2014). The neuro- genesis of the hippocampal DG plays a critical role in the formation of hippocampal-dependent spatial learning and memory function (Dupret et al. 2008; Stone et al. 2011).
+
+Neurogenesis is a complicated process that includes neural stem cell (NSC) proliferation, neuronal or astrocytic differ- entiation, and migration of newborn neurons. NSCs, which are located in the hilus/subgranular zone (SGZ) of hippocampal DG, partially begin to differentiate into neurons or astrocytes, while others retain the ability to divide. Some of the newly generated granule neurons can migrate into the granule cell layer (GCL) and functionally incorporate into the hip- pocampal circuit (granule neurons–CA3–CA1 loop) (Vado- daria and Jessberger 2014; van Praag et al. 2002).
+
+Postnatal neurogenesis in the DG may be sensitive to outside stimulation, such as hypoxia–ischemia, hyperoxia, and stress (Bartley et al. 2005; Belnoue et al. 2013; Porzionato et al. 2013). In recent years, there has been increasing research into the effect of anesthetics on post- natal neurogenesis in the DG (Fang et al. 2012; Erasso et al. 2013; Nie et al. 2013; Stratmann et al. 2009). How- ever, the effects of ketamine on neonatal hippocampal neurogenesis in vivo have not been reported. The present study aims to explore the effects of ketamine on postnatal neurogenesis in the hippocampal DG in vivo.
+
+Materials and Methods
+
+Animal Treatment
+
+All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical
+
+123
+
+Neurotox Res (2016) 30:185–198
+
+University. The timed-pregnant Sprague–Dawley rats were housed in a temperature-controlled (22–23 (cid:3)C) room on a 12 h:12 h light:dark cycle (light on at 8:00 AM) with free access to food and water. The PND-7 male rat pups (11–14 g) were randomly assigned to ketamine-treated and control groups. In the treated group, ketamine was diluted in 0.9 % normal saline, and PND-7 rats were intraperi- toneally administered with 40 mg/kg doses of ketamine in four injections at 1 h intervals (40 mg/kg 9 4 injections). Control rats received an equal volume of normal saline. Temperature probes were used to facilitate control of temperature at 36.5 ± 1 (cid:3)C using computer-controlled heater/cooler plates integrated into the floor of the cham- ber. Between each injection, animals were returned to their chamber to help maintain body temperature and reduce stress.
+
+BrdU Injections
+
+All animals received an intraperitoneal injection of BrdU (5-bromo-2-deoxyuridine; Sigma) at a dosage of 100 mg/ kg after ketamine anesthesia according to the following experimental schedule.
+
+Experiment 1: To evaluate the effect of ketamine on the proliferation and differentiation of NSCs in the DG during the BGS, the PND-7 rats received a single intraperitoneal injection of BrdU on PND-7, 13, and 20 after ketamine treatment. The animals were then anesthetized and fixed by perfusion at 24 h after each BrdU injection. The experi- mental protocol is described in Tables 1a and 2.
+
+Experiment 2: To exclude the GFAP/BrdU double- positive cells that were proliferative astrocytes, the PND-7 rats received a single intraperitoneal injection of BrdU on PND-7, 13, and 20 after exposure to treatment. The animals were then perfused at 3 h after each BrdU injection. The experimental protocol is detailed in Tables 1b and 2.
+
+Experiment 3: To determine the effect of ketamine on the migration of newborn granule neurons in the DG, the PND-7 rats received three consecutive BrdU injections on PND-7, 8, and 9 after exposure to treatment. At 28 and 35 days after the last BrdU injection, the animals were anesthetized and fixed by perfusion. The experimental protocol is described in Table 1c and 2.
+
+Cell Apoptotic Assays
+
+Nestin/caspase-3 and GFAP/caspase-3 double-immunoflu- orescence staining was utilized to detect whether ketamine could induce the apoptosis of NSCs or astrocytes. At 12 h after the end of control and ketamine-anesthesia treatment, the neonatal rats were anesthetized and fixed by perfusion (n = 5 per group).
+
+Neurotox Res (2016) 30:185–198
+
+187
+
+Table 1 Experimental design
+
+Total no. of BrdU injections
+
+Postnatal day on which BrdU was administered
+
+Survival (day) after the last BrdU injection
+
+a. Experiment 1 (n = 5)
+
+Effect of ketamine on the proliferation and differentiation
+
+1
+
+7
+
+1
+
+of NSCs in the DG of PND-7 rats
+
+1
+
+13
+
+1
+
+1
+
+20
+
+1
+
+b. Experiment 2 (n = 5)
+
+To exclude the GFAP/BrdU double-positive cells that were
+
+1
+
+7
+
+3
+
+proliferative astrocytes
+
+1 1
+
+13 20
+
+3 3
+
+c. Experiment 3 (n = 5)
+
+Effect of ketamine on the migration of newborn granule
+
+3
+
+7–9
+
+28
+
+neurons in the DG of PND-7 rats
+
+3
+
+7–9
+
+35
+
+Table 2 Immunolabeling
+
+Targeted process
+
+NSC proliferation
+
+Neuronal differentiation
+
+Astrocytic differentiation
+
+Astrocytic proliferation
+
+IF stain
+
+Nestin/BrdU b-tubulin III/BrdU
+
+GFAP/BrdU
+
+GFAP/BrdU
+
+these steps. Blocking of nonspecific epitopes with 10 % donkey serum in PBS with 0.3 % Triton-X for 2 h at room temperature preceded incubation overnight at 4 (cid:3)C with the primary antibodies listed in Table 3 in PBS with 0.3 % Triton-X. On the next day, the sections were incubated with the appropriate secondary fluorescent antibodies (In- vitrogen Carlsbad, CA) for 2 h at room temperature.
+
+Migration of newborn granule neurons
+
+IF immunofluorescence
+
+Tissue Preparation and Immunofluorescence
+
+NeuN/BrdU
+
+Astrocytic development was detected by using GFAP single-labeled staining. The sections were incubated over- night at 4 (cid:3)C with a fluorescent antibody for the GFAP (Table 3). After three washes in PBS, sections were incu- bated with secondary fluorescent antibody (Invitrogen) for 2 h at room temperature.
+
+At the indicated time point, animals were deeply anes- thetized and then transcardially perfused with 0.9 % nor- mal saline followed by 4 % paraformaldehyde. The brains were removed, postfixed overnight in 4 % paraformalde- hyde, and placed in 30 % sucrose until sunk. The coronal sections of brain were cut consecutively at a thickness of 30 lm when the hippocampus was initially exposed. The fifteenth section was taken and stored in PBS. According to the Atlas of the Developing Rat Brain and previous reports (Ashwell and Paxinos 2008; Paxinos and Watson 1986), the positions of hippocampus coronal sections selected in to the our study were about 2.20–2.25 mm posterior bregma at PND-8 rats, about 2.35–2.40 mm posterior to the bregma at PND-14 rats, about 2.50–2.55 mm posterior to the bregma at PND-21 rats, and about 2.75–2.85 mm posterior to the bregma at PND-37 and PND-44 rats, respectively.
+
+For Nestin/BrdU, b-tubulin III/BrdU, GFAP/BrdU, and NeuN/BrdU double-immunofluorescence the BrdU antigen was exposed by incubating the sections in 2-normal hydrochloric acid for 30 min at 37 (cid:3)C and then washed three times with PBS for 5 min between each of
+
+staining,
+
+To characterize the phenotype of cell apoptosis, brain sections were analyzed by double-labeled staining. The sections were incubated overnight at 4 (cid:3)C with the appro- priate primary antibodies listed in Table 3. After three washes with PBS, the sections were incubated with the suitable secondary fluorescent antibodies (Invitrogen) for 2 h at room temperature.
+
+A skilled pathologist blinded to the study conditions examined the labeled sections using a laser scanning con- focal microscope (Fluoview 1000, Olympus). The number of single- or double-positive cells in the hippocampal DG was quantified using Image-Pro Plus software.
+
+Western Blot Analysis
+
+Thirty and thirty-seven days after the control or ketamine- anesthesia treatment, the animals were decapitated, and the hippocampal DG tissue was dissected carefully with ana- tomic microscope (leica EZ4HD). The harvested hip- pocampal tissues were homogenized on ice using lysate buffer plus protease inhibitors. The lysates were cen- trifuged at 14,000 rpm for 15 min at 4 (cid:3)C and were
+
+123
+
+188
+
+Neurotox Res (2016) 30:185–198
+
+Table 3 Primary antibodies
+
+Antibody name
+
+Specificity
+
+Host species
+
+Dilution rates
+
+Company
+
+Nestin b-tubulin III
+
+Neural stem cells
+
+Newborn neurons
+
+Rabbit
+
+Rabbit
+
+1:100
+
+1:200
+
+Abcam
+
+Abcam
+
+GFAP
+
+Astrocytes
+
+Rabbit
+
+1:200
+
+Millipore
+
+NeuN
+
+Mature neurons
+
+Mouse
+
+1:400
+
+Millipore
+
+BrdU
+
+Newly generated cells
+
+Mouse
+
+1:1000
+
+Sigma
+
+BrdU
+
+Newly generated cells
+
+Rabbit
+
+1:500
+
+Abcam
+
+Caspase-3
+
+Cell apoptosis
+
+Mouse
+
+1:100
+
+Santa Cruz
+
+resolved by 12 % polyacrylamide gel electrophoresis, and the target proteins were transferred to nitrocellulose membranes. The blots were incubated with blocking buffer for 2 h at room temperature and then incubated for 24 h at 4 (cid:3)C with the primary antibodies: rabbit anti-GFAP anti- body (1:1000, Millipore) and GAPDH. The membranes were then incubated with appropriate secondary alkaline antibody donkey phosphatase-conjugated (1:10,000, Abcam) for 1 h. The band intensity was quan- tified using Image J software (n = 5 per group).
+
+anti-rabbit
+
+Morris Water Maze Test
+
+Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, China).
+
+Statistical Analysis
+
+The statistical analysis was conducted using SPSS 13.0, and the graphs were created using GraphPad Prism 5. The data were analyzed using Mann-Whitney U test. The interaction between time and group factors in a two-way ANOVA was used to analyze the difference of escape latency between rats in the control group and rats treated with ketamine in the MWM. The data are presented as the mean ± SD, and p \ 0.05 was considered statistically significant.
+
+The hippocampal-dependent spatial memory abilities were tested by using the Morris water maze (MWM). Different set of rats were tested 2 months after administration of ketamine on PND-7. A circular, black painted pool (180 cm diameter, 50 cm deep) was filled with water to a depth of 30 cm. The water temperature was maintained at 25 ± 1 (cid:3)C. An invisible platform (10 cm diameter) was submerged 1 cm below the water surface and placed in the center of the III quadrant which was determined with four starting locations called I, II, III, and IV at equal distance on the edge of the pool. During five consecutive days, the experiments were conducted in a dark and quiet laboratory, all the rats were trained four times per day, the starting positions were random for each rat. When the rat found the platform, the rat was allowed to stay on it for 30 s. If a rat did not find the platform within 120 s, the rat would be guided gently to the place and allowed to stay on it for 30 s, and the latency time to find the hidden platform was recorded as 120 s. The average time from four trials rep- resented as the daily result for the rat. On the sixth day, the hidden platform was removed, and the rat was placed in the opposite quadrant. Rats were allowed to swim freely for 120 s. The numbers the rat swam to cross the previous platform area, and the times the rat stayed in the target quadrant within 120 s were recorded. Each animal’s path was tracked by a computerizing video system. After every trial, each rat was placed in a heater plates for 1 to 2 min until dry before being returned to its chamber. The data were analyzed using software for the MWM (Jiangsu
+
+Results
+
+Ketamine Anesthesia in Postnatal Rats Induced Inhibition of NSC Proliferation in the Hippocampal DG
+
+As shown in Fig. 1d, e, the percentage (8 ± 1.04 %) and density (11 ± 1.08 lm2) of Nestin?/BrdU? cells in the ketamine-treated group were significantly decreased compared to those in the control group (14 ± 1.30 %; 20 ± 2.22 lm2) 1 day (PND-8) after exposure to keta- mine. This suppressive effect of ketamine on NSC pro- liferation was also found at 7 days (PND-14: 10 ± 0.77 vs. 14 ± 1.45 %; 15 ± 1.29 vs. 19 ± 2.08 lm2) after (PND-21: anesthesia 18 ± vs. 11 ± 1.32 1.75 lm2) after anesthesia. Typical immunofluorescence pictures are shown in Fig. 1a, b, c. In addition, we found that there were no significant differences in NSC prolif- eration at different time points (PND-8, 14, and 21) in either the control groups or the ketamine-treated groups. These data indicated that ketamine could significantly inhibit the proliferation of NSCs in the hippocampal DG of neonatal rats for at least 1 week and that the ability of NSC proliferation could recover after anesthesia.
+
+but vs.
+
+disappeared 13 ± 1.13 %;
+
+at
+
+14 days 17 ± 2.44
+
+at 2 weeks
+
+123
+
+Neurotox Res (2016) 30:185–198
+
+Fig. 1 Effect of ketamine on the proliferation of NSCs in the hippocampal dentate gyrus (DG) of neonatal rats. The NSCs were labeled with primary antibodies against Nestin (green) and BrdU (red). The immunoreactive cells were visualized using a laser scanning confocal microscope (a, b and c; magnification: 9200); the scale bar is 100 lm. The filled arrows point to
+
+Ketamine Anesthesia in Postnatal Rats Promoted the Neuronal Differentiation of NSCs in the Hippocampal DG
+
+Figure 2d, e show that the percentage (18 ± 2.27 %) and density (23 ± 3.74 lm2) of b-tubulin III?/BrdU? cells were significantly increased in the ketamine-treated group com- pared to those in the control group (12 ± 2.07 %;
+
+189
+
+Nestin/BrdU double-labeled cells. The ratio of Nestin?/BrdU? cells to Nestin? cells was calculated (d). The Y axis ‘‘(lm2)’’ represents the density of Nestin?/BrdU? cells in the DG (e). Data are presented as the mean ± SD (n = 5). **p\0.01 versus control group. GCL granule cell layer; ML molecular layer; PCL polymorphic cell layer (Color figure online)
+
+17 ± 3.25/lm2) 1 day (PND-8) after exposure to ketamine. This stimulating effect of ketamine on neuronal differenti- ation of NSCs was also assessed at 7 days (PND-14: 16 ± 2.24 vs. 11 ± 1.25 %; 21 ± 3.01 vs. 16 ± 2.05 lm2) after anesthesia but was not detected at 14 days (PND-21: 12 ± 1.90 vs. 12 ± 2.34 %; 18 ± 2.28 vs. 17 ± 2.92 lm2) after anesthesia. Typical immunofluorescence pictures are shown in Fig. 2a, b, c. Moreover, we did not observe
+
+123
+
+190
+
+significant differences in the neuronal differentiation of NSCs at different time points (PND-8, 14 and 21) in either the control groups or the ketamine-treated groups. These data indicated that ketamine could significantly promote the neuronal differentiation of NSCs in the hippocampal DG of neonatal rats for at least 1 week, and this stimulating effect finally disappeared by 2 weeks after anesthesia.
+
+Fig. 2 Effect of ketamine on the neuronal differentiation of NSCs in the hippocampal dentate gyrus (DG) of neonatal rats. The newborn neurons were labeled with primary antibodies against b-tubulin III (green) and BrdU (red). The immunoreactive cells were visualized using a laser scanning confocal microscope (a, b and c; magnification: 9200); the scale bar is 100 lm. The filled arrows point to b-tubulin
+
+123
+
+Neurotox Res (2016) 30:185–198
+
+Ketamine Anesthesia in Postnatal Rats Attenuated the Astrocytic Differentiation of NSCs in the Hippocampal DG
+
+Similar to the effect of ketamine on NSC proliferation, the percentage (11 ± 0.89 %) and density (10 ± 1.46 lm2) of GFAP?/BrdU? cells were significantly decreased in the
+
+III/BrdU double-labeled cells. The ratio of b-tubulin III?/BrdU? cells to BrdU? cells was calculated (d). The Y axis ‘‘(lm2)’’ represents the density of b-tubulin III?/BrdU? cells in the DG (e). Data are presented as the mean ± SD (n = 5). *p\0.05, **p\0.01 versus control group. GCL granule cell layer, ML molecular layer, PCL polymorphic cell layer (Color figure online)
+
+Neurotox Res (2016) 30:185–198
+
+Fig. 3 Effect of ketamine on the astrocytic differentiation of NSCs in the hippocampal dentate gyrus (DG) of neonatal rats. The newborn astrocytes were labeled with primary antibodies against GFAP (green) and BrdU (red). The immunoreactive cells were visualized using a laser scanning confocal microscope (a, b and c; magnification: 9200); the scale bar is 100 lm. The filled arrows point to GFAP/ BrdU double-labeled cells. The ratio of GFAP?/BrdU? cells to BrdU? cells was calculated (d). The Y axis ‘‘(lm2)’’ represents the density of GFAP?/BrdU? cells in the DG (e). To exclude that GFAP/
+
+ketamine-treated group compared to those in the control group (15 ± 1.30 %; 17 ± 2.07 lm2) 1 day (PND-8) after exposure to ketamine. This inhibitory effect of ketamine on astrocytic differentiation of NSCs was also detected at
+
+191
+
+BrdU double-positive cells were the proliferative astrocytes, another set of animals was perfused and sacrificed at 3 h after BrdU injection on PND-7, 13, and 20. The proliferative astrocytes were also stained with primary antibodies against GFAP and BrdU, and the density of GFAP/BrdU double-labeled cells was calculated (f). Data are presented as the mean ± SD (n = 5). *p\0.05, **p\0.01 vs. control group. GCL granule cell layer, ML molecular layer, PCL polymorphic cell layer (Color figure online)
+
+7 days (PND-14: 13 ± 1.40 vs. 15 ± 0.97 %; 12 ± 2.11 vs. 16 ± 1.88 lm2) after anesthesia but disappeared at 14 days (PND-21: 14 ± 1.57 vs. 14 ± 1.39 %; 13 ± 0.91 vs. 14 ± 0.82 lm2) after anesthesia (Fig. 3d, e). Typical
+
+123
+
+192
+
+immunofluorescence pictures are shown in Fig. 3a, b, c. Moreover, we did not observe significant differences in the astrocytic differentiation of NSCs at different time points (PND-8, 14 and 21) in either the control groups or the ketamine-treated groups. These data suggested that keta- mine could also inhibit the astrocytic differentiation of NSCs in the hippocampal DG of neonatal rats for at least 1 week, and this inhibitory effect finally disappeared by 2 weeks after anesthesia.
+
+To exclude the GFAP/BrdU double-positive cells that were proliferative astrocytes, the animals were perfused at 3 h after BrdU injection on PND-7, 13, and 20. We found only a small amount of GFAP/BrdU double-positive cells in both the control and ketamine groups at the three time points, and there were no significant differences in the proliferation of matured astrocytes between the ketamine and control groups (Fig. 3f).
+
+The NSCs and Astrocytes in the Hippocampal DG of Neonatal Rats are Resistant to Ketamine-Induced Cell Apoptosis
+
+To investigate the effects of ketamine on the apoptosis of NSCs and astrocytes in the hippocampal DG of neonatal rats, we analyzed the nestin/caspase-3 and GFAP/caspase-3 double-positive cells in the hippocampal DG using double- immunofluorescence staining 12 h after the end of keta- mine anesthesia. The results showed that there were no significant changes in the numbers of nestin/caspase-3 or GFAP/caspase-3 double-positive cells in either the control or ketamine groups. These results suggested that the dosage and duration of ketamine used in our experiment could not induce the apoptosis of NSCs and astrocytes in the hip- pocampal DG (Fig. 4a, b).
+
+Ketamine Anesthesia in Postnatal Rats Inhibited the Migration of Newborn Neurons in the GCL of the Hippocampal DG
+
+According to previous research (Kempermann et al. 2003; Esposito et al. 2005), the hippocampal GCL can be divided into four zones (SGZ, GCL1, GCL2, and GCL3) from the inside to outside of the GCL. The newborn neurons dif- ferentiated from NSCs could migrate from the SGZ to different locations throughout the GCL. The experimental is shown in Fig. 5a. To better visualize the protocol migration of the newly generated neurons in the GCL, we examined the NeuN?/BrdU? cells in the GCL 28 days (PND-37) and 35 days (PND-44) after the last BrdU injection using double-immunofluorescence staining.
+
+According to our findings, ketamine could significantly increase the rate of BrdU-positive neurons in the SGZ compared to the total BrdU-positive neurons in the GCL
+
+123
+
+Neurotox Res (2016) 30:185–198
+
+Fig. 4 Effect of ketamine on the apoptosis of NSCs and astrocytes in the hippocampal dentate gyrus of neonatal rats. The PND-7 rats were perfused and sacrificed at 12 h after four injections of 40 mg/kg ketamine at 1-h intervals. The apoptosis of NSCs and astrocytes is shown with nestin/caspase-3 (a) and GFAP/caspase-3 (b) double- staining (magnification: 9400; the scale bar immunofluorescence layer, PCL is 50 lm). GCL granule cell polymorphic cell layer
+
+layer, ML molecular
+
+PND-44: vs. (PND-37: 74 ± 6.11 vs. 26 ± 10.28 %) (Fig. 5d); the rate of BrdU- positive neurons in GCL1 compared to the total BrdU- positive neurons in the GCL was significantly decreased in the ketamine group compared to that in control group on PND-37 (24 ± 10.61 vs. 46 ± 12.57 %) and PND-44 (21 ± 9.65 vs. 51 ± 4.65 %) (Fig. 5e). The rate of BrdU- positive neurons in GCL2 compared to the total BrdU- positive cells in the GCL was significantly decreased in the ketamine group compared to that in control group on PND- 37 (5 ± 7.36 vs. 16 ± 5.06 %) and PND-44 (5 ± 6.85 vs. 23 ± 7.45 %) (Fig. 5f). The rate of BrdU-positive neurons in GCL3 compared to the total BrdU-positive neurons in the GCL showed no significant difference between the control and ketamine groups on PND-37 and PND-44. Typical immunofluorescence pictures are shown in Fig. 5b, c. Taken together, that neonatal ketamine exposure could inhibit the migration of postna- tally generated neurons in the GCL of the hippocampal DG and restrict them inside the GCL.
+
+70 ± 16.73
+
+36 ± 9.31 %;
+
+these results suggest
+
+Neurotox Res (2016) 30:185–198
+
+Fig. 5 Effect of ketamine on the migration of newborn neurons in the hippocampal dentate gyrus (DG) of neonatal rats. Experimental protocol (a). Representative photographs from a laser scanning confocal microscope are shown (b, c; magnification: a, e 9200; b– d and f–h 9400); the scale bar is 50 lm (a, b). The NeuN (green)/ BrdU (red) double-positive cells distributed in the GCL. The filled
+
+Ketamine Anesthesia in Postnatal Rats Inhibited the Growth of Astrocytes in the Hippocampal DG
+
+The normal migration of newborn neurons in the hip- pocampal DG is dependent on the development of
+
+193
+
+arrows point to the double-positive cells. The percentage of BrdU- positive cells expressing NeuN in each cell layers of GCL was calculated (d, e, f). Data are presented as the mean ± SD (n = 5). *p\0.05, **p\0.01 versus control group. SGZ subgranular zone, GCL granule cell layer (Color figure online)
+
+astrocytes in this area, which play a supporting role in the migration of newborn neurons. The experimental protocol is presented in Fig. 6a. Our results showed that ketamine could restrain the growth of radial glial cells in the hip- pocampal DG on PND-37 and PND-44, and the density of
+
+123
+
+194
+
+Fig. 6 Effect of ketamine on the development of radial glia in the hippocampal dentate gyrus (DG). Experimental protocol (a). Repre- sentative photographs from a laser scanning confocal microscope are shown (b; magnification: a, c, e and g 9200; b, d, f and h 9400); the scale bars are 100 lm (a) and 50 lm (b). The density of GFAP
+
+GFAP-positive cells in the hippocampal DG was signifi- cantly reduced in the ketamine-treated group compared to that in the control group (PND-37: 176 ± 9.96 vs. 230 ± 9.95 lm2; PND-44: 193 ± 12.62 vs. 244 ± 10.97 lm2)
+
+123
+
+Neurotox Res (2016) 30:185–198
+
+positive cells in the DG was calculated (c). The expression level of GFAP in the DG was measured by Western blot analysis at the same time points (d, e). Data are presented as the mean ± SD (n = 5). **p\0.01 versus control group. GCL granule cell layer, ML molecular layer, PCL polymorphic cell layer (Color figure online)
+
+(Fig. 6c). Typical immunofluorescence pictures are shown in Fig. 6b.
+
+The hippocampus tissues of rats on PND-37 and PND- 44 were used for Western blot analysis. Quantification of
+
+Neurotox Res (2016) 30:185–198
+
+the Western blot showed that the ketamine anesthesia induced less visible bands representing GFAP compared with the control group at these two time points (PND-37: 57 ± 4.06 vs. 89 ± 3.44 %; PND-44: 76 ± 6.88 vs. 93 ± 3.41 %; Fig. 6d, e).
+
+that neonatal ketamine exposure had a significantly inhibitory effect on the growth of radial glial cells, which may be an important reason for the inhibition of migration of newborn neurons.
+
+Together,
+
+these data suggest
+
+Neonatal Ketamine Exposure Caused Spatial Memory Impairment in the Adult Stage
+
+Figure 7a–d showed the memory and learning performance of rats in 2 months old. The latency to find the hidden platform of two groups rats had a reduced tendency as training progressed, which indicated that the animals were learning from practice of everyday. However, during the five training days, the latency to locate the hidden platform in ketamine group was significantly longer than that in control group (p \ 0.05; Fig. 7a), indicating neonatal
+
+Fig. 7 Anesthesia with ketamine in neonatal rats at postnatal day 7 (PND-7) induces learning and memory impairment in the adult stage. Ketamine anesthesia significantly increased the latency time of rat swimming in the Morris water maze (MWM) as compared with the control group (a). The times that the rats stayed in the target quadrant within 120 s was significantly reduced in ketamine group than that in
+
+195
+
+ketamine exposure could induce significantly impairment in learning and memory functions during the adult stage. In the memory retrieval tests, the times that the rats stayed in the target quadrant within 120 s was significantly reduced in ketamine group than that in control group (28 ± 9.02 vs. 44 ± 7.80 %; Fig. 7b). Also, the numbers of crossing over the previous platform site within 120 s was significantly in control group reduced in ketamine group than that (2 ± 0.75 vs. 5 ± 1.41; Fig. 7c). The typical track chart were shown in Fig. 7d. These data suggested that exposing ketamine (40 mg/kg 9 4 injections) to PND-7 rats could cause hippocampal-dependent neurocognitive impairment in the adult stage.
+
+Discussion
+
+Widespread and growing research has reported that keta- mine has neurotoxic effects on the developing animal brain (Ikonomidou et al. 1999; Liu et al. 2011; Paule et al. 2011; Zou et al. 2009), and its safety in pediatric anesthesia has
+
+control group (b). The numbers of crossing over the previous platform site within 120 s was significantly reduced in ketamine group than that in control group (c). Typical path chart of space exploration were exhibited (d). Data are presented as mean ± SD (n = 6). *p\0.05, **p\0.01 versus control group (Color figure online)
+
+123
+
+196
+
+been the subject of extensive concern for anesthesiologists and the public, based on the evidence that ketamine may have an association with neurocognitive impairment in children (Pfenninger et al. 2002; Wilder et al. 2009). However, the causal link between neuron death in the developing animal brain induced by anesthetics and long- term hippocampal-dependent neurocognitive deficits has not been elucidated. It is therefore of interest for us to explore the other mechanisms that can explain the hip- pocampal-dependent neurocognitive dysfunction caused by neonatal ketamine exposure.
+
+The substantial neurogenesis in the hippocampal DG lasts for life in animals and humans (Abrous et al. 2005). the production of During the process of neurogenesis, granule cells may change dynamically with age. In the rat, the granule cells in the DG are generated from the 14th day of gestation until the adult stage, and approximately 80 % of the granule cells are produced postnatally with a peak around seven days after birth (Altman and Bayer 1990). The accumulated results have demonstrated that the factors interfering with neuron production (e.g., postnatal or adult) may have a significant impact on hippocampus-dependent function (Young et al. 1999; Kempermann and Gage 2002). However, only some newborn neurons can be selected by the DG and allowed to migrate into the normal position of the GCL to meet the functional demand (Dupret et al. 2007; Kee et al. 2007). Hence, neurogenesis in the DG plays a crucial role in the normal of structure and function of the hippocampus.
+
+Numerous studies have suggested that NMDA-R plays an important role in regulating the neurogenesis of the hippocampal DG (Joo et al. 2007; Kitayama et al. 2004; Luk et al. 2003). However, the effects of blocking NMDA- R on the neurogenesis of the hippocampal DG are con- troversial (Nacher et al. 2001; Nacher and McEwen 2006; Arvidsson et al. 2001). Ketamine, as an NMDA-R inhi- bitor, was reported to inhibit the proliferation of NSCs isolated from the SVZ in the rat fetal cortex and enhance its neuronal differentiation in a previous in vitro study (Dong et al. 2012); however, its effect on postnatal neurogenesis in the hippocampal DG has not been studied in vivo. Hence, it might provide a new perspective to study the neonatal neurotoxicity of ketamine.
+
+BrdU, a classical tool for the detection of cell fate, was used to test neurogenesis. The scheme and dose of BrdU administration in our tests was based on previous experi- ments (Guidi et al. 2005; Zhang et al. 2014). We first observed the change in NSC proliferation and differentia- tion in the DG within two weeks after ketamine anesthesia. Our results showed that ketamine could significantly inhi- bit the proliferation of NSCs with decreased numbers of Nestin/BrdU double-positive cells. It was also found that the astrocytic differentiation of NSCs was markedly
+
+123
+
+Neurotox Res (2016) 30:185–198
+
+attenuated with a decreased number of GFAP/BrdU dou- ble-positive cells, while the neuronal differentiation of NSCs was obviously promoted with an increased number of b-tubulin III/BrdU double-positive cells. Our present results are partially consistent with the reports of Dong et al. (Dong et al. 2012). In addition, these effects of ketamine on the proliferation and differentiation of NSCs could last at least 1 week but disappeared 2 weeks after neonatal ketamine exposure. It is known that mature astrocytes can proliferate after exposure to some types of stimulation, such as stroke (Barreto et al. 2011). To exclude the proliferative mature astrocytes from the newly differentiated astrocytes, GFAP/BrdU double-labeling immunostaining was performed 3 h after the BrdU injec- tion, by which time BrdU had been adequately incorpo- rated newly differentiated astrocytes had not been generated. It was found that only a small number of mature astrocytes were capable of proliferating in the hippocampus of neonatal rats, and ketamine did not significantly promote or suppress the proliferation of mature astrocytes. There was no sig- nificant difference in the number of GFAP/BrdU double- positive cells between the control and ketamine groups. Therefore, it was determined that the GFAP/BrdU double- positive cells detected at 24 h after the BrdU injection could represent the newborn astrocytes differentiated from NSCs. In addition, to observe the effect of ketamine on the apoptosis of NSCs and astrocytes in the DG of neonatal rats, the nestin?/caspase-3? and GFAP?/caspase-3? cells were measured using double-labeled immunofluorescence. The results showed that neither nestin/caspase-3 nor GFAP/caspase-3 double-positive cells were found in the control or ketamine groups. Although neuron apoptosis has been demonstrated to be induced by neonatal exposure to ketamine, the present dosage and duration of ketamine were unable to induce the apoptosis of NSCs and astrocytes in the DG of neonatal rats. Thus, it is suggested that the reduced numbers of nestin/BrdU double-positive cells and GFAP/BrdU double-positive cells were not caused by cell death after ketamine exposure.
+
+into
+
+the
+
+proliferative
+
+cells,
+
+but
+
+the
+
+It is necessary for the newly differentiated neurons to migrate into the GCL of hippocampal DG to exert normal function. The abnormal migration of newborn granule neurons in the hippocampal DG is associated with hip- pocampal-specific cognitive deficits (Manning et al. 2012). The present study showed that ketamine could markedly inhibit the migration of newborn neurons with a decreased percentage of NeuN/BrdU double-positive cells in each layer of the GCL in the hippocampal DG both at PND-37 and PND-44. Further study indicated that the number of GFAP- positive cells and the expression of GFAP in hippocampal DG were significantly reduced in the ketamine group com- pared to the control group. Our findings suggest that the
+
+Neurotox Res (2016) 30:185–198
+
+reduced expression of GFAP may be caused by suppressing the astrocytic differentiation of NSCs after neonatal keta- mine exposure. The inhibitory effect of ketamine on the growth of astrocytes may result in abnormally positioned newborn neurons within the GCL during neuronal migration because astrocytes play a support role in the migration of newborn neurons (Sibbe et al. 2009).
+
+A previous study reported that colchicine injection into the DG caused the impairment in hippocampal-dependent spatial memory, but the lesion was limited to the DG rather than other hippocampal regions (Keith et al. 2007). This result suggested that the DG damage alone could produce a hippocampal-type neurocognitive dysfunction. According to the present study, neonatal ketamine exposure induced a significant alteration of neurogenesis in the hippocampal DG, which may be an important reason leading to abnor- malities in the structure of the hippocampus. It might have a close association with the ketamine-induced neurocog- nitive impairment.
+
+The mechanisms by which ketamine induce the inter- ference of neurogenesis in the hippocampal DG remain to be determined. In our previous in vitro study, suppressing Ca2?-PKCa-ERK1/2 signaling pathway may be involved in this inhibitory effect of ketamine on hippocampal NSCs proliferation (Yu-Qing et al. 2014). Thus, our future studies will include exploring whether ketamine exposure affects the hippocampal neurogenesis process through interfering with the calcium signaling pathway in vivo study.
+
+In summary, neonatal ketamine exposure could interfere the hippocampal DG, with postnatal neurogenesis of including the inhibition of NSC proliferation and astrocytic differentiation, the promotion of neuronal differentiation, the inhibition of astrocytic growth, and neuronal migration in the GCL. These findings may account for the adult hippocampal-dependent dysfunction induced by neonatal ketamine exposure.
+
+neurocognitive
+
+Acknowledgments This work was supported by the National Nat- ural Science Foundation of China (81171013), the Key Subject of Colleges and Universities Natural Science Foundation of Jiangsu Province (10KJA320052).
+
+Compliance with Ethical Standards
+
+Conflict of interest The authors have declared that no competing interests exist.
+
+References
+
+Abrous DN, Koehl M, Le Moal M (2005) Adult neurogenesis: from precursors to network and physiology. Physiol Rev 85:523–569 Altman J, Bayer SA (1990) Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods. J Comp Neurol 301:365–381
+
+197
+
+Arvidsson A, Kokaia Z, Lindvall O (2001) N-methyl-D-aspartate receptor-mediated increase of neurogenesis in adult rat dentate gyrus following stroke. Eur J Neurosci 14:10–18
+
+Asadi P, Ghafouri HB, Yasinzadeh M, Kasnavieh SM, Modirian E (2013) Ketamine and atropine for pediatric sedation: a prospec- tive double-blind randomized controlled trial. Pediatr Emerg Care 29:136–139
+
+Ashwell KWS, Paxinos G (2008) Atlas of the developing rat nervous
+
+system. Elsevier, San Diego
+
+Barreto GE, Sun X, Xu L, Giffard RG (2011) Astrocyte proliferation following stroke in the mouse depends on distance from the infarct. PLoS ONE 6:e27881
+
+Bartley J, Soltau T, Wimborne H, Kim S, Martin-Studdard A et al (2005) BrdU-positive cells in the neonatal mouse hippocampus following hypoxic-ischemic brain injury. BMC Neurosci 6:15
+
+Belnoue L, Grosjean N, Ladeveze E, Abrous DN, Koehl M (2013) Prenatal stress inhibits hippocampal neurogenesis but spares olfactory bulb neurogenesis. PLoS ONE 8:e72972
+
+Byrnes ML, Reynolds JN, Brien JF (2001) Effect of prenatal ethanol exposure during the brain growth spurt of the guinea pig. Neurotoxicol Teratol 23:355–364
+
+Dong C, Rovnaghi CR, Anand KJ (2012) Ketamine alters the neurogenesis of rat cortical neural stem progenit or cells. Crit Care Med 40:2407–2416
+
+Dupret D, Fabre A, Dobrossy MD, Panatier A, Rodriguez JJ et al learning depends on both the addition and
+
+(2007) Spatial removal of new hippocampal neurons. PLoS Biol 5:e214 Dupret D, Revest JM, Koehl M, Ichas F, De Giorgi F et al (2008) Spatial relational memory requires hippocampal adult neuroge- nesis. PLoS ONE 3:e1959
+
+Erasso DM, Camporesi EM, Mangar D, Saporta S (2013) Effects of isoflurane or propofol on postnatal hippocampal neurogenesis in young and aged rats. Brain Res 1530:1–12
+
+Esposito MS, Piatti VC, Laplagne DA, Morgenstern NA, Ferrari CC et al (2005) Neuronal differentiation in the adult hippocampus recapitulates 25: 10074–10086
+
+embryonic
+
+development.
+
+J Neurosci
+
+Fang F, Xue Z, Cang J (2012) Sevoflurane exposure in 7-day-old rats affects neurogenesis, neurodegeneration and neurocognitive function. Neurosci Bull 28:499–508
+
+Guerra GG, Robertson CM, Alton GY, Joffe AR, Cave DA et al (2011) Neurodevelopmental outcome following exposure to sedative and analgesic drugs for complex cardiac surgery in infancy. Paediatr Anaesth 21:932–941
+
+Guidi S, Ciani E, Severi S, Contestabile A, Bartesaghi R (2005) Postnatal neurogenesis in the dentate gyrus of the guinea pig. Hippocampus 15:285–301
+
+Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J et al (1999) Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 283:70–74
+
+Joo JY, Kim BW, Lee JS, Park JY, Kim S et al (2007) Activation of NMDA receptors increases proliferation and differentiation of hippocampal neural progenitor cells. J Cell Sci 120:1358–1370 Kee N, Teixeira CM, Wang AH, Frankland PW (2007) Preferential incorporation of adult-generated granule cells into spatial memory networks in the dentate gyrus. Nat Neurosci 10: 355–362
+
+Keith JR, Wu Y, Epp JR, Sutherland RJ (2007) Fluoxetine and the dentate gyrus: memory, recovery of function, and electrophys- iology. Behav Pharmacol 18:521–531
+
+Kempermann G, Gage FH (2002) Genetic influence on phenotypic differentiation in adult hippocampal neurogenesis. Brain Res Dev Brain Res 134:1–12
+
+Kempermann G, Gast D, Kronenberg G, Yamaguchi M, Gage FH (2003) Early determination and long-term persistence of adult-
+
+123
+
+198
+
+generated new neurons in the hippocampus of mice. Develop- ment 130:391–399
+
+Kitayama T, Yoneyama M, Tamaki K, Yoneda Y (2004) Regulation of neuronal differentiation by N-methyl-D-aspartate receptors expressed in neural progenitor cells isolated from adult mouse hippocampus. J Neurosci Res 76:599–612
+
+Liu F, Paule MG, Ali S, Wang C (2011) Ketamine-induced neurotoxicity and changes in gene expression in the developing rat brain. Curr Neuropharmacol 9:256–261
+
+Lledo PM, Alonso M, Grubb MS (2006) Adult neurogenesis and functional plasticity in neuronal circuits. Nat Rev Neurosci 7:179–193
+
+Luk KC, Kennedy TE, Sadikot AF (2003) Glutamate promotes proliferation of striatal neuronal progenitors by an NMDA receptor-mediated mechanism. J Neurosci 23:2239–2250 Luskin MB (1993) Restricted proliferation and migration of postna- tally generated neurons derived from the forebrain subventricular zone. Neuron 11:173–189
+
+Manning EE, Ransome MI, Burrows EL, Hannan AJ (2012) Increased adult hippocampal neurogenesis and abnormal migration of adult-born granule neurons is associated with hippocampal- specific cognitive deficits in phospholipase C-beta1 knockout mice. Hippocampus 22:309–319
+
+Mongiat LA, Schinder AF (2011) Adult neurogenesis and the plasticity of the dentate gyrus network. Eur J Neurosci 33:1055–1061 Nacher J, McEwen BS (2006) The role of N-methyl-D-asparate
+
+receptors in neurogenesis. Hippocampus 16:267–270
+
+Nacher J, Rosell DR, Alonso-Llosa G, McEwen BS (2001) NMDA receptor antagonist treatment induces a long-lasting increase in the number of proliferating cells, PSA-NCAM-immunoreactive granule neurons and radial glia in the adult rat dentate gyrus. Eur J Neurosci 13:512–520
+
+Nie H, Peng Z, Lao N, Dong H, Xiong L (2013) Effects of sevoflurane on self-renewal capacity and differentiation of cultured neural stem cells. Neurochem Res 38:1758–1767
+
+Paule MG, Li M, Allen RR, Liu F, Zou X et al (2011) Ketamine anesthesia during the first week of life can cause long-lasting cognitive deficits in rhesus monkeys. Neurotoxicol Teratol 33:220–230
+
+Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates,
+
+vol 2. Academic Press, Sydney
+
+Pfenninger EG, Durieux ME, Himmelseher S (2002) Cognitive impairment after small-dose ketamine isomers in comparison to equianalgesic racemic ketamine in human volunteers. Anesthe- siology 96:357–366
+
+123
+
+Neurotox Res (2016) 30:185–198
+
+Porzionato A, Macchi V, Zaramella P, Sarasin G, Grisafi D et al (2013) Effects of postnatal hyperoxia exposure on the rat dentate Funct gyrus 220(1):229–247
+
+and
+
+subventricular
+
+zone. Brain
+
+Struct
+
+Sibbe M, Forster E, Basak O, Taylor V, Frotscher M (2009) Reelin and Notch1 cooperate in the development of the dentate gyrus. J Neurosci 29:8578–8585
+
+Stone SS, Teixeira CM, Zaslavsky K, Wheeler AL, Martinez-Canabal A et al (2011) Functional convergence of developmentally and adult-generated granule cells in dentate gyrus circuits supporting hippocampus-dependent memory. Hippocampus 21:1348–1362 Stratmann G, May LD, Sall JW, Alvi RS, Bell JS et al (2009a) Effect of hypercarbia and isoflurane on brain cell death and neurocog- nitive rats. Anesthesiology 110:849–861
+
+dysfunction
+
+in
+
+7-day-old
+
+Stratmann G, Sall JW, May LD, Bell JS, Magnusson KR et al (2009b) Isoflurane differentially affects neurogenesis and long-term neurocognitive function in 60-day-old and 7-day-old rats. Anesthesiology 110:834–848
+
+Vadodaria KC, Jessberger S (2014) Functional neurogenesis in the
+
+adult hippocampus: then and now. Front Neurosci 8:55
+
+van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD et al (2002) Functional neurogenesis in the adult hippocampus. Nature 415:1030–1034
+
+Wilder RT, Flick RP, Sprung J, Katusic SK, Barbaresi WJ, Mickelson C, Gleich SJ, Schroeder DR, Weaver AL, Warner DO (2009) Early exposure to anesthesia and learning disabilities in a population-based birth cohort. Anesthesiology 110:796–804 Young D, Lawlor PA, Leone P, Dragunow M, During MJ (1999) Environmental enrichment inhibits spontaneous apoptosis, pre- vents seizures and is neuroprotective. Nat Med 5:448–453 Yu-Qing Wu, Liang Tuo, Huang He et al (2014) Ketamine inhibits proliferation of neural stem cell from neonatal rat hippocampus in vitro. Cell Physiol Biochem 34:1792–1801
+
+Zhang K, Zhao T, Huang X, Wu LY, Wu K et al (2014) Notch1 mediates postnatal neurogenesis in hippocampus enhanced by intermittent hypoxia. Neurobiol Dis 64:66–78
+
+Zou X, Patterson TA, Divine RL, Sadovova N, Zhang X et al (2009a) Prolonged exposure to ketamine increases neurodegeneration in the developing monkey brain. Int J Dev Neurosci 27:727–731
+
+Zou X, Patterson TA, Sadovova N, Twaddle NC, Doerge DR et al (2009b) Potential neurotoxicity of ketamine in the developing rat brain. Toxicol Sci 108:149–158

new_pdfs/10.1007_s12640-018-9877-3.txt

@@ -0,0 +1,305 @@
+Neurotoxicity Research (2018) 34:188–197 https://doi.org/10.1007/s12640-018-9877-3
+
+ORIGINAL ARTICLE
+
+Neonatal Exposure to Low-Dose (1.2%) Sevoflurane Increases Rats’ Hippocampal Neurogenesis and Synaptic Plasticity in Later Life
+
+Xi Chen 1 & Xue Zhou 1 & Lu Yang 1 & Xu Miao 1 & Di-Han Lu 1 & Xiao-Yu Yang 1 & Zhi-Bin Zhou 1 & Wen-Bin Kang 1 & Ke-Yu Chen 1 & Li-Hua Zhou 2 & Xia Feng 1
+
+Received: 26 October 2017 / Revised: 7 January 2018 / Accepted: 26 January 2018 / Published online: 9 February 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018
+
+Abstract The increasing usage of general anesthetics on young children and infants has drawn extensive attention to the effects of these drugs on cognitive function later in life. Recent animal studies have revealed improvement in hippocampus-dependent perfor- mance after lower concentrations of sevoflurane exposure. However, the long-term effects of low-dose sevoflurane on the developing brain remain elusive. On postnatal day (P) 7, rats were treated with 1.2% sevoflurane (1.2% sevo group), 2.4% sevoflurane (2.4% sevo group), and air control (C group) for 6 h. On P35–40, rats’ hippocampus-dependent learning and memory was tested using the Morris water maze. Cognition-related and synapse-related proteins in the hippocampus were measured using Western blotting on P35. On the same day, neurogenesis and synapse ultrastructure were evaluated using immunofluorescence and transmission electron microscopy (TEM). On P35, the rats neonatally exposed to 1.2% sevoflurane showed better behavioral results than control rats, but not in the 2.4% sevo group. Exposure to 1.2% sevoflurane increased the number of 5′-bromo-2- deoxyuridine (BrdU)-positive cells in the dentate gyrus and improved both synaptic number and ultrastructure in the hippocam- pus. The expression levels of BDNF, TrkB, postsynaptic density (PSD)-95, and synaptophysin in the hippocampus were also increased in the 1.2% sevo group. In contrast, no significant changes in neurogenesis or synaptic plasticity were observed between the C group and the 2.4% sevo group on P35. These results showed that exposure of the developing brain to a low concentration of sevoflurane for 6 h could promote spatial learning and memory function, along with increased hippocampal neurogenesis and synaptic plasticity, in later life.
+
+Keywords Sevoflurane . Hippocampus . Cognitive function . Neurogenesis . Synaptic plasticity
+
+Introduction
+
+The developing brain is vulnerable to environmental influ- ences including general anesthetics (Loepke and Soriano 2008; Mellon et al. 2007). The widespread and prevalent use
+
+Xi Chen and Xue Zhou contributed equally to this work.
+
+Li-Hua Zhou
+
+zhoulih@mail.sysu.edu.cn
+
+Xia Feng
+
+fengxiar@sina.com
+
+1 Department of Anaesthesiology, The First Affiliated Hospital of Sun
+
+Yat-Sen University, No. 58 Zhongshan Road 2, Guangzhou 510080, Guangdong, People’s Republic of China 2 Department of Anatomy, Zhongshan School of Medicine, Sun
+
+of anesthesia in children makes its safety a major health issue of interest. Sevoflurane is a volatile anesthetic that is common- ly used, particularly in clinical pediatric anesthesia, because it is better tolerated than many other anesthetics and has excel- lent results (Goa et al. 1999). Accumulating evidence suggests that neonatal rodent exposure to higher concentrations of sevoflurane could induce developmental neurotoxicity, in- cluding long-term learning disabilities, degeneration of neu- rons, and impairment of synaptic plasticity (Feng et al. 2012; Ishizeki et al. 2008; Tao et al. 2016). However, recent studies have shown that lower concentrations of sevoflurane have neuroprotective effects (Chen et al. 2015; Payne et al. 2005). These complicated results suggest that different parameters such as concentration, timing, and exposure duration of sevoflurane are critical to the final outcomes and reflect the complexity of the effects on the central nervous system. However, the effect of low-concentration sevoflurane during the neonatal period on learning and memory ability in later life
+
+Yat-Sen University, No. 74 Zhongshan Road 2, Guangzhou 510080, Guangdong, People’s Republic of China
+
+Neurotox Res (2018) 34:188–197
+
+has been unclear. The present work aimed to fill a current gap in the literature by investigating whether exposure to a low concentration (1.2%) of sevoflurane during early postnatal life impacts learning and memory ability later in life.
+
+Here, we exposed rats at postnatal day (P) 7 to air or to 1.2 or 2.4% sevoflurane for 6 h. During the juvenile stage, we compared the effects of lower and higher concentrations of sevoflurane on anesthetic-induced hippocampus-dependent learning and memory ability, neurogenesis, and synaptic plas- ticity in the hippocampal area.
+
+Methods
+
+Ethical Approval
+
+The use of rats in this study was approved by the Institutional Animal Care and Use Committee at Sun Yat-sen University (Guangzhou, China). All experiments were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and ARRIVE guidelines. Sprague-Dawley multiparous dams (n = 31) with litters con- taining male pups (n = 135) were purchased from Experimental Animal Center of Sun Yat-sen University, China. We only used male offspring to exclude the influence of estrogen on the biochemical data and neurocognitive func- tions. The pups from postnatal day 0 (P0) to P20 were housed with the dams in a 12-h:12-h light:dark cycle (light from 07:00 to 19:00), and room temperature (RT) was maintained at 21 ± 1 °C. On P21, the pups were weaned and housed 4–6 per cage in a standard environment.
+
+Anesthesia
+
+SD rats at P7 (weight 14–16 g) were randomly divided into the air-treated control (C group), the 1.2% sevoflurane-exposed (1.2% sevo group), and the 2.4% sevoflurane-exposed (2.4% sevo group). Rats in the 1.2% sevo group and the 2.4% sevo group were placed in a plastic container and exposed to 1.2 or 2.4% sevoflurane continuously for 6 h, using air as a carrier, with a total gas flow of 2 L min−1. A nasopharyngeal airway tube was put in their mouth to prevent apnea and hypoxia when the rats stopped moving in the container. During expo- sure, the temperature inside the container was maintained at 30 °C using an external heating device (NPS-A3 heating de- vice, Midea Co., Guangdong, China) and a hot water bag on the bottom of the container with a constant temperature main- tained between 30 and 35 °C. The concentrations of sevoflurane, oxygen, and carbon dioxide in the chamber were monitored by a gas monitor (Detex-Ohmeda, Louisville, CO, USA). During exposure, an investigator monitored the rats’ spontaneous respiratory frequency and skin color every 5 min to detect any apnea or hypoxia. The rats were immediately
+
+exposed to air and excluded from the experiment if these symptoms were detected. Sevoflurane administration was ter- minated 6 h later, and the rats were exposed only to air. When the rats were moving freely again, they were placed back into their maternal cages. Rats in the C group were exposed to the same container as the rats in the 1.2% sevo and 2.4% sevo group but were exposed to air alone for 6 h.
+
+Arterial Blood Gas Analysis
+
+We performed arterial blood analysis in order to exclude the influence of respiratory or metabolic disorder. The arterial blood samples from the C, 1.2% sevo, and 2.4% sevo groups were obtained from the left cardiac ventricle immediately after removal from the maternal cage (n = 5 in each group) at the end of anesthesia. They were analyzed immediately after col- lection using a blood gas analyzer (Gem Premier 3000, US). We analyzed the pH, arterial carbon dioxide tension (PaCO2), arterial oxygen tension (PaO2), and blood glucose levels of the arterial blood samples.
+
+Morris Water Maze Test
+
+On P35, the rats were tested for spatial learning and memory ability using the Morris water maze (MWM). Three groups of rats (n = 10 in each group, weight 90–100 g) were tested on the MWM, which consists of two different tests including hidden platform acquisition and a probe trial test, at P35– P40 using the Water Maze Tracking System (MT-200; Chengdu, China). A white platform (12 cm diameter) was submerged in a circular pool (160 cm in diameter, 50 cm in height) that was filled with warm water (23 ± 2 °C). The pool, located in a room with no windows, was virtually divided into four quadrants. A video camera connected to the computer running the tracking software was suspended above the pool and captured the rats’ movements for analysis. At P35, before the test, a single habituation trial was performed without the platform; in this trial, the rats were placed in the water for 120 s. In the hidden platform acquisition test, performed at P36–P39, each rat was placed, facing the wall of the pool, in one of the four quadrants and allowed to swim freely in search of the escape platform for a maximum of 120 s. The experi- ment was repeated with four trials per day for four consecutive days. The average escape latency time (latency to reach the platform) was measured to evaluate spatial learning ability. At P40, a probe trial test was performed by removing the plat- form and releasing the rats into the water for 120 s. We calcu- lated the time spent in the quadrant that previously contained the target and the frequency of crossing the former location of the platform. The rats were dried and placed back into a heated cage after completing each test.
+
+189
+
+190
+
+Western Blot Analysis
+
+On P10 and 28, rats (n = 5 in each group at each sacrifice time point) were sacrificed by rapid decapitation, and the bilateral hippocampus areas were harvested and stored at − 80 °C until use. Protein was extracted using RIPA lysis buffer (Keygen Biotech, Nanjing, China). The amount of protein in each hip- pocampal tissues was measured using a protein assay kit (BCA, Pierce, Thermo, USA). Polyacrylamide-SDS gels with an equal amount of 50-μg load in each lane were electropho- resed, and the proteins transferred onto PVDF membranes (Millipore, Carrigtwohill, Ireland). The blots were blocked with 5% skim milk in Tris-buffered saline (150 mM NaCl, 0.1% TWEEN 20, 20 mM Tris, pH 7.4) for 1 h and then incubated overnight at 4 °C with anti-BDNF (1:1000, Novusbio, USA), anti-TrkB (1:800, Millipore, Ireland), anti- postsynaptic density (PSD-95) (1:2000, Abcam, England), and anti-synaptophysin (1:20,000, Abcam, England) primary antibodies. After rinsing, membranes were probed with corre- sponding secondary antibodies at RT for 2 h. Immunoreactive bands were detected with an enhanced chemiluminescence detection system (Bio-Rad, USA). A β-actin antibody (1:1000, ABclonal, China) was used to normalize for sample loading and transfer. The intensities of the bands were densitometrically quantified using ImageJ.
+
+BrdU Injections and Immunofluorescence
+
+For the 5′-bromo-2-deoxyuridine (BrdU) injections, we followed the methods as previously described (Chen et al. 2015; Tozuka et al. 2005). BrdU has been described as a marker of neurogenesis and can incorporate into DNA only during the S-phase of the mitotic process (Kee et al. 2002). BrdU (Sigma, America) was dissolved in normal saline (10 mg mL−1) and injected at a dosage of 300 mg/kg. To investigate the effects of 1.2% sevoflurane on cellular prolif- eration, we performed a single injection of BrdU i.p. 24 h after sevoflurane exposure. Three days later, the rats were perfused, and their brains were processed for immunofluorescence. To investigate the effects of 1.2% sevoflurane on the survival of newborn cells, we performed a single injection of BrdU i.p. 24 h before sevoflurane exposure. Four weeks (28 days) after the BrdU injection, the rats were perfused, and their brains were processed for immunofluorescence.
+
+For morphological examination, rats were deeply anesthe- tized with chloral hydrate at P10 and P35 (n = 5 in each group at each sacrifice time point, 80–100 g) and then transcardially perfused with 0.9% normal saline at RT followed by a fixative solution of 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M PBS (pH 7.4) at 4 °C. The brains were harvested, postfixed in 4% paraformaldehyde for 8 h, and subsequently soaked in 30% sucrose until they sank. Consecutive frozen coronal sections of the hippocampus were
+
+Neurotox Res (2018) 34:188–197
+
+cut at a thickness of 30 μm. Every fifth section of the consec- utive sections was processed by BrdU staining. DNA was first denatured by incubation with 2 N HCl for 30 min at 37 °C followed by a 15-min wash in 0.1 M boric acid (pH 8.5), with three 10-min washes in 0.01 M PBS before each step. The sections were blocked in 3% BSA and 0.4% Triton X-100 for 2 h at RT before being incubated with primary antibody (rat anti-BrdU, 1:200, ab6326, Abcam, UK) in 1% BSA over- night at 4 °C. Then, the sections were incubated with second- ary antibody (Cy3 goat anti-rat IgG, 1:200, KGAB018, Keygentee, China) for 2 h at RT. Fluorescence was detected with a fully automatic fluorescence microscope (Olympus BX63, Japan). An observer who was blinded to group assign- ment was responsible for counting the number of BrdU- positive cells at × 200 magnification. Total cell counts were divided by the total number of sections for analysis.
+
+Transmission Electron Microscopy
+
+TEM was used to assess synaptic plasticity in the hippo- campus after exposure to treatment (n = 5 in each group) at P35. Twenty-eight days after exposure to treatment, the rats were perfused transcardially with 50 mL of 0.9% normal saline, followed by 50 mL of a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde (Sigma- Aldrich, G6257, USA) in 0.1 M PBS. Approximately 1 mm3 of tissue per rat was dissected from the hippocam- pus and fixed in 2% glutaraldehyde for 2 h at 4 °C. The tissues were rinsed in 0.1 M cacodylate buffer and postfixed with 1% osmium tetroxide for 2 h. Then, the tissue was rinsed with distilled water before undergoing dehydration in a graded ethanol series. Subsequently, the tissue was infiltrated overnight at 4 °C using a mixture of half acetone and half resin. The tissue was embedded in resin 24 h later and then cured fully as follows: 37 °C overnight, 45 °C for 12 h, and 60 °C for 24 h. After that, 70-nm sections were cut and stained with 3% uranyl ace- tate for 20 min and 0.5% lead citrate for 5 min. Ultrastructural changes in synapses in the hippocampus were observed under TEM. Five pictures of each subre- gion per ultrathin section (five rats in total per group) were taken at each of two magnifications: × 13,500 and × 37,000. All pictures taken at × 13,500 magnification were used to observe the number of synapses, and all pictures taken at × 37,000 magnification were used to measure the thickness of the postsynaptic density and the width of the synaptic cleft. The number of synapses was expressed as the average number of synapses in each picture taken at × 13,500. The thickness of the postsynap- tic density and the width of the synaptic were expressed as the average values for all synapses in all pictures taken at × 37,000, as described. We measured the distances using the image analysis software ImageJ.
+
+Neurotox Res (2018) 34:188–197
+
+191
+
+Statistical Analysis
+
+The results were expressed as the mean ± standard deviation (SD) for each group. The statistical tests were conducted using the computerized statistical package SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism Software version 5.0 (GraphPad Software, Inc., San Diego, CA, USA). The arterial blood data were analyzed using Student’s t test. One-way ANOVA was used to evaluate differences in the quantities of hippocampal proteins, numbers of BrdU-positive cells and synapses, and ultrastructure parameters of synapses among groups. Unpaired t tests and two-way ANOVA were used to analyze the results of the MWM. Each experiment was per- formed at least three times. A value of P < 0.05 was consid- ered statistically significant.
+
+which indicated that the rats were learning from practice every day. However, from the third training day, the la- tency to locate the hidden platform in the 1.2% sevo group was significantly shorter than that in the C group and of the 2.4% sevo group (all P < 0.001 vs. C group; Fig. 1a). In the probe trial, the time spent in the target quadrant in the 1.2% sevo group (5.22 ± 2.30) was longer than that in the C (4.08 ± 2.50) group and in the 2.4% sevo group (4.19 ± 2.21; P < 0.01, Fig. 1b). Moreover, the frequency of passing through the target quadrant was significantly higher in the 1.2% sevo group (4.50 ± 2.396) than that in the C group (3.17 ± 1.76) and the 2.4% sevo group (2.92 ± 1.53, Fig. 1c). There were no significant differences in the rats’ swimming speeds (C, 23.44 ± 0.99 cm s−1; 1.2% sevo, 24.01 ± 0.81 cm s−1; 2.4% sevo, 24.43 ± 1.02 cm s−1, according to one-way ANOVA, P = 0.63) among the three groups.
+
+Results
+
+Sevoflurane Does Not Cause Respiratory or Metabolic Disorder
+
+1.2% Sevoflurane Increased the Number of BrdU-Positive Cells in DG
+
+During sevoflurane exposure period, none of the rats appeared with apnea or hypoxia. Arterial blood analysis was used to exclude the influence of respiratory or metabolic disorder. No rats died during the exposure period. Compared with the control group, there were no significant changes in the pH, PaCO2, PaO2, or arterial blood glucose levels before or after exposure in the 1.2% sevo group or the 2.4% sevo group (Table 1).
+
+1.2% Sevoflurane Increased Spatial Learning and Memory Development Later in Life
+
+Four weeks after sevoflurane exposure, the spatial learn- ing and memory was measured by the MWM test de- scribed in the BMethods.^ The results (Fig. 1) showed that the latency to find the hidden platform decreased gradu- ally day by day as training progressed in the three groups,
+
+To test the level of neurogenesis in the DG, we used BrdU to label proliferative cells as an indicator of neurogenesis. The im- munofluorescence images showed that BrdU-labeled cells were present in all three groups on both P10 and P35, which means that hippocampal neurogenesis was active in the young rats. In addition, the neurogenesis level was higher on P10 (Fig. 2a–c) than that on P35 (Fig. 2d–f) in all the groups. The data from immunofluorescence staining demonstrated that 1.2% sevoflurane significantly increased the number of BrdU- positive cells on both P10 and P35. Statistical testing showed that the number of BrdU-positive cells was significantly larger in the 1.2% sevo group than that in the C group and the 2.4% sevo group on P10 (C, 254 ± 37.92; 1.2% sevo, 408 ± 35.85; 2.4% sevo, 245 ± 34.24; P < 0.0001, Fig. 2g) and P35 (C, 30.83 ± 2.85; 1.2% sevo, 35.83 ± 2.14; 2.4% sevo, 28.50 ± 2.35; P = 0.0004, Fig. 2h). No significant difference was found between the C group and the 2.4% sevo group.
+
+Table 1 Arterial blood analysis (N = 5 in each group)
+
+Groups
+
+C
+
+1.2% sevo
+
+2.4% sevo
+
+P value
+
+pH PaCO2 (kPa) PaO2 (kPa) Glucose (mmol L−1)
+
+7.40 ± 0.05 3.57 ± 0.38 13.38 ± 0.55 5.6 ± 0.8
+
+7.39 ± 0.08 3.56 ± 0.52 13.42 ± 0.51 5.3 ± 0.7
+
+7.37 ± 0.09 3.58 ± 0.45 13.35 ± 0.60 5.5 ± 0.5
+
+0.70 0.82 0.92 0.69
+
+Neonatal exposure to a low or high concentration of sevoflurane does not lead to significant cardiorespiratory dysfunction. Arterial blood gas analysis revealed no significant difference in any of the measured parameters among the three groups (t test, all P values > 0.05) PaCO2 arterial carbon dioxide tension, PaO2 arterial oxygen tension, Glucose blood glucose levels, C control, 1.2% sevo 1.2% sevoflurane-exposed group, 2.4% sevo 2.4% sevoflurane-exposed group
+
+192
+
+Fig. 1 Exposure to 1.2%, but not 2.4%, sevoflurane in neonatal rats on P7 induces spatial learning and memory changes in the juvenile stage. a In the MWM, the 1.2% sevo group had a significantly shorter latency than the C group to reach the platform. b The numbers of rats that reached the target quadrant within 120 s were significantly increased in the 1.2% sevo group compared with those in the C group. c The frequency of crossing the former location of the target platform within 120 s was significantly increased in the 1.2% sevo group compared with that in the C group. Data are presented as the mean ± SD (n = 10 in each group). *P < 0.05 versus C group; **P < 0.01 versus C group; ***P < 0.001 versus C group. C, control; 1.2% sevo, 1.2% sevoflurane-exposed group; 2.4% sevo, 2.4% sevoflurane-exposed group
+
+Hippocampal Synaptic Changes in Hippocampus
+
+Expression of Synapse-Associated Proteins in the Hippocampus
+
+Two typical synaptic proteins in the hippocampus were visualized by SDS-PAGE and immunoblotting with corresponding antibod- ies for PSD-95 and synaptophysin (SYN). The optical density ratios of the band intensities of the studied proteins normalized to β-actin were expressed as fold changes in the three groups.
+
+Compared with the level of PSD-95 in the hippocampus of the C group, the PSD-95 protein in the hippocampus of the 1.2% sevo group was significantly increased on P35 (158.6% of control, P = 0.0066), but there was no significant change in the 2.4% sevo group (128.5% of control, P = 0.73). Moreover, as with the higher level of PSD-95 observed in the 1.2% sevo group, the SYN protein level in the hippocampus of the 1.2% sevo group at P35 was also increased (231.6% of control, P = 0.0082) compared with that of the C group (Fig. 3). However, the SYN protein levels showed no significant change in the 2.4% sevo group compared with those in the C group (197.6% of control, P = 0.50).
+
+Ultrastructural Changes in Hippocampal Synapses
+
+The number of synapses and the synaptic ultrastructure of the hippocampus were examined using TEM 4 weeks after
+
+Neurotox Res (2018) 34:188–197
+
+sevoflurane exposure. Compared with the C and 2.4% sevo groups, the 1.2% sevo group showed an increase in the num- ber of synapses in the hippocampus (C, 13.0 ± 2.74; 1.2% sevo, 17.4 ± 2.80; 2.4% sevo, 10.80 ± 1.92; P = 0.0042 of control, Fig. 4a–c, g). The statistical analysis showed a signif- icant difference in ultrastructure changes: we found a notice- ably narrower synaptic cleft width and greater PSD thickness in the 1.2% sevo group than in either of the other groups (Table 2). No differences were found between the C and 2.4% sevo groups in the number of synapses, synaptic cleft width, or PSD thickness.
+
+1.2% Sevoflurane Increased the Levels of BDNF and TrkB in the Hippocampus
+
+To assess the neuroprotective effects of low-dose sevoflurane on the developing brain, the levels of the neurogenesis- and synaptic plasticity-related proteins BDNF and TrkB (Lu et al. 2013; Sairanen et al. 2005) (Hariri et al. 2003) were examined by Western blotting at P35. The optical density ratios of the band intensities of the studied proteins normalized to β-actin were expressed as fold changes for the three groups. At P35, the expression levels of BDNF and TrkB protein in the hip- pocampus of the 1.2% sevo group were significantly in- creased compared with those of the C group (BDNF 63.5% of control, P = 0.0063; TrkB 49.8% of control, P = 0.0002) and the 2.4% sevo group (BDNF 54.2% of control, P = 0.90;
+
+Neurotox Res (2018) 34:188–197
+
+Fig. 2 Neonatal exposure to 1.2% sevoflurane increased neuronal neurogenesis in the hippocampal DG on P10 and P35. a–c BrdU- positive cells on P10 in sevoflurane-treated and untreated rats. d–e BrdU-positive cells on P35 in sevoflurane-treated and untreated rats. g,
+
+TrkB 29.8% of control, P = 0.64; Fig. 3a–c). There was no significant difference between the C group and the 2.4% sevo group in these proteins.
+
+Discussion
+
+Sevoflurane is one of the most commonly used anesthetics in neonatal and pediatric anesthesia practice. The safety of the clinical application of sevoflurane in young children is still unclear. The present work aimed to find out whether exposure to a lower concentration (1.2%) of sevoflurane during early postnatal life impacts learning and memory ability later in life. In the current study, we selected 1.2% sevoflurane as the lower dose because this is the lowest sub-anesthetic dose that can prevent rats’ movement in response to a slight stimulus in neonatal rats and did not inhibit respiration. But beyond that, 1.2% sevoflurane is comparable to be used in the clinical setting. Using the MWM, we found that exposure of neonatal rats to 1.2% sevoflurane for 6 h improved their hippocampus- dependent learning and memory ability. In addition, changes were found in the number of BrdU-positive cells in the DG and the number of synapses, synaptic cleft width, and
+
+h Summary data for the experiment at P10 and P35. Scale bar represents 50 μm. ***P < 0.001 versus C group; ****P < 0.0001 versus C group. C, control; 1.2% sevo, 1.2% sevoflurane-exposed group; 2.4% sevo, 2.4% sevoflurane-exposed group
+
+postsynaptic density thickness in the hippocampus of the low-dose group. Our observations indicated that increased neurogenesis and synaptic plasticity in the hippocampus caused by low-dose sevoflurane might induce changes in neu- robehavioral function later in life.
+
+The MWM test in the current study demonstrated that neo- natal exposure to 1.2% sevoflurane for 6 h could facilitate the spatial learning and memory ability of rats later in life. Our result was consistent with previous studies showing that neo- natal exposure to sevoflurane in rodents has no potential to harm their neurobehavioral function in adulthood (Callaway et al. 2012; Liang et al. 2010). More remarkably, Chen et al. found that a subclinical dose of sevoflurane could promote hippocampal neurogenesis in neonatal rats and facilitate den- tate gyrus-dependent learning (Chen et al. 2015). Furthermore, in vitro studies confirmed that a lower dose of sevoflurane could promote the self-renewal capacity and dif- ferentiation of cultured neural cells (Nie et al. 2013; Yang et al. 2017). Our results showed that a higher concentration of sevoflurane had no deleterious effects on learning and mem- ory. However, some studies have found apparently contradic- tory results, indicating that neonatal exposure to a high dose of sevoflurane in rodents and nonhuman primates produces
+
+193
+
+194
+
+Fig. 3 Exposure to 1.2% sevoflurane increased BDNF, TrkB, PSD-95, and SYN levels in the hippocampus. a Representative immunoblots for the expression levels of BDNF and TrkB in the hippocampus 4 weeks after sevoflurane exposure. b, c Quantification of BDNF and TrkB normalized to β-actin (n = 5 per group). d Representative immunoblots for the expression levels of PSD-95 and SYN in the hippocampus 4 weeks after sevoflurane exposure. e, f Quantification of PSD-95 and
+
+neurobehavioral defects persisting into adulthood (Haseneder et al. 2009; Jevtovic-Todorovic et al. 2003). We assume that this discrepancy is due to the use of different animal models and behavioral tests.
+
+Recent studies have confirmed that a subclinical concen- tration of sevoflurane can enhance the proliferation of cultured neural stem cells (NSCs) (Nie et al. 2013). Our immunofluo- rescence histochemistry results also showed that 1.2% sevoflurane exposure increased the number of BrdU-positive cells at both P10 and P35, indicating a positive effect on neurogenesis. This finding is consistent with a previous study, which demonstrated that a sub-anesthetic dose of sevoflurane led to a significant increase in neurogenesis in neonatal rats (Chen et al. 2015). Furthermore, high concentrations and mul- tiple exposures to sevoflurane anesthesia during the neonatal period are considered to be associated with a reduction in neurogenesis (Fang et al. 2017; Lee et al. 2017). These results suggest that sevoflurane exerts dual effects on cognitive func- tion and neurogenesis depending on the dose and duration. In addition, a previous study showed that BDNF plays an impor- tant role in regulating the basal level of neurogenesis in the dentate gyrus (Lee et al. 2002), and those newly generated cells can mature into functional neurons in the mammalian brain (van Praag et al. 2002). In our study, compared with
+
+Neurotox Res (2018) 34:188–197
+
+SYN normalized to β-actin (n = 5 per group). Data are expressed as the mean ± SD. One-way ANOVA: BDNF F = 7.24, P = 0.0063; TrkB F = 17.92, P = 0.0002; PSD-95 F= 7.84, P = 0.0066; SYN F = 7.357, P = 0.0082; *P < 0.05 versus C group, **P < 0.01 versus C group, ***P < 0.001 versus C group, #P < 0.05 versus 2.4% sevo group, and ##P < 0.05 versus 2.4% sevo group. C, control; 1.2% sevo, 1.2% sevoflurane-exposed group; 2.4% sevo, 2.4% sevoflurane-exposed group
+
+expression in the C group and the 2.4% sevo group, hippo- campal BDNF and TrkB protein expression in the 1.2% sevo group was prominently increased after exposure to sevoflurane for 28 days. These results suggest a critical role of BDNF signaling and neurogenesis in hippocampus- dependent learning and memory. In support of this possibility, BDNF signaling has been shown to improve cognitive func- tion (Hariri et al. 2003), and the process of neurogenesis may be a substrate for learning and memory (van Praag et al. 2002). We showed here that the improvement in learning and mem- ory after exposure to low-dose sevoflurane might be linked to neurogenesis via increases in the expression of BDNF and TrkB.
+
+In addition, we observed that 6 h of low-dose sevoflurane exposure could augment the number of synapses and improve the synaptic ultrastructure in the hippocampus. Previous in- vestigations have reported that changes in the number and function of synapses can cause changes in synaptic plasticity and thereby affect learning and memory (Martin et al. 2000). Using TEM to analyze the number and the ultrastructure of synapses in the hippocampus, we found ultrastructural chang- es in hippocampal synapses after low-dose sevoflurane expo- sure. Furthermore, we analyzed the synaptic plasticity by mea- suring two synaptic marker proteins: synaptophysin (a
+
+Neurotox Res (2018) 34:188–197
+
+195
+
+Fig. 4 Effects of sevoflurane on the synaptic ultrastructure of the hippocampus on P35 as visualized by TEM. a–c Representative images show the difference in the number of synapses per unit volume across the three groups (red arrows count the number of synapse). Scale bars = 1 μm. d–f Representative images show the differences in the synaptic
+
+interfaces across the three groups. Scale bars = 200 nm. Magnification is × 13,500 (a–c) and × 37,000 (d–f). Summary data for the experiment are presented in g. **P < 0.001 versus C group; ##P < 0.01 versus 2.4% sevo group. C, control; 1.2% sevo, 1.2% sevoflurane-exposed group; 2.4% sevo, 2.4% sevoflurane-exposed group
+
+presynaptic marker) and PSD-95 (a postsynaptic marker) (Head et al. 2009). The expression levels of synaptophysin and PSD-95 were significantly higher in the 1.2% sevo group than those in the other two groups. Recent studies have dem- onstrated that early exposure to high-dose of sevoflurane can induce neurotoxicity by decreasing the expression of synaptophysin and PSD-95 in the hippocampus (Wang et al. 2013; Zheng et al. 2013) and lead to greater synaptic loss and ultrastructural damage (Amrock et al. 2015). However, these studies noted that high-dose sevoflurane produced neurotoxic effects related to synaptic plasticity damage. Whether the
+
+neuroprotective effect of low-dose sevoflurane is connected to synaptic changes remains unknown. The current study gives us an indication that low-dose sevoflurane exposure exerts a neuroprotective effect on the developing brain and that effect may relate to the improvement in synaptic plasticity.
+
+BDNF is an important, well-studied neurotrophin that carries out a variety of neurotrophic and neuroprotective func- tions in the developing brain (Gray et al. 2013). A consider- able body of research indicates that the role of BDNF signal- ing in hippocampus-dependent learning and memory is
+
+Table 2 Structural parameters of the synaptic interface in the hippocampus (N = 10 synapses)
+
+Groups
+
+C
+
+1.2% sevo
+
+2.4% sevo
+
+P value
+
+PSD thickness (nm) Synaptic cleft width (nm)
+
+32.38 ± 4.69 20.00 ± 1.60
+
+41.39 ± 4.32**** 13.31 ± 1.11****
+
+31.76 ± 3.36 18.64 ± 1.714
+
+P < 0.0001 P < 0.0001
+
+Data are presented as the mean ± SEM N the number of synapses, PSD postsynaptic density ****P < 0.0001 vs. C (one-way ANOVA)
+
+196
+
+important both in humans and in experimental animals (Hariri et al. 2003; Lee et al. 2004; Tyler et al. 2002). Moreover, growing evidence suggests a more nuanced role for BDNF signaling in learning and memory, in which it acts primarily as a facilitator of synaptic plasticity and neuronal survival (Gray et al. 2013). This study showed that BDNF and TrkB protein expression in the hippocampus prominently increased after long-term exposure to low-dose sevoflurane compared with high-dose sevoflurane exposure or no exposure. It is plausible that increased hippocampal expression of BDNF and TrkB may play a mechanistic role in the behavioral per- formance improvement induced by low-dose sevoflurane. Therefore, we hypothesize that the observed improvement in neurogenesis and synaptic plasticity may be connected with BDNF expression and TrkB signaling.
+
+Our study has some limitations. First, our experiment just observed a phenomenon and tendency that the improvement both in cognitive function and in neurogenesis/synaptic plas- ticity followed by 1.2% sevoflurane exposure; we did not demonstrate a definite connection between them. This limita- tion may weaken our evidence regarding the causal link be- tween cognitive function and hippocampal neurogenesis/ synaptic plasticity. This is the first step that we have observed the tendency, but the exact mechanism needs more investiga- tions, which will be also reported by related articles in future. Second, we did not investigate the effects of low-dose sevoflurane on other domains of cognitive function; we fo- cused only on learning and memory function because it is the major domain of cognitive function. However, the data from the current study suggest that low-dose sevoflurane exposure could facilitate learning and memory; this possibility merits further studies to undercover the underlying mechanisms.
+
+In conclusion, our findings demonstrate that neonatal ex- posure to low-dose sevoflurane improves hippocampus- dependent learning and memory later in life. This effect may be connected to improved hippocampal neurogenesis and syn- aptic plasticity. If exposure of young patients to lower doses of sevoflurane can promote learning and memory, the selection of this anesthetic and dose range can serve as a new strategy to improve outcomes for children who must undergo anesthesia. However, further clinical studies will need to confirm this possibility.
+
+Funding Information This work was supported by the grants from the National Natural Science Foundation of China (No. 81571032 to Xia Feng and No. 81701047 to Xue Zhou).
+
+Compliance with Ethical Standards
+
+The use of rats in this study was approved by the Institutional Animal Care and Use Committee at Sun Yat-sen University (Guangzhou, China). All experiments were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and ARRIVE guidelines.
+
+Neurotox Res (2018) 34:188–197
+
+Conflict of Interest The authors declare that they have no conflict of interest.
+
+References
+
+Amrock LG, Starner ML, Murphy KL, Baxter MG (2015) Long-term effects of single or multiple neonatal sevoflurane exposures on rat hippocampal ultrastructure. Anesthesiology 122(1):87–95. https:// doi.org/10.1097/ALN.0000000000000477
+
+Callaway JK, Jones NC, Royse AG, Royse CF (2012) Sevoflurane anes- thesia does not impair acquisition learning or memory in the Morris water maze in young adult and aged rats. Anesthesiology 117(5): 1091–1101. https://doi.org/10.1097/ALN.0b013e31826cb228 Chen C, Shen FY, Zhao X, Zhou T, Xu DJ, Wang ZR, Wang YW (2015) Low-dose sevoflurane promotes hippocampal neurogenesis and fa- cilitates the development of dentate gyrus-dependent learning in neonatal rats. ASN neuro 7(2):175909141557584. https://doi.org/ 10.1177/1759091415575845
+
+Fang F, Song R, Ling X, Peng M, Xue Z, Cang J (2017) Multiple sevoflurane anesthesia in pregnant mice inhibits neurogenesis of fetal hippocampus via repressing transcription factor Pax6. Life Sci 175:16–22. https://doi.org/10.1016/j.lfs.2017.03.003
+
+Feng X, Liu JJ, Zhou X, Song FH, Yang XY, Chen XS, Huang WQ, Zhou LH, Ye JH (2012) Single sevoflurane exposure decreases neuronal nitric oxide synthase levels in the hippocampus of developing rats. Br J Anaesth 109(2):225–233. https://doi.org/10.1093/bja/aes121 Goa KL, Noble S, Spencer CM (1999) Sevoflurane in paediatric anaes-
+
+thesia: a review. Paediatr Drugs 1:127–153
+
+Gray JD, Milner TA, McEwen BS (2013) Dynamic plasticity: the role of glucocorticoids, brain-derived neurotrophic factor and other trophic factors. Neuroscience 239:214–227. https://doi.org/10.1016/j. neuroscience.2012.08.034
+
+Hariri A, Goldberg T, Mattay V, Kolachana B, Callicott J, Egan M, Weinberger D (2003) Brain-derived neurotrophic factor val(66)met polymorphism affects human memory-related hippocampal activity and predicts memory performance. J Neurosci 23:6690–6694 Haseneder R, Kratzer S, von Meyer L, Eder M, Kochs E, Rammes G (2009) Isoflurane and sevoflurane dose-dependently impair hippo- campal long-term potentiation. Eur J Pharmacol 623(1-3):47–51. https://doi.org/10.1016/j.ejphar.2009.09.022
+
+Head E, Corrada MM, Kahle-Wrobleski K, Kim RC, Sarsoza F, Goodus M, Kawas CH (2009) Synaptic proteins, neuropathology and cog- nitive status in the oldest-old. Neurobiol Aging 30(7):1125–1134. https://doi.org/10.1016/j.neurobiolaging.2007.10.001
+
+Ishizeki J, Nishikawa K, Kubo K, Saito S, Goto F (2008) Amnestic concentrations of sevoflurane inhibit synaptic plasticity of hippo- campal CA1 neurons through gamma-aminobutyric acid-mediated mechanisms. Anesthesiology 108:447–456
+
+Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF (2003) Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci : Off J Soc Neurosci 23(3):876–882
+
+Kee N, Sivalingam S, Boonstra R, Wojtowicz JM (2002) The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis. J Neurosci Methods 115(1):97–105. https://doi.org/10.1016/S0165- 0270(02)00007-9
+
+Lee J, Duan W, Mattson MP (2002) Evidence that brain-derived neuro- trophic factor is required for basal neurogenesis and mediates, in part, the enhancement of neurogenesis by dietary restriction in the hippocampus of adult mice. J Neurochem 82:1367–1375
+
+Neurotox Res (2018) 34:188–197
+
+Lee JL, Everitt BJ, Thomas KL (2004) Independent cellular processes for hippocampal memory consolidation and reconsolidation. Science (New York, NY) 304:839–843
+
+Lee S, Chung W, Park H, Park H, Yoon S, Park S, Park J, Heo JY, Ju X, Yoon SH, Kim YH, Ko Y (2017) Single and multiple sevoflurane exposures during pregnancy and offspring behavior in mice. Paediatr Anaesth 27(7):742–751. https://doi.org/10.1111/pan.13139 Liang G, Ward C, Peng J, Zhao Y, Huang B, Wei H (2010) Isoflurane causes greater neurodegeneration than an equivalent exposure of sevoflurane in the developing brain of neonatal mice. Anesthesiology 112(6):1325–1334. https://doi.org/10.1097/ALN. 0b013e3181d94da5
+
+Loepke AW, Soriano SG (2008) An assessment of the effects of general anesthetics on developing brain structure and neurocognitive func- tion. Anesth Analg 106(6):1681–1707. https://doi.org/10.1213/ane. 0b013e318167ad77
+
+Lu B, Nagappan G, Guan X, Nathan PJ, Wren P (2013) BDNF-based synaptic repair as a disease-modifying strategy for neurodegenera- tive diseases. Nat Rev Neurosci 14(6):401–416. https://doi.org/10. 1038/nrn3505
+
+Martin SJ, Grimwood PD, Morris RG (2000) Synaptic plasticity and memory: an evaluation of the hypothesis. Annu Rev Neurosci 23(1):649–711. https://doi.org/10.1146/annurev.neuro.23.1.649 Mellon RD, Simone AF, Rappaport BA (2007) Use of anesthetic agents in neonates and young children. Anesth Analg 104(3):509–520. https://doi.org/10.1213/01.ane.0000255729.96438.b0
+
+Nie H, Peng Z, Lao N, Dong H, Xiong L (2013) Effects of sevoflurane on self-renewal capacity and differentiation of cultured neural stem cells. Neurochem Res 38:1758–1767
+
+Payne RS, Akca O, Roewer N, Schurr A, Kehl F (2005) Sevoflurane- induced preconditioning protects against cerebral ischemic neuronal damage in rats. Brain Res
+
+Sairanen M, Lucas G, Ernfors P, Castren M, Castren E (2005) Brain- derived neurotrophic factor and antidepressant drugs have different but coordinated effects on neuronal turnover, proliferation, and sur- vival in the adult dentate gyrus. J Neurosci : Off J Soc Neurosci 25: 1089–1094
+
+Tao G, Luo Y, Xue Q, Li G, Tan Y, Xiao J, Yu B (2016) Docosahexaenoic acid rescues synaptogenesis impairment and long-term memory def- icits caused by postnatal multiple sevoflurane exposures. Biomed Res Int 2016:4062579
+
+Tozuka Y, Fukuda S, Namba T, Seki T, Hisatsune T (2005) GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells. Neuron 47:803–815
+
+Tyler WJ, Alonso M, Bramham CR, Pozzo-Miller LD (2002) From ac- quisition to consolidation: on the role of brain-derived neurotrophic factor signaling in hippocampal-dependent learning. Learning Mem (Cold Spring Harbor, NY) 9:224–237
+
+van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD, Gage FH (2002) Functional neurogenesis in the adult hippocampus. Nature 415(6875):1030–1034. https://doi.org/10.1038/4151030a
+
+Wang SQ, Fang F, Xue ZG, Cang J, Zhang XG (2013) Neonatal sevoflurane anesthesia induces long-term memory impairment and decreases hippocampal PSD-95 expression without neuronal loss. Eur Rev Med Pharmacol Sci 17:941–950
+
+Yang Z, Lv J, Li X, Meng Q, Yang Q, Ma W, Li Y, Ke ZJ (2017) Sevoflurane decreases self-renewal capacity and causes c-Jun N- terminal kinase-mediated damage of rat fetal neural stem cells. Sci Rep 7:46304. https://doi.org/10.1038/srep46304
+
+Zheng H, Dong Y, Xu Z, Crosby G, Culley DJ, Zhang Y, Xie Z (2013) Sevoflurane anesthesia in pregnant mice induces neurotoxicity in fetal and offspring mice. Anesthesiology 118(3):516–526. https:// doi.org/10.1097/ALN.0b013e3182834d5d
+
+197

new_pdfs/10.1016_S1995-7645(14)60066-3.txt

@@ -0,0 +1,605 @@
+Asian Pacific Journal of Tropical Medicine (2014)407-411
+
+Contents lists available at ScienceDirect
+
+Asian Pacific Journal of Tropical Medicine
+
+journal homepage:www.elsevier.com/locate/apjtm
+
+Document heading
+
+doi: 10.1016/S1995-7645(14)60066-3
+
+Effect of propofol and ketamine anesthesia on cognitive function and immune function in young rats Yan-Li Cao, Wei Zhang*, Yan-Qun Ai, Wen-Xia Zhang, Yi Li
+
+Department of Anesthesiology, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China
+
+A R T I C L E I N F O A B S T R A C T
+
+Article history:
+
+Received 10 November 2013 Received in revised form 15 December 2013 Accepted 15 February 2014 Available online 20 May 2014
+
+Keywords:
+
+Propofol Ketamine Cognitive function Immune function
+
+Objective:
+
+To investigate the effects of propofol and ketamine on the cognitive function and A total of 80 young rats were randomly divided into immune function in young rats. four groups: Control group, ketamine group (experimental group A), propofol group (experimental group B), ketamine and propofol group (experimental group C). All rats had continuous injection for three times, serum IL-2, IL-4 and IL-10 and whole brain IL-1毬 level, hippocampal neuronal apoptosis level were measured. The cognitive ability in rats was tested by water maze. Results: Water maze test showed on the 1st d, the maze test latency of the control group, the experimental group B and the experimental group C water were decreased gradually; Compared with the control group after 3 days, the latency of the experimental group A, experimental group B and experimental group C were all decreased, the crossing circle times were also reduced. Hippocampal neuron apoptosis were (2.3依1.7)%, (14.7依6.9)%, (4.2依3.3)%, (10.2依4.8)% in control group, experimental group A, experimental group B and experimental group C, respectively. The neurons apoptosis of experimental group A was significantly increased. The serum IL-4 and IL- 10 of the experimental group A, experimental group B and experimental group C after anesthesia were significantly higher than the control group. The whole brain IL-1毬 of the experimental group A, experimental group B and experimental group C were significantly lower than the control Propofol can reduce anesthesia effect of ketamine on the cognitive function group. and immune function in the young rats.
+
+Method:
+
+Conclusions:
+
+1. Introduction
+
+Intravenous general anesthetics not only has good analgesic effect, but also has no significant side effects on inhibiting respiration, which is widely used in clinical surgical treatment[1]. The clinical application showed that the use of general anesthesia can cause recent cognitive impairment and mental ill effect and other adverse reactions. Neonatal ’ and infant s central nervous system and immune system are still in the developmental stage and particularly sensitive to the external environment. The mechanisms of anesthesia
+
+effects on the central nervous system and immune system are still not very clear[2]. Therefore, this experiment aims to provide the basis for clinical by the application of ketamine and the composite application of propofol and ketamine on the cognitive function and immune function in young rats.
+
+2. Materials and methods
+
+2.1. Animals
+
+Corresponding author: Wei Zhang, Chief Physician, Department of Anesthesiology,
+
+First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China. Tel: 15038068026 E-mail: caoyanli530@163.com Foundation project: It is supported by Youth Innovation Fund of The First Affiliated Hospital of Zhengzhou University (2012-2015) and National Natural Science Foundation(81200909).
+
+A total of 80 healthy 7-day-old SD rats, male or female, weighing 12-18 g were selected. All animals were provided by XX University Experimental Animal Center, and were kept in a constant temperature 25 , constant humidity 40% -50% environment, and had freely drank autoclaved water.
+
+℃
+
+407
+
+Yan-Li Cao et al./Asian Pacific Journal of Tropical Medicine (2014)407-411
+
+408 2.2. Main reagents and instruments
+
+Optical microscope was purchased from Japanese Nikon company,German Leica Microtome was purchased from Dalian Dajian Medical Devices Co., Ltd., Micro pipette and homogenizer were purchased from the German Eppendorf Company, -80 refrigerator were purchased from China Haier Company. TUNEL assay kit was purchased from Roche Company, IL-4, IL-1毬 and IgE radioimmunoassay kit were purchased from Wuhan Boster Biological Engineering Co., Ltd., Ketamine (100 mg/10 mL) were purchased from Jiangsu Hengrui Limited Company, propofol injection (200 mg/20 mL) were purchased from Sichuan Shule Pharmaceutical Corporation. Experimental animal cages, precision electronic balance, 0.9% saline solution, hematoxylin, eosin staining solution were provided by the laboratory.
+
+℃
+
+2.3. Experimental methods
+
+2.3.1. Experimental animal model and grouping methods
+
+A total of 80 young rats were randomly divided into four groups (the control group, experimental group A, =20). All experimental group B, experimental group C) ( young rats received the adaptive breeding for 1 week in animal room. The animals in the control group received 0.9% saline l mL by intraperitoneal injection every 2 h, continuous for 3 times. The animals in experiment group A received 80 mg/kg ketamine l mL by intraperitoneal injection every 2 h, continuous for 3 times. The animals in experiment group B received 80 mg/kg propofol 1 mL by intraperitoneal injection every 2 h, continuous for 3 times. The animals in experiment group C received 80 mg/kg ketamine and propofol 1 mL by intraperitoneal injection every 2 h, continuous for 3 times. The injection volume was 1 mL, and if it was less than l mL it was supplemented by saline. Half of rats in each group were randomly sacrificed after 15 min of anesthesia, the other half underwent Morris water maze test 3 weeks later. All died or abandoned animals in midway were supplemented by modeling again.
+
+n
+
+selected. The heart was exposed by thoracotomy, the perfusion needle was inserted to the ascending aorta from the left ventricle, and fixed. The right auricle was cut. It was washed at 4 saline by perfusion needle until the effluent of the right atrium was clear. Then it was fixed by 4% paraformaldehyde phosphate buffer. Hippocampal was isolated from the brain tissue when the body tissues and organs were hard, they were paraffin-embedded and cut. Neuronal apoptosis detection was performed by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) method. TUNEL-positive cells showed brown particles in the nucleus. Six horizons were randomly selected and average optical density was measured. Positive intensity and the apoptotic index were calculated. × The formula was as follow: apoptotic index (AI) = MOD Area% 100, MOD represents the average gray level; area% represents the percentage of the total positive nucleus area in the total nucleus area. The other half young rats cerebral was obtained quickly by sterile opening cranium, and brain tissue was mixed with ice normal saline by homogenizer. 10% brain homogenate was prepared at 4 , and centrifuged at 3 000 r/min for 15 min. The supernatant was stored at -80 for test. Whole brain IL-1毬 levels were detected by ELISA.
+
+℃
+
+×
+
+℃
+
+℃
+
+2.3.4. Morris water maze test
+
+Behavior of rats was observed by Morris water maze[3]. Round tank has four quadrants. A black platform was fixed at the fourth quadrant, located 1 cm underwater. The rats were put into the water of a randomly select quadrant, swim tracks of the rats were recorded with a camera. How long rats find the platform is the latency. After this test, the platform was removed and the rats were put into water from the same water-entering point, the times of crossing the former platform were measured.
+
+2.4. Statistical analysis
+
+2.3.2. Immune parameters detection
+
+Using heparinization disposable 5 mL sterile syringe, 2 mL blood was obtained by percutaneous puncture at the point of maximal impulse and then it was injected into sterile EP , it was centrifuged at 3 000 r/min at tube. After 30 min at 4 low temperature for 10 min. Serum was separated and stored at -80 for the test. Serum IL-2, IL-4 and IL-10 levels were detected by ELISA.
+
+℃
+
+℃
+
+Data were expressed as mean依SD values and analyzed with SPSS 13.0 software. After the variance test, the difference between two groups was compared with single factor analysis <0.05 was considered as statistical significant of variance. difference.
+
+P
+
+3. Results
+
+2.3.3. Brain tissue specimen collection, preparation and indicators test
+
+3.1. Serum IL-2, IL-4 and IL-10 and whole brain IL-1毬 level
+
+After blood collection, half of the young rats were randomly
+
+The serum IL-2 in each groups showed no significant
+
+Yan-Li Cao et al./Asian Pacific Journal of Tropical Medicine (2014)407-411
+
+P
+
+>0.05). The serum IL-4 and IL-10 of the difference ( experimental group A, experimental group B and experimental group C were significantly higher than <0.05). There was significant the normal control group ( difference in the serum IL-4 and IL-10 levels between experimental group C and experimental group A ( <0.05). The whole brain IL-1毬 level of the experimental group A, experimental group B and experimental group C were significantly lower than the normal control group ( <0.05). There was significant difference in whole brain IL-1毬 level P between experimental group C and experimental group A ( <0.05) (Table 1).
+
+P
+
+P
+
+P
+
+in the experimental group A, experimental group B and experimental group C, which was statistically significant different from that of the control group ( <0.05). Compared with the experimental group A, the latency on the 3rd day was significant different from that of the experimental group B and experimental group C ( <0.05). Compared with the control group, there was significant difference in the crossing circle times from experimental group A, experimental group B and experimental group C ( <0.05). Compared with the experimental group A, the crossing circle times of the there was significant difference in the crossing circle times from experimental group B and experimental group C ( <0.05) (Table 3).
+
+P
+
+P
+
+P
+
+P
+
+3.2. Hippocampal neurons apoptosis
+
+Hippocampal neurons apoptosis of the experimental group A was (14.7依6.9)%, which was significantly increased than <0.05); The apoptosis that of the control group [(2.3依1.7)%] ( rate of the experimental group B was (4.2依3.3)%, which >0.05), but had no significant difference from NS group ( significantly lower than that of experimental group A ( <0.05); The apoptosis rate of the experimental group C was (10.2依 4.8)%, which was significantly increased compared with the control group ( <0.05), but significantly decreased compared with the experimental group A (
+
+P
+
+P
+
+P
+
+P
+
+P
+
+<0.05).
+
+3.3. Rat behavior observation
+
+With the changes of time, the latency of control group, experimental group B and experimental group C were decreased gradually, there was significant difference between that on 1st d and 3rd d ( <0.05). The latency of experimental group A was not changed significantly P >0.05); The latency after 3 days gradually decreased (
+
+P
+
+4. Discussion
+
+Postoperative cognitive dysfunction attracts increasing attention. Numerous studies have shown the use of narcotic drugs is closely related the understanding dysfunction. Hippocampal neuron is considered to be major neurons which involved in long-term memory. Damage in the neurons synapse structure can significantly affect the ability of learning and memory in rats. Postsynaptic membrane receptor also involved in cognitive function of rats as an important information carrier, and that the pathogenesis of Alzheimer s patients is related with the decreased expression of receptor [4-11]. The immune changes under ’ attention. anesthetized stress of rats also attract researchers Anesthetic ketamine and propofol were involved in the regulation of the central nervous system by inhibiting postsynaptic membrane receptors, but the changes of the cognitive function and immune function and the mechanism is still not clear. Thus we anesthetized the young rats in this
+
+’
+
+Table 1
+
+Serum IL-2, IL-4 and IL-10 and whole brain IL-1毬 level of rats. Indexes IL-2 (ng/mL) IL-4 (ng/mL) IL-10 (ng/mL) IL-1毬 (ngL) Note: * Compared with the control group,
+
+Control group 0.5依0.1 0.4依0.1 0.7依0.2 115.4依15.3 P
+
+Experimental group A 0.4依0.1 * 1.5依0.5 * 3.1依0.8 * 93.1依10.2
+
+△
+
+<0.05,
+
+Compared with experimental group A,
+
+eExperimental group B 0.4依0.1 * 1.3依0.5 * 2.6依0.6 * 97.1依18.6
+
+P
+
+<0.05.
+
+Experimental group C 0.4依0.1 △ * 1.0依0.3 △ * 1.8依0.5 △ * 112.8依18.4
+
+Table 2
+
+Water maze test results.
+
+Indexes
+
+Control group Experimental group A Experimental group B Experimental group C
+
+Note: * Compared with the control group,
+
+1 d 112.7依24.4 115.0依24.5 114.2依26.2 109.9依25.3 P
+
+<0.05,
+
+△
+
+Latency(s) 2 d 89.5依15.4 105.9依20.6 92.6依16.8 90.3依20.4
+
+Compared with experimental group A,
+
+P
+
+3 d * 53.2依12.1 △ 104.3依17.3 △ * 65.0依13.1 △ * 72.4依11.6
+
+<0.05.
+
+Crossing circle times
+
+6.2依2.3 1.2依0.6 3.5依2.1 3.2依2.2
+
+△
+
+△
+
+△
+
+409
+
+Yan-Li Cao et al./Asian Pacific Journal of Tropical Medicine (2014)407-411
+
+410
+
+study and then explore the effect of ketamine and propofol on the cerebral development and the immune system. As a classical neurological behavior methods, morris water maze has become the standard mode to study the memory mechanism[12]. Therefore, this experiment also chose this classic mode to explore the effect of anesthetics on the cognitive function in rats. This study showed that the latency at three days is not significantly shorter after the use of anesthetics ketamine, and the times of cross the flat is far less than the normal control group, which suggested the ketamine anesthesia can reduce the spatial learning and memory ability in young rats .The latency at three days is significantly shorter than 1 day of young rats after the use of anesthetics propofol, but the exploration time was significantly longer than the normal control group. The times of cross the flat is significantly increased than the normal control group, which suggested the ketamine anesthesia can reduce the spatial learning and memory ability in young rats, but the degree is significantly less than the ketamine group. Studies have found that ketamine abusers have a long damaged memory, and its mechanism is related to damaged hippocampal neurons, which prompt some neuronal apoptosis of rats. Once propofol does not have an impact on cognitive function in rats , but the use of many drugs can cause propofol nerve degeneration and affect the brain development[13-18]. The spatial learning and memory ability in young rats of ketamine & propofol group is similar to the propofol group, the latency is significantly shorter than the ketamine group, and the times of cross the flat is also significantly increased, which suggested that the ketamine & propofol anesthesia can reduce the effect ketamine on cognitive function. The apoptosis rate of ketamine & propofol group is significantly lower than the ketamine group by the study of hippocampal neuronal apoptosis, which is accordance with the change of spatial learning and memory ability in young rats. That showed propofol can reduce ketamine-induced apoptosis in hippocampal neurons. These results indicate that the mechanism that propofol can ’ cognitive reduce the effect of ketamine anesthesia on rats lies in hippocampal neuronal apoptosis. With the increase in hippocampal neuronal apoptosis, the long-term learning and memory dysfunction are more obvious[19-23]. Numerous studies also believes that the use of propofol in humans and animals have the cerebral protective effect. Its protective mechanism may be related with reducing cerebral metabolic rate of oxygen and intracranial pressure and reducing excitatory amino acid glutamate neurotransmitter release, blocking glutamate pathways, reducing neurotoxicity induced pathological damage, the lipid peroxidation effect, preventing protein denaturation and release of inflammatory
+
+mediators and preventing secondary damage neuronal cells; reducing the formation of free radicals[24-26]. Immunity is one of the main effects of the stress response. Immune function in young rats still in the developmental stage, which is sensitive the stimulation of external factors. Generally it is believed that the impact of stress on the immune system is mainly suppression and regulation. T lymphocytes are the most important component which constitute the immune system. Good immune function of the body needs to maintain a moderate level of response and homeostasis. Th1 cells and Th2 cells is critical in maintaining the balance of immune function. Once there is Th cell subsets imbalance, that is Thl/Th2 ratio and functional imbalance, will lead to immune dysfunction[27,28], which usually expressed as the abnormal secretion of the activation factor of various inflammatory cell. This study suggests that the anesthetic of each group has little effect on IL-2 secretion, but the serum IL-4 and IL-10 were significantly increased, while the whole brain IL-1毬 were significantly decreased. Studies suggest that stress response of the adult rats is different from that in the young rats. Adult rats have a strong adaptability,while the young rats have long-lasting effects to the stress response and have a continuing influence. IL-1毬 is a cytokines produced by a variety of cells in a infection and inflammation state, which have a wide range of physiological effects. It often called the central stress-mediated factors.When there is systemic stress responses, central IL-1毬 may showed high expression[29,30]. In summary, ketamine can inhibit the cognitive function in young rats and has toxic effects on hippocampal neurons. In combination with propofol, it can reduce neurotoxicity and protect the brain tissue. Ketamine can inhibit the immune function in young rats, while propofol can reduce ’ s inhibition to the immune function .However, the ketamine there are still some gaps between the animal studies and human clinical trials. If it is consistent with human trials still need further research.
+
+Conflict of interest statement
+
+We declare that we have no conflict of interest.
+
+References
+
+[1] Chen S. Clinical comparative study of propofol and isoflurane Strait
+
+on elderly patientscognitive function after surgery. Pharmaceutical J
+
+24
+
+2012;
+
+(6): 127-128.
+
+[2] Gan RH, Xu LL, Xie AQ, Tian S, Yang ZY. Influence of isoflurane
+
+Yan-Li Cao et al./Asian Pacific Journal of Tropical Medicine (2014)407-411
+
+411
+
+and propofol on cognitive of aged patients. 23
+
+(6): 1104-1106.
+
+Western Med
+
+2011;
+
+once or repeatedly on the brain against global cerebral ischemia-
+
+Chin J Anesthesiol
+
+26
+
+reperfusion injury in rats.
+
+2006;
+
+(8): 720-
+
+[3] Sun HJ, Liang SJ, Zhang LW. Effects of propofol and isoflurane J
+
+anesthesia on elderly patients cognitive function after surgery. Knotty
+
+9
+
+2010;
+
+(10): 773-774.
+
+[4] Marsden KC, Shemesh A, Bayer KU, Carroll RC. Selective
+
+2+
+
+translocation of Ca 栻alpha) to inhibitory synapses. 107
+
+/calmodulin protein kinase IIalpha (CaMK
+
+Proc Natl Acad Sci USA
+
+2010;
+
+723.
+
+[19] Zhao DM, Li QC, Diao HL, Liu HF, Wang LM, Huang F. Effects
+
+of propofol pretreatment on learning and memory ability and J
+
+’
+
+hippocampus injury of Alzheimer Binzhou Med School
+
+s disease-like model rats.
+
+30
+
+2007;
+
+(6): 407-411.
+
+[20] Erasso DM, Camporesi EM, Mangar D, Saporta S. Effects of
+
+(47): 20559-20564.
+
+[5] Gustin RM, Shonesy BC, Robinson SL, Rentz TJ, Baucum AJ,
+
+isoflurane or propofol on postnatal hippocampal eurogenesis in
+
+Brain Res
+
+1530
+
+young and aged rats.
+
+2013;
+
+: 1-12.
+
+Jalan-Sakrikar N, et al. Loss of Thr286 phosphorylation disrupts synaptic CaMK栻 毩 targeting, NMDAR activity and behavior in pre-adolescent mice.
+
+Mol Cell Neurosci
+
+47
+
+2011;
+
+(4): 286-292.
+
+[6] Chen NM, Ding L. Propofol to the elderly colon cancer S100毬 Chin Rural
+
+expression and cognitive function after radical surgery. Med
+
+19
+
+2012;
+
+(8): 13-14.
+
+[21] Mirski MA, Lewin JJ, Ledroux S, Thompson C, Murakami
+
+P, Zink EK, et al. Cognitive improvement during continuous
+
+sedation in critically ill, awake and responsive patients: the Acute Intensive Care Med
+
+Neurological ICU Sedation Trial (ANIST).
+
+36
+
+2010;
+
+(9): 1505-1513.
+
+[22] Erasso DM, Chaparro RE, Quiroga Del Rio CE, Karlnoski R,
+
+[7] Xu XD, Zhang LC. Experimental research of propofol on learning
+
+Chin Pract Med
+
+8 (12): 7-9. 2013;
+
+and memory function.
+
+[8] Wu D. The influence of Sevoflurane and propofol general
+
+Camporesi EM, Saporta S. Quantitative assessment of new cell
+
+proliferation in the dentate gyrus and learning after isoflurane 2 ;
+
+Brain Res
+
+orpropofol anesthesia in young and aged rats.
+
+2012;
+
+anesthesia on elderly patients after anesthesia emergence time and
+
+Chin Pharmaceutical
+
+22
+
+cognitive function.
+
+2013;
+
+(5): 95-96.
+
+ë te S, Leydet J, Echenne B, Rivier F, et al. [9] Meyer P, Langlois C, So Unexpected neurological sequelae following propofol anesthesia in Brain Dev
+
+32
+
+infants: Three case reports.
+
+2010;
+
+(10): 872-878.
+
+1441: 38-46.
+
+[23] Schoen J, Husemann L, Tiemeyer C, Lueloh A, Sedemund-
+
+Adib B, Berger KU, et al. Cognitive function after sevoflurane- vs
+
+. propofol-based anaesthesia for on-pump cardiac surgery: a Br J Anaesth
+
+106
+
+randomized controlled trial.
+
+2011;
+
+(6): 840-850.
+
+[10] Xiao J,Zheng LM, Wang ML, et al. Effects of disoprofol and
+
+[24] Soukup J, Selle A, Wienke A, Steighardt J, Wagner NM, Kellner
+
+isoflurane on postoperative cognitive dysfunction in the elderly. Chin Modern Doctor
+
+48
+
+2010;
+
+(11): 80-81.
+
+P. Efficiency and safety of inhalative sedation with sevoflurane in
+
+comparison to an intravenous sedation concept with propofol in
+
+[11] Sharma S, Rakoczy S, Brown-Borg H. Assessment of spatial
+
+Life Sci
+
+23
+
+memory in mice.
+
+2010;
+
+(17-18): 521-536.
+
+intensive care patients: study protocol for a randomized controlled
+
+Trials
+
+10
+
+trial.
+
+2012;
+
+(13): 135.
+
+[12] Jiang Y, Zhou ZJ, Chen P, Huang F, Xian YS, Wang YL, et
+
+[25] Lewis LD, Weiner VS, Mukamel EA, Donoghue JA, Eskandar EN,
+
+al.Effects of multiple doses of propofol on cognitive function in
+
+Chin J Anesthesiol
+
+28
+
+neonatal rats.
+
+2008;
+
+(11): 1007-1009.
+
+[13] Jiang Y, Gao J, Chen P, Li QY. Effects of propofol on pyramidal J Chongqing
+
+neuron apoptosis in hippocampus of neonatal rats. Med Univ
+
+34
+
+2009;
+
+(3): 304-307.
+
+[14] Xiao B, Han F, Shi YX. Expression changes of CaMKII 毩 and 2009;
+
+Progr Anatomical Sci
+
+pCaMKII in the PTSD rat amygdala. 15
+
+(4): 396-399.
+
+[15] Chen YF. The effect of propofol on cell apoptosis after freezing
+
+Xianning Coll J
+
+21
+
+injury rats.
+
+2007;
+
+(6): 474-475.
+
+Madsen JR, et al. Rapid fragmentation of neuronal networks at the Proc Natl Acad Sci
+
+onset of propofol-induced unconsciousness. USA
+
+109
+
+2012;
+
+(49): E3377-E3386.
+
+[26] Ye X, Lian Q, Eckenhoff MF, Eckenhoff RG, Pan JZ. Differential PLoS
+
+general anesthetic effects on microglial cytokine expression. One
+
+8 (1): e52887. 2013;
+
+[27] Erdogan MA, Demirbilek S, Erdil F, Aydogan MS, Ozturk E,
+
+Togal T, et al. The effects of cognitive impairment on anaesthetic
+
+Eur J Anaesthesiol
+
+29
+
+requirement in the elderly.
+
+2012;
+
+(7): 326-
+
+331.
+
+[16] Kuai JK, Han LC, Chai W. The long-term changes and
+
+[28] Luo J, Min S, Wei K, Li P, Dong J, Liu YF. Propofol protects
+
+mechanism of cognitive function after ketamine anesthesia in
+
+Chin Med Sci
+
+16
+
+neonatal rats.
+
+2007;
+
+(9): 789-791.
+
+[17] Song SL, Wang JM. Experimental research about the effect of
+
+against impairment of learning-memory and imbalance of
+
+hippocampal Glu/GABA induced by electroconvulsive shock in
+
+J Anesth
+
+25
+
+depressed rats.
+
+2011;
+
+(5): 657-665.
+
+monosodium gutamate on the ability of learning and memory and
+
+Guangzhou Med
+
+the behaviour in open field in rats.
+
+2008;
+
+39
+
+(1):
+
+[29] Rezaei F, Nasseri K, Esfandiari GR, Sadeghi SM, Fathie M,
+
+Gharibi F. Remifentanil added to propofol for induction of
+
+1-3.
+
+[18] Li YC, Li Y, Wang YS, Li JQ, Li ZX, Li EY, et al. Protective
+
+anesthesia can reduce reorientation time after electroconvulsive
+
+J ECT
+
+28
+
+therapy in patients with severe mania.
+
+2012;
+
+(2): 124-
+
+effects of preconditioning with different doses of propofol given
+
+127.

new_pdfs/10.1016_j.bbrc.2021.03.063.txt

@@ -0,0 +1,259 @@
+Biochemical and Biophysical Research Communications 553 (2021) 65e71
+
+Contents lists available at ScienceDirect
+
+Biochemical and Biophysical Research Communications
+
+j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y b b r c
+
+Maternal anesthesia with sevoflurane during the mid-gestation induces social interaction deficits in offspring C57BL/6 mice Qingcai Chen a, 1, Wei Chu b, 1, Rui Sheng b, Shaoyong Song a, Jianping Yang a, Fuhai Ji a, ** Xin Jin a, * a Department of Anesthesiology, First Affiliated Hospital of Soochow University, Suzhou, 215006, China b Department of Pharmacology, Soochow University School of Pharmaceutical Science, Suzhou, 215123, China
+
+,
+
+a r t i c l e i n f o
+
+a b s t r a c t
+
+Article history: Received 17 January 2021 Accepted 11 March 2021 Available online 20 March 2021
+
+Keywords: Anesthesia Sevoflurane Neurotoxicity Sociability Preference for social novelty Three-chambered social paradigm
+
+Sevoflurane anesthesia in pregnant mice could induce neurotoxicity in the developing brain and disturb learning and memory in the offspring mice. Whether it could impair social behaviors in the offspring mice is uncertain. Therefore, we assessed the neurobehavioral effect of in-utero exposure to sevoflurane on social interaction behaviors in C57BL/6 mice. The pregnant mice were anesthetized with 2.5% sevo- flurane in 100% oxygen for 2 h, and their offspring mice were tested in three-chambered social paradigm, which includes three 10-min sessions of habituation, sociability, and preference for social novelty. At the juvenile age, the offspring mice showed abnormal sociability, as proved by not taking more time sniffing at the stranger 1 mouse compared with the empty enclosure (108.5 ± 25.4 vs. 108.2 ± 44.0 s, P ¼ 0.9876). Meanwhile, these mice showed impaired preference for social novelty, as evidenced by not taking more time sniffing at the stranger 2 compared with the stranger 1 mouse (92.1 ± 52.2 vs. 126.7 ± 50.8 s, P ¼ 0.1502). At the early adulthood, the offspring mice retrieved the normal sociability (145.6 ± 33.2 vs. 76.0 ± 31.8 s, P ¼ 0.0001), but remained the impaired preference for social novelty (100.6 ± 29.3 vs. 118.0 ± 47.9 s, P ¼ 0.3269). Collectively, these results suggested maternal anesthesia with sevoflurane could induce social interaction deficits in their offspring mice. Although the disturbance of sociability could be recoverable, the impairment of preference for social novelty could be long-lasting.
+
+© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+1. Introduction
+
+Preclinical studies suggested that anesthetic agents could induce the neurotoxicity in developing brain and cause neurobehavioral changes in adulthood [1e4]. Anesthetic-induced developmental neurotoxicity could be attributed to multiple factors, including anesthetic agents, anesthesia regimen (i.e., concentration and dura- tion), and brain vulnerability [2,5]. Sevoflurane is commonly used in parturient undergoing non-obstetric surgery, which incurs many concerns on neurodevelopmental consequences.
+
+environmental toxicants including anesthetic agents should play an important role [8,9]. It was reported that in-utero exposure to isoflurane could impair the spatial working memory of the rat [5]. Therefore, we hypothesized that in-utero exposure to sevoflurane could induce social interaction deficit in offspring C57BL/6 mice. To verify this possibility, we performed maternal anesthesia with sev- oflurane in pregnant mice on gestational day 14. Next, we conducted social interaction test in their offspring mice at one- and two-month- old. The main objective was to determine whether in-utero exposure to sevoflurane was induce social interaction deficit in offspring mice.
+
+In recent years, children with autistic spectrum disorders are increasing. The core symptom of autism is social interaction deficit [6,7], but the neuropathological mechanism remains uncertain. The combined effect of genetic predisposition and early exposure to
+
+2. Methods and materials
+
+2.1. Animals
+
+Corresponding author. ** Corresponding author.
+
+E-mail addresses: jifuhai@hotmail.com (F. Ji), jinxin@suda.edu.cn (X. Jin).
+
+1 These authors contributed equally to this work (Q.C.C. and W.C.).
+
+This study was approved by the Institutional Animal Care and Use Committee of Soochow University. Adult C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Suzhou, China). One male and four female mice were housed per
+
+https://doi.org/10.1016/j.bbrc.2021.03.063 0006-291X/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+Q. Chen, W. Chu, R. Sheng et al.
+
+cage for breeding the offspring mice. Six pregnant mice on gesta- tional day 14 were randomly assigned to receive either 2.5% sevo- flurane in 100% oxygen or just 100% oxygen as the control. Their offspring mice were correspondingly assigned as the testing mice. Several pregnant mice without any treatment were chosen to produce the offspring mice as the stranger mice. The pups were fostered by their own dams till weaning on postnatal day 21. All mice were raised in a controlled condition (21e22 (cid:2)C, 12 h light/ dark cycle, light on at 7 a.m.), with access to standard mouse chow and water ad libitum.
+
+2.2. Maternal anesthesia
+
+A clinically-retired anesthesia machine was used to supply one- way gas flow. A transparent plastic box (20 L (cid:3) 20 W (cid:3) 6 H cm) was used as the anesthetizing chamber, with three holes for gas inflow, gas outflow, and gas monitoring. A heating-pad was placed un- derneath the anesthetizing chamber to keep mice warm during anesthesia. Sevoflurane anesthesia on pregnant mice in this study was strictly performed by the protocols of previous study [10], in which arterial blood pressure and blood gas analysis were demonstrated within normal limits. The pregnant mice retained spontaneous respiration during inhalational anesthesia. Sevo- flurane was washed out with pure oxygen for 15 min, and the pregnant mice with right reflex were put back to home cages.
+
+2.3. Social apparatus
+
+The three-chambered social box (40L (cid:3) 60W (cid:3) 22H cm) with two enclosures (7D (cid:3) 15H cm) was used for social interaction test (Fig. 1AeC). An improved video-tracking system programed by ANY-maze (Stoelting Co., USA) was used to capture the movement of mouse. Given the testing mouse initiates social approaching to the stranger mouse by nose-to-nose or nose-to-tail sniffing (Fig. 1D), the animal’s head was tracked by the video-tracking system. Four behavioral parameters was automatically measured by ANY-maze program, including the time sniffing at the enclosure and number of sniffs at the enclosure, the time exploring in the side-chamber and number of entries into side chamber. Specifically, “at the enclosure” is defined as the mouse head entering an area of 3 cm around the enclosure.
+
+2.4. Social interaction test
+
+The offspring mice (N ¼ 17 Control, 9 males and 8 females; N ¼ 14 Sevoflurane, 9 males and 5 females) were tested at one- and two-month-old (i.e., the juvenile and early-adult age). In advance, the testing mouse was housed single for 1-h isolation in the behavioral room. The stranger mice were the identical background, same gender and similar age as the testing mouse, and they had exactly no contact before. Social interaction test is composed of three 10-min sessions of habituation, sociability, and preference for social novelty. Firstly, the testing mouse was allowed to freely explore in social box with two doorways opening. Next, an unfa- miliar conspecific (Stranger 1) was introduced into one enclosure, and the testing mouse was allowed to sniff the stranger 1 or explore the empty enclosure (Fig. 1E, G). After that, another unfamiliar conspecific (Stranger 2) was introduced into the other enclosure, and the testing mouse was allowed to sniff the stranger 1 and stranger 2 (Fig. 1F, H). Placement of the stranger 1 on the left or right side was systematically altered between trials, and social apparatus was cleaned after each trial to minimize olfactory disturbance.
+
+66
+
+Biochemical and Biophysical Research Communications 553 (2021) 65e71
+
+2.5. Statistical analysis
+
+Data were shown as Mean ± SD. Graphpad Prism 5.0 software (San Diego, USA) was used for statistical analyses. Social data ob- tained from the left and right side were mutually exclusive in each 10-min session and they were normally distributed by Kolmogorov-Smirnov tests. Therefore, two-tailed paired t-test was used to compare the side preferences (stranger 1 vs. the opposite). Two-way repeated measures (RM) ANOVAs were used to analyze the interaction effects of treatment (control or sevoflurane) (cid:3) side (stranger 1 or the opposite). Based on the preliminary study, a sample size of more than 5 (sociability) and 13 (preference for so- cial novelty) could lead to a 90% power to detect a difference in side preference with 5% type I error. P values less than 0.05 (*), 0.01 (**) and 0.001 (***) were considered statistically significant.
+
+3. Results
+
+3.1. Maternal anesthesia with sevoflurane induced abnormal sociability in the offspring mice at one-month-old
+
+The offspring mice in the control showed normal sociability, as proved by taking more time sniffing (Fig. 2A left, 167.7 ± 55.4 vs. 102.5 ± 35.4 s, P ¼ 0.001) and sniffing more frequently (Fig. 2B left, 42.9 ± 16.2 vs. 27.9 ± 9.1, P ¼ 0.0078) at the enclosure containing the stranger 1, as compared to the empty enclosure. Meanwhile, these testing mice spent more time exploring in the chamber with stranger 1 than in the empty chamber (Fig. 2C left, 298.5 ± 60.7 vs. 215.0 ± 45.8 s, P ¼ 0.0034). However, the offspring mice undergoing in-utero exposure to sevoflurane showed no side preference be- tween the stranger 1 and empty side, in terms of the time sniffing (Fig. 2A right, 108.5 ± 25.4 vs. 108.2 ± 44.0 s, P ¼ 0.9876) or the number of sniffs (Fig. 2B right, 43.2 ± 11.7 vs. 44.4 ± 12.9, P ¼ 0.8089) at the enclosure, or the time exploring in the chamber (Fig. 2C right, 240.2 ± 42.5 vs. 237.5 ± 50.7 s, P ¼ 0.9122).
+
+In addition, two-way ANOVAs showed the significant interaction effects of treatment (control or sevoflurane) (cid:3) side (the stranger 1 or empty side), in terms of the time taken sniffing (Fig. 2A, F ¼ 7.7070, P ¼ 0.0095), the number of sniffs (Fig. 2B, F ¼ 5.3030, P ¼ 0.0287) and the time spent exploring (Fig. 2C, F ¼ 5.4560, P ¼ 0.0266). As for the number of entries into the chamber, the offspring mice in either group displayed no significant differences between two sides (Fig. 3D), which indicated the similar probabilities of exploration in the left and right chamber. Together, these data suggested that maternal anesthesia with 2.5% sevoflurane could induce the socia- bility deficit in their offspring mice at juvenile age.
+
+3.2. Maternal anesthesia with sevoflurane induced abnormal preference for social novelty in the offspring mice at one-month-old
+
+The offspring mice in the control showed normal preference for social novelty, as evidenced by taking more time sniffing (Fig. 2E left, 92.9 ± 33.5 vs. 193.6 ± 61.1 s, P < 0.0001) and sniffing more frequently (Fig. 2F left, 26.7 ± 6.8 vs. 45.6 ± 20.7, P ¼ 0.0013) at the enclosure containing stranger 2, as compared to the stranger 1. Meanwhile, these testing mice spent more time exploring in the chamber with stranger 2 than in the chamber with stranger 1 (Fig. 2G left, 194.5 ± 33.4 vs. 323.8 ± 54.5 s, P < 0.0001). However, the offspring mice undergoing in-utero exposure to sevoflurane showed no side preference between the stranger 2 and stranger 1 mouse, in terms of the time sniffing (Fig. 2E right, 92.1 ± 52.2 vs. 126.7 ± 50.8 s, P ¼ 0.1502) or the number of sniffs (Fig. 2F right, 29.1 ± 12.5 vs. 35.0 ± 11.2, P ¼ 0.2354) at the enclosure, or the time exploring in the chamber (Fig. 2G right, 204.8 ± 77.5 vs. 286.0 ± 88.6 s, P ¼ 0.0785).
+
+Q. Chen, W. Chu, R. Sheng et al.
+
+Biochemical and Biophysical Research Communications 553 (2021) 65e71
+
+Fig. 1. Social interaction test in the three-chambered social box. (A) Schematic view of the three-chambered social box with two video-cameras hung right above two enclosures. (B) Schematic view of two enclosures in the left and right chambers. (C) One testing mouse is exploring in the social box with two doorways opening. (D) One testing mouse is sniffing the stranger mouse in the manner of nose-to-nose or nose-to-tail. (E) One testing mouse shows normal sociability as proved by preferring the stranger 1 mouse to the empty enclosure. (F) The testing mouse shows normal preference for social novelty as evidenced by preferring the stranger 2 to stranger 1 mouse. (G) One testing mouse shows impaired sociability as proved by no side preference between the stranger 1 mouse and the empty enclosure. (H) The testing mouse shows impaired preference for social novelty as evidenced by no side preference between the stranger 2 and stranger 1 mouse. Unit of dimension: millimeters. E ¼ the empty enclosure, S1 ¼ Stranger 1, S2 ¼ Stranger 2.
+
+Additionally, two-way ANOVAs showed the significant interac- tion effect of treatment (control or sevoflurane) (cid:3) side (stranger 2 or stranger 1) in terms of time taken sniffing at the enclosure (Fig. 2E and F ¼ 5.248, P ¼ 0.0294). As for the number of entries into the chamber, the offspring mice in either group displayed no sig- nificant differences between two sides (Fig. 2H), which reflected the equal opportunities of exploration in the left and right chamber. Together, these data suggested that maternal anesthesia with 2.5% sevoflurane could impair the preference for social novelty in their offspring mice at juvenile age.
+
+showed normal sociability, as evidenced by taking more time sniffing the stranger 1 over the empty enclosure (Fig. 3A right, 145.6 ± 33.2 vs. 76.0 ± 31.8 s, P ¼ 0.0001). This recovery of sociability was sup- ported by the number of sniffs at the enclosure (Fig. 3B right, 57.0 ± 16.9 vs. 35.7 ± 6.2, P ¼ 0.0009) and the time spent exploring in the chamber (Fig. 3C right, 305.0 ± 43.2 vs. 185.1 ± 35.7 s, P < 0.0001). In addition, two-way ANOVAs detected no interaction effect of treatment (cid:3) side in either parameter (Fig. 3AeC), which supported the recovery of sociability in a certain extent. Collectively, these data suggested that the offspring mice exposed to sevoflurane in-utero could retrieve normal sociability at early-adulthood.
+
+3.3. The offspring mice exposed to sevoflurane in-utero retrieved normal sociability at two-month-old
+
+The offspring mice undergoing fetal exposure to sevoflurane
+
+67
+
+Q. Chen, W. Chu, R. Sheng et al.
+
+Biochemical and Biophysical Research Communications 553 (2021) 65e71
+
+Fig. 2. The offspring mice undergoing in-utero exposure to sevoflurane show abnormal sociability and impaired preference for social novelty at one-month-old. The offspring mice exposed to sevoflurane in-utero show no side preference between the stranger 1 mouse and the empty side, in terms of time sniffing at the enclosure (A, right), number of sniffs (B, right), and time exploring in the chamber (C, right). Additionally, these offspring mice show no side preference between the stranger 2 and stranger 1 mouse, in terms of time sniffing at the enclosure (E, right), number of sniffs (F, right), and time exploring in the chamber (G, right). As for the number of entries into chamber, there are not significant differences between two sides in the session of either sociability (D) or preference for social novelty (H). Data are expressed as Mean ± SD. N ¼ 17 Control and 14 Sevoflurane. **P < 0.01, ***P < 0.001.
+
+3.4. The offspring mice exposed to sevoflurane in-utero remained abnormal preference for social novelty at two-month-old
+
+The offspring mice undergoing fetal exposure to sevoflurane
+
+showed again no side preference between the stranger 2 and stranger 1 mouse, in terms of the time sniffing (Fig. 3E right, 100.6 ± 29.3 vs. 118.0 ± 47.9 s, P ¼ 0.3269) or the number of sniffs (Fig. 3F right, 42.9 ± 15.6 vs. 52.2 ± 14.1, P ¼ 0.1074) at the enclosure,
+
+68
+
+Q. Chen, W. Chu, R. Sheng et al.
+
+Biochemical and Biophysical Research Communications 553 (2021) 65e71
+
+Fig. 3. The offspring mice undergoing in-utero exposure to sevoflurane retrieve normal sociability, but remained abnormal preference for social novelty at two-month-old. The offspring mice exposed to sevoflurane in-utero take more time sniffing (A, right) and sniff more frequently (B, right) at the enclosure containing stranger 1, and they spend more time exploring in the chamber with stranger 1 (C, right), as compared to the empty side. However, these offspring mice show no side preference between the stranger 2 and stranger 1 mouse, in terms of time sniffing at the enclosure (E, right), number of sniffs (F, right), and time exploring in the chamber (G, right). As for the number of entries into chamber, there are not significant differences between two sides in the session of either sociability (D) or preference for social novelty (H). Data are expressed as Mean ± SD. N ¼ 17 Control and 14 Sevoflurane. *P < 0.05, ***P < 0.001.
+
+or the time exploring in the chamber (Fig. 3G right, 248.7 ± 64.6 vs. 250.7 ± 67.3 s, P ¼ 0.9558). Collectively, these data suggested that the offspring mice exposed to sevoflurane in-utero could not re- covery the normal preference for social novelty at early-adulthood,
+
+indicating that maternal anesthesia with 2.5% sevoflurane might cause the long-term impairment of social memory in offspring mice.
+
+69
+
+Q. Chen, W. Chu, R. Sheng et al.
+
+4. Discussions
+
+Clinical anesthesia facilitates a variety of surgical procedures by bringing patients into an unconscious and painless state, which is usually regarded as a safe and reversible process [11]. However, it may not be the truth in two extremes of life, including the pediatric and geriatric patients [12,13]. A great number of pregnant women are receiving non-obstetric surgeries and fetal intervention pro- cedures under general anesthesia [1]. As many anesthetic agents are lipophilic, they can easily cross placenta and cause fetal expo- sure to anesthetics. Several studies have suggested anesthetic- induced developmental neurotoxicity, ranging from human em- bryonic stem cells in vitro to rodents and non-human primates in vivo [14e18]. It was reported that sevoflurane anesthesia in pregnant mice on gestation day 14 induced the learning and memory impairment in offspring mice [10]. Therefore, we anes- thetized the pregnant mice on gestational day 14, and tested social interaction behaviors of the offspring mice at one- and two-month- old. As a result, we found that maternal anesthesia with 2.5% sev- oflurane for 2 h was able to induce social deficits in offspring mice. In recent decades, children with autism spectrum disorders are largely increasing [7,8,19]. Autism is clincally diagnosed by social interaction deficit, communicative impairment, and repetitive stereotyped behaviors [20e22]. As a core symptom of autism, many preclinical studies were conducted to determine the mechanism of social interaction deficit [23,24]. A three-chambered social para- digm is well-designed to test social behaviors in mouse model [25,26]. Two video-cameras were hung right above two enclosures, which could montage two video-images and capture the moment of testing mice in ANY-maze program.
+
+In this study, we examined two biological profiles of testing mice, including sociability and preference for social novelty. In detail, sociability, reflecting social affiliation, is primarily judged by the testing mouse taking more time sniffing the stranger 1 over the empty enclosure. Similarly, preference for social novelty, reflecting social recognition memory, is largely judged by the testing mouse taking more time sniffing the stranger 2 over the stranger 1. In early studies, side preferences were mainly determined by the time spent exploring in the left or right chamber [25,27]. The testing mice might spend much time wandering in side chambers, instead of directly interacting with the stranger mouse. Then, the time sniffing the stranger mouse was used to reflect side preference in social studies [22,24,28e30]. Meanwhile, the number of sniffs acted as an auxiliary parameter, like the entries into open arms in elevated plus maze [5,31,32].
+
+In this study, we found that the offspring mice undergoing in- utero exposure to sevoflurane showed abnormal sociability at ju- venile age, whereas these offspring mice retrieved normal socia- bility at early-adulthood. The disturbance of sociability in offspring mice displayed in a time-dependent manner, and the retrieving of sociability might be explained by an environmental stimuli. It was reported ameliorate sevoflurane-induced neurotoxicity and reverse learning and memory impairment in mice [10,33].
+
+that
+
+environmental
+
+enrichment
+
+can
+
+In contrast, the offspring mice exposed to sevoflurane in-utero showed abnormal preference for social novelty at juvenile age, and these mice remained this abnormality at early-adulthood. This long-term impairment of social recognition memory was in line with sevoflurane-induced impairment in learning and memory, as indicated by water-maze task or fear-conditioning test [10,33,34]. Notably, sevoflurane anesthesia in this study was conducted by the protocols of previous studies, in which blood gas analysis denied any hypoxemia in anesthetized mice [10,35]. Admittedly, this study has several limitations. Firstly, we did not investigate social inter- action behaviors of the offspring mice at more time points. Given
+
+70
+
+Biochemical and Biophysical Research Communications 553 (2021) 65e71
+
+the recovery of sociability at two-month-old, it was possible that the testing mice undergoing in-utero exposure to sevoflurane could regain normal preference for social novelty in later life or amelio- rated by environmental stimuli [10]. However, our findings have detected the detrimental effects of in-utero exposure to sevo- flurane on social behaviors of offspring mice. Secondly, it should be cautious to extrapolate our findings in mice to human being. Given that the gestational period is 21 days in mice, the anesthesia duration of 2 h in mice (nearly 27 h in humans) is much longer than the average time of sevoflurane anesthesia in humans. In addition, the sequence and timing of neurodevelopmental processes are different between species. For instance, the neurogenesis and neural migration is predominant in the 2nd trimester for humans, but in the 3rd trimester for rodents [1]. Thirdly, we had no idea of whether the anesthetized dams might have difficulties in fostering pups. To rule out this confounding factor, we will arrange cross- nursing between the anesthetized and control dams in future study. Finally, we did not investigate the underlying mechanisms of social like the altered expression of special receptor proteins in developing brain. How- ever, we will explore the detailed mechanism of social deficits in offspring mice based on the current observations, and determine specific brain regions including the anesthetic-related GABA metabolism and social memory-related NMDA expression [5].
+
+interaction deficits in offspring mice,
+
+In conclusion, maternal anesthesia with sevoflurane during the interaction deficits in the mid-gestation could induce social offspring mice. In particular, the sociability of the offspring mice could be abnormal at juvenile age, and it could return normally at early-adulthood. However, the preference for social novelty of these mice could be impaired for much longer time.
+
+Declaration of competing interest
+
+The authors declare no conflict of interest.
+
+Acknowledgements
+
+This study was supported by grants from Natural Science
+
+Foundation of China (81471835).
+
+Appendix A. Supplementary data
+
+Supplementary data to this article can be found online at
+
+https://doi.org/10.1016/j.bbrc.2021.03.063.
+
+References
+
+[1] A. Palanisamy, Maternal anesthesia and fetal neurodevelopment, Int. J. Obstet.
+
+Anesth. 21 (2012) 152e162.
+
+[2] X. Shen, Y. Dong, Z. Xu, H. Wang, C. Miao, S.G. Soriano, D. Sun, M.G. Baxter, Y. Zhang, Z. Xie, Selective anesthesia-induced neuroinflammation in devel- oping mouse brain and cognitive impairment, Anesthesiology 118 (2013) 502e515.
+
+[3] Y. Takaenoki, Y. Satoh, Y. Araki, M. Kodama, R. Yonamine, S. Yufune, T. Kazama, Neonatal exposure to sevoflurane in mice causes deficits in maternal behavior later in adulthood, Anesthesiology 120 (2014) 403e415.
+
+[4] M. Satomoto, Y. Satoh, K. Terui, H. Miyao, K. Takishima, M. Ito, J. Imaki, Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice, Anesthesiology 110 (2009) 628e637. [5] A. Palanisamy, M.G. Baxter, P.K. Keel, Z. Xie, G. Crosby, D.J. Culley, Rats exposed to isoflurane in utero during early gestation are behaviorally abnormal as adults, Anesthesiology 114 (2011) 521e528.
+
+[6] J.N. Crawley, Designing mouse behavioral tasks relevant to autistic-like be-
+
+haviors, Ment. Retard. Dev. Disabil. Res. Rev. 10 (2004) 248e258.
+
+[7] S.S. Moy, J.J. Nadler, Advances in behavioral genetics: mouse models of autism,
+
+Mol. Psychiatr. 13 (2008) 4e26.
+
+[8] J.J. Schwartzer, C.M. Koenig, R.F. Berman, Using mouse models of autism spectrum disorders to study the neurotoxicology of gene-environment in- teractions, Neurotoxicol. Teratol. 36 (2013) 17e35.
+
+[9] P.L. Oliver, K.E. Davies, Interaction between environmental and genetic factors
+
+Q. Chen, W. Chu, R. Sheng et al.
+
+modulates schizophrenic endophenotypes in the Snap-25 mouse mutant blind-drunk, Hum. Mol. Genet. 18 (2009) 4576e4589.
+
+[10] H. Zheng, Y. Dong, Z. Xu, G. Crosby, D.J. Culley, Y. Zhang, Z. Xie, Sevoflurane anesthesia in pregnant mice induces neurotoxicity in fetal and offspring mice, Anesthesiology 118 (2013) 516e526.
+
+[11] H.M. Holtby, Neurological
+
+injury and anesthetic neurotoxicity following neonatal cardiac surgery: does the head rule the heart or the heart rule the head, Future Cardiol. 8 (2012) 179e188.
+
+[12] G.K. Istaphanous, J. Howard, X. Nan, E.A. Hughes, J.C. McCann, J.J. McAuliffe, S.C. Danzer, A.W. Loepke, Comparison of the neuroapoptotic properties of equipotent anesthetic concentrations of desflurane, isoflurane, or sevoflurane in neonatal mice, Anesthesiology 114 (2011) 578e587.
+
+[13] J. Jiang, H. Jiang, Effect of the inhaled anesthetics isoflurane, sevoflurane and desflurane on the neuropathogenesis of Alzheimer’s disease (review), Mol. Med. Rep. 12 (2015) 3e12.
+
+[14] X. Lei, Q. Guo, J. Zhang, Mechanistic insights into neurotoxicity induced by anesthetics in the developing brain, Int. J. Mol. Sci. 13 (2012) 6772e6799.
+
+[15] C. Wang, Advanced pre-clinical research approaches and models to studying
+
+pediatric anesthetic neurotoxicity, Front. Neurol. 3 (2012) 142.
+
+[16] X. Bai, D. Twaroski, Z.J. Bosnjak, Modeling anesthetic developmental neuro- toxicity using human stem cells, Semin. CardioThorac. Vasc. Anesth. 17 (2013) 276e287.
+
+[17] D. Twaroski, Z.J. Bosnjak, X. Bai, MicroRNAs: new players in anesthetic- induced developmental neurotoxicity, Pharm. Anal. Acta 6 (2015) 357. [18] Z.J. Bosnjak, Y. Yana, S. Canfield, M.Y. Muravyevaa, C. Kikuchia, C. Wellsc, J. Corbettd, X. Baia, Ketamine induces toxicity in human neurons differenti- ated from embryonic stem cells via mitochondrial apoptosis pathway, Curr. Drug Saf. 7 (2012) 106e119.
+
+[19] F.I. Roullet, J.K. Lai, J.A. Foster, In utero exposure to valproic acid and autism–a current review of clinical and animal studies, Neurotoxicol. Teratol. 36 (2013) 47e56.
+
+[20] C.B. Boylan, M.E. Blue, C.F. Hohmann, Modeling early cortical serotonergic
+
+deficits in autism, Behav. Brain Res. 176 (2007) 94e108.
+
+[21] B.L. Pearson,
+
+J.K. Bettis, K.Z. Meyza, L.Y. Yamamoto, D.C. Blanchard, R.J. Blanchard, Absence of social conditioned place preference in BTBR Tþtf/J mice: relevance for social motivation testing in rodent models of autism, Behav. Brain Res. 233 (2012) 99e104.
+
+[22] K.K. Chadman, Fluoxetine but not risperidone increases sociability in the BTBR mouse model of autism, Pharmacol. Biochem. Behav. 97 (2011) 586e594. [23] A.C. Felix-Ortiz, K.M. Tye, Amygdala inputs to the ventral hippocampus bidi- rectionally modulate social behavior, J. Neurosci. 34 (2014) 586e595. [24] S.S. Moy, J.J. Nadler, N.B. Young, R.J. Nonneman, A.W. Grossman, D.L. Murphy, A.J. D’Ercole, J.N. Crawley, T.R. Magnuson, J.M. Lauder, Social approach in
+
+71
+
+Biochemical and Biophysical Research Communications 553 (2021) 65e71
+
+genetically engineered mouse lines relevant to autism, Gene Brain Behav. 8 (2009) 129e142.
+
+[25] S.S. Moy, J.J. Nadler, A. Perez, R.P. Barbaro, J.M. Johns, T.R. Magnuson, J. Piven, J.N. Crawley, Sociability and preference for social novelty in five inbred strains: an approach to assess autistic-like behavior in mice, Gene Brain Behav. 3 (2004) 287e302.
+
+[26] J.N. Crawley, T. Chen, A. Puri, R. Washburn, T.L. Sullivan, J.M. Hill, N.B. Young, J.J. Nadler, S.S. Moy, L.J. Young, H.K. Caldwell, W.S. Young, Social approach behaviors in oxytocin knockout mice: comparison of two independent lines tested in different laboratory environments, Neuropeptides 41 (2007) 145e163.
+
+[27] J.J. Nadler, S.S. Moy, G. Dold, D. Trang, N. Simmons, A. Perez, N.B. Young, R.P. Barbaro, J. Piven, T.R. Magnuson, J.N. Crawley, Automated apparatus for quantitation of social approach behaviors in mice, Gene Brain Behav. 3 (2004) 303e314.
+
+[28] S.S. Moy, V.D. Nikolova, N.V. Riddick, L.K. Baker, B.H. Koller, Preweaning sensorimotor deficits and adolescent hypersociability inGrin1Knockdown mice, Dev. Neurosci. 34 (2012) 159e173.
+
+[29] S.S. Moy, R.J. Nonneman, N.B. Young, G.P. Demyanenko, P.F. Maness, Impaired sociability and cognitive function in Nrcam-null mice, Behav. Brain Res. 205 (2009) 123e131.
+
+[30] S.S. Moy, R.J. Nonneman, G.O. Shafer, V.D. Nikolova, N.V. Riddick, K.L. Agster, L.K. Baker, D.J. Knapp, Disruption of social approach by MK-801, amphet- amine, and fluoxetine in adolescent C57BL/6J mice, Neurotoxicol. Teratol. 36 (2013) 36e46.
+
+[31] S.S. Moy,
+
+J.J. Nadler, N.B. Young, A. Perez, L.P. Holloway, R.P. Barbaro, J.R. Barbaro, L.M. Wilson, D.W. Threadgill, J.M. Lauder, T.R. Magnuson, J.N. Crawley, Mouse behavioral tasks relevant to autism: phenotypes of 10 inbred strains, Behav. Brain Res. 176 (2007) 4e20.
+
+[32] K. Kent, V. Arientyl, M.M. Khachatryan, R.I. Wood, Oxytocin induces a condi- J. Neuroendocrinol. 25 (2013)
+
+tioned social preference in female mice, 803e810.
+
+[33] Y. Zhao, K. Chen, X. Shen, Environmental enrichment attenuated sevoflurane- induced neurotoxicity through the PPAR-gamma signaling pathway, BioMed Res. Int. 2015 (2015) 107e149.
+
+[34] W. Chung, S. Park, J. Hong, S. Park, S. Lee, J. Heo, D. Kim, Y. Ko, Sevoflurane exposure during the neonatal period induces long-term memory impairment but not autism-like behaviors, Paediatr. Anaesth. 25 (2015) 1033e1045. [35] Y. Dong, G. Zhang, B. Zhang, R.D. Moir, W. Xia, E.R. Marcantonio, D.J. Culley, G. Crosby, R.E. Tanzi, Z. Xie, The common inhalational anesthetic sevoflurane induces apoptosis and increases beta-amyloid protein levels, Arch. Neurol. 66 (2009) 620e631.

new_pdfs/10.1016_j.bbrc.2022.01.022.txt

@@ -0,0 +1,257 @@
+Biochemical and Biophysical Research Communications 593 (2022) 129e136
+
+Contents lists available at ScienceDirect
+
+Biochemical and Biophysical Research Communications
+
+j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / y b b r c
+
+Neonatal exposure to sevoflurane impairs preference for social novelty in C57BL/6 female mice at early-adulthood
+
+Huayue Liu a, c, 1, Xiaowen Meng a, c, 1, Yixuan Li b, Shiwen Chen b, Yumeng Ji b, , Xin Jin a, c, * Shaoyong Song a, d, Fuhai Ji a, c, ** a Institute of Anesthesiology, Soochow University, Suzhou, 215006, PR China b Suzhou Medical College of Soochow University, Suzhou, 215123, PR China c Department of Anesthesiology, First Affiliated Hospital of Soochow University, Suzhou, 215006, PR China d Department of Pain Medicine, Dushu Lake Hospital Affiliated to Soochow University, Suzhou, 215124, PR China
+
+a r t i c l e i n f o
+
+a b s t r a c t
+
+Article history: Received 23 December 2021 Accepted 8 January 2022 Available online 12 January 2022
+
+Keywords: Sevoflurane Sociability Preference for social novelty Anesthesia Autism
+
+Social interaction deficit is core symptom of children with autism, owing to interaction of genetic pre- disposition and environmental toxins. Sevoflurane could induce neurotoxicity in developing brain in rodent models. This study aims to investigate whether sevoflurane anesthesia in neonatal period could impair social behaviors in male and female mice. Twenty-eight male and thirty-one female mice were randomly assigned to receive 3.0% sevoflurane or 60% oxygen on postnatal day 6. They were tested for social interaction behaviors at one- and two-month-old. In addition, the cortex and hippocampus of neonatal mice undergoing sevoflurane anesthesia were harvested for immunoblotting analysis. As a result, both male and female mice undergoing sevoflurane anesthesia showed strong sociability and weak preference for social novelty at juvenile age. In addition, the male mice developed normal pref- erence for social novelty at early-adulthood; However, the female mice remained weak preference for social novelty. Furthurmore, sevoflurane anesthesia could decrease the levels of PSD95 but not Neuroligin-1 in the hippocampus but not cortex of neonatal mice. In conclusion, sevoflurane anesthesia in neonatal period could disturb development of social memory and impair preference for social novelty in female mice at early-adulthood, with the potential mechanism of decreasing PSD95 expression in the hippocampus of C57BL/6 mice. © 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
+
+1. Introduction
+
+Children with autism spectrum disorders are largely increasing and the ratio of boys and girls is nearly 4:1 all over the world [1,2]. The core symptom of autism is social interaction deficit, with the potential mechanism of genetic predisposition and environmental toxicants [3,4]. Meanwhile, some anesthetics are reported to induce neurotoxicity in the developing brain [5,6]. Sevoflurane, an inha- is commonly used in pediatric anesthesia. lational anesthetic,
+
+Abbreviations: ASD, autism spectrum disorders; PND, postnatal day; PSD95,
+
+postsynaptic density-95.
+
+Corresponding author. Department of Anesthesiology, Suzhou, 215006, PR
+
+China. ** Corresponding author. Department of Anesthesiology, Suzhou, 215006, PR China.
+
+Preclinical studies suggested that neonatal anesthesia with sevo- flurane could impair learning and memory in rodents [7,8]. In particular, neonatal exposure to 3% sevoflurane for 6 h in mice could cause learning deficit in fear conditioning test and social deficit in open field cage [9]. However, it is uncertain whether sevoflurane could impair biological profiles of social affiliation and social memory in mice, and whether the male and female mice would behave differently.
+
+Sevoflurane could produce anesthetic effect by stimulating GABA receptors and inducing an imbalance of excitatory and inhibitory neurotransmission [10]. Although anesthetists are taking advantage of sevoflurane for rapid onset time and short duration, pediatric patients are taking risk of sevoflurane-induced develop- mental neurotoxicity and behavioral abnormality. Therefore, we set out to investigate whether neonatal anesthesia with sevoflurane could disturb sociability and preference for social novelty in male and female mice at juvenile age and early-adulthood.
+
+E-mail addresses: jifuhai@hotmail.com (F. Ji), jinxin@suda.edu.cn (X. Jin). 1 These authors contributed equally to this work (H. Y. Liu and X.W. Meng).
+
+We hypothesized that neonatal exposure to sevoflurane could
+
+https://doi.org/10.1016/j.bbrc.2022.01.022 0006-291X/© 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. 0/).
+
+H. Liu, X. Meng, Y. Li et al.
+
+induce social interaction deficit in mice. To verify this hypothesis, we firstly anesthetized C57BL/6 mice with 3.0% sevoflurane in 60% oxygen for 2 h on postnatal day 6. Next, we tested social behaviors of the male and female mice at one- and two-month-old. Finally, we examined the levels of Neuroligin-1 and PSD95 in the cortex and hippocampus of sevoflurane-exposed neonatal mice.
+
+2. Methods
+
+2.1. Animals
+
+This study was approved by the Institutional Animal Care and Use Committee at Soochow University (Suzhou, Jiangsu, China). Twenty-four female and six male C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Jiangsu, China) for producing next generation of mice. On postnatal day (PND) 21, the offspring mice were separated from dams and housed 4e5 per cage by gender. Four male and four female mice were specifically used as the stranger mice, which were trained to stay calmly in the enclosure before social interaction test. All the mice were raised with free access to food and water in a controlled environment (room temperature 21e22 (cid:1)C, 12/12 h light/dark cycle, and light on at 7 a.m.).
+
+2.2. Anesthesia
+
+Inc.) was employed to supply a consistent concentration of anesthetic gas. A sealed plastic box (20 L (cid:3) 20 W (cid:3) 6 H cm) was used as the anes- thetizing chamber, which was drilled with three holes for gas inflow, gas outflow and gas monitoring. An electric heater was placed underneath the anesthetizing chamber to keep neonatal mice warm during anesthesia. A gas analyzer (Datex-Ohmeda, Inc.) was applied to adjust gas concentrations. On postnatal day 6, the neonatal mice were randomly assigned into two groups. Twenty- eight mice (17 males and 11 female) received 3.0% sevoflurane in 60% oxygen for 2 h (Sevo), and thirty-one mice (11 males and 20 female) inhaled merely 60% oxygen for 2 h (Oxyg). Sex of each mouse and amount of each group were not identified until weaning on PND 21. These subject mice were tested for social interaction behavior at one- and two-month-old.
+
+A retired anesthesia machine (Datex-Ohmeda,
+
+Another battery of neonatal mice were treated with the air condition (control) or 60% oxygen (oxyg) or 3.0% sevoflurane in 60% oxygen (sevo) for 2 h on PND 6, and then killed for harvesting brain tissues 24 h after treatment. Sevoflurane anesthesia was strictly performed by the protocols of previous studies [11,12], in which all neonatal mice could spontaneously breath during general anes- thesia, and their arterial blood pressure and blood gas analysis showed within normal limits. The vapor for releasing sevoflurane was turned off at the end of anesthesia, and the residual anesthetic was washed out with 60% oxygen for 15 min. Finally, these pups were smeared with own bedding and sent back to their dams.
+
+2.3. Social interaction paradigm
+
+Social interaction test is performed with the three-chambered social box, with three chambers (40 L (cid:3) 20 W (cid:3) 22 H cm) and two enclosures (7 ID (cid:3) 15 H cm). The floor is painted grey to pro- vide a high contrast with the testing mice. Grid bars of the enclo- sure allow direct contacting between the subject and stranger mice. A novel video-tracking system was developed by hanging two video-cameras right above two enclosures. Thereby, two video- images were integrated into one with the montage effect in ANY- maze program (Stoelting Co., USA). The subject mouse initiates social interaction with the stranger mouse by nose-to-nose or nose-
+
+130
+
+Biochemical and Biophysical Research Communications 593 (2022) 129e136
+
+to-tail sniffing, thus the animal's head is tracked by the ANY-maze program.
+
+2.4. Social interaction test
+
+First of all, the stranger mice were transferred into behavioral room and hidden 2 m away from social apparatus. Each subject mouse was taken into behavioral room about 45 min before social interaction test. In the first session (Habituation, 10-min), the sub- ject mouse was gently placed into the middle chamber, and allowed to freely explore in three chambers. In the second session (Socia- bility, 10-min), the subject mouse was guided into the middle chamber and transiently confined there. An unfamiliar conspecific (Stranger 1) was introduced into one enclosure, the subject mouse was allowed to explore in three chambers and sniff at two enclo- sures containing Stranger 1 or not. In the third session (Preference for social novelty 10-min), the subject mouse was again confined into the middle chamber. Another unfamiliar conspecific (Stranger 2) was introduced into the other enclosure, and the subject mouse was allowed to explore in three chambers and sniff at two enclo- sures containing Stranger 2 or Stranger 1. Placement of Stranger 1 on left and right side were balanced between trials, and two stranger mice were the same gender as the subject mice.
+
+Sociability is characteristic of the mouse taking more time sniffing its conspecific mouse compared with an inanimate object. Preference for social novelty is characteristic of the mouse taking more time sniffing an unfamiliar mouse compared with a familiar one. Four parameters were measured for judging social choice, including 1) time sniffing at the enclosure, 2) number of sniffs, 3) time exploring in the chamber, and 4) number of entries. Sniffing time at the enclosure was primary outcome, number of sniffs at the enclosure and time exploring in the chamber were secondary outcomes. In social interaction test, “at the enclosure” is defined as the head of mouse entering an area about 3 cm around the enclo- sure, as described in similar social study [13]. And “in the chamber” is defined as the head of mouse entering into the chamber.
+
+2.5.
+
+Immunoblotting analysis
+
+The brain tissues of neonatal mice were harvested on dry ice at 24 h after treatment. Next, the cortex and hippocampus were ho- mogenized on ice using the immunoprecipitation buffer plus pro- tease inhibitor. And then, the lysates were centrifuged at 15,000 rpm for 30 min at 4 (cid:1)C. After that, the lysates were quan- tified for total protein by the bicinchoninic acid (BCA) protein assay kit (MultiSciences Biotech Co., Ltd. Cat: PQ0012, Lot: A91041). Finally, western blot was performed by the protocols to analyze protein levels in cortex and hippocampus. Neuroligin-1 antibody (1:1000; Santa Cruz Biotechnology, Inc.) was used to detect neuroligin-1 (101 kDa). PSD-95 antibody (1:1000; Cell Signaling Technology, Inc.) was used to detect PSD-95 (95 kDa). Antieb-actin (1:5000; Sigma) was used to detect b-actin (42 kDa).
+
+2.6. Statistical analysis
+
+Data were expressed as Mean ± SD. Statistical analyses were performed by using GraphPad Prism 5.0 (San Diego, USA). Data representing social behavior of testing mice were normally distributed by Kolmogorov-Smirnov test. Data of each mouse from the left or right side were mutually exclusive, and two-tailed paired t-test was used to determine side preference, which was supported by other social studies [14,15]. Student's t-test was used to assess differences in the levels of Neuroligin-1 and PSD95 expression in cortex and hippocampus of mice. P values less than 0.05 (*), 0.01 (**) and 0.001 (***) were considered statistically significant.
+
+H. Liu, X. Meng, Y. Li et al.
+
+3. Results
+
+3.1. Both male and female subject mice show strong sociability and weak preference for social novelty at one-month-old, and sevoflurane anesthesia on postnatal day 6 could not influence the biologic profiles of social affiliation and social memory in the juvenile mice
+
+The subject mice undergoing anesthesia with 3.0% sevoflurane in 60% oxygen on postnatal day 6, both males and females, showed strong sociability at one-month-old, as proved by taking more time (Fig. 1A, 183.3 ± 51.6 vs 73.6 ± 32.6 s, P < 0.0001 Male; 250.7 ± 72.7 vs 71.1 ± 33.3 s, P ¼ 0.0002 Female) approaching the stranger 1 mouse, sniffing more frequently (Fig. 1B, 53.6 ± 21.1 vs 22.9 ± 10.6 , P < 0.0001 Male; 49.0 ± 15.9 vs 21.8 ± 9.4, P ¼ 0.0024 Female) at the enclosure containing stranger 1, and spending more time exploring (Fig. 1C, 348.3 ± 56.7 vs 170.6 ± 47.4 s, P < 0.0001 Male; 377.5 ± 71.4 vs 161.5 ± 58.8 s, P ¼ 0.0002 Female) in the chamber with stranger 1, as compared with the empty side. Collectively, the male and fe- male mice, exposed to sevoflurane in the neonatal period, showed the well-developed social affiliation at the juvenile age.
+
+However, the subject mice undergoing anesthesia with 3.0% sevoflurane in 60% oxygen on postnatal day 6, either males or fe- males, showed weak preference for social novelty at one-month- old, as evidenced by taking no more time (Fig. 1E, 105.2 ± 50.7 vs 151.8 ± 72.3 s, P ¼ 0.068 Male; 139.6 ± 55.8 vs 171.5 ± 68.1 s, P ¼ 0.3189 Female) approaching the stranger 2 mouse, sniffing no more frequently (Fig. 1F, 31.0 ± 13.0 vs 37.2 ± 14.4, P ¼ 0.3085 Male; 33.1 ± 11.8 vs 36.2 ± 7.0, P ¼ 0.4993 Female) at the enclosure containing stranger 2, or spending no more time exploring (Fig. 1G, 229.8 ± 90.3 vs 271.9 ± 80.2 s, P ¼ 0.3126 Male; 246.2 ± 54.3 vs 282.8 ± 63.1 s, P ¼ 0.3063 Female) in the chamber with stranger 2, as compared with the stranger 1 side. Together, the male and female mice, exposed to sevoflurane in neonatal period, showed the undevel- oped social memory at the juvenile age.
+
+3.2. The male subject mice, but not the female, showed normal preference for social novelty at two-month-old, and sevoflurane anesthesia on postnatal day 6 could impair the development of social memory of female mice at the early-adulthood
+
+The subject mice undergoing anesthesia with 3.0% sevoflurane in 60% oxygen on postnatal day 6, both males and females, showed strong sociability at two-month-old, as proved by taking more time (Fig. 2A, 196.2 ± 50.8 vs. 59.3 ± 24.3 s, P < 0.0001 Male; 189.9 ± 60.4 vs. 58.4 ± 23.8 s, P ¼ 0.0028 Female) approaching the stranger 1 mouse, sniffing more frequently (Fig. 2B, 66.5 ± 17.3 vs. 28.4 ± 10.8, P < 0.0001 Male; 58.3 ± 13.6 vs. 30.4 ± 6.9, P ¼ 0.0051 Female) at the enclosure containing stranger 1, and spending more time exploring (Fig. 2C, 347.4 ± 55.6 vs. 160.0 ± 43.1 s, P < 0.0001 Male; 324.6 �� 65.2 vs. 175.8 ± 43.8 s, P ¼ 0.008 Female) in the chamber with stranger 1, as compared with the empty side. Collectively, the male and female mice, exposed to sevoflurane in the neonatal period, showed the sustained social affiliation at the early- adulthood.
+
+Meanwhile, the male subject mice, but not the females, under- going anesthesia with 3.0% sevoflurane in 60% oxygen on postnatal day 6 showed normal preference for social novelty at two-month- old, as evidenced by taking more time (Fig. 2E, 102.0 ± 42.0 vs. 146.6 ± 59.3 s, P ¼ 0.0463 Male; 101.5 ± 34.7 vs. 138.0 ± 49.9 s, P ¼ 0.2462 Female) approaching the stranger 2 mouse, sniffing more frequently (Fig. 2F, 37.1 ± 12.2 vs. 53.1 ± 15.0, P ¼ 0.0102 Male; 40.3 ± 10.3 vs. 53.1 ± 26.0, P ¼ 0.3074 Female) at the enclosure con- taining stranger 2, or spending more time exploring (Fig. 2G, 211.2 ± 58.3 vs. 284.3 ± 59.9 s, P ¼ 0.0254 Male; 228.9 ± 65.3 vs. 269.8 ±
+
+131
+
+Biochemical and Biophysical Research Communications 593 (2022) 129e136
+
+64.3 s, P ¼ 0.4307 Female) in the chamber with stranger 2, as compared with the stranger 1 side. Together, the male mice exposed to sevoflurane in the neonatal period showed the already- developed social memory at the early-adulthood, but the female mice remained undeveloped social memory at the early-adulthood.
+
+3.3. Neonatal exposure to 3.0% sevoflurane in 60% oxygen, as well as inhalation of 60% oxygen, decreased PSD95 levels in the hippocampus, but not the cortex, of neonatal mice
+
+In the cortex (Fig. 3A) and the hippocampus (Fig. 3B) of neonatal mice, the immunoblotting displayed that anesthesia with 3.0% sevoflurane (lanes 9-12) and inhalation of 60% oxygen (lanes 5-8) did not change the levels of bands representing Neuroligin-1 expression, as compared with the control (lanes 1-4), Quantifica- tion of Western blot, based on the ratio of Neuroligin-1 levels to b- Actin levels, did not show that neonatal exposure to sevoflurane change the levels of Neuroligin-1 in the cortex (Fig. 3C, 100.0±33.1 vs. 92.7±14.0, P¼0.6975 grey) or the hippocampus (Fig. 3D, 100.0±19.3 vs. 84.8±20.0, P¼0.3155 grey) of the neonatal mice, as compared with the control (white) .
+
+The immunoblotting displayed that anesthesia with 3.0% sevo- flurane (lanes 9-12) and inhalation of 60% oxygen (lanes 5-8) did not change the levels of bands representing PSD95 expression in the cortex (Fig. 3E) of neonatal mice, as compared with the control (lanes 1-4). Quantification of Western blot did not show that neonatal exposure to sevoflurane change the levels of PSD95 in the cortex (Fig. 3G, 100.0±23.4 vs. 120.1±27.9, P¼0.3113 grey), as compared with the control (white). However, the immunoblotting displayed that anesthesia with 3.0% sevoflurane (lanes 9-12) and inhalation of 60% oxygen (lanes 5-8) decreased the levels of bands representing PSD95 expression in the hippocampus (Fig. 3F) of neonatal mice, as compared with the control (lanes 1-4). Quanti- fication of Western blot decreased that neonatal exposure to sev- oflurane change the levels of PSD95 in the cortex (Fig. 3H, 100.0±35.9 vs. 49.2±10.8, P¼0.035 grey), as compared with the control (white).
+
+4. Discussions
+
+This study was to explore whether neonatal anesthesia with sevoflurane could induce social interaction deficit in C57BL/6 mice, and we performed the experiment with several important con- siderations and preparations. Firstly, we conducted inhalational anesthesia with 3.0% sevoflurane in 60% oxygen for 2 h as in similar studies [16e18], and this regimen of anesthesia in neonatal mice was designed to mimic clinical anesthesia in pediatric patients. Secondly, we employed the three-chambered social paradigm to assess social behaviors of the subject mice [19e21] which could reflect two biological profiles of sociability and preference for social novelty [22]. Thirdly, the subject mice were arranged for social interaction tests at one- and two-month-old, as the juvenile age and early-adulthood were considered to be two critical periods of brain development in human [13,23]. Finally, we tested social interaction behaviors of male and female mice respectively, in or- der to investigate interaction effects of anesthesia and sex on social behaviors of the mice. Thereby, these results were adequate to determine whether neonatal exposure to sevoflurane could induce social interaction deficit in C57BL/6 mice.
+
+Our data suggested that both male and female mice, in the three-chambered social test, showed strong sociability and weak preference for social novelty at one-month-old, indicating the robust social affiliation with the conspecific and underdevelopment of social memory at the juvenile age. However, the male mice un- dergoing either sevoflurane anesthesia or oxygen control displayed
+
+H. Liu, X. Meng, Y. Li et al.
+
+Biochemical and Biophysical Research Communications 593 (2022) 129e136
+
+Fig. 1. The juvenile mice anesthetized with 3.0% sevoflurane in 60% oxygen on PND 6, either male or female, show strong sociability but weak preference for social novelty at one-month-old. In the sociability test, the subject mice in either group, take more time sniffing the stranger 1 mouse (A), sniff more frequently at the enclosure containing stranger 1 (B), and spend more time exploring in the chamber with stranger 1 (C), as compared to the empty side. In the test of social novelty preference, the subject mice in either group, take no more time sniffing the stranger 2 mouse (E), sniff no more frequently at the enclosure containing stranger 2 (F), or spend no more time exploring in the chamber with stranger 2 (G), as compared to the stranger 1 side. (D, H) There are not significant differences between two sides in the number of entries into the chamber. Data are expressed as Mean ± SD. N ¼ 11 Oxygen and 17 Sevoflurane for males, 20 Oxygen and 11 Sevoflurane for females. Paired t-test, two-side. *P < 0.05, **P < 0.01, ***P < 0.001.
+
+132
+
+H. Liu, X. Meng, Y. Li et al.
+
+Biochemical and Biophysical Research Communications 593 (2022) 129e136
+
+Fig. 2. The early-adult female mice, anesthetized with 3.0% sevoflurane in 60% oxygen on PND 6, show strong sociability but weak preference for social novelty at two- month-old. In the sociability test, the subject mice in either group, take more time sniffing the stranger 1 mouse (A), sniff more frequently at the enclosure containing stranger 1 (B), and spend more time exploring in the chamber with stranger 1 (C), as compared to the empty side. In the test of social novelty preference, the male mice take more time sniffing the stranger 2 mouse (E), sniff more frequently at the enclosure containing stranger 2 (F), and spend more time exploring in the chamber with stranger 2 (G), as compared to the stranger 1 side. However, the female mice undergoing neonatal anesthesia with sevoflurane show no significant difference (E, F and G, right) between the stranger 2 and stranger 1 side. (D, H) There are not significant differences between two sides in the number of entries into the chamber. Data are expressed as Mean ± SD. N ¼ 11 Oxygen and 15 Sevoflurane for males, 12 Oxygen and 7 Sevoflurane for females. Paired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
+
+133
+
+H. Liu, X. Meng, Y. Li et al.
+
+Biochemical and Biophysical Research Communications 593 (2022) 129e136
+
+Fig. 3. Neonatal exposure to 3.0% sevoflurane in 60% oxygen or only 60% oxygen decrease the level of PSD95 in hippocampus, but not cortex, of the female mice. Western blot analysis displays that neither oxygen (lanes 5e8) nor sevoflurane (lanes 9e12) decreases the level of Neuroligin-1 in either cortex (A) or hippocampus (B) of the female mice, as compared to the control condition (lanes 1e4). Quantification of Western blot shows that neither oxygen (black bar) nor sevoflurane (grey bar) decreases the level of Neuroligin-1 in either cortex (C) or hippocampus (D), as compared to the control condition (white bar). Meanwhile, western blot analysis displays that both oxygen (lanes 5e8) and sevoflurane (lanes 9e12) decreases the level of PSD95 in hippocampus (F) but not cortex (E) of the female mice, as compared to the control condition (lanes 1e4). Quantification of Western blot shows that either oxygen (black bar) or sevoflurane (grey bar) decreases the level of PSD95 in hippocampus (H) but not cortex (G), as compared to the control condition (white bar). N ¼ 4 in each, Student's t-test. *P < 0.05.
+
+134
+
+H. Liu, X. Meng, Y. Li et al.
+
+normal preference for social novelty at two-month-old, indicating the well-development of social memory at the early-adulthood. Meanwhile, the female mice undergoing neonatal exposure to sevoflurane remained very weak preference for social novelty, indicating the developmental retardation of social memory at the early-adulthood. Taken together, these results demonstrated that sevoflurane anesthesia in neonatal period could impair preference for social novelty of the female mice at the early-adulthood. In addition, the developmental neurotoxicity of sevoflurane anes- thesia could be partial to female but not male, and neurobehavioral abnormality could be partial to social memory but not social affiliation.
+
+Three-chambered social paradigm was widely used for social interaction test in mouse model, which could examine sociability and preference for social novelty [15,24]. Sociability was defined as the propensity of testing mice to spend more time exploring in the chamber containing Stranger 1 than in the empty chamber [14,23]. Preference for social novelty was defined as the propensity of testing mice to spend more time exploring in the chamber con- taining Stranger 2 than in the chamber containing Stranger 1 [24,25]. Because the testing mouse might take too much time wandering and self-grooming, the time exploring in the chamber could not totally represent its real choice [26]. Therefore, we measured time sniffing at the enclosure, as the primary outcome, for accessing social choice of mice. Meanwhile, time exploring in the chamber and number of sniffs at the enclosure were used as the secondary outcomes. Besides, number of entries to side chamber was considered to be an internal control of general activities.
+
+In the sociability tests, both male and female mice, either the control or the sevoflurane-exposed, took much more time sniffing Stranger 1 compared with the empty enclosure at one- and two- month-old. These results suggested that 1) social affiliation with conspecific could be a stable and basic instinct of mice, and 2) neonatal exposure to sevoflurane could not impair the sociability of male and female mice. In the social novelty preference tests, both male and female mice, either the control or the sevoflurane- exposed, did not take more time sniffing Stranger 2 compared with Stranger 1 at one-month-old, and the male but not female mice preferred Stanger 2 to Stranger 1 at two-month-old. These results suggested that 1) social memory of the mice could be un- stable and superior neurocognitive function of mice [22,24,25], and 2) neonatal exposure to sevoflurane could selectively impair pref- erence for social novelty of female, but not male, mice at the early- adulthood.
+
+In this study, the male mice showed normal preference for novel conspecific at the early-adulthood, rather than at the juvenile age and this phenomenon suggested that social recognition memory was age-dependent in subject mice [26e28]. Meanwhile, the fe- male mice undergoing sevoflurane anesthesia, but not the control, showed weak preference for novel conspecific at the early- adulthood, and this abnormality suggested that sevoflurane- induced neurotoxicity could disturb neurodevelopmental process of social memory in female mice. In clinical phenotypes, children diagnosed as autism spectrum disorders are gender-biased, with more boys outweighing girls. We had no idea of the sexual discrepancy between social deficit in human being and social memory impairment in mouse model, and the potential explana- tion should be based on the development of body and brain. In terms of biological dimorphism, the boys could lag behind the girls in mental and physical development in adolescence, while the fe- male mice should weigh less than male mice in juvenile and early- adulthood.
+
+It was reported that sevoflurane anesthesia could impair learning and memory by inducing neuronal apoptosis and inhib- iting synapse plasticity [11,29,30], which were in accordance with
+
+135
+
+Biochemical and Biophysical Research Communications 593 (2022) 129e136
+
+disturbance of social memory in female mice undergoing sevo- flurane anesthesia in our study. Next, we performed immunoblot- ing analysis to determine whether sevoflurane anesthesia in neonatal mice could affect the expression of Neuroligin-1 and PSD95 in the brain tissues. As a result, immunobloting analysis suggested that sevoflurane could not change the levels of Neuroligin-1 in the cortex and hippocampus of neonatal mice. However, we found that sevoflurane could decrease the levels of PSD95 in the hippocampus of neonatal mice. This result suggested the sevoflurane-induced neurotoxicity in developing brain of neonatal mice, although the detailed signaling pathways and neuropathologic mechanisms were unclear at this moment. Chung et al. reported that neonatal exposure to sevoflurane could cause the long-term memory impairment in C57BL/6J mice but not autism-like features [7], indicating the complication of social behavior and recognition memory in mice. In future study, we will explore the neuropathologic mechanisms of social memory impairment, and assess the inconformity of normal preference for social novlety in the oxygen-controlled mice with decreased levels of PSD95 in the hippocampus of neonatal mice.
+
+limitations. Firstly, we found the impairment of social memory in female mice undergoing sevo- flurane anesthesia, and then performed immunobloting analysis in brain tissue of sex-mixed mice, because it was difficult to distin- guish male and female mice in neonatal period. Secondly, we had only anesthetized neonatal mice with 3% sevoflurane for 2 h, and this regimen mimicking clinical anesthesia could be close to lower- limit. In future, we will anesthetize neonatal mice with 3% sevo- flurane for 6 h or 2 h for three times, to explore the accumulative impact of sevoflurane-induced neurotoxicity on social behaviors in mice. Thirdly, we performed 3.0% sevoflurane anesthesia in 60% oxygen in neonatal mice in animal study, but we had no idea of whether 60% oxygen itself could produce negative effects in developing brain. In future, we will determine the effect of 60% oxygen on social interaction behaviors of mice.
+
+This study has several
+
+In conclusion, C57BL/6 mice have two biological profiles of so- ciability and preference for social novelty, which were dominated by robust social affiliation and gradually-developed social memory. Neonatal exposure to sevoflurane could not impair sociability and preference for social novelty in male mice; however it could disturb preference for social novelty in female mice, with the potential mechanism of decreasing PSD95 levels in the hippocampus.
+
+Declaration of competing interest
+
+The authors declare no competing financial interests.
+
+Acknowledgements
+
+This work was supported by the National Natural Science Foundation of China (82001126 to S.Y.S, 82072130 and 81873925 to F.H.J), Natural Science Foundation of Jiangsu Province (BK20191171 to F.H.J).
+
+Appendix A. Supplementary data
+
+Supplementary data to this article can be found online at
+
+https://doi.org/10.1016/j.bbrc.2022.01.022.
+
+References
+
+[1] B.L. Pearson,
+
+J.K. Bettis, K.Z. Meyza, L.Y. Yamamoto, D.C. Blanchard, R.J. Blanchard, Absence of social conditioned place preference in BTBR Tþtf/J mice: relevance for social motivation testing in rodent models of autism, Behav. Brain Res. 233 (1) (2012) 99e104. Jul 15.
+
+[2] K.K. Chadman, Fluoxetine but not risperidone increases sociability in the BTBR
+
+H. Liu, X. Meng, Y. Li et al.
+
+mouse model of autism, Pharmacol. Biochem. Behav. 97 (3) (2011) 586e594. Jan.
+
+[3] J.J. Schwartzer, C.M. Koenig, R.F. Berman, Using mouse models of autism spectrum disorders to study the neurotoxicology of gene-environment in- teractions, Neurotoxicol. Teratol. 36 (2013) 17e35. Mar-Apr.
+
+[4] N. Kratsman, D. Getselter, E. Elliott, Sodium butyrate attenuates social behavior deficits and modifies the transcription of inhibitory/excitatory genes in the frontal cortex of an autism model, Neuropharmacology 102 (2016) 136e145. Mar.
+
+[5] M.E. McCann, S.G. Soriano, General anesthetics in pediatric anesthesia: in- fluences on the developing brain, Curr. Drug Targets 13 (7) (2012) 944e951. injury and anesthetic neurotoxicity following neonatal cardiac surgery: does the head rule the heart or the heart rule the head, Future Cardiol. 8 (2) (2012) 179e188.
+
+[6] H.M. Holtby, Neurological
+
+[7] W. Chung, S. Park, J. Hong, et al., Sevoflurane exposure during the neonatal period induces long-term memory impairment but not autism-like behaviors, Paediatr. Anaesth. 25 (10) (2015) 1033e1045. Oct.
+
+[8] J. Liu, X. Zhang, W. Zhang, G. Gu, P. Wang, Effects of sevoflurane on young male adult C57BL/6 mice spatial cognition, PLoS One 10 (8) (2015), e0134217. [9] M. Satomoto, Y. Satoh, K. Terui, et al., Neonatal exposure to sevoflurane in- duces abnormal social behaviors and deficits in fear conditioning in mice, Anesthesiology 110 (2009) 628e637.
+
+[10] J. Jiang, H. Jiang, Effect of the inhaled anesthetics isoflurane, sevoflurane and desflurane on the neuropathogenesis of Alzheimer's disease (review), Mol. Med. Rep. 12 (1) (2015) 3e12. Jul.
+
+[11] Y. Lu, X. Wu, Y. Dong, Z. Xu, Y. Zhang, Z. Xie, Anesthetic sevoflurane causes neurotoxicity differently in neonatal naïve and alzheimer disease transgenic mice, Anesthesiology 112 (6) (2010) 1404e1416.
+
+[12] Y. Takaenoki, Y. Satoh, Y. Araki, et al., Neonatal exposure to sevoflurane in mice causes deficits in maternal behavior later in adulthood, Anesthesiology 120 (2) (2014) 403e415.
+
+[13] B.D. Semple, S.A. Canchola, L.J. Noble-Haeusslein, Deficits in social behavior emerge during development after pediatric traumatic brain injury in mice, J. Neurotrauma 29 (17) (2012) 2672e2683. Nov 20.
+
+[14] G. Riedel, S.H. Kang, D.Y. Choi, B. Platt, Scopolamine-induced deficits in social memory in mice: reversal by donepezil, Behav. Brain Res. 204 (1) (2009) 217e225. Dec 1.
+
+[15] J.N. Crawley, T. Chen, A. Puri, et al., Social approach behaviors in oxytocin knockout mice: comparison of two independent lines tested in different laboratory environments, Neuropeptides 41 (3) (2007) 145e163. Jun. [16] Y. Zhao, K. Chen, X. Shen, Environmental enrichment attenuated sevoflurane- induced neurotoxicity through the PPAR-gamma signaling pathway, BioMed
+
+136
+
+Biochemical and Biophysical Research Communications 593 (2022) 129e136
+
+Res. Int. 2015 (2015) 107e149.
+
+[17] M.H. Ji, L.L. Qiu, J.J. Yang, et al., Pre-administration of curcumin prevents neonatal sevoflurane exposure-induced neurobehavioral abnormalities in mice, Neurotoxicology 46 (2015) 155e164. Jan.
+
+[18] Y. Dong, G. Zhang, B. Zhang, et al., The common inhalational anesthetic sev- oflurane induces apoptosis and increases beta-amyloid protein levels, Arch. Neurol. 66 (5) (2009) 620e631. May.
+
+[19] S.S. Moy, H.T. Ghashghaei, R.J. Nonneman, et al., Deficient NRG1-ERBB signaling alters social approach: relevance to genetic mouse models of schizophrenia, J. Neurodev. Disord. 1 (4) (2009) 302e312. Dec.
+
+[20] S.S. Moy, R.J. Nonneman, N.B. Young, G.P. Demyanenko, P.F. Maness, Impaired sociability and cognitive function in Nrcam-null mice, Behav. Brain Res. 205 (1) (2009) 123e131. Dec 14.
+
+[21] S.S. Moy, J.J. Nadler, N.B. Young, et al., Social approach in genetically engi- neered mouse lines relevant to autism, Gene Brain Behav. 8 (2) (2009) 129e142. Mar.
+
+[22] J.N. Crawley, Designing mouse behavioral tasks relevant to autistic-like be-
+
+haviors, Ment. Retard. Dev. Disabil. Res. Rev. 10 (4) (2004) 248e258.
+
+[23] S.S. Moy, R.J. Nonneman, G.O. Shafer, et al., Disruption of social approach by MK-801, amphetamine, and fluoxetine in adolescent C57BL/6J mice, Neuro- toxicol. Teratol. 36 (2013) 36e46. Mar-Apr.
+
+[24] S.S. Moy, J.J. Nadler, A. Perez, et al., Sociability and preference for social novelty in five inbred strains: an approach to assess autistic-like behavior in mice, Gene Brain Behav. 3 (5) (2004) 287e302.
+
+[25] B.L. Pearson, E.B. Defensor, D.C. Blanchard, R.J. Blanchard, C57BL/6J mice fail to exhibit preference for social novelty in the three-chamber apparatus, Behav. Brain Res. 213 (2) (2010) 189e194. Dec 1.
+
+[26] J.J. Nadler, S.S. Moy, G. Dold, et al., Automated apparatus for quantitation of social approach behaviors in mice, Gene Brain Behav. 3 (5) (2004) 303e314. Oct.
+
+[27] O. Kaidanovich-Beilin, T. Lipina, I. Vukobradovic, J. Roder, J.R. Woodgett,
+
+Assessment of social interaction behaviors, JoVE : JoVE 25 (48) (2011). Feb.
+
+[28] K. Kent, V. Arientyl, M.M. Khachatryan, R.I. Wood, Oxytocin induces a condi- tioned social preference in female mice, J. Neuroendocrinol. 25 (9) (2013) 803e810. Sep.
+
+[29] T. Tagawa, S. Sakuraba, K. Kimura, A. Mizoguchi, Sevoflurane in combination with propofol, not thiopental, induces a more robust neuroapoptosis than sevoflurane alone in the neonatal mouse brain, J. Anesth. 28 (6) (2014) 815e820. Dec.
+
+[30] X.D. Han, M. Li, X.G. Zhang, Z.G. Xue, J. Cang, Single sevoflurane exposure increases methyl-CpG island binding protein 2 phosphorylation in the hip- pocampus of developing mice, Mol. Med. Rep. 11 (1) (2015) 226e230. Jan.

new_pdfs/10.1016_j.bcp.2012.06.001.txt

@@ -0,0 +1,311 @@
+Biochemical Pharmacology 84 (2012) 558–563
+
+Contents lists available at SciVerse ScienceDirect
+
+Biochemical Pharmacology
+
+j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o c h e m p h a r m
+
+Fetal exposure to high isoflurane concentration induces postnatal memory and learning deficits in rats
+
+Fei-Juan Kong a, Lei-Lei Ma b, Wen-Wen Hu c, Wen-Na Wang b, Hui-Shun Lu c,*, Shu-Ping Chen a,**
+
+a Department of Anesthesiology, First People’s Hospital of Hangzhou, PR China b Department of Anesthesiology, Second Affiliated Hospital, School of Medicine, Zhejiang University, PR China c Department of Anesthesiology, Women’s Hospital, School of Medicine, Zhejiang University, PR China
+
+A R T I C L E
+
+I N F O
+
+A B S T R A C T
+
+Article history: Received 9 May 2012 Accepted 1 June 2012 Available online 15 June 2012
+
+Keywords: Isoflurane Fetal rats Memory and learning deficits Neuron apoptosis Synaptic plasticity
+
+We developed a maternal fetal rat model to study the effects of isoflurane-induced neurotoxicity on the fetuses of pregnant rats exposed in utero. Pregnant rats at gestational day 14 were exposed to 1.3 or 3% isoflurane for 1 h. At postnatal day 28, spatial learning and memory of the offspring were examined using the Morris Water Maze. The apoptosis was evaluated by caspase-3 immunohistochemistry in the hippocampal CA1 region. Simultaneously, the ultrastructure changes of synapse in the hippocampal CA1 and dentate gyrus region were observed by transmission electron microscopy (TEM). The 3% isoflurane treatment group showed significantly longer escape latency, less time spent in the third quadrant and fewer original platform crossings in the Morris Water Maze test, significantly increased number and optical densities of caspase-3 neurons. This treatment also produced remarkable changes in synaptic ultrastructure compared with the control and the 1.3% isoflurane groups. There were no differences in the Morris Water Maze test, densities of caspase-3 positive cells, or synaptic ultrastructure between the control and 1.3% isoflurane groups. High isoflurane concentration (3%) exposure during pregnancy caused spatial memory and learning impairments and more neurodegeneration in the offspring rats compared with control or lower isoflurane concentrations.
+
+(cid:2) 2012 Elsevier Inc. All rights reserved.
+
+1. Introduction
+
+prior neurodevelopmental studies focused on postnatal subjects rather than on the fetuses.
+
+Inhalation anesthetics such as isoflurane have been widely used in recent years in clinical and research practices. A growing body of evidence both in animals [1–3], and humans [4–6] supports the view that exposure to anesthetics early in life causes neurohis- topathologic changes and long-term cognitive impairments. Our recent study also demonstrated gestational exposure to a clinically relevant concentration of isoflurane could cause neuron apoptosis, changes of synaptic structure, and postnatal spatial memory and learning impairments in the offspring rats [7,8]. Anesthesia given to immature rodents causes cognitive dysfunction, raising the possibility that the same might be true for millions of human fetuses, neonates and infants undergoing surgical procedures under general anesthesia each year. Nevertheless, the majority of
+
+Corresponding author at: Department of Anesthesiology, Women’s Hospital, School of Medicine, Zhejiang University, No. 1 Bachelor Road, Hangzhou 310006, PR China. Tel.: +86 571 87065701x2410; fax: +86 571 87061878. ** Corresponding author at: Department of Anesthesiology, First People’s Hospital of Hangzhou, No. 261 Huansha Road, Hangzhou, 310006, PR China. Tel.: +86 571 87065701x10448; fax: +86 571 87914773. E-mail address: kongfeijuan@163.com (S.-P. Chen). 0006-2952/$ – see front matter (cid:2) 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2012.06.001
+
+Many pregnant women, fetuses, and infants are exposed to a variety of anesthetic agents for surgical or diagnostic procedures each year. Pregnant women sometimes undergo general anesthe- sia during their pregnancy for surgeries unrelated to the delivery, such as fetal and non-obstetric surgeries, especially during mid- gestation [9,10]. It is estimated that some 1–2% of pregnant women in developed countries undergo anesthesia during their pregnancy for surgery unrelated to the delivery [11].
+
+Since most general anesthetic agents are lipophilic and cross the placenta easily [12], the developing fetal brains will be exposed to anesthetics as well. We have previously shown that 1.3% isoflurane administered during pregnancy produced detectable effects to the rat pups [7,8]. Furthermore, in some cases, such as fetal surgery to correct various congenital malformations during mid-gestation, the fetal brain can be exposed to 2–3 times (2.5–3 Minimal Alveolar Concentration (MAC)) higher than clinically relevant concentrations of inhalation anesthetics to relax uterine smooth muscle and provide adequate anesthesia [10,13]. Fetal surgery is relatively new and rare, however, it is a rapidly growing and evolving area, and may become standard therapy for most disabling malformations that are currently treated in young infants [13,14]. Given the dose-dependent neurodegenerative properties
+
+F.-J. Kong et al. / Biochemical Pharmacology 84 (2012) 558–563
+
+of anesthetics, we hypothesized that high isoflurane concentra- tions normally used during fetal surgery causes spatial memory and in offspring.
+
+learning
+
+impairments and more neurodegeneration
+
+Here, we studied the potential effects of different isoflurane concentrations on neuroapoptosis and cognitive function on the offspring of pregnant rats exposed to anesthesia at gestational day 14. the changes of synaptic ultrastructure in the hippocampal area used transmission electron microscopy (TEM).
+
+In addition, we
+
+investigated
+
+2. Materials and methods
+
+2.1. Animals
+
+2.3.1. Place trials
+
+The place trials were performed at postnatal day 29 for 4 days to determine the rats’ ability to obtain spatial information. At postnatal day 28, rats were tested for their ability to swim to a visible platform through a 30-s swimming training. A dark black curtain surrounded the pool to prevent confounding visual cues. All rats received 4 trials per day in each of the four quadrants of the swimming pool. On each trial, rats were placed in a fixed position into the swimming pool facing the wall. They were allotted 120 s to find the platform upon which they sat for 20 s before being removed from the pool. If a rat did not find the platform within 120 s, the rat was gently guided to the platform and allowed to remain there for 20 s. For all training trials, swim speed and the time to reach the platform (escape latency) were recorded. The less time it took a rat to reach the platform, the better the learning ability. We took the average of four trials as the escape latency each day.
+
+All of the animals were treated according to the guidelines of the Guide for the Care and Use of Laboratory Animals (China Ministry of Health). The Laboratory Animal Care Committee of Zhejiang University approved all experimental procedures and protocols. All efforts were made to minimize the number of animals used and their suffering. The dams were housed in polypropylene cages, and the room temperature was maintained at 22 8C, with a 12-h light–dark cycle. The dams at gestational day 14 were used for all experiments, because this time corresponds approximately to mid-gestation in humans [15,16], the period when most non-obstetric surgeries and fetal interventions are performed [9,10].
+
+2.3.2. Probe trials
+
+Probe trials were conducted immediately after the four-day period to evaluate memory retention capabilities. The probe trials involved the submerged platform of the third quadrant from the pool and allowing the rats to swim for 120 s in any of the four quadrants of the swimming pool. Time spent in the third quadrant and the number of original platform crossing in the third quadrant was recorded.
+
+2.4. Transmission electron microscopy
+
+2.2. Anesthesia exposure
+
+The dams were randomly divided into three groups: control, low concentration of isoflurane (1.3%), and high concentration of isoflurane (3%) treatment groups (n = 8). The dams were placed in plastic containers resting in water baths with a constant tempera- ture of 38 8C. In these boxes, pregnant rats in isoflurane treatment groups were exposed to 1.3 or 3% isoflurane (Lot 826005U, Abbott Laboratories Limited, USA) in a humidified 30% oxygen carrier gas for 1 h; the control group was exposed to simply humidified 30% oxygen without any inhalational anesthetic for 1 h. We chose 1.3% because it represents 1 MAC in the pregnant rats [17], and 3% is equal to (cid:2)2 MAC. The determination of anesthetic duration based on our preliminary study which indicated that maternal physiological states remained stable throughout a 1-h isoflurane exposure. The isoflurane concentration, oxygen and carbon dioxide levels in the box were monitored with an agent gas monitor (Vamos, Drager Medical AG & Co. KgaA, Germany). Otherwise, control and experimental animals were under treatment and environment. Arterial blood gases (ABG) and blood glucose were measured at the end of the 1-h anesthetic exposure. The rectal temperature was maintained at 37 (cid:3) 0.5 8C. After exposure, all the dams were returned to their cages and allowed to deliver naturally. The postnatal body weights of the rat pups were monitored.
+
+the same
+
+2.3. Memory and learning studies
+
+Four rat pups (2 females and 2 males) from each dam were selected to determine cognitive function at postnatal day 28 with a Morris Water Maze test with minor modifications [1]. A round pool (diameter, 150 cm; depth, 50 cm) was filled with warm (24 8C) opaque water to a height of 1.5 cm above the top of the movable clear 15-cm-diameter platform in the third quadrant. A video tracking system recorded the swimming motions of animals, and the data were analyzed using motion-detection software for the Morris Water Maze (Actimetrics Software, Evanston, IL, USA). After every trial, each rat was wiped before returning to its regular cage, kept warm and allowed free access to food.
+
+After the Morris Water Maze test, six pups per group were anesthetized with a lethal dose of Nembutal. The thoracic cavities were opened and perfused intracardially with 100 mL of normal saline. Then the hippocampus, including CA1 and dentate gyrus area, of each rat was taken out immediately. Immersion fixation was completed on tissues about 1 mm3 from the hippocampus. Samples were rinsed in cold phosphate-buffered saline (PBS) and placed in 2.5% glutaraldehyde at 4 8C for 4 h. The tissue was rinsed in buffer and post-fixed with 1% osmium tetroxide for 1 h. Then, the tissue was rinsed with distilled water before undergoing a graded ethanol dehydration series and was infiltrated using a mixture of half propylene oxide and half resin overnight. Twenty- four hours later, the tissue was embedded in resin. 120 nm sections were cut and stained with 4% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Ultrastructure changes of synapse in the hippocampus were observed under a transmission electron microscope (Philips Tecnai 10, Holland).
+
+2.5. Tissue section preparation
+
+After the Morris Water Maze test, two pups from each dam were anesthetized by intraperitoneal injection of a lethal dose of Nembutal. The aorta was cannulated and the animal was firstly perfused with 200 mL of normal saline, then with 250 mL of 4% formaldehyde (freshly made from paraformaldehyde) for 20– 30 min. The fixed brain was then removed from the cranial cavity and post-fixed overnight in the same fixative at 4 8C. The tissues were embedded in paraffin, and transverse paraffin sections containing the hippocampal area were mounted on silanecoated slides. Sections were deparaffinaged and rehydrated. Then the sections were treated for antigen retrieval with 10.2 mmol/L sodium citrate buffer, pH 6.1, for 20 min at 95 8C for immunohis- tochemistry.
+
+2.6. Immunohistochemistry for caspase-3
+
+Caspase-3 positive cells were measured in the hippocampal immunohistochemical methods described
+
+CA1 region, using
+
+559
+
+560
+
+F.-J. Kong et al. / Biochemical Pharmacology 84 (2012) 558–563
+
+previously [7,8]. The brain region was chosen because is particularly vulnerable to anesthesia-induced neurodegeneration [1] and is important to memory and learning. Briefly, the sections mentioned above were washed in 0.01 M PBS containing 0.3% Triton X-100 (pH 7.4, PBS-T), followed by blocking in 5% normal goat serum in 0.01 M PBS. The sections were then incubated in the primary antibodies rabbit polyclonal against anti-caspase-3 (1:200, Santa Cruz Biotechnology, USA) overnight at 4 8C. After a thorough wash in PBS, sections were incubated with biotinylated goat anti-rabbit IgG antibody (1:200, Wuhan Boster Biological Technology, Ltd., China) for 2 h at room temperature, followed by avidin–biotin–peroxidase complex solution (ABC, 1:100, Wuhan Boster Biological Technology, Ltd., China) for 2 h at room temperature. Immunolabeling was visualized with 0.05% diami- nobenzdine (DAB, Wuhan Boster Biological Technology, Ltd., China) plus 0.3% H2O2 in PBS and the reaction was stopped by rinsing the slides with 0.2 M Tris–HCl. Sections were mounted onto 0.02% poly-L-lysinecoated slides and allowed to dry at room temperature. Then the sections were dehydrated through a graded series of alcohols, cleared in xylene and finally coverslipped. Rat Immunoglobulin IgG (1:200, Biomeda Corporation, USA) was used instead of primary antibody as a negative control. Other chemicals used in this study were provided by Cell Signaling Technology (Beverly, MA). Three sections from hippocampal CA1 region of each animal were randomly selected and images were photographed under 400(cid:4) magnification in 3 visual fields/per section, the caspase-3 positive neurons were counted in the same area. The optical densities of caspase-3 positive neurons were measured quantitatively using Image-Pro Plus version 6.0 (Media Cybernet- ics, Inc., Silver Spring, USA). The optical density of caspase-3 positive cells in a particular brain region was calculated by dividing the integrated optical density of caspase-3 positive cells by the area of that brain region.
+
+it
+
+treatment groups on any measured variables for ABG values and blood glucose levels. Taking these measures reduces the possibility that isoflurane-induced neurodegeneration in the fetal brains was caused by physiologic side effects (e.g. hypoglycemia, hypoxia and hypercapnia). All pups were viable and there were no significant differences in growth rate of the rat pups among the three groups (P0, 7.23 (cid:3) 0.55, 7.24 (cid:3) 0.49 and 7.18 (cid:3) 0.67 g; P28, 102.26 (cid:3) 3.45, 103.19 (cid:3) 4.15 and 101.78 (cid:3) 4.15 g, in control, 1.3% and 3% isoflurane- exposed pups, respectively).
+
+2.7. Statistical analysis
+
+All data were presented as mean (cid:3) S.E.M. Results of weight of postnatal rat pups and place trials of postnatal rats were analyzed using 2-way ANOVA for repeated measurements. Other data were analyzed using one-way ANOVA, followed by Tukey post hoc multiple comparison tests. A P value of <0.05 was considered statistically significant. All statistical tests and graphs were performed or generated, respectively, using Graph-Pad Prism Version 4.0 (Graph- Pad Prism Software, Inc., CA, USA).
+
+3. Results
+
+3.1. Physiologic parameters
+
+As shown in Table 1, ABG values and blood glucose levels were within the normal physiologic range. There were no significant differences between the low and high concentrations of isoflurane
+
+Table 1 Maternal physiological parameters during isoflurane exposure.
+
+0 h
+
+1 h
+
+1.3% Iso
+
+3% Iso
+
+1.3% Iso
+
+3% Iso
+
+pH PaCO2 (mmHg) PaO2 (mmHg) SaO2 (%) Glucose (mg/dl)
+
+7.45 (cid:3) 0.02 40.8 (cid:3) 2.01 159 (cid:3) 5.46 96.7 (cid:3) 1.3 114 (cid:3) 18
+
+7.41 (cid:3) 0.02 40.6 (cid:3) 1.64 162 (cid:3) 4.25 95.3 (cid:3) 1.1 116 (cid:3) 20
+
+Values are mean (cid:3) S.E.M. n = 8 for each group. Iso = isoflurane; PaCO2 = arterial carbon dioxide tension; SaO2 = arterial oxygen saturation.
+
+7.44 (cid:3) 0.02 43.6 (cid:3) 2.65 163 (cid:3) 6.98 96.1 (cid:3) 0.9 115 (cid:3) 21
+
+7.36 (cid:3) 0.01 43.9 (cid:3) 3.17 166 (cid:3) 5.45 95.4 (cid:3) 1.2 114 (cid:3) 16
+
+tension; PaO2 = arterial oxygen
+
+Fig. 1. Rats exposed to isoflurane in utero at high concentration (3%) impaired postnatal memory and learning ability in the Morris Water Maze test. (A) Place trial demonstrating the latency for offspring rats to reach platform measuring spatial information acquisition. (B and C) Probe trial demonstrating the number of original platform crossing (B) and the time spent in the third quadrant (C) measuring memory retention capabilities. Iso, isoflurane. Data represent mean (cid:3) S.E.M. of 32 postnatal rats from 8 pregnant mothers (n = 8) in each group. *P < 0.05 compared with both control and 1.3% Iso.
+
+F.-J. Kong et al. / Biochemical Pharmacology 84 (2012) 558–563
+
+3.2. Morris Water Maze test
+
+Pups whose mother anesthetized with 3% isoflurane showed a significantly worse performance in the water maze. As shown in Fig. 1A, pups in all groups showed a rapid decrease in latency. While the pups of the 3% isoflurane group spent more time to find the platform than those of 1.3% isoflurane group and control group in the place trial (F(2,63.969) = 4.715, P < 0.05). Swimming speeds
+
+were also analyzed during place trials, and no differences were observed among three groups. In the probe test, the number of crossing over the former platform location in 3% isoflurane-treated pups was fewer than the others (F(2,29) = 5.265, P < 0.05; Fig. 1B), as well as the time spent in the third quadrant where the platform (F(2,29) = 4.417, P < 0.05; Fig. 1C). There were no located significantly differences either in the place trial or in the probe test between the control and 1.3% isoflurane groups.
+
+Fig. 2. Rats exposed to isoflurane in utero at high concentration (3%) increased apoptosis in the hippocampus CA1 region. (Aa) Caspase-3 immunohistochemical staining in control pups (cid:4)400. (Ab) Caspase-3 immunohistochemical staining in 1.3% isoflurane-exposed pups (cid:4)400. (Ac) Caspase-3 immunohistochemical staining in 3% isoflurane- exposed pups (cid:4)400. (B) The number (Ba) and optical density (Bb) of caspase-3 positive neurons in each group. Iso, isoflurane. Data represent mean (cid:3) S.E.M. of 48 sections of 16 postnatal rats from 8 pregnant mothers (n = 8) in each group. **P < 0.01 compared to control, ***P < 0.001 compared with both control and 1.3% Iso, ### P < 0.001 compared to 1.3% Iso. Scale bar = 50 mm.
+
+Fig. 3. Ultrastructural changes of synapse in the CA1 and dentate gyrus area of hippocampus under TEM. (A) TEM showed that 3% isoflurane (c) significantly decreased the number of synapses in pups compared to control (a) and 1.3% isoflurane (b) group (magnification, (cid:4)6200). Scale bar = 2 mm. (B) Higher magnification image (magnification, (cid:4)24,000) showed widened synaptic cleft and disintegration of postsynaptic densities in utero. Arrows = synaptic cleft; arrowheads = postsynaptic densities. Scale bar = 0.5 mm.
+
+in pups after 3%
+
+isoflurane exposure
+
+561
+
+562
+
+F.-J. Kong et al. / Biochemical Pharmacology 84 (2012) 558–563
+
+3.3. Immunoreactivity assay
+
+Table 2 The hippocampal synaptic structural parameters among groups.
+
+The 1.3% isoflurane for 1 h did not significantly affect the number and optical densities of caspase-3 positive neurons in the hippocampal CA1 region of the pups when compared with the control (Fig. 2Aa and b). However, 3% isoflurane for 1 h significantly increased caspase-3 number (222% increase over the control, F(2,27) = 13.55, P < 0.001; Fig. 2Ac and Ba) and optical densities in the CA1 region of the hippocampus (129% increase over the control, F(2,27) = 8.353, P < 0.01; Fig. 2Ac and Bb).
+
+Control
+
+1.3% isoflurane
+
+3% isoflurane
+
+Numerical density (N/mm3) Width of synaptic cleft (nm) Postsynaptic density (nm)
+
+2.45 (cid:3) 0.18 24.91 (cid:3) 2.01 76.59 (cid:3) 7.41
+
+2.41 (cid:3) 0.20 24.09 (cid:3) 2.16 75.52 (cid:3) 6.25
+
+1.44 (cid:3) 0.17* 33.26 (cid:3) 2.65* 50.25 (cid:3) 6.98*
+
+Data represent mean (cid:3) S.E.M. of 6 postnatal rats from 8 pregnant mothers (n = 8) in each group.
+
+P < 0.05 compared with both control and 1.3% isoflurane.
+
+3.4. Ultrastructure changes in synapse of hippocampus
+
+Synapses with postsynaptic densities, an inerratic synaptic cleft and a presynaptic vas were clearly visible in the control pups (Fig. 3Aa and Ba). The structure of synapse in the hippocampal CA1 and dentate gyrus region of pups whose mothers received 1.3% isoflurane treatment impaired (Fig. 3Ab and Bb). However, in the 3% isoflurane-treated pups, the number of synapses decreased in the dentate gyrus and CA1 area, while a widened synaptic cleft, thinned postsynaptic densities and loss of a presynaptic vas were observed (F(2,26) = 5.406, P < 0.05; Fig. 3Ac and Bc, Table 2).
+
+for 1 h was not significantly
+
+4. Discussion
+
+In the present study, we employed a new model, a maternal fetal rat model, to study the behavioral and neurotoxic effects of exposure to different isoflurane concentrations. The outcome of our study shows that 1 h of isoflurane anesthesia at a high concentration (3%) in pregnant rats impaired postnatal spatial memory and learning in the offspring rats, whereas pups that received low (1.3%) concentrations behaved similarly to control pups. Moreover, there was a tendency of increased apoptosis observed at the hippocampal in pups subject to high concentration of isoflurane anesthesia, as well as remarkable impairments of synaptic ultrastructure in the hippocampal CA1 and dentate gyrus region.
+
+level
+
+neurodegeneration, even cognitive deficits of their offspring. However, the effects of anesthesia used during the development of fetal brains on postnatal memory and learning ability are controversial, with transient improvement [21], no effects [22] and permanent impairment [1,7,8] all being reported. These discre- pancies could be due to methodological differences, species differences (rats vs. mice), pharmacological differences (isoflurane vs. sevoflurane), differences in anesthetic concentrations (0.5–2 MAC), or differences in anesthetic durations (1–6 h). Last but not the isoflurane exposure. Since different neurodevelopmental events are performed in their timing relative to gestational age, it is expected that the vulnerability of the brain to the adverse effects of the anesthetic agents would be different depending on the time of exposure. Correspondingly, behavioral outcome varies as a function of the neurodevelopmental events occurring at the time of exposure. Altered neurodevelopmental programming in utero, cognitive deficits, psychiatric disturbances, and other diseases may occur [23–25]. The time of isoflurane exposure in the current study corresponds approximately to mid- gestation in human, and studies in several animal species suggest that susceptibility limited to a brain developmental state corresponding to the human second trimester of pregnancy. Together with our previous study [7,8], these results suggest that whether isoflurane induces neurodegeneration in the fetal rat brain or subsequent cognitive impairments depending on the time of isoflurane exposure (mid-gestation), higher concentrations (3% or around 2 MAC for 1 h), and longer anesthetic durations (1.3% for 6 h). These results are consistent with the dose- and time- dependent toxic effects of isoflurane in tissue cultures [26,27] and newborn animals [1].
+
+least
+
+is the time of
+
+is
+
+important aspects of cognitive function. The Water Maze protocol evaluates long-term/reference memory that involves a sequence of specific molecular processes in the CA1 area of the hippocampus [18]. The place trials were performed to obtain spatial information and the probe trials were conducted to evaluate memory retention capabilities. Our results showed that prenatal exposure to isoflurane at a high concentration (3%) displayed deficits in postnatal spatial learning and memory capabilities in pups as manifested by the longer escape latency to reach the platform, the fewer times of original platform crossing and the less time spent in the target quadrant in the Morris Water Maze test. The lack of differences in swimming speeds of all groups excluded the possibility that sensorimotor disturbances in any of the groups could have influenced the learning and memory deficits observed in our study. These behavioral changes are unlikely to be associated with an indirect deleterious effect of isoflurane on pregnancy because maternal physiological parameters during isoflurane anesthesia were normal, and there were no differences in non-cognitive variables, such as litter size, viability, and weight among the three groups, further suggesting that the fetal rat brain was impaired by maternally administered 3% isoflurane.
+
+Learning and memory are
+
+to determine
+
+the rats’ ability
+
+The observation of impaired performance in a spatial learning and memory test after high concentration anesthesia in agreement with previous studies focusing on the developing neonatal brains of rodents [1,19] and the fetal brains of guinea pigs [20]. Taken together, these findings clearly reinforce the idea that high concentrations of isoflurane anesthesia are capable of causing
+
+is
+
+The tendency of increased apoptosis in the hippocampal CA1 region in pups exposed to 3% isoflurane is in agreement with the impaired performance of these rats in the water maze test. The hippocampus plays a major role in spatial learning and memory [28], and its synaptic plasticity is altered by the majority of agents used in general anesthesia [1,7,8]. It is widely recognized that there is a relationship between hippocampal synaptic plasticity and learning and memory [29–31]. In our study, 1.3% isoflurane treatment did not significantly affect normal structure of synapses. However, 3% induced sharp changes of synaptic ultrastructure in the dentate gyrus and CA1 area characterized by the decreased synapse number, the widened synaptic cleft and the thinned postsynaptic densities. The synaptic cleft is a region of information transmission among neurons and plays an important role in the dynamics of synaptic activity. The postsynaptic density is the material basis of synaptic efficacy. The thickness of postsynaptic densities and the ability of learning and memory training and memory retention go hand in hand [30,32]. A decreased number of synapses, a widened synaptic cleft and thinned postsynaptic densities changed synaptic activity. Taken together, we speculate that 3% isoflurane for 1 h significantly increased apoptosis in the CA1 area of hippocampus, leading to impairments in synapse structure and function and consequent damage in synaptic plasticity, and finally to spatial learning and memory deficits.
+
+isoflurane
+
+F.-J. Kong et al. / Biochemical Pharmacology 84 (2012) 558–563
+
+The mechanisms of inhalation anesthetic-mediated neurode- generation in the developing brain are still not clear. There is a hypothesis that inhalational anesthetics, such as isoflurane, might induce cell death processes through activation of g-aminobutyric acid type A receptors and/or inhibition of N-methyl-D-aspartate (NMDA) receptors in the developing brain [1,33,34], although this view has not been established definitively. Our recent studies suggest that CHOP and caspase-12-mediated ER stress-induced cell death appear to be the major mediators of anesthesia- mediated apoptotic cellular death [7]. In addition, we also demonstrate that inhalational anesthetics induce spatial memory and learning impairments through the down-regulation of GAP-43 and NPY in the hippocampus [8], the up-regulation of C/EBP homologous transcription factor protein (CHOP) and caspase-12 [7], and consequent impairments in synaptic plasticity [7,8]. Given the rapid development of fetal surgery [13,14] and the recent concerns over the possible harmful implications of the various anesthetic drugs at various stages of neurodevelopment, it is in urgent need of a better understanding of how maternal general anesthesia affects the developing fetal brain. A better understand- ing of the mechanisms of clinically relevant anesthetic neurotox- icity will help us to define the scope of the problem in humans and develop strategies that will minimize the possible harmful effects of general anesthesia to patients.
+
+[7] Kong FJ, Xu LH, He DQ, Zhang XM, Lu HS. Effects of gestational isoflurane exposure on postnatal memory and learning in rats. Eur J Pharmacol 2011; 670:168–74.
+
+[8] Kong FJ, Tang YW, Lou AF, Chen H, Xu LH, Zhang XM, et al. Effects of isoflurane exposure during pregnancy on postnatal memory and learning in offspring rats. Mol Biol Rep 2012;39:4849–55.
+
+[9] Goodman S. Anesthesia for nonobstetric surgery in the pregnant patient.
+
+Semin Perinatol 2002;26:136–45.
+
+[10] Tran KM. Anesthesia for fetal surgery. Semin Fetal Neonatal Med 2010;15:40–5. [11] Crowhurst JA. Anaesthesia for non obstetric surgery during pregnancy. Acta
+
+Anaesthesiol Belg 2002;53:295–7.
+
+[12] Dwyer R, Fee JP, Moore J. Uptake of halothane and isoflurane by mother and
+
+baby during caesarean section. Br J Anaesth 1995;74:379–83.
+
+[13] Myers LB, Cohen D, Galinkin J, Gaiser R, Kurth CD. Anesthesia for fetal surgery.
+
+Paediatr Anaesth 2002;12:569–78.
+
+[14] Goldsmith MF, Straus SE, Kupfer C, Lenfant C, Collins F, Hodes RJ, et al. 2020
+
+vision: NIH heads foresee the future. JAMA 1999;282:2287–90.
+
+[15] Clancy B, Darlington RB, Finlay BL. Translating developmental time across
+
+mammalian species. Neuroscience 2001;105:7–17.
+
+[16] Clancy B, Kersh B, Hyde J, Darlington RB, Anand KJ, Finlay BL. Web-based method for translating neurodevelopment from laboratory species to humans. Neuroinformatics 2007;57:9–94.
+
+[17] Mazze RI, Rice SA, Baden JM. Halothane, isoflurane, and enflurane MAC in pregnant and nonpregnant female and male mice and rats. Anesthesiology 1985;62:339–41.
+
+[18] Izquierdo I, Medina JH, Vianna MR, Izquierdo LA, Barros DM. Separate mecha-
+
+nisms for short- and long-term memory. Behav Brain Res 1999;103:1–11.
+
+[19] Ma D, Williamson P, Januszewski A, Nogaro MC, Hossain M, Ong LP, et al. Xenon mitigates isoflurane-induced neuronal apoptosis in the developing rodent brain. Anesthesiology 2007;106:746–53.
+
+Acknowledgments
+
+[20] Rizzi S, Carter LB, Ori C, Jevtovic-Todorovic V. Clinical anesthesia causes perma- nent damage to the fetal guinea pig brain. Brain Pathol 2008;18:198–210. [21] Li Y, Liang G, Wang S, Meng Q, Wang Q, Wei H. Effects of fetal exposure to isoflurane on postnatal memory and learning in rats. Neuropharmacology 2007;53:942–50.
+
+We thank Xiao-Ming Zhang, M.D., Ph.D. (Associate Professor, Institute of Anatomy and Cell Biology, School of Medicine, Zhejiang University, China) for technical support and Shu Han, M.D., Ph.D. (Associate Professor, Institute of Anatomy and Cell Biology, School of Medicine, Zhejiang University, China) for his statistical assistance and thought-provoking discussions. Our work was supported by Medical and Health Research Fund of Health Department of Zhejiang Provincial, China (No. 2010KYA129).
+
+[22] McClaine RJ, Uemura K, de la Fuente SG, Manson RJ, Booth JV, White WD, et al. General anesthesia improves fetal cerebral oxygenation without evidence of subsequent neuronal injury. J Cereb Blood Flow Metab 2005;25:1060–9. [23] Gluckman PD, Hanson MA, Bateson P, Beedle AS, Law CM, Bhutta ZA, et al. Towards a new developmental synthesis: adaptive developmental plasticity and human disease. Lancet 2009;373:1654–7.
+
+[24] Gluckman PD, Hanson MA, Cooper C, Thornburg KL. Effect of in utero and early- life conditions on adult health and disease. N Engl J Med 2008;359:61–73. [25] Le Pen G, Gourevitch R, Hazane F, Hoareau C, Jay TM, Krebs MO. Peri-pubertal maturation after developmental disturbance: a model for psychosis onset in the rat. Neuroscience 2006;143:395–405.
+
+[26] Wei H, Liang G, Yang H, Wang Q, Hawkins B, Madesh M, et al. The common inhalational anesthetic isoflurane induces apoptosis via activation of inositol 1,4,5-trisphosphate receptors. Anesthesiology 2005;108:251–60.
+
+References
+
+[1] Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zor- umski CF, et al. Early exposure to common anesthetic agents causes wide- spread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 2003;23:876–82.
+
+[2] Culley DJ, Baxter M, Yukhananov R, Crosby G. The memory effects of general anesthesia persist for weeks in young and aged rats. Anesth Analg 2003; 96:1004–9.
+
+[3] Culley DJ, Baxter MG, Yukhananov R, Crosby G. Longterm impairment of acquisition of a spatial memory task following isoflurane-nitrous oxide anes- thesia in rats. Anesthesiology 2004;100:309–14.
+
+[4] Wilder RT, Flick RP, Sprung J, Katusic SK, Barbaresi WJ, Mickelson C, et al. Early exposure to anesthesia and learning disabilities in a population-based birth cohort. Anesthesiology 2009;110:796–804.
+
+[5] Kalkman CJ, Peelen L, Moons KG, Veenhuizen M, Bruens M, Sinnema G, et al. Behavior and development in children and age at the time of first anesthetic exposure. Anesthesiology 2009;110:805–12.
+
+[27] Yang H, Liang G, Hawkins BJ, Madesh M, Pierwola A, Wei HF. Inhalational anesthetics induce cell damage by disruption of intracellular calcium homeo- stasis with different potencies. Anesthesiology 2008;109:243–50.
+
+[28] Broadbent NJ, Squire LR, Clark RE. Spatial memory, recognition memory, and
+
+the hippocampus. Proc Natl Acad Sci U S A 2004;101:14515–20.
+
+[29] Sametsky EA, Disterhoft JF, Geinisman Y, Nicholson DA. Synaptic strength and postsynaptically silent synapses through advanced aging in rat hippocampal CA1 pyramidal neurons. Neurobiol Aging 2010;31:813–25.
+
+[30] Thompson JV, Sullivan RM, Wilson DA. Developmental emergence of fear learning corresponds with changes in amygdale synaptic plasticity. Brain Res 2008;1200:58–65.
+
+[31] Gruart A, Munoz MD, Delgado-Garcia JM. Involvement of the CA3–CA1 syn- apse in the acquisition of associative learning in behaving mice. J Neurosci 2006;26:1077–87.
+
+[32] Ziff EB. Enlightening the postsynaptic density. Neuron 1997;19:1163–74. [33] Olney JW, Young C, Wozniak DF, Jevtovic-Todorovic V, Ikonomidou C. Do pediatric drugs cause developing neurons to commit suicide? Trends Phar- macol Sci 2004;25:135–9.
+
+[6] Monk TG, Weldon BC, Garvan CW, Dede DE, van der Aa MT, Heilman KM, et al. Predictors of cognitive dysfunction after major noncardiac surgery. Anesthe- siology 2008;108:18–30.
+
+[34] Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikranian K, et al. Blockade of NMDA receptors and apoptotic neurodegeneration in the devel- oping brain. Science 1999;283:70–4.
+
+563

new_pdfs/10.1016_j.biopha.2016.01.034.txt

@@ -0,0 +1,475 @@
+Biomedicine & Pharmacotherapy 78 (2016) 322–328
+
+Available
+
+online
+
+at
+
+ScienceDirect www.sciencedirect.com
+
+Original article
+
+Neuronal apoptosis may not contribute to the long-term cognitive dysfunction induced by a brief exposure to 2% sevoflurane in developing rats
+
+Yi Lu, Yan Huang, Jue Jiang, Rong Hu, Yaqiong Yang, Hong Jiang*, Jia Yan*
+
+Department of Anesthesiology, Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University, School of Medicine, 639 Zhi Zao Ju Road, Shanghai 200011, China
+
+A R T I C L E
+
+I N F O
+
+A B S T R A C T
+
+Article history: Received 7 November 2015 Accepted 26 January 2016
+
+Keywords: Anesthesia Sevoflurane Neurotoxicity Neurodevelopment Apoptosis Developing brain
+
+Background: Sevoflurane is an inhaled anesthetic commonly used in the pediatric. Recent animal studies suggest that early exposure to high concentration of sevoflurane for a long duration can induce neuroapoptosis and later cognitive dysfunction. However, the neurodevelopmental impact induced by lower concentration and shorter exposure duration of sevoflurane is unclear. To investigate whether early exposure to 2% concentration of sevoflurane for a short duration (clinically relevant usage of sevoflurane) can also induce neuroapoptosis and later cognitive dysfunction. Methods: Rat pups were subjected to control group, 2% sevoflurane for 3 h and 3% sevoflurane for 6 h. TUNEL assay and apoptotic enzyme cleaved caspase-3 measured by western blot were used for detection of neuronal apoptosis in frontal cortex and CA1 region of hippocampus 24 after sevoflurane treatment. Long-term cognitive function was evaluated by Morris water maze and passive avoidance test as the rats grew up. Results: The apoptotic levels in frontal cortex and CA1 region were significantly increased after rats exposed to 3% sevoflurane for 6 h (P 0.05), but not 2% sevoflurane for 3 h (P > 0.05). Exposure to both 2% sevoflurane for 3 h and 3% sevoflurane for 6 h could cause long-term cognitive dysfunction and animals exposed to 3% sevoflurane for 6 h exhibited worse neurodevelopmental outcomes (P Conclusion: It was suggested that neuronal apoptosis might not contribute to long-term cognitive dysfunction induced by 2% concentration and short exposure time of sevoflurane. Our findings also suggested that the mechanisms of sevoflurane-induced neurodevelopmental impact might be various, depending on the concentration and exposure duration. ã
+
+<
+
+<
+
+0.05).
+
+2016 Elsevier Masson SAS. All rights reserved.
+
+1. Introduction
+
+Over the past two decades, a series of animal experiments have indicated that various general anesthetics may be neurotoxic to the developing brain [1–5]. Thus, the potential for anesthetics-induced developmental neurotoxicity is of concern to the anesthesiologists, surgeons and parents of children undergoing surgery. The risks of childhood anesthesia have recently emerged as a public health concern [6,7]. In 2009, the U.S. Food and Drug Administration (FDA) established a partnership with the International Anesthesia Research Society (IARS) SmartTot to offer funds for preclinical and clinical studies concerning anesthetics-related neurodevelopmen- tal and
+
+issues
+
+[8]. Moreover,
+
+the Pediatric Anesthesia
+
+NeuroDevelopment Assessment (PANDA) study team holds a biennial scientific symposia to review recent preclinical and clinical data related to anesthetic neurotoxicity [9,10].
+
+inhaled anesthetics [3]. Sevoflurane is a commonly used inhaled anaes- thetic for the induction and maintenance of general anesthesia during surgery. Because it has the advantage of a low blood–gas partition coefficient and pungency, sevoflurane is widely used as a pediatric anesthetic. Recently, several animal studies reported that early-life long (6–9 h) exposure to high concentrations (3–4%) of sevoflurane could cause neuronal apoptosis and subsequent long- term cognitive impairment [11–15]. However, 3 h exposure to the lower concentrations (1–2%) of sevoflurane more closely approx- imates typical general pediatric anesthetic episodes for anesthesia maintenance [16]. Whether lower concentrations and a shorter duration of exposure to sevoflurane can induce neuronal apoptosis and later cognitive impairment is unclear.
+
+Every year, millions of children are exposed to
+
+Corresponding authors.
+
+E-mail addresses: mzkyanj@163.com (J. Yan), dr_hongjiang@163.com (H. Jiang).
+
+http://dx.doi.org/10.1016/j.biopha.2016.01.034 0753-3322/ ã
+
+2016 Elsevier Masson SAS. All rights reserved.
+
+Y. Lu et al. / Biomedicine & Pharmacotherapy 78 (2016) 322–328
+
+323
+
+The immature human brain is most vulnerable to neurotoxic agents during the (BGS), which begins at mid- gestation and continues for 2–3 years after birth [17–19]. In rodents, the window of vulnerability to neurotoxic agents occurs first 2–3 weeks after birth [20,21]. In this primarily during the study, we aimed to investigate whether 3 h of exposure to 2% sevoflurane, as used in clinical practice, has pro-apoptotic effects on the developing brain and impairs the long-term cognitive function in rats in the same manner as prolonged exposure to high concentrations.
+
+“brain growth spurt”
+
+2. Materials and methods
+
+2.1. Animals
+
+in rodents occurs on postnatal day (PND) 7 [22], Sprague-Dawley (SD) PND7 rats weighing 14–18 g, provided by the Animal Center of Shanghai Jiao Tong University School of Medicine (Shanghai, China) were used in this study. The housing and treatment of the animals were in accordance with the National Institutes of Health guidelines for animal experimentation and approved by the institutional animal care and use committee. The animals were kept on a 12-h light/dark cycle (light from 7 am to 7 pm) with room temperature (23
+
+Because peak
+
+anesthesia-induced neurodegeneration
+
+(cid:1)
+
+1 (cid:3)C).
+
+Animals were killed by lethal injection of pentobarbital at the time of blood sampling.
+
+2.4. Analysis of apoptotic levels
+
+2.4.1. TUNEL assay of brain
+
+Twenty-four hours after sevoflurane exposure, six rats from each group (n = 6) were anesthetized with sodium pentobarbital fixed, dehydrated and made into and the brains were perfused, paraffin sections (5 mm), as described previously [24]. Apoptotic cells in the brain sections were detected by TUNEL Assay using the FragELTM DNA Fragmentation Detection Kit (Merck, Darmstadt, Germany), according to the manufacturer’s protocol. Briefly, brain sections were permeabilized with proteinase K (20 mg/ml) at room temperature for 20 min. Endogenous peroxidase was inactivated by 3% H2O2. Specimens were incubated for 1.5 h with terminal deoxynucleotidyl transferase (TdT) labelling reaction mixture, and apoptotic cells were visualized with 3,30-diaminobenzidine (DAB), and normal nuclei were counterstained with methyl green. Because to anesthetics at PND7 and the hippocampus is closely related to learning and memory [25], the number of apoptotic neurons in the frontal cortex and the CA1 region of the hippocampus was quantified. We selected two random viewing fields (400(cid:5)) per region (frontal cortex and CA1) from one brain section per animal for analysis in a double blinded manner.
+
+the cerebral cortex
+
+reaches peak vulnerability
+
+2.2. Sevoflurane exposure
+
+Rat pups were separated from their mothers for acclimatization prior to sevoflurane exposure. Pups from the same litter were randomly allocated to three different groups. Totally, ninety PND7 rats were included in this study (n = 30 for each group). Rats in the control group received 100% oxygen for 6 h in a chamber at 37 (cid:3)C. Rats in the other two groups were exposed to either 2% sevoflurane (SEVOFRANE1, Osaka, (Sevo1 group) or 3% sevoflurane for 6 h (Sevo2 group) under 100% oxygen in the same chamber at 37 (cid:3)C as described previously [13]. The concentration of sevoflurane in the chamber was monitored and maintained by a flow to the vaporizer as we described previously [23]. The gas chamber was 2 l/min. We chose these treatments because 3 h exposure to 2% seveflurane more closely approximates typical general pediatric anesthetic episodes for anesthesia maintenance [16] and 6 h exposure to 3% sevoflurane can cause neuronal apoptosis in developing animals [11,12,14,15].
+
+Japan)
+
+for 3 h
+
+2.3. Arterial blood gas analysis
+
+2.4.2. Western blot
+
+Apoptosis was also assessed using western blot to quantify cleaved caspase-3 (Cl-Csp3) in all groups (n = 6). Briefly, tissue samples of the frontal cortex and CA1 region were collected from three groups twenty-four hours after sevoflurane exposure. Tissues were lysed in a buffer containing a protease inhibitor cocktail (Calbiochem, San Diego, CA, USA) and homogenated. The homogenate was centrifuged and the supernatant was collected for further analysis. Protein concentrations were measured by BCA Protein Assay Kit (Novagen, San Diego, CA, USA). Equal amounts of protein were boiled in loading buffer (Beyotime, Beijing, China) and separated by 10% polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose, and the blots were probed overnight with anti-cleaved caspase-3 (1:200, Millipore, Darm- stadt, Germany) and b-actin antibodies (1:500, internal standard, Santa Cruz, San Diego, CA, USA) at 4 (cid:3)C. Primary antibodies were visualized using secondary antibodies conjugated to horseradish peroxidase (Santa Cruz, San Diego, CA, USA) and ECL reagent (Pierce, Rockford, IL, USA). Quantitative analysis of Cl-Csp3 was normalized to b-actin using the Quantity One software.
+
+To determine adequacy of ventilation and oxygenation, arterial blood samples (n = 6) were obtained from the left cardiac ventricle in each group at the end of anesthesia, and the samples were immediately analyzed by a blood gas analyzer (Radiometer, ABL800, Denmark). We compared the pH, pO2, pCO2, oxygen saturation (sO2), and the concentrations of blood glucose (Glu), (cid:4)) among the groups. lactic acid (Lac) and bicarbonate (HCO3
+
+2.5. Neurologic assessment
+
+2.5.1. Morris water maze
+
+To assess neurodevelopmental outcomes, particularly the learning and memory functions of juveniles, rats from all groups
+
+Table 1 Arterial blood gas analysis for the three groups.
+
+pH
+
+pCO2 (mmHg)
+
+pO2 (mmHg)
+
+sO2 (%)
+
+Lac (mmol/l)
+
+HCO3
+
+(cid:4) (mmol/l)
+
+Glu (mmol/l)
+
+Control 2%Sevo 3h 3%Sevo 6h
+
+7.463 7.417 7.404
+
+(cid:1) (cid:1) (cid:1)
+
+0.030 0.025 0.045
+
+29.4 30.6 32.3
+
+(cid:1) (cid:1) (cid:1)
+
+1.2 1.1 1.5
+
+117.5 113.9 108.0
+
+(cid:1) (cid:1) (cid:1)
+
+4.8 6.9 5.4
+
+99.6 99.5 99.3
+
+(cid:1) (cid:1) (cid:1)
+
+0.1 0.1 0.2
+
+1.7 1.8 1.9
+
+(cid:1) (cid:1) (cid:1)
+
+0.2 0.2 0.2
+
+20.2 21.2 21.0
+
+(cid:1) (cid:1) (cid:1)
+
+0.5 0.7 0.8
+
+5.5 5.5 5.9
+
+(cid:1) (cid:1) (cid:1)
+
+0.3 0.4 0.4
+
+aP
+
+<
+
+0.05 compared to the control group.
+
+324
+
+Y. Lu et al. / Biomedicine & Pharmacotherapy 78 (2016) 322–328
+
+Fig. 1. TUNEL assay for apoptosis of frontal cortex and CA1 region of hippocampus in control and treatment groups. (A) Representative images of frontal cortex and CA1 region. TUNEL-positive apoptotic nuclei were stained by brown and normal nuclei were stained by cyan. Scale bar = 50 mm. (B) Quantification of apoptotic nuclei in the frontal cortex and CA1 region. *P
+
+<
+
+0.05 compared to control group, #P
+
+<
+
+0.05 compared to Sevo1 group.
+
+Fig. 2. Western blot analysis of apoptotic enzyme Cl-Csp3 from frontal cortex and CA1 region of hippocampus in control and treatment groups. (A) Representative blots of Cl- Csp3. b-actin was used as the internal standard. (B) Quantification of the target protein expression levels. *P 0.05 compared to Sevo1 group.
+
+<
+
+0.05 compared to control group, #P
+
+<
+
+Y. Lu et al. / Biomedicine & Pharmacotherapy 78 (2016) 322–328
+
+Fig. 3. Morris water maze test for the evaluation of learning and memory when the rats grew up to adolescence in control and treatment groups. (A) Statistical analysis of finding the former platform. (C) Representative traces of the paths latency in swum by the rats after the end of probe trials. The white circle in left lower quadrant represents the removed platform. (D) Statistical analysis of the number of times the former platform was crossed by the rats. (E) Statistical analysis of the percentage of time spent by the rats in the target quadrant. *P 0.05 compared to control group, #P
+
+finding the hidden platform on trial days. (B) Statistical analysis of swimming distance before
+
+<
+
+<
+
+0.05 compared to Sevo1 group.
+
+were subjected to Morris water maze after reaching 6 weeks of age (n = 12), as previously described [24]. Briefly, a circular pool (1.6 m diameter, 60 cm height) was used for the water maze, and a submerged platform (10 cm diameter, 2 cm below the surface of fixed position in the pool. The water the water) was located at a 1 (cid:3)C. Probe trials were conducted twice (cid:1) temperature was set at 23 five consecutive days. In the trials, rats were trained to a day for finding swim to and locate the hidden platform. The time spent in the hidden platform and the swimming distance before reaching the platform were recorded. After the probe trials, the platform was removed, and the rats were allowed to swim freely for 120 s: the number of times that the former platform was crossed and the percentage of time spent in the target quadrant were determined. The entire behavioral test was recorded and analyzed using a MS-
+
+type Water Maze Video analysis system (Chengdu Instrument Ltd., Chengdu, China). Finally, to investigate cognitive function during development, the passive avoidance test was performed at 3 months.
+
+2.5.2. Passive avoidance test
+
+The passive avoidance test was performed as previously described [26]. The apparatus used for the passive avoidance test included a behavioral stimulation controller and a video shuttle box (Chengdu Instrument Ltd., Chengdu, China). The test relies the natural preference of rats for darkness. Briefly, on the first trial day, the rats were placed in the illuminated compartment after 2 min of habituation to the dark compartment and allowed to re-enter the dark compartment. On the following day, an electric foot shock was
+
+325
+
+326
+
+Y. Lu et al. / Biomedicine & Pharmacotherapy 78 (2016) 322–328
+
+floor of the dark compartment after the delivered through the grid rats entered. Twenty-four hours later, the retention of passive avoidance was determined by comparing the time elapsed prior to re-entry into the dark compartment with the arbitrary maximum time of 180 s.
+
+2.6. Statistical analysis
+
+(cid:1)
+
+SEM. SAS 9.2 (SAS Institute Inc., Cary, North Carolina, USA) was used for statistical analysis. One-way ANOVA was used to determine statistically significant differences between the three groups, and Tukey’s post hoc analysis was performed to correct for multiple comparisons when applicable. Statistical significance was accepted as P
+
+All data are expressed as the mean
+
+<
+
+0.05.
+
+3. Results
+
+3.1. Neonatal sevoflurane exposure does not induce metabolic or respiratory distress
+
+indicate any signs of metabolic or respiratory distress in animals exposed to 2% sevoflurane for 3 h or 3% sevoflurane for 6 h, and the pH, pO2, (cid:4) did not differ significantly among pCO2, sO2, Glu, Lac and HCO3 the three groups (Table 1). Thus, there is little probability of brain injury induced by metabolic or respiratory distress during sevoflurane anesthesia.
+
+Arterial blood gas analysis did not
+
+3.2. Exposure to 2% sevoflurane for 3 h did not induce neuroapoptosis in the developing brain
+
+0.05, Fig. 3C–E). These parameters were further decreased in 0.05, Fig. 3C–E). Long-term cognitive function of the three groups was also evaluated by the passive avoidance test when these rats became adults (3 months old). The results showed that the latency to re- enter the dark compartment was significantly decreased in both the Sevo1 and Sevo2 groups, with the Sevo2 rats displaying the findings demonstrated a shortest latency (P long-term following early exposure to both 2% sevoflurane for 3 h and 3% sevoflurane for 6 h.
+
+<
+
+(P the Sevo2 group, compared to the Sevo1 group (P
+
+<
+
+<
+
+0.05, Fig. 4). These learning and memory deficiencies
+
+4. Discussion
+
+Sevoflurane is a volatile anesthetic that is frequently used in children as a sole agent or intravenous anesthetics. It is believed to be a g-aminobutyrate acid agonist (GABAA) [8,27]. Recently, animal experiments have suggested that by promoting neuronal apoptosis, sevoflurane is neurotoxic to the immature brain [3,11,12,14,28]. However, the experimental animals in these studies generally underwent a prolonged exposure to high concentration of sevoflurane by inhalation, which is unusual for clinical practice. In this study, we provided in vivo evidence that a clinically-relevant concentration and exposure duration of sevo- flurane (2% for 3 h) did not enhance neuronal apoptosis in two specific regions of the developing brain, whereas long-term cognitive deficiency still existed. This finding is important because the results indicated that enhanced neuronal apoptosis may not contribute to the long-term cognitive dysfunction induced by sevoflurane in normal clinical applications. Other potential mechanisms of sevoflurane-induced long-term cognitive deficien- cy should be studied to address the possible neurotoxicity.
+
+in conjunction with
+
+To study whether the clinically-relevant usage of sevoflurane (2% for 3 h) can induce neuroapoptosis similar to prolonged (6 h) exposure to high concentration in the developing brain, we assayed the apoptotic levels of the frontal cortex and CA1 region of the hippocampus after different treatments by using TUNEL assay and detection of the apoptotic enzyme Cl-Csp3 through western blotting. The TUNEL assay demonstrated no significant differences in apoptotic neuronal cells in either the frontal cortex or CA1 region of rats exposed to 2% sevoflurane (P > 0.05). However, exposure to 3% sevoflurane for 6 h increased the numbers of apoptotic cells in both the frontal cortex and CA1 region, compared with the control and Sevo1 groups (P
+
+(3%) of sevoflurane
+
+for 3 h versus
+
+the controls
+
+<
+
+0.05, Fig. 1).
+
+In addition, there was no significant difference between the Sevo1 and control groups in Cl-Csp3 protein expression analyzed by western blot (P > 0.05). However, Cl-Csp3 expression was significantly increased in the Sevo2 group (P
+
+<
+
+0.05, Fig. 2).
+
+To study apoptotic effects, it is important to obtain results from more than one independent method (preferably several methods) before drawing a conclusion [29]. Thus, to assess apoptosis, we performed not only TUNEL assays but also detection of the apoptotic enzyme Cl-Csp3. Moreover, two independent behavioral tests were conducted to evaluate long-term cognitive outcomes after the rats reached adulthood. The results of the two assays were equivalent.
+
+Early exposure to sevoflurane in rats results in significant concentration and exposure duration-dependent impairment in adulthood memory [13]. In this study, we found that early exposure to 3% sevoflurane for 6 h induced worse neurodevelop- mental outcomes than 2% sevoflurane for 3 h. Our results also confirmed the dose- and time-dependent neurotoxic effect of sevoflurane on the developing brain. Moreover, we demonstrated that a lower inhaled concentration and shorter exposure duration to sevoflurane long-term cognitive dysfunction, although no enhancement of neuronal apoptosis
+
+in early
+
+life could cause
+
+3.3. Exposure to both 2% sevoflurane for 3 h and 3% sevoflurane for 6 h early in life can cause long-term cognitive dysfunction
+
+To investigate the effects of early sevoflurane exposure on long- first subjected the rats from the control term cognitive function, we and treatment groups to the Morris water maze test when they were 6 weeks old. The result showed that the latency and swimming distance were significantly increased in rats from the Sevo1 and Sevo2 groups on trial day 4 and 5, compared to control group (P 0.05, Fig. 3A and B). Moreover, it took the Sevo2 group find the platform on trial day 4 and 5, compared to the more time to Sevo1 group (P 0.05, Fig. 3A). The rats from Sevo2 group also swam further than did the Sevo1 group on trial day 4 and 5 (P 0.05, Fig. 3B). The number of times that the rats crossed the platform and percentage of time spent in the target quadrant were significantly decreased in both the Sevo1 and Sevo2 groups
+
+<
+
+<
+
+<
+
+Fig. 4. Passive avoidance test for the cognitive function the rats grew up to adulthood in control and treatment groups. *P 0.05 compared to control group, #P
+
+<
+
+<
+
+0.05 compared to Sevo1 group.
+
+Y. Lu et al. / Biomedicine & Pharmacotherapy 78 (2016) 322–328
+
+was found. There may be different mechanisms responsible for the neurotoxicity induced by the varying treatment conditions of sevoflurane.
+
+The mechanism of neurodevelopmental impairment mediated by a brief exposure to lower concentration of sevoflurane is still not clear. Wu et al. suggested that physiological disturbance may contribute to sevoflurane-induced long-term learning and memo- ry dysfunction immature rats [30]. However, under our sevoflurane treatment conditions, no metabolic or respiratory distress was found in PND7 rats via arterial blood gas analysis. Therefore, physiological disturbance may be excluded in this study. Although the mechanism of neurotoxicity caused by sevoflurane is not fully understood, several recent studies provide clues to possible novel mechanisms of sevoflurane-induced neurotoxicity. First, Hu et al. suggested that by increasing tau protein expression, sevoflurane could change the arrangement of the microtubule cytoskeleton in the hippocampus of the developing rat brain [31]. Second, several researchers have found that early sevoflurane exposure had an impact on rat neuronal ultrastructure compo- nents, such as dendritic spines, synapses and mitochondria [28,32]. However, these studies did not demonstrate whether these mechanisms were associated with the long-term neuro- developmental impact induced by sevoflurane. Therefore, further studies are needed to explore the relationship between neuronal injury mechanisms and neurodevelopmental outcomes.
+
+in
+
+induce apoptotic neurodegeneration in the infant mouse brain, Br. J. Pharmacol. 146 (2005) 189–197.
+
+[2] M.M. Straiko, C. Young, D. Cattano, C.E. Creeley, H. Wang, D.J. Smith, et al.,
+
+Lithium protects against anesthesia-induced developmental neuroapoptosis, Anesthesiology 110 (2009) 862–868.
+
+[3] L. Pellegrini, Y. Bennis, L. Velly, I. Grandvuillemin, P. Pisano, N. Bruder, et al.,
+
+Erythropoietin protects newborn rat against sevoflurane-induced neurotoxicity, Paediatr. Anaesth. 24 (2014) 749–759.
+
+[4] H. Zheng, Y. Dong, Z. Xu, G. Crosby, D.J. Culley, Y. Zhang, et al., Sevoflurane
+
+anesthesia in pregnant mice induces neurotoxicity in fetal and offspring mice, Anesthesiology 118 (2013) 516–526.
+
+[5] W.-Y. Wang, Y. Luo, L.-J. Jia, S.-F. Hu, X.-K. Lou, S.-L. Shen, et al., Inhibition of
+
+aberrant cyclin-dependent kinase 5 activity attenuates isoflurane neurotoxicity in the developing brain, Neuropharmacology 77 (2014) 90–99.
+
+[6] K. Servick, Biomedical research: researchers struggle to gauge risks of
+
+childhood anesthesia, Science 346 (2014) 1161–1162.
+
+[7] A.W. Loepke, T.G. Hansen, Is this your (paediatric patient’s) brain on
+
+(anaesthetic) drugs? The search for a potential neurological phenotype of anaesthesia-related neurotoxicity in humans, Eur. J. Anaesthesiol. 32 (2015) 298–300.
+
+[8] B.A. Rappaport, S. Suresh, S. Hertz, A.S. Evers, B.A. Orser, Anesthetic
+
+neurotoxicity–clinical implications of animal models, N. Engl. J. Med. 372 (2015) 796–797.
+
+[9] T.L. Miller, R. Park, L.S. Sun, Report of the third PANDA symposium on
+
+anesthesia and neurodevelopment in children, J. Neurosurg. Anesthesiol. 24 (2012) 357–361.
+
+[10] T.L. Miller, R. Park, L.S. Sun, Report of the fourth PANDA Symposium on
+
+Anesthesia and Neurodevelopment in Children, J. Neurosurg. Anesthesiol. 26 (2014) 344–348.
+
+[11] H. Zhou, S. Li, X. Niu, P. Wang, J. Wang, M. Zhang, Protective effect of FTY720 against sevoflurane-induced developmental neurotoxicity in rats, Cell Biochem. Biophys. 67 (2013) 591–598.
+
+limitations. Although we provided evidence that apoptosis was not responsible for the long-term cognitive dysfunction induced by 3 h exposure to 2% sevoflurane, the mechanism was not demonstrated in our study, as described above. Furthermore, due to interspecies variability, experimental animal models may not completely represent the pathophysiologi- cal processes in humans. There are inherent limitations to translate preclinical data to human practice. However, the animal data should not be ignored.
+
+Our work has several
+
+5. Conclusion
+
+[12] W.Y. Wang, R. Yang, S.F. Hu, H. Wang, Z.W. Ma, Y. Lu, N-stearoyl-L-tyrosine
+
+ameliorates sevoflurane induced neuroapoptosis via MEK/ERK1/2 MAPK signaling pathway in the developing brain, Neurosci. Lett. 541 (2013) 167–172.
+
+[13] X. Shen, Y. Liu, S. Xu, Q. Zhao, X. Guo, R. Shen, et al., Early life exposure to
+
+sevoflurane impairs adulthood spatial memory in the rat, Neurotoxicology 39 (2013) 45–56.
+
+[14] X. Lei, W. Zhang, T. Liu, H. Xiao, W. Liang, W. Xia, et al., Perinatal
+
+supplementation with omega-3 polyunsaturated fatty acids improves sevoflurane-induced neurodegeneration and memory impairment in neonatal rats, PLoS One 8 (2013) e70645.
+
+[15] W.Y. Wang, H. Wang, Y. Luo, L.J. Jia, J.N. Zhao, H.H. Zhang, et al., The effects of
+
+metabotropic glutamate receptor 7 allosteric agonist N,N'- dibenzhydrylethane-1,2-diamine dihydrochloride on developmental sevoflurane neurotoxicity: role of extracellular signal-regulated kinase 1 and 2 mitogen-activated protein kinase signaling pathway, Neuroscience 205 (2012) 167–177.
+
+In this study, we have shown that early exposure to both 2% sevoflurane impair adulthood learning and memory function but only exposure to 3% sevoflurane for 6 h could reinforce neuronal apoptosis. We therefore concluded that neuronal apoptosis might not contribute to the long-term cognitive dysfunction induced by a brief exposure to a in developing rats. Moreover, the mechanisms of sevoflurane-induced neurodevelopmental impact might be vari- ous, depending on the concentration and exposure duration. Further studies regarding the mechanism for the sevoflurane- induced neurodevelopmental impact are urgently needed to develop methods to protect the immature brain.
+
+for 3 h and 3% sevoflurane
+
+for 6 h may
+
+lower concentration of sevoflurane
+
+findings suggested
+
+the
+
+that
+
+[16] X. Zou, T.A. Patterson, N. Sadovova, N.C. Twaddle, D.R. Doerge, X. Zhang, et al., Potential neurotoxicity of ketamine in the developing rat brain, Toxicol. Sci. 108 (2009) 149–158.
+
+[17] H. Hayashi, P. Dikkes, S.G. Soriano, Repeated administration of ketamine may lead to neuronal degeneration in the developing rat brain, Paediatr. Anaesth. 12 (2002) 770–774.
+
+[18] C. Ikonomidou, P. Bittigau, C. Koch, K. Genz, F. Hoerster, U. Felderhoff-Mueser,
+
+et al., Neurotransmitters and apoptosis in the developing brain, Biochem. Pharmacol. 62 (2001) 401–405.
+
+[19] L. Sun, Early childhood general anaesthesia exposure and neurocognitive
+
+development, Br. J. Anaesth. 105 (2010) i61–i68.
+
+[20] A. Fredriksson, T. Archer, H. Alm, T. Gordh, P. Eriksson, Neurofunctional deficits and potentiated apoptosis by neonatal NMDA antagonist administration, Behav. Brain Res. 153 (2004) 367–376.
+
+[21] F. Liu, M.G. Paule, S. Ali, C. Wang, Ketamine-induced neurotoxicity and changes in gene expression in the developing rat brain, Curr. Neuropharmacol. 9 (2011) 256–261.
+
+[22] A.W. Loepke, F.X. McGowan Jr., S.G. Soriano, CON: the toxic effects of
+
+Conflict of interest
+
+anesthetics in the developing brain: the clinical perspective, Anesth. Analg. 106 (2008) 1664–1669.
+
+[23] H. Jiang, Y. Huang, H. Xu, Y. Sun, N. Han, Q.F. Li, Hypoxia inducible factor-1alpha
+
+No conflicts of interest declared.
+
+is involved in the neurodegeneration induced by isoflurane in the brain of neonatal rats, J. Neurochem. 120 (2012) 453–460.
+
+[24] J. Yan, Y. Huang, Y. Lu, J. Chen, H. Jiang, Repeated administration of ketamine
+
+Acknowledgment
+
+can induce hippocampal neurodegeneration and long-term cognitive impairment via the ROS/HIF-1alpha pathway in developing rats, Cell. Physiol. Biochem. 33 (2014) 1715–1732.
+
+This work was supported by research funds from Shanghai Municipal Commission of Health and Family Planning (grant number: 201440356).
+
+[25] J.H. Yon, J. Daniel-Johnson, L.B. Carter, V. Jevtovic-Todorovic, Anesthesia
+
+induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways, Neuroscience 135 (2005) 815–827.
+
+[26] M. Ozarowski, P.L. Mikolajczak, A. Bogacz, A. Gryszczynska, M. Kujawska, J.
+
+References
+
+Jodynis-Liebert, et al., Rosmarinus officinalis L. leaf extract improves memory impairment and affects acetylcholinesterase and butyrylcholinesterase activities in rat brain, Fitoterapia 91 (2013) 261–271.
+
+[1] C. Young, V. Jevtovic-Todorovic, Y.Q. Qin, T. Tenkova, H. Wang, J. Labruyere,
+
+et al., Potential of ketamine and midazolam, individually or in combination, to
+
+[27] B.H. Lee, O.D. Hazarika, G.R. Quitoriano, N. Lin, J. Leong, H. Brosnan, et al., Effect of combining anesthetics in neonates on long-term cognitive function, Int. J. Dev. Neurosci. 37 (2014) 87–93.
+
+327
+
+328
+
+Y. Lu et al. / Biomedicine & Pharmacotherapy 78 (2016) 322–328
+
+[28] L.G. Amrock, M.L. Starner, K.L. Murphy, M.G. Baxter, Long-term effects of single or multiple neonatal sevoflurane exposures on rat hippocampal ultrastructure, Anesthesiology 122 (2015) 87–95.
+
+[31] Z.Y. Hu, H.Y. Jin, L.L. Xu, Z.R. Zhu, Y.L. Jiang, R. Seal, Effects of sevoflurane on the expression of tau protein mRNA and Ser396/404 site in the hippocampus of developing rat brain, Paediatr. Anaesth. 23 (2013) 1138–1144.
+
+[29] D.R. Schultz, W.J. Harrington Jr., Apoptosis: programmed cell death at a
+
+[32] A. Briner, M. De Roo, A. Dayer, D. Muller, W. Habre, L. Vutskits, Volatile
+
+molecular level, Semin. Arthritis Rheum. 32 (2003) 345–369.
+
+[30] B. Wu, Z. Yu, S. You, Y. Zheng, J. Liu, Y. Gao, et al., Physiological disturbance may contribute to neurodegeneration induced by isoflurane or sevoflurane in 14 day old rats, PLoS One 9 (2014) e84622.
+
+anesthetics rapidly increase dendritic spine density in the rat medial prefrontal cortex during synaptogenesis, Anesthesiology 112 (2010) 546–556.

