updates on offline

20230808-AI coding-1st round/1027 – Li 2016_test.txt

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+PMID: 28430606 PMCID: PMC5464800 DOI: 10.18632/oncotarget.15405
+MATERIALS AND METHODS
+Animals
+Male and female Wistar rats, three months of age, weighing 200 ± 20 g, were purchased from the Animal Experimental Center of the Second Affiliated Hospital of the Harbin Medical University (Harbin, China). Prior to the experiment, rats were quarantined for two weeks at the Northeast Agricultural University (Harbin, China). All experiments were performed in accordance with the guidelines outlined by the Ethical Committee for Animal Experiments (Northeast Agricultural University, Harbin, China).
+
+Mating and drug administration
+Thirty-six Wistar rats were divided into 12 cages (one male and two females per cage) with an iron mesh at the bottom. On the next morning the vaginal suppository was investigated through the iron mesh. When sperm was detected, female rats were annotated as pregnant at day 0 (P0). The female rats were anesthetized via intravenous ketamine injection (200 mg/Kg) for 3 h on P14 [55]. The total volume of ketamine stayed below 2 mL/100 mg. Ketamine-treated offspring were recorded as K group, while individuals within the control group were recorded as C group. The first day after birth was recorded as B0. During B25-B30, Morris water maze task, contextual and cued fear conditioning, and olfactory tasks were used to test learning and memory capacity (n = 120, 5/dam, Figure ​Figure11).
+Sample collections
+Rat pups were sacrificed at B30 via cervical dislocation, and were recovered to collect brain tissue for Nissl staining (n = 24, 1/dam), Golgi staining (n = 24, 1/dam), and western blotting (n = 72, 3/dam). A subset of their hippocampuses were quickly dispensed on ice, put into a freezing tube, and frozen in liquid nitrogen, while other tissues were preserved in 10% formalin.

20230808-AI coding-1st round/1027 – Li 2017.txt

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+PMID: 28430606 PMCID: PMC5464800 DOI: 10.18632/oncotarget.15405
+MATERIALS AND METHODS
+Animals
+Male and female Wistar rats, three months of age, weighing 200 ± 20 g, were purchased from the Animal Experimental Center of the Second Affiliated Hospital of the Harbin Medical University (Harbin, China). Prior to the experiment, rats were quarantined for two weeks at the Northeast Agricultural University (Harbin, China). All experiments were performed in accordance with the guidelines outlined by the Ethical Committee for Animal Experiments (Northeast Agricultural University, Harbin, China).
+
+Mating and drug administration
+Thirty-six Wistar rats were divided into 12 cages (one male and two females per cage) with an iron mesh at the bottom. On the next morning the vaginal suppository was investigated through the iron mesh. When sperm was detected, female rats were annotated as pregnant at day 0 (P0). The female rats were anesthetized via intravenous ketamine injection (200 mg/Kg) for 3 h on P14 [55]. The total volume of ketamine stayed below 2 mL/100 mg. Ketamine-treated offspring were recorded as K group, while individuals within the control group were recorded as C group. The first day after birth was recorded as B0. During B25-B30, Morris water maze task, contextual and cued fear conditioning, and olfactory tasks were used to test learning and memory capacity (n = 120, 5/dam, Figure ​Figure11).
+Sample collections
+Rat pups were sacrificed at B30 via cervical dislocation, and were recovered to collect brain tissue for Nissl staining (n = 24, 1/dam), Golgi staining (n = 24, 1/dam), and western blotting (n = 72, 3/dam). A subset of their hippocampuses were quickly dispensed on ice, put into a freezing tube, and frozen in liquid nitrogen, while other tissues were preserved in 10% formalin.
+
+Nissl's staining
+Coronal brain sections were cut in a vibratome (Leica VT1200S, Germany) after the brains were postfixed in the same fixative. To ensure matching of hippocampal sections between groups, we used anatomical landmarks provided by the brain atlas. The selected brain sections were stained with 0.5% cresyl violet and we selected three 104 μm2 areas for examination with a light microscope (Leica DFC420, Germany) to count neuron numbers in the CA1 and CA3 regions of the hippocampus.
+
+Golgi staining
+Golgi-Cox staining was utilized to obtain hippocampal dendritic spine density via the FD Rapid GolgiStainTM Kit (FD Neuro Technologies Inc), following the manufacturer's instructions. Coronal tissue sections of 150 μm thickness were cut at room temperature, using a vibratome (Leica VT1200S, Germany) and then, they were put on gelatin coated slides. Subsequently, slides were dehydrated with a gradient of 50%, 75%, 95%, to 100% ethanol and cleared in xylene, then the specimens were prepared with slide coverslips and sealed with Permount. The slides were then examined in detail with a light microscope (Leica DFC420, Germany). We analyzed the stained spine, using techniques similar to those described in previous study [56]. Five pyramidal neurons were analyzed that were well-impregnated and clearly distinguishable from others in each hippocampus (20 × objective lens). Five segments of 10 μm of apical and basal dendrites respectively, were randomly selected from each pyramidal neuron for inspection (via 200 × oil immersion lens) to quantify the density of spines. Spinal density of secondary apical and basal dendrites was analyzed at proximal segments emerging at more than 50 μm distance from the soma of the hippocampal CA1 neurons. All of these spines were required to exhibit a clearly distinguishable base or origin and were isolated from neighboring dendrites. Spine density was calculated per 10 μm of dendritic length. The open-source ImageJ 1.48 r Java image-viewing software and Adobe Photoshop CC 2015 were used to calibrate the scale and enlarge the segments of the spines. An investigator blinded to the experimental condition completed all analyses.
+
+Morris water maze test
+Place navigation trials
+To test hippocampal-dependent spatial cognition, rats were trained in the standard morris water maze with a hidden platform [57]. A white escape platform (12 cm diameter) was submerged in a circular pool (160 cm diameter, at a 50 cm depth), filled with warm (23–25°C) opaque water. At B25-29, each rat pup underwent four trial sessions per day (60–70 min inter-trial interval) for five consecutive days. Each trial consisted of releasing the rat into the water, facing the outer edge of the pool at one of the quadrants (in random sequence) and permitting the animal to escape to the platform. They received four trials per day of training in search for the submerged and unmarked platform, with trial durations of 60 s on the platform at the end of trials. All trials were videotaped, and the swimming paths of rats were recorded with the ANY-maze video tracking system (Stoelting Co., IL, USA), which enabled us to measure the time taken (latency) to find the platform (s), as well as other behavioral information obtained during this spatial reference memory test. The animals were dried and placed beneath a heating lamp after completion of each test.
+
+Spatial probe test
+A probe trial was performed 1 d after the last trial at B30 where the platform was removed from the pool to assess memory retention for the location of the platform. During the 60 s test trial, we recorded and analyzed the swimming speed (cm/s), the swimming path tracks, and the number of entries into the platform quadrant zone.
+
+Contextual and cued fear conditioning
+Conditioning training on day one consisted of placing the rat pups in the chamber and exposing the animals to a mild footshock paired with an auditory cue. The rat pup was brought from the home cage to the testing room and placed into the conditioning chamber. It had 3 min to explore the novel environment. The auditory cue (a 90 dB tone) was sounded for approximately 30 sec. A stimulus light within the wall of the chamber may also be illuminated. During the last seconds of the auditory signal, an unconditioned aversive stimulus, a mild footshock in the range of 0.25 to 0.5 mA, was administered through the grid floor for 2 sec. The number of seconds spent freezing in the test chamber on the training day was considered the control measure of unconditioned fear. The rat pup was left in the conditioning chamber for 1 min after the last pairing, during which the association between the aversive stimulus and the properties of the conditioning chamber was further established. The rat pup was then returned to its home cage.
+
+Testing on day 2 began approximately 24 hours after the conditioning session. The rat pup was returned to the same conditioning chamber and scored for bouts of freezing behavior. No footshock was administered on day two. The number of seconds spent freezing in the identical test chamber on day two was considered the measure of contextually conditioned fear, i.e., freezing within identical context. Freezing was defined as a lack of movement other than respiration. Presence or absence of freezing behavior was generally recorded by an investigator, who was blinded to the experimental condition, taking a note every 10 sec for 5 min, for a maximum total score of 30 freezing bouts. The rat pup was then returned to its home cage.
+
+The second phase of testing began an hour later. A further testing chamber with very different properties provided the altered context. Changing the sensory cues as much as possible was essential so that the rat pup perceives the novel context as unrelated to the conditioning chamber. Such as triangle-shaped test chamber with different lighting was used and lemon juice was painted on the walls, while a different investigator wore gloves and a lab coat of different texture than on the training day. Freezing behavior was scored for 3 min. Contextual discrimination of fear conditioning was quantified by comparing the number of freezing bouts in the same contextual environment to the number of freezing bouts in the novel contextual environment.
+
+At the end of the first 3 min, the tone that was presented on training day one (was well as the light stimulus cue if used on day one) was presented in the novel context environment. Freezing behavior was scored for the next 3 min in the presence of the sound (and light) cues. Cued conditioning was calculated via comparison of the number of freezing bouts in the novel context environment in the presence of the cue with the number of freezing bouts in the novel context environment in the absence of the cue (Figure ​(Figure5a5a).
+
+Olfactory task
+This task was designed to investigate the olfactory learning and memory abilities [58]. For this experiment, two holes (3 cm diameter and 4.5 cm deep) were used. A polypropylene swab, embedded in a fine plastic mesh and containing 20 μL of diluted odors (1:10) was placed at the bottom of each hole and covered with wood shavings. The acquisition test (one session) consisted of one odor (either limonene or carvone, Sigma-Aldrich) being presented in both holes for a 5 min period. In a preliminary experiment, with simultaneous presentation of the same pair of odors (one odor in each hole) in a one-trial test, rat pups spent the same amount of time exploring either hole, indicating no preference for one of the two odors. The recall test consisted of a 3 min session in which one hole was odorized with the previously presented odor, while the other hole was odorized with a new odor (Figure ​(Figure6a).6a). The delay between acquisition and recall tests was 60 min. During the recall test, the cumulated exploration time of each hole was converted as the percentage of the total exploration time of both holes. Rat pups were considered to have remembered the familiar odor when they spent less time exploring the hole containing it, in relation to the time spent exploring the hole containing the new odor. Equal exploration times for both holes during the recall test were considered to indicate that rat pup did not remember the familiar odor. Both odors were used alternatively during acquisition or recall and presented randomly in each of the two holes to avoid place preference bias (Figure ​(Figure6a6a).
+
+Cell culture and drug treatment
+PC12 cells were obtained from the Northeast Agricultural University, Harbin, China. The cells were cultured in DMEM medium (Gibco), supplemented with 10% (v/v) FBS, penicillin/streptomycin (100 U/mL; 100 μg/mL) at 37°C under an atmosphere of 5% CO2 and 95% air. The cells were seeded in 6-well plates with 2-9 × 105 cells/well or 96-well plates with 2-9 × 104 cells/well, and the culture medium was changed daily. Cells were pretreated for 3 h with Protein Kinase A (PKA) inhibitor (H89, 10 μM, H group), Extracellular Regulated Protein Kinases (ERK) inhibitor (SCH772984, 10 μM, S group), PKA inhibitor + ERK inhibitor (S+H group), DMSO (solvent of inhibitors, D group), and ketamine (K group).
+
+Cell counting kit-8 (CCK-8) assay
+Cell viability was detected via the CCK8 assay (Beyotime Institute of Biotechnology, Suzhou, Jiangsu, China). Following the indicated treatments, CCK8 solution (10 μl) was added to each well (96-well plates). Then, the cells were cultured at 37°C for one further hour. The optical density of each well was measured at 450 nm with a Bio-Tek microplate reader (Bio-Tek Instruments, Thermo Fisher Scientific, Winooski, VT).
+
+WB
+150 μg of protein were separated via 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (HybondTM-C Extra, GE Healthcare) via electroblotting. After washing, membranes were blocked with 3% (w/v) BSA (biotopped) for 4 h at room temperature and incubated overnight at 4°C in BSA with antibodies that are specific for Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII), p-CaMKII, CaMKIV, p-CaMKIV, ERK, p-ERK, PKA, CREB, p-CREB (1.5:1000, EnoGene), p-PKA, and Brain Derived Neurotrophic Factor (BDNF, 1:1000, abcam). Membranes were washed thrice with PBS containing 0.1% Tween and then incubated for 1 h at room temperature either with a horseradish peroxidase-conjugated secondary antibody (Goat anti-Rabbit IgG Antibody HRP (ABIN) or a goat anti-Mouse IgG Antibody HRP (Sigma)) in BSA.
+
+Data analysis
+All data were analyzed with GraphPad Prism 7.0 (GraphPad Software Inc., USA) via one-way ANOVA, followed by Turkey's Post Hoc test or unpaired two-tailed Student t-test. Values were considered to be statistically significant for P < 0.05. Data are presented as means ± standard deviation unless otherwise noted.

20230808-AI coding-1st round/1043 – Gao 2021.txt

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+PMID: 34645094 DOI: 10.31083/j.jin2003065
+2. Methods
+2.1 Animals
+Six adult female Sprague-Dawley (SD) rats, weighing 180–220 g, were raised with free diet and water intake in polypropylene cages for 7 days. Then the female SD rats were mated with male SD rats with sexual experience at 7:00 PM after adaptive feeding. Vaginal smears were performed the next morning and pregnancy day 0, G0, was defined by sperm detection. The pregnant rats were randomly divided into two groups: a control group (control, n = 3) and a sevoflurane group (SeV, n = 3). The six female SD rats were raised to G14.5 (middle pregnancy).
+
+2.2 Anesthesia
+On pregnancy day 14.5, the rats allocated to sevoflurane exposure were put inside a 30 cm 
+×
+ 20 cm 
+×
+ 120 cm box. A mixture of oxygen and sevoflurane (2 L/min with 4% sevoflurane) was delivered through an inlet port connected to a vaporizer, while a gas analyzer installed on a second port allowed monitoring of anesthetic gas concentration. Pregnant rats are more sensitive to sevoflurane and a minimum alveolar concentration (MAC) of 2.4% in healthy adult rats [27], so the concentration of sevoflurane (3%) is equivalent to 1.3 MAC to maintain a surgical level of anesthesia. The rats in the control group inhaled oxygen (2 L/min). However, limited to anesthesia machine conditions that it is not completely airtight, the inhalation concentration of sevoflurane should be 4% in order to reach 1.8 MAC to maintain a surgical level of anesthesia in the SeV group. The inhalation time is 3 hours in the SeV group. During the procedure, the skin color of the rats’ mouths, noses, limbs, and respiratory amplitudes and frequencies were observed to avoid hypoxia respiratory depression. After anesthesia, the rats were sent back to their cages after the righting reflex was recovered. After sevoflurane anesthetization, an arterial blood gas analysis was performed to assess gas exchange and glycemic status in female rats. The site of blood sampling was left heart artery. If there was a significant derangement, e.g., severe hypoxemia, these female rats were no longer involved in the follow-up experiments. No female rats were excluded in the study. Then the rat offspring were reared and delivered naturally.
+
+2.3 Tissue section preparation
+Three offspring were randomly selected from each group with one offspring/dam. The ex vivo brain samples of the offspring rats were harvested at the day 30 of postpartum (P30) for histology, immunostaining and Golgi staining. Rats in both the control and SeV groups were executed and perfused through the left ventricle with precooling saline followed by 4% paraformaldehyde in 0.01 M phosphate buffered saline (PBS) pH 7.35. The brain tissue of the rats was taken and post-fixed for 24 hours for paraffin and frozen sections.
+
+To analyze the status of the NRG1–ErbB4 pathway in the interneurons, the expression levels of NRG1 and ErbB4 in LII and III of the ECT were examined via immunohistochemistry. The NRG1–ErbB4 pathway plays an important role in the genesis, migration, differentiation, maturation, and neurotransmitter synthesis of GABAergic interneurons. We assumed that the number of GABAergic interneurons the LII/LIII ECT of offspring in could be affected by alterations of the NRG1–ErbB4 pathway. Therefore, we label GABAergic interneurons with PV to represent PV interneurons. We also used glutamic acid decarboxylase 67 (GAD67) to label GABAergic interneuron (the key enzyme of GABA neurotransmitter synthesis) positive cells to represent the total GABAergic interneurons in the LII/LIII ECT. The expression levels of PV and GAD67 in LII and LIII of the ECT were examined via immunohistochemistry. That is, Interneurons were identified by immunoreactivity to PV and GAD67. To investigate whether NRG1–ErbB4 pathway changes in offspring after prenatal sevoflurane exposure affect the formation of subunits during maturation, we detected NMDA receptor subunit 2A (NR2A) and NMDA receptor subunit 2B (NR2B) by immunofluorescence. The expression levels of NR2A and NR2B in LII and LIII of the ECT were examined via immunohistochemistry to analyze the status of NMDA receptors in the ECT. There is a fixed pattern of neurite growth in the developing brain. We assumed that prenatal sevoflurane exposure could affect the inherent growth pattern of dendrites and dendritic spines in pyramidal neurons through NRG1–ErbB4 alterations. Therefore, we used Golgi silver staining to investigate the length of dendrites and the number of branches and dendritic spines. Golgi staining was performed to analyze the total dendrite length, number of dendritic branches, spatial distribution of dendrites, and density of dendritic spines in the pyramidal neurons in the ECT.
+
+2.4 Histology and immunohistochemistry
+The coronal sections of the brain were deparaffinized, rehydrated, and immersed in 3% H
+2
+O
+2
+ at room temperature for 30 min. Antigens were retrieved in a 0.01 mol/L citric buffer (pH 6.0) at 97 
+∘
+C for 15 min. The coronal sections were cooled down for 1 h before being blocked by 10% bovine serum albumin (BSA) solution. Staining with diluted primary antibodies was conducted at 4 
+∘
+C overnight (for at least 18 h). The primary antibodies included rabbit anti rat NRG-1 (1:1000, Cat. No Ab191139, Abcam, Cambridge, UK), rabbit anti rat ErbB4 (1:250, Cat. No Sc-283, Santa Cruz, Dallas, Texas, USA), rabbit anti rat NR2A (1:1000, Cat. No cell signaling technology, Massachusetts, USA), rabbit anti rat NR2B (1:1000, Cat. No 06-600, Millipore, Massachusetts, USA), mice anti rat PV (1:1000, Cat. No #2886709, Millipore), and rat anti rat GAD67 (1:2500, Cat. No. MAB5406, Millipore, Massachusetts, USA). After being washed by 0.1% PBST for three times (5 min), the sections were stained with diluted second antibodies at room temperature for 2 h and kept in a dark place. After washed by 0.1% PBST for three times (5 min), the sections were counterstained with hematoxylin, dehydrated with ethanol and mounted with coverslips. Then the expression levels of NRG1, ErbB4, PV, GAD67, NR2, A and NR2B were examined using a fluorescence microscope (Leica DM6000B, Germany). The results were shown as positive cells/sections. In each rat, we randomly select 5–6 coronal sections to count the cells to avoid error resulting from the section status. The brain area sections (ECT) we selected for immunohistochemical section is fixed. There is an inward concave angle under the area of the ECT, which is used to locate the central cortex and reduce the error. The size of the ECT in this part of the rat brain is relatively fixed, so the randomly selected sections can be regarded as roughly the same size which is comparable.
+
+2.5 Golgi stain
+150 
+μ
+m-thick frozen brain sections were obtained from control and SeV rats. Golgi–Cox staining was performed using the FD Rapid Golgi stain kit (Cat. NO. PK401, FD NeuroTechnologies, Inc. Columbia, USA) according to the manufacturer’s protocols. Ten well-individualized pyramidal neurons in LII and LIII of the ECT were randomly selected from each rat. Sequential optical sections of 1392 
+×
+ 1040 pixels were taken at 1.5 
+μ
+m intervals along the z-axis (Leica, DMi8 + DFC7000J, Germany). The Imaris software (BitPlane AG, Zurich, Switzerland) was used for tridimensional reconstruction. The total dendrite length, number of dendritic branches, and spatial distribution of dendrites in the pyramidal neurons of the ECT were estimated using Sholl analysis [28]. To measure the density of dendritic spines, a straight dendrite was scanned on the z-axis using a 100 
+×
+ objective microscope. A 40-
+μ
+m long dendrite was randomly intercepted with image J 1.46r (National institute of health, Bethesda, Maryland, USA). The number of synaptic spines was counted and the density of synaptic spines (spines/10 
+μ
+m) was calculated. At least 10 terminal dendrites were selected for each sample.
+
+2.6 Statistical analysis
+All the data was expressed as mean 
+±
+ standard deviation. JMP software version 16.0 (SAS Institute, Cary, NC, USA) was used for statistical processing. All parameters were tested for normal distribution using the Kolmogorov-Smirnov test. Two independent-sample t tests were conducted used to compare the parameters differences between the control and sevoflurane groups, including NRG1, ErbB4, PV, GAD67, NR2B and NR2A. Dendrites were analyzed with Kruskal-Wallis test (Sholl analysis) and Steel Dwass post hoc test using JMP software version 16.0 (SAS Institute, Cary, NC, USA) [28]. It was considered that a difference was statistically significant when P 
+<
+ 0.05. GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) software was used to make drawings.

20230808-AI coding-1st round/1054 – Takaenoki 2014.txt

@@ -0,0 +1,53 @@
+PMID: 24061597 DOI: 10.1097/ALN.0000435846.28299.e7
+Materials and Methods
+Animals
+All experiments were conducted according to the institutional ethical guidelines for animal experiments of the National Defense Medical College and were approved by the Committee for Animal Research at National Defense Medical College (Tokorozawa, Saitama, Japan). Inbred C57BL/6 mice were used in this study and maintained as described previously.5 
+
+Anesthesia and Hydrogen Treatment
+Sevoflurane anesthesia was carried out as described previously.5  In brief, on postnatal day 6 (P6), pups were placed in a humid chamber immediately after removal of mice from the maternal cage. A 3% concentration of sevoflurane was administered in 30% oxygen as the carrier gas. Control mice were exposed to 30% oxygen. Hydrogen gas (1.3%) was supplied as described previously.30  Total gas flow rate was 2 l/min.
+
+Mouse Study Design
+In each experiment, siblings from the same litter were randomly allocated into one of the following groups so that each group was balanced on littermate. No obvious differences (e.g., body size and weight) were observed within the litters, and there was no significant difference in mean body weight among the groups (data not shown).
+
+Survival rate of delivered pups: control, sevoflurane, and sevoflurane + hydrogen groups (n = 17–19 dams for each group); a minimum biologically important difference was set at a 30% decrease from the baseline level in the control group.
+
+Pup exchange test: control and sevoflurane groups (n = 6 dams for each group); a minimum biologically important difference was set at a 30% decrease from the baseline level in the control group.
+
+Behavioral studies: control, sevoflurane, and sevoflurane + hydrogen groups (n = 10–11 dams for each group); the primary outcome measure was latencies for pup retrieval; in the pup retrieval test, a minimum biologically important difference was set at a 30% increase from the baseline level in the control group.
+
+Hormonal assay: control, hydrogen, sevoflurane, and sevoflurane + hydrogen groups (n = 4–5 dams for each group); a minimum biologically important difference was set as 30% decrease from the baseline level in the control group.
+
+Immunohistochemical study: control and sevoflurane groups (n = 5 dams for each group); a minimum biologically important difference was set at a 30% decrease from the baseline level in the control group.
+
+In total, we prepared 160 female pups, which received anesthesia or hydrogen treatment at P6 (55 of control, 56 of sevoflurane, 40 of sevoflurane + hydrogen, and 9 of hydrogen groups). Among them, eight pups with sevoflurane and one pup with sevoflurane + hydrogen died during the treatment. Then, these siblings from the same litter were reunited and cohoused till the experiment (mice were similarly caged and housed in all groups). At 3 weeks of age, mice were weaned and allowed to further mature. At 7–9 weeks of age, female mice were mated with healthy males that had not been exposed to any anesthetic. Among them, 23 female mice did not get pregnant (eight of control, six of sevoflurane, five of sevoflurane + hydrogen, and four of hydrogen groups) and 1 control mouse died due to failure of delivery. These mice were excluded from the final analysis. Thus, for first delivery experiments, we used 46 control dams, 42 sevoflurane-treated dams, 34 sevoflurane + hydrogen–treated dams, and 5 hydrogen-treated dams. These mice were allocated as described above (1–5 in this section).
+
+Among them, some dams were further analyzed for behavioral studies of parous dams: the same sets of mice for behavioral studies in first-time delivery were reused in behavioral studies in second-time (parous) delivery (control: 7 for survival rate and 11 for behavioral studies; sevoflurane-treated: 8 for survival rate and 10 for behavioral studies).
+
+For paternal study experiments, 26 age-matched male mice were either subjected to anesthesia (n = 13) or control (n = 13) treatment at P6 (no mice died during the treatment). Siblings from the same litter were allocated into each group almost equally (i.e., groups were balanced on littermate).
+
+Oxytocin and Vasopressin Assay
+Plasma concentrations of oxytocin and vasopressin in dams at 10 weeks of age were examined by enzyme-linked immunosorbent assay using commercially available kits (Oxytocin enzyme-linked immunosorbent assay kit and arg8-Vasopressin enzyme-linked immunosorbent assay kit; Enzo Life Sciences, Farmingdale, NY). Assays were performed according to the manufacturer’s instructions. Blood samples were collected from the inferior vena cava within 6 h after parturition.
+
+Immunohistochemical Study
+Immunohistochemical studies using the anti-c-Fos antibody (rabbit polyclonal; sc-52; Santa Cruz Biotechnology, Santa Cruz, CA) were performed as previously described.30  Samples were obtained within 6 h after parturition. The numbers of immunoreactive cells were counted by an observer blinded to the groups.
+
+Behavioral Studies
+On the morning of parturition, maternal behaviors were examined. Maternal behavioral studies using first-time mothers were performed at 10–12 weeks of age. The same sets of female mice were reused in the maternal behavioral studies for second-time (parous) mothers: those mice were mated again at 19–25 weeks of age, and maternal behaviors were examined at 22–28 weeks of age. Paternal behavioral studies using male mice were performed at 11 weeks of age. Survival rate (percentage of the number of pups at the indicated day compared with that at birth) was recorded until P6. In each experiment, observation was made by the same observer who was blinded to the groups. All apparatus used in this study was made by O’Hara & CO., LTD. (Tokyo, Japan).
+
+Evaluation of Maternal Behavior
+Pregnant females were individually housed for a few days before parturition and examined for maternal behavior on the morning of parturition. The number of pups with milk in their digestive tract and that of poorly cleaned pups (with placenta, amniotic membrane, or umbilical cords) was recorded on that day. Nest quality was also evaluated at the same time using the score system described previously31  with some modifications: grade 3, shaped like a deep hollow surrounded by high banks; grade 2, a hollow with medium-height banks; grade 1, flat with low banks, but still discrete; grade 0, no depression in bedding with no banks. Each new dam was also evaluated for time spent crouching over pups and the percentage of newborns scattered for 20 min with minimal disturbance as described previously.32  The percentage of scattered pups was expressed as a percentage with respect to time. We calculated the percentage of scatter as follows for each pup: (duration of scatter/total time observed (20 min) × 100). We then calculated the average for each group. These evaluations were carried out before the pup retrieval test.
+
+Pup Retrieval Test
+The pup retrieval test was performed essentially as described previously.14  Before the test, pups were separated from dams for 30 min. At the beginning, each mouse was put in one corner of a cage and three of her pups were placed in different corners of the same cage. The cages were continuously observed for 10 min with minimal disturbance. Latencies to sniff a pup for the first time and to return each pup to the nest were evaluated.
+
+Evaluation of Parental Behavior
+Parental behavior of virgin male mice toward pups was evaluated for 20 min. At the beginning, each mouse was put in one corner of a cage and three new born pups were placed in different corners of the same cage as described in the pup retrieval test. Latencies to sniff a pup for the first time and the numbers of males which committed attacks toward pups were evaluated. If any of the pups was attacked during the test, all pups were removed immediately and this subject was considered as “attack.”
+
+Pup Exchange Test
+The pup exchange test was conducted as described previously with some modifications.14  Pups born to a female dam couple (a dam with sevoflurane exposure at P6 and a control), which were born on the same day, were exchanged within 12 h after delivery. The number of surviving pups was evaluated for 6 days after birth.
+
+Olfactory Test
+The olfactory test was conducted as described previously.3 
+
+Statistical Analysis
+Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA). Comparisons of the means of each group were performed using Student t test, one-way ANOVA followed by Bonferroni post hoc test, and two-way ANOVA followed by Bonferroni post hoc test. Comparisons of the survival rate until P6 were performed using a log-rank (Mantel-Cox) test. We did not exclude any data in this study. P values of less than 0.05 were considered statistically significant. Values are presented as the mean ± SEM in bar graphs.

20230808-AI coding-1st round/1107 – Boctor 2008.txt

@@ -0,0 +1,18 @@
+PMID: 18667523 PMCID: PMC2721666 DOI: 10.1093/toxsci/kfn152
+MATERIALS AND METHODS
+Animals Sprague-Dawley dams (n = 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°C ± 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 μl) 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.
+
+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 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° incline board. Each pup was allowed 60 s to complete a 180° 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.
+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.
+Forelimb hang behavior. To analyze the latency to fall, a Cox Proportional Hazards model was run using SAS (SAS Version 9.1, SAS Institute Inc., Cary, NC) with treatment, sex, and PND as factors.

20230808-AI coding-1st round/1134 – Guan 2019.txt

@@ -0,0 +1,9 @@
+PMID: 31257533 PMCID: PMC6625379 DOI: 10.3892/mmr.2019.10397
+Materials and methods
+Rat HPC model All animal procedures were conducted with the approval of the Animal Care and Use Committee of Guangxi Medical University (Nanning, China). Seven-day-old (P7) male Sprague-Dawley pups (average body weight, 10–15 g, n=70) were identified and numbered using picric acid, which were revealed to the investigator only after the completion of experiments and analyses. All pups were housed in a temperature-controlled room (22±1°C) with a 12-h light/dark schedule. H89 (Selleck Chemicals) and Sp-cAMP (Sigma-Aldrich; Merck KGaA) were prepared in 5 µl double-distilled water. The experimental set-up is illustrated in Fig. 2 (n=10) and the following experimental groupings were used: i) Normal saline group (NS group) received intraperitoneal injections of an equal volume of normal saline; ii) propofol group (P group) received intraperitoneal injections of 100 mg/kg propofol; iii) following the propofol treatment as in the P group, the propofol + Sp-cAMP group (P+Sp-cAMP group) received intracerebroventricular injections of 20 nmol/5 µl Sp-cAMP (a cAMP-dependent protein kinase agonist); iv) HPC+P group rats were placed in a chamber containing 8% oxygen and 92% nitrogen for 10 min, and the pups were subsequently exposed to room air for a further 10 min, and following five HPC cycles, the rats received an intraperitoneal injection of 100 mg/kg propofol; v) HPC+P +H89 group was exposed to 5 µmol/5 µl H89 [a protein kinase A (PKA) inhibitor] by intracerebroventricular injections, followed by the same protocol as in the HPC+P group; vi) the remaining pups in the two blank test groups received intracerebroventricular injections of dimethyl sulfoxide (D-ICV group) or normal saline (NS-ICV group). All pups were sacrificed according to standard protocols (100 mg/kg intraperitoneal sodium pentobarbital). Brain tissue slices were prepared for immunohistochemistry and the levels of PKA, CREB, phosopho (p)-CREB, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 were evaluated by western blotting. Morphological and structural changes were evaluated by haematoxylin and eosin (H&E) staining and transmission electron microscopy.
+Intraventricular injections As aforementioned, rats were anesthetized with sodium pentobarbital and centralized coordinates of anterior fontanel (x=0, y=0, z=0) using stereotaxic apparatus (Ryward Life Technology Co., Ltd.), the sterile cannula was implanted at AP-2 mm (front and posterior), MLR-1.5 mm (left and right of the midline), and H-2 mm (depth from the left ventricle, x=−1.0 mm, y=2 mm, z=0). After positioning, the skull was drilled, and then Sp-cAMP (5 µl) or H89 (5 µl) was slowly injected at rate of 0.1 µl/min. The blank groups following the same protocol with an equal volume of DMSO or normal saline.
+ELISA The intracellular concentrations of adenylyl cyclase in the pups was determined by ELISA according to the instructions of the assay manufacturer (cat. no. S0026; Beyotime Institute of Biotechnology).
+Western blot analysis All pups were sacrificed to harvest the brain tissue. The protein was extracted by RIPA Lysis Buffer (Beijing Solarbio Science & Technology Co., Ltd.) and protein concentration measured using a bicinchoninic acid protein assay (Biotype Biotech Co.). The mass of protein loaded per lane was 20 µl. Equal amounts of proteins were loaded onto 12% SDS-polyacrylamide gels. Electrophoresed proteins were transferred to polyvinylidene difluoride membranes (0.22-µm pore size; EMD Millipore). The membranes were blocked using 5% bovine serum albumin (blocking buffer) for 2 h at room temperature and incubated with the following primary antibodies overnight at 4°C: β-tubulin (1:2,000; cat. no. 48885), caspase-3 (1:1,000; cat. no. 48658) and cleaved caspase-3 (1:1,000; cat. no. 29034; all from Signalway Antibody) Bcl-2 (1:1,000; cat no. ab196495; Abcam), Bax (cat. no. 27727) PKA (cat. no. 5842S), CREB (cat. no. 9197S) and p-CREB (cat. no. 9198S) (all 1:1,000; from Cell Signaling Technology, Inc.), and GAPDH (1:10,000; cat. no. 10494-1-AP; Proteintech, Inc.). The membranes were washed three times with Tris-buffered saline 1% Tween-20 (TBST; pH 7.4) and then incubated in horseradish peroxidase-conjugated secondary antibody (1:10,000; cat. no. 134658; LI-COR Biosciences) for 2 h at room temperature (23–25°C) and washed three times with TBST. The bands were developed using an Odyssey infrared imaging system (LI-COR Biosciences) and evaluated using densitometric analysis (ImageJ 1.52 h, National Institutes of Health).
+H&E and immunohistochemical staining Morphological and structural changes were observed by H&E staining. Tissues were fixed in 4% ice-cold paraformaldehyde at 4°C for 2 h and paraffin-embedded sections were obtained. The paraffin sections were dewaxed in xylene for 15 min and rehydrated using graded ethanol. The sections were immersed in haematoxylin for 30 sec and then subjected to antigen retrieval using 0.01 mol/l sodium citrate and incubated with 10% normal goat serum at room temperature for 30 min to block nonspecific binding, followed by incubation with the primary antibodies against PKA C and p-CREB (cat. nos. 5842S and 9198S, 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight. The sections were incubated with streptavidin-horseradish peroxidase at room temperature for 30 min and then stained with 0.05% 3,3-diaminobenzidine substrate, followed by counterstaining with 1% haematoxylin at 37°C for 30 sec. The sections were observed using a microscope (Olympus BX53; Olympus Corporation) and four fields of the hippocampus were randomly selected in every section which represented the areas of interest and the positive cells were counted using Image-Pro Plus version 6.0 software (Media Cybernetics Inc.).
+Electron microscopy The ultrastructures of neurocytes were observed by transmission electron microscopy (HITACHI H-7650; Hitachi, Ltd.). Briefly, 2.5% glutaraldehyde solution was perfused into the rats, and the tissues were fixed in 1% OsO4 at 4°C for 1 h, dehydrated in increasing concentrations of ethanol and embedded in Epon. Then, the samples were sectioned into semi-thin slices (1 µm) and stained with 1% uranyl acetate and 5% uranyl acetate at 37°C for 20 min. The ultrastructures of the entire mitochondria were measured by manually measuring length using Image Pro Plus (version 6.0.0.260, Media Cybernetics, Inc.).
+Statistical analysis Data are presented as the mean ± standard error, and were analysed using SPSS version 17.0 (SPSS, Inc.) and GraphPad Prism 5 software (GraphPad Software Inc.). Multiple comparisons were performed using one-way analysis of variance (ANOVA), followed by Dunnett's post hoc test, as appropriate. P<0.05 was considered to indicate a statistically significant difference.

