DOI,Citation ID,First author,Year,animal type,exposure age,behavior test: Y/N,intervention1,intervention2 （anesthetics only),genetic chain,content,Question 1,Question 1_original_sentences,Question 2,Question 2_original_sentences,Question 3,Question 3_original_sentences,intervention_1,Question 4intervention_1_original_sentences,intervention_2,Question 4intervention_2_original_sentences,Question 5,Question 5_original_sentences,Question 6,Question 6_original_sentences,Question 7,Question 7_original_sentences,Question 8,Question 8_original_sentences,Question 9,Question 9_original_sentences,Question 10,Question 10_original_sentences,correct_1,correct_2,correct_3,correct_4,correct_5,correct_6,fn
10.1016/j.biopha.2018.09.111,279.0,Bi,2018,rats,postnatal day 7,Y,sevoflurane,none,sprague dawley,"Biomedicine & Pharmacotherapy 108 (2018) 1469–1476

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Original article

Sevoflurane induces neurotoxicity in the developing rat hippocampus by upregulating connexin 43 via the JNK/c-Jun/AP-1 pathway

T

Congjie Bia,b, Qiuping Caib, Yangyang Shana, Fan Yanga, Shiwei Suna, Xiuying Wua, Hongtao Liua,⁎ a Department of Anesthesiology, Shengjing Hospital, China Medical University, Shenyang, China b Department of Anesthesiology, Dalian Central Hospital, Dalian, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Connexin 43 Sevoflurane Neurotoxicity MAPK JNK AP-1

As one of the most popular anesthetics, sevoflurane is widely used in pediatric anesthesia. Unfortunately, an increasing number of studies have demonstrated that sevoflurane has potential neurotoxic effects on the de- veloping brain and cognition, even in adolescence. Connexin 43 (Cx43) has been documented to contribute to cognitive dysfunction. The present study hypothesized that Cx43 may participate in sevoflurane-induced neu- roinjury and investigated the underlying mechanisms in young Sprague Dawley (SD) rats. Seven-day-old SD rats (P7) were exposed to 3% sevoflurane for 4 h. The levels of Cx43,mitogen-activated protein kinase (MAPK) signaling pathway components(including total and phosphorylated p38, extracellular signal-regulated kinase (ERK), and c-Jun n-terminal kinase (JNK) and activator protein 1(AP-1) transcription factors (including total and phosphorylated c-Fos, and c-Jun) were assessed by Western blot analysis. Neuronal apoptosis was detected using immunohistochemistry (IHC). The Morris water maze (MWM) was performed to evaluate cognitive function from P28 to P33. The results showed that anesthesia with 3% sevoflurane for 4 h increased Cx43 levels in the rat hippocampus from 6 h to 3 d, and compared with sevoflurane exposure in the control group rats, exposure in P7 SD rats also increased the ratios of phosphorylated JNK to JNK and, phosphorylated c-Jun to c-Jun in the hippocampus from 6 h to 3 d. All these effects could be alleviated by pretreatment with the JNK inhibitor SP600125 (10 mg/kg). Neuroapoptosis was similarly increased from 6 h to 1 d after inhaled sevoflurane ex- posure. Finally, the MWM indicated that sevoflurane could increase the escape latency and, decrease the number of platform crossings from P28 to P33. Overall, our findings suggested that sevoflurane increased Cx43 ex- pression and induced to apoptosis by activating the JNK/c-Jun signaling pathway in the hippocampus of P7 rats. This finding may reveal a new strategy for preventing sevoflurane-induced neuronal dysfunction.

1. Introduction

As the most popular inhalation anesthetic, sevoflurane has many merits, including low pungency, a sweet smell and a low blood/gas partition coefficient. Despite these benefits, sevoflurane has been re- ported to exert potentially neurotoxic effects on the developing brain and to induce neuronal apoptosis. Moreover, in humans, reports in- dicate that children exposed to anesthesia in early life have a poten- tially increased incidence of learning deficits in adolescence [1,2]. The associated mechanisms may include neuroapoptosis, neuroinflamma- tion, reactive oxygen species accumulation, neurotransmitter dis- turbances, and changes in synaptic plasticity [3–6]. Unfortunately, there are still few clinical interventions and treatments available to prevent these potential neuronal dysfunctions.

In the central nervous system (CNS), glial cells, especially astro- cytes, express large amounts of connexins (Cxs). To date, 20 and 21 isoforms of Cxs have been identified in mice and humans, respectively. Among these Cxs, Cx43 is the most abundant protein expressed in as- trocytes [7]. Cxs can form homomeric or heteromeric hexamers on cellular plasma membranes. These structures are also called connexons or hemichannels and, can align and dock with other connexons on neighboring cells to form gap junction (GJ) channels [8–10]. Previous studies have demonstrated that sevoflurane-induced cytotoxic effects were related to the Cx43 protein in many tissues. Hirata et al, [11] suggested that sevoflurane reduced phosphorylated-Cx43 protein levels during myocardial ischemia and led arrhythmias in adult rats. Huang F et al, [12] found that sevoflurane induced cytotoxicity in rat liver BRL- 3 A cells by reducing Cx32 but not Cx43 protein. However, few reports

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Corresponding author. E-mail address: 2895364359@qq.com (H. Liu).

https://doi.org/10.1016/j.biopha.2018.09.111 Received 30 May 2018; Received in revised form 11 September 2018; Accepted 19 September 2018 0753-3322/ © 2018 Published by Elsevier Masson SAS.

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have examined the effects of sevoflurane on the Cx43 protein in the developing brain.

Mitogen-activated protein kinases (MAPKs) are a family of serine- threonine protein kinases that include three major members: extra- cellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal ki- nase (JNK). MAPK signaling cascades play pivotal roles in both normal and pathological conditions, such as nervous system development [13], neurodegeneration [14], sensation [15]and brain inflammation [16]. Recent studies also showed that MAPK pathways are associated with anesthetic-induced neurotoxicity. Shinya Yufune et al. [17] observed that the suppression of ERK phosphorylation through oxidative stress was involved in the mechanism that underlies sevoflurane-induced toxicity in the developing brain. Another study [18] found that both the JNK and p38 MAPK pathways participate in protection against iso- flurane-induced neuroapoptosis by dexmedetomidine in the hippo- campus of neonatal rats. An important target of MAPK is activator protein 1 (AP-1), which is a key regulator of the response to environ- mental stress [19]. AP-1 is a dimeric transcription factor that consists of basic leucine-zipper (bZIP) proteins, mainly from the Fos and Jun fa- milies. AP-1 proteins are targets of the mitogen-activated protein (MAP) kinase cascade (Fig. 1). To date, there are no reports about the effects of inhaled anesthetics on Cx43 expression and its relationship with post- operative cognitive dysfunction in the developing brain. Therefore, we hypothesized that exposure to sevoflurane during development would increase the expression of Cx43 and cleaved-caspase-3 in the hippo- campus through the modulation of Ap-1/MAPK cascades.

2. Materials and methods

2.1. Reagents

The following anesthetics and substances were used: sevoflurane (Abbott, Wiesbaden, Germany), anti-Cx43, and anti-GAPDH(Sigma- Aldrich, St. Louis, MO, USA). All other reagents were purchased from Cell Signaling Technology (Boston, MA) unless otherwise specified.

2.2. Ethical approval

The present study was approved by the animal care and ethics committee of ShengJing Hospital of China Medical University (Shenyang, China) and was performed in accordance with the National Institutes of Health Guide for the Use of Laboratory Animals.

2.3. Study animals

A total of 30 Sprague –Dawley (SD) rats(10 males, 20 females), weighting 220–250 g, were purchased from Liaoning Changsheng Bio- Technology Co., Ltd. The rats were housed under a 14 : 10 constant light –dark cycle with free access to water and food for one week at

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kinase Fig. transduction pathway and (MAPK) transcriptional the Activator regulation of Protein-1 (AP-1) pathway. MAPK pathways are activated by stimuli and three major pathways (ERK, JNK,p38) were phosphorylated, then enter the nucleus directly to regulate tran- scription factors such as AP-1. AP-1 comprises Jun and Fos family members, which play a pivotal role in regulating gene transcription in various biologic processes.

1. Mitogen-activated

protein

signal

room temperature (24 ± 1 °C), and then male and female rats were caged at a ratio of 1:2. The female rats were housed in individual cages when they were confirmed to be pregnant until they delivered natu- rally. The day of birth was noted as postnatal day 0 (P0). Postnatal day 7 (P7) male or female rat pups (sex hormones have on effect on the experimental results from 7 day to 14 day because SD rats are in their infancy in this period) weighing 14–18 g, were used in this study.

2.4. Anesthetic exposure

P7 rat pups were separated from their mothers and placed in a glass chamber (20 × 12 × 10 cm) resting in a water bath to maintain a constant environmental temperature of 38 °C. Pups from a different litter were randomly allocated to two groups. In the chamber, the rats were exposed to either 3% sevoflurane in a 30% oxygen carrier gas (balanced with nitrogen) or a carrier gas without sevoflurane for 4 h. The induction flow rates were 6 l/min for the first 5 min for induction and then 1 l/min for maintenance. The concentrations of sevoflurane, oxygen and carbon dioxide in the chamber were measured by a gas analyzer (Datex Cardiocap II, Datex-Ohmeda, Madison, WI, USA), and the rectal temperature of the pups was maintained at 37 ± 0.5 °C. The anesthetized pups were recovered in 30% oxygen for 20 min and re- turned to their mothers’ cages until the next procedure. For the inter- vention studies, we administered an inhibitor to the rats via an in- the traperitoneal experiments were performed in a blinded manner.

injection 2 h before sevoflurane anesthesia. All

2.5. Hippocampus harvesting and protein level quantification

At the end of anesthesia, five pups from each group were randomly selected and killed by decapitation at 6 h, 1 d, 3 d and 7 d. The hip- pocampus of each pup was harvested and then stored at −80 °C until use. We lysed the harvested hippocampus in ice-cold radio immuno precipitation assay (RIPA) buffer containing protease inhibitors (10 mM Tris-HCl, PH 7.4, 150 mm NaCl, 2 mM EDTA, 0.5% Nonider P-40, 1 μg/ ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A) and a phe- nylmethylsulfonyl fluoride solution(1 mM), as previously described [20,36]. The lysates were then collected and centrifuged at 1880018,800×g (Micro 21R, Thermo, Germany) for 30 min at 4 °C. We used a bicinchoninic acid (BCA) protein assay kit (Pierce, Iselin, NJ) to quantify the amount of protein.

2.6. Experimental protocol

Two experiments were performed. Experiment one, included two group, the sevoflurane group and the control group. After anesthetic exposure and in accordance with the above method, five of twenty P7 rat pups in each group were randomly sacrificed at 6 h, 1 d, 3 d and 7 d after the experimental intervention. The expression levels of Cx43, total

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and phosphorylated MAPKs, and total and phosphorylated c-Jun and c- Fos were tested with Western blots (Fig. 2).

In experiment two, according to the MAPK signal defined in ex- periment one, one or several corresponding inhibitors were injected intraperitoneally 2 h before sevoflurane exposure. Only the ratio of phosphorylated JNK to JNK was increased in MAPK signal after sevo- flurane exposure in experiment one (Fig. 4), so the JNK inhibitor SP600125 10mg/Kg was finally injected intraperitoneally 2 h before sevoflurane exposure without other inhibitors of ERK and p38. Rat pups in the sevoflurane and control groups received either an inhibitor or an equal volume of DMSO including control, control + SP600125, sevo- flurane and sevoflurane + SP600125. After gas exposure, at least ten P7 rat pups (five were used for Western blots and five were used for immunohistochemical analyses) in four groups were sacrificed at 6h, 1d, 3d and 7d.

2.7. Western blots

Fifty micrograms of each protein sample was separated by 12.5% sodium dodecyl electrophoresis (SDS‑PAGE); using a semidry blotting apparatus(Bio-Rad Laboratories, Munich, Germany), the proteins were electrotransferred to ni- trocellulose membranes (Millipore Corp., Eschborn, Germany) and then incubated overnight at 4 °C with the appropriate primary antibodies: anti-Cx43 (1:1000; SAB4501175), anti-ERK1/2 (1:1000; 4695), anti- phospho-ERK1/2 (1:1000; 4370), anti-JNK (1:1000; 9252), anti- phospho-JNK (1:2000; 9251), anti-p38 MAPK (1:1000; 8690), anti- phospho-p38 MAPK (1:1000; 4511), anti-c-Jun (1:1000; 9165), anti- phospho-c-Jun (1:1000; 3270), anti-c-Fos (1:1000; 2250), anti- phospho-c-Fos (1:1000; 5348) and anti-GAPDH (1:1000; A9169). Then, the respective secondary antibodies conjugated to horseradish perox- idase (HRP) were added for 2 h followed by three washes. The positive reactive bands were detected by Amersham enhanced chemilumines- cence (ECL) reagents. The blots were scanned using an Amersham Image 600 scanner (GE Healthcare Life Sciences), and the protein band density was quantified using ImageJ software. Protein expression levels were evaluated by the GAPDH ratio.

sulfate-polyacrylamide

gel

2.8. Immunohistochemical analysis of cleaved caspase-3

Caspase-3 positive cells were detected using immunohistochemistry (IHC). Five rats in each group were euthanized by transcardial perfu- sion with saline, followed immediately by 4% paraformaldehyde at 6 h, 1d, 3d and 7d. Then, the whole brains were harvested, postfixed in 4% paraformaldehyde, embedded in paraffin, and cut into 3.5um-thick sections. These tissue sections were then baked, deparaffinized, rehy- drated, and quenched of endogenous peroxides. A primary antibody against activated caspase-3 (1:200 dilution, catalog no. 9662; Cell Signaling Technology) was applied and incubated at 4 °C overnight followed by a 40-min incubation with a biotinylated goat antirabbit antibody (1:500 dilution; Santa Cruz Biotechnology). Hippocampal CA1 region, a region of the brain vital for memory formation, was colorized with diaminobenzidine solution for 8 min and counterstained with he- matoxylin. The sections were observed using an E100 microscope (Nikon Corporation, Japan, 400× magnification), with 3 randomly chosen fields imaged per slide. One slide per animal was prepared and counted in five rats. Caspase-3 positive cells were counted manually in each hippocampal slide vision.

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Fig. 2. Schematic representation of the ex- perimental protocol.

2.9. Morris water maze (MWM)

To assess neurodevelopmental outcomes in adolescence, particu- larly learning and memory functions, twenty-four rats from four groups and control + SP600125, including sevoflurane + SP600125, were subjected to the MWM after 28 d (six rats in each group), as previously described. 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 the water) was located at a fixed position in the pool. The water tem- perature was set at 23 ± 1 °C. Escape latency trials were conducted once per day for five consecutive days. In the trials, the rats were trained to swim to and locate the hidden platform. After every trial, each mouse was placed in a holding cage under a hair dryer for 5 min to dry before returning to its regular cage. The time spent finding the hidden platform and the swimming distance before reaching the plat- form were recorded. After the escape latency trials, the platform was removed, and the rats were allowed to swim freely for 90 s; the number of times that the former platform was crossed was determined. The entire behavioral test was recorded and analyzed using an Noldus Ethovision XT video analysis system (Netherland).

control,

sevoflurane

Each rat was placed on the platform in the center of the MWM for 30 s and, then released into the water from an assigned release point. The rat was allowed to swim for 90 s or until it landed on the platform. If the rat failed to reach the platform within 90 s, it was placed on the platform for an additional 10 s. The swimming distance and the time required to reach the platform were recorded using video tracking and analyzed by MWM software. After the MWM test, all twenty-four rats were sacrificed without biochemical analysis.

2.10. Statistical analysis

Data was analyzed using GraphPad Prism 6 software (version 6.0; Graphpad Software, Inc.). Statistical significance was determined by Two-way ANOVA followed by Tukey multiple comparison tests as ap- propriate. Interaction between time and group factors in a two-way ANOVA with repeated measurements was used to analyze the differ- ence of learning curves (based on escape latency) in the MWM. At least three individual trials were performed for each experiment and data represented as mean ± SEM. P < 0.05 was considered statistically significant. Specific p values are indicated in figure legends.

3. Results

3.1. Sevoflurane markedly increased Cx43 expression in the hippocampus in vivo

The expression of the Cx43 protein in the hippocampus of P7 SD rats increased naturally and gradually with age in the control group during the observation period from 6 h to 7 d; this period is the rapid devel- opment stage of the CNS in SD rats. Compared with that of the control group, sevoflurane exposure for 4 h significantly increased Cx43 ex- pression from 6 h to 3 d after exposure. After 3d, Cx43 expression in the treatment group was consistent with that of the control group. Also, sevoflurane-induced upregulation of Cx43 protein was most sig- nificantly at 1d and the effect was almost disappeared at 7d (Fig. 3).

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Fig. 3. The effects of sevoflurane exposure in P7 SD rats on Cx43 expression in the hippocampus. (A) Western blotting results show that anesthesia with 3% sevoflurane for 4 h in P7 SD rats increases Cx43 expression in the hippocampus from 6 h to 3 d compared to the control condition. (B) Quantification of the Western blot results shows the differences between sevoflurane and control group in 6 h, 1d, 3d and 7d for the Cx43 expression in the rat hippocampus. (C) Quantification of the Western blot results shows the differences between 6 h, 1d, 3d and 7d for the Cx43 expression in the sevoflurane and control group. N = 5 in each group. *P < 0.05 and **P < 0.01 compared with the control group; #P < 0.05 and ##P < 0.01 compared with 6h, $P < 0.05 and $$P < 0.01 compared with 1d, & P < 0.05 and && P < 0.01 compared with 3d in each group.

Fig. 4. The effect of sevoflurane on the phosphorylation of the MAPKs p38, JNK, and ERK1/2. (A) Western blotting results show that sevoflurane anesthesia in P7 SD rats does not significantly alter the ratio of phosphorylated p38 to p38 or, the ratio of phosphorylated extracellular signal-regulated kinase (ERK1/2) to ERK1/2 in the hippocampus compared to those in the control rats. However, compared to that of the control rats, 3% sevoflurane exposure for 4 h in P7 SD rats increased the ratio of phosphorylated JNK to JNK in the hippocampus from 6 h to 3 d after exposure. (B) Quantification of the Western blotting results shows that sevoflurane increases the ratio of phosphorylated JNK to JNK in the rat hippocampus compared to that in the control rats. (C) Quantification of the Western blot results shows the differences between 6 h, 1d, 3d and 7d for the Cx43 expression in the sevoflurane and control group. N = 5 in each group. *P < 0.05 and **P < 0.01 compared with the control group; #P < 0.05 and ##P < 0.01 compared with 6h, $$P < 0.01 compared with 1d in each group.

3.2. Sevoflurane modulated Cx43 expression by activating the JNK/c-Jun pathway

To study the signaling pathway by which sevoflurane regulates Cx43 expression, we evaluated the effect of sevoflurane on the activa- tion of MAPKs, including ERK1/2, JNK and p38. As shown in Fig. 4, compared with the control group, after 4 h of sevoflurane exposure, the phosphorylation of JNK was increased from 6 h to 3 d. However, the phosphorylation levels of ERK1/2 and p38 were similar in the two groups at all time points. This finding indicated that the Cx43 function of P7 SD pups exposed to sevoflurane may be regulated by the JNK signaling pathway. Meanwhile, the ratios of phosphorylated p38 to p38 and phosphorylated ERK1/2 to ERK1/2 were similar between 6 h, 1d, 3d and 7d. But the ratio of phosphorylated JNK to JNK was increased naturally from 6 h to 7d, just like Cx43 protein in the control group. Furthermore, to confirm the effect of sevoflurane on c-Jun and c-Fos, which are AP-1 subunits that are regulated by MAPKs, we also tested the expression of phosphorylated and total c-Fos and c-Jun. The results suggested that the sevoflurane-induced increase in the expression of Cx43 occurred via the JNK/MAPK signaling pathway and the regulation of c-Jun/AP-1 nuclear translocation (Figs. 4 and 5).

3.3. Pretreatment with the JNK inhibitor SP600125 alleviated sevoflurane- induced Cx43 upregulation and inhibited JNK/c-Jun activity

4 h, so we just blocked the JNK without ERK or p38 signaling pathway using 10 mg/kg SP600125 (5% DMSO + 30%PEG 300 + 5%Tween 80 + ddH2O),a JNK inhibitor, by intraperitoneal injection at 2 h before 3% sevoflurane exposure. All P7 SD rats were sacrificed 1 d after sevo- flurane anesthesia because the expression of phosphor-JNK/JNK and phosphor-c-Jun/c-Jun peaks at P8, and we assessed the effects of SP600125 on sevoflurane anesthesia-induced c-Jun/JNK activation and Cx43 expression in the hippocampus of SD rats. Immunoblotting showed that SP600125 treatment alone did not significantly alter the levels of Cx43, phospho-c-Jun/c-Jun or phospho-JNK/JNK compared with the levels observed in the control rats. However, SP600125 treatment reduced the expression of Cx43, phospho-c-Jun/c-Jun and phospho-JNK/JNK in the hippocampus after sevoflurane anesthesia (Fig. 6).

3.4. Sevoflurane increased the number of cells with cleaved caspase-3 in the hippocampus of P7 rats

Because cleaved caspase-3 is an essential apoptotic regulator, we measured the expression of cleaved caspase-3 in the rat hippocampus with IHC. Compared to the number of cells in the control rats, sevo- flurane exposure significantly increased the number of cells with cleaved caspase-3 expression from 6 h to 1 d. Intraperitoneal pretreat- ment with the JNK inhibitor SP600125 at a concentration of 10 mg/kg provided a protective effect by significantly decreasing the amount of cleaved caspase-3. However, SP600125 alone did not reduce the

Only phosphor-JNK/JNK was elevated after exposure sevoflurane

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Fig. 5. The effect of sevoflurane on activator protein-1 (AP-1) in the hippocampus of P7 SD rats. (A) Western blotting results show that anesthesia with 3% sevoflurane for 4 h in P7 SD rats affects AP-1 phosphorylation in the hippocampus. Compared with the levels in the control rats, sevoflurane exposure in P7 SD rats significantly increased phosphorylated c-Jun levels in the hippocampus from 6 h to 3 d, and did not affect phosphorylated c-Fos in either group. The total c-Jun and c- Fos levels were similar in the two groups. (B) Quantification of the Western blotting results shows that sevoflurane increases the ratio of phosphorylated c-Jun to c- Jun in the hippocampus of the rats as compared to that in the control rats. (C) Quantification of the Western blot results shows the differences between 6 h, 1d, 3d and 7d for the Cx43 expression in the sevoflurane and control group. N = 5 in each group. *P < 0.05 and **P < 0.01 compared with the control group; #P < 0.05 compared with 6h in each group.

amount of cleaved caspase-3 in the hippocampus compared with that of the control. Also, from 6 h to 7 d, as observed for the expression of Cx43, cleaved caspase-3 also increased gradually in the hippocampus of P7 SD pups, and the same effect was observed after intraperitoneal pretreatment with SP600125. Sevoflurane exposure could increase apoptosis in P7 SD rats hippocampus at 1d and also could be sig- nificantly weakened by SP600125 (Fig. 7).

These data suggested that sevoflurane could affect hippocampus func- tion in P7 SD rats (in adolescence) and that SP600125 could attenuate sevoflurane anesthesia-induced cognitive impairments in young SD rats.

4. Discussion

3.5. Postnatal sevoflurane exposure reduces learning and memory ability

The procedure used for the behavior study is shown in Fig. 8. Rats in the sevoflurane group needed significantly more time to find the sub- merged platform on trial day 5 than did rats from the other three groups in the escape latency portion of the MWM (P < 0.05, Fig. 8A). Rats from the sevoflurane + SP600125 and SP600125 groups were com- parable to the control rats in terms of the latency to find the submerged platform (P > 0.05, Fig. 8A). In the probe trial, the animals in the sevoflurane group crossed the platform less frequently (P < 0.05), and this effect was improved by treatment with SP600125 (P > 0.05, Fig. 8B). Additionally, there were no significant differences in the average swimming speeds among the four groups (results not shown).

The current study demonstrated that anesthesia with 3% sevo- flurane for 4 h caused a significant upregulation of Cx43 in the hippo- campus of P7 SD rats. This increase was regulated by the activation of a MAPK signaling pathway, the JNK pathway, which subsequently in- creased AP-1 activity via the translocation of c-Jun from the cytosol to the nucleus. Our IHC studies showed that 3% sevoflurane increased the expression of cleaved caspase-3 in the hippocampus from 6 h to 1 d. This result is consistent with a previous study [21]. The expression of Cx43 and cleaved caspase-3 recovered to normal levels at 3 d after 3% sevoflurane anesthesia. SP600125, a JNK inhibitor, could decrease the upregulation of Cx43, c-Jun/AP-1 and cleaved caspase-3 in the hippo- campus after sevoflurane exposure and improve rat cognitive impair- ment after 30 d, which demonstrated that the Cx43 and JNK/c-Jun/AP- 1 pathway may be involved in sevoflurane-induced neuroinjury.

Fig. 6. Effects of SP600125 on JNK/c-Jun activity and Cx43. Western blotting results show that 10 mg/kg SP600125 can significantly inhibit Cx43 expression and the JNK/c-Jun signaling pathway in the hippocampus of P7 SD rats 1 d after anesthesia with 3% sevoflurane for 4 h (A, B). N = 5 in each group. *P < 0.05 compared with the control group.

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Fig. 7. Anesthesia with 3% sevoflurane for 4 h in P7 SD rats increases apoptosis in the hippocampus, as indicated by cleaved caspase-3 positive cells. Images were taken at 400× magnification. (A) Apoptosis increased gradually from 6 h to 7 d in the control group, as indicated by the cleaved caspase-3-positive cell ratio in the hippocampus of P7 SD rats. (B) After exposure to 3% sevoflurane for 4 h in P7 SD rats, apoptosis in the hippocampus was obviously increased from 6 h to 1 d compared with the levels in the control group. Apoptosis rates were similar from 3 d to 7 d after 3% sevoflurane anesthesia exposure. (C) The JNK inhibitor, SP600125, alone did not increase apoptosis compared with that of the control group. (D) SP600125 obviously attenuated sevoflurane-induced apoptosis in the hippocampus from 6 h to 1 d after 3% sevoflurane anesthesia for 4 h in P7 SD rats. (E) Statistical results for the cleaved caspase-3 positive cell ratio between the control, control + SP600125, sevoflurane and sevoflurane + SP600125 groups in the hippocampus of P7 SD rats after exposure to 3% sevoflurane for 4 h. (F) Statistical results for the cleaved caspase-3 positive cell ratio between 6 h, 1d, 3d and 7d in the hippocampus of P7 SD rats after exposure to 3% sevoflurane for 4 h in four groups. N = 5 in each group. *P < 0.05 and **P < 0.01 compared with the control group; #P < 0.05 and ##P < 0.01 compared with 6h, $P < 0.05 and $$P < 0.01 compared with 1d, &P < 0.05 and && P < 0.01 compared with 3d in each group.

Accumulating clinical data suggest that anesthetics, including N- methyl-D-aspartate (NMDA) antagonists and/or γ-amino-butyric acid (GABA) agonists, can cause neurotoxicity in neonates [22–24]. Un- fortunately, to date, how to prevent these neural injuries remains controversial [25]. During the rapid development of the nervous system, neurons are sensitive to interference from the synaptic en- vironment. At this time, contact with anesthetics can activate astro- cytes, interfere with the synapse development environment, and cause neurotoxicity, resulting in synapse malformation, neuronal apoptosis and injury. Experiments have shown that the toxicity of narcotic drugs to mammals is consistent with synapse formation periods [26].

Although there are no definitive conclusions about inhalation an- esthesia-induced toxicity, the potential mechanisms may include neu- roapoptosis, neuroinflammation, reactive oxygen species accumulation, neurotransmitter disturbances, and changes in synaptic plasticity [3,5,6]. Therefore, we hypothesized that the neurotoxicity of anes- thetics in developing rats may be related to their effects on the devel- opment of synapses.

In the CNS, astrocytes can support, isolate, and nourish nerve cells. In recent years, increasing evidence indicated that astrocytes play im- portant roles, such as roles in synaptic plasticity, information trans- mission, nervous system damage, inflammation, learning and memory.

Fig. 8. Anesthesia with 3% sevoflurane was administered for 4 h in P7 SD rats, and cogni- tive behavior was tested with the MWM from day 28 to day 33. (A) There were no obvious differences among the four groups from day 1 to day 4. The rats in the sevoflurane group needed significantly more time to find the submerged platform on trial day 5 than did the rats in the other three groups in the escape latency portion of the MWM. (B) The rats in the sevoflurane group crossed the platform fewer times than did the rats in the other three groups. N = 6 in each group. *P < 0.05 com- pared with the control group.

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As the most expressed connexin in astrocytes, Cx43 could directly affect these functions of astrocytes.

The Cx43 protein can not only form a GJ channel, facilitating direct material exchange between two adjacent cells, but also form hemi- channels, resulting in the destruction of cell membrane integrity and the formation of a direct connection between the cytoplasm and the extracellular space. Generally, GJ channels which help to maintain neuronal homeostasis under normal conditions [30], have been thought to exert protective effects on the CNS. Conversely, hemichannels are in the closed state under normal conditions, but open when the CNS suf- fers damage, leading to intracellular glutamate, ATP, and calcium ion release from cells, accelerating the spread of toxins and eventually causing neuronal death [31]. When the CNS is damaged, the expression of the Cx43 protein increases, and a large number of Cx43 proteins initiate the formation of hemichannels, leading to intercellular com- munication and the destruction of cell integrity. Several studies have suggested potential associations between astrocytic Cx43 and synaptic transmission, plasticity and information processing [32–35].

Since there is a clear link between anesthetic neuroinjury and the function of the Cx43 protein, we can assume that Cx43 plays an im- portant role in sevoflurane-induced neurotoxicity. Moreover, the neu- rotoxicity of sevoflurane is dose-dependent and time-dependent. Shen et al. [36] showed that exposure to 3% sevoflurane for 2 h daily for 3 d increased the proinflammatory cytokine, IL-6, in the hippocampus of 6- day-old mice and increased cognitive impairment in adolescence. In the current study, we observed that Cx43 expression was increased in the hippocampus of P7 rats from 6 h to 1 d after exposure to sevoflurane for 4 h. In the control group, the expression of the Cx43 and p-JNK protein in the hippocampus increased naturally and gradually from P7 to P14. This phenomenon may be due to the observation period. When humans and animals are born, the CNS is not fully developed and must undergo a stage called the brain growth spurt. In rats, the brain growth spurt occurs approximately 1–14 days after birth. During this period, neu- rons, astrocytes, dendrites and axons grow rapidly, and large numbers of synapses and complex neural networks form during the short term. At the same time, the old neural network is removed to form a more effective neural loop. During this period, the CNS can affect protein synthesis. We observed that the natural growth of Cx43 and p-JNK from P7 to P14 may be related to the time of brain outgrowth.

MAPK is a serine-threonine protein kinase, that can transmit ex- tracellular signals into intracellular spaces, and AP-1 is regulated by the MAPK signaling cascade after its activation by external stimuli. The AP- 1 transcription factor is associated with tumor proliferation during the early period. Recently, AP-1 was found to have additional functions beyond its effects on tumors, including roles in apoptosis, inflamma- tion, differentiation and wound healing. AP-1 can be activated by various environmental stress factors, and it then activates MAPK cas- cades and enhances AP-1 activity by triggering the phosphorylation of distinct substrates. In addition, previous reports have demonstrated that JNK is a stress-activated kinase and represents one of the most im- portant MAPKs in intracellular signaling pathways. Consistent with results from previous studies [18], our observations illustrated that one of the MAPK signaling pathways, the JNK pathway, could function as an upstream mediator of Cx43 in the hippocampus of P7 SD pups. We then determined whether the JNK/AP-1 signaling cascade could parti- cipate in Cx43 upregulation after sevoflurane anesthesia. The results revealed that the inhibition of the JNK pathway reversed the sevo- flurane-induced upregulation of Cx43 and c-Jun/AP-1 phosphorylation. This conclusion provides a new strategy for treating sevoflurane-in- duced injury in the developing brain.

The activation of cleaved caspase-3 is always considered the most important indicator of apoptosis. In the present study, we also eval- uated apoptosis by immunohistochemical staining. We found that se- voflurane exposure led to widespread neuroapoptosis in the hippo- campus from 6 h to 1 d in P7 SD rats, which is consistent with previous In addition, pretreatment with the JNK inhibitor, studies [4,38].

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SP600125, could decrease cleaved caspase-3 levels in the hippocampus of P7 SD pups. It is reasonable to hypothesize that there is a causal relationship between Cx43 and cleaved caspase-3. Moreover, this pos- tulation was supported by our present study, in which Cx43 expression was ameliorated significantly, accompanied by a significant reduction in cleaved caspase-3 levels, after treatment with SP600125. Thus, our data suggested that Cx43 is involved in sevoflurane-induced neuroin- jury. This result provides the first evidence that the inhibition of the JNK signaling pathway can reduce the expression of Cx43; the reduc- tion of Cx43 expression also reduced hippocampal apoptosis and im- proved long-term biological behaviors in P7 SD rats.

The relationship between sevoflurane-induced neuroinjury and MAPK kinases has been widely studied, but the results are inconsistent. Previous studies have indicated that sevoflurane suppressed the phos- phorylation of ERK1/2, but did not influence JNK phosphorylation following a 6 h exposure [15,39–41]. However, Wang et al, [42] de- monstrated that p-JNK and p-p38 kinase levels were significantly up- regulated following sevoflurane exposure compared to those of the control group, and the sevoflurane-increased ERK1/2 levels were not as high as the JNK levels in the P7 rats hippocampus. Yang et al, [43] observed that the JNK pathway might play a key role in the decreased survival of fetal neural stem cells (FNSCs) induced by inhaling 4.8% sevoflurane. JNK inhibition by the specific inhibitor SP600125 rescued FNSCs from cell damage and increased neuronal survival. Compared to these studies, the marked difference in our study could be due to the time points of observation. Most the changes in the expression of MAPK proteins were only evaluated from 0 to 6 h following sevoflurane ex- posure and were not observed over a longer duration. To date, few studies have observed sevoflurane-induced neuroinjury occurring from P7 to P14 in the developing rat hippocampus.

The present study has several limitations. First, in this study, the assays used to measure Cx43 predominately identified a single band, and we were unable to determine the phosphorylation status of Cx43. Because Cx43 can be phosphorylated by various kinases, the activation of a specific kinase may not correlate with Cx43 phosphorylation in our in- models. Second, we injected the JNK inhibitor, SP600125, traperitoneally instead of intracerebroventricularly. SP600125, with a molecular weight of 220.23 kD, has high ester solubility and is gen- erally dissolved with DMSO. It can be easily absorbed intraperitoneally. Importantly, JNK was obviously inhibited after intraperitoneal injec- tion of SP600125 in the P7 rat hippocampus in our study. Therefore we believe that intraperitoneally injected SP600125 can cross the BBB. Finally, we did not investigate whether there were differences in ar- terial blood gas between the control and sevoflurane groups. However, a previous study [44] showed that anesthesia with 3% sevoflurane for 4 h did not significantly change the values of pH, partial pressure of oxygen and partial pressure of carbon dioxide compared with those of the control group.

5. Conclusions

Overall, for the first time, we demonstrated that sevoflurane ex- posure in P7 SD rats could induce Cx43 upregulation and apoptosis by activating the JNK/c-Jun/AP-1 signaling pathway in the hippocampus of developing rats and could cause cognitive decline in adolescence. The JNK inhibitor SP600125 could inhibit the JNK/c-Jun/AP-1 sig- naling pathway and alleviate sevoflurane-induced Cx43 expression and apoptosis, improving learning and memory functions after sevoflurane exposure in the brains of developing rats.

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rotoxicity differently in neonatal naïve and Alzheimer disease transgenic mice, Anesthesiology 112 (2010) 1404–1416.",rats,['Seven-day-old SD rats (P7) were exposed to 3% sevoflurane for 4 h.'],postnatal day 7,['Seven-day-old SD rats (P7) were exposed to 3% sevoflurane for 4 h.'],Y,['The Morris water maze (MWM) was performed to evaluate cognitive function from P28 to P33.'],sevoflurane,"['Pups from a different litter were randomly allocated to two groups. In the chamber, the rats were exposed to either 3% sevoflurane in a 30% oxygen carrier gas (balanced with nitrogen) or a carrier gas without sevoflurane for 4 h.']",none,[],sprague dawley,['Seven-day-old SD rats (P7) were exposed to 3% sevoflurane for 4 h.'],"This study investigates the mechanism behind sevoflurane-induced neurotoxicity in the developing rat hippocampus, focusing on the role of connexin 43 and the JNK/c-Jun/AP-1 pathway.",['The present study hypothesized that Cx43 may participate in sevoflurane-induced neuroinjury and investigated the underlying mechanisms in young Sprague Dawley (SD) rats.'],The study introduces the use of the JNK inhibitor SP600125 to alleviate sevoflurane-induced neurotoxicity.,['All these effects could be alleviated by pretreatment with the JNK inhibitor SP600125 (10 mg/kg).'],The findings suggest a new strategy for preventing sevoflurane-induced neuronal dysfunction by targeting the JNK/c-Jun/AP-1 pathway.,['This finding may reveal a new strategy for preventing sevoflurane-induced neuronal dysfunction.'],The study's limitations include the inability to determine the phosphorylation status of Cx43 and the method of administering the JNK inhibitor SP600125.,"['First, in this study, the assays used to measure Cx43 predominately identified a single band, and we were unable to determine the phosphorylation status of Cx43.', 'Second, we injected the JNK inhibitor, SP600125, intraperitoneally instead of intracerebroventricularly.']",Potential applications include the development of therapeutic interventions targeting the JNK/c-Jun/AP-1 pathway to mitigate sevoflurane-induced neurotoxicity in pediatric anesthesia.,['This finding may reveal a new strategy for preventing sevoflurane-induced neuronal dysfunction.'],True,True,True,True,True,True,10.1016/j.biopha.2018.09.111
10.1093/toxsci/kfn152,1107.0,Boctor,2008,rats,postnatal day 7,Y,ketamine,none,sprague dawley,"TOXICOLOGICAL SCIENCES 106(1), 172–179 (2008) doi:10.1093/toxsci/kfn152 Advance Access publication July 30, 2008

Neonatal PCP Is More Potent than Ketamine at Modifying Preweaning Behaviors of Sprague-Dawley Rats

Sherin Y. Boctor, Cheng Wang, and Sherry A. Ferguson1

Department of Interdisciplinary Biomedical Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and Division of Neurotoxicology, National Center for Toxicological Research/US Food and Drug administration, Jefferson, Arkansas 72079

Received May 2, 2008; accepted July 10, 2008

Treatment with N-methyl-D-aspartate (NMDA) receptor antag- onists, such as ketamine (KET) or phencyclidine (PCP), can trigger apoptotic neurodegeneration in neonatal rodents; however, little is known about the behavioral alterations resulting from such treatment. Here, rats were sc treated with saline; 10 mg/kg PCP on postnatal days (PNDs) 7, 9, and 11; 20 mg/kg KET (six injections every 2 h on PND 7); or a regimen of ketamine and 250 mg/kg L-carnitine (KLC) both administered on PND 7 with additional 250 mg/kg doses of L-carnitine given on PNDs 8–11. Postinjection, the home cage behavior of each pup was categorized on PNDs 7–11. Slant board and forelimb hang behaviors were examined on PNDs 8–11 and 12–16, respectively. The initial KET or KLC injections on PND 7 elevated abnormal home cage activity (i.e., paresis and paddling); however, KLC pup behavior returned to normal by the fourth injection, indicating the protective effects of L-carnitine against NMDA antagonist toxicity. PCP treatment caused sub- stantial abnormal home cage activity on each injection day (PNDs 7, 9, and 11). Latencies to turn on the slant board were significantly longer on PND 8 for KET- and PCP-treated pups and PND 10 for PCP-treated pups. On PND 12, the forelimb hang time of PCP-treated pups was significantly shorter. Body weight was decreased on PNDs 8–18 in PCP-treated pups and PNDs 8–10 in KET-treated pups. These data indicate that developmental NMDA antagonist treatment causes short-term behavioral alter- ations which appear related to motor coordination and may be cerebellar in nature. Furthermore, single PCP injections appear more potent at altering behavior than multiple injections of KET. Key Words: ketamine; phencyclidine; neurodegeneration;

(dizocilpine), phencyclidine (PCP), or ketamine (KET) results in increased neuronal degeneration (Hayashi et al., 2002; Ikonomidou et al., 1999; Scallet et al., 2004; Wang and Johnson, 2005; Wang et al., 2001). Specifically, multiple injections of 20 mg/kg KET caused increased numbers of Fluoro-Jade, transferase–mediated dUTP nick-end labeling, and silver-stained cells in the hip- pocampus, thalamus, subiculum, caudate nucleus, and frontal, cingulate, parietal, and retrosplenial cortices (Ikonomidou et al., 1999; Scallet et al., 2004). Single injections of the same or higher doses (25–75 mg/kg) do not appear to cause similar neuronal cell death (Hayashi et al., 2002; Scallet et al., 2004).

terminal deoxynucleotidyl

Such NMDA antagonist–induced neurodegeneration has been shown to result in behavioral deficits as well. For example, neonatal PCP or MK-801 treatment causes later sensorimotor gating deficits as measured by prepulse inhibition (Harris et al., 2003; Wang et al., 2001) and impairs Morris water maze performance in juvenile and adult rats (Sircar, 2003; Sircar and Rudy, 1998). Neonatal PCP treatment has been described to cause increased sensitivity to later PCP treatment as well as transient deficits in spatial alternation performance (Wang et al., 2001). Repeated neonatal MK-801 treatment results in long-term deficits in radial-arm maze performance (Kawabe et al., 2007). Those behavioral deficits imply that the observed neurodegen- eration following developmental NMDA antagonist treatment has long-term effects.

forelimb hang; negative geotaxis; pup behavior.

The excitatory neurotransmitter glutamate activates iono- tropic (a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, kainate, and N-methyl-D-aspartate [NMDA]) and metabotropic (G protein linked) receptors and is essential for neuronal differentiation, migration, and survival (reviewed by Meldrum, 2000). Treatment of postnatal day (PND) 7 rats with non- competitive NMDA receptor antagonists such as MK-801

1 To whom correspondence should be addressed at Division of Neurotoxicology, National Center for Toxicological Research/FDA, 3900 NCTR Drive, Jefferson, AR 72079. Fax 870-543-7181. E-mail: sherry.ferguson@fda.hhs.gov.

long-term behavioral effects after developmental NMDA antagonist treatment led us to hypoth- there may be acute effects observable during esize that treatment. Here, the behavioral effects of PCP or KET were evaluated using those treatment regimens previously shown to produce significant neurodegeneration. Rat pups were treated on PND 7 with KET at 2-h intervals for six injections or on PNDs 7, 9, and 11 with PCP. As a preliminary exploration, the potential protective effects of L-carnitine were measured in KET-treated rats since L-carnitine appears to prevent glutamate neurotoxicity (Felipo et al., 1994) and neurodegeneration in the frontal cortex of PND 7 rats (Zou et al., 2008). Home cage behavior of the pups was rated using a comprehensive scoring system on PNDs 7–11 after each treatment. Slant board (negative

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Published by Oxford University Press 2008.

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ACUTE PCP OR KETAMINE EFFECTS IN NEONATAL RATS

geotaxis; PNDs 8–11) and forelimb hang (PNDs 12–16) behaviors were examined to assess potential early neurotoxicant- induced dysfunctions.

MATERIALS AND METHODS

Animals

Sprague-Dawley dams (n ¼ 48) had normal vaginal births and on the day of birth (PND 0), each litter was separated by sex, and four males and four females were randomly selected so that each litter was culled to eight. The dams with their natural litters (culled to four/sex/litter) were obtained on PND 0 from the breeding colony at the National Center for Toxicological Research (NCTR/ FDA). Each dam was individually housed in a standard polycarbonate cage lined with wood chip bedding and provided with ad libitum food (NIH-31, Purina Mills, St Louis, MO) and water. The colony room was maintained at 22(cid:1)C ± 1(cid:1)C (mean ± SE) and 45–55% humidity on a 12-h light/dark cycle (7:00 A.M.–7:00 P.M.). Each pup was paw tattooed on PND 1 and also identified with a nontoxic marker on the dorsal side and tail tip on PND 4. All animal procedures followed the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and were approved in advance by the NCTR Institutional Animal Care and Use Committee.

Treatment

Ketamine hydrochloride (100 mg/ml solutions as Ketaset, Fort Dodge Animal Health, Fort Dodge, IA) was diluted with saline to produce 2 mg/ml solutions. PCP (NIDA, Bethesda, MD) and L-carnitine (Sigma-Aldrich Corp., St Louis, MO) were dissolved in 0.9% saline. Ketamine hydrochloride (400 ll) and L-carnitine (500 mg) were diluted with 10 ml of saline to produce 40 mg/ml KET and 500 mg/ml L-carnitine solutions, respectively. These solutions were combined in a 50 ml conical tube to obtain the KLC dose (250 mg/kg L-carnitine and 20 mg/kg ketamine) injected on PND 7. Solutions were made weekly and kept refrigerated. The sc injections were done using a 25-gauge needle.

The within-litter treatment (one pup/sex/treatment/litter) was a particularly important aspect of the experimental design since it is well recognized that differences in maternal care can affect offspring behavior (Barron and Riley, 1985; Fleming et al., 1999) and, at least in rats, pup behavior determines some aspects of maternal care (Marino et al., 2002). Thus, similar to that described by Zissen et al. (2007), overall maternal care was controlled at the litter level in that each dam cared for a litter which contained pups of all treatment groups. However, as noted by Zissen et al. (2007), this cannot prevent or control for differential treatment of individual pups by the dam.

Home Cage Pup Behavior

To determine the immediate effects of treatment, home cage behavior was assessed on PNDs 7–11. At each treatment time, the dam was placed in a holding cage. Each pup was then identified and when indicated, injected. Those pups not injected (e.g., PCP-treated pups at 8:00 A.M., 10:00 A.M., 2:00 P.M., and 4:00 P.M. on PND 7 and on PNDs 8 and 10 as well as the KET-treated pups on PNDs 8–11) were handled in a manner similar to the injected pups. Time of the last injection/handling for each litter was recorded, and the dam was returned to the home cage. Time from dam removal to replacement into the home cage was less than 120 s. At 5, 14, 23, and 32 min posttreatment, the behavior of each pup was assessed by one of two experimenters blind to treatment. Thus, there were four observations at five of the six treatment times on PND 7 (i.e., pups were observed after injections/handling at 8:00 A.M., 10:00 A.M., 12:00 P.M., 2:00 P.M., and 4:00 P.M., but not after the 6:00 P.M. injection/ handling time). On PNDs 8–11, there were four observations following the 12:00 P.M. treatment time. Each pup was categorized as exhibiting one of 12 different behaviors (see Table 1) which were based on a previous scoring system (Goodwin and Barr, 2005). Only one behavior/pup/observation time was recorded.

Slant Board Behavior (Negative Geotaxis)

Vestibular system integrity and Motor coordination were examined using a slant board test as previously described (Adams et al., 1985). Briefly, between 7:30 and 9:00 A.M. on PNDs 8–11, the dam was removed and each pup was placed on its ventral side with its nose pointing toward the lower end of a sandpaper-covered 45(cid:1) incline board. Each pup was allowed 60 s to complete a 180(cid:1) turn. One trial/day was conducted, and the latency to turn or fall from the apparatus was recorded by a tester blind to treatment conditions.

Forelimb Hang Behavior

Muscle strength/coordination was examined using a forelimb hang test as previously described (Cada et al., 2000). Briefly, between 7:30 and 10:00 A.M. on PNDs 12–16, the dam was removed and each pup was placed on a taut string stretched between two blocks of wood spaced 46 cm apart and 41 cm above a padded surface. One trial/day was conducted, and the latency to fall was recorded (maximum 60 s) by a tester blind to treatment conditions.

Statistical Analyses

Body weight. Offspring body weights were compared using ANOVAs with factors of treatment (control, KET, PCP, and KLC), sex, and the repeated measure of PND (JMP, Version 7.0; SAS Institute Inc., Cary, NC). Tukey post hoc tests were used to further analyze significant main effects or interactions.

Treatment assignment was based on PND 4 body weight such that all groups had similar average body weights prior to treatment. The four groups were (1) 10 mg/kg PCP at 12:00 P.M. on PNDs 7, 9, and 11; (2) six injections of 20 mg/kg KET on PND 7 (8:00 A.M.–6:00 P.M.), separated by 2-h intervals; (3) six injections of 20 mg/kg KET and 250 mg/kg L-carnitine on PND 7 (8:00 A.M.– 6:00 P.M.), separated by 2-h intervals followed by 250 mg/kg L-carnitine at 12:00 P.M. on PNDs 8–11; and (4) six injections of saline at on PND 7 (8:00 A.M.– 6:00 P.M.), separated by 2-h intervals followed by saline at 12:00 P.M. on PNDs 8–11. The doses and treatment regimens were based on previous reports indicating that similar treatments caused neurodegeneration in rats (Ikonomidou et al., 1999; Scallet et al., 2004; Wang et al., 2001). The L-carnitine dose was based on studies of its protective effects against 1-methyl-phenylpyridinium ion–induced apoptosis (Wang et al., 2007). Thus, for each of the 48 litters, 1 male and 1 female were assigned to each treatment resulting in 48 pups/sex/ treatment.