new_pdfs/10.1016_j.bja.2018.04.034.txt

@@ -0,0 +1,343 @@
+British Journal of Anaesthesia, 121 (2): 406e416 (2018)
+
+doi: 10.1016/j.bja.2018.04.034
+
+Advance Access Publication Date: 5 June 2018
+
+Neuroscience and Neuroanaesthesia
+
+Role of epigenetic mechanisms in transmitting the effects of neonatal sevoflurane exposure to the next
+
+generation of male, but not female, rats L.-S. Ju1, J.-J. Yang1, T. E. Morey1, N. Gravenstein1,2, C. N. Seubert1, J. L. Resnick3, J.-Q. Zhang4 and A. E. Martynyuk1,2,*
+
+1Department of Anesthesiology, University of Florida College of Medicine, Gainesville, FL, USA, 2The McKnight Brain Institute, University of Florida College of Medicine, Gainesville, FL, USA, 3Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL, USA and 4Department of Anesthesiology, Zhengzhou University, Zhengzhou, China
+
+Corresponding author. E-mail: amartynyuk@anest.ufl.edu
+
+This article is accompanied by an editorial: A poisoned chalice: the heritage of parental anaesthesia exposure by Vutskits et al., Br J Anesth 2018:121:337e339, doi: 10.1016/j.bja.2018.05.013.
+
+Abstract
+
+Background: Clinical studies report learning disabilities and attention-deficit/hyperactivity disorders in those exposed to general anaesthesia early in life. Rats, primarily males, exposed to GABAergic anaesthetics as neonates exhibit behav- ioural abnormalities, exacerbated responses to stress, and reduced expression of hypothalamic K (Kcc2). The latter is implicated in development of psychiatric disorders, including male predominant autism spectrum disorders. We tested whether parental early life exposure to sevoflurane, the most frequently used anaesthetic in pae- diatrics, affects the next generation of unexposed rats. Methods: Offspring (F1) of unexposed or exposed to sevoflurane on postnatal day 5 Sprague-Dawley rats (F0) were subjected to behavioural and brain gene expression evaluations. Results: Male, but not female, progeny of sevoflurane-exposed parents exhibited abnormalities in behavioural testing and Kcc2 expression. Male F1 rats of both exposed parents exhibited impaired spatial memory and expression of hip- pocampal and hypothalamic Kcc2. Offspring of only exposed sires had abnormalities in elevated plus maze and prepulse inhibition of startle, but normal spatial memory and impaired expression of hypothalamic, but not hippocampal, Kcc2. In contrast to exposed F0, their progeny exhibited normal corticosterone responses to stress. Bisulphite sequencing revealed increased CpG site methylation in the Kcc2 promoter in F0 sperm and F1 male hippocampus and hypothalamus that was in concordance with the changes in Kcc2 expression in specific F1 groups. Conclusions: Neonatal exposure to sevoflurane can affect the next generation of males through epigenetic modification of Kcc2 expression, while F1 females are at diminished risk.
+
+þ
+
+(cid:2)
+
+(cid:2)
+
+2Cl
+
+Cl
+
+Keyword: anesthesia; DNA methylation; heredity; neurodevelopmental disorders; pediatrics
+
+Editorial decision: 2 May 2018; Accepted: 2 May 2018 © 2018 British Journal of Anaesthesia. Published by Elsevier Ltd. All rights reserved. For Permissions, please email: permissions@elsevier.com
+
+exporter
+
+406
+
+Editor’s key points
+
+(cid:5) Early exposure to general anaesthetics can result in persistent cognitive dysfunction in adult animals, but effects on their offspring are unknown.
+
+(cid:5) Offspring of rats exposed to sevoflurane as neonates were investigated for behavioural abnormalities, changes in brain gene expression and deoxyribonucleic acid methylation in the genes’ promoters.
+
+(cid:5) Adult male, but not female, progeny of rats neonatally exposed to sevoflurane exhibited abnormalities in epigenetic regulation, gene expression and behaviour.
+
+Most retrospective epidemiological studies of neurocognitive function in older children who had general anaesthesia early in life have found significant deficiencies.1 Considering the compelling animal data, the US Food and Drug Administration recommended avoiding, when possible, anaesthesia in chil- dren <3 yr old, and emphasised the pressing need for further research.2 The full range of neonatal anaesthesia-induced abnormalities, the mechanisms involved, and the role of sex remain poorly understood even in exposed animals.3
+
+We have found that rats exposed as neonates to sevoflurane, propofol, or etomidate, anaesthetics with clinically important effects on GABA type A receptors (GABAAR), exhibit behavioural deficiencies and exacerbated hypothalamic-pituitary adrenal (HPA) axis responses to stress.4e8 These anaesthetic-induced abnormalities are greater in male rats and reminiscent of those induced by excessive postnatal stress.9e11 Anaesthetic- enhanced GABAAR signalling, which is depolarising/stimula- tory during early life because of a high Na (NKCC1)/ co-transporter ratio,12e14 could play an K important role in initiating and mediating these abnormalities. Thus, NKCC1 inhibition before anaesthesia was protective, whereas anaesthetised neonatal rats had hypothalamic upre- gulated Nkcc1 and downregulated Kcc2 messenger ribonucleic acid (mRNA) concentrations even in adulthood.7,8
+
+þ
+
+þ
+
+(cid:2)
+
+K
+
+2Cl
+
+þ
+
+(cid:2)
+
+(cid:2)
+
+2Cl
+
+(KCC2) Cl
+
+During the second postnatal week, GABAAR-mediated neuronal signalling undergoes a fundamental transition from predominantly depolarising/stimulatory to inhibitory caused by concomitant developmental downregulation of NKCC1 and, most importantly, upregulation of neuron-specific KCC2. This shift is brain region- and sex-dependent, occurring earlier in females.12e14 Anaesthetic-induced delay in the developmental NKCC1/KCC2 ratio maturation could have serious conse- quences for brain functioning as delay/impairment in NKCC1/ KCC2 ratio maturation has been linked to neuropsychiatric disorders, including autism spectrum disorders (ASD) and schizophrenia, which predominate in males.15e17 A growing number of studies point to co-occurrence of ASD and attention-deficit/hyperactivity disorder (ADHD). Thus, 50e70% of those with ASD exhibit ADHD symptoms, whereas 15e25% of children with ADHD have symptoms of ASD.18 Importantly, clinical studies report significant increases in ADHD in those who had medical procedures early in life that required expo- sure to general anaesthesia, with repeated exposures being a prognostic factor for more severe outcome.2
+
+Recent studies in rodents demonstrate that the develop- mental effects of excessive stress early in life can be carried to the next generation or beyond, presumably by epigenetic mechanisms such as non-coding RNAs and deoxyribonucleic
+
+Multigenerational developmental effects of sevoflurane - 407
+
+acid (DNA) methylation.19e21 We have found that rats exposed as neonates to sevoflurane exhibited increased expression of hippocampal DNA methyltransferases, in addition to abnor- malities at the synaptic and behavioural levels.22 These en- zymes catalyse DNA methylation at the 5 position of cytosine residues adjacent to guanines (CpG sites), typically leading to long-term transcriptional repression. To investigate whether neonatal exposure to sevoflurane affects exposed parents and their unexposed progeny, neonatal male and female rats were exposed to 6 h of anaesthesia with sevoflurane, and their progeny were tested for inherited behavioural and molecular alterations.
+
+0
+
+Methods
+
+Animals
+
+All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee. Sprague-Dawley rats were housed under controlled illumina- tion (12-h light/dark, lights on at 7:00AM) and temperature (23e24 C) with free access to food and water. Within 24 h of delivery, litters were culled to 12 pups. At 21 postnatal days (P21), pups were weaned and housed in sex-matched groups of two for the rest of the study.
+
+(cid:3)
+
+Treatment groups
+
+The P5 male and female rat pups were kept in a temperature- controlled chamber (37ºC) with a continuous supply of 30% (cid:2)1) during anaesthesia with 6 vol% oxygen in air (1.5 L min sevoflurane for 3 min for induction and 2.1 vol% sevoflurane for 357 min as maintenance (sevoflurane group). Previously, we have shown that blood glucose and gas levels after 2.1% sevo- flurane for 6 h were in the normal range.4 Control F0 animals were subjected to animal facility rearing only (control group). The F0 male and female rats were sequentially evaluated on the elevated plus maze (EPM) starting on P60, for prepulse inhibition (PPI) of the acoustic startle response on P70, and for corticosterone responses to physical restraint for 30 min on (cid:4)P160 followed by isolation of brain and gamete tissue sam- ples for further analyses (Fig. 1). Twenty-four F0 males and 24 females were mated on ~P90 to produce the F1 generation. F0 breeders were randomised into one of the following four groups for mating: 1) control malesþcontrol females (con- M*con-F); 2) exposed malesþcontrol females (sevo-M*con-F); 3) control malesþexposed females (con-M*sevo-F); and 4) exposed malesþexposed females (sevo-M*sevo-F). The female was kept alone throughout the entire gestation and post- partum rearing periods. The F1 rats, 144 in total [n¼18 per sex (two) per group (four)], which were subjected to facility rearing only, were evaluated in the EPM starting on P60, PPI of startle on P70, Morris water maze (MWM) testing starting on P79, and for the corticosterone responses to restraint for 30 min on (cid:4)P90, followed by isolation of brain tissue samples for further analyses. A separate cohort of F1 rats was sacrificed on P5 to collect brain tissue for bisulphite sequencing.
+
+Basal and stress-induced activity of the HPA axis Blood samples (~300 mL) were collected at rest and 10, 60, and 120 min after the restraint, as previously described.7 Serum corticosterone was measured using commercial ELISA kits (Cayman Chemical Company, Ann Arbor, MI, USA) following the manufacturer’s instructions.7,8
+
+408 -
+
+Ju et al.
+
+Fig 1. Study design. EPM, elevated plus maze; PPI, prepulse inhibition; MWM, Morris water maze.
+
+Behavioural tests
+
+The EPM, acoustic startle response, PPI of startle, and MWM tests were performed as previously described.4e8
+
+Tissue collection
+
+vector with the TOPO TA cloning kit for sequencing (Life Technologies, Carlsbad, CA, USA). Miniprep was performed on each positive clone using ZR Plasmid Miniprep kit (Zymo Research). Sanger sequencing was done by Genewiz (South Plainfield, NJ, USA) using M13R primers. The DNA methylation status of all CpG sites was analysed using Benchling Molecular Biology 2.0 Software (Benchling, San Francisco, CA, USA).
+
+Adult rats were anaesthetised with sevoflurane and decapi- tated. Whole brains were removed and immediately put in a stainless steel adult rat brain slicer matrix with 0.5 mm coro- nal section slice intervals (Zivic Instruments, Pittsburgh, PA, USA). Hypothalamic paraventricular nucleus (PVN) tissue was punched out with a 1-mm ID glass capillary tube. The hippo- campus was isolated from the respective slices. Tissues were placed in vials filled with RNAlater solution (Invitrogen, Carlsbad, CA, USA) and stored at (cid:2)80 C. Sperm were isolated from the caudal epididymis of adult males and stored at (cid:2)80 C. After separation from the adipose tissues, ovaries were stored at (cid:2)80(cid:3)C.
+
+(cid:3)
+
+(cid:3)
+
+Analyses of mRNA levels for Nkcc1, Kcc2, and glucocorticoid receptors (Gr)
+
+The mRNA levels for Nkcc1, Kcc2 in the PVN of the hypothala- mus and hippocampus, and for Gr in the hippocampus were analysed via qRTePCR as previously described.7,8
+
+Bisulphite sequencing
+
+Genomic DNA was extracted from the sperm pellet and ovaries of adult F0 rats and from hippocampal and hypotha- lamic tissues of P5 F1 rats using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). The sodium bisulfite conversion was performed with EZ DNA Methylation kits (Zymo Research, Irvine, CA, USA) following the manufacturer’s instructions. The primers (Nkcc1: forward: GAGAGGAGTTTATAGGGTT; reverse: AACCCTAC(A/G)CTAACCAACCTC; Kcc2: forward: GATTGTAAGTGTTTTTATTATTGAGTTGTATATT; reverse: AATAAACTTTTCCCCTTTTATACCC) were designed for the bisulfite-converted DNA sequences, using previously pub- lished sequences.23,24 PCR amplification was performed with HotStar Taq (Qiagen). Amplicons were cloned into pCR4-TOPO
+
+Statistical analysis
+
+Values are reported as mean (standard deviation). Statistical analyses were carried out on raw data using SigmaPlot 13.0 software (Systat Software, Inc., San Jose, CA, USA). To assess differences in total corticosterone concentration, EPM behav- iour and gene expression for Nkcc1, Kcc2, and Gr, t-test and one way analysis of variance (ANOVA) were used for F0 and F1 generations, respectively. Two way ANOVA with experimental groups and time as the independent variables was run to analyse changes in serum corticosterone concentrations at rest and at three time points after the restraint. Two way ANOVA was used to analyse the PPI data, with the treatment and prepulse intensity as independent variables, and the MWM latencies to escape data, with experimental groups and days of training as the independent variables. One-way ANOVA was used to analyse time spent in the target quad- rant and numbers of crossings during the MWM probe test. Two way measures ANOVA with treatment as ‘between’- subject factor and CpG site as ‘within’-subject factor was used to analyse the frequency methylation of CpG sites. Multiple pairwise comparisons were done with the Holm-Sidak method. All comparisons were run as two-tailed tests. A P value <0.05 was considered significant. The sample sizes in this study were based on previous experience with the same experimental techniques.6-8
+
+Results
+
+Neuroendocrine and behavioural abnormalities in F0 rats
+
+Adult F0 rats, exposed to sevoflurane as neonates, had significantly higher total corticosterone responses to restraint
+
+Multigenerational developmental effects of sevoflurane - 409
+
+stress compared with F0 controls [males, t(8)¼(cid:2)8.09, P<0.001; and females, t(8)¼(cid:2)3.05, P¼0.015]. These increases in cortico- sterone responses were because of higher concentrations of corticosterone 10 min after restraint (P<0.001, males, Fig. 2a and b; and P<0.001, females, Fig. 2c and d).
+
+The F0 male rats, exposed to sevoflurane as neonates, spent a shorter time in open arms [t(20)¼2.67, P¼0.015, Fig. 2e] and travelled shorter distances during the EPM test [t(20)¼2.27, P¼0.034, Fig. 2f]. In F0 females, there was no significant between-subjects effect of neonatal sevoflurane exposure on time spent in open arms and distance travelled during the EPM test (Fig. 2g and h).
+
+There were significant effects of neonatal exposure to sevoflurane on PPI of startle in adult F0 rats [F(1,66)¼14.80, P<0.001, males, Fig. 2i; and F(1,66)¼9.13, P¼0.004, females, Fig. 2j]. Startle stimuli by themselves caused similar responses in the control and sevoflurane groups of F0 male and female rats.
+
+Hypothalamic and hippocampal Nkcc1/Kcc2 mRNA ratios in F0 rats
+
+The F0 male rats from the sevoflurane group had increased Nkcc1 mRNA levels [t(11)¼(cid:2)3.29, P¼0.007, Fig. 3a] and decreased Kcc2 mRNA levels [t(11)¼2.24, P¼0.047, Fig. 3b] in the PVN of the hypothalamus, resulting in significantly increased Nkcc1/Kcc2 mRNA ratios [t(11)¼(cid:2)6.97, P<0.001, Fig. 3c]. The F0 female rats from the sevoflurane group had increased Nkcc1 mRNA levels [t(10)¼(cid:2)2.91, P¼0.016, Fig. 3d], but not significantly altered Kcc2 mRNA levels (Fig. 3e) in the PVN of the hypothalamus. Still, the resulting Nkcc1/Kcc2 mRNA ratios in sevoflurane exposed F0 females were increased [t(10)¼(cid:2)3.17, P¼0.01, Fig. 3f]. In the hippocampus of F0 male rats from the sevoflurane group, only Kcc2 mRNA levels were reduced [t(10)¼4.17, P¼0.002, Fig. 3h]. Overall, changes in hippocampal Kcc2 mRNA and Nkcc1 mRNA resulted in an increased Nkcc1/Kcc2 mRNA ratio in F0 males [t(10)¼(cid:2)3.27, P¼0.008, Fig. 3i]. In contrast, hippocampal mRNA
+
+Fig 2. Adult F0 rats, exposed to sevoflurane on postnatal Day 5, exhibited exacerbated corticosterone responses to physical restraint for 30 min, impaired behaviour in the elevated plus maze (EPM) and reduced prepulse inhibition (PPI) of startle. Shown are the respective concentrations of serum corticosterone across each collection point, and the total corticosterone response in male (a, b) and female (c, d) rats. To assess differences in total corticosterone concentrations, area under the curve in respect to ground (concentrations of cortico- sterone at rest were taken as a ground), was calculated. Data are means [standard deviation (SD)] from five rats per treatment group. (eeh) Shown are time (%) spent in open arms of the EPM and distance travelled by male (e, f) and female (g, h) rats. Data are means (SD) from 11 male and 12 female rats per treatment group. (i, j) Shown are %PPI responses in male (i) and female (j) rats. Data are means (SD) from 12 rats per treatment group. Colour coding in (eeh) is applicable to all figures. *P<0.05 vs control.
+
+410 -
+
+Ju et al.
+
+þ
+
+þ
+
+(cid:2)
+
+þ
+
+(cid:2)
+
+(Kcc2) and glucocorticoid receptors (Gr) in the paraventricular nucleus (PVN) of the Fig 3. Gene expression of Na hypothalamus and hippocampus of adult F0 rats, exposed to sevoflurane on postnatal Day 5. Shown are the respective levels of Nkcc1 messenger ribonucleic acid (mRNA), Kcc2 mRNA and the resulting Nkcc1/Kcc2 mRNA ratios in the PVN of the hypothalamus of males (aec) and females (def) and in the hippocampus of males (gei) and females (jel). Data normalised against control are means [standard deviation (SD)] from a minimum of six rats per treatment group (n¼7, male sevoflurane group, hypothalamus). (m, n) Shown are levels of Gr mRNA in the hippocampus of male (m) and female (n) rats. Data normalised against control are means (SD) from six rats per treatment group. Colour coding in m and n is applicable to all figures. *P<0.05 vs control.
+
+K
+
+2Cl
+
+(Nkcc1), K
+
+2Cl
+
+levels for Nkcc1, Kcc2, and Nkcc1/Kcc2 were similar in control and sevoflurane exposed F0 female rats (Fig. 3jel). The hip- pocampal levels of Gr mRNA were similar in control and sev- oflurane exposed F0 male (Fig. 3m) and female rats (Fig. 3n).
+
+Behavioural abnormalities and corticosterone responses to stress in F1 rats
+
+Serum concentrations of corticosterone in male and female rats from the F1 generation were not different among all experimental groups within the same sex (Fig. 4aed).
+
+In F1 males, there was a significant between-subjects effect of parental neonatal exposure to sevoflurane on time spent in
+
+open arms [F(3,67)¼3.51, P¼0.02; Fig. 4e], but there was no sig- nificant effect on distance travelled (Fig. 4f) during the EPM test. Only F1 male progeny of exposed males and unexposed females spent shorter time in open arms of the EPM. The time spent in open arms and distance travelled during the EPM test were not different amongst all experimental groups of F1 fe- male rats (Fig. 4g and h).
+
+There was a significant effect of parental sevoflurane exposure on PPI of startle responses in F1 male rats [F(3,204)¼9.19, P<0.001; Fig. 4i]. Only male progeny of exposed sires exhibited reduced PPI of startle at PP3 (P¼0.014 vs F1 males of con-M*con-F), and PP6 (P¼0.007 vs F1 males of con- M*con-F). There was no significant treatment effect on PPI of
+
+Multigenerational developmental effects of sevoflurane - 411
+
+Fig 4. F1 male, but not female, offspring of sires exposed to sevoflurane on postnatal Day 5, exhibit behavioural abnormalities, while both F1 females and F1 males had normal corticosterone responses to stress. Shown are the respective concentrations of serum corticosterone across each collection point, and the total corticosterone responses in male (a, b) and female (c, d) F1 rats. To assess differences in total corticosterone concentrations, area under the curve in respect to ground (concentrations of corticosterone at rest were taken as a ground), was calculated. Data are means [standard deviation (SD)] from six animals per treatment group. Shown are % of time spent in open arms of the elevated plus maze (EPM) and distance travelled by male (e, f) and female (g, h) F1 rats. Data are means (SD) typically from 18 animals per treatment group (n¼17, male con-M*con-F group). (i, j) Shown are %PPI responses at prepulse intensity (PP) of 3 dB, 6 dB, and 12 dB in male (i) and female (j) F1 rats. Data are means (SD) from 18 rats per treatment group. (k) Plots showing the values of escape latencies during the 5-day training period from P80 to P84 for F1 male rats. (l, m) Histograms showing the time spent in the target quadrant and the number of times the rat crossed the previous location of the escape platform. (nep) Shown are respective data for F1 female rats collected during the Morris water maze (MWM) tests. Data are means (SD) from 18 animals per treatment group. Colour coding in (eeh) is applicable to all figures. *P<0.05 vs F1 males from the con-M*con-F group.
+
+412 -
+
+Ju et al.
+
+startle in F1 female rats (Fig. 4j). The startle amplitudes were similar among all experimental groups of F1 male and F1 fe- male rats.
+
+Hypothalamic and hippocampal Nkcc1/Kcc2 mRNA ratios and hippocampal Gr mRNA levels in F1 rats
+
+In males, the MWM test showed no significant between- subjects effect of parental sevoflurane exposure on the escape latencies across the 5-day training period, but there was a significant within-subjects effect of day of training [F(4,272)¼30.03, P<0.001; Fig. 4k]. There were significant effects of parental sevoflurane exposure on time in the target quad- rant [F(3,68)¼2.75, P¼0.049; Fig. 4l] and times of crossing over the platform [F(3,68)¼3.06, P¼0.034; Fig. 4m]. Only male offspring of both exposed parents spent significantly shorter time in the target quadrant (P¼0.04 vs F1 males of con-M*con-F) and made less crossings over the former platform (P¼0.04 vs F1 males of con-M*con-F). There were no significant group effects in the MWM tests of F1 female rats (Fig. 4nep).
+
+In F1 males there was a significant between-subjects effect of sevoflurane exposure on Nkcc1 mRNA levels parental [F(3,22)¼4.55, P¼0.013, Fig. 5a], Kcc2 mRNA levels [F(3,22)¼13.53, P<0.001, Fig. 5b], and the Nkcc1/Kcc2 mRNA ratios [F(3,22)¼5.68, P¼0.005, Fig. 5c] in the PVN of the hypothalamus. In contrast, F1 females showed no such between-subjects effects in the PVN of the hypothalamus (Fig. 5def).
+
+In the hippocampus of F1 males, there was no significant between-subject effect of parental neonatal sevoflurane exposure on Nkcc1 mRNA levels (Fig. 5g), but there was a sig- nificant effect on Kcc2 mRNA levels [F(3,20)¼3.55, P¼0.03, Fig. 5h] and thus the Nkcc1/Kcc2 mRNA ratio [F(3,22)¼5.52, P¼0.006, Fig. 5i]. In the hippocampus of F1 females, there were no
+
+þ
+
+þ
+
+(cid:2)
+
+þ
+
+(cid:2)
+
+(Kcc2) and glucocorticoid receptors (Gr) in the paraventricular nucleus (PVN) of the Fig 5. Gene expression for Na hypothalamus and hippocampus of F1 rats. Shown are the respective levels of Nkcc1 messenger ribonucleic acid (mRNA), Kcc2 mRNA, and the resulting Nkcc1/Kcc2 mRNA ratios in the PVN of the hypothalamus of F1 males (aec) and F1 females (def) and in the hippocampus of F1 males (gei) and F1 females (jel). Data normalised against control are means [standard deviation (SD)] from at least six rats per treatment group (n¼7, male con-M*con-F and sevo-M*con-F groups). (m, n) Shown are levels of Gr mRNA in the hippocampus of male (m) and female (n) rats. Data normalised against control are means (SD) from six rats per treatment group. Colour coding in (m) and (n) is applicable to all figures. *P<0.05 vs F1 males from the con-M*con-F group.
+
+K
+
+2Cl
+
+(Nkcc1), K
+
+2Cl
+
+Multigenerational developmental effects of sevoflurane - 413
+
+Fig 6. Methylation in promoter region of Kcc2 gene in sperm and ovary deoxyribonucleic acid (DNA) of F0 rats and in the hypothalamus and hippocampus DNA of F1 rats. (A) Bisulphite sequencing of CpG sites in the Kcc2 gene of nine clones from four individual sperm (A,aec) and ovary (A,def) DNA samples isolated from sevoflurane-exposed and control F0 rats. Heat maps show DNA methylation status of CpG sites in the promoter region of the Kcc2 gene in sperm (A,a) and ovaries (A,d) of F0 rats. Red cells show methylated sites. X axisdCpG sites; Y axisdclones. Histograms showing methylation frequency at each CpG site (A,bdmales; A,ddfemales) and DNA methylation level at all six CpG sites (A,cdmales; A,fdfemales). (B) Shown are the DNA methylation status of CpG sites, methylation frequencies at each CpG site and DNA methylation level at all six CpG sites in the Kcc2 gene of 9e10 clones from the hypothalamus of F1 male rats (B,aec) and F1 female rats (B,def). (C) The results of similar analyses as in (B) for hippocampus of F1 rats. Data are means (standard deviation) from four rats per y P<0.05 vs all treatment group (n¼5, hypothalamus samples isolated from male rats). *P<0.05 vs F1 males from the con-M*con-F group. other treatment groups. Colour coding in A,c is applicable to A,b; in A,f to A,e; in B,c,f to B,b,e; in C,c,f to C,b,e.
+
+significant between-subjects effects of parental neonatal sev- oflurane exposure on Cl
+
+(cid:2)
+
+transporter mRNA (Fig. 5jel).
+
+There was significant between-subjects effect of parental neonatal sevoflurane exposure on the hippocampal Gr mRNA levels in F1 males [F(3,20)¼7.44, P¼0.002, Fig. 5m], but not in F1 females (Fig. 5n).
+
+ovaries of control and sevoflurane-exposed adult female rats (Fig. 6A,def). Greater methylation changes might be present in oocytes, a minor fraction of the cells in the ovary.
+
+There was significant effect of parental treatment on the frequencies of CpG site methylation in the hypothalamus in F1 male [F(3,96)¼32.09, P<0.001, Fig. 6B,aec], but not female prog- eny (Fig. 6B,def).
+
+DNA methylation in the Kcc2 gene promoter
+
+In sperm of F0 rats there was significant effect of treatment [F(1,36)¼59.06, P<0.001, Fig. 6A,aec] and within-subjects effect of CpG site [F(5,36) ¼ 37.80, P<0.001] on methylation frequency. There was a trend but no significant difference between CpG site methylation frequency in the Kcc2 gene promoter in
+
+The CpG site methylation frequency in the hippocampus of F1 male rats was largest if both parents were exposed to sev- oflurane as neonates [F(3,72)¼96.83, P<0.001, Fig. 6C,aec], while F1 females were not significantly affected (Fig. 6C,def). We did not detect significant differences in the frequency of CpG site methylation in the promoter in the Nkcc1 gene in F0 sperm of control and rats exposed to sevoflurane as neonates.
+
+414 -
+
+Ju et al.
+
+Discussion
+
+A single exposure of neonatal rats to sevoflurane, the most frequently used general anaesthetic in paediatrics, led to sig- nificant behavioural abnormalities and changes in DNA methylation not only in exposed rats in adulthood, but also in their adult male offspring that were never exposed to sevo- flurane. These effects of sevoflurane were strongly sex- dependent. The findings that male offspring only, but not female littermates, were affected indicate that it is unlikely that sevoflurane-induced abnormalities are transmitted to the next generation through sevoflurane-altered behaviour of the exposed dams. Furthermore, in the EPM and PPI of startle behavioural tests, male offspring of control females and exposed males were the only experimental group that exhibited significant abnormalities, even though F0 males did not have a direct contact with their progeny. These findings, together with increased hypothalamic and hippocampal Nkcc1/Kcc2 mRNA ratios in exposed parents and their male offspring and similarly increased DNA methylation of the Kcc2 gene promoter in the sperm of F0 exposed sires and hypo- thalamic and hippocampal tissues of their male, but not fe- male progenies, strongly support involvement of epigenetic mechanisms in the effects of parental neonatal exposure to sevoflurane to the next generation.
+
+The similarities between the developmental effects of exposure to GABAergic anaesthetics4e8 and perinatal stress9e11 early in life suggests similarities in the underlying mechanisms of both phenomena. Recent studies in rodents also report heritable multigenerational effects of perinatal stress.19e21 Similar to our findings of normal corticosterone responses to physical restraint in progeny of sevoflurane- exposed parents, Morgan and Bale19 found normal cortico- sterone responses in offspring of prenatally stressed males and control females. Similar to our findings that only males were affected by neonatal exposure of their parents to sev- oflurane, developmental effects of paternal prenatal stress were detected in male offspring only.19 Among plausible explanations for normal corticosterone responses in male offspring of the exposed rats could be increased expression of Grs in the hippocampus, PVN, pituitary, or all three consistent with our finding of increased concentrations of Grs mRNA in the hippocampus of F1 male rats where only one parent had been exposed to sevoflurane. The GRs mediate the negative feedback of corticosterone on HPA axis activity.25
+
+Male offspring of exposed male F0/unexposed female F0 exhibited reductions in PPI of acoustic startle and in time spent in open arms of the EPM. These PPI and EPM abnor- malities were accompanied by greater increases in hypotha- lamic PVN Nkcc1/Kcc2 mRNA ratios. Also, male progeny of exposed male F0/unexposed female F0 had significantly higher CpG methylation frequencies in the promoter of the Kcc2 gene in the hypothalamus. In contrast, abnormalities in spatial memory during the MWM test, a standardised and widely used behavioural test that strongly correlates with hippocampal synaptic plasticity,26 were most prominent in male offspring when both parents were exposed. Again, consistent with behavioural findings, the greatest increase in hippocampal Nkcc1/Kcc2 mRNA ratio was found in male offspring of this group. Furthermore, this group had significantly higher CpG methylation in the promoter of the Kcc2 gene in the hippo- campus. Together, these findings support an important role of epigenetic mechanisms in the mediation of heritable
+
+developmental effects of early life exposure to sevoflurane. Another novel observation is that a delay or postponement in the developmental maturation in the Nkcc1/Kcc2 ratio in the PVN of the hypothalamus can selectively affect EPM and PPI behaviour with no significant effect on MWM behaviour, while impaired developmental maturation of the Nkcc1/Kcc2 ratio in the hippocampus can have profound consequences for MWM behaviour, with no significant effects on EPM and PPI behav- iour. In future studies it will be important to elucidate how closely the observed changes in gene expression in each spe- cific experimental group translate to changes in respective protein concentrations.
+
+Why male offspring of exposed male F0/unexposed female F0 exhibit significant deficiencies in the EPM and PPI of startle tests, especially when compared with offspring of both exposed parents, remains to be elucidated, as does why only male progeny were affected. A greater HPA axis response to stress in exposed F0 males,6 as opposed to greater stress re- sponses in naı¨ve females,27 suggest that anaesthesia alters postnatal brain sex differentiation, at least as it relates to HPA axis function. The primary female sex steroid hormone 17b- oestradiol, synthesised in neonatal brain through aromati- sation of testis-derived testosterone, directs brain sexual differentiation by organisational actions during a critical period.28 Of relevance, 17b-oestradiol is known to down- regulate neuronal Kcc2 expression.13,14 It would be important to explore whether sex-dependent developmental effects of sevoflurane include effects on brain sexual differentiation. Even though an increase in the Nkcc1 mRNA level was detected only in the hypothalamus of one group of second generation male rats, the male progenies of exposed male F0/ unexposed female F0, it remains to be elucidated how such an increase in hypothalamic Nkcc1 mRNA level was passed to the next generation, as we were not able to detect significant changes in the methylation pattern of the Nkcc1 gene pro- moter in sperm of exposed F0 males.
+
+In summary, our results demonstrate for the first time that neurobehavioural abnormalities induced by neonatal expo- sure to the general anaesthetic sevoflurane can be transmitted to the next generation in a complex, sex- and brain region- specific mode through epigenetic mechanisms. This basic science study deals with a complex biological phenomenon of intergenerational heritability of the effects of environmental factors, in general, and with a newly uncovered potentially important translational problem (i.e. intergenerational heri- tability of the effects of early in life general anaesthesia exposure). Mechanisms of sevoflurane-induced sex- and brain region-specific effects across two generations are exciting and challenging topics for future studies. To further substantiate translational applicability of this phenomenon, additional animal studies using different neonatal anaesthesia para- digms that more broadly model stages of human postnatal brain development at the time of anaesthesia exposure and duration of anaesthesia exposure in young human patients will be needed.
+
+Authors’ contributions
+
+Designed research: A.E.M., L.-S.J., T.E.M., N.G., C.N.S., J.L.R., J.-Q.Z. Performed research: L.-S.J., J.-J.Y. Analysed data: L.-S.J., J.-J.Y., A.E.M. Wrote the paper: A.E.M., T.E.M., N.G., C.N.S., J.-Q.Z. Approved the final manuscript: all authors.
+
+Acknowledgements
+
+The authors thank B. Setlow, J. Li, and X. Yang for helpful advice and technical assistance.
+
+Declaration of interest
+
+The authors declare that they have no conflicts of interest.
+
+Funding
+
+National Institutes of Health (R01NS091542, R01NS091542-S to A.E.M.), I. Heermann Anesthesia Foundation, Inc (to J.L-S.), the Jerome H. Modell Endowed Professorship (to N.G.), and the National Natural Science Foundation of China (U1404807, 81771149 to J.Z.).
+
+References
+
+1. Ing C, Sun M, Olfson M, et al. Age at exposure to surgery and anesthesia in children and association with mental disorder diagnosis. Anesth Analg 2017; 125: 1988e98
+
+2. U.S. Food and Drug Administration. FDA drug safety communication: FDA review results in new warnings about using general anesthetics and sedation drugs in young children and pregnant women. Available from: https://www.fda.gov/ Drugs/DrugSafety/ucm532356.htm. [Accessed 25 August 2017]
+
+3. Sanders RD, Hassell J, Davidson AJ, Robertson NJ, Ma D. Impact on neuro- development: an update. Br J Anaesth 2013; 110(Suppl 1): i53e72 anaesthetics surgery and of
+
+4. Edwards DA, Shah HP, Cao W, Gravenstein N, Seubert CN, Martynyuk AE. Bumetanide alleviates epileptogenic and neurotoxic effects of sevoflurane in neonatal rat brain. Anesthesiology 2010; 112: 567e75
+
+5. Cao W, Pavlinec C, Gravenstein N, Seubert CN, Martynyuk AE. Roles of aldosterone and oxytocin in ab- normalities caused by sevoflurane anesthesia in neonatal rats. Anesthesiology 2012; 117: 791e800
+
+6. Xu C, Tan S, Zhang J, et al. Anesthesia with sevoflurane in neonatal rats: developmental neuroendocrine abnormal- ities and alleviating effects of the corticosteroid and Cl(-) importer antagonists. Psychoneuroendocrinology 2015; 60: 173e81
+
+7. Ju LS, Yang JJ, Gravenstein N, et al. Role of environmental stressors in determining the developmental outcome of neonatal anesthesia. Psychoneuroendocrinology 2017; 81: 96e104
+
+8. Yang J, Ju L, Jia M, et al. Subsequent maternal separation exacerbates neurobehavioral in rats neonatally exposed to sevoflurane anesthesia. Neurosci Lett 2017; 661: 137e42 abnormalities
+
+9. Furukawa M, Tsukahara T, Tomita K, et al. Neonatal maternal separation delays the GABA excitatory-to- inhibitory functional switch by inhibiting KCC2 expres- sion. Biochem Biophys Res Commun 2017; 493: 1243e9 10. O’Malley D, Dinan TG, Cryan JF. Neonatal maternal sepa- ration in the rat impacts on the stress responsivity of central corticotropin-releasing factor receptors in adult- hood. Psychopharmacology (Berl) 2011; 214: 221e9
+
+Multigenerational developmental effects of sevoflurane - 415
+
+11. Veerawatananan B, Surakul P, Chutabhakdikul N. Maternal restraint stress delays maturation of cation- chloride cotransporters and GABAA receptor subunits in the hippocampus of rat pups at puberty. Neurobiol Stress 2015; 3: 1e7
+
+12. Ben-Ari Y. The GABA excitatory/inhibitory developmental journey. Neuroscience 2014; 279: sequence: a personal 187e219
+
+13. Perrot-Sinal TS, Sinal CJ, Reader
+
+JC, Speert DB, McCarthy MM. Sex differences in the chloride cotrans- porters, NKCC1 and KCC2, in the developing hypothala- mus. J Neuroendocrinol 2007; 19: 302e8
+
+14. Nunez JL, McCarthy MM. Resting intracellular calcium concentration, depolarizing GABA and possible role of local estradiol synthesis in the developing male and fe- male hippocampus. Neuroscience 2008; 158: 623e34
+
+15. Tang X, Kim J, Zhou L, et al. KCC2 rescues functional deficits in human neurons derived from patients with Rett syndrome. Proc Natl Acad Sci USA 2016; 113: 751e6
+
+16. Merner ND, Mercado A, Khanna AR, et al. Gain-of-function missense variant in SLC12A2, encoding the bumetanide- sensitive NKCC1 cotransporter, identified in human schizophrenia. J Psychiatr Res 2016; 77: 22e6
+
+17. Lemonnier E, Villeneuve N, Sonie S, et al. Effects of bumetanide on neurobehavioral function in children and adolescents with autism spectrum disorders. Transl Psy- chiatry 2017; 7, e1056
+
+18. Antshel KM, Zhang-James Y, Wagner KE, Ledesma A, Faraone SV. An update on the comorbidity of ADHD and ASD: a focus on clinical management. Expert Rev Neurother 2016; 16: 279e93
+
+19. Morgan CP, Bale TL. Early prenatal stress epigenetically programs dysmasculinization in second-generation offspring via the paternal lineage. J Neurosci 2011; 31: 11748e55
+
+20. Bohacek J, Farinelli M, Mirante O, et al. Pathological brain plasticity and cognition in the offspring of males sub- jected to postnatal traumatic stress. Mol Psychiatry 2015; 20: 621e31
+
+21. Rodgers AB, Morgan CP, Leu NA, Bale TL. Transgenera- tional epigenetic programming via sperm microRNA re- capitulates effects of paternal stress. Proc Natl Acad Sci USA 2015; 112: 13699e704
+
+22. Ju LS, Jia M, Sun J, et al. Hypermethylation of hippocampal synaptic plasticity-related genes is involved in neonatal sevoflurane exposure-induced cognitive impairments in rats. Neurotox Res 2016; 29: 243e55
+
+23. Cho HM, Lee HA, Kim HY, Han HS, Kim IK. Expression of Naþ-Kþ -2Cl- cotransporter 1 is epigenetically regulated during postnatal development of hypertension. Am J Hypertens 2011; 24: 1286e93
+
+24. Yeo M, Berglund K, Hanna M, et al. Bisphenol A delays the perinatal chloride shift in cortical neurons by epigenetic effects on the Kcc2 promoter. Proc Natl Acad Sci USA 2013; 110: 4315e20
+
+25. Sapolsky RM, Krey LC, McEwen BS. The neuroendocri- nology of stress and aging: the glucocorticoid cascade hypothesis. Endocr Rev 1986; 7: 284e301
+
+26. Vorhees CV, Williams MT. Value of water mazes for assessing spatial and egocentric learning and memory in rodent basic research and regulatory studies. Neurotoxicol Teratol 2014; 45: 75e90
+
+416 -
+
+Ju et al.
+
+27. Iwasaki-Sekino A, Mano-Otagiri A, Ohata H, Yamauchi N, Shibasaki T. Gender differences in corticotropin and corticosterone secretion and corticotropin-releasing fac- tor mRNA expression in the paraventricular nucleus of the the hypothalamus and the central nucleus of
+
+amygdala in response to footshock stress or psychological stress in rats. Psychoneuroendocrinology 2009; 34: 226e37 28. McCarthy MM, Nugent BM, Lenz KM. Neuroimmunology and neuroepigenetics in the establishment of sex differ- ences in the brain. Nat Rev Neurosci 2017; 18: 471e84
+
+Handling editor: H.C. Hemmings Jr

new_pdfs/10.1016_j.brainres.2015.10.050.txt

@@ -0,0 +1,653 @@
+b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7
+
+Available online at www.sciencedirect.com
+
+www.elsevier.com/locate/brainres
+
+Research Report Sevoflurane postconditioning improves long-term learning and memory of neonatal hypoxia-ischemia brain damage rats via the PI3K/Akt-mPTP pathway
+
+Zhongmeng Laia, Liangcheng Zhanga,n Qingxiu Xua
+
+, Jiansheng Sua, Dongmiao Caib,
+
+aDeparment of Anesthesiology, Fujian Medical University Union Hospital, 29 Xin-Quan Road, Fuzhou 350001, PR China bDeparment of Anesthesiology, The First Affiliated Hospital of Xiamen University, 55 Zhen-Hai Road, Xiamen 3610003, PR China
+
+a r t i c l e i n f o
+
+a b s t r a c t
+
+Article history:
+
+Accepted 16 October 2015
+
+Available online 2 November 2015
+
+Background: Volatile anesthetic postconditioning has been documented to provide neuro- protection in adult animals. Our aim was to investigate whether sevoflurane postcondi- tioning improves long-term learning and memory of neonatal hypoxia-ischemia brain
+
+Keywords: Sevoflurane postconditioning Neonatal rat
+
+Hypoxic-ischemic brain damage
+
+Long-term learning and memory
+
+PI3K/Akt pathway
+
+Mitochondrial permeability
+
+transition pore
+
+damage (HIBD) rats, and whether the PI3K/Akt pathway and mitochondrial permeability
+
+transition pore (mPTP) opening participate in the effect.
+
+Methods: Seven-day-old Sprague-Dawley rats were subjected to brain HI and randomly allocated to 10 groups (n ¼24 each group) and treated as follows: (1) Sham, without hypoxia-ischemia; (2) HI/Control, received cerebral hypoxia-ischemia; (3) HIþAtractylo- side (Atr), (4) HIþCyclosporin A (CsA), (5) HIþsevoflurane (Sev), (6) HIþSevþ LY294002 (9) HIþSevþAtr, and (LY), (10) HIþSevþCsA. Twelve rats in each group underwent behavioral testing and their brains were harvested for hippocampus neuron count and morphology study.
+
+(7) HIþSevþ L-NAME (L-N),
+
+(8) HIþSevþ SB216763 (SB),
+
+Brains of the other 12 animals were harvested 24 h after intervention to examine the expression of Akt, p-Akt, eNOS, p-eNOS, GSK-3β, p-GSK-3β by Western bolting and mPTP opening. Results: Sevoflurane postconditioning significantly improved the long-term cognitive performance of the rats, increased the number of surviving neurons in CA1 and CA3
+
+hippocampal regions, and protected the histomorphology of the left hippocampus. These effects were abolished by inhibitors of PI3K/eNOS/GSK-3β. Although blocking mPTP opening simulated sevoflurane postconditioning-induced neuroprotection, it failed to enhance it.
+
+Abbreviations: HIBD, hypoxia-ischemia brain damage; mPTP, mitochondrial permeability transition pore; eNOS, endothelial nitric
+
+oxide synthase; mPTP, mitochondrial permeability transition pore; Atr, Atractyloside; CsA, Cyclosporin A;
+
+TTC,
+
+triphenyltertrazolium chloride; Sev,
+
+sevoflurane; EL, Escape latency; HE, hematoxylin–eosin; BSA, ovine serum albumin;
+
+least significant difference; PCA, principal components analysis.
+
+SD, n Corresponding author. Fax: þ86 59183346181. E-mail addresses: guodh1981@163.com (Z. Lai), zhanglc6@163.com (L. Zhang), sjs2028@163.com (J. Su),
+
+standard deviation; LSD,
+
+caidongmiao@hotmail.com (D. Cai), 18201019@qq.com (Q. Xu).
+
+http://dx.doi.org/10.1016/j.brainres.2015.10.050 0006-8993/& 2015 Elsevier B.V. All rights reserved.
+
+26
+
+b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7
+
+Conclusions: Sevoflurane postconditioning exerts a neuroprotective effect against HIBD in neonatal rats via PI3K/Akt/eNOS and PI3K/Akt/GSK-3β pathways, and blockage of mPTP opening may be involved in attenuation of histomorphological injury.
+
+& 2015 Elsevier B.V. All rights reserved.
+
+1.
+
+Introduction
+
+As a key part in learning and memory adjustment, the hippocampus, including the CA1, CA3, and DG regions, participates in integrating transferred outside information into the nerve center (Chan et al., 2010; Morris et al., 2012). The CA1 region in particular, is highly sensitive to hypoxia- ischemia brain damage (HIBD) (Hopkins and Haaland, 2004). In neonates HIBD may result from perinatal asphyxia, birth injury, and neonatal cardiac surgery, and is a common cause of neonatal death and neurobehavioral impairments (Fan et al., 2005). HIBD may cause apoptosis or necrosis of hippocampal neurons by a cascade of damaging reactions, thus impairing learning and memory (Zhang et al., 2002; Vannucci RC1 and Vannucci, 2001). Therefore, protection of hippocampal neurons is of great significance in prevention and treatment of HIBD.
+
+of cerebral impairment, Akt activation after cerebral ischemia is part of the endogenous protection process and the PI3K/Akt pathway participates in sevoflurane protection against cere- bral ischemia-reperfusion injury, (Zitta et al., 2010; Ye et al., 2012) while neuronal mitochondria undergo permeability transition (Nieminen et al., 1996). Emerging evidence has demonstrated that sevoflurane postconditioning reduces cer- ebral HI-induced brain tissue loss via mitochondrial KATP channels, (Ren et al., 2014) and improves short-term learning and memory after focal cerebral ischemia-reperfusion injury in adult rats via inhibition of neuronal apoptosis through the PI3K/Akt pathway (Wang et al., 2010). However, studies have not examined the long-term learning and memory of neona- tal HIBD rats that received sevoflurane postconditioning, the role of PI3K/Akt-mPTP pathway in the process, and related signal-regulating downstream Akt kinases.
+
+A method to provide this protective effective is ischemic or pharmacological postconditioning, which has been docu- mented to protect tissues and organs by making cells more tolerant of oxygen deficit (Peng et al., 2012; Danielisová et al., 2008; Hu et al., 2013). In cardiac studies, this postconditioning reduces calcium overload, the oxidative stress response, and ATP consumption in cells and mitochondria, thus protecting the cardiac muscle (Tsang et al., 2004; Lim et al., 2007; Lemoine et al., 2010; Fang et al., 2010). The mechanism involves activation of the Phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) pathway, possible activation of kinases including the downstream proteins of Akt, such as glycogen synthase kinase 3 (GSK-3β), P70S6 kinase (P70S6K), and endothelial nitric oxide synthase (eNOS), suppression of mitochondrial permeability transition pore (mPTP) opening, and opening potassium channels (Tsang et al., 2004; Lim et al., 2007; Lemoine et al., 2010; Fang et al., 2010). In studies
+
+Therefore, the purposes of current study were to examine the potential protective effect of sevoflurane postcondition- ing on long-term cognitive performance in neonatal HIBD rats, the related underlying mechanism, and the role of mPTP opening in this process.
+
+2.
+
+Results
+
+Sevoflurane postconditioning improves non-spatial
+
+2.1. and spatial learning and memory
+
+Compared with the HI/Control group, sevoflurane postcondi- tioning increased the DIs of the postconditioning groups (Po0.01), which were reduced by the LY294002 (a PI3K inhibitor), L-NAME (an eNOS inhibitor), and SB216763 (a GSK-3β inhibitor) (Po0.05). Atr did not worsening the DIs of the HI/Control group (P40.05), but it decreased the DIs of
+
+Fig. 1 – Experimental protocol and animal grouping. Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L- NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane. P indicates postnatal age in days.
+
+b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7
+
+27
+
+Fig. 2 – Results of the novel object recognition test. Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane. Comparisons between groups were using one-way ANOVA with Fisher's LSD as post-hoc procedure. The detailed sample size, mean7SD are listed in Supplementary table S1.
+
+Table 1 – Results of the place navigation study.
+
+Day 1
+
+Day 2
+
+Day 3
+
+Day 4
+
+Day 5
+
+Strategy (%)
+
+Sham HI/Control HIþSev HIþSevþLY HIþSevþL-N HIþSevþSB HIþAtr HIþSevþAtr HIþCsA HIþSevþCsA
+
+46.0078.04 74.1578.72 55.5277.45 70.2078.26 67.8377.40 67.9079.67 74.5079.24 74.17710.28 ▲ 55.9178.71 54.8778.64
+
+▲n
+
+n
+
+▲
+
+n
+
+n
+
+n
+
+n
+
+n
+
+35.0976.20 68.3879.01 42.3877.11 66.66710.72 n 64.7878.02 63.02710.15 n 69.3079.56 68.7979.69 39.5675.57 39.8874.93
+
+▲n
+
+n
+
+▲
+
+n
+
+n
+
+n
+
+▲
+
+27.3875.60 56.3376.69 34.1075.69 53.7176.48 49.3876.61 49.2777.29 58.5679.34 56.5278.29 33.9276.83 32.7676.97
+
+▲n
+
+n
+
+▲
+
+n
+
+▲n
+
+▲n
+
+n
+
+n
+
+▲
+
+16.7873.86 47.3475.87 19.5473.22 45.4675.50 39.5875.33 38.5475.49 49.7475.05 48.5074.94 18.6673.18 18.6073.91
+
+▲
+
+n
+
+▲
+
+n
+
+▲n
+
+▲n
+
+n
+
+n
+
+▲
+
+14.3872.22 38.2273.93 16.2672.90 37.2573.43 32.3572.30 36.8673.59 39.2573.52 39.3173.35 15.8172.56 15.3772.52
+
+▲
+
+n
+
+▲
+
+n
+
+▲n
+
+n
+
+n
+
+n
+
+▲
+
+223(92.92) 164(74.55) 208(86.67) 175(79.55) 187(77.92) 171(77.73) 159(72.27) 162(73.64) 214(89.17) 217(90.42)
+
+▲n
+
+n
+
+▲
+
+n
+
+n
+
+n
+
+n
+
+n
+
+▲
+
+▲
+
+▲
+
+▲
+
+▲
+
+▲
+
+▲
+
+Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane. Data are presented as mean7standard deviation. ▲ Po0.05 when compared with HI/Control group. n Po0.05 when compared with HIþSev group.
+
+HIþSev group (Po0.01). Cyclosporine A itself improved the DIs of HI/Control group (Po0.01), but produced no significant improvement in the HIþSev group (P40.05) (Fig. 2).
+
+Results of the Morris water maze test are shown in Table 1
+
+and Fig. 3. When compared with that of the HI/Control group
+
+and of the groups receiving inhibitors (LY294002, L-NAME,
+
+groups, while marginal strategy and random strategy were dominant in the HI/Control, HIþSevþLY/L-N/SB, HIþAtr, and In space exploration, no significant HIþSevþAtr groups. difference in swimming speed was found between the groups (Fig. 3B; F ¼ 0.504, P ¼0.869). Principal component analysis, including cross number (X1), probe time (X2), and probe
+
+SB216763), the EL of the groups receiving Sev or CsA or both was significantly shortened (Po0.05). Atr eliminated the protective effect of Sev (Po0.05), although it did not worsen HI injury itself (P40.05). CsA alone shortened the EL, but did not enhance the protective effect of Sev (P40.05). Straight strategy and tendency strategy were the primary movement strategies in the Sham, HIþSev, HIþCsA, and HIþSevþCsA
+
+length (X3) on the original platform quadrant, showed that the initial eigenvalues λ 1 was 2.469, and cumulative percent ¼ so the first principal was 82.296%, þ0.376X3 can be extracted to represent the 0.360X1 comprehensive index of rat's spatial memory (Fig. 3A). The result showed that sevoflurane postconditioning significantly improved the score of HI/Control group (Po0.01), which was
+
+component Z1
+
+þ0.367X2
+
+28
+
+b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7
+
+Fig. 3 – Results of the space exploration test. Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane. Comparisons between groups were using one-way ANOVA with Fisher’s LSD as post-hoc procedure. The detailed sample size, mean7SD are listed in Supplementary table S2.
+
+respectively decreased by LY294002, L-NAME, and SB216763 (Po0.01). Atr did not reduce the score of HI/Control group (P ¼0.870), but decreased that of HIþSev group (Po0.01). The combined treatment (HIþSevþCsA) did not increase the score of HIþSev group (P¼ 0.414), but CsA alone increased the score of HI/Control group (Po0.01).
+
+Sevoflurane postconditioning alleviates neuronal
+
+2.2. damage and loss
+
+2.4.
+
+Ca2þ
+
+induces mPTP opening
+
+A decreasing tendency in optical density at 540 nm (OD540) was seen in the CaCl2-induced mPTP opening time in all groups. mPTP opening was expressed as a reduction in OD540 during a 5 min period (△OD540/min). In the HIþSev, HIþCsA, and HIþSevþCsA groups the decreases were lower than in the HI/Control, HIþAtr, and HIþSevþAtr groups, respectively (Po0.01). In the HIþSevþLY, HIþSevþL-N, and HIþSevþSB groups, the decreases were higher than in HIþSev group (Po0.05) (Fig. 8).
+
+In the Sham group, neuronal degeneration and neuronal In the HI/Control, apoptosis were occasionally observed. HIþAtr, and HIþSevþAtr groups obvious pyramidal layer thinning (only 1–2 layers), cell body shrinkage and deformity, disordered cell arrangement, gliocyte proliferation, capillary edema, disconnected neighboring neurons, obvious neuronal apoptosis and loss, and low density were observed. In the HIþSev, HIþCsA, and HIþSevþCsA groups a denser pyramidal layer distribution (3–4 aligned layers), hyperchromatic cyto- plasm, regular cell arrangement, clear structure, only mini- mal few discrete neuronal loss, and remarkably reduced cell apoptosis and cellular atrophy were observed. In the groups given the inhibitors (LY294002, L-NAME, and SB216763), 2–3 layer derangement, cell body shrinkage and deformity, glial cell proliferation, cell connection loss, and obvious neuron apoptosis and neuron absence were seen (Figs. 4 and 5). The surviving neuron density of the CA1 and CA3 regions are shown in Fig. 6.
+
+Sevoflurane postconditioning increases the expression
+
+2.3. of p-Akt, p-eNOS, and p-GSK-3β in the left hippocampus
+
+No significant difference in t-Akt/eNOS/GSK-3β expression was found in all groups (Fig. 7). The expressions of p-Akt/ eNOS/GSK-3β were higher in the HIþSev than in the HI/ Control group (Po0.05), and significantly decreased in the groups receiving inhibitors when compared with the HIþSev group (Po0.05).
+
+3.
+
+Discussion
+
+The results of this study using the classical Rice–Vannucci sevoflurane postconditioning model improves long-term learning and memory in neonatal HIBD rats by reducing hippocampal neurons loss; (2) activation of the PI3K/Akt/eNOS and PI3K/Akt/GSK-3β pathways is involved in the neuroprotection provided by sevoflurane postcondi- tioning; and (3) blocking mPTP opening plays an important role in this effect.
+
+showed that:
+
+(1)
+
+Morphological changes of brain tissues are direct indexes to measure HI-induced brain damage. The key brain devel- opment periods of rats are from 1 to 2 days before birth to 2 weeks after birth (Schousboe et al., 2004). Therefore, we intervened on postnatal day 7 and observed structural changes on day 42. The result showed that neuronal degen- eration and neuronal apoptosis were occasionally observed in CA1 and CA3 region in the Sham group, while HI/Control group experienced obvious damage. This finding is consistent with that of other study, (Kumral et al., 2004) and indicates that neonatal HIBD may cause ongoing damage to the hippocampus of rats, which lasts to puberty (postnatal day 35). The CA1 and CA3 regions protected by sevoflurane postconditioning showed denser pyramidal layer distribu- tion, hyperchromatic cytoplasm, regular cell arrangement, clear structure, only minimal neuronal loss, and remarkably reduced cell apoptosis and cellular atrophy, indicating that
+
+b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7
+
+Fig. 4 – Hematoxylin and eosin staining of left hippocampal CA1 region neurons in 42 day old rats (magnification, 200 (cid:2) , 400 (cid:2) ; scale bars¼10 lm). Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane.
+
+29
+
+30
+
+b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7
+
+Fig. 5 – Hematoxylin and eosin staining of left hippocampal CA3 region neurons in 42 day old rats (magnification, 200 (cid:2) ,400 (cid:2) ; scale bars¼10 lm). Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane.
+
+b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7
+
+Fig. 6 – Number of surviving neurons in the left hippocampal CA1 and CA3 regions. Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane. Comparisons between groups were using one-way ANOVA with Fisher’s LSD as post-hoc procedure. The detailed sample size, mean7SD are listed in Supplementary table S3.
+
+sevoflurane postconditioning is capable of protecting the hippocampus from HIBD.
+
+is a hippocampus-dependent cognition (Ennaceur and Delacour, 1988). The novel object recognition test reflects non-spatial learning and memory ability, and can test instant, short- term, and long-term memory retention ability. Rats in the HI/ Control group showed much lower new object recognition than rats in the Sham group at all time points. Presumably, the instant, short-term, and long-term non-spatial memory abilities of HIBD rats were damaged. Sevoflurane postcondi- tioning, however, improved their non-spatial memory ability but the indexes did not reach the levels of the Sham group indicating that sevoflurane postconditioning alone is incap- able of completely eliminating damage to non-spatial mem- ory ability caused by cerebral HI damage.
+
+In rats,
+
+the habit of exploring new things
+
+opening, of mPTPs are key factors that determine whether injury is reversible or irreversible. Mitochondrial permeability transition is a common pathway shared by death and apoptosis of (Kroemer and Reed, 2000; Juhaszova et al., 2009). Sevoflurane and isoflurane postcondi- tioning against cerebral ischemia-reperfusion injury involve inhibition of mPTP opening (Feng et al., 2005; Pagel et al., 2006). Interestingly, neuronal mitochondria undergo perme- ability transition as well, (Nieminen et al., 1996) and inhibi- tion of mPTP activity has become a novel neuroprotection strategy (Kristal et al., 2004). Crucial factors for mPTP opening are mitochondrial calcium overload, ATP depletion, and oxidative stress, and these are exactly what occur in the brains of neonatal HIBD rats during hypoxia-reoxygenation. Thus, sevoflurane postconditioning-conferred neuroprotec- tion against HIBD may also act on mPTP opening.
+
+injured cells
+
+The Morris water maze is a classic method testing hippocampus-related spatial learning and memory ability (Rodríguez et al., 2003). The Morris water maze test showed that sevoflurane postconditioning improved learning effi- ciency, learning strategy, and long-term spatial memory as compared to the HI/Control group. However, sevoflurane postconditioning did not improve these measures to the level of the Sham group.
+
+Research has proven that ischemic injury is a dynamic process, and that rodents continue to loose neurons weeks after cerebral ischemia (Li et al., 1995; Du et al., 1996). This loss is especially characterized by brain damage during the growth period (Hu et al., 2000). Our research showed that compared with the Sham group, rats in the HI/Control group had obvious hippocampal neuron loss and more serious apoptosis, which is consistent with the findings of the aforementioned studies. Neurons almost completely depend on mitochondria-supplied ATP to maintain their function, so the state of mitochondria is an important factor that deter- mines whether injured neurons survive (Kann et al., 2005; Mancuso et al., 2007). mPTPs are highly conductive nonspe- cific channels across the outer mitochondrial and inner mitochondrial membrane. Opening, and the degree of
+
+In the current study, the HIþSev group showed signifi- cantly lower mPTP activity, reduced long-term hippocampal pathological injuries, and improved behaviors as compared with the HI/Control group. Atr, a mPTP-specific opener, did not worsen hippocampal neuron damage or harm long-term learning and memory by itself, but appeared to reverse the neuroprotection of sevoflurane postconditioning. Although CsA, a mPTP-specific blocker, mimicked the neuroprotection provided by sevoflurane postconditioning, failed to enhance the effect of sevoflurane postconditioning. These results suggest that sevoflurane postconditioning possibly protects the brain by blocking the mitochondrial permeability transition and reducing metabolic energy disorder and neu- ron damage in the hippocampus and other tissues of HIBD rats.
+
+it
+
+Endothelial nitric oxide synthase (eNOS), a downstream kinase of Akt, can activate the release of eNO and inhibit platelet aggregation and generation of superoxides in vessels so as to protect endothelial function and promote heman- giectasis and neovascularization, (Rikitake et al., 2002) effi- ciently preventing secondary lower perfusion and the damage to vascular endothelial cells caused by oxygen- derived free radicals during reperfusion period after HIBD.
+
+31
+
+32
+
+b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7
+
+Fig. 7 – Western blot analysis. Protein expression of p-Akt/p-eNOS/p-GSK-3β and t-Akt/t-eNOS/ t-GSK-3β (A) in the left hippocampus in the Sham, HI/Control, HIþSev, and HIþSevþLY groups were determined 24 hours after the intervention. (B and C) Relative density of p-Akt and t-Akt, respectively. β-Actin served as the internal control. Error bars represent standard error of the mean. Statistical comparisons were performed with the Student's t-test. #Po0.05 compared with HI/Control. *Po0.05 compared with HIþSev group. Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane.
+
+The latter 2 are key processes in the occurrence and devel- opment of HIBD. Some researchers believe that NO is involved in mediating the preconditioning and postcondi- tioning effects of volatile anesthetics like sevoflurane and isoflurane, and it remarkably reduces myocardial infarction size in ischemia/reperfusion models (Tessier-Vetzel et al., 2006; Lamberts et al., 2009). Rastaldo et al. (2007) have shown that endogenous NO may activate PKG via cGMP, and ulti- mately affect the human body by inhibiting mPTP opening. The NO/cGMP pathway is one of the important molecular mechanisms by which volatile anesthetics like sevoflurane exert their effects (Johns, 1996). Other studies have shown that GSK-3β takes part in the transduction of several intra- cellular signaling pathways, genetic transcription and trans- lation, embryogenesis, and neuronal death and apoptosis Jope and Johnson, 2004; Chong (Balaraman et al., 2006;
+
+et al., 2007). Furthermore, the PI3K/Akt/GSK-3β pathway provides negative feedback and promotes cell survival by phosphorylating ser9 of GSK-3β, and mediating GSK-3β activ- ity inhibition (Pap and Cooper, 1998; Duarte et al., 2008).
+
+Ischemic/pharmacological preconditioning and postcondi- tioning produce cardioprotective effects through inhibiting mPTP activity and reducing apoptosis via the PI3K/Akt/GSK- 3β signaling pathway (Juhaszova et al., 2009; Feng et al., 2005; Gomez et al., 2008; Zhu et al., 2010). The concentration of GSK-3β in the central nervous system is extremely high, especially in the hippocampus (Hidenori et al., 2006). The expression level of GSK-3β reaches a peak during late preg- nancy and early after birth, and is closely related to develop- ment and reconstruction of neurons (Takahashi et al., 1994; Leroy and Brion, 1999). We found that the P13K pathway was activated to induce more phosphorylation of Akt, eNOS, and
+
+b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7
+
+of p90rsk2 under SB216763 treatment is necessary to further elucidate the mechanism in the future.
+
+Fig. 8 – Alterations of mitochondrial permeability transition pore opening. Atr, atractyloside; CsA, Cyclosporin A; HI, hypoxia-ischemia; L-N, L-NAME; LY, LY294002; SB, SB216763; Sev, sevoflurane. Comparisons between groups were using one-way ANOVA with Fisher's LSD as post-hoc procedure. The detailed sample size, mean7SD are listed in Supplementary table S4.
+
+GSK-3β in the HIþSev group than in the HI/Control, specific inhibitors LY294002, L-NAME, and SB216763 blocked sevoflur- ane postconditioning-induced expression of p-Akt, p-eNOS, and p-GSK-3β, respectively, and mPTP activity was greatly increased in the corresponding groups, neutralizing the effect of sevoflurane postconditioning on HIBD. These findings indicate that sevoflurane postconditioning may confer neu- roprotection against HIBD by inhibiting mitochondrial per- meability transition via the PI3K/Akt/eNOS and PI3K/Akt/ GSK-3β pathways. Although sevoflurane-induced activation of PI3K/Akt has been confirmed to provide heart and cerebral protections in several studies (Ye et al., 2012; Zhang et al., 2014), however, the underlying mechanism of sevoflurane- induced activation of PI3K/Akt is still unknown. It will be interesting to indentify the upstream components of the signaling pathway(s) that exert sevoflurane-induced activa- tion of PI3K/Akt. Previous studies have suggested that PI3K/ AKT/eNOS and PI3K/Akt/GSK-3β pathways may regulate mPTP through PKC-epsilon, reactive oxygen species, Ca2þ and mitochondrial ATP-dependent Kþ channels (Ren et al., 2014; Juhaszova et al., 2004; Pravdic et al., 2009). But the detailed mechanism is still not fully understood and is worthy of further investigation.
+
+There are some limitations to this study. Arterial blood gas analysis was not performed during cerebral HI. Some researchers believe that cerebral HI itself, to a certain degree, can promote proliferation of cortical neurons and hippocam- pal neural precursor cells in neonatal rats, (Bartley et al., 2005) while sevoflurane lowers the cerebral metabolic rate, so both play a role in the recovery of neurological function. The result that Art neutralized the neuroprotective effect of sevoflurane postconditioning may partially be due to its effect of inhibiting energy generation rather than blocking mPTP. CsA may be incapable of blocking mPTP opening when mitochondria are seriously injured, and its neuroprotective effect may be dose-dependent. Whether combining different concentrations of sevoflurane and different doses of CsA can protect against HIBD to different degrees requires future study. We did not perform 2,3,5-triphenyltertrazolium chlor- ide (TTC) staining to determine brain infarct volume. Lastly, other pathways such as PI3K/Akt/mTOR or ERK1/2 were not studied.
+
+In conclusion, sevoflurane postconditioning may improve long-term learning and memory of neonatal HIBD rats pos- sibly by blocking mPTP opening and reducing neuron death and apoptosis in the hippocampus via PI3K/Akt/eNOS and PI3K/Akt/GSK-3β pathways.
+
+4.
+
+Experimental procedures
+
+4.1.
+
+Animals
+
+A total of 240 male and female clean Sprague-Dawley rats, 7 days of age and weighting 12–16 g, (Shanghai Slac Labora- tory Animal Co., Ltd., China) were used in this study. They were housed and treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 80-23, revised in 2011). All rats were maintained under standard laboratory temperature and humidity and a 12 day/night cycle (8 am/8 pm), and were allowed free access to food and water. The study was approved by the Experimental Animal Care Committee of the Fujian Medical University Union Hospital, and efforts were made to minimize the number of animals used and their suffering.
+
+4.2.
+
+Experimental protocol
+
+It is noteworthy that sevoflurane postconditionin caused phosphorylation of GSK-3β at Ser9 and its inhibition, while SB216763 blocked sevoflurane-induced phosphorylation of GSK-3β and neuroprotection in our study. These two results seem conflicted since both Sev postconditioning and SB216763 treatment inhibits GSK-3β. However, previous stu- dies have consistently reported that SB216763 reduced the phosphorylation levels of GSK-3β Ser9 (possibly via inhibition of p90rsk2 (Zhang et al., 2003; Lochhead et al., 2001; Liang and Chuang, 2007), a kinase of GSK-3β Ser9), indicating the inhibition effect of SB216763 on GSK-3β has a complicated mechanism which eventually blocked the sevoflurane- induced protection in this study. Examining kinase activity
+
+The animals were randomly allocated into 10 groups (n¼ 24 per group; Fig. 1): (1) Sham, without hypoxia-ischemia; (2) HI/ Control, received cerebral hypoxia-ischemia; (3) HIþAtractylo- side (Atr), (4) HIþCyclosporin A (CsA), treated like the control and respectively injected with Atr (10 mg/kg) and CsA (5 mg/ kg); (5) HIþsevoflurane (Sev), treated like the control and (6) HIþSevþLY, received sevoflurane postconditioning; (7) HIþSevþL-N, (10) HIþSevþCsA, treated like the HIþSev group and respectively injected with LY294002 (0.3 mg/kg), L-NAME (10 mg/kg), SB216763 (0.2 mg/kg), Atr (10 mg/kg), and CsA (5 mg/kg). LY294002, L-NAME, and SB216763 are specific blockers of
+
+(8) HIþSevþSB,
+
+(9) HIþSevþAtr,
+
+33
+
+34
+
+b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7
+
+Akt, eNOS, and GSK-3β, respectively. Atr and CsA open and close, respectively, mPTPs. In each group, the brains of the rats that received behavioral testing (from 32-days-old to 42- days-old; n¼ 12 per group) were harvested for determination of hippocampal neuron count and morphology study, and the brains of the other 12 rats were harvested 24 h after the intervention for Western blot analysis, and study of mito- chondrial permeability transition pore opening.
+
+4.3.
+
+Cerebral HI model and sevoflurane postconditioning
+
+The cerebral HI model was adapted from a procedure described previously (Ren et al., 2014). Briefly, the rats were anesthetized with pentobarbital sodium (0.5–1%, 40–50 mg/ kg, intraperitoneal), and their left common carotid arteries were permanently ligated with a double 7–0 surgical silk; the arteries in the Sham group, however, were not ligated. A dose of 5 mL of 0.1% DMSO or drug (LY294002, L-NAME, SB216763, Atr, CsA) with 0.1% DMSO was injected into the left lateral ventricle immediately after the surgery as previously described (Satoh and Onoue, 2005). After waking, the rats were returned to their cages with the mothers for 1.5–2.5 h, – and then placed in a chamber containing humidified 8% O2 92% N2 for 2 h. The air temperature in the chamber was maintained at 36.571 1C. The chamber was then exposed to room air for 15–20 min. For sevoflurane postconditioning, the animals were placed in a chamber containing 2.5% sevoflur- –70% N2 for 30 min after cerebral HI injury. After ane in 30% O2 waking, the neonates were cleaned with 75% alcohol and returned to their mothers.
+
+4.4.
+
+Novel object recognition test
+
+The rats were evaluated with a nonspatial object recognition memory task 25 days after the intervention as described by Ennaceur and Delacour (1988) and Bruel-Jungerman et al. (2005). Briefly, for the first 3 days, after being comforted and stroked, each animal was put into an open chamber made of black plexiglas (80 (cid:2) 80 (cid:2) 60 cm3) for a 5 min acclimation and the test was conducted on the fourth day. Before the test, the animals received a 5 min training in the chamber containing 2 different objects (a white cube and a red cylinder) fixed at adjacent angles with a spacing of 10 cm from the field wall. Rats were put into the chamber with their backs turned towards the objects and allowed to explore the chamber freely for 5 minutes. Exploratory behavior can be identified when rats touch the objects with their noses or put their noses at places within 2 cm of the objects. To test memory storage, the white cube was kept in the chamber and the red cylinder was replaced by a blue semisphere. Exploratory time of new (T2) and old (T1) objects within 5 min was recorded and memorization ability of the rats was assessed by discrimina- þT2). The blue semisphere was replaced tion index: DI¼ T2/(T1 by a green prism 3 h after training, and the green prism was replaced by a yellow irregular shape 24 h after the training. The time each rat used to explore new and old objects was recorded for calculating DI. The DIs at 5 min, 3 h, and 24 h after the training (DI0 h, DI3 h, DI24 h) represent the instant, short-term, and long-term memory, respectively. Data with total exploration time less than 20 s were excluded from
+
+statistic analysis. The field was always provided with even light, and the objects and fields were cleaned with 75% ethanol after each testing.
+
+4.5.
+
+Morris water maze test
+
+After the novel object recognition test, the Morris water maze was used to test spatial learning and memory (Peng et al., 2012; Jiang et al., 2004). Briefly, a black circular pool (120 cm in diameter, 50 cm in height) was filled with water (2571 1C) to a depth of 25 cm and located in a quiet room. Chinese ink was added to make the water opaque. The water maze was conceptually divided into 4 quadrants, and a hyaline platform (10 cm in diameter) was submerged 1 cm below the surface of the water at the midpoint of the third quadrant. In the place navigation trial, each rat underwent 4 successive trials a day for 5 days for memory acquisition training, with a 15 min interval between trials for the rat to recover physically. The sequence of water-entry points differed each day, but the location of the platform was constant. Escape latency (EL) to find the platform was measured up to a maximum of 120 s. On locating the platform, the rat was left there for 15 s before the next trial. If the rats failed to locate the platform within 120 s, it was guided to the platform and allowed to stay there for 15 s. Latency and the search strategies, including straight strategy, tendency strategy, marginal strategy, and random strategy, were recorded for each trial. Twenty-four hours after the last training session, a space exploration trial was performed. The platform was removed from the pool and rats were allowed to swim freely for 60 s. Four indexes were calculated: (1) the time spent by the rats in the third quadrant in which the platform was hidden during acquisition trials; (2) the number of rats crossing exactly over the original position of the platform; (3) the search path in the target quadrant; (4) the total movement distance. Search speed was calculated by total movement distance divided by 120 cm/s. All trials were videotaped by a camera located 2 m above the water surface and computer analyzed.
+
+4.6.
+
+Histology of left hippocampal neurons
+
+After the behavioral studies, rats were anesthetized with pentobarbital, transcardialy perfused with 200 mL of 4 1C heparin saline solution and then with 300 mL of 4% paraf- ormaldehyde. Left hippocampus was made into a wax block according to Paxinos–Waston methods. Continual coronal sections (4 mm in thickness) at approximately 3.3 mm caudal to bregma were obtained, and subjected to hematoxylin– eosin (HE) staining. The sections were examined by an observer blinded to the rat group assignment. Neurons microscopically showed a clear boundary, a round or an oval shape, a smooth cell membrane, basophilic cytoplasm (Nissl body), a large and round nucleus, a clear nuclear membrane and a large and round nucleolus will be defined as surviving neurons. Apoptotic neurons will not be regarded as surviving ones. Surviving neurons in pyramidal cell layer of the CA1 and CA3 regions were counted (n/mm) by two investigators blind to experimental conditions, and a count was deter- mined by averaging the total of 5 sections.
+
+b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7
+
+4.7. Western blot analysis
+
+Acknowledgments
+
+Proteins were separated on a 12% SDS-PAGE gel, and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, USA). The membrane was blocked using 5% nonfat milk and incubated with a mouse anti-p-Akt, t-Akt, p-eNOS, t-eNOS, p-GSK-3β, and t-GSK-3β monoclonal antibody (mAb) (Cell Signaling Technology, Beverly, MA, USA) or a mouse anti-β- actin mAb (Sigma, USA). The proteins were visualized and quantified using ECL reagents (Pierce, IL, USA).
+
+This work was supported by grants from Department of Edu- cation, Fujian Province (type A) (Grant number: JA12159) in part, and the Science Foundation of the Fujian Province, China (Grant No. 2015J01465).
+
+Appendix A.
+
+Supplementary material
+
+4.8.
+
+mPTP opening assay
+
+Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres. 2015.10.050
+
+Preparation of mitochondria was adapted from a procedure described previously (Wu et al., 2006). All procedures were carried out in the cold (0–4 1C). Hippocampal pieces were placed in isolation buffer (250 mmol/L sucrose, 210 mmol/L mannitol, 1 mmol/L K-EDTA, 10 mmol/L Tris–HCl, pH 7.4) and homogenized (10 mL buffer/g). The homogenate was imme- diately centrifuged at 2000g for 3 min. The supernatant was centrifuged again at 2000g for 3 min, the second supernatant was decanted and centrifuged at 12,000g for 8 min, and the resulting supernatant was decanted and resuspended in isolation buffer without K-EDTA. The suspension was cen- trifuged at 12,000g for 10 min and the resulting mitochondrial pellet was resuspended in the same buffer. Mitochondrial protein concentration was quantified according to the Brad- ford's method using 1 g/mL bovine serum albumin (BSA) as standard. Purity and integrity of isolated mitochondria were confirmed by neutral red-Janus green B staining (Sigma, USA). Isolated mitochondria from the hippocampus (0.5 mg protein) was resuspended in swelling buffer (71 mmol/L sucrose, 215 mmol/L mannitol, and 10 mmol/L sodium succinate in 5 mmol/L HEPES, pH 7.4) to a final volume of 2 mL, and incubated at 25 1C for 2 min. mPTP-induced mitochondrial swelling was confirmed by 5 min incubation with the strong mPTP inhibitor CsA before addition of CaCl2, and was mea- sured with a spectrophotometer (Beckman DU800, USA) as a reduction in optical density at 540 nm (OD540) (Kristal and Brown, 1999; Baines et al., 2003).
+
+4.9.
+
+Statistical analysis
+
+r e f e r e n c e s
+
+Chan, R.H., Song, D., Goonawardena, A.V., Bough, S., Sesay, J.,
+
+Hampson, R.E., et al., 2010. Changes of hippocampal CA3–CA1 population nonlinear dynamics across different training ses- sions in rats performing a memory-dependent task. Conf. Proc. IEEE Eng. Med. Biol. Soc. 2010, 5464–5467.
+
+Morris, A.M., Churchwell, J.C., Kesner, R.P., Gilbert, P.E., 2012.
+
+Selective lesions of the dentate gyrus produce disruptions in place learning for adjacent spatial locations. Neurobiol. Learn. Mem. 97, 326–331.
+
+Hopkins, R.O., Haaland, K.Y., 2004. Neuropsychological and neu- ropathological effects of anoxic or ischemic induced brain injury. J. Int. Neuropsychol. Soc. 10, 957–961.
+
+Fan, L.W., Lin, S., Pang, Y., Lei, M., Zhang, F., Rhodes, P.G., et al.,
+
+2005. Hypoxia-ischemia induced neurological dysfunction and brain injury in the neonatal rat. Behav. Brain Res. 165, 80–90.
+
+Zhang, C., Shen, W., Zhang, G., 2002. N-methyl-D-aspartate
+
+receptor and L-type voltage-gated Ca2þ suppress the release of cytochrome and the expression of proeaspase-3 in rat hippocampus after global brain ischemia. Neurosci. Lett. 328, 265–268.
+
+channel antagonists
+
+Vannucci RC1, Brucklacher R.M., Vannucci, S.J., 2001. Intracellular
+
+calcium accumulation during the evolution of hypoxic- ischemic brain damage in the immature rat. Brain 126, l17–l20. Peng, B., Guo, Q.L., He, Z.J., Ye, Z., Yuan, Y.J., Wang, N., et al., 2012. Remote ischemic postconditioning protects the brain from global cerebral ischemia/reperfusion injury by up-regulating endothelial nitric oxide synthase through the PI3K/Akt path- way. Brain Res. 1445, 92–102.
+
+All data were presented as mean7standard deviation (SD). For comparison between multiple groups, data were analyzed by one-way ANOVA. When a statistical difference was deter- mined by ANOVA, the least significant difference (LSD) procedure was applied. The percentage of search strategies were examined by the Mann–Whitney method, and repetitive measure ANOVA was used to measure mean EL at different time points. Spatial probe trial data were analyzed by one- way ANOVA and principal components analysis (PCA). All analyses were performed with SPSS 13.0 for Windows, and a value of Po0.05 was considered significant.
+
+Danielisova´ , V., Gottlieb, M., Ne´ methova´ , M., Burda, J., 2008.
+
+Effects of bradykinin postconditioning on endogenous anti- oxidant enzyme activity after transient forebrain ischemia in rat. Neurochem. Res. 33, 1057–1064.
+
+Hu, X., Xie, C., He, S., Zhang, Y., Li, Y., Jiang, L., 2013. Remifentanil
+
+postconditioning improves global cerebral ischemia induced spatial learning and memory deficit in rats via inhibition of neuronal apoptosis through the PI3K signaling pathway. Neurol. Sci. 34, 1955–1962.
+
+Tsang, A., Hausenloy, D.J., Mocanu, M.M., Yellon, D.M., 2004.
+
+Postconditioning: a form of “modified reperfusion” protects the myocardium by activating the phosphatidylinositol 3- kinase-Akt pathway. Circ. Res. 95, 230–232.
+
+Lim, S.Y., Davidson, S.M., Hausenloy, D.J., Yellon, D.M., 2007.
+
+Competing interests
+
+Preconditioning and postconditioning: the essential role of the mitochondrial permeability transition pore. Cardiovasc. Res. 75, 530–535.
+
+Lemoine, S., Zhu, L., Beauchef, G., Lepage, O., Babatasi, G.,
+
+The authors report no conflict of interest.
+
+Ivascau, C., et al., 2010. Role of 70-kDa ribosomal protein S6
+
+35
+
+36
+
+b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7
+
+kinase, nitric oxide synthase, glycogen synthase kinase-3 beta, and mitochondrial permeability transition pore in desflurane-induced postconditioning in isolated human right atria. Anesthesiology. 112, 1355–1363.
+
+Fang, N.X., Yao, Y.T., Shi, C.X., Li, L.H., 2010. Attenuation of
+
+Feng, J., Lucchinetti, E., Ahuja, P., Pasch, T., Perriard, J.C., Zaugg, M., 2005. Isoflurane postconditioning prevents opening of the mitochondrial permeability transition pore through inhibition of glycogen synthase kinase 3beta. Anesthesiology 103, 987–995.
+
+ischemia reperfusion injury by sevoflurane postconditioning involves protein kinase B and glycogen synthase kinase 3 beta activation in isolated rat hearts. Mol. Biol. Rep. 37, 3763–3769.
+
+Zitta, K., Meybotun, P., Bein, B., Ohnesorge, H., Steinfath, M.,
+
+Scholz, J., et al., 2010. Cytoprotective effects of the volatile anesthetic sevoflurane is highly dependent on timing and duration of Sevoflurane conditioning: fimings from a human, in-vitro hypoxia model. Eur. J. Pharmacol. 22, 475–480. Ye, Z., Guo, Q., Xia, P., Wang, N., Wang, E., Yuan, Y., 2012.
+
+Pagel, P.S., Krolikowski, J.G., Neff, D.A., Weihrauch, D., Bienen- graeber, M., Kersten, J.R., et al., 2006. Inhibition of glycogen synthase kinase enhances isoflurane-induced protection against myocardial infarction during early reperfusion in vivo. Anesth. Analg. 102, 1348–1354.
+
+Kristal, B.S., Stavrovskaya, I.G., Narayanan, M.V., Krasnikov, B.F., Brown, A.M., Beal, M.F., et al., 2004. The mitochondrial per- meability transition as a target for neuroprotection. J. Bioe- nergy Biomembr. 36, 309–312.
+
+Sevoflurane postconditioning involves an up-regulation of HIF-1alpha and HO-1 expression via PI3K/Akt pathway in a rat model of focal cerebral ischemia. Brain Res. 1463, 63–74. Nieminen, A.L., Petrie, T.G., Lemasters, J.J., Selman, W.R., 1996.
+
+Rikitake, Y., Hirata, K., Kawashima, S., Ozaki, M., Takahashi, T.,
+
+Ogawa, W., et al., 2002. Involvement of endothelial nitric oxide in sphingosine-1-phosphate-induced angiogenesis. Arterios- cler. Thromb. Vasc. Biol. 22, 108–114.
+
+Cyclosporin A delays mitochondrial depolarization induced by N-methyl-D-aspanate in cortical neurons: evidence of the mitochondrial permeability transition. Neuroscience 75, 993–997.
+
+Ren, X., Wang, Z., Ma, H., Zuo, Z., 2014. Sevoflurane postcondi- tioning provides neuroprotection against brain hypoxia- ischemia in neonatal rats. Neurol. Sci. 35, 1401–1404.
+
+Wang, J.K., Yu, L.N., Zhang, F.J., Yang, M.J., Yu, J., Yan, M., et al.,
+
+2010. Postconditioning with sevoflurane protects against focal cerebral ischemia and reperfusion injury via PI3K/Akt path- way. Brain Res. 1357, 142–151.
+
+Tessier-Vetzel, D., Tissier, R., Waintraub, X., Ghaleh, B., Berdeaux,
+
+A., 2006. Isoflurane inhaled at the onset of reperfusion potentiates the cardioprotective effect of ischemic postcon- ditioning through a NO-dependent mechanism. J. Cardiovasc. Pharmacol. 47, 487–492.
+
+Lamberts, R.R., Onderwater, G., Hamdani, N., Vreden, M.J.,
+
+Steenhuisen, J., Eringa, E.C., et al., 2009. Reactive oxygen species-induced stimulation of 5 AMP-activated protein kinase mediates sevoflurane-induced cardioprotection. Cir- culation 120, 10–15.
+
+Rastaldo, R., Pagliaro, P., Cappello, S., Penna, C., Mancardi, D.,
+
+Schousboe, A., Sarup, A., Bak, L.K., Waagepetersen, H.S., Larsson, O.M., 2004. Role of astrocytic transport processes in glutama- tergic and GABAergic neurotransmission. Neurochem. Int. 45, 521–527.
+
+Kumral, A., Uysal, N., Tugyan, K., Sonmez, A., Yilmaz, O., Gok- men, N., et al., 2004. Erythropoietin improves long-term spatial memory deficits and brain injury following neonatal hypoxia-ischemia in rats. Behav. Brain Res. 153, 77–86. Ennaceur, A., Delacour, J., 1988. A new one-trial test for neuro- biological studies of memory in rats. Behav. Brain Res. 31, 47–59.
+
+Rodrı´guez, V.M., Carrizales, L., Mendoza, M.S., Fajardo, O.R.,
+
+Giordano, M., 2003. Effects of sodium arsenite exposure on development and behavior in the rat. Neurotoxicol. Teratol. 24, 743–750.
+
+Li, Y., Chopp, M., Jiang, N., Yao, F., Zaloga, C., 1995. Temporal
+
+profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat. J. Cereb. Blood Flow Metab. 15, 389–397.
+
+Du, C., Hu, R., Csernansky, C.A., Hsu, C.Y., Choi, D., 1996. Very
+
+delayed infarction after mild focal cerebral ischemia: a role for apoptosis. J. Cereb. Blood Flow Metab. 16, 195–201.
+
+Westerhof, N., et al., 2007. Nitric oxide and cardiac function. Life Sci. 81, 779–793.
+
+Johns, R.A., 1996. Nitric oxide, cyclic guanosine momophosphate,
+
+and the anesthetic state. Anesthesiology 85, 457–459. Balaraman, Y., Limaye, A.R., Levey, A.I., Srinivasan, S., 2006.
+
+Glycogen synthase kinase-3beta and Alzheimer’s disease: pathophysiological and therapeutic significance. Cell Mol. Life Sci. 63, 1226–1235.
+
+Jope, R.S., Johnson, G.V., 2004. The glamour and gloom of glyco- gen synthase kinase-3. Trends Biochem. Sci. 29, 95–102.
+
+Chong, Z.Z., Li, F., Maiese, K., 2007. The pro-survival pathways of mTOR and protein kinase B target glycogen synthase kinase- 3beta and nuclear factor-kappa B to foster endogenous microglial cell protection. J. Mol. Med. 19, 263–272.
+
+Pap, M., Cooper, G.M., 1998. Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway. J. Biol. Chem. 273, 19929–19932.
+
+Duarte, A.I., Santos, P., Oliveira, C.R., Santos, M.S., Rego, A.C., 2008. Insulin neuroprotection against oxidative stress is mediated by Akt and GSK-3beta signaling pathways and changes in protein expression. Biochim. Biophys. Acta 1783, 994–1002.
+
+Hu, B.R., Liu, C.L., Ouyang, Y., Blomgren, K., Siesjo¨ , B.K., 2000.
+
+Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation. Cereb. Blood Flow Metab. 20, 1294–1300.
+
+Kann, O., Kovacs, R., Njunting, M., Behrens, C.J., Ota´ hal, J.,
+
+Lehmann, T.N., et al., 2005. Metabolic dysfunction during neuronal activation in the ex vivo hippocampus from chronic epileptic rats and humans. Brain 128, 2396–2407.
+
+Gomez, L., Paillard, M., Thibault, H., Derumeaux, G., Ovize, M., 2008. Inhibition of GSK3beta by postconditioning is required to prevent opening of the mitochondrial permeability transi- tion pore during reperfusion. Circulation 117, 2761–2768. Zhu, J., Rebecchi, M.J., Tan, M., Glass, P.S., Brink, P.R., Liu, L., 2010. Age-associated differences in activation of Akt/GSK-3beta signaling pathways and inhibition of mitochondrial perme- ability transition pore opening in the rat heart. J. Gerontol. A Biol. 65, 611–619.
+
+Mancuso, C., Scapagini, G., Curro` , D., Giuffrida Stella, A.M., De Marco, C., Butterfield, D.A., et al., 2007. Mitochondrial dys- function free radical generation and cellular stress response in neurodegenerative disorders. Front. Biosci. 12, 1107–1123. Kroemer, G., Reed, J.C., 2000. Mitochondrial control of cell death.
+
+Hidenori, E., Chikako, N., Hiroshi, K., 2006. Activation of the Akt/ GSK-3β signaling pathway mediates survival of vulnerable hippocampal neurons after transient global cerebral ischemia in rat. J. Cereb. Blood Flow Metabol. 26, 1479–1489.
+
+Nat. Med. 6, 513–519.
+
+Juhaszova, M., Zorov, D.B., Yaniv, Y., Nuss, H.B., Wang, S., Sollott, S.J., 2009. Role of glycogen synthase kinase-3beta in cardio- protection. Circ. Res. 104, 1240–1252.
+
+Takahashi, M., Tomizawa, K., Kato, R., Sato, K., Uchida, T., Fujita, S.C., et al., 1994. Localization and developmental changes of tau protein kinase I/glycogen synthase kinase-3 beta in rat brain. J. Neurochem. 63, 245–255.
+
+b r a i n r e s e a r c h 1 6 3 0 ( 2 0 1 6 ) 2 5 – 3 7
+
+Leroy, K., Brion, J.P., 1999. Developmental expression and locali- zation of glycogen synthase kinase-3beta in rat brain. J. Chem. Neuroanat. 16, 279–293.
+
+Zhang, J., Wang, C., Yu, S., Luo, Z., Chen, Y., Liu, Q., et al., 2014. Sevoflurane postconditioning protects rat hearts against ischemia-reperfusion injury via the activation of PI3K/AKT/ mTOR signaling. Sci. Rep. 4, 7317.
+
+Bartley, J., Soltau, T., Wimborne, H., Kim, S., Martin-Studdard, A., Hess, D., et al., 2005. Brd U-positive cells in the neonatal mouse hippocampus following hypoxic-ischemic brain injury. BMC Neurosci. 6, 15.
+
+Satoh, J., Onoue, H., 2005. Nogo-A and nogo receptor expression in demyelinating lesions of multiple sclerosis. J. Neuropathol. Exp. Neurol. 64, 129–138.
+
+Juhaszova, M., Zorov, D.B., Kim, S.H., Pepe, S., Fu, Q., Fishbein, K. W., et al., 2004. Glycogen synthase kinase-3beta mediates convergence of protection signaling to inhibit the mitochon- drial permeability transition pore. J. Clin. Invest. 113, 1535–1549.
+
+Bruel-Jungerman, E., Laroche, S., Rampon, C., 2005. New neurons
+
+in the dentate gyrus are involved in the expression of enhanced long-term memory following environmental enrichment. Eur. J. Neurosci. 21, 513–521.
+
+Jiang, M.L., Han, T.Z., Pang, W., Li, L., 2004. Gender-and age-
+
+Pravdic, D., Sedlic, F., Mio, Y., Vladic, N., Bienengraeber, M.,
+
+Bosnjak, Z.J., 2009. Anesthetic-induced preconditioning delays opening of mitochondrial permeability transition pore via protein Kinase C-epsilon-mediated pathway. Anesthesiology 111, 267–274.
+
+Zhang, F., Phiel, C.J., Spece, L., Gurvich, N., Klein, P.S., 2003.
+
+Inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3) in response to lithium. Evidence for autoregulation of GSK-3. J. Biol. Chem. 278, 33067–33077.
+
+specific impairment of rat performance in the Morris water maze following prenatal exposure to an MRI magnetic field. Brain Res. 995, 140–144.
+
+Wu, L., Shen, F., Lin, L., Zhang, X., Bruce, I.C., Xia, Q., 2006. The neuroprotection conferred by activating the mitochondrial ATP sensitive Kþ channel is mediated by inhibiting the mitochondrial permeability transition pore. Neurosci. Lett. 402, 184–189.
+
+Kristal, B.S., Brown, A.M., 1999. Apoptogenic ganglioside GD3
+
+Lochhead, P.A., Coghlan, M., Rice, S.Q., Sutherland, C., 2001.
+
+Inhibition of GSK-3 selectively reduces glucose-6-phosphatase and phosphatase and phosphoenolypyruvate carboxykinase gene expression. Diabetes 50, 937–946.
+
+Liang, M.H., Chuang, D.M., 2007. Regulation and function of
+
+glycogen synthase kinase-3 isoforms in neuronal survival. J. Biol. Chem. 282, 3904–3917.
+
+directly induces the mitochondrial permeability transition. Biol. Chem. 274, 23169–23175.
+
+Baines, C.P., Song, C.X., Zheng, Y.T., Wang, G.W., Zhang, J., Wang, O.L., et al., 2003. Protein kinase C-epsilon interacts with and inhibits the permeability transition pore in cardiac mito- chondria. Circ. Res. 92, 873–880.
+
+37

new_pdfs/10.1016_j.ejphar.2011.08.050.txt

@@ -0,0 +1,289 @@
+European Journal of Pharmacology 670 (2011) 168–174
+
+Contents lists available at SciVerse ScienceDirect
+
+European Journal of Pharmacology
+
+j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / e j p h a r
+
+Behavioural Pharmacology Effects of gestational isoflurane exposure on postnatal memory and learning in rats Feijuan Kong a, 1, Linhao Xu b, 2, Daqiang He b, 2, Xiaoming Zhang b, 2, Huishun Lu a,⁎ a Department of Anesthesiology, Women's Hospital, School of Medicine, Zhejiang University, China b Institute of Anatomy and Cell Biology, School of Medicine, Zhejiang University, China
+
+a r t i c l e
+
+i n f o
+
+a b s t r a c t
+
+Article history: Received 16 July 2011 Received in revised form 15 August 2011 Accepted 27 August 2011 Available online 14 September 2011
+
+Keywords: Isoflurane Memory and learning impairment Hippocampus C/EBP homologous protein Caspase-12 Neuron apoptosis
+
+A maternal fetal rat model was developed to study the effects of gestational isoflurane exposure on postnatal memory and learning and investigate the potential mechanisms. Pregnant rats at gestational day 14 were ex- posed to 1.3% isoflurane for 4 h. Spatial learning and memory of the offspring were examined using the Mor- ris Water Maze. The expression levels of C/EBP homologous transcription factor protein (CHOP) and caspase- 12 in the hippocampus of the pups were determined by immunohistochemistry and western blot analysis. Simultaneously, the ultrastructure changes of synapse in the hippocampal CA1 and dentate gyrus region were also observed by transmission electron microscopy (TEM). Prenatal exposure to isoflurane impaired postnatal spatial memory and learning in the offspring rats as shown by the longer escape latency and the fewer times of original platform crossing in the Morris Water Maze test. The number of CHOP and caspase- 12 positive neurons significantly increased by 138% and 147% respectively in the hippocampus of isoflur- ane-exposed pups, as well as the levels of CHOP and caspase-12 protein. Furthermore, TEM studies showed changes of synaptic ultrastructure in isoflurane-exposed hippocampus characterized by the decreased synap- se number, the widened synaptic cleft and the thinned postsynaptic densities. These results demonstrate that gestational exposure to a clinically relevant concentration of isoflurane could cause neuron apoptosis, changes of synaptic structure, and postnatal spatial memory and learning impairments in offspring. Our study further showed that the up-regulation of CHOP and caspase-12 may contribute to isoflurane-induced neuron apoptosis.
+
+© 2011 Elsevier B.V. All rights reserved.
+
+1. Introduction
+
+Many pregnant women, fetuses, and infants are exposed to a variety of anesthetic agents for surgical or diagnostic procedures each year. Pregnant women sometimes undergo general anesthesia during their pregnancy for surgeries unrelated to the delivery, such as fetal and non-obstetric surgeries, especially during midgestation (Goodman, 2002; Tran, 2010). Since most general anesthetic agents are lipophilic and cross the placenta easily (Dwyer et al., 1995), the developing fetal brains will be exposed to anesthetics as well. Inhalation anesthetics such as isoflurane have been widely used in recent years in clinical and research practices. Preclinical studies demonstrate that early exposure to anesthetic agents causes neuroapoptosis and long-term cognitive im- pairments (Culley et al., 2004; Jevtovic-Todorovic et al., 2003; Ma et al., 2007), and recent clinical studies support the possibility (DiMaggio et al., 2008; Kalkman et al., 2009; Wilder et al., 2009). These observations
+
+⁎ Corresponding author at: Department of Anesthesiology, Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, Bachelor Road 1, 310006, PR China. Tel.: + 86 571 87061501 2410; fax: + 86 571 87061878.
+
+E-mail address: lig08010915@163.com (H. Lu).
+
+1 Postal address: Department of Anesthesiology, Women's Hospital, School of Medi-
+
+cine, Zhejiang University, Hangzhou, Bachelor Road 1, 310006, PR China.
+
+raise concerns about the potentially deleterious effects of general anes- thesia in the human fetus, neonate, and infant. Nevertheless, the major- ity of prior neurodevelopmental studies focused on postnatal subjects rather than on the fetuses. In this study, we hypothesized that gestation- al exposure to isoflurane during maternal anesthesia may have deleteri- ous effects on the fetal brain that leads to postnatal spatial memory and learning impairments in the offspring rats.
+
+The cellular and molecular mechanisms of anesthetics-mediated neurotoxicity remain unclear. Previous studies indicate that endo- plasmic reticulum (ER) stress is associated with a range of diseases, including ischemia/reperfusion injury, neurodegeneration, and dia- betes (Oyadomari and Mori, 2004), making ER stress a probable in- stigator of pathological cell death and dysfunction. At least three pathways contribute to ER stress-mediated cell death: transcription activation of the C/EBP homologous transcription factor (CHOP) (Oyadomari and Mori, 2004), activation of the IRE1-tumor necrosis factor receptor-associated factor (TRAF2) pathway (Matsukawa et al., 2004) and activation of ER-resident caspase-12 (Nakagawa and Yuan, 2000; Nakagawa et al., 2000). CHOP, a member of the C/ EBP transcription factor family, is induced by ER stress and thus causes apoptosis. Caspase-12, an ER-specific caspase, participates in apoptosis under ER stress. In the current study, we hypothesized that CHOP and caspase-12 play a role in the mechanisms of isoflur- ane-induced neuron apoptosis.
+
+2 Postal address: Institute of Anatomy and Cell Biology, School of Medicine, Zhejiang
+
+University, Hangzhou, Yuhangtang Road 388, 310058, PR China.
+
+0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.08.050
+
+F. Kong et al. / European Journal of Pharmacology 670 (2011) 168–174
+
+169
+
+In the present study, using a maternal fetal rat model, we tested the capacity for learning and memory in pups of fetal exposure to iso- flurane with the Morris Water Maze. Then we used transmission elec- tron microscopy (TEM) to investigate synaptic ultrastructure changes in the hippocampal area. We also measured the levels of CHOP and caspase-12 protein in the hippocampal area, and analyzed their rela- tionship with isoflurane-induced neuron apoptosis.
+
+2. Materials and methods
+
+2.1. Animals
+
+All of the animals were treated according to the guidelines of the Guide for the Care and Use of Laboratory Animals (China Ministry of Health). The Laboratory Animal Care Committee of Zhejiang Universi- ty approved all experimental procedures and protocols. All efforts were made to minimize the number of animals used and their suffer- ing. The dams were housed in polypropylene cages, and the room temperature was maintained at 22 °C, with a 12-hour light–dark cycle. The dams at gestational day 14 were used for all experiments, because this time corresponds approximately to midgestation in humans (Clancy et al., 2001, 2007), the period when most nonobste- tric surgeries and fetal interventions are performed (Goodman, 2002; Tran, 2010).
+
+2.2. Anesthesia exposure
+
+Ten dams were randomly divided into a control and an isoflurane group (n = 5). The dams were placed in plastic containers resting in water baths with a constant temperature of 38 °C. In these boxes, the dams were either exposed to 1.3% isoflurane (Lot 826005U, AB- BOTT, USA) in a humidified 30% oxygen carrier gas or simply humid- ified 30% oxygen without any inhalational anesthetic for 4 h. We chose 1.3% as the anesthetic concentration because it represents 1 minimum alveolar concentration (MAC) in the pregnant rats (Mazze et al., 1985). The determination of anesthetic duration based on our preliminary study which indicated that maternal physiological states remained stable throughout a 4-hour isoflurane exposure. The isoflurane concentration in the box was monitored with an agent gas monitor (Vamos, Drager Medical AG & Co. KgaA). Otherwise, control and experimental animals were under the same treatment and envi- ronment. During isoflurane anesthesia, arterial blood gases and blood glucose were measured at the end of the 4-hour anesthetic exposure. The rectal temperature was maintained at 37 ± 0.5 °C. After exposure, the dams were returned to their cages and allowed to deliver natural- ly. The postnatal body weights of the rat pups were monitored.
+
+2.3. Memory and learning studies
+
+Four rat pups (2 females and 2 males) from each dam were select- ed to determine cognitive function at postnatal day 28 with a Morris Water Maze test with minor modifications (Jevtovic-Todorovic et al., 2003). A round pool (diameter, 150 cm; depth, 50 cm) was filled with
+
+Fig. 1. Effects of rats exposed to isoflurane on postnatal memory and learning ability. (A) Place trial demonstrating the latency for offspring rats to reach platform measuring spatial information acquisition. (B) Probe trial demonstrating the number of original platform crossing measuring memory retention capabilities. *P b 0.05 compared with control.
+
+warm (24 °C) opaque water to a height of 1.5 cm above the top of the movable clear 15-cm-diameter platform in the third quadrant. A video tracking system recorded the swimming motions of animals, and the data were analyzed using motion-detection software for the Morris Water Maze (Actimetrics Software, Evanston, IL, USA). After every trial, each rat was wiped before returning to its regular cage, keeping warm and free diet.
+
+2.3.1. Place trials
+
+The place trials were performed at postnatal day 29 for 4 days to determine the rats' ability to obtain spatial information. At postnatal day 28, the rats were made to know the existence of the platform through a 30-second swimming training. A dark black curtain sur- rounded the pool to prevent confounding visual cues. All rats received 4 trials per day in each of the four quadrants of the swimming pool. On each trial, rats were placed in a fixed position into the swimming pool facing the wall. They were allotted 120 s to find the platform upon which they sat for 20 s before being removed from the pool. If a rat did not find the platform within 120 s, the rat was gently guided to the platform and allowed to remain there for 20 s. For all training trials, swim speed and the time to reach the platform (escape latency) were recorded. The less time it took a rat to reach the platform, the
+
+Table 1 Maternal physiological parameters during isoflurane anesthesia.
+
+0 h
+
+4 h
+
+Control
+
+1.3% isoflurane
+
+Control
+
+1.3% isoflurane
+
+pH PaCO2 (mm Hg) PaO2 (mm Hg) SaO2 (%) Glucose (mg/dl)
+
+7.43 ± 0.02 35.8 ± 2.47 169.5 ± 4.32 95.4 ± 1.1 113 ± 21
+
+7.43 ± 0.01 36.7 ± 1.34 168.2 ± 6.19 94.2 ± 1.2 115 ± 16
+
+7.41 ± 0.02 36.6 ± 2.39 166.2 ± 6.41 95.1 ± 0.9 116 ± 22
+
+7.39 ± 0.01 37.9 ± 3.25 165.3 ± 5.32 94.6 ± 1.1 115 ± 12
+
+1.3% isoflurane did not affect arterial blood gas values and blood glucose levels significantly. PaCO2 = arterial carbon dioxide tension; PaO2 = arterial oxygen tension; SaO2 = arterial oxygen saturation.
+
+170
+
+F. Kong et al. / European Journal of Pharmacology 670 (2011) 168–174
+
+better the learning ability. We took the average of four trials as the es- cape latency each day.
+
+2.3.2. Probe trials
+
+Probe trials were conducted immediately after the four-day period to evaluate memory retention capabilities. The probe trials involved removing the submerged platform from the pool and allowing the rats to swim for 120 s in any of the four quadrants of the swimming pool. Time spent in the third quadrant and the number of original platform crossing in the third quadrant was recorded.
+
+immediately. Immersion fixation was completed on tissues about 1 mm3 from the hippocampus. Samples were rinsed in cold phosphate-buffered saline (PBS) and placed in 2.5% glutaraldehyde at 4 °C for 4 h. The tissue was rinsed in buffer and post-fixed with 1% osmium tetroxide for 1 h. Then, the tissue was rinsed with distilled water before undergoing a grad- ed ethanol dehydration series and was infiltrated using a mixture of half propylene oxide and half resin overnight. Twenty-four hours later, the tis- sue was embedded in resin. 120 nm sections were cut and stained with 4% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Ultrastruc- ture changes of synapse in the hippocampus were observed under a transmission electron microscope (Phliphs Tecnai 10, Holland).
+
+2.4. Transmission electron microscopy
+
+2.5. Tissue section preparation
+
+After the Morris Water Maze test, three pups per group were anesthe- tized with a lethal dose of Nembutal. The thoracic cavities were opened and perfused intracardially with 100 mL of normal saline. Then the hippo- campus, including CA1 and dentate gyrus area, of each rat was taken out
+
+After the Morris Water Maze test, two pups from each dam were anesthetized by intraperitoneal injection of a lethal dose of Nembutal. The aorta was cannulated and the animal was firstly perfused with
+
+Fig. 2. The expression of CHOP and caspase-12 increased significantly in the hippocampus of isoflurane-exposed pups showed by immuno-reaction. (Aa) CHOP immunohistochem- ical staining in control pups × 400. (Ab) CHOP immunohistochemical staining in isoflurane-exposed pups × 400. (Ac) Caspase-12 immunohistochemical staining in control pups × 400. (Ad) Caspase-12 immunohistochemical staining in isoflurane-exposed pups × 400. The number and optical density of the CHOP (B) and caspase-12 (C) positive neurons were compared between the control and 1.3% isoflurane treatment groups. **P b 0.01 compared to control. Scale bar = 50 μm.
+
+F. Kong et al. / European Journal of Pharmacology 670 (2011) 168–174
+
+200 mL of normal saline, then with 250 mL of 4% formaldehyde (freshly made from paraformaldehyde) for 20–30 min. The fixed brain was then removed from the cranial cavity and post-fixed over- night in the same fixative at 4 °C. The tissues were embedded in par- affin, and transverse paraffin sections containing the hippocampal area (5 mm thick) were mounted on silanecoated slides. Sections were deparaffinaged and rehydrated. Then the sections were treated for antigen retrieval with 10.2 mmol/L sodium citrate buffer, pH 6.1, for 20 min at 95 °C for immunohistochemistry.
+
+2.6. Immunohistochemistry analysis
+
+The sections mentioned above were washed in 0.01 M PBS con- taining 0.3% Triton X-100 (pH 7.4, PBS-T), followed by blocking in 5% normal goat serum in 0.01 M PBS. The sections were then incubat- ed in the primary antibodies rabbit polyclonal against anti-CHOP or caspase-12 (1:100, Santa Cruz Biotechnology, USA) overnight at 4 °C. After a thorough wash in PBS, sections were incubated with bio- tinylated goat anti-rabbit IgG antibody (1:200, Boster, China) for 2 h at room temperature, followed by avidin–biotin–peroxidase complex solution (ABC, 1:100, Boster) for 2 h at room temperature. Immunola- beling was visualized with 0.05% diaminobenzdine (DAB) plus 0.3% H2O2 in PBS and the reaction was stopped by rinsing the slides with 0.2 M Tris–HCl. Sections were mounted onto 0.02% poly-L-lysine- coated slides and allowed to dry at room temperature. Then the sec- tions were dehydrated through a graded series of alcohols, cleared in xylene and finally coverslipped. Rat Immunoglobulin IgG (1:200, Biomeda Corporation, USA) was used instead of primary antibody as a negative control. Three sections from each animal were selected at random and images were photographed under 400× magnification in 3 visual fields/per section, the CHOP and caspase-12 positive neu- rons were counted in the same area. The optical densities of CHOP and caspase-12 positive neurons were measured quantitatively using NIH image software (ImageJ, National Institutes of Health, Be- thesda, MD).
+
+and graphs were performed or generated, respectively, using Graph- Pad Prism Version 4.0 (GraphPad Prism Software, Inc. CA, USA).
+
+3. Results
+
+3.1. Physiologic parameters
+
+As shown in Table 1, ABG values and blood glucose levels were within the normal physiologic range. There were no significant differ- ences in ABG values and blood glucose levels before and after expo- sure in both the control group and the 1.3% isoflurane treatment group. All pups were viable and there were no significant differences in growth rate of the rat pups between the two groups (data not shown).
+
+3.2. Morris Water Maze test
+
+As shown in Fig. 1A, pups in both groups showed a rapid decrease in latency, while the pups of the isoflurane group spent more time to find the platform than those of control group in the place trial (P b 0.05). Swimming speeds were also analyzed during place trials, and no differences were observed between the two groups (data not shown). In the probe test, the number of crossing over the former platform location in isoflurane-treated pups was fewer than the cor- responding control animals (Fig. 1B, P b 0.05), but the time spent in the third quadrant where the platform located has no difference (data not shown).
+
+2.7. Western blot analysis
+
+After Morris Water Maze test, two pups from each pregnant moth- er were anesthetized with a lethal dose of Nembutal. Then their tho- racic cavities were opened and perfused intracardially with 100 mL of normal saline. Hippocampus, including CA1 and dentate gyrus field, of each rat was taken out immediately to obtain fresh tissue speci- mens. Protein concentration was determined by the BCA method using bovine serum albumin as the standard. Protein samples (50 μg) were separated by 12% sodium dodecyl sulfate polyacryl- amide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellu- lose membrane. The membranes were blocked by nonfat dry milk buffer for 2 h and then incubated overnight at 4 °C with primary an- tibody against CHOP or caspase-12 (1:500, Santa Cruz Biotechnology, USA). The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies and developed with ECL kit. The optical densities of bands were quantitatively analyzed using Bio-Rad Quantity One 4.6.2 (Bio-Rad Laboratories, USA). The results were expressed as a relative density. Equal protein loading in each lane was confirmed by hybridization with a 1:2000 dilution of β- actin antibody (Santa Cruz Biotechnology, USA).
+
+2.8. Statistical analysis
+
+All data were presented as mean ± S.E.M. Results of weight of postnatal rat pups and place trials of postnatal rats were analyzed using 2-way ANOVA for repeated measurements. Other data were an- alyzed using Student's t-test for comparison of two groups. A P value of b0.05 was considered statistically significant. All statistical tests
+
+Fig. 3. The levels of CHOP and caspase-12 protein remarkably increased in the hippo- campus of isoflurane-exposed pups. (A) Representative changes of CHOP and cas- pase-12 by western blot analysis. (B, C) The quantified CHOP (B) and caspase-12 (C) bands were normalized to the loading control β-actin. **P b 0.01, ***P b 0.001 com- pared to control.
+
+171
+
+172
+
+F. Kong et al. / European Journal of Pharmacology 670 (2011) 168–174
+
+3.3. Immunoreactivity assay
+
+In the isoflurane-exposed pups, the expression of CHOP in the hip- pocampal CA1 and dentate gyrus area increased compared with the control (Fig. 2A a and b). The caspase-12 expression displayed the same tendency of increase in the hippocampal area in the isoflur- ane-treated pups (Fig. 2Ac and d). Quantitative analysis of the num- ber and optical densities of CHOP and caspase-12 positive neurons for the whole hippocampal CA1 and dentate gyrus area showed that their immunoreactivity was increased compared with the control (Fig. 2B and C, P b 0.01).
+
+3.4. CHOP and caspase-12 protein levels
+
+Consistent with the findings of immunohistochemistry studies, western blot analysis showed that the levels of CHOP and caspase- 12 protein markedly increased in the hippocampal region of isoflur- ane-exposed pups when compared with the control (Fig. 3, P b 0.01).
+
+3.5. Ultrastructure changes in synapse of hippocampus
+
+Synapses with postsynaptic densities, an inerratic synaptic cleft and a presynaptic vas were clearly visible in the control pups (Fig. 4A and C). In contrast, in the isoflurane-treated pups, the num- ber of synapses decreased in the dentate gyrus and CA1 area, while a widened synaptic cleft, thinned postsynaptic densities and loss of a presynaptic vas were observed (Fig. 4).
+
+4. Discussion
+
+demonstrate that gestational exposure to a clinically relevant concen- tration of isoflurane causes postnatal spatial memory and learning impairments in the offspring rats. Moreover, neuron apoptosis and changes of synaptic structure were also observed at the hippocampal level in pups subject to isoflurane.
+
+Our present work confirmed that the levels of CHOP and caspase- 12 increased at hippocampal level in isoflurane-exposed rats, as indi- cated by the significant increase in the amount and densities of CHOP and caspase-12-positive cells, as well as the levels of CHOP and cas- pase-12 protein. Neuronal cell death after general anesthesia has re- cently been documented in several immature animal models. Some studies proposed that inhalational anesthetics, such as isoflurane, in- duced cell death processes through activation of γ-aminobutyric acid and inhibition of N-methyl-D-aspartate receptors (Ikonomidou et al., 1999; Olney et al., 2004). However, the mechanisms of the effect are not clear or fully understood. Recent advances indicate that ER re- sponses play a pivotal role in cellular apoptosis after exposure to var- ious stresses, such as hypoxia, calcium dysregulation and oxidative stress (Larner et al., 2005; Schroder and Kaufman, 2005). C/EBP ho- mologous protein (CHOP), also known as GADD153 (growth arrest- and DNA damage-inducible gene 153), is a member of the C/EBP fam- ily of bZIP transcription factors, and its low expression under normal conditions is induced to high levels by ER stress. The role of CHOP in ER stress-induced apoptosis has been illustrated in Chop−/− mice (Oyadomari et al., 2001; Zinszner et al., 1998). Caspase-12 has been proposed as a key mediator of ER stress-induced apoptosis (Szegezdi et al., 2003). CHOP activation occurs concomitantly with the activa- tion of caspase-12, and activated caspase-12 in turn produces activa- tion of the caspase cascade (Rao et al., 2002). Caspase-12 activation is mediated mainly by calpain, which is released from the ER membrane by tumor necrosis factor receptor-associated factor. Subsequently, caspase-12 interacts with caspase-9, which forms part of the ‘intrinsic’ apoptotic pathway, leading to activation of the executer caspase-3.
+
+In the present study, we employed a new model, a maternal fetal rat model, to study the behavioral and neurotoxic effects of exposure to anesthetics and investigate the potential mechanisms. Our results
+
+Fig. 4. Ultrastructural changes of synapse in the CA1 and dentate gyrus area of hippocampus under TEM. (A, C) control pups, (B, D) isoflurane-exposed pups.
+
+F. Kong et al. / European Journal of Pharmacology 670 (2011) 168–174
+
+Therefore, CHOP and caspase-12-mediated ER stress-induced cell death appear to be the major mediators of anesthesia-mediated apoptotic cellu- lar death.
+
+Learning and memory are important aspects of cognitive func- tion. Our results showed that prenatal exposure to isoflurane dis- played deficits and memory capabilities in pups as manifested by the longer escape latency and the fewer times of original platform crossing in the Morris Water Maze test. The lack of differences in swimming speeds be- tween the two groups suggested that the learning and memory def- icits observed in our study were not due to sensorimotor disturbances. Consistent with previous studies in maternal fetal rat models, these findings indicate that rats exposed to anesthetics in utero during fetal neurodevelopment is capable of causing be- havioral abnormalities in adolescent animals (Chalon et al., 1981; Palanisamy et al., 2011). However, the effects of anesthesia used during the development of fetal brains on postnatal memory and learning ability are controversial, with transient improvement (Li et al., 2007), no effects (McClaine et al., 2005) and permanent im- pairment (Chalon et al., 1981; Palanisamy et al., 2011) all being reported. These discrepancies could be due to methodological dif- ferences, species differences (rats vs. mice), pharmacological differ- ences in anesthetic concentrations (0.5–2 MAC), or differences in anesthetic durations (1–6 h). Last but not the least is the time of isoflurane exposure. Since different neurodevelopmental events are performed in their timing relative to gestational age, it is expected that the vulnerabil- ity of the brain to the adverse effects of the anesthetic agents would be different depending on the time of exposure. Correspond- ingly, behavioral outcome varies as a function of the neurodevelop- mental events occurring at the time of exposure. The time of isoflurane exposure in the current study corresponds approximately to midgestation in human, and studies in several animal species suggest that susceptibility is limited to a brain developmental state corresponding to the human second trimester of pregnancy.
+
+in postnatal
+
+spatial
+
+learning
+
+(isoflurane vs.
+
+sevoflurane), differences
+
+Acknowledgments
+
+We thank Shu Han, M.D., Ph.D. (Associate Professor, Institute of Anatomy and Cell Biology, School of Medicine, Zhejiang University, China) for the technical support and thought-provoking discus- sions. Our work was supported by the Medical and Health Re- search Fund of Health Department of Zhejiang Provincial, China (no. 2010KYA129).
+
+References
+
+Chalon, J., Tang, C.K., Ramanathan, S., Eisner, M., Katz, R., Turndorf, H., 1981. Exposure to halothane and enflurane affects learning function of murine progeny. Anesth. Analg. 60, 794–797.
+
+Clancy, B., Darlington, R.B., Finlay, B.L., 2001. Translating developmental time across
+
+mammalian species. Neuroscience 105, 7–17.
+
+Clancy, B., Kersh, B., Hyde, J., Darlington, R.B., Anand, K.J., Finlay, B.L., 2007. Web-based method for translating neurodevelopment from laboratory species to humans. Neuroinformatics 5, 79–94.
+
+Culley, D.J., Baxter, M.G., Yukhananov, R., Crosby, G., 2004. Long-term impairment of acquisition of a spatial memory task following isoflurane-nitrous oxide anesthesia in rats. Anesthesiology 100, 309–314.
+
+DiMaggio, C.J., Sun, L., Kakavouli, A., Li, G., 2008. Exposure to anesthesia and the risk of developmental and behavioral disorders in young children. Anesthesiology 109, A1415.
+
+Dwyer, R., Fee, J.P., Moore, J.M., 1995. Uptake of halothane and isoflurane by mother
+
+and baby during caesarean section. Br. J. Anaesth. 74, 379–383.
+
+Goodman, S., 2002. Anesthesia for nonobstetric surgery in the pregnant patient. Semin.
+
+Perinatol. 26, 136–145.
+
+Gruart, A., Munoz, M.D., Delgado-Garcia, J.M., 2006. Involvement of the CA3–CA1 syn- apse in the acquisition of associative learning in behaving mice. J. Neurosci. 26, 1077–1087.
+
+Ikonomidou, C., Bosch, F., Miksa, M., Bittigau, P., Vockler, J., Dikranian, K., Tenkova, T.I., Stefovska, V., Turski, L., Olney, J.W., 1999. Blockade of NMDA receptors and apopto- tic neurodegeneration in the developing brain. Science 283, 70–74.
+
+Jevtovic-Todorovic, V., Hartman, R.E., Izumi, Y., Benshoff, N.D., Dikranian, K., Zorumski, C.F., Olney, J.W., Wozniak, D.F., 2003. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J. Neurosci. 23, 876–882.
+
+In the present study, our results demonstrated that the number of synapses decreased significantly in the dentate gyrus and CA1 area with widened synaptic clefts, thinned postsynaptic densities and a loss of the presynaptic vas in the isoflurane-exposed rats. It is widely recognized that there is a relationship between hippocampal synaptic plasticity and memory (Gruart et al., 2006; Sametsky et al., 2010; Thompson et al., 2008). The synaptic cleft is a region of information transmission among neurons and plays an important role in the dy- namics of synaptic activity. The thickness of postsynaptic densities and the ability of learning and memory training and memory reten- tion go hand in hand. These changes impaired synapse normal struc- ture and resulted in interruption of synaptic connections. Our results suggest that the interruption of synaptic connections may be a mech- anism which further leads to changes of synaptic plasticity and conse- quent memory and learning impairments.
+
+Kalkman, C.J., Peelen, L., Moons, K.G., Veenhuizen, M., Bruens, M., Sinnema, G., de Jong, T.P., 2009. Behavior and development in children and age at the time of first anes- thetic exposure. Anesthesiology 110, 805–812.
+
+Larner, S.F., Hayes, R.L., Wang, K.K., 2005. Unfolded protein response after neuro-
+
+trauma. J. Neurotrauma 23, 807–829.
+
+Li, Y., Liang, G., Wang, S., Meng, Q., Wang, Q., Wei, H., 2007. Effects of fetal exposure to isoflurane on postnatal memory and learning in rats. Neuropharmacology 53, 942–950.
+
+Ma, D., Williamson, P., Januszewski, A., Nogaro, M.C., Hossain, M., Ong, L.P., Shu, Y., Franks, N.P., Maze, M., 2007. Xenon mitigates isoflurane-induced neuronal apoptosis in the developing rodent brain. Anesthesiology 106, 746–753.
+
+Matsukawa, J., Matsuzawa, A., Takeda, K., Ichijo, H., 2004. The ASK1-MAP kinase cas-
+
+cades in mammalian stress response. J. Biochem. 136, 261–265.
+
+Mazze, R.I., Rice, S.A., Baden, J.M., 1985. Halothane, isoflurane, and enflurane MAC in pregnant and nonpregnant female and male mice and rats. Anesthesiology 62, 339–341.
+
+McClaine, R.J., Uemura, K., de la Fuente, S.G., Manson, R.J., Booth, J.V., White, W.D., Campbell, K.A., McClaine, D.J., Benni, P.B., Eubanks, W.S., Reynolds, J.D., 2005. General anesthesia improves fetal cerebral oxygenation without evidence of subse- quent neuronal injury. J. Cereb. Blood Flow Metab. 25, 1060–1069.
+
+Nakagawa, T., Yuan, J., 2000. Cross-talk between two cysteine protease families: activa-
+
+tion of caspase-12 by calpain in apoptosis. J. Cell Biol. 150, 887–894.
+
+In summary, the current findings demonstrate that prenatal expo- sure to anesthetic drugs during a critical period of neural develop- ment causes neuron apoptosis, changes of synaptic structure, and postnatal spatial memory and learning impairments in the offspring rats. We speculate that the up-regulation of CHOP and caspase-12 may contribute to neural cell apoptosis, leading to damage in synapse number and function and consequent impairments in synaptic plas- ticity, all of which would contribute to the long-term neurocognitive decline. With the gradual rise in the occurrence of fetal and non-ob- stetric surgery during pregnancy under general anesthesia, it is im- perative that further animal studies into the mechanism as well as clinical studies defining human susceptibility are both urgently need- ed. A better understanding of the inhalational anesthetics mecha- nisms will help us to guide clinical trials aiming to define the scope of the problem in humans and may lead to preventive and therapeu- tic strategies.
+
+Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B.A., Yuan, J., 2000. Caspase- 12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amy- loidbeta. Nature 403, 98–103.
+
+Olney, J.W., Young, C., Wozniak, D.F., Jevtovic-Todorovic, V., Ikonomidou, C., 2004. Do pediatric drugs cause developing neurons to commit suicide? Trends Pharmacol. Sci. 25, 135–139.
+
+Oyadomari, S., Mori, M., 2004. Roles of CHOP/GADD153 in endoplasmic reticulum
+
+stress. Cell Death Differ. 11, 381–389.
+
+Oyadomari, S., Takeda, K., Takiguchi, M., Gotoh, T., Matsumoto, M., Wada, I., Akira, S., Araki, E., Mori, M., 2001. Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway. Proc. Natl. Acad. Sci. U. S. A. 98, 10845–10850.
+
+Palanisamy, A., Baxter, M.G., Keel, P.K., Xie, Z., Crosby, G., Culley, D.J., 2011. Rats ex- posed to isoflurane in utero during early gestation are behaviorally abnormal as adults. Anesthesiology 114, 521–528.
+
+Rao, R.V., Castro-Obregon, S., Frankowski, H., Schuler, M., Stoka, V., del Rio, G., Bredesen, D.E., Ellerby, H.M., 2002. Coupling endoplasmic reticulum stress to the cell death program. An Apaf-1-independent intrinsic pathway. J. Biol. Chem. 277, 21836–21842. Sametsky, E.A., Disterhoft, J.F., Geinisman, Y., Nicholson, D.A., 2010. Synaptic strength and postsynaptically silent synapses through advanced aging in rat hippocampal CA1 pyramidal neurons. Neurobiol. Aging 31, 813–825.
+
+173
+
+174
+
+F. Kong et al. / European Journal of Pharmacology 670 (2011) 168–174
+
+Schroder, M., Kaufman, R.J., 2005. The mammalian unfolded protein response. Annu.
+
+Rev. Biochem. 74, 739–789.
+
+Szegezdi, E., Fitzgerald, U., Samali, A., 2003. Caspase-12 and ER-stress-mediated
+
+apoptosis: the story so far. Ann. N. Y. Acad. Sci. 1010, 186–194.
+
+Wilder, R.T., Flick, R.P., Sprung, J., Katusic, S.K., Barbaresi, W.J., Mickelson, C., Gleich, S.J., Schroeder, D.R., Weaver, A.L., Warner, D.O., 2009. Early exposure to anesthesia and learning disabilities in a population-based birth cohort. Anesthesiology 110, 796–804.
+
+Thompson, J.V., Sullivan, R.M., Wilson, D.A., 2008. Developmental emergence of fear learning corresponds with changes in amygdala synaptic plasticity. Brain Res. 1200, 58–65.
+
+Zinszner, H., Kuroda, M., Wang, X., Batchvarova, N., Lightfoot, R.T., Remotti, H., Stevens, J.L., Ron, D., 1998. CHOP is implicated in programmed cell death in response to im- paired function of the endoplasmic reticulum. Genes Dev. 12, 982–995.
+
+Tran, K.M., 2010. Anesthesia for fetal surgery. Semin. Fetal Neonatal Med. 15, 40–45.

new_pdfs/10.1016_j.ijdevneu.2019.04.002.txt

@@ -0,0 +1,295 @@
+International Journal of Developmental Neuroscience 75 (2019) 19–26
+
+Contents lists available at ScienceDirect
+
+International Journal of Developmental Neuroscience
+
+journal homepage: www.elsevier.com/locate/ijdevneu
+
+Dexmedetomidine reduced sevoflurane-induced neurodegeneration and long-term memory deficits in neonatal rats
+
+T
+
+Toru Goyagi
+
+Department of Anesthesia and Intensive Care Medicine, Akita University Graduate School of Medicine, 1-1-1 Hondo, Akita, Akita 010-8543, Japan
+
+A R T I C L E I N F O
+
+A B S T R A C T
+
+Keywords: Sevoflurane Anesthesia Dexmedetomidine Cognitive function Neonate Neural toxicity
+
+Exposure to sevoflurane and other inhalational anesthetics can induce neurodegeneration in the developing brain. Although dexmedetomidine (DEX) has provided neuroprotection against hypoxic ischemic injury, rela- tively little is known about whether it has the neuroprotective effects against anesthetic-induced neurodegen- eration. This study examined whether DEX improves the long-term cognitive dysfunction observed after ex- posure of neonatal rats to 3% sevoflurane. Seven-day-old rats received intraperitoneal saline (DEX 0) or DEX (6.6, 12.5, 25 μg/kg) 30 min before exposure to 3% sevoflurane with 21% oxygen for 4 h (n = 10 per group). The pups in the control group received only DEX 25 μg/kg without anesthesia. The escape latency in the Morris water maze was significantly increased in the DEX 0 group compared with the sham and control group, and the escape latency, but not the swimming path length, was significantly shorter at post-natal day 47 in the DEX 25 than in the DEX 0 group. The percent time spent in the quadrant was significantly decreased in the DEX 0 group compared with the sham and control group, and the percent time spent in the quadrant was significantly in- creased in the DEX 25 group compared with the DEX 0 groups. The freezing times of the DEX 0 and 6.6 groups were significantly decreased compared with those in the sham, control and DEX 25 groups. The number of NeuN- positive cells in the CA1 region was significantly decreased in the DEX 0 and 6.6 groups compared with the sham, control and DEX 25 groups. These findings indicate pre-treatment with DEX may improve long-term cognitive function and ameliorate the neuronal degeneration induced by sevoflurane exposure in neonatal rats.
+
+1. Introduction
+
+Exposure of the immature brain to general anesthetics causes structural and functional alterations, subsequently resulting in the neurodegenerations (Disma et al., 2016; Stratmann, 2011; Walters and Paule, 2017). Sevoflurane, an inhalational anesthetic, is widely used in pediatric anesthesia. Many experimental studies have shown exposure of the developing brain to sevoflurane causes neurological impairment in later adulthood (Amrock et al., 2015; Fang et al., 2012; Zheng et al., 2013). Furthermore, clinical reports have shown long-term behavioral impairments and memory dysfunction in infants and children after anesthesia exposure (Davidson et al., 2016; Sun et al., 2016; Warner et al., 2018). There is concern that infants and children could be af- fected if they receive general anesthesia for a prolonged period and multiple times during the neonatal and infant term (Andropoulos and Greene, 2017). As general anesthesia is necessary for surgery, the in- vestigation of method to ameliorate anesthesia-induced neurotoxicity is urgently needed.
+
+adjuvant of anesthesia to provide analgesia and sedation in the pre- operative and postoperative periods (Kamibayashi, 2000). These effects are useful for the prevention of postoperative delirium (Duan et al., 2018). Moreover, DEX protects organs in various injury models in- cluding ischemia (Dahmani et al., 2005; Engelhard et al., 2002; Eser et al., 2008; Goyagi et al., 2009; Zhu et al., 2013), inflammation (Vincent Degos et al., 2013), and traumatic injury (Schoeler et al., 2012), owing to its anti-apoptotic effects (Engelhard et al., 2002), its ability to decrease caspase-3 elevation (Dahmani et al., 2005; Eser et al., 2008), its effects on the phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways (Zhu et al., 2013) via the α2A and imidazole 1 receptors (Ma et al., 2004). Pharmacological preventive strategies against neurodegeneration induced by general anesthesia have been introduced (Disma et al., 2016; Walters and Paule, 2017). DEX is likely to be a protective com- pound that could be used to prevent or ameliorate anesthesia-induced neurodegeneration (Walters and Paule, 2017). Based on previous stu- dies, DEX can ameliorated the neuroapoptosis and cognitive dysfunc- tion induced by exposure of the developing brain to ketamine (Duan et al., 2014), isoflurane (Li et al., 2014; Sanders et al., 2010, 2009),
+
+Dexmedetomidine (DEX) is an α2-adrenoceptor agonist that has sedative and analgesic effects, and has been widely used clinically as an
+
+E-mail address: tgoyagi@doc.med.akita-u.ac.jp.
+
+https://doi.org/10.1016/j.ijdevneu.2019.04.002 Received 31 January 2019; Received in revised form 28 March 2019; Accepted 3 April 2019
+
+Available online 05 April 2019 0736-5748/ © 2019 ISDN. Published by Elsevier Ltd. All rights reserved.
+
+T. Goyagi
+
+sevoflurane (Perez-Zoghbi et al., 2017) and propofol (Lv et al., 2017; Wang et al., 2016; Xiao et al., 2018). Moreover, DEX has neuropro- tective effects in the hypoxic-ischemic neonatal brain (Ren et al., 2016; Zhou et al., 2018) and in an acute hyperoxic neonatal rat model (Endesfelder et al., 2017; Sifringer et al., 2015). In contrast, DEX does not ameliorate the injury induced by sevoflurane, and induces neural apoptosis in neonatal rats (Lee et al., 2017). Although many reports have shown that DEX is protective against anesthesia-induced neuro- degeneration, few reports have shown high mortality rates under high- dose DEX with sevoflurane anesthesia in neonatal rats (Lee et al., 2017; Perez-Zoghbi et al., 2017). Thus, the efficiency of DEX against sevo- flurane-induced neurodegeneration is not fully understood.
+
+We hypothesized that DEX administration could ameliorate the neurodegeneration triggered by sevoflurane in the developing rat brain and improve cognitive function in the long-term. This study examined the effects of DEX on rat brain histological changes, of through the assessment of persistent normal cells, and cognitive function in later life following perinatal sevoflurane exposure.
+
+2. Material and methods
+
+All animal protocols were approved by the animal research com- mittee of Akita University, Japan (Approval number: a-1-2625). Seven- day-old (P7) Wistar rats (male and female) rat pups (body weight, 12–15 g) were used in this study. Animals were housed under standard conditions (12 h light/12 h dark cycle at 22 °C) in the Animal Research Laboratory at Akita University. All efforts to reduce the number of animals and their suffering were made. The animals were randomly divided into 6 groups (n = 10 per group) as follows: no anesthesia and no injection (sham), no anesthesia and intraperitoneal 25 μg/kg DEX (control), intraperitoneal saline (DEX 0), intraperitoneal 6.6 μg/kg DEX intraperitoneal 12.5 μg/kg DEX (DEX 12.5), and in- (DEX 6.6), traperitoneal 25 μg/kg DEX (DEX 25).
+
+After 30 min intraperitoneal injection on P7, the pups were put into a plastic chamber, exposed to 3% sevoflurane with 2 L/min of 21% oxygen for 4 h, and returned to their mother’s cage. The oxygen and sevoflurane concentration were measured using a gas analysis system (GE Healthcare BioSciences, Pittsburgh, PA). The chamber was main- tained at 30 ± 1 °C using an infrared heat lamp during the exposure.
+
+2.1. Cognitive tests
+
+2.1.1. Morris water maze
+
+Spatial memory retention was examined using the Morris water maze by blinded observer as described previously (Goyagi, 2018). At P27 – P29, acquisition trials were executed 4 times per day for 3 suc- cessive days. The latency and the swimming path length to reach the hidden platform were measured using a video image motion analyzer (DVTrack DVT-11; Muromachi Kikai Co. Ltd, Tokyo, Japan). If the rat could not reach the hidden platform within 90 s, it was placed on the platform for 30 s during an acquisition trial. At P47 – P49, retention trials were executed 4 times per day. If the rats failed to find the platform within 90 s, the latency was regarded as 90 s. In this study, a probe trial was not done during the acquisition trials.
+
+2.1.2. Fear conditioning test
+
+Fear conditioning was performed to evaluate contextual memory retention using the fear conditioning system (MK-450RSQ; Muromachi Kikai Co., Ltd, Tokyo, Japan) as described previously (Goyagi, 2018). The apparatus consisted of a clear rectangular Plexiglas box with a floor of for the delivery of electric currents. At P42, the rats were placed on the cleaned parallel metallic rods to be accustomed to new environment for 1 min, before they were presented with a 70-dB white noise for 30 s A mild foot shock (0.4-mA) was administered through the metallic rods during the last 1 s of the tone presentation. The tone-shock pairing was repeated once per minute for the next 2 min. The rats were left in
+
+20
+
+International Journal of Developmental Neuroscience 75 (2019) 19–26
+
+Fig. 1. Morris water maze results 3 weeks after sevoflurane exposure. (A) Escape latency to reach the platform, (B) swimming path length to the platform, and (C) swimming speed were measured. Days 1, 2, and 3 of the acquisition trials were P27, P28, and P29, respectively. Although the escape latency on day 3 significantly decreased in all groups compared with the latency on day 1, no significant differences were found among the groups on days 1–3, whereas the swimming path length in the sham group was significantly shorter than that in the other groups. The data are expressed as means ± SDs (n = 10). DEX, dexmedetomidine.
+
+the cage for an additional 60 s before returned to their cage. At P49, cued fear memory was tested by placing rats into an unrelated en- vironment for 90 s without any tone and presenting the auditory cue for a further 60 s used for conditioning. Freezing time was measured by the percent of time during the tone presentation using a video image mo- tion analyzer (DVTrack DVT-11; Muromachi Kikai Co. Ltd, Tokyo, Japan).
+
+2.2. Histological analyses
+
+2.2.1. Neuronal nuclei staining
+
+After finished the water maze task and fear conditioning test at P49, the rats’ brains were removed and embedded in paraffin following the perfusion of heparinized saline then 150 mL of 4% paraformaldehyde in phosphate buffer (pH 7.4) to use further neuronal nuclei (NeuN) stain, as described previously (Goyagi, 2018). In brief, 3-μm-thick serial transverse sections were incubated with a mouse monoclonal antibody to NeuN antigen (NeuN; 1:100 diluted in blocking solution; Millipore Corporation, Temecula, CA) for 10 min at 37 °C. Immunodetection was performed using avidin-horse radish peroxidase complexes with
+
+T. Goyagi
+
+Fig. 3. Fear conditioning test results. The freezing time in response to the conditioned stimulus tone on P49 was significantly longer in the DEX 25 groups compared with the DEX 0 and 6.6 groups. The freezing time was significantly decreased in the DEX 0 group compared with the sham and the control group. * P < 0.05 vs sham, † P < 0.05 vs control, and ‡ P < 0.05 vs DEX 25. The data are expressed as means ± SD (n = 10). DEX, dexmedetomidine.
+
+biotinylated antibodies to rabbit and mouse IgG (MILLIPORE IHC Se- lectR Immunoperoxidase Secondary Detection System; Millipore Cor- poration), with diaminobenzidine. Then we counterstained those with hematoxylin. The NeuN-positive cells express as mature typical neurons after growth. We counted the number of NeuN-positive cells in bilateral 500 μm × 300 μm areas in the CA1 hippocampus, amygdala, and cer- ebral cortical layer 3, as described previously (Goyagi, 2018).
+
+2.2.2. Positive cell density map (PCDM)
+
+The PCDM was made as described previously (Goyagi, 2018; Wada et al., 2006). In brief, the composite image was FFT- bandpass-filtered using the Image J program (National Institute of Health, Bethesda, MD) to eliminate low-frequency drifts (> 20 pixels [50 μm]) and high-fre- quency noises (< 1 pixel [2.5 μm]). The PDCM was made with a
+
+21
+
+International Journal of Developmental Neuroscience 75 (2019) 19–26
+
+Fig. 2. Morris water maze results 6 weeks after sevoflurane exposure. (A) Escape latency to reach the platform, (B) swimming path length, (C) swimming speed, and (D) percent time to reach the quadrant were measured on day 1 (P47). The escape latency was significantly longer in the DEX 0 group than in the sham and the control group, and significantly shorter in the DEX 25 group than in the DEX 0 group. No significant differences were observed in swim- ming path length among the groups. The swimming speed in the DEX 0 and 12.5 groups was significantly decreased compared with that in the sham group. The percent time spent in the quadrant was significantly decreased in the DEX 0 group compared with the sham and the control group. However, it was significantly increased in the DEX 25 group compared with the DEX 0 group. * P < 0.05 vs sham, † P < 0.05 vs control, and ‡ P < 0.05 vs DEX 25. The data are expressed as means ± SD (n = 10). DEX, dexmedetomidine.
+
+custom-made program using MATLAB (MathWorks INC., Natick, MA) (Wada et al., 2006), then adjusted for each section automatically and enumeration of NeuN-positive cells in each 100 μm × 100 μm square section. Finally, the normalized PCDMs were seen as averaged for each group (Fig. 6 A). As mentioned our previous study (Goyagi, 2018), the PCDMs were analyzed whether the DEX-treated groups showed in- creased NeuN cell density compared with the DEX 0 group. The areas were mapped as colored to indicate significantly increased normal neurons in blocks where the P value was less than 0.05 (Fig. 6B).
+
+2.3. Statistical analysis
+
+The escape latency, the swimming speed, the swimming path length, the freezing time, and the number of NeuN-positive cells are expressed as means ± standard deviation (SD). Comparisons of these variables among the groups were performed using a one-way or two- way analysis of variance (ANOVA) for multiple comparisons followed by Bonferroni post hoc tests. Each PCDM using a Gaussian filter of the block size (SD = 100 μm) was analyzed using t-tests for each block. Differences with p-values less than 0.05 were considered statistically significant. We performed all analyses using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA).
+
+3. Results
+
+3.1. DEX improves memory retention in the Morris water maze
+
+The escape latencies and swimming path lengths to the hidden platform in the acquisition trials during postnatal day 27–29 (P27–29) are shown in Fig. 1. No statistically significant differences among the groups were found for the DEX treated, sham and control groups, in- dicating that all rats acquired the task equally well. As shown in Fig. 2, the escape latency was significantly increased in the DEX 0 group compared with the sham, the control and the DEX 25 group, and the escape latency, but not the swimming path length, was significantly shorter in the DEX 25 group than in the DEX 0 group at P47. In addi- the swimming speed in the DEX 0 and 12.5 groups was tion,
+
+T. Goyagi
+
+International Journal of Developmental Neuroscience 75 (2019) 19–26
+
+Fig. 4. NeuN staining of brain sections from sevoflurane-exposed rats. Representative NeuN staining at -3 ± 0.2 mm from bregma is shown. NeuN-positive cells in the entire image at lower magnification (A), CA1 hippocampus at higher magnification (B), amygdala at higher magnification (C), and cortex at higher magnification (D) are shown for each group. Squares indicate the measuring field in the CA1, amygdala, and cortex. Scale bar: 3 mm (A) or 100 μm (B, C, and D). DEX, dexmedetomidine.
+
+22
+
+T. Goyagi
+
+Fig. 5. Numbers of NeuN-positive cells in the hippocampus, amygdala, and cortex of sevoflurane-exposed rats. The number of NeuN-positive cells in the CA1 region of the hippocampus was decreased in the DEX 0 group compared with the sham, control, DEX 12.5 and 25 groups. The number of NeuN-positive cells was increased in the DEX 12.5 and 25 groups compared with the DEX 0 group (A). The number of NeuN- positive cells in the amygdala was decreased in the DEX 0 group compared with the sham and the control group, and increased in the DEX 12.5 and 25 groups compared with the DEX 0 group (B). The number of NeuN-positive cells in the cortex was decreased in the DEX 0 group compared with the sham and the control group (C). * P < 0.05 vs Sham, † P < 0.05 vs control, ‡ P < 0.05 vs DEX 25, § P < 0.05 vs DEX 12.5. The data are expressed as means ± SD (n = 10). DEX, dexmedetomidine.
+
+significantly decreased compared with that in the sham group. The percent time spent in the quadrant was significantly decreased in the DEX 0 group compared with the sham and the control group, and the percent time spent in the quadrant was significantly increased in the DEX 25 group compared with the DEX 0 group. This indicates that the DEX 25 improved the escape latency, the swimming speed, and he percent time spent in the quadrant.
+
+3.2. Fear conditioning test
+
+The freezing times in response to the conditioned stimulus tone were significantly decreased in the DEX 0 and 6.6 groups compared with the sham, the control and DEX 25 groups. The data indicate that the DEX 25 group had a freezing time that was similar to that in the sham group. (Fig. 3).
+
+23
+
+International Journal of Developmental Neuroscience 75 (2019) 19–26
+
+3.3. DEX attenuates the reduction in neuronal numbers in anesthesia- exposed brains
+
+Fig. 4 shows NeuN staining of brain sections from the 5 groups at -3 mm caudally to bregma. The number of NeuN-positive cells per 0.15 mm2 in the hippocampal CA1 region was significantly decreased in the DEX 0 and 6.6 groups compared with the sham, the control and the DEX 25 groups. In addition, the DEX 12.5 and 25 groups had an increased number of positive cells compared with those in the DEX 0 group (Fig. 5A). The number of NeuN-positive cells per 0.15 mm2 in the amygdala was significantly decreased in the DEX 0 group compared with the sham and the control group, and the number of cells in the DEX 12.5 and 25 groups were significantly increased compared with those in the DEX 0 group, suggesting that DEX 12.5 and 25 increased the number of intact cells in the amygdala (Fig. 5B). The number of NeuN-positive cells per 0.15 mm2 in the cortex was significantly de- creased in the DEX 0 group compared with the sham and the control group (Fig. 5C).
+
+3.4. PCDM and statistical parameter mapping of positive cell density
+
+Fig. 6A shows the NeuN PCDM for each group. The positive cell density tended to increase in the cortex. DEX-treated rats showed an increase in the hippocampal and cortical PCDM compared with the DEX 0 group. The differences among the groups are shown in detail in Fig. 6B. DEX-treated rats had significantly increased NeuN-positive cell densities when compared with those in the DEX 0 group. Increased NeuN expression was observed in almost all cortical and hippocampal areas, indicating that the numbers of normal cells in the DEX 12.5 and 25 groups were profoundly higher than those in the DEX 0 group.
+
+4. Discussion
+
+Our study shows that the pre-treatment of neonatal rats with DEX before exposure to sevoflurane anesthesia improved cognitive function, and increased the number of intact neurons in the cortex, hippocampus, and amygdala in the adulthood, indicating that DEX attenuates the neural degeneration induced by exposure of the developing rat brain to sevoflurane.
+
+Although the doses of DEX used in this study were 6.6, 12.5, and 25 μg/kg, we did not find a dose-response relationship. The rats in the DEX 25 group obtained the maximum effects compared with those in the other groups, as seen in the water maze, fear conditioning test, and histological results. These results were consistent with previous similar studies (Li et al., 2014; Sanders et al., 2010, 2009), though inconsistent with others (Lee et al., 2017; Perez-Zoghbi et al., 2017). In previous studies, DEX ameliorated the neuroapoptosis and cognitive dysfunction induced by exposure of the developing brain to isoflurane (Li et al., 2014; Sanders et al., 2010, 2009), sevoflurane (Perez-Zoghbi et al., 2017), and propofol (Lv et al., 2017; Wang et al., 2016; Xiao et al., 2018). Sanders et. al. showed that DEX 25, 50, and 75 μg/kg prevents cortical apoptosis in vitro and in vivo, whereas higher doses of DEX do not further increase the protection against isoflurane-induced injury in the cortex (Sanders et al., 2010). In contrast, the study from Perez- Zoghbi et al. showed that co-administration of DEX (1 μg/kg) during 2.5% sevoflurane exposure for 6 h provided significant neuroprotection, as measured by a reduction in the number of caspase-3 positive cells in several brain regions, whereas DEX at doses of 10, and 25 μg/kg in combination with sevoflurane increased mortality (Perez-Zoghbi et al., 2017).
+
+In contrast to previous studies, the aim of this study was to measure cognitive function, using the fear conditioning test, and the number of intact neurons in the brain during adulthood, as in our previous study (Goyagi, 2018). We only measured the freezing time to tone but did not use contextual stimuli. The amygdala contributes to the acquisition of conditioned fear responses to a cue, whereas the amygdala and the
+
+T. Goyagi
+
+International Journal of Developmental Neuroscience 75 (2019) 19–26
+
+Fig. 6. Positive cell density map (PCDM) and statistical parameter mapping of positive cell densities in the brains of sevoflurane-exposed rats. (A) The NeuN PCDM for each group is shown. (B) Statistical parametric mapping showing the areas (red) where the DEX-treated groups yielded significantly higher NeuN-positive cell densities than the DEX 0 group, indicating the numbers of normal cells in the DEX-treated group were higher than in the DEX 0 group. DEX, dexmedetomidine. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
+
+hippocampus contribute to the acquisition of contextual conditioned fear responses (Phillips and LeDoux, 1992; Selden et al., 1991; Stanton, 2000). In this study, we measured the number of NueN-positive cells in both the amygdala and CA1 hippocampus. The cells in both regions increased in the DEX 12.5 and 25 groups, which was consistent with the behavioral results. The behavioral and histological results from this study suggested that administration of 12.5 and 25 μg/kg DEX
+
+improved learning and memory following sevoflurane-induced neuro- logical impairment in the developing brain.
+
+Neural toxicity induced by exposure to general anesthesia in neo- natal rodents has been shown to lead to neurological impairments in adulthood (Disma et al., 2016; Stratmann, 2011; Walters and Paule, 2017). Numerous animal studies have reported that anesthetic neuro- toxicity occurs via several pathways (Acharya et al., 2015; Jevtovic-
+
+24
+
+T. Goyagi
+
+Todorovic, 2018; Shen et al., 2013; Sinner et al., 2014). We have pre- viously reported that the use of oxygen as a carrier gas at concentra- tions higher than 21% ameliorates sevoflurane-induced neurodegen- eration. (Goyagi, 2018). Therefore, we used air as a carrier for sevoflurane anesthesia to minimize the neuroprotective effects of oxygen. According to the results of a previous study, co-administration of high dose DEX (5, 25 and 50 μg/kg) and 2.5% sevoflurane in rat pups causes high mortality rates (Perez-Zoghbi et al., 2017), and DEX pro- duces significant cellular degeneration and apoptosis in primary sen- sory brain regions (Pancaro et al., 2016), though we did not find the degeneration induced by DEX administration alone (control group) in this study. Although we also used 3% sevoflurane in this study, 25 μg/ kg DEX did not cause a high rate of mortality. This difference may be because of the different methodology used and the environment. Fur- ther studies are needed to clarify the neurotoxic effects of DEX in neonatal brain, with or without sevoflurane anesthesia.
+
+This study has several limitations. First, the rat pups were main- tained under spontaneous ventilation during sevoflurane anesthesia. Since sevoflurane depresses ventilation in a dose-dependent manner, hypercapnia may be seen during the anesthesia exposure. Although we did not measure the blood gas analysis in this study, there was no significant difference in the rate of hypercapnia among the groups in our previous study (Goyagi, 2018). Therefore, hypercapnia was not examined here. Second, the rat pups received sevoflurane with 21% oxygen in this study. According to our previous study, rat pups that receive sevoflurane with 21% oxygen exhibit a profound increase in neurodegeneration compared with those that receive 30% oxygen (Goyagi, 2018). DEX has neuroprotective effects against hypoxic brain and spinal insult (Dahmani et al., 2005; Engelhard et al., 2002; Eser et al., 2008; Goyagi et al., 2009; Goyagi and Tobe, 2014; Ma et al., 2004; Zhu et al., 2013), and in animal neonatal models, particularly in the hypoxic-ischemic neonatal brain (Ren et al., 2016; Zhou et al., 2018). It is possible that DEX might be protective against the hypoxic effects of 21% oxygen rather than the neurotoxicity induced by sevo- flurane here. Although a carrier gas with 21% oxygen might increase the neurodegeneration induced by sevoflurane anesthesia in the neo- natal brain compared with higher oxygen concentrations, DEX ame- liorated the neurodegeneration induced by sevoflurane exposure with 21% oxygen in this study, as previously described (Perez-Zoghbi et al., 2017). Third, we used the Morris water maze and fear conditioning test to measure neurocognitive memory function in later adulthood in this study. As there are many tests for cognitive function in rats, further studies are warranted to measure the effects on other type of cognitive function. Fourth, the mechanisms underlying the protective effects of DEX were not clear from this study. Based on a previous study, DEX may attenuate the reduction in the expression of anti-apoptotic sig- naling pathways mediated by Bcl-2 and phosphor-ERK1/2 induced by isoflurane anesthesia, in a similar manner to that of neonatal brain injury (Li et al., 2014). Moreover, DEX has anti-apoptotic effects (Engelhard et al., 2002), decreases caspase-3 elevation (Dahmani et al., 2005; Eser et al., 2008) and attenuates the activation of the PI3k/Akt/ glycogen synthase kinase (GSK)3beta pathway in propofol-induced neuroapoptosis (Lv et al., 2017; Wang et al., 2016; Xiao et al., 2018). Further research is warranted in order to ascertain the precise neuro- protective mechanisms of DEX against neural toxicity induced by an- esthesia exposure in the neonatal brain.
+
+5. Conclusion
+
+Four-hour administration of sevoflurane anesthesia in neonatal rats caused significant cognitive impairment in adulthood. Administration of a single dose of 25 μg/kg DEX before sevoflurane exposure improved cognitive functions, and maximum effects were observed at this dose compared with the other doses. Also, DEX attenuates the reduction in neuronal numbers in sevoflurane-exposed brains. DEX is likely to be useful as a neuroprotective agent in pediatric anesthesia.
+
+25
+
+International Journal of Developmental Neuroscience 75 (2019) 19–26
+
+Ethics approval
+
+The animal experiments were performed according to international ethical standards and approved by the research ethics committee of Akita University (a-1-2625).
+
+Funding
+
+This study was supported in part by a Grant-in-Aid for Scientific
+
+Research (C), JSPS KAKENHI Grant Number JP 24592291.
+
+Conflict of interest
+
+The authors declare no conflicts of interest.
+
+Acknowledgements
+
+I would like to thank Mr. Yoshitsugu Tobe and Dr. Yoko Masaki, Ph.D. from the Department of Anesthesia and Intensive Care Medicine, Akita Graduate School of Medicine, for technical assistance. I am also grateful to Dr. Makoto Wada, M.D., Ph.D. from the Department of Rehabilitation for Brain Functions, National Rehabilitation Center for Persons with Disabilities, for technical advice and the use of his soft- ware.
+
+References
+
+Acharya, N.K., Goldwaser, E.L., Forsberg, M.M., Godsey, G.A., Johnson, C.A., Sarkar, A., DeMarshall, C., Kosciuk, M.C., Dash, J.M., Hale, C.P., Leonard, D.M., Appelt, D.M., Nagele, R.G., 2015. Sevoflurane and Isoflurane induce structural changes in brain vascular endothelial cells and increase blood-brain barrier permeability: possible link to postoperative delirium and cognitive decline. Brain Res. 1620, 29–41.
+
+Amrock, L.G., Starner, M.L., Murphy, K.L., Baxter, M.G., 2015. Long-term effects of single or multiple neonatal sevo urane exposures on rat hippocampal ultrastructure. Anesthesiology 122, 87–95.
+
+Andropoulos, D.B., Greene, M.F., 2017. Anesthesia and developing brains — implications
+
+of the FDA warning. N. Engl. J. Med. 376, 905–907.
+
+Dahmani, S., Rouelle, D., Gressens, P., Mantz, J., 2005. Effects of dexmedetomidine on hippocampal focal adhesion kinase tyrosine phosphorylation in physiologic and is- chemic conditions. Anesthesiology 103, 969–977.
+
+Davidson, A.J., Disma, N., de Graaff, J.C., Withington, D.E., Dorris, L., Bell, G., Stargatt, R., Bellinger, D.C., Schuster, T., Arnup, S.J., Hardy, P., Hunt, R.W., Takagi, M.J., Giribaldi, G., Hartmann, P.L., Salvo, I., Morton, N.S., von Ungern Sternberg, B.S., Locatelli, B.G., Wilton, N., Lynn, A., Thomas, J.J., Polaner, D., Bagshaw, O., Szmuk, P., Absalom, A.R., Frawley, G., Berde, C., Ormond, G.D., Marmor, J., McCann, M.E., 2016. Neurodevelopmental outcome at 2 years of age after general anaesthesia and awake-regional anaesthesia in infancy (GAS): an international multicentre, rando- mised controlled trial. Lancet 387, 239–250.
+
+Degos, Vincent, Le charpentier, Tifenn, Chhor, Vibol, Brissaud, Olivier, Lebon, Sophie, Schwendimann, Leslie, Bednareck, Nathalie, Passemard, Sandrine, Mantz, Jean, Gressens, Pierre, 2013. Neuroprotective effects of dexmedetomidine against gluta- mate agonist-induced neuronal cell death are related to increased astrocyte brain- derived neurotrophic factor expression. Anesthesiology 118, 1123–1132.
+
+Disma, N., Mondardini, M.C., Terrando, N., Absalom, A.R., Bilotta, F., 2016. A systematic
+
+review of methodology applied during preclinical anesthetic neurotoxicity studies: important issues and lessons relevant to the design of future clinical research. Paediatr. Anaesth. 26, 6–36.
+
+Duan, X., Li, Y., Zhou, C., Huang, L., Dong, Z., 2014. Dexmedetomidine provides neu- roprotection: impact on ketamine-induced neuroapoptosis in the developing rat brain. Acta Anaesthesiol. Scand. 58, 1121–1126.
+
+Duan, X., Coburn, M., Rossaint, R., Sanders, R.D., Waesberghe, J.V., Kowark, A., 2018. Efficacy of perioperative dexmedetomidine on postoperative delirium: systematic review and meta-analysis with trial sequential analysis of randomised controlled trials. Br. J. Anaesth. 121, 384–397.
+
+Endesfelder, S., Makki, H., von Haefen, C., Spies, C.D., Buhrer, C., Sifringer, M., 2017.
+
+Neuroprotective effects of dexmedetomidine against hyperoxia-induced injury in the developing rat brain. PLoS One 12, e0171498.
+
+Engelhard, K., Werner, C., Kaspar, S., Möllenberg, O., Blobner, M., Bachl, M., Kochs, E., 2002. Effect of the alpha2-agonist dexmedetomidine on cerebral neurotransmitter concentrations during cerebral ischemia in rats. Anesthesiology 96, 450–457. Eser, O., Fidan, H., Sahin, O., Cosar, M., Yaman, M., Mollaoglu, H., Songur, A., Buyukbas, S., 2008. The influence of dexmedetomidine on ischemic rat hippocampus. Brain Res. 1218, 250–256.
+
+Fang, F., Xue, Z., Cang, J., 2012. Sevoflurane exposure in 7-day-old rats affects neuro- genesis, neurodegeneration and neurocognitive function. Neurosci. Bull. 28, 499–508.
+
+Goyagi, T., 2018. The additional oxygen as a carrier gas during long-duration sevoflurane
+
+T. Goyagi
+
+exposure ameliorate the neuronal apoptosis and improve the long-term cognitive function in neonatal rats. Brain Res. 1678, 220–230.
+
+Goyagi, T., Tobe, Y., 2014. Dexmedetomidine improves the histological and neurological outcomes 48 h after transient spinal ischemia in rats. Brain Res. 1566, 24–30. Goyagi, T., Nishikawa, T., Tobe, Y., Masaki, Y., 2009. The combined neuroprotective
+
+effects of lidocaine and dexmedetomidine after transient forebrain ischemia in rats. Acta Anaesthesiol. Scand. 53, 1176–1183.
+
+Jevtovic-Todorovic, V., 2018. Exposure of developing brain to general anesthesia: What is
+
+the animal evidence? Anesthesiology 128, 832–839.
+
+Kamibayashi, T.M.M., 2000. Clinical uses of alpha 2-adrenergic agonists. Anesthesiology
+
+93, 1345–1349.
+
+Lee, J.R., Lin, E.P., Hofacer, R.D., Upton, B., Lee, S.Y., Ewing, L., Joseph, B., Loepke, A.W., 2017. Alternative technique or mitigating strategy for sevoflurane-induced neuro- degeneration: a randomized controlled dose-escalation study of dexmedetomidine in neonatal rats. Br. J. Anaesth. 119, 492–505.
+
+Li, Y., Zeng, M., Chen, W., Liu, C., Wang, F., Han, X., Zuo, Z., Peng, S., 2014.
+
+Dexmedetomidine reduces isoflurane-induced neuroapoptosis partly by preserving PI3K/Akt pathway in the hippocampus of neonatal rats. PLoS One 9, e93639. Lv, J., Wei, Y., Chen, Y., Zhang, X., Gong, Z., Jiang, Y., Gong, Q., Zhou, L., Wang, H., Xie, Y., 2017. Dexmedetomidine attenuates propofol-induce neuroapoptosis partly via the activation of the PI3k/Akt/GSK3beta pathway in the hippocampus of neonatal rats. Environ. Toxicol. Pharmacol. 52, 121–128.
+
+Ma, D., Hossain, M., Rajakumaraswamy, N., Arshad, M., Sanders, R.D., Franks, N.P.,
+
+Maze, M., 2004. Dexmedetomidine produces its neuroprotective effect via the alpha 2A-adrenoceptor subtype. Eur. J. Pharmacol. 502, 87–97.
+
+Pancaro, C., Segal, B.S., Sikes, R.W., Almeer, Z., Schumann, R., Azocar, R.J., Marchand, J.E., 2016. Dexmedetomidine and ketamine show distinct patterns of cell degenera- tion and apoptosis in the developing rat neonatal brain. J. Matern. Fetal. Neonatal. Med. 29, 3827–3833.
+
+Perez-Zoghbi, J.F., Zhu, W., Grafe, M.R., Brambrink, A.M., 2017. Dexmedetomidine- mediated neuroprotection against sevoflurane-induced neurotoxicity extends to several brain regions in neonatal rats. Br. J. Anaesth. 119, 506–516.
+
+Phillips, R.G., LeDoux, J.E., 1992. Differential contribution of amygdala and hippo-
+
+campus to cued and contextual fear conditioning. Behav. Neurosci. 106, 274–285. Ren, X., Ma, H., Zuo, Z., 2016. Dexmedetomidine postconditioning reduces brain injury after brain hypoxia-ischemia in neonatal rats. J. Neuroimmune Pharmacol. 11, 238–247.
+
+Sanders, R.D., Xu, J., Shu, Y., Januszewski, A., Halder, S., Fidalgo, A., Sun, P., Hossain, M., Ma, D., Maze, M., 2009. Dexmedetomidine attenuates isoflurane-induced neu- rocognitive impairment in neonatal rats. Anesthesiology 110, 1077–1085.
+
+Sanders, R.D., Sun, P., Patel, S., Li, M., Maze, M., Ma, D., 2010. Dexmedetomidine pro- vides cortical neuroprotection: impact on anaesthetic-induced neuroapoptosis in the rat developing brain. Acta Anaesthesiol. Scand. 54, 710–716.
+
+Schoeler, M., Loetscher, P.D., Rossaint, R., Fahlenkamp, A.V., Eberhardt, G., Rex, S., Weis, J., Coburn, M., 2012. Dexmedetomidine is neuroprotective in an in vitro model for traumatic brain injury. BMC Neurol. 12, 20.
+
+Selden, N.R., Everitt, B.J., Jarrard, L.E.R., T W, 1991. Complementary roles for the
+
+26
+
+International Journal of Developmental Neuroscience 75 (2019) 19–26
+
+amygdala and hippocampus in aversive conditioning to explicit and contextual cues. Neuroscience 42, 335–350.
+
+Shen, X., Dong, Y., Xu, Z., Wang, H., Miao, C., Soriano, S.G., Sun, D., Baxter, M.G., Zhang, Y., Xie, Z., 2013. Selective anesthesia-induced neuroinflammation in developing mouse brain and cognitive impairment. Anesthesiology 118, 502–515.
+
+Sifringer, M., von Haefen, C., Krain, M., Paeschke, N., Bendix, I., Buhrer, C., Spies, C.D., Endesfelder, S., 2015. Neuroprotective effect of dexmedetomidine on hyperoxia-in- duced toxicity in the neonatal rat brain. Oxid. Med. Cell. Longev. 2015, 1–10. Sinner, B., Becke, K., Engelhard, K., 2014. General anaesthetics and the developing brain:
+
+an overview. Anaesthesia 69, 1009–1022.
+
+Stanton, M.E., 2000. Multiple memory systems, development and conditioning. Behav.
+
+Brain Res. 110, 25–37.
+
+Stratmann, G., 2011. Neurotoxicity of anesthetic drugs in the developing brain. Anesth.
+
+Analg. 113, 1170–1179.
+
+Sun, L.S., Li, G., Miller, T.L., Salorio, C., Byrne, M.W., Bellinger, D.C., Ing, C., Park, R., Radcliffe, J., Hays, S.R., DiMaggio, C.J., Cooper, T.J., Rauh, V., Maxwell, L.G., Youn, A., McGowan, F.X., 2016. Association between a single general anesthesia exposure before age 36 months and neurocognitive outcomes in later childhood. JAMA 315, 2312–2320.
+
+Wada, M., Yoshimi, K., Higo, N., Ren, Y.R., Mochizuki, H., Mizuno, Y., Kitazawa, S., 2006. Statistical parametric mapping of immunopositive cell density. Neurosci. Res. 56, 96–102.
+
+Walters, J.L., Paule, M.G., 2017. Review of preclinical studies on pediatric general an- esthesia-induced developmental neurotoxicity. Neurotoxicol. Teratol. 60, 2–23. Wang, Y., Wu, C., Han, B., Xu, F., Mao, M., Guo, X., Wang, J., 2016. Dexmedetomidine
+
+attenuates repeated propofol exposure-induced hippocampal apoptosis, PI3K/Akt/ Gsk-3beta signaling disruption, and juvenile cognitive deficits in neonatal rats. Mol. Med. Rep. 14, 769–775.
+
+Warner, D.O., Zaccariello, M.J., Katusic, S.K., Schroeder, D.R., Hanson, A.C., Schulte, P.J., Buenvenida, S.L., Gleich, S.J., Wilder, R.T., Sprung, J., Hu, D., Voigt, R.G., Paule, M.G., Chelonis, J.J., Flick, R.P., 2018. Neuropsychological and behavioral outcomes after exposure of young children to procedures requiring general anesthesia: the mayo anesthesia safety in kids (MASK) study. Anesthesiology 129, 89–105. Xiao, Y., Zhou, L., Tu, Y., Li, Y., Liang, Y., Zhang, X., Lv, J., Zhong, Y., Xie, Y., 2018. Dexmedetomidine attenuates the propofol-induced long-term neurotoxicity in the developing brain of rats by enhancing the PI3K/Akt signaling pathway. Neuropsychiatr. Dis. Treat. 14, 2191–2206.
+
+Zheng, S.Q., An, L.X., Cheng, X., Wang, Y.J., 2013. Sevoflurane causes neuronal apoptosis and adaptability changes of neonatal rats. Acta Anaesthesiol. Scand. 57, 1167–1174. Zhou, X.M., Liu, J., Wang, Y., Zhang, M.H., 2018. Silencing of long noncoding RNA MEG3 enhances cerebral protection of dexmedetomidine against hypoxic-ischemic brain damage in neonatal mice by binding to miR-129-5p. J. Cell. Biochem. 28, 1–11. Zhu, Y.M., Wang, C.C., Chen, L., Qian, L.B., Ma, L.L., Yu, J., Zhu, M.H., Wen, C.Y., Yu,
+
+L.N., Yan, M., 2013. Both PI3K/Akt and ERK1/2 pathways participate in the pro- tection by dexmedetomidine against transient focal cerebral ischemia/reperfusion injury in rats. Brain Res. 1494, 1–8.

new_pdfs/10.1016_j.neulet.2013.04.008.txt

@@ -0,0 +1,277 @@
+Neuroscience Letters 545 (2013) 17– 22
+
+Contents lists available at SciVerse ScienceDirect
+
+Neuroscience Letters
+
+j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / n e u l e t
+
+JNK pathway may be involved in isoflurane-induced apoptosis in the hippocampi of neonatal rats
+
+Yujuan Li a,∗,1, Fei Wang a,1, Chuiliang Liu b, Minting Zeng a, Xue Han a, Tao Luo c, Wei Jiang c, Jie Xu c, Huaqiao Wang c,∗∗
+
+a Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China b Department of Anesthesiology, ChanCheng Central Hospital, Foshan 528031, China c Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China
+
+h i g h l i g h t s
+
+We investigated the effects of JNK pathway on isoflurane-induced neuroapoptosis. • SP600125 reduced isoflurane-induced apoptosis in the hippocampi of neonatal rats. • Isoflurane-induced activation of JNK and c-Jun was inhibited by SP600125. • SP600125 reversed isoflurane-induced decrease of Bcl-xL. • SP600125 maintained Akt activation.
+
+a r t i c l e
+
+i n f o
+
+a b s t r a c t
+
+Article history: Received 14 February 2013 Received in revised form 30 March 2013 Accepted 1 April 2013
+
+Keywords: Apoptosis Anesthetics volatile – isoflurane C-Jun N-terminal kinase Caspase-3 Hippocampus
+
+Previous studies have demonstrated that isoflurane, a commonly used volatile anesthetic, can induce widespread apoptosis in the neonatal animal brains and result in persistent cognitive impairment. Isoflurane-induced cytosolic Ca2+ overload and activation of mitochondrial pathway of apoptosis may be involved in this neurodegeneration. The c-Jun N-terminal kinase (JNK) signaling can regulate the expression of the Bcl-2 family members that modulates mitochondrial membrane integrity. Therefore, we hypothesize that JNK signaling pathway activation contributes to isoflurane-induced apoptosis in the brain. In this study, Sprague-Dawley neonatal rats at postnatal day 7 were exposed to 1.1% isoflurane or (cid:2)g or the vehicle was intraventricularly air for 4 h. The JNK inhibitor SP600125 at 5 administered before the exposure. Neuronal apoptosis in the hippocampi of neonatal rats was detected by TUNEL 6 h after isoflurane or air exposure. The protein expression of phospho-JNK, phospho-c-Jun, and caspase-3 as well as the antiapoptotic protein Bcl-xL and Akt/glycogen synthase kinase (GSK)-3(cid:3) pathway was detected by Western blotting. Isoflurane significantly increased apoptotic cells in the hippocampal CA1, CA3, and DG regions. The JNK inhibitor SP600125 dose-dependently inhibited isoflurane-induced neuronal apoptosis and increase of caspase-3 and phospho-JNK. SP600125 also attenuated isoflurane- induced down-regulation of Bcl-xL and maintained the activated Akt level to increase the phosphorylation of GSK-3(cid:3) at Ser9. Our results indicate that JNK activation contributes to isoflurane-induced neuroapopto- sis in the developing brain. Maintaining Bcl-xL and Akt activation may be involved in the neuroprotective effects of SP600125.
+
+(cid:2)g, 10
+
+(cid:2)g, 20
+
+(cid:2)g, 30
+
+© 2013 Elsevier Ireland Ltd. All rights reserved.
+
+Abbreviations: Akt, protein kinase B; AP-1, activator-protein 1; Bcl-2, B cell lymphoma/lewkmia-2; CNS, central nervouse system; DG, dentate gyrus; GSK-3(cid:3), glycogen
+
+synthase kinase 3(cid:3); i.c.v, intracerebroventricular; JNK, c-jun N-terminal kinase; PI3K, phosphatidylinositol 3 kinase; TUNEL, TdT-mediated dUTP nick end labeling.
+
+∗
+
+Corresponding author at: Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, No. 107 Yanjiang West Road, Guangzhou 510120, China.
+
+Tel.: +86 02081332060; fax: +86 02081332833. ∗∗
+
+Corresponding author at: Department of Anatomy and Neurobiology, Zhongshan School of Medicine, Sun Yat-sen University, No. 74 Zhongshan Second Road, Guangzhou
+
+510080, China. Tel.: +86 02084111676; fax: +86 02084112545.
+
+(M. Zeng), hanmingxuan@rocketmail.com (X. Han), luotao20080808@163.com (T. Luo), jiangweijw@yahoo.com.cn (W. Jiang), xujie@mail.sysu.edu.cn (J. Xu), wanghq@mail.sysu.edu.cn (H. Wang).
+
+E-mail addresses: yujuan 04@yahoo.com.cn
+
+(Y. Li), 1995wangfei@sina.com.cn
+
+(F. Wang),
+
+lcl1204@yahoo.com.cn
+
+(C. Liu), minting19@163.com
+
+1 Both these authors contributed equally to this work.
+
+0304-3940/$ – see front matter © http://dx.doi.org/10.1016/j.neulet.2013.04.008
+
+2013 Elsevier Ireland Ltd. All rights reserved.
+
+18
+
+Y. Li et al. / Neuroscience Letters 545 (2013) 17– 22
+
+1. Introduction
+
+Exposure to anesthetics has been associated with widespread apoptotic neurodegeneration in the developing brains and per- sistent cognitive in animals [5,16,23] Moreover, nociceptive stimuli, such as formalin subcutaneous injection or surgical incision, further augment apoptosis and cognitive impair- ment induced by anesthetics in developing rats [24]. Some clinical retrospective studies have found that anesthesia and surgery in children younger than 4 years increase their probability of devel- oping disabilities in reading, writing and mathematics learning [15,30]. These reports have led to concerns about the possible detrimental effects of anesthesia and sedation in the pediatric pop- ulation.
+
+impairment
+
+Isoflurane is a commonly used volatile anesthetic. Current stud- ies have suggested that isoflurane causes severe neuroapoptosis in both developing animal brains and primary neuronal cells [5,16,29]. Isoflurane induces neuronal apoptosis and degeneration via [Ca2+]i (cid:4)-aminobutyric acid (GABA)A overload through the opening of the receptor-mediated synaptic voltage-dependent calcium channels (VDCCs) and the excessive Ca2+ release from the endoplasmic reticulum via activation of inositol-1,4,5-trisphosphate (IP3) recep- tors [35,36]. Isoflurane-induced [Ca2+]i overload not only activates mitochondrial pathway of apoptosis [29,33], but also is linked to the activation of c-Jun N-terminal kinase (JNK) because the phosphory- lation of c-Jun is prevented by antagonizing IP3 receptors [5]. The regulation of mitochondrial membrane integrity and the release of apoptogenic factors from mitochondria are tightly controlled by the proteins of Bcl-2 family [34]. The JNK signaling plays a pivotal role in mediating neuronal apoptosis through direct regulation of the expression of Bcl-2 family members and activation of the acti- vator protein 1 (AP-1) transcription factor family member c-Jun [1] that provides indirect transcriptional regulation of the Bcl-2 family members [14,18], including down-regulation of the antiapoptotic proteins Bcl-2 and Bcl-xL [8,14].
+
+(cid:2)g SP600125 or 12% DMSO only. The injection was performed 30 (cid:2)l as described before [6] under isoflurane anesthesia with a 5 microsyringe and 0.4 mm external diameter needle. The location of injection was 2.0 mm rostral, 1.5 mm lateral to the lambda and 2.0 mm deep to the skull surface of rats. The injection solution of (cid:2)l/min. The accuracy 5 of i.c.v. injection was verified by methylene blue in our preliminary experiments. All animals were sacrificed 6 h after termination of gas exposure and their hippocampi were used for Western blot- ting (n = 6) or TdT-mediated dUTP nick end labeling (TUNEL) with fluorescent dye (n = 6).
+
+(cid:2)l/rat was infused at a constant rate of 2.5
+
+For Western blotting studies, rat pups were anaesthetized with isoflurane and then sacrificed by decapitation. Hippocampi of rats −80 ◦C until were isolated immediately on ice and then stored at used. Western blotting was performed as we have described pre- viously [20]. In brief, the protein concentrations of samples were determined using the BCA protein assay (Bio-Rad,Herts, UK). Sixty micrograms of each sample were subjected to Western blot analy- sis using the following primary antibodies: anti-cleaved caspase-3 at 1:2000 dilution, anti-phospho-JNK at 1:2000 dilution, anti-JNK at 1:2000 dilution, anti-phospho-c-Jun at 1:1000 dilution, anti- phospho-Akt (Ser 473) at 1:2000 dilution, anti-Akt at 1:5000 dilution, anti- phospho-GSK-3(cid:3) (Ser 9) at 1:2000 dilution, anti- GSK-3(cid:3) at 1:2000 dilution, anti-Bcl-xL at 1:2000 dilution and anti-(cid:3)-actin at 1:2000 dilution. All antibodies were purchased from Cell Signaling Technology Company, USA. Images were scanned by an Image Master II scanner (GE Healthcare) and were analyzed using Image Quant TL software (v2003.03, GE Healthcare). The band signals of phospho-JNK, phospho-Akt and phospho-GSK-3(cid:3) were normalized to their total JNK, Akt and GSK-3(cid:3) from the same sam- ples. The band signals of other interesting proteins were normalized (cid:3)-actin and the results in each group were normalized to those of to that of corresponding control group.
+
+Protein kinase B (Akt), a serine/threonine kinase, also plays a prominent role in regulating neuronal survival. Once Akt is acti- vated, it inhibits apoptosis through inactivating Bad and glycogen synthase kinase 3(cid:3) (GSK-3(cid:3)) by phosphorylation [21,25]. Recent studies show that there is a potential crosstalk between JNK and Akt signaling, Akt signaling is involved in the apoptotic effect of JNK [10,31]. SP600125, a selective JNK inhibitor [2], has showed neu- roprotective effects in several neurodegenerative diseases [17,26]. Whether JNK signaling pathway contributes to isoflurane-induced neuroapoptosis remains underdetermined. In this study, we inves- tigated the effects of SP600125 on isoflurane-induced neuronal apoptosis and the expression of the antiapoptotic proteins Bcl-xL and Akt in the hippocampi of neonatal rats.
+
+For TUNEL studies, rat pups were anaesthetized with isoflu- rane and perfused transcardially with 4% paraformaldehyde. Their (cid:2)m thickness. brains were paraffin embedded and sectioned at 6 (cid:2)m apart) As we described before [19], four or five sections (200 for each animal at the same plane of the hippocampus were chosen for detecting apoptosis using TUNEL fluorescent method (Promega, Madision, WI, USA). The slides were protected from direct light during experiment. Hoechst was used to stain nuclei. The TUNEL positive cells in CA1, CA3 and dentate gyrus (DG) areas of hippocam- pus were analyzed immediately with NIS-Elements BR imaging processing and analysis software (Nikon Corporation, Japan). The densities of the TUNEL positive cells in CA1, CA3 and DG were cal- culated by dividing the number of TUNEL positive cells by the area of that brain region.
+
+2. Materials and methods
+
+All animal procedures were in compliance with the NIH Guide for the Use of Laboratory Animals and approved by the Animal Care and Use Committee of Sun Yat-sen University. Seven-day-old (P7) Sprague-Dawley rat pups (Guangdong Medical Laboratory Animal ± 3 g were exposed to 1.1% isoflu- Co, China) with body weight at 16 rane (about 0.5 MAC in P7 rats [22]) for 4 h to induce neuronal apoptosis, or to air in a temperature-controlled chamber as we described before [21]. The concentrations of anesthetic gas, oxy- gen and carbon dioxide (CO2) in the chamber were measured by a gas analyzer (Datex-Ohmeda, Madison, WI).
+
+Four doses of SP600125 (Selleck Chemicals LLC, Houston, TX, (cid:2)g) or 12% dimethyl sulfoxide (DMSO) as USA) (5, 10, 20 or 30 the vehicle were administered by intracerebroventricular (i.c.v.) injection 15 min before isoflurane exposure. Some rats received
+
+±
+
+SEM. The Graphpad Prism 4.0 soft- ware was used to conduct the statistical analyses. A two-tailed P value of less than 0.05 was considered statistically significant. One way ANOVA with Newman–Keuls Multiple Comparison Test was used when data was normally distributed and had equal variances. Otherwise, non-parametric test with Dunn’s Multiple Comparisons was used to compare the density of TUNEL positive cells as well as the relative protein abundance data among groups in Western blots.
+
+Data are presented in mean
+
+3. Results
+
+Our preliminary experiments for arterial blood gas monitoring showed that the neonatal rats had no hypoglycemia and acidosis during isoflurane exposure. Neuronal apoptosis in the hippocam- pal CA1, CA3 and DG regions of P7 rats were detected by TUNEL (Fig. 1). Isoflurane increased the number of apoptotic cells by 498% in CA1 (P < 0.01), 214% in CA3 (P < 0.001) and 217% in DG (P < 0.001)
+
+Y. Li et al. / Neuroscience Letters 545 (2013) 17– 22
+
+Fig. 1. JNK/SAPK inhibitor SP600125 inhibited the increase of isoflurane-induced TUNEL positive cells in the hippocampi of P7 rats. Representative images of TUNEL in the (cid:2)m. (C) Quantification of TUNEL hippocampal CA1 region (A) and CA3 region (B). Green staining indicated TUNEL-positive cells, blue staining indicated nuclear. Scan bar = 50 (cid:2)g SP600125; Iso: isoflurane. **P < 0.01, ***P < 0.001, vs. group DMSO; #P < 0.05, ##P < 0.01, ###P < 0.001, positive cells in the hippocampal CA1, CA3 and DG regions. SP30: 30 vs. group SP30; (cid:2)P < 0.05, (cid:2)(cid:2)(cid:2)P < 0.001, vs. group Iso + DMSO. Mean ± SEM, n = 6/group. (For interpretation of the references to color in figure legend, the reader is referred to the web version of the article.)
+
+(cid:2)g as compared to sham controls. The JNK inhibitor SP600125 at 30 inhibited the increase of isoflurane-induced neuronal apoptosis by 84% in CA1 (P < 0.05), by 84% in CA3 (P < 0.001) and 71% in DG (P < 0.05). In addition, we detected the change of cleaved caspase- 3 protein expression in the hippocampus (Fig. 2). Isoflurane with or without DMSO increased the expression of cleaved caspase-3 by 174.6% (P < 0.001) or 187.2% (P < 0.001), respectively. SP600125 dose-dependently decreased the expression of cleaved caspase-3. (cid:2)g, inhibited isoflurane-induced All doses of SP600125, except for 5 increase of cleaved caspase-3 by 71.3% (P < 0.01), 84.5% (P < 0.001) and 95.5% (P < 0.001), respectively (Fig. 2B). SP600125 alone neither increased the expression of cleaved caspase-3 nor the number of apoptotic cells in hippocampus.
+
+Isoflurane increased phospho-JNK at 46 kd by 38.7% (P < 0.05) as (cid:2)g inhibited phospho- compared to sham controls. SP600125 at 30 rylation of the 46 kd (P < 0.001) and 54 kd (P < 0.01) JNK (Fig. 2C). In accord with JNK activation, isoflurane increased the expression of phospho-c-Jun by 47.1% (P < 0.001) and decreased the expression of (cid:2)g significantly Bcl-xL protein by 40.4% (P < 0.05). SP600125 at 30 reversed the isoflurane-induced expression change of phospho-c- Jun (P < 0.01) (Fig. 3A and B) and Bcl-xL (P < 0.05) (Fig. 3C and D).
+
+SP600125 attenuated this inhibition by 70.0% (P < 0.05) (Fig. 3E and F). Isoflurane did not significantly influence the protein expression of phospho-GSK-3(cid:3), while isoflurane combined with SP600125 sig- nificantly increased its expression compared with control (P < 0.05) (Fig. 3G and H).
+
+4. Discussion
+
+Isoflurane is a commonly used volatile anesthetic during human surgery. Previous studies have demonstrated that it increases neuroapoptosis and induces long-term cognitive dysfunction in developing animals [5,16,29]. The present study demonstrates for the first time the neuroprotective effect of the JNK inhibitor SP600125 against neurodegeneration induced by isoflurane, as evi- denced by diminishing isoflurane-induced activation of caspase-3 and formation of apoptotic cells in the hippocampi of neonatal rats. SP600125 significantly inhibited isoflurane-induced increase of phosphorylation of JNK and c-Jun, downregultion of Bcl-xL, and decrease of Akt activation, which may be involved in its neuropro- tective effects.
+
+To investigate whether Akt/GSK signaling is involved in the antiapoptotic effect of SP600125, we measured proteins expres- sion of phospho-Akt and phospho-GSK-3(cid:3). Isoflurane inhibited the expression of phospho-Akt protein by 55.2% (P < 0.001), while
+
+Isoflurane, when used in low doses or for short periods, induces small to moderate increases in [Ca2+]i by activating ryanodine and IP3 receptor in endoplasmic reticulum of neuron, which trig- gers important survival signals including phosphorylation of Akt and Bcl-2 families. These signals play very important roles in the
+
+19
+
+20
+
+Y. Li et al. / Neuroscience Letters 545 (2013) 17– 22
+
+Fig. 2. SP600125 dose-dependently inhibited isoflurane-induced increase of caspase-3 and phosphorylation of JNK in the hippocampi of P7 rats. (A) Representative Western (cid:2)g SP600125; blots of caspase-3, phospho-JNK and JNK; (B and C) the quantitative analysis of cleaved caspase-3 (B) and phospho- JNK (C). Con: control; Iso: isoflurane; SP5: 5
+
+(cid:3)
+
+(cid:2)g SP600125; SP20: 20
+
+(cid:2)g SP600125; SP30: 30
+
+(cid:2)g SP600125. *P < 0.05, ***P < 0.001, vs. group Con; #P < 0.05, ##P < 0.01, vs. group DMSO;
+
+SP10: 10 (cid:2)(cid:2)P < 0.01, (cid:2)(cid:2)(cid:2)P < 0.001, vs. group Iso + DMSO; (cid:2)P < 0.05, (cid:2)(cid:2)P < 0.01, (cid:2)(cid:2)(cid:2)P < 0.001, vs. group Iso. Mean
+
+P < 0.05, vs. group SP30;
+
+±
+
+SEM, n = 6/group.
+
+neuroprotection of isoflurane preconditioning against ischemia or hypoxia in rat brain [3,4]. While when isoflurane is used in high doses or for long periods, especially if developing neurons are exposed to isoflurane, it will induce [Ca2+]i overload and results in neuronal apoptosis.
+
+The JNK signaling pathway is implicated in neuronal apo- ptosis such as ischemia/reperfusion and ethanol [9,11–13]. In the present study, our results suggest that JNK signaling also involves in isoflurane- induced neuronal apoptosis. The JNK pathways include nuclear pathway and non-nuclear pathway [11,12]. Activated JNK phos- phorylates nuclear substrate, the transcription factor c-Jun, which leads to increase of AP-1 transcription activity to modulate tran- scription of genes related to apoptosis. On the other hand, activated JNK regulates the activation of non-nuclear substrates including Bcl-2 family members [11,12]. Our current results showed that SP600125 pretreatment prevented isoflurane-induced increase of phosphorylation of JNK and c-Jun as well as increase of caspase- 3, which suggest that activated JNK nuclear pathway is involved in isoflurane-induced neuronal apoptosis. Sevoflurane, another inhaled anesthetic, also leads to an increase of phospho-JNK and apoptosis in neonatal rat brain [27,28]. However, SP600125 did not
+
+triggered by
+
+several brain
+
+injury
+
+stimuli,
+
+attenuate sevoflurane-induced apoptosis [27], which indicates that isoflurane and sevoflurane may induce neuroapoptosis in develop- ing brain by different mechanisms.
+
+The antiapoptotic protein Bcl-xL is widely expressed in the central nervous system (CNS), which enhances cell survival by maintaining mitochondrial membrane integrity and inhibits cytochrome c release [34]. Anesthesia cocktail containing isoflu- rane, nitrous oxide (N2O) and midazolam can downregulate Bcl-xL expression to induce neurotoxicity in developing rat brains [33]. In this context, we observed that isoflurane alone also caused a decreased expression of Bcl-xL in the hippocampi of P7 rats, and that this decrease was blocked by SP606125, thus preventing the mitochondrial membrane alteration and neuronal apoptosis. This result is in agreement with previous studies that suggest that JNK signaling promotes apoptosis possibly via transcriptional regula- tion of Bcl-2 family gene, including Bcl-xL [8,14,18]. Our results indicate that inhibition of Bcl-xL expression is a critical step in the isoflurane-induced apoptosis pathway and that this effect is dependent on JNK activation.
+
+Prosurvival pathways, such as Akt, may be inactivated dur- ing the apoptotic process [25,32]. Our experiments showed that isoflurane inhibited Akt phosphorylation while SP600125
+
+Y. Li et al. / Neuroscience Letters 545 (2013) 17– 22
+
+Fig. 3. SP600125 prevented isoflurane-induced increase of phospho-c-Jun and decrease of BcL-xL, and maintained activated Akt level and increased phosphorylation of GSK-3(cid:3) (G); (B, D, F and H) the quantitative analysis of phospho-c-Jun (B), Bcl-xL (D), phospho-Akt (F) and phospho-GSK-3(cid:3) (cid:2)g SP600125. *P < 0.05, ***P < 0.001, vs. group DMSO; (cid:2)P < 0.05, (cid:2)(cid:2)P < 0.01, vs. group Iso + DMSO. Mean
+
+in hippocampus of P7 rats. (A, C, E and G) Representative Western blots of phospho-c-Jun (A), Bcl-xL (C), phospho-Akt (E) and phospho-GSK-3(cid:3)
+
+(H). Iso: isoflurane; SP30: 30
+
+±
+
+SEM, n = 6/group.
+
+maintained the level of activated Akt. This result is supported by the evidence that phospho-GSK-3(cid:3) at Ser9 (inactivated form), one of the Akt phosphorylation site, is increased. This result is in agreement with previous studies that show that there is poten- tial crosstalk between JNK and Akt signaling [10,31]. It is possible that SP600125 maintain the mitochondrial membrane integrity by increasing Bcl-xL expression, thus prevents [Ca2+]i overload induced by isoflurane. However, it should be noted that isoflu- rane inhibited the expression of phospho-Akt but did not influence the expression of phospho-GSK-3(cid:3) at Ser9. The possible reason for this discrepancy may include activation of other intracellular mechanisms as a response to isoflurane, which consequently could also regulate the phosphorylation of GSK-3(cid:3). In our other exper- iments, we found isoflurane also activated p38 mitogen-activated protein kinase. A recent study suggests p38 can increase the
+
+phosphorylation of GSK-3(cid:3) at Ser 9 [7]. Thus, it is possible that phospho-GSK-3(cid:3) stays unchangeable due to the effects of isoflu- rane on Akt and p38. This effect on Akt may explain, in part, the antiapoptotic effects of SP600125 against isoflurane-induced neu- ronal cell apoptosis in developing rat brain. Our experiments only observed short-time effects of SP600125 in the hippocampi of neonatal rats, additional studies are needed to test the impact of SP600125 on isoflurane-induced cognitive dysfunction.
+
+5. Conclusions
+
+In conclusion, our results are consistent with the hypothesis that the JNK inhibitor SP600125 potentially attenuates isoflurane- induced neuroapoptosis in the hippocampi of developing rats by inhibiting isoflurane-induced JNK activation, the subsequent JNK
+
+21
+
+22
+
+Y. Li et al. / Neuroscience Letters 545 (2013) 17– 22
+
+dependent activation of c-Jun, suppression of Bcl-xL expression, and activation of caspase-3. In addition, maintenance of activated Akt may also be involved in the antiapoptotic effect of SP600125. Our findings suggest that JNK signaling pathway activation is cru- cial for isoflurane-induced neuroapoptosis.
+
+[15] C.J. Kalkman, L. Peelen, K.G. Moons, M. Veenhuizen, M. Bruens, G. Sinnema, T.P. de Jong, Behavior and development in children and age at the time of first anesthetic exposure, Anesthesiology 110 (2009) 805–812.
+
+[16] F. Kong, L. Xu, D. He, X. Zhang, H. Lu, Effects of gestational isoflurane exposure on postnatal memory and learning in rats, Eur. J. Pharmacol. 670 (2011) 168–174. [17] C.Y. Kuan, R.E. Burke, Targeting the JNK signaling pathway for stroke and Parkinson’s diseases therapy, Curr. Drug Targets CNS Neurol. Disord. 4 (2005) 63–67.
+
+Acknowledgments
+
+The study was supported by NSFC (30700787) and the GNSF (S2011010004558) to Yujuan Li; NSTFG (2008B030301320 and 2010B031600070), and STFG (2012J410076) to Huaqiao Wang.
+
+[18] C. Li, G. Xing, M. Dong, L. Zhou, J. Li, G. Wang, D. Zou, R. Wang, J. Liu, Y. Niu, Beta- asarone protection against beta-amyloid-induced neurotoxicity in PC12 cells via JNK signaling and modulation of Bcl-2 family proteins, Eur. J. Pharmacol. 635 (2010) 96–102.
+
+[19] Y. Li, G. Liang, S. Wang, Q. Meng, Q. Wang, H. Wei, Effects of fetal exposure to isoflurane on postnatal memory and learning in rats, Neuropharmacology 53 (2007) 942–950.
+
+References
+
+[20] Y. Li, C. Liu, Y. Zhao, K. Hu, J. Zhang, M. Zeng, T. Luo, W. Jiang, H. Wang, Sevoflurane induces short-term changes in proteins in the cerebral cortices of developing rats, Acta Anaesthesiol. Scand. 3 (2013) 380–390.
+
+[1] A. Behrens, M. Sibilia, E.F. Wagner, Amino-terminal phosphorylation of c-Jun regulates stress-induced apoptosis and cellular proliferation, Nat. Genet. 21 (1999) 326–329.
+
+[21] H.R. Luo, H. Hattori, M.A. Hossain, L. Hester, Y. Huang, W. Lee-Kwon, M. Donowitz, E. Nagata, S.H. Snyder, Akt as a mediator of cell death, Proc. Natl. Acad. Sci. U.S.A. 100 (2003) 11712–11717.
+
+[2] B.L. Bennett, D.T. Sasaki, B.W. Murray, E.C. O’Leary, S.T. Sakata, W. Xu, J.C. Leis- ten, A. Motiwala, S. Pierce, Y. Satoh, S.S. Bhagwat, A.M. Manning, D.W. Anderson, S.P600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 13681–13686.
+
+[3] P.E. Bickler, C.S. Fahlman, The inhaled anesthetic, isoflurane, enhances Ca2+- dependent survival signaling in cortical neurons and modulates MAP kinases, apoptosis proteins and transcription factors during hypoxia, Anesth. Analg. 103 (2006) 419–429.
+
+[4] P.E. Bickler, X. Zhan, C.S. Fahlman,
+
+Isoflurane preconditions hippocam- pal neurons against oxygen-glucose deprivation: role of intracellular Ca2+ and mitogen-activated protein kinase signaling, Anesthesiology 103 (2005) 532–539.
+
+[22] G. Orliaguet, B. Vivien, O. Langeron, B. Bouhemad, P. Coriat, B. Riou, Minimum alveolar concentration of volatile anesthetics in rats during postnatal matura- tion, Anesthesiology 95 (2001) 734–739.
+
+[23] M.G. Paule, M. Li, R.R. Allen, F. Liu, X. Zou, C. Hotchkiss, J.P. Hanig, T.A. Patterson, W.J. Slikker, C. Wang, Ketamine anesthesia during the first week of life can cause long-lasting cognitive deficits in rhesus monkeys, Neurotoxicol. Teratol. 33 (2011) 220–230.
+
+[24] Y. Shu, Z. Zhou, Y. Wan, R.D. Sanders, M. Li, C.K. Pac-Soo, M. Maze, D. Ma, Nociceptive stimuli enhance anesthetic-induced neuroapoptosis in the rat developing brain, Neurobiol. Dis. 45 (2012) 743–750.
+
+[25] G. Song, G. Ouyang, S. Bao, The activation of Akt/PKB signaling pathway and
+
+cell survival, J. Cell. Mol. Med. 9 (2005) 59–71.
+
+[5] A.M. Brambrink, A.S. Evers, M.S. Avidan, N.B. Farber, D.J. Smith, X. Zhang, G.A. Dissen, C.E. Creeley, J.W. Olney, Isoflurane-induced neuroapoptosis in the neonatal rhesus macaque brain, Anesthesiology 112 (2010) 834–841.
+
+[6] Y. Cheng,
+
+J.M. Gidday, Q. Yan, A.R. Shah, D.M. Holtzman, Marked factor injury, Ann. Neurol. 41 (1997)
+
+age-dependent neuroprotection by brain-derived neurotrophic against neonatal hypoxic-ischemic brain 521–529.
+
+[7] C.H. Choi, B.H. Lee, S.G. Ahn, S.H. Oh, Proteasome inhibition-induced p38 MAPK/ERK signaling regulates autophagy and apoptosis through the dual phosphorylation of glycogen synthase kinase 3beta, Biochem. Biophys. Res. Commun. 418 (2012) 759–764.
+
+[8] R. Chu, M. Upreti, W.X. Ding, X.M. Yin, T.C. Chambers, Regulation of Bax by c- Jun NH2-terminal kinase and Bcl-xL in vinblastine-induced apoptosis, Biochem. Pharmacol. 78 (2009) 241–248.
+
+[9] J. Fan, G. Xu, D.J. Nagel, Z. Hua, N. Zhang, G. Yin, A model of ischemia and reperfusion increases JNK activity, inhibits the association of BAD and 14-3- 3, and induces apoptosis of rabbit spinal neurocytes, Neurosci. Lett. 473 (2010) 196–201.
+
+[10] A. Fornoni, A. Pileggi, R.D. Molano, N.Y. Sanabria, T. Tejada, J. Gonzalez- Quintana, H. Ichii, L. Inverardi, C. Ricordi, R.L. Pastori, Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation, Dia- betologia 51 (2008) 298–308.
+
+[26] W. Wang, L. Shi, Y. Xie, C. Ma, W. Li, X. Su, S. Huang, R. Chen, Z. Zhu, Z. Mao, Y. Han, M. Li, S.P600125, a new JNK inhibitor, protects dopaminergic neurons in the MPTP model of Parkinson’s disease, Neurosci. Res. 48 (2004) 195–202. [27] W.Y. Wang, H. Wang, Y. Luo, L.J. Jia, J.N. Zhao, H.H. Zhang, Z.W. Ma, Q.S. Xue, B.W. Yu, The effects of metabotropic glutamate receptor 7 allosteric ago- nist N,N(cid:4)-dibenzhydrylethane-1,2-diamine dihydrochloride on developmental sevoflurane neurotoxicity: role of extracellular signal-regulated kinase 1 and 2 mitogen-activated protein kinase signaling pathway, Neuroscience 205 (2012) 167–177.
+
+[28] Y. Wang, Y. Cheng, G. Liu, X. Tian, X. Tu, J. Wang, Chronic exposure of gestation rat to sevoflurane impairs offspring brain development, Neurol. Sci. 33 (2012) 535–544.
+
+[29] H. Wei, B. Kang, W. Wei, G. Liang, Q.C. Meng, Y. Li, R.G. Eckenhoff, Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently, Brain Res. 1037 (2005) 139–147.
+
+[30] R.T. Wilder, R.P. Flick, J. Sprung, S.K. Katusic, W.J. Barbaresi, C. Mickelson, S.J. Gleich, D.R. Schroeder, A.L. Weaver, D.O. Warner, Early exposure to anesthesia and learning disabilities in a population-based birth cohort, Anesthesiology 110 (2009) 796–804.
+
+[31] M. Yeste-Velasco, J. Folch, G. Casadesus, M.A. Smith, M. Pallas, A. Camins, Neuro- protection by c-Jun NH2-terminal kinase inhibitor SP600125 against potassium deprivation-induced apoptosis involves the Akt pathway and inhibition of cell cycle reentry, Neuroscience 159 (2009) 1135–1147.
+
+[11] Q.H. Guan, D.S. Pei, Q.G. Zhang, Z.B. Hao, T.L. Xu, G.Y. Zhang, The neuro- protective action of SP600125, a new inhibitor of JNK, on transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 via nuclear and non-nuclear pathways, Brain Res. 1035 (2005) 51–59.
+
+[12] Q.H. Guan, D.S. Pei, Y.Y. Zong, T.L. Xu, G.Y. Zhang, Neuroprotection against inhibitor of c-Jun N-terminal ischemic brain kinase (JNK) via nuclear and non-nuclear pathways, Neuroscience 139 (2006) 609–627.
+
+injury by a small peptide
+
+[13] J.Y. Han, E.Y. Jeong, Y.S. Kim, G.S. Roh, H.J. Kim, S.S. Kang, G.J. Cho, W.S. Choi, C-jun N-terminal kinase regulates the interaction between 14-3-3 and Bad in ethanol-induced cell death, J. Neurosci. Res. 86 (2008) 3221–3229.
+
+[14] H.S. Jeong, H.Y. Choi, T.W. Choi, B.W. Kim, J.H. Kim, E.R. Lee, S.G. Cho, Dif- ferential regulation of the antiapoptotic action of B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma extra long (Bcl-xL) by c-Jun N-terminal protein kinase (JNK) 1-involved pathway in neuroglioma cells, Biol. Pharm. Bull. 31 (2008) 1686–1690.
+
+[32] G. Yin, L.Y. Li, M. Qu, H.B. Luo, J.Z. Wang, X.W. Zhou, Upregulation of AKT attenuates amyloid-beta-induced cell apoptosis, J. Alzheimers Dis. 25 (2011) 337–345.
+
+[33] J.H. Yon, J. Daniel-Johnson, L.B. Carter, V. Jevtovic-Todorovic, Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways, Neuroscience 135 (2005) 815–827.
+
+[34] H. Zhao, M.A. Yenari, D. Cheng, R.M. Sapolsky, G.K. Steinberg, Bcl-2 overex- pression protects against neuron loss within the ischemic margin following experimental stroke and inhibits cytochrome c translocation and caspase-3 activity, J. Neurochem. 85 (2003) 1026–1036.
+
+[35] Y. Zhao, G. Liang, Q. Chen, D.J. Joseph, Q. Meng, R.G. Eckenhoff, M.F. Eck- enhoff, H. Wei, Anesthetic-induced neurodegeneration mediated via inositol 1,4,5-trisphosphate receptors, J. Pharmacol. Exp. Ther. 333 (2010) 14–22. [36] Y.L. Zhao, Q. Xiang, Q.Y. Shi, S.Y. Li, L. Tan, J.T. Wang, X.G. Jin, A.L. Luo, GABAergic excitotoxicity injury of the immature hippocampal pyramidal neurons’ expo- sure to isoflurane, Anesth. Analg. 113 (2011) 1152–1160.