20230808-AI coding-1st round/1187 – Huang 2018.txt

@@ -0,0 +1,14 @@
+PMID: 29325538 PMCID: PMC5765622 DOI: 10.1186/s12871-018-0471-2
+Methods
+This study was approved by the Ethics Committee of Affiliated Shengjing Hospital of China Medical University, and specific pathogen free SD pregnant rats weighing 380–420 g were purchased from the Experimental Animal Center of Affiliated Shengjing Hospital of China Medical University. Animals were housed at 22–24 °C, 40–60% humidity with a 12-h light /dark cycle and had free access to food and water. Rats at the gestational age of 21 days (E21) were used in subsequent experiments. According to the isoflurane dose, rats were divided into 3 groups: the Iso1 group (1.3% isoflurane), the Iso2 group (2.0% isoflurane) and the control group (0% isoflurane; O2).
+
+In the absence of anesthesia, intratracheal intubation was difficult in the control group. Thus, all the rats retained spontaneous breathing and did not receive intratracheal intubation. Inhalation of isoflurane at a high concentration may inhibit respiration and cause hypoxia. Thus, in our pilot study, pregnant rats at the gestational age of 20 days (E20) were anesthetized intraperitoneally with pentobarbital sodium and catheter indwelling was done in the right carotid artery; rats were then allowed to recover at room temperature. At E21, rats were placed in a box filled with prefilled gas according to the following groups: 50% O2 was administered in the control group; 1.3% isoflurane was administered in the Iso1 group (50% oxygen, balanced with nitrogen); 2.0% isoflurane was administered in the Iso2 group (50% oxygen, balanced with nitrogen). All rats were retained spontaneous breathing and exposed in the box for 3 h (the concentrations of isoflurane and oxygen were monitored). The mean arterial blood pressure was continuously monitored via a catheter in the carotid artery, and arterial gas analysis was performed hourly. The results showed that inhalation of isoflurane at 1.3% or 2.0% had no influence on the arterial gas and mean arterial blood pressure. Rats used in pilot study will not be used for formal study.
+
+In this study, a total of 48 rats at E21 were randomly assigned into 3 groups and exposed to isoflurane at the predesigned concentration for 3 h. Animals were allowed to recover at room temperature and housed until they delivered. The number of fetuses was recorded, and healthy male neonatal rats were used in the experiments. At day 28 after birth (P28), the male offsprings were randomly assigned into two groups: one for Morris water maze (MWM) test to evaluate memory and learning and the other one were housed until day 90 after birth (P90) to receive the same MWM test.
+
+MWM test used a round swimming pool sized 150 cm in diameter and 60 cm in height with a platform sized 10 cm in diameter in the maze. The removable platform was 1.5 cm lower than the water surface. The visual cues (a variety of figures) on the maze’s inner wall remained unchanged during the study. Training and examination were performed in the water at 20 °C. After each examination, rats were dried under a lamp and returned to the cages.
+
+Place navigation test was performed for consecutive 5 days. In brief, platform were placed in a quadrant (the 4th quadrant in this study). At predesigned time point, rats were placed in a random quadrant (once for each quadrant). If the rat found the platform within 90s, it was allowed to stay on the platform for 15 s and then placed out of the pool. The spatial navigation test was performed on the 6th day to evaluate memory. In brief, the platform was removed, rats were placed in a random quadrant and the swimming trajectory was recorded within 90s. In the test, the proportion of swimming distance in the platform quadrant to the total swimming distance and the times of crossing the platform were calculated. The swimming distance in the platform quadrant reflects spatial localization and the times of crossing the platform reflects the accuracy of spatial memory. Before training, the platform was visible above the water surface, which may exclude rats with visual defects that were unable to find the platform. In addition, rats with poor performance in the test, such as those could not find the hidden platform and swam along the wall, were also excluded from this study.
+
+Two hours after the spatial navigation test, rats were intraperitoneally anesthetized with pentobarbital sodium. Half of each group of the rats were used to collect brain and followed by the separation of hippocampus. The hippocampus was weighed and lysed for total protein extraction. Samples were then stored at −80 °C for later use. Western blotting was performed to detect the protein expression of CREB and p-CREB in the hippocampus. The half of the rats were transcardially perfused with 4% paraformaldehyde and the brain was collected and fixed in 4% paraformaldehyde. Immunohistochemistry was performed to detect CREB and p-CREB expression. (Fig. 1).
+The neonatal rats were randomly assigned into different groups to reduce variation. We normalized CREB and p-CREB protein expression in control group as 1. CREB and p-CREB expression in the Iso1 and Iso2 group was compared with the controls. All data are expressed as mean ± standard deviation. Statistical analyses were performed by using SPSS software (version 21.0; IBM, Corp., Armonk, NY, USA). One-Way ANOVA was used to compare the means between groups. A value of P < 0.05 indicated significance.

20230808-AI coding-1st round/1209 – Li 2013.txt

@@ -0,0 +1,11 @@
+PMID: 23603260 DOI: 10.1016/j.neulet.2013.04.008
+2. Materials and methods
+All animal procedures were in compliance with the NIH Guide for the Use of Laboratory Animals and approved by the Animal Care and Use Committee of Sun Yat-sen University. Seven-day-old (P7) Sprague-Dawley rat pups (Guangdong Medical Laboratory Animal Co, China) with body weight at 16 ± 3 g were exposed to 1.1% isoflurane (about 0.5 MAC in P7 rats [22]) for 4 h to induce neuronal apoptosis, or to air in a temperature-controlled chamber as we described before [21]. The concentrations of anesthetic gas, oxygen and carbon dioxide (CO2) in the chamber were measured by a gas analyzer (Datex-Ohmeda, Madison, WI).
+
+Four doses of SP600125 (Selleck Chemicals LLC, Houston, TX, USA) (5, 10, 20 or 30 μg) or 12% dimethyl sulfoxide (DMSO) as the vehicle were administered by intracerebroventricular (i.c.v.) injection 15 min before isoflurane exposure. Some rats received 30 μg SP600125 or 12% DMSO only. The injection was performed as described before [6] under isoflurane anesthesia with a 5 μl microsyringe and 0.4 mm external diameter needle. The location of injection was 2.0 mm rostral, 1.5 mm lateral to the lambda and 2.0 mm deep to the skull surface of rats. The injection solution of 5 μl/rat was infused at a constant rate of 2.5 μl/min. The accuracy of i.c.v. injection was verified by methylene blue in our preliminary experiments. All animals were sacrificed 6 h after termination of gas exposure and their hippocampi were used for Western blotting (n = 6) or TdT-mediated dUTP nick end labeling (TUNEL) with fluorescent dye (n = 6).
+
+For Western blotting studies, rat pups were anaesthetized with isoflurane and then sacrificed by decapitation. Hippocampi of rats were isolated immediately on ice and then stored at −80 °C until used. Western blotting was performed as we have described previously [20]. In brief, the protein concentrations of samples were determined using the BCA protein assay (Bio-Rad,Herts, UK). Sixty micrograms of each sample were subjected to Western blot analysis using the following primary antibodies: anti-cleaved caspase-3 at 1:2000 dilution, anti-phospho-JNK at 1:2000 dilution, anti-JNK at 1:2000 dilution, anti-phospho-c-Jun at 1:1000 dilution, anti-phospho-Akt (Ser 473) at 1:2000 dilution, anti-Akt at 1:5000 dilution, anti- phospho-GSK-3β (Ser 9) at 1:2000 dilution, anti-GSK-3β at 1:2000 dilution, anti-Bcl-xL at 1:2000 dilution and anti-β-actin at 1:2000 dilution. All antibodies were purchased from Cell Signaling Technology Company, USA. Images were scanned by an Image Master II scanner (GE Healthcare) and were analyzed using Image Quant TL software (v2003.03, GE Healthcare). The band signals of phospho-JNK, phospho-Akt and phospho-GSK-3β were normalized to their total JNK, Akt and GSK-3β from the same samples. The band signals of other interesting proteins were normalized to those of β-actin and the results in each group were normalized to that of corresponding control group.
+
+For TUNEL studies, rat pups were anaesthetized with isoflurane and perfused transcardially with 4% paraformaldehyde. Their brains were paraffin embedded and sectioned at 6 μm thickness. As we described before [19], four or five sections (200 μm apart) for each animal at the same plane of the hippocampus were chosen for detecting apoptosis using TUNEL fluorescent method (Promega, Madision, WI, USA). The slides were protected from direct light during experiment. Hoechst was used to stain nuclei. The TUNEL positive cells in CA1, CA3 and dentate gyrus (DG) areas of hippocampus were analyzed immediately with NIS-Elements BR imaging processing and analysis software (Nikon Corporation, Japan). The densities of the TUNEL positive cells in CA1, CA3 and DG were calculated by dividing the number of TUNEL positive cells by the area of that brain region.
+
+Data are presented in mean ± SEM. The Graphpad Prism 4.0 software was used to conduct the statistical analyses. A two-tailed P value of less than 0.05 was considered statistically significant. One way ANOVA with Newman–Keuls Multiple Comparison Test was used when data was normally distributed and had equal variances. Otherwise, non-parametric test with Dunn's Multiple Comparisons was used to compare the density of TUNEL positive cells as well as the relative protein abundance data among groups in Western blots.

20230808-AI coding-1st round/1256 – Lin 2018.txt

@@ -0,0 +1,30 @@
+PMID: 29461008 PMCID: PMC5908131 DOI: 10.1111/jcmm.13524
+2. MATERIALS AND METHODS
+2.1. Drugs
+All drugs were prepared just before use: propofol (Diprivan; AstraZeneca UK limited, Italy: jc393, 20 mL: 200 mg); 20% intralipid (2B6061; Baxter, Deerfield, IL, USA); SAHA (Selleck Chemicals LLC, Houston, TX, USA). HGN antisense was synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. Senegenin (purity ≥ 98%) was purchased from Nanjing SenBeiJia Biological Technology Co., Ltd. (Jiangsu province, China).
+
+Anti‐β‐actin and anti‐rabbit IgG secondary antibody were obtained from Cell Signaling Technology (Cell Signaling Tech, MA, USA). Anti‐CREB (Phospho S133), anti‐NMDAR2B, anti‐HDAC2, antisynaptophysin, anti‐Ac‐H4K12 and anti‐Ac‐H3K14 antibodies were purchased from Abcam (Abcam, Cambridge, MA, USA). Anti‐HGN antibody was synthesized by Kitgen Bio‐tech Co., Ltd.(Zhejiang province, China).
+
+2.2. Animals
+The protocol in this study was approved by the institutional review board of the First Affiliated Hospital of Nanchang University on the Use of Animals in Research and Teaching. All the methods in this study were performed in co‐ordination with relevant guidelines and regulations. Sprague Dawley (SD) rats were purchased from the animal science research department of the Jiangxi Traditional Chinese Medicine College (JZDWNO: 2011‐0030; Nanchang, Jiangxi,China). The learning and memory functions of the parental rats were assessed using the Morris water maze (MWM) system before mating, so that to minimize the hereditary difference. Animals were housed separately under standard laboratory conditions with 12:12 light/dark cycle, 24 ± 1°C and had free access to tap water. Two female rats in cages with one male rat per cage for mating. Pregnancy was diagnosed by the sign of vaginal plug.
+
+2.3. Drug treatment
+On E7, pregnant rats received intravenous infusion of propofol (n = 10 dams) with the rate of 20 mg kg−1 h−1 for 4 hours, equal volume of saline (n = 10 dams) or intralipid (n = 5 dams), respectively.
+
+Electrocardiograms, saturation of pulse oximetry (SpO2) and tail non‐invasive blood pressure were continuously monitored during maternal propofol exposure. Using heating lamp and temperature controller to monitor the rectal temperature and keep it at 37 ± 0.5°C. Arterial blood sampling from lateral caudal artery for blood gas analysis at the end of propofol anaesthesia. If the total time of SpO2 <95% and/or the systolic blood pressure <80% of the baseline in excess of 5 minutes, the pregnant rat was got rid of the study, and other pregnant rats were chosen to supply the sample size, so as to exclude the interfering effect of maternal hypotension or hypoxia on cognitive function in the pup rats.
+
+After delivery, the offspring rats born to the same pregnant rat were randomly subdivided into the SAHA, SEN, HGNA group and their relative control groups (DMSO, NS(1) and NS(2) group, respectively; Figure ​Figure1).1). It has been proved that the acetylation level of histone in hippocampus obviously increased 2 hour after the administration of HDAC inhibitor.27 Therefore, 90 mg kg−1 SAHA (HDAC inhibitor), at a concentration of 0.6 μmol L−1 dissolved into dimethyl sulphoxide (DMSO) was injected to the offspring in SAHA group by the intraperitoneal route at 2 hours before each MWM trial. The same volume of DMSO was given to the DMSO group. Senegenin, a kind of Chinese medicine, was proved to up‐regulate the expression of NR2B mRNA and protein, thus to mitigate cognitive dysfunction.28 So, 15 mg kg−1 Senegenin and equal volume of saline were given intraperitoneally at 2 hours before each MWM trial to SEN or NS(1) groups, respectively. HGN antisense oligonucleotide (0.25 nmol μL−1, 1.5 μL) or normal saline (1.5 μL) was injected to offspring's hippocampus in HGNA or NS(2) group as previously described,18, 29 once daily for seven consecutive days before MWM trial.
+2.4. Morris water maze test
+Spatial learning and memory were assessed by the MWM test from post‐natal day 30 (P30) to P36 according to previously described5, 30 with SLY‐WMS Morris water maze test system (Beijing Sunny Instruments Co. Ltd., Beijing, China). Briefly, the trials start at 9 o'clock in the morning in the MWM system with the pool was filled with water to a height of 1.0 cm above the top of a 15‐cm‐diameter platform, in the second quadrant (target quadrant), and the water maintained at 24 ± 1°C. The training trial was performed once a day for six consecutive days. In each training trial, offspring rats were placed in the water facing the wall of the pool in the third quadrant, the farthest one from the target quadrant. The animals were allowed to search for the hidden platform or for 120 seconds. They were allowed to remain on the platform for 30 seconds when they found the platform and the time for the animal to find the platform was recorded as escape latency (indicating learning ability). For those who did not find the platform within 120 seconds, the animals were gently guided to the platform and allowed to stay there for 30 seconds, and their escape latency was recorded as 120 seconds. At the end of the reference training (P37), the platform was removed. The offspring rats were allowed to perform spatial probe test (memory function test) for 120 seconds. Times across the platform (platform crossing times, indicate memory function), the swimming trail and speed were automatically recorded by the system. The mean value of the platform crossing times, escape latency and speed of the offspring born to the same pregnant rats was taken as the final results.
+
+2.5. Brain hippocampus harvest
+The day after the MWM test, rats were anaesthetized with isoflurane and killed by cervical dislocation. Hippocampus tissues were harvested and stored in Eppendorf tubes that had been treated with 1% DEPC and were stored at −80°C (for Western blot analyses) or immersed in 4% paraformaldehyde (for immunofluorescence assay).
+
+2.6. Western blot analysis
+The hippocampus (n = 6, with three male and three female offspring rats from each group) were homogenized on ice in lysis buffer containing a protease inhibitors cocktail. Protein concentration was determined by the bicinchoninic acid protein assay kit. Protein samples (20 μg) were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a PVDF membrane. The membranes were blocked by non‐fat dry milk buffer for 1.5 h and then incubated overnight at 4°C with antihistone H3 (acetyl K14) (1:2000), antihistone H4 (acetyl K12) (1:10000), anti‐NMDAR2B (1:1000), anti‐HGN (1:1000), antisynaptophysin (1:10000) and anti‐β‐actin (1:2000), respectively. Thereafter, the membranes were washed three times with TBS‐T buffer for 15 minutes and incubated with the horseradish peroxidase (HRP)‐conjugated secondary antibody for 2 hours at room temperature. The immune complexes were washed three times with TBS‐T buffer and detected using the ECL system (Millipore Corporation, MA, USA). The images of Western blot products were collected and analysed by ImageJ 1.50i (Wayne Rasband, National Institutes of Health, USA). The density of observed protein band was normalized to that of β‐actin in the same sample. The results of offspring from all the other group were then normalized to the average values of normal saline control offspring (control+NS group) in the same Western blot. The mean expression level of all of the offspring born to the same mother rat in the same group was calculated as the final expression level of the observed proteins.
+
+2.7. Immunofluorescence staining
+Immunofluorescence staining was used to assess HDAC2 and phospho‐CREB in the hippocampus of offspring rats after the MWM test. Hippocampus from offspring rats (n = 6, with three male and three female offspring rats from each group) were fixated in paraformaldehyde. Five‐μm frozen sections of the hippocampus were used for the immunofluorescence staining. The sections were incubated with anti‐HDAC2 (1:300) and anti‐CREB (1:100) dissolved in 1% bovine serum albumin in phosphate‐buffered saline at 4°C overnight. Then, the sections were incubated with fluorescent‐conjugated anti‐rabbit secondary antibody (1:300) for 1 hour in the dark at room temperature. Negative control sections were incubated with PBS as a substitute for primary antibody. Finally, the sections were wet mounted and viewed immediately using a inverted fluorescence microscope (200×) (Olympus, Japan). The target protein was red, and nuclei were blue. The proteins of HDAC2 and p‐CREB were excited by the green light, while the DAPI was performed by UV blue light. All images were recorded at 10 × 20× (Exp Acq‐700mmm, Offset Acq‐1, Gain Acq‐1, Gamma Acq‐300). The density of HDCA2 and p‐CREB staining was conducted on the images using Image‐Pro Plus 6.0 (Media Cybernetics Inc., USA). The images were converted it into black and white pictures. After intensity calibration, hippocampal CA1 area was chosen to analyse and the integrated optical density (IOD) was measured. IOD/Area was calculated as the protein expression level.
+
+2.8. Statistical analysis
+All analyses were performed with SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Data from escape latency in the MWM test were subjected to a repeated measures two‐way analysis of variance (RM two‐way ANOVA) and were followed by least significant difference t (LSD‐t) analysis when a significant overall between‐subject factor was found (P < 0.05). Data from Western blot and immunofluorescence staining results were subjected to one‐way ANOVA analysis. All data well provided for any of the variables. The LSD t test was used to determine the difference between groups. Statistical significance was declared at P < .05.

20230808-AI coding-1st round/1395 – Liu 2018.txt

@@ -0,0 +1,7 @@
+PMID: 29434807 PMCID: PMC5776508 DOI: 10.3892/etm.2017.5651
+Materials and methods
+Animals The current study was approved by the Animal Care Committee at Sun Yat-sen University (Guangzhou, China) and performed in accordance with the National Institutes of Health Guide for the Use of Laboratory Animals (27). A total of 24 C57BL/6 male mouse pups, aged 7 days (P7) and weighing 3.5–4.5 g were obtained from Guangdong Medical Laboratory Animal Center (Guangdong, China; permission no. SCXK2011-0029). The pups were housed in the same cage as their mothers and were kept under temperature-controlled environmental conditions (26°C) on a 14:10 constant light-dark cycle until P7. The mother mice had free access to food and water. The mouse pups at P7 were exposed to 2.6% sevoflurane (Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) for 6 h [~1.0 minimal alveolar concentration (MAC) in P7 mice] in 50% oxygen in a temperature-controlled chamber, following a previously described protocol (n=12) (17). The control mice were exposed to normal air for 6 h under the same condition (n=12). The concentrations of anesthetic gas, oxygen and carbon dioxide in the chamber were measured using a gas analyzer (Datex-Ohmeda; GE Healthcare, Chicago, IL, USA). All animals were sacrificed 2 h following termination of sevoflurane/oxygen exposure and their cortices were used for western blotting (sevoflurane group, n=6; control group, n=6) or TdT-mediated dUTP nick end labeling (TUNEL) with fluorescent dye (sevoflurane group, n=6; control group, n=6).
+Tissue preparation Half of the mice in each group were used for western blotting and half of the mice for TUNEL studies. For western blotting, mouse pups were anaesthetized by inhaling 3% of sevoflurane until loss of the righting reflex (LORR), which indicated the mice had lost consciousness. Then the mice were sacrificed by decapitation. Cortices were isolated immediately on ice and then stored at −80°C until use. For TUNEL studies, mouse pups were sacrificed by inhaling 3% of sevoflurane until LORR and perfused transcardially with ice-cold normal saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min at 4°C. Their brains were post-fixed in the same fixative for 48 h at 4°C, and then paraffin embedded and sectioned into 6-µm-thick sections. As described in previous studies (15,16,25), at least three sections in the same plane of the hippocampus for each animal were selected to detect cells that exhibited positive TUNEL staining; all sections used in TUNEL were 100 µm apart and the sections were according to Figures 129–131 in the Atlas of the Developing Mouse Brain (28).
+Western blotting Western blotting was performed as previously described (15,16,25). Briefly, the protein concentration in each sample was determined using a BCA protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Sample proteins (40 µg/lane) were separated on 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology, Shanghai, China) in Tris-buffered saline with Tween-20 (TBST) at room temperature for 1 h. Membranes were subsequently incubated at 4°C overnight with the following primary antibodies: Anti-cleaved caspase-3 (cat no. 9664) at 1:2,000 dilution, anti-α-fodrin (which contain SBDP145 and SBDP120 fragments; cat no. 2122) at 1:2,000 dilution, anti-phosphorylated-(p)-JNK (cat no. 4668) at 1:2,000 dilution, anti-JNK (cat no. 9252) at 1:2,000 dilution, anti-p-ERK1/2 (cat no. 4376) at 1:1,000 dilution, anti-ERK1/2 (cat no. 4695) at 1:1,000 dilution, anti-p-P38 (cat no. 4631) at 1:1,000 dilution, anti-P38 (cat no. 9212) at 1:1,000 dilution, anti-p-CREB (cat no. 9198) at 1:1,000 dilution, anti-p-nuclear factor-κB (NF-κB) (cat no. 3033) at 1:1,000 dilution, anti-p-Akt (Ser 473) (cat no. 4060) at 1:2,000 dilution, anti-Akt (cat no. 4685) at 1:5,000 dilution, anti-p-GSK-3β (Ser 9) (cat no. 5558) at 1:2,000 dilution, anti-GSK-3β (cat no. 9315) at 1:2,000 dilution, anti-p-CRMP-2 (Thr 514) (cat no. 9397) at 1:2,000 dilution, anti-CRMP-2 (cat no. 9393) at 1:2,000 dilution and anti-β-actin (cat no. 3700) at 1:2,000 dilution (all Cell Signaling Technology, Inc., Danvers, MA, USA) and anti-p-GSK-3β (Ty 216) (cat no. ab75745; Abcam, Cambridge, USA) at 1:2,000 dilution. The membranes were washed with TBST three times and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse IgG, cat no. A0216; goat anti-rabbit IgG, cat no. A0208; 1:2,000; Beyotime Institute of Biotechnology) at room temperature for 1 h. The membranes were washed with TBST three times and visualized using an enhanced chemiluminescence detection system (cat no. 34580; Thermo Fisher Scientific, Inc.). Images were scanned using an Image Master II scanner (GE Healthcare) and were analyzed using Image Quant TL software (v2003.03, GE Healthcare). The band signals of p-ERK1/2, p-JNK, p-p38, p-Akt, p-GSK-3 and p-CRMP-2 were normalized to the bands of total ERK1/2, JNK, p38, Akt, GSK-3β and CRMP-2 from the same samples. The band signals of the other proteins were normalized to those of β-actin and the results in each group were normalized to that of the corresponding control group.
+TUNEL assay TUNEL was performed following a previously described protocol (15,16). A Dead End™ fluorometric TUNEL system (Promega Corporation, Madison, WI, USA) was used and staining following the manufacturer's protocol. Briefly, TUNEL labeling was conducted with a mix of 45 µl equilibration buffer, 5 µl nucleotide mix and 1 µl recombinant terminal deoxynucleotidyl transferase (rTdT) enzyme in a humidified, lucifugal chamber for 1 h at 37°C, and then Hoechst 33258 (H-33258; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to stain nuclei for 10 min at room temperature. The sections were protected by anti-Fade solution and mounted on glass coverslips with clear nail polish sealing the edges. Slides were protected from direct light during the experiment. The images of TUNEL positive cells in the retrosplenial cortex (RS), frontal cortex (FC) and parietal association cortex (PtA) areas were acquired by Ti-S inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan) and analyzed using NIS-Elements Basic Research imaging processing and analysis software (version 3.0; Nikon Corporation). The density of TUNEL positive cells in the three cortical regions was calculated by dividing the number of TUNEL positive cells by the area of that brain region.
+Statistical analysis Sample size was calculated using PASS 11 software (NCSS, LLC, Kaysville, UT, USA) to achieve 80% power at a significance level of P<0.05. All data were determined to be normally distributed using the Shapiro-Wilk test and had no significant heterogeneity of variance as detected by Levene's test. GraphPad Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA) was used to conduct all statistical analyses. Data were presented as mean ± standard deviation and were analyzed by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

20230808-AI coding-1st round/1455 – Lu 2018.txt

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+PMID: 29731813 PMCID: PMC5920718 DOI: 10.3892/etm.2018.5950
+Materials and methods
+Animal model All experiments were performed according to the guidelines of the Guide for the Care and Use of Laboratory Animals (22) and were approved by the Institutional Animal Care and Use Committee of Ruijin Hospital Affiliated to Shanghai Jiaotong University (Shanghai, China). A total of 174 C57BL/6 mice (sex ratio, 1:1), were provided by the Model Animal Research Center of Nanjing University (Nanjing, China). They were housed in polypropylene cages (5 or 6 animals per cage) and kept at a 12 h light-dark cycle at room temperature (21–24°C) in 55% humidity for 7 days prior to testing. All animals had free access to food and water.
+Experimental protocols There were two experimental protocols used based on the sevoflurane concentration used in previous studies (23,24) and 1.3 and 2.6% sevoflurane was used in the present study. For protocol one, 36 mice were randomly assigned into 3 groups with 12 mice in each group: The 2.6 and 1.3% sevoflurane groups and the control group (exposed to 30% O2). Following exposure to sevoflurane or O2 for 6 h, the mice from all 3 groups were sacrificed by intraperitoneal injection of 1.5% pentobarbital sodium (375 mg/kg) (Dalian Idery Biotechnology Co., Ltd., Dalian, China). Hippocampal tissue samples from these mice were collected to measure the expression of caspase-3 using immunohistochemistry, the cleavage of PARP by western blotting, and levels of BDNF, Ntrk2, pro-BDNF, p75NTR and PKB/Akt by ELISA. To evaluate whether hypoxia and respiratory depression occurred in mice during anesthesia, blood gas analysis was performed in another 78 mice, which were randomly assigned into 3 groups: 2.6% sevoflurane (n=36), 1.3% sevoflurane (n=36) and control (n=6) groups. The mice in the 1.3 and 2.6% sevoflurane groups were divided into subgroups based on the length of time they were exposed to sevoflurane (1, 2, 3, 4, 5 and 6 h), with 6 mice in each subgroup.
+For protocol two, a total of 60 mice were randomly assigned into 3 groups with 20 mice in each group: 2.6, 1.3% sevoflurane and control groups. Following exposure to sevoflurane for 4 weeks, the MWM test was performed in half of the mice in each group. The MWM test was conducted on the remaining mice at week 12.
+
+Sevoflurane exposure As stated in a previous study (25), animals were placed in a temperature-controlled (37–38°C) transparent anesthetic chamber that was connected to an anesthetic gas monitor (Datex-Ohmeda S/5, Datex-Ohmeda; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). For mice in the 1.3 and 2.6% sevoflurane groups, mixed gas (5% sevoflurane and 30% O2) was pre-aerated at a flow rate of 10 l/min until the concentration of sevoflurane reached 5% in the chamber and prior to placing mice in the chamber. Subsequently, these mice were placed into the chamber immediately. Following maintenance of 5% sevoflurane for 30 sec, mice were exposed to 1.3 or 2.6% sevoflurane for the indicated time periods (1–6 h), during which 30% O2 was continually gassed into the chamber at a flow rate of 3 l/min. For mice in the control group, 30% O2 alone was aerated into the chamber for 6 h, with a flow rate of 3 l/min.
+Blood gas analysis The mice were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital (50 mg/kg). Then blood samples (0.2 ml) were obtained from the left ventricle by cardiac puncture, after which the mice were sacrificed by intraperitoneal injection of 1.5% sodium pentobarbital (375 mg/kg). The partial pressure of oxygen (PaO2), partial pressure of carbon dioxide (PaCO2) and arterial oxygen saturation (SaO2) were detected using a portable blood gas analyzer (OPTI Medical Systems Inc., Roswell, GA, USA).
+Tissue sample collection Following sevoflurane exposure, all the mice were sacrificed by intraperitoneal injection of 1.5% pentobarbital sodium (375 mg/kg). The brain was then rapidly removed and the complete hippocampus was dissected. Hippocampal tissue samples were stored at −80°C prior to use in laboratory experiments.
+Immunohistochemistry The hippocampal tissues were fixed overnight in 4% paraformaldehyde at 4°C. The hippocampal slices (5-µm-thick) were subsequently prepared using a vibrating tissue slicer (Campden Instruments, Ltd., Loughborough, UK). Immunohistochemical staining was performed as previously described (26,27). Briefly, slices were incubated with hydrogen peroxide in methanol to block endogenous peroxidase activity and 10% normal goat serum (cat. no. C0265; Beyotime Institute of Biotechnology, Haimen, China) to reduce non-specific antibody binding prior to immunohistochemical staining. Slices were then incubated with a rabbit anti-caspase-3 antibody (1:200; cat. no. AC033; Beyotime Institute of Biotechnology) at 4°C for 12 h, followed by three washes with PBS. Subsequently, these slices were incubated with secondary antibody (1:4,000; cat. no. A0562; biotinylated goat anti-rabbit antibody; Beyotime Institute of Biotechnology) for 30 min at 37°C. Following washing with PBS, immunoreactivity was visualized using the streptavidin-peroxidase complex and 3,3′-diaminobenzidine (both from Beyotime Institute of Biotechnology). A DM5000B light microscope (Leica Microsystems GmBH, Wetzlar, Germany) was used to observe and collect images. The image analysis software Image Pro Plus version 4.0 (Media Cybernetics, Inc., Rockville, MD, USA) was used to count the number of caspase-3 positive cells.
+Western blotting The preparation of hippocampus protein extraction was performed as previously described (28,29). Total proteins were extracted with radioimmunoprecipitation assay buffer [1% Triton X-100, 50 mM Tris, (pH 7.4), 150 mM NaCl, M, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA and 1% sodium deoxycholate]. Following 13,000 × g centrifugation at 4°C for 20 min, the supernatant was used for western blotting (30,31). The BCA method was used to assay protein concentrations. In brief, hippocampal tissue proteins were separated by 10% SDS polyacrylamide gel electrophoresis and then electrotransferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat powdered milk for 1 h at 25°C. The proteins were probed with rabbit anti-PARP antibodies (1:200, cat. no. AP102) or rat anti-GAPDH antibodies (1:5,000, cat. no. AG019) overnight at 4°C. Then, goat anti-rabbit (1:4,000; cat. no. A0208) or goat anti-rat (1:4,000; cat. no. A0192) horseradish peroxidase-conjugated secondary antibodies were used for 2 h incubation at room temperature (all from Beyotime Institute of Biotechnology). Proteins were visualized by an enhanced chemiluminescence method and analyzed with the Dolphin-Doc Plus Gel Documentation system (version 1141002; Wealtec Corp., Sparks, NV, USA). This procedure was repeated twice for all 3 groups. The relative level of PARP was presented as the band intensity and normalized to the corresponding band intensities of GAPDH.
+ELISA The method of hippocampus protein extraction mentioned above was also used for ELISA. The levels of BDNF, Ntrk2, pro-BDNF, p75NTR and PKB/Akt were measured using an ELISA kit (cat. no. EK0312; Wuhan Boster Bio-Engineering Co., Ltd., Wuhan, China) according to the manufacturer's instructions. Briefly, protein samples were added to the enzyme label plate and incubated for 1.5 h at 37°C. Next, the biotin-labeled antibodies were added for 1 h incubation at 37°C. Following washing, 30 min incubation with avidin peroxidase complex was conducted at 37°C. Color was developed using 3,3′,5,5′-tetramethylbenzidine following 20 min incubation at 37°C. Following reaction termination with a ‘stop’ solution, the products were measured at 450 nm using a microplate spectrophotometer (Spectramax 190; Molecular Devices LLC, Sunnyvale, CA, USA). All samples were assayed in duplicate and the readings were normalized to the amount of standard protein.
+Behavioral studies Prior to the MWM test, mice received 2 min of touch for 5 days to avoid the fear to touch during the test. The MWM test was performed as previously described (32,33), with minor modifications. The round pool (diameter, 122 cm) was filled with warm water, made opaque by the addition of titanium dioxide and an escape platform was placed in the northwest quadrant and hidden 0.5 cm below the surface of the water. The MWM test was performed on 7 consecutive days (6 days for training and 1 day for the probe test). Briefly, mice received 4 training sessions daily for 6 consecutive days. Each trial began from a different point and ended when the mice found the platform. The time from beginning to end was considered to be the time of escape latency. If mice could not find the platform within 90 sec, the time of escape latency was recorded as 90 sec. If mice found the platform within 90 sec, the real time from beginning to end was recorded as the time of escape latency. The swim rate during training was also recorded. On day 7, the probe test was performed by allowing the mice to swim for 60 sec in the absence of the platform. During 60 sec swimming, the time spent in the northwest quadrant and platform site crossovers was recorded and analyzed using the MWM JLBehv-FCS video analysis system (DigBehv-MG; Shanghai Jiliang Software Technology Co., Ltd., Shanghai, China).
+Statistical analysis All data are presented as the mean ± standard error of the mean. A repeated measures analysis of variance (ANOVA) was used to measure the differences within groups over time. Meanwhile, one-way ANOVA was applied for comparison among groups (2.6, 1.3% sevoflurane and control groups), followed by Student Newman-Keuls post hoc test. The correlation between the swim rate and time of escape latency was identified using the Pearson Correlation coefficient. For all the analysis, P<0.05 was used to indicate a statistically significant difference. Additionally, SPSS 11.5 (SPSS, Inc., Chicago, IL, USA) was used for the analysis of the present study.

20230808-AI coding-1st round/1730 – Lai 2016.txt

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+PMID: 26541582 DOI: 10.1016/j.brainres.2015.10.050
+4. Experimental procedures
+4.1. Animals
+A total of 240 male and female clean Sprague-Dawley rats, 7 days of age and weighting 12–16 g, (Shanghai Slac Laboratory Animal Co., Ltd., China) were used in this study. They were housed and treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 80-23, revised in 2011). All rats were maintained under standard laboratory temperature and humidity and a 12 day/night cycle (8 am/8 pm), and were allowed free access to food and water. The study was approved by the Experimental Animal Care Committee of the Fujian Medical University Union Hospital, and efforts were made to minimize the number of animals used and their suffering.
+
+4.2. Experimental protocol
+The animals were randomly allocated into 10 groups (n=24 per group; Fig. 1): (1) Sham, without hypoxia-ischemia; (2) HI/Control, received cerebral hypoxia-ischemia; (3) HI+Atractyloside (Atr), (4) HI+Cyclosporin A (CsA), treated like the control and respectively injected with Atr (10 mg/kg) and CsA (5 mg/kg); (5) HI+sevoflurane (Sev), treated like the control and received sevoflurane postconditioning; (6) HI+Sev+LY, (7) HI+Sev+L-N, (8) HI+Sev+SB, (9) HI+Sev+Atr, (10) HI+Sev+CsA, treated like the HI+Sev group and respectively injected with LY294002 (0.3 mg/kg), L-NAME (10 mg/kg), SB216763 (0.2 mg/kg), Atr (10 mg/kg), and CsA (5 mg/kg). LY294002, L-NAME, and SB216763 are specific blockers of Akt, eNOS, and GSK-3β, respectively. Atr and CsA open and close, respectively, mPTPs. In each group, the brains of the rats that received behavioral testing (from 32-days-old to 42-days-old; n=12 per group) were harvested for determination of hippocampal neuron count and morphology study, and the brains of the other 12 rats were harvested 24 h after the intervention for Western blot analysis, and study of mitochondrial permeability transition pore opening.
+
+4.3. Cerebral HI model and sevoflurane postconditioning
+The cerebral HI model was adapted from a procedure described previously (Ren et al., 2014). Briefly, the rats were anesthetized with pentobarbital sodium (0.5–1%, 40–50 mg/kg, intraperitoneal), and their left common carotid arteries were permanently ligated with a double 7–0 surgical silk; the arteries in the Sham group, however, were not ligated. A dose of 5 µL of 0.1% DMSO or drug (LY294002, L-NAME, SB216763, Atr, CsA) with 0.1% DMSO was injected into the left lateral ventricle immediately after the surgery as previously described (Satoh and Onoue, 2005). After waking, the rats were returned to their cages with the mothers for 1.5–2.5 h, and then placed in a chamber containing humidified 8% O2–92% N2 for 2 h. The air temperature in the chamber was maintained at 36.5±1 °C. The chamber was then exposed to room air for 15–20 min. For sevoflurane postconditioning, the animals were placed in a chamber containing 2.5% sevoflurane in 30% O2–70% N2 for 30 min after cerebral HI injury. After waking, the neonates were cleaned with 75% alcohol and returned to their mothers.
+
+4.4. Novel object recognition test
+The rats were evaluated with a nonspatial object recognition memory task 25 days after the intervention as described by Ennaceur and Delacour (1988) and Bruel-Jungerman et al. (2005). Briefly, for the first 3 days, after being comforted and stroked, each animal was put into an open chamber made of black plexiglas (80×80×60 cm3) for a 5 min acclimation and the test was conducted on the fourth day. Before the test, the animals received a 5 min training in the chamber containing 2 different objects (a white cube and a red cylinder) fixed at adjacent angles with a spacing of 10 cm from the field wall. Rats were put into the chamber with their backs turned towards the objects and allowed to explore the chamber freely for 5 minutes. Exploratory behavior can be identified when rats touch the objects with their noses or put their noses at places within 2 cm of the objects. To test memory storage, the white cube was kept in the chamber and the red cylinder was replaced by a blue semisphere. Exploratory time of new (T2) and old (T1) objects within 5 min was recorded and memorization ability of the rats was assessed by discrimination index: DI=T2/(T1+T2). The blue semisphere was replaced by a green prism 3 h after training, and the green prism was replaced by a yellow irregular shape 24 h after the training. The time each rat used to explore new and old objects was recorded for calculating DI. The DIs at 5 min, 3 h, and 24 h after the training (DI0 h, DI3 h, DI24 h) represent the instant, short-term, and long-term memory, respectively. Data with total exploration time less than 20 s were excluded from statistic analysis. The field was always provided with even light, and the objects and fields were cleaned with 75% ethanol after each testing.
+
+4.5. Morris water maze test
+After the novel object recognition test, the Morris water maze was used to test spatial learning and memory (Peng et al., 2012, Jiang et al., 2004). Briefly, a black circular pool (120 cm in diameter, 50 cm in height) was filled with water (25±1 °C) to a depth of 25 cm and located in a quiet room. Chinese ink was added to make the water opaque. The water maze was conceptually divided into 4 quadrants, and a hyaline platform (10 cm in diameter) was submerged 1 cm below the surface of the water at the midpoint of the third quadrant. In the place navigation trial, each rat underwent 4 successive trials a day for 5 days for memory acquisition training, with a 15 min interval between trials for the rat to recover physically. The sequence of water-entry points differed each day, but the location of the platform was constant. Escape latency (EL) to find the platform was measured up to a maximum of 120 s. On locating the platform, the rat was left there for 15 s before the next trial. If the rats failed to locate the platform within 120 s, it was guided to the platform and allowed to stay there for 15 s. Latency and the search strategies, including straight strategy, tendency strategy, marginal strategy, and random strategy, were recorded for each trial. Twenty-four hours after the last training session, a space exploration trial was performed. The platform was removed from the pool and rats were allowed to swim freely for 60 s. Four indexes were calculated: (1) the time spent by the rats in the third quadrant in which the platform was hidden during acquisition trials; (2) the number of rats crossing exactly over the original position of the platform; (3) the search path in the target quadrant; (4) the total movement distance. Search speed was calculated by total movement distance divided by 120 cm/s. All trials were videotaped by a camera located 2 m above the water surface and computer analyzed.
+
+4.6. Histology of left hippocampal neurons
+After the behavioral studies, rats were anesthetized with pentobarbital, transcardialy perfused with 200 mL of 4 °C heparin saline solution and then with 300 mL of 4% paraformaldehyde. Left hippocampus was made into a wax block according to Paxinos–Waston methods. Continual coronal sections (4 µm in thickness) at approximately 3.3 mm caudal to bregma were obtained, and subjected to hematoxylin–eosin (HE) staining. The sections were examined by an observer blinded to the rat group assignment. Neurons microscopically showed a clear boundary, a round or an oval shape, a smooth cell membrane, basophilic cytoplasm (Nissl body), a large and round nucleus, a clear nuclear membrane and a large and round nucleolus will be defined as surviving neurons. Apoptotic neurons will not be regarded as surviving ones. Surviving neurons in pyramidal cell layer of the CA1 and CA3 regions were counted (n/mm) by two investigators blind to experimental conditions, and a count was determined by averaging the total of 5 sections.
+
+4.7. Western blot analysis
+Proteins were separated on a 12% SDS-PAGE gel, and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, USA). The membrane was blocked using 5% nonfat milk and incubated with a mouse anti-p-Akt, t-Akt, p-eNOS, t-eNOS, p-GSK-3β, and t-GSK-3β monoclonal antibody (mAb) (Cell Signaling Technology, Beverly, MA, USA) or a mouse anti-β-actin mAb (Sigma, USA). The proteins were visualized and quantified using ECL reagents (Pierce, IL, USA).
+
+4.8. mPTP opening assay
+Preparation of mitochondria was adapted from a procedure described previously (Wu et al., 2006). All procedures were carried out in the cold (0–4 °C). Hippocampal pieces were placed in isolation buffer (250 mmol/L sucrose, 210 mmol/L mannitol, 1 mmol/L K-EDTA, 10 mmol/L Tris–HCl, pH 7.4) and homogenized (10 mL buffer/g). The homogenate was immediately centrifuged at 2000g for 3 min. The supernatant was centrifuged again at 2000g for 3 min, the second supernatant was decanted and centrifuged at 12,000g for 8 min, and the resulting supernatant was decanted and resuspended in isolation buffer without K-EDTA. The suspension was centrifuged at 12,000g for 10 min and the resulting mitochondrial pellet was resuspended in the same buffer. Mitochondrial protein concentration was quantified according to the Bradford׳s method using 1 g/mL bovine serum albumin (BSA) as standard. Purity and integrity of isolated mitochondria were confirmed by neutral red-Janus green B staining (Sigma, USA). Isolated mitochondria from the hippocampus (0.5 mg protein) was resuspended in swelling buffer (71 mmol/L sucrose, 215 mmol/L mannitol, and 10 mmol/L sodium succinate in 5 mmol/L HEPES, pH 7.4) to a final volume of 2 mL, and incubated at 25 °C for 2 min. mPTP-induced mitochondrial swelling was confirmed by 5 min incubation with the strong mPTP inhibitor CsA before addition of CaCl2, and was measured with a spectrophotometer (Beckman DU800, USA) as a reduction in optical density at 540 nm (OD540) (Kristal and Brown, 1999, Baines et al., 2003).
+
+4.9. Statistical analysis
+All data were presented as mean±standard deviation (SD). For comparison between multiple groups, data were analyzed by one-way ANOVA. When a statistical difference was determined by ANOVA, the least significant difference (LSD) procedure was applied. The percentage of search strategies were examined by the Mann–Whitney method, and repetitive measure ANOVA was used to measure mean EL at different time points. Spatial probe trial data were analyzed by one-way ANOVA and principal components analysis (PCA). All analyses were performed with SPSS 13.0 for Windows, and a value of P<0.05 was considered significant.