Body Weight

Body weights of the offspring were recorded on PNDs 4, 7, 8, 9, 10, 11, and 18. On PNDs 8–11, body weights were recorded after behavioral testing and prior to treatment.

Home cage pup behavior. Data from the five observation times on PND 7 (8:00 A.M., 10:00 A.M., 12:00 P.M., 2:00 P.M., and 4:00 P.M.) were analyzed separately from the single observation time on PNDs 8–11 (12:00 P.M.). Six behaviors were categorized as abnormal activity: fast activity, paddling, partial paddling, paresis, partial paresis, wall climbing. To analyze abnormal activity, each pup at each observation at each time was assigned a ‘‘1’’ if it exhibited any of the six abnormal behaviors or a ‘‘0’’ for any other behavior. Generalized linear models with a log link and Poisson distribution were used to analyze the counts for each of the two data sets (PND 7 only and PNDs 8–11) with factors of treatment, observation time (e.g., 8:00 A.M., 10:00 A.M.) (PND 7 analysis only), minutes posttreatment (e.g., 5, 14, 23, or 32 min), and sex.

Slant board behavior. Each pup could exhibit one of three outcomes: a successful turn within 60 s, a fall from the apparatus within 60 s, or an incomplete turn. A failure was categorized as a fall or an incomplete turn. The odds of failure were analyzed using a generalized linear model with repeated measures and a binomial distribution and logit link function. To analyze the latency to turn time, a Cox Proportional Hazards model was run in SAS (SAS Version 9.1; SAS Institute Inc.) using treatment, sex, and PND as factors. Pups that fell or did not complete the turn were accounted for in this analysis by adjusting the empirical distribution function.

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TABLE 1 Behavior

Behaviora

Definition

No. of instancesb

Quiet

No movement except for twitches

5929

and respiration

Immobile

No movement except for respiration

234

with the head up

Activity Fast activity Paddling

Walking, sniffing, and rearing Running On dorsal side while moving forelimbs and hind limbs

Partial paddling Maternal interference Dam interacted with pup

On dorsal side with three limbs moving

3028 3 418

340 241

FIG. 1. Body weight on PNDs 4–18, averaged over sex. The larger graph shows body weight on PNDs 4–11 while the inset graph shows PND 18 body weight. *Indicates the PCP-treated group weighed significantly less than controls. †Indicates the KET-treated group weighed significantly less than controls.

(e.g., licking, moving, or stepping on pup)

Paresis

On ventral side with hind limbs moving and stiff forelimbs

129

Partial paresis

Grooming Wall climbing

Unidentifiable

On ventral side with hind limbs moving

and one stiff forelimb

Grooming any part of the body Standing on hind limbs against cage side while forelimbs are alternately moving back and forth Pup could not be identified

271

1 15

3175

tests of the significant sex by PND interaction (F6,2244 ¼ 3.62, p < 0.002) demonstrated that males were significantly heavier than females on PND 18 only (p < 0.05) (mean ± SE for PND 18 males and females: 40.0 ± 0.5 and 38.4 ± 0.5 g, respectively). There was neither a main effect of sex nor a significant inter- action of treatment with sex.

(typically, as a result of nursing and inability to identify via paw tattoo or markings)

Home Cage Pup Behavior

aThese categories and the scoring system were modified from Goodwin and

Barr (2005).

bThe total number of observations for PNDs 7–11 home cage behavior was 13,244 (four posttreatment observations [5, 14, 23, and 32 min posttreatment] at each of five observation times on PND 7 (8:00 A.M., 10:00 A.M., 12:00 P.M., 2:00 P.M., and 4:00 P.M.), four posttreatment observations at each single treatment time on PNDs 9–11, maximum eight pups each of 48 litters).

Forelimb hang behavior. To analyze the latency to fall, a Cox Pro- portional Hazards model was run using SAS (SAS Version 9.1, SAS Institute Inc., Cary, NC) with treatment, sex, and PND as factors.

PND 7 abnormal behavior. There was a significant in- teraction of treatment with observation time (p < 0.0001) (Fig. 2, large graph). KET-treated pups had elevated levels of abnormal activity at each of the five observation times (p < 0.001) compared with controls. KLC-treated pups exhibited higher abnormal activity levels at 8:00 A.M., 10:00 A.M., and 12:00 P.M. relative to controls (p < 0.02); by 2:00 P.M., however, their abnormal behavior was within the range of controls. Although the PCP-treated pups had somewhat higher counts of abnormal activity at 10:00 A.M. (p < 0.004), the PCP treatment at 12:00 P.M. caused a significant increase in abnormal activity levels (p < 0.0001) which remained elevated (p < 0.0001) throughout the last two observation times (i.e., 2:00 and 4:00 P.M.).

RESULTS

Mortality and Body Weight

There were three deaths. One male KET pup died after the 2:00 P.M. PND 7 home cage behavior observation and before the 4:00 P.M. PND 7 treatment. One male KLC pup died on PND 8 after the 12:00 P.M. home cage behavior observation time and another male KLC pup died on PND 9 after the 12:00 P.M. home cage observation time.

Body weight analysis revealed a significant interaction of treatment with PND (F18,2244 ¼ 75.66, p < 0.0001) (Fig. 1). Post hoc tests indicated that there were no significant body weight differences on PND 4 or 7 (before treatment). However, on PNDs 8, 9, and 10, the KET-treated group weighed less than the control group (p < 0.05). On PNDs 8–18, the PCP-treated group weighed less than the control group (p < 0.05). Post hoc

PNDs 8–11 abnormal behavior. A significant interaction of treatment with PND (p < 0.0001) indicated that PCP-treated pups exhibited elevated levels of abnormal activity on PNDs 9 and 11 (i.e., treatment days) relative to all other treatment groups (p < 0.0001) (Fig. 2, inset graph). Levels of abnormal behavior in KET-, KLC-treated, and control pups did not differ from one another on PNDs 8–11, nor did PCP-treated pups exhibit significant levels of abnormal behavior on PNDs 8 and 10 (i.e., days on which handling, but no PCP treatment, occurred).

Slant Board Behavior (Negative Geotaxis)

Analysis of the odds of failure (falling or incomplete turn) indicated a significant interaction of treatment by PND (v2 ¼ 32.05, df ¼ 9, p < 0.0002) (data not shown). Post hoc tests

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FIG. 2. Level of abnormal home cage pup behavior on PNDs 7–11. The larger graph shows abnormal behavior on PND 7 while the inset graph shows abnormal behavior on PNDs 8–11. *Indicates higher levels of abnormal activity in the PCP-treated group relative to the control group. †Indicates higher levels of abnormal activity in the KET-treated group relative to the control group. ‡Indicates higher levels of abnormal activity in the KLC-treated group relative to the control group.

on each PND indicated that on PND 8, the KET- and PCP- treated groups had increased odds of failure (p < 0.05) (mean ± SE for control, KET-, KLC-, and PCP-treated groups: 0.19 ± 0.04, 0.39 ± 0.05, 0.21 ± 0.04, and 0.63 ± 0.05 proportion failing to turn, respectively). On PND 10, the PCP-treated group again had increased odds of failure (p < 0.05) (mean ± SE for control, KET-, KLC-, and PCP-treated groups: 0.17 ± 0.04, 0.21 ± 0.04, 0.22 ± 0.04, and 0.39 ± 0.05 proportion failing to turn, respectively). On PND 11, the proportion failing to turn was less than 0.25; however, relative to the control group, the KLC-treated group was more likely to fail (p < 0.05) (mean ± SE for control, KET-, KLC-, and PCP-treated groups: 0.13 ± 0.03, 0.16 ± 0.04, 0.24 ± 0.04 and 0.14 ± 0.04, respectively).

For illustrative purposes, Figures 3A–3D shows the pro- portion of each treatment group making a successful turn during the time allotted on PNDs 8–11. Analysis of latency to turn indicated a significant interaction of treatment by PND (v2 ¼ 68.69, df ¼ 9, p < 0.0001). Post hoc tests indicated that on PND 8, KET- and PCP-treated pups had longer latencies to turn than controls (p < 0.05) (mean ± SE for control, KET, KLC, and PCP groups: 16.8 ± 1.5, 25.4 ± 2.1, 17.2 ± 1.4, and 40.4 ± 2.0 s, respectively) (see also Fig. 3A). On PND 10, the PCP-treated group had a longer latency to turn (p < 0.05) (mean ± SE for control, KET, KLC, and PCP groups: 12.0 ± 1.0, 13.0 ± 1.5, 13.8 ± 1.7, and 25.3 ± 2.3 s, respectively) (see also Fig. 3C). Neither the main effect of sex nor any interactions with sex were significant.

Forelimb Hang Behavior

For illustrative purposes, Figures 4A–4E show the pro- portion of each treatment group that fell from the forelimb hang apparatus during the time allotted on PNDs 12–16. Analysis of latency to fall indicated a significant interaction of treatment with PND (v2 ¼ 27.78, df ¼ 12, p < 0.006). Post hoc tests revealed that on PND 12, relative to controls, the PCP-treated group had a shorter latency to fall (p < .05) (mean ± SE for

control, KET-, KLC-, and PCP-treated groups: 15.5 ± 1.2, 14.5 ± 1.1, 15.1 ± 1.2, and 13.0 ± 1.1, respectively) (see also Fig. 4A). On PND 16, the KLC- and PCP-treated groups had longer latencies to fall (p < 0.05) (mean ± SE for control, KET-, KLC-, and PCP-treated groups: 12.0 ± 0.9, 14.6 ± 1.3, 15.0 ± 1.2, 15.6 ± 1.3, respectively) (see also Fig. 4D). There was neither a significant main effect of sex nor any significant interactions with sex.

DISCUSSION

NMDA receptor antagonists can affect psychological and physiological functions in humans, and it is becoming clear that the musculoskeletal system can be altered as well (Brunson et al., 2001; Kakizawa et al., 2000; Wolff and Winstock, 2006). In the current study, neonatal rats were treated with KET, PCP, or ketamine þ L-carnitine (KLC) and assessed for home cage pup behavior, slant board behavior, and forelimb hang time on PNDs 7–11, 8–11, and 12–16, respectively. The single 10 mg/kg PCP injection on PNDs 7, 9, and 11 decreased body weight gain and produced high levels of abnormal activity as well as altered slant board and forelimb hang behavior. The PCP effects appeared more severe than those caused by the prolonged 20 mg/kg KET treatment on PND 7. These data suggest that three injections of 10 mg/kg PCP over PNDs 7–11 were more potent at modifying preweaning behaviors than were repeated injections of 20 mg/kg KET on PND 7. The addition of L-carnitine to the KET treatment regimen appeared to ameliorate KET-induced adverse effects on body weight, home cage pup behavior, and slant board behavior. These results more completely describe the acute effects of NMDA antagonist treatment in rat pups and may be associated with the neuronal degeneration known to occur with similar treatment.

Increased levels of abnormal home cage behavior occurred within 5–32 min after the initial PCP and KET injections on

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FIG. 3. Proportion of each treatment group making a successful slant board turn during the allotted 60 s. Each of the four panels (A–D) represents a single test day and is similar to a typical ‘‘survival curve.’’ Here, the ‘‘survival’’ estimates were inverted such that higher curves correspond to a higher proportion of animals making successful turns (e.g., on PND 8, approximately 43% of the control group had turned successfully at the end of 10 s). Note that a smaller proportion of the PCP-treated group successfully turned within the allotted 60-s trial on PNDs 8 and 10. See text for description of statistically significant effects.

PND 7. These abnormal behaviors were primarily paddling and paresis; other behaviors such as wall climbing rarely occurred. This is the first study to quantitatively describe the acute behavioral effects of developmental NMDA antagonist treat- ment; however, paddling has been described in PND 7 mice treated with 40 mg/kg KET (Young et al., 2005) and in adult rats treated with 2–15 mg/kg PCP (Chen et al., 1959; Sturgeon et al., 1979). Further, KET can cause paresis, ataxia, and temporary paralysis in humans (Wolff and Winstock, 2006). Thus, the specific types of abnormal activity induced in neo- natal rats by the NMDA antagonists here resemble those described for similarly treated adults.

level of KET-induced abnormal behavior occurred after the initial injection (i.e., at 8:00 A.M.) on PND 7. Subsequently, levels declined at each 2-h observation time even though each was accompanied by an additional 20 mg/kg injection. Still, the rate of decline was shallow enough such that abnormal behavior levels at the fifth treatment (i.e., 4:00 P.M.) remained elevated. By 12:00 P.M. on the following day, there were no measurable levels of abnormal home cage behavior in the KET-treated group. This pattern of initially high levels of PND 7 abnormal behavior with a decline throughout the day was very similar to that exhibited by the KLC group, albeit to a lesser extent. While the initial injection of ketamine with L-carnitine elevated abnormal behavior levels, the rate of decline throughout the remainder of the day was fairly steep

The highest

and by the fourth injection (i.e., 2:00 P.M.), abnormal behavior levels were comparable to and remained at control levels. Thus, while the initial KLC treatment was associated with an increase in abnormal home cage behavior, the addition of L-carnitine somewhat ameliorated the effects of KET. Neither KET nor KLC had lasting effects on home cage pup behavior.

The effects of PCP treatment on home cage pup behavior differed quantitatively, but not qualitatively, from those of KET and KLC. Specifically, the single PND 7 injection elevated abnormal behavior levels well above those of the initial KET injection, and 4 h postinjection, abnormal behavior levels were still high. Further, while there appeared to be a type of tolerance to repeated KET treatment on PND 7, PCP treatment on PNDs 9 and 11 elevated abnormal behaviors to the same extent as the initial injection on PND 7. Still, similar to KET and KLC treatments, the effects of PCP on home cage pup behavior had no lasting effects since level of abnormal home cage behavior was indistinguishable from controls on PNDs 8 and 10. These differences in levels of home cage abnormal activity may be related to differences in the half-lives of KET and PCP. In adult rats, iv treatment with PCP results in a half-life of 3–5 h (Shelnutt et al., 1999) whereas a half-life of less than 1 h is obtained with iv or im treatment with KET (Edwards et al., 2002; Williams et al., 2004). Thus, it is not surprising that PCP-induced abnormal home cage behavior was still evident 4 h posttreatment on PND 7.

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FIG. 4. Proportion of each treatment group falling from the forelimb hang apparatus during the allotted 60 s. Each of the five panels (A–E) represents a single test day and is similar to a typical ‘‘survival curve.’’ Here, the ‘‘survival’’ estimates were inverted such that higher curves correspond to a higher proportion of animals falling (e.g., on PND 8, approximately 27% of the control group had fallen at the end of 10 s). Note the shorter fall latencies in the PCP-treated group on PND 12 and the longer latencies to fall in the PCP- and KLC-treated groups on PND 16. See text for statistically significant effects.

The slant board and forelimb hang behaviors measured here are typical of those utilized in developmental neurotoxicity studies and were included since developmental PCP treatment has been shown to cause later motor coordination deficits (Sircar, 2003; Sircar and Rudy, 1998; Wang et al., 2001; Wiley et al., 2003). Although home cage pup behavior on PND 8 (conducted approximately 4 h after slant board behavior tests) indicated no abnormal behavior in the PCP- and KET-treated pups, both groups were slower to turn on the slant board on that day. Further, PCP-treated pups were slower to turn on PND 10, but not on PNDs 9 and 11, days on which the test was conducted prior to PCP treatment. This pattern indicates that the behavioral effects of the PCP treatment were still detectable via slant board behavior approximately 20, but not 44, h later. PCP, but not KET, treatment appeared to affect muscle strength as latency to fall in the forelimb hang test was decreased on PND 12. Finally, the repeated saline injections appeared not to affect behavior since slant board and forelimb hang results of the control group here were similar to those of previous studies (Ferguson et al., 2003).

There is some evidence of sexual dimorphism in the effects of PCP and anesthetics such as isoflurane and phenobarbital. The half-life of PCP is longer in adult female Sprague-Dawley rats than in males after iv treatment, and this appears to be due

to sex differences in metabolism (Shelnutt et al., 1999). On the other hand, neonatal treatment with isoflurane or phenobarbital resulted in more severe neurotoxicity in male Sprague-Dawley rats when examined at adulthood (Rothstein et al., 2008). There was no evidence of sexual dimorphism in response to the KET, KLC, or PCP treatments in the preweaning assessments conducted in the present study; however, assessments contin- ued through adulthood, and it is possible that sex differences in sensitivity to these compounds may be apparent after puberty. These NMDA antagonist-induced behavioral alterations may be related to increased cerebellar apoptosis. While apoptotic elimination of cerebellar granule cells is a natural process in rodent pups (Wood et al., 1993), inadequate stimulation of these cells or insufficient mossy fiber connections may increase the rate of apoptotic granule cell elimination. For example, treatment with the competitive NMDA receptor antagonist CGP 39551 on PNDs 7–11 increased DNA fragmentation levels in the inner granule layer and caspase-3 (a proapoptotic protease) levels in the rodent cerebellum (Monti and Contest- abile, 2000). Further, developmental treatment with non- competitive NMDA receptor antagonists such as MK-801 or KET resulted in increased numbers of multiple climbing fiber innervations in the cerebellum, increased apoptotic cerebellar cell death, and mild motor coordination impairments in mice

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(Kakizawa et al., 2000; Rudin et al., 2005). Thus, the KET and PCP treatments here are likely to have altered cerebellar devel- opment which may have resulted in impaired motor coor- dination expressed as paddling and paresis, longer slant board turn latencies, and shorter forelimb fall latencies.

the position or opinions of the FDA nor does mention of trade names or commercial products constitute endorsement or recommendation for use. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the FDA.

The mechanism by which L-carnitine ameliorated the adverse effects of KET treatment on body weight and behavior is not clear nor is it clear what effects L-carnitine may have had irrespective of the effects of KET. A group treated solely with L-carnitine (i.e., without KET) was not included as the focus of the study was on the effects of KET and PCP. The protective effects of L-carnitine may be mitochondrially mediated since L-carnitine protects against age-dependent mitochondrial decay (Hagen et al., 2002). NMDA antagonist treatment causes neuronal cell death through the Bax-cytochrome c-caspase pathway (Yoon et al., 2003) in which Bax is translocated into the outer mitochondrial membrane. Thus, L-carnitine may have prevented the KET-induced behavioral modifications by pro- hibiting the release of cytochrome c which would prevent caspase activation and apoptosis. As evidence, L-carnitine inhibits apoptosis in murine MC3T3-E1 osteoblastic cells by decreasing the release of cytochrome c and the activation of caspase-9 and caspase-3 (Xie et al., 2007).

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FUNDING

Interagency Agreement #224-93-0001 between the National Center for Toxicological Research (NCTR) / Food and Drug Administration (FDA) and the National Institute for Environ- / National Toxicology mental Health Sciences (NIEHS) Program (NTP).

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ACKNOWLEDGMENTS

The authors would like to thank Brett Thorn and Jacob Wulff of the Division of Personalized Medicine and Nutrition/ NCTR for their excellent statistical analyses. The authors would also like to thank Natalya Sadovova of Toxicologic Pathology Associates, the Bionetics Corporation, as well as Joseph (Ruda) Pollard and Melody Smith for their superb technical help. This document has been reviewed in accordance with United States Food and Drug Administration (FDA) policy and approved for publication. Approval does not signify that the contents necessarily reflect

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Wang, C. Z., and Johnson, K. M. (2005). Differential effects of acute and subchronic administration on phencyclidine-induced neurodegeneration in the perinatal rat. J. Neurosci. Res. 81, 284–292.

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Labruyere, J., and Olney, J. W. midazolam, degeneration in the infant mouse brain. Br. J. Pharmacol. 146, 189–197. Zissen, M. H., Zhang, G., McKelvy, A., Propst, J. T., Kendig, J. J., and Sweitzer, S. M. (2007). Tolerance, opioid-induced allodynia and withdrawal associated allodynia in infant and young rats. Neuroscience 144, 247–262. Zou, X., Sadovova, N., Patterson, T. A., Divine, R. L., Hotchkiss, C. E., Ali, S. F., Hanig, J. P., Paule, M. G., Slikker, W., Jr., and Wang, C. (2008). The effects of L-carnitine on the combination of, inhalation anesthetic-induced developmental, neuronal apoptosis in the rat frontal cortex. Neuroscience 151, 1053–1065.

individually or in combination,

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o n 1 2 F e b r u a r y 2 0 2 4",rats,['Neonatal PCP Is More Potent than Ketamine at Modifying Preweaning Behaviors of Sprague-Dawley Rats'],postnatal day 7,"['10 mg/kg PCP on postnatal days (PNDs) 7, 9, and 11; 20 mg/kg KET (six injections every 2 h on PND 7);']",Y,"['Slant board and forelimb hang behaviors were examined on PNDs 8–11 and 12–16, respectively.']",ketamine,"['Treatment with N-methyl-D-aspartate (NMDA) receptor antag- onists, such as ketamine (KET) or phencyclidine (PCP),']",phencyclidine,"['Treatment with N-methyl-D-aspartate (NMDA) receptor antag- onists, such as ketamine (KET) or phencyclidine (PCP),']",sprague dawley,['Neonatal PCP Is More Potent than Ketamine at Modifying Preweaning Behaviors of Sprague-Dawley Rats'],Little is known about the behavioral alterations resulting from NMDA antagonist treatment in neonatal rodents.,"['Treatment with N-methyl-D-aspartate (NMDA) receptor antagonists, such as ketamine (KET) or phencyclidine (PCP), can trigger apoptotic neurodegeneration in neonatal rodents; however, little is known about the behavioral alterations resulting from such treatment.']",None,[],These data suggest that three injections of 10 mg/kg PCP over PNDs 7–11 were more potent at modifying preweaning behaviors than were repeated injections of 20 mg/kg KET on PND 7.,['These data suggest that three injections of 10 mg/kg PCP over PNDs 7–11 were more potent at modifying preweaning behaviors than were repeated injections of 20 mg/kg KET on PND 7.'],None,[],None,[],True,True,True,True,False,True,10.1093/toxsci/kfn152
10.1093/bja/aet073,794.0,Boscolo,2013,rats,postnatal day 7,Y,"midazolam, isoflurane, nitrous oxide",none,sprague dawley,"British Journal of Anaesthesia 110 (S1): i47–i52 (2013) Advance Access publication 24 April 2013 . doi:10.1093/bja/aet073

Mitochondrial protectant pramipexole prevents sex-specific long-term cognitive impairment from early anaesthesia exposure in rats

A. Boscolo1,2, C. Ori2, J. Bennett3,4, B. Wiltgen5 and V. Jevtovic-Todorovic1*

1 Department of Anaesthesiology, University of Virginia, Charlottesville, VA, USA 2 Department of Anaesthesiology and Pharmacology, University of Padua, Padua, Italy 3 Parkinson’s Disease Centre and 4 Department of Neurology, Virginia Commonwealth University, Richmond, VA, USA 5 Department of Psychology, University of Virginia, Charlottesville, VA, USA

Corresponding author. E-mail: vj3w@virginia.edu

Editor’s key points

† Neonatal exposure to a combination of general anaesthetics results in developmental neurotoxicity and cognitive impairment in adult rats.

† The cognitive impairment could be prevented by co-treatment with the mitochondrial targeted anti-oxidant pramipexole.

† Female rats were more

vulnerable to anaesthesia-induced cognitive impairment, and thus might benefit more by such protective therapy.

Background. Exposure to general anaesthesia during critical stages of brain development results in long-lasting cognitive impairment. Co-administration of protective agents could minimize the detrimental effects of anaesthesia. Co-administration of R(+)pramipexole integrity, (PPX), a synthetic aminobenzothiazol derivative that restores mitochondrial prevents anaesthesia-induced mitochondrial and neuronal damage and prevents early development of cognitive impairment. Here, we determine the protective effects of PPX into late adulthood in male and female rats.

Methods. Postnatal day 7 rats of both sexes were exposed to mock anaesthesia or combined midazolam, nitrous oxide, and isoflurane anaesthesia for 6 h with or without PPX. Cognitive abilities were assessed between 5 and 7 months of age using Morris water maze spatial navigation tasks.

Results. Examination of spatial reference memory revealed that female, but not male, neonatal rats exposed to anaesthesia showed slowing of acquisition rates, which was significantly improved with PPX treatment. Examination of memory retention revealed that both male and female anaesthesia-treated rats have impaired memory retention performance compared with sham controls. Co-treatment with PPX resulted in improvement in memory retention in both sexes.

Conclusion. PPX provides long-lasting protection against cognitive impairment known to occur when very young animals are exposed to anaesthesia during the peak of brain development. Anaesthesia-induced cognitive impairment appears to be sex-specific with females being more vulnerable than males, suggesting that they could benefit more from early prevention.

Keywords: anaesthetics volatile, neonates; oxygen, toxicity; recovery, cognitive

isoflurane; anaesthetics gases, nitrous oxide; memory;

Accepted for publication: 14 February 2013

Both animal and human data suggest that exposure to general anaesthesia (GA) during critical stages of brain de- velopment results in long-lasting cognitive impairment.1 – 7 This problem begs the question of how GA can be used safely in the very young. One possibility would be co-administration of protective agents to minimize the detrimental effects of GA.

In an effort to address this possibility, we previously exam- ined the effects of early exposure to anaesthesia on mito- chondria as their proper functioning and morphogenesis are crucial for timely neuronal development. We found that mito- chondria are very vulnerable to GA-induced toxicity and are

one of the first intracellular organelles targeted by anaes- thetics.7 Injured mitochondria cause substantial up-regulation of free oxygen radicals, which, in turn, lead to lipid peroxida- tion in cellular membranes and neuronal loss in vulnerable brain regions.7 As neurones have high oxygen requirements and relative deficiency in oxidative defences, they are highly sensitive to excessive free oxygen radical production, suggest- ing that GA-induced mitochondrial damage and ensuing oxi- dative stress could be one of the key mechanisms leading to neuronal damage during early stages of brain development. Motivated by this finding we examined the free oxygen scav- enger, R(+)pramipexole (PPX), a synthetic aminobenzothiazol

& The Author [2013]. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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derivative that restores mitochondrial integrity,8 and found that when administered at the time of GA exposure, PPX pre- vents GA-induced mitochondrial and neuronal damage and prevents the development of cognitive impairment in young adult rats.7

To assess whether the protective effect of PPX is long- lasting and sex-specific we followed animals into late adult- hood (5–7 months of age) and compared benefits of PPX on cognitive performance of male and female rats exposed to GA at the peak of brain development (7 days of age).

Methods

We exposed postnatal day 7 (P7) Sprague-Dawley rats of both sexes to one of four treatment protocols: (i) sham controls (mock GA-vehicle, 0.1% dimethyl sulfoxide+21% oxygen for 6 h); (ii) GA-treated (midazolam, 9 mg kg21, i.p.; single injection immediately before administration of 0.75% isoflurane+75% nitrous oxide+24% oxygen for 6 h); (iii) GA+PPX-treated (PPX, 1 mg kg21 i.p.; four doses—at 9 h before, immediately before, immediately after, and 9 h after 6 h of GA); and (iv) PPX alone (same dosing regimen but mock GA). At the end of the treatments, rat pups were reunited with their mothers. Rats were housed using standard housing on a 12 h light/dark cycle with ad libitum access to food and water. All experiments were approved by the Animal Care and Use Committee of the University of Virginia Health System and were done in accordance with the Public Health Service’s Policy on Human Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used.

R(+)PPX doses and the dosing regimen were selected based on results from our prior work7 and the half-life of R(+)PPX which is estimated to be 8–12 h9 [i.e. R(+)PPX was dosed (cid:2)every half-life around the time of anaesthesia exposure]. An adult rodent dose of 1 mg kg21 is equivalent to a human dose of 0.2 mg kg21.9 10 Thus, these doses are very small and clinically feasible. Based on high-pressure liquid chromatography assays, R(+)PPX was .99.9% chem- ically and .99% enantiomerically pure.11

We found no differences among the groups in general ap- pearance or body weight over the next 7 months (data not shown). Cognitive abilities were assessed between 5 and 7 months of age using the Morris water maze test with the adult size pool (180 cm inner diameter).1 To examine their ability to swim, animals were tested in cued trials using a visible platform that was switched to a new location for each trial. During the place trials, rats were tested on their ability to learn the location of a platform (submerged, not visible), which remained in the same location during all trials. Two acquisition place trials were performed, each four blocks long (2 days per block). Probe trials were per- formed after each acquisition place trial (after blocks four and nine). During these trials, the platform was removed and times and patterns of swimming were analysed with special attention focused on time spent in the target quadrant.

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Data were analysed by analysis of variance using treat- ment and sex as between-subject variables and blocks of trials as within-subject variables. Pairwise comparisons were done after analysis of significant treatment effects and P-values exceeding Bonferroni corrected levels were noted. We considered P,0.05 to be statistically significant.

Results To assess whether GA-induced impairment in performance could be attributable to confounding visual or motor impair- ments, we performed a visual platform trial. The data shown in Figure 1 confirm that there were no differences in the time it took male or female rats from each group to reach a visible platform raised 1 cm above the water level (n¼4 trials).

The examination of spatial reference memory capabilities in the same animals during place trials (submerged platform, fixed location different from that used in the visual platform trials), revealed that female, but not male, rats exposed to

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Fig 1 Neonatal anaesthesia exposure has no effect on visual and motor performance in Morris water maze. (A) There was no differ- ence in the time it took male rats from each group (sham con- trols, anaesthesia—GA, GA+PPX, and PPX alone) to reach a visible platform raised above the water level (visual platform trial) in any of the four trials. (B) There was no difference in the time it took female rats from each group (sham controls, anaes- thesia—GA, GA+PPX, and PPX alone) to reach a visible platform raised above the water level in any of the four trials. The number of animals in each experimental group and for each sex is indicated in the graphs.

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Fig 2 Neonatal anaesthesia exposure has a long-lasting effect on spatial reference learning that is sex-specific and preventable by timely treatment with PPX. Male (A and C) and female (B and D) rats were tested at 5–7 months of age for their ability to learn the location of a sub- merged (not visible) platform during place trials. We used escape path length (A and B) and time to escape to the platform (C and D) as two main measures of spatial reference learning. Male rats exposed to GA showed no signs of impaired acquisition rates in terms of the escape path length (except during the third block of trials, *P,0.05) (A) or the time to escape to the platform (C). Spatial learning of PPX+GA male rats was indistinguishable from controls. Female rats exposed to GA showed significant slowing of acquisition rates in terms of the escape path length (B) and the time to the platform (D) (*P,0.05; **P,0.01; and ***P,0.001). It took GA-treated animals until the end of training (ninth block of trials) to perform similarly to controls. Females treated with PPX+GA performed much like sham controls throughout the testing period (P.0.05) (B and D). The timing of the probe trials after the last place trials is indicated in blocks 5 and 10. (The number of animals in each experimental group and for each sex is indicated in the graphs.) PPX only animals behaved much like controls (n¼9 males and 10 females; data not shown).

GA showed significant slowing of acquisition rates in terms of the escape path length (Fig. 2A and B) and the time to the platform (Fig. 2C and D); it was not until the very end of train- ing (Day 9) that GA-treated females (B and D) improved and performed similarly to controls. PPX treatment offered signifi- cant protection of cognitive function; females treated with PPX+GA performed much like sham controls throughout the testing period (P.0.05) (B and D). As GA-treated males did not show significantly impaired performance (A and C) (except on escape path length during the third block of trials), the protective effect of PPX on place trials in males was less impressive. PPX only animals behaved much like controls (n¼9 males and 10 females; data not shown).

To assess memory retention, we used probe trials wherein the submerged platform was removed from the pool (after the last place trials—after blocks four and nine as indicated

in Fig. 2). Hence, the animals were forced to rely on learned visual cues to find the quadrant where the platform had been (target quadrant). Both male and female GA-treated rats demonstrated impaired memory retention performance compared with sham controls (Fig. 3). Control male (A) and female rats (B) spent significantly more time during the second probe trial in the target quadrant (P,0.01) and significantly more time in the target quadrant compared with other three quadrants (P,0.001), which sug- gests a significant level of spatial memory retention. GA-treated male (C) and female (D) animals had no prefer- ence for any quadrant in the first or second probe trials despite previous training. Their behaviour could be described best as aimless swimming from one quadrant to another, suggesting a complete lack of retention and learning. In male (E) and female (F) animals exposed to GA+PPX

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Fig 3 Neonatal anaesthesia exposure has long-lasting effect on retention in both sexes and was preventable by timely treatment with PPX. Retention was examined using probe trials where animals were forced to rely on learned visual cues to find the quadrant where the platform had been (target quadrant). Control male (A) and female rats (B) spent significantly more time during the second probe trial in the target quad- rant (**P,0.01) and significantly more time in the target quadrant compared with the other three quadrants (***P,0.001). GA-treated male (C) and female (D) animals had no preference for any of the quadrants in the first or second probe trials despite previous training. GA+PPX-treated male (E) and female (F) animals spent significantly more time in the target quadrant during the second probe trial (females, *P,0.05) and significantly more time in the target quadrant compared with the other three quadrants [(E) males, ***P,0.001; (F) females, *P,0.05]. This behaviour was very similar to that of sham controls. The number of animals in each experimental group and for each gender is indicated in the graphs. PPX only animals behaved much like controls (n¼9 males and 10 females; data not shown).

treatment, in the memory retention shown as significantly more time spent in the target quadrant during the second probe trial (females, P,0.05) and significantly more time in the target quadrant compared with the other three quadrants (males, P,0.001; females, P,0.05), thus displaying learning behaviour that resembled sham control animals. PPX only animals behaved much like controls (n ¼ 9 males and 10 females; data not shown).

there was an improvement

Swimming speeds also were analysed during cued and place trials. No differences were observed (data not shown), further arguing that swimming performance deficits in were not GA-treated rats.

responsible for place-learning impairment

Discussion

Cognitive impairment is known to occur when very young animals are exposed to anaesthesia during the peak of brain development.1 – 7 Our previous work showed that GA integrity and function resulting in impairs mitochondrial

increased oxygen free radicals, lipid peroxidation, and neur- onal deletion.7 12 PPX was tested as a potential protective agent because it scavenges free oxygen radicals inside mitochondria and protects mitochondrial integrity.13 14 PPX readily crosses the blood–brain barrier and is concentrated in the brain where it is taken up and most highly concentrated in mitochondria.15 Previously, we showed that PPX decreases production of free oxygen radicals and protects neuronal sur- vival while protecting against GA-induced cognitive impair- ment in rats when assessed in early adolescence.7 Here, we show that this protection is long lasting because it can be detected in late adulthood as well.

Retention memory was impaired by GA administration at P7 (the peak of synaptogenesis) in both sexes, but adminis- tration of PPX around the time of anaesthesia exposure com- pletely prevented the development of this deficit, as demonstrated by normal cognitive performance in adulthood (5–7 months of age). GA exposure at P7 caused more detri- mental long-term effects on spatial learning in female than in male rats compared with sex-specific controls; this too was prevented by PPX.

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Anaesthesia and sex-specific learning impairment

Available evidence regarding sex differences in Morris water maze performance is conflicting. Some studies have shown learning,16 17 that males have an advantage in spatial whereas others suggest that this advantage disappears when females undergo additional training aimed at alleviating the stress response.18 19 However, newer evidence shows no base- line sex difference in Morris water maze performance.20 Simi- larly, we observed no difference in spatial learning between sham control males and females.

The effect of sex on GA-induced developmental neurotox- icity remains poorly understood. An earlier study by Rothstein and colleagues21 suggests that although exposure to GA during very early stages of synaptogenesis (postnatal day 0) resulted in significant impairment in cognitive abilities in both sexes, male rats were more affected than females when assessed in very young adulthood (1.5 months of age). The reasons for sex differences on GA-induced cognitive impairments over the course of adolescence and adulthood are yet to be deciphered, although a direct correlation between a decrease in performance in the Morris water maze and a decrease in hippocampal volume has been reported.21

The fact that spatial reference memory performance can be confounded by stress18 19 suggests the possibility that female-specific long-term impairment of spatial reference memory could be attributable to an anaesthesia-modified stress response that is female-specific. It is reasonable to propose that this could be caused by complex influences on hormonal cycling and mitochondrial development as a result of an early exposure to anaesthesia, although this notion needs to be examined critically.

We conclude that early exposure to GA causes long-term cognitive impairment that can be prevented by timely ad- ministration of PPX. As females appear to be more affected than males when studied in later adulthood, we suggest that they might benefit more from early prevention.

Acknowledgement

The authors thank Nathan Naughton for technical assistance.

Declaration of interest J.B. holds multiple patents protecting the use of R(+)PPX and receives royalty payments from those patents. Patents pro- tecting use of R(+)PPX have been licensed to Knopp Bios- ciences and Biogen-Idec. Neither company provided support for this study or was involved in data collection, data analysis nor manuscript preparation.

Funding

This work was supported by the NIH/NICHD, grant HD 44517 (V.J.-T.), John E. Fogarty Award TW007423-128322 (V.J.-T.), an American Recovery and Reinvestment Act supplement from the NIH/NICHD grant HD 44517S (V.J.-T.), March of Dimes National Award (V.J.-T.) and a Harold Carron

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endowment (V.J.-T.). V.J.-T. was an Established Investigator of the American Heart Association.

Authors’ role

The roles of each author in the study and preparation of the manuscript:

A.B.—data collection, data analysis, data interpretation, study design, drafting the article, providing intellectual content, final approval of the manuscript;

C.O.—providing intellectual content, final approval of the

manuscript;

J.B.—providing PPX for the study, providing intellectual content, assistance with revisions, final approval of the manuscript;

B.W.—providing Morris water maze facility, study design, data analysis and interpretation, providing intellectual content, final approval of the manuscript;

V.J.-T.—conception and design of the study, data inter- pretation and analysis, drafting the article, revising it, provid- ing intellectual content, final approval of the manuscript.

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11 Alavian KN, Dworetzky SI, Bonanni L, et al. Effects of dexprami- pexole on brain mitochondrial conductances and cellular bio- energetic efficiency. Brain Res 2012; 1446: 1– 11

12 Sanchez V, Feinstein SD, Lunardi N, et al. General anaesthesia causes long-term impairment of mitochondrial morphogenesis and synaptic transmission in developing rat brain. Anesthesiology 2011; 115: 992– 1002

13 Cassarino DS, Fall CP, Smith TS, Bennett JP Jr. Pramipexole reduces reactive oxygen species production in vivo and in vitro and inhibits the mitochondrial permeability transition produced by neurotoxin methylpyridinium ion. J Neurochem 1998; 71: 295– 301

the

parkinsonian

14 Zou L, Xu J, Jankovic J, He Y, Appel SH, Le W. Pramipexole inhibits lipid peroxidation and reduces injury in the substantia nigra induced by the dopaminergic neurotoxin 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine in C57BL/6 mice. Neurosci Lett 2000; 281: 167–70

15 Danzeisen R, Schwalenstoecker B, Gillardon F, et al. Targeted antioxidative and neuroprotective properties of the dopamine agonist pramipexole and its nondopaminergic enantiomer [(+)2-amino-4,5,6,7-tetrahydro-6-propylamino- SND919CL2x benzathiazole dihydrochloride]. J Pharmacol Exp Ther 2006; 316: 189–99

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16 Brandeis R, Brandys Y, Yehuda S. The use of the Morris water maze in the study of memory and learning. Int J Neurosci 1989; 48: 29–69

17 Jonasson Z. Meta-analysis of

in rodent models of learning and memory: a review of behavioural and biological data. Neurosci Biobehav Rev 2005; 28: 811– 25

sex differences

18 Perrot-Sinal TS, Kostenuik MA, Ossenkopp KP, Kavaliers M. Sex dif- ferences in performance in the Morris water maze and the effects of initial nonstationary hidden platform training. Behav Neurosci 1996; 110: 1309– 20

19 Simpson J, Kelly JP. An investigation of whether there are sex differences in certain behavioural and neurochemical rat. Behav Brain Res 2012; 229: parameters 289 – 300

in the

20 Mazor A, Matar MA, Kaplan Z, Kozlovsky N, Zohar J, Cohen H. Gender-related qualitative differences in baseline and post-stress anxiety responses are not reflected in the incidence of criterion- based PTSD-like behaviour patterns. World J Biol Psychiatry 2009; 10: 856–69

21 Rothstein S, Simkins T, Nun˜ ez JL. Response to neonatal anaesthe- sia: effect of sex on anatomical and behavioural outcome. Neuro- science 2008; 152: 959– 69

Handling editor: H. C. Hemmings",rats,"['Postnatal day 7 rats of both sexes were exposed to mock anaesthesia or combined midazolam, nitrous oxide, and isoflurane anaesthesia for 6 h with or without PPX.']",postnatal day 7,"['Postnatal day 7 rats of both sexes were exposed to mock anaesthesia or combined midazolam, nitrous oxide, and isoflurane anaesthesia for 6 h with or without PPX.']",Y,['Cognitive abilities were assessed between 5 and 7 months of age using Morris water maze spatial navigation tasks.'],midazolam,"['Postnatal day 7 rats of both sexes were exposed to mock anaesthesia or combined midazolam, nitrous oxide, and isoflurane anaesthesia for 6 h with or without PPX.']",nitrous oxide,"['Postnatal day 7 rats of both sexes were exposed to mock anaesthesia or combined midazolam, nitrous oxide, and isoflurane anaesthesia for 6 h with or without PPX.']",sprague dawley,['We exposed postnatal day 7 (P7) Sprague-Dawley rats of both sexes to one of four treatment protocols.'],The study addresses the issue of long-term cognitive impairment from early anaesthesia exposure and tests the protective effects of PPX.,"['Here, we determine the protective effects of PPX into late adulthood in male and female rats.']",None,[],"The findings suggest that PPX provides long-lasting protection against cognitive impairment from early anaesthesia exposure, with implications for both sexes but particularly females.",['PPX provides long-lasting protection against cognitive impairment known to occur when very young animals are exposed to anaesthesia during the peak of brain development.'],None,[],The potential application is the use of PPX as a protective agent against cognitive impairment from early anaesthesia exposure.,['The cognitive impairment could be prevented by co-treatment with the mitochondrial targeted anti-oxidant pramipexole.'],True,True,True,False,False,True,10.1093/bja/aet073
10.1093/bja/aes121,505.0,Feng,2012,rats,postnatal day 7,N,sevoflurane,none,sprague dawley,"British Journal of Anaesthesia 109 (2): 225–33 (2012) Advance Access publication 25 April 2012 . doi:10.1093/bja/aes121

NEUROSCIENCES AND NEUROANAESTHESIA

Single sevoflurane exposure decreases neuronal nitric oxide synthase levels in the hippocampus of developing rats

X. Feng1†, J. J. Liu1†, X. Zhou1, F. H. Song2, X. Y. Yang1, X. S. Chen1, W. Q. Huang1*, L. H. Zhou2* and J. H. Ye3 1 Department of Anaesthesiology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, PR China 2 Department of Anatomy, Zhong Shan School of Medicine, Sun Yat-sen University, Guangzhou, PR China 3 Department of Anaesthesiology, New Jersey Medical School, Newark, NJ, USA

Corresponding authors. E-mail: sumshwq@gmail.com (WQH) and zhoulih@mail.sysu.edu.cn (LHZ)

Editor’s key points

† General anaesthesia is

implicated in developmental neurotoxicity in neonatal animals and in subsequent cognitive dysfunction.

Background. The use of general anaesthetics in young children and infants has raised concerns regarding the adverse effects of these drugs on brain development. Sevoflurane might have harmful effects on the developing brain; however, these effects have not been well investigated.

Methods. Postnatal day 7 (P7) Sprague– Dawley rats were continuously exposed to 2.3% sevoflurane for 6 h. We used the Fox battery test and Morris water maze (MWM) to examine subsequent neurobehavioural performance. Cleaved caspase-3 and neuronal nitric oxide synthase (nNOS) were quantified by immunoblotting, and the Nissl staining was used to observe the histopathological changes in the hippocampus.

† The effects of sevoflurane

on cell death and neurobehavioural performance were studied in neonatal rats.

† A single 6 h exposure

Results. A single 6 h sevoflurane exposure at P7 rats resulted in increased cleaved caspase-3 expression and decreased nNOS levels in the hippocampus, and induced the loss of pyramidal neurones in the CA1 and CA3 subfields of the hippocampus at P7–8. These changes were accompanied by temporal retardation of sensorimotor reflexes. However, neither the Fox battery test at P1–21 nor the MWM test at P28–32 showed differences between the air- and sevoflurane-treated groups.

produced early evidence of neuronal death in the hippocampus, but had no effect on subsequent neurobehavioural performance.

Conclusions. Although early exposure to sevoflurane increases activated caspase-3 expression and neuronal loss and decreases nNOS in the neonatal hippocampus, it does not affect subsequent neurobehavioural performances in juvenile rats.

Keywords: anaesthetic, sevoflurane; caspase 3; hippocampus; memory; neuronal nitric oxide synthase; sevoflurane

Accepted for publication: 3 March 2012

Animal and human brains grow rapidly during early develop- ment, making them especially vulnerable to environmental influences, including general anaesthetics. As anaesthetic and surgical technology advances, the administration of general including premature anaesthetics to very young children, babies, is becoming increasingly common. Accumulating evi- dence suggests that general anaesthetics administered to neo- natal animals including widespread degeneration of neurones and long-term abnormal social behaviour and cognitive dysfunction.1 – 3 This has raised significant concerns among anaesthesiologists, neuroscientists, parents, and the media regarding the safety of general anaes- thetics in infants and neonates.

can have deleterious effects,

Sevoflurane is one of the most frequently used inhalation anaesthetics. It has been used during child labour4 and is

especially suitable for infants and children because of its low blood gas partition coefficient, rapid onset and offset, aromatic odour, and low pungency into the airway.5 Several studies6 7 and case reports8 have shown epileptiform EEG and seizure ac- tivity during induction of anaesthesia with sevoflurane. A previ- ous study reported that no significant neurodegeneration in primary cortical neurones was found in rats exposed to sevo- flurane at clinically relevant concentrations.9 Conversely, Sato- moto and colleagues3 showed that sevoflurane results in learning deficits and autism-like abnormal social behaviours in neonatal mice. Moreover, a study in human volunteers sug- gested that sevoflurane disrupts human emotional memory processing.10 These results raise the possibility that sevoflurane might be harmful to the developing brain. However, the effects of sevoflurane on immature brains have not been well explored.

† These authors contributed equally to this work.

& The Author [2012]. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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Neuronal nitric oxide synthase (nNOS) is the predominant NOS isoform in the central nervous system (CNS). During neuronal development, nNOS is transcriptionally induced in CNS.11 Furthermore, nNOS in the hippocampus is localized at sites of neuronal proliferation and migration.12 Nitric oxide, the product of nNOS, and its downstream effectors regulate cell proliferation and differentiation in the develop- ing nervous system by playing a neurotrophic role in neuronal cell–cell communication.13 14 In the hippocampus, nNOS plays an important role in synaptic plasticity and is involved in learning and memory.15

Recent evidence suggests that during the period of rapid brain development, enhanced degeneration of neurones trig- gered by anaesthetics is caused by increased intracellular free calcium,16 a potent activator of nNOS.17 Caspase-3, a member of the cysteine-aspartic acid protease (caspase) family, which is essential for normal brain development. The sequential activation of caspases plays a central role in the execution phase of apoptosis,18 and cleaved caspase-3 indicates the endogenous levels of activated caspase-3, a marker of cell apoptosis.19 20

To investigate the potential neurotoxicity induced by sevo- rats to sevoflurane and flurane, we exposed neonatal assessed the protein expression levels of nNOS and cleaved caspase-3 in the developing hippocampus. We also exam- ined neurobehavioural performance to determine whether a single sevoflurane exposure had deleterious effects on long-term cognitive function.

Methods

Animals

The use of animals in this study was approved by the Institu- tional Animal Care and Use Committee at Sun Yat-sen Uni- versity (Guangzhou, Guangdong, China). All efforts were made to minimize the number of animals used and their suf- fering. Male Sprague–Dawley (SD) rats were obtained from the Experimental Animal Centre of Sun Yat-sen University. The rats were housed under a 12 h light–dark cycle (light from 07:00 to 19:00) at 20– 228C. In addition, the rats were given ad libitum access to water and food. A total of 19 litters consisting of 99 male pups were used in this study. Each experimental condition had its own group of littermate controls to minimize variability in the rate of apoptosis.21

Sevoflurane exposure

Rats at postnatal day 7 (P7, 16–17 g) were randomly divided into a sevoflurane-treated group (51 rats) and an air-treated control group (48 rats). Rats in the sevoflurane-treated group were placed in a plastic container and continuously exposed to 2.3% sevoflurane for 6 h using air as a carrier with a gas flow of 2 litre min21. During sevoflurane exposure, the con- tainer was heated to 388C (NPS-A3 heated device, Midea, Co., Guangdong, China). Sevoflurane, oxygen, and carbon dioxide in the chamber were monitored using a gas monitor (Detex-Ohmeda, Louisville, KY, USA). After 6 h, the rats were exposed to air only and, when able to move

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freely, were placed back into their maternal cages. During sevoflurane exposure, an investigator monitored respiratory frequency and skin colour; if signs of apnoea or hypoxaemia were detected, the rat was immediately exposed to air and excluded from the experiment. Rats in the control group were placed into the container and were exposed to air only for 6 h.