new_pdfs/10.1016_j.neuropharm.2007.09.005.txt

@@ -0,0 +1,725 @@
+Available online at www.sciencedirect.com
+
+Neuropharmacology 53 (2007) 942e950
+
+www.elsevier.com/locate/neuropharm
+
+Effects of fetal exposure to isoflurane on postnatal memory and learning in rats
+
+Yujuan Li a,b, Ge Liang a, Shouping Wang a,b, Qingcheng Meng a, Qiujun Wang a,c, Huafeng Wei a,*
+
+a Department of Anesthesiology and Critical Care, University of Pennsylvania, 305 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104, USA b Department of Anesthesia, Second Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510120, China c Department of Anesthesiology, The Third Clinical Hospital, Hebei Medical University, Shijiazhuang 050051, China
+
+Received 9 March 2007; received in revised form 9 August 2007; accepted 10 September 2007
+
+Abstract
+
+In a maternal fetal rat model, we investigated the behavioral and neurotoxic effects of fetal exposure to isoflurane. Pregnant rats at gestational day 21 were anesthetized with 1.3% isoflurane for 6 h. Apoptosis was quantified in the hippocampus and cortex at 2 and 18 h after exposure in the fetal brain and in the postnatal day 5 (P5) pup brain. Spatial memory and learning of the fetal exposed pups were examined with the Morris Water Maze at juvenile and adult ages. Rat fetal exposure to isoflurane at pregnancy day 21 through maternal anesthesia significantly decreased spontaneous apoptosis in the hippocampal CA1 region and in the retrosplenial cortex at 2 h after exposure, but not at 18 h or at P5. Fetal ex- posure to isoflurane did not impair subsequent juvenile or adult postnatal spatial reference memory and learning and, in fact, improved spatial memory in the juvenile rat. These results show that isoflurane exposure during late pregnancy is not neurotoxic to the fetal brain and does not impair memory and learning in the juvenile or adult rat. (cid:2) 2007 Elsevier Ltd. All rights reserved.
+
+Keywords: Anesthetics; Fetus; Developing brain; Apoptosis; Memory; Learning
+
+1. Introduction
+
+Anesthetics cause neurotoxicity in a concentration and time dependent manner in in vitro neuronal models (Chang and Chou, 2001; Eckenhoff et al., 2004; Kim et al., 2006; Kvolik et al., 2005; Loop et al., 2005; Matsuoka et al., 2001; Wei et al., 2005; Wise-Faberowski et al., 2005; Xie et al., 2006, 2007). Relatively fewer studies have investigated the neuro- toxic effects of anesthetics in in vivo models. Isoflurane expo- sure at a clinically relevant concentration (0.75%) for 6 h during postnatal development in rats caused persistent memory and learning deficits, which was associated with widespread neuronal apoptosis (Jevtovic-Todorovic et al., 2003; Yon
+
+Corresponding author. Tel.: þ1 215 662 3193; fax: þ1 215 349 5078. E-mail address: weih@uphs.upenn.edu (H. Wei).
+
+et al., 2005). Neurons in the developing brain are specifically vulnerable to isoflurane neurotoxicity (Jevtovic-Todorovic et al., 2003). However, the mechanisms for isoflurane neuro- toxicity are unknown.
+
+Since anesthetics easily cross the placenta, the developing fetal brain will be exposed to inhaled anesthetics, such as iso- flurane, when pregnant women require surgery. In some cases, such as fetal surgery to correct various congenital malforma- tions during mid-gestation (18e25 weeks) (Myers et al., 2002), the fetal brain can be exposed to 2e3 times (2.5e3 minimal alveolar concentration (MAC)) higher than normal concentrations of inhaled anesthetics, in order to relax uterine smooth muscle and provide adequate anesthesia (Cauldwell, 2002; Myers et al., 2002). Although fetal surgery is relatively new, it is a rapidly growing and evolving area, and may be- come standard therapy for most disabling malformations that
+
+0028-3908/$ - see front matter (cid:2) 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2007.09.005
+
+Y. Li et al. / Neuropharmacology 53 (2007) 942e950
+
+are currently treated in young infants (Goldsmith et al., 1999; Myers et al., 2002). Because most fetal surgeries in humans are performed during mid-gestation, it is important and urgent to know if the anesthetics used cause damage to the develop- ing brain and subsequent postnatal memory problems and learning disabilities.
+
+The aim of the current study was to determine whether exposure to clinically relevant concentrations of isoflurane during prenatal development causes neuronal apoptosis and postnatal learning and memory deficits.
+
+2. Materials and methods
+
+2.1. Animals
+
+Institute of Animal Care and Use Committee (IACUC) at the University of Pennsylvania approved all experimental procedures and protocols used in this study. All efforts were made to minimize the number of animals used and their suffering. SpragueeDawley pregnant rats (Charles River Laboratories, Inc Wilmington, MA) were housed in polypropylene cages and the room temper- ature was maintained at 22 (cid:2)C, with a 12 h lightedark cycle. Pregnant rats at gestation day 21 (E21) were used for all experiments because it approximately corresponds to mid-gestation in human beings according to the theory of brain growth spurt (Dobbing and Sands, 1979; Jevtovic-Todorovic et al., 2003), and is a common time for most fetal surgeries (18e25 weeks) (Myers et al., 2002). We have designed the following three related studies: (1) pilot study; (2) neurodegeneration study; (3) finally a behavioral study. A pilot study was first conducted to find the highest concentration of isoflurane not accompanied by significant arterial blood gas (ABG) and mean arterial blood pressure (MABP) changes in the mothers. A neurodegeneration study was used to determine the appearance of apoptosis by detection of caspase-3 and TUNEL (terminal de- oxynucleotidyl transferase biotin-dUTP nick end labeling) positive cells in the fetal brain (2 and 18 h post-exposure) or neonatal brain at postnatal day 5 (P5). We have chosen the above time points to detect apoptosis in fetal or newborn brains, based on previously published work (Jevtovic-Todorovic et al., 2003). The behavioral study was performed to investigate the effects of fetal exposure to isoflurane on postnatal memory and learning. The pregnant rats used in each study were not reused in the other two studies. Within each study described above, animals were randomly divided into either isoflurane treatment or sham control groups. Pregnant rats in the isoflurane treatment groups inhaled isoflurane for 6 h, while those in the sham control group only inhaled a carrier gas (30% oxygen, balanced with nitrogen) for 6 h under the same experimental
+
+conditions. The distribution of pregnant rats and pups in all three groups is illustrated in Fig. 1.
+
+2.2. Anesthetic exposure
+
+Isoflurane is used clinically at a wide range of concentrations (about 0.2e 3%), depending on the presence of other kinds of anesthetics or narcotics and the type and duration of surgery. As isoflurane neurotoxicity is concentration- dependent (Jevtovic-Todorovic et al., 2003; Wei et al., 2005), a primary goal of this study was to investigate if the highest isoflurane concentration used clin- ically is harmful to the fetal brain. Due to our concern that the physiological side effects of these drugs would contaminate the interpretation, we conducted a pilot study to determine the highest anesthetic concentration we could use without invasive support (tracheal intubation and ventilation) that would not significantly affect arterial blood gas (ABG) and mean arterial blood pressure (MABP) in the mothers, and then used this concentration in the subsequent formal study. We wanted to avoid tracheal intubation, as it could possibly af- fect the hemodynamics of pregnant rats and the apoptosis in the fetal brains. In addition, this makes it more difficult to set up the sham control groups without anesthesia. In the pilot study, five pregnant rats were initially anesthetized with 2% isoflurane in 30% oxygen via a snout cone for approximately 1 h and the right femoral artery was catheterized for blood sample collection and measure- ment of MABP by a pressure transducer/amplifier (AD Instruments Inc., Colorado Springs, CO, USA). The rats were recovered for 2 h and then ex- posed to isoflurane, starting at 1.5% in a humidified carrier gas of 30% oxygen, balance nitrogen for 6 h in a monitored chamber in hood. The pregnant rats breathed spontaneously without intubation or other support while being warmed using a deltaphase isothermal pad (Braintree Scientific Inc, Braintree, MA, USA). The rectal temperature was maintained (Fisher Scientific, Pitts- burgh, PA, USA) at 37 (cid:3) 0.5 (cid:2)C. We monitored isoflurane concentration in the chamber using IR absorbance (Ohmeda 5330, Detex-Ohmeda, Louisville, CO, USA). Arterial blood (0.1 ml) from previously placed femoral arterial catheter was collected and ABG determined every 2 h for up to 6 h by an ABG analyzer (Nova Biomedical, Waltham, MA, USA). Blood glucose was simultaneously measured with a glucometer (ACCU-CHECK Advantage, Roche Diagnostics Corporations, Indianapolis, IN, USA). Control rats were exposed only to humidified 30% O2 balanced by N2 (carrier gas for isoflurane in the treatment group) for 6 h in the same chamber under the same experi- mental conditions as in the treatment group. Because one pregnant rat treated with 1.5% isoflurane showed obvious acidemia (which reversed after termina- tion of anesthesia), we decreased the isoflurane concentration to 1.3%, and subsequently found no significant changes in the ABG or MABP between the treatment group and the sham control group (Table 1). Therefore, 1.3% iso- flurane was used in the ensuing neurodegeneration and behavioral studies.
+
+Total Pregnant Rats (44)
+
+Pilot Study (8)
+
+Neurodegeneration Study (21)
+
+Behavioral Study (15)
+
+Control (4) 1.3% Isoflurane (4)
+
+Control (10)
+
+1.3% Isoflurane (11)
+
+Control (7)
+
+1.3% Isoflurane (8)
+
+2 Hr (Mother 5) (Fetus 12)
+
+18 Hr (Mother 5) (Fetus 14)
+
+2 Hr (Mother 5) (Fetus 13)
+
+18 Hr (Mother 6) (Fetus 13)
+
+Behavioral study Pups (26)
+
+Neurodeg- eneration study P5 (7)
+
+Behavioral study Pups (31)
+
+Neurodeg- eneration study P5 (8)
+
+Fig. 1. Nomogram illustrating distribution of pregnant mother and their fetus or postnatal rats among different studies.
+
+943
+
+944
+
+Y. Li et al. / Neuropharmacology 53 (2007) 942e950
+
+In the behavioral study, pregnant rats were treated with 1.3% isoflurane (n ¼ 8) or carrier gas (sham controls, n ¼ 7) for 6 h. The monitoring was the same as that in the pilot study except that femoral artery catheters were not placed. After the exposures, the animals were returned to their cages and the rat pups were delivered naturally. Four rat pups from each pregnant mother were raised to P28 (Juvenile) and P118 (adult), and then used to deter- mine memory and learning ability with a Morris Water Maze (MWM). Two rat pups from the control group and one from the isoflurane group died unexpect- edly, leaving a total of 26 and 31 rat pups in the control and isoflurane treat- ment groups respectively (Fig. 1).
+
+corresponding to figure 96 of the rat fetal brain atlas (Paxinos et al., 1990) were chosen for detection of apoptosis by caspase-3 immunohistochemistry and TUNEL staining. In the initial examination of brains sections from the neurodegeneration study, we noticed that apoptosis was most apparent in the hippocampus CA1 region and the retrosplenial cortex, and thus we chose these two brain regions to quantify apoptosis.
+
+2.5. Immunohistochemistry for caspase-3
+
+In the fetal brain apoptosis study, pregnant rats were treated with either 1.3% isoflurane or carrier gas for 6 h. At 2 and 18 h after exposure, the rat pups were delivered by C-section under sodium pentobarbital (100 mg/kg, i.p.) anesthesia. The fetal brains were removed and snap frozen for immuno- histochemical analysis. Two fetal brains from each pregnant rat were studied. In addition, newborn brains from the rat pups born to the pregnant rats in the behavioral study group (one pup from each pregnant rat, treatment n ¼ 8, con- trol n ¼ 7) were also obtained at postnatal day 5 and prepared for the apoptosis study at P5 (Fig. 1).
+
+2.3. Measurement of isoflurane concentration in the brain tissues
+
+To confirm that the isoflurane concentration in the fetal brain correlated with the inhaled concentration and brain concentration in the pregnant mothers, we measured the brain isoflurane concentrations in the fetus and the mother simultaneously in one rat. Briefly, after the pregnant rat was ex- posed to 1.3% isoflurane for 6 h, the brains of both mother and fetuses were removed and the brain tissue was immediately placed into 4 ml of 0.02 M phosphate buffer (with 1 mM halothane as internal standard) and homogenized in a glass homogenizer. The homogenate was centrifuged (30,000 (cid:4) g at 4 (cid:2)C for 30 min), the supernatant collected and then loaded onto C18 cartridge that had been conditioned with 2 ml methanol and washed with water, for solid phase extraction. The final sample was eluted with 0.5 ml solution of methanol and 2-propanol (vol:vol 2:1) with 0.1% trifluoroacetic acid. All procedures were performed in the cool room (4 (cid:2)C). A 250 ml aliquot of each final elute was injected into a high performance liquid chromatography (HPLC) system, equipped with a refractive index monitor, for quantitation.
+
+Caspase-3 positive cells were detected using immunohistochemical methods described previously (Gown and Willingham, 2002). Briefly, brain incubated in 3% hydrogen peroxide in methanol for sections were first 20 min to quench endogenous peroxidase activity. Sections were then incu- bated with blocking solution containing 10% normal goat serum in 0.1% phos- phate buffered saline with 0.1% Tween 20 (PBST) for 1 h at room temperature after washing with 0.1% PBST. The anti-activated caspase-3 primary antibody (1/200, Cell Signaling Technology, Inc Danvers, MA, USA) was then applied in blocking solution and incubated at 4 (cid:2)C overnight. Tissue sections were bio- tinylated with goat anti-rabbit antibody (1/200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) in 0.1% PBST for 40 min, followed by incubation with the avidinebiotinylated peroxidase complex (Vectostain ABC-Kit, Vector Lab, Burlingame, CA, USA) for 40 min. Tissue sections were colorized with diaminobenzidine (DAB, Vector Laboratories, Burlingame, CA, USA) for 8 min and counterstained with modified hematoxylin. Negative control sec- tions were incubated in blocking solution that did not contain primary anti- body. Images were acquired and assessed at 200(cid:4) using IP lab 7.0 software linked to an Olympus IX70 microscope (Olympus Corporation, Japan) equip- ped with a Cooke SensiCam camera (Cooke Corporation, Romulus, MI, USA). Three brain tissue sections at 10 mm corresponding to the Atlas of the Devel- oping Rat Brain, Figure 96 (Paxinos et al., 1990) were chosen from each an- imal and analyzed for caspase-3 positive cells in the two brain regions. Two persons blinded to the treatments counted the total number of caspase-3 pos- itive cells in the hippocampal CA1 region and retrosplenial cortex. The areas of entire hippocampal CA1 region and retrosplenial cortex were defined ac- cording to the Atlas of the Developing Rat Brain, Figure 96 (Paxinos et al., 1990) and the area measured using IPLab Suite v3.7 imaging processing and analysis software (Biovision Technologies, Exton, PA, http://www.Bio- Vis.com). The density of caspase-3 positive cells in a particular brain region was calculated by dividing the number of caspase-3 positive cells by the area of that brain region.
+
+2.4. Tissue preparation
+
+After treatment with isoflurane or carrier gas alone (control), pregnant rats were anesthetized with sodium pentobarbital intraperitoneally (i.p. 100 mg/kg) at either 2 or 18 h after the end of isoflurane exposure, and the fetuses removed by cesarean section. Likewise, postnatal pups at day 5 (P5) were given the same dose of sodium pentobarbital. All fetuses and pups were then perfused transcardially with ice-cold normal saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were then removed and post-fixed overnight in the same fixative at 4 (cid:2)C, and cryoprotected in 30% (wt/vol) sucrose in 0.1 M phosphate buffer (pH 7.4) at 4 (cid:2)C for 24 h. Thereaf- ter, the brains were frozen in isopentane at (cid:5)20 (cid:2)C and stored at (cid:5)80 (cid:2)C until use. Serial coronal sections (10 mm) were cut in a cryostat (DolbeyeJamison Optical Company, Inc., Pottstown, PA, USA), mounted on gelatin-coated slides and stored at (cid:5)80 (cid:2)C. Coronal brain sections from the same brain
+
+2.6. TUNEL for DNA fragmentation
+
+Three brain sections (10 mm) adjacent to the sections used for caspase-3 detection were used for TUNEL staining using the DeadEnd(cid:3) Colorimetric TUNEL System Kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocol (Gavrieli et al., 1992). Briefly, sections were permeabilized by proteinase K solution (20 mg/ml) for 8 min, incubated in equilibration buffer for 10 min and the terminal deoxynucleotidyl transferase (TdT) and biotinylated nucleotide were added to the section and incubated in a humidified chamber at 37 (cid:2)C for 1 h. The reaction was then stopped, fol- lowed by incubation with horseradish peroxidase-labeled streptavidin, colori- zation with DAB/ H2O2 and counterstained with modified hematoxylin. For treated with DNase I positive-controls,
+
+the tissue sections were first
+
+Table 1 Isoflurane (1.3%) did not affect arterial blood pressure and blood gas significantly
+
+Baseline
+
+2 h
+
+4 h
+
+6 h
+
+Control
+
+1.3% Iso
+
+Control
+
+1.3% Iso
+
+Control
+
+1.3% Iso
+
+Control
+
+1.3% Iso
+
+pH PaCO2 PaO2 (cid:5) HCO3 MABP
+
+7.46 (cid:3) 0.02 36.7 (cid:3) 2.80 158 (cid:3) 6.98 25.6 (cid:3) 2.38 117 (cid:3) 7.42
+
+7.46 (cid:3) 0.03 32.8 (cid:3) 0.64 162 (cid:3) 4.67 23.3 (cid:3) 1.57 119 (cid:3) 0.56
+
+7.47 (cid:3) 0.01 33.4 (cid:3) 1.39 149 (cid:3) 9.08 24.4 (cid:3) 1.15 106 (cid:3) 9.61
+
+7.44 (cid:3) 0.02 37.2 (cid:3) 4.33 159 (cid:3) 2.12 25.5 (cid:3) 2.02 90 (cid:3) 6.67
+
+7.45 (cid:3) 0.01 35.8 (cid:3) 3.43 166 (cid:3) 5.45 25.2 (cid:3) 2.84 102 (cid:3) 4.06
+
+7.39 (cid:3) 0.01 39.9 (cid:3) 4.95 156 (cid:3) 8.67 24.2 (cid:3) 2.53 89 (cid:3) 6.68
+
+7.49 (cid:3) 0.00 37.5 (cid:3) 0.90 163 (cid:3) 5.45 28.3 (cid:3) 2.70 106 (cid:3) 4.81
+
+7.39 (cid:3) 0.02 36.6 (cid:3) 2.20 154 (cid:3) 9.43 23 (cid:3) 2.50 89 (cid:3) 7.33
+
+Values are mean (cid:3) SD. n ¼ 4 for each group. Iso, isoflurane; MABP, mean arterial blood pressure.
+
+Y. Li et al. / Neuropharmacology 53 (2007) 942e950
+
+(1000 U/ml, pH 7.6) for 10 min at room temperature to initiate breakdown of DNA. Incubation of sections in reaction buffer without TdT provided negative controls. Images were acquired, and TUNEL quantitation performed as de- scribed above for caspase-3.
+
+rats (P115) received only one block of trials each day for 5 days using a new plat- form location in an effort to increase task difficulty and improve test sensitivity.
+
+2.7.4. Probe trials
+
+2.7. Spatial reference memory and learning performance
+
+2.7.1. Morris Water Maze (MWM)
+
+Pregnant rats were allowed to deliver after the isoflurane treatment and 4 pups per litter (2 females and 2 males) were raised. The body weights of the rat pups were recorded at P0, P3, P5, P11, P17 and P28 to determine growth rate. We determined spatial reference memory and learning with the MWM as reported previously with some modification (Jevtovic-Todorovic et al., 2003). A schematic of the experimental paradigm is shown in Fig. 2. A round, fiberglass pool, 150 cm in diameter and 60 cm in height, was filled with water to a height of 1.5 cm above the top of the movable clear 15 cm diameter platform. The pool was located in a room with numerous visual cues (including com- puters, posters and desks) that remained constant during the studies. Water was kept at 20 (cid:2)C and opacified with titanium dioxide throughout all training and testing. A video tracking system recorded the swimming motions of animals and the data were analyzed using motion-detection software for the MWM (Ac- timetrics Software, Evanston, IL, USA). After every trial, each rat was placed in a holding cage, under an infrared heat lamp, before returning to its regular cage.
+
+2.7.2. Cued trials
+
+Probe trials were conducted after the last place trials for the juveniles (P36) and adults (P119) to evaluate memory retention capabilities. After all rats com- pleted the last place trial on the fifth day, the platform was removed from the water maze and rat was started to swim in the quadrant opposite to one the plat- form was placed before. The rats were allowed to swim for 60 s during each probe trial and the time the rats spent in each quadrant was recorded. The per- centage of the swimming time spent in the target (probe) quadrant where the platform was placed before was calculated. The time spent in the target quadrant compared to other quadrants was an indication of memory retention.
+
+2.7.5. Learning to reach criterion test
+
+After the last probe test for the adult rats, the animals performed the learn- ing to reach criterion test during the next 9 days as described previously (Chen et al., 2000). The experimental procedure was similar to the place trial except that the platform location was changed. For each rat, the platform was moved between nine different locations set up by the computer. Each rat received up to eight trials per day. In order to advance to the next platform location, each rat had to reach the criterion of three successive trials with escape latency of 20 s or less. If a rat reached a criterion in 8 or less trials, a new platform lo- cation would be selected the following day. The numbers of learned platforms and the number of trials used to reach the criteria were recorded and com- pared. The number of platforms learned and the number of trials to reach a cri- terion indicated the learning ability of the rats.
+
+The cued trials were performed only for postnatal rats at P28 and P29 (28 rats in control group and 31 rats in treatment group) to determine whether any non- cognitive performance impairments (e.g. visual impairments and/or swimming difficulties) were present, which might affect performance on the place or probe trials. A white curtain surrounded the pool to prevent confounding visual cues. All rats received 4 trials per day. On each trial, rats were placed in a fixed position in the swimming pool facing the wall and were allowed to swim to a platform with a rod (cue) 20 cm above water level randomly placed in any of the 4 quad- rants of the swimming pool. They were allotted 60 s to find the platform upon which they sat for 30 s before being removed from the pool. If a rat did not find the platform within 60 s, the rat was gently guided to the platform and al- lowed to remain there for 30 s. The time for each rat to reach the cued platform and the swim speed was recorded and the data at P28/29 were analyzed.
+
+2.8. Statistical analysis
+
+To reduce variance from different size litters, we averaged the data from all fetal or postnatal rats from the same mother and considered them as a single sample. Results of weight gain of postnatal rat pups, ABG and MABP of preg- nant rats and place trials of postnatal rats were analyzed using 2-way ANOVA for repeated measurements. Data for immunohistochemistry, TUNEL and other behavioral studies were analyzed using Student’s t-test for comparison of two groups or by ANOVA followed by Fisher’s post hoc multiple compar- ison tests for those with more than two groups. In all experiments, difference were considered statistically significant at P < 0.05.
+
+2.7.3. Place trials
+
+3. Results
+
+After completion of cued trials, we used the same rats to perform the place trials to determine the rat’s ability to learn the spatial relationship between distant cues and the escape platform (submerged, no cue rod), which remained in the same location for all place trials. The starting points were random for each rat. The time to reach the platform was recorded for each trial. The less time it took a rat to reach the platform, the better the learning ability. The juvenile rats (P32) received two blocks of trials (two trials per block, 30 s apart, 60 s max- imum for each trial and 2 h rest between blocks) each day for 5 days. The adult
+
+3.1. Comparison of basic physiological variables and brain isoflurane concentrations between 1.3% isoflurane treatment and sham control groups
+
+In the pilot study, the pregnant rats at E21, initially exposed to 1.5% isoflurane for 3 h, developed respiratory acidosis
+
+E21
+
+P0
+
+P28 P29 P32
+
+P36
+
+P115
+
+P119
+
+P120
+
+P128
+
+Delivery Rat pups
+
+Cued Trials 4 trials per day
+
+Place Trials 2 blocks per day 2 trials per block
+
+Place Trials 1 block per day 2 trials perblock
+
+Learning to Reach Criterion Test
+
+Anesthetic Exposures Pregnant rats at gestation day 21
+
+Probe trial after last Place Trial
+
+Probe trial after last Place Trial
+
+Fig. 2. Schematic time-line of Morris Water Maze tests paradigm. E21, pregnant rats at gestational day 21; P0, postnatal day 0.
+
+945
+
+946
+
+Y. Li et al. / Neuropharmacology 53 (2007) 942e950
+
+(PaCO2 increased from 32 to 55 mmHg) and hypoxia (PaO2 decreased from 179 to 82 mmHg). When the isoflurane con- centration was reduced to 1.3%, for up to 6 h (2, 4 and 6 h), there were no significant changes in any of the ABG variables as compared to the controls (Table 1). MABP decreased slightly beginning at 2 h compared to the same animal’s base- line, but was not significantly different from the control group. Therefore, 1.3% isoflurane for 6 h was used in the subsequent formal study. In addition, there were no significant differences in the blood glucose levels before and after exposure in both the control group (113 (cid:3) 28 mg/dl vs. 116 (cid:3) 29 mg/dl; n ¼ 4; P > 0.05) and the 1.3% isoflurane treatment group (117 (cid:3) 17 mg/dl vs. 118 (cid:3) 14 mg/dl; n ¼ 8; P > 0.05). In the behavioral study, there were no significant differences between the two groups on growth rate measured by weight gain in rats from postnatal day 0 (P0) to P28 (data not shown). The concentration of isoflurane in the brain of a pregnant rat was 0.42 mmol/g after exposure to 1.3% isoflurane for 6 h, which was indistinguishable from that of the fetal brain (0.40 mmol/g) measured at the same time.
+
+3.2. Isoflurane inhibited spontaneous neuronal apoptosis in fetus brains
+
+We determined the degree of apoptosis by counting caspase- 3 positive and TUNEL-positive cells in different brain regions at 2 h and 18 h after isoflurane treatment and then at postnatal day 5. There was spontaneous apoptosis represented by caspase-3 positive or TUNEL positive cells in the developing fetal brain (Figs. 3 and 4). The caspase-3 positive cells were concentrated in the dorsal midline of the fetal brain along its rostralecaudal axis. There were no significant differences bet- ween control and treatment groups in the areas of hippocampus CA1 region and the retrosplenial cortex (data not shown) deter- mined at 2, 18 h and P5. Compared to the sham control group, 1.3% isoflurane treatment significantly decreased spontaneous apoptosis determined by the density of apoptotic cells in both the hippocampus CA1 region and in retrosplenial cortex at 2 h after treatment, but no differences were seen at 18 h after treatment or at P5 (Fig. 3B,C and 4B,C). Isoflurane treatment significantly decreased the density of caspase-3 and TUNEL positive cells by 80% (P < 0.001) and 81% (P < 0.001) respec- tively in the hippocampal CA1 region (Fig. 3B,C) and by 82% (P < 0.001) and 87% (P < 0.01) respectively in retrosplenial cortex (Fig. 4B,C) at 2 h after isoflurane treatment.
+
+reference learning ability in the same animals using the place tri- als, the escape latency to platform was analyzed with two-factor ANOVAwith treatment as a between subjects factor and block as a repeated measure. This analysis yields a main difference in block (P < 0.0001 at P32e36 and P115e119). However, nei- ther the main effect of treatment nor the interaction between treatment and block were significant at P32e36 or at P115e 119 (Fig. 5A,B). The results indicated that the performance dur- ing the place trials improved as training progressed but the there was no significant difference between the two groups at juvenile (P32e36) or at adult (P115e119) ages. We further tested the learning ability in the same adult rats using a more rigorous pro- tocoldthe learning to reach criterion test. They all performed equally well in learning as shown in the numbers of platform reached (Fig. 5C) and took the same number of trials to reach a criterion at each platform (Fig. 5D), suggesting a similar learn- ing ability between the two groups. After the place test, the same postnatal rats were used in a probe test to determine memory re- tention. The juvenile postnatal rats born to the mothers treated with 1.3% isoflurane for 6 h had significantly improved reten- tion of memory (P < 0.05) by spending a greater percentage of time swimming in the probe quadrant as compared with the corresponding control animals (Fig. 6). The swim speed in both groups was not different (data not shown), further indicat- ing the improvement of retention memory was not caused by the rats’ swimming ability as showed in cued trials. However, when the probe test was repeated in adult rats (P119), there was no sig- nificant difference in the percentage of time spent in the probe quadrant between the two groups (Fig. 6). These data suggest that any improvement in memory during juvenile age did not last into the adult age.
+
+4. Discussion
+
+Our initial hypothesis was that isoflurane exposure during pregnancy would cause apoptosis in the fetal brain and subse- quent postnatal memory and learning disabilities. This hypoth- esis was based on recent work which showed isoflurane exposure during early postnatal development resulted in an in- crease in apoptosis and subsequent behavioral impairment (Jevtovic-Todorovic et al., 2003). In contrast, we found that isoflurane exposure during late pregnancy transiently inhibited spontaneous apoptosis in the fetal brain and does not impair memory and learning in the juvenile or adult rat.
+
+3.3. Effect of fetal exposure to isoflurane on memory and learning
+
+Using the MWM, we examined the effect of prenatal isoflur- ane exposure on the memory and learning ability in postnatal rats at different ages. In the cued trials, there was no significant difference in the latency of swimming to a visible platform be- tween the control group of 26 postnatal rats from 7 pregnant mothers (n ¼ 7) and the isoflurane treatment group of 31 postna- tal rats from eight pregnant mothers (n ¼ 8) (18.5 (cid:3) 1.86 s vs. 18 (cid:3) 2.22 s; P > 0.05). When we compared the spatial
+
+The fact that isoflurane did not induce apoptosis in the pre- natal rat brain is consistent with a recent study (McClaine et al., 2005), showing no evidence for a neurotoxic effect of the com- bination of isoflurane/midazolam/sodium thiopental exposure to fetal sheep. Spontaneous apoptosis in the fetal developing brains may be a normal process that shapes the brains as it ma- tures. It seemed that spontaneous apoptosis significantly de- creased as the brain matured especially at postnatal day 5 (Figs. 3 and 4), which was consistent with another report (White and Barone, 2001). The inhibition of spontaneous apoptosis in the fetus developing brains by isoflurane determined 2 h after treatment was not expected. Although the significance of the transient inhibition of spontaneous apoptosis by isoflurane is
+
+Y. Li et al. / Neuropharmacology 53 (2007) 942e950
+
+A
+
+(a)
+
+Control, Caspase-3
+
+(b)
+
+1.3% Isoflurane, Caspase-3
+
+(c)
+
+Control, TUNEL
+
+(d)
+
+1.3% Isoflurane, TUNEL
+
+B
+
+2
+
+m m s
+
+/
+
+l l
+
+250
+
+200
+
+***
+
+Control
+
+1.3% Iso
+
+C
+
+2
+
+m m s
+
+/
+
+250
+
+200
+
+***
+
+Control
+
+1.3% Iso
+
+e C e v
+
+i t i
+
+150
+
+l l
+
+e C e v
+
+150
+
+s o P 3 - e s a p s a C
+
+100
+
+50
+
+i t i
+
+s o P L E N U T
+
+100
+
+50
+
+0
+
+0
+
+2h
+
+18h Hippocampus CA1
+
+P5
+
+2h
+
+18h Hippocampus CA1
+
+P5
+
+Fig. 3. In utero isoflurane inhibited spontaneous apoptosis in the hippocampus CA1 determined 2 h after treatment. (A) Arrows indicate caspase-3 positive cells and TUNEL-labeled cells in the hippocampus CA1 region of normal control fetal brains (Aa and Ac) or 2 h after isoflurane treatment (Ab and Ad) at E21. (B and C). The comparison between the density of caspase-3 positive cells (B) and TUNEL-positive cells (C) in the hippocampus CA1 region at different times after iso- flurane treatment. Data represent mean (cid:3) SE of 12e14 fetus rat brains from 5e6 pregnant mothers in either the control group or the isoflurane treatment group at 2 and 18 h after isoflurane treatment (see Fig. 1 for the detailed distribution of rats). Seven postnatal rats from 7 pregnant mothers (n ¼ 7) and 8 postnatal rats from 8 pregnant mothers (n ¼ 8) from the behavioral study were used for the neurodegeneration study at P5 (see Fig. 1). Data represent mean (cid:3) SE. ***P < 0.001 versus control. Iso, isoflurane. Scale bar 50 mm.
+
+unknown from this study, we speculate this will not affect brain development significantly. Our observation of postnatal rats in the behavioral study did not note obvious physical or behavioral differences between control and isoflurane treatment groups, except the transient memory improvement in the isoflurane treated rats. Nevertheless, the results from this study suggest
+
+that isoflurane exposure to pregnant rats does not induce apo- ptosis in the developing fetal brain and does not impair the memory and learning of their offspring.
+
+Our results would appear to be at odds with those observed by Jevtovic-Todorovic et al. (2003). However, in that study, ne- onates and not fetuses were exposed to isoflurane, and the
+
+947
+
+948
+
+Y. Li et al. / Neuropharmacology 53 (2007) 942e950
+
+A
+
+(a)
+
+Control, Caspase-3
+
+(b)
+
+1.3% Isoflurane, Caspase-3
+
+(c)
+
+Control, TUNEL
+
+(d)
+
+1.3% Isoflurane, TUNEL
+
+B
+
+35
+
+Control
+
+C
+
+35
+
+Control
+
+2
+
+m m / s l l e C e v i t i
+
+30
+
+25
+
+20
+
+***
+
+1.3% Iso
+
+2
+
+m m / s l l e C e v i t i
+
+30
+
+25
+
+20
+
+**
+
+1.3% Iso
+
+s o P 3 - e s a p s a C
+
+15
+
+10
+
+5
+
+s o P L E N U T
+
+15
+
+10
+
+5
+
+0
+
+0
+
+2h
+
+18h Retrosplenial Cortex
+
+P5
+
+2h
+
+18h Retrosplenial Cortex
+
+P5
+
+Fig. 4. Isoflurane inhibited spontaneous apoptosis in the retrosplenial cortex of fetal brains 2 h after treatment. (A) Arrows indicate caspase-3 positive cells and TUNEL-labeled cells in the retrosplenial cortex of normal control fetal brains (Aa and Ac) or 2 h after isoflurane treatment (Ab and Ad) at E21. The density of caspase-3 positive cells (B) and TUNEL-positive cells (C) in the retrosplenial cortex area at different times after isoflurane treatment were compared between the control and 1.3% isoflurane treatment groups. Data represent mean (cid:3) SE of 12e14 postnatal rats from 5e6 pregnant mothers in either control group or the iso- flurane treatment group at 2 and 18 h after isoflurane treatment (see Fig. 1 for the detailed distribution of rats). Seven postnatal rats from 7 pregnant mothers (n ¼ 7) and 8 postnatal rats from 8 pregnant mothers (n ¼ 8) from the behavioral study were used for the neurodegeneration study at P5 (see Fig. 1). Data represent mean (cid:3) SE. ***P < 0.001, **P < 0.01, compared to control. Iso, isoflurane. Scale bar 50 mm.
+
+Y. Li et al. / Neuropharmacology 53 (2007) 942e950
+
+A
+
+50
+
+Control
+
+40B
+
+Control
+
+) s d n o c e S
+
+( y c n e t a L
+
+40
+
+30
+
+20
+
+10
+
+1.3% Iso
+
+) s d n o c e S
+
+(
+
+y c n e t a L
+
+30
+
+20
+
+10
+
+1.3% Iso
+
+0
+
+0
+
+0
+
+1
+
+4 Day of Test (P32-36)
+
+2
+
+3
+
+5
+
+0
+
+1
+
+4 Day of Test (P115-119)
+
+2
+
+3
+
+5
+
+10C m r o f t a P d e n r a e L f o r e b m u N
+
+8
+
+l
+
+6
+
+4
+
+2
+
+D
+
+a
+
+i r e t i r C h c a e R o t
+
+s l a i r T
+
+10
+
+8
+
+6
+
+4
+
+2
+
+Control
+
+1.3% Iso
+
+0
+
+control
+
+1.3% Iso
+
+0
+
+0
+
+1
+
+2
+
+3 platform location
+
+4
+
+5
+
+6
+
+7
+
+8
+
+9
+
+Fig. 5. Isoflurane in utero did not affect postnatal learning ability in juvenile or adult rats. Spatial learning and memory performance were determined using the Morris Water Maze place test paradigm in postnatal juvenile (A) and adult (B) rats and using the learning to reach criteria test in the P120e128 adult (C and D). Data represent mean (cid:3) SE of 26 postnatal rats from 7 pregnant mothers (n ¼ 7) in the control group or 31 postnatal rats from 8 pregnant mothers (n ¼ 8) in the isoflurane treatment group. Iso, isoflurane.
+
+interpretation was that the vulnerability of isoflurane-induced apoptosis in the developing brains is correlated to the period of synaptogenesis (Jevtovic-Todorovic et al., 2003; Yon et al., 2005). If true, then the fetal rat is not expected to be as vulner- able as the neonatal rat, which is consistent with the results of
+
+50
+
+
+
+Control
+
+t n a r d a u q e b o r p n
+
+40
+
+30
+
+20
+
+1.3% Iso
+
+i
+
+e m
+
+i t
+
+%
+
+10
+
+0
+
+P36
+
+P119
+
+Day of Test
+
+Fig. 6. Isoflurane significantly increased spatial retention memory in postnatal juvenile but not adult rats. Probe test was performed at postnatal day 36 (P36) and 119 (P119) after the last place trial. Data represent mean (cid:3) SE of 26 post- natal rats born from 7 pregnant mothers (n ¼ 7) in the control group or 31 postnatal rats born from 8 pregnant mothers (n ¼ 8) in the isoflurane treatment group. *P < 0.05 compared to control. Iso, isoflurane.
+
+our study. However, it should be noted that although vulnerabil- ity to isoflurane-induced apoptosis seems to coincide with the period of synaptogenesis (Hansen et al., 2004; Ikonomidou et al., 2001; Jevtovic-Todorovic et al., 2003), there exists no di- rect evidence showing that synaptogenesis is itself altered, or is causally linked to the subsequent cognitive changes. Toxic ef- fects of isoflurane have also been found in the adult and aged brains (Culley et al., 2003, 2004), when of course little in the way of synaptogenesis is occurring.
+
+Isoflurane has long been considered to be cytoprotective against ischemia in the heart and brain (Sakai et al., 2007; Warner, 2000). In previous studies, we and others (Kudo et al., 2001; Wei et al., 2005) have shown that the concentration and time required for isoflurane to induce apoptosis in cultured neurons was greater than that used clinically in patients. It is possible that sub-apoptotic exposure to isoflurane induces pro- tection, analogous to that induced by hypoxia. Accordingly, our preliminary unpublished data have shown that preconditioning of rat primary cortical neurons with 2 MAC isoflurane for 1 h abolished the neurotoxicity induced by a subsequent exposure to 2 MAC isoflurane for 24 h. Thus, it is possible that our use of isoflurane at low ‘‘clinical’’ concentration in the pregnant rat may precondition the fetal brain against spontaneous apo- ptosis in the current study. Therefore, it remains possible that the higher anesthetic concentrations used in fetal surgery (w3% isoflurane) may produce neurotoxicity in the fetal brain.
+
+949
+
+950
+
+Y. Li et al. / Neuropharmacology 53 (2007) 942e950
+
+Other limitations of this study are that we did not expose the mother/fetus to isoflurane at other time points during the pregnancy, so it is possible that we missed the time point when the fetus might be more vulnerable to isoflurane neuro- toxicity. Further, we avoided tracheal intubation in this study in an attempt to minimize confounding variables. This pre- vented us from testing the effects of isoflurane at concentra- tions up to 3% often used during fetal surgery in humans (Cauldwell, 2002; Myers et al., 2002).
+
+cleaved caspase 3. Journal of Histochemistry and Cytochemistry 50, 449e454.
+
+Hansen, H.H., Briem, T., Dzietko, M., Sifringer, M., Voss, A., Rzeski, W., Zdzisinska, B., Thor, F., Heumann, R., Stepulak, A., Bittigau, P., Ikonomidou, C., 2004. Mechanisms leading to disseminated apoptosis fol- lowing NMDA receptor blockade in the developing rat brain. Neurobiol- ogy of Disease 16, 440e453.
+
+Ikonomidou, C., Bittigau, P., Koch, C., Genz, K., Hoerster, F., Felderhoff- Mueser, U., Tenkova, T., Dikranian, K., Olney, J.W., 2001. Neurotransmit- ters and apoptosis in the developing brain. Biochemical Pharmacology 62, 401e405.
+
+In summary, prenatal exposure to isoflurane at a concentra- tion commonly used for the maintenance of general anesthesia during late pregnancy in rats does not appear to be neurotoxic to the fetal brain and does not impair memory and learning in the postnatal juvenile or adult rat.
+
+Acknowledgments
+
+Jevtovic-Todorovic, V., Hartman, R.E.,
+
+Izumi, Y., Benshoff, N.D., Dikranian, K., Zorumski, C.F., Olney, J.W., Wozniak, D.F., 2003. Early ex- posure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. Journal of Neu- roscience 23, 876e882.
+
+Kim, H., Oh, E., Im, H., Mun, J., Yang, M., Khim, J.Y., Lee, E., Lim, S.H., Kong, M.H., Lee, M., Sul, D., 2006. Oxidative damages in the DNA, lipids, and proteins of rats exposed to isofluranes and alcohols. Toxicology 220, 169e178.
+
+We acknowledge discussions with Drs Roderic Eckenhoff, Randall Pittman and Maryellen Eckenhoff and the technical support of Dr Min Li, University of Pennsylvania. We thank Dr Alex Loeb for editorial assistance. This study was supported by March of Dimes Birth Defects Foundation Research Grant (12-FY05-62, PI: Huafeng Wei), and partially Institute of General Medical supported by the National Science (NIGMS) K08 grant (1-K08-GM-073224-01, PI: Huafeng Wei), and the Research Fund at the Department of Anesthesiology and Critical Care, University of Pennsylvania (PI: Huafeng Wei).
+
+Kudo, M., Aono, M., Lee, Y., Massey, G., Pearlstein, R.D., Warner, D.S., 2001. Effects of volatile anesthetics on N-methyl-D-aspartate excitotoxicity in primary rat neuronaleglial cultures. Anesthesiology 95, 756e765.
+
+Kvolik, S., Glavas-Obrovac, L., Bares, V., Karner, I., 2005. Effects of inhala- tion anesthetics halothane, sevoflurane, and isoflurane on human cell lines. Life Sciences 77, 2369e2383.
+
+Loop, T., Dovi-Akue, D., Frick, M., Roesslein, M., Egger, L., Humar, M., Hoetzel, A., Schmidt, R., Borner, C., Pahl, H.L., Geiger, K.K., Pannen, B.H., 2005. Volatile anesthetics induce caspase-dependent, mito- chondria-mediated apoptosis in human T lymphocytes in vitro. Anesthesi- ology 102, 1147e1157.
+
+Matsuoka, H., Kurosawa, S., Horinouchi, T., Kato, M., Hashimoto, Y., 2001. Inhalation anesthetics induce apoptosis in normal peripheral lymphocytes in vitro. Anesthesiology 95, 1467e1472.
+
+References
+
+Cauldwell, C.B., 2002. Anesthesia for fetal surgery. Anesthesiology Clinics of
+
+McClaine, R.J., Uemura, K., de la Fuente, S.G., Manson, R.J., Booth, J.V., White, W.D., Campbell, K.A., McClaine, D.J., Benni, P.B., Eubanks, W.S., Reynolds, J.D., 2005. General anesthesia improves fetal cerebral oxygenation without evidence of subsequent neuronal injury. Journal of Cerebral Blood Flow and Metabolism 25, 1060e1069.
+
+North America 20, 211e226.
+
+Myers, L.B., Cohen, D., Galinkin, J., Gaiser, R., Kurth, C.D., 2002. Anaesthe-
+
+Chang, Y.C., Chou, M.Y., 2001. Cytotoxicity of halothane on human gingival
+
+sia for fetal surgery. Paediatric Anaesthesia 12, 569e578.
+
+fibroblast cultures in vitro. Journal of Endodontics 27, 82e84.
+
+Paxinos, G., Tork, I., Teccot, L.H., Valentino, K., 1990. Atlas of the Develop-
+
+Chen, G., Chen, K.S., Knox, J., Inglis, J., Bernard, A., Martin, S.J., Justice, A., McConlogue, L., Games, D., Freedman, S.B., Morris, R.G., 2000. A learn- ing deficit related to age and beta-amyloid plaques in a mouse model of Alzheimer’s disease. Nature 408, 975e979.
+
+ing Rat Brain. Academic Press.
+
+Sakai, H., Sheng, H., Yates, R.B., Ishida, K., Pearlstein, R.D., Warner, D.S., 2007. Isoflurane provides long-term protection against focal cerebral ische- mia in the rat. Anesthesiology 106, 92e99.
+
+Culley, D.J., Baxter, M., Yukhananov, R., Crosby, G., 2003. The memory ef- fects of general anesthesia persist for weeks in young and aged rats. Anes- thesia and Analgesia 96, 1004e1009.
+
+Culley, D.J., Baxter, M.G., Yukhananov, R., Crosby, G., 2004. Long-term impairment of acquisition of a spatial memory task following isoflurane- nitrous oxide anesthesia in rats. Anesthesiology 100, 309e314.
+
+Dobbing, J., Sands, J., 1979. Comparative aspects of the brain growth spurt.
+
+Early Human Development 3, 79e83.
+
+Warner, D.S., 2000. Isoflurane neuroprotection e a passing fantasy, again? An-
+
+esthesiology 92, 1226e1228.
+
+Wei, H., Kang, B., Wei, W., Liang, G., Meng, Q.C., Li, Y., Eckenhoff, R.G., 2005. Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently. Brain Research 1037, 139e147.
+
+White, L.D., Barone, S., 2001. Qualitative and quantitative estimates of apo- ptosis from birth to senescence in the rat brain. Cell Death and Differen- tiation 8, 345e356.
+
+Eckenhoff, R.G., Johansson, J.S., Wei, H., Carnini, A., Kang, B., Wei, W., Pidikiti, R., Keller, J.M., Eckenhoff, M.F., 2004. Inhaled anesthetic en- hancement of amyloid-beta oligomerization and cytotoxicity. Anesthesiol- ogy 101, 703e709.
+
+Wise-Faberowski, L., Zhang, H., Ing, R., Pearlstein, R.D., Warner, D.S., 2005. Isoflurane-induced neuronal degeneration: an evaluation in orga- notypic hippocampal slice cultures. Anesthesia and Analgesia 101, 651e657.
+
+Gavrieli, Y., Sherman, Y., Bensasson, S.A., 1992. Identification of pro- grammed cell-death in situ via specific labeling of nuclear-DNA fragmen- tation. Journal of Cell Biology 119, 493e501.
+
+Goldsmith, M.F., Straus, S.E., Kupfer, C., Lenfant, C., Collins, F., Hodes, R.J., Gordis, E., Fauci, A.S., Alexander, D., Battey, J.F., Slavkin, H.C., Leshner, A.I., Olden, K., Hyman, S.E., Fischbach, G.D., 1999. 2020 vi- sion: NIH heads foresee the future. Journal of the American Medical As- sociation 282, 2287e2290.
+
+Gown, A.M., Willingham, M.C., 2002. Improved detection of apoptotic cells in archival paraffin sections: immunohistochemistry using antibodies to
+
+Xie, Z., Dong, Y., Maeda, U., Alfille, P., Culley, D.J., Crosby, G., Tanzi, R.E., 2006. The common inhalation anesthetic isoflurane induces apoptosis and increases amyloid beta protein levels. Anesthesiology 104, 988e994. Xie, Z., Dong, Y., Maeda, U., Moir, R.D., Xia, W., Culley, D.J., Crosby, G., Tanzi, R.E., 2007. The inhalation anesthetic isoflurane induces a vicious cycle of apoptosis and amyloid beta-protein accumulation. Journal of Neu- roscience 27, 1247e1254.
+
+Yon, J.H., Daniel-Johnson, J., Carter, L.B., Jevtovic-Todorovic, V., 2005. An- esthesia induces neuronal cell death in the developing rat brain via the in- trinsic and extrinsic apoptotic pathways. Neuroscience 135, 815e827.

new_pdfs/10.1016_j.ntt.2020.106890.txt

@@ -0,0 +1,495 @@
+Neurotoxicology and Teratology 80 (2020) 106890
+
+Contents lists available at ScienceDirect
+
+Neurotoxicology and Teratology
+
+journal homepage: www.elsevier.com/locate/neutera
+
+Regions of the basal ganglia and primary olfactory system are most sensitive to neurodegeneration after extended sevoflurane anesthesia in the perinatal rat
+
+☆
+
+T
+
+Susan M. Burks, John F. Bowyer, Jennifer L. Walters, John C. Talpos
+
+⁎
+
+National Center for Toxicological Research, 3900 NCTR Rd, Jefferson, AR 72079, United States of America
+
+A R T I C L E I N F O
+
+A B S T R A C T
+
+Keywords: Neurotoxicity Development Fluoro-Jade Hypoxia Indusium griseum
+
+Extended general anesthesia early in life is neurotoxic in multiple species. However, little is known about the temporal progression of neurodegeneration after general anesthesia. It is also unknown if a reduction in natural cell death, or an increase in cell creation, occurs as a form of compensation after perinatal anesthesia exposure. The goal of this study was to evaluate markers of neurodegeneration and cellular division at 2, 24, or 72 h after sevoflurane (Sevo) exposure (6 h) in fully oxygenated postnatal day (PND) 7 rats. Neurodegeneration was ob- served in areas throughout the forebrain, while the largest changes (fold increase above vehicle) were observed in areas associated with either the primary olfactory learning pathways or the basal ganglia. These regions included the indusium griseum (IG, 25-fold), the posterior dorso medial hippocampal CA1 (17-fold), bed nucleus of the stria terminalis (Bed Nuclei STM, 5-fold), the shell of the nucleus accumbens (Acb, 5-fold), caudate/ putamen (CPu, 5-fold), globus pallidus (GP, 9-fold) and associated thalamic (11-fold) and cortical regions (5- fold). Sevo neurodegeneration was minimal or undetectable in the ventral tegmentum, substantia nigra, and most of the hypothalamus and frontal cortex. In most brain regions where neurodegeneration was increased 2 h post Sevo exposure, the levels returned to < 4-fold above control levels by 24 h. However, in the IG, CA1, GP, anterior thalamus, medial preoptic nucleus of the hypothalamus (MPO), anterior hypothalamic area (AHP), and the amygdaloid nuclei, neurodegeneration at 24 h was double or more than that at 2 h post exposure. Anesthesia exposure causes either a prolonged period of neurodegeneration in certain brain regions, or a distinct secondary degenerative event occurs after the initial insult. Moreover, regions most sensitive to Sevo neurodegeneration did not necessarily coincide with areas of new cell birth, and new cell birth was not consistently affected by Sevo. The profile of anesthesia related neurotoxicity changes with time, and multiple mechanisms of toxicity may exist in a time-dependent fashion.
+
+1. Introduction
+
+Early in life, the brain of mammalian species undergoes a period of rapid growth known as the brain growth spurt (BGS) (Dobbing and Sands, 1979; Workman et al., 2013). During this time, the brain is thought to be more vulnerable to toxic insults such as general an- esthesia exposure (Eriksson, 1997; Ikonomidou, 2009; Rice and Barone, 2000). Anesthesia related neurotoxicity in animal models of human neonatal brain development was first established by Olney and collea- gues after ketamine or nitrous oxide exposure in the rat (Jevtovic- Todorovic et al., 2001). Since then, markers of neurotoxicity have been anesthetic. This reported with every FDA approved general
+
+phenomenon has been observed in multiple species including rats (Jevtovic-Todorovic et al., 2000; Scallet et al., 2004), mice (Istaphanous et al., 2011; Zheng et al., 2013b), nonhuman primates (Brambrink et al., 2010; Paule et al., 2011; Slikker Jr. et al., 2007; Talpos et al., 2019), nematodes (Gentry et al., 2013; Na et al., 2017), and zebrafish (Guo et al., 2015; Kanungo et al., 2013). The clinical relevance of these findings has been debated (Vutskits and Culley, 2019). However, sev- eral large scale retrospective studies have shown that multiple ex- posures to anesthesia within the first two years of life is associated with an increased incidence of learning disabilities and attention-deficit hyperactivity disorder (Flick et al., 2011; Sprung et al., 2012; Wilder et al., 2009), as well as an increased use of psychotropic drugs to treat a
+
+☆
+
+The information in these materials is not a formal dissemination of information by the Food and Drug Administration (FDA) and does not represent agency
+
+position or policy.
+
+⁎
+
+Corresponding author. E-mail address: John.Talpos@fda.hhs.gov (J.C. Talpos).
+
+https://doi.org/10.1016/j.ntt.2020.106890 Received 13 December 2019; Received in revised form 10 April 2020; Accepted 29 April 2020
+
+Available online 12 May 2020 0892-0362/ Published by Elsevier Inc.
+
+S.M. Burks, et al.
+
+variety of conditions (Ing et al., 2020). It seems that early life exposure to general anesthesia increases the likelihood of individuals having cognitive differences during development (Ing and Brambrink, 2019). Perinatal anesthesia related neurotoxicity is well established in animal models of human use (Brambrink et al., 2012; Brambrink et al., 2010; Ikonomidou, 2009; Jevtovic-Todorovic et al., 2001; Paule et al., 2011; Slikker Jr. et al., 2007; Talpos et al., 2019; Walters and Paule, 2017). However, the temporal progression of this neurotoxicity has not been described. For example, the pattern of neurodegeneration (post- exposure) that develops over time and across brain regions has not been determined. Also, the temporal aspects of any “compensatory” effects that involve increased birth of new cells in the days after insult are unknown. Addressing these knowledge gaps will help in understanding the mechanisms behind anesthesia related neurotoxicity and in de- termining the clinical relevance of animal models to human perinatal anesthesia exposure.
+
+More than 100 laboratory animal studies have described the effects of prolonged anesthetic exposure to neonates in various species. While several studies evaluate toxicity in multiple brain structures (Deng et al., 2014; Lee et al., 2017; Perez-Zoghbi et al., 2017; Rizzi et al., 2008), most focus on one or two areas of interest. This makes it difficult to determine which areas of the brain are most vulnerable to anesthesia related neurotoxicity and to resolve discrepancies within the literature (Brambrink et al., 2012; Brambrink et al., 2010; Zhang et al., 2016; Zou et al., 2009). Moreover, by focusing on specific brain regions, we cannot determine if neurotoxicity is caused by anesthetic drugs acting on in- dividual cells or is the result of disrupted activity at a network level.
+
+Another obstacle in understanding the nature of anesthesia-related neurotoxicity is the focus on a single time point to assess neurodegen- eration. Most studies quantify neurodegeneration several hours after the ending of exposure. In some ways, this approach is logical as many markers of neurotoxicity, such as Fluoro-jade C (FJC) or caspase-based stains, are ephemeral in nature. However, assessing markers of toxicity at a single timepoint assumes neurodegeneration happens at the same pace and via a single mechanism throughout the brain. A lack of ap- propriate neuronal stimulation can increase apoptosis (Kilb et al., 2011; Lewin and Barde, 1996). Accordingly, a “second wave” of neurode- generation in brain regions heavily innervated by areas effected in the initial insult might be expected at later time points. Conversely, internal mechanisms to ameliorate early increases in neurodegeneration within a region may only be observable at later time points. For example, a decrease in baseline apoptosis or neurogenesis may be observed in animals exposed to anesthesia to compensate for earlier damage (Jiang et al., 2016). There may be a decrease in the number of newborn cells (neuronal or glial) in regions with high levels of neurodegeneration due to loss of neurotrophic factor(s) that would have been released by dying neurons (Lewin and Barde, 1996). It is unlikely that moments where the highest level of neurodegeneration are observed are optimal to detect endogenous compensation.
+
+Sevo is currently the most frequently used general anesthetic in humans, and its neurotoxic potential has been well described (Amrock et al., 2015; Brioni et al., 2017; Delgado-Herrera et al., 2001; Fang et al., 2012; Lerman and Johr, 2009; Pellegrini et al., 2014; Walters and Paule, 2017; Zheng et al., 2013a). Accordingly, the primary goal of this study was to determine regional and temporal patterns of acute neu- rodegeneration that occurs in the rat forebrain following perinatal an- esthesia exposure in fully oxygenated animals. To do this, PND 7 rats were exposed to Sevo (2.5% in 75% oxygen/25% nitrogen carrier gas for 6 h). Animals were sacrificed at 2, 24, or 72 h after cessation of Sevo. This enabled the determination of how markers of neurodegen- eration (FJC) differed by region and changed with time. Throughout this study our primary endpoint was on the incidence of FJC positive cells. We selected FJC because it is one of the most commonly used methods to study degenerating neurons, making it an excellent mark of neurotoxic insult. It is effective at highlighting areas of the brain that have been impacted by general anesthesia and serves to effectively
+
+2
+
+Neurotoxicology and Teratology 80 (2020) 106890
+
+demonstrate areas of potential interest. However, an increased in- cidence in neurodegeneration is only one of the many changes that have been observed in the brains of animals exposed to general anesthesia early in life. Early life exposure has been shown to decrease dendritic spine densities (Briner et al., 2011), alter development of GABAeric networks (Osterop et al., 2015), influence neurogenesis (Stratmann et al., 2010), alter neurotransmitter receptor densities (Zurek et al., 2014), and induce neuroinflammation (Zheng et al., 2013a). Any one of these changes may cause the observed changes in cognition and beha- vior that have been reported.
+
+2. Methods
+
+2.1. Animals
+
+Pregnant Sprague-Dawley rats (Charles Rivers, USA) arrived on gestational day 5. Litters were culled to four males and four females on PND 4. A within-litter treatment design was used to evaluate the effect of Sevo. Four animals, 2 males and 2 females, were selected from each litter. One animal of each sex was randomly assigned to the Sevo group with the other being assigned to the vehicle condition. Three animals per sex were assigned to each treatment / timepoint combination (N = 72). Multiple stains were used on tissue from the same animal. Two animals were removed from the 72 h Sevo group. One animal was removed for failing to meet inclusion criteria for oxygenation (no hy- poxic animals were included in the study), and a second animal died between exposure and sacrifice. Rats were housed in a light (12 h/12 h light/dark cycle) and temperature (22 ± 2 °C) controlled vivarium and given free access to food and water (NIH41 laboratory animal diet, Envigo, Madison, WI). All animal procedures were carried out in ac- cordance with the Guide for the Care and Use of Laboratory Animals. Animal use and procedures were approved by the NCTR Institutional Animal Care and Use Committee (IACUC), which has full NIH-OLAW accreditation. Animals were housed in the NCTR facility in isolator top boxes with wooden chip bedding and ad libitum food and water.
+
+2.2. Study design
+
+On PND 7, rats were exposed to vehicle gas alone (75% oxygen/25% nitrogen) or 2.5% Sevo (in vehicle gas) for 6 h. During anesthesia ex- posure, each pup was placed in an individual airtight acrylic chamber and the selected gas mixture was delivered at a flow rate of 0.75–1 L/ min (Walters et al., 2020). The concentration of Sevo was set using a commercial gas analyzer (Riken, USA). Surface body temperature was collected prior to and every 2 h following the start of Sevo exposure using an infrared thermometer (Micro-Epsilon, Ortenburg, Germany). Heating plates located beneath each chamber were used to maintain body temperatures at baseline levels. In addition, arterial oxygen sa- turation (SpO2), breath rate, heart rate, and pulse distention were monitored in each pup continuously using a pulse oximeter (Starr Life Sciences Corp, USA). The average SPO2 value was calculated every 30 min (Supplemental Table 1); if an individual rat's SPO2 fell below 85% during any of the 30 min intervals, it was excluded from the study. Upon recovery, the pups were removed from the chambers, rubbed with bedding material from their home cage, and returned to their dams. Control animals were treated the same as the experimental group ex- cept they were not exposed to anesthesia and there SPO2 was not monitored.
+
+Pups were sacrificed 2 h (PND 7), 24 h (PND 8), and 72 h (PND 10) after the cessation of Sevo or vehicle gas exposure. Briefly, the rats were deeply anesthetized with pentobarbital and transcardially perfused with 0.9% heparinized saline followed by 10% neutral buffered for- malin. Brains were removed and post-fixed in 10% neutral buffered formalin for 24 h, cryoprotected in 20% sucrose until they sank, and subsequently frozen on dry ice and stored at −80 °C. Tissue was cut into 30 μm thick coronal sections using a cryostat, stored in 0.08%
+
+S.M. Burks, et al.
+
+sodium azide in PBS for up to two weeks and then transferred to freezing solution (0.02 M phosphate buffer (pH 7.4) containing 25% (v/ v) glycerol and 30% (v/v) ethylene glycol) until processed for im- munohistochemistry or histology.
+
+2.3. FJC immunolabeling
+
+For FJC labeling, a modified method (Bowyer et al., 2018b; Schmued et al., 2005) was used. Briefly, sections of interest were re- moved from freezing solution and rinsed three times in 0.1 M phosphate buffer (PB, pH 7.4) for 1 min. Sections were then mounted on gelatin coated slides in 0.005 M PB (pH 7.4) and dried at 50 °C for 2 h. Sub- sequently, slides were immersed for: 3 min in basic alcohol, 2 min in 70% ETOH, 2 min in Millipore water, 11 min in 0.06% potassium permanganate, 2 min in Millipore water, 10 min in FJC (0.00001% in 0.1% glacial acetic acid), and three 2 min washes in Millipore water. Slides were then dried at 50 °C for 5–10 min, cleared with xylene for 1 min, and cover-slipped with DPX mounting media.
+
+2.4. Mki67 immunolabeling
+
+A buffer of 0.1 M PB (pH 7.4) containing 0.4% Triton X-100 was used in all the steps involving free floating sections agitated on an or- bital shaker. Sections containing regions of interest were initially wa- shed in buffer three times (15 min each) to remove excess freezing solution. After a 30 min pre-incubation in 4% normal goat serum, the sections were incubated in 4% serum and chicken polyclonal antibody to Mki67 (1:2000, EnCor Biotechnology, USA) for 1 to 2 h at room temperature followed by 18 to 24 h at 5 °C. Sections were then washed three times for 15 min and incubated in a biotinylated goat anti-chicken antibody (1:350, Invitrogen, USA) for 1 h at room temperature. The sections were then washed three times (15 min per wash) and incubated in Streptavidin TRITC (1:200, Jackson ImmunoResearch, USA) for 1 h. The sections were then washed three times (15 min per wash) and mounted on Superfrost Plus slides (Thermo Fisher Scientific, USA) and dried at room temperature for ≥12 h in the dark. Finally, the slides were cleared in xylene and cover-slipped with DPX mounting medium.
+
+2.5. NeuN immunolabeling
+
+Sections containing the four regions (IG, anterior CPu (CPua), anterior thalamus, and CA1) with the highest per mm2 levels of FJC labeled cells were immunolabeled with an antibody to NeuN in con- junction with DAB visualization. Sections were washed in 0.1 M PB (pH 7.4) for 15 min and then incubated in 0.1 M PB containing 0.05% H2O2 for 10 min to suppress the endogenous peroxidases. From this point on, except for the last step of 3,3′-diaminobenzidine (DAB) pro- cessing, incubation and washing solutions consisted of 0.1 M PB con- taining 0.25% Triton X-100. Sections were then washed three times for 5 min. Following a 20 min pre-incubation in 5% normal goat serum, the sections were incubated in rabbit anti-NeuN (1:1000, Abcam, USA) antibody for 18 to 24 h at room temperature. Sections were then wa- shed three times for 5 min and incubated in a biotinylated goat anti- rabbit antibody (1:300, Thermo Fisher Scientific, USA) for 2 h. The signal was then amplified using the avidin and biotinylated horseradish peroxidase macromolecular complex (Vector Laboratories, USA) and visualized with 0.5 mg/mL of DAB in Tris-HCl buffer. Sections were washed twice for 5 min in Tris-HCl, mounted, and dried on a slide warmer for ≥12 h. Finally, the slides were cleared in xylene and cover- slipped with DPX mounting medium.
+
+2.6. Thionine staining
+
+Thionine staining was performed to verify brain regions. Sections from regions where the highest levels of FJC staining were observed from PND 7, 8 and 10 were mounted from 0.1 M PB (pH 7.4) on
+
+3
+
+Neurotoxicology and Teratology 80 (2020) 106890
+
+Superfrost Plus slides (Thermo Fisher Scientific, USA) and dried at 55 °C for 15 min. They were then immersed in double distilled water for 4 min. Subsequently, the sections were immersed in a solution of 0.1% thionine acetate (Sigma-Aldrich, USA) in double distilled water for 8 min. The sections were then transferred through two washes of water (2 min each) followed by 70% ethanol in water (2 min), 95% ethanol (2 min) and 100% ethanol (2 min). The sections were then transferred to xylene for ≥2 min and cover-slipped as described above.
+
+2.7. Image capturing and analysis
+
+Imaging of brain tissue was conducted using a Nikon Eclipse Ni microscope equipped with digital cameras (Photometrics, USA; Nikon, USA). FJC, Mki67, NeuN, and thionine labeling were quantified in the somatosensory cortex, motor cortex, CPu, thalamus, CA1 region of the hippocampus, septum and amygdala at 10× magnification using NIS Elements AR automated software (Nikon, USA). Brain regions were defined in accordance with brain atlases for adult and neonatal rats (Paxinos and Watson, 2014; Ramachandra and Subramanian, 2011).
+
+2.8. Stereological analysis
+
+The brain regions in which the highest levels of FJC positive neu- rons were identified with the aid of adult and neonatal atlases as guides. Given the absence of a complete neonatal atlas, these locations were verified using thionine stained sections from the same regions that the FJC sections were taken to determine neurodegeneration from the pups sacrificed at PND 7, 8 and 10 [see Fig. 1]. Subsequent to identifying these regions, unbiased stereological estimates of positively im- munolabeled cells/structures were performed from images that were captured with Photometrics (fluorescent) or Nikon (brightfield) digital cameras using NIS elements AR software for analysis.
+
+Unless otherwise stated, six animals were used for each treatment condition; however, only 4 animals reached inclusion criteria under the 72 h Sevo condition. For each animal included, one instance of each region was utilized. When more than one instance of a region was available for an animal, the average count for that region, in that an- imal, was utilized for statistics. Therefore, N is reflective of the number of animals included in each treatment group. All FJC, NeuN and Mki67 cell counts represented are calculated from both hemispheres, and thus represent the region as a whole. Data were normalized by the total area of the counted region of interest and expressed as number of label positive cells per mm2. All animals were given a unique ID that was not indicative of treatment prior to analysis. The investigator who took images and conducted immunohistochemical analysis was unaware of treatment conditions and post-exposure intervals at the time of analysis. FJC images were taken at 10× using a FITC filter and Photometrics camera. FJC positive cells were counted in NIS Elements AR by re- stricting the area to ≥4μm2 and ≤ 75 μm2, MinFret ≥1.93 μm, and SumIntensity ≥102 and ≤ 2,876,455. Threshold settings were as fol- lows: Smooth 5×, Clean 2×, Fill holes ON, Separate 1×. Binning was set at 8. Mki67 images were taken at 20× using TRITC filter, ND4 filter, and Photometrics camera. Mki67 positive cells were counted in NIS Elements AR by restricting Area ≥ 24 μm2 and ≤ 300 μm2, Width ≥ 2.29 μm, and MeanIntensity ≥229 and ≤ 65,535. Threshold settings were as follows: Smooth OFF, Clean OFF, Fill holes OFF, Separate 4×. Binning was set at 8. NeuN images were taken at 10× using brightfield imaging and a Nikon camera. Images of NeuN positive cells in the CPua and anterior thalamus were first processed by a medium equalization accuracy strength of 30, followed by Fourier transform noise reduction at 0.880 and detail enhancement at 0.045 with average intensity maintained. NeuN positive cells were then counted in NIS Elements AR by restricting area ≥ 0.42 μm2 and ≤ 2276.45μm2, threshold set to intensity with the following set- tings: Smooth OFF, Clean 1×, Fill holes OFF, Separate 4×. Binning was set at 8. For the densely populated regions of CA1 and IG, to get
+
+S.M. Burks, et al.
+
+4
+
+Neurotoxicology and Teratology 80 (2020) 106890
+
+(caption on next page)
+
+S.M. Burks, et al.
+
+Neurotoxicology and Teratology 80 (2020) 106890
+
+Fig. 1. Coronal brain sections showing regions with highest levels of increased neurodegeneration after Sevo anesthesia. The left most column (A1, +2.76 from bregma through G1, −4.80 from bregma) shows coronal sections in adult rats (Paxinos and Watson, 2014) that correspond to coronal sections in the PND 7 (A2 to G2), PND 8 (A3 to G3) and PND 10 (A4 to G4). The gold color highlights superimposed on the adult sections correspond to the regions in the neonates where neurodegeneration was highest. The regions in neonates do not correspond perfectly with the adults, as seen in the septal, posterior cortical and midbrain regions. The lateral septum is not highlighted in gold because it occurs rostral to C1, at 0.24 mm from bregma, while the retromammillary decussation and ventral tegmental area (rostral), and substantia nigra, reticular part and dorsal tier are not shown because they are caudal of bregma −4.80 mm. Image G1 is a composite of images from the adult diencephalon (bregma −3.96 mm) and cortex / hippocampus (bregma −4.80). This was necessary to provide an adequate representation of the more caudal aspects of the PND 7–10 rat brain. Abbreviations for the gold highlighted brain regions of interest in A1 through G1 are: A29c-1 = more anterior region of the retrosplenial cortex A29c-2 = more posterior region of the retrosplenial cortex Acb = accumbens shell AHP = anterior hypothalamic area, posterior part AIV & LO = agranular insular cortex ventral + lateral orbital cortex Anterior Thalamus = VA, VL and intralaminar nuclei of the thalamus BMA & MeA = basomedial amygdaloid nucleus + medial amygdaloid nuclei ST = Bed Nuclei STM; lateral + medial division of bed nucleus of the stria terminalis CA1 = field CA1 of hippocampus CG Ctx = cingulate cortex CPua = caudate/putamen, ~ 2.16 mm bregma CPub = caudate/putamen, ~ −0.12 to −0.24 mm bregma DLG = dorsal lateral geniculate nucleus GP = lateral globus pallidus IG = indusium griseum, hippocampal rudiment M1 & M2 = primary + secondary motor cortex, layer I & II M2 = secondary motor cortex, layer I & II MPO = medial preoptic nucleus of the hypothalamus PMCo = posteromedial cortical amygdaloid nucleus VTA = retromammillary decussation + ventral tegmental area, rostral S1BF = primary somatosensory cortex, barrel field SNR & SNCD = substantia nigra, reticular part + dorsal tier.
+
+Fig. 2. Regions with the highest levels of FJC labeling at 2 h after Sevo. The average ± SEM of FJC positive cells after 2 h Sevo, normalized to 1 mm2 area, are displayed in de- creasing order. N = 6, however for the Bed Nuclei STM, AHP, and SNR & SNCD regions, N = 5 due to proces- sing/regional complications. As the sections used were 30 μm thick, the total number of degenerating neurons per 1 mm3 would be 40× that shown for each region. Abbreviations present in Fig. 2 are the same as used in Fig. 1. The blue background indicates that the region is associated with the primary olfactory learning system while the tan background indicates that the region is part of the basal ganglia and motor movement system. The arrow points to the values related to the SNR and SNCD. **P ≤ 0.002, ***P ≤ 0.0002, ****P ≤ 0.00002.
+
+accurate cellular discrimination of NeuN, images were first processed by Fourier transform noise reduction at 0.543 and detail enhancement at 0.034 with average intensity maintained. Subsequently, a green component contrast was applied with the following settings: Low = 0, High = 177, Gamma =5. Then the positive cells were counted using the same settings as for CPua and anterior thalamus.
+
+2.9. Statistical analysis
+
+Results are presented as mean ± SEM. The 2, 24, and 72 h groups were analyzed using separate two-way ANOVAs (region and treatment). Post-hoc analysis was performed using a Sidak's post-hoc test (alpha = 0.02), or unpaired t-tests, (effect of sacrifice interval; alpha = 0.05). All analyses were performed in GraphPad Prism version 6 (GraphPad Software, Inc.; USA). Due to processing/regional compli- cations, in certain circumstances N of 6 was not available. The Bed
+
+5
+
+S.M. Burks, et al.
+
+6
+
+Neurotoxicology and Teratology 80 (2020) 106890
+
+(caption on next page)
+
+S.M. Burks, et al.
+
+Neurotoxicology and Teratology 80 (2020) 106890
+
+Fig. 3. Visual presentation of FJC labeling in three regions with high levels of FJC labeling at 2 h after Sevo exposure. Representative micrographs of FJC labeling in the control and Sevo animals for the CPua, Acb, and M1 & M2 are shown 2 h after Sevo. Both high and low magnifications are shown; the red arrows point to the same location on the two different magnifications (indicated by the red magnification bars). The larger FJC labeled structures in the three regions ranged from 2 to 8 μm2.
+
+Fig. 4. Regions with highest levels of FJC labeling at 24 h after Sevo. The regions with the highest increases in (> 4 and ≤ 75 μm2) FJC structures present at 24 h after Sevo are shown on the x- axis. The average of means along with the SEMs shown on the y-axis are nor- malized to 1 mm2 area for both control and Sevo groups. Abbreviations present are the same as used in Fig. 1. The blue background indicates that the region is associated with the primary olfactory learning system while the tan back- ground indicates that the region is part of the basal ganglia and motor move- ment system. The arrow points to the bars related to the SNR and SNCD. ****P ≤ 0.00002.
+
+the
+
+numbers
+
+of
+
+large
+
+Nuclei STM, AHP, and SNR & SNCD regions of the 2 h post Sevo ani- mals, N = 5 (Fig. 2). For the 24 h post Sevo animals in the Anterior Thalamus, Bed Nuclei STM, A29c-1, S1BF, BMA & MeA, VTA, MPO, and SNR & SNCD regions, N = 5 (Figs. 4 and 9). For the 24 h post Sevo animals in the GP and AHP, N = 4 (Fig. 4). For NeuN, 24 h post Sevo controls were N = 4 in the IG (Fig. 7). Controls for Mki67 at the anterior thalamus where N = 5 (Fig. 9). In the CPua, 24 h post Sevo and control animals were N = 4 for Mki67 (Fig. 9).
+
+3. Results
+
+Preliminary data identified over ten specific brain regions in which the density of FJC labeling within the region was at least three-fold increased over other brain regions. FJC labeling within these regions, as well as some regions which have significant synaptic connections to the identified regions, was then conducted to determine the statistical dif- ferences between regions over the three timepoints (PND 7 at 2 h post Sevo, PND 8 at 24 h post Sevo, and PND 10 at 72 h post Sevo).
+
+Regions highlighted in gold are superimposed over coronal sections (+2.76 to-4.8 mm from bregma) of an adult rat brain in Fig. 1, in- dicating increased FJC labeling due to Sevo. Corresponding thionine- stained brain sections from neonatal pups at PND 7, 8 and 10 are shown in the remaining three columns. The anatomy of the brain regions in the adult versus neonate coronal sections approximately correspond. However, more posteriorly, the corpus collosum appears to extend further back with respect to the midbrain. Thus, there is an apparent 1 mm discrepancy caudally at −5.0 mm from bregma on PND 7–10. At this age, the neocortex and the hippocampal morphology agrees with a position at −4.8 to −5.0 mm from the bregma in adults while the midbrain shown corresponds to about −4.0 mm in adults. This dis- crepancy is less pronounced at PND 10. In the sections more rostral, the correspondence seems to be uniform up to +2.78 mm from bregma. Note that the lateral septum is not highlighted in Fig. 1 because it
+
+occurs rostral to C1, at 0.24 mm from bregma. As can be seen in Fig. 1, the size of the coronal sections in the neonatal brain increase about 30% from PND 7 to 10.
+
+Increased FJC labeling was determined by evaluating the number of FJC-labeled neurons per mm2 in brain regions of the Sevo or control group. Two hours after Sevo, neurodegeneration was most prominent in brain regions associated with the primary olfactory learning system (Fig. 2, blue), as well as the basal ganglia and thalamic and cortical regions related to motor movement (Fig. 2, tan). The regions of the primary olfactory learning system included: the indusium griseum (IG), lateral septum, accumbens shell (Acb), posterior hippocampal CA1 (lighter color indicates looser association), bed nucleus of the stria terminalis (Bed Nuclei STM), posteromedial cortical nuclei of the amygdala (PMCo), and the amygdaloid nucleus (BMa & MeA). In the basal ganglia associated brain regions, layer I and II of the anterior motor cortex (M1 & M2), anterior thalamus, lateral globus pallidus (GP), and caudate/putamen (CPua,b, both anterior medial and more posterior ventral) were affected. The retrosplenial cortex (A29c-1 and A29c-2), barrel fields of cortical primary somatosensory (S1BF), sec- ondary motor cortex (M2), and cingulate cortex (CG Ctx) were the other regions most affected by Sevo. The total number of FJC-labeled neurons per region, irrespective of its total area, are found in Supplemental Fig. 1. (Because the areas of regions analyzed varied greatly, the ab- solute numbers per region are greater in the regions of the anterior thalamus and CPu, which encompass larger total areas. From that standpoint, the regions of the anterior thalamic nuclei (−1.2 to 3.0) and CPub had the greatest number of FJC labeled neurons followed by CA1, PMCo, Bed Nuclei STM, A29c-1, A29c-2, and Acb.) FJC labeling in the substantia nigra (SNR & SNCD) and ventral tegmental area (VTA) was minimal (Fig. 2 and Supplemental Fig. 2). Also, there was very little labeling in the frontal cortex at +4.2 mm from bregma (Supplemental Fig. 1). Representative micrographs show Sevo exposure increases FJC labeling for the CPua, Acb, and M2 (Fig. 3).
+
+7
+
+S.M. Burks, et al.
+
+Twenty-four h after Sevo, the FJC labeling was within 3-fold of control in half the brain regions (Fig. 4). There was still a 3 to 6-fold increase in the numbers of FJC labeling in the Bed Nuclei STM, Acb and more anterior medial CPu (CPua) relative to control at 24 h post Sevo.
+
+8
+
+Neurotoxicology and Teratology 80 (2020) 106890
+
+Fig. 5. Visual presentation of FJC labeling in three regions with high levels of neurodegeneration 24 h after Sevo exposure. Representative micrographs for neurodegeneration/FJC labeling in the control and Sevo groups for the IG, Bed Nuclei STM and CA1 are shown at 24 h after Sevo. Both high and low magnifications are shown; the red arrows point to the same location on the two different magnifications as indicated by the red magnification bars. FJC labeling in the CA1 is present in objects approaching 9 μm in diameter with a pronounced layer of FJC labeled puncta above in the region of fibers of passage. FJC-labeled objects of the same size are seen in the IG with a light interspersion of puncta. FJC-labeled objects present in the Bed Nuclei STM are ≤8 μm with very few puncta being present.
+
+However, after 24 h, in the IG, posterior CA1, anterior thalamic nuclei, and GP, there was still 25.7, 17, 11.7, and 9.8-fold, respectively, higher levels of FJC labeling compared to control. There was very little effect of Sevo on FJC labeling in most of the hippocampus except for the striking increases in CA1, caudally just before the appearance of the subiculum where the morphology of the cortex and hippocampus cor- respond to the CA1 region present from about −4.5 to −5.0 mm from bregma (Fig. 5). High levels of FJC labeling can be seen in the lateral dorsal (both AD and LDVL) central medial (CM) and ventromedial (VM) nuclei of the thalamus (Fig. 6). The levels of FJC labeling in all brain regions were within 3.8-fold or less at 72 h after Sevo compared to control except for the M1 & M2 motor cortex at −0.6 mm from bregma (Supplemental Fig. 3). Interestingly, high levels of FJC labeled struc- tures of the size of degenerating neurons were seen at 72 h after Sevo in two of the four rats evaluated (Supplemental Fig. 4). These are most likely images of neurons dying within the last 24 h.
+
+The total number of surviving neurons per brain region (at PND 7 and 8) with the high increases in FJC labeling were determined by using NeuN immunolabeling, which will detect most but not all neurons (Mullen et al., 1992). Micrographs of these NeuN labeled regions can be found in Supplemental Fig. 5. The number of NeuN labeled cells within a region in one section and the number of FJC cells in an adjacent section for the IG, CPua, anterior thalamus and CA1 (Fig. 5) were de- termined. A single section was not as feasible for use for dual staining due to the fragility of the PND 7 and 8 sections and the densely packed NeuN neurons labeled with DAB obscured the FJC labeled cells. The percentage of FJC labeling relative to NeuN labeled cells within a re- gion after Sevo or vehicle gas at 2 h (Fig. 7) was calculated via the following method [(total FJC positive cells per 1mm2 / total NeuN positive cells per 1mm2)*100]. Unfortunately, enough tissue did not remain at the 24 h condition to determine the total number of NeuN positive cells. However, the number of NeuN positive cells were little changed between the 2 and 72 h conditions (15% difference for the IG). Accordingly, the number of NeuN positive cells at the 2 h condition was also used for the 24 h condition.
+
+The newly-born cells within regions were determined at 2 h and 24 h using antibodies to Mki67 to determine if the regions with more intense Sevo-related neurodegeneration (FJC labeling) had any obvious connection with regions of new cell birth. There were appreciable numbers of Mki67 labeled nuclei in the CPua, Acb, S1BF, and LDVL (Fig. 8). The regions of mitotic activity are identified at 10×; enlarged Mki67 nuclei about to separate are also shown at 20× for clarity. The numbers of Mki67 labeling nuclei per mm2 were determined in both control and Sevo groups at 2 and 24 h in the CPua, anterior thalamus, and VM nuclei of the thalamus (Fig. 9). It was expected that if the newborn cells, which would not be neuronal, in these regions were dying, that there would be fewer Mki67 labeled nuclei in the Sevo group at 2 h. At 24 h they might be either: 1) decreased due to con- tinued degeneration or, 2) increased due to compensation for loss at 2 h. However, effects of Sevo were variable between the thalamus and CPua, with no clear evidence that Mki67 labeling was altered by Sevo. Mki67 labeled cells were present in high numbers in all the regions with high FJC labeling.
+
+S.M. Burks, et al.
+
+4. Discussion
+
+PND 7 rats were exposed to Sevo general anesthesia after which, regions of the forebrain were quantified with FJC, and how the regional pattern of neurodegeneration varied with time was determined. Pathways of the basal ganglia and portions of the olfactory learning system were most affected by Sevo. Remarkably, in some brain regions as many as 10% of the total neurons appeared to be dying 24 h after Sevo exposure (IG; Fig. 7). We also observed significant neurodegen- eration in areas of the brain, such as the IG (Figs. 2 and 4), and in other areas that failed to reach statistical significance, such as the lateral septum and the bed nucleus STM that have been previously overlooked. In contrast to previous studies (Lee et al., 2017; Perez-Zoghbi et al., 2017; Zhou et al., 2016), we observed clear, but spatially restricted, damage to the CA1 region of the hippocampus. By 72 h signs of neu- rodegeneration were greatly reduced. The reduced profile of hippo- campal damage observed here may be caused by the exclusion of ani- mals that did not maintained adequate oxygenation throughout the course of exposure. Transient levels of low oxygen during prolonged Sevo exposure may result in a more pervasive pattern of neurodegen- eration within the hippocampus.
+
+9
+
+Neurotoxicology and Teratology 80 (2020) 106890
+
+Fig. 6. Visual presentation of FJC labeling in four thalamic nuclei with high levels of in- creased FJC labeling at 24 h after Sevo exposure. The large top panel shows the entire thalamus in one hemisphere. The third ventricle and the ventral aspects of the hippocampus are present at the very top of the panel. The panel is a composite of twenty-four micrographs taken at 10× magnification. FJC labeling was prominent in the lateral dorsal thalamic nucleus, ven- trolateral portion (LDVL), anterodorsal portion (AD), ventromedial (VM) and central medial (CM) nuclei of the thalamus as shown in the bottom four panels. Magnification is the same for all four and represented in the far-left panel. White boxes highlight the areas in the lower panels. FJC labeling is present in objects up to ⩰ 9 μm in diameter with a light interspersion of puncta.
+
+Several regions of the forebrain showed elevated levels of FJC staining 2 h after Sevo exposure (Fig. 2). The brain regions affected could be roughly split into two groups: those related to the basal ganglia and motor movement (anterior thalamus, CPua,b, GP, M1& M2, GP, M2 and possibly S1BF (Gerfen and Surmeier, 2011; Ikemoto et al., 2015) and regions that have been associated with the primary olfactory learning pathway (IG, CA1, Bed Nuclei STM, Acb, lateral septum, BMa & MeA and PMCo (Shipley and Adamek, 1984)) as well as “spatial cognition” (CA1, (Gilbert et al., 2001; Kesner et al., 2004) (Gallagher and Chiba, 1996), fear learning (BMa & MeA, and PMco (Gallagher and Chiba, 1996)), reward and addiction (Acb (Di Chiara, 2002)), response to stress (Bed Nuclei STM (Choi et al., 2008)) and many other aspects of behavior. Elevated levels of FJC, although not statistically significant, were also detected in other areas known to be important to learning and memory such as A29c-1 and A29c-2 (Nelson et al., 2018; Todd et al., 2019). By 24 h after Sevo (Fig. 4), the same general regional pattern was seen in the basal ganglia pathway and the primary olfactory learning pathways in the top twelve brain regions. Moreover, neuro- degeneration was again detected in A29c-1 and A29c-2 as well as the AHP.
+
+Most studies investigating anesthesia related neurotoxicity evaluate
+
+S.M. Burks, et al.
+
+Fig. 7. Percentage of munoreactive cells at 2 and 24 h after Sevo. The percentage of FJC labeled cellular structures within a region in Sevo and control groups was indirectly calculated by determining the numbered of NeuN labeled cells within a region in one section and the number of FJC cells in an adjacent section for the IG, anterior CPu anterior thalamus and CA1. The per- centage of large (> 4 and ≤ 75 μm2 in area) FJC structures relative to NeuN labeled cells present at 2 and 24 h after Sevo or in control are shown. The mean percentages along with SEMs are displayed. ** indicates P ≤ 0.0001.
+
+large FJC structures present relative to NeuN im-
+
+animals at a single time point. In contrast, we evaluated Sevo related neurodegeneration at 2, 24, and 72 h after the cessation of exposure. Neurodegeneration was detected subsequent to what is seen at 2 h and regions experiencing prolonged neurodegeneration and/or those un- dergoing a “second wave” of neurodegeneration were identified. Some of the regions associated with the basal ganglia pathway and the pri- mary olfactory learning pathway showed signs of either a prolonged neurodegeneration (CPua) or a second neurodegenerative event (IG, CA1, and thalamus). These regions had multifold increases in FJC staining at 24 h compared to 2 h. In contrast, the M1 & M2, PMCo, CPub, and cingulate more rapidly returned to control, or near control levels. For example, the PMCo and CPub have levels of FJC staining approximately 5-fold higher than control at 2 h, but levels were es- sentially normal at 24 h post exposure. These data indicate there are regional differences in sensitivity to anesthesia related neurotoxicity, and that timing of neurotoxicity differs in a region dependent manner. One explanation for this phenomenon could be delayed scavenging
+
+10
+
+Neurotoxicology and Teratology 80 (2020) 106890
+
+of the dead neurons in brain regions with high neurodegeneration; that is, the brain could be rate-limited in the clearance of dead neurons. Dead neurons in excess of the threshold could be misinterpreted as dying at a later point. However, “Delayed scavenging” was not ob- served in the Acb and CPub. These regions had high levels of neuro- degeneration at 2 h, but 5-fold drops in the number of degenerating neurons labeled with FJC at 24 h. Moreover, delayed scavenging cannot explain the actual increase in degenerating neurons in the IG, anterior thalamus, and CA1 at 24 h compared to 2 h. These data indicate the initial loss of neurons in some regions associated with the basal ganglia and the primary olfactory learning pathway is followed by a “second wave” of cell death. Endogenous neurodegeneration might increase because of the loss of cellular inputs triggered by the “first wave” of neurodegeneration. Alternatively, Sevo exposure may cause a second wave of neurodegeneration at 24 h via a fundamentally different me- chanism and with a different temporal profile from that observed at 2 h. Regardless of the mechanism, the prospect of a distinct second wave of neurodegeneration increases the difficulty in ameliorating anesthesia related neurodegeneration and highlights the importance of con- sidering multiple time points when studying anesthesia related neuro- degeneration.
+
+It is unclear why Sevo causes an increase in FJC positive cells in some areas, while leaving others unaffected. It is not as simple as an abundance of newly divided cells. Many areas of the brain where ele- vated numbers of FJC positive cells were observed were past their periods of “peak” neurogenesis. For example, neurogenesis is thought to peak at post-conception day (PCD) 17 in the Acb (Clancy et al., 2009), PCD 16 in the CA1 (Wyss and Sripanidkulchai, 1985) (Workman et al., 2013), and PCD 14 in the CPu (Clancy et al., 2009; Workman et al., 2013). In the CA1 we observed about 5% of all cells being FJC positive (Fig. 7) even though neurogenesis in the region is greatly reduced by PCD 20 (Wyss and Sripanidkulchai, 1985). These data therefore suggest that a cell being recently born is not enough to make it vulnerable to anesthesia related neurotoxicity.
+
+Another possible explanation for certain areas being more sensitive to Sevo is the high prevalence of GABAA receptors. While the IG does have high levels of GABAA receptor mRNA (PND 5 rats (Poulter et al., 1992)), so does the dentate gyrus (Poulter et al., 1992) where little to no evidence of neurodegeneration was detected. Some of the neuro- degeneration may be the consequence of a lack of organized stimula- tory input caused by anesthesia exposure. For example, previous work has demonstrated an increase in degenerating cells in the substantia nigra (bilateral) 1–4 days after an excitatory striatal lesion (unilateral) in the PND 7 rat (Macaya et al., 1994). Similarly, stimulatory activity is required for normal cortical development (Kilb et al., 2011; Lewin and Barde, 1996). However, it is unclear if a lack of organized stimulation could so rapidly impact neurodegeneration and cause an increase in FJC staining 2 h post Sevo cessation. If that is indeed the case, it would ultimately translate into lasting behavioral changes when normal sti- mulation has been restored. Clearly, additional studies are required.
+
+Another possibility is that some neuron types, more abundant in certain regions, are more vulnerable to insult in adults and neonates. For example, the basal ganglia associated regions of the thalamus, which are shown here to be sensitive to Sevo, are also sensitive to cell death as a result of thiamine deficiency and methamphetamine in adult rodents (Bowyer et al., 2008; Bowyer et al., 2018b). Except for the retrosplenial (A29c) and motor cortex, all brain regions where Sevo resulted in high levels of neurodegeneration in the perinatal rat also display pronounced neurotoxicity in adult rodents exposed to me- thamphetamine. Most of the potential mechanisms behind the hy- perthermic and excitatory neurotoxicity produced by amphetamines and seizures would not be thought to occur during anesthesia. How- ever, blood flow disruption within regions where neurodegeneration occurs has been observed with methamphetamine and amphetamine exposure, and likely triggers the seizures and neurodegeneration pro- duced (Bowyer et al., 2018a). Sevoflurane can induce vasodilation
+
+S.M. Burks, et al.
+
+Neurotoxicology and Teratology 80 (2020) 106890
+
+Fig. 8. a,b. Mki67 immunolabeling labeling in the CPua, Acb, S1BF and LDVL at 2 h after Sevo anesthesia (8a) or control (8B). Cells undergoing the process of mitosis in the CPua, Acb, S1BF and LDVL at 2 h after Sevo exposure were labeled using Mki67. The white boxes indicate where mitosis has just occurred with two new nuclei appearing in the CPua, Acb, and S1BF (a). In the LDVL, it appears some time has passed since the original nucleus has divided (a). Magnification is the same for all four regions and is shown in the two CPua panels.
+
+(Matta et al., 1999; Sakata et al., 2019). It is therefore possible that, in the perinatal rats exposed to Sevo, a deficiency in brain blood-flow occurs in regions with pronounced neurodegeneration. Young animals may lack the physiological robustness needed to overcome periods of abnormal regional blood flow.
+
+FJC is the most recent and most selective fluorescent ligand/label developed to detect neurodegeneration, it can label the soma, den- drites, axons and terminals of degenerating neurons in adult animals (Schmued et al., 2005). However, in perinatal animals, the Fluoro Jade (versions b and c) labeling is primarily observed as circular structures ranging from 2 to ≤10 μm in diameter with very little or no labeling of the dendrites and axons as seen in the present study and previously (Scallet et al., 2004). The location, morphology and size of the larger FJC structures seen in perinatal animals after Sevo exposure coincides well with the TUNEL data, which detects apoptotic degeneration by labeling the degraded fragmenting DNA (Scallet et al., 2004; Schmued et al., 2005). The ligand(s) generated during neurodegeneration that covalently bind to FJC is unknown, but it is likely highly positively charged. It is not known why dendrites and soma (other than the nu- cleus) were not labeled with FJC in perinates.
+
+In normal perinates, FJC labeling is present among the degrading DNA and histones found in or surrounding the collapsing nucleus during the apoptotic process. From this study and previous research,
+
+most of the cells labeled with FJC are likely neurons (Scallet et al., 2004; Schmued et al., 2005). Sevo exposure did not decrease the Mki67 labeling, which indicates that most of the cells observed dividing were not neuronal in nature. However, this assumes that Mki67 antibodies cannot label FJC labeled neurons owing to degradation of the Mki67 protein. In the present study, the FJC labeling in the hippocampal CA1, IG and some thalamic nuclei showed morphological similarities with adult FJC labeling. FJC labeled the entire soma and a few proximal dendrites. In the CA1 region, FJC also labeled fibers of passage residing about the degenerating neurons. It is not clear why this occurred only in these regions, but it could be due to a different type of neurodegen- erative process other than classic apoptosis, or that the neurons affected in this area were further along in their differentiation to the “adult” state.
+
+During the BGS, new cells are continually created while others are degenerating via normal apoptotic processes. This dynamic state has led some to question the clinical relevance of anesthesia related death during the BGS. Hypothetically, endogenous amelioration of anesthesia related cell death could occur by either decreasing the rate of apoptosis, or by increasing the creation of new cells. Several areas of the brain did have a transient reduction in FJC levels at either 24 or 72 h post ex- posure, suggesting potential compensation. Yet, it is unlikely that the decline in endogenous neurodegeneration is enough to compensate for
+
+11
+
+S.M. Burks, et al.
+
+Fig. 9. Effects of Sevo on Mki67 labeling in the CPu and thalamus. The numbers of Mki67 labeled cells in the CPua, anterior thalamus and VM nuclei of the thalamus are shown in the control and Sevo groups at the 2 h and 24 h time points. No statistical significance was observed.
+
+the multiple fold increase in neurodegeneration that occurred due to Sevo exposure. Similarly, Mki67, a non-specific nuclear marker of the birth/mitosis of all cells (Brown and Gatter, 1990; von Bohlen und Halbach, 2011) was used to determine if more cells were born in the areas of greatest neurodegeneration. There was no convincing evidence of an increase in new cell birth (Mki67 labeling) in regions where Sevo exposure increased neurodegeneration the most (anterior thalamus, Figs. 4 and 9). Although not statistically significant, some brain regions trended toward elevated levels of Mki67 binding under the control condition (e.g., CPu and thalamus, Fig. 9), as well as increased neuro- degeneration after Sevo exposure. The brain does not appear to meaningfully increase the supply of viable neurons within the first 72 h
+
+12
+
+Neurotoxicology and Teratology 80 (2020) 106890
+
+of Sevo exposure. In most of these regions, neuronal cell birth is thought to have ended by PND 7–8 (Feliciano and Bordey, 2013). In future re- search, it will be important to consider the developmental fate of cells born in the brain in the hours after Sevo insult.
+
+Many of the brain areas where neurodegeneration was observed are crucial for cognition, and prolonged general anesthesia can cause lasting behavioral changes. Surprisingly, with an independent cohort of rats treated under near identical conditions, we saw no significant ef- fects of Sevo exposure when tested during adulthood on a battery of operant tests (Walters et al., 2020). Behavioral effects of these ex- posures were limited to an altered locomotor activity response after an amphetamine challenge. It is possible that we may have detected more robust effects of Sevo if we evaluated Morris Water Maze (MWM) or a radial arm maze (RAM) performance. Many reports have previously shown animals treated with anesthesia early in life to have altered performance on those tasks in adulthood (Walters and Paule, 2017). However, the MWM and RAMs are both dependent upon a functioning hippocampus (Morris et al., 1982; Olton et al., 1978), and we observed less damage to the hippocampus than reported in other studies. We included only those animals that maintained adequate oxygenation throughout the entirety of the exposure, and we observed less extensive neurodegeneration in the hippocampus, potentially because of this. The diffuse nature of the insult, with generally fewer than 1% of neurons dying in any area (notable exceptions) may not have been sufficient to impair performance on operant based tasks of cognition (Walters et al., 2020).
+
+While still controversial, an ever-growing body of evidence in- dicates that extended exposure to general anesthesia, including Sevo, early in life has the potential to be neurotoxic. Here, we confirmed that in adequately oxygenated animals, Sevo exposure caused neurodegen- eration but to a lesser extent in some brain regions (e.g., hippocampus) than previously seen. Moreover, our comprehensive assessment of the brain highlights additional regions that may be vulnerable to anesthesia related neuronal degeneration. Most striking was the damage seen in the IG; over 10% of the total cells stained positive for FJC at 24 h post- exposure. By including a time course, we also established that neuro- degeneration does not progress at a uniform pace after Sevo exposure. The temporal pattern of FJC staining also suggests a second wave of degeneration at 24 h, likely driven by a different mechanism than that which caused degeneration at 2 h. The realization that anesthesia re- lated neurodegeneration does not occur at a uniform pace, and that damage may be more spread than initially assumed, is crucial for the future study of mechanisms of and treatments for anesthesia related neurotoxicity.
+
+Supplementary data to this article can be found online at https://
+
+doi.org/10.1016/j.ntt.2020.106890.
+
+Funding
+
+This work was funded by NCTR Protocol E07601.01.
+
+Declaration of competing interest
+
+The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ- ence the work reported in this paper.
+
+Acknowledgements
+
+The authors would like to thank James Raymick for his assistance
+
+with tissue processing.
+
+References
+
+Amrock, L.G., Starner, M.L., Murphy, K.L., Baxter, M.G., 2015. Long-term effects of single or multiple neonatal sevoflurane exposures on rat hippocampal ultrastructure.
+
+S.M. Burks, et al.
+
+Anesthesiology 122 (1), 87–95.
+
+Bowyer, J.F., Thomas, M.T., Schmued, L.C., Ali, S.F., 2008. Brain region-specific neuro- degenerative profiles showing the relative importance of amphetamine dose, hy- perthermia, seizures and the blood-brain barrier. Ann. N. Y. Acad. Sci. 1139, 127–139.
+
+Bowyer, J.F., Tranter, K.M., Robinson, B.L., Hanig, J.P., Faubion, M.G., Sarkar, S., 2018a. The time course of blood brain barrier leakage and its implications on the progression of methamphetamine-induced seizures. Neurotoxicology 69, 130–140.
+
+Bowyer, J.F., Tranter, K.M., Sarkar, S., Hanig, J.P., 2018b. Microglial activation and vascular responses that are associated with early thalamic neurodegeneration re- sulting from thiamine deficiency. Neurotoxicology 65, 98–110.
+
+Brambrink, A.M., Evers, A.S., Avidan, M.S., Farber, N.B., Smith, D.J., Zhang, X., Dissen, G.A., Creeley, C.E., Olney, J.W., 2010. Isoflurane-induced neuroapoptosis in the neonatal rhesus macaque brain. Anesthesiology 112 (4), 834–841.
+
+Brambrink, A.M., Evers, A.S., Avidan, M.S., Farber, N.B., Smith, D.J., Martin, L.D., Dissen, G.A., Creeley, C.E., Olney, J.W., 2012. Ketamine-induced neuroapoptosis in the fetal and neonatal rhesus macaque brain. Anesthesiology 116 (2), 372–384. Briner, A., Nikonenko, I., De Roo, M., Dayer, A., Muller, D., Vutskits, L., 2011.
+
+Developmental stage-dependent persistent impact of propofol anesthesia on dendritic spines in the rat medial prefrontal cortex. Anesthesiology 115 (2), 282–293. Brioni, J.D., Varughese, S., Ahmed, R., Bein, B., 2017. A clinical review of inhalation anesthesia with sevoflurane: from early research to emerging topics. J. Anesth. 31 (5), 764–778.
+
+Brown, D.C., Gatter, K.C., 1990. Monoclonal antibody Ki-67: its use in histopathology.
+
+Histopathology 17 (6), 489–503.
+
+Choi, D.C., Evanson, N.K., Furay, A.R., Ulrich-Lai, Y.M., Ostrander, M.M., Herman, J.P., 2008. The anteroventral bed nucleus of the stria terminalis differentially regulates hypothalamic-pituitary-adrenocortical axis responses to acute and chronic stress. Endocrinology 149 (2), 818–826.
+
+Clancy, B., Teague-Ross, T.J., Nagarajan, R., 2009. Cross-species analyses of the cortical
+
+GABAergic and subplate neural populations. Front. Neuroanat. 3, 20.
+
+Delgado-Herrera, L., Ostroff, R.D., Rogers, S.A., 2001. Sevoflurance: approaching the
+
+ideal inhalational anesthetic. A pharmacologic, pharmacoeconomic, and clinical re- view. CNS Drug Rev 7 (1), 48–120.
+
+Deng, M., Hofacer, R.D., Jiang, C., Joseph, B., Hughes, E.A., Jia, B., Danzer, S.C., Loepke, A.W., 2014. Brain regional vulnerability to anaesthesia-induced neuroapoptosis shifts with age at exposure and extends into adulthood for some regions. Br. J. Anaesth. 113 (3), 443–451.
+
+Di Chiara, G., 2002. Nucleus accumbens shell and core dopamine: differential role in
+
+behavior and addiction. Behav. Brain Res. 137 (1–2), 75–114.
+
+Dobbing, J., Sands, J., 1979. Comparative aspects of the brain growth spurt. Early Hum.
+
+Dev. 3 (1), 79–83.
+
+Eriksson, P., 1997. Developmental neurotoxicity of environmental agents in the neonate.
+
+Neurotoxicology 18 (3), 719–726.
+
+Fang, F., Xue, Z., Cang, J., 2012. Sevoflurane exposure in 7-day-old rats affects neuro- genesis, neurodegeneration and neurocognitive function. Neurosci. Bull. 28 (5), 499–508.
+
+Feliciano, D.M., Bordey, A., 2013. Newborn cortical neurons: only for neonates? Trends
+
+Neurosci. 36 (1), 51–61.
+
+Flick, R.P., Katusic, S.K., Colligan, R.C., Wilder, R.T., Voigt, R.G., Olson, M.D., Sprung, J., Weaver, A.L., Schroeder, D.R., Warner, D.O., 2011. Cognitive and behavioral out- comes after early exposure to anesthesia and surgery. Pediatrics 128 (5), e1053–e1061.
+
+Gallagher, M., Chiba, A.A., 1996. The amygdala and emotion. Curr. Opin. Neurobiol. 6
+
+(2), 221–227.
+
+Gentry, K.R., Steele, L.M., Sedensky, M.M., Morgan, P.G., 2013. Early developmental
+
+exposure to volatile anesthetics causes behavioral defects in Caenorhabditis elegans. Anesth. Analg. 116 (1), 185–189.
+
+Gerfen, C.R., Surmeier, D.J., 2011. Modulation of striatal projection systems by dopa-
+
+mine. Annu. Rev. Neurosci. 34, 441–466.
+
+Gilbert, P.E., Kesner, R.P., Lee, I., 2001. Dissociating hippocampal subregions: double dissociation between dentate gyrus and CA1. Hippocampus 11 (6), 626–636.
+
+Guo, P., Huang, Z., Tao, T., Chen, X., Zhang, W., Zhang, Y., Lin, C., 2015. Zebrafish as a model for studying the developmental neurotoxicity of propofol. J. Appl. Toxicol. 35 (12), 1511–1519.
+
+Ikemoto, S., Yang, C., Tan, A., 2015. Basal ganglia circuit loops, dopamine and motiva-
+
+tion: a review and enquiry. Behav. Brain Res. 290, 17–31.
+
+Ikonomidou, C., 2009. Triggers of apoptosis in the immature brain. Brain and
+
+Development 31 (7), 488–492.
+
+Ing, C., Brambrink, A.M., 2019. Mayo Anesthesia Safety in Kids continued: two new studies and a potential redirection of the field. Br. J. Anaesth. 122 (6), 716–719. Ing, C., Ma, X., Sun, M., Lu, Y., Wall, M.M., Olfson, M., Li, G., 2020. Exposure to surgery and anesthesia in early childhood and subsequent use of attention deficit hyper- activity disorder medications. Anesth. Analg. https://doi.org/10.1213/ANE. 0000000000004619. 31923004.
+
+Istaphanous, G.K., Howard, J., Nan, X., Hughes, E.A., McCann, J.C., McAuliffe, J.J.,
+
+Danzer, S.C., Loepke, A.W., 2011. Comparison of the neuroapoptotic properties of equipotent anesthetic concentrations of desflurane, isoflurane, or sevoflurane in neonatal mice. Anesthesiology 114 (3), 578–587.
+
+Jevtovic-Todorovic, V., Benshoff, N., Olney, J.W., 2000. Ketamine potentiates cere-
+
+brocortical damage induced by the common anaesthetic agent nitrous oxide in adult rats. Br. J. Pharmacol. 130 (7), 1692–1698.
+
+Jevtovic-Todorovic, V., Wozniak, D.F., Benshoff, N.D., Olney, J.W., 2001. A comparative evaluation of the neurotoxic properties of ketamine and nitrous oxide. Brain Res. 895 (1–2), 264–267.
+
+Jiang, Y., Tong, D., Hofacer, R.D., Loepke, A.W., Lian, Q., Danzer, S.C., 2016. Long-term
+
+13
+
+Neurotoxicology and Teratology 80 (2020) 106890
+
+fate mapping to assess the impact of postnatal isoflurane exposure on hippocampal progenitor cell productivity. Anesthesiology 125 (6), 1159–1170.
+
+Kanungo, J., Cuevas, E., Ali, S.F., Paule, M.G., 2013. Ketamine induces motor neuron toxicity and alters neurogenic and proneural gene expression in zebrafish. J. Appl. Toxicol. 33 (6), 410–417.
+
+Kesner, R.P., Lee, I., Gilbert, P., 2004. A behavioral assessment of hippocampal function
+
+based on a subregional analysis. Rev. Neurosci. 15 (5), 333–351.
+
+Kilb, W., Kirischuk, S., Luhmann, H.J., 2011. Electrical activity patterns and the func-
+
+tional maturation of the neocortex. Eur. J. Neurosci. 34 (10), 1677–1686.
+
+Lee, J.R., Lin, E.P., Hofacer, R.D., Upton, B., Lee, S.Y., Ewing, L., Joseph, B., Loepke, A.W., 2017. Alternative technique or mitigating strategy for sevoflurane-induced neuro- degeneration: a randomized controlled dose-escalation study of dexmedetomidine in neonatal rats. Br. J. Anaesth. 119 (3), 492–505.
+
+Lerman, J., Johr, M., 2009. Inhalational anesthesia vs total intravenous anesthesia (TIVA)
+
+for pediatric anesthesia. Paediatr. Anaesth. 19 (5), 521–534.
+
+Lewin, G.R., Barde, Y.A., 1996. Physiology of the neurotrophins. Annu. Rev. Neurosci. 19,
+
+289–317.
+
+Macaya, A., Munell, F., Gubits, R.M., Burke, R.E., 1994. Apoptosis in substantia nigra
+
+following developmental striatal excitotoxic injury. Proc. Natl. Acad. Sci. U. S. A. 91 (17), 8117–8121.
+
+Matta, B.F., Heath, K.J., Tipping, K., Summors, A.C., 1999. Direct cerebral vasodilatory
+
+effects of sevoflurane and isoflurane. Anesthesiology 91 (3), 677–680.
+
+Morris, R.G., Garrud, P., Rawlins, J.N., O'Keefe, J., 1982. Place navigation impaired in
+
+rats with hippocampal lesions. Nature 297 (5868), 681–683.
+
+Mullen, R.J., Buck, C.R., Smith, A.M., 1992. NeuN, a neuronal specific nuclear protein in
+
+vertebrates. Development 116 (1), 201–211.
+
+Na, H.S., Brockway, N.L., Gentry, K.R., Opheim, E., Sedensky, M.M., Morgan, P.G., 2017. The genetics of isoflurane-induced developmental neurotoxicity. Neurotoxicol. Teratol. 60, 40–49.
+
+Nelson, A.J.D., Hindley, E.L., Vann, S.D., Aggleton, J.P., 2018. When is the rat retro-
+
+splenial cortex required for stimulus integration? Behav. Neurosci. 132 (5), 366–377.
+
+Olton, D.S., Walker, J.A., Gage, F.H., 1978. Hippocampal connections and spatial dis-
+
+crimination. Brain Res. 139 (2), 295–308.
+
+Osterop, S.F., Virtanen, M.A., Loepke, J.R., Joseph, B., Loepke, A.W., Vutskits, L., 2015.
+
+Developmental stage-dependent impact of midazolam on calbindin, calretinin and parvalbumin expression in the immature rat medial prefrontal cortex during the brain growth spurt. Int. J. Dev. Neurosci. 45, 19–28.
+
+Paule, M.G., Li, M., Allen, R.R., Liu, F., Zou, X., Hotchkiss, C., Hanig, J.P., Patterson, T.A., Slikker Jr., W., Wang, C., 2011. Ketamine anesthesia during the first week of life can cause long-lasting cognitive deficits in rhesus monkeys. Neurotoxicol. Teratol. 33 (2), 220–230.
+
+Paxinos, G., Watson, C., 2014. The Rat Brain in Stereotaxic Coordinates, 7th ed. Elsevier. Pellegrini, L., Bennis, Y., Velly, L., Grandvuillemin, I., Pisano, P., Bruder, N., Guillet, B., 2014. Erythropoietin protects newborn rat against sevoflurane-induced neurotoxi- city. Paediatr. Anaesth. 24 (7), 749–759.
+
+Perez-Zoghbi, J.F., Zhu, W., Grafe, M.R., Brambrink, A.M., 2017. Dexmedetomidine- mediated neuroprotection against sevoflurane-induced neurotoxicity extends to several brain regions in neonatal rats. Br. J. Anaesth. 119 (3), 506–516.
+
+Poulter, M.O., Barker, J.L., O’Carroll, A.M., Lolait, S.J., Mahan, L.C., 1992. Differential and transient expression of GABAA receptor alpha-subunit mRNAs in the developing rat CNS. J. Neurosci. 12 (8), 2888–2900.
+
+Ramachandra, R., Subramanian, T., 2011. Atlas of the Neonatal Rat Brain. CRC Press. Rice, D., Barone .Jr., S., 2000. Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ. Health Perspect. 108 (Suppl. 3), 511–533.
+
+Rizzi, S., Carter, L.B., Ori, C., Jevtovic-Todorovic, V., 2008. Clinical anesthesia causes permanent damage to the fetal guinea pig brain. Brain Pathol. 18 (2), 198–210. Sakata, K., Kito, K., Fukuoka, N., Nagase, K., Tanabe, K., Iida, H., 2019. Cerebrovascular reactivity to hypercapnia during sevoflurane or desflurane anesthesia in rats. Korean J Anesthesiol 72 (3), 260–264.
+
+Scallet, A.C., Schmued, L.C., Slikker Jr., W., Grunberg, N., Faustino, P.J., Davis, H., Lester, D., Pine, P.S., Sistare, F., Hanig, J.P., 2004. Developmental neurotoxicity of ketamine: morphometric confirmation, exposure parameters, and multiple fluorescent labeling of apoptotic neurons. Toxicol. Sci. 81 (2), 364–370.
+
+Schmued, L.C., Stowers, C.C., Scallet, A.C., Xu, L., 2005. Fluoro-Jade C results in ultra
+
+high resolution and contrast labeling of degenerating neurons. Brain Res. 1035 (1), 24–31.
+
+Shipley, M.T., Adamek, G.D., 1984. The connections of the mouse olfactory bulb: a study
+
+using orthograde and retrograde transport of wheat germ agglutinin conjugated to horseradish peroxidase. Brain Res. Bull. 12 (6), 669–688.
+
+Slikker Jr., W., Zou, X., Hotchkiss, C.E., Divine, R.L., Sadovova, N., Twaddle, N.C.,
+
+Doerge, D.R., Scallet, A.C., Patterson, T.A., Hanig, J.P., Paule, M.G., Wang, C., 2007. Ketamine-induced neuronal cell death in the perinatal rhesus monkey. Toxicol. Sci. 98 (1), 145–158.
+
+Sprung, J., Flick, R.P., Katusic, S.K., Colligan, R.C., Barbaresi, W.J., Bojanic, K., Welch, T.L., Olson, M.D., Hanson, A.C., Schroeder, D.R., Wilder, R.T., Warner, D.O., 2012. Attention-deficit/hyperactivity disorder after early exposure to procedures requiring general anesthesia. Mayo Clin. Proc. 87 (2), 120–129.
+
+Stratmann, G., Sall, J.W., May, L.D., Loepke, A.W., Lee, M.T., 2010. Beyond anesthetic properties: the effects of isoflurane on brain cell death, neurogenesis, and long-term neurocognitive function. Anesth. Analg. 110 (2), 431–437.
+
+Talpos, J.C., Chelonis, J.J., Li, M., Hanig, J.P., Paule, M.G., 2019. Early life exposure to extended general anesthesia with isoflurane and nitrous oxide reduces responsivity on a cognitive test battery in the nonhuman primate. Neurotoxicology 70, 80–90.
+
+Todd, T.P., Fournier, D.I., Bucci, D.J., 2019. Retrosplenial cortex and its role in cue-
+
+specific learning and memory. Neurosci. Biobehav. Rev. 107, 713–728.
+
+S.M. Burks, et al.
+
+von Bohlen und Halbach, O., 2011. Immunohistological markers for proliferative events, gliogenesis, and neurogenesis within the adult hippocampus. Cell Tissue Res. 345 (1), 1–19.
+
+Vutskits, L., Culley, D.J., 2019. GAS, PANDA, and MASK: no evidence of clinical anes-
+
+thetic neurotoxicity!. Anesthesiology 131 (4), 762–764.
+
+Walters, J.L., Paule, M.G., 2017. Review of preclinical studies on pediatric general an- esthesia-induced developmental neurotoxicity. Neurotoxicol. Teratol. 60, 2–23. Walters, J.L., Chelonis, J.J., Fogle, C.M., Ferguson, S.A., Sarkar, S., Paule, M.G., Talpos, J.C., 2020. Acetyl-L-carnitine does not prevent neurodegeneration in a rodent model of prolonged neonatal anesthesia. Neurotoxicol. Teratol. https://www.ncbi.nlm.nih. gov/pubmed/32376384.
+
+Wilder, R.T., Flick, R.P., Sprung, J., Katusic, S.K., Barbaresi, W.J., Mickelson, C., Gleich, S.J., Schroeder, D.R., Weaver, A.L., Warner, D.O., 2009. Early exposure to anesthesia and learning disabilities in a population-based birth cohort. Anesthesiology 110 (4), 796–804.
+
+Workman, A.D., Charvet, C.J., Clancy, B., Darlington, R.B., Finlay, B.L., 2013. Modeling transformations of neurodevelopmental sequences across mammalian species. J. Neurosci. 33 (17), 7368–7383.
+
+Wyss, J.M., Sripanidkulchai, B., 1985. The development of Ammon’s horn and the fascia dentata in the cat: a [3H]thymidine analysis. Brain Res. 350 (1–2), 185–198.
+
+14
+
+Neurotoxicology and Teratology 80 (2020) 106890
+
+Zhang, X., Shen, F., Xu, D., Zhao, X., 2016. A lasting effect of postnatal sevoflurane an- esthesia on the composition of NMDA receptor subunits in rat prefrontal cortex. Int. J. Dev. Neurosci. 54, 62–69.
+
+Zheng, S.Q., An, L.X., Cheng, X., Wang, Y.J., 2013a. Sevoflurane causes neuronal apop- tosis and adaptability changes of neonatal rats. Acta Anaesthesiol. Scand. 57 (9), 1167–1174.
+
+Zheng, H., Dong, Y., Xu, Z., Crosby, G., Culley, D.J., Zhang, Y., Xie, Z., 2013b. Sevoflurane anesthesia in pregnant mice induces neurotoxicity in fetal and offspring mice. Anesthesiology 118 (3), 516–526.
+
+Zhou, X., Li, W., Chen, X., Yang, X., Zhou, Z., Lu, D., Feng, X., 2016. Dose-dependent effects of sevoflurane exposure during early lifetime on apoptosis in hippocampus and neurocognitive outcomes in Sprague-Dawley rats. Int J Physiol Pathophysiol Pharmacol 8 (3), 111–119.
+
+Zou, X., Patterson, T.A., Divine, R.L., Sadovova, N., Zhang, X., Hanig, J.P., Paule, M.G.,
+
+Slikker Jr., W., Wang, C., 2009. Prolonged exposure to ketamine increases neuro- degeneration in the developing monkey brain. Int. J. Dev. Neurosci. 27 (7), 727–731. Zurek, A.A., Yu, J., Wang, D.S., Haffey, S.C., Bridgwater, E.M., Penna, A., Lecker, I., Lei, G., Chang, T., Salter, E.W., Orser, B.A., 2014. Sustained increase in alpha5GABAA receptor function impairs memory after anesthesia. J. Clin. Invest. 124 (12), 5437–5441.

new_pdfs/10.1021_acschemneuro.0c00106.txt

@@ -0,0 +1,203 @@
+Downloaded via JOHNS HOPKINS UNIV on November 20, 2023 at 16:49:37 (UTC).
+
+See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
+
+Research Article
+
+pubs.acs.org/chemneuro
+
+PDE‑7 Inhibitor BRL-50481 Reduces Neurodegeneration and Long- Term Memory Deficits in Mice Following Sevoflurane Exposure Yingle Chen, Shunyuan Li,* Xianmei Zhong, Zhenming Kang, and Rulei Chen
+
+Cite This: ACS Chem. Neurosci. 2020, 11, 1353−1358
+
+Read Online
+
+ACCESS
+
+Article Recommendations
+
+Metrics & More
+
+ABSTRACT: Sevoflurane, one of the most commonly used anesthetic agents, has been demonstrated to induce widespread neurodegeneration in the developing brain. We aimed to evaluate the protective effects of a PDE-7 inhibitor (BRL-50481) against the neurotoxic effects of sevoflurane on the developing nervous system. Spatial learning and memory in sevoflurane-treated mice were examined using the Morris water maze test, and neuroprotective effects of PDE-7 inhibitor (BRL-50481) against sevoflurane- induced impairments were evaluated. Our results showed that sevoflurane treatment markedly induced neurodegeneration and impaired long-term memory in neonatal mice. Notably, BRL-50481 coadministration could significantly attenuate sevoflurane- induced learning and memory defects, prevent deterioration of recognition memory, and protect against neuron apoptosis. Mechanistically, BRL-50481 administration suppressed sevoflurane-induced neurodegenerative disorders through restoring cAMP and activating cAMP/CREB signaling in the hippocampus. PDE7 inhibitor may be a potential therapeutic agent for sevoflurane- induced neurodegeneration and long-term memory deficits. KEYWORDS: Sevoflurane, neurodegeneration, long-term memory deficits, PDE-7 inhibitor, cAMP/CREB signaling
+
+■ INTRODUCTION
+
+rats exhibited significantly dose- and duration-dependent neurodegeneration with various doses and durations of sevoflurane treatments.12 Amrock et al. evaluated the neuro- degenerative effects of single or multiple doses of 2.5% sevoflurane administration using neonatal rats and found that 2 h exposure resulted in severe synaptic loss and dramatic apoptotic cell death in many brain regions.11
+
+Pregnant women, newborns, and infants are often exposed to anesthetic agents during childbirth or for surgical procedures. It has been demonstrated that the administration of anesthetic reagents is toxic to the developing brain and causes widespread neurodegeneration and long-term deficits in learning and behavior.1−5 Hence, it is of crucial importance to study the effects of anesthetics on the developing nervous system and discover effective therapeutic treatments. Sevoflurane, also called fluoromethyl,
+
+The cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) has been extensively implicated in neurogenesis, survival, proliferation, and differ- entiation.13−16 As a transcriptional factor, CREB is activated by
+
+is one of the most commonly used volatile anesthetic agents for the induction and maintenance of general anesthesia.6 It is useful for infants and children due to its rapid induction, fast recovery, and less irritation to the airway.6,7 However, numerous studies have reported that neonatal administration of sevoflurane induced widespread neurological disorders, including neurodegenera- tion, deficits learning tasks, and long-term potentiation inhibition.8−12 Zheng et al. found that neonatal
+
+Received: February 25, 2020 Accepted: March 25, 2020 Published: April 9, 2020
+
+in spatial
+
+© 2020 American Chemical Society
+
+1353
+
+https://dx.doi.org/10.1021/acschemneuro.0c00106 ACS Chem. Neurosci. 2020, 11, 1353−1358
+
+ACS Chemical Neuroscience
+
+pubs.acs.org/chemneuro
+
+Research Article
+
+Figure 1. Sevoflurane does not affect learning and memory of neonatal mouse at early stages. Two weeks after sevoflurane exposure, spatial learning and memory were examined by Morris water maze test on Day1 (P21), Day 2 (P22), and Day 3 (P23). (A) Delay time to reach the platform of indicated groups. (B) Swimming path length before reaching the platform of indicated groups. (C) Percent of time spent in the target quadrant in a probe test of indicated groups. n = 8 for each time point. Data represent mean ± SD.
+
+Figure 2. BRL-50481 attenuates sevoflurane induced learning and memory defects. Eight weeks after sevoflurane exposure, spatial learning and memory were examined by Morris water maze test on Day 1 (P63), Day 2 (P64), and Day 3 (P65). (A) Delay time to reach the platform of indicated groups. (B) Swimming path length before reaching the platform of indicated groups. (C) Percent of time spent in the target quadrant in a probe test of indicated groups. n = 8 for each time point. Data represent mean ± SD, *P < 0.05, #P < 0.001 compared with sham group.
+
+cAMP-dependent phosphorylation on Ser 133, which triggers the transcription of downstream targets including BDNF, TrkB, and c-Fos, and eventually regulates neurogenesis.16−18 Recently, Xiong et al. reported that sevoflurane-nitrous oxide learning and memory deficiencies anesthesia-induced spatial were associated with the cAMP/CREB signaling inhibition in rats.10
+
+The phosphodiesterase 7 (PDE-7) is a cAMP-specific metallophosphohydrolase enzyme, which suppresses the cAMP/CREB signaling pathway though catalyzing cAMP to the inactive form 5′AMP.4,19,20 Accumulating evidence have showed that PDE-7 inhibitors emerge as promising candidates for promoting neuron survival, improving cognitive symptoms, and treating memory deficits.21−24 BRL-50481 is a PDE-7 specific inhibitor, which can decrease animals’ anxiety levels, promote oligodendrocyte precursor differentiation, inhibit neuroinflammation, and protect against spinal cord in- jury.21,22,24,25 However, the roles of BRL-50481 on sevoflur- ane-induced neurodegenerative disorders remain unknown. In the present study, we demonstrated the neuroprotective effects of PDE7 inhibitor, BRL-50481, against sevoflurane-induced neurodegeneration and long-term memory deficits. Noticeably, BRL-50481 attenuated sevoflurane-induced learning and memory defects, prevented deterioration of recognition memory, and protected against neuron apoptosis through activating the cAMP/CREB signaling, suggesting a potential
+
+therapeutic role of PDE7 inhibitors in the treatment of sevoflurane-induced neurodegenerative disorders.
+
+■ RESULTS AND DISCUSSION
+
+Sevoflurane is a commonly used volatile anesthetic agent due to it exhibiting rapid induction, fast recovery, and less irritation to the airway.6,7 However, accumulative research and clinical data have demonstrated that neonatal administration of sevoflurane may cause widespread neurological disor- ders.8−12,16 Recently, PDE-7 inhibitors have emerged as promising candidates for promoting neuron survival, improv- ing cognitive symptoms, and treating memory deficits.4,19−27 However, little is known about whether PDE-7 inhibitors can attenuate sevoflurane-induced neurodegenerative disorders. Here, we utilized a PDE-7 specific inhibitor, BRL-50481, to demonstrate the preventive effect of PDE-7 inhibitors on sevoflurane-induced neurodegeneration and long-term memo- ry deficits.
+
+Neither Sevoflurane nor the BRL-50481 Affects the Spatial Learning and Memory Ability of Neonatal Mice at Early Stage. P7 neonatal pups undergo extensive neurogenesis to develop episodic memory and establish hippocampal learning. Therefore, they are very sensitive to neurotoxic influences at this stage.8,11,12,16 Based on this, we used P7 mouse pups to perform anesthesia administration to evaluate the neurotoxic effects of sevoflurane exposure. We first
+
+1354
+
+https://dx.doi.org/10.1021/acschemneuro.0c00106 ACS Chem. Neurosci. 2020, 11, 1353−1358
+
+ACS Chemical Neuroscience
+
+investigated the spatial learning and memory ability in P7 pups after 4 h sevoflurane exposure and then evaluated the neuroprotective effects of PDE-7 inhibitor (BRL-50481) through coadministration with sevoflurane (Figure 1). As shown in Figure 1A, the time of delay to find the platform was measured on postnatal days 21, 22, and 23 (Day 1, Day 2, and Day 3) after 4 h sevoflurane exposure on P7 pups, which was comparable to that of the sham group. Moreover, the low or high dose PDE-7 inhibitor (BRL-50481) injected groups showed the same pattern compared to sham and control group. Similarly, there was no difference in the swimming path length and percentage of time stay in the target quadrant (Figure 1B and C). These data suggested that neither sevoflurane nor the PDE-7 inhibitor affected the spatial learning and memory ability of mice at the early developmental stage.
+
+BRL-50481 Attenuates Sevoflurane-Induced Learn- ing and Memory Defects in Neonatal Mouse. We next monitored the spatial learning and memory ability of 9 week postnatal mice (P63−P65) using the Morris water maze test. As shown in Figure 2, 4 h sevoflurane exposure (B0 group) induced marked neurocognitive deficiency compared to the sham and control groups. The delay time to find the platform and swimming path length were significantly prolonged for th B0 group (Figure 2A and B). The sevoflurane-induced memory retention defect was observed using the probe test, where the B0 group spent the least amount of time in the target quadrant (Figure 2C). Although the PDE-7 inhibitor BRL-50481 alone (control group) did not exhibit enhanced neurocognition and memory retention compared to sham the sevoflurane-induced learning and (control vs sham), memory defects were significantly attenuated by a high-dose (5 mg/kg) BRL-50481 injection (B5 vs B0), which was not rescued by the low-dose (1 mg/kg) BRL-50481 administration (B1 vs B0). We observed improved escape latency (Figure 2A and C) and shorter swimming length (Figure 2B) in the control and B5 groups, which indicted that a higher dose of PDE-7 inhibitor could significantly attenuate sevoflurane- induced learning and memory defects.
+
+Interestingly, we found that the spatial learning and memory ability of the pups receiving 4 h sevoflurane exposure was similar to that of the sham group at an early stage (P21−P23). However, 8 weeks after sevoflurane exposure, these mice exhibited significant neurodegenerative disorders compared to the sham group (P63−P65). We speculated that this phenotypic trait might be caused by progressive neuro- degeneration. There was no or less deficit observed at the early stage (2 weeks), but the deficits were evident at 8 weeks after sevoflurane exposure. In line with our findings, other groups observed similar results. Keith et al. demonstrated that cell-death-induced hippocampal DG deficit was improved over 6 weeks.28 Fang et al. treated the 7 day old rats with sevoflurane exposure and found altered neurodegeneration, neurocognitive function, and neurogenesis at 6 weeks instead of 2 weeks after exposure.9 Further studies need to be performed to address the cause of this delay in the onset of cognitive deficit.
+
+BRL-50481 Prevents Sevoflurane-Induced Deteriora- tion of Recognition Memory in Neonatal Mouse. To further investigate the recognition memory defects induced by sevoflurane, we performed the novel object recognition test, which utilizes the natural tendency of rodents to spend more time to explore a novel object than a familiar one. The results the sevoflurane-treated group (B0) and showed that
+
+pubs.acs.org/chemneuro
+
+Research Article
+
+sevoflurane plus low-dose BRL-50481 injected group (B1) indeed exhibited a malfunction in the memory of the mice, they barely remembered the object that they were supposed to be familiar (Figure 3). In contrast, the high-dose BRL-50481
+
+Figure 3. BRL-50481 prevents sevoflurane induced deterioration of recognition memory. (A) Exploration time of indicated group spent with an old object and new object. (B) Discrimination index of recognizing the new vs old object of an indicated group. n = 8 for each group. Data represent mean ± SD, *P < 0.05 compared with sham.
+
+injected group (B5) prevented this malfunction and displayed the recognition ability similar to that of the sham and control groups (Figure 3). The above results suggested that a higher dose of PDE-7 inhibitor prevented sevoflurane-induced deterioration of recognition memory.
+
+BRL-50481 Protects against Sevoflurane-Induced Apoptosis in Hippocampus. To further evaluate the neurocognitive deficits after 4 h of sevoflurane exposure, we performed caspase-3 (CA3) IHC staining in the hippocampal CA1 and dentate gyrus (DG) regions. The degenerated neurons were labeled by CV3 and are shown in Figure 4A.
+
+Figure 4. BRL-50481 protects against sevoflurane induced apoptosis in hippocampus. (A) Representative images of cleaved caspase-3 immunohistochemical staining in the hippocampal CA1 region. Arrows indicate the cleaved caspase-3 positive cells. (B, C) Auantitative statistic of degenerated neurons of indicated groups in hippocampal CA1 (B) and (C) DG regions. n = 8 for each group. Data represent mean ± SD, *P < 0.05, #P < 0.001 compared with Sham.
+
+Apoptotic cell density (AC-3 cells mm−2) increased about 10- fold in CA1 and DG regions in the sevoflurane-treated group (B0) compared to the sham group (Figure 4B and C). Notably, coadministration of high-dose BRL-50481 with sevoflurane (B5) strikingly reduced sevoflurane-induced apoptosis (Figure 4B and C).
+
+1355
+
+https://dx.doi.org/10.1021/acschemneuro.0c00106 ACS Chem. Neurosci. 2020, 11, 1353−1358
+
+ACS Chemical Neuroscience
+
+BRL-50481 Suppresses Sevoflurane-Induced Neuro- degeneration through Restoring Hippocampal cAMP/ CREB Signaling. We next measured the pCREB and total CREB protein levels in each group. Although there was no significant difference in total CREB protein levels after sevoflurane exposure (Figure 5A and B), pCREB protein
+
+Figure 5. BRL-50481 administration rescues sevoflurane induced pCREB downregulation. (A) Immunoblots of lysates from indicated hippocampus to show the protein levels of total CREB, pCREB, and β-actin. (B, C) Relative protein levels of total (B) CREB and (C) pCREB normalized by β-actin. n = 8 for each group. Data represent mean ± SD, *P < 0.05 compared with sham.
+
+expression in the B0 group was significantly decreased (Figure 5A and C). Accordingly, coadministration of high-dose PDE-7 inhibitor BRL-50481 with sevoflurane (B5) could restore pCREB to the sham group’s level. These results indicated that sevoflurane impaired the cycle of pCREB and decreased the pCREB level and the PDE-7 inhibitor might be involved in this cycle to block the inhibitory effect of sevoflurane.
+
+CREB phosphorylation can be triggered by cAMP accumulation. We observed decreased pCREB expression after sevoflurane exposure in the above results; next, we asked whether cAMP was involved in the sevoflurane-induced neurodegeneration and long-term memory deficits. The ELISA the cAMP levels were significantly results showed that the sevoflurane-treated decreased in the hippocampus of group (B0 in Figure 6). Accordingly, coadministration of high-dose PDE-7 inhibitor BRL-50481 with sevoflurane (B5) could restore cAMP to the sham group’s level. Thus, the results suggested that sevoflurane exposure suppressed the
+
+Figure 6. BRL-50481 administration facilitates cAMP accumulation after sevoflurane exposure. The hippocampal cAMP levels of indicated groups were determined by using a mouse cAMP ELISA kit. Data represent mean ± SD, *P < 0.05 compared with sham.
+
+pubs.acs.org/chemneuro
+
+Research Article
+
+cAMP accumulation and in turn inhibited the cAMP/CREB signaling, whereas BRL-50481 prevented this inhibitory effect and restored cAMP/CREB signaling in the hippocampus.
+
+Increasing the intracellular cAMP levels appears to favor the survival and differentiation of neurons, such as oligodendroglial and Schwann cells.13−15,18,25,29,30 PDE-7 catalyzes cAMP to the inactive form and then suppresses the cAMP/CREB signaling pathway. study, we demonstrated the neuroprotective effects of PDE7 inhibitor against sevoflurane- induced neurotoxicity through activating the cAMP/CREB signaling pathway. Notably, the rescued neurodegenerative disorders were observed in the high-dose BRL-50481-treated improve- including prevention of neurodegeneration, group, ment in learning and memory, reduction of apoptosis in the hippocampus, restoration of cAMP, and activation of the cAMP/CREB signaling pathway. In agreement with the neuroprotective function of PDE7 inhibitors, Valdes-Moreno et al. found that PDE7 inhibitors improved feeding and anxiety behaviors of rats through increasing the accumbal and hypothalamic thyrotropin-releasing hormone expression.24 Medina-Rodriguez et al. revealed that PDE7 inhibitor treatment could accelerate human oligodendrocyte precursor differentiation and survival.25 Paterniti et al. demonstrated that PDE7 inhibitor administration could significantly reduce the degree of spinal cord inflammation, tissue injury, and levels of TNF-α, IL-6, COX-2, and iNOS.21 Collectively, these results strongly suggested that PDE7 inhibitors could promote neurogenesis and improve neurodegenerative disorders. Specifically, the PDE7 inhibitor BRL-50481 is a potential drug candidate to be further studied for the treatment of sevoflurane-induced neurodegeneration.
+
+In this
+
+This study demonstrated the neuroprotective effects of PDE7 inhibitor, BRL-50481, on sevoflurane-induced neuro- degeneration. Mechanistically, BRL-50481 administration significantly attenuated sevoflurane-induced learning and memory defects, deterioration of recognition memory, and neuron apoptosis through activating the cAMP/CREB signal- ing in the hippocampus. These findings suggested that PDE7 inhibitor BRL-50481 is a potential drug candidate for the treatment of sevoflurane-induced neurodegenerative disorders.
+
+■ MATERIALS AND METHODS
+
+Animals and Treatments. Seven day old C57BL/6 male mice (Beijing Vital River Company, Beijing, China) were used in this study. The mice were bred and maintained in the animal care facility following the standard rearing conditions of 12 h light and 12 h dark. All mouse studies were performed following the guidelines established by the Institutional Animal Care and Use Committee in Quanzhou First Hospital Affiliated to Fujian Medical University (QFH2017jb43i).
+
+BRL-50481 (Tocris Bioscience, Bristol, United Kingdom) was dissolved in 2.5% dimethyl sulfoxide (Sigma, St. Louis, MO) with 0.9% NaCl and injected intraperitoneally into pups before subjecting them to sevoflurane, with a vehicle injection as control. Thirty minutes later, the injected pups were put into a semiclosed chamber and exposed to 3% sevoflurane for 4 h. After exposure, pups were returned to the parents’ cages and monitored for health status until the following tests.
+
+The pups were randomly divided into five groups as follows:
+
+Sham: vehicle intraperitoneal injection; Control: 5 mg/kg BRL-50481 intraperitoneal injection; B0: Sevoflurane anesthesia, vehicle intraperitoneal injection; B1: Sevoflurane anesthesia, 1 mg/kg BRL-50481 intra- peritoneal injection;
+
+1356
+
+https://dx.doi.org/10.1021/acschemneuro.0c00106 ACS Chem. Neurosci. 2020, 11, 1353−1358
+
+ACS Chemical Neuroscience
+
+B5: Sevoflurane anesthesia, 5 mg/kg BRL-50481 intra- peritoneal injection.
+
+Each group contained 10 pups.
+
+Morris Water Maze Test and Analysis. The spatial memory ability of control and treated mice was determined using the Morris water maze test developed by Richard Morris.31 In brief, a 160 cm diameter and 60 cm high circular tank was filled with water at 30 cm high. The water temperature was maintained at 22 °C. A 12 cm diameter circular platform was submerged 1 cm below the water surface in the center of one of the four virtual quadrants.32
+
+The control and treated mice were trained four times per day for 6 days. The mouse was released into the water, and it navigated to reach the platform. The maximum swimming time of the tested mouse was 80 s. If the mouse could escape to the refuge within 60 s, the delay to find the platform time was recorded as 60 s. Mice were allowed to stay on the platform for 15 s, and then they were sent to their cages under a heat lamp to maintain their core temperature. The escape latency was recorded by a tracking system, and data were analyzed using ViewPoint video tracking system (ViewPoint Behavior Technology, Civrieux, France). Three daily trials were averaged for each animal.32 Immunohistochemistry (IHC) Analyses. Mice were euthanized and perfused with cold phosphate-buffered saline and 4% paraformaldehyde immediately. The brains were fixed with 4% paraformaldehyde overnight and then cryoprotected by immersion in 30% sucrose at 4 °C for 48 h. Coronal sections (25 μm) were cut using a manual rotary microtome (Leica, Wetzlar, Germany).
+
+The caspase-3 IHC staining was performed as previously described.33 The cleaved caspase-3 antibody (ab13847) was purchased from Abcam (Cambridge, MA).
+
+Immunoblotting Analyses. Frozen hippocampus homogenates were lysed using radioimmunoprecipitation buffer (Bioequip, Shanghai, China). The samples were subjected to immunoblotting analysis as described previously.33 The pCREB (Ser133, #9198, 1:1000 dilution) and CREB (#9197, 1:2000 dilution) primary antibodies were ordered from Cell Signaling Technology (Danvers, MA), and the internal control β-actin antibody was ordered from Abcam (ab8226, 1:2000 dilution).
+
+cAMP Concentration Assay. cAMP levels were measured using the mouse cAMP ELISA kit (ab133051, Biocompare, South San Francisco, CA) following the manufacturer’s instructions.
+
+Statistical Analysis. Statistical analyses were carried out by using the SPSS 11.0 package. Differences between groups were analyzed using analysis of variance (ANOVA) or two-sample t test with Bonferroni correction. All data represent mean ± standard deviation (SD). Statistical significance thresholds were set at *P < 0.05.
+
+■ AUTHOR INFORMATION
+
+Corresponding Author
+
+Shunyuan Li − Department of Anesthesiology, Quanzhou First Hospital Affiliated to Fujian Medical University, Quanzhou 362000, Fujian, China; Email: cylfj@126.com
+
+orcid.org/0000-0003-1778-8386;
+
+Authors
+
+Yingle Chen − Department of Anesthesiology, Quanzhou First Hospital Affiliated to Fujian Medical University, Quanzhou 362000, Fujian, China
+
+Xianmei Zhong − Department of Anesthesiology, Quanzhou First Hospital Affiliated to Fujian Medical University, Quanzhou 362000, Fujian, China
+
+Zhenming Kang − Department of Anesthesiology, Quanzhou First Hospital Affiliated to Fujian Medical University, Quanzhou 362000, Fujian, China
+
+Rulei Chen − Department of Anesthesiology, Quanzhou First Hospital Affiliated to Fujian Medical University, Quanzhou 362000, Fujian, China
+
+Complete contact information is available at:
+
+pubs.acs.org/chemneuro
+
+Research Article
+
+https://pubs.acs.org/10.1021/acschemneuro.0c00106
+
+Author Contributions Did the experiments and analyzed the data: Y.C., S.L., X.Z., Z.K., R.C. Designed the study and wrote the manuscript: S.L. All authors approved the final submission. Funding This work was supported by the Natural Science Foundation Project of Fujian Province (#2018J01200). Notes The authors declare no competing financial interest.
+
+■ REFERENCES
+
+(1) Warner, D. O., Zaccariello, M. J., Katusic, S. K., Schroeder, D. R., Hanson, A. C., Schulte, P. J., Buenvenida, S. L., Gleich, S. J., Wilder, R. T., Sprung, J., Hu, D., Voigt, R. G., Paule, M. G., Chelonis, J. J., and Flick, R. P. (2018) Neuropsychological and Behavioral Outcomes after Exposure of Young Children to Procedures Requiring General Anesthesia: The Mayo Anesthesia Safety in Kids (MASK) Study. Anesthesiology 129, 89−105. (2) Andropoulos, D. B. (2018) Effect of Anesthesia on the Developing Brain: Infant and Fetus. Fetal Diagn Ther 43, 1−11. (3) Glatz, P., Sandin, R. H., Pedersen, N. L., Bonamy, A. K., Eriksson, L. I., and Granath, F. (2017) Association of Anesthesia and Surgery During Childhood With Long-term Academic Performance. JAMA Pediatr 171, e163470. (4) Bollen, E., and Prickaerts, J. (2012) Phosphodiesterases in neurodegenerative disorders. IUBMB Life 64, 965−970. (5) Ing, C., Wall, M. M., DiMaggio, C. J., Whitehouse, A. J. O., Hegarty, M. K., Sun, M., von Ungern-Sternberg, B. S., Li, G., and Sun, L. S. (2017) Latent Class Analysis of Neurodevelopmental Deficit After Exposure to Anesthesia in Early Childhood. J. Neurosurg Anesthesiol 29, 264−273. (6) De Hert, S., and Moerman, A. (2015) Sevoflurane. F1000Research 4, 626. (7) Goa, K. L., Noble, S., and Spencer, C. M. (1999) Sevoflurane in paediatric anaesthesia: a review. Paediatr Drugs 1, 127−153. (8) Satomoto, M., Satoh, Y., Terui, K., Miyao, H., Takishima, K., Ito, M., and Imaki, J. (2009) Neonatal Exposure to Sevoflurane Induces Abnormal Social Behaviors and Deficits in Fear Conditioning in Mice. Anesthesiology 110, 628−637. (9) Fang, F., Xue, Z., and Cang, J. (2012) Sevoflurane exposure in 7- day-old rats affects neurogenesis, neurodegeneration and neuro- cognitive function. Neurosci. Bull. 28, 499−508. (10) Xiong, W.-X., Zhou, G.-X., Wang, B., Xue, Z.-G., Wang, L., Sun, H.-C., and Ge, S.-J. (2013) Impaired spatial learning and memory after sevoflurane-nitrous oxide anesthesia in aged rats is associated with down-regulated cAMP/CREB signaling. PLoS One 8, e79408− e79408. (11) Amrock, L. G., Starner, M. L., Murphy, K. L., and Baxter, M. G. (2015) Long-term effects of single or multiple neonatal sevoflurane exposures on rat hippocampal ultrastructure. Anesthesiology 122, 87− 95. (12) Zheng, S. Q., An, L. X., Cheng, X., and Wang, Y. J. (2013) Sevoflurane causes neuronal apoptosis and adaptability changes of neonatal rats. Acta Anaesthesiol. Scand. 57, 1167−1174. (13) Nakagawa, S., Kim, J. E., Lee, R., Chen, J., Fujioka, T., Malberg, J., Tsuji, S., and Duman, R. S. (2002) Localization of phosphorylated cAMP response element-binding protein in immature neurons of adult hippocampus. J. Neurosci. 22, 9868−9876. (14) Fujioka, T., Fujioka, A., and Duman, R. S. (2004) Activation of cAMP signaling facilitates the morphological maturation of newborn neurons in adult hippocampus. J. Neurosci. 24, 319−328. (15) Ao, H., Ko, S. W., and Zhuo, M. (2006) CREB activity maintains the survival of cingulate cortical pyramidal neurons in the adult mouse brain. Mol. Pain 2, 15.
+
+1357
+
+https://dx.doi.org/10.1021/acschemneuro.0c00106 ACS Chem. Neurosci. 2020, 11, 1353−1358
+
+ACS Chemical Neuroscience
+
+(16) Ding, M.-l., Ma, H., Man, Y.-g., and Lv, H.-y. (2017) Protective effects of a green tea polyphenol, epigallocatechin-3-gallate, against sevoflurane-induced neuronal apoptosis involve regulation of CREB/ BDNF/TrkB and PI3K/Akt/mTOR signalling pathways in neonatal mice. Can. J. Physiol. Pharmacol. 95, 1396−1405. (17) Chrivia, J. C., Kwok, R. P., Lamb, N., Hagiwara, M., Montminy, M. R., and Goodman, R. H. (1993) Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365, 855−859. (18) Kida, S., and Serita, T. (2014) Functional roles of CREB as a positive regulator in the formation and enhancement of memory. Brain Res. Bull. 105, 17−24. (19) Chevalier, E., Lagente, V., Dupont, M., Fargeau, H., Palazzi, X., Richard, V., Dassaud, M., Fric, M., Coupe, M., Carre, C., Leduc, S., Bernardelli, P., Vergne, F., Berna, P., and Bertrand, C. P. (2012) Lack of involvement of type 7 phosphodiesterase in an experimental model of asthma. Eur. Respir. J. 39, 582−588. (20) Morales-Garcia, J. A., Echeverry-Alzate, V., Alonso-Gil, S., Sanz- SanCristobal, M., Lopez-Moreno, J. A., Gil, C., Martinez, A., Santos, A., and Perez-Castillo, A. (2017) Phosphodiesterase7 Inhibition Activates Adult Neurogenesis in Hippocampus and Subventricular Zone In Vitro and In Vivo. Stem Cells 35, 458−472. (21) Paterniti, I., Mazzon, E., Gil, C., Impellizzeri, D., Palomo, V., Redondo, M., Perez, D. I., Esposito, E., Martinez, A., and Cuzzocrea, S. (2011) PDE 7 inhibitors: new potential drugs for the therapy of spinal cord injury. PLoS One 6, e15937. (22) Redondo, M., Zarruk, J. G., Ceballos, P., Pérez, D. I., Pérez, C., Perez-Castillo, A., Moro, M. A., Brea, J., Val, C., Cadavid, M. I., Loza, M. I., Campillo, N. E., Martínez, A., and Gil, C. (2012) Neuroprotective efficacy of quinazoline type phosphodiesterase 7 inhibitors in cellular cultures and experimental stroke model. Eur. J. Med. Chem. 47, 175−185. (23) Morales-Garcia, J. A., Alonso-Gil, S., Gil, C., Martinez, A., Santos, A., and Perez-Castillo, A. (2015) Phosphodiesterase 7 inhibition induces dopaminergic neurogenesis in hemiparkinsonian rats. Stem Cells Transl. Med. 4, 564−575. (24) Valdes-Moreno, M. I., Alcantara-Alonso, V., Estrada-Camarena, E., Mengod, G., Amaya, M. I., Matamoros-Trejo, G., and de Gortari, P. (2017) Phosphodiesterase-7 inhibition affects accumbal and hypothalamic thyrotropin-releasing hormone expression, feeding and anxiety behavior of rats. Behav. Brain Res. 319, 165−173. (25) Medina-Rodriguez, E. M., Arenzana, F. J., Pastor, J., Redondo, M., Palomo, V., Garcia de Sola, R., Gil, C., Martinez, A., Bribian, A., and de Castro, F. (2013) Inhibition of endogenous phosphodiesterase 7 promotes oligodendrocyte precursor differentiation and survival. Cell. Mol. Life Sci. 70, 3449−3462. (26) Pekkinen, M., Ahlstrom, M. E. B., Riehle, U., Huttunen, M. M., and Lamberg-Allardt, C. J. E. (2008) Effects of phosphodiesterase 7 inhibition by RNA interference on the gene expression and differentiation of human mesenchymal stem cell-derived osteoblasts. Bone 43, 84−91. (27) Safavi, M., Baeeri, M., and Abdollahi, M. (2013) New methods for the discovery and synthesis of PDE7 inhibitors as new drugs for neurological and inflammatory disorders. Expert Opin. Drug Discovery 8, 733−751. (28) Keith, J. R., Wu, Y., Epp, J. R., and Sutherland, R. J. (2007) Fluoxetine and the dentate gyrus: memory, recovery of function, and electrophysiology. Behav. Pharmacol. 18, 521−531. (29) Li, Q. Q., Shi, G. X., Yang, J. W., Li, Z. X., Zhang, Z. H., He, T., Wang, J., Liu, L. Y., and Liu, C. Z. (2015) Hippocampal cAMP/PKA/ CREB is required for neuroprotective effect of acupuncture. Physiol. Behav. 139, 482−490. (30) Wang, H., Xu, J., Lazarovici, P., Quirion, R., and Zheng, W. (2018) cAMP Response Element-Binding Protein (CREB): A Possible Signaling Molecule Link in the Pathophysiology of Schizophrenia. Front. Mol. Neurosci. 11, 255. (31) Morris, R. (1984) Developments of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 11, 47−60.
+
+pubs.acs.org/chemneuro
+
+Research Article
+
+(32) Vorhees, C. V., and Williams, M. T. (2006) Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat. Protoc. 1, 848−858. (33) Guo, L., Zhang, P., Chen, Z., Xia, H., Li, S., Zhang, Y., Kobberup, S., Zou, W., and Lin, J. D. (2017) Hepatic neuregulin 4 signaling defines an endocrine checkpoint for steatosis-to-NASH progression. J. Clin. Invest. 127, 4449−4461.
+
+1358
+
+https://dx.doi.org/10.1021/acschemneuro.0c00106 ACS Chem. Neurosci. 2020, 11, 1353−1358

new_pdfs/10.1097_01.anes.0000291447.21046.4d.txt

@@ -0,0 +1,649 @@
+Anesthesiology 2007; 107:963–70
+
+Copyright © 2007, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc.
+
+Isoflurane Preconditioning Improves Long-term Neurologic Outcome after Hypoxic–Ischemic Brain Injury in Neonatal Rats Ping Zhao, M.D., Ph.D.,* Longyun Peng, M.D., Ph.D.,† Liaoliao Li, M.D., Ph.D.,† Xuebing Xu, M.D., Ph.D.,‡ Zhiyi Zuo, M.D., Ph.D.§
+
+Background: Preconditioning the brain with relatively safe drugs seems to be a viable option to reduce ischemic brain injury. The authors and others have shown that the volatile anesthetic isoflurane can precondition the brain against isch- emia. Here, the authors determine whether isoflurane precon- ditioning improves long-term neurologic outcome after brain ischemia.
+
+Methods: Six-day-old rats were exposed to 1.5% isoflurane for 30 min at 24 h before the brain hypoxia–ischemia that was induced by left common carotid arterial ligation and then ex- posure to 8% oxygen for 2 h. The neuropathology, motor coor- dination, and learning and memory functions were assayed 1 month after the brain ischemia. Western analysis was per- formed to quantify the expression of the heat shock protein 70, Bcl-2, and survivin 24 h after isoflurane exposure.
+
+Results: The mortality was 45% after brain hypoxia–isch- emia. Isoflurane preconditioning did not affect this mortality. However, isoflurane preconditioning attenuated ischemia-in- duced loss of neurons and brain tissues, such as cerebral cortex and hippocampus in the survivors. Isoflurane also improved the motor coordination of rats at 1 month after ischemia. The learning and memory functions as measured by performance of Y-maze and social recognition tasks in the survivors were not affected by the brain hypoxia–ischemia or isoflurane precondi- tioning. The expression of Bcl-2, a well-known antiapoptotic protein, in the hippocampus is increased after isoflurane expo- sure. This increase was reduced by the inhibitors of inducible nitric oxide synthase. Inducible nitric oxide synthase inhibition also abolished isoflurane preconditioning–induced neuropro- tection.
+
+Conclusions: Isoflurane preconditioning improved the long- term neurologic outcome after brain ischemia. Inducible nitric oxide synthase may be involved in this neuroprotection.
+
+PERINATAL hypoxic–ischemic brain injury is estimated to occur in 1 of 4,000 births.1 Most of the survivors (approximately 60%) have long-term neurologic or cog- nitive disability.1–3 Because of the huge impact on hu- man health and financial burden on our society, finding methods to reduce ischemic brain injury has been a
+
+focus of medical research. Many interventions have been explored for potential neuroprotection. However, clini- cally practical methods to reduce ischemic brain injury have not been well established yet.
+
+One of the important advances on ischemic brain in- jury research in the recent years is the recognition that ischemic injury is a dynamic process characterized by ongoing neuronal loss for a long period of time after ischemia (for weeks in rodents).4,5 Various methods or approaches have been shown to be neuroprotective in animal studies. However, few of them are effective in improving neurologic outcome in clinical studies. One of the possible reasons for this phenomenon is that previous animal studies often examined the neurologic outcome a few days after the brain ischemia and that human studies frequently evaluated neurologic outcome a few months later. It is now a well-known phenomenon that some of the protective methods may just delay cell death after brain ischemia.6 – 8 Therefore, it is important to evaluate the long-term neuroprotective effects of a method in preclinical studies.
+
+Pretreatment of various organs, including brain, with brief episodes of ischemia has been shown to reduce injury after a prolonged episode of ischemia.9 This phe- nomenon is called ischemic preconditioning. Various other stimuli, such as hypoxia and hypothermia, have been shown to induce preconditioning effects.10 –13 However, the utility of the preconditioning effects in- duced by these stimuli in clinical practice is questionable because of the danger or the complex biologic effects of the stimuli. We and others have shown that isoflurane can induce preconditioning effects in the brain.14 –16 Isoflurane is a commonly used volatile anesthetic and has been safely used in clinical practice for decades. We designed this study to test the hypothesis that isoflurane preconditioning can improve long-term neurologic out- come after brain ischemia.
+
+Research Associate, † Postdoctoral Research Fellow, Department of Anes- thesiology, § Associate Professor, Departments of Anesthesiology, Neuroscience, and Neurological Surgery, University of Virginia. ‡ Postdoctoral Research Fel- low, Departments of Anesthesiology, University of Virginia and The First Munic- ipal People’s Hospital of Guangzhou, Guangzhou, China. Received from the Department of Anesthesiology, University of Virginia, Charlottesville, Virginia. Submitted for publication March 20, 2007. Accepted for publication August 9, 2007. Supported by grant Nos. R01 GM065211 and R01 NS045983 (to Dr. Zuo) from the National Institutes of Health, Bethesda, Mary- land. Drs. Peng and Li contributed equally to the project, and both can be considered as second authors of the article.
+
+Address correspondence to Dr. Zuo: Department of Anesthesiology, Univer- sity of Virginia Health System, 1 Hospital Drive, P.O. Box 800710, Charlottesville, Virginia 22908-0710. zz3c@virginia.edu. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. ANESTHESIOLOGY’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
+
+Materials and Methods
+
+The animal protocol was approved by the institutional Animal Care and Use Committee of the University of Virginia, Charlottesville, Virginia. All animal experiments were conducted in accordance with the National Insti- tutes of Health Guide for the Care and Use of Labora- tory Animals (National Institutes of Health publication No. 80-23) revised in 1996. All reagents unless specified
+
+Anesthesiology, V 107, No 6, Dec 2007
+
+963
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 0 7 6 9 6 3 6 5 5 5 2 7 0 0 0 0 5 4 2 - 2 0 0 7 1 2 0 0 0 - 0 0 0 1 6 p d
+
+/
+
+/
+
+/
+
+/
+
+.
+
+f
+
+b y g u e s t
+
+o n 2 0 N o v e m b e r 2 0 2 3
+
+964
+
+below were obtained from Sigma Chemical (St. Louis, MO).
+
+Neonatal Cerebral Hypoxia–Ischemia Model Cerebral hypoxia–ischemia was induced as we previ- ously described.17 Briefly, 7-day-old male and female Sprague-Dawley rats were anesthetized by isoflurane in 30% O2–70% N2, and their left common carotid arteries were permanently ligated with a double 7-0 surgical silk. The rats were allowed to awake and were returned to their cages with the mothers for 3 h. The neonates were then placed in a chamber containing humidified 8% O2–92% N2 for 2 h at 37°C. The air temperature in the chamber was continuously monitored and maintained at 37°C. The chamber was then opened to room air for 15 min, and the animals were returned to their cages.
+
+Isoflurane Preconditioning and Study Groups Six-day-old rats were placed in a chamber containing 1.5% isoflurane carried by 30% O2–70% N2 for 30 min at 24 h before the cerebral hypoxia–ischemia. The neo- nates usually started to feed within 30 min after the isoflurane application. In the first set of experiments, six groups of neonates were studied: (1) control, (2) 1.5% isoflurane treatment only, (3) cerebral hypoxia–isch- emia, (4) 1.5% isoflurane pretreatment and then cerebral hypoxia–ischemia, (5) 1 mg/kg N-(3-(aminomethyl)ben- zyl)acetamidine (1400 W; BIOMOL Research Laborato- ries Inc., Plymouth Meeting, PA) injected intraperitone- ally 24 h before cerebral hypoxia–ischemia, and (6) 1 mg/kg 1400 W injected intraperitoneally 30 min before the isoflurane pretreatment and then cerebral hypoxia– ischemia. Neonates from the same mother were assigned to these six experimental conditions. Neonates in groups 2, 4, and 6 were pretreated with isoflurane, whereas the others from the same mother were placed in a chamber containing 30% O2–70% N2 but no isoflu- rane for 30 min and were assigned to groups 1, 3, and 5. In the second set of experiments, four groups of rats were studied: (1) control, (2) 1.5% isoflurane treatment, (3) 200 mg/kg aminoguanidine administered intraperito- neally 30 min before the isoflurane treatment, and (4) 1 mg/kg 1400 W injected intraperitoneally 30 min before the isoflurane treatment. Aminoguanidine and 1400 W were dissolved in normal saline, and the injected volume was from 0.16 to 0.2 ml per rat. Rats in the control group and isoflurane treatment only group received 0.2 ml normal saline at the corresponding times. Aminoguani- dine and 1400 W are inducible nitric oxide synthase (iNOS) inhibitors that have been shown to inhibit iNOS activity in rat brain18 and iNOS-mediated neuroprotec- tion induced by isoflurane and prenatal hypoxic precon- ditioning at the regimen used in this study.11,17 The rat brains were harvested 24 h after isoflurane treatment for Western analysis.
+
+Anesthesiology, V 107, No 6, Dec 2007
+
+ZHAO ET AL.
+
+Mortality and Body Weight Monitoring Death during the period from the onset of cerebral hypoxia–ischemia to 1 month afterward was recorded, and the mortality rate was calculated. Rat body weights were measured just before and 1 month after the cere- bral hypoxia–ischemia.
+
+Brain Histopathology Brain histopathologic evaluation was performed in rats in the first set of experiments. One month (30 days) after the cerebral hypoxia–ischemia, rats were euthanized by isoflurane and transcardially perfused with 30 ml saline. Brains were removed and stored in 4% phosphate-buff- ered paraformaldehyde for 4 h at room temperature. Eight-micrometer-thick cryostat coronal sections at ap- proximately 3.3 mm caudal to bregma were obtained and subjected to Nissl staining. These sections were examined by an observer blinded to the group assign- ment of the sections. The cerebral cortical and hip- pocampal areas in each of the hemispheres were mea- sured by using National Institutes of Health Image 1.60 (Bethesda, MD). The area ratio of the cerebral cortex and hippocampus in the left hemisphere to those in the right hemisphere was calculated and used to reflect brain tissue loss in the left hemisphere after brain hypoxia– ischemia. Neuronal density in the perirhinal cortex was determined as follows. A reticle (approximately 0.034 mm2) was used to count cells in the same size area. Nissl staining–positive cells were counted in the area. Three determinations, each on different locations in the left perirhinal cortex, were performed and averaged to yield a single number (density of the neurons) for the brain region of each individual rat. The neuronal density in the right perirhinal cortex was determined in the same way. The neuronal density ratio in the left/right perirhinal cortex was then calculated to measure the neuronal loss after brain hypoxia–ischemia.
+
+Motor Coordination Evaluation This evaluation was performed just before the rats were killed for brain histopathology. Rats were placed on a rotarod whose speed increased from 4 to 40 rpm in 5 min. The latency and the speed of rats’ falling off the rod were recorded. Each rat was tested three times, and the speed–latency index (latency in seconds (cid:1) speed in rpm) for each trial was calculated. The mean index value of the three trials was used to reflect the motor coordi- nation functions of each rat.
+
+Y Maze and Social Recognition The Y-maze and social recognition tests were per- formed as described previously19 at 1 day before the rats were killed for brain histopathology. During Y-maze test, rats were placed in the center of a symmetrical Y maze and were allowed to explore freely in the maze for 8 min. The total number and sequence of arms entered
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 0 7 6 9 6 3 6 5 5 5 2 7 0 0 0 0 5 4 2 - 2 0 0 7 1 2 0 0 0 - 0 0 0 1 6 p d
+
+/
+
+/
+
+/
+
+/
+
+.
+
+f
+
+b y g u e s t
+
+o n 2 0 N o v e m b e r 2 0 2 3
+
+ISOFLURANE PRECONDITIONING–INDUCED NEUROPROTECTION
+
+were recorded. An arm was entered if the hind paws of the rat were completely in the arm. The percentage alternation that was the percentage of the number of entry into all three arms in the maximum possible alter- nations (the total number of arms entered divided by 3) was calculated for each rat.
+
+The social recognition task was tested by placing a test rat in a clean acrylic cage. A male juvenile (3- to 4-week- old) rat was placed into the cage with the test rat for 2 min. The two rats were separated for 3 h and were placed together again for 2 min. The duration of social investigation of the juvenile rat by the test rat during the two 2-min periods was recorded. Social investigation behaviors include direct contact with the juvenile for inspection and close following ((cid:2) 1 cm) of the juvenile. If there was any aggressive encounter between the rats, the experiments were terminated and the data were excluded from analysis. The ratio of duration of the social investigation during the second 2-min period in the duration of the first 2-min period was calculated to measure the social recognition memory.
+
+Western Blot Analysis Cerebral cortex and hippocampus were dissected from the rats in the second set of experiments and were sonicated in ice-cold 20 mM Tris-HCl (pH 7.5) containing 5 mM Mg Cl2, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 (cid:1)g/ml aprotinin, 1 mM DL-dithiothreitol, and 2 mM sodium orthovanadate. The sample was centri- fuged at 1,000g at 4°C for 10 min. The protein concen- trations in the supernatants were determined by the Lowry assay using a protein assay kit. Equal protein samples (50 (cid:1)g per lane) were separated by 12% sodium dodecyl sulfate–polyacrylamide gels and then electro- transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). The primary antibodies were rabbit poly- clonal anti– heat shock protein 70 (HSP70) antibody (1: 1,500 dilution, catalog No. SPA-812; Stressgen, Victoria, British Columbia, Canada), antisurvivin antibody (1:500 dilution, catalog No. S8191), antiactin antibody (1:2,000 dilution, catalog No. A2066), and mouse monoclonal anti–Bcl-2 antibody (1:2,000 dilution, catalog No. sc-509; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Protein bands were visualized by the enhanced chemilumines- cence detection method with reagents from Amersham Pharmacia Biotech (Piscataway, NJ). The protein band volumes were quantified by a densitometry with Image- Quant 5.0 Windows NT software (Molecular Dynamics, Sunnyvale, CA). The volumes of Bcl-2, HSP70, and sur- vivin protein bands were normalized to those of actin to control for errors in protein sample loading and trans- ferring during the Western blot analysis. The results in the groups after isoflurane exposure were then normal- ized to those of control animals.
+
+Statistical Analysis Our previous study showed that a 2-h left hemisphere hypoxia–ischemia reduced the weight of left brain hemi- sphere by approximately 30% and isoflurane precondi- tioning decreased this brain loss to approximately 10% with an SD of approximately 12% when the brains were examined at 7 days after the brain hypoxia–ischemia.17 Based on these results, it was estimated that 7 rats per group would be needed to detect the protective effects (brain loss reduction/brain pathology) of isoflurane pre- conditioning with a desired power of 80% at an (cid:2) level of 0.05 by t test. However, this sample estimate was used only as a reference in the experimental design of this study because of the obvious differences in the duration of observation after brain hypoxia–ischemia (1 week vs. 1 month) and outcome parameters between this study and our previous study.17
+
+Data are presented as mean (cid:3) SD. Results of hip- pocampal and cortical area ratio, neuronal density ratio, speed–latency index, percentage of alternation, and the ratio of the investigation times of the different study groups were compared by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls (SNK) method or by one-way ANOVA on ranks followed by the Dunn method as appropriate. The Western blot data were analyzed by one-way ANOVA on ranks followed by the Dunn method. The mortality rates among groups were analyzed by Z test. The comparison of body weight among groups was performed by ANOVA for repeated measures followed by the SNK method. P (cid:2) 0.05 was considered significant. All statistical analyses were per- formed with SigmaStat (Systat Software, Inc., Point Rich- mond, CA).
+
+Results
+
+General characteristics of various study groups in the first set of experiments are presented in table 1. The mortality was 45% in rats that had brain hypoxia–isch- emia for 2 h. This mortality was not significantly altered by isoflurane preconditioning or application of 1400 W. The body weight of 7-day-old neonates (just before the brain hypoxia–ischemia) and 37-day-old neonates (30 days after the brain hypoxia–ischemia) among all study groups including control rats was not different (table 1). There were significant differences among the various groups of rats in the left brain loss/damage as assessed grossly (fig. 1) or by the ratio of left/right cerebral cor- tical area (F(5, 45) (cid:4) 13.82, P (cid:2) 0.001), hippocampal area (F(5, 45) (cid:4) 10.98, P (cid:2) 0.001), and neuronal density in the perirhinal cortex (F(5, 45) (cid:4) 15.61, P (cid:2) 0.001) (fig. 2). Brain hypoxia–ischemia caused significant brain loss/damage in the left hemisphere assessed at 30 days after the injury (compared with control group by SNK method, q (cid:4) 7.75, P (cid:2) 0.001 for cortical area; q (cid:4) 8.36,
+
+Anesthesiology, V 107, No 6, Dec 2007
+
+965
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 0 7 6 9 6 3 6 5 5 5 2 7 0 0 0 0 5 4 2 - 2 0 0 7 1 2 0 0 0 - 0 0 0 1 6 p d
+
+/
+
+/
+
+/
+
+/
+
+.
+
+f
+
+b y g u e s t
+
+o n 2 0 N o v e m b e r 2 0 2 3
+
+966
+
+ZHAO ET AL.
+
+Table 1. General Characteristics
+
+Fate after Brain Hypoxia–Ischemia
+
+Body Weight of the Survivors, g
+
+Dead
+
+Survived
+
+Mortality, %
+
+7 days old
+
+37 days old
+
+Control 1.5% Iso HI 1.5% Iso (cid:6) HI 1400 W (cid:6) HI 1400 W (cid:6) 1.5% Iso (cid:6) HI
+
+0 0 25 38 9 9
+
+24 23 30 30 9 9
+
+0 0 45* 56* 50* 50*
+
+13.7 (cid:3) 1.9 13.7 (cid:3) 1.4 14.3 (cid:3) 2.0 14.4 (cid:3) 2.0 14.9 (cid:3) 1.7 14.9 (cid:3) 1.2
+
+143 (cid:3) 30 150 (cid:3) 35 161 (cid:3) 23 144 (cid:3) 35 156 (cid:3) 13 153 (cid:3) 5
+
+Values (body weight) are mean (cid:3) SD. * P (cid:2) 0.05 compared with control. 1400 W (cid:4) 1 mg/kg N-(3-(aminomethyl)benzyl)acetamidine injected intraperitoneally; HI (cid:4) brain hypoxia–ischemia that was induced by left common carotid artery ligation plus hypoxia with 8% O2 for 2 h at 37°C to 7-day-old rats; Iso (cid:4) isoflurane for 30 min.
+
+P (cid:2) 0.001 for hippocampal area; and q (cid:4) 9.49, P (cid:2) 0.001 for neuronal density in the perirhinal cortex) (figs. 1 and 2). This hypoxia-ischemia–induced brain loss/dam- age was significantly attenuated by preconditioning with 1.5% isoflurane (comparison between hypoxia–ischemia and isoflurane preconditioning plus hypoxia–ischemia by SNK method, q (cid:4) 3.69, P (cid:4) 0.033 for cortical area; q (cid:4) 3.87, P (cid:4) 0.043 for hippocampal area; and q (cid:4) 4.40,
+
+P (cid:4) 0.016 for neuronal density in the perirhinal cortex) (fig. 2). These results indicated that isoflurane precondi- tioning improved neuropathology even at 1 month after brain hypoxia–ischemia. This isoflurane precondition- ing–induced improvement was attenuated by the iNOS inhibitor 1400 W (fig. 1 and 2), suggesting a role of iNOS in this protection.
+
+Fig. 1. Brain hypoxia-ischemia–induced brain tissue loss. Rep- resentative brains of 37-day-old rats with various treatments. 1400 W (cid:1) 1 mg/kg N-(3-(aminomethyl)benzyl)acetamidine in- jected intraperitoneally 30 min before the isoflurane exposure or 24 h before the cerebral hypoxia–ischemia; Con (cid:1) control; HI (cid:1) cerebral hypoxia–ischemia that was induced by left com- mon carotid artery ligation plus hypoxia with 8% O2 for 2 h at 37°C to 7-day-old rats; Iso (cid:1) 1.5% isoflurane for 30 min.
+
+The motor coordination functions as reflected by the speed–latency index in the rotarod test among the rats in various groups were statistically different (F(5, 112) (cid:4) 8.66, P (cid:2) 0.001) (fig. 3). The brain hypoxia–ischemia impaired motor coordination functions (compared with control group by SNK method, q (cid:4) 4.53, P (cid:4) 0.005). This impairment was significantly attenuated by isoflu- rane preconditioning (comparison between hypoxia– ischemia and isoflurane preconditioning plus hypoxia– ischemia by SNK method, q (cid:4) 3.26, P (cid:4) 0.023) (fig. 3), suggesting that isoflurane preconditioning improved mo- tor functions after brain ischemia. The iNOS inhibitor 1400 W abolished this improvement caused by isoflu- rane preconditioning (comparison between isoflurane preconditioning plus hypoxia–ischemia and 1400 W plus isoflurane preconditioning plus hypoxia–ischemia by SNK method, q (cid:4) 5.28, P (cid:4) 0.002), indicating the role of iNOS in the isoflurane preconditioning–induced motor coordination improvement. There was no differ- ence among the rats from various groups in the perfor- mance of Y maze (by one-way ANOVA on ranks, H5 (cid:4) 7.82, P (cid:4) 0.166) or the social recognition tasks (by one-way ANOVA on ranks, H5 (cid:4) 4.29, P (cid:4) 0.509) (fig. 3), suggesting that brain hypoxia–ischemia or isoflurane treatment did not affect the performance of rats in the Y-maze and social recognition tasks. Of note, the total numbers of arms entered by control rats and rats with cerebral hypoxia–ischemia only were 15 (cid:3) 9 and 16 (cid:3) 12, respectively (P (cid:5) 0.05), suggesting that cerebral hypoxia–ischemia did not impair the motor functions severely enough to affect the performance of rats in the Y-maze test.
+
+Western analysis showed that there was significant difference in Bcl-2 expression in the hippocampus
+
+Anesthesiology, V 107, No 6, Dec 2007
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 0 7 6 9 6 3 6 5 5 5 2 7 0 0 0 0 5 4 2 - 2 0 0 7 1 2 0 0 0 - 0 0 0 1 6 p d
+
+/
+
+/
+
+/
+
+/
+
+.
+
+f
+
+b y g u e s t
+
+o n 2 0 N o v e m b e r 2 0 2 3
+
+ISOFLURANE PRECONDITIONING–INDUCED NEUROPROTECTION
+
+Fig. 2. Isoflurane preconditioning re- duced brain hypoxia-ischemia–induced neuropathology. (A) Representative coro- nal sections at approximately 3.3 mm caudal to bregma from 37-day-old rats and after Nissl staining. (B) Area ratio of left/right cerebral cortex. (C) Area ratio of left/right hippocampus. (D) Neuronal density ratio in the left/right perirhinal cortex. Results are mean (cid:2) SD (n (cid:1) 6 –11). * P < 0.05 compared with control. # P < 0.05 compared with cerebral hy- poxia–ischemia only. 1400 W (cid:1) 1 mg/kg N-(3-(aminomethyl)benzyl)acetamidine injected intraperitoneally 30 min before the isoflurane exposure or 24 h before the cerebral hypoxia–ischemia; Con (cid:1) control; HI (cid:1) cerebral hypoxia–ischemia that was induced by left common carotid artery ligation plus hypoxia with 8% O2 for 2 h at 37°C to 7-day-old rats; Iso (cid:1) 1.5% isoflurane for 30 min.
+
+among the various groups (by one-way ANOVA on ranks, H3 (cid:4) 13.18, P (cid:4) 0.004). Isoflurane significantly in- creased the expression of Bcl-2 in the hippocampus (comparison between control and isoflurane exposure groups by Dunn method, Q (cid:4) 2.98, P (cid:2) 0.05). This increased expression was decreased by aminoguanidine and 1400 W, two iNOS inhibitors, suggesting that isoflu- rane-induced Bcl-2 expression was iNOS dependent (fig. 4). Isoflurane exposure did not change the expression of survivin and HSP70 in the hippocampus or cerebral cortex (fig. 4).
+
+Discussion
+
+These results suggest that isoflurane preconditioning im- proves the long-term neurologic outcome after brain ischemia.
+
+We used 1.5% isoflurane in this study. This concentra- tion was the highest concentration that did not signifi- cantly affect the blood gases and pH but induced pre- conditioning effects on the brain in our previous study using the same animal model.17 One minimum alveolar concentration (the concentration to inhibit 50% of sub- jects to respond to surgical stimuli) of isoflurane is 1.12% and 1.15%, respectively, for adult rats and humans22,23 and 1.6% for human neonates.24 Therefore, the isoflu- rane concentration used in this study is clinically rele- vant.
+
+Early studies showed infarct maturation after 2 days in rat models of stroke.20,21 Studies in the recent years have shown that ischemic injury is a dynamic process charac- terized by ongoing neuronal loss for at least 14 days after ischemia in rodents.4,5 Protective methods, such as postinjury mild hypothermia, reduced brain injury eval- uated a few days after the ischemia but did not show protection when the evaluation was performed 1 month after the brain ischemia.7,8 These studies underscore the importance of examining the long-term neurologic out- come of any protective methods. Our study showed that isoflurane preconditioning reduced cerebral cortical and loss and improved motor coordination hippocampal functions at 1 month after the brain hypoxia–ischemia.
+
+We ligated one common carotid artery and then ex- posed 7-day-old neonates to 8% oxygen to induce hy- poxic–ischemic brain injury. This is a widely used animal model to simulate human perinatal brain ischemia.25 The maturation of the brain in the 7-day-old rat is similar to that of human newborn brain.25,26 Perinatal brain isch- emia in human is often caused by brain ischemia super- imposed on severe systemic hypoxia.27 Therefore, the brain injury in the animal model used in our study shares many features of the human perinatal brain injury.
+
+We monitored the mortality and body weight gain to measure the general well being of the rats in each study group. The hypoxic–ischemic injury caused 45% mortal- ity and isoflurane preconditioning did not affect this
+
+Anesthesiology, V 107, No 6, Dec 2007
+
+967
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 0 7 6 9 6 3 6 5 5 5 2 7 0 0 0 0 5 4 2 - 2 0 0 7 1 2 0 0 0 - 0 0 0 1 6 p d
+
+/
+
+/
+
+/
+
+/
+
+.
+
+f
+
+b y g u e s t
+
+o n 2 0 N o v e m b e r 2 0 2 3
+
+968
+
+Fig. 3. Isoflurane preconditioning improved motor coordina- tion after brain hypoxia–ischemia. (A) Speed–latency index of 37-day-old rats with various treatments in the rotarod test. (B) Percentage of alternation of 37-day-old rats with various treat- ments in the Y-maze test. (C) Ratio of the investigation times in the second trial over those in the first trial. Results are mean (cid:2) SD (n (cid:1) 9 –30). * P < 0.05 compared with control. # P < 0.05 compared with cerebral hypoxia–ischemia only. ^ P < 0.05 compared with isoflurane preconditioning plus cerebral hy- poxia–ischemia. 1400 W (cid:1) 1 mg/kg N-(3-(aminomethyl)benzy- l)acetamidine injected intraperitoneally 30 min before the isoflurane exposure or 24 h before the cerebral hypoxia–isch- emia; HI (cid:1) cerebral hypoxia–ischemia that was induced by left common carotid artery ligation plus hypoxia with 8% O2 for 2 h at 37°C to 7-day-old rats; Iso (cid:1) 1.5% isoflurane for 30 min.
+
+mortality rate. These results are consistent with those of our previous report.17 The body weights of the survivors at the end of the study in all four groups are not signif- icantly different, suggesting the body weight gain is not a sensitive parameter to reflect the degree of brain injury in this animal model. These results are also similar to our previous data.17
+
+We quantified hypoxia-ischemia–induced brain loss by the cortical and hippocampal area and neuronal density in the perirhinal cortex at approximately 3.3 mm caudal to bregma. The section at this level was used to reflex the brain injury because this section contains hippocam-
+
+Anesthesiology, V 107, No 6, Dec 2007
+
+ZHAO ET AL.
+
+pus and the degree of brain injury was similar in sections from 1 mm rostral to 5 mm caudal to bregma in this brain hypoxia–ischemia model.28 We chose to count neuronal density in the perirhinal cortex because this brain region is easily recognized and localization of this region in brain sections can be very accurate. Blood supply of the perirhinal cortex is mainly from the ipsilateral internal carotid artery. Therefore, significant neuronal injury in the perirhinal cortex was anticipated in our newborn rats after the cerebral hypoxia–ischemia. Our study showed that the cortical and hippocampal area and the neuronal density in the perirhinal cortex of the ischemic hemisphere were significantly decreased by the hypoxi- a–ischemia and this decrease was attenuated by isoflu- rane preconditioning. These results are strong evidence that isoflurane preconditioning improves neuropatho- logical outcome at 1 month after brain hypoxia–isch- emia.
+
+Two types of neurologic functions were monitored in our study. The motor coordination functions of the rats were assayed by the rotarod test. Hypoxic–ischemic brain injury significantly reduced the duration and speed that the rats could stay on the rotarod compared with control rats, suggesting that these rats had impaired motor coordination functions. Rats preconditioned by isoflurane before the hypoxic–ischemic injury per- formed better than rats subjected to hypoxic–ischemic injury only. Therefore, isoflurane preconditioning im- proves not only the neuropathologic outcome but also neurologic functions after brain ischemia.
+
+The learning and memory functions were evaluated by the Y-maze and social recognition tasks. These two tasks are very sensitive for measuring early learning and mem- ory deficits.19 The social recognition tests rats to identify and remember con-specifics, whereas the spontaneous alternation Y maze assesses spatial working memory. Because these two tasks measure hippocampus-depen- dent learning and memory functions19 and the hip- pocampus in the ischemic hemisphere in our model was obviously injured, one would expect that the rats after hypoxic–ischemic brain injury would have had worse performance than did control rats in the Y-maze and social recognition tasks. To our surprise, there was no significant difference in the performance of these two tasks among the six groups of rats. Poor performance on water maze tasks that examine long-term spatial learning and reference memory was found with the neonatal rats after hypoxic–ischemic brain injury.29 However, the per- formance of those rats on eight-arm maze tasks that test long-term reference memory and short-term working memory was not significantly different from the control rats in the same study.29 The reasons for the apparent discrepancy of the findings from water maze and eight- arm maze tasks in this previous study and the obvious brain structure injury and the maintained learning and memory functions assayed by the Y-maze and social
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 0 7 6 9 6 3 6 5 5 5 2 7 0 0 0 0 5 4 2 - 2 0 0 7 1 2 0 0 0 - 0 0 0 1 6 p d
+
+/
+
+/
+
+/
+
+/
+
+.
+
+f
+
+b y g u e s t
+
+o n 2 0 N o v e m b e r 2 0 2 3
+
+ISOFLURANE PRECONDITIONING–INDUCED NEUROPROTECTION
+
+Fig. 4. The effects of isoflurane on the expression of heat shock protein 70 (HSP70), Bcl-2, and survivin proteins in the cerebral cortex and hippocampus of 7-day-old rats. Six-day-old rats were exposed to 1.5% isoflurane for 30 min, and the cerebral cortex and hippocampus were removed for Western analysis at 24 h after the isoflurane exposure. (A and C) A representative film image of the bands. (B and D) The graphic presentation of HSP70, Bcl-2, and survivin protein abundance quantified by integrating the volume of bands from 5–10 rats for each experimental condition and normalizing the data by those of actin. Values in graphs are mean (cid:2) SD of the fold changes over the controls, with the controls being set as 1. * P < 0.05 compared with controls. # P < 0.05 compared with isoflurane only. 1400 W (cid:3) Iso (cid:1) 1 mg/kg N-(3-(aminomethyl)benzyl)acetamidine (1400 W) injected intraperitoneally 30 min before the isoflurane exposure; Ag (cid:3) Iso (cid:1) 200 mg/kg aminoguanidine injected intraperitoneally at 30 min before the isoflurane exposure; Iso (cid:1) 1.5% isoflurane for 30 min.
+
+recognition tasks in our study are not known. Y-maze, social recognition, and eight-arm tasks are nonstressful, and the water maze test is stressful. One explanation for the discrepancy is that rats after hypoxic–ischemic brain injury can compensate well with various mechanisms, such as through the functions of the nonischemic hemi- sphere, in performing nonstressful tasks but not perform well in the stressful tasks that measure the learning and memory functions.
+
+It has been proposed that delayed neuroprotection that occurs a few hours after the application of precon- ditioning stimuli involves synthesis of protective pro- teins.30 Bcl-2 can reduce ischemia-induced increase of mitochondrial membrane permeability and cytochrome c release from the mitochondrion.31 The released cyto- chrome c will bind with protease-activating factor 1 to form the apoptosome that will activate caspase 9. This process ultimately will activate caspase 3 to induce cell apoptosis. Bcl-2 can bind with the C terminus of pro- tease-activating factor 1 to inhibit the association of caspase 9 with protease-activating factor 1.32–34 Thus, Bcl-2, via acting on various steps, inhibits apoptosis and is a protective protein. Our results showed that rats exposed to isoflurane had an increased Bcl-2 in the hippocampus and this increase was inhibited by amino- guanidine and 1400 W, two iNOS inhibitors. Isoflurane preconditioning–induced neuroprotection observed 1 week after the brain ischemia was shown to be iNOS
+
+dependent in our previous study using the same animal model.17 In this study, 1400 W abolished the isoflurane preconditioning–induced neuroprotection. Therefore, our results suggest that the increased Bcl-2 expression contributes to the neuroprotection induced by isoflu- it is rane preconditioning. Consistent with this idea, known that brain injury in the neonatal brain hypoxia– ischemia model is caused, at least partly, by apopto- sis.35,36 Our results also suggest a link between iNOS and Bcl-2. Nitric oxide can induce Bcl-2 expression.37 Nitric oxide produced by iNOS can activate signal transducer and activator of transcription 3,38 a transcription factor that increases Bcl-2 expression.39 These previous stud- ies, along with the results presented here, suggest that iNOS is a signaling molecule upstream of Bcl-2 to medi- ate isoflurane preconditioning–induced neuroprotec- tion.
+
+Survivin is a member of the inhibitor of apoptosis gene family and may be through its association with caspases to inhibit apoptosis.40,41 Our results showed that isoflu- rane exposure did not alter the expression of survivin in the cerebral cortex and hippocampus of rats, suggesting that isoflurane preconditioning–induced neuroprotec- tion does not involve survivin. Isoflurane exposure also did not change the expression of HSP70. HSP70 is mo- lecular chaperons for damaged proteins and can be in- to provide protec- duced by various stress stimuli tion.42,43 Our results indicate that HSP70 is not involved
+
+Anesthesiology, V 107, No 6, Dec 2007
+
+969
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 0 7 6 9 6 3 6 5 5 5 2 7 0 0 0 0 5 4 2 - 2 0 0 7 1 2 0 0 0 - 0 0 0 1 6 p d
+
+/
+
+/
+
+/
+
+/
+
+.
+
+f
+
+b y g u e s t
+
+o n 2 0 N o v e m b e r 2 0 2 3
+
+970
+
+in the isoflurane preconditioning–induced neuroprotec- tion. However, caution should be exercised regarding the suggestion of noninvolvement of survivin and HSP70 in the isoflurane preconditioning–induced neuroprotec- tion because the expression change of these two pro- teins may occur at other time points after isoflurane preconditioning. We chose to quantify protein expres- sion at 24 h after isoflurane exposure in this study be- cause we subjected the rats to the brain hypoxic–isch- emic injury at this time point and reasoned that the involved protective proteins should be expressed at this time to reduce brain injury.
+
+In summary, we have shown that preconditioning with isoflurane at a clinically relevant concentration can in- duce a long-lasting neuroprotection in rats. This effect may involve an increased expression of Bcl-2. Because isoflurane is a commonly used and relatively safe drug, our finding may have implications in clinical situations that brain ischemia occurs as a planned or anticipated event, such as perceived difficult labor with potential newborn brain ischemia, newborns for open heart sur- gery and newborns with high risks of intraventricular or periventricular hemorrhage.
+
+References
+
+1. Lynch JK, Nelson KB: Epidemiology of perinatal stroke. Curr Opin Pediatr
+
+2001; 13:499–505
+
+2. Sran SK, Baumann RJ: Outcome of neonatal strokes. Am J Dis Child 1988;
+
+142:1086–8
+
+3. Sreenan C, Bhargave R, Robertson CM: Cerebral infarction in the term new-born: Clinical presentation and long-term outcome. J Pediatr 2000; 137: 351–5
+
+4. Li Y, Chopp M, Jiang N, Yao F, Zaloga C: Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 1995; 15:389–97
+
+5. Du C, Hu R, Csernansky CA, Hsu CY, Choi D: Very delayed infarction after mild focal cerebral ischemia: A role for apoptosis? J Cereb Blood Flow Metab 1996; 16:195–201
+
+6. Kawaguchi M, Kimbro JR, Drummond JC, Cole DJ, Kelly PJ, Patel PM: Isoflurane delays but does not prevent cerebral infarction in rats subjected to focal ischemia. ANESTHESIOLOGY 2000; 92:1335–42
+
+7. Dietrich WD, Busto R, Alonso O, Globus MY, Ginsberg MD: Intraischemic but not postischemic brain hypothermia protects chronically following global forebrain ischemia in rats. J Cereb Blood Flow Metab 1993; 13:541–9
+
+8. Trescher WH, Ishiwa S, Johnston MV: Brief post-hypoxic-ischemic hypo-
+
+thermia markedly delays neonatal brain injury. Brain Dev 1997; 19:326–38
+
+9. Nandagopal K, Dawson TM, Dawson VL: Critical role for nitric oxide signaling in cardiac and neuronal ischemic preconditioning and tolerance. JPET 2001; 297:474–8
+
+10. Gidday JM, Shah AR, Maceren RG, Wang Q, Pelligrino DA, Holtzman DM, Park TS: Nitric oxide mediates cerebral ischemic tolerance in a neonatal rat model of hypoxic preconditioning. J Cereb Blood Flow Metab 1999; 19:331–40 11. Zhao P, Zuo Z: Prenatal hypoxia-induced adaptation and neuroprotection that is inducible nitric oxide synthase-dependent. Neurobiol Dis 2005; 20:871–80 12. Yuan H-B, Huang Y, Zheng S, Zuo Z: Hypothermic preconditioning in- creases survival of Purkinje neurons in rat cerebellar slices after an in vitro simulated ischemia. ANESTHESIOLOGY 2004; 100:331–7
+
+13. Yuan HB, Huang Y, Zheng S, Zuo Z: Hypothermic preconditioning reduces Purkinje cell death possibly by preventing the over-expression of inducible nitric oxide synthase in rat cerebellar slices after an in vitro simulated ischemia. Neuroscience 2006; 142:381–9
+
+14. Zheng S, Zuo Z: Isoflurane preconditioning reduces Purkinje cell death in
+
+an in vitro model of rat cerebellar ischemia. Neuroscience 2003; 118:99–106
+
+15. Zheng S, Zuo Z: Isoflurane preconditioning induces neuroprotection
+
+Anesthesiology, V 107, No 6, Dec 2007
+
+ZHAO ET AL.
+
+against ischemia via activation of p38 mitogen-activated protein kinase. Mol Pharmacol 2004; 65:1172–80
+
+16. Kapinya KJ, Lowl D, Futterer C, Maurer M, Waschke K, Isaev NK, Dirnagl U: Tolerance against ischemic neuronal injury can be induced by volatile anes- thetics and is inducible NO synthase dependent. Stroke 2002; 33:1889–98
+
+17. Zhao P, Zuo Z: Isoflurane preconditioning induces neuroprotection that is inducible nitric oxide synthase-dependent in the neonatal rats. ANESTHESIOLOGY 2004; 101:695–702
+
+18. Nagayama M, Zhang F, Iadecola C: Delayed treatment with aminoguani- dine decreases focal cerebral ischemic damage and enhances neurologic recov- ery in rats. J Cereb Blood Flow Metab 1998; 18:1107–13
+
+19. Ohno M, Sametsky EA, Younkin LH, Oakley H, Younkin SG, Citron M, Vassar R, Disterhoft JF: BACE1 deficiency rescues memory deficits and cholin- ergic dysfunction in a mouse model of Alzheimer’s disease. Neuron 2004; 41: 27–33
+
+20. Garcia J, Yoshida Y, Chen H, Li Y, Zhang Z, Lian J, Chen S, Chopp M: Progression from ischemic injury to infarct following middle cerebral artery occlusion in the rat. Am J Pathol 1993; 142:623–35
+
+21. Lin T, He Y, Wu G, Khan M, Hsu C: Effect of brain edema on infarct volume
+
+in a focal cerebral ischemia model in rats. Stroke 1993; 24:117–21
+
+22. Orliaguet G, Vivien B, Langeron O, Bouhemad B, Coriat P, Riou B: Mini- mum alveolar concentration of volatile anesthetics in rats during postnatal mat- uration. ANESTHESIOLOGY 2001; 95:734–9
+
+23. Eger EI: Uptake and distribution, Anesthesia. Edited by Miller RD. Phila-
+
+delphia, Churchill Livingstone, 2000, pp 74 –95
+
+24. LeDez KM, Lerman J: The minimum alveolar concentration (MAC) of
+
+isoflurane in preterm neonates. ANESTHESIOLOGY 1987; 67:301–7
+
+25. Hagberg H, Bona E, Gilland E, Puka-Sundvall M: Hypoxia-ischaemia model in the 7-day-old rat: Possibilities and shortcomings. Acta Paediatr Suppl 1997; 422:85–8
+
+26. Dobbing J, Sands J: The brain growth spurt in various mammalian species.
+
+Early Hum Dev 1979; 3:79–84
+
+27. Johnston MV: Neonatal hypoxic-ischemic brain insults and their mecha- nisms, New Concepts in Cerebral Ischemia. Edited by Simon SA, Nicolelis MAL. Boca Raton, CRC Press, 2002, pp 31– 61
+
+28. Ma D, Hossain M, Chow A, Arshad M, Battson RM, Sanders RD, Mehmet H, Edwards AD, Franks NP, Maze M: Xenon and hypothermia combine to provide neuroprotection from neonatal asphyxia. Ann. Neurol 2005; 58:182–93
+
+29. Ikeda T, Mishima K, Yoshikawa T, Iwasaki K, Fujiwara M, Xia YX, Ikenoue T: Selective and long-term learning impairment following neonatal hypoxic- ischemic brain insult in rats. Behav Brain Res 2001; 118:17–25
+
+30. Dirnagl U, Simon RP, Hallenbeck JM: Ischemic tolerance and endogenous
+
+neuroprotection. Trends Neurosci 2003; 26:248–54
+
+31. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD: The release of cytochrome c from mitochondria: A primary site for Bcl-2 regulation of apopto- sis. Science 1997; 275:1132–6
+
+32. Hu Y, Benedict MA, Wu D, Inohara N, Nunez G: Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation. Proc Natl Acad Sci U S A 1998; 95:4386–91
+
+33. Huang DC, Adams JM, Cory S: The conserved N-terminal BH4 domain of Bcl-2 homologues is essential for inhibition of apoptosis and interaction with CED-4. EMBO J 1998; 17:1029–39
+
+34. Pan G, O’Rourke K, Dixit VM: Caspase-9, Bcl-XL, and Apaf-1 form a ternary
+
+complex. J Biol Chem 1998; 273:5841–5
+
+35. Nakajima W, Ishida A, Lange MS, Gabrielson KL, Wilson MA, Martin LJ, Blue ME, Johnston MV: Apoptosis has a prolonged role in the neurodegeneration after hypoxic ischemia in the newborn rat. J. Neurosci 2000; 20:7994–8004
+
+36. Gill R, Soriano M, Blomgren K, Hagberg H, Wybrecht R, Miss MT, Hoefer S, Adam G, Niederhauser O, Kemp JA, Loetscher H: Role of caspase-3 activation in cerebral ischemia-induced neurodegeneration in adult and neonatal brain. J Cereb Blood Flow Metab 2002; 22:420–30
+
+37. Nishikawa M, Takeda K, Sato EF, Kuroki T, Inoue M: Nitric oxide regulates energy metabolism and Bcl-2 expression in intestinal epithelial cells. Am J Physiol 1998; 274:G797–801
+
+38. Hierholzer C, Kalff JC, Billiar TR, Bauer AJ, Tweardy DJ, Harbrecht BG: Induced nitric oxide promotes intestinal inflammation following hemorrhagic shock. Am J Physiol Gastrointest Liver Physiol 2004; 286:G225–33
+
+39. Alas S, Emmanouilides C, Bonavida B: Inhibition of interleukin 10 by rituximab results in down-regulation of bcl-2 and sensitization of B-cell non- Hodgkin’s lymphoma to apoptosis. Clin Cancer Res 2001; 7:709–23
+
+40. Altieri DC: Survivin in apoptosis control and cell cycle regulation in
+
+cancer. Prog Cell Cycle Res 2003; 5:447–52
+
+41. Kobayashi K, Hatano M, Otaki M, Ogasawara T, Tokuhisa T: Expression of a murine homologue of the inhibitor of apoptosis protein is related to cell proliferation. Proc Natl Acad Sci U S A 1999; 96:1457–62
+
+42. Kelly S, Yenari MA: Neuroprotection: Heat shock proteins. Curr Med Res
+
+Opin 2002; 18 (suppl 2):s55–60
+
+43. Giffard RG, Yenari MA: Many mechanisms for hsp70 protection from
+
+cerebral ischemia. J Neurosurg Anesthesiol 2004; 16:53–61
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 0 7 6 9 6 3 6 5 5 5 2 7 0 0 0 0 5 4 2 - 2 0 0 7 1 2 0 0 0 - 0 0 0 1 6 p d
+
+/
+
+/
+
+/
+
+/
+
+.
+
+f
+
+b y g u e s t
+
+o n 2 0 N o v e m b e r 2 0 2 3

new_pdfs/10.1097_ALN.0000000000002904.txt

@@ -0,0 +1,779 @@
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+HHS Public Access Author manuscript Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Published in final edited form as:
+
+Anesthesiology. 2019 November ; 131(5): 1077–1091. doi:10.1097/ALN.0000000000002904.
+
+Early Postnatal Exposure to Isoflurane Disrupts Oligodendrocyte Development and Myelin Formation in the Mouse Hippocampus.
+
+Qun Li, Ph.D., Reilley P. Mathena, B.S., Jing Xu, M.D., O’Rukevwe N. Eregha, B.A., Jieqiong Wen, B.S., Cyrus D. Mintz, M.D., Ph.D. Department of Anesthesiology and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, MD.
+
+Abstract
+
+Background: Early postnatal exposure to general anesthetics may interfere with brain development. We tested the hypothesis that isoflurane causes a lasting disruption in myelin development via actions on the mammalian target of rapamycin (mTOR) pathway.
+
+Methods: Mice were exposed to 1.5% isoflurane for 4 hours at postnatal day 7. The mTOR inhibitor, rapamycin, or the pro-myelination drug, clemastine, were administered on days 21-35. Mice underwent Y-maze and novel object position recognition tests (n=12 per group) on days 56-62 or were sacrificed for either immunohistochemistry (n=8 per group), Western blotting (n=8 per group) at day 35 or were sacrificed for electron microscopy at day 63.
+
+Results: Isoflurane exposure increased the percentage of pS6+ oligodendrocytes in fimbria of hippocampus from 22±7% to 51±6% (p<0.0001). In Y-maze testing, isoflurane-exposed mice did not discriminate normally between old and novel arms, spending equal time in both (50±5% old: 50±5% novel, p=0.999), indicating impaired spatial learning. Treatment with clemastine restored discrimination, as evidenced by increased time spent in the novel arm (43±6% old:57±6% novel, p<0.001) and rapamycin had a similar effect (44±8% old:56±8% novel; p<0.001). Electron microscopy shows a reduction in myelin thickness as measured by an increase in g-ratio from 0.76±0.06 for controls to 0.79±0.06 for the isoflurane group (p<0.001). Isoflurane exposure followed by rapamycin treatment resulted in a g-ratio (0.75±0.05) that did not differ significantly from the control value (p=0.426). Immunohistochemistry and Western blotting show that isoflurane acts on oligodendrocyte precursor cells to inhibit both proliferation and differentiation. DNA methylation and expression of a DNA methyl transferase 1 is reduced in oligodendrocyte precursor cells after isoflurane treatment. Effects of isoflurane on oligodendrocyte precursor cells were abolished by treatment with rapamycin.
+
+Corresponding Author: Cyrus D. Mintz, M.D., Ph.D., Johns Hopkins University School of Medicine Department of Anesthesiology and Critical Care Medicine, 720 Rutland Ave., Ross 370, Baltimore, MD 21205, 917-733-0422, cmintz2@jhmi.edu. Clinical Trial Number and Registry URL: Not applicable.
+
+Prior Presentations: Title: Early Postnatal Exposure to General Anesthesia Disrupts Oligodendrocyte Development and Myelin Formation in Hippocampus. Presentation 1: The Sixth Pediatric Anesthesia and NeuroDevelopment Assessment (PANDA) Symposium: Moderated Poster Discussion. Saturday, 2:00-3:00 pm, April 14th, 2018. New York. Presentation 2: The Forty Eighth Society for Neuroscience (SfN) Annual Meeting. Poster Presentation (#204.15). Sunday 1:00-5:00 pm. November 4, 2018. San Diego, CA
+
+Conflicts of Interest: The authors declare no competing interests.
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+Conclusions: Early postnatal exposure to isoflurane in mice causes lasting disruptions of oligodendrocyte development in the hippocampus via actions on the mTOR pathway.
+
+Summary Statement:
+
+Early (postnatal day 7) isoflurane exposure in mice disrupts oligodendrocyte development and myelin formation in hippocampal white matter via activation of mTOR and alterations of in DNA methylation levels.
+
+Introduction
+
+Modern general anesthesia allows the safe performance of several hundred million surgical procedures annually. 1 However, there is growing concern that some vulnerable categories of patients, particularly young children, geriatric patients, and individuals with underlying brain disorders, may be at risk of lasting cognitive dysfunction.2-4 While conclusive evidence of anesthetic neurotoxicity has not been established in human studies, some animal studies have shown that exposure to general anesthetics in early development cause impaired neurocognitive performance5-8 and that the peak period of behavioral and cognitive vulnerability to general anesthetics in rodents occurs in early postnatal life.9-11 Based on these studies, the U.S. Food and Drug Administration issued a warning that lengthy or repeated exposure to general anesthetics and sedative drugs from the third trimester of prenatal development through the first three years of life may cause lasting impairment in the cognitive function.12 The molecular and cellular mechanisms underlying this phenomenon remain poorly understood.
+
+Most studies investigating anesthetic neurotoxicity have focused on neuronal development. 7,13 However, brain function is also dependent on the myelin-forming oligodendrocytes, which undergo critical developmental events during the putative window of vulnerability. Myelination involves proliferation of oligodendrocyte progenitor cells, differentiation of oligodendrocyte progenitor cells into mature oligodendrocytes, and ensheathment of axons. Myelin is critical for neurotransmission in the CNS and disruptions of myelin function are associated with neurological and psychiatric disorders.14,15 In this study, we test the hypothesis that early postnatal exposure to isoflurane affects oligodendrocyte development and myelin formation in the hippocampus in an in vivo mouse model. To establish the extent to which isoflurane-induced deficits can be attributed to impaired myelination, we employed clemastine, an antimuscarinic drug approved drug for multiple sclerosis therapy, which promotes oligodendrocyte differentiation and myelination and reverses the phenotype of several murine disease models involving demylelination.16,17
+
+Exposure to general anesthetics has been shown to impact molecular signaling pathways implicated in the dynamic maintenance of cellular homeostasis and development.13 Recently, the mammalian target of rapamycin (mTOR) signaling has emerged as a critical integrator of activity of nerve cells and synaptic inputs that in turn affect many cellular metabolic processes.18 Studies have implicated mTOR signaling in neurodevelopmental and neuropsychiatric disorders.19 We previously showed that isoflurane disrupts development of newborn hippocampal neurons and synaptic formation via activation of the mTOR pathway. 7, 20 In this study, we further investigate the role of mTOR in isoflurane-induced
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 2
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+neurotoxicity in mouse oligodendrocyte development and myelination using mTOR activity markers and rapamycin, an mTOR pathway inhibitor. We explored the effects on axon- oligodendrocyte precursor synapses, which are thought to be critical for turning oligodendrocyte development to match neuronal activity.21-23 Activity in the mTOR pathway mediates DNA methylation in neurons 24 and cancer cells,25 and it has been reported that DNA methylation is a well-recognized epigenetic modification that regulates oligodendrocyte development and is necessary for efficient myelin formation.26-28 Thus, the effect of general anesthetics and mTOR activation on DNA methylation level in oligodendrocytes has also been investigated.
+
+Materials and Methods
+
+Animal paradigm and experimental timeline.
+
+A total of 120 (61 male and 59 female) immature C57BL/6 mice (body weight = 4.4±0.9 g. at postnatal day 7) were used in this study. 84 (44 male and 40 female) of them were randomly selected for the rapamycin experiment and 36 (17 male and 19 female) for the clemastine experiment. Sex was not factored into research design as a biological variable. Both sexes were equally represented in all experiments. All study protocols involving mice were approved by the Animal Care and Use Committee at the Johns Hopkins University and conducted in accordance with the NIH guidelines for care and use of animals. Experimental procedures followed the modified protocols from a previously published journal.7
+
+At postnatal day 7, animals were exposed to isoflurane or room air for 4 hours. From postnatal days 21-35, half of the isoflurane-exposed mice were injected (i.p.) bi-daily with rapamycin (n=28 per group) or fed daily with clemastine through gastric gavage (n=12 per group). The other half were injected with vehicle of rapamycin or fed with vehicle of clemastine.
+
+For the rapamycin experiment, a subset of mice from each group were sacrificed at postnatal day 35 for immunohistochemistry (n=8 per group) or Western blotting (n=8 per group). The remaining mice underwent behavioral testing for spatial learning and memory functions between postnatal days 56-62 (n=12 for each group). After behavior tests, two mice from each group were processed for electron microscopy at postnatal day 63. Only behavior tests were conducted for clemastine feeding experiment (n=12 for each group) (Fig. 1A).
+
+Isoflurane exposure.
+
+At postnatal day 7, two-thirds of the mice were evenly distributed across littermate groups and were randomly selected for isoflurane exposure. The other one-third of the mice stayed in room air as a naïve control. Volatile anesthesia exposure was accomplished using a Supera tabletop portable non-rebreathing anesthesia machine. 3% isoflurane mixed in 100% oxygen was initially delivered in a closed chamber for 3-5 min and after loss of righting reflex, animals were transferred to the specially designed plastic tubes. A heating pad (36.5ºC) was placed underneath the exposure setup. The mice were exposed to 1.5% isoflurane carried in 100% oxygen for 4 hours. A calibrated flowmeter was used to deliver oxygen at a flow rate of 5 L/min and an agent specific vaporizer was used to deliver isoflurane. During isoflurane
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 3
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+exposure, mice were monitored for change in physiological state using the non-invasive MouseOx plus instrument (STARR Life Sciences, Holliston, MA, USA). A collar clip connected to the instrument was placed on the neck and a temperature probe placed on the skin of the abdomen. Ten-minute readings with 1-hour intervals were taken. Data was collected in four time-points and averaged for each case. The skin temperature (34.1±0.8ºC), pulse distention (168.9±36.6 μm), heart rate (376.8±94.1 bpm), breath rate (77.4±35.8 brpm), and oxygen saturation (99.3±0.3%) were recorded. After the isoflurane exposure, mice were returned to their moms together with their littermates upon regaining righting reflex. All animals (100%) survived the isoflurane exposure.7
+
+Rapamycin injection.
+
+A total of 84 mice were equally divided into three groups: 1) naïve control; 2) isoflurane exposure plus vehicle; and 3) isoflurane plus rapamycin injection. From postnatal days 21-35, half of the isoflurane-exposed mice (group 3; n=28 per group) were injected intraperitoneally with 0.2% rapamycin dissolved in vehicle solution and the other half with vehicle only (group 2; n=28 per group). Vehicle consisted of 5% Tween 80 (Sigma Aldrich, St. Louis, MO, USA), 10% polyethylene glycol 400 (Sigma-Aldrich, St. Louis, MO, USA), and 8% ethanol in saline. Mice received 100 μl rapamycin or vehicle for each injection at 48 hour intervals from postnatal days 21-35. 7
+
+Clemastine feeding.
+
+In this experiment, 36 animals were also equally divided into three groups as above. Clemastine (Tocris Bioscience, Bristol, UK) was dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) at 10 mg/ml followed by further dilution in ddH2O into 1 mg/ml. From postnatal days 21-35, half of the isoflurane exposed mice (n=12 for each group) were fed clemastine (10 mg/kg) daily via gastric gavage using plastic feeding tubes (gauge 22; Instech, Plymouth Meeting, PA, USA), and the other half (n=12 for each group) were fed same volume of 10% DMSO as vehicle.16,17
+
+Behavior tests.
+
+The novel object position recognition test and Y-maze test were performed at the last week of the survival period (postnatal days 56-62).7 Experimenters were blinded to condition when behavioral tests were carried out and quantified.
+
+1). Novel object position recognition test: The test was assessed in a 27.5 cm × 27.5 cm × 25 cm opaque chamber. During the pre-test day (day 1), each mouse was habituated to the chamber and allowed to explore 2 identical objects (glass bottles, 2.7 cm diameter, 12 cm height, and colored paper inside) for 15 minutes. The mouse was then returned to its home cage for a retention period of 24 hours. On the test day (day 2), the mouse was reintroduced to the chamber and presented with one object that stayed in the same position (old position) while the other object was moved to a new position (novel position). A five-minute period of movement and interaction with the objects was recorded with a video camera that was mounted above the chamber and exploratory behavior was measured by a blinded observer. Exploratory behavior was defined as touching the object with snouts. The numbers of exploratory contacts with the novel object and with the old
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 4
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+object were respectively recorded, and the ratios over the total exploratory contact numbers were calculated.
+
+2). Y-maze test: In the pre-test phase (day 1), mice explored and habituated in the start arm (no visual cue) and 1 out of 2 possible choice arms with overt visual cue (old arm) for 15 minutes. This was followed by the recognition phase (day 2) 24 hours later, in which the animals could move freely in the three arms and choose between the 2 choice arms (old arm and novel arm) after being released from the start arm. The timed trials (5 minutes) were video recorded as well as graded by an observer blinded to the conditions for exploration time in each choice arm and the percentages over total exploratory time were calculated.
+
+Immunohistochemistry.
+
+During postnatal days 30-35, 5-bromo-2’-deoxyuridine (Abcam, Cambridge, UK) was injected intraperitoneally at 50mg/kg daily in animals randomly selected from three groups (n=8 for each group). At postnatal day 35, mice were perfused with 40 ml 4% paraformaldehyde in PBS. Brains were removed and post-fixed at 4ºC overnight, followed by 30% sucrose in PBS at 4°C for 48 hours. The brains were coronally sectioned in 40 μm thickness using a freezing microtome. For each brain, 72 sections containing fimbria were collected in a 24-well tissue culture plate and they were divided into twelve wells in a rotating order (6 sections per well). Seven wells of sections were immunostained for: (1) phospho-S6 and adenomatous polyposis coli; (2) 5-bromo-2’-deoxyuridine and neural/glial antigen 2 ; (3) adenomatous polyposis coli and platelet-derived growth factor receptor alpha; (4) vesicular glutamate transporter 1 and neural/glial antigen 2; (5) myelin basic protein; (6) DNA methyltransferase 1 and Olig2 (oligodendrocyte transcription factor marker); (7) 5- methylcytosine (5-mC) and adenomatous polyposis coli. For 5-bromo-2’-deoxyuridine staining, sections were pretreated with 2N HCl to denature DNA (37°C; 45min), and with 2 × 15min borate buffer (pH 8.5) to neutralize the HCl. After 3×10min PBS washing, sections were blocked in 10% normal goat serum and 0.1% triton X-100 for 60min, followed by primary antibody incubation at 4ºC overnight. Primary antibodies used in this study were: rabbit anti-phospho-S6 (1:1,000; Cell Signaling, Boston, MA, USA), mouse anti-5- bromo-2’-deoxyuridine (1:200; Abcam, Cambridge, UK), rabbit anti-neural/glial antigen 2 (1:200; Millipore, Burlington, MA, USA), mouse anti-adenomatous polyposis coli (1:2,000; Millipore, Burlington, MA, USA), mouse anti-myelin basic protein (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti- platelet-derived growth factor receptor alpha (1:500; Lifespan Bio, Seattle, WA, USA), mouse anti- vesicular glutamate transporter 1 (1;200; Abcam, Cambridge, UK), mouse anti-DNA methyltransferase 1 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Olig2 (1:2,000; Abcam, Cambridge, UK), and rabbit anti-5-methylcytosine (1:2,500; Abcam, Cambridge, UK). After 3×10min washes in PBS, sections were incubated with secondary antibodies for 2 hours: Alexa 488 conjugated goat anti-rabbit IgG (1:300; Invitrogen, Eugene, OR, USA) mixed with Cy3 conjugated goat anti-mouse IgG (1:600; Jackson ImmunoResearch Labs, West Grove, PA, USA), or Alexa 488-goat anti-mouse IgG (1:300; Invitrogen, Eugene, OR, USA) mixed with Cy3 conjugated goat anti-rabbit IgG (1:600; Jackson ImmunoResearch labs, West Grove, PA, USA). After 3×10min PBS washes, sections were mounted onto slides, air-dried, and cover-slipped.21
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 5
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+Cell counting and immuno-fluoresce intensity analysis in fimbria
+
+The sections were observed and imaged using a Leica 4000 confocal microscope (Wetzlar, Germany). All single- or double- immunolabeled cells within hippocampal fimbria area were counted using ImageJ with cell counter plugin (NIH, Bethesda, MD, USA). The criteria for counting mTOR active oligodendrocytes required a cell to have both phospho- S6+ (in red channel) and adenomatous polyposis coli+ (in green channel) cytoplasm and merged image of double labeled cells appeared yellow color (Fig. 1B). Proliferating oligodendrocyte progenitor cells and 5-methylcytosine+ oligodendrocytes were counted for cells that have 5-bromo-2’-deoxyuridine+ or 5-methylcytosine + nuclei and neural/glial antigen 2+ or adenomatous polyposis coli+ cytoplasm. However, both DNA methyltransferase 1 and Olig2 reactivity were seen in nuclei. Identification of excitatory axon-oligodendrocyte progenitor cell synapses involved vesicular glutamate transporter 1+ terminal boutons closely apposing on the surface of neural/glial antigen 2+ oligodendrocyte progenitor cells. For adenomatous polyposis coli and platelet-derived growth factor receptor alpha double-stained sections, almost no double-labeled cells were seen, which means these two markers label cells in different oligodendrocyte development stages without overlapping.
+
+Images containing fimbria were taken at 20x magnification in red (Cy3), green (Alexa 488), and merged channels. All single- (Cy3+ or Alexa488+) and double-labeled cells in fimbria were counted. Images were opened and initialized in ImageJ. The fimbria area was outlined using the ‘‘Freehand’’ tool. “Plugins”, “Analysis”, and ‘‘Cell Counter’’ tools were selected, and each labeled cell inside was clicked, with which each counted cell was marked preventing the same cell from being counted twice. The numbers of counted cells were automatically recorded. The ratio of a specific marker labeled oligodendrocytes (such as yellow-colored phospho-S6+/ adenomatous polyposis coli+ cells over all green adenomatous polyposis coli+ cells in Fig. 1B) was calculated. For each case, numbers from 12 fimbria images (6 sections, both sides) were averaged. There was almost no double staining for adenomatous polyposis coli and platelet-derived growth factor receptor alpha. We the used ratio of adenomatous polyposis coli+ over platelet-derived growth factor receptor alpha+ cells to evaluate the maturation of oligodendrocyte lineage cells. For axon- oligodendrocyte progenitor cells synapse, five neural/glial antigen 2 positive cells from each image (60 cells for each case) were randomly selected and photos were taken in a higher magnification (40x). Every vesicular glutamate transporter 1+ terminal boutons apposing on each selected cell were counted with imageJ and average numbers were calculated.
+
+The fluorescence intensity of myelin basic protein immunoreactivity in fimbria were also quantitatively analyzed using ImageJ. Photos of the fimbria area from immunostained sections were taken at 20x magnification. Identical photo exposure was set for all groups. The image was opened with ImageJ and outline of fimbria was drawn with “Freehand” tool. The “set measurements” was selected from the analyze menu and “integrated density” was activated. A region in lateral ventricle was selected as background. The final myelin basic protein intensity of fimbria area equals measured density minus background.
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 6
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+Western blotting.
+
+Eight animals from each group were quickly perfused with cold saline on day 35. From the medial aspect of the hemisphere, the hippocampus was exposed and separated from brain tissue. Fimbria located in the ventrolateral side of the hippocampus were easily identified by bright white color under dissection microscope, and then removed with fine forceps. Fimbria tissue was lysed in the lysis buffer, homogenized with a bullet bender (Next Advance, Troy, NY, USA), and centrifuged. The supernatant was taken and stored in −80°C. The next day, samples were prepared with 1:1 denaturing sample buffer (Bio-Rad, Hercules, CA, USA), boiled for 5 min, and run on 4-12% Bis-Tris Protein Gels (Invitrogen, Carlsbad, CA, USA) in running buffer (Invitrogen, Carlsbad, CA, USA) with 150 volts for about 1 hour. The proteins were transferred to nitrocellulose blotting membranes (Invitrogen, Carlsbad, CA, USA). Blots were probed with anti-neural/glial antigen 2 (1:200; Millipore, Burlington, MA, USA), anti-NK2 homeobox 2 (1:200; Abcam, Cambridge, UK), anti-myelin basic protein (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-DNA methyltransferase 1 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-β-actin antibodies (1:1,000; Cell Signaling Technology, Boston, MA, USA). The membranes with the primary antibodies were stored in 4°C overnight. After incubation in secondary antibodies (1:2,000; Cell Signaling Technology, Boston, MA, USA) for 1 hour, blots were visualized using ECL western blotting substrate kit (Pierce Biotechnology, Waltham, MA, USA). Images were acquired using ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA) and were quantitated with ImageJ (NIH, Bethesda, MD, USA). First, the images were opened using File>Open. The rectangles around all lanes (each lane includes bands for detected marker and β-actin) were drawn by choosing “Rectangular Selection”. Then proceeding to “Analyze>Gels>Plot Lanes”, peaks were generated representing the density of bands, followed by clicking “Straight Line” tool to enclose the peaks and selecting the “Wand” tool to highlight the peaks. After this, Analyze>Gels>Label Peaks was used to get numbers for peak area (band intensity). The ratios of band density of oligodendrocyte lineage markers over β-actin were calculated.21
+
+Electron microscopy.
+
+Two animals from each group were perfused with 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) plus 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in PBS at postnatal day 63 (after behavior tests) and post-fixed at 4°C for 1 week. Brains containing fimbria were dissected into small blocks (2mm × 2mm × 2mm). The blocks were placed into 1% OsO4 (Electron Microscopy Sciences, Hatfield, PA, USA) for 1 hour, stained in 0.5% uranyl acetate (Electron Microscopy Sciences, Hatfield, PA, USA) overnight, and dehydrated in a series of alcohols followed by propylene oxide for 3 hours. After being infiltrated with a 1:1 mixture of propylene oxide and EMBed-812 embedding resin (Electron Microscopy Sciences, Hatfield, PA, USA) for 3 hours, the blocks were embedded with the same resin in the plastic templates at 60ºC overnight.21 Parasagittal semi-thin sections (1 μm) were cut and stained with 1% Toluidine blue for preliminary light microscopy observation. Then, 90 nm ultrathin sections were cut, picked up on Forvar- coated slotted grids, and stained with 0.5% uranyl acetate and 0.5% lead citrate (Electron Microscopy Sciences, Hatfield, PA, USA). Thin sections were observed and imaged with a Hitachi 7600 transmission electron microscope (Chiyoda, Tokyo, Japan). For each case, 10
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 7
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+photos were randomly photographed at 20,000×. The thickness of myelin was quantitatively measured by determining g-ratio, which was calculated by dividing the diameter of the axon by the diameter of the entire myelinated fiber as previously described. ImageJ (NIH, Bethesda, MD, USA) was used by first opening ultrastructural images. The scale was set according to the scale bar in the images by selecting “Analyze>Set Scale”. The “straight line tool” was selected to measure axonal caliber and diameter of myelinated axons. One hundred axons per group (two animals, fifty from each) were randomly selected and quantitatively analyzed (n=100).16
+
+Statistical analysis.
+
+The statistics were performed with GraphPad Prism 6 (La Jolla, CA, USA) program. The sample size was based on our previous experience with this design. No a priori statistical power calculation was conducted. Normal distribution was verified using the D’Agostino Pearson test. Data for immunohistochemistry, Western blotting, and electron microscopy were analyzed using one-way analysis of variance (ANOVA). The factor of variable was comparisons among groups (control vs. isoflurane plus vehicle vs. isoflurane plus rapamycin). The behavior tests were analyzed with two-way ANOVA. For this analysis, the second factor was animal’s choice between old vs. novel positions (or arms) and only the values for this variable in each individual group were compared. The Tukey post hoc test was employed for intergroup comparisons. The two-tailed test was set according to convention. The criteria for significant difference was set a priori at p<0.05. In this study, all results were expressed as mean ± standard deviation (SD). The sample size “n” represents the number of animals for each group. Only exception is g-ratio analysis with electron microscopy in which “n” indicates the number of randomly selected axons from two mice per group (n=100). This analysis way is extensively applied for g-ratio study.16 Because all animals survived tests, there were no missing data in this study. No exclusions for outliers were made in this study. In some experiments, the sample size was increased in response to peer review.
+
+Results
+
+Effect of early isoflurane exposure on mTOR activity in oligodendrocytes in hippocampal fimbria.
+
+All experiments compared the three groups as follows: naïve control, isoflurane exposure plus vehicle, and isoflurane exposure plus treatment (rapamycin or clemastine). We first assayed for activity in the mTOR pathway in oligodendrocytes by double labeling for phospho-S6, a reliable reporter of activity in this pathway,29 and adenomatous polyposis coli, a standard marker for oligodendrocyte. In the control group, 22±7% adenomatous polyposis coli positive oligodendrocytes in fimbria were also immuno-labeled for phospho- S6. There was a profound increase in the percentage of phospho-S6+/ adenomatous polyposis coli+ cells over adenomatous polyposis coli+ cells to 51±6% in isoflurane exposure mice (p<0.0001). However, this increase of phospho-S6+/ adenomatous polyposis coli+ cells over adenomatous polyposis coli+ cells was prevented by treatment with rapamycin (32±12%, p=0.001). These data indicate early exposure of a general anesthetic
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 8
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+agent causing a lasting increase in the activity of the mTOR signaling pathway in the oligodendrocytes of hippocampus white matter, and rapamycin attenuates this increase.
+
+Effect of isoflurane exposure on spatial learning.
+
+Next, we asked whether isoflurane exposure impairs spatial learning and memory behaviors using the novel objective position recognition test (Fig. 2A) and Y-maze test (Fig. 2B), and whether treatment with rapamycin or clemastine restores these functions in our exposure paradigm.
+
+1). Rapamycin study: In the novel object position recognition test, control animals made 58±8% contacts with the object that had been repositioned as compared to 42±8% contacts with the unchanged object (p<0.0001). The isoflurane-exposed mice made essentially equal contacts at both objects (53±6% vs. 47±6% times; p=0.398). Rapamycin treatment restored the performance to near control levels (55±8% vs. 45±8% times; p=0.016) (Fig. 2C). In the Y-maze test, control animals exhibited a higher percentage of exploration time in novel arm (58±5% exploration time) compared to the old one (42±5% exploration time) (p<0.0001). Isoflurane-exposed animals without rapamycin treatment had equal exploration times in both arms (50±5% vs. 50±5% time duration; p=0.999), and rapamycin treatment restored performance in this task (56±8% vs. 44±8% time duration; p<0.001) (Fig. 2D).
+
+2). Clemastine study: In the novel object recognition test, control animals made more contacts with the object in the novel position (57±8% vs. 43±8% times; p=0.007), but isoflurane exposed animals exhibited no exploration preference (51±10% vs. 49±10% times; p=0.998). Clemastine treatment increased the difference near the control cases (56±7% vs. 44±7% times; p=0.028) (Fig. 2E). Similarly, in the Y-maze test, unlike controls (58±6% vs. 42±6% time duration; p<0.0001), isoflurane exposed mice spent identical time in both old and novel arms (51±7% vs. 49±7% time duration; p=0.999), and this effect of isoflurane was reversed by clemastine treatment (57±6% vs. 43±6% time duration; p<0.001) (Fig. 2F).
+
+Effects of isoflurane on oligodendrocyte development.
+
+In order to measure the proliferation of oligodendrocyte progenitor cells, the brain tissue was immunolabeled with antibodies against 5-bromo-2’-deoxyuridine and an oligodendrocyte progenitor cells marker, neural/glial antigen 2. We examined proliferating oligodendrocyte progenitor cells by counting 5-bromo-2’-deoxyuridine and neural/glial antigen 2 double- labeled cells in fimbria. We counted 49±14% neural/glial antigen 2 positive oligodendrocyte progenitor cells in control, and 27±9% neural/glial antigen 2 positive cells in isoflurane exposed animals were 5-bromo-2’-deoxyuridine positive (p=0.001). This ratio number increased to 47±7% (p=0.003) in isoflurane plus rapamycin injection group (Fig. 3A). Interestingly, we found that neural/glial antigen 2 expression from Western blot in the isoflurane exposure group (83±20% intensity over β-actin) is slightly lower than the control (87±15%, p=0.882) and rapamycin treatment groups (89±17%, p=0.819), but there is no statistical difference among groups (Fig. 3B). However, expression level of NK2 homeobox 2, a transcription factor that identifies oligodendrocyte differentiation, was downregulated by isoflurane (67±16% vs. 32±10% intensity over β-actin; p=0.001) and rapamycin attenuated
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 9
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+this effect (58±22% % intensity over β-actin; p=0.015) (Fig. 3B). We then performed double-immunolabeling for adenomatous polyposis coli and the oligodendrocyte progenitor cell marker platelet-derived growth factor receptor alpha, and applied oligodendrocyte/ oligodendrocyte progenitor cell ratio as a parameter to evaluate the oligodendrocyte differentiation in fimbria. 30 The ratio of the number of adenomatous polyposis coli positive mature oligodendrocytes over the number of platelet-derived growth factor receptor alpha labeled oligodendrocyte progenitor cells in isoflurane exposed mice (233±43%) revealed lower than in control (347±70% cells; p=0.002), and rapamycin treatment increases this ratio (338±52% cells; p=0.003) (Fig. 3C).
+
+To test the effects of isoflurane on axon-oligodendrocyte progenitor cell synapses, we identified excitatory axon-oligodendrocyte progenitor cell synapses in the fimbria as puncta that were immunopositive for vesicular glutamate transporter 1, which were closely apposed on neural/glial antigen 2+ cell bodies. We found that the number of axon-oligodendrocyte progenitor cell synapses on each oligodendrocyte progenitor cell in isoflurane exposure (0.8±0.6 vGlut1+ terminals per OPC) showed a statistically significant reduction compared to control (2.6±1.2 terminals per cell; p=0.001) and rapamycin treatment rescued these synapses (2.2±0.7 per cell; p=0.008) from isoflurane exposure (Fig. 4).
+
+Effects of isoflurane exposure on myelination.
+
+To test for changes in myelination after anesthesia exposure, we measured fluoresce intensity of immunolabeling for the myelin basic protein in fimbria. We found an immuno- intensity reduction in isoflurane exposure (70±18% intensity over control) compared to control conditions (100±17% control; p=0.006), which were partially restored with rapamycin treatment (92±17% rapamycin treatment; p=0.041) (Fig. 5A). We then conducted Western blot from fimbria tissue to confirm this finding. The band intensity of myelin basic protein over β-actin showed a statistically significant decrease by isoflurane exposure (110±30% vs. 60±19% intensity ratio; p=0.002) and expression of myelin basic protein was restored with rapamycin treatment (100±26% intensity ratio; p=0.013) (Fig. 5B). For further confirmation, we conducted electron microscopy to test for changes in the thickness of myelin wraps after isoflurane exposure. The quantitative analysis revealed a statistically significant increase of g-ratio (thinner myelin sheath) in isoflurane exposed animals than control (0.76±0.06 vs. 0.79±0.06 g-ratio; p<0.001) and rapamycin treatment reversed this difference (0.75±0.05 g-ratio; p<0.0001) (Fig. 5C).
+
+Effects of isoflurane exposure on DNA methylation in oligodendrocytes.
+
+We asked if early exposure to isoflurane has a lasting effect on DNA methylation levels in oligodendrocytes. We observed that 60±15% of Olig2+ cells (a transcription factor marker for oligodendrocyte lineage cells) in control conditions were double-labeled with DNA methyltransferase 1 in fimbria as compared to only 42±12% Olig2+ cells (p=0.027) in the isoflurane exposure group. Rapamycin treatment following isoflurane exposure increased the ratio of DNA methyltransferase 1+/Olig2+ over Olig2+ cells (62±11% ratio; p=0.01) (Fig. 6A). Western blots were conducted which confirmed that DNA methyltransferase 1 expression is reduced with isoflurane treatment (25±8% intensity ratio over β-actin) compared to control (58±21% intensity ratio over β-actin; p<0.001) and a partial recovery
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 10
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+Discussion
+
+results from rapamycin treatment (49±9% ratio; p=0.006) (Fig. 6B). In order to determine whether changes in DNA methyltransferase 1 levels have functional significance, we assayed levels of 5-methylcytosine, which is a product of DNA methyltransferase 1 mediated DNA methylation, in oligodendrocytes. Isoflurane exposure decreased the ratio of 5-methylcytosine positive nuclei over adenomatous polyposis coli labeled oligodendrocytes (33±13% ratio) relative to control (52±13% ratio; p=0.006), and rapamycin treatment reversed this decrease (48±7% ratio; p=0.031) (Fig. 6C).
+
+In this study, we report that early postnatal exposure to isoflurane in mice causes a substantial disruption of oligodendrocyte development and myelination in fimbria of the hippocampus, which is the predominant bundle of efferent axonal fibers from the hippocampus. Proliferation and differentiation of oligodendrocyte progenitor cells are chronically impaired by early isoflurane exposure, as is the formation of synaptic connections between oligodendrocyte progenitor cells and axons. This results in a measurable loss of myelin in the fimbria. Proper connections and communications between hippocampus and the neocortex are critical for performing the cognitive and psychological functions.14, 31, 32 Myelination is essential in establishing connectivity in the growing brain by facilitating rapid and synchronized information transfer across the nervous system. Once thought of as solely a passive insulator, myelin is now understood to be actively involved in the function and development of the CNS.33 Abnormal myelination of axons disrupts the communications between brain regions, and it has been reported that myelin deficits in the hippocampus cause cognitive and psychological disorders.34-36 Previous studies have shown an acute increase in apoptosis of oligodendrocytes, the myelin forming glial cells, with early exposure to isoflurane,37, 38 but our findings demonstrate a lasting effect of anesthesia on oligodendrocyte proliferation and differentiation that results in a decrease in myelination in the hippocampal fimbria.
+
+The process of oligodendrocyte development occurs based on an intrinsic program that is modulated by neurotransmitters and electrical activity in CNS.39-41 Axonal terminals release glutamate as a transmitter at not only axonal terminals but also at discrete sites along axons in white matter.42 By acting on AMPA or NMDA receptors expressed on oligodendrocyte progenitor cells, glutamate increases the downstream phosphorylation of the cAMP response element binding protein and release of calcium from intracellular stores,43 thereby promoting oligodendrocyte progenitor cell proliferation and differentiation. As an agonist of GABA-A and glycine receptors,44 as well as a potential NMDA inhibitor,45 isoflurane suppresses excitatory neurotransmission.46 It raises the possibility that a profound direct action on oligodendrocyte progenitor cells, which express both GABA and NMDA receptors, is caused by isoflurane during a critical period in development and that this may alter the developmental program, thus resulting in deficits in myelination. An alternative or complementary explanation may be an indirect effect mediated by the electrochemical synapses that occur between axonal terminals and oligodendrocyte progenitor cells (axon- oligodendrocyte progenitor cell synapses). Formation of glutamatergic axon- oligodendrocyte progenitor cell synapses plays an important role in promoting activity- dependent oligodendrocyte development and maintenance.21-23 The chronic, lasting effects
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 11
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+of isoflurane on neuronal synapses that we have previously shown may translate into reduced activity at axon- oligodendrocyte progenitor cell synapses, thus leading to suppression of oligodendrocyte progenitor cell development. A previous study showed that plasticity of axon-oligodendrocyte progenitor cell synapses is highly dependent on electrical activity.21 The data in this study further confirms that exposure of isoflurane reduces the number of excitatory (vesicular glutamate transporter 1 positive) axon-oligodendrocyte progenitor cell synapses in hippocampus, which is likely a key mechanism of general anesthetic-induced hypomyelination.
+
+Our previous work has indicated that exposure to isoflurane disrupts the development of hippocampal neurons generated in the early postnatal period by inappropriately increasing activity in the mTOR pathway and we found that both behavioral and histological changes could be reversed by pharmacologic mTOR inhibition.7 In the present study, as in neurons, we observe a lasting alteration in the tone of mTOR signaling in oligodendrocytes in the hippocampal fimbria for a protracted period after isoflurane exposure which appears to be integral to the developmental disruption. We found a substantial improvement in phenotype with rapamycin treatment in this study as well. The mTOR pathway is an intracellular signaling pathway that regulates cellular activities including proliferation, differentiation, apoptosis, metabolism, transmitter release, and other biological processes.18 In the past decade, many studies have implicated mTOR signaling in CNS developmental and neuropsychiatric disorders.19 Two structurally and functionally distinct mTOR-containing complexes have been identified in oligodendrocytes. The first, mTOR complex 1 (mTORC1), contains the adaptor protein Raptor, which influences myelin basic protein expression via an alternative mechanism and is sensitive to the drug rapamycin. The second complex, mTORC2, contains Ritor, and it is thought to control myelin gene expression at mRNA level and is relatively rapamycin insensitive.47
+
+The mTOR pathway itself plays a complex role in myelination; mTOR activity can either enhance or suppress oligodendrocyte development depending on the context. A study using a mouse line with oligodendrocyte-specific knockdown of mTOR in CNS has provided evidence that mTOR is essential for oligodendrocyte development and myelination.48 Inhibition of mTOR via rapamycin in cultured adult oligodendrocyte progenitor cells or in a mouse model starting at 6 weeks of age results in oligodendrocyte differentiation deficits along with reduced expression of major myelin proteins and mRNAs.49-51 In contrast, activation of mTOR induced by tuberous sclerosis complex −1 or 2 gene mutations in early oligodendrocyte progenitor cells caused white matter abnormalities, including myelin deficits in CNS.52-54 A bidirectional action of the PI3K-Akt-mTOR axis in myelination has also been reported in studies of the PNS. If tuberous sclerosis complex −1 deletion occurs in early developmental stages in Schwann cells, mTOR hyperactivity arrests the process by which Schwann cells ensheathe axons. If mTOR activity is increased in Schwann cells after they have begun wrapping around axons, there is actually in increase in myelination.55 Thus, oligodendrocyte development and myelination may be dependent on precise balance and timing in mTOR signaling. Either increasing or decreasing levels of mTORC1 activity interferes with oligodendrocyte differentiation and causes potentially causes hypomyelination.56
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 12
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+While we do not yet have a clear picture of how changes in mTOR activity act on oligodendrocytes, our data indicate changes in DNA methylation as a promising direction. Recent work shows developmental anesthetic toxicity may involve epigenetic modulation 57 and that DNA methylation plays an important role in regulating oligodendrocyte progenitor cell proliferation and differentiation.26-28 Intriguingly, mTOR signaling has been shown to negatively regulate DNA methylation via an action on DNA methyltransferase 1,24, 25 suggesting a possible connection between mTOR signaling and oligodendrocyte development. This finding is consistent with our data showing that isoflurane exposure and concomitant increases in mTOR signaling lead to a decrease in DNA methyltransferase 1 expression and DNA methylation in developing oligodendrocytes, and that both of these changes are reversible with rapamycin treatment.
+
+We propose that oligodendrocytes should be further studied both as a potential target in anesthetic neurotoxicity and as a model system in which to further explore the interplay of anesthetics, mTOR signaling, and DNA methylation. Our work is limited by the rodent model, which has well-known confounds related to anesthetic administration in very young, small mice in which physiologic monitoring and control of respiratory function is challenging. In particular, we have chosen to use 100% oxygen as a carrier for isoflurane, which has the beneficial effect of preventing hypoxia in our model system, but which raises the possibility of a combined effect of isoflurane and hyperoxia damage that cannot be fully controlled for in our experimental model. While it is indeed the case that supplemental oxygen is frequently used in pediatric anesthesia practice it would be ideal to avoid confounds presented by hyperoxia and other physiologic issues via studies in large animal models and in cell culture models and we hope further work in this area will be undertaken in these systems.
+
+Acknowledgments:
+
+This work was funded by the US National Institutes of Health, Bethesda, MD (5R01GM120519 to C.D.M.) and by the Johns Hopkins Department of Anesthesiology and Critical Care (StAAR award to C.D.M.)
+
+Funding Statement: This work was funded by the US National Institutes of Health, Bethesda, MD. (5R01GM120519 to C.D.M).
+
+References
+
+1. Weiser TG, Regenbogen SE, Thompson KD, Haynes AB, Lipsitz SR, Berry WR, & Gawande AA (2008). An estimation of the global volume of surgery: a modelling strategy based on available data. Lancet (London, England), 372(9633), 139–144.
+
+2. Eckenhoff JE (1953). Relationship of anesthesia to postoperative personality changes in children. AMA Am J Dis Child, 86(5), 587–591. [PubMed: 13103772]
+
+3. Davidson AJ, Disma N, de Graaff JC, Withington DE, Dorris L, Bell G, Stargatt R, Bellinger DC, Schuster T, Arnup SJ, Hardy P, Hunt RW, Takagi MJ, Giribaldi G, Hartmann PL, Salvo I, Morton NS, von Ungern Sternberg BS, Locatelli BG, Wilton N, Lynn A, Thomas JJ, Polaner D, Bagshaw O, Szmuk P, Absalom AR, Frawley G, Berde C, Ormond GD, Marmor J, McCann ME, GAS consortium. (2016). Neurodevelopmental outcome at 2 years of age after general anaesthesia and awake-regional anaesthesia in infancy (GAS): an international multicentre, randomised controlled trial. Lancet (London, England), 387(10015), 239–250.
+
+4. Sun LS, Li G, Miller TL, Salorio C, Byrne MW, Bellinger DC, Ing C, Park R, Radcliffe J, Hays SR, DiMaggio CJ, Cooper TJ, Rauh V, Maxwell LG, Youn A, McGowan FX (2016). Association
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 13
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+Between a Single General Anesthesia Exposure Before Age 36 Months and Neurocognitive Outcomes in Later Childhood. JAMA, 315(21), 2312–2320. [PubMed: 27272582]
+
+5. Coleman K, Robertson ND, Dissen GA, Neuringer MD, Martin LD, Cuzon Carlson VC, Kroenke C, Fair D, Brambrink AM (2017). Isoflurane Anesthesia Has Long-term Consequences on Motor and Behavioral Development in Infant Rhesus Macaques. Anesthesiology, 126(1), 74–84. [PubMed: 27749311]
+
+6. Alvarado MC, Murphy KL, & Baxter MG (2017). Visual recognition memory is impaired in rhesus monkeys repeatedly exposed to sevoflurane in infancy. Br J Anaesth, 119(3), 517–523. [PubMed: 28575197]
+
+7. Kang E, Jiang D, Ryu YK, Lim S, Kwak M, Gray CD, Xu M, Choi JH, Junn S, Kim J, Xu J, Schaefer M, Johns RA, Song H, Ming GL, Mintz CD (2017). Early postnatal exposure to isoflurane causes cognitive deficits and disrupts development of newborn hippocampal neurons via activation of the mTOR pathway. PLoS Biol, 15(7), e2001246. [PubMed: 28683067]
+
+8. Shen X, Liu Y, Xu S, Zhao Q, Guo X, Shen R, & Wang F (2013). Early life exposure to sevoflurane impairs adulthood spatial memory in the rat. Neurotoxicology, 39, 45–56. [PubMed: 23994303] 9. Stratmann G, Sall JW, May LDV, Bell JS, Magnusson KR, Rau V, Visrodia KH, Alvi RS, Ku B, Lee MT, Dai R (2009). Isoflurane differentially affects neurogenesis and long-term neurocognitive function in 60-day-old and 7-day-old rats. Anesthesiology, 110(4), 834–848. [PubMed: 19293705] 10. Pontén E, Fredriksson A, Gordh T, Eriksson P, & Viberg H (2011). Neonatal exposure to propofol affects BDNF but not CaMKII, GAP-43, synaptophysin and tau in the neonatal brain and causes an altered behavioural response to diazepam in the adult mouse brain. Behav Brain Res, 223(1), 75–80. [PubMed: 21540061]
+
+11. Zhu C, Gao J, Karlsson N, Li Q, Zhang Y, Huang Z, Li H, Kuhn HG, Blomgren K (2010). Isoflurane anesthesia induced persistent, progressive memory impairment, caused a loss of neural stem cells, and reduced neurogenesis in young, but not adult, rodents. J Cereb Blood Flow Metab: Official Journal of the International Society of Cerebral Blood Flow and Metabolism, 30(5), 1017– 1030.
+
+12. Center for Drug Evaluation and Research. “Drug Safety and Availability – FDA Drug Safety Communication: FDA Review Results in New Warnings about Using General Anesthetics and Sedation Drugs in Young Children and Pregnant Women.” US Food and Drug Administration Home Page. https://www.fda.gov/Drugs/DrugSafety/ucm532356.htm
+
+13. Vutskits L, & Xie Z (2016). Lasting impact of general anaesthesia on the brain: mechanisms and relevance. Nat Rev Neurosci, 17(11), 705–717. [PubMed: 27752068]
+
+14. Nickel M, & Gu C (2018). Regulation of Central Nervous System Myelination in Higher Brain Functions. Neural Plast, 2018, 1–12.
+
+15. Chiaravalloti ND, DeLuca J. Cognitive impairment in multiple sclerosis. Lancet Neurol. 2008;7(12):1139–1151. [PubMed: 19007738]
+
+16. Liu J, Dupree JL, Gacias M, Frawley R, Sikder T, Naik P, & Casaccia P (2016). Clemastine Enhances Myelination in the Prefrontal Cortex and Rescues Behavioral Changes in Socially Isolated Mice. J Neurosci, 36(3), 957–962. [PubMed: 26791223]
+
+17. Li Z, He Y, Fan S, & Sun B (2015). Clemastine rescues behavioral changes and enhances remyelination in the cuprizone mouse model of demyelination. Neurosci Bull, 31(5), 617–625. [PubMed: 26253956]
+
+18. Laplante M, & Sabatini DM (2012). mTOR signaling in growth control and disease. Cell, 149(2), 274–293. [PubMed: 22500797]
+
+19. Costa-Mattioli M, & Monteggia LM (2013). mTOR complexes in neurodevelopmental and neuropsychiatric disorders. Nat Neurosci, 16(11), 1537–1543. [PubMed: 24165680] 20. Xu J, Mathena RP, Xu M, Wang Y, Chang C, Fang Y, Zhang P, Mintz CD (2018). Early Developmental Exposure to General Anesthetic Agents in Primary Neuron Culture Disrupts Synapse Formation via Actions on the mTOR Pathway. Int J Mol Sci, 19(8), 2183.
+
+21. Li Q, Houdayer T, Liu S, & Belegu V (2017). Induced Neural Activity Promotes an Oligodendroglia Regenerative Response in the Injured Spinal Cord and Improves Motor Function after Spinal Cord Injury. J Neurotrauma, 34(24), 3351–3361. [PubMed: 28474539]
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 14
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+22. Bergles DE, & Richardson WD (2015). Oligodendrocyte Development and Plasticity. Cold Spring Harb Perspect Biol, 8(2), a020453. [PubMed: 26492571]
+
+23. Gautier HO, Evans KA, Volbracht K, James R, Sitnikov S, Lundgaard I, James F, Lao-Peregrin C, Reynolds R, Franklin RJ, Káradóttir RT (2015). Neuronal activity regulates remyelination via glutamate signalling to oligodendrocyte progenitors. Nat Commun, 6(1), 8518. [PubMed: 26439639]
+
+24. Zhang X, He X, Li Q, Kong X, Ou Z, Zhang L, Gong Z, Long D, Li J, Zhang M, Ji W, Zhang W, Xu L, Xuan A (2017). PI3K/AKT/mTOR Signaling Mediates Valproic Acid-Induced Neuronal Differentiation of Neural Stem Cells through Epigenetic Modifications. Stem Cell Reports, 8(5), 1256–1269. [PubMed: 28494938]
+
+25. Wang C, Wang X, Su Z, Fei H, Liu X, & Pan Q (2015). The novel mTOR inhibitor Torin-2 induces autophagy and downregulates the expression of UHRF1 to suppress hepatocarcinoma cell growth. Oncol Rep, 34(4), 1708–1716. [PubMed: 26239364]
+
+26. Moyon S, Huynh JL, Dutta D, Zhang F, Ma D, Yoo S, Lawrence R, Wegner M, John GR, Emery B, Lubetzki C, Franklin R, Fan G, Zhu J, Dupree JL, Casaccia P (2016). Functional Characterization of DNA Methylation in the Oligodendrocyte Lineage. Cell Reports, 15(4), 748–760. [PubMed: 27149841]
+
+27. Moyon S, Ma D, Huynh JL, Coutts DJC, Zhao C, Casaccia P, & Franklin RJM (2017). Efficient Remyelination Requires DNA Methylation. eNeuro, 4(2), ENEURO.0336-16.2017.
+
+28. Moyon S, & Casaccia P (2017). DNA methylation in oligodendroglial cells during developmental myelination and in disease. Neurogenesis (Austin, Tex.), 4(1), e1270381.
+
+29. Zhou M, Li W, Huang S, Song J, Kim JY, Tian X, Kang E, Sano Y, Liu C, Balaji J, Wu S, Zhou Y, Zhou Y, Parivash SN, Ehninger D, He L, Song H, Ming GL, Silva AJ (2013). mTOR Inhibition ameliorates cognitive and affective deficits caused by Disc1 knockdown in adult-born dentate granule neurons. Neuron, 77(4), 647–654. [PubMed: 23439118]
+
+30. Assinck P, Duncan GJ, Plemel JR, Lee MJ, Stratton JA, Manesh SB, Liu J, Ramer LM, Kang SH, Bergles DE, Biernaskie J, Tetzlaff W (2017). Myelinogenic Plasticity of Oligodendrocyte Precursor Cells following Spinal Cord Contusion Injury. J Neurosci, 37(36), 8635–8654. [PubMed: 28760862]
+
+31. Preston AR, & Eichenbaum H (2013). Interplay of hippocampus and prefrontal cortex in memory. Curr Biol, 23(17), R764–73. [PubMed: 24028960]
+
+32. Frankland PW, & Bontempi B (2005). The organization of recent and remote memories. Nat Rev Neurosci, 6(2), 119–130. [PubMed: 15685217]
+
+33. Fields RD (2014). Neuroscience. Myelin--more than insulation. Science (New York, N.Y.), 344(6181), 264–266.
+
+34. Sacco R, Bisecco A, Corbo D, Della Corte M, d’Ambrosio A, Docimo R, Gallo A, Esposito F, Esposito S, Cirillo M, Lavorgna L, Tedeschi G, Bonavita S (2015). Cognitive impairment and memory disorders in relapsing–remitting multiple sclerosis: the role of white matter, gray matter and hippocampus. J Neurol, 262(7), 1691–1697. [PubMed: 25957638]
+
+35. Damjanovic D, Valsasina P, Rocca MA, Stromillo ML, Gallo A, Enzinger C, Hulst HE, Rovira A, Muhler N, De Stefano N, Bisecco A, Fazekas F, Arevalo MJ, Yousry TA, Filippi M (2017). Hippocampal and Deep Gray Matter Nuclei Atrophy Is Relevant for Explaining Cognitive Impairment in MS: A Multicenter Study. AJNR Am J Neuroradiol, 38(1), 18–24. [PubMed: 27686487]
+
+36. Chen BH, Park JH, Lee T-K, Song M, Kim H, Lee JC, Kim YM, Lee CH, Hwang IK, Kang IJ, Yan BC, Won MH, Ahn JH (2018). Melatonin attenuates scopolamine-induced cognitive impairment via protecting against demyelination through BDNF-TrkB signaling in the mouse dentate gyrus. Chem Biol Interact, 285, 8–13. [PubMed: 29476728]
+
+37. Brambrink AM, Back SA, Riddle A, Gong X, Moravec MD, Dissen GA, Creeley CE, Dikranian KT, Olney JW (2012). Isoflurane-induced apoptosis of oligodendrocytes in the neonatal primate brain. Ann Neurol, 72(4), 525–535. [PubMed: 23109147]
+
+38. Jiang D, Lim S, Kwak M, Ryu YK, & Mintz CD (2015). The changes of oligodendrocytes induced by anesthesia during brain development. Neural Regen Res, 10(9), 1386–1387. [PubMed: 26604888]
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 15
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+39. Stevens B, Porta S, Haak LL, Gallo V, & Fields RD (2002). Adenosine: a neuron-glial transmitter promoting myelination in the CNS in response to action potentials. Neuron, 36(5), 855–868. [PubMed: 12467589]
+
+40. Barres BA, & Raff MC (1993). Proliferation of oligodendrocyte precursor cells depends on electrical activity in axons. Nature, 361(6409), 258–260. [PubMed: 8093806]
+
+41. Gibson EM, Purger D, Mount CW, Goldstein AK, Lin GL, Wood LS, Inema I, Miller SE, Bieri G, Zuchero JB, Barres BA, Woo PJ, Vogel H, Monje M (2014). Neuronal activity promotes oligodendrogenesis and adaptive myelination in the mammalian brain. Science (New York, N.Y.), 344(6183), 1252304.
+
+42. Kukley M, Capetillo-Zarate E, & Dietrich D (2007). Vesicular glutamate release from axons in white matter. Nat Neurosci, 10(3), 311–320. [PubMed: 17293860]
+
+43. Redondo C, López-Toledano MA, Lobo MVT, Gonzalo-Gobernado R, Reimers D, Herranz AS, Paino CL, Bazán E (2007). Kainic acid triggers oligodendrocyte precursor cell proliferation and neuronal differentiation from striatal neural stem cells. J Neurosci Res, 85(6), 1170–1182. [PubMed: 17342781]
+
+44. Grasshoff C, & Antkowiak B (2006). Effects of isoflurane and enflurane on GABAA and glycine receptors contribute equally to depressant actions on spinal ventral horn neurones in rats. Br J Anaesth, 97(5), 687–694. [PubMed: 16973644]
+
+45. Petrenko AB, Yamakura T, Sakimura K, & Baba H (2014). Defining the role of NMDA receptors in anesthesia: are we there yet? Eur J Pharmacol, 723, 29–37. [PubMed: 24333550]
+
+46. Baumgart JP, Zhou Z-Y, Hara M, Cook DC, Hoppa MB, Ryan TA, & Hemmings HC (2015). Isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx, not Ca2+-exocytosis coupling. Proc Natl Acad Sci U S A, 112(38), 11959–11964. [PubMed: 26351670]
+
+47. Jacinto E, Loewith R, Schmidt A, Lin S, Rüegg MA, Hall A, & Hall MN (2004). Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat Cell Bio, 6(11), 1122– 1128. [PubMed: 15467718]
+
+48. Wahl SE, McLane LE, Bercury KK, Macklin WB, & Wood TL (2014). Mammalian target of rapamycin promotes oligodendrocyte differentiation, initiation and extent of CNS myelination. J Neurosci, 34(13), 4453–4465. [PubMed: 24671992]
+
+49. Guardiola-Diaz HM, Ishii A, & Bansal R (2012). Erk1/2 MAPK and mTOR signaling sequentially regulates progression through distinct stages of oligodendrocyte differentiation. Glia, 60(3), 476– 486. [PubMed: 22144101]
+
+50. Tyler WA, Gangoli N, Gokina P, Kim HA, Covey M, Levison SW, & Wood TL (2009). Activation of the Mammalian Target of Rapamycin (mTOR) Is Essential for Oligodendrocyte Differentiation. J Neurosci, 29(19), 6367–6378. [PubMed: 19439614]
+
+51. Narayanan SP, Flores AI, Wang F, & Macklin WB (2009). Akt signals through the mammalian target of rapamycin pathway to regulate CNS myelination. J Neurosci, 29(21), 6860–6870. [PubMed: 19474313]
+
+52. Jiang M, Liu L, He X, Wang H, Lin W, Wang H, Yoon SO, Wood TL, Lu QR (2016). Regulation of PERK–eIF2α signalling by tuberous sclerosis complex-1 controls homoeostasis and survival of myelinating oligodendrocytes. Nat Commun, 7(1), 12185. [PubMed: 27416896]
+
+53. Scholl T, Mühlebner A, Ricken G, Gruber V, Fabing A, Samueli S, Gröppel G, Dorfer C, Czech T, Hainfellner JA, Prabowo AS, Reinten RJ, Hoogendijk L, Anink JJ, Aronica E, Feucht M (2017). Impaired oligodendroglial turnover is associated with myelin pathology in focal cortical dysplasia and tuberous sclerosis complex. Brain Pathol, 27(6), 770–780. [PubMed: 27750396]
+
+54. Carson RP, Kelm ND, West KL, Does MD, Fu C, Weaver G, McBrier E, Parker B, Grier MD, Ess KC (2015). Hypomyelination following deletion of Tsc2 in oligodendrocyte precursors. Ann Clin Transl Neurol, 2(12), 1041–1054. [PubMed: 26734657]
+
+55. Figlia G, Norrmén C, Pereira JA, Gerber D, & Suter U (2017). Dual function of the PI3K-Akt- mTORC1 axis in myelination of the peripheral nervous system. ELife, 6.
+
+56. Lebrun-Julien F, Bachmann L, Norrmén C, Trötzmüller M, Köfeler H, Rüegg MA, Hall MN, Suter U (2014). Balanced mTORC1 activity in oligodendrocytes is required for accurate CNS myelination. J Neurosci, 34(25), 8432–8448. [PubMed: 24948799]
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 16
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+57. Dalla Massara L, Osuru HP, Oklopcic A, Milanovic D, Joksimovic SM, Caputo V, DiGruccio MR, Ori C, Wang G, Todorovic SM, Jevtovic-Todorovic V (2016). General Anesthesia Causes Epigenetic Histone Modulation of c-Fos and Brain-derived Neurotrophic Factor, Target Genes Important for Neuronal Development in the Immature Rat Hippocampus. Anesthesiology, 124(6), 1311–1327. [PubMed: 27028464]
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 17
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+Fig. 1. (A) Experimental timeline. A total of 120 mice (84 for rapamycin injection and 36 for clemastine feeding experiments) were used in this study. At postnatal day 7, two-thirds of the mice were exposed to isoflurane carried for 4 hours and the other one-third of the animals remained in room air as naïve control. From postnatal days 21-35, isoflurane- exposed mice were injected (i.p.) with rapamycin or vehicle at 48 hour intervals; or daily fed with clemastine or vehicle. Mice were sacrificed at postnatal day 35 for immunohistochemistry and Western blotting, or at postnatal day 63 for electron microscopy. The novel objective position recognition test and Y-maze test were performed during postnatal days 57-62. (B). Effect of early isoflurane exposure on mTOR pathway activity in oligodendrocytes of hippocampus fimbria. Coronal brain sections from control, isoflurane exposure, and isoflurane plus rapamycin groups were immunostained with adenomatous polyposis coli (APC; green) and phosphor-S6 (pS6; red) antibodies. Arrows indicate phospho-S6+ and adenomatous polyposis coli+ double-labeled cells (yellow) in merged images. Scale bar=10 μm. The histogram shows quantitative results. In isoflurane-exposed mice, the ratio of phospho-S6+/ adenomatous polyposis coli+ over adenomatous polyposis coli+ cells is dramatically increased compared to control and this increase is reversed with rapamycin treatment. (n=8 for each group; One-way ANOVA; **: p<0.01; ****: p<0.0001). Iso: isoflurane. Error bars represent SD.
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 18
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+Fig. 2. Isoflurane impairs cognitive functions via mTOR activity. (A). Novel object recognition test. On day 1, mice were allowed to explore two identical objects in an opaque chamber. On day 2, one object was moved to a novel position. Exploratory behavior was defined as the number of object-contacting with snouts. (B). Y-maze test. On day 1, mice habituated in the start arm and one choice arm. On day 2, the animal could choose between two arms. Exploration time in both arms was respectively recorded. (C). Novel object recognition test for rapamycin study. Control animals made more contact with the object in the novel position than that in the old position. Isoflurane-exposed mice have identical contacts for both positions. Rapamycin treatment restores performance to near control levels. (D). Y- maze study. Control animals stayed in the novel arm for longer time than in the old arm. Isoflurane-exposed animals stayed in both arms for same time. Rapamycin treatment reversed this ratio to near control. (E). Novel object recognition test for clemastine study. Control animals spent more time exploring the object in the novel position, but isoflurane- exposed animals exhibited no exploration preference. Clemastine treatment increased difference near the level of control animals. (F). Y-maze test. Similarly, isoflurane mice spent identical time for both arms, but this effect of isoflurane was reversed by feeding clemastine. The statistics was Two-way ANOVA (n=12 for each group; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; ns: no significance). Error bars: SD.
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 19
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+Fig. 3. The effect of isoflurane exposure and rapamycin treatment on oligodendrocyte development in fimbria. (A). Oligodendrocyte progenitor cell proliferation was detected with 5-bromo-2’- deoxyuridine and neural/glial antigen 2 double-immunolabeling. A reduction in 5-bromo-2’- deoxyuridine +/ neural/glial antigen 2+ cells in isoflurane-exposed animals was observed compared to the control. This number was increased in isoflurane plus rapamycin injection group. Scale bar=20μm. (B). Western blot data indicated that the neural/glial antigen 2 level was not altered by isoflurane exposure and rapamycin administration. Expression of NK2 homeobox 2, a transcript factor for oligodendrocyte differentiation, was downregulated by isoflurane and rapamycin treatment attenuated this effect. (C). Oligodendrocyte differentiation was analyzed with lineage tracing using immunohistochemistry. The ratio of adenomatous polyposis coli+ mature oligodendrocyte number over platelet-derived growth factor receptor alpha+ oligodendrocyte progenitor cells in isoflurane-exposed mice revealed reduction compared to control, and rapamycin treatment increases the ratio. Scale bar=20μm. (n=8 for each group; One-way ANOVA; *: p<0.05; **: p<0.01; ns: no significance). Iso: isoflurane. Error bars indicate SD.
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 20
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+Fig. 4. The effect of isoflurane exposure and rapamycin treatment on the numbers of excitatory axon-oligodendrocyte progenitor cell synapses in fimbria. The vesicular glutamate transporter 1+ axon-oligodendrocyte progenitor cell synapses were identified with terminals apposing on neural/glial antigen 2+ oligodendrocyte progenitor cells (arrows indicate these synapses). The number in isoflurane exposure mice was lower than control and rapamycin treatment rescued these axon- oligodendrocyte progenitor cell synapses. Scale bar=5μm. (A). Control. (B). Isoflurane exposure plus vehicle group (C). Isoflurane exposure with rapamycin treatment. (D). Graph showing quantitative data (n=8 for each group; One-way ANOVA; **: p<0.01). Iso: isoflurane. Error bars equal SD.
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 21
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+Fig. 5. The effect of isoflurane exposure and rapamycin treatment on myelination in fimbria. (A). Myelination was quantitatively analyzed with myelin basic protein immunostaining. The myelin basic protein intensity in isoflurane-exposed mice was reduced compared to control conditions and is increased with rapamycin treatment. For this analysis, the same exact photo setting was performed for all groups. * in photos: CA3 of hippocampus; fi: fimbria of hippocampus; LV: lateral ventricle. Scale bar=200μm. (B). Western blot data indicated expression of myelin basic protein was decreased by isoflurane exposure and then elevated by rapamycin treatment. (C). Electron microscopy analysis was performed in fimbria parasagittal ultrathin sections. The ratio of axonal caliber over diameter of myelinated fiber (g-ratio) was increased in isoflurane-exposed animals (it means decreased myelin thickness) compared to control, and rapamycin treatment reversed this change. Scale bar=0.5μm. n=8 for each group in (A), (B), and n=100 for each group in (C). One-way ANOVA; *: p<0.05; **: p<0.01. Iso: isoflurane. Error bars indicate SD.
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 22
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+A u t h o r
+
+M a n u s c r i p t
+
+Li et al.
+
+Fig. 6. The effect of isoflurane exposure and rapamycin treatment on DNA methylation level in oligodendrocytes. (A). DNA methylation level examined with DNA methyltransferase 1 and Olig2 double immunolabeling. The percentage of DNA methyltransferase 1+/Olig2+ over Olig2+ nuclei in isoflurane-exposed mice was lower than control and rapamycin increased this ratio. Scale bar=25 μm. (B). Western blot data revealed isoflurane dramatically decreased the DNA methyltransferase 1 level and rapamycin increased DNA methyltransferase 1. (C). 5-methylcytosine, the product of DNA methylation catalyzed by DNA methyltransferase 1, was detected in nuclei of adenomatous polyposis coli+ oligodendrocytes. The ratio of 5-methylcytosine +/ adenomatous polyposis coli + over adenomatous polyposis coli+ cells showed a statistically significant decrease with isoflurane exposure and rapamycin injection reversed this decrease. Scale bar=25μm. (n=8 for each group; One-way ANOVA; *: p<0.05; **: p<0.01; ***: p<0.001). Iso: isoflurane. Error bars represent SD.
+
+Anesthesiology. Author manuscript; available in PMC 2020 November 01.
+
+Page 23