20230808-AI coding-1st round/214 – Wen 2021.txt

@@ -0,0 +1,27 @@
+PMID: 33791908 DOI: 10.1007/s11064-021-03301-5
+
+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 provided with ad libitum access to water and food. All neonatal 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 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 temperature, 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 instruction 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 gentle 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 minutes. 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 density 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.
+
+Isoflurane Exposure In vitro
+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) dissolved in DMSO was added to ISO+Rapa group 1 h before isoflurane exposure, bringing the final concentration of rapamycin and DMSO in the culture medium to 100 nM and 0.1% respectively. The same volume of DMSO was added to both the CON and ISO groups. Cell-coated plates for the ISO and ISO+Rapa groups were placed in humidified, sealable 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 ℃ incubator 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 transcardially 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 solution (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 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-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 secondary antibodies Alex Fluor488 anti‐rabbit antibody (1:200,
+
+Jackson ImmunoResearch, West Grove, PA, USA), Cy5 anti-mouse antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA), and 4′, 6-diamidino-2-phenylindole (DAPI, 1:5000). After a final 3 more washes with PBS, sections were mounted on slides with 2.5% PVA/DABCO Mounting Media.
+
+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-Synapsin-1 (1:200, EMD Millipore, Burlington, MA, USA ), rabbit anti-PSD-95 (1:250, EMD Millipore, Burlington, MA, USA), rabbit anti-Gephyrin (1:800, Abcam, Cambridge, MA, USA), rabbit anti-pAKT (1:50, Cell Signaling 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, Cambridge, MA, USA ). After 3 washes with PBS, neurons were incubated for 2 h at room temperature with secondary antibodies Alexa Fluor488 anti‐rabbit antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA), Cy5 anti-mouse antibody (1:200, Jackson ImmunoResearch, 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.
+
+Imaging and Analyzing
+For evaluating synaptogenesis in vivo, 5 sections representing 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, Germany), 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 different coronal level of the dentate gyrus were picked randomly from each animal by an investigator blind to condition. 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 signaling 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.
+
+Statistical Analysis
+Results were expressed as mean± SEM. Data were analyzed by GraphPad Prism 8 (GraphPad, San Diego, CA, USA). Data were analyzed using two-way analysis of variance (ANOVA) with Tukey’s test for multiple comparisons. Statistical significance was set a priori at p <0.05.

20230808-AI coding-1st round/230 – Obradovic 2018.txt

@@ -0,0 +1,23 @@
+PMID: 28840469 PMCID: PMC5808855 DOI: 10.1007/s12035-017-0730-0
+Materials and Methods
+Animals
+We used 7-day-old (PND7) CD-1 mice (Harlan Laboratories, Indianapolis, IN) for all experiments. We chose this age because 1) it is when rodents are most vulnerable to GA-induced developmental neurotoxicity [16] and, 2) it falls before developmental pruning of the IPB begins [15]. Our ketamine anesthesia protocol was as follows: experimental mouse pups were exposed to 6h of ketamine anesthesia and controls were exposed to 6h of mock anesthesia (vehicle) injected I.M. During anesthesia, pups were carefully monitored. After the administration of anesthesia, mice were reunited with their mothers until sacrifice (from P8 until P65). The weaning was done at P21 using the standard protocol. At the desired age mice were divided randomly into two groups: one group for assessing expression of pro- and the mature form of BDNF using the Western blotting technique and the second group for morphometric studies of IPB development. Our randomization process was designed to provide each group with roughly equal representation of pups from each dam.
+
+The experiments were approved by the Animal Use and Care Committees at the University of Colorado the Office of Animal Resources (OLAR), Aurora, Colorado and the Animal Use and Care Committees of the University of Virginia, Charlottesville, Virginia. The experiments were done in accordance with the Public Health Service's Policy on Humane Care and Use of Laboratory Animals. Efforts were made to minimize the number of animals used while being able to conduct meaningful statistical analyses.
+
+Anesthesia administration
+To achieve general anesthesia state, we used a ketamine protocol known to cause significant developmental neurotoxicity in PND7 mice whereby mouse pups received a total of four doses of ketamine, at 75 mg/kg, IM every 90 minutes so that the loss of righting reflex and lack of response to tail pinch could be maintained for 6 hours [14]. For control animals, saline was administered using the same volume and administration schedule. During entire anesthesia procedure, animals were kept away from their mother and were housed in standard, tightly closed mice cages, with free air flow through air filters. Animals were kept in close proximity to each other in the cages, so they could preserve, even under anesthesia, important olfactory cues and stimuli, necessary to bust and sustain their metabolic output. During the experiment, we carefully monitored animals and measured environmental temperature in their breeding cages. We established that the ambient temperature in the breeding cage was around 37.0±1°C. Considering that animals at this age are very sensitive to change in body temperature, they were kept under constant ambient temperature maintained with heating pads conveniently set up around the cages. The ambient temperature was assessed at frequent time intervals using the thermometer.
+
+Western blot studies
+For BDNF protein quantification, we dissected the hippocampus immediately after the brains were removed from the individual pups using a dissecting scope (10× magnification). Tissue was collected on ice and was snap-frozen in liquid nitrogen immediately. The protein concentration of the lysates was determined with the Total Protein kit using the Bradford method (Cayman Chemical, Ann Arbor, MI). Approximately 10-25 μg of total protein was heat- denatured, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) through 4-20% Tris-glycine polyacrylamide gradient gels (BioRad, Hercules, CA). Separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked at room temperature for 1h in 3% bovine serum albumin (BSA) followed by incubation at 4°C overnight with primary antibody, anti-BDNF (1:500, Alomone Labs, Jerusalem, Israel), and anti-β-actin antibody (1:10000 Sigma Aldrich, USA) as a loading control.
+
+Membranes were incubated for 1h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies - goat anti-rabbit or goat anti-mouse IgG (1:10000, Santa Cruz, Dallas, TX). Three washes with 0.3% Tween-20 in Tris-buffered saline were performed between all steps. Immunoreactivity was detected using enhanced chemiluminescence substrate (Super Signal west Femto; Thermo Scientific, UT). Images were captured using GBOX (Chemi XR 5, Syngene, MD) and gels were analyzed densitometrically with the computerized image analysis program ImageQuant 5.0 (GE Healthcare, Life Sciences, Piscataway, NJ).
+
+Histological Preparation
+Mice were deeply anesthetized with 2% isoflurane and immediately perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (at pH 7.4). Brains were extracted and immersed in fresh 4% paraformaldehyde and incubated at 4°C for additional 2-3 days before being embedded in agar. Briefly, brain coronal sections (50μm thickness) were cut using vibratome. Tissue sections were blocked with 1× TBS contains normal goat serum 5%, 1% BSA and 0.1% triton X-100 for 1h at room temperature before incubated with primary antibodies against calbindin (anti-calbindin D-28K antibody, 1:1000; Gene Tex, CA, USA) overnight at 4°C. Free floating sections were then washed three times with TBS, and then incubated with corresponding HRP-conjugated secondary antibodies (1:200) at room temperature for 2h. Tissue sections were mounted on glass slides and air dried. For detection, we used DAB Peroxidase substrate kit (Vector Laboratories) following manufacturer's instructions.
+
+Histological Morphometric Assessment
+The morphometric analyses of IBP developmental shortening (from PND10 until PND65 in both control and ketamine-treated mice) were performed using coronal hippocampal slices (50μm) cut from bregma -1.34mm to -2.30mm (as determined using a mouse brain atlas). The images were scanned at 20× magnification using an Aperio Scanscope XT digital slide scanner (Aperio Technologies Inc., Vista, CA) at University of Virginia, Charlottesville, VA and at University of Colorado, Aurora, CO. The hippocampal area in digital sections (.svs file) was extracted at 600μm scale to convert to a .tiff file and was spatially calibrated using 1000 μm2 grid prior to quantifying using Image-Pro Plus 7.0 software (Media Cybernetics, MD). The morphometric approach used to evaluate so called ‘normalized length of IPB’, which takes into consideration individual variability and developmental growth of hippocampus. The IPB length was approximated from the tip of the inferior blade of the dentate granule cell layer (“a”). The length of CA3 was approximated from the tip of the inferior blade to the apex of the curvature of the CA3 pyramidal cell layer (“b”). Normalized IPB length was taken as a ratio between “a” and “b”. The values from serial sections (n=3-6 serial sections per animal from 6-7 animals per age group) were averaged to provide a single data point and is presented as normalized IPB length. The results from different age groups were statistically analyzed by t test and between both groups by Two-way ANOVA using Graph Pad Prism 5.01 software (Graph Pad, CA). The experimenters were blinded to the experimental condition.
+
+Statistical analysis
+Comparisons among groups were made using one-way and two-way ANOVAs followed by Tukey's post hoc test. Using the standard version of GraphPad Prism 5.01 software (Media Cybernetics, Inc, Bethesda, MD), we considered p<0.05 to be statistically significant. All data are presented as mean ±SD or mean ±SEM. The sample sizes reported throughout the Results and in the Figure Legends were based on previous experience.

20230808-AI coding-1st round/234 – Chen 2021.txt

@@ -0,0 +1,17 @@
+PMID: 33756347 DOI: 10.1016/j.bbrc.2021.03.063
+2. Methods and materials
+2.1. Animals
+This study was approved by the Institutional Animal Care and Use Committee of Soochow University. Adult C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Suzhou, China). One male and four female mice were housed per cage for breeding the offspring mice. Six pregnant mice on gestational day 14 were randomly assigned to receive either 2.5% sevoflurane in 100% oxygen or just 100% oxygen as the control. Their offspring mice were correspondingly assigned as the testing mice. Several pregnant mice without any treatment were chosen to produce the offspring mice as the stranger mice. The pups were fostered by their own dams till weaning on postnatal day 21. All mice were raised in a controlled condition (21–22 °C, 12 h light/dark cycle, light on at 7 a.m.), with access to standard mouse chow and water ad libitum.
+
+2.2. Maternal anesthesia
+A clinically-retired anesthesia machine was used to supply one-way gas flow. A transparent plastic box (20 L × 20 W × 6 H cm) was used as the anesthetizing chamber, with three holes for gas inflow, gas outflow, and gas monitoring. A heating-pad was placed underneath the anesthetizing chamber to keep mice warm during anesthesia. Sevoflurane anesthesia on pregnant mice in this study was strictly performed by the protocols of previous study [10], in which arterial blood pressure and blood gas analysis were demonstrated within normal limits. The pregnant mice retained spontaneous respiration during inhalational anesthesia. Sevoflurane was washed out with pure oxygen for 15 min, and the pregnant mice with right reflex were put back to home cages.
+
+2.3. Social apparatus
+The three-chambered social box (40L × 60W × 22H cm) with two enclosures (7D × 15H cm) was used for social interaction test (Fig. 1A–C). An improved video-tracking system programed by ANY-maze (Stoelting Co., USA) was used to capture the movement of mouse. Given the testing mouse initiates social approaching to the stranger mouse by nose-to-nose or nose-to-tail sniffing (Fig. 1D), the animal’s head was tracked by the video-tracking system. Four behavioral parameters was automatically measured by ANY-maze program, including the time sniffing at the enclosure and number of sniffs at the enclosure, the time exploring in the side-chamber and number of entries into side chamber. Specifically, “at the enclosure” is defined as the mouse head entering an area of 3 cm around the enclosure.
+
+
+2.4. Social interaction test
+The offspring mice (N = 17 Control, 9 males and 8 females; N = 14 Sevoflurane, 9 males and 5 females) were tested at one- and two-month-old (i.e., the juvenile and early-adult age). In advance, the testing mouse was housed single for 1-h isolation in the behavioral room. The stranger mice were the identical background, same gender and similar age as the testing mouse, and they had exactly no contact before. Social interaction test is composed of three 10-min sessions of habituation, sociability, and preference for social novelty. Firstly, the testing mouse was allowed to freely explore in social box with two doorways opening. Next, an unfamiliar conspecific (Stranger 1) was introduced into one enclosure, and the testing mouse was allowed to sniff the stranger 1 or explore the empty enclosure (Fig. 1E, G). After that, another unfamiliar conspecific (Stranger 2) was introduced into the other enclosure, and the testing mouse was allowed to sniff the stranger 1 and stranger 2 (Fig. 1F, H). Placement of the stranger 1 on the left or right side was systematically altered between trials, and social apparatus was cleaned after each trial to minimize olfactory disturbance.
+
+2.5. Statistical analysis
+Data were shown as Mean ± SD. Graphpad Prism 5.0 software (San Diego, USA) was used for statistical analyses. Social data obtained from the left and right side were mutually exclusive in each 10-min session and they were normally distributed by Kolmogorov-Smirnov tests. Therefore, two-tailed paired t-test was used to compare the side preferences (stranger 1 vs. the opposite). Two-way repeated measures (RM) ANOVAs were used to analyze the interaction effects of treatment (control or sevoflurane) × side (stranger 1 or the opposite). Based on the preliminary study, a sample size of more than 5 (sociability) and 13 (preference for social novelty) could lead to a 90% power to detect a difference in side preference with 5% type I error. P values less than 0.05 (∗), 0.01 (∗∗) and 0.001 (∗∗∗) were considered statistically significant.

20230808-AI coding-1st round/243 – Xiao 2017.txt

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+PMID: 29249940 PMCID: PMC5715384 DOI: 10.3389/fncel.2017.00373
+Materials and Methods
+Animals
+Male and female C57/BL6 mice were provided by the Third Military Medical University and housed under a 12 h light/dark cycle in a temperature-controlled room with free access to food and water. All the experimental procedures were performed in accordance with the guidelines for laboratory animal care and use and were approved by Third Military Medical University. Each litter was kept together with its mother throughout the experiment, except for the brief intervals of separation required for the daily injections. At least five mice in each group were analyzed for immunofluorescence staining and three mice for western blot.
+
+Drug Treatment
+The day of birth was designated postnatal day 0 (P0). On P7, pups received a vehicle or propofol injection intraperitoneally (i.p.) at a subanesthetic dose of 30 or 60 mg/kg (Cattano et al., 2008; Yang B. et al., 2014), according to our previous study (Huang et al., 2016). The same volume of intralipid was administered i.p. as a vehicle control for propofol. All the neonatal mice were grouped randomly with a random number table base for similar body weight.
+
+To explore the morphological changes in the Purkinje cells, Bergmann glia and granule neuron, pups were sacrificed 24 h (P8) after drug treatment. To evaluate whether propofol affected the radial migration of the granule neurons, a single-dose BrdU injection (50 mg/kg i.p., dissolved in saline) was administered to the pups at P8, which was 1 day after injection with propofol or vehicle. Pups were sacrificed 2 days after the BrdU injection (P10).
+
+To maintain a mouse body temperature of 37°C, the pups were primitively anesthetized in their home cage and then transferred to a Thermocare® ICS therapy warmer unit (Thermocare, Incline Village, NV, USA) after being sedated to keep warm in all the experiments. Meanwhile, mouse normal skin color and respiration were observed.
+
+Immunofluorescence
+The dissected cerebella were soaked in 4% paraformaldehyde for 24 h. For cryosections, the tissues (P8) were embedded and sectioned in the sagittal plane at 30 μm. The remaining tissues (P8) and tissues collected on P10 were embedded in paraffin and sagittal sections (5 μm thickness) were collected. Cryosections were used for all the immunofluorescence staining for P8 and paraffin sections were used for Hematoxylin-eosin (HE) staining and BrdU immunofluorescence staining. The sections were pretreated with 3% bovine serum albumin (BSA) (37°C, 1 h) to block non-specific binding and 0.3% Triton X-100 (37°C, 30 min) to increase permeability. Then, the sections were incubated with the following primary antibodies in 1% BSA (4°C, 18 h): (1) mouse anti-calbindin D-28K (CB) (1:1000, Swant, Bellinzona, Switzerland); (2) rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) (1:200, Merck Millipore, Darmstadt, Germany); (3) rabbit polyclonal anti-brain lipid binding protein (BLBP) (1:400, Merck Millipore, Darmstadt, Germany); and (4) mouse anti-neuronal nuclei (NeuN) (1:200, Merck Millipore, Darmstadt, Germany). One percent BSA served as the negative control. After three washing steps with phosphate-buffered saline (PBS, pH 7.4), the sections were incubated with the following secondary antibodies in PBS (room temperature (RT), 3 h): (1) Alexa Fluor 488-conjugated anti-mouse IgG (1:400, Jackson ImmunoResearch, West Grove, PA, USA) for CB and NeuN staining; and (2) cy3-conjugated anti-rabbit IgG (1:400, Jackson ImmunoResearch, West Grove, PA, USA) for BLBP and GFAP staining. All the sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) and then mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA). For BrdU staining, the paraffin sections were deparaffinized in xylene, rehydrated in graded alcohol and processed for antigen retrieval by boiling in citrate buffer (pH 6.0) for 5 min. After incubation in 2 M HCl (37°C, 30 min) and 0.3% Triton X-100 (37°C, 30 min), the sections were exposed to mouse anti-BrdU IgG (37°C for 2 h and then RT for 22 h) (1:600, BD Pharmingen™, Palo Alto, CA, USA) in 1% BSA, followed by the cy3-conjugated anti-mouse IgG secondary antibody (RT, 3 h) (1:400, Jackson ImmunoResearch, West Grove, PA, USA) and DAPI counterstaining. Fluorescence micrographs of the whole parasagittal cerebellar slices were acquired under a Zeiss (Oberkochen, Germany) Axiovert microscope equipped with a Zeiss AxioCam digital color camera connected to the Zeiss Axiovision 3.0 system. The pictures of the Purkinje dendrite and Bergmann fiber contact points were taken with a TCS-SP8 (Leica, Germany) laser scanning confocal microscope connected to a LAS AF Lite system. A z-stack of images, consisting of 6 image planes taken at 1 μm interval was obtained (for a total stack depth of 5 μm). The 5 μm z-stack was taken from the middle of the section to minimize the potential artificial bias.
+
+Western Blot
+Cerebella were harvested on P8 and then isolated and homogenized in ice-cold RIPA Lysis buffer (Beyotime, Shanghai, China). After centrifuging the lysates (15,000× g, 5 min at 4°C), the protein concentration was calculated using the Bicinchoninic Acid Kit (Beyotime, Shanghai, China). Then, 50 μg of protein from each sample was separated by 10% SDS-polyacrylamide electrophoresis (120 min 80 V) and then transferred to a nitrocellulose (NC) membrane (90 min at 210 mA). The membranes were incubated in 5% fat-free milk in Tris-buffered saline containing 0.1% Tween 20 (3 h at RT). Membranes were then incubated with the following primary antibodies (4°C, overnight): (1) hamster monoclonal anti-Notch1 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA); (2) rabbit polyclonal anti-Jagged1 (1:500, Santa Cruz Biotechnology, USA); (3) mouse anti-β-actin (1:1000, Cell Cwbio, Beijing, China); and (4) rabbit anti-GAPDH (1:1000, Cell Cwbio, China), followed by the following peroxidase-conjugated secondary antibodies (RT, 2 h): (1) goat anti-mouse IgG (1:1000, Santa Cruz Biotechnology, USA); (2) goat anti-rabbit IgG (1:1000, Santa Cruz Biotechnology, USA); and (3) goat anti-Syrian hamster IgG (1:1000, Santa Cruz Biotechnology, USA). All the bands were exposed to X-ray films (Kodak, Rochester, NY, USA), detected using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL, USA), and analyzed with the Gel-Pro analyzer (Quantity One 4.0; Bio-Rad Laboratories, Hercules, CA, USA). Quantification of Jagged1 and Notch1 were normalized to the internal reference protein β-actin or GAPDH, and then normalized to the control values.
+
+Quantification
+The quantification was obtained from regional analysis of lobe IX. All the sections were taken from similar medial-lateral position within the cerebellum, and each count area was chosen from the same field by the middle of the lobe IX. Calbindin-positive cells were analyzed along the long axis for 500 μm in the middle, and the dendrite length of Purkinje cells was evaluated by measuring the primary dendrite from the soma up to the surface of the ML (three Purkinje dendrites were measured per picture). Number of the NeuN-positive granule neurons were analyzed in the center region of the IGL in lobe IX along the long axis (unit area 2000 μm2). The number of BLBP- and GFAP-positive Bergmann fibers was counted from a 100-μm length in the middle area of lobe IX according to our previous methods (Yamada et al., 2000; Eiraku et al., 2005; Yang Y. et al., 2014). To analyze the astrocytes in the deep white matter, we compared the intensity of the GFAP-positive cells and fibers. Both the background integrated optical density (IOD) and surveyed area (same center area of the white matter from each group) were acquired, and the relative optical density (ROD) was calculated by subtracting the background from the IOD of the positive staining (Bao et al., 2017). Contact points between the calbindin-positive Purkinje cells and GFAP-positive Bergmann fibers were defined as where the tips of growing Purkinje cell dendrites were aligned parallel and attached directly to the rod-like domain of Bergmann fibers, entering the base of the overlying EGL, as previously reported (Yamada et al., 2000; Yamada and Watanabe, 2002; Lordkipanidze and Dunaevsky, 2005). Points were counted per image (212.5-μm length) at the interface between the EGL and ML. Only the yellow dots at the end of the dendrites in the direction of the Bergmann fiber were included, while the crossed ones were excluded in case of false positive. For quantifying granule neuron migration, BrdU-labeled cells were counted in a rectangular box (200 μm width and about 100 cells were counted) extending from the pial surface to the end of the IGL; this value was expressed as a percentage of the total number of BrdU-labeled cells. At least five sections were analyzed in each mouse and five mice from each group. All the quantitative statistics were performed blind to the experimental treatment.
+
+Statistical Analysis
+All the data were presented as the mean ± standard deviation and analyzed using one-way analysis of variance (ANOVA) followed by Fisher’s protected least- significant difference post hoc test or a least-significant difference multiple-comparison. The differences were statistically significant when the P value was less than 0.05. Statistical analysis was performed using the SPSS 19.0 software (SPSS Inc., Chicago, IL, USA).

20230808-AI coding-1st round/248 – Goyagi 2019.txt

@@ -0,0 +1,22 @@
+PMID: 30959098 DOI: 10.1016/j.ijdevneu.2019.04.002
+2 Material and methods
+All animal protocols were approved by the animal research committee of Akita University, Japan (Approval number: a-1-2625). Seven-day-old (P7) Wistar rats (male and female) rat pups (body weight, 12–15 g) were used in this study. Animals were housed under standard conditions (12 h light/12 h dark cycle at 22 °C) in the Animal Research Laboratory at Akita University. All efforts to reduce the number of animals and their suffering were made. The animals were randomly divided into 6 groups (n = 10 per group) as follows: no anesthesia and no injection (sham), no anesthesia and intraperitoneal 25 μg/kg DEX (control), intraperitoneal saline (DEX 0), intraperitoneal 6.6 μg/kg DEX (DEX 6.6), intraperitoneal 12.5 μg/kg DEX (DEX 12.5), and intraperitoneal 25 μg/kg DEX (DEX 25).
+
+After 30 min intraperitoneal injection on P7, the pups were put into a plastic chamber, exposed to 3% sevoflurane with 2 L/min of 21% oxygen for 4 h, and returned to their mother's cage. The oxygen and sevoflurane concentration were measured using a gas analysis system (GE Healthcare BioSciences, Pittsburgh, PA). The chamber was maintained at 30 ± 1 °C using an infrared heat lamp during the exposure.
+
+Cognitive tests
+2.1.1 Morris water maze
+Spatial memory retention was examined using the Morris water maze by blinded observer as described previously (Goyagi, 2018). At P27 – P29, acquisition trials were executed 4 times per day for 3 successive days. The latency and the swimming path length to reach the hidden platform were measured using a video image motion analyzer (DVTrack DVT-11; Muromachi Kikai Co. Ltd, Tokyo, Japan). If the rat could not reach the hidden platform within 90 s, it was placed on the platform for 30 s during an acquisition trial. At P47 – P49, retention trials were executed 4 times per day. If the rats failed to find the platform within 90 s, the latency was regarded as 90 s. In this study, a probe trial was not done during the acquisition trials.
+
+2.1.2 Fear conditioning test
+Fear conditioning was performed to evaluate contextual memory retention using the fear conditioning system (MK-450RSQ; Muromachi Kikai Co., Ltd, Tokyo, Japan) as described previously (Goyagi, 2018). The apparatus consisted of a clear rectangular Plexiglas box with a floor of for the delivery of electric currents. At P42, the rats were placed on the cleaned parallel metallic rods to be accustomed to new environment for 1 min, before they were presented with a 70-dB white noise for 30 s A mild foot shock (0.4-mA) was administered through the metallic rods during the last 1 s of the tone presentation. The tone-shock pairing was repeated once per minute for the next 2 min. The rats were left in the cage for an additional 60 s before returned to their cage. At P49, cued fear memory was tested by placing rats into an unrelated environment for 90 s without any tone and presenting the auditory cue for a further 60 s used for conditioning. Freezing time was measured by the percent of time during the tone presentation using a video image motion analyzer (DVTrack DVT-11; Muromachi Kikai Co. Ltd, Tokyo, Japan).
+
+Histological analyses
+2.2.1 Neuronal nuclei staining
+After finished the water maze task and fear conditioning test at P49, the rats’ brains were removed and embedded in paraffin following the perfusion of heparinized saline then 150 mL of 4% paraformaldehyde in phosphate buffer (pH 7.4) to use further neuronal nuclei (NeuN) stain, as described previously (Goyagi, 2018). In brief, 3-μm-thick serial transverse sections were incubated with a mouse monoclonal antibody to NeuN antigen (NeuN; 1:100 diluted in blocking solution; Millipore Corporation, Temecula, CA) for 10 min at 37 °C. Immunodetection was performed using avidin-horse radish peroxidase complexes with biotinylated antibodies to rabbit and mouse IgG (MILLIPORE IHC SelectR Immunoperoxidase Secondary Detection System; Millipore Corporation), with diaminobenzidine. Then we counterstained those with hematoxylin. The NeuN-positive cells express as mature typical neurons after growth. We counted the number of NeuN-positive cells in bilateral 500 μm × 300 μm areas in the CA1 hippocampus, amygdala, and cerebral cortical layer 3, as described previously (Goyagi, 2018).
+
+2.2.2 Positive cell density map (PCDM)
+The PCDM was made as described previously (Goyagi, 2018; Wada et al., 2006). In brief, the composite image was FFT- bandpass-filtered using the Image J program (National Institute of Health, Bethesda, MD) to eliminate low-frequency drifts (>20 pixels [50 μm]) and high-frequency noises (<1 pixel [2.5 μm]). The PDCM was made with a custom-made program using MATLAB (MathWorks INC., Natick, MA) (Wada et al., 2006), then adjusted for each section automatically and enumeration of NeuN-positive cells in each 100 μm × 100 μm square section. Finally, the normalized PCDMs were seen as averaged for each group (Fig. 6 A). As mentioned our previous study (Goyagi, 2018), the PCDMs were analyzed whether the DEX-treated groups showed increased NeuN cell density compared with the DEX 0 group. The areas were mapped as colored to indicate significantly increased normal neurons in blocks where the P value was less than 0.05 (Fig. 6B).
+
+Statistical analysis
+The escape latency, the swimming speed, the swimming path length, the freezing time, and the number of NeuN-positive cells are expressed as means ± standard deviation (SD). Comparisons of these variables among the groups were performed using a one-way or two-way analysis of variance (ANOVA) for multiple comparisons followed by Bonferroni post hoc tests. Each PCDM using a Gaussian filter of the block size (SD = 100 μm) was analyzed using t-tests for each block. Differences with p-values less than 0.05 were considered statistically significant. We performed all analyses using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA).