Arterial blood gas analysis

Arterial blood analysis was performed on P7–8 rats (16–17 g) from the sevoflurane- and air-treated groups.1 22 Arterial blood samples were obtained from the left cardiac ventricle immediately after removal from the maternal cage (0 h, n¼3 in each subgroup) or after anaesthesia (6 h, n¼3 in each subgroup). Samples were transferred into heparinized glass capillary tubes and analysed immediately by a blood gas analyser (Gem premier 3000). The pups were killed by decapitation at the time of blood sampling and the analysis of each sample was repeated at least three times.

Behavioural studies

The Fox battery test was used to assess the cerebral matur- ation of P1 –21 rats,23 24 and the Morris water maze (MWM) was used to test spatial learning and memory performance in P28 –32 rats.25 26

The Fox battery test

Fox battery tests were conducted on 12 rats from P1 to P21 (4–55 g) daily between 08:30 and 23:00 which corresponded to the rats’ active period, as described in previous studies.23 24 At P7, rats were randomly divided into sevoflurane-treated (n¼6) and the air-treated groups (n¼6) that were exposed to sevoflurane or to air for 6 h, respectively. The Fox battery test was performed after the rats had fully recovered from an- aesthesia and were able to move freely as described.23 24 The time of the appearance (days) of the eye opening, incisor eruption, limb grasp, crossed extensor reflex, negative geo- taxis reflex, and gait reflex was recorded for each rat. Add- itionally, the time needed to achieve the righting reflex, the negative geotaxis reflex, and the gait reflex was recorded. The maximum angle at which the animals could maintain the position on an inclined board test for 5 s was also documented.

MWM test Based on a previous study,25 we performed the MWM test on P28 (80 –100 g) rats using the Water Maze Tracking System (TME; Chengdu, China) with minor modifications (1984).26 This test was conducted on both sevoflurane-treated (n¼9) and air-treated groups (n¼6). The MWM consisted of a grey circular tank (100 cm diameter, 50 cm in depth), which was surrounded by several visual cues. Immediately before the test, the tank was filled with water [22 (1)8C] to a height of 30 cm. The tank was equally divided into the target (T, where a plastic platform was submerged), right (R), opposite (O), and left (L) quadrants, with four starting

Sevoflurane decreases nNOS in developing hippocampus

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locations that were equidistant from the rim. We conducted memory-acquisition trials (training) four times daily for 5 days. A single adaptation trial (without the platform) was performed and the rats were released into the pool for 60 s in the absence of any escape platform on day 0. On the follow- ing 4 days (days 1–4, Place Navigation), two blocks of tests (morning 08:30 –11:00 and afternoon 14:30 –15:00) were per- formed with four trials per block per day for each rat. In each trial, the rat was placed into the water facing the wall from one of the four starting points. The escape latency (time to find the submerged platform), the swimming route to reach the platform, and the swimming speed were measured by a computer-operated video tracking system. Once the rat had reached the platform, it was allowed to remain on the plat- form for 15 s for orientation purposes. Those rats that failed to independently find the escape platform within 60 s were placed on to the escape platform by the experimenter. The rats were removed afterwards to rest in a heated cage until the next trial. Four daily trials were averaged for each animal. On day 5, the memory retention tests (Spatial Probe) were performed in the absence of a submerged plat- form in the tank. The rat was placed into the water, facing the wall from one of the four starting points. Within 120 s, both the time spent in each quadrant and the swimming route were recorded. The rats were then removed from the tank and placed back into the heated cage.

Western blot analysis

Immunoblotting was performed on hippocampi obtained from 48 P7–8 rats (16 –17 g) as previously described.27 28 Briefly, rats were killed by decapitation at 0, 2, 6, and 24 h after 6 h sevoflurane or air treatments, with six rats at each time point per treatment. The rat brain was quickly dissected, and the hippocampus was quickly removed and homogenized in 100 mg ml21 RIPA Lysis Buffer (She- nergy Biocolor Co., China) with 1% (v/v) PMSF (Shenergy Biocolor Co., China). The homogenate was centrifuged at 13 000g for 20 min at 48C, and the supernatant was sepa- rated and stored at 2808C until further use. The proteins extracted from the hippocampus were separated on a 10% gel by electrophoresis and transferred on to polyviny- lidene fluoride membranes (Pall Co., USA). The blots were then incubated with anti-cleaved caspase-3 (1:1000, rabbit polyclonal, Asp175; Cell Signaling Technology, Inc.,

USA) or anti-b-actin (1:2000, mouse monoclonal; Santa Cruz Biotechnology, USA) antibodies. The changes in the protein expression levels of nNOS using an anti-nNOS antibody (1:500, mouse monoclonal; Santa Cruz Biotech- nology, USA) were examined using the ECL-PLUS system (CWBIO, China) and imaged. The b-actin levels were used as a loading control. Optical density was measured by analysing scanned images using the Image J software (NIH, USA). Changes in protein expression ratio (compared with b-actin) were determined by optical density measurements (n¼3 for each rat hippocampus sample).

Histopathological examination

Sevoflurane-treated (n¼6) and air-treated (n¼6) rats (P7 –8, 16–17 g) were killed for the Nissl staining at 6 h after a 6 h exposure to either sevoflurane or air. Animals were anaes- thetized with a lethal dose of 10% chloral hydrate and trans- cardially perfused with saline through the left cardiac ventricle until the liver and lungs were cleared of blood, fol- lowed by 4% paraformaldehyde in 0.1 M PB (NaH2PO4.2H2O 2.96 g, Na2HPO4.12H2O 29 g dissolved in 1000 ml water, PH 7.4). The perfusion lasted for 15– 25 min. The brains were removed and incubated overnight in the same fixative. Paraffin blocks of brain tissue (0.5 mm thick) included sec- tions of the hippocampus at different levels along the septo- temporal axis and associated areas.29 Coronal hippocampal sections 5 mm in thickness were Nissl-stained, and examined under a light microscope (Nikon ECLIPSE, 50i, Japan) to study the morphological changes of pyramidal neurones in the CA1 and CA3 regions of the hippocampus. We counted cells (n¼3 for each imaged from three sections per animal group). Nissl-positive cells were counted only if the structures were of the appropriate size and shape, possessed a Nissl- positive nucleus and cytoplasmic Nissl-positive particles. The number of Nissl-positive neurones in the pyramidal cell layers of the bilateral CA1 regions was counted at ×400 magnification by two individuals in a blinded manner.30 Questionable structures were examined under ×1000 mag- nification and were not counted if identification remained uncertain.

Statistical analysis

Values are presented as mean (SEM). The SPSS 13.0 software was used for statistical analysis. We tested for normality

Table 1 Arterial blood analysis. 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 in the sevoflurane and control groups to air (t-test, all P-values.0.05). PaCO2 , arterial carbon dioxide tension; PaO2 , arterial oxygen tension

Time (h)

n

Arterial blood analysis

pH

PaCO2 (kPa)

PaO2 (kPa)

Glucose (mmol litre21)

Control

0 6

3 3

7.39 (0.08) 7.38 (0.07)

3.59 (0.43) 3.56 (0.45)

13.4 (0.52) 13.3 (0.60)

5.5 + 0.4 5.3 + 0.6

Sevoflurane

0 6

3 3

7.39 (0.04) 7.34 (0.06)

3.58 (0.39) 3.61 (0.46)

13.4 (0.44) 13.3 (0.59)

5.4 + 0.7 5.2 + 0.5

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using the Shapiro–Wilk test and homogeneity of variance by Levene’s test. Comparisons of means between two groups were performed using Student’s t-test or the Wilcoxon W-test. Statistical significance was assessed using multivari- ate analysis of variance followed by the Bonferroni multiple comparison testing. When appropriate, 2×2 comparisons were made using a least significant difference test. P-values of ≤0.05 were considered statistically significant.

Results

Neurobehavioural development of neonatal rats

Physical development

Arterial blood analysis showed that pH, carbon dioxide tension, oxygen tension, and glucose levels were not signifi- cantly different between sevoflurane- and air-treated animal groups (Table 1). Pups treated with sevoflurane appeared pink during the 6 h of gas exposure. The appearance of eye opening or incisor eruption did not differ between sevoflurane- and air-treated animal groups (Table 2).

Table 2 Appearance of physical and neurological signs in control or sevoflurane-exposed rats. The values represent the mean (SEM) in days

Days of appearance

Control (n56)

Sevoflurane (n56)

Eye opening

12.2 (0.09)

12.2 (0.15)

Incisor eruption

5.6 (0.90)

5.6 (0.30)

Forelimb grasp

2.4 (0.30)

2.5 (0.34)

Hindlimb grasp

10.8 (0.90)

9.3 (0.30)

Crossed extensor reflex

12.4 (0.40)

11.3 (0.30)

Negative geotaxis 208

5.4 (0.10)

5.4 (0.05)

Negative geotaxis 458

10.4 (1.00)

10.0 (1.00)

Gait reflex

11.1 (1.02)

10.0 (0.83)

Reflexes and motor development

In the Fox battery test on P1–21 neonatal rats (Table 2), the appearance of hindlimb grasp and extensor reflexes was slightly delayed, but no significant differences were detected in the sevoflurane-treated group compared with the air- treated group. The appearance of the forelimb grasp reflex, negative geotaxis reflex, or the gait reflex was not altered in sevoflurane-treated rats (Table 2). From P1 to P21, the time required by the pups to achieve the righting reflex (Fig. 1A), negative taxis (Fig. 1B), and gait reflexes (Fig. 1C) decreased with age in both the air- and sevoflurane-treated groups. Although sevoflurane-treated rats at P7 and P9–12 displayed slightly lower performance in the righting reflex (Fig. 1A) and displayed a 208 negative geotaxis (Fig. 1B),

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P9 P10 P11 P12 P13 P14 P15 P16 P17

P10 P11 P12 P13 P14 P17

P21 P28

P10 P11 P12 P13 P14 P15 P16 P17 P18 P19

Fig 1 Effects of sevoflurane exposure at P7 on sensorimotor neurobehaviour of neonatal rats from P1 to P28 in the Fox battery test. Daily performance in the righting reflex (A); in the negative geotaxis on a 208-tilted (B), and 458-tilted board (C); and in the gait reflex (D). The time to achieve the righting reflex (in seconds) is expressed as mean (SEM). (E) Daily performance on the inclined board test. The results are expressed as mean (SEM) angle.

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Sevoflurane decreases nNOS in developing hippocampus

these differences were not statistically significant compared with air-treated rats in their performances of the 408 nega- tive geotaxis reflex (Fig. 1C), the gait reflex (Fig. 1D), or the inclined board test (Fig. 1E). The performance of sevoflurane- treated rats in the Fox battery test returned to the level of air-treated control rats at P13 (Fig. 1A– E).

Spatial learning and memory development

During place navigation in the MWM test, P28–32 rats had an escape latency that was longer in the first trial (Fig. 2A) and shorter in the last trial (Fig. 2B) for every rat tested (n¼15) in air- and sevoflurane-treated groups. This indicated the en- hancement of escape acquisition (Fig. 2C). No significant differ- ences were found between these two groups in escape latency at each time point (Fig. 2C). During the spatial bias of the MWM test, the average percentage of the swimming distance of sevoflurane-treated rats (Fig. 2D) compared with that of air- treated control rats (Fig. 2E) was 38.4% (13.2%) [mean (SEM)] vs 44.5% (6.1%) in target, 21.3% (7.5%) vs 23.8% (5.5%) in right, 19.3% (9.6%) vs 12.8% (0.7%) in opposite, and 21.0% (6.9%) vs 19.0% (8.4%) in the left quadrants. Statistical ana- lyses showed that the average percentage of the swimming distance in the target quadrant was significantly longer than in any of the other quadrants of either sevoflurane- or air- treated control groups (all P,0.05, Fig. 2F). The percentage of the swimming distance in sevoflurane-treated rats was not sig- nificantly different from that of air-treated control rats in every quadrant, including the target quadrant (all P.0.05, Fig. 2F). The swimming speed measured during the place navigation trial was not significantly different between sevoflurane-treated [13.1 (1.9) m min21)] and air-treated control rats [12.5 (2.2) m min21]. This ruled out the possibility that differences obtained during place navigation and spatial probe trials were due to physical impairments.

Sevoflurane-induced expression of cleaved caspase-3 and reduction in nNOS protein in neonatal rat hippocampus

Expression levels of cleaved caspase-3 (Fig. 3A) and nNOS (Fig. 3B) were examined by western blotting at four time points (0, 2, 6, and 24 h) after 6 h of sevoflurane or air expos- ure (Fig. 3C and D). Cleaved caspase-3 protein expression levels in the hippocampus of sevoflurane-treated rats signifi- cantly increased from 0 to 6 h, then declined, and was sub- sequently maintained at a level higher than that of control rats at 24 h (Fig. 3A). A quantitative study showed that ex- pression levels of cleaved caspase-3 in the hippocampus of sevoflurane-treated rats were significantly higher than in air- treated control rats at each time point (all P,0.01, Fig. 3C). Expression of cleaved caspase-3 in sevoflurane-treated rats was significantly higher at 2 and 6 h, compared with any other time point (all P,0.01, Fig. 3C). There were no signifi- cant differences between the 2 and 6 h time points (all P.0.05, Fig. 3C).

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T R O L

Sevo

Control

Fig 2 Effects of sevoflurane exposure at P7 on spatial learning and memory abilities of P28 – 32 rats in the MWM test. The learn- ing and memory abilities of sevoflurane- (n¼9) and air-treated (control, n¼9) rats were assessed using place navigation (A – C) and spatial probe trials (D – F). A representative route of a control rat to reach the escape platform on day 1 (A) and day 4 (B). (C) The latencies to reach the escape platform in the 4 day place navigation trials, which show no significant differences between the control and sevoflurane groups. The results are expressed as the mean (SEM) time in seconds. (D and E) A repre- sentative route of a sevoflurane-treated rat (D) and a control rat (E) in search of the escape platform on day 5. T, target quad- rant; R, O, L, quadrants which were to the right, opposite, or left of the target quadrant. (F) A comparison of the average percent- age of the swimming distance in each quadrant of the MWM for the sevoflurane and control groups. The target quadrant (T) in the spatial probe trial demonstrates the retention ability of P,0.05 compared spatial learning and memory in MWM. P,0.05 com- with the T quadrant within the sevoflurane group; pared with the T quadrant within the control group. There were no significant differences between the control and sevoflurane groups in any of the quadrants of the MWM.



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0

C0h C2h C6h C24h S0h S2h S6h S24h

C0h C2h C6h C24h S0h S2h S6h S24h

Fig 3 Western blot analysis showed that sevoflurane exposure induced an increase in cleaved caspase-3, but a reduction in the nNOS at P7. (A and B) Samples were obtained from the hippocampi of rats subjected to air (control, lanes 1–4) or sevoflurane treatment (lanes 2– 5, respect- ively) at 0, 2, 6, and 24 h after treatment. Representative graphs of the effects of sevoflurane on cleaved caspase-3 and nNOS levels are shown. (C) Expression of cleaved caspase-3 in the sevoflurane group was significantly higher than that in the control group at each time point. The highest levels of cleaved caspase-3 expression occurred at 6 h, then 2, 0, and 24 h after sevoflurane exposure. There was no significant difference between each time point. (D) The expression of nNOS in the sevoflurane group at 24 h after exposure was significantly lower than that in the control group. The lowest level of nNOS occurred at 24 h, which was significantly lower than at other time points. There were no differences between each control group. (C and D) †P,0.05 vs control, 0 h; P,0.05 vs sevoflurane, 0 h; A P,0.05 vs sevoflurane, 2 h;

W

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S

P,0.05 vs sevoflurane, 6 h.

Sevoflurane treatment induced a higher level of cleaved caspase-3 and nNOS protein were lower in sevoflurane- treated rats from 0 to 24 h compared with control (Fig. 3B). Quantitative studies showed that levels of nNOS in the hippo- campus of sevoflurane-treated rats were lower than in the control group at each time point (all P,0.01, Fig. 3D). In the sevoflurane-treated group, nNOS levels were the highest at 0 h but the lowest at 24 h (Fig. 3D). The difference in nNOS expression was significant between all time points of sevoflurane-treated subgroups (all P,0.05, Fig. 3D).

Discussion In this study, we used 2.3% sevoflurane as the highest con- centration that did not inhibit respiration and circulation in rat pups under our experimental conditions, compared with 3.28%, which is the minimum alveolar concentration of sevo- flurane in neonatal rats.31 Thus, 2.3% sevoflurane is compar- able with that used in the clinical setting. The arterial blood analysis further confirmed that none of the rats suffered from apnoea or hypoxaemia during the 6 h sevoflurane exposure.

Sevoflurane results in histopathological changes in neonatal rat hippocampus

The Nissl staining revealed neuronal morphology changes that were apparent in the subfields of the hippocampus at 6 h after sevoflurane exposure. Compared with pyramidal neurones in the hippocampus of air-treated control rats (Fig. 4A– D), there were remarkable neuropathological changes including neuronal loss and nucleus shrinkage in the CA1 region (Fig. 4E and F). Oedema resulting from the neuronal cell bodies and cytoplasmic Nissl body loss were also found in the CA3 region (Fig. 4G and H) of the hippocam- pus of sevoflurane-treated rats. Statistical analysis showed a significant decrease in the density of healthy pyramidal neu- rones in CA1 and CA3 regions of the hippocampus in sevoflurane-treated rats compared with control rats (all P,0.05, Fig. 4I). These results indicate that sevoflurane ex- posure induced a reduction in the number of healthy pyram- idal neurones in the hippocampus.

Neurones in the developing brain appear to be specifically vulnerable to neurotoxicity induced by general anaesthetics.1 Several studies have shown that sevoflurane exposure of neonatal animals significantly increases neurone apoptosis in several brain regions.3 32 33 Consistent with these findings, this study demonstrated that a single sevoflurane exposure to neonatal (P7) rats significantly increased the loss of pyr- amidal neurones in both the CA1 and CA3 subfields of the hippocampus. This study also showed that nNOS expression in hippocampal neurones was significantly decreased after sevoflurane exposure. We found that down-regulation of nNOS peaked at 24 h and up-regulation of activated caspase-3 peaked at 2–6 h after sevoflurane exposure. Because caspases are considered inducers of apoptotic cell death,3 32 33 our data suggest that the reduction in nNOS might from sevoflurane-induced death of nNOS- positive neurones. Our results are consistent with results of a previous stress reduces the density of nNOS-positive neurones in the fascia

result

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Fig 4 Sevoflurane exposure-induced histopathological changes in the hippocampus of P7 neonatal rats at 6 h after the 6 h sevoflurane treat- ment as shown by the Nissl staining. (A – E) Representative photomicrographs of coronal sections of the hippocampus of P7 rats (magnification, ×100, scale bar, 50 mm in A, C, E, and G; magnification, ×400, scale bar, 200 mm in B, D, F, and H). The Nissl-stained normal pyramidal neurones in the CA1 (A and B) and CA3 (C and D) regions of air-treated rats. Histopathological changes of the Nissl-stained pyramidal neurones in the CA1 (E and F) and CA3 (G and H) regions of the hippocampus of sevoflurane-treated rats. (I) Comparison of the Nissl-positive pyramidal neurones in the CA1 and CA3 regions of the hippocampus between sevoflurane- and air-treated rats. Each value represents the mean (SEM) (n¼5 rats per group). *P,0.05 compared with air-treated controls in the same region.

dentata and Ammon’s horn of rats.21 However, Cattano and colleagues found that nitrous oxide induces a pro-apoptotic effect, which is associated with increased levels of intracellu- lar free calcium, and followed by the up-regulation of nNOS and p53 in neonatal rat brain.34 35 These differences might be due to tissue-specificity of nNOS gene expression in re- sponse to anaesthetics. While Cattano and colleagues ana- lysed nNOS mRNA expression levels in the forebrain, we investigated nNOS protein expression levels in the hippocam- pus. Importantly, when applied alone, nitrous oxide failed to induce robust neuronal cell death,36 37 whereas sevoflurane, which potentiates g-aminobutyric acid type A (GABAA) recep- tors,38 exhibits deleterious effects including widespread de- generation of neurones and long-term abnormal social behaviour and cognitive dysfunction when applied to neo- natal animals.1 3 39 Therefore, these two inhaled anaes- thetics might affect cellular survival and cell death via different pathways, which could induce changes in nNOS expression.

hippocampus around P7–8, there were no differences in be- havioural performances in the MWM test at P28–32. This was consistent with findings obtained in P7–12 rats on the day of sevoflurane exposure, where sevoflurane-exposed pups did not show lower performance in sensorimotor reflexes. Our results are consistent with previous studies that showed that prenatal methylenedioxymethamphetamine-exposed pups displayed lower performances in the Fox battery test from P2 to P12 and were able to completely recover by P19 to P28.24 40 Recovery of neurobehavioural performance of neo- natal pups indicates a high degree of plasticity of neonatal rat brain.24 40 This hypothesis was further supported by our present results from the MWM test, which confirmed that sevoflurane exposure at P7 does not affect spatial learning and memory activities at P28–32. Several previous reports also indicated that sevoflurane exposure induced only short- term dysfunctions of learning and memory.41 42 Previous studies showed that a reduction in nNOS expression contri- butes to deficits in hippocampal-dependent learning and long- term potentiation,34 35 and deletion of the nNOS gene in mice resulted in abnormal social behaviour and impaired remote

Despite the increase in activated caspase-3 expression, the decrease in nNOS expression, and the neuronal loss in neonatal

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spatial memory.43 Spatial learning and memory are complex, and involve different brain areas such as the cerebellum, striatum, cerebral cortex, amygdala, and hippocampus. The hippocampus has been extensively studied as a part of the brain system responsible for learning and memory, the function of which can be specifically detected by the MWM test.25 Given that sevoflurane exposure can lead to persistent learning deficits in fear conditioning,3 which is thought to mainly depend upon amygdala function, we assume that spatial learning and memory functions are less vulnerable to sevoflurane-induced neurotoxicity. Finally and most importantly, our data show that sevoflurane-induced activation of caspase-3 was temporary (peaked at 6 h, but declined at 24 h). It is possible that nNOS protein expression in the hippocampus might have returned to levels that support learning and memory functions at P28–32. Taken together, the roles of these factors in learning, memory, and cognitive dysfunction are still unknown and warrant further study.

spatial

In summary, this study demonstrated that a single 6 h ex- posure of 2.3% sevoflurane in neonatal rats induced neuro- apoptosis and a reduction in nNOS protein expression in the hippocampus within 24 h of sevoflurane exposure. However, sevoflurane exposure did not cause persistent changes in sen- sorimotor neurobehavioural performance. Moreover, sevoflur- ane did not affect learning and memory abilities of treated rats at juvenile ages. Given the serious implications for in particular, clinical public health, trials, are needed to determine effects of the exposure of sevoflurane in early life on learning and memory abilities.

further investigations,

Acknowledgement

We appreciate the technical help of Dr Jing Li at the Depart- ment of Anaesthesiology, New Jersey Medical School, NJ, USA.

Declaration of interest None declared.

Funding This work was supported by research grants obtained from the National Science Foundation Council of China (81070995; 31171290; 31140050), the Guangdong Science Foundation (2010B031600037), National Institutes of Health, and the Na- tional Institute on Alcohol Abuse and Alcoholism, USA (AA016964, AA016618).

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26 Li D, Wang J, Yew DT, Lucy Forster E, Yao Z. Age-related altera- tions of Nestin-immunoreactive neurons in rat basal forebrain with aged memory deficit. Neurochem Int 2008; 53: 270–7 27 Uebing A, Arvanitis P, Li W, et al. Effect of pregnancy on clinical status and ventricular function in women with heart disease. Int J Cardiol 2010; 139: 50– 9

28 Wang J, Yan L, Zhao X, Wu W, Zhou LH. The diversity of nNOS gene expression in avulsion-injured spinal motoneurons among laboratory rodents. Nitric Oxide 2010; 22: 37 –42

29 Ananth C, Dheen ST, Gopalakrishnakone P, Kaur C. Distribution of NADPH-diaphorase and expression of nNOS, N-methyl-D-aspar- tate receptor (NMDAR1) and non-NMDA glutamate receptor (GlutR2) genes in the neurons of the hippocampus after domoic rats. Hippocampus 2003; 13: acid-induced lesions in adult 260– 72

30 He FQ, Qiu BY, Zhang XH, et al. Tetrandrine attenuates spatial memory impairment and hippocampal neuroinflammation via inhi- biting NF-kappaB activation in a rat model of Alzheimer’s disease induced by amyloid-beta(1–42). Brain Res 2011; 1384: 89–96 31 Orliaguet G, Vivien B, Langeron O, Bouhemad B, Coriat P, Riou B. Minimum alveolar concentration of volatile anesthetics in rats during postnatal maturation. Anesthesiology 2001; 95: 734– 9

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35 Zhang Y, Li N, Yang Z. Perinatal food restriction impaired spatial learning and memory behavior and decreased the density of nitric oxide synthase neurons in the hippocampus of adult male rat offspring. Toxicol Lett 2010; 193: 167–72

36 Ma D, Williamson P, Januszewski A, et al. Xenon mitigates isoflurane-induced neuronal apoptosis in the developing rodent brain. Anesthesiology 2007; 106: 746– 53

37 Maze M, Fujinaga M. Recent advances in understanding the actions

and toxicity of nitrous oxide. Anaesthesia 2000; 55: 311–4

38 Mihic SJ, Ye Q, Wick MJ, et al. Sites of alcohol and volatile anaes- thetic action on GABA(A) and glycine receptors. Nature 1997; 389: 385–9

39 Mellon RD, Simone AF, Rappaport BA. Use of anesthetic agents in neonates and young children. Anesth Analg 2007; 104: 509– 20 40 Vaccarino FM, Ment LR. Injury and repair in developing brain. Arch

Dis Child Fetal Neonatal Ed 2004; 89: F190– 2

41 Liu XS, Xue QS, Zeng QW, et al. Sevoflurane impairs memory con- solidation in rats, possibly through inhibiting phosphorylation of glycogen synthase kinase-3beta in the hippocampus. Neurobiol Learn Mem 2010; 94: 461– 7

42 Wiklund A, Granon S, Faure P, Sundman E, Changeux JP, Eriksson LI. Object memory in young and aged mice after sevo- flurane anaesthesia. Neuroreport 2009; 20: 1419–23

43 Tanda K, Nishi A, Matsuo N, et al. Abnormal social behavior, hyperactivity, impaired remote spatial memory, and increased D1-mediated dopaminergic signaling in neuronal nitric oxide syn- thase knockout mice. Mol Brain 2009; 2: 19

233",rats,['Postnatal day 7 (P7) Sprague– Dawley rats were continuously exposed to 2.3% sevoflurane for 6 h.'],postnatal day 7,['Postnatal day 7 (P7) Sprague– Dawley rats were continuously exposed to 2.3% sevoflurane for 6 h.'],Y,['We used the Fox battery test and Morris water maze (MWM) to examine subsequent neurobehavioural performance.'],sevoflurane,['Postnatal day 7 (P7) Sprague– Dawley rats were continuously exposed to 2.3% sevoflurane for 6 h.'],none,[],sprague dawley,['Postnatal day 7 (P7) Sprague– Dawley rats were continuously exposed to 2.3% sevoflurane for 6 h.'],The study investigates the effects of sevoflurane on neuronal nitric oxide synthase levels and neurobehavioural performance in developing rats.,"['The use of general anaesthetics in young children and infants has raised concerns regarding the adverse effects of these drugs on brain development. Sevoflurane might have harmful effects on the developing brain; however, these effects have not been well investigated.']",None,[],The findings suggest early exposure to sevoflurane increases activated caspase-3 expression and neuronal loss but does not affect subsequent neurobehavioural performances in juvenile rats.,"['Although early exposure to sevoflurane increases activated caspase-3 expression and neuronal loss and decreases nNOS in the neonatal hippocampus, it does not affect subsequent neurobehavioural performances in juvenile rats.']",None,[],None,[],True,True,False,True,True,True,10.1093/bja/aes121
10.1038/s41598-021-85125-5,966.0,Hogarth,2021,rats,postnatal day 7,N,sevoflurane,none,sprague dawley,"OPEN

Singular and short‑term anesthesia exposure in the developing brain induces persistent neuronal changes consistent with chronic neurodegenerative disease

Kaley Hogarth1,2, Ramesh Babu Vanama1,2, Greg Stratmann3 & Jason T. Maynes1,2,4*

The potential adverse impact of inhalational anesthetics on the developing brain was highlighted by the addition of a medication warning by the U.S. Food and Drug Administration for their use in the pediatric population. To investigate mechanisms by which early life anesthesia exposure could induce long‑term neuronal dysfunction, we exposed rats to 1 minimum alveolar concentration sevoflurane at 7 days of life. The animals were raised normally until adulthood (P300) prior to sacrifice and analysis of cortical tissue structure (TEM), mitochondrial quality control and biogenesis pathways (Western blot, ELISA, ADP/ATP content), and markers of oxidative stress, proteotoxicity and inflammation (Western blot, ELISA). We found that early life anesthesia exposure led to adverse changes in mitochondrial quality maintenance pathways, autophagy and mitochondrial biogenesis. Although there was an escalation of oxidative stress markers and an increase in the nuclear localization of stress‑ related transcription factors, cellular redox compensatory responses were blunted, and oxidative phosphorylation was reduced. We found upregulation of mitochondrial stress and proteotoxicity markers, but a significant reduction of mitochondrial unfolded protein response end‑effectors, contributing to an increase in inflammation. Contrary to acute exposure, we did not find an increase in apoptosis. Our findings suggest that a limited, early exposure to anesthesia may produce lasting cellular dysfunction through the induction of a sustained energy deficient state, resulting in persistent neuroinflammation and altered proteostasis/toxicity, mimicking aspects of chronic neurodegenerative diseases.

In 2016, the Food and Drug Administration (FDA) added a medication warning label to inhalational anesthetics, indicating a potential risk for adverse long-term neurocognitive outcomes with repeated or extended exposures in young children and in pregnant mothers (with risk to the unborn fetus)1. This warning is scientifically based on a combination of retrospective clinical studies, generally highlighting the risks to young patients receiving more than one anesthetic, but these studies are unable to inform on specific mechanisms since the anesthetic regi- mens were not controlled2–5. In a recent prospective, multisite trial, the effect of a single general anesthesia (GA) exposure was compared to neuraxial anesthesia6. The results from this trial agree with the previously described retrospective studies, in that a single, short exposure to GA in an otherwise healthy young child likely does not have an adverse effect. While investigating the potential effect of duration (or number) of anesthetics, these clinical studies do not interrogate causative pharmaceuticals or physiologic processes, in pathways that could plausibly result in the long temporal association between drug exposure and the observed cognitive phenotype. To address this association, pre-clinical investigations have attempted to elucidate an underlying mechanism, controlling for variables which may also affect neurocognitive development and confound retrospective clini- cal studies. Two early studies in rat models illustrated that anesthetic agents acutely induced widespread neu- ronal apoptosis7,8, findings which were later extended to non-human primates9. Similarly, small animal models

1Division of Molecular Medicine, SickKids Research Institute, Toronto, Canada. 2Department of Anesthesia and Pain Medicine, Hospital for Sick Children, 555 University Ave., Toronto, ON M5G 1X8, Canada. 3Department of Anesthesia and Perioperative Medicine, University of California, San Francisco, San Francisco, USA. 4Department of Anesthesiology and Pain Medicine, University of Toronto, Toronto, Canada. *email: jason.maynes@sickkids.ca

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illustrated that early exposure to inhalational anesthetic agents could adversely affect cognition and learning10–13. The potential mechanisms explored include gross neuronal apoptosis14,15, dendritic pruning16, changes to neuro-/ synaptogenesis17, mitochondrial dysfunction and the generation of reactive oxygen species (ROS)18, alteration to brain derived neurotrophic factor/p75NTR signalling19, and AMPA receptor modulation20, among others. However, most of the observed cellular, tissue or organism changes are in very close proximity to the anesthetic administration, making the link to long-term neurocognitive function challenging to elucidate, and not allowing for the description of techniques or therapies that might mitigate the adverse effects.

To address this knowledge gap, we utilized our previously described animal model of neurocognitive develop- ment, where sevoflurane exposure in early life (P7) was shown to affect learning and induce long-term adverse neurocognitive changes11,13,21,22. Through analysis of cortical tissue structure (TEM), mitochondrial quality con- trol and biogenesis pathways (Western blot, ELISA, ADP/ATP content), and markers of oxidative stress, pro- teotoxicity and inflammation (Western blot, ELISA), we illustrate that a singular anesthetic exposure produces adverse changes that persist into adulthood. The observed changes include the induction of enduring neuroin- flammation and mitochondrial injury, mimicking aspects of chronic neurodegenerative diseases, providing a potentially addressable mechanism for the associated temporally distant neurological dysfunction.

Results Adult cortical neuron mitochondrial ultrastructure and size are altered after infant sevoflu‑ rane exposure. Mitochondria undergo the antagonistic dynamic processes of fission and fusion in order to appropriately respond to cellular metabolic needs and external stimuli, and for repair processes23. To examine if an early exposure to inhalational anesthetics could alter this structure–function relationship in adult neuronal mitochondria, we examined the organelle’s ultrastructure using electron microscopy (Fig. 1a,b). We found that mitochondria from adult animals with sevoflurane exposure had a significantly smaller average area (37% reduc- tion in size, p = 0.01) and an altered, swollen cristae architecture (Fig. 1a–c). The fragmented mitochondrial population contrasts with the fused and dilated mitochondria noted after acute inhalational anesthetic exposure, indicating a temporal evolution of mitochondrial morphology24.

To elucidate a mechanism by which sevoflurane exposure could alter mitochondrial morphology, we exam- ined the level of proteins involved in mitochondrial fusion and fission. The primary driver of mitochondrial fission is the GTPase Dynamin-related Protein-1 (DRP1). We found that total DRP1 levels were reduced in sevo- flurane treated brains, but that the ratio of activated (phospho-Ser616) to total DRP1 was profoundly increased (77% increase, p = 0.006), indicating a higher level of fission enzyme activity (Fig. 2a,b). DRP1 does not make physical contact with the mitochondrial membrane, rather it relies on adapter proteins to facilitate membrane cleavage. We found that inhalational anesthesia exposure significantly elevated the level of Mitochondrial Fission Factor (MFF) (60% increase, p = 0.04), the DRP1 adapter primarily responsible for membrane cleavage under cell stress (Fig. 2c). Since mitochondrial morphology is a balance between fusion and fission, we also examined proteins involved in the fusion process. We found that the two main fusion proteins, OPA1 (inner membrane) and MFN2 (outer membrane), have reduced levels after sevoflurane exposure (with a reduction in both the short (OPA-S) and long (OPA-L) forms of OPA-1) (Fig. 3). The reduction in OPA1-L is consistent with the observation of impaired cristae structure, as this protein is vital in maintenance of cristae folding and architecture25. These changes collectively represent a long-term shift in the mitochondrial morphological regulatory network, towards a fragmented mitochondrial phenotype in the sevoflurane exposed animals.

Early sevoflurane exposure adversely affects adult mitochondrial protein homeostasis, orga‑ nelle biogenesis, and oxidative phosphorylation. To determine if the changes we observed in orga- nelle morphology were accompanied by alterations to mitochondrial activity, we interrogated metrics commonly associated with the chronic mitochondrial dysfunction as typically observed in chronic neurological diseases and aging26. We found that mitochondria from animals exposed to inhalational anesthesia in infancy had a higher proportion of mitochondrial protein components encoded by nuclear genes (i.e. succinate dehydrogenase subunit A, SDHA) compared to those encoded by the mitochondrial genome (i.e. mitochondrially encoded cytochrome C oxidase subunit 1, mtCO1) (Fig. 2d), a sign of altered mitochondrial proteostasis and synthetic function26,27. This finding was supported by a reduction of mitochondrial genome component ND1 relative to nuclear GAPDH (a higher ΔCt value, indicating a lower mitochondrial DNA content) (Fig. 1d) and a reduction in mitogenic transcriptional regulators (SirT1, ERRα and PGC1-α) in the sevoflurane exposed group (Fig. 4). As obligate aerobes, neurons are particularly dependent on mitochondrial derived ATP as they are unable to sup- plement their energetic demand with glycolysis28. In the sevoflurane exposed rats, we found an elevation of the ADP/ATP ratio, indicating a reduction in the available cellular energy (Fig. 1e, Supplemental Figure 1).

Mitochondria are both a primary generator of ROS, and a main site of ROS-induced damage. In cortical tissue from animals with early sevoflurane exposure, we found a higher level of 4-HNE lipid adducts (1.9-fold increase, p = 0.04), a sign of chronic ROS damage (Fig. 5a), similar to those previously noted with acute anesthesia exposure10,29. As a corollary, we found elevated nuclear localization of transcription factors known to combat oxidative stress, including HIF, NFR2 and FOXO3 (Fig. 6a). Interestingly, these changes were not accompanied by an increase in the total level of SOD2, a mitochondrial anti-oxidant protein (Fig. 5b,c). Overall, our findings are consistent with disease states that induce chronic neuronal dysfunction, namely adverse changes to mito- chondrial protein synthesis, energy production, mitochondrial biogenesis, and an elevation in long-term ROS generation and oxidative damage.

Exposure to sevoflurane impairs the mitochondrial unfolded protein response (mtUPR). The maintenance of proteostasis and the handling of misfolded proteins through the mitochondrial unfolded pro-

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Figure 1. Infant (P7) sevoflurane exposure alters mitochondrial ultrastructure and energetic status in adult rat cortical tissue. Transmission electron microscopy (TEM) imaging illustrates significant changes to mitochondrial ultrastructure including swollen cristae (*), dilated rough endoplasmic reticulum (arrows) and a decreased average mitochondrial size (increase in fragmented phenotype) with infant sevoflurane exposure. (a) representative P7 sevoflurane-exposed adult rat cortical brain TEM image, (b) littermate control adult rat cortical brain TEM image. In cortical tissue, sevoflurane treatment resulted in a reduction in (c) average mitochondrial size (area, from TEM) and (d) relative cortical mitochondrial DNA content (ΔCt comparing mitochondrial ND1 to nuclear GAPDH). (e) Sevoflurane reduces energy availability, illustrated through an increased ADP/ATP. TEM magnification ×20,000, scale bars represent 500 nm. Values represent means ± SEM, with 6 animals in each group. Statistical significance between groups indicated with (*) for p-value < 0.05, and (**) for p-value < 0.01.

tein response (mtUPR) is an important determinant of mitochondrial health and sustained neuronal function. In sevoflurane-exposed animals, we found that 14 of 16 tested mtUPR-associated transcription factors had an enriched nuclear concentration, compared to unexposed animals (Fig. 6a). The increase in mtUPR- and mito- chondria stress-associated transcription factors was accompanied by a decrease in measured end-effectors of the mtUPR, including HSP60 (61% reduction, p = 0.03) and ClpP (40% reduction, p = 0.04) (Fig. 6b,c). Interestingly, the decrease in HSP60 was in the context of a 3.2-fold increase in the nuclear concentration of the transcription factor responsible for its expression (HSF), illustrating significant problems with the governance of this quality control and stress response pathway. Dysregulation of the mtUPR is consistent with our observed mitonuclear protein imbalance described above26.

Sevoflurane exposure increases neuroinflammatory mediators. Neuroinflammation is a feature common in many disease states with chronic neuronal dysfunction30. To determine the potential role of neuronal inflammation in our model, we quantified the amount of inflammatory cytokines in sevoflurane exposed and

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Figure 2. Sevoflurane exposure at P7 promotes mitochondrial fission and induces mitonuclear protein imbalance in the cortical tissue of adult rats. Sevoflurane exposure reduced the total level of the pro-fission GTPase DRP1 (a), but dramatically increased the level of the activated, phosphorylated form (pSer616-DRP1) (b). Similarly, the pro-fission DRP1 adapter MFF was elevated with sevoflurane exposure (c). Sevoflurane altered mitochondrial homeostasis, with an increase in nuclear-encoded protein components of the electron transport chain (Succinate Dehydrogenase Complex A-SDHA) relative to the mitochondrial-encoded components (Mitochondrial Cytochrome C Oxidase 1-mtCO1), adversely altering the mitonuclear protein balance (lower proportion of mitochondrial-encoded proteins)27. (d). Values represent means ± SEM, with 6 animals in each group. Statistical significance between groups indicated with (*) for p-value < 0.05, and (**) for p-value < 0.01.

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Figure 3. Protein mediators of mitochondrial fusion are reduced following P7 sevoflurane exposure. Pro-fusion proteins essential for inner- (OPA1 (a–c)), and outer- (MFN-2 (d)) mitochondrial membrane association are reduced in sevoflurane exposed tissue. While both the long and short forms of OPA1 were reduced, the ratio of long:short was increased, indicating an alteration to fusion activation. Values represent means ± SEM, with 6 animals in each group. Statistical significance between groups indicated with (*) for p-value < 0.05. All samples derived from same experiment and gels/blots processed in parallel.

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Figure 4. Adult cortical tissue demonstrates a lower level of mitochondrial biogenic regulators following infant sevoflurane exposure. Western blot analysis indicates sevoflurane exposed samples had a decrease in transcription factors responsible for mitogenic signaling/mitochondrial biogenesis (ERRα (a), PGC1-α (b)) and transcriptional regulation (SirT1 (c)), consistent with the reduction of cellular mitochondrial content and quality control. Values represent means ± SEM, with 6 animals in each group. Statistical significance between groups indicated with (*) for p-value < 0.05. All samples derived from same experiment and gels/blots processed in parallel.

Figure 5. Adult rat cortex demonstrates long-term oxidative damage following P7 sevoflurane exposure. Lipid peroxidation adducts (4-HNE) were increased in sevoflurane exposed rat brains (a), indicating persistent oxidative stress. However, SOD2 has not increased, indicating the lack of a compensatory mitochondrial anti- oxidant response (b,c). Values represent means ± SEM, with 6 animals in each group. Statistical significance between groups indicated with (*) for p-value < 0.05. All samples derived from same experiment and gels/blots processed in parallel.

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Figure 6. Sevoflurane exposure induces persistent alterations to the mitochondrial unfolded protein response (mtUPR). Quantification of nuclear-localized transcription factors reveals an increase in those responsible for mitochondrial stress response and mtUPR pathways after infant sevoflurane exposure (dashed bars), relative to non-exposed animals (black bars) (a). The observed changes in transcription factors were reflected in the levels of enzymes involved in the mitochondrial protein quality control (ClpP) (b) and folding (HSP-60) (c). Values represent means ± SEM, with 6 animals in each group. Statistical significance between groups indicated with (*) for p-value < 0.05, and (**) for p-value < 0.01.

control cortical tissue using an ELISA. We observed significant increases in four pro-inflammatory cytokines: Interleukin 6 (IL-6), Tumor Necrosis Factor alpha (TNFα), CCL5, and Macrophage Inflammatory Protein (MIP/ CCL3) in the sevoflurane-exposed rats (Fig. 7a). As validation for the ELISA, we also performed Western blot analysis with TNFα, providing confirmatory results (Fig. 7b,c).

Sevoflurane exposure reduces autophagosomal clearance and apoptotic signalling. Previous reports investigating acute or subacute exposures to sevoflurane have found significant and widespread neu- ronal apoptosis7. We investigated whether apoptotic cell death, and associated organelle quality control pathways (autophagy), were altered when the point of analysis was distal to the anesthesia exposure. Contrary to previous findings, we did not observe significant differences in cleaved (activated) caspase-3 between control and exposed brains (Supplemental Figure 2B). We did find a significant decrease in active LC3 in sevoflurane-exposed ani-

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Figure 7. Early developmental exposure to sevoflurane increases the abundance of inflammatory cytokines in cortical rat tissue. Sevoflurane exposure increased the level of neuroinflammatory markers by ELISA (a) and Western blot (b,c), compared to controls. Values represent means ± SEM, with 6 animals in each group. Statistical significance between groups indicated with (*) for p-value < 0.05, and (**) for p-value < 0.01.

mals (17% decrease, p = 0.04) (Supplemental Figure 2A), indicating a reduction in autophagic activation and flux. In line with these findings, we observed an increase in the secreted neurotrophic factor cleaved (mature) brain-derived neurotropic factor (mBDNF), with no overall change in the uncleaved protein (proBDNF), and no observable difference in the levels of MEF2-A or MEF2-C, as transcriptional regulators of neurodevelopment and neuronal survival (Supplemental Figure 3). These results indicate that, despite the observed adverse changes in mitochondrial and inflammatory markers with anesthetic exposure, there is no evidence of continued cell death and an actual decrease in the turnover of damaged organelles. These findings do not rule out an increase in apoptosis at the exact time of drug exposure.

Discussion General anesthetics are widely administered to facilitate the safe completion of surgeries in the pediatric popula- tion, but concerns about their effects on the developing brain have led to a recent FDA warning. How anesthetic drugs may affect neuronal activity at a time distant to the drug exposure is not known. To address this question, we have utilized our rodent model of neurodevelopment and illustrated that sevoflurane exposure in infancy leads to adverse neurophysiological changes in the adult animal, including persistent changes to mitochondrial structure and function, protein quality control, increased oxidative stress, and neuroinflammation (Fig. 8). The observed alterations to cellular physiology reveal notable similarities with chronic neurodegenerative diseases, including a blunting of compensatory stress pathways, impairment of oxidative metabolism, impairments to mitochondrial biogenesis, and increased proteostasis/proteotoxicity.

Gas anesthetics are known to have an acute adverse effect on mitochondrial function, through multiple mechanisms, including the direct inhibition of mitochondrial respiratory chain activity31. We have described an additional chronic toxicity after an acute exposure, leading to a persistent stress state. Despite sevoflurane resulting in a lower cellular mitochondrial content, we found impaired mitogenic signalling with reductions in PGC1α, SirT1 and ERRα levels, considered essential compensatory mechanisms to rectify reduced mitochondrial productivity and to promote the genesis of new, healthy mitochondria32. At the protein level, we saw a shift in the

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Figure 8. Early-life sevoflurane exposure can induce perpetuating neuronal dysfunction in the adult brain. Our findings indicate that anesthesia exposure in the developing brain adversely alters cellular energetic function through changes to mitochondrial morphology and function, resulting in a lasting adverse shift in mitochondrial quality control and regeneration/biogenesis pathways. This mechanism allows poorly functional organelles to persist, inducing proteotoxicity, oxidative stress, and neuroinflammation, creating a cell non- autonomous propagation of the phenotype, potentially through Cdk1.Image created using BioRender.com.

mitochondrial fusion/fission balance, favoring smaller mitochondria, which are known to be less energetically efficient, as demonstrated by the increase in ADP/ATP ratio33. These smaller mitochondria would normally be cleared or repaired, but the observed decreased rate of autophagy would allow dysfunctional mitochondria to persist34. Mitochondrial architecture is intimately related to function, and our structural aberrations are logically consistent with the functional impairments described here and elsewhere35.

Despite significant increases in the transcription factors that regulate oxidative stress responses, protein misfolding pathways, and mitochondrial stress responses, we observed a lack of functional (proteomic) compen- sation in these areas. A lack of anti-oxidant response proteins in the face of oxidative stress has previously been observed following acute GA exposure, which led to the suggestion that SOD2 may represent a specific anesthesia target10,29. Alteration of the mtUPR is consistent with our previously published findings that illustrated how gas anesthetics alter protein folding and induce endoplasmic reticulum stress36. Many neurological disorders exhibit intracellular depositions of misfolded protein aggregates, and increases in mtUPR transcriptional regulators is one of the earliest phenotypic changes observed in chronic neurodegenerative diseases37. In Parkinson’s disease models, chronic activation of the mtUPR results in a time-dependent increase in mitochondrial dysfunction38. Similarly, we find that the nuclear transcriptional response is congruent with a stressed environment (retrograde response), but the resultant mitochondrial proteome (anterograde response) is inadequately protective39.

We did not observe any significant differences in cortical neuronal density or active apoptosis between con- trol and sevoflurane-exposed animals. Our findings are in contrast to the widespread and cell type non-specific apoptosis observed in in vitro and in vivo models of (sub)acute anesthesia exposure (reviewed in31). It may be that apoptosis occurred in our model immediately after drug exposure, but without persistence into the adult brain where we still observe changes to cellular function, reducing the likelihood that apoptosis contributes signifi- cantly to the adult phenotype. It is possible that cell death during acute exposure could reduce brain development and neuronal connectivity, although organ plasticity and repair could compensate for such effects. In line with this idea, previous reports have indicated that when cognitive phenotypes are directly correlated to measured neuronal apoptosis, the phenotype can recover within days40. In isolated mouse neurons exposed to isoflurane, apoptosis was induced through an increase in proBDNF (p75NTR activation), at the expense of neurotrophic mature BDNF levels (TrkB activation)41. Conversely, we found an increase in mature BDNF with sevoflurane exposure, possibly explaining the lack of apoptosis. While a pro-survival environment might be adaptive, per- petuating the existence of dysfunctional cells could be disadvantageous for developing synapse architecture.