new_pdfs/10.1097_ALN.0000435846.28299.e7.txt

@@ -0,0 +1,487 @@
+Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/930985/ on 06/09/2018
+
+Neonatal Exposure to Sevoflurane in Mice Causes Deficits in Maternal Behavior Later in Adulthood
+
+Yumiko	Takaenoki,	M.D.,	Yasushi	Satoh,	Ph.D.,	Yoshiyuki	Araki,	M.D.,	Mitsuyoshi	Kodama,	M.D.,	Ph.D.,		 Ryuji	Yonamine,	M.D.,	Shinya	Yufune,	M.D.,	Tomiei	Kazama,	M.D.,	Ph.D.
+
+ABSTRACT
+
+Background: In animal models, exposure to general anesthetics induces widespread increases in neuronal apoptosis in the developing brain. Subsequently, abnormalities in brain functioning are found in adulthood, long after the anesthetic exposure. These abnormalities include not only reduced learning abilities but also impaired social behaviors, suggesting pervasive deficits in brain functioning. But the underlying features of these deficits are still largely unknown. Methods: Six-day-old C57BL/6 female mice were exposed to 3% sevoflurane for 6 h with or without hydrogen (1.3%) as part of the carrier gas mixture. At 7–9 weeks of age, they were mated with healthy males. The first day after parturition, the maternal behaviors of dams were evaluated. The survival rate of newborn pups was recorded for 6 days after birth. Results: Female mice that received neonatal exposure to sevoflurane could mate normally and deliver healthy pups similar to controls. But these dams often left the pups scattered in the cage and nurtured them very little, so that about half of the pups died within a couple of days. Yet, these dams did not show any deficits in olfactory or exploratory behaviors. Notably, pups born to sevoflurane-treated dams were successfully fostered when nursed by control dams. Mice coadministered of hydrogen gas with sevoflurane did not exhibit the deficits of maternal behaviors. Conclusion: In an animal model, sevoflurane exposure in the developing brain caused serious impairment of maternal behav- iors when fostering their pups, suggesting pervasive impairment of brain functions including innate behavior essential to spe- cies survival. (Anesthesiology 2014; 120:403-15)
+
+A CCUMULATING evidence indicates that exposure
+
+What We Already Know about This Topic
+
+to general anesthetics at clinically effective concentra- tions induces widespread increases in neuronal apoptosis in the developing brain of a variety of animals ranging from rodents to rhesus monkeys.1–10 Furthermore, long after the anesthetic exposure, learning deficits are manifested later in adulthood1–5 even though a significant increase in neuro- nal apoptosis is no longer evident.11 The primary cause of these learning deficits in the adults is not fully understood, which hinders identification of the underlying pathophysiol- ogy. The impairment of brain function caused by neonatal exposure to sevoflurane is not specific to the learning deficit. We previously reported that neonatal exposure to sevoflu- rane induced a disturbance in social behaviors in mice that resembles those observed in subjects with autism.3 This evi- dence suggests that pervasive deficits in brain functioning may be induced. However, there is not enough evidence to support this hypothesis.
+
+Anesthetic	exposure	to	neonatal	animals	results	in	increased	 programmed	cell	death	in	the	brain	and	altered	neurocognitive	 development
+
+The	effects	of	this	exposure	on	innate	behavior	are	relatively unexplored
+
+What This Article Tells Us That Is New
+
+Female	 mouse	 pups	 exposed	 to	 sevoflurane	 anesthesia	 ex- hibited	deficits	in	classic	maternal	behaviors	after	delivery,	an	 effect	which	was	prevented	by	coadministration	of	the	antioxi- dant,	hydrogen	gas	with	sevoflurane
+
+Previous	anesthesia	exposure	did	not	alter	oxytocin	or	vaso- pressin	release	in	the	maternal	mice	after	delivery
+
+behavioral task for mice in normal laboratory conditions. Although it is believed that maternal behavior is influenced by hormones,15,16 accumulating evidence suggests a specific hormonal condition is not necessary to induce maternal behaviors. For instance, even nonpregnant nulliparous mice can exhibit maternal behaviors when extensively exposed to pups17 although they are rarely maternal spontaneously and actively avoid pups.18 This evidence indicates that sensory stimuli provided by newborns are important in the rapid onset of maternal behaviors in mammals.19–22
+
+Animals must adapt rapidly to changing environmental conditions. In mammals, pregnant females undergo fun- damental behavioral changes: the pattern of care by moth- ers to their offspring, which is called maternal behavior, is induced in female mothers. Maternal behavior is critical in rodents because pups are born deaf and blind. Neural mechanisms for maternal behavior have been studied most extensively in rodents,12–14 and it may be the most complex
+
+Recently, we found a high rate of mortality in pups born to female mice that were exposed to sevoflurane in their early
+
+Submitted for publication November 9, 2012. Accepted for publication August 29, 2013. From the Department of Anesthesiology, National
+
+Defense Medical College, Tokorozawa, Saitama, Japan. Copyright © 2013, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins. Anesthesiology 2014; 120:403-15
+
+Anesthesiology, V 120 • No 2
+
+403
+
+February 2014
+
+Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/930985/ on 06/09/2018
+
+Maternal Behavior in Sevoflurane-treated Mice
+
+stage after birth. In this study, to examine the deficient sur- vival of pups, we investigated whether maternal behavior was impaired in these dams. These mice showed serious impair- ment of maternal behaviors when they fostered their pups although parturition was normal, which severely impaired the survival of their offspring. We hypothesized two possible mechanisms for impairment of maternal behaviors in these dams: circulating neurohormones, vasopressin and oxytocin, which are related to maternal behaviors,23–26 could have been altered by neonatal exposure to general anesthetics. Alter- natively, activation of central circuits underlying maternal behaviors could have been altered without a change in release of vasopressin and oxytocin into the circulation. Accumu- lating evidence suggests the existence of central circuit for maternal behaviors in the medial preoptic area (MPOA) in the rostral hypothalamus.19,27,28 In addition, we hypoth- esized that neonatal exposure to sevoflurane could have altered maternal behavior by a mechanism reversible by anti- oxidant: antioxidant reportedly mitigated behavioral deficits caused by neonatal exposure to general anesthetics.29,30
+
+important difference was set at a 30% decrease from the baseline level in the control group.
+
+3. Behavioral studies: control, sevoflurane, and sevo- flurane + hydrogen groups (n = 10–11 dams for each group); the primary outcome measure was latencies for pup retrieval; in the pup retrieval test, a minimum bio- logically important difference was set at a 30% increase from the baseline level in the control group.
+
+4. Hormonal assay: control, hydrogen, sevoflurane, and sevoflurane + hydrogen groups (n = 4–5 dams for each group); a minimum biologically important difference was set as 30% decrease from the baseline level in the control group. Immunohistochemical study: control and sevoflurane groups (n = 5 dams for each group); a minimum bio- logically important difference was set at a 30% decrease from the baseline level in the control group. 5. In total, we prepared 160 female pups, which received anesthesia or hydrogen treatment at P6 (55 of control, 56 of sevoflurane, 40 of sevoflurane + hydrogen, and 9 of hydrogen groups). Among them, eight pups with sevoflurane and one pup with sevoflurane + hydrogen died during the treatment. Then, these siblings from the same litter were reunited and cohoused till the experiment (mice were similarly caged and housed in all groups). At 3 weeks of age, mice were weaned and allowed to further mature. At 7–9 weeks of age, female mice were mated with healthy males that had not been exposed to any anesthetic. Among them, 23 female mice did not get pregnant (eight of control, six of sevoflurane, five of sevoflurane + hydrogen, and four of hydrogen groups) and 1 control mouse died due to failure of delivery. These mice were excluded from the final analysis. Thus, for first delivery experiments, we used 46 control dams, 42 sevoflu- rane-treated dams, 34 sevoflurane + hydrogen–treated dams, and 5 hydrogen-treated dams. These mice were allocated as described above (1–5 in this section).
+
+Materials and Methods Animals All experiments were conducted according to the insti- tutional ethical guidelines for animal experiments of the National Defense Medical College and were approved by the Committee for Animal Research at National Defense Medi- cal College (Tokorozawa, Saitama, Japan). Inbred C57BL/6 mice were used in this study and maintained as described previously.5
+
+Anesthesia and Hydrogen Treatment Sevoflurane anesthesia was carried out as described previously.5 In brief, on postnatal day 6 (P6), pups were placed in a humid chamber immediately after removal of mice from the maternal cage. A 3% concentration of sevoflurane was administered in 30% oxygen as the carrier gas. Control mice were exposed to 30% oxygen. Hydrogen gas (1.3%) was supplied as described previously.30 Total gas flow rate was 2 l/min.
+
+Among them, some dams were further analyzed for behavioral studies of parous dams: the same sets of mice for behavioral studies in first-time delivery were reused in behavioral studies in second-time (parous) delivery (control: 7 for survival rate and 11 for behavioral studies; sevoflurane- treated: 8 for survival rate and 10 for behavioral studies).
+
+Mouse Study Design In each experiment, siblings from the same litter were ran- domly allocated into one of the following groups so that each group was balanced on littermate. No obvious differ- ences (e.g., body size and weight) were observed within the litters, and there was no significant difference in mean body weight among the groups (data not shown).
+
+For paternal study experiments, 26 age-matched male mice were either subjected to anesthesia (n = 13) or control (n = 13) treatment at P6 (no mice died during the treatment). Siblings from the same litter were allocated into each group almost equally (i.e., groups were balanced on littermate).
+
+1. Survival rate of delivered pups: control, sevoflurane, and sevoflurane + hydrogen groups (n = 17–19 dams for each group); a minimum biologically important dif- ference was set at a 30% decrease from the baseline level in the control group.
+
+Oxytocin and Vasopressin Assay Plasma concentrations of oxytocin and vasopressin in dams at 10 weeks of age were examined by enzyme-linked immu- nosorbent assay using commercially available kits (Oxytocin enzyme-linked immunosorbent assay kit and arg8-Vasopres- sin enzyme-linked immunosorbent assay kit; Enzo Life Sci- ences, Farmingdale, NY). Assays were performed according
+
+2. Pup exchange test: control and sevoflurane groups (n = 6 dams for each group); a minimum biologically
+
+Anesthesiology 2014; 120:403-15
+
+404
+
+Takaenoki et al.
+
+Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/930985/ on 06/09/2018
+
+PERIOPERATIVE MEDICINE
+
+to the manufacturer’s instructions. Blood samples were col- lected from the inferior vena cava within 6 h after parturition.
+
+sniff a pup for the first time and to return each pup to the nest were evaluated.
+
+Immunohistochemical Study Immunohistochemical studies using the anti-c-Fos antibody (rabbit polyclonal; sc-52; Santa Cruz Biotechnology, Santa Cruz, CA) were performed as previously described.30 Sam- ples were obtained within 6 h after parturition. The num- bers of immunoreactive cells were counted by an observer blinded to the groups.
+
+Evaluation of Parental Behavior Parental behavior of virgin male mice toward pups was eval- uated for 20 min. At the beginning, each mouse was put in one corner of a cage and three new born pups were placed in different corners of the same cage as described in the pup retrieval test. Latencies to sniff a pup for the first time and the numbers of males which committed attacks toward pups were evaluated. If any of the pups was attacked during the test, all pups were removed immediately and this subject was considered as “attack.”
+
+Behavioral Studies On the morning of parturition, maternal behaviors were examined. Maternal behavioral studies using first-time mothers were performed at 10–12 weeks of age. The same sets of female mice were reused in the maternal behavioral studies for second-time (parous) mothers: those mice were mated again at 19–25 weeks of age, and maternal behav- iors were examined at 22–28 weeks of age. Paternal behav- ioral studies using male mice were performed at 11 weeks of age. Survival rate (percentage of the number of pups at the indicated day compared with that at birth) was recorded until P6. In each experiment, observation was made by the same observer who was blinded to the groups. All appara- tus used in this study was made by O’Hara & CO., LTD. (Tokyo, Japan).
+
+Pup Exchange Test The pup exchange test was conducted as described previ- ously with some modifications.14 Pups born to a female dam couple (a dam with sevoflurane exposure at P6 and a control), which were born on the same day, were exchanged within 12 h after delivery. The number of surviving pups was evaluated for 6 days after birth.
+
+Olfactory Test The olfactory test was conducted as described previously.3
+
+Statistical Analysis Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA). Comparisons of the means of each group were performed using Student t test, one-way ANOVA followed by Bonferroni post hoc test, and two-way ANOVA followed by Bonferroni post hoc test. Comparisons of the survival rate until P6 were performed using a log-rank (Mantel-Cox) test. We did not exclude any data in this study. P values of less than 0.05 were considered statistically significant. Values are presented as the mean ± SEM in bar graphs.
+
+Evaluation of Maternal Behavior Pregnant females were individually housed for a few days before parturition and examined for maternal behavior on the morning of parturition. The number of pups with milk in their digestive tract and that of poorly cleaned pups (with pla- centa, amniotic membrane, or umbilical cords) was recorded on that day. Nest quality was also evaluated at the same time using the score system described previously31 with some modifications: grade 3, shaped like a deep hollow surrounded by high banks; grade 2, a hollow with medium-height banks; grade 1, flat with low banks, but still discrete; grade 0, no depression in bedding with no banks. Each new dam was also evaluated for time spent crouching over pups and the percentage of newborns scattered for 20 min with minimal disturbance as described previously.32 The percentage of scat- tered pups was expressed as a percentage with respect to time. We calculated the percentage of scatter as follows for each pup: (duration of scatter/total time observed (20 min) × 100). We then calculated the average for each group. These evalua- tions were carried out before the pup retrieval test.
+
+Results Survival Was Significantly Impaired in Pups Born to Mothers Exposed to Sevoflurane in Their Early Stage after Birth Female mice were exposed to 3% sevoflurane for 6 h and allowed to mature. These female mice appeared to grow nor- mally and could bear pups. But we found that about half of their pups died within 2 days after birth, whereas pups born to control mice (without exposure to sevoflurane) showed more than 80% survival rate at 6 days after birth (fig. 1). A log-rank analysis confirmed the difference, indicating a significantly lower survival rate in pups from sevoflurane- treated dams compared with those from control dams (P < 0.0001). The number of delivered pups in sevoflurane- treated dams was not significantly differ from control dams (sevoflurane group, n = 110 from 17 dams; control group, n = 124 from 19 dams), indicating that parturition was nor- mal in sevoflurane-treated dams.
+
+Pup Retrieval Test The pup retrieval test was performed essentially as described previously.14 Before the test, pups were separated from dams for 30 min. At the beginning, each mouse was put in one corner of a cage and three of her pups were placed in differ- ent corners of the same cage. The cages were continuously observed for 10 min with minimal disturbance. Latencies to
+
+Anesthesiology 2014; 120:403-15
+
+405
+
+Takaenoki et al.
+
+Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/930985/ on 06/09/2018
+
+Maternal Behavior in Sevoflurane-treated Mice
+
+returned to the nest by a dam using mouth grip. Sevoflu- rane-treated dams displayed a significantly longer latency to retrieve the pups than control dams (fig. 3, A–C; t test, sevoflurane-treated dams vs. control dams, t = 2.25, P < 0.05 [first retrieval]; t = 2.33, P < 0.05 [second retrieval]; and t = 2.78, P < 0.05 [complete retrieval]). The impairment of retrieval was not caused by failure of the dams to detect the pups, because latency to approach and sniff a pup for the first time was indistinguishable between sevoflurane-treated and control dams (fig. 3D; t test, sevoflurane-treated dams vs. control dams, t = 0.52, P > 0.05). Furthermore, the olfac- tory test showed that olfactory function was also normal in sevoflurane-treated dams (fig. 3E; t test, sevoflurane-treated dams vs. control dams, t = 0.10, P > 0.05). These results indi- cated that exploratory and investigative behaviors toward pups were normal in sevoflurane-treated dams, yet they were unable to effectively perform maternal behaviors.
+
+Fig. 1. Neonatal exposure to sevoflurane in female mice caused insufficient survival of their pups. Pup survival rate was assessed for 6 days using 110 (born to sevoflurane- treated dams [n = 17]) and 124 pups (born to control dams [n = 19]). About half of the pups born to sevoflurane-treated dams died, whereas pups born to control dams showed >80% surviving rate.
+
+Maternal Nurturing Was Impaired in Mice Exposed to Sevoflurane in Their Early Stage after Birth The number of pups that did not have milk in their diges- tive tracts was increased in pups born to sevoflurane-treated dams when compared with pups born to control dams (fig. 2, A and B). However, the ratio of poorly cleaned pups was indistinguishable between them (fig. 2C).
+
+Survival Deficits of the Pups Lay Entirely with Dams The reduced pup survival was likely caused by the impair- ment of maternal behaviors in sevoflurane-treated dams. To confirm whether the high mortality rate of pups born to sevo- flurane-treated dams was caused by dams or pups, we carried out the pup exchange test. In this test, pups born to sevoflu- rane-treated dams were successfully fostered when nursed by control dams (fig. 4A). But, more than half of pups born to control dams died when nursed by sevoflurane-treated dams (fig. 4A). A log-rank analysis indicated that the survival rate of pups nurtured by sevoflurane-treated dams was significantly lower than the survival rate of pups nurtured by control dams during the 6 days after birth (P < 0.0001). In addition, the pups born to 3% sevoflurane-treated dams but nursed by control dams had milk in their digestive tracts, whereas pups born to controls but nursed by sevoflurane-treated mice did not (fig. 4B). Thus, we concluded that the sevoflurane-treated dams caused the survival deficit of the pups.
+
+Furthermore, to assess whether the excess mortality of pups born to sevoflurane-treated dams was caused by defects in nurturing, we investigated maternal behaviors in sevoflu- rane-treated dams. Around the time of delivery, mice usu- ally prepare a high-walled, corner nest, which is significantly different from the flat, centrally located, sleeping pad of the nonpregnant female.20 Because these features are characteris- tic of maternal females, the evaluation of nest quality is often used as an indicator of maternal behavior.20 We found that sevoflurane-treated dams exhibited incomplete nest building compared with control dams (fig. 2, D–G). Comparison of the nest quality scores confirmed the difference, indicating the significant difference between sevoflurane-treated dams and controls (fig. 2G; t test, t = 3.32, P < 0.01). In addition, we examined the ratio of scattered pups as another indicator of maternal behavior.32,33 We found that the ratio of scat- tered pups out of the nest was significantly higher in the sevoflurane-treated dams (fig. 2H; t test, sevoflurane-treated group vs. control group, t = 2.17, P < 0.05).
+
+Maternal Behavior Was Not Impaired in Second-time Parous Dams that Were Exposed to Sevoflurane in Their Early Stage after Birth Subsequent to the first delivery, mice usually show a perma- nently enhanced rate of induction of maternal behaviors20 although the underlying mechanism is largely unknown. Therefore, we investigated whether maternal behaviors were also impaired in second-time parous mice that were exposed to sevoflurane at P6. We found that pup survival rate was indistinguishable between the sevoflurane-treated second-time parous dams and the second-time parous con- trol dams during the 6 days after birth (fig. 5). A log-rank analysis indicated that the survival rate of pups nurtured by sevoflurane-treated second-time parous dams was not sig- nificantly different from those of control dams during the 6 days after birth (P > 0.05). Majority of pups born to parous dams exposed to sevoflurane were cleaned and had milk in their digestive tracts, similar to pups born to parous control
+
+When all pups are in the nest, the dam normally hov- ers over them, allowing pups to suckle (crouching). We found that sevoflurane-treated dams exhibited a significantly shorter duration of crouching compared with control dams (fig. 2I; t test, sevoflurane-treated dams vs. control dams, t = 3.84, P < 0.01).
+
+Maternal behavior was further evaluated by the pup retrieval test, which is frequently used to measure maternal behavior.14,32,34,35 In the test, we monitored the response of dams to three pups placed in different corners of the cage for 10 min. Pups that wander from the nest are usually
+
+Anesthesiology 2014; 120:403-15
+
+406
+
+Takaenoki et al.
+
+Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/930985/ on 06/09/2018
+
+PERIOPERATIVE MEDICINE
+
+A
+
+B
+
+C
+
+D
+
+E
+
+F
+
+G
+
+H
+
+I
+
+Fig. 2. Neonatal exposure to sevoflurane in mice caused deficits in maternal behaviors later in adulthood. The aspects of maternal behaviors were assessed on the day of parturition. The same sets of mice were used in all tests shown in figure 2 (control dams, n = 11; sevoflurane-treated dams, n = 10). (A) Pups nursed by control dams (Cont) had milk in their digestive tract (arrow), whereas pups nursed by sevoflurane-treated dams (Sevo) did not. (B) Ratio of pups with or without milk in the digestive tract. (C) Ratio of cleaned or poorly cleaned pups. (D–F) Representative images of nests made by sevoflurane-treated mice and controls. (D) A control dam built a crater-like hollow with high banks, and no pups were scattered outside of the nest. (E) When the dam was removed from the nest, pups under the crouching dam in the deep bottom were apparent. Sevoflurane-treated dams built much shallower or flat nests. (F) Significantly more pups were scattered outside of the nest. (G) The nest quality scores (grade 0–3). (H) Percentage of scattered pups outside of the nest. (I) Time spent crouching over the pups in the nest. *P < 0.05, **P < 0.01 (t test).
+
+dams (fig. 6, A and B). Sevoflurane-treated parous dams also exhibited nest quality scores similar to parous control dams (fig. 6C; t test, sevoflurane-treated dams vs. control dams, t = 0.61, P > 0.05). The ratio of scattered pups was not sig- nificantly different between sevoflurane-treated parous dams and parous control dams (fig. 6D; t test, sevoflurane-treated group vs. control group, t = 0.93, P > 0.05). Time spent crouching was also indistinguishable between them (fig. 6E; t test, sevoflurane-treated dams vs. control dams, t = 0.36, P > 0.05).
+
+t = 0.07, P > 0.05 [first retrieval]; t = 0.06, P > 0.05 [second retrieval]; and t = 0.04, P > 0.05 [complete retrieval]). The latency to approach and sniff a pup for the first time was also indistinguishable between them (fig. 7B; t test, sevoflurane- treated dams vs. control dams, t = 0.83, P > 0.05). These results indicated that maternal behaviors of parous dams were indistinguishable regardless of sevoflurane exposure in their early stage after birth.
+
+Parental Behaviors Were Impaired in Male Mice That Were Exposed to Sevoflurane in Their Early Stage after Birth Accumulating evidence suggests that the neural circuit for maternal behavior exists in males as well as female mice.20
+
+In the pup retrieval test, sevoflurane-treated parous dams displayed latencies to retrieve the pups similar to controls (fig. 7A; t test, sevoflurane-treated dams vs. control dams,
+
+Anesthesiology 2014; 120:403-15
+
+407
+
+Takaenoki et al.
+
+Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/930985/ on 06/09/2018
+
+Maternal Behavior in Sevoflurane-treated Mice
+
+A
+
+B
+
+A
+
+B
+
+C
+
+Fig. 4. The pup exchange test demonstrated that the re- duced survival of pups lay entirely with dams. Pups were cross-fostered by sevoflurane-treated dams and control dams. (A) Six paired sevoflurane-treated and control dams that produced six to nine pups each were exchanged within 12 h after delivery. Pup survival rate was assessed for 6 days using 44 (born to sevoflurane-treated dams but nursed by control dams) and 45 pups (born to control dams but nursed by sevoflurane-treated dams). (B) Pups born to sevoflurane- treated dams had milk in the digestive tract (arrow) when nursed by control mice (Cont), whereas pups born to con- trol dams had no milk in the digestive tract when nursed by sevoflurane-treated dams (Sevo).
+
+D
+
+E
+
+Hydrogen Coadministration Mitigated Impairments of Maternal Behavior Caused by Sevoflurane Exposure in Their Early Stage after Birth We recently showed that the antioxidative effects of molecu- lar hydrogen gas suppressed neurotoxicity caused by neo- natal exposure to anesthetics in the developing brain.30 Hydrogen gas can be easily supplied as part of the carrier gas mixture during anesthesia. Thus, we sought to investigate whether coadministration of hydrogen gas with sevoflurane could effectively suppress impairments of maternal behaviors caused by neonatal anesthetic exposure.
+
+Fig. 3. Pup retrieval responses were impaired in sevoflurane- exposed, first-time dams on the day of parturition. Same sets of dams for figure 2 were used. At the beginning, each dam was put in one corner of a cage and three of her pups were placed in different corners of the same cage. (A and B) Representative images of dams in the retrieval test at 2 min. (A) Control dams retrieved all pups within a couple of minutes. (B) But majority of pups born to sevoflurane-treated dams were still scattered out- side of the nest at 2 min (right). (C) Latency to retrieve each pup by sevoflurane-treated dams or control dams. (D) Latency to sniff a pup for the first time. (E) The olfactory test. *P < 0.05 (t test).
+
+The survival deficits of pups born to dams exposed to sevoflurane at P6 was prevented by coadministration of 1.3% hydrogen gas with sevoflurane (fig. 9). A log-rank anal- ysis indicated that pup survival rate was indistinguishable between the control dams and the sevoflurane + hydrogen– treated dams (P > 0.05). Furthermore, a log-rank analysis
+
+In some rodents, behavior toward the young is essentially the same for each sex.36,37 To investigate whether parental behavior is impaired by neonatal exposure to sevoflurane, sevoflurane-treated virgin male mice were exposed to three newborn pups for 20 min. The latency to approach and sniff a pup for the first time was indistinguishable regard- less of neonatal exposure to sevoflurane (fig. 8A; t test, sevo- flurane-treated male vs. control male, t = 0.37, P > 0.05). But we observed that some of sevoflurane-treated males bit pups within a few minutes after exposure to newborn pups, whereas control males did not commit attacks (fig. 8B). Thus, nurturing behaviors were impaired in male mice simi- lar to first-time mothers.
+
+Fig. 5. Maternal behavior was not impaired in parous dams that were exposed to sevoflurane at P6. Pup survival rate was assessed for 6 days using 59 pups (born to sevoflurane-treat- ed parous [second-time] dams [n = 8]) and 48 (born to control parous [second-time] dams [n = 7]).
+
+Anesthesiology 2014; 120:403-15
+
+408
+
+Takaenoki et al.
+
+Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/930985/ on 06/09/2018
+
+PERIOPERATIVE MEDICINE
+
+A
+
+B
+
+C
+
+D
+
+E
+
+Fig. 6. Maternal behaviors in sevoflurane-treated parous dams were normal. The aspects of maternal behaviors were assessed on the day of parturition. Same sets of mice were used in all tests shown in figure 6 (parous control dams, n = 11; sevoflurane- treated parous dams, n = 10). (A) The ratio of pups with or without milk in the digestive tract. (B) The ratio of cleaned or poorly cleaned pups. (C) The nest quality scores (grade 0–3). (D) Percentage of scattered pups outside of the nest. (E) Time spent crouching over the pups in the nest.
+
+also indicated that the pup survival rate of the sevoflurane + hydrogen–treated dams was significantly higher than that of the sevoflurane-treated dams (P < 0.0001).
+
+between them (fig. 10B). Furthermore, we found that the sevoflurane + hydrogen–treated dams exhibited a normal nest-building score (fig. 10C), a normal ratio of scattered pups in their home cage (fig. 10D), and a normal duration of crouching (fig. 10E) compared with those of the con- trol dams. A one-way ANOVA followed by Bonferroni post hoc test confirmed these findings, indicating no significant
+
+The number of pups that did not have milk in their digestive tracts was indistinguishable between the control group and sevoflurane + hydrogen–treated dams (fig. 10A). The ratio of poorly cleaned pups was also indistinguishable
+
+A
+
+B
+
+0
+
+Fig. 7. Pup retrieval responses were normal in sevoflurane-treated parous mice. Same sets of dams for figure 6 were used. (A) Latency to retrieve each pup by sevoflurane-treated parous dams and parous control dams. (B) Latency to sniff a pup for the first time.
+
+Anesthesiology 2014; 120:403-15
+
+409
+
+Takaenoki et al.
+
+Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/930985/ on 06/09/2018
+
+Maternal Behavior in Sevoflurane-treated Mice
+
+Plasma Concentrations of Oxytocin and Vasopressin Were Not Significantly Changed in Sevoflurane-treated Dams Oxytocin and vasopressin are known to be implicated in the induction of maternal behaviors.23–26 It is possible that neo- natal exposure to anesthetics might alter the release of these hormones, leading to the impairment of maternal behav- ior. Therefore, we sought to examine whether the plasma concentration levels of oxytocin and vasopressin could be disrupted in sevoflurane-treated dams by the use of enzyme- linked immunosorbent assay. Furthermore, to exclude the possibility that the exposure to hydrogen gas per se counter- acts perturbations in hormonal levels, we also examined the plasma concentration levels of these hormones in hydrogen- treated mice. We found that there was no significant change in plasma oxytocin concentration on the day of parturition when comparing control, sevoflurane, hydrogen, and sevo- flurane + hydrogen groups (fig. 12A). A two-way ANOVA confirmed this, indicating no significant main effect of sevo- flurane treatment (F = 0.005, P > 0.05) and of hydrogen treatment (F = 0.012, P > 0.05). The interaction between sevoflurane and hydrogen treatment was not significant as well (F = 0.446, P > 0.05).
+
+A
+
+B
+
+Fig. 8. Parental behaviors were impaired in male mice that were exposed to sevoflurane at P6. Each mouse (control group: n = 13; sevoflurane-treated group: n = 13) was put in one corner of a cage, and three pups from healthy dams were placed in different corners of the same cage. (A) Latency to sniff a pup for the first time. (B) Percentage of attacking and not attacking male mice.
+
+differences between sevoflurane + hydrogen–treated dams and controls in these tests (fig. 10, C–E; F and P values are presented below each panel; post hoc test, P > 0.05 for each test). In the pup retrieval test, the sevoflurane + hydro- gen–treated dams performed similar to the control dams (fig. 11A). A one-way ANOVA followed by Bonferroni post hoc test confirmed this, indicating no significant difference between sevoflurane + hydrogen–treated dams and controls in each retrieval (fig. 11A; F and P values are presented below each panel; post hoc test, P > 0.05 for each retrieval). We did not detect significant differences in the analysis for laten- cies to approach and to sniff a pup for the first time among groups (fig. 11B; one-way ANOVA, F = 0.19, P > 0.05). Olfaction abilities were also indistinguishable among groups (fig. 11C; one-way ANOVA, F = 0.008, P > 0.05). Together, it can be concluded that concomitant hydrogen inhalation significantly mitigated impairment of maternal behaviors caused by neonatal exposure to sevoflurane.
+
+Similarly, there was no significant change in plasma vaso- pressin concentration on the day of parturition when com- paring control, sevoflurane, hydrogen, and sevoflurane + hydrogen groups (fig. 12B). A two-way ANOVA confirmed this, indicating no significant main effect of sevoflurane treatment (F = 0.018, P > 0.05) and of hydrogen treat- ment (F = 1.194, P > 0.05). The interaction between sevo- flurane and hydrogen treatment was not significant as well (F = 0.206, P > 0.05). Therefore, we concluded that neither neonatal exposure to sevoflurane nor to hydrogen altered the blood concentration levels of oxytocin and vasopressin when fostering their pups.
+
+The Number of c-Fos–Immunopositive Cells Decreased in the MPOA in Sevoflurane-treated Dams Modification of neurons in the MPOA in the rostral hypo- thalamus is required to express maternal behaviors.19,27,28 It was reported that when a mouse takes care of pups, c-Fos is induced in the MPOA.38,39 Thus, we set out to quantify the number of c-Fos–immunopositive cells in the MPOA from maternal dams when fostering their pups. In sevoflurane- treated dams, the number of c-Fos–immunopositive cells was significantly reduced in the MPOA when compared with control dams (fig. 13, A and B; t test, sevoflurane-treated dams vs. control dams, t = 6.92, P < 0.001). This finding suggested that neonatal exposure to sevoflurane disrupted neural mechanism to induce maternal behavior.
+
+Fig. 9. Hydrogen coadministration mitigated the deficient sur- vival rate in pups caused by sevoflurane exposure to dams in their early stage after birth. Pup survival rate was assessed for 6 days using 124 (born to control dams [n = 19]), 110 (born to sevoflurane-treated dams [n = 17]), and 128 pups (born to sevoflurane + hydrogen-treated dams [n = 19]). The same data for the control group and sevoflurane-treated group in figure 1 are reused.
+
+Discussion
+
+In this study, we showed that sevoflurane exposure in the developing brain of female mice caused serious impair- ment of maternal behaviors when fostering their pups although parturition was normal. Furthermore, survival
+
+Anesthesiology 2014; 120:403-15
+
+410
+
+Takaenoki et al.
+
+Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/930985/ on 06/09/2018
+
+PERIOPERATIVE MEDICINE
+
+A
+
+B
+
+C
+
+D
+
+E
+
+Fig. 10. Hydrogen coadministration mitigated impairments of maternal behavior caused by sevoflurane exposure to dams in their early stage after birth. The aspects of maternal behaviors were assessed on the day of parturition. Same sets of mice were used in all tests shown in figure 10 (control dams, n = 11; sevoflurane-treated dams, n = 10; sevoflurane + hydrogen coadmin- istered dams, n = 10). The same data for the control dams and sevoflurane-treated dams in figure 2 are reused. (A) Ratio of pups with or without milk in the digestive tract. (B) Ratio of cleaned or poorly cleaned pups. (C) The nest quality score (grade 0–3). (D) Percentage of scattered pups outside of the nest. (E) Time spent crouching over the pups in the nest. Comparisons were performed using a one-way ANOVA followed by Bonferroni post hoc test. F and P values are presented below each panel. *P < 0.05, **P <0.01, ***P < 0.001.
+
+(placentophagia).20 Because these behaviors are char- acteristic of maternal females, some researchers classify them as one of maternal behaviors. However, from the dam’s perspective, afterbirth materials contain attractive substances such as placental opioid-enhancing factor that potentiate the antihyperalgesic properties of endogenous opioids.20 Thus, cleaning pup is not a maternal behavior in the sense of pup-directed caretaking behavior.
+
+rate was severely impaired in pups born to sevoflurane- treated dams. Pup exchange test showed that the sur- vival impairment of the pups lay entirely with dams. Taken together, deficits in maternal behavior of sevoflu- rane-treated dams caused the impaired survival of their offspring. The deficits in maternal behavior in sevoflu- rane-treated virgin females were not attributed to the secondary effect of other changes such as locomotor or olfactory functions because exploratory and investiga- tive behaviors toward pups were normal in these dams. It should be noted that all dams, irrespective of sevo- flurane treatment, promptly approached pups when the pups were placed outside the nest, indicating that neo- natal exposure to sevoflurane might not cause an inabil- ity to detect sensory cues emanating from pups. We did not find significant difference in pup-cleaning behavior between sevoflurane-treated dams and controls. At deliv- ery, normal dams usually devote an inordinate amount of attention to birth material and ingest the afterbirth
+
+We found that blood concentrations of oxytocin and vasopressin were not significantly changed in sevoflurane- treated dams compared with controls when fostering their pups. However, some studies reported that neonatal expo- sure to general anesthetics increased proinflammatory cyto- kines and stress hormones shortly after the anesthesia.40–44 Because some hormones are known to play important roles in neuronal development,45,46 we cannot exclude the pos- sibility that neonatal exposure to general anesthetics could cause impairment to hormone dynamics shortly after the anesthesia, which might affect neuronal development.
+
+Anesthesiology 2014; 120:403-15
+
+411
+
+Takaenoki et al.
+
+Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/930985/ on 06/09/2018
+
+Maternal Behavior in Sevoflurane-treated Mice
+
+A
+
+A
+
+B
+
+Fig. 12. Plasma concentrations of oxytocin and vasopressin were not significantly changed in sevoflurane-treated dams. (A) Levels of plasma oxytocin concentration (control dams, n = 5; sevoflurane-treated dams, n = 4; sevoflurane and hy- drogen–treated dams, n = 5; and hydrogen-treated dams, n = 5). (B) Levels of plasma vasopressin (control dams, n = 5; sevoflurane-treated dams, n = 4; sevoflurane and hydrogen– treated dams, n = 4; and hydrogen-treated dams, n = 5).
+
+B
+
+C
+
+density protein-95 in the hippocampus.40 Because postsyn- aptic density protein-95 is a candidate molecule implicated in synaptic plasticity,49,50 neonatal exposure to anesthetics might impair synaptic plasticity, which is required to mod- ulate neuronal circuit. Thus, one might speculate that the impairment of the hippocampus was involved in the impair- ment of maternal behaviors in sevoflurane-treated dams. Fur- thermore, it was reported that lesions in the hippocampus disrupted maternal behavior in rats although the underlying mechanism was largely unknown.51 However, some studies reported that limbic structures including the hippocampus are important but not essential for maternal behavior.52–54 One might also speculate that the defect in maternal behav- iors was secondary to learning or memory deficits in sevoflu- rane-treated mice. However, a defect in maternal behavior is not necessarily indicative of these deficits. Indeed, there are mice that have deficits in maternal behaviors but not in learn- ing and memory. For instance, mice lacking the immediate early gene fosB showed deficits in maternal behaviors but not in learning ability.13
+
+Fig. 11. Pup retrieval responses were mitigated in sevoflu- rane + hydrogen–treated dams. Same sets of dams for fig- ure 10 were used. The same data for the control dams and sevoflurane-treated dams in figure 3 are reused. (A) Latency to retrieve pups by sevoflurane-treated dams, sevoflurane + hydrogen–treated dams and control dams. (B) Latency to sniff a pup for the first time. (C) The olfactory test. Comparisons were performed using a one-way ANOVA followed by Bonfer- roni post hoc test. F and P values are presented below each panel. *P < 0.05, **P < 0.01.
+
+We found that the number of c-Fos–immunoreactive cells was significantly decreased in the MPOA from sevo- flurane-treated dams when compared with controls. Accu- mulating evidence indicates that the MPOA in the rostral hypothalamus plays critical roles in the induction of mater- nal behavior.19,27,28 Stimulation from pups converges on the MPOA, and it activates neurons in the MPOA. Then, the activated neurons induce the expression of transcription fac- tors such as c-Fos and its homolog FosB. These transcription factors are known to be essential for the facilitation of mater- nal behaviors.13,31,34 Thus, the reduced induction of c-Fos in the MPOA suggested that neonatal exposure to anesthet- ics impaired neuronal mechanism that plays critical role in maternal behavior.
+
+How does neonatal exposure to general anesthetics cause impairment of maternal behaviors in mice? Although the molecular, cellular, and neurological mechanisms are largely unknown, accumulating evidence indicated that expression of maternal behaviors seems to require change of the neu- ral circuit in the brain.34 Generally, once virgin females are sensitized by extensive exposure to pups, maternal behaviors last for at least several days without further pup exposure. Therefore, the experience of pup exposure may be important to elicit changes in the neural circuit for maternal behav- iors, which modify maternal responsiveness in a long-lasting manner. Taking this into consideration, one possible expla- nation for the impairment of maternal behavior in sevoflu- rane-treated mice is that the neural circuit indispensable for this type of behavior might be stunted in these mice. But the mothering experience at the first delivery might add an additional redundant route, via memory, to the process of activating the maternal neural circuit. Thus, second-time
+
+The hippocampus plays critical roles in multiple brain functions including working memory, which is required to do complex cognitive tasks.47,48 Hippocampus is known to be vulnerable to neonatal exposure to general anesthetics. For instance, it was reported that neonatal exposure to general anesthetics reportedly decreased the expression of postsynaptic
+
+Anesthesiology 2014; 120:403-15
+
+412
+
+Takaenoki et al.
+
+Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/930985/ on 06/09/2018
+
+PERIOPERATIVE MEDICINE
+
+A
+
+B
+
+Fig. 13. The number of c-Fos–immunopositive cells in the medial preoptic area (MPOA) decreased in sevoflurane-treated dams on the day of parturition. (A) Immunohistochemical staining for c-Fos in the MPOA (control dams: n = 5; sevoflurane-treated dams: n = 5). Scale bars: 250 μm. (B) Quantification of the immunohistochemical staining. ***P < 0.001 (t test).
+
+parous dams could foster pups irrespective of sevoflurane exposure. An alternative explanation is that stimuli ema- nating from newborn pups might not sufficiently activate the maternal neural circuit in the sevoflurane-treated mice: because of the failure to change the neural circuit effectively in sevoflurane-treated dams, they could not overcome the threshold to express maternal behavior. However, the par- tial change of the maternal neural circuit induced at the first delivery might contribute to overcome the threshold at the second delivery. In both cases, the adaptation to induce maternal behavior might depend on finely tuned neuronal mechanisms in the circuit for maternal behavior. It should be noted that the capacity to learn still remained in sevo- flurane-treated mice because maternal behavior deficit was no longer apparent after the birth of the second litter. This is consistent with a report that environmental enrichment reversed anesthetic-induced memory impairments in the rat developing brain almost completely even when instituted with substantial delay.55
+
+Although there are interpretative limitations to translate ani- mal models to humans, an understanding of the neurobio- logical basis for the deficits of maternal behavior caused by neonatal exposure to sevoflurane is important in psychiatric medicine and would be helpful for ensuring safer anesthesia in pediatric medicine.
+
+In conclusion, sevoflurane exposure in the mouse devel- oping brain causes serious impairment of maternal behav- iors when the females foster their pups although parturition is normal. We previously showed that neonatal exposure to sevoflurane caused impairments of social behaviors that resemble those observed in autism.3 Together with the previous reports, it may be concluded that in an animal model, neonatal exposure to general anesthetics causes pervasive deficits in brain functioning including even an innate behavior that is essential to species survival. Further molecular neuropathological investigations are necessary to fully explain the diverse behavioral alterations caused by neonatal exposure to general anesthetics.
+
+It was reported that oxidative stress was involved in anesthetic-induced neurotoxicity in the developing brain.29 Hydrogen has recently received attention as an effective antioxidant because of its small size and electrically neutral properties, enabling it to reach target organs easily, to diffuse across cell membranes rapidly, and to penetrate the blood– brain barrier for the protection of neurons.56 We previously showed that coadministation of hydrogen gas significantly suppressed the increase in neuroapoptosis and subsequently mitigated the deficits in social behaviors as well as learn- ing deficits caused by neonatal exposure to sevoflurane.30 In the current study, we show that coadministration of hydrogen gas significantly reduced impairment of mater- nal behaviors caused by neonatal exposure to sevoflurane, suggesting further potential of hydrogen coadministration for therapeutic use.
+
+Acknowledgments The authors thank Ms. Kiyoko Takamiya and Mrs. Yuko Ogura (Department of Anesthesiology, National Defense Medical College, Tokorozawa, Saitama, Japan) for excellent technical help in this study.
+
+This work was supported by Japan Society for the Pro- motion of Science ( JSPS; Tokyo, Japan); grant numbers 22500304, 23791734, 25861404, and 25293331.
+
+Competing Interests The authors declare no competing interests.
+
+Correspondence Address correspondence to Dr. Satoh: Department of Anes- thesiology, National Defense Medical College, 3-2 Namiki, Tokorozawa 359–8513, Japan. wndlt3@gmail.com. Informa- tion on purchasing reprints may be found at www.anesthe- siology.org or on the masthead page at the beginning of this issue. ANeSTheSIOlOgY’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
+
+Similarities in the maternal behaviors of humans and rodents suggest an existence of a general neural circuit for these behaviors. In humans, child abuse and neglect are global problems with serious life-long consequences, includ- ing increased likelihood for a wide range of mental disorders.
+
+Anesthesiology 2014; 120:403-15
+
+413
+
+Takaenoki et al.
+
+Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/930985/ on 06/09/2018
+
+Maternal Behavior in Sevoflurane-treated Mice
+
+nulliparous and parturient females. Physiol Behav 1981; 27:863–8
+
+References 1. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF: Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 2003; 23:876–82
+
+19. Numan M: Motivational systems and the neural circuitry of maternal behavior in the rat. Dev Psychobiol 2007; 49:12–21 20. Kristal MB: The biopsychology of maternal behavior in non- human mammals. ILAR J 2009; 50:51–63
+
+2. Stratmann G, Sall JW, May LD, Bell JS, Magnusson KR, Rau V, Visrodia KH, Alvi RS, Ku B, Lee MT, Dai R: Isoflurane differ- entially affects neurogenesis and long-term neurocognitive function in 60-day-old and 7-day-old rats. ANESTHESIOLOGY 2009; 110:834–48
+
+21. Stern JM, Lonstein JS: Neural mediation of nursing and related maternal behaviors. Prog Brain Res 2001; 133:263–78 22. Brunelli SA, Shair HN, Hofer MA: Hypothermic vocalizations of rat pups (Rattus norvegicus) elicit and direct maternal search behavior. J Comp Psychol 1994; 108:298–303
+
+3. Satomoto M, Satoh Y, Terui K, Miyao H, Takishima K, Ito M, Imaki J: Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice. ANESTHESIOLOGY 2009; 110:628–37
+
+23. Bick J, Dozier M: Mothers’ and children’s concentrations of oxytocin following close, physical interactions with bio- logical and non-biological children. Dev Psychobiol 2010; 52:100–7
+
+4. Fredriksson A, Pontén E, Gordh T, Eriksson P: Neonatal exposure to a combination of N-methyl-D-aspartate and gamma-aminobutyric acid type A receptor anes- thetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. ANESTHESIOLOGY 2007; 107:427–36
+
+24. van Leengoed E, Kerker E, Swanson HH: Inhibition of post- partum maternal behaviour in the rat by injecting an oxyto- cin antagonist into the cerebral ventricles. J Endocrinol 1987; 112:275–82
+
+25. Lee HJ, Macbeth AH, Pagani JH, Young WS III: Oxytocin: The great facilitator of life. Prog Neurobiol 2009; 88:127–51 26. Bosch OJ, Neumann ID: Brain vasopressin is an important regulator of maternal behavior independent of dams’ trait anxiety. Proc Natl Acad Sci U S A 2008; 105:17139–44
+
+5. Kodama M, Satoh Y, Otsubo Y, Araki Y, Yonamine R, Masui K, Kazama T: Neonatal desflurane exposure induces more robust neuroapoptosis than do isoflurane and sevoflu- rane and impairs working memory. ANESTHESIOLOGY 2011; 115:979–91
+
+27. Numan M: A neural circuitry analysis of maternal behavior in the rat. Acta Paediatr Suppl 1994; 397:19–28
+
+6. Brambrink AM, Evers AS, Avidan MS, Farber NB, Smith DJ, Zhang X, Dissen GA, Creeley CE, Olney JW: Isoflurane- induced neuroapoptosis in the neonatal rhesus macaque brain. ANESTHESIOLOGY 2010; 112:834–41
+
+28. Numan M: Hypothalamic neural circuits regulating maternal responsiveness toward infants. Behav Cogn Neurosci Rev 2006; 5:163–90
+
+29. Boscolo A, Starr JA, Sanchez V, Lunardi N, DiGruccio MR, Ori C, Erisir A, Trimmer P, Bennett J, Jevtovic-Todorovic V: The abolishment of anesthesia-induced cognitive impairment by timely protection of mitochondria in the developing rat brain: The importance of free oxygen radicals and mitochon- drial integrity. Neurobiol Dis 2012; 45:1031–41
+
+7. Cattano D, Young C, Straiko MM, Olney JW: Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain. Anesth Analg 2008; 106:1712–4
+
+8. Palanisamy A, Baxter MG, Keel PK, Xie Z, Crosby G, Culley DJ: Rats exposed to isoflurane in utero during early gestation are behaviorally abnormal as adults. ANESTHESIOLOGY 2011; 114:521–8
+
+30. Yonamine R, Satoh Y, Kodama M, Araki Y, Kazama T: Coadministration of hydrogen gas as part of the carrier gas mixture suppresses neuronal apoptosis and subsequent behavioral deficits caused by neonatal exposure to sevoflu- rane in mice. ANESTHESIOLOGY 2013; 118:105–13
+
+9. Johnson SA, Young C, Olney JW: Isoflurane-induced neuro- apoptosis in the developing brain of nonhypoglycemic mice. J Neurosurg Anesthesiol 2008; 20:21–8
+
+10. Slikker W Jr, Zou X, Hotchkiss CE, Divine RL, Sadovova N, Twaddle NC, Doerge DR, Scallet AC, Patterson TA, Hanig JP, Paule MG, Wang C: Ketamine-induced neuronal cell death in the perinatal rhesus monkey. Toxicol Sci 2007; 98:145–58 11. Loepke AW, Istaphanous GK, McAuliffe JJ III, Miles L, Hughes EA, McCann JC, Harlow KE, Kurth CD, Williams MT, Vorhees CV, Danzer SC: The effects of neonatal isoflurane exposure in mice on brain cell viability, adult behavior, learning, and memory. Anesth Analg 2009; 108:90–104
+
+31. Kuroda KO, Meaney MJ, Uetani N, Kato T: Neurobehavioral basis of the impaired nurturing in mice lacking the immedi- ate early gene FosB. Brain Res 2008; 1211:57–71
+
+32. Takayanagi Y, Yoshida M, Bielsky IF, Ross HE, Kawamata M, Onaka T, Yanagisawa T, Kimura T, Matzuk MM, Young LJ, Nishimori K: Pervasive social deficits, but normal parturition, in oxytocin receptor-deficient mice. Proc Natl Acad Sci U S A 2005; 102:16096–101
+
+33. Brooks LR, Le CD, Chung WC, Tsai PS: Maternal behavior in transgenic mice with reduced fibroblast growth factor recep- tor function in gonadotropin-releasing hormone neurons. Behav Brain Funct 2012; 8:47
+
+12. Numan M, Numan MJ, Marzella SR, Palumbo A: Expression of c-fos, fos B, and egr-1 in the medial preoptic area and bed nucleus of the stria terminalis during maternal behavior in rats. Brain Res 1998; 792:348–52
+
+34. Kuroda KO, Meaney MJ, Uetani N, Fortin Y, Ponton A, Kato T: ERK-FosB signaling in dorsal MPOA neurons plays a major role in the initiation of parental behavior in mice. Mol Cell Neurosci 2007; 36:121–31
+
+13. Brown JR, Ye H, Bronson RT, Dikkes P, Greenberg ME: A defect in nurturing in mice lacking the immediate early gene fosB. Cell 1996; 86:297–309
+
+14. Satoh Y, Endo S, Nakata T, Kobayashi Y, Yamada K, Ikeda T, Takeuchi A, Hiramoto T, Watanabe Y, Kazama T: ERK2 con- tributes to the control of social behaviors in mice. J Neurosci 2011; 31:11953–67
+
+35. Liu HX, Lopatina O, Higashida C, Fujimoto H, Akther S, Inzhutova A, Liang M, Zhong J, Tsuji T, Yoshihara T, Sumi K, Ishiyama M, Ma WJ, Ozaki M, Yagitani S, Yokoyama S, Mukaida N, Sakurai T, Hori O, Yoshioka K, Hirao A, Kato Y, Ishihara K, Kato I, Okamoto H, Cherepanov SM, Salmina AB, Hirai H, Asano M, Brown DA, Nagano I, Higashida H: Displays of paternal mouse pup retrieval following commu- nicative interaction with maternal mates. Nat Commun 2013; 4:1346
+
+15. Rosenblatt JS, Mayer AD, Giordano AL: Hormonal basis dur- ing pregnancy for the onset of maternal behavior in the rat. Psychoneuroendocrinology 1988; 13:29–46
+
+16. Mann PE, Bridges RS: Lactogenic hormone regulation of maternal behavior. Prog Brain Res 2001; 133:251–62
+
+17. Rosenblatt JS: Nonhormonal basis of maternal behavior in the rat. Science 1967; 156:1512–4
+
+36. Lonstein JS, De Vries GJ: Sex differences in the parental behavior of rodents. Neurosci Biobehav Rev 2000; 24:669–86 37. Wynne-Edwards KE, Timonin ME: Paternal care in rodents: Weakening support for hormonal regulation of the transition
+
+18. Fleming AS, Luebke C: Timidity prevents the virgin female rat from being a good mother: Emotionality differences between
+
+Anesthesiology 2014; 120:403-15
+
+414
+
+Takaenoki et al.
+
+Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/930985/ on 06/09/2018
+
+PERIOPERATIVE MEDICINE
+
+to behavioral fatherhood in rodent animal models of bipa- rental care. Horm Behav 2007; 52:114–21
+
+47. Saxe MD, Battaglia F, Wang JW, Malleret G, David DJ, Monckton JE, Garcia AD, Sofroniew MV, Kandel ER, Santarelli L, Hen R, Drew MR: Ablation of hippocampal neurogenesis impairs contextual fear conditioning and synaptic plasticity in the dentate gyrus. Proc Natl Acad Sci U S A 2006; 103:17501–6 48. Jones MW: A comparative review of rodent prefrontal cortex and working memory. Curr Mol Med 2002; 2:639–47
+
+38. Calamandrei G, Keverne EB: Differential expression of Fos protein in the brain of female mice dependent on pup sen- sory cues and maternal experience. Behav Neurosci 1994; 108:113–20
+
+39. Numan M, Numan MJ: Expression of Fos-like immunoreac- tivity in the preoptic area of maternally behaving virgin and postpartum rats. Behav Neurosci 1994; 108:379–94
+
+49. Ehrlich I, Klein M, Rumpel S, Malinow R: PSD-95 is required for activity-driven synapse stabilization. Proc Natl Acad Sci U S A 2007; 104:4176–81
+
+40. Shen X, Dong Y, Xu Z, Wang H, Miao C, Soriano SG, Sun D, Baxter MG, Zhang Y, Xie Z: Selective anesthesia-induced neuroinflammation in developing mouse brain and cognitive impairment. ANESTHESIOLOGY 2013; 118:502–15
+
+50. Béïque JC, Andrade R: PSD-95 regulates synaptic transmis- sion and plasticity in rat cerebral cortex. J Physiol 2003; 546(Pt 3):859–67
+
+41. Patanella AK, Zinno M, Quaranta D, Nociti V, Frisullo G, Gainotti G, Tonali PA, Batocchi AP, Marra C: Correlations between peripheral blood mononuclear cell production of BDNF, TNF-alpha, IL-6, IL-10 and cognitive performances in multiple sclerosis patients. J Neurosci Res 2010; 88:1106–12 42. Tan EK, Chan LL: Neurovascular compression syndromes and hypertension: Clinical relevance. Nat Clin Pract Neurol 2007; 3:416–7
+
+51. Kimble DP, Rogers L, Hendrickson CW: Hippocampal lesions disrupt maternal, not sexual, behavior in the albino rat. J Comp Physiol Psychol 1967; 63:401–7
+
+52. Lamb ME: Physiological mechanisms in the control of mater- nal behavior in rats: A review. Psychol Bull 1975; 82:104–19 53. Steele MK, Rowland D, Moltz H: Initiation of maternal behav- ior in the rat: Possible involvement of limbic norepinephrine. Pharmacol Biochem Behav 1979; 11:123–30
+
+43. van Gool WA, van de Beek D, Eikelenboom P: Systemic infection and delirium: When cytokines and acetylcholine collide. Lancet 2010; 375:773–5
+
+54. Slotnick BM: Disturbances of maternal behavior in the rat following lesions of the cingulate cortex. Behaviour 1967; 29:204–36
+
+44. Nishiyama T, Yamashita K, Yokoyama T: Stress hormone changes in general anesthesia of long duration: Isoflurane- nitrous oxide vs sevoflurane-nitrous oxide anesthesia. J Clin Anesth 2005; 17:586–91
+
+55. Shih J, May LD, Gonzalez HE, Lee EW, Alvi RS, Sall JW, Rau V, Bickler PE, Lalchandani GR, Yusupova M, Woodward E, Kang H, Wilk AJ, Carlston CM, Mendoza MV, Guggenheim JN, Schaefer M, Rowe AM, Stratmann G: Delayed environmental enrichment reverses sevoflurane-induced memory impair- ment in rats. ANESTHESIOLOGY 2012; 116:586–602
+
+45. Thompson CC, Potter GB: Thyroid hormone action in neural development. Cereb Cortex 2000; 10:939–45
+
+46. Grober MS, Winterstein GM, Ghazanfar AA, Eroschenko VP: The effects of estradiol on gonadotropin-releasing hor- mone neurons in the developing mouse brain. Gen Comp Endocrinol 1998; 112:356–63
+
+56. Ohsawa I, Ishikawa M, Takahashi K, Watanabe M, Nishimaki K, Yamagata K, Katsura K, Katayama Y, Asoh S, Ohta S: Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med 2007; 13:688–94
+
+Anesthesiology 2014; 120:403-15
+
+415
+
+Takaenoki et al.

new_pdfs/10.1097_ALN.0b013e3181974fa2.txt

@@ -0,0 +1,791 @@
+Anesthesiology 2009; 110:628 –37
+
+Copyright © 2009, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc.
+
+Neonatal Exposure to Sevoflurane Induces Abnormal Social Behaviors and Deficits in Fear Conditioning in Mice Maiko Satomoto, M.D.,* Yasushi Satoh, Ph.D.,† Katsuo Terui, M.D., Ph.D.,‡ Hideki Miyao, M.D., Ph.D.,§ Kunio Takishima, Ph.D.,(cid:1) Masataka Ito, M.D., Ph.D.,# Junko Imaki, M.D., Ph.D.**
+
+Background: Neonatal exposure to anesthetics that block N- methyl-D-aspartate receptors and/or hyperactivate (cid:1)-aminobu- tyric acid type A receptor has been shown to cause neuronal degeneration in the developing brain, leading to functional deficits later in adulthood. The authors investigated whether exposure of neonatal mice to inhaled sevoflurane causes defi- cits in social behavior as well as learning disabilities.
+
+Methods: Six-day-old C57BL/6 mice were exposed to 3% sevoflurane for 6 h. Activated cleaved caspase-3 immunohisto- chemical staining was used for detection of apoptosis. Cognitive functions were tested by pavlovian conditioned fear test. Social behavior was tested by social recognition and interaction tests. Results: Neonatal exposure to sevoflurane significantly in- creased the number of apoptotic cells in the brain immediately after anesthesia. It caused persistent learning deficits later in adulthood as evidenced by decreased freezing response in both contextual and cued fear conditioning. The social recognition test demonstrated that mice with neonatal exposure to sevoflu- rane did not develop social memory. Furthermore, these mice showed decreased interactions with a social target compared with controls in the social interaction test, indicating a social interaction deficit. The authors did not attribute these abnor- malities in social behavior to impairments of general interest in novelty or olfactory sensation, because they did not detect sig- nificant differences in the test for novel inanimate object inter- action or for olfaction.
+
+Conclusions: This study shows that exposure of neonatal mice to inhaled sevoflurane could cause not only learning def- icits but also abnormal social behaviors resembling autism spectrum disorder.
+
+MANY pregnant women, newborns, and infants are ex- posed to a variety of anesthetic agents to prevent pain during childbirth or for surgical procedures. Anesthetic agents sometimes have to be administered during an important period of brain growth, the brain growth spurt period, which occurs from the last 3 months of pregnancy until approximately 2 yr after birth (in hu- mans) or during the first 2 weeks after birth (in mice and rats).1– 4 To minimize risks to the fetus or neonates, it is necessary to study the effect of anesthetics not only in
+
+terms of teratogenicity, but also on the developing ner- vous system.
+
+Recently, it has been demonstrated that neonatal ad- ministration of anesthetics induced widespread neuro- degeneration and severe deficits in spatial learning tasks in rodents.5,6 Jevtovic-Todorovic et al.5 reported that neonatal exposure to a cocktail of anesthetics that are commonly used in pediatric surgery induced brain cell death 15 times more frequently than in control rat brains, and that these animals developed learning prob- lems later in adulthood. Fredriksson et al.6 reported that coadministration of an N-methyl-D-aspartate (NMDA) receptor antagonist with (cid:1)-aminobutyric acid type A (GABAA) receptor agonists synergistically potentiated neonatal brain cell death and resulted in functional def- icits in adult mice, although the underlying mechanism is not fully understood. The most thoroughly investi- gated drug that has NMDA antagonist and GABAA agonist property is ethanol, which induces fetal alcohol syn- drome if the fetus is exposed during the brain growth spurt.3,7 Although detrimental effects of anesthetics on cognitive function have been reported, to our knowl- edge, few studies have investigated the effects of anes- thetics on social behavior. Therefore, we designed the current study to investigate the potential risks of neona- tal exposure to anesthetics to cause social abnormalities. Sevoflurane (2,2,2-trifluoro-1-[trifluoromethyl]ethyl flu- oromethyl ether) is one of the most frequently used volatile anesthetics for induction and maintenance of general anesthesia during surgery and cesarean delivery because of its low blood gas partition coefficient and low pungency. It is especially useful for infants and children because of its properties of rapid induction and recovery together with less irritation to the airway.8 Sevoflurane has been shown to enhance GABAA receptors9 and block NMDA receptors, although more research is necessary to better characterize its effects on NMDA receptors.10 In this investigation, we studied the potential risks of neo- natal exposure to sevoflurane to cause social abnormal- ities and cognitive deficits in mice.
+
+Postgraduate Student, # Associate Professor, ** Professor, Department of Developmental Anatomy and Regenerative Biology, † Assistant Professor, (cid:1) Pro- fessor, Department of Biochemistry, National Defense Medical College. ‡ Asso- ciate Professor, Department of Obstetric Anesthesia, § Professor and Chairman, Department of Anesthesiology, Saitama Medical Center, Saitama Medical Univer- sity, Kawagoe, Saitama, Japan. Received from the Department of Developmental Anatomy and Regenerative Biology, National Defense Medical College, Tokorozawa, Saitama, Japan. Submit- ted for publication May 24, 2008. Accepted for publication November 10, 2008. Supported in part by the Ministry of Defense of Japan, Tokyo, Japan (Drs. Satoh and Imaki). There are no financial relationships between any of the authors and any commercial organization with a vested interest in the outcome of the study.
+
+Address correspondence to Dr. Satoh: Department of Biochemistry, National Defense Medical College, 3-2 Namiki, Tokorozawa 359-8513, Japan. ys@ ndmc.ac.jp. Information on purchasing reprints may be found at www.anesthesiology. org or on the masthead page at the beginning of this issue. ANESTHESIOLOGY’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
+
+Materials and Methods
+
+The experiments were approved by the Committee for Animal Research at National Defense Medical College (Tokorozawa, Saitama, Japan). Pregnant C57BL/6 mice were purchased from SLC (SLC Japan Inc., Shizuoka, Japan). The animals were illuminated with a 12-h light– dark cycle (light from 07:00 to 19:00), and room tem-
+
+Anesthesiology, V 110, No 3, Mar 2009
+
+628
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 1 0 3 6 2 8 3 6 8 3 7 1 0 0 0 0 5 4 2 - 2 0 0 9 0 3 0 0 0 - 0 0 0 3 1 p d
+
+/
+
+/
+
+/
+
+/
+
+f
+
+b y g u e s t
+
+o n 3 0 M a r c h 2 0 2 1
+
+SEVOFLURANE NEUROTOXICITY IN THE DEVELOPING BRAIN
+
+perature was maintained at 21° (cid:1) 1°C. At the age of 3 weeks, the mice were weaned and housed in groups of 4 animals in a room. Mice had ad libitum access to water and food.
+
+Previous studies reported that there is litter variability in the rate of apoptosis that occurs spontaneously in neonate mice.11 Therefore, a balanced number of con- trol and experimental animals were drawn from the same litters, so that each experimental condition had its own group of littermate controls. Only the male off- spring were used in this study. A total of 51 litters, 101 control and 103 treated pups, were used in this study.
+
+Anesthesia Treatment Postnatal day 6 (P6) male mice were placed in an acrylic box and exposed to 3% sevoflurane or no anes- thetics for 6 h. The total gas flow was 2 l/min, using air as a carrier. During anesthetic exposure, the mice were kept warm on a plate heated to 38°C. Control and ex- perimental animals were under the same treatment and environment except that the control animals were ex- posed only to air.
+
+Arterial Blood Gas Analysis Arterial blood analysis was performed essentially as described previously.5,12 Briefly, the pups underwent a quick arterial blood sampling from the left cardiac ven- tricle, and the samples were transferred into heparinized glass capillary tubes. A single sample (55 (cid:2)l) was ana- lyzed immediately after blood collection by blood gas analyzer (ABL800; Radiometer, Copenhagen, Denmark). Samples were obtained immediately after removal from the maternal cage (0 h) or at the end of anesthesia (6 h). At the time of blood sampling, the experiments were terminated by decapitation.
+
+Histopathologic Studies Animals from both treatment and control groups were perfused transcardially with 0.1 M phosphate buffer con- taining 4% paraformaldehyde immediately after 6 h of sevoflurane anesthesia, and then the brains were ex- posed to immersion fixation for 24 h at 4°C. The brains were histologically analyzed using paraffin-embedded sections (5 (cid:2)m thick). For immunohistochemistry, anti– active caspase-3 antiserum (D175; Cell Signaling Tech- nology, Beverly, MA) was used at dilutions of 1:400 in antibody diluent (Dako, Glostrup, Denmark). Before to use, sections were dewaxed in xylene and hydrated using a graded series of ethanol. Antigenic retrieval was performed by immersing mounted tissue sections in 0.01 mM sodium citrate (pH 6.0) and heating in an autoclave (121°C) for 5 min. Deparaffinized sections were blocked for endogenous peroxidase activity as described previ- ously,13 followed by blocking with a nonspecific staining blocking reagent (Dako) for 1 h to reduce background staining. The sections were then incubated overnight in a humidified chamber at 4°C. Subsequently, peroxidase- conjugated secondary antibody (DAKO En Vision (cid:2) sys- tem; Dako) and 3,3-diaminobenzine-tetrachloride (DAB; Vector Laboratories, Burlingame, CA) were used accord- ing to the manufacturer’s instructions. Finally, the sec- tions were counterstained with Nissl. Activated caspase- 3–positive cells were counted by the investigator who was blinded to the treatment conditions.
+
+transferase–mediated de- oxyuridine 5-triphosphate– biotin nick end labeling stain- ing was performed using an in situ apoptosis detection kit (ApopTag fluorescein; CHEMICON, Temecula, CA) accord- ing to the manufacturer’s protocol. Sections were counter- stained with 4=,6-diamidino-2-phenylindole (DAPI). Fluores- cein was histochemically examined with a fluorescent microscope (TE-2000E; Nikon, Tokyo, Japan) equipped with interlined charge-coupled device camera (DS-U1; Nikon).
+
+Terminal deoxynucleotidyl
+
+Laser Color Doppler Cerebral blood flow (CBF) was measured by a laser- Doppler blood perfusion imager (Peri Scan PIM II; Per- imed, Stockholm, Sweden). Mice were taken out of the chamber before and every hour during anesthetic treat- ment and were placed face down on the floor while being continuously exposed to sevoflurane via a tube with its opening positioned at the nose of the animals. Their head skins were peeled for scanning CBF, and data were captured using appropriate software (LDPIwin ver- sion 2.6; Lisca, Linko¨ping, Sweden). The perfusion re- sponse is presented in arbitrary perfusion units. Because the arbitrary perfusion units values are not absolute blood flow, the magnitude of the difference in perfusion was calculated as the ratio between the area of maxi- mum peak perfusion and areas of baseline perfusion. Arbitrary perfusion unit values were compared between anesthetized animals and those with mock anesthesia at baseline and at 1-h intervals for 6 h.
+
+Preparation of Protein Extracts Mice forebrain was quickly removed and were homog- enized in four volumes of 20 mM Tris-HCl, pH 7.4, 2 mM EDTA, protease inhibitor cocktail (Complete, Roche Di- agnostics, Penzberg, Germany), and phosphatase inhib- itors (20 mM glycerophosphate, 1 mM Na3VO4, 2 mM NaF). After homogenization, a portion of each sample was immediately frozen at (cid:3)80°C. The rest of the ho- mogenate was centrifuged at 15,000g for 30 min at 4°C. The supernatant solutions were separated and stored at (cid:3)80°C until use. The amount of protein in each sample was measured using a protein assay kit (BCA; Pierce, Rockford, IL).
+
+Western Blot Analysis The homogenate proteins were subjected to sodium do- decyl sulfate polyacrylamide gel electrophoresis. The pro-
+
+Anesthesiology, V 110, No 3, Mar 2009
+
+629
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 1 0 3 6 2 8 3 6 8 3 7 1 0 0 0 0 5 4 2 - 2 0 0 9 0 3 0 0 0 - 0 0 0 3 1 p d
+
+/
+
+/
+
+/
+
+/
+
+f
+
+b y g u e s t
+
+o n 3 0 M a r c h 2 0 2 1
+
+630
+
+teins were transferred onto polyvinylidene fluoride mem- branes (Immobilon-P; Millipore, Bedford, MA). The blots were immunoreacted with anti– cleaved poly(adenosine diphosphate–ribose) polymerase (PARP; 1:1,000, rabbit polyclonal, Asp214; Cell Signaling) or anti–(cid:3)-actin (1:5,000, mouse monoclonal, AC-15; Sigma, St. Louis, MO) antibod- ies, and the protein bands were visualized by chemilumi- nescence detection system (SuperSignal West Pico; Pierce).
+
+Behavioral Studies As described previously for CBF and histopathologic stud- ies, some sets of mice for behavioral studies were exposed to 3% or 0% sevoflurane for 6 h at P6. They were allowed to mature, and at the appropriate ages, sevoflurane and control mice underwent behavioral tests, namely, open- field, elevated plus-maze, Y-maze, fear conditioning, social recognition, social interaction, olfactory, and novelty tests. The movement of each mouse was monitored and analyzed using a computer-operated video tracking system (SMART, Barcelona, Spain). In the tasks using apparatus with arms, arm entry was counted when all four legs of the animal entered each arm. The apparatus was cleaned after each trial. All apparatus used in this study were made by O’Hara & Co., Ltd. (Tokyo, Japan).
+
+Open-field Test. Emotional responses to a novel en- vironment were measured by an open-field test using 8-week-old mice, by a previously described method.14 Activity was measured as the total distance traveled (meters) in 10 min.
+
+Elevated Plus-maze Test. The elevated plus-maze test was performed as previously described.14 The elevated plus maze consisted of two open arms (25 (cid:4) 5 cm) and two enclosed arms being elevated to a height of 50 cm above the floor. Normally, mice prefer a closed environ- ment to an open area. Mouse behavior was recorded during a 10-min test period. The percentage of time spent in the open arms was used as an index of anxiety- like behavior. Mice used for the test were aged 8 weeks. Spontaneous Alternation in the Y-maze Test. This study was performed as previously described.14 This study allowed us to assess spatial working memory. The symmetrical Y maze made of acrylic consists of three arms (25 (cid:4) 5 cm) separated by 120° with 15-cm-high transparent walls. Each mouse was placed in the center of the Y maze, and the mouse was allowed to freely explore the maze for 8 min. The sequence and the total number of arms entered were recorded. The percentage of alternation is the number of triads containing entries into all three arms divided by the maximum possible number of alternations (total number of arm entries minus 2) (cid:4) 100. Mice used for the test were aged 11 weeks. The motion of the animals was manually recorded.
+
+Fear Conditioning Test. This is a simple and sensi- tive test of hippocampal-dependent and hippocampal- independent learning as previously described.14 Briefly,
+
+Anesthesiology, V 110, No 3, Mar 2009
+
+SATOMOTO ET AL.
+
+the conditioning trial for contextual and cued fear con- ditioning consisted of a 5-min exploration period fol- lowed by three conditioned stimulus– unconditioned stimulus pairings separated by 1 min each: uncondi- tioned stimulus, 1 mA foot shock intensity, 1 s duration; conditioned stimulus, 80 db white noise, 20 s duration; unconditioned stimulus was delivered during the last seconds of conditioned stimulus presentation. A contex- tual test was performed in the conditioning chamber for 5 min in the absence of white noise at 24 h after condi- tioning. A cued test (for the same set of mice) was performed by presentation of a cue (80 db white noise, 3 min duration) in alternative context with distinct visual and tactile cues. The rate of freezing response (absence of movement in any parts of the body during 1 s) was scored automatically and used to measure fear memory. The test was performed on mice of two different age groups: 8 weeks or between 14 and 17 weeks.
+
+Social Recognition Test. Social recognition test was conducted as described previously.15 We transferred 18- week-old mice from group to individual housing for 7 days before testing to permit establishment of a home cage territory. Testing began when a stimulus female mouse was introduced into the home cage of each male mouse for 1-min confrontation. At the end of the 1-min trial, the stimulus animal was removed and returned to an individual cage. This sequence was repeated for four trials with 10-min intertrial intervals, and each stimulus was introduced to the same male resident in all four trials. In a fifth trial, another stimulus mouse was intro- duced to a resident male mouse.
+
+Social Interaction Test. Caged social interaction for social versus inanimate targets was performed in an open field using two cylinder cages allowing olfactory and minimal tactile interaction as described previo- usly.16 The cylinder cages were 10 cm in height, with a bottom diameter of 9 cm and bars spaced 7 mm apart. Olfactory Test. Fifteen-week-old mice were habitu- ated to the flavor of a novel food (blueberry cheese) for 3 days before testing. On the fourth day, after 24 h of food deprivation, a piece of blueberry cheese was buried under 2 cm of bedding in a clean cage. The mice were placed in the cage, and the time required to find the food was measured manually.
+
+Novelty Test. Activity was measured as the total du- ration of interaction with an inanimate novel object (red tube) in 10 min.
+
+The same set of mice underwent social recognition (at 19 weeks of age), social interaction (at 14 weeks of age), olfactory (at 15 weeks of age), and novelty (at 15 weeks of age) tests. In other analyses, each test was conducted with a new set of animals.
+
+Statistical Analysis Statistical analysis was performed using Statview soft- ware (SAS, Cary, NC). Comparisons of the means of two
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 1 0 3 6 2 8 3 6 8 3 7 1 0 0 0 0 5 4 2 - 2 0 0 9 0 3 0 0 0 - 0 0 0 3 1 p d
+
+/
+
+/
+
+/
+
+/
+
+f
+
+b y g u e s t
+
+o n 3 0 M a r c h 2 0 2 1
+
+SEVOFLURANE NEUROTOXICITY IN THE DEVELOPING BRAIN
+
+631
+
+Table 1. Arterial Blood Gas Analysis
+
+Arterial Blood Gas
+
+Time, h
+
+n
+
+pH
+
+PacO2, mmHg
+
+PaO2, mmHg
+
+SaO2, %
+
+Sham operation
+
+3% Sevoflurane
+
+0 6 0 6
+
+8 8 8 8
+
+7.40 (cid:1) 0.09 7.38 (cid:1) 0.04 7.32 (cid:1) 0.08 7.46 (cid:1) 0.06
+
+26.0 (cid:1) 4.6 29.4 (cid:1) 6.2 26.9 (cid:1) 4.6 27.5 (cid:1) 6.9
+
+100.2 (cid:1) 5.7 95.6 (cid:1) 10.0 98.5 (cid:1) 6.9 80.9 (cid:1) 4.8
+
+95.8 (cid:1) 1.1 96.1 (cid:1) 1.0 95.4 (cid:1) 1.1 95.4 (cid:1) 0.8
+
+Neonatal exposure to sevoflurane does not induce significant cardiorespiratory dysfunction. Analysis of arterial blood gas revealed no significant differences in any of the measured parameters between mice exposed for 6 h to sevoflurane and control (sham operation) exposed to air for 6 h (t test, all P values (cid:5) 0.05). PaCO2
+
+(cid:6) arterial carbon dioxide tension; PaO2
+
+(cid:6) arterial oxygen tension; SaO2
+
+(cid:6) arterial oxygen saturation.
+
+groups were performed using the Student t test. In the Y-maze task, comparisons of group performance relative to random levels were performed using the one-sample t test. Data of the social recognition task were analyzed by repeated-measures two-way analysis of variance. Values are presented as mean (cid:1) SEM.
+
+Results
+
+Neonatal Exposure to Sevoflurane Did Not Induce Significant Disturbance in Ventilation, Oxygenation, or CBF To examine the effect of neonatal exposure to sevoflu- rane, we exposed P6 mice to 3% sevoflurane for 6 h. Hypoxia is a known cause of neuronal cell death.17 To assess the adequacy of ventilation and oxygenation, we examined the blood gas data in mice during the anesthe- sia. Control samples were obtained from pups exposed to air during the same period. We found that pH, arterial carbon dioxide tension, arterial oxygen tension, and arterial oxygen saturation did not differ significantly from sham control (table 1). These results, together with
+
+the fact that pups looked pink throughout the 6 h of gas exposure, led us to conclude that it was unlikely that apoptosis in this protocol was caused by hypoxia/ hypoventilation.
+
+Further, to assess the adequacy of cerebral perfu- sion, we measured CBF during anesthesia using a laser-Doppler blood perfusion imager. Control mice were exposed to air for corresponding period of the sevoflurane treated mice. Anesthesia treatment with sevoflurane did not affect CBF compared with control mice at any point during the 6 h of anesthesia (figs. 1A and B).
+
+Neonatal Exposure to Sevoflurane Induced Extensive Apoptotic Neurodegeneration Sevoflurane anesthesia significantly increased cleaved caspase-3 apoptosis in the mice immediately after expo- sure (table 2 and figs. 2– 4). Figures 2B and D showed that the increased apoptosis was most robust in the caudate/putamen, retrosplenial cortex, dorsal hip- pocampal commissure, and neocortex in the brains of pups with sevoflurane exposure. In other sections, thal-
+
+A
+
+Fig. 1. Neonatal exposure to sevoflurane (Sevo) did not induce hypoperfusion of the brain. (A) Representative images of laser color Doppler for cerebral blood flow in a control mouse (upper panel) and in a sevoflurane exposed mouse (lower panel). The degree of perfusion is shown by the color code, with red repre- senting high perfusion and blue repre- senting lower perfusion. No different pat- terns of cerebral blood flow were observed between control and anesthe- tized mice throughout the 6-h exposure period. (B) Time course of cerebral blood flow measured by laser color Doppler during 6 h of 3% sevoflurane administra- tion. The degree of perfusion is pre- sented in arbitrary perfusion units. There was no difference between sevoflurane and control groups during the 6-h period (control, n (cid:2) 3; 3% sevoflurane, n (cid:2) 4). Scale bar: 5 mm.
+
+B
+
+Anesthesiology, V 110, No 3, Mar 2009
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 1 0 3 6 2 8 3 6 8 3 7 1 0 0 0 0 5 4 2 - 2 0 0 9 0 3 0 0 0 - 0 0 0 3 1 p d
+
+/
+
+/
+
+/
+
+/
+
+f
+
+b y g u e s t
+
+o n 3 0 M a r c h 2 0 2 1
+
+632
+
+Table 2. Brain Regions in Which Sevoflurane-induced Neurodegeneration Was Heavily Concentrated
+
+Brain Region
+
+Severity, Fold Increase
+
+CA1 (hippocampus) CA3 (hippocampus) Dentate gyrus Dorsal hippocampal commissure Frontal cortex Temporal cortex Amygdala Caudate/putamen Mammillary complex Retrosplenial cortex Subiculum Pontine nuclei Inferior colliculus Thalamus
+
+4.8 29.7 32.5 65.2 66.7 148.0 51.0 47.7 21.1 166.5 121.5 38.8 32.1 376.7
+
+Severity of damage is expressed as the fold increase (i.e., how many times greater) in the density of degenerating neurons labeled by activated cleaved caspase-3 immunohistochemical staining in the sevoflurane-treated brain (n (cid:6) 6 mice) compared with the rate of degeneration in the same region of control brain (n (cid:6) 6 mice).
+
+amus, subiculum, inferior colliculus, and pontine nuclei were also shown to be damaged severely (figs. 3B and C). Figure 4B indicated that severe neuronal damage occurred in the extrahippocampal circuit, which is be- lieved to be important for mediating learning and mem- ory functions,18 including dorsal hippocampal commis- sure as well as thalamus and retrosplenial cortex. Apoptosis were also observed in amygdala (fig. 4D), hippocampus (fig. 4F), frontal cortex (fig. 4H), and mam- millary complex (fig. 4J). These data indicated that the apoptotic response to sevoflurane was robust and fol- lowed a pattern that was characteristic of the pattern reported for other anesthetic drugs or ethanol.18
+
+It was reported that caspase-3 cleavage may occur under a condition that is a nonapoptotic event,19 raising questions about the reliability of cleaved caspase-3 im- munostaining for detecting cell death. Therefore, to ver- ify that the cleaved caspase-3 immunoreactivity repre- sent authentic apoptosis, we also performed terminal deoxynucleotidyl transferase–mediated deoxyuridine 5-triphosphate– biotin nick end labeling as another inde- pendent measure of apoptotic cell death. We found the same pattern of staining as observed by cleaved caspase-3 staining (figs. 4L, N, P, and R).
+
+Further, to verify that the previously described immu- nohistochemically detectable reactivity represented au- thentic apoptosis and to quantify the apoptosis re- sponse, we examined cortex extracts from control and sevoflurane-treated pups by Western blot analysis using antibody specific for cleaved PARP. PARP is one of the main cleavage targets of caspase-3 in vivo, and the cleav- age is readily detected in many apoptosis model.20 West- ern immunoblotting with anticleaved PARP antibody de- tected immunoreactivity in sevoflurane-exposed pup
+
+Anesthesiology, V 110, No 3, Mar 2009
+
+SATOMOTO ET AL.
+
+Fig. 2. The apoptotic response to sevoflurane (Sevo) was robust in pup brain. Light microscopic views of the mouse brain after exposure to room air for 6 h (A and C) and to 3% sevoflurane for 6 h (B and D). Sections were immunochemically stained to reveal caspase-3 activation (A–D). Black dots represent caspase- 3–positive cells, which indicate apoptosis. A substantially higher density of cleaved caspase-3–positive profile is present in sevoflurane-treated brain. cp (cid:2) caudate/putamen; dhc (cid:2) dorsal hippocampal commissure; rs (cid:2) retrosplenial cortex. Scale bars: 1 mm.
+
+extracts, whereas the band was under detection level in control pub brain extracts (fig. 4S).
+
+General Behavior Was Normal in Mice with Neonatal Exposure to Sevoflurane To examine responses to a novel environment, mice with neonatal exposure to sevoflurane were assayed in an open-field test. These mice did not differ from control animals in their exploratory behavior (fig. 5A; t test, t (cid:6) 1.24, P (cid:5) 0.05). To study whether anxiety-related behav- ior of mice with neonatal exposure to sevoflurane was affected, mice underwent an elevated plus-maze test. Anxiety-related behavior was assessed by the percentage of time spent in the open arms of the test equipment. Anesthetized mice did not differ significantly in the per- centage of time spent in the open arms (fig. 5B; t test, t (cid:6) 0.76, P (cid:5) 0.05). These results indicate that the emotional state of mice with neonatal exposure to sevoflurane did not differ grossly from controls under the conditions of this study.
+
+Further, to examine whether exposure of the develop- ing brain to sevoflurane was associated with changes in
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 1 0 3 6 2 8 3 6 8 3 7 1 0 0 0 0 5 4 2 - 2 0 0 9 0 3 0 0 0 - 0 0 0 3 1 p d
+
+/
+
+/
+
+/
+
+/
+
+f
+
+b y g u e s t
+
+o n 3 0 M a r c h 2 0 2 1
+
+SEVOFLURANE NEUROTOXICITY IN THE DEVELOPING BRAIN
+
+being no difference compared with control (fig. 5C; t test, t (cid:6) 1.20, P (cid:5) 0.05). This result suggests that spatial working memory was not affected by exposure of the developing brain to sevoflurane.
+
+Neonatal Exposure to Sevoflurane Induced Deficits in Contextual and Cued Fear Conditioning To assess the influence of neonatal exposure to sevoflurane on long-term memory, mice underwent con- textual/cued fear conditioning. In this paradigm, mice learn to associate previous neutral auditory cues and the apparatus (context) with electric foot shock in a single training session, such that robust long-term memory was established for an experimental context (hippocampus dependent) and an auditory cue (hippocampus indepen- dent).21,22 Long-term memory was assessed based on the freezing reaction of the mice in response to the context or the conditioned cue. The freezing response to the same context in mice with neonatal exposure to sevoflu- rane was reduced significantly compared with controls after a 24-h retention delay at 8 weeks of age (fig. 6A; t test, t (cid:6) 3.10, P (cid:7) 0.01). The response of mice with sevoflurane to the cued fear conditioning was also re- duced significantly after a 48-h retention delay compared with control mice (fig. 6B; t test, t (cid:6) 3.16, P (cid:7) 0.01). It was reported that exposure of infant mice to ethanol induced neuroapoptosis and subsequent memory im- pairments that were very severe at P30 and less severe at P75.18 This result provided evidence favoring the inter- pretation that recovery of some type of learning func- tions might occur in later adulthood in ethanol-treated mice. Therefore, further to examine whether the neona- tal exposure to sevoflurane causes permanent neurocog- nitive deficits in mice and how it evolves over time, we undertook an assessment of hippocampal function at later time point using another set of mice.
+
+Fig. 3. Thalamus and other regions were also severely damaged after neonatal exposure to sevoflurane (Sevo). Sagittal (A and B) and coronal (C) views of the mouse brain after exposure to room air for 6 h (A) and to 3% sevoflurane for 6 h (B and C) as described in figure 2. A substantially higher density of cleaved caspase-3–positive profile is present in thalamus (th) as well as subiculum (s), inferior colliculus (ic), and pontine nuclei (pn). rs (cid:2) retrosplenial cortex. Scale bars: 1 mm.
+
+spatial working memory, mice underwent a Y-maze spontaneous alternation task. Working memory refers to a cognitive function that provides concurrent temporary storage and manipulation of the information necessary for complex cognitive tasks. This test examines whether mice remember the position of the arm selected in the preceding choice. Mice with and without sevoflurane exposure performed this task with 64.3 (cid:1) 7.3% and 60.0 (cid:1) 8.3% correct choices, respectively, which were well above the expected results of random choices (ran- dom choice (cid:6) 50%; one-sample t test, P (cid:7) 0.05), there
+
+We found that neonatal exposure to sevoflurane caused deficits in the fear conditioning test at 14 –17 weeks of age similar to the results at 8 weeks. The freezing response of mice with sevoflurane was reduced significantly in contextual tests compared with that of controls after a 24-h retention delay (fig. 6C; t test, t (cid:6) 3.48, P (cid:7) 0.01). The freezing response of sevoflurane- exposed mice to cued fear was also reduced significantly compared with that of controls after a 48-h retention delay at 14 –17 weeks of age (fig. 6D; t test, t (cid:6) 2.11, P (cid:7) 0.05). These results strongly suggested that exposure of P6 mice to sevoflurane caused hippocampal-depen- dent and -independent neurocognitive deficits that per- sisted for relatively long time periods (at least from 8 to 14 –17 weeks of age) of the mice’s lifespan.
+
+Neonatal Exposure to Sevoflurane Induced Abnormal Social Interaction Mice are a social species and exhibit social interaction behavior.23 Therefore, we investigated whether mice
+
+Anesthesiology, V 110, No 3, Mar 2009
+
+633
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 1 0 3 6 2 8 3 6 8 3 7 1 0 0 0 0 5 4 2 - 2 0 0 9 0 3 0 0 0 - 0 0 0 3 1 p d
+
+/
+
+/
+
+/
+
+/
+
+f
+
+b y g u e s t
+
+o n 3 0 M a r c h 2 0 2 1
+
+634
+
+with neonatal exposure to sevoflurane display abnormal social behaviors. First, we investigated social memory, which depends predominantly on olfactory cues. This ability is needed for social familiarity and can be identi- fied as a consistent decrease in olfactory investigation during repeated encounters with a female in the social recognition test. Control mice showed a significant de- cline in the time spent in investigating a female with subsequent presentation of the same female in trials 3 and 4, as compared with trial 1 (fig. 7A). This decrease was not due to a general decline in olfactory investiga-
+
+A
+
+B
+
+C
+
+Anesthesiology, V 110, No 3, Mar 2009
+
+SATOMOTO ET AL.
+
+Fig. 4. Inhaled sevoflurane increased ap- optosis in several regions of the brain. Light microscopic views of the mouse brain after exposure to room air for 6 h (A, C, E, G, and I) and to 3% sevoflurane (Sevo) for 6 h (B, D, F, H, and J). Higher density of cleaved caspase-3–positive profile was present in extrahippocampal circuit (A and B), amygdala (C and D), hippocampus (E and F), frontal cortex (G and H), and mammillary complex (I and J). Scale bars: 1 mm in A and B, 50 (cid:3)m in C–J, and 100 (cid:3)m in I and J. (K–R) Termi- nal deoxynucleotidyl transferase–medi- ated deoxyuridine 5-triphosphate– biotin nick end labeling (TUNEL; L, N, P, and R) showed similar pattern of neuroapopto- sis to cleaved caspase-3 staining. Sections stained with 4=,6-dia- were midino-2-phenylindole (DAPI; K, M, O, and Q). Representative images of cortex (K, L, O, and P) and caudate (M, N, Q, and R) are shown. Scale bars: 500 (cid:3)m. (S) Poly(adenosine diphosphate–ribose) poly- merase (PARP) was cleaved after neonatal exposure to sevoflurane. Protein extracts of control (exposure to room air for 6 h) and sevoflurane-exposed cortex were prepared and analyzed for cleaved PARP immunoreac- tivity on Western blot. Representative blot from three independent results (from three pairs of pups) was shown. (cid:4)-Actin reactivity was used as a protein loading control. dhc (cid:2) dorsal hippocampal commissure; rs (cid:2) retro- splenial cortex; th (cid:2) thalamus.
+
+counter
+
+tion, because presentation of a novel female during trial 5 resulted in a similar amount of investigation as trial 1 with the original female. In contrast, mice with neonatal exposure to sevoflurane showed high levels of sustained investigation at each encounter with the same female and the same level of investigation when presented with a new female at trial 5, significantly different from the response of controls (analysis of variance, F (cid:6) 14.51, P (cid:7) 0.001 [between control and sevoflurane administra- tion]; F (cid:6) 28.34, P (cid:7) 0.0001 [between trials]; F (cid:6) 16.64, P (cid:7) 0.0001 [interaction between trials and sevoflurane
+
+Fig. 5. Behavioral effects of neonatal sevoflu- rane exposure were assessed by the open- field test (total distance traveled in 10 min; control, n (cid:2) 10; sevoflurane, n (cid:2) 11; A), ele- vated plus-maze test (percentage of time spent in open arms; control, n (cid:2) 18; sevoflu- rane, n (cid:2) 20; B), and Y-maze test (percentage of correct alternation response; control, n (cid:2) 10; sevoflurane, n (cid:2) 9; C). No significant dif- ferences were observed between mice with neonatal sevoflurane exposure and controls in these tests.
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 1 0 3 6 2 8 3 6 8 3 7 1 0 0 0 0 5 4 2 - 2 0 0 9 0 3 0 0 0 - 0 0 0 3 1 p d
+
+/
+
+/
+
+/
+
+/
+
+f
+
+b y g u e s t
+
+o n 3 0 M a r c h 2 0 2 1
+
+SEVOFLURANE NEUROTOXICITY IN THE DEVELOPING BRAIN
+
+A
+
+B
+
+A
+
+B
+
+C
+
+D
+
+C
+
+D
+
+Fig. 6. Neonatal exposure of mice to sevoflurane (Sevo) induced impaired memory performance in both contextual and cued tests. (A and B) Contextual and cued tests at 8 weeks of age. (A) Freezing response was measured in the context before shock (basal freezing) and in the conditioning chamber (contextual fear response) 24 h after conditioning (control, n (cid:2) 8; sevoflurane, n (cid:2) 8). (B) Freezing response (for the same set of mice as in A) was measured in an alternative context without auditory cue (basal freezing after conditioning) or with cue 2 days after conditioning. (C and D) Contextual and cued tests for another set of mice at a different age (14 –17 weeks of age) as in A and B (control, n (cid:2) 9; sevoflurane, n (cid:2) 9). For all figures, asterisks represent statistical difference (* P < 0.05, ** P < 0.01).
+
+administration]; fig. 7A). These data suggest that mice with neonatal exposure to sevoflurane do not develop social memory.
+
+In a test for social versus inanimate preference, control mice spent significantly more time interacting with the social target than with the inanimate target (fig. 7B; t test, t (cid:6) 7.30, P (cid:7) 0.0001). In contrast, mice with neonatal exposure to sevoflurane spent a similar amount of time interacting with both targets (fig. 7B; t test, t (cid:6) 1.77, P (cid:5) 0.05). Furthermore, mice with neonatal exposure to sevoflurane exhibited decreased interaction with a social target compared with controls (fig. 7B; t test, t (cid:6) 2.38, P (cid:7) 0.05), indicating a social interaction deficit. We did not attribute the abnormalities in social recognition and inter- action to impairment in general interest in novelty or olfac- tory sensation, because we did not detect significant differ- ences between groups in tests for novel inanimate object interaction (fig. 7C; t test, t (cid:6) 0.21, P (cid:5) 0.05) or for olfaction (fig. 7D; t test, t (cid:6) 0.12, P (cid:5) 0.05). Therefore, it can be concluded that mice with neonatal exposure to sevoflurane demonstrated deficits in social behavior.
+
+Discussion
+
+This study showed that single administration of sevoflurane to neonatal mice caused a significant in-
+
+Fig. 7. Neonatal exposure to sevoflurane (Sevo) induced abnor- mal social behavior in adulthood. (A) Olfactory investigations in mice with neonatal exposure to sevoflurane were used for social recognition test. Social memory by male mice was mea- sured as the difference in anogenital investigation. Data depict the amount of time allocated to investigating the same female during each of four successive 1-min trials. A fifth trial depicts the response to a new female. Asterisks represent statistical differences (*** P < 0.001) between each trial compared with the first trial (control, n (cid:2) 17; sevoflurane, n (cid:2) 18). (B) When exposed to caged social and inanimate targets (social interac- tion test) in an open field, control mice showed a normal preference for the social target over an inanimate target, whereas the difference of interaction time between both targets was not significant in mice with neonatal exposure to sevoflu- rane. Furthermore, mice with neonatal exposure to sevoflurane spent significantly less time interacting with the social target compared with controls (control, n (cid:2) 17; sevoflurane, n (cid:2) 18). Asterisks represent statistical differences (* P < 0.05, *** P < 0.001). (C) Time spent interacting with a novel inanimate object was not significantly affected by neonatal exposure to sevoflu- rane (control, n (cid:2) 17; sevoflurane, n (cid:2) 18). (D) Mice with neonatal exposure to sevoflurane did not show significant dif- ferences from controls in latency to find a buried treat after overnight food deprivation (control, n (cid:2) 17; sevoflurane, n (cid:2) 18).
+
+crease in neuroapoptosis in the brain compared with littermate controls exposed only to air. Consistent with other recent evidence that apoptotic neurodegeneration can be induced by exposure during the brain growth spurt to drugs that block NMDA receptors and/or hyper- activate GABAA receptors,5,6,18 neonatal exposure to sevoflurane was shown to induce widespread apoptosis in several major brain regions, leading to impaired learn- ing later in adulthood. This finding for sevoflurane is consistent with recent finding by Johnson et al.,24 who found that a neonatal exposure to isoflurane triggers a significant neuroapoptosis response in the mouse brain. Our results of fear conditioning strongly suggested that exposure of P6 mice to sevoflurane caused learning deficits, although we could not rule out the possibility of the effects of sevoflurane on sensitization despite true conditioning to the auditory cue. Furthermore, this
+
+Anesthesiology, V 110, No 3, Mar 2009
+
+635
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 1 0 3 6 2 8 3 6 8 3 7 1 0 0 0 0 5 4 2 - 2 0 0 9 0 3 0 0 0 - 0 0 0 3 1 p d
+
+/
+
+/
+
+/
+
+/
+
+f
+
+b y g u e s t
+
+o n 3 0 M a r c h 2 0 2 1
+
+636
+
+study also showed that exposure of neonatal mice to inhaled sevoflurane caused deficits in social behavior. To our knowledge, this is the first study to show that single administration of sevoflurane, which is a commonly used anesthetic in pediatric surgery throughout the world, causes a robust neuroapoptosis response in infant mouse brain and behavioral deficits in both cognitive and social spheres. The minimum alveolar anesthetic concentra- tion that prevents purposeful movement in response to supramaximal noxious stimulation in 50% of animals (minimum alveolar concentration) of sevoflurane in hu- man neonates is 3.3 (cid:1) 0.2%.8 Therefore, the concentra- tion of sevoflurane (3%) used in this mice study would be comparable to clinically used ranges.
+
+Most anesthetics act via NMDA and/or GABAA recep- tors. It was demonstrated that neonatal coadministration of an NMDA antagonist and a GABAA agonist was much more detrimental than either of these used alone.5,6 Although the precise mechanism for this is yet to be understood, this evidence suggested that more severe neurodegeneration was induced when both NMDA and GABAA receptors were simultaneously altered in the developing brain. Therefore, an anesthetic that has NMDA antagonist and GABAA agonist properties would be of concern when administered to the developing brain.
+
+Autism spectrum disorders (ASDs) are a group of com- mon neuropsychiatric disorders characterized primarily by impairments in social, communicative, and behavioral functioning,25 of unknown mechanism. Epidemiologic studies have shown that the prevalence of ASDs is 3– 6 per 1,000 children. If neonatal exposure to anesthetics induces deficits in social behavior, a causal link between ASDs and neonatal exposure to anesthetics could be suggested, because deficits in social behaviors are a core feature of ASDs. Our findings revealed that neonatal exposure to sevoflurane induced deficits in social mem- interaction in mice. These tests were ory and social thought to be core paradigms to test autistic behavior in mice and have been used to measure autistic behavior in other ASD models as well.16,17,26,27 This study is the first to indicate the potential risk of general anesthetics to induce disturbances in social behaviors that resembles those seen in ASDs.
+
+However, there is a caveat that the relevance of these mouse findings to the human situation is unknown and requires clarification. It is too early to say whether anes- thetics have the same effect in humans. There may be species differences in the detrimental effects of anes- thetic agents on the developing brain. Physicians could reduce any potential risks by limiting the duration of anesthetic administration in neonates.
+
+In any case, the current results suggest the potential hazards of neonatal sevoflurane exposure in causing so- cial behavioral alterations. We would like to insist on the need for further research to determine whether a corre-
+
+Anesthesiology, V 110, No 3, Mar 2009
+
+SATOMOTO ET AL.
+
+lation exists between anesthesia exposure during devel- opment and ASDs in human populations. What would be the potential association between the sevoflurane-in- duced apoptosis and sevoflurane-induced changes in so- cial behaviors? Several authors have proposed that glu- tamate and GABAergic system disturbance in cortical network in ASDs may be characterized by an imbalance between excitation and inhibition in neuronal net- works.28,29 Such a change may lead to hyperexcitability or unstable neuronal networks, which may alter oscilla- tory rhythms in brain.30,31 The excitatory/inhibitory bal- ance in cortical networks may be controlled by the relative numbers and activities of glutamatergic and GABAergic neurons.29 Indeed, reduced GABAergic inhi- bition by mutations of genes encoding subunits of the GABAA receptors is associated with ASDs.28,29,32 Neural loss by a drug that may violate NMDA and GABAA recep- tors in the critical period, seen in the current study, might interfere with the developmental mechanisms pat- terning the balance between excitation and inhibition system and cause ASD-like behaviors.
+
+Our results showed that neuronal degeneration was particularly severe in several of the specific brain regions that comprise the extrahippocampal circuit, which is believed to be important for mediating learning and memory functions. This pattern was similar to the pat- tern for neonatal exposure to ethanol, which has NMDA antagonist and GABAA agonist properties.18 These pat- tern of brain damage and subsequent learning deficits are described in ethanol-treated mice.18 In this regard, it is noteworthy that there is evidence that prenatal expo- sure to ethanol may be a factor in social difficulties in humans.33 Further including electrophysi- ologic study, will be needed to address the molecular mechanisms that explain the relevance between neona- tal exposure to sevoflurane and deficits in social behav- ior. It would also be necessary to study whether other drugs that cause neuroapoptosis in the critical period would induce deficits in social behavior.
+
+research,
+
+The authors thank Kyoko Takeuchi, Ph.D. (Assistant Professor), Kenji Miura, Ph.D. (Lecturer), Kazuyo Kuroki (Technician, all from the Department of Devel- opmental Anatomy and Regenerative Biology, National Defense Medical College, Tokorozawa, Saitama, Japan), Kunihito Takahashi, Ph.D., (Senior Researcher, Hakuju Institute for Health Science Co. Ltd., Tokyo, Japan), and Tatsuyo Hara- sawa (Technician, Central Research Laboratory, National Defense Medical Col- lege) for the participation in this study; and Kouichi Fukuda, Ph.D. (Associate Professor, Center for Laboratory Animal Science, National Defense Medical Col- lege), for the assistance in animal administration.
+
+References
+
+1. Bayer SA, Altman J, Russo RJ, Zhang X: Timetables of neurogenesis in the human brain based on experimentally determined patterns in the rat. Neurotoxi- cology 1993; 14:83–144
+
+2. Rice D, Barone S Jr: Critical periods of vulnerability for the developing nervous system: Evidence from humans and animal models. Environ Health Perspect 2000; 108:511–33
+
+3. Olney JW, Tenkova T, Dikranian K, Qin YQ, Labruyere J, Ikonomidou C: Ethanol-induced apoptotic neurodegeneration in the developing C57BL/6 mouse brain. Brain Res Dev Brain Res 2002; 133:115–26
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+f /
+
+1 1 0 3 6 2 8 3 6 8 3 7 1 0 0 0 0 5 4 2 - 2 0 0 9 0 3 0 0 0 - 0 0 0 3 1 p d
+
+/
+
+/
+
+/
+
+/
+
+f
+
+b y g u e s t
+
+o n 3 0 M a r c h 2 0 2 1
+
+SEVOFLURANE NEUROTOXICITY IN THE DEVELOPING BRAIN
+
+637
+
+4. Dobbing J, Sands J: Comparative aspects of the brain growth spurt. Early
+
+Hum Dev 1979; 3:79–83
+
+5. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF: Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 2003; 23:876–82
+
+6. Fredriksson A, Ponte´n E, Gordh T, Eriksson P: Neonatal exposure to a combination of N-methyl-D-aspartate and (cid:1)-aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behav- ioral deficits. ANESTHESIOLOGY 2007; 107:427–36
+
+7. Ikonomidou C, Bittigau P, Ishimaru MJ, Wozniak DF, Koch C, Genz K, Price MT, Stefovska V, Ho¨rster F, Tenkova T, Dikranian K, Olney JW: Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome. Science 2000; 287: 1056–60
+
+M, Lang D, Speer A, Olney JW, Ikonomidou C: Apoptotic neurodegeneration following trauma is markedly enhanced in the immature brain. Ann Neurol 1999; 45:724–35
+
+18. Wozniak DF, Hartman RE, Boyle MP, Vogt SK, Brooks AR, Tenkova T, Young C, Olney JW, Muglia LJ: Apoptotic neurodegeneration induced by ethanol in neonatal mice is associated with profound learning/memory deficits in juve- niles followed by progressive functional recovery in adults. Neurobiol Dis 2004; 17:403–14
+
+19. Rosado JA, Lopez JJ, Gomez-Arteta E, Redondo PC, Salido GM, Pariente JA: Early caspase-3 activation independent of apoptosis is required for cellular func- tion. J Cell Physiol 2006; 209:142–52
+
+20. Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC: Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 1994; 371:346–7
+
+8. Lerman J, Sikich N, Kleinman S, Yentis S: The pharmacology of sevoflurane
+
+21. Kim JJ, Fanselow MS: Modality-specific retrograde amnesia of fear. Science
+
+in infants and children. ANESTHESIOLOGY 1994; 80:814–24
+
+1992; 256:675–7
+
+9. Nishikawa K, Harrison NL: The actions of sevoflurane and desflurane on the gamma-aminobutyric acid receptor type A: Effects of TM2 mutations in the alpha and beta subunits. ANESTHESIOLOGY 2003; 99:678–84
+
+22. Phillips RG, LeDoux JE: Differential contribution of amygdala and hip- pocampus to cued and contextual fear conditioning. Behav Neurosci 1992; 106:274–85
+
+10. Hollmann MW, Liu HT, Hoenemann CW, Liu WH, Durieux ME: Modulation of NMDA receptor function by ketamine and magnesium, part II: interactions with volatile anesthetics. Anesth Analg 2001; 92:1182–91
+
+11. Young C, Jevtovic-Todorovic V, Qin YQ, Tenkova T, Wang H, Labruyere J, Olney JW: Potential of ketamine and midazolam, individually or in combination, to induce apoptotic neurodegeneration in the infant mouse brain. Br J Pharmacol 2005; 146:189–97
+
+12. Lu LX, Yon JH, Carter LB, Jevtovic-Todorovic V: General anesthesia acti- vates BDNF-dependent neuroapoptosis in the developing rat brain. Apoptosis 2006; 11:1603–15
+
+13. Ito M, Nakashima M, Tsuchida N, Imaki J, Yoshioka M: Histogenesis of the intravitreal membrane and secondary vitreous in the mouse. Invest Ophthalmol Vis Sci 2007; 48:1923–30
+
+14. Satoh Y, Endo S, Ikeda T, Yamada K, Ito M, Kuroki M, Hiramoto T, Imamura O, Kobayashi Y, Watanabe Y, Itohara S, Takishima K: Extracellular signal-regulated kinase 2 (ERK2) knockdown mice show deficits in long-term memory; Erk2 has a specific function in learning and memory. J Neurosci 2007; 27:10765–76
+
+23. Murcia CL, Gulden F, Herrup K: A question of balance: A proposal for new
+
+mouse models of autism. Int J Dev Neurosci 2005; 23:265–75
+
+24. Johnson SA, Young C, Olney JW: Isoflurane-induced neuroapoptosis in the developing brain of nonhypoglycemic mice. J Neurosurg Anesthesiol 2008; 20:21–8
+
+25. American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, 4th ed. Washington, DC, American Psychiatric Association, 2000
+
+26. Moretti P, Bouwknecht JA, Teague R, Paylor R, Zoghbi HY: Abnormalities of social interactions and home-cage behavior in a mouse model of Rett syn- drome. Hum Mol Genet 2005; 14:205–20
+
+27. Lijam N, Paylor R, McDonald MP, Crawley JN, Deng CX, Herrup K, Stevens KE, Maccaferri G, McBain CJ, Sussman DJ, Wynshaw-Boris A: Social interaction and sensorimotor gating abnormalities in mice lacking Dvl1. Cell 1997; 90:895– 905
+
+28. Polleux F, Lauder JM: Toward a developmental neurobiology of autism.
+
+Ment Retard Dev Disabil Res Rev 2004; 10:303–17
+
+29. Rubenstein JL, Merzenich MM: Model of autism: Increased ratio of excita-
+
+15. Jin D, Liu HX, Hirai H, Torashima T, Nagai T, Lopatina O, Shnayder NA, Yamada K, Noda M, Seike T, Fujita K, Takasawa S, Yokoyama S, Koizumi K, Shiraishi Y, Tanaka S, Hashii M, Yoshihara T, Higashida K, Islam MS, Yamada N, Hayashi K, Noguchi N, Kato I, Okamoto H, Matsushima A, Salmina A, Munesue T, Shimizu N, Mochida S, Asano M, Higashida H: CD38 is critical for social behavior by regulating oxytocin secretion. Nature 2007; 446:41–5
+
+tion/inhibition in key neural systems. Genes Brain Behav 2003; 5:255–67
+
+30. Uhlhaas PJ, Singer W: Neural synchrony in brain disorders: Relevance for
+
+cognitive dysfunctions and pathophysiology. Neuron 2006; 52:155–68
+
+31. Buzsa´ki G, Draguhn A: Neuronal oscillations in cortical networks. Science
+
+2004; 304:1926–9
+
+32. Hussman JP: Suppressed GABAergic inhibition as a common factor in
+
+16. Kwon CH, Luikart BW, Powell CM, Zhou J, Matheny SA, Zhang W, Li Y, Baker SJ, Parada LF: Pten regulates neuronal arborization and social interaction in mice. Neuron 2006; 50:377–88
+
+17. Bittigau P, Sifringer M, Pohl D, Stadthaus D, Ishimaru M, Shimizu H, Ikeda
+
+suspected etiologies of autism. Autism Dev Disord 2001; 2:247–8
+
+33. Bishop S, Gahagan S, Lord C: Re-examining the core features of autism: A comparison of autism spectrum disorder and fetal alcohol spectrum disorder. J Child Psychol Psychiatry 2007; 48:1111–21 /
+
+33. Bishop S, Gahagan S, Lord C: Re-examining the core features of autism: A comparison of autism spectrum disorder and fetal alcohol spectrum disorder. J Child Psychol Psychiatry 2007; 48:1111–21 /
+
+Anesthesiology, V 110, No 3, Mar 2009
+
+D o w n o a d e d
+
+l
+
+f r o m h
+
+t t
+
+p
+
+: / /
+
+p u b s . a s a h q o r g a n e s t h e s o o g y / a r t i c e - p d
+
+.
+
+/
+
+i
+
+l
+
+l
+
+1 1 0 3 6 2 8 3 6 8 3 7 1 0 0 0 0 5 4 2 - 2 0 0 9 0 3 0 0 0 - 0 0 0 3 1 p d
+
+/
+
+/
+
+/
+
+f
+
+b y g u e s t
+
+o n 3 0 M a r c h 2 0 2 1

new_pdfs/10.1097_ALN.0b013e31819daedd.txt

@@ -0,0 +1,303 @@
+Anesthesiology 2009; 110:1077– 85
+
+Copyright © 2009, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc.
+
+Dexmedetomidine Attenuates Isoflurane-induced Neurocognitive Impairment in Neonatal Rats Robert D. Sanders, B.Sc., M.B.B.S., F.R.C.A.,* Jing Xu, M.D.,† Yi Shu, B.Sc.,‡ Adam Januszewski, B.Sc., M.B.B.S.,§ Sunil Halder, B.Sc., M.B.B.S.,(cid:1) Antonio Fidalgo, M.Sc.,‡ Pamela Sun, B.Sc.,# Mahmuda Hossain, Ph.D.,** Daqing Ma, M.D., Ph.D.,†† Mervyn Maze, M.B., Ch.B., F.R.C.P., F.R.C.A., F.Med.Sci.‡‡
+
+Background: Neuroapoptosis is induced by the administra- tion of anesthetic agents to the young. As (cid:1)2 adrenoceptor signaling plays a trophic role during development and is neu- roprotective in several settings of neuronal injury, the authors investigated whether dexmedetomidine could provide func- tional protection against isoflurane-induced injury.
+
+Methods: Isoflurane-induced injury was provoked in organo- typic hippocampal slice cultures in vitro or in vivo in postnatal day 7 rats by a 6-h exposure to 0.75% isoflurane with or without dexmedetomidine. In vivo, the (cid:1)2 adrenoceptor antagonist ati- pamezole was used to identify if dexmedetomidine neuropro- tection involved (cid:1)2 adrenoceptor activation. The (cid:2)-amino-bu- tyric-acid type A antagonist, gabazine, was also added to the organotypic hippocampal slice cultures in the presence of isoflurane. Apoptosis was assessed using cleaved caspase-3 im- munohistochemistry. Cognitive function was assessed in vivo on postnatal day 40 using fear conditioning.
+
+Results: In vivo dexmedetomidine dose-dependently pre- vented isoflurane-induced injury in the hippocampus, thala- mus, and cortex; this neuroprotection was attenuated by treat- ment with atipamezole. Although anesthetic treatment did not affect the acquisition of short-term memory, isoflurane did induce long-term memory impairment. This neurocognitive deficit was prevented by administration of dexmedetomidine, which also inhibited isoflurane-induced caspase-3 expression in organotypic hippocampal slice cultures in vitro; however, gabazine did not modify this neuroapoptosis.
+
+Conclusion: Dexmedetomidine attenuates isoflurane-induced injury in the developing brain, providing neurocognitive pro- tection. Isoflurane-induced injury in vitro appears to be inde- pendent of activation of the (cid:2)-amino-butyric-acid type A recep- tor. If isoflurane-induced neuroapoptosis proves to be a clinical problem, administration of dexmedetomidine may be an im- portant adjunct to prevent isoflurane-induced neurotoxicity.
+
+ANESTHESIA has recently been associated with wide- spread apoptotic neurodegeneration in the neonatal rat brain with persistent functional neurocognitive impair-
+
+ment, exemplified by impaired memory formation.1– 4 This discovery has led to concern about the possible detrimental effects of anesthesia and sedation in the pediatric population. The observed apoptotic neurode- generation mimics the neuronal injury of fetal alcohol syndrome5 and is thought to be secondary to impaired neurotransmission during a critical period of synapto- genesis that triggers so-called neuronal suicide. Indeed, there is significant evidence that preventing synaptic neurotransmission causes deleterious long-term central nervous system changes,6 with synaptic neurotransmis- sion critical to avoid synaptic pruning and apoptosis of activity-deprived neurons.7,8
+
+Generically, anesthetic agents are thought to inhibit synaptic neurotransmission by potentiating (cid:1)-amino- butyric-acid type A (GABAA) receptors, inhibiting gluta- mate N-methyl-D-aspartate (NMDA) channels or activat- ing two-pore potassium channels.9 The net result leads to cellular hyperpolarization and a reduction in neuronal activity. However, during development, this artificial silenc- ing of synapses is thought to induce an apoptotic cascade via disruption of the action of trophic factors, notably brain-derived neurotrophic factor,2,3 phosphorylated ex- tracellular signal-regulated protein kinase 1 and 2 (pERK),2 and phosphorylated-cyclic-adenosine monophosphate (AMP) response element binding protein with subse- quent stimulation of the intrinsic apoptotic cascade.4,10 The intrinsic cascade results in cytochrome C release and Bax signaling to activate the caspase enzymes that provoke cell death by apoptosis.4,10,11 Subsequently, ex- trinsic apoptotic signaling may also be activated.10 These toxic effects have now been established after as little as 60 min of below 1 minimum alveolar concentration of isoflurane in the 7-day-old rat12; thus, a relationship be- tween anesthesia, neuroapoptosis and cognitive dys- function has been established.
+
+Academic Clinical Fellow, ‡ Doctoral Student, § House Officer, # Medical Student, ** Research Technician, †† Senior Lecturer, ‡‡ Sir Ivan Magill Professor of Anaesthesia, Department of Anaesthetics, Pain Medicine and Intensive Care, Imperial College London, United Kingdom; (cid:1) Honorary Research Fellow, Imperial College London, and Specialty Trainee, Department of Anaesthetics, Reading General Hospital, Reading, United Kingdom; † Attending Physician, Department of Anesthesiology, Gongli Hospital, Pudong, Shanghai, China. Professor Maze has been a consultant for Abbott Laboratories, Abbott Park, Illinois, to facilitate registration of dexmedetomidine in the United States. Received from the Department of Anaesthetics, Pain Medicine and Intensive Care, Imperial College London, United Kingdom. Submitted for publication January 23, 2008. Accepted December 2, 2008. Supported by Chelsea and Westminster Healthcare NHS Trust, London, United Kingdom, and the Westmin- ster Medical School Research Trust, London, United Kingdom.
+
+Address correspondence to Professor Maze: Magill Department of Anaesthet- ics, Intensive Care and Pain Medicine, Imperial College London, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH. m.maze@ imperial.ac.uk. Information on purchasing reprints may be found at www. anesthesiology.org or on the masthead page at the beginning of this issue. ANESTHESIOLOGY’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
+
+The NMDA antagonist ketamine (20 mg kg(cid:1)1 and above) and the GABAergic agonist midazolam (9 mg kg(cid:1)1) both induce apoptotic neurodegeneration in in- fant mice13 despite having different mechanisms of an- esthetic action. This has significant implications for pedi- atric anesthesia as these drugs are used for premedication, sedation or analgesia in several clinical settings. Further- more, as these agents have differing mechanisms of anes- thetic action, yet induce this neuroapoptosis, it has been argued that it is the anesthetic state that produces the injury.11 To date, only one exception to this rule has been identified, the noble anesthetic gas xenon, which prevented isoflurane-induced toxicity.4 However, xenon
+
+Anesthesiology, V 110, No 5, May 2009
+
+1077
+
+1078
+
+is currently not widely available; therefore, we have been seeking to identify alternative methods to amelio- rate this toxicity.
+
+Early in life, (cid:2)2 adrenoceptors are thought to play a trophic role in central nervous system signaling,14,15 with endogenous norepinephrine activating cellular survival mechanisms such as the Ras-Raf-pERK pathway.16,17 Acti- vation of this Ras-Raf-pERK pathway has been associated with neuroprotection against the apoptosis induced by NMDA antagonists in the young.2 Dexmedetomidine also increases the expression of the antiapoptotic proteins mdm2 and bcl-2 in a model of adult ischemic cerebral injury18; in vitro, it has been shown to upregulate brain- derived neurotrophic factor, phosphorylated-cyclic-AMP response element binding protein, and pERK signal- ing.16,19,20 However, it is not known whether modifica- tion of these proteins represents a true antiapoptotic effect of dexmedetomidine or whether these findings were merely a correlate of increased cellular survival. Herein, we show that dexmedetomidine protects against anesthetic-induced apoptosis in vivo and in vitro, indi- cating that it does possess antiapoptotic qualities. Im- portantly, we again establish that isoflurane injury provokes a long-term neurocognitive deficit and then demonstrate that this functional deficit can be atten- uated by dexmedetomidine.
+
+Materials and Methods
+
+The study protocol was approved by the Home Office (London, United Kingdom) and conforms to the United Kingdom Animals (Scientific Procedures) Act of 1986.
+
+In Vitro Experiments Organotypic hippocampal slices were derived from postnatal day 8 or 9 C57Bl/6 mice pups (Harlan Labora- tories, Huntingdon, United Kingdom) and cultured by the interface method21,22 with some modifications. In brief, the brain was quickly dissected and placed in ice-cooled (4°C) dissection solution. All stages of slice preparation were performed under sterile and ice-cooled conditions. Excess tissue (including the cerebellum, ol- factory bulbs, and meninges) was removed, and the brain was cut into 400-(cid:3)m sagittal slices using a McIl- lwain Tissue Chopper (Mickle Laboratory, Cambridge, United Kingdom). Under a dissecting microscope and avoiding contact with the hippocampus, the slices were separated using fine forceps. Slices containing the intact hippocampus were selected and positioned onto 30-mm- diameter semiporous cell culture inserts (five slices per insert) (Falcon; Becton Dickinson Labware, Millipore, Bedford, MA) and placed in a six-well tissue culture tray (Multiwell; Falcon, Becton Dickinson Labware). Eagle minimum essential medium enhanced with heat-inacti- vated horse serum (1.5 ml) was then transferred to each well.
+
+Anesthesiology, V 110, No 5, May 2009
+
+SANDERS ET AL.
+
+The slices were incubated for 24 h in humidified air at 37°C, enriched with 5% carbon dioxide. The culture medium was replaced the next day with fresh, temper- ature-equilibrated medium before exposure to gas treat- ments. The groups of slices (n (cid:2) 15 per group) were assigned to control (air (cid:3) 5% carbon dioxide), dexme- detomidine 1 (cid:3)M, gabazine 50 (cid:3)M, 0.75% isoflurane, 0.75% isoflurane (cid:3) dexmedetomidine 1 (cid:3)M, and 0.75% isoflurane (cid:3) gabazine 50 (cid:3)M.
+
+All subsequent gas exposure occurred in a specially con- structed exposure chamber as previously described.23 The gases, warmed by a water bath, were delivered in the headspace above the slices by a standard anesthetic machine at 2–3 l/min, and concentrations were moni- tored with an S/5 spirometry module (Datex-Ohmeda, Bradford, United Kingdom). After 3– 4 min of gas flow, the chambers were sealed and placed in a 37°C incuba- tor for 6 h (Galaxy R Carbon Dioxide Chamber; Wolf Laboratories, Pocklington, York, United Kingdom). After exposure, the slices were returned to the incubator for a further 12 h of culture to allow for suitable caspase-3 expression and then fixed overnight in 4% paraformal- dehyde and subsequently immersed in 30% sucrose for a further 24 h at 4°C before slicing with a cryostat.
+
+In Vivo Experiments Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane in 25% oxygen or air in a temperature-controlled chamber (n (cid:2) 6 per group). Three doses of saline or dexmedetomidine (1, 10, or 25 (cid:3)g/kg) were administered by intraperitoneal injection over the 6-h exposure (at 0, 2, and 4 h). One group received 0.75% isoflurane, 25 (cid:3)g/kg dexmedetomidine, and 500 (cid:3)g/kg nonselective (cid:2)2 adrenoceptor antagonist atipamezole in 3 doses over the 6-h exposure (n (cid:2) 4 per group). An additional three doses of 75 (cid:3)g/kg dexme- detomidine in air were given to establish at extreme doses of dexmedetomidine whether apoptosis could be induced (n (cid:2) 6 per group).
+
+The animals were sacrificed (with 100 mg/kg sodium pentobarbital by intraperitoneal injection) at the end of gas exposure and perfused transcardially with heparin- ized saline followed by 4% paraformaldehyde in 0.1 M buffer. After removal of the brain and storage overnight at 4°C in paraformaldehyde, it was transferred to 30% sucrose solution with phosphate buffer and 1% sodium azide and kept at 4°C until the brains were sectioned and stained immunohistochemically for caspase-3.
+
+Immunohistochemistry For the in vitro experiments, the slices were sectioned at 25-(cid:3)m intervals using a cryostat, and the inner sec- tions were mounted onto Super Plus-coated glass slides (VWR International, Lutterworth, United Kingdom). The sections were allowed to dry at 37°C for 24 h and then immunostained while adherent to the slides. Concerning
+
+DEXMEDETOMIDINE INHIBITS ISOFLURANE-INDUCED INJURY
+
+the in vivo experiments, the brain was sliced at 30-(cid:3)m intervals beginning at (cid:1)3.6 mm from the bregma, the sections were then transferred to a six-well plate con- taining phosphate-buffered saline. Sections were dried at 37°C for 24 h and then immunostained while adherent to the slides, before preincubation with hydrogen 0.3% peroxidase in methanol for 30 min and then rinsed in phosphate-buffered saline. The sections were then incu- bated overnight at 4°C with rabbit anti-cleaved caspase-3 (1:2,500; New England Biolab, Hitchin, United King- dom) and then washed three times in phosphate-buff- ered saline with 3% Triton at room temperature. Biotin- ylated secondary antibodies (1:200; Sigma, St. Louis, MO) and the avidin-biotin-peroxidase complex (Vector Laboratories, Orton Southgate, Peterborough, United Kingdom) were applied. The sections were again washed in phosphate-buffered saline before incubating with 0.02% 3,3=-diaminobenzidine with nickel ammo- nium sulfate in 0.003% hydrogen peroxide (DAB kit, Vector Laboratories). The sections were dehydrated through a gradient of ethanol solutions (70 –100%) and then mounted (floating section) and covered with a cover slip.
+
+Neurocognitive Evaluation Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane in 25% oxygen or air in a temperature-controlled chamber (n (cid:2) 6 per group). Three doses of saline or 25 (cid:3)g/kg dexmedetomidine were administered by intraperitoneal injection over the 6-h exposure (at 0, 2, and 4 h). The animals were al- lowed to mature until postnatal day 40 and then tested for hippocampal-dependent memory and learning func- tion in a previously reported contextual fear-condition- ing behavioral paradigm24 in which the rats were taken from the vivarium in the behavioral room on the first test day and allowed to sit undisturbed in their homecage for 10 min. Once placed in the conditioning chamber, the rats were allowed 198 s of exploration.
+
+The conditioning chamber was cubic (30 cm (cid:4) 24 cm (cid:4) 21 cm; Med Associates, Inc., St. Albans, VT) and had a white opaque back wall, aluminum sidewalls, and a clear polycarbonate front door. The conditioning box had a removable grid floor and waste pan. Between each rat, the box was cleaned with an almond-scented solution and dried thoroughly. The grid floor contained 36 stain- less steel rods (diameter, 3 mm) spaced 8 mm center to center. When placed in the chamber, the grid floor made contact with a circuit board through which a scrambled shock was delivered. During training and context test- ing, a standard HEPA filter provided background white noise of 65 db.
+
+Afterwards, all animals received 6 cycles of 214 s of trace fear conditioning. The tone was presented for 16 s (2 kHz) followed by a trace interval of 18 s and subse- quent foot shock (2 s, 0.85 mA). The rats were removed
+
+from the conditioning chamber 198 s after the last shock and returned to their home cage. The total time of the acquisition phase was 26 min. Acquisition time was defined as the time spent immobile after a shock divided by the intertrial interval. On the next day, trained rats were exposed to the same acquisition environment but received neither tone nor shock for 8 min (context test). The percentage of time an animal froze during the 8-min observation periods was calculated as the number of observations judged to be freezing divided by the total number of observations in 8 min (i.e., 60 observations). Freezing time was assessed using VideoFreeze software (Med Associates Inc., Burlington, VT); therefore, the assessment can be considered objective. The percentage of freezing time (context results) and the area under curve were derived from plots between the percentage freezing time and trial time in the tone test and were used for statistical comparison (mean (cid:5) SD, n (cid:2) 6 per group).
+
+Statistical Analyses The number of caspase-3–positive neurons in the cor- tex, thalamus, and hippocampus in each brain slice were counted by an observer blinded to the experimental protocol. Four brain slices were counted per animal. The immunohistochemical and behavioral data are presented as mean (cid:5) SD. Statistical analyses was performed by ANOVA followed by post hoc Newman Keuls testing using the INSTAT (London, United Kingdom) program. P (cid:6) 0.05 was set as significant.
+
+Results
+
+All animals survived the in vivo experiments. Isoflu- rane induced neuroapoptosis throughout the cortex, thalamus, and hippocampus reflected by the increase in the number of caspase-3–positive cells observed (fig. 1, A–C). Isoflurane (0.75% (cid:3) saline) increased caspase-3 expression relative to air ((cid:3) saline) controls in the cor- tex from 44 (cid:5) 7 to 270 (cid:5) 34 cells (P (cid:6) 0.05; fig. 1D), in the hippocampus from 8 (cid:5) 3 to 80 (cid:5) 11 cells (P (cid:6) 0.05; fig. 1E), and in the thalamus from 4 (cid:5) 2 to 62 (cid:5) 15 cells (P (cid:6) 0.05; fig. 1F). In contrast dexmedetomidine in the presence of air did not increase cellular caspase-3 ex- pression relative to controls (fig. 1, E–F).
+
+Dexmedetomidine (1–25 (cid:3)g/kg) provided dose-dependent neuroprotection reducing isoflurane-induced caspase-3 ex- pression in the cortex (186 (cid:5) 23 to 129 (cid:5) 29 cells; P (cid:6) 0.05) relative to isoflurane (270 (cid:5) 34 cells), hippocam- pus (28 (cid:5) 11 to 15 (cid:5) 5 cells; P (cid:6) 0.05) relative to isoflurane (80 (cid:5) 11 cells), and thalamus (21 (cid:5) 6 to 9 (cid:5) 4 cells; P (cid:6) 0.05) relative to isoflurane (62 (cid:5) 15 cells). The addition of 25 (cid:3)g/kg dexmedetomidine provided the most potent protection that was significantly better than 1 or 10 (cid:3)g/kg dexmedetomidine (P (cid:6) 0.05) in each
+
+Anesthesiology, V 110, No 5, May 2009
+
+1079
+
+1080
+
+brain area. In the hippocampus and thalamus, but not the cortex, 25 (cid:3)g/kg dexmedetomidine reduced the injury induced by isoflurane to baseline (P (cid:7) 0.05 vs. Air (cid:3) Saline). Reversal of dexmedetomidine neuroprotection by the (cid:2)2 adrenoceptor antagonist, atipamezole, in the hippocampus (P (cid:6) 0.05; fig. 1E), thalamus, and cortex (nonsignificant) indicates that this effect is at least partly mediated by (cid:2)2 adrenoceptors in these regions.
+
+Consistent with previous data,4 6 hours of 0.75% isoflu- rane also induced neuroapoptosis in organotypic hip- pocampal slice cultures, increasing caspase-3 expression by 44% (control 18 (cid:5) 6 vs. isoflurane 26 (cid:5) 11 cells; P (cid:6) 0.01; fig. 2, A and B). This effect was reversed by addi- tion of 1 (cid:3)M dexmedetomidine (20 (cid:5) 7 cells; P (cid:6) 0.05, fig. 2C), reducing caspase-3 expression to within 10% of controls. As reported previously,25 gabazine itself was nontoxic (showing caspase-3 expression 92% of air-sa- line treated controls; P (cid:7) 0.05); it did not attenuate isoflurane-induced apoptosis (P (cid:7) 0.05 vs.. isoflurane; fig. 2, D and E).
+
+At postnatal day 40, neonatal treatment with isoflu- induced
+
+rane-saline, but not air-dexmedetomidine,
+
+Anesthesiology, V 110, No 5, May 2009
+
+SANDERS ET AL.
+
+Fig. 1. Dexmedetomidine (Dex) inhibits isoflurane-induced neuroapoptosis in vivo. Seven-day-old rats were exposed to air or isoflurane (0.75%) for 6 h with injections of saline or intraperitoneal dexmedetomidine given three times at 0, 2, and 4 h. (A) Photomicrograph of a cor- tical section from an animal exposed to air and three doses of saline over 6 h stained immunohistochemically for caspase-3. (B) A similar photomicrograph of a cortical section from an animal exposed to isoflu- rane and three doses of saline over 6 h. (C) Photomicrograph of a cortical section from an animal exposed to isoflurane and three doses of 1 (cid:3)g/kg dexmedetomi- dine over 6 h stained immunohisto- chemically for caspase-3. (D) Histogram showing the number of caspase-3–posi- tive cortical neurons against interven- tion. (E) Histogram showing the number of caspase-3–positive hippocampal neu- rons against intervention. (F) Histogram showing the number of caspase-3–posi- tive thalamic neurons against interven- tion. The interventions include: Air (cid:4) Sa- line intraperitoneal (Air/Sal), Air (cid:4) Dex 25 (cid:3)g/kg (Air/Dex25), Air (cid:4) Dex 75 (cid:3)g/kg (Iso/Dex75), Isoflurane (cid:4) saline (Iso/Sal), Iso (cid:4) Dex 1 (cid:3)g/kg (Iso/Dex1), Iso (cid:4) Dex 10 (cid:3)g/kg (Iso/Dex10), Iso (cid:4) Dex 25 (cid:3)g/kg (Iso/Dex25), Iso (cid:4) Dex 25 (cid:3)g/kg (cid:4) Atipamezole 500 (cid:3)g/kg (Iso/ Dex25/Atp); n (cid:5) 4 – 6 per group. * (cid:5) P < 0.05 versus Air (cid:4) Sal; ** (cid:5) P < 0.001 versus Air (cid:4) Sal; # (cid:5) P < 0.05 versus Iso (cid:4) Sal; (cid:4) (cid:5) P < 0.01 versus Iso (cid:4) Sal; ˆ (cid:5) P < 0.001 versus Iso (cid:4) Sal; § (cid:5) P < 0.05 versus Iso (cid:4) Dex 25; ˜ (cid:5) P < 0.05 versus Iso (cid:4) Dex1 or Iso (cid:4) Dex10.
+
+neurocognitive impairment as assessed by context fear conditioning (a marker of long-term memory); however, none of the groups exhibited any deficit in the acquisition phase (indicating no deficit in short- term memory; fig. 3A). The percentage freezing time in the contextual fear-conditioning experiment was 48 (cid:5) 5% in controls (air-saline), 45 (cid:5) 11% with air- dexmedetomidine–treated animals, and 29 (cid:5) 7% with isoflurane-saline–treated animals (fig. 3B). Dexmedeto- midine ameliorated the neurocognitive impairment in- duced by isoflurane; percentage freezing time 46 (cid:5) 9% with isoflurane-dexmedetomidine–treated animals.
+
+Discussion
+
+Isoflurane induced widespread cerebral neuroapopto- sis in neonatal rat pups with subsequent long-term neu- rocognitive impairment of the animals. As the injury occurred in the neonatal period and animal training and testing followed this injury, this indicates impairment in learning and memory consistent with a significant hip-
+
+DEXMEDETOMIDINE INHIBITS ISOFLURANE-INDUCED INJURY
+
+isoflurane treatment. Importantly, in contrast to isoflu- rane (and other agents such as midazolam and ket- amine13), dexmedetomidine itself lacks neurotoxicity even at extremely high doses such as 75 (cid:3)g/kg dexme- detomidine (a dose that is 75 times the ED50 for hypno- sis27). Dexmedetomidine also did not induce neurocog- nitive impairment at the clinically relevant dose of 25 (cid:3)g/kg. Although dexmedetomidine could also attenuate isoflurane-induced neuroapoptosis in organotypic hip- pocampal slice cultures, gabazine did not reverse this effect, suggesting that isoflurane’s neuroapoptotic effect is not mediated by GABAA receptors.
+
+Caveats These results indicate that dexmedetomidine can in- hibit neuroapoptosis provoked by isoflurane in vitro and in vivo; however, several caveats need to be raised before further interpretation of our data. Previous re- ports have shown that the apoptosis involved neurons, and the injured cells in our study morphologically ap- pear to be neurons. Therefore we assume the dying cells are neurons; this is supported by our data showing a neurocognitive deficit induced by isoflurane. In addition, our marker of apoptosis and cell death, caspase-3 expres- sion, has been previously validated in this model of anesthetic-injury.1– 4 Although we (and others1) have correlated the apoptosis observed with the neurocogni- tive deficits induced by isoflurane in neonatal rats, we still cannot exclude that other mechanisms (such as effects on neurogenesis or synaptic function) do not contribute to the pathogenesis or the protection af- forded by dexmedetomidine.
+
+Fig. 2. Dexmedetomidine (Dex) inhibits isoflurane-induced neuroapoptosis in vitro. C57Bl/6 mice pup organotypic hip- pocampal cultures were exposed to (A) air (cid:4) 5% carbon dioxide (control), (B) isoflurane 0.75% (Iso), (C) isoflurane 0.75% (cid:4) Dex 1 (cid:3)M (Iso (cid:4) Dex), and (D) isoflurane (cid:4) gabazine 50 (cid:3)M (Iso (cid:4) Gab) for 6 h and then stained for caspase-3 using immuno- histochemistry. Quantified data are presented in section E. * (cid:5) P < 0.01 versus Control; ** (cid:5) P < 0.001 versus Control; # (cid:5) P < 0.05 versus Iso and P < 0.01 versus Iso (cid:4) Gab.
+
+pocampal lesion.24,26 These data support previous ex- periments showing a significant hippocampal injury af- ter anesthetic treatment.1 Dexmedetomidine provided neuroprotection against isoflurane-induced neuroapop- tosis in a dose-dependent manner, acting via activation of (cid:2)2 adrenoceptors (as atipamezole reversed dexme- detomidine’s neuroprotective effect). Crucially, dexme- detomidine prevented the neurocognitive sequelae of
+
+Despite data showing that the hypnotic, analgesic, and neuroprotective effects of dexmedetomidine primarily relate to activation of the (cid:2)2A adrenoceptor,28,29 atipam- ezole significantly inhibited dexmedetomidine’s neuro- protective effect only in the hippocampus. Although there was a trend to a reversal in effect in the thalamus and cortex, atipamezole did not significantly alter dexmedetomidine protection in these regions. This may indicate alternate receptor targets in these regions, such as imidazoline receptors,30 but we suspect a type II error may also account for these findings.
+
+Minimal disturbances in arterial blood gases1,3 have been reported in previous studies; however, the poten- tial to induce hypoglycemia in these animals during the anesthetic period is of concern,31 but this has also been shown not to occur.12 Indeed, it is possible that the addition of dexmedetomidine to isoflurane could exac- erbate both the cardiovascular and respiratory depres- sion of the anesthetic state; however, it is also conceiv- able that high doses of dexmedetomidine may have increased either blood pressure (via activation of (cid:2)2B adrenoceptors) or glucose (via (cid:2)2A adrenoceptors). Therefore, we also conducted an in vitro experiment to control for the potential confounding effects of hypoxia,
+
+Anesthesiology, V 110, No 5, May 2009
+
+1081
+
+1082
+
+glucose, and temperature dysregulation using the orga- notypic hippocampal slice culture model. We employed the latter experimental paradigm because synapses re- main intact, which is imperative because inhibition of synaptic neurotransmission is hypothesized as critical to the injury. It is known that isoflurane is directly neurotoxic in the organotypic hippocampal slice culture model.4,32 We used a single dose of dexmedetomidine (1 (cid:3)M) in our organotypic hippocampal slice culture studies and did not corroborate the extensive dose response curve that was obtained in vivo because the aim in this experiment was to identify whether dexmedetomidine was acting via a direct or physiologic mechanism. These data suggest that dexmedetomidine can prevent the isoflurane injury by direct action within the central nervous system.
+
+It should also be noted that dexmedetomidine, al- though neuroprotective, does not entirely reverse the isoflurane injury in the cortex (despite prominent neu- roprotection of the thalamus and hippocampus being observed). However, our neurocognitive tests did not uncover an isoflurane-associated deficit in memory ac- quisition that typically depends on a functional prefron- tal cortex.33 Therefore, despite significant apoptosis in the cortex, our study suggests the cortex is not function- ally impaired after isoflurane treatment (0.75% for 6 h),
+
+Anesthesiology, V 110, No 5, May 2009
+
+SANDERS ET AL.
+
+Fig. 3. Cognitive function assessed by trace fear conditioning. Seven-day-old Sprague-Dawley rat pups were exposed with air or 0.75% isoflurane (Iso) in ox- ygen with or without saline or dexme- detomidine (Dex) treatment for 6 h. They were allowed to live up to 40 days and then tested for hippocampal-dependent memory and learning function. (A) The plot of the mean percentage of freezing time of acquisition against six test trials of trace fear conditioning (day 1). (B) The mean of the percentage of freezing time (context results) obtained from trace fear conditioning (day 2). Mean (cid:6) SD (n (cid:5) 6); * (cid:5) P < 0.05 versus other groups.
+
+but we do suggest that the effects of other doses of isoflurane still require investigation. In monkeys, ket- amine injury is primarily involved the cortex rather than subcortical structures34; it is possible that, if observed in humans, cortical apoptosis induced by anesthetics may be the predominant injury. Further tests of cortex-based neurocognitive function in rodents and primates should be conducted before this injury is dismissed.
+
+A final difficulty plaguing all preclinical studies is the ability to extrapolate across species; in this regard, differ- ential interspecies vulnerability to isoflurane injury may be apparent. Indeed, recent data have suggested that monkey brains may be less vulnerable to ketamine injury than ro- dent brains.34 However, isoflurane may be more potent at inducing apoptosis than ketamine, especially because the injury is apparent after subanesthetic isoflurane concentra- tions lasting only 1 h.12 Whether anesthetic-induced neu- rotoxicity is a clinical problem requires further investiga- tion, including studies involving monkeys and ultimately humans; while we await these answers, we need to strive to obtain a safe anesthetic therapy.
+
+Mechanism of Dexmedetomidine Neuroprotection In these studies, we have explored whether dexme- detomidine is antiapoptotic (as suggested, but not di-
+
+DEXMEDETOMIDINE INHIBITS ISOFLURANE-INDUCED INJURY
+
+rectly investigated by many preclinical studies). We pro- pose that administration of an (cid:2)2 adrenoceptor agonist during the critical phase of synaptogenesis activates the endogenous postsynaptic norepinephrine-mediated tro- phic system, which couples to a pERK-Bcl-2 pathway to produce its antiapoptotic effect.19,20,21 Further studies will probe the involvement of this pathway in vivo. Despite our data showing a role for (cid:2)2 adrenoceptor activation in dexmedetomidine neuroprotection, other potential receptor subtypes, such as the imidazoline re- ceptors, can upregulate pERK and are activated by dexmedetomidine,30 thus providing an alternative mech- anism for dexmedetomidine’s neuroprotective effect.
+
+Mechanism of Isoflurane Neurotoxicity Interestingly, gabazine could not reverse the isoflu- rane-induced neurodegeneration despite the hypothesis that, by potentiating GABAA receptor activity, isoflurane inhibits neurotransmission detrimentally during the crit- ical period of synaptogenesis.11 Thus, GABAA receptor activation may not be critical for isoflurane-induced neu- rotoxicity, although it is of interest that GABAA receptor antagonists can attenuate isoflurane’s neuroprotective effect.25,35 While GABAA receptor activation remains an important target for anesthesia, especially for intrave- nous anesthetics such as propofol, its role in haloge- nated volatile anesthesia is less clear.36,37 Therefore, it would appear that activation of the GABAA receptor is important for isoflurane neuroprotection but not neces- sarily critical for toxicity. Whether GABAA receptor antag- onism can attenuate propofol-induced neurotoxicity38 will be of interest because it reverses the propofol anesthetic state.39 However, GABAA receptors are not involved in the neurotoxicity; therefore, it may be possible to design a safe anesthetic agent for use in the young. Interest- ingly, a difference in the ability of sevoflurane and isoflu- rane to induce apoptosis has also been observed previ- ously,40 although preliminary evidence suggests that sevoflurane, similar to isoflurane, also induces neuro- apoptosis in the neonatal rat brain.41
+
+Another receptor target that may be responsible for the isoflurane injury is the NMDA receptor, which plays a critical role in neurodevelopment.8 Each of the neuro- apoptotic-inducing anesthetics, including isoflurane, ket- amine, and MK-801, inhibit the NMDA receptor subtype of the glutamate receptor.1–5,10 –13 An exception to this rule is xenon, another NMDA receptor antagonist, which produces protection against isoflurane-induced injury rather than neuroapoptosis in the neonatal rat brain.4 We consider it likely that xenon exerts an antiapoptotic effect independent of its action at the NMDA receptor. It is of interest that both (cid:2)2 adrenoceptor agonists and xenon can attenuate the injury produced by NMDA an- tagonists in the adult brain42,43; therefore, despite differ- ences in the morphology of the adult and neonatal tox- icity, we cannot discount overlapping mechanisms of
+
+injury. In addition, we have not as yet evaluated whether a neuroprotective cocktail of xenon and dexmedetomi- dine can be employed to further reduce isoflurane tox- icity because they provide synergistic protection against neonatal hypoxic-ischemic injury.44
+
+Neurocognitive Effects Our results from our fear conditioning paradigm sup- port the previous reports of neurocognitive impairment in adult rats after neonatal anesthesia.1,45 Fear condition- ing consists of placing a rat or a mouse in a chamber and giving one or more mild electric foot shocks. After a shock, the animal becomes immobile (freezing), a natu- ral response to fear that can be used as an indication of memory formation. Complex neuronal circuitry involv- ing the frontal cortex, hippocampus, periaqueductal gray, and rostral ventral medulla underlie the acquisition and retention of fear conditioning.24 Notably, a damaged hippocampus is unable to process the incoming stimuli producing a memory deficit.26
+
+Interestingly, all groups displayed normal learning as- the animals sessed by the acquisition of memory; showed increasing levels of freezing across the training tones, with freezing levels post-shock on sixth pairing approximately 70%. This indicates a normal short-term memory, a function predominantly involving the pre- frontal cortex.31 In the context assessment, 24 h after acquisition training, animals exposed to isoflurane and saline displayed less freezing when compared to naive controls, indicating a neurocognitive deficit. Thus isoflu- rane-treated animals showed an abnormal response to contextual fear conditioning, indicating a severe hip- pocampal lesion24,26 consistent with previous reports.1 However, our experiments employed a much lower dose of anesthetic than in the previous studies (0.75% isoflurane vs. 0.75% isoflurane plus 75% nitrous oxide and 9 mg/kg midazolam). Even with subanesthetic dos- ing, the potential for functional neurocognitive deficit is apparent. In contrast, dexmedetomidine alone did not induce any memory deficit. Furthermore, the addition of dexmedetomidine to isoflurane reversed the neurocog- nitive compromise induced by isoflurane. This is of crit- ical importance because dexmedetomidine is the first agent to be shown to reverse the neurocognitive dys- function provoked by isoflurane.
+
+Dexmedetomidine is widely available and has an ex- panding role in pediatric clinical practice; therefore, if anesthetic-induced neurodegeneration is proven to be a clinical problem, we may already have available a thera- peutic intervention that can be employed in this setting where necessary. In situations where dexmedetomidine is not available, another (cid:2)2 agonist, clonidine, could be a candidate, although further studies are warranted because, although atipamezole significantly reversed dexmedetomi- dine’s neuroprotective effect in the hippocampus, the pro- tection afforded in the thalamus and cortex were not sig-
+
+Anesthesiology, V 110, No 5, May 2009
+
+1083
+
+1084
+
+nificantly attenuated; we cannot be sure that all (cid:2)2 adrenoceptor agonists will afford this protection.
+
+Clinical Implications Clinically, no information has detailed the extent of anesthetic-induced neonatal neurodegeneration in hu- mans. In terms of neurodevelopment, a 7-day-old rat pup represents the peak of the synaptogenic period, but this period extends from birth to up to 2–3 yr in humans; therefore, the window of vulnerability may be greater in humans.46,47 One cannot advocate withholding anesthe- sia or analgesia during early human life on the basis of these findings because of the harm that this can do.47–50 However, if anesthetic-induced neurodegeneration is re- vealed as a clinical problem for pediatric anesthesia, administration of an (cid:2)2 adrenoceptor agonist during the anesthesia maybe prudent. Thus, this study has uncov- ered a plausible and promising novel application of a widely available class of drugs that may significantly affect the safety of clinical practice.
+
+The use of (cid:2)2 adrenoceptor agonists in pediatric prac- tice is expanding as a result of their potent sedative/ hypnotic qualities, analgesic action, potential organ-pro- tective effects, reduction in postoperative nausea and vomiting and delirium, and relative lack of respiratory side effects.51,52 Their use in neonatal practice requires evaluation based on these factors.51 In the future, their organ-protective, including neuroprotective, effects may be of importance to the provision of safe, balanced pediatric anesthesia.47
+
+References
+
+1. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF: Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 2003; 23:876–82
+
+2. Hansen HH, Briem T, Dzietko M, Sifringer M, Voss A, Rzeski W, Zdzisinska B, Thor F, Heumann R, Stepulak A, Bittigau P, Ikonomidou C: Mechanisms leading to disseminated apoptosis following NMDA receptor blockade in the developing rat brain. Neurobiol Dis 2004; 16:440–53
+
+3. Lu LX, Yon JH, Carter LB, Jevtovic-Todorovic V: General anesthesia activates BDNF-dependent neuroapoptosis in the developing rat brain. Apoptosis 2006; 11:1603–15
+
+4. Ma D, Williamson P, Januszewski A, Nogaro MC, Hossain M, Ong LP, Shu Y, Franks NP, Maze M: Xenon mitigates isoflurane-induced neuronal apoptosis in the developing rodent brain. ANESTHESIOLOGY 2007; 106:746–53
+
+5. Ikonomidou C, Bittigau P, Ishimaru MJ, Wozniak DF, Koch C, Genz K, Price MT, Stefovska V, Horster F, Tenkova T, Dikranian K, Olney JW: Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome. Science 2000; 287: 1056–60
+
+6. Jain N, Florence SL, Qi HX, Kaas JH: Growth of new brainstem connections in adult monkeys with massive sensory loss. Proc Natl Acad Sci U S A. 2000; 97:5546–50
+
+7. Hestrin S: Developmental regulation of NMDA receptor-mediated synaptic
+
+currents at a central synapse. Nature 1992; 357:686–9
+
+8. Hardingham GE, Bading H: The Yin and Yang of NMDA receptor signaling.
+
+Trends Neurosci 2003; 26:81–9
+
+9. Franks NP, Lieb WR: Molecular and cellular mechanisms of general anaes-
+
+thesia. Nature 1994; 367:607–14.
+
+10. Yon JH, Daniel-Johnson J, Carter LB, Jevtovic-Todorovic V: Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience 2005; 135:815–27
+
+11. Olney JW, Young C, Wozniak DF, Ikonomidou C, Jevtovic-Todorovic V: Anesthesia-induced developmental neuroapoptosis. Does it happen in humans? ANESTHESIOLOGY 2004; 101:273–5
+
+Anesthesiology, V 110, No 5, May 2009
+
+SANDERS ET AL.
+
+12. Johnson SA, Young C, Olney JW: Isoflurane-induced neuroapoptosis in the developing brain of nonhypoglycemic mice. J Neurosurg Anesthesiol 2008; 20:21–8
+
+13. Young C, Jevtovic-Todorovic V, Qin YQ, Tenkova T, Wang H, Labruyere J, Olney JW: Potential of ketamine and midazolam, individually or in combination, to induce apoptotic neurodegeneration in the infant mouse brain. Br J Pharmacol 2005; 146:189–97
+
+14. Winzer-Serhan UH, Leslie FM: Expression of alpha2A adrenoceptors during
+
+rat neocortical development. J Neurobiol 1999; 38:259–69
+
+15. Song ZM, Abou-Zeid O, Fang YY: Alpha2a adrenoceptors regulate phos- phorylation of microtubule-associated protein-2 in cultured cortical neurons. Neuroscience 2004; 123:405–18
+
+16. Wang Q, Lu R, Zhao J, Limbird LE: Arrestin serves as a molecular switch, linking endogenous alpha2-adrenergic receptor to SRC-dependent, but not SRC- independent, ERK activation. J Biol Chem 2006; 281:25948–55
+
+17. Philipp M, Brede ME, Hadamek K, Gessler M, Lohse MJ, Hein L: Placental alpha(2)-adrenoceptors control vascular development at the interface between mother and embryo. Nat Genet 2002; 31:311–5
+
+18. Engelhard K, Werner C, Eberspa¨cher E, Bachl M, Blobner M, Hildt E, Hutzler P, Kochs E: The effect of the alpha 2-agonist dexmedetomidine and the N-methyl-D-aspartate antagonist S((cid:3))-ketamine on the expression of apoptosis- regulating proteins after incomplete cerebral ischemia and reperfusion in rats. Anesth Analg 2003; 96:524–31
+
+19. Dahmani S, Paris A, Jannier V, Hein L, Rouelle D, Scholz J, Gressens P, Desmonts JM, Mantz J: Dexmedetomidine increases hippocampal phosphory- lated extracellular signal-regulated protein kinase 1 and 2 content by an alpha 2-adrenoceptor-independent mechanism: Evidence for the involvement of imida- zoline I1 receptors. ANESTHESIOLOGY 2008; 108:457–66
+
+20. Dahmani S, Rouelle D, Gressens P, Mantz J: Effect of dexmedetomidine on brain derived neurotrophic factor expression in rat hippocampal slices (ab- stract). ANESTHESIOLOGY 2006; 105:A681
+
+21. Gahwiler BH: Organotypic monolayer cultures of nervous tissue. J Neuro-
+
+sci Methods 1981; 4:329–42
+
+22. Stoppini L, Buchs PA, Muller D: A simple method for organotypic cultures
+
+of nervous tissue. J Neurosci Methods 1991; 37:173–82
+
+23. Wilhelm S, Ma D, Maze M, Franks NP: Effects of xenon on in vitro and in
+
+vivo models of neuronal injury. ANESTHESIOLOGY 2002; 96:1485–91
+
+24. Quinn JJ, Loya F, Ma QD, Fanselow MS: Dorsal hippocampus NMDA receptors differentially mediate trace and contextual fear conditioning. Hip- pocampus 2005; 15:665–74
+
+25. Ma D, Hossain M, Rajakumaraswamy N, Franks NP, Maze M: Combination of xenon and isoflurane produces a synergistic protective effect against oxygen- glucose deprivation injury in a neuronal-glial co-culture model. ANESTHESIOLOGY 2003; 99:748–51
+
+26. Fanselow MS: Contextual fear, gestalt memories, and the hippocampus.
+
+Behavioural Brain Res 2000; 110:73–81
+
+27. Sanders RD, Giombini M, Ma D, Ohashi Y, Hossain M, Fujinaga M, Maze M: Dexmedetomidine exerts dose-dependent age-independent antinociception but age-dependent hypnosis in Fischer rats. Anesth Analg 2003; 100:1295–302
+
+28. Lakhlani PP, MacMillan LB, Guo TZ, McCool BA, Lovinger DM, Maze M, Limbird LE: Substitution of a mutant alpha2a-adrenergic receptor via “hit and run” gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo. Proc Natl Acad Sci U S A 1997; 94:9950–5 29. Ma D, Hossain M, Rajakumaraswamy N, Arshad M, Sanders RD, Franks NP, Maze M: Dexmedetomidine produces its neuroprotective effect via the (cid:2)2A- adrenoceptor subtype. Eur J Pharmacol 2004; 502:87–97
+
+30. Dahmani S, Paris A, Hein L, Gressens P, Mantz J: Dexmedetomidine activates ERK 1 & 2 via a non-(cid:2)2 adrenoceptors-mediated pathway (abstract). ANESTHESIOLOGY 2007:A12
+
+31. Loepke AW, McCann JC, Kurth CD, McAuliffe JJ: The physiologic effects
+
+of isoflurane anesthesia in neonatal mice. Anesth Analg 2006; 102:75–80
+
+32. Wise-Faberowski L, Zhang H, Ing R, Pearlstein RD, Warner DS: Isoflurane- induced neuronal degeneration: An evaluation in organotypic hippocampal slice cultures. Anesth Analg 2005; 101:651–7
+
+33. Blum S, Hebert AE, Dash PK: A role for the prefrontal cortex in recall of
+
+recent and remote memories. Neuroreport 2005; 17:341–4
+
+34. Slikker W Jr, Zou X, Hotchkiss CE, Divine RL, Sadovova N, Twaddle NC, Doerge DR, Scallet AC, Patterson TA, Hanig JP, Paule MG, Wang C: Ketamine- induced neuronal cell death in the perinatal rhesus monkey. Toxicol Sci 2007; 98:145–58
+
+35. Elsersy H, Mixco J, Sheng H, Pearlstein RD, Warner DS: Selective gamma- aminobutyric acid type A receptor antagonism reverses isoflurane ischemic neuroprotection. ANESTHESIOLOGY 2006; 105:81–90
+
+36. Jurd R, Arras M, Lambert S, Drexler B, Siegwart R, Crestani F, Zaugg M, Vogt KE, Ledermann B, Antkowiak B, Rudolph U: General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit. FASEB J 2003; 17:250–2
+
+37. Sonner JM, Zhang Y, Stabernack C, Abaigar W, Xing Y, Laster MJ: GABA(A) receptor blockade antagonizes the immobilizing action of propofol but not ketamine or isoflurane in a dose-related manner. Anesth Analg 2003; 96:706–12 38. Cattano D, Young C, Olney J: Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain (abstract). ANESTHESIOLOGY 2007; 106: A1984
+
+DEXMEDETOMIDINE INHIBITS ISOFLURANE-INDUCED INJURY
+
+39. Nelson LE, Lu J, Guo T, Saper CB, Franks NP, Maze M: The sedative component of anesthesia is mediated by GABA(A) receptors in an endogenous sleep pathway. Nat Neurosci 2002; 5:979–84
+
+combination of N-methyl-D-Aspartate and gamma-aminobutyric acid type A re- ceptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioural deficits. ANESTHESIOLOGY 2007; 107:427–36
+
+40. Wei H, Kang B, Wei W, Liang G, Meng QC, Li Y, Eckenhoff RG: Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently. Brain Res 2005; 1037:139–47
+
+41. Satomoto M, Satoh Y, Terui K, Imaki J: Inhaled sevoflurane induces apoptosis in the developing brain and behavioural abnormality in mice (abstract). ANESTHESIOLOGY 2008; 109:A26
+
+42. Marrs W, Kuperman J, Avedian T, Roth RH, Jentsch JD: Alpha-2 adreno- ceptor activation inhibits phencyclidine-induced deficits of spatial working mem- ory in rats. Neuropsychopharmacology 2005; 30:1500–10
+
+46. Dobbing J, Sands J: Comparative aspects of the brain growth spurt. Early
+
+Hum Dev 1979; 3:79–83
+
+47. Sanders RD, Ma D, Brooks P, Maze M: Balancing paediatric anaesthesia: Preclinical insights into analgesia, hypnosis, neuroprotection and neurotoxicity. Br J Anaesth 2008; 101:597–609
+
+48. Anand KJS, Hickey PR: Pain and its effects in the human neonate and fetus.
+
+N Engl J Med 1987; 317:1321–29
+
+49. Porter FL, Grunau RE, Anand KJ: Long-term effects of pain in infants. J Dev
+
+43. Nagata A, Nakao Si S, Nishizawa N, Masuzawa M, Inada T, Murao K, Miyamoto E, Shingu K: Xenon inhibits but N(2)O enhances ketamine-induced c-Fos expression in the rat posterior cingulate and retrosplenial cortices. Anesth Analg 2001; 92:362–8
+
+44. Rajakumaraswamy N, Ma D, Hossain M, Sanders RD, Franks NP, Maze M: Neuroprotective interaction produced by xenon and dexmedetomidine on in vitro and in vivo neuronal injury models. Neurosci Lett 2006; 409:128–33
+
+45. Fredriksson A, Ponten E, Gordh T, Eriksson P: Neonatal exposure to a
+
+Behav Pediatr 1999; 20:253–61
+
+50. Taddio A, Katz J, Ilersich AL, Koren G: Effect of neonatal circumcision on pain response during subsequent routine vaccination. Lancet 1997; 349:599–603 51. Sanders RD, Maze M: Alpha2-adrenoceptor agonists. Curr Opin Investig Drugs 2007; 8:25–33
+
+52. Bergendahl H, Lonnqvist PA, Eksborg S: Clonidine: An alternative to benzodiazepines for premedication in children. Curr Opin Anaesthesiol 2005; 18:608–13
+
+Anesthesiology, V 110, No 5, May 2009
+
+1085

new_pdfs/10.1097_ALN.0b013e3182834d5d.txt

@@ -0,0 +1,1011 @@
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+NIH Public Access Author Manuscript Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Published in final edited form as:
+
+Anesthesiology. 2013 March ; 118(3): 516–526. doi:10.1097/ALN.0b013e3182834d5d.
+
+Sevoflurane anesthesia in pregnant mice induces neurotoxicity in fetal and offspring mice
+
+Hui Zheng, M.D., Ph.D.1,# [Research fellow], Yuanlin Dong, M.D., M.S.2,# [Senior Research Technologist], Zhipeng Xu, M.D., Ph.D.3 [Research fellow], Gregory Crosby, M.D.4 [Associate Professor], Deborah J. Culley, M.D.5 [Associate Professor], Yiying Zhang, M.D., M.S.6 [Research fellow], and Zhongcong Xie, M.D., Ph.D.7,* [Associate Professor] 1Geriatric Anesthesia Research Unit, Department of Anesthesia, Critical Care and Pain Medicine; Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129-2060; Associate Professor, Department of Anesthesiology, Beijing Chest Hospital, Capital Medical University, Beijing, P. R. China
+
+2Geriatric Anesthesia Research Unit, Department of Anesthesia, Critical Care and Pain Medicine; Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts
+
+3Geriatric Anesthesia Research Unit, Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts
+
+4Department of Anesthesia, Brigham & Women’s Hospital and Harvard Medical School, Boston, Massachusetts
+
+5Department of Anesthesia, Brigham & Women’s Hospital and Harvard Medical School Boston, MA 02115
+
+6Geriatric Anesthesia Research Unit, Department of Anesthesia, Critical Care and Pain Medicine; Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts
+
+7Geriatric Anesthesia Research Unit, Department of Anesthesia, Critical Care and Pain Medicine; Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts
+
+Abstract
+
+Background—Each year over 75,000 pregnant women in the United States undergo anesthesia care. We set out to assess the effects of anesthetic sevoflurane in pregnant mice on neurotoxicity and learning and memory in fetal and offspring mice.
+
+Methods—Pregnant mice (gestation stage day 14) and mouse primary neurons were treated with 2.5% sevoflurane for 2 h and 4.1% sevoflurane for 6 h, respectively. Brain tissues of both fetal and offspring mice (postnatal day 31), and the primary neurons were harvested and subjected to
+
+Corresponding author. Zhongcong Xie, M.D., Ph.D., Associate Professor of Anesthesia, Geriatric Anesthesia Research Unit, Department of Anesthesia, Critical Care and Pain Medicine; Massachusetts General Hospital and Harvard Medical School; 149 13th St., Room 4310; Charlestown, MA 02129-2060, (T) 617-724-9308; (F) 617-643-9277; zxie@partners.org. #Hui Zheng and Yuanlin Dong contributed equally to the work. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Summary Statement: Sevoflurane anesthesia in pregnant mice induced increases in interleukin-6 levels, reductions in synaptic marker postsynaptic density-95 and synaptophysin levels, caspase-3 activation, and learning and memory impairment in fetal and offspring mice.
+
+Work attributed to: the Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital and Harvard Medical School.
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+Western blot and immunhistochemistry to assess interleukin-6, synaptic markers postsynaptic density-95 and synaptophysin, and caspase-3 levels. Separately, learning and memory function in the offspring mice was determined in the Morris Water Maze.
+
+Results—Sevoflurane anesthesia in pregnant mice induced caspase-3 activation, increased interleukin-6 levels [256% ± 50.98 (mean ± SD) vs. 100% ± 54.12, P = 0.026], and reduced postsynaptic density-95 (61% ± 13.53 vs. 100% ± 10.08, P = 0.036) and synaptophysin levels in fetal and offspring mice. The sevoflurane anesthesia impaired learning and memory in offspring mice at postnatal day 31. Moreover, interleukin-6 antibody mitigated the sevoflurane-induced reduction in postsynaptic density-95 levels in the neurons. Finally, environmental enrichment attenuated the sevoflurane-induced increases in interleukin-6 levels, reductions of synapse markers, and learning and memory impairment.
+
+Conclusion—These results suggest that sevoflurane may induce detrimental effects in fetal and offspring mice, which can be mitigated by environmental enrichment. These findings should promote more studies to determine the neurotoxicity of anesthesia in the developing brain.
+
+Introduction
+
+Anesthesia neurotoxicity in the developing brain has been investigated in animals and in humans, and has become a major health issue of interest to both the medical community1 and the public2. Anesthesia and surgery may induce neurodevelopment impairment and cognitive dysfunction in children [reviewed in3]. In preclinical studies, anesthesia has been shown to induce neurotoxicity and learning and memory impairment in young animals [4, reviewed in5].
+
+Each year over 75,000 pregnant women in the United States have non-obstetric surgery and fetal intervention procedures under anesthesia6. Anesthesia neurotoxicity in the developing brain could happen in the fetus because: (1) brain development starts as early as the second trimester of pregnancy; (2) anesthesia can induce neurotoxicity in both adult and young mice, and most general anesthetics are lipophilic and thus cross placenta easily; (3) moreover, uterine exposure to ethanol, valproic acid, and anesthetic isoflurane have been shown to induce behavioral abnormalities in adulthood [7, reviewed in8]. It remains largely to be determined, however, whether anesthesia in pregnant mice can induce neurotoxicity in fetal mice (the developing brain), and neurotoxicity and learning and memory impairment in offspring mice after birth.
+
+Sevoflurane is currently the most commonly used inhalation anesthetic. Previous studies have shown that anesthesia with 2.5% sevoflurane for 2 h can induce neurotoxicity in the brain tissues of adult (5-month-old) mice without statistically significant alteration in the values of blood pressure and blood gas9. We therefore determined whether the same sevoflurane anesthesia in pregnant mice could induce neurotoxicity and learning and memory impairment in fetal and offspring mice. Finally, we investigated whether environmental enrichment (EE), a complex living milieu that has been shown to improve learning and memory10–12, could ameliorate the sevoflurane anesthesia-induced detrimental effects.
+
+Materials and Methods
+
+Mice anesthesia
+
+The protocol was approved by the Massachusetts General Hospital Standing Committee (Boston, Massachusetts) on the Use of Animals in Research and Teaching. Three month-old C57BL/6J female mice (The Jackson Laboratory, Bar Harbor, ME) were mated with male mice. The pregnant mice were identified and then housed individually. The offspring mice
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 2
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+were weaned 21 days after birth. Animals were kept in a temperature-controlled (22 – 23 °C) room under a 12-h light/dark period (light on at 7:00 AM); standard mouse chow and water were available ad libitum. At gestation stage day 14 (G14), the pregnant mice were randomly assigned to an anesthesia or control group. Mice randomized to the anesthesia group received 2.5% sevoflurane in 100% oxygen for 2 h in an anesthetizing chamber. The control group received 100% oxygen at an identical flow rate for 2 h in an identical chamber as described in our previous studies9. The mice breathed spontaneously, and concentrations of anesthetic and oxygen were measured continuously (Ohmeda, Tewksbury, MA). Temperature of the anesthetizing chamber was controlled to maintain rectal temperature of the animals at 37 ± 0.5 °C. Mean arterial blood pressure was not measured in these mice because the same sevoflurane anesthesia was shown not to alter the values of blood pressure and blood gas in our previous studies9. Anesthesia was terminated by discontinuing sevoflurane and placing the animals in a chamber containing 100% oxygen until 20 min after return of the righting reflex. The anesthesia with 2.5% sevoflurane (about 1.1 minimum alveolar concentration) for two hours in mice was employed to demonstrate whether clinically relevant sevoflurane anesthesia in pregnant mice, which had been shown to induce neurotoxicity in adult mice9, could also induce neurotoxicity in fetal mice and then neurobehavioral deficits in offspring mice. Twenty pregnant mice were included in the experiments, which generated a sufficient number of fetal mice for the biochemistry studies (n = 6 per arm), and offspring mice for the biochemistry (n = 6 per arm) and behavioral studies (n = 15 per arm). Our pilot studies showed a mean difference of 1.5 (3 vs. 1.5) in platform crossing times, an standard deviation (SD) of 1.8 in the control group and 1.3 in the anesthesia group. From the pilot study, we also estimated a mean difference of 150% (250% vs. 100%) in IL-6 levels in brain tissues, an SD of 51 in the control group and 54 in the anesthesia group. Assuming this study would have similar effect sizes, a sample size of 6 per arm for the biochemistry studies and a sample size of 15 per arm for the behavioral studies would lead to 90% or larger power to detect the differences using two-sample t-test with 5% type 1 error.
+
+Mouse primary neurons
+
+The protocol was approved by the Massachusetts General Hospital Standing Committee (Boston, Massachusetts) on the Use of Animals in Research and Teaching. The harvest of neurons was performed as described in our previous studies13,14. Seven to 10 days after harvesting, the neurons were treated with 4.1% sevoflurane for 6 h as described in our previous studies9. The treatment with 4.1% sevoflurane for 6 h was used to determine whether the sevoflurane anesthesia, which can induce cytotoxicity9, could also reduce levels of postsynaptic density-95 (PSD-95), the marker for synapse. The interleukin (IL)-6 antibody (10 µg/ml) was administrated to the neurons one hour before the sevoflurane treatment. The neurons were harvested at the end of the anesthesia and were subjected to Western blot analysis.
+
+Brain tissue harvest and protein level quantification
+
+Immediately following the sevoflurane anesthesia, we performed a cesarean section to extract the fetal mice and harvested their brain tissues. We also used decapitation to kill postnatal day (P) 31 offspring mice and harvested their brain tissues. Separate groups of mice were used for the Western blot analysis and the immunohistochemistry studies, respectively. For the Western blot analysis, the harvested brain tissues were homogenized on ice using immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid, 0.5% Nonidet P-40) plus protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A) as described in our previous studies15. The lysates were collected, centrifuged at 12,000 rapid per minute for 15 min, and quantified for total proteins with bicinchoninic acid protein assay kit (Pierce, Iselin, NJ)15.
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 3
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+Western blot analysis
+
+Western blot analysis was performed using the methods described in our previous studies15. Whole cerebral hemispheres were used for Western blot analysis because there would be an insufficient amount of hippocampus tissues from the fetal mice for Western blot analysis. IL-6 antibody (1:1,000 dilution, Abcam, Cambridge, MA) was used to recognize IL-6 (24 kDa). PSD-95 antibody (1:1,000, Cell Signaling, Danvers, MA) was used to detect PSD-95 (95 kDa). A caspase-3 antibody (1:1000 dilution; Cell Signaling Technology) was used to recognize full-length caspase-3 (35 – 40 kDa) and caspase-3 fragment (17–20 kDa) resulting from cleavage at aspartate position 175. Antibody anti-β-Actin (1:10,000, Sigma, St. Louis, MO) was used to detect β-Actin (42 kDa). Western blot quantification was performed as described by Xie et al.16. Briefly, signal intensity was analyzed using a Bio-Rad (Hercules, CA) image program (Quantity One). We quantified the Western blots in two steps, first using β-Actin levels to normalize (e.g., determining the ratio of IL-6 to β-Actin amount) protein levels and control for loading differences in the total protein amount. Second, we presented changes in protein levels in mice or neurons undergoing sevoflurane anesthesia as a percentage of those in the control group. 100% of protein level changes refer to control levels for the purpose of comparison to experimental conditions.
+
+The quantification of Western blot was based not only on the images presented in figures, but also the images not presented in the figures in order to have adequate effect size (e.g., n = 6 in biochemistry studies)15.
+
+Immunohistochemistry
+
+Immunohistochemistry was performed using the methods described in our previous studies17. P31 offspring mice were anesthetized with sevoflurane briefly (2.5% sevoflurane for 4 min) and perfused transcardially with heparinized saline followed by 4% paraformaldehyde in 0.1M phosphate buffer at pH 7.4. The anesthesia with 2.5% sevoflurane for 4 min in mice provided adequate anesthesia for the perfusion procedure without causing statistically significant changes in blood pressure and blood gas according to our previous studies9. Mouse brain tissues were removed and kept at 4 °C in paraformaldehyde. Five µm frozen sections from the mouse brain hemispheres were used for the immunohistochemistry staining17. The sections were incubated with the primary antibody synaptophysin (Sigma, 1:500) dissolved in 1% bovine serum albumin in phosphate buffered saline at 4 °C overnight. The next day, the sections were exposed to secondary antibody [Alexa Fluor 594 goat anti-rabbit IgG (H+L), Invitrogen, Grand Island, NY). Finally, the sections were wet mounted and viewed immediately using a fluorescence microscope (60 X). We used the mouse hippocampus in the studies of immunohistochemistry density quantification to determine whether sevoflurane anesthesia can induce neurotoxicity in the hippocampus. The photos were taken and an investigator who was blind to the experimental design counted the density of synaptophysin using Image J Version 1.38 (National Institutes of Health, Bethesda, MD)17.
+
+Morris Water Maze (MWM)
+
+A round steel pool, 150 cm in diameter and 60 cm in height, was filled with water to a height of 1.0 cm above the top of a 15-centimeter diameter platform. The pool was covered with a black curtain and was located in an isolated room with four visual cues on the wall of pool. Water was kept at 20 °C and opacified with titanium dioxide. The P31 offspring mice were tested in the MWM four trials per day for 7 days. Each of the mice was put in the pool to search for the platform and the starting points were random for each mouse. When the mouse found the platform, the mouse was allowed to stay on it for 15 s. If a mouse did not find the platform within a 90-s period, the mouse was gently guided to the platform and allowed to stay on it for 15 s. A video tracking system recorded the swimming motions of
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 4
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+the animals, and the data were analyzed using motion-detection software for the MWM (Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, P.R. China). At the end of the reference training (P37), the platform was removed from the pool and the mouse was placed in the opposite quadrant. Mice were allowed to swim for 90 s and the times the mouse swam to cross the platform area was recorded (platform cross times). Mouse body temperature was maintained by active heating as described by Bianchi et al.18. Specifically, after every trial, each mouse was placed in a holding cage under a heat lamp for 1 to 2 min until dry before returning to its regular cage.
+
+Environmental enrichment
+
+The EE in the current experiment was created in a large cage (70 × 70 × 46 centimeter) that included 5 – 6 toys (e.g., wheels, ladders, and small mazes) as described in previous studies with modification10,11. The pregnant mice were put in the EE everyday for two hours before delivery. The pregnant mice delivered offspring mice at G21. Then, the mother and the babies were put in the EE again everyday for two hours from P4 to P30. The objects were changed two to three times a week to provide newness and challenge.
+
+Statistics
+
+The nature of the hypothesis testing was two-tailed. Data were expressed as mean ± SD. The data for platform crossing time were not normally distributed, thus were expressed as median and interquartile range (IQR). The number of samples varied from 6 to 15, and the samples were normally distributed except platform crossing time (tested by normality test, data not shown). Two-way ANOVA was used to determine the interaction of IL-6 antibody and sevoflurane treatment, and interaction of EE and sevoflurane anesthesia. Interaction between time and group factors in a two way ANOVA with repeated measurements was used to analyze the difference of learning curves (based on escape latency) between mice in the control group and mice treated with anesthesia in the MWM. Multiple comparisons in escape latency of MWM were adjusted using Bonferroni method (with 7 tests, and threshold of 0.05/7 = 0.0071) (*). There were no missing data for the variables of MWM (escape latency and platform crossing time) during the data analysis. Student two-sample t-test was used to determine the difference between the sevoflurane and control conditions on levels of IL-6, PSD-95, and synaptophysin. Finally, the Mann-Whitney test was used to determine the difference between the sevoflurane and control conditions on platform crossing times. P values less than 0.05 (*, # and ^) and 0.01 (**, ## and ^^) were considered statistically significant. SAS software version 9.2 (Cary, NC) was used to analyze the data.
+
+Results
+
+Sevoflurane anesthesia in pregnant mice induced learning and memory impairment in offspring mice
+
+The pregnant mice were either treated with 2.5% sevoflurane anesthesia for 2 h or under the control condition at gestation stage day 14 (G14). The mice delivered offspring mice at G21, and the offspring mice were tested in the MWM from P31 to P37. Comparison of the time that each mouse took to reach a platform during reference training (escape latency) showed that there was a statistically significant interaction between time and group based on escape latency in the MWM between mice following the control condition and mice that were given sevoflurane anesthesia (fig. 1A, ^ P = 0.012, two-way ANOVA with repeated measurement). Comparison of the number of times that each mouse crossed the location of an absent platform at the end of reference training (platform crossing times) indicated that there was a non-significant difference in the platform crossing times between the control condition and the sevoflurane anesthesia (fig. 1B, P = 0.051, Mann-Whitney test,
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 5
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+sevoflurane: median = 1 and IQR = 1 to 3 versus control: median = 2 and IQR = 2 to 4.5). There was no statistically significant difference in mouse swimming speed between the sevoflurane anesthesia and the control group (data not shown). Taken together, these data suggest that sevoflurane anesthesia in pregnant mice may induce learning and memory impairment in offspring mice.
+
+Sevoflurane anesthesia in pregnant mice induced neurotoxicity in fetal mice
+
+Given that the sevoflurane anesthesia in pregnant mice can induce learning and memory impairment in offspring mice, we assessed the effects of sevoflurane anesthesia on the levels of proinflammatory cytokine IL-6, PSD-95, and caspase-3 activation, the neurotoxicity of which may represent underlying mechanisms of learning and memory impairment19–28. The pregnant mice received anesthesia with 2.5% sevoflurane for 2 h or the control condition at G14. We harvested the brain tissues of the fetal mice at the end of the experiment, and these tissues were subjected to Western blot analysis. Immunoblotting of IL-6 showed that the sevoflurane anesthesia induced more visible bands representing IL-6 as compared to the control condition (fig. 2A). There was no statistically significant difference in β-Actin levels between the control condition and the sevoflurane anesthesia. Quantification of the Western blot showed that the sevoflurane anesthesia increased IL-6 levels in the brain tissues of fetal mice as compared to the control condition: 256% ± 50.98 versus 100% ± 54.12, * P = 0.026 (fig. 2B).
+
+Next, we investigated the effects of the sevoflurane anesthesia in pregnant mice on levels of PSD-95, the marker of synapse, in the brain tissues of the fetal mice. Immunoblotting of PSD-95 showed that the sevoflurane anesthesia in pregnant mice produced less visible bands representing PSD-95 in the Western blot as compared to the control condition (fig. 2C). Quantification of the Western blot showed that the sevoflurane anesthesia in pregnant mice reduced PSD-95 levels in the brain tissues of fetal mice as compared to the control condition: 61% ± 13.53 versus 100% ± 10.08, * P = 0.036 (fig. 2D). Finally, we assessed effects of the sevoflurane anesthesia in pregnant mice on caspase-3 activation in the brain tissues of fetal mice. Caspase-3 immunoblotting showed that the sevoflurane anesthesia in pregnant mice increased levels of caspase-3 fragment without statistically significant changes in the levels of FL caspase-3 in the brain tissues of fetal mice (fig. 2E). The quantification of the Western blot, based on the ratio of caspase-3 fragment to FL-caspase-3, revealed that the sevoflurane anesthesia in pregnant mice induced caspase-3 activation as compared to control condition (fig. 2F): ** P = 0.0075, 198% ± 35 versus 100% ± 21.
+
+Taken together, these results suggest that anesthesia with 2.5% sevoflurane for 2 h in pregnant mice may induce neurotoxicity, including increases in proinflammatory cytokine levels, a reduction in synapse marker numbers, and caspase-3 activation in fetal mice, which may then lead to learning and memory impairment.
+
+Sevoflurane anesthesia in pregnant mice reduced synaptophysin levels in the hippocampus of offspring mice
+
+Given that sevoflurane anesthesia may cause acute neurotoxicity in fetal mice and learning and memory impairment in offspring mice at a later time, e.g., P31, we assessed the effects of the sevoflurane anesthesia on levels of IL-6 and synapse markers in the hippocampus of P31 mice. Immunohistochemistry analysis showed that the sevoflurane anesthesia reduced levels of synaptophysin, the synapse marker29, in the hippocampus of P31 mice (fig. 3A). Quantification of the immunohistochemistry image showed that the sevoflurane anesthesia decreased levels of synaptophysin: 77% ± 14.00 versus 100% ± 16.73, ** P = 0.0003 (fig. 3B). These results suggest that the sevoflurane anesthesia in pregnant mice may induce synaptic loss at a later time, e.g., P31, leading to learning and memory impairment.
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 6
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+The sevoflurane-induced reduction in PSD-95 level was dependent on the sevoflurane- induced increases in IL-6 level
+
+Given that the sevoflurane anesthesia increased IL-6 levels and decreased PSD-95 levels in brain tissues of fetal mice at G14, we then determined their potential association in mouse primary neurons. Treatment with 4.1% sevoflurane for 6 h reduced PSD-95 levels in mouse primary neurons as compared to the control condition (fig. 4A). The treatment with sevoflurane reduced PSD-95 levels as compared to the control condition, but IL-6 antibody mitigated the sevoflurane-induced reduction in PSD-95 levels, evidenced by more visible bands representing PSD-95 following the treatment of sevoflurane plus IL-6 antibody than following the treatment of sevoflurane plus saline (fig. 4A). Quantification of the Western blot showed that the sevoflurane treatment reduced PSD-95 levels (20% ± 4.58 vs. 100% ± 19, ** P = 0.001) and IL-6 antibody mitigated the sevoflurane anesthesia-induced reduction in PSD-95 levels: 36% ± 8.33 versus 20% ± 4.58, * P = 0.035 (fig. 4B). Two-way ANOVA indicated that there was an interaction between IL-6 antibody and sevoflurane, and that IL-6 antibody mitigated the sevoflurane-induced reduction in PSD-95 levels: ^^ P = 0.003 (fig. 4B). These results suggest that the sevoflurane-induced reduction in PSD-95 level may be dependent on the sevoflurane-induced increases in IL-6 level. Interestingly, IL-6 antibody also reduced PSD-95 levels in the primary neurons (fig. 4A and 4B).
+
+EE attenuated the sevoflurane anesthesia-induced learning and memory impairment in offspring mice
+
+EE has been shown to improve learning and memory30,31, and we therefore assessed whether EE can ameliorate the sevoflurane anesthesia-induced learning and memory impairment. Two-way ANOVA with repeated measurement analysis showed that there was a statistically significant interaction between time and group based on escape latency between mice following sevoflurane anesthesia plus standard environment (SE) and sevoflurane anesthesia plus EE, and EE mitigated the sevoflurane anesthesia-induced increases in escape latency of mice swimming in the MWM (^^ P = 0.0004, fig. 5A). Sevoflurane anesthesia plus EE also increased the platform crossing times of mice in the MWM as compared to sevoflurane anesthesia plus SE (** P = 0.003, Mann-Whitney test, fig. 5B, sevoflurane plus EE: median = 4 and IQR = 3.75 to 4.25 versus sevoflurane plus SE: median = 1 and IQR = 1 to 3). EE alone did not alter escape latency nor platform crossing times of mice swimming in the MWM (fig. 5C and 5D). Two-way ANOVA with repeated measurement analysis showed that there was no statistically significant interaction between time and group based on escape latency between mice following the control condition plus standard environment (SE) and control condition plus EE in the MWM (P = 0.345, fig. 5C), although there was a statistically significant group main effect based on escape latency between mice following the control condition plus SE and control condition plus EE (P = 0.009), figure 5C.
+
+Finally, the mice swimming speed in the MWM among all of these conditions were not different (data not shown). Taken together, these data suggest that EE may ameliorate the learning and memory impairment in the offspring mice that is caused by the sevoflurane anesthesia in the pregnant mice. These results are consistent with the findings that EE ameliorates cognitive deficits10,11.
+
+EE mitigated the sevoflurane-induced increase in IL-6 levels and reduction in levels of PSD-95 and synaptophysin in the brain tissues of offspring mice
+
+Given EE can ameliorate the sevoflurane anesthesia-induced learning and memory impairment, and synapse loss is the pathological finding closely associated with cognitive dysfunction and dementia24, we determined the effects of EE on the sevoflurane anesthesia- induced alterations of IL-6 and synapse marker PSD-95 and synaptophysin levels in the
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 7
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+Discussion
+
+brain tissues of offspring mice. IL-6 immunoblotting showed that sevoflurane anesthesia in pregnant mice increased IL-6 levels in the brain tissues of P31 offspring mice, and EE mitigated the effects (fig. 6A). The quantification of the Western blot illustrated that the sevoflurane anesthesia increased IL-6 levels: 250% ± 77 versus 100% ± 25, * P = 0.0032. EE mitigated the sevoflurane-induced increase in IL-6 levels: 89% ± 17 versus 250% ± 77, # P = 0.016 (fig. 6B). There was no statistically significant difference in IL-6 levels between the control condition plus SE and control condition plus EE (fig. 6C). Immunoblotting of PSD-95 showed that sevoflurane anesthesia in pregnant mice decreased PSD-95 levels in the brain tissues of P31 offspring mice, and EE mitigated the sevoflurane anesthesia-induced reduction in PSD-95 levels in the brain tissues of offspring mice examined at P31 (fig. 6D and 6E, ## P = 0.0046): 102 ± 3.23 (sevoflurane plus EE) versus 38% ± 19.39 (sevoflurane plus SE) versus 100% ± 20.6 (control plus SE). There was a higher level of PSD-95 in the control plus EE as compared to the control plus SE (fig. 6F). Immunohistochemistry staining showed that sevoflurane anesthesia in pregnant mice decreased synaptophysin levels in the brain tissues of P31 offspring mice as compared to the control condition (fig. 6G and 6H, ** P = 0.00003): 77% ± 17 versus 100% ± 21, EE mitigated the sevoflurane anesthesia-induced reduction in synaptophysin levels in the brain tissues of offspring mice at P31 (fig. 6G and 6H, ## P = 0.000001): 77% ± 17 (sevoflurane plus SE) versus 141% ± 36.44 (Sevoflurane plus EE). Collectively, these results suggest that EE may rescue the sevoflurane anesthesia- induced neuroinflammation and synaptic loss, leading to amelioration of the sevoflurane anesthesia-induced learning and memory impairment.
+
+The widespread and growing use of anesthesia in the developing brain makes its safety a major health issue of interest [1, reviewed in3]. This has become a matter of even greater concern with the evidence that anesthesia and surgery may induce neurodevelopment impairment in children, and that anesthetics are neurotoxic in young animals [reviewed in3]. Many pregnant women in the United States have nonobstetric surgery and fetal intervention procedures under anesthesia each year6,32. We therefore determined whether anesthesia with sevoflurane in pregnant mice could induce detrimental effects in fetal mice and offspring mice. We chose sevoflurane in the studies because sevoflurane is currently the most commonly used inhalation anesthetic, although sevoflurane might be less toxic than isoflurane33. Moreover, the effects of isoflurane in pregnant mice on behavioral changes in offspring mice have been determined7.
+
+Sevoflurane anesthesia in pregnant mice induced learning and memory impairment in offspring mice at P31 (fig. 1). The same sevoflurane anesthesia induced acute neurotoxicity as evidenced by the increased levels of proinflammatory cytokine IL-6, reduced levels of synapse marker PSD-95, and caspase-3 activation in the brain tissues of fetal mice (fig. 2). The sevoflurane anesthesia in pregnant mice also increased IL-6 levels, and decreased levels of PSD-95 and synaptophysin in the brain tissues of P31 offspring mice (fig. 6). Proinflammatory cytokine IL-6 can be released by the microglia cells during their activation, fueling neuroinflammation and leading to cognitive dysfunction34–36 and mild cognitive impairment (MCI)37 in medical and surgical patients38. PSD-95 is a postsynaptic marker39,40. The reduction of PSD-95 has been shown to be associated with decreases in synapse number or synaptic loss, a part of the mechanisms underlying AD-associated dementia and impairment of learning and memory [23,24, reviewed in25]. In the in vitro studies, IL-6 antibody attenuated the sevoflurane-induced reduction in PSD-95 levels, which suggests that the sevoflurane-induced increase in IL-6 levels may lead to reduction in PSD-95 levels. Taken together, these data suggest that sevoflurane may increase neuroinflammation, e.g., increase in IL-6 levels, which causes a reduction in synapse number, leading to learning and memory impairment. Future studies, including
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 8
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+determination of whether antiinflammation medicine(s) can rescue the sevoflurane anesthesia-induced synaptic loss and impairment of learning and memory, are warranted to further test this hypothesis.
+
+IL-6 antibody itself reduced PSD-95 levels in the primary neurons (fig. 4A and 4B). This could be due to IL-6 antibody only mitigating the effects associated with IL-6 accumulation, e.g., mitigating a reduction in PSD-95 levels. In the absence of IL-6 accumulation, however, the IL-6 antibody may have nonspecific effects. The exact mechanisms of these effects remain to be determined.
+
+The sevoflurane anesthesia induced caspase-3 activation, increases in IL-6 levels, and a reduction in PSD-95 levels 2 h after the anesthesia in the brain tissues of fetal mice, which occurred more rapidly than in the brain tissues of adult mice (6 h)9. These data suggest that fetal mice might be more vulnerable to neurotoxicity than adult mice.
+
+The mechanisms by which anesthetics induce neuroinflammation remain to be determined. Anesthetics have been shown to increase cytosolic calcium levels41–44. The elevation of cytosolic calcium is associated with increased levels of proinflammatory cytokines45, potentially through activation of nuclear factor-κB signaling pathway46–49. Activated nuclear factor-κB translocates to the nucleus where it binds to the promoter region of multiple genes, including cytokine genes46–50. Thus, the future studies will include determining whether anesthetics can increase calcium levels in neurons and microglia cells to trigger generation of proinflammatory cytokine, e.g., IL-6, through nuclear factor-κB signaling pathway.
+
+EE, consisting of social interaction and novel stimulation, may result in various neuroplastic changes, including increased hippocampal neurons51, improved spatial abilities and enhanced dendritic growth52, increased neurogenesis53, and increased nerve growth factor54 after brain injury. EE has also been shown to improve learning and memory function10–12. We found that EE ameliorated the sevoflurane anesthesia-induced learning and memory impairment, and mitigated the sevoflurane anesthesia-induced increase in IL-6 levels and reduction in synaptic markers (fig. 5 and 6). These results suggest that EE may rescue the sevoflurane anesthesia-induced neuroinflammation and synaptic loss, leading to improvement of the sevoflurane anesthesia-induced impairment of learning and memory.
+
+The studies have several limitations. First, we did not determine the long-term (e.g., 3 to 6 months) effects of sevoflurane anesthesia on learning and memory function, however, the current findings were able to illustrate the effects of sevoflurane anesthesia on behavioral changes (e.g., spatial learning and memory impairment) and the potential underlying cellular mechanisms (e.g., caspase activation, increases in IL-6 levels and synaptic loss). Second, we only focused on one proinflammatory cytokine, IL-6, in the experiments because IL-6 has been shown to contribute to learning and memory impairment. Sevoflurane anesthesia in pregnant mice may also induce other changes (e.g., microglia activation) in the brain tissues of fetal mice consistent with neuroinflammation, which need to be investigated in future studies.
+
+It is unknown whether the anesthesia itself contributes to the clinically observed cognitive impairment, or the need for anesthesia/surgery is a marker for other unidentified factors that contribute. In order to either rule in or rule out the contribution of anesthesia, we will determine whether anesthesia alone can induce neuroinflammation and learning and memory in young mice. Our established preclinical mouse model will be used to determine whether anesthetic alone can induce detrimental effects (e.g., learning and memory impairment, and neuroinflammation) in young animals (developing brain), to reveal the underlying mechanisms, and to explore targeted interventions. Moreover, nociceptive
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 9
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+stimuli such as surgical incision and pain with formalin have been shown to potentiate the anesthetic-induced neurotoxicity and neurobehavioral deficits55. The future studies may also include assessing whether other perioperative factors, e.g., hypothermia and hypotension, can potentiate anesthesia-induced neurotoxicity and neurobehavioral deficits.
+
+In conclusion, clinically relevant sevoflurane anesthesia in pregnant mice can induce acute neurotoxicity, including increases in IL-6 levels, reductions in synapse marker PSD-95 and caspase-3 activation, in the brain tissues of fetal mice. The same sevoflurane anesthesia in pregnant mice also induced long-term detrimental effects, including reductions in synapse marker PSD-95 and synaptophysin, and impairment of learning and memory in offspring mice at 31 days after the birth. These results suggest that sevoflurane anesthesia in pregnant mice may induce neuroinflammation, caspase activation and synaptic loss, leading to learning and memory impairment. Finally, EE may be able to rescue the sevoflurane anesthesia-induced learning and memory impairment by mitigating the sevoflurane anesthesia-induced synaptic loss and neuroinflammation. These findings will promote more research in anesthesia neurotoxicity in the developing brain, especially mechanistic studies.
+
+Acknowledgments
+
+Funding: This research was supported by R21AG029856, R21AG038994 and R01 GM088801 from National Institutes of Health, Bethesda, Maryland, and from Cure Alzheimer’s Fund, Wellesley, Massachusetts to Zhongcong Xie. The cost of anesthetic sevoflurane was generously provided by the Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts.
+
+The authors thank Hui Zheng, Ph.D. (Assistant Professor of Medicine, Biostatistics Center, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts) for the advice and help in the data analysis of the studies.
+
+Reference
+
+1. Rappaport B, Mellon RD, Simone A, Woodcock J. Defining safe use of anesthesia in children. N Engl J Med. 2011; 364:1387–1390. [PubMed: 21388302]
+
+2. Belluck P. F.D.A to Study Whether Anesthesia Poses Cognitive Risks in Young Children. The New Yorker Time. 2011 Mar 9. Science.
+
+3. Sun L. Early childhood general anaesthesia exposure and neurocognitive development. Br J Anaesth. 2010; 105(Suppl 1):i61–i68. [PubMed: 21148656]
+
+4. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci. 2003; 23:876–882. [PubMed: 12574416]
+
+5. Creeley CE, Olney JW. The young: Neuroapoptosis induced by anesthetics and what to do about it. Anesth Analg. 2010; 110:442–448. [PubMed: 19955510]
+
+6. Kuczkowski KM. Nonobstetric surgery during pregnancy: What are the risks of anesthesia? Obstet Gynecol Surv. 2004; 59:52–56. [PubMed: 14707749]
+
+7. Palanisamy A, Baxter MG, Keel PK, Xie Z, Crosby G, Culley DJ. Rats exposed to isoflurane in utero during early gestation are behaviorally abnormal as adults. Anesthesiology. 2011; 114:521– 528. [PubMed: 21307768]
+
+8. Reitman E, Flood P. Anaesthetic considerations for non-obstetric surgery during pregnancy. Br J Anaesth. 2011; 107(Suppl 1):i72–i78. [PubMed: 22156272]
+
+9. Dong Y, Zhang G, Zhang B, Moir RD, Xia W, Marcantonio ER, Culley DJ, Crosby G, Tanzi RE, Xie Z. The common inhalational anesthetic sevoflurane induces apoptosis and increases beta- amyloid protein levels. Arch Neurol. 2009; 66:620–631. [PubMed: 19433662]
+
+10. Hoffman AN, Malena RR, Westergom BP, Luthra P, Cheng JP, Aslam HA, Zafonte RD, Kline AE. Environmental enrichment-mediated functional improvement after experimental traumatic
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 10
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+brain injury is contingent on task-specific neurobehavioral experience. Neurosci Lett. 2008; 431:226–230. [PubMed: 18162321]
+
+11. Kline AE, Wagner AK, Westergom BP, Malena RR, Zafonte RD, Olsen AS, Sozda CN, Luthra P, Panda M, Cheng JP, Aslam HA. Acute treatment with the 5-HT(1A) receptor agonist 8-OH-DPAT and chronic environmental enrichment confer neurobehavioral benefit after experimental brain trauma. Behav Brain Res. 2007; 177:186–194. [PubMed: 17166603]
+
+12. Sozda CN, Hoffman AN, Olsen AS, Cheng JP, Zafonte RD, Kline AE. Empirical comparison of typical and atypical environmental enrichment paradigms on functional and histological outcome after experimental traumatic brain injury. J Neurotrauma. 2010; 27:1047–1057. [PubMed: 20334496]
+
+13. Zhen Y, Dong Y, Wu X, Xu Z, Lu Y, Zhang Y, Norton D, Tian M, Li S, Xie Z. Nitrous oxide plus isoflurane induces apoptosis and increases beta-amyloid protein levels. Anesthesiology. 2009; 111:741–752. [PubMed: 19741497]
+
+14. Zhang Y, Dong Y, Wu X, Lu Y, Xu Z, Knapp A, Yue Y, Xu T, Xie Z. The mitochondrial pathway of anesthetic isoflurane-induced apoptosis. J Biol Chem. 2010; 285:4025–4037. [PubMed: 20007710]
+
+15. Zhang Y, Xu Z, Wang H, Dong Y, Shi HN, Culley DJ, Crosby G, Marcantonio ER, Tanzi RE, Xie Z. Anesthetics isoflurane and desflurane differently affect mitochondrial function, learning, and memory. Ann Neurol. 2012; 71:687–698. [PubMed: 22368036]
+
+16. Xie Z, Culley DJ, Dong Y, Zhang G, Zhang B, Moir RD, Frosch MP, Crosby G, Tanzi RE. The common inhalation anesthetic isoflurane induces caspase activation and increases amyloid beta- protein level in vivo. Ann Neurol. 2008; 64:618–627. [PubMed: 19006075]
+
+17. Wu X, Lu Y, Dong Y, Zhang G, Zhang Y, Xu Z, Culley DJ, Crosby G, Marcantonio ER, Tanzi RE, Xie Z. The inhalation anesthetic isoflurane increases levels of proinflammatory TNF-alpha, IL-6, and IL-1beta. Neurobiol Aging. 2012; 33:1364–1378. [PubMed: 21190757]
+
+18. Bianchi SL, Tran T, Liu C, Lin S, Li Y, Keller JM, Eckenhoff RG, Eckenhoff MF. Brain and behavior changes in 12-month-old Tg2576 and nontransgenic mice exposed to anesthetics. Neurobiol Aging. 2008; 29:1002–1010. [PubMed: 17346857]
+
+19. Wan Y, Xu J, Ma D, Zeng Y, Cibelli M, Maze M. Postoperative impairment of cognitive function in rats: A possible role for cytokine-mediated inflammation in the hippocampus. Anesthesiology. 2007; 106:436–443. [PubMed: 17325501]
+
+20. Reichenberg A, Yirmiya R, Schuld A, Kraus T, Haack M, Morag A, Pollmacher T. Cytokine- associated emotional and cognitive disturbances in humans. Arch Gen Psychiatry. 2001; 58:445– 452. [PubMed: 11343523]
+
+21. Sparkman NL, Buchanan JB, Heyen JR, Chen J, Beverly JL, Johnson RW. Interleukin-6 facilitates lipopolysaccharide-induced disruption in working memory and expression of other proinflammatory cytokines in hippocampal neuronal cell layers. J Neurosci. 2006; 26:10709– 10716. [PubMed: 17050710]
+
+22. Weaver JD, Huang MH, Albert M, Harris T, Rowe JW, Seeman TE. Interleukin-6 and risk of cognitive decline: MacArthur studies of successful aging. Neurology. 2002; 59:371–378. [PubMed: 12177370]
+
+23. Hongpaisan J, Sun MK, Alkon DL. PKC ε activation prevents synaptic loss, Aβ elevation, and cognitive deficits in alzheimer's disease transgenic mice. J Neurosci. 2011; 31:630–643. [PubMed: 21228172]
+
+24. Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Katzman R. Physical basis of cognitive alterations in Alzheimer's disease: Synapse loss is the major correlate of cognitive impairment. Ann Neurol. 1991; 30:572–580. [PubMed: 1789684]
+
+25. Querfurth HW, LaFerla FM. Alzheimer's disease. N Engl J Med. 2010; 362:329–344. [PubMed: 20107219]
+
+26. Burguillos MA, Deierborg T, Kavanagh E, Persson A, Hajji N, Garcia-Quintanilla A, Cano J, Brundin P, Englund E, Venero JL, Joseph B. Caspase signalling controls microglia activation and neurotoxicity. Nature. 2011; 472:319–324. [PubMed: 21389984]
+
+27. Wan Y, Xu J, Meng F, Bao Y, Ge Y, Lobo N, Vizcaychipi MP, Zhang D, Gentleman SM, Maze M, Ma D. Cognitive decline following major surgery is associated with gliosis, β-amyloid
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 11
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+accumulation, and tau phosphorylation in old mice. Crit Care Med. 2010; 38:2190–2198. [PubMed: 20711073]
+
+28. Terrando N, Monaco C, Ma D, Foxwell BM, Feldmann M, Maze M. Tumor necrosis factor-α triggers a cytokine cascade yielding postoperative cognitive decline. Proc Natl Acad Sci U S A. 2010; 107:20518–20522. [PubMed: 21041647]
+
+29. Calhoun ME, Jucker M, Martin LJ, Thinakaran G, Price DL, Mouton PR. Comparative evaluation of synaptophysin-based methods for quantification of synapses. J Neurocytol. 1996; 25:821–828. [PubMed: 9023727]
+
+30. Bouet V, Freret T, Dutar P, Billard JM, Boulouard M. Continuous enriched environment improves learning and memory in adult NMRI mice through theta burst-related-LTP independent mechanisms but is not efficient in advanced aged animals. Mech Ageing Dev. 2011; 132:240–248. [PubMed: 21530571]
+
+31. Veena J, Srikumar BN, Mahati K, Bhagya V, Raju TR, Shankaranarayana Rao BS. Enriched environment restores hippocampal cell proliferation and ameliorates cognitive deficits in chronically stressed rats. J Neurosci Res. 2009; 87:831–843. [PubMed: 19006089]
+
+32. Van De Velde M, De Buck F. Anesthesia for non-obstetric surgery in the pregnant patient. Minerva Anestesiol. 2007; 73:235–240. [PubMed: 17473818]
+
+33. Liang G, Ward C, Peng J, Zhao Y, Huang B, Wei H. Isoflurane causes greater neurodegeneration than an equivalent exposure of sevoflurane in the developing brain of neonatal mice. Anesthesiology. 2010; 112:1325–1334. [PubMed: 20460994]
+
+34. Teeling JL, Perry VH. Systemic infection and inflammation in acute CNS injury and chronic neurodegeneration: Underlying mechanisms. Neuroscience. 2009; 158:1062–1073. [PubMed: 18706982]
+
+35. van Gool WA, van de Beek D, Eikelenboom P. Systemic infection and delirium: When cytokines and acetylcholine collide. Lancet. 2010; 375:773–775. [PubMed: 20189029]
+
+36. Willard LB, Hauss-Wegrzyniak B, Wenk GL. Pathological and biochemical consequences of acute and chronic neuroinflammation within the basal forebrain cholinergic system of rats. Neuroscience. 1999; 88:193–200. [PubMed: 10051200]
+
+37. Schuitemaker A, Dik MG, Veerhuis R, Scheltens P, Schoonenboom NS, Hack CE, Blankenstein MA, Jonker C. Inflammatory markers in AD and MCI patients with different biomarker profiles. Neurobiol Aging. 2009; 30:1885–1889. [PubMed: 18378357]
+
+38. Hudetz JA, Gandhi SD, Iqbal Z, Patterson KM, Pagel PS. Elevated postoperative inflammatory biomarkers are associated with short- and medium-term cognitive dysfunction after coronary artery surgery. J Anesth. 2011; 25:1–9. [PubMed: 21061037]
+
+39. Takeuchi M, Hata Y, Hirao K, Toyoda A, Irie M, Takai Y. SAPAPs. A family of PSD-95/SAP90- associated proteins localized at postsynaptic density. J Biol Chem. 1997; 272:11943–11951. [PubMed: 9115257]
+
+40. Liu Q, Trotter J, Zhang J, Peters MM, Cheng H, Bao J, Han X, Weeber EJ, Bu G. Neuronal LRP1 knockout in adult mice leads to impaired brain lipid metabolism and progressive, age-dependent synapse loss and neurodegeneration. J Neurosci. 2010; 30:17068–17078. [PubMed: 21159977] 41. Zhang G, Dong Y, Zhang B, Ichinose F, Wu X, Culley DJ, Crosby G, Tanzi RE, Xie Z. Isoflurane-
+
+induced caspase-3 activation is dependent on cytosolic calcium and can be attenuated by memantine. J Neurosci. 2008; 28:4551–4560. [PubMed: 18434534]
+
+42. Zhang J, Dong Y, Xu Z, Zhang Y, Pan C, McAuliffe S, Ichinose F, Yue Y, Liang W, Xie Z. 2- deoxy-D-glucose attenuates isoflurane-induced cytotoxicity in an in vitro cell culture model of H4 human neuroglioma cells. Anesth Analg. 2011; 113:1468–1475. [PubMed: 21965367]
+
+43. Wei H, Liang G, Yang H, Wang Q, Hawkins B, Madesh M, Wang S, Eckenhoff RG. The common inhalational anesthetic isoflurane induces apoptosis via activation of inositol 1,4,5-trisphosphate receptors. Anesthesiology. 2008; 108:251–260. [PubMed: 18212570]
+
+44. Yang H, Liang G, Hawkins BJ, Madesh M, Pierwola A, Wei H. Inhalational anesthetics induce cell damage by disruption of intracellular calcium homeostasis with different potencies. Anesthesiology. 2008; 109:243–250. [PubMed: 18648233]
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 12
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+45. Kim D, Cho SH, Kim JS, Jo SH, Lee SJ, Kim KT, Choi SY. Human astrocytic bradykinin B(2) receptor modulates zymosan-induced cytokine expression in 1321N1 cells. Peptides. 2010; 31:101–107. [PubMed: 19854233]
+
+46. Meffert MK, Chang JM, Wiltgen BJ, Fanselow MS, Baltimore D. NF-κB functions in synaptic signaling and behavior. Nat Neurosci. 2003; 6:1072–1078. [PubMed: 12947408]
+
+47. Vexler ZS, Yenari MA. Does inflammation after stroke affect the developing brain differently than adult brain? Dev Neurosci. 2009; 31:378–393. [PubMed: 19672067]
+
+48. Baeuerle PA, Henkel T. Function and activation of NF-κB in the immune system. Annu Rev Immunol. 1994; 12:141–179. [PubMed: 8011280]
+
+49. Schneider A, Martin-Villalba A, Weih F, Vogel J, Wirth T, Schwaninger M. NF-κB is activated and promotes cell death in focal cerebral ischemia. Nat Med. 1999; 5:554–559. [PubMed: 10229233]
+
+50. Neumann M, Naumann M. Beyond IkappaBs: Alternative regulation of NF-κB activity. FASEB J. 2007; 21:2642–2654. [PubMed: 17431096]
+
+51. Kempermann G, Kuhn HG, Gage FH. More hippocampal neurons in adult mice living in an enriched environment. Nature. 1997; 386:493–495. [PubMed: 9087407]
+
+52. Leggio MG, Mandolesi L, Federico F, Spirito F, Ricci B, Gelfo F, Petrosini L. Environmental enrichment promotes improved spatial abilities and enhanced dendritic growth in the rat. Behav Brain Res. 2005; 163:78–90. [PubMed: 15913801]
+
+53. Olson AK, Eadie BD, Ernst C, Christie BR. Environmental enrichment and voluntary exercise massively increase neurogenesis in the adult hippocampus via dissociable pathways. Hippocampus. 2006; 16:250–260. [PubMed: 16411242]
+
+54. Torasdotter M, Metsis M, Henriksson BG, Winblad B, Mohammed AH. Environmental enrichment results in higher levels of nerve growth factor mRNA in the rat visual cortex and hippocampus. Behav Brain Res. 1998; 93:83–90. [PubMed: 9659990]
+
+55. Shu Y, Zhou Z, Wan Y, Sanders RD, Li M, Pac-Soo CK, Maze M, Ma D. Nociceptive stimuli enhance anesthetic-induced neuroapoptosis in the rat developing brain. Neurobiol Dis. 2012; 45:743–750. [PubMed: 22075165]
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 13
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+Final Boxed Summary Statement
+
+What we know about this topic
+
+The effects of maternal exposure to sevoflurane to fetal neurotoxicity and neurobehavioral outcome are controversial
+
+What new information this study provides
+
+Sevoflurane may induce detrimental effects in fetal and offspring mice, which can be mitigated by environmental enrichment.
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 14
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+Figure 1. Anesthesia with 2.5% sevoflurane for two hours in pregnant mice at gestation stage day 14 (G14) induces learning and memory impairment in offspring mice tested at postnatal day (P)31 A. Sevoflurane anesthesia increases escape latency time of mice swimming in the Morris Water Maze (MWM) as compared to the control condition. Two way ANOVA with repeated measurement analysis shows that there is statistically significant interaction between time and group based on escape latency between mice following the control condition and mice following the sevoflurane anesthesia in the MWM (^ P = 0.012). * indicates that there is a statistically significant difference in the escape latency between the control group and the sevoflurane group. B. Sevoflurane anesthesia reduces the platform
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 15
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+crossing times of mice swimming in the MWM as compared to the control condition (P = 0.051, Mann-Whitney test, median = 1 and IQR = 1 to 3 versus control: median = 2 and IQR = 2 to 4.5). G, gestation stage day; P, postnatal day; MWM, Morris Water Maze; ANOVA, analysis of variance; IQR, interquartile range. n = 15.
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 16
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+Figure 2. Anesthesia with 2.5% sevoflurane for two hours in pregnant mice at G14 increases IL-6 levels, decreases PSD-95 levels and induce caspase-3 activation in the brain tissues of fetal mice A. Sevoflurane anesthesia increases IL-6 levels in the brain tissues of fetal mice as compared to the control condition in Western blot analysis. There is no statistically significant difference in the amounts of β-Actin in the mouse brain tissues following the sevoflurane anesthesia or control condition. B. Quantification of the Western blot shows that sevoflurane anesthesia increases IL-6 levels in the mouse brain tissues as compared to the control condition (* P = 0.026). C. The sevoflurane anesthesia reduces PSD-95 levels in the brain tissues of fetal mice as compared to the control condition in Western blot analysis.
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 17
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+There is no statistically significant difference in the amounts of β-Actin in the mouse brain tissues following the sevoflurane anesthesia or control condition. D. Quantification of the Western blot shows that sevoflurane anesthesia reduces PSD-95 levels in the mouse brain tissues as compared to the control condition (* P = 0.036). E. The sevoflurane anesthesia induces caspase-3 activation in the brain tissues of fetal mice as compared to the control condition in Western blot analysis. There is no statistically significant difference in the amounts of β-Actin in the mouse brain tissues following the sevoflurane anesthesia or control condition. F. Quantification of the Western blot shows that sevoflurane anesthesia induces caspase-3 activation in the mouse brain tissues as compared to the control condition (** P = 0.0075). G, gestation stage day; IL-6, interleukin-6; PSD, postsynaptic density-95; FL, full length. n = 6.
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 18
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+Figure 3. Anesthesia with 2.5% sevoflurane for two hours in pregnant mice at G14 decreases synaptophysin levels in the hippocampus of offspring mice examined at P31 A. Sevoflurane anesthesia decreases synaptophysin levels in the brain tissues of offspring mice as compared to the control condition in immunohistochemistry analysis. B. Quantification of the immunohistochemistry image shows that sevoflurane anesthesia decreases synaptophysin levels in the mouse brain tissues as compared to the control condition (** P = 0.0003). G, gestation stage day; P, postnatal day. n = 6.
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 19
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+Figure 4. IL-6 antibody mitigates the sevoflurane-induced reduction in PSD-95 levels in mouse primary neurons A. Treatment with 4.1% sevoflurane for six hours (lanes 7 to 9) reduces PSD-95 levels as compared to the control condition (lanes 1 to 3). The treatment of IL-6 antibody (lanes 10 – 12) mitigates the sevoflurane-induced reduction in PSD-95 levels. There is no statistically significant difference in the amounts of β-Actin in the mouse primary neurons following the treatments of sevoflurane, IL-6 antibody or control condition. B. Quantification of the Western blot shows that sevoflurane treatment decreases PSD-95 levels as compared to the control condition (** P = 0.001). Treatment with sevoflurane plus IL-6 antibody leads to a lesser degree of reduction in PSD-95 levels as compared to treatment with sevoflurane plus
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 20
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+saline (# P = 0.035). Two-way ANOVA shows that there is an interaction of IL-6 antibody and sevoflurane, and that IL-6 antibody mitigates the sevoflurane-induced reduction in PSD-95 levels (^^ P = 0.003). IL-6, interleukin-6; PSD-95, postsynaptic density-95; ANOVA, analysis of variance. n = 6.
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 21
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+Figure 5. Environmental enrichment (EE) attenuates the sevoflurane-induced learning and memory impairment in offspring mice A. Two way ANOVA with repeated measurement analysis shows that there is a statistically significant interaction between time and group based on escape latency between mice following sevoflurane anesthesia plus SE and sevoflurane anesthesia plus EE (^^ P = 0.0004). * indicates that there is a statistically significant difference in the escape latency between the sevoflurane plus SE group and the sevoflurane plus EE group. B. Mann- Whitney test shows that the platform crossing time of mice swimming in the MWM following the sevoflurane anesthesia plus EE is longer than that of mice following the sevoflurane anesthesia plus SE (** P = 0.003, sevoflurane plus EE: median = 4 and IQR = 3.75 to 4.25 versus sevoflurane plus SE: median = 1 and IQR = 1 to 3). C. ANOVA shows that there is no statistically significant interaction between time and group based on escape latency of mice swimming in the MWM between the control condition plus SE and the control condition plus EE (P = 0.345, N.S.). D. Mann-Whitney test shows that there is no statistically significant difference in platform crossing time of mice swimming in the MWM between the control condition plus SE and the control condition plus EE (P = 0.499, N.S., control condition plus EE: median = 3 and IQR = 2 to 4 versus control condition plus SE: median = 2 and IQR = 2 to 4.25). MWM, Morris Water Maze; ANOVA, analysis of variance; IQR, interquartile range; SE, standard environment; EE, environmental enrichment. n = 15.
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 22
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+Figure 6. EE mitigates the sevoflurane-induced increase in IL-6 levels, and reduction in levels of PSD-95 and synaptophysin in mouse brain tissues A. Sevoflurane anesthesia plus SE increases IL-6 levels as compared to the control condition plus SE. Sevoflurane anesthesia plus EE leads to lower levels of IL-6 as compared to the sevoflurane anesthesia plus SE. There is no statistically significant difference in β-Actin levels among the above treatments B. Quantification of the Western blot shows that the sevoflurane anesthesia plus SE increases IL-6 levels as compared to the control condition (* P = 0.032), and EE mitigates the sevoflurane anesthesia-induced increase in IL-6 levels (## P = 0.016). C. There is no statistically significant difference in IL-6 levels between control plus EE and control plus SE. D. Sevoflurane anesthesia plus SE reduces PSD-95 levels as compared to the control condition plus SE. Sevoflurane anesthesia plus EE leads to higher levels of PSD-95 as compared to the sevoflurane anesthesia plus SE. There is no statistically significant difference in β-Actin levels among the above treatments E. Quantification of the Western blot shows that the sevoflurane anesthesia plus SE reduces PSD-95 levels as compared to the control condition (* P = 0.019), and EE mitigates the sevoflurane anesthesia-induced reduction in PSD-95 levels (## P = 0.0046). F. The PSD-95 level increases following control plus EE as compared to control plus SE. G. Sevoflurane anesthesia plus SE leads to a reduction in synaptophysin levels in the brain tissues of offspring mice as compared to the control condition in the immunohistochemistry analysis. EE mitigates the sevoflurane anesthesia-induced reduction in synaptophysin levels. The
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 23
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Zheng et al.
+
+synaptophysin level increases following control plus EE as compared to control plus SE. H. Quantification of the immunohistochemistry image shows that sevoflurane anesthesia plus SE leads to a reduction in synaptophysin levels in the brain tissues of offspring mice as compared to the control condition plus SE (black bar, **P = 0.00003). Both EE plus control condition (net bar, ** P = 0.0013) or sevoflurane (gray bar, ** P = 0.000001) cause higher synaptophysin levels in the brain tissues of offspring mice as compared to the control condition (white bar). Finally, there is an interaction between EE and sevoflurane anesthesia that EE mitigates the sevoflurane anesthesia-induced reduction in synaptophysin levels in the hippocampus of offspring mice (^^ P = 0.00005). PSD-95, postsynaptic density-95; IL-6, interleukin-6; SE, standard environment; EE, environmental enrichment. n = 6.
+
+Anesthesiology. Author manuscript; available in PMC 2014 March 01.
+
+Page 24