20230808-AI coding-1st round/263 – Li 2019.txt

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+PMID: 31436548 PMCID: PMC6800770 DOI: 10.1097/ALN.0000000000002904
+Materials and Methods
+Animal paradigm and experimental timeline.
+A total of 120 (61 male and 59 female) immature C57BL/6 mice (body weight = 4.4±0.9 g. at postnatal day 7) were used in this study. 84 (44 male and 40 female) of them were randomly selected for the rapamycin experiment and 36 (17 male and 19 female) for the clemastine experiment. Sex was not factored into research design as a biological variable. Both sexes were equally represented in all experiments. All study protocols involving mice were approved by the Animal Care and Use Committee at the Johns Hopkins University and conducted in accordance with the NIH guidelines for care and use of animals. Experimental procedures followed the modified protocols from a previously published journal.7
+
+At postnatal day 7, animals were exposed to isoflurane or room air for 4 hours. From postnatal days 21-35, half of the isoflurane-exposed mice were injected (i.p.) bi-daily with rapamycin (n=28 per group) or fed daily with clemastine through gastric gavage (n=12 per group). The other half were injected with vehicle of rapamycin or fed with vehicle of clemastine.
+
+For the rapamycin experiment, a subset of mice from each group were sacrificed at postnatal day 35 for immunohistochemistry (n=8 per group) or Western blotting (n=8 per group). The remaining mice underwent behavioral testing for spatial learning and memory functions between postnatal days 56-62 (n=12 for each group). After behavior tests, two mice from each group were processed for electron microscopy at postnatal day 63. Only behavior tests were conducted for clemastine feeding experiment (n=12 for each group) (Fig. 1A).
+Isoflurane exposure.
+At postnatal day 7, two-thirds of the mice were evenly distributed across littermate groups and were randomly selected for isoflurane exposure. The other one-third of the mice stayed in room air as a naïve control. Volatile anesthesia exposure was accomplished using a Supera tabletop portable non-rebreathing anesthesia machine. 3% isoflurane mixed in 100% oxygen was initially delivered in a closed chamber for 3-5 min and after loss of righting reflex, animals were transferred to the specially designed plastic tubes. A heating pad (36.5ºC) was placed underneath the exposure setup. The mice were exposed to 1.5% isoflurane carried in 100% oxygen for 4 hours. A calibrated flowmeter was used to deliver oxygen at a flow rate of 5 L/min and an agent specific vaporizer was used to deliver isoflurane. During isoflurane exposure, mice were monitored for change in physiological state using the non-invasive MouseOx plus instrument (STARR Life Sciences, Holliston, MA, USA). A collar clip connected to the instrument was placed on the neck and a temperature probe placed on the skin of the abdomen. Ten-minute readings with 1-hour intervals were taken. Data was collected in four time-points and averaged for each case. The skin temperature (34.1±0.8ºC), pulse distention (168.9±36.6 μm), heart rate (376.8±94.1 bpm), breath rate (77.4±35.8 brpm), and oxygen saturation (99.3±0.3%) were recorded. After the isoflurane exposure, mice were returned to their moms together with their littermates upon regaining righting reflex. All animals (100%) survived the isoflurane exposure.7
+
+Rapamycin injection.
+A total of 84 mice were equally divided into three groups: 1) naïve control; 2) isoflurane exposure plus vehicle; and 3) isoflurane plus rapamycin injection. From postnatal days 21-35, half of the isoflurane-exposed mice (group 3; n=28 per group) were injected intraperitoneally with 0.2% rapamycin dissolved in vehicle solution and the other half with vehicle only (group 2; n=28 per group). Vehicle consisted of 5% Tween 80 (Sigma Aldrich, St. Louis, MO, USA), 10% polyethylene glycol 400 (Sigma-Aldrich, St. Louis, MO, USA), and 8% ethanol in saline. Mice received 100 μl rapamycin or vehicle for each injection at 48 hour intervals from postnatal days 21-35. 7
+
+Clemastine feeding.
+In this experiment, 36 animals were also equally divided into three groups as above. Clemastine (Tocris Bioscience, Bristol, UK) was dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) at 10 mg/ml followed by further dilution in ddH2O into 1 mg/ml. From postnatal days 21-35, half of the isoflurane exposed mice (n=12 for each group) were fed clemastine (10 mg/kg) daily via gastric gavage using plastic feeding tubes (gauge 22; Instech, Plymouth Meeting, PA, USA), and the other half (n=12 for each group) were fed same volume of 10% DMSO as vehicle.16,17
+
+Behavior tests.
+The novel object position recognition test and Y-maze test were performed at the last week of the survival period (postnatal days 56-62).7 Experimenters were blinded to condition when behavioral tests were carried out and quantified.
+
+1). Novel object position recognition test: The test was assessed in a 27.5 cm × 27.5 cm × 25 cm opaque chamber. During the pre-test day (day 1), each mouse was habituated to the chamber and allowed to explore 2 identical objects (glass bottles, 2.7 cm diameter, 12 cm height, and colored paper inside) for 15 minutes. The mouse was then returned to its home cage for a retention period of 24 hours. On the test day (day 2), the mouse was reintroduced to the chamber and presented with one object that stayed in the same position (old position) while the other object was moved to a new position (novel position). A five-minute period of movement and interaction with the objects was recorded with a video camera that was mounted above the chamber and exploratory behavior was measured by a blinded observer. Exploratory behavior was defined as touching the object with snouts. The numbers of exploratory contacts with the novel object and with the old object were respectively recorded, and the ratios over the total exploratory contact numbers were calculated.
+2). Y-maze test: In the pre-test phase (day 1), mice explored and habituated in the start arm (no visual cue) and 1 out of 2 possible choice arms with overt visual cue (old arm) for 15 minutes. This was followed by the recognition phase (day 2) 24 hours later, in which the animals could move freely in the three arms and choose between the 2 choice arms (old arm and novel arm) after being released from the start arm. The timed trials (5 minutes) were video recorded as well as graded by an observer blinded to the conditions for exploration time in each choice arm and the percentages over total exploratory time were calculated.
+Immunohistochemistry.
+During postnatal days 30-35, 5-bromo-2’-deoxyuridine (Abcam, Cambridge, UK) was injected intraperitoneally at 50mg/kg daily in animals randomly selected from three groups (n=8 for each group). At postnatal day 35, mice were perfused with 40 ml 4% paraformaldehyde in PBS. Brains were removed and post-fixed at 4ºC overnight, followed by 30% sucrose in PBS at 4°C for 48 hours. The brains were coronally sectioned in 40 μm thickness using a freezing microtome. For each brain, 72 sections containing fimbria were collected in a 24-well tissue culture plate and they were divided into twelve wells in a rotating order (6 sections per well). Seven wells of sections were immunostained for: (1) phospho-S6 and adenomatous polyposis coli; (2) 5-bromo-2’-deoxyuridine and neural/glial antigen 2 ; (3) adenomatous polyposis coli and platelet-derived growth factor receptor alpha; (4) vesicular glutamate transporter 1 and neural/glial antigen 2; (5) myelin basic protein; (6) DNA methyltransferase 1 and Olig2 (oligodendrocyte transcription factor marker); (7) 5-methylcytosine (5-mC) and adenomatous polyposis coli. For 5-bromo-2’-deoxyuridine staining, sections were pretreated with 2N HCl to denature DNA (37°C; 45min), and with 2 × 15min borate buffer (pH 8.5) to neutralize the HCl. After 3×10min PBS washing, sections were blocked in 10% normal goat serum and 0.1% triton X-100 for 60min, followed by primary antibody incubation at 4ºC overnight. Primary antibodies used in this study were: rabbit anti-phospho-S6 (1:1,000; Cell Signaling, Boston, MA, USA), mouse anti-5-bromo-2’-deoxyuridine (1:200; Abcam, Cambridge, UK), rabbit anti-neural/glial antigen 2 (1:200; Millipore, Burlington, MA, USA), mouse anti-adenomatous polyposis coli (1:2,000; Millipore, Burlington, MA, USA), mouse anti-myelin basic protein (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti- platelet-derived growth factor receptor alpha (1:500; Lifespan Bio, Seattle, WA, USA), mouse anti- vesicular glutamate transporter 1 (1;200; Abcam, Cambridge, UK), mouse anti-DNA methyltransferase 1 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Olig2 (1:2,000; Abcam, Cambridge, UK), and rabbit anti-5-methylcytosine (1:2,500; Abcam, Cambridge, UK). After 3×10min washes in PBS, sections were incubated with secondary antibodies for 2 hours: Alexa 488 conjugated goat anti-rabbit IgG (1:300; Invitrogen, Eugene, OR, USA) mixed with Cy3 conjugated goat anti-mouse IgG (1:600; Jackson ImmunoResearch Labs, West Grove, PA, USA), or Alexa 488-goat anti-mouse IgG (1:300; Invitrogen, Eugene, OR, USA) mixed with Cy3 conjugated goat anti-rabbit IgG (1:600; Jackson ImmunoResearch labs, West Grove, PA, USA). After 3×10min PBS washes, sections were mounted onto slides, air-dried, and cover-slipped.21
+
+Cell counting and immuno-fluoresce intensity analysis in fimbria
+The sections were observed and imaged using a Leica 4000 confocal microscope (Wetzlar, Germany). All single- or double- immunolabeled cells within hippocampal fimbria area were counted using ImageJ with cell counter plugin (NIH, Bethesda, MD, USA). The criteria for counting mTOR active oligodendrocytes required a cell to have both phospho-S6+ (in red channel) and adenomatous polyposis coli+ (in green channel) cytoplasm and merged image of double labeled cells appeared yellow color (Fig. 1B). Proliferating oligodendrocyte progenitor cells and 5-methylcytosine+ oligodendrocytes were counted for cells that have 5-bromo-2’-deoxyuridine+ or 5-methylcytosine + nuclei and neural/glial antigen 2+ or adenomatous polyposis coli+ cytoplasm. However, both DNA methyltransferase 1 and Olig2 reactivity were seen in nuclei. Identification of excitatory axon-oligodendrocyte progenitor cell synapses involved vesicular glutamate transporter 1+ terminal boutons closely apposing on the surface of neural/glial antigen 2+ oligodendrocyte progenitor cells. For adenomatous polyposis coli and platelet-derived growth factor receptor alpha double-stained sections, almost no double-labeled cells were seen, which means these two markers label cells in different oligodendrocyte development stages without overlapping.
+
+Images containing fimbria were taken at 20x magnification in red (Cy3), green (Alexa 488), and merged channels. All single- (Cy3+ or Alexa488+) and double-labeled cells in fimbria were counted. Images were opened and initialized in ImageJ. The fimbria area was outlined using the ‘‘Freehand’’ tool. “Plugins”, “Analysis”, and ‘‘Cell Counter’’ tools were selected, and each labeled cell inside was clicked, with which each counted cell was marked preventing the same cell from being counted twice. The numbers of counted cells were automatically recorded. The ratio of a specific marker labeled oligodendrocytes (such as yellow-colored phospho-S6+/ adenomatous polyposis coli+ cells over all green adenomatous polyposis coli+ cells in Fig. 1B) was calculated. For each case, numbers from 12 fimbria images (6 sections, both sides) were averaged. There was almost no double staining for adenomatous polyposis coli and platelet-derived growth factor receptor alpha. We the used ratio of adenomatous polyposis coli+ over platelet-derived growth factor receptor alpha+ cells to evaluate the maturation of oligodendrocyte lineage cells. For axon- oligodendrocyte progenitor cells synapse, five neural/glial antigen 2 positive cells from each image (60 cells for each case) were randomly selected and photos were taken in a higher magnification (40x). Every vesicular glutamate transporter 1+ terminal boutons apposing on each selected cell were counted with imageJ and average numbers were calculated.
+
+The fluorescence intensity of myelin basic protein immunoreactivity in fimbria were also quantitatively analyzed using ImageJ. Photos of the fimbria area from immunostained sections were taken at 20x magnification. Identical photo exposure was set for all groups. The image was opened with ImageJ and outline of fimbria was drawn with “Freehand” tool. The “set measurements” was selected from the analyze menu and “integrated density” was activated. A region in lateral ventricle was selected as background. The final myelin basic protein intensity of fimbria area equals measured density minus background.
+
+Western blotting.
+Eight animals from each group were quickly perfused with cold saline on day 35. From the medial aspect of the hemisphere, the hippocampus was exposed and separated from brain tissue. Fimbria located in the ventrolateral side of the hippocampus were easily identified by bright white color under dissection microscope, and then removed with fine forceps. Fimbria tissue was lysed in the lysis buffer, homogenized with a bullet bender (Next Advance, Troy, NY, USA), and centrifuged. The supernatant was taken and stored in −80°C. The next day, samples were prepared with 1:1 denaturing sample buffer (Bio-Rad, Hercules, CA, USA), boiled for 5 min, and run on 4-12% Bis-Tris Protein Gels (Invitrogen, Carlsbad, CA, USA) in running buffer (Invitrogen, Carlsbad, CA, USA) with 150 volts for about 1 hour. The proteins were transferred to nitrocellulose blotting membranes (Invitrogen, Carlsbad, CA, USA). Blots were probed with anti-neural/glial antigen 2 (1:200; Millipore, Burlington, MA, USA), anti-NK2 homeobox 2 (1:200; Abcam, Cambridge, UK), anti-myelin basic protein (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), anti-DNA methyltransferase 1 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-β-actin antibodies (1:1,000; Cell Signaling Technology, Boston, MA, USA). The membranes with the primary antibodies were stored in 4°C overnight. After incubation in secondary antibodies (1:2,000; Cell Signaling Technology, Boston, MA, USA) for 1 hour, blots were visualized using ECL western blotting substrate kit (Pierce Biotechnology, Waltham, MA, USA). Images were acquired using ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA) and were quantitated with ImageJ (NIH, Bethesda, MD, USA). First, the images were opened using File>Open. The rectangles around all lanes (each lane includes bands for detected marker and β-actin) were drawn by choosing “Rectangular Selection”. Then proceeding to “Analyze>Gels>Plot Lanes”, peaks were generated representing the density of bands, followed by clicking “Straight Line” tool to enclose the peaks and selecting the “Wand” tool to highlight the peaks. After this, Analyze>Gels>Label Peaks was used to get numbers for peak area (band intensity). The ratios of band density of oligodendrocyte lineage markers over β-actin were calculated.21
+
+Electron microscopy.
+Two animals from each group were perfused with 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) plus 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in PBS at postnatal day 63 (after behavior tests) and post-fixed at 4°C for 1 week. Brains containing fimbria were dissected into small blocks (2mm × 2mm × 2mm). The blocks were placed into 1% OsO4 (Electron Microscopy Sciences, Hatfield, PA, USA) for 1 hour, stained in 0.5% uranyl acetate (Electron Microscopy Sciences, Hatfield, PA, USA) overnight, and dehydrated in a series of alcohols followed by propylene oxide for 3 hours. After being infiltrated with a 1:1 mixture of propylene oxide and EMBed-812 embedding resin (Electron Microscopy Sciences, Hatfield, PA, USA) for 3 hours, the blocks were embedded with the same resin in the plastic templates at 60ºC overnight.21 Parasagittal semi-thin sections (1 μm) were cut and stained with 1% Toluidine blue for preliminary light microscopy observation. Then, 90 nm ultrathin sections were cut, picked up on Forvar-coated slotted grids, and stained with 0.5% uranyl acetate and 0.5% lead citrate (Electron Microscopy Sciences, Hatfield, PA, USA). Thin sections were observed and imaged with a Hitachi 7600 transmission electron microscope (Chiyoda, Tokyo, Japan). For each case, 10 photos were randomly photographed at 20,000×. The thickness of myelin was quantitatively measured by determining g-ratio, which was calculated by dividing the diameter of the axon by the diameter of the entire myelinated fiber as previously described. ImageJ (NIH, Bethesda, MD, USA) was used by first opening ultrastructural images. The scale was set according to the scale bar in the images by selecting “Analyze>Set Scale”. The “straight line tool” was selected to measure axonal caliber and diameter of myelinated axons. One hundred axons per group (two animals, fifty from each) were randomly selected and quantitatively analyzed (n=100).16
+
+Statistical analysis.
+The statistics were performed with GraphPad Prism 6 (La Jolla, CA, USA) program. The sample size was based on our previous experience with this design. No a priori statistical power calculation was conducted. Normal distribution was verified using the D’Agostino Pearson test. Data for immunohistochemistry, Western blotting, and electron microscopy were analyzed using one-way analysis of variance (ANOVA). The factor of variable was comparisons among groups (control vs. isoflurane plus vehicle vs. isoflurane plus rapamycin). The behavior tests were analyzed with two-way ANOVA. For this analysis, the second factor was animal’s choice between old vs. novel positions (or arms) and only the values for this variable in each individual group were compared. The Tukey post hoc test was employed for intergroup comparisons. The two-tailed test was set according to convention. The criteria for significant difference was set a priori at p<0.05. In this study, all results were expressed as mean ± standard deviation (SD). The sample size “n” represents the number of animals for each group. Only exception is g-ratio analysis with electron microscopy in which “n” indicates the number of randomly selected axons from two mice per group (n=100). This analysis way is extensively applied for g-ratio study.16 Because all animals survived tests, there were no missing data in this study. No exclusions for outliers were made in this study. In some experiments, the sample size was increased in response to peer review.

20230808-AI coding-1st round/268 – Zheng 2013.txt

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+PMID: 23314109 PMCID: PMC3580035 DOI: 10.1097/ALN.0b013e3182834d5d
+Materials and Methods
+Mice Anesthesia
+The protocol was approved by the Massachusetts General Hospital Standing Committee (Boston, Massachusetts) on the Use of Animals in Research and Teaching. Three-month-old C57BL/6J female mice (The Jackson Laboratory, Bar Harbor, ME) were mated with male mice. The pregnant mice were identified and then housed individually. The offspring mice were weaned 21 days after birth. Animals were kept in a temperature-controlled (22°–23°C) room under a 12-h light/dark period (light on at 7:00 AM); standard mouse chow and water were available ad libitum . At gestational day (G) 14, the pregnant mice were assigned randomly to an anesthesia group or a control group. Mice randomized to the anesthesia group received 2.5% sevoflurane in 100% oxygen for 2 h in an anesthetizing chamber. The control group received 100% oxygen at an identical flow rate for 2 h in an identical chamber as described in our previous studies.9The mice breathed spontaneously, and concentrations of anesthetic and oxygen were measured continuously (Datex-Ohmeda Inc., Tewksbury, MA). The temperature of the anesthetizing chamber was controlled to maintain rectal temperature of the animals at 37° ± 0.5°C. Mean arterial blood pressure was not measured in these mice because the same sevoflurane anesthesia was shown not to alter the values of blood pressure and blood gas in our previous studies.9Anesthesia was terminated by discontinuing sevoflurane and placing the animals in a chamber containing 100% oxygen until 20 min after return of the righting reflex. The anesthesia with 2.5% sevoflurane (approximately 1.1 minimum alveolar concentration) for 2 h in mice was used to demonstrate whether clinically relevant sevoflurane anesthesia in pregnant mice, which had been shown to induce neurotoxicity in adult mice,9could also induce neurotoxicity in fetal mice and then neurobehavioral deficits in offspring mice. Twenty pregnant mice were included in the experiments, which generated a sufficient number of fetal mice for the biochemistry studies (n = 6 per arm), and offspring mice for the biochemistry (n = 6 per arm) and behavioral studies (n = 15 per arm). Our pilot studies showed a mean difference of 1.5 (3 vs.  1.5) in platform crossing times, with an SD of 1.8 in the control group and 1.3 in the anesthesia group. From the pilot study, we also estimated a mean difference of 150% (250% vs.  100%) in interleukin (IL)-6 levels in brain tissues, with an SD of 51 in the control group and 54 in the anesthesia group. Assuming this study would have similar effect sizes, a sample size of 6 per arm for the biochemistry studies and a sample size of 15 per arm for the behavioral studies would lead to a 90% or larger power to detect the differences using two-sample Student t  test with 5% type I error.
+
+Mouse Primary Neurons
+The protocol was approved by the Massachusetts General Hospital Standing Committee on the Use of Animals in Research and Teaching. The harvest of neurons was performed as described in our previous studies.13,14Seven to 10 days after harvesting, the neurons were treated with 4.1% sevoflurane for 6 h as described in our previous studies.9The treatment with 4.1% sevoflurane for 6 h was used to determine whether the sevoflurane anesthesia, which can induce cytotoxicity,9could also reduce levels of postsynaptic density-95 (PSD-95), the marker for synapse. The IL-6 antibody (10 μg/ml) was administrated to the neurons 1 h before the sevoflurane treatment. The neurons were harvested at the end of anesthesia and were subjected to Western blot analysis.
+
+Brain Tissue Harvest and Protein Level Quantification
+Immediately after the sevoflurane anesthesia, we performed a cesarean section to extract the fetal mice and harvested their brain tissues. We also used decapitation to kill postnatal day (P) 31 offspring mice and harvested their brain tissues. Separate groups of mice were used for the Western blot analysis and the immunohistochemistry studies, respectively. For the Western blot analysis, the harvested brain tissues were homogenized on ice using immunoprecipitation buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid, and 0.5% Nonidet P-40) plus protease inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A) as described in our previous studies.15The lysates were collected, centrifuged at 12,000 rpm for 15 min, and quantified for total proteins with bicinchoninic acid protein assay kit (Pierce Technology Co., Iselin, NJ).15 
+
+Western Blot Analysis
+Western blot analysis was performed using the methods described in our previous studies.15Whole cerebral hemispheres were used for Western blot analysis because there would be an insufficient amount of hippocampus tissues from the fetal mice for Western blot analysis. IL-6 antibody (1:1,000 dilution; Abcam, Cambridge, MA) was used to recognize IL-6 (24 kDa). PSD-95 antibody (1:1,000; Cell Signaling Technology, Danvers, MA) was used to detect PSD-95 (95 kDa). A caspase-3 antibody (1:1,000 dilution; Cell Signaling Technology) was used to recognize full-length caspase-3 (35–40 kDa) and caspase-3 fragment (17–20 kDa) resulting from cleavage at aspartate position 175. Antibody anti–β-actin (1:10,000; Sigma, St. Louis, MO) was used to detect β-actin (42 kDa). Western blot quantification was performed as described by Xie et al.  16Briefly, signal intensity was analyzed using a Bio-Rad (Hercules, CA) image program (Quantity One). We quantified the Western blots in two steps. First, we used β-actin levels to normalize (e.g. , determining the ratio of IL-6 to β-actin amount) protein levels and control for loading differences in the total protein amount. Second, we presented changes in protein levels in mice or neurons undergoing sevoflurane anesthesia as a percentage of those in the control group. One hundred percent of protein level changes refer to control levels for the purpose of comparison with experimental conditions.
+
+The quantification of Western blot was based not only on the images presented in figures but also on the images not presented in the figures to have adequate effect size (e.g. , n = 6 in biochemistry studies).15 
+
+Immunohistochemistry
+Immunohistochemistry was performed using the methods described in our previous studies.17P31 offspring mice were anesthetized with sevoflurane briefly (2.5% sevoflurane for 4 min) and perfused transcardially with heparinized saline followed by 4% paraformaldehyde in 0.1M phosphate buffer at pH 7.4. The anesthesia with 2.5% sevoflurane for 4 min in mice provided adequate anesthesia for the perfusion procedure without causing statistically significant changes in blood pressure and blood gas according to our previous studies.9Mouse brain tissues were removed and kept at 4°C in paraformaldehyde. Five-micron frozen sections from the mouse brain hemispheres were used for the immunohistochemistry staining.17The sections were incubated with the primary antibody synaptophysin (1:500; Sigma) dissolved in 1% bovine serum albumin in phosphate-buffered saline at 4°C overnight. The next day, the sections were exposed to secondary antibody (Alexa Fluor 594 goat anti-rabbit IgG [H+L]; Invitrogen, Grand Island, NY). Finally, the sections were wet mounted and viewed immediately using a fluorescence microscope (60×). We used the mouse hippocampus in the studies of immunohistochemistry density quantification to determine whether sevoflurane anesthesia can induce neurotoxicity in the hippocampus. The photographs were taken and an investigator who was blind to the experimental design counted the density of synaptophysin using ImageJ version 1.38 (National Institutes of Health, Bethesda, MD).17 
+
+Morris Water Maze
+A round steel pool, 150 cm in diameter and 60 cm in height, was filled with water to a height of 1.0 cm above the top of a 10-cm diameter platform. The pool was covered with a black curtain and was located in an isolated room with four visual cues on the wall of the pool. Water was kept at 20°C and opacified with titanium dioxide. The P31 offspring mice were tested in the Morris water maze (MWM) four times per day for 7 days. Each of the mice was put in the pool to search for the platform, and the starting points were random for each mouse. When the mouse found the platform, the mouse was allowed to stay on it for 15 s. If a mouse did not find the platform within a 90-s period, the mouse was gently guided to the platform and allowed to stay on it for 15 s. A video tracking system recorded the swimming motions of the animals, and the data were analyzed using motion-detection software for the MWM (Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China). At the end of the reference training (P37), the platform was removed from the pool and the mouse was placed in the opposite quadrant. Mice were allowed to swim for 90 s and the times the mouse swam to cross the platform area was recorded (platform crossing times). Mouse body temperature was maintained by active heating as described by Bianchi et al .18Specifically, after every trial, each mouse was placed in a holding cage under a heat lamp for 1 to 2 min until dry before being returned to its regular cage.
+
+Environmental Enrichment
+The EE in the current experiment was created in a large cage (70 × 70 × 46 cm) that included five or six toys (e.g ., wheels, ladders, and small mazes) as described in previous studies, with modification.10,11The pregnant mice were put in the EE every day for 2 h before delivery. The pregnant mice delivered offspring mice at G21. Then, the mother and the babies were put in the EE again every day for 2 h from P4 to P30. The objects were changed two to three times per week to provide newness and challenge.
+
+Statistical Analysis
+The nature of the hypothesis testing was two-tailed. Data were expressed as mean ± SD. The data for platform crossing time were not distributed normally and thus were expressed as median and interquartile range (IQR). The number of samples varied from 6–15, and the samples were distributed normally, with the exception of platform crossing time (tested by normality test, data not shown). Two-way ANOVA was used to determine the interaction of IL-6 antibody and sevoflurane treatment, and the interaction of EE and sevoflurane anesthesia. Interaction between time and group factors in a two-way ANOVA with repeated measurements was used to analyze the difference of learning curves (based on escape latency) between mice in the control group and mice treated with anesthesia in the MWM. Multiple comparisons in escape latency of MWM were adjusted using the Bonferroni method (with seven tests and a threshold of 0.05/7 = 0.0071). There were no missing data for the variables of MWM (escape latency and platform crossing time) during the data analysis. The Student two-sample t  test was used to determine the difference between the sevoflurane and control conditions on levels of IL-6, PSD-95, and synaptophysin. Finally, the Mann–Whitney U  test was used to determine the difference between the sevoflurane and control conditions on platform crossing times. Values of P < 0.05 were considered statistically significant. SAS software version 9.2 (SAS Institute Inc., Cary, NC) was used to analyze the data.

20230808-AI coding-1st round/269 – Lee 2014.txt

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+PMID: 25165850 PMCID: PMC4148240 DOI: 10.1371/journal.pone.0105340
+Methods
+Subjects
+All experiments were conducted with approval from the Institutional Animal Care and Use Committee at the University of California, San Francisco. Five Sprague-Dawley dams with litters of postnatal day 6 (P6) pups from were obtained from Charles River Laboratories (Gilroy, CA). Each litter contained only males and was culled to ten pups. In total, the males were taken from at least ten different litters. On P7, animals from each litter were randomly assigned to control and treatment groups. They were weaned at P23 and housed three per cage under standard lab housing with 12 h light/dark cycle. Animals were food restricted (access to food only during light cycle) for tasks involving object recognition to increase activity and object exploration.
+
+Anesthesia
+Anesthesia was delivered as described previously [14], [30], [31]. Briefly, animals in the treatment groups received either isoflurane or desflurane as a single agent in air and oxygen (FiO250%) at 1 Minimum Alveolar Concentration [27] for four hours. MAC was determined by tail clamping every 15 minutes, and anesthetic concentration was adjusted accordingly, so that on average 50% of animals would move in response to clamping (Fig. 1). 12 out of 18 animals anesthetized with isoflurane survived to undergo behavioral testing, and 13 out of 18 animals anesthetized with desflurane survived and underwent behavioral testing. Control animals were concurrently placed in an anesthesia glove box of the same material and conditions without being exposed to anesthesia or tail clamping. Animals were kept on a warming blanket, and temperatures were measured using an infrared laser thermometer and maintained with a goal of 35°C.
+Histology
+Brains from the two anesthetized groups and the control group (n = 10 per group) were assessed for acute neuronal death. Twelve hours after anesthesia, animals were transcardially perfused with cold 4% paraformaldehyde in phosphate-buffered saline and brains were removed, postfixed, and sunk in sucrose solution. They were then sliced into 60 micron-thick slices and every other slice was mounted and stained with FluoroJade C, a marker specific for neurodegeneration [32], [33] (FJC, 0.001%, Millipore, Billerica, MA). FJ-positive cells were counted using Nikon Eclipse 80i microscope under 20X magnification in each slice containing the structure of interest. Structures included in analysis were the anterodorsal (AD), anteroventral (AV), laterodorsal (LD), and anteromedial (AM) thalamic nuclei, as well as CA1-3 regions of the hippocampus and the dentate gyrus.
+
+Object Recognition Tasks
+Object recognition was assessed using similar arrangements as others [19], [28]. Behavior testing occurred during the light phase of the circadian cycle between 0800 and 1700 hrs in two separate arenas, hereafter referred to as contexts, of identical size (61 cm square base, walls 50 cm high). Context 1 had yellow walls with a base covered in wood-effect vinyl lining, and context 2 had black walls with a black plastic base. Different visual cues were placed on the walls of each context. A video camera (SONY HDR-CX190) was mounted 2 meters above the testing area for recording and observing subjects. For each task, except the allocentric object-location task, subjects were placed into contexts in the same location and facing the south wall (away from the objects). Beginning at P42, subjects were habituated to the two contexts prior to testing by being placed individually into the context for 5 min per day for 4 consecutive days. All animals underwent all behavioral tasks. Subjects were tested on the same day for any given task and in the same sequence of tasks. All tasks were performed in the order presented in subsequent weeks, except for the first two (novel object and object-place) which were performed in the same week. The order of testing during the day was counterbalanced among groups.
+
+Investigation of an object was defined as sniffing or placing the nose within 1 cm of and oriented toward the object. Subjects were recorded, and observers blinded to group assignment were used to determine investigation times. Object investigation times during the initial exposure for each task were compared to assess for possible confounding effects of varying investigation times on the ability to recognize objects. All objects and testing arenas were wiped with 70% ethanol between testing.
+
+Novel Object Recognition Testing began at P48 with novel object recognition. A single trial was performed for each animal consisting of “exposure” and “test” phases separated by a two-minute delay (Fig. 2A). During the exposure, subjects were placed into the context and allowed to explore two identical objects for four minutes. After the delay, they were placed into the same context for three minutes with one of the objects replaced with a novel object. Half of the subjects were tested in each context with the location (left or right) of the novel object counterbalanced among subjects.
+Object-Place Recognition Subjects were tested in their ability to recognize an object and its location. Two trials were performed, and investigation times were totaled for the two trials. In the exposure, two different objects were presented in a context for four minutes. After a two-minute delay, two identical copies of one of the previous objects were presented in the same context for three minutes (Fig. 2B). Both objects were equally familiar, but one now occupied a different location within the context.
+Allocentric Object-Place Recognition For the previous task, subjects were always introduced into the context facing the wall (south wall) opposite the two objects (Fig. 2C). In the allocentric version of the task, for the initial exposure, subjects were again placed into the context facing the south wall. In the test phase, however, the entry point was varied and half of the subjects were introduced facing either the east or west wall (Fig. 2C). Two trials were performed and the entry point was randomized among subjects.
+Object-Context Recognition Subjects were assessed in their ability to recognize an object with a particular context. The task required two separate exposures, each lasting four minutes and separated by a two-minute delay (Fig. 2D). In the first exposure, a pair of identical objects was presented in a context. Next, subjects were placed in a different context with a different pair of objects. In the test phase, lasting three minutes, subjects were placed into a context with one of each previously encountered object. Thus, one object was presented in the same context as before, while the other object appeared within a context in which it had not been explored. Two trials were conducted, and the test phase occurred in opposite contexts for each trial (Fig. 2D).
+Object-Place-Context Recognition Subjects were tested in their ability to recognize an object with its location and context (Fig. 2E). In the first exposure, two different objects were presented within a context. Next, subjects were placed in the opposite context with the same two objects and their locations reversed. Thus, after two exposures, each object was observed in both contexts and locations (left and right). In the test phase, two identical copies of either of the previous objects were presented in a context. The location and context associated with one object were familiar, while the other “displaced” object appeared in a location and context in which it had not been observed. Two trials were conducted with the test phase occurring in opposite contexts for each trial (Fig. 2E).
+Social Behavior and Social Recognition
+Following object recognition, animals were given unrestricted access to food. Social interaction and recognition were assessed using a discrimination paradigm one week after completing object recognition testing at P80. In the exposure, the subject was presented with a caged stimulus animal and a novel object for five minutes. This arrangement evaluates social behavior by determining whether subjects spend more time investigating the stimulus animal or object7. After a sixty-minute delay, subjects were presented simultaneously with the same “familiar” animal and a novel animal for three minutes. Recognition of the previously encountered animal was demonstrated by decreased investigation of the familiar target relative to the novel one.
+
+Same-sex juvenile conspecifics were used as stimulus animals. Male pups five weeks of age were housed individually one week prior to testing. Investigation of the stimulus animal was defined as sniffing or direct contact with the subject’s nose or paws. Investigation of the novel object was defined as sniffing or placing the nose within 1 cm of and oriented toward object.
+
+Statistical Analysis
+Data were analyzed using Prism 6 Software for Mac OSX (GraphPad Software Inc., San Diego, CA). Data were assessed for normal distribution using the D’Agostino and Pearson test. Parametric tests were used for normally distributed data; otherwise, nonparametric tests were used for analysis. All comparisons used a two-tail test and a P value less than 0.05 was considered statistically significant.
+
+Total FluoroJade-positive cells for each brain region were compared among the groups – control, desflurane, isoflurane – using one-way ANOVA for parametric data or the Kruskal-Wallis test for nonparametric data. Bonferroni’s post-test with multiple comparisons was used following one-way ANOVA, and Dunn’s post-test was used with the Kruskal-Wallis test. The fold-increase in neuronal death was determined for each structure by dividing the total FJ-positive cells for all anesthetized animals (n = 20) by the average number of FJ-positive cells per structure for control animals (n = 10).
+
+Recognition tasks were first assessed by comparing the investigation times of each target using paired tests for each group. Paired t-test was used for normally distributed data, and nonparametric data were analyzed with the Wilcoxon matched-pairs rank test. Also, to identify possible confounding effects of varying investigation times on subsequent object/animal recognition, the times during the exposure phase were compared between the groups using either one-way ANOVA with Bonferroni’s post-test or the Kruskal-Wallis test with Dunn’s post-test.
+
+In addition, a “discrimination index” (DI) was calculated and represents the relative time spent exploring each target (eg. Familiar versus Novel). To calculate DI, the time spent investigating the familiar target was subtracted from the time spent on the novel target, and this was divided by the total time spent investigating the two (eg. DI = (Novel-Familiar)/(Total Time)). This value was compared to a theoretical value of zero using one sample t-test to assess whether a preference was shown for one of the objects, and a positive DI indicates preference for the novel aspect of the task. For each task, DI of control animals was compared against DI of all anesthetized animals. Also, within the group of anesthetized animals, the DI of desflurane-treated subjects was compared with that of isoflurane-treated subjects. These comparisons were made using either unpaired t-test for parametric data or the Mann Whitney test for nonparametric data.

20230808-AI coding-1st round/276 – Peng 2014.txt

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+PMID: 25099925 PMCID: PMC4169313 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 provided ad libitum. Thirty-eight postnatal day 7 (P7) rats were used for the ELISA 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 determined by a power analysis. We anticipated 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, 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 min and then 1.5% ISO for 6 h the following day (1.5% ISO), or preconditioned (PC) with a 30 min 1.5% ISO exposure and then exposed to 1.5% ISO for 6 h the following day (PC + 1.5% ISO). The control animals were exposed to carrier gas (30% oxygen balanced in nitrogen) for 30 min and then carrier gas again for 6 h the following day in the plexiglass chambers but not in the water bath. Exposure to ISO for 30 min alone at P7 has been shown not to be detrimental12 and thus this control group was not included. In order to maintain a steady state of anesthetic gas and to prevent accumulation of expired carbon dioxide within the chamber, we used 6 liters 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 studies.4,15,24 Two rats died during exposure to 1.5% ISO for 6 hrs, 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 2–3% ISO. Blood (0.1 ml) was collected from the left ventricle and centrifuged to separate the plasma. We measured levels of S100β, a neuronal injury marker, using Sangtec 100 ELISA kits (DiaSorinInc, Stillwater, MN) following the manufacturer’s protocol and as we described previously.25 Briefly, 50 µl of plasma from each rat was placed in each well of a 96-well-plate and mixed with 150 µl of tracer from the kit, and incubated for 2 hours. Afterwards, 3,3’,5,5’tetramethylbenzidine substrate and stop solution were added to each well. The optical density was read at 450 nm. The sensitivity 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 anesthetized 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. At the time of the assay, the brain tissue 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 (Cell Signaling #9664), caspase-12 (Cell Signaling #2202), or Beclin-1 (Cell Signaling #3495). The density was measured by Quantity One software (BIO-RAD version 4.5.0) and GS-800 Densitometer (BIO-RAD, Hercules, CA) and the data are expressed as the percent of control of the means from 1 blot per animal per group.
+
+Immunohistochemistry
+Immunohistochemical localization of caspase-3 was performed 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 perfused with ice cold saline before the brains were removed, fixed with 4% paraformaldehyde, cryprototected in 30% sucrose, frozen in isopentane and stored at −80°C. Coronal cryosections (10µm) were incubated in 3% hydrogen peroxide, 10% normal goat serum and cleaved caspase-3 antibody (1:400; Cell Signaling Technology, #9664) overnight at room temperature. The next day, the sections were incubated with Alexa Fluor® 594 goat anti-rabbit IgG and coverslipped using ProLong® Gold Antifade Reagent containing the nuclear stain, DAPI (Invitrogen). 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 counted in the CA1 region of the hippocampus and the adjacent 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. Differences were considered statistically significant at p<0.01.

20230808-AI coding-1st round/279 – Bi 2018.txt

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+PMID: 30372849
+DOI: 10.1016/j.biopha.2018.09.111
+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 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 naturally. 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 returned to their mothers’ cages until the next procedure. For the intervention studies, we administered an inhibitor to the rats via an intraperitoneal injection 2 h before sevoflurane anesthesia. All the experiments were performed in a blinded manner.
+
+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 hippocampus 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 phenylmethylsulfonyl 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 and phosphorylated MAPKs, and total and phosphorylated c-Jun and c-Fos were tested with Western blots (Fig. 2).
+
+Fig. 2
+Download : Download high-res image (114KB)
+Download : Download full-size image
+Fig. 2. Schematic representation of the experimental protocol.
+
+In experiment two, according to the MAPK signal defined in experiment 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 sevoflurane 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, sevoflurane 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 sulfate-polyacrylamide gel electrophoresis (SDS‑PAGE); using a semidry blotting apparatus(Bio-Rad Laboratories, Munich, Germany), the proteins were electrotransferred to nitrocellulose 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 peroxidase (HRP) were added for 2 h followed by three washes. The positive reactive bands were detected by Amersham enhanced chemiluminescence (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.
+
+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 perfusion 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, rehydrated, 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 hematoxylin. 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.
+
+2.9. Morris water maze (MWM)
+To assess neurodevelopmental outcomes in adolescence, particularly learning and memory functions, twenty-four rats from four groups including control, control + SP600125, sevoflurane and 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 temperature 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 platform 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).
+
+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 appropriate. Interaction between time and group factors in a two-way ANOVA with repeated measurements was used to analyze the difference of learning curves (based on escape latency) 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.

20230808-AI coding-1st round/282 – Ozer 2017.txt

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+PMID: 28814087 DOI: 10.4149/BLL_2017_017
+Materials and methods The preclinical animal study was conducted at the Firat University Experimental Research Centre in December 2012. After receiving approval from the institutional Animal Experimentation Ethics Committee, seven-day-old male Wistar-Albino rats were obtained from the Experimental Research Centre. The Helsinki Universal Declaration of Animal Rights was followed at every stage of the study. The rats were kept in the rooms with ambient temperature of 22–24 °C and with 12/12 hour day/night cycle. Except for the time it took for the experimental tests, the subjects were kept in the same cage with their mothers until postnatal day 21 (PN21). After the 21st day, the rats were put in separate cages and fed with standard rat chow and tap water. Using permuted block randomisation methods, the subjects were divided into the two groups: Group C acted as the control and did not receive any anaesthesia, and in Group S, anaesthesia was achieved with 2.3 % sevofl urane in 50 % oxygen (O2 )-air mixture. The concentration of sevofl urane was adjusted according to the tail test. The tail test was applied every 15 minutes. The middle 1/3 of the tail was clamped, and if there was response, sevofl urane concentration was increased 15 %. All subjects were put in a plastic, transparent anaesthesia chamber that was connected to the anaesthesia device and was ventilated with 4 L/min fl ow and 50 % O2-air mixture. Immediately after the six-hour administration of anaesthesia, oxygen arterial blood gases were evaluated in 3 subjects from each group. At the end of the application period, half of the subjects were sacrifi ced to determine the early effects of sevofl urane (Group SE and Group CE), while the rest of the subjects were sacrifi ced 6 weeks after the application to determine the late effects of sevofl urane (Group SL and Group CL). In addition, the levels of serum BDNF, brain tissue BDNF and caspase 3 were evaluated for all subjects. The anaesthesia chamber was heated from the outside during the entire experiment to prevent hypothermia. Glucose and saline were administered intradermally to prevent hypoglycaemia and hypovolaemia. Subjects that experienced discolouration of the skin (cyanosis) or a decrease in respiratory rate that did not improve with stimuli, were excluded from the study.
+The study’s primary outcomes were serum BDNF levels, cortex and hippocampal BDNF levels and neurocognitive status. Secondary outcomes were cortex and hippocampal caspase 3 levels. Blood samples were taken at decapitation phase into serum separator tube to evaluate serum BDNF. Blood samples were centrifuged at 1000 x g for 15 minute and stored at –20 °C. Serum BDNF levels were measured by the enzyme-linked immunosorbent assay (ELISA) method (EK0308, Boster Biological Technology, Ltd.). Brain tissue samples were kept for 24 hours in 10 % formaldehyde prepared with phosphate buffer saline. Brain tissue samples were taken for routine tissue processing for immunohistochemical (IHC) examination procedure following the sagittal reduction process. Brain tissue was divided at the midline on the sagittal plane. The BDNF levels (Abcam, ab108319, Cambridge, UK) and caspase 3 levels (Abcam, ab13847, Cambridge, UK) were assessed with IHC in brain hemispheres (0 – no staining, 1 – mild, 2 – moderate, 3 – severe). The behaviour, anxiety states and spatial learning abilities of the subjects during the long-term period (6 weeks later) were evaluated by using the plus arm test and the Morris water test, respectively. Open arm avoidance index was calculated according to the formula 100 – ((% of the time spent in the open arms + % of the entrance to the open arms)/2), while the total locomotor activity was calculated based on line crossings + rearing. Swimming tests were done 4 times a day for a period of 4 days. The test was repeated 2 days after training and the time it took to reach the platform (latency) and the time spent in the platform quadrant after the platform was removed were recorded. The subjects that completed the swimming test were put back into their heated cages. Those conducting the experiment knew, which group of subjects they were dealing with, but those evaluating biochemical, IHC and neurocognitive tests did not know, which samples and subjects belonged to which group. SPSS 15 was used for a statistical evaluation. Nonparametric methods were used for all variables because the sample size was small. Kruskal–Wallis test was used for one-way analysis of variance (ANOVA) of nonparametric data, therefore median values were calculated instead of mean. When it was determined not to the equal of medians with Kruskal–Wallis test, Mann–Whitney U test was used for post-hoc multiple comparisons. The escape latency within the group was evaluated by Wilcoxon test. The correlation between parameters was assessed by Spearman correlation test and p < 0.05 was considered signifi cant.