Our observed molecular alterations parallel those found in common neurodegenerative disorders. This similarity is intriguing, as a potential relationship exists between general anesthetic exposure and the age of onset of neurodegenerative disease, though this association remains an area of active investigation42,43. Neuro- inflammation is a hallmark of chronic neurodegenerative disease, with common elevations of IL-6 and TNFα, two cytokines increased in our samples. We observed significant elevation of the chemokine CCL5, previously illustrated to be elevated in Alzheimer’s and Parkinson’s, with a linear correlation to clinical severity in the latter disease44. The cytokine elevations observed in sevoflurane exposed animals are consistent with the increase in the nuclear localization of the NFR2 and XBP transcription factors (Fig. 6a), involved in inflammation-mediated stress mitigation responses45. The mitochondrial fragmentation exhibited in neurodegenerative disorders is usually associated with an increase in DRP1 activity, suggesting a common pathway related to mitochondrial morphology. The activating phosphorylation on DRP1 that we detected, pSer616, is a Cdk1-dependent site, a kinase known to be involved in the onset and evolution of neurodegenerative diseases28. Inflammatory cytokines

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can activate Cdk1, but their production is also regulated by the kinase, creating a cell non-autonomous method to expand dysfunctional signalling46. We postulate that anesthesia-induced metabolic and mitochondrial dys- function could result in persistent upregulation of stress pathways, producing a vicious cycle of self-propagating neuroinflammation and further metabolic deficiency (Fig. 8). This includes persistent upregulation of DRP1 phosphorylation, production of inflammatory cytokines, and a deficiency in oxidative phosphorylation with an increase in ROS. Using these pathways, an early exposure to a GA could promote continued neuronal dysfunc- tion and result in a persistent cognitive phenotype, similar to neurodegenerative diseases where chronic levels of misfolded proteins result in energetic failure and cellular oxidative stress. Gas general anesthetics may induce a similar proteotoxic stress with a self-perpetuating cycle of damage, inflammation and neuronal dysfunction. Further work is needed to determine if this pathway is unique, or only more noticed, in the developing brain.

The obligate dependence of neurons on mitochondria to meet their high energy demand makes neuronal activity particularly suspectable to mitochondrial dysfunction. While the results presented here are discussed in this context, it is important to note that our methodology precludes the ability to attribute all findings specifically to effects on neurons. Adverse impact on other cell types, most notably glial cells which have a high abundance and functional role in the cortex, likely also contributes to the phenotype.

In conclusion, our results indicate that exposure to sevoflurane during a developmentally sensitive period results in persistent cortical impairment through the induction of molecular changes consistent with an energy deficient state, neuroinflammation and altered proteostasis, without proper compensatory responses. Our study supports the idea that therapeutic agents aimed to reduce energetic dysfunction at the time of surgery may rep- resent viable options to mitigate developmental toxicity, and that markers of neuroinflammation may be useful to acutely determine anesthetic effects.

Materials and methods Experimental animals. Experiments were performed using male Sprague Dawley rats (total N = 12), ran- domly assigned to experimental (sevoflurane exposed) and control groups. Experimental and control animals were equally co-populated and maintained in identical environments, including food and water, temperature and light/dark cycles, to the best of our ability. There was no animal mortality during the entire time of observa- tion. All experiments were approved by the Institutional Animal Care and Use Committee of the University of California (San Francisco, California) and performed in accordance with national and institutional guidelines for animal care. All animal experiments were carried out according to the ARRIVE 2.0 guidelines, as described in the relevant section.

Sevoflurane exposure. Anesthetic exposure was performed according to our previously described protocol13. Briefly, on postnatal day 7 (P7), rat pups were randomly assigned to either exposed to 1 minimum alveolar concentration of sevoflurane (corresponding to ~ 5% atm), or identical environment without anesthetic agent as sham controls (6 animals per group) for a total of 4 h (FiO2 = 0.5), as we performed previously11,13,21,22,47. P7 was used as the exposure time point as it represents the period of peak synaptogenesis and the most vulner- able period for GA mediated neurotoxicity in rodent models48.

Animal euthanization and tissue collection. Ten months following anesthetic exposure (P300), all animals were euthanized following deep anesthetization with isoflurane (greater than 5% with loss of pedal pain reflex) by subsequent transcardial perfusions of 0.9% saline and 4% paraformaldehyde in 0.1 M phosphate buff- ered saline (pH 7.4). Cortical brain regions were immediately placed in preservation solution (0.21 M mannitol, 0.07 M sucrose and 20% DMSO) and flash frozen with ethanol and dry ice. Samples were stored at − 80 °C and shipped to the Hospital for Sick Children for downstream analysis. Following tissue collection, all experiments were performed blinded until grouping for analysis.

Transmission electron microscopy (TEM). All TEM for mitochondrial ultrastructural analysis was per- formed by the Pathology Lab facility at the Hospital for Sick Children, Toronto, Canada. Images were captured on a JEOL JEM1011 (JEOL, Inc., Peabody, MA) microscope. TEM images (20,000× magnification) were ana- lyzed using unbiased automated object identification in ImageJ49. For mitochondrial size analysis, mitochondrial area was blindly quantified in a minimum of ten random microscope fields, per sample.

Quantitative polymerase chain reaction (qPCR). To analyze variations in mitochondrial content, we performed qPCR on isolated genomic DNA, as previously described50. Briefly, genomic DNA was isolated from 10 mg of cortical mouse tissue using Tissue Genomic DNA Mini Kit (Geneaid, New Taipei City, Taiwan), as per manufacturer’s instructions. Each 20 μL qPCR reaction contained 10 μL SsoFast EvaGreen PCR Supermix (Bio-Rad), 0.8 μL forward and reverse mitochondrial primers (10 μM) (amplifying the ND1 region of the mito- chondrial genome) (Supplemental Table 2), 3 μL genomic DNA (10 ng/μL), and sterile water to the final volume. Reactions were performed using a CFX96 Real Time PCR instrument (Bio-Rad), using CFX Manager Software to determine Cq values. Mitochondrial DNA content (ND1) was normalized to GAPDH using a ΔCq method, providing a relative cellular mitochondrial genome content50.

ADP/ATP ratio assay. As an indicator of cellular energy status, ADP/ATP ratio was performed using the Abcam Bioluminescent ADP/ATP Ratio Assay Kit and performed according to manufacturer’s instructions (ab65313, Cambridge, MA, USA). In brief, 100 µL of ATP Reaction Mix (1× ATP Monitoring Enzyme in Nucle- otide Releasing Buffer) was loaded on a 96-well plate, followed by a baseline plate reading. To measure ATP

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levels, 100 µg of cortical lysate was then added to each well, followed by 2 min incubation and plate read. Finally, to determine ADP levels, 100 µL ADP (1× ADP Converting Enzyme in Nucleotide Releasing Buffer) was then added to each well and incubated for 2 min, followed by a final plate reading. ADP and ATP content quantified using a standard curve. Bioluminescence measured on Varioskan LUX Plate Reader (Thermo Scientific, MA, USA).

Western blots. All Western blots were performed on cortical lysate, which was prepared by placing 20 mg of tissue into a 1.5 mL microcentrifuge tube containing a 5 mm stainless steel bead (Qiagen), 300 μL cold 1× RIPA buffer and 1× cOmplete Protease Inhibitor Cocktail (Sigma-Aldrich). Samples were loaded into the Tis- sueLyser II (Qiagen), and subjected to 2 cycles of agitation of 2 min at 20 Hz. Following homogenization, an additional 200 μL of RIPA was added, and samples were agitated for 2 h at 4 °C, followed by 20 min centrifuga- tion at 15,000×g. Supernatants were collected, and protein content quantified using Pierce BCA Protein Assay Kit (Thermo Scientific). Cellular lysates were separated on a 4–12% (w/v) gradient SDS-PAGE gels (Genscript, Piscataway, NJ), before transfer to 0.2 μm PVDF membranes with a Trans-Blot Turbo Transfer System (0.8A and 25V) (Bio-Rad, Hercules, CA). Membranes were blocked (TBS-T + 5% skim milk) for 1 h, followed by overnight incubation with a primary antibody (TBS-T + 1% skim milk) at 4 °C (see Supplemental Table 1). Membranes were washed in TBS-T for 15 min prior to incubating in species specific horseradish peroxidase (HRP)-linked secondary antibody (TBS-T + 1% skim milk) for 1 h. Protein levels were determined by imaging on a Gel-Doc XRSystem (Bio-Rad) following detection with the Enhanced Chemiluminescence System (GE Life Sciences, Mississauga, Canada). The optical densities obtained were analysed with ImageJ, with GAPDH used as a loading control49,51,52. All samples derived from same experiment and gels/blots were processed in parallel.

4‑Hydroxynonenal (HNE) adduct ELISA. To examine oxidative damage in the cortical tissue, we quan- tified the abundance of 4-HNE protein adducts using the OxiSelect HNE Adduct Competitive ELISA Kit, as per manufacturer’s specifications (Product STA-838-T, Cell BioLabs, San Diego, CA, USA). Briefly, ~ 500 μg of total protein from lysed cell extracts in RIPA buffer were plated on an HNE-conjugated ELISA plate, followed by the addition of an anti-HNE polyclonal antibody and HRP-conjugated secondary antibody. The HNE adduct content was quantified spectrophotometrically at 450 nm, with comparison to an HNE-BSA standard curve, and normalized to total protein loading.

Mitochondrial unfolded protein response (mtUPR) profile. To quantify mtUPR activation, we determined the nuclear localization of mtUPR-related transcription factors (TF) using the Mitochondrial UPR TF Activation Profiling Plate, as per manufacturer’s instructions (including AP1, ATF4, C/EBP, CHOP, E2F1, FOXO3, HIF, HSF, MEF2, Nfκβ, NFR1, NFR2/ARE, p53, SATB, TFEB and XBP) (Product FA-1006, Signosis, Santa Clara, CA, USA). Briefly, nuclear extracts were isolated from 50 mg of brain tissue using the Nuclear Extraction Kit (Signosis). Following isolation, 15 μg of nuclear extracts were mixed with biotin-labeled DNA probes (containing TF consensus sequences) resulting in TF:DNA probe complexes. Following spin-column purification, DNA probes were removed, and the resulting TF extracts were added to a pre-coated plate with TF specific complementary sequences. TF content was detected with streptavidin-HRP conjugate.

Inflammatory cytokine ELISA. Inflammatory cytokines were quantified using the Rat Inflammation ELISA Strip Assay, as per manufacturer’s instructions (Product EA-1201, Signosis, Santa Clara, CA). Briefly, cortical tissue was lysed in Lysis Buffer and 10 µg total protein loaded into wells coated with primary antibodies against inflammatory cytokines (TNFα, IL-6, IL-1α, IL-1β, IFNγ, MCP-1, CCL3 and CCL5). Following incu- bation with enzyme-linked antibodies, HRP substrate was added to produce a colorimetric response, which was quantified spectrophotometrically at 450 nm. Cytokine quantification was derived using a standard curve (Product EA-1202, Signosis).

Statistical analysis. Unless otherwise noted, all results are presented as means ± SEM. Following a Kol- mogorov–Smirnov test for normalcy, statistical significance was determined using either un-paired Student’s t-test with Welsh correction or un-paired Mann–Whitney U-test, as appropriate. Where more than one com- parison was performed, significance was determined using an ANOVA, using the Dunnett method for multiple comparison correction. Statistical significance was calculated using GraphPad Prism 6.0 using a p-value of < 0.05 as a cut-off for significance.

Received: 27 June 2020; Accepted: 25 February 2021

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Acknowledgements The authors would like to thank the members of the Department of Anesthesia and Pain Medicine at the Hos- pital for Sick Children for the protection of research time for the study investigators, and Sandra Singhroy for technical assistance. JTM would also like to thank the Wasser Family and SickKids Foundation as the holder of the Wasser Chair in Anesthesia and Pain Medicine.

Author contributions K.H. performed a majority of data curation, formal analysis, methodology and writing—original draft. R.B.V. provided a minority of data curation and methodology. G.S. provided a minority of project conceptualization, supervision, validation and writing—review and editing. J.T.M. provided a majority of project conceptualization, funding acquisition, project administration, supervision, validation and writing—review and editing.

Competing interests The authors declare no competing interests.

Additional information Supplementary Information The online version contains supplementary material available at https ://doi. org/10.1038/s4159 8-021-85125 -5.

Correspondence and requests for materials should be addressed to J.T.M.

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Scientific Reports | (2021) 11:5673 |

https://doi.org/10.1038/s41598-021-85125-5

13",rats,['we exposed rats to 1 minimum alveolar concentration sevoflurane at 7 days of life.'],postnatal day 7,['we exposed rats to 1 minimum alveolar concentration sevoflurane at 7 days of life.'],N,[],sevoflurane,['we exposed rats to 1 minimum alveolar concentration sevoflurane at 7 days of life.'],none,[],sprague dawley,"['Experiments were performed using male Sprague Dawley rats (total N = 12), randomly assigned to experimental (sevoflurane exposed) and control groups.']",This study addresses the long-term neurocognitive effects of early life anesthesia exposure by investigating mitochondrial dysfunction and neuroinflammation as underlying mechanisms.,"['To address this association, pre-clinical investigations have attempted to elucidate an underlying mechanism, controlling for variables which may also affect neurocognitive development and confound retrospective clinical studies.']",The study utilizes a rodent model to investigate the long-term effects of early life anesthesia exposure on mitochondrial function and neuroinflammation.,"['To address this knowledge gap, we utilized our previously described animal model of neurocognitive development, where sevoflurane exposure in early life (P7) was shown to affect learning and induce long-term adverse neurocognitive changes.']","The article argues that early exposure to anesthesia may produce lasting cellular dysfunction, mimicking aspects of chronic neurodegenerative diseases, which could have significant implications for pediatric anesthesia practices.","['Our findings suggest that a limited, early exposure to anesthesia may produce lasting cellular dysfunction through the induction of a sustained energy deficient state, resulting in persistent neuroinflammation and altered proteostasis/toxicity, mimicking aspects of chronic neurodegenerative diseases.']","The study's limitations include its reliance on animal models, which may not fully replicate human neurodevelopmental processes.","['The obligate dependence of neurons on mitochondria to meet their high energy demand makes neuronal activity particularly suspectable to mitochondrial dysfunction. While the results presented here are discussed in this context, it is important to note that our methodology precludes the ability to attribute all findings specifically to effects on neurons.']",Potential applications include the development of therapeutic agents to mitigate developmental toxicity of anesthesia and the use of neuroinflammation markers to determine anesthetic effects.,"['In conclusion, our results indicate that exposure to sevoflurane during a developmentally sensitive period results in persistent cortical impairment through the induction of molecular changes consistent with an energy deficient state, neuroinflammation and altered proteostasis, without proper compensatory responses. Our study supports the idea that therapeutic agents aimed to reduce energetic dysfunction at the time of surgery may represent viable options to mitigate developmental toxicity, and that markers of neuroinflammation may be useful to acutely determine anesthetic effects.']",True,True,True,True,True,True,10.1038/s41598-021-85125-5
10.1159/000369698,570.0,Huang,2015,rats,postnatal day 7,N,ketamine,none,sprague dawley,"315

Accepted: November 17, 2014

1421-9778/15/0351-0315$39.50/0

This is an Open Access article licensed under the terms of the Creative Commons Attribution- NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.

Original Paper

Ketamine Interferes with the Proliferation and Differentiation of Neural Stem Cells in the Subventricular Zone of Neonatal Rats

He Huanga Lu Liua Bing Lic Pan-Pan Zhaoa Chun-Mei Xua Yang-Zi Zhua Cheng-Hua Zhoub Yu-Qing Wua

aJiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, bJiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical College, Xuzhou, cDepartment of Anesthesiology, Jiangning Hospital of Chinese Medicine, Nanjing, P R. China

Keywords Ketamine • Neural stem cell • Proliferation • Differentiation • Subventricular zone

Abstract Background: Previous studies have shown ketamine can alter the proliferation and differentiation of neural stem cells (NSCs) in vitro. However, these effects have not been entirely clarified in vivo in the subventricular zone (SVZ) of neonatal rats. The present study was designed to investigate the effects of ketamine on the proliferation and differentiation of NSCs in the SVZ of neonatal rats in vivo. Methods: Postnatal day 7 (PND-7) male Sprague- Dawley rats were administered four injections of 40 mg/kg ketamine at 1-h intervals, and then 5-bromodeoxyuridine (BrdU) was injected intraperitoneally at PND-7, 9 and 13. NSC proliferation was assessed with Nestin/BrdU double-labeling immunostaining. Neuronal and astrocytic differentiation was evaluated with β-tubulin III/BrdU and GFAP/BrdU double-labeling immunostaining, respectively. The expressions of nestin, β-tubulin III and GFAP were measured using Western blot analysis. The apoptosis of NSCs and astrocytes in the SVZ of neonatal rats was evaluated using nestin/caspase-3 and GFAP/caspase-3 double-labeling immunostaining. Results: Neonatal ketamine exposure significantly reduced the number of nestin/BrdU and GFAP/BrdU double-positive cells in the SVZ. Meanwhile, the expressions of nestin and GFAP in the SVZ from the ketamine group were significantly decreased compared those in the control group. Still, no double-positive cells for nestin/caspase-3 and GFAP/caspase-3 were found after ketamine exposure. In addition, the neuronal differentiation of NSCs in the SVZ was markedly promoted by ketamine with an increased number of β-tubulin III/BrdU double-positive cells and enhanced expression of β-tubulin III. These effects of ketamine on the NSCs in the SVZ often lasted at least 1 week after ketamine anesthesia. Conclusion: In the present study, it was demonstrated that ketamine could alter neurogenesis by inhibiting the proliferation of NSCs, suppressing their differentiation into astrocytes and promoting the neuronal differentiation of the NSCs in the SVZ of neonatal rats during a critical period of their neurodevelopment.

Copyright © 2015 S. Karger AG, Basel

Dr. Yu-Qing Wu and Dr. Cheng-Hua Zhou

Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College Tongshan Road 209, Xuzhou 221004 (PR China) and Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical College Tongshan Road 209, Xuzhou 221004 (PR China) E-Mail xymzyqwu@126.com, E-Mail chzhou77@163.com

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Introduction

Ketamine, a N-methyl-D-aspartate receptor (NMDA-R) ion channel blocker, is widely used in anesthesia, analgesia and sedation during the neonatal period [1, 2]. However, ketamine has been found to cause neuronal apoptosis and neurological function deficits. The conclusions of animal and clinical research have generated worry regarding the safety of using ketamine in the pediatric population [3-9].

The developing brain experiences a critical period called the brain growth spurt (BGS), which begins at the end of pregnancy in rodents and extends to the first 2-3 weeks after birth [10]. In humans, the corresponding period starts at the last trimester of pregnancy and continues to 2 years after birth. Substantial neurogenesis occurs in this period, which is coordinated by NSC proliferation, differentiation, migration, survival and synaptogenesis. This critical phase is essential to normal brain structure and functional development [11]. Neurogenesis occurs in several regions during brain development, especially in the SVZ, a restricted neurogenesis region that exists throughout life in the brain beside the hippocampal dentate gyrus (DG). Neurogenesis in the SVZ and DG can be stimulated by many factors, including stroke [12]. One or more of the events that are required for neurogenesis, such as NSC proliferation and neuronal differentiation, play an important role in compensating for lost neurons.

Although the neonatal neuroapoptosis induced by ketamine has been demonstrated by increasing number of studies, the effects of ketamine on neurogenesis have not been completely clarified. A recent in vitro study showed that ketamine altered the proliferation and differentiation of rat cortical neural stem progenitor cells [13]. However, the effects of ketamine on neonatal neurogenesis in vivo remain to be investigated. Given the importance of SVZ neurogenesis to the structural and functional development of the brain, the effects of ketamine on the proliferation and differentiation of the NSCs in the SVZ of neonatal rats were investigated in this study.

Materials and Methods

Animals treatment

All the animal experiments were approved by the Institutional Animal Care and Use Committee of XuZhou Medical College. Timed-pregnant Sprague-Dawley rats were housed at 24°C on a 12-hr:12-hr light:dark cycle with free access to food and water. The PND-7 male rats (11-14g) selected from all the pups were used in the experiments. These rats were randomly assigned to control groups and ketamine groups. Ketamine was diluted in 0.9 % normal saline. PND-7 rats in treated group were administered intraperitoneally by four injections of 40 mg/kg ketamine with 1h intervals. Animals in control group received equal volume of saline at the same time points. Custommade temperature probes were used to facilitate control of temperature at 36.5 ± 1°C using computer-controlled heater/cooler plates integrated into the floor of chamber. Between each injection animals were returned to their chamber to help maintain body temperature and reduce stress.

BrdU injections

After anesthesia, neonatal rats received a single intraperitoneal injection of BrdU (5-bromo-2- deoxyuridine; Sigma, 100 mg/kg) in 0.9% NaCl solution at PND-7, 9 and 13. The animals were fixed by perfusion at 3 h after BrdU injection to observe the proliferation of matured astrocytes or at 24 h after BrdU injection to observe the proliferation and differentiation of the NSCs. The detailed experimental protocol is listed in Table 1.

Tissue preparation and double-immunofluorescence

The animals were anesthetized and then transcardially perfused after BrdU injection. The coronal sections of the brain were cut on a microtome at a thickness of 30 μm. When the SVZ was initially exposed, the five consecutive coronal sections were cut and discarded, and then the next three consecutive coronal sections were cut and selected for the double-labeled immunofluorescence of nestin/BrdU, β-tubulin III/

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Table 1.

Experimental Protocol (n=5). The interval to perfusion refers to the time from the

BrdU injection to transcardiac perfusion. IF = immunofluorescence

Table 2.

Primary Antibodies. List of primary antibodies, their suppliers,

the animal used to raise the antibodies in, and the working dilution

BrdU and GFAP/BrdU, respectively. This procedure was repeated five times. The sections were incubated with 50% formamide in PBS for 2 h at 65°C and then in 2 normal hydrochloric acid incubation for 30 min at 45°C, followed by 3 washes with PBS for 10 min. The blocking of nonspecific epitopes with 10% donkey serum in PBS with 0.3% Triton-X for 2 h at RT preceded overnight incubation at 4°C, with the appropriate primary antibody listed in Table 2 in PBS with 0.3% Triton-X. After 3 washes with PBS, the sections were incubated with suitable secondary fluorescent antibodies (Alexa488-labeled donkey anti-rabbit and Alexa594-labeled donkey anti-mouse; 1:200; Invitrogen) for 2 h at room temperature. The sections were observed by a skilled pathologist blinded to this research using image stacks on a laser scanning confocal microscope (Fluoview 1000, Olympus). Evaluation of cell apoptosis

To evaluate the effect of ketamine on apoptosis in the NSCs and astrocytes, a double-immunofluorescence detection of nestin/caspase-3 and GFAP/caspase-3 was performed. The animals were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde at 12 h after the end of ketamine anesthesia. Then, the brain was removed, postfixed over-night in 4% paraformaldehyde and placed in 30% sucrose until it sunk. Coronal sections of the brain were cut on a microtome. When the SVZ was initially exposed, the coronal sections of the brain were cut consecutively at a thickness of 30 μm. The tenth section was picked up and stored in PBS for double-label immunofluorescence. The sections were blocked with 10% donkey serum in PBS with 0.3% Triton-X for 2 h at RT and then incubated overnight at 4°C with the appropriate primary antibody listed in Table 2 in PBS with 0.3% Triton-X. After being washed with PBS 3 times, the sections were incubated with the suitable secondary fluorescent antibodies for 2 h at room temperature.

Western blot analysis

The expressions of nestin, β-tubulin III and GFAP were measured using Western blot analysis. Briefly, the brain tissues from the subventricular zone (SVZ) were homogenized with lysis buffer and protease inhibitors (Beyotime, China). The lysates were centrifuged at 14000 rpm for 15 min at 4°C. Equal amounts of the proteins (25μg) were resolved on a sodium dodecyl sulfate 10% or 12% polyacrylamide gel, and the separated 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 with the primary antibodies against nestin (1:1000, Abcam), β-tubulin III (1:1000, Abcam), GFAP (1:1000, Millipore) and GAPDH. Then, the membranes were incubated with appropriate secondary antibodies for 1 h. The immunoreactive bands were visualized with a chemiluminescence detection system. The band intensity was quantified using Image J software.

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Fig. 1.

Effect of ketamine on the proliferation of NSCs in the SVZ of neonatal rats. The PND-7 rats were ex- posed to 4 injections of 40 mg/kg ketamine at 1-h intervals. Then, the rats received a single intraperitoneal injection of BrdU (100 mg/kg) immediately after anesthesia or at PND-9 and PND-13. The animals were sacrificed at PND-8, 10 or 14. The NSCs were labeled by primary antibodies against nestin (Green) and BrdU (Red). The immunoreactive cells were visualized using a laser scanning confocal microscope (A; Ma- gnification 1: ×200, 2: ×400). The arrows point to nestin/BrdU double-labeled cells. The density of nestin/ BrdU double-positive cells was counted (B). The expression level of nestin was measured using Western blot analysis (C). The data are presented as the means ± SD (n=5). At the same time point: ** <0.01, vs control group. SVZ = subventricular zone; LV = lateral ventricle.

P

Statistical analysis

The data are presented as the means ± SD. The statistical analysis and the graphs were completed using GraphPad Prism 5. The significant differences between the groups were analyzed with an unpaired two-tailed t-test or one-way ANOVA. P<0.05 was considered statistically significant.

Results

Ketamine inhibits the proliferation of NSCs in the SVZ of neonatal rats

+ + cells in the ketamine group /BrdU As shown in Fig. 1A and B, the density of Nestin 2 ) was significantly decreased compared to that in the control group (107±6/ (36±1/μm 2 ) 24 h (PND-8) after exposure to ketamine. This suppressive effect of ketamine on NSC μm 2 proliferation was also found at 3 days (PND-10; 39±4 vs 107±8/μm ) and 7 days (PND- 2 ) after anesthesia. Western blot analysis also showed that the 14; 41±3 vs 100±13/μm

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Fig. 2.

Effect of ketamine on the neuronal differentiation of NSCs in the SVZ of neonatal rats. The PND-7 rats were exposed to four injections of 40 mg/kg ketamine at 1-h intervals. Then, the rats received a sin- gle intraperitoneal injection of BrdU (100 mg/kg) immediately after anesthesia or at PND-9 and PND-13. The animals were sacrificed at PND-8, 10 or 14. The newborn neurons were labeled by primary antibodies against neuronal skeleton protein β-tubulin III (Green) and BrdU (Red). The immunoreactive cells were visualized using a laser scanning confocal microscope (A; Magnification 1: ×200, 2: ×400). The arrows poin- ted to β-tubulin III/BrdU double-labeled cells. The density of β-tubulin III/BrdU double-positive cells was counted (B). The expression level of β-tubulin III was measured using Western blot analysis (C). The data are presented as the means ± SD (n=5). At the same time point: * <0.01, vs control group. SVZ = subventricular zone; LV = lateral ventricle.

P

P

<0.05, **

expression of nestin was significantly reduced at 1, 3 and 7 days after ketamine anesthesia compared to that in the control group (Fig. 1C). There were no significant differences in the NSC proliferation at different time points (PND-8, 10 and 14) in either the control groups or the ketamine-treated groups. Ketamine promotes the neuronal differentiation of NSCs in the SVZ of neonatal rats

+ + cells in the ketamine group (38±3/ /BrdU It was shown that the density of β-tubulin III 2 2 μm ) 24 h ) was significantly increased compared to that in the control group (34±2/μm (PND-8) after exposure to ketamine. This stimulant effect of ketamine on the neuronal 2 differentiation of NSCs was also found at 3 days (PND-10; 38±1 vs 34±1/μm ) and 7 days 2 (PND-14; 39±1 vs 35±2/μm ) after ketamine anesthesia (Fig. 2A and B). The expression of β-tubulin III also showed a significant increase in the ketamine group compared to the control group at PND-8, 10 and 14 by Western blot analysis (Fig. 2C). There were no significant differences in the neuronal differentiation of the NSCs at different time points (PND-8, 10 and 14) in either the control groups or the ketamine-treated groups.

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Fig. 3.

Effect of ketamine on the astrocytic differentiation of NSCs in the SVZ of neonatal rats. The PND-7 rats were exposed to four injections of 40 mg/kg ketamine at 1-h intervals. Then, the rats received a single intraperitoneal injection of BrdU (100 mg/kg) immediately after anesthesia or at PND-9 and PND-13. The

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animals were sacrificed at PND-8, 10 or 14. 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; Magnification 1: ×200, 2: ×400). The arrows pointed to GFAP/BrdU double-labeled cells. The density of GFAP/BrdU double-positive cells was counted (B). The expression level of GFAP was measured using Western blot analysis (C). To observe the proliferation of matured astrocytes, the animals were perfused and sacrificed at 3 h after each injection of BrdU. The proliferative astrocytes were also stai- ned with primary antibodies against GFAP and BrdU (D and E; Magnification: ×400). The data are presented <0.01, vs control group. SVZ = subventricular zone; LV as the means ± SD (n=5). At the same time point: ** = lateral ventricle. Fig. 4.

P

Effect of ketamine on the apoptosis of NSCs and astrocytes in the SVZ of neo- natal 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 apoptotic NSCs (A) or astrocytes (B) were stained with primary antibodies against nestin, GFAP and caspase-3. SVZ = subven- tricular zone; LV = lateral ventricle.

Ketamine attenuates the astrocytic differentiation of NSCs in the SVZ of neonatal rats

+ + cells in the ketamine treatment /BrdU 2 ) was significantly decreased compared to that in the control group (33±2/ group (7±1/μm 2 the μm ) 1 day (PND-8) after ketamine anesthesia. This suppressive effect of ketamine on 2 ) and astrocytic differentiation of NSCs was also found at 3 days (PND-10; 9±2 vs 32±3/μm 2 7 days (PND-14; 10±1 vs 33±1/μm ) after ketamine treatment. The Western blot analysis of GFAP also showed a significant decrease in the ketamine group compared to the control group at PND-8, 10 and 14 (Fig. 3C). There were no significant differences in the astrocytic differentiation of the NSCs at different time points (PND-8, 10 and 14) in either the control groups or the ketamine-treated groups. In addition, Fig. 3D and E show that only a small number of GFAP/BrdU double positive cells were found in both the control and ketamine groups at the time points of 3 h after each injection of BrdU. There were no significant differences in the proliferation of matured astrocytes between the ketamine and control groups at different time points (3 h after BrdU injection on PND-7, 9 and 13).

As shown in Fig. 3A and B, the density of GFAP

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The NSCs and astrocytes in the SVZ of neonatal rats are resistant to ketamine-induced cell apoptosis

To evaluate the effects of ketamine on the apoptosis of NSCs and astrocytes in the SVZ of neonatal rats, we measured nestin/caspase-3 and GFAP/caspase-3 double-positive cells by double-labeled immunofluorescence at 12 h after the end of ketamine anesthesia. The result showed that there were no significant nestin/caspase-3 or GFAP/caspase-3 double-positive cells in either the control or ketamine groups. These findings suggest that the present dosage and duration of ketamine was unable to induce apoptosis in the NSCs and astrocytes (Fig. 4).

Discussion

Ketamine, a N-methyl-D-aspartate (NMDA) receptor inhibitor, is widely used in infants and children for anesthesia and sedation [14]. Recent studies have reported that ketamine can induce neuronal apoptosis in the neonatal mammalian brain, which is usually considered to contribute to behavioral abnormalities that may arise during adulthood [3-5, 7]. However, some studies have suggested that neonatal anesthesia-induced neuroapoptosis is not the only factor that contributes to sustained cognitive deficits [15].

Neurogenesis is established during gestation in most brain regions and is nearly completed before birth except in two regions: the SVZ and the hippocampal DG. The normal development of these two regions is very important to learning and memory [16-21]. The process of neurogenesis involves the proliferation of NSCs, the neuronal and astrocytic differentiation of NSCs, and their migration and functional integration into the neural circuit. In this complicated process, the proliferation and differentiation of NSCs are not only initial but also extremely crucial.

Numerous studies showed that NMDA-R plays an important role in regulating the proliferation and differentiation of NSCs derived from the hippocampal DG [22-27]. However, the role of NMDA-R in the neurogenesis of SVZ has not been widely studied [28-30] and the consequences of blocking NMDA-R are controversial, partly because of the different animal models and various brain regions studied.

During the S phase of the cell cycle, BrdU is able to substitute for thymine, bearing a critical significance in the study of cellular dynamics. The dosage of BrdU used is based on that of a previous study [31]. Nestin is a commonly used marker of NSCs that is stably expressed from PND-1 to PND-28 in the SVZ [30]. The time points observed in our experiment fell exactly within this period. Of note, GFAP, which is an astrocyte marker, was also able to be expressed in the NSCs. However, the NSCs in this brain region did not initially express GFAP, and later, the NSCs gradually expressed this protein in the SVZ during the adult stage [32, 33]. It is known that mature astrocytes can proliferate after exposure to stimulation, such as a stroke. To exclude the proliferation of other cells, the proliferative NSCs were double-labeled with the neural stem cell marker nestin and the proliferation marker BrdU in the present study. The results indicated that ketamine is able to significantly suppress the proliferation of NSCs in the SVZ of neonatal rats at 1, 3 and 7 days after ketamine anesthesia (PND-8, 10 and 14).

The astrocytic differentiation of NSCs was also found to be markedly attenuated, reflected by a decreased number of GFAP/BrdU double-positive cells, whereas the neuronal differentiation of NSCs in the SVZ was obviously promoted, reflected by an increased number of β-tubulin III/BrdU double-positive cells. Because the NSCs in the SVZ only gradually express GFAP during the adult stage instead of the neonatal period, the GFAP/BrdU double positive cells in our experiment should not include those proliferative NSCs with a GFAP- positive label. In addition, to exclude the proliferation of mature astrocytes, GFAP/BrdU double-labeling immunostaining was performed 3 h after the BrdU injection, at which time BrdU had been adequately incorporated into the proliferative cells but newly differentiated astrocytes had not been generated. Only a small amount of mature astrocytes were found

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to be capable of proliferating in the SVZ of neonatal rats, and ketamine did not significantly promote or suppress the proliferation of mature astrocytes. Therefore, it was considered that the GFAP/BrdU double-positive cells detected at 24 h after the BrdU injection could represent the newborn astrocytes differentiated from NSCs.

A previous in vitro study showed that ketamine significantly inhibited the proliferation of NSCs isolated from SVZ in the rat fetal cortex and enhanced its neuronal differentiation [13]. Another in vitro study found that the proliferation of NSCs from the SVZ was markedly suppressed by MK-801, an NMDA-R antagonist [34]. These reports were coincident with our present results in vivo.

The toxicity of ketamine may cause cell death rather than directly inhibiting proliferation. + + The present study showed that neither nestin cells were /caspase-3 obviously found in the control or ketamine groups. Although neuronal apoptosis has been demonstrated to be induced by neonatal exposure to ketamine [4, 5], the present dosage and duration of ketamine could not significantly induce the apoptosis of NSCs and astrocytes in the SVZ of neonatal rats. It is thus 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.

+ + nor GFAP /caspase-3

To explore the duration of the ketamine effect, we observed the proliferation and differentiation of NSCs at 1, 3 and 7 days after ketamine anesthesia. We found that the alterations in the proliferation and differentiation of NSCs induced by ketamine occurred as early as one day after anesthesia (PND-8) and was still obvious at 7 days after ketamine exposure (PND-14). A recent in vivo study reported that, after blocking NMDA-R by MK-801, the number of proliferative cells in the SVZ of neonatal rats markedly decreased at the age of PND-7 and PND-14 [30]. This result was in agreement with our study.

Normally, newly generated neurons in the SVZ migrate along the rostral migratory stream (RMS) to the olfactory bulb (OB), where some of them join in the existing neural circuits; neurogenesis in OB plays a crucial role in long-term olfactory memory [35]. Our study indicated that ketamine significantly inhibited NSC proliferation and astrocytic differentiation while enhancing neuronal differentiation during the development of SVZ, which may result in the abnormalities in the number and proportion of neurons and astrocytes, thus disturbing the structure and formation of neuronal circuits in the cortical layers and olfactory bulb and possibly leading to alterations of the brain functions. Evidence has shown that hippocampus-dependent neurocognitive functions in the adult stage can be affected by neonatal exposure to ketamine [8, 36, 37]. However, the effect of ketamine on olfactory cognitive function has barely been studied. It is unknown whether neonatal ketamine anesthesia could contribute to clinically observed olfactory cognitive impairment in the adult stage. Our findings that ketamine interferes with the activity of NSCs in the SVZ during the postnatal period suggest that neonatal exposure to ketamine may be closely associated with olfactory cognitive disorders.

inhibit the proliferation of NSCs, to suppress their differentiation into astrocytes, and to promote the neuronal differentiation of NSCs in the SVZ of neonatal rats during the critical period of neurodevelopment. Given the importance of NSC proliferation and differentiation to neurogenesis, it is possible that the neonatal ketamine exposure may interfere with the neurogenic processes of the SVZ in the developing brain.

In summary, this study demonstrated that ketamine

is able to

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81171013), the Postgraduate Scientific Research and Innovation project of Jiangsu Province (CXLX13_997), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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o n 1 2 F e b r u a r y 2 0 2 4",rats,['Postnatal day 7 (PND-7) male Sprague- Dawley rats were administered four injections of 40 mg/kg ketamine at 1-h intervals.'],postnatal day 7,['Postnatal day 7 (PND-7) male Sprague- Dawley rats were administered four injections of 40 mg/kg ketamine at 1-h intervals.'],N,[],ketamine,['Postnatal day 7 (PND-7) male Sprague- Dawley rats were administered four injections of 40 mg/kg ketamine at 1-h intervals.'],none,[],sprague dawley,['Postnatal day 7 (PND-7) male Sprague- Dawley rats were administered four injections of 40 mg/kg ketamine at 1-h intervals.'],"The study investigates the effects of ketamine on the proliferation and differentiation of NSCs in the SVZ of neonatal rats in vivo, which has not been entirely clarified previously.","['However, these effects have not been entirely clarified in vivo in the subventricular zone (SVZ) of neonatal rats.']",None,[],"The study demonstrates that ketamine can alter neurogenesis by inhibiting the proliferation of NSCs, suppressing their differentiation into astrocytes, and promoting neuronal differentiation, which may impact neurodevelopmental processes.","['In the present study, it was demonstrated that ketamine could alter neurogenesis by inhibiting the proliferation of NSCs, suppressing their differentiation into astrocytes and promoting the neuronal differentiation of the NSCs in the SVZ of neonatal rats during a critical period of their neurodevelopment.']",None,[],None,[],True,True,True,True,True,True,10.1159/000369698
10.1016/j.neuropharm.2014.03.011,359.0,Lee,2014,rats,postnatal day 7,Y,isoflurane,none,sprague dawley,"Neuropharmacology 83 (2014) 9e17

Contents lists available at ScienceDirect

Neuropharmacology

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 r o p h a r m

Isoflurane exposure in newborn rats induces long-term cognitive dysfunction in males but not females

Bradley H. Lee a, John Thomas Chan a, Ekaterina Kraeva b, Katherine Peterson c, Jeffrey W. Sall a, *

a Department of Anesthesia and Perioperative Care, University of California, San Francisco, 513 Parnassus Ave., Box 0542, Med Sci S261, San Francisco, CA 94143, USA b University of Arizona College of Medicine, 550 E Van Buren St., Phoenix, AZ 85004, USA c University of Rochester, 500 Wilson Blvd., Rochester, NY 14627, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 December 2013 Received in revised form 19 March 2014 Accepted 22 March 2014 Available online 1 April 2014

Keywords: Anesthetics Isoflurane Sex Toxicity Memory

Volatile anesthetics are used widely for achieving a state of unconsciousness, yet these agents are incompletely understood in their mechanisms of action and effects on neural development. There is mounting evidence that children exposed to anesthetic agents sustain lasting effects on learning and memory. The explanation for these behavioral changes remains elusive, although acute neuronal death after anesthesia is commonly believed to be a principal cause. Rodent models have shown that isoflurane exposure in newborns induces acute neuroapoptosis and long-term cognitive impairment. However, the assessment of predisposing factors is lacking. We investigated the role of sex by delivering isoflurane to postnatal day (P)7 male and female Sprague Dawley rats for 4 h. Brain cell death was assessed 12 h later using FluoroJade C staining in the thalamus, CA1e3 regions of hippocampus, and dentate gyrus. Behavior was assessed separately using a series of object recognition tasks and a test of social memory beginning at P38. We found that isoflurane exposure significantly increased neuronal death in each brain region with no difference between sexes. Behavioral outcome was also equivalent in simple novel object recognition. However, only males were impaired in the recognition of objects in different locations and contexts. Males also exhibited deficient social memory while females were intact. The profound behavioral impairment in males relative to females, in spite of comparable cell death, suggests that males are more susceptible to long-term cognitive effects and this outcome may not be exclusively attributed to neuronal death.

(cid:1) 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Anesthetic agents are instrumental in their ability to induce an unconscious state, devoid of pain and awareness, and millions of children undergo anesthesia each year as a routine part of their medical care. However, the safety of anesthesia in this population is a concern and prior retrospective studies have noted a correlation between anesthesia exposure and learning disabilities (Flick et al., 2011; Wilder et al., 2009). In addition, various animal models have shown that early anesthetic exposure results in significant long-term behavioral deficits (Gentry et al., 2013; Jevtovic- Todorovic et al., 2003; Brambrink et al., 2010).

The reason behind these behavioral changes remains unknown, although acute neuronal injury is widely perceived to be an un- derlying cause. Numerous animal experiments have documented neuronal death following exposure to volatile anesthetics (Jevtovic- Todorovic et al., 2003; Stratmann et al., 2009a; Satomoto et al., factors influencing cell death have been 2009). researched extensively, including duration of anesthesia and the specific anesthetic agent that is delivered (Stratmann et al., 2009a; Istaphanous et al., 2011; Ramage et al., 2013). Various mechanisms have even been proposed to account for the process of anesthetic- induced neuroapoptosis (Lu et al., 2006; Head et al., 2009). Nevertheless, the precise correlation between neuronal death and behavioral phenotype is undetermined, and although neuronal death is certainly alarming it has not been determined to be causative.

In addition,

Corresponding author. Tel.: þ1 415 476 0322; fax: þ1 415 476 8841. E-mail address: sallj@anesthesia.ucsf.edu (J.W. Sall).

Because no adequate explanation exists, the final phenotype of behavior remains an indispensable tool for investigating anesthetic

http://dx.doi.org/10.1016/j.neuropharm.2014.03.011 0028-3908/(cid:1) 2014 Elsevier Ltd. All rights reserved.

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B.H. Lee et al. / Neuropharmacology 83 (2014) 9e17

effects. Many questions, such as the types of memory that are affected and factors that predispose to cognitive dysfunction, are most aptly explored using behavioral models. Also, concerns of early anesthesia leading to autism-like or impaired social behavior have been raised and begun to be explored in animals (Satomoto et al., 2009). With mounting evidence of the detrimental effects of anesthetics on the developing brain, these questions become increasingly important to address.

Among potential risk factors, the role of sex has not been studied. Human gender differences in outcomes following trau- matic and ischemic brain injury are well known (Roof and Hall, 2000; Ottochian et al., 2009) and are also seen in prematurity and neonatal stroke (Kesler et al., 2004; Mayoral et al., 2009). Epidemiologic studies in humans investigating cognitive outcome after early anesthesia exposure suggest that males may be more susceptible to long-term effects (DiMaggio et al., 2011; Hansen et al., 2011; Jevtovic-Todorovic et al., 2013), although methodo- logical issues preclude us from identifying a true difference. We proposed to determine the influence of sex on two well-known outcomes e brain cell death and behavior.

Fig. 1. Group assignment. At postnatal day 7, male and female rats were randomized to control or isoflurane groups for separate experiments investigating neuronal death and behavior. Neuronal death was assessed 12 h after anesthesia. Separate groups of ani- mals underwent behavioral testing beginning at P38. The number of animals in each group (n) is also presented.

between 0800 and 1700 h. Animals were food restricted for tasks involving object recognition. Access to food was limited to the light cycle in order to increase activity and object exploration during the testing period.

Exposure to isoflurane, a common inhaled volatile anesthetic, has repeatedly been shown to cause acute neuronal death and persistent cognitive dysfunction in newborn male rodents (Jevtovic-Todorovic et al., 2003; Stratmann et al., 2009a), but it is unknown how females are affected. This is because most studies use only male rodents or do not separately assess male and female subjects. In rodent models, effects of anesthesia have been studied following exposure during a time of peak brain development and synaptogenesis, typically postnatal day 7 (Jevtovic-Todorovic et al., 2003; Olney et al., 2000; Stratmann et al., 2009b; Shih et al., 2012). Cell death occurs acutely in the period immediately following anesthesia (Jevtovic-Todorovic et al., 2003; Istaphanous et al., 2011; Shih et al., 2012), and the thalamus and hippocampus are areas known to be susceptible to extensive neurodegeneration (Jevtovic- Todorovic et al., 2003; Satomoto et al., 2009; Shih et al., 2012). Long-term behavior is separately assessed in adolescence or adulthood using a range of tasks (Jevtovic-Todorovic et al., 2003; Stratmann et al., 2009a; Satomoto et al., 2009; Ramage et al., 2013). In this study, we investigate sex-specific outcomes after anes- thesia by assessing acute neurodegeneration after isoflurane exposure in the thalamus, CA1e3 regions of hippocampus, and dentate gyrus, as well as evaluating behavior with a series of object recognition and social memory tasks. In female rats, certain behavioral tests that use aversive stimuli or induce stress can be influenced by hormone cycling (Boscolo et al., 2013; Simpson and Kelly, 2012; Marcondes et al., 2001). To minimize these effects, we assessed behavior using spontaneous recognition tasks that rely on rodents’ natural preference for novel stimuli. Moreover, the associative memory used in these tests has been shown to be sensitive to lesions in hippocampal and thalamic circuits (Eacott and Norman, 2004; Langston and Wood, 2010; Cross et al., 2012).

2.2. Anesthesia

Male and female subjects were separately anesthetized for a duration of four hours as we have previously described (Stratmann et al., 2009c). Briefly, isoflurane was delivered into the anesthetic chamber, and gas concentrations were continu- ously monitored. The isoflurane concentration was initially set to 4% (time ¼ 0 min) and subsequently maintained at 1 Minimum Alveolar Concentration (MAC, the concentration required to prevent movement in 50% of subjects in response to a painful stimulus, Fig. 2). Every 15 min after induction, a supramaximal pain stimulus was produced by applying an alligator clamp to each rat’s tail. Movement was defined as any gross movement other than breathing, and the percent of animals that moved in response to tail-clamping was calculated. Isoflurane concentration was then adjusted to maintain 50% response to the stimulus. Control animals were treated identically without tail-clamping or administration of anesthetic. Animals in the anesthesia chamber were kept on a warming blanket and the temperature was measured every 15 min using infrared thermometer, and the position and heating were adjusted to maintain normothermia.

2.3. Histology

Brains from male and female treatment and control groups (n ¼ 10 per group) were assessed for acute neuronal death. Twelve hours after anesthesia, animals were anesthetized and 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 highly specific for neuro- degeneration (FJC, 0.001%, Millipore, Billerica, MA). FJ-positive cells were counted using Nikon Eclipse 80i microscope under 20(cid:3) magnification in each slice con- taining the structure of interest. Structures included in analysis were the ante- rodorsal (AD), anteroventral (AV), laterodorsal (LD), and anteromedial (AM) thalamic nuclei, as well as CA1e3 regions of the hippocampus and the dentate gyrus. Because the sex of newborns rats is often ambiguous, genetic screening was used to confirm sex as described elsewhere (Miyajima et al., 2009). Briefly, DNA was isolated from tissue samples, and Sex-determining region Y (Sry, male-specific) and beta actin (autosomal) gene sequences were amplified by polymerase chain reaction (PCR) using Taq DNA polymerase (G-Biosciences, St. Louis, MO) and primers ob- tained from Eurofins MWG Operon (Huntsville, AL). After isolation of genomic DNA, PCR products were subjected to electrophoresis in 2% agarose gel, and males were identified by presence of two separate bands and females with a single band.

2. Materials and methods

2.1. Subjects

2.4. Object recognition tasks

All experiments were conducted with approval from the Institutional Animal Care and Use Committee at the University of California, San Francisco. Sprague Dawley dams with litters containing male-only and female-only pups were obtained from Charles River Laboratories (Gilroy, CA). On postnatal day (P)7, animals were randomly assigned to control or treatment groups (Fig. 1). Following treatment, subjects were either killed and fixed for histology or cross fostered between dams. At P21, before reaching sexual maturity, each animal’s sex was assessed and they were separated into groups by sex. Control and treatment animals were kept together in clean acrylic cages with bedding changed weekly and ad libitum access to food and water. Cages for both sexes were kept in the same room within the animal care facility with 12 h lightedark cycle and regulation of temperature (18e25 (cid:2)C) and humidity (45e65%). At P30, they were housed in pairs with one treatment and one control animal per cage. All behavioral testing occurred during the light cycle

Testing occurred similar to the paradigm used by others (Eacott and Norman, 2004; Langston and Wood, 2010). Male and female subjects were assessed using the same testing area and objects. Testing arenas and objects were wiped with 70% ethanol between subjects. Object recognition testing took place in two separate testing arenas, hereafter referred to as “contexts”, of identical size (61 cm square base, walls 50 cm high). The two were distinct in their appearance and texture to allow testing of context-specific memory. Context 1 had yellow walls and a base covered in wood-effect vinyl lining, while context 2 had black walls and a black plastic base. Visual cues were placed on three different walls within each context. Animals were introduced into the contexts facing the same direction and in the same location, and subjects were habituated to the contexts prior to testing. Each object was validated to avoid object bias. Investigation of an object was defined as sniffing or placing the nose within 1 cm of and oriented toward the object. Subjects

B.H. Lee et al. / Neuropharmacology 83 (2014) 9e17

Fig. 2. MAC and isoflurane concentration in subjects. In separate experiments, male and female P7 rats were anesthetized to determine MAC and compare the potency of isoflurane. These experiments provided a larger sample size to compare anesthetic effects, and comparison between sexes shows no difference in the concentration and MAC values. The subjects in this study underwent the same anesthetic model for ex- periments of neurodegeneration and behavior.

were video recorded and reviewed by blinded observers to determine investigation times.