new_pdfs/10.1097_ALN.0b013e318289bc9b.txt

@@ -0,0 +1,925 @@
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+NIH Public Access Author Manuscript Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Published in final edited form as:
+
+Anesthesiology. 2013 May ; 118(5): . doi:10.1097/ALN.0b013e318289bc9b.
+
+Early Exposure to General Anesthesia Disturbs Mitochondrial Fission and Fusion in the Developing Rat Brain
+
+Annalisa Boscolo, M.D.*, Desanka Milanovic, Ph.D.†, John A. Starr, B.S.‡, Victoria Sanchez, B.S.§, Azra Oklopcic, B.S.||, Laurie Moy#, C Carlo Ori, M.D.**, Alev Erisir, M.D., Ph.D.††, and Vesna Jevtovic-Todorovic, M.D., Ph.D.‡‡ *Research Associate, Department of Anesthesiology, University of Virginia Heath System, Charlottesville, Virginia, and Department of Anesthesiology and Pharmacology, University of Padua, Padua, Italy
+
+†Research Associate, Department of Anesthesiology, University of Virginia Heath System, and The Institute for Biological Research “Sinisa Stankovic,” University of Belgrade, Belgrade, Serbia
+
+‡Medical Student, Department of Anesthesiology, University of Virginia Heath System
+
+§Graduate Student, Department of Anesthesiology, University of Virginia Heath System, and Neuroscience Graduate Program, University of Virginia, Charlottesville, Virginia
+
+||Technician, Department of Anesthesiology, University of Virginia Heath System
+
+#Undergraduate Student, Department of Anesthesiology, University of Virginia Heath System
+
+**Professor, Department of Anesthesiology and Pharmacology, University of Padua
+
+††Associate Professor, Neuroscience Graduate Program, University of Virginia, and Department of Psychology, University of Virginia
+
+‡‡Professor, Department of Anesthesiology, University of Virginia Heath System, and Department of Psychology, University of Virginia
+
+Abstract
+
+Background—General anesthetics induce apoptotic neurodegeneration in the developing mammalian brain. General anesthesia (GA) also causes significant disturbances in mitochondrial morphogenesis during intense synaptogenesis. Mitochondria are dynamic organelles that undergo remodeling via fusion and fission. The fine balance between these two opposing processes determines mitochondrial morphometric properties, allowing for their regeneration and enabling normal functioning. As mitochondria are exquisitely sensitive to anesthesia-induced damage, we examined how GA affects mitochondrial fusion/fission.
+
+Methods—Seven-day-old rat pups received anesthesia containing a sedative dose of midazolam followed by a combined nitrous oxide and isoflurane anesthesia for 6 h.
+
+Results—GA causes 30% upregulation of reactive oxygen species (n = 3–5 pups/group), accompanied by a 2-fold downregulation of an important scavenging enzyme, superoxide dismutase (n = 6 pups/group). Reactive oxygen species upregulation is associated with impaired mitochondrial fission/fusion balance, leading to excessive mitochondrial fission. The imbalance
+
+Copyright © 2013, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins. Address correspondence to: Dr. Jevtovic-Todorovic: Department of Anesthesiology, University of Virginia Health System, PO Box 800710, Charlottesville, Virginia 22908. vj3w@virginia.edu. Information on purchasing reprints may be found at www.anesthesiology.org or on the masthead page at the beginning of this issue. Anesthesiology’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+between fission and fusion is due to acute sequestration of the main fission protein, dynamin- related protein 1, from the cytoplasm to mitochondria, and its oligomerization on the outer mitochondrial membrane. These are necessary steps in the formation of the ring-like structures that are required for mitochondrial fission. The fission is further promoted by GA-induced 40% downregulation of cytosolic mitofusin-2, a protein necessary for maintaining the opposing process, mitochondrial fusion (n = 6 pups/group).
+
+Conclusions—Early exposure to GA causes acute reactive oxygen species upregulation and disturbs the fine balance between mitochondrial fission and fusion, leading to excessive fission and disturbed mitochondrial morphogenesis. These effects may play a causal role in GA-induced developmental neuroapoptosis.
+
+Recent animal and emerging human data suggest that general anesthetics commonly used in pediatric medicine could be damaging to the developing nervous system. The neurotoxic effects are described as apoptotic in nature1–4 and are accompanied by severe and long- lasting disturbances in synaptogenesis.5–8 It appears that the impairment of synaptic development involves not only deletion of the existing synapses, but also a disturbance in the formation of novel synapses.9
+
+Proper morphogenesis, function, and regional distribution of mitochondria are crucially important in the development and function of immature synapses and, consequently, for the formation of functional brain circuitries. Our recent studies indicate that general anesthesia (GA) causes statistically significant decrease in synapses, and disturbances in mitochondrial morphogenesis in the vicinity of synaptic connections, thus pointing at mitochondria as organelles likely to be responsible for anesthesia-induced impairment of neuronal development and synaptic function.10 Furthermore, we previously reported that the general anesthetic isoflurane, when combined with midazolam and nitrous oxide, causes apoptotic neurodegeneration that is, in part, mitochondria dependant.4 These findings collectively suggest that mitochondria could be an important and early target for GA-induced impairment of neuronal development and synaptogenesis.
+
+Mitochondria are highly dynamic. Their ability to provide adequate support to the developing neurons relies on constant remodeling via fusion and fission.11 A fine dynamic balance between these two opposing processes depends on the physiological and metabolic requirements of a neuron. Overactive fission leads to mitochondrial fragmentation, whereas overactive fusion leads to undue mitochondrial enlargement. Both phenomena may cause impaired mitochondrial function.
+
+Fusion and fission in mammalian neurons are controlled by many proteins. A protein of particular interest in the control of fission is an important member of the dynamin superfamily of proteins, dynamin-related protein 1 (Drp-1), which mediates the remodeling of the inner and outer mitochondrial membranes.12,13 Drp-1 translocates to the mitochondrial outer membrane and polymerizes to form a ring-like structure that enables mitochondrial division. A protein of particular interest in the control of fusion is mitofusin-2 (Mfn-2), a member of the Mfn family of proteins.11 Mfn-2 stabilizes the interaction between two adjacent mitochondria. 14 Interestingly, Mfn-2 also controls mitochondrial oxidative metabolism and the redox state of a neuron,15 a function that was of interest in view of our recently published findings, suggesting that GA causes upregulation of reactive oxygen species (ROS).16
+
+We examined the acute in vivo effects of GA on the dynamic balance between mitochondrial fission and fusion, two key processes in mitochondrial proliferation, regeneration, and function. We administered a routine anesthesia cocktail containing isoflurane, nitrous oxide, and midazolam to rats during the intense stage of their brain development (at postnatal day
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 2
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+[P] 7). We confirmed that acute anesthesia exposure results in an imbalance of ROS homeostasis, caused, in part, by modulation of the function of scavenging enzymes. We also discovered that GA causes downregulation of the fusion protein Mfn-2 and translocation of a fission protein, Drp-1, from cytoplasm to mitochondria, followed by the enhancement of Drp-1 oligomerization, ultimately leading to excessive mitochondrial fission.
+
+Materials and Methods
+
+Animals
+
+Sprague–Dawley rat pups (Harlan Laboratories, Indianapolis, IN) at P7 were used for all experiments. This postnatal day is when rat pups are most vulnerable to anesthesia-induced neuronal damage.4 Our routine anesthesia protocol was used as previously described.10 Briefly, experimental rat pups were exposed to 6 h of anesthesia and controls were exposed to 6 h of mock anesthesia (vehicle + air). After the administration of anesthesia, rats were randomly divided into three groups: one group for ultrastructural analysis of the subiculum using electron microscopy, one group for assessing expression of several proteins using the Western blotting technique, and one group for functional studies of superoxide dismutase (SOD) and catalase activity using ELISA. Rat pups assigned for histological studies were reunited with their mothers and killed 24 h postanesthesia (at P8). Rat pups assigned for Western blot and ELISA studies were killed immediately postanesthesia (at P7).
+
+The experiments were approved by the Animal Use and Care Committee of the University of Virginia Health System, Charlottesville, Virginia, and were performed in accordance with the Public Health Service’s Policy on Human Care and Use of Laboratory Animals. Efforts were made to minimize the number of animals used.
+
+Anesthesia
+
+We used our routine anesthesia protocol as previously described.10,16 Briefly, nitrous oxide and oxygen were delivered using a calibrated flowmeter. Isoflurane was administered using an agent-specific vaporizer that delivers a set percentage of anesthetic into the anesthesia chamber. Midazolam (Sigma–Aldrich Chemical, St. Louis, MO) was dissolved in 0.1% dimethyl sulfoxide just before administration. For control animals, 0.1% dimethyl sulfoxide was used alone. To administer a specific concentration of nitrous oxide/oxygen and isoflurane in a highly controlled environment, an anesthesia chamber was used. Rats were kept normothermic and normoxic while glucose homeostasis was maintained within normal limits throughout the experiment, as previously described.17,18 For control experiments, air was substituted for the gas mixture. After initial equilibration of the nitrous oxide/oxygen/ isoflurane or air atmosphere inside the chamber, the composition of the chamber gas was analyzed by infrared analyzer (Datex Ohmeda, Madison, WI) to establish the concentrations of nitrous oxide, isoflurane, carbon dioxide, and oxygen. P7 rat pups received a single injection of midazolam (9 mg/kg, intraperitoneally) followed by 6 h of nitrous oxide (75%), isoflurane (0.75%), and oxygen (approximately 24%). Thus, the measured fraction of inspired oxygen in both control and experimental conditions was 0.21–0.24. Several studies have shown that this protocol causes significant developmental neuroapoptosis.1,4,10,16,19
+
+Histopathologic Studies
+
+On P8, each pup was deeply anesthetized with phenobarbital (65 mg/kg, intraperitoneally) (University of Virginia Pharmacy, Charlottesville, Virginia). Perfusion and fixation of brain tissue were performed as previously described.10,16 Briefly, the left ventricle was cannulated, the descending aorta was clamped, and an initial flush was carried out with Tyrodes solution (30–40 ml) (Sigma–Aldrich Chemical). For morphometric analyses of pyramidal neurons, we perfused with paraformaldehyde (2%) and glutaraldehyde (2%).
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 3
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+After the perfusion, we removed the rats’ brains and stored them in the same fixative overnight. Both control and experimental pups were perfused by an experienced experimenter on the same day, using the same solution to assure uniform tissue fixation. Any brain considered to have been inadequately perfused was not processed for electron microscopy analysis. Our routine electron microscopy protocol has been described elsewhere.6,10 Briefly, fixed brains were coronally sectioned (50–75 μm thick) with a DTK-1000 microslicer (Ted Pella, Tools for Science and Industry, Redding, CA). The subiculum was localized as described in anatomical maps,20 fixed in 2% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA), stained with 4% uranyl acetate (Electron Microscopy Sciences), and embedded in aclar sheets using epon–araldite resins. The subiculum was then dissected from the aclar sheets and embedded in BEEM capsules (Electron Microscopy Sciences). To prepare capsules for microtome cutting (Sorvall MT-2 microtome, Ivan Sorvall, Norwalk, CT), the tips were manually trimmed so that ultrathin slices (silver interference color, 600–900 Å) could be cut using a diamond knife (Diatome, Hatfield, PA). Ultrathin sections were placed on grids and examined using a 1230 TEM electron microscope (Carl Zeiss, Oberkochen, Germany).
+
+Morphometric Analyses
+
+Our protocol for morphometric analyses of mitochondria was previously described.10 Briefly, as the cytoplasmic soma of pyramidal neurons cannot be captured in their entirety with a single photo frame at such high magnification (×6,000–×12,000), we took multiple sequential pictures using a 16-megapixel digital camera (SIA-12C digital cameras, Scientific Instruments and Applications, Duluth, GA), then tiled them seamlessly together to make a mosaic of one whole cell body. We analyzed 5 neurons from each animal (n = 4 pups/group) for a total of 20 neurons in the control group and anesthesia-treated group each. For statistical analysis, we used n = 4 pups/group after we obtained the average from five neurons in each pup. From these mosaic pictures, the cytoplasmic and mitochondrial areas were measured using Image-Pro Plus 6.1 computer software (MediaCybernetics, Bethesda, MD). The number of animals necessary for these complex and time-consuming ultrastructural histological studies was determined based on our previously published studies (n = 4 pups in each group from four different litters. Equal numbers of male and female pups were used for each experimental condition. Control and experimental pups were equally represented from each litter).6,10 The investigator analyzing electron micrographs was blinded to the experimental conditions.
+
+Catalase and SOD Activity Assays
+
+Control and experimental groups of rats were killed immediately postanesthesia, and the subicular and thalamic brain tissues were removed quickly. The tissues were homogenized in 20 mm HEPES buffer at pH 7.2, containing 1 mm EDTA, mannitol, and sucrose per gram of brain tissue (for SOD) or cold phosphate-buffered saline (for catalase) with 1mm EDTA. Upon centrifugation at 1,500g, followed by centrifugation at 10,000g at 4°C, the supernatants were collected and the assays were carried out at 25°C. SOD activity was assayed using a commercially available kit (Superoxide Dismutase Assay kit, Cayman Chemical, Ann Arbor, MI) that can detect the activity of all three forms of SOD—Cu/Zn-, Mn-, and Fe-SOD—as absorbance at 440–460 nm. Catalase activity was assayed using a commercially available kit (OxiSelect Catalase Activity Assay kit, Cell Biolabs Inc., San Diego, CA) with absorbance detected at 520 nm. The assays were performed following the manufacturer’s instructions using a microplate reader (VersaMax, Molecular Devices, Chicago, IL). Total protein was measured for each sample on the day of the assay using a commercially available protein determination kit (Bradford method) (Cayman Chemical, Ann Arbor, MI). The activities of SOD and catalase were expressed in arbitrary units per
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 4
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+milligram protein. The group sizes for each experimental condition are indicated in the figure legends.
+
+Subcellular Fractionation
+
+Mainly subicular tissues (with some thalamic contamination) were collected immediately postanesthesia. Briefly, the tissues were cut in small pieces and gently homogenized in ice- chilled Dounce homogenizers (20 strokes) using isotonic extraction buffer A from a Mitochondrial Isolation Kit (Sigma-Aldrich Co.) with protease inhibitor cocktail (Roche, Indianapollis, IN). The homogenates were centrifuged at 1,000g for 5 min to remove unbroken cells and nuclei. Supernatants were transferred into new tubes and centrifuged at low speed (3,500g for 10 min) to yield mitochondria-enriched fractions without lysosome and peroxisome contamination. Supernatants were removed and centrifuged at 70,000g to obtain pure cytosol fractions, and the mitochondria-enriched pellets were carefully resuspended and washed again in 1× extraction buffer A. The mitochondrial fractions then were re-pelleted by centrifugation at 1,000 and 3,500g for 5 and 10 min, respectively. Proteins from the mitochondria-enriched pellets were extracted by vortexing for 1 min in lysis buffer containing 20 mm Tris-HCl at pH 8.0, 137 mm NaCl, 10% glycerol, 1% nonidet P-40, and 2 mm EDTA. Following centrifugation at 13,000g, the supernatants were removed and protein concentration in the pellets was determined using the bicinchoninic acid micro- protein assay (Micro BCA protein assay kit, Pierce Inc., Rockford, IL). Subcelullar fractionation was performed at 4°C. The pellets are considered to contain the “heavy” mitochondrial fraction—enriched with mitochondria with substantially diminished presence of lysosomes and peroxisomes, which are common contaminants of this fraction.
+
+Western Blotting
+
+The protein concentration of the lysates was determined with the Total Protein kit (Sigma– Aldrich Chemical Co.). For separation of Mfn-2 and Drp-1 monomers, protein samples (30 μg per lane) were heat-denaturized in 2× Laemmli sample buffer, electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel, and transferred to a nitrocellulose membrane (Hybond ECL, Amersham International, Buckinghamshire, United Kingdom). To investigate Drp-1 oligomerization, the boiling step was omitted and 60 μg of samples were subjected to nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (without β-merkaptoethanol or dithlothreltol in 2× loading buffer) on 8% acrylamide gel21 and transferred to a polyvinylidene difluoride membrane (Millipore, Danvers, MA).
+
+The membranes subsequently were incubated and probed with the anti-Drp-1 primary antibody or anti-Mfn 2 antibody at a dilution of 1:1000 each (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in Tris-buffered saline–Tween overnight, followed by the incubation with the appropriate secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Inc.). Immunoreactive bands were visualized using enhanced chemiluminescence (Pierce, Inc.). β-ACTIN (1:10,000, Sigma-Aldrich) and porin (1:2500, Invitrogen, Eugene, OR) were used as loading controls for cytosolic and mitochondrial fraction, respectively. The molecular size of the proteins of interest was determined by comparison to pre-stained protein markers (BioRad, Hercules, CA). All gels were densitometrically analyzed in GBOX-chemi (Syngene, Frederick, MD) using the computerized image analysis program ImageQuant 5.0 (GE Heathcare, Life Sciences, Piscataway, NJ). Data were analyzed first as a ratio between the protein of interest and β- actin (or porin) and expressed as a percent change from a control density. The group sizes for each experimental condition are indicated in the figure Legends.
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 5
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+Spectrophotometric Detection of ROS
+
+Control and experimental groups of rats were killed immediately postanesthesia, and the subicular and thalamic brain tissues were quickly removed. ROS were measured as hydrogen peroxide using the horseradish peroxidase-linked spectrophotometric assay kit according to the manufacturer’s instructions (Amplex Red, Invitrogen). Briefly, extracted brain mitochondria samples (120 μg) were added to a 96-well plate containing 100 μl of reaction buffer consisting of 0.1 U/ml of the horseradish peroxidase, 50 μm Amplex UltraRed, and 1 μl of dimethyl sulfoxide. Reactions were incubated at room temperature for 30 min and protected from light. Resorufin absorptions were followed at 560 nm using a VersaMax tunable microplate reader (Molecular Devices, Chicago, IL). Hydrogen peroxide levels are expressed in arbitrary units (per milligram protein). The group sizes for each experimental condition are indicated in the figure legends.
+
+Statistical Analysis
+
+Single comparisons among groups were made using an unpaired two-tailed t test. When ANOVA with repeated measures was needed, the Bonferroni correction was used to help maintain prescribed alpha levels (e.g., 0.05). Histograms in cumulative frequency analysis were compared with chi-square-test. Using the standard version of GraphPad Prism 5.01 software (Media Cybernetics, Inc., Bethesda, MD), we considered P < 0.05 to be statistically significant. All the data are presented as mean + SEM. No experimental data were missing or lost to statistical analysis.
+
+Results
+
+GA Induces Excessive Mitochondrial Fission in Developing Neurons
+
+Our ultrastructural analysis of mitochondrial morphology in subicular pyramidal neurons revealed that anesthesia-treated animals contain numerous small round mitochondria displaying globular morphology 24 h postanesthesia exposure (on P8). Compared to controls (fig. 1A), it appeared that anesthesia-treated subicular neurons contained significantly more mitochondria (fig. 1B). The mitochondrial matrix was pale and showed signs of swelling. Although the inner and outer membranes appeared somewhat intact, the cristae seemed distorted and difficult to discern (fig. 1C), suggesting ultrastructural damage to mitochondria undergoing excessive fission.
+
+To quantify the observed effect, which suggested that GA may increase mitochondrial density, we performed detailed morphometric analysis of each mitochondrion and determined mitochondrial density in the soma of pyramidal subicular neurons. We calculated mitochondrial density by counting the number of mitochondrial profiles per unit area (μm2) of cytoplasmic soma in each pyramidal neuron. We found that there were approximately 30% more mitochondrial profiles in experimental neurons compared to controls (* P = 0.0179) (fig. 2A). However, when the sum of mitochondrial areas was presented as a percent of the cytoplasmic area of pyramidal neurons, we found that mitochondria in the control and experimental neurons occupied approximately the same percent of the cytoplasmic soma (P = 0.8067) (fig. 2B), suggesting that the higher density of mitochondrial profiles could be due to enhanced fission, resulting in a shift of mitochondrial pool from a larger to a smaller category (n = 4 control and four experimental pups). To examine this notion, we performed frequency distribution analysis by grouping mitochondria by their area and counting the number of mitochondria in each bin as shown in figure 2C. We found that there were significantly more mitochondria smaller than 0.16 μm2 in experimental animals when compared to controls (P < 0.001, horizontal bar), suggesting a leftward shift in mitochondrial size due to GA treatment. Indeed, when we performed cumulative frequency analysis (in percentage), designed to take into account the differences
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 6
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+in overall mitochondrial number in control versus experimental neurons, we found a leftward shift toward the smaller category (fig. 2D). For example, although mitochondria smaller than 0.012 μm2 were detected in GA-treated pyramidal neurons, none that small could be detected in control neurons. In addition, more than 50% of mitochondria in GA- treated neurons were smaller than 0.1 μm2, whereas about 30% were found in that size category in control animals. This finding suggests that GA may enhance mitochondrial fission.
+
+GA Causes Excessive Accumulation of ROS
+
+To begin to understand the mechanism(s) by which anesthesia may cause excessive mitochondrial fission, we examined whether anesthesia exposure causes undue accumulation of ROS. This idea stems from the fact that oxidative stress, implicated in promoting fission and inhibiting fusion,21 may disturb the fine balance between these two processes crucial for proper mitochondrial remodeling.11 We measured ROS with a kit that detects hydrogen peroxide in fresh brain homogenate obtained from P7 rats immediately after 6 h of anesthesia. As shown in figure 3, the level of ROS in experimental animals was significantly increased (about 30%) compared to that in sham controls (*P = 0.0357) (n = 3 rat pups in the control group; n = 5 rat pups in the experimental group), suggesting that anesthesia promotes significant ROS accumulation.
+
+GA Acutely Impairs SOD but not Catalase Activity
+
+As excessive ROS accumulation could be the result of ineffective scavenging machinery (responsible for maintaining ROS levels within normal limits), we set out to examine whether anesthesia has an acute effect on two important scavenging enzymes, SOD and catalase. We measured their activities in fresh brain homogenate obtained from P7 rat pups immediately after 6 h of anesthesia or sham treatment. The activity of SOD and catalase was expressed in units per milligram of protein. As shown in figure 4, there was a significant, 2- fold decrease in SOD activity immediately after anesthesia treatment compared to that in sham controls (fig. 4A) (**P = 0.0011; n = 6 pups in control group; n = 6 pups in experimental group). When we measured the activity of catalase, we found no change in the experimental groups compared to that in the sham controls (fig. 4B) (P = 0.6631; n = 6 pups in control group; n = 6 pups in experimental group).
+
+GA Modulates Expressions of Mfn-2 and Drp-1 Proteins, Two Important Regulators of Mitochondrial Fusion and Fission, Respectively
+
+In view of our findings that anesthesia may cause excessive mitochondrial fission and inappropriate ROS accumulation, and the fact that oxidative stress can lead to disturbances of fine balance between mitochondrial fusion and fission, we assessed whether anesthesia modulates the expression of two key proteins responsible for maintaining mitochondrial dynamics, Mfn-2 and Drp-1. As Mfn-2 and Drp-1 proteins are involved in active remodeling of the outer and inner mitochondrial membranes, and could be localized in cytoplasm or be sequestered in the mitochondrial membrane, we measured their expression in both cytosolic and mitochondrial compartments. As shown in figure 5, there was about a 40% decrease in cytosolic Mfn-2 protein in GA-treated animals compared to that in sham controls (*P = 0.026; n = 6 pups in control group; n = 6 pups in experimental group) (fig. 5A). However, we found a slight but nonsignificant difference (P = 0.075) between Mfn-2 expression in the mitochondrial fractions of GA-treated versus sham controls (fig. 5B) (n = 9 pups in the control group; n = 9 pups in the experimental group).
+
+As shown in figure 6, we detected a significant decrease (about 40%) (***P < 0.0001) in Drp-1 protein expression in cytosol from GA-treated versus sham-treated animals (fig. 6A) (n = 11 pups in the control group; n = 11 pups in the experimental group), and a significant
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 7
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+Discussion
+
+increase (about 50%) (***P = 0.0002) in Drp-1 protein expression in the mitochondrial fraction of GA-treated versus sham-treated animals (fig. 6B), suggesting substantial translocation of Drp-1 to the mitochondrial membranes (n = 10 pups in the control group; n = 10 pups in the experimental group).
+
+Once Drp-1 is translocated from the cytoplasm to mitochondrial membranes, the Drp-1 monomer undergoes self-assembly (i.e., oligomerization) on the mitochondrial outer membrane, a step that allows the formation of the ring-like structure necessary for mitochondrial fission.12,13 As our findings suggest that anesthesia causes oxidative stress and may promote Drp-1 translocation to mitochondria, and in view of the fact that the oxidative stress can cause Drp-1 oligomerization, we set out to examine whether anesthesia promotes excessive formation of Drp-1 oligomers, which could, in part, explain enhanced mitochondrial fission. Indeed, we found that anesthesia increases the amount of the oligomerized form of Drp-1 protein by about 45% compared to that in controls (fig. 7; **P = 0.0037; n = 7 pups in the control group; n = 7 pups in the experimental group).
+
+Early exposure to GA causes acute upregulation of ROS that is, in part, due to downregulation of SOD activity and lack of compensatory modulation of catalase activity. ROS upregulation is associated with impaired mitochondrial fission and fusion. This could be due to differential modulation of mitochondrial fission/fusion proteins. On the one hand, GA causes a decrease in Drp-1 protein in the cytoplasm due to an apparent translocation to mitochondria, with subsequent Drp-1 oligomerization on the outer mitochondrial membrane, a necessary step in the formation of the ring-like structures and fission. On the other hand, a lack of compensatory modulation of Mfn-2, a protein necessary for mitochondrial fusion, tips the fine equilibrium toward excessive mitochondrial fission (fig. 8). Based on previous reports suggesting that the GA causes massive developmental neuroapoptosis,1,4,18,19 and on others suggesting that excessive mitochondrial fission is associated with apoptotic cell death,21–23 we propose that excessive mitochondrial fission may be important for GA- induced developmental neurotoxicity.
+
+Here we confirm that GA causes acute ROS upregulation.16 Moreover, we propose that a GA-induced disturbance in the neuronal redox state is likely caused by an imbalance between ROS production and ROS scavenging. Because mitochondria exposed to GA undergo excessive fission, and because unbalanced fission may lead to mitochondrial dysfunction11,21 resulting in excessive ROS generation, it is possible that overproduction of ROS is the likely cause of ROS upregulation as dysfunctional mitochondria could be the greatest intracellular source of ROS. Indeed, Barsoum et al.24 have shown that persistent mitochondrial fission leads to mitochondrial dysfunction and excessive production of ROS, one of the earliest signs of disturbed homeostasis leading to neuronal cell death.
+
+Nevertheless, a fully functional scavenging system is also very important. For example, in order to manage a substantial increase in production of superoxide ions (normally generated with a rate constant that is already 3–8 times that of superoxide decomposition by SOD), the activity of SOD has to increase substantially.25 However, we show that GA induces statistically significant decrease in SOD activity, thus placing ROS homeostasis in double jeopardy: impaired scavenging in the setting of enhanced ROS production. The GA effect on scavenging enzymes seems to be selective. Despite a decrease in SOD activity, the activity of catalase is spared. Although the reason for this selective effect remains unclear, the lack of a compensatory increase in catalase activity in the setting of upregulated levels of its substrate, hydrogen peroxide, may worsen the acute oxidative stress in developing neurons.
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 8
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+Although GA induces differential effects on the activity of these scavenging enzymes, it remains to be determined whether GA has an effect on their protein content.
+
+It remains unclear whether GA-induced mitochondrial fission is the main cause of acute oxidative stress (especially as impaired mitochondria could be a powerful source of ROS) or its outcome, as mitochondria are also an important target of ROS. Disturbed redox state of the neuron regulates the translocation of Drp-1 from the cytosol to mitochondria and its oligomerization in vitro, thus directing mitochondrial dynamics toward excessive fission.21 Here we show that in vivo upregulation of ROS is associated with similar changes in Drp-1. Although the mechanism of Drp-1 oligomerization in vivo remains unsettled, previous in vitro studies have suggested that oxidative stress promotes the formation of thiol cross-links from cysteine residues in Drp-1, which could, in turn, promote the formation of Drp-1 oligomers.26
+
+In some neurodegenerative diseases27 and some forms of acute brain injury,28 there is a shift toward excessive mitochondrial fission, leading to neuronal death. Consequently, Drp-1 inhibitors such as the Mdivi family of compounds were shown to prevent mitochondrial fission, loss of mitochondrial membrane potential, and neuronal death both in vitro and in vivo.28 In fact, Drp1 translocation to mitochondrial membrane and subsequent mitochondrial fission were the key features that preceded neuronal death. Although the safety profile of presently available Drp-1 antagonists in very young animals remains to be established, it is possible that these agents may offer some benefit in protecting against GA-induced disturbance in mitochondrial fission as Drp-1 may be an important cellular target of GA- induced developmental neurotoxicity.
+
+Although the focus of this study was not on following the neuronal fate during excessive mitochondrial fission, we and other researchers previously have reported that early exposure to GA enhances developmental neuroapoptosis by massive activation of caspase-3.1,3,4,9,18,19 As excessive fission is considered to be an early occurrence during apoptosis, 22,23 the question remains whether apoptosis is caused by fission or whether fission is its consequence. Based on our previously published findings, one would suggest the former conclusion. We know that our GA protocol causes early cytochrome c leak, resulting in caspase-3 and -9 activation and apoptotic neurodegeneration that is considered to be intrinsic (mitochondria)-dependent.4 Although the exact timing of these processes vis- à-vis mitochondrial fission remains to be deciphered, we suggest that GA-induced mitochondrial fission promotes acute cytochrome c leak, leading to caspase activation. Some earlier reports support this view. For example, dominant negative mutants of Drp-1, which antagonize mitochondrial fission, have been shown to block cytochrome c release, apoptotic activation, and cell death.22,24,29,30 In addition, overexpression of the Mfn family of proteins (that promote mitochondrial fusion) has been known to curtail cytochrome c release and inhibit apoptosis. 31 However, other reports suggest that mitochondrial fission is an epiphenomenon that accompanies apoptosis. For instance, overexpression of the anti- apoptotic protein Bcl-xL (known to protect mitochondrial membrane) was demonstrated to block cytochrome c release in vitro, but did not block mitochondrial fission. This suggests that mitochondrial fission may not be the causal event in apoptosis-associated cytochrome c release.32 Regardless of what view is accepted, GA-induced ROS upregulation disturbs fusion/fission balance and promotes excessive mitochondrial fission, as we have shown here, while promoting excessive cytochrome c release and caspases 9 and -3 activation, as we have shown previously.4
+
+We know that the long-term effects of early exposure to GA are manifested as mitochondrial enlargement.10 Here we report that the acute effects of GA are manifested as a decrease, not an increase, in mitochondrial size. Although it remains to be examined whether GA has
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 9
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+long-term effects on fission/fusion machinery, possibly causing excessive fusion long after the initial exposure occurs, it is more likely that the mitochondria undergoing undue fission become vulnerable to fragmentation of cristae and inner mitochondrial membranes. This could, in turn, cause significant impairment of mitochondrial membrane integrity, leading to “leakiness” and swelling. Supporting this notion is our earlier report describing that mitochondrial swelling in addition to derangement and fragmentation of cristae indeed occurs when mitochondria are examined two weeks postanesthesia.10 In addition, a report by Barsoum et al.24 showed that excessive fission can impair the integrity of mitochondrial cristae often referred to as “cristae remodeling.”30,33
+
+Although our study was not designed for live imaging of mitochondrial dynamics in terms of mitochondrial localization, antero versus retrograde transport along the neuronal axon, or speed of movement, it is possible that the disturbed fission and fusion could cause impaired mitochondrial agility and dynamics. Well-designed in vitro studies in which neurons can be examined during anesthesia exposure using time lapse confocal imaging will be needed to confirm this notion.
+
+The concern regarding the maintenance of physiological homeostasis during anesthesia brings into focus the potential role of hypoxia in anesthesia-induced ROS upregulation. Indeed hypoxia may cause a significant rise in ROS in ischemic neurons, which could in turn initiate mitochondrial injury and neuronal death.34 As our earlier studies using the same GA protocol have ruled out the occurrence of hypoxia,1,4,18 it is unlikely that the GA- induced ROS upregulation we report herein is due to inadequate oxygenation.
+
+The role of Mfn-2 protein extends beyond controlling fusion. Mfn-2 modulates metabolism via electron transport chain complexes I, IV, and V35,36 and controls oxygen consumption and electrochemical potential.15 Hence, Mfn-2 plays an important role in controlling the redox and metabolic state of the cell. Due to its role in curtailing oxidative stress and cytochrome c release, while interacting with anti-apoptotic bcl family of proteins,37 Mfn-2 may be crucial for maintaining mitochondrial integrity. Consequently, Mfn-2 is regarded as an important therapeutic target for the treatment of diseases caused by disturbed mitochondrial homeostasis and morphogenesis.38 Indeed some studies have shown that compensatory upregulation of Mfn-2 is the key to blocking the upregulation of ROS and mitochondrial fragmentation. 39,40 Because of the vital role of Mfn-2 in mitochondrial and neuronal function and its apparent downregulation by GA, we suggest that Mfn-2 could be an important target for preventive strategies aimed at curtailing GA-induced developmental neuroapoptosis.
+
+Although our anesthesia protocol is a reliable model for studying developmental neurodegeneration, it is based on the use of anesthetics in combination. Therefore, despite the fact that clinical anesthesia commonly relies on the use of more than one anesthetic to achieve the desired effect, we were unable to examine the relative contribution of each agent. Further studies with individual anesthetics could decipher their relative importance.
+
+In conclusion, early exposure to GA impairs mitochondrial morphogenesis and disturbs fission/fusion. This is accompanied by the disturbance of neuronal scavenging machinery and excessive ROS accumulation.
+
+Acknowledgments
+
+This study was supported by the National Institute of Health/The Eunice Kennedy Shriver National Institute of Child and Human Development 44517 (to Vesna Jevtovic-Todorovic) and 44517-S (to Vesna Jevtovic-Todorovic), Bethesda, Maryland; Harold Carron endowment (to Vesna Jevtovic-Todorovic), University of Virginia, Charlottesville, Virginia; John E. Fogarty Award 007423-128322 (to Vesna Jevtovic-Todorovic), National Institute
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 10
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+References
+
+of Health, Bethesda, Maryland; and March of Dimes National Award (to Vesna Jevtovic-Todorovic). Vesna Jevtovic-Todorovic was an Established Investigator of the American Heart Association (Dallas, Texas), National Award.
+
+The authors thank Jan Redick, B.S., Laboratory Director of the Advanced Microscopy Facility at the University of Virginia Health System, Charlottesville, Virginia, for technical assistance with electron microscopy and data analyses. The authors thank Shawn D. Feinstein, B.S. (Medical Student, Virginia Commonwealth University School of Medicine, Richmond, Virginia), and Kirsten Rose, Ph.D. (Research Associate, Department of Anesthesiology, University of Virginia Health System), for technical assistance with mitochondrial analysis and Western blot studies, respectively.
+
+1. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci. 2003; 23:876–82. [PubMed: 12574416]
+
+2. Rizzi S, Carter LB, Ori C, Jevtovic-Todorovic V. Clinical anesthesia causes permanent damage to the fetal guinea pig brain. Brain Pathol. 2008; 18:198–210. [PubMed: 18241241]
+
+3. Slikker W Jr, Zou X, Hotchkiss CE, Divine RL, Sadovova N, Twaddle NC, Doerge DR, Scallet AC, Patterson TA, Hanig JP, Paule MG, Wang C. Ketamine-induced neuronal cell death in the perinatal rhesus monkey. Toxicol Sci. 2007; 98:145–58. [PubMed: 17426105]
+
+4. Yon JH, Daniel-Johnson J, Carter LB, Jevtovic-Todorovic V. Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience. 2005; 135:815–27. [PubMed: 16154281]
+
+5. Head BP, Patel HH, Niesman IR, Drummond JC, Roth DM, Patel PM. Inhibition of p75 neurotrophin receptor attenuates isoflurane-mediated neuronal apoptosis in the neonatal central nervous system. Anesthesiology. 2009; 110:813–25. [PubMed: 19293698]
+
+6. Lunardi N, Ori C, Erisir A, Jevtovic-Todorovic V. General anesthesia causes long-lasting disturbances in the ultrastructural properties of developing synapses in young rats. Neurotox Res. 2010; 17:179–88. [PubMed: 19626389]
+
+7. Briner A, De Roo M, Dayer A, Muller D, Habre W, Vutskits L. Volatile anesthetics rapidly increase dendritic spine density in the rat medial prefrontal cortex during synaptogenesis. Anesthesiology. 2010; 112:546–56. [PubMed: 20124985]
+
+8. Briner A, Nikonenko I, De Roo M, Dayer A, Muller D, Vutskits L. Developmental stage-dependent persistent impact of propofol anesthesia on dendritic spines in the rat medial prefrontal cortex. Anesthesiology. 2011; 115:282–93. [PubMed: 21701379]
+
+9. Lemkuil BP, Head BP, Pearn ML, Patel HH, Drummond JC, Patel PM. Isoflurane neurotoxicity is mediated by p75NTR-RhoA activation and actin depolymerization. Anesthesiology. 2011; 114:49– 57. [PubMed: 21169791]
+
+10. Sanchez V, Feinstein SD, Lunardi N, Joksovic PM, Boscolo A, Todorovic SM, Jevtovic-Todorovic V. General anesthesia causes long-term impairment of mitochondrial morphogenesis and synaptic transmission in developing rat brain. Anesthesiology. 2011; 115:992–1002. [PubMed: 21909020] 11. Chan DC. Mitochondrial fusion and fission in mammals. Annu Rev Cell Dev Biol. 2006; 22:79–
+
+99. [PubMed: 16704336]
+
+12. Yoon Y, Pitts KR, McNiven MA. Mammalian dynamin-like protein DLP1 tubulates membranes. Mol Biol Cell. 2001; 12:2894–905. [PubMed: 11553726]
+
+13. Smirnova E, Griparic L, Shurland DL, van der Bliek AM. Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. Mol Biol Cell. 2001; 12:2245–56. [PubMed: 11514614]
+
+14. Koshiba T, Detmer SA, Kaiser JT, Chen H, McCaffery JM, Chan DC. Structural basis of mitochondrial tethering by mitofusin complexes. Science. 2004; 305:858–62. [PubMed: 15297672]
+
+15. Chen H, Chomyn A, Chan DC. Disruption of fusion results in mitochondrial heterogeneity and dysfunction. J Biol Chem. 2005; 280:26185–92. [PubMed: 15899901]
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 11
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+16. Boscolo A, Starr JA, Sanchez V, Lunardi N, DiGruccio MR, Ori C, Erisir A, Trimmer P, Bennett J, Jevtovic-Todorovic V. The abolishment of anesthesia-induced cognitive impairment by timely protection of mitochondria in the developing rat brain: The importance of free oxygen radicals and mitochondrial integrity. Neurobiol Dis. 2012; 45:1031–41. [PubMed: 22198380]
+
+17. Jevtovic-Todorovic V, Benshoff N, Olney JW. Ketamine potentiates cerebrocortical damage induced by the common anaesthetic agent nitrous oxide in adult rats. Br J Pharmacol. 2000; 130:1692–8. [PubMed: 10928976]
+
+18. Lu LX, Yon JH, Carter LB, Jevtovic-Todorovic V. General anesthesia activates BDNF-dependent neuroapoptosis in the developing rat brain. Apoptosis. 2006; 11:1603–15. [PubMed: 16738805] 19. Yon JH, Carter LB, Reiter RJ, Jevtovic-Todorovic V. Melatonin reduces the severity of anesthesia- induced apoptotic neurodegeneration in the developing rat brain. Neurobiol Dis. 2006; 21:522–30. [PubMed: 16289675]
+
+20. Paxinos, G.; Watson, C. The Rat Brain in Stereotaxic Coordinates. Australia: Academic Press; 1944.
+
+21. Tian C, Murrin LC, Zheng JC. Mitochondrial fragmentation is involved in methamphetamine- induced cell death in rat hippocampal neural progenitor cells. PLoS ONE. 2009; 4:e5546. [PubMed: 19436752]
+
+22. Frank S, Gaume B, Bergmann-Leitner ES, Leitner WW, Robert EG, Catez F, Smith CL, Youle RJ. The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis. Dev Cell. 2001; 1:515–25. [PubMed: 11703942]
+
+23. Youle RJ, Karbowski M. Mitochondrial fission in apoptosis. Nat Rev Mol Cell Biol. 2005; 6:657–
+
+63. [PubMed: 16025099]
+
+24. Barsoum MJ, Yuan H, Gerencser AA, Liot G, Kushnareva Y, Gräber S, Kovacs I, Lee WD, Waggoner J, Cui J, White AD, Bossy B, Martinou JC, Youle RJ, Lipton SA, Ellisman MH, Perkins GA, Bossy-Wetzel E. Nitric oxide-induced mitochondrial fission is regulated by dynamin- related GTPases in neurons. EMBO J. 2006; 25:3900–11. [PubMed: 16874299]
+
+25. Crichton, RR.; Ward, RJ. Oxidative Stress and Redox Active Metal Ions. In: Crichton, RR.; Ward, RJ., editors. Metal-based Neurodegeneration: From Molecular Mechanisms to Therapeutic Strategies. Vol. Chapter 2. John Wiley & Sons; 2006. p. 21-51.
+
+26. Costantini P, Belzacq AS, Vieira HL, Larochette N, de Pablo MA, Zamzami N, Susin SA, Brenner C, Kroemer G. Oxidation of a critical thiol residue of the adenine nucleotide translocator enforces Bcl-2-independent permeability transition pore opening and apoptosis. Oncogene. 2000; 19:307– 14. [PubMed: 10645010]
+
+27. Reddy PH, Reddy TP, Manczak M, Calkins MJ, Shirendeb U, Mao P. Dynamin-related protein 1 and mitochondrial fragmentation in neurodegenerative diseases. Brain Res Rev. 2011; 67:103–18. [PubMed: 21145355]
+
+28. Grohm J, Kim SW, Mamrak U, Tobaben S, Cassidy-Stone A, Nunnari J, Plesnila N, Culmsee C. Inhibition of Drp1 provides neuroprotection in vitro and in vivo. Cell Death Differ. 2012; 19:1446–58. [PubMed: 22388349]
+
+29. Breckenridge DG, Stojanovic M, Marcellus RC, Shore GC. Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol. J Cell Biol. 2003; 160:1115–27. [PubMed: 12668660] 30. Germain M, Mathai JP, McBride HM, Shore GC. Endoplasmic reticulum BIK initiates DRP1- regulated remodelling of mitochondrial cristae during apoptosis. EMBO J. 2005; 24:1546–56. [PubMed: 15791210]
+
+31. Wu S, Zhou F, Zhang Z, Xing D. Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins. FEBS J. 2011; 278:941–54. [PubMed: 21232014]
+
+32. Sheridan C, Delivani P, Cullen SP, Martin SJ. Bax- or Bak-induced mitochondrial fission can be uncoupled from cytochrome C release. Mol Cell. 2008; 31:570–85. [PubMed: 18722181]
+
+33. Scorrano L. Opening the doors to cytochrome c: Changes in mitochondrial shape and apoptosis. Int J Biochem Cell Biol. 2009; 41:1875–83. [PubMed: 19393761]
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 12
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+34. Abramov AY, Scorziello A, Duchen MR. Three distinct mechanisms generate oxygen free radicals in neurons and contribute to cell death during anoxia and reoxygenation. J Neurosci. 2007; 27:1129–38. [PubMed: 17267568]
+
+35. Bach D, Pich S, Soriano FX, Vega N, Baumgartner B, Oriola J, Daugaard JR, Lloberas J, Camps M, Zierath JR, Rabasa-Lhoret R, Wallberg-Henriksson H, Laville M, Palacín M, Vidal H, Rivera F, Brand M, Zorzano A. Mitofusin-2 determines mitochondrial network architecture and mitochondrial metabolism. A novel regulatory mechanism altered in obesity. J Biol Chem. 2003; 278:17190–7. [PubMed: 12598526]
+
+36. Pich S, Bach D, Briones P, Liesa M, Camps M, Testar X, Palacín M, Zorzano A. The Charcot- Marie-Tooth type 2A gene product, Mfn2, up-regulates fuel oxidation through expression of OXPHOS system. Hum Mol Genet. 2005; 14:1405–15. [PubMed: 15829499]
+
+37. Delivani P, Adrain C, Taylor RC, Duriez PJ, Martin SJ. Role for CED-9 and Egl-1 as regulators of mitochondrial fission and fusion dynamics. Mol Cell. 2006; 21:761–73. [PubMed: 16543146]
+
+38. Jahani-Asl A, Cheung EC, Neuspiel M, MacLaurin JG, Fortin A, Park DS, McBride HM, Slack RS. Mitofusin 2 protects cerebellar granule neurons against injury-induced cell death. J Biol Chem. 2007; 282:23788–98. [PubMed: 17537722]
+
+39. Yu T, Robotham JL, Yoon Y. Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology. Proc Natl Acad Sci USA. 2006; 103:2653–8. [PubMed: 16477035]
+
+40. Li J, Liu X, Wang H, Zhang W, Chan DC, Shi Y. Lysocardiolipin acyltransferase 1 (ALCAT1) controls mitochondrial DNA fidelity and biogenesis through modulation of MFN2 expression. Proc Natl Acad Sci USA. 2012; 109:6975–80. [PubMed: 22509026]
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 13
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+What We Already Know about This Topic
+
+
+
+Common general anesthetics induce apoptotic neurodegeneration in the developing mammalian brain and disturb mitochondrial morphogenesis during synaptogenesis and fission
+
+What This Article Tells Us That Is New
+
+
+
+Early exposure to general anesthetics causes acute reactive oxygen species upregulation and disturbs the fine balance between mitochondrial fission and fusion, implicating yet another causal role for general anesthetics-induced developmental neuroapoptosis
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 14
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+Fig. 1. Anesthesia causes acute ultrastructural changes in mitochondria of pyramidal subicular neurons of 8-day-old rats. (A) Mitochondria in the cytoplasm of subicular pyramidal neurons from sham control animals resemble long tubules with intact inner and outer membranes and numerous cristae tightly packed inside healthy looking matrix. (B) Mitochondria in the cytoplasm of subicular pyramidal neurons from anesthesia- treated animals are numerous. The mitochondria are round, small, and display globular morphology 24 h postanesthesia exposure (on P8). Their matrix is pale and shows the signs of swelling. Although the inner and outer membranes appear somewhat intact, the cristae seem distorted and difficult to discern (C). N = nucleus.
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 15
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+Fig. 2. Anesthesia induces excessive fission of mitochondria in the soma of pyramidal subicular neurons of 8-day-old rats. (A) Mitochondrial density was assessed by counting the number of mitochondrial profiles per unit area (μm2) of cytoplasmic soma in each pyramidal neuron. There are approximately 30% more mitochondrial profiles in anesthesia-treated neurons compared with controls (*P = 0.0179). (B) The summation of mitochondrial areas, presented as a percent of the cytoplasmic area of pyramidal neurons, reveals that mitochondria in the control and experimental neurons occupy approximately the same area of the cytoplasmic soma (P = 0.8067). (C) Frequency distribution analysis by grouping of mitochondrial area indicates that there are significantly more mitochondria smaller than 0.16 μm2 (indicated with horizontal bar) in experimental animals compared with controls (P < 0.001). (D) Cumulative frequency analysis (in percentage), designed to take into account the differences in overall mitochondrial number in control vs. experimental neurons, indicates a leftward shift toward smaller mitochondria after anesthesia treatment, with over 50% of mitochondria in the category of lesser than 0.1 μm2. In addition, mitochondria smaller than 0.012 μm2 were detected in the anesthesia-treated pyramidal neurons, whereas none that small could be detected in the control neurons (n = 4 control and four experimental pups, five neurons from each pup).
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 16
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+Fig. 3. Anesthesia causes acute reactive oxygen species upregulation. Reactive oxygen species were measured in fresh homogenates of subicular and thalamic tissues obtained from P7 rats immediately after 6 h of anesthesia or sham treatment using a kit that detects hydrogen peroxide as described in Methods. We found that the level of reactive oxygen species in anesthesia-treated animals was increased significantly (about 30%) compared to that in sham controls (*P < 0.0357) (n = 3 rat pups in control group; n = 5 rat pups in the experimental group). P7 = postnatal day 7.
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 17
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+Fig. 4. Anesthesia acutely impairs superoxide dismutase (SOD) but not catalase activity. The activities of SOD and catalase were measured in fresh homogenates of subicular and thalamic tissues obtained from P7 rat pups immediately after 6 h of anesthesia or sham treatment and are expressed in units per milligram of protein. (A) We found a significant 2- fold decrease in SOD activity immediately after anesthesia treatment compared to that in sham controls (**P = 0.0011) (n = 6 pups in the control group; n = 6 pups in experimental group). (B) There was no difference in catalase activity between the sham control and experimental groups (P = 0.6631) (n = 6 pups in the control group; n = 6 pups in the experimental group). P7 = postnatal day 7.
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 18
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+Fig. 5. Anesthesia decreases expression of mitofusin-2 (Mfn-2) in the cytosolic fraction. The expression of Mfn-2 protein was estimated from Western blotting in fresh cytosolic and mitochondrial fractions of subicular and thalamic tissues obtained from P7 rats immediately postanesthesia or sham treatment. The protein levels are estimated from Western blotting as percent change from sham controls after normalization to β-actin (cytosolic fraction) or porin (mitochondrial fraction). (A) In the anesthesia- treated group (Treat), Mfn-2 protein expression in the cytosolic fraction was decreased by about 40% compared to that in the sham controls (Cont) (*P = 0.026) (n = 6 pups in the control group; n = 6 pups in the experimental group). (B) In the anesthesia-treated group (Treat), Mfn-2 protein expression in the mitochondrial fraction was approximately the same as that in the experimental group compared to that in the sham controls (Cont) (P = 0.0745) (n = 9 pups in control group; n = 9 pups in experimental group). The molecular mass standards (in kDa) are shown at the right of the representative Western blots (C: cytosolic Mfn-2; D: mitochondrial Mfn-2). (*) Indicates a nonspecific band detected by anti-Mfn2 antibody. P7 = postnatal day 7.
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 19
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+Fig. 6. Anesthesia decreases expression of Drp-1 in the cytosolic fraction and increases Drp-1 in the mitochondrial fraction. The expression of Drp-1 protein was estimated by Western blotting in fresh cytosolic and mitochondrial fractions of subicular and thalamic tissues obtained from P7 rat pups immediately postanesthesia or sham treatment. The protein levels were expressed as percent change from sham controls after normalization to β-actin (cytosolic fraction) or porin (mitochondrial fraction). (A) In the anesthesia-treated group (Treat), Drp-1 protein expression in the cytosolic fraction was significantly decreased compared to sham controls (Cont) (***P < 0.0001) (n = 11 pups in control group; n = 11 pups in experimental group). (B) In the anesthesia-treated group (Treat), Drp-1 protein expression in the mitochondrial fraction was significantly increased compared to that in sham controls (Cont) (***P = 0.0002) (n = 10 pups in the control group; n = 10 pups in the experimental group). The molecular mass standards (in kDa) are shown at the right of the representative Western blots (C = cytosolic Drp-1; D = mitochondrial Drp-1). (*) Indicates alternate splice variant typically recognized by antibody against Drp-1. Drp-1 = dynamin-related protein 1; P7 = postnatal day 7.
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 20
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+Fig. 7. Anesthesia enhances Drp-1 oligomerization in the mitochondria. The samples for Drp-1 oligomer analysis were subjected to a nonreducing gel sodium dodecyl sulfate- polyacrylamide gel electrophoresis. Anesthesia (Treat) increases the protein content of the oligomerized form of Drp-1 in the mitochondrial fraction by about 45% compared to sham controls (Cont) (**P = 0.0037; n = 7 pups in the control group; n = 7 pups in the experimental group). The molecular mass standards (in kDa) are shown at the right of the representative Western blots. Drp-1 = dynamin-related protein 1.
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 21
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Boscolo et al.
+
+Fig. 8. Proposed pathways that may be responsible for the excessive mitochondrial fission caused by an early exposure to anesthesia. Anesthesia causes downregulation of superoxide dismutase activity accompanied by a lack of compensatory modulation of catalase activity, and these effects are associated with reactive oxygen species upregulation. Elevated reactive oxygen species differentially modulate mitochondrial fission and fusion. They are suggested to induce acute downregulation of Drp-1 protein in the cytoplasm due to its translocation to mitochondria, followed by its oligomerization on the outer mitochondrial membrane, a necessary step in the formation of the ring-like structures required for mitochondrial fission. General anesthesia also causes acute downregulation of mitofusin-2 (Mfn-2), a protein necessary for mitochondrial fusion, thus tipping the fine equilibrium between fission and fusion toward excessive mitochondrial fission. Mitochondria that undergo excessive fission are less functional and more likely to generate excessive amounts of reactive oxygen species, thus further promoting reactive oxygen species upregulation in the setting of downregulated superoxide dismutase activity. In addition, down-regulation of Mfn-2 in the cytoplasm disturbs the redox balance in the neuron, leading to additional reactive oxygen species accumulation. Drp-1 = dynamin-related protein 1.
+
+Anesthesiology. Author manuscript; available in PMC 2014 January 03.
+
+Page 22

new_pdfs/10.1097_EJA.0b013e328330d453.txt

@@ -0,0 +1,723 @@
+Original article 181
+
+The effect of ketamine on N-methyl-D-aspartate receptor subunit expression in neonatal rats Li-Chun Hana, Li-nong Yaob, Sheng-xi Wuc, Yong-hui Yangb, Li-Xian Xua,M and Wei Chaib,M
+
+X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C 3 V C 1 y 0 a b g g Q Z X d g G 2 M w Z L e = o n 1 1 2 0 2 0 2 3
+
+r
+
+i
+
+i
+
+l
+
+f
+
+j
+
+l
+
+I
+
+/
+
+/
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / e a n a e s t h e s o o g y
+
+j
+
+i
+
+l
+
+b y B h D M 5 e P H K a v 1 z E o u m 1 Q N 4 a + k J L h E Z g b s I H o 4
+
+f
+
+t
+
+f
+
+Background and objective Ketamine has been widely used in paediatric anaesthesia but its influence on development in infants and toddlers still remains unclear. In order to elucidate the influence of ketamine on brain development in neonatal rats, semiquantitative reverse transcriptase PCR, quantitative reverse transcriptase PCR and immunohistochemistry assays were performed to detect the expression of N-methyl-D-aspartate receptor subtypes expression.
+
+Methods Seven-day-old rats were divided into two random groups. All of them were injected with ketamine intraperitoneally at postnatal day (PND) 7; one group was sacrificed at PND 7, but the other group was sacrificed at PND 28. Each group was divided into five random subgroups.
+
+Results In the semiquantitative reverse transcriptase PCR and quantitative reverse transcriptase PCR experiments, ketamine treatment caused a marked increase in mRNA expression in all subtypes at PND 7 and in NR2A subtypes at PND 28. Immunohistochemistry results indicated that NR2A, 2B and 2C receptor protein increased
+
+significantly at PND 7, and NR2A receptor protein increased at PND 28.
+
+Conclusions Exposure to ketamine resulted in an increase in N-methyl-D-aspartate receptor subunits at PND 7, and this increase persisted to PND 28 in NR2A. Eur J Anaesthesiol 27:181–186 Q 2010 European Society of Anaesthesiology.
+
+European Journal of Anaesthesiology 2010, 27:181–186
+
+Keywords: hippocampus, immunohistochemistry, ketamine, N-methyl-D- aspartate, quantitative reverse transcriptase PCR, semiquantitative reverse transcriptase PCR
+
+aDepartment of Anesthesiology, School of Stomatology, bDepartment of Anesthesiology, Tangdu Hospital and cDepartment of Anatomy, Histology and Embryology, K. K. Leung Brain Research Centre, Fourth Military Medical University, Xi’an, PR China
+
+Correspondence to Dr Li-Xian Xu, Department of Anesthesiology, School of Stomatology, Fourth Military Medical University, Xi’an 710032, PR China E-mail: kqmzk@fmmu.edu.cn
+
+Correspondence to Dr Wei Chai, Department of Anesthesiology, Tangdu Hospital, Fourth Military Medical University, Xi’an, 710038, PR China E-mail: tdmzka@fmmu.edu.cn
+
+Received 12 February 2009 Revised 14 July 2009 Accepted 14 July 2009
+
+Introduction Ketamine is used as a general paediatric anaesthetic for surgical procedures in infants and toddlers. It has been reported that it blocks excitatory synaptic transmission by acting as a noncompetitive N-methyl-D-aspartate (NMDA) receptor ion channel blocker [1].
+
+NMDA receptors play important roles in excitatory synap- tic transmission, in brain cell migration, differentiation, survival and activity-dependent synaptic plasticity under- lying learning and memory [2]. Ikonomidou et al. [3] was the first to demonstrate that the NMDA receptor antagonists MK-801 and ketamine induce neuroapoptosis in several encephalic regions in rats at postnatal day (PND) 7 after treatment for 8 h. Since then, groups have verified that NMDA receptor antagonists can provoke neuroapop- tosis in many encephalic regions [4–6].
+
+Many studies have shown that NMDA receptor anta- gonists can change the expression of NMDA receptor subtypes. In the present study, in order to investigate the influence of ketamine on brain development in neonatal
+
+rats, we aimed to show that ketamine, administered as a classic general anaesthetic agent for short-term anaesthe- sia caused changes in the expression of NMDA receptor subtypes NR1, NR2A, NR2B and NR2C at the mRNA level using quantitative reverse transcriptase (qRT) PCR and semiquantitative reverse transcriptase (sqRT) PCR techniques and at the protein level using immuno- histochemistry.
+
+Materials and methods Animals Seven-day-old male and female Sprague Dawley rats (body weight 11.1–17.5 g) were housed in plastic cages with their mothers and maintained on a 12 : 12 h light/ dark cycle at 22–258C ambient temperature with food and water available ad libitum for the mothers. All of the experimental procedures were approved by the Animal Use and Care Committee for Research and followed the ethical guidelines for investigation of experimental pain in conscious animals [7].
+
+M Dr Li-Xian Xu and Dr Wei Chai contributed equally to the writing of this article.
+
+Rats (n ¼ 40) were divided into two random groups. In one group (n ¼ 20) the rats were injected with ketamine
+
+0265-0215 (cid:1) 2010 Copyright European Society of Anaesthesiology
+
+DOI:10.1097/EJA.0b013e328330d453
+
+Copyright © European Society of Anaesthesiology. Unauthorized reproduction of this article is prohibited.
+
+182 European Journal of Anaesthesiology 2010, Vol 27 No 2
+
+X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C 3 V C 1 y 0 a b g g Q Z X d g G 2 M w Z L e = o n 1 1 2 0 2 0 2 3
+
+r
+
+i
+
+i
+
+l
+
+f
+
+j
+
+l
+
+I
+
+/
+
+/
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / e a n a e s t h e s o o g y
+
+j
+
+i
+
+l
+
+b y B h D M 5 e P H K a v 1 z E o u m 1 Q N 4 a + k J L h E Z g b s I H o 4
+
+f
+
+t
+
+f
+
+Table 1 Summary of experimental protocol
+
+PND 7
+
+C K1 K2
+
+Control group 100 mg kg(cid:1)1 ketamine persistently for 6 h 100 mg kg(cid:1)1 discontinuously for three times for 2 h; the three times were given once every other day
+
+K3 K4
+
+50 mg kg(cid:1)1 ketamine persistently for 6 h 50 mg kg(cid:1)1 discontinuously for three times for 2 h; the three times were given once every other day
+
+PND 28
+
+C K1 K2
+
+Control group 100 mg kg(cid:1)1 ketamine persistently for 6 h 100 mg kg(cid:1)1 discontinuously for three times for 2 h; the three times were given once every other day
+
+K3 K4
+
+50 mg kg(cid:1)1 ketamine persistently for 6 h 50 mg kg(cid:1)1 discontinuously for three times for 2 h; the three times were given once every other day
+
+intraperitoneally (i.p.) at PND 7 [8] and sacrificed within 24 h. In the other group (n ¼ 20) the rats were also injected with ketamine at PND 7 and were sacrificed at PND 28. Each group was divided into five random subgroups (n ¼ 4 per subgroup). The control group received 0.9% physio- logical saline. The other four groups received i.p. injec- tions of ketamine (K1–K4) [9] (see Table 1).
+
+Semiquantitative reverse-transcriptase PCR and quantitative real-time reverse-transcriptase PCR All animals from the different groups (n ¼ 4) were killed by decapitation under ether anaesthesia. sqRT-PCR was used to qualitatively assess the effect of NMDA subtype receptor expression. qRT-PCR was then applied in order to further quantify the observed effects. Table 2 sum- marizes information about the oligonucleotide primers used in this study. All primer sequences were checked in GenBank (National Center for Biotechnology Infor- mation, Bethesda, Maryland, USA) to avoid inadvertent sequence homologies. b-actin was used as an internal control. Animals were decapitated under ether anaesthe- sia, and the hippocampus was quickly dissected out and frozen at (cid:1)808C until use. Total RNA was isolated using Trizol reagent (Invitrogen, Virginia, USA), according to the manufacturer’s instructions, and then reverse transcribed with Moloney murine leukaemia virus (M-MLV) reverse transcriptase (Invitrogen) and oligo(dT)12–18 primers.
+
+For sqRT-PCR, a PCR reaction mixture containing 10 mmol l(cid:1)1 Tris (pH 8.3), 50 mmol l(cid:1)1 KCI, 1.5 mmol l(cid:1)1 MgCI2, 100 ml of deoxyribonucleotide triphosphate
+
+(dNTP), 2.5 units of Taq DNA polymerase (Takara, Kyoto, Japan), 0.5 ml of synthesized cDNA and 20 pmol of each sense and antisense primer pair. The PCR reac- tion was performed for 30 cycles using a PTC-100 Pro- grammed Thermal Controller (MJ Research, Watertown, Massachusetts, USA) as follows: 1 min at 938C, 30 s at appropriate annealing temperature (Table 2) and 1 min at 728C, with 1 min of 938C treatment before starting the thermal cycles, and, finally, an 8 min extension at 728C was conducted. PCR was performed simultaneously on control and experimental rat samples, with the internal controls (b-actin) running in parallel with the examined mRNAs. In all reverse transcriptase PCR experiments including negative controls, in which template RNA or reverse transcriptase was omitted, no PCR product was detected. Ten microlitres of each PCR product was electrophoresed on a 3% agarose gel containing ethidium bromide. Resulting gel bands were visualized in an ultraviolet (UV) transilluminator, and images were cap- tured with an eight-bit charge coupled device (CCD) camera (Ultra-Violet Products, Upland, California, USA).
+
+Quantitative PCR was set up using SYBR Green- containing premix from Takara. The reverse transcrip- tase reaction product (100 ng) was amplified in a 25 ml reaction with 12.5 SYBR Premix EX Taq (Takara, Shiga, Japan). Samples were heated to 908C for 30 s, and then amplified for 40 cycles consisting of 958C for 15 s and 608C for 15 s. Relative quantification of NMDA subtype receptors was performed by a comparative threshold cycle method. All data are expressed as mean (cid:2) SEM. Experimental groups were compared by analysis of var- iance. P values of less than 0.05 were considered to be statistically significant.
+
+Immunohistochemistry Rats in the control group and those in the K1 and K3 subgroups (n ¼ 4), which were sacrificed on PND 7 or PND 28, were perfused transcardially with 100 ml of 0.01 mol l(cid:1)1 PBS (pH 7.4), followed by 100 ml of 4% (w/v) paraformal- dehyde and 75% (v/v) saturated picric acid in 0.1 mol l(cid:1)1 phosphate buffer (pH 7.4). The brains were then removed immediately and placed into the same fresh fixative for an additional 2 h at 48C. Subsequently, the brains were
+
+Table 2 Oligonucleotide primers used in the quantitative reverse transcriptase PCR and semiquantitative reverse transcriptase PCR experiments
+
+Subunits
+
+Subunits primer sequences
+
+Expected size (bp)
+
+Annealing temperature (8C)
+
+R1
+
+NR2A
+
+NR2B
+
+NR2C
+
+b-actin
+
+50-ATGGCATCATCGGACTTCAG-30 50-GGGCTCTTGGTGGATTGTCA-30 50-ATTCATCCCTTCGTTGGTTG-30 50-GCTATGGGCAGGCAGAGAAG-30 50-GTGGGCACTGAGGACTTGTT-30 50-TGTACGACATCAGCGAGGAC-30 50-TCGTATTCCTCCAGCACCTT-30 50-GATCCAGCCACTCACCGTAG-30 50-TGGTGGGTATGGGTCAGAAGGACTC-30 50-CATGGCTGGGGTGTTGAAGGTCTCA-30
+
+431
+
+395
+
+319
+
+300
+
+265
+
+58
+
+56
+
+56.3
+
+55
+
+57.3
+
+Copyright © European Society of Anaesthesiology. Unauthorized reproduction of this article is prohibited.
+
+Effect of ketamine on NMDA receptor subunit expression Han et al. 183
+
+X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C 3 V C 1 y 0 a b g g Q Z X d g G 2 M w Z L e = o n 1 1 2 0 2 0 2 3
+
+r
+
+i
+
+i
+
+l
+
+f
+
+j
+
+l
+
+I
+
+/
+
+/
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / e a n a e s t h e s o o g y
+
+j
+
+i
+
+l
+
+b y B h D M 5 e P H K a v 1 z E o u m 1 Q N 4 a + k J L h E Z g b s I H o 4
+
+f
+
+t
+
+f
+
+placed into 30% (w/v) sucrose solution in 0.1 mol l(cid:1)1 phos- phate buffer (pH 7.4) overnight at 48C (the sucrose solution contained 0.02% NaN3), and then cut serially into 30 mm thick coronal sections by the use of a freezing microtome (Kryostat 1720; Leitz, Mannheim, Germany). The sections were placed into five different dishes accord- ing to their numerical order while cutting (e.g. sections 1 and 7 in dish 1; sections 2 and 8 in dish 2; sections 3 and 9 in dish 3; sections 4 and 10 in dish 4; and sections 5 and 11 in dish 5). Each dish usually contained 28–32 sections. All sections were washed carefully with 0.01 mol l(cid:1)1 PBS. The sections in the first three dishes were used for immu- nohistochemistry for NR2A–2C. Briefly, the sections were incubated at 48C sequentially with: a mixture of rabbit anti-NR2A, 2B and 2C serum (1 : 200 dilution; Elek mol- nar) for 24 h; biotinylated goat antirabbit immunoglobulin G (1 : 200 dilution; Vector) for 2 h; and avidin-labelled horseradish peroxidase compound (1 : 100 dilution; Vector) for 1 h. The diluent used for all antibodies was 0.05 mol l(cid:1)1 PBS containing 5% (v/v) normal donkey serum, 0.5% (v/v) Triton X-100, 0.05% (w/v) sodium azide (NaN3) and 0.25% (w/v) carrageenan (pH 7.3). In the fourth dish, normal rabbit serum was used instead of rabbit anti-NR2A, 2B and 2C serum, and the following steps were the same as mentioned above. The fifth dish was used for Nissl stain- ing in order to locate a positive construction. The sections were rinsed at least three times in 0.01 mol l(cid:1)1 PBS (pH 7.4) after each incubation, for at least 10 min. The sections were coloured with diaminobenzidine (DAB) and H2O2, then sections were mounted onto clean glass slides, air dried and cover-slipped with a mixture of 50% (v/v) glycerin and 2.5% (w/v) triethylene diamine (antifading agent) in 0.01 mol l(cid:1)1 PBS. Finally, the sections were studied under a microscope.
+
+Fig. 1
+
+) n
+
+i t c a - β / ( s l e v e l
+
+A N R m e v i t a l e R
+
+120
+
+100
+
+80
+
+60
+
+40
+
+20
+
+0
+
+120
+
+100
+
+80
+
+60
+
+40
+
+20
+
+0
+
+120
+
+100
+
+80
+
+60
+
+40
+
+20
+
+C
+
+C
+
+#
+
+#
+
+#
+
+
+
+
+
+
+
+K1
+
+$
+
+K1
+
+
+
+K2
+
+NR1
+
+
+
+$
+
+K2
+
+NR2A
+
+
+
+
+
+K3
+
+
+
+K3
+
+
+
+$
+
+7d 28d
+
+
+
+K4
+
+$
+
+
+
+K4
+
+
+
+0
+
+Results Semiquantitative reverse-transcriptase PCR and quantitative real-time reverse-transcriptase PCR Reverse transcriptase PCR revealed mRNA expression of NR1, NR2A, NR2B and NR2C receptor subtypes as well as the b-actin in the rat hippocampus. The size of the bands for each receptor corresponded to the expected cDNA fragment size based on the choice of oligonucleo- tide primers (Table 2).
+
+120
+
+100
+
+80
+
+60
+
+40
+
+C
+
+#
+
+K1
+
+
+
+K2
+
+NR2B
+
+
+
+K3
+
+
+
+K4
+
+
+
+20
+
+Ketamine treatment caused a marked increase in NR1, NR2A, NR2B and NR2C mRNA expressions at PND 7 when compared with the control (P < 0.05; Figs 1 and 2). At PND 28, ketamine treatment resulted in a different pattern of NMDA receptor mRNA expression from that at PND 7. Moreover, we also observed that the expres- sion of NR2A mRNA was significantly increased not only at PND 7 but also at PND 28 (P < 0.05; Figs 1 and 2), but no significant change was observed in NR1, NR2B or NR2C mRNA expression at PND 28 when compared with the control group (P > 0.05; Figs 1 and 2). More- over, no change was found among groups K1–K4, so we
+
+0
+
+C
+
+K1
+
+K2
+
+K3
+
+K4
+
+NR2C
+
+Histogram summary for relative expression levels of NR1, NR2A, NR2B and NR2C mRNA in the hippocampus of PND 7 and PND 28 rats after administration of ketamine (n ¼ 16, mean (cid:2) SE). C, control group; K1– K4, ketamine-treated groups. (cid:3)P < 0.05, compared with control group at PND 7; $P < 0.05, compared with control group at PND 28; and #P < 0.05, control group at PND 7 compared with the control group at PND 28. PND, postnatal day.
+
+Copyright © European Society of Anaesthesiology. Unauthorized reproduction of this article is prohibited.
+
+184 European Journal of Anaesthesiology 2010, Vol 27 No 2
+
+X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C 3 V C 1 y 0 a b g g Q Z X d g G 2 M w Z L e = o n 1 1 2 0 2 0 2 3
+
+r
+
+i
+
+i
+
+l
+
+f
+
+j
+
+l
+
+I
+
+/
+
+/
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / e a n a e s t h e s o o g y
+
+j
+
+i
+
+l
+
+b y B h D M 5 e P H K a v 1 z E o u m 1 Q N 4 a + k J L h E Z g b s I H o 4
+
+f
+
+t
+
+f
+
+Fig. 2
+
+Electrophoresis strip of NR1, NR2A, NR2B and NR2C mRNA expression in the hippocampus of PND 7 and PND 28 rats after administration of ketamine. The expression of b-actin mRNA was used as an internal control. C, control group; K1–K4, ketamine-treated groups (see Materials and methods). PND, postnatal day.
+
+concluded that the dosage and schedule of exposure might not influence the effects of ketamine.
+
+In addition, NR1, NR2A and NR2C subtype receptor mRNA expression at PND 28 exhibited a significant increase when compared with PND 7 in the control group, but NR2B exhibited a reverse trend (P < 0.05; Figs 1 and 2).
+
+subgroups was higher than that in the control group: NR2A (control, 1.7 (cid:2) 0.1; K1, 7.2 (cid:2) 3.5; K3, 9.9 (cid:2) 4.3), NR2B (control, 1.2 (cid:2) 0.1; K1, 8.8 (cid:2) 3.4; K3, 9.5 (cid:2) 4.5) and NR2C (control, 3.8 (cid:2) 1.5; K1, 16.2 (cid:2) 6.2; K3, 16.8 (cid:2) 6.8). At PND 28, only the NR2A subtype receptor expression was significantly increased in the K1 and K3 groups com- pared with the control group: NR2A (control, 10.8 (cid:2) 3.2; K1, 28.5 (cid:2) 4.5; K3, 32.2 (cid:2) 5.4). There was no significant difference between the K1 and K3 subgroups.
+
+Discussion In the present study, we observed that neonatal rats receiving ketamine, administered for short periods and at moderate doses, could upregulate the NR1, NR2A, 2B and 2C receptor subtype mRNA and NR2A, 2B and 2C receptor subtype protein at PND 7, and the NR2A recep- tor mRNA and protein expression increase persisted to PND 28. It is well accepted that NMDA receptor antagonists can induce an increase in some NMDA recep- tor subtype mRNA expression during critical periods of [10,11]. Chronic treatment of cultured development neurons from neonatal rat brains with amino-phosphono- pentanoate 5 (AP-5), an NMDA receptor antagonist, increased NR2B mRNA expression, as well as NR1 and NR2A/B polypeptides [12]. Also, increased expression of excitatory amino acid receptor subunit mRNA may con- tribute to the enhanced vulnerability to excitotoxic injury that has been observed after MK-801 treatment [13].
+
+Previous studies [14,15] showed that NMDA receptors participated in central nervous system (CNS) regulation and appeared to regulate the excitatory synaptic trans- mission and synaptic plasticity underlying learning and in glutamate transmission, memory. Abnormalities particularly involving overstimulation of NMDA recep- tors, have been implicated in apoptosis, abnormal axonal arborization and aberrant CNS development [16]. In 2002, Olney [17] observed that NMDA receptor anta- gonists interfered with CNS development and caused abnormalities in morphology and function. Neonatal rats receiving AP-5 or MK-801 during the first 2 weeks of life developed abnormal axonal arborizations in the retinal connections to the superior colliculus, interfering with normal visual responses [18]. Neurons with NMDA receptors are exquisitely sensitive to overstimulation, and they are similarly sensitive to understimulation during synaptogenesis. Too much NMDA receptor stimulation triggers excitotoxic neurodegeneration, but too little triggers apoptotic neurodegeneration [17].
+
+Immunohistochemistry of NR2A, 2B and 2C subtype protein Representative photomicrographs of NR2A, 2B and 2C subtype receptor staining and statistical analysis with the Student’s t-test among the groups are presented in Fig. 3. At PND 7, NR2A, 2B and 2C subtype receptor expression was observed mainly in the hippocampus, and the corresponding receptor expression in the K1 and K3
+
+Ketamine treatment may alter glutamatergic synaptic transmission through NMDA receptors and contributes to the upregulation of NMDA receptor mRNA expres- sion, but how increased expression of excitatory amino acid receptor subunit mRNA contributes to excitotoxic injury and neuroapoptosis remains controversial. Some researchers have found that glutamate binding to NMDA
+
+Copyright © European Society of Anaesthesiology. Unauthorized reproduction of this article is prohibited.
+
+Effect of ketamine on NMDA receptor subunit expression Han et al. 185
+
+Fig. 3
+
+X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C 3 V C 1 y 0 a b g g Q Z X d g G 2 M w Z L e = o n 1 1 2 0 2 0 2 3
+
+r
+
+i
+
+i
+
+l
+
+f
+
+j
+
+l
+
+I
+
+/
+
+/
+
+w w . c o m / e a n a e s t h e s o o g y
+
+j
+
+i
+
+l
+
+b y B h D M 5 e P H K a v 1 z E o u m 1 Q N 4 a + k J L h E Z g b s I H o 4
+
+f
+
+t
+
+f
+
+Representative photomicrographs of brain sections showing N-methyl-D-aspartate receptor subtype neurons in the CA1 region of the hippocampus in rat brains in the control (a, d, g), K1 (b, e, h) and K3 (c, f, i) groups. (m and n) The statistical analysis of NMDA receptor subtype neurons in different groups on PND 7 and PND 28. Ketamine (K1 and K3) produced much higher expressions of NR2A, 2B and 2C on PND 7, and NR2A on PND 28 than the control groups; scale bar ¼ 100 mm in (l) for (a)–(l). (cid:3)Groups K1 and K3 are statistically significantly different (P < 0.05) from group C in the CA1 region on PND 7 and PND 28. NMDA, N-methyl-D-aspartate; PND, postnatal day.
+
+Copyright © European Society of Anaesthesiology. Unauthorized reproduction of this article is prohibited.
+
+186 European Journal of Anaesthesiology 2010, Vol 27 No 2
+
+X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C 3 V C 1 y 0 a b g g Q Z X d g G 2 M w Z L e = o n 1 1 2 0 2 0 2 3
+
+r
+
+i
+
+i
+
+l
+
+f
+
+j
+
+l
+
+I
+
+/
+
+/
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / e a n a e s t h e s o o g y
+
+j
+
+i
+
+l
+
+b y B h D M 5 e P H K a v 1 z E o u m 1 Q N 4 a + k J L h E Z g b s I H o 4
+
+f
+
+t
+
+f
+
+receptors caused Ca2þ influx that activates second mes- sengers, thus regulating neuronal migration, differen- tiation and synaptic plasticity [19–22]. High concen- trations of glutamate resulted in too much Ca2þ influx, causing excitotoxicity. Here, we showed that ketamine could increase mRNA expression of NMDA receptor subtypes. However, if this block is eliminated, the increased expression of NMDA receptors might result in increased glutamate binding, thus inducing excessive Ca2þ influx. This altered level of Ca2þ influx could lead to neuroapoptosis. Further studies on ketamine-induced changes in apoptosis and Ca2þ-binding proteins are necessary to elucidate this possibility.
+
+Glutamate and NMDA receptors mediate a variety of complicated biological processes such as induction, generation, differentiation, apoptosis, migration, synaptic formation and neural network establishment [2,14– 15,17,23]. Synaptic and extrasynaptic NMDA receptors have fundamentally different effects on the fate of neurons. Synaptic NMDA receptors promote survival, whereas extrasynaptic NMDA receptors trigger neuronal degeneration and cell death [24,25]. Therefore, an increase in the expression of some NMDA receptor subunits might result in changes in subunit composition and possibly influence all of these developmental pro- cesses. Although the mechanisms underlying upregula- tion of expression were not clear, Wang et al. [26] suggested that increased NMDA receptor expression might be due to an increased rate of transcription or decreased rate of degradation.
+
+Taken together, these studies suggest that neonatal animals receiving chronic NMDA receptor antagonists developed abnormal neuronal structure and altered CNS function. Our present study demonstrated that ketamine, administered for short periods and at clinical application doses, induced changes in NMDA receptor subunit com- position and increased some NMDA receptor subtype expressions in neonatal rats, and this effect persisted to PND 28. The expressions of NMDA receptor subunits showed a period specificity at both the transcriptional and translational levels. Ketamine as a kind of NMDA recep- tor antagonist might change the period-specific expres- sion of some NMDA receptor subunits.
+
+3 Ikonomidou C, Olney JW, Bosch F, et al. Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 1999; 283:70–74.
+
+4 Jevtovic-Todorovic V, Hartman RE, Izumi Y, et al. Early exposure to common
+
+anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 2003; 23:876–882.
+
+5 Luk KC, Kennedy TE, Sadikot AF. Glutamate promotes proliferation of
+
+striatal neuronal progenitors by an NMDA receptor-mediated mechanism. J Neurosci 2003; 23:2239–2250.
+
+6 Mellon RD, Simone AF, Rappaport BA. Use of anesthetic agents in neonates and young children. Anesth Analg 2007; 104:509–520. 7 Zimmermann M. Ethical guidelines for investigations of experimental pain in
+
+conscious animals. Pain 1983; 16:109–110.
+
+8 Mickly GA, Remmers-Roeber DR, Crouse C. Ketamine blocks a taste- mediated conditioned motor response in perinatal rats. Pharmacol Biochem Behav 2000; 66:547–552.
+
+9 Wolfensohn S, Lloyd M. Handbook of laboratory animal management and
+
+welfare. Oxford, UK: Wiley-Blackwell Inc.; 2003; 185–186.
+
+10 Trevisan L, Fitzgerald LW, Brose N, et al. Chronic ingestion of ethanol up- regulates NMDAR1 receptor subunit immunoreactivity in rat hippocampus. J Neurochem 1994; 62:1635–1638.
+
+11 Hinoi E, Fujimori S, Nakamura Y, et al. Constitutive expression of
+
+heterologous N-methyl-D-aspartate receptor subunits in rat adrenal medulla. J Neurosci Res 2002; 68:36–45.
+
+12 Follesa P, Ticku MK. NMDA receptor upregulation: molecular studies in cultured mouse cortical neurons after chronic antagonist exposure. Neuroscience 1996; 16:2172–2178.
+
+13 Wilson MA, Kinsman SL, Johnston MV. Expression of NMDA receptor subunit mRNA after MK-801 treatment in neonatal rats. Dev Brain Res 1998; 109:211–220.
+
+14 Kato K, Li ST, Zorumski CF. Modulation of long-term potentiation induction
+
+in the hippocampus by NMDA-mediated presynaptic inhibition. Neuroscience 1999; 92:1261–1272.
+
+15 Hudspith MJ. Glutamate: a role in normal brain function, anesthesia,
+
+analgesia and CNS injury. Br J Anesth 1997; 78:731–747.
+
+16 Haberny KA, Paule MG, Scallet AC, et al. Ontogeny of the N-methyl-D-
+
+aspartate (NMDA) receptor system and susceptibility to neurotoxicity. Toxicol Sci 2002; 68:9–17.
+
+17 Olney JW. New insights and new issues in developmental neurotoxicology.
+
+Neurotoxicology 2002; 23:659–668.
+
+18 Simon DK. NMDA receptor antagonists disrupt the formation of a
+
+mammalian neural map. Proc Natl Acad Sci U S A 1992; 89:10593– 10597.
+
+19 Akopian A, Witkovsky P. Calcium and retinal function. Mol Neurobiol 2002;
+
+2:113–132.
+
+20 Holt M, Cooke A, Wu MM, et al. Bulk membrane retrieval in the synaptic terminal of retinal bipolar cells. J Neurosci 2003; 23:1329–1339. 21 Weiler R, Janssen BU. Spinule-type neurite outgrowth from horizontal cells
+
+during light adaptation in the carp retina: an actin-dependent process. J Neurocytol 1993; 22:129–139.
+
+22 Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodeling. Nat Rev Mol Cell Biol 2003; 4:517–529.
+
+23 Behar TN, Scott CA, Greene CL, et al. Glutamate acting at NMDA
+
+receptors stimulates embryonic cortical neuronal migration. J Neurosci 1999; 19:4449–4461.
+
+24 Wittmann M, Bengtson CP, Bading H. Extrasynaptic NMDA receptors:
+
+mediators of excitotoxic cell death. Pharmacol Cereb Ischemia 2004;253–266.
+
+25 Jiang Q, Gu Z, Zhang G, et al. NMDA receptor activation results in
+
+regulation of extracellular signal-regulated kinases by protein kinases and phosphatases in glutamate-induced neuronal apoptotic-like death. Brain Res 2000; 887:285–292.
+
+26 Wang C, Sadovova N, Fu X, et al. The role of the N-methyl-D-aspartate
+
+Acknowledgement This work was supported by grants from the National Natural Science Foundation of China (nos 30470556 and 30570683).
+
+receptor in katemine-induced apoptosis in rat forebrain culture. Neuroscience 2005; 132:967–977.
+
+References 1 Yamamura T, Harada K, Okamura A, Kemmotsu O. Is the site of action of ketamine anesthesia the N-methyl-D-aspartate receptor? Anesthesiology 1990; 72:704–710.
+
+2 Komuro H, Rakic P. Modulation of neuronal migration by NMDA receptors.
+
+Science 1993; 260:95–97.
+
+Copyright © European Society of Anaesthesiology. Unauthorized reproduction of this article is prohibited.

new_pdfs/10.1111_jcmm.13524.txt

@@ -0,0 +1,1281 @@
+Received: 22 September 2017 | Accepted: 12 December 2017 DOI: 10.1111/jcmm.13524
+
+O R I G I N A L A R T I C L E
+
+Propofol exposure during early gestation impairs learning and memory in rat offspring by inhibiting the acetylation of histone
+
+Jiamei Lin1,2 | Shengqiang Wang1 | Yunlin Feng1 | Weihong Zhao1 | Weilu Zhao1 | Foquan Luo1
+
+| Namin Feng1
+
+1Department of Anesthesiology, the First Affiliated Hospital, Nanchang University, Nanchang, China
+
+Abstract
+
+Propofol is widely used in clinical practice, including non-obstetric surgery in preg-
+
+2Department of Anesthesiology, the Eastern Hospital of the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
+
+nant women. Previously, we found that propofol anaesthesia in maternal rats during
+
+the third trimester (E18) caused learning and memory impairment to the offspring
+
+Correspondence Foquan Luo Email: lfqjxmc@outlook.com
+
+rats, but how about the exposure during early pregnancy and the underlying mecha-
+
+nisms? Histone acetylation plays an important role in synaptic plasticity.
+
+study, propofol was administered to the pregnant rats in the early pregnancy (E7).
+
+In this
+
+Funding information Natural Science Foundation of Jiangxi Province, Grant/Award Number: 20132BAB205022, 20171ACB20030; National Natural Science Foundation of China, Grant/Award Number: 81060093, 81460175
+
+The learning and memory function of the offspring were tested by Morris water
+
+maze (MWM) test on post-natal day 30. Two hours before each MWM trial, histone
+
+deacetylase 2 (HDAC2) inhibitor, suberoylanilide hydroxamic acid (SAHA), Senegenin
+
+(SEN, traditional Chinese medicine), hippyragranin (HGN) antisense oligonucleotide
+
+(HGNA) or vehicle were given to the offspring. The protein levels of HDAC2, acety-
+
+lated histone 3 (H3) and 4 (H4), cyclic adenosine monophosphate (cAMP) response
+
+element-binding protein (CREB), N-methyl-D-aspartate receptor (NMDAR) 2 subunit B (NR2B), HGN and synaptophysin in offspring’s hippocampus were determined by
+
+Western blot or immunofluorescence test.
+
+It was discovered that infusion with
+
+propofol in maternal rats on E7 leads to impairment of learning and memory in off-
+
+spring,
+
+increased the protein levels of HDAC2 and HGN, decreased the levels of
+
+acetylated H3 and H4 and phosphorylated CREB, NR2B and synaptophysin. HDAC2
+
+inhibitor SAHA, Senegenin or HGN antisense oligonucleotide reversed all the
+
+changes. Thus, present results indicate exposure to propofol during the early gesta- tion impairs offspring’s learning and memory via inhibiting histone acetylation.
+
+SAHA, Senegenin and HGN antisense oligonucleotide might have therapeutic value
+
+for the adverse effect of propofol.
+
+K E Y W O R D S
+
+histone deacetylase, learning and memory, pregnancy, propofol
+
+Jiamei Lin and Shengqiang Wang contributed equally to this work (co-first author).
+
+- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine. 2600 | wileyonlinelibrary.com/journal/jcmm J Cell Mol Med. 2018;22:2600–2611.
+
+15824934, 2018, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.13524 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+| 2601
+
+LIN ET AL.
+
+1 |
+
+I N T R O D U C T I O N
+
+mainly targeting HDAC2, probably has therapeutic potentialities for the learning impairment caused by neurodegenerative diseases.22-24
+
+Histone deacetylase inhibitors facilitated synaptic plasticity and
+
+Accumulating evidence indicates general anaesthetics exposure dur-
+
+memory by promoting the combination of CREB with CREB-binding
+
+ing pregnancy may cause neurotoxic effects and induce persistent cognitive dysfunction of offspring rats.1-3 Propofol is commonly used in pregnancy for non-obstetric surgery. Xiong et al4 showed that
+
+protein (CBP) domain, which subsequently activate CREB-mediated transcription.25-27 Our early researches showed that anaesthesia dur-
+
+ing early gestation damaged the neurons and reduced the expression
+
+anaesthesia with propofol on gestational day 18 (E18) associated
+
+of NR2B in hippocampus, thus leading to learning and memory impairments in offspring rats.11,12 In this study, we aim to investigate
+
+with the up-regulation of caspase-3 and the loss of neurons, as well
+
+as associated with the down-regulation synaptophysin expression in offspring rats’ hippocampus and caused persistent spatial
+
+whether histone acetylation involves in the cognitive function
+
+learning
+
+impairment induced by propofol anaesthesia during early pregnancy.
+
+impairment in offspring. Our previous study showed that propofol
+
+anaesthesia in the second trimester inhibits the cognitive function of
+
+the offspring that is related to down-regulation of the protein levels
+
+2 | M A T E R I A L S A N D M E T H O D S
+
+of brain-derived neurotrophic factor (BDNF) and synaptophysin in offspring hippocampus.5 Exposure to propofol for 5 hour caused
+
+2.1 | Drugs
+
+death of neurons and oligodendrocytes in foetal and neonatal NHP brain.6 However, little attention was paid to the early stage of gesta- tion, which is equivalent to the early pregnancy of human.7 It is
+
+All drugs were prepared just before use: propofol (Diprivan; AstraZe-
+
+Italy:
+
+jc393, 20 mL: 200 mg); 20% intralipid
+
+neca UK limited,
+
+(2B6061; Baxter, Deerfield, IL, USA); SAHA (Selleck Chemicals LLC,
+
+reported that 0.75% to 2% gestational women have to experience non-obstetric surgery due to various medical problems.8 This number
+
+Houston, TX, USA). HGN antisense was synthesized by Sangon Bio- tech (Shanghai, China) Co., Ltd. Senegenin (purity ≥ 98%) was pur-
+
+is increasing with the development of laparoscopic technique, and
+
+chased from Nanjing SenBeiJia Biological Technology Co., Ltd.
+
+the most common surgical procedure performed in the early preg- nancy is laparoscopy.9 It is reported that about 28% of the non- obstetric surgeries occurred in the first trimester.10 Our earlier stud-
+
+(Jiangsu province, China).
+
+Anti-b-actin and anti-rabbit
+
+IgG secondary antibody were
+
+obtained from Cell Signaling Technology (Cell Signaling Tech, MA,
+
+ies demonstrated that propofol, ketamine, enflurane,
+
+isoflurane or
+
+USA). Anti-CREB (Phospho S133), anti-NMDAR2B, anti-HDAC2,
+
+sevoflurane anaesthesia in the early pregnancy inhibits the cognitive
+
+antisynaptophysin, anti-Ac-H4K12 and anti-Ac-H3K14 antibodies
+
+function, damages hippocampal neurons, reduces NR2B mRNA and increased HGN mRNA levels in offspring rats’ hippocampus,11-14 but
+
+were purchased from Abcam (Abcam, Cambridge, MA, USA). Anti-
+
+HGN antibody was synthesized by Kitgen Bio-tech Co., Ltd.(Zhejiang
+
+the underlying pathogenesis needs to be clarified.
+
+province, China).
+
+is considered the cellular mecha- nism of memory formation and plays a role in synaptic plasticity.15
+
+Long-term potentiation (LTP)
+
+NR2B is an important positive regulator of learning and memory by promoting synaptic plasticity and LTP.16,17 The balance between
+
+2.2 | Animals
+
+The protocol in this study was approved by the institutional review
+
+positive and negative learning and memory-regulating genes and pro-
+
+board of the First Affiliated Hospital of Nanchang University on the
+
+teins is key to the formation, maintenance, as well as retrieval of
+
+Use of Animals in Research and Teaching. All the methods in this
+
+memory. HGN is a negative regulating protein that highly expresses
+
+study were performed in co-ordination with relevant guidelines and
+
+in hippocampus, acting suppression/clearance function in memory regulating.18 Inhibiting HGN by antisense oligonucleotide induces an
+
+regulations. Sprague Dawley (SD) rats were purchased from the ani-
+
+mal science research department of the Jiangxi Traditional Chinese
+
+increase in performance of Morris water maze and LTP. This indi-
+
+Medicine College (JZDWNO: 2011-0030; Nanchang, Jiangxi,China).
+
+cates that HGN negatively regulates synaptic plasticity and LTP and
+
+The learning and memory functions of
+
+the parental
+
+rats were
+
+plays negative regulating role in the formation and maintenance of
+
+assessed using the Morris water maze (MWM) system before mating,
+
+memory.
+
+so that to minimize the hereditary difference. Animals were housed
+
+Persistent changes in synapses, which based on appropriate gene
+
+separately under standard laboratory conditions with 12:12 light/ dark cycle, 24 (cid:1) 1°C and had free access to tap water. Two female
+
+transcription and subsequent protein synthesis, are the structural basis of learning and memory processes.19 Both compact chromatin
+
+rats in cages with one male rat per cage for mating. Pregnancy was
+
+structure and the accessibility of DNA to target genes can be modu-
+
+diagnosed by the sign of vaginal plug.
+
+lated by chromatin remodelling, in particular, histone tail acetylation, thus to regulate their expression.20,21 Histone acetylation regulates
+
+by acetyltransferases (HATs) and histone deacetylases (HDACs).
+
+2.3 | Drug treatment
+
+HATs serve as transcriptional activators, whereas HDACs serve as
+
+rats received intravenous infusion of propo- (n = 10 dams) with the rate of 20 mg kg(cid:3)1 h(cid:3)1 for 4 hours,
+
+On E7, pregnant
+
+transcriptional repressors. Increased HDAC activity had been linked
+
+fol
+
+to neurodegeneration. Growing evidence indicated that SAHA, which
+
+15824934, 2018, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.13524 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+2602 |
+
+LIN ET AL.
+
+equal volume of saline (n = 10 dams) or intralipid (n = 5 dams),
+
+previously described,18,29 once daily for seven consecutive days
+
+before MWM trial.
+
+respectively.
+
+Electrocardiograms, saturation of pulse oximetry (SpO2) and tail
+
+non-invasive blood pressure were continuously monitored during
+
+2.4 | Morris water maze test
+
+maternal propofol exposure. Using heating lamp and temperature
+
+the rectal
+
+to monitor
+
+Spatial learning and memory were assessed by the MWM test from post-natal day 30 (P30) to P36 according to previously described5,30
+
+temperature and keep it at controller 37 (cid:1) 0.5°C. Arterial blood sampling from lateral caudal artery for
+
+with SLY-WMS Morris water maze test system (Beijing Sunny Instru- ments Co. Ltd., Beijing, China). Briefly, the trials start at 9 o’clock in
+
+blood gas analysis at the end of propofol anaesthesia. If the total time of SpO2 <95% and/or the systolic blood pressure <80% of the
+
+the morning in the MWM system with the pool was filled with
+
+baseline in excess of 5 minutes, the pregnant rat was got rid of the
+
+study, and other pregnant rats were chosen to supply the sample
+
+water to a height of 1.0 cm above the top of a 15-cm-diameter plat-
+
+size, so as to exclude the interfering effect of maternal hypotension
+
+form, in the second quadrant (target quadrant), and the water main- tained at 24 (cid:1) 1°C. The training trial was performed once a day for
+
+or hypoxia on cognitive function in the pup rats.
+
+six consecutive days. In each training trial, offspring rats were placed
+
+After delivery, the offspring rats born to the same pregnant rat
+
+were randomly subdivided into the SAHA, SEN, HGNA group and
+
+in the water facing the wall of the pool in the third quadrant, the
+
+their relative control groups (DMSO, NS(1) and NS(2) group, respec-
+
+farthest one from the target quadrant. The animals were allowed to
+
+tively; Figure 1). It has been proved that the acetylation level of his-
+
+search for the hidden platform or for 120 seconds. They were
+
+after
+
+increased 2 hour
+
+the tone in hippocampus obviously administration of HDAC inhibitor.27 Therefore, 90 mg kg(cid:3)1 SAHA (HDAC inhibitor), at a concentration of 0.6 lmol L(cid:3)1 dissolved into dimethyl sulphoxide (DMSO) was injected to the offspring in SAHA
+
+allowed to remain on the platform for 30 seconds when they found
+
+the platform and the time for the animal to find the platform was
+
+recorded as escape latency (indicating learning ability). For those
+
+who did not find the platform within 120 seconds, the animals were
+
+group by the intraperitoneal route at 2 hours before each MWM
+
+gently guided to the platform and allowed to stay there for 30 sec-
+
+onds, and their escape latency was recorded as 120 seconds. At the
+
+trial. The same volume of DMSO was given to the DMSO group.
+
+Senegenin, a kind of Chinese medicine, was proved to up-regulate
+
+end of the reference training (P37), the platform was removed. The
+
+offspring rats were allowed to perform spatial probe test (memory
+
+the expression of NR2B mRNA and protein, thus to mitigate cogni- tive dysfunction.28 So, 15 mg kg(cid:3)1 Senegenin and equal volume of saline were given intraperitoneally at 2 hours before each MWM
+
+function test) for 120 seconds. Times across the platform (platform
+
+crossing times,
+
+indicate memory function), the swimming trail and
+
+speed were automatically recorded by the system. The mean value
+
+trial to SEN or NS(1) groups, respectively. HGN antisense oligonu- cleotide (0.25 nmol lL(cid:3)1, 1.5 lL) or normal saline (1.5 lL) was injected to offspring’s hippocampus in HGNA or NS(2) group as
+
+of the platform crossing times, escape latency and speed of the off-
+
+spring born to the same pregnant rats was taken as the final results.
+
+F I G U R E 1 Experimental design. Pregnant dams were exposed to Propofol, 20% Intralipid or normal saline on E7, and the offspring were treated with SAHA, Senegenin, HGNA or vehicles two hours before behavioural testing. The number in parentheses represents the number of animals: F, female; M, male; SAHA, suberoylanilide hydroxamic acid, also known as vorinostat; DMSO, dimethyl sulphoxide; SEN, Senegenin; NS(1), Normal saline intraperitoneal injection; NS(2), Normal saline intrahippocampus injection; HGNA, HGN antisense oligonucleotide
+
+15824934, 2018, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.13524 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+| 2603
+
+LIN ET AL.
+
+green light, while the DAPI was performed by UV blue light. All images were recorded at 10 9 209 (Exp Acq-700mmm, Offset Acq-
+
+2.5 | Brain hippocampus harvest
+
+1, Gain Acq-1, Gamma Acq-300). The density of HDCA2 and p-
+
+The day after the MWM test, rats were anaesthetized with isoflu-
+
+rane and killed by cervical dislocation. Hippocampus tissues were
+
+CREB staining was conducted on the images using Image-Pro Plus
+
+harvested and stored in Eppendorf tubes that had been treated with 1% DEPC and were stored at (cid:3)80°C (for Western blot analyses) or
+
+6.0 (Media Cybernetics Inc., USA). The images were converted it into
+
+black and white pictures. After intensity calibration, hippocampal
+
+CA1 area was chosen to analyse and the integrated optical density
+
+immersed in 4% paraformaldehyde (for immunofluorescence assay).
+
+(IOD) was measured. IOD/Area was calculated as the protein expres-
+
+sion level.
+
+2.6 | Western blot analysis
+
+The hippocampus (n = 6, with three male and three female offspring
+
+2.8 | Statistical analysis
+
+rats from each group) were homogenized on ice in lysis buffer con-
+
+All analyses were performed with SPSS 17.0 software (SPSS, Inc.,
+
+taining a protease inhibitors cocktail. Protein concentration was
+
+Chicago, IL, USA). Data from escape latency in the MWM test were
+
+determined by the bicinchoninic acid protein assay kit. Protein sam- ples (20 lg) were separated by sodium dodecyl sulphate polyacry-
+
+subjected to a repeated measures two-way analysis of variance (RM
+
+lamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF
+
+two-way ANOVA) and were followed by least significant difference t
+
+membrane. The membranes were blocked by non-fat dry milk buffer for 1.5 h and then incubated overnight at 4°C with antihistone H3
+
+(LSD-t) analysis when a significant overall between-subject factor was found (P < 0.05). Data from Western blot and immunofluores-
+
+cence staining results were subjected to one-way ANOVA analysis.
+
+(acetyl K14)
+
+(1:10000), anti-
+
+(1:2000), antihistone H4 (acetyl K12)
+
+NMDAR2B antisynaptophysin (1:10000) and anti-b-actin (1:2000), respectively. Thereafter, the
+
+All data well provided for any of the variables. The LSD t test was
+
+anti-HGN (1:1000),
+
+(1:1000),
+
+used to determine the difference between groups. Statistical signifi- cance was declared at P < .05.
+
+membranes were washed three times with TBS-T buffer for 15 min-
+
+utes and incubated with the horseradish peroxidase (HRP)-conju-
+
+gated secondary antibody for 2 hours at room temperature. The
+
+3 | R E S U L T S
+
+immune complexes were washed three times with TBS-T buffer and
+
+detected using the ECL system (Millipore Corporation, MA, USA).
+
+3.1 | Physiological parameters of maternal propofol anaesthesia
+
+The images of Western blot products were collected and analysed
+
+(Wayne Rasband, National
+
+Institutes of Health,
+
+by ImageJ 1.50i
+
+During propofol infusion, the maternal body temperature, respiratory
+
+USA). The density of observed protein band was normalized to that of b-actin in the same sample. The results of offspring from all the
+
+rate, arterial oxygen saturation, heart rate and non-invasive blood
+
+other group were then normalized to the average values of normal saline control offspring (control+NS group) in the same Western blot.
+
+pressure were continuously monitored and recorded every five min-
+
+utes. No significant change in these physiological parameters had
+
+The mean expression level of all of the offspring born to the same
+
+been seen during propofol exposure (4 hours). Tail artery blood was
+
+collected from pregnant rats for blood gas analysis after propofol perfusion, and no significant difference (P > .05) was observed
+
+mother rat in the same group was calculated as the final expression
+
+level of the observed proteins.
+
+(Table 1). These results suggested that propofol has no side effect
+
+indicating the
+
+on the physiological parameters in pregnant rats,
+
+2.7 |
+
+Immunofluorescence staining
+
+Immunofluorescence staining was used to assess HDAC2 and phos-
+
+pho-CREB in the hippocampus of offspring rats after the MWM test. Hippocampus from offspring rats (n = 6, with three male and three
+
+T A B L E 1 Maternal arterial blood gas at the end of propofol exposure or normal saline (n = 10, mean (cid:1) SD)
+
+female offspring rats from each group) were fixated in paraformalde- hyde. Five-lm frozen sections of the hippocampus were used for
+
+Normal Saline exposure pregnant rats
+
+Propofol exposure pregnant rats
+
+Indexes
+
+the immunofluorescence staining. The sections were incubated with
+
+7.39 (cid:1) 0.04
+
+7.38 (cid:1) 0.05
+
+pH
+
+anti-HDAC2 (1:300) and anti-CREB (1:100) dissolved in 1% bovine serum albumin in phosphate-buffered saline at 4°C overnight. Then,
+
+94.00 (cid:1) 3.52
+
+97.17 (cid:1) 3.49
+
+PO2 (mm Hg)
+
+45.33 (cid:1) 2.88
+
+the sections were incubated with fluorescent-conjugated anti-rabbit
+
+44.83 (cid:1) 5.78
+
+PCO2 (mm Hg) HCO(cid:3) K+ (mmol L(cid:3)1) Na+ (mmol L(cid:3)1) Ca2+ (mmol L(cid:3)1)
+
+3 (mmol L(cid:3)1)
+
+secondary antibody (1:300) for 1 hour in the dark at room tempera-
+
+27.95 (cid:1) 3.21
+
+26.68 (cid:1) 2.32
+
+ture. Negative control sections were incubated with PBS as a substi-
+
+3.48 (cid:1) 0.29
+
+3.47 (cid:1) 0.39
+
+tute for primary antibody. Finally, the sections were wet mounted
+
+141.67 (cid:1) 1.03
+
+140.83 (cid:1) 1.47
+
+and viewed immediately using a inverted fluorescence microscope (2009)
+
+1.38 (cid:1) 0.05
+
+1.34 (cid:1) 0.03
+
+(Olympus, Japan). The target protein was red, and nuclei
+
+9.18 (cid:1) 0.99
+
+9.57 (cid:1) 0.55
+
+Glu
+
+were blue. The proteins of HDAC2 and p-CREB were excited by the
+
+15824934, 2018, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.13524 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+2604 |
+
+LIN ET AL.
+
+results of offspring rats in this study are likely caused directly by
+
+the data of offspring from normal saline and intralipid infusion group
+
+into one control group in the following data analysis. Propofol expo-
+
+propofol rather than secondary effects of maternal propofol infusion.
+
+sure increased escape latency, while decreased platform crossing
+
+times in offspring compared to the saline control condition (Fig- ure 3C,D, P < .05),
+
+3.2 | Physical features of the offspring
+
+indicating propofol anaesthesia on E7 impairs
+
+spatial learning and memory in offspring.
+
+The birth rate (total number of neonates born to each mother rat), sur-
+
+vival rate (survived more than 30 days), gender ratio (the ratio of
+
+SAHA, Senegenin and HGN antisense oligonucleotide have been
+
+shown to improve learning and memory by facilitating histone acety-
+
+females to males) and the average weight of the offspring on day P30
+
+lation, increasing NR2B expression and inhibiting HGN expression, respectively.18,27,28 Therefore, we assessed whether they can amelio-
+
+in propofol exposure group were not significantly different from nor-
+
+mal saline control group (Figure 2). Dyskinesia was not observed in
+
+either of the two groups. These results indicate that maternal propofol
+
+rate the learning and memory impairment caused by propofol expo-
+
+anaesthesia at the early pregnant stage (E7) has no significant effects
+
+sure during pregnancy. Based on the previous discovery on the pharmacodynamics,18,27,28 SAHA or Senegenin was intraperitoneally
+
+on physical development of offspring rats, indicating the differences in
+
+injected into the offspring 2 hours before each MWM test, while
+
+learning and memory observed in this study are caused by propofol
+
+HGN antisense oligonucleotide was injected into hippocampus
+
+exposure during pregnancy rather than physical differences.
+
+2 hours before each MWM test. The results showed that SAHA,
+
+Senegenin or HGN antisense oligonucleotide treatment ameliorated
+
+3.3 | and the ameliorating effect of SAHA, Senegenin and HGN antisense oligonucleotide
+
+Impaired learning and memory in offspring
+
+the cognitive function deficit caused by propofol exposure during pregnancy (Figure 4A-F, P < .05). SAHA, Senegenin or HGN anti-
+
+sense oligonucleotide had no obvious effect on the learning and
+
+There was no obvious difference in offspring between normal saline
+
+memory in offspring that had not exposed to propofol during preg-
+
+and intralipid infusion group (Figure 3C,D). Therefore, we merged
+
+nancy (Figure 4A-F).
+
+F I G U R E 2 Maternal propofol exposure had no effect on the physical features of the offspring rats. The physical features of the offspring rats between propofol exposure and normal saline control group had no significant difference (P > .05). A, The birth rate (average litter size, total number of neonates born to each mother rat). B, Survival rate of offspring (survived more than 30 days). C, Gender ratio (the ratio of females to males, gender composition). D, The average weight of the offspring on day P30
+
+15824934, 2018, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.13524 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+| 2605
+
+LIN ET AL.
+
+F I G U R E 3 Maternal Propofol exposure impaired learning and memory in offspring. Post-natal thirty days (P30), the learning and memory were assessed using the Morris water maze (mean (cid:1) SD). A, Escape latency (indicating learning ability) among control groups. B, Platform crossing times (indicating memory ability) among control groups. C, Propofol exposure increased escape latency in offspring compared to the saline control condition (*P < .05). No statistically significant difference was observed between the saline control and intralipid group. D, Propofol exposure decreased platform crossing times in offspring compared to the saline control condition (*P < .05). No significant difference was observed between the saline control and intralipid group
+
+F I G U R E 4 SAHA, SEN and HGNA treatment mitigated the learning and memory impairment (mean (cid:1) SD). A, Propofol exposure increased the escape latency in offspring compared to the control condition (*P < .05), and SAHA treatment significantly reversed the effect (#P < .05). B, SEN treatment significantly reversed the effect (#P < .05). C, HGNA treatment significantly reversed the effect (#P < .05). D, Propofol exposure decreased the platform crossing times in offspring compared with the control condition (*P<.05), and SAHA treatment reversed the effect (#P < .05). E, SEN treatment reversed the effect caused by propofol exposure (#P < .05). F, HGNA treatment reversed the effect caused by propofol exposure (#P < .05), and SAHA, SEN and HGNA treatment had no significant effect on learning and memory in offspring that were not exposed to propofol during pregnancy. Error bar = SD
+
+(HDACs) and histone acetyltransferases (HATs).33,34 HATs acetylate
+
+3.4 | Reduced histone acetylation levels and the mitigating effect of SAHA,Senegenin and HGN antisense oligonucleotide
+
+multiple lysine residues on histones, and different acetylated sites
+
+result in different downstream biological effects. H3K14 and H4K12
+
+acetylation have been shown to play a crucial part in learning, mem- ory and synaptic plasticity.35 The results showed that propofol expo-
+
+Histone deacetylation was implicated in memory impairments.31,32
+
+The acetylation of histone is regulated by histone deacetylases
+
+sure during pregnancy up-regulated HDAC2 protein expression in
+
+15824934, 2018, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.13524 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+2606 |
+
+LIN ET AL.
+
+offspring rat’s hippocampus (Figure 6, P < .05), whereas decreased
+
+and maintenance.37 NR2B is critical positive regulating factor,38 while HGN is considered as an important
+
+recognized as
+
+the acetylation levels of H3K14 and H4K12 significantly (Figure 5, P < .05). SAHA, Senegenin and HGN antisense oligonucleotide alle- viated these changes (Figures 5 and 6, P < .05). These results indi-
+
+factor.18 The
+
+negative
+
+results
+
+in this
+
+study
+
+showed that
+
+propofol anaesthesia during pregnancy resulted in decrease in NR2B protein (Figure 8A, P < .05), while increased the level of HGN protein (Figure 8B, P < .05), resulted in decreased ratio of NR2B/HGN in offspring rats’ hippocampus (Figure 8C, P < 0.05).
+
+cate that propofol anaesthesia during pregnancy inhibits histone acetylation in offspring rats’ hippocampus, which could be alleviated
+
+by SAHA, Senegenin or HGN antisense oligonucleotide.
+
+The ratio of NR2B/HGN was reversed significantly by SAHA,
+
+or HGN antisense
+
+oligonucleotide
+
+Senegenin P < .05).
+
+(Figure 8A-C,
+
+3.5 | Decreased phosphorylated CREB levels in hippocampus and the mitigating effect of SAHA, Senegenin and HGN antisense oligonucleotide
+
+3.7 | Down-regulated expression of synaptophysin in the hippocampus of offspring rats and the improving effect of SAHA, Senegenin and HGN antisense oligonucleotide
+
+Phosphorylation of CREB is recognized as a molecular marker of mem- ory processing in the hippocampus for spatial learning.36 Therefore, we
+
+investigated the phosphorylation of CREB in this study. The results
+
+showed that propofol anaesthesia during pregnancy resulted in decrease in phospho-CREB protein in offspring rats’ hippocampus.
+
+Synaptophysin plays an important
+
+role in the exocytosis of
+
+SAHA, Senegenin or HGN antisense oligonucleotide treatment allevi- ated the effects (Figure 7, P < .05). These results suggest that propofol
+
+synaptic vesicles and acknowledged as a marker of synaptic density.39 Synapse loss is closely associated with cognitive dys- function and learning impairment.40,41 The results showed that the
+
+anaesthesia during pregnancy on E7 can down-regulate the phosphory-
+
+lation of CREB in hippocampus of the offspring, whereas SAHA, Sene-
+
+protein level of synaptophysin in maternal propofol exposure
+
+lower
+
+than control condition (Figure 9),
+
+group was
+
+indicating
+
+genin or HGN antisense oligonucleotide ameliorates this effect.
+
+maternal propofol exposure on E7 impairs the synaptic plasticity in offspring rats’ hippocampus, whereas the level of synaptophysin
+
+3.6 | Decreased the ratio of NR2B/HGN in offspring rat’s hippocampus and the reversing effect of SAHA, Senegenin or HGN antisense oligonucleotide
+
+in SAHA, Senegenin or HGN antisense oligonucleotide-treated
+
+group was higher than propofol exposure group (Figure 9), sug-
+
+gesting that SAHA, Senegenin and HGN antisense oligonucleotide
+
+The balance between positive and negative regulating factors
+
+can reverse the down-regulated expression of
+
+synaptophysin
+
+of learning and memory plays a key role in the memory obtain
+
+caused by propofol.
+
+F I G U R E 5 Maternal propofol exposure reduced the level of histone acetylation and the reversed effect of SAHA, SEN and HGNA treatment. Acetylation level of H3K14 and H4K12 was detected by Western blot (mean (cid:1) SD). Maternal exposure to propofol decreased the acetylation level of H3K14 and H4K12 in offspring compared to the control condition (P < .001), and SAHA treatment significantly increased acetylated H3K14 (P = .016) and H4K12 (P = .003) levels; SEN treatment significantly increased acetylated H3K14 (P = .012) and H4K12 (P = .002) levels; HGNA treatment significantly increased acetylated H3K14 (P = .042) and H4K12 (P = .029) levels. The protein levels of acetylated H3K14 and H4K12 in propofol + SAHA, propofol + SEN or propofol + HGNA group were not significantly different from those in control + NS group (P > .05)
+
+15824934, 2018, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.13524 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+| 2607
+
+LIN ET AL.
+
+F I G U R E 6 Maternal propofol exposure increased the level of HDAC2 and the reversed effect of SAHA, SEN and HGNA treatment. HDAC2 protein level was determined by immunofluorescence (mean (cid:1) SD). Maternal exposure to propofol up-regulated the expression of HDAC2 protein in offspring compared to the control condition (P = .001). SAHA treatment significantly inhibited the expression of HDAC2 protein (P = .029); SEN treatment significantly decreased HDAC2 protein level (P = .032); HGNA treatment significantly decreased HDAC2 protein level (P = .006). The protein levels of acetylated HDAC2 in propofol+SAHA, propofol + SEN or propofol + HGNA group were not significantly different from those in control + NS group (P > .05)
+
+F I G U R E 7 Maternal propofol exposure decreased the level of phospho-CREB and the reversed effect of SAHA, SEN and HGNA treatment. Phospho-CREB protein level was determined by immunofluorescence (mean (cid:1) SD). Maternal exposure to propofol decreased the expression of phospho-CREB protein in offspring compared to the control condition (P < .001). SAHA, SEN and HGNA treatment significantly increased phospho-CREB protein level (P = .006, P = .006, P = .016, respectively). The protein levels of phospho-CREB in propofol + SAHA, propofol + SEN or propofol + HGNA group were not significantly different from those in control + NS group (P > .05)
+
+4 | D I S C U S S I O N
+
+H3K14 and H4K12 and the phosphorylation of CREB, down-regu-
+
+lates the expression of NR2B, up-regulates the expression of HGN
+
+The current study findings suggest that pregnant rats propofol
+
+and decreases the ration of NR2B/HGN and the expression of
+
+anaesthesia on E7 impairs the learning and memory in offspring rats,
+
+synaptophysin. SAHA, Senegenin and HGN antisense oligonucleotide
+
+increases the expression of HDAC2,
+
+inhibits the acetylation of
+
+ameliorate all these changes.
+
+15824934, 2018, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.13524 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+2608 |
+
+LIN ET AL.
+
+F I G U R E 8 Maternal propofol exposure broken the balance between NR2B and HGN and the mitigating effect of SAHA, SEN and HGNA treatment. Expression of NR2B and HGN protein was detected by Western blot (mean (cid:1) SD). A, Maternal exposure to propofol decreased the NR2B protein level (P < .001). B, Maternal exposure to propofol increased the HGN protein level (P < .001). C, The ratio of NR2B/HGN was significantly reduced in offspring compared with the control condition (P < .001). SAHA, SEN and HGNA treatment significantly increased NR2B protein level and decreased HGN protein level and reversed the ratio of NR2B/HGN (P < .05). The protein levels of NR2B and HGN and the ratio of NR2B/ HGN in propofol + SAHA, propofol + SEN or propofol + HGNA group were not significantly different from those in control + NS group (P > .05)
+
+Maternal body temperature, respiratory rate, saturation of pulse
+
+the 2nd trimester of pregnancy) may cause learning deficits in the rat offspring.42 Our previous study showed that ketamine, propofol
+
+oximetry, heart rate and non-invasive blood pressure were continu-
+
+ously monitored during propofol exposure, and no obvious abnor-
+
+and enflurane anaesthesia during early gestation (on gestation day 7) induced learning and memory impairment in offspring rats,11-14 asso-
+
+mality was observed. Furthermore, maternal artery blood gases were
+
+analysed after the 4 hours propofol infusion and showed no signifi-
+
+ciated with hippocampal neuron injury, NR2B receptor subunit
+
+cant change (Table 1). Moreover, there was no significant difference
+
+reduction and increased level of HGN mRNA.
+
+in birth rate, offspring survival rate, the ratio of sex or basic physical
+
+How does propofol anaesthesia during pregnancy impair the
+
+development of offspring between propofol and saline group. These results suggested that the impaired learning and memory of the rats’
+
+learning and memory in offspring? The consolidation and mainte-
+
+nance of memory require specific genes expression, and histone
+
+acetylation promotes the expression of these genes, while histone deacetylation represses their expression.21,43 Histone deacetylases
+
+offspring may be not caused by pathological disorders but caused by
+
+the pregnant rats propofol anaesthesia in the current study.
+
+Several animal studies showed that anaesthetics exposure during gestation induced apoptosis in foetal brain.1,2,40 Xiong et al and our
+
+(HDACs) inhibit the expression of these genes, while histone acetyl- transferases (HATs) promote their expression.44 Among the HDACs,
+
+HDAC2 was implicated in learning and memory, it negatively regu-
+
+previous study showed that prenatal propofol exposure resulted in learning and memory deficit in offspring.4,5 While these studies
+
+lates synaptic plasticity and memory process by suppressing memory specific genes’ expression, and loss function of HDAC2 facilitates synaptic plasticity and learning and memory.32 Graff et al showed
+
+mainly focused on the second and third trimester, there is little
+
+information in relation to the effect of propofol anaesthesia during
+
+early pregnancy on the cognitive function in offspring. Because some
+
+that HDAC2 overexpression reduced the histone acetylation of his-
+
+tone and inhibited the expression of memory specific genes. HDAC2
+
+of non-obstetric surgeries during pregnancy occurred in the first tri- mester,10 our current study mainly focus on gestation day 7, which distinct with the exposure time-point in previous studies,4-6 the dif-
+
+is significantly enriched near the histones of genes shown to play a
+
+key role in learning, memory and synaptic plasticity, such as H2B
+
+lysine (K) 5, H3K14, H4K5, and H4K12. Reversing the build- up of
+
+ferent exposure time-point may alter the vulnerability to general
+
+anaesthetics for
+
+the developing brain. Halothane and enflurane
+
+HDAC2 by short-hairpin-RNA -mediated knockdown activated these
+
+exposure on gestation day 6 and 10 (amount to the early and early
+
+genes, reinstated structural and synaptic plasticity and abolished the
+
+15824934, 2018, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.13524 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+| 2609
+
+LIN ET AL.
+
+synaptic plasticity and long-term potential (LTP). Not only the activa-
+
+tion of positive regulatory mechanisms that favour memory storage
+
+but also the removal of inhibitory constraints that prevent memory storage are required for long-lasting of synaptic plasticity.37 Negative
+
+regulators play an important role in the formation and maintenance
+
+of memory. Hippyragranin (HGN) is a protein which expresses in rat hippocampus and involves in negative memory regulation.18 Down-
+
+regulation of HGN by antisense oligonucleotide in the hippocampal
+
+CA1 region caused enhanced learning and memory as well as ele-
+
+vated LTP. Therefore, we hypothesize that the balance between the
+
+positive regulator NR2B and negative regulator HGN plays a pivotal
+
+role in the learning and memory process. Present study showed that
+
+Senegenin treatment reversed the protein levels of NR2B, HGN and
+
+the ratio of NR2B/HGN, as well as enhanced learning and memory,
+
+which in accordance with the previous research that Senegenin
+
+attenuates cognitive impairment by up-regulating expression of hip- pocampal NR2B expression in rats.28 While treatment with HGN
+
+antisense oligonucleotide inhibited the expression of HGN protein,
+
+reversed the ratio of NR2B/HGN and the learning and memory impairment as previous report.18
+
+Transcription factor CREB (cAMP response element-binding pro-
+
+tein) shows an important role in synaptic plasticity underlying learn- ing memory.48,49 CREB is a critical mediator of cAMP- and calcium-
+
+F I G U R E 9 Maternal propofol exposure decreased the expression of synaptophysin and the reversed effect of SAHA, SEN and HGNA treatment. Synaptophysin level was determined by Western blot (mean (cid:1) SD). Maternal exposure to propofol decreased the expression of synaptophysin in offspring compared to the control condition (P = .002). SAHA, SEN and HGNA treatment significantly increased synaptophysin protein level (P = .026, P = .007, P = .027, respectively). The protein levels of synaptophysin in propofol + SAHA, propofol + SEN or propofol + HGNA group were not significantly different from those in control + NS group (P > .05)
+
+inducible transcription, whereas the phosphorylation of serine 133
+
+is its main
+
+in its kinase-inducible domain (KID)
+
+(phospho-Ser133)
+
+transactivating form. Phospho-Ser133 plays a role in CREB to bind the KIX domain of the coactivators CBP and p300 (CBP/p300).50 Vecsey et al25 demonstrated that enhancement of hippocampus-
+
+dependent memory and synaptic plasticity by HDAC inhibitors was
+
+relied on the binding of CREB and CREB-binding protein (CBP),
+
+which induced robust activation of gene transcription afterwards.
+
+The activity of CREB is essential to the gene transcription of NR2B,
+
+neurodegeneration-associated memory impairments. Abolished the memory impairments in connection with neurodegeneration.35 Our
+
+and expression of NR2B relies on the binding of p-CREB to its bind- ing site at the promoter of the NR2B gene.51 Fujita et al27 have
+
+demonstrated that HDAC inhibitor up-regulated the expression of
+
+isoflurane anaesthesia during
+
+earlier study suggested that maternal
+
+acetylated histones and NR2B mRNA in the hippocampus, and up-
+
+third trimester impairs the spatial
+
+learning and memory of the off-
+
+spring rats, and its mechanism in connection with the up-regulation
+
+regulated expression of acetylated histones was accompanied by
+
+enhanced binding of p-CREB to its binding site at the promoter of the NR2B gene.27 These findings indicated that HDAC inhibitor pro-
+
+of HDAC2 mRNA and subsequent inhibits the expression of CREB mRNA and NR2B, while HDAC2 inhibition reversed these changes.30
+
+motes learning and memory by increasing the acetylation of histone
+
+Consistent to our previous study, our results suggest that maternal
+
+propofol anaesthesia on E7 impairs learning and memory in offspring
+
+and the phosphorylation of CREB, and subsequent increase of NR2B
+
+rats, causes the overexpression of HDAC2 and inhibits the acetyla-
+
+expression. Our previous study has demonstrated that isoflurane
+
+tion of H3K14 and H4K12, and these effects were reversed by
+
+anaesthesia during the third trimester impaired learning and memory in offspring rats via “HDAC2-CREB-NR2B” pathway.30
+
+SAHA. Senegenin and HGN antisense oligonucleotide treatment also
+
+showed similar effects.
+
+Synaptophysin is a synaptic protein marker and provides a struc- tural basis for synaptic plasticity.52 Decrease in synaptophysin is implicated in learning and memory impairment.1,4,5 Graff et al35
+
+NMDA receptors play a crucial role in neuronal development and circuit formation. Subunit NR2B is critical to learning and memory.45
+
+It is reported that the enhancement of pre-frontal cortical long-term
+
+demonstrated that HDAC2 overexpression reduced synaptophysin
+
+potentiation (LTP) and working memory via the up-regulate expres- sion of NR2B specifically in the forebrain region.46 While decreased
+
+protein level and caused memory impairments, HDAC2 inhibition
+
+reversed the effects. As synaptophysin is one of the CREB target genes,53 we detected the expression of synaptophysin in the present
+
+expression of NR2B subunit suppressed NMDA-dependent long- learning.47 Therefore,
+
+term potentiation (LTP) and impaired spatial
+
+study. The results showed that propofol anaesthesia during preg- nancy reduced the protein level of synaptophysin in offspring’s
+
+NR2B acts as a positive regulator in memory process by promoting
+
+15824934, 2018, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.13524 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+2610 |
+
+LIN ET AL.
+
+C O N F L I C T O F I N T E R E S T S
+
+hippocampus, whereas SAHA, Senegenin and HGN antisense
+
+oligonucleotide mitigated the reduced synaptophysin levels; mean-
+
+The authors declare that they have no conflict of interest.
+
+while, the increased expression of synaptophysin was companied
+
+with decreased HDAC2 protein level, increased histone acetylation
+
+A U T H O R C O N T R I B U T I O N S
+
+and CREB phosphorylation level. The BDNF-TrkB signalling pathway
+
+is one of the downstream regulating targets of histone acetylation,
+
+F.Q.L. and J.M.L. conceived and designed the experiments. J.M.L.,
+
+so BDNF-TrkB signalling pathway may be one of the underlying
+
+Y.L.F. and S.Q.W. performed the experiments. J.M.L and F.Q.L. anal-
+
+downstream mechanisms of learning and memory deficits induced by
+
+ysed the data. J.M.L. contributed reagents/materials/analysis tools.
+
+propofol exposure during early gestation.
+
+It
+
+is confirmed that
+
+J.M.L and F.Q.L. wrote the article. All the authors read and approved
+
+HDAC2 up-regulation will impair BDNF-TrkB signalling pathway and results in cognitive impairments induced by isoflurane.54 Our previ-
+
+the final manuscript.
+
+ous study also verified the role of BDNF-TrkB signalling pathway in
+
+O R C I D
+
+the cognitive deficits induced by propofol during late pregnant stage.5 Whether BDNF-TrkB signalling pathway involves in the
+
+Foquan Luo
+
+http://orcid.org/0000-0003-0106-0710
+
+learning and memory impairments induced by maternal propofol
+
+anaesthesia needs to be explored in future study.
+
+Present study has several limitations. First, we had not accessed
+
+R E F E R E N C E S
+
+the pathological changes of neurons in the foetal brains immediately
+
+1. Zheng H, Dong Y, Xu Z, et al. Sevoflurane anesthesia in pregnant mice induces neurotoxicity in fetal and offspring mice. Anesthesiol- ogy. 2013;118:516-526.
+
+after maternal propofol exposure and during various period of brain
+
+in the present
+
+development (e.g., post-natal day 1 to 10). Second,
+
+study, we only used MWM to evaluate learning and memory.
+
+2. Zhao T, Li Y, Wei W, et al. Ketamine administered to pregnant rats in the second trimester causes long-lasting behavioral disorders in offspring. Neurobiol Dis. 2014;68:145-155.
+
+Although MWM is recognized as an appropriate way to evaluate the
+
+spatial learning and memory in rodents, to provide a more compre-
+
+3. Palanisamy A, Baxter MG, Keel PK, et al. Rats exposed to isoflurane in utero during early gestation are behaviorally abnormal as adults. Anesthesiology. 2011;114:521-528.
+
+hensive assessment of learning and memory in rat offspring, multiple
+
+behavioural test such as open field test, step-through test and the
+
+fear conditioning test should be used in future study. Third, we have
+
+4. Xiong M, Li J, Alhashem HM, et al. Propofol exposure in pregnant rats induces neurotoxicity and persistent learning deficit in the off- spring. Brain Sci. 2014;4:356-375.
+
+explored the underlying mechanisms only from hippocampus. Mater-
+
+nal propofol exposure may also affect other brain regions, such as cortex, thalamus and hypothalamus regions. Li et al55 found that
+
+5. Liang Z, Luo F, Zhao W, et al. Propofol exposure during late stages of pregnancy impairs learning and memory in rat offspring via J Cell Mol Med. 2016;20:1920-1931. the BDNF-TrkB signalling pathway.
+
+propofol anaesthesia in pregnant rats induced caspase-3 activation
+
+and microglial response in foetal rats. They found that the activated
+
+6. Creeley C, Dikranian K, Dissen G, et al. Propofol-induced apoptosis of neurones and oligodendrocytes in fetal and neonatal rhesus maca- que brain. Br J Anaesth. 2013;110:i29-i38.
+
+caspase-3-positive cells were abundant and heavily concentrated in the cortex, thalamus and hypothalamus regions.55 Whether maternal
+
+propofol anaesthesia will affect the histone acetylation in other brain
+
+7. Clancy B, Darlington RB, Finlay BL. Translating developmental time
+
+regions should be studied. We had only evaluated the short-term
+
+across mammalian species. Neuroscience. 2001;105:7-17.
+
+8. Goodman S. Anesthesia for nonobstetric surgery in the pregnant
+
+therapeutic effects of SAHA, Senegenin and HGNA on behaviour
+
+patient. Semin perinatal. 2002;26:136-145.
+
+performance and proteins. The long-term or long-lasting therapeutic
+
+9. F€orster S, Reimer T, Rimbach S, et al. CAMIC recommendations for laparoscopy in non-obstetric indications during pregnancy. surgical Zentralbl Chir. 2016;141:538-544.
+
+effects of these drugs on learning and memory deficits and protein
+
+expression changes caused by propofol exposure on E7 should be
+
+10. Fardiazar Z, Derakhshan I, Torab R, et al. Maternal-neonatal out- come in pregnancies with non-obstetric laparotomy during preg- nancy. Pak J Biol Sci. 2014;17:260-265.
+
+evaluated in future study.
+
+Taken together, the results of the present study suggest that
+
+propofol anaesthesia during first trimester causes learning and mem-
+
+11. Qin Z, Foquan L, Weilu Z, et al. Effect of prolonged anesthesia with propofol during early pregnancy on cognitive function of offspring rats. Chin J Anesthesiol. 2014;34:1051-1053.
+
+ory deficit in offspring rats by inhibiting histone acetylation. SAHA,
+
+Senegenin and HGN antisense oligonucleotide can ameliorate these
+
+12. Fo-quan L, Jun-wu L, Shu-xin T, et al. Effect of inhalation of enflu- rane in early pregnancy on the expression of NR2B in the hip- pocampus of offspring of rats. Chin J Anesthesiol. 2011;31:1076- 1078.
+
+impairments.
+
+A C K N O W L E D G E M E N T S
+
+13. Li Gang Z, Wei-lu L. Fo-quan. Effect of ketamine anesthesia in early pregnancy on the c-fos mRNA and c-jun mRNA expression in off- spring of rats. Chin J Anesthesiol. 2010;30:1333-1335.
+
+We thank other members of the laboratory for valuable discussion
+
+and technical help. This research was supported by National Natural
+
+14. Bing-da L, Fuo-quan L, Wei-lu Z, et al. Effect of ketamine anesthesia in early pregnancy on expression of hippyragranin mRNA in hip- pocampus in offspring of rats. Chin J Anesthesiol. 2012;32:1334- 1336.
+
+Science Foundation of China (NO. 81460175, 81060093) and Natu-
+
+Jiangxi Province of China
+
+ral Science Foundation of
+
+(NO.
+
+20171ACB20030, 20132BAB205022).
+
+15824934, 2018, 5, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.13524 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+| 2611
+
+LIN ET AL.
+
+36. Mizuno M, Yamada K, Maekawa N, Saito K. CREB phosphorylation as a molecular marker of memory processing in the hippocampus for spatial learning. Behav Brain Res. 2002;133:135-141.
+
+15. Barria A, Malinow R. NMDA receptor subunit composition controls synaptic plasticity by regulating binding to CaMKII. Neuron. 2005;48:289-301.
+
+16. Williams JM, Gu(cid:2)evremont D, Kennard JT, et al. Long-term regulation of n-methyl-d-aspartate receptor subunits and associated synaptic proteins following hippocampal synaptic plasticity. Neuroscience. 2003;118:1003-1013.
+
+37. Abel T, Martin KC, Bartsch D, Kandel ER. Memory suppressor genes: inhibitory constraints on the storage of long-term memory. Science. 1998;279:338-341.
+
+38. Bliss TV. Young receptors make smart mice. Nature. 1999;401:25-
+
+17. Miwa H, Fukaya M, Watabe AM, et al. Functional contributions of synaptically localized NR2B subunits of the NMDA receptor to synaptic transmission and long-term potentiation in the adult mouse CNS. J Physiol. 2008;586:2539-2550.
+
+27.
+
+39. Voigt T, De Lima A, Beckmann M. Synaptophysin immunohistochem- istry reveals inside-out pattern of early synaptogenesis in ferret cerebral cortex. J Comp Neurol. 1993;330:48-64.
+
+40. Kong F, Xu L, He D, et al. Effects of gestational isoflurane exposure J Pharmacol. on postnatal memory and learning in rats. Eur 2011;670:168-174.
+
+18. Zhang XH, Zhang H, Tu Y, et al. Identification of a novel protein for memory regulation in the hippocampus. Biochem Biophys Res Com- mun. 2005;334:418-424.
+
+41. Terry RD, Masliah E, Salmon DP, et al. Physical basis of cognitive alterations in Alzheimer’s disease: synapse loss is the major correlate of cognitive impairment. Ann Neurol. 1991;30:572-580.
+
+19. Sutton MA, Schuman EM. Dendritic protein synthesis, synaptic plas-
+
+ticity, and memory. Cell. 2006;127:49-58.
+
+20. Goldberg AD, Allis CD, Bernstein E. Epigenetics: a landscape takes
+
+shape. Cell. 2007;128:635-638.
+
+42. Chalon J, Tang CK, Ramanathan S, et al. Exposure to halothane and enflurane affects learning function of murine progeny. Anesth Analg. 1981;60:794-797.
+
+21. Penney J, Tsai LH. Histone deacetylases in memory and cognition.
+
+Sci Signal. 2014;7:re12.
+
+22. Singh P, Thakur MK. Reduced recognition memory is correlated with decrease in DNA methyltransferase1 and increase in histone deacetylase2 protein expression in old male mice. Biogerontology. 2014;15:339-346.
+
+43. Kandel ER. The molecular biology of memory storage: a dialogue
+
+between genes and synapses. Science. 2001;294:1030-1038.
+
+44. Graff J, Tsai LH. Histone acetylation: molecular mnemonics on the
+
+chromatin. Nat Rev Neurosci. 2013;14:97-111.
+
+23. Agudelo M, Gandhi N, Saiyed Z, et al. Effects of alcohol on histone deacetylase 2 (HDAC2) and the neuroprotective role of trichostatin A (TSA). Alcohol Clin Exp Res. 2011;35:1550-1556.
+
+45. Sepulveda FJ, Bustos FJ, Inostroza E, et al. Montecino M, van Zun- dert B. Differential roles of NMDA Receptor Subtypes NR2A and NR2B in dendritic branch development and requirement of RasGRF1. J Neurophysiol. 2010;103:1758-1770.
+
+24. Wagner FF, Zhang YL, Fass DM, et al. Kinetically Selective Inhibitors of Histone Deacetylase 2 (HDAC2) as Cognition Enhancers. Chem Sci. 2015;6:804-815.
+
+46. Cui Y, Jin J, Zhang X, et al. Forebrain NR2B overexpression facilitat- ing the prefrontal cortex long-term potentiation and enhancing working memory function in mice. PLoS One. 2011;6:e20312.
+
+25. Vecsey CG, Hawk JD, Lattal KM, et al. Histone deacetylase inhibi- tors enhance memory and synaptic plasticity via CREB: CBP-depen- dent transcriptional activation. J Neurosci. 2007;27:6128-6140. 26. Fass DM, Reis SA, Ghosh B, et al. Crebinostat: a novel cognitive enhan- cer that inhibits histone deacetylase activity and modulates chromatin- mediated neuroplasticity. Neuropharmacology. 2013;64:81-96.
+
+47. Clayton DA, Mesches MH, Alvarez E, et al. A hippocampal NR2B defi- cit can mimic age-related changes in long-term potentiation and spa- tial learning in the Fischer 344 rat. J Neurosci. 2002;22:3628-3637. 48. Sakamoto K, Karelina K, Obrietan K. CREB: a multifaceted regulator of neuronal plasticity and protection. J Neurochem. 2011;116:1-9. 49. Kida S. A functional role for CREB as a positive regulator of memory formation and LTP. Exp Neurobiol. 2012;21:136-140.
+
+27. Fujita Y, Morinobu S, Takei S, et al. Vorinostat, a histone deacetylase inhibitor, facilitates fear extinction and enhances expression of the hippocampal NR2B-containing NMDA receptor gene. J Psychiatr Res. 2012;46:635-643.
+
+50. Xu W, Kasper LH, Lerach S, et al. Individual CREB-target genes dic- tate usage of distinct cAMP-responsive coactivation mechanisms. EMBO J. 2007;26:2890-2903.
+
+28. Xie W, Yang Y, Gu X, et al. Senegenin attenuates hepatic ischemia- reperfusion induced cognitive dysfunction by increasing hippocampal NR2B expression in rats. PLoS One. 2012;7:e45575.
+
+51. Rani CS, Qiang M, Ticku MK. Potential role of cAMP response ele- ment-binding protein in ethanol-induced N-methyl-D-aspartate receptor 2B subunit gene transcription in fetal mouse cortical cells. Mol Pharmacol. 2005;67:2126-2136.
+
+29. Hou Q, Gao X, Zhang X, et al. SNAP-25 in hippocampal CA1 region is involved in memory consolidation. Eur J Neuorsci. 2004;20:1593- 1603.
+
+52. Sheng M, Kim MJ. Postsynaptic signaling and plasticity mechanisms.
+
+30. Luo F, Hu Y, Zhao W, et al. Maternal Exposure of Rats to Isoflurane during Late Pregnancy Impairs Spatial Learning and Memory in the Offspring by Up-Regulating the Expression of Histone Deacetylase 2. PLoS ONE. 2016;11:e0160826.
+
+Science. 2002;298:776-780.
+
+53. Lonze BE, Ginty DD. Function and regulation of CREB family tran- scription factors in the nervous system. Neuron. 2002;35:605-623. 54. Ji M1, Dong L, Jia M, et al. Epigenetic enhancement of brain-derived neurotrophic factor signaling pathway improves cognitive impair- ments induced by isoflurane exposure in aged rats. Mol Neurobiol 2014;50:937-944.
+
+31. Peleg S, Sananbenesi F, Zovoilis A, et al. Altered histone acetylation in mice. is associated with age-dependent memory impairment Science. 2010;328:753-756.
+
+32. Guan JS, Haggarty SJ, Giacometti E, et al. HDAC2 negatively regu- plasticity. Nature. formation synaptic and lates memory 2009;459:55-60.
+
+55. Li J, Xiong M, Nadavaluru PR, et al. Dexmedetomidine attenuates neurotoxicity induced by prenatal propofol exposure. J Neurosurg Anesthesiol. 2016;28:51-64.
+
+33. Berndsen CE, Denu JM. Catalysis and substrate selection by his- Struct Biol. acetyltransferases. Curr Opin tone/protein lysine 2008;18:682-689.
+
+How to cite this article: Lin J, Wang S, Feng Y, et al.
+
+Propofol exposure during early gestation impairs learning and
+
+34. Haberland M, Montgomery RL, Olson EN. The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nat Rev Genet. 2009;10:32-42.
+
+memory in rat offspring by inhibiting the acetylation of histone. J Cell Mol Med. 2018;22:2600–2611.
+
+35. Gr€aff J, Rei D, Guan JS, et al. An epigenetic blockade of cognitive functions in the neurodegenerating brain. Nature. 2012;483:222- 226.
+
+https://doi.org/10.1111/jcmm.13524

new_pdfs/10.1111_pan.12263.txt

@@ -0,0 +1,335 @@
+Pediatric Anesthesia ISSN 1155-5645
+
+O R I G I N A L A R T I C L E
+
+Effects of sevoflurane on the expression of tau protein mRNA and Ser396/404 site in the hippocampus of developing rat brain Zhi-yong Hu1, Hai-yan Jin1, Li-li Xu2, Zhi-rui Zhu1, Yi-lei Jiang1 & Robert Seal3
+
+1 Department of Anesthesiology, The Children’s Hospital, School of Medicine, Zhejiang University, Hangzhou, China 2 Department of Anesthesiology, The Second Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, China 3 Department of Anesthesia and Pain Medicine, University of Alberta and Stollery Children’s Hospital, Edmonton, AB, Canada
+
+Keywords phosophorulation; tau; sevoflurane; neonatal
+
+Summary
+
+Correspondence Zhi-yong Hu, Department of Anesthesiology, The Children’s Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Email: huzhiyong777@126.com
+
+Section Editor: Andrew Davidson
+
+Accepted 18 August 2013
+
+doi:10.1111/pan.12263
+
+Background: General anesthesia induces a transient hyperphosphorylation of tau protein that is associated with neurotoxicity in neonatal rats, but the mechanism remains unknown. The current study sought to investigate the effects of sevoflurane on the levels of tau phosphorylation at phosphor- Ser396/404 and total tau mRNA in the hippocampus of neonatal rats. Materials and Methods: Thirty-six 7-day-old rats were randomly exposed for 6 h to either 3% sevoflurane (S) or air (NC) as a placebo. They were sacri- ficed at 1, 7 and 14 days after the anesthesia, respectively, and thus assigned to S1d, S7d, S14d, NC1d, NC7d, and NC14d groups (n = 6). Their brain tissues were harvested and then subjected to histopathologic, Western blot and real- time polymerase chain reaction analysis. Results: Microtubule cytoskeletons were arranged in neat parallel rows in rats exposed only to air, whereas the microtubules were arranged in a disor- derly and intermittent (nonparallel) fashion in rats exposed to sevoflurane. The levels of tau mRNA in the S1d and S7d groups were significantly higher than those in the NC1d and NC7d groups. There was no significant differ- ence in the levels of tau mRNA between the S14d and NC14d groups. The levels of tau protein at Ser404 in the S1d, S7d, and S14d groups were signifi- cantly higher than those in NC1d, NC7d, and NC14d groups. The levels of tau protein at Ser396 in the S1d, and S7d groups were significantly higher than those in the NC1d, and NC7d groups, while there was no significant difference in the levels of tau protein at Ser396 between the S14d group and the NC14d group, respectively. Conclusion: In rat hippocampus, sevoflurane was associated with microtubu- lar disarray as well as increased levels of tau mRNA and excessive phosphor- ylation of tau protein at Ser396 and Ser404. This implicates that sevoflurane may induce neurotoxicity.
+
+Introduction
+
+In humans, anesthetic agents sometimes have to be administered during the brain growth spurt period that occurs between the third trimester and the age of approx- imately 2 years. This time period is equivalent to the first week after birth in mice and rats. Recently, it has been demonstrated in rodents that neonatal administration of
+
+anesthetics induced widespread neurodegeneration and severe deficits in spatial learning tasks (1,2). The underly- ing mechanism is not fully understood. To minimize risks, the risk of anesthesia in neonates, it is necessary to undertake further study to assess the effects of anesthet- ics on the developing nervous system.
+
+(2,2,2-trifluoro-1-[trifluoromethyl]ethyl fluoromethyl ether) is one of the most frequently used
+
+Sevoflurane
+
+1138
+
+© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144
+
+14609592, 2013, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pan.12263 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+Z. Hu et al.
+
+Effects of sevoflurane on the expression of tau protein
+
+volatile anesthetics for the induction and maintenance of general anesthesia during surgery because of its low blood–gas partition coefficient and low pungency. In infants and children, these properties convey the benefit of rapid induction and recovery as well as less irritation to the airway. Sevoflurane has been shown to enhance GABAA receptors (3) and to block NMDA receptors, although more research is necessary to better character- ize its effects on NMDA receptors (4).
+
+drawn from the same litters, so that each experimental condition had its own group of littermate controls. All animals were kept in standard animal cages under con- ventional housing conditions (12-h light-dark cycle, 22°C), with ad libitum access to food and water. All experimental procedures were in accordance with the Guidance Suggestions for the Care and Use of Labora- tory Animals, formulated by the Ministry of Science and Technology of China (8).
+
+Tau protein is a highly soluble microtubule-associated protein (MAP). In humans, these proteins are mostly found in neurons compared with nonneuronal cells. One of tau’s main functions is to modulate the stability of axonal microtubules. Hyperphosphorylation of the tau protein (tau inclusions, pTau) can result in the self- assembly of tangles of paired helical filaments (PHFs) and straight filaments, which are involved in the patho- genesis of Alzheimer’s disease (AD) and other tauopa- thies. Several other studies (5,6) have shown that sevoflurane may induce apoptosis in the brain tissues of neonatal mice. It has also been associated with increased tau phosphorylation through specific kinase activation and with spatial memory deficits. These data support a correlation between exposures to anesthetic agents and cognitive decline. Tau is hyperphosphorylated in PHFs, and specific phosphorylation sites have been implicated in the loss of tau’s association with the membrane cortex during AD disease state, including Ser 199/202, Thr 231, and Ser 396/404. Over-activation of proline-directed kinases such as cyclin-dependent kinase 5(CDk5) and glycogen synthase kinase 3(GSK3) has been implicated in the aberrant phosphorylation of tau at the proline- directed site (7). The hippocampal formation is essential for the processing of episodic memories for autobio- graphical events that happen in unique spatiotemporal contexts. Distinct regions, layers, and cells of the hippo- campal formation exhibit different profiles of structural and molecular development during early postnatal life.
+
+Anesthesia treatment
+
+Our research protocol was approved by the institutional animal research review board of Zhejiang University (Zju201301-1-02-021). Thirty-six 7-day-old male rats were allocated by computer-generated random numbers to a 6 h exposure in an anesthesia chamber with either 3% sevoflurane (H20100586; Abbott, Chicago, IL, USA) plus 60% oxygen (group S) or air as a normal control (group NC). Sevoflurane was delivered into the chamber by an agent-specific vaporizer. All anesthetized rats breathed spontaneously and underwent heart rate monitoring. As well, body temperature was monitored with a rectal probe and maintained between 36.0°C and 37.0°C by means of a heating pad. Rats were sacrificed at 1, 7 and 14 days following exposure, respectively, and were thus assigned to sevoflurane group (S1d, S7d, S14d groups, n = 6 in each) and normal control group (NC1d, NC7d, NC14d groups, n = 6 in each). Their brains were removed immediately after death and then frozen in dry ice and stored at (cid:1)70°C until used.
+
+Arterial blood gas analysis
+
+To determine the adequacy of ventilation, arterial blood was sampled immediately after removal from the mater- nal cage (0 h) or at the end of anesthesia (6 h) by obtain- ing a single sample (100 ll) from the left carotid artery using a 24 gauge SURFLO (Terumo, Tokyo, Japan) catheter. Bicarbonate concentration (millimoles per liter), oxygen saturation (%), pH, paCO2 (mmHg), and paO2 (mmHg) were measured immediately after blood collection, using a Nova Biomedical blood gas apparatus (ABL800; Radiometer, Copenhagen, Denmark).
+
+In this investigation, we examined the roles of Cdk5 and GSK3 in tau hyperphosphorylation in neonatal rat hippocampus induced by sevoflurane. We also assessed the effect of sevoflurane on the levels of tau phosphory- lation at phosphor-Ser396/404 and tau mRNA in the hippocampus of neonatal rat.
+
+Materials and methods
+
+Examination of microtubule structure by electron microscopy
+
+Animals
+
+The hippocampus was removed and cut into 1 mm3 fragments. These were then fixed in 2.5% glutaraldehyde for 2 hours followed by 1% osmium tetroxide (pH 7.3–7.4) for 1–2 h. After fixation, the samples were rinsed with buffer for 20 min, dehydrated, soaked, and
+
+Thirty-six neonatal male Wistar rats aged 7 days were purchased from Zhejiang Academy of Medical Science (Hangzhou, China) (SYXK(zhe)2005-0072). A balanced number of control and experimental animals were
+
+© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144
+
+1139
+
+14609592, 2013, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pan.12263 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+Z. Hu et al.
+
+Effects of sevoflurane on the expression of tau protein
+
+embedded. An ultra-thin slicer was used to cut slices of 1–10 lm thickness that were then stained and viewed under electron microscopy (model CM10; Philips, Ein- dhoven, captured through a CCD camera (model C4742-95; Hamamatsu, Bridgewater, NJ, USA) and Advantage CCD Camera System software (Advanced Microscopy Techniques Corporation, Danvers, MA, USA).
+
+the SYBR Green PCR signal was confirmed by melting curve analysis. Acquired data were analyzed by LIGHTCY- CLE 2000 software 3.5 (Roche). The Ct value of each gene was normalized against that of GAPDH. Tau pri- mer sequences were as follows: tau-sense 5′ACC CCG CCA GGA GTT TGA C-3′, tau-antisense 5′-GAT CTT CGC CCC CGT TTG-3′ 244 bp, GAPDH-sense 5′- CTA CAA TGA GCT GCG TGT GGC-3′, GAPDH- antisense 5′-CAG GTC CAG ACG CAG GAT GGC-3′ 207 bp.
+
+the Netherlands).
+
+Images were
+
+Western blotting analysis for tau pSer396 and pSer404
+
+All data are expressed as mean (cid:3) SD. SPSS 12.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Numerical data including Oxygen saturation, PaO2, PaCO2, pH, and the levels of tau phosphorylation at phosphor-Ser396/404 and tau mRNA between groups were analyzed by the Student’s t-test, and intragroup numerical data were analyzed by repeated measures ANOVA. Statistical significance was accepted as P < 0.05.
+
+For the Western blot analysis, samples (80 lg protein) were prepared using neonatal rat hippocampal tissue. These were mixed with sample buffer, separated by 10% SDS-PAGE and electroblotted to a nitro cellulose mem- brane. The membrane was blocked for 1 hour at room temperature with blocking solution (5% nonfat milk in Tris-buffered saline with Tween 20 [TBST]). Blots were then incubated overnight at 4°C with the specific rat monoclonal antibodies anti-pSer396 (sc-101815) and anti-pSer40 (sc-12952) (1 : 200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or b-actin anti- body (Santa Cruz Biotechnology). The samples were then washed three times and incubated with a horserad- ish peroxidase-labeled second antibody rabbit anti-rat IgG (1 : 2000 dilution; GE Healthcare, Shanghai, China) for 1 h at room temperature prior to visualiza- tion with a chemiluminescence detection technique (Su- perSignal West Pico Chemiluminescent Substrates; Pierce Biotechnology, Rockford, IL, USA). Densitomet- ric techniques were performed to quantify the protein band absorbance (GEL-PRO ANALYZER software; Bio-Rad Laboratories, Hercules, CA, USA) and expressed as rel- ative densitometric units of the corresponding control.
+
+Results
+
+The results of arterial blood gas analysis
+
+To assess the effects of selected anesthetics on the devel- oping brain, we exposed the rats to sevoflurane for 6 h. There were no signs of metabolic or respiratory distress. Oxygen saturation, PaO2, PaCO2, and pH did not differ significantly comparing with the control animals exposed to air for 6 h (Table 1).
+
+The observation of microtubes by electron microscopy
+
+The microtubule cytoskeleton was arranged in neat rows and parallel to each other in the NC group, whereas the microtubules were arranged in a disorderly and inter- mittent fashion and were not parallel to each other in group S (Figure 1).
+
+Tau assay and quantitative real-time PCR
+
+Total RNA was isolated from sevoflurane group and control group. Hippocampus neurons using the RNA- easy mini kit (Takara, Dalian, China) according to the manufacture’s instruction. First-strand cDNA was syn- thesized from 5 lg of total RNA using the Super Script III first-strand synthesis kit (Takara) and random hex- amer system (Roche, Shanghai, China). Quantification of the target genes was performed with Power SYBR Green PCR master mix kit (ABI, Carlsbad, CA, USA) in Bio-Rad MX3000P real-time PCR system according to the manufacturer’s instructions. Triplicate quantita- tive reverse transcription PCRs were carried out for each sample. The PCR amplification cycles were as follows: initial denaturation at 95°C for 15 min, followed by 40 cycles with denaturation at 95°C for 20 s, and annealing-extension at 60°C for 35s. The specificity of
+
+The expression of tau mRNA in neonatal rat hippocampus tissues
+
+The levels of tau mRNA in the S1d and S 7d groups were significantly higher than those in the NC1d and NC7d groups(P < 0.05). There was no significant difference in the levels of tau mRNA between S14d and NC14d groups (P > 0.05; Figure 2).
+
+The expression of tau protein Ser396 site in neonatal rat hippocampus issues
+
+The levels of tau protein at Ser396 in S1d, and S7d groups were significantly higher than those in NC1d, and NC7d groups (P < 0.05). There were no significant difference
+
+1140
+
+© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144
+
+14609592, 2013, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pan.12263 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+Z. Hu et al.
+
+Effects of sevoflurane on the expression of tau protein
+
+Table 1 Arterial blood gas analysis. Neonatal exposure to 3% sevoflurane does not induce significant cardiorespiratory dysfunction. Analysis of arterial blood gas revealed no significant differences in any of the measured parameters between rats exposed for 6 h to sevoflurane and control rats exposed to air for 6 h (t-test, all P values > 0.05)
+
+Arterial blood gas
+
+PaO2, mmHg
+
+PaCO2, mmHg
+
+pH
+
+SaO2
+
+Time, h
+
+98.6 (cid:3) 0.3 99.3 (cid:3) 0.7 98.6 (cid:3) 0.4 98.2 (cid:3) 0.8
+
+98.6 (cid:3) 8.5 99.1 (cid:3) 10.2 98.7 (cid:3) 6.3 98.2 (cid:3) 12.5
+
+39.9 (cid:3) 5.7 40.2 (cid:3) 3.9 41.2 (cid:3) 4.4 42.5 (cid:3) 7.2
+
+7.41 (cid:3) 0.03 7.40 (cid:3) 0.04 7.43 (cid:3) 0.06 7.42 (cid:3) 0.03
+
+Exposed to air for 6 h (n = 18) Exposed to 3% sevoflurane for 6 h (n = 18)
+
+0 6 0 6
+
+PaCO2, arterial carbon dioxide tension; PaO2, arterial oxygen tension; SaO2, arterial oxygen saturation.
+
+(b)
+
+(a)
+
+(c)
+
+(d)
+
+Figure 1 Electron microscopic examination of hippocampal neurons from 7-day-old rats exposed for 6 h in an anesthesia chamber to either 3% sevoflurane in 60% oxygen or air. Anesthesia caused dis- array of microtubule in rat hippocampus. Neonatal rats (7 day) were exposed to air (a) or 3% sevoflurane plus 60% oxygen (b–d) for 6 h, then the hippocampal tissues were taken and examined by electron microscopy. After exposure to air for 6 h (a), the microtubules were
+
+arranged in neat parallel rows. After exposure to 3% sevoflurane plus 60% oxygen for 6 h (b–d), the microtubules were arranged in a disor- derly and intermittent fashion and were not in parallel with each other (b), or became disrupted, indistinct and have lost the normal order of arrangement (c,d). Arrows indicate the microtubule structures. Scale bars: 0.2 lm. (magnification 960 000).
+
+and tau mRNA in neonatal rat hippocampus following the administration of 3% sevoflurane for 6 h. We found that sevoflurane induced increased levels of tau mRNA at 1 and 7 days as well as producing excessive phosphor- ylation of tau protein at Ser404 at 1, 7, and 14 days and Ser396 at 1 and 7 days in neonatal rat hippocampus. These results suggest that sevoflurane may induce neuro- toxicity in neonatal rats. This is consistent with recent evidence that different types of anesthetic agents, includ- ing sevoflurane, promote tau phosphorylation (6). Furthermore, in our study on electron microscopy, we observed that hippocampal neuron microtubules were significantly changed and became disorganized follow- ing sevoflurane exposure.
+
+in the levels of tau protein at Ser396 between the S14d and the NC14d groups, respectively (P > 0.05; Figure 3).
+
+The expression of tau protein Ser404site in neonatal rat hippocampus tissues
+
+The levels of tau protein at Ser404 in S1d, S7d, and S14d groups were significantly higher than those in NC1d, NC7d, and NC14d groups. (P < 0.05; Figure 4).
+
+Discussion
+
+In this study, we investigated the effects of sevoflurane on the levels of tau phosphorylation at phosphor-Ser396/404
+
+© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144
+
+1141
+
+14609592, 2013, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pan.12263 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+Z. Hu et al.
+
+Effects of sevoflurane on the expression of tau protein
+
+We chose sevoflurane anesthesia because a recent study by Satomoto et al. (9) indicated that anesthesia with 3% sevoflurane plus 60% oxygen for 6 h does not significantly alter blood gas and brain blood flow. At
+
+the same time, we hypothesized that this high concentra- tion of sevoflurane anesthesia would be sufficient to demonstrate changes in tau phosphorylation at phos- phor-Ser396/404 and tau mRNA in the brain tissues of neonatal rats.
+
+The tau protein is the taylorism end product of selective montaging from a single gene designated microtubule-associated in humans (10). Its primary function is to regulate the stabilization of axonal microtubules, and it has two means of dominating microtubule stability: isoforms and phosphorylation. Moreover, hyperphosphoryla- tion of the tau protein (tau inclusions, p-tau) can lead to the self-assembly of tangles of paired helical fila- ments and straight filaments involved in the pathogen- esis of Alzheimer’s disease and other tauopathies (11). In other neurodegenerative diseases, the deposition of assemblages substantial in certain tau isoforms has been found. When misfolded, this otherwise extraordi- narily soluble protein can constitute exceedingly insol- uble assemblages resulting in a few neurodegenerative diseases. Recent research (5) suggests that tau protein
+
+(MAPT)
+
+protein
+
+tau
+
+Figure 2 The expression of tau mRNA (mean (cid:3) SD). After exposure to 3% sevoflurane in 60% oxygen for 6 h, the levels of tau RNA in the S1d and S7d groups were significantly higher than those in the NC1d and NC7d groups (P < 0.05). There was no significant difference in the levels of tau mRNA between S14d and NC14d groups (P > 0.05). There were 6 rats in each group at each time point. *P < 0.05 compared with the control group.
+
+(a)
+
+(b)
+
+respectively (P > 0.05). *P < 0.05, compared with the control group. (a) The expression of p-tau protein Ser396 site by Western blot analy- sis; S, sevoflurane group; NC, control group. (b) Quantitative expres- sion of p-tau protein Ser396 site. The data are expressed as mean (cid:3) SD (ratio to b-actin). There were six rats from each group at each time point.
+
+Figure 3 The expression of p-tau protein Ser396 site in neonatal rat hippocampus tissues. After exposure to 3% sevoflurane in 60% oxygen for 6 h, the levels of tau protein at Ser396 in the S1d and S7d groups were significantly higher than those in the NC1d and NC7d groups (P < 0.05). There were no significant differences in the levels of tau protein at Ser396 between the S14d and the NC14d groups,
+
+(a)
+
+(b)
+
+Figure 4 The expression of p-tau protein pSer404 in neonatal rat hip- pocampus tissues. After exposure to 3% sevoflurane plus 60% oxy- gen for 6 h, the levels of tau protein at Ser404 in S1d, S7d, and S14d groups were significantly higher than those in NC1d, NC7d, and NC14d groups (P < 0.05). *P < 0.05, compared with the control group. (a)
+
+The expression of p-tau protein Ser404 site by western blot analysis; S, sevoflurane group; NC, control group. (b) quantitative expression of p-tau protein Ser404 site. The data are expressed as mean (cid:3) SD (ratio to b-actin).There were six rats from each group at each time point.
+
+1142
+
+© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144
+
+14609592, 2013, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pan.12263 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+Z. Hu et al.
+
+Effects of sevoflurane on the expression of tau protein
+
+might be discharged extracellularly by an exosome- based mechanism in Alzheimer’s disease and in anes- thetic neurotoxicity in neonatal animals with similar geriatric neuroapoptosis. Lunardi et al. found that anesthesia caused long-lasting ultrastructural dis- array in the subicular neuropil and mitochondria of 21-day-old rats. Head et al. (13) showed that isoflura- ne significantly decreased the number of synapses in the hippocampus compared with baseline in postnatal day (PND) 5 mice. Our electron microscopic finding in hippocampal neurons of microtubular disorganiza- tion supports the finding of Planel et al. (14). They found that exposure to isoflurane at clinically relevant doses led to increased levels of phospho-tau, increased insoluble aggregated forms of tau and detachment of tau from microtubules. these It microtubular structure changes may destroy the stabil- ity of microtubules, damage axonal transport and eventually led to neuroapoptosis.
+
+of nontransgenic mice. Some phosphorylation sites have been linked to specific aspects of tau pathology such as the sequestration of normal tau, the inhibition of tau MT binding, and the promotion of tau aggre- gation. Planel et al. (14) also demonstrated that in JNLP3 mice, a mouse model of tauopathy expressing P301L mutant tau that exposure to 1.3% isoflurane for 4 h increased tau phosphorylation at the AT8, CP13(Ser202), pS262, MC6, and PHF-1 epitopes. Recently, Tan et al. (19) confirmed these results in rats and demonstrated that 1.5% isoflurane for 2 h resulted in tau hyperphosphorylation at the Thr205 and Ser396 epitopes in the hippocampus and attrib- uted this effect to anesthesia-induced hypothermia. Our study showed that tau protein at Ser404 was excessively hyperphosphorylated after and 14 days, and at Ser396 after 1 and 7 days in neonatal rat hippocampus following sevoflurane anesthesia. Therefore, consistent with the findings of Van der Jeugd, sevoflurane may lead to neuronal apoptosis in the developing rodent brain through tau hyperphosph- orylation at the Ser396 and Ser404 sites. (20).
+
+(12)
+
+1,
+
+is probable that
+
+7,
+
+The hippocampus is involved in learning and in con- solidation of explicit memories from short-term memory to cortical memory storage for the long term; its precise role in memory storage remains an active area of research and is beyond the scope of this research. Recent investigations have shown both that activation of a mutant tau gene in mice results in neuronal loss, brain atrophy, and memory impairment; and that inhibition of the mutant tau gene leads to cessation of neuronal loss, decreased atrophy of brain, and improvement in memory (15). Increased tau phosphorylation following anesthesia has also been observed with sodium pento- barbital, ketamine, or urethane (16). Yan et al. (5) examined the effects of sevoflurane on caspase-3 activa- tion and Ablevels in the brain tissues of neonatal mice and concluded sevoflurane may induce neurotoxicity. Our research showed that in the newborn rats sevoflura- ne induced increased levels of tau mRNA at 1 and 7 days followed by a decline by day 14. This suggests that a mechanism of sevoflurane-induced neurotoxicity could occur through activation of mutant tau genes.
+
+This study has some limitations. Firstly, a 6-h-long exposure to sevoflurane in a 7-day-old rat pup has potentially small clinical relevance. Secondly, we did not correlate our findings with testing for learning and mem- ory defects. Thirdly, our observations of phosphoryla- tion of tau protein and microtubular disarray are suggestive, but not conclusive about the possible mecha- nisms for sevoflurane neurotoxicity. Finally, compara- bility of our work with those of others is confounded by differences in experimental animal ages, test parameters, and the dose, duration and method of administration of sevoflurane. Future studies will help elucidate these mechanisms.
+
+In conclusion, we have shown that sevoflurane, the most commonly used neonatal general anesthetic, can induce an increase in the levels of tau mRNA as well as excessive phosphorylation of tau protein at Ser396 and Ser404 in neonatal rat hippocampus. As well, we observed electron microscopic evidence of microtubular disarray in the hippocampus of sevoflurane-exposed rats. These findings suggest that sevoflurane anesthesia (up to 3%) may be neurotoxic in neonatal rats. These findings should support the need for further studies to determine the potential neurotoxicity of sevoflurane anesthesia in the developing brain of animals and humans.
+
+Phosphorylation of tau is adjusted by a host of kin- ases, containing PKN, a serine/threonine kinase, which phosphorylates tau, giving rise to destruction of microtubule organization. Because the hyperphosph- orylated tau was situated at the PHF-1(S396/S404) and threonine (T) T231 positions (17) in the intraneu- the PHF-1(S396/ rofibrillary tangles S404) was chosen to as the marker of sevoflurane- induced neurotoxicity in our study. Planel et al. (18) observed anesthesia induced by chloral hydrate, sodium pentobarbital, or isoflurane resulted in a robust hyperphosphorylation of tau at PHF-1 (Ser396/Ser404) epitopes in the brain
+
+stage,
+
+(NFT)
+
+short-term (30–60 min)
+
+that
+
+Acknowledgments
+
+This work was the Ministry of Education, Zhejiang, China (Y201017446),
+
+supported by the project of
+
+© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144
+
+1143
+
+14609592, 2013, 12, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pan.12263 by Johns Hopkins University, Wiley Online Library on [20/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
+
+Z. Hu et al.
+
+Effects of sevoflurane on the expression of tau protein
+
+the Bureau of (Y201121392) and the project of Chinese Medicine, Zhejiang, China (2011ZA067) and the Project of Medical Technology, Zhejiang, China (2013ZDA011), (2013KYB193). The Authors wish to thank the Key Laboratory for Diagnosis and Therapy of Neonatal Diseases and the Key Laboratory of
+
+Reproductive Genetics, Zhejiang University, Ministry of Education, Zhejiang, China, for their support.
+
+Conflict of interest
+
+No conflicts of interest declared.
+
+References
+
+15 Santacruz K, Lewis J, Spires T et al. Tau suppression in a neurodegenerative mouse model improves memory function. Science 2005; 309: 476–481.
+
+7 Shahani N, Brandt R. Functions and mal- functions of the tau proteins. Cell Mol Life Sci 2002; 59: 1168–1680.
+
+1 Jevtovic-Todorovic V, Hartman RE, Izumi Y et al. Early exposure to common anes- thetic agents causes widespread neurodegen- eration in the developing rat brain and persistent learning deficits. J Neurosci 2003; 23: 876–882.
+
+8 The Ministry of Science and Technology of the People s Republic of China. Guidance Suggestions for the Care and Use of Labora- tory Animals. 2006-09-30.
+
+16 Holscher C, van Aalten L, Sutherland C.
+
+Anaesthesia generates neuronal insulin resis- tance by inducing hypothermia. BMC Neuro- sci 2008; 9: 100.
+
+2 Fredriksson A, Ponte′n E, Gordh T et al. Neonatal exposure to a combination of N-methyl-D-aspartate and gamma- aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegenera- tion and persistent behavioral deficits. Anesthesiology 2007; 107: 427–436.
+
+9 Satomoto M, Satoh Y, Terui K et al.
+
+17 Augustinack JC, Schneider A, Mandelkow EM et al. Specific tau phosphorylation sites correlate with severity of neuronal cytopa- thology in Alzheimer’s disease. Acta Neuro- pathol 2002; 103: 26–35.
+
+Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice. Anesthesiology 2009; 110: 628–637.
+
+10 Sultan A, Nesslany F, Violet M et al.
+
+Nuclear tau, a key player in neuronal DNA protection. J Biol Chem 2011; 286: 4566–4575.
+
+3 Nishikawa K, Harrison NL. The actions of sevoflurane and desflurane on the gamma- aminobutyric acid receptor type A: effects of TM2 mutations in the alpha and beta subun- its. Anesthesiology 2003; 99: 678–684. 4 Hollmann MW, Liu HT, Hoenemann CW
+
+18 Planel E, Richter KE, Nolan CE et al. Anes- thesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia. J Neurosci 2007; 27: 3090–3097.
+
+11 Iqbal K, Liu F, Gong CX et al. Tau in
+
+Alzheimer disease and related tauopathies. Curr Alzheimer Res 2010; 7: 656–664. 12 Lunardi N, Ori C, Erisir A et al. General
+
+19 Tan W, Cao X, Wang J et al. Tau hyper-
+
+et al. Modulation of NMDA receptor func- tion by ketamine and magnesium, part II: interactions with volatile anesthetics. Anesth Analg 2001; 92: 1182–1191.
+
+phosphorylation is associated with memory impairment after exposure to 1.5% isoflurane without temperature maintenance in rats. Eur J Anaesthesiol 2010; 27: 835–841.
+
+Anesthesia causes long-lasting disturbances in the ultrastructural properties of develop- ing synapses in young rats. Neurotox Res 2010; 17: 179–188.
+
+5 Lu Y, Wu X, Dong Y et al. Anesthetic sevo- flurane causes neurotoxicity differently in neonatal na€ıve and Alzheimer disease trans- genic mice. Anesthesiology 2010; 112: 1404–1416.
+
+20 Van der Jeugd A, Ahmed T, Burnouf S et al. Hippocampal tauopathy in tau transgenic mice coincides with impaired hippocampus- dependent learning and memory, and attenuated late-phase long-term depression of synaptic transmission. Neurobiol Learn Mem 2011; 95: 296–304.
+
+13 Head BP, Patel HH, Niesman IR et al. Inhi- bition of p75 Neurotrophin receptor attenu- ates Isoflurane-mediated neuronal apoptosis in the neonatal central nervous system. Anes- thesiology 2009; 110: 813–825.
+
+6 Le Freche H, Brouillette J, Fernandez-
+
+14 Planel E, Bretteville A, Liu L et al. Accelera- tion and persistence of neurofibrillary pathol- ogy in a mouse model of tauopathy following anesthesia. FASEB J 2009; 23: 2595–2604.
+
+Gomez FJ et al. Tau phosphorylation and sevoflurane anesthesia: an association to postoperative cognitive impairment. Anes- thesiology 2012; 116: 779–787.
+
+1144
+
+© 2013 John Wiley & Sons Ltd Pediatric Anesthesia 23 (2013) 1138–1144

new_pdfs/10.1186_s12871-018-0471-2.txt

@@ -0,0 +1,227 @@
+Huang et al. BMC Anesthesiology (2018) 18:5 DOI 10.1186/s12871-018-0471-2
+
+R E S E A R C H A R T I C L E
+
+Open Access
+
+Influence of isoflurane exposure in pregnant rats on the learning and memory of offsprings Wei Huang, Yunxia Dong, Guangyi Zhao, Yuan Wang, Jingjing Jiang and Ping Zhao*
+
+Abstract
+
+Background: About 2% of pregnant women receive non-obstetric surgery under general anesthesia each year. During pregnancy, general anesthetics may affect brain development of the fetus. This study aimed to investigate safe dosage range of isoflurane. Methods: Forty-eight SpragueDawley (SD) pregnant rats were randomly divided into 3 groups and inhaled 1.3% isoflurane (the Iso1 group), 2.0% isoflurane (the Iso2 group) and 50% O2 alone (the control group) for 3 h, respectively. Their offsprings were subjected to Morris water maze at day 28 and day 90 after birth to evaluate learning and memory. The expression of cAMP-response element binding protein (CREB) and phosphorylated cAMP-response element binding protein (p-CREB) was detected in the hippocampus dentate gyrus. Results: Less offsprings of Iso2 group were able to cross the platform than that of the control group (P < 0.05). Accordingly, the Iso2 offsprings expressed p-CREB mainly in the subgranular zone in contrast to the whole granular cell layer of hippocampus dentate gyrus as detected in the Iso1 and control offsprings; the expression level of pCREB was also lower in the Iso2 than Iso1 or control offsprings (P < 0.05). Conclusion: Inhalation of isoflurane at 1.3% during pregnancy has no significant influence on learning and memory of the offspring; exposure to isoflurane at 2.0% causes damage to spatial memory associated with inhibition of CREB phosphorylation in the granular cell layer of hippocampus dentate gyrus.
+
+Keywords: Isoflurane, CREB, Pregnancy, Memory, Offsprings
+
+Background Approximately, more than 2% of pregnant women receive non-obstetric surgery under general anesthesia [1, 2]. In humans, brain development mainly occurs in the fetal period when the proliferation, differentiation and migra- tion of neurons and the formation and modification of synapses as well as myelin are very active. Thus, during that time, the fetal development of central nervous system is extremely vulnerable to both internal and external en- vironmental changes and neurons without formation of synapses will become apoptotic [3, 4]. General anesthesia during pregnancy may affect brain development of the fetus and their learning abilities.
+
+However, there is no guideline for isoflurane usage during pregnancy due to lack of clinical studies [5]. In 1985, Uemura et al. first found that the fetus exposed to halothane affected synaptic development in the neonatal brains [6], which have confirmed by increasing evidence [7, 8]. It was proposed that anesthetics used in general anesthesia increase the apoptosis of immature neurons, causing damage to the nervous system in fetus [9]. To date, a variety of studies have shown that high concen- tration of anesthetics in general anesthesia cause dam- ages to nervous system, but these anesthetics at a clinical or subclinical concentration on fetal brain development is unclear.
+
+the influence of
+
+Correspondence: mzekcd@sj-hospital.org Department of Anesthesiology, Shengjing Hospital of China Medical University, No. 36 SanHao Street, HePing District, ShenYang, Liaoning Province, People’s Republic of China
+
+Therefore, it is important to investigate the influence of general anesthesia on brain development of offspring in order to guide anesthesia in pregnant women receiving non-obstetric surgery. In the present study, pregnant rats
+
+© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
+
+Huang et al. BMC Anesthesiology (2018) 18:5
+
+were exposed to isoflurane at different concentrations and subjected their offsprings to the behavior study, aiming to investigate the influence of isoflurane exposure during pregnancy on the memory and learning abilities of the offspring as well as to explore the range of safe doses, which may provide evidence for the clinical use and inves- tigations of anesthetics. We hypothesize that isoflurane inhalation during pregnancy compromises the offspring’s learning abilities and memory in a concentration- dependent manner.
+
+Methods This study was approved by the Ethics Committee of Affiliated Shengjing Hospital of China Medical University, and specific pathogen free SD pregnant rats weighing 380–420 g were purchased from the Experimental Animal Center of Affiliated Shengjing Hospital of China Medical University. Animals were housed at 22–24 °C, 40–60% hu- midity with a 12-h light /dark cycle and had free access to food and water. Rats at the gestational age of 21 days (E21) were used in subsequent experiments. According to the isoflurane dose, rats were divided into 3 groups: the Iso1 group (1.3% isoflurane), the Iso2 group (2.0% isoflur- ane) and the control group (0% isoflurane; O2).
+
+In the absence of anesthesia, intratracheal intubation was difficult in the control group. Thus, all the rats retained spontaneous breathing and did not receive intra- tracheal intubation. Inhalation of isoflurane at a high con- centration may inhibit respiration and cause hypoxia. Thus, in our pilot study, pregnant rats at the gestational age of 20 days (E20) were anesthetized intraperitoneally with pentobarbital sodium and catheter indwelling was done in the right carotid artery; rats were then allowed to recover at room temperature. At E21, rats were placed in a box filled with prefilled gas according to the following groups: 50% O2 was administered in the control group; 1.3% isoflurane was administered in the Iso1 group (50% oxygen, balanced with nitrogen); 2.0% isoflurane was ad- ministered in the Iso2 group (50% oxygen, balanced with nitrogen). All rats were retained spontaneous breathing and exposed in the box for 3 h (the concentrations of iso- flurane and oxygen were monitored). The mean arterial blood pressure was continuously monitored via a catheter in the carotid artery, and arterial gas analysis was performed hourly. The results showed that inhalation of isoflurane at 1.3% or 2.0% had no influence on the arterial gas and mean arterial blood pressure. Rats used in pilot study will not be used for formal study.
+
+In this study, a total of 48 rats at E21 were randomly assigned into 3 groups and exposed to isoflurane at the pre- designed concentration for 3 h. Animals were allowed to re- cover at room temperature and housed until they delivered. The number of fetuses was recorded, and healthy male neo- natal rats were used in the experiments. At day 28 after birth
+
+Page 2 of 7
+
+(P28), the male offsprings were randomly assigned into two groups: one for Morris water maze (MWM) test to evaluate memory and learning and the other one were housed until day 90 after birth (P90) to receive the same MWM test.
+
+MWM test used a round swimming pool sized 150 cm in diameter and 60 cm in height with a platform sized 10 cm in diameter in the maze. The removable platform was 1.5 cm lower than the water surface. The visual cues (a variety of figures) on the maze’s inner wall remained un- changed during the study. Training and examination were performed in the water at 20 °C. After each examination, rats were dried under a lamp and returned to the cages.
+
+Place navigation test was performed for consecutive 5 days. In brief, platform were placed in a quadrant (the 4th quadrant in this study). At predesigned time point, rats were placed in a random quadrant (once for each quadrant). If the rat found the platform within 90s, it was allowed to stay on the platform for 15 s and then placed out of the pool. The spatial navigation test was performed on the 6th day to evaluate memory. In brief, the platform was removed, rats were placed in a random quadrant and the swimming trajectory was recorded within 90s. In the test, the proportion of swimming distance in the platform quadrant to the total swimming distance and the times of crossing the platform were calculated. The swimming dis- tance in the platform quadrant reflects spatial localization and the times of crossing the platform reflects the accur- acy of spatial memory. Before training, the platform was visible above the water surface, which may exclude rats with visual defects that were unable to find the platform. In addition, rats with poor performance in the test, such as those could not find the hidden platform and swam along the wall, were also excluded from this study.
+
+Two hours after the spatial navigation test, rats were intraperitoneally anesthetized with pentobarbital sodium. Half of each group of the rats were used to collect brain and followed by the separation of hippocampus. The hippocampus was weighed and lysed for total protein extraction. Samples were then stored at −80 °C for later use. Western blotting was performed to detect the protein expression of CREB and p-CREB in the hippocampus. The half of the rats were transcardially perfused with 4% paraformaldehyde and the brain was collected and fixed in 4% paraformaldehyde. Immunohistochemistry was per- formed to detect CREB and p-CREB expression. (Fig. 1).
+
+The neonatal rats were randomly assigned into different groups to reduce variation. We normalized CREB and p- CREB protein expression in control group as 1. CREB and p-CREB expression in the Iso1 and Iso2 group was com- pared with the controls. All data are expressed as mean ± standard deviation. Statistical analyses were performed by using SPSS software (version 21.0; IBM, Corp., Armonk, NY, USA). One-Way ANOVA was used to compare the means between groups. A value of P < 0.05 indicated significance.
+
+Huang et al. BMC Anesthesiology (2018) 18:5
+
+Page 3 of 7
+
+Fig. 1 Study protocol. The E21 pregnant rats were randomized to inhalation with isoflurane 1.3%, 2.0% or O2. After the pregnant rats gave birth, the male offspring rats were randomized to day 28 after birth (P28) and day 90 after birth (P90), followed by MWM (place navigation 5 days and spatial navigation on the 6th day). At 2 h after the spatial navigation of MWM, detect the protein expression of CREB and p-CREB in the hippocampus
+
+Results A total of 48 pregnant rats were used in this study, and eventually 316 male neonatal rats were used in the subse- quent experiments. There were 52, 51 and 54 rats at day 28 after birth in the control, Iso1 and Iso2 group, respect- ively; there were 54, 51 and 54 rats at day 90 after birth in the control, Iso1 and Iso2 group, respectively (Table 1).
+
+There was no significant difference in the percentage of swimming distance in platform quadrant (IV quadrant) among three groups (P > 0.05). The times of crossing the platform in the Iso2 group was significantly lower than in the control group (P < 0.05) (Figs. 2 and 3).
+
+CREB expression in the granule cell layer of the hippo- campus dentate gyrus was comparable among the three groups (P > 0.05). p-CREB expression was mainly found in the whole granule cell layer of the hippocampus dentate gyrus in the control and Iso1 group, but mainly found in subgranular zone (SGZ) in the Iso2 group. In addition, p- CREB expression in the Iso2 group was significantly lower than in the Iso1 and control group (Fig. 4).
+
+CREB expression was similar among the three groups (P > 0.05). In addition, there was no marked difference in
+
+Table 1 The male offsprings in each group
+
+p-CREB expression between the Iso1 and control group (P > 0.05), however, p-CREB expression in the Iso2 group was significantly lower than in the control group (P < 0.05) (Fig. 5).
+
+Discussion The growth and development of central nervous system are very complex in mammals. A substantial proportion of neurons undergo apoptosis during normal develop- ment. In synaptic plasticity phase, the nervous system is extremely sensitive to the internal and external environ- ments and neurons that don’t form synapses will undergo apoptosis [3, 4]. Human brain development occurs mainly in fetus and mature slowly after birth [10], which is differ- ent from other species. For example, the nervous system of small rodents is largely immature at birth and rapidly developed after birth. Thus, in an animal study, brain development should be temporally equivalent to that in humans. It has been shown that brain development of rats at E21 is equivalent to that of human fetus at the gesta- tional age of 12–16 weeks the second trimester [10, 11]. In this study, pregnant SD rats at E21 were exposed to iso- flurane to mimic anesthesia on pregnant woman in the second trimester; isoflurane at 1.3% and 2.0% is equivalent to 1 and 1.5 MAC, respectively [12, 13].
+
+P28
+
+Control 53-1a
+
+Iso1
+
+51
+
+P90
+
+Total
+
+54 107-1a
+
+51
+
+102
+
+a1 rat in the control group was excluded due to poor performance in MWM test
+
+Iso2
+
+54
+
+54
+
+108
+
+The behavior MWM test is often employed as an ef- fective tool to evaluate spatial learning and memory of rodents [14, 15]. In the spatial navigation test, the ratio of swimming distance in the platform quadrant to the total swimming distance reflects the capability of spatial
+
+Huang et al. BMC Anesthesiology (2018) 18:5
+
+Fig. 2 The track of MWM space exploration experiment at P28 of offsprings. a: The green circle in the diagram is the platform, the red line is the trajectories of rats. b: The times of crossing the platform. c: The percentage of platform quadrant. *: P < 0.05 vs. Control
+
+Fig. 3 The track of MWM space exploration experiment at P90 of offsprings. a: The green circle in the diagram is the platform, the red line is the trajectories of rats. b: The times of crossing the platform. c: The percentage of platform quadrant. *: P < 0.05 vs. Control
+
+Page 4 of 7
+
+Huang et al. BMC Anesthesiology (2018) 18:5
+
+Page 5 of 7
+
+Fig. 4 CREB and p-CREB expression in the granule cell layer of the hippocampus dentate gyrus of offspring rats. a: Arrow points positive cells. b: The P28 offspring rats. c: The P90 offspring rats. *: P < 0.05 vs. Control
+
+location, and the times of crossing the platform reflects the accuracy of spatial memory. Our results showed that the ratio of swimming distance was comparable in the young (28 days) and adult (90 days) rats among the three groups, but the times of crossing the platform in the Iso2 group was significantly less than in the other two groups, indicating that isoflurane at a high concentration compromises the accuracy of spatial memory in rats but has little influence on their spatial localization.
+
+The hippocampus is crucial to learning and memory [16–18]. The dentate gyrus in hippocampus is responsible for cognition and location navigation and transduces signals from the inner olfactory cortex to other regions of the hippocampus [19–21]. CREB is an important nuclear pro- tein expressed widely in the cortex and hippocampus of adult rats. The dentate gyrus has the highest expression of
+
+CREB in the hippocampus [22]. CREB plays important roles in neurogenesis, synaptic formation, learning and memory [23, 24]; it regulates the transcription of a large number of genes, such as brain derived neurotrophic factor, c-fos, synaptic I and Ca/calmodulin-dependent protein kinases kinases or CaM kinases [25, 26], to form new synapses and gain long-term memory. Increased CREB expression and/or activity promotes memory formation [27, 28], and reduces CREB expression and/or activity in- hibits memory formation [29–31]. Phosphorylated CREB was detected in cortical neurons with plasticity formation and hippocampal neurons after long-term potentiation stimulation and neurobehavioral training [32]. In addition, injection of CREB at dorsal hippocampus in mice was found to improve spatial memory in water maze test, but injection of the CREB variant that was unable to be
+
+Huang et al. BMC Anesthesiology (2018) 18:5
+
+Page 6 of 7
+
+Fig. 5 Western blotting of CREB and p-CREB expression in the hippocampus dentate gyrus of offspring rats. a and c: The P28 offspring rats. b and d: The P90 offspring rats. *: P < 0.05 vs. Control
+
+phosphorylated at ser133 deteriorated spatial memory of these mice [33]. Increased p-CREB enhanced memory and cognitive abilities in mice [34]. Therefore, CREB phosphor- ylation contributes to the formation of memory. In this study, our results showed that p-CREB expression in the Iso2 group was significantly lower than in the control group and Iso1 group, which is consistent with the findings from MWM test. These findings confirm the crucial role of CREB phosphorylation in the formation of memory [33].
+
+The cortex at the hippocampus dentate gyrus can be di- layer and vided into the molecular layer, granular cell polymorphic cell layer. Immunohistochemistry showed that CREB was expressed mainly in the granule cell layer of the dentate gyrus in offspring rats. In the control group and Iso1 group, p-CREB was expressed in the whole gran- ule cell layer of the dentate gyrus, but its expression was only detectable in the subgranular zone (SGZ) in the Iso2 group. In 1998, Eriksson et al. confirmed neurogenesis in the dentate gyrus of humans for the first time [35]. Since then, increasing evidence has indicated a neural stem cell region in mammalian brain that is localized between the granular cell layer and hilus region with a size of 50– 100 μm [36]. The region is also known as the subgranular zone. Neurons in the SGZ may differentiate into mature granular cells, some intermediate neurons, and glial cells, which are finally integrated into the granular cell layer [37–39]. These cells then form synapses, playing import- ant roles in learning and memory. In the present study, the results showed that p-CREB was mainly expressed in SGZ of the dentate gyrus in the Iso2 group. Our results indicates that inhalation of isoflurane at a high concentra- tion affects CREB phosphorylation in fetal brain without altering the CREB expression, which leads to compro- mised learning and memory. The new neural stem cells in SGZ in adulthood are not affected by the anesthetic and
+
+may further differentiate into granular cells and join the granular cell layer. Thus, the expression of CREB and p- CREB in SGZ remained unchanged. However, we could not exclude that isoflurane inhalation during pregnancy has little influence on neural stem cells in SGZ.
+
+In the present study, pregnant rats were exposed to iso- flurane for 3 h, which is equivalent to 48 h general anesthesia in humans. Other harmful stimulation was not employed aiming to reduce other confounding factors. it is rare that pregnant However, women received anesthesia without surgery or surgery is performed under anesthesia for several weeks. Thus, al- though our results indicate that isoflurane has influence on neural development, we usually will not expect the equivalent conditions in general clinical practice.
+
+in clinical practice,
+
+Conclusion Inhalation of isoflurane at 1.3% during pregnancy has no sig- nificant influence on learning and memory of the offspring in rats; exposure to isoflurane at 2.0% during pregnancy af- fects the accuracy of spatial memory of the offspring, but has little influence on spatial localization, which is associated to inhibition of CREB phosphorylation in the granular cell layer of the dentate gyrus in the hippocampus of fetal rats.
+
+Abbreviations CREB: CAMP-response element binding protein; E21: Gestational age of 21 days; MWM: Morris water maze; P28: Day 28 after birth; P90: Day 90 after birth; p-CREB: Phosphorylated cAMP-response element binding protein; SD pregnant rats: SpragueDawley pregnant rats; SGZ: Subgranular zone
+
+Acknowledgements Not Applicable.
+
+Funding The study was funded by Natural Science Foundation of China (81671311) and Natural science fund of Liaoning province (2015020467).
+
+Huang et al. BMC Anesthesiology (2018) 18:5
+
+Availability of data and materials The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.
+
+Authors’ contributions YD and YW carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. GY and JJ participated in the design of the study, carried out immunoassays and performed the statistical analysis. WH and PZ conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.
+
+Ethics approval and consent to participate This study was approved by the Ethics Committee of Affiliated Shengjing Hospital of China Medical University. Reference number for the ethics approval is 2017PS335K.
+
+Consent for publication Not Applicable.
+
+Competing interests The authors declare that they have no competing interests.
+
+Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
+
+Received: 14 August 2017 Accepted: 4 January 2018
+
+References 1.
+
+Reitman E, Flood P. Anaesthetic considerations for non-obstetric surgery during pregnancy. Br J Anaesth. 2011;107:i72–8. Cheek TG, Baird E. Anesthesia for nonobstetric surgery: maternal and fetal considerations. Clin Obstet Gynecol. 2012;52:535–45. Kong FJ, Ma LL, WW H, Wang WN, HS L, Chen SP. Fetal exposure to high isoflurane concentration induces postnatal memory and learning deficits in rats. Biochem Pharmacol. 2012;84:558–63. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci. 2003;23:876–82. Cunningham F, Leveno K, Bloom S, Hauth J, Rouse D, Spong C. Williams obstetrics [M]. New York: McGraw-Hill Professional; 2009. Uemura E, Levin ED, Bowman RE. Effects of halothane on synaptogenesis and learning behavior in rats. Exp Neurol. 1985;89:520–9.
+
+2.
+
+3.
+
+4.
+
+5.
+
+6.
+
+7. Wang Y, Li Y, Xing Q, Han XG, Dong X, Lu Y, Zhou M. Sevoflurane anesthesia in pregnant rats negatively affects nerve function in offspring potentially via inhibition of the Wnt/β-catenin pathway. Mol Med Rep. 2017;15:2753–9. Fang F, Song R, Ling X, Peng M, Xue Z, Cang J. Multiple sevoflurane anesthesia in pregnant mice inhibits neurogenesis of fetal hippocampus via repressing transcription factor Pax6. Life Sci. 2017;175:16–22. 9. Wang Y, Cheng Y, Liu G, Tian X, Tu X, Wang J. Chronic exposure of gestation rat to sevoflurane impairs offspring brain development. Neurol Sci. 2012;33:535–44.
+
+8.
+
+10. Palanisamy A. Maternal anesthesia and fetal neurodevelopment. Int J Obstet Anesth. 2012;21:152–62.
+
+11. Clancy B, Darlington RB, Finlay BL. Translating developmental time across mammalian species. Neuroscience. 2001;105:7–17.
+
+12. Yoo KY, Lee JC, Yoon MH, Shin MH, Kim SJ, Kim YH, Song TB, Lee J. The effects of volatile anesthetics on spontaneous contractility of isolated human pregnant uterine muscle: a comparison among sevoflurane, desflurane, isoflurane, and halothane. Anesth Analg. 2006;103:443–7. 13. Zhai WH, Zhao J, Huo SP, Chen XG, Li YD, Zhang ZL, LL Y, Song S, Wang QJ. Mechanisms of cytotoxicity induced by the anesthetic isoflurane: the role of inositol 1, 4, 5-trisphosphate receptors. Genet Mol Res. 2015;14:6929–42. 14. Callaway JK, Jones NC, Royse AG, Royse CF. Memory impairment in rats
+
+after desflurane anesthesia is age and dose dependent. J Alzheimers Dis. 2015;44:995–1005.
+
+15. Barrientos RM, Kitt MM, D'Angelo HM, Watkins LR, Rudy JW, Maier SF. Stable, long-term, spatial memory in young and aged rats achieved with a one day Morris water maze training protocol. Learn Mem. 2016;23:699–702.
+
+Page 7 of 7
+
+16. Rosi S, Andres-Mach M, Fishman KM, Levy W, Ferquson RA, Fike JR. Cranial irradiation alters the behaviorally induced immediate-early gene arc ( activity- regulated cytoskeleton-associated protein). Cancer Res. 2008;68:9763–70. 17. Mack ML, Preston AR. Decisions about the past are guided by reinstatement of specific memories in the hippocampus and perirhinal cortex. NeuroImage. 2016; 127:144–57. Slee EA, Adrain C, Martin SJ. Executioner caspase-3,-6,and-7 perform distinct,non-redundant roles during the demolition phase of apoptosis. J Biol Chem. 2001;276:7320–6.
+
+18.
+
+19. Gonçalves JT, Schafer ST, Gage FH. Adult neurogenesis in the hippocampus: from stem cells to behavior. Cell. 2016;167:897–914.
+
+20. Mazumder S, Plesca D, Almasan A. Caspase-3 activation is a critical determinant of genotoxic stress-induced apoptosis. Methods Mol Biol. 2008;414:13–21. 21. de Vasconcellos AP, Zugno AI, Dos Santos AH, Nietto FB, Crema LM, Goncalves M, Franzon R, de Souza Wyse AT, da Rocha ER, Dalmaz C. Na+, K+ -ATPase activity is reduced in hippocampus of rats submitted to an experimental model of depression: effect of chronic lithium treatment and possible involvement in learning deficits. Neurobiol Learn Mem. 2005;84:102–10. 22. Yu W, Zhang L, Han TZ, Jiang ML. Distribution of p-CREB in hippocampal formation of adult rats. J Fourth Mil Med Univ. 2004;25:874–6. 23. Han JH, Kushner SA, Yiu AP, Cole CJ, Matynia A, Brown RA, Neve RL, Guzowski JF, Silva AJ, Josselyn SA. Neuronal competition and selection during memory formation. Science. 2007;316:457–60.
+
+24. Mizuno M, Yamada K, Maekawa N, Saito K, Seishima M, Nabeshima TCREB. Phosphorylation as a molecular marker of memory processing in the hippocampus for spatial learning. Behav Brain Res. 2002;133:135–41.
+
+25. Deisseroth K, Bito H, Tsien RW. Signaling from synapse to nucleus: postsynaptic CREB phosphorylation during multiple forms of hippocampal synaptic plasticity. Neuron. 1996;16:89–101.
+
+26. Zhu DY, Lau L, Liu SH, Wei JS, Activation LYM. Of cAMP-response-element- bingding protein (CREB) after focal cerebral ischemia stimulates neurogenesis in the adult dentate gyrus. Proc Natl Acad Sci U S A. 2004;101:9453–7.
+
+27. Pittenger C, Huang YY, Paletzki RF, Bourtchouladze R, Scanlin H, Vronskaya S, Kandel ER. Reversible inhibition of CREB/ ATF transcription factors in region CA1 of the dorsal hippocampus disrupts hippocampus-dependent spatial memory. Neuron. 2002;34:447–62.
+
+28. Hinoi E, Balcar VJ, Kuramoto N, Nakamichi N, Yoneda Y. Nuclear transcription factors in the hippocampus. Prog Neurobiol. 2002;68:145–65. 29. Putignano E, Lonetti G, Cancedda L, Ratto G, Costa M, Maffei L, Pizzorusso T.
+
+30.
+
+31.
+
+32.
+
+33.
+
+34.
+
+35.
+
+Developmental downregulation of histone posttranslational modifications regulates visual cortical plasticity. Neuron. 2007;53:747–59. Kida S, Josselyn SA, Peña de Ortiz S, Kogan JH, Chevere I, Masushige S, Silva AJCREB. Required for the stability of new and reactivated fear memories. Nat Neurosci. 2002;5:348–55. Todorovski Z, Asrar S, Liu J, Saw NM, Joshi K, Cortez MA, Snead OC 3rd, Xie W, Jia Z. LIMK1 regulates long-term memory and synaptic plasticity via the transcriptional factorCREB. Mol Cell Biol. 2015;35:1316–28. Taubenfeld SM, Wiig KA, Monti B, Dolan B, Pollonini G, Alberini CM. Fornix- dependent induction of hippocampal CCAAT enhancer-binding protein [beta] and [delta] co-localizes with phosphorylated cAMP response element-binding protein and accompanies long-term memory consolidation. J Neurosci. 2001;21:84–91. Sekeres MJ, Neve RL, Frankland PW, Josselyn SA. Dorsal hippocampal CREB is both necessary and sufficient for spatial memory. Learn Mem. 2010;17:280–3. Lee Y, Kim J, Jang S, Administration OS. Of Phytoceramide enhances memory and upregulates the expression of pCREB and BDNF in hippocampus of mice. Biomol Ther (Seoul). 2013;21:229–33. Eriksson PS, Perfilieva E, Björk-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH. Neurogenesis in the adult human hippocampus. Nat Med. 1998;4:1313–7. Traiffort E, Ferent J. Neural stem cells and notch signaling. Med Sci (Paris). 2015;31:1115–25.
+
+36.
+
+37. Garcia AD, Doan NB, Imura T, Bush TG, Sofroniew MV. GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain. Nat Neurosci. 2004;7:1233–41. Lepousez G, Nissant A, Lledo PM. Adult neurogenesis and the future of the rejuvenating brain circuits. Neuron. 2015;86:387–401. Kee N, Teixeira CM, Wang AH, Frankland PW. Preferential incorporation of adult-generated granule cells into spatial memory networks in the dentate gyrus. Nat Neurosci. 2007;10:355–62.
+
+37. Garcia AD, Doan NB, Imura T, Bush TG, Sofroniew MV. GFAP-expressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain. Nat Neurosci. 2004;7:1233–41. Lepousez G, Nissant A, Lledo PM. Adult neurogenesis and the future of the rejuvenating brain circuits. Neuron. 2015;86:387–401. Kee N, Teixeira CM, Wang AH, Frankland PW. Preferential incorporation of adult-generated granule cells into spatial memory networks in the dentate gyrus. Nat Neurosci. 2007;10:355–62.
+
+38.