20230808-AI coding-1st round/299 – Lin 2016.txt

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+PMID: 27688943 PMCID: PMC5036436 DOI: 10.1002/brb3.514
+Materials and Methods
+Treatment with sevoflurane
+C57/BL6 mice were used throughout the study, which was approved by the SUNY Downstate IACUC. A total of nine litters of mice were used to establish the approximate MAC for P7 mice. A separate set of 11 litters of mice were used for treatment without tail clamp and mice from this group were used for behavioral tests later on in life. At P7, all male pups from each litter (ranging from 2 to 6 pups) were randomly assigned to either the sevo or the no sevo (control) group, while the female pups remained with the dam. During a 2‐h treatment period, pups from the sevo group were separated from the dam and exposed to sevo in a 40% oxygen (O2) and 60% nitrogen (N2) gas mixture (GTS‐WELCO, Newark Distribution, Morrisville, PA). These pups were placed on a 37°C heating pad to prevent hypothermia during treatment. A pulse oximeter sensor (MSTAT 4 mm, Kent Scientific Corporations, Torrington, CN) was placed on one of the hind paws of the pup and measurements for heart rate (HR) and blood oxygen saturation (SpO2) were recorded every 5 min. To establish the approximate MAC of sevo on P7 mice, each treatment of sevo consisted of two mice and tail clamp was done every 10 min. The sevo concentration was adjusted to a higher concentration if both mice moved during tail clamp, adjusted to a lower concentration when neither mouse moved during tail clamp, and no adjustment was made when only one mouse responded to stimuli. This was our approach to determine that with a given sevo concentration, 50% of mice did not respond to stimuli. The sevo concentration was recorded every 5 min since that was the time interval that we used to record peripheral capillary oxygen saturation (SpO2) and HR. The pups from the control group were also separated from the dam and exposed only to 40% O2 and 60% N2. At the end of the 2‐h treatment the pups were returned to their home cage and reunited with their dams. All pups were then reared and weaned following standard institution procedures.
+
+Behavior tests
+The mice that were involved in the behavior tests had undergone a 2‐h sevo or no sevo treatment at P7 without tail clamping. They were reared and group housed under standard conditions. The sevo‐treated mice were marked to distinguish them from the no‐sevo–treated mice within a litter. We examined at most one to two litters of mice at a time for each behavior, with at least 1 week of resting time in between different behavioral tests. The behavior tests were given sequentially for the active place avoidance (APA), reciprocal social interaction, and olfaction habituation/dishabituation. After completion of these tests, we then introduced three‐chamber interaction, open field, and novel object recognition (NOR). All behavioral apparatus were assembled and remained in their original locations throughout the entire duration of the project. The APA test was done on mice starting at the age of P27. All other behaviors were conducted on mice within the age range of 1.5–5 months old. The following reasons contributed to variation in the number of mice used for some tests. First, we were not able to examine all treated mice on the APA due to irreparable malfunctioning of the APA apparatus. Therefore, we introduced NOR as a second cognition test on mice that had not been used for the APA. Second, some mice were not included in testing if they were not within the age range at the time of the test, specifically the second group of tests such as three‐chamber interaction, open field, and NOR. Besides the APA and the open field, all other tests that required manual scoring were first videotaped and then scored by experimenters who were blind to the treatment status of the mice.
+
+Locomotion and anxiety‐like behavior
+Open field test An open field apparatus was used to assess the general physical and anxiety‐like performance of the mice based on their ambulatory locomotion in the arena (Crawley 1985). In a well‐lit novel room, each mouse was given 30 min to explore the open field arena. Locomotion activities such as distance and time in different compartments of the arena were automatically measured using a computerized tracking apparatus (Versadat, Versamax, Groovy, CA).
+Learning and memory‐like behavior
+Active place avoidance test The APA test is a hippocampus‐dependent spatial memory test. A rotating arena consisting of a circular platform (40 cm diameter) was placed in the center of a dimly lit room. The mouse was trained to avoid a 60° shock zone, which could be defined within a region of the room identified by multiple visual cues (Fenton et al. 1998; Wesierska et al. 2005). Two‐day trials were performed as described previously (Burghardt et al. 2012). Briefly, the mouse was given 10 min for each trial with at least a 50‐min intertrial interval. The locomotion of the mouse was tracked by computer‐based software that analyzed images from an overhead camera and delivered shocks appropriately (Tracker, Bio‐Signal Group Corp., Brooklyn, NY). A brief constant current foot shock (500 msec, 60 Hz, 0.2 mA) across pairs of rods was delivered to the shock zone upon entrance of the mouse. Track analysis software (Bio‐Signal Group Corp.) was used to compute the number of times that the mouse entered the shock zone.
+Novel object recognition test This is a two consecutive day test examining learning and memory‐like behavior on adult male mice (3–5 months of age; Leger et al. 2013). The test was conducted in a room with dim lighting. Day 1 is considered the familiarization phase. Mice were individually habituated in a standard open field apparatus for 10 min. They were then taken out of the arena briefly and two identical glass bottles filled with pink silica gel were placed in the center of the arena. The glass bottles were positioned 5 inches from each other such that the mouse can travel freely across the center of the arena without obstruction. The mouse was then put back in the arena and allowed 10 min to become familiar with the two identical objects. Day 2 is the test phase. One of the glass bottles is taken out of the arena and replaced with a yellow laboratory tube rack (H 6.5, W 3.5, D 2 inches) as a novel object. The holes on the sides of the tube rack were taped to prevent the mouse from climbing on them during the experiment. The same mouse was placed in the arena for 10 min in an identical manner as day 1 and allowed 10 min of exploration time. The times spent sniffing and interacting with (attempting to climb up or jump on or at) the familiar and the novel objects were scored for each mouse.
+Social interactions
+Reciprocal social interaction The subject mouse (P7 sevo treated or no sevo control) was transferred from his home cage to a new cage with fresh bedding and allowed to habituate to the cage for 10 min. At the end of this 10‐min session, a novel male target mouse (that had not undergone treatment during P7) of similar age was introduced into the same cage. The subject and the target mice were allowed to interact for 10 min. The amount of time that the subject mouse spent interacting with the target mouse (push–crawl/following behavior), self‐grooming, and exploring the arena and the total time the mouse was mobile were scored manually (Silverman et al. 2010).
+Three‐chamber interaction A three‐chamber apparatus made of clear plexiglass was used for this study (Nadler et al. 2004). The apparatus is divided into three equally sized compartments (H 9.5, W 8, D 16 inches). First, the subject mouse was habituated for 10 min in the center chamber. Then the doors that give access to the left and right sides of the chamber were opened allowing the subject mouse to freely explore all three chambers for 10 min. During this time, the novel target mouse was habituated under a wire pencil cup on a separate tabletop. After 10 min of three‐chamber exploration, the doors were closed and the subject mouse was briefly confined in the center chamber. During this time, we set up the three‐chamber apparatus such that the novel target mouse was placed on one side of the chamber and a novel empty pencil cup on the other side. A weighted plastic cup was placed on the top of each pencil holder to prevent the subject mouse from climbing on the top of it. The doors were then opened to allow the subject mouse to explore the three chambers for 10 min.
+Communication
+Olfaction habituation/dishabituation The mouse was transferred to a new cage containing a thin layer of fresh bedding and a hole for inserting a cotton tipped swab. After a 10‐min habituation period in the new cage, the mouse was presented with nonsocial and social odors. Each odor was presented for three consecutive times; the order of presentation was water, almond extract (1:100, Spice Supreme), orange extract (1:100, McCormick), mouse socials 1 and 2. The mouse social odors were taken by wiping in a zigzag pattern across the bottom surface of different cages for odors 1 and 2; each cage housed unfamiliar mice of the same sex and strain. Each presentation of odor lasted 2 min. The amount of time that the mouse spent sniffing the cotton swab, including nose poking, chewing, sniffing, and close proximity (2 cm) of the nose to cotton swab was scored (Silverman et al. 2010).
+Statistical analysis
+All statistical analysis was done using GraphPad Prism 5.0 (GraphPad, San Diego, CA). Data with one variable such as the open field and the reciprocal social interaction were analyzed by t test. Data with two variables such as the APA, the NOR, the three‐chamber interaction, and the olfaction habituation/dishabituation were analyzed by two‐way ANOVA, followed by Bonferroni posttests.

20230808-AI coding-1st round/307 – Kong 2011.txt

@@ -0,0 +1,31 @@
+PMID: 21930122 DOI: 10.1016/j.ejphar.2011.08.050
+2. Materials and methods
+2.1. Animals
+All of the animals were treated according to the guidelines of the Guide for the Care and Use of Laboratory Animals (China Ministry of Health). The Laboratory Animal Care Committee of Zhejiang University approved all experimental procedures and protocols. All efforts were made to minimize the number of animals used and their suffering. The dams were housed in polypropylene cages, and the room temperature was maintained at 22 °C, with a 12-hour light–dark cycle. The dams at gestational day 14 were used for all experiments, because this time corresponds approximately to midgestation in humans (Clancy et al., 2001, Clancy et al., 2007), the period when most nonobstetric surgeries and fetal interventions are performed (Goodman, 2002, Tran, 2010).
+
+2.2. Anesthesia exposure
+Ten dams were randomly divided into a control and an isoflurane group (n = 5). The dams were placed in plastic containers resting in water baths with a constant temperature of 38 °C. In these boxes, the dams were either exposed to 1.3% isoflurane (Lot 826005U, ABBOTT, USA) in a humidified 30% oxygen carrier gas or simply humidified 30% oxygen without any inhalational anesthetic for 4 h. We chose 1.3% as the anesthetic concentration because it represents 1 minimum alveolar concentration (MAC) in the pregnant rats (Mazze et al., 1985). The determination of anesthetic duration based on our preliminary study which indicated that maternal physiological states remained stable throughout a 4-hour isoflurane exposure. The isoflurane concentration in the box was monitored with an agent gas monitor (Vamos, Drager Medical AG & Co. KgaA). Otherwise, control and experimental animals were under the same treatment and environment. During isoflurane anesthesia, arterial blood gases and blood glucose were measured at the end of the 4-hour anesthetic exposure. The rectal temperature was maintained at 37 ± 0.5 °C. After exposure, the dams were returned to their cages and allowed to deliver naturally. The postnatal body weights of the rat pups were monitored.
+
+2.3. Memory and learning studies
+Four rat pups (2 females and 2 males) from each dam were selected to determine cognitive function at postnatal day 28 with a Morris Water Maze test with minor modifications (Jevtovic-Todorovic et al., 2003). A round pool (diameter, 150 cm; depth, 50 cm) was filled with warm (24 °C) opaque water to a height of 1.5 cm above the top of the movable clear 15-cm-diameter platform in the third quadrant. A video tracking system recorded the swimming motions of animals, and the data were analyzed using motion-detection software for the Morris Water Maze (Actimetrics Software, Evanston, IL, USA). After every trial, each rat was wiped before returning to its regular cage, keeping warm and free diet.
+
+2.3.1. Place trials
+The place trials were performed at postnatal day 29 for 4 days to determine the rats' ability to obtain spatial information. At postnatal day 28, the rats were made to know the existence of the platform through a 30-second swimming training. A dark black curtain surrounded the pool to prevent confounding visual cues. All rats received 4 trials per day in each of the four quadrants of the swimming pool. On each trial, rats were placed in a fixed position into the swimming pool facing the wall. They were allotted 120 s to find the platform upon which they sat for 20 s before being removed from the pool. If a rat did not find the platform within 120 s, the rat was gently guided to the platform and allowed to remain there for 20 s. For all training trials, swim speed and the time to reach the platform (escape latency) were recorded. The less time it took a rat to reach the platform, the better the learning ability. We took the average of four trials as the escape latency each day.
+
+2.3.2. Probe trials
+Probe trials were conducted immediately after the four-day period to evaluate memory retention capabilities. The probe trials involved removing the submerged platform from the pool and allowing the rats to swim for 120 s in any of the four quadrants of the swimming pool. Time spent in the third quadrant and the number of original platform crossing in the third quadrant was recorded.
+
+2.4. Transmission electron microscopy
+After the Morris Water Maze test, three pups per group were anesthetized with a lethal dose of Nembutal. The thoracic cavities were opened and perfused intracardially with 100 mL of normal saline. Then the hippocampus, including CA1 and dentate gyrus area, of each rat was taken out immediately. Immersion fixation was completed on tissues about 1 mm3 from the hippocampus. Samples were rinsed in cold phosphate-buffered saline (PBS) and placed in 2.5% glutaraldehyde at 4 °C for 4 h. The tissue was rinsed in buffer and post-fixed with 1% osmium tetroxide for 1 h. Then, the tissue was rinsed with distilled water before undergoing a graded ethanol dehydration series and was infiltrated using a mixture of half propylene oxide and half resin overnight. Twenty-four hours later, the tissue was embedded in resin. 120 nm sections were cut and stained with 4% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Ultrastructure changes of synapse in the hippocampus were observed under a transmission electron microscope (Phliphs Tecnai 10, Holland).
+
+2.5. Tissue section preparation
+After the Morris Water Maze test, two pups from each dam were anesthetized by intraperitoneal injection of a lethal dose of Nembutal. The aorta was cannulated and the animal was firstly perfused with 200 mL of normal saline, then with 250 mL of 4% formaldehyde (freshly made from paraformaldehyde) for 20–30 min. The fixed brain was then removed from the cranial cavity and post-fixed overnight in the same fixative at 4 °C. The tissues were embedded in paraffin, and transverse paraffin sections containing the hippocampal area (5 mm thick) were mounted on silanecoated slides. Sections were deparaffinaged and rehydrated. Then the sections were treated for antigen retrieval with 10.2 mmol/L sodium citrate buffer, pH 6.1, for 20 min at 95 °C for immunohistochemistry.
+
+2.6. Immunohistochemistry analysis
+The sections mentioned above were washed in 0.01 M PBS containing 0.3% Triton X-100 (pH 7.4, PBS-T), followed by blocking in 5% normal goat serum in 0.01 M PBS. The sections were then incubated in the primary antibodies rabbit polyclonal against anti-CHOP or caspase-12 (1:100, Santa Cruz Biotechnology, USA) overnight at 4 °C. After a thorough wash in PBS, sections were incubated with biotinylated goat anti-rabbit IgG antibody (1:200, Boster, China) for 2 h at room temperature, followed by avidin–biotin–peroxidase complex solution (ABC, 1:100, Boster) for 2 h at room temperature. Immunolabeling was visualized with 0.05% diaminobenzdine (DAB) plus 0.3% H2O2 in PBS and the reaction was stopped by rinsing the slides with 0.2 M Tris–HCl. Sections were mounted onto 0.02% poly-l-lysine-coated slides and allowed to dry at room temperature. Then the sections were dehydrated through a graded series of alcohols, cleared in xylene and finally coverslipped. Rat Immunoglobulin IgG (1:200, Biomeda Corporation, USA) was used instead of primary antibody as a negative control. Three sections from each animal were selected at random and images were photographed under 400× magnification in 3 visual fields/per section, the CHOP and caspase-12 positive neurons were counted in the same area. The optical densities of CHOP and caspase-12 positive neurons were measured quantitatively using NIH image software (ImageJ, National Institutes of Health, Bethesda, MD).
+
+2.7. Western blot analysis
+After Morris Water Maze test, two pups from each pregnant mother were anesthetized with a lethal dose of Nembutal. Then their thoracic cavities were opened and perfused intracardially with 100 mL of normal saline. Hippocampus, including CA1 and dentate gyrus field, of each rat was taken out immediately to obtain fresh tissue specimens. Protein concentration was determined by the BCA method using bovine serum albumin as the standard. Protein samples (50 μg) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membranes were blocked by nonfat dry milk buffer for 2 h and then incubated overnight at 4 °C with primary antibody against CHOP or caspase-12 (1:500, Santa Cruz Biotechnology, USA). The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies and developed with ECL kit. The optical densities of bands were quantitatively analyzed using Bio-Rad Quantity One 4.6.2 (Bio-Rad Laboratories, USA). The results were expressed as a relative density. Equal protein loading in each lane was confirmed by hybridization with a 1:2000 dilution of β-actin antibody (Santa Cruz Biotechnology, USA).
+
+2.8. Statistical analysis
+All data were presented as mean ± S.E.M. Results of weight of postnatal rat pups and place trials of postnatal rats were analyzed using 2-way ANOVA for repeated measurements. Other data were analyzed using Student's t-test for comparison of two groups. A P value of < 0.05 was considered statistically significant. All statistical tests and graphs were performed or generated, respectively, using GraphPad Prism Version 4.0 (GraphPad Prism Software, Inc. CA, USA).

20230808-AI coding-1st round/341 – Huang 2016.txt

@@ -0,0 +1,34 @@
+PMID: 26966008 DOI: 10.1007/s12640-016-9615-7
+Materials and Methods
+Animal Treatment
+All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. The timed-pregnant Sprague–Dawley rats were housed in a temperature-controlled (22–23 °C) room on a 12 h:12 h light:dark cycle (light on at 8:00 AM) with free access to food and water. The PND-7 male rat pups (11–14 g) were randomly assigned to ketamine-treated and control groups. In the treated group, ketamine was diluted in 0.9 % normal saline, and PND-7 rats were intraperitoneally administered with 40 mg/kg doses of ketamine in four injections at 1 h intervals (40 mg/kg × 4 injections). Control rats received an equal volume of normal saline. 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 the chamber. Between each injection, animals were returned to their chamber to help maintain body temperature and reduce stress.
+
+BrdU Injections
+All animals received an intraperitoneal injection of BrdU (5-bromo-2-deoxyuridine; Sigma) at a dosage of 100 mg/kg after ketamine anesthesia according to the following experimental schedule.
+
+Experiment 1: To evaluate the effect of ketamine on the proliferation and differentiation of NSCs in the DG during the BGS, the PND-7 rats received a single intraperitoneal injection of BrdU on PND-7, 13, and 20 after ketamine treatment. The animals were then anesthetized and fixed by perfusion at 24 h after each BrdU injection. The experimental protocol is described in Tables 1a and 2.
+Experiment 2: To exclude the GFAP/BrdU double-positive cells that were proliferative astrocytes, the PND-7 rats received a single intraperitoneal injection of BrdU on PND-7, 13, and 20 after exposure to treatment. The animals were then perfused at 3 h after each BrdU injection. The experimental protocol is detailed in Tables 1b and 2.
+
+Experiment 3: To determine the effect of ketamine on the migration of newborn granule neurons in the DG, the PND-7 rats received three consecutive BrdU injections on PND-7, 8, and 9 after exposure to treatment. At 28 and 35 days after the last BrdU injection, the animals were anesthetized and fixed by perfusion. The experimental protocol is described in Table 1c and 2.
+
+Cell Apoptotic Assays
+Nestin/caspase-3 and GFAP/caspase-3 double-immunofluorescence staining was utilized to detect whether ketamine could induce the apoptosis of NSCs or astrocytes. At 12 h after the end of control and ketamine-anesthesia treatment, the neonatal rats were anesthetized and fixed by perfusion (n = 5 per group).
+
+Tissue Preparation and Immunofluorescence
+At the indicated time point, animals were deeply anesthetized and then transcardially perfused with 0.9 % normal saline followed by 4 % paraformaldehyde. The brains were removed, postfixed overnight in 4 % paraformaldehyde, and placed in 30 % sucrose until sunk. The coronal sections of brain were cut consecutively at a thickness of 30 μm when the hippocampus was initially exposed. The fifteenth section was taken and stored in PBS. According to the Atlas of the Developing Rat Brain and previous reports (Ashwell and Paxinos 2008; Paxinos and Watson 1986), the positions of hippocampus coronal sections selected in our study were about 2.20–2.25 mm posterior to the bregma at PND-8 rats, about 2.35–2.40 mm posterior to the bregma at PND-14 rats, about 2.50–2.55 mm posterior to the bregma at PND-21 rats, and about 2.75–2.85 mm posterior to the bregma at PND-37 and PND-44 rats, respectively.
+
+For Nestin/BrdU, β-tubulin III/BrdU, GFAP/BrdU, and NeuN/BrdU double-immunofluorescence staining, the BrdU antigen was exposed by incubating the sections in 2-normal hydrochloric acid for 30 min at 37 °C and then washed three times with PBS for 5 min between each of these steps. Blocking of nonspecific epitopes with 10 % donkey serum in PBS with 0.3 % Triton-X for 2 h at room temperature preceded incubation overnight at 4 °C with the primary antibodies listed in Table 3 in PBS with 0.3 % Triton-X. On the next day, the sections were incubated with the appropriate secondary fluorescent antibodies (Invitrogen Carlsbad, CA) for 2 h at room temperature.
+Astrocytic development was detected by using GFAP single-labeled staining. The sections were incubated overnight at 4 °C with a fluorescent antibody for the GFAP (Table 3). After three washes in PBS, sections were incubated with secondary fluorescent antibody (Invitrogen) for 2 h at room temperature.
+
+To characterize the phenotype of cell apoptosis, brain sections were analyzed by double-labeled staining. The sections were incubated overnight at 4 °C with the appropriate primary antibodies listed in Table 3. After three washes with PBS, the sections were incubated with the suitable secondary fluorescent antibodies (Invitrogen) for 2 h at room temperature.
+
+A skilled pathologist blinded to the study conditions examined the labeled sections using a laser scanning confocal microscope (Fluoview 1000, Olympus). The number of single- or double-positive cells in the hippocampal DG was quantified using Image-Pro Plus software.
+
+Western Blot Analysis
+Thirty and thirty-seven days after the control or ketamine-anesthesia treatment, the animals were decapitated, and the hippocampal DG tissue was dissected carefully with anatomic microscope (leica EZ4HD). The harvested hippocampal tissues were homogenized on ice using lysate buffer plus protease inhibitors. The lysates were centrifuged at 14,000 rpm for 15 min at 4 °C and were resolved by 12 % polyacrylamide gel electrophoresis, and the target proteins were transferred to nitrocellulose membranes. The blots were incubated with blocking buffer for 2 h at room temperature and then incubated for 24 h at 4 °C with the primary antibodies: rabbit anti-GFAP antibody (1:1000, Millipore) and GAPDH. The membranes were then incubated with appropriate secondary alkaline phosphatase-conjugated donkey anti-rabbit antibody (1:10,000, Abcam) for 1 h. The band intensity was quantified using Image J software (n = 5 per group).
+
+Morris Water Maze Test
+The hippocampal-dependent spatial memory abilities were tested by using the Morris water maze (MWM). Different set of rats were tested 2 months after administration of ketamine on PND-7. A circular, black painted pool (180 cm diameter, 50 cm deep) was filled with water to a depth of 30 cm. The water temperature was maintained at 25 ± 1 °C. An invisible platform (10 cm diameter) was submerged 1 cm below the water surface and placed in the center of the III quadrant which was determined with four starting locations called I, II, III, and IV at equal distance on the edge of the pool. During five consecutive days, the experiments were conducted in a dark and quiet laboratory, all the rats were trained four times per day, the starting positions were random for each rat. When the rat found the platform, the rat was allowed to stay on it for 30 s. If a rat did not find the platform within 120 s, the rat would be guided gently to the place and allowed to stay on it for 30 s, and the latency time to find the hidden platform was recorded as 120 s. The average time from four trials represented as the daily result for the rat. On the sixth day, the hidden platform was removed, and the rat was placed in the opposite quadrant. Rats were allowed to swim freely for 120 s. The numbers the rat swam to cross the previous platform area, and the times the rat stayed in the target quadrant within 120 s were recorded. Each animal’s path was tracked by a computerizing video system. After every trial, each rat was placed in a heater plates for 1 to 2 min until dry before being returned to its chamber. The data were analyzed using software for the MWM (Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, China).
+
+Statistical Analysis
+The statistical analysis was conducted using SPSS 13.0, and the graphs were created using GraphPad Prism 5. The data were analyzed using Mann-Whitney U test. The interaction between time and group factors in a two-way ANOVA was used to analyze the difference of escape latency between rats in the control group and rats treated with ketamine in the MWM. The data are presented as the mean ± SD, and p < 0.05 was considered statistically significant.

20230808-AI coding-1st round/359 – Lee 2014.txt

@@ -0,0 +1,32 @@
+PMID: 24704083 PMCID: PMC4077337 DOI: 10.1016/j.neuropharm.2014.03.011
+2. Materials and methods
+2.1. Subjects
+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 light–dark cycle and regulation of temperature (18–25 °C) and humidity (45–65%). At P30, they were housed in pairs with one treatment and one control animal per cage. All behavioral testing occurred during the light cycle 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.
+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 continuously monitored. The isoflurane concentration was initially set to 4% (time = 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 = 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 neurodegeneration (FJC, 0.001%, Millipore, Billerica, MA). FJ-positive cells were counted using Nikon Eclipse 80i microscope under 20× magnification in each slice containing the structure of interest. Structures included in analysis were the anterodorsal (AD), anteroventral (AV), laterodorsal (LD), and anteromedial (AM) thalamic nuclei, as well as CA1–3 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 obtained 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.
+
+2.4. Object recognition tasks
+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 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 object–place–context 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 recorded and later reviewed.
+
+2.5. Social behavior and social recognition
+Social interaction and recognition were assessed using a discrimination paradigm. 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. Investigation 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'Agostino-Pearson test. Parametric tests were used for normally distributed data; 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 (object–place–context 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 = (Novel − 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 Kruskal–Wallis 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 Kruskal–Wallis 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 determined for each structure by dividing the total FJ-positive cells of each treatment animal (n = 20) by the average number of FJ-positive cells per structure for the whole control group (n = 20).

20230808-AI coding-1st round/365 – Bercker 2009.txt

@@ -0,0 +1,42 @@
+10.1007/s12640-009-9063-8
+Materials and Methods
+The experiments were performed according to the guidelines of the German Animal Protection Law and were approved by the Berlin State authorities.
+
+Wistar rat pups were purchased from the Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin BgVV, Berlin, Germany.
+
+Experimental Protocol
+Six-day-old Wistar rats received either intraperitoneal (i.p.) injections of propofol or underwent inhalational anesthesia with sevoflurane and were separated from their mother during the experimental phase. In every litter animals were randomized either for anesthesia or controls.
+
+For propofol anesthesia doses of 30 mg/kg body weight were given every 90 min up to a cumulative dose of 90 mg/kg. For gas administration rats were placed into an incubation chamber (Billups Rothenberg Inc., Del Mar, USA), which was connected to an anesthesia system (F.Stephan GmBH, Gackenbach) for 6 h. Carbon dioxide and sevoflurane concentrations were monitored using a gas monitor (Datex Ohmeda, GE Healthcare, Munich, Germany). To avoid rebreathing of carbon dioxide, inspired CO2 concentration was continuously monitored and kept below 1 vol% by adjusting the fresh gas flow. Pilot studies established that sevoflurane concentrations between 3 and 5% maintained sufficient depth of anesthesia, as determined by lack of reaction to a painful stimulus. However, it was also observed that skin color changed and respiratory diminished in some animals, suggesting respiratory insufficiency. Accordingly, animals were closely monitored during the experiment and sevoflurane concentration adjusted between 3 and 5 vol% to maintain normal skin color and adequate respiratory efforts.
+
+The animals were observed for another 90 min until they were awake and active to be returned to their mother after the last injection and 6 h of gas application respectively. To maintain body temperature and prevent hypothermia, animals were placed on a heating device. During anesthesia respiratory frequency and skin color were observed to detect apnea and hypoxia. If bradypnoe occurred, rats received a pain stimulus, if breathing did not restart or resuscitation efforts were necessary rats were excluded from further processing and analysis. Verum and control animals received injections of PÄD II cristalloid/glucose (50 g/l) solution (Fresenius-Kabi, Bad Homburg, Germany) to prevent hypoglycemia and hypovolemia. Control animals were separated from the mother for the same period as the anesthetized animals and received injections of the crystalloid/glucose solution as well. In order to reduce the amount of laboratory animals, the control animals of the propofol group were pooled with control animals from the sevoflurane group. Therefore, sham injections have not been performed. However, all experiments were performed using the same experimental settings and laboratories during the same time.
+
+For perfusion fixation animals were killed with an injection of an overdose of chloral hydrate 24 h after starting anesthesia. A solution of PBS (phosphate buffered saline) mixed with heparine (Thrombophob 25,000, Heparin-Natrium; Nordmark Arzneimittel GmbH, Uetersen, Germany) was injected slowly into the heart and ascending aorta in order to wash out the blood from the vessels. Afterwards, rats were perfused with a solution containing paraformaldehyde 4% (Merck, Darmstadt, Germany) with cacodylate buffer (Sigma, Deisenhofen, Germany) for 10 min (De Olmos cupric silver staining).
+
+Histology
+To visualize degenerating cells, coronal sections (70 μm) of the whole brain were cut on a vibratome and stained with silver nitrate and cupric nitrate (De Olmos and Ingram 1971). This technique stains degenerating cells dying via an apoptotic or non-apoptotic mechanism. Degenerating cells were identified by their distinct dark appearance due to silver impregnation.
+
+Quantification of Damage
+Quantification of brain damage was assessed in the frontal, parietal, cingulate, retrosplenial cortex, caudate nucleus (mediodorsal part), thalamus (laterodorsal, mediodorsal, and ventral nuclei), septum, dentate gyrus, hypothalamus, cornu ammonis field CA1, and subiculum in silver stained sections by estimating mean numerical densities (Nv) of degenerating cells (Gundersen et al. 1988). An unbiased counting frame (0.05 mm × 0.05 mm: dissector height 0.07 mm) and a high aperture objective were used for sampling. The Nv for each brain region was determined with 8–10 dissectors. Regional Nv values from 17 brain regions were summed to give a total score for degenerating neurons for each brain. Figure 1 shows representative silver stained brain regions.
+
+Fig. 1
+figure 1
+Light microscopic overviews of silver-stained transverse sections picturing neurodegenerative changes in 6-day-old rats. The images of the rat thalamus show examples 24 h after treatment a after propofol treatment, b sevoflurane treatment, and c for controls. Degenerated neurons are pictured as small dark dots
+
+Full size image
+Behavioral Task
+For behavioral testing n = 13 propofol treated, n = 7 sevoflurane treated animals and n = 6 controls were anesthetized as described above. Only male pups were used. At the beginning of behavioral testing, animals were 7 weeks old. Animals were group-housed (4–5 animals per cage) under standard laboratory conditions (22 ± 2°C room temperature) with an artificial 12 h light-dark cycle (lights on 6.00–18.00 h). Rats had access to food (Altromin 1326, Lage, Germany) and water ad libitum. They were acclimated to the animal unit for at least 2 weeks before testing. One hour prior to testing rats were transferred to a quiet anteroom. All experiments were performed between 8.00 and 13.00 h and only male animals were tested. In order to avoid possible carry-over effects, a pause of 7 days, during which the animals were left undisturbed in their home cages, was introduced between the hole board and water maze task.
+
+Morris Water Maze (MWM) Test
+The water maze task was conducted by using the same experimental design as described previously (Bert et al. 2002). A blue circular tank (diameter 200 cm, 60 cm deep) was filled up with water (21 ± 1°C) to a height of 42 cm. The tank was surrounded by several visual cues and was indirectly illuminated (120 lx at the centre of the pool).
+
+For a single adaptation trial (day 0, without platform), the rats were released into the pool for 90 s with no escape platform present. On the following 8 days (day 1–8, place version) a transparent platform (16 × 16 cm) was submerged 1.5 cm below the surface in the middle of one of four virtual quadrants which was according to adaptation trial neither preferred nor avoided by the rat. Each day the animals were lowered into the water facing the wall from three different starting points (left, opposite, and right from the platform quadrant). Animals that did not find the escape platform within 90 s were placed onto it by the experimenter. All rats were allowed to remain on the platform for 30 s for orientation and were afterwards removed to rest for 60 s in a heated cage until the next trial. For each trial the escape latency to reach the platform was measured by a computerized tracking system (TSE VideoMot, Version 1.43, Bad Homburg, Germany). For each animal the three daily trials were averaged. On day 9 the escape platform was removed (spatial probe) and the time spent in each quadrant during a single 90 s trial was registered. On day 10 (cued version) the platform was elevated 1 cm above water level, signaled by a white cylinder (diameter 3 cm and 4 cm high), and moved to the quadrant opposite to the initial quadrant. This test was performed to assess the motivation to escape from the water and sensor-motor integrity. The testing procedure and recorded parameter during the cued version were the same as for the hidden platform version of the task (Morris 1984).
+
+Hole Board Test
+The hole board apparatus consisted of a square box (50 × 50 cm) made of grey Perspex with 16 equally spaced holes (diameter 2.5 cm), and was situated in a sound-attenuated chamber. The behavior of rats was monitored by an overhead installed video camera which was linked to a computerized tracking system (TSE VideoMot2, Bad Homburg, Germany). The test was conducted on two consecutive days. On both days rats were placed in the centre of the apparatus and observed for 10 min. The numbers of nose pokes and rearings as well as the distance traveled were recorded. After each animal the box was cleaned with 2-propanol 30%. Habituation to the apparatus was defined as a significant reduction of nose pokes, rearings and locomotor activity from the 1st to the 2nd day (Voits et al. 1995).
+
+Blood Gas Analysis
+To exclude severe hypoxia, hypercapnia or lactic acidosis, a blood gas analysis was performed for example in one animal of each group by transcutaneous puncture of the left ventricle. The probe was analyzed by a blood gas analyzer (Radiometer ABL series, Radiometer, Copenhagen, Denmark).
+
+Statistical Analysis
+The Kolmogorov Smirnov test was used to test for normal distribution. The results of the sum scores were compared using the Mann–Whitney U test between controls and propofol as well as between controls and sevoflurane. The place version data of the water maze test were analyzed by two-way ANOVAs on repeated measures followed by Holm-Sidak method for post-hoc multiple pair-wise comparisons. The spatial probe and the cued version data were analyzed by one-way ANOVAs followed by Holm-Sidak post-hoc tests. The data of the hole board test were analyzed by paired t-tests. Differences were considered to be significant if P < 0.05.

20230808-AI coding-1st round/3744 – Li 2021.txt

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+PMID: 33437390 PMCID: PMC7791508
+Materials and methods
+Materials and reagents
+Sevoflurane was purchased from Henry Schein, Inc. (Melville, NY, USA), and insulin (Humulin R U-100) from Eli Lily (Indianapolis, IN, USA). Primary antibodies used in this study are listed in Table 1. Peroxidase-conjugated anti-mouse and anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). The enhanced chemiluminescence (ECL) kit was from Pierce (Rockford, IL, USA). Other chemicals were from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
+Animals and animal treatments
+The breeding pairs of C57BL/6J mice were initially obtained from Jackson Laboratory (New Harbor, ME, USA). The mice were bred in our air-conditioned animal facility and housed with a 12/12 hr light/dark cycle and with ad libitum access to food and water. The housing, breeding, and animal experiments were approved by the Institutional Animal Care and Use Committee of the New York State Institute for Basic Research in Developmental Disabilities and were in accordance with the PHS Policy on Human Care and Use of Laboratory Animals (revised March 15, 2010).
+
+Induction of anesthesia was carried out by placing neonatal mice at the age of postnatal (P) days 7 in an anesthesia chamber (25 cm × 15 cm × 13 cm) filled with 5% sevoflurane in a mixture of O2 and N2 (50%/50%). The sevoflurane concentration was reduced to 2.5% after the induction period of 3 min and was maintained for 6 hr. The air flow rate was 0.9-1.0 L/min during anesthesia. A small petri dish of water was placed into the anesthesia chamber to maintain moisture. At the end of anesthesia, the sevoflurane was turned off, and the mouse pups were kept in the same chamber with O2 and N2 for one hour to allow their recovery from anesthesia. A warm pad was placed in the anesthesia chamber to maintain the body temperature of the neonatal mice to 35-36°C during the procedure. After they awakened from anesthesia, the mouse pups were returned to their parents’ cages. Neonatal mice of control groups were removed from the parents’ cages and left in the experiment room for the same periods of time as the anesthetized group.
+
+Neonatal mice received a total of 7.0 μl insulin (140 mU/mouse) or saline treatment through intranasal delivery 30 min before the beginning of anesthesia. The manual intranasal administration method was modified from that for adult mice reported previously [23]. Briefly, the P7 mouse pups were held in a supine position in hand, and 1.0 μl insulin or saline was delivered into the left nare by using a 2.5-μl Eppendorf pipette. The pups were given 15-20 sec to allow the fluid to be inhaled before repeating the administration six times.
+
+Neonatal mice (P7, both male and female) from various litters were randomly assigned into four groups: (1) control (Con) group, which received intranasal administration of saline instead of insulin and were not anesthetized; (2) sevoflurane (Sevo) group, which received intranasal saline followed by anesthesia with sevoflurane; (3) sevoflurane plus insulin (Sevo+Ins) group, which received both; and (4) control insulin (Ins) group, which received insulin but not sevoflurane. Mouse pups at the age of P7 with body weight less than 3.0 grams were excluded from the study. A total of 10 and 6 mouse pups were included in each group and time point for Western blot analyses and immunohistochemistry, respectively. To eliminate any potential bias caused by litter variations, a similar number of mouse pups from each litter was assigned to each group, and each group included pups from several litters.
+
+Western blot analysis
+The mouse pups were sacrificed by decapitation, and the forebrains were removed and homogenized in pre-chilled buffer containing 50 mM Tris-HCl (pH 7.4), 50 mM GlcNAc, 20 µM UDP, 2.0 mM EGTA, 2.0 mM Na3VO4, 50 mM NaF, 20 mM glycerophosphate, 0.5 mM AEBSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 4 µg/ml pepstatin A. Protein concentrations of the homogenates were determined by using the Pierce 660-nm Protein Assay (Rockford, IL, USA). The homogenate samples were resolved by 10% SDS-PAGE and electro-transferred onto Immobilon-P membrane (Millipore, Bedford, MA, USA). The blots were then probed with primary antibodies and developed with the corresponding horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescent kit.
+
+Immunofluorescence
+Mouse brains were immersion-fixed in 4% paraformaldehyde at 4°C for 24 hr, followed by dehydration in 30% sucrose at 4°C for 48 hr. Coronal brain sections (40-µm thick) were cut by using a freezing sliding microtome. The sections were stored in antifreeze solution, consisting of glycerol, ethylene glycol, and phosphate-buffered saline (PBS) at the ratio of 3:3:4, at -20°C till immunofluorescence staining at a later time.
+
+Coronal mouse brain sections at the same plate, as evidenced by the identical hippocampal size and structure in the sections, were chosen for immunofluorescence studies. The brain sections were first washed with PBS three times, 15 min each, followed by incubation in 0.5% Triton X-100 in PBS for 20 min. The sections were then washed with PBS for another 10 min and blocked in PBS containing 5% normal goat serum and 0.1% Triton X-100 for 30 min, followed by incubation overnight at 4°C with antibody against cleaved caspase-3. After washing with PBS again, the sections were incubated with Alexa 488-conjugated goat anti-mouse IgG (1:1000) at room temperature for 2 hr. The sections were washed for a last time, mounted, and cover-slipped by using Prolong ® gold anti-fade mountant (Invitrogen, Carlsbad, CA, USA). The immunostaining was analyzed by using a laser scanning confocal microscope (PCM 200, Nikon). The immuno-positive cells were counted manually from three sections per mouse brain and six brains per group.
+
+Statistical analysis
+The quantitative data were analyzed by one-way ANOVA plus post hoc test, if applicable, by using Graphpad. All data are presented as means ± SEM, and P < 0.05 was considered statistically significant.