All subjects underwent the full series of testing in the order presented here with one trial per day. The subjects’ order of testing also rotated each day so that the timing of behavioral testing was counterbalanced among subjects and groups. Testing began at postnatal day 38 (P38) with novel object recognition (Fig. 3). Subjects were assessed in their ability to recall a previously encountered object. A single trial was performed, and half of the subjects were tested in context 1 and the other half in context 2. During the “exposure”, the subject was placed into the context and explored two identical objects for four minutes. Following a two-minute delay, in the “test” phase, the animal was placed into the same context with one of the previous objects replaced with a novel object. The location (left or right) of the novel object within each context was counterbalanced among subjects. For each task, object investigation times during the initial exposure were compared, given possible confounding effects of varying investigation times on object recognition in the subsequent test phase.

Using object recognition as the premise, the tasks were then made increasingly complex. By using different objects and varying the locations and contexts in which they were presented, subjects were assessed in their ability to associate an object with a particular location, context, or combination of location and context. The arrangement used to assess each of these associative memory tasks is presented in Fig. 3.

In the final task of objecteplaceecontext recognition, control female subjects were identified as having increased object investigation during the exposure, thereby potentially conferring an advantage in subsequent object recognition. The following set of trials (Trials 3 and 4) were therefore performed while controlling for investigation times. Subjects were observed during the exposure with a goal of 15 s of investigation per object. Animals remained in the context for a minimum of two minutes and a maximum of five minutes to ensure adequate familiarization to the context. After the two-minute mark, if they reached the required investigation times, then they were removed. The test phase lasted four minutes and was recor- ded and later reviewed.

Fig. 3. Object recognition tasks. A) Novel object recognition. Two identical objects are presented in the exposure phase, and one of these objects is replaced with a novel object (*) in the test phase. B) Objecteplace recognition. Two different objects are presented followed by two identical objects. In the test phase, one object (*) appears in a new location within the context. C) Objectecontext recognition. Two separate pairs of objects are presented in two different contexts so that each object is associated with a context. In the test phase, one object (*) appears within a context in which it has not been explored. D) Objecteplaceecontext recognition. Two different objects are pre- sented in Exposure 1. These same objects are presented in a different context with their locations switched in Exposure 2. Thus, after two exposures, each object is seen both locations and contexts. In the test phase, two identical objects are presented in either context, so that one object (*) is presented in a novel configuration of place and context. There are four total configurations with two of these beginning in context 1 (Setup 1) and the other two in context 2 (Setup 2).

2.5. Social behavior and social recognition

Social interaction and recognition were assessed using a discrimination para- digm. In the “exposure” phase, the subject was presented with a caged stimulus animal alongside an empty cage for five minutes. This arrangement evaluates social interaction by determining whether subjects appropriately spend more time investigating the social target (Satomoto et al., 2009). After a sixty-minute delay, the subject was presented simultaneously with the same “familiar” stimulus animal and a novel animal for three minutes. Social recognition is demonstrated by decreased investigation of the familiar target relative to the novel one.

Same-sex juvenile conspecifics were used as stimulus animals. Male and female pups five weeks of age were housed individually one week prior to testing. Inves- tigation was defined as any direct contact with the subject’s nose or paws, as well as sniffing toward any part of the juvenile including the tail if it extended outside of the cage. Investigation of the empty cage was defined as sniffing or placing the nose within 1 cm of and oriented toward the cage, and excluded using the cage as a support during rearing.

2.6. 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’Ag- ostino-Pearson test. Parametric tests were used for normally distributed data;

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otherwise, a nonparametric test was used. All comparisons used a two-tail test and a P value less than 0.05 was considered statistically significant.

Subjects were evaluated in their ability to recognize familiar stimuli, reflected by the relative time spent investigating two separate targets. For the final task (objecteplaceecontext recognition), times from Trials 1 and 2 were combined for analysis, and Trials 3 and 4 were assessed together. The ratio paired t-test was used to compare normally distributed data, and nonparametric data were analyzed with the Wilcoxon matched-pairs rank test. In addition, a “discrimination index” (DI) was calculated, representing the time spent investigating the novel target relative to the familiar target. 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 (eg. DI ¼ (Novel (cid:4) Familiar)/(Total Time)). DI provides a single value and therefore allows analysis by two-way ANOVA to compare effects of treatment or sex.

To identify and control for possible confounding effects of varying investigation times on subsequent object/animal recognition, the investigation times during the exposure phase were compared between the groups. These times were compared using one-way ANOVA for normally distributed data and KruskaleWallis test for nonparametric data. Bonferonni’s post-test with multiple comparisons was used following one-way ANOVA, and Dunn’s post-test was used with the KruskaleWallis test.

Two-way ANOVA was used to assess the effects of sex and treatment on neuronal death. Neuronal death for each brain region was compared using two-way ANOVA and Bonferroni post-test. The fold-increase in neuroapoptosis was deter- mined for each structure by dividing the total FJ-positive cells of each treatment animal (n ¼ 20) by the average number of FJ-positive cells per structure for the whole control group (n ¼ 20).

3. Results

P < 0.0001), and dentate gyrus (F(1,36) ¼ 41.17, P < 0.0001). The fold- increase in cell death for each brain region is shown in Fig. 4G.

3.2. Behavior group assignment and anesthesia

In separate series of experiments, survival and blood gas ana- lyses (pH, pO2, pCO2) were performed and found to be the same in P7 male and female rats and similar to results reported previously (Ramage et al., 2013). In addition, we assessed MAC using pooled data from multiple rounds of anesthesia in separate male and fe- male groups. There was no difference identified in the anesthetic requirement or MAC between sexes (Fig. 2). Therefore, at P7, groups of male and female animals were obtained from the vendor and assigned to control or treatment groups for behavioral experi- ments. Twelve pups were assigned to each male or female control group and eighteen pups to each male or female anesthesia group. Given the anticipated mortality, eighteen rats were anesthetized in each male and female group with the goal of yielding 11e12 treatment subjects per group. The mortality of 27% for all anes- thetized subjects is consistent with prior results (Stratmann et al., 2009a; Ramage et al., 2013; Stratmann et al., 2009b), although the difference in mortality for separately anesthetized groups yielded a greater number of treatment female rats. In addition, the sex of several pups was incorrectly assigned by the vendor, resulting in final group sizes of 8 male control, 16 female control, 8 male treatment, and 18 female treatment animals (Fig. 1).

3.1. Brain cell death occurs similarly in males and females

There was increased neuronal death acutely in male and female treatment groups relative to the two control groups for each brain region with no significant effect of sex on the extent of cell death (Fig. 4AeF). Isoflurane resulted in markedly increased cell death in the anterodorsal thalamus (F(1,36) ¼ 57.41, P < 0.0001), ante- roventral thalamus (F(1,36) ¼ 53.98, P < 0.0001), anteromedial thalamus (F(1,36) ¼ 110.5, P < 0.0001), laterodorsal thalamus (F(1,36) ¼ 92.56, P < 0.0001), hippocampus (F(1,36) ¼ 38.92,

3.3. Object discrimination remains intact in both sexes

All groups were able to distinguish familiar and novel objects evidenced by greater investigation of the novel object (control male P ¼ 0.0151, treatment male P ¼ 0.023, control female P < 0.0001, treatment female P < 0.0001, ratio paired t-test of familiar vs. novel object, Fig. 5A). Two-way ANOVA of the Discrimination Index (DI), which represents the time spent investigating the novel target

Fig. 4. Neuronal death results. AeF) There is increased cell death in each brain region for both male and female animals immediately following anesthesia (n ¼ 10 per group). The extent of cell death in treatment males and females is similar. Representative images (FJC staining 12 h post-anesthesia) from male and female brains at 20(cid:3) magnification are displayed alongside graphs comparing total FluoroJade-positive cells for each structure. G) The average fold-increases in neuronal death relative to controls are shown. The fold- increase is determined from pooled data of all anesthetized subjects relative to the average of the control subjects.

B.H. Lee et al. / Neuropharmacology 83 (2014) 9e17

Fig. 5. A) Novel object recognition. In the task, subjects are tested in their ability to recognize a familiar object independent of location or context. All groups were able to recognize and distinguish the two objects reflected by increased investigation of the novel object. B) The discrimination index (DI), representing the time spent investigating the novel object relative to the familiar one, was similar among the groups. C) Objecteplace recognition. This task requires the subject to identify an object and its previous location within the context. Treatment males were the only subjects unable to recognize the object and its spatial location. D) Two-way ANOVA with Bonferroni post-test identified the female treatment DI as significantly higher than male treatment DI. E) Objectecontext recognition. Subjects are tested in their ability to recognize an object and the context in which it appeared. All but the male treatment group successfully recognized an object with its associated context. F) The male treatment DI was lower than the female treatment DI but did not reach statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. ¼ not significant.

relative to the familiar one, demonstrated no effect of treatment (F(1,45) ¼ 0.0005, P ¼ 0.98) or sex (F(1,45) ¼ 0.002, P ¼ 0.96, Fig. 5B). There was no difference among groups in time spent investigating the objects during the initial exposure (P ¼ 0.29, KruskaleWallis test).

familiar vs. out-of-context object, Fig. 5E). DI was unaffected by treatment or sex (F(1,46) ¼ 3.2, P ¼ 0.08; F(1,46) ¼ 1.09, P ¼ 0.3; two- way ANOVA). Investigation times of the initial exposures were combined for analysis, and one-way ANOVA revealed no difference among groups (P ¼ 0.25).

3.4. Males are impaired in objecteplace, objectecontext, and objecteplaceecontext recognition while females are unaffected

All groups but the male treatment group were able to recognize an object and its spatial location. Subjects, except male treatment animals, preferentially explored the object occupying a novel location (control male P ¼ 0.027, treatment male P ¼ 0.73, control female P ¼ 0.0028, treatment female P < 0.0001, ratio paired t-test of familiar vs. novel location, Fig. 5C). Two-way ANOVA of DI did not demonstrate a significant effect of treatment (F(1,44) ¼ 0.49, P ¼ 0.48) or sex (F(1,44) ¼ 2.88, P ¼ 0.09), although further investi- gation comparing treatment groups by sex revealed that the female treatment DI was significantly higher than the male treatment DI (P ¼ 0.041, Bonferroni post-test, Fig. 5D). This difference occurred despite increased object exploration by the male treatment group during the initial exposure (P ¼ 0.0011, KruskaleWallis test; male treatment vs. female treatment, P ¼ 0.02, Dunn’s post-test).

Male treatment subjects were also impaired in objectecontext recognition (P ¼ 0.69, ratio paired t-test of familiar vs. out-of- context, Fig. 5E). All other groups spent more time investigating the out-of-context object (control male P ¼ 0.015, control female P < 0.0001, treatment female P ¼ 0.012, ratio paired t-test of

The male treatment group was the only one impaired in objecte placeecontext recognition, as well. The investigation times for Trials 1 and 2 were combined for analysis, and all groups but the male treatment group spent more time investigating the “dis- placed” object (control male P ¼ 0.0034, treatment male P ¼ 0.20, control female P < 0.0001, treatment female P ¼ 0.013; ratio paired t-test of familiar vs. displaced object, Fig. 6A). Comparison of DI by two-way ANOVA found a significant interaction between treatment and sex (F(1,46) ¼ 8.9, P ¼ 0.004), and further evaluation identified the male treatment DI as significantly lower than female treatment DI (P ¼ 0.01, Bonferonni post-test, Fig. 6B). In this task, the female control group spent more time investigating the objects during the exposure than the male treatment group (P ¼ 0.041, one-way ANOVA, Bonferonni post-test).

Because increased object exploration during the exposure leads to deeper encoding of memory, we repeated the experiment while controlling for investigation time. Times were combined for anal- ysis in trials 3 and 4, and once again the male treatment group was unable to distinguish between familiar and novel configurations of object, place, and context (control male P ¼ 0.048, treatment male P ¼ 0.1239, control female P ¼ 0.0002, treatment female P ¼ 0.001, ratio paired t-test of familiar vs. displaced object, Fig. 6C). The fe- male treatment DI was higher than male treatment DI although this

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Fig. 6. Objecteplaceecontext recognition. A) In the final test of object recognition, animals were evaluated in their memory of an object, its spatial position, and the context in which it appeared. Only the male treatment group was unable to recognize the object with its associated location and context. B) Two-way ANOVA identified a significant interaction of treatment and sex, and the treatment female DI was greater than the treatment male DI. C) A second set of trials was performed for the task because of differences in investigation time between groups during initial exposure, possibly conferring an advantage to females in subsequent recognition. Even when controlling for investigation time during the exposure, female treatment animals demonstrated successful object recognition while male treatment subjects were again impaired. D) In trials 3 and 4, the female treatment DI was again higher than male treatment DI but did not reach statistical significance.

did not reach statistical significance (F(1,46) ¼ 3.3, P ¼ 0.07; F(1,46) ¼ 0.69, P ¼ 0.41; two-way ANOVA, Fig. 6D). As expected, no difference was found between groups for the exposure phase (P ¼ 0.43, one-way ANOVA).

3.5. Both sexes display normal social investigatory behavior but only males have impaired social memory

All animals demonstrated normal social investigatory behavior and spent much more time with the social target than the empty cage (all P < 0.0001, ratio paired t-test of social target vs. empty cage, Fig. 7A). There was no difference in total time spent investi- gating the stimulus animal among the four groups (P ¼ 0.39, one- way ANOVA), reflecting equal motivation and interest regardless of treatment or sex.

Treatment males were the only subjects with deficient social memory and could not distinguish familiar and novel stimulus animals following a one-hour delay (P ¼ 0.32, ratio paired t-test of familiar vs. novel, Fig. 7B). All other groups preferentially explored the novel stimulus animal (control male P ¼ 0.037, control female P ¼ 0.0202, treatment female P < 0.0001, ratio paired t-test of familiar vs. novel, Fig. 7B). The male treatment DI was also signif- icantly lower than the female treatment DI (F(1,46) ¼ 6.5, P ¼ 0.04; two-way ANOVA, Bonferonni post-test, Fig. 7C).

observation not only identifies a worse behavioral outcome in males but also challenges the theory that neuronal death has a causative relationship.

The lack of studies investigating sex differences after anesthesia exposure may be due, in part, to intrinsic difficulties faced when comparing subjects with species and sex-specific differences in memory. In rats, females generally outperform males in tasks of object recognition, while males are better at tasks of spatial learning and memory (Saucier et al., 2008). Studies in mice are variable with some reporting either no difference or male superi- ority in object recognition (Frick and Gresack, 2003; Benice et al., 2006), while others find females to be better in object discrimi- nation (Bettis and Jacobs, 2012, 2009). An investigation in Long- Evans rats shows that age and sex could influence social recogni- tion ability, although estrous cycle appeared to have no effect (Markham and Juraska, 2007). The differences we find between treatment animals of each sex may be attributed partially to innate variability in learning and memory; however, this alone likely does not account for the significant disparity in behavior. By using a series of tasks with increasing difficulty we are able to make comparisons along a spectrum, and it is doubtful that memory in females is robust enough to completely mask an insult to the developing brain, while males are unable to distinguish between targets in any task relying on associative or social memory.

4. Discussion

The major finding of this study is that while neuronal death occurs similarly in the hippocampus and thalamic nuclei of both sexes immediately following isoflurane exposure, there is a pro- found difference in behavioral outcomes. Males are impaired in multiple tasks that rely on allocentric cues, association of contex- tual details, and social recognition. Anesthetized females, mean- while, were unaffected in these tasks. This suggests that males and females follow distinct paths of neural and cognitive development after an early anesthetic-mediated effect on the brain. Both male and female rats exhibit extensive neuronal death, yet drastic behavioral impairment is manifested only in male subjects. This

Male impairment in memory may be related to early anesthetic effects on the developing brain. Associative memory involves learning relationships among distinct elements and is necessary for understanding the significance of combinations of cues (Aggleton et al., 2007; Mumby et al., 2002). Hippocampal and thalamic le- sions in rats affect associative memory and cause impaired learning of spatial relationships between elements (Mumby et al., 2002; Piterkin et al., 2008) and objecteplace recognition (Cross et al., 2012). Social recognition relies on the interaction of multiple brain regions and hormones, including oxytocin and vasopressin (Bluthe et al., 1990; Landgraf et al., 1995; Lukas et al., 2011). The lateral septum contains high numbers of vasopressin receptors (De Vries and Buijs, 1983) and has reciprocal connections to higher order brain regions including the thalamus and hippocampus (Caffe

B.H. Lee et al. / Neuropharmacology 83 (2014) 9e17

synapse formation (Bath et al., 2013; Bibel and Barde, 2000), and estrogen has been shown to increase the expression of BDNF (Lu et al., 2006; Sato et al., 2007; Blurton-Jones and Tuszynski, 2006; Begliuomini et al., 2007). Anesthetics not only cause cell death but have been shown to result in significant neuroinflammation (Shen et al., 2013), in addition to changes in cell signaling (Masaki et al., 2004), stem cell proliferation (Sall et al., 2009), and synapse for- mation (Lunardi et al., 2010; Briner et al., 2011); these effects could be mitigated by protective properties of estrogen and progesterone (Ishihara et al., 2013; Brinton et al., 1997; Liu et al., 2008; Murphy et al., 1998; Schwarz and McCarthy, 2008). Due to hormonal dif- ferences, females may be more resilient in their ability to recover and form proper connections after anesthesia-induced effects on neural development. This might explain why females in our study were unaltered while males were profoundly impaired in tests of both associative memory and social recognition.

The timing of isoflurane exposure in the rats at postnatal day 7 occurs during a period of peak neural development and synapto- genesis, which overlaps with a corresponding stage of development in humans in the late 3rd trimester through the first several months of life depending on the specific brain region of interest (Rice and Barone, 2000). The subjects in our study were tested during adolescence, which is when most retrospective human trials identify a cognitive deficit in children after anesthesia (Flick et al., 2011; Wilder et al., 2009). Thus, the timing of anesthetic expo- sure and subsequent cognitive outcomes are consistent between species, which suggests that similar processes of neural develop- ment may be affected in both. Still, rodents are simply a model, and it is important to understand the differences in the overall complexity and timing of development between species when interpreting the experimental findings in animals and how they apply to humans.

Fig. 7. A) Social interaction. In the exposure, the subjects were simultaneously pre- sented with a caged same-sex juvenile and an inanimate object, and all subjects dis- played normal social behavior and spent more time with the social target. B) Social recognition. Animals were tested in their ability to recognize a previously encountered juvenile using a discrimination model. In the test phase, subjects were presented simultaneously with a familiar and novel juvenile. All groups except the male treat- ment group were able to recognize the familiar animal, evidenced by the decreased investigation of the familiar relative to the novel animal. C) Discrimination index. Two- way ANOVA identified the treatment female DI as significantly higher than the treatment male DI. *P < 0.05, **P < 0.01, ***P < 0.001.

et al., 1987; Bielsky and Young, 2004). Anesthesia exposure early in life causing brain cell death, which is particularly profound in the thalamus (Jevtovic-Todorovic et al., 2003; Stratmann et al., 2009a; Satomoto et al., 2009; Shih et al., 2012), may potentially contribute to the behavioral deficits we observed. Unlike Satomoto et al., we did not observe abnormal social behavior, and both male and female treatment subjects displayed the same social interac- tion as controls; therefore, our findings do not support a correlation between anesthesia and autism-like behavior, and the impairment in social recognition is more likely a consequence of memory encoding than innate social behavior.

Importantly, females suffer a similar extent of cell death yet do not display behavioral deficits. A possible explanation is that fe- males have improved recovery following neurotoxic effects of volatile anesthetics, especially since many studies find protective effects of hormones that are expressed predominantly in females (Ishihara et al., 2013; Brinton et al., 1997; Liu et al., 2008). Brain- derived neurotrophic factor (BDNF) is a secreted protein involved in the survival, growth, and development of neurons and proper

There are certain other limitations to consider when interpret- ing our results. While a deficit was not identified in the female treatment subjects, this does not necessarily mean that females were entirely unaffected. A behavioral deficit might be revealed by increasing the difficulty of the tasks (for instance, prolonging delay between exposure and memory retrieval). Other factors that may play a role in the cognitive outcomes of male and female subjects in the study include possible differences between sexes in their response to food restriction, activity patterns during the day, as well as hormone cycling, which might influence behavioral findings and should be kept in mind. 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. Finally, there are inherent limitations in anesthetizing P7 animals that expose them to physiologic changes, notably that of hypercarbia (Stratmann et al., 2009a). Although physiologic parameters are difficult to reliably monitor or control in P7 rodents, blood gas analyses (pH, pO2, pCO2) can be measured and reveal no difference between males and females, and hyper- carbia alone does not predispose subjects to memory deficits (Stratmann et al., 2009a).

The mechanisms by which anesthetics alter long-term behavior remain unknown. Within the nervous system, processes of brain cell death, changes in synapses, and alteration of stem cell function have been implicated in behavioral deficits. To date, no studies have conclusively tied behavior to a neuro-anatomic or neuro-chemical change. The results of this study suggest that brain cell death alone is not sufficient to account for the cognitive dysfunction since both sexes had equivalent cell death. Future studies exploring sex- specific outcomes will help elucidate these effects and lead to a better understanding of the roles of sex and hormones on brain development and protection.

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Funding and disclosure

This work was supported by NIH Grant Award GM086511 and UCSF Department of Anesthesia and Perioperative Care Hamilton Award to Dr. Jeffrey Sall.

Author contributions

Frick, K.M., Gresack, J.E., 2003. Sex differences in the behavioral response to spatial and object novelty in adult C57BL/6 mice. Behav. Neurosci. 117, 1283e1291. 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, 185e189.

Hansen, T.G., Pedersen,

J.C., Morton, N.S., Christensen, K., 2011. Academic performance in adolescence after inguinal hernia repair in infancy: a nationwide cohort study. Anesthesiology 114, 1076e1085.

J.K., Henneberg, S.W., Pedersen, D.A., Murray,

JS and BL designed the project, analyzed data, and wrote the first draft of the manuscript. JC, EK, KP, and JL designed and analyzed data from cell death and behavioral experiments. All authors contributed to the final draft of the manuscript.

Head, B.P., Patel, H.H., Niesman, I.R., Drummond, J.C., Roth, D.M., Patel, P.M., 2009. Inhibition of p75 neurotrophin receptor attenuates isoflurane-mediated neuronal apoptosis in the neonatal central nervous system. Anesthesiology 110, 813e825.

Ishihara, Y., Kawami, T., Ishida, A., Yamazaki, T., 2013. Allopregnanolone-mediated protective effects of progesterone on tributyltin-induced neuronal injury in rat hippocampal slices. J. Steroid Biochem. Mol. Biol. 135, 1e6.

Acknowledgments

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, 578e587.

Funding provided by grant award GM086511 to JWS from Na- tional institutes of Health, Bethesda, MD. UCSF Department of Anesthesia and Perioperative Care Hamilton Award to JWS. The authors declare no competing financial interests.

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Stratmann, G., May, L.D., Sall, J.W., Alvi, R.S., Bell, J.S., Ormerod, B.K., Rau, V., Hilton, Lee, M.T., Visrodia, K.H., Ku, B., Zusmer, E.J., Guggenheim, J., Firouzian, A., 2009a. Effect of hypercarbia and isoflurane on brain cell death and neurocognitive dysfunction in 7-day-old rats. Anesthe- siology 110, 849e861.

J.F., Dai, R.,

Stratmann, G., Sall, J.W., May, L.D., Bell, J.S., Magnusson, K.R., Rau, V., Visrodia, K.H., Alvi, R.S., Ku, B., Lee, M.T., Dai, R., 2009b. Isoflurane differentially affects neu- rogenesis and long-term neurocognitive function in 60-day-old and 7-day-old rats. Anesthesiology 110, 834e848.

Stratmann, G., Sall, J.W., Eger 2nd, E.I., Laster, M.J., Bell, J.S., May, L.D., Eilers, H., Krause, M., Heusen, F., Gonzalez, H.E., 2009c. Increasing the duration of iso- flurane anesthesia decreases the minimum alveolar anesthetic concentration in 7-day-old but not in 60-day-old rats. Anesth. Analg. 109, 801e806.

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. Anes- thesiology 110, 796e804.

17",rats,['We investigated the role of sex by delivering isoflurane to postnatal day (P)7 male and female Sprague Dawley rats for 4 h.'],postnatal day 7,['We investigated the role of sex by delivering isoflurane to postnatal day (P)7 male and female Sprague Dawley rats for 4 h.'],Y,['Behavior was assessed separately using a series of object recognition tasks and a test of social memory beginning at P38.'],isoflurane,['We investigated the role of sex by delivering isoflurane to postnatal day (P)7 male and female Sprague Dawley rats for 4 h.'],none,[],sprague dawley,['We investigated the role of sex by delivering isoflurane to postnatal day (P)7 male and female Sprague Dawley rats for 4 h.'],The study addresses the issue of sex differences in cognitive dysfunction following anesthetic exposure in newborn rats.,"['Among potential risk factors, the role of sex has not been studied.']",The study presents innovations in methodology by assessing sex-specific outcomes after anesthesia exposure in rats.,"['In this study, we investigate sex-specific outcomes after anes- thesia by assessing acute neurodegeneration after isoflurane exposure in the thalamus, CA1e3 regions of hippocampus, and dentate gyrus, as well as evaluating behavior with a series of object recognition and social memory tasks.']","The article argues the impact of findings by suggesting that males are more susceptible to long-term cognitive effects of anesthesia, which may not be exclusively attributed to neuronal death.","['The profound behavioral impairment in males relative to females, in spite of comparable cell death, suggests that males are more susceptible to long-term cognitive effects and this outcome may not be exclusively attributed to neuronal death.']",Limitations include the inherent difficulties in comparing subjects with species and sex-specific differences in memory and the potential influence of factors like food restriction and hormone cycling on behavioral findings.,"['While a deficit was not identified in the female treatment subjects, this does not necessarily mean that females were entirely unaffected.', 'Other factors that may play a role in the cognitive outcomes of male and female subjects in the study include possible differences between sexes in their response to food restriction, activity patterns during the day, as well as hormone cycling.']",Potential applications include the need for further research into sex-specific neuroprotective strategies and the implications for pediatric anesthesia practices.,['Future studies exploring sex- specific outcomes will help elucidate these effects and lead to a better understanding of the roles of sex and hormones on brain development and protection.'],True,True,True,True,True,True,10.1016/j.neuropharm.2014.03.011
10.3892/etm.2017.5004,4315.0,Ling,2017,rats,postnatal day 7,Y,sevoflurane,none,sprague dawley,"3824

EXPERIMENTAL AND THERAPEUTIC MEDICINE 14: 3824-3830, 2017

Sevoflurane exposure in postnatal rats induced long‑term cognitive impairment through upregulating caspase‑3/cleaved‑poly (ADP‑ribose) polymerase pathway

YUNZHI LING1*, XIAOHONG LI1*, LI YU2, QISHENG LIANG1, XUEWU LIN1, XIAODI YANG3, HONGTAO WANG4 and YE ZHANG5

1Department of Anesthesiology, First Affiliated Hospital of Bengbu Medical College, Anhui, Hefei 233004; Departments of 2Laboratory Medicine, 3Parasitology, and 4Immunology, Bengbu Medical College, Anhui, Hefei 233030; 5Department of Anesthesiology, Second Affiliated Hospital of Anhui Medical University, Anhui, Hefei 230601, P.R. China

Received September 8, 2016; Accepted May 16, 2017

DOI: 10.3892/etm.2017.5004

Abstract. The association of anesthetic exposure in infants or young children with the long-term impairment of neuro- logic functions has been reported previously; however, the underlying mechanisms remain largely unknown. In order to identify dysregulated gene expression underlying long-term cognitive impairment caused by sevoflurane exposure at the postnatal stage, the present study initially performed behavioral tests on adult Wistar rats, which received 3% sevoflurane at postnatal day 7 (P7) for different time course. Subsequently, transcriptome profiling of hippocampal tissues from experimental and control rats was performed. Significant impairment of the working memory was observed in adult rats with sevoflurane exposure for 4‑6 h, when compared with the control rats. The results indicated that a total of 264 genes were aberrantly expressed (51 downregulated and 213 upregulated; fold change >2.0; P<0.05; false discovery rate <0.05) in the hippocampus of experimental adult rats compared with those from control rats. Particularly, the expression of caspase-3 gene (CASP3), encoding caspase‑3 protein, presented the most significant upregulation, which was further validated by quan- titative polymerase chain reaction and immunohistochemical analysis. Further analysis revealed that CASP3 expression

level was negatively correlated with the rats' spatial working memory performance, as indicated by the Y-maze test. The level of cleaved‑poly (ADP‑ribose) polymerase (PARP), a substrate of caspase-3, was also increased in the hippocampus of experimental adult rats. Thus, the present study revealed that upregulation of caspase-3/cleaved-PARP may be involved in long-term cognitive impairment caused by sevoflurane exposure in infants, which may be useful for the clinical prevention of cognitive impairment.

Introduction

Although anesthesia can protect patients undergoing surgical procedures from significant pain, recent studies have demonstrated that exposure to inhaled anesthetics, such as sevoflurane and isoflurane, induced neuropathological altera- tions in experimental animals (1,2). In addition, several lines of evidence from animal models have suggested that excessive exposure to volatile anesthetics may cause irreversible and long-term behavioral changes resembling autism spectrum disorders (3). In clinical practice, sevoflurane is the most widely used inhaled anesthetics, particularly for cesarean section and surgical procedures in infants and young children (4,5); thus, the present study focused on sevoflurane.

Correspondence to: Dr Li Yu, Department of Laboratory Medicine, Bengbu Medical College, 2600 East Sea Road, Anhui, Hefei 233030, P.R. China E‑mail: yuli95@163.com

Dr Ye Zhang, Department of Anesthesiology, Second Affiliated Hospital of Anhui Medical University, 678 Furong Road, Anhui, Hefei 230601, P.R. China E‑mail: zhang_ye011@163.com

Contributed equally

In humans, the association of anesthetic exposure in infants or young children with the long-term impairment of neurologic functions has been reported in several retrospective clinical studies. For instance, Kalkman et al (6) observed that chil- dren with exposure to anesthetic agents at an age <24 months presented more behavioral disturbances in comparison with those who were exposed to anesthetics after the age of 2 years. Similarly, Wilder et al (7) demonstrated that children receiving two or more types of anesthetics at the same time were at an increased risk of developing learning disabilities, with a hazard ratio of 1.59, subsequent to retrospectively analyzing a cohort of 5,357 children.

Key words: sevoflurane, caspase‑3, cleaved‑poly (ADP‑ribose) polymerase

long-term cognitive

impairment,

To date, numerous dysregulated biological processes have been identified for underpinning the pathologic basis of neurologic function impairment caused by sevoflurane expo- sure. For instance, Zhang et al (8) reported that inhalation of

LING et al: POSTNATAL SEVOFLURANE EXPOSURE AND LONG-TERM COGNITIVE IMPAIRMENT

sevoflurane may reduce synaptotagmin‑1 protein levels in the hippocampus, which then reduced the efficiency of synaptic transmission, thus resulting in memory impairment in rats. In one of our previous studies, decreased PSD95 expression in the medial prefrontal cortex was also observed in mice with cognitive impairment induced by sevoflurane (9). Furthermore, Xiong et al (10) reported that the CREB signaling pathway was inhibited in aged rats following exposure to sevoflurane. However, it is unknown whether these dysregulated biological processes are implicated in sevoflurane‑induced long‑term neurologic functional impairment. Systematic investiga- tion of the aberrantly expressed genes in animals exhibiting neurological dysfunctions due to exposure to sevoflurane at postnatal age is also lacking.

The hippocampus is a major component of the central nervous system, which is located inside the medial temporal lobe. The hippocampus structure contains two main parts, namely Ammon's horn and the dentate gyrus, as well as four relatively independent parts, including cornu ammonis 1 (CA1), CA2, CA3 and CA4. The hippocampus serves a crucial role in regulating the cognitive function (11). A previous study demonstrated that the hippocampus is vulnerable to adverse events, including hypoxemia, exposure to inhaled anesthetics and surgical trauma (12). Evidence from magnetic resonance imaging studies indicated that decreased hippocampal volume is significantly associated with mild cognitive impairment in various neuropsychiatric diseases (13,14).

nitrogen at a flow rate of 10 l/min. Next, the rats were exposed to 3% sevoflurane for the specified time period at a rate of 1.5 l/min. During the anesthesia process, the concentrations of sevoflurane, carbon dioxide and oxygen in the gas mixture were monitored with an anesthetic gas monitor (Datex‑Ohmeda S/5; GE Healthcare Life Sciences, Chicago, IL, USA). Rats were breathing spontaneously during anesthesia. Anesthesia was ended by discontinuing the anesthetics, and then rats were housed in normal conditions until 12 weeks old, at which time behavioral tests were performed.

Behavioral experiments. As described in our previous study (9), three tests were conducted in sequence, including the elevated plus‑maze (EPM), O‑maze and Y‑maze. For each behavioral test, the movement tracks of experimental rats were recorded by a video‑tracking software (Any‑Maze version 5.1; Stoelting Co., Wood Dale, IL, USA) and analyzed by an additional researcher who was blinded to the experi- mental protocols. All the test were performed during the dark phase (active period of rats) between 1 a.m. and 4 p.m. The experimental details of EPM test, O-maze and Y-maze were as described in previous studies (9,17‑19). Briefly, the EPM and O-maze tests were used to assess the anxiety-like behavior in rodents, while the Y-maze test was used to investigate the immediate spatial working memory (a pattern of manifestation of cognitive function) of rodents.

In the present study, it was first investigated whether expo- sure to sevoflurane at a postnatal age caused later behavioral alterations in Wistar rats. Next, gene profiling was performed in hippocampus samples from Wistar rats with or without sevoflurane exposure during developmental stages. The study also attempted to explore the underlying molecular mecha- nisms of long-term neural impairment caused by exposure to sevoflurane.

Materials and methods

Animals. According to previous observations (15), a total fo 49, male Wistar rats (14.54±1.52 g) at postnatal day 7 (P7) were selected for experimental analyses. The Wistar rats at P7 were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). Rats were housed in poly- propylene cages under a 12-h alternating light/dark cycle, with food and water supplied ad libitum in the institutional animal facilities. All the experimental protocols were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Bengbu Medical College (Anhui, China) and performed according to the Guide for the Care and Use of Laboratory Animals (16). All efforts were made to minimize animal suffering and to reduce the number of animals used.

Anesthesia methods. A total of 48 Wistar rats at P7 were randomly divided into four groups (n=12), including the 0, 2, 4 and 6‑h treatment group, in which Wistar rats were exposed to 3% sevoflurane for 0, 2, 4 and 6 h, respectively. For anes- thesia, Wistar rats were placed in a temperature-controlled (37±0.5˚C) plexiglas anesthesia chamber. First, the rats were subject to 5% sevoflurane exposure for 30 sec, provided in a gas mixture of 5% carbon dioxide, 21% oxygen and balanced

Gene expression microarray analysis. Rats were anaes- thetized by isoflurane with an induction dosage of 4%, maintained at 2% (RuiTaibio, Beijing, China) and decapi- tated to obtain the hippocampus, which was then stored at ‑80˚C until RNA extraction. Total RNA was extracted from the hippocampal tissues using a standard TRIzol reagent (catalogue no. 15596026; Thermo Fisher Scientific, Inc., Waltham, MA, USA) as described previously (9). In brief, after the tissue was homogenized, 0.3 ml TRIzol was added to each sample. Then, 0.3 ml 100% chloroform (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) was added to stratify the sample solution; transfer the aqueous phase containing the RNA to a new tube. Finally, 0.5 ml isopro- panol was added to the aqueous phase to precipitate the total RNA. The quality and concentration of the RNA samples were then assessed at he absorbance ratios of A260/280 and A260/230 using a NanoDrop ND‑1000 spectrophotometer (Thermo Fisher Scientific, Inc.), and samples were denatured by 2% agarose gel electrophoresis.

The whole transcription profile of all mRNAs targeted by the microarray for each sample was determined using an Affymetrix Rat Genome U34 Array (Thermo Fisher Scientific, Inc.). Sample labeling and array hybridization were performed according to the manufacturer's instructions with minor modifications. In order to analyze the gene expres- sion, CapitalBio Corporation (Beijing, China) completed the following steps: Briefly, mRNA was purified from total RNA following the removal of rRNA using mRNA-ONLY™ Eukaryotic mRNA Isolation kit (Epicentre, Madison, WI, USA). Next, each sample was amplified and transcribed into fluorescent cDNA along the entire length of the transcripts without 3' bias using random primers (catalogue no. 79236; Qiagen, Hilden, Germany). Subsequent to purification with

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an RNeasy Mini kit (Qiagen, Hilden, Germany), the labeled cDNAs were hybridized with the specific probes on the Array. The hybridized arrays were washed, fixed and scanned at 5 mm/pixel resolutions with an Agilent DNA microarray scanner (G2505C; Agilent Technologies, Inc., Santa Clara, CA, USA).

Upon collection of signal, technical quality control was performed using dChip version 2005 (Affymetrix; Thermo Fisher Scientific, Inc.) with the default settings. Expression data were normalized by quantile normalization and the robust multichip average algorithm, as previously described (20). Probe-level files were generated following normalization. According to the fold change (FC) analysis (FC >2.0) and false discovery rate (FDR) analysis (FDR <0.05), differen- tially expressed genes were identified through FC filtering according to the predetermined P‑value threshold for signifi- cant differences (set at P<0.05).

saline, incubated for at least 48 h in 4% paraformaldehyde (Sigma‑Aldrich; Merck, Darmstadt, Germany) and embedded in paraffin. Next, the paraffin-embedded tissues were sectioned into 4‑µm slices, and sections with the hippo- campus structure were used for immunohistochemical analyses. Slices were incubated with rabbit anti-caspase-3 primary antibody (Catalogue no. AC030; Beyotime Institute of Biotechnology; 1:200) at 4˚C overnight, then incubated with biotinylated anti‑rabbit secondary antibody (catalogue no. A0277; Beyotime Institute of Biotechnology; 1:1,000) for 30 min at 37˚C, and immunoreactivity was then visu- alized by addition of a streptavidin-peroxidase complex and 3,3'‑diaminobenzidine (both from Beyotime Institute of Biotechnology). Counterstaining was performed with hematoxylin (Zhongshan Golden Bridge, Beijing, China). Subsequent to each incubation step, slices were washed with Tris‑buffered saline/Tween 20 three times for 5 min each. All images were captured using an Axioskop fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).

Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Total RNA was reversely transcribed into cDNA using a PrimeScript RT Reagent kit with gDNA Eraser (Takara Biotechnology Co., Ltd., Dalian, China) following the manufacturer's instructions, as previously described (21). Next, qPCR was performed using a SYBR Green PCR kit (Takara Biotechnology Co., Ltd.) on a CFX96 Real‑Time PCR Detection System (Bio‑Rad Laboratories, Inc., Hercules, CA, USA). The PCR conditions included an initial step at 95˚C for 5 min, followed by 40 cycles of annealing and extension, prior to quantification at 95˚C for 15 sec and 60˚C for 30 sec. Each cDNA sample was analyzed in triplicate, in a final volume of 25 µl, containing 1 µl cDNA, 400 nM of the forward and reverse gene‑specific primers (1 µl), 12.5 µl 2x SYBR Green master mix (catalogue no. 639676; Takara Biotechnology Co., Ltd.) and 10.5 µl distilled water. The relative gene expression level was quantified based on the cycle threshold values (22) and normalized to the reference gene, which was glyceraldehyde 3‑phosphate dehydrogenase (GAPDH). Primer sequences used were listed as follows: KIF2A: F 5'‑ATT TTC TCT CAT TGA CCT GGC TG‑3', R 5'‑ACT CCT TGA GTG CTA AAA GGC‑3'; RYBP: F 5'‑CGA CCA GGC CAA AAA GAC AAG‑3', R 5'‑CAC ATC GCA GAT GCT GCA T-3'; DOCK7: F 5'‑CCA TCT GGA AGC GCC TTT G‑3', R 5'‑ACG ATG ATC TCT AGC GTG TCT‑3'; CDC40: F 5'‑CTC TAG CTG CTT CGT ATG GCT‑3', R 5'‑CAA GTG CAT GAG AGA GTC CGC-3'; PTBP3: F 5'‑CCA GCC ATT GGA TTT CCT CAA‑3', R 5'‑AAA AAG CCC ATG TGG TGT GAT A-3'; NRTN: F 5'‑GGG CTA CAC GTC GGA TGA G‑3', R 5'‑CCA GGT CGT AGA TGC GGA TG‑3'; TMEM205: F 5'‑CAC TTG CTG GTC TTG TCT GGT‑3', R 5'‑GGA GAC GTG AAA ATA GAC TGG G-3'; SLC39A3: F 5'‑GGT GGC GTA TTC CTG GCT AC‑3', R 5'‑CTG CTC CAC GAA CAC AGT GA‑3'; CDCA3: F 5'‑GAG TAG CAG ACC CTC GTT CAC‑3', R 5'‑TCT CTA CCT GAA TAG GAG TGC G-3'; KIF1C: F 5'‑AGT GTG GGT TTG TGT GTA TGA G‑3', R 5'‑CCA GCA TCG CAC CAT GTA GA‑3'; CAS P3: F 5'‑ATG GAG AAC AAC AAA ACC TCA GT‑3', R 5'‑TTG CTC CCA TGT ATG GTC TTT AC‑3'; GAP DH: F 5'‑AGG TCG GTG TGA ACG GAT TTG 3', R 5'‑TGT AGA CCA TGT AGT TGA GGT CA-3'.

Immunohistochemical assay. Following sacrifice, the entire rat brain was rapidly removed, washed with phosphate-buffered

Western blotting. The preparation of hippocampal tissues for protein extraction was performed as described in previous studies (23,24). Briefly, total proteins were extracted using the radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). The hippocampus tissue proteins were separated by 10% sodium dodecyl sulfate-polyacryl- amide gel electrophoresis and then electrotransferred to a nitrocellulose membrane. After blocking with 5% non‑fat milk for 1 h at room temperature and being washed three times (10 min each) using 1x TBST, the membrane was incubated with primary antibodies at 4˚C overnight and then with horseradish peroxidase-conjugated secondary antibody (Beyotime Institute of Biotechnology) at room tempera- ture for 2 h. The primary antibodies used were as follows: Rabbit anti‑cleaved‑poly (ADP‑ribose) polymerase (PARP) antibody (catalogue no. AP102; 1:200) and rat anti‑GAPDH antibody (catalogue no. AG019; 1:5,000; both from Beyotime Institute of Biotechnology). Subsequently, the target proteins were visualized by an enhanced chemiluminescence method (catalogue no. W1001, Promega Corporation, Madison, WI, USA) and analyzed with the Gel Image Documentation System (Wealtec Corp., Sparks, NV, USA). The relative level of PARP was normalized to that of GAPDH, as presented by band intensity.

Statistical analysis. All data are presented as the mean ± standard error of the mean, and all statistical analyses were conducted using GraphPad Prism (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) and SPSS (version 17.0; SPSS, Inc., Chicago, IL, USA) software. For behavioral tests and RT‑qPCR results, one‑way analysis of variance (ANOVA) was applied to compare intergroup differences with Bonferroni post hoc tests. The correlation analysis was performed using Pearson's correlation coefficients. For western blotting and immunohistochemistry results, two-tailed Student's t-test was applied for comparison. A P‑value of <0.05 was considered to indicate differences that were statistically significant. Any additional experimental data not provided in the current study are indicated by ‘data not shown’ and can be obtained upon request.

LING et al: POSTNATAL SEVOFLURANE EXPOSURE AND LONG-TERM COGNITIVE IMPAIRMENT

Results

Sevoflurane exposure at P7 causes long‑term spatial working memory impairment in adult rats. In order to investigate the long‑term effect of sevoflurane exposure on cognitive func- tions, the infant rats at P7 were exposed to 3% sevoflurane for different time durations (0, 2, 4 or 6 h), and behavioral tests were then performed when the rats were 12-weeks-old. As shown in Fig. 1A, there was no significant difference in the exploration time, manifested as the time spent in the open arms of the maze, in the EPM test between rats receiving 3% sevoflurane exposure and those without exposure (one‑way ANOVA, P>0.05 for each comparison with the 0 h group). Similarly, there was also no difference in the total activity, which was demonstrated by the total number of arm entries, among groups receiving 3% sevoflurane exposure for different time lengths (one‑way ANOVA, P>0.05 vs. the 0 h group; Fig. 1B). Furthermore, in the O‑maze test, no difference was observed in the latency to enter the anxiety-associated bright compartment and in the total time spent in the open sector (one‑way ANOVA, P>0.05; Fig. 1C and D).

As for the Y-maze test, in which rats had to select between two lanes when facing the Y-shaped track, rats receiving 3% sevoflurane exposure during the early postnatal stage for 4 and 6 h exhibited significantly reduced spontaneous alterations in arm entries when compared with those without sevoflurane exposure (Fig. 1E; one‑way ANOVA, P<0.05). By contrast, there was no significant difference between rats with 3% sevo- flurane exposure for 2 h and control rats with 0‑h exposure (P=0.594). In addition, it was observed that longer time of sevoflurane exposure may induce more severe spatial working memory impairment, but the difference was not statistically significant (6 h vs. 4 h groups). However, the number of arm entries presented no significant difference among groups, suggesting that the spatial working memory impairment may not be due to weakened movements (P>0.05 vs. the 0 h group; Fig. 1F).

Gene profiling is dysregulated in the hippocampus of adult rats exposed to sevoflurane at P7. As shown in Fig. 1E, expo- sure to 3% sevoflurane for 6 h at P7 caused the most severe spatial working memory impairment in aged rats; therefore, rats from this group were selected for the following analyses. In order to explore the molecular pathways underlying the sevoflurane-induced long-term spatial working memory impairment, transcriptome analysis of the hippocampus, the major structure participating in the regulation of cognitive functions, was first performed in adult rats from the 6‑h group (n=3). Meanwhile, normal rats (n=3) that did not receive 3% sevoflurane exposure were used as the controls.

A total of 7,127 probes were detected, among which 264 genes demonstrated significantly different expression levels between rats from the 6 h group and the controls (fold change >2.0, P<0.05 and FDR <0.05; data not shown). Among the dysregulated 264 genes, 51 were downregulated and 213 were upregulated. In order to validate the accuracy of mRNA array, a total of 10 dysregulated mRNAs, including 5 upregulated genes, namely KIF2A, RYBP, DOCK7, CDC40 and PTBP3, and 5 downregulated genes, namely NRTN, TMEM205, SLC39A3, CDCA3 and KIF1C, were randomly selected for

Figure 1. Behavioral tests of adult rats exposed to 3% sevoflurane for 0, 2, 4 and 6 h at postnatal day 7. (A) The time spent in the open arms of the maze (proportion) and (B) number of total arm entries were investigated by the EPM test. (C) The latency to enter the open arms (sec) and (D) time spent in the open arms (sec) were examined in O‑maze test. (E) Spontaneous alternations (%), and (F) number of arms visited per minute were analyzed by Y‑maze test. No significant differences were observed in the EPM and O‑maze tests between rats with and without 3% sevoflurane exposure. In the Y‑maze test, exposure to 3% sevoflurane for 4 and 6 h led to decreased spontaneous alternations, but did not disturb the number of arms visited/min. Data are expressed as the mean ± standard deviation (n=12 per group; one‑way analysis of variance and Bonferroni's post hoc test). *P<0.05 and **P<0.01 vs. the 0 h group. n.s., not significant difference with 0 h group; EPM, elevated plus-maze.

further verification using RT‑qPCR. All the 10 selected genes presented consistent expression levels in terms of the regula- tory direction with the results extracted from mRNA array (data not shown).

Increased caspase‑3 gene (CASP3) expression is correlated with long‑term spatial working memory performance of rats exposed to sevoflurane at P7. Specifically, the expression of CASP3 (encoding caspase‑3 protein) was observed to be the most significantly upregulated gene from microarray data obtained in the current study (FC=2.53). This gene has previ- ously been implicated in the impairment due to exposure to (25,26). Therefore, the current study selected this gene for subsequent analysis. Firstly, RT-qPCR was performed to validate whether CASP3 was indeed upregulated in the hippocampus of experimental rats when compared with healthy rats. As shown in Fig. 2A, CASP3 expression level

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Figure 2. Expression of caspase‑3 was upregulated in the hippocampus of adult rats exposed to 3% sevoflurane for 6 h at P7 compared with control rats without exposure. (A) Quantitative polymerase chain reaction of CASP3 expression in the hippocampus of adult rats. Immunohistochemical staining of caspase-3 in the hippocampus of adult rats without 3% sevoflurane exposure is shown at (B) x4 and (C) x20 magnification. Immunohistochemical staining of caspase‑3 in the hippocampus of adult rats exposed to 3% sevoflurane for 6 h at postnatal day 7 is demonstrated at (D) x4 and (E) x20 magnification. Scale bar, 50 (at x4 magnification) or 100 µm (at x20 magnification). Caspase‑3 positive cells were indicated by arrows. (F) Statistical analysis of caspase‑3 positive cells in the hippocampus of adult rats. Data are expressed as the mean ± standard error (n=3 per group; two‑tailed Student's t‑test). ***P<0.001 vs. control (0 h) group. CASP3, caspase-3 gene.

in the hippocampus of rats with exposure to sevoflurane was 1.97 times higher than those without sevoflurane exposure (P<0.001). Immunohistochemical analysis of the hippocampal tissue sections also validated this observation. As shown in Fig. 2B‑F, the number of caspase‑3 positive cells in the hippocampus of experimental rats was approximately 4 times higher in comparison with those in healthy rats (P<0.001).