new_pdfs/10.1213_ANE.0000000000000030.txt

@@ -0,0 +1,745 @@
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+NIH Public Access Author Manuscript Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Published in final edited form as:
+
+Anesth Analg. 2014 June ; 118(6): 1284–1292. doi:10.1213/ANE.0000000000000030.
+
+Subclinical Carbon Monoxide Limits Apoptosis in the Developing Brain After Isoflurane Exposure
+
+Ying Cheng and Richard J. Levy, MD Division of Anesthesiology and Pain Medicine, Children’s National Medical Center, The George Washington University School of Medicine and Health Sciences, Washington, DC.
+
+Abstract
+
+BACKGROUND—Volatile anesthetics cause widespread apoptosis in the developing brain. Carbon monoxide (CO) has antiapoptotic properties, and exhaled endogenous CO is commonly rebreathed during low-flow anesthesia in infants and children, resulting in subclinical CO exposure. Thus, we aimed to determine whether CO could limit isoflurane-induced apoptosis in the developing brain.
+
+METHODS—Seven-day-old male CD-1 mouse pups underwent 1-hour exposure to 0 (air), 5, or 100 ppm CO in air with or without isoflurane (2%). We assessed carboxyhemoglobin levels, cytochrome c peroxidase activity, and cytochrome c release from forebrain mitochondria after exposure and quantified the number of activated caspase-3 positive cells and TUNEL positive nuclei in neocortex, hippocampus, and hypothalamus/thalamus.
+
+RESULTS—Carboxyhemoglobin levels approximated those expected in humans after a similar time-weighted CO exposure. Isoflurane significantly increased cytochrome c peroxidase activity, cytochrome c release, the number of activated caspase-3 cells, and TUNEL positive nuclei in the forebrain of air-exposed mice. CO, however, abrogated isoflurane-induced cytochrome c peroxidase activation and cytochrome c release from forebrain mitochondria and decreased the number of activated caspase-3 positive cells and TUNEL positive nuclei after simultaneous exposure with isoflurane.
+
+CONCLUSIONS—Taken together, the data indicate that CO can limit apoptosis after isoflurane exposure via inhibition of cytochrome c peroxidase depending on concentration. Although it is unknown whether CO directly inhibited isoflurane-induced apoptosis, it is possible that low-flow
+
+Copyright © 2014 International Anesthesia Research Society
+
+Address correspondence to Richard J. Levy, MD, Division of Anesthesiology and Pain Medicine, Children’s National Medical Center, 111 Michigan Ave., NW, Washington, DC 20010. rlevy@cnmc.org.. DISCLOSURES Name: Ying Cheng Contribution: This author helped collect data, analyzed the data, and contributed in study design. Attestation: Ms. Cheng attests to the integrity of the data and the analysis and approves the final manuscript. She is the archival author. Name: Richard J. Levy, MD. Contribution: This author designed the study, analyzed the data, and prepared the manuscript. Attestation: Dr. Levy attests to the integrity of the data and the analysis and approves the final manuscript. This manuscript was handled by: Gregory J. Crosby, MD.
+
+The authors declare no conflicts of interest.
+
+Reprints will not be available from the authors.
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+anesthesia designed to target rebreathing of specific concentrations of CO may be a desired strategy to develop in the future in an effort to prevent anesthesia-induced neurotoxicity in infants and children.
+
+Anumber of commonly used anesthetic drugs cause widespread neuronal apoptosis in the developing mammalian brain.1–5 Vulnerability coincides with the period of synaptogenesis, and anesthesia-induced neurotoxicity has been shown to result in significant neuron loss, behavioral impairments, and cognitive deficits in a variety of newborn animal models.6,7 Although a causal relationship in humans has yet to be demonstrated, evidence indicating an association between anesthesia exposure and cognitive and behavioral disorders in young children continues to emerge.8–10 Thus, there is a need to develop protective strategies to prevent potential anesthesia-induced neurodegeneration in infants and children.
+
+The exact upstream mechanisms that initiate anesthesia-induced neurotoxicity are not completely understood; however, downstream, the process is mediated by the mitochondrial pathway of apoptosis.6,11 After anesthetic exposure, Bax translocates to the outer mitochondrial membrane, resulting in mitochondrial permeabilization, release of cytochrome c, widespread caspase-3 activation, and DNA fragmentation.6 Upstream of this phenomenon, cytochrome c is bound to cardiolipin on the inner mitochondrial membrane via electrostatic and hydrophobic interactions.12 Cytochrome c has peroxidase activity and, in the presence of hydrogen peroxide, oxidizes cardiolipin to hydroperoxycardiolipin.12 This mobilizes cytochrome c from the inner membrane and permits it to be released after permeabilization of the outer mitochondrial membrane.
+
+Carbon monoxide (CO) is a colorless and odorless gas that has antiapoptotic properties.13–18 CO prevents apoptosis by binding to the cytochrome c-cardiolipin complex and inhibiting cytochrome c peroxidase activity.12,19 This prevents oxidation of cardiolipin, mobilization and release of cytochrome c, and subsequent caspase activation. It has been demonstrated that brief exposure to low concentrations of CO inhibits developmental programmed cell death in vivo in the forebrain of newborn mice.19 It is important to note that infants and children are routinely exposed to CO during low-flow anesthesia when rebreathing is permitted.20,21 The source of CO in this setting is likely exhaled endogenous CO generated via heme catabolism.21 Because exhaled CO is not scavenged or removed from the anesthesia breathing circuit, during low-flow anesthesia, patients rebreathe exhaled CO and experience a subclinical CO exposure.21,22 In this work, we aimed to determine whether the antiapoptotic effects of subclinical concentrations of inspired CO could limit anesthesia- induced neuronal apoptosis. We demonstrated that CO exposure limits apoptosis in a variety of brain regions in newborn mice exposed to isoflurane by inhibiting cytochrome c peroxidase activity and subsequent cytochrome c release. These findings are clinically relevant and could have implications for the development of low-flow anesthesia as a standard paradigm to target low CO concentration exposures in infants and children to prevent anesthesia-induced neurotoxicity.
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 2
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+METHODS
+
+Animal Exposures
+
+The care of the animals in this study was in accordance with National Institutes of Health and Institutional Animal Care and Use Committee guidelines. Study approval was granted by the Children’s National Medical Center. Sixto 8-week-old CD-1 pregnant female mice (20–30 grams) were acquired (Charles River, Wilmington, MA) to yield newborn pups. CD-1 mice were chosen because pups have been shown to reliably demonstrate neuronal changes consistent with human neonatal injury in specific experimental models.23 On postnatal day 7 (P7), we exposed male CD-1 mouse pups to 0 ppm CO (air), 5 ppm CO in air, or 100 ppm CO in air with and without isoflurane (2%) for 1 hour in a 7-L Plexiglas chamber (25 × 20 × 14 cm). The 3 experimental CO cohorts represented: negative control (0 ppm CO), low concentration subclinical CO (5 ppm), and high concentration subclinical CO (100 ppm). The chamber had a port for fresh gas inlet and a port for gas outlet that was directed to a fume hood exhaust using standard suction tubing. Specific concentrations of CO in air (premixed gas H-cylinders, Air Products, Camden, NJ) were verified using an electrochemical sensing CO detector (Monoxor III, Bacharach, Anderson, CA). Designated CO mixtures were delivered through the variable bypass isoflurane vaporizer and exposure chamber at a flow rate of 8 to 12 L/min. Mice were kept warm with an infrared heating lamp (Cole-Parmer, Court Vernon Hills, IL). P7 was chosen because synaptogenesis peaks at day 7 in rodents and is completed by the second or third week of life.24,25 One hour exposure to 2% isoflurane has been shown to activate brain capsase-3 in 7-day-old mice and is a brief anesthetic exposure.26 After exposure, pups were placed with their respective dams. Eighty- four newborn mice were evaluated.
+
+Carboxyhemoglobin (COHb) Levels
+
+COHb levels were measured immediately after 1-hour exposure. At the time of euthanasia, after pentobarbital injection (150 mg/kg, intraperitoneal), 200 μL blood was sampled from the left ventricle and COHb measured via 6 wavelength co-oximetry (Radiometer Osm3 Hemoximeter, Copenhagen, Denmark, range 0–100 ± 0.2%). Five animals per cohort were evaluated.
+
+Activated Caspase-3 Immunohistochemistry
+
+Five hours after exposure, following euthanasia with pentobarbital injection (150 mg/kg, ip), the brain was perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) via left ventricle injection for 30 minutes and then postfixed in additional fixative solution for 24 hours at 4°C. Serial sections were cut at a thickness of 6 μm in the coronal plane through the cerebral hemispheres beginning at −1.7 mm from bregma and 2.1 mm from interaural, and individual sections were slide mounted. Immunohistochemistry was performed on 3 to 4 nonserial nonadjacent sections using polyclonal antirabbit activated caspase-3 (Cell Signaling Technology, Beverly, MA), biotinylated secondary antibody (goat antirabbit, Cell Signaling Technology), and developed with diaminobenzidine. Nuclei were counterstained with hematoxylin. The number of activated caspase-3 positive cells per square millimeter was quantified at ×10 magnification in neocortex (primary and secondary somatosensory and auditory neocortices), hippocampus (dentate gyrus, CA1, CA2, and CA3 regions), and
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 3
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+hypothalamic/thalamic region (laterodorsal, mediodorsal, ventromedial, ventrolateral, ventroposteromedial, ventroposterolateral thalamic nuclei, ventromedial hypothalamic nucleus, peduncular part of the lateral hypothalamus, and the central anterior hypothalamic area) of both hemispheres in 3 to 4 animals per group. Brain regions were defined in accordance with Mouse Brain Atlas.27,28
+
+Terminal Deoxynucleotidyl Transferase-Mediated UTP Nick End-Labeling Staining
+
+Five hours after exposure, following euthanasia, the brain was perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) via left ventricle injection for 30 minutes and then postfixed in additional fixative solution for 24 hours at 4°C. Paraffin- embedded brain sections were cut into 6-μm sections in the coronal plane through the cerebral hemispheres beginning at −1.7 mm from bregma, 2.1 mm from interaural, slide mounted, and stained for terminal deoxynucleotidyl transferase-mediated UTP nick end- labeling (TUNEL). Sections were incubated in 0.5% Triton at room temperature, followed by proteinase K at 37°C, then immersed in terminal deoxynucleotidyl transferase (TdT) buffer (30 mmol/L Tris-HCl buffer, pH 7.2, 140 mmol/L sodium cacodylate, and 1 mmol/L cobalt chloride) at room temperature. This was followed by incubation with TdT and biotin-16-dUTP for 60 minutes at 37°C. The reaction was terminated with TB buffer (300 mmol/L sodium chloride with 30 mmol/L sodium citrate) at room temperature, followed by immersion in 3% hydrogen peroxide and 2% fetal bovine serum at room temperature. The sections were then covered with an Avidin Biotin Complex (1:200 dilution) for 30 minutes at room temperature, incubated with FITCAvidin D for detection, and counterstained with DAPI. The numbers of TUNEL positive nuclei in neocortex, hippocampus, and hypothalamic/thalamic region (identical regions as for activated caspase-3) were quantified at ×10 magnification in 3 to 4 nonserial sections per mouse, and 3 to 4 mice per cohort were evaluated. Brain regions were defined in accordance with Mouse Brain Atlas.27,28
+
+Cytochrome C Peroxidase Activity
+
+Immediately after 1-hour exposure, cytochrome c was extracted from fresh mitochondria as previously described.29 Isolated forebrain mitochondria (20 mg/mL) were suspended in a hypotonic 0.015 M KCl solution for 10 minutes on ice and then centrifuged at 105,000g for 15 minutes at 4°C. The pellet was resuspended in 0.15 M KCl solution for 10 minutes on ice and then centrifuged again at 105,000g for 15 minutes at 4°C. The supernatant was collected and cytochrome c content quantified with spectrophotometry. The peroxidase activity of 0.5 to 1 μM cytochrome c was determined by measuring the rate of oxidation of 50 μM 2,2 azinobis-(2-ethylbenzthiazoline-6-sulfonate) (ABTS) in 10 mM potassium phosphate buffer (pH 7.4) at 415 nm (ε415 = 3.6 × 104 M−1 cm−1) after the addition of hydrogen peroxide.30 Five animals per cohort were evaluated.
+
+Heme C Determination
+
+Immediately after 1-hour exposure, forebrain mitochondria and cytosol were isolated by differential centrifugation.31 As previously described, forebrain was harvested and homogenized in ice-cold H medium (70 mM sucrose, 220 mM mannitol, 2.5 mM Hepes, pH 7.4 and 2 mM EDTA).31 The homogenate was spun at 1500g for 10 minutes at 4°C.
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 4
+
+′-
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+Supernatant was removed and centrifuged at 10,000g for 10 minutes at 4°C. Cytosolic supernatant was collected, and pellet was resuspended in H medium and centrifuged again at 10,000g for 10 minutes at 4°C. Pellet was again resuspended in H medium, and mitochondrial and cytosolic protein concentrations subsequently determined using the method of Lowry.31
+
+Mitochondrial and cytosolic heme c content were calculated from the difference in spectra (dithionate/ascorbate reduced minus air-oxidized) of mitochondria or cytosolic protein (0.5– 1 mg) solubilized in 10% lauryl maltoside using an absorption coefficient of 20.5 mM−1 cm−1 at 550 to 535 nm.32,33 Five animals per cohort were evaluated.
+
+Statistical Analysis
+
+Sample sizes for each end point were chosen based on previous work.19 Our previous study used 8 animals per cohort for COHb and heme c determination, 3 to 4 animals per cohort for activated caspase-3 and TUNEL assessment, and 5 animals per cohort for measurement of cytochrome c peroxidase activity, and data followed normal probability distribution.19 For this work, sample sizes were based on the number of animals needed to detect a 30% difference from air-exposed control values with a power of 80 based on an α of 0.01. Data are presented as mean ± SE. To assess statistical significance, we performed pairwise comparisons in an analysis of variance design using Tukey test.
+
+RESULTS
+
+Brief Subclinical CO Exposure With and Without Isoflurane in Newborn Mouse Pups
+
+To investigate the effects of subclinical CO on isoflurane-induced neuronal apoptosis, we exposed 7-day-old male CD-1 mouse pups to 0 ppm CO (air), 5 ppm CO (low concentration subclinical exposure), or 100 ppm CO (upper limit of subclinical exposure) for 1 hour with and without isoflurane (2%). P7 was chosen because synaptogenesis peaks on day 7 in rodents and is completed by the second or third week of life.24,25 The exposure time was chosen because inspiring isoflurane for 1 hour has been shown to activate brain capsase-3 in 7-day-old mice and is a clinically relevant anesthetic duration in infants and children.26
+
+COHb levels increased significantly in the cohort exposed to 100 ppm CO without isoflurane with a trend toward significance in animals exposed to 100 ppm with isoflurane (Fig. 1). There was no significant difference in COHb levels in cohorts exposed to 5 ppm CO compared with air-exposed control values (Fig. 1). Isoflurane exposure had no independent effect on COHb levels (P = 0.34 [95% CI, 1.45 ± 0.05]) (Fig. 1). It is important to note that the resultant COHb levels measured after CO exposure in all groups approximated levels expected in humans after a similar time-weighted exposure (e.g., mean COHb of 3.27% in humans after 1-hour exposure to 100 ppm CO, 95% CI ± 0.12) (Fig. 1).34–36 Furthermore, these levels were well below values known to result in tissue hypoxia (70% COHb) and were markedly less than levels known to elicit signs and symptoms in humans (10% COHb).13,37 Thus, 1-hour exposure to 5 or 100 ppm CO in 7-day-old mouse pups was a subclinical CO exposure.
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 5
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+Brief Subclinical CO Exposure Inhibits Neuronal Apoptosis in Isoflurane-Exposed Mice
+
+Commonly used anesthetics cause widespread apoptosis in the developing mammalian brain.1–5 Thus, we determined the effect of CO on isoflurane-induced neuronal apoptosis in the neocortex, hippocampus, and hypothalamic and thalamic regions by assessing for activated caspase-3 with immunohistochemistry and TdT-mediated TUNEL staining on slide-mounted brain sections. Pups were evaluated 5 hours after simultaneous exposure to either 0 ppm CO (air), 5 ppm CO, or 100 ppm CO with or without isoflurane on P7.
+
+Consistent with previous work, 1-hour exposure to isoflurane in air significantly increased the number of activated caspase-3 positive cells and TUNEL positive nuclei in all brain regions examined compared with nonisoflurane air-exposed animals (Figs. 2 and 3).26 CO significantly decreased the number of activated caspase-3 positive cells and TUNEL positive nuclei in virtually every brain region of both cohorts exposed to CO with isoflurane compared with mice exposed to isoflurane in air (Figs. 2 and 3). It is important to note that activated caspase-3 and the number of TUNEL positive nuclei were at or below air-exposed levels in all brain regions evaluated after 100 ppm CO and isoflurane exposure. In addition, there appeared to be a concentration-dependent CO effect on the number of activated caspase-3 positive cells in the hypothalamus/thalamus and TUNEL positive nuclei in the neocortex and hippocampus of isoflurane-exposed mice.
+
+Exposure to CO without isoflurane had variable effects on activated caspase-3 and the number of TUNEL positive nuclei compared with air-exposed controls (Figs. 2 and 3). Differences trended toward significance between CO-exposed cohorts without isoflurane in the number of TUNEL positive nuclei in the hippocampus and hypothalamus/thalamus of 100 ppm CO exposed mice vs animals exposed to 5 ppm CO (Fig. 3).
+
+CO Inhibits Forebrain Cytochrome C Peroxidase Activity and Isoflurane-Induced Cytochrome C Release from Mitochondria
+
+Peroxidation of cardiolipin is an upstream event that is important for cytochrome c release and initiation of the mitochondrial apoptosis pathway.12 CO can bind to the cytochrome c- cardiolipin complex and inhibit cytochrome c peroxidase activity, thereby preventing oxidation of cardiolipin and mobilization and release of cytochrome c.12,19 Thus, we extracted cytochrome c from forebrain mitochondria immediately after 1-hour exposure to either 0 ppm CO (air), 5 ppm CO, or 100 ppm CO with or without isoflurane on P7 and measured peroxidase activity of cytochrome c using spectrophotometry. To assess for cytochrome c release, we measured the amount of heme c (the heme moiety of cytochrome c) in forebrain mitochondrial and cytosolic fractions immediately after exposure.
+
+Isoflurane either significantly increased or trended toward a significant increase in forebrain cytochrome c peroxidase activity after 1-hour exposure in air compared with all nonisoflurane-exposed cohorts (Fig. 4). All isoflurane-exposed cohorts demonstrated significantly higher or a trend toward significantly higher cytochrome c peroxidase activity compared with nonisoflurane CO matched cohorts (Fig. 4). CO exposure significantly decreased forebrain cytochrome c peroxidase activity in isoflurane and nonisoflurane- exposed cohorts in a concentration-dependent manner (Fig. 4). CO-mediated inhibition of
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 6
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+cytochrome c peroxidase resulted in enzyme activities that were below that of air-exposed control values in all CO-exposed cohorts (Fig. 4).
+
+After isoflurane exposure for 1 hour in air, heme c levels decreased significantly in the mitochondrial fraction and increased significantly in the cytosolic fraction compared with nonisoflurane air-exposed controls, suggesting increased cytochrome c release in the forebrain after exposure (Fig. 5). CO exposure without isoflurane trended toward a significant increase in the amount of heme c in forebrain mitochondria in 5 ppm exposed animals compared with air-exposed controls (Fig. 5A). Cytosolic heme c content in forebrain decreased significantly in both cohorts exposed to CO without isoflurane compared with air-exposed controls, indicating inhibition of cytochrome c release (Fig. 5B). Exposure to CO with isoflurane resulted in significantly increased heme c levels within forebrain mitochondria of 100 ppm exposed mice and significantly decreased heme c in cytosol of both CO-exposed cohorts compared with animals exposed to isoflurane alone (Fig. 5). Decreases in cytosolic heme c after CO exposure with isoflurane were dose- dependent, and levels were at or below air-exposed control values (Fig. 5B). Mitochondrial heme c levels in animals exposed to isoflurane with 100 ppm CO were equivalent to levels seen in forebrain mitochondria of controls exposed to air alone (Fig. 5A). Taken together, the data suggest that CO inhibits forebrain cytochrome c peroxidase and, depending on concentration, can decrease isoflurane-induced cytochrome c release.
+
+DISCUSSION
+
+Our findings are consistent with previous work and support the concept that isoflurane causes neurotoxicity in the developing mammalian brain via activation of the intrinsic apoptosis pathway.6,11 In addition, we demonstrate for the first time that, upstream from cytochrome c release, isoflurane increases forebrain cytochrome c peroxidase activity. It is important to note that subclinical concentrations of CO inhibited isoflurane-induced cytochrome c peroxidase activation in a dose-dependent manner and decreased the release of cytochrome c, limiting apoptosis in the developing brain after exposure to isoflurane.
+
+Because hydrogen peroxide is required for induction of cytochrome c peroxidase, increased peroxidase activity likely resulted from isoflurane-induced oxidative stress.38,39 Although we did not measure free radicals as part of this study, isoflurane and other anesthetic drugs generate reactive oxygen, nitrogen species, and hydrogen peroxide in developing neurons, hippocampus, subiculum, and thalamus.40–42 In addition, cytochrome c peroxidase activity has been shown to increase in the forebrain of mice during oxidative stress, and cytochrome c has been developed as a biosensor for hydrogen peroxide detection.43,44 Thus, isoflurane exposure likely increased hydrogen peroxide production that, in turn, enhanced the peroxidase activity of cytochrome c.38
+
+Finding increased cytochrome c peroxidase activity after isoflurane exposure is significant because it uncovers a potential target for therapeutic intervention. Peroxidase activation is necessary and critical for mobilization and release of cytochrome c from mitochondria during apoptosis.12 Release of cytochrome c is often considered the “point of no return” in the pathway.45 Thus, targeting the peroxidase activity of cytochrome c is logical and
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 7
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+inhibiting cytochrome c peroxidase could prevent cytochrome c release during proapoptotic stimuli such as with anesthetic exposure. In support of this concept, low concentrations of inhaled CO (50–500 ppm) have been shown to prevent apoptosis in endothelium, vascular smooth muscle, liver, lung tissue during hyperoxia, sepsis, and ischemia-reperfusion.14–18 In previous work, we demonstrated that brief exposure to low CO concentrations can inhibit cytochrome c peroxidase in vivo and impair programmed cell death in the developing forebrain of newborn mice.19 Here we demonstrate that CO, inspired at subclinical concentrations, inhibits activation of cytochrome c peroxidase during isoflurane exposure and impairs release of cytochrome c.
+
+CO prevents apoptosis by binding to the cytochrome c-cardiolipin complex and inhibiting cytochrome c peroxidase activity.12 This prevents oxidation of cardiolipin and subsequent release of cytochrome c.12 Although it is possible that CO may exert its prosurvival response via a variety of other mechanisms, our data suggest that abrogation of isoflurane- induced apoptosis may be due to CO-mediated inhibition of cytochrome c peroxidase activation. This is supported by finding concentration-dependent responses to subclinical CO exposure within different aspects of the intrinsic apoptosis pathway that we assayed (cytochrome c peroxidase, cytochrome c release, activation of caspase-3, and DNA breakage [TUNEL]).
+
+Although the data indicate that low concentrations of CO can inhibit apoptosis in the developing brain, we have not conclusively shown that CO directly prevents isoflurane- induced neuronal apoptosis. This is important because the CO effect could simply be a generalized, nonspecific phenomenon. Thus, it is possible that CO-mediated inhibition of apoptosis and anesthetic-induced apoptosis are 2 distinct processes that occur simultaneously and independently within the developing brain but not necessarily in the same cells. If these 2 processes are mutually exclusive, then certain cell populations would undergo apoptosis after exposure to isoflurane while totally different cell populations, destined to die via developmental programmed cell death, would survive due to CO- mediated inhibition of the intrinsic apoptosis pathway. Although the total number of neurons undergoing apoptosis in this scenario would be relatively decreased compared with isoflurane exposure alone, the impact on neurodevelopment could be devastating. This is because we have previously demonstrated that exposure to low concentrations of CO in the absence of an anesthetic prevents natural programmed cell death in the neocortex and hippocampus of 10-day-old mice and impairs neurocognitive development.19 Thus, although CO has antiapoptotic properties, its unchecked effects in the developing brain could be equally as deleterious as the effects of anesthetics alone.
+
+Furthermore, brief, low concentration CO exposures can also cause oxidative stress.46 Such undesired effects could enhance apoptosis during development and could explain our findings regarding activated caspase-3 and TUNEL in the cohorts exposed to CO for 1 hour without isoflurane. So it is possible that CO has the potential to exert proapoptotic effects that could act synergistically with isoflurane. Thus, before implementing subclinical CO exposure in everyday clinical practice, its safety and efficacy must be established given the fact that its pro- and antiapoptotic effects could have adverse consequences in the developing brain.
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 8
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+The only way to prove that CO directly prevents the proapoptotic effect of isoflurane is to evaluate neurocognitive function and behavior after exposure. The fact that we have not included such an assessment in this work is a major limitation of the study. However, this will be the focus of future work. Thus, until we determine neurodevelopmental outcome after combined exposure to CO with isoflurane, we cannot draw definitive conclusions about the benefit of CO during an anesthetic.
+
+Regarding clinical toxicity, elevated COHb levels are formed after exposure to high concentrations of CO and can interfere with tissue oxygen delivery by impairing oxygen binding to and dissociation from hemoglobin.37 Overt clinical toxicity manifests from tissue hypoxia when COHb levels are higher than 70%, and signs and symptoms first appear when COHb levels are higher than 10%.13,37 Acute exposure to CO concentrations larger than 800 ppm can rapidly cause brain injury, cerebral edema, coma, and death, while brief exposure to 220 ppm results in headache, dizziness, and impaired judgment.34 Exposure to concentrations <120 ppm does not elicit any appreciable clinical symptoms.34 Thus, short- term exposure to 5 or 100 ppm CO is a non–life-threatening subclinical exposure, and the resultant COHb levels are well below values that cause tissue hypoxia and clinical signs and symptoms.
+
+CO is endogenously produced, and infants and children routinely inspire subclinical concentrations of CO when rebreathing is permitted during low-flow general anesthesia.20,21,47 In previous work, we found that children inspire an average of 2 to 3 ppm CO and as much as 18 ppm CO during low-flow anesthesia.20,21 This resulted in an increase in COHb by an average of 0.2% up to 0.5% from baseline after 1 hour of low-flow anesthesia.20 In the current work, we found a similar increase in COHb after 1-hour exposure to 5 ppm CO and a 3% to 4% increase after exposure to 100 ppm CO. Thus, exposing newborn mice to 5 ppm CO with isoflurane mimics a subclinical CO exposure during lowflow anesthesia at a time point in late infancy.
+
+Here we found that 5 ppm CO limited apoptosis after isoflurane exposure while 100 ppm CO maintained levels of apoptosis at or below control values. These findings suggest that higher concentrations of subclinical CO may be necessary to completely offset the proapoptotic response to isoflurane. Thus, it is possible that levels of CO encountered with rebreathing during routine lowflow anesthesia may limit isoflurane-induced neurotoxicity but may not be adequate to completely prevent it. However, more investigation is necessary before such conclusions can be made.
+
+Targeting cytochrome c peroxidase to offset the oxidative stress of an anesthetic exposure is a novel and intriguing concept. Given that CO is commonly rebreathed during lowflow anesthesia and readily diffuses across the blood-brain barrier to gain access to the mitochondrial inner membrane, it has the potential to be developed as an antiapoptotic agent to prevent anesthesia-induced neurotoxicity.48 However, before routine application of CO as such a therapeutic agent, neurodegeneration needs to be definitively shown to occur in children after anesthetic exposure, CO must be shown to directly inhibit isoflurane-induced apoptosis, and the safety of subclinical CO exposure during development needs to be established. With further work, however, it is possible that low-flow anesthesia, intended to
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 9
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+result in low concentration CO exposure, may be established as a standard paradigm designed to protect the developing brain of the infants and children we care for.
+
+Acknowledgments
+
+Funding: None.
+
+REFERENCES
+
+1. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF. Early exposure to common anesthetic agents causes widespread neurode-generation in the developing rat brain and persistent learning deficits. J Neurosci. 2003; 23:876–82. [PubMed: 12574416]
+
+2. Stefovska VG, Uckermann O, Czuczwar M, Smitka M, Czuczwar P, Kis J, Kaindl AM, Turski L, Turski WA, Ikonomidou C. Sedative and anticonvulsant drugs suppress postnatal neurogenesis. Ann Neurol. 2008; 64:434–45. [PubMed: 18991352]
+
+3. Istaphanous GK, Loepke AW. General anesthetics and the developing brain. Curr Opin Anaesthesiol. 2009; 22:368–73. [PubMed: 19434780]
+
+4. Brambrink AM, Evers AS, Avidan MS, Farber NB, Smith DJ, Zhang X, Dissen GA, Creeley CE, Olney JW. Isoflurane-induced neuroapoptosis in the neonatal rhesus macaque brain. Anesthesiology. 2010; 112:834–41. [PubMed: 20234312]
+
+5. Istaphanous GK, Howard J, Nan X, Hughes EA, McCann JC, McAuliffe JJ, Danzer SC, Loepke AW. Comparison of the neuroapoptotic properties of equipotent anesthetic concentrations of desflurane, isoflurane, or sevoflurane in neonatal mice. Anesthesiology. 2011; 114:578–87. [PubMed: 21293251]
+
+6. Olney JW, Young C, Wozniak DF, Ikonomidou C, Jevtovic-Todorovic V. Anesthesia-induced developmental neuroapoptosis. Does it happen in humans? Anesthesiology. 2004; 101:273–5. [PubMed: 15277906]
+
+7. Rizzi S, Ori C, Jevtovic-Todorovic V. Timing versus duration: determinants of anesthesia-induced developmental apoptosis in the young mammalian brain. Ann N Y Acad Sci. 2010; 1199:43–51. [PubMed: 20633108]
+
+8. Wilder RT, Flick RP, Sprung J, Katusic SK, Barbaresi WJ, Mickelson C, Gleich SJ, Schroeder DR, Weaver AL, Warner DO. Early exposure to anesthesia and learning disabilities in a population- based birth cohort. Anesthesiology. 2009; 110:796–804. [PubMed: 19293700]
+
+9. DiMaggio C, Sun LS, Kakavouli A, Byrne MW, Li G. A retrospective cohort study of the association of anesthesia and hernia repair surgery with behavioral and developmental disorders in young children. J Neurosurg Anesthesiol. 2009; 21:286–91. [PubMed: 19955889]
+
+10. Flick RP, Katusic SK, Colligan RC, Wilder RT, Voigt RG, Olson MD, Sprung J, Weaver AL, Schroeder DR, Warner DO. Cognitive and behavioral outcomes after early exposure to anesthesia and surgery. Pediatrics. 2011; 128:e1053–61. [PubMed: 21969289]
+
+11. Yon JH, Daniel-Johnson J, Carter LB, Jevtovic-Todorovic V. Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience. 2005; 135:815–27. [PubMed: 16154281]
+
+12. Kapetanaki SM, Silkstone G, Husu I, Liebl U, Wilson MT, Vos MH. Interaction of carbon monoxide with the apoptosis-inducing cytochrome c-cardiolipin complex. Biochemistry. 2009; 48:1613–9. [PubMed: 19183042]
+
+13. Kao LW, Nañagas KA. Carbon monoxide poisoning. Emerg Med Clin North Am. 2004; 22:985–
+
+1018. [PubMed: 15474779]
+
+14. Zhou H, Liu J, Pan P, Jin D, Ding W, Li W. Carbon monoxide inhalation decreased lung injury via anti-inflammatory and anti-apoptotic effects in brain death rats. Exp Biol Med (Maywood). 2010; 235:1236–43. [PubMed: 20810760]
+
+15. Song R, Kubo M, Morse D, Zhou Z, Zhang X, Dauber JH, Fabisiak J, Alber SM, Watkins SC, Zuckerbraun BS, Otterbein LE, Ning W, Oury TD, Lee PJ, McCurry KR, Choi AM. Carbon
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 10
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+monoxide induces cytoprotection in rat orthotopic lung transplantation via anti-inflammatory and anti-apoptotic effects. Am J Pathol. 2003; 163:231–42. [PubMed: 12819027]
+
+16. Bernardini C, Zannoni A, Bacci ML, Forni M. Protective effect of carbon monoxide pre- conditioning on LPS-induced endothelial cell stress. Cell Stress Chaperones. 2010; 15:219–24. [PubMed: 19693705]
+
+17. Sarady JK, Zuckerbraun BS, Bilban M, Wagner O, Usheva A, Liu F, Ifedigbo E, Zamora R, Choi AM, Otterbein LE. Carbon monoxide protection against endotoxic shock involves reciprocal effects on iNOS in the lung and liver. FASEB J. 2004; 18:854–6. [PubMed: 15001560]
+
+18. Wang X, Wang Y, Kim HP, Nakahira K, Ryter SW, Choi AM. Carbon monoxide protects against hyperoxia-induced endothelial cell apoptosis by inhibiting reactive oxygen species formation. J Biol Chem. 2007; 282:1718–26. [PubMed: 17135272]
+
+19. Cheng Y, Thomas A, Mardini F, Bianchi SL, Tang JX, Peng J, Wei H, Eckenhoff MF, Eckenhoff RG, Levy RJ. Neurodevelopmental consequences of sub-clinical carbon monoxide exposure in newborn mice. PLoS One. 2012; 7:e32029. [PubMed: 22348142]
+
+20. Levy RJ, Nasr VG, Rivera O, Roberts R, Slack M, Kanter JP, Ratnayaka K, Kaplan RF, McGowan FX Jr. Detection of carbon monoxide during routine anesthetics in infants and children. Anesth Analg. 2010; 110:747–53. [PubMed: 20185653]
+
+21. Nasr V, Emmanuel J, Deutsch N, Slack M, Kanter J, Ratnayaka K, Levy R. Carbon monoxide re- breathing during low-flow anaesthesia in infants and children. Br J Anaesth. 2010; 105:836–41. [PubMed: 20947594]
+
+22. Woehlck HJ. Carbon monoxide rebreathing during low flow anesthesia. Anesth Analg. 2001; 93:516–7. [PubMed: 11473890]
+
+23. Farahani R, Kanaan A, Gavrialov O, Brunnert S, Douglas RM, Morcillo P, Haddad GG. Differential effects of chronic intermittent and chronic constant hypoxia on postnatal growth and development. Pediatr Pulmonol. 2008; 43:20–8. [PubMed: 18041750]
+
+24. Rice D, Barone S Jr. Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ Health Perspect. 2000; 108(Suppl 3):511–33. [PubMed: 10852851]
+
+25. Sanno H, Shen X, Kuru N, Bormuth I, Bobsin K, Gardner HA, Komljenovic D, Tarabykin V, Erzurumlu RS, Tucker KL. Control of postnatal apoptosis in the neocortex by RhoA-subfamily GTPases determines neuronal density. J Neurosci. 2010; 30:4221–31. [PubMed: 20335457] 26. Johnson SA, Young C, Olney JW. Isoflurane-induced neuroapoptosis in the developing brain of
+
+nonhypoglycemic mice. J Neurosurg Anesthesiol. 2008; 20:21–8. [PubMed: 18157021]
+
+27. Franklin, KBJ.; Paxinos, G. The Mouse Brain in Stereotaxic Coordinates. Academic Press; San Diego, CA: 1997.
+
+28. Paxinos, G.; Watson, C. Atlas of the Developing Mouse Brain: At E17.5, PO, and P6. Academic Press; San Diego, CA: 2007.
+
+29. Jacobs EE, Sanadi DR. The reversible removal of cytochrome c from mitochondria. J Biol Chem. 1960; 235:531–4. [PubMed: 14406362]
+
+30. Kim NH, Jeong MS, Choi SY, Kang JH. Peroxidase activity of cytochrome c. bull. Korean Chem Soc. 2004; 25:1889–92.
+
+31. Piel DA, Gruber PJ, Weinheimer CJ, Courtois MR, Robertson CM, Coopersmith CM, Deutschman CS, Levy RJ. Mitochondrial resuscitation with exogenous cytochrome c in the septic heart. Crit Care Med. 2007; 35:2120–7. [PubMed: 17855825]
+
+32. Ozawa T, Tanaka M, Shimomura Y. Crystallization of the middle part of the mitochondrial electron transfer chain: cyto-chrome bc1-cytochrome c complex. Proc Natl Acad Sci U S A. 1980; 77:5084–6. [PubMed: 6254056]
+
+33. Tanaka M, Ogawa N, Ihara K, Sugiyama Y, Mukohata Y. Cytochrome aa(3) in Haloferax volcanii. J Bacteriol. 2002; 184:840–5. [PubMed: 11790755]
+
+34. Winter PM, Miller JN. Carbon monoxide poisoning. JAMA. 1976; 236:1502. [PubMed: 989121] 35. Peterson JE, Stewart RD. Absorption and elimination of carbon monoxide by inactive young men. Arch Environ Health. 1970; 21:165–71. [PubMed: 5430002]
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 11
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+36. Stewart RD, Peterson JE, Baretta ED, Bachand RT, Hosko MJ, Herrmann AA. Experimental human exposure to carbon mon-oxide. Arch Environ Health. 1970; 21:154–64. [PubMed: 5430001]
+
+37. Gorman D, Drewry A, Huang YL, Sames C. The clinical toxicology of carbon monoxide. Toxicology. 2003; 187:25–38. [PubMed: 12679050]
+
+38. Puchkov MN, Vassarais RA, Korepanova EA, Osipov AN. Cytochrome c produces pores in cardiolipin-containing planar bilayer lipid membranes in the presence of hydrogen peroxide. Biochim Biophys Acta. 2013; 1828:208–12. [PubMed: 23085196]
+
+39. Kim H, Oh E, Im H, Mun J, Yang M, Khim JY, Lee E, Lim SH, Kong MH, Lee M, Sul D. Oxidative damages in the DNA, lipids, and proteins of rats exposed to isofluranes and alcohols. Toxicology. 2006; 220:169–78. [PubMed: 16442689]
+
+40. Bai X, Yan Y, Canfield S, Muravyeva MY, Kikuchi C, Zaja I, Corbett JA, Bosnjak ZJ. Ketamine
+
+enhances human neural stem cell proliferation and induces neuronal apoptosis via reactive oxygen species-mediated mitochondrial pathway. Anesth Analg. 2013; 116:869–80. [PubMed: 23460563] 41. Matsuoka H, Watanabe Y, Isshiki A, Quock RM. Increased production of nitric oxide metabolites in the hippocampus under isoflurane anaesthesia in rats. Eur J Anaesthesiol. 1999; 16:216–24. [PubMed: 10234490]
+
+42. Boscolo A, Milanovic D, Starr JA, Sanchez V, Oklopcic A, Moy L, Ori CC, Erisir A, Jevtovic- Todorovic V. Early exposure to general anesthesia disturbs mitochondrial fission and fusion in the developing rat brain. Anesthesiology. 2013; 118:1086–97. [PubMed: 23411726]
+
+43. Cheng Y, Corbin JG, Levy RJ. Programmed Cell Death is Impaired in Fmr1mutant mice. Dev Neuroscience. 2013; 35:347–58.
+
+44. De Wael K, Bashir Q, Van Vlierberghe S, Dubruel P, Heering HA, Adriaens A. Electrochemical determination of hydrogen peroxide with cytochrome c peroxidase and horse heart cytochrome c entrapped in a gelatin hydrogel. Bioelectrochemistry. 2012; 83:15–8. [PubMed: 21889423]
+
+45. Ferraro E, Pulicati A, Cencioni MT, Cozzolino M, Navoni F, di Martino S, Nardacci R, Carrì MT, Cecconi F. Apoptosomedeficient cells lose cytochrome c through proteasomal degradation but survive by autophagy-dependent glycolysis. Mol Biol Cell. 2008; 19:3576–88. [PubMed: 18550800]
+
+46. Thom SR, Ischiropoulos H. Mechanism of oxidative stress from low levels of carbon monoxide. Res Rep Health Eff Inst. 1997; 80:1–19. [PubMed: 9476263]
+
+47. Hayashi M, Takahashi T, Morimatsu H, Fujii H, Taga N, Mizobuchi S, Matsumi M, Katayama H, Yokoyama M, Taniguchi M, Morita K. Increased carbon monoxide concentration in exhaled air after surgery and anesthesia. Anesth Analg. 2004; 99:444–8. [PubMed: 15271722]
+
+48. Sutherland BA, Harrison JC, Nair SM, Sammut IA. Inhalation gases or gaseous mediators as neuroprotectants for cerebral ischaemia. Curr Drug Targets. 2013; 14:56–73. [PubMed: 23170797]
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 12
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+Figure 1. Carboxyhemoglobin (COHb) levels after carbon monoxide (CO) exposure with and without isoflurane. COHb levels were measured immediately after CO exposure with (+) and without (−) isoflurane. Values are expressed as percentage (%) COHb means plus standard error. N = 5 animals per cohort. *P < 0.05 vs air and 5 ppm CO cohorts. †P < 0.01 vs air and 5 ppm CO cohorts.
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 13
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+Figure 2. Activated caspase-3 after carbon monoxide (CO) exposure with and without isoflurane. Immunohistochemistry for activated caspase-3 was performed on coronal sections 5 hours after exposure. (A) Representative sections imaged at ×10 from somatosensory neocortex (NC), hippocampus (HC), and hypothalamic/thalamic region (H/T) obtained after 1-hour exposure to air (0 ppm CO), 5 ppm CO, or 100 ppm CO with (+) and without (−) isoflurane are depicted. Arrowheads indicate activated caspase-3 stained cells. Activated caspase-3 positive cells undergoing degeneration within the boxed area in each section are magnified in the inset. CA1, CA2, dentate gyrus (DG) regions of HC are labeled. Scale bars, 100 μm. Quantification of activated caspase-3 stained cells in (B) neocortex (C) hippocampus and (D) hypothalamic/thalamic region are demonstrated. Values are expressed as means plus standard error. N = 3–4 animals per cohort. *P < 0.05 vs 0 ppm CO − isoflurane. †P < 0.01 vs 0 ppm CO − isoflurane. ^P < 0.001 vs 0 ppm CO − isoflurane. @ P < 0.05 vs 0 ppm CO + isoflurane. ‡P < 0.01 vs 0 ppm CO + isoflurane. #P < 0.001 vs 0 ppm CO + isoflurane. $P < 0.05 vs 5 ppm CO + isoflurane.
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 14
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+Figure 3. Apoptosis after carbon monoxide (CO) exposure with and without isoflurane. TUNEL assays were performed on coronal sections 5 hours after exposure. (A) Representative sections imaged at ×10 from somatosensory neocortex (NC), hippocampus (HC), and hypothalamic/thalamic region (H/T) obtained after 1-hour exposure to air (0 ppm CO), 5 ppm CO, or 100 ppm CO with (+) and without (−) isoflurane are depicted. Green TUNEL positive nuclei are visible. CA1, CA2, dentate gyrus (DG) regions of HC are labeled. Scale bars, 100 μm. Quantification of total TUNEL positive nuclei from NC, HC, and H/T in 3–4 nonserial coronal sections are demonstrated in (B) neocortex (C) hippocampus and (D) hypothalamic/thalamic region. Values are expressed as means plus standard error. N = 3–4 animals per cohort. *P < 0.05 vs 0 ppm CO − isoflurane. †P < 0.01 vs 0 ppm CO − isoflurane. ^P < 0.001 vs 0 ppm CO − isoflurane. @ P < 0.05 vs 0 ppm CO + isoflurane. ‡P < 0.01 vs 0 ppm CO + isoflurane. % P < 0.05 vs 5 ppm CO − isoflurane. &P < 0.01 vs 5 ppm CO − isoflurane. $P < 0.05 vs 5 ppm CO + isoflurane.?P < 0.05 vs 100 ppm CO − isoflurane.
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 15
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+Figure 4. Cytochrome c peroxidase activity after carbon monoxide (CO) exposure with and without isoflurane. Steady-state cytochrome c peroxidase activity immediately after 1-hour exposure is shown. Values are expressed as means plus standard error. N = 5 animals per cohort. *P < 0.05 vs 0 ppm CO − isoflurane, P < 0.001 vs 5 ppm CO − isoflurane, vs 100 ppm CO − isoflurane. †P < 0.01 vs 0 ppm CO − isoflurane. ‡P < 0.001 vs 0 ppm CO − isoflurane. ^P < 0.05 vs 5 ppm CO − isoflurane. @P < 0.01 vs 5 ppm CO − isoflurane. #P < 0.05 vs 100 ppm CO − isoflurane.?P < 0.01 vs 0 ppm CO + isoflurane. $P < 0.01 vs 5 ppm CO + isoflurane. %P < 0.001 vs 0 ppm CO + isoflurane.
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 16
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+N H P A A u t h o r
+
+I
+
+
+
+M a n u s c r i p t
+
+Cheng and Levy
+
+Figure 5. Cytochrome c release after carbon monoxide (CO) exposure with and without isoflurane. Heme c content within (A) mitochondria and (B) cytosol is demonstrated. Values are expressed as means plus standard error. N = 5 animals per cohort. *P < 0.05 vs 0 ppm CO − isoflurane. †P < 0.01 vs 0 ppm CO − isoflurane. #P < 0.05 vs 5 ppm CO + isoflurane. ‡P < 0.001 vs 0 ppm CO − isoflurane. ^P < 0.01 vs 0 ppm CO and 5 ppm + isoflurane. @P < 0.01 vs 5 ppm CO − isoflurane.
+
+Anesth Analg. Author manuscript; available in PMC 2014 June 01.
+
+Page 17

new_pdfs/10.1213_ANE.0b013e318281e988.txt

@@ -0,0 +1,639 @@
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+Pediatric Neuroscience
+
+Section Editors: Peter J. Davis/Gregory J. Crosby
+
+Characterization and Quantification of Isoflurane- Induced Developmental Apoptotic Cell Death in Mouse Cerebral Cortex
+
+George K. Istaphanous, MD,*† Christopher G. Ward, MD,*† Xinyu Nan, BS,* Elizabeth A. Hughes, BS,* John C. McCann, BS,* John J. McAuliffe, MD, MBA,*† Steve C. Danzer, PhD,*† and Andreas W. Loepke, MD, PhD*†
+
+BACKGROUND: Accumulating evidence indicates that isoflurane and other, similarly acting anes- thetics exert neurotoxic effects in neonatal animals. However, neither the identity of dying corti- cal cells nor the extent of cortical cell loss has been sufficiently characterized. We conducted the present study to immunohistochemically identify the dying cells and to quantify the fraction of cells undergoing apoptotic death in neonatal mouse cortex, a substantially affected brain region. METHODS: Seven-day-old littermates (n = 36) were randomly assigned to a 6-hour exposure to either 1.5% isoflurane or fasting in room air. Animals were euthanized immediately after exposure and brain sections were double-stained for activated caspase 3 and one of the following cellular markers: Neuronal Nuclei (NeuN) for neurons, glutamic acid decarboxylase (GAD)65 and GAD67 for GABAergic cells, as well as GFAP (glial fibrillary acidic protein) and S100β for astrocytes. RESULTS: In 7-day-old mice, isoflurane exposure led to widespread increases in apoptotic cell death relative to controls, as measured by activated caspase 3 immunolabeling. Confocal analy- ses of caspase 3–labeled cells in cortical layers II and III revealed that the overwhelming majority of cells were postmitotic neurons, but some were astrocytes. We then quantified isoflurane- induced neuronal apoptosis in visual cortex, an area of substantial injury. In unanesthetized control animals, 0.08% ± 0.001% of NeuN-positive layer II/III cortical neurons were immuno- reactive for caspase 3. By contrast, the rate of apoptotic NeuN-positive neurons increased at least 11-fold (lower end of the 95% confidence interval [CI]) to 2.0% ± 0.004% of neurons imme- diately after isoflurane exposure (P = 0.0017 isoflurane versus control). In isoflurane-treated animals, 2.9% ± 0.02% of all caspase 3–positive neurons in superficial cortex also coexpressed GAD67, indicating that inhibitory neurons may also be affected. Analysis of GABAergic neurons, however, proved unexpectedly complex. In addition to inducing apoptosis among some GAD67- immunoreactive neurons, anesthesia also coincided with a dramatic decrease in both GAD67 (0.98 vs 1.84 ng/mg protein, P < 0.00001, anesthesia versus control) and GAD65 (2.25 ± 0.74 vs 23.03 ± 8.47 ng/mg protein, P = 0.0008, anesthesia versus control) protein levels. CONCLUSIONS: Prolonged exposure to isoflurane increased neuronal apoptotic cell death in 7-day-old mice, eliminating approximately 2% of cortical neurons, of which some were identified as GABAergic interneurons. Moreover, isoflurane exposure interfered with the inhibitory nervous system by downregulating the central enzymes GAD65 and GAD67. Conversely, at this age, only a minority of degenerating cells were identified as astrocytes. The clinical relevance of these findings in animals remains to be determined.
+
+(Anesth Analg 2013;116:845–54)
+
+From the Departments of *Anesthesia and †Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio.
+
+Accepted for publication November 27, 2012.
+
+Supported in part by 2 mentored research grants from the Foundation for Anesthesia Education and Research (FAER) to G. K. I. and to C. G. W., men- tor A. W. L.
+
+General anesthetics, such as isoflurane, are used in
+
+millions of children around the world.1 However, preclinical studies demonstrating increased brain cell death after anesthetic exposure in developing animals have raised serious concerns about their safe use in the very young.2–8
+
+This report was previously presented, in part, at the 2010 International Anes- thesia Research Society meeting.
+
+George K. Istaphanous, MD, is currently affiliated with the Department of Anesthesiology, Children’s Hospital Los Angeles, Los Angeles, CA. Christopher G. Ward, MD, is currently affiliated with the Department of Anesthesiology and Critical Care, Children’s Hospital of Philadelphia, Philadelphia, PA.
+
+The authors declare no conflicts of interest.
+
+Reprints will not be available from the authors.
+
+Address correspondence to Andreas Loepke, MD, PhD, Department of Anes- thesia, Cincinnati Children’s Hospital Medical Center, ML2001, 3333 Burnet Ave., Cincinnati, OH 45229. Address e-mail to pedsanesthesia@gmail.com.
+
+Copyright © 2013 International Anesthesia Research Society. DOI: 10.1213/ANE.0b013e318281e988
+
+Anesthesia-induced brain cell death is widespread and at least some of the dying cells are neurons. However, whether and to what extent neurons versus non-neuronal cells are affected by isoflurane exposure is unclear. Although previous studies have convincingly demonstrated the presence of increased brain cell death in neonatal animals using a variety of cell death markers, such as caspase 3, Fluoro Jade B, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling), and cupric silver stains, these markers were not specific to a particular brain cell type and have not been routinely combined with phenotypic markers for
+
+April 2013 • Volume 116 • Number 4
+
+www.anesthesia-analgesia.org 845
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+specific cell types.2,3,5,9 Accordingly, previous studies have relied on cell morphology alone to identify cells vulnerable to anesthesia-induced degeneration such as neurons. Most of these studies have localized cell death to cortical and subcortical gray matter, where the predominant cell types are neurons and glia. Determining the relative extent to which these 2 cell types are affected by anesthesia exposure will be particularly important for predicting the functional consequences of anesthesia-induced cell loss because the different cell types possess vastly different regenerative capacities. Whereas glial cell proliferation can occur throughout adulthood, neuronal proliferation becomes increasingly restricted as the brain matures.10
+
+In the case of neuronal cell loss, it is also unclear which neuronal subtypes are most vulnerable. Answering this question is of particular importance for understanding anesthesia-induced cell death because of the phenomenon’s particular pattern of distribution. In contrast to other types of brain injury, such as ischemia in which a majority of cells in an affected region are destroyed, anesthetics induce widespread, scattered cell loss. Dying cortical brain cells are found immediately adjacent to apparently unaffected cells, suggesting that intrinsic differences among brain cells con- fer distinct vulnerabilities; however, the nature of these dif- ferences is not known.
+
+Accordingly, the present study quantitatively and quali- tatively characterized the cellular phenotype of susceptible brain cells in the superficial layers of neocortex, a brain region substantially affected by isoflurane-induced apop- totic cell death in neonatal mice. This was accomplished by using specific immunohistochemical markers for neurons, inhibitory interneurons, and astrocytes, and by comparing these findings with naturally occurring apoptosis in fasted, unanesthetized littermates, in order to provide insights into the selectivity, potential mechanisms, and consequences of anesthesia-induced cell death.
+
+METHODS All procedures were approved by the Institutional Animal Care and Use Committee and conformed to the guidelines for ethical treatment of animals. Efforts were made to mini- mize the number of animals used. Breeding pairs of male CD1 and female C57BL/6 mice were housed in a 12/12- hour light-dark cycle at 22°C with free access to food and water. This hybrid was selected because they exhibit robust anesthesia-induced apoptosis with acceptable survival.3
+
+For protein analyses, a separate set of animals (n = 22) was treated and euthanized as described above. The left hemispheres were cut into 4 coronal sections, frozen in liquid nitrogen, and stored at −80°C until use. At a later date, sections of neocortex around bregma −3 mm were separated with a razor blade on dry ice and then homog- enized twice in cell lysis buffer solution for approximately 10 seconds each time at 4°C. The homogenate was then centrifuged at 13,000 rpm using a refrigerated microcen- trifuge (Fresco centrifuge, Sorvall, Buckinghamshire, UK). The supernatant was removed and used for testing for the specific proteins.
+
+Immunohistochemistry Slide-mounted brain sections were blocked for 1 hour in nor- mal goat serum, followed by incubation in rabbit antiactivated caspase 3 polyclonal antibodies (1:100, 9661L; Cell Signaling, Danvers, MA) for 18 hours at −4°C, combined with one of the following antibodies: (1) mouse anti– Neuronal Nuclei (NeuN) monoclonal antibodies (NeuN, 1:500, Chemicon, MAB377; Millipore, Billerica, MA), (2) mouse antiglutamate decarboxylase isoform 67 (antiglutamic acid decarboxylase [GAD]67, 1:2000, MAB5406; Chemicon), (3) mouse anti- S100β (1:500, CB1040; Millipore), or (4) chicken antiglial fibrillary acidic protein (GFAP) (1:500, AB5541; Chemicon). Sections were then rinsed in blocker and incubated in Alexa Fluor 488 goat antirabbit secondary antibodies (1:200, A11034, Molecular Probes Inc.; Invitrogen, Carlsbad, CA) for 4 hours at 20°C, combined with either Alexa Fluor 594 goat antimouse (1:250, A11032, Molecular Probes) or Alexa Fluor 594 goat antichicken (1:250, A11042, Molecular Probes) sec- ondary antibodies, as appropriate for the primary antibody species. After immunostaining, sections were dehydrated in an ascending ethanol series, cleared in xylenes, and mounted with Krystalon (EMD, Gibbstown, NJ).
+
+Identification of Cellular Phenotype To determine the phenotype of degenerating cells, brain sections from anesthesia-treated and control animals, cor- responding to Bregma −2.46 to −2.70 (figures 51–53 in the mouse brain atlas by Paxinos and Franklin11) and double- immunostained for caspase 3 and NeuN or triple stained for caspase 3, S100β, and GFAP, were examined by an observer unaware of group assignment. NeuN and S100β stains can- not be combined in the same section, because both second- ary antibodies are raised in the same species.
+
+Isoflurane Treatment For caspase 3 immunohistochemistry, 7-day-old CD1 and C57BL/6 hybrid littermates (n = 14) were randomly assigned to a 6-hour exposure to 1.5% isoflurane (approxi- mately 0.6 minimum alveolar concentration in these mice) in 30% oxygen (anesthesia, n = 8) or to 6 hours in room air (control, n = 6). Immediately after treatment, animals were euthanized with an overdose of ketamine, acepromazine, and xylazine. Brains were immersion-fixed in 4% parafor- maldehyde in phosphate-buffered saline (pH 7.4), postfixed overnight at 4°C, and cryopreserved in 25% sucrose. Brains were snap frozen and 40-μm coronal sections were cut on a cryostat (Thermo Electronics, Kalamazoo, MI). Sections were mounted to charged slides and stored at −80°C until use.
+
+Caspase 3 immunostaining was excited using the 488-nm laser line, and emission wavelengths between 510 and 540 nm were collected to identify caspase-positive cells in layers II/III from retrosplenial cortex to piriform cortex using an SP5 confocal microscope set up on a DMI6000 stand (Leica Microsystems, Wetzlar, Germany) equipped with a 63× objective (NA 1.4). This region was selected because it has repeatedly demonstrated increased numbers of apoptotic cells in immature rodents.2,3 Immunostaining for NeuN, S100β, or GFAP was excited using the 543-nm laser line, and emission wavelengths between 600 and 650 nm were collected. Confocal optical sections were collected through the midpoint of the caspase 3–positive cell (pinhole = 1 Airy unit). Data are expressed as the percentage of caspase 3–immunoreactive cells that were also NeuN- or GFAP-positive, respectively.
+
+846
+
+www.anesthesia-analgesia.org
+
+ANesthesiA & ANAlgesiA
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+Quantification of Apoptotic Cells Using the Optical Dissector Method Further quantification of the effects of isoflurane exposure on cortical neurons and on GABAergic interneurons was performed as previously described.3,8 Briefly, confocal image stacks of caspase 3/GAD67 double labeling were collected at 1-µm increments through the entire Z-depth of the tissue (40 μm) using 1× optical zoom. Six image stacks were collected from layers II/III of visual cortex, corresponding to figures 51 to 53 in the mouse brain atlas by Paxinos and Franklin,11 from each animal, as follows: for each hemisphere, 3 adja- cent confocal image stack frames were collected beginning 750 μm from the midline and moving laterally (Leica SP5, 63× 1.4 NA objective, 1-μm steps). Image stacks, which were 120 × 120 µm in dimension for NeuN and 240 × 240 µm for GAD67, because of the significantly lower cellular density for the latter stain compared with NeuN, were transferred to Neurolucida software (v7.50.4; MBF Bioscience, Williston, VT) for analysis. Using the optical dissector method, an observer unaware of group assignment quantified the respective numbers of NeuN-positive or GAD67-positive cells, the corresponding number of caspase 3–positive cells, and the number of caspase 3/GAD67 or caspase 3/NeuN double-positive cells in each field.12,13 Cells were considered positive if their fluorescence intensity was 2 times or greater than the background intensity. Counts from all 6 respective image stacks were averaged for each animal.
+
+Quantification of GAD67 and GAD65 Expression Using Competitive Enzyme-Linked Immunosorbent Assay We used a competitive enzyme-linked immunosorbent assay to quantify the expression of the two γ-aminobutyric acid A (GABAA) synthesizing enzymes, GAD67 and GAD65. Rat antiglutamate decarboxylase isoform 67 (Anti GAD67, 1:5000, 671-C; Alpha Diagnostics Inc., San Antonio, TX) and goat antiglutamate decarboxylase isoform 65 (Anti GAD65, 1:32,000, Ab67725; Abcam, Cambridge, MA) antibodies were incubated overnight with the homogenized cortical tissue samples. These bound antibody/antigen complexes were then added to a GAD67 or GAD65 antigen-coated well blocked with 5% bovine serum albumin. Rabbit antirat and rabbit antigoat secondary antibodies were added to GAD67 and GAD65 complexes, respectively. The secondary anti- bodies were covalently bound to horseradish peroxidase, an enzyme that cleaves the peroxide in the chromophore 3,3′,5,5′-tetramethylbenzidine. This enzyme activation turned on the chromophore and emitted a blue signal, which when treated with 2 M sulfuric acid turned to a yellow color, which was measured at 450 nm using a spectrophotometer (Jenway Genova Life Science Spectrophotometer; Bibby Scientific Limited, Staffordshire, UK). Absorbancy was then compared with a standard curve allowing for the determi- nation of the isoforms’ concentrations.
+
+Statistical Analysis All sample sizes for group assignment were made a priori. For each animal, the total NeuN-positive cells were counted over the 6 fields. The number of caspase 3/NeuN dou- ble-positive cells was defined as an event. The data were
+
+April 2013 • Volume 116 • Number 4
+
+normalized to events (caspase 3/NeuN double-positive cells) per 400 NeuN-positive cells counted, the lower end of cells encountered in each animal, to avoid extrapolation. Gross inspection of the raw data revealed that caspase 3 activation in NeuN-positive cells was a rare event with a mean incidence of 2.4% and a maximal incidence of 3.6% in the anesthesia-treated animals. This event rate met the criteria for analysis using the Poisson distribution.
+
+The Poisson mean event rate, λ, and its 95% CI were deter- mined using the MATLAB® function [lambdahat,lambdaci] = poissfit(data,alpha). The vector “data” represented the number of events per 400 counted NeuN cells for each ani- mal in the group of interest and α = (1 − CI). The mean event rates, λ, derived from the MATLAB function, were used to construct probability distribution function curves for the 2 groups (see Appendix).
+
+The raw event counts were used to compute the ratio of events in the anesthesia-treated group to the control group using equations 6 and 7 in Graham et al.14 This method was used as an independent means to assess the mean event ratio and to determine the 95% CI for the event ratio.
+
+All other data are presented as means ± SEM. Group comparisons were made using the Mann-Whitney U test. Statistical calculations were analyzed using Stata/IC 10.1 for Mac OS X (Stata Corp., College Station, TX). Statistical significance was accepted at P < 0.05.
+
+RESULTS Neonatal Isoflurane Exposure Increases Apoptosis Throughout the Developing Mouse Brain In 7-day-old mice, isoflurane exposure led to widespread qualitative increases in apoptotic cell death relative to controls, as measured by caspase 3 immunolabeling. Although cellu- lar degeneration was observed in many brain regions, such as thalamus, striatum, and hippocampus, superficial cortical cell layers II and III comprised the highest density of apoptotic cells (Fig. 1), consistent with previous findings.3,8 Accordingly, quantitative analyses focused on this region in an effort to char- acterize the cellular phenotype and to quantify apoptotic cell death after isoflurane exposure in an area of “maximal” injury.
+
+Neurons Are Preferentially Lost After Isoflurane Exposure To reveal the cellular specificity of isoflurane-induced apoptosis, caspase 3 labeling was combined with either NeuN immunohistochemistry, which labels postmitotic neurons, or S100β and GFAP immunohistochemistry, which primarily label glial cells, the 2 predominant cell classes in the neocortex. Confocal analyses in cortical layers II and III revealed that 98% ± 0.6% of all caspase 3–labeled cells colocalized with NeuN in isoflurane-treated mice (Figs. 2 and 3). In contrast, 0.3% ± 0.26% and 6.6% ± 1% of degenerating cells were GFAP- and S100β-positive, respectively (Figs. 3 and 4), suggesting that isoflurane overwhelmingly affects cortical, postmitotic neurons immediately after exposure.
+
+Although far fewer cells were caspase 3 immunoreac- tive in control animals, similar relative percentages of the dying cells expressed NeuN (98% ± 2% [95% CI 92.4, 103.5] or 98% ± 0.6% [95% CI 96.9, 99.8] for control or anesthesia,
+
+www.anesthesia-analgesia.org
+
+847
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+respectively) or GFAP (0% [95% CI 0, 0] or 0.3% ± 0.26% [95% CI −0.3, 0.8], for control or anesthesia, respectively). These percentages followed the rare event rate, as outlined below and were indistinguishable from anesthesia-treated animals in terms of neuronal versus astrocytic cells (P = 0.18 or P ≈ 1 for NeuN or GFAP colocalization, respectively, comparing anesthesia with control). This suggests that, although cell loss was substantially higher after anesthesia exposure, the cell type being lost, predominantly neurons, was similar to naturally occurring cell death, as observed in control animals.
+
+Figure 1. Isoflurane exposure induces signifi- cant apoptotic brain cell death in neocortex of neonatal mice. Representative photomicrograph obtained with laser confocal microscopy demon- strating activation of caspase 3 (green dots, a marker of apoptosis) in superficial layers II/III of neocortex (arrowheads) and layer IV/V (arrows) in (A) unanesthetized, fasted control animals, or (B) after a 6-hour exposure to 1.5% isoflurane on day 7 of life. Scale bar = 200 µm.
+
+Isoflurane Exposure Increases Apoptosis at Least 11-Fold in Superficial Cortical Neurons Because the majority of dying cells in superficial cortical layers were identified as neurons, based on colocalization with NeuN, we further quantified isoflurane-induced neuronal apoptosis among cells in the visual cortex, using the optical dissector method. In unanesthetized, fasted animals, 0.08% ± 0.001% of NeuN-positive layer II/III cortical neurons were immunoreactive for caspase 3, undergoing natural apoptosis. By contrast, the rate of apoptotic NeuN-positive neurons increased to 2.0% ± 0.004% of all postmitotic neurons immediately after isoflurane exposure (P = 0.0017 isoflurane versus control; Fig. 3). The average number of cells counted on a group basis was 590 ± 47 for the control group and 582 ± 39 for the anesthesia group; these values were not significantly different. Two caspase 3/NeuN double-positive cells were observed in the control group for an event rate of 0.23 per 400 NeuN-positive cells. A total of 95 double-positive cells were observed in the anesthesia-treated animals, yielding an event rate of 8.77 per 400 NeuN-positive cells. The mean (95% CI) for the ratio of events was 38.8 (10.5, 143). Using the Poisson statistics, the 95% CIs for λ ranged from 0.015 to 1.05 (mean = 0.23) caspase 3/NeuN double-positive cells per 400 NeuN-positive cells for the control animals, and from 6.64 to 10.83 (mean = 8.55) in the anesthesia-treated animals. The predicted ratio of events, comparing anesthesia-treated animals with controls, on the basis of mean λ was 37.2. Conversely, the probability of observing no events per 400 NeuN-positive cells in the control group was P = 0.79, whereas the probability of observing 1 event was P = 0.18. For the anesthesia-treated group, 8 events has the maximal probability of being observed (P = 0.137) (see Appendix). The central 50% of the probability mass for the event ratio lies between 24 and 62; thus, 50% of the time the observed ratio of caspase 3/NeuN double-positive cells per X (an arbitrary large number) NeuN-positive cells in anesthesia- treated to control animals will be between 24 and 62. The limits were narrower than, but close to, the limits that would be computed using the Wald large number approximation.15
+
+Figure 2. Postmitotic neurons are affected by apoptotic cell death after isoflurane exposure in neonatal mice. Representative photo- micrograph demonstrating neocortical cells stained for the apop- totic marker–activated caspase 3 (green, top), the neuronal marker Neuronal Nuclear antigen (NeuN) (red, middle), and a merged image of the 2 stains (bottom) in a mouse brain after a 6-hour exposure to 1.5% isoflurane on day 7 of life. Cells labeled for activated caspase 3 and NeuN (yellow, arrows) are identified as postmitotic neurons undergoing apoptosis, compared with cells only expressing caspase 3 (arrowheads). The left column represents a 90-degree rotation on the y-axis confirming colocalization of both markers in a single neuron (yellow). Scale bar = 10 µm.
+
+Isoflurane Exposure Increases Apoptosis in GABAergic Cortical Neurons Although many caspase 3/NeuN double-positive neurons in anesthesia-treated animals appeared to be principal cells based on morphological criteria (pyramidal structure
+
+848
+
+www.anesthesia-analgesia.org
+
+ANesthesiA & ANAlgesiA
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+Figure 3. Anesthesia-induced and physiological apoptosis over- whelmingly affect postmitotic neurons in neonatal mice. Graphical quantification of the differential effects of isoflurane-induced apop- tosis (left pie chart, Anesthesia) or natural apoptosis (right, No Anesthesia) on neurons (Neuronal Nuclear antigen, NeuN) and astro- cytes (glial fibrillary acidic protein, GFAP) in superficial neocortex in 7-day-old mice after a 6-hour exposure to 1.5% isoflurane or 6 hours of fasting. Isoflurane induces apoptotic cell death in 2% of brain cells in layer II/III of neocortex, compared with only 0.08% succumb- ing to physiological apoptosis in 7-day-old mice. Both after anes- thesia and in unanesthetized animals, the overwhelming majority of affected cells are postmitotic neurons (98% NeuN+), whereas astro- cytes are substantially less affected (0.3% GFAP+ or 6.6% S100β+; data not shown). *P < 0.01 compared with no anesthesia.
+
+with radially oriented apical and basal dendrites; Figs. 1 and 2), caspase 3–immunoreactive cells with bipolar or stellate morphologies were occasionally observed, sug- gesting that interneurons are also affected during isoflu- rane exposure. To determine whether this was indeed the case, caspase 3 immunolabeling was combined with immu- nohistochemistry for the GABAergic interneuron marker GAD67. In isoflurane-treated animals, 2.9% ± 0.02% of all caspase 3–positive neurons in superficial cortex also coexpressed GAD67 (Fig. 5). Expressed as the percent- age of GABAergic interneurons that were affected by the anesthetic exposure, almost 28% of all GAD67-positive cells were also immunoreactive for caspase 3, imply- ing that a significant percentage of the GAD67 popu- lation underwent apoptosis after isoflurane exposure. Further analysis, however, led us to interpret these
+
+Figure 4. Anesthesia-induced apoptosis does not substantially affect astrocytes. Representative photomicrograph after a 6-hour exposure to 1.5% isoflurane in a 7-day-old mouse and demonstrating the astroglial cell markers glial fibrillary acidic protein (GFAP) (red, top left), S100β (blue, top right), the apoptotic marker–activated caspase 3 (green, bottom left), and the 3 channels merged (bot- tom right). A very small minority of caspase 3–expressing, apoptotic cells colocalized GFAP and S100β (GFAP+/S100β+, double arrows), unequivocally identifying them as astrocytes. The great majority of GFAP+/S100β+ cells (asterisk) and GFAP−/S100β+ cells (double arrowheads) did not contribute to the pool of apoptotic cells. Some apoptotic, caspase 3–positive cells expressing S100β, but lacking GFAP (single arrow), exhibited a nonastrocytic morphology raising the possibility that they were not astrocytes. The great majority of apoptotic cells were identified as neurons, coexpressing Neuronal Nuclear antigen (not shown) but lacking GFAP as well as S100β (single arrowhead). Scale bar = 20 µm.
+
+April 2013 • Volume 116 • Number 4
+
+numbers cautiously. Specifically, an estimation of the number of GAD67-immunoreactive somatic profiles revealed a significant reduction in the density of neurons with labeled soma in anesthesia-treated animals relative to controls. Accordingly, the reduced denominator in anes- thesia-treated animals would bias the observed percen- tile toward overestimating the effect on the total neuronal population, but underestimating the effect on GABAergic neurons.
+
+Isoflurane Exposure Reduces GAD67 and GAD65 Expression in GABAergic Interneurons in Superficial Cortex To confirm the observation of reduced GAD67 labeling, con- focal image stacks were collected from visual cortex, and the optical dissector method was used to quantify the number
+
+www.anesthesia-analgesia.org
+
+849
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+of GAD67-positive cells, caspase 3–positive cells, and dou- ble-positive cells. After isoflurane exposure, the density of GAD67-immunoreactive cell bodies was reduced in anesthe- sia-treated animals by a factor of 3, relative to unanesthetized, fasted littermates (1.2 ± 0.4 · 103 vs 3.9 ± 0.7 · 103 cells/mm3, P = 0.011). However, in accordance with our other results, the density of all caspase 3–positive cells (both GAD67-positive and -negative) was increased 17-fold in these sections, com- paring anesthesia animals with control (11.2 ± 2.1 · 103 vs 0.66 ± 0.03 · 103 cells/mm3, P = 0.01) (Fig. 6).
+
+Enzyme-linked immunosorbent assay results mirrored the immunohistochemical findings. Isoflurane exposure caused a decrease in GAD67 expression compared with control (29.17 ± 11.10 vs 119.21 ± 11.23 ng/mg protein, P < 0.00001). The other isoform of the GABA-synthesizing enzyme, GAD65, was also decreased after anesthesia expo- sure compared with the control group (2.25 ± 0.74 vs 23.03 ± 8.47 ng/mg protein, P = 0.0008), whereas β-actin, a ubiq- uitous isoform of actin, was found to be 24.60 ± 4.60 ng/mg protein (CI 14.0, 35.2) in control animals versus 27.99 ± 3.63 ng/mg protein (CI 19.1, 36.9) in anesthetized animals (P = 0.8432 anesthesia versus control).
+
+DISCUSSION Prolonged exposure to the inhaled anesthetic isoflurane has been shown to trigger widespread brain cell death in several in vivo and in vitro neonatal animal models2–9,16–20 and to lead to subsequent long-term neurocognitive impairment,2,4,21 raising serious concerns regarding the safe use of isoflurane and similarly acting drugs in neonates.22–24 Several animal studies have localized cell death to cortical and subcortical gray matter, where the predominant cell types are neurons and glia, and have observed a predilection for the superficial cortical layers, peaking in 7-day-old rodents.2–4 However, the cellular phenotype of susceptible cells has not been immunohistochemically identified and the absolute extent of cell death has not been quantified; instead, previous studies have solely relied on morphological criteria for identification and have described apoptotic cell death as a percentage increase of physiological apoptosis.
+
+In this regard, the present study in 7-day-old mice intro- duces 4 key findings. First, the great majority of cortical brain cells, 98%, eliminated immediately after a neonatal exposure to isoflurane were postmitotic neurons, as identified by NeuN expression. Second, isoflurane led to the demise of 2% of all cortical NeuN-positive neurons in layer II/III, a region con- sistently exhibiting substantial apoptosis, which represented an at least 11-fold increase over physiological apoptosis observed in unanesthetized, fasted littermates. Third, despite the disparate rates of apoptosis observed in anesthetized and unanesthetized animals, postmitotic neurons, and not astrocytes, were the predominant affected cell type in both groups. This observation suggests that anesthetic neurotoxic- ity may target the same cell population vulnerable to normal, developmentally regulated cell death. Finally, isoflurane led to neuroapoptosis in a segment of GABAergic interneurons and was associated with a decrease in the expression of the main GABA-synthesizing enzymes, suggesting that isoflu- rane may, at least transiently, interfere with proper inhibitory function in the developing brain.
+
+Figure 5. GABAergic interneurons undergo apoptotic cell death after isoflurane exposure in neonatal mice. Representative photomicro- graph illustrating labeling of neocortical cells in layers II/III with the apoptotic marker–activated caspase 3 (green, top), interneuronal marker glutamic acid decarboxylase (GAD)67 (red, middle), and the 2 channels merged (bottom) in a 7-day-old mouse after a 6-hour exposure to 1.5% isoflurane. Colocalization of activated caspase 3 and GAD67 signifies a GABAergic interneuron undergoing apop- totic cell death (arrow), whereas adjacent GAD67-positive cells are seemingly unaffected (arrowheads). The left column represents a 90-degree rotation on the y-axis confirming colocalization of both markers in a single neuron (yellow). Scale bar = 10 µm.
+
+Isoflurane Substantially Increases Apoptosis Among Postmitotic Neurons, but Not Astrocytes NeuN is a neuron-specific protein that is found in the nuclei of neuronal cell types of the central nervous system, signify- ing postmigratory status and is absent from glial cells.25 In the murine neocortex, NeuN is first expressed in subplate neu- rons, the first cortical neurons to develop, starting at embry- onic day 17.5 and does not reach adult levels until 16 days postnatally.25,26 Demonstrating that 98% of cells expressing the apoptotic marker caspase 3 also coexpressed the neuro- nal marker NeuN, the present study unequivocally identified the cortical cells primarily affected by cellular death imme- diately after an isoflurane exposure as postmitotic neurons. This finding is somewhat surprising, given the fact that sus- ceptibility to anesthesia-induced, cortical cytotoxicity has historically been found to be limited to very young animals, peaking at postnatal day 7, and dramatically subsiding after 10 days of age in small rodents.27 However, our results extend observations in neonatal rats, demonstrating that isoflurane does not seem to induce cellular death in neuronal progeni- tor cells.28 Nevertheless, given that many susceptible cells were found in the superficial cortical layer II and that mam- malian cortex forms in an inside-out pattern, whereby newly
+
+850
+
+www.anesthesia-analgesia.org
+
+ANesthesiA & ANAlgesiA
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+Figure 6. Isoflurane exposure leads to apoptotic cell death in a sig- nificant fraction of GABAergic interneurons as well as to a decrease in the GABAergic neuronal density in neocortex of neonatal mice. Graphical quantification of the differential effects of isoflurane- induced apoptosis (left pie chart, Anesthesia) or natural apoptosis (right, No Anesthesia) on GABAergic, glutamic acid decarboxylase (GAD)67-positive neurons in superficial neocortex of 7-day-old mice after a 6-hour exposure to 1.5% isoflurane or 6 hours of fasting, respectively. Isoflurane induces apoptotic cell death in 28% of GAD67-positive brain cells in layer II/III of neocortex, compared with physiological apoptosis in 2.4% of cells in fasted 7-day-old control mice. However, isoflurane exposure also significantly reduced the density of GAD67-positive neurons, which may lead to an underesti- mation of the affected percentage of GABAergic neurons. *P < 0.01 and †P < 0.02 compared with no anesthesia.
+
+generated neurons migrate past the earlier generated cells to form more superficial layers,29 the present findings sug- gest that susceptibility for anesthesia-induced cell death is increased in relatively young, postmitotic neurons.
+
+Another startling observation of the present study in post- natal day 7 mice was the dramatically lower rate of cell death, less than 0.5% and 7% of apoptotic cells expressed GFAP and S100β, respectively, observed among astrocytes, which are part of the glial family of brain cells and represent the other large portion of cortical cells. Astrocytes serve to protect, nurture, and support neurons by producing trophic factors, regulate neurotransmitters and ion concentrations, remove toxins and debris, mediate synaptogenesis, and contribute to synapse elimination as well as structural neuronal plastic- ity.30,31 This finding does not exclude that these cells are not involved in neuronal degeneration via proapoptotic signals, such as observed during brain ischemia.32 It is also conceiv- able that astrocytes may undergo more pronounced apoptotic cell death at a later time point compared with neurons, similar to experimental models of brain ischemia.33 Although GFAP is a widely accepted marker for astrocytes, it has also been found to be expressed in some neuronal progenitors.34 S100β is most frequently found in astrocytes, but has also been found in some neurons and oligodendrocytes.33,35 Moreover, some of the S100β-positive cells in our study may have exhibited neu- ronal morphology (Fig. 4); therefore, we cannot exclude that some of these cells may not have been astroglia, which may help explain the apparent overlap in the cell counts.
+
+Previous studies in neonatal rats have demonstrated an up to 68-fold increase in apoptotic brain cell death after an isoflurane-based anesthetic exposure.2 However, the absolute percentage of cells affected by this phenomenon remained unknown. Apoptotic cell death is an integral part of normal brain maturation, eliminating 50% to 70% of neurons and progenitor cells during the extent of central nervous system development, which spans over several weeks in small rodents.36,37 Accordingly, at any given time point, only a small fraction of cells undergo physiological apoptotic cell death. In 5-day-old mice, apoptotic cell death has been found to occur in 0.07% of cortical neurons.38 Similarly, the present study observed caspase 3 labeling, a marker for apoptotic cell death, in <0.08% of fasted 7-day-old control mice. Conversely, immediately after a 6-hour exposure to isoflurane, the percentage of neurons undergoing apoptotic cell death increased at least 11-fold to approximately 2% of
+
+April 2013 • Volume 116 • Number 4
+
+all neurons in the superficial layers of visual cortex. This region was selected for quantification because anesthesia- induced cell loss has repeatedly exhibited a substantial predilection for cells in superficial neocortex. Other less- susceptible brain regions not examined here would likely demonstrate a lower percentage of apoptotic cells.
+
+The long-term effects of the elimination of up to 2% of neurons in neocortex and other brain regions on subsequent brain structure and function remain speculative. The tem- porary increase in neuroapoptosis observed here could pos- sibly be offset by increased subsequent neurogenesis, similar to observations after postnatal hypoxia,39 or by subsequent decreases in naturally occurring apoptosis. The number of neocortical neurons peaks in 16-day-old mice and decreases by 30% thereafter,26 suggesting that the developing mouse brain may have sufficient reserve capacity to absorb a 2% neuronal loss. Consistent with this interpretation, a previous study by our group did not detect a significant decrease in adult corti- cal neuronal density after a similar neonatal isoflurane expo- sure,3 although a 2% reduction would be difficult to detect even with the most robust cell-counting techniques. Neurocognitive abnormalities were also absent in these animals, again suggest- ing that any deficits, if present, are subtle.3 However, the small quantity of the eliminated brain cells does not exclude the pos- sibility of long-term network disruptions, because loss of even a small number of neurons with important function or during critical periods for brain development may have a significant impact on the subsequent development of neural networks.
+
+Isoflurane Exposure Leads to Apoptotic Cell Death in GABAergic Interneurons and to Decreased Expression of GAD65 and GAD67 GABA, the main inhibitory neurotransmitter in the central nervous system, is synthesized by 2 isoforms of the enzyme GAD, which are located on 2 different genes and demonstrate dissimilar location and function.40 GAD67, the predominant isoform responsible for >90% of GABA production, is located in the cytosol, and thought to function as a trophic factor,41 whereas GAD65 is located at the nerve terminals, specifically responding to short-term demands for GABA, such as during neurotransmission.42 GAD67 immunohistochemistry revealed a 12-fold increase in the number of caspase 3/GAD67 double- positive cells in anesthesia-exposed animals. This finding sug- gests that isoflurane leads to the demise of a significant amount of GABAergic neurons immediately after exposure, as also
+
+www.anesthesia-analgesia.org
+
+851
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+recently observed in newborn rats.43 Similarly, exposure to other GABA agonists, muscimol and propofol have also been previ- ously found to induce GABAergic neuronal death in immature rat telencephalon cultures.44 More mature cells treated with these GABA agonists, however, were less vulnerable to long- term effects, suggesting that susceptibility is age-dependent.44
+
+In the present study, prolonged exposure to isoflurane led to a significant decrease in both isoforms of the GABA- synthesizing enzyme. These reductions in GAD expres- sion could be explained by either isoflurane-induced neuronal cell death of GAD-containing (i.e., GABAergic) neurons or by a drug-induced downregulation or cleav- age of the enzyme. Although the present study found a 12-fold increase in the number of apoptotic GABAergic neurons after isoflurane exposure, potentially explaining the observed reduction in GAD expression, the disparate reductions in the 2 GAD isoforms, by 50% for GAD67 and by >90% for GAD65, suggest that exposure to anesthesia might have additional functional effects, beyond cell death, on GABAergic interneurons. Previous findings of decreased GAD enzyme activity after treatment with the GABAA agonists vigabatrin, propofol, and muscimol support this hypothesis.44,45 This downregulation could be explained by an isoflurane-induced, excitotoxic cleavage of GAD65 and GAD67, as previously reported in cultured hippocampal GABAergic neurons.46 Regardless of the mechanism, given the fact that interneurons comprise 12% to 15% of all corti- cal neurons in adult rodents,47 the permanent loss of even a small number of GABAergic interneurons or prolonged interference with their function could potentially have pro- found effects on the normal balance of excitation and inhibi- tion in the developing brain. Given the anesthesia-induced alterations in GAD expression, however, the absolute frac- tion of GABAergic neurons eliminated during isoflurane exposure is difficult to assess, because both the numera- tor, the number of surviving GABAergic neurons, and the denominator in this equation, GAD67 expression, changed. Because astrocytes are also a source for GABA and con- tain GAD67,48 we cannot exclude that glia may have had a role in the observed changes in GAD expression. However, converging lines of evidence lead us to believe that the anes- thesia-induced reduction in GAD expression observed in our experiments was predominantly mediated by neurons, rather than astrocytes. First, we found that both GAD67 and GAD65 expressions were decreased after anesthetic exposure; the combination of GAD65 and GAD67 is only expressed in neurons, whereas astrocytes have only been shown to express GAD67.48 Second, although some GAD67–positively stained cells could have been astrocytes, it is more likely that they were GABAergic neurons, because cellular apoptosis over- whelmingly affected cells expressing the neuronal marker NeuN, and to a much lesser degree the astroglial markers GFAP or S100β, suggesting that alterations in GAD expression may have also predominantly occurred in neurons.
+
+Human applicability of the present findings in animals remains unresolved. Although histopathological studies cannot be performed in healthy children after exposure to general anesthetics, several epidemiological studies have returned conflicting results. Some have detected an association between exposure to anesthesia and surgery early in life and subsequent behavioral or learning abnormalities,49,50 whereas others have
+
+not observed any deleterious effects in children exposed to anesthetics and sedatives during vulnerable periods in their brain development.51–53 The present study used clinically relevant doses of isoflurane, approximating 0.6 minimum alveolar concentration,8 for a relatively long exposure period, which may be outside of the normal clinical practice, to create a measurable effect, because the injury is exposure time– dependent and dose-dependent.9 The maturational stage of the human brain equivalent to the mice used in the present study remains controversial; older data suggest that postnatal day 7 mice are comparable to human infants,54 whereas more contemporary studies indicate mouse brain development at this stage to be closer to human fetuses at midgestation.55
+
+In conclusion, a 6-hour exposure to clinical doses of iso- flurane increased neuronal apoptotic cell death in 7-day- old mice, killing approximately 2% of cortical neurons, of which some were identified as GABAergic interneurons. Conversely, astrocytes were substantially less affected by iso- flurane exposure at this age. Moreover, isoflurane exposure dramatically decreased expression of both isoforms of the GABA-synthesizing enzyme GAD, which indicates that the anesthetic drug may interfere with proper inhibitory function in the developing brain. The permanence of these findings, however, remains unknown. Additional studies will need to identify the phenomenon’s selectivity and molecular mecha- nisms to determine its applicability to pediatric anesthesia. E
+
+APPENDIX
+
+Supplemental Figure 1. Significantly higher rate of neuronal apop- tosis observed in isoflurane-exposed animals compared with unanesthetized littermates. Cumulative probability curves using the Poisson distribution of rare events and depicting the number of apoptotic neurons (NeuN+/caspase 3+ cells) per 400 neurons (NeuN+ cells) for 7-day-old mice exposed to 1.5% isoflurane for 6 hours (filled circles) or fasted in room air (filled squares). The event rate λ was normalized to 400 NeuN+ cells, the lower end of the num- ber of cells counted in each animal, to avoid extrapolation. Graphs were calculated using the MATLAB® (MathWorks, Natick, MA) func- tion [lambdahat,lambdaci] = poissfit(data,alpha) in an iterative man- ner. The vector ‘data’ represented the number of events per 400 counted NeuN+ cells for each animal in the group of interest and alpha was the confidence interval. The output was used to construct a cumulative probability curve for the Poisson mean event rate for both treatment groups.
+
+852
+
+www.anesthesia-analgesia.org
+
+ANesthesiA & ANAlgesiA
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+DISCLOSURES Name: George K. Istaphanous, MD. Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript. Attestation: George K. Istaphanous has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Name: Christopher G. Ward, MD. Contribution: This author helped conduct the study. Attestation: Christopher G. Ward has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Name: Xinyu Nan, BS. Contribution: This author helped conduct the study. Attestation: Xinyu Nan has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Name: Elizabeth A. Hughes, BS. Contribution: This author helped conduct the study. Attestation: Elizabeth A. Hughes has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Name: John C. McCann, BS. Contribution: This author helped conduct the study. Attestation: John C. McCann has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Name: John J. McAuliffe, MD, MBA. Contribution: This author helped analyze the data and write the manuscript. Attestation: John J. McAuliffe has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Name: Steve C. Danzer, PhD. Contribution: This author helped design the study and write the manuscript. Attestation: Steve C. Danzer has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Name: Andreas W. Loepke, MD, PhD. Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript. Attestation: Andreas W. Loepke has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files. This manuscript was handled by: Gregory J. Crosby, MD.
+
+REFERENCES 1. DeFrances CJ, Cullen KA, Kozak LJ. National Hospital Discharge Survey: 2005 annual summary with detailed diagno- sis and procedure data. Vital Health Stat 2007;13:1–209
+
+2. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF. Early expo- sure to common anesthetic agents causes widespread neurode- generation in the developing rat brain and persistent learning deficits. J Neurosci 2003;23:876–82
+
+3. Loepke AW, Istaphanous GK, McAuliffe JJ III, Miles L, Hughes EA, McCann JC, Harlow KE, Kurth CD, Williams MT, Vorhees CV, Danzer SC. The effects of neonatal isoflurane exposure in mice on brain cell viability, adult behavior, learning, and mem- ory. Anesth Analg 2009;108:90–104
+
+April 2013 • Volume 116 • Number 4
+
+4. Stratmann G, May LD, Sall JW, Alvi RS, Bell JS, Ormerod BK, Rau V, Hilton JF, Dai R, Lee MT, Visrodia KH, Ku B, J, Firouzian A. Effect of Zusmer EJ, Guggenheim hypercarbia and isoflurane on brain cell death and neurocog- nitive dysfunction in 7-day-old rats. Anesthesiology 2009;110: 849–61
+
+5. Brambrink AM, Evers AS, Avidan MS, Farber NB, Smith DJ, Zhang X, Dissen GA, Creeley CE, Olney JW. Isoflurane- induced neuroapoptosis in the neonatal rhesus macaque brain. Anesthesiology 2010;112:834–41
+
+6. Rizzi S, Ori C, Jevtovic-Todorovic V. Timing versus dura- tion: determinants of anesthesia-induced developmental apoptosis in the young mammalian brain. Ann NY Acad Sci 2010;1199:43–51
+
+7. Lemkuil BP, Head BP, Pearn ML, Patel HH, Drummond JC, Patel PM. Isoflurane neurotoxicity is mediated by p75NTR- RhoA activation and actin depolymerization. Anesthesiology 2011;114:49–57
+
+8. Istaphanous GK, Howard J, Nan X, Hughes EA, McCann JC, McAuliffe JJ, Danzer SC, Loepke AW. Comparison of the neu- roapoptotic properties of equipotent anesthetic concentrations of desflurane, isoflurane, or sevoflurane in neonatal mice. Anesthesiology 2011;114:578–87
+
+9. Johnson SA, Young C, Olney JW. Isoflurane-induced neuro- apoptosis in the developing brain of nonhypoglycemic mice. J Neurosurg Anesthesiol 2008;20:21–8
+
+10. Henschel O, Gipson KE, Bordey A. GABAA receptors, anes- thetics and anticonvulsants in brain development. CNS Neurol Disord Drug Targets 2008;7:211–24
+
+11. Paxinos G, Franklin KB. The Mouse Brain in Stereotaxic Coordinates. 2nd ed. San Diego: Academic Press, 2001
+
+12. West MJ, Slomianka L, Gundersen HJ. Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat Rec 1991;231:482–97
+
+13. West MJ. Stereological methods for estimating the total number of neurons and synapses: issues of precision and bias. Trends Neurosci 1999;22:51–61
+
+14. Graham PL, Mengersen K, Morton AP. Confidence limits for the ratio of two rates based on likelihood scores: non-iterative method. Stat Med 2003;22:2071–83
+
+15. Rothman KJ, Greenland S. Modern Epidemiology. 2nd ed. Philadelphia: Lippincott-Raven, 1998
+
+16. Wei H, Kang B, Wei W, Liang G, Meng QC, Li Y, Eckenhoff RG. Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently. Brain Res 2005;1037:139–47
+
+17. Wise-Faberowski L, Zhang H, Ing R, Pearlstein RD, Warner DS. Isoflurane-induced neuronal degeneration: an evalua- tion in organotypic hippocampal slice cultures. Anesth Analg 2005;101:651–7
+
+18. Wei H, Liang G, Yang H. Isoflurane preconditioning inhib- ited isoflurane-induced neurotoxicity. Neurosci Lett 2007;425: 59–62
+
+19. Ma D, Williamson P, Januszewski A, Nogaro MC, Hossain M, Ong LP, Shu Y, Franks NP, Maze M. Xenon mitigates isoflurane- induced neuronal apoptosis in the developing rodent brain. Anesthesiology 2007;106:746–53
+
+20. Head BP, Patel HH, Niesman IR, Drummond JC, Roth DM, Patel PM. Inhibition of p75 neurotrophin receptor attenuates isoflurane-mediated neuronal apoptosis in the neonatal central nervous system. Anesthesiology 2009;110:813–25
+
+21. Stratmann G, Sall JW, May LD, Bell JS, Magnusson KR, Rau V, Visrodia KH, Alvi RS, Ku B, Lee MT, Dai R. Isoflurane dif- ferentially affects neurogenesis and long-term neurocognitive function in 60-day-old and 7-day-old rats. Anesthesiology 2009;110:834–48
+
+22. Jevtovic-Todorovic V, Olney JW. PRO: anesthesia-induced developmental neuroapoptosis—status of the evidence. Anesth Analg 2008;106:1659–63
+
+23. Loepke AW, McGowan FX Jr, Soriano SG. CON: the toxic effects of anesthetics in the developing brain: the clinical perspective. Anesth Analg 2008;106:1664–9
+
+www.anesthesia-analgesia.org
+
+853
+
+24. Loepke AW. Developmental neurotoxicity of sedatives and anesthetics: a concern for neonatal and pediatric critical care medicine? Pediatr Crit Care Med 2010;11:217–26
+
+42. Erlander MG, Tobin AJ. The structural and functional heteroge- neity of glutamic acid decarboxylase: a review. Neurochem Res 1991;16:215–26
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+25. Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specific nuclear protein in vertebrates. Development 1992;116:201–11 26. Lyck L, Krøigård T, Finsen B. Unbiased cell quantification reveals a continued increase in the number of neocortical neurones during early post-natal development in mice. Eur J Neurosci 2007;26:1749–64
+
+27. Yon JH, Daniel-Johnson J, Carter LB, Jevtovic-Todorovic V. Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience 2005;135:815–27
+
+28. Sall JW, Stratmann G, Leong J, McKleroy W, Mason D, Shenoy S, Pleasure SJ, Bickler PE. Isoflurane inhibits growth but does not cause cell death in hippocampal neural precursor cells grown in culture. Anesthesiology 2009;110:826–33
+
+29. Rakic P. Mode of cell migration to the superficial layers of fetal monkey neocortex. J Comp Neurol 1972;145:61–83
+
+30. Aschner M, Sonnewald U, Tan KH. Astrocyte modulation of neurotoxic injury. Brain Pathol 2002;12:475–81
+
+31. Stevens B. Neuron-astrocyte signaling in the development and plasticity of neural circuits. Neurosignals 2008;16:278–88
+
+32. Anderson MF, Blomstrand F, Blomstrand C, Eriksson PS, Nilsson M. Astrocytes and stroke: networking for survival? Neurochem Res 2003;28:293–305
+
+33. Yu AC, Wong HK, Yung HW, Lau LT. Ischemia-induced apop- tosis in primary cultures of astrocytes. Glia 2001;35:121–30 34. Yang Q, Hamberger A, Khatibi N, Stigbrand T, Haglid KG. Presence of S-100 beta in cholinergic neurones of the rat hind- brain. Neuroreport 1996;7:3093–9
+
+35. Vives V, Alonso G, Solal AC, Joubert D, Legraverend C. Visualization of S100B-positive neurons and glia in the cen- tral nervous system of EGFP transgenic mice. J Comp Neurol 2003;457:404–19
+
+36. Oppenheim RW. Cell death during development of the nervous system. Annu Rev Neurosci 1991;14:453–501
+
+37. Rakic S, Zecevic N. Programmed cell death in the developing human telencephalon. Eur J Neurosci 2000;12:2721–34
+
+38. Hodge RD, D’Ercole AJ, O’Kusky JR. Insulin-like growth fac- tor-I (IGF-I) inhibits neuronal apoptosis in the developing cere- bral cortex in vivo. Int J Dev Neurosci 2007;25:233–41
+
+39. Fagel DM, Ganat Y, Silbereis J, Ebbitt T, Stewart W, Zhang H, Ment LR, Vaccarino FM. Cortical neurogenesis enhanced by chronic perinatal hypoxia. Exp Neurol 2006;199:77–91
+
+40. Buddhala C, Hsu CC, Wu JY. A novel mechanism for GABA synthesis and packaging into synaptic vesicles. Neurochem Int 2009;55:9–12
+
+41. Kanaani J, Kolibachuk J, Martinez H, Baekkeskov S. Two dis- tinct mechanisms target GAD67 to vesicular pathways and pre- synaptic clusters. J Cell Biol 2010;190:911–25
+
+43. Zhou ZW, Shu Y, Li M, Guo X, Pac-Soo C, Maze M, Ma D. The glutaminergic, GABAergic, dopaminergic but not cholinergic neurons are susceptible to anaesthesia-induced cell death in the rat developing brain. Neuroscience 2011;174:64–70
+
+44. Honegger P, Pardo B, Monnet-Tschudi F. Muscimol-induced death of GABAergic neurons in rat brain aggregating cell cul- tures. Brain Res Dev Brain Res 1998;105:219–25
+
+45. Honegger P, Matthieu JM. Selective toxicity of the general anes- thetic propofol for GABAergic neurons in rat brain cell cultures. J Neurosci Res 1996;45:631–6
+
+46. Baptista MS, Melo CV, Armelão M, Herrmann D, Pimentel DO, Leal G, Caldeira MV, Bahr BA, Bengtson M, Almeida RD, Duarte CB. Role of the proteasome in excitotoxicity-induced cleavage of glutamic acid decarboxylase in cultured hippocam- pal neurons. PLoS One 2010;5:e10139
+
+47. Beaulieu C. Numerical data on neocortical neurons in adult rat, with special reference to the GABA population. Brain Res 1993;609:284–92
+
+48. Lee M, Schwab C, McGeer PL. Astrocytes are GABAergic cells that modulate microglial activity. Glia 2011;59:152–65
+
+49. Wilder RT, Flick RP, Sprung J, Katusic SK, Barbaresi WJ, Mickelson C, Gleich SJ, Schroeder DR, Weaver AL, Warner DO. Early exposure to anesthesia and learning disabilities in a population-based birth cohort. Anesthesiology 2009;110: 796–804
+
+50. DiMaggio C, Sun LS, Li G. Early childhood exposure to anes- thesia and risk of developmental and behavioral disorders in a sibling birth cohort. Anesth Analg 2011;113:1143–51
+
+51. Rozé JC, Denizot S, Carbajal R, Ancel PY, Kaminski M, Arnaud C, Truffert P, Marret S, Matis J, Thiriez G, Cambonie G, André M, Larroque B, Bréart G. Prolonged sedation and/or analge- sia and 5-year neurodevelopment outcome in very preterm infants: results from the EPIPAGE cohort. Arch Pediatr Adolesc Med 2008;162:728–33
+
+52. Bartels M, Althoff RR, Boomsma DI. Anesthesia and cognitive performance in children: no evidence for a causal relationship. Twin Res Hum Genet 2009;12:246–53
+
+53. Guerra GG, Robertson CM, Alton GY, Joffe AR, Cave DA, Dinu IA, Creighton DE, Ross DB, Rebeyka IM; Western Canadian Complex Pediatric Therapies Follow-up Group. Neurodevelopmental outcome following exposure to sedative and analgesic drugs for complex cardiac surgery in infancy. Paediatr Anaesth 2011;21:932–41
+
+54. Dobbing J, Sands J. Quantitative growth and development of human brain. Arch Dis Child 1973;48:757–67
+
+55. Clancy B, Darlington RB, Finlay BL. Translating developmental time across mammalian species. Neuroscience 2001;105:7–17
+
+854
+
+www.anesthesia-analgesia.org
+
+ANesthesiA & ANAlgesiA

new_pdfs/10.1213_ANE.0b013e3182a8c709.txt

@@ -0,0 +1,499 @@
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+Neonatal Exposure to Sevoflurane Causes Significant Suppression of Hippocampal Long-Term Potentiation in Postgrowth Rats
+
+Rui Kato, MD, PhD,* Kaori Tachibana, MD, PhD,* Naoki Nishimoto, PhD,† Toshikazu Hashimoto, MD, PhD,* Yosuke Uchida, MD,* Ryoko Ito, MD,* Kenkichi Tsuruga, MD,* Koichi Takita, MD, PhD,* and Yuji Morimoto, MD, PhD*
+
+BACKGROUND: The inhaled anesthetic sevoflurane is commonly used for neonates in the clinical setting. Recent studies have indicated that exposure of neonatal rodents to sevoflu- rane causes acute widespread neurodegeneration and long-lasting neurocognitive dysfunction. Although acute toxic effects of sevoflurane on cellular viability in the hippocampus have been reported in some studies, little is known about the effects of neonatal sevoflurane exposure on long-term hippocampal synaptic plasticity, which has been implicated in the processes of learn- ing and memory formation. Our study is the first to examine the long-term electrophysiological impact of neonatal exposure to a clinically relevant concentration of sevoflurane. METHODS: On postnatal day 7, rats were exposed to sevoflurane (1% or 2% for 2 hours) with oxygen. To eliminate the influence of blood gas abnormalities caused by sevoflurane-induced respiratory suppression, a group of rats were exposed to a high concentration of carbon dioxide (8% for 2 hours) to duplicate respiratory disturbances caused by 2% sevoflurane exposure. RESULTS: Exposure of neonatal rats to 2% sevoflurane for 2 hours caused significant suppres- sion of long-term potentiation (LTP) induction in the postgrowth period. There was no significant difference between the control group and the CO2-exposed group in LTP induction, indicating that sevoflurane-induced LTP suppression was not caused by blood gas abnormalities. CONCLUSION: Our present findings indicate that neonatal exposure to sevoflurane at a higher concentration can cause alterations in the hippocampal synaptic plasticity that persists into adulthood.
+
+(Anesth Analg 2013;117:1429–35)
+
+Neonatal exposure to anesthetics has been shown to
+
+induce acute neuronal cell death and long-lasting behavioral abnormalities in animal models includ- ing primates.1–6 Jevtovic-Todorovic et al.1 reported that isoflurane, individually or in combination with other anes- thetics, caused neurodegeneration, deficits of hippocampal electrophysiological function, and persistent memory and learning impairments in rats. Fredriksson et al.2 reported that neonatal exposure to a combination of thiopental or propofol and ketamine caused a synergistic neurotoxic effect in mice. According to those reports, anesthetics that activate γ-aminobutyric acid-A (GABA) receptors and block N-methyl-d-aspartate (NMDA)-type glutamate receptors have neurotoxic effects in developing animals.6,7
+
+Sevoflurane is one of the most commonly used inhaled anesthetics for neonates in the clinical setting. It activates GABA receptors (GABAR) and blocks NMDA receptors,8,9
+
+From the *Department of Anesthesiology and Critical Care Medicine and †Di- vision of Clinical Trial Management, Center for Translational Research, Hok- kaido University Graduate School of Medicine, Sapporo, Japan.
+
+and several lines of study have shown that it causes neu- rodegeneration in the central nervous system in neona- tal rodents and long-lasting neurocognitive dysfunction, including learning disabilities.3,10–13 The effect of acute anes- thetic toxicity on cellular viability has been well studied; however, the mechanism of long-lasting learning disabili- ties is still uncertain.
+
+Hippocampal synaptic plasticity has been implicated in many learning and memory processes.14–18 Therefore, 1 possible mechanism that underlies the learning disability found after neonatal exposure to sevoflurane is assumed to be impairments in hippocampal synaptic plasticity. In this study, we aimed to characterize the electrophysiologi- cal alterations caused by neonatal sevoflurane exposure by investigating in vivo long-term potentiation (LTP) induc- tion in the hippocampal CA1 region. We also evaluated the effects of hypercapnia and acidosis using a carbon dioxide (CO2) exposure model because respiratory disturbances caused by anesthesia are unavoidable in small animals. The present study is the first to examine the long-term electro- physiological impact of exposing neonatal rats to clinically relevant concentrations of sevoflurane.
+
+Accepted for publication August 2, 2013.
+
+Funding: This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan Society for the Promotion of Science. (Grants-in- Aid for Scientific Research 22591699, 20791060)
+
+The authors declare no conflicts of interest.
+
+Reprints will not be available from the authors.
+
+Address correspondence to Rui Kato, MD, PhD, Department of Anesthesiol- ogy and Critical Care Medicine, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-Ku, Sapporo 060-8638, Japan. Address e-mail to katorui@med.hokudai.ac.jp.
+
+Copyright © 2013 International Anesthesia Research Society DOI: 10.1213/ANE.0b013e3182a8c709
+
+METHODS Animals The experiments were approved by the Committee for Animal Research of the Hokkaido University Graduate School of Medicine. Pregnant Wistar/ST rats were obtained from Shizuoka Laboratory Animal Center (Hamamatsu, Japan). The animals were housed in a room maintained at 22°C to 25°C with a 12-hour light-dark cycle (light from
+
+December 2013 • Volume 117 • Number 6
+
+www.anesthesia-analgesia.org 1429
+
+ReseaRch RepoRt
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+Neonatal Sevoflurane Exposure Impairs Long-Term Potentiation Induction
+
+06:00 to 18:00) and were given free access to food and water. Rats were handled in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Hokkaido University Graduate School of Medicine.
+
+Gas Exposure At postnatal day 7, male pups were divided into 4 treat- ment groups as follows: animals exposed to (1) 1% sevo- flurane with oxygen (O2), (2) 2% sevoflurane with O2, (3) 8% CO2 with O2 to mimic hypercapnia and acidosis caused by 2% sevoflurane exposure, or (4) 100% O2 as a control. Rat pups were placed in an acrylic chamber and exposed to warmed, humidified gas for 2 hours. The total gas flow was 3 L/min to maintain a stable gas concentration. The concentrations of sevoflurane, CO2, and O2 in the chamber were continuously monitored using an anesthetic gas moni- tor (5250RGM, Datex-Ohmeda GE Healthcare, Chalfont St. Giles, United Kingdom). The gas temperature in the cham- ber was maintained at 27.5°C to 28.6°C, measured using a TR-200 (F.S.T., Foster City, CA), and the rats’ body tempera- tures were maintained at 35.1°C to 36.5°C, measured using a Thermofocus® (Tecnimed, Tokyo, Japan). To avoid litter variability, equal numbers of rat pups from each litter were randomly assigned to the 4 treatment groups. After gas exposure, rat pups were brought up in an austere environ- ment in standardized cages to avoid the influence of envi- ronmental enrichment on later neurocognitive functions.13
+
+Arterial Blood Gas Assessment To assess respiratory disturbances caused by sevoflurane exposure, arterial blood gas assessment was performed as described previously.19,20 At postnatal day 7, rats underwent arterial blood sampling from the left ventricle via transtho- racic puncture under local subcutaneous anesthesia (0.05 mL of 1% lidocaine) at 30, 60, 90, or 120 minutes during 2 hour treatments. Since taking even a single sample of blood could be fatal for the rats, blood sampling was conducted only once for each rat. A small amount of blood (<100 μL) was collected and analyzed immediately using a blood gas analyzer (ABL510, Radiometer Medical, Brønshøj, Denmark) that measured pH, partial pressure of CO2, and O2 (Paco2 and Pao2 in mm Hg, respectively). In all cases, the procedure was completed within 1 minute in the chamber.
+
+Electrophysiological Study Between postnatal days 63 and 70, an electrophysiologi- cal study was performed following our reported method.21 Rats were anesthetized with 1% halothane in a mixture of 21% O2 and 79% nitrogen through a tracheal catheter, and their lungs mechanically ventilated (SN-480–7, Shinano, Tokyo, Japan). They were placed in a stereotaxic apparatus with the bregma and lambda in the same horizontal plane, and their body temperatures were maintained at 37°C ± 0.5°C with a heating pad throughout the recording period. The concentration of halothane and expired CO2 tension was continuously monitored through a tracheal catheter using an anesthetic gas monitor (5250RGM, Datex-Ohmeda GE Healthcare), and the expired CO2 tension was main- tained between 35 and 45 mm Hg. A monopolar recording electrode was inserted into the pyramidal cell body layer
+
+1430
+
+www.anesthesia-analgesia.org
+
+of the hippocampal CA1 region (5.0 mm posterior and 3.0 mm lateral to the bregma and approximately 2.2 mm ven- tral to the dura), to record extracellular population spike amplitude (PSA) (Fig. 1A). A bipolar stimulating electrode was inserted into the ipsilateral Schaffer collaterals (3.0 mm posterior and 1.5 mm lateral to the bregma, and 2.8 mm ventral to the dura) to deliver cathodal stimulus (fre- quency 0.1 Hz, pulse duration 250 μs) (Fig. 1A). A single electrical stimulus evoked action potentials in the Schaffer collaterals, resulting in activation of pyramidal cells of the CA1 region, and extracellular PSA was recorded (MacLab, ADInstruments, Sydney, Australia). We measured the PSA following our previous method.21 Briefly, the PSA was defined as the absolute voltage of a vertical line running from the population spike minimum to its intersection with a line tangential to the population spike onset and popula- tion spike offset. To adjust the test stimulus, changes in PSA caused by varied stimulus intensity were recorded, and the intensity of the test stimulus was then fixed to produce a half-maximal response for each rat. After establishing a sta- ble baseline for 30 minutes, LTP was induced by applying high-frequency stimulation (HFS; 10 trains at 1 Hz each, composed of 8 pulses at 400 Hz), at the same intensity as the test stimulation. Then relative ratios of PSAs before and after the HFS were then plotted every 5 minutes for 60 minutes after HFS. On completion of the experiment, small lesions were made using a direct electric current (5 µA for 30 seconds) at the tips of the recording and stimulating electrodes. The positions of the electrodes were examined histologically (Fig. 1) according to methods in similar pre- vious reports.22,23 Histological confirmation of the locations of the electrodes showed misplacement of the electrodes in 2 rats in each group, so the data obtained from those elec- trodes were excluded from the following assessment. All
+
+Figure 1. Positions of stimulating and recording electrodes (A). The triangle shows the position of the tip of the stimulating electrode, which was located 3 mm posterior and 1.5 mm lateral to the bregma and 2.8 mm under the dura. The circle shows the position of the tip of the recording electrode, which was located 5 mm posterior and 3 mm lateral to the bregma and 2.2 mm under the dura. The locations of the electrodes were examined using a post hoc histological test (B, C). The arrow shows the tip of the stimulating electrode located at the Schaffer collateral (B), and the arrowhead shows the recording electrode located in the pyramidal cell layer of the CA1 region (C).
+
+aNesthesia & aNalgesia
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+data were collected by investigators who were blinded to treatment assignment.
+
+Statistical Methods All statistical analyses were performed using GraphPad Prism version 5.04 (GraphPad Software, Inc., San Diego, CA). First, we confirmed normal distribution of the obtained data by the Shapiro-Wilk test and a normal-probability plot. Data obtained in arterial blood gas analysis were analyzed using 1-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test for each time point. In the comparisons of LTP data, we used 2-way repeated-mea- sures ANOVA with treatment and time as factors to assess the interaction, and we used 1-way ANOVA followed by Bonferroni multiple comparison test to evaluate differences among the groups at each time point. All averaged data are presented as mean ± SEM. Probability values (P) of <5% were considered significant.
+
+RESULTS Anesthetic Effect of Neonatal Sevoflurane Exposure Neonatal 2% sevoflurane exposure caused immediate loss of spontaneous movement and righting reflex but pre- served the response to tail pinch. Neonatal 2% sevoflurane also reduced respiratory rate throughout treatment. In the group exposed to 1% sevoflurane, spontaneous movement decreased, but the righting reflex, tail pinch response and frequency of breath were not disturbed. CO2 exposure caused no remarkable change. After treatment, all rats recovered completely.
+
+Arterial Blood Gas Analysis The results from blood gas analysis are summarized in Table 1. In the CO2 and 2% sevoflurane groups, there were significant respiratory disturbances from hypercapnia and respiratory acidosis throughout treatment compared with the control and 1% sevoflurane groups, whereas 1% sevo- flurane exposure caused no significant changes compared with no exposure in the control group. We could not find significant differences in Pao2, Paco2, or pH between the CO2 and 2% sevoflurane-exposed groups, that is, the hyper- capnia and acidosis observed in the 2% sevoflurane group were the same in the CO2 group. Hypoxia was not observed during treatment in any group.
+
+Neonatal Exposure to 2% Sevoflurane Caused Significant Suppression of Hippocampal LTP Induction After the establishment of stable baseline PSAs, the groups (N = 8 for each) were subjected to HFS. PSAs were signifi- cantly augmented in the control and CO2 groups, and LTP was induced as shown in Figure 2A. The maximal responses were 214.1% ± 29.8%, 218.6% ± 25.4%, 170.7% ± 20.1%, and 130.9% ± 10.5% in the control, CO2, 1% sevoflurane, and 2% sevoflurane groups, respectively. Two-way repeated-mea- sures ANOVA revealed significant differences among the treatments (F = 3.90; P = 0.019) but not among time courses (F = 1.86; P = 0.058). There was no interaction among the groups (F = 0.70; P = 0.825). The LTP data between 15 and
+
+December 2013 • Volume 117 • Number 6
+
+60 minutes after HFS were used for this analysis because LTP responses were unstable for 10 minutes after HFS. LTP induction in the 2% sevoflurane group was significantly suppressed compared with the control and CO2 groups at 5, 10, 15, 20, 25, 30, 35, and 40 minutes (Fig. 2B). There were no significant differences among the other groups. However, the statistical power of the analysis between the control and 1% sevoflurane groups might have been insuf- ficient from our sample size. As shown in Table 2, the width of Bonferroni adjusted 95% confidence interval for the mean difference between these groups was greater com- pared with the mean difference at each time point. Further research may be necessary to detect the difference caused by 1% sevoflurane exposure. These results indicated that neonatal 2% sevoflurane exposure caused significant long- lasting suppression of hippocampal LTP induction, but neo- natal 8% CO2 exposure with O2 did not cause any significant electrophysiological changes.
+
+DISCUSSION In this study, we showed that neonatal exposure to 2% sevoflurane caused significant suppression of LTP induc- tion in an in vivo electrophysiological study of adult rats. Hypercapnia and acidosis, which mimicked the sevoflu- rane-induced respiratory disturbances, had no influence on LTP induction.
+
+In the present study, we used 1% halothane to anesthe- tize rats during hippocampal LTP measurement. In vivo electrophysiological study under halothane anesthesia is a well-established procedure in adult rodents.24,25 Halothane anesthesia provides a more stabilized anesthetic depth during experiments compared with the intermittent IV or intraperitoneal injection of anesthetics. Although halothane has been shown to depress CA1 population spikes and LTP induction when applied to hippocampal slices,26 exposure to halothane in vivo does not block the induction of hip- pocampal LTP.27 The discrepancy between these findings in vitro and in vivo could reflect differences in the balance between excitatory and inhibitory circuitry. Thus, halothane has been used successfully for in vivo studies of hippocam- pal synaptic plasticity in rodents, including our previous studies.21,24,25,28,29 To avoid the possible modulation of hippo- campal synaptic activity by halothane-induced physiologi- cal changes (e.g., hypoxia and ischemia), we used intensive respiratory management in our in vivo electrophysiological studies.21,28,29 Respiratory management by tracheal intuba- tion, mechanical ventilation, and monitoring of end-tidal CO2 enabled us to control halothane-induced respiratory depression. Halothane also suppresses circulation; how- ever, our preliminary study showed that 1% halothane had relatively little influence on femoral arterial pressure in adult rats (data not shown). Thus, we assume that halo- thane anesthesia in this in vivo study had minimal electro- physiological and physiological influence on hippocampal synaptic activity.
+
+learn- ing impairments after neonatal exposure to anesthetics remain poorly understood. Although acute widespread cell death has been observed,1,30,31 several studies have shown increased neurogenesis and apparent anatomical recovery
+
+The critical mechanisms that contribute to
+
+www.anesthesia-analgesia.org 1431
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+Neonatal Sevoflurane Exposure Impairs Long-Term Potentiation Induction
+
+Table 1. Arterial Blood Gas Analysis Group Control
+
+Time (min) 30 60 90 120 30 60 90 120 30 60 90 120 30 60 90 120
+
+CO2
+
+1% Sevoflurane
+
+2% Sevoflurane
+
+N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
+
+pH 7.43 ± 0.04 7.36 ± 0.03 7.37 ± 0.02 7.38 ± 0.02 7.18 ± 0.03a 7.16 ± 0.03a 7.14 ± 0.02a 7.20 ± 0.02a 7.41 ± 0.02b 7.40 ± 0.04b 7.36 ± 0.01b 7.34 ± 0.05b 7.18 ± 0.03ac 7.23 ± 0.0ac 7.17 ± 0.03ac 7.18 ± 0.03ac
+
+Paco2 (mm Hg) 37.9 ± 5.4 47.2 ± 7.3 44.0 ± 3.0 46.5 ± 2.6 76.3 ± 4.9a 88.7 ± 5.8a 81.5 ± 5.0a 79.7 ± 6.2a 39.1 ± 2.8b 38.7 ± 3.3b 41.6 ± 1.0b 53.9 ± 5.6b 77.5 ± 5.7ac 79.1 ± 3.8ac 81.7 ± 2.8ac 89.9 ± 5.1ac
+
+Pao2 (mm Hg) 414.0 ± 51.6 414.2 ± 30.5 369.2 ± 53.7 350.6 ± 51.8 296.8 ± 70.9 200.1 ± 24.7 215.2 ± 45.8 237.5 ± 51.2 405.1 ± 77.0 358.4 ± 94.6 360.2 ± 64.4 366.9 ± 66.1 337.6 ± 28.4 226.9 ± 52.8 200.2 ± 28.9 235.4 ± 40.5
+
+Data from arterial blood gas analysis. All groups were composed of 5 rats. Blood samples were collected from the left ventricle every 30 minutes during gas exposure. Statistical analysis revealed that neonatal 2% sevoflurane anesthesia and 8% CO2 exposure caused significant hypercapnia and acidosis in rats. There were no significant differences between the control group and 1% sevoflurane group or the CO2 group and 2% sevoflurane group for pH and Paco2. Hypoxia was not observed in any group. The acidosis and hypercapnia observed in the 2% sevoflurane group were well mimicked by the CO2 group. Data are presented as mean ± SEM. aVersus control group. bVersus CO2 group. cVersus 1% sevoflurane group. P < 0.05.
+
+Figure 2. Neonatal 2% sevoflurane anesthesia suppressed long-term potentiation (LTP) induction in the postgrowth period. Specimen record- ings (A) and time course responses of population spike amplitude (PSA) to 60 minutes after high-frequency stimulation (HFS) (B). Two-way repeated-measures ANOVA revealed that there was no interaction between treatments and time course changes, whereas there were signifi- cant differences between treatments during the period from 15 to 60 minutes after HFS. Statistical analysis revealed that there were signifi- cant differences between the 2% sevoflurane group and the control and CO2 groups at 5, 10, 15, 20, 25, 30, 35, and 40 minutes after HFS. All groups were composed of 8 rats. Data are shown as mean ± SEM. *Versus control group, †Versus CO2 group.
+
+after acute histopathological damage induced by neonatal anesthesia.5,20 In those studies, rat pups that were exposed to anesthesia showed minimal to no histopathological damage as adults. This suggests that the developing brain may have a high capacity for self-repair of morphological
+
+changes. This evidence led us to hypothesize that there might be additional, or even alternative, mechanisms that could mediate anesthetic-induced long-term learning defi- cits and other cognitive dysfunctions besides the immediate morphological changes in the hippocampus.
+
+1432
+
+www.anesthesia-analgesia.org
+
+aNesthesia & aNalgesia
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+Table 2. Bonferroni Adjusted 95% Confidence Intervals for the Mean Difference Between Treatments in Figure 2B
+
+Control versus CO2
+
+Time points (min)
+
+15 20 25 30 35 40 45 50 55 60
+
+Lower CL (%) −65.09 −61.71 −79.29 −72.26 −69.94 −70.10 −91.01 −86.49 −70.41 −83.53 Control versus 1% sevoflurane
+
+Difference (%) 12.97 11.84 −2.97 9.2 3.92 8.29 −8.37 −2.1 13.92 2.72
+
+Upper CL (%) 91.04 85.4 73.36 90.65 77.78 86.69 74.28 82.29 98.24 88.98
+
+Time points (min)
+
+15 20 25 30 35 40 45 50 55 60
+
+Lower CL (%) −124.87 −122.88 −123.25 −125.13 −115.66 −119.30 −135.91 −131.13 −116.65 −130.26 Control versus 2% sevoflurane
+
+Difference (%) −46.81 −49.33 −46.93 −43.67 −41.81 −40.9 −53.27 −46.74 −32.33 −44.01
+
+Upper CL (%) 31.26 24.22 29.4 37.78 32.05 37.49 29.38 37.66 52 42.24
+
+Time points (min)
+
+15 20 25 30 35 40 45 50 55 60
+
+Difference (%) −90.49 −89.36 −91.08 −90.68 −87.1 −83.76 −89.11 −84.98 −72.2 −88.02
+
+Lower CL (%) −168.55 −162.91 −167.41 −172.13 −160.96 −162.16 −171.76 −169.37 −156.52 −174.27
+
+Upper CL (%) −12.43 −15.80 −14.76 −9.23 −13.24 −5.37 −6.46 −0.59 12.13 −1.76
+
+Bonferroni adjusted 95% confidence interval (95% CI) for the mean difference by the treatment combination between groups in Figure 2B. Mean difference (Difference), lower confidence limits (lower CL), and upper CL between groups at each time point was listed. 95% CI for the mean difference showed that there was a statistical difference in the control group versus 2% Sevoflurane group, and there was no statistical significance difference in the control group versus CO2 group and in the control group versus 1% Sevoflurane group at each time point. However, in the control group versus CO2 group and in the control group versus 1% Sevoflurane group, the statistical power might be insufficient from our sample size, because the width of 95% CI was greater compared with the mean difference between groups. Further, research is required to detect the difference.
+
+One possible reason for anesthetic-induced neurotoxicity is the potential effect of anesthetics on synaptic morphology and function. Previously, we reported that the GABAergic anesthetic pentobarbital, administered at P7, induced long- lasting suppression of hippocampal synaptic plasticity as well as learning disturbances.21 Commonly used anesthet- ics with GABAergic or antiglutamatergic properties such as midazolam, isoflurane, and nitrous oxide, when combined, also suppress hippocampal LTP in adulthood after admin- istration at P7.1 Thus, hippocampal LTP might be associated with the pathophysiology of neonatal anesthetic-related learning deficits remaining in adulthood.
+
+During synaptogenesis, signals via NMDA and/or GABA receptors mediate neuronal survival, migration, and synaptic formation.32,33 It has been reported that abnormal
+
+December 2013 • Volume 117 • Number 6
+
+or excess signals of those receptors may have a long- lasting negative influence over synaptic maturation.34,35 Because sevoflurane has GABAergic and/or antiglutama- tergic properties,8,9 neonatal sevoflurane anesthesia pas- sively modulates those synaptic developmental processes and induces abnormal synaptic formation or ectopic neu- ronal cell distribution that leads to impairment of syn- aptic plasticity such as induction of hippocampal LTP in the CA1 region. We have reported that administration of dexmedetomidine, an α2A adrenergic agonist, causes no impairment in hippocampal synaptic plasticity28 and has no adverse effect on cognitive functions in adulthood.36 The absence of neurotoxicity in rodents treated neonatally with dexmedetomidine, which has no GABAergic or glu- tamatergic properties, may suggest that pharmacological intervention via GABAergic and/or glutamatergic neuro- transmission might play a crucial role in the impairment of hippocampal neural functions, including hippocampal synaptic plasticity. However, further study is needed to clarify the mechanisms underlying sevoflurane-induced LTP impairment shown in this study.
+
+Despite several conflicting reports,1,3,31 it has been well documented that neonatal rodents develop respiratory suppression and subsequent blood gas abnormalities dur- ing anesthetic exposure.21,37,38 Loepke et al.20 pointed out the possible involvement of such blood gas abnormalities in anesthetic-induced neurotoxicity observed in neonatal rodents. Yet the role of blood gas abnormalities in neurode- velopment has remained uncertain. Recently, we attempted to evaluate the influence of blood gas abnormalities in P7 rat pups on their later hippocampal function.29 In that study, P7 rat pups exposed to 13% CO2 under 21% O2 showed blood gas abnormalities along with hypoxia, hypercapnia, and metabolic acidosis, which inhibited hippocampal LTP induction in the postgrowth period.29 Importantly, com- pared with acidosis only, prolonged metabolic acidosis with hypoxia has been thought to carry a higher risk for neuro- nal deficits.39 Furthermore, studies performed in clinical set- tings have demonstrated a correlation between the degree of metabolic acidosis and neonatal neurological outcome.40 This evidence led us to suspect that neither hypercapnia nor low pH, but the coexistence of hypoxia and progres- sive metabolic acidosis during CO2 exposure, could have profound effects on hippocampal synaptic plasticity in later life. In light of this evidence, we used 100% O2 as a carrier gas in the current study. We needed 8% CO2 with 100% O2 to mimic respiratory disturbance and subsequent blood gas abnormalities caused by 2% sevoflurane exposure; this CO2 exposure protocol caused no impairment in later LTP induc- tion, suggesting that respiratory disturbances caused by neonatal exposure to 2% sevoflurane had negligible influ- ence on hippocampal synaptic plasticity in later life. This finding also led us to speculate that hypercapnia and low pH during development, without hypoxia and possible metabolic acidosis, had less effect on hippocampal synaptic plasticity in later life.
+
+In conclusion, our present findings indicate that expo- sure to 2% sevoflurane causes persistent suppression of LTP induction. This persistent change in synaptic plastic- ity after neonatal sevoflurane exposure may be one of the
+
+www.anesthesia-analgesia.org 1433
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+Neonatal Sevoflurane Exposure Impairs Long-Term Potentiation Induction
+
+mechanisms underlying sevoflurane-induced neurocogni- tive dysfunction. E
+
+DISCLOSURES Name: Rui Kato, MD, PhD. Contribution: This author designed the study, conducted almost all experiments with some other authors, and wrote the manuscript. Attestation: Rui Kato attests to the integrity of the original data and the analysis reported in this manuscript. Yuji Morimoto is the archival author. Name: Kaori Tachibana, MD, PhD. Contribution: This author helped in designing the study and conducting experiments. Name: Naoki Nishimoto, PhD. Contribution: This author helped in statistical analysis. Name: Toshikazu Hashimoto, MD, PhD. Contribution: This author helped in designing the study and preparing the manuscript. Name: Yosuke Uchida, MD. Contribution: This author helped in preparing the manuscript. Name: Ryoko Ito, MD. Contribution: This author helped in electrophysiological tests. Name: Kenkichi Tsuruga, MD Contribution: This author helped in preparing the manuscript. Name: Koichi Takita, MD, PhD. Contribution: This author helped in electrophysiological tests and preparing the manuscript. Name: Yuji Morimoto, MD, PhD. Contribution: This author helped in designing the study and preparing the manuscript. Attestation: Yuji Morimoto is the archival author of this manuscript. This manuscript was handled by: Gregory J. Crosby, MD.
+
+REFERENCES 1. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF. Early expo- sure to common anesthetic agents causes widespread neurode- generation in the developing rat brain and persistent learning deficits. J Neurosci 2003;23:876–82
+
+2. Fredriksson A, Pontén E, Gordh T, Eriksson P. Neonatal expo- sure to a combination of N-methyl-D-aspartate and gamma- aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. Anesthesiology 2007;107:427–36
+
+3. Satomoto M, Satoh Y, Terui K, Miyao H, Takishima K, Ito M, Imaki J. Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice. Anesthesiology 2009;110:628–37
+
+4. Slikker W Jr, Zou X, Hotchkiss CE, Divine RL, Sadovova N, Twaddle NC, Doerge DR, Scallet AC, Patterson TA, Hanig JP, Paule MG, Wang C. Ketamine-induced neuro- nal cell death in the perinatal rhesus monkey. Toxicol Sci 2007;98:145–58
+
+5. Stratmann G, May LD, Sall JW, Alvi RS, Bell JS, Ormerod BK, Rau V, Hilton JF, Dai R, Lee MT, Visrodia KH, Ku B, Zusmer EJ, Guggenheim J, Firouzian A. Effect of hyper- carbia and isoflurane on brain cell death and neurocogni- tive dysfunction in 7-day-old rats. Anesthesiology 2009;110: 849–61
+
+6. Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vöckler J, Dikranian K, Tenkova TI, Stefovska V, Turski L, Olney JW. Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 1999;283:70–4
+
+7. Olney JW, Young C, Wozniak DF, Jevtovic-Todorovic V, Ikonomidou C. Do pediatric drugs cause developing neurons to commit suicide? Trends Pharmacol Sci 2004;25:135–9
+
+1434
+
+www.anesthesia-analgesia.org
+
+8. Hollmann MW, Liu HT, Hoenemann CW, Liu WH, Durieux ME. Modulation of NMDA receptor function by ketamine and magnesium. Part II: interactions with volatile anesthetics. Anesth Analg 2001;92:1182–91
+
+9. Nishikawa K, Harrison NL. The actions of sevoflurane and desflurane on the gamma-aminobutyric acid receptor type A: effects of TM2 mutations in the alpha and beta subunits. Anesthesiology 2003;99:678–84
+
+10. Bercker S, Bert B, Bittigau P, Felderhoff-Müser U, Bührer C, Ikonomidou C, Weise M, Kaisers UX, Kerner T. Neurodegeneration in newborn rats following propofol and sevoflurane anesthesia. Neurotox Res 2009;16:140–7
+
+11. Edwards DA, Shah HP, Cao W, Gravenstein N, Seubert CN, Martynyuk AE. Bumetanide alleviates epileptogenic and neurotoxic effects of sevoflurane in neonatal rat brain. Anesthesiology 2010;112:567–75
+
+12. Kodama M, Satoh Y, Otsubo Y, Araki Y, Yonamine R, Masui K, Kazama T. Neonatal desflurane exposure induces more robust neuroapoptosis than do isoflurane and sevoflurane and impairs working memory. Anesthesiology 2011;115:979–91
+
+13. Shih J, May LD, Gonzalez HE, Lee EW, Alvi RS, Sall JW, Rau V, Bickler PE, Lalchandani GR, Yusupova M, Woodward E, Kang H, Wilk AJ, Carlston CM, Mendoza MV, Guggenheim JN, Schaefer M, Rowe AM, Stratmann G. Delayed environmental enrichment reverses sevoflurane-induced memory impairment in rats. Anesthesiology 2012;116:586–602
+
+14. Morris RG, Garrud P, Rawlins JN, O’Keefe J. Place naviga- tion impaired in rats with hippocampal lesions. Nature 1982;297:681–3
+
+15. Burgess N, Maguire EA, O’Keefe J. The human hippocampus and spatial and episodic memory. Neuron 2002;35:625–41 16. Bliss TV, Collingridge GL. A synaptic model of memory: long- term potentiation in the hippocampus. Nature 1993;361:31–9 17. Gruart A, Muñoz MD, Delgado-García JM. Involvement of the CA3-CA1 synapse in the acquisition of associative learning in behaving mice. J Neurosci 2006;26:1077–87
+
+18. Whitlock JR, Heynen AJ, Shuler MG, Bear MF. Learning induces long-term potentiation in the hippocampus. Science 2006;313:1093–7
+
+19. Lu LX, Yon JH, Carter LB, Jevtovic-Todorovic V. General anes- thesia activates BDNF-dependent neuroapoptosis in the devel- oping rat brain. Apoptosis 2006;11:1603–15
+
+20. Loepke AW, Istaphanous GK, McAuliffe JJ 3rd, Miles L, Hughes EA, McCann JC, Harlow KE, Kurth CD, Williams MT, Vorhees CV, Danzer SC. The effects of neonatal isoflurane exposure in mice on brain cell viability, adult behavior, learning, and mem- ory. Anesth Analg 2009;108:90–104
+
+21. Tachibana K, Hashimoto T, Kato R, Tsuruga K, Ito R, Morimoto Y. Long-lasting effects of neonatal pentobarbital administration on spatial learning and hippocampal synaptic plasticity. Brain Res 2011;1388:69–76
+
+22. Ohashi S, Matsumoto M, Otani H, Mori K, Togashi H, Ueno K, Kaku A, Yoshioka M. Changes in synaptic plastic- ity in the rat hippocampo-medial prefrontal cortex pathway induced by repeated treatments with fluvoxamine. Brain Res 2002;949:131–8
+
+23. Tachibana K, Kato R, Tsuruga K, Takita K, Hashimoto T, Morimoto Y. Altered synaptic transmission in rat anterior cingulate cortex following peripheral nerve injury. Brain Res 2008;1238:53–8
+
+24. Thiels E, Xie X, Yeckel MF, Barrionuevo G, Berger TW. NMDA receptor-dependent LTD in different subfields of hippocampus in vivo and in vitro. Hippocampus 1996;6:43–51
+
+25. Yeckel MF, Berger TW. Spatial distribution of potentiated syn- apses in hippocampus: dependence on cellular mechanisms and network properties. J Neurosci 1998;18:438–50
+
+26. MacIver MB, Tauck DL, Kendig JJ. General anaesthetic modi- fication of synaptic facilitation and long-term potentiation in hippocampus. Br J Anaesth 1989;62:301–10
+
+27. Pearce RA, Stringer JL, Lothman EW. Effect of volatile anes- thetics on synaptic transmission in the rat hippocampus. Anesthesiology 1989;71:591–8
+
+28. Tachibana K, Hashimoto T, Kato R, Uchida Y, Ito R, Takita K, Morimoto Y. Neonatal administration with dexmedetomidine
+
+aNesthesia & aNalgesia
+
+H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l
+
+i
+
+Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n
+
+r
+
+i
+
+i
+
+l
+
+1 1 / 2 0 / 2 0 2 3
+
+D o w n o a d e d
+
+l
+
+f r o m h t t p : / / j o u r n a s . l
+
+l
+
+w w . c o m / a n e s t h e s a - a n a g e s a
+
+i
+
+l
+
+b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I
+
+f
+
+does not impair the rat hippocampal synaptic plasticity later in adulthood. Paediatr Anaesth 2012;22:713–9
+
+29. Tachibana K, Hashimoto T, Takita K, Ito R, Kato R, Morimoto Y. Neonatal exposure to high concentration of carbon dioxide produces persistent learning deficits with impaired hippocampal synaptic plasticity. Brain Res 2013; 1507:83–90
+
+30. Istaphanous GK, Howard J, Nan X, Hughes EA, McCann JC, McAuliffe JJ, Danzer SC, Loepke AW. Comparison of the neu- roapoptotic properties of equipotent anesthetic concentrations of desflurane, isoflurane, or sevoflurane in neonatal mice. Anesthesiology 2011;114:578–87
+
+31. Liang G, Ward C, Peng J, Zhao Y, Huang B, Wei H. Isoflurane causes greater neurodegeneration than an equivalent expo- sure of sevoflurane in the developing brain of neonatal mice. Anesthesiology 2010;112:1325–34
+
+32. Komuro H, Rakic P. Modulation of neuronal migration by NMDA receptors. Science 1993;260:95–7
+
+33. Behar TN, Smith SV, Kennedy RT, McKenzie JM, Maric I, Barker JL. GABA(B) receptors mediate motility signals for migrating embryonic cortical cells. Cereb Cortex 2001;11:744–53
+
+34. De Roo M, Klauser P, Briner A, Nikonenko I, Mendez P, Dayer A, Kiss JZ, Muller D, Vutskits L. Anesthetics rapidly promote
+
+December 2013 • Volume 117 • Number 6
+
+synaptogenesis during a critical period of brain development. PLoS One 2009;4:e7043
+
+35. Koyama R, Tao K, Sasaki T, Ichikawa J, Miyamoto D, Muramatsu R, Matsuki N, Ikegaya Y. GABAergic excitation after febrile sei- zures induces ectopic granule cells and adult epilepsy. Nat Med 2012;18:1271–8
+
+36. Sanders RD, Sun P, Patel S, Li M, Maze M, Ma D. Dexmedetomidine provides cortical neuroprotection: impact on anaesthetic-induced neuroapoptosis in the rat developing brain. Acta Anaesthesiol Scand 2010;54:710–6
+
+37. Danneman PJ, Mandrell TD. Evaluation of five agents/meth- ods for anesthesia of neonatal rats. Lab Anim Sci 1997;47:386–95 38. Loepke AW, McCann JC, Kurth CD, McAuliffe JJ. The physi- ologic effects of isoflurane anesthesia in neonatal mice. Anesth Analg 2006;102:75–80
+
+39. Low JA, Galbraith RS, Muir DW, Killen HL, Pater EA, Karchmar EJ. Factors associated with motor and cognitive deficits in chil- dren after intrapartum fetal hypoxia. Am J Obstet Gynecol 1984;148:533–9
+
+40. Hanrahan JD, Cox IJ, Edwards AD, Cowan FM, Sargentoni J, Bell JD, Bryant DJ, Rutherford MA, Azzopardi D. Persistent increases in cerebral lactate concentration after birth asphyxia. Pediatr Res 1998;44:304–11
+
+www.anesthesia-analgesia.org 1435

new_pdfs/10.1371_journal.pbio.2001246.txt

@@ -0,0 +1,477 @@
+SHORT REPORTS
+
+Early postnatal exposure to isoflurane causes cognitive deficits and disrupts development of newborn hippocampal neurons via activation of the mTOR pathway
+
+a1111111111 a1111111111 a1111111111 a1111111111 a1111111111
+
+Eunchai Kang1,2☯, Danye Jiang3☯, Yun Kyoung Ryu3☯, Sanghee Lim3, Minhye Kwak3, Christy D. Gray3, Michael Xu3, Jun H. Choi1¶, Sue Junn1, Jieun Kim1, Jing Xu3, Michele Schaefer3, Roger A. Johns3, Hongjun Song1,2,4, Guo-Li Ming1,2,4, C. David Mintz3*
+
+1 Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 2 Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 3 Department of Anesthesiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 4 The Solomon Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America
+
+OPEN ACCESS
+
+☯ These authors contributed equally to this work. ¶Author Jun Choi was unavailable at the time of acceptance for publication to confirm his authorship contributions. On his behalf, all other authors have reported his contributions to the best of their knowledge. * cmintz2@jhmi.edu
+
+Citation: Kang E, Jiang D, Ryu YK, Lim S, Kwak M, Gray CD, et al. (2017) Early postnatal exposure to isoflurane causes cognitive deficits and disrupts development of newborn hippocampal neurons via activation of the mTOR pathway. PLoS Biol 15(7): e2001246. https://doi.org/10.1371/journal. pbio.2001246
+
+Received: September 30, 2016
+
+Accepted: June 2, 2017
+
+Published: July 6, 2017
+
+Copyright: © 2017 Kang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
+
+Data Availability Statement: All relevant data are within the paper and its Supporting Information files.
+
+Funding: Johns Hopkins ACCM Department anesthesiology.hopkinsmedicine.org (grant number StAAR) to CDM. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. NIH- NIGMS www.nih.gov (grant number 1R01GM120519-01 and 1K08GM104329-01) to CDM. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. NIH www.nih.gov
+
+Abstract
+
+Clinical and preclinical studies indicate that early postnatal exposure to anesthetics can lead to lasting deficits in learning and other cognitive processes. The mechanism underlying this phenomenon has not been clarified and there is no treatment currently available. Recent evidence suggests that anesthetics might cause persistent deficits in cognitive func- tion by disrupting key events in brain development. The hippocampus, a brain region that is critical for learning and memory, contains a large number of neurons that develop in the early postnatal period, which are thus vulnerable to perturbation by anesthetic exposure. Using an in vivo mouse model we demonstrate abnormal development of dendrite arbors and dendritic spines in newly generated dentate gyrus granule cell neurons of the hippo- campus after a clinically relevant isoflurane anesthesia exposure conducted at an early postnatal age. Furthermore, we find that isoflurane causes a sustained increase in activity in the mechanistic target of rapamycin pathway, and that inhibition of this pathway with rapa- mycin not only reverses the observed changes in neuronal development, but also substan- tially improves performance on behavioral tasks of spatial learning and memory that are impaired by isoflurane exposure. We conclude that isoflurane disrupts the development of hippocampal neurons generated in the early postnatal period by activating a well-defined neurodevelopmental disease pathway and that this phenotype can be reversed by pharma- cologic inhibition.
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+1 / 18
+
+Anesthetic toxicity and mTOR
+
+(grant number NS048271 and MH105128) to GLM. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. NIH www.nih.gov (grant number NS047344) to HS. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
+
+Competing interests: The authors have declared that no competing interests exist.
+
+Abbreviations: BDNF, brain-derived neurotrophic factor; DCG, dentate gyrus granule cell; DIV, day in vitro; DISC1, disrupted in schizophrenia 1; GFP, green florescent protein; IP, intraperitoneal; mTOR, mechanistic target of rapamycin; P, postnatal day; PI3K-Akt, phosphoinositide 3 kinase-protein kinase B; pS6, phosphor-S6.
+
+Author summary
+
+The United States Food and Drug Administration has recently warned that exposure to anesthetic and sedative drugs during the third trimester of prenatal development and dur- ing the first 3 years of life may cause lasting impairments in cognitive function. The mech- anisms by which this undesirable side effect occurs are unknown. In this manuscript, we present evidence in mice that early developmental exposure to isoflurane, a canonical gen- eral anesthetic, disrupts the appropriate development of neurons in the hippocampus, a brain region associated with learning and memory. Isoflurane also causes up-regulation of the mechanistic target of rapamycin (mTOR) pathway, a signaling system that has been associated with other neurodevelopmental cognitive disorders. Treatment with an inhibi- tor of the mTOR pathway after isoflurane exposure normalizes neuronal development and also ameliorates the impairments in learning induced by isoflurane. We conclude that early exposure to isoflurane can cause learning deficits via actions on the mTOR pathway, and that this mechanism represents a potentially druggable target to minimize the side effects of anesthetics on the developing brain.
+
+Introduction
+
+Several large retrospective analyses link exposure to anesthetics and surgery within the first 3 years of life with subsequent effects on cognitive function, as measured by worsened perfor- mance on school assessments, an increase in billing codes relevant to learning disorders, and deficits in neuropsychological testing [1–3]. It is difficult to separate the effects of surgery, anesthesia, and comorbidity in clinical studies. However, multiple independent investigations conducted in rodent models using different anesthetics and varying exposure paradigms in the absence of surgery indicate that early developmental exposure to general anesthetic agents results in lasting impairment on behavioral measures of neurocognitive function, predomi- nantly in the domain of learning and memory [4–12]. While 2 recent clinical studies give some reassurance that short, single exposures in healthy children may not have dramatic conse- quences [13,14], clear evidence of lasting cognitive deficits was detected recently in a carefully conducted study of a somewhat longer clinically relevant anesthetic exposure in nonhuman primates [15]. Thus, there are serious concerns in the anesthesiology, surgery, and pediatrics literature that anesthetic exposure may result in worsened cognitive outcomes for some unknown fraction of the hundreds of thousands of children under age 4 who undergo surgery each year [16–18]. In response to these findings, the US Food and Drug Administration recently issued a drug safety communication warning that anesthetic exposure may pose risks to brain development and calling for further research on this topic. The molecular and cellular mechanisms underlying this phenomenon have yet to be clearly elucidated, and no prophylac- tic or treatment strategies exist.
+
+Much of the literature on the effects of anesthetic exposure on brain function focuses on the
+
+potential for anesthetics to activate apoptotic cell death pathways in neurons [6,19], but more recent work has led to the novel hypothesis that anesthetics cause lasting effects on cognitive function via sublethal effects on critical processes in neuronal development [20]. In humans, the neural circuitry underlying higher brain functions, such as learning, is primarily estab- lished between the second trimester and early childhood [21], a period that includes the win- dow of putative vulnerability to anesthetics identified in epidemiologic studies [18]. During this time, critical ongoing developmental events are occurring in many neurons of the hippo- campus, including growth of dendritic arbors and generation of dendritic spines, which are
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+2 / 18
+
+Anesthetic toxicity and mTOR
+
+the postsynaptic elements of excitatory synapses [22]. There are substantial differences in developmental timelines in the different species in which the effects of early postnatal anesthe- sia exposure on cognitive function have been studied, but one notable common feature is the generation and development of a large percentage of the dentate gyrus granule cell (DGC) neurons in the hippocampus [23], a structure that is critical to cognitive functions, including learning and memory. Thus, in this study we investigated the effects of anesthesia exposure on dendritic arbor and spine development in early postnatally generated DGCs, which may be an important target population and may also serve as a model for postnatal neuron development in other brain regions.
+
+We employed a retrovirus-mediated labeling method in intact mice to examine the devel- opment of dendrite arbors and dendritic spines in DGCs in vivo after exposure to a clinically relevant dose of isoflurane. This approach allows morphological analyses of a uniform and well-studied population of neurons, the DGCs, at a single cell level in vivo [24]. We find that early postnatal exposure to isoflurane results in a substantial and lasting disruption of dendritic arborization and spine development. Isoflurane was found to over-activate the mechanistic target of rapamycin (mTOR) pathway, a signaling system critical for normal development, which has been implicated in neurodevelopmental disorders in which cognitive function is affected, including autism and fragile X mental retardation [25,26]. Strikingly, the adverse effects of isoflurane on both dendrite morphology and behavioral tests of learning can be reversed with rapamycin, an mTOR inhibitor. Our findings reveal a novel mechanism by which anesthetics disrupt brain development that has been implicated in other neurodevelop- mental disorders and that is potentially reversible via drug therapy.
+
+Results and discussion
+
+In order to investigate the effects of anesthetics on dentate gyrus neuron development in vivo, we employed stereotaxic injection to deliver a retrovirus expressing green florescent protein (GFP) to label newly generated dentate gyrus neurons [24]. Injections were conducted at post- natal day (P) 15; on P18, the animals were exposed to isoflurane, a canonical halogenated ether vapor anesthetic. The dose of isoflurane exposure (1.5%) falls well within clinically relevant parameters, as the minimum alveolar concentrations of isoflurane ranges between 1.6% and 1.8% in children between ages 0 and 4 [27]. A 4 hour-exposure duration was selected based on clinical data, which showed that significant learning deficits in children are associated with more than 2 hours of anesthetic exposure [3]. All exposed mice survived and recovered readily, and results of physiologic monitoring of sentinel animals are shown in S1 Table. Tissue was collected for morphological studies at P30. A flow diagram of these experiments is shown in Fig 1A.
+
+We sought to determine whether exposure to anesthetics during development alters neuro-
+
+nal structure in newborn DGCs lasting fashion. Previous investigations have been potentially confounded by an inability to determine the developmental stage at which any given neuron under analysis was affected by anesthetics, given the nonhomogenous timeline of neuronal development that occurs even within discrete brain regions. In our model, the labeled DGCs, which have fully definable structure due to GFP expression that allows for easy analysis of morphology (Fig 1B), represent a cohort of cells with a uniform birthdate, all of which were exposed to anesthetics at the same point in their developmental timeline. Examination of den- dritic structure revealed a striking finding: compared to neurons in unexposed littermate con- trols, labeled neurons in isoflurane-exposed animals exhibit an 83% increase in total dendritic arbor length at P30 (p < 0.005; Fig 1C–1E). To further elucidate this phenomenon, we con- ducted a Sholl analysis, which revealed a significant increase in dendrite arbor complexity with
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+3 / 18
+
+Anesthetic toxicity and mTOR
+
+Fig 1. Isoflurane exposure results in overgrowth of dendritic arbors. (A) A schematic diagram of isoflurane exposure procedure for morphology examination. (B) Sample confocal image of dentate gyrus granule cell (DGCs) infected with retrovirus expressing green florescent protein (GFP) (scale bar: 100 μm). Representative confocal images (C) and tracings (D) of individual control and isoflurane-exposed GFP+ neurons at postnatal day (P) 30 exhibiting overgrowth in the isoflurane group relative to control conditions (scale bar: 10 μm for both C and D). Summaries of total dendritic length (E) and Sholl analysis of dendritic complexity (F) of GFP+ neurons show marked overgrowth of dendritic arbors. Numbers associated with bar graph indicate the number of neurons examined from at least 5 animals per group. The same groups of neurons were examined in (E) and (F). Values represent mean ± SEM (**p < 0.01; Student t test for E and *p < 0.0001 ANOVA for F). Underlying data in S1 Data under Fig 1F tab.
+
+https://doi.org/10.1371/journal.pbio.2001246.g001
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+4 / 18
+
+Anesthetic toxicity and mTOR
+
+isoflurane exposure (p < 0.0001; Fig 1F). This finding seems to represent an acceleration of dendrite growth, because dendritic length and complexity in the isoflurane group no longer differs from controls at P60 (S1A–S1D Fig). Branch number is unaffected at either time point (S1E Fig). Cell positioning within the dentate gyrus is unaffected (S1F Fig), suggesting no defi- cits in migration, but soma size is significantly increased with isoflurane exposure at P30, but not P60 (S1G Fig), further suggesting an abnormal acceleration in DGC growth.
+
+The change in timing of dendritic development resulting from anesthetic exposure repre-
+
+sents a novel and surprising effect of anesthetics on the developing brain. In vitro studies of axon growth suggest that volatile anesthetics such as isoflurane may slow the growth of axons and prepolarized neurites [28,29], but axons and dendrites have substantial differences in their developmental properties [30]. A cell culture study that specifically examined dendrites found that exposure to propofol, but not midazolam, at 1 day in vitro (DIV) caused a lasting suppres- sion of dendritic growth in GABAergic neurons [31]. While the timing of exposure and mea- surement loosely resembles our model, the difference in anesthetic agents and the lack of an in vivo context may explain the disparate findings. Furthermore, the DGCs are primarily gluta- matergic and have properties quite distinct from the GABAergic interneurons population [32]. The only other study to assess the effects of anesthetics on dendrites in vivo found no acute change in the dendritic arbors of prefrontal cortex pyramidal neurons in P16 rats 6 hours after isoflurane exposure, but did not examine longer-term effects [33]. Thus, it is unclear whether the transient dendritic hypertrophy we observed might generalize beyond the DGCs exposed early in their development. Abnormalities in dendritic arbor development may have a profound impact on the function of a neuron via effects on the neuron’s synaptic field and pathologic overgrowth of dendrites has been hypothesized as a component of human neu- rodevelopmental diseases such as autism and schizophrenia [34]. Overgrowth of dendritic arbors has been observed in some animal models of Fragile-X syndrome [35] and autism [36]. However, we cannot determine whether the phenomenon that we observed is a cause of neu- ronal dysfunction or simply an epiphenomenon or adaptive response.
+
+We next asked whether isoflurane exposure results in long-term deficits in learning poten-
+
+tially attributable to a disruption of the function of the DGCs in which we have detected a morphological abnormality. Animals were exposed to isoflurane 1.5% for 4 hours at P18 and evaluated for deficits in the object-place recognition and the Y-maze tests of spatial learning at P60 (Fig 2A). Both of these tasks are highly sensitive to alterations in the function of even small numbers of dentate gyrus neurons [37]. In the object-place recognition test, control animals spend significantly more time exploring objects in novel positions, but isoflurane- exposed animals exhibit no exploration preference (Fig 2B, S2A and S2C Fig). Similarly, in the Y-maze test, unlike controls, isoflurane-exposed mice do not exhibit a preference for explora- tion of the newly available arm (Fig 2C, S2B and S2D Fig). These data demonstrate that isoflur- ane exposure results in a lasting reduction in performance on the tasks of spatial learning that are dependent on the hippocampus and potentially sensitive to disruption of the development of the dentate gyrus.
+
+Next, we asked whether the observed changes in behavior after anesthetic exposure could be attributed to a lasting change in synapses of the DGCs. We used the retrovirus-mediated labeling method to quantify the density of dendritic spine formations at P60, the age at which behavioral testing took place (Fig 2A). Dendritic spines are dynamic, actin-dependent struc- tures that are critical for learning and memory functions [38]. Spines have range of morpholo- gies traditionally classified as stubby, thin, and mushroom shape. We found a small, but significant decrease in the total density of spines (12% decrease, p < 0.05) in anesthesia- exposed groups, and a very striking 39% decrease in the density of mushroom spines (p < 0.001; Fig 2D, S2E and S2F Fig). No significant change was seen in the density of stubby
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+5 / 18
+
+Anesthetic toxicity and mTOR
+
+Fig 2. Isoflurane exposure impairs spatial learning and causes a loss of dendritic spines in dentate gyrus neurons. (A) A schematic diagram of isoflurane exposure procedure for behavior tests and spine analysis. Shown in (B and C) are summaries of the object-place recognition test (B) and the Y- maze test (C) (Control n = 12, Isoflurane n = 11; **p < 0.01, Student t test). (D) Representative processed confocal images of dendritic spines of control and isoflurane-exposed green florescent protein positive (GFP) neurons at postnatal day (P) 60 (scale bar: 2 μm). Shown on right are summary plots of total and mushroom class dendritic spine density, revealing a striking loss of mature spines. Numbers associated with the bar graph indicate the number of dendritic segments examined from at least 5 mice from each group, a total of 2,586 spines in the control group and 2,818 spines in the isoflurane group were analyzed (*p < 0.05; ****p < 0.0001, Student t test). Underlying data in S1 Data under Fig 2B-D tab.
+
+https://doi.org/10.1371/journal.pbio.2001246.g002
+
+or thin spines (S2G and S2H Fig). Stubby spines are thought to be immature, thin spines are highly plastic and often transient unless converted into mushroom morphology, and mush- room spines typically represent long-lasting, stable synaptic connections [39]. The reduction in mushroom spine number suggests a substantial loss of synapses that could reasonably account for the reduced performance in spatial learning.
+
+Our finding of a reduction in spine density in the cohort of labeled DGCs is in keeping with
+
+an increasing body of work suggests that relatively immature neurons exposed to anesthetics may suffer a long-lasting loss of synaptic connections. Studies from 2 different groups in rats found that early postnatal exposure to either sevoflurane alone or a combination of isoflurane, midazolam, and nitrous oxide resulted in a long-term reduction in the number of synaptic profiles measured by quantitative electron microscopy in the hippocampal CA1 and subicu- lum areas, respectively [40,41]. The hippocampus is a relatively late developing structure [42],
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+6 / 18
+
+Anesthetic toxicity and mTOR
+
+and thus during early postnatal life, it has numerous neurons that are still undergoing active dendrite arborization and spine formation. In support of the hypothesis that developing neu- rons may be vulnerable to anesthesia-induced synapse loss, a long-term study of the effects of single dose propofol exposure in rats found a decrease in spines in the medial prefrontal cortex of rats exposed at P5 and measured at P90 [43]. In striking contrast, exposure at P15 actually caused an increase in spine number [43], suggesting a notable difference in vulnerability that occurs with neuronal maturation. If developmental exposure to anesthetics can cause a lasting or even permanent loss of synaptic connections in key brain regions such as the hippocampus and pre-frontal cortex this event may represent a perturbation of the development of key brain circuitry, which, in turn, could explain an ongoing loss of cognitive function.
+
+A common feature shared by several neurodevelopmental disorders with phenotypes remi-
+
+niscent of what we have observed in neurons exposed to anesthesia during development is an alteration in signaling in the mTOR pathway [44]. To determine whether activity in the mTOR system is altered by an early exposure to anesthetics we conducted quantitative fluores- cence immunohistochemistry using an antibody against phospho-S6 (pS6), a reliable reporter of activity in this pathway [37]. We exposed mice to isoflurane at 1.5% for 4 hours and mea- sured pS6 immunoreactivity in the DGC layer. We found an increase of greater than 2-fold in pS6 intensity at P30 (p < 0.0005; Fig 3A, S3 Fig), which was still evident at P60 (S4 Fig). This demonstrates a substantial and lasting upregulation of activity in the mTOR pathway in the dentate gyrus during the period in which we have observed morphological alterations.
+
+We next asked whether increased activity in the mTOR pathway is required for the isoflur- ane-induced deficits in spatial learning that we observed previously. Mice were exposed to iso- flurane 1.5% for 4 hours on P18, given intraperitoneal (IP) injections either of vehicle control or 20 mg/kg rapamycin, a pharmacologic inhibitor of mTOR, every other day between P21 and P29, and then assayed for spatial learning via behavioral testing (Fig 3B). To confirm that our rapamycin treatment effectively suppressed isoflurane-mediated activity in the mTOR pathway, we tested for pS6 immunoreactivity in the dentate gyrus of animals exposed to iso- flurane and then treated with rapamycin. We found that rapamycin treatment significantly reduced pS6 immunoreactivity compared to isoflurane and that levels were comparable to untreated controls (Fig 3A).
+
+Subsequently, we tested whether blocking mTOR activation induced by isoflurane could rescue the morphological disruptions and behavioral deficits observed after isoflurane treat- ment. First, we tested the effects of mTOR inhibition on isoflurane-induced dendrite growth acceleration. We found that rapamycin treatment after isoflurane significantly reduces total dendritic length compared with the control group (p < 0.05) and that dendritic length in the isoflurane plus rapamycin group is not significantly different from controls (Fig 3C). Sholl analysis indicates that rapamycin treatment after isoflurane results in arbor complexity that is more similar to what is measured with control conditions than with isoflurane alone (Fig 3D). Rapamycin treatment alone has no effect on spatial learning (S5A–S5D Fig), but rapamycin treatment after isoflurane exposure restores performance to near control levels in both the object-place recognition and Y-maze tests (Fig 3E and 3F and S5C–S5F Fig). Subsequently, we assayed the numbers of dendritic spines in the retrovirus-labeled DGCs exposed to isoflurane with and without rapamycin treatment. We find no significant difference in the total dendritic spine density between the vehicle and rapamycin groups exposed to isoflurane (Fig 3G and S5G and S5H Fig). However, when only the mushroom spines are considered, we find an increase in spine density in the rapamycin group compared to the vehicle treated group (p < 0.0001) (Fig 3G). There is no significant difference in mushroom spine density between the control group that did not receive isoflurane and the isoflurane plus rapamycin group (Fig 3G). By contrast stubby spine density appears to be reduced by isoflurane and rapamycin
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+7 / 18
+
+Anesthetic toxicity and mTOR
+
+Fig 3. Isoflurane exposure leads to aberrant activation of the mechanistic target of rapamycin (mTOR) signaling pathway, and pharmacological inhibition of the mTOR activities rescues deficits in behavioral tests and loss of spines. (A) Representative confocal images of phospho-S6 (pS6) immunofluorescence at postnatal day (P) 30 in the dentate gyrus showing an increase in labeling in the isoflurane plus vehicle (Iso/V) group relative to controls and a return to baseline in the group exposed to isoflurane and subsequently treated with rapamycin, designated Iso/R. The upper panels
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+8 / 18
+
+Anesthetic toxicity and mTOR
+
+are original confocal images with DAPI in blue and pS6 labeling in red, and the lower panels are processed for quantification with black pS6 signal on white background (ML, molecular layer; DG, dentate gyrus; HI, hilus, scale bar: 50 μm). Also shown in (A) quantification of normalized pS6 expression in the dentate gyrus granule cell layer (***p < 0.001, ANOVA, numbers in each bar represent n for images analyzed). (B) Schematic diagram of rapamycin treatment for behavior tests and spine analysis. Summaries of total dendritic length (C) and Sholl analysis of dendritic complexity (D) of GFP+ neurons show a rescue of normal dendritic arbor length and complexity with Iso/R. Values represent mean ± SEM (*p < 0.05, **p < 0.01; ANOVA for C; *p < 0.0001 ANOVA for D). Numbers in each bar represent number of cells analyzed per group, minimum of 5 animals per group). Summaries of object-place recognition test (E) and Y-maze test (F) for Iso/V and Iso/R show a recovery to near control performance with Iso/R. (Control n = 10, Iso/V n = 11, Iso/R n = 11; *: p < 0.05; **: p < 0.01, Student t test). (G) Representative confocal images of dendritic spines at P60. Scale bar: 2 μm. Shown on right are summary plots of total and mature dendritic spine density. Numbers associated with bar graph indicate the number of dendritic segments examined, a total of 2,586 spines in the control group, 1,831 spines in the isoflurane plus vehicle group, and 2,999 spines in the isoflurane plus rapamycin group were analyzed (****p < 0.0001; ns: non-significant; ANOVA, numbers in each bar represent n of dendritic segments analyzed per group, minimum of 5 animals per group). Underlying data in S1 Data under Fig 3A-G.
+
+https://doi.org/10.1371/journal.pbio.2001246.g003
+
+treatment relative to isoflurane alone, and no significant differences are measured in thin spines (S5I and S5J Fig). Thus, our data suggest that rapamycin, by inhibiting the mTOR path- way, prevents an isoflurane-induced reduction in stable synaptic connections.
+
+Taken together, our findings indicate that isoflurane causes a sustained increase in activity
+
+in the mTOR pathway that leads to dendrite growth acceleration and either synapse loss or reduced synapse formation in DGCs. Superficially, our results are at odds with a previous study, showing no activation of mTOR in the hippocampus after sevoflurane anesthesia [45], but in the other study, measurements were taken hours after exposure, whereas in the current study we made measurements 1 to 2 weeks later, with a goal of elucidating longer term effects on neuronal development. The mTOR pathway is an intriguing potential mechanism of injury, as it has been implicated both in normal functions in brain development and it is disarrayed in a wide-range of human neurodevelopmental disease [46]. The mTOR pathway is involved in normal development of dendrites and synapses through its actions, integrating signals from the phosphoinositide 3 kinase-protein kinase B (PI3K-Akt) system, which is influenced by both activity and neurotrophic growth factors, such as brain-derived neurotrophic factor (BDNF), that act via tyrosine kinase receptors [47,48]. Downstream mediators of mTOR that influence synaptogenesis include actions on mitochondrial function, lipid synthesis, and trans- lational control via the mTOR1 complex and RhoGTPase actions on the cytoskeleton via the mTOR2 complex [47,48]. Enhanced activity in the mTOR pathway induced by knockdown of disrupted in schizophrenia 1 (DISC1) in newly generated DGCs in adult animals causes accel- erated development of dendrites, similar to what we have seen, but it is accompanied by an increase in spine formation [37,49], which stands in apparent contrast to the spine decrease seen in our model. However, several key differences exist between the models that may explain this discrepancy: (1) our study follows the neurons in question for a much longer period, and thus it is possible that overgrowth leads to spine loss over a sufficient length of time; (2) in the DISC1 study, only the studied cohort of newborn DGCs was affected, whereas in our model isoflurane may exert an effect on the surrounding cells as well as the labeled cells; (3) the influ- ence of the DISC1 knockdown was permanent, whereas in our model isoflurane is given tran- siently and its effects may therefore be manifested differently over time; and (4) we observe overgrowth at P30, which is no longer apparent at P60, and it is possible that early acceleration of growth followed by slowing may induce synaptic loss as a result of a disruption of the nor- mal timing of dendritic arbor growth relative to dendritic spine growth. Additionally, it should be noted that the effects of changes in mTOR signaling may depend on context and on activity in other systems. For instance, Kumar et al. showed that transient inhibition of mTOR, which alone decreases spine formation, could actually increase formation of mushroom spines in a
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+9 / 18
+
+Anesthetic toxicity and mTOR
+
+developmental model when it was accompanied by activation of the PI3K-Akt system or treat- ment with BDNF [50]. In this model, an increase in mushroom spines is accompanied by a decrease in filopodial protrusions that the authors interpret as a destabilization or regression of synapses. Isoflurane and other anesthetics act on multiple targets in developing neurons, and thus understanding their actions on spine and synapse formation will require a full inves- tigation of how each component of the signaling systems that underlie this process is affected. Given the complexity of the mTOR pathway, the effects of a lasting change in the activity of this pathway are difficult to predict. A sustained increase in mTOR pathway tone certainly has the potential to powerfully alter neurotransmission in the dentate gyrus, as evidenced by the appearance of epileptiform activity in mice with selective deletions of phosphatase and tensin homolog, an mTOR pathway inhibitor, in DGCs [51]. Thus, we hypothesize that isoflurane- induced changes in mTOR signaling have the potential to disrupt the course of neuronal devel- opment in the dentate gyrus and perhaps in other brain areas in such a way as to disrupt cogni- tive function. Even if our findings do not generalize to other cell types and brain regions, they still have significant implications given that substantial populations of DGCs are generated in rodents [52], nonhuman primates [53], and humans [52] during the hypothesized period of susceptibility to anesthesia-induced cognitive deficits in each of these species and these neu- rons are critical for learning across species. Furthermore, our findings suggest the possibility that harmful effects of mTOR overactivation could be prevented. Complex neurodevelopmen- tal cognitive disorders like autism, in which the pathophysiology may involve changes in mTOR pathway activity that stem from a combination of genetic and environmental factors occurring at unknown times during development, present great challenges in designing an mTOR targeted therapy [54]. By contrast, anesthetic effects on cognitive function result from a brief toxic insult at a known time, and therefore might be more amenable to treatment. Thus, our discovery of a novel, reversible mechanism of injury in developmental anesthetic neuro- toxicity has translational potential that can be explored in future studies.
+
+Methods
+
+Ethics
+
+All study protocols involving mice were approved by the Animal Care and Use Committee at the Johns Hopkins University (protocol MO14M315) and conducted in accordance with the NIH guidelines for care and use of animals.
+
+Animals
+
+C57BL/6 mice were housed in a temperature- and humidity-controlled room with a 12:12 hour light:dark cycle, and provided with ad libitum access to water and food. Both sexes were equally represented in all experiments. No animals were excluded.
+
+Isoflurane treatment and physiologic monitoring of sentinel animals
+
+P18 mouse littermates were randomly assigned to 2 groups. In Group 1 (isoflurane), mice were exposed to 1.5% isoflurane carried in 100% oxygen for 4 hours. A calibrated flowmeter was used to deliver oxygen at a flow rate of 5 L/min and an agent-specific vaporizer was used to deliver isoflurane. In Group 2 (control), mice were exposed to room air for 4 hours. Animals were returned to their cages together with their littermates upon regaining righting reflex. Mice were continually monitored and recorded for skin temperature, heart rate, and oxygen saturation during the 4-hour isoflurane treatment (PhysioSuite; Kent Scientific, Torrington,
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+10 / 18
+
+Anesthetic toxicity and mTOR
+
+CT). Intracardiac puncture was used to collect left ventricular blood samples from selected sentinel animals, and those confirmed to be arterial are reported.
+
+Production and stereotaxic injection of engineered retroviruses
+
+Engineered self-inactivating murine retroviruses were used to express GFP under Ubiquitin promotor (pSUbGW vector) specifically in proliferating cells and their progeny [55,56]. High titers of engineered retroviruses (1 x 109 unit/ml) were produced by cotransfection of retroviral vectors and VSVG into HEK293gp cells followed by ultracentrifugation of viral supernatant as previously described [24,49,55–57]. After induction with a single ketamine injection (50mg/ kg), high titers of GFP-expressing retroviruses were stereotaxically injected into the P15 mice dentate gyrus through a 32-gauge microsyringe (Hamilton Robotics, Reno, NV) at 2 sites of the following coordinates relative to the bregma (mm): AP: −2.2, ML: ±2.2, DV: −2.4. The ret- rovirus-containing solution was injected at a rate of 0.025 μl/min for a total of 0.5 μl per site. After infusion, the microsyringe was left in place for an additional 5 minutes to ensure full virus diffusion and to minimize backflow. After surgery, mice were monitored for general health every day until full recovery. In order to test for a possible confound related to the use of ketamine anesthesia, pS6 immunoreactivity in the dentate gyrus was quantified at P30 in naïve control animals and compared to pS6 immunoreactivity in animals doses with ketamine as above. No significant difference is seen in pS6 levels between these groups (S6 Fig).
+
+Immunostaining
+
+After transcardial perfusion fixation with 4% paraformaldehyde/PBS, brains were sliced trans- versely (50 μm thick) with microtome and processed for immunohistochemistry. Primary antibodies, including goat anti-GFP (Rockland, 1:1000) and chicken anti-GFP (Millipore, 1:1000) were used. Immunofluorescence was performed with a combination of Alexa Fluor 488- or Alexa Fluor 594-labeled anti-goat, anti-chicken, or anti-rabbit secondary antibodies (1:250) and 4´,6´-diaminodino-2-phenylindole (DAPI, 1:5000). For analysis of pS6 levels, pri- mary antibodies against pS6-Ser235/236 (rabbit, 1:1000, Cell Signaling) were used. Effective immunostaining of pS6 required an antigen retrieval protocol as previously described [58]. Briefly, sections were incubated in target retrieval solution (DAKO) in 85˚C for 20 minutes followed by washing with PBS for t3 times before the incubation with primary antibody.
+
+Imaging and analyses
+
+Images were acquired on a confocal system (Zeiss LSM 710 or Leica SPE) and morphological analyses were carried out as previously described [24,49,55,56,58,59]. Images for dendritic and spine morphology were deconvoluted with Auto Quant X (Media Cybernetics, Rockville, MD) using the blind algorithm, which employs an iteratively refined theoretical PSF. No further processing was performed prior to image analysis. For visualization, brightness, and contrast levels were adjusted using Image J (NIH). For analysis of dendritic development, three-dimen- sional (3D) reconstructions of entire dendritic processes of each GFP+ neuron were obtained from Z-series stacks of confocal images using excitation wavelength of 488 nm at high magnifi- cation (x 40 lens with 0.7x optical zoom). The two-dimensional (2D) projection images were traced with NIH Image J plugin, NeuronJ. All GFP+ DGCs with largely intact, clearly identifi- able dendritic trees were analyzed for total dendritic length. The measurements did not include corrections for inclinations of dendritic process and therefore represented projected lengths. Sholl analysis for dendritic complexity was carried out by counting the number of dendrites that crossed a series of concentric circles at 10 μm intervals from the cell soma using ImageJ (NIH). For complete 3D reconstruction of spines, consecutive stacks of images were
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+11 / 18
+
+Anesthetic toxicity and mTOR
+
+acquired using an excitation wavelength of 488 nm at high magnification (x 63 lens with 5x optical zoom) to capture the full depth of dendritic fragments (20–35 μm long, 40~70 dendritic fragments in each condition analyzed) and spines using a confocal microscope (Zeiss, Oberko- chen. Germany). Confocal image stacks were deconvoluted using a blind deconvolution method (Autoquant X; Media Cybernetics, Rockville, MD). The structure of dendritic frag- ments and spines was traced using 3D Imaris software using a “fire” heatmap and a 2D x–y orthoslice plane to aid visualization (Bitplane, Belfast, UK). Dendritic fragments were traced using automatic filament tracer, whereas dendritic spines were traced by means of an autopath method with the semiautomatic filament tracer (diameter; min: 0.1, max: 2.0, contrast: 0.8). For spine classification, a custom MatLab (MathWorks, Natick, MA) script was used based on the algorithm; stubby: length (spine) <1.5 and max width (head)<mean_width (neck) (cid:3)1.2; mushroom: max width (head) >mean width (neck) (cid:3)1.2 and max_width (head) >0.3; if the spine was not classified as mushroom or stubby, it was defined as long-thin. Axonal bouton volume from axonal fragments was measured by using 3D Imaris software and using a magic wand menu (Bitplane, Belfast, UK) after deconvolution. For analysis of pS6 levels, the sections were processed in parallel and images were acquired using the identical settings, (Zeiss LSM 710, 20X lens). Fluorescence intensity was measured within the granular cell layer using Ima- geJ (NIH) and the value was normalized to background signal in the same image. These data were then subsequently normalized to the area of the dentate gyrus granule layer as defined by DAPI staining. All experiments were carried out in a blind fashion to experimental conditions.
+
+Behavioral tests
+
+Sixty-day-old mice housed in groups (5 mice per cage) were handled for at least 2 minutes per day for 3 days before the start of the behavioral experiments. All behavioral tests were per- formed during the light phase of the cycle between 8:00AM and 6:00PM. Experimenters were blind to the samples when behavioral tests were carried out and quantified. The numbers of mice per condition are indicated in the figure legends.
+
+Object-place recognition test. Object-place recognition was performed as previously described [37]. Briefly, the test was assessed in a 27.5 cm × 27.5 cm × 25 cm opaque chamber with a prominent cue on 1 of the walls. Each mouse was habituated to the chamber for 15 min- utes daily for 2 days. During the training phrase, each mouse was allowed to explore 2 identical objects (glass bottle, 2.7 cm diameter, 12 cm height, and colored paper inside) for 10 minutes. The mouse was then returned to its home cage for a retention period of 24 hours. The mouse was reintroduced to the training context and presented with 1 object that stayed in the same position as during training while the other object was moved to a new position. Movement and interaction with the objects was recorded with a video camera that was mounted above the chamber and exploratory behavior was measured by a blinded observer. Exploratory behavior was defined as sniffing, licking, or touching the object while facing the object.
+
+Y-maze test.
+
+In the Y-maze test, mice were released from the start arm (no visual cue)
+
+and allowed to habituate to only 1 out of 2 possible choice arms (overt visual cue) for 15 min- utes. This was followed at 24 hours later by the recognition phrase in which the animal could choose between the 2 choice arms after being released from the start arm. The timed trials (5 minutes) were video recorded as well as graded by an observer blind to condition for total exploration time in each choice arm.
+
+Rapamycin treatment
+
+P21 mouse littermates were given IP injections of rapamycin (Sigma-Aldrich, St. Louis, MO) prepared from a stock solution (25 mg/ml in 100% ethanol, stored at -20˚C) diluted to a final
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+12 / 18
+
+Anesthetic toxicity and mTOR
+
+concentration of 4% (v/v) ethanol in the vehicle. Vehicle consisted of 5% Tween 80 (Sigma- Aldrich, St. Louis, MO) and 10% polyethylene glycol 400 (Sigma-Aldrich, St. Louis, MO) as pre- viously described [58,60,61]. Both rapamycin- and vehicle-treated mice received the same vol- ume for each injection (200 μl). Mice received treatments at 48 hour intervals from P21 to P29.
+
+Statistics
+
+Results are expressed as mean ± SEM. A one-tailed Student t test or ANOVA with Bonferroni test for intergroup comparisons were used for most statistical comparisons between groups as described in the figure legends using Prism Software (Graphpad Software Inc, La Jolla, CA). For Sholl analysis ANOVA was used at each point to test for differences between distributions. All data examined with parametric tests were determined to be normally distributed, and the criteria for statistical significance was set a priori at p < 0.05. Sample sizes were predicted based on experience from previous similar work [24]. All relevant data are available from the authors.
+
+Supporting information
+
+S1 Fig. A dense field of isoflurane and control group dendrites is shown for P30 and P60 to illustrate the overgrowth phenonenom (scale bar: 50μm). (A). Neurolucida tracings of P60 neurons suggest that the overgrowth does not persist at P60 (scale bar: 20μm) (B), and quanti- tative analysis by dendrite length measurement (C) Sholl analysis (D) do not show significant differences between control and isoflurane groups at P60. Isoflurane exposure does not sub- stantially alter DGC distribution. The bar graph in E shows positioning of control and isoflur- ane-exposed newborn DGCs in the dentate gyrus at P30 and P60. Layers 1, 2, and 3 refer to the inner, middle, and outer layers of granule cells in the dentate gyrus, respectively; layer 4 refers to the molecular layer. Soma size of DGCs is significantly increased at P30 ((cid:3): p<0.01 Student’s t-test), but not at P60 as show in F. To determine whether isoflurane increases branch number, we counted branch points in each dendritic arbor of the labeled neurons. No significant difference was found at either P30 or P60 (G). For all bar graphs, numbers on each barindicate the number of neurons examined from at least four mice from per group. Underly- ing data in S1 Data under Fig S1C-G. (TIF)
+
+S2 Fig. Absolute values for exploration time during the object-place recognition (A) and the Y-maze tests (B) are shown at 24 h after training (Object place-recognition: Control n = 12, Iso n = 11, (cid:3)p < 0.05, Student’s t-test; Y-maze: Control n = 12, Iso n = 11; (cid:3)p < 0.05, Student’s t-test). Individual data points are shown for the object-place recognition (C) and Y-maze tasks (D) The number of spines counted as a function of the length of each dendritic fragment on which they were counted is represented graphically for total spines (E) and mushroom spines (F) for the isoflurane (red) and control (black) groups. Dendritic spine density measurements for stubby and thin spine morphologies in control and isoflurane conditions are shown in (G) and (H), respectively. No significant differences were seen in either of these morphological groups. A reduction in stubby spine density is seen with isoflurane and a further reduction with isoflurane and rapamycin, and no significant difference is measured in thin spine density between any of the conditions ((cid:3)(cid:3) p < 0.01 (cid:3)(cid:3)(cid:3)p < 0.001 ANOVA). (TIF)
+
+S3 Fig. A tiled reconstruction of a representative confocal image of an entire dentate gyrus at P40 with immunohiostochemistry for pS6 and counterstaining for DAPI for control and isoflurane-exposed is shown (blue: DAPI; red: pS6). Scale bar: 100 μm. (TIF)
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+13 / 18
+
+Anesthetic toxicity and mTOR
+
+S4 Fig. Immunoreactivity for pS6 measured at P60 is significantly increased after isoflur- ane treatment, and is reduced with rapamycin treatment. ((cid:3)(cid:3)(cid:3)p < 0.001, (cid:3)(cid:3)(cid:3)(cid:3)p < 0.0001, ANOVA, numbers in each bar represent n for images analyzed) Scale bar: 50 μm. Underlying data in S1 Data file under Fig S4. (TIF)
+
+S5 Fig. As an additional control tests of spatial learning were performed on animals treated with rapamycin only, in the absence of isoflurane to test for possible effects or rapamycin independent of anesthesia-induced deficits. No change in performance relative to control is measured with rapamycin in either object place-recognition (A) or Y-maze test paradigms (B). These results are also presented in the context of the control, isoflurane, and isoflurane plus rapamycin groups (C,object place recognition; D, Y-maze). Individual data points are shown for the object-place recognition (E) and Y-maze tasks (F). The number of spines counted as a function of the length of each dendritic fragment on which they were counted is represented graphically for total spines (G) and mature spines (H) for the isoflurane (red) and control (black) groups. Dendritic spine density measurements for stubby and thin spine morphologies in control and isoflurane conditions are shown in (I) and (J), respectively. Underlying data in S1 Data under Fig S5A-J. (TIF)
+
+S6 Fig. All animals used in experiments requiring stereotaxic injection of retrovirus, including both controls and isoflurane exposed groups, were anesthetized with small doses of ketamine to facilitate the surgery. To test for a possible confounding effect of ketamine, levels of pS6 labeling in the dentate gyrus were measured in naïve controls and in animals that received ketamine only. No significant difference pS6 immunoreactivity is seen between the two groups (Student’s t-test). Numbers on each bar indicate the number for images analyzed from at least five mice from per group. Scale bar: 25 μm. Underlying data in S1 Data under Fig S6. (TIF)
+
+S1 Table. Data describing the physiologic response to anesthesia from is presented from a cohort of sentinel animals. As in experimental protocols, mouse pups on postnatal day 18 (P18) were induced with Isoflurane 3% in oxygen until loss of righting reflex, and anesthesia was maintained at 1.5% in oxygen for 4h while the animals were spontaneously ventilating. Heart rate, oxyhemoglobin saturation, and skin surface temperature were measured with the Kent Scientific PhysioSuite hourly, and values obtained throughout a given hour were aver- aged (T1A). Data are presented in T1A as the mean ± SEM (n = 4 readings taken in 6 sentinel animals). At the end of the protocol animals were sacrificed, and blood samples were obtained by attempted cannulation of the left ventricle. Due to technical limitations we were not able to obtain an arterial sample for all animals. The value for partial pressure of oxygen for arterial samples is shown as is the blood glucose concentration for all samples (T1B). Underlying data in S1 Data under Fig 3A–3G. Underlying data in S1 Data under Fig S1T. (TIF)
+
+S1 Data. Source data. Cited in figure legends in manuscript. (XLSX)
+
+Acknowledgments
+
+We would like to acknowledge the helpful contributions of Sunu Kim (technical assistance) and Allan Gottschalk (critical commentary).
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+14 / 18
+
+Anesthetic toxicity and mTOR
+
+Author Contributions
+
+Conceptualization: Yun Kyoung Ryu, Roger A. Johns, Hongjun Song, Guo-Li Ming, C. David
+
+Mintz.
+
+Funding acquisition: C. David Mintz.
+
+Investigation: Eunchai Kang, Danye Jiang, Yun Kyoung Ryu, Sanghee Lim, Minhye Kwak,
+
+Christy D. Gray, Michael Xu, Jun H. Choi, Sue Junn, Jieun Kim, C. David Mintz.
+
+Methodology: Eunchai Kang, Yun Kyoung Ryu, Hongjun Song, Guo-Li Ming, C. David
+
+Mintz.
+
+Project administration: C. David Mintz.
+
+Resources: Roger A. Johns, Hongjun Song, Guo-Li Ming, C. David Mintz.
+
+Supervision: Eunchai Kang, Yun Kyoung Ryu, Roger A. Johns, Hongjun Song, C. David
+
+Mintz.
+
+Visualization: C. David Mintz.
+
+Writing – original draft: Eunchai Kang, Danye Jiang, Christy D. Gray, Michele Schaefer, C.
+
+David Mintz.
+
+Writing – review & editing: Michael Xu, Jing Xu, Michele Schaefer, Roger A. Johns, Hongjun
+
+Song, C. David Mintz.
+
+References 1. DiMaggio C, Sun LS, Kakavouli A, Byrne MW, Li G. A retrospective cohort study of the association of
+
+anesthesia and hernia repair surgery with behavioral and developmental disorders in young children. J Neurosurg Anesthesiol. 2009; 21(4): 286–291. https://doi.org/10.1097/ANA.0b013e3181a71f11 PMID: 19955889
+
+2.
+
+Ing C, Dimaggio C, Whitehouse A, Hegarty MK, Brady J, von Ungern-Sternberg B, et al. Long-term Dif- ferences in Language and Cognitive Function After Childhood Exposure to Anesthesia. Pediatrics. 2012 Aug; peds.2011-3822; https://doi.org/10.1542/peds.2011-3822 PMID: 22908104
+
+3. Wilder RT, Flick RP, Sprung J, Katusic SK, Barbaresi WJ, Mickelson C, et al. Early exposure to anes- thesia and learning disabilities in a population-based birth cohort. Anesthesiology. 2009; 110(4): 796– 804. https://doi.org/10.1097/01.anes.0000344728.34332.5d PMID: 19293700
+
+4. Erasso DM, Chaparro RE, Quiroga Del Rio CE, Karlnoski R, Camporesi EM, Saporta S. Quantitative assessment of new cell proliferation in the dentate gyrus and learning after isoflurane or propofol anes- thesia in young and aged rats. Brain Res. 2012; 1441: 38–46. https://doi.org/10.1016/j.brainres.2011. 11.025 PMID: 22297171
+
+5. Huang L, Cichon J, Ninan I, Yang G. Post-anesthesia AMPA receptor potentiation prevents anesthesia- induced learning and synaptic deficits. Sci Transl Med. 2016; 8 (344): 344ra385.
+
+6.
+
+Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, et al. Early expo- sure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci. 2003; 23(3): 876–882. PMID: 12574416
+
+7.
+
+Lee BH, Chan JT, Hazarika O, Vutskits L, Sall JW. Early exposure to volatile anesthetics impairs long- term associative learning and recognition memory. PLoS ONE. 2014;9(8): e105340. https://doi.org/10. 1371/journal.pone.0105340 PMID: 25165850
+
+8.
+
+Levin ED, Uemura E, Bowman RE. Neurobehavioral toxicology of halothane in rats. Neurotoxicol Tera- tol. 1991; 13(4): 461–470. PMID: 1921926
+
+9. Ramage TM, Chang FL, Shih J, Alvi RS, Quitoriano GR, Rau V, et al. Distinct long-term neurocognitive outcomes after equipotent sevoflurane or isoflurane anaesthesia in immature rats. Br J Anaesth. 2013; 110 Suppl 1: i39–46.
+
+10. Satomoto M, Satoh Y, Terui K, Miyao H, Takishima K, Ito M, et al. Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice. Anesthesiology. 2009 Mar; 110(3): 628–637. https://doi.org/10.1097/ALN.0b013e3181974fa2 PMID: 19212262
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+15 / 18
+
+Anesthetic toxicity and mTOR
+
+11. Shih J, May LD, Gonzalez HE, Lee EW, Alvi RS, Sall JW, et al. Delayed environmental enrichment reverses sevoflurane-induced memory impairment in rats. Anesthesiology. 2012 Mar; 116(3): 586– 602. https://doi.org/10.1097/ALN.0b013e318247564d PMID: 22354242
+
+12.
+
+Zhu C, Gao J, Karlsson N, Li Q, Zhang Y, Huang Z, et al. Isoflurane anesthesia induced persistent, pro- gressive memory impairment, caused a loss of neural stem cells, and reduced neurogenesis in young, but not adult, rodents. J Cereb Blood Flow Metab. 2010 May; 30(5): 1017–1030. https://doi.org/10. 1038/jcbfm.2009.274 PMID: 20068576
+
+13. Davidson AJ, Disma N, de Graaff JC, Withington DE, Dorris L, Bell G, et al. Neurodevelopmental out- come at 2 years of age after general anaesthesia and awake-regional anaesthesia in infancy (GAS): an international multicentre, randomised controlled trial. Lancet. 2016; 387(10015): 239–250. https://doi. org/10.1016/S0140-6736(15)00608-X PMID: 26507180
+
+14. Sun LS, Li G, Miller TL, Salorio C, Byrne MW, Bellinger DC; et al. Association Between a Single General Anesthesia Exposure Before Age 36 Months and Neurocognitive Outcomes in Later Childhood. JAMA. 2016 Jun; 315(21): 2312–2320. https://doi.org/10.1001/jama.2016.6967 PMID: 27272582
+
+15. Raper J, Alvarado MC, Murphy KL, Baxter MG. Multiple Anesthetic Exposure in Infant Monkeys Alters Emotional Reactivity to an Acute Stressor. Anesthesiology. 2015 Nov; 123(5): 1084–1092. https://doi. org/10.1097/ALN.0000000000000851 PMID: 26313293
+
+16. Hays SR, Deshpande JK. Newly postulated neurodevelopmental risks of pediatric anesthesia: theories that could rock our world. J Urol. 2013 Apr; 189(4): 1222–1228. https://doi.org/10.1016/j.juro.2012.11. 090 PMID: 23178900
+
+17. Kuehn BM. FDA considers data on potential risks of anesthesia use in infants, children. JAMA. 2011 May; 305(17): 1749–1750, 1753. https://doi.org/10.1001/jama.2011.546 PMID: 21540413
+
+18. Rappaport BA, Suresh S, Hertz S, Evers AS, Orser BA. Anesthetic neurotoxicity—clinical implications of animal models. N Engl J Med. 2015 Feb; 372: 796–797. https://doi.org/10.1056/NEJMp1414786 PMID: 25714157
+
+19.
+
+Istaphanous GK, Ward CG, Nan X, Hughes EA, McCann JC, McAuliffe JJ, et al. Characterization and quantification of isoflurane-induced developmental apoptotic cell death in mouse cerebral cortex. Anesth Analg. 2013 Apr; 116(4): 845–854. https://doi.org/10.1213/ANE.0b013e318281e988 PMID: 23460572
+
+20. Wagner M, Ryu YK, Smith SC, Mintz CD. Review: effects of anesthetics on brain circuit formation. J Neurosurg Anesthesiol. 2014 Oct; 26(4): 358–362. https://doi.org/10.1097/ANA.0000000000000118 PMID: 25144504
+
+21.
+
+Tau GZ, Peterson BS. Normal development of brain circuits. Neuropsychopharmacology. 2010 Jan; 35 (1): 147–168. https://doi.org/10.1038/npp.2009.115 PMID: 19794405
+
+22.
+
+Lefebvre JL, Sanes JR, Kay JN. Development of Dendritic Form and Function. Annu Rev Cell Dev Biol. 2015; 31: 741–777. https://doi.org/10.1146/annurev-cellbio-100913-013020 PMID: 26422333
+
+23. Kang E, Berg DA, Furmanski O, Jackson WM, Ryu YK, Gray CD, et al. Neurogenesis and developmen- tal anesthetic neurotoxicity. Neurotoxicol Teratol. 2017 Mar; 60:33–39. https://doi.org/10.1016/j.ntt. 2016.10.001 PMID: 27751818
+
+24. Ge S, Goh EL, Sailor KA, Kitabatake Y, Ming GL, Song H. GABA regulates synaptic integration of newly generated neurons in the adult brain. Nature. 2006 Feb; 439(7076): 589–593. https://doi.org/10. 1038/nature04404 PMID: 16341203
+
+25. Dolen G, Bear MF. Fragile x syndrome and autism: from disease model to therapeutic targets. J Neuro- dev Disord. 2009 Jun; 1(2): 133–140. https://doi.org/10.1007/s11689-009-9015-x PMID: 21547712
+
+26.
+
+Takei N, Nawa H. mTOR signaling and its roles in normal and abnormal brain development. Front Mol Neurosci. 2014 Apr; 7: 28. https://doi.org/10.3389/fnmol.2014.00028 PMID: 24795562
+
+27. Cameron CB, Robinson S, Gregory GA. The minimum anesthetic concentration of isoflurane in chil- dren. Anesth Analg. 1984 Apr; 63(4): 418–420. PMID: 6703367
+
+28. Mintz CD, Smith SC, Barrett KM, Benson DL. Anesthetics interfere with the polarization of developing cortical neurons. J Neurosurg Anesthesiol. 2012; 24(4): 368–375. https://doi.org/10.1097/ANA. 0b013e31826a03a6 PMID: 23085784
+
+29. Ryu YK, Khan S, Smith SC, Mintz CD. Isoflurane impairs the capacity of astrocytes to support neuronal development in a mouse dissociated coculture model. J Neurosurg Anesthesiol. 2014 Oct; 26(4): 363– 368. https://doi.org/10.1097/ANA.0000000000000119 PMID: 25191957 30. Jan YN, Jan LY. The control of dendrite development. Neuron. 2003 Oct; 40(2): 229–242. PMID: 14556706
+
+31. Vutskits L, Gascon E, Tassonyi E, Kiss JZ. Clinically relevant concentrations of propofol but not midazo- lam alter in vitro dendritic development of isolated gamma-aminobutyric acid-positive interneurons. Anesthesiology. 2005 May; 102(5): 970–976. PMID: 15851884
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+16 / 18
+
+Anesthetic toxicity and mTOR
+
+32. Amaral DG, Scharfman HE, Lavenex P. The dentate gyrus: fundamental neuroanatomical organization (dentate gyrus for dummies). Prog Brain Res. 2007; 163: 3–22. https://doi.org/10.1016/S0079-6123 (07)63001-5 PMID: 17765709
+
+33. Briner A, De Roo M, Dayer A, Muller D, Habre W, Vutskits L. Volatile anesthetics rapidly increase den- dritic spine density in the rat medial prefrontal cortex during synaptogenesis. Anesthesiology. 2010 Mar; 112(3): 546–556. https://doi.org/10.1097/ALN.0b013e3181cd7942 PMID: 20124985
+
+34.
+
+Jan YN, Jan LY. Branching out: mechanisms of dendritic arborization. Nat Rev Neurosci. 2010 May; 11(5): 316–328. https://doi.org/10.1038/nrn2836 PMID: 20404840
+
+35. Galvez R, Smith RL, Greenough WT. Olfactory bulb mitral cell dendritic pruning abnormalities in a mouse model of the Fragile-X mental retardation syndrome: further support for FMRP’s involvement in dendritic development. Brain Res Dev Brain Res. 2005 Jun; 157(2): 214–216. https://doi.org/10.1016/j. devbrainres.2005.03.010 PMID: 15878626
+
+36.
+
+Jiang M, Ash RT, Baker SA, Suter B, Ferguson A, Park J, et al. Dendritic arborization and spine dynam- ics are abnormal in the mouse model of MECP2 duplication syndrome. J Neurosci. 2013 Dec; 33(50): 19518–19533. https://doi.org/10.1523/JNEUROSCI.1745-13.2013 PMID: 24336718
+
+37.
+
+Zhou M, Li W, Huang S, Song J, Kim JY, Tian X, et al. mTOR Inhibition ameliorates cognitive and affec- tive deficits caused by Disc1 knockdown in adult-born dentate granule neurons. Neuron. 2013 Feb; 77(4): 647–654. https://doi.org/10.1016/j.neuron.2012.12.033 PMID: 23439118
+
+38. Grienberger C, Chen X, Konnerth A. Dendritic function in vivo. Trends Neurosci. 2015 Jan; 38(1): 45–
+
+54. https://doi.org/10.1016/j.tins.2014.11.002 PMID: 25432423
+
+39. Bourne J, Harris KM. Do thin spines learn to be mushroom spines that remember? Curr Opin Neurobiol. 2007 Jun; 17(3): 381–386. https://doi.org/10.1016/j.conb.2007.04.009 PMID: 17498943
+
+40. Amrock LG, Starner ML, Murphy KL, Baxter MG. Long-term effects of single or multiple neonatal sevo- flurane exposures on rat hippocampal ultrastructure. Anesthesiology. 2015 Jan; 122(1): 87–95. https:// doi.org/10.1097/ALN.0000000000000477 PMID: 25289484 Lunardi N, Hucklenbruch C, Latham JR, Scarpa J, Jevtovic-Todorovic V. Isoflurane impairs immature astroglia development in vitro: the role of actin cytoskeleton. J Neuropathol Exp Neurol. 2011 Apr; 70(4): 281–291. https://doi.org/10.1097/NEN.0b013e31821284e9 PMID: 21412172
+
+40. Amrock LG, Starner ML, Murphy KL, Baxter MG. Long-term effects of single or multiple neonatal sevo- flurane exposures on rat hippocampal ultrastructure. Anesthesiology. 2015 Jan; 122(1): 87–95. https:// doi.org/10.1097/ALN.0000000000000477 PMID: 25289484 Lunardi N, Hucklenbruch C, Latham JR, Scarpa J, Jevtovic-Todorovic V. Isoflurane impairs immature astroglia development in vitro: the role of actin cytoskeleton. J Neuropathol Exp Neurol. 2011 Apr; 70(4): 281–291. https://doi.org/10.1097/NEN.0b013e31821284e9 PMID: 21412172
+
+42. Semple BD, Blomgren K, Gimlin K, Ferriero DM, Noble-Haeusslein LJ. Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species. Prog Neu- robiol. 2013; 106–107: 1–16. https://doi.org/10.1016/j.pneurobio.2013.04.001 PMID: 23583307
+
+43. Briner A, Nikonenko I, De Roo M, Dayer A, Muller D, Habre W, et al. Developmental Stage-dependent persistent impact of propofol anesthesia on dendritic spines in the rat medial prefrontal cortex. Anesthe- siology. 2010; 11: 282–293.
+
+44. Costa-Mattioli M, Monteggia LM. mTOR complexes in neurodevelopmental and neuropsychiatric disor- ders. Nat Neurosci. 2013; 16: 1537–1543. https://doi.org/10.1038/nn.3546 PMID: 24165680
+
+45.
+
+Tan H, Li CL, Zhang L, Yang ZJ, Zhao X, Wang YW. Sevoflurane inhibits the phosphorylation of ribo- somal protein S6 in neonatal rat brain. Int J Clin Exp Med. 2015; 8(9): 14816–14826. PMID: 26628963
+
+46.
+
+Lipton JO, Sahin M. The neurology of mTOR. Neuron. 2014 Oct; 84: 275–291. https://doi.org/10.1016/ j.neuron.2014.09.034 PMID: 25374355
+
+47. Switon K, Kotulska K, Janusz-Kaminska A, Zmorzynska J, Jaworski J. Molecular neurobiology of mTOR. Neuroscience. 2017 Jan; 341: 112–153. https://doi.org/10.1016/j.neuroscience.2016.11.017 PMID: 27889578
+
+48. Swiech L, Perycz M, Malik A, Jaworski J. Role of mTOR in physiology and pathology of the nervous sys- tem. Biochim Biophys Acta. 2008 Jan; 1784(1): 116–132. https://doi.org/10.1016/j.bbapap.2007.08. 015 PMID: 17913600
+
+49. Duan X, Chang JH, Ge S, Faulkner RL, Kim JY, Kitabatake Y, et al. Disrupted-In-Schizophrenia 1 regu- lates integration of newly generated neurons in the adult brain. Cell. 2007 Sept; 130(6): 1146–1158. https://doi.org/10.1016/j.cell.2007.07.010 PMID: 17825401
+
+50. Kumar V, Zhang MX, Swank MW, Kunz J, Wu GY. Regulation of dendritic morphogenesis by Ras- PI3K-Akt-mTOR and Ras-MAPK signaling pathways. J Neurosci. 2005 Dec; 25 (4): 11288–11299.
+
+51. Pun RY, Rolle IJ, Lasarge CL, Hosford BE, Rosen JM, Uhl JD; et al. Excessive activation of mTOR in postnatally generated granule cells is sufficient to cause epilepsy. Neuron. 2012 Sep; 75(6): 1022– 1034. https://doi.org/10.1016/j.neuron.2012.08.002 PMID: 22998871
+
+52. Altman J, Bayer SA. Migration and distribution of two populations of hippocampal granule cell precur- sors during the perinatal and postnatal periods. J Comp Neurol. 1990 Nov; 301(3): 365–381. https:// doi.org/10.1002/cne.903010304 PMID: 2262596
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+17 / 18
+
+Anesthetic toxicity and mTOR
+
+53. Rakic P, Nowakowski RS. The time of origin of neurons in the hippocampal region of the rhesus mon- key. J Comp Neurol. 1981 Feb; 196(1): 99–128. https://doi.org/10.1002/cne.901960109 PMID: 7204668
+
+54. Sato A. mTOR, a Potential Target to Treat Autism Spectrum Disorder. CNS Neurol Disord Drug Tar- gets. 2016; 15(5): 533–543. https://doi.org/10.2174/1871527315666160413120638 PMID: 27071790
+
+55. Kang E, Burdick KE, Kim JY, Duan X, Guo JU, Sailor KA, et al. Interaction between FEZ1 and DISC1 in regulation of neuronal development and risk for schizophrenia. Neuron. 2011 Nov; 72(4): 559–571. https://doi.org/10.1016/j.neuron.2011.09.032 PMID: 22099459
+
+56.
+
+Zhang H, Kang E, Wang Y, Yang C, Yu H, Wang Q, et al. Brain-specific Crmp2 deletion leads to neuro- nal development deficits and behavioural impairments in mice. Nat Commun. 2016; 7: https://doi.org/ 10.1038/ncomms11773 PMID: 27249678
+
+57. Song J, Sun J, Moss J, Wen Z, Sun GJ, Hsu D, et al. Parvalbumin interneurons mediate neuronal cir- cuitry-neurogenesis coupling in the adult hippocampus. Nat Neurosci. 2013 Dec; 16(12): 1728–1730. https://doi.org/10.1038/nn.3572 PMID: 24212671
+
+58. Kim JY, Duan X, Liu CY, Jang MH, Guo JU, Pow-anpongkul N, et al. DISC1 regulates new neuron development in the adult brain via modulation of AKT-mTOR signaling through KIAA1212. Neuron. 2009 Sep; 63(6): 761–773. https://doi.org/10.1016/j.neuron.2009.08.008 PMID: 19778506 59. Jang MH, Bonaguidi MA, Kitabatake Y, Sun J, Song J, Kang E, et al. Secreted frizzled-related protein 3 regulates activity-dependent adult hippocampal neurogenesis. Cell Stem Cell. 2013 Feb; 12(2): 215– 223. https://doi.org/10.1016/j.stem.2012.11.021 PMID: 23395446
+
+60. Kim JY, Liu CY, Zhang F, Duan X, Wen Z, Song J, et al. Interplay between DISC1 and GABA signaling regulates neurogenesis in mice and risk for schizophrenia. Cell. 2012 Mar; 148(5): 1051–1064. https:// doi.org/10.1016/j.cell.2011.12.037 PMID: 22385968
+
+61. Mao Y, Ge X, Frank CL, Madison JM, Koehler AN, Doud MK, et al. Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell. 2009 Mar; 136(6): 1017–1031. https://doi.org/10.1016/j.cell.2008.12.044 PMID: 19303846
+
+PLOS Biology | https://doi.org/10.1371/journal.pbio.2001246 July 6, 2017
+
+18 / 18

new_pdfs/10.1371_journal.pone.0070645.txt

@@ -0,0 +1,449 @@
+Perinatal Supplementation with Omega-3 Polyunsaturated Fatty Acids Improves Sevoflurane- Induced Neurodegeneration and Memory Impairment in Neonatal Rats
+
+Xi Lei1, Wenting Zhang2, Tengyuan Liu3, Hongyan Xiao1, Weimin Liang1, Weiliang Xia3*, Jun Zhang1*
+
+1 Department of Anesthesiology, Huashan Hospital, Fudan University, Shanghai, P. R. China, 2 National Key Laboratory of Medical neurobiology, Fudan University, Shanghai, P. R. China, 3 School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiaotong University, Shanghai, P. R. China
+
+Abstract
+
+Objectives: To investigate if perinatal Omega-3 polyunsaturated fatty acids (n-3 PUFAs) supplementation can improve sevoflurane-induced neurotoxicity and cognitive impairment in neonatal rats.
+
+Methods:Female Sprague-Dawley rats (n = 3 each group) were treated with or without an n-3 PUFAs (fish oil) enriched diet from the second day of pregnancy to 14 days after parturition. The offspring rats (P7) were treated with six hours sevoflurane administration (one group without sevoflurane/prenatal n-3 PUFAs supplement as control). The 5- bromodeoxyuridine (Brdu) was injected intraperitoneally during and after sevoflurane anesthesia to assess dentate gyrus (DG) progenitor proliferation. Brain tissues were harvested and subjected to Western blot and immunohistochemistry respectively. Morris water maze spatial reference memory, fear conditioning, and Morris water maze memory consolidation were tested at P35, P63 and P70 (n = 9), respectively.
+
+Results:Six hours 3% sevoflurane administration increased the cleaved caspase-3 in the thalamus, parietal cortex but not hippocampus of neonatal rat brain. Sevoflurane anesthesia also decreased the neuronal precursor proliferation of DG in rat hippocampus. However, perinatal n-3 PUFAs supplement could decrease the cleaved caspase-3 in the cerebral cortex of neonatal rats, and mitigate the decrease in neuronal proliferation in their hippocampus. In neurobehavioral studies, compared with control and n-3 PUFAs supplement groups, we did not find significant spatial cognitive deficit and early long-term memory impairment in sevoflurane anesthetized neonatal rats at their adulthood. However, sevoflurane could impair the immediate fear response and working memory and short-term memory. And n-3 PUFAs could improve neurocognitive function in later life after neonatal sevoflurane exposure.
+
+Conclusion: Our study demonstrated that neonatal exposure to prolonged sevoflurane could impair the immediate fear response, working memory and short-term memory of rats at their adulthood, which may through inducing neuronal apoptosis and decreasing neurogenesis. However, these sevoflurane-induced unfavorable neuronal effects can be mitigated by perinatal n-3 PUFAs supplementation.
+
+Citation: Lei X, Zhang W, Liu T, Xiao H, Liang W, et al. (2013) Perinatal Supplementation with Omega-3 Polyunsaturated Fatty Acids Improves Sevoflurane- Induced Neurodegeneration and Memory Impairment in Neonatal Rats. PLoS ONE 8(8): e70645. doi:10.1371/journal.pone.0070645
+
+Editor: Georges Chapouthier, Universite´ Pierre et Marie Curie, France
+
+Received April 25, 2013; Accepted June 20, 2013; Published August 13, 2013
+
+Copyright: (cid:2) 2013 Lei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
+
+Funding: This work was supported by Natural Science Foundation of China (to Jun Zhang, Grant No. 81171020). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
+
+Competing Interests: The authors have declared that no competing interests exist.
+
+E-mail: weiliangxia@gmail.com (WX); snapzhang@yahoo.com.cn (JZ)
+
+Introduction
+
+Sevoflurane is one of the most frequently used volatile general anesthetic agents used during surgical procedures. It is especially useful for pediatric anesthesia because sevoflurane allows rapid induction and recovery and is less irritating to the airway than other inhaled anesthetics [1]. Recent evidence demonstrates that volatile anesthetics can induce neuronal apoptosis [2,3], affect in vitro and in vivo [4], and disturb long-term neurogenesis neurocognitive function in 7-day-old rats [5]. Although several studies report sevoflurane is less cytotoxic than isoflurane and desflurane, sevoflurane exposure in neonates reportedly increases
+
+risk for neurodevelopmental impairments in animal models [6–9]. Given its clinical relevance and potential for unfavorable outcomes in pediatric anesthesia, we sought to substantiate sevoflurane’s putative neurotoxic effects, and develop a strategy to prevent sevoflurane-induced neurodevelopmental impairment in neonatal rats.
+
+Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are essential dietary nutrients that play critical roles in brain development and function. Their contributions to learning and memory are well documented with maternal n-3 PUFA supplementation during gestation [10]. Prenatal n-3 PUFAs supplementation confers long- term neuroprotection against neonatal hypoxia-ischemic injury via
+
+PLOS ONE | www.plosone.org
+
+1
+
+August 2013 | Volume 8 |
+
+Issue 8 | e70645
+
+N-3 PUFAs Improve Neurotoxicity of Sevoflurane
+
+anti-inflammatory actions [11], and attenuates hyperoxia-induced neuronal apoptosis in the developing brain [12]. Conversely, n-3 PUFAs deficiency altered neurogenesis in embryonic [13] and adult [14] rat brains. They also may exert effects in human neurodegenerative conditions. In a randomized double-blind trial, n-3 PUFAs administration demonstrated positive effects in a small group of Alzheimer’s patients [15]. The effect of n-3 PUFAs on postnatal anesthetic-induced neurotoxicity in the developing brain, however, has never been studied. We hypothesized that n-3 PUFAs supplementation during pregnancy and lactation could protect against neurotoxicity in neonatal rats exposed to sevo- flurane anesthesia.
+
+Materials and Methods
+
+Animal/Anesthesia treatment
+
+The rats used in the present study were obtained from the Animal Care Center of Fudan University. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee, Fudan University. One-day pregnant female Sprague Dawley rats (weight 220–250 g) were randomly assigned to one of the three groups: control, sevoflurane, or sevoflurane with n-3 PUFAs (n = 3 per group). Fish oil, the main source of n-3 PUFAs (Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA)), was extracted from the capsule (1000 mg/capsule that containing 180 mg EPA and 120 mg DHA, Puritan’s Pride, Bohemia, NY, USA) and added to food. Pregnant dams in the sevoflurane and control groups were fed a regular laboratory rodent diet with a low n-3 PUFAs concentration (0.5% of total fatty acid), whereas the sevoflurane with n-3 PUFAs group were fed the same diet, but supplemented with n-3 PUFAs (15 mg fish oil/g regular diet) from day 2 of pregnancy to 14 days after parturition. Dams were given free access to food and water, and dams for all groups were kept under identical housing conditions with a 12-h light cycle. On postnatal day 7 (P7), in the sevoflurane and the rat pups sevoflurane with n-3 PUFAs groups received sevoflurane anesthe- sia.
+
+P7 rats were placed in a sealed box ventilated with 3% sevoflurane in 60% oxygen and treated for 6 h. The temperature in the sealed box was maintained at 33–35uC. The total survival percentage of P7 rats after 6-h anesthesia was 88.4%; the likely cause of death was respiration depression. After anesthesia, the pups were returned to the dams. Control rat pups were placed in the same box without sevoflurane exposure and under identical experimental conditions. The flow chart for the experimental protocol is summarized in Figure 1.
+
+Blood gas analysis
+
+gases. Blood was percutaneously aspirated from the left cardiac ventricle after 0, 2, 4, and 6 h of anesthesia (n = 3 per time point). From these samples, we measured partial pressures of carbon dioxide and oxygen, pH, and blood lactate and glucose levels with a Radiometer ABL 800 blood gas analyzer (Radiometer, Copenhagen, Denmark).
+
+BrdU injection
+
+sevoflurane affects progenitor cell proliferation in the S-phase of the cell cycle, bromodeoxyuridine ((+)-59-bromo-29-deoxyuridine [BrdU]; 97%; Sigma-Aldrich, St. in 0.9% sterile saline solution was injected Louis, MO, USA) intraperitoneally using the procedure described by Wojtowicz [16]. The first dose (150 mg/kg) was administered immediately before sevoflurane treatment, and the three subsequent injections (50 mg/kg BrdU) were given at 24-h intervals following sevo- flurane anesthesia.
+
+To determine whether
+
+Tissue preparation and immunohistochemistry
+
+Animals were deeply anesthetized with chloral hydrate and then transcardially perfused with 0.9% saline followed by 4% parafor- maldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4. The brains were removed, postfixed overnight in 4% paraformalde- hyde/PBS, and placed in 30% sucrose until they sank in the solution. Coronal sections (30 mm) were cut on a microtome (Leica CM1900 UV, Wetzlar, Germany) and every sixth section was stored in 30% sucrose containing 30% ethylene glycol to stain BrdU or cleaved caspase-3.
+
+For immunocytochemical detection of BrdU-labeled nuclei, DNA was denatured to expose the antigen for incubation with 2 N hydrochloric acid for 30 min at 37uC, followed by neutralization with two 10-min incubation periods in 0.5 M boric acid (pH 8.5) at room temperature (RT). Sections were subjected to three 10- min washes in PBS with 0.3% Triton-X with 10 min between each wash. Nonspecific epitopes were blocked with 1% serum for 30 min at RT, and were incubated overnight at 4uC with either BrdU (1:100; BD Pharmingen, Franklin Lakes, NJ, USA) or cleaved caspase-3 (1:1,000; Cell Signaling, Danvers, MA, USA) antibody in PBS and 1% serum. On day 2, the sections were incubated with the appropriate secondary fluorescent antibodies (Alexa Fluor 488, 1:200; Invitrogen, Carlsbad, CA, USA) for 2 h at RT, in PBS. Nuclear counterstaining was performed with 49,6-diamidino-2-phenylin- dole (1:500; Beyotime Institute of Biotechnology, Haimen, China), which was followed by mounting and coverslipping with an aqueous mounting medium. Images were acquired with a microscope (Leica DM2500). BrdU- or cleaved caspase-3-positive cells were counted in a blinded manner at 620 magnification [17]. Questionable structures were excluded from the count if their identification remained uncertain under 640 magnification.
+
+followed by three 5-min washes
+
+Twelve naı¨ve P7 rats that that did not participate in other experiments were used to assess the effect of sevoflurane on blood
+
+Figure 1. Schematic timeline of the experimental procedure. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) supplementation began in dams from pregnancy day 2 to 14 days after parturition or until the day when the brains of their offspring were harvested. The neonatal rats were exposed to 3% sevoflurane (Sevo) for 6 h at their seventh day (P7). The behavioral tests including Morris water maze spatial reference memory, fear conditioning, and Morris water maze memory consolidation on P35–38, P63–64, and P70–79, respectively. P = postnatal day. doi:10.1371/journal.pone.0070645.g001
+
+PLOS ONE | www.plosone.org
+
+2
+
+August 2013 | Volume 8 |
+
+Issue 8 | e70645
+
+Western blot analysis
+
+The cerebral cortex, thalamus, and hippocampus were harvest- ed 18 h after sevoflurane treatment. The brain tissues were homogenized in RIPA buffer (Millipore, Temecula, CA, USA) containing complete protease inhibitor cocktail and 2 mM phenylmethylsulfonyl fluoride. The lysates were collected and centrifuged at 12,000 rpm for 30 min at 4uC. After the protein samples were quantified using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA), 60 mg of each sample was electrophoresed through a 14% sodium dodecyl sulfate-polyacryl- amide gel and wet electrotransferred to 0.45-mm nitrocellulose membranes (Millipore). The blots were incubated overnight at 4uC with a polyclonal anti-cleaved caspase-3 antibody, and then incubated with a rabbit anti-mouse polyclonal horseradish peroxidase-conjugated secondary antibody (1:5,000; Epitomics, Hangzhou, Zhejiang Province, China) at RT for 1 h. Protein signals were detected using an enhanced chemiluminescence detection system (Pierce Biotechnology). A b-actin antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to normalize sample loading and transfer. Band intensities were densitometrically quantified using Gel-Pro Analyzer (Media Cybernetics, Bethesda, MD, USA).
+
+Neurobehavioral tests
+
+in the neurobehavioral tests to exclude estrogen influences on neurocog- nitive evaluations. The water maze setup in spatial reference
+
+We used only male offspring (n = 9 per group)
+
+Figure 2. Morris water maze setup. Numbers: platform location; Letters: drop location. In the spatial reference memory task, the platform location is in the middle of one of four virtual quadrants (2). In the probe training session, the rat are released from four pseudor- andomly assigned points (D,E,H and G) which provide two short and two medium swims to the platform location per session. In the probe test session, the drop location (F) is at the opposite of original platform. In the memory consolidation task, the platform location are quarter-way between the center of the maze and the wall of the tank on the border of two quadrants (1) or within a quadrant (4), or in the center of the maze (3) or in the middle of one of four virtual quadrants (2). The drop location was pseudorandomly varied to incorporate one short, one medium, and one long swim to platform. doi:10.1371/journal.pone.0070645.g002
+
+PLOS ONE | www.plosone.org
+
+3
+
+N-3 PUFAs Improve Neurotoxicity of Sevoflurane
+
+memory task and memory consolidation task was shown in Figure 2.
+
+Morris water maze spatial reference memory. Probe training: Rats trained for 4 consecutive days (postnatal days 35– 38, P35–38) in the Morris water maze following treatment with a vehicle or 3% sevoflurane for 6 h. A platform (10.3-cm diameter) was submerged in a circular pool (180-cm diameter, 50-cm depth) filled with warm (23–25uC) opaque water. Rats performed two training sessions each day. In each session, rats performed four trials in which they were released from one of four pseudor- andomly assigned release points while facing the tank wall. This provided two short and two medium swims per session. Animals were allowed 60 s to locate the hidden platform, and if they failed to find the hidden platform in the allotted time, the investigator guided the animal to the platform. In either case, the rats were removed from the platform after 15 s. Training sessions were conducted until the rats could locate the hidden platform in less than 15 s in at least five sessions (average time per session). All trials were videotaped, and rat swim paths were recorded with ANY-maze video tracking system (Stoelting Co., Wood Dale, IL, USA), which allowed us to measure the time taken (latency) to find the platform(s), as well as other behavioral information obtained during the spatial reference memory test. The animals were dried and placed beneath a heating lamp after completing each test.
+
+test: A probe trial was performed with the platform removed from the tank to assess memory retention for the hidden platform location. Probe trials were administered 1 day after the last training session (P38). During the 60-s probe trial, we determined the number of entries into the platform quadrant zone, the swimming speed (cm/s), the total distance (cm), and the in the target quadrant relative versus the other time spent quadrants.
+
+Probe
+
+Fear conditioning test. Rats underwent fear conditioning tests on postnatal days 63–64 (P63–64). Every time four rats randomly chosen from three groups were trained in each session. Rats were placed in plastic chambers with a grid floor constructed from 19 stainless steel bars (4-mm diameter, spaced every 16 mm). The floors were connected to a shock delivery system (Coulbourn, Whitehall, PA, USA), and electrical shocks were delivered through illuminated with the stainless overhead fluorescent bulbs, and a ventilation fan provided background noise (65 db). The training context was considered the appearance, odor, and texture of the environment (chamber and room) in which the rats were trained. After a 3-min baseline exploratory period, rats were presented with three auditory tones (2,000 Hz, 90 db) that were followed 1 min later by an electric shock (1 mA, 2 s). We quantified the rats’ fear response with freezing, which is an innate defensive fear response in rodents and a reliable measure of learned fear. Freezing was defined as the lack of movement, except for respiration. We examined rats in the fear condition test the day after they first received the electrical shock to determine whether they showed fear to the training context or the auditory tone. For the context test, rats were placed in the chamber where they were trained on the previous day. The rats remained in the chamber for 8 min, without an auditory tone or shock. For the tone test, rats were transported in groups to a context chamber with black boards covering the walls. Rats were allowed a 3-min exploratory period before three 30-s tones were played (2,000 Hz, 90 db, separated by 60 s). Rats were removed from the chamber 30 s after the tone presentation. The order of the context and tone tests was counterbalanced so that half of each treatment group first was tested for context and then for tone, whereas the other half of the treatment group was tested in the reverse order. FreezeView software (Coulbourn) was used to score
+
+steel bars. The chamber was
+
+August 2013 | Volume 8 |
+
+Issue 8 | e70645
+
+each animal’s freezing behavior separately for the training period and the context and tone tests, which were expressed as a percentage.
+
+Morris water maze memory consolidation. Working mem- ory (WM): On postnatal day 70 (P70), the testing room was rearranged by repositioning the water tank and adding new spatial cues. The platform was submerged 1.5 cm below the water surface in one of four designated platform positions. From P70 onward, one session was conducted per day. Each session began with a 60-s free swim (performance not scored) in which rats explored the maze, and was followed by a 1-min rest interval and three subsequent scored trials. Rats that found the platform during the free swim were allowed to rest on the platform for 15 s. Rats that failed to find the platform during the free swim were guided to the platform and remained there for 15 s. After the free swim, three trials were administered in which the rat was released from one of six pseudorandomly chosen locations that faced the tank wall. The platform location was identical for all animals in a session, but the drop location was pseudorandomly varied to incorporate one short, one medium, and one long swim. Training sessions were administered until the session average for finding the hidden platform was less than 15 s. The latency for reaching the platform was recorded by the ANY-maze video tracking system.
+
+Short-term memory (STM) and early long-term memory (ELTM): When the WM latencies of rats in task were plateaued on postnatal day 77 (P77), we increased the delay between the free swim and the subsequent trials. The delay was extended from 1 min on P77 to 1 h on postnatal day 78 (P78) to test STM, and then to 4 h on P79 to test ELTM. Performances on the last trial after the free swim on P77 (1-min delay), P78 (1-h delay), and P79 (4-h delay) were used as measures of WM, STM, and ELTM, respectively.
+
+Statistical methods
+
+All data are presented as mean 6 standard deviation. We performed two-tailed t to determine differences in cleaved caspase-3 immunohistochemistry and blood gas parameters between the control and sevoflurane groups. We used a one-way analysis of variance followed by Newman-Keuls post hoc tests to determine differences among groups for interactions between n-3 PUFAs or sevoflurane and cleaved caspase-3 activation, BrdU quantification, or neurobe- havioral tests. For all tests, p,0.05 was considered statistically significant.
+
+tests
+
+(assuming equal variances)
+
+Results
+
+Blood gas analysis
+
+We assessed blood gas and biochemical changes in P7 rats treated with continuous 3% sevoflurane in 60% oxygen for 0, 2, 4, or 6 h. The partial pressures of carbon dioxide and oxygen, pH, and blood glucose and lactate levels at each time point are shown in Figure 3. We observed that prolonged sevoflurane anesthesia caused hypercarbia, but not hypoxemia, which could be due to respiration depression.
+
+Neuronal apoptosis
+
+Caspase-3 is a ubiquitously distributed caspase, and its activation strongly suggests cellular apoptosis [18]. Eighteen hours showed after sevoflurane anesthesia induction, neonatal rats greater amounts of cleaved caspase-3 immunoreactivity in the (Figure 4K–M) parietal cortex (Figure 4A–E) and thalamus compared to controls. Our results sevoflurane dramatically increased the incidence of apoptosis in thalamic (median 46-fold increase compared with control). neurons
+
+indicate that
+
+PLOS ONE | www.plosone.org
+
+4
+
+N-3 PUFAs Improve Neurotoxicity of Sevoflurane
+
+Although sevoflurane treatment slightly increased apoptosis in cornu ammonis (CA) 1 and 3 hippocampal regions (Figure 4F–J), these changes were not significant.
+
+n-3 PUFAs attenuate sevoflurane-induced neuronal apoptosis
+
+We examined cortical extracts with western blots to verify activated caspase-3 immunofluorescence represented apoptosis, and to quantify the apoptotic response. Cleaved caspase-3 immunoblotting confirmed that 3% sevoflurane treatment for 6 h increased caspase-3 activity in the parietal cortex of neonatal supplementation significantly rat pups. Perinatal n-3 PUFAs ameliorated cleaved caspase-3 expression in the parietal cortex (Figure 5) of offspring rats that underwent sevoflurane anesthesia.
+
+n-3 PUFAs reverse sevoflurane-induced inhibition of hippocampal neuronal proliferation
+
+Newly generated BrdU-labeled cells were observed in the dentate gyrus (DG) subgranular zone following immunofluores- cence labeling. Sevoflurane decreased the number and fluores- cence intensity of BrdU-labeled cells in the DG compared with controls, but these changes were attenuated by perinatal n-3 PUFAs supplementation (Figure 6). These findings suggest that perinatal n-3 PUFAs supplementation can reverse sevoflurane- induced neuronal proliferation inhibition in neonatal rat hippo- campus.
+
+n-3 PUFAs improve neonatal sevoflurane-induced neurobehavioral deficits in adulthood
+
+Sevoflurane anesthesia and n-3 PUFAs supplementation did not affect escape latencies during the six probe training sessions in the Morris water maze (Figure 7A). Similarly, sevoflurane anesthesia did not affect the frequency required to cross the platform region (Figure 7B) or the swimming distance (Figure 7C) during the probe the trial. Finally, n-3 PUFAs supplementation did not affect sevoflurane-induced reduction in swimming speed (Figure 7D).
+
+In the fear conditioning training session, the postshock freezing response in rats with neonatal exposure to sevoflurane was significantly decreased compared with controls for shocks 1 and 2. Maternal n-3 PUFAs significantly increased postshock freezing in the offspring. Postshock freezing was similar across all groups for the third tone/shock pairing (Figure 7E). To assess the influence of neonatal exposure to sevoflurane on ELTM, rats underwent contextual/cued fear conditioning tests. In this paradigm, we failed to find any differences after one training day (Figure 7F–G). These results indicate that sevoflurane has no effect, and n-3 PUFAs did not further improve ELTM in the fear conditioning test.
+
+supplementation, however,
+
+Memory consolidation describes the transition from unstable memories to stable memories. At least four different stages of memory consolidation are distinguished, three of which are assessed here: WM (minutes), STM (minutes to hours), and ELTM (greater than 3 h). We did not assess remote long-term memory because this process requires delays between memory formation and recall that span weeks to months. In the 1-min delay training session, sevoflurane increased escape latencies in the sixth and eighth sessions, whereas supplementation decreased the escape latencies in sevoflurane-treated animals in the fourth, sixth, and eighth sessions (Figure 7H–I). In the 1-h delay session (session 9), sevoflurane-treated rats had significantly longer escape latencies than controls, and sevoflurane-treated rats pre-treated with fish oil had significantly lower escape latencies than sevoflurane-treated rats (Figure 7J). Sevoflurane anesthesia
+
+fish oil
+
+August 2013 | Volume 8 |
+
+Issue 8 | e70645
+
+N-3 PUFAs Improve Neurotoxicity of Sevoflurane
+
+Figure 3. Arterial blood gas and biochemical analysis. A: pH; B: PaCO2; C: PaO2; D: Blood lactate concentration; E: Blood glucose concentration. FiO2: fraction of inspired oxygen; PaCO2: partial pressure of carbon dioxide; PaO2: partial pressure of oxygen. Sevo treatment could decrease pH (A) due to hypercarbia (B) and increase the blood glucose concentration; n = 3 at each time. doi:10.1371/journal.pone.0070645.g003
+
+impaired performance in a spatial recognition memory task when 1-min and 1-h delays were introduced between memory encoding and memory retrieval, and perinatal n-3 PUFAs supplementation alleviated this deficit. These results indicate that sevoflurane can impair WM and STM, but n-3 PUFAs can alleviate these impairments. Interestingly, there was no significant difference among groups in escape latency at the 4-h delay session (session 10, Figure 7K), indicating that sevoflurane may not affect ELTM.
+
+Discussion
+
+Main findings
+
+The major findings in this study were as follows: (1) sevoflurane exposure induced neuronal apoptosis in rat pups, (2) sevoflurane exposure decreased hippocampal neuron proliferation in neonates, (3) sevoflurane exposure in neonates impaired STM two months later, and (4) perinatal n-3 PUFA supplementation protects neurons against all three of these sevoflurane-induced changes.
+
+neuronal apoptosis. Compared with littermate controls, rat pups ex- posed to 3% sevoflurane for 6 h on P7 (third trimester-equivalent in humans) showed increased apoptotic neurodegeneration in
+
+Effects
+
+of
+
+sevoflurane
+
+anesthesia
+
+on
+
+PLOS ONE | www.plosone.org
+
+5
+
+August 2013 | Volume 8 |
+
+Issue 8 | e70645
+
+PLOS ONE | www.plosone.org
+
+6
+
+N-3 PUFAs Improve Neurotoxicity of Sevoflurane
+
+August 2013 | Volume 8 |
+
+Issue 8 | e70645
+
+N-3 PUFAs Improve Neurotoxicity of Sevoflurane
+
+Figure 4. The effects of neonatal sevoflurane exposure on caspase-3 expression. Immunofluorescence revealed the effects of the 6-h 3% sevoflurane exposure on cleaved caspase-3 expression in neonatal rat brains at P7 (n = 3 in each group). The photomicrographs (56) of cleaved caspase-3 in the parietal cortex in control group (A) and in the Sevo group (B); C: The photomicrographs (56with 206inset) of cleaved caspase-3 in the parietal cortex in control group (C) and in the Sevo group (D); Quantification of cleaved caspase-3 in parietal cortex (control vs. Sevo, p = 0.0389) (E); The photomicrographs (106) of cleaved caspase-3 in the CA1 (F), CA3 (H) of controls and CA1 (G), CA3 (I) in Sevo group; Quantification of cleaved caspase-3 in hippocampus region (control vs. Sevo, NS) (J); Photomicrographs (106) of cleaved caspase-3 in the thalamus in control group (K) and in Sevo group (L); Quantification of cleaved caspase-3 in the thalamus region (control vs. Sevo, p = 0.0002) (M). doi:10.1371/journal.pone.0070645.g004
+
+major brain regions that are important for learning and memory. Neuronal apoptosis was partly attributed to hypercarbia caused by sevoflurane-induced respiratory depression, although a previous indicated that hypercarbia does not cause significant report neurocognitive impairment [19]. Some studies indicate sevoflur- ane can induce neuronal apoptosis in neonatal rodents, but most of these studies examined apoptosis only in neocortical [20,21] or hippocampal [22] tissue. We found that neonatal sevoflurane exposure induced caspase-3 activation in the cortex and thalamus, but not the hippocampus. Similarly, Zhu et al. [4] reported that isoflurane had no obvious effect on hippocampal cell death in P14 rats. Anesthetic treatment therefore may have differential effects on neurons at various developmental stages.
+
+on sevoflurane neurogenesis. Neonatal neurogenesis begins when cells prolif- erate and ends when cells migrate and integrate into a neuronal circuit as a functional neuron. is widely believed that neurogenesis enables hippocampal plasticity and new memories [23]. In addition to neuronal apoptosis, several recent studies correlate alterations in neurogenesis with cognitive performance [24,25]. Hippocampal neurogenesis initiated after volatile anesthesia in P7 rats [26–28], and the mechanisms underlying this phenomenon have been reviewed [29]. The effect of general anesthesia on neurogenesis, however, is controversial. Whether anesthesia stimulates or depresses neurogenesis appears to depend on the duration, concentration [30], and type of anesthetic administrated [31,32], as well as the animal model used [27] and the experimental conditions [33]. We found that 3% sevoflurane treatment for 6 h significantly decreased the number of BrdU+ cells in the hippocampus DG in P7 rats. Because this inhibition of proliferation can persist the neural in DG can be greatly reduced and affect progenitor pool subsequent neurogenesis [4]. Why sevoflurane anesthesia influ- ences proliferation without causing neuronal apoptosis in the developing hippocampus is unknown. Nevertheless, the sensitivity of the neonatal central nervous system to sevoflurane may affect
+
+Effects
+
+of
+
+anesthesia
+
+It
+
+is
+
+for more than 4 weeks,
+
+brain areas differently, depending on the survival, proliferation, differentiation, and migration patterns of neurons for each region.
+
+Effects of sevoflurane anesthesia on learning and memory
+
+Both juvenile and adult rodents exposed to volatile anesthetics during gestational or neonatal development can show learning and memory impairments [6,34]. We employed a series of neurobe- havioral tests and also found that sevoflurane exposure in neonates resulted in long-term neurocognitive sequelae. Interestingly, we did not observe significant effects on traditional ‘‘hippocampus- dependent’’ ELTM 8 weeks after sevoflurane anesthesia, but sevoflurane did significantly reduce the immediate fear response to the tone/shock pairings in the fear conditioning test. In addition, sevoflurane impaired spatial memory in the Morris water maze memory consolidation test when the delay between memory acquisition and retrieval was extended from 1 min to 1 h, but not to 4 h. This suggests that sevoflurane impaired WM and STM, rather than ELTM. Collectively, tests suggest that sevoflurane has negative effects on WM and STM, but not ELTM. Conversely, Kodama et al. [8] demonstrated that sevoflurane treatment did not impair WM in neonates. The brain regions that were most affected by neuronal apoptosis, changes in memory-related signaling [35], and protein production [36] with sevoflurane exposure are important for learning and memory, which may explain this discrepancy between the results from Kodama et al and ours [37]. Although we examined three brain regions that play major roles in the early stages of learning [38] and the three types of memory [39], other regions also participate in these cognitive functions. Shih et al [7] attributed neurocog- nitive dysfunction to acute sevoflurane-induced neuron death, especially in the thalamus, which was the most severely affected region. In that study, however, the authors did not examine the effect of anesthesia on neurogenesis in the neonatal brain. Consistent with the findings in previous studies, we found that sevoflurane decreased neuronal proliferation in the developing
+
+these neurobehavioral
+
+Figure 5. Perinatal n-3 PUFAs supplementation attenuates 6-h 3% sevoflurane-induced neuronal apoptosis. Cortical cleaved caspase-3 expression in neonatal brain was examined with Western blot (A); Quantification of cleaved caspase-3 (one-way ANOVA, Newman-Keul post hoc test, F = 6.286, p = 0.0274), *p,0.05 control (n = 3) vs. Sevo (n = 3); #p,0.05 Sevo vs. Sevo+n-3 PUFAs (n = 4) (B). doi:10.1371/journal.pone.0070645.g005
+
+PLOS ONE | www.plosone.org
+
+7
+
+August 2013 | Volume 8 |
+
+Issue 8 | e70645
+
+N-3 PUFAs Improve Neurotoxicity of Sevoflurane
+
+Figure 6. Perinatal n-3 PUFAs supplementation increases the attenuation of neuronal cells proliferation caused by neonatal exposure to 3% sevoflurane for 6-h in dentate gyrus (DG) region of neonatal hippocampus. BrdU was examined at the DG region by immunofluorescence (A); Quantification of BrdU at the DG region of neonatal hippocampus (one-way ANOVA, Newman-Keul post hoc test, F = 26.07, p = 0.0011), *p,0.05 control vs. Sevo; ###p,0.05 Sevo vs. Sevo+n-3 PUFAs; &&p,0.01 control vs. Sevo+n-3 PUFAs (B). n = 3 in each group. doi:10.1371/journal.pone.0070645.g006
+
+hippocampus. Similarly, a previous study reported that inhibition interfered with memory function [40]. We of neurogenesis speculate that sevoflurane-induced neuron death and failed proliferation in the developing central nervous system reduce brain volume and the number of synaptic connections, and disrupts brain plasticity, which is similar to the effects of alcohol on brain [41]. Neurons lost to apoptosis can be compensated via neurogenesis, as has been reported in stroke cases [42]. However, these newly generated neurons can it integrate into an existing brain network and function in learning and memory.
+
+is more essential
+
+that
+
+commonly have been used as a daily supplement for pregnant and lactating women as it benefits neurodevelopmental outcomes [44]. Therefore, we decided to test whether n-3 PUFAs could improve sevoflurane-induced brain dysfunction. Our data revealed that n-3 PUFA dietary supplementation to dams led to significant and prolonged neuroprotection in offspring rats that received neonatal sevoflurane exposure. This is the first report of n-3 PUFAs exerting protective effects against sevoflurane-induced cognitive impair- ment in rats, specifically by attenuating apoptosis and improving neuronal proliferation.
+
+sevoflurane-induced neurotoxicity and behavioral deficits. Considering volatile anesthetics can impair brain function in rodents, finding interven- tions that improve or prevent sevoflurane-induced memory deficits in the developing brain might provide insight into the neurotoxic mechanisms of volatile anesthetics. Although some drugs can protect against anesthesia-induced neurotoxicity [43], n-3 PUFAs
+
+n-3
+
+PUFAs
+
+protect
+
+against
+
+Furthermore, there were significantly more BrdU+ immunore- active cells in the hippocampus DG when n-3 PUFA supplemen- tation was combined with sevoflurane anesthesia, than with sevoflurane anesthesia alone. Compared with the control group, the robust effect of n-3 PUFAs on neurogenesis does not appear to promote performance improvements in the neurobehavioral tests. One possible explanation for the proliferative cells do not survive [45], differentiate into mature
+
+this observation is
+
+that
+
+PLOS ONE | www.plosone.org
+
+8
+
+August 2013 | Volume 8 |
+
+Issue 8 | e70645
+
+PLOS ONE | www.plosone.org
+
+9
+
+N-3 PUFAs Improve Neurotoxicity of Sevoflurane
+
+August 2013 | Volume 8 |
+
+Issue 8 | e70645
+
+N-3 PUFAs Improve Neurotoxicity of Sevoflurane
+
+Figure 7. Perinatal n-3 PUFAs supplementation improves neonatal sevoflurane exposure induced neurobehavioral impairment at adulthood (n = 9 each group). A–D Morris water maze spatial reference memory. Latency to platform in learning phase (A); Frequency to across the platform region (B); Swimming distance during the probe trial (C); Swimming speed during the probe trial (D); **p = 0.0019 control vs. Sevo. Fear conditioning (E–G). Post shock freezing (E): post shock 1 (F = 29.437, p = 0.0041, one-way ANOVA, Newman-Keul post hoc test, *p,0.05 control vs. Sevo; #p,0.05 Sevo vs. Sevo+n-3 PUFAs); post shock 2 (F = 10.3, p = 0.0033, one-way ANOVA, Newman-Keul post hoc test, *p,0.05 control vs. Sevo group; #p,0.05 Sevo vs. Sevo+n-3 PUFAs). Tone freezing (F); Context freezing (G); Morris water maze memory consolidation (H–K): Latency to platform in learning phase (H); Escape latency at 1-min delay (I) F = 10.25, p = 0.0031, one-way ANOVA, Newman-Keul post hoc test,**p,0.01 control vs. Sevo, ##p,0.01 Sevo vs. Sevo+n-3 PUFAs; Escape latency at 1-h delay (J); F = 13.70, p = 0.0014, one-way ANOVA, Newman-Keul post hoc test, **p,0.01 control vs. Sevo ##p,0.01 Sevo vs. Sevo+n-3 PUFAs; Escape latency at 4-h delay (K). doi:10.1371/journal.pone.0070645.g007
+
+neurons, or effectively integrate into circuits for learning and memory. A longer observational period is needed to assess these possibilities.
+
+Although we show that n-3 PUFAs have anti-apoptotic effects and neurogenesis-promoting properties, the molecular signaling involved in these mechanisms are unclear. Zhang and coworkers showed that n-3 PUFAs could confer long-term neuroprotection against hypoxic-ischemic brain injury by suppressing the inflam- matory response [11], and proinflammatory factors are believed to be one of the pro-apoptosis factors that contributes to volatile anesthetic-induced neurotoxicity [46]. In addition, maternal feeding of DHA significantly prevented stress-induced oxidative damage, apoptosis, and mitochondrial metabolism dysfunction in the hippocampus of offspring [47]. Wu et al [48] found that dietary n-3 PUFAs could normalize brain-derived neurotrophic factor (BDNF) levels in a rat model of traumatic brain injury, which is important for neuronal survival, differentiation, and function. It is possible that maternal n-3 PUFA supplementation during pregnancy could protect against postnatal reduction of brain neurotrophins in offspring [49]. Thus, n-3 PUFAs could alleviate neuronal apoptosis via regulating the inflammatory response, restoring BDNF imbalance [50], and/or decreasing reactive oxygen species levels [51] induced by sevoflurane in developing neurons. On the other hand, neurogenesis impairment might be caused by an anesthetic-induced decrease in trophic support (e.g., reduced BDNF levels), and maternal n-3 PUFA supplementation during pregnancy can protect against postnatal reduction of brain neurotrophins (BDNF and nerve growth factor) in offspring [52], which may promote neurogenesis in the developing brain. Collectively, these data support the hypothesis important contributions to the that n-3 PUFAs make several developing nervous system that help to prevent learning and memory impairments [11,48,53].
+
+n-3 PUFAs themselves. Secondly, we did not measure the fatty acid contents in brain and plasma of the rats at several stages (e.g., P0, P14, P38, P64, P79). In a previous study, we have found perinatal n-3 PUFAs supplementation increased functional poly- unsaturated fatty acid composition in brain cortical tissue in neonatal rats at postnatal 14 day with the same dietary feeding protocol used in this study [11]. The results from that study suggest increased n-3PUFAs levels in the brain at P14 are important for neuroprotection in the perinatal period, and later in life. Finally, we did not investigate whether the number of immature neurons was affected by postnatal sevoflurane exposure in P7–10 male rats. Even so, 80–90% of proliferating cells in the hippocampus DG differentiated into mature neurons; therefore, a decrease in BrdU+ cell numbers suggest neonatal sevoflurane exposure influences hippocampal neurogenesis in postnatal rats at P7–10. A recent study showed that exposure to general anesthetics during development appears to influence the percentage of neurons in a new cell population [54]. Therefore, future work should examine which cell types are affected by sevoflurane anesthesia by immunolabeling with specific neuronal cell markers.
+
+Conclusion
+
+Sevoflurane exposure in neonates results in neuronal apoptosis and impaired proliferation, both of which can cause neurocogni- tive disabilities later in life. In addition, our results provide evidence that perinatal n-3 PUFA supplementation can improve neurocognitive deficits, possibly by reducing neuronal apoptosis and neurogenesis impairment in the developing brain. Neverthe- less, it is critical to recognize that rodent brain development is fundamentally different from that of humans, and the present results might not be directly translated into clinical practice. Hence, further investigations are warranted to fully understand the effects of n-3 PUFAs on general anesthetic-induced neurotoxicity.
+
+Limitations
+
+There were some limitations to the present study. Firstly, we did not include an experimental group that received n-3 PUFAs during the perinatal period, without sevoflurane exposure. Nevertheless, our primary aim was to determine whether n-3 PUFAs mitigated sevoflurane-induced neurotoxicity and subse- quent cognitive impairment, rather than determine the effects of
+
+Author Contributions
+
+Conceived and designed the experiments: JZ WX. Performed the experiments: XL WZ HX TL. Analyzed the data: XL. Contributed reagents/materials/analysis tools: XL WZ JZ WX. Wrote the paper: XL JZ WL. Approved final manuscript: JZ.
+
+References
+
+1. Patel SS, Goa K (1996) Sevoflurane: a review of its pharmacodynamic and pharmacokinetic properties and its clinical use in general anaesthesia. Drugs 51: 658–700. Johnson SA, Young C, Olney JW (2008) Isoflurane-induced neuroapoptosis in the developing brain of nonhypoglycemic mice. J Neurosurg Anesthesiol 20: 21– 28.
+
+2.
+
+5. Stratmann G, Sall JW, May LDV, Bell JS, Magnusson KR, et al. (2009) Isoflurane differentially affects neurogenesis and long-term neurocognitive function in 60-day-old and 7-day-old rats. Anesthesiology 110: 834–848.
+
+6. Satomoto M, Satoh Y, Terui K, Miyao H, Takishima K, et al. (2009) Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice. Anesthesiology 110: 628–637.
+
+3. Xie Z, Dong Y, Maeda U, Alfille P, Culley DJ, et al. (2006) The common inhalation anesthetic isoflurane induces apoptosis and increases amyloid b protein levels. Anesthesiology 104: 988–994.
+
+7. Shih J, May LDV, Gonzalez HE, Lee EW, Alvi RS, et al. (2012) Delayed environmental enrichment reverses sevoflurane-induced memory impairment in rats. Anesthesiology 116: 586–602.
+
+4. Zhu C, Gao J, Karlsson N, Li Q, Zhang Y, et al. (2010) Isoflurane anesthesia induced persistent, progressive memory impairment, caused a loss of neural stem cells, and reduced neurogenesis in young, but not adult, rodents. J Cereb Blood Flow Metab 30: 1017–1030.
+
+8. Kodama M, Satoh Y, Otsubo Y, Araki Y, Yonamine R, et al. (2011) Neonatal desflurane exposure induces more robust neuroapoptosis than do isoflurane and sevoflurane and impairs working memory. Anesthesiology 115: 979–991.
+
+PLOS ONE | www.plosone.org
+
+10
+
+August 2013 | Volume 8 |
+
+Issue 8 | e70645
+
+9. Wei H, Kang B, Wei W, Liang G, Meng QC, et al. (2005) Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently. Brain Res 1037: 139–147.
+
+10. Olsen SF, Secher NJ, Tabor A, Weber T, Walker JJ, et al. (2005) Randomised clinical trials of fish oil supplementation in high risk pregnancies. BJOG 107: 382–395.
+
+11. Zhang W, Hu X, Yang W, Gao Y, Chen J (2010) Omega-3 polyunsaturated fatty acid supplementation confers long-term neuroprotection against neonatal hypoxic–ischemic brain injury through anti-inflammatory actions. Stroke 41: 2341–2347.
+
+12. Tuzun F, Kumral A, Ozbal S, Dilek M, Tugyan K, et al. (2012) Maternal prenatal omega-3 fatty acid supplementation attenuates hyperoxia-induced apoptosis in the developing rat brain. Int J Dev Neurosci 30: 315–323.
+
+13. Bertrand PC, O’Kusky JR, Innis SM (2006) Maternal dietary (n-3) fatty acid deficiency alters neurogenesis in the embryonic rat brain. J Nutr 136: 1570– 1575.
+
+14. Beltz BS, Tlusty MF, Benton JL, Sandeman DC (2007) Omega-3 fatty acids upregulate adult neurogenesis. Neurosci Lett 415: 154–158.
+
+15. Freund-Levi Y, Eriksdotter-Jonhagen M, Cederholm T, Basun H, Faxen-Irving G, et al. (2006) omega-3 Fatty acid treatment in 174 patients with mild to moderate Alzheimer disease: OmegAD Study: a randomized double-blind trial. Arch Neurol 63: 1402–1408.
+
+16. Wojtowicz JM, Kee N (2006) BrdU assay for neurogenesis in rodents. Nat Protoc 1: 1399–1405.
+
+17. He FQ, Qiu BY, Zhang XH, Li TK, Xie Q, et al. (2011) Tetrandrine attenuates spatial memory impairment and hippocampal neuroinflammation via inhibiting NF-kB activation in a rat model of Alzheimer’s disease induced by amyloid-b (1– 42). Brain Res 1384: 89–96.
+
+18. Gown AM, Willingham MC (2002) Improved detection of apoptotic cells in archival paraffin sections: immunohistochemistry using antibodies to cleaved caspase 3. J Histochem Cytochem 50: 449–454.
+
+19. Stratmann G, May LDV, Sall JW, Alvi RS, Bell JS, et al. (2009) Effect of hypercarbia and isoflurane on brain cell death and neurocognitive dysfunction in 7-day-old rats. Anesthesiology 110: 849–861. Istaphanous GK, Howard J, Nan X, Hughes EA, McCann JC, et al. (2011) the neuroapoptotic properties of equipotent anesthetic Comparison of concentrations of desflurane, isoflurane, or sevoflurane in neonatal mice. Anesthesiology 114: 578–587.
+
+20.
+
+21. Lu Y, Wu X, Dong Y, Xu Z, Zhang Y, et al. (2010) Anesthetic sevoflurane causes neurotoxicity differently in neonatal naı¨ve and Alzheimer’s disease transgenic mice. Anesthesiology 112: 1404–1416.
+
+22. Zhou H, Li S, Niu X, Wang P, Wang J, et al. (2013) Protective effect of FTY720 against sevoflurane-induced developmental neurotoxicity in rats. Cell Biochem Biophys 1–8: Epub ahead of print.
+
+23. Denis-Donini S, Dellarole A, Crociara P, Francese MT, Bortolotto V, et al. (2008) Impaired adult neurogenesis associated with short-term memory defects in NF-kappaB p50-deficient mice. J Neurosci 28:3911–3919. 24. Bianchi P, Ciani E, Guidi S, Trazzi S, Felice D, et al. (2010) Early pharmacotherapy restores neurogenesis and cognitive performance in the Ts65Dn mouse model for Down syndrome. J Neurosci 30:8769–8779.
+
+25. Zhao C, Deng W, Gage FH (2008) Mechanisms and functional implications of adult neurogenesis. Cell 132: 645–660.
+
+26. Fang F, Xue Z, Cang J (2012) Sevoflurane exposure in 7-day-old rats affects neurogenesis, neurodegeneration and neurocognitive function. Neuroscience Bulletin: Epub ahead of print.
+
+27. Stratmann G, Sall JW, May LD, Bell JS, Magnusson KR, et al. (2009) Isoflurane differentially affects neurogenesis and long-term neurocognitive function in 60- day-old and 7-day-old rats. Anesthesiology 110: 834–848.
+
+28. Stratmann G, Sall JW, May LD, Loepke AW, Lee MT (2010) Beyond anesthetic properties: the effects of isoflurane on brain cell death, neurogenesis, and long- term neurocognitive function. Anesth Analg 110: 431–437.
+
+29. Lei X, Guo Q, Zhang J (2012) Mechanistic Insights into Neurotoxicity Induced by Anesthetics in the Developing Brain. Int J Mol Sci 13: 6772–6799.
+
+30. Zhao X, Yang Z, Liang G, Wu Z, Peng Y, et al. (2013) Dual effects of isoflurane on proliferation, differentiation, and survival in human neuroprogenitor cells. Anesthesiology 118: 537–549.
+
+31. Dallasen RM, Bowman JD, Xu Y, Hashimoto M, Katakura M, et al. (2011) Isoflurane does not cause neuroapoptosis but reduces astroglial processes in young adult mice. Med Gas Res 1: 27.
+
+32. Tung A, Herrera S, Fornal CA, Jacobs BL (2008) The effect of prolonged anesthesia with isoflurane, propofol, dexmedetomidine, or ketamine on neural cell proliferation in the adult rat. Anesth Analg 106: 1772–1777.
+
+PLOS ONE | www.plosone.org
+
+11
+
+N-3 PUFAs Improve Neurotoxicity of Sevoflurane
+
+33. Engelhard K, Winkelheide U, Werner C, Kluge J, Eberspa¨ cher E, et al. (2007) Sevoflurane affects neurogenesis after forebrain ischemia in rats. Anesth Analg 104: 898–903.
+
+34. Loepke AW, Istaphanous GK, McAuliffe JJ 3rd, Miles L, Hughes EA, et al. (2009) The effects of neonatal isoflurane exposure in mice on brain cell viability, adult behavior, learning, and memory. Anesth Analg 108: 90–104.
+
+35. Liu XS, Xue QS, Zeng QW, Li Q, Liu J, et al. (2010) Sevoflurane impairs memory consolidation in rats, possibly through inhibiting phosphorylation of glycogen synthase kinase-3b in the hippocampus. Neurobiol Learn Mem 94: 461–467.
+
+36. Alkire MT, Guzowski JF (2008) Hypothesis: Suppression of memory protein formation underlies anesthetic-induced amnesia. Anesthesiology 109: 768–770. 37. Aggleton JP, O’Mara SM, Vann SD, Wright NF, Tsanov M, et al. (2010) Hippocampal–anterior thalamic pathways for memory: uncovering a network of direct and indirect actions. Eur J Neurosci 31: 2292–2307.
+
+38. Winocur G (1985) The hippocampus and thalamus: their roles in short-and long-term memory and the effects of interference. Behav Brain Res 16: 135–152. Izquierdo I, Medina JH, Vianna MRM, Izquierdo LA, Barros DM (1999) Separate mechanisms for short-and long-term memory. Behav Brain Res 103: 1–11. 39.
+
+40. Winocur G, Wojtowicz JM, Sekeres M, Snyder JS, Wang S (2006) Inhibition of neurogenesis interferes with hippocampus-dependent memory function. Hippo- campus 16: 296–304.
+
+41. Richardson HN, Chan SH, Crawford EF, Lee YK, Funk CK, et al.(2009) Permanent impairment of birth and survival of cortical and hippocampal proliferating cells following excessive drinking during alcohol dependence. Neurobiol Dis 36:1–10. Jin K, Wang X, Xie L, Mao XO, Zhu W, et al. (2006) Evidence for stroke- induced neurogenesis in the human brain. Proc Natl Acad Sci U S A 103: 13198–13202. 42.
+
+43. Yon JH, Carter LB, Reiter RJ, Jevtovic-Todorovic V (2006) Melatonin reduces the severity of anesthesia-induced apoptotic neurodegeneration in the develop- ing rat brain. Neurobiol Dis 21: 522–530. Innis SM (2007) Dietary (n-3) fatty acids and brain development. J Nutr 137: 855–859.
+
+44.
+
+45. Takasawa KI, Kitagawa K, Yagita Y, Sasaki T, Tanaka S, et al. (2002) Increased proliferation of neural progenitor cells but reduced survival of newborn cells in the contralateral hippocampus after focal cerebral ischemia in rats. J Cereb Blood Flow Metab 22: 299–307.
+
+46. Wu X, Lu Y, Dong Y, Zhang G, Zhang Y, et al.
+
+(2012) The inhalation anesthetic isoflurane increases levels of proinflammatory TNF-a, IL-6, and IL- 1b. Neurobiol Aging 33: 1364–1378.
+
+47. Feng Z, Zou X, Jia H, Li X, Zhu Z, et al. (2012) Maternal docosahexaenoic acid feeding protects against impairment of learning and memory and oxidative stress in prenatally stressed rats: possible role of neuronal mitochondria metabolism. Antioxid Redox Signal 16: 275–289.
+
+48. Wu A, Ying Z, Gomez-Pinilla F (2004) Dietary omega-3 fatty acids normalize BDNF levels, reduce oxidative damage, and counteract learning disability after traumatic brain injury in rats. J Neurotrauma 21: 1457–1467.
+
+49. Sable PS, Dangat KD, Joshi AA, Joshi SR (2012) Maternal omega 3 fatty acid supplementation during pregnancy to a micronutrient imbalanced diet protects postnatal reduction of brain neurotrophins in the rat offspring. Neuroscience 217: 46–55.
+
+50. Lu LX, Yon JH, Carter LB, Jevtovic-Todorovic V (2006) General anesthesia in the developing rat brain. activates BDNF-dependent neuroapoptosis Apoptosis 11: 1603–1615.
+
+51. Zhang J, Dong Y, Xu Z, Zhang Y, Pan C, et al. (2011) 2-Deoxy-D-glucose attenuates isoflurane-induced cytotoxicity in an in vitro cell culture model of H4 human neuroglioma cells. Anesth Analg 113: 1468–1475.
+
+52. Sable PS, Dangat DK, Joshi AA, Joshi SR (2012) Maternal omega 3 fatty acid supplementation during pregnancy to a micronutrient-imbalanced diet protects postnatal reduction of brain neurotrophins in the rat offspring. Neuroscience 217: 46–55.
+
+53. McNamara RK, Carlson SE (2006) Role of omega-3 fatty acids in brain development and function: potential implications for the pathogenesis and prevention of psychopathology. Prostaglandins Leukot Essent Fatty Acids 75: 329–349.
+
+54. Dong CRC, Anand KJ (2012) Ketamine alters the neurogenesis of rat cortical neural stem progenitor cells. Crit Care Med 40: 2407–2416.
+
+August 2013 | Volume 8 |
+
+Issue 8 | e70645