20230808-AI coding-1st round/384 – Kang 2017.txt

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+PMID: 28683067 PMCID: PMC5500005 DOI: 10.1371/journal.pbio.2001246
+Methods
+Ethics
+All study protocols involving mice were approved by the Animal Care and Use Committee at the Johns Hopkins University (protocol MO14M315) and conducted in accordance with the NIH guidelines for care and use of animals.
+
+Animals
+C57BL/6 mice were housed in a temperature- and humidity-controlled room with a 12:12 hour light:dark cycle, and provided with ad libitum access to water and food. Both sexes were equally represented in all experiments. No animals were excluded.
+
+Isoflurane treatment and physiologic monitoring of sentinel animals
+P18 mouse littermates were randomly assigned to 2 groups. In Group 1 (isoflurane), mice were exposed to 1.5% isoflurane carried in 100% oxygen for 4 hours. A calibrated flowmeter was used to deliver oxygen at a flow rate of 5 L/min and an agent-specific vaporizer was used to deliver isoflurane. In Group 2 (control), mice were exposed to room air for 4 hours. Animals were returned to their cages together with their littermates upon regaining righting reflex. Mice were continually monitored and recorded for skin temperature, heart rate, and oxygen saturation during the 4-hour isoflurane treatment (PhysioSuite; Kent Scientific, Torrington, CT). Intracardiac puncture was used to collect left ventricular blood samples from selected sentinel animals, and those confirmed to be arterial are reported.
+
+Production and stereotaxic injection of engineered retroviruses
+Engineered self-inactivating murine retroviruses were used to express GFP under Ubiquitin promotor (pSUbGW vector) specifically in proliferating cells and their progeny [55,56]. High titers of engineered retroviruses (1 x 109 unit/ml) were produced by cotransfection of retroviral vectors and VSVG into HEK293gp cells followed by ultracentrifugation of viral supernatant as previously described [24,49,55–57]. After induction with a single ketamine injection (50mg/kg), high titers of GFP-expressing retroviruses were stereotaxically injected into the P15 mice dentate gyrus through a 32-gauge microsyringe (Hamilton Robotics, Reno, NV) at 2 sites of the following coordinates relative to the bregma (mm): AP: −2.2, ML: ±2.2, DV: −2.4. The retrovirus-containing solution was injected at a rate of 0.025 μl/min for a total of 0.5 μl per site. After infusion, the microsyringe was left in place for an additional 5 minutes to ensure full virus diffusion and to minimize backflow. After surgery, mice were monitored for general health every day until full recovery. In order to test for a possible confound related to the use of ketamine anesthesia, pS6 immunoreactivity in the dentate gyrus was quantified at P30 in naïve control animals and compared to pS6 immunoreactivity in animals doses with ketamine as above. No significant difference is seen in pS6 levels between these groups (S6 Fig).
+
+Immunostaining
+After transcardial perfusion fixation with 4% paraformaldehyde/PBS, brains were sliced transversely (50 μm thick) with microtome and processed for immunohistochemistry. Primary antibodies, including goat anti-GFP (Rockland, 1:1000) and chicken anti-GFP (Millipore, 1:1000) were used. Immunofluorescence was performed with a combination of Alexa Fluor 488- or Alexa Fluor 594-labeled anti-goat, anti-chicken, or anti-rabbit secondary antibodies (1:250) and 4ʹ,6ʹ-diaminodino-2-phenylindole (DAPI, 1:5000). For analysis of pS6 levels, primary antibodies against pS6-Ser235/236 (rabbit, 1:1000, Cell Signaling) were used. Effective immunostaining of pS6 required an antigen retrieval protocol as previously described [58]. Briefly, sections were incubated in target retrieval solution (DAKO) in 85°C for 20 minutes followed by washing with PBS for t3 times before the incubation with primary antibody.
+
+Imaging and analyses
+Images were acquired on a confocal system (Zeiss LSM 710 or Leica SPE) and morphological analyses were carried out as previously described [24,49,55,56,58,59]. Images for dendritic and spine morphology were deconvoluted with Auto Quant X (Media Cybernetics, Rockville, MD) using the blind algorithm, which employs an iteratively refined theoretical PSF. No further processing was performed prior to image analysis. For visualization, brightness, and contrast levels were adjusted using Image J (NIH). For analysis of dendritic development, three-dimensional (3D) reconstructions of entire dendritic processes of each GFP+ neuron were obtained from Z-series stacks of confocal images using excitation wavelength of 488 nm at high magnification (x 40 lens with 0.7x optical zoom). The two-dimensional (2D) projection images were traced with NIH Image J plugin, NeuronJ. All GFP+ DGCs with largely intact, clearly identifiable dendritic trees were analyzed for total dendritic length. The measurements did not include corrections for inclinations of dendritic process and therefore represented projected lengths. Sholl analysis for dendritic complexity was carried out by counting the number of dendrites that crossed a series of concentric circles at 10 μm intervals from the cell soma using ImageJ (NIH). For complete 3D reconstruction of spines, consecutive stacks of images were acquired using an excitation wavelength of 488 nm at high magnification (x 63 lens with 5x optical zoom) to capture the full depth of dendritic fragments (20–35 μm long, 40~70 dendritic fragments in each condition analyzed) and spines using a confocal microscope (Zeiss, Oberkochen. Germany). Confocal image stacks were deconvoluted using a blind deconvolution method (Autoquant X; Media Cybernetics, Rockville, MD). The structure of dendritic fragments and spines was traced using 3D Imaris software using a “fire” heatmap and a 2D x–y orthoslice plane to aid visualization (Bitplane, Belfast, UK). Dendritic fragments were traced using automatic filament tracer, whereas dendritic spines were traced by means of an autopath method with the semiautomatic filament tracer (diameter; min: 0.1, max: 2.0, contrast: 0.8). For spine classification, a custom MatLab (MathWorks, Natick, MA) script was used based on the algorithm; stubby: length (spine) <1.5 and max width (head)<mean_width (neck) *1.2; mushroom: max width (head) >mean width (neck) *1.2 and max_width (head) >0.3; if the spine was not classified as mushroom or stubby, it was defined as long-thin. Axonal bouton volume from axonal fragments was measured by using 3D Imaris software and using a magic wand menu (Bitplane, Belfast, UK) after deconvolution. For analysis of pS6 levels, the sections were processed in parallel and images were acquired using the identical settings, (Zeiss LSM 710, 20X lens). Fluorescence intensity was measured within the granular cell layer using ImageJ (NIH) and the value was normalized to background signal in the same image. These data were then subsequently normalized to the area of the dentate gyrus granule layer as defined by DAPI staining. All experiments were carried out in a blind fashion to experimental conditions.
+
+Behavioral tests
+Sixty-day-old mice housed in groups (5 mice per cage) were handled for at least 2 minutes per day for 3 days before the start of the behavioral experiments. All behavioral tests were performed during the light phase of the cycle between 8:00am and 6:00pm. Experimenters were blind to the samples when behavioral tests were carried out and quantified. The numbers of mice per condition are indicated in the figure legends.
+
+Object-place recognition test Object-place recognition was performed as previously described [37]. Briefly, the test was assessed in a 27.5 cm × 27.5 cm × 25 cm opaque chamber with a prominent cue on 1 of the walls. Each mouse was habituated to the chamber for 15 minutes daily for 2 days. During the training phrase, each mouse was allowed to explore 2 identical objects (glass bottle, 2.7 cm diameter, 12 cm height, and colored paper inside) for 10 minutes. The mouse was then returned to its home cage for a retention period of 24 hours. The mouse was reintroduced to the training context and presented with 1 object that stayed in the same position as during training while the other object was moved to a new position. Movement and interaction with the objects was recorded with a video camera that was mounted above the chamber and exploratory behavior was measured by a blinded observer. Exploratory behavior was defined as sniffing, licking, or touching the object while facing the object.
+Y-maze test In the Y-maze test, mice were released from the start arm (no visual cue) and allowed to habituate to only 1 out of 2 possible choice arms (overt visual cue) for 15 minutes. This was followed at 24 hours later by the recognition phrase in which the animal could choose between the 2 choice arms after being released from the start arm. The timed trials (5 minutes) were video recorded as well as graded by an observer blind to condition for total exploration time in each choice arm.
+Rapamycin treatment
+P21 mouse littermates were given IP injections of rapamycin (Sigma-Aldrich, St. Louis, MO) prepared from a stock solution (25 mg/ml in 100% ethanol, stored at -20°C) diluted to a final concentration of 4% (v/v) ethanol in the vehicle. Vehicle consisted of 5% Tween 80 (Sigma-Aldrich, St. Louis, MO) and 10% polyethylene glycol 400 (Sigma-Aldrich, St. Louis, MO) as previously described [58,60,61]. Both rapamycin- and vehicle-treated mice received the same volume for each injection (200 μl). Mice received treatments at 48 hour intervals from P21 to P29.
+
+Statistics
+Results are expressed as mean ± SEM. A one-tailed Student t test or ANOVA with Bonferroni test for intergroup comparisons were used for most statistical comparisons between groups as described in the figure legends using Prism Software (Graphpad Software Inc, La Jolla, CA). For Sholl analysis ANOVA was used at each point to test for differences between distributions. All data examined with parametric tests were determined to be normally distributed, and the criteria for statistical significance was set a priori at p < 0.05. Sample sizes were predicted based on experience from previous similar work [24]. All relevant data are available from the authors.

20230808-AI coding-1st round/3879 – Chen 2020.txt

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+PMID: 32271540 DOI: 10.1021/acschemneuro.0c00106
+Materials and Methods
+ARTICLE SECTIONSJump To
+Animals and Treatments
+Seven day old C57BL/6 male mice (Beijing Vital River Company, Beijing, China) were used in this study. The mice were bred and maintained in the animal care facility following the standard rearing conditions of 12 h light and 12 h dark. All mouse studies were performed following the guidelines established by the Institutional Animal Care and Use Committee in Quanzhou First Hospital Affiliated to Fujian Medical University (QFH2017jb43i).
+BRL-50481 (Tocris Bioscience, Bristol, United Kingdom) was dissolved in 2.5% dimethyl sulfoxide (Sigma, St. Louis, MO) with 0.9% NaCl and injected intraperitoneally into pups before subjecting them to sevoflurane, with a vehicle injection as control. Thirty minutes later, the injected pups were put into a semiclosed chamber and exposed to 3% sevoflurane for 4 h. After exposure, pups were returned to the parents’ cages and monitored for health status until the following tests.
+The pups were randomly divided into five groups as follows:
+Sham: vehicle intraperitoneal injection;
+Control: 5 mg/kg BRL-50481 intraperitoneal injection;
+B0: Sevoflurane anesthesia, vehicle intraperitoneal injection;
+B1: Sevoflurane anesthesia, 1 mg/kg BRL-50481 intraperitoneal injection;
+B5: Sevoflurane anesthesia, 5 mg/kg BRL-50481 intraperitoneal injection.
+Each group contained 10 pups.
+Morris Water Maze Test and Analysis
+The spatial memory ability of control and treated mice was determined using the Morris water maze test developed by Richard Morris. (31) In brief, a 160 cm diameter and 60 cm high circular tank was filled with water at 30 cm high. The water temperature was maintained at 22 °C. A 12 cm diameter circular platform was submerged 1 cm below the water surface in the center of one of the four virtual quadrants. (32)
+The control and treated mice were trained four times per day for 6 days. The mouse was released into the water, and it navigated to reach the platform. The maximum swimming time of the tested mouse was 80 s. If the mouse could escape to the refuge within 60 s, the delay to find the platform time was recorded as 60 s. Mice were allowed to stay on the platform for 15 s, and then they were sent to their cages under a heat lamp to maintain their core temperature. The escape latency was recorded by a tracking system, and data were analyzed using ViewPoint video tracking system (ViewPoint Behavior Technology, Civrieux, France). Three daily trials were averaged for each animal. (32)
+Immunohistochemistry (IHC) Analyses
+Mice were euthanized and perfused with cold phosphate-buffered saline and 4% paraformaldehyde immediately. The brains were fixed with 4% paraformaldehyde overnight and then cryoprotected by immersion in 30% sucrose at 4 °C for 48 h. Coronal sections (25 μm) were cut using a manual rotary microtome (Leica, Wetzlar, Germany).
+The caspase-3 IHC staining was performed as previously described. (33) The cleaved caspase-3 antibody (ab13847) was purchased from Abcam (Cambridge, MA).
+Immunoblotting Analyses
+Frozen hippocampus homogenates were lysed using radioimmunoprecipitation buffer (Bioequip, Shanghai, China). The samples were subjected to immunoblotting analysis as described previously. (33) The pCREB (Ser133, #9198, 1:1000 dilution) and CREB (#9197, 1:2000 dilution) primary antibodies were ordered from Cell Signaling Technology (Danvers, MA), and the internal control β-actin antibody was ordered from Abcam (ab8226, 1:2000 dilution).
+cAMP Concentration Assay
+cAMP levels were measured using the mouse cAMP ELISA kit (ab133051, Biocompare, South San Francisco, CA) following the manufacturer’s instructions.
+Statistical Analysis
+Statistical analyses were carried out by using the SPSS 11.0 package. Differences between groups were analyzed using analysis of variance (ANOVA) or two-sample t test with Bonferroni correction. All data represent mean ± standard deviation (SD). Statistical significance thresholds were set at *P < 0.05.

20230808-AI coding-1st round/407 – Istaphanous 2013.txt

@@ -0,0 +1,31 @@
+PMID: 23460572 DOI: 10.1213/ANE.0b013e318281e988
+METHODS
+All procedures were approved by the Institutional Animal Care and Use Committee and conformed to the guidelines for ethical treatment of animals. Efforts were made to minimize the number of animals used. Breeding pairs of male CD1 and female C57BL/6 mice were housed in a 12/12-hour light-dark cycle at 22°C with free access to food and water. This hybrid was selected because they exhibit robust anesthesia-induced apoptosis with acceptable survival.3
+
+Isoflurane Treatment
+For caspase 3 immunohistochemistry, 7-day-old CD1 and C57BL/6 hybrid littermates (n = 14) were randomly assigned to a 6-hour exposure to 1.5% isoflurane (approximately 0.6 minimum alveolar concentration in these mice) in 30% oxygen (anesthesia, n = 8) or to 6 hours in room air (control, n = 6). Immediately after treatment, animals were euthanized with an overdose of ketamine, acepromazine, and xylazine. Brains were immersion-fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4), postfixed overnight at 4°C, and cryopreserved in 25% sucrose. Brains were snap frozen and 40-μm coronal sections were cut on a cryostat (Thermo Electronics, Kalamazoo, MI). Sections were mounted to charged slides and stored at −80°C until use.
+
+For protein analyses, a separate set of animals (n = 22) was treated and euthanized as described above. The left hemispheres were cut into 4 coronal sections, frozen in liquid nitrogen, and stored at −80°C until use. At a later date, sections of neocortex around bregma −3 mm were separated with a razor blade on dry ice and then homogenized twice in cell lysis buffer solution for approximately 10 seconds each time at 4°C. The homogenate was then centrifuged at 13,000 rpm using a refrigerated microcentrifuge (Fresco centrifuge, Sorvall, Buckinghamshire, UK). The supernatant was removed and used for testing for the specific proteins.
+
+Immunohistochemistry
+Slide-mounted brain sections were blocked for 1 hour in normal goat serum, followed by incubation in rabbit antiactivated caspase 3 polyclonal antibodies (1:100, 9661L; Cell Signaling, Danvers, MA) for 18 hours at −4°C, combined with one of the following antibodies: (1) mouse anti–Neuronal Nuclei (NeuN) monoclonal antibodies (NeuN, 1:500, Chemicon, MAB377; Millipore, Billerica, MA), (2) mouse antiglutamate decarboxylase isoform 67 (antiglutamic acid decarboxylase [GAD]67, 1:2000, MAB5406; Chemicon), (3) mouse anti-S100β (1:500, CB1040; Millipore), or (4) chicken antiglial fibrillary acidic protein (GFAP) (1:500, AB5541; Chemicon). Sections were then rinsed in blocker and incubated in Alexa Fluor 488 goat antirabbit secondary antibodies (1:200, A11034, Molecular Probes Inc.; Invitrogen, Carlsbad, CA) for 4 hours at 20°C, combined with either Alexa Fluor 594 goat antimouse (1:250, A11032, Molecular Probes) or Alexa Fluor 594 goat antichicken (1:250, A11042, Molecular Probes) secondary antibodies, as appropriate for the primary antibody species. After immunostaining, sections were dehydrated in an ascending ethanol series, cleared in xylenes, and mounted with Krystalon (EMD, Gibbstown, NJ).
+
+Identification of Cellular Phenotype
+To determine the phenotype of degenerating cells, brain sections from anesthesia-treated and control animals, corresponding to Bregma −2.46 to −2.70 (figures 51–53 in the mouse brain atlas by Paxinos and Franklin11) and double-immunostained for caspase 3 and NeuN or triple stained for caspase 3, S100β, and GFAP, were examined by an observer unaware of group assignment. NeuN and S100β stains cannot be combined in the same section, because both secondary antibodies are raised in the same species.
+
+Caspase 3 immunostaining was excited using the 488-nm laser line, and emission wavelengths between 510 and 540 nm were collected to identify caspase-positive cells in layers II/III from retrosplenial cortex to piriform cortex using an SP5 confocal microscope set up on a DMI6000 stand (Leica Microsystems, Wetzlar, Germany) equipped with a 63× objective (NA 1.4). This region was selected because it has repeatedly demonstrated increased numbers of apoptotic cells in immature rodents.2,3 Immunostaining for NeuN, S100β, or GFAP was excited using the 543-nm laser line, and emission wavelengths between 600 and 650 nm were collected. Confocal optical sections were collected through the midpoint of the caspase 3–positive cell (pinhole = 1 Airy unit). Data are expressed as the percentage of caspase 3–immunoreactive cells that were also NeuN- or GFAP-positive, respectively.
+
+Quantification of Apoptotic Cells Using the Optical Dissector Method
+Further quantification of the effects of isoflurane exposure on cortical neurons and on GABAergic interneurons was performed as previously described.3,8 Briefly, confocal image stacks of caspase 3/GAD67 double labeling were collected at 1-µm increments through the entire Z-depth of the tissue (40 μm) using 1× optical zoom. Six image stacks were collected from layers II/III of visual cortex, corresponding to figures 51 to 53 in the mouse brain atlas by Paxinos and Franklin,11 from each animal, as follows: for each hemisphere, 3 adjacent confocal image stack frames were collected beginning 750 μm from the midline and moving laterally (Leica SP5, 63× 1.4 NA objective, 1-μm steps). Image stacks, which were 120 × 120 µm in dimension for NeuN and 240 × 240 µm for GAD67, because of the significantly lower cellular density for the latter stain compared with NeuN, were transferred to Neurolucida software (v7.50.4; MBF Bioscience, Williston, VT) for analysis. Using the optical dissector method, an observer unaware of group assignment quantified the respective numbers of NeuN-positive or GAD67-positive cells, the corresponding number of caspase 3–positive cells, and the number of caspase 3/GAD67 or caspase 3/NeuN double-positive cells in each field.12,13 Cells were considered positive if their fluorescence intensity was 2 times or greater than the background intensity. Counts from all 6 respective image stacks were averaged for each animal.
+
+Quantification of GAD67 and GAD65 Expression Using Competitive Enzyme-Linked Immunosorbent Assay
+We used a competitive enzyme-linked immunosorbent assay to quantify the expression of the two γ-aminobutyric acid A (GABAA) synthesizing enzymes, GAD67 and GAD65. Rat antiglutamate decarboxylase isoform 67 (Anti GAD67, 1:5000, 671-C; Alpha Diagnostics Inc., San Antonio, TX) and goat antiglutamate decarboxylase isoform 65 (Anti GAD65, 1:32,000, Ab67725; Abcam, Cambridge, MA) antibodies were incubated overnight with the homogenized cortical tissue samples. These bound antibody/antigen complexes were then added to a GAD67 or GAD65 antigen-coated well blocked with 5% bovine serum albumin. Rabbit antirat and rabbit antigoat secondary antibodies were added to GAD67 and GAD65 complexes, respectively. The secondary antibodies were covalently bound to horseradish peroxidase, an enzyme that cleaves the peroxide in the chromophore 3,3′,5,5′-tetramethylbenzidine. This enzyme activation turned on the chromophore and emitted a blue signal, which when treated with 2 M sulfuric acid turned to a yellow color, which was measured at 450 nm using a spectrophotometer (Jenway Genova Life Science Spectrophotometer; Bibby Scientific Limited, Staffordshire, UK). Absorbancy was then compared with a standard curve allowing for the determination of the isoforms’ concentrations.
+
+Statistical Analysis
+All sample sizes for group assignment were made a priori. For each animal, the total NeuN-positive cells were counted over the 6 fields. The number of caspase 3/NeuN double-positive cells was defined as an event. The data were normalized to events (caspase 3/NeuN double-positive cells) per 400 NeuN-positive cells counted, the lower end of cells encountered in each animal, to avoid extrapolation. Gross inspection of the raw data revealed that caspase 3 activation in NeuN-positive cells was a rare event with a mean incidence of 2.4% and a maximal incidence of 3.6% in the anesthesia-treated animals. This event rate met the criteria for analysis using the Poisson distribution.
+
+The Poisson mean event rate, λ, and its 95% CI were determined using the MATLAB® function [lambdahat, lambdaci] = poissfit(data, alpha). The vector “data” represented the number of events per 400 counted NeuN cells for each animal in the group of interest and α = (1 − CI). The mean event rates, λ, derived from the MATLAB function, were used to construct probability distribution function curves for the 2 groups (see Appendix).
+
+The raw event counts were used to compute the ratio of events in the anesthesia-treated group to the control group using equations 6 and 7 in Graham et al.14 This method was used as an independent means to assess the mean event ratio and to determine the 95% CI for the event ratio.
+
+All other data are presented as means ± SEM. Group comparisons were made using the Mann-Whitney U test. Statistical calculations were analyzed using Stata/IC 10.1 for Mac OS X (Stata Corp., College Station, TX). Statistical significance was accepted at P < 0.05.

20230808-AI coding-1st round/408 – Cao 2014.txt

@@ -0,0 +1,95 @@
+PMID: 25063071 DOI: 10.1016/S1995-7645(14)60066-3
+2. Materials and methods
+2.1. Animals
+A total of 80 healthy 7-day-old SD rats, male or female,
+weighing 12-18 g were selected. All animals were provided
+by XX University Experimental Animal Center, and were
+kept in a constant temperature 25 ℃, constant humidity
+40% -50% environment, and had freely drank autoclaved
+water.
+2.2. Main reagents and instruments
+Optical microscope was purchased from Japanese Nikon
+company,German Leica Microtome was purchased from
+Dalian Dajian Medical Devices Co., Ltd., Micro pipette and
+homogenizer were purchased from the German Eppendorf
+Company, -80 ℃ refrigerator were purchased from China
+Haier Company. TUNEL assay kit was purchased from Roche
+Company, IL-4, IL-1毬 and IgE radioimmunoassay kit were
+purchased from Wuhan Boster Biological Engineering Co.,
+Ltd., Ketamine (100 mg/10 mL) were purchased from Jiangsu
+Hengrui Limited Company, propofol injection (200 mg/20 mL)
+were purchased from Sichuan Shule Pharmaceutical
+Corporation. Experimental animal cages, precision electronic
+balance, 0.9% saline solution, hematoxylin, eosin staining
+solution were provided by the laboratory.
+2.3. Experimental methods
+2.3.1. Experimental animal model and grouping methods
+A total of 80 young rats were randomly divided into
+four groups (the control group, experimental group A,
+experimental group B, experimental group C) (n=20). All
+young rats received the adaptive breeding for 1 week in
+animal room. The animals in the control group received 0.9%
+saline l mL by intraperitoneal injection every 2 h, continuous
+for 3 times. The animals in experiment group A received 80 mg/kg
+ketamine l mL by intraperitoneal injection every 2 h,
+continuous for 3 times. The animals in experiment group B
+received 80 mg/kg propofol 1 mL by intraperitoneal injection
+every 2 h, continuous for 3 times. The animals in experiment
+group C received 80 mg/kg ketamine and propofol 1 mL by
+intraperitoneal injection every 2 h, continuous for 3 times.
+The injection volume was 1 mL, and if it was less than l mL it
+was supplemented by saline. Half of rats in each group were
+randomly sacrificed after 15 min of anesthesia, the other half
+underwent Morris water maze test 3 weeks later. All died
+or abandoned animals in midway were supplemented by
+modeling again.
+2.3.2. Immune parameters detection
+Using heparinization disposable 5 mL sterile syringe, 2 mL
+blood was obtained by percutaneous puncture at the point
+of maximal impulse and then it was injected into sterile EP
+tube. After 30 min at 4 ℃, it was centrifuged at 3 000 r/min at
+low temperature for 10 min. Serum was separated and stored
+at -80 ℃ for the test. Serum IL-2, IL-4 and IL-10 levels
+were detected by ELISA.
+2.3.3. Brain tissue specimen collection, preparation and
+indicators test
+After blood collection, half of the young rats were randomly
+perfusion needle was inserted to the ascending aorta from
+the left ventricle, and fixed. The right auricle was cut. It
+was washed at 4 ℃ saline by perfusion needle until the
+effluent of the right atrium was clear. Then it was fixed
+by 4% paraformaldehyde phosphate buffer. Hippocampal
+was isolated from the brain tissue when the body tissues
+and organs were hard, they were paraffin-embedded
+and cut. Neuronal apoptosis detection was performed by
+terminal deoxynucleotidyl transferase-mediated nick end
+labeling (TUNEL) method. TUNEL-positive cells showed
+brown particles in the nucleus. Six horizons were randomly
+selected and average optical density was measured.
+Positive intensity and the apoptotic index were calculated.
+The formula was as follow: apoptotic index (AI) = MOD ×
+Area% × 100, MOD represents the average gray level; area%
+represents the percentage of the total positive nucleus area
+in the total nucleus area. The other half young rats cerebral
+was obtained quickly by sterile opening cranium, and brain
+tissue was mixed with ice normal saline by homogenizer.
+10% brain homogenate was prepared at 4 ℃, and centrifuged
+at 3 000 r/min for 15 min. The supernatant was stored at
+-80 ℃ for test. Whole brain IL-1毬 levels were detected by
+ELISA.
+2.3.4. Morris water maze test
+Behavior of rats was observed by Morris water maze[3].
+Round tank has four quadrants. A black platform was fixed
+at the fourth quadrant, located 1 cm underwater. The rats
+were put into the water of a randomly select quadrant,
+swim tracks of the rats were recorded with a camera. How
+long rats find the platform is the latency. After this test, the
+platform was removed and the rats were put into water from
+the same water-entering point, the times of crossing the
+former platform were measured.
+2.4. Statistical analysis
+Data were expressed as mean依SD values and analyzed with
+SPSS 13.0 software. After the variance test, the difference
+between two groups was compared with single factor analysis
+of variance. P<0.05 was considered as statistical significant
+difference.

20230808-AI coding-1st round/415 – Ju 2020.txt

@@ -0,0 +1,22 @@
+PMID: 32047423 PMCID: PMC6997293 DOI: 10.3389/fncel.2020.00004
+Materials and Methods
+Animals
+All experiments were approved by the relevant Committees of Chungnam National University, Daejeon, South Korea (CNU-01135). C57BL/6J mice were maintained in a specific pathogen-free (SPF) room maintained at 22°C, with a 12 h light/dark cycle, and fed ad libitum. Animals received anesthesia during the light cycle. This research adheres to the ARRIVE (Animal Research: Reporting in vivo Experiments) guidelines.
+
+Anesthesia
+PND 16/17 mice were randomly divided into three groups: control, sevoflurane, and sevoflurane plus rapamycin groups. Mice in the sevoflurane and sevoflurane plus rapamycin groups were placed in a 1-l plastic chamber and exposed to a constant flow of fresh gas [fraction of inspired oxygen (FiO2) 0.4, 4 L/min] containing 2.5% sevoflurane for 2 h. Full recovery was confirmed 30 min after discontinuing sevoflurane. Control mice were treated identically but without sevoflurane. The anesthesia chamber was placed in a 36°C water bath to maintain a constant temperature. Carbon dioxide and sevoflurane were monitored using an S/5 compact anesthetic monitor and a mCAiO gas analyzer module (Datex-Ohmeda, Helsinki, Finland).
+
+Rapamycin Treatment
+Rapamycin (LC Laboratories, Woburn, MA, USA) was reconstituted in ethanol at a concentration 10 μg/μl and then diluted in 5% Tween-80 (Sigma–Aldrich, St. Louis, MO, USA) and 5% PEG-400 (Sigma–Aldrich, St. Louis, MO, USA), as described (Chen et al., 2009). Mice in the sevoflurane plus rapamycin group were each administered three intraperitoneal injections of rapamycin (5 mg/kg) at 24 h intervals prior to sevoflurane exposure, whereas mice in the control and sevoflurane groups were injected with an identical volume of vehicle.
+
+Western blotting
+Whole-brain samples were obtained from the mice 24 h after sevoflurane exposure. Mice were exposed to carbon dioxide before brain extraction, and each whole brain was homogenized with a tissue grinder in RIPA lysis buffer [ELPIS-BIOTECH, Daejeon, South Korea, 100 mM Tris–hydrochloride (pH 8.5), 200 mM NaCl, 5 mM EDTA, and 0.2% sodium dodecyl sulfate], containing phosphatase and protease inhibitor cocktails (Sigma–Aldrich). After centrifuging the homogenized samples at 12,000× g for 15 min at 4°C, the supernatants were decanted and their protein concentrations were measured using the Bradford assay (Bio-Rad, Hercules, CA, USA). Samples (20 μg) were electrophoresed on SDS PAGE gels, and transferred to nitrocellulose membranes (pore size, 0.2 μm; Amersham Protran®, GE Healthcare, Buckinghamshire, UK) at 200 mA for 2 h. The membranes were blocked for 1 h with Tris-buffered saline-Tween 20 [10 mM Tris–hydrochloride (pH 7.6), 150 mM NaCl, and 0.1% Tween 20], containing 3% bovine serum albumin (BSA), followed by incubation with primary antibodies and the appropriate secondary antibodies coupled to horseradish peroxidase. Specific antibody-labeled proteins were detected using the enhanced chemiluminescence system (WEST-ZOL plus; iNtRON BioTechnology, Seongnam, South Korea). Primary antibodies included antibodies to phospho-mTOR(S2448), mTOR (Cell Signaling Technology, Danvers, MA, USA), postsynaptic density 90 (PSD95; Neuromab, Davis, CA, USA), GAD65 (Abcam, Cambridge, UK), NDUFB8 (a mitochondrial complex I subunit; Santa Cruz Biotechnology, Santa Cruz, TX, USA), COX4 (a mitochondrial complex IV subunit; Novus Biologicals, Centennial, CO, USA) and actin (Santa Cruz Biotechnology, Santa Cruz, TX, USA). Antibodies against GluA1 (1193) and GluA2 (1195) have been described previously (Kim et al., 2009).
+
+Oxygen Consumption Rate
+Mitochondria were isolated from brain tissues 24 h after sevoflurane exposure, as previously described (Chung et al., 2017a). Each brain was homogenized in a mitochondrial isolation buffer [70 mM sucrose, 210 mM mannitol, 5 mM HEPES, 1 mM EGTA, and 0.5% (w/v) fatty acid–free BSA (pH 7.2)] with a Teflon-glass homogenizer (Thomas Fisher Scientific, Swedesboro, NJ, USA). After centrifugation at 600× g for 10 min at 4°C and at 17,000× g for 10 min at 4°C, the mitochondrial fraction was resuspended in a mitochondrial isolation buffer. Protein concentration was measured by the Bradford assay (Bio-Rad), and 20 μg aliquots of protein were diluted with 50 μl mitochondrial assay solution [70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, 0.2% (w/v) fatty acid–free BSA, 10 mM succinate, and 2 μM rotenone (pH 7.2)] and seeded in an XF-24 plate (Seahorse Bioscience, North Billerica, MA, USA). The plates were centrifuged at 2,000× g for 20 min at 4°C using a swinging bucket microplate adaptor (Eppendorf, Hamburg, Germany); 450 μl mitochondrial assay buffer was added to each plate, and the plates were maintained at 37°C for 8–10 min. Each plate was transferred to a Seahorse XF-24 extracellular flux analyzer (Seahorse Bioscience) and the oxygen consumption rate (OCR) was measured at five stages: stage I (basal level); stage II, following the addition of adenosine diphosphate (ADP); stage III, following the addition of oligomycin, a mitochondrial oxidative phosphorylation (OXPHOS) complex 5 inhibitor; stage IV, following the addition of carbonyl cyanide m-chlorophenyl hydrazine (CCCP), a mitochondrial OXPHOS complex 4 inhibitor; and stage V, following the addition of antimycin A, a mitochondrial OXPHOS complex 3 inhibitor. OCR was automatically calculated and recorded using Seahorse XF-24 software (Seahorse Bioscience).
+
+Electrophysiology
+Whole-cell voltage-clamp recordings of pyramidal neurons in the CA1 region of the hippocampus were obtained as described (Chung et al., 2015a). Twenty-four hours after exposure to sevoflurane or fresh gas, sagittal slices of the hippocampus (300 μm) were prepared in ice-cold dissection buffer (212 mM sucrose, 25 mM NaHCO3, 5 mM KCl, 1.25 mM NaH2PO4, 10 mM d-glucose, 2 mM sodium pyruvate, 1.2 mM sodium ascorbate, 3.5 mM MgCl2, and 0.5 mM CaCl2) aerated with 95% O2/5% CO2, using a VT1200S vibratome (Leica, Arrau, Switzerland). Slices were transferred immediately to a 32°C chamber containing artificial cerebrospinal fluid (aCSF: 125 mM NaCl, 25 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 10 mM d-glucose, 1.3 mM MgCl2, and 2.5 mM CaCl2, continuously aerated with 95% O2/5% CO2) and incubated for 30 min. Glass capillaries were filled with two kinds of internal solutions. For miniature excitatory postsynaptic current (mEPSC) recordings, the glass capillaries were filled with an internal solution containing 117 mM CsMeSO4, 10 mM tetraethylammonium chloride, 8 mM NaCl, 10 mM HEPES, 5 mM QX-314-Cl, 4 mM Mg-adenosine triphosphate (ATP), 0.3 mM Na-guanosine triphosphate, and 10 mM EGTA; for miniature inhibitory postsynaptic current (mIPSC) recordings, the glass capillaries were filled with an internal solution containing 115 mM CsCl, 10 mM tetraethylammonium chloride, 8 mM NaCl, 10 mM HEPES, 5 mM QX-314-Cl, 4 mM Mg-ATP, 0.3 mM Na-guanosine triphosphate, and 10 mM EGTA. Whole-cell recordings were performed under visual control (BX50WI; Olympus, Tokyo, Japan) with a multi clamp 700A amplifier (Molecular Devices, San Jose, CA, USA). Data were acquired with Clampex 9.2 (Molecular Devices, San Jose, CA, USA) and analyzed using Clampfit 9 software (Molecular Devices, San Jose, CA, USA).
+
+Statistical Analysis
+The sample size was determined based on previous experience or as previously described (Chung et al., 2015b, 2017b). All statistical analyses were performed using R statistical software (3.1.2: R Core Team, Austria). All continuous variables were tested to determine whether they met conditions of normality and homogeneity of variance. One-way ANOVA with post hoc Tukey HSD test was performed when both conditions were met, Welch’s ANOVA with post hoc Tukey HSD test was performed when homogeneity of variance was unmet, and the Kruskal–Wallis test with post hoc Dunn’s test was performed if normality was unmet. P < 0.05 was considered statistically significant. Statistical results are presented as Supplementary Statistics.

20230808-AI coding-1st round/4315 – Ling 2017.txt

@@ -0,0 +1,14 @@
+PMID: 29042986 PMCID: PMC5639422 DOI: 10.3892/etm.2017.5004
+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 polypropylene 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 anesthesia, 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 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 experimental 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.
+Gene expression microarray analysis Rats were anaesthetized by isoflurane with an induction dosage of 4%, maintained at 2% (RuiTaibio, Beijing, China) and decapitated 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 isopropanol 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 expression, 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 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), differentially expressed genes were identified through FC filtering according to the predetermined P-value threshold for significant differences (set at P<0.05).
+
+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′-ATTTTCTCTCATTGACCTGGCTG-3′, R 5′-ACTCCTTGAGTGCTAAAAGGC-3′; RYBP: F 5′-CGACCAGGCCAAAAAGACAAG-3′, R 5′-CACATCGCAGATGCTGCAT-3′; DOCK7: F 5′-CCATCTGGAAGCGCCTTTG-3′, R 5′-ACGATGATCTCTAGCGTGTCT-3′; CDC40: F 5′-CTCTAGCTGCTTCGTATGGCT-3′, R 5′-CAAGTGCATGAGAGAGTCCGC-3′; PTBP3: F 5′-CCAGCCATTGGATTTCCTCAA-3′, R 5′-AAAAAGCCCATGTGGTGTGATA-3′; NRTN: F 5′-GGGCTACACGTCGGATGAG-3′, R 5′-CCAGGTCGTAGATGCGGATG-3′; TMEM205: F 5′-CACTTGCTGGTCTTGTCTGGT-3′, R 5′-GGAGACGTGAAAATAGACTGGG-3′; SLC39A3: F 5′-GGTGGCGTATTCCTGGCTAC-3′, R 5′-CTGCTCCACGAACACAGTGA-3′; CDCA3: F 5′-GAGTAGCAGACCCTCGTTCAC-3′, R 5′-TCTCTACCTGAATAGGAGTGCG-3′; KIF1C: F 5′-AGTGTGGGTTTGTGTGTATGAG-3′, R 5′-CCAGCATCGCACCATGTAGA-3′; CAS P3: F 5′-ATGGAGAACAACAAAACCTCAGT-3′, R 5′-TTGCTCCCATGTATGGTCTTTAC-3′; GAP DH: F 5′-AGGTCGGTGTGAACGGATTTG3′, R 5′-TGTAGACCATGTAGTTGAGGTCA-3′.
+Immunohistochemical assay Following sacrifice, the entire rat brain was rapidly removed, washed with phosphate-buffered 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 hippocampus 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 visualized 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).
+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-polyacrylamide 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 temperature 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.