Since the spatial working memory was impaired more severely in rats exposed to sevoflurane for 6 h as compared with rats exposed for 2 and 4 h, or compared with the control group, it was speculated that the alteration of spatial working memory performance may partly be due to the fluctuated CASP3 expression. Supporting our hypothesis, further analysis revealed that the CASP3 mRNA level was negatively correlated with the spatial working memory in experimental rats (n=36; r2=0.279, P=0.043; Fig. 3). Insufficient negative association between CASP3 mRNA level and spatial working memory performance in experimental rats suggested that genes than other CASP3 may also be involved in the dysregulation of spatial working memory impairment.

Cleaved‑PARP level is increased in the hippocampus of adult rats exposed to sevoflurane at P7. As a substrate of caspase-3, PARP has been demonstrated to also be associ- ated with long-term memory impairment. Therefore, the present study examined whether PARP was cleaved by caspase-3 in experimental rats by western blotting. As shown in Fig. 4, the relative level of cleaved‑PARP was significantly

Figure 3. CASP3 expression was negatively correlated with spatial working memory in adult rats with exposure to 3% sevoflurane at postnatal day 7. CASP3, caspase-3 gene.

increased in adult rats with exposure to sevoflurane for 6 h at P7, when compared with those without sevoflurane exposu re ( P= 0. 0 02), f u r t her con f i r m i ng t hat t he caspase-3/PARP pathway may be involved in long-term memory impairment.

Discussion

In the present study, the long-term impairment effect on cogni- tive functions of sevoflurane exposure at the postnatal stage was validated through in vivo animal experiments, which is

LING et al: POSTNATAL SEVOFLURANE EXPOSURE AND LONG-TERM COGNITIVE IMPAIRMENT

Figure 4. Protein level of cleaved‑PARP was upregulated in the hippocampus of rats receiving 3% sevoflurane at postnatal day 7 compared with the control rats. (A) Images of western blotting; (B) statistical analysis of the relative level of cleaved‑PARP in each group. **P<0.01 vs. control (0 h) group (two‑tailed Student's t‑test). PARP, poly (ADP‑ribose) polymerase; P7, postnatal day 7.

in accordance to the observations of previous studies (6,15). In particular, significant spatial working memory impairment was detected in adult rats exposed to 3% sevoflurane for 6 h at P7, while rats receiving 3% sevoflurane exposure for 2 h or without exposure did not present impairment. However, the underlying molecular pathways of the long-term cognitive impairment induced by 3% sevoflurane exposure in postnatal rats remain unclear.

Supporting the results of the present study, several previous studies have indicated that exposure to sevoflurane for 6 h significantly upregulated the expression of caspase‑3 in the hippocampus of postnatal rats (27), while upregulation of caspase-3 may lead to memory and cognitive impairment though mediating neural apoptosis in the hippocampus. As a substrate of caspase-3, PARP can be cleaved by caspase-3, and increased cleaved-PARP levels have been observed to be asso- ciated with long‑term memory impairment (28). As such, the expression data of caspase-3 and PARP suggest that apoptosis serves an important role in the cognitive impairment caused by exposure to 3% sevoflurane in postnatal rats. Apoptosis is a genetically controlled mechanism of cell death involved in the regulation of tissue homeostasis. Typically, B‑cell lymphoma‑2 (Bcl‑2) and caspase‑3 are the main factors inhibiting apoptosis and the final executive protein in apoptosis, respectively, while Bcl‑2‑associated X protein may induce apoptosis by increasing the mitochondrial membrane permeability (29). However, other molecular pathways also underlie the neurological damage caused by exposure to anesthetics. One such example is the Rho/Rho-kinase pathway. According to the study by Lemkuil et al (30), isoflurane induced neurotoxicity in rats by activating p75NTR-RhoA, while inhibition of the activity of RhoA reversed the neurotoxicity of isoflurane. In addition, Pearn et al (31) reported that p75NTR and RhoA kinase activa- tion were involved in propofol-induced apoptosis in developing neurons in vitro and in vivo. The present study added novel results regarding the roles of caspase-3 and cleaved-PARP in sevoflurane exposure‑induced cognitive impairment, and highlighted the importance of caspase-3 and cleaved PARP in the pathogenesis of nervous system dysfunction.

sevoflurane exposure for <2 h did not have an evident effect. Consistent with our results, Han et al (15) reported that expo- sure to 2% sevoflurane at postnatal days 7‑9 (2 h per day) may induce significantly impaired cognitive function in rats reaching adulthood, whereas receiving 2% sevoflurane for 2 h for only 1 day (at P7) did not result in impaired cognitive function in adult rats. Shen et al (32) also identified that sevoflurane exerted a dose‑dependent neurotoxicity on the neurons. More specifi- cally, rats in the developmental stages (P7) did not present cognitive impairment and nerve inflammation due to exposure to 3% sevoflurane for 2 h at one time, while those inhaling 3% sevoflurane for 2 h per day for 3 consecutive days exhibited marked impairment of cognition. Furthermore, Peng et al (33) revealed that low concentration of sevoflurane (1.5%) may not cause significant cognitive impairment even with exposure for as long as 3 days (2 h per day), while high concentration of sevoflurane (3%) for only 2 h was sufficient to induce severe cognitive impairment. Therefore, the present study, along with other similar studies, suggested that minimizing the use dosage and concentration of anesthetics, particularly sevoflurane, in clinical practice may greatly reduce the neurological damage.

Several limitations in the current study need to be acknowledged. Firstly, due to limitation of financial support, gene profiling analyses were only performed on rats from the 6 h experimental and control groups, but not on those receiving sevoflurane exposure for 2 or 4 h. Since the degree of cognitive impairment varies among different experimental groups, it may be meaningful to analyze the difference of gene profiling among groups. In addition, the apoptosis of neurons from the hippocampus of experimental and control groups was not detected. Since caspase-3 upregulation has been widely reported to be associated with apoptosis, the apoptosis of neurons from the hippocampus would be more severe in experimental groups, and even exacerbated along with the time of exposure to 3% sevoflurane; however, this requires further investigation.

Another interesting phenomenon observed in the current study is that 3% sevoflurane exposure for >4 h at P7 was able to impair the cognitive functions of adult rats, while 3%

In conclusion, the present study identified multiple genes, which may be involved in long-term cognitive impairment caused by exposure to sevoflurane. The upregulation of caspase-3/cleaved-PARP specifically may reveal a novel mechanism, which may be useful for the clinical prevention of cognitive impairment.

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EXPERIMENTAL AND THERAPEUTIC MEDICINE 14: 3824-3830, 2017

Acknowledgements

16. Barthold SW, Bayne K and Davis M: Washington: National Academy Press, 2011.

The current study was supported by the Anhui Education Department (grant no. KJ2015B004by) and the Bengbu Medical College Innovation Grant (grant no. BYKY1424ZD).

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29. Negrín G, Rubio S, Marrero MT, Quintana J, Eiroa JL, Triana J and Estévez F: The eudesmanolide tanapsin from Tanacetum oshanahanii and its acetate induce cell death in human tumor cells through a mechanism dependent on reactive oxygen species. Phytomedicine 22: 385‑393, 2015.

30. Lemkuil BP, Head BP, Pearn ML, Patel HH, Drummond JC and Patel PM: Isoflurane neurotoxicity is mediated by p75NTR‑RhoA activation and actin depolymerization. Anesthesiology 114: 49‑57, 2011.

31. Pearn ML, Hu Y, Niesman IR, Patel HH, Drummond JC, Roth DM, Akassoglou K, Patel PM and Head BP: Propofol neurotoxicity is mediated by p75 neurotrophin receptor activa- tion. Anesthesiology 116: 352‑361, 2012.

13. Apostolova LG, Green AE, Babakchanian S, Hwang KS, Chou YY, Toga AW and Thompson PM: Hippocampal atrophy and ventricular enlargement in normal aging, mild cognitive impairment (MCI), and Alzheimer Disease. Alzheimer Dis Assoc Disord 26: 17‑27, 2012.

14. Zhang J, Yu C, Jiang G, Liu W and Tong L: 3D texture analysis on MRI images of Alzheimer’s disease. Brain Imaging Behav 6: 61‑69, 2012.

32. 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.

33. Peng S, Zhang Y, Sun DP, Zhang DX, Fang Q and Li GJ: The effect of sevoflurane anesthesia on cognitive function and the expression of Insulin-like Growth Factor-1 in CA1 region of hippocampus in old rats. Mol Biol Rep 38: 1195‑1199, 2011.

15. Han T, Hu Z, Tang Y, Shrestha A, Ouyang W and Liao Q: Inhibiting Rho kinase 2 reduces memory dysfunction in adult rats exposed to sevoflurane at postnatal days 7‑9. Biomed Rep 3: 361‑364, 2015.",rats,"['In order to investigate the long‑term effect of sevoflurane exposure on cognitive func- tions, the infant rats at P7 were exposed to 3% sevoflurane for different time durations (0, 2, 4 or 6 h), and behavioral tests were then performed when the rats were 12-weeks-old.']",postnatal day 7,"['In order to investigate the long‑term effect of sevoflurane exposure on cognitive func- tions, the infant rats at P7 were exposed to 3% sevoflurane for different time durations (0, 2, 4 or 6 h), and behavioral tests were then performed when the rats were 12-weeks-old.']",Y,"['Behavioral experiments. As described in our previous study (9), three tests were conducted in sequence, including the elevated plus‑maze (EPM), O‑maze and Y‑maze.']",sevoflurane,"['In order to investigate the long‑term effect of sevoflurane exposure on cognitive func- tions, the infant rats at P7 were exposed to 3% sevoflurane for different time durations (0, 2, 4 or 6 h), and behavioral tests were then performed when the rats were 12-weeks-old.']",none,[],wistar,"['In order to investigate the long‑term effect of sevoflurane exposure on cognitive func- tions, the infant rats at P7 were exposed to 3% sevoflurane for different time durations (0, 2, 4 or 6 h), and behavioral tests were then performed when the rats were 12-weeks-old.']","The study addresses the underlying mechanisms of long-term cognitive impairment caused by sevoflurane exposure in infants, which remain largely unknown.","['The association of anesthetic exposure in infants or young children with the long-term impairment of neuro- logic functions has been reported previously; however, the underlying mechanisms remain largely unknown.']",None,[],"The study reveals that upregulation of caspase-3/cleaved-PARP may be involved in long-term cognitive impairment caused by sevoflurane exposure in infants, which may be useful for the clinical prevention of cognitive impairment.","['Thus, the present study revealed that upregulation of caspase-3/cleaved-PARP may be involved in long-term cognitive impairment caused by sevoflurane exposure in infants, which may be useful for the clinical prevention of cognitive impairment.']","Gene profiling analyses were only performed on rats from the 6 h experimental and control groups, not on those receiving sevoflurane exposure for 2 or 4 h. The apoptosis of neurons from the hippocampus of experimental and control groups was not detected.","['Firstly, due to limitation of financial support, gene profiling analyses were only performed on rats from the 6 h experimental and control groups, but not on those receiving sevoflurane exposure for 2 or 4 h.', 'In addition, the apoptosis of neurons from the hippocampus of experimental and control groups was not detected.']",None,[],True,True,True,True,True,False,10.3892/etm.2017.5004
10.1016/j.brainres.2014.02.008,618.0,Liu,2014,rats,postnatal day 7,N,sevoflurane,none,sprague dawley,"b r a i n r e s e a r c h 1 5 5 7 ( 2 0 1 4 ) 1 – 1 1

Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report Early exposure to sevoflurane inhibits Ca2þ activity in hippocampal CA1 pyramidal neurons of developing rats

channels

Aili Liua,b, Yize Lic,d, Tao Tane, Xin Tiana,b,f,n

aSchool of Biomedical Engineering, Tianjin Medical University, Tianjin 300070, China bLaboratory of Neurobiology in Medicine, Research Center of Basic Medical Science, Tianjin Medical University, Tianjin 300070, China cDepartment of Anesthesiology, Tianjin Medical University General Hospital, Tianjin 300070, China dTianjin Research Institute of Anesthesiology, Tianjin 300070, China eMinistry of Education Key Laboratory of Child Development and Disorders, and Chongqing Key Laboratory of Translational Medical Research in Cognitive Development and Learning and Memory Disorders, Children's Hospital of Chongqing Medical University, Chongqing 400014, China fTianjin Neurological Institute, Tianjin 300070, China

a r t i c l e i n f o

a b s t r a c t

Article history:

Accepted 4 February 2014

Available online 8 February 2014

Keywords: Sevoflurane Inhibit Activity of Ca2þ The period of rapid brain

channels

Sevoflurane is one of inhalation anesthetics and has been commonly used in obstetric and pediatric anesthesia. The widespread use of sevoflurane in newborns and infants has made its safety a health issue of concern. Voltage-gated Ca2þ channels (VGCCs) play an important role in neuronal excitability and are essential for normal brain development. However, the role of sevoflurane on regulating Ca2þ brain development is still not well understood.

channels during the period of rapid

The aim of this study is to explore the effects of sevoflurane on voltage-gated Ca2þ channels for hippocampal CA1 pyramidal neurons during the period of rapid brain

development

development. 1-week-old Sprague-Dawley rats were randomly divided into 3 groups: control group, 2.1% sevoflurane group (exposed to 2.1% sevoflurane for 6 h) and 3% sevoflurane group (exposed to 3% sevoflurane for 6 h). Whole-cell patch clamp technique was used. I–V curve, steady-state activation and inactivation curves of Ca2þ channels were studied in rats of the both 3 treated groups at 5 different ages (1 week, 2 weeks, 3 weeks,

4 and 5 weeks old).

After anesthesia with sevoflurane at 1-week-old rats, Ca2þ

channels current density was significantly decreased at week 1 and week 2 (po0.01). And 3% sevoflurane exposure resulted in a rightward shift in steady-state activation curve at week 1 and week 2, as well as the inactivation curve from week 1 to week 3. However, the 2.1% sevoflurane-induced

Abbreviations: HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EGTA, ethylene glycol tetraacetic acid; 4-AP,

4-aminopyridine; TEA-Cl, Newman–Keuls

tetrathylammonium chlorine; TTX,

tetrodotoxin; ATP, adenosine 50-triphosphate; SNK, Student–

n Corresponding author at: School of Biomedical Engineering, Tianjin Medical University, Tianjin 300070, China. Fax: þ86 22 83336951. E-mail addresses: liuzitong.aili@163.com (A. Liu), liyizelisa@126.com (Y. Li), tantao_tijmu@126.com (T. Tan),

tianx@tijmu.edu.cn (X. Tian).

http://dx.doi.org/10.1016/j.brainres.2014.02.008 0006-8993 & 2014 Elsevier B.V. All rights reserved.

2

b r a i n r e s e a r c h 1 5 5 7 ( 2 0 1 4 ) 1 – 1 1

rightward shift was only found in steady-state inactivation curve of Ca2þ 1 and week 2. Both the slope factor (k) of Ca2þ increased by 3% sevoflurane at week 1 (po0.05).

channels at week

channels activation and inactivation curves

Therefore, early exposure to sevoflurane persistently inhibits Ca2þ

channels activity in hippocampal CA1 pyramidal neurons of developing rats but the development of Ca2þ level at juvenile age. Moreover, the inhibition of 3% channels recovers to normal sevoflurane on VGCCs is greater than that of 2.1% sevoflurane.

& 2014 Elsevier B.V. All rights reserved.

1.

Introduction

As one of commonly used inhalation anesthetics, sevoflurane has been widely used in obstetric and pediatric anesthesia for its hemodynamic stability, short-lasting action and excellent respiratory tolerance (Edwards et al., 2010; Gibert et al., 2012). However, there may be some side effects caused by sevoflur- ane. The epileptiform EEG and seizure activity have been observed during sevoflurane anesthesia in children (Conreux et al., 2001; Constant et al., 2005; Vakkuri et al., 2001). And a previous study suggested that sevoflurane would block human motional memory (Alkire et al., 2008). Several studies have reported that sevoflurane induces social behavior abnormality and learning deficits in mice (Jevtovic-Todorovic et al., 2003; Satomoto et al., 2009). Meanwhile, increasing evidence indi- cates that sevoflurane exposure leads to widespread neuroa- poptosis and cognitive impairment on immature brain (Kodama et al., 2011; Istaphanous et al., 2011; Shen et al., 2013). This has raised serious concerns among anaesthesiolo- gists, neuroscientists and parents regarding the safety of sevoflurane on brain development.

et al., 2012; Gong et al., 2012; Rithalia et al., 2004). However, the role of sevoflurane on regulating VGCCs during the period of rapid brain development is still not well understood.

To explore the critical period of development, we first recorded voltage-gated calcium currents in hippocampal CA1 pyramidal neurons by using whole-cell patch clamp techni- que. To determine whether the sevoflurane-induced persis- tent inhibition on VGCCs would occur during development, we further assessed the effects of 6-h 3% sevoflurane expo- sure on I–V curve, steady-state activation and inactivation curves of VGCCs. Finally, we investigated whether the inhibi- tion of sevoflurane on VGCCs was concentration-dependent by evaluating the properties of VGCCs after a lower concen- tration (2.1%) of sevoflurane administration with the same treatment time (6 h).

2.

Results

2.1.

Isolated hippocampal neurons

Developing brain is sensitive and vulnerable to general anesthetics (Liang et al., 2010). Sevoflurane might have harmful effects on the developing brain. Following numerous children investigations, Kalkman et al. (2009) has found out that early exposure to anesthetics before 2 years old can induce an increased risk of cognitive dysfunction in children. Moreover, extensive studies indicate that prolonged exposure to sevoflurane on neonatal rat results in widespread neuroa- poptosis and later spatial memory damage (Fang et al., 2012; Istaphanous et al., 2011; Wang et al., 2012). However, the mechanisms of sevoflurane-mediated neuronal death and cognitive impairment on immature brain are largely unknown. Hence, the effects of sevoflurane on developing brains need to be further explored.

Voltage-gated calcium channels (VGCCs) are involved in regulating Ca2þ signaling in brain (Norris et al., 2010) and play an important role in neurotransmitter release, synaptic plas- ticity, neuronal excitability and gene expression (Li et al., 2007; Zhang et al., 2009). The alteration in Ca2þ signaling influences neurogenesis, neurodegeneration and neurocognitive function (Berridge, 2011, 2010). Meanwhile, the modulation of Ca2þ channel activity has a key function on neurons survival and the viability of hippocampal neurons might be reduced by the dysfunction of Ca2þ channels (Li et al., 2007). Therefore, VGCCs are essential for normal brain development. Several studies have shown that VGCCs can be inhibited by sevoflurane (Eckle

The hippocampal pyramidal neurons in Fig. 1A with a smooth and bright appearance and no visible organelles were selected for recording. It was hard to perform whole-cell patch clamp recording on the cell with reduced viability, whose appearance was granular (Fig. 1B). The neurons adopted in control and sevoflurane groups were showed in Fig. 1C and D.

2.2. pyramidal neurons during the period of rapid brain development

Changes of VGCCs properties in hippocampal CA1

Control group were used to evaluate the changes of VGCCs properties in hippocampal CA1 pyramidal neurons during the period of rapid brain development. The inward slowly- inactivating Ca2þ current was evoked by 250 ms depolarizing voltage steps from (cid:2) 60 mV toþ40 mV in 10 mV increment. The Ca2þ currents can be blocked completely by 0.5 mmol/L CdCl2 (Fig. 2E).

The current–voltage (I–V) relationship curve of Ca2þ

current showed that potential-dependent Ca2þ current was activated from pulse potential of (cid:2) 40 mV and reached its maximal amplitude at approximatelyþ10 mV in control 1 w but 0 mV in others (Fig. 2A). The current density in control group was calculated to assess the changes in Ca2þ currents (Table 1). With the increases of rat age from 1 week to 5 weeks, the current density increased gradually and there was a significant

b r a i n r e s e a r c h 1 5 5 7 ( 2 0 1 4 ) 1 – 1 1

Fig. 1 – Isolated hippocampal neurons. (A) Healthy pyramidal neuron from rat hippocampus. (B) Neuron with reduced viability in the hippocampus. (C) Healthy pyramidal neuron from rats in control group. (D) Healthy pyramidal neuron from rats in sevoflurane group.

difference on the maximal current density between control 2 w and control 3 w groups (Po0.01) (Fig. 2B). The maximal current density in control 5 w was greater than that of control 4 w, but no significant difference was found between these two groups (P40.05). That indicates that the relatively rapid growth of VGCCs may occur between week 2 and week 3 in rat. To further investigate the VGCCs properties, we plotted the steady-state activation and inactivation curves fitted by Boltzmann equation. The significant leftward shift was showed in the steady-state activation and inactivation curves (Fig. 2C and D). Steady-state activation current of VGCCs was obtained by 250 ms depolarizing potentials from (cid:2) 60 mV to þ30 mV in 10 mV increment. The inset showed the traces of VGCCs steady-state activation current. The values of half- activation potential (V1/2) and activation slope factor (k) in control group can be seen in Table 2. The gradually reduced V1/2 imply that VGCCs activated at more hyperpolarized voltages when rats growing older. The V1/2 was significantly different between control 1 w and control 2 w groups (Po0.01), but no significant differences had been detected between control 2 w and control 3 w groups, control 3 w and control 4 w groups, as well as between control 4 w and control 5 w

groups (P40.05). The significant difference in k only existed between control 3 w and control 4 w groups (Po0.05).

Additionally, a 250 ms test pulse at þ10 mV was applied after 500 ms pre-pulse potential ranging from (cid:2)60 mV to þ30 mV in 10 mV increment to assess steady-state inactiva- tion parameters. The gradually reduced values of half- inactivation potential (V1/2) in control groups suggest that the VGCCs also inactivated at more hyperpolarized voltages with the increment of rat ages (Table 3). These differences in V1/2 between control 1 w and control 2 w groups, control 2 w and control 3 w groups, as well as control 3 w and control 4 w groups, control 4 w and control 5 w groups were not statisti- cally significant (P40.05). And there was no distinct variation tendency in inactivation slope factor (k).

Effects of sevoflurane on VGCCs in hippocampal CA1

2.3. pyramidal neurons during the period of rapid brain development

Moreover, we evaluated the I–V curve, steady-state activat- ion and inactivation curves of VGCCs in hippocampal CA1 pyramidal neurons after 2.1% and 3% sevoflurane exposure.

3

4

b r a i n r e s e a r c h 1 5 5 7 ( 2 0 1 4 ) 1 – 1 1

Fig. 2 – Changes on properties of VGCCs in rat hippocampal CA1 pyramidal neurons during the period of rapid brain development. (A) Average current–voltage (I–V) relationship curve of VGCCs in control group. Inset showed the depolarizing stimulation pulses of Ca2þ currents. (B) The amplitude of I–V relationship curves in 5 different ages. (C) Steady-state activation curve of VGCCs in control group. Insets showed the command steps and traces of steady-state activation currents. (D) Steady- state inactivation curve of VGCCs in control group. Insets showed the command steps and traces of steady-state inactivation currents. (E) Single stimulation pulses and traces of Ca2þ currents before and after the presence of 0.5 mmol/L CdCl2. All data represents as mean7SEM,

n Po0.01, n ¼ 10.

Rats were separated into 5 different age groups: group 1 w, group 2 w, group 3 w, group 4 w and group 5 w.

Sevoflurane decreases the amplitude of I–V curve for

2.3.1. VGCCs The peak amplitude of I–V curves appeared at the same in both control group and two membrane potential

sevoflurane-treated groups (Fig. 3A–E, þ10 mV in group 1 w, 0 mV in group 2 w, group 3 w, group 4 w and group 5 w). However, as compared with the control group, sevoflurane decreased the amplitude of I–V relationship curves of VGCCs at week 1, week 2 and week 3.

Table 1 shows the values of maximal current density in the both 3 treated groups. Statistical analysis showed

b r a i n r e s e a r c h 1 5 5 7 ( 2 0 1 4 ) 1 – 1 1

Table 1 – The maximal current density in each groupa.

Group (w)

Maximal current density (pA/pF)

Control

2.1% Sevo

3% Sevo

1 2 3 4 5

9.5670.63 10.8470.24 14.0870.93 15.0070.84 17.2270.59

n 6.8870.24 n 8.4270.22 14.0370.34 15.1170.87 17.2070.69

3.6870.55 6.2070.11 12.1770.34 14.7570.72 17.1570.66

a All data represents as mean7SEM. n Po0.01 vs control group, n ¼10.

Table 2 – The values of half-activation potential (V1/2) and activation slope factor (k) in each groupa.

Group (w)

V1/2 (mV)

k

Control

2.1% Sevo

3% Sevo

Control

2.1% Sevo

3% Sevo

1 2 3 4 5

(cid:2) 7.8970.48 (cid:2) 10.8470.44 (cid:2) 11.9070.44 (cid:2) 11.9770.39 (cid:2) 11.6370.66

(cid:2)6.5670.29 (cid:2)9.6670.52 (cid:2)11.8370.50 (cid:2)11.5870.48 (cid:2)11.3870.58

(cid:2)4.1470.52# (cid:2)9.0070.58# (cid:2)10.7970.56 (cid:2)11.497 0.59 (cid:2)11.3370.56

6.9670.43 6.3270.40 6.2670.39 5.3770.34 5.2670.59

6.9670.26 7.1870.47 5.8170.45 5.2670.43 5.7270.42

9.3870.48 7.4070.52 6.3270.50 5.8870.53 5.2870.47

a All data represents as mean7SEM. n

Po0.01 vs control group.

# Po0.05 vs control group, n ¼10.

Table 3 – The values of half-inactivation potential (V1/2) and inactivation slope factor (k) in each groupa.

Group (w)

V1/2 (mV)

k

Control

2.1% Sevo

3% Sevo

Control

2.1% Sevo

3% Sevo

1 2 3 4 5

(cid:2)15.6071.46 (cid:2)17.7471.24 (cid:2)18.7271.33 (cid:2)18.5271.36 (cid:2)18.1871.29

(cid:2)14.1871.98# (cid:2)15.6671.15# (cid:2)18.3471.69 (cid:2)18.3171.18 (cid:2)18.2771.78

(cid:2) 8.6571.80 (cid:2) 14.2371.46 (cid:2) 16.1970.94 (cid:2) 17.5571.12 (cid:2) 18.3171.85

n

n

n

10.2571.43 8.6571.15 9.4771.27 10.2071.32 10.2171.26

10.0871.93 8.6671.07 9.4371.61 10.1771.14 10.1371.73

14.6471.06 8.7271.30 9.8570.91 9.8271.04 10.3871.82

a All data represents as mean7SEM. n Po0.01 vs control group. # Po0.05 vs control group, n ¼10.

significant differences in the maximal current density between 2.1% sevoflurane and control groups, 3% sevoflurane and control groups, as well as 2.1% and 3% sevoflurane- treated groups at week 1 and week 2 (Po0.01) (Fig. 3F). Although the maximal current density in 3% sevoflurane 3 w was lower than that of control 3 w, 3% sevoflurane did not result in significant decrease in maximal current density at week 3 (P40.05).

–IS)/IC, IC is the current density of control group, IS is the current density of sevoflurane group) of 5 different age groups were calculated to express intui- tively the reduction of sevoflurane exposure on Ca2þ currents. Sevoflurane inhibited Ca2þ currents at week 1, week 2 and week 3 (Fig. 3G). And 3% sevoflurane induced a greater degree of inhibition on Ca2þ

The inhibition rates ((IC

currents than 2.1% sevoflurane.

Sevoflurane exposure delays the steady-state activation

2.3.2. and inactivation of VGCCs Sevoflurane exposure decreased the amplitude of voltage- gated Ca2þ channels activation currents as compared with the control group at week 1 and week 2 (Fig. 4). In addition, the half-activation potential (V1/2) of steady-state activation curve was significantly shifted to the more depolarized potential by 3% sevoflurane in group 1 w and group 2 w (Po0.05) (Table 2). And 3% sevoflurane caused significant increase in slope factor (k) at week 1 (Po0.01). But no statistical difference could be found between 2.1% sevoflur- ane and control groups (P40.05).

The amplitude of steady-state inactivation currents for VGCCs in sevoflurane exposure group were significantly different from control group at week 1, week 2 and week 3

n

n

n

n

5

6

b r a i n r e s e a r c h 1 5 5 7 ( 2 0 1 4 ) 1 – 1 1

Fig. 3 – Effects of sevoflurane (Sevo) on current–voltage (I–V) relationship curves of VGCCs in rat hippocampal CA1 pyramidal neurons during the period of rapid brain development. (A) I–V relationship curve of VGCCs in group 1 w. (B) I–V relationship curve in group 2 w. (C) I–V relationship curve in group 3 w. (D) I–V relationship curve in group 4 w. (E) I–V relationship curve in group 5 w. (F) The amplitude of I–V relationship curves for VGCCs in 5 different age groups. (G) The inhibition rates of sevoflurane on maximal current density. All data represents as mean7SEM,

n Po0.01, n ¼10.

b r a i n r e s e a r c h 1 5 5 7 ( 2 0 1 4 ) 1 – 1 1

Fig. 4 – Effects of sevoflurane (Sevo) on steady-state activation of VGCCs in rat hippocampal CA1 pyramidal neurons during the ¼1(cid:2) {1þexp period of rapid brain development. The activation curves fitting was constructed according to Boltzman equation: G/Gmax (cid:2) Vrev), I is the peak current at Vm, Vrev is the reversal potential.) is the activated conductance of [(Vm whole-cell channels at Vm, Gmax is the maximal activated conductance of whole-cell channels at different Vm, Vm is the depolarizing pulse potential, V1/2 is the half-activation potential, and k is the activation slope factor. (A) steady-state activation curve of VGCCs in group 1 w. Insets showed the traces of steady-state activation currents. (B) steady-state activation curve of VGCCs in group 2 w. (C) steady-state activation curve in group 3 w. (D) steady-state activation curve in group 4 w. (E) steady-state activation curve in group 5 w. All data represents as mean7SEM, n¼ 10.

(cid:2) 1, where G (G¼ I/(Vm

(cid:2) V1/2)/k]}

7

8

b r a i n r e s e a r c h 1 5 5 7 ( 2 0 1 4 ) 1 – 1 1

Fig. 5 – Effects of sevoflurane (Sevo) on steady-state inactivation of VGCCs in rat hippocampal CA1 pyramidal neurons during the period of rapid brain development. The inactivation curves fitting was constructed according to Boltzman equation: (cid:2) 1, where I is the peak current at Vm, Imax is the maximal peak current at different Vm, Vm is the I/Imax depolarizing pre-pulse potential, V1/2 is the half-inactivation potential, and k is the inactivation slope factor. (A) steady-state inactivation curve of VGCCs in group 1 w. Insets showed the traces of steady-state inactivation currents. (B) steady-state inactivation curve of VGCCs in group 2 w. (C) steady-state inactivation curve in group 3 w. (D) steady-state inactivation curve in group 4 w. (E) steady-state inactivation curve in group 5 w. All data represents as mean7SEM, n¼ 10.

¼ {1þexp[(Vm

(cid:2) V1/2)/k]}

b r a i n r e s e a r c h 1 5 5 7 ( 2 0 1 4 ) 1 – 1 1

(Fig. 5). Compared to control group, 3% sevoflurane-treated rats showed significant increases on half-inactivation poten- tial (V1/2) of steady-state inactivation curve in group 1 w, group 2 w and group 3 w (Po0.01) and inactivation slope factor (k) in group 1 w (Po0.01) (Table 3). There was signifi- cant shift in V1/2 to the more depolarized potential in group 1 w and group 2 w (Po0.05) after 2.1% sevoflurane adminis- tration without significant change in k (P40.05).

In all, 3% sevoflurane exposure induced a significant rightward shift in steady-state activation curve at week 1 and week 2, as well as the inactivation curve from week 1 to week 3. However, the 2.1% sevoflurane-induced rightward shift was only found in the steady-state inactivation curve of VGCCs at week 1 and week 2.

3.

Discussion

After birth, the brain of rats will grow rapidly to mature during the first two weeks of life (Loepke and Soriano, 2008). Meanwhile, In normal rat, our study demonstrated that the peak amplitude of maximal Ca2þ current increased gradually with a significant difference between week 2 and week 3, and reached to a dynamic stability at week 3, week 4 and week 5. Besides, there was significant leftward shift in the steady- state activation and inactivation curves from week 1 to week 3. These findings suggest that the critical period of develop- ment for VGCCs in normal rat may occur between week 2 and week 3.

hyperpolarization of the membrane and inhibition of neuro- nal excitability. That supports the hypothesis that general anesthetics may act through some special ion channels, which serve as possible cellular targets for general anes- thetics, to inhibit neuronal excitability (Brosnan and Thiesen, 2012; Eckle et al., 2012; Franks, 2008).

Although early exposure to sevoflurane delayed the nor- mal increase of voltage-gated Ca2þ currents, the steady-state activation and inactivation occurring at week 1, week 2 and week 3, the development of VGCCs recovered to normal level at week 4 and week 5. That finding consistent with several studies about effects of sevoflurane on developing rat or mice. Early exposure to sevoflurane results in neuroinflam- mation and neuronal apoptosis in brain tissue of neonatal rat and mice, but no abnormal neurobehavioral performances are detected at their juvenile age (Fang et al., 2012; Feng et al., 2012). Further studies are needed to explore the mechanism underlying sevoflurane-induced suppression on activity of VGCCs in hippocampal CA1 pyramidal neurons reduces gradually and disappears following the increases of rat age. In summary, this study demonstrated that early exposure to sevoflurane on neonatal rat inhibited VGCCs activity and the development of VGCCs recovered to normal level at juvenile age. Thus, VGCCs can be modulated and inhibited persistently by sevoflurane. And the degree of inhibition may depend on the concentration of sevoflurane. Findings in this study would provide support for safer application of sevo- flurane in newborns and infants.

As described previously (Lu et al., 2010; Satomoto et al., 2009; Seubert et al., 2013), we chose 3% and 2.1% sevoflurane to investigate the sevoflurane-induced alterations on I–V curve, steady-state activation and inactivation curves of VGCCs in rat hippocampal CA1 pyramidal neurons during the period of rapid brain development. Several studies have significantly shown depresses the amplitude of Ca2þ currents (Gong et al., 2012; Rithalia et al., 2004). In agreement with that finding, we found that the peak amplitude of I–V curve significantly reduced at week 1 and week 2 when compared with the control condi- tions. The maximal current density was decreased at week 1 by 62% (Po0.01), 28% (Po0.01) and at week 2 by 43% (Po0.01), 22% (Po0.01) by 3% sevoflurane and 2.1% sevoflurane, respec- tively. Even more importantly, the current density in sevo- flurane groups at week 2 was below the normal level at week 1. Furthermore, after a 6 h exposure to 3% sevoflurane, VGCCs steady-state activation were significantly delayed at week 1, week 2 and recovered to normal level at week 3, week 4 and week 5. However, that changes in Ca2þ channel activation curve were not found in 2.1% sevoflurane groups. Finally, our study showed that 3% sevoflurane significantly delayed the VGCCs steady-state inactivation at week 1, week 2 and week 3, and these delay only occurred at week 1 and week 2 in 2.1% sevoflurane groups. Our data indicate that a lower concentra- tion (2.1%) of sevoflurane with the same treatment time (6 h) have less inhibitory effects on VGCCs than 3% sevoflurane.

sevoflurane

that

administration

VGCCs are an important pathway for extracellular calcium entry (Zhang et al., 2009). The sevoflurane-induced reduction on Ca2þ currents may result in decreased opening probability of VGCCs and a less influx of Ca2þ , which leads to

4.

Experimental procedures

4.1.

Animals

This experiment was approved by the Institutional Animal Care and Use Committee at Tianjin Medical University. Sprague-Dawley (SD) rats (both male and female) were obtained from the Experimental Animal Centre of Tianjin Medical University, Tianjin, China. Rats were housed under 12 h light-dark cycles with free access to food and water at room temperature (21–24 1C). 1-week-old SD rats were ran- domly assigned to control group, 2.1% sevoflurane group and 3% sevoflurane group to receive gas exposure. After gas exposure, electrophysiological recording were performed on rats of 5 different ages (from 1 week to 5 weeks).

4.2.

Sevoflurane exposure

The rats were placed in a home-made anesthetic induction chamber using an electric blanket to maintain the tempera- ture at 35–37 1C, and calcium lime was spread on the bottom of the chamber to absorb carbon dioxide. Rats in 3% sevo- flurane group and 2.1% sevoflurane group were respectively exposed to 3% and 2.1% sevoflurane for 6 h using oxygen as gas carrier with a gas flow of 4 L/min, while the rats in control group breathed independently oxygen with a gas flow of 4 L/min for 6 h in the same induction chamber. During sevoflurane exposure, the gas concentration of carbon dioxide, oxygen and sevoflurane in the chamber were monitored by gas monitor (Detex-Ohmeda, Louisville, KY, USA). After anesthesia, rats were

9

10

b r a i n r e s e a r c h 1 5 5 7 ( 2 0 1 4 ) 1 – 1 1

Table 4 – Arterial blood analysis.

Group

Time (h)

pH

PaO2 (mmHg)

PaCO2 (mmHg)

Control

0 6

7.4170.05 7.3870.07

99.775.1 100.176.3

27.575.2 26.674.3

2.1% Sevo

0 6

7.4070.03 7.4070.05

10076.1 99.575.4

27.774.9 27.975.1

3% Sevo

0 6

7.4070.08 7.3970.04

98.675.8 99.575.9

28.274.8 26.975.9

exposed to oxygen only until they recovered from anesthetic. Then, all of rats were placed back into the maternal cages.

4.5.

Electrophysiological recording

4.3.

Blood gas analysis

As described previously (Feng et al., 2012; Lu et al., 2006), we performed blood gas analysis on rats of the both 3 treated groups before anesthesia exposure (0 h) and at the end of 6 h of anesthesia (6 h). Three pups of each group were used for that analysis. Arterial blood samples were collected from the left cardiac ventricle and immediately transferred into the blood gas analyzer (GEM Premier 3000, Instrumentation laboratory, Bedford, MA) to measure the blood pH, PaCO2 (arterial carbon dioxide tension) and PaO2 (arterial oxygen tension). The values of blood pH, PaCO2 and PaO2 in the sevoflurane groups did not significantly differ from that values in control group (Table 4).

4.4. neurons

Acute dissociation of hippocampal CA1 pyramidal

In this study, hippocampal pyramidal neurons were acutely isolated as described previously by Zhang et al. (2009) with some modifications. Briefly, rat was decapitated and the brain was rapidly removed, iced for 1–2 min in 0–4 1C artificial þ5%CO2 gas. cerebrospinal fluid (ACSF) bubbled with 95%O2 ACSF solution contains 120 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 and 10 mM Glucose, pH 7.4, with HCl and NaOH. The hippocam- pus was separated out and cut into 400–600 μm thick slices on home-made ACSF ice pillow. The slices were then placed into þ5%CO2 to incubate for 1 h at 32 1C. ACSF bubbled with 95%O2 The CA1 region of hippocampus was dissected out using 1 mL syringe needle under stereomicroscope and transferred into ACSF containing 0.67 mg/mL Pronase XIV (Sigma) saturated þ5%CO2 gas to dissociate for 20–45 min at 32 1C with 95%O2 (1 w:20 min; 2 w:30 min; 3 w/4 w:40 min; 5 w:45 min). After enzyme digestion, slices were washed three times in stan- dard extracellular fluid bubbled with 100% O2 gas and dis- sociated by a graded series of fire-polished Pasteur pipettes. The standard extracellular fluid contains 130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM Glucose and 10 mM HEPES, pH 7.4, with HCl and NaOH. Then, the cell suspension was plated into a 35 mm Lux Petri dish containing oxygen saturated standard extracellular fluid. And the neurons were allowed to settle to the button of the dish before recording.

In current experiment, as described in detail (Taketo and Yoshioka, 2000), whole-cell patch clamp recordings were performed to record voltage-gated calcium channel currents at room temperature (21–24 1C). Recording pipettes were pulled from borosilicate glass capillaries on P-97 horizontal puller (Sutter Instrument Company, USA). The resistance of pipettes filled with intra-pipette solution was 3–5 MΩ. Intra- pipette solution contains 110 mM CsCl, 30 mM TEA-Cl, 10 mM EGTA, 10 mM HEPES and 3 mM ATP, PH¼ 7.3, with CsOH and HCl.

Under inverted microscope (BX51-WI, Olympus, Japan), we selected randomly pyramidal neurons with a smooth and bright appearance and no visible organelles for recording. During the recording, neurons were bathed in an extracellular solution, which contains 110 mM Choline chloride, 20 mM TEA-Cl, 10 mM 4-AP, 1 mM MgCl2, 10 mM BaCl2, 10 mM HEPES, 10 mM Glucose and 0.001 mM TTX, PH ¼7.4, with HCl. Voltage-clamp recordings were obtained with Axon patch 200B patch-clamp amplifier (Molecular Devices, Foster City, CA, USA) and Digidata 1440 interface (Molecular Devices, Foster City, CA, USA). After establishing a gigaseal (42 GΩ), the membrane was broken to form whole-cell configuration. Series resistance and membrane capacitance were routinely compensated by 60–80%, leakage and capacity currents were subtracted on-line using a P/4 protocol. The data were acquired using pCLAMP 10.0 (Molecular Devices, Foster City, CA, USA) running on a computer. Recording signals were low- pass filtered at 2 kHz and digitized at 10 kHz. In this study, whole-cell recording was voltage-clamped at (cid:2)50 mV. Data were obtained from 10 to 14 cells per group.

4.6.

Data analysis

Current density was calculated by dividing current at mem- brane potential by the cell-membrane capacitance (i.e. pA/pF). All data were analyzed by pCLAMP Clampfit 10.0 (Molecular Devices, Foster City, CA, USA), Origin 6.0 (OriginLab Corp, Northampton, MA, USA). The data were presented as mean- s7standard error of the mean (S.E.M.), one way ANOVA followed by SNK post hoc test (SPSS, Chicago, IL) were used for statistical analysis. Significant level was set to 0.05.

Disclosure statement

No author of this paper has a conflict of interest to declare.

b r a i n r e s e a r c h 1 5 5 7 ( 2 0 1 4 ) 1 – 1 1

Acknowledgments

This study was supported by the National Natural Science Foundation of China (Nos. 91132722 and 61375113).

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11",rats,"['1-week-old Sprague-Dawley rats were randomly divided into 3 groups: control group, 2.1% sevoflurane group (exposed to 2.1% sevoflurane for 6 h) and 3% sevoflurane group (exposed to 3% sevoflurane for 6 h).']",postnatal day 7,"['1-week-old Sprague-Dawley rats were randomly divided into 3 groups: control group, 2.1% sevoflurane group (exposed to 2.1% sevoflurane for 6 h) and 3% sevoflurane group (exposed to 3% sevoflurane for 6 h).']",N,[],sevoflurane,['Research Report Early exposure to sevoflurane inhibits Ca2+ activity in hippocampal CA1 pyramidal neurons of developing rats'],none,[],sprague dawley,"['1-week-old Sprague-Dawley rats were randomly divided into 3 groups: control group, 2.1% sevoflurane group (exposed to 2.1% sevoflurane for 6 h) and 3% sevoflurane group (exposed to 3% sevoflurane for 6 h).']",The role of sevoflurane on regulating Ca2+ channels during the period of rapid brain development is still not well understood.,"['However, the role of sevoflurane on regulating Ca2+ channels during the period of rapid brain development is still not well understood.']",None,[],Early exposure to sevoflurane persistently inhibits Ca2+ channels activity in hippocampal CA1 pyramidal neurons of developing rats but the development of Ca2+ channels recovers to normal level at juvenile age.,"['Therefore, early exposure to sevoflurane persistently inhibits Ca2+ channels activity in hippocampal CA1 pyramidal neurons of developing rats but the development of Ca2+ channels recovers to normal level at juvenile age.']",None,[],None,[],True,True,True,True,True,True,10.1016/j.brainres.2014.02.008
10.1007/s11064-015-1529-x,460.0,Liu,2015,rats,postnatal day 7,N,sevoflurane,none,sprague dawley,"Neurochem Res (2015) 40:788–799 DOI 10.1007/s11064-015-1529-x

O R I G I N A L P A P E R

Altered Metabolomic Profiles May Be Associated with Sevoflurane-Induced Neurotoxicity in Neonatal Rats

Bin Liu • Yuechao Gu • Hongyan Xiao • Xi Lei • Weimin Liang • Jun Zhang

Received: 19 November 2014 / Revised: 23 January 2015 / Accepted: 28 January 2015 / Published online: 7 February 2015 (cid:2) Springer Science+Business Media New York 2015

Abstract Experimental studies demonstrate that inhaled anesthetics can cause neurodegeneration and neurobehav- ioral dysfunctions. Evidence suggests changes in cerebral metabolism following inhaled anesthetics treatment can perturb cerebral homeostasis, which may be associated with their induced neurotoxicity. Seven-day-old rat pups were divided into two groups: control group (Group C) and sevoflurane group (Group S, 3 % sevoflurane exposure for 6 h). Gas chromatography–mass spectrometry (GC–MS) was used for analyzed differential metabolites of cerebral cortex in both groups, Also western blot, flow cytometry, enzymatic methods and electron microscopy were per- formed in various biochemical and anatomical assays. Sevoflurane exposure significantly elevated caspase-3 acti- vation and ROS levels, decreased mitochondrial cardiolipin

contents, and changed cellular ultrastructure in the cerebral cortex. Correspondingly, these results corroborated the GC– MS findings which showed altered metabolic pathways of glucose, amino acids, and lipids, as well as intracellular an- tioxidants and osmolyte systems in neonatal brain following prolonged exposure to high sevoflurane concentration. Our data indicate that sevoflurane anesthesia causes significant oxidative stress, neuroapoptosis, and cellular ultrastructure damage which is associated with altered brain metabotype in the neonatal rat. Our study also confirmed that GC–MS is a strategic and complementary platform for the metabolomic characterization of sevoflurane-induced neurotoxicity in the developing brain.

Keywords Metabolome (cid:2) Sevoflurane (cid:2) Developing brain (cid:2) Gas chromatography (cid:2) Neurotoxicity

Bin Liu and Yuechao Gu have contributed equally to this work.

Introduction

B. Liu (cid:2) H. Xiao (cid:2) X. Lei (cid:2) W. Liang (cid:2) J. Zhang (&) Department of Anesthesiology, Huashan Hospital, Fudan University, No. 12 Wulumuqi Middle Road, Jin’an District, Shanghai 200040, People’s Republic of China e-mail: snapzhang@aliyun.com

B. Liu e-mail: lbzaixuzhou@126.com

H. Xiao e-mail: hongyanxiao@yahoo.cn

Mounting experimental studies demonstrate that commonly used general anesthetics, especially inhaled anesthetics, could trigger widespread apoptotic neurodegeneration in the developing brain, and cause long-term neurobehavioral abnormalities in rodents [1–5] and nonhuman primates [6, 7]. However, the cellular mechanisms of general anes- thetics-induced neurotoxicity are not fully clarified.

X. Lei e-mail: anesthesia2006xi@163.com

W. Liang e-mail: chiefliang@yahoo.com.cn

Y. Gu Department of Anesthesiology, Shanghai Cancer Hospital, Fudan University, Shanghai 200032, People’s Republic of China e-mail: yuechaogu@126.com

General anesthesia can greatly influence cerebral meta- bolism in many ways [8–10]: cerebral blood flow-meta- bolism uncoupling, cerebral reduction metabolic oxygen rate, glucose utilization, and oxidative adenosine triphosphate (ATP) production rate in animals and humans in a dose-dependent manner. Given their in- fluence on brain metabolism, anesthetics may perturb cerebral homeostasis, and thus may be associated with

in

regional

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inhaled anesthetic-induced neurotoxicity. Therefore, it is to understand the effects of inhaled vitally important anesthetics on cerebral metabolism in the developing brain. Although anesthesia disrupts many metabolic processes, like those mentioned above, the effects of other metabolic pathways in the brain remain unknown. Metabolomics re- cently has been introduced to study the small-molecule metabolite profiles of biological organisms [11]. This method can be applied to elucidate changes of metabolites in the brain of animals exposed to neurotoxins and provide information to identify early and differential markers for disease [12].

By using proton magnetic resonance spectroscopy (1- HMRS), Makaryus et al. [13] analyzed cerebral metabolites in rats and children [14] during general anesthesia and found that inhaled and intravenous anesthetics produce distinct meta- bolomic profiles in some brain regions such as parietal cortex and hippocampus. Here we hypothesize the alternation in cerebral metabolism underlie potential changes in anatomical/ biochemical characteristics after inhaled anesthetics treat- ment, and these neurochemical sequelae may be involved in inhaled anesthetic-induced neurotoxicity [15]. To test our hypothesis, we applied gas chromatography–mass spec- trometry (GC–MS) analysis to assay brain metabolites in neonatal rats, intended to identify the early metabolic phe- notypes following prolonged exposure to high sevoflurane concentration. Sevoflurane, a commonly used inhaled anes- thetic in clinical paediatric anesthesia, has been reported to have neurotoxic property on developing brain. Further, we examined apoptotic markers, oxidative status, and ultra- structural properties in a neonatal rat model to gain a better insight sevoflurane-induced neurotoxicity.

into the pathogenesis of

Materials and Methods

Chemicals and Reagents for Metabolomic Analysis

Methanol (pesticide residue grade), bis-(trimethylsilyl)-tri- fluoroacetamide (BSTFA) plus 1 % trimethylchlorosilane (TMCS) (REGIS Technologies Inc. Morton Grove, IL, USA), and amino acid standard solution were purchased from Sigma- Aldrich (St. Louis, MO, USA). L-2-chlorophenylalanine (in- ternal standard) was obtained from Shanghai Hengbai Biotech Co. Ltd. (Shanghai, China). All other chemicals and reagents were purchased from Anpel Company (Shanghai, China). Distilled water was prepared using the Milli-Q Reagent-Water System (Millipore, MA, USA).