new_pdfs/10.1371_journal.pone.0105340.txt

@@ -0,0 +1,473 @@
+Early Exposure to Volatile Anesthetics Impairs Long-Term Associative Learning and Recognition Memory
+
+Bradley H. Lee1, John Thomas Chan1, Obhi Hazarika1, Laszlo Vutskits2, Jeffrey W. Sall1*
+
+1 Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco, California, Unites States of America, 2 Department of Anesthesiology, Pharmacology and Intensive Care, University Hospital of Geneva, Geneva, Switzerland
+
+Abstract
+
+Background: Anesthetic exposure early in life affects neural development and long-term cognitive function, but our understanding of the types of memory that are altered is incomplete. Specific cognitive tests in rodents that isolate different memory processes provide a useful approach for gaining insight into this issue.
+
+Methods:Postnatal day 7 (P7) rats were exposed to either desflurane or isoflurane at 1 Minimum Alveolar Concentration for 4 h. Acute neuronal death was assessed 12 h later in the thalamus, CA1-3 regions of hippocampus, and dentate gyrus. In separate behavioral experiments, beginning at P48, subjects were evaluated in a series of object recognition tests relying on associative learning, as well as social recognition.
+
+Results: Exposure to either anesthetic led to a significant increase in neuroapoptosis in each brain region. The extent of neuronal death did not differ between groups. Subjects were unaffected in simple tasks of novel object and object-location recognition. However, anesthetized animals from both groups were impaired in allocentric object-location memory and a more complex task requiring subjects to associate an object with its location and contextual setting. Isoflurane exposure led to additional impairment in object-context association and social memory.
+
+Conclusion:Isoflurane and desflurane exposure during development result in deficits in tasks relying on associative learning and recognition memory. Isoflurane may potentially cause worse impairment than desflurane.
+
+Citation: Lee BH, Chan JT, Hazarika O, Vutskits L, Sall JW (2014) Early Exposure to Volatile Anesthetics Impairs Long-Term Associative Learning and Recognition Memory. PLoS ONE 9(8): e105340. doi:10.1371/journal.pone.0105340
+
+Editor: Yael Abreu-Villac¸a, Universidade do Estado do Rio de Janeiro, Brazil
+
+Received April 30, 2014; Accepted July 18, 2014; Published August 28, 2014
+
+Copyright: (cid:1) 2014 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
+
+Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All data are within the paper and supporting files.
+
+Funding: Funding for the study was provided through National Institutes of Health Grant GM086511 to JWS, the University of California San Francisco Department of Anesthesia and Perioperative Care Hamilton Award to JWS, and by Swiss National Science Foundation Grant 31003A_130625 to LV. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
+
+Competing Interests: The authors have declared that no competing interests exist.
+
+Email: sallj@anesthesia.ucsf.edu
+
+Introduction
+
+Every day, anesthetics are used around the world in newborns and infants who undergo medical procedures. There is growing concern that anesthetics can significantly alter the developing brain, and animal models have shown that exposure to anesthetics at an early age lead to neuronal death and long-term cognitive dysfunction [1–3]. Epidemiologic studies suggest that humans are also susceptible to long-term cognitive effects after anesthesia [4,5]. Our knowledge of cognitive effects in humans has been, until recently [6], limited to retrospective studies that typically assess global tests of learning and behavior [4,5,7,8]. For instance, most of identify cognitive or learning disabilities by evaluating databases for individuals with diagnostic codes for unspecified delays, behavioral disorders, language or speech problems [7,8], or through IQ and achievement tests [4,5]. Because these studies examine generalized learning problems, they contribute minimally to our understanding of the memory processes that underlie the cognitive impairment.
+
+these epidemiologic studies
+
+An important challenge in the study of anesthetic neurotoxicity is developing a model by which cognitive effects in animals can be translated to humans. Memory processing is highly conserved across rodent and human species [9]. In particular, hippocampal memory functions are very similar between rats and humans [9], and the hippocampus is crucial in spatial encoding, associative learning, and recognition memory in both rats and humans [9– 12].
+
+Rodent models therefore provide valuable insight into the types of memory that may be affected in humans. However, behavioral studies are prone to using overlapping models for evaluating learning and memory. Many studies use similar tests, such as the Morris water maze [2,13–15], because they have consistently identified a cognitive deficit. Identifying impairment in specific memory processes, such as recognition and associative memory, in animal models will provide insight into effects in humans and may help guide future assessments of learning and memory in children, as has recently been reported [6].
+
+Recognition memory, which is a subtype of declarative in humans for recalling different events,
+
+memory,
+
+is crucial
+
+PLOS ONE | www.plosone.org
+
+1
+
+August 2014 | Volume 9 |
+
+Issue 8 | e105340
+
+objects, and people [16,17]. It has been shown that animals also have episodic-like memory that can be demonstrated through tests involving memory for ‘‘what,’’ ‘‘where,’’ and ‘‘when’’ details of an event. This was first described in birds [18] and more recently in rodents [12,19–22], and models have since been developed to examine recognition memory in various ways [20,23–25]. recognition memory Furthermore, many studies processes rely on the hippocampus and thalamus [19,26], which are areas of neuronal degeneration following anesthesia [2,14].
+
+find that
+
+The present study was designed to evaluate the effects of two commonly used volatile anesthetics – isoflurane and desflurane – on specific learning and memory processes following neonatal exposure. After delivering 1 Minimum Alveolar Concentration [27] of either anesthetic for 4 hours at postnatal day 7 (P7), subjects were evaluated in a set of recognition tasks involving associative memory, as well as social memory, that have been shown to be sensitive to lesions in hippocampal and thalamic circuits [19,28,29].
+
+Methods
+
+Subjects
+
+from the Institutional Animal Care and Use Committee at the University of California, San Francisco. Five Sprague-Dawley dams with litters of postnatal day 6 (P6) pups from were obtained from Charles River Laboratories (Gilroy, CA). Each litter contained only males and was culled to ten pups. In total, the males were taken from at least ten different litters. On P7, animals from each litter were randomly assigned to control and treatment groups. They were weaned at P23 and housed three per cage under standard lab housing with 12 h light/dark cycle. Animals were food restricted (access to food only during light cycle) for tasks involving object recognition to increase activity and object exploration.
+
+All experiments were conducted with approval
+
+Anesthesia
+
+Anesthesia was delivered as described previously [14,30,31]. Briefly, animals in the treatment groups received either isoflurane or desflurane as a single agent in air and oxygen (FiO250%) at 1 Minimum Alveolar Concentration [27] for four hours. MAC was determined by tail clamping every 15 minutes, and anesthetic concentration was adjusted accordingly, so that on average 50% of animals would move in response to clamping (Fig. 1). 12 out of 18 animals anesthetized with isoflurane survived to undergo behav- ioral testing, and 13 out of 18 animals anesthetized with desflurane survived and underwent behavioral testing. Control animals were concurrently placed in an anesthesia glove box of the same material and conditions without being exposed to anesthesia or tail clamping. Animals were kept on a warming blanket, and temperatures were measured using an infrared laser thermometer and maintained with a goal of 35uC.
+
+Histology
+
+Brains from the two anesthetized groups and the control group (n = 10 per group) were assessed for acute neuronal death. Twelve hours after anesthesia, animals were transcardially perfused with cold 4% paraformaldehyde in phosphate-buffered saline and brains were removed, postfixed, and sunk in sucrose solution. They were then sliced into 60 micron-thick slices and every other slice was mounted and stained with FluoroJade C, a marker specific for neurodegeneration [32,33] (FJC, 0.001%, Millipore, Billerica, MA). FJ-positive cells were counted using Nikon Eclipse 80i microscope under 20X magnification in each slice containing
+
+PLOS ONE | www.plosone.org
+
+2
+
+Anesthetic Effects on Memory
+
+Figure 1. MAC of isoflurane and desflurane. Anesthetics were separately delivered to P7 rats in air and oxygen (FiO2 50%) as previously described6, 9. Tail-clamping occurred every 15 minutes, and anesthetic concentration was adjusted to 1 MAC. As before6, 9, MAC decreases with increasing duration of anesthesia for both agents. doi:10.1371/journal.pone.0105340.g001
+
+the structure of interest. Structures included in analysis were the anterodorsal (LD), and anteromedial (AM) thalamic nuclei, as well as CA1-3 regions of the hippocampus and the dentate gyrus.
+
+(AD), anteroventral
+
+(AV),
+
+laterodorsal
+
+Object Recognition Tasks
+
+Object recognition was assessed using similar arrangements as others [19,28]. Behavior testing occurred during the light phase of the circadian cycle between 0800 and 1700 hrs in two separate arenas, hereafter referred to as contexts, of identical size (61 cm square base, walls 50 cm high). Context 1 had yellow walls with a base covered in wood-effect vinyl lining, and context 2 had black walls with a black plastic base. Different visual cues were placed on the walls of each context. A video camera (SONY HDR-CX190) was mounted 2 meters above the testing area for recording and observing subjects. For each task, except the allocentric object- location task, subjects were placed into contexts in the same (away from the objects). location and facing the south wall Beginning at P42, subjects were habituated to the two contexts prior to testing by being placed individually into the context for 5 min per day for 4 consecutive days. All animals underwent all behavioral tasks. Subjects were tested on the same day for any given task and in the same sequence of tasks. All tasks were performed in the order presented in subsequent weeks, except for the first two (novel object and object-place) which were performed in the same week. The order of testing during the day was counterbalanced among groups.
+
+Investigation of an object was defined as sniffing or placing the nose within 1 cm of and oriented toward the object. Subjects were recorded, and observers blinded to group assignment were used to determine investigation times. Object investigation times during the initial exposure for each task were compared to assess for possible confounding effects of varying investigation times on the ability to recognize objects. All objects and testing arenas were wiped with 70% ethanol between testing.
+
+Novel Object Recognition. Testing began at P48 with novel object recognition. A single trial was performed for each animal consisting of ‘‘exposure’’ and ‘‘test’’ phases separated by a two- minute delay (Fig. 2A). During the exposure, subjects were placed into the context and allowed to explore two identical objects for four minutes. After the delay, they were placed into the same context for three minutes with one of the objects replaced with a
+
+August 2014 | Volume 9 |
+
+Issue 8 | e105340
+
+Anesthetic Effects on Memory
+
+Figure 2. Object recognition. For each task, except allocentric object-place recognition, subjects are introduced at and facing the wall away from the objects. (A) Novel object recognition. Two identical objects are presented in the exposure, and one (right) is replaced with a novel object in the test phase. (B) Object-place recognition. Two different objects are presented, followed by two identical objects. In the test phase, the right object appears in a novel location within the context. (C) In the allocentric version of object-place recognition, subjects are again introduced at and facing the south wall (S) in the exposure. However, for the test phase, subjects are placed at and facing either the east (E) or west (W) wall. (D) Object-context recognition. Two different pairs of objects are presented in two different contexts, so each object is associated with a particular context. In the test phase, one object (right object, top row; left object, bottom row) appears within a context in which it has not been explored. (E) Object-place-context recognition. Two different objects are first presented in a context. The object locations are then reversed and presented in a different context. Thus, after two exposures, each object is seen in both contexts and both locations (left and right). In the test phase, two objects are presented in either context, so one (right object, top row; left object, bottom row) appears in a novel configuration of place and context. doi:10.1371/journal.pone.0105340.g002
+
+novel object. Half of the subjects were tested in each context with the location (left or right) of the novel object counterbalanced among subjects.
+
+Object-Place Recognition. Subjects were tested in their ability to recognize an object and its location. Two trials were performed, and investigation times were totaled for the two trials. In the exposure, two different objects were presented in a context for four minutes. After a two-minute delay, two identical copies of one of the previous objects were presented in the same context for three minutes (Fig. 2B). Both objects were equally familiar, but one now occupied a different location within the context.
+
+two different objects were (Fig. 2E). presented within a context. Next, subjects were placed in the opposite context with the same two objects and their locations reversed. Thus, after two exposures, each object was observed in both contexts and locations (left and right). In the test phase, two identical copies of either of the previous objects were presented in a context. The location and context associated with one object were familiar, while the other ‘‘displaced’’ object appeared in a location and context in which it had not been observed. Two trials were conducted with the test phase occurring in opposite contexts for each trial (Fig. 2E).
+
+In the first exposure,
+
+Allocentric Object-Place Recognition. For the previous task, subjects were always introduced into the context facing the wall (south wall) opposite the two objects (Fig. 2C). In the allocentric version of the task, for the initial exposure, subjects were again placed into the context facing the south wall. In the test phase, however, the entry point was varied and half of the subjects were introduced facing either the east or west wall (Fig. 2C). Two trials were performed and the entry point was randomized among subjects.
+
+Object-Context Recognition. Subjects were assessed in their ability to recognize an object with a particular context. The task required two separate exposures, each lasting four minutes and separated by a two-minute delay (Fig. 2D). In the first exposure, a pair of identical objects was presented in a context. Next, subjects were placed in a different context with a different pair of objects. In the test phase, lasting three minutes, subjects were placed into a context with one of each previously encountered object. Thus, one object was presented in the same context as before, while the other object appeared within a context in which it had not been explored. Two trials were conducted, and the test phase occurred in opposite contexts for each trial (Fig. 2D).
+
+Social Behavior and Social Recognition
+
+Following object recognition, animals were given unrestricted access to food. Social interaction and recognition were assessed using a discrimination paradigm one week after completing object recognition testing at P80. In the exposure, the subject was presented with a caged stimulus animal and a novel object for five minutes. This arrangement evaluates social behavior by deter- spend more time investigating the mining whether subjects stimulus animal or object7. After a sixty-minute delay, subjects were presented simultaneously with the same ‘‘familiar’’ animal and a novel animal the previously encountered animal was demonstrated by decreased investigation of the familiar target relative to the novel one.
+
+for three minutes. Recognition of
+
+Same-sex juvenile conspecifics were used as stimulus animals. Male pups five weeks of age were housed individually one week prior to testing. Investigation of the stimulus animal was defined as sniffing or direct contact with the subject’s nose or paws. Investigation of the novel object was defined as sniffing or placing the nose within 1 cm of and oriented toward object.
+
+Object-Place-Context Recognition. Subjects were tested in their ability to recognize an object with its location and context
+
+PLOS ONE | www.plosone.org
+
+3
+
+August 2014 | Volume 9 |
+
+Issue 8 | e105340
+
+Anesthetic Effects on Memory
+
+Figure 3. Neuronal death by group. A to F) Exposure to either anesthetic – desflurane or isoflurane – led to significantly increased neuronal death in each brain region. The degree of neurodegeneration was similar in desflurane and isoflurane-treated subjects. Sample images from brains at 20X magnification are shown alongside graphs comparing total cell death for each structure. G) The average increases in neuronal death relative to controls are shown. *P,0.05. doi:10.1371/journal.pone.0105340.g003
+
+Statistical Analysis
+
+Data were analyzed using Prism 6 Software for Mac OSX (GraphPad Software Inc., San Diego, CA). Data were assessed for normal distribution using the D’Agostino and Pearson test. Parametric tests were used for normally distributed data; otherwise, nonparametric tests were used for analysis. All comparisons used a two-tail test and a P value less than 0.05 was considered statistically significant.
+
+Total FluoroJade-positive cells for each brain region were compared among the groups – control, desflurane, isoflurane – using one-way ANOVA for parametric data or the Kruskal-Wallis test for nonparametric data. Bonferroni’s post-test with multiple comparisons was used following one-way ANOVA, and Dunn’s post-test was used with the Kruskal-Wallis test. The fold-increase in neuronal death was determined for each structure by dividing the total FJ-positive cells for all anesthetized animals (n = 20) by the average number of FJ-positive cells per structure for control animals (n = 10).
+
+the groups using either one-way ANOVA with Bonferroni’s post- test or the Kruskal-Wallis test with Dunn’s post-test.
+
+In addition, a ‘‘discrimination index’’ (DI) was calculated and (eg. represents the relative time spent exploring each target Familiar versus Novel). To calculate DI, the time spent investigating the familiar target was subtracted from the time spent on the novel target, and this was divided by the total time investigating the two (eg. DI = (Novel-Familiar)/(Total spent Time)). This value was compared to a theoretical value of zero using one sample t-test to assess whether a preference was shown for one of the objects, and a positive DI indicates preference for the novel aspect of the task. For each task, DI of control animals was compared against DI of all anesthetized animals. Also, within the group of anesthetized animals, the DI of desflurane-treated subjects was compared with that of isoflurane-treated subjects. These comparisons were made using either unpaired t-test for parametric data or the Mann Whitney test for nonparametric data.
+
+Recognition tasks were first assessed by comparing the investigation times of each target using paired tests for each group. Paired t-test was used for normally distributed data, and nonparametric data were analyzed with the Wilcoxon matched- pairs rank test. Also, to identify possible confounding effects of varying investigation times on subsequent object/animal recogni- tion, the times during the exposure phase were compared between
+
+Results
+
+Increased neuronal death occurs similarly in desflurane and isoflurane-treated animals
+
+There was increased neuronal death in each brain region in animals exposed to either desflurane or isoflurane relative to the control animals (Fig. 3). No difference in the extent of cell death
+
+PLOS ONE | www.plosone.org
+
+4
+
+August 2014 | Volume 9 |
+
+Issue 8 | e105340
+
+Anesthetic Effects on Memory
+
+was identified between the two anesthetized groups. Anesthetic exposure resulted in significantly increased cell death in the (P = 0.0001, one-way ANOVA; control vs. des hippocampus P = 0.0002, control vs. iso P = 0.99, Bonferroni), dentate gyrus (P = 0.0003, one-way ANOVA; control vs. des P = 0.0002, control vs. iso P = 0.03, des vs. iso P = 0.16, Bonferroni), anterodorsal thalamus (P,0.0001, one-way ANOVA; control vs. des P,0.0001, control vs. iso P = 0.0007, des vs. iso P = 0.98, Bonferroni), anteromedial thalamus (P,0.0001, one-way ANOVA; control vs. des P,0.0001, control vs. iso P,0.0001, des vs. iso P = 0.99, Bonferroni), anteroventral thalamus (P,0.0001, iso Kruskal-Walli P = 0.001, des vs. iso P = 0.99, Dunn’s), and laterodorsal thalamus (P,0.0001, one-way ANOVA; control vs. des P,0.0001, control vs. iso P,0.0001, des vs. iso P = 0.99, Bonferroni). The relative fold-increase in cell death for each brain region is shown in Figure 3G.
+
+iso P = 0.0015, des vs.
+
+test; control vs. des P,0.0001, control vs.
+
+Novel Object and Object-Place Recognition are Unaffected
+
+subjects (novel object P = 0.9, unpaired t-test; object-place P = 0.3, Mann Whitney test) or between desflurane and isoflurane subjects (novel object P = 0.83, unpaired t-test; object-place P = 0.64, Mann Whitney test).
+
+Isoflurane but not desflurane treated animals are impaired in object-context and social recognition
+
+Only the isoflurane group was impaired in the ability to associate an object with its context and spent similar amounts of time with each object in this task (control P = 0.001, Wilcoxon test familiar vs. novel context; desflurane P = 0.006, isoflurane P = 0.2, paired t-test, Fig. 5A). DIs of control and desflurane subjects were greater than zero but not in isoflurane-treated subjects (control P = 0.004, desflurane P = 0.04, isoflurane P = 0.95, one sample t- test, Fig. 5B). Comparison of DI between control and anesthetized subjects did not reveal a difference (P = 0.094, unpaired t-test, Fig. 5B). Within the anesthetized group, DI did not differ significantly between desflurane and isoflurane-treated subjects (P = 0.32, unpaired t-test). Exploration times in the exposure phases of the object-context task were similar for the three groups (P = 0.6, one-way ANOVA).
+
+Subjects from each group were able to distinguish familiar and novel objects, revealed by increased investigation times of the novel object (control P = 0.006, desflurane P = 0.01, isoflurane P = 0.0003; paired t-test familiar vs. novel, Fig. 4A). Object-place recognition was also intact in each group, and animals spent more location (control P = 0.006, time with the object desflurane P = 0.001, isoflurane P = 0.0008, paired t-test familiar vs. novel location, Fig. 4C). There was no difference in object exploration times among groups during the exposure for either task (novel object P = 0.5, one-way ANOVA, object-place P = 0.2, Kruskal-Wallis).
+
+in a novel
+
+Discrimination Indexes [3] for all subjects were greater than recognition (control P = 0.007, zero for both novel object desflurane P = 0.002, isoflurane P = 0.002, one sample t-test, Fig. 4B) and object-place recognition (control P = 0.01, desflurane P = 0.001, isoflurane P = 0.001, one sample t-test, Fig. 4D). No differences in DI were identified between control and anesthetized
+
+Isoflurane animals also had impaired social memory while desflurane animals were unaffected when comparing social target (control P = 0.0009, desflurane P = 0.002, investigation times isoflurane P = 0.08; paired t-test familiar vs. novel animal, Fig. 5C). DIs of control and desflurane subjects were greater than zero (control P = 0.0009, desflurane P = 0.002, one sample t-test, Fig. 5D), although isoflurane DI did not differ significantly from zero (P = 0.064, one sample t-test, Fig. 5D). No difference between DI was identified in control vs. anesthetized groups (P = 0.84, unpaired t-test). the isoflurane DI was lower than desflurane DI although it did not reach statistical significance (P = 0.17, unpaired t-test). In the exposure of the social recognition task, animals from all groups displayed normal social behavior and spent significantly greater time investigating the social target relative to the object (all P, 0.0001, paired t-test object vs. social target).
+
+In the subset of anesthetized subjects,
+
+Figure 4. Novel object and object-place recognition. A) Subjects all demonstrated successful object recognition and preferentially explored the novel object. B) Each group’s DI was significantly greater than zero, and there was no difference in DIs. C) Subjects were also able to identify an object in a novel location, demonstrated by a relative increase in investigation of that object. D) Again, DIs for all subjects were greater than zero with no differences identified. *P,0.05, **P,0.01, ***P,0.001, CON = control, DES = desflurane, ISO = isoflurane. doi:10.1371/journal.pone.0105340.g004
+
+PLOS ONE | www.plosone.org
+
+5
+
+August 2014 | Volume 9 |
+
+Issue 8 | e105340
+
+Anesthetic Effects on Memory
+
+Figure 5. Object-context and social recognition. A) Isoflurane-treated animals were impaired in associating an object with a particular context. Animals exposed to desflurane, on the other hand, recognized when an object appeared in a different context and spent more time with that object. B) The DI for anesthetized subjects in this task did not differ from zero, and, within this group, only the desflurane DI significantly exceeded zero. C) Desflurane-treated subjects also had no change in social recognition ability, spending more time with the novel animal, while isoflurane-treated animals had deficient social memory. D) DI for both control and anesthetized animals exceeded zero, although DI for the subset of isoflurane-treated subjects did not. *P,0.05, **P,0.01, ***P,0.001, n.s. = not significant. doi:10.1371/journal.pone.0105340.g005
+
+Anesthetized subjects are impaired in allocentric object- place and object-place-context recognition
+
+Animals from both isoflurane and desflurane groups were impaired in object recognition when the entry site was varied in the allocentric version of the object-place task (control P = 0.001, desflurane P = 0.08, paired t-test familiar vs. novel; isoflurane P = 0.2, Wilcoxon test, Fig. 6A). The control DI was greater than zero (P = 0.0004, one sample t-test, Fig. 6B), while neither desflurane nor isoflurane DI differed from zero (desflurane P = 0.094, isoflurane P = 0.31, one sample t-test, Fig. 6B). DI of control animals was also significantly greater than that of anesthetized subjects (P = 0.024, unpaired t-test), although no difference was detected in the subset of desflurane and isoflurane- treated animals (P = 0.95, unpaired t-test).
+
+Anesthetized subjects from both groups were also unable to distinguish objects task (control P = 0.04, desflurane P = 0.5, paired t-test familiar vs. displaced; isoflurane P = 0.8, Wilcoxon test, Fig. 6C). Only the control DI exceeded zero in this task (control P = 0.021, desflurane P = 0.71, isoflurane P = 0.7, one sample t-test, Fig. 6D). Control DI was again significantly greater than DI for anesthetized subjects (P = 0.04, unpaired t-test), and no difference was found between desflurane and isoflurane DIs (P = 0.59, unpaired t-test). Investi- gation times during the exposures were similar between groups for each task (allocentric object-place P = 0.1, object-place-context P = 0.7, one-way ANOVA). The summary of all behavioral testing is presented in Table 1, where each group is evaluated whether they demonstrate a preference for the novel portion of the task by recognizing a familiar set of stimuli.
+
+in the object-place-context
+
+Discussion
+
+The main finding of this study is that exposure to the volatile anesthetics isoflurane and desflurane causes impairment in tasks relying on specific cognitive processes of associative learning and recognition memory. After exposure to 1 MAC of either
+
+anesthetic for 4 hours during the early postnatal period, adult subjects could identify a novel object and recognize changes in an location. However, anesthetized animals were object’s spatial unable to recognize an object’s location when they entered the testing arena from a different vantage point or perform a complex task requiring the integration of object, place, and context details. In addition, isoflurane-treated subjects were impaired in context- specific object recognition and exhibited deficient social memory. The behaviors assessed in this study provide valuable insight into the types of learning affected by neonatal anesthesia exposure. The object recognition tasks performed here rely on spatial memory, but they also require associative processing to encode the relationships among distinct elements encountered during a given exposure [28,34,35]. Both control and treatment animals easily recognize a novel object, but animals that were anesthetized on P7 begin to show impairment when presented with objects that were previously in a different location or context, suggesting problems with associative learning. The impairment in the allocentric object-place task may also be related to spatial memory, because the animals are able to identify objects when relying on egocentric cues but struggle when forced to rely on allocentric cues.
+
+Episodic memory is associative in nature, and memory formation relies in large part on our ability to link new experiences and items with closely related ideas, facts, and the environment or context in which we learn them [36]. Clearly, a problem forming associations and relationships would affect memory encoding over time. Furthermore, within the broad domain of episodic memory, recognition memory is a specific type of memory that, according to the dual process model, is comprised of recollection and familiarity [26,36]. It is likely that impairment in the object recognition and in associative memory tasks could also result recollection, a process underlying recognition memory [19,28]. We recently reported deficits in recollection in both rodents and children after anesthesia at an early age [6]. Persistent problems with associative and recognition memory in children would have important consequences for learning and development throughout
+
+from a deficit
+
+PLOS ONE | www.plosone.org
+
+6
+
+August 2014 | Volume 9 |
+
+Issue 8 | e105340
+
+Anesthetic Effects on Memory
+
+Figure 6. Allocentric object-place and object-place-context recognition. A) Exposure to isoflurane or desflurane led to impairment in identifying an object’s location when the site of entry into the context was changed. The varied entry points forced subjects to rely on allocentric cues to identify the object’s location. B) DI of control animals was significantly greater than that of anesthetized subjects. Neither desflurane nor isoflurane DI significantly exceeded zero. C) Isoflurane and desflurane-treated subjects were also impaired in recognition of an object that required association of its place and context. D) Again, control DI was greater than anesthetized DI. Neither subset of anesthetized subjects – desflurane or isoflurane – had DI greater than zero. *P,0.05, **P,0.01, n.s. = not significant. doi:10.1371/journal.pone.0105340.g006
+
+adolescence. The precise cognitive domains that may be impaired in children and how these effects manifest later in life is still unclear, and these are important areas of future investigation.
+
+Isoflurane has been used in numerous studies to investigate the effects of anesthesia and many labs have reported cell death and behavioral changes after isoflurane exposure [1–3,14,30]. The effects associated with desflurane, though, are less well described. Similar to other volatile anesthetics, desflurane in neonates has been shown to induce cell death [37,38]. However, few studies of behavior have been performed, and only one of these has demonstrated cognitive impairment [38]. Kodama and colleagues found that mice exposed to desflurane later developed problems with short-term and long-term memory [38]. In our present study, we demonstrate impairment in desflurane-treated animals using two separate tasks that involve associative learning. Together, these behavioral results show that desflurane, like isoflurane [2,14,30] and sevoflurane [13,30,39], alters long-term cognitive behavior.
+
+Isoflurane-treated animals were impaired in two additional behavioral suggesting a distinct outcome from those anesthetized with desflurane. Others have also identified distinct outcomes using different anesthetic agents [37,38,40,41], although the reason underlying these behavioral findings is unclear. The types of memory involved in this series of behavioral testing are processed in the medial including temporal hippocampus and dentate gyrus, as well as the anterior thalamus and prefrontal cortex [26],[42], and we identified increased neurodegeneration in each of these brain regions. However, the observation of distinct behavioral outcomes occurred in the setting of a similar extent of neuronal injury. The discrepancy between histologic and behavioral findings suggest that, although neuronal death may play a role in determining behavioral phenotype, other effects on neural development likely contribute, as well. In fact, there is evidence that volatile anesthetics can alter synaptogenesis and dendritic spine density even in the absence of cell death [43]. In addition, anesthetics have been shown to result in significant
+
+tasks,
+
+lobe [19,28],
+
+Table 1. Summary of behavioral testing.
+
+Discrimination Index for task greater than zero?
+
+Control
+
+DES
+
+ISO
+
+Novel Object Recognition
+
+Yes
+
+Yes
+
+Yes
+
+Object-Place Recognition
+
+Yes
+
+Yes
+
+Yes
+
+Object-Context Recognition
+
+Yes
+
+Yes
+
+No
+
+Social Recognition
+
+Yes
+
+Yes
+
+No
+
+Allocentric Object-Place Recognition
+
+Yes
+
+No
+
+No
+
+Object-Place-Context Recognition
+
+Yes
+
+No
+
+No
+
+For each test, recognition of a familiar set of stimuli results in preferential exploration of the novel aspect of the task. Discrimination Index (DI) represents the time spent with the novel object or animal relative to the familiar one, and DI significantly greater than zero demonstrates successful recognition in the task. doi:10.1371/journal.pone.0105340.t001
+
+PLOS ONE | www.plosone.org
+
+7
+
+August 2014 | Volume 9 |
+
+Issue 8 | e105340
+
+neuroinflammation [41], changes in cell signaling [44], and stem cell proliferation [45,46]. It is likely that anesthetic effects on these processes of brain development contribute to the ultimate cognitive outcome.
+
+Isoflurane-treated animals also had difficulty with social recognition which is more likely related to long-term memory processes than their capacity for social interaction. Unlike previous reports [39], we found all animals behaved similarly during the exposure portion of the test, spending much more time with a novel animal than an object. In fact, throughout these experiments the treatment groups demonstrated a difference in none of suggests exploration time during the exposure phase. This anesthetic exposure does not alter investigatory or social behavior, motivation, or attention.
+
+Limitations
+
+The purpose of this study is to evaluate two separate anesthetics using outcomes of cell death and behavior. We cannot make conclusive remarks regarding mechanisms underlying cognitive impairment, and separate studies are needed to better understand these processes. Also, a comprehensive analysis of neuronal death was not undertaken, and it is possible that other brain regions show a difference. The hippocampus and thalamus were chosen, however, because of their underlying role in the investigated behavior.
+
+Social recognition is based on olfaction in rodents [47] and we did not perform a separate experiment to exclude impaired olfaction as the basis for deficient social recognition in our subjects. However, we have previously determined that anesthetic exposure does not impair olfaction [6]. Isoflurane-treated subjects displayed typical social behavior in each part of the test, suggesting impaired recognition was due to effects on memory rather than interest,
+
+References
+
+1. Gentry KR, Steele LM, Sedensky MM, Morgan PG (2013) Early developmental exposure to volatile anesthetics causes behavioral defects in Caenorhabditis elegans. Anesth Analg 116: 185–189. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, et al. (2003) Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 23: 876–882.
+
+2.
+
+3. Brambrink AM, Evers AS, Avidan MS, Farber NB, Smith DJ, et al. (2010) Isoflurane-induced neuroapoptosis in the neonatal rhesus macaque brain. Anesthesiology 112: 834–841.
+
+4. Flick RP, Katusic SK, Colligan RC, Wilder RT, Voigt RG, et al. (2011) Cognitive and behavioral outcomes after early exposure to anesthesia and surgery. Pediatrics 128: e1053–1061.
+
+5. Wilder RT, Flick RP, Sprung J, Katusic SK, Barbaresi WJ, et al. (2009) Early exposure to anesthesia and learning disabilities in a population-based birth cohort. Anesthesiology 110: 796–804.
+
+6. Stratmann G, Lee J, Sall JW, Lee B, Alvi BS, et al. (2014) Effect of general anesthesia in infancy on long-term recognition memory in humans and rats. Neuropsychopharmacology 39. EPub ahead of print.
+
+7. DiMaggio C, Sun LS, Kakavouli A, Byrne MW, Li G (2009) A retrospective cohort study of the association of anesthesia and hernia repair surgery with behavioral and developmental disorders in young children. J Neurosurg Anesthesiol 21: 286–291.
+
+8. DiMaggio C, Sun LS, Li G (2011) Early childhood exposure to anesthesia and risk of developmental and behavioral disorders in a sibling birth cohort. Anesth Analg 113: 1143–1151.
+
+9. Bunsey M, Eichenbaum H (1996) Conservation of hippocampal memory function in rats and humans. Nature 379: 255–257.
+
+10. Eichenbaum H, Yonelinas AP, Ranganath C (2007) The medial temporal lobe and recognition memory. Annu Rev Neurosci 30: 123–152.
+
+11. Burgess N, Maguire EA, O’Keefe J (2002) The human hippocampus and spatial and episodic memory. Neuron 35: 625–641.
+
+12. Fortin NJ, Wright SP, Eichenbaum H (2004) Recollection-like memory retrieval in rats is dependent on the hippocampus. Nature 431: 188–191.
+
+13. Shih J, May LD, Gonzalez HE, Lee EW, Alvi RS, et al.
+
+(2012) Delayed environmental enrichment reverses sevoflurane-induced memory impairment in rats. Anesthesiology 116: 586–602.
+
+PLOS ONE | www.plosone.org
+
+8
+
+Anesthetic Effects on Memory
+
+motivation, or olfaction. Also, although the rats were tested serially, they did not show any signs of decreasing interest with the objects as we used objects that appeared novel to the subjects in each trial. In fact, the exploration times remained very similar across the tests from first to last.
+
+There are numerous studies documenting effects of gestational and early life stress on long-term behavior [48,49]. Because the animals were shipped, rather than bred in the housing facility, it is possible that they were exposed to early life stress that may affect aspects of behavior. Although effects of stress are likely evenly distributed amongst behavior groups, these considerations should be taken into account when interpreting behavioral results.
+
+Finally, the cognitive outcomes from the two anesthetics appear to be different; however, it is possible that the two anesthetics were not entirely equal in depth in spite of being adjusted to 1 MAC. This must be taken into consideration when attempting to make direct comparisons between the two volatile anesthetics.
+
+Conclusion
+
+Neonatal exposure to isoflurane and desflurane led to impair- ment in object recognition tasks relying on spatial and associative memory. These findings provide evidence that anesthetics can affect distinct cognitive processes that are fundamental to learning and memory in rodents, as well as humans.
+
+Author Contributions
+
+Conceived and designed the experiments: BHL JTC OH LV JWS. Performed the experiments: BHL JTC OH LV JWS. Analyzed the data: BHL JTC OH LV JWS. Contributed reagents/materials/analysis tools: BHL JTC OH LV JWS. Contributed to the writing of the manuscript: BHL JTC OH LV JWS.
+
+14. Stratmann G, May LD, Sall JW, Alvi RS, Bell JS, et al.
+
+(2009) Effect of hypercarbia and isoflurane on brain cell death and neurocognitive dysfunction in 7-day-old rats. Anesthesiology 110: 849–861.
+
+15. Boscolo A, Ori C, Bennett J, Wiltgen B, Jevtovic-Todorovic V (2013) Mitochondrial protectant pramipexole prevents sex-specific long-term cognitive impairment from early anaesthesia exposure in rats. Br J Anaesth.
+
+16. Yonelinas AP (2001) Components of episodic memory: the contribution of recollection and familiarity. Philos Trans R Soc Lond B Biol Sci 356: 1363– 1374.
+
+17. Brown MW, Aggleton JP (2001) Recognition memory: what are the roles of the perirhinal cortex and hippocampus? Nat Rev Neurosci 2: 51–61.
+
+18. Clayton NS, Dickinson A (1998) Episodic-like memory during cache recovery by scrub jays. Nature 395: 272–274.
+
+19. Eacott MJ, Norman G (2004) Integrated memory for object, place, and context in rats: a possible model of episodic-like memory? J Neurosci 24: 1948–1953.
+
+20. Eacott MJ, Easton A, Zinkivskay A (2005) Recollection in an episodic-like memory task in the rat. Learn Mem 12: 221–223.
+
+21. Dere E, Huston JP, De Souza Silva MA (2005) Episodic-like memory in mice: simultaneous assessment of object, place and temporal order memory. Brain Res Brain Res Protoc 16: 10–19.
+
+22. Kart-Teke E, De Souza Silva MA, Huston JP, Dere E (2006) Wistar rats show episodic-like memory for unique experiences. Neurobiol Learn Mem 85: 173– 182.
+
+23. Eichenbaum H, Fortin N, Sauvage M, Robitsek RJ, Farovik A (2010) An animal model of amnesia that uses Receiver Operating Characteristics (ROC) analysis to distinguish recollection from familiarity deficits in recognition memory. Neuropsychologia 48: 2281–2289.
+
+24. Easton A, Eacott MJ (2010) Recollection of episodic memory within the medial temporal lobe: behavioural dissociations from other types of memory. Behav Brain Res 215: 310–317.
+
+25. Sauvage MM (2010) ROC in animals: uncovering the neural substrates of recollection and familiarity in episodic recognition memory. Conscious Cogn 19: 816–828.
+
+26. Aggleton JP, Dumont JR, Warburton EC (2011) Unraveling the contributions of the diencephalon to recognition memory: a review. Learn Mem 18: 384–400. 27. Macbeth AH, Edds JS, Young WS 3rd (2009) Housing conditions and stimulus females: a robust social discrimination task for studying male rodent social recognition. Nat Protoc 4: 1574–1581.
+
+August 2014 | Volume 9 |
+
+Issue 8 | e105340
+
+28. Langston RF, Wood ER (2010) Associative recognition and the hippocampus: differential effects of hippocampal lesions on object-place, object-context and object-place-context memory. Hippocampus 20: 1139–1153.
+
+29. Cross L, Brown MW, Aggleton JP, Warburton EC (2012) The medial dorsal thalamic nucleus and the medial prefrontal cortex of the rat function together to support associative recognition and recency but not item recognition. Learn Mem 20: 41–50.
+
+30. Ramage TM, Chang FL, Shih J, Alvi RS, Quitoriano GR, et al. (2013) Distinct long-term neurocognitive outcomes after equipotent sevoflurane or isoflurane anaesthesia in immature rats. Br J Anaesth.
+
+31. Stratmann G, Sall JW, Eger EI 2nd, Laster MJ, Bell JS, et al. (2009) Increasing the duration of isoflurane anesthesia decreases the minimum alveolar anesthetic concentration in 7-day-old but not in 60-day-old rats. Anesth Analg 109: 801– 806.
+
+32. Schmued LC, Stowers CC, Scallet AC, Xu L (2005) Fluoro-Jade C results in ultra high resolution and contrast labeling of degenerating neurons. Brain Res 1035: 24–31.
+
+33. Wang L, Liu YH, Huang YG, Chen LW (2008) Time-course of neuronal death in the mouse pilocarpine model of chronic epilepsy using Fluoro-Jade C staining. Brain Res 1241: 157–167.
+
+34. Aggleton JP, Sanderson DJ, Pearce JM (2007) Structural hippocampus. Hippocampus 17: 723–734.
+
+34. Aggleton JP, Sanderson DJ, Pearce JM (2007) Structural hippocampus. Hippocampus 17: 723–734.
+
+35. Mumby DG, Gaskin S, Glenn MJ, Schramek TE, Lehmann H (2002) Hippocampal damage and exploratory preferences in rats: memory for objects, places, and contexts. Learn Mem 9: 49–57.
+
+36. Mayes A, Montaldi D, Migo E (2007) Associative memory and the medial temporal lobes. Trends Cogn Sci 11: 126–135. Istaphanous GK, Howard J, Nan X, Hughes EA, McCann JC, et al. (2011) the neuroapoptotic properties of equipotent anesthetic Comparison of concentrations of desflurane, isoflurane, or sevoflurane in neonatal mice. Anesthesiology 114: 578–587.
+
+36. Mayes A, Montaldi D, Migo E (2007) Associative memory and the medial temporal lobes. Trends Cogn Sci 11: 126–135. Istaphanous GK, Howard J, Nan X, Hughes EA, McCann JC, et al. (2011) the neuroapoptotic properties of equipotent anesthetic Comparison of concentrations of desflurane, isoflurane, or sevoflurane in neonatal mice. Anesthesiology 114: 578–587.
+
+38. Kodama M, Satoh Y, Otsubo Y, Araki Y, Yonamine R, et al. (2011) Neonatal desflurane exposure induces more robust neuroapoptosis than do isoflurane and sevoflurane and impairs working memory. Anesthesiology 115: 979–991.
+
+PLOS ONE | www.plosone.org
+
+9
+
+Anesthetic Effects on Memory
+
+39. Satomoto M, Satoh Y, Terui K, Miyao H, Takishima K, et al. (2009) Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice. Anesthesiology 110: 628–637.
+
+40. Liang G, Ward C, Peng J, Zhao Y, Huang B, et al. (2010) Isoflurane causes greater neurodegeneration than an equivalent exposure of sevoflurane in the developing brain of neonatal mice. Anesthesiology 112: 1325–1334.
+
+41. Shen X, Dong Y, Xu Z, Wang H, Miao C, et al. (2013) Selective anesthesia- induced neuroinflammation in developing mouse brain and cognitive impair- ment. Anesthesiology 118: 502–515.
+
+42. Aggleton JP, Hunt PR, Nagle S, Neave N (1996) The effects of selective lesions within the anterior thalamic nuclei on spatial memory in the rat. Behav Brain Res 81: 189–198.
+
+43. Briner A, De Roo M, Dayer A, Muller D, Habre W, et al. (2010) Volatile anesthetics rapidly increase dendritic spine density in the rat medial prefrontal cortex during synaptogenesis. Anesthesiology 112: 546–556.
+
+44. Masaki E, Kawamura M, Kato F (2004) Attenuation of gap-junction-mediated signaling facilitated anesthetic effect of sevoflurane in the central nervous system of rats. Anesth Analg 98: 647–652, table of contents.
+
+45. Sall JW, Stratmann G, Leong J, McKleroy W, Mason D, et al. (2009) Isoflurane inhibits growth but does not cause cell death in hippocampal neural precursor cells grown in culture. Anesthesiology 110: 826–833.
+
+46. Lin N, Moon TS, Stratmann G, Sall JW (2013) Biphasic change of progenitor proliferation in dentate gyrus after single dose of isoflurane in young adult rats. J Neurosurg Anesthesiol 25: 306–310.
+
+47. Tang AC, Reeb BC, Romeo RD, McEwen BS (2003) Modification of social memory, hypothalamic-pituitary-adrenal axis, and brain asymmetry by neonatal novelty exposure. J Neurosci 23: 8254–8260.
+
+48. Koenig JI, Elmer GI, Shepard PD, Lee PR, Mayo C, et al. (2005) Prenatal exposure to a repeated variable stress paradigm elicits behavioral and neuroendocrinological changes in the adult offspring: potential relevance to schizophrenia. Behav Brain Res 156: 251–261.
+
+49. Weinstock M (2001) Alterations
+
+induced by gestational
+
+stress
+
+in brain
+
+morphology and behaviour of the offspring. Prog Neurobiol 65: 427–451.
+
+August 2014 | Volume 9 |
+
+Issue 8 | e105340

new_pdfs/10.1371_journal.pone.0160826.txt

@@ -0,0 +1,455 @@
+RESEARCH ARTICLE
+
+Maternal Exposure of Rats to Isoflurane during Late Pregnancy Impairs Spatial Learning and Memory in the Offspring by Up-Regulating the Expression of Histone Deacetylase 2
+
+a11111
+
+Foquan Luo1‡*, Yan Hu1,2‡, Weilu Zhao1, Zhiyi Zuo3, Qi Yu1, Zhiyi Liu1, Jiamei Lin1, Yunlin Feng1, Binda Li4, Liuqin Wu4, Lin Xu1
+
+1 Department of Anesthesiology, the First Affiliated Hospital, Nanchang University, Nanchang 33006, China, 2 Department of Anesthesiology, Jiangxi Province Traditional Chinese Medicine Hospital, Nanchang 33006, China, 3 Department of Anesthesiology, University of Virginia, Charlottesville, VA, 22908, United States of America, 4 Department of Anesthesiology, Jiangxi Province Tumor Hospital, Nanchang 330006, China
+
+‡ These authors are co-first authors on this work. * lfqjxmc@outlook.com
+
+OPEN ACCESS
+
+Citation: Luo F, Hu Y, Zhao W, Zuo Z, Yu Q, Liu Z, et al. (2016) Maternal Exposure of Rats to Isoflurane during Late Pregnancy Impairs Spatial Learning and Memory in the Offspring by Up-Regulating the Expression of Histone Deacetylase 2. PLoS ONE 11 (8): e0160826. doi:10.1371/journal.pone.0160826
+
+Editor: Huafeng Wei, University of Pennsylvania, UNITED STATES
+
+Received: September 22, 2015
+
+Accepted: June 6, 2016
+
+Published: August 18, 2016
+
+Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
+
+Data Availability Statement: All relevant data are within the paper.
+
+Funding: FL received the funding from the national natural science foundation of China (NO.81460175, NO.81060093), http://www.nsfc.gov.cn/; from the natural science foundation of Jiangxi province of China (NO.20122BAB205012, NO. 20132BAB205022), http://www.jxstc.gov.cn/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
+
+Abstract
+
+Increasing evidence indicates that most general anesthetics can harm developing neurons and induce cognitive dysfunction in a dose- and time-dependent manner. Histone deacety- lase 2 (HDAC2) has been implicated in synaptic plasticity and learning and memory. Our previous results showed that maternal exposure to general anesthetics during late preg- nancy impaired the offspring’s learning and memory, but the role of HDAC2 in it is not known yet. In the present study, pregnant rats were exposed to 1.5% isoflurane in 100% oxygen for 2, 4 or 8 hours or to 100% oxygen only for 8 hours on gestation day 18 (E18). The offspring born to each rat were randomly subdivided into 2 subgroups. Thirty days after birth, the Morris water maze (MWM) was used to assess learning and memory in the off- spring. Two hours before each MWM trial, an HDAC inhibitor (SAHA) was given to the off- spring in one subgroup, whereas a control solvent was given to those in the other subgroup. The results showed that maternal exposure to isoflurane impaired learning and memory of the offspring, impaired the structure of the hippocampus, increased HDAC2 mRNA and downregulated cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) mRNA, N-methyl-D-aspartate receptor 2 subunit B (NR2B) mRNA and NR2B pro- tein in the hippocampus. These changes were proportional to the duration of the maternal exposure to isoflurane and were reversed by SAHA. These results suggest that exposure to isoflurane during late pregnancy can damage the learning and memory of the offspring rats via the HDAC2-CREB -NR2B pathway. This effect can be reversed by HDAC2 inhibition.
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+1 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+Competing Interests: The authors have declared that no competing interests exist.
+
+Introduction
+
+Increasing evidence indicates that most general anesthetics are harmful to developing neurons and cause cognitive deficits in a dose- and time- dependent manner. Previous study [1] reported that exposure of pregnant rats to low concentrations of isoflurane (1.3%) for 6 hours did not cause neurodegeneration in the fetal brain or affect learning and memory in the off- spring. However, in a similar animal model, exposure to high concentrations of isoflurane (3%) for only 1 hour caused significant neurodegeneration in fetal brain [2], suggesting a dose- dependent effect of isoflurane neurotoxicity. The majority of general anesthetics are lipophilic and can easily cross the placental barrier. About 0.5% to 2% of pregnant women will suffer non-obstetric surgery [3–5], and most of these procedures (up to 73%) must be completed under general anesthesia [6]. More than 75,000 pregnant women in the United States and 5,700 to 7,600 pregnant women in the European Union undergo non-obstetric surgery each year [7]. However, little is known regarding the effects of maternal exposure to general anes- thetics during late pregnancy on the offspring’s subsequent learning and memory. Data from Sweden showed that among 5,405 patients who had non-obstetric surgery during pregnancy, 23% had procedures during the third trimester [4]. Most of the published studies about isoflur- ane showed a protective effect on the brain, however our previous studies showed that maternal exposure to propofol, ketamine, enflurane, isoflurane or sevoflurane during early gestation could cause learning and memory deficits and showed time-dependent effects [8]. A recent ani- mal study indicated that rats exposed to isoflurane in utero at a time that corresponds to the second trimester in humans exhibited impaired spatial memory [9]. However, rats exposed to isoflurane on gestational day 21(E21) showed no neurotoxicity to the fetal brain, and no learn- ing and memory impairments in the juvenile or adult rats [1].
+
+Synaptic plasticity is critical to memory formation and storage [10]. Histone acetylation has been implicated in synaptic plasticity and learning and memory [11–13]. Histone deacetylase (HDAC) inhibitors can reinstate learning and promote the retrieval of long-term memory in animals with massive nerve degeneration [14]. These findings suggested that HDAC inhibition may provide a therapeutic avenue for memory impairment caused by neurodegenerative dis- eases. Among HDAC family members, HDAC2 functions in modulating synaptic plasticity and producing long-lasting changes to neural circuits, which in turn negatively regulate learn- ing and memory [15]. The hyperphosphorylation of HDAC2 decreases the phosphorylation of cAMP response-element binding (CREB) protein, leading to a decrease in the CREB protein levels [16]. The administration of SAHA increased the levels of acetylated histones, accompa- nied by enhanced binding of phospho-CREB (p-CREB) to its binding site in the promoter of the NR2B gene, a subunit of N-methyl-D-aspartic (NMDA) receptors. This effect led to increased NR2B protein levels in the rat hippocampus, thus facilitating fear extinction [17]. Thus, HDAC2 modulates learning and memory by inhibiting CREB expression and down-reg- ulating the expression of NR2B. Isoflurane can induce repression of contextual fear memory in 3-month-old mice by reducing histone acetylation in the hippocampus, an effect that can be rescued by the HDAC inhibitor sodium butyrate [18]. Neonatal mice repeatedly exposed to isoflurane also showed repression of contextual fear memory [19].
+
+Many pregnancies include non-obstetric surgery during the late pregnancy due to diverse medical conditions, such as acute appendicitis, symptomatic cholelithiasis, and trauma [20– 22]. Increasing reports suggested that any trimester of pregnancy should not be considered as a contraindication to surgery, and many non-obstetric surgeries can be safely performed in the third trimester [20–27]. Prospective clinical studies showed that approximately 27.6% of appendectomies performed during pregnancy were done in the third trimester, and none of the children exhibited any developmental delay during a 47.2-month (range from 13 to 117
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+2 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+months) follow-up time after delivery [28], however learning and memory was not evaluated in these children. The effect of maternal exposure to isoflurane on learning and memory and its mechanism is not well understood. Therefore, the present study was designed to explore the effects of maternal exposure of rats to isoflurane during late pregnancy (corresponding to the human third trimester) on learning and memory in the offspring. Further, we hypothesized that the detrimental effects of isoflurance on learning and memory are mediated through changes in the HDAC2-CREB-NR2B pathway, which we explored by administration of an SAHA.
+
+Experimental Procedures
+
+Subjects
+
+This protocol was approved by the institutional review board of the First Affiliated Hospital of Nanchang University on the Use of Animals in Research and Teaching. Seventy-day-old female Sprague-Dawley (SD) rats (maternal rats) were supplied by the animal science research department of the Jiangxi Traditional Chinese Medicine College (JZDWNO: 2011–0030). The learning and memory functions of the parental rats were assessed with the MWM before mat- ing. Female rats were then housed with a male rat (2 female: 1 male rat per cage) for mating. Pregnant rats were identified and divided into the isoflurane exposure 2h (I2), 4h (I4), 8h (I8) and control (C) groups (n = 10 per group) based on the MWM test results to minimize the effects of maternal differences in learning and memory.
+
+Anesthesia
+
+On E18, gravid rats in the I2, I4 and I8 groups were exposed to 1.5% isoflurane (Abbott labo- ratories Ltd, Worcester, MA, USA) in 100% oxygen for 2, 4 and 8 hours, respectively, while those in the control group received 100% oxygen only. Electrocardiogram, saturation of pulse oximetry, and the respiratory rate of the rats as well as the inhaled concentration of iso- flurane were monitored continuously with a Datex-Ohmeda ULT-I analyzer. The tail inva- sive blood pressure was monitored intermittently. The rectal temperature was maintained at 37 ± 0.5°C with heating pads. The exposure time began from the loss of the righting reflex. The depth and rate of breath was monitored. The exposure durations were selected because different lengths of surgeries are performed [29], and neuronal damage or apoptosis reaches a maximum when general anesthetic exposure time reaches 6 to 8 hours [30]. Our prelimi- nary study showed that maternal exposure to 1.5% isoflurane for 8 hours did not significantly change blood pressure, blood glucose or venous blood gases. The concentration of isoflurane was selected because 1.5% isoflurane in 100% oxygen equals approximately 1 MAC (mini- mum alveolar concentration) in gestating rats and caused righting reflex loss in our prelimi- nary studies. At the end of the exposure time, all of the rats were exposed to 100% oxygen for 30 min for anesthesia recovery in an anesthesia chamber (40 × 40 × 25 cm). If the cumulative <95% and/or the systolic blood pressure (SBP) decreased by more than 20% of time of SpO2 baseline more than 5 minutes, the dam would be excluded from the study, and another dam was selected to supplement the sample size, thereby excluding the harmful effect of maternal ischemia or hypoxia on offspring rats. Furthermore, to clarify whether exposure to isoflurane caused a significant effect on the internal environment of maternal rats, 10 additional rats at gestational day 18 were selected. Five were exposed to 1.5% isoflurane in 100% oxygen for 8 hours, and the other five were exposed to 100% oxygen for 8 hours. Femoral vein blood was harvested for blood gas analysis.
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+3 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+Morris Water Maze (MWM) Test
+
+The age of P30 in rat corresponds to preschool age in human [31]. Therefore, we evaluated the spatial learning and memory of the offspring begining on P30 with MWM according previous report [32]. All of the offspring were acclimated to the experimental environment for 30 min before testing. The Morris water maze is a black circular steel pool with a diameter of 150 cm and a height of 60 cm, filled with 24 ± 1°C water to a depth of 20 cm. A circular escape platform of 10 cm in diameter was submerged 1 cm below the water surface in the second quadrant. The swimming trail and speed of the rats was automatically recorded by the SLY-WMS Morris water maze test system (Beijing Sunny Instruments Co. Ltd., Beijing, China). The escape latency (time needed to find the platform), platform crossing times (number of times the rat swam across the submerged platform), and the target quadrant traveling time (time spent in the platform-hidden quadrant) were recorded automatically by the test system. The tests were begun at 9:00 am, one time per day for seven consecutive days. Each offspring rat was put into the pool to search for the platform one time per day for six days (training trial). The starting point was in the third quadrant, the farthest quadrant from the platform-hidden quadrant (the second quadrant in the present study, named the target quadrant). The rats were placed in the water facing the wall of the pool. The same starting point was used for each rat (with a colour marker on the pool wall). The animals were allowed to stay on the platform for 30 seconds when they found the platform. If an animal could not find the platform within 120 s, the escape latency was recorded as 120 s for that trial. The animal was then guided to the platform and allowed to stay on it for 30 s. On the seventh day, the platform was removed. Rats were allowed to swim for 120s to test their memory (platform-crossing times and target quadrant traveling time). The mean of the latencies, platform-crossing times and target quadrant traveling time of the offspring rats born by the same mother rat were calculated as the final results.
+
+The offspring born to the same dam in each group were subdivided into the SAHA sub- group (I2S, I4S, I8S and CS subgroup) and the non-SAHA subgroup (I2N, I4N, I8N and CN subgroup) (Fig 1). Two hours before each MWM test, 90 mg/kg SAHA (Selleck Chemicals, Houston, TX, USA), at a concentration of 0.6 μM in dimethyl sulfoxide (DMSO) was given intraperitoneally to the offspring in the SAHA subgroups. An equal volume of DMSO was given to the rats in the non-SAHA subgroups. We selected 2 h before each MWM trial as the administration time point for SAHA based on the fact that 2 h after SAHA administration, the expression of NR2B increased in the hippocampus of Sprague-Dawley rats by enhancing his- tone acetylation, thus facilitating fear extinction [17].
+
+Transmission Electron Microscopy
+
+The offspring were anesthetized with isoflurane at 24 h after the MWM test and then eutha- nized by cervical dislocation. Left hippocampus tissues were harvested quickly (in 1 minute) on ice and cut into small pieces of 1 mm3. The hippocampal pieces were immersed in 2.5% glu- taraldehyde in 0.1 mol/l phosphate buffer (pH 7.4) at 4°C for 3 hours, rinsed three times in 0.1 M PBS (phosphate buffered saline), fixed in 1% osmium tetroxide at 4°C for 2 h, dehydrated, embedded, cut into ultrathin sections of 50–70 nm, stained by saturated uranium acetate and lead citrate and observed by transmission electron microscopy.
+
+Real Time Polymerase Chain Reaction (RT- PCR)
+
+Total RNA of the hippocampus was extracted with Trizol reagent (Invitrogen) according to the manufacturer’s protocol. The mRNA concentration was measured (OD 260 nm) with a spec- trophotometer (Nanophotometer P, MPLEN Co., Germany). Reverse transcription was per- formed with 1 μg total RNA using a Prime ScriptTM RT reagent Kit with gDNA Eraser (Perfect
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+4 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+Fig 1. Experimental design. Pregnant dams were exposed to 1.5% isoflurane in 100% oxygen or to 100% oxygen alone for the times indicated on E18 and the offspring were treated with 90 mg/kg SAHA (ip) or vehicle (DMSO) 2 hours before behavioral testing. The number in parentheses represents the number of animals: F = female, M = male; DMSO = dimethyl sulfoxide; SAHA = suberanilohydroxamic acid, also known as vorinostat; TEM = transmission electron microscopy.
+
+doi:10.1371/journal.pone.0160826.g001
+
+Real Time; RR047A, TaKaRa BIO Inc., Japan). The cDNA sample was amplified by a real time PCR instrument (ABI7500), with SYBR Premix Ex TaqTM (Tli RNaseH Plus; Code: RR820A, TaKaRa Co., Japan). β-actin was chosen as a reference gene. The length of both the HDAC2 product and the CREB product is 94 bp, whereas the length of the NR2B product is 103 bp, and the length of the β-actin product is 150 bp. PCR amplification was performed with the fol- lowing cycling parameters: one cycle of 95°C for 30 s followed by 40 cycles of 95°C for 5 s, 60°C for 34 s, 95°C for 15 s, 60°C for 1 min and 95°C for 15 s.
+
+The ABI7500 instrument automatically analyzed the fluorescence signal and converted it to the Ct value, using β-actin as a housekeeping gene and the Ct value of group C as the compara- tive object. Single-product amplification was confirmed by melting curve and gel electrophore- sis analysis. The expression levels of HDAC2, CREB and NR2B mRNA were normalized to β- actin mRNA and the values of the control group. The mean mRNA expression level of all of the offspring born to the same mother rat was calculated as the final expression level of mRNA.
+
+Western Blot
+
+Total protein was extracted by lysing the hippocampus (one offspring from each dam) in lysis buffer (Thermo Scientific, Rockford, IL, USA) containing a protease inhibitors cocktail (Sigma-Aldrich). Total protein (50 μg/ lane) was separated on a polyacrylamide gel and then transferred onto PVDF membranes. The membranes were blocked with Protein-Free T20 Blocking Buffer (Thermo Scientific) for 1 h at room temperature and incubated with rabbit polyclonal anti-NR2B antibody (Cell signaling Technology, 1:500) or rabbit polyclonal anti-β actin antibody (Cell Signaling Technology, 1:500) overnight at 4°C. After incubation with goat anti-rabbit HRP-conjugated IgG, the protein complex was revealed with enhanced chemilumi- nescence reagents (Pierce, IL, USA) and quantified by Genesnap version 7.08. The density of
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+5 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+NR2B protein band was normalized to that of β-actin in the same sample. The results from iso- flurane exposed offspring were then normalized to the average values of control offpring in the same western blot. The mean expression level of all of the offspring born to the same mother rat was calculated as the final expression level of NR2B protein.
+
+Statistical Analysis
+
+All values shown represent the mean ± SEM. The escape latency was subjected to a two-way repeated measures ANOVA (RM ANOVA) with prenatal treatment as a between-litters inde- pendent factor and day as a repeated factor. When an initial ANOVA showed main effects of the factors as well as significant interactions among the factors, post hoc comparisons were conducted by the least significant difference (LSD) t test. The mRNA and protein data, plat- form crossing times, and target quadrant traveling time were analyzed by one-way ANOVA, and followed by LSD t test when a significant difference was found in groups (p < 0.05). Results are considered statistically significant at p < 0.05.
+
+Results
+
+Isoflurane Exposure Does Not Alter Maternal Blood Gases
+
+To clarify whether exposure to 1.5% isoflurane for 8 hours causes significant changes to the internal environment during late pregnancy, 10 gravid rats on E18 were used. We continuously monitored the saturation of pulse oximetry during anesthesia. As a matter of convenience, femoral vein blood gas analysis was used to evaluate whether isoflurane exposure would cause changes in acid-base balance or serum electrolytes in maternal rats. All of the indices of venous blood gases showed no significant changes after an 8-hour exposure to 1.5% isoflurane com- pared with rats exposed to oxygen only (Table 1). These results indicate that exposure to 1.5% isoflurane for 8 hours on E18 does not cause significant metabolic changes to pregnant rats.
+
+Impaired Learning and Memory in Rat Offspring and the Ameliorating Effect of SAHA
+
+The results of MWM showed that the offspring in isoflurane exposure group had to spend more time finding the platform than the control group. At the third MWM trial, the escape latency in the I2N, I4N or I8N groups was longer than the control group (p < 0.05). The escape latency increased with the increase of isoflurane exposure time. The escape latency in
+
+Table 1. The effect of isoflurane exposure on femoral venous blood gas and electrolytes in maternal rats.
+
+Indexes
+
+pH
+
+PO2 (mmHg) PCO2 (mmHg) - (mmol/L) HCO3 BE(B) (mmol/L) Ca2+ (mmol/L) K+ (mmol/L) Na+ (mmol/L)
+
+100% oxygen 7.39±0.03 46.33±6.15 51.5±3.62 31.13±0.45 3.75±1.33 1.24±0.20 4.51±0.64 135.50±1.22
+
+1.5% isoflurane+100% oxygen 7.39±0.23 49.50±4.93 50.50±12.79 27.85±4.78 3.20±0.80 1.41±0.06 4.8±0.50 134.25±0.96
+
+Rats were exposed to oxygen or isoflurane + oxygen for 8 hour and monitored continuously. Final values were recorded at the end of the 8 hour period. n = 5.
+
+doi:10.1371/journal.pone.0160826.t001
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+6 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+5
+
+p < 0.05). At the 6th
+
+I8N group was longer than control group at 3rd, 4th, 5th and 6th trial ( training trial, the escape latency in the I8N group was significantly longer than the I2N or I4N group (# p < 0.05) (Fig 2A). The offspring in isoflurane exposure group spent less time traveling in the platform hidden quadrant. The target quadrant traveling time in I8N group was less than CN group ((cid:2) p < 0.05), I2N and I4N group (# p < 0.05). The offspring in iso- flurane exposure group swam across the location where the platform hidden less than control group, especially those in I8N group. The platform-crossing times in I8N group was less than CN group ((cid:2) p < 0.05), I2N and I4N group (# p < 0.05, Fig 2B and 2C). These results indicate
+
+Fig 2. Maternal isoflurane exposure impaired learning and memory in offspring: Offspring of rats exposed to isoflurane on gestation day 18 (E18) for 2h (I2N), 4h (I4N) and 8h (I8N) respectively. Thirty days postneaonatal (P30), the learning and memory was assessed using the Morris water maze: (a) Escape latency (time to find the hidden platform). At the third trial, the escape latency in the I2N, I4N or I8N group was significant longer than the control group (* p < 0.05); The escape latency increased with the increase of isoflurane exposure time. The escape latency in I8N group was significant longer than control group at 3rd, 4th, 5th and 6th trial (5 p < 0.05). At the 6th training trial, the escape latency in the I8N group was significantly longer than the I2N or I4N group (# p < 0.05); (b) Target quadrant traveling time. The offspring in isoflurane exposure group spent less time traveling in the platform hidden quadrant (target quadrant). The target quadrant traveling time in I8N group was significant less than CN group (* p < 0.05), I2N and I4N group (# p < 0.05); (c) Platform crossing times. The offspring in isoflurane exposure group swam across the location where the platform hidden (platform-crossing times) less than control group, especially those in I8N group. The platform-crossing times in I8N group was significant less than CN group (* p < 0.05), I2N and I4N group (# p < 0.05). CN = control group.
+
+doi:10.1371/journal.pone.0160826.g002
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+7 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+Fig 3. HDAC2 inhibition alleviated the impaired learning caused by maternal isoflurane exposure. SAHA potentiates the learning ability of normal rats, the escape latencey in SAHA treated normal offspring was shorter than normal control offspring at 2nd, 4th and 5th trial (* p < 0.05, Fig 3a); The escape latency in I2S, I4S and I8S group were shorter than their relative control groups (I2N, I4N and I8N group respectivly), but had no statistical differences (p > 0.05, Fig 3b, c and d). The escape latency in I8S group was longer than normal control group (CN group) at 3rd, 4th and 5th trial (p < 0.05, Fig 3e). S = SAHA treated subgroup; N = non—SAHA treated subgroup.
+
+doi:10.1371/journal.pone.0160826.g003
+
+that maternal exposure to isoflurane of approximately 1 MAC (1 MAC of isoflurane for rats on gestational day 14–16 is 1.4%) [33, 34] can impair learning and memory in the offspring. To study whether the learning and memory impairment caused by maternal isoflurane exposure could be reversed by HDAC inhibitor, SAHA was given to the offspring before each MWM trial. The escape latencey in SAHA treated normal offspring was shorter than control group at 2nd, 4th and 5th trial ((cid:2) p < 0.05, Fig 3A). The escape latency in I2S, I4S and I8S group were shorter than their relative control groups (I2N, I4N and I8N group respectivly), but had no statistical differences (p > 0.05, Fig 3B, 3C and 3D). The escape latency in I8S group was longer than normal control group (CN group) at 3rd, 4th and 5th trial (p < 0.05, Fig 3E). The target quadrant traveling time in SAHA treated sugroup was more than relative non-SAHA subgroup (p < 0.05, Fig 4A). The traveling time in I2S, I4S and I8S group was not significantly different from that in CN group (Fig 4A). The platform-crossing times in SAHA treated sugroups increased compared with their relative non-SAHA subgroups (Fig 4B). But the plat- form-crossing times in I8S subgroup was still less than CN group (p < 0.05, Fig 4B). These
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+8 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+Fig 4. HDAC2 inhibition reversed the memory impairment caused by maternal isoflurane exposure. (a) Target quadrant traveling time. The target quadrant traveling time in SAHA treated sugroup was significant more than relative non-SAHA subgroups (* p < 0.05); The traveling time in I2S, I4S and I8S group was not significantly different from that in CN group. (b) Platform crossing times. The platform-crossing times in SAHA treated sugroups increased compared with their relative non-SAHA subgroups, but had no statistical differences. The platform-crossing times in I8S subgroup was still less than CN group (* p < 0.05).
+
+doi:10.1371/journal.pone.0160826.g004
+
+results indicate that SAHA can alleviate the learning and memory impairment caused by iso- flurane exposure, but cannot completely reverse the impairment when the exposure time was 8 hours.
+
+Maternal Isoflurane Exposure Disrupted Ultrastructural Features of Hippocampal Neurons in Offspring
+
+Ultrastructural changes in hippocampal neurons were evaluated by electron microscopy. Maternal isoflurane exposure impaired the structure of the hippocampus when the exposure time was more than 4 hours. The ultrastructure in group I2N showed no obvious differences compared to group CN. When the exposure time was lengthened to 4 hours, neuron number decreased, nuclei became irregular, cytoplasmic area decreased, mitochondrial number decreased, and we observed evidence of disordered mitochondrial cristae. The quantity of rough endoplasmic reticulum, ribosome and Golgi apparatus decreased, and the ribosomes exhibited degranulation. When the isoflurane exposure time was prolonged to 8 hours, all of these changes became more prominent. We observed fewer neurons with dilated intercellular space. Dissolved mitochondrial cristae and swollen Golgi apparatus could be observed in group I8. HDAC inhibition alleviated all the hippocampal impairments caused by isoflurane expo- sure, but the neurons number had no obvious change (Fig 5).
+
+Increased HDAC2 mRNA Expression Caused By Isoflurane and the Reversed Effect of SAHA
+
+Maternal isoflurnae exposure increased the expression levels of HDAC2 mRNA in the hippo- campus of the offspring rats (p < 0.05; Fig 6A). SAHA reversed the expression of HDAC2 mRNA in the hippocampus. The expression levels of HDAC2 mRNA in the CS, I2S, I4S and I8S groups were lower than those in the CN, I2N, I4N and I8N groups respectively (p < 0.05;
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+9 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+Fig 5. HDAC2 inhibition alleviated the hippocampal ultrastructure impairment caused by maternal isoflurane exposure (transmission electron microscopy, ×6000). The hippocampal ultrastructure showed apparent abnormality with the increase of isoflurane exposure time. The ultrastructure showed no differences compared to the control group when isoflurane exposure time was 2h (I2N). When isoflurane exposure time lengthen to 4h, the neuron number decreased, the nuclei became irregular, cytoplasmic area decreased, mitochondrial number decreased, and we observed evidence of disordered mitochondrial cristae. The quantity of rough endoplasmic reticulum, ribosome and Golgi apparatus decreased, and the ribosomes exhibited degranulation (I4N). When the exposure time prolonged to 8h, all of these changes became more prominent, there were fewer neurons with dilated intercellular space. Dissolved mitochondrial cristae and swollen Golgi apparatus could be observed (I8N). HDAC inhibition alleviated the impairments, but did not increase the neuronal number (I4S and I8S group).
+
+doi:10.1371/journal.pone.0160826.g005
+
+Fig 6B). These results indicate that HDAC2 inhibition can reverse the overexpression of HDAC2 mRNA in offspring caused by maternal isoflurane exposure during late pregnancy.
+
+Downregulated CREB mRNA Expression and the Reversed Effect of SAHA
+
+The expression levels of CREB mRNA in the hippocampus of the offspring in the I2N, I4N and I8N groups were significantly lower than those in the CN group (p < 0.05; Fig 7A). These results indicate that maternal isoflurane exposure during late pregnancy can inhibit the expres- sion of CREB mRNA in offspring. The CREB mRNA levels in the I8N group were significantly lower than in the I2N group and I4N group (p < 0.05; Fig 7A), suggesting that prolonged expo- sure to isoflurane during late pregnancy has a more profound effect on inhibiting CREB mRNA expression. The expression levels of CREB mRNA in the CS group were higher than those in the CN group (p < 0.05; Fig 7B). This finding indicates that SAHA can potentiate the expression of CREB mRNA in the hippocampus of the offspring rats. The expression levels of CREB mRNA in the I2S, I4S and I8S groups were significantly higher than those in the I2N, I4N and I8N groups respectively (p < 0.05; Fig 7B). These results indicate that SAHA can reverse the inhibiting effect of maternal isoflurane exposure on CREB mRNA expression off- spring rats. However, the expression level of CREB mRNA in the I8S group was still lower than CN group (p < 0.05, Fig 7B). This means that SAHA cannot completely reverse the inhibiting effect of maternal isoflurane exposure on the expression of CREB mRNA when the exposure time is 8 hours.
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+10 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+Fig 6. HDAC2 inhibition reversed the overexpression of HDAC2 mRNA caused by maternal isoflurane exposure. The expression levels of HDAC2 mRNA in offspring hippocampus were detected by real time PCR (RT—PCR). The levels of mRNA were normalized to that of β-actin in the same sample and then normalized to the average values of control offspring in the same RT-PCR. The mean value of the mRNA expression level of all of the offspring born to the same mother rat was calculated as the final expression level of mRNA. (a) maternal isoflurane exposure potentiated the expression of HDAC2 mRNA. The HDAC2 mRNA levels in the offpsring hippocampus in I2N, I4N and I8N group were higher than normal control group (CN group, * p < 0.05). (b) SAHA reversed the overexpression of HDAC2 mRNA. The HDAC2 mRNA levels in SAHA treated subgroup were higher than non-SAHA subgroups (* p < 0.05).
+
+doi:10.1371/journal.pone.0160826.g006
+
+Downregulated Expression of NR2B and the Reversed Effect of SAHA
+
+The expression levels of NR2B mRNA in the hippocampus of the offspring in the I2N, I4N and I8N groups were lower than those in the CN group (p < 0.05; Fig 8A3). The expression levels of NR2B mRNA in the I4N and I8N groups were lower than those in the I2N group (p <0.05; Fig 8A3). The changes of NR2B protein expression levels were similar to NR2B mRNA levels (Fig 8B3). These results indicate that maternal isoflurane exposure during late pregnancy
+
+Fig 7. HDAC2 inhibition reversed the downregulated expression of CREB mRNA caused by maternal isoflurane exposure: (a) Isoflurane exposure downregulated CREB mRNA expression. The CREB mRNA levels in the offspring hippocampus in isoflurane exposed group were lower than control group (* p < 0.05). With the increase of isoflurane exposure time, the downregulated effect became more obvious. The CREB mRNA levels in I8N group were higher than I2N group (# p < 0.05). (b) SAHA reversed the down-regulation of CREB mRNA expression. Compared with relative non-SAHA subgroup, the levels of CREB mRNA in SAHA treated sugroup increased (* p < 0.05). But the CREB mRNA levels in I8S subgroup were still lower than normal control group (# p < 0.05). This indicates that SAHA cannot completely reverse the downregulated effect caused by isoflurane when the exposure time prolonged to 8 hours.
+
+doi:10.1371/journal.pone.0160826.g007
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+11 / 18
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+12 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+Fig 8. HDAC2 inhibition reversed the downregulated expression of NR2B caused by maternal isoflurane exposure. (A1) Amplification plot of NR2B and β-actin mRNA; (A2) Agarose gel electrophoresis images of NR2B and β-actin mRNA; (A3) Maternal isoflurane exposure downregulated the expression of NR2B mRNA (mean ± SE): The levels of NR2B mRNA in isoflurane exposure group (I2N, I4N, I8N) were lower than CN group (* p < 0.05). The NR2B mRNA levels in I4N and I8N group were lower than I2N group (# p < 0.05); (A4) SAHA reversed the downregulated expression of NR2B mRNA (mean ± SE): The levels of NR2B mRNA in I2S, I4S and I8S subgroup were higher than I2N, I4N and I8N subgroup respectively (* p < 0.05). The NR2B mRNA levels in I8S subgroup were lower than CN group (# p < 0.05). (B1) NR2B protein western blot images; (B2) Maternal isoflurane exposure downregulated the expression of NR2B protein (mean ± SD): The protein levels of NR2B protein were lower than CN group (# compared with I2N group, p<0.05b); (B3) HDAC2 inhibition reversed the downregulated expression of NR2B protein: The protein levels of NR2B in I2S, I4S and I8S subgroups were higher than I2N, I4N and I8N sugroups respectively. The levels of NR2B protein in I8S subgroup were lower than CN group (# p < 0.001). This indicates that SAHA cannot completely reverse the downregulated effect caused by isoflurane when the exposure time prolonged to 8 hours.
+
+doi:10.1371/journal.pone.0160826.g008
+
+inhibits the expression of NR2B in the hippocampus of the offspring rats and that prolonged isoflurane exposure can exacerbate these changes. The expression levels of NR2B protein and mRNA in the CS, I2S, I4S and I8S groups were higher than those in the CN, I2N, I4N and I8N groups respectively (Fig 8A4 and 8B3). These results indicate that SAHA can reverse the inhib- iting effect of maternal isoflurane exposure on NR2B expression in the hippocampus of the off- spring. However, the mRNA and protein levels of NR2B in the I8S group were still lower than those in the CN group (Fig 8A4 and 8B3). Thus, HDAC2 inhibition cannot completely reverse the inhibiting effects of maternal isoflurane exposure on the expression of NR2B when the exposure time is 8 hours.
+
+Discussion
+
+The present study provides preclinical evidence that maternal rat exposure to isoflurane during late pregnancy impairs the spatial learning and memory in their offspring. The behavioural abnormality was associated with hippocampal neuronal damage, overexpression of HDAC2 mRNA and the subsequent downregulation of CREB mRNA and NR2B in hippocampus. This finding is similar to the results described in a previous report, which showed that maternal exposure to 1.4% isoflurane for 4 hours on gestational day 14 impairs the offspring rats’ spatial memory acquisition [9]. The results of the present study are different from another report showing that isoflurane exposure during late pregnancy was not neurotoxic to the fetal brain and did not impair learning and memory in juvenile or adult offspring [1]. Isoflurane neurotoxicity is concentration-dependent [35]. General anesthetics can be neuroprotective and neurotoxic, depending on the levels, timing and mode of exposure. Isoflurane exerts multiple effects on neuronal stem cell survival, proliferation and differentiation. Short exposures to low isoflurane concentrations promote proliferation and differentiation of ReNcell CX cells, whereas prolonged exposure to high isoflurane concentrations induced significant cell damage [36]. This may be one of the critical reasons that our result is different from the report by Li et al. [1]. Our study involved 1.5% isoflurane, approximately 1 MAC, which is higher than the concentration used in the previous study (1.3%) [1]. The different exposure timing may be another reason for the differences between two experiments. The exposure time point in our experiment was E18 day, whereas that in previous study was E21 [1]. Therefore, the develop- mental maturity of the neurons was different between two experiments, which may result in different vulnerability to isoflurane [37]. In the present study, the dams were divided into dif- ferent groups based on their learning and memory results tested before pregnancy. This was meant to exclude the effects of genetic factors on the learning and memory of the offspring and may have facilitated the significant differences in learning and memory obtained between the control and isoflurane exposure groups.
+
+There was no obvious dyskinesia in offspring. There was no difference in swimming speed (automatically record by MWM system) of the offspring among groups. We had not evaluated the anxiety of offspring rats in present study. But previous study had revealed that rats exposed
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+13 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+to isoflurane in utero on E14 have reduced anxiety compared with controls [9]. Thus the differ- ences in learning and memory results were not caused by abnormal motor function or anxiety in offspring. The femoral vein blood gas analysis showed that exposure to this concentration of isoflurane for 8 hours had no significant effect on the blood gases and electrolytes of the dams (Table 1). Normal maternal body temperature was maintained during the isoflurane exposure process. Dams were removed from the study if they exhibited a cumulative time of more than 5 minutes with SpO2 damaging effect on learning and memory was not induced by physiological disturbances caused by isoflurane.
+
+<95% or >20% decrease in systolic blood pressure (SBP). Therefore, the
+
+How does isoflurane exposure damage the spatial learning and memory function in off- spring? Transmission electron microscopy results showed that neuron number and ultrastruc- ture in offspring hippocampus had been impaired (Fig 5). HDAC inhibitors could alleviate the impairments, thus improving learning and memory. These data suggest that maternal exposure to isoflurane during late pregnancy harms hippocampal neurons, thus impairing learning and memory in the offspring. Previous results showed that prenatal exposure to 1.3% isoflurane for 4 hours on gestational day 14 led to impaired synaptic ultrastructure in the hippocampus of the offspring and thus causing poor learning and memory [38].
+
+Recently, histone acetylation, which is regulated by histone deacetylases (HDAC), has been
+
+implicated in memory formation [39–43]. Increasing histone-tail acetylation can facilitate learning and memory [12, 13]. Further studies showed that HDAC2, but not HDAC1, decreases dendritic spine density, synapse number, synaptic plasticity and memory formation [15]. HDAC2 regulates learning and memory via the transcription factor CREB and the recruitment of the transcriptional coactivator and histone acetyltransferase CREB-binding pro- tein (CBP) via the CREB-binding domain of CBP [44]. The inhibition of HDAC can modulate hippocampal-dependent long-term memory in a CBP-dependent manner [45]. Inhibiting HDAC increases the levels of acetylated histones and phospho-CREB (p-CREB), which enhances the binding of p-CREB to its binding site at the promoter of the NR2B gene, thus increasing the expression of NR2B in the hippocampus [17]. Thus, HDAC2 impairs learning and memory through a pathway involving HDAC2-CREB-NR2B. NR2B is critical to the for- mation and maintenance of learning and memory [46–48]. It is undetermined whether mater- nal isoflurane exposure during late pregnancy damage the spatial learning and memory in offspring via this pathway. The hippocampus is a critical structure for learning and memory. Thus, the expression levels of HDAC2, CREB, and NR2B mRNA and NR2B protein in the hip- pocampus of the offspring were analyzed in the present study. The results showed that mater- nal isoflurane exposure during late pregnancy increased the expression of HDAC2 mRNA, decreased CREB mRNA and NR2B mRNA and protein in the hippocampus of the offspring. This is similar to a previous report that maternal anesthesia with ketamine on G14 downregu- lated the expression of NR2B in the hippocampus of offspring [49]. These results indicate that maternal isoflurane exposure during late pregnancy causes the over-expression of HDAC2, thereby inhibiting the expression of CREB mRNA, resulting in downregulation of NR2B in the hippocampus of the offspring rats. These effects lead to impaired learning and memory in the offspring. NMDA receptor blockade acts critical role in determining whether neurons are reversibly injured or are driven to cell death by isoflurane [50]. Thus, these results indicate that maternal isoflurane exposure during late pregnancy can damage the learning and memory of the offspring rats via the HDAC2-CREB -NR2B pathway.
+
+Further supporting the role of HDAC2, CREB and NR2B in the learning and memory dys-
+
+function of the offspring, we showed that SAHA (an HDAC inhibitor that mainly inhibits HDAC2) treatment 2 hours before each Morris water maze trial reversed the impaired learning and memory and the alterations in expression of HDAC2, CREB and NR2B mRNA and
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+14 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+protein in the hippocampus. Many lines of evidence showed that HDAC inhibitors that mainly inhibit HDAC2 potentiate learning and memory. The class I HDAC inhibitor RGFP963 can enhance the consolidation of cued fear extinction [51]. Kinetically selective HDAC2 inhibitors rescued the memory deficits in mice with neurodegenerative disease by increasing H4K12 and H3K9 histone acetylation in hippocampal neurons [52]. SAHA facilitated fear extinction of rats by enhancing the expression of hippocampal NR2B-containing NMDA receptors. The lev- els of acetylated histones in the hippocampus increased significantly 2 hours after SAHA administration and were accompanied by enhanced binding of p-CREB to its binding site at the promoter of the NR2B gene [17], resulted in the increase of of NR2B mRNA levels, but not NR1 or NR2A mRNA. Therefore, we administered SAHA to the offspring 2 hours before every water maze trial. It is impossible to know whether the learning and memory improvements caused by SAHA in animals exposed to isoflurane are due to its general compensatory effects or a specific reversal of the effects of isoflurane. However, the effects of SAHA on animals exposed to isoflurane, along with the effects of isoflurane on HDAC, suggest that isoflurane may act on HDAC to affect learning and memory. The results of the present study provided the first demonstration that an HDAC inhibitor reverses the learning and memory impair- ments caused by maternal isoflurane exposure during late pregnancy. β-amyloid protein accu- mulation, caspase activation [53, 54], inositol 1,4,5-trisphosphate (IP3) receptor activation [36, 55] and calcium dysregulation [56] are critical pathological mechanisms in the neurotoxicity caused by isoflurane. It remains to be clarified whether these mechanisms are involved in the learning and memory impairment caused by maternal isoflurane exposure.
+
+The maintenance of normal cognition is known to require precise excitatory–inhibitory (E/ I) balance [57]. Disrupted NMDA-receptor signaling may be a molecular substrate common to a number of neurodevelopmental, neuropsychiatric disorders [58]. NR2B is an excitatory receptor and plays a critical role in the maintenance and formation of normal learning and memory. NR2B signaling can be maintained at a normal range to keep the brain in E/I balance. The rats in the CS group in the current study were normal offspring that had not exposed to isoflurane during pregnancy and had normal levels of inhibitory receptors. It is possible that they could maintain normal NR2B function by autoregulation or other pathways. NR2B pro- tein expression in the I2, I4 and I8 groups had been inhibited by maternal isoflurane exposure. Therefore, the expression levels of NR2B protein in these rats could be increased by SAHA via the HDAC2-CREB-NR2B pathway [16, 17] to maintain the E/I balance. Protein levels are dependant on numerous factors, including gene transcription, translation and the number and functional state of cells that produce the protein. Maybe there are some unknow factors affect- ing the mRNA translating to protein, thus result in the increasing degree of NR2B protein was different from that of NR2B mRNA in the groups of I2S, I4S and I8S. Future studies are needed to determine if there are other factors affecting the translation of mRNA to protein in this pathway.
+
+The present study only assessed the ultrastructural and molecular changes in the offspring
+
+brains after the behaviour test. The acute changes in the fetal brains immediately or several hours after isoflurane exposure had not been evaluated. Whether pretreatment with SAHA prior to isoflurane exposure will block all the harmful effects caused by isoflurane in offspring is not know yet. These questions require further clarification.
+
+Taken together, the results in the present study suggested that maternal exposure of rats to
+
+isoflurane during late pregnancy impairs the spatial learning and memory of the offspring. This effect was associated with damage to hippocampal neurons, the overexpression of HDAC2 mRNA and the subsequent downregulation of CREB mRNA and NR2B. HDAC2 inhibition improved the impaired learning and memory of the offspring induced by maternal exposure to isoflurane.
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+15 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+Author Contributions
+
+Conceived and designed the experiments: FL WZ.
+
+Performed the experiments: YH QY BL LW ZL LX YF JL.
+
+Analyzed the data: FL ZZ.
+
+Contributed reagents/materials/analysis tools: ZL LX.
+
+Wrote the paper: FL ZZ YF.
+
+References 1.
+
+Li Y, Liang G, Wang S, Meng Q, Wang Q, Wei H. Effects of fetal exposure to isoflurane on postnatal memory and learning in rats. Neuropharmacology. 2007; 53(8):942–50. PMID: 17959201
+
+2. Wang S, Peretich K, Zhao Y, Liang G, Meng Q, Wei H. Anesthesia-induced neurodegeneration in fetal rat brains. Pediatr Res. 2009; 66(4):435–40. PMID: 20016413 doi: 10.1203/PDR.0b013e3181b3381b
+
+3. Kort B, Katz VL, Watson WJ. The effect of nonobstetric operation during pregnancy. Surg Gynecol Obstet. 1993; 177(4):371–6. PMID: 8211581
+
+4. Mazze RI, Källén B. Reproductive outcome after anesthesia and operation during pregnancy: a registry study of 5405 cases. Am J Obstet Gynecol. 1989; 161(5):1178–85. PMID: 2589435
+
+5. Brodsky JB, Cohen EN, Brown BW Jr, Wu ML, Whitcher C. Surgery during pregnancy and fetal out- come. Am J Obstet Gynecol. 1980; 138(8):1165–7. PMID: 7446625
+
+6. Baldwin EA, Borowski KS, Brost BC, Rose CH. Antepartum nonobstetrical surgery at (cid:3)23 weeks’ ges- tation and risk for preterm delivery. Am J Obstet Gynecol. 2015; 212(2):232.e1–5. PMID: 25218955
+
+7. Van De Velde M, De Buck F. Anesthesia for non-obstetric surgery in the pregnant patient. Minerva Anestesiol. 2007; 73(4):235–40. PMID: 17473818
+
+8.
+
+Zhang Qin,Luo Foquan,Zhao Weilu,Li Bingda,Tang Yang,Hu Yan.Effect of prolonged anesthesia with propofol during early pregnancy on cognitive function of offspring rats. Chin J Anesthesiol, 2014; 34(9): 1051–1053. doi: 10.3760/cma.j.issn.0254-1416.2014.09.005
+
+9. Palanisamy A, Baxter MG, Keel PK, Xie Z, Crosby G, Culley DJ. Rats exposed to isoflurane in utero during early gestation are behaviorally abnormal as adults. Anesthesiology. 2011; 114(3):521–8. PMID: 21307768 doi: 10.1097/ALN.0b013e318209aa71
+
+10. Guan Z, Giustetto M, Lomvardas S, Kim JH, Miniaci MC, Schwartz JH, et al. Integration of long-term- memory-related synaptic plasticity involves bidirectional regulation of gene expression and chromatin structure. Cell. 2002; 111(4):483–93. PMID: 12437922
+
+11. Korzus E, Rosenfeld MG, Mayford M. CBP histone acetyltransferase activity is a critical component of memory consolidation. Neuron. 2004; 42(6):961–72. PMID: 15207240
+
+12.
+
+Levenson JM, O'Riordan KJ, Brown KD, Trinh MA, Molfese DL, Sweatt JD. Regulation of histone acety- lation during memory formation in the hippocampus. J Biol Chem. 2004; 279(39):40545–59. PMID: 15273246
+
+13. Vecsey CG, Hawk JD, Lattal KM, Stein JM, Fabian SA, Attner MA, et al. Histone deacetylase inhibitors enhance memory and synaptic plasticity via CREB:CBP-dependent transcriptional activation. J Neu- rosci. 2007; 27(23):6128–40. PMID: 17553985
+
+14.
+
+Fischer A, Sananbenesi F, Wang X, Dobbin M, Tsai LH. Recovery of learning and memory is associ- ated with chromatin remodelling. Nature. 2007; 447(7141):178–82. PMID: 17468743
+
+15. Guan JS, Haggarty SJ, Giacometti E, Dannenberg JH, Joseph N, Gao J, et al. HDAC2 negatively regu- lates memory formation and synaptic plasticity. Nature. 2009; 459(7243):55–60. PMID: 19424149 doi: 10.1038/nature07925
+
+16. Almeida S, Cunha-Oliveira T, Laço M, Oliveira CR, Rego AC. Dysregulation of CREB activation and histone acetylation in 3-nitropropionic acid-treated cortical neurons: prevention by BDNF and NGF. Neurotox Res. 2010; 17(4):399–405. PMID: 19779956 doi: 10.1007/s12640-009-9116-z 17. Fujita Y, Morinobu S, Takei S, Fuchikami M, Matsumoto T, Yamamoto S, et al. SAHA, a histone deace- tylase inhibitor, facilitates fear extinction and enhances expression of the hippocampal NR2B-contain- ing NMDA receptor gene. J Psychiatr Res. 2012; 46(5):635–43. PMID: 22364833 doi: 10.1016/j. jpsychires.2012.01.026
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+16 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+18.
+
+Zhong T, Qing QJ, Yang Y, Zou WY, Ye Z, Yan JQ, et al. Repression of contexual fear memory induced by isoflurane is accompanied by reduction in histone acetylation and rescued by sodium butyrate. Br J Anaesth. 2014; 113(4):634–43. PMID: 24838805 doi: 10.1093/bja/aeu184
+
+19.
+
+Zhong T, Guo Q, Zou W, Zhu X, Song Z, Sun B, et al. Neonatal isoflurane exposure induces neurocog- nitive impairment and abnormal hippocampal histone acetylation in mice. PLoS One. 2015; 10(4): e0125815. PMID: 25928815 doi: 10.1371/journal.pone.0125815
+
+20. Barraco RD, Chiu WC, Clancy TV, Como JJ, Ebert JB, Hess LW, et al. EAST Practice Management Guidelines Work Group.Practice management guidelines for the diagnosis and management of injury in the pregnant patient: the EAST Practice Management Guidelines Work Group. J Trauma. 2010; 69 (1):211–4. PMID: 20622592 doi: 10.1097/TA.0b013e3181dbe1ea
+
+21. Machado NO, Machado LS. Laparoscopic cholecystectomy in the third trimester of pregnancy: report of 3 cases. Surg Laparosc Endosc Percutan Tech. 2009; 19(6):439–41. PMID: 20027085 doi: 10. 1097/SLE.0b013e3181c30fed
+
+22. Upadhyay A, Stanten S, Kazantsev G, Horoupian R, Stanten A. Laparoscopic management of a nonob- stetric emergency in the third trimester of pregnancy. Surg Endosc. 2007; 21(8):1344–8. PMID: 17285387
+
+23. Buser KB. Laparoscopic surgery in the pregnant patient:results and recommendations. JSLS. 2009; 13(1):32–5. PMID: 19366538
+
+24. Qaiser R, Black P. Neurosurgery in pregnancy. Semin Neurol. 2007; 27(5):476–81. PMID: 17940927 25. Holthausen UH, Mettler L, Troidl H. Pregnancy: A contraindication? World J Surg.1999; 23(8):856–62. PMID: 10415212
+
+26. Affleck DG, Handrahan DL, Egger MJ, Price RR. The laparoscopic management of appendicitis and cholelithiasis during pregnancy. Am J Surg.1999; 178(6):523–9. PMID: 10670865
+
+27. Meshikhes AN. Successful laparoscopic cholecystectomy in the third trimester of pregnancy. Saudi Med J. 2008; 29(2):291–2. PMID: 18246244
+
+28. Choi JJ, Mustafa R, Lynn ET, Divino CM. Appendectomy during pregnancy: follow-up of progeny. J Am Coll Surg. 2011; 213(5):627–32. PMID: 21856183 doi: 10.1016/j.jamcollsurg.2011.07.016
+
+29. Rohan D, Buggy DJ, Crowley S, Ling FK, Gallagher H, Regan C, et al. Increased incidence of postoper- ative cognitive dysfunction 24 hr after minor surgery in the elderly. Can J Anaesth. 2005; 52(2):137–42. PMID: 15684252
+
+30. Wang C, Anastasio N, Popov V, Leday A, Johnson KM. Blockade of N-methyl-D-aspartate receptors by phencyclidine causes the loss of corticostriatal neurons. Neuroscience. 2004; 125(2):473–83. PMID: 15062989
+
+31. Rodier PM. Chronology of neuron development: animal studies and their clinical implications. Dev Med Child Neurol. 1980; 22(4): 525–45. PMID: 7409345
+
+32. Bing-da LI,Fuo—quan LUO,Wei—lu ZHAO,Yang TANG,Qin ZHANG,Yan HU.Effect of ketamine anes- thesia in early pregnancy on expression of hippyragranin mRNA in hippocampus in offsprings of rats. Chin J Anesthesiol, 2012, 32(11): 1334–1336. doi: 10.3760/cma.j.issn.0254-1416.2012.11.013
+
+33. Mazze RI, Fujinaga M, Rice SA, Harris SB, Baden JM. Reproductive and teratogenic effects of nitrous oxide, halothane, isoflurane and enflurane in Sprague-Dawley rats. Anesthesiology. 1986; 64(3):339– 44. PMID: 3954129
+
+34. Mazze RI, Rice SA, Baden JM. Halothane, isoflurane and enflurane MAC in pregnant and nonpregnant female and male mice and rats. Anesthesiology. 1985; 62(3):339–41. PMID: 3977116
+
+35. Wei H, Kang B, Wei W, Liang G, Meng QC, Li Y, et al. Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently. Brain Res. 2005; 1037(1–2):139–47. PMID: 15777762
+
+36.
+
+Zhao X, Yang Z, Liang G, Wu Z, Peng Y, Joseph DJ, et al. Dual effects of isoflurane on proliferation, dif- ferentiation, and survival in human neuroprogenitor cells. Anesthesiology. 2013; 118(3):537–49. PMID: 23314167 doi: 10.1097/ALN.0b013e3182833fae
+
+37. Hofacer RD, Deng M, Ward CG, Joseph B, Hughes EA, Jiang C, et al. Cell age-specific vulnerability of neurons to anesthetic toxicity. Ann Neurol. 2013; 73(6):695–704. PMID: 23526697 doi: 10.1002/ana. 23892
+
+38. Kong F, Xu L, He D, Zhang X, Lu H. Effects of gestational isoflurane exposure on postnatal memory and learning in rats. Eur J Pharmacol. 2011; 670(1):168–74. PMID: 21930122 doi: 10.1016/j.ejphar. 2011.08.050
+
+39. Morris MJ, Mahgoub M, Na ES, Pranav H, Monteggia LM. Loss of histone deacetylase 2 improves working memory and accelerates extinction learning. J Neurosci. 2013; 33(15):6401–11. PMID: 23575838 doi: 10.1523/JNEUROSCI.1001-12.2013
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+17 / 18
+
+Maternal Exposure to Isoflurane Impairs Memory in Offspring by HDAC2
+
+40. Mahgoub M, Monteggia LM. A role for histone deacetylases in the cellular and behavioral mechanisms underlying learning and memory. Learn Mem. 2014; 21(10):564–8. PMID: 25227251 doi: 10.1101/lm. 036012.114
+
+41. Penney J, Tsai LH. Histone deacetylases in memory and cognition. Sci Signal. 2014; 7(355):re12. PMID: 25492968 doi: 10.1126/scisignal.aaa0069
+
+42.
+
+Fass DM, Reis SA, Ghosh B, Hennig KM, Joseph NF, Zhao WN, et al. Crebinostat: a novel cognitive enhancer that inhibits histone deacetylase activity and modulates chromatin-mediated neuroplasticity. Neuropharmacology. 2013; 64:81–96. PMID: 22771460 doi: 10.1016/j.neuropharm.2012.06.043
+
+43. Maddox SA, Schafe GE. Epigenetic alterations in the lateral amygdala are required for reconsolidation of a Pavlovian fear memory. Learn Mem. 2011; 18(9):579–93. PMID: 21868438 doi: 10.1101/lm. 2243411
+
+44. Bambah-Mukku D, Travaglia A, Chen DY, Pollonini G, Alberini CM. A positive autoregulatory BDNF feedback loop via C/EBPβ mediates hippocampal memory consolidation. J Neurosci. 2014; 34 (37):12547–59. PMID: 25209292 doi: 10.1523/JNEUROSCI.0324-14.2014
+
+45. Haettig J, Stefanko DP, Multani ML, Figueroa DX, McQuown SC, Wood MA. HDAC inhibition modu- lates hippocampus-dependent long-term memory for object location in a CBP-dependent manner. Learn Mem. 2011; 18(2):71–9. PMID: 21224411 doi: 10.1101/lm.1986911
+
+46. Bliss TV. Young receptors make smart mice. Nature. 1999; 401(6748):25–7. PMID: 10485698
+
+47.
+
+Tang YP, Shimizu E, Dube GR, Rampon C, Kerchner GA, Zhuo M, et al. Genetic enhancement of learn- ing and memory in mice. Nature. 1999; 401(6748):63–9. PMID: 10485705
+
+48. Plattner F, Hernández A, Kistler TM, Pozo K, Zhong P, Yuen EY, et al. Memory enhancement by target- ing Cdk5 regulation of NR2B. Neuron. 2014; 81(5):1070–83. PMID: 24607229 doi: 10.1016/j.neuron. 2014.01.022
+
+49.
+
+Zhao T, Li Y, Wei W, Savage S, Zhou L, Ma D. Ketamine administered to pregnant rats in the second tri- mester causes long-lasting behavioral disorders in offspring. Neurobiol Dis. 2014; 68:145–55. PMID: 24780497 doi: 10.1016/j.nbd.2014.02.009
+
+50.
+
+Jevtovic-Todorovic V, Beals J, Benshoff N, Olney JW. Prolonged exposure to inhalational anesthetic nitrous oxide kills neurons in adult rat brain. Neuroscience. 2003; 122(3):609–16. PMID: 14622904
+
+51. Bowers ME, Xia B, Carreiro S, Ressler KJ. The Class I HDAC inhibitor RGFP963 enhances consolida- tion of cued fear extinction. Learn Mem. 2015; 22(4):225–31. PMID: 25776040 doi: 10.1101/lm. 036699.114
+
+52. Wagner FF, Zhang YL, Fass DM, Joseph N, Gale JP, Weïwer M, et al. Kinetically Selective Inhibitors of Histone Deacetylase 2 (HDAC2) as Cognition Enhancers. Chem Sci. 2015; 6(1):804–15. PMID: 25642316
+
+53. Pan C, Xu Z, Dong Y, Zhang Y, Zhang J, McAuliffe S, et al. The potential dual effects of anesthetic iso- flurane on hypoxia-induced caspase-3 activation and increases in β-site amyloid precursor protein- cleaving enzyme levels. Anesth Analg. 2011; 113(1):145–52. PMID: 21519046 doi: 10.1213/ANE. 0b013e3182185fee
+
+54. Sun Y, Zhang Y, Cheng B, Dong Y, Pan C, Li T, et al. Glucose may attenuate isoflurane-induced cas- pase-3 activation in H4 human neuroglioma cells. Anesth Analg. 2014; 119(6):1373–80. PMID: 25068691 doi: 10.1213/ANE.0000000000000383
+
+55. Wei H, Liang G, Yang H, Wang Q, Hawkins B, Madesh M, et al. The common inhalational anesthetic isoflurane induces apoptosis via activation of inositol 1,4,5-trisphosphate receptors. Anesthesiology. 2008; 108(2):251–60. PMID: 18212570 doi: 10.1097/01.anes.0000299435.59242.0e
+
+56. Wei H. The role of calcium dysregulation in anesthetic-mediated neurotoxicity. Anesth Analg. 2011; 113(5):972–4. PMID: 22021793 doi: 10.1213/ANE.0b013e3182323261
+
+57. Sohal VS, Zhang F, Yizhar O, Deisseroth K. Parvalbumin neurons and gamma rhythms enhance corti- cal circuit performance. Nature. 2009; 459(7247): 698–702. PMID: 19396159 doi: 10.1038/ nature07991
+
+58. Gandal MJ, Sisti J, Klook K, Ortinski PI, Leitman V, Liang Y, Thieu T, Anderson R, Pierce RC, Jonak G, Gur RE, Carlson G, Siegel SJ. GABAB-mediated rescue of altered excitatory-inhibitory balance, gamma synchrony and behavioral deficits following constitutive NMDAR-hypofunction. Transl Psychia- try. 2012; 2:e142. PMID: 22806213 doi: 10.1038/tp.2012.69
+
+PLOS ONE | DOI:10.1371/journal.pone.0160826 August 18, 2016
+
+18 / 18

new_pdfs/10.18632_oncotarget.15405.txt

@@ -0,0 +1,391 @@
+Research Paper: Neuroscience Ketamine administered pregnant rats impair learning and memory in offspring via the CREB pathway
+
+Xinran Li1, Cen Guo1, Yanan Li1, Lina Li1, Yuxin Wang1, Yiming Zhang1, Yue Li1, Yu Chen1, Wenhan Liu1 and Li Gao1 1 College of Veterinary Medicine, Northeast Agricultural University, Harbin, China
+
+Correspondence to: Li Gao, email: gaoli43450@163.com
+
+Keywords: CREB pathway, ketamine, learning and memory, pregnant rats, rat offspring, Neuroscience
+
+Received: December 21, 2016
+
+Accepted: January 27, 2017
+
+Published: February 16, 2017
+
+Copyright: Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
+
+ABSTRACT
+
+Ketamine has been reported to impair the capacity for learning and memory. This study examined whether these capacities were also altered in the offspring and investigated the role of the CREB signaling pathway in pregnant rats, subjected to ketamine-induced anesthesia. On the 14th day of gestation (P14), female rats were anesthetized for 3 h via intravenous ketamine injection (200 mg/Kg). Morris water maze task, contextual and cued fear conditioning, and olfactory tasks were executed between the 25th to 30th day after birth (B25-30) on rat pups, and rats were sacrificed on B30. Nerve density and dendritic spine density were examined via Nissl’s and Golgi staining. Simultaneously, the contents of Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII), p-CaMKII, CaMKIV, p-CaMKIV, Extracellular Regulated Protein Kinases (ERK), p-ERK, Protein Kinase A (PKA), p-PKA, cAMP-Response Element Binding Protein (CREB), p-CREB, and Brain Derived Neurotrophic Factor (BDNF) were detected in the hippocampus. We pretreated PC12 cells with both PKA inhibitor (H89) and ERK inhibitor (SCH772984), thus detecting levels of ERK, p-ERK, PKA, p-PKA, p-CREB, and BDNF. The results revealed that ketamine impaired the learning ability and spatial as well as conditioned memory in the offspring, and significantly decreased the protein levels of ERK, p-ERK, PKA, p-PKA, p-CREB, and BDNF. We found that ERK and PKA (but not CaMKII or CaMKIV) have the ability to regulate the CREB-BDNF pathway during ketamine-induced anesthesia in pregnant rats. Furthermore, ERK and PKA are mutually compensatory for the regulation of the CREB-BDNF pathway.
+
+INTRODUCTION
+
+Ketamine abuse causes more severe problems during pregnancy [1]. In addition, between 0.75% and 2% of pregnant women require surgery either related to the pregnancy or to unrelated medical problems [2]. Furthermore, with the popularity of minimally invasive surgery, surgery during pregnancy has become increasingly widespread [3]. Therefore, the risks of anesthetic administration (such as ketamine) for the fetus have become more important. Unfortunately, only few reports investigated the effects of generally utilized anesthesia on neurodevelopmental consequences for a fetus prior to birth [4-6].
+
+It is well known that the hippocampus plays a central role for learning and memory processes [7], and
+
+consequently, this is a known target for drug regulation. Ketamine is a high affinity uncompetitive antagonist of voltage dependent N-Methyl-D-aspartic Acid Receptor (NMDAR) and has been used for decades as a dissociative anesthetic that also appears to be a useful tool in psychiatric research [8]. Ketamine can attenuate learning and memory impairment, particularly for short- term memory [9]. The cAMP-Response Element Binding Protein (CREB) has been reported to be involved in the learning and memory deficits, caused by ketamine [10].
+
+Ketamine can enter the fetus through the placental barrier, where it may exert a stronger impact on the fetus since the fetal brain is still in a stage of development. Even normal use of ketamine may affect the fetus. Therefore, this study examined whether the abilities of learning and memory were altered in the offspring as well as the
+
+32433
+
+ketamine-induced effect on CREB signaling pathways in ketamine-induced pregnant rats on gestational day 14.
+
+Morris water maze test
+
+RESULTS
+
+Nissl’s staining
+
+We selected three 104 μm2 areas to conduct a neuron count in both the CA1 (Figure 2c-2d) and CA3 (Figure 2e- 2f) regions of the hippocampus. As shown in Figure 2a-2g, the cell density of K group decreased by 20.5% compared to C group (p < 0.05).
+
+Morris water maze test data revealed that rat pups had suffered from a significant main effect on escape latency during spatial training on testing days 2-3 (Figure 4a). In contrast, the escape latency did not reveal any significant differences between the C group and the K group during place navigation trials on day 4-5 (p = 0.223) and spatial probe tests (p = 0.062, Figure 4b). It is worth noting that no significant differences in animals’ swimming speeds were detected (C group: 25.00 ± 1.96 cm/s and K group: 26.00 ± 1.33 cm/s, according to a one- way ANOVA: F = 0.191, p = 0.827) between the C and K group (Figure 4c-4d).
+
+Golgi staining
+
+Contextual and cued fear conditioning
+
+Fully impregnated CA1 pyramidal cells can be detected via Golgi staining, and the spines of the apical dendrites can be analyzed under a light microscope using a 200 × oil immersion objective. We randomly selected 10 μm apical (Figure 3a-3b) and basal (Figure 3c-3d) from the same neurological level to count the number of dendritic spines. Only the density of apical and basal dendrites was determined in our study, as several different types of spines were not always clearly visible (e.g., thin, mushroom, or branched dendrites). Spine density decreased by 21.6% in the K group compared to the C group (P < 0.05, Figure 3g).
+
+Contextual and cued fear conditioning is a standard fear conditioning task that measures the ability of rat pups to learn and remember an association between an averse experience and environmental cues. In contextual and cued fear conditioning, a significant difference was found in CS (p < 0.05), while no significant difference was found between K and C group in other tests (Figure 5b).
+
+Olfactory tasks
+
+As shown in Figure 6b, during the acquisition stage, no significant differences were found in the investigation time of Hole 1 and Hole 2, indicating that rats had no
+
+Figure 1: Mating and drug administration. The Vaginal suppository (b) and sperm (c) were observed and female rats were defined as pregnant at day 0 (P0). Female rats were anesthetized via intravenous ketamine injection on P14. The first day after birth was recorded as B0. During B25-B30, behavioral testing was utilized to test the learning and memory capacities (a).
+
+32434
+
+Figure 2: Nissl’s staining was utilized to observe neuronal cells. a. and b. Areas of 104 μm2 were selected and neuron numbers were counted in the CA1 c. and d. and CA3 e. and f. regions of the hippocampus. g. Cells within K group decreased compared to C group (p < 0.05).
+
+32435
+
+Figure 3: Golgi staining revealed hippocampal dendritic spine density. 10 μm of apical a. and b. and basal c. and d. dendrites were randomly selected from each pyramidal neuron for inspection (via 200 × oil immersion lens) to quantify spinal density. e. Spinal density decreased by 21.6% in K group compared to C group (p < 0.05).
+
+32436
+
+Table 1: Results of CCK-8 test Concentration of ketamine (μg/mL) Cell viability
+
+1 17%
+
+0.9 34%
+
+0.8 39%
+
+0.7 43%
+
+0.6 49%
+
+0.5 53%
+
+0.4 64%
+
+0.3 75%
+
+0.2 76%
+
+specific preference for carvone or limonene. During the recall stage, no significant differences were found in the investigation time of novel odor between K group and C group.
+
+Ketamine exposure affects protein expression in the hippocampus
+
+Results of the CCK-8 test
+
+To evaluate the optimum concentration of ketamine for PC12 cells, a CCK8 assay was performed. PC12 cells were exposed to ketamine at different concentrations (0.2 μg/mL, 0.3 μg/mL, 0.4 μg/mL, 0.5 μg/mL, 0.6 μg/ mL, 0.7 μg/mL, 0.8 μg/mL, 0.9 μg/mL, 1μg/mL) for 3 h. According to Table 1, the 50% cell viability of ketamine for PC12 cells was 0.6 μg/mL.
+
+To examine whether ketamine treatment has the ability to alter the protein expression of learning and memory related proteins, we measured protein levels of CaMKII, p-CaMKII, CaMKIV, p-CaMKIV, ERK, p-ERK, PKA, p-PKA, p-CREB, and BDNF in the rat hippocampus. As shown in Figure 7 and in comparison to the values of each corresponding C group, no significant difference was found in the protein levels of CaMKII, p-CaMKII, CaMKIV, and p-CaMKIV. However, the protein levels of ERK, p-ERK, PKA, p-PKA, p-CREB, and BDNF had significantly decreased (p < 0.05) to 91.6%, 71.1%, 74.5%, 92.5%, 67.4%, and 64.2% of their original values, respectively, while the CREB protein level significantly increased (p < 0.05) to 129% (Figure 8).
+
+Figure 4: To test hippocampus-dependent spatial cognition, rats were trained in the standard morris water maze. Rat pups showed significant main effects in their escape latency during spatial training during testing days 2-3 a. During the 60 s probe trial, we recorded and analyzed the swimming path tracks c. and d.; however, no significant differences were detected in the spatial probe test b.
+
+32437
+
+Figure 5: Contextual and cued fear conditioning is a fear conditioning task that measures the ability of a rat to learn and remember an association between an aversive experience and environmental cues. a. Experimental process of contextual and cued fear conditioning. b. A significant difference was found in CS between K and C group.
+
+32438
+
+Ketamine exposure affects protein expression in PC12 cells
+
+No significant difference was found between C
+
+and D group. Compared to C group, the protein levels of ERK, p-ERK, PKA, p-PKA, CREB, p-CREB, and BDNF decreased significantly (p < 0.05): p-ERK decreased to 70.9% in S group (p < 0.05), p-PKA decreased to 74.4% in H group (p < 0.05), and the protein levels of p-ERK,
+
+Figure 6: Olfactory discrimination tasks are excellent measures of learning and memory in rats. The acquisition test (one session) consisted of presentation of one odor (limonene or carvone), presented in both holes. The recall test consisted of a 3 min session in which one hole was odorized with the previously presented odor, while the other hole with a novel odor (a). There was no significant difference in the investigation time of novel odor between K group and C group (b).
+
+32439
+
+p-PKA, p-CREB, and BDNF decreased to 53.7%, 68.1%, 72.7%, and 66.1% (p < 0.05). The protein levels of p-ERK decreased by 24.3% in S+H group compared to S group, while the p-PKA protein levels decreased by 8.5% in S+H group compared to H group (Figure 9).
+
+DISCUSSION
+
+The morris water maze task, contextual and cued fear conditioning, and olfactory tasks enable the evaluation of learning and memory of spatial, conditioned, and odor cues in rat pups (Figure 4-6). Nerve density and dendritic spine density decreased, which all influenced the nerve conduction efficiency as well as learning ability and memory capacity [6, 11]. However, during the anaesthetization process, anesthetic stress, oxygen saturation, gastrointestinal tract squeezing the uterus, change of placental blood flow, supine position, oppressing blood vessels, and other factors all impact on fetal rats. To exclude these effects from our analysis, we cultured PC12 cells, exploring the effect of ketamine on the CREB pathway.
+
+The prevalence of substance abuse in pregnant women is similar to that of the general population, resulting in an increased fetal exposure rate during the most vulnerable period of neurodevelopment and organogenesis. Many pregnant women are exposed to various types of anesthetics for surgery or diagnostic procedures every year. Furthermore, numerous women will also undergo surgery during pregnancy, unrelated to childbirth. Consensus is that fetal exposure to alcohol is harmful. Prenatal alcohol exposure may induce abnormal brain development as well as decrease the capacity for learning and memory [12, 13]. Similar to alcohol, anticonvulsants, sedatives (such as ketamine), or narcotics can pass through the placental barrier and for ketamine in particular, researches showed its ability to impair the capacity for learning and memory [14, 15]. Evidence links early exposure to anesthesia with cognitive impairment [16]. In addition, ketamine is also one of the most commonly used drugs in pediatric clinical anesthesia and its reported influence on learning and memory has always been of clinical concern. Moreover, ketamine is a frequently abuse drug in the public, which
+
+Figure 7: Ketamine exposure affects protein expression in the hippocampus. No signifcant difference was found in the protein levels of CaMKII. a., p-CaMKII b., CaMKIV c., and p-CaMKIV d.
+
+32440
+
+includes pregnant women [17]. Zhang et al. suggests that repeated ketamine exposure induced long-term cognitive impairment via increased NOX2 [18]. Experimental evidence indicates that the NMDAR antagonist ketamine impairs cognition [19]. Prolonged ketamine exposure in neonates at anesthetic doses has been reported to cause long-term impairments of learning and memory [20]. Furthermore, ketamine decreased p-CREB in the hippocampus [21, 22], and decreased levels of BDNF [23]. CREB has been demonstrated to be involved in learning and memory deficits caused by ketamine [10, 22]. These findings raise concern about potential adverse effects of ketamine exposure to fetuses and infants.
+
+In our study, the ratio of P-CREB/total CREB was decreased in the rat hippocampus. The function of CREB is dominantly regulated by phosphorylation at Ser133, which results in the activation of gene transcription [24]. The Phosphorylated CREB protein recruits the transcriptional activator CREB-binding protein (CBP), thus stimulating the transcription of CRE-regulated genes that are involved in neurogenesis and neuritogenesis [25]. We therefore hypothesize that P-CREB may be responsible for compensatory increases in CREB protein levels; however, further testing is required to confirm this hypothesis. p-CREB promotes immediate early genes such as the c-fos gene via interaction with the CRE sequence located within promotor regions (TGACGTCA) [26]. Genetic deletion of CREB selectively impaired the hippocampus-dependent spatial memory of mice subjected to the Morris water maze [27], which coincided
+
+with our results. Furthermore, CREB phosphorylation is a necessary step in the process leading to the generation of new dendritic spines [27]. In addition, the cAMP–CREB signaling cascade is critical for the generation of new neurons in the rodent hippocampus, also facilitating their subsequent morphological maturation [28].
+
+Several findings have shown that the dysregulation of involved in cognitive impairment [29]. The neurons of the hippocampus of aged animals showed a down-regulation of BDNF and p-CREB expression, associated with learning and memory impairment [30, 31], which was also similar with our result. In this study, BDNF and p-CREB revealed the same tendency (Figure 9g-9h). BDNF has also been reported to elicit rapid action potentials, thus influencing neuronal excitability, and it has a demonstrated role in activity-dependent synaptic plasticity events such as long-term potentiation, learning tasks, and memory [32, 33]. BDNF is involved in structural remodeling, neuronal plasticity, and synaptic restructuring [34, 35]. Several signaling pathways,
+
+the CREB-BDNF cascade has been
+
+those involving CaMKII, CaMKIV, ERK, and PKA, have been associated with the regulation of de novo protein synthesis in the context of synaptic plasticity, converging on the phosphorylation of CREB at Ser133 residue (Figure 10). It is generally accepted that ketamine blocks NMDAR, thus mediating the neurotransmission of postsynaptic receptors [36]. NMDAR in turn mediates the release of neurotransmitters (such as acetylcholine, dopamine, GABA, and NE), and regulates the levels of sodium and
+
+including
+
+Figure 8: Ketamine exposure affects protein expression in the hippocampus. Protein levels of ERK a., p-ERK b., PKA c., p-PKA d., p-CREB f., and BDNF g. signifcantly decreased in the hippocampus (p < 0.05) to 91.6%, 71.1%, 74.5%, 92.5%, 67.4%, and 64.2%, of their initial values, while the CREB e. protein level signifcantly increased to 129%..
+
+32441
+
+calcium. The increased association between CaMK II and CREB, followed by phosphorylation of CREB in response to Wnt5a stimulation was suppressed in a minimal hepatic encephalopathy rat model [37]. Ca2+ signaling not only plays a critical role in regulating apoptosis and autophagy [38], but also affects CREB phosphorylation. Cohen reported that CREB phosphorylation also proceeds with slow, sigmoid kinetics, that are rate-limited due to the paucity of CaMKIV, protecting against saturation of phospho-CREB as a response to increased firing rates and elevated Ca2+ transients [39]. Interestingly, no significance was found in the protein levels of CaMKII, p-CaMKII, CaMKIV, and p-CaMKIV in rat pups (Figure 7). Liu’s research demonstrated that low-intensity pulsed ultrasound increased the intracellular concentration of calcium and enhanced protein levels of CaMKII and
+
+CaMKIV; however, it did not promote the activation of CREB [40]. CaMKII was markedly decreased following a stress-priming methamphetamine-induced conditioned place preference reinstatement test; however, p-CREB expressions in the medial prefrontal cortex were increased [41]. Guo reported that ERK and CREB phosphorylation was not mediated by CaMK [42]. Therefore, we speculate here that the effect of ketamine on CaMKII and CaMKIV can only be sustained within a window of time following anesthesia. Consequently, it did not have long-term effects on neurodevelopment, but a proving experiment is still required.
+
+To explore whether ERK or PKA influence the phosphorylation of CREB, we cultured PC12 with both a ERK and a PKA inhibitor (SCH772984 and H89). PKA phosphorylates and activates CREB, which then
+
+Figure 9: Ketamine exposure affects protein expression in PC12 cells. C: Control group; D: D group, DMSO (solvent of inhibitors); K: K group, ketamine; S: S group, SCH772984 (ERK inhibitor); H: H group, H89 (PKA inhibitor); S+H: S+H group (PKA inhibitor + ERK inhibitor). a. No significant difference was found between C and D group. Compared to the C group, the protein levels of ERK b., p-ERK c., PKA d., p-PKA e., CREB f., p-CREB g., and BDNF h. decreased significantly (p < 0.05).
+
+32442
+
+binds to the CRE domain on DNA and in turn activates genes that are involved in the process of learning and memorization; however, ketamine inhibits this process [10]. N-acetylserotonin appears to partly restore the ketamine-induced decrease of ERK and BDNF to control levels [43]. Phosphorylation of CREB at Ser133 can be catalyzed via a number of protein kinases, including cAMP-dependent PKA [44]. The ERK1/2 are members of the mitogen activated protein kinase (MAPK) family and are necessary for cell growth, differentiation, survival, molecular information processing, and stabilization of structural changes in dendritic spines [45, 46]. When treated with SCH772984 or H89 alone, no change
+
+in the protein levels of p-CREB and BDNF were observed; however, these decreased when treated with the SCH772984 or H89 (Figure 9g-9h). This explains how ERK and PKA can regulate the phosphorylation of CREB. Furthermore, when ERK or PKA do not participate in this process, ERK and PKA have the ability to replace each other, thus independently regulating the phosphorylation of CREB. Lin identified the involvement of cAMP/PKA and ERK dependent CREB signaling pathways in the luteolin-mediated miR-132 expression and neuritogenesis of PC12 cells [47]. Won demonstrated that DA-9801 exerts its beneficial effects of stimulating neurite outgrowth through the ERK1/2-CREB pathway
+
+Figure 10: CREB pathway. Several signaling pathways, including those involving ERK, and PKA have been associated with the regulation of de novo protein synthesis in the context of synaptic plasticity, converging on the phosphorylation of CREB at Ser133 residue.
+
+32443
+
+in PC12 cells [48]. Behavioral analyses of animals with altered ERK signaling have revealed a central involvement of this cascade in learning and memory [49], and it has been reported that ERK activity was decreased in dentate gyrus of aged rats, which did not sustain LTP [50, 51]. Coccomyxa gloeobotrydiformis (CGD) significantly increased ERK and CREB phosphorylation in the hippocampus, suggesting that the learning and memory- enhancing effects of CGD might be associated with the ERK/CREB pathway [52]. Li et al. reported that pretreatment with resveratrol effectively restored synaptic plasticity in chronic cerebral hypoperfusion rats both functional and structural via PKA-CREB activation [28]. The levels of PKA and cAMP were increased in the rat hippocampus following a step-down inhibitory avoidance task [53]. Furthermore, transgenic mice with the inhibitory regulatory subunit of PKA were impaired in their long- term memory abilities due to contextual fear conditioning [54].
+
+as C group. The first day after birth was recorded as B0. During B25-B30, Morris water maze task, contextual and cued fear conditioning, and olfactory tasks were used to test learning and memory capacity (n = 120, 5/dam, Figure 1).
+
+Sample collections
+
+Rat pups were sacrificed at B30 via cervical dislocation, and were recovered to collect brain tissue for Nissl staining (n = 24, 1/dam), Golgi staining (n = 24, 1/ dam), and western blotting (n = 72, 3/dam). A subset of their hippocampuses were quickly dispensed on ice, put into a freezing tube, and frozen in liquid nitrogen, while other tissues were preserved in 10% formalin.
+
+Nissl’s staining
+
+In summary, the present study investigated learning as well as spatial and conditioned memory of rat pups, following ketamine anesthesia during pregnancy. Moreover, ERK and PKA, but not CaMKII or CaMKIV, can regulate the CREB-BDNF pathway in this animal model. Furthermore, ERK and PKA in the regulation of CREB-BDNF pathway are mutually compensating.
+
+MATERIALS AND METHODS
+
+Coronal brain sections were cut in a vibratome (Leica VT1200S, Germany) after the brains were postfixed in the same fixative. To ensure matching of hippocampal sections between groups, we used anatomical landmarks provided by the brain atlas. The selected brain sections were stained with 0.5% cresyl violet and we selected three 104 μm2 areas for examination with a light microscope (Leica DFC420, Germany) to count neuron numbers in the CA1 and CA3 regions of the hippocampus.
+
+Golgi staining
+
+Animals
+
+Male and female Wistar rats, three months of age, weighing 200 ± 20 g, were purchased from the Animal Experimental Center of the Second Affiliated Hospital of the Harbin Medical University (Harbin, China). Prior to the experiment, rats were quarantined for two weeks at the Northeast Agricultural University (Harbin, China). All experiments were performed in accordance with the guidelines outlined by the Ethical Committee for Animal Experiments (Northeast Agricultural University, Harbin, China).
+
+Mating and drug administration
+
+Thirty-six Wistar rats were divided into 12 cages (one male and two females per cage) with an iron mesh at the bottom. On the next morning the vaginal suppository was investigated through the iron mesh. When sperm was detected, female rats were annotated as pregnant at day 0 (P0). The female rats were anesthetized via intravenous ketamine injection (200 mg/Kg) for 3 h on P14 [55]. The total volume of ketamine stayed below 2 mL/100 mg. Ketamine-treated offspring were recorded as K group, while individuals within the control group were recorded
+
+to obtain hippocampal dendritic spine density via the FD Rapid GolgiStainTM Kit (FD Neuro Technologies Inc), following instructions. Coronal tissue sections of 150 μm thickness were cut at room temperature, using a vibratome (Leica VT1200S, Germany) and then, they were put on gelatin coated slides. Subsequently, slides were dehydrated with a gradient of 50%, 75%, 95%, to 100% ethanol and cleared in xylene, then the specimens were prepared with slide coverslips and sealed with Permount. The slides were then examined in detail with a light microscope (Leica DFC420, Germany). We analyzed the stained spine, using techniques similar to those described in previous study [56]. Five pyramidal neurons were analyzed that were well-impregnated and clearly distinguishable from others in each hippocampus (20 × objective lens). Five segments of 10 μm of apical and basal dendrites respectively, were randomly selected from each pyramidal neuron for inspection (via 200 × oil immersion lens) to quantify the density of spines. Spinal density of secondary apical and basal dendrites was analyzed at proximal segments emerging at more than 50 μm distance from the soma of the hippocampal CA1 neurons. All of these spines were required to exhibit a
+
+Golgi-Cox staining was utilized
+
+the manufacturer’s
+
+32444
+
+clearly distinguishable base or origin and were isolated from neighboring dendrites. Spine density was calculated per 10 μm of dendritic length. The open-source ImageJ 1.48 r Java image-viewing software and Adobe Photoshop CC 2015 were used to calibrate the scale and enlarge the segments of the spines. An investigator blinded to the experimental condition completed all analyses.
+
+Morris water maze test
+
+illuminated. During the last seconds of the auditory signal, an unconditioned aversive stimulus, a mild footshock in the range of 0.25 to 0.5 mA, was administered through the grid floor for 2 sec. The number of seconds spent freezing in the test chamber on the training day was considered the control measure of unconditioned fear. The rat pup was left in the conditioning chamber for 1 min after the last pairing, during which the association between the aversive stimulus and the properties of the conditioning chamber was further established. The rat pup was then returned to its home cage.
+
+Place navigation trials
+
+To test hippocampal-dependent spatial cognition, rats were trained in the standard morris water maze with a hidden platform [57]. A white escape platform (12 cm diameter) was submerged in a circular pool (160 cm diameter, at a 50 cm depth), filled with warm (23–25 °C) opaque water. At B25-29, each rat pup underwent four trial sessions per day (60–70 min inter-trial interval) for five consecutive days. Each trial consisted of releasing the rat into the water, facing the outer edge of the pool at one of the quadrants (in random sequence) and permitting the animal to escape to the platform. They received four trials per day of training in search for the submerged and unmarked platform, with trial durations of 60 s on the platform at the end of trials. All trials were videotaped, and the swimming paths of rats were recorded with the ANY- maze video tracking system (Stoelting Co., IL, USA), which enabled us to measure the time taken (latency) to find the platform (s), as well as other behavioral information obtained during this spatial reference memory test. The animals were dried and placed beneath a heating lamp after completion of each test.
+
+Spatial probe test
+
+Testing on day 2 began approximately 24 hours after the conditioning session. The rat pup was returned to the same conditioning chamber and scored for bouts of freezing behavior. No footshock was administered on day two. The number of seconds spent freezing in the identical test chamber on day two was considered the measure of contextually conditioned fear, i.e., freezing within identical context. Freezing was defined as a lack of movement other than respiration. Presence or absence of freezing behavior was generally recorded by an investigator, who was blinded to the experimental condition, taking a note every 10 sec for 5 min, for a maximum total score of 30 freezing bouts. The rat pup was then returned to its home cage.
+
+The second phase of testing began an hour later. A further testing chamber with very different properties provided the altered context. Changing the sensory cues as much as possible was essential so that the rat pup perceives the novel context as unrelated to the conditioning chamber. Such as triangle-shaped test chamber with different lighting was used and lemon juice was painted on the walls, while a different investigator wore gloves and a lab coat of different texture than on the training day. Freezing behavior was scored for 3 min. Contextual discrimination of fear conditioning was quantified by comparing the number of freezing bouts in the same contextual environment to the number of freezing bouts in the novel contextual environment.
+
+A probe trial was performed 1 d after the last trial at B30 where the platform was removed from the pool to assess memory retention for the location of the platform. During the 60 s test trial, we recorded and analyzed the swimming speed (cm/s), the swimming path tracks, and the number of entries into the platform quadrant zone.
+
+Contextual and cued fear conditioning
+
+At the end of the first 3 min, the tone that was presented on training day one (was well as the light stimulus cue if used on day one) was presented in the novel context environment. Freezing behavior was scored for the next 3 min in the presence of the sound (and light) cues. Cued conditioning was calculated via comparison of the number of freezing bouts in the novel context environment in the presence of the cue with the number of freezing bouts in the novel context environment in the absence of the cue (Figure 5a).
+
+Conditioning training on day one consisted of placing the rat pups in the chamber and exposing the animals to a mild footshock paired with an auditory cue. The rat pup was brought from the home cage to the testing room and placed into the conditioning chamber. It had 3 min to explore the novel environment. The auditory cue (a 90 dB tone) was sounded for approximately 30 sec. A stimulus light within the wall of the chamber may also be
+
+Olfactory task
+
+This task was designed to investigate the olfactory learning and memory abilities [58]. For this experiment, two holes (3 cm diameter and 4.5 cm deep) were used. A polypropylene swab, embedded in a fine plastic mesh and containing 20 µL of diluted odors (1:10) was placed at
+
+32445
+
+the bottom of each hole and covered with wood shavings. The acquisition test (one session) consisted of one odor (either limonene or carvone, Sigma-Aldrich) being presented in both holes for a 5 min period. In a preliminary experiment, with simultaneous presentation of the same pair of odors (one odor in each hole) in a one-trial test, rat pups spent the same amount of time exploring either hole, indicating no preference for one of the two odors. The recall test consisted of a 3 min session in which one hole was odorized with the previously presented odor, while the other hole was odorized with a new odor (Figure 6a). The delay between acquisition and recall tests was 60 min. During the recall test, the cumulated exploration time of each hole was converted as the percentage of the total exploration time of both holes. Rat pups were considered to have remembered the familiar odor when they spent less time exploring the hole containing it, in relation to the time spent exploring the hole containing the new odor. Equal exploration times for both holes during the recall test were considered to indicate that rat pup did not remember the familiar odor. Both odors were used alternatively during acquisition or recall and presented randomly in each of the two holes to avoid place preference bias (Figure 6a).
+
+WB
+
+150 µg of protein were separated via 10% SDS- polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (HybondTM-C Extra, GE Healthcare) via electroblotting. After washing, membranes were blocked with 3% (w/v) BSA (biotopped) for 4 h at room temperature and incubated overnight at 4 °C in BSA with antibodies that are specific for Ca2+/Calmodulin- Dependent Protein Kinase II (CaMKII), p-CaMKII, CaMKIV, p-CaMKIV, ERK, p-ERK, PKA, CREB, p-CREB (1.5:1000, EnoGene), p-PKA, and Brain Derived Neurotrophic Factor (BDNF, 1:1000, abcam). Membranes were washed thrice with PBS containing 0.1% Tween and then incubated for 1 h at room temperature either with a horseradish peroxidase-conjugated secondary antibody (Goat anti-Rabbit IgG Antibody HRP (ABIN) or a goat anti-Mouse IgG Antibody HRP (Sigma)) in BSA.
+
+Data analysis
+
+Cell culture and drug treatment
+
+PC12 cells were obtained from the Northeast Agricultural University, Harbin, China. The cells were cultured in DMEM medium (Gibco), supplemented with 10% (v/v) FBS, penicillin/streptomycin (100 U/mL; 100 μg/mL) at 37 °C under an atmosphere of 5% CO2 and 95% air. The cells were seeded in 6-well plates with 2-9 × 105 cells/well or 96-well plates with 2-9 × 104 cells/well, and the culture medium was changed daily. Cells were pretreated for 3 h with Protein Kinase A (PKA) inhibitor (H89, 10 μM, H group), Extracellular Regulated Protein Kinases (ERK) inhibitor (SCH772984, 10 μM, S group), PKA inhibitor + ERK inhibitor (S+H group), DMSO (solvent of inhibitors, D group), and ketamine (K group).
+
+All data were analyzed with GraphPad Prism 7.0 (GraphPad Software Inc., USA) via one-way ANOVA, followed by Turkey’s Post Hoc test or unpaired two-tailed Student t-test. Values were considered to be statistically significant for P < 0.05. Data are presented as means ± standard deviation unless otherwise noted.
+
+Authors contribution
+
+XL and LG designed the study. XL, CG, and LG designed the behavioral testing. XL, CG, YL, LL, XW, and YZ collected data for behavioral testing. XL, YL, YC, and WL processed the brain tissue. XL and YL collected and analyzed the data. XL, YL, and CG interpreted the data. XL wrote and edited the manuscript. All authors critically reviewed the content and approved the final version for publication.
+
+Cell counting kit-8 (CCK-8) assay
+
+ACKNOWLEDGMENTS
+
+Cell viability was detected via the CCK8 assay (Beyotime Institute of Biotechnology, Suzhou, Jiangsu, China). Following the indicated treatments, CCK8 solution (10 μl) was added to each well (96-well plates). Then, the cells were cultured at 37 °C for one further hour. The optical density of each well was measured at 450 nm with a Bio-Tek microplate reader (Bio-Tek Instruments, Thermo Fisher Scientific, Winooski, VT).
+
+This study was supported by the National Natural
+
+Science Foundation of China (31572580 and 31372491).
+
+CONFLICTS OF INTEREST
+
+There is no conflict of interest.
+
+REFERENCES
+
+1. Rofael HZ, Turkall RM, Abdel-Rahman MS. Immunomodulation by cocaine and ketamine in postnatal rats. Toxicology. 2003; 188: 101-14. doi: 10.1016/S0300- 483X(03)00081-7.
+
+32446
+
+2. Goodman S. Anesthesia for nonobstetric surgery in the pregnant patient. Seminars in perinatology. 2002; 26: 136- 45. dio: 10.1053/sper.2002.32203.
+
+Fulton S, Parrott AC. Motor delays in MDMA (ecstasy) exposed infants persist to 2 years. Neurotoxicol Teratol. 2016; 54: 22-8. doi: 10.1016/j.ntt.2016.01.003.
+
+3. Cheek TG, Baird E. Anesthesia for nonobstetric surgery: maternal and fetal considerations. Clinical obstetrics and gynecology. 2009; 52: 535-45. doi: 10.1097/ GRF.0b013e3181c11f60.
+
+14. Zhang YH, Zhang J, Song JN, Xu X, Cai JS, Zhou Y, Gao JG. The PI3K-AKT-mTOR pathway activates recovery from general anesthesia. Oncotarget. 2016; 7: 40939-52. doi: 10.18632/oncotarget.10172.
+
+4. Kong FJ, Ma LL, Hu WW, Wang WN, Lu HS, Chen SP. Fetal exposure to high isoflurane concentration induces postnatal memory and learning deficits in rats. Biochemical pharmacology. 2012; 84: 558-63. doi: 10.1016/j. bcp.2012.06.001.
+
+5. Zheng H, Dong Y, Xu Z, Crosby G, Culley DJ, Zhang Y, Xie Z. Sevoflurane anesthesia in pregnant mice induces neurotoxicity in fetal and offspring mice. The Journal of the American Society of Anesthesiologists. 2013; 118: 516-26. doi: 10.1097/ALN.0b013e3182834d5d.
+
+6. Zhao T, Li Y, Wei W, Savage S, Zhou L, Ma D. Ketamine administered to pregnant rats in the second trimester causes long-lasting behavioral disorders in offspring. Neurobiol Dis. 2014; 68: 145-55. doi: 10.1016/j.nbd.2014.02.009.
+
+7. Squire LR. Memory and the hippocampus: a synthesis from findings with rats, monkeys, and humans. Psychol Rev. 1992; 99: 195-231. doi: org/10.1037/0033-295X.99.2.195.
+
+15. Weed MR, Bookbinder M, Polino J, Keavy D, Cardinal RN, Simmermacher-Mayer J, Cometa FN, King D, Thangathirupathy S, Macor JE, Bristow LJ. Negative Allosteric Modulators Selective for The NR2B Subtype of The NMDA Receptor Impair Cognition in Multiple Domains. Neuropsychopharmacology. 2016; 41: 568-77. doi: 10.1038/npp.2015.184.
+
+16. Wilder RT, Flick RP, Sprung J, Katusic SK, Barbaresi WJ, Mickelson C, Gleich SJ, Schroeder DR, Weaver AL, Warner DO. Early exposure to anesthesia and learning disabilities in a population-based birth cohort. Anesthesiology. 2009; 110: 796-804. doi: 10.1097/01. anes.0000344728.34332.5d.
+
+17. Rofael HZ, Turkall RM, Abdel-Rahman MS. Immunomodulation by cocaine and ketamine in postnatal rats. Toxicology. 2003; 188: 101-14. doi: org/10.1016/ S0300-483X(03)00081-7.
+
+8. Wang LQ, Liu SZ, Wen X, Wu D, Yin L, Fan Y, Wang Y, Chen WR, Chen P, Liu Y, Lu XL, Sun HL, Shou W, et al. Ketamine-mediated afferent-specific presynaptic transmission blocks in low-threshold and sex-specific subpopulation of myelinated Ah-type baroreceptor neurons of rats. Oncotarget. 2015; 6: 44108-22. doi: 10.18632/ oncotarget.6586.
+
+9. Chen Q, Min S, Hao X, Peng L, Meng H, Luo Q, Chen J, Li X. Effect of Low Dose of Ketamine on Learning Memory Function in Patients Undergoing Electroconvulsive Therapy-A Randomized, Double-Blind, Controlled Clinical Study. The Journal of ECT. 2016. doi: 10.1097/ YCT.0000000000000365.
+
+10. Peng S, Yang X, Liu GJ, Zhang XQ, Wang GL, Sun HY. From the camp pathway to search the ketamine-related learning and memory. Eur Rev Med Pharmacol Sci. 2015; 19: 161-4. WOS: 000352211700025.
+
+18. Zhang H, Sun XR, Wang J, Zhang ZZ, Zhao HT, Li HH, Ji MH, Li KY, Yang JJ. Reactive Oxygen Species-mediated Loss of Phenotype of Parvalbumin Interneurons Contributes to Long-term Cognitive Impairments After Repeated Neonatal Ketamine Exposures. Neurotox Res. 2016; 30: 593-605. doi: 10.1007/s12640-016-9653-1.
+
+19. Trevlopoulou A, Touzlatzi N, Pitsikas N. The nitric oxide donor sodium nitroprusside attenuates recognition memory deficits and social withdrawal produced by the NMDA receptor antagonist ketamine and induces anxiolytic-like behaviour in rats. Psychopharmacology (Berl). 2016; 233: 1045-54. doi: 10.1007/s00213-015-4181-x.
+
+20. Stevens RA, Butler BD, Kokane SS, Womack AW, Lin Q. Neonatal inhibition of Na+-K+-2Cl—cotransporter prevents ketamine induced spatial learning and memory impairments. Neurotoxicol Teratol. 2016. doi: 10.1016/j. ntt.2016.11.001.
+
+11. Tang X, Wu D, Gu LH, Nie BB, Qi XY, Wang YJ, Wu FF, Li XL, Bai F, Chen XC, Xu L, Ren QG, Zhang ZJ. Spatial learning and memory impairments are associated with increased neuronal activity in 5XFAD mouse as measured by manganese-enhanced magnetic resonance imaging. Oncotarget. 2016;7:57556-57570. doi: 10.18632/ oncotarget.11353.
+
+21. Reus GZ, Abaleira HM, Titus SE, Arent CO, Michels M, da Luz JR, dos Santos MA, Carlessi AS, Matias BI, Bruchchen L, Steckert AV, Ceretta LB, Dal-Pizzol F, et al. Effects of ketamine administration on the phosphorylation levels of CREB and TrKB and on oxidative damage after infusion of MEK inhibitor. Pharmacol Rep. 2016; 68: 177-84. doi: 10.1016/j.pharep.2015.08.010.
+
+12. Fish EW, Holloway HT, Rumple A, Baker LK, Wieczorek LA, Moy SS, Paniagua B, Parnell SE. Acute alcohol exposure during neurulation: Behavioral and brain structural consequences in adolescent C57BL/6J mice. Behav Brain Res. 2016; 311: 70-80. doi: 10.1016/j.bbr.2016.05.004.
+
+22. Kempf SJ, Metaxas A, Ibanez-Vea M, Darvesh S, Finsen B, Larsen MR. An integrated proteomics approach shows synaptic plasticity changes in an APP/PS1 Alzheimer’s mouse model. Oncotarget. 2016; 7: 33627-48. doi: 10.18632/oncotarget.9092.
+
+13. Singer LT, Moore DG, Min MO, Goodwin J, Turner JJ,
+
+23. Liu WX, Wang J, Xie ZM, Xu N, Zhang GF, Jia M, Zhou ZQ, Hashimoto K, Yang JJ. Regulation of glutamate
+
+32447
+
+transporter 1 via BDNF-TrkB signaling plays a role in the anti-apoptotic and antidepressant effects of ketamine in chronic unpredictable stress model of depression. Psychopharmacology (Berl). 2016; 233: 405-15. doi: 10.1007/s00213-015-4128-2.
+
+24. Montminy MR, Gonzalez GA, Yamamoto KK. Regulation of cAMP-inducible genes by CREB. Trends in neurosciences. 1990; 13: 184-8. doi: 10.1016/0166- 2236(90)90045-C.
+
+35. Alonso M, Medina JH, Pozzo-Miller L. ERK1/2 activation is necessary for BDNF to increase dendritic spine density in hippocampal CA1 pyramidal neurons. Learning & Memory. 2004; 11: 172-8. doi: 10.1101/lm.67804.
+
+36. Rodriguez-Munoz M, Sanchez-Blazquez P, Merlos M, Garzon-Nino J. Endocannabinoid control of glutamate therapeutic potential and NMDA consequences of dysfunction. Oncotarget. 2016; 7:55840- 55862. doi: 10.18632/oncotarget.10095. receptors: the
+
+25. Spencer JP, Vauzour D, Rendeiro C. Flavonoids and cognition: the molecular mechanisms underlying their behavioural effects. Archives of biochemistry and biophysics. 2009; 492: 1-9. doi: 10.1016/j.abb.2009.10.003.
+
+37. Ding S, Xu Z, Yang J, Liu L, Huang X, Wang X, Zhuge Q. The Involvement of the Decrease of Astrocytic Wnt5a in the Cognitive Decline in Minimal Hepatic Encephalopathy. Mol Neurobiol. 2016. doi: 10.1007/s12035-016-0216-5.
+
+26. Berkowitz LA, Riabowol KT, Gilman MZ. Multiple sequence elements of a single functional class are required for cyclic AMP responsiveness of the mouse c-fos promoter. Mol Cell Biol. 1989; 9: 4272-81. doi: 10.1128/ MCB.9.10.4272.
+
+38. Liu F, Li ZF, Wang ZY, Wang L. Role of subcellular in regulating apoptosis and calcium redistribution autophagy in cadmium-exposed primary rat proximal tubular cells. J Inorg Biochem. 2016; 164: 99-109. doi: 10.1016/j.jinorgbio.2016.09.005.
+
+27. Bourtchuladze R, Frenguelli B, Blendy J, Cioffi D, Schutz G, Silva AJ. Deficient long-term memory in mice with a targeted mutation of the cAMP-responsive element- binding protein. Cell. 1994; 79: 59-68. doi: 10.1016/0092- 8674(94)90400-6.
+
+28. Li H, Wang J, Wang P, Rao Y, Chen L. Resveratrol Reverses the Synaptic Plasticity Deficits in a Chronic Cerebral Hypoperfusion Rat Model. J Stroke Cerebrovasc Dis. 2016; 25: 122-8. doi: 10.1016/j.jstrokecerebrovasdis.2015.09.004.
+
+29. Zhao Y, Xiao M, He W, Cai Z. Minocycline upregulates cyclic AMP response element binding protein and brain- derived neurotrophic factor in the hippocampus of cerebral ischemia rats and improves behavioral deficits. Neuropsychiatr Dis Treat. 2015; 11: 507-16. doi: 10.2147/ NDT.S73836.
+
+30. Lim YY, Villemagne VL, Laws SM, Ames D, Pietrzak RH, Ellis KA, Harrington KD, Bourgeat P, Salvado O, Darby D. BDNF Val66Met, Aβ amyloid, and cognitive decline in preclinical Alzheimer’s disease. Neurobiology of Aging. 2013; 34: 2457-64. doi: 10.1016/j. neurobiolaging.2013.05.006.
+
+31. Guerrieri D, van Praag H. Exercise-mimetic AICAR transiently benefits brain function. Oncotarget. 2015; 6: 18293-313. doi: 10.18632/oncotarget.4715.
+
+32. Nacmias B, Piccini C, Bagnoli S, Tedde A, Cellini E, Bracco L, Sorbi S. Brain-derived neurotrophic factor, apolipoprotein E genetic variants and cognitive performance in Alzheimer’s disease. Neuroscience letters. 2004; 367: 379-83. doi: 10.1016/j.neulet.2004.06.039.
+
+33. Fossati P, Radtchenko A, Boyer P. Neuroplasticity: from MRI symptoms. European Neuropsychopharmacology. 2004; 14: S503-S10. doi: 10.1016/j.euroneuro.2004.09.001. to depressive
+
+39. Cohen SM, Ma H, Kuchibhotla KV, Watson BO, Buzsaki G, Froemke RC, Tsien RW. Excitation-Transcription Coupling in Parvalbumin-Positive Interneurons Employs a Novel CaM Kinase-Dependent Pathway Distinct from Excitatory Neurons. Neuron. 2016; 90: 292-307. doi: 10.1016/j.neuron.2016.03.001.
+
+40. Liu SH, Lai YL, Chen BL, Yang FY. Ultrasound Enhances the Expression of Brain-Derived Neurotrophic Factor in Astrocyte Through Activation of TrkB-Akt and Calcium- CaMK Signaling Pathways. Cereb Cortex. 2016. doi: 10.1093/cercor/bhw169.
+
+41. Han WY, Du P, Fu SY, Wang F, Song M, Wu CF, Yang JY. Oxytocin via its receptor affects restraint stress-induced methamphetamine CPP reinstatement in mice: Involvement of the medial prefrontal cortex and dorsal hippocampus glutamatergic system. Pharmacol Biochem Behav. 2014; 119: 80-7. doi: 10.1016/j.pbb.2013.11.014.
+
+42. Guo Y, Feng P. OX2R activation induces PKC-mediated ERK and CREB phosphorylation. Exp Cell Res. 2012; 318: 2004-13. doi: 10.1016/j.yexcr.2012.04.015.
+
+43. Choudhury A, Singh S, Palit G, Shukla S, Ganguly S. Administration of N-acetylserotonin and melatonin alleviate chronic ketamine-induced behavioural phenotype accompanying BDNF-independent dependent converging cytoprotective mechanisms in the hippocampus. Behav Brain Res. 2016; 297: 204-12. doi: 10.1016/j. bbr.2015.10.027. and
+
+44. Hagiwara M, Brindle P, Harootunian A, Armstrong R, Rivier J, Vale W, Tsien R, Montminy M. Coupling of hormonal stimulation and transcription via the cyclic AMP- responsive factor CREB is rate limited by nuclear entry of protein kinase A. Molecular and cellular biology. 1993; 13: 4852-9. doi: 10.1128/MCB.13.8.4852.
+
+34. Duman R. Pathophysiology of depression: the concept of synaptic plasticity. European psychiatry. 2002; 17: 306-10. doi: 10.1016/S0924-9338(02)00654-5.
+
+45. Ivanov A, Pellegrino C, Rama S, Dumalska I, Salyha Y, Ben-Ari Y, Medina I. Opposing role of synaptic and extrasynaptic NMDA receptors in regulation of the extracellular signal-regulated kinases (ERK) activity
+
+32448
+
+in cultured rat hippocampal neurons. The Journal of physiology. 2006; 572: 789-98. doi: 10.1113/ jphysiol.2006.105510.
+
+46. Sindreu CB, Scheiner ZS, Storm DR. Ca 2+-stimulated adenylyl cyclases regulate ERK-dependent activation of MSK1 during fear conditioning. Neuron. 2007; 53: 79-89. doi: 10.1016/j.neuron.2006.11.024
+
+53. Bernabeu R, Bevilaqua L, Ardenghi P, Bromberg E, Schmitz P, Bianchin M, Izquierdo I, Medina JH. Involvement of hippocampal cAMP/cAMP-dependent protein kinase signaling pathways in a late memory consolidation phase of aversively motivated learning in rats. Proceedings of the National Academy of Sciences. 1997; 94: 7041-6.
+
+47. Lin LF, Chiu SP, Wu MJ, Chen PY, Yen JH. Luteolin induces microRNA-132 expression and modulates neurite outgrowth in PC12 cells. PLoS One. 2012; 7: e43304. doi: 10.1371/journal.pone.0043304.
+
+54. Abel T, Nguyen PV, Barad M, Deuel TA, Kandel ER, Bourtchouladze R. Genetic demonstration of a role for PKA in the late phase of LTP and in hippocampus-based long-term memory. Cell. 1997; 88: 615-26. doi: 10.1016/ S0092-8674(00)81904-2.
+
+48. Won JH, Ahn KH, Back MJ, Ha HC, Jang JM, Kim HH, Choi SZ, Son M, Kim DK. DA-9801 promotes neurite outgrowth via ERK1/2-CREB pathway in PC12 cells. Biol Pharm Bull. 2015; 38: 169-78. doi: 10.1248/bpb.b14-00236.
+
+49. Atkins CM, Selcher JC, Petraitis JJ, Trzaskos JM, Sweatt JD. The MAPK cascade is required for mammalian associative learning. Nature neuroscience. 1998; 1: 602-9. doi: 10.1038/2836.
+
+50. McGahon B, Maguire C, Kelly A, Lynch M. Activation of p42 mitogen-activated protein kinase by arachidonic acid and trans-1-amino-cyclopentyl-1, 3-dicarboxylate impacts on long-term potentiation in the dentate gyrus in the rat: analysis of age-related changes. Neuroscience. 1999; 90: 1167-75. doi: 10.1016/S0306-4522(98)00528-4.
+
+51. Drulis-Fajdasz D, Wojtowicz T, Wawrzyniak M, Wlodarczyk J, Mozrzymas JW, Rakus D. Involvement of cellular metabolism in age-related LTP modifications in rat hippocampal slices. Oncotarget. 2015; 6: 14065-81. doi: 10.18632/oncotarget.4188.
+
+55. Clancy B, Darlington RB, Finlay BL. Translating developmental species. Neuroscience. 2001; 105: 7-17. doi: 10.1016/S0306- 4522(01)00171-3. across mammalian time
+
+56. Xiao H, Liu B, Chen Y, Zhang J. Learning, memory and synaptic plasticity in hippocampus in rats exposed to sevoflurane. International Journal of Developmental Neuroscience. 2016; 48: 38-49. doi: 10.1016/j. ijdevneu.2015.11.001.
+
+57. Morris R, Garrud P, Rawlins J, O’Keefe J. Place navigation impaired in rats with hippocampal lesions. Nature. 1982; 297: 681-3.
+
+58. Mandairon N, Sacquet J, Garcia S, Ravel N, Jourdan F, Didier A. Neurogenic correlates of an olfactory discrimination task in the adult olfactory bulb. European journal of neuroscience. 2006; 24: 3578-88. doi: 10.1111/j.1460-9568.2006.05235.x.
+
+52. Sun L, Jin Y, Dong L, Sui HJ, Sumi R, Jahan R, Hu D, Li Z. Coccomyxa Gloeobotrydiformis Improves Learning and Memory in Intrinsic Aging Rats. Int J Biol Sci. 2015; 11: 825-32. doi: 10.7150/ijbs.10861.
+
+32449

new_pdfs/10.31083_j.jin2003065.txt

@@ -0,0 +1,313 @@
+e c n e i c s o r u e N
+
+e v i t a r g e t n I
+
+f o
+
+l a n r u o J
+
+Original Research
+
+Prenatal sevoflurane exposure causes abnormal development of the entorhinal cortex in rat offspring
+
+Ying Gao1, Tianyun Zhao1, Yanxin Chen2, Zhixiang Sun3, Junming Lu2, Ziwen Shi1, Xingrong Song1,*
+
+1Department of Anesthesiology, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, 510623 Guangzhou, Guangdong, China 2Department of Anesthesiology, Guangdong Hospital of Traditional Chinese Medicine (The Second Affiliated Hospital of Guangzhou University of
+
+Traditional Chinese Medicine), 510120 Guangzhou, Guangdong, China 3Department of Anesthesiology, Shanghai University of Medicine & Health Sciences Affiliated Zhoupu Hospital, 201318 Shanghai, China
+
+Correspondence: songxingrong@gwcmc.org (Xingrong Song)
+
+DOI:10.31083/j.jin2003065 This is an open access article under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/).
+
+Submitted: 14 March 2021 Revised: 26 March 2021 Accepted: 12 May 2021 Published: 30 September 2021
+
+As a gamma-aminobutyric acid type A receptor agonist sevoflurane is a common general anesthetic used in anesthesia and affects the neural development in offspring. We hypothesized that sevoflurane could regulate interneurons via the neuregulin-1-epidermal growth factor receptor-4 (NRG1–ErbB4) pathway in the entorhinal cortex (ECT) of the middle pregnancy. Six female rats in middle pregnancy (14.5 days of pregnancy) were randomly and equally divided into sevoflurane (SeV) and control groups. The rats in the SeV group were exposed to 4% sevoflurane for 3 hours. The expression levels of NRG1 and ErbB4, parvalbumin (PV) and glutamic acid decarboxylase (GAD67), and N-methyl-D-aspartate receptor subunit 2A (NR2A) and subunit 2B (NR2B) in offspring were examined through immunohis- tochemistry. The pyramidal neurons in the ECT were examined via Golgi staining. The levels of NRG1 and ErbB4 were significantly de- creased (P < 0.01) and the levels of PV and GAD67 (interneurons) were found to be decreased in the SeV group (P < 0.01). The level of NR2B was found to be increased while the level of NR2A being decreased in the SeV group (P < 0.01). The development of pyra- midal neurons was abnormal in the SeV group (P < 0.05). Conclu- sively, prenatal sevoflurane exposure could lead to the disturbance of the interneurons by activating the NRG1–ErbB4 pathway and sub- sequently result in abnormal development of pyramidal neurons in middle pregnancy. Prenatal sevoflurane exposure in middle preg- nancy could be potentially harmful to the neural development of rat offspring. This study may reveal a novel pathway in the influence mechanism of sevoflurane on rat offspring.
+
+middle pregnancy is a “busy period of neural development” in which neurons proliferate, migrate, and differentiate [2]. Anesthetics commonly used in clinical practice have been found to mainly affect two major neurotransmitter recep- tors, N -methyl-D-aspartate (NMDA) receptors and gamma- aminobutyric acid (GABA) receptors, during the develop- ment of the central nervous system (CNS). Exposure to gen- eral anesthetics before and after birth could affect brain devel- opment. A warning against prolonged and repeated exposure to general anesthetics during pregnancy was proposed by the American Food and Drug Administration. Sevoflurane, as a GABA type A (GABAA) receptor agonist/enhancer [3] is a common general anesthetic used in anesthesia for pregnant women. Hence, prenatal sevoflurane exposure during middle pregnancy could affect the neural development of offspring.
+
+As an excitatory transmitter in the early stage of neural development, GABA is a key neurotransmitter in the devel- opment of the brain that plays an important role in the pro- liferation and migration of neurons [4, 5]. When neurons migrate to the target cortex, the process of neuronal migra- tion is terminated by contact of GABA receptor. With the development of the brain, the GABAA receptor changes from excitability to inhibition and depolarization to hyperpolariza- tion [4, 5]. As a GABAA receptor agonist, sevoflurane could play an important role in the proliferation and migration of neurons.
+
+Keywords
+
+Sevoflurane; Interneurons; Middle pregnancy; NRG1–ErbB4; Entorhinal cortex
+
+1. Introduction
+
+About 0.75–2% of pregnant women need non-obstetric surgery every year [1]. Middle pregnancy (namely, second trimester) is considered to be a safe period for surgical anes- thesia. With the development of fetal surgery and laparo- scopic technology, an increasing number of pregnant women undergo surgery under general anesthesia during middle pregnancy [1]. However, recently, studies have shown that
+
+The “vulnerability window” of neurotoxicity of general anesthetics is mainly at the peak of brain nerve cell prolif- eration, migration, or synaptic development (i.e., during the middle or late stage of pregnancy) [2]. In middle pregnancy, the fetus’ brain is extremely sensitive to changes in the en- vironment. Middle pregnancy is a critical period for brain nerve development (neuron proliferation, migration, and the formation of neural connections), and most of these events occur in middle pregnancy [6]. In addition to unavoidable emergency operations in middle pregnancy, selective oper- ations are also mostly carried out in this period. Prena-
+
+J. Integr. Neurosci. 2021 vol. 20(3), 613-622 ©2021 The Author(s). Published by IMR Press.
+
+tal sevoflurane exposure in middle pregnancy may affect fe- tal neural development and cause neural dysfunction in off- spring.
+
+Interneurons, located with GABAA receptors, can control and regulate the pyramidal neurons in the entorhinal cortex (ECT). The layers II and III (LII and LIII) pyramidal neu- rons of the ECT send out perforating fibers to transmit in- formation to the hippocampus, and the hippocampus forms memories by editing and storing information [7]. The hip- pocampus is the key center of brain association, learning, and memory. Thus, interference with interneurons may lead to learning and memory dysfunction. However, previous re- lated studies have been mainly focused on ultrastructural and functional impairments of pyramidal neurons after sevoflu- rane exposure [8–11]. Relatively little attention has been paid to interneurons.
+
+Neuregulin-1 (NRG1), a member of the epidermal growth factor family, plays an important role in promoting the phos- phorylation of epidermal growth factor receptor-4 (ErbB4) [12, 13]. ErbB4 receptors have a variety of isomers, includ- ing ErbB (1–4). ErbB4 is highly expressed in the brain and has a strong affinity for NRG1. Most ErbB4 receptors are ex- pressed on parvalbumin (PV) interneurons, which account for 40% of interneurons [12–14]. The NRG1–ErbB4 path- way can regulate and interact with the secretion function of GABAA receptors [15]. The NRG1–ErbB4 pathway plays a great role in the occurrence, migration, and synaptic plas- ticity of neurons [16–18] by regulating the composition of interneurons’ subunit and the activity of receptor [19–21]. Furthermore, alterations of the NRG1–ErbB4 pathway could regulate key receptors connected to learning and memory ability, such as NMDA receptors [22, 23]. Moreover, ex- posure to general anesthetics can interfere with the key pro- cesses of dendritic growth and the development of pyramidal neurons [24]. Li et al. [25] found that disruption of NRG1– ErbB4 signaling in the PV-positive interneurons caused cog- nitive impairment in rats after exposure to isoflurane. Hence, it is possible that sevoflurane can induce neural dysfunction in offspring by regulating GABAA receptors in the PV in- terneurons of the ECT through the NRG1–ErbB4 pathway. Consequently, the influenced NRG1–ErbB4 pathway could further regulate interneurons and pyramidal neurons and af- fect the information transmission function of the ECT.
+
+Herein, we hypothesize that sevoflurane could regulate in- terneurons via the NRG1–ErbB4 pathway in the ECT of off- spring during the middle pregnancy. Gestational day 14.5 in pregnant rats is similar to middle pregnancy in humans [26]. In this study, pregnant mice were exposed to sevoflurane in middle pregnancy. The status of the NRG1–ErbB4 pathway, interneurons, NMDA receptors, and the dendrite morphol- ogy of pyramidal neurons were examined to test the hypothe- sis. Discovering the mechanism of sevoflurane-induced neu- rotoxicity is of great significance for guiding the standardized clinical use of general anesthetics and research into toxicity prevention and treatment.
+
+614
+
+2. Methods 2.1 Animals
+
+Six adult female Sprague-Dawley (SD) rats, weighing 180–220 g, were raised with free diet and water intake in polypropylene cages for 7 days. Then the female SD rats were mated with male SD rats with sexual experience at 7:00 PM after adaptive feeding. Vaginal smears were performed the next morning and pregnancy day 0, G0, was defined by sperm detection. The pregnant rats were randomly divided into two groups: a control group (control, n = 3) and a sevoflurane group (SeV, n = 3). The six female SD rats were raised to G14.5 (middle pregnancy).
+
+2.2 Anesthesia
+
+On pregnancy day 14.5, the rats allocated to sevoflurane exposure were put inside a 30 cm × 20 cm × 120 cm box. A mixture of oxygen and sevoflurane (2 L/min with 4% sevoflu- rane) was delivered through an inlet port connected to a va- porizer, while a gas analyzer installed on a second port al- lowed monitoring of anesthetic gas concentration. Pregnant rats are more sensitive to sevoflurane and a minimum alve- olar concentration (MAC) of 2.4% in healthy adult rats [27], so the concentration of sevoflurane (3%) is equivalent to 1.3 MAC to maintain a surgical level of anesthesia. The rats in the control group inhaled oxygen (2 L/min). However, lim- ited to anesthesia machine conditions that it is not completely airtight, the inhalation concentration of sevoflurane should be 4% in order to reach 1.8 MAC to maintain a surgical level of anesthesia in the SeV group. The inhalation time is 3 hours in the SeV group. During the procedure, the skin color of the rats’ mouths, noses, limbs, and respiratory amplitudes and frequencies were observed to avoid hypoxia respiratory de- pression. After anesthesia, the rats were sent back to their cages after the righting reflex was recovered. After sevoflu- rane anesthetization, an arterial blood gas analysis was per- formed to assess gas exchange and glycemic status in female rats. The site of blood sampling was left heart artery. If there was a significant derangement, e.g., severe hypoxemia, these female rats were no longer involved in the follow-up experi- ments. No female rats were excluded in the study. Then the rat offspring were reared and delivered naturally.
+
+2.3 Tissue section preparation
+
+Three offspring were randomly selected from each group with one offspring/dam. The ex vivo brain samples of the off- spring rats were harvested at the day 30 of postpartum (P30) for histology, immunostaining and Golgi staining. Rats in both the control and SeV groups were executed and perfused through the left ventricle with precooling saline followed by 4% paraformaldehyde in 0.01 M phosphate buffered saline (PBS) pH 7.35. The brain tissue of the rats was taken and post-fixed for 24 hours for paraffin and frozen sections.
+
+To analyze the status of the NRG1–ErbB4 pathway in the interneurons, the expression levels of NRG1 and ErbB4 in LII and III of the ECT were examined via immunohisto- chemistry. The NRG1–ErbB4 pathway plays an important
+
+Volume 20, Number 3, 2021
+
+role in the genesis, migration, differentiation, maturation, and neurotransmitter synthesis of GABAergic interneurons. We assumed that the number of GABAergic interneurons the LII/LIII ECT of offspring in could be affected by alter- ations of the NRG1–ErbB4 pathway. Therefore, we label GABAergic interneurons with PV to represent PV interneu- rons. We also used glutamic acid decarboxylase 67 (GAD67) to label GABAergic interneuron (the key enzyme of GABA neurotransmitter synthesis) positive cells to represent the to- tal GABAergic interneurons in the LII/LIII ECT. The expres- sion levels of PV and GAD67 in LII and LIII of the ECT were examined via immunohistochemistry. That is, Interneurons were identified by immunoreactivity to PV and GAD67. To investigate whether NRG1–ErbB4 pathway changes in off- spring after prenatal sevoflurane exposure affect the forma- tion of subunits during maturation, we detected NMDA re- ceptor subunit 2A (NR2A) and NMDA receptor subunit 2B (NR2B) by immunofluorescence. The expression levels of NR2A and NR2B in LII and LIII of the ECT were examined via immunohistochemistry to analyze the status of NMDA receptors in the ECT. There is a fixed pattern of neurite growth in the developing brain. We assumed that prenatal sevoflurane exposure could affect the inherent growth pat- tern of dendrites and dendritic spines in pyramidal neurons through NRG1–ErbB4 alterations. Therefore, we used Golgi silver staining to investigate the length of dendrites and the number of branches and dendritic spines. Golgi staining was performed to analyze the total dendrite length, number of dendritic branches, spatial distribution of dendrites, and den- sity of dendritic spines in the pyramidal neurons in the ECT.
+
+2.4 Histology and immunohistochemistry
+
+The coronal sections of the brain were deparaffinized, re- hydrated, and immersed in 3% H2O2 at room temperature for 30 min. Antigens were retrieved in a 0.01 mol/L citric buffer (pH 6.0) at 97 C for 15 min. The coronal sections were cooled down for 1 h before being blocked by 10% bovine serum albumin (BSA) solution. Staining with diluted primary antibodies was conducted at 4 C overnight (for at least 18 h). The primary antibodies included rabbit anti rat NRG-1 (1:1000, Cat. No Ab191139, Abcam, Cambridge, UK), rab- bit anti rat ErbB4 (1:250, Cat. No Sc-283, Santa Cruz, Dal- las, Texas, USA), rabbit anti rat NR2A (1:1000, Cat. No cell signaling technology, Massachusetts, USA), rabbit anti rat NR2B (1:1000, Cat. No 06-600, Millipore, Massachusetts, USA), mice anti rat PV (1:1000, Cat. No #2886709, Milli- pore), and rat anti rat GAD67 (1:2500, Cat. No. MAB5406, Millipore, Massachusetts, USA). After being washed by 0.1% PBST for three times (5 min), the sections were stained with diluted second antibodies at room temperature for 2 h and kept in a dark place. After washed by 0.1% PBST for three times (5 min), the sections were counterstained with hema- toxylin, dehydrated with ethanol and mounted with cov- erslips. Then the expression levels of NRG1, ErbB4, PV, GAD67, NR2, A and NR2B were examined using a fluores- cence microscope (Leica DM6000B, Germany). The results
+
+
+
+
+
+Volume 20, Number 3, 2021
+
+were shown as positive cells/sections. In each rat, we ran- domly select 5–6 coronal sections to count the cells to avoid error resulting from the section status. The brain area sec- tions (ECT) we selected for immunohistochemical section is fixed. There is an inward concave angle under the area of the ECT, which is used to locate the central cortex and reduce the error. The size of the ECT in this part of the rat brain is rela- tively fixed, so the randomly selected sections can be regarded as roughly the same size which is comparable.
+
+2.5 Golgi stain
+
+150 µm-thick frozen brain sections were obtained from control and SeV rats. Golgi–Cox staining was performed using the FD Rapid Golgi stain kit (Cat. NO. PK401, FD NeuroTechnologies, Inc. Columbia, USA) according to the manufacturer’s protocols. Ten well-individualized pyrami- dal neurons in LII and LIII of the ECT were randomly se- lected from each rat. Sequential optical sections of 1392 × 1040 pixels were taken at 1.5 µm intervals along the z-axis (Leica, DMi8 + DFC7000J, Germany). The Imaris software (BitPlane AG, Zurich, Switzerland) was used for tridimen- sional reconstruction. The total dendrite length, number of dendritic branches, and spatial distribution of dendrites in the pyramidal neurons of the ECT were estimated using Sholl analysis [28]. To measure the density of dendritic spines, a straight dendrite was scanned on the z-axis using a 100 × objective microscope. A 40-µm long dendrite was randomly intercepted with image J 1.46r (National institute of health, Bethesda, Maryland, USA). The number of synaptic spines was counted and the density of synaptic spines (spines/10 µm) was calculated. At least 10 terminal dendrites were se- lected for each sample.
+
+2.6 Statistical analysis
+
+All the data was expressed as mean ± standard deviation. JMP software version 16.0 (SAS Institute, Cary, NC, USA) was used for statistical processing. All parameters were tested for normal distribution using the Kolmogorov-Smirnov test. Two independent-sample t tests were conducted used to compare the parameters differences between the control and sevoflurane groups, including NRG1, ErbB4, PV, GAD67, NR2B and NR2A. Dendrites were analyzed with Kruskal- Wallis test (Sholl analysis) and Steel Dwass post hoc test using JMP software version 16.0 (SAS Institute, Cary, NC, USA) [28]. It was considered that a difference was statistically sig- nificant when P < 0.05. GraphPad Prism 5.0 (GraphPad Soft- ware, San Diego, CA, USA) software was used to make draw- ings.
+
+3. Results 3.1 Prenatal sevoflurane exposure down regulates the level of NRG1–ErbB4 in LII/LIII of the ECT in offspring
+
+The results showed that the expression of NRG1 in the SeV group was significantly lower than that in the control group (control group: 9.94 ± 4.26, SeV group: 3.72 ± 2.08, P < 0.01) (Fig. 1). The ErbB4 level in SeV group was also
+
+615
+
+Fig. 1. Exposure of sevoflurane to maternal rats impaired the NRG1–ErbB4 signaling pathway in the LII/LIII entorhinal cortex of offspring. Inverted fluorescence microscopy showed that the NRG1 and ErbB4 positive cells were stained with green fluorescence. The top picture shows the field of vision under the 100 × light microscope of the inverted fluorescence microscope. We used the inverted fluorescence microscope to scan the whole picture of immunofluorescence staining of 100 × rat brain slices. In the whole picture, the area in the white box is pyramidal neurons in the LII/LIII entorhinal cortex. In the control (A and C) and sevoflurane (B and D) groups, A and B show the NRG1 positive cells in the LII/LIII entorhinal cortex of the P30 progeny. C and D show the ErbB4 positive cells. The numbers were significantly decreased in the sevoflurane group compared to the control group (E and F). Note: The symbol ‘**’ represented there was significant difference between groups under the significant level of 0.01.
+
+significantly lower than that in the control group (control group: 20.11 ± 12.67, SeV: 6.47 ± 3.41, P < 0.01) (Fig. 1).
+
+3.3 Prenatal exposure to sevoflurane leads to the abnormal expressions of NMDA receptor subunits in LII/LIII of the ECT of offspring
+
+3.2 Prenatal sevoflurane exposure reduces the number of interneurons in the LII/LIII ECT of offspring
+
+The results showed that the number of PV positive cells in the SeV group was significantly lower than that in control group (control group: 23.41 ± 1.01, SeV group: 7.41 ± 0.82, P < 0.01) (Fig. 2). The number of GAD67 positive cells in the SeV group decreased significantly (control group: 17.44 ± 4.63, SeV group: 10.05 ± 3.17, P < 0.01) (Fig. 2).
+
+The results were as follows (Fig. 3): compared to the control group, the number of NR2A positive cells in the SeV group was decreased (control group: 24.00 ± 10.83, SeV group: 11.40 ± 10.10, P < 0.05) while the number of NR2B positive cells was significantly increased (control group: 15.61 ± 5.14, SeV group: 38.21 ± 8.50, P < 0.01).
+
+616
+
+Volume 20, Number 3, 2021
+
+Fig. 2. The GABAergic interneurons of offspring in the LII/LIII entorhinal cortex were significantly reduced. PV and GAD67 positive cells were stained with red fluorescence, so in the inverted fluorescence microscope at 20X magnification, these cells are indicated by red fluorescence. (A) The PV positive cells in the LII/LIII entorhinal cortex of the P30 offspring in the control group. (B) PV immunofluorescence (red) on a P30 offspring rat in the sevoflurane group. (C) GAD67 immunofluorescence (red) on a P30 offspring rat in the control group. (D) GAD67 immunofluorescence (red) on a P30 offspring rat in the sevoflurane group. (E) The numbers of PV cells were significantly decreased in the sevoflurane compared to the control group. (F) The numbers of GAD67
+
+cells were significantly decreased in the sevoflurane group compared to the control group. Note: The symbol ‘**’ represented there was significant difference between groups under the significant level of 0.01.
+
+3.4 Prenatal sevoflurane exposure results in the abnormal development of the dendrites of pyramidal neurons
+
+The results showed that compared to that in the control group, the number of dendritic spines of the pyramidal neu- rons in LII/LIII of the ECT in the SeV group was significantly increased (control group: 8.88 ± 1.83, SeV group: 11.86 ± 1.27, P < 0.01), the total length of dendrites in the SeV group was significantly lower than that in the control group (con- trol group: 3819.32 ± 614.99, SeV: 2978.45 ± 577.31, P < 0.01), and the number of dendrite branches in the SeV group was significantly lower than that in control group (control group: 38.24 ± 4.66, SeV group: 32.22 ± 6.88, P < 0.01) (see Fig. 4). Sholl analysis showed that the spatial distribution of dendrites was abnormal (P < 0.05, Fig. 4).
+
+4. Discussion
+
+We previously speculated that prenatal sevoflurane, as an exogenous GABAA receptor agonist, down regulated the NRG1–ErbB4 signaling pathway and that this change could lead to the disturbance of interneurons and the abnormal de- velopment of the dendrites of pyramidal neurons. The re- sults of this study basically confirmed our hypothesis. This could be helpful for the standardization of the clinical use of sevoflurane and its toxicity prevention and treatment.
+
+With the development of surgical technology and fetal surgery, an increasing number of pregnant women need to be exposed to general anesthetics. Sevoflurane mainly acts as GABAA receptor agonist/enhancer and has sedative, anal-
+
+gesic, and muscle relaxant effects. Bartolini et al. [29] found that the inhalation of sevoflurane in 2MAC can inhibit uter- ine muscle contraction in a dose-dependent manner. Thus, based on its inhibition of uterine contraction, which can pre- vent premature delivery, sevoflurane is the most widely used inhalation anesthetic during pregnancy. Sevoflurane easily impacts fetuses though the placenta [30]. Zheng et al. [9] found that with the inhalation of 2.5% sevoflurane for 2 hours and 4.1% sevoflurane for 6 hours, the offspring of pregnant rat showed decreased learning and memory ability, accompa- nied by the release of inflammatory agents in the hippocam- pus and abnormal synaptic development. In recent years, a large number of studies have shown that sevoflurane it neurotoxic to the developing brain, which can lead to in- creased neuronal apoptosis, the inhibition of proliferation, neuronal development disorders, and long-term neurobe- havioral abnormalities [31, 32]. However, most of the au- thors of these studies were focus on the hippocampus, which is related to learning and memory, and mainly studies the ul- trastructural and functional impairment of projection neu- rons. Little attention has been paid to LII/LIII of the ECT, which is the key area of hippocampal information input. In LII/LIII of the ECT, interneurons, characterized as GABAer- gic neurons, could be regulated by sevoflurane. Impacts on the NRG1–ErbB4 pathway in interneurons may further reg- ulate interneurons themselves and pyramidal neurons and af- fect the information transmission functions of the ECT.
+
+Volume 20, Number 3, 2021
+
+617
+
+Fig. 3. Maternal exposure of sevoflurane leads to impaired maturation of NMDA receptors in offspring. NR2A and NR2B positive cells are indicated by green fluorescence. (A) The NR2A positive cells in the LII/LIII entorhinal cortex of the P30 offspring in the control group. (B) NR2A immunofluorescence (green) on a P30 offspring rat in the sevoflurane group. (C) NR2B immunofluorescence (green) on a P30 offspring rat in the control group. (D) NR2B immunofluorescence (green) on a P30 offspring rat in the sevoflurane group. (E) The numbers of NR2A cells were significantly decreased in the sevoflurane compared to the control group. (F) The numbers of NR2B cells were significantly increased in the sevoflurane compared to the control group. Note: The
+
+symbol ‘**’ represented there was significant difference between groups under the significant level of 0.01.
+
+In this study, we simulated clinical practice. The preg- nant rats were anesthetized with 4% sevoflurane for 3 hours at 14.5 days of gestation to reach a MAC of 1.8 (lower than normal rats, considering that pregnant rats are more sensitive to sevoflurane), which was equivalent to that of most surgical operations for pregnant females. We found that sevoflurane, as an exogenous GABAA receptor agonist, could down reg- ulate the NRG1–ErbB4 signaling pathway (Fig. 2). NRG1 is highly expressed in mammalian embryos and decreases with age. It involves many aspects of neural development, includ- ing neuronal migration, survival, axon projection, myelin sheath development, synaptic formation and the regulation of neurotransmitter receptor expression [13, 16, 33–37]. Pre- vious studies have revealed that the NRG1–ErbB4 pathway is necessary for the generation and migration of intermedi- ate neurons originating from the medial ganglionic eminence (MGE) region [16, 38–40]. The impairment of the NRG1– ErbB4 pathway can affect the migration of PV interneurons from the MGE region. PV interneurons account for 40% of interneurons, and there was a decrease of 30–50% of in-
+
+terneurons in ErbB4 gene knockout rats. We administered sevoflurane anesthesia during middle pregnancy, which is the critical period of the development of the generation and mi- gration of the embryonic interneurons. In this period, the MGE area produces PV interneurons, which migrate to the edge of the cortex and subventricular area and then radially to the target cortex [29]. Our results also showed that the NRG1–ErbB4 pathway was down regulated along with a de- crease in the number of interneurons. Therefore, the de- crease in the number of interneurons in LII/LIII of the ECT in the SeV group could have been due to the inhibition of the formation of interneurons by the decreased NRG1–ErbB4 level. At the same time, the decreased NRG1–ErbB4 blocked interneurons’ migration to LII/LIII of the ECT.
+
+The NMDA receptor (NMDAR) is mainly composed of two NR1 and two NR2 subunits. The distribution of NM- DAR subunits (NR1, NR2, NR3) also changes with the pro- cess of neurodevelopment. NR1 begins to increase after birth until puberty and reaches a peak level in the third week af- ter birth. NR2B and NR2D are the main subunits of NR2 in
+
+618
+
+Volume 20, Number 3, 2021
+
+Fig. 4. Sevoflurane exposure disturbs the maturation of pyramidal neurons in the LII/LIII entorhinal cortex of offspring. Six P30 brains in each group were processed for Golgi–Cox impregnation, and pyramidal neurons in the LII/LIII entorhinal cortex were studied in the control (examples in A) and sevoflurane (examples in C) groups. The left panels (A and C) show two examples of Golgi-impregnated neurons. The total branch number and dendritic length were significantly decreased in the sevoflurane group compared to the control group (E and F) (n = 24 neurons in each group). In addition, higher spine density was observed in the sevoflurane group had than the control group (see B, D, and H) (n = 24 dendrites in each group). Sholl analysis showed that the
+
+complexity of dendritic trees was lower in the sevoflurane group than the control group (G). Note: The symbol ‘*’ represented there was significant difference between groups under the significant level of 0.05. The symbol ‘**’ represented there was significant difference between groups under the significant level of 0.01.
+
+the embryonic stage. The expression of NR2 changes signif- icantly in the two weeks after birth. NR2A begins to par- tially replace NR2B until the brain matures. The develop- ment of NMDAR subunits marks the transition from im- mature to mature neurons and is closely related to the de- velopment of learning and memory ability. In our experi- ment, exposure to sevoflurane was found to decrease the ex- pression of NR2A and increase the expression of NR2B in offspring, which is consistent with the findings of previous studies [31, 32]. NRG1–ErbB4 changes could increase the phosphorylation of NR2B by Fyn and, thus, reduce the in- ternalization of NR2B and delay the transition from NR2B to NR2A. NRG1–ErbB4 can regulate not only the release and
+
+activity of GABAergic neurotransmitters but also the exci- tatory synapses on inhibitory neurons (interneurons). The intracellular segment of the ErbB4 protein contains post- synaptic postsynaptic dens 95 (PSD-95)/DiscsLarge/zonula occludens protein-1 (ZO1) (PDZ) domains, which are an- chored to the postsynaptic membrane by interacting with other proteins (such as PSD-95) that also contain PDZ [41– 43]. Thus, ErbB4 regulates the function of the NMDAR by the connection of PDZ [41–43]. The NRG1–ErbB4 pathway phosphorylates the phosphorylation site of NR2B through a member of the Src family of Fyn (SRC/Fyn), which blocks the internalization of NR2B by protein AP-2 [44, 45]. In this way, the NRG1–ErbB4 pathway increases the expression of
+
+Volume 20, Number 3, 2021
+
+619
+
+NR2B on the postsynaptic membrane [44, 45]. Thus, the de- creased NRG1–ErbB4 level results in increased NR2B and de- creased NR2A levels (Fig. 3). This alteration may lead to the abnormal development of learning and memory ability.
+
+Pyramidal neurons are responsible for information trans- mission, and their function is very important. Therefore, it has always been a research hotspot in the topic of the neu- rotoxicity of general anesthetics during development. In- terneurons play an important role in regulating pyramidal neurons. We found that prenatal sevoflurane exposure in middle pregnancy resulted in a significant increase in the number of dendritic spines, a significant decrease in the to- tal length of dendrites, and an abnormal spatial distribution of dendrites in offspring (Fig. 4). There could be three pos- sible mechanism behind the increased number of dendritic spines. One explanation is that the decrease in the number of interneurons leads to the weakening of their regulation of pyramidal neurons, resulting in abnormal numbers of den- dritic spines. The second explanation is that the change of the NRG1–ErbB4 pathway could regulate the dendritic spines of pyramidal neurons. Barros et al. [35] found that the num- ber of pyramidal dendritic spines decreased after ErbB4 and ErbB2 knockout in the nervous system. However, the num- ber of pyramidal dendritic spines did not decrease in the ex- periment of ErbB4 knockout by pyramidal cells [44, 46]. It is suggested that ErbB2 may play a role of functional com- pensation, and there is an over-compensation in the num- ber of pyramidal dendritic spines [37, 47]. The third ex- planation is that the NR2B subunit of NMDAR combines with PSD-95 and calmodulin-dependent kinase II (CaMKII), which activates a series of downstream signaling pathways and leads to an increase in the number of dendritic spines [48]. The NR2B/PSD95/kalirin-7 pathway is very impor- tant in the development of neuronal dendritic spines. Re- cent studies have shown that PSD-95, kalirin-7, and NR2B form a complex with a postsynaptic membrane through the PDZ domain. NR2B can activate kalirin-7, and then activate Rac1, a downstream RhoGTPase family member [49]. In this way, NR2B could dynamically regulate actin cytoskeleton re- arrangement, promote the growth of dendritic spines, induce the formation of spinous structures in neuronal bodies, and cause the excessive formation of dendritic spines in pyramidal neurons and interneurons [49]. The decrease of total length and branches of dendrites in offspring may be explained by the change of the NRG1–ErbB4 signaling pathway and NR2B subunit, which mainly involve the growth and pruning of dendrites [36, 37]. Further, the plasticity of dendritic spines affects learning and memory function, especially the forma- tion of long-term memory [50, 51].
+
+The initial experimental hypothesis has been verified in this study. However, to test the hypothesis, this study focused on the study of the ECT with a concentration on the memory transfer station. Thus, this study did not involve an investi- gation of the dentate gyrus area of the hippocampus, which was directly projected by pyramidal neurons in the ECT. The
+
+620
+
+time (3 h) for sevoflurane exposure may be too long in ac- tual clinical situation. However, despite the hard exploratory work we’ve done, there is a few results we assumed except for that sevoflurane exposed to fetal brain for at least 3 hours or neonatal brains for more than 6 hours were neurotoxic. These results suggested that the safe concentration and ex- posure time in most clinical practice is safe.
+
+5. Conclusions
+
+Prenatal sevoflurane exposure in middle pregnancy could lead to the disorder of the interneurons by activating the GABAA receptor and its NRG1–ErbB4 pathway. In this way, prenatal sevoflurane leads to the abnormal dendrite devel- opment of the pyramidal neurons in the ECT of offspring rats and, thus, may interfere with the process of information transmission from the ECT to the hippocampus. This study indicated a possible novel neurotoxic pathway in the influ- ence of sevoflurane on ECT of rat offspring. In clinical prac- tice, the concentration of sevoflurane is much lower than the concentration in this study because of the addition of other auxiliary drugs such as opioid sedatives. Secondly, because of the advancement of surgical procedures, most non-obstetric procedures during pregnancy do not require 3 hours. There- fore, the current clinical use of sevoflurane is safe. How- ever, prolonged exposure to high concentrations of sevoflu- rane still needs to be alert to neurotoxicity.
+
+Abbreviations
+
+gamma- aminobutyric acid; CNS, central nervous system; GABAA, GABA type A; LII and LIII, layers II and III; NRG1, Neuregulin-1; ErbB4, epidermal growth factor receptor-4; PV, parvalbumin; SD, Sprague–Dawley; SeV, sevoflu- rane; MAC, minimum alveolar concentration; P30, the day 30 of postpartum; PBS, phosphate buffered saline; GAD67, glutamic acid decarboxylase 67; NR2A, NMDA receptor subunit 2A; NR2B, NMDA receptor subunit 2B; BSA, bovine serum albumin; MGE, medial ganglionic eminence region; NMDAR, NMDA receptor; PDZ, PSD- 95/DiscsLarge/ZO1; PSD-95, postsynaptic dens 95; ZO1, onula occludens protein-1; SRC/Fyn, Src family of Fyn; CaMKII, calmodulin-dependent kinase II.
+
+NMDA, N -methyl-D-aspartate;
+
+GABA,
+
+Author contributions
+
+XS, YG and TZ designed research; YG, TZ, YC, ZS, ZS and JL performed experiments; YC analyzed data; YG wrote the paper; TZ and XS critically revised the paper.
+
+Ethics approval and consent to participate
+
+This experiment was approved by the Animal Ethics Committee of Guangzhou Medical University, and the re- searchers strictly followed the relevant provisions of the “Guidelines for the Care and Use of Laboratory Animals” is- sued by the National Institutes of Health in 1996 (ethic code: 2016-029).
+
+Volume 20, Number 3, 2021
+
+Acknowledgment
+
+We thank Xiaolong Zeng for assistance in manuscript re-
+
+vision preparation.
+
+Funding
+
+This work was granted by the National Natural Science
+
+Foundation of China (Granted no. 81870823).
+
+Conflict of interest
+
+The authors declare no conflict of interest.
+
+References [1] Okeagu CN, Anandi P, Gennuso S, Hyatali F, Stark CW, Prab- hakar A, et al. Clinical management of the pregnant patient under- going non-obstetric surgery: Review of guidelines. Best Practice & Research Clinical Anaesthesiology. 2020; 34: 269–281.
+
+[2] Jevtovic-Todorovic V, Absalom AR, Blomgren K, Brambrink A, Crosby G, Culley DJ, et al. Anaesthetic neurotoxicity and neuro- plasticity: an expert group report and statement based on the BJA Salzburg Seminar. British Journal of Anaesthesia. 2013; 111: 143– 151.
+
+[3] Grasshoff C, Antkowiak B. Propofol and Sevoflurane Depress Spinal Neurons in Vitro via Different Molecular Targets. Anes- thesiology. 2004; 101: 1167–1176.
+
+[4] Ben-Ari Y. Excitatory actions of gaba during development: the na- ture of the nurture. Nature Reviews Neuroscience. 2002; 3: 728– 739.
+
+[5] Wang DD, Kriegstein AR. Defining the role of GABA in cortical development. Journal of Physiology. 2009; 587: 1873–1879. [6] Palanisamy A. Maternal anesthesia and fetal neurodevelopment. International Journal of Obstetric Anesthesia. 2012; 21: 152–162. [7] Bjarnadottir M, Misner DL, Haverfield-Gross S, Bruun S, Hel- gason VG, Stefansson H, et al. Neuregulin1 (NRG1) Signaling through Fyn Modulates NMDA Receptor Phosphorylation: Dif- ferential Synaptic Function in NRG1+/- Knock-Outs Compared with Wild-Type Mice. Journal of Neuroscience. 2007; 27: 4519– 4529.
+
+[8] Istaphanous GK, Ward CG, Nan X, Hughes EA, McCann JC, McAuliffe JJ, et al. Characterization and quantification of isoflurane-induced developmental apoptotic cell death in mouse cerebral cortex. Anesthesia and Analgesia. 2013; 116: 845–854. [9] Zheng H, Dong Y, Xu Z, Crosby G, Culley D, Zhang Y, et al. Sevoflurane Anesthesia in Pregnant Mice Induces Neurotoxicity in Fetal and Offspring Mice. Anesthesiology. 2013; 118: 516–526. [10] Briner A, De Roo M, Dayer A, Muller D, Habre W, Vutskits L. Volatile anesthetics rapidly increase dendritic spine density in the rat medial prefrontal cortex during synaptogenesis. Anesthesiol- ogy. 2010; 112: 546–556.
+
+[11] Zhao T, Li Y, Wei W, Savage S, Zhou L, Ma D. Ketamine admin- istered to pregnant rats in the second trimester causes long-lasting behavioral disorders in offspring. Neurobiology of Disease. 2014; 68: 145–155.
+
+[12] Corfas G, Roy K, Buxbaum JD. Neuregulin 1-erbB signaling and the molecular/cellular basis of schizophrenia. Nature Neuro- science. 2004; 7: 575–580.
+
+[13] Mei L, Xiong W. Neuregulin 1 in neural development, synaptic plasticity and schizophrenia. Nature Reviews. Neuroscience. 2008; 9: 437–452.
+
+[14] Liang D, Fan F, Ding W, Fang Y, Hu L, Lei B, et al. Increased Seizure Susceptibility for Rats Subject to Early Life Hypoxia might be Associated with Brain Dysfunction of NRG1-ErbB4 Signaling in Parvalbumin Interneurons. Molecular Neurobiology. 2020; 57: 5276–5285.
+
+[15] Harrison PJ, Weinberger DR. Schizophrenia genes, gene expres- sion, and neuropathology: on the matter of their convergence. Molecular Psychiatry. 2005; 10: 40–68.
+
+Volume 20, Number 3, 2021
+
+[16] Anton ES, Ghashghaei HT, Weber JL, McCann C, Fischer TM, Cheung ID, et al. Receptor tyrosine kinase ErbB4 modulates neu- roblast migration and placement in the adult forebrain. Nature Neuroscience. 2004; 7: 1319–1328.
+
+[17] Ozaki M, Tohyama K, Kishida H, Buonanno A, Yano R, Hashikawa T. Roles of neuregulin in synaptogenesis between mossy fibers and cerebellar granule cells. Journal of Neuroscience Research. 2000; 59: 612–623.
+
+[18] Zhao JJ, Lemke G. Selective disruption of neuregulin-1 function in vertebrate embryos using ribozyme-tRNA transgenes. Devel- opment. 1998; 125: 1899–1907.
+
+[19] Gu Z, Jiang Q, Fu AKY, Ip NY, Yan Z. Regulation of NMDA re- ceptors by neuregulin signaling in prefrontal cortex. Journal of Neuroscience. 2005; 25: 4974–4984.
+
+[20] Ozaki M, Sasner M, Yano R, Lu HS, Buonanno A. Neuregulin-beta induces expression of an NMDA-receptor subunit. Nature. 1997; 390: 691–694.
+
+[21] Konradi C, Heckers S. Molecular aspects of glutamate dysregula- tion: implications for schizophrenia and its treatment. Pharma- cology & Therapeutics. 2003; 97: 153–179.
+
+[22] Gabrieli D, Schumm SN, Vigilante NF, Meaney DF. NMDA Receptor Alterations after Mild Traumatic Brain Injury Induce Deficits in Memory Acquisition and Recall. Neural Computation. 2021; 33: 67–95.
+
+[23] Howe T, Blockeel AJ, Taylor H, Jones MW, Bazhenov M, Malerba P. NMDA receptors promote hippocampal sharp-wave ripples and the associated coactivity of CA1 pyramidal cells. Hippocam- pus. 2020; 30: 1356–1370.
+
+[24] Bartolini G, Ciceri G, Marín O. Integration of GABAergic in- terneurons into cortical cell assemblies: lessons from embryos and adults. Neuron. 2013; 79: 849–864.
+
+[25] Li X, Su F, Ji M, Zhang G, Qiu L, Jia M, et al. Disruption of hippocampal neuregulin 1-ErbB4 signaling contributes to the hippocampus-dependent cognitive impairment induced by isoflu- rane in aged mice. Anesthesiology. 2014; 121: 79–88.
+
+[26] Clancy B, Darlington RB, Finlay BL. Translating developmental
+
+time across mammalian species. Neuroscience. 2001; 105: 7–17.
+
+[27] Ihmsen H, Schywalsky M, Plettke R, Priller M, Walz F, Schwilden H. Concentration-effect relations, prediction probabilities (Pk), and signal-to-noise ratios of different electroencephalographic parameters during administration of desflurane, isoflurane, and sevoflurane in rats. Anesthesiology. 2008; 108: 276–285.
+
+[28] Sholl DA. Dendritic organization in the neurons of the visual and motor cortices of the cat. Journal of Anatomy. 1953; 87: 387–406. [29] Bartolini G, Ciceri G, Marín O. Integration of GABAergic in- terneurons into cortical cell assemblies: lessons from embryos and adults. Neuron. 2013; 79: 849–864.
+
+[30] Chai D, Cheng Y, Jiang H. Fundamentals of fetal toxicity relevant to sevoflurane exposures during pregnancy. International Journal of Developmental Neuroscience. 2019; 72: 31–35.
+
+[31] Wang W, Jia L, Luo Y, Zhang H, Cai F, Mao H, et al. Location- and Subunit-Specific NMDA Receptors Determine the Developmen- tal Sevoflurane Neurotoxicity through ERK1/2 Signaling. Molec- ular Neurobiology. 2016; 53: 216–230.
+
+[32] Zhang X, Shen F, Xu D, Zhao X. A lasting effect of postnatal sevoflurane anesthesia on the composition of NMDA receptor subunits in rat prefrontal cortex. International Journal of Devel- opmental Neuroscience. 2016; 54: 62–69.
+
+[33] Falls DL. Neuregulins: functions, forms, and signaling strategies.
+
+Experimental Cell Research. 2003; 284: 14–30.
+
+[34] Buonanno A, Fischbach GD. Neuregulin and ErbB receptor sig- naling pathways in the nervous system. Current Opinion in Neu- robiology. 2001; 11: 287–296.
+
+[35] Barros CS, Calabrese B, Chamero P, Roberts AJ, Korzus E, Lloyd K, et al. Impaired maturation of dendritic spines without disor- ganization of cortical cell layers in mice lacking NRG1/ErbB sig- naling in the central nervous system. Proceedings of the National Academy of Sciences. 2009; 106: 4507–4512.
+
+[36] Fazzari P, Paternain AV, Valiente M, Pla R, Luján R, Lloyd K, et
+
+621
+
+al. Control of cortical GABA circuitry development by Nrg1 and ErbB4 signalling. Nature. 2010; 464: 1376–1380.
+
+[37] Cooper MA, Koleske AJ. ErbB4 localization to interneurons: clearer insights into schizophrenia pathology. Biological Psychi- atry. 2011; 70: 602–603.
+
+[38] Buonanno A. The neuregulin signaling pathway and schizophre- nia: from genes to synapses and neural circuits. Brain Research Bulletin. 2010; 83: 122–131.
+
+[39] Fisahn A, Neddens J, Yan L, Buonanno A. Neuregulin-1 modulates hippocampal gamma oscillations: implications for schizophrenia. Cerebral Cortex. 2009; 19: 612–618.
+
+[40] Flames N, Long JE, Garratt AN, Fischer TM, Gassmann M, Birchmeier C, et al. Short- and long-range attraction of corti- cal GABAergic interneurons by neuregulin-1. Neuron. 2004; 44: 251–261.
+
+[41] Garcia RA, Vasudevan K, Buonanno A. The neuregulin recep- tor ErbB-4 interacts with PDZ-containing proteins at neuronal synapses. Proceedings of the National Academy of Sciences. 2000; 97: 3596–3601.
+
+[42] Krivosheya D, Tapia L, Levinson JN, Huang K, Kang Y, Hines R, et al. ErbB4-neuregulin signaling modulates synapse development and dendritic arborization through distinct mechanisms. Journal of Biological Chemistry. 2008; 283: 32944–32956.
+
+[43] Huang YZ, Won S, Ali DW, Wang Q, Tanowitz M, Du QS, et al. Regulation of neuregulin signaling by PSD-95 interacting with ErbB4 at CNS synapses. Neuron. 2000; 26: 443–455.
+
+[44] Wang JQ, Guo M, Jin D, Xue B, Fibuch EE, Mao L. Roles of sub- unit phosphorylation in regulating glutamate receptor function. European Journal of Pharmacology. 2014; 728: 183–187.
+
+622
+
+[45] Nakazawa T, Komai S, Tezuka T, Hisatsune C, Umemori H, Semba K, et al. Characterization of Fyn-mediated tyrosine phos- phorylation sites on GluR epsilon 2 (NR2B) subunit of the N - methyl-D-aspartate receptor. Journal of Biological Chemistry. 2001; 276: 693–699.
+
+[46] Alkire MT, Gruver R, Miller J, McReynolds JR, Hahn EL, Cahill L. Neuroimaging analysis of an anesthetic gas that blocks human emotional memory. Proceedings of the National Academy of Sci- ences. 2008; 105: 17221727.
+
+[47] Yin D, Sun X, Bean JC, Lin TW, Sathyamurthy A, Xiong W, et al. Regulation of spine formation by ErbB4 in PV-positive interneu- rons. Journal of Neuroscience. 2013; 33: 19295–19303.
+
+[48] Tolias KF, Bikoff JB, Burette A, Paradis S, Harrar D, Tavazoie S, et al. The Rac1-GEF Tiam1 couples the NMDA receptor to the activity-dependent development of dendritic arbors and spines. Neuron. 2005; 45: 525–538.
+
+[49] Penzes P, Jones KA. Dendritic spine dynamics–a key role for
+
+kalirin-7. Trends in Neurosciences. 2008; 31: 419–427.
+
+[50] Xu T, Yu X, Perlik AJ, Tobin WF, Zweig JA, Tennant K, et al. Rapid formation and selective stabilization of synapses for endur- ing motor memories. Nature. 2009; 462: 915–919.
+
+[51] Tschida K, Mooney R. Deafening Drives Cell-Type-Specific Changes to Dendritic Spines in a Sensorimotor Nucleus Impor- tant to Learned Vocalizations. Neuron. 2012; 73: 1028–1039.
+
+Volume 20, Number 3, 2021

new_pdfs/10.3389_fncel.2017.00373.txt

@@ -0,0 +1,485 @@
+ORIGINAL RESEARCH published: 22 November 2017 doi: 10.3389/fncel.2017.00373
+
+Propofol Exposure in Early Life Induced Developmental Impairments in the Mouse Cerebellum
+
+Rui Xiao 1,2, Dan Yu 1,2, Xin Li 2, Jing Huang 1, Sheng Jing 1, Xiaohang Bao 1, Tiande Yang 1* and Xiaotang Fan 2*
+
+1Department of Anesthesiology, Xinqiao Hospital, Third Military Medical University, Chongqing, China, 2Department of Developmental Neuropsychology, Third Military Medical University, Chongqing, China
+
+Edited by: Tycho Hoogland, Erasmus Medical Center, Netherlands
+
+Reviewed by: Hiroshi Nishiyama, University of Texas at Austin, United States Karen M. Smith, University of Louisiana at Lafayette, United States
+
+Propofol is a widely used anesthetic in the clinic while several studies have demonstrated that propofol exposure may cause neurotoxicity in the developing brain. However, the effects of early propofol exposure on cerebellar development are not well understood. Propofol (30 or 60 mg/kg) was administered to mice on postnatal day (P)7; Purkinje cell dendritogenesis and Bergmann glial cell development were evaluated on P8, and granule neuron migration was analyzed on P10. The results indicated that exposure to propofol on P7 resulted in a significant reduction in calbindin-labeled Purkinje cells and their dendrite length. Furthermore, propofol induced impairments in Bergmann glia development, which might be involved in the delay of granule neuron migration from the external granular layer (EGL) to the internal granular layer (IGL) during P8 to P10 at the 60 mg/kg dosage, but not at the 30 mg/kg dosage. Several reports have suggested that the Notch signaling pathway plays instructive roles in the morphogenesis of Bergmann glia. Here, it was revealed that propofol treatment decreased Jagged1 and Notch1 protein levels in the cerebellum on P8. Taken together, exposure to propofol during the neonatal period impairs Bergmann glia development and may therefore lead to cerebellum development defects. Our results may aid in the understanding of the neurotoxic effects of propofol when administrated to infants.
+
+Keywords: propofol, cerebellum, development, neurotoxicity, mouse
+
+Correspondence: Tiande Yang 31011@sina.com Xiaotang Fan fanxiaotang2005@163.com
+
+Received: 08 September 2017 Accepted: 09 November 2017 Published: 22 November 2017
+
+Citation: Xiao R, Yu D, Li X, Huang J, Jing S, Bao X, Yang T and Fan X (2017) Propofol Exposure in Early Life Induced Developmental Impairments in the Mouse Cerebellum. Front. Cell. Neurosci. 11:373. doi: 10.3389/fncel.2017.00373
+
+INTRODUCTION
+
+The cerebellum is characterized by laminated structures, and its abnormal morphogenesis may lead to deficits related to disorders such as Dandy-Walker Malformations, Joubert Syndrome and other congenital spinocerebellar ataxias (Millen and Gleeson, 2008). An increasing number of recent studies have suggested that anesthesia may be neurotoxic to the brain and lead to various long-term behavioral disorders, especially in the infants (Olney et al., 2002; Stargatt et al., 2006; Patel and Sun, 2009; DiMaggio et al., 2011; Reddy, 2012; Sinner et al., 2014).
+
+Abbreviations: BLBP, Brain lipid binding protein; BrdU, 5-Bromo-2(cid:48)-deoxyuridine; BSA, Bovine serum albumin; CB, Calbindin; DAPI, 4(cid:48),6-diamidino-2-phenylindole; EGL, External granular layer; GFAP, Glial fibrillary acidic protein; HE, Hematoxylin-eosin; IGL, Internal granular layer; IOD, Integrated optical density; i.p., intraperitoneally; ML, Molecular layer; NC, Nitrocellulose; NeuN, Neuronal nuclei; PBS, Phosphate-buffered saline; PCL, Purkinje cell layer; ROD, Relative optical density; RT, Room temperature.
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+1
+
+November 2017 | Volume 11 | Article 373
+
+Xiao et al.
+
+function depends on well-organized neuronal connections and the integration of afferent and efferent fibers into the cerebellar circuitry. The mouse cerebellar cortex has a well-defined architecture consisting of the following three major layers: (1) the molecular layer (ML); (2) the Purkinje cell layer (PCL); and (3) the granule layer (Voogd and Glickstein, 1998). To establish proper lamination and circuitry, important events such as neuronal differentiation, morphogenesis, and migration need to be precisely regulated during cerebellar development (Altman and Winfree, 1977; Buffo and Rossi, 2013). Mouse cerebellar development continues until 3 weeks after birth. During this postnatal period, cerebellar cells undergo sequential development steps in spatially well-defined regions. Purkinje cells are the principal neurons, and are transformed from a stellate morphology into their essential dendritic structures between the first and second weeks (Eccles, 1970; Sotelo and Rossi, 2013). At the early postnatal stage, granule cell precursors are the most abundant in the external granular layer (EGL), followed by an inward radial migration along the Bergmann glial radial fibers to their destination, the internal granule layer (IGL; Komuro et al., 2001; Buffo and Rossi, 2013). By the end of the third postnatal week, the EGL disappeared and three well-defined neuronal layers have formed (Qiu et al., 2010).
+
+Proper cerebellar
+
+Propofol (6,2 diisopropylphenol) is an anesthetic that works through the activation of gamma amino butyric acid A (GABAA) (NMDA) and block of N-methyl-D-aspartate receptors glutamate receptors (Franks and Lieb, 1994; Irifune et al., 2003). It is widely used in the clinic for induction and maintenance of general anesthesia and conscious sedation, especially in neurosurgery, for its unique benefit on cerebral physiology including reduction in cerebral blood flow, intracranial pressure and cerebral metabolism (Diaz and Kaye, 2017). The increasing utilization of propofol as a drug of abuse is of high concern, and a public health threat, especially for developing fetuses. Studies in rodents have confirmed that propofol exposure caused toxic effects in the developing brain. Consistent with other reports, we previously found that propofol administration during early postnatal life suppressed hippocampal neurogenesis (Huang et al., 2016). Additionally, propofol has been implicated in causing movement disorders since 30 years ago, which strongly suggests that propofol may damage the cerebellum (Dingwall, 1987; Zabani and Vaghadia, 1996; Bendiksen and Larsen, 1998; Brooks, 2008). Recent studies have also indicated that propofol depressed Purkinje cell activity and affected the cerebellum circuitry (Jin R. et al., 2015; Jin W. Z. et al., 2015; Lee K. Y. et al., 2015). However, little is known regarding the impact of propofol exposure on the development of the cerebellar neuronal population.
+
+In this study, newborn mice at P7 were administered a single injection of 30 or 60 mg/kg of propofol or vehicle of equal volume to explore the effects of propofol on Purkinje cell dendritogenesis and Bergmann glia development. The migration of newborn granule cells in the EGL was evaluated with a 5-bromodeoxyuridine (BrdU) labeling protocol. As the Notch signaling pathway has been indicated to play crucial roles in the regulation of development of Bergmann
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+2
+
+Propofol and Cerebellar Development
+
+its involvement in the underlying mechanism on the glia, neurotoxic effects by propofol during the early stage were also explored.
+
+MATERIALS AND METHODS
+
+Animals Male and female C57/BL6 mice were provided by the Third Military Medical University and housed under a 12 h light/dark cycle in a temperature-controlled room with free access to food and water. All the experimental procedures were performed in accordance with the guidelines for laboratory animal care and use and were approved by Third Military Medical University. Each litter was kept together with its mother throughout the experiment, except for the brief intervals of separation required for the daily injections. At least five mice in each group were analyzed for immunofluorescence staining and three mice for western blot.
+
+Drug Treatment The day of birth was designated postnatal day 0 (P0). On P7, pups injection intraperitoneally (i.p.) at a subanesthetic dose of 30 or 60 mg/kg (Cattano et al., 2008; Yang B. et al., 2014), according to our previous study (Huang et al., 2016). The same volume of intralipid was administered i.p. as a vehicle the neonatal mice were grouped control randomly with a random number table base for similar body weight.
+
+received a vehicle or propofol
+
+for propofol. All
+
+To explore the morphological changes in the Purkinje cells, Bergmann glia and granule neuron, pups were sacrificed 24 h (P8) after drug treatment. To evaluate whether propofol affected the radial migration of the granule neurons, a single-dose BrdU injection (50 mg/kg i.p., dissolved in saline) was administered to the pups at P8, which was 1 day after injection with propofol or vehicle. Pups were sacrificed 2 days after the BrdU injection (P10).
+
+To maintain a mouse body temperature of 37◦C, the pups were primitively anesthetized in their home cage and then transferred to a Thermocare(cid:114) ICS therapy warmer unit (Thermocare, Incline Village, NV, USA) after being sedated to keep warm in all the experiments. Meanwhile, mouse normal skin color and respiration were observed.
+
+Immunofluorescence The dissected cerebella were soaked in 4% paraformaldehyde for 24 h. For cryosections, the tissues (P8) were embedded and sectioned in the sagittal plane at 30 µm. The remaining tissues (P8) and tissues collected on P10 were embedded in paraffin and sagittal sections (5 µm thickness) were collected. Cryosections were used for all the immunofluorescence staining for P8 and paraffin sections were used for Hematoxylin-eosin (HE) staining and BrdU immunofluorescence staining. The sections were pretreated with 3% bovine serum albumin (BSA) (37◦C, 1 h) to block non-specific binding and 0.3% Triton X-100 (37◦C, 30 min) to increase permeability. Then, the sections were incubated with the following primary antibodies in 1% BSA (4◦C,
+
+November 2017 | Volume 11 | Article 373
+
+Xiao et al.
+
+18 h): (1) mouse anti-calbindin D-28K (CB) (1:1000, Swant, Bellinzona, Switzerland); (2) rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) (1:200, Merck Millipore, Darmstadt, Germany); (3) rabbit polyclonal anti-brain lipid binding protein (BLBP) (1:400, Merck Millipore, Darmstadt, Germany); and (4) mouse anti-neuronal nuclei (NeuN) (1:200, Merck Millipore, Darmstadt, Germany). One percent BSA served as the negative control. After three washing steps with phosphate-buffered saline (PBS, pH 7.4), the sections were incubated with the following secondary antibodies in PBS (room temperature (RT), 3 h): (1) Alexa Fluor 488-conjugated anti-mouse IgG (1:400, Jackson ImmunoResearch, West Grove, PA, USA) for CB and NeuN staining; and (2) cy3-conjugated anti-rabbit IgG (1:400, Jackson ImmunoResearch, West Grove, PA, USA) for BLBP and GFAP staining. All the sections were counterstained with 4(cid:48),6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) and then mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). For BrdU staining, the paraffin sections were deparaffinized in xylene, rehydrated in graded alcohol and processed for antigen retrieval by boiling in citrate buffer (pH 6.0) for 5 min. After incubation in 2 M HCl (37◦C, 30 min) and 0.3% Triton X-100 (37◦C, the sections were exposed to mouse anti-BrdU 30 min), IgG (37◦C for 2 h and then RT for 22 h) (1:600, BD PharmingenTM, Palo Alto, CA, USA) in 1% BSA, followed by the cy3-conjugated anti-mouse IgG secondary antibody (RT, 3 h) (1:400, Jackson ImmunoResearch, West Grove, PA, USA) and DAPI counterstaining. Fluorescence micrographs of the whole parasagittal cerebellar slices were acquired under a Zeiss (Oberkochen, Germany) Axiovert microscope equipped with a Zeiss AxioCam digital color camera connected to the Zeiss Axiovision 3.0 system. The pictures of the Purkinje dendrite and Bergmann fiber contact points were taken with a TCS-SP8 (Leica, Germany) laser scanning confocal microscope connected to a LAS AF Lite system. A z-stack of images, consisting of 6 image planes taken at 1 µm interval was obtained (for a total stack depth of 5 µm). The 5 µm z-stack was taken from the middle of the section to minimize the potential artificial bias.
+
+Western Blot Cerebella were harvested on P8 and then isolated and homogenized in ice-cold RIPA Lysis buffer (Beyotime, Shanghai, China). After centrifuging the lysates (15,000× g, 5 min at 4◦C), the protein concentration was calculated using the Bicinchoninic Acid Kit (Beyotime, Shanghai, China). Then, 50 µg of protein from each sample was separated by 10% SDS-polyacrylamide electrophoresis (120 min 80 V) and then transferred to a nitrocellulose (NC) membrane (90 min at 210 mA). The membranes were incubated in 5% fat-free milk in Tris-buffered saline containing 0.1% Tween 20 (3 h at RT). Membranes were then incubated with the following primary antibodies (4◦C, overnight): (1) hamster monoclonal anti-Notch1 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); (2) rabbit polyclonal anti-Jagged1 (1:500, Santa Cruz Biotechnology, USA); (3) mouse anti- β-actin (1:1000, Cell Cwbio, Beijing, China); and (4) rabbit
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+3
+
+Propofol and Cerebellar Development
+
+followed by anti-GAPDH (1:1000, Cell Cwbio, China), the following peroxidase-conjugated secondary antibodies (RT, 2 h): (1) goat anti-mouse IgG (1:1000, Santa Cruz Biotechnology, USA); IgG (1:1000, Santa Cruz Biotechnology, USA); and (3) goat anti-Syrian hamster IgG (1:1000, Santa Cruz Biotechnology, USA). All the bands were exposed to X-ray films (Kodak, Rochester, NY, USA), detected using an enhanced chemiluminescence detection kit IL, USA), and analyzed (Quantity One 4.0; Bio-Rad with the Gel-Pro analyzer Laboratories, Hercules, CA, USA). Quantification of Jagged1 and Notch1 were normalized to the internal reference protein β-actin or GAPDH, and then normalized to the control values.
+
+(2) goat
+
+anti-rabbit
+
+(Pierce, Rockford,
+
+Quantification The quantification was obtained from regional analysis of lobe IX. All the sections were taken from similar medial- lateral position within the cerebellum, and each count area was chosen from the same field by the middle of the lobe IX. Calbindin-positive cells were analyzed along the long axis for 500 µm in the middle, and the dendrite length of Purkinje cells was evaluated by measuring the primary dendrite from the soma up to the surface of the ML (three Purkinje dendrites were measured per picture). Number of the NeuN-positive granule neurons were analyzed in the center the IGL in lobe IX along the long axis (unit region of area 2000 µm2). The number of BLBP- and GFAP-positive Bergmann fibers was counted from a 100-µm length in the middle area of lobe IX according to our previous methods (Yamada et al., 2000; Eiraku et al., 2005; Yang Y. et al., 2014). To analyze the astrocytes in the deep white matter, we compared the intensity of the GFAP-positive cells and fibers. Both the background integrated optical density (IOD) and surveyed area (same center area of the white matter from each group) were acquired, and the relative optical density (ROD) was calculated by subtracting the background from the IOD of the positive staining (Bao et al., 2017). Contact points between the calbindin-positive Purkinje cells and GFAP-positive Bergmann fibers were defined as where the tips of growing Purkinje cell dendrites were aligned parallel and attached directly to the rod-like domain of Bergmann fibers, entering the base of the overlying EGL, as previously reported (Yamada et al., 2000; Yamada and Watanabe, 2002; Lordkipanidze and Dunaevsky, 2005). Points were counted per image (212.5-µm length) at the interface between the EGL and ML. Only the yellow dots at the end of the dendrites in the direction of the Bergmann fiber were included, while the crossed ones were excluded in case of false positive. For quantifying granule neuron migration, BrdU-labeled cells were counted in a rectangular box (200 µm width and about 100 cells were counted) extending from the pial surface to the end of the IGL; this value was expressed as a percentage of the total number of BrdU-labeled cells. At least five sections were analyzed in each mouse and five mice from each group. All the quantitative statistics were performed blind to the experimental treatment.
+
+November 2017 | Volume 11 | Article 373
+
+Xiao et al.
+
+Propofol and Cerebellar Development
+
+FIGURE 1 | Propofol treatment did not alter the formation of the cerebellum at P8. (A–C) The folia structure of the cerebellum at P8 is revealed by Hematoxylin-eosin (HE) staining. (D–F) Magnifications of panels (A–C) show the structure of lobe IX. (G–I) Magnified of the area identified by the black boxes in panels (E–H) show the external granular layer (EGL), molecular layer (ML) and internal granule layer (IGL) of lobe lobe IX. (J) Propofol treatment did not alter the morphologies or cerebellar areas at P8 between the groups. (K) Comparison of relatively identical areas from lobe IX show no obvious differences in the thickness of the EGL at P8 between the groups. Data are presented as the mean ± SD (n = 4). Scale bar: (A–C): 500 µm; (D–F): 100 µm; and (G–I): 25 µm.
+
+Statistical Analysis All the data were presented as the mean ± standard deviation and analyzed using one-way analysis of variance (ANOVA) followed by Fisher’s protected least- significant difference post hoc test or a least-significant difference multiple-comparison. The differences were statistically significant when the P value was less than 0.05. Statistical analysis was performed using the SPSS 19.0 software (SPSS Inc., Chicago, IL, USA).
+
+RESULTS
+
+Propofol Treatment Does Not Alter Cerebellum Formation Folia structure revealed by the HE staining on the sagittal vermal sections from the cerebellum was similar in all groups at P8 (Figures 1A–I). There were no significant alterations in the areas (Vehicle 1.94 ± 0.10 mm2, Pro 30 mg/kg or
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+4
+
+November 2017 | Volume 11 | Article 373
+
+Xiao et al.
+
+Propofol and Cerebellar Development
+
+FIGURE 2 | Propofol treatment decreased the number of Purkinje cells and depressed the dendrite length at P8. (A–C) Calbindin-stained cerebellar Purkinje cells from the (A) Vehicle, (B) Propofol (30 mg/kg) and (C) Propofol (60 mg/kg) groups. (D–F) Magnified images of Panels (A–C) show the calbindin-positive cells and their dendrites in lobe IX. (G) Quantification of the number of calbindin-positive cells in the purkinje cell layer (PCL). (H) Quantification of the primary Purkinje dendrite length. Data are presented as the mean ± SD (n = 5). Scale bar: (A–C): 200 µm and (D–F): 50 µm. ∗P < 0.05.
+
+60 mg/kg 1.98 ± 0.24 mm2 or 2.00 ± 0.21 mm2, respectively, P > 0.05; n = 4; Figure 1J). In the same area of lobe IX (Figures 1D–F), EGL thickness was not altered by propofol treatment (Vehicle 31.39 ± 2.46 µm; Pro 30 mg/kg or 60 mg/kg 31.48 ± 2.08 µm or 33.75 ± 1.69 µm, respectively, P > 0.05; n = 4; Figure 1K).
+
+30 mg/kg propofol and pups treated with vehicle. However, pups treated with 60 mg/kg propofol had decreased calbindin- positive cells compared to the pups treated with vehicle (Vehicle 22.04 ± 1.69 and Pro 60 mg/kg 19.20 ± 1.00, P < 0.05; n = 5; Figure 2G). The Purkinje dendrite length was also shortened by 60 mg/kg propofol treatment (Vehicle 74.33 ± 18.77 µm and Pro 60 mg/kg 50.85 ± 8.12 µm, P < 0.05; n = 5; Figure 2H).
+
+Propofol Treatment Decreases the Number of and Dendrite Outgrowth from Purkinje Cells in a Dose-Dependent Manner Purkinje cells were revealed by their specific marker calbindin. In comparably identical middle areas from lobe IX in the cerebellum (Figures 2A–F), there was no significant difference in the number of calbindin-positive cells (Figure 2G) and the Purkinje dendrite length (Figure 2H) between the pups treated with
+
+Propofol Treatment Does Not Alter NeuN-Positive Granule Neurons in the IGL NeuN is a specific marker for granule neurons located in the IGL of the cerebellum. In comparably center areas of the IGL from lobe IX in the cerebellum (Figures 3A–F), propofol treatment at both doses of 30 mg/kg and 60 mg/kg did not alter the number
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+5
+
+November 2017 | Volume 11 | Article 373
+
+Xiao et al.
+
+Propofol and Cerebellar Development
+
+FIGURE 3 | Propofol treatment did not affect the NeuN-positive cells in the IGL at P8. (A–C) NeuN-stained cerebellar granule neurons from the (A) Vehicle, (B) Propofol (30 mg/kg) and (C) Propofol (60 mg/kg) groups. (D–F) Magnified images of panels (A–C) show the NeuN-positive cells in lobe IX. (G) Quantification of the NeuN positive cells in the IGL. Data are presented as the mean ± SD (n = 5). Scale bar: (A–C): 200 µm and (D–F): 50 µm.
+
+of NeuN-positive cells in the IGL when compared to vehicle treatment (Vehicle 20.08 ± 1.25, Pro 30 mg/kg or 60 mg/kg 20.96 ± 1.45 or 19.56 ± 1.44, respectively, P > 0.05; n = 5; Figure 3G).
+
+Propofol Treatment Suppresses Bergmann Glial Cell Filiform Processes and Affects the Astrocyte Phenotype Bergmann glia was a specialized form of astrocyte, derived from radial glial cells (Xu et al., 2013). During the first week of postnatal development, Bergmann glia were located
+
+among Purkinje cells and extended fibers into the ML directing the distal growth of the Purkinje cell dendritic tree (Voogd and Glickstein, 1998; Yamada and Watanabe, 2002). To determine whether propofol treatment affected Bergmann glial shafts, we observed Bergmann glial organizational structure in lobe IX of the cerebellum through immunostaining assays with specific antibodies for BLBP and GFAP. The radial from the BLBP-positive Bergmann fibers were processes found to extend to the surface of the cerebellum, and BLBP-positive Bergmann soma aligned roughly in a single layer next to Purkinje cells (Figures 4A–C). The BLBP-stained treatment radial
+
+fibers were decreased following propofol
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+6
+
+November 2017 | Volume 11 | Article 373
+
+Xiao et al.
+
+Propofol and Cerebellar Development
+
+FIGURE 4 | Propofol treatment suppressed Bergmann glial cell filiform processes at P8. (A–C) Brain lipid binding protein (BLBP)-stained Bergmann glia fibers in lobe IX from the (A) Vehicle, (B) Propofol (30 mg/kg) and (C) Propofol (60 mg/kg) groups. (D–F) Glial fibrillary acidic protein (GFAP)-stained Bergmann glia fibers in lobe IX from the (D) Vehicle, (E) Propofol (30 mg/kg) and (F) Propofol (60 mg/kg) groups. (G) Quantification of the number of BLBP-positive fibers in the ML. (H) Quantification of the number of GFAP-positive fibers in the ML. (I) Quantification of the optical density of GFAP-positive staining in the white matter. Data are presented as the mean ± SD (n = 5). Scale bar: (A–F): 50 µm. ∗P < 0.05 and ∗∗P < 0.01.
+
+(30 or 60 mg/kg) compared with the vehicle-treated group (Figure 4G). Bergmann glial morphology was further assessed by immunostaining histological sections of the cerebella with antibodies directed at GFAP (Figures 4D–F). There were no significant differences in the GFAP-labeled Bergmann glial processes between the propofol (30 mg/kg)-treated and vehicle-treated groups, whereas the number of radial shafts was significantly decreased after propofol administration at the 60 mg/kg dose; and the images displayed hyperplastic astrocytes in the deep white matter (Figures 4H,I). The results showed that propofol promoted a great disturbance in radial glia phenotypic differentiation and Bergmann glia filiform processes that influenced the formation of the radial scaffold.
+
+defined as dendritic tips in parallel and closely stuck to the Bergmann glia fibers at the interface between the ML and EGL (Figures 5J–L). Immunolabeling for calbindin-positive dendrites and GFAP-positive fibers revealed that the number of contact points was significantly decreased due to the high propofol treatment at 60 mg/kg compared with the vehicle- treated group (Vehicle 19.50 ± 1.40 and Pro 60 mg/kg 16.58 ± 0.90, P < 0.01; n = 5; Figures 5J–M). The number of contact points was not affected after administration of 30 mg/kg propofol to the mice. Arrows showed the tips of calbindin-immunopositive dendrites are intimately attached to the rod-like shaft of Bergmann fiber contacting domains. These data indicated that the attachments of the Purkinje cells on Bergmann fibers were disrupted after propofol injection, which subsequently resulted in Purkinje cell dendritogenesis disorder.
+
+Propofol Treatment Disrupts the Contacts between Purkinje Cells and Bergmann Glial Cells Purkinje cells make contacts with Bergmann glia through the connections between Purkinje cell dendrites and Bergmann glia fibers in the ML (Figures 5A–I). Connections were
+
+Propofol Treatment Delays the Migration of Granule Neurons from the EGL to IGL HE staining on sagittal vermal sections from the cerebellum was used to analyze the folia structure of the cerebellum at
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+7
+
+November 2017 | Volume 11 | Article 373
+
+Xiao et al.
+
+Propofol and Cerebellar Development
+
+FIGURE 5 | Propofol treatment disrupted the contacts between Purkinje cells and Bergmann glial cells at P8. (A–L) Immunolabeling for calbindin (green), GFAP (red), 4(cid:48),6-diamidino-2-phenylindole (DAPI) (blue), their merged images and respective high-resolution images of the merges in the cerebellar lobe IX. (A–C) Calbindin-stained Purkinje cells from the (A) Vehicle, (B) Propofol (30 mg/kg) and (C) Propofol (60 mg/kg) groups. (D–F) GFAP-stained Bergmann glial cell fibers from the (D) Vehicle, (E) Propofol (30 mg/kg) and (F) Propofol (60 mg/kg) groups. (G–I) The merged images showing the calbindin staining, GFAP staining and DAPI in the PCL. (J–L) Magnified images of panels (G–I) show the relationship between the calbindin-positive cells and GFAP-positive cells. The arrows indicate that the tips of calbindin-immunopositive dendrites are intimately attached to the rod-like shaft of Bergmann fiber contacting domains (M) Quantification of the numbers of contact points between the GFAP-positive fibers and calbindin-positive cells around the border between the ML and EGL in the identical lobe of the cerebellum. Data are presented as the mean ± SD (n = 5). Scale bar: (A–I): 50 µm and (J–L): 25 µm. ∗∗P < 0.01.
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+8
+
+November 2017 | Volume 11 | Article 373
+
+Xiao et al.
+
+Propofol and Cerebellar Development
+
+FIGURE 6 | Propofol treatment increased the thickness of the EGL at P10. (A–C) The folia structure of the cerebellum is revealed by HE staining from the (A) Vehicle, (B) Propofol (30 mg/kg) and (C) Propofol (60 mg/kg) groups. (D–F) Magnified images of panels (A–C) show the structure of lobe IX. (G–I) Magnified areas identified by the black boxes in panels (D–F) show the EGL, ML and IGL, respectively, from lobe IX. (J) Propofol treatment did not alter the morphology or cerebellar area at P10 between the groups. (K) Comparison of relatively identical areas from lobe IX show that propofol treatment increases the thickness of the EGL at P10 compared with the vehicle-treated mice. Data are presented as the mean ± SD (n = 5). Scale bar: (A–C): 500 µm, (D–F): 100 µm, and (G–I): 25 µm. ∗∗P < 0.01.
+
+P10 in all the groups (Figures 6A–I). Propofol treatment did not alter the area size at P10 (Vehicle 2.84 ± 0.35 mm2; Pro 30 or 60 mg/kg 2.91 ± 0.33 mm2 or 2.89 ± 0.14 mm2,
+
+respectively, P > 0.05; n = 5; Figure 6J). In identical areas of lobe IX (Figures 6D–F), propofol treatment at both 30 and 60 mg/kg increased the thickness of the EGL compared with
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+9
+
+November 2017 | Volume 11 | Article 373
+
+Xiao et al.
+
+Propofol and Cerebellar Development
+
+FIGURE 7 | Propofol treatment suppressed the radial migration of the granule neurons from the EGL to IGL. Granule neurons were labeled with BrdU in vivo at P8 and the cerebella were harvested at P10 (A–C). Sections were counterstained with DAPI (blue). (D) Quantification of the percentage of BrdU-positive cells in EGL, ML, or IGL to the total BrdU-positive cells at P10. (E) Quantification of the total number of BrdU-positive cells in EGL, ML and IGL at P10. Data are presented as the mean ± SD (n = 5). Scale bar: (A–C): 50 µm. ∗P < 0.05 and ∗∗P < 0.01.
+
+the vehicle-treated group (Vehicle 30.66 ± 0.96 µm; Pro 30 or 60 mg/kg 34.06 ± 1.27 µm or 34.73 ± 0.43 µm, respectively, P < 0.01; n = 5; Figures 6G–I,K).
+
+the post-mitotic cells in the EGL migrate to their destination in the IGL. The correct positioning of these cells is essential for the final cytoarchitecture and in particular for the three well-defined laminations. It has been suggested that Bergmann glial provided the scaffold for granule neuron migration (Buffo and Rossi, 2013; Xu et al., 2013). BrdU birthdating was used to evaluate the effect of propofol on further granule neuron migration. At P10, the total number of BrdU positive cells was not changed by Propofol treatment (Vehicle 99.05 ± 14.77; Pro 30 or 60 mg/kg 107.40 ± 12.05 or 94.03 ± 16.26, respectively, P > 0.05; n = 5; Figure 7E) and there was no significant difference in the percent of BrdU-positive cells in the ML in the propofol-treated mice compared to the vehicle-treated mice (Figures 7A–C). While the statistical analysis revealed that propofol treatment (30 or 60 mg/kg) increased the percent of BrdU-positive cells in the EGL by 3% or 7.3%, respectively, compared to the vehicle-treated group (Figure 7D). Meanwhile, the percentage of BrdU-positive cells in the IGL was also decreased significantly by propofol treatment (60 mg/kg) compared with the vehicle- treated mice; in contrast, there were no changes due to propofol administration at the 30 mg/kg dosage (Figure 7D). These results indicate that propofol treatment did not suppress the proliferation, but the migration of granule neurons from the EGL to IGL.
+
+During postnatal development of
+
+the cerebellum,
+
+Propofol Treatment Down-Regulates the Jagged1/Notch Pathway in the Cerebellum It has been reported that BLBP protein is a direct target of Notch signaling (Anthony et al., 2005) and active Notch1 signaling is involved in radial fiber formation of Bergmann glia (Xu et al., 2013). The loss of Jagged1, a ligand for Notch signaling, induced a reduction in the number of Bergmann glia cells and affected their morphology (Tanaka and Marunouchi, 2003; Weller et al., 2006). We found that Jagged1 and Notch1 levels were considerably decreased in the the cerebella at P8 following exposure to propofol at 30 mg/kg dose compared to vehicle treatment; a further reduction was detected after treatment with the 60 mg/kg dose (Figure 8A). Quantitative analysis showed that propofol treatment (30 or 60 mg/kg) decreased Jagged1 expression by 20% or 32%, respectively, compared with the vehicle-treated group (Figure 8B); Notch1 protein levels were decreased by 22% and 45%, respectively (Figure 8C). These results suggested that propofol impaired the morphogenesis and phenotypic differentiation of Bergmann glia, potentially via Jagged1/Notch1 signaling.
+
+DISCUSSION
+
+In this study, we studied the effects of propofol exposure on cerebellar development during early life. Our results demonstrated that a single injection of propofol at a dosage
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+10
+
+November 2017 | Volume 11 | Article 373
+
+Xiao et al.
+
+Propofol and Cerebellar Development
+
+FIGURE 8 | Propofol treatment induced down-regulation of the Jagged1/Notch pathway in the cerebellum at P8. (A) Representative western blotting for the Jagged1 and Notch1 proteins from the cerebella in each group. (B) Densitometric quantification of Jagged1. Jagged1 protein levels in the Propofol (30 mg/kg)- and Propofol (60 mg/kg)-treated groups were significantly lower than in the vehicle-treated group. (C) Densitometric quantification of Notch. Notch1 protein levels in the Propofol (30 mg/kg)- and Propofol (60 mg/kg)-treated groups were significantly lower than in the vehicle-treated group. ∗P < 0.05 and ∗∗P < 0.01.
+
+of 60 mg/kg led to reduced Purkinje cell dendritogenesis, cells migration, and suppressed radial retarded granule glia phenotypic differentiation to Bergmann glia cells. The unbalanced transformational process demonstrated by decreased glial fibers in the ML and increased GFAP-positive astrocytes in the white matter may be due to inhibition of Notch signaling. Propofol at a lower dose of 30 mg/kg resulted in a less pronounced interruption in cerebellar development, with no significant influences on Purkinje cell morphogenesis.
+
+Purkinje neurons are GABAergic neurons and considered the sole output neuron in the to grow postnatally. As circuit, Purkinje neurons are also the major cerebellar cell group that integrate motor coordination and learning (Van Der Giessen et al., 2008; Lee R. X. et al., 2015). In rodents, Purkinje neurons exhibit cytoarchitectural changes characterized by highly branched dendritic trees during first 2 weeks after birth (Tanaka, 2009). Our previous studies together with other reports have demonstrated that
+
+Purkinje neurons are extremely vulnerable to the neurotoxic effects of EtOH exposure during the early postnatal period (Yang Y. et al., 2014). Emerging evidence has found that propofol administration to neonatal animals caused significant loss in the hippocampus (Han et al., 2015; Huang cell et al., 2016), a typical laminated structure development investigation, we demonstrated that postnatally. propofol exposure significantly decreased Purkinje neurons as assessed by calbindin in the cerebellum at P8 in a dose-dependent manner. We further confirmed that propofol reduced administration into neonatal mice the length of cerebellar Purkinje neuron dendrites in a dose-dependent manner. Taken together, these results indicated that propofol treatment impaired dendritic growth in Purkinje neurons.
+
+In this
+
+significantly
+
+Bergmann glia, which are normally located in the PCL, extend radial fibers stretching from the cell body towards the pial surface. The radial glial cells originate from the ventricular neuroepithelium (VN), migrate and differentiate to
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+11
+
+November 2017 | Volume 11 | Article 373
+
+Xiao et al.
+
+all cerebellar glia, including the Bergmann glia, a specialized subtype of the astrocyte (Buffo and Rossi, 2013). It has been indicated that Bergmann glial cells are arranged around the cell bodies of Purkinje neurons at P8 (Xu et al., 2013). Both BLBP and GFAP are specific markers for Bergmann glia, and immunofluorescence staining showed that the number of Bergmann glial fibers in mice exposed to propofol was significantly decreased and that the radial fibers could not extend to the pial surface. Moreover, GFAP-positive, star-shaped astrocytes were increased in the IGL and deep white matter of the cerebella after propofol treatment at the 60 mg/kg dose. These data implied that propofol might accelerate the transformation of radial glial cells into astrocytic phenotype, with star-shaped bushy processes, rather than Bergmann glia with filiform processes. Indeed, Bergmann glia extend long radial fibers in synchrony with the growth of Purkinje cell dendrites during postnatal development. Thus, Bergmann glial fibers may specifically contribute to Purkinje cell dendrite development (Bellamy, 2006). Recent studies indicated that Bergmann fibers enwrapped the synapses in parallel and climbing fibers interact with Purkinje cells affecting Purkinje cell dendrite arborization (Yamada et al., 2000; Lordkipanidze and Dunaevsky, 2005). Consistent with previous reports, we noticed that suppressed Bergmann glial cell filiform processes and their alignment from propofol exposure led to decreased attachments between Purkinje cell dendritic tips and glial fibers loss fibers. Hence, might contribute to propofol-induced suppressed Purkinje cell dendritogenesis.
+
+it
+
+inferred that Bergmann glial
+
+laminated postnatal cerebellar cortex structures is achieved through the directional migration of committed granule neurons along the Bergmann glial radial fibers from the EGL to their destination in the IGL (Sillitoe and Joyner, 2007; Qiu et al., 2010). The intimate structural associations between granule neurons and Bergmann glial fibers are crucial for granule neuron migration. Moreover, several abnormalities in the Bergmann glial fiber radial scaffold structure have been shown to cause granule cell migration alterations (Shetty et al., 1994; Qu and Smith, 2005; Yue et al., 2005; Lin et al., 2009; Nguyen et al., 2013). Propofol induced a thicker EGL and increased number of granule cells remaining in the EGL, which suggested that granule cell migration was retarded. We found that granule cell migration from the EGL to IGL was markedly suppressed by using BrdU birthdating. The lower efficiency of migration could be due to physical impediments to the Bergmann fibers. It is also possible that some granule neurons migrate normally along adequate fibers; hence, only a subpopulation is dramatically retarded in their migration.
+
+The creation of
+
+several transcription factors and nuclear receptors (Xu et al., 2013). It has been reported that the Notch signaling pathway was an important factor involved in Bergmann glial differentiation and maturation (Lutolf et al., 2002; Tanaka and Marunouchi, 2003). Additionally, the Notch signaling pathway regulates early events in radial formation through direct cell-cell contacts and is necessary for neuronal-induced radial glia formation (Weinmaster, 1997; Gaiano et al., 2000). An in vitro study has
+
+Bergmann glia development
+
+is
+
+regulated by
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+12
+
+Propofol and Cerebellar Development
+
+confirmed that Notch pathway activation induced cerebellar astroglia to adopt radial glia morphologies (Patten et al., 2003). Many studies have demonstrated that Notch or Jagged ablation in mice promoted a reduced number of Bergmann glia and abnormal Bergmann processes, which led to further deceleration of granule cell migration and impaired Purkinje cell development (Lutolf et al., 2002; Weller et al., 2006; Komine et al., 2007). In this study, we found that both Notch1 and Jagged1 levels were decreased by propofol treatment, even at the low dose, indicating that the Notch signaling pathway was active early in the inhibition of Bergmann glia development induced by propofol exposure.
+
+It has been raised that mechanism of propofol toxicity in immature neurons was GABAergic via the GABAA receptor (Kahraman et al., 2008). GABAA receptor is widely distributing in the cerebellum, including granule cells, Bergmann cells, Purkinje cells, stellate/basket cells and so on (Laurie et al., 1992). What’s more, the GABAA receptors in Bergmann glia express more in the early development period and then decrease in the adulthood (Müller et al., 1994). It highly matches the enhanced activity of Bergmann glia in granule cell migration and synapse formation or remodeling in the early time. Riquelme et al. (2002) found 50% of the Purkinje dendritic spines with the neuronal GABAA receptors were wrapped by Bergmann glia fibers contained GABAA receptors. Ango et al. (2008) further confirmed the dendritic-targeting GABAergic stellate axons are guided to Purkinje dendrites by the Bergmann fibers scaffold and crucial in the physiological control of synaptic integration in postsynaptic neurons. GABAA receptor indeed plays an important role in Bergmann glia function. Therefore, the influence of propofol on Bergmann glia or the whole cerebellum via GABAA receptor deserved more consideration, and the relationship between GABAA receptor and Notch pathway needed to be further explored.
+
+In conclusion, our data indicate that propofol treatment during the early postnatal time significantly impaired Bergmann glial cell development and filiform processes, and led to and granule inhibition of Purkinje cells migration afterwards. This the that cerebellum is sensitive to neurotoxicity induced by high doses of propofol. These findings suggest that propofol used in neonates or young children should be monitored more carefully.
+
+cell morphogenesis
+
+study indicates
+
+AUTHOR CONTRIBUTIONS
+
+RX and DY conducted the experiments, collected and analyzed the data and drafted the manuscript; XL, JH, SJ and XB contributed to acquisition and analysis of data; TY and XF designed the experiments, supervised the project and revised the manuscript.
+
+ACKNOWLEDGMENTS
+
+This study was supported by the National Natural Science Foundation of China (No. 81371197).
+
+November 2017 | Volume 11 | Article 373
+
+Xiao et al.
+
+REFERENCES
+
+Altman, J., and Winfree, A. T. (1977). Postnatal development of the cerebellar cortex in the rat. V. Spatial organization of purkinje cell perikarya. J. Comp. Neurol. 171, 1–16. doi: 10.1002/cne.901710102
+
+Ango, F., Wu, C., Van der Want,
+
+J., Wu, P., Schachner, M., and J. Huang, Z. J. (2008). Bergmann glia and the recognition molecule CHL1 organize GABAergic axons and direct innervation of Purkinje cell dendrites. PLoS Biol. 6:e103. doi: 10.1371/journal.pbio.0060103
+
+Anthony, T. E., Mason, H. A., Gridley, T., Fishell, G., and Heintz, N. (2005). Brain lipid-binding protein is a direct target of Notch signaling in radial glial cells. Genes Dev. 19, 1028–1033. doi: 10.1101/gad.1302105
+
+Bao, X., Cai, Y., Wang, Y., Zhao, J., He, X., Yu, D., et al. (2017). Liver X receptor β is involved in formalin-induced spontaneous pain. Mol. Neurobiol. 54, 1467–1481. doi: 10.1007/s12035-016-9737-1
+
+Bellamy, T. C. (2006). Interactions between Purkinje neurones and Bergmann glia.
+
+Cerebellum 5, 116–126. doi: 10.1080/14734220600724569
+
+Bendiksen, A., and Larsen, L. M. (1998). Convulsions, ataxia and hallucinations following propofol. Acta Anaesthesiol. Scand. 42, 739–741. doi: 10.1111/j.1399- 6576.1998.tb05312.x
+
+Brooks, D. E. (2008). Propofol-induced movement disorders. Ann. Emerg. Med.
+
+51, 111–112. doi: 10.1016/j.annemergmed.2007.08.023
+
+Buffo, A., and Rossi, F. (2013). Origin, lineage and function of cerebellar glia. Prog.
+
+Neurobiol. 109, 42–63. doi: 10.1016/j.pneurobio.2013.08.001
+
+Cattano, D., Young, C., Straiko, M. M., and Olney, J. W. (2008). Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain. Anesth. Analg. 106, 1712–1714. doi: 10.1213/ane.0b013e318172ba0a
+
+Diaz, J. H., and Kaye, A. D. (2017). Death by propofol. J. La. State Med. Soc. 169,
+
+28–32.
+
+DiMaggio, C., Sun, L. S., and Li, G. (2011). Early childhood exposure to anesthesia and risk of developmental and behavioral disorders in a sibling birth cohort. Anesth. Analg. 113, 1143–1151. doi: 10.1213/ANE.0b013e3182147f42
+
+Dingwall, A. E. (1987). Oculogyric crisis after day case anaesthesia. Anaesthesia
+
+42:565. doi: 10.1111/j.1365-2044.1987.tb04081.x
+
+Eccles, J. C. (1970). Neurogenesis and morphogenesis in the cerebellar cortex. Proc.
+
+Natl. Acad. Sci. U S A 66, 294–301. doi: 10.1073/pnas.66.2.294
+
+Eiraku, M., Tohgo, A., Ono, K., Kaneko, M., Fujishima, K., Hirano, T., et al. (2005). DNER acts as a neuron-specific Notch ligand during Bergmann glial development. Nat. Neurosci. 8, 873–880. doi: 10.1038/nn1492
+
+Franks, N. P., and Lieb, W. R. (1994). Molecular and cellular mechanisms of
+
+general anaesthesia. Nature 367, 607–614. doi: 10.1038/367607a0 (2000). Radial glial
+
+Gaiano, N., Nye,
+
+identity is promoted by Notch1 signaling in the murine forebrain. Neuron 26, 395–404. doi: 10.1016/s0896-6273(00)81172-1
+
+J. S., and Fishell, G.
+
+Han, D., Jin, J., Fang, H., and Xu, G. (2015). Long-term action of propofol on cognitive function and hippocampal neuroapoptosis in neonatal rats. Int. J. Clin. Exp. Med. 8, 10696–10704.
+
+Huang,
+
+Jing, S., Chen, X., Bao, X., Du, Z., Li, H., et al. (2016). Propofol administration during early postnatal life suppresses hippocampal neurogenesis. Mol. Neurobiol. 53, 1031–1044. doi: 10.1007/s12035-014-9052-7 Irifune, M., Takarada, T., Shimizu, Y., Endo, C., Katayama, S., Dohi, T., et al. (2003). Propofol-induced anesthesia in mice is mediated by γ-aminobutyric acid-A and excitatory amino acid receptors. Anesth. Analg. 97, 424–429. doi: 10.1213/01.ane.0000059742.62646.40
+
+J.,
+
+Jin, R., Liu, H., Jin, W. Z., Shi, J. D., Jin, Q. H., Chu, C. P., et al. (2015). Propofol depresses cerebellar Purkinje cell activity via activation of GABAA and glycine receptors in vivo in mice. Eur. J. Pharmacol. 764, 87–93. doi: 10.1016/j.ejphar. 2015.06.052
+
+Jin, W. Z., Shi, J. D., Liu, H., Lan, Y., Chu, C. P., and Qiu, D. L. (2015). Effects of propofol on the dynamic properties of sensory information processing in the mouse cerebellar cortical molecular layer in vivo. Pharmacology 96, 271–277. doi: 10.1159/000441006
+
+Kahraman, S., Zup, S. L., McCarthy, M. M., and Fiskum, G.
+
+(2008). GABAergic mechanism of propofol toxicity in immature neurons. J. Neurosurg. Anesthesiol. 20, 233–240. doi: 10.1097/ANA.0b013e31817ec34d
+
+Komine, O., Nagaoka, M., Watase, K., Gutmann, D. H., Tanigaki, K., Honjo, T., et al. (2007). The monolayer formation of Bergmann glial cells is regulated
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+13
+
+Propofol and Cerebellar Development
+
+by Notch/RBP-J signaling. Dev. Biol. 311, 238–250. doi: 10.1016/j.ydbio.2007. 08.042
+
+Komuro, H., Yacubova, E., Yacubova, E., and Rakic, P. (2001). Mode and tempo of tangential cell migration in the cerebellar external granular layer. J. Neurosci. 21, 527–540.
+
+Laurie, D. J., Seeburg, P. H., and Wisden, W. (1992). The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. J. Neurosci. 12, 1063–1076.
+
+Lee, R. X., Huang, J. J., Huang, C., Tsai, M. L., and Yen, C. T. (2015). Plasticity of cerebellar Purkinje cells in behavioral training of body balance control. Front. Syst. Neurosci. 9:113. doi: 10.3389/fnsys.2015.00113
+
+Lee, K. Y., Kim, Y. I., Kim, S. H., Park, H. S., Park, Y. J., Ha, M. S., et al. (2015). Propofol effects on cerebellar long-term depression. Neurosci. Lett. 609, 18–22. doi: 10.1016/j.neulet.2015.09.037
+
+Lin, Y., Chen, L., Lin, C., Luo, Y., Tsai, R. Y., and Wang, F. (2009). Neuron- derived FGF9 is essential for scaffold formation of Bergmann radial fibers and migration of granule neurons in the cerebellum. Dev. Biol. 329, 44–54. doi: 10.1016/j.ydbio.2009.02.011
+
+Lordkipanidze, T., and Dunaevsky, A. (2005). Purkinje cell dendrites grow in
+
+alignment with Bergmann glia. Glia 51, 229–234. doi: 10.1002/glia.20200
+
+Lutolf, S., Radtke, F., Aguet, M., Suter, U., and Taylor, V. (2002). Notch1 is required for neuronal and glial differentiation in the cerebellum. Development 129, 373–385.
+
+Millen, K. J., and Gleeson, J. G. (2008). Cerebellar development and disease. Curr.
+
+Opin. Neurobiol. 18, 12–19. doi: 10.1016/j.conb.2008.05.010 J. M., Grosche,
+
+Müller, T., Fritschy,
+
+J., Pratt, G. D., Möhler, H., and Kettenmann, H. (1994). Developmental regulation of voltage-gated K+ channel and GABAA receptor expression in Bergmann glial cells. J. Neurosci. 14, 2503–2514.
+
+Nguyen, H., Ostendorf, A. P., Satz,
+
+J. S., Westra, S., Ross-Barta, S. E., Campbell, K. P., et al. (2013). Glial scaffold required for cerebellar granule cell migration is dependent on dystroglycan function as a receptor for basement membrane proteins. Acta Neuropathol. Commun. 1:58. doi: 10.1186/2051- 5960-1-58
+
+Olney, J. W., Wozniak, D. F., Jevtovic-Todorovic, V., Farber, N. B., Bittigau, P., and Ikonomidou, C. (2002). Drug-induced apoptotic neurodegeneration in the developing brain. Brain Pathol. 12, 488–498. doi: 10.1111/j.1750-3639.2002. tb00467.x
+
+Patel, P., and Sun, L. (2009). Update on neonatal anesthetic neurotoxicity: insight into molecular mechanisms and relevance to humans. Anesthesiology 110, 703–708. doi: 10.1097/ALN.0b013e31819c42a4
+
+Patten, B. A., Peyrin,
+
+(2003). Sequential signaling through Notch1 and erbB receptors mediates radial glia differentiation. J. Neurosci. 23, 6132–6140.
+
+J. M., Weinmaster, G., and Corfas, G.
+
+Qiu, Z., Cang, Y., and Goff, S. P. (2010). Abl family tyrosine kinases are essential for basement membrane integrity and cortical lamination in the cerebellum. J. Neurosci. 30, 14430–14439. doi: 10.1523/JNEUROSCI.2861 -10.2010
+
+Qu, Q., and Smith, F. I. (2005). Neuronal migration defects in cerebellum of the Largemyd mouse are associated with disruptions in Bergmann glia organization and delayed migration of granule neurons. Cerebellum 4, 261–270. doi: 10.1080/14734220500358351
+
+Reddy, S. V. (2012). Effect of general anesthetics on the developing brain.
+
+J. Anaesthesiol. Clin. Pharmacol. 28, 6–10. doi: 10.4103/0970-9185.92426
+
+Riquelme, R., Miralles, C. P., and De Blas, A. L. (2002). Bergmann glia GABAA receptors concentrate on the glial processes that wrap inhibitory synapses. J. Neurosci. 22, 10720–10730.
+
+Shetty, A. K., Burrows, R. C., Wall, K. A., and Phillips, D. E. (1994). Combined pre- and postnatal ethanol exposure alters the development of Bergmann glia in rat cerebellum. Int. J. Dev. Neurosci. 12, 641–649. doi: 10.1016/0736- 5748(94)90016-7
+
+Sillitoe, R. V., and Joyner, A. L. (2007). Morphology, molecular codes and circuitry produce the three-dimensional complexity of the cerebellum. Annu. Rev. Cell Dev. Biol. 23, 549–577. doi: 10.1146/annurev.cellbio.23.090506.123237
+
+Sinner, B., Becke, K., and Engelhard, K. (2014). General anaesthetics and the developing brain: an overview. Anaesthesia 69, 1009–1022. doi: 10.1111/anae. 12637
+
+November 2017 | Volume 11 | Article 373
+
+Xiao et al.
+
+Sotelo, C., and Rossi, F. (2013). ‘‘Purkinje cell migration and differentiation,’’ in Handbook of the Cerebellum and Cerebellar Disorders, eds M. Manto, J. D. Schmahmann, F. Rossi, D. L. Gruol and N. Koibuchi (Dordrecht: Springer Netherlands), 147–178.
+
+Stargatt, R., Davidson, A. J., Huang, G. H., Czarnecki, C., Gibson, M. A., Stewart, S. A., et al. (2006). A cohort study of the incidence and risk factors for negative behavior changes in children after general anesthesia. Paediatr. Anaesth. 16, 846–859. doi: 10.1111/j.1460-9592.2006.01869.x
+
+Tanaka, M. (2009). Dendrite formation of cerebellar Purkinje cells. Neurochem.
+
+Res. 34, 2078–2088. doi: 10.1007/s11064-009-0073-y
+
+Tanaka, M., and Marunouchi, T. (2003). Immunohistochemical localization of Notch receptors and their ligands in the postnatally developing rat cerebellum. Neurosci. Lett. 353, 87–90. doi: 10.1016/s0304-3940(03)01068-1
+
+Van Der Giessen, R. S., Koekkoek, S. K., van Dorp, S., De Gruijl, J. R., Cupido, A., Khosrovani, S., et al. (2008). Role of olivary electrical coupling in cerebellar motor learning. Neuron 58, 599–612. doi: 10.1016/j.neuron.2008.03.016
+
+Voogd, J., and Glickstein, M. (1998). The anatomy of the cerebellum. Trends
+
+Neurosci. 21, 370–375. doi: 10.1016/S0166-2236(98)01318-6
+
+Weinmaster, G. (1997). The ins and outs of notch signaling. Mol. Cell. Neurosci. 9,
+
+91–102. doi: 10.1006/mcne.1997.0612
+
+Weller, M., Krautler, N., Mantei, N., Suter, U., and Taylor, V. (2006). Jagged1 ablation results in cerebellar granule cell migration defects and depletion of Bergmann glia. Dev. Neurosci. 28, 70–80. doi: 10.1159/000090754
+
+Xu, H., Yang, Y., Tang, X., Zhao, M., Liang, F., Xu, P., et al. (2013). Bergmann glia function in granule cell migration during cerebellum development. Mol. Neurobiol. 47, 833–844. doi: 10.1007/s12035-013-8405-y
+
+Yamada, K., Fukaya, M., Shibata, T., Kurihara, H., Tanaka, K., Inoue, Y., et al. (2000). Dynamic transformation of Bergmann glial fibers proceeds in correlation with dendritic outgrowth and synapse formation of cerebellar Purkinje cells. J. Comp. Neurol. 418, 106–120. doi: 10.1002/(sici)1096- 9861(20000228)418:1<106::aid-cne8>3.0.co;2-n
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+14
+
+Propofol and Cerebellar Development
+
+Yamada, K., and Watanabe, M. (2002). Cytodifferentiation of Bergmann glia and its relationship with Purkinje cells. Anat. Sci. Int. 77, 94–108. doi: 10.1046/j. 0022-7722.2002.00021.x
+
+Yang, B., Liang, G., Khojasteh, S., Wu, Z., Yang, W., Joseph, D., et al. (2014). Comparison of neurodegeneration and cognitive impairment in neonatal mice exposed to propofol or isoflurane. PLoS One 9:e99171. doi: 10.1371/journal. pone.0099171
+
+Yang, Y., Tang, Y., Xing, Y., Zhao, M., Bao, X., Sun, D., et al. (2014). Activation of is protective against ethanol-induced developmental impairment of Bergmann glia and Purkinje neurons in the mouse cerebellum. Mol. Neurobiol. 49, 176–186. doi: 10.1007/s12035-013 -8510-y
+
+liver X receptor
+
+Yue, Q., Groszer, M., Gil, J. S., Berk, A. J., Messing, A., Wu, H., et al. (2005). PTEN deletion in Bergmann glia leads to premature differentiation and affects laminar organization. Development 132, 3281–3291. doi: 10.1242/dev. 01891
+
+Zabani, I., and Vaghadia, H. (1996). Refractory dystonia during propofol anaesthesia in a patient with torticollis-dystonia disorder. Can. J. Anaesth. 43, 1062–1064. doi: 10.1007/bf03011910
+
+Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
+
+Copyright © 2017 Xiao, Yu, Li, Huang, Jing, Bao, Yang and Fan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
+
+November 2017 | Volume 11 | Article 373