20230808-AI coding-1st round/435 – Li 2019.txt

@@ -0,0 +1,30 @@
+PMID: 31263401 PMCID: PMC6585163 DOI: 10.3389/fncel.2019.00251
+Materials and Methods
+Animal Protocols
+We performed all the experimental protocols according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23). The animal procedures were approved by the Animal Care and Use Committee of Xi’an Jiaotong University and designed to minimize the number and suffering of rats used. PND 7 and embryonic day 18–19 Sprague-Dawley rats were obtained from Laboratory Animal Centre of Xi’an Jiaotong University.
+
+Morris Water Maze
+The spatial learning and memory function of rats after ketamine exposure were tested by MWM experiments as described in a previous study (Shen et al., 2013). Specifically, PND 42–47 rats (n = 10 per group) were trained for place trials and spatial probe tests in a large tank (diameter: 150 cm, depth: 60 cm), which was filled to a depth of 32 cm of warm water (maintained around 25 ± 1°C) and divided into four quadrants. A platform (diameter: 12 cm, height: 30 cm) was placed in the center of the third quadrant (the target) and submerged approximately 2 cm beneath the water surface. We poured milk powder into the water to make the water opaque. We conducted the place trials at PND 42–46 with 4 trials daily at the same time point and performed the probe trials on PND 47 after 5 days’ training. The swimming of rats during the tests was recorded by a video tracking system installed above the tank. In place trials, rats were placed into four quadrants (spaced 20 min apart) to swim freely for a maximum of 120 s. If the rats could not find the platform within 120 s, they were allowed to stay on the platform for 20 s to observe the environment by guiding. The time for rats to reach the platform and swimming speed were recorded. In probes trials, the platform was removed and the rats were put into the first quadrant and allowed to swim for 120 s. The times of rats crossing the original platform were recorded.
+
+Anesthetic Exposure in vivo and Tissue Preparation
+The PND 7 rats, weighing 13–18 g, were housed with their mother and maintained at a temperature of 24°C in a 12 h/12 h light/dark cycle with free access to food and water. We assigned the rats randomly into three groups (28 rats from 7 nests in each group, 4 pups per nest): (i) the rats in control group received equal volume of normal saline by intraperitoneal injection as ketamine solution at corresponding time points; (ii) the rats in ketamine group received 40 mg/kg ketamine, diluted in normal saline and administrated by intraperitoneal injection (ketamine, Sigma–Aldrich Inc. St. Louis, MO, United States), the initial injection was considered to be the loading dose, 30% of it was injected at approximately 40 min intervals to maintain the anesthesia for 4 h (Lu et al., 2017); (iii) the rats in the 17β-estradiol group received 17β-estradiol (17β-estradiol, Tocris, Minneapolis, MN, United States; DMSO, Sigma–Aldrich, St Louis, MO, United States) dissolved in dimethylsulfoxide (DMSO) at a concentration of 100 ug/ml, 100 ug/kg 17β-estradiol administered intraperitoneally 8 h, 1 h prior to and 3 h after ketamine’s initial injection (Liu et al., 2007). During anesthesia, all pups were kept on an electric blanket with the temperature set at 36.5 ± 1°C to maintain body temperature and reduce stress. We observed the respiratory rate, skin color, and body movement of rats carefully and tested the voluntary movement by clamping the pup tails. Pulse oxygen saturation (SpO2) was detected by attaching the infant pulse oximetry probes to the rat abdomen. After the anesthesia, the pups received BrdU (50 mg/kg, intraperitoneal injection) every 24 h for 7 consecutive days. On PND 14, the rats were decapitated and the brain tissues of SVZ and SGZ were harvested to detect neurogenesis.
+
+At 12 h after anesthesia, rat pups (n = 6 per group, captured randomly) were sacrificed by decapitation. Both the brain tissue from SVZ and SGZ were isolated immediately on ice and the stored at −80°C until use for western blotting. The rats (n = 6 per group, captured randomly) were sacrificed and perfused transcardially with 0.9% saline 7 days after anesthesia, followed by cold 4% paraformaldehyde in PBS. Then the harvested brains were postfixed in 4% paraformaldehyde overnight at 4°C and dehydrated in 30% sucrose solution for 3–4 days, as we described previously (Lu et al., 2017). The brain tissue from bregma +0.2 mm to bregma −6.0 mm was the region of interest, which were cut into 16 μm coronary tissue slices by freezing microtome (SLEE, Germany). These brain slices were collected and used for future immunohistochemistry staining. The rest of the rat pups (n = 10 per group) were bred for behavior study at adulthood.
+
+Immunohistochemistry
+Immunohistochemistry was used to evaluate NSC proliferation in SVZ and SGZ by BrdU staining. Firstly, the brain slices were incubated with 2 N HCl for 30 min to denaturate the DNA at 37°C. After being incubated with 0.1 mol L−1 boric acid (pH 8.5) for 10 min at room temperature followed by three times washing with 0.1 M PBS, the slices were blocked by 2% goat serum and 0.3% Triton X-100 for 2 h at room temperature, then incubated with the mouse monoclonal anti-BrdU antibody (1:200, Abcam, United Kingdom) at 4°C overnight. The next day, after three washings with 0.1 M PBS, the slices were incubated with tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibodies for 2 h at room temperature. BrdU-positive cells were counted within defined regions of interest in the SVZ and SGZ. In total, the mean numbers of BrdU-positive cells of six brain slices for each rat, spaced approximately 200 μm apart, were examined by the observer blindly. For each slice, five regions were captured by fluorescence microscopy (BX51, Olympus, Tokyo, Japan), and the planar area enclosed by each region was 50 × 50 μm. The edges of the captured regions were defined according to structural details to ensure the fields did not overlap (Zhang et al., 2009). The density of positive cells was presented as the total number of BrdU-positive cells in the SVZ and SGZ.
+
+NSC Culture
+Primary cultured NSCs were obtained from the cortex of rat at embryonic day 18–19 under sterile conditions. Briefly, the forebrain portion was isolated and placed in ice-cold Hank’s solution (without Mg2+ and Ca2+, Gibco, Carlsbad, CA, United States). The tissues were then dissociated and triturated mechanically by a fire-polished Pasteur pipette softly. After centrifugation, the isolated cells were collected and re-suspended in free-serum DMEM/F12 medium (Gibco, Carlsbad, CA, United States) which was supplemented with 2% B27 (Gibco, Carlsbad, CA, United States), 20 ng/ml EGF (Gibco, Carlsbad, CA, United States), 20 ng/ml bFGF (Gibco, Carlsbad, CA, United States), and 100 U/ml penicillin and phytomycin. Cells were cultured for 7 days to form enough neurospheres and then passaged at a density of 2 × 105 cells/ml followed by collection and dissociation as previously described by Reynolds and Weiss (Reynolds et al., 1992). Half of the medium was changed every 3 days. After the second passage, the cells were ready for future experiments. For identification assessment, the cells after passage were seeded onto 100 μg/mL poly-L-lysine-coated coverslips and cultured in differentiating medium that contained 100× N2 supplement, 100× B27 supplement, and 1% fetal bovine serum (FBS, Gibco, Carlsbad, CA, United States) in DMEM/F12 (without b-FGF) for 7 days.
+
+Drug Exposure and Neurogenesis Analysis in vitro
+The cells were assigned to the following groups: control group, ketamine group, and 17β-estradiol group. No drug treatment was added to the control group. NSCs in the ketamine group were exposed to 100 μM ketamine for 24 h. NSCs in the 17β-estradiol group were pretreated with 17β-estradiol (100 nM) for 30 min and then 100 μM of ketamine was added to the culture medium for 24 h. For proliferative analysis, NSCs were seeded on cover slips which were pre-coated with 100 μg/mL poly-L-lysine and incubated with BrdU for the last 4 h. Following being fixed with 4% paraformaldehyde, the cells were stained with BrdU antibody (1:200, Abcam, United Kingdom) and DAPI. As for neuronal differentiation analysis, after being exposed to ketamine with or without 17β-estradiol for 24 h, the cells were seeded on cover slips which were pre-coated with 100 μg/mL poly-L-lysine and incubated with differentiating medium for 7 days, then the cells were harvested for immunohistochemical staining. The cells were labeled with β-tubulin III antibody (1:500; Sigma-Aldrich Inc. St. Louis, MO, United States). Briefly, 5–7 randomly selected fields were captured in each coverslip, and the numbers of β-tubulin III-positive cells were counted (at least 200 cells per test case). Data were collected from three independent experiments.
+
+Cell Apoptosis Test
+We used terminal dUTP nick-end Labeling (TUNEL) assay to detect cell apoptosis. Briefly, after passage, the dissociated cells were exposed to ketamine with or without 17β-estradiol for 24 h. After the treatments, cells were fixed with 4% paraformaldehyde for 15 min. The TUNEL assay was performed according to the instruction of in situ Cell Death Detection Kit (Roche Inc. Roche, Mannheim, Germany). Data were collected from three independent experiments.
+
+Western Blot Analysis
+Brain tissues from the SVZ and SGZ of rats (n = 6 per group) at 12 h after anesthesia and cell cultures at 24 h following drug exposure were subjected to Western blot analyses as described in our previous studies (Lu et al., 2017). Briefly, the tissues were lysed by RIPA lysis buffer with protease and phosphatase inhibitors. The lysates were homogenized with an electric homogenizer and maintained on ice for 15 min. After being centrifuged for 15 min at 14000 rpm at 4°C, the supernatant was aspirated and the resulting lysates were placed in a new tube. We used the BCA protein assay kit to examine the protein concentrations. Bovine serum albumin (BSA) was used as a standard. An equal amount of the resulting lysate was resolved by sodium dodecyl sulfate-polyacrylamide gel and the separated proteins were transferred to polyvinylidene fluoride membranes. After being blocked for 1 h at room temperature, the membranes were then incubated with appropriate dilutions of primary antibodies at 4°C overnight. The used antibodies included anti-caspase-3 (cleaved, 17 KDa, 1:1000, Cell Signal Technology Inc. Beverly, MA, United States), anti-phosophorylated GSK-3β (p-GSK-3β, 1:1000, Cell Signal Technology Inc. Beverly, MA, United States), anti-GSK-3β (1:1000, Cell Signal Technology Inc. Beverly, MA, United States), and anti-β-actin (1:1000, Cell Signal Technology Inc., Beverly, MA, United States). The following day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit or anti-mouse) for 2 h at room temperature. After being enhanced by chemiluminescence (ECL), the signals were then exposed to X-ray films. Each band in the Western blot represented an independent experiment and at least three independent experiments were conducted. Data were expressed as the ratio to optical density (OD) values of the corresponding controls. The Western blots were quantified as described in our previous study.
+
+Statistical Analysis
+Data obtained from the study were presented as mean ± SEM. Every data point represented a mean for each animal in a single case. SigmaPlot 12.0 was used for all statistical analysis. Data were tested and then confirmed with normality and equal variance criteria. A one-way analysis of variance (ANOVA) following the post hoc Holm-Sidak method was used to analyze the differences among different groups. A two-tailed probability value P < 0.05 was considered statistically significant.

20230808-AI coding-1st round/437 – Chen 2018.txt

@@ -0,0 +1,27 @@
+PMID: 29427282 DOI: 10.1007/s12640-018-9877-3
+Methods
+Ethical Approval
+The use of rats in this study was approved by the Institutional Animal Care and Use Committee at Sun Yat-sen University (Guangzhou, China). All experiments were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and ARRIVE guidelines. Sprague-Dawley multiparous dams (n = 31) with litters containing male pups (n = 135) were purchased from Experimental Animal Center of Sun Yat-sen University, China. We only used male offspring to exclude the influence of estrogen on the biochemical data and neurocognitive functions. The pups from postnatal day 0 (P0) to P20 were housed with the dams in a 12-h:12-h light:dark cycle (light from 07:00 to 19:00), and room temperature (RT) was maintained at 21 ± 1 °C. On P21, the pups were weaned and housed 4–6 per cage in a standard environment.
+
+Anesthesia
+SD rats at P7 (weight 14–16 g) were randomly divided into the air-treated control (C group), the 1.2% sevoflurane-exposed (1.2% sevo group), and the 2.4% sevoflurane-exposed (2.4% sevo group). Rats in the 1.2% sevo group and the 2.4% sevo group were placed in a plastic container and exposed to 1.2 or 2.4% sevoflurane continuously for 6 h, using air as a carrier, with a total gas flow of 2 L min−1. A nasopharyngeal airway tube was put in their mouth to prevent apnea and hypoxia when the rats stopped moving in the container. During exposure, the temperature inside the container was maintained at 30 °C using an external heating device (NPS-A3 heating device, Midea Co., Guangdong, China) and a hot water bag on the bottom of the container with a constant temperature maintained between 30 and 35 °C. The concentrations of sevoflurane, oxygen, and carbon dioxide in the chamber were monitored by a gas monitor (Detex-Ohmeda, Louisville, CO, USA). During exposure, an investigator monitored the rats’ spontaneous respiratory frequency and skin color every 5 min to detect any apnea or hypoxia. The rats were immediately exposed to air and excluded from the experiment if these symptoms were detected. Sevoflurane administration was terminated 6 h later, and the rats were exposed only to air. When the rats were moving freely again, they were placed back into their maternal cages. Rats in the C group were exposed to the same container as the rats in the 1.2% sevo and 2.4% sevo group but were exposed to air alone for 6 h.
+
+Arterial Blood Gas Analysis
+We performed arterial blood analysis in order to exclude the influence of respiratory or metabolic disorder. The arterial blood samples from the C, 1.2% sevo, and 2.4% sevo groups were obtained from the left cardiac ventricle immediately after removal from the maternal cage (n = 5 in each group) at the end of anesthesia. They were analyzed immediately after collection using a blood gas analyzer (Gem Premier 3000, US). We analyzed the pH, arterial carbon dioxide tension (PaCO2), arterial oxygen tension (PaO2), and blood glucose levels of the arterial blood samples.
+
+Morris Water Maze Test
+On P35, the rats were tested for spatial learning and memory ability using the Morris water maze (MWM). Three groups of rats (n = 10 in each group, weight 90–100 g) were tested on the MWM, which consists of two different tests including hidden platform acquisition and a probe trial test, at P35–P40 using the Water Maze Tracking System (MT-200; Chengdu, China). A white platform (12 cm diameter) was submerged in a circular pool (160 cm in diameter, 50 cm in height) that was filled with warm water (23 ± 2 °C). The pool, located in a room with no windows, was virtually divided into four quadrants. A video camera connected to the computer running the tracking software was suspended above the pool and captured the rats’ movements for analysis. At P35, before the test, a single habituation trial was performed without the platform; in this trial, the rats were placed in the water for 120 s. In the hidden platform acquisition test, performed at P36–P39, each rat was placed, facing the wall of the pool, in one of the four quadrants and allowed to swim freely in search of the escape platform for a maximum of 120 s. The experiment was repeated with four trials per day for four consecutive days. The average escape latency time (latency to reach the platform) was measured to evaluate spatial learning ability. At P40, a probe trial test was performed by removing the platform and releasing the rats into the water for 120 s. We calculated the time spent in the quadrant that previously contained the target and the frequency of crossing the former location of the platform. The rats were dried and placed back into a heated cage after completing each test.
+
+Western Blot Analysis
+On P10 and 28, rats (n = 5 in each group at each sacrifice time point) were sacrificed by rapid decapitation, and the bilateral hippocampus areas were harvested and stored at − 80 °C until use. Protein was extracted using RIPA lysis buffer (Keygen Biotech, Nanjing, China). The amount of protein in each hippocampal tissues was measured using a protein assay kit (BCA, Pierce, Thermo, USA). Polyacrylamide-SDS gels with an equal amount of 50-μg load in each lane were electrophoresed, and the proteins transferred onto PVDF membranes (Millipore, Carrigtwohill, Ireland). The blots were blocked with 5% skim milk in Tris-buffered saline (150 mM NaCl, 0.1% TWEEN 20, 20 mM Tris, pH 7.4) for 1 h and then incubated overnight at 4 °C with anti-BDNF (1:1000, Novusbio, USA), anti-TrkB (1:800, Millipore, Ireland), anti-postsynaptic density (PSD-95) (1:2000, Abcam, England), and anti-synaptophysin (1:20,000, Abcam, England) primary antibodies. After rinsing, membranes were probed with corresponding secondary antibodies at RT for 2 h. Immunoreactive bands were detected with an enhanced chemiluminescence detection system (Bio-Rad, USA). A β-actin antibody (1:1000, ABclonal, China) was used to normalize for sample loading and transfer. The intensities of the bands were densitometrically quantified using ImageJ.
+
+BrdU Injections and Immunofluorescence
+For the 5′-bromo-2-deoxyuridine (BrdU) injections, we followed the methods as previously described (Chen et al. 2015; Tozuka et al. 2005). BrdU has been described as a marker of neurogenesis and can incorporate into DNA only during the S-phase of the mitotic process (Kee et al. 2002). BrdU (Sigma, America) was dissolved in normal saline (10 mg mL−1) and injected at a dosage of 300 mg/kg. To investigate the effects of 1.2% sevoflurane on cellular proliferation, we performed a single injection of BrdU i.p. 24 h after sevoflurane exposure. Three days later, the rats were perfused, and their brains were processed for immunofluorescence. To investigate the effects of 1.2% sevoflurane on the survival of newborn cells, we performed a single injection of BrdU i.p. 24 h before sevoflurane exposure. Four weeks (28 days) after the BrdU injection, the rats were perfused, and their brains were processed for immunofluorescence.
+
+For morphological examination, rats were deeply anesthetized with chloral hydrate at P10 and P35 (n = 5 in each group at each sacrifice time point, 80–100 g) and then transcardially perfused with 0.9% normal saline at RT followed by a fixative solution of 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M PBS (pH 7.4) at 4 °C. The brains were harvested, postfixed in 4% paraformaldehyde for 8 h, and subsequently soaked in 30% sucrose until they sank. Consecutive frozen coronal sections of the hippocampus were cut at a thickness of 30 μm. Every fifth section of the consecutive sections was processed by BrdU staining. DNA was first denatured by incubation with 2 N HCl for 30 min at 37 °C followed by a 15-min wash in 0.1 M boric acid (pH 8.5), with three 10-min washes in 0.01 M PBS before each step. The sections were blocked in 3% BSA and 0.4% Triton X-100 for 2 h at RT before being incubated with primary antibody (rat anti-BrdU, 1:200, ab6326, Abcam, UK) in 1% BSA overnight at 4 °C. Then, the sections were incubated with secondary antibody (Cy3 goat anti-rat IgG, 1:200, KGAB018, Keygentee, China) for 2 h at RT. Fluorescence was detected with a fully automatic fluorescence microscope (Olympus BX63, Japan). An observer who was blinded to group assignment was responsible for counting the number of BrdU-positive cells at × 200 magnification. Total cell counts were divided by the total number of sections for analysis.
+
+Transmission Electron Microscopy
+TEM was used to assess synaptic plasticity in the hippocampus after exposure to treatment (n = 5 in each group) at P35. Twenty-eight days after exposure to treatment, the rats were perfused transcardially with 50 mL of 0.9% normal saline, followed by 50 mL of a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde (Sigma-Aldrich, G6257, USA) in 0.1 M PBS. Approximately 1 mm3 of tissue per rat was dissected from the hippocampus and fixed in 2% glutaraldehyde for 2 h at 4 °C. The tissues were rinsed in 0.1 M cacodylate buffer and postfixed with 1% osmium tetroxide for 2 h. Then, the tissue was rinsed with distilled water before undergoing dehydration in a graded ethanol series. Subsequently, the tissue was infiltrated overnight at 4 °C using a mixture of half acetone and half resin. The tissue was embedded in resin 24 h later and then cured fully as follows: 37 °C overnight, 45 °C for 12 h, and 60 °C for 24 h. After that, 70-nm sections were cut and stained with 3% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Ultrastructural changes in synapses in the hippocampus were observed under TEM. Five pictures of each subregion per ultrathin section (five rats in total per group) were taken at each of two magnifications: × 13,500 and × 37,000. All pictures taken at × 13,500 magnification were used to observe the number of synapses, and all pictures taken at × 37,000 magnification were used to measure the thickness of the postsynaptic density and the width of the synaptic cleft. The number of synapses was expressed as the average number of synapses in each picture taken at × 13,500. The thickness of the postsynaptic density and the width of the synaptic were expressed as the average values for all synapses in all pictures taken at × 37,000, as described. We measured the distances using the image analysis software ImageJ.
+
+Statistical Analysis
+The results were expressed as the mean ± standard deviation (SD) for each group. The statistical tests were conducted using the computerized statistical package SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism Software version 5.0 (GraphPad Software, Inc., San Diego, CA, USA). The arterial blood data were analyzed using Student’s t test. One-way ANOVA was used to evaluate differences in the quantities of hippocampal proteins, numbers of BrdU-positive cells and synapses, and ultrastructure parameters of synapses among groups. Unpaired t tests and two-way ANOVA were used to analyze the results of the MWM. Each experiment was performed at least three times. A value of P < 0.05 was considered statistically significant.

20230808-AI coding-1st round/460 – Liu 2015.txt

@@ -0,0 +1,34 @@
+PMID: 25663300 DOI: 10.1007/s11064-015-1529-x
+Materials and Methods
+Chemicals and Reagents for Metabolomic Analysis
+Methanol (pesticide residue grade), bis-(trimethylsilyl)-trifluoroacetamide (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 (internal 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 University. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee, Fudan University. According to the flow chart of the experimental 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 °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 °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 performed 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 (Radiometer, Copenhagen, Denmark).
+
+GC–MS Analysis
+Prior to metabolic profiling, frontal cortex (n = 6/group) were homogenized with 50 μL L-2-chlorophenylalanine in a 2-mL centrifuge tube. Then, 0.4 mL methanol-chloroform (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 °C and 400 μL 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 temperature of 50 °C for approximately 30 min using the TurboVap nitrogen evaporator (Caliper Life Science, Hopkinton, MA). Afterward, 100 µL of anhydrous toluene (stored with sodium sulfate) was added to each of the dried tissues. Following 1 min of vortex-mixing, the samples 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 μL MOX reagent was added to the dried samples, vortex-mixed for 2 min, and incubated at 37 °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 μL of BSTFA (with 1 % TMCS) to each sample, vortex-mixed for 2 min, and incubated at 70 °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 μm × 250-μm inner diameter, 0.25-μm film thickness; J&W Scientific, Folsom, CA, USA). A 1-μL 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 °C for 2 min, then raised to 240 °C at a rate of 5 °C/min, and finally to 290 °C at a rate of 10 °C/min for 11 min. The injection, transfer line, and ion source temperatures were 280, 270, and 220 °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, http://www.nist.gov/index.html) and Fiehn databases. The peaks with similarity index of more than 70 % were selected and named the putative metabolite identities.
+
+Multivariate Data Analysis
+The resulting GC–MS data were first processed by normalizing peak area of each analyte based on total integral area calculation performed using an in-house script (Microsoft Office Excel). All processed data were then mean-centered and unit-variance scaled before they were subjected 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 μL from each of the five samples belonging to the test groups. QC samples were analyzed at constant intervals to ensure that the data acquisition 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 complete protease inhibitor cocktail and 2 mM phenylmethylsulfonyl fluoride. The lysates were collected and centrifuged at 12,000 rpm for 30 min at 4 °C. After the protein samples were quantified using a BCA Protein Assay 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 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-μm mesh. The chemiluminescent probe with flow cytometry technique was used for detection of intracellular ROS level according to a previously described method [19]. Briefly, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) probe was added to the cell suspensions at a final concentration of 10 μmol/L and incubated at 37 °C, protected from light for 1 h, followed by flow cytometry (FACSCanto, BD Biosciences, San Jose, CA, USA) measurement. The formation of the oxidized fluorescent derivative 2′,7′-dichlorofluorescein (DCF) was monitored with excitation light at 488 nm and emission light at 525 nm, and normalized by protein concentration. By quantifying fluorescence intensity of DCF, the ROS levels in both groups were calculated.
+
+Mitochondrial Cardiolipin Assay
+Extraction of mitochondria was performed using the mitochondria isolation kit (Shanghai Genmed Scientifics Inc., China). Briefly, the frontal cortex was lysed in precooled centrifuge tubes and disrupted by 80 passes in the homogenizer with a tight fitting Dounce homogenizer. The homogenate was then centrifuged for 10 min at 1,500g at 4 °C. The mitochondria-rich supernatant was then collected and centrifuged for 10 min at 10,000g at 4 °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 °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 cardiolipin of freshly isolated mitochondria [20]. Briefly, reagents (90 μL) from cardilopin assay kits (Shanghai GenMed Scientifics Inc., Shanghai, China) was added into mitochondrial sample (10 μL) on the microplate. The microplate was gently shaken and incubated in a dark room for 20 min at room temperature. Then fluorescence intensity 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 normalized by protein concentration.
+
+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 approximately 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 °C, immersed in propylene oxide, and embedded in Araldite 502 resin at 60 °C. Ultrathin (0.5 μm) 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 10,000× magnification.
+
+Statistical Analysis
+We performed one-way ANOVA to determine differences in caspase-3 activation and cardiolipin contents, and independent 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.

20230808-AI coding-1st round/462 – Liu 2022.txt

@@ -0,0 +1,23 @@
+PMID: 35063768 DOI: 10.1016/j.bbrc.2022.01.022
+2. Methods
+2.1. Animals
+This study was approved by the Institutional Animal Care and Use Committee at Soochow University (Suzhou, Jiangsu, China). Twenty-four female and six male C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Jiangsu, China) for producing next generation of mice. On postnatal day (PND) 21, the offspring mice were separated from dams and housed 4–5 per cage by gender. Four male and four female mice were specifically used as the stranger mice, which were trained to stay calmly in the enclosure before social interaction test. All the mice were raised with free access to food and water in a controlled environment (room temperature 21–22 °C, 12/12 h light/dark cycle, and light on at 7 a.m.).
+
+2.2. Anesthesia
+A retired anesthesia machine (Datex-Ohmeda, Inc.) was employed to supply a consistent concentration of anesthetic gas. A sealed plastic box (20 L × 20 W × 6 H cm) was used as the anesthetizing chamber, which was drilled with three holes for gas inflow, gas outflow and gas monitoring. An electric heater was placed underneath the anesthetizing chamber to keep neonatal mice warm during anesthesia. A gas analyzer (Datex-Ohmeda, Inc.) was applied to adjust gas concentrations. On postnatal day 6, the neonatal mice were randomly assigned into two groups. Twenty-eight mice (17 males and 11 female) received 3.0% sevoflurane in 60% oxygen for 2 h (Sevo), and thirty-one mice (11 males and 20 female) inhaled merely 60% oxygen for 2 h (Oxyg). Sex of each mouse and amount of each group were not identified until weaning on PND 21. These subject mice were tested for social interaction behavior at one- and two-month-old.
+
+Another battery of neonatal mice were treated with the air condition (control) or 60% oxygen (oxyg) or 3.0% sevoflurane in 60% oxygen (sevo) for 2 h on PND 6, and then killed for harvesting brain tissues 24 h after treatment. Sevoflurane anesthesia was strictly performed by the protocols of previous studies [11,12], in which all neonatal mice could spontaneously breath during general anesthesia, and their arterial blood pressure and blood gas analysis showed within normal limits. The vapor for releasing sevoflurane was turned off at the end of anesthesia, and the residual anesthetic was washed out with 60% oxygen for 15 min. Finally, these pups were smeared with own bedding and sent back to their dams.
+
+2.3. Social interaction paradigm
+Social interaction test is performed with the three-chambered social box, with three chambers (40 L × 20 W × 22 H cm) and two enclosures (7 ID × 15 H cm). The floor is painted grey to provide a high contrast with the testing mice. Grid bars of the enclosure allow direct contacting between the subject and stranger mice. A novel video-tracking system was developed by hanging two video-cameras right above two enclosures. Thereby, two video-images were integrated into one with the montage effect in ANY-maze program (Stoelting Co., USA). The subject mouse initiates social interaction with the stranger mouse by nose-to-nose or nose-to-tail sniffing, thus the animal's head is tracked by the ANY-maze program.
+
+2.4. Social interaction test
+First of all, the stranger mice were transferred into behavioral room and hidden 2 m away from social apparatus. Each subject mouse was taken into behavioral room about 45 min before social interaction test. In the first session (Habituation, 10-min), the subject mouse was gently placed into the middle chamber, and allowed to freely explore in three chambers. In the second session (Sociability, 10-min), the subject mouse was guided into the middle chamber and transiently confined there. An unfamiliar conspecific (Stranger 1) was introduced into one enclosure, the subject mouse was allowed to explore in three chambers and sniff at two enclosures containing Stranger 1 or not. In the third session (Preference for social novelty 10-min), the subject mouse was again confined into the middle chamber. Another unfamiliar conspecific (Stranger 2) was introduced into the other enclosure, and the subject mouse was allowed to explore in three chambers and sniff at two enclosures containing Stranger 2 or Stranger 1. Placement of Stranger 1 on left and right side were balanced between trials, and two stranger mice were the same gender as the subject mice.
+
+Sociability is characteristic of the mouse taking more time sniffing its conspecific mouse compared with an inanimate object. Preference for social novelty is characteristic of the mouse taking more time sniffing an unfamiliar mouse compared with a familiar one. Four parameters were measured for judging social choice, including 1) time sniffing at the enclosure, 2) number of sniffs, 3) time exploring in the chamber, and 4) number of entries. Sniffing time at the enclosure was primary outcome, number of sniffs at the enclosure and time exploring in the chamber were secondary outcomes. In social interaction test, “at the enclosure” is defined as the head of mouse entering an area about 3 cm around the enclosure, as described in similar social study [13]. And “in the chamber” is defined as the head of mouse entering into the chamber.
+
+2.5. Immunoblotting analysis
+The brain tissues of neonatal mice were harvested on dry ice at 24 h after treatment. Next, the cortex and hippocampus were homogenized on ice using the immunoprecipitation buffer plus protease inhibitor. And then, the lysates were centrifuged at 15,000 rpm for 30 min at 4 °C. After that, the lysates were quantified for total protein by the bicinchoninic acid (BCA) protein assay kit (MultiSciences Biotech Co., Ltd. Cat: PQ0012, Lot: A91041). Finally, western blot was performed by the protocols to analyze protein levels in cortex and hippocampus. Neuroligin-1 antibody (1:1000; Santa Cruz Biotechnology, Inc.) was used to detect neuroligin-1 (101 kDa). PSD-95 antibody (1:1000; Cell Signaling Technology, Inc.) was used to detect PSD-95 (95 kDa). Anti–β-actin (1:5000; Sigma) was used to detect β-actin (42 kDa).
+
+2.6. Statistical analysis
+Data were expressed as Mean ± SD. Statistical analyses were performed by using GraphPad Prism 5.0 (San Diego, USA). Data representing social behavior of testing mice were normally distributed by Kolmogorov-Smirnov test. Data of each mouse from the left or right side were mutually exclusive, and two-tailed paired t-test was used to determine side preference, which was supported by other social studies [14,15]. Student's t-test was used to assess differences in the levels of Neuroligin-1 and PSD95 expression in cortex and hippocampus of mice. P values less than 0.05 (∗), 0.01 (∗∗) and 0.001 (∗∗∗) were considered statistically significant.

20230808-AI coding-1st round/470 – Kong 2012.txt

@@ -0,0 +1,28 @@
+PMID: 22705347 DOI: 10.1016/j.bcp.2012.06.001
+2. Materials and methods
+2.1. Animals
+All of the animals were treated according to the guidelines of the Guide for the Care and Use of Laboratory Animals (China Ministry of Health). The Laboratory Animal Care Committee of Zhejiang University approved all experimental procedures and protocols. All efforts were made to minimize the number of animals used and their suffering. The dams were housed in polypropylene cages, and the room temperature was maintained at 22 °C, with a 12-h light–dark cycle. The dams at gestational day 14 were used for all experiments, because this time corresponds approximately to mid-gestation in humans [15], [16], the period when most non-obstetric surgeries and fetal interventions are performed [9], [10].
+
+2.2. Anesthesia exposure
+The dams were randomly divided into three groups: control, low concentration of isoflurane (1.3%), and high concentration of isoflurane (3%) treatment groups (n = 8). The dams were placed in plastic containers resting in water baths with a constant temperature of 38 °C. In these boxes, pregnant rats in isoflurane treatment groups were exposed to 1.3 or 3% isoflurane (Lot 826005U, Abbott Laboratories Limited, USA) in a humidified 30% oxygen carrier gas for 1 h; the control group was exposed to simply humidified 30% oxygen without any inhalational anesthetic for 1 h. We chose 1.3% because it represents 1 MAC in the pregnant rats [17], and 3% is equal to ∼2 MAC. The determination of anesthetic duration based on our preliminary study which indicated that maternal physiological states remained stable throughout a 1-h isoflurane exposure. The isoflurane concentration, oxygen and carbon dioxide levels in the box were monitored with an agent gas monitor (Vamos, Drager Medical AG & Co. KgaA, Germany). Otherwise, control and experimental animals were under the same treatment and environment. Arterial blood gases (ABG) and blood glucose were measured at the end of the 1-h anesthetic exposure. The rectal temperature was maintained at 37 ± 0.5 °C. After exposure, all the dams were returned to their cages and allowed to deliver naturally. The postnatal body weights of the rat pups were monitored.
+
+2.3. Memory and learning studies
+Four rat pups (2 females and 2 males) from each dam were selected to determine cognitive function at postnatal day 28 with a Morris Water Maze test with minor modifications [1]. A round pool (diameter, 150 cm; depth, 50 cm) was filled with warm (24 °C) opaque water to a height of 1.5 cm above the top of the movable clear 15-cm-diameter platform in the third quadrant. A video tracking system recorded the swimming motions of animals, and the data were analyzed using motion-detection software for the Morris Water Maze (Actimetrics Software, Evanston, IL, USA). After every trial, each rat was wiped before returning to its regular cage, kept warm and allowed free access to food.
+
+2.3.1. Place trials
+The place trials were performed at postnatal day 29 for 4 days to determine the rats’ ability to obtain spatial information. At postnatal day 28, rats were tested for their ability to swim to a visible platform through a 30-s swimming training. A dark black curtain surrounded the pool to prevent confounding visual cues. All rats received 4 trials per day in each of the four quadrants of the swimming pool. On each trial, rats were placed in a fixed position into the swimming pool facing the wall. They were allotted 120 s to find the platform upon which they sat for 20 s before being removed from the pool. If a rat did not find the platform within 120 s, the rat was gently guided to the platform and allowed to remain there for 20 s. For all training trials, swim speed and the time to reach the platform (escape latency) were recorded. The less time it took a rat to reach the platform, the better the learning ability. We took the average of four trials as the escape latency each day.
+
+2.3.2. Probe trials
+Probe trials were conducted immediately after the four-day period to evaluate memory retention capabilities. The probe trials involved the submerged platform of the third quadrant from the pool and allowing the rats to swim for 120 s in any of the four quadrants of the swimming pool. Time spent in the third quadrant and the number of original platform crossing in the third quadrant was recorded.
+
+2.4. Transmission electron microscopy
+After the Morris Water Maze test, six pups per group were anesthetized with a lethal dose of Nembutal. The thoracic cavities were opened and perfused intracardially with 100 mL of normal saline. Then the hippocampus, including CA1 and dentate gyrus area, of each rat was taken out immediately. Immersion fixation was completed on tissues about 1 mm3 from the hippocampus. Samples were rinsed in cold phosphate-buffered saline (PBS) and placed in 2.5% glutaraldehyde at 4 °C for 4 h. The tissue was rinsed in buffer and post-fixed with 1% osmium tetroxide for 1 h. Then, the tissue was rinsed with distilled water before undergoing a graded ethanol dehydration series and was infiltrated using a mixture of half propylene oxide and half resin overnight. Twenty-four hours later, the tissue was embedded in resin. 120 nm sections were cut and stained with 4% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Ultrastructure changes of synapse in the hippocampus were observed under a transmission electron microscope (Philips Tecnai 10, Holland).
+
+2.5. Tissue section preparation
+After the Morris Water Maze test, two pups from each dam were anesthetized by intraperitoneal injection of a lethal dose of Nembutal. The aorta was cannulated and the animal was firstly perfused with 200 mL of normal saline, then with 250 mL of 4% formaldehyde (freshly made from paraformaldehyde) for 20–30 min. The fixed brain was then removed from the cranial cavity and post-fixed overnight in the same fixative at 4 °C. The tissues were embedded in paraffin, and transverse paraffin sections containing the hippocampal area were mounted on silanecoated slides. Sections were deparaffinaged and rehydrated. Then the sections were treated for antigen retrieval with 10.2 mmol/L sodium citrate buffer, pH 6.1, for 20 min at 95 °C for immunohistochemistry.
+
+2.6. Immunohistochemistry for caspase-3
+Caspase-3 positive cells were measured in the hippocampal CA1 region, using immunohistochemical methods described previously [7], [8]. The brain region was chosen because it is particularly vulnerable to anesthesia-induced neurodegeneration [1] and is important to memory and learning. Briefly, the sections mentioned above were washed in 0.01 M PBS containing 0.3% Triton X-100 (pH 7.4, PBS-T), followed by blocking in 5% normal goat serum in 0.01 M PBS. The sections were then incubated in the primary antibodies rabbit polyclonal against anti-caspase-3 (1:200, Santa Cruz Biotechnology, USA) overnight at 4 °C. After a thorough wash in PBS, sections were incubated with biotinylated goat anti-rabbit IgG antibody (1:200, Wuhan Boster Biological Technology, Ltd., China) for 2 h at room temperature, followed by avidin–biotin–peroxidase complex solution (ABC, 1:100, Wuhan Boster Biological Technology, Ltd., China) for 2 h at room temperature. Immunolabeling was visualized with 0.05% diaminobenzdine (DAB, Wuhan Boster Biological Technology, Ltd., China) plus 0.3% H2O2 in PBS and the reaction was stopped by rinsing the slides with 0.2 M Tris–HCl. Sections were mounted onto 0.02% poly-l-lysinecoated slides and allowed to dry at room temperature. Then the sections were dehydrated through a graded series of alcohols, cleared in xylene and finally coverslipped. Rat Immunoglobulin IgG (1:200, Biomeda Corporation, USA) was used instead of primary antibody as a negative control. Other chemicals used in this study were provided by Cell Signaling Technology (Beverly, MA). Three sections from hippocampal CA1 region of each animal were randomly selected and images were photographed under 400× magnification in 3 visual fields/per section, the caspase-3 positive neurons were counted in the same area. The optical densities of caspase-3 positive neurons were measured quantitatively using Image-Pro Plus version 6.0 (Media Cybernetics, Inc., Silver Spring, USA). The optical density of caspase-3 positive cells in a particular brain region was calculated by dividing the integrated optical density of caspase-3 positive cells by the area of that brain region.
+
+2.7. Statistical analysis
+All data were presented as mean ± S.E.M. Results of weight of postnatal rat pups and place trials of postnatal rats were analyzed using 2-way ANOVA for repeated measurements. Other data were analyzed using one-way ANOVA, followed by Tukey post hoc multiple comparison tests. A P value of <0.05 was considered statistically significant. All statistical tests and graphs were performed or generated, respectively, using Graph-Pad Prism Version 4.0 (GraphPad Prism Software, Inc., CA, USA).