Animals and Anesthesia

Sprague–Dawley (SD) rats used in the present study were obtained from the Animal Care Center of Fudan

789

University. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee, Fu- dan University. According to the flow chart of the ex- perimental protocol (Fig. 1), the rat pups (body weight: 12.1 ± 0.1 g) at postnatal day 7 (P7) were divided into two groups: control (Group C) and sevoflurane-treated (Group S).

P7 rats in group S were placed in a sealed chamber ventilated with 3 % sevoflurane in 100 % oxygen for 6 h and sevoflurane concentration was continuously measured through a gas sample line by using a monitor (Datex Ohmeda S/5, Helsinki, Finland) whereas those in group C were placed in a similar chamber for 6 h under identical experimental conditions without sevoflurane exposure. The temperature in the sealed chamber was maintained at 33–35 (cid:3)C with a heating pad. The total survival percentage of P7 rats in group S after 6-h anesthesia was 90 %. After treatment, the rat pups were returned to their dams for lactation. The rats in the same litter were used for each experiment and they were sacrificed by rapid decapitation at 12 h after sevoflurane exposure. The frontal cortex was harvested and stored at -80 (cid:3)C until use. The preparations of the brain samples and the number of animals used were described in their methods, respectively.

Blood Gas Analysis

P7 rats (n = 4 each group) were used to assess the effect of sevoflurane treatment on arterial blood gases. Arterial blood sampling from the left cardiac ventricle was per- formed immediately after the end of sevoflurane anesthesia according to the previous described method [16].We measured partial pressures of carbon dioxide (PaCO2) and oxygen (PaO2), pH, and blood lactate and glucose levels with a Radiometer ABL 800 blood gas analyzer (Ra- diometer, Copenhagen, Denmark).

GC–MS Analysis

Prior to metabolic profiling, frontal cortex (n = 6/group) were homogenized with 50 lL L-2-chlorophenylalanine in a 2-mL centrifuge tube. Then, 0.4 mL methanol-chloro- form (3:1, V:V) as extraction liquid was added to each homogenate. After 2 min of vortex-mixing, the samples were centrifuged at 12,000 rpm for 10 min at 4 (cid:3)C and 400 lL of supernatant from each sample was transferred into a new 2-mL glass tube. The supernatants of cortical samples were concentrated to complete dryness at a tem- perature of 50 (cid:3)C for approximately 30 min using the (Caliper Life Science, TurboVap nitrogen evaporator Hopkinton, MA). Afterward, 100 lL of anhydrous toluene (stored with sodium sulfate) was added to each of the dried tissues. Following 1 min of vortex-mixing, the samples

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Group C

100% O2 treatment for 6h

P7 rats

Group S

3% sevoflurane treatment in 100% O2 for 6h

Fig. 1 Flow chart of experimental protocol

were evaporated to dryness using the evaporator to ensure the complete elimination of any traces of water which might interfere with the subsequent GC–MS analysis. Then, 80 lL MOX reagent was added to the dried samples, vortex-mixed for 2 min, and incubated at 37 (cid:3)C for at least 2 h as a methoximation step. Derivatization reaction aimed to increase the volatility of polar metabolites was then initiated by adding 100 lL of BSTFA (with 1 % TMCS) to each sample, vortex-mixed for 2 min, and incubated at 70 (cid:3)C for 60 min. Following the incubation, each sample was vortex-mixed for 2 min and carefully transferred to the autosampler vials for subsequent GC–MS analysis [17].

GC–MS analysis was performed on an Agilent 7890A gas chromatography system coupled with an Agilent 5975C mass spectrometer (Agilent, USA). The system utilized a DB-5MS capillary column coated with 5 % diphenyl cross-linked with 95 % dimethylpolysiloxane (30 lm 9 250-lm inner diameter, 0.25-lm film thickness; J&W Scientific, Folsom, CA, USA). A 1-lL aliquot of the analyte was injected in splitless mode. Helium was used as the carrier gas, the front inlet purge flow was 3 mL/min, and the gas flow rate through the column was 1 mL/min. The initial temperature was kept at 80 (cid:3)C for 2 min, then raised to 240 (cid:3)C at a rate of 5 (cid:3)C/min, and finally to 290 (cid:3)C at a rate of 10 (cid:3)C/min for 11 min. The injection, transfer line, and ion source temperatures were 280, 270, and 220 (cid:3)C, respectively. The energy was -70 eV in electron impact mode. The mass spectrometry data were acquired in full-scan mode with the m/z range of 20–600 at a rate of 100 spectra per second after a solvent delay of 492 s. Chroma TOF4.3X software of LECO Corporation were used to acquire mass spectrometric data [18]. Mass spectra of all detected compounds were compared with spectra in the National Institute of Standards and Technology (NIST,

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Cleaved caspase-3 expression

(Western Blot)

ROS level measurement

Frontal cortex harvest

(Flow Cytometry)

Cardiolipin contents

12 hours

(Microplate reader method)

Cellular ultrastructure

(Electron microscopic analysis)

Metabolomic profiles

(GC-MS analysis)

http://www.nist.gov/index.html) and Fiehn databases. The peaks with similarity index of more than 70 % were se- lected and named the putative metabolite identities.

Multivariate Data Analysis

The resulting GC–MS data were first processed by nor- malizing peak area of each analyte based on total integral area calculation performed using an in-house script (Mi- crosoft Office Excel). All processed data were then mean- centered and unit-variance scaled before they were sub- jected to principal component analysis (PCA) (version 11.5, SIMCA-P software, Umetrics, Umea, Sweden) to identify clustering trend, as well as detect and exclude outliers. Quality control (QC) samples for cortical tissues were prepared by randomly pooling 5 lL from each of the five samples belonging to the test groups. QC samples were analyzed at constant intervals to ensure that the data ac- quisition for GC/MS metabolic profiling was reproducible for all samples. Variable importance in the projection (VIP) cutoff value was defined as 1.00.

Western Blot Analysis

The frontal cortical tissues were homogenized in RIPA buffer (Millipore, Temecula, CA, USA) containing com- plete protease inhibitor cocktail and 2 mM phenylmethyl- collected and sulfonyl fluoride. The centrifuged at 12,000 rpm for 30 min at 4 (cid:3)C. After the protein samples were quantified using a BCA Protein As- say Kit (Pierce Biotechnology, Rockford, IL, USA), the cleaved caspase-3 expression was detected by western blot analysis according to our previous method [4]. Data were expressed as mean ± SD. The changes were presented as a

lysates were

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percentage of those of the control group. One-hundred- percent of caspase-3 activation refers to control level for the purpose of comparison to that in Group S.

Measurement of ROS Levels

The frontal cortex was cleaned in PBS and dissociated in trypsin solution, and stopped using DMEM solution. A single-cell suspension was obtained by using a 70-lm mesh. The chemiluminescent probe with flow cytometry technique was used for detection of intracellular ROS level according to a previously described method [19]. Briefly, 20,70-dichlorofluorescin diacetate (DCFH-DA) probe was added to the cell suspensions at a final concentration of 10 lmol/L and incubated at 37 (cid:3)C, protected from light for 1 h, followed by flow cytometry (FACSCanto, BD Bio- sciences, San Jose, CA, USA) measurement. The formation 20,70-dichlo- of rofluorescein (DCF) was monitored with excitation light at 488 nm and emission light at 525 nm, and normalized by protein concentration. By quantifying fluorescence inten- the ROS levels in both groups were sity of DCF, calculated.

the

oxidized fluorescent

derivative

Mitochondrial Cardiolipin Assay

Extraction of mitochondria was performed using the mi- tochondria isolation kit (Shanghai Genmed Scientifics Inc., China). Briefly, the frontal cortex was lysed in precooled centrifuge tubes and disrupted by 80 passes in the ho- mogenizer with a tight fitting Dounce homogenizer. The homogenate was then centrifuged for 10 min at 1,500g at 4 (cid:3)C. The mitochondria-rich supernatant was then col- lected and centrifuged for 10 min at 10,000g at 4 (cid:3)C. The mitochondrial pellets were then washed with 2 mL of preservation medium (25 mmol/L potassium phosphate; 5 mmol/L MgCl2, pH 7.2) and centrifuged for 5 min at 10,000g at 4 (cid:3)C. Purified mitochondrial samples were freeze-thawed three times and suspended to 5.5 mg/mL in PBS before use. The mitochondrial cardilopin contents were quantified by the microplate reader method using the high affinity 10-N-nonyl acridine orange (NAO) for car- diolipin of freshly isolated mitochondria [20]. Briefly, reagents (90 lL) from cardilopin assay kits (Shanghai GenMed Scientifics Inc., Shanghai, China) was added into mitochondrial sample (10 lL) on the microplate. The mi- croplate was gently shaken and incubated in a dark room for 20 min at room temperature. Then fluorescence inten- sity was measured with excitation light at 580 nm and emission light at 630 nm. The cardilopin contents were expressed as relative fluorescence unit (RFU) and nor- malized by protein concentration.

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Electron Microscopy

The rat brain was perfused with normal saline solution followed by phosphate-buffered 2.5 % glutaraldehyde and 4 % paraformaldehyde 12 h after sevoflurane treatment, then the frontal cortex was sliced into sections of ap- proximately 1 mm2, and kept in the same glutaraldehyde solution for 12 h at room temperature. Samples were postfixed in 1 % osmium tetroxide for 2 h, dehydrated in a series of alcohol solutions at 4 (cid:3)C, immersed in propylene oxide, and embedded in Araldite 502 resin at 60 (cid:3)C. Ul- trathin (0.5 lm) sections were placed on grids and stained with uranyl acetate and lead citrate before examination with a transmission electron microscope (Philips CM-120, Eindhoven, The Netherlands). The organelles of neuronal cells were observed and imaged at 10,0009 magnification.

Statistical Analysis

We performed one-way ANOVA to determine differences in caspase-3 activation and cardiolipin contents, and in- dependent Student’s t test to compare the difference in arterial blood gas analysis and ROS levels. Independent t tests with Welch’s correction were then used for statistical comparison of discriminant metabolite levels between Group C and Group S, which determined for sevoflurane- induced alteration of metabolic profiling in neonatal rat model. The significance level was set at p \ 0.05.

Results

Blood Gas Analysis

The Table 1 showed the results of arterial blood gas ana- lysis and biochemical parameters from neonate rats in both groups immediately after sevoflurane treatment. Data from arterial blood analysis revealed that 6 h sevoflurane anes- in thesia

did

not

induce

significant

disturbances

Table 1 Arterial blood gas and biochemical parameters in control and anesthetized neonatal rats (n = 4/group)

Group C

Group S

PH PaO2 (mmHg) PaCO2 (mmHg) Blood glucose (mmol/L)

7.41 ± 0.12

398.7 ± 53.4

38.8 ± 5.2

5.58 ± 1.56

7.19 ± 0.08*

175.0 ± 10.8

81.9 ± 8.4*

6.33 ± 0.75

Blood lactate (mmol/L)

0.35 ± 0.04

0.42 ± 0.10

Data presented as Mean ± SD. Statistic analysis with independent student t test, compared with Group C * p \ 0.05

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oxygenation, blood glucose and lactate. However, com- pared with Group C, exposure to 3 % sevoflurane for 6 h caused significant hypercapnia and acidosis in neonatal rats.

Sevoflurane Alters the Metabolomic Profiles

Data Processing and Pattern Recognition

The representative GC–MS chromatogram of a cortical sample of a sevoflurane-treated P7 rat depicting the peaks of discriminate metabolites is shown in Fig. 2 with the major metabolites noted.

PCA of brain samples indicated that the cerebral cortex in sevoflurane-treated and control neonatal rats formed two distinct clusters on the scores plot. In order to obtain a higher level of group separation and get a better understanding of the variables responsible for classification, a supervised partial least squares discriminant analysis (PLS-DA) was employed, and spectral profiles were readily distinguished between Group S and Group C (Fig. 3a). PLS-DA is com- monly used for classification purposes and biomarker se- lection in metabolomics studies. If a statistically significant discrimination between two groups can be found, then the model parameters can be interpreted for their discriminating power and metabolic biomarkers can be found. The classi- fication parameters from the software were R2X = 0.710, R2Y = 0.975, and Q2 = 0.834, which were stable and good to fitness and prediction. A sevenfold cross-validation was used to estimate the robustness and the predictive ability of our model. To further validate our model, we performed 200 permutations, and the R2 and the Q2 intercept values were (0.0, 0.679) and (0.0, 0.091) for the cerebral cortex (Fig. 3b), which indicates that the PLS-DA models in this study have good or excellent fitness and predictive abilities. These

Fig. 2 The typical spectrum from frontal cortex extract in a neonatal rat. NAA N-acetyl-L- aspartic acid

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results demonstrate that GC–MS-based metabolomic ana- lysis is well-suited to detect sevoflurane-induced brain metabolic alterations in neonatal rat.

Metabolic Profiling

GC–MS analysis provided the metabolic profiles of neonatal brains in both groups. A set of discriminant brain metabolites for distinguishing sevoflurane-treated rats from control ones in the PLS-DA model was identified. The 26 metabolites were picked out by PLS-DA (VIP [ 1) and t test (p value \0.05), and 16 metabolites were selected for their correlation with mole- cular and structural aspects of sevoflurane-induced neuro- toxicity, involving in energetic metabolism, neuronal apoptosis, oxidative stress, and cell swelling. They were used to make the heatmap (Fig. 4) and summarized in Table 2 with their classifications. These metabolites were related to metabolic pathways of glucose, amino acids and lipids (phospholipids), as well as intracellular antioxidants and osmolyte systems. Next, we determined whether the changes we found in metabolism underlie potential chan- ges in anatomical/biochemical features (neuroapoptosis, oxidative stress, and cellular ultrastructure damage) after sevoflurane treatment.

that was

responsible

Sevoflurane Increases Neuronal Apoptosis

Caspase-3 activation is an indicator of cellular apoptosis [21]. Compared with Group C, 3 % sevoflurane treatment for 6 h significantly increased cleaved caspase-3 levels in cerebral cortex of Group S (Fig. 5). Our results suggest prolonged exposure to high sevoflurane concentration dramatically increased the incidence of apoptosis in the cerebral cortex of neonatal rat.

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Fig. 3 Data processing and pattern recognition of metabolomic analysis between the two groups. a Partial least squares projection to latent structures and discriminant analysis (PLS-DA) scores plot of cerebral cortex in neonatal rats. The statistical data analysis resulted

Fig. 4 Heatmap of differential metabolites between Group S and Group C. Heatmap showing differential metabolites from cerebral cortex in Group S paired with metabolites in Group C. Each row represents one metabolite, and each column represents one tissue sample. The relative metabolite level is depicted according to the indicates color

scale.

Red

indicates

upregulation;

green

Sevoflurane Increases Oxidative Stress

To test oxidative stress status, we measured total ROS levels after sevoflurane treatment. We found that the total

793

in a cluster formation at the two treatments. The replicates (n = 6/ each group) represent untreated controls (Group C) and high concentration sevoflurane exposure (Group S). b Validation of the model with a permutation test

downregulation. 3.0, 0, and -3.0 are fold changes in the correspond- ing spectrum. CC1-CC6 represent tissues 1–6 from Group C, whereas CH1-CH6 represent tissues 1–6 from Group S. The different metabolites clearly self-segregated into Group C and Group S clusters. Independent t test with Welch’s correction (significance at p \ 0.05)

ROS level in cerebral cortex significantly increased after 3 % sevoflurane treatment for 6 h, compared with that in Group C (Fig. 6). To further evaluate the effect of in- creased generation of ROS after sevoflurane exposure, we

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Table 2 marker metabolites found in GC/MS analysis of brain samples of sevoflurane exposure and control neonatal rats

Metabolites

RT (min)

Chemical class

Identified by

% Change of Group S from control

p value

Similarity

VIP

Lactic acid

6.35012

Organic acid

Fiehn databases

23.0

0.016203

904

1.44735

Sarcosine

7.28777

Amino acid

Fiehn databases

19.4

0.033741

985

3.2138

Noradrenaline Proline

11.3467 12.1510

neurotransmitter Amino acid

Fiehn databases Fiehn databases

31.5 -33.1

0.041111 0.011095

937 935

4.29261 2.18524

Succinic acid

12.6381

Dicarboxylic acid

Fiehn databases

53.1

0.006009

904

3.14927

Total creatine

18.5306

Organic acid

Fiehn databases

20.8

0.037678

800

1.0311

Glutamine

19.3965

Amino acid

Fiehn databases

58.8

0.008508

828

1.53816

Glutamic acid

20.1758

Amino acid

Fiehn databases

30.0

0.002963

839

1.26475

Taurine N-acetyl-L-aspartic acid O-Phosphorylethanolamine

20.6277

21.0327

23.7303

Organic acid

Amino acid

Sphingolipid

Fiehn databases

Fiehn databases

NIST

35.6

33.2

24.2

0.008136

0.002821

0.004955

745

879

852

1.30407

2.8725

3.22187

Dehydroascorbic acid

24.9923

Hydroxy acid

Fiehn databases

33.0

0.00668

889

1.94691

Ascorbate

27.0483

Hydroxy acid

Fiehn databases

64.0

0.003216

724

2.22106

Tryptophan

31.8846

Amino acid

Fiehn databases

60.0

0.017015

919

1.16551

Oleic acid

32.0865

Organic acid

NIST

170.0

0.001782

792

1.00714

Inosine

37.1657

Nucleoside

Fiehn databases

43.3

0.002179

861

3.1662

RT retention time, VIP variable importance in the projection

these two findings suggest that oxidative stress is increased in the neonatal brain exposed to sevoflurane.

Ultrastructural Features in Cerebral Cortex After Sevoflurane Exposure

Fig. 5 Neonatal exposure to sevoflurane increases neuroapoptosis. a The cortical cleaved caspase-3 expression in neonatal brain was examined with western blot. b Quantification of cleaved caspase-3 (one-way ANOVA, F = 0.607, p = 0.004), *p \ 0.05 Group C (100.0 ± 11.4 %, n = 3) versus Group S (687.2 ± 25.1 %, n = 3)

In Group S, electron micrographs of the cerebral cortex showed ultrastructural features of typical cellular swelling. We observed hallmarks of cellular swelling in Group S, including local swelling, is recognized by a pale and watery cytoplasm under electron microscopic analysis, and swollen organelles, including dilated rough endoplasmic reticulum (RER) fragments (Fig. 8a), and increased vacuolated and swollen mitochondria (Fig. 8b); these changes were ob- served in both cell bodies and the neuropil of bilateral frontal cortex in Group S, but not in Group C. Interestingly, sevoflurane treatment also significantly decreased the num- ber of synaptic vesicles at axon terminals (Fig. 8c).

Discussion

measured isolated mitochondrial cardiolipin contents of the cerebral cortex. Cardiolipin, a unique mitochondrial phos- pholipid involved in mitochondrial energy metabolism, is a critical target of mitochondrially-generated ROS and reg- ulates signaling events related to apoptosis [22]. We dis- covered that cardiolipin contents in Group S were greatly reduced when compared to Group C (Fig. 7). Combined

The present study aimed at investigating metabolic changes occurring in the cortical region of neonatal rat brain after prolonged exposure to high concentration sevoflurane us- ing GC–MS analysis. P7 rat, a common animal mode used for studying inhaled anesthetic-induced neurotoxicity, was applied in this study. As we know, frontal cortex helps mediate encoding in episodic memory and retrieval from

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Fig. 6 Neonatal exposure to sevoflurane increases ROS levels. The cortical ROS levels in neonatal brain were examined with flow cytometry. Left is formation of oxidized fluorescent derivative 20,70- dichlorofluorescein (DCF) monitored with flow cytometry. Right is comparison of relative fluorescence intensity of DCF between Group

Fig. 7 Neonatal exposure to sevoflurane reduces mitochondrial cardiolipin contents. The mitochondrial cardiolipin contents in neonatal brain were determined enzymatically in Group C (100 ± 20.2 %, n = 4) and Group S (64.4 ± 6.9 %, n = 4). The relative fluorescence unit represents relative mitochondrial cardiolipin contents in cerebral cortex. *p \ 0.05 Group C versus Group S(One- way ANOVA, F = 8.45, p = 0.023)

episodic memory (working memory), and is involved in many higher cognitive functions [23]. The neuronal apoptosis and structure damage at early birth period in this brain region could influence its later development and adulthood. subsequently Therefore, frontal cortex was selected as a target brain region. Previous studies have showed that changes in metabolic profiling during general anesthesia in human and rodent brains [13, 14]. Because long-term neurobehaviour impairment has been reported following inhaled anesthetic

neurocognitive

function

in

795

C (1795.5 ± 114.1, n = 5) and Group S(2,153.7 ± 103.8, n = 7). The relative fluorescence intensity represents relative ROS levels in cerebral cortex. *p \ 0.05 Group C versus Group S (independent student t test, t value = 2.296, p = 0.045)

exposure [1, 3, 4], the effects of anesthesia on cerebral metabolism is assumed also persist for a relative long time. However, the characterization of cerebral metabolomics during recovery from anesthesia remain unclear, the brain samples harvested 12 h after sevoflurane anesthesia were used for this purpose.

We found that alteration in metabolite levels in the frontal cortex of neonatal rats in response to sevoflurane exposure. These changes may underlie sevoflurane-induced oxidative stress, cellular ultrastructure damage, and neu- roapoptosis that we observed under the same anesthetic exposure in the developing brain. Previous studies have implicated that regional abnormalities of brain metabolites were associated with some neurological disorders [24]. Here, we found GC–MS-based metabolomics analysis, to the best of our knowledge, is first time used to characterize effects of anesthetic agents on the developing rodent brain. The PLS-DA model robustly differentiated the metabolic profiles in the sevoflurane group from the control group. The method was subsequently applied to elucidate char- acteristic metabolites, which has the potential to map per- turbations of neurochemical changes in the developing brain [25]. This can prove to be a useful technique for investigating brain metabolic changes on exposure to general anesthetics. Remarkably, we found differential metabolites identified with altered metabolism of glucose, amino acids, and lipids, as well as intracellular antioxidants and osmolyte systems. The changes in metabolomics pro- vided potential evidences of sevoflurane-induced develop- mental neurotoxicity including ultrastructural damage, oxidative stress and neuroapoptosis.

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Fig. 8 Ultrastructural changes in cerebral cortex following 3 % sevoflurane treatment for 6 h. Compared with control, neonatal exposure to sevoflurane results in dilated RER fragments (a),

Cellular Swelling

A number of factors have been implicated in cellular swelling [26]. One important factor is mitochondrial dys- function, in particular the opening of the mitochondrial permeability transition (mPT) pore [27], characterized by an increase in permeability of the inner mitochondrial membrane to small solutes (ions and molecules, reducing equivalents) [28]. The mPT may lead to osmotic swelling of the mitochondrial matrix [29], mitochondrial dysfunc- tion, defective oxidative phosphorylation, impaired ATP synthesis, and the generation of free radicals [30] which can subsequently lead to cell swelling. Myo-inosine and taurine are thought to regulate intracellular volume and

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increased vacuolated and swollen mitochondria (b) and reduction in number of synaptic vesicles (c) in neurons (magnification 910,000)

osmolarity [31]. Our GS-MS results showed a decrease in concentration of both these intracellular osmolytes in Group S, indicating alterations in intracellular osmolarity. Osmotic changes, which are associated with an influx of extracellular water into the intracellular compartment leading to cell swelling and irreversible cell damage [32], are thus likely caused by prolonged exposure to high sevoflurane concentration. This was found by electron micrographs of cerebral cortex that revealed swelling of the RER and mitochondria, similar to results in the frontal cortex following N2O (70 %) combined with isoflurane (1 %) treatment for 8 h in nonhuman primates [6]. The cellular edema could be deleterious to neuronal and glial functioning.

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Oxidative Stress

ROS generation is part of the normal cellular metabolism in brain. However, the imbalance between production of ROS and the protective effect of the antioxidant system responsible for their neutralization and removal result in oxidative stress. Our metabolomic analysis revealed re- duced intracellular levels of nonenzymatic antioxidants, ascorbate and dehydroascorbic acid, in sevoflurane-ex- posed neonatal brain, which may result from their con- sumption against ROS overload. Previous studies found that inhaled anesthetics induced neuroapoptosis through overproduction of ROS [33, 34]. In this study, we found prolonged exposure to high sevoflurane concentration sig- nificantly increased ROS levels and reduced mitochondrial cardiolipin contents, one of markers of oxidative stress in the cerebral cortex of neonatal rat. Cardiolipin is a unique tetra-acyl phospholipid that is found almost exclusively in the inner mitochondrial membrane where it is required for optimal mitochondrial function. It is particularly suscepti- ble to ROS attack due to its high content of unsaturated fatty acids, resulting in increased membrane lipid per- oxidation and reduced efficiency in mitochondrial oxida- tive considerable evidences suggest that an imbalance between oxidants and antioxidants play a role in the pathogenesis of several neurological disorders, such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis et al. [36, 37]. Sev- eral studies demonstrated that supplement of antioxidants, for example, melatonin [38], and omega-3 fatty acids [4] or ROS scavenger EUK-134 [39] could protect neuronal cells from anesthetics-induced neurotoxicity. From the per- spective of metabolomics, impaired intracellular an- tioxidants system may play a role in reduced ability to remove free radicals and subsequent occurrence of oxida- tive stress.

phosphorylation

[35]. There

are

Neuroapoptosis

In our metabolomic analysis, we found that sevoflurane significantly reduced O-phosphorylethanolamine (75.8 % of control), an ethanolamine derivative that is used to construct sphingomyelins, and led to a compensatory in- crease in oleic acid (2.7 fold of control). Also, decline of N-acetyl-L-aspartic acid (NAA) levels, a marker of neu- ronal viability, in Group S implies neuronal loss following sevoflurane treatment. All these results seem to suggest that prolonged exposure to high sevoflurane concentration could increase neuroapoptosis or inhibit neuronal growth [40]. Correspondingly, activation of caspase-3 in frontal cortex in Group S suggests increase in neuronal apoptosis after exposure to 3 % sevoflurane for 6 h, which is con- sistent with results in previous findings. The number

797

reduction of neurons and inhibition of cell proliferation in the neonatal period could lead to behavioral deficits in adulthood.

Metabolism Inhibition

As one of the most metabolically active organs, the brain depends on a continuous supply of energy to stabilize ionic homeostasis, energy consuming biochemical reactions, and physiological processes [41]. Hence, even a slight impair- ment in energy metabolism can have dramatic effects on the brain. Due to difference in affected tissues, glucose levels in ABG analysis seem not significantly change, however, the metabolomic results showed intermediates including lactate (77.0 % of control) and succinic acid (46.9 % of control) levels in the glucose metabolic path- way significantly decreased 12 h after sevoflurane anes- thesia in Group S compared with Group C. As a result, the total creatine pool, including creatine and high-energy phosphocreatine, also reduced to 79.2 % of control. Its depletion could increase cellular vulnerability to insuffi- ciency of ATP synthesis, causing cellular dysfunction [42]. In addition to as an indicator of neuronal damage, NAA has also been reported as a biomarker for mitochondrial status [43], hence a decrease in NAA might also be linked to mitochondrial dysfunction and altered neuronal energy metabolism [44]. Correspondingly, electron microscopy analysis suggested possible damaged mitochondria after sevoflurane treatment, exhibiting increased vacuolation and swelling. As the mitochondrion is often described as the ‘‘cellular power plant’’, its dysgenesis is one of the first signs of neuronal dysfunction [45], and has widespread consequences on many critical cellular events including essential roles in ROS level upregulation and apoptosis.

As well, altered amino acid metabolism in the neonatal brain may also play a role in sevoflurane-induced neuro- toxicity. Prolonged exposure to high sevoflurane concen- tration significantly reduced levels of glutamine (41.2 % of control), glutamic acid (70.0 %), aspartic acid (75.7 %), and proline (66.9 %). Given their intermediate roles in the Krebs cycle [46], this decrease paralleled the reduction of lactate and succinic acid levels. Since these amino acids are also involved in the synthesis of proteins, peptides, and fatty acids, reduction of their levels suggests neuronal growth inhibition in developing brain. The changes in glutamine–glutamate, noradrenaline, and tryptophan levels suggest glutamatergic, noradrenergic, and serotoninergic neurotransmission may be affected in Group S. Glutamate, playing a decisive role in the consolidation of memory, is primarily of neuronal origin [47]; therefore, decrease in glutamate levels, which might be due to leakage of gluta- mate from damaged neurons (may account for the reduced number of synaptic vesicles we observed at the electron

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microscopy level), changed glutamate receptor function- ing, altered synaptic activity, abnormal glutamine–gluta- mate cycling or dysfunctional glutamate transport, might result in altered learning and memory functions of the brain. These findings suggest altered synaptic transmission that may contribute to abnormal synaptic plasticity and cognitive impairments observed in rats treated with inhaled anesthetics during the neonatal period. Another non- essential amino acid sarcosine, a glycine transporter I in- hibitor, also decreased compared with Group C, which may be involved in recognition and memory impairments [48]. Therefore our results also suggest pathways involved in neuronal energy metabolism can be investigated as therapeutic targets for inhaled anesthetic-induced acute widespread neurodegeneration.

Study Limitations and Conclusion

There are some limitations to our study. First, blood gas abnormalities including hypercarbia and oxygenation de- pression were found during sevoflurane exposure. Previ- ous report indicated inhaled anesthetic-induced brain cell death may be partly caused by these effects [49], which may also produce profound metabolic effects on the neonatal brain. We cannot exclude this possibility, as we did not include a carbon dioxide inhalation group. Second, a time series of metabolomic analysis following anesthe- sia could better help to understand a time-course for sevoflurane-induced activation of the different metabolic pathways in developing brain. And finally, we performed the metabolomic analysis on the frontal cortex only. Metabolic changes in other susceptible brain regions, for example, other cerebral cortex or hippocampus, may also be involved in sevoflurane-induced neurotoxicity. Also, as anesthetic exposure differentially induces apoptotic cell death in different brain regions [50],uncorroborated as- sumption that sevoflurane affects metabolism equally in all brain cells renders the association of the observed metabolomic changes and caspase-3 expression may be speculative in this study.

In conclusion, GC–MS-based metabolomic analysis can be used for studying anesthetic effects on the developing brain. The inhalational anesthetic sevoflurane treatment produced a significant metabolic signature in neonatal brains, altering metabolic pathways of glucose, lipids, and amino acids, intracellular antioxidant and osmolyte sys- tems. These metabolic interactions in the developing brain may be of particular importance during its growth spurt period since they are essential in neuronal survival, neu- rogenesis, and even neurotransmission in the brain. Im- portantly, GC–MS can be used to screen for metabolites altered by anesthetics, which can complement anatomical and functional approaches. Although we did not use these

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differential metabolites to predict biochemical/cellular damages following sevoflurane treatment, metabolomic analysis which incorporates other analysis, such as pro- teomics, can better interpret our findings in this study.

Acknowledgments This work was supported by Natural Science Foundation of China (to Jun Zhang, No. 81171020). Thanks for as- sistance of Biotree Biotechnology Co. Ltd (Shanghai, China) on GC– MS analysis.

Conflict of interest Bin Liu, Yuechao Gu, Hongyan Xiao, Xi Lei, Weimin Liang and Jun Zhang reported no conflicts of interest.

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50. Deng M, Hofacer RD, Jiang C, Joseph B, Hughes EA, Jia B, Danzer SC, Loepke AW (2014) Brain regional vulnerability to anaesthesia-induced neuroapoptosis shifts with age at exposure and extends into adulthood for some regions. Br J Anaesth 113:443–451

123",rats,"['Seven-day-old rat pups were divided into two groups: control group (Group C) and sevoflurane group (Group S, 3 % sevoflurane exposure for 6 h).']",postnatal day 7,"['Seven-day-old rat pups were divided into two groups: control group (Group C) and sevoflurane group (Group S, 3 % sevoflurane exposure for 6 h).']",N,[],sevoflurane,"['Seven-day-old rat pups were divided into two groups: control group (Group C) and sevoflurane group (Group S, 3 % sevoflurane exposure for 6 h).']",none,[],sprague dawley,['Sprague–Dawley (SD) rats used in the present study were obtained from the Animal Care Center of Fudan'],None,[],GC–MS is a strategic and complementary platform for the metabolomic characterization of sevoflurane-induced neurotoxicity in the developing brain.,['Our study also confirmed that GC–MS is a strategic and complementary platform for the metabolomic characterization of sevoflurane-induced neurotoxicity in the developing brain.'],None,[],"Blood gas abnormalities including hypercarbia and oxygenation depression were found during sevoflurane exposure, which may also produce profound metabolic effects on the neonatal brain.","['There are some limitations to our study. First, blood gas abnormalities including hypercarbia and oxygenation depression were found during sevoflurane exposure.']",None,[],True,True,True,True,True,True,10.1007/s11064-015-1529-x
10.1213/ANE.0000000000000380,276.0,Peng,2014,rats,postnatal day 7,N,isoflurane,none,sprague dawley,"H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l

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Neuroscience in Anesthesiology and Perioperative Medicine

Section Editor: Gregory J. Crosby

Anesthetic Preconditioning Inhibits Isoflurane-Mediated Apoptosis in the Developing Rat Brain

Jun Peng, MD, Julie K. Drobish, MD, Ge Liang, MD, Zhen Wu, MD, Chunxia Liu, MD, Donald J. Joseph, PhD, Hossam Abdou, BS, Maryellen F. Eckenhoff, PhD, and Huafeng Wei, MD, PhD

BACKGROUND: We hypothesized that preconditioning (PC) with a short exposure to isoflurane (ISO) would reduce neurodegeneration induced by prolonged exposure to ISO in neonatal rats, as previously shown in neuronal cell culture. METHODS: We randomly divided 7-day-old Sprague-Dawley rats into 3 groups: control, 1.5% ISO, and PC + 1.5% ISO. The control group was exposed to carrier gas (30% oxygen balanced in nitrogen) for 30 minutes and then to carrier gas again for 6 hours the following day. The 1.5% ISO group was exposed to carrier gas for 30 minutes and then to 1.5% ISO for 6 hours the fol- lowing day. The PC + 1.5% ISO group was preconditioned with a 30-minute 1.5% ISO exposure and then exposed to 1.5% ISO for 6 hours the following day. Blood and brain samples were col- lected 2 hours after the exposures for determination of neurodegenerative biomarkers, includ- ing caspase-3, S100β, caspase-12, and an autophagy biomarker Beclin-1. RESULTS: Prolonged exposure to ISO significantly increased cleaved caspase-3 expression in the cerebral cortex of 7-day-old rats compared with the group preconditioned with ISO and the controls using Western blot assays. However, significant differences were not detected for other markers of neuronal injury. CONCLUSIONS: The ISO-mediated increase in cleaved caspase-3 in the postnatal day 7 rat brain is ameliorated by PC with a brief anesthetic exposure, and differences were not detected in other markers of neuronal injury. (Anesth Analg 2014;119:939–46)

Isoflurane (ISO) is a widely used general anesthetic

for both adult and pediatric surgeries. Many studies have been performed to elucidate the harms and ben- efits of ISO on neurons1–9 and in the developing brain.4,10–12 Depending on the circumstances, ISO has been reported to have both neurotoxic and neuroprotective effects.

A large number of in vivo studies have shown that ISO causes apoptosis in the developing brain of various species of animals4,10,11,13–15 and that subsequent learning and mem- ory are impaired.10,11 Furthermore, ISO has also been shown to be toxic in various cell culture models.2,4,16,17

From the Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

Jun Peng, MD, is currently affiliated with Department of Anesthesia, Second Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.

Zhen Wu, MD, is currently affiliated with Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.

Chunxia Liu, MD, is currently affiliated with Department of Anesthesiology, China-Japan Friendship Hospital, Beijing, China.

Accepted for publication June 13, 2014.

Other studies have also shown that ISO can have neuro- protective effects. In particular, when ISO is used as a pre- conditioning drug, it can provide neuroprotection against various hypoxic-ischemic insults to the developing rodent brain.12,18–21 Previous in vitro work from our laboratory, and others, has shown that preconditioning with ISO has a protective effect on neuronal cell cultures subsequently exposed to ISO for a longer duration.2 However, inhibition of ISO-induced neuronal apoptosis during brain develop- ment by preconditioning has not yet been examined.

Given that ISO has been a successful preconditioning drug against subsequent anesthetic exposure for devel- oping neurons in vitro2 and also against brain infarction induced by hypoxia and/or ischemia in vivo,12,18,20,22,23 we hypothesized that preconditioning with a short exposure to ISO would reduce neurodegeneration induced by a pro- longed exposure to ISO in an animal model. We assessed the effects of 1.5% ISO exposure for 6 hours on apoptotic biomarkers in 7-day-old rats and then determined whether preconditioning with a short exposure to 1.5% ISO for 30 minutes changed the apoptotic response.

Funding: Supported by National Institute of General Medicine (NIGMS), National Institutes of Health (GM-073224, GM084979, GM084979-02S1 to HW), Bethesda, MD, March of Dimes Birth Defects Foundation Research Grant ( 12-FY08-167 to HW), White Plains, NY, Research Fund at the Department of Anesthesiology and Critical Care, University of Pennsylvania (to HW), Philadelphia, PA.

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The authors declare no conflicts of interest.

This report was previously presented, in part, at the Society for Neuroscience, 2010, San Diego, CA. Program

157.6.

Reprints will not be available from the authors.

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Address correspondence to Huafeng Wei, MD, PhD, Department of Anes- thesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, 305 John Morgan Bldg, 3620 Hamilton Walk, Philadelphia, PA 19104. Address e-mail to Huafeng.Wei@uphs.upenn.edu.

Copyright © 2014 International Anesthesia Research Society DOI: 10.1213/ANE.0000000000000380

METHODS Animals The experimental procedures and protocols used in this study were approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania. All efforts were made to minimize the number of animals used and their suffering. Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were housed with a 12-hour light–dark cycle at 22°C, with food and water pro- vided ad libitum. Thirty-eight postnatal day 7 (P7) rats were used for the enzyme-linked immunosorbent assay (ELISA)

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RESEARCH REPORT

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Preconditioning Reduces Neonatal Anesthetic-Induced Apoptosis

and Western blots and 11 for immunohistochemistry, with approximately equal numbers of male and female rat pups randomly assigned to each condition.

Anesthesia Exposure The groups of rats were exposed to treatments in parallel. The minimum number of rats in each group was deter- mined by a power analysis. We anticipated that a large effect size would be clinically significant and chose an effect size of 1.3, and using the desired statistical level of 0.8 and probability level of 0.05, we determined a minimum sample size per group (2-tailed hypothesis) of 11 animals. P7 rats were placed in plexiglass chambers resting in a 37°C water bath to maintain a constant environmental temperature. The rat pups were exposed in these chambers to carrier gas (30% oxygen balanced in nitrogen) for 30 minutes and then to 1.5% ISO for 6 hours the following day (1.5% ISO) or pre- conditioned (PC) with a 30-minute 1.5% ISO exposure and then exposed to 1.5% ISO for 6 hours the following day (PC + 1.5% ISO). The control animals were exposed to carrier gas (30% oxygen balanced in nitrogen) for 30 minutes and then to carrier gas again for 6 hours the following day in the plexiglass chambers but not in the water bath. Exposure to ISO for 30 minutes alone at P7 has been shown not to be detrimental,12 and thus this control group was not included. To maintain a steady state of anesthetic gas and to prevent accumulation of expired carbon dioxide within the chamber, we used 6 L of total gas flow throughout the experiments. The ISO, oxygen, and carbon dioxide levels in the chamber were monitored using IR absorbance (Ohmeda 5330, Datex- Ohmeda, Louisville, CO) as described in our previous stud- ies.4,15,24 Two rats died during exposure to 1.5% ISO for 6 hours, 1 from the ISO alone group and the other from the PC plus ISO group.

Determination of Plasma S100β Two hours after the completion of the anesthetic treatment, P7 rats from the control, 1.5% ISO, and PC + 1.5% ISO groups were deeply anesthetized with 2% to 3% ISO. Blood (0.1 mL) was collected from the left ventricle and cen- trifuged to separate the plasma. We measured levels of S100β, a neuronal injury marker, using Sangtec 100 ELISA kits (DiaSorin Inc., Stillwater, MN) following the manufac- turer’s protocol and as we described previously.25 Briefly, 50 μL plasma from each rat was placed in each well of a 96-well plate and mixed with 150 μL tracer from the kit and incubated for 2 hours. Afterward, 3,3′,5,5′tetramethyl- benzidine substrate and stop solution were added to each well. The optical density was read at 450 nm. The sensi- tivity was determined by plotting the standard curve and then measuring concentrations of the samples from the standard curve.

Western Blot Assays Western blots were performed as we described previously.15,24 Two hours after the ISO exposure, after the mice were anesthe- tized and blood samples collected from the heart (see above), the mice were perfused with ice-cold saline through the heart and the parietal cortex dissected, frozen in liquid nitrogen, and stored at −80°C. At the time of the assay, the brain tissue

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from the P7 rat cortical tissue was thawed and homogenized and the total protein concentrations were quantified. The proteins were then separated by 12% gel electrophoresis and were transferred to a nitrocellulose membrane. The blots were incubated with an antibody against cleaved caspase-3 9664; Cell Signaling Technology, Danvers, (Cell Signaling 2202), or Beclin-1 (Cell MA), caspase-12 (Cell Signaling # Signaling 3495). The density was measured by Quantity # One software (BIO-RAD version 4.5.0, BIO-RAD, Hercules, CA) and GS-800 Densitometer (BIO-RAD), and the data are expressed as the percent of control of the means from 1 blot per animal per group.

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Immunohistochemistry Immunohistochemical localization of caspase-3 was per- formed in a separate group of P7 rats, as previously described.15 Briefly, 2 hours after the ISO exposure, P7 pups were deeply anesthetized with ISO and transcardially per- fused with ice-cold saline before the brains were removed, fixed with 4% paraformaldehyde, cryoprotected in 30% sucrose, frozen in isopentane, and stored at −80°C. Coronal cryosections (10 μm) were incubated in 3% hydrogen perox- ide, 10% normal goat serum, and cleaved caspase-3 antibody (1:400; Cell Signaling 9664) overnight at room temperature. The next day, the sections were incubated with Alexa Fluor® 594 goat anti-rabbit IgG (Life Technologies, Grand Island, NY) and coverslipped using ProLong® Gold Antifade Reagent containing the nuclear stain, 4’,6-diamidino-2-phenylindole (Invitrogen, Life Technologies). Quantitative imaging was conducted on an Olympus IX70 microscope equipped with a Cooke SensiCam camera (Applied Scientific Instrumentation, Eugene, OR) and IP lab 4.0 software (Biovision Technologies, Exton, PA). Caspase-positive and total number of cells were

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Figure 1. Preconditioning (PC) inhibits the increase of cleaved cas- pase-3 induced by isoflurane (ISO). A, Representative Western blot of cleaved caspase-3 in the cerebral cortex after 6-hour exposure to ISO (1.5% ISO) or PC with 1.5% ISO before the same 6-hour ISO exposure (PC + 1.5% ISO) at postnatal day 7. B, Quantitative anal- ysis of the Western blots of caspase-3, normalized to β-actin as percent of control, showed that prolonged exposure to 1.5% ISO significantly increased cleaved caspase-3 compared with controls (P = 0.0004), while PC (PC + 1.5% ISO) significantly ameliorated the increase compared with 1.5% ISO (P = 0.0009). The PC + 1.5% ISO group was also significantly different from controls (P = 0.0007). N = 16 for all groups, P < 0.001. Data are presented as box plots of the means with whiskers (minimum to maximum).

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ANESTHESiA & ANAlgESiA

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Table 1. P Values (95% Confidence Intervals) of the Western Blot and Immunohistochemical Analyses

Control versus isoflurane

Control versus preconditioning + isoflurane

Isoflurane versus preconditioning + isoflurane

Western blots Caspase-3 S100β Caspase-12 Beclin-1

0.0004 (−1310 to −428.7) 0.19 (−147.4 to 7.044) 0.96 (−79.41 to 48.46) 0.71 (−59.91 to 44.18)

0.0007 (−777.8 to 103.8) 0.49 (−101.0 to 53.43) 0.27 (−44.11 to 83.76) 0.57 (−38.61 to 63.09)

0.0009 (91.69 to 973.2)

0.34 (−23.46 to 116.2) 0.17 (−28.64 to 99.24) 0.53 (−30.75 to 70.96)

Immunohistochemistry Caspase-3 cortex Caspase-3 hippocampus

0.034 (−7.155 to −1.245) 0.034 (−4.346 to −1.804)

0.049 (−3.880 to 2.030) 0.21 (−1.696 to 0.846)

0.021 (0.539 to 6.011) 0.021 (1.474 to 3.826)

Table 2. Means ± SEs of the Western Blot and Immunohistochemical Analyses

Control versus isoflurane

Control versus preconditioning + isoflurane

Isoflurane versus preconditioning + isoflurane

Western blots Caspase-3 S100β Caspase-12 Beclin-1

100 ± 36.03 100 ± 6.26 100 ± 13.78 100 ± 16.63

1106 ± 219.7 170 ± 30.03 115.5 ± 22.06 107 ± 15.40

489.9 ± 116.3 123.8 ± 9.97 80.17 ± 15.79 87.76 ± 10.55

Immunohistochemistry

Caspase-3 cortex Caspase-3 hippocampus

0.4 ± 0.058

0.2 ± 0.116

4.600 ± 0.984

3.275 ± 0.335

1.325 ± 0.357

0.625 ± 0.290

detected in the number of caspase-3–positive cells in the ISO-exposed group compared with controls or the PC group in the cortex or the hippocampus (Fig. 2, B and C; Tables 1 and 2). These data support the claim that PC, with a short exposure to ISO, before a prolonged ISO exposure, significantly reduces the ISO-mediated apoptotic neuro- degeneration in the developing rat brain using Western blot assays.

The level of neurodegeneration was further studied by determining the plasma levels of S100β, a marker of neuro- nal injury, in P7 rats exposed to 1.5% ISO for 6 hours, with and without ISO PC (Fig. 3). The ELISA assay did not reveal significant differences in plasma S100β levels between these groups (Tables 1 and 2).

counted in the CA1 region of the hippocampus and the adja- cent parietal cortex at ×20 magnification. The brain sampled and analyzed in parietal cortex was the same region used in the Western blot from the opposite brain hemisphere. The mean number of cells was calculated from 3 sections per animal and the data expressed as the percentage of caspase- 3–positive cells in each region.

Statistical Analysis All data were analyzed using the Mann-Whitney U test to determine between-group differences and exact P values using STATA statistical software (StataCorp LP, College Station, TX). Differences were considered statistically sig- nificant at P < 0.01.

RESulTS Preconditioning Significantly Reduces ISO’s Apoptotic Effect This study tested the effects of PC, with a short exposure to ISO before a prolonged exposure to ISO, on apoptotic neurodegeneration in postnatal rats. The level of apopto- sis was evaluated by determining cleaved caspase-3 levels in the cerebral cortex using Western blot immunoassays (Fig. 1A). The cleaved caspase-3 levels, expressed as per- cent of control, in the P7 rats exposed to ISO alone were significantly higher than the PC group (P = 0.0009) or the controls (P = 0.0004; Fig. 1B; Tables 1 and 2). In addition, the animals with PC with ISO were also significantly dif- ferent from controls (P = 0.0007; Fig. 1B; Tables 1 and 2). When the P7 cortex and hippocampus were quantita- tively analyzed for the immunohistochemical localiza- tion of caspase-3 (Fig. 2), significant differences were not

The effect of PC on the apoptotic pathway was also examined by caspase-12 activation in the developing brain. Western blot analysis of caspase-12 levels in the P7 cerebral cortex (Fig. 4A) after either a 6-hour exposure to 1.5% ISO or PC with ISO before the prolonged exposure were not sig- nificantly different from controls (Fig. 4B; Tables 1 and 2).

Effect of ISO Treatment on Autophagy Autophagy, after anesthetic exposures, with and without PC, was examined in the P7 developing brain by measuring Beclin-1 levels. Western blot analysis (Fig. 5A) showed that a 6-hour ISO exposure did not significantly change Beclin-1 levels (Fig. 5B; Tables 1 and 2).

DISCuSSION This study provides new evidence that PC with ISO, before a long ISO exposure, significantly decreases ISO-mediated apoptosis in the developing brain. This finding is based on a significant reduction in the caspase-3 levels using Western blot assays. However, significant differences were not detected in caspase-3 levels in the cerebral cortex and hippocampus after either ISO exposure or PC. Other mark- ers of neuronal injury, S100β, caspase-12, and Beclin-1, were not significantly affected by either the prolonged ISO exposure or PC. While we have been able to exclude a large effect of these markers, it is possible that we could not detect significant effects due to our small sample size (e.g., Figs. 2 and 3).