new_pdfs/10.3389_fncel.2019.00251.txt

@@ -0,0 +1,495 @@
+fncel-13-00251
+
+June 12, 2019
+
+Time: 17:26
+
+# 1
+
+ORIGINAL RESEARCH published: 13 June 2019 doi: 10.3389/fncel.2019.00251
+
+17β-Estradiol Treatment Attenuates Neurogenesis Damage and Improves Behavior Performance After Ketamine Exposure in Neonatal Rats
+
+Weisong Li1, Huixian Li1, Haidong Wei1, Yang Lu1, Shan Lei1, Juan Zheng1, Haixia Lu2, Xinlin Chen2, Yong Liu2 and Pengbo Zhang1*
+
+1 Department of Anesthesiology, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China, 2 Institute of Neurobiology, National Key Academic Subject, Physiology of Xi’an Jiaotong University, Xi’an, China
+
+Ketamine exposure disturbed normal neurogenesis in the developing brain and resulted in subsequent neurocognitive deficits. 17β-estradiol provides robust neuroprotection in a variety of brain injury models in animals of both sexes and attenuates neurodegeneration induced by anesthesia agents. In the present study, we aimed to investigate whether 17β-estradiol could attenuate neonatal ketamine exposure- disturbed neurogenesis and behavioral performance. We treated 7-day-old (Postnatal day 7, PND 7) Sprague-Dawley rats and neural stem cells (NSCs) with either normal saline, ketamine, or 17β-estradiol before/after ketamine exposure, respectively. At PND 14, the rats were decapitated to detect neurogenesis in the subventricular zone (SVZ) and subgranular zone (SGZ) of the hippocampus by immunofluorescence staining. The proliferation, neuronal differentiation, and apoptosis of NSCs were assessed by immunohistochemistry method and TUNEL assay, respectively. The protein levels of cleaved caspase-3 in vivo in addition to GSK-3β and p-GSK-3β in vitro were examined by western blotting. Spatial learning and memory abilities were assessed by Morris water maze (MWM) test at PND 42–47. Ketamine exposure decreased cell proliferation in the SVZ and SGZ, inhibited NSC proliferation and neuronal differentiation, promoted NSC apoptosis and led to adult cognitive deficits. Furthermore, ketamine increased cleaved caspase-3 in vivo and decreased the expression of p-GSK-3β in vitro. Treatment with 17β-estradiol could attenuate ketamine-induced changes both in vivo and in vitro. For the first time we showed that 17β-estradiol alleviated ketamine-induced neurogenesis inhibition and cognitive dysfunction in the developing rat brain. Moreover, the protection of 17β-estradiol was associated with GSK-3β.
+
+Edited by: Hung-Ming Chang, Taipei Medical University, Taiwan
+
+Reviewed by: Yuriko Iwakura, Niigata University, Japan Hari Prasad Osuru, University of Virginia, United States
+
+Correspondence: Pengbo Zhang zhpbo@xjtu.edu.cn
+
+Specialty section: This article was submitted to Cellular Neurophysiology, a section of the journal Frontiers in Cellular Neuroscience
+
+Received: 25 December 2018 Accepted: 20 May 2019 Published: 13 June 2019
+
+Citation: Li W, Li H, Wei H, Lu Y, Lei S, Zheng J, Lu H, Chen X, Liu Y and Zhang P (2019) 17β-Estradiol Treatment Attenuates Neurogenesis Damage and Improves Behavior Performance After Ketamine Exposure in Neonatal Rats. Front. Cell. Neurosci. 13:251. doi: 10.3389/fncel.2019.00251
+
+Keywords: neural stem cells, ketamine, neurotoxicity, 17β-estradiol, p-GSK-3β
+
+INTRODUCTION
+
+Brain growth spurt (BGS) is critical for the normal development of the central nervous system. Substantial neurogenesis occurs in this period, which is characterized by abundant neural stem cell (NSC) changes including cell proliferation, differentiation, migration, and connection of neural cells (Muramatsu et al., 2007). In rodents, this period lasts from birth to the first 2 weeks of life
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+June 2019 | Volume 13 | Article 251
+
+1
+
+fncel-13-00251
+
+June 12, 2019
+
+Time: 17:26
+
+# 2
+
+17β-Estradiol and Neuroprotection
+
+Li et al.
+
+MATERIALS AND METHODS
+
+(Ponten et al., 2012). Since the developing brain is vulnerable to exogenous substrates during BGS, toxic insults may induce impairment in learning and memory abilities in functional adulthood (Eriksson, 1997; Eriksson et al., 2000). It was speculated that neurogenesis damage at an early age would cause cognitive impairment (Kang et al., 2017).
+
+Animal Protocols We performed all the experimental protocols according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23). The animal procedures were approved by the Animal Care and Use Committee of Xi’an Jiaotong University and designed to minimize the number and suffering of rats used. PND 7 and embryonic day 18–19 Sprague-Dawley rats were obtained from Laboratory Animal Centre of Xi’an Jiaotong University.
+
+Ketamine is a non-competitive blocker of N-methyl-D- aspartate (NMDA) receptor and is commonly used in pediatric anesthesia. Recent studies have shown that ketamine inhibits NSC proliferation and disturbs normal neurogenesis (Scallet et al., 2004; Lu et al., 2017), causes neuroapoptosis and neurodegeneration in the developing brain, which may ultimately lead to long-term neurocognitive and memory dysfunctions (Paule et al., 2011; Sabbagh et al., 2012). Although some underlying molecular signals such as the PKC/ERK1/2 pathway, reactive oxygen species-mediated mitochondria dysfunction, and glycogen synthase kinase 3β were speculated to be involved in pathophysiological abnormality induced by ketamine exposure (Huang et al., 2012; Bai et al., 2013; Liu et al., 2013), specific adjunctive therapy aiming to mitigate these negative effect of ketamine is still lacking.
+
+Morris Water Maze The spatial learning and memory function of rats after ketamine exposure were tested by MWM experiments as described in a previous study (Shen et al., 2013). Specifically, PND 42–47 rats (n = 10 per group) were trained for place trials and spatial probe tests in a large tank (diameter: 150 cm, depth: 60 cm), which was filled to a depth of 32 cm of warm water (maintained around 25 ± 1◦C) and divided into four quadrants. A platform (diameter: 12 cm, height: 30 cm) was placed in the center of the third quadrant (the target) and submerged approximately 2 cm beneath the water surface. We poured milk powder into the water to make the water opaque. We conducted the place trials at PND 42–46 with 4 trials daily at the same time point and performed the probe trials on PND 47 after 5 days’ training. The swimming of rats during the tests was recorded by a video tracking system installed above the tank. In place trials, rats were placed into four quadrants (spaced 20 min apart) to swim freely for a maximum of 120 s. If the rats could not find the platform within 120 s, they were allowed to stay on the platform for 20 s to observe the environment by guiding. The time for rats to reach the platform and swimming speed were recorded. In probes trials, the platform was removed and the rats were put into the first quadrant and allowed to swim for 120 s. The times of rats crossing the original platform were recorded.
+
+17β-estradiol is a principal female hormone, which provides in robust neuroprotection in many brain injury models both sexes and attenuates neurodegeneration induced by anesthesia agents (McCullough and Hurn, 2003; Li et al., 2014). 17β-estradiol also plays a role as a potent modulator for physiological neurogenesis. NSCs derived from embryos and adults both express estrogen receptor a (ERa) and estrogen receptor b (ERb) (Brännvall et al., 2002). 17β-estradiol not only promotes the proliferation and neuronal differentiation of embryonic NSCs (Brännvall et al., 2002; Isgor and Watson, 2005; Kishi et al., 2005), but also regulates the migration of embryonic neuroblasts via ERb (Wang et al., 2003). 17β-estradiol synaptic transmission, and post-stroke neurogenesis (Garcia-Segura et al., 2001; Zheng et al., 2012). However, whether 17β-estradiol administration protects the developing brain from ketamine- caused neurogenesis cognitive dysfunction remains unclear.
+
+also
+
+enhances
+
+axonal
+
+sprouting,
+
+impairment and improves
+
+Anesthetic Exposure in vivo and Tissue Preparation The PND 7 rats, weighing 13–18 g, were housed with their mother and maintained at a temperature of 24◦C in a 12 h/12 h light/dark cycle with free access to food and water. We assigned the rats randomly into three groups (28 rats from 7 nests in each group, 4 pups per nest): (i) the rats in control group received equal volume of normal saline by intraperitoneal injection as ketamine solution at corresponding time points; (ii) the rats in ketamine group received 40 mg/kg ketamine, diluted in normal saline and administrated by intraperitoneal injection (ketamine, Sigma–Aldrich Inc. St. Louis, MO, United States), the initial injection was considered to be the loading dose, 30% of it was injected at approximately 40 min intervals to maintain the anesthesia for 4 h (Lu et al., 2017); (iii) the rats in the 17β-estradiol group received 17β-estradiol (17β-estradiol, Tocris, Minneapolis, MN, United States; DMSO, Sigma–Aldrich, St Louis, MO, United States) dissolved in dimethylsulfoxide (DMSO) at a concentration of 100 ug/ml, 100 ug/kg 17β-estradiol
+
+As a serine/threonine kinase, glycogen synthase kinase (GSK) 3β plays an important role in multiple fundamental functions of cell in the developing brain, including neurogenesis, apoptosis, cell cycle, cytoskeletal integrity, and axon growth (Hur and Zhou, 2010; Kandimalla et al., 2016). Exposure to ketamine decreased GSK-3β phosphorylation and induced neurotoxicity both in the NSCs and neurons of the neonatal rat brains (Bai et al., 2013; Liu et al., 2013; Huang et al., 2015; Lu et al., 2017). Increasing GSK-3β phosphorylation attenuated ketamine-induced neurogenesis disorder and neural cell injury (Lu et al., 2017, 2018). Interestingly, GSK-3β is also a downstream target of estradiol signaling (Shi et al., 2013; Wu et al., 2014). In the present study, we aimed to figure out whether 17β-estradiol could attenuate neurogenesis damage and cognitive dysfunction induced by ketamine exposure. We also investigated whether the GKS-3β signaling pathway participated in the protective effects of 17β-estradiol on ketamine-induced injury in neurogenesis.
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+June 2019 | Volume 13 | Article 251
+
+2
+
+fncel-13-00251
+
+June 12, 2019
+
+Time: 17:26
+
+# 3
+
+17β-Estradiol and Neuroprotection
+
+Li et al.
+
+solution (without Mg2+ and Ca2+, Gibco, Carlsbad, CA, United States). The tissues were then dissociated and triturated mechanically by a fire-polished Pasteur pipette softly. After centrifugation, the isolated cells were collected and re-suspended in free-serum DMEM/F12 medium (Gibco, Carlsbad, CA, United States) which was supplemented with 2% B27 (Gibco, Carlsbad, CA, United States), 20 ng/ml EGF (Gibco, Carlsbad, CA, United States), 20 ng/ml bFGF (Gibco, Carlsbad, CA, United States), and 100 U/ml penicillin and phytomycin. Cells were cultured for 7 days to form enough neurospheres and then passaged at a density of 2 × 105 cells/ml followed by collection and dissociation as previously described by Reynolds and Weiss (Reynolds et al., 1992). Half of the medium was changed every 3 days. After the second passage, the cells were ready for future experiments. For identification assessment, the cells after passage were seeded onto 100 µg/mL poly-L-lysine-coated coverslips and cultured in differentiating medium that contained 100× N2 supplement, 100× B27 supplement, and 1% fetal bovine serum (FBS, Gibco, Carlsbad, CA, United States) in DMEM/F12 (without b-FGF) for 7 days.
+
+administered intraperitoneally 8 h, 1 h prior to and 3 h after ketamine’s initial injection (Liu et al., 2007). During anesthesia, all pups were kept on an electric blanket with the temperature set at 36.5 ± 1◦C to maintain body temperature and reduce stress. We observed the respiratory rate, skin color, and body movement of rats carefully and tested the voluntary movement by clamping the pup tails. Pulse oxygen saturation (SpO2) was detected by attaching the infant pulse oximetry probes to the rat abdomen. After the anesthesia, the pups received BrdU (50 mg/kg, intraperitoneal injection) every 24 h for 7 consecutive days. On PND 14, the rats were decapitated and the brain tissues of SVZ and SGZ were harvested to detect neurogenesis.
+
+At 12 h after anesthesia, rat pups (n = 6 per group, captured randomly) were sacrificed by decapitation. Both the brain tissue from SVZ and SGZ were isolated immediately on ice and the stored at −80◦C until use for western blotting. The rats (n = 6 per group, captured randomly) were sacrificed and perfused transcardially with 0.9% saline 7 days after anesthesia, followed by cold 4% paraformaldehyde in PBS. Then the harvested brains were postfixed in 4% paraformaldehyde overnight at 4◦C and dehydrated in 30% sucrose solution for 3–4 days, as we described previously (Lu et al., 2017). The brain tissue from bregma +0.2 mm to bregma −6.0 mm was the region of interest, which were cut into 16 µm coronary tissue slices by freezing microtome (SLEE, Germany). These brain slices were collected and used for future immunohistochemistry staining. The rest of the rat pups (n = 10 per group) were bred for behavior study at adulthood.
+
+Drug Exposure and Neurogenesis Analysis in vitro The cells were assigned to the following groups: control group, ketamine group, and 17β-estradiol group. No drug treatment was added to the control group. NSCs in the ketamine group were exposed to 100 µM ketamine for 24 h. NSCs in the 17β-estradiol group were pretreated with 17β-estradiol (100 nM) for 30 min and then 100 µM of ketamine was added to the culture medium for 24 h. For proliferative analysis, NSCs were seeded on cover slips which were pre-coated with 100 µg/mL poly-L-lysine and incubated with BrdU for the last 4 h. Following being fixed with 4% paraformaldehyde, the cells were stained with BrdU antibody (1:200, Abcam, United Kingdom) and DAPI. As for neuronal differentiation analysis, after being exposed to ketamine with or without 17β-estradiol for 24 h, the cells were seeded on cover slips which were pre-coated with 100 µg/mL poly-L-lysine and incubated with differentiating medium for 7 days, then the cells were harvested for immunohistochemical staining. The cells were labeled with β-tubulin III antibody (1:500; Sigma-Aldrich Inc. St. Louis, MO, United States). Briefly, 5–7 randomly selected fields were captured in each coverslip, and the numbers of β-tubulin III-positive cells were counted (at least 200 cells per test case). Data were collected from three independent experiments.
+
+Immunohistochemistry Immunohistochemistry was used to evaluate NSC proliferation in SVZ and SGZ by BrdU staining. Firstly, the brain slices were incubated with 2 N HCl for 30 min to denaturate the DNA at 37◦C. After being incubated with 0.1 mol L−1 boric acid (pH 8.5) for 10 min at room temperature followed by three times washing with 0.1 M PBS, the slices were blocked by 2% goat serum and 0.3% Triton X-100 for 2 h at room temperature, then incubated with the mouse monoclonal anti-BrdU antibody (1:200, Abcam, United Kingdom) at 4◦C overnight. The next day, after three washings with 0.1 M PBS, the slices were incubated with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibodies for 2 h at room temperature. BrdU-positive cells were counted within defined regions of interest in the SVZ and SGZ. In total, the mean numbers of BrdU-positive cells of six brain slices for each rat, spaced approximately 200 µm apart, were examined by the observer blindly. For each slice, five regions were captured by fluorescence microscopy (BX51, Olympus, Tokyo, Japan), and the planar area enclosed by each region was 50 × 50 µm. The edges of the captured regions were defined according to structural details to ensure the fields did not overlap (Zhang et al., 2009). The density of positive cells was presented as the total number of BrdU-positive cells in the SVZ and SGZ.
+
+Cell Apoptosis Test (TUNEL) We used terminal dUTP nick-end Labeling assay after passage, the dissociated cells were exposed to ketamine with or without 17β-estradiol for 24 h. After the treatments, cells were fixed with 4% paraformaldehyde for 15 min. The TUNEL assay was performed according to the instruction (Roche Inc. Roche, of Mannheim, Germany). Data were collected from three independent experiments.
+
+to detect
+
+cell
+
+apoptosis. Briefly,
+
+NSC Culture Primary cultured NSCs were obtained from the cortex of rat at embryonic day 18–19 under sterile conditions. Briefly, the forebrain portion was isolated and placed in ice-cold Hank’s
+
+in situ Cell Death Detection Kit
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+June 2019 | Volume 13 | Article 251
+
+3
+
+fncel-13-00251
+
+June 12, 2019
+
+Time: 17:26
+
+# 4
+
+17β-Estradiol and Neuroprotection
+
+Li et al.
+
+Western Blot Analysis Brain tissues from the SVZ and SGZ of rats (n = 6 per group) at 12 h after anesthesia and cell cultures at 24 h following drug exposure were subjected to Western blot analyses as described in our previous studies (Lu et al., 2017). Briefly, the tissues were lysed by RIPA lysis buffer with protease and phosphatase inhibitors. The lysates were homogenized with an electric homogenizer and maintained on ice for 15 min. After being centrifuged for 15 min at 14000 rpm at 4◦C, the supernatant was aspirated and the resulting lysates were placed in a new tube. We used the BCA protein assay kit to examine the protein concentrations. Bovine serum albumin (BSA) was used as a standard. An equal amount of the resulting lysate was resolved by sodium dodecyl sulfate-polyacrylamide gel and the separated proteins were transferred to polyvinylidene fluoride membranes. After being blocked for 1 h at room temperature, the membranes were then incubated with appropriate dilutions of primary antibodies at 4◦C overnight. The used antibodies included anti- caspase-3 (cleaved, 17 KDa, 1:1000, Cell Signal Technology Inc. Beverly, MA, United States), anti-phosophorylated GSK-3β (p-GSK-3β, 1:1000, Cell Signal Technology Inc. Beverly, MA, United States), anti-GSK-3β (1:1000, Cell Signal Technology Inc. Beverly, MA, United States), and anti-β-actin (1:1000, Cell Signal Technology Inc., Beverly, MA, United States). The following day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit or anti-mouse) for 2 h at room temperature. After being enhanced by chemiluminescence (ECL), the signals were then exposed to X-ray films. Each band in the Western blot represented an independent experiment and at least three independent experiments were conducted. Data were expressed as the ratio to optical density (OD) values of the corresponding controls. The Western blots were quantified as described in our previous study.
+
+to the ketamine group, the escape latency was decreased in the 17β-estradiol group on both trial days 3 and 4 [Figure 1C; F(2,48) = 62.35; p < 0.05]. The times to pass over the target platform and time spent in target area are shown in Figures 1D,E. Compared with the control group, rats in the ketamine group had less time to pass over the target platform and spent less time in the target area [F(2,72) = 89.64; p < 0.05]. In contrast, compared with the ketamine group, rats in the 17β-estradiol group passed over the target platform more frequently and spent more time in the target quadrant [F(2,60) = 70.35; p < 0.05]. Collectively, these results showed that ketamine exposure in neonatal rats would induce cognitive impairment in adulthood and that pretreatment with 17β-estradiol could attenuate this defect.
+
+17β-Estradiol Enhanced Proliferation and Reduced Apoptosis of Cells in the SVZ and SGZ Following Ketamine Exposure BrdU incorporation was used to assess cell proliferation. As shown in Figure 2, BrdU-positive cells were distributed in the SVZ and SGZ among all groups. Less BrdU-positive cells were found in the SVZ and SGZ of the ketamine group. Increased BrdU-positive cells were detected in both regions of the control and 17β-estradiol groups. Quantitative analysis showed that the number of BrdU-positive cells in SVZ and in SGZ of ketamine group declined significantly when compared to the control group [F(2,36) = 31.97; p < 0.01, in SVZ and F(2,60) = 6.031; p < 0.01 in SGZ]. The number of BrdU-positive cells in the 17β-estradiol group was significantly higher than that of the ketamine group both in SVZ and in SGZ [F(2,36) = 31.97; p < 0.05 in SVZ and F(2,60) = 6.031; p < 0.05 in SGZ], respectively. However, it was less than that of the control group in SVZ [F(2,36) = 31.97; p < 0.05], but not in SGZ [F(2,60) = 6.031; p > 0.05] (Figure 2G). Considering the important role of cleaved caspase-3 in cell apoptosis (Moosavi et al., 2012), the protein levels of cleaved caspase-3 in the SVZ and SGZ 12 h after ketamine exposure were examined by Western blotting. When compared to the control group, where the levels of cleaved caspase-3 were relatively low, ketamine exposure increased the cleaved caspase- 3 expressions in both regions [F(2,6) = 8.627; p < 0.05, in SVZ and F(2,6) = 9.742; p < 0.05 in SGZ]. However, pretreatment with 17β-estradiol decreased protein expressions of cleaved caspase-3 in the SVZ and SGZ of neonatal rats after ketamine exposure[F(2,6) = 8.627; p < 0.05, in SVZ and F(2,6) = 9.742; p < 0.05 in SGZ] (Figure 2H).
+
+Statistical Analysis Data obtained from the study were presented as mean ± SEM. Every data point represented a mean for each animal in a single case. SigmaPlot 12.0 was used for all statistical analysis. Data were tested and then confirmed with normality and equal variance criteria. A one-way analysis of variance (ANOVA) following the post hoc Holm-Sidak method was used to analyze the differences among different groups. A two-tailed probability value P < 0.05 was considered statistically significant.
+
+RESULTS
+
+17β-Estradiol Rescued Proliferation and Apoptosis of NSCs Following Ketamine Exposure While It Reversed the Decrease of Neuron Production Induced by Ketamine To identify the characteristics of cultured cells, we first stained the cells with NSC marker nestin 3 days after seeding. The results showed that both in suspension and adherent culture most of the cells were nestin-positive. The percentages of nestin-positive cells were 96.0 ± 2.1 and
+
+17β-Estradiol Improved Ketamine-Induced Decline of Learning and Memory The MWM test results are shown in Figure 1. There was no significant difference in the swimming speed among the control, ketamine, and 17β-estradiol groups [Figure 1B; F(2,36) = 8.956; p > 0.05]. When compared to the control group, the escape latency was increased in the ketamine group on trial day 3 [Figure 1C; F(2,48) = 50.65; p < 0.05]. However, when compared
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+June 2019 | Volume 13 | Article 251
+
+4
+
+fncel-13-00251
+
+June 12, 2019
+
+Time: 17:26
+
+# 5
+
+17β-Estradiol and Neuroprotection
+
+Li et al.
+
+FIGURE 1 | Diagram of the study and results of Morris water maze trials. (A) The diagram of the timeline of the study. (B) Swimming speed comparison between three groups during the training. There was no difference among three groups. (C) Comparison of the latency to reach the hidden platform. ∗p < 0.05 compared with control group, #p < 0.05 compared with ketamine group. (D) Comparison of the numbers to pass over the target platform. ∗p < 0.05 compared with control group, #p < 0.05 compared with ketamine group. (E) Comparison of the time spent in target quadrant. ∗p < 0.05 compared with control group, #p < 0.05 compared with ketamine group. Data are present as the means ± SEM. n = 6 in each group.
+
+95.0 ± 2.0%, respectively (Figures 3A,B). After incubation the cultured cells with differentiating medium for 7 days, expressed neuronal marker β-tubulin III (22.3 ± 1.9%, Figures 3C,D) and astrocytes marker glial fibrillary acidic (59.8 ± 3.2%, Figures 3C,D). Taken protein (GFAP) together, the cells used in this study were NSCs.
+
+cells following ketamine exposure. There was no significant difference between the 17β-estradiol group and the control group [F(2,42) = 148.6; p > 0.05] (Figure 3H). These findings indicated that 17β-estradiol rescued the proliferation of NSCs exposed to ketamine.
+
+by differentiation of NSCs was immunofluorescence staining of β-tubulin III (Figures 3I–K). Ketamine exposure for 24h decreased the percentage of β-tubulin III-positive inhibited neuronal indicating ketamine production from NSCs. However, co-administration with 17β-estradiol reversed the effects of ketamine on neuronal production [F(2,42) = 60.36; p < 0.05] (Figure 3L, p < 0.05). There was no significant difference between the 17β-estradiol
+
+Neuronal
+
+assessed
+
+these data suggested that
+
+To examine the proliferation of NSCs in vitro, BrdU incorporation method was used (Figures 3E–G). It was shown that the number of BrdU-positive cells was decreased after exposure to ketamine for 24 h when compared to the control group [F(2,42) = 148.6; p < 0.05]. However, pretreatment with 17β-estradiol increased the number of BrdU-positive
+
+cells,
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+June 2019 | Volume 13 | Article 251
+
+5
+
+fncel-13-00251
+
+June 12, 2019
+
+Time: 17:26
+
+# 6
+
+17β-Estradiol and Neuroprotection
+
+Li et al.
+
+FIGURE 2 | The proliferative changes in the SVZ and SGZ. (A–F) Representative images of BrdU immunoreactive cells (red) in the SVZ and SGZ 7 days after anesthesia. Scale bar = 100 µm. (G) Quantification of BrdU-positive cells following different treatment. ∗p < 0.05 compared with control group, #p < 0.05 compared with ketamine group. (H) The expression and quantification of caspase-3 by western blotting in the SVZ and SGZ 12 h after anesthesia. ∗p < 0.05 compared with control group, #p < 0.05 compared with ketamine group. Data are presented as the means ± SEM. n = 6 in each group.
+
+group and the control group [F(2,42) = 60.36; p > 0.05]. These findings indicated that 17β-estradiol rescued the decrease of neuronal production from NSCs exposed to ketamine.
+
+control condition. However, pretreatment with 17β-estradiol significantly reduced NSC apoptosis induced by ketamine exposure [F(2,42) = 27.81; p < 0.05] (Figure 3P).
+
+We used western blotting to determine related molecules involved in ketamine-induced damage and 17β-estradiol- elicited neuroprotection (Figures 4A,B). The results showed that ketamine exposure for 24h increased cleaved caspase- 3 expression [F(2,24) = 76.59; p < 0.05] and decreased
+
+Next, we used TUNEL staining to assess the apoptosis of NSCs, aiming to investigate whether 17β-estradiol could reduce NSC apoptosis induced by ketamine exposure (Figures 3M–O). After ketamine exposure, the number of TUNEL positive cells was obviously increased when compared with the
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+June 2019 | Volume 13 | Article 251
+
+6
+
+fncel-13-00251
+
+June 12, 2019
+
+Time: 17:26
+
+# 7
+
+17β-Estradiol and Neuroprotection
+
+Li et al.
+
+FIGURE 3 | Identification of cultured cells and the proliferation, differentiation as well as apoptosis of NSCs following different treatment. (A) Images of nestin (red) immunoreactive neurosphere. (B) Images of nestin (red) immunoreactive NSCs. (C) Images of NSCs differentiating into neurons (red) and astrocyte (green). Scale bar = 100 µm. (D) Rate of specific cellular phenotype to total cells (DAPI, blue). (E–G) Representative images of BrdU immunoreactive cells (red) in control, ketamine, and 17β-estradiol group, respectively. Scale bar = 100 µm. (H) Quantification of BrdU-positive cells following different treatment. ∗p < 0.05 compared with control group, #p < 0.05 compared with ketamine group. (I–K) Representative images of β-tubulinimmunoreactive cells (red) in control, ketamine, and 17β-estradiol group, respectively. Scale bar = 50 µm. (L) Quantification of β-tubulin III -positive cells following different treatment. ∗p < 0.05 compared with control group, #p < 0.05 compared with ketamine group. (M–O) Representative images of TUNEL immunoreactive cells (green) in control, ketamine, and 17β-estradiol groups, respectively. Scale bar = 50 µm. (P) Quantification of TUNEL-positive cells following different treatment. ∗p < 0.05 compared with control group, #p < 0.05 compared with ketamine group. Data were collected from three independent experiments and are presented as the means ± SEM.
+
+that GSK-3β and caspase-3 were These findings involved in ketamine-induced apoptosis and 17β-estradiol elicited protection.
+
+p-GSK-3β expression [F(2,42) = 43.28; p < 0.05] in NSCs compared to the control. Pretreatment with 17β-estradiol decreased the levels of cleaved caspase-3 [F(2,24) = 76.59; p < 0.05] and prevented the deregulation of p-GSK-3β expressions [F(2,42) = 43.28; p < 0.05] in NSCs exposed to ketamine for 24 h. However, there were no differences in the protein expressions of p-GSK-3β [F(2,42) = 43.28; p > 0.05] and cleaved caspase-3 [F(2,24) = 76.59; p > 0.05] between the control group and 17β-estradiol group (Figures 4C,D).
+
+suggest
+
+DISCUSSION
+
+In the present study, our data showed that treatment with 17β-estradiol improved neonatal ketamine exposure-induced
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+June 2019 | Volume 13 | Article 251
+
+7
+
+fncel-13-00251
+
+June 12, 2019
+
+Time: 17:26
+
+# 8
+
+17β-Estradiol and Neuroprotection
+
+Li et al.
+
+FIGURE 4 | Detection of cleaved caspase-3 and pGSK-3β by western blotting. (A,B) Representative images of western blotting analysis of the cleaved caspase-3 and pGSK-3β in NSCs 24 h following drug exposure. (C,D) Quantification of cleaved caspase-3 and pGSK-3β expression normalized to β-actin following different treatment. ∗p < 0.05 compared with control group, #p < 0.05 compared with ketamine group. Data were collected from three independent experiments and are expressed as the ratio to optical density (OD) values of the corresponding controls.
+
+maze at an older age than that used in this study (Li et al., 2014). These findings indicate a potential strategy for prevention of ketamine-induced neurocognitive deficits.
+
+cognitive deficits and mitigated ketamine-caused changes in cellular proliferation and apoptosis in the SVZ and SGZ. Treatment with 17β-estradiol rescued neurogenesis and reduced apoptosis of NSCs exposed to ketamine in vitro, in which GSK-3β might play a role in 17β-estradiol-elicited protection.
+
+Many studies showed that ketamine caused neuroapoptosis and neurodegeneration in the developing brain, which may finally induce learning and memory disabilities in adults (Fredriksson et al., 2007; Paule et al., 2011; Huang et al., 2012; Moosavi et al., 2012; Sabbagh et al., 2012). However, few studies reported the effect of ketamine on neurogenesis and its long-term outcome in vivo. In mammals, new neurons are generated continuously to certain brain areas throughout life. These neurons are differentiated from NSCs located primarily in the SVZ and SGZ. Proliferation and/or survival of NSCs are the basic events in neurogenesis. Considering the significance of neurogenesis during the BGS period, we evaluated the NSC proliferation and survival using BrdU labeling and apoptosis analysis. It was shown that ketamine inhibited the cell proliferation in the neurogenesis regions of neonatal rats, indicating that neonatal ketamine exposure might impair neurogenesis. This is consistent with previous studies (Huang et al., 2015, 2016; Dong et al., 2016). Interestingly, we observed that 17β-estradiol alleviates cellular proliferating changes induced by ketamine in neurogenesis regions of neonatal rats, indicating that 17β-estradiol enhanced NSC proliferation following ketamine exposure during brain development, which is a benefit in vitro
+
+The toxicity of general anesthesia on the developing brain has raised concern in recent years. It is a consensus that repeated exposure to general anesthetic before 3 years of age is harmful to the developing brain (Hu et al., 2017). Ketamine is a commonly used anesthetic in pediatric clinics. It has been proven that ketamine can lead to long-lasting cognitive impairments in rodent and primate models (Fredriksson et al., 2007; Paule et al., 2011; Huang et al., 2012; Moosavi et al., 2012; Zhao et al., 2014). Unfortunately, there has been no safe and effective measure to prevent this deficit until now. To evaluate whether 17β-estradiol could improve the learning and memory ability of adult rats that were subjected to ketamine exposure during the neonatal stage, the Morris water maze was used in this study. The rats in the 17β-estradiol group passed over the target platform more frequently and spent more time in the target area compared with the ketamine group, suggesting that 17β-estradiol ameliorates long-lasting cognitive dysfunction in rodents who received ketamine in the early stage of life. Consistently, Li et al. also reported that 17β-estradiol attenuated long-term cognitive impairments in developing rats though they used higher doses of ketamine and 17β-estradiol and performed the Morris water
+
+for neurogenesis. Furthermore, our
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+June 2019 | Volume 13 | Article 251
+
+8
+
+fncel-13-00251
+
+June 12, 2019
+
+Time: 17:26
+
+# 9
+
+17β-Estradiol and Neuroprotection
+
+Li et al.
+
+study demonstrated that 17β-estradiol rescued proliferation and neuronal production of NSCs exposed to ketamine. Whether anesthetic-induced neurogenesis inhibition contributes to any disabilities in learning and memory functions remains unknown. Our recent work showed that restoration of neurogenesis in SVZ and SGZ in neonatal rats could improve the neonatal ketamine exposure-induced adult spatial learning and memory deficits (Lu et al., 2017). In this study, treatment with 17β-estradiol mitigated ketamine-induced changes in neurogenesis and cognitive deficits. Li et al. reported that ketamine induced neuroapoptosis in the prefrontal cortex of the developing brain and caused long- term cognitive dysfunction in adulthood, and treatment with 17β-estradiol attenuated ketamine-induced damages (Li et al., 2014). Our previous work showed that ketamine exposure did not increase the rate of TUNEL-positive cells in the frontal cortex (Lu et al., 2017). The discrepancy might be due to dose of drugs, regions of interest and endpoints of observation. Collectively, our findings supported a causal link between neurogenesis damage and cognitive dysfunction in neurodevelopmental toxicity of anesthetics.
+
+of NSCs was investigated in the study. Later studies should select more indexes to detect differentiation of NSCs in vitro. Thirdly, GSK-3β is the target of numerous molecules, but we did not investigate the upstream and downstream signal of GSK-3β caused by ketamine or 17β-estradiol, and further studies are needed.
+
+CONCLUSION
+
+In conclusion, using a model of neurotoxicity following ketamine exposure to neonatal rats, we found that ketamine exposure increased apoptosis and decreased proliferation of cells in the SVZ and SGZ, leading to a decline in spatial learning and memory abilities in adulthood. Administration of 17β-estradiol enhanced neurogenesis by decreasing apoptosis and increasing proliferation of NSCs. Furthermore, GSK-3β might be an important molecule that is involved in this process. The present findings provide an experimental basis for the use of 17β-estradiol as a therapeutic drug against the development neurotoxicity of ketamine. Clinically, the results suggest 17β-estradiol may help to prevent ketamine- induced developmental neurotoxicity and GSK-3β may become a molecular target for its treatment. Nevertheless, a much work still needs to be done before clinical use, because a more specific treatment target should be developed and translated to humans.
+
+Apoptosis is a critical procedure during the development of the neural system, which occurs at various developmental stages from neurogenesis to adulthood (Hara et al., 2018). However, exogenous insult-induced apoptosis had a lasting impact on neurogenesis in the developing brain (Sokolowski et al., 2013). Maintenance of the homeostasis of neurogenesis provides basis for normal brain structure and function. As an antagonist of non-competitive NMDA receptor, ketamine induced NSC apoptosis by activation of GSK-3β (Lu et al., 2018). It was reported that ketamine induced neuroapoptosis in the prefrontal cortex accompanied by the downregulation of 17β-estradiol, BDNF, and p-Akt in neonatal rats, and treatment with 17β-estradiol attenuated ketamine-induced injuries (Li et al., 2014). We found that treatment with 17β-estradiol decreased NSC apoptosis and increased p-GSK-3β levels, indicating that 17β-estradiol decreased NSC apoptosis by inactivation of GSK- 3β. Further study needs to be done to elucidate how ketamine or 17β-estradiol affect GSK-3β phosphorylation in the NSCs. NSCs express ERa and ERb (Brännvall et al., 2002). 17β-estradiol promotes NSC proliferation and differentiation into neurons (Brännvall et al., 2002; Isgor and Watson, 2005; Kishi et al., 2005). In the present study, whether the protection of 17β-estradiol on ketamine-induced neurogenesis damage is merely due to the increase in NSC proliferation and neuronal differentiation or the decrease in NSC apoptosis needs to be further studied.
+
+ETHICS STATEMENT
+
+This in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23). The protocol was approved by the Animal Care and Use Committee of Xi’an Jiaotong University.
+
+study was
+
+carried out
+
+AUTHOR CONTRIBUTIONS
+
+PZ, WL, and HuL conceived and designed the experiments. WL, HuL, YaL, SL, and JZ performed the experiments. HW, SL, and XC analyzed the data. HaL and YoL contributed with the Materials and Methods, and critically revised the manuscript. All authors wrote the manuscript.
+
+FUNDING
+
+Although some important discoveries were revealed by these studies, it is worth emphasizing that several limitations exist. Firstly, NSC differentiation, neuronal apoptosis, or migration in the developing brain were not detected because our main aim for this study was to observe whether anesthesia doses of ketamine could inhibit NSC proliferation, induce NSC apoptosis in neonatal rats, and cause cognitive deficits in adults as well as whether treatment with 17β-estradiol could attenuate these changes. Further study should be done to reveal NSC differentiation, neuronal apoptosis, or migration in the developing brain exposed to ketamine with or without 17β-estradiol. Secondly, only in vitro neuronal differentiation
+
+This work was supported by the National Natural Science Foundation of China (81071071 and 81171247), Key Scientific Innovation Team of Shaanxi Province and Technological (2014KCT-22), and Science and Technology Development Project of Shaanxi Province (2013KTCL03-09).
+
+ACKNOWLEDGMENTS
+
+The thank authors reading the manuscript.
+
+Prof. Malgorzata Garstka
+
+for
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+June 2019 | Volume 13 | Article 251
+
+9
+
+fncel-13-00251
+
+June 12, 2019
+
+Time: 17:26
+
+# 10
+
+17β-Estradiol and Neuroprotection
+
+Li et al.
+
+REFERENCES
+
+Liu, J. R., Baek, C., Han, X. H., Shoureshi, P., and Soriano, S. G. (2013). Role of glycogen synthase kinase-3beta in ketamine-induced developmental neuroapoptosis in rats. Br. J. Anaesth. 110(Suppl. 1), i3–i9. doi: 10.1093/bja/ aet057
+
+Bai, X., Yan, Y., Canfield, S., Muravyeva, M. Y., Kikuchi, C., Zaja, I., et al. (2013). Ketamine enhances human neural stem cell proliferation and induces neuronal apoptosis via reactive oxygen species-mediated mitochondrial pathway. Anesth. Analg. 116, 869–880. doi:10.1213/ANE.0b013e31828 60fc9
+
+Liu, R., Wang, X., Liu, Q., Yang, S. H., and Simpkins, J. W. (2007). Dose dependence and therapeutic window for the neuroprotective effects of 17beta-estradiolwhen doi: administered after cerebral 10.1016/j.neulet.2007.01.074
+
+ischemia. Neurosci. Lett. 415, 237–241.
+
+Brännvall, K., Korhonen,
+
+L.,
+
+and
+
+Lindholm, D.
+
+(2002). Estrogen- stem cell proliferation and doi:10.1006/mcne.2002.
+
+receptor-dependent differentiation. Mol. Cell Neurosci. 21, 512–520. 1194
+
+regulation of neural
+
+Lu, P., Lei, S., Li, W., Lu, Y., Zheng, J., Wang, N., et al. (2018). Dexmedetomidine protects neural stem cells from ketamine-induced injury. Cell Physiol. Biochem. 47, 1377–1388. doi: 10.1159/000490823
+
+Dong, C., Rovnaghi, C. R., and Anand, K. J. (2016). Ketamine exposure during embryogenesis inhibits cellular proliferation in rat fetal cortical neurogenic regions. Acta Anaesthesiol. Scand. 60, 579–587. doi: 10.1111/aas.12689
+
+Lu, Y., Giri, P. K., Lei, S., Zheng, J., Li, W., Wang, N., et al. (2017). Pretreatment with minocycline restores neurogenesis in the subventricular zone and subgranular zone of the hippocampus after ketamine exposure in neonatal rats. Neuroscience 352, 144–154. doi: 10.1016/j.neuroscience.2017.03.057
+
+Eriksson, P. (1997). Developmental neurotoxicity of environmental agents in the
+
+neonate. Neurotoxicology 18, 719–726.
+
+McCullough, L. D., and Hurn, P. D.
+
+(2003). Estrogen and ischemic neuroprotection: an integrated view. Trends Endocrinol. Metab. 14, 228–235. doi: 10.1016/s1043-2760(03)00076-6
+
+Eriksson, P., Ankarberg, E., and Fredriksson, A. (2000). Exposure to nicotine during a defined period in neonatal life induces permanent changes in brain nicotinic receptors and in behaviour of adult mice. Brain Res. 853, 41–48. doi: 10.1016/s0006-8993(99)02231-3
+
+Moosavi, M., Yadollahi Khales, G., Rastegar, K., and Zarifkar, A. (2012). The effect of sub-anesthetic and anesthetic ketamine on water maze memory acquisition, consolidation and retrieval. Eur. J. Pharmacol. 677, 107–110. doi: 10.1016/j. ejphar.2011.12.021
+
+Fredriksson, A., Ponten, E., Gordh, T., and Eriksson, P. (2007). Neonatal exposure to a combination of N-methyl-D-aspartate and gamma-aminobutyric acid type a receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. Anesthesiology 107, 427–436. doi: 10.1097/01. anes.0000278892.62305.9c
+
+Muramatsu, R., Ikegaya, Y., Matsuki, N., and Koyama, R. (2007). Neonatally born granule cells numerically dominate adult mice dentate gyrus. Neuroscience 148, 593–598. doi: 10.1016/j.neuroscience.2007.06.040
+
+Garcia-Segura, L. M., Azcoitia, I., and DonCarlos, L. L. (2001). Neuroprotection by estradiol. Prog. Neurobiol. 63, 29–60. doi: 10.1016/s0301-0082(00)00025-3 Hara, Y., Sudo, T., Togane, Y., Akagawa, H., and Tsujimura, H. (2018). Cell death in neural precursor cells and neurons before neurite formation prevents the emergence of abnormal neural structures in the Drosophila optic lobe. Dev. Biol. 436, 28–41. doi: 10.1016/j.ydbio.2018.02.004
+
+Paule, M. G., Li, M., Allen, R. R., Liu, F., Zou, X., and Hotchkiss, C. H. (2011). Ketamine anesthesia during the first week of life can cause long- lasting cognitive deficits in rhesus monkeys. Neurotoxicol. Teratol. 33, 220–230. doi: 10.1016/j.ntt.2011.01.001
+
+Ponten, E., Viberg, H., Gordh, T., Eriksson, P., and Fredriksson, A. (2012). Clonidine abolishes the adverse effects on apoptosis and behaviour after neonatal ketamine exposure in mice. Acta Anaesth. Scand. 56, 1058–1065. doi: 10.1111/j.1399-6576.2012.02722.x
+
+Hu, D., Flick, R. P., Zaccariello, M. J., Colligan, R. C., Katusic, S. K., Schroeder, D. R., et al. (2017). Association between exposure of young children to procedures requiring general anesthesia and learning and behavioral outcomes in a population-based birth cohort. Anesthesiology 127, 227–240. doi: 10.1097/ ALN.0000000000001735
+
+Reynolds, B. A., Tetzlaff, W., and Weiss, S. (1992). A multipotent EGF- responsive cell produces neurons and astrocytes. J. Neurosci. 12, 4565–4574. doi: 10.1523/jneurosci.12-11-04565. 1992
+
+striatal
+
+embryonic progenitor
+
+Huang, H., Liu, C. M., Sun, J., Hao, T., Xu, C. M., Wang, D., et al. (2016). Ketamine affects the neurogenesis of the hippocampal dentate gyrus in 7- day-old Rats. Neurotox. Res. 30, 185–198. doi: 10.1007/s12640-016-9615- 9617
+
+Sabbagh, J. J., Heaney, C. F., Bolton, M. M., Murtishaw, A. S., and Kinney, J. W. (2012). Examination of ketamine-induced deficits in sensorimotor gating and spatial learning. Physiol. Behav. 107, 355–363. doi: 10.1016/j.physbeh.2012.0 8.007
+
+Huang, H., Liu, L., Li, B., Zhao, P. P., Xu, C. M., Zhu, Y. Z., et al. (2015). Ketamine interferes with the proliferation and differentiation of neural stem cells in the subventricular zone of neonatal rats. Cell Physiol. Biochem. 35, 315–325. doi: 10.1159/000369698
+
+Scallet, A. C., Schmued, L. C., Slikker, W., Grunberg, N., Faustino, P. J., Davis, H., et al. (2004). Developmental neurotoxicity of ketamine: morphometric labeling of confirmation, exposure parameters, and multiple fluorescent apoptotic neurons. Toxicol. Sci. 81, 364–370. doi: 10.1093/toxsci/kfh224
+
+Huang, L., Liu, Y., Jin, W., Ji, X., and Dong, Z. (2012). Ketamine potentiates hippocampal neurodegeneration and persistent learning and memory impairment through the PKC gamma-ERK signaling pathway in the developing brain. Brain Res. 1476, 164–171. doi: 10.1016/j.brainres.2012.07.059
+
+Shen, X., Dong, Y., Xu, Z., Wang, H., Miao, C., Soriano, S. G., et al. (2013). Selective anesthesia-induced neuroinflammation in developing mouse brain and cognitive impairment. Anesthesiology 118, 502–515. doi: 10.1097/ALN. 0b013e3182834d77
+
+Hur, E. M., and Zhou, F. Q. (2010). GSK3 signalling in neural development. Nat.
+
+Rev. Neurosci. 11, 539–551. doi: 10.1038/nrn2870
+
+Shi, C., Wu, F., Zhu, X. C., and Xu, J. (2013). Incorporation of beta-sitosterol into the membrane increases resistance to oxidative stress and lipid peroxidation via estrogen receptor-mediated PI3K/GSK3beta signaling. Biochim. Biophys. Acta 1830, 2538–2544. doi: 10.1016/j.bbagen.2012.12.012
+
+Isgor, C., and Watson, S. J. (2005). Estrogen receptor alpha and beta mRNA expressions by proliferating and differentiating cells in the adult rat dentate gyrus and subventricular zone. Neuroscience 134, 847–856. doi: 10.1016/j. neuroscience.2005.05.008
+
+Sokolowski, K., Obiorah, M., Robinson, K., McCandlish, E., Buckley, B., and DiCicco-Bloom, E. (2013). Neural stem cell apoptosis after low-methylmercury exposures loss and in postnatal hippocampus produce persistent cell adolescent memory deficits. Dev. Neurobiol. 73, 936–949. doi: 10.1002/dneu.2 2119
+
+Kandimalla, R., Thirumala, V., and Reddy, P. H. (2016). Is Alzheimer’s disease a type 3 diabetes? a critical appraisal. Biochim. Biophys. Acta 1863, 1078–1089. doi: 10.1016/j.bbadis.2016.08.018
+
+Kang, E., Berg, D. A., Furmanski, O., Jackson, W. M., Ryu, Y. K., Gray, C. D., et al. (2017). Neurogenesis and developmental anesthetic neurotoxicity. Neurotoxicol. Teratol. 60, 33–39. doi: 10.1016/j.ntt.2016.10.001
+
+Wang, L., Andersson, S., Warner, M., and Gustafsson, J. A. (2003). Estrogen receptor (ER)beta knockout mice reveal a role for ERbeta in migration of cortical neurons in the developing brain. Proc. Natl. Acad. Sci. U.S.A. 100, 703–708. doi: 10.1073/pnas.242735799
+
+Kishi, Y., Takahashi, J., Koyanagi, M., Morizane, A., Okamoto, Y., and Horiguchi, S. (2005). Estrogen promotes differentiation and survival of dopaminergic neurons derived from human neural stem cells. J. Neurosci. Res. 79, 279–286. doi: 10.1002/jnr.20362
+
+Wu, J., Williams, D., Walter, G. A., Thompson, W. E., and Sidell, N. (2014). Estrogen increases Nrf2 activity through activation of the PI3K pathway in MCF-7 breast cancer cells. Exp. Cell Res. 328, 351–360. doi: 10.1016/j.yexcr. 2014.08.030 Zhang, P., Li,
+
+Li, J., Wang, B., Wu, H., Yu, Y., Xue, G., and Hou, Y. (2014). 17beta-estradiol attenuates ketamine-induced neuroapoptosis and persistent cognitive deficits in the developing brain. Brain Res. 1593, 30–39. doi: 10.1016/j.brainres.2014.0 9.013
+
+J., Liu, Y., Chen, X., Kang, Q., Zhao,
+
+J., et al.
+
+(2009). and
+
+Human neural
+
+stem cell
+
+transplantation attenuates
+
+apoptosis
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+June 2019 | Volume 13 | Article 251
+
+10
+
+fncel-13-00251
+
+June 12, 2019
+
+Time: 17:26
+
+# 11
+
+17β-Estradiol and Neuroprotection
+
+Li et al.
+
+Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
+
+improves neurological Anaesthesiol. 024.x
+
+functions after cerebral 1184–1191. doi: 53,
+
+ischemia in rats. Acta 10.1111/j.1399-6576.2009.02
+
+Scand.
+
+Zhao, T., Li, Y., Wei, W., Savage, S., Zhou, L., and Ma, D. (2014). Ketamine administered to pregnant rats in the second trimester causes long-lasting behavioral disorders in offspring. Neurobiol. Dis. 68, 145–155. doi: 10.1016/j. nbd.2014.02.009
+
+Copyright © 2019 Li, Li, Wei, Lu, Lei, Zheng, Lu, Chen, Liu and Zhang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
+
+Zheng, J., Zhang, P., Li, X., Lei, S., Li, W., He, X., et al. (2012). Post-stroke estradiol treatment enhances neurogenesis in the subventricular zone of rats after permanent focal cerebral ischemia. Neuroscience 231, 82–90. doi: 10.1016/ j.neuroscience.2012.11.042
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+June 2019 | Volume 13 | Article 251
+
+11

new_pdfs/10.3389_fncel.2020.00004.txt

@@ -0,0 +1,465 @@
+ORIGINAL RESEARCH published: 28 January 2020 doi: 10.3389/fncel.2020.00004
+
+The mTOR Inhibitor Rapamycin Prevents General Anesthesia-Induced Changes in Synaptic Transmission and Mitochondrial Respiration in Late Postnatal Mice
+
+Xianshu Ju 1,2,3†, Min Jeong Ryu 1†, Jianchen Cui 1,2,3, Yulim Lee 1,2,3, Sangil Park 4, Boohwi Hong 4,5, Sungho Yoo 4, Won Hyung Lee 4,5, Yong Sup Shin 4,5, Seok-Hwa Yoon 4,5, Gi Ryang Kweon 1,2, Yoon Hee Kim 4,5, Youngkwon Ko 4,5, Jun Young Heo 1,2,3* and Woosuk Chung 2,4,5*
+
+Edited by:
+
+Julie S. Haas, Lehigh University, United States
+
+1Department of Biochemistry, Chungnam National University School of Medicine, Daejeon, South Korea, 2Department of Medical Science, Chungnam National University School of Medicine, Daejeon, South Korea, 3Infection Control Convergence Research Center, Chungnam National University School of Medicine, Daejeon, South Korea, 4Department of Anesthesia and Pain Medicine, Chungnam National University Hospital, Daejeon, South Korea, 5Department of Anesthesia and Pain Medicine, Chungnam National University School of Medicine, Daejeon, South Korea
+
+Reviewed by: Se-Young Choi, Seoul National University, South Korea Seok-Kyu Kwon, Korea Institute of Science and Technology (KIST), South Korea
+
+Correspondence: Jun Young Heo junyoung3@gmail.com Woosuk Chung woosuk119@gmail.com
+
+†These authors have contributed equally to this work
+
+Received: 06 August 2019 Accepted: 09 January 2020 Published: 28 January 2020
+
+Citation: Ju X, Ryu MJ, Cui J, Lee Y, Park S, Hong B, Yoo S, Lee WH, Shin YS, Yoon S-H, Kweon GR, Kim YH, Ko Y, Heo JY and Chung W (2020) The mTOR Inhibitor Rapamycin Prevents General Anesthesia-Induced Changes in Synaptic Transmission and Mitochondrial Respiration in Late Postnatal Mice. Front. Cell. Neurosci. 14:4. doi: 10.3389/fncel.2020.00004
+
+Preclinical animal studies have continuously reported the possibility of long-lasting neurotoxic effects after general anesthesia in young animals. Such studies also show that the neurological changes induced by anesthesia in young animals differ by their neurodevelopmental stage. Exposure to anesthetic agents increase dendritic spines and induce sex-dependent changes of excitatory/inhibitory synaptic transmission in late postnatal mice, a critical synaptogenic period. However, the mechanisms underlying these changes remain unclear. Abnormal activation of the mammalian target of rapamycin (mTOR) signaling pathway, an important regulator of neurodevelopment, has also been shown to induce similar changes during neurodevelopment. Interestingly, previous studies show that exposure to general anesthetics during neurodevelopment can activate the mTOR signaling pathway. This study, therefore, evaluated the role of mTOR signaling after exposing postnatal day (PND) 16/17 mice to sevoflurane, a widely used inhalation agent in pediatric patients. We first confirmed that a 2-h exposure of 2.5% sevoflurane could induce widespread mTOR phosphorylation in both male and female mice. Pretreatment with the mTOR inhibitor rapamycin not only prevented anesthesia-induced mTOR phosphorylation, but also the increase in mitochondrial respiration and male-dependent enhancement of excitatory synaptic transmission. However, the changes in inhibitory synaptic transmission that appear after anesthesia in female mice were not affected by rapamycin pretreatment. Our results suggest that mTOR inhibitors may act as potential therapeutic agents for anesthesia-induced changes in the developing brain.
+
+Keywords: general anesthesia, mTOR, neurodevelopment, neurotoxicity, synaptic transmission
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+1
+
+January 2020 | Volume 14 | Article 4
+
+Ju et al.
+
+INTRODUCTION
+
+Preclinical report possible neurotoxic effects from anesthesia in young rodents, sheep, and non-human primates (Olutoye et al., 2016; Jevtovic-Todorovic, 2018). The United States Food and Drug Administration (U.S. FDA) has therefore published warnings regarding the repeated or prolonged use of anesthesia in children under age 3 years. Fortunately, recent clinical studies strongly suggest that a single, short exposure to anesthetic does not affect neurodevelopment (O’Leary and Warner, 2017; Warner et al., 2018; McCann et al., 2019). However, there are still concerns regarding multiple anesthetic exposures (Warner et al., 2018; Zaccariello et al., 2019).
+
+animal
+
+studies
+
+continuously
+
+anesthesia-induced neurotoxicity depends on their neurodevelopmental stage. While anesthesia induces neuronal cell death in neonatal mice, the same anesthetics induces excitatory/inhibitory imbalance in late postnatal mice (Briner et al., 2010; Chung et al., 2017a; Ju et al., 2019). Importantly, excitatory/inhibitory imbalance has been linked to diverse neurodevelopmental disorders (Meredith, 2015; Lee et al., 2017). Because most procedures requiring anesthesia in humans are performed during the postnatal period, these anesthesia-induced changes in late postnatal mice may be of great importance, as the neurodevelopment of mice during this stage may be equivalent to the neurodevelopment of human infants (Workman et al., 2013). However, the mechanisms underlying anesthesia-induced changes in late postnatal mice are still not completely understood. of
+
+Previous
+
+studies
+
+also
+
+show that
+
+a serine/threonine functions controls including protein synthesis, energy metabolism, cell survival, autophagy and mitochondria biogenesis in peripheral tissues. In the nervous system, mTOR pathway regulates axonal sprouting, axonal regeneration and myelination, ion channel and receptor expression, and dendritic spine growth (Bockaert and Marin, 2015; Huber et al., 2015). Previous studies also show that activation of mTOR enhances synaptic activity by promoting AMPA receptor synthesis and expression at the cell surface (Wang et al., 2006; Ran et al., 2013). In addition, mTOR regulates dendritic spine development and formation (Tavazoie et al., 2005; Lee et al., 2011), and excitatory synaptic transmission (Tang et al., 2002; Cammalleri et al., 2003). As exposure to anesthetics increase synaptic proteins, dendritic imbalance spinogenesis, (Briner et al., 2010; Chung et al., 2017a; Ju et al., 2019), it is highly possible that mTOR signaling is involved with these anesthesia-induced changes. Indeed, previous studies show that anesthetics increase mTOR signaling in various ages (Li et al., 2010, 2017; Zhang et al., 2014; Kang et al., 2017). For example, injection of ketamine in adult mice was found to induce mTOR activation, accompanied by increased spinogenesis and excitatory synaptic transmission (Li et al., 2010). Isoflurane induction of anesthesia in postnatal day (PND) 15 mice was found to induce long-lasting mTOR pathway activation in the dentate gyrus, leading to changes in dendritic arbors, dendritic spines numbers, and impaired learning and memory (Kang
+
+The mammalian
+
+target
+
+rapamycin
+
+(mTOR),
+
+kinase,
+
+intra-cellular
+
+and induce
+
+excitatory/inhibitory
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+2
+
+Rapamycin Prevents Neurological Changes
+
+et al., 2017). These changes were prevented by the mTOR pathway inhibitor rapamycin. However, the exact role of mTOR after general anesthesia in late postnatal mice has not been sufficiently evaluated.
+
+To evaluate the role of mTOR signaling following anesthesia in late postnatal mice, PND 16/17 mice were exposed to 2.5% sevoflurane (the most widely used inhalation agent in pediatric patients) for 2 h. Administration of sevoflurane to PND 16/17 mice has been shown to increase dendritic spine formation, to alter mitochondrial function, and to induce sex-dependent changes in excitatory/inhibitory synaptic transmission (Chung et al., 2017a; Ju et al., 2019). Based on previous findings, this study hypothesized that sevoflurane-induced changes in late postnatal mice could be prevented by inhibiting the mTOR pathway with rapamycin.
+
+MATERIALS AND METHODS
+
+Animals All experiments were approved by the relevant Committees of Chungnam National University, Daejeon, South Korea (CNU-01135). C57BL/6J mice were maintained in a specific pathogen-free (SPF) room maintained at 22◦C, with a 12 h light/dark cycle, and fed ad libitum. Animals received anesthesia during the light cycle. This research adheres to the ARRIVE (Animal Research: Reporting in vivo Experiments) guidelines.
+
+Anesthesia PND 16/17 mice were randomly divided into three groups: control, sevoflurane, and sevoflurane plus rapamycin groups. Mice in the sevoflurane and sevoflurane plus rapamycin groups were placed in a 1-l plastic chamber and exposed to a constant flow of fresh gas [fraction of inspired oxygen (FiO2) 0.4, 4 L/min] containing 2.5% sevoflurane for 2 h. Full recovery was confirmed 30 min after discontinuing sevoflurane. Control mice were treated identically but without sevoflurane. The anesthesia chamber was placed in a 36◦C water bath to maintain a constant temperature. Carbon dioxide and sevoflurane were monitored using an S/5 compact anesthetic monitor and a mCAiO gas analyzer module (Datex-Ohmeda, Helsinki, Finland).
+
+Rapamycin Treatment Rapamycin (LC Laboratories, Woburn, MA, USA) was reconstituted in ethanol at a concentration 10 µg/µl and then diluted in 5% Tween-80 (Sigma–Aldrich, St. Louis, MO, USA) and 5% PEG-400 (Sigma–Aldrich, St. Louis, MO, USA), as described (Chen et al., 2009). Mice in the sevoflurane plus rapamycin group were each administered three intraperitoneal injections of rapamycin (5 mg/kg) at 24 h intervals prior to sevoflurane exposure, whereas mice in the control and sevoflurane groups were injected with an identical volume of vehicle.
+
+Western blotting Whole-brain samples were obtained from the mice 24 h after sevoflurane exposure. Mice were exposed to carbon
+
+January 2020 | Volume 14 | Article 4
+
+Ju et al.
+
+dioxide before brain extraction, and each whole brain was homogenized with a tissue grinder in RIPA lysis buffer [ELPIS-BIOTECH, Daejeon, South Korea, 100 mM Tris–hydrochloride (pH 8.5), 200 mM NaCl, 5 mM EDTA, and 0.2% sodium dodecyl sulfate], containing phosphatase and protease (Sigma–Aldrich). After cocktails centrifuging the homogenized samples at 12,000× g for 15 min at 4◦C, the supernatants were decanted and their protein concentrations were measured using the Bradford assay (Bio-Rad, Hercules, CA, USA). Samples (20 µg) were electrophoresed on SDS PAGE gels, and transferred to (pore size, 0.2 µm; Amersham nitrocellulose membranes Protran(cid:114), GE Healthcare, at Buckinghamshire, UK) 200 mA for 2 h. The membranes were blocked for 1 h with Tris-buffered saline-Tween 20 [10 mM Tris–hydrochloride (pH 7.6), 150 mM NaCl, and 0.1% Tween 20], containing followed by incubation 3% bovine serum albumin (BSA), with primary antibodies and the appropriate secondary Specific antibodies antibody-labeled proteins were detected using the enhanced chemiluminescence iNtRON BioTechnology, Seongnam, South Korea). Primary antibodies included antibodies to phospho-mTOR(S2448), mTOR (Cell Signaling Technology, Danvers, MA, USA), postsynaptic density 90 (PSD95; Neuromab, Davis, CA, USA), GAD65 (Abcam, Cambridge, UK), NDUFB8 (a mitochondrial complex I subunit; Santa Cruz Biotechnology, Santa Cruz, TX, USA), COX4 (a mitochondrial complex IV subunit; Novus Biologicals, Centennial, CO, USA) and actin (Santa Cruz Biotechnology, Santa Cruz, TX, USA). Antibodies against GluA1 (1193) and GluA2 (1195) have been described previously (Kim et al., 2009).
+
+inhibitor
+
+coupled
+
+to horseradish peroxidase.
+
+system (WEST-ZOL
+
+plus;
+
+Oxygen Consumption Rate Mitochondria were isolated from brain tissues 24 h after sevoflurane exposure, as previously described (Chung et al., 2017a). Each brain was homogenized in a mitochondrial isolation buffer [70 mM sucrose, 210 mM mannitol, 5 mM HEPES, 1 mM EGTA, and 0.5% (w/v) fatty acid–free BSA (pH 7.2)] with a Teflon-glass homogenizer (Thomas Fisher Scientific, Swedesboro, NJ, USA). After centrifugation at 600× g for 10 min at 4◦C and at 17,000× g for 10 min at 4◦C, the mitochondrial fraction was resuspended in a mitochondrial isolation buffer. Protein concentration was measured by the Bradford assay (Bio-Rad), and 20 µg aliquots of protein were diluted with 50 µl mitochondrial assay solution [70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, 0.2% (w/v) fatty acid–free BSA, 10 mM succinate, and 2 µM rotenone (pH 7.2)] and seeded in an XF-24 plate (Seahorse Bioscience, North Billerica, MA, USA). The plates were centrifuged at 2,000× g for 20 min at 4◦C using a swinging bucket microplate adaptor (Eppendorf, Hamburg, Germany); 450 µl mitochondrial assay buffer was added to each plate, and the plates were maintained at 37◦C for 8–10 min. Each plate was transferred to a Seahorse XF-24 extracellular flux analyzer (Seahorse Bioscience) and the oxygen consumption rate (OCR) was measured at five stages: stage I (basal level);
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+3
+
+Rapamycin Prevents Neurological Changes
+
+following the addition of adenosine diphosphate stage II, (ADP); stage III, following the addition of oligomycin, a mitochondrial oxidative phosphorylation (OXPHOS) complex 5 inhibitor; stage IV, following the addition of carbonyl cyanide m-chlorophenyl hydrazine (CCCP), a mitochondrial OXPHOS complex 4 inhibitor; and stage V, following the addition of antimycin A, a mitochondrial OXPHOS complex 3 inhibitor. OCR was automatically calculated and recorded using Seahorse XF-24 software (Seahorse Bioscience).
+
+Electrophysiology Whole-cell voltage-clamp recordings of pyramidal neurons in the CA1 region of the hippocampus were obtained as described (Chung et al., 2015a). Twenty-four hours after exposure to sevoflurane or fresh gas, sagittal slices of the hippocampus (300 µm) were prepared in ice-cold dissection buffer (212 mM sucrose, 25 mM NaHCO3, 5 mM KCl, 1.25 mM NaH2PO4, 10 mM d-glucose, 2 mM sodium pyruvate, 1.2 mM sodium ascorbate, 3.5 mM MgCl2, and 0.5 mM CaCl2) aerated with 95% O2/5% CO2, using a VT1200S vibratome (Leica, Arrau, Switzerland). Slices were transferred immediately to a 32◦C chamber containing artificial cerebrospinal fluid (aCSF: 125 mM NaCl, 25 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 10 mM d-glucose, 1.3 mM MgCl2, and 2.5 mM CaCl2, continuously aerated with 95% O2/5% CO2) and incubated for 30 min. Glass capillaries were filled with two kinds of internal solutions. For miniature excitatory postsynaptic current (mEPSC) recordings, the glass capillaries were filled with an internal solution containing 117 mM CsMeSO4, 10 mM tetraethylammonium chloride, 8 mM NaCl, 10 mM HEPES, 5 mM QX-314-Cl, 4 mM Mg-adenosine triphosphate (ATP), 0.3 mM Na-guanosine triphosphate, and 10 mM EGTA; for miniature inhibitory postsynaptic current (mIPSC) recordings, the glass capillaries were filled with an internal solution containing 115 mM CsCl, 10 mM tetraethylammonium chloride, 8 mM NaCl, 10 mM HEPES, 5 mM QX-314-Cl, 4 mM Mg-ATP, 0.3 mM Na-guanosine triphosphate, and 10 mM EGTA. Whole-cell recordings were performed under visual control (BX50WI; Olympus, Tokyo, Japan) with a multi clamp 700A amplifier (Molecular Devices, San Jose, CA, USA). Data were acquired with Clampex 9.2 (Molecular Devices, San Jose, CA, USA) and analyzed using Clampfit 9 software (Molecular Devices, San Jose, CA, USA).
+
+Statistical Analysis The sample size was determined based on previous experience or as previously described (Chung et al., 2015b, 2017b). All statistical analyses were performed using R statistical software (3.1.2: R Core Team, Austria). All continuous variables were tested to determine whether they met conditions of normality and homogeneity of variance. One-way ANOVA with post hoc Tukey HSD test was performed when both conditions were met, Welch’s ANOVA with post hoc Tukey HSD test was performed when homogeneity of variance was unmet, and the Kruskal–Wallis test with post hoc Dunn’s test was performed if normality was unmet. P < 0.05 was considered
+
+January 2020 | Volume 14 | Article 4
+
+Ju et al.
+
+Rapamycin Prevents Neurological Changes
+
+FIGURE 1 | Rapamycin prevents sevoflurane-induced activation of mammalian target of rapamycin (mTOR) signaling in male and female postnatal day (PND) 16/17 mice. (A) Time schedule for experiments. PND 14/15 mice were intraperitoneally injected with vehicle or rapamycin once daily for 3 days. On day 3 (PND 16/17), the mice were exposed to 2.5% sevoflurane anesthesia for 2 h. On day 4, mice were sacrificed and their whole brains were extracted. (B–E) Western blot of whole-brain samples obtained 24 h after sevoflurane exposure. (B,D) Western blotting with antibodies specific for phosphorylated and total mTOR and actin (loading control) in male and female mice. (C,E) Quantification of mean ± SD protein band intensity in panels (B,D; n = 4 or 5 per group; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).
+
+statistically significant. Statistical Supplementary Statistics.
+
+results are presented as
+
+RESULTS
+
+Sevoflurane Exposure in PND 16/17 Mice Induces Widespread Activation of the mTOR Signaling Pathway sevoflurane induces widespread activation To assess level of mTOR of in phosphorylation was measured by western blotting whole brain samples obtained 24 h after sevoflurane exposure. We also evaluated whether three daily injections of rapamycin prior to sevoflurane exposure could prevent mTOR phosphorylation (Figure 1A). Because we had found that sevoflurane changes were sex-dependent, mTOR phosphorylation was separately measured in male and female mice. Sevoflurane exposure enhanced phosphorylation of mTOR in both male and female mice, and such phosphorylation was injection sexes (Figures 1B–E).
+
+that
+
+the mTOR signaling pathway,
+
+the
+
+blocked
+
+in
+
+both
+
+by
+
+rapamycin
+
+Rapamycin Treatment Prevents a Sevoflurane-Induced Increase of Mitochondrial Function in PND 16/17 Mice We previously respiration continuously increases for up to 9 h after sevoflurane exposure in PND 16/17 mice (Chung et al., 2017a). Mitochondrial respiration, which is conducted by assembled mitochondrial and nuclear-originated proteins, produces ATP by consuming oxygen. To determine the association between mTOR signaling and sevoflurane-induced changes in mitochondrial respiration, the amounts of the OXPHOS complex subunit proteins NADH:
+
+reported that mitochondrial
+
+Ubiquinone Oxidoreductase Subunit B8 (NDUFB8; subunit of OXPHOS complex I) and cytochrome c oxidase subunit 4 (COX4; subunit of OXPHOS complex IV) were measured 24 h after sevoflurane exposure. Sevoflurane increased the level of NDUFB8, but not of COX4, only in female mice. The increase was inhibited by preinjection with rapamycin (Figures 2A–D). To assess mitochondrial respiration 24 h after sevoflurane exposure, we also measured the OCR in mitochondria isolated from whole brains (Figures 2E–H). Sevoflurane exposure increased basal OCR (stage I) only in female mice, but increased ADP-induced OCR (stage II), oligomycin induced ATP production (stage III), and maximal OCR (stage IV) in both male and female mice. These changes were prevented by rapamycin pretreatment in both male and female mice (Figures 2F,H). Taken together, sevoflurane- induced changes in mitochondrial function in an mTOR dependent manner.
+
+these
+
+results
+
+showed that
+
+Rapamycin Treatment Prevents a Sevoflurane-Induced Increase of AMPA Receptor Subunit GluA2 in PND 16/17 Male Mice We previously reported that a single exposure of PND 16/17 mice to sevoflurane affects the level of expression of synaptic molecules 6 h later (Chung et al., 2017a; Ju et al., 2019). To confirm longer-lasting changes in expression and to determine the role of mTOR signaling, western blotting was performed 24 h after sevoflurane exposure. The expression of GluA2 was significantly increased only in male mice, while there were no significant changes in female mice (Figure 3). The increase in GluA2 expression was inhibited by rapamycin pretreatment, suggesting that the mTOR pathway is also associated with changes in protein expression after exposure to sevoflurane in male mice.
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+4
+
+January 2020 | Volume 14 | Article 4
+
+Ju et al.
+
+Rapamycin Prevents Neurological Changes
+
+FIGURE 2 | Rapamycin prevents sevoflurane-induced increases in mitochondrial function and oxidative phosphorylation (OXPHOS) complexes in PND 16/17 mice. (A–D) Whole-brain samples obtained 24 h after sevoflurane exposure. (A,C) Western blotting with antibodies specific for the OXPHOS complex subunits NDUFB8 (OXPHOS complex I) and COX4 (OXPHOS complex IV) and actin (loading control) in male and female mice. (B,D) Quantification of mean ± SD protein band intensity in panels (A,C; n = 4 or 5 per group; n.s., not significant, ∗p < 0.05, ∗∗∗p < 0.01). (E–H) Oxygen consumption rate (OCR) of mitochondria isolated from the whole brain 24 h after sevoflurane exposure. The substrate was used by adding succinate to the mitochondrial assay buffer. Adenosine diphosphate (ADP) was used to stimulate adenosine triphosphate (ATP) turnover and ATP generation was measured with oligomycin (Oligo). Maximal OCR was assessed using carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and non-mitochondrial OCR was measured using antimycin A (AA; n = 4 or 5 per group; n.s., not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Values are presented as mean ± SD.
+
+Rapamycin Treatment Prevents Sevoflurane-Induced Changes of Excitatory Synaptic Transmission in Male Mice But Does Not Prevent Changes of Inhibitory Synaptic Transmission in Female Mice Exposure of PND 16/17 mice to sevoflurane was shown to induce acute, sex-dependent changes in various brain regions
+
+Ju et al., 2019). To extend these (Chung et al., 2017a; findings, we assessed changes of excitatory/inhibitory synaptic transmission in CA1 pyramidal neurons in the hippocampus 24 h after sevoflurane exposure. Sevoflurane increased mEPSC frequency only in male mice (Figure 4), an increase blocked by preinjection of rapamycin (Figures 4A,B). In contrast, sevoflurane affected mIPSC frequency only in female mice (Figure 5), but these changes were unaffected by preinjection of rapamycin (Figures 5C,D). These results suggest that only the
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+5
+
+January 2020 | Volume 14 | Article 4
+
+Ju et al.
+
+Rapamycin Prevents Neurological Changes
+
+FIGURE 3 | Rapamycin prevents the sevoflurane-induced increase in the expression of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit GluA2 in PND 16/17 male mice. (A–D) Western blotting of whole-brain samples obtained 24 h after sevoflurane exposure for expression of excitatory {postsynaptic density 95 [PSD95], GluA1, GluA2} and inhibitory (GAD65) synaptic proteins. Actin was used as the loading control. (B,D) Mean ± SD protein band intensity in panels (A,C; n = 4 or 5 per group; n.s., not significant, ∗∗p < 0.01).
+
+sex-dependent changes in excitatory synaptic transmission are mTOR dependent.
+
+DISCUSSION
+
+Few studies to date have analyzed the mechanisms underlying changes in synaptic transmission after exposure to anesthesia in late postnatal mice. To gain further insight and to identify a possible molecular target for preventing such anesthesia-induced changes, we focused on the mTOR pathway due to the fact that mTOR signaling regulates mitochondrial function (Ramanathan and Schreiber, 2009; Morita et al., 2015, 2017), dendritic spine formation, AMPA receptor synthesis, and excitatory synaptic transmission (Bockaert and Marin, 2015). Our results indicate that the mTOR pathway is associated with the changes that occur after anesthetic exposure in late postnatal male mice.
+
+function has been shown to be involved with dendritic spine formation and synaptic transmission (Li et al., 2004; Guo et al., 2017; Rossi and Pekkurnaz, increases spinogenesis and induces change in synaptic transmission, we previously suggested the compensatory increase of mitochondria
+
+Neuronal mitochondrial
+
+2019).
+
+Since
+
+anesthetic
+
+exposure
+
+function as a possible mechanism (Chung et al., 2017a). However, we were unable to provide a mechanism by which sevoflurane increased mitochondrial function. Previous studies have suggested that the mTOR complex stimulates the synthesis of mitochondrial components by regulating translation (Morita et al., 2013). In our present study, we also suggest that mTOR regulates in mitochondrial function by showing that rapamycin pretreatment efficiently blocks the sevoflurane-induced increases in mitochondrial OCR in both male and female mice. While our results suggest that mTOR may regulate the sevoflurane-induced neurological changes through mitochondrial activation, many studies also show that mTOR signaling itself is an important regulator of dendritic spine formation, AMPA receptor synthesis, and excitatory synaptic transmission (Bockaert and Marin, 2015). Thus, the anesthesia-induced changes in a mitochondrial-independent fashion as well. However, considering previous evidence function with regarding the significance of mitochondrial neurological changes, it is highly possible that mTOR regulates the sevoflurane-induced changes through both mitochondrial- dependent and independent pathways.
+
+sevoflurane-induced
+
+changes
+
+inhibition of
+
+the mTOR pathway can prevent
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+6
+
+January 2020 | Volume 14 | Article 4
+
+Ju et al.
+
+Rapamycin Prevents Neurological Changes
+
+FIGURE 4 | Rapamycin prevents the increase in excitatory synaptic transmission in PND 16/17 male mice 24 h after sevoflurane exposure. (A,B) Frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in male mice 24 h after sevoflurane exposure with/without rapamycin pretreatment (control: 20 cells from three mice; sevoflurane: 16 cells from three mice; rapamycin: 19 cells from three mice; n.s., not significant, ∗∗p < 0.01). (C,D) Frequency and amplitude of mEPSC in female mice 24 h after sevoflurane exposure with/without rapamycin pretreatment (control: 20 cells from three mice; sevoflurane: 18 cells from three mice; rapamycin: 19 cells from three mice; n.s., not significant). Values are presented as mean ± SD.
+
+variable a in neuroscience (Shansky and Woolley, 2016; Bale and Epperson, 2017; Torres-Rojas and Jones, 2018), and has been shown to affect anesthesia-induced neurotoxicity during neurodevelopment in young animals (Boscolo et al., 2013; Ju et al., 2019). Unfortunately, the majority of clinical studies have been performed with male patients (Lin et al., 2017), and the clinical significance of sex is still in need of further evaluation. Several sex-dependent changes were also discovered in our present study. First, while sevoflurane activates mTOR in both sexes, mTOR-dependent increases in AMPA receptor subunit expression and excitatory synaptic transmission only occurred in male mice. This may be due to sex-dependent differences in the downstream signaling of mTOR. A previous study has shown different downstream activities between male and female mice in non-neural tissues. For instance, when compared to female mice, male mice showed decreased basal mTORC1 activity in the liver and heart tissue, while the basal mTORC2 activity was increased in muscle tissue (Baar et al., 2016). Sevoflurane-induced mTOR activation may result in male-dependent changes due to differences in mTOR downstream signaling in male and female mice. Second, sevoflurane exposure induced different mitochondrial changes
+
+Sex is
+
+recognized as
+
+valuable biological
+
+between sexes. When measured 24 h after sevoflurane exposure, male mice displayed increases of ADP induced OCR, maximal OCR and ATP production without changes in basal OCR and OXPHOS subunit protein levels. However, all stages of the mitochondrial OCR and OXPHOS subunit protein levels were still increased in female mice. Such discrepancies between male and female mice may be due to the differences in mitochondrial respiratory function, morphology, and reactive oxygen species (ROS) homeostasis (Khalifa et al., 2017). Previous studies also show sex-dependent differences in mitochondrial biogenesis after oxygen-glucose deprivation and reoxygenation (Sharma et al., 2014). Another interesting fact is that sevoflurane-induced activation of the mTOR pathway can sex-dependently affect the expression of a specific set of proteins, as female mice show more significant changes for a mitochondrial protein, but male mice display more difference in an excitatory synaptic protein. While this may be due to distinct downstream mTOR signaling between male and female mice as mentioned above, distinct gene expression (sexually dimorphic genes) and epigenetic sex differences may also be involved (Yang et al., 2006; McCarthy and Nugent, 2015).
+
+An important factor in our study was the duration of rapamycin treatment. Initially, the experiments involved a single
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+7
+
+January 2020 | Volume 14 | Article 4
+
+Ju et al.
+
+Rapamycin Prevents Neurological Changes
+
+FIGURE 5 | Rapamycin does not prevent the decrease in inhibitory synaptic transmission in PND 16/17 female mice 24 h after sevoflurane exposure. (A,B) Frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) in male mice 24 h after sevoflurane exposure with/without rapamycin pretreatment (control: 20 cells from four mice; sevoflurane: 22 cells from four mice; rapamycin: 22 cells from four mice; n.s., not significant). (C,D) Frequency and amplitude of mIPSC in female mice 24 h after sevoflurane exposure with/without rapamycin pretreatment (control: 23 cells from four mice; sevoflurane: 21 cells from four mice; rapamycin: 21 cells from four mice; n.s., not significant, ∗p < 0.05, ∗∗p < 0.01).
+
+injection of rapamycin (Supplementary Figure S1). Although this single injection restored the phosphorylation level of mTOR, it did not prevent the sevoflurane-induced increase in mEPSC frequency in male mice. We next used a daily injection of rapamycin (5 mg/kg/day) for three consecutive days based on previous studies (Zeng et al., 2009; Huang et al., 2010; Hartman et al., 2012). Unlike a single injection, multiple rapamycin injections were capable of preventing the sevoflurane- induced increase in excitatory synaptic transmission. mTOR acts by forming two distinct protein complexes, mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). Despite rapamycin being an mTOR inhibitor, prolonged rapamycin treatment is required to block mTORC2. One possible explanation is that blockade of mTORC2 by multiple rapamycin injections is required to block changes in excitatory synaptic transmission.
+
+the female-dependent changes in inhibitory synaptic transmission after sevoflurane exposure seems to be irrelevant of mTOR signaling, as rapamycin pretreatment did not affect inhibitory synaptic transmission in both male and female mice. Also, the changes of inhibitory synaptic transmission observed 24 h
+
+Another
+
+interesting finding of our
+
+study is
+
+that
+
+than the changes after sevoflurane exposure was different observed shortly after anesthesia induction (Chung et al., 2017a; Ju et al., 2019). Whereas mIPSC frequency increased in female mice 6 h after sevoflurane exposure (Ju et al., 2019), mIPSC frequency decreased 24 h after exposure. Genetic mouse models of neurodevelopmental disorders have also shown reversed changes in synaptic transmission during development (Chung et al., 2019). Although these reversed changes may be due to compensation mechanisms, more studies focusing on the mechanism behind time-dependent changes in inhibitory synaptic transmission in female mice are required.
+
+limitations. Although the oxygen concentration during anesthesia was relatively low (FiO2 0.4), thereby avoiding oxygen toxicity, we are unable to rule out the effects of slight changes in arterial carbon dioxide (PaCO2) and blood pH after exposure to sevoflurane for 2 h (Chung et al., 2017a). Another the inconsistency of brain regions among experiments. Western blot and mitochondrial experiments were performed using to confirm widespread whole-brain samples, enabling us changes function. in mTOR signaling and mitochondrial However, electrophysiology experiments were performed using
+
+This study has several
+
+limitation was
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+8
+
+January 2020 | Volume 14 | Article 4
+
+Ju et al.
+
+only hippocampal neurons. We have previously reported that sevoflurane exposure induces similar, but slightly different, changes in different brain regions (Chung et al., 2017a; Ju et al., 2019). Since the importance of sex regarding sevoflurane- induced changes were more thoroughly addressed in the hippocampus, we evaluated the possible sex-dependent effects of rapamycin in the hippocampus (CA1 region).
+
+In conclusion, exposure of PND 16/17 mice to sevoflurane enhanced induces mTOR phosphorylation, excitatory function mitochondrial synaptic transmission. Although further regarding the mechanism behind inhibitory synaptic changes in female mice are necessary, our results suggest that mTOR inhibitors may be potential therapeutic agents for anesthesia-induced changes during neurodevelopment.
+
+to and male-dependent studies
+
+leading
+
+DATA AVAILABILITY STATEMENT
+
+All datasets generated for this study are included in the article/Supplementary Material.
+
+ETHICS STATEMENT
+
+The animal study was reviewed and approved by Committees of Chungnam National University, Daejeon, South Korea (CNU-01135). Written informed consent was obtained
+
+REFERENCES
+
+Baar, E. L., Carbajal, K. A., Ong, I. M., and Lamming, D. W. (2016). Sex- and tissue- specific changes in mTOR signaling with age in C57BL/6J mice. Aging Cell 15, 155–166. doi: 10.1111/acel.12425
+
+Bale, T. L., and Epperson, C. N. (2017). Sex as a biological variable: who, what, when, why and how. Neuropsychopharmacology 42, 386–396. doi: 10.1038/npp. 2016.215
+
+Bockaert, J., and Marin, P. (2015). mTOR in brain physiology and pathologies.
+
+Physiol. Rev. 95, 1157–1187. doi: 10.1152/physrev.00038.2014
+
+Boscolo, A., Ori, C., Bennett, J., Wiltgen, B., and Jevtovic-Todorovic, V. (2013). Mitochondrial protectant pramipexole prevents sex-specific long-term cognitive impairment from early anaesthesia exposure in rats. Br. J. Anaesth. 110, i47–52. doi: 10.1093/bja/aet073
+
+Briner, A., De Roo, M., Dayer, A., Muller, D., Habre, W., and Vutskits, L. (2010). Volatile anesthetics rapidly increase dendritic spine density in the rat medial prefrontal cortex during synaptogenesis. Anesthesiology 112, 546–556. doi: 10.1097/aln.0b013e3181cd7942
+
+Cammalleri, M., Lutjens, R., Berton, F., King, A. R., Simpson, C., Francesconi, W., et al. (2003). Time-restricted role for dendritic activation of the mTOR-p70S6K pathway in the induction of late-phase long-term potentiation in the CA1. Proc. Natl. Acad. Sci. U S A 100, 14368–14373. doi: 10.1073/pnas. 2336098100
+
+Chen, C., Liu, Y., Liu, Y., and Zheng, P. (2009). mTOR regulation and therapeutic rejuvenation of aging hematopoietic stem cells. Sci. Signal. 2:ra75. doi: 10.1126/scisignal.2000559
+
+Chung, C., Ha, S., Kang, H., Lee, J., Um, S. M., Yan, H., et al. (2019). Early correction of N-Methyl-D-Aspartate receptor function improves autistic-like social behaviors in adult Shank2−/− mice. Biol. Psychiatry 85, 534–543. doi: 10.1016/j.biopsych.2018.09.025
+
+Chung, W., Choi, S. Y., Lee, E., Park, H., Kang,
+
+J., Park, H., et al. (2015a). Social deficits in IRSp53 mutant mice improved by NMDAR and mGluR5 suppression. Nat. Neurosci. 18, 435–443. doi: 10.1038/nn.3927
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+9
+
+Rapamycin Prevents Neurological Changes
+
+from the owners for the participation of their animals in this study.
+
+AUTHOR CONTRIBUTIONS
+
+XJ and MR: helped design, development, execution of experiments and preparation of manuscript. SP and BH: helped perform the statistics. JC, YL and SY: helped execution of experiments. WL, YS, S-HY, GK, YHK and YK: helped design and development of experiments, oversight. WC and JH: helped design, development and execution of experiments, oversight, preparation of manuscript.
+
+FUNDING
+
+This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT) (NRF-2018R1C1B6003139, NRF-2017R1A5A2015385, NRF-2019M3E5D1A02068575) and by the research fund of Chungnam national university hospital (2019-1569-01).
+
+SUPPLEMENTARY MATERIAL
+
+The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel.2020.000 04/full#supplementary-material.
+
+Chung, W., Park, S., Hong, J., Park, S., Lee, S., Heo, J., et al. (2015b). Sevoflurane exposure during the neonatal period induces long-term memory impairment but not autism-like behaviors. Paediatr. Anaesth. 25, 1033–1045. doi: 10.1111/pan.12694
+
+Chung, W., Ryu, M.
+
+J. Y., Lee, S., Yoon, S., Park, H., et al. (2017a). Sevoflurane exposure during the critical period affects synaptic transmission and mitochondrial respiration but not long-term behavior in mice. Anesthesiology 126, 288–299. doi: 10.1097/aln.00000000000 01470
+
+J., Heo,
+
+Chung, W., Yoon, S., and Shin, Y. S.
+
+(2017b). Multiple exposures of sevoflurane during pregnancy induces memory impairment in young female offspring mice. Korean J. Anesthesiol. 70, 642–647. doi: 10.4097/kjae.2017. 70.6.642
+
+Guo, L., Tian, J., and Du, H. (2017). Mitochondrial dysfunction and synaptic transmission failure in Alzheimer’s disease. J. Alzheimers Dis. 57, 1071–1086. doi: 10.3233/jad-160702
+
+Hartman, A. L., Santos, P., Dolce, A., and Hardwick, J. M. (2012). The mTOR inhibitor rapamycin has limited acute anticonvulsant effects in mice. PLoS One 7:e45156. doi: 10.1371/journal.pone.0045156
+
+Huang, X., Zhang, H., Yang, J., Wu, J., Mcmahon, J., Lin, Y., et al. (2010). Pharmacological inhibition of the mammalian target of rapamycin pathway suppresses acquired epilepsy. Neurobiol. Dis. 40, 193–199. doi: 10.1016/j.nbd. 2010.05.024
+
+Huber, K. M., Klann, E., Costa-Mattioli, M., and Zukin, R. S. (2015). Dysregulation of mammalian target of rapamycin signaling in mouse models of autism. J. Neurosci. 35, 13836–13842. doi: 10.1523/jneurosci.2656-15.2015
+
+Jevtovic-Todorovic, V. (2018). Exposure of developing brain to general anesthesia: what is the animal evidence? Anesthesiology 128, 832–839. doi: 10.1097/aln. 0000000000002047
+
+Ju, X., Jang, Y., Heo, J. Y., Park, J., Yun, S., Park, S., et al. (2019). Anesthesia affects excitatory/inhibitory synapses during the critical synaptogenic period in the hippocampus of young mice: importance of sex as a biological variable. Neurotoxicology 70, 146–153. doi: 10.1016/j.neuro.2018.11.014
+
+January 2020 | Volume 14 | Article 4
+
+Ju et al.
+
+Kang, E., Jiang, D., Ryu, Y. K., Lim, S., Kwak, M., Gray, C. D., et al. (2017). Early postnatal exposure to isoflurane causes cognitive deficits and disrupts development of newborn hippocampal neurons via activation of the mTOR pathway. PLoS Biol. 15:e2001246. doi: 10.1371/journal.pbio.2001246
+
+Khalifa, A. R., Abdel-Rahman, E. A., Mahmoud, A. M., Ali, M. H., Noureldin, M., Saber, S. H., et al. (2017). Sex-specific differences in mitochondria biogenesis, morphology, respiratory function and ROS homeostasis in young mouse heart and brain. Physiol. Rep. 5:e13125. doi: 10.14814/phy2.13125
+
+Kim, M. H., Choi,
+
+J. H., Paik, S. K., et al. (2009). Enhanced NMDA receptor-mediated synaptic transmission, enhanced long-term potentiation and impaired learning and memory in mice lacking IRSp53. J. Neurosci. 29, 1586–1595. doi: 10.1523/jneurosci.4306- 08.2009
+
+J., Yang,
+
+J., Chung, W., Kim,
+
+Lee, C. C., Huang, C. C., and Hsu, K. S. (2011). Insulin promotes dendritic spine and synapse formation by the PI3K/Akt/mTOR and Rac1 signaling pathways. Neuropharmacology 61, 867–879. doi: 10.1016/j.neuropharm.2011.06.003
+
+Lee, E., Lee,
+
+(2017). Excitation/inhibition imbalance in animal models of autism spectrum disorders. Biol. Psychiatry 81, 838–847. doi: 10.1016/j.biopsych.2016.05.011
+
+J., and Kim, E.
+
+Li, N., Lee, B., Liu, R. J., Banasr, M., Dwyer, J. M., Iwata, M., et al. (2010). mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists. Science 329, 959–964. doi: 10.1126/science.1190287 Li, X., Wu, Z., Zhang, Y., Xu, Y., Han, G., and Zhao, P. (2017). Activation of autophagy contributes to sevoflurane-induced neurotoxicity in fetal rats. Front. Mol. Neurosci. 10:432. doi: 10.3389/fnmol.2017.00432
+
+Li, Z., Okamoto, K., Hayashi, Y., and Sheng, M. (2004). The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses. Cell 119, 873–887. doi: 10.1016/j.cell.2004.11.003
+
+Lin, E. P., Lee, J. R., Lee, C. S., Deng, M., and Loepke, A. W. (2017). Do anesthetics harm the developing human brain? An integrative analysis of animal and human studies. Neurotoxicol. Teratol. 60, 117–128. doi: 10.1016/j.ntt.2016. 10.008
+
+McCann, M. E., De Graaff, J. C., Dorris, L., Disma, N., Withington, D., Bell, G., et al. (2019). Neurodevelopmental outcome at 5 years of age after general anaesthesia or awake-regional anaesthesia in infancy (GAS): an international, multicentre, randomised, controlled equivalence trial. Lancet 393, 664–677. doi: 10.1016/S0140-6736(18)32485-1
+
+McCarthy, M. M., and Nugent, B. M. (2015). At the frontier of epigenetics of brain sex differences. Front. Behav. Neurosci. 9:221. doi: 10.3389/fnbeh.2015. 00221
+
+Meredith, R. M. (2015). Sensitive and critical periods during neurotypical and aberrant neurodevelopment: a framework for neurodevelopmental disorders. Neurosci. Biobehav. Rev. 50, 180–188. doi: 10.1016/j.neubiorev.2014.12.001 Morita, M., Gravel, S. P., Chenard, V., Sikstrom, K., Zheng, L., Alain, T., et al. (2013). mTORC1 controls mitochondrial activity and biogenesis through 4E- BP-dependent translational regulation. Cell Metab. 18, 698–711. doi: 10.1016/j. cmet.2013.10.001
+
+Morita, M., Gravel, S. P., Hulea, L., Larsson, O., Pollak, M., St-Pierre, J., et al. (2015). mTOR coordinates protein synthesis, mitochondrial activity and proliferation. Cell Cycle 14, 473–480. doi: 10.4161/15384101.2014.991572 Morita, M., Prudent, J., Basu, K., Goyon, V., Katsumura, S., Hulea, L., et al. (2017). mTOR controls mitochondrial dynamics and cell survival via MTFP1. Mol. Cell 67, 922.e925–935.e925. doi: 10.1016/j.molcel.2017.08.013
+
+O’Leary, J. D., and Warner, D. O. (2017). What do recent human studies the association between anaesthesia in young children 458–464.
+
+tell us about and neurodevelopmental outcomes? Br. doi: 10.1093/bja/aex141
+
+J. Anaesth.
+
+119,
+
+Olutoye, O. A., Sheikh, F., Zamora, I. J., Yu, L., Akinkuotu, A. C., Adesina, A. M., et al. (2016). Repeated isoflurane exposure and neuroapoptosis in the midgestation fetal sheep brain. Am. J. Obstet. Gynecol. 214, 542.e541–542.e548. doi: 10.1016/j.ajog.2015.10.927
+
+Ramanathan, A., and Schreiber, S. L. (2009). Direct control of mitochondrial function by mTOR. Proc. Natl. Acad. Sci. U S A 106, 22229–22232. doi: 10.1073/pnas.0912074106
+
+Frontiers in Cellular Neuroscience | www.frontiersin.org
+
+10
+
+Rapamycin Prevents Neurological Changes
+
+Ran, I., Gkogkas, C. G., Vasuta, C., Tartas, M., Khoutorsky, A., Laplante, I., et al. (2013). Selective regulation of GluA subunit synthesis and AMPA
+
+receptor-mediated synaptic function and plasticity by the translation repressor 4E-BP2 in hippocampal pyramidal cells. J. Neurosci. 33, 1872–1886. doi: 10.1523/jneurosci.3264-12.2013
+
+Rossi, M. J., and Pekkurnaz, G. (2019). Powerhouse of the mind: mitochondrial plasticity at the synapse. Curr. Opin. Neurobiol. 57, 149–155. doi: 10.1016/j. conb.2019.02.001
+
+Shansky, R. M., and Woolley, C. S. (2016). Considering sex as a biological variable will be valuable for neuroscience research. J. Neurosci. 36, 11817–11822. doi: 10.1523/jneurosci.1390-16.2016
+
+Sharma, J., Johnston, M. V., and Hossain, M. A. (2014). Sex differences in mitochondrial biogenesis determine neuronal death and survival in response to oxygen glucose deprivation and reoxygenation. BMC Neurosci. 15:9. doi: 10.1186/1471-2202-15-9
+
+Tang, S. J., Reis, G., Kang, H., Gingras, A. C., Sonenberg, N., and Schuman, E. M. (2002). A rapamycin-sensitive signaling pathway contributes to long-term synaptic plasticity in the hippocampus. Proc. Natl. Acad. Sci. U S A 99, 467–472. doi: 10.1073/pnas.012605299
+
+Tavazoie, S. F., Alvarez, V. A., Ridenour, D. A., Kwiatkowski, D. J., and Sabatini, B. L. (2005). Regulation of neuronal morphology and function by the tumor suppressors Tsc1 and Tsc2. Nat. Neurosci. 8, 1727–1734. doi: 10.1038/nn1566
+
+Torres-Rojas, C., and Jones, B. C. (2018). Sex differences in neurotoxicogenetics.
+
+Front. Genet. 9:196. doi: 10.3389/fgene.2018.00196
+
+Wang, Y., Barbaro, M. F., and Baraban, S. C. (2006). A role for the mTOR pathway in surface expression of AMPA receptors. Neurosci. Lett. 401, 35–39. doi: 10.1016/j.neulet.2006.03.011
+
+Warner, D. O., Zaccariello, M. J., Katusic, S. K., Schroeder, D. R., Hanson, A. C., Schulte, P. J., et al. (2018). Neuropsychological and behavioral outcomes after exposure of young children to procedures requiring general anesthesia: the mayo anesthesia safety in kids (MASK) study. Anesthesiology 129, 89–105. doi: 10.1097/ALN.0000000000002232
+
+Workman, A. D., Charvet, C. J., Clancy, B., Darlington, R. B., and Finlay, B. L. (2013). Modeling transformations of neurodevelopmental sequences across mammalian species. J. Neurosci. 33, 7368–7383. doi: 10.1523/JNEUROSCI. 5746-12.2013
+
+Yang, X., Schadt, E. E., Wang, S., Wang, H., Arnold, A. P., Ingram-Drake, L., et al. (2006). Tissue-specific expression and regulation of sexually dimorphic genes in mice. Genome Res. 16, 995–1004. doi: 10.1101/gr.5217506
+
+Zaccariello, M. J., Frank, R. D., Lee, M., Kirsch, A. C., Schroeder, D. R., Hanson, A. C., et al. (2019). Patterns of neuropsychological changes after the mayo general anaesthesia in young children: secondary analysis of anesthesia safety in kids study. Br. J. Anaesth. 122, 671–681. doi: 10.1016/j.bja. 2019.01.022
+
+Zeng, L. H., Rensing, N. R., and Wong, M. (2009). The mammalian target of rapamycin signaling pathway mediates epileptogenesis in a model of temporal lobe epilepsy. J. Neurosci. 29, 6964–6972. doi: 10.1523/jneurosci.0066-09.2009 J., Wang, C., Yu, S., Luo, Z., Chen, Y., Liu, Q., et al. (2014). Sevoflurane postconditioning protects rat hearts against ischemia-reperfusion injury via the activation of PI3K/AKT/mTOR signaling. Sci. Rep. 4:7317. doi: 10.1038/srep07317
+
+Zhang,
+
+Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
+
+Copyright © 2020 Ju, Ryu, Cui, Lee, Park, Hong, Yoo, Lee, Shin, Yoon, Kweon, Kim, Ko, Heo and Chung. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
+
+January 2020 | Volume 14 | Article 4

new_pdfs/10.3892_etm.2017.5651.txt

@@ -0,0 +1,221 @@
+2066
+
+EXPERIMENTAL AND THERAPEUTIC MEDICINE 15: 2066-2073, 2018
+
+Influence of sevoflurane exposure on mitogen‑activated protein kinases and Akt/GSK‑3β/CRMP‑2 signaling pathways in the developing rat brain
+
+YAFANG LIU1*, CHUILIANG LIU2*, MINTING ZENG3, XUE HAN1, KUN ZHANG1, YANNI FU1, JUE LI1 and YUJUAN LI1
+
+1Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510120; 2Department of Anesthesiology, Chancheng Center Hospital, Guangdong Medical College, Foshan, Guangdong 528030; 3Department of Anesthesiology, Guangzhou Women and Children's Medical Centre of Guangzhou Medical University, Guangzhou, Guangdong 510523, P.R. China
+
+Received October 19, 2016; Accepted October 20, 2017
+
+DOI: 10.3892/etm.2017.5651
+
+Abstract. Prolonged exposure to volatile anesthetics causes neurodegeneration in developing animal brains. However, their underlying mechanisms of action remain unclear. The current study investigated the expression of proteins associated with the mitogen-activated protein kinases (MAPK) and protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β)/collapsin response mediator protein 2 (CRMP-2) signaling pathways in the cortices of neonatal mice following exposure to sevoflurane. Seven-day-old (P7) neonatal C57BL/6 mice were randomly divided into 2 groups and either exposed to 2.6% sevoflurane or air for 6 h. Terminal deoxyribonucleotide transferase medi- ated dUTP nick end labeling (TUNEL) staining, as well as the expression of activated caspase-3 and α-fodrin, was used to detect neuronal apoptosis in the cortices of mice. MAPK signaling pathways were investigated by detecting the expres- sion of phosphorylated (p-) extracellular signal-regulated kinase 1/2 (ERK1/2), p-cyclic adenosine monophosphate response element-binding protein (CREB), p-p38, p-nuclear factor (NF-κB) and p-c-Jun N-terminal kinase (p-JNK). Akt/GSK-3β/CRMP-2 signaling pathways were assessed by detecting the expression of p-Akt, p-GSK-3β and p-CRMP-2 in the cortices of P7 mice 2 h following exposure to sevoflu- rane. The results demonstrated that sevoflurane significantly
+
+increased the apoptosis of cells in the retrosplenial cortex (RS), frontal cortex (FC) and parietal association cortex (PtA), increased the expression of cleaved caspase-3 expression and promoted the formation of 145 kDa and 120 kDa fragments from α‑fodrin. Sevoflurane inhibited the phosphorylation of ERK1/2 and CREB, stimulated the phosphorylation of p38 and NF-κB, but did not significantly affect the phosphorylation of JNK. Furthermore, sevoflurane inhibited the phosphorylation of Akt, decreased the phosphorylation of GSK-3β at ser9 and increased the phosphorylation of CRMP2 at Thr514. These results suggest that multiple signaling pathways, including ERK1/2, P38 and Akt/GSK-3β/CRMP-2 may be involved in sevoflurane‑induced neuroapoptosis in the developing brain.
+
+Introduction
+
+Exposure to general anesthetics during brain development may induce widespread apoptotic neurodegeneration in various mammalian species (1-4). Sevoflurane is an inhaled anesthetic commonly used in the clinic, particularly in pediatric medicine, due to its minimal airway reactivity and low blood/gas partition coefficient (5). Previous studies have indicated that sevoflurane causes biochemical changes, including apoptosis, amyloid-β accumulation and neuroinflammation in the hippocampus or cortex, and induces hippocampus-dependent and -independent cognitive dysfunction in developing mice (4,6,7). However, its underlying mechanisms of action remain unknown.
+
+Correspondence to: Professor Yujuan Li or Dr Jue Li, Department of Anesthesiology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107 Yanjiang West Road, Guangzhou, Guangdong 510120, P.R. China E-mail: yujuan_04@hotmail.com E-mail: leejue@foxmail.com
+
+Contributed equally
+
+Key words: apoptosis, sevoflurane, mitogen-activated protein kinase, protein kinase B, developing brain
+
+Mitogen-activated protein kinases (MAPKs) are a family of serine-threonine protein kinases that consist of three major members: Extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK and c-Jun N-terminal kinases (JNK) (8). MAPK signaling cascades serve crucial cellular roles under normal and pathological conditions, including nervous system develop- ment and neurodegeneration (9,10). Activation of the JNK and p38 pathways may contribute to apoptosis whereas the activa- tion of ERK1/2 induces cell survival following central nervous system injury (11). ERK1/2-dependent phosphorylation of the cyclic adenosine monophosphate response element-binding protein (CREB) may lead to the transcriptional upregulation
+
+LIU et al: INFLUENCE OF SEVOFLURANE ON MAPK AND Akt/GSK-3β/CRMP-2 SIGNALING PATHWAYS
+
+of the anti-apoptotic proteins Bcl-2 and brain-derived neuro- trophic factor, which promote the survival and differentiation of neurons (12,13). It has been demonstrated that the transient suppression of ERK phosphorylation in neonatal mice causes marked apoptosis of brain cells and has profound long-term effects on brain function, including a reduction in long-term potentiation and memory impairments, and exhibiting no pref- erence for interacting with animate vs. inanimate objects (14). Previous studies demonstrated that the inhaled anesthetic isoflurane suppresses ERK phosphorylation and increases the phosphorylation of p38, MAPK and JNK in the hippo- campus of neonatal rats. Additionally, isoflurane increases neuronal apoptosis by activating the JNK and p38 MAPK pathways (15,16). However, it remains unclear exactly how sevoflurane affects the MAPK pathway.
+
+Glycogen synthase kinase-3β (GSK-3β) functions in a wide range of cellular processes, including cell proliferation, differentiation, motility and apoptosis (17-19). It is one of the most important downstream targets of the protein kinase B (Akt) signaling pathway. Akt phosphorylating serine at posi- tion 9 in GSK-3β inhibits GSK-3β activity (20). In neurons, GSK-3β is involved in neuronal microtubule dynamics and determines axon/dendrite polarity by phosphorylating the downstream targets of GSK-3β, such as collapsin response mediator protein 2 (CRMP-2) (17,21,22). CRMP2 is involved in neuronal differentiation and axon growth via the binding of CRMP-2 and tubulin, which promotes microtubule assembly (23). In cultured neurons, CRMP2 has been demon- strated to be critical in axon specification, elongation and branching, thereby establishing and maintaining neuronal polarity (23). CRMP-2 has also been proved to co-localize with Numb and regulate Numb-mediated endocytosis, which is associated with axon growth (24). The binding of CRMP-2 to tubulin is inhibited following the phosphorylation of CRMP-2 by GSK-3β (21). Furthermore, it has been demon- strated that the Akt/GSK-3β/CRMP-2 signaling pathway serves important roles in the establishment of axonal-dendritic polarity in vitro (21) and in mediating axonal injury in the neonatal rat brain following hypoxia-ischemia in vivo (22). The inhibition of Akt signaling serves a critical role in isoflurane-induced neuroapoptosis in developing rats (25). Tao et al (26) also demonstrated that sevoflurane anesthesia stimulates Tau phosphorylation and activates GSK-3β in the hippocampus of young mice, causing cognitive impairment. However, it remains unknown how sevoflurane affects the Akt/GSK-3β/CRMP-2 pathway.
+
+To determine the molecular mechanisms of neurotoxicity induced by anesthesia with sevoflurane, the current study investigated changes in the expression of proteins in the MAPK and Akt/GSK-3β/CRMP-2 signaling pathways in the cortices of 7-day-old neonatal mice.
+
+Materials and methods
+
+Animals. The current study was approved by the Animal Care Committee at Sun Yat-sen University (Guangzhou, China) and performed in accordance with the National Institutes of Health Guide for the Use of Laboratory Animals (27). A total of 24 C57BL/6 male mouse pups, aged 7 days (P7) and weighing 3.5-4.5 g were obtained from Guangdong Medical
+
+Laboratory Animal Center (Guangdong, China; permission no. SCXK2011-0029). The pups were housed in the same cage as their mothers and were kept under temperature-controlled environmental conditions (26˚C) on a 14:10 constant light‑dark cycle until P7. The mother mice had free access to food and water. The mouse pups at P7 were exposed to 2.6% sevo- flurane (Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) for 6 h [~1.0 minimal alveolar concentration (MAC) in P7 mice] in 50% oxygen in a temperature-controlled chamber, following a previously described protocol (n=12) (17). The control mice were exposed to normal air for 6 h under the same condition (n=12). The concentrations of anesthetic gas, oxygen and carbon dioxide in the chamber were measured using a gas analyzer (Datex-Ohmeda; GE Healthcare, Chicago, IL, USA). All animals were sacrificed 2 h following termination of sevoflurane/oxygen exposure and their cortices were used for western blotting (sevoflurane group, n=6; control group, n=6) or TdT-mediated dUTP nick end labeling (TUNEL) with fluorescent dye (sevoflurane group, n=6; control group, n=6).
+
+Tissue preparation. Half of the mice in each group were used for western blotting and half of the mice for TUNEL studies. For western blotting, mouse pups were anaesthetized by inhaling 3% of sevoflurane until loss of the righting reflex (LORR), which indicated the mice had lost consciousness. Then the mice were sacrificed by decapitation. Cortices were isolated immediately on ice and then stored at ‑80˚C until use. For TUNEL studies, mouse pups were sacrificed by inhaling 3% of sevoflurane until LORR and perfused transcardially with ice-cold normal saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min at 4˚C. Their brains were post‑fixed in the same fixative for 48 h at 4˚C, and then paraffin embedded and sectioned into 6‑µm‑thick sections. As described in previous studies (15,16,25), at least three sections in the same plane of the hippocampus for each animal were selected to detect cells that exhibited positive TUNEL staining; all sections used in TUNEL were 100 µm apart and the sections were according to Figures 129-131 in the Atlas of the Developing Mouse Brain (28).
+
+Western blotting. Western blotting was performed as previ- ously described (15,16,25). Briefly, the protein concentration in each sample was determined using a BCA protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Sample proteins (40 µg/lane) were separated on 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology, Shanghai, China) in Tris-buffered saline with Tween-20 (TBST) at room tempera- ture for 1 h. Membranes were subsequently incubated at 4˚C overnight with the following primary antibodies: Anti-cleaved caspase-3 (cat no. 9664) at 1:2,000 dilution, anti-α-fodrin (which contain SBDP145 and SBDP120 fragments; cat no. 2122) at 1:2,000 dilution, anti-phosphorylated-(p)-JNK (cat no. 4668) at 1:2,000 dilution, anti-JNK (cat no. 9252) at 1:2,000 dilution, anti-p-ERK1/2 (cat no. 4376) at 1:1,000 dilu- tion, anti-ERK1/2 (cat no. 4695) at 1:1,000 dilution, anti-p-P38 (cat no. 4631) at 1:1,000 dilution, anti-P38 (cat no. 9212) at 1:1,000 dilution, anti-p-CREB (cat no. 9198) at 1:1,000 dilu- tion, anti-p-nuclear factor-κB (NF-κB) (cat no. 3033) at 1:1,000
+
+2067
+
+2068
+
+EXPERIMENTAL AND THERAPEUTIC MEDICINE 15: 2066-2073, 2018
+
+Figure 1. Sevoflurane increased the number of TUNEL positive cells in the cortices of P7 mice. Representative images of TUNEL staining in the (A) RS and FC regions of the cortices. Green staining indicated TUNEL‑positive cells, blue staining indicated nuclear staining (magnification, x100). The white arrows indicate the TUNEL‑positive cells. (B) Quantification of TUNEL positive cells in the RS, FC and PtA regions of the cortices. All results are presented as the mean ± standard deviation of the mean (n=6). ***P<0.001 vs. CON. TUNEL, Terminal deoxyribonucleotide transferase mediated dUTP nick end labeling; Con, control group; Sevo, sevoflurane group; RS, retrosplenial cortex; FC, frontal cortex; PtA, parietal association cortex; P7, 7‑day‑old neonatal mice.
+
+dilution, anti-p-Akt (Ser 473) (cat no. 4060) at 1:2,000 dilu- tion, anti-Akt (cat no. 4685) at 1:5,000 dilution, anti-p-GSK-3β (Ser 9) (cat no. 5558) at 1:2,000 dilution, anti-GSK-3β (cat no. 9315) at 1:2,000 dilution, anti-p-CRMP-2 (Thr 514) (cat no. 9397) at 1:2,000 dilution, anti-CRMP-2 (cat no. 9393) at 1:2,000 dilution and anti-β-actin (cat no. 3700) at 1:2,000 dilution (all Cell Signaling Technology, Inc., Danvers, MA, USA) and anti-p-GSK-3β (Ty 216) (cat no. ab75745; Abcam, Cambridge, USA) at 1:2,000 dilution. The membranes were washed with TBST three times and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse IgG, cat no. A0216; goat anti-rabbit IgG, cat no. A0208; 1:2,000; Beyotime Institute of Biotechnology) at room temperature for 1 h. The membranes were washed with TBST three times and visualized using an enhanced chemiluminescence detection system (cat no. 34580; Thermo Fisher Scientific, Inc.). Images were scanned using an Image Master II scanner (GE Healthcare) and were analyzed using Image Quant TL software (v2003.03, GE Healthcare). The band signals of p-ERK1/2, p-JNK, p-p38, p-Akt, p-GSK-3 and p-CRMP-2 were normalized to the bands of total ERK1/2, JNK, p38, Akt, GSK-3β and CRMP-2 from the same samples. The band signals of the other proteins were normalized to those of β-actin and the results in each group were normalized to that of the corresponding control group.
+
+solution and mounted on glass coverslips with clear nail polish sealing the edges. Slides were protected from direct light during the experiment. The images of TUNEL positive cells in the retrosplenial cortex (RS), frontal cortex (FC) and parietal association cortex (PtA) areas were acquired by Ti-S inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan) and analyzed using NIS-Elements Basic Research imaging processing and analysis software (version 3.0; Nikon Corporation). The density of TUNEL positive cells in the three cortical regions was calculated by dividing the number of TUNEL positive cells by the area of that brain region.
+
+Statistical analysis. Sample size was calculated using PASS 11 software (NCSS, LLC, Kaysville, UT, USA) to achieve 80% power at a significance level of P<0.05. All data were determined to be normally distributed using the Shapiro‑Wilk test and had no significant heterogeneity of variance as detected by Levene's test. GraphPad Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA) was used to conduct all statistical analyses. Data were presented as mean ± standard deviation and were analyzed by Student's t‑test. P<0.05 was considered to indicate a statistically significant difference.
+
+Results
+
+TUNEL assay. TUNEL was performed following a previ- ously described protocol (15,16). A Dead End™ fluorometric TUNEL system (Promega Corporation, Madison, WI, USA) was used and staining following the manufacturer's protocol. Briefly, TUNEL labeling was conducted with a mix of 45 µl equilibration buffer, 5 µl nucleotide mix and 1 µl recombi- nant terminal deoxynucleotidyl transferase (rTdT) enzyme in a humidified, lucifugal chamber for 1 h at 37˚C, and then Hoechst 33258 (H-33258; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to stain nuclei for 10 min at room temperature. The sections were protected by anti-Fade
+
+Sevoflurane induces neuroapoptosis by activating caspase‑3 and calpain in the cortices of developing mice. The results of preliminary experiments for arterial blood gas monitoring in the current study demonstrated that neonatal mice exhibited no hypoglycemia and acidosis during sevoflurane exposure. Neuronal apoptosis in the cortical RS, FC and PtA regions of P7 mice were detected by TUNEL (Fig. 1). Sevoflurane increased the number of apoptotic cells by 338.37% in RS, 409.78% in FC and 360.94% in PtA compared with controls (all P<0.001). In addition, changes in the expres- sion of cleaved caspase-3 and α-fodrin (α-II-Spectrin) in the
+
+LIU et al: INFLUENCE OF SEVOFLURANE ON MAPK AND Akt/GSK-3β/CRMP-2 SIGNALING PATHWAYS
+
+Figure 2. Sevoflurane increased the expression of cleaved caspase‑3, SBDP145 and SBDP120 fragments of α-fodrin in the cortices of P7 mice. Representative western blots of (A) cleaved caspase-3 and (B) α-fodrin. Quantitative analysis of (C) cleaved caspase-3, (D) SBDP145 and (E) SBDP120 expression. All results are presented as the mean ± standard deviation of the mean (n=6). **P<0.01 and ***P<0.001, vs. Con. CON, control group; Sevo, sevoflurane group; P7, 7‑day‑old neonatal mice.
+
+Figure 3. Sevoflurane inhibited the phosphorylation of ERK1/2 and CREB, increased the phosphorylation of p38 and NF‑κB, and did not alter JNK activa- tion in the cortex of P7 mice. Representative Western blots of (A) ERK1/2, p-ERK1/2 and p-CREB, (D) p-p38, P-38 and p-NF-κB and (G) JNK and p-JNK. Quantitative analysis of (B) p-ERK1/2 (44 and 42 kDa), (C) p-CREB, (E) p-p38, (F) p-NF-κB (F) and (H) p-JNK. The results are presented as the mean ± stan- dard deviation of the mean (n=6). *P<0.05, **P<0.01, ***P<0.001 vs. CON. CON, control; SEVO, sevoflurane; NF‑κB, nuclear factor κB; p-, phosphorylated; CREB, cyclic adenosine monophosphate response element-binding protein; JNK, c-Jun N-terminal kinase; ERK1/2, extracellular signal-regulated kinase 1/2.
+
+cortices of mice were assessed by western blotting (Fig. 2). Sevoflurane anesthesia significantly increased the expression of cleaved caspase‑3 protein expression by 11.66‑fold. (P<0.01;
+
+Fig. 2A and C). To examine whether sevoflurane anesthesia influences calpain activity, the expression of α-fodrin in the cortex was measured. Cleavage of the 320 kDa full-length
+
+2069
+
+2070
+
+EXPERIMENTAL AND THERAPEUTIC MEDICINE 15: 2066-2073, 2018
+
+Figure 4. Sevoflurane inhibited the activity of the Akt/GSK‑3β/CRMP2 pathway. Sevoflurane reduced the phosphorylation of Akt and GSK‑3β (Ser 9) and increased phosphorylation of GSK-3β (Ty216) and CRMP2 (Thr 514) in the cortex of P7 mice. (A) Representative western blots of p-Akt, p-GSK-3β (Ser 9), p-GSK-3β (Ty216) and p-CRMP2 (Thr 514); Quantitative analysis of (B) p-Akt, (C) p-GSK-3β (Ser 9), (D) p-GSK-3β (Ty216) and (E) p-CRMP2 (Thr 514). Results are presented as the mean ± standard deviation of the mean (n=6). **P<0.01 vs. Con. P7, 7‑day‑old neonatal mice; Con, control group; Sevo, sevoflurane group; p-, phosphorylated; GSK-3β, glycogen synthase kinase-3β; Akt, protein kinase B; CRMP2, collapsin response mediator protein 2.
+
+α-fodrin by calpain leads to the formation of the 145 kDa frag- ment (known as Spectrin breakdown product 145; SBPD145), which served as a relative measure of calpain activity, whereas the appearance of an additional 120 kDa fragment (also known as Spectrin breakdown product 120; SBPD120) served as an indicator of caspase-3 activity (29). The amount of the 145 kDa and 120 kDa protein fragment significantly increased by 140.1% (P<0.01; Fig. 2B and D) and 324.3% (P<0.001; Fig. 2B and E), respectively, immediately following termina- tion of sevoflurane anesthesia.
+
+cortex following anesthesia with sevoflurane. By contrast, sevoflurane significantly increased the expression of p‑p38 by 204.5% (P<0.001; Fig. 3D and E) and its downstream substrate p-NF-κB by 58.9% (P<0.01; Fig. 3D and F). The expression of p-JNK remained unchanged in the cortices following exposure to sevoflurane (P=0.0665; Fig. 3G and H). Furthermore, the expression of ERK1/2, JNK and p38 exhibited no significant differences between sevoflurane and control rats (data not shown).
+
+The effect of sevoflurane on MAPK signaling pathways. Sevoflurane significantly decreased the expression of p-ERK1/2 at 44 kD by 73.9% and p-ERK1/2 at 42 kD by 47.0% (P<0.001; Fig. 3A and B). To further analyze ERK1/2 activity, the phosphorylation of CREB, one of the substrates of ERK1/2, was examined. A 22.6% decrease (P<0.05; Fig. 3A and C) in p-CREB expression was observed in the
+
+The effects of sevoflurane on the Akt/GSK‑3β/CRMP‑2 pathway. To determine whether the Akt/GSK-3β/CRMP-2 signaling pathway is involved in sevoflurane‑induced neuroap- otosis, the expression of Akt, GSK-3β and CRMP-2, and their phosphorylation were assessed in the cortex following anes- thesia with sevoflurane. Western blot analysis demonstrated that sevoflurane inhibits Akt activity as, following sevoflurane treatment; the expression of p‑Akt was significantly reduced
+
+LIU et al: INFLUENCE OF SEVOFLURANE ON MAPK AND Akt/GSK-3β/CRMP-2 SIGNALING PATHWAYS
+
+by 58.9% compared with the control (P<0.01; Fig. 4A and B). Sevoflurane activated GSK-3β by significantly reducing the expression of p-GSK-3β at Ser 9 by 21.45% (P<0.01; Fig. 4A and C) and significantly increasing the expression of p-GSK-3β at Ty216 by 28.02% (P<0.01; Fig. 4A and D). Furthermore, the expression of p-CRMP2 at Thr 514, which reflects GSK-3β activity, was significantly increased by 198.42% following sevoflurane anesthesia compared with the control (P<0.01; Fig. 4A and E). The expression of Akt, GSK-3β and CRMP‑2 did not differ significantly between rats in the sevoflurane and control groups (data not shown).
+
+Discussion
+
+is responsible for the late phase of depolarization-stimulated CREB phosphorylation and the CaMK cascade regulates the early transient phase (38). The results of the current study demonstrate that sevoflurane decreases ERK phosphorylation and the phosphorylation of downstream CREB, indicating that sevoflurane may inhibit CREB activation by decreasing the phosphorylation of ERK. It has been demonstrated that the suppression of ERK phosphorylation is critically involved in the mechanism underlying sevoflurane‑induced toxicity in the developing brain and that lithium or N-stearoyl-L-tyrosine attenuates anesthetic-induced neuroapoptosis by upregulating the ERK pathway. The results of the present study indicate that the ERK-CREB pathway may be involved in sevoflu- rane-induced neurotoxicity and cognitive dysfunctions.
+
+In the current study, it was demonstrated that 6 h exposure to 2.6% sevoflurane significantly increases neuronal apoptosis in the cortices of P7 mice. The activation of calpain and caspase-3 contributed to this neuronal apoptosis. Sevoflurane suppressed the phosphorylation of ERK1/2 and CREB, and promoted the phosphorylation of p38 and NF-κB, but did not influence JNK phosphorylation following 6-h exposure. Furthermore, sevoflurane inhibited the activity of the Akt/GSK‑3β/CRMP-2 pathway by reducing the phosphorylation of Akt and GSK-3β, and increasing the phosphorylation of CRMP-2.
+
+It has been demonstrated that sevoflurane induces neuro- apoptosis, which is dependent on the depth of the anesthesia and is time- and brain region-specific (7,30,31). Previous studies have indicated that isoflurane induces more neurotox- icity than equivalent doses of sevoflurane (25,32). Exposure of P7 rats to 1% sevoflurane for 2 h did not result in severe neuroapoptosis, whereas increased concentrations of sevoflu- rane caused neuroapoptosis or cognitive dysfunction in the developing brain (31,33). In the current study, it was demon- strated that exposure of P7 rats to 2.6% sevoflurane (~1 MAC) activates capsase-3 dependent apoptosis, as indicated by the increase in cleaved 19/17-kDa caspase-3 subunits and genera- tion of the α-fodrin 120 kDa fragments (31,33). Furthermore, the relative activity of calpain, manifested as changes in the level of proteolytic fragment in the 145 kDa of α-fodrin, was also increased following sevoflurane exposure in the cortices of P7 rats. Calpains are a family of cysteine proteases activated by calcium and autolytic processing. It has been demonstrated that the pathological activation of calpain leads to cytoskeletal protein breakdown, the loss of structural integrity, dysfunctions of axonal transport and eventually, neuronal cell death (34). Activated calpain may also bind to the apoptosis induced factor (AIF) and thus mediate the caspase-independent apop- totic pathway (35). Indeed, our previous study determined that sevoflurane increases the expression of AIF in the cortex of P7 rats (36). Therefore, it is possible that sevoflurane induces neuroapoptosis by activating the caspase-dependent and -inde- pendent pathways.
+
+Sevoflurane induces abnormal social behaviors and cogni- tive dysfunctions in developing animals. CREB activation by phosphorylation is essential in the process of memory forma- tion and maintenance by inducing the expression of genes that are essential for learning (13). Hardingham et al (37) demon- strated that depolarization-stimulated CREB phosphorylation is dependent on activation of the ERK and calcium-dependent protein kinase (CaMK) signaling pathways. The ERK pathway
+
+Neuroinf lammation contributes to volatile anes- thetic‑induced cognitive deficits and it has been demonstrated that isoflurane induces learning impairment by activating the NF-κB pathway and upregulating the expression of hippo- campal interleukin-1β in rodents (39,40). Sevoflurane also increases levels of the pro‑inflammatory cytokine interleukin‑6 and tumor necrosis factor-α in the developing mouse brain (7). Anti‑inflammatory therapy significantly attenuated the cogni- tive impairments induced by sevoflurane in young and aged rats (7,41). Our previous study demonstrated that isoflurane induces neuroapoptosis by activating the p38-NF-κB signaling pathway in the brain of developing rats (16). In the present study, it was demonstrated that sevoflurane activates the p38 pathway, as demonstrated by the increase in the phosphoryla- tion of p38 and its downstream substrate NF-κB. However, further studies are required to identify whether the p38-NF-κB pathway is involved sevoflurane‑induced neuroapoptosis and neuroinflammation. Unlike isoflurane, sevoflurane does not promote the phosphorylation of JNK, which is consistent with the results of a study by Wang et al (42) demonstrating that inhibition of JNK does not attenuate sevoflurane-induced neuroapoptosis. Furthermore, sevoflurane induces astrocytic dysfunction by inactivating the JAK/STAT pathway in the hippocampus of neonatal rats (43). These results suggest that the inhibition of the JNK pathway may contribute to neurotox- icity of sevoflurane.
+
+The use of general anesthesia may increase the risk of Alzheimer's disease (AD). Isoflurane or sevoflurane promotes AD neuropathogenesis by inducing caspase activation, accumulation of β-amyloid (Aβ) and overt tau hyperphosphory- lation (5,44,45). GSK-3 activation is a critical step in the cascade of detrimental events that occur during the development in AD, preceding the neurofibrillary tangles and neuronal death path- ways (19). It has been demonstrated that sevoflurane induces Tau phosphorylation and GSK-3β activation in the hippocampus of developing mice (26). GSK-3β catalytic kinase activity is regu- lated by the differential phosphorylation of serine/threonine residues, including Ser 21 and Ser 9, which have an inhibitory effect, and tyrosine residues such as Tyr 279 and Tyr 216, which have an activating effect. CRMP-2, a phospho-protein involved in axonal outgrowth and microtubule dynamics, is aberrantly phosphorylated at Thr514 by GSK-3β in the brain of patients with AD (46). In the current study, sevoflurane decreased the phosphorylation of Akt and its downstream protein GSK-3β at Ser9 and also enhanced the phosphorylation of GSK-3β at Ty216, suggesting that sevoflurane activates GSK-3β. This
+
+2071
+
+2072
+
+EXPERIMENTAL AND THERAPEUTIC MEDICINE 15: 2066-2073, 2018
+
+effect was confirmed by the effect of sevoflurane on CRMP2 phosphorylation at Thr514, which is one of the downstream sites of GSK-3β. Wang et al (47) suggested that the suppression of CRMP-2 hyperphosphorylation ameliorates β-amyloid-induced cognitive dysfunction and hippocampal axon degeneration. However, it remains unknown whether the suppression of CRMP-2 hyperphosphorylation ameliorates neurotoxicity of sevoflurane in the developing brain.
+
+7. Shen X, Dong Y, Xu Z, Wang H, Miao C, Soriano SG, Sun D, Baxter MG, Zhang Y and Xie Z: Selective anesthesia-induced neuroinflammation in developing mouse brain and cognitive impairment. Anesthesiology 118: 502-515, 2013.
+
+8. Ravingerová T, Barancík M and Strnisková M: Mitogen-activated protein kinases: A new therapeutic target in cardiac pathology. Mol Cell Biochem 247: 127-138, 2003.
+
+9. Roux PP and Blenis J: ERK and p38 MAPK-activated protein kinases: A family of protein kinases with diverse biological func- tions. Microbiol Mol Biol Rev 68: 320-344, 2004.
+
+The current study had several limitations. Firstly, the changes in the expression of proteins were only evaluated 2 h following sevoflurane exposure and were not observed over a longer duration, which may improve understanding regarding the changes in the expression of these proteins. Additionally, specific inhibitors of p38 or GSK‑3β were not used. These may be useful in determining the exact mechanism of some signaling pathways following exposure to sevoflurane. Additionally, synaptic morphology and the behavior of animals following exposure to sevoflurane was not assessed in the current study. These evaluations may contribute to clarifying the effects of these pathways on sevoflurane‑induced long‑time cognitive impairment.
+
+In conclusion, the results of the current study demonstrated that sevoflurane induces changes in the expression of proteins that serve an important role in the brain development of neonatal animals. The proteins identified suggest that sevo- flurane may disturb neuronal migration, differentiation and energy metabolism in the brains of neonatal rats, which may contribute to its neurodegenerative effects.
+
+Acknowledgements
+
+10. Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K and Cobb MH: Mitogen -activated protein (MAP) kinase pathways: Regulation and physiological functions. Endocr Rev 22: 153-183, 2001.
+
+11. Liu AL, Wang XW, Liu AH, Su XW, Jiang WJ, Qiu PX and Yan GM: JNK and p38 were involved in hypoxia and reoxy- genation-induced apoptosis of cultured rat cerebellar granule neurons. Exp Toxicol Pathol 61: 137-143, 2009.
+
+12. Riccio A, Ahn S, Davenport CM, Blendy JA and Ginty DD: Mediation by a CREB family transcription factor of NGF-dependent survival of sympathetic neurons. Science 286: 2358-2361, 1999.
+
+13. Lonze BE and Ginty DD: Function and regulation of CREB family transcription factors in the nervous system. Neuron 35: 605-623, 2002.
+
+14. Yufune S, Satoh Y, Takamatsu I, Ohta H, Kobayashi Y, Takaenoki Y, Pagès G, Pouysségur J, Endo S and Kazama T: Transient Blockade of ERK Phosphorylation in the critical period causes autistic phenotypes as an adult in mice. Sci Rep 5: 10252, 2015.
+
+15. Li Y, Wang F, Liu C, Zeng M, Han X, Luo T, Jiang W, Xu J and Wang H: JNK pathway may be involved in isoflurane‑induced apoptosis in the hippocampi of neonatal rats. Neurosci Lett 545: 17-22, 2013.
+
+16. Liao Z, Cao D, Han X, Liu C, Peng J, Zuo Z, Wang F and Li Y: Both JNK and P38 MAPK pathways participate in the protection by dexmedetomidine against isoflurane‑induced neuroapoptosis in the hippocampus of neonatal rats. Brain Res Bull 107: 69-78, 2014. 17. Yoshimura T, Kawano Y, Arimura N, Kawabata S, Kikuchi A and Kaibuchi K: GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity. Cell 120: 137-149, 2005.
+
+The present study was supported by National Natural Science Foundation of China (grant no. 81371259), the Natural Science Foundation of Guangdong Province (grant no. 2016A030313251), the Science and Technology Program of Gagungdong Province (grant nos. 2014A020212147 and A2013206), the Science and Technology Planning Project of Guangzhou, China (grant no. 201605122118121) and the Medical Science and Technology Program of Foshan (grant no. 20130841).
+
+18. Martin M, Rehani K, Jope RS and Michalek SM: Toll -like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nat Immunol 6: 777-784, 2005. 19. Takashima A: GSK-3 is essential in the pathogenesis of Alzheimer's disease. J Alzheimers Dis 9 (3 Suppl): S309-S317, 2006.
+
+20. Cross DA, Alessi DR, Cohen P, Andjelkovich M and Hemmings BA: Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378: 785-789, 1995. 21. Yoshimura T, Arimura N, Kawano Y, Kawabata S, Wang S and Kaibuchi K: Ras regulates neuronal polarity via the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway. Biochem Biophys Res Commun 340: 62-68, 2006.
+
+References
+
+22. Xiong T, Tang J, Zhao J, Chen H, Zhao F, Li J, Qu Y, Ferriero D and Mu D: Involvement of the Akt/GSK-3β/CRMP-2 pathway in axonal injury after hypoxic-ischemic brain damage in neonatal rat. Neuroscience 216: 123-132, 2012.
+
+1. Brambr in k A M, Evers AS, Avidan MS, Fa rber N B, Smith DJ, Zhang X, Dissen GA, Creeley CE and Olney JW: Isoflurane-induced neuroapoptosis in the neonatal rhesus macaque brain. Anesthesiology 112: 834-841, 2010.
+
+2. Zheng H, Dong Y, Xu Z, Crosby G, Culley DJ, Zhang Y and Xie Z: Sevoflurane anesthesia in pregnant mice induces neuro- toxicity in fetal and offspring mice Anesthesiology 118: 516-526, 2013.
+
+3. Paule MG, Li M, Allen RR, Liu F, Zou X, Hotchkiss C, Hanig JP, Patterson TA, Slikker W Jr and Wang C: Ketamine anesthesia during the first week of life can cause long‑lasting cognitive deficits in rhesus monkeys. Neurotoxicol Teratol 33: 220-230, 2011.
+
+4. Zhang X, Xue Z and Sun A: Subclinical concentration of sevoflu- rane potentiates neuronal apoptosis in the developing C57BL/6 mouse brain. Neurosci Lett 447: 109-114, 2008.
+
+5. Goa KL, Noble S and Spencer CM: Sevoflurane in paediatric anaesthesia: A review. Paediatr Drugs 1: 127-153, 1999.
+
+6. Dong Y, Zhang G, Zhang B, Moir RD, Xia W, Marcantonio ER, Culley DJ, Crosby G, Tanzi RE and Xie Z: The common inha- lational anesthetic sevoflurane induces apoptosis and increases beta-amyloid protein levels. Arch Neurol 66: 620-631, 2009.
+
+23. Inagaki N, Chihara K, Arimura N, Menager C, Kawano Y, Matsuo N, Nishimura T, Amano M and Kaibuchi K: CRMP-2 induces axons in cultured hippocampal neurons. Nat Neurosci 4: 781-782, 2001.
+
+24. Nishimura T, Fukata Y, Kato K, Yamaguchi T, Matsuura Y, Kamiguchi H and Kaibuchi K: CRMP-2 regulates polarized Numb-mediated endocytosis for axon growth. Nat Cell Biol 5: 819-826, 2003.
+
+25. Li Y, Zeng M, Chen W, Liu C, Wang F, Han X, Zuo Z and Peng S: Dexmedetomidine reduces isoflurane‑induced neuroapoptosis partly by preserving PI3K/Akt pathway in the hippocampus of neonatal rats. PLoS One 9: e93639, 2014.
+
+26. Tao G, Zhang J, Zhang L, Dong Y, Yu B, Crosby G, Culley DJ, Zhang Y and Xie Z: Sevoflurane induces tau phosphorylation and glycogen synthase kinase 3β activation in young mice. Anesthesiology 121: 510-527, 2014.
+
+27. National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals: Guide for the Care and Use of Laboratory Animals. 8th edition. National Academies Press, Washington, DC, 2011.
+
+28. Paxinos G, Halliday G, Watson C, Koutcherov Y and Wang H: Atlas of the developing mouse brain at E17.5, P0 and P6. Academic Press, New York, NY, 2006.
+
+LIU et al: INFLUENCE OF SEVOFLURANE ON MAPK AND Akt/GSK-3β/CRMP-2 SIGNALING PATHWAYS
+
+29. Vanags DM, Pörn-Ares MI, Coppola S, Burgess DH and Orrenius S: Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis. J Biol Chem 271: 31075-31085, 1996.
+
+30. Zheng SQ, An LX, Cheng X and Wang YJ: Sevoflurane causes neuronal apoptosis and adaptability changes of neonatal rats. Acta Anaesthesiol Scand 57: 1167-1174, 2013.
+
+31. Xiao H, Liu B, Chen Y and Zhang J: Learning, memory and synaptic plasticity in hippocampus in rats exposed to sevoflurane. Int J Dev Neurosci 48: 38-49, 2016.
+
+32. Liang G, Ward C, Peng J, Zhao Y, Huang B and Wei H: Isoflurane causes greater neurodegeneration than an equivalent exposure of sevoflurane in the developing brain of neonatal mice. Anesthesiology 112: 1325-1334, 2010.
+
+33. Wang SQ, Fang F, Xue ZG, Cang J and Zhang XG: Neonatal sevoflurane anesthesia induces long-term memory impairment and decreases hippocampal PSD-95 expression without neuronal loss. Eur Rev Med Pharmacol Sci 17: 941-950, 2013.
+
+34. Vosler PS, Brennan CS and Chen J: Calpain-mediated signaling mechanisms in neuronal injury and neurodegeneration. Mol Neurobiol 38: 78-101, 2008.
+
+40. Li ZQ, Rong XY, Liu YJ, Ni C, Tian XS, Mo N, Chui DH and Guo XY: Activation of the canonical nuclear factor-κB pathway is involved in isoflurane‑induced hippocampal interleukin‑1β elevation and the resultant cognitive deficits in aged rats. Biochem Biophys Res Commun 438: 628-634, 2013.
+
+41. Gong M, Chen G, Zhang XM, Xu LH, Wang HM and Yan M: Parecoxib mitigates spatial memory impairment induced by sevoflurane anesthesia in aged rats. Acta Anaesthesiol Scand 56: 601-607, 2012.
+
+42. Wang WY, Wang H, Luo Y, Jia LJ, Zhao JN, Zhang HH, Ma ZW, Xue QS and Yu BW: The effects of metabotropic glutamate receptor 7 allosteric agonist N, N'-dibenzhydrylethane-1, 2-diamine dihydrochloride on developmental sevoflurane neurotoxicity: Role of extracellular signal-regulated kinase 1 and 2 mitogen-activated protein kinase signaling pathway. Neuroscience 205: 167-177, 2012.
+
+43. Wang W, Lu R, Feng DY and Zhang H: Sevoflurane Inhibits Glutamate-Aspartate Transporter and Glial Fibrillary acidic protein expression in hippocampal astrocytes of neonatal rats through the Janus Kinase/Signal Transducer and activator of transcription (JAK/STAT) pathway. Anesth Analg 123: 93-102, 2016.
+
+35. Lu JR, Lu WW, Lai JZ, Tsai FL, Wu SH, Lin CW and Kung SH: Calcium flux and calpain‑mediated activation of the apoptosis-inducing factor contribute to enterovirus 71-induced apoptosis. J Gen Virol 94: 1477-1485, 2013.
+
+36. Li Y, Liu C, Zhao Y, Hu K, Zhang J, Zeng M, Luo T, Jiang W and Wang H: Sevoflurane induces short‑term changes in proteins in the cerebral cortices of developing rats. Acta Anaesthesiol Scand 57: 380-390, 2013.
+
+44. Liu XS, Xue QS, Zeng QW, Li Q, Liu J, Feng XM and Yu BW: Sevoflurane impairs memory consolidation in rats, possibly through inhibiting phosphorylation of glycogen synthase kinase-3β in the hippocampus. Neurobiol Learn Mem 94: 461-467, 2010.
+
+45. Dong Y, Wu X, Xu Z, Zhang Y and Xie Z: Anesthetic isoflurane increases phosphorylated tau levels mediated by caspase activa- tion and Aβ generation. PLoS One 7: e39386, 2012.
+
+37. Hardingham GE, Arnold FJ and Bading H: Nuclear calcium signaling controls CREB-mediated gene expression triggered by synaptic activity. Nat Neurosci 4: 261-267, 2001.
+
+38. Wu GY, Deisseroth K and Tsien RW: Activity-dependent CREB phosphorylation: Convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway. Proc Natl Acad Sci USA 98: 2808-2813, 2001.
+
+39. Cao L, Li L, Lin D and Zuo Z: Isoflurane induces learning impairment that is mediated by interleukin 1β in rodents. PLoS One 7: e51431, 2012.
+
+46. Ni MH, Wu CC, Chan WH, Chien KY and Yu JS: GSK-3 medi- ates the okadaic acid‑induced modification of collapsin response mediator protein-2 in human SK-N-SH neuroblastoma cells. J Cell Biochem 103: 1833-1848, 2008.
+
+47. Wang Y, Yin H, Li J, Zhang Y, Han B, Zeng Z, Qiao N, Cui X, Lou J and Li J: Amelioration of β-amyloid-induced cognitive dysfunction and hippocampal axon degeneration by curcumin is associated with suppression of CRMP-2 hyperphosphorylation. Neurosci Lett 557: 112-117, 2013.
+
+2073

new_pdfs/10.3892_etm.2018.5950.txt

@@ -0,0 +1,365 @@
+EXPERIMENTAL AND THERAPEUTIC MEDICINE 15: 4097-4104, 2018
+
+Sevoflurane-induced memory impairment in the postnatal developing mouse brain
+
+ZHIJUN LU1, JIHUI SUN1, YICHUN XIN1, KEN CHEN1, WEN DING1 and YUJIA WANG2
+
+1Department of Anesthesia, Rui Jin Hospital Luwan Branch, Shanghai Jiao Tong University School of Medicine, Shanghai 200020; 2Intensive Care Unit, Shanghai Jing'an District Shibei Hospital, Shanghai 200443, P.R. China
+
+Received March 31, 2016; Accepted March 6, 2017
+
+DOI: 10.3892/etm.2018.5950
+
+Abstract. The aim of the present study was to confirm that sevoflurane induces memory impairment in the postnatal developing mouse brain and determine its mechanism of action. C57BL/6 mice 7 days old were randomly assigned into a 2.6% sevoflurane (n=68), a 1.3% sevoflurane (n=68) and a control (n=38) group. Blood gas analysis was performed to evaluate hypoxia and respiratory depression during anes- thesia in 78 mice. Measurements for expression of caspase‑3 by immunohistochemistry, cleavage of poly adenosine diphosphate‑ribose polymerase (PARP) by western blotting, as well as levels of brain‑derived neurotrophic factor (BDNF), tyrosine kinase receptor type 2 (Ntrk2), pro‑BDNF, p75 neuro- trophin receptor (p75NTR) and protein kinase B (PKB/Akt) by enzyme-linked immunosorbent assay were performed in the hippocampus of 12 mice from each group. A total of 60 mice underwent the Morris water maze (MWM) test. Results from the MWM test indicated that the time spent in the northwest quadrant and platform site crossovers by mice in the 2.6 and 1.3% sevoflurane groups was significantly lower than that of the control group. Meanwhile, levels of caspase‑3 and cleaved PARP in the 2.6 and 1.3% sevoflurane groups were significantly higher than that in the control group. Levels of pro‑BDNF and p75NTR were significantly increased and the level of PKB/Akt was significantly decreased following exposure to 2.6% sevoflurane. Finally, the memory of post- natal mice was impaired by sevoflurane, this was determined using a MWM test. Therefore, the results of the current study suggest that caspase‑3 induced cleavage of PARP, as well as pro‑BDNF, p75NTR and PKB/Akt may be important in sevoflurane-induced memory impairment in the postnatal developing mouse brain.
+
+Correspondence to: Dr Yujia Wang, Intensive Care Unit, Shanghai Jing'an District Shibei Hospital, 4500 Gonghexin Road, Shanghai 200443, P.R. China E-mail: yujiawangwww@hotmail.com
+
+Introduction
+
+Sevoflurane is a widely used inhalational anesthetic agent that is commonly used in cesarean sections (1) and in operations on infants and young children (2,3). However, the neurotoxic effect of sevoflurane can damage the developing brain and thereby influence long‑term learning and memory in animals (4‑6). Thus, the use of sevoflurane in cesarean delivery or opera- tion for infants and young children may be detrimental to the brain development of infants and children, and influence the memory and cognitive function during childhood. Currently, sevoflurane-induced memory impairment is a method of evaluating sevoflurane-induced damage in the developing brain (5,6). However, the mechanism of sevoflurane‑induced memory impairment remains unclear.
+
+Previous studies have demonstrated that 6 h sevoflu- rane exposure may significantly increase the expression of caspase‑3, which is a marker of neural apoptosis (7), in the hippocampi of postnatal rats (8) and neonatal mice (9). Furthermore, it has been determined that neural apoptosis in the hippocampus is associated with impaired memory and cognitive function (10,11). However, to the best of our knowledge, there have been no studies identifying the role of caspase‑3 in sevoflurane‑induced memory impairment in the developing brain. Poly adenosine diphosphate-ribose polymerase (PARP), a substrate of caspase‑3, is associated with long‑term memory (12‑14). Thus, it was suggested that PARP may be cleaved by overexpressed caspase‑3 in the sevoflurane-treated developing brain. In addition, brain‑derived neurotrophic factor (BDNF), cleaved from a precursor of BDNF (pro‑BDNF) and tyrosine kinase receptor type 2 (Ntrk2, also known as TrkB), both important in the survival and growth of neurons (15,16), are also corre- lated with learning and memory (17). Furthermore, it has been reported that the p75 neurotrophin receptor (p75NTR) has marked effects on hippocampal function: Knockout of p75NTR enhanced spatial memory in adult mice (18). Thus, p75NTR may also be involved in sevoflurane‑induced memory impairment in the developing brain. Furthermore, protein kinase B (PKB/Akt, a serine/threonine kinase) has been found to serve a role in numerous pathways that are associated with memory (19,20).
+
+Key words: sevoflurane, memory impairment, postnatal mouse, hippocampus
+
+Emergency cesarean sections are performed on pregnant women in their third trimester gestation when required and
+
+4098
+
+LU et al: SEVOFLURANE EXPOSURE IN POSTNATAL MICE
+
+sevoflurane is often used as an anesthetic during this proce- dure. However, the use of sevoflurane during a cesarean section may affect brain development and cause memory impairment in postnatal infants. Thus, 7-day-old mice, equivalent to a human third trimester gestation (21), were used in the current study to investigate the effect of sevoflu- rane on the memory of postnatal infants. In the present study, levels of caspase‑3, cleaved PARP, BDNF, Ntrk2, pro‑BDNF, p75NTR and PKB/Akt proteins were investigated in the hippocampi of postnatal mice following 6 h sevoflurane exposure to identify the mechanism of sevoflurane‑induced memory impairment in the developing brain. The memory of these postnatal mice was assessed using a Morris water maze (MWM) test at weeks 4 and 12 following sevoflurane exposure to confirm the effect of sevoflurane on memory impairment in postnatal mice.
+
+Sevoflurane exposure. As stated in a previous study (25), animals were placed in a temperature‑controlled (37‑38˚C) transparent anesthetic chamber that was connected to an anes- thetic gas monitor (Datex‑Ohmeda S/5, Datex‑Ohmeda; GE Healthcare Bio‑Sciences, Pittsburgh, PA, USA). For mice in the 1.3 and 2.6% sevoflurane groups, mixed gas (5% sevoflurane and 30% O2) was pre‑aerated at a flow rate of 10 l/min until the concentration of sevoflurane reached 5% in the chamber and prior to placing mice in the chamber. Subsequently, these mice were placed into the chamber immediately. Following main- tenance of 5% sevoflurane for 30 sec, mice were exposed to 1.3 or 2.6% sevoflurane for the indicated time periods (1‑6 h), during which 30% O2 was continually gassed into the chamber at a flow rate of 3 l/min. For mice in the control group, 30% O2 alone was aerated into the chamber for 6 h, with a flow rate of 3 l/min.
+
+Materials and methods
+
+Animal model. All experiments were performed according to the guidelines of the Guide for the Care and Use of Laboratory Animals (22) and were approved by the Institutional Animal Care and Use Committee of Ruijin Hospital Affiliated to Shanghai Jiaotong University (Shanghai, China). A total of 174 C57BL/6 mice (sex ratio, 1:1), were provided by the Model Animal Research Center of Nanjing University (Nanjing, China). They were housed in polypropylene cages (5 or 6 animals per cage) and kept at a 12 h light‑dark cycle at room temperature (21‑24˚C) in 55% humidity for 7 days prior to testing. All animals had free access to food and water.
+
+Experimental protocols. There were two experimental protocols used based on the sevoflurane concentration used in previous studies (23,24) and 1.3 and 2.6% sevoflurane was used in the present study. For protocol one, 36 mice were randomly assigned into 3 groups with 12 mice in each group: The 2.6 and 1.3% sevoflurane groups and the control group (exposed to 30% O2). Following exposure to sevoflurane or O2 for 6 h, the mice from all 3 groups were sacrificed by intraperitoneal injection of 1.5% pentobarbital sodium (375 mg/kg) (Dalian Idery Biotechnology Co., Ltd., Dalian, China). Hippocampal tissue samples from these mice were collected to measure the expression of caspase‑3 using immunohistochemistry, the cleavage of PARP by western blotting, and levels of BDNF, Ntrk2, pro‑BDNF, p75NTR and PKB/Akt by ELISA. To evaluate whether hypoxia and respi- ratory depression occurred in mice during anesthesia, blood gas analysis was performed in another 78 mice, which were randomly assigned into 3 groups: 2.6% sevoflurane (n=36), 1.3% sevoflurane (n=36) and control (n=6) groups. The mice in the 1.3 and 2.6% sevoflurane groups were divided into subgroups based on the length of time they were exposed to sevoflurane (1, 2, 3, 4, 5 and 6 h), with 6 mice in each subgroup.
+
+For protocol two, a total of 60 mice were randomly assigned into 3 groups with 20 mice in each group: 2.6, 1.3% sevoflu- rane and control groups. Following exposure to sevoflurane for 4 weeks, the MWM test was performed in half of the mice in each group. The MWM test was conducted on the remaining mice at week 12.
+
+Blood gas analysis. The mice were anesthetized by intraperi- toneal injection of 1.5% sodium pentobarbital (50 mg/kg). Then blood samples (0.2 ml) were obtained from the left ventricle by cardiac puncture, after which the mice were sacrificed by intraperitoneal injection of 1.5% sodium pentobarbital (375 mg/kg). The partial pressure of oxygen (PaO2), partial pressure of carbon dioxide (PaCO2) and arte- rial oxygen saturation (SaO2) were detected using a portable blood gas analyzer (OPTI Medical Systems Inc., Roswell, GA, USA).
+
+Tissue sample collection. Following sevoflurane exposure, all the mice were sacrificed by intraperitoneal injection of 1.5% pentobarbital sodium (375 mg/kg). The brain was then rapidly removed and the complete hippocampus was dissected. Hippocampal tissue samples were stored at ‑80˚C prior to use in laboratory experiments.
+
+Immunohistochemistry. The hippocampal tissues were fixed overnight in 4% paraformaldehyde at 4˚C. The hippo- campal slices (5‑µm‑thick) were subsequently prepared using a vibrating tissue slicer (Campden Instruments, Ltd., Loughborough, UK). Immunohistochemical staining was performed as previously described (26,27). Briefly, slices were incubated with hydrogen peroxide in methanol to block endogenous peroxidase activity and 10% normal goat serum (cat. no. C0265; Beyotime Institute of Biotechnology, Haimen, China) to reduce non‑specific antibody binding prior to immunohistochemical staining. Slices were then incubated with a rabbit anti‑caspase‑3 antibody (1:200; cat. no. AC033; Beyotime Institute of Biotechnology) at 4˚C for 12 h, followed by three washes with PBS. Subsequently, these slices were incubated with secondary antibody (1:4,000; cat. no. A0562; biotinylated goat anti-rabbit antibody; Beyotime Institute of Biotechnology) for 30 min at 37˚C. Following washing with PBS, immunoreactivity was visualized using the streptav- idin‑peroxidase complex and 3,3'‑diaminobenzidine (both from Beyotime Institute of Biotechnology). A DM5000B light microscope (Leica Microsystems GmBH, Wetzlar, Germany) was used to observe and collect images. The image analysis software Image Pro Plus version 4.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used to count the number of caspase‑3 positive cells.
+
+EXPERIMENTAL AND THERAPEUTIC MEDICINE 15: 4097-4104, 2018
+
+Western blotting. The preparation of hippocampus protein extraction was performed as previously described (28,29). Total proteins were extracted with radioimmunoprecipitation assay buffer [1% Triton X‑100, 50 mM Tris, (pH 7.4), 150 mM NaCl, M, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA and 1% sodium deoxycholate]. Following 13,000 x g centrifu- gation at 4˚C for 20 min, the supernatant was used for western blotting (30,31). The BCA method was used to assay protein concentrations. In brief, hippocampal tissue proteins were separated by 10% SDS polyacrylamide gel electrophoresis and then electrotransferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat powdered milk for 1 h at 25˚C. The proteins were probed with rabbit anti‑PARP antibodies (1:200, cat. no. AP102) or rat anti‑GAPDH anti- bodies (1:5,000, cat. no. AG019) overnight at 4˚C. Then, goat anti‑rabbit (1:4,000; cat. no. A0208) or goat anti‑rat (1:4,000; cat. no. A0192) horseradish peroxidase‑conjugated secondary antibodies were used for 2 h incubation at room temperature (all from Beyotime Institute of Biotechnology). Proteins were visualized by an enhanced chemiluminescence method and analyzed with the Dolphin‑Doc Plus Gel Documentation system (version 1141002; Wealtec Corp., Sparks, NV, USA). This procedure was repeated twice for all 3 groups. The rela- tive level of PARP was presented as the band intensity and normalized to the corresponding band intensities of GAPDH.
+
+end was recorded as the time of escape latency. The swim rate during training was also recorded. On day 7, the probe test was performed by allowing the mice to swim for 60 sec in the absence of the platform. During 60 sec swimming, the time spent in the northwest quadrant and platform site crossovers was recorded and analyzed using the MWM JLBehv‑FCS video analysis system (DigBehv‑MG; Shanghai Jiliang Software Technology Co., Ltd., Shanghai, China).
+
+Statistical analysis. All data are presented as the mean ± stan- dard error of the mean. A repeated measures analysis of variance (ANOVA) was used to measure the differences within groups over time. Meanwhile, one-way ANOVA was applied for comparison among groups (2.6, 1.3% sevoflurane and control groups), followed by Student Newman‑Keuls post hoc test. The correlation between the swim rate and time of escape latency was identified using the Pearson Correlation coefficient. For all the analysis, P<0.05 was used to indicate a statistically significant difference. Additionally, SPSS 11.5 (SPSS, Inc., Chicago, IL, USA) was used for the analysis of the present study.
+
+Results
+
+ELISA. The method of hippocampus protein extraction mentioned above was also used for ELISA. The levels of BDNF, Ntrk2, pro‑BDNF, p75NTR and PKB/Akt were measured using an ELISA kit (cat. no. EK0312; Wuhan Boster Bio‑Engineering Co., Ltd., Wuhan, China) according to the manufacturer's instructions. Briefly, protein samples were added to the enzyme label plate and incubated for 1.5 h at 37˚C. Next, the biotin‑labeled antibodies were added for 1 h incubation at 37˚C. Following washing, 30 min incubation with avidin peroxidase complex was conducted at 37˚C. Color was developed using 3,3',5,5'‑tetramethylbenzidine following 20 min incubation at 37˚C. Following reaction termination with a ‘stop’ solution, the products were measured at 450 nm using a microplate spectrophotometer (Spectramax 190; Molecular Devices LLC, Sunnyvale, CA, USA). All samples were assayed in duplicate and the readings were normalized to the amount of standard protein.
+
+Behavioral studies. Prior to the MWM test, mice received 2 min of touch for 5 days to avoid the fear to touch during the test. The MWM test was performed as previously described (32,33), with minor modifications. The round pool (diameter, 122 cm) was filled with warm water, made opaque by the addition of titanium dioxide and an escape platform was placed in the northwest quadrant and hidden 0.5 cm below the surface of the water. The MWM test was performed on 7 consecutive days (6 days for training and 1 day for the probe test). Briefly, mice received 4 training sessions daily for 6 consecutive days. Each trial began from a different point and ended when the mice found the platform. The time from beginning to end was considered to be the time of escape latency. If mice could not find the platform within 90 sec, the time of escape latency was recorded as 90 sec. If mice found the platform within 90 sec, the real time from beginning to
+
+Results of blood gas analysis. The PaO2, PaCO2 and SaO2 values remained stable in the 2.6 and 1.3% sevoflurane and control groups following treatment. There were no significant differences identified among groups and the PaO2, PaCO2 and SaO2 values did not notably change with increasing time periods of sevoflurane exposure (Table I).
+
+Sevoflurane increases caspase‑3 expression. Significantly more caspase‑3 positive cells were found in the 2.6 and 1.3% sevoflurane groups compared with the control group (P<0.05). Meanwhile, the number of positive cells in the 2.6% sevo- flurane group was significantly higher than that of the 1.3% sevoflurane group (P<0.05; Fig. 1).
+
+Sevoflurane promotes the cleavage of PARP. Relative levels of cleaved PARP in the 2.6% (1.552±0.178) and 1.3% (1.376±0.157) sevoflurane groups were significantly increased following sevoflurane exposure compared with the control group (0.729±0.106; P<0.001). However, there was no signifi- cant difference in the level of cleaved PARP (P>0.05) detected in the 2.6 and 1.3% sevoflurane groups (P>0.05; Fig. 2).
+
+Effect of sevoflurane on BDNF, Pro‑BDNF, TrkB, Akt/PKB and p75NTR. According to ELISA, 2.6% sevoflurane signifi- cantly increased the expression of Pro‑BDNF compared with the control group (2.6% sevoflurane group, 3,146.32±47.96 vs. control group, 2,817.17±47.96; P<0.05). Furthermore, the level of Akt/PKB was significantly decreased following 6 h exposure to 2.6% sevoflurane, compared with the control group (2.6% sevoflurane group, 1,263.50±27.08 vs. control group, 1,557.35±59.87; P<0.05). In addition, levels of p75NTR in the 2.6% (119.40±2.58) and 1.3% (119.04±1.45) sevoflurane groups were significantly higher than those in the control group (108.34±3.77; P<0.05). However, there were no signifi- cant differences in the levels of BDNF and TrkB (P>0.05) among groups (Table II).
+
+4099
+
+4100
+
+LU et al: SEVOFLURANE EXPOSURE IN POSTNATAL MICE
+
+Table I. Results of blood gas analysis during sevoflurane exposure.
+
+Time and concentration of sevoflurane exposure
+
+PaO2 (mmHg)
+
+PaCO2 (mmHg)
+
+SaO2
+
+Before exposure (control group) 1 h of 2.6% 2 h of 2.6% 3 h of 2.6% 4 h of 2.6% 5 h of 2.6% 6 h of 2.6% 1 h of 1.3% 2 h of 1.3% 3 h of 1.3% 4 h of 1.3% 5 h of 1.3% 6 h of 1.3%
+
+105±2 103±2 98±4 100±2 99±2 103±3 100±2 103±2 99±1 99±2 102±3 100±3 99±2
+
+21.8±0.8 23.6±0.7 23.0±0.9 23.6±1.2 22.5±0.9 22.0±0.9 23.0±1.0 23.9±0.7 23.3±0.9 24.1±1.5 23.6±0.9 23.6±0.9 23.0±1.1
+
+99.0±0.3 98.3±0.4 97.4±0.6 98.0±0.5 97.4±0.9 98.2±0.4 99.0±0.5 98.1±0.4 97.2±0.6 97.9±0.5 98.6±0.5 97.6±0.4 98.1±0.5
+
+A repeated measures ANOVA was used to assess the differences of data at different time points. The one-way ANOVA method was applied for comparison among groups. No significant differences were observed among the data at different time points and among the three groups. Data are presented as the mean ± standard error of the mean. n=6. PaO2, partial pressure of oxygen; PaCO2, partial pressure of carbon dioxide; SaO2, arterial oxygen saturation. ANOVA, analysis of variance.
+
+Figure 1. Expression of caspase‑3 in hippocampus cells. (A‑F) Images of immunohistochemical staining. (A and B) Control; (C and D) 1.3% sevoflurane and (E and F) 2.6% sevoflurane groups. A, C and E, magnification, x4, scale bar, 50 µm; B, D and F: magnification, x20, scale bar, 100 µm. The boxes in A, C and E indicate the area of caspase‑3 positive cells, and parts B, D and F are these boxes at a higher magnification. The arrows in B, D and F indicate caspase‑3 positive cells. (G) The numbers of caspase‑3 positive cells in the hippocampal CA1 region in each group; *P<0.05, compared with the control group; #P<0.05, compared with the 1.3% sevoflurane group.
+
+Effect of sevoflurane on mouse memory. As presented in Table III, the time of escape latency significantly decreased as duration time increased in each group during 6 days training at weeks 4 and 12 following sevoflurane exposure (P<0.05). Moreover, the time of escape latency on days 4, 5 and 6 in the 2.6 and 1.3% sevoflurane groups were all significantly higher than that of the control group 4 weeks following sevoflurane exposure (P<0.05). Meanwhile, no significant difference in time of escape latency on days 1, 2 and 3 was observed between
+
+the 2.6 and 1.3% sevoflurane groups 4 weeks following sevo- flurane exposure. However, only the time of escape latency on day 6 of training in the 2.6% sevoflurane group was signifi- cantly higher than that of the control group 12 weeks following sevoflurane exposure (P<0.05). Furthermore, no significant difference among groups was revealed in the other times of escape latency (P>0.05) and there was no significant correla- tion between the time of escape latency and swim rate (r>0; P>0.05) observed in the present study.
+
+EXPERIMENTAL AND THERAPEUTIC MEDICINE 15: 4097-4104, 2018
+
+4101
+
+Table II. Levels of BDNF, Pro‑BDNF, TrkB, Akt/PKB and p75NTR in the hippocampal tissues in each group.
+
+Group
+
+BDNF (pg/ml)
+
+Pro‑BDNF (pg/ml)
+
+TrkB (pg/ml)
+
+Akt/PKB (pg/ml)
+
+p75NTR (pg/ml)
+
+2.6% sevoflurane 1.3% sevoflurane Control P-valueb
+
+1,198.69±31.12 1,188.00±18.02 1,129.50±34.15 0.128
+
+3,146.32±47.96a 2,938.97±113.63 2,817.17±47.96 0.039
+
+711.39±20.37 759.39±13.78 717.60±18.89 0.139
+
+1,263.50±27.08a 1,459.45±23.00 1,557.35±59.87 <0.001
+
+119.40±2.58a 119.04±1.45a 108.34±3.77 0.013
+
+aP<0.05 vs. the control using Student Newman‑Keuls post hoc test. bP‑value was calculated from 2.6%, 1.3% sevoflurane and control groups by the one‑way analysis of variance method. Data are presented as the mean ± standard error of the mean. n=12. BDNF, brain‑derived neuro- trophic factor; TrkB, tyrosine kinase receptor type 2; Akt/PKB, protein kinase B; pro‑BDNF, a precursor of BDNF; p75NTR, p75 neurotrophin receptor; PKB/Akt, protein kinase B.
+
+Discussion
+
+In the present study, the results of the MWM test determined that the memory of mice in the 2.6 and 1.3% sevoflurane groups were significantly weakened compared with that in the control group. These results provide evidence for sevoflurane‑induced memory impairment in the developing brain of postnatal mice, suggesting that the use of sevoflurane during cesarean section may damage the brain development of postnatal infants. However, the time spent in the northwest quadrant and platform site crossovers was not significantly decreased by 2.6% sevoflurane exposure compared with that in the 1.3% sevoflurane group, apart from the platform site crossovers at the 12th week after sevoflurane exposure. Thus, there may not be a dose‑dependent effect in sevoflurane‑induced memory impairment for postnatal mice.
+
+Figure 2. Levels of cleaved PARP in the hippocampus tissue in each group. (A) Images of western blotting; (B) Relative levels of cleaved PARP in each group. *P<0.05, compared with the control group. PARP, poly adenosine diphosphate-ribose polymerase.
+
+The results of the probe test revealed that the time spent in the northwest quadrant (week 4: 2.6% sevoflurane group, 0.04±0.03 sec; 1.3% sevoflurane group, 0.19±0.09 sec; control group, 0.88±0.21 sec; and week 12: 2.6% sevoflurane group, 0.23±0.11 sec; 1.3% sevoflurane group, 1.00±0.27 sec and control group, 15.32±3.62 sec) and number of platform site crossovers (week 4: 2.6% sevoflurane group, 0.25±0.16; 1.3% sevoflurane group, 0.63±0.32; control group, 2.38±0.65; and week 12: 2.6% sevoflurane group, 0.67±0.33; 1.3% sevo- flurane group, 2.63±0.71; control group, 4.67±1.18) in the 2.6 and 1.3% sevoflurane groups were significantly lower compared with the control group (P<0.05) at weeks 4 and 12 after sevoflurane exposure. Moreover, platform site cross- overs were significantly decreased following 6 h exposure to 2.6% sevoflurane compared with the 1.3% sevoflurane group 12 weeks after sevoflurane exposure (P<0.05; Table IV).
+
+Consistent with previous studies (8,9), caspase‑3 expression was significantly increased by sevoflurane in the present study. Furthermore, the results of the present study provided evidence for an association between caspase‑3 and sevoflurane‑induced memory impairment in postnatal mice. Meanwhile, levels of cleaved PARP in the 2.6 and 1.3% sevoflurane groups were significantly higher than that in the control group. It has been reported that the spatial memory of rats with twice-repeated cerebral ischemia could be significantly improved by decreasing levels of PARP and caspase‑3 (34). Furthermore, increased expression of caspase‑3 and cleavage of PARP are associated with neuronal apoptosis in hippocampal tissue (35,36), which is a major mechanism of memory impairment. Therefore, this evidence indicates that caspase‑3 induced cleavage of PARP may result in neuronal apoptosis in the hippocampus and lead to memory impairment in postnatal mice.
+
+The expression of Akt/PKB and pro‑BDNF in the hippocampal tissue was also significantly altered by 2.6% sevoflurane. It was reported that the cleavage of pro‑BDNF is important in the formation of memories (37). The present study indicated that cleavage of pro‑BDNF may be repressed by sevoflurane, leading to accumulation of pro‑BDNF in the hippocampal tissue. Overall, pro‑BDNF is cleaved and changed into BDNF, which is mediated by the activation of phosphatidylinositol 3‑kinase (PI3K), in BDNF‑dependent spatial memory formation (38). PI3K can then phosphorylate Akt/PKB. Inhibition of Akt phosphorylation exacerbates memory deficits in a rat model of Alzheimer's disease (39).
+
+4102
+
+LU et al: SEVOFLURANE EXPOSURE IN POSTNATAL MICE
+
+Table III. Results of 6 days training in Morris water maze test.
+
+A, Changes of time of escape latency at 4th week (sec)
+
+Day
+
+2.6% sevoflurane group
+
+1.3% sevoflurane group
+
+Control group
+
+P‑valuec
+
+1 2 3 4 5 6 P-valued
+
+84.56±3.02 85.66±2.70 81.96±6.13 76.48±4.85a 85.50±2.52a,b 69.17±5.56a,b 0.009
+
+73.94±4.98 74.98±4.85 71.88±6.45 65.99±5.34a 67.22±4.89a 63.27±6.14a <0.001
+
+82.58±4.15 78.61±4.03 70.99±5.96 50.19±5.72 45.55±4.87 35.97±4.49 <0.001
+
+0.396 0.269 0.233 0.003 <0.001 <0.001
+
+B, Changes of time of escape latency at 12th week (sec)
+
+Day
+
+2.6% sevoflurane group
+
+1.3% sevoflurane group
+
+Control group
+
+P‑valuec
+
+1 2 3 4 5 6 P-valued
+
+81.68±3.58 71.96±4.98 54.40±5.19 55.23±5.48 53.23±5.84 52.42±5.46a,b <0.001
+
+77.27±4.88 71.36±5.63 53.39±4.92 44.25±5.53 36.68±4.49 27.34±4.14 <0.001
+
+72.61±5.29 69.71±6.12 67.75±5.16 60.81±5.59 42.09±5.42 32.73±5.12 <0.001
+
+0.265 0.532 0.351 0.123 0.075 0.001
+
+aP<0.05 vs. the control using Student Newman‑Keuls post hoc test, bP<0.05 vs. the 1.3% sevoflurane group using Student Newman‑Keuls post hoc test. cP‑value was calculated from 2.6%, 1.3% sevoflurane and control groups by the one‑way analysis of variance method. dP-value was calculated from time of escape latency (days 1, 2, 3, 4, 5, 6) by a repeated measures analysis of variance. Data are presented as the mean ± standard error of the mean. n=10.
+
+Table IV. Time spent in the northwest quadrant and platform site crossovers in each group.
+
+A, Results of the 4th week
+
+Results of probe test
+
+2.6% sevoflurane group
+
+1.3% sevoflurane group
+
+Control group
+
+P‑valuec
+
+Time spent in the northwest quadrant (sec) Platform site crossovers (times)
+
+0.04±0.03a
+
+0.25±0.16a
+
+0.19±0.09a
+
+0.63±0.32a
+
+0.88±0.21
+
+2.38±0.65
+
+0.001
+
+0.003
+
+B, Results of the 12th week
+
+Results of probe test
+
+2.6% sevoflurane group
+
+1.3% sevoflurane group
+
+Control group
+
+P‑valuec
+
+Time spent in the northwest quadrant (sec) Platform site crossovers (times)
+
+0.23±0.11a
+
+0.67±0.33a,b
+
+1.00±0.27a
+
+2.63±0.71a
+
+15.32±3.62
+
+4.67±1.18
+
+<0.001
+
+0.006
+
+aP<0.05 vs. the control using a Student Newman‑Keuls post hoc test. bP<0.05 vs. the 1.3% sevoflurane group using Student Newman‑Keuls post hoc test. cP‑value was calculated from 2.6%, 1.3% sevoflurane and control groups by the one‑way analysis of variance method. Results are presented as the mean ± standard error of the mean. n=10.
+
+Thus, activation of PI3K may be inhibited by sevoflurane and thereby decrease levels of phosphorylated Akt, as well as inhibit cleavage of pro‑BDNF. Akt phosphorylation and
+
+activation of PI3K was not assessed in the present study, which is a limitation. However, it was speculated that the decrease in Akt levels observed in the present study was caused by the
+
+EXPERIMENTAL AND THERAPEUTIC MEDICINE 15: 4097-4104, 2018
+
+feedback inhibition of phosphorylated Akt. Further studies are required to consider the phosphorylation of Akt and the activa- tion of PI3K in this potential mechanism.
+
+In addition, the results also demonstrated that p75NTR was upregulated following sevoflurane exposure in postnatal mice. It has been reported that the pro-form of nerve growth factor (proNGF)‑induced neuronal apoptosis is dependent on p75NTR in Alzheimer's disease (40). The balance of TrkA/p75NTR signaling is associated with (‑)‑epigallocatechin‑3‑gallate ameliorated learning and memory deficits in APP/PS1 trans- genic mice (41). Furthermore, the balance of TrkA/p75NTR may be regulated by proNGF in the hippocampus (42). Thus, the balance of TrkA/p75NTR in the present study may be broken by sevoflurane, thereby impairing the ability of post- natal mice to form memories. ProNGF may serve a role in the sevoflurane‑induced increase of p75NTR, promoting neuronal apoptosis and memory impairment. However, the effect of sevoflurane expression on TrkA and proNGF in postnatal mice is unknown. Further studies are required to investigate this effect systematically.
+
+4. Liu F, Rainosek SW, Frisch‑Daiello JL, Patterson TA, Paule MG, Slikker W Jr, Wang C and Han X: Potential adverse effects of prolonged sevoflurane exposure on developing monkey brain: From abnormal lipid metabolism to neuronal damage. Toxicol Sci 147: 562-572, 2015.
+
+5. Tagawa T, Sakuraba S, Kimura K and Mizoguchi A: Sevoflurane in combination with propofol, not thiopental, induces a more robust neuroapoptosis than sevoflurane alone in the neonatal mouse brain. J Anesth 28: 815-820, 2014.
+
+6. Zheng S, Chen X, Wang Y and An L: Effects of sevoflurane on brain neuroapoptosis and ability of long-term learning and memory in newborn rats. Beijing Da Xue Xue Bao 47: 674-678, 2015 (In Chinese).
+
+7. Baydas G, Reiter R, Akbulut M, Tuzcu M and Tamer S: Melatonin inhibits neural apoptosis induced by homocysteine in hippo- campus of rats via inhibition of cytochrome c translocation and caspase‑3 activation and by regulating pro‑and anti‑apoptotic protein levels. Neuroscience 135: 879‑886, 2005.
+
+8. Zhou X, Song FH, He W, Yang XY, Zhou ZB, Feng X and Zhou LH: Neonatal exposure to sevoflurane causes apoptosis and reduces nNOS protein expression in rat hippocampus. Mol Med Rep 6: 543‑546, 2012.
+
+9. Istaphanous GK, Howard J, Nan X, Hughes EA, McCann JC, McAuliffe JJ, Danzer SC and Loepke AW: Comparison of the neuroapoptotic properties of equipotent anesthetic concentra- tions of desflurane, isoflurane, or sevoflurane in neonatal mice. Anesthesiology 114: 578-587, 2011.
+
+Additionally, it has been demonstrated that cleavage of pro‑BDNF is essential for long‑term hippocampal plasticity (38). Thus, the inhibition of pro‑BDNF cleavage in mice exposed to sevoflurane may affect hippocampal plasticity in postnatal mice. Meanwhile, the increased expression of caspase‑3 may induce neuronal apoptosis in the hippocampal tissue of postnatal mice in the present study. Thus, sevoflurane may discourage the plas- ticity of the hippocampus and promote neuronal apoptosis in a developing brain by inhibiting the cleavage of pro‑BDNF and upregulating caspase‑3 in postnatal mice.
+
+In the present study, hypoxia and respiratory depression were also evaluated in postnatal mice during sevoflurane exposure. The results identified that there were no significant differences among groups. However, in a previous study by Schlünzen et al (43), the mean PaCO2 and total CBF decreased, which may be induced by sevoflurane anesthesia and may cause hyperventilation. A limitation of the present study was the inability to confirm whether the sevoflurane anesthesia lead to hyperventilation. Further studies are required to inves- tigate the hyperventilation caused by sevoflurane anesthesia.
+
+In conclusion, the present study demonstrated that sevo- flurane‑induced memory impairment may be associated with neuronal apoptosis by inhibiting the cleavage of pro‑BDNF, as well as increasing caspase‑3 and p75NTR levels in the postnatal developing mouse brain.
+
+Acknowledgements
+
+The present study was supported by the Natural Science Foundation of Shanghai (grant no. 11ZR1423200)
+
+References
+
+10. Albasser MM, Amin E, Lin TC, Iordanova MD and Aggleton JP: Evidence that the rat hippocampus has contrasting roles in object recognition memory and object recency memory. Behav Neurosci 126: 659-669, 2012.
+
+11. Chambers RA, Potenza MN, Hoffman RE and Miranker W: Simulated apoptosis/neurogenesis regulates lea r ning and memory capabilities of adaptive neural networks. Neuropsychopharmacology 29: 747-758, 2004.
+
+12. Sung YJ and Ambron RT: PolyADP -ribose polymerase-1 (PARP‑1) and the evolution of learning and memory. Bioessays 26: 1268-1271, 2004.
+
+13. Cohen‑Armon M, Visochek L, Katzoff A, Levitan D, Susswein AJ, Klein R, Valbrun M and Schwartz JH: Long‑term memor y requires polyADP‑ribosylation. Science 304: 1820-1822, 2004.
+
+14. Goldberg S, Visochek L, Giladi E, Gozes I and Cohen‑Armon M: PolyADP-ribosylation is required for long-term memory formation in mammals. J Neurochem 111: 72-79, 2009.
+
+15. Zhou B, Cai Q, Xie Y and Sheng ZH: Snapin recruits dynein to BDNF‑TrkB signaling endosomes for retrograde axonal trans- port and is essential for dendrite growth of cortical neurons. Cell Rep 2: 42-51, 2012.
+
+16. Stansfield KH, Pilsner JR, Lu Q, Wright RO and Guilarte TR: Dysregulation of BDNF‑TrkB signaling in developing hippo- campal neurons by Pb(2+): Implications for an environmental basis of neurodevelopmental disorders. Toxicol Sci 127: 277-295, 2012.
+
+17. Lai H, Zhao H, Zeng L and Lv Y: BDNF and TrkB mRNA expression in aged hippocampus neurons and their relationship with learning and memory. Prog Anatomical Sci 10: 353‑356, 2004 (In Chinese).
+
+18. Barrett GL, Reid CA, Tsafoulis C, Zhu W, Williams DA, Paolini AG, Trieu J and Murphy M: Enhanced spatial memory and hippocampal long-term potentiation in p75 neurotrophin receptor knockout mice. Hippocampus 20: 145-152, 2010.
+
+19. Yao D, He X, Wang J and Zhao Z: Effects of PI3K/Akt signaling pathway on learning and memory abilities in neonatal rats with hypoxic‑ischemic brain damage. Zhongguo Dang Dai Er Ke Za Zhi 13: 424‑427, 2011 (In Chinese).
+
+20. Zhang JF, Zhou W and Wang T: Effects of propofol on studying and memory dysfunction and cortex trkb/akt pathway in focal cerebral ischemia/reperfusion injury of rats. Herald Med 2: 008, 2012 (In Chinese).
+
+1. Erden V, Erkalp K, Yangin Z, Delatioglu H, Kiroglu S, Ortaküz S and Ozdemir B: The effect of labor on sevoflurane requirements during cesarean delivery. Int J Obstet Anesth 20: 17-21, 2011. 2. Vora KS, Shah VR, Patel D, Modi MP and Parikh GP: Sevoflurane versus propofol in the induction and maintenance of anaesthesia in children with laryngeal mask airway. Sri Lanka J Child Health 43: 77‑83, 2014.
+
+21. Sadrian B, Subbanna S, Wilson DA, Basavarajappa B and Saito M: Lithium prevents long-term neural and behavioral pathology induced by early alcohol exposure. Neuroscience 206: 122‑135, 2012.
+
+22. National Research Council: Guide for the Care and Use of Laboratory Animals. National Academies Press, Washington, DC, pp1072‑1073, 2011.
+
+3. Lerman J, Sikich N, Kleinman S and Yentis S: The pharma- cology of sevoflurane in infants and children. Anesthesiology 80: 814-824, 1994.
+
+23. Wiklund A, Granon S, Faure P, Sundman E, Changeux JP and Eriksson LI: Object memory in young and aged mice after sevo- flurane anaesthesia. Neuroreport 20: 1419‑1423, 2009.
+
+4103
+
+4104
+
+LU et al: SEVOFLURANE EXPOSURE IN POSTNATAL MICE
+
+24. Yue T, Shanbin G, Ling M, Yuan W, Ying X and Ping Z: Sevoflurane aggregates cognitive dysfunction and hippocampal oxidative stress induced by β‑amyloid in rats. Life Sci 143: 194-201, 2015.
+
+25. Zhang F, Feng X, Zeng Q, Wang B, Wilhelmsen K, Li Q, Cao X and Yu B: Sevoflurane induced amnesia inhibits hippocampal Arc expression partially through 5-hydroxytryptamine-7 recep- tors in the bilateral basolateral amygdala in rats. Neurosci Lett 562: 13‑18, 2014.
+
+26. Zhong T, Guo Q, Zou W, Zhu X, Song Z, Sun B, He X and Yang Y: Neonatal isoflurane exposure induces neurocognitive impairment and abnormal hippocampal histone acetylation in mice. Plos One 10: e0125815, 2015.
+
+27. Musumeci G, Castrogiovanni P, Loreto C, Castorina S, Pichler K and Weinberg AM: Post‑traumatic caspase‑3 expression in the adjacent areas of growth plate injury site: A morphological study. Int J Mol Sci 14: 15767‑15784, 2013.
+
+28. Ge HW, Hu WW, Ma LL and Kong FJ: Endoplasmic reticulum stress pathway mediates isoflurane‑induced neuroapoptosis and cognitive impairments in aged rats. Physiol Behav 151: 16‑23, 2015. 29. Lin D and Zuo Z: Isoflurane induces hippocampal cell injury and cognitive impairments in adult rats. Neuropharmacology 61: 1354‑1359, 2011.
+
+30. Cao J, Wang Z, Mi W and Zuo Z: Isoflurane unveils a critical role of glutamate transporter type 3 in regulating hippocampal GluR1 trafficking and context‑related learning and memory in mice. Neuroscience 272: 58-64, 2014.
+
+31. Li XM, Su F, Ji MH, Zhang GF, Qiu LL, Jia M, Gao J, Xie Z and Yang JJ: Disruption of hippocampal neuregulin 1-ErbB4 signaling contributes to the hippocampus-dependent cognitive impairment induced by isoflurane in aged mice. Anesthesiology 121: 79‑88, 2014.
+
+35. Comim CM, Barichello T, Grandgirard D, Dal‑Pizzol F, Quevedo J and Leib SL: Caspase‑3 mediates in part hippocampal apoptosis in sepsis. Mol Neurobiol 47: 394‑398, 2013.
+
+36. Gerace E, Masi A, Resta F, Felici R, Landucci E, Mello T, Pellegrini‑Giampietro DE, Mannaioni G and Moroni F: PARP‑1 activation causes neuronal death in the hippocampal CA1 region by increasing the expression of Ca(2+)‑permeable AMPA recep- tors. Neurobiol Dis 70: 43‑52, 2014.
+
+37. Barnes P and Thomas KL: Proteolysis of proBDNF is a key regulator in the formation of memory. PLoS One 3: e3248, 2008. 38. Mizuno M, Yamada K, Takei N, Tran MH, He J, Nakajima A, Nawa H and Nabeshima T: Phosphatidylinositol 3‑kinase: A molecule mediating BDNF‑dependent spatial memory forma- tion. Mol Psychiatry 8: 217‑224, 2003.
+
+39. Shaerzadeh F, Motamedi F and Khodagholi F: Inhibition of akt phosphorylation diminishes mitochondrial biogenesis regulators, tricarboxylic acid cycle activity and exacerbates recognition memory deficit in rat model of Alzheimer's disease. Cell Mol Neurobiol 34: 1223‑1233, 2014.
+
+40. Pedraza CE, Podlesniy P, Vidal N, Arévalo JC, Lee R, Hempstead B, Ferrer I, Iglesias M and Espinet C: Pro‑NGF isolated from the human brain affected by Alzheimer's disease induces neuronal apoptosis mediated by p75NTR. Am J Pathol 166: 533‑543, 2005. 41. Liu M, Chen F, Sha L, Wang S, Tao L, Yao L, He M, Yao Z, Liu H, Zhu Z, et al: (‑)‑Epigallocatechin‑3‑gallate ameliorates learning and memory deficits by adjusting the balance of TrkA/p75NTR signaling in APP/PS1 transgenic mice. Mol Neurobiol 49: 1350‑1363, 2014.
+
+42. Fortress AM, Buhusi M, Helke KL and Granholm AC: Cholinergic degeneration and alterations in the TrkA and p75NTR balance as a result of pro‑NGF injection into aged rats. J Aging Res 2011: 460543, 2011.
+
+32. Cao Y, Ni C, Li Z, Li L, Liu Y, Wang C, Zhong Y, Cui D and Guo X: Isoflurane anesthesia results in reversible ultrastructure and occludin tight junction protein expression changes in hippocampal blood-brain barrier in aged rats. Neurosci Lett 587: 51-56, 2015. 33. Li Z, Cao Y, Li L, Liang Y, Tian X, Mo N, Liu Y, Li M, Chui D and Guo X: Prophylactic angiotensin type 1 receptor antagonism confers neuroprotection in an aged rat model of postoperative cognitive dysfunction. Biochem Biophys Res Commun 449: 74-80, 2014.
+
+43. Schlünzen L, Vafaee M, Juul N and Cold G: Regional cerebral blood flow responses to hyperventilation during sevoflurane anaesthesia studied with PET. Acta Anaesthesiol Scand 54: 610-615, 2010. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) License.
+
+34. Hatip‑Al‑Khatib I, Iwasaki K, Chung EH, Egashira N, Mishima K and Fujiwara M: Inhibition of poly (ADP‑ribose) polymerase and caspase‑3, but not caspase‑1, prevents apoptosis and improves spatial memory of rats with twice-repeated cerebral ischemia. Life Sci 75: 1967-1978, 2004.

new_pdfs/10.3892_mmr.2014.2751.txt

@@ -0,0 +1,167 @@
+226
+
+MOLECULAR MEDICINE REPORTS 11: 226-230, 2015
+
+Single sevoflurane exposure increases methyl‑CpG island binding protein 2 phosphorylation in the hippocampus of developing mice
+
+XIAO‑DAN HAN, MIN LI, XIAO‑GUANG ZHANG, ZHANG‑GANG XUE and JING CANG
+
+Department of Anesthesia, Zhongshan Hospital, Fudan University, Shanghai 200032, P.R. China
+
+Received December 16, 2013; Accepted June 9, 2014
+
+DOI: 10.3892/mmr.2014.2751
+
+Abstract. Sevoflurane is an inhaled anesthetic that is widely used in clinical practice, particularly for pediatric anesthesia. Previous studies have suggested that sevoflurane may induce neurotoxicity in the brains of neonatal mice. In the present study, the possible mechanism of neurodegeneration induced by sevoflurane in the developing brain, and the possibility that memantine treatment is able to reverse this phenomenon, were investigated. On postnatal day 7 (P7) C57BL/6 mice were continuously exposed to 1.5% sevoflurane for 2 h following pre‑injection of saline or memantine. Methyl‑CpG island binding protein 2 (MeCP2), cAMP response element‑binding protein (CREB) and brain‑derived neurotrophic factor (BDNF) expression in the hippocampus was measured by western blotting. Exposure to 1.5% sevoflurane resulted in increased MeCP2 phosphorylation in the hippocampus, which was reversed by memantine injection. However, neither CREB phosphorylation nor BDNF expression were significantly altered by sevoflurane treatment. The current study indicated that sevoflurane causes neurotoxicity in the developing brain, and that this may be attributed to increased MeCP2 phosphor- ylation in the hippocampus. It was also demonstrated that this neurotoxicity can be prevented by the N‑methyl‑D‑aspartate glutamate receptor inhibitor memantine.
+
+Introduction
+
+persistent learning deficits (1,2). Loepke et al (3) observed that isoflurane led to apoptotic neurodegeneration, and Head et al (4) elucidated the relevant signaling pathways. Satomoto et al (5) demonstrated that exposing neonatal mice to sevoflurane results in learning deficits, in addition to autism‑like abnormal social behavior. These studies together demonstrate that sevoflurane is harmful to the developing brain.
+
+Methyl‑CpG island binding protein 2 (MeCP2) is a transcriptional repressor and is important in neuron matura- tion and normal brain function (6). Studies have suggested that MeCP2 is closely associated with NMDA receptors in the brain (7,8). MeCP2 is a nuclear protein that selectively binds methylated DNA, then recruits other proteins to form an inhibition complex, thereby inhibiting the expression of various genes (9,10). MeCP2 gene mutations can cause the neurodevelopmental disorder Rett syndrome (11), which can lead to cognitive impairment, motor disability and repetitive stereotyped hand movements. Up to 80% of those affected with Rett syndrome experience seizures. A number of studies have hypothesized that these symptoms may be due to defects in experience‑dependent synapse maturation (12,13), where normal synapse connections fail to establish in the critical period. These studies have demonstrated that MeCP2 is critical in the maturation of the nervous system and the normal functioning of nerve cells.
+
+Sevoflurane is widely used in the clinic, particularly in pediatric anesthesia. However, infants brains are at a stage at which important changes are occurring, including synapse formation and axon and dendrite growth; this renders them strongly susceptible to environmental influences, such as general anesthetics. A number of studies have demonstrated that early exposure to a variety of anesthetics can cause widespread neurodegeneration in the developing brain and
+
+cAMP response element‑binding protein (CREB) is a nuclear regulatory factor that may regulate gene transcrip- tion following phosphorylation of serine 133, thus enhancing the expression of multiple target genes. One study indicated that CREB is the major transcription regulatory factor of brain‑derived neurotrophic factor (BDNF), and thus increases BDNF expression. This is a crucial process in the survival and differentiation of neurons (14). Another study demonstrated that isoflurane anesthesia induces neuronal apoptosis by affecting the formation of neuronal synapses in the develop- mental stages, potentially via the BDNF‑p75‑RhoA signaling pathway (4).
+
+Correspondence to: Dr Jing Cang, Department of Anesthesia, Building 10, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, P.R. China E‑mail: cangj_jcang@163.com
+
+Key words: sevoflurane, neurotoxicity, P7 mice, MeCP2, memantine, hippocampus
+
+Memantine is a derivative of amantadine, and acts on the glutamatergic system by inhibiting NMDA receptors. It is involved in neuroprotection and acts to preserve normal synaptic function. It is approved for use in moderate‑to‑severe Alzheimer's disease, by the FDA and the European Medicines Agency, and has been demonstrated to produce positive effects on cognition, mood and behavior (15,16).
+
+HAN et al: SINGLE SEVOFLURANE EXPOSURE INCREASES MECP2 PHOSPHORYLATION
+
+To investigate the molecular mechanism of neurodegen- eration induced by sevoflurane, neonatal mice were exposed to sevoflurane and the protein expression levels of CREB and BDNF were assessed in the developing hippocampus. Protein expression levels of MeCP2 and the effect of pre‑injected memantine were also examined, in order to determine whether memantine is able to reverse the neurodegeneration.
+
+Materials and methods
+
+Animals and sevoflurane exposure. All animal experiments were performed using protocols approved by the institutional animal use and care committee of the Zhongshan Hospital, Fudan University (Shanghai, China). At postnatal day 7 (P7), male C57BL/6 mice (weight, 3‑5 g; Shanghai Laboratory Animal Center, Shanghai, China) were randomly divided into a sevoflurane‑treated group (n=6) and an air‑treated control group (n=6) for analysis of the effects of sevoflurane on CREB phosphorylation, BDNF expression and MeCP2 phosphorylation levels. Mice were placed in a plastic container and continuously exposed to 1.5% sevoflurane (Maruishi Pharmaceutical Co., Osaka, Japan) in air, or to air alone for 2 h, with a gas flow of 2 l/min. For further experiments, male C57BL/6 mice at postnatal day 7 (P7) were randomly divided into four groups: The sevoflurane‑saline group (sevo group, n=7); the air‑saline group (control group, n=6); the sevoflurane‑memantine group (sevo+mem group, n=7); and the air‑memantine group (mem group, n=6). Mice received 1 mg/kg saline or memantine intraperitoneally prior to sevo- flurane or air treatment. The mice were then placed in a plastic container and continuously exposed to 1.5% sevoflurane in air or to air alone for 2 h, with a gas flow of 2 l/min. During expo- sure to sevoflurane or air, the container was heated to 37˚C with a heating pad. The concentrations of sevoflurane, oxygen and carbon dioxide in the container were monitored with a gas monitor (Datex Cardiocap Ⅱ, Datex‑Ohmeda, Madison, WI, USA). Following exposure to sevoflurane or air, the mice were returned to their cages. The mice were housed six per cage and maintained on a 12 h light/dark cycle with access to food and water ad libitum. Two hours post‑exposure the mice were sacrificed by decapitation, and their hippocampi were removed.
+
+Sodium dodecyl sulphate (SDS) was added to the samples prior to boiling for 10 min at 100˚C. Equal quantities of protein (15 µg) were used to detect the expression of the proteins of interest. Samples were electrophoresed on 10 or 15% SDS polyacrylamide gel, blotted onto polyvinylidine fluoride membranes (Bio‑Rad Laboratories, Hercules, CA, USA) and then incubated with the following antibodies overnight at 4˚C: Anti‑phospho‑CREB (ser133), (cat no. 06‑519, EMD Millipore, Billerica, MA, USA) 1:4,000 dilution in 5% non‑fat milk; anti‑CREB (cat no. MAB5432, Millipore) 1:5,000 dilution in 5% non‑fat milk; anti‑BDNF (cat no. AB1779SP, Millipore) 1:1,000 dilution in 5% non‑fat milk; anti‑MeCP2 (cat no. 3456P, Cell Signaling Technology, Danvers, MA, USA) 1:4,000 dilution in 5% non‑fat milk; anti‑phospho‑MeCP2‑S421 (cat no. AP3693a, Abgent Biotech, Suzhou, China) 1:2,000 dilution in 5% non‑fat milk); and anti‑actin (cat no. A5441, Sigma‑Aldrich, St. Louis, MO, USA) 1:10,000 dilution in 5% non‑fat milk. The following day, the blots were incubated for 1 h at room temperature with horseradish peroxidase‑conjugated secondary goat anti‑rabbit or goat anti‑mouse immunoglob- ulin G (Kangchen, Shanghai, China), 1:5,000 dilution in 5% non‑fat milk. Immunoreactive bands were visualized using Amersham ECL Prime Western Blotting Detection kit (cat NO.RPN2232; GE Healthcare, Chalfont St. Giles, UK). The protein signals were quantified using Quantity One software and a GS‑800 Calibrated Imaging Densitometer (Bio‑Rad Laboratories) and normalized to a corresponding internal reference: CREB for the exression of p‑CREB‑S133, MeCP2 for P‑MeCP2‑S421 and actin for BDNF.
+
+Statistical analysis. All data are presented as the mean ± stan- dard error. Data were analyzed using the unpaired Student's t‑test in Origin software, version 7.5 (OriginLab, Northampton, MA, USA). P<0.05 was considered to represent a statistically significant difference.
+
+Results
+
+Arterial blood gas analyses. Arterial blood samples were obtained from the left cardiac ventricle of the mice immedi- ately after exposure to sevoflurane, and were transferred to heparinized glass capillary tubes. Blood pH, partial pressure of carbon dioxide in mmHg (PaCO2), partial pressure of oxygen in mmHg (PaO2), lactate (Lac), and bicarbonate (HCO3) were analyzed immediately after blood collection using a GEM Premier 3000 analyzer (Instrumentation Laboratory, Lexington, MA, USA).
+
+Protein extraction and western blot analysis. Resected hippocampi were placed into 1.5‑ml centrifuge tubes and preserved in liquid nitrogen. All methods were conducted on ice. An NE‑PER Nuclear and Cytoplasmic Extraction kit (cat no.78835; Thermo Fisher Scientific, Waltham, MA, USA) was used to extract protein samples. All steps were conducted according to the manufacturer's instructions.
+
+Sevoflurane does not induce metabolic or respiratory dete- rioration. Blood gas analyses indicated that there was no deterioration in respiration or metabolism in the animals following a 2‑h sevoflurane exposure. All parameters were tested, including PH, PaCO2, PaO2, oxygen saturation, Lac and HCO3. No significant differences in any of the parameters were detected between the sevoflurane and the control group (P>0.05; Table I).
+
+Sevoflurane does not cause changes in CREB phosphoryla- tion and BDNF expression in the hippocampus. Western blot analyses of hippocampal CREB phosphorylation and BDNF expression levels were performed 2 h following exposure to sevoflurane or air. The results indicated that there were no significant differences in levels of CREB phosphorylation (Fig. 1) and BDNF (Fig. 2) expression between the sevoflu- rane‑treated (n=6) and air‑treated (n=6) groups.
+
+Sevoflurane treatment increases MeCP2 phosphorylation at the serine 421 loci in the hippocampus. Western blot analyses of hippocampal MeCP2 phosphorylation in the
+
+227
+
+228
+
+MOLECULAR MEDICINE REPORTS 11: 226-230, 2015
+
+Table I. Arterial blood gas analysis.
+
+Arterial blood gas
+
+Control (n=6)
+
+Sevoflurane (n=6)
+
+pH PaCO2 (mmHg) PaO2 (mmHg) SaO2 (%) Lac (mmol/l) HCO3 (mmol/l)
+
+7.4±0.05 28.4±3.0 91.0±6.8 93.6±2.6 1.1±0.5 23.8±1.6
+
+7.38±0.04 30.1±4.0 88.8±5.6 92.7±2.5 1.2±0.4 24.1±1.5
+
+Exposure to sevoflurane did not induce significant metabolic or respiratory impairment. Analysis of arterial blood gas revealed no significant differences in any of the measured parameters between the sevoflurane group and the control one (t‑test, P>0.05). Pa, partial pressure; Sa, saturation.
+
+Figure 3. Sevofl urane treatment leads to a signifi cant increase in hippo‑ 3. Sevoflurane treatment leads to a significant increase in hippo- campal P‑MeCP2‑S421 expression levels. *P<0.05, sevoflurane‑treated group vs. control group at P7. MeCP2, methyl‑CpG island binding protein 2.
+
+Figure 1. Sevoflurane treatment did not cause significant changes in hip- pocampal CREB phosphorylation in mouse hippocampi at postnatal day 7. P>0.05, sevoflurane group (n=6) vs. the control group (n=6). CREB, cAMP response element‑binding protein.
+
+Figure 4. Memantine treatment results in normalized P‑MeCP2‑S421 expres- sion levels following sevoflurane treatment. Sevoflurane‑treated (n=7) mice exhibited a significant increase in hippocampal P‑MeCP2‑S421 expression levels (*P<0.05 vs. control group, n=6). The sevo + mem group (n=7) exhib- ited a significant decrease in hippocampal P‑MeCP2‑S421 expression levels (*P<0.05 vs. sevo group). Significantly different levels of P‑MeCP2‑S421 expression were not identified in the mem group (n=6) when compared with the control group (n=6) (P>0.05). MeCP2, methyl‑CpG island binding protein 2.
+
+Figure 2. Sevoflurane treatment did not cause significant changes in hip- pocampal BDNF expression levels in mouse hippocampi at postnatal day 7. P>0.05, sevoflurane group (n=6) vs. control group (n=6). BDNF, brain‑derived neurotrophic factor.
+
+sevoflurane‑treated and control groups were performed 2 h following exposure to sevoflurane or air. The results indi- cated that sevoflurane‑treated mice exhibited an increase
+
+in hippocampal MeCP2 phosphorylation at serine 421 loci (P‑MeCP2‑S421), compared with phosphorylation in the hippocampi of the control mice. There expression level of P‑MeCP2‑S421 was increased in the sevoflurane‑treated group (n=6) compared with the control group(n=6) (P<0.05; Fig. 3).
+
+Sevoflurane increases the MeCP2 phosphorylation at the serine 421 loci in the hippocampus, and pre‑injection of memantine reverses this phenomenon. Western blot analyses of hippocampal MeCP2 phosphorylation were performed 2 h following exposure to sevoflurane or air. Compared with the control group (air and saline, n=6), the sevo group (sevoflurane
+
+HAN et al: SINGLE SEVOFLURANE EXPOSURE INCREASES MECP2 PHOSPHORYLATION
+
+and saline, n=7) exhibited a significant increase in hippo- campal P‑MeCP2‑S421 expression levels (P<0.05; Fig. 4). Compared with the sevo group, the sevo+mem group exhib- ited a significant decrease in hippocampal P‑MeCP2‑S421 levels (P<0.05; Fig. 4). No significant difference was detected between the mem group (memantine and saline, n=6) and the control group (n=6).
+
+for synaptic NMDA receptors than extrasynaptic NMDA receptors. Thus, it exhibits an important role in neuroprotec- tion, while preserving normal synaptic function (26). In the present study, memantine was able to reverse the increase in MeCP2 phosphorylation in the hippocampus following sevoflurane exposure. This demonstrates that memantine may have a protective effect against neurodegeneration induced by sevoflurane exposure.
+
+Discussion
+
+In the present study, it was demonstrated that P‑MeCP2‑S421 expression levels in hippocampi resected at P7 increased in mice exposed to 1.5% sevoflurane for 2 h, and that meman- tine pre‑injection was able to reverse this increase. However, sevoflurane did not cause significant changes in CREB phos- phorylation and BDNF expression levels in the hippocampus. A dose of 1.5% sevoflurane, which did not inhibit respira- tion and circulation in mouse pups, was selected for the current study. Arterial blood analyses confirmed that none of the mice experienced hypoxemia or hypercapnia during the 2‑h sevoflurane exposure; there were no significant differences in any of the tested parameters between the sevoflurane group and the control group. These results exclude the possibility of hypoxemia and hypercapnia affecting the outcome of the following experiments.
+
+In conclusion, the results of the present study demonstrated that P‑MeCP2‑S421 expression levels in the hippocampus increased in P7 mice exposed to 1.5% sevoflurane for 2 h, and pre‑injected memantine reversed this increase. Sevoflurane did not cause changes in CREB phosphorylation or BDNF expression levels in the hippocampus. Future investigation of MeCP2 and NMDA receptors is required in order to further investigate their effects on the central nervous system during its development.
+
+Acknowledgements
+
+The current study was supported by grants from the National Natural Science Foundation of China (grant no. 81100796).
+
+References
+
+A previous study demonstrated that early exposure to sevoflurane causes widespread neurodegeneration in the developing brain (17). However, the exact mechanism of action underlying the effect of sevoflurane remains unknown. The results of the present study may provide a possible explana- tion for sevoflurane‑mediated neurodegeneration, as they suggested that MeCP2 may be important in neuronal degen- eration following neonatal sevoflurane exposure.
+
+The γ‑a m inobutyr ic acid type A (GA BA) and N‑methyl‑D‑aspartate glutamate (NMDA) receptors are essential for the development of an ordered neural map (18,19), and are important in the alteration of synaptic transmis- sion. Neurotransmitters or compounds that act on them may contribute to the impairment of brain development and synaptogenesis (20,21). MeCP2 links closely with NMDA receptors in the brain, and NMDA receptors (particularly NR2A) are essential for visual cortical function in the absence of MeCP2 (22). The activity‑dependent expression of another NMDA subunit (NR2B) is mediated by MeCP2‑dependent epigenetic regulation (23). Thus, as indicated in the present study, MeCP2 may regulate NMDA receptors, leading to various effects on brain function in mice. MeCP2 phos- phorylation at serine 421 loci is a key signal, which may cause downstream changes in the signaling pathway and influence the central nervous system. Previous studies indicated that CDKL5 is a target of MeCP2 in the brain, and is regulated by DNA methylation (24). MeCP2 can also interact with some microRNAs to regulate brain function (25). Further study of these aspects is required in order to figure out whether and how microRNAs are involved in the regulation of neuroplas- ticity in the brain.
+
+1. Jevtovic‑Todorovic V, Hartman RE, Izumi Y, et al: Early exposure to common anesthetic agents causes widespread neurodegen- eration in the developing rat brain and persistent learning deficits. J Neurosci 23: 876‑882, 2003.
+
+2. Liang G, Ward C, Peng J, Zhao Y, Huang B and Wei H: Isoflurane causes greater neurodegeneration than an equivalent exposure of sevoflurane in the developing brain of neonatal mice. Anesthesiology 112: 1325‑1334, 2010.
+
+3. Loepke AW, Istaphanous GK, McAuliffe JR, et al: The effects of neonatal isoflurane exposure in mice on brain cell viability, adult behavior, learning, and memory. Anesth Analg 108: 90‑104, 2009. 4. Head BP, Patel HH, Niesman IR, Drummond JC, Roth DM and Patel PM: Inhibition of p75 neurotrophin receptor attenuates isoflurane‑mediated neuronal apoptosis in the neonatal central nervous system. Anesthesiology 110: 813‑825, 2009.
+
+5. Satomoto M, Satoh Y, Terui K, et al: Neonatal exposure to sevo- flurane induces abnormal social behaviors and deficits in fear conditioning in mice. Anesthesiology 110: 628‑637, 2009.
+
+6. Kishi N and Macklis JD: MECP2 is progressively expressed in post‑migratory neurons and is involved in neuronal maturation rather than cell fate decisions. Mol Cell Neurosci 27: 306‑321, 2004.
+
+7. Nguyen MV, Du F, Felice CA, et al: MeCP2 is critical for main- taining mature neuronal networks and global brain anatomy during late stages of postnatal brain development and in the mature adult brain. J Neurosci 32: 10021‑10034, 2012.
+
+8. Kron M, Howell CJ, Adams IT, et al: Brain activity mapping in Mecp2 mutant mice reveals functional deficits in forebrain circuits, including key nodes in the default mode network, that are reversed with ketamine treatment. J Neurosci 32: 13860‑13872, 2012.
+
+9. Cohen S, Gabel HW, Hemberg M, et al: Genome‑wide activity‑dependent MeCP2 phosphorylation regulates nervous system development and function. Neuron 72: 72‑85, 2011.
+
+10. Fuks F, Hurd PJ, Wolf D, Nan X, Bird AP and Kouzarides T: The methyl‑CpG‑binding protein MeCP2 links DNA methylation to histone methylation. J Biol Chem 278: 4035‑4040, 2003.
+
+11. Bienvenu T and Chelly J: Molecular genetics of Rett syndrome: when DNA methylation goes unrecognized. Nat Rev Genet 7: 415‑426, 2006.
+
+Memantine is an amantadine derivative and NMDA receptor inhibitor. It has been used to treat Alzheimer's disease and is accepted to be safer than other NMDA receptor inhibi- tors, as a therapeutic dose of memantine has a greater affinity
+
+12. Zhou Z, Hong EJ, Cohen S, et al: Brain‑specific phosphorylation of MeCP2 regulates activity‑dependent Bdnf transcription, dendritic growth, and spine maturation. Neuron 52: 255‑269, 2006.
+
+13. Li H, Zhong X, Chau KF, Williams EC and Chang Q: Loss of activity‑induced phosphorylation of MeCP2 enhances synapto- genesis, LTP and spatial memory. Nat Neurosci 14: 1001‑1008, 2011.
+
+229
+
+230
+
+MOLECULAR MEDICINE REPORTS 11: 226-230, 2015
+
+14. Tao X, Finkbeiner S, Arnold DB, Shaywitz AJ and Greenberg ME: Ca2+ influx regulates BDNF transcription by a CREB family transcription factor‑dependent mechanism. Neuron 20: 709‑726, 1998.
+
+21. Yon JH, Daniel‑Johnson J, Carter LB and Jevtovic‑Todorovic V: Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience 135: 815‑827, 2005.
+
+15. Areosa Sastre ASF and McShane R: Memantine for dementia. The Cochrane Library. 2006.
+
+16. Mount C and Downton C: Alzheimer disease: progress or profit? Nat Med 12: 780‑784, 2006.
+
+17. Zhang X, Xue Z and Sun A: Subclinical concentration of sevo- flurane potentiates neuronal apoptosis in the developing C57BL/6 mouse brain. Neurosci Lett 447: 109‑114, 2008.
+
+22. Durand S, Patrizi A, Quast KB, et al: NMDA receptor regulation prevents regression of visual cortical function in the absence of Mecp2. Neuron 76: 1078‑1090, 2012.
+
+23. Lee S, Kim W, Ham BJ, Chen W, Bear MF and Yoon BJ: Activity‑dependent N R2B expression is mediated by MeCP2‑dependent epigenetic regulation. Biochem Biophys Res Commun 377: 930‑934, 2008.
+
+18. Simon DK, Prusky GT, O'Leary DD and Constantine‑Paton M: N‑methyl‑D‑aspartate receptor antagonists disrupt the formation of a mammalian neural map. Proc Natl Acad Sci USA 89: 10593‑10597, 1992.
+
+19. Hardingham GE, Fukunaga Y and Bading H: Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut‑off and cell death pathways. Nat Neurosci 5: 405‑414, 2002. 20. Johnson SA, Young C and Olney JW: Isoflurane‑induced neuro- apoptosis in the developing brain of nonhypoglycemic mice. J Neurosurg Anesthesiol 20: 21‑28, 2008.
+
+24. Carouge D, Host L, Aunis D, Zwiller J and Anglard P: CDKL5 is a brain MeCP2 target gene regulated by DNA methylation. Neurobiol Dis 38: 414‑424, 2010.
+
+25. Im HI, Hollander JA, Bali P and Kenny PJ: MeCP2 controls BDNF expression and cocaine intake through homeostatic inter- actions with microRNA‑212. Nat Neurosci 13: 1120‑1127, 2010. 26. Xia P, Chen HS, Zhang D and Lipton SA: Memantine prefer- entially blocks extrasynaptic over synaptic NMDA receptor currents in hippocampal autapses. J Neurosci 30: 11246‑11250, 2010.