Caspase-3 is one of the final mediators of the apoptotic pathway and is a well-established biomarker of apoptosis. The most important aspect of our results is that ISO PC decreased caspase-3 levels induced by a prolonged ISO exposure in the developing brain. In a similar study, Shu et al26 showed that xenon pretreatment before a combined

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Figure 3. Effects of isoflurane (ISO) preconditioning (PC) on S100β in plasma. No significant differences were found in plasma S100β levels with enzyme-linked immunosorbent assays from postnatal day 7 (P7) rats after exposure to 1.5% ISO for 6 hours with (P = 0.34) or without PC (P = 0.19) or with PC (PC + 1.5% ISO) compared with controls (P = 0.049). Controls, n = 9; 1.5% ISO, n = 13; PC + 1.5% ISO, n = 13. Data are presented as box plots of the means with whiskers (minimum to maximum).

ISO/nitrous oxide exposure decreased apoptosis, while nitrous oxide PC had no effect. Furthermore, we have previ- ously shown that sevoflurane PC can also inhibit neuronal cell death induced by prolonged exposure to ISO.2 Thus, more studies are necessary to determine the mechanism for the protective effect of PC so that novel approaches can be developed to mimic this effect.

Anesthetics have been shown to be both neuroprotec- tive and neurotoxic in the developing brain, and thus con- centrations and durations must be considered in pediatric

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Figure 2. Isoflurane (ISO) precondi- tioning (PC) significantly inhibits neu- ronal apoptosis induced by prolonged exposure to ISO in the cerebral cortex and hippocampus. (A), Representative images of immunostaining for cleaved caspase-3 in the postnatal day 7 (P7) rat cerebral cortex (left panels) and hippocampus (right panels) of con- trols (top panels), after ISO exposure (middle panels), and PC with a brief anesthetic exposure before the ISO exposure (PC + ISO; bottom panels). Apoptotic cells are stained for cas- pase-3 (red), and total cells are stained with 4’,6-diamidino-2-phenylindole (blue). The insets indicate the areas sampled in the cortex and hippocam- pus for quantitation. Scale bar, 25 μm. Quantitative analysis of the percentage of cleaved caspase-3–positive cells in (B) the cerebral cortex and (C) the hip- pocampal CA1 region. No significant differences were found in the number of caspase-3–positive cells in the cor- tex in the ISO (P = 0.034) or PC + ISO (0.049) groups compared with controls or between the ISO and PC groups (P = 0.021) or in the hippocampus (P = 0.034, P = 0.21, P = 0.021, respectively). Data represent the mean of 3 adjacent brain sections per animal, n = 4 animals for both experimental groups and n = 3 for controls. Data are presented as box plots of the means with whiskers (minimum to maximum).

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Figure 4. Effects of isoflurane (ISO) on cleaved caspase-12 in the cerebral cortex. A, Representative Western blot of cleaved cas- pase-12 in the cerebral cortex after 6-hour exposure to ISO (1.5% ISO) or preconditioning (PC) with 1.5% ISO before the same 6-hour ISO exposure (PC + 1.5% ISO). B, Quantitation of caspase-12 levels, normalized to β-actin as percent of control, showed no significant differences in the levels of cleaved caspase-12 in the postnatal day 7 (P7) brain between groups exposed to 1.5% ISO for 6 hours com- pared with controls (P = 0.96) or compared with ISO PC (P = 0.17) or the PC group compared with controls (P = 0.27). Controls, n = 9; 1.5% ISO, n = 9; PC + 1.5% ISO, n = 9. Data are presented as box plots of the means with whiskers (minimum to maximum).

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Figure 5. Effects of isoflurane (ISO) exposure on Beclin-1 in the cere- bral cortex. A, Representative Western blot of Beclin-1 levels in the cerebral cortex after a 6-hour exposure to ISO (1.5% ISO) or precon- ditioning (PC) with 1.5% ISO before the same 6-hour ISO exposure (PC + 1.5% ISO). B, Quantitative analysis of Beclin-1, normalized to β-actin as percent of control, showed that 1.5% ISO for 6 hours, with (P = 0.53) or without ISO PC (P = 0.71), or PC compared with controls (P = 0.57) did not significantly affect the levels of Beclin-1 in the cerebral cortex at postnatal day 7 (P7). Controls, n = 10; 1.5% ISO, n = 10; PC + 1.5% ISO, n = 11. Data are presented as box plots of the means with whiskers (minimum to maximum).

anesthetic practice. Our previous study suggested that prolonged exposure to sevoflurane can induce neuronal damage in vitro.27 The IV anesthetic, propofol, has also been shown to be both neurotoxic28,29 and neuroprotective against brain damage induced by ischemia and other stress factors.30,31 Therefore, it is important to investigate the dose and time responses of anesthetic-induced effects in the developing brain to use their neuroprotective features but minimize their neurotoxic effects.

S100β, a dimeric cytosolic calcium-binding protein released by glial cells, is a biomarker of blood–brain bar- rier dysfunction32 and overall brain distress.33 It has been studied clinically as a biomarker for traumatic brain injury and hypoxic-ischemic brain injury.34,35 We have previously studied S100β in the developing fetal rat brain and found that an in utero exposure to 3% ISO for 1 hour resulted in higher levels of S100β in the plasma of fetal rats when compared with controls.25 Furthermore, we showed an increase in plasma S100β after exposure to a subclinical concentration of ISO in neonatal mice.15 Though the cur- rent study did not show a significant difference in S100β, S100β may be a useful biomarker to detect anesthetic- mediated damage in the developing brain as indicated above, although further studies are needed to investigate its role in pediatric patients.

Caspase-12, part of the apoptotic pathway, is activated by disruption of the calcium homeostasis in the endoplasmic reticulum (ER).36 Anesthetics have been shown to cause cal- cium dysregulation in the ER via multiple mechanisms.37 In immature hippocampal neurons, ISO exposure was shown to enhance γ-aminobutyric acid–induced intracellular cal- cium increase, which was blocked by dantrolene, indicating that ISO exposure causes ryanodine receptor–dependent calcium release from the ER,17 which is consistent with our

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previous studies in different types of neurons.5 Other stud- ies also suggest that ISO exposure during brain develop- ment causes increased activation of inositol triphosphate receptors (InsP3R), resulting in increased calcium release from the ER leading to cell damage and neurodegenera- tion.3,4,27,38 Furthermore, caspase-12–positive neurons in the hippocampus were significantly increased in fetal rats exposed to 1.3% ISO for 4 hours.39 Caspase-12 has also been shown to indirectly activate caspase-3 in the neuronal apop- totic pathway.40 Although a previous study suggested that ISO-induced neuroapoptosis during brain development in rodents involved both intrinsic and extrinsic pathways,41 it is not clear whether the caspase-12–dependent pathway is also involved. In this study, we did not find significant cas- pase-12 activation after the ISO exposures, possibly because our ISO concentration may not have been high enough to cause ER stress and caspase-12–dependent neuroapoptosis. Further dose-dependent and exposure duration studies are needed to clarify this question.

Beclin-1, a protein required for autophagosome forma- tion, is an important regulator and biomarker of autophagy activity.42–44 Interestingly, autophagy may have both ben- eficial and harmful effects on the brain, depending on the experimental conditions. Autophagy appears to be essential to both ischemic and hyperbaric oxygen PC before cerebral ischemia.45 Furthermore, reducing Beclin-1 levels has been shown to exacerbate neurodegeneration in Alzheimer dis- ease models, while overexpression of Beclin-1 can prevent neuronal cell death.46 Autophagy activity can be regulated by InsP3R activity, while ISO has been shown to activate InsP3R and cause cell apoptosis by overactivation of InsP3R.47,48 In addition, autophagy activity may be an upstream regulator of apoptosis, and excessive autophagy may lead to cell death by apoptosis. The effects of InsP3R activity on autophagy depend on the level of InsP3R activation, which then deter- mines whether the effect will be protective or toxic. While this study did not find an effect on Beclin-1 expression at P7 after exposure to 1 concentration of ISO, further studies are needed to investigate the effects of general anesthetics on cell autophagy, and therefore neuroprotection or neurotoxic- ity, especially in the developing brain.

While we have not yet discovered the mechanism by which ISO PC prevents ISO-induced apoptosis, other groups have studied ISO PC before cerebral ischemia and have linked several mechanisms to this process. One study found that ISO PC caused a decrease in glutamate receptor activa- tion,49 and another found that it decreased protein aggrega- tion.50 Changes in the expression of various genes have also been discovered, but the significance of these genetic changes has not been fully determined.51–53 Furthermore, ISO may provide PC neuroprotection by causing a moderate increase of cytosolic calcium concentrations via adequate activation of the InsP3R calcium channel.54,55 While we recognize that ISO PC for brain ischemia is not the same as for a subsequent anesthetic exposure, it is possible that there may be similari- ties in the cascade of events and consequences. One of the limitations of the current study is that we only investigated a few of the potential mechanisms underlying the dual effects of neuroprotection and neurotoxicity caused by ISO. Future

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experiments could continue to address the many other mech- anisms that are likely involved in this process.

The long-term behavioral adaptations to the effects of preadolescent drug exposures are more permanent com- pared with the same exposures later in life,56 with the peak period of anesthetic-induced apoptosis occurring during synaptogenesis.57 This vulnerable period during rat devel- opment is between approximately postnatal days 2 and 14 and between postconception day 153 and postnatal day 288 in the human, based on a species prediction model recently developed to correlate the timing of neural events between species.58 Translating exact developmental milestones between rats and humans is complicated. Postnatal brain maturation in the rat encompasses many developmental events, such as neurogenesis, neuronal migration, synapto- genesis, and apoptosis, the extent of which varies greatly, depending on the brain region of interest.59,60

The present study tested the hypothesis that PC with ISO can prevent apoptosis caused by a prolonged exposure to ISO, and our results with caspase-3, but not other mark- ers of neuronal injury, support this hypothesis and indicate that in vivo ISO PC is neuroprotective, while a prolonged exposure to ISO is neurotoxic during early postnatal brain development. It is important to determine the optimal con- centration and duration range for anesthetic exposures dur- ing postnatal brain development, which has implications for our pediatric patients. E

DISClOSuRES Name: Jun Peng, MD. Contribution: This author helped design the study, conduct the study, and analyze the data. Attestation: Jun Peng has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Name: Julie K. Drobish, MD. Contribution: This author helped with the writing of the manuscript. Attestation: Julie K. Drobish has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Name: Ge Liang, MD. Contribution: This author helped design the study, conduct the study, and analyze the data. Attestation: Ge Liang has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Name: Zhen Wu, MD. Contribution: This author helped conduct the study and ana- lyze the data. Attestation: Zhen Wu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Name: Chunxia Liu, MD. Contribution: This author helped conduct the study and ana- lyze the data. Attestation: Chunxia Liu has seen the original study data, reviewed the analysis of the data, and approved the final manuscript. Name: Donald J. Joseph, PhD. Contribution: This author helped conduct the study and ana- lyze the data. Attestation: Donald J. Joseph reviewed the analysis of the data and approved the final manuscript. Name: Hossam Abdou, BS.

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Contribution: This author helped with the writing of the manuscript. Attestation: Hossam Abdou reviewed the analysis of the data and approved the final manuscript. Name: Maryellen F. Eckenhoff, PhD. Contribution: This author helped with the writing of the manuscript. Attestation: Maryellen F. Eckenhoff reviewed the analysis of the data and approved the final manuscript. Name: Huafeng Wei, MD, PhD. Contribution: This author helped design the study, analyze the data, and write the manuscript. Attestation: Huafeng Wei has seen the original study data, reviewed the analysis of the data, approved the final manu- script, and is the author responsible for archiving the study files. This manuscript was handled by: Gregory J. Crosby, MD.

ACKNOWlEDGMENTS The authors would like to thank Rebecca Speck, PhD, MPH, Department of Anesthesiology and Critical Care, University of Pennsylvania, for advice and assistance with the statistical analyses.

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ANESTHESiA & ANAlgESiA",rats,"['We randomly divided 7-day-old Sprague-Dawley rats into 3 groups: control, 1.5% ISO, and PC + 1.5% ISO.']",postnatal day 7,"['We randomly divided 7-day-old Sprague-Dawley rats into 3 groups: control, 1.5% ISO, and PC + 1.5% ISO.']",N,[],isoflurane,['Anesthetic Preconditioning Inhibits Isoflurane-Mediated Apoptosis in the Developing Rat Brain'],none,[],sprague dawley,"['We randomly divided 7-day-old Sprague-Dawley rats into 3 groups: control, 1.5% ISO, and PC + 1.5% ISO.']",Inhibition of ISO-induced neuronal apoptosis during brain development by preconditioning has not yet been examined.,"['However, inhibition of ISO-induced neuronal apoptosis during brain development by preconditioning has not yet been examined.']",None,[],The ISO-mediated increase in cleaved caspase-3 in the postnatal day 7 rat brain is ameliorated by PC with a brief anesthetic exposure.,"['The ISO-mediated increase in cleaved caspase-3 in the postnatal day 7 rat brain is ameliorated by PC with a brief anesthetic exposure, and differences were not detected in other markers of neuronal injury.']",None,[],None,[],True,True,True,True,True,True,10.1213/ANE.0000000000000380
10.1007/s11064-021-03301-5,214.0,Wen,2021,mice,postnatal day 7,N,isoflurane,none,fmr1-ko,"Neurochemical Research (2021) 46:1577–1588 https://doi.org/10.1007/s11064-021-03301-5

ORIGINAL PAPER

Early Isoflurane Exposure Impairs Synaptic Development in Fmr1 KO Mice via the mTOR Pathway

Jieqiong Wen1,3 · Jing Xu2,3 · R. Paige Mathena3 · Jun H. Choi3 · C. David Mintz3

Received: 19 January 2021 / Revised: 23 February 2021 / Accepted: 17 March 2021 / Published online: 31 March 2021 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract General anesthetics (GAs) may cause disruptions in brain development, and the effect of GA exposure in the setting of pre- existing neurodevelopmental disease is unknown. We tested the hypothesis that synaptic development is more vulnerable to GA-induced deficits in a mouse model of fragile X syndrome than in WT mice and asked whether they were related to the mTOR pathway, a signaling system implicated in both anesthesia toxicity and fragile X syndrome. Early postnatal WT and Fmr1-KO mice were exposed to isoflurane and brain slices were collected in adulthood. Primary neuron cultures isolated from WT and Fmr1-KO mice were exposed to isoflurane during development, in some cases treated with rapamycin, and processed for immunohistochemistry at maturity. Quantitative immunofluorescence microscopy was conducted for synaptic markers and markers of mTOR pathway activity. Isoflurane exposure caused reduction in Synpasin-1, PSD-95, and Gephyrin puncta that was significantly lower in Fmr1-KO mice than in WT mice. Similar results were found in cell culture, where synapse loss was ameliorated with rapamycin treatment. Early developmental exposure to isoflurane causes more profound synapse loss in Fmr1- KO than WT mice, and this effect is mediated by a pathologic increase in mTOR pathway activity.

Keywords Anesthesia · Brain Development · mTOR · Fmr1

Introduction

In late 2016, the U.S. Food and Drug Administration issued a warning that repeated or prolonged exposure (more than 3 hours) of general anesthesia and sedatives for children under 3 years of age and pregnant women in their third trimester may impair neurodevelopment [1]. This announcement was based on a wealth of animal research primarily in rodents [2–10], but also in nonhuman primates [11–13] showing that early developmental exposure to general anesthesia disrupts a wide range of neurodevelopmental processes and results in lasting cognitive deficits as measured by behavioral test- ing. It was also based on a number of epidemiologic studies

C. David Mintz cmintz2@jhmi.edu

showing that lengthy or multiple general anesthetics were associated with worsened outcomes related to cognition and/ or behavior [14–18]. Recent clinical trials have given some reassurance that short anesthetics for surgery in healthy children do not have negative effects on measures of intel- ligence at least during early childhood [19–21], although lingering concerns still exist related to secondary outcomes focused on behavioral even in these studies [22]. A striking feature of nearly all the literature describing both human and animal research in this area is the focus on the effects of anesthetics on the normal brain. The effects of anesthet- ics as neurotoxins in a brain potentially rendered vulnerable by disease or injury, a scenario which is not uncommon in clinical practice, are largely unexplored. In the experiments described in this manuscript, we explore the question of whether neurodevelopmental injury from anesthesia expo- sure might be worsened in the context of pre-existing disease that adversely impacts brain development.

1 Department of Anesthesiology, The Second Affiliated

Hospital of Xi’an Jiaotong University, Xi’an 710004, China 2 Department of Anesthesiology, The First Affiliated Hospital

of Xi’an Jiaotong University, Xi’an 710061, China

3 Department of Anesthesiology and Critical Care Medicine,

The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

Fragile X syndrome (FXS) is the most common genetic cause of intellectual disability. It is caused by mutations in the Fmr1 gene which codes the fragile mental retardation 1 protein (FMRP). FMRP is widely expressed in the brain and can be detected in soma, dendrite, and synapses of

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neuronas. It is believed to be critically involved in neural plasticity, via its actions regulating the synthesis of syn- aptic proteins [23, 24]. It is commonly studied with the Fmr1 knockout (KO) mouse model, which exhibits notable alterations in synaptic structure and function, as well as displaying behavioral abnormalities including repetitive behaviors, anxiety, and hyperreactivity [25, 26]. Early developmental exposure to general anesthetics have been shown to disrupt the structure and function of synapses in the hippocampus, neocortex, and other brain regions e.g. [27–33]. The combined effects on synaptic development of anesthetic exposure and FXS are unknown.

The molecular mechanisms underlying FXS pathology involve the disruption of numerous systems that control neuronal development, including changes in the mamma- lian target of rapamycin (mTOR) pathway [34, 35]. The mTOR protein is a serine/threonine protein kinase that plays a central role in a complex system which integrates a wide range of intra and extracellular inputs to control cel- lular metabolism [36]. It is critically important for normal neuronal development and alterations in mTOR signaling have been implicated in a wide range of neurodevelop- mental disease [37–40]. Studies of both human tissue and of mouse models of FXS suggest that an inappropriate increase in activity in the mTOR pathway may play an important causative role in the pathology of this disorder [41–43]. Previous work from our group and others sug- gests that early developmental exposure to general anes- thetics may cause a pathological upregulation in mTOR pathway signaling that in turn leads to disruptions in brain development and that some phenotypic elements of devel- opmental anesthetic neurotoxicity can be ameliorated with pharmacologic inhibition of the mTOR pathway [44–48]. Taken together, these data suggest the possibility that early developmental anesthetic exposure has the potential to worsen aberrant mTOR signaling in FXS patients.

Results

Isoflurane Worsens the Impaired Synaptogenesis Observed in Fmr1 KO Mice

To investigate the effects of developmental anesthesia expo- sure on synaptogenesis, wild type (WT) and Fmr1 KO mice were treated with 1.5% isoflurane for 4hrs on P7 and brains were harvested at P60 to detect lasting changes (Fig. 1a). Brain sections representing the hippocampal dentate gyrus were collected and fluorescently immunolabeled with anti- bodies against Synapsin-1 (universal pre-synaptic marker), PSD-95 (excitatory post-synaptic marker), and Gephyrin (inhibitory postsynaptic marker). We chose to focus on the dentate gyrus as it continues to develop postnatally in rodents, non-human primates, and humans during the pro- posed window of susceptibility to anesthetic toxicity, and we have previously shown it is susceptible to synaptic pathology as a result of early postnatal anesthetic exposure [44, 49].

Confocal microscopy was used to image synaptic markers and the fluorescence intensity of the markers and the number of synpatic puncta identified by each marker were measured. Fluorescence intensity is a measure of expression of the pro- tein the marker is raised against and puncta number serves as a measure of the number of synapses of the type represented by that marker. The dentate gyrus was identified with

DAPI staining (Fig. 2a). Representative images of high- powered fields with Synapsin-1, PSD-95, and Gephyrin immunolabeling that were used for analysis are shown in Fig. 2b, c, d, respectively. We found that early isoflurane exposure significantly decreased the intensity and puncta number of Synapsin- 1, PSD-95, and Gephyrin in the WT

Here we test the overall hypothesis that early develop- mental exposure to general anesthesia worsens synaptic pathology observed in FXS. We further ask whether this may be due to a synergistic effect on the mTOR pathway. To this end, we have conducted in vivo and in vitro immu- nohistochemistry experiments in the Fmr1 KO mouse model using synaptic markers and mTOR pathway activ- ity markers as well as pharmacologic inhibition of mTOR pathway activity.

Fig. 1 A schematic diagram of isoflurane exposure in vivo and in vitro. a Mice were exposed to 1.5% isoflurane for 4hrs on P7 and brain tissue was collected for immunofluorescence staining on P60. b Primary neurons were exposed to 1.8% isoflurane for 4 h on 5DIV with 100 nM rapamycin added into WT-Rapa and KO-Rapa groups 1 hr before isoflurane exposure. Cells were fixed for immunofluores- cence staining on 12DIV

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Fig. 2 Isoflurane inhibits synaptogenesis in vivo and the loss increases significantly in Fmr1 KO mice. (Aa) The white arrows indicate the imaged locations of synaptic markers (green) in dentate gyrus, shaped using DAPI (blue) (scale bar = 100 μm). (Ab) The representative immunofluorescence image of Gephyrin (green) and DAPI (blue) in dentate gyrus (scale bar = 50 μm). b–d The repre- sentative immunofluorescence images of Synapsin-1, PSD-95, and Gephyrin respectively (green), (scale bars = 5 μm). e After isoflurane exposure, the puncta number of Synapsin-1 significantly decreased in the WT and KO groups, and the puncta number of Synapsin-1 in

the KO- ISO group was smaller compared to the WT-ISO group. f After isoflurane exposure, the puncta number of PSD-95 significantly decreased in the WT and KO groups, and the puncta number of PSD-95 in the KO-ISO group was smaller compared to the WT-ISO group. g After isoflurane exposure, the puncta number of Gephyrin in the KO-CON group and KO-ISO group was lower compared to the WT-CON group and WT-ISO group, respectively. (n = 30 fields per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA with Tukey´s multiple comparisons test for e, f, g)

group (Fig. 2). In Fmr1 KO mice, isoflurane exposure resulted in no change in fluorescence intensity with Syn- apsin-1 and PSD-95 markers (Fig. 2b, c), yet showed a significant decrease in intensity in Gephyrin signal, which was also significantly lower than the intensity of Gephyrin signal in the WT-isoflurane group (Fig. 2d). Puncta number for all three synaptic markers was significantly reduced by isoflurane exposure in both WT and Fmr1 KO mice, and the number of puncta in the Fmr1 KO-isoflurane group was sig- nificantly reduced compared to WT-isoflurane (Fig. 2e–g). These data indicate that the number of all synapses, includ- ing excitatory and inhibitory synapses, in the dentate gyrus

are reduced in Fmr1 KO relative to WT, and that the degree of synapse loss in Fmr1 KO mice is worsened by early developmental isoflurane exposure relative to what is seen in WT mice exposed to isoflurane.

Isoflurane Increases Activity in the mTOR Signaling Pathway in Fmr1 KO Mice

In order to test the effect of early developmental anesthetic exposure on the mTOR signaling pathway in the setting of FXS, we conducted quantitative fluorescence immunohis- tochemistry in the same pool of brain sections described in

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2.1 above. Immunolabeling was conducted with antibodies specific for the active phosphyorylated states of key pro- teins in the mTOR pathway, including p-AKT, p-mTOR, and p-S6. DAPI staining was used to identify the dentate gyrus, and co-immunolabeling was conducted with parvalbumin to identify inhibitory interneurons, which in preliminary data were the most likely to exhibit changes in mTOR pathway activity markers (Fig. 3a). Confocal microscopy was con- ducted in the dentate gyrus region and the density of cells that exhibited positive labeling for each phospo-protein was measured. Representative images for each marker are shown

in Fig. 3b–d. The density of p-AKT and p-S6 positive cells were significantly higher in Fmr1 KO mice than WT mice in the absence of isoflurane exposure (Fig. 3e–g). With isoflu- rane exposure, there was a significant increase in the density of cells positive for all mTOR pathway activity markers in the WT group and a significant increase in the Fmr1 KO group both relative to the Fmr1 KO control group and to the WT isoflurane group (Fig. 3e–g).

These data indicate that pathologic upregulation of mTOR in Fmr1 KO is enhanced by early developmental exposure to to isoflurane.

Fig. 3 Isoflurane over activates the mTOR signaling pathway in Fmr1 KO mice. a The representatively magnified images of positive cells the representative image shows p-S6 (green), DAPI (blue), and PV (red). b–d) The representative immunofluorescence images of p-AKT, p- mTOR, and p-S6 in dentate gyrus, p-AKT/p-mTOR/p-S6 (green), DAPI (blue), and PV (red) (scale bars = 50 μm), all of the arrows indicate the positive cells and the hallow arrows indicate the magnified cells shown in lower left corner. e KO-ISO group had a higher density of p-AKT positive cells compared to KO-CON and

WT-ISO groups. f After isoflurane exposure, the density of p-mTOR positive cells increased in WT and KO groups, and KO-ISO group had a higher density of p-AKT positive cells compared to WT-ISO groups. g After isoflurane exposure, the density of p-S6 positive cells increased in WT and KO groups, and KO-ISO group had a higher density of p-S6 positive cells compared to WT-ISO groups. (n = 20 fields per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA with Tukey´s multiple comparisons test for e, f, g)

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Isoflurane Causes Increased Loss of Synapses in Cultured Neurons Derrived from Fmr1 KO Neurons and Effects on Synpatogenesis can be Mitigated via Pharmacologic Inhibition of the mTOR

that the effects of isoflurane and Fmr1 mutation on synapse formation are medaited via the mTOR pathway.

Isoflurane Increases Activity in mTOR Neurons Cultured from Fmr1 KO Mice

Next, we chose to explore our underlying hypothesis in pri- mary neuronal culture. We did this for several reasons: 1. Anesthetic exposure in early postnatal mice can be com- plicated by alterations in physiology and homeostasis, and while we have previously shown that our exposure system does not cause very substantial disruptions [44], cell culture models do not have these limitations at all; 2. Anesthetic exposure impacts the development of both neurons and glia [3, 50] which have interrelated and co-dependent develop- mental processes that cannot be separated out easily in vivo, whereas in cell culture it is possible to study the effects on neurons in isolation; 3. Dentate gyrus neurons have some notable differences from other neuronal cell types, and our cell culture model, comprised of cortical neurons, represents a different and more diverse population, thus allowing us to ask whether our findings are generalizable. Neurons were isolated from WT and Fmr1 KO mice and exposed with isoflurane at 5DIV, the period of time when synapses begin to develop. In some experiments, rapamycin was used for pharmacologic inhibition. Cells were fixed and processed for quantitative fluorescence immunocytochemistry and images were taken via confocal microscopy for analysis (Fig. 1b).

First, we asked whether the effects on synapse numbers we observed were replicated in primary cell culture, using the same panel of synaptic marker antibodies. Figure 4a shows an example of a neuron, with DAPI to note the cell body, MAP-2 to identify the dendrites and confirm neuronal cell type, and Gephryin as an example of a synaptic marker. Results were obtained by analyzing intensity of synaptic marker labeling and counting puncta number on dendritic segments, and representative images of these segments are shown for Synapsin-1, PSD-95, and Gephyrin are shown in Fig. 4b–d, respectively. The number of Synapsin-1, PSD-95, and Gephyrin puncta in both WT and Fmr1 KO groups were all significantly decreased with isoflurane exposure as com- pared to carrier gas controls. The isoflurane- exposed Fmr1 KO group had significantly fewer puncta positive for Syn- apsin-1, PSD-95, and Gephryin than the WT group exposed to isoflurane. When neurons were treated with rapamycin, the puncta number of all three synaptic markers was signifi- cantly increased compared to the isoflurane-alone condition and it is restored to levels near baseline (Fig. 4e–g). The intensity of Synapsin-1 and PSD-95 decreased in the WT group and KO group after isoflurane, respectively (Fig. 2). These findings indicate that the synaptogenesis in Fmr1 KO neurons is impaired, and the loss of synapses is worsened by isoflurane exposure. Furthermore, treatment with rapamycin appears to prevent synapse loss, supporting the conclusion

Finally, we employed primary neuron culture to test the hypothesis that isoflurane exposure results in increased activity in the mTOR pathway relative to Fmr1 KO alone. To do so, we used immunocytochemistry and confocal micros- copy to measure the percentage of neurons that were positive for the p-AKT, p-mTOR, and p-S6 markers. We included a rapamycin treatment condition to verify that these markers were all indicative of increased activity in the mTOR path- way in the culture model.

Representative examples of immunolabeling for each mTOR pathway activity marker along with immunolabe- ling for MAP-2 to define neurons being measured are shown in Fig. 5a. A higher percentage of Fmr1 KO neurons were positive for p-AKT, p-mTOR, and p-S6 than were positive for these markers in the WT group (Fig. 5b–d). With isoflu- rane exposure, the percent of neurons positive for all of the mTOR pathway markers were increased both in the WT and Fmr1 KO groups, and the percentage of cells positive for p-mTOR and p-S6 was significantly greater in the Fmr1 KO isoflurane group than in the WT isoflurane-exposed group (Fig. 5b–d). In both WT and Fmr1 KO cultures, treatment with rapamycin singificantly reduced the percentage of neu- rons positive for p-AKT, p-mTOR, and p-S6 (Fig. 5b, c, d). These findings support the conclusion that early devel- pomental isoflurane exposure further upregulates signaling in the mTOR pathway in neurons with the Fmr1 mutation.

Discussion

In the present study, using both in vivo and in vitro model systems we found that early developmental isoflurane exposure interfered with synapse development in normal mice and that this effect was more pronounced in Fmr1 KO mice. Based on data showing upregulation in mTOR signaling markers with isoflurane exposure and mitiga- tion of the effects of isoflurane on synapses loss with an mTOR pathway inhibitor, we conclude that the mechanism involves a pathological increase in mTOR pathway activity. The CNS pathology of the Fmr1 KO mutation in mice has been well documented, thus making this an ideal model for the exploration of potentially synergistic effects of develop- mental anesthetic neurotoxicity in genetic disorders of brain development.

For example, synaptic pathology in Fmr1 KO mice has been well described. Compared with wild- type mice, the density of dendritic spines of vertebral neurons in the 2/3

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Fig. 4 Isoflurane inhibits synaptogenesis in vitro and exacerbates the impairments on Fmr1 KO neurons. a The representative immunofluo- rescence images of synaptic marker Gephyrin (green), DAPI (blue), and MAP2 (red) in neurons at 12DIV. The white box in the figure showed the location of the segments picked in each neuron for imag- ing the segment and nucleus are 20 μm apart (scale bar = 20 μm). b–d The representative images of Synapsin-1, PSD-95, and Gephy- rin in dendrites, Synapsin-1/PSD-95/Gephyrin (green), MAP2 (red) (scale bars = 5 μm). e The puncta number of Synapsin-1 significantly decreased in WT and KO groups after isoflurane exposure and could be reversed when added with rapamycin. The KO- ISO group had a

lower expression compared with the WT-ISO group. f The puncta number of PSD-95 significantly decreased after isoflurane exposure in WT and KO groups could be reversed when added with rapamycin. The KO-ISO had smaller puncta number than the WT- ISO group. g The puncta number of Gephyrin significantly decreased after isoflu- rane exposure in WT and KO groups could be reversed when added with rapamycin. The KO-ISO group had smaller number compared to the WT-ISO group. (n = 40 fields per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA with Tukey´s multi- ple comparisons test for e, f, g)

cortical layer of Fmr1 KO mice was increased, while the vol- ume of dendritic spines was smaller [51]. Through diolistic labeling and three- dimensional reconstruction, it has been shown that the density of dendritic spines increased, but the density of mature dendritic spines decreased in the hip- pocampal CA1 region of Fmr1 KO mice in P60 [52]. In general, studies of the effects of anesthetics on synapses have shown a decrease in synapse numbers with early developmental exposure [4, 6, 53], and there do not seem to

be reports of increased synapse numbers. Our work in the dentate gyrus, a hippocampal area which does have notable differences with the CA fields of the hippocampus, shows a particularly profound loss of mature dendritic spines [44], which might be interpreted as a feature in common with Fmr1 KO pathology. Whether proceeding via the same mechanism, as we hypothesize here, or not, a combina- tion of Fmr1 KO and early developmental anesthetic expo- sure would have the potential to profoundly reduce mature

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Fig. 5 Isoflurane activated the mTOR signaling pathway in vitro, and there was a hyperactivation in Fmr1 KO neurons compared with WT neurons. a The representative immunofluorescence images of p-AKT/ p-mTOR /p-S6 (green), MAP2 (red), and DAPI (blue) in neurons at 12DIV. White arrows indicate positive cells (scale bars = 50 μm). b After isoflurane exposure, the density of p- AKT positive cells increased in the WT and KO groups and reduced to baseline in KO group when rapamycin added. c After isoflurane exposure, the den- sity of p-mTOR positive cells increased in WT and KO groups and

reduced to baseline when rapamycin added. The KO-ISO group had a higher density of p-mTOR positive cells compared to the WT- ISO group. d After isoflurane exposure, the density of p-S6 posi- tive cells increased in WT and KO groups and reduced to baseline when rapamycin added. The KO-ISO had a higher density of p-S6 positive cells compared to the WT-ISO + V group. (n = 40 fields per group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA with Tukey´s multiple comparisons test for b, c, d)

dendritic spines. Given the importance of dendritic spines as the postsynaptic element of excitatory synapses in the cortex and hippocampus, an additive or synergistic effect on synap- tic loss has the potential to be devastating for brain function. Both the Fmr1 KO mutation and developmental anes- thetic neurotoxicity result in other characteristic pathological phenotypes besides synaptic disarray. Each condition has a

distinct effect on cell proliferation and survival. Fmr1 KO mice have substantially disordered brain cytoarchitecture, including altered cortical lamination, which is characterized by loss of both excitatory and inhibitory neurons [54].

This is accompanied by an increase in some populations of both oligodendrocytes and astrocytes [54]. The Fmr1 KO mutation has also been shown to inhibit neurogenesis

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in the hippocampal dentate gyrus [55]. The mechanisms underlying these deficits have not been established, but each is likely to contribute to the abnormal cognitive and behavioral phenotype observed in the Fmr1 KO mouse model of FXS [56].

Developmental anesthetic neurotoxicity also has well described phenotypes involving cell survival and prolif- eration. An acute increase in the levels of apoptotic death of neurons and other brain cell types has been reported as one of the major consequences of early postnatal anesthetic exposure [57–59], although it is unclear if it ultimately results in lasting decreases in neuronal cell numbers. Fur- thermore, several lines of evidence indicate that neuronal stem cell proliferation is inhibited, perhaps in a lasting fash- ion, by early anesthetic exposure [2, 60–62]. Our study did not examine effects on cell proliferation and survival, and we cannot predict what the outcome of combining genetic and toxic pathological effects might be, however it seems reasonable to predict that they would be more profoundly disruptive than either alone.

Our present study focused on the overarching hypoth- esis that early developmental isoflurane exposure and Fmr1 KO share a common potential mechanism that would be expected to disrupt brain development, namely a pathologi- cal upregulation of signaling in the mTOR pathway. Our previous work and that of others points to a chronic upregu- lation in mTOR pathway signaling that lasts for some time after the anesthetic exposure and which has been shown to play a causative role in resulting pathologies as evidenced by amelioration of these pathologies with pharmacologic inhibition of mTOR signaling [45, 47, 48, 63]. The myr- iad of phenotypic and resulting cognitive and behavioral abnormalities in FXS are likely attributable to multiple mechanisms and we do not seek to argue that mTOR is the only or the most important among them. However, there is substantial evidence of disrupted mTOR signaling associ- ated with FXS [34, 35, 41–43]. Also, our findings in this manuscript support the hypothesis of aberrant mTOR sign- aling in the Fmr1 KO mouse model of FXS. This begs the question of what neurodevelopmental consequences would be expected to arise from two distinct but convergent dis- ruptions of mTOR function during brain development. The answer is too complex to determine hypothetically, as the mTOR pathway regulates such a diverse array of develop- mental processes, including but not limited to regulation of neural stem and progenitor cell development, neuronal cell growth and differentiation, and the formation and mainte- nance of synapses [37, 40]. We predicted and found last- ing disruptions in synapse formation in the experiments of this manuscript, and we further predict that a wide array of abnormal developmental phenotypes will be uncovered with further exploration. Whether these findings would translate to human biology and what the consequences of lengthy or

repeated anesthetic exposure in FXS patients are can only be determined through patient-centered studies.

Our study has several limitations that should be noted. As with all animal models, the Fmr1 KO mouse is not a per- fect representation of FXS and anesthetic exposure in small rodents presents challenges in maintaining normal physiol- ogy to the degree that would be possible in human patients in an operating room setting, which is one reason we chose to replicate key findings in a cell culture environment. The Fmr1 KO occurs only in male mice, and thus if sex effects exert an effect on the interaction between disorders or brain development generally and anesthesia toxicity, they cannot be detected by our study. Also, because of the complexity of the mTOR pathway, both measurement of changes in it and pharmacologic interventions on it are invariably less than straightforward. However, we believe that in spite of any limitations, the data presented here represent a compelling argument to begin to consider whether inadvertent iatrogenic harm, which may in part be preventable, can result from the use of anesthetic and sedative drugs in patients with FXS and other developmental brain disorders. On this basis, we advocate for further exploration of this topic, including via clinical studies.

Methods

Animals

The C57BL/6 WT mice and heterozygous Fmr1 KO (HET) mice were purchased from the Jackson Laboratory. Mice were housed in a temperature-controlled and humidity- controlled room with a 12:12 h light: dark cycle and pro- vided with ad libitum access to water and food. All neo- natal offspring used in this study were a result of mating WT male mice with Fmr1 HET female mice. Samples from toe clipping on postnatal day 5 were sent to Transnetyx for genotyping. All protocols involving mice were approved by the Animal Care and Use Committee at the Johns Hopkins University and were conducted in accordance with the NIH guidelines for care and use of animals.

Isoflurane Exposure In vivo

P7 mice were randomly divided into two experimental groups: an isoflurane exposure group and a control group. All mice in the isoflurane exposure group underwent an induction period, in which they were exposed with 3% isoflurane (Baxter Healthcare, Cooperation, Deerfield, IL, USA) for 3 min or until loss of righting reflex, whichever was first. The isoflurane group mice were exposed to 1.5% isoflurane carried in 50% oxygen continuously for 4 h via a nosecone designed to minimize rebreathing of exhaled

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gases. For the control group, mice were separated from dams and exposed to room air for 4 h. During all exposures, mice were placed under a heat lamp and monitored for skin tem- perature, oxygen saturation, heart rate, and oxygen saturation (MouseOx, Starr Life Sciences, Oakmont, PA). Mice were returned to their home cage dam upon regaining righting reflex.

Primary Neuron Culture

Primary neurons were isolated from the dissected cortex of P0-P1 mice as described previously [63]. Due to the timing of tissue harvest, the genotype of each neonatal mouse was unknown so the brain tissue of each mouse was collected separately. Tail samples of the mice were sent to Transnetyx for genotyping to select WT and KO cells needed for the follow-up experiments. Neonatal mice were decapitated, and the heads were placed in 75% ethanol, then transferred to cold Hank’s Balanced Salt Solution (HBSS) without calcium and magnesium (Gibco, Carlsbad, CA, USA). The brains were collected after the skin and skull were removed. The cortex was isolated under a dissecting microscope, and the meninges were removed completely. Following the instruc- tion of the papain kit (Worthington Biochemical CoRapa, Lakewood, NJ, USA), the cortex was digested in 20 units/ ml papain and 0.005% DNAase at 37℃ for 30 minutes and albumin-ovomucoid inhibitor solution was added to stop the digestion. Tissue was further dissociated through gen- tle repeated pipetting. After allowing undissociated tissue to settle to the bottom of the tube, the supernatant liquid was collected and centrifugated at 1000 rpm for 5 min- utes. Then, the cell pellet was resuspended in neurobasal medium (Gibco, Carlsbad, CA, USA) supplemented by 2% B27 (Gibco, Carlsbad, CA, USA), 0.5mM GlutaMax (Gibco, Carlsbad, CA, USA), and Penicillin-Streptomycin (100 U/mL) (Gibco, Carlsbad, CA, USA). After counting and adjusting the cell density, cells were plated at a den- sity of 16×104 cells/ml in 24-well plates with 12mm glass coverslips coated with 0.05mg/ml Poly-D- Lysine (Corning, NY, USA). Cells were incubated in a humidified atmosphere maintained at 37°C, 5% CO2/95% air, and half of the media was changed every 2 days.

to both the CON and ISO groups. Cell-coated plates for the ISO and ISO+Rapa groups were placed in humidified, seal- able chamber, a 15 min equilibration period was performed, in which 1.8% Isoflurane (Baxter Healthcare, Cooperation, Deerfield, IL, USA) in the carrier gas (5% CO2, 21% O2 and 74%N2) was continuously delivered. After this 15 min equilibration, the chamber was tightly sealed containing the 1.8% isoflurane in carrier gas and was placed in a 37 ℃ incu- bator for 4 h. For the CON group, the same procedure was repeated except cells only received the carrier gas for 4 h. For medium changes, fresh drug was added to maintain the appropriate concentration of vehicle and rapamycin.

Immunofluorescence Staining and Imaging

At P60, mice were anesthetized with isoflurane and transcar- dially perfused with cold PBS for brain tissue collection. Samples were blinded with code for further analysis. After postfixation with 4% paraformaldehyde (PFA) overnight and dehydration with 30% sucrose for 3–4 days, coronal sections containing the dentate gyrus from the hippocampus were obtained using a microtome. Sections were 50μm thickness, and after collection, they were stored in antifreeze media at − 20℃. For immunohistochemistry, sections were rinsed 3 times with PBS for 5 min and incubated in blocking solu- tion (5% donkey serum with 0.1% Triton X-100 in PBS) for 1 hour at room temperature. Sections were incubated with primary antibodies at 4 ℃ overnight: rabbit anti-Synapsin-1 (1:200, EMD Millipore, Burlington, MA, USA), rabbit anti- PSD-95 (1:200, EMD Millipore, Burlington, MA, USA), rabbit anti-Gephyrin (1:200, Abcam, Cambridge, MA, USA), rabbit anti-pAKT (1:50, Cell Signaling Technol- ogy, Danvers, MA, USA), rabbit anti-pS6 (1:1000, Fisher Scientific, Hampton, NH, USA), rabbit anti-pmTOR (1:50, Cell Signaling Technology, Danvers, MA, USA), and mouse anti-parvalbumin (PV) (1:1000, Swant, Marly, Fribourg, Switzerland). After another 3 washes in PBS, sections were incubated for 2 hours at room temperature with the second- ary antibodies Alex Fluor488 anti‐rabbit antibody (1:200,

Isoflurane Exposure In vitro

Jackson ImmunoResearch, West Grove, PA, USA), Cy5 anti-mouse antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA), and 4′, 6-diamidino-2-phenylin- dole (DAPI, 1:5000). After a final 3 more washes with PBS, sections were mounted on slides with 2.5% PVA/DABCO Mounting Media.

At 5DIV, the cell-coated plates were randomly divided into three groups: control group (CON), isoflurane group (ISO), and isoflurane with 100nM rapamycin group (ISO+Rapa). Rapamycin (Sigma- Aldrich Inc, St. Louism, MO, USA) dis- solved in DMSO was added to ISO+Rapa group 1 h before isoflurane exposure, bringing the final concentration of rapa- mycin and DMSO in the culture medium to 100 nM and 0.1% respectively. The same volume of DMSO was added

At 12DIV, cells on coverslips were fixed with 4% PFA for 20 min at room temperature. After washing with PBS 3 times, neurons were incubated in blocking solution (5% donkey serum with 0.1% Triton X-100 in PBS) for 1 h at room temperature. Then, the neurons were incubated with primary antibodies at 4 ℃ overnight: rabbit anti-Synap- sin-1 (1:200, EMD Millipore, Burlington, MA, USA ), rabbit anti-PSD-95 (1:250, EMD Millipore, Burlington,

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MA, USA), rabbit anti-Gephyrin (1:800, Abcam, Cam- bridge, MA, USA), rabbit anti-pAKT (1:50, Cell Sign- aling Technology, Danvers, MA, USA), rabbit anti-pS6 (1:1000, Fisher Scientific, Hampton, NH, USA), rabbit anti-pmTOR (1:50, Cell Signaling Technology, Danvers, MA, USA), and mouse anti-MAP2 (1:200, Abcam, Cam- bridge, MA, USA ). After 3 washes with PBS, neurons were incubated for 2 h at room temperature with sec- ondary antibodies Alexa Fluor488 anti‐rabbit antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA), Cy5 anti-mouse antibody (1:200, Jackson Immu- noResearch, West Grove, PA, USA), and 4′, 6-diamidino- 2-phenylindole (DAPI, 1:5000). Following 3 more washes with PBS, coverslips were mounted on slides with 2.5% PVA/DABCO Mounting Media. Mounted coverslips were labeling in code to facilitate blinding of further experimentation.

Statistical Analysis

Results were expressed as mean± SEM. Data were ana- lyzed by GraphPad Prism 8 (GraphPad, San Diego, CA, USA). Data were analyzed using two-way analysis of vari- ance (ANOVA) with Tukey’s test for multiple compari- sons. Statistical significance was set a priori at p <0.05.

Conclusion

Early isoflurane exposure aggravates the defects of synap- togenesis in Fmr1 KO animals, the adverse effects might be mediated by mTOR signaling pathway.

Acknowledgements This study is supported in part by the Chinese Scholarship Council (Second Affiliated Hospital of Xi’an Jiaotong University) and the NIH (Johns Hopkins University School of Medi- cine—Grant Nos. R01GM137213 and R01GM120519).

Imaging and Analyzing

For evaluating synaptogenesis in vivo, 5 sections repre- senting different coronal level of the dentate gyrus were picked randomly from each animal. For each section, 3 images were randomly taken in the dentate gyrus defined by DAPI staining by an experimenter blind to condition. For evaluating synaptogenesis in vitro, 5 neurons were picked randomly from the 4 quadrants and the center of each coverslip. The image of each dendrite segment defined by MAP2 immunolabeling was taken 20 μm apart from the nucleus defined by DAPI immunolabeling. Representative images were taken using a 63 × 1.0 N.A. objective with an additional 5.0x magnification lens under a Leica SP8 confocal microscope (Leica, Wetzlar, Ger- many), and the settings were consistent for each capture. Synaptic puncta were quantified using ImageJ software (NIH, Bethesda, MD, USA). For evaluating the activation of mTOR signaling in vivo, 5 sections representing dif- ferent coronal level of the dentate gyrus were picked ran- domly from each animal by an investigator blind to condi- tion. Images of the dentate gyrus were taken using a 20 × 1.0 N.A. objective with an additional 0.75x magnification lens on a Leica SP8 confocal microscope (Leica, Wetzlar, Germany). For evaluating the activation of mTOR sign- aling in vitro, 5 fields were picked randomly from the 4 quadrants and center of each coverslip by an investigator blind to condition, and images were taken with a 20 × 1.0 N.A. objective. Cell counts to determine the proportion of cells positive for markers being analyzed were conducted using ImageJ software (NIH, Bethesda, MD, USA). All imaging and analysis were conducted by an investigator blind to the conditions.

Author Contributions Data curation: JW and JX; formal analysis: JW and JHC; funding acquisition: JW and CDM; methodology: JW, JX, RPM, JHC, and CDM; software: JW; supervision: CDM; writing— original draft: JW, JX, and CDM; Writing—review and editing: JX, RPM, and CDM All authors reviewed the final manuscript.

Data Availability The datasets are available from the corresponding author upon reasonable request.

Declarations

Conflict of interests The authors declare no competing interests.

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Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

58. Jevtovic-Todorovic V (2018) Exposure of developing brain to general anesthesia: What is the animal evidence? Anesthesiology 128:832–839",mice,['Early postnatal WT and Fmr1-KO mice were exposed to isoflurane and brain slices were collected in adulthood.'],postnatal day 7,['mice were exposed to 1.5% isoflurane for 4hrs on P7 and brains were harvested at P60'],N,[],isoflurane,['mice were exposed to 1.5% isoflurane for 4hrs on P7 and brains were harvested at P60'],none,[],fmr-1 ko,['The C57BL/6 WT mice and heterozygous Fmr1 KO (HET) mice were purchased from the Jackson Laboratory.'],The study explores whether neurodevelopmental injury from anesthesia exposure might be worsened in the context of pre-existing disease that adversely impacts brain development.,"['In the experiments described in this manuscript, we explore the question of whether neurodevelopmental injury from anesthesia exposure might be worsened in the context of pre-existing disease that adversely impacts brain development.']",None,[],"The study concludes that early developmental exposure to isoflurane causes more profound synapse loss in Fmr1-KO than WT mice, mediated by a pathologic increase in mTOR pathway activity.","['Early developmental exposure to isoflurane causes more profound synapse loss in Fmr1- KO than WT mice, and this effect is mediated by a pathologic increase in mTOR pathway activity.']",The study's limitations include the Fmr1 KO mouse not being a perfect representation of FXS and the challenges of anesthetic exposure in small rodents.,"['As with all animal models, the Fmr1 KO mouse is not a perfect representation of FXS and anesthetic exposure in small rodents presents challenges in maintaining normal physiology.']",None,[],True,True,True,True,True,False,10.1007/s11064-021-03301-5