20230808-AI coding-1st round/4738 – Cao 2015.txt

@@ -0,0 +1,31 @@
+PMID: 25052764 DOI: 10.1002/cbin.10349
+Materials and methods
+Animals
+C57BL/6 mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). The in vivo induction of ketamine-related hippocampal neurotoxicity was done at 2 weeks. Quantitative real time PCR of miR-34 family was used at 3 weeks, as was hippocampal injection of lentivirual vector of miR-34c. For analyses of TUNEL staining and Western blotting, 2-month old mice were used. All experimental procedures were reviewed and approved by the Animal Care Committee at the first affiliated Hospital of XinXiang Medical College.
+
+Induction of ketamine-related hippocampal neurotoxicity
+The in vivo protocol to induce ketamine-related hippocampal neurotoxicity was done as before with slight modifications (Hayashi et al., 2002; Huang et al., 2012, Liu et al., 2012). Young C57BL/6 mice, postnatal 14 days, were intraperitoneally administrated with repeated dosage of 75 mg/kg ketamine per day for six consecutive days (n = 28). Normal saline was injected in the control group of mice (n = 25).
+
+RNA isolation and reverse transcription
+Hippocampal RNA was isolated with Trizol reagent (In Vitrogen, Carlsbad, CA, USA). Briefly, mice were anesthetized and decapitated. Hippocampal samples were retrieved and homogenized at 1 mL Trizol/0.1 g tissue. The quantity of RNA was assessed by spectrophotometry followed by 1% agarose gel electrophoresis. Total RNA was treated with 10 U of RNase free DNase I, and reverse transcription (RT) was done in a total volume of 20 μL with random hexamer primers using a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). cDNA was stored at −20°C until further use.
+
+Quantitative RT-PCR
+Expression of miR-34a, miR-34b, miR-34c, and house-keeping gene GAPDH were measured by TaqMan microRNA RT-PCR on the ABI 7900 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Expression profiles of each gene were quantified using corresponding standard curves. End-point RT-PCR of miR-34a, miR-34b, miR-34c, and GAPDH used 50 ng of total RNA with a mirVana RT-PCR miRNA Detection Kit (Ambion, Austin, Texas, USA). PCR products were separated and visualized on a 4% agarose gel. Each sample was run in triplicate and a mean value of each Ct triplicate was used.
+
+Lentivirus production and transduction
+To downregulate miR-34c, the coding sequence for a 2’-O-methyl oligonucleotide of miR-34c inhibitor was UCCGUCACAUCAAUCGACUAACG, and the non-specific control antisense sequence was UACUCUUUCUAGGAGGUUGUUAUU (Yu et al., 2012). These two sequences were amplified and cloned into pCDH-CMV-MCS-EF1-coGFP for in vivo gene transfer, resulting in a miR-34c inhibitor vector (lenti-miR34c-I) and miR-34c non-specific control vector (lenti-miR34c-C) (System Biosciences, Mountain View, CA, USA). The lentivirual expression vectors and pPACK packaging vector were co-transfected into 293T cells, and viral particles were collected and concentrated to high titer.
+
+Hippocampal injection
+One day after the 6-day ketamine treatment, the injections of lent viruses were performed on the right side of the cortex. A tiny hole was drilled above hippocampus and a Hamilton syringe was used to inject 2 μL of lentivirus of miR-34c inhibitor (lenti-miR34c-I, 20 μM, n = 17) or non-specific control (lenti-miR34c-C, 20 μM, n = 14) at the coordinates assessed from bregma and skull surface: anteroposterior −2.0 mm, lateral +1.5 mm, and vertical −1.5 mm. After injection, the incision was quickly sealed with dental cement.
+
+Western blotting
+Western blotting analysis was conducted at 2 months. Four mice with Lenti-miR34c-I injection and four mice with Lenti-miR34c-C injection were included in this analysis. Forty micrograms of hippocampal protein were collected and separated on an 8% NuPage Gel with MES buffer (Invitrogen, Carlsbad, CA, USA) and transferred to a polyvinylidene difluoride membrane. Primary antibody dilutions included 1:500 BCL2 (Santa Cruz, USA), 1:100 phosphorylated-PKC (p-PKC) (Sant Cruz Biotechnologies, Santa Cruz, CA, USA), 1:100 phosphorylated-ERK (p-ERK) (Sant Cruz Biotechnologies, Santa Cruz, CA, USA), and 1:1,000 β-actin (Cell Signaling, Danvers, MA, USA). Membranes were then incubated in primary antibody in Odyssey Blocking Buffer at 4°C for 24 h, followed by three washes in 0.1% PBS-T and 1 h incubation at RT with 1:1,000 secondary antibodies. The films were visualized and quantified on the Odyssey Infrared Imaging Center (Li-Cor, Lincoln, NE, USA).
+
+TUNEL staining for hippocampal apoptosis
+Hippocampal slices (350 μm) were prepared for terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining to detect the apoptosis, using an In Situ Cell Death Detection Kit according to manufacturer's protocol (Roche, Branchburg, NJ, USA). Five mice with Lenti-miR34c-I injection and 5 mice with Lenti-miR34c-C injection were included in the analysis. Hippocampal CA1 region was examined under a fluorescent scope. The apoptotic CA1 neurons were identified based on their size, location and immuno-reaction to TUNEL staining. The average number of the apoptotic neurons per 0.01 mm2 was measured and compared between control hippocampi and hippocampi treated with miR-34c inhibitor.
+
+Morris water maze (MWM) testing
+The MWM testing was carried out 1 month after hippocampal transfection of miR-34c knockdown. Eight mice with Lenti-miR34c-I injection and 5 mice with Lenti-miR34c-C injection were included in this analysis. In a large circular tank with a transparent platform (10 cm × 10 cm), warm water at 26°C was added to submerge the platform 1 cm below the surface. Visual cues of color paints were used to aid mice in locating the platform. The mice were given training sessions four times per day for one week before final testing. In each training session, the mice were put in the maze to locate the platform in 2 min followed by resting on the platform for 30 s. If mice did not locate the platform in 2 min, they were aided with flashing lights to the platform with 30 s of rest on top of the platform. On the final day of examination, the average swimming time and swimming distance were compared between control mice and the mice with miR-34c knockdown.
+
+Statistical analysis
+Statistic analysis was conducted with SPSS software (version 11.0). The measured data were presented as mean ± standard deviations. The statistical differences were measured with a Student's t-test, and the significance set at P < 0.05.

20230808-AI coding-1st round/4902 – Ren 2014.txt

@@ -0,0 +1,15 @@
+PMID: 24705859 DOI: 10.1007/s10072-014-1726-4
+Methods
+All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Virginia (Charlottesville, VA). All surgical and experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publications number 80-23) revised in 2011. Efforts were made to minimize the number of animals used and their suffering.
+
+Neonatal brain hypoxia–ischemia modal
+Brain HI was performed in 7-day-old male and female Sprague–Dawley rats as described previously [10, 11]. In brief, neonates were anesthetized by isoflurane and their left common carotid arteries were permanently ligated with a double 7-0 surgical silk. The procedure lasted <5 min. After surgery, neonates were returned to the cages with their mothers for 3 h. The neonates were then placed in a chamber filled with humidified 8 % oxygen–92 % nitrogen for 2 h at 37 °C. The oxygen concentration and temperature in the chamber were continuously monitored.
+
+Drug application
+The neonates were randomly divided into the following groups: (1) control, (2) brain HI, (3) brain HI and postconditioning with 1, 2 and 3 % sevoflurane, (4) brain HI and 5-HD treatment (10 mg/kg) and (5) brain HI, 5-HD treatment and postconditioning with 2 % sevoflurane. Sevoflurane postconditioning was performed by exposing neonates to various concentrations of sevoflurane in 30 % O2 for 1 h immediately after brain HI. Neonates of brain HI alone group were placed in a chamber flushed with 30 % O2 for 1 h. The mitochondrial KATP channel inhibitor 5-HD was dissolved in normal saline and administered intraperitoneally just before the start of brain HI. The dose of 5-HD was based on a previous study in which intraperitoneal injection of 10 mg/kg 5-HD blocked ischemic preconditioning-induced protection [12].
+
+Brain injury/tissue loss quantification
+After 7 days of the brain HI, rats were sacrificed under deep isoflurane anesthesia and then their brains were harvested as described previously [11, 13]. The hindbrain was removed from cerebral hemispheres and bilateral hemispheres were weighed separately. The weight ratio of left to right hemispheres was calculated.
+
+Statistical analysis
+The results are presented as mean ± SD (n ≥ 6). Statistical analysis was performed by one-way analysis of variance followed by the Tukey’s test. A P ≤ 0.05 was considered statistically significant.

20230808-AI coding-1st round/505 – Feng 2012.txt

@@ -0,0 +1,24 @@
+PMID: 22535834 PMCID: PMC3393078 DOI: 10.1093/bja/aes121
+Methods
+Animals
+The use of animals in this study was approved by the Institutional Animal Care and Use Committee at Sun Yat-sen University (Guangzhou, Guangdong, China). All efforts were made to minimize the number of animals used and their suffering. 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 20–22°C. 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 min−1. During sevoflurane exposure, the container was heated to 38°C (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 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, n=3 in each subgroup) or after anaesthesia (6 h, n=3 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 maturation 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 (n=6) and the air-treated groups (n=6) 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 anaesthesia 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 geotaxis reflex, and gait reflex was recorded for each rat. Additionally, 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 (n=9) and air-treated groups (n=6). 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)°C] 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 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 following 4 days (days 1–4, Place Navigation), two blocks of tests (morning 08:30–11:00 and afternoon 14:30–15:00) were performed 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 platform 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 platform 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 ml−1 RIPA Lysis Buffer (Shenergy Biocolor Co., China) with 1% (v/v) PMSF (Shenergy Biocolor Co., China). The homogenate was centrifuged at 13 000g for 20 min at 4°C, and the supernatant was separated and stored at −80°C until further use. The proteins extracted from the hippocampus were separated on a 10% gel by electrophoresis and transferred on to polyvinylidene 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-β-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 Biotechnology, USA) were examined using the ECL-PLUS system (CWBIO, China) and imaged. The β-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 β-actin) were determined by optical density measurements (n=3 for each rat hippocampus sample).
+
+Histopathological examination
+Sevoflurane-treated (n=6) and air-treated (n=6) 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 anaesthetized with a lethal dose of 10% chloral hydrate and transcardially perfused with saline through the left cardiac ventricle until the liver and lungs were cleared of blood, followed 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 sections of the hippocampus at different levels along the septotemporal axis and associated areas.29 Coronal hippocampal sections 5 μm 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 imaged from three sections per animal (n=3 for each 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 magnification 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 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 multivariate 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.

20230808-AI coding-1st round/5173 – Liu 2012.txt

@@ -0,0 +1,28 @@
+PMID: 19352168 DOI: 10.1097/ALN.0b013e31819daedd
+Materials and Methods
+The study protocol was approved by the Home Office (London, United Kingdom) and conforms to the United Kingdom Animals (Scientific Procedures) Act of 1986.
+
+In Vitro  Experiments
+Organotypic hippocampal slices were derived from postnatal day 8 or 9 C57Bl/6 mice pups (Harlan Laboratories, Huntingdon, United Kingdom) and cultured by the interface method21,22with some modifications. In brief, the brain was quickly dissected and placed in ice-cooled (4°C) dissection solution. All stages of slice preparation were performed under sterile and ice-cooled conditions. Excess tissue (including the cerebellum, olfactory bulbs, and meninges) was removed, and the brain was cut into 400-μm sagittal slices using a McIllwain Tissue Chopper (Mickle Laboratory, Cambridge, United Kingdom). Under a dissecting microscope and avoiding contact with the hippocampus, the slices were separated using fine forceps. Slices containing the intact hippocampus were selected and positioned onto 30-mm-diameter semiporous cell culture inserts (five slices per insert) (Falcon; Becton Dickinson Labware, Millipore, Bedford, MA) and placed in a six-well tissue culture tray (Multiwell; Falcon, Becton Dickinson Labware). Eagle minimum essential medium enhanced with heat-inactivated horse serum (1.5 ml) was then transferred to each well.
+
+The slices were incubated for 24 h in humidified air at 37°C, enriched with 5% carbon dioxide. The culture medium was replaced the next day with fresh, temperature-equilibrated medium before exposure to gas treatments. The groups of slices (n = 15 per group) were assigned to control (air + 5% carbon dioxide), dexmedetomidine 1 μm, gabazine 50 μm, 0.75% isoflurane, 0.75% isoflurane + dexmedetomidine 1 μm, and 0.75% isoflurane + gabazine 50 μm.
+
+All subsequent gas exposure occurred in a specially constructed exposure chamber as previously described.23The gases, warmed by a water bath, were delivered in the headspace above the slices by a standard anesthetic machine at 2–3 l/min, and concentrations were monitored with an S/5 spirometry module (Datex-Ohmeda, Bradford, United Kingdom). After 3–4 min of gas flow, the chambers were sealed and placed in a 37°C incubator for 6 h (Galaxy R Carbon Dioxide Chamber; Wolf Laboratories, Pocklington, York, United Kingdom). After exposure, the slices were returned to the incubator for a further 12 h of culture to allow for suitable caspase-3 expression and then fixed overnight in 4% paraformaldehyde and subsequently immersed in 30% sucrose for a further 24 h at 4°C before slicing with a cryostat.
+
+In Vivo  Experiments
+Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane in 25% oxygen or air in a temperature-controlled chamber (n = 6 per group). Three doses of saline or dexmedetomidine (1, 10, or 25 μg/kg) were administered by intraperitoneal injection over the 6-h exposure (at 0, 2, and 4 h). One group received 0.75% isoflurane, 25 μg/kg dexmedetomidine, and 500 μg/kg nonselective α2adrenoceptor antagonist atipamezole in 3 doses over the 6-h exposure (n = 4 per group). An additional three doses of 75 μg/kg dexmedetomidine in air were given to establish at extreme doses of dexmedetomidine whether apoptosis could be induced (n = 6 per group).
+
+The animals were sacrificed (with 100 mg/kg sodium pentobarbital by intraperitoneal injection) at the end of gas exposure and perfused transcardially with heparinized saline followed by 4% paraformaldehyde in 0.1 m buffer. After removal of the brain and storage overnight at 4°C in paraformaldehyde, it was transferred to 30% sucrose solution with phosphate buffer and 1% sodium azide and kept at 4°C until the brains were sectioned and stained immunohistochemically for caspase-3.
+
+Immunohistochemistry
+For the in vitro  experiments, the slices were sectioned at 25-μm intervals using a cryostat, and the inner sections were mounted onto Super Plus-coated glass slides (VWR International, Lutterworth, United Kingdom). The sections were allowed to dry at 37°C for 24 h and then immunostained while adherent to the slides. Concerning the in vivo  experiments, the brain was sliced at 30-μm intervals beginning at −3.6 mm from the bregma, the sections were then transferred to a six-well plate containing phosphate-buffered saline. Sections were dried at 37°C for 24 h and then immunostained while adherent to the slides, before preincubation with hydrogen 0.3% peroxidase in methanol for 30 min and then rinsed in phosphate-buffered saline. The sections were then incubated overnight at 4°C with rabbit anti-cleaved caspase-3 (1:2,500; New England Biolab, Hitchin, United Kingdom) and then washed three times in phosphate-buffered saline with 3% Triton at room temperature. Biotinylated secondary antibodies (1:200; Sigma, St. Louis, MO) and the avidin-biotin-peroxidase complex (Vector Laboratories, Orton Southgate, Peterborough, United Kingdom) were applied. The sections were again washed in phosphate-buffered saline before incubating with 0.02% 3,3′-diaminobenzidine with nickel ammonium sulfate in 0.003% hydrogen peroxide (DAB kit, Vector Laboratories). The sections were dehydrated through a gradient of ethanol solutions (70–100%) and then mounted (floating section) and covered with a cover slip.
+
+Neurocognitive Evaluation
+Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane in 25% oxygen or air in a temperature-controlled chamber (n = 6 per group). Three doses of saline or 25 μg/kg dexmedetomidine were administered by intraperitoneal injection over the 6-h exposure (at 0, 2, and 4 h). The animals were allowed to mature until postnatal day 40 and then tested for hippocampal-dependent memory and learning function in a previously reported contextual fear-conditioning behavioral paradigm24in which the rats were taken from the vivarium in the behavioral room on the first test day and allowed to sit undisturbed in their homecage for 10 min. Once placed in the conditioning chamber, the rats were allowed 198 s of exploration.
+
+The conditioning chamber was cubic (30 cm × 24 cm × 21 cm; Med Associates, Inc., St. Albans, VT) and had a white opaque back wall, aluminum sidewalls, and a clear polycarbonate front door. The conditioning box had a removable grid floor and waste pan. Between each rat, the box was cleaned with an almond-scented solution and dried thoroughly. The grid floor contained 36 stainless steel rods (diameter, 3 mm) spaced 8 mm center to center. When placed in the chamber, the grid floor made contact with a circuit board through which a scrambled shock was delivered. During training and context testing, a standard high efficiency particulate air filter (HEPA) filter provided background white noise of 65 db.
+
+Afterwards, all animals received 6 cycles of 214 s of trace fear conditioning. The tone was presented for 16 s (2 kHz) followed by a trace interval of 18 s and subsequent foot shock (2 s, 0.85 mA). The rats were removed from the conditioning chamber 198 s after the last shock and returned to their home cage. The total time of the acquisition phase was 26 min. Acquisition time was defined as the time spent immobile after a shock divided by the intertrial interval. On the next day, trained rats were exposed to the same acquisition environment but received neither tone nor shock for 8 min (context test). The percentage of time an animal froze during the 8-min observation periods was calculated as the number of observations judged to be freezing divided by the total number of observations in 8 min (i.e. , 60 observations). Freezing time was assessed using VideoFreeze software (Med Associates Inc., Burlington, VT); therefore, the assessment can be considered objective. The percentage of freezing time (context results) and the area under curve were derived from plots between the percentage freezing time and trial time in the tone test and were used for statistical comparison (mean ± SD, n = 6 per group).
+
+Statistical Analyses
+The number of caspase-3–positive neurons in the cortex, thalamus, and hippocampus in each brain slice were counted by an observer blinded to the experimental protocol. Four brain slices were counted per animal. The immunohistochemical and behavioral data are presented as mean ± SD. Statistical analyses was performed by ANOVA followed by post hoc  Newman Keuls testing using the INSTAT (London, United Kingdom) program. P < 0.05 was set as significant.

20230808-AI coding-1st round/520 – Burks 2020.txt

@@ -0,0 +1,27 @@
+PMID: 32413489 DOI: 10.1016/j.ntt.2020.106890
+2. Methods
+2.1. Animals
+Pregnant Sprague-Dawley rats (Charles Rivers, USA) arrived on gestational day 5. Litters were culled to four males and four females on PND 4. A within-litter treatment design was used to evaluate the effect of Sevo. Four animals, 2 males and 2 females, were selected from each litter. One animal of each sex was randomly assigned to the Sevo group with the other being assigned to the vehicle condition. Three animals per sex were assigned to each treatment / timepoint combination (N = 72). Multiple stains were used on tissue from the same animal. Two animals were removed from the 72 h Sevo group. One animal was removed for failing to meet inclusion criteria for oxygenation (no hypoxic animals were included in the study), and a second animal died between exposure and sacrifice. Rats were housed in a light (12 h/12 h light/dark cycle) and temperature (22 ± 2 °C) controlled vivarium and given free access to food and water (NIH41 laboratory animal diet, Envigo, Madison, WI). All animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals. Animal use and procedures were approved by the NCTR Institutional Animal Care and Use Committee (IACUC), which has full NIH-OLAW accreditation. Animals were housed in the NCTR facility in isolator top boxes with wooden chip bedding and ad libitum food and water.
+
+2.2. Study design
+On PND 7, rats were exposed to vehicle gas alone (75% oxygen/25% nitrogen) or 2.5% Sevo (in vehicle gas) for 6 h. During anesthesia exposure, each pup was placed in an individual airtight acrylic chamber and the selected gas mixture was delivered at a flow rate of 0.75–1 L/min (Walters et al., 2020). The concentration of Sevo was set using a commercial gas analyzer (Riken, USA). Surface body temperature was collected prior to and every 2 h following the start of Sevo exposure using an infrared thermometer (Micro-Epsilon, Ortenburg, Germany). Heating plates located beneath each chamber were used to maintain body temperatures at baseline levels. In addition, arterial oxygen saturation (SpO2), breath rate, heart rate, and pulse distention were monitored in each pup continuously using a pulse oximeter (Starr Life Sciences Corp, USA). The average SPO2 value was calculated every 30 min (Supplemental Table 1); if an individual rat's SPO2 fell below 85% during any of the 30 min intervals, it was excluded from the study. Upon recovery, the pups were removed from the chambers, rubbed with bedding material from their home cage, and returned to their dams. Control animals were treated the same as the experimental group except they were not exposed to anesthesia and there SPO2 was not monitored.
+
+Pups were sacrificed 2 h (PND 7), 24 h (PND 8), and 72 h (PND 10) after the cessation of Sevo or vehicle gas exposure. Briefly, the rats were deeply anesthetized with pentobarbital and transcardially perfused with 0.9% heparinized saline followed by 10% neutral buffered formalin. Brains were removed and post-fixed in 10% neutral buffered formalin for 24 h, cryoprotected in 20% sucrose until they sank, and subsequently frozen on dry ice and stored at −80 °C. Tissue was cut into 30 μm thick coronal sections using a cryostat, stored in 0.08% sodium azide in PBS for up to two weeks and then transferred to freezing solution (0.02 M phosphate buffer (pH 7.4) containing 25% (v/v) glycerol and 30% (v/v) ethylene glycol) until processed for immunohistochemistry or histology.
+
+2.3. FJC immunolabeling
+For FJC labeling, a modified method (Bowyer et al., 2018b; Schmued et al., 2005) was used. Briefly, sections of interest were removed from freezing solution and rinsed three times in 0.1 M phosphate buffer (PB, pH 7.4) for 1 min. Sections were then mounted on gelatin coated slides in 0.005 M PB (pH 7.4) and dried at 50 °C for 2 h. Subsequently, slides were immersed for: 3 min in basic alcohol, 2 min in 70% ETOH, 2 min in Millipore water, 11 min in 0.06% potassium permanganate, 2 min in Millipore water, 10 min in FJC (0.00001% in 0.1% glacial acetic acid), and three 2 min washes in Millipore water. Slides were then dried at 50 °C for 5–10 min, cleared with xylene for 1 min, and cover-slipped with DPX mounting media.
+
+2.4. Mki67 immunolabeling
+A buffer of 0.1 M PB (pH 7.4) containing 0.4% Triton X-100 was used in all the steps involving free floating sections agitated on an orbital shaker. Sections containing regions of interest were initially washed in buffer three times (15 min each) to remove excess freezing solution. After a 30 min pre-incubation in 4% normal goat serum, the sections were incubated in 4% serum and chicken polyclonal antibody to Mki67 (1:2000, EnCor Biotechnology, USA) for 1 to 2 h at room temperature followed by 18 to 24 h at 5 °C. Sections were then washed three times for 15 min and incubated in a biotinylated goat anti-chicken antibody (1:350, Invitrogen, USA) for 1 h at room temperature. The sections were then washed three times (15 min per wash) and incubated in Streptavidin TRITC (1:200, Jackson ImmunoResearch, USA) for 1 h. The sections were then washed three times (15 min per wash) and mounted on Superfrost Plus slides (Thermo Fisher Scientific, USA) and dried at room temperature for ≥12 h in the dark. Finally, the slides were cleared in xylene and cover-slipped with DPX mounting medium.
+
+2.5. NeuN immunolabeling
+Sections containing the four regions (IG, anterior CPu (CPua), anterior thalamus, and CA1) with the highest per mm2 levels of FJC labeled cells were immunolabeled with an antibody to NeuN in conjunction with DAB visualization. Sections were washed in 0.1 M PB (pH 7.4) for 15 min and then incubated in 0.1 M PB containing 0.05% H2O2 for 10 min to suppress the endogenous peroxidases. From this point on, except for the last step of 3,3′-diaminobenzidine (DAB) processing, incubation and washing solutions consisted of 0.1 M PB containing 0.25% Triton X-100. Sections were then washed three times for 5 min. Following a 20 min pre-incubation in 5% normal goat serum, the sections were incubated in rabbit anti-NeuN (1:1000, Abcam, USA) antibody for 18 to 24 h at room temperature. Sections were then washed three times for 5 min and incubated in a biotinylated goat anti-rabbit antibody (1:300, Thermo Fisher Scientific, USA) for 2 h. The signal was then amplified using the avidin and biotinylated horseradish peroxidase macromolecular complex (Vector Laboratories, USA) and visualized with 0.5 mg/mL of DAB in Tris-HCl buffer. Sections were washed twice for 5 min in Tris-HCl, mounted, and dried on a slide warmer for ≥12 h. Finally, the slides were cleared in xylene and cover-slipped with DPX mounting medium.
+
+2.6. Thionine staining
+Thionine staining was performed to verify brain regions. Sections from regions where the highest levels of FJC staining were observed from PND 7, 8 and 10 were mounted from 0.1 M PB (pH 7.4) on Superfrost Plus slides (Thermo Fisher Scientific, USA) and dried at 55 °C for 15 min. They were then immersed in double distilled water for 4 min. Subsequently, the sections were immersed in a solution of 0.1% thionine acetate (Sigma-Aldrich, USA) in double distilled water for 8 min. The sections were then transferred through two washes of water (2 min each) followed by 70% ethanol in water (2 min), 95% ethanol (2 min) and 100% ethanol (2 min). The sections were then transferred to xylene for ≥2 min and cover-slipped as described above.
+
+2.7. Image capturing and analysis
+Imaging of brain tissue was conducted using a Nikon Eclipse Ni microscope equipped with digital cameras (Photometrics, USA; Nikon, USA). FJC, Mki67, NeuN, and thionine labeling were quantified in the somatosensory cortex, motor cortex, CPu, thalamus, CA1 region of the hippocampus, septum and amygdala at 10× magnification using NIS Elements AR automated software (Nikon, USA). Brain regions were defined in accordance with brain atlases for adult and neonatal rats (Paxinos and Watson, 2014; Ramachandra and Subramanian, 2011).
+
+2.8. Stereological analysis
+The brain regions in which the highest levels of FJC positive neurons were identified with the aid of adult and neonatal atlases as guides. Given the absence of a complete neonatal atlas, these locations were verified using thionine stained sections from the same regions that the FJC sections were taken to determine neurodegeneration from the pups sacrificed at PND 7, 8 and 10 [see Fig. 1]. Subsequent to identifying these regions, unbiased stereological estimates of positively immunolabeled cells/structures were performed from images that were captured with Photometrics (fluorescent) or Nikon (brightfield) digital cameras using NIS elements AR software for analysis.

20230808-AI coding-1st round/531 – Li 2007.txt

@@ -0,0 +1,41 @@
+PMID: 17959201 PMCID: PMC2170454 DOI: 10.1016/j.neuropharm.2007.09.005
+2. Materials and methods
+2.1. Animals
+Institute of Animal Care and Use Committee (IACUC) at the University of Pennsylvania approved all experimental procedures and protocols used in this study. All efforts were made to minimize the number of animals used and their suffering. Sprague–Dawley pregnant rats (Charles River Laboratories, Inc Wilmington, MA) were housed in polypropylene cages and the room temperature was maintained at 22 °C, with a 12 h light–dark cycle. Pregnant rats at gestation day 21 (E21) were used for all experiments because it approximately corresponds to mid-gestation in human beings according to the theory of brain growth spurt (Dobbing and Sands, 1979, Jevtovic-Todorovic et al., 2003), and is a common time for most fetal surgeries (18–25 weeks) (Myers et al., 2002).
+
+We have designed the following three related studies: (1) pilot study; (2) neurodegeneration study; (3) finally a behavioral study. A pilot study was first conducted to find the highest concentration of isoflurane not accompanied by significant arterial blood gas (ABG) and mean arterial blood pressure (MABP) changes in the mothers. A neurodegeneration study was used to determine the appearance of apoptosis by detection of caspase-3 and TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) positive cells in the fetal brain (2 and 18 h post-exposure) or neonatal brain at postnatal day 5 (P5). We have chosen the above time points to detect apoptosis in fetal or newborn brains, based on previously published work (Jevtovic-Todorovic et al., 2003). The behavioral study was performed to investigate the effects of fetal exposure to isoflurane on postnatal memory and learning. The pregnant rats used in each study were not reused in the other two studies. Within each study described above, animals were randomly divided into either isoflurane treatment or sham control groups. Pregnant rats in the isoflurane treatment groups inhaled isoflurane for 6 h, while those in the sham control group only inhaled a carrier gas (30% oxygen, balanced with nitrogen) for 6 h under the same experimental conditions. The distribution of pregnant rats and pups in all three groups is illustrated in Fig. 1.
+2.2. Anesthetic exposure
+Isoflurane is used clinically at a wide range of concentrations (about 0.2–3%), depending on the presence of other kinds of anesthetics or narcotics and the type and duration of surgery. As isoflurane neurotoxicity is concentration-dependent (Jevtovic-Todorovic et al., 2003, Wei et al., 2005), a primary goal of this study was to investigate if the highest isoflurane concentration used clinically is harmful to the fetal brain. Due to our concern that the physiological side effects of these drugs would contaminate the interpretation, we conducted a pilot study to determine the highest anesthetic concentration we could use without invasive support (tracheal intubation and ventilation) that would not significantly affect arterial blood gas (ABG) and mean arterial blood pressure (MABP) in the mothers, and then used this concentration in the subsequent formal study. We wanted to avoid tracheal intubation, as it could possibly affect the hemodynamics of pregnant rats and the apoptosis in the fetal brains. In addition, this makes it more difficult to set up the sham control groups without anesthesia. In the pilot study, five pregnant rats were initially anesthetized with 2% isoflurane in 30% oxygen via a snout cone for approximately 1 h and the right femoral artery was catheterized for blood sample collection and measurement of MABP by a pressure transducer/amplifier (AD Instruments Inc., Colorado Springs, CO, USA). The rats were recovered for 2 h and then exposed to isoflurane, starting at 1.5% in a humidified carrier gas of 30% oxygen, balance nitrogen for 6 h in a monitored chamber in hood. The pregnant rats breathed spontaneously without intubation or other support while being warmed using a deltaphase isothermal pad (Braintree Scientific Inc, Braintree, MA, USA). The rectal temperature was maintained (Fisher Scientific, Pittsburgh, PA, USA) at 37 ± 0.5 °C. We monitored isoflurane concentration in the chamber using IR absorbance (Ohmeda 5330, Detex-Ohmeda, Louisville, CO, USA). Arterial blood (0.1 ml) from previously placed femoral arterial catheter was collected and ABG determined every 2 h for up to 6 h by an ABG analyzer (Nova Biomedical, Waltham, MA, USA). Blood glucose was simultaneously measured with a glucometer (ACCU-CHECK Advantage, Roche Diagnostics Corporations, Indianapolis, IN, USA). Control rats were exposed only to humidified 30% O2 balanced by N2 (carrier gas for isoflurane in the treatment group) for 6 h in the same chamber under the same experimental conditions as in the treatment group. Because one pregnant rat treated with 1.5% isoflurane showed obvious acidemia (which reversed after termination of anesthesia), we decreased the isoflurane concentration to 1.3%, and subsequently found no significant changes in the ABG or MABP between the treatment group and the sham control group (Table 1). Therefore, 1.3% isoflurane was used in the ensuing neurodegeneration and behavioral studies.
+In the behavioral study, pregnant rats were treated with 1.3% isoflurane (n = 8) or carrier gas (sham controls, n = 7) for 6 h. The monitoring was the same as that in the pilot study except that femoral artery catheters were not placed. After the exposures, the animals were returned to their cages and the rat pups were delivered naturally. Four rat pups from each pregnant mother were raised to P28 (Juvenile) and P118 (adult), and then used to determine memory and learning ability with a Morris Water Maze (MWM). Two rat pups from the control group and one from the isoflurane group died unexpectedly, leaving a total of 26 and 31 rat pups in the control and isoflurane treatment groups respectively (Fig. 1).
+
+In the fetal brain apoptosis study, pregnant rats were treated with either 1.3% isoflurane or carrier gas for 6 h. At 2 and 18 h after exposure, the rat pups were delivered by C-section under sodium pentobarbital (100 mg/kg, i.p.) anesthesia. The fetal brains were removed and snap frozen for immunohistochemical analysis. Two fetal brains from each pregnant rat were studied. In addition, newborn brains from the rat pups born to the pregnant rats in the behavioral study group (one pup from each pregnant rat, treatment n = 8, control n = 7) were also obtained at postnatal day 5 and prepared for the apoptosis study at P5 (Fig. 1).
+
+2.3. Measurement of isoflurane concentration in the brain tissues
+To confirm that the isoflurane concentration in the fetal brain correlated with the inhaled concentration and brain concentration in the pregnant mothers, we measured the brain isoflurane concentrations in the fetus and the mother simultaneously in one rat. Briefly, after the pregnant rat was exposed to 1.3% isoflurane for 6 h, the brains of both mother and fetuses were removed and the brain tissue was immediately placed into 4 ml of 0.02 M phosphate buffer (with 1 mM halothane as internal standard) and homogenized in a glass homogenizer. The homogenate was centrifuged (30,000 × g at 4 °C for 30 min), the supernatant collected and then loaded onto C18 cartridge that had been conditioned with 2 ml methanol and washed with water, for solid phase extraction. The final sample was eluted with 0.5 ml solution of methanol and 2-propanol (vol:vol 2:1) with 0.1% trifluoroacetic acid. All procedures were performed in the cool room (4 °C). A 250 μl aliquot of each final elute was injected into a high performance liquid chromatography (HPLC) system, equipped with a refractive index monitor, for quantitation.
+
+2.4. Tissue preparation
+After treatment with isoflurane or carrier gas alone (control), pregnant rats were anesthetized with sodium pentobarbital intraperitoneally (i.p. 100 mg/kg) at either 2 or 18 h after the end of isoflurane exposure, and the fetuses removed by cesarean section. Likewise, postnatal pups at day 5 (P5) were given the same dose of sodium pentobarbital. All fetuses and pups were then perfused transcardially with ice-cold normal saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were then removed and post-fixed overnight in the same fixative at 4 °C, and cryoprotected in 30% (wt/vol) sucrose in 0.1 M phosphate buffer (pH 7.4) at 4 °C for 24 h. Thereafter, the brains were frozen in isopentane at −20 °C and stored at −80 °C until use. Serial coronal sections (10 μm) were cut in a cryostat (Dolbey–Jamison Optical Company, Inc., Pottstown, PA, USA), mounted on gelatin-coated slides and stored at −80 °C. Coronal brain sections from the same brain corresponding to figure 96 of the rat fetal brain atlas (Paxinos et al., 1990) were chosen for detection of apoptosis by caspase-3 immunohistochemistry and TUNEL staining. In the initial examination of brains sections from the neurodegeneration study, we noticed that apoptosis was most apparent in the hippocampus CA1 region and the retrosplenial cortex, and thus we chose these two brain regions to quantify apoptosis.
+
+2.5. Immunohistochemistry for caspase-3
+Caspase-3 positive cells were detected using immunohistochemical methods described previously (Gown and Willingham, 2002). Briefly, brain sections were first incubated in 3% hydrogen peroxide in methanol for 20 min to quench endogenous peroxidase activity. Sections were then incubated with blocking solution containing 10% normal goat serum in 0.1% phosphate buffered saline with 0.1% Tween 20 (PBST) for 1 h at room temperature after washing with 0.1% PBST. The anti-activated caspase-3 primary antibody (1/200, Cell Signaling Technology, Inc Danvers, MA, USA) was then applied in blocking solution and incubated at 4 °C overnight. Tissue sections were biotinylated with goat anti-rabbit antibody (1/200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) in 0.1% PBST for 40 min, followed by incubation with the avidin–biotinylated peroxidase complex (Vectostain ABC-Kit, Vector Lab, Burlingame, CA, USA) for 40 min. Tissue sections were colorized with diaminobenzidine (DAB, Vector Laboratories, Burlingame, CA, USA) for 8 min and counterstained with modified hematoxylin. Negative control sections were incubated in blocking solution that did not contain primary antibody. Images were acquired and assessed at 200× using IP lab 7.0 software linked to an Olympus IX70 microscope (Olympus Corporation, Japan) equipped with a Cooke SensiCam camera (Cooke Corporation, Romulus, MI, USA). Three brain tissue sections at 10 μm corresponding to the Atlas of the Developing Rat Brain, Figure 96 (Paxinos et al., 1990) were chosen from each animal and analyzed for caspase-3 positive cells in the two brain regions. Two persons blinded to the treatments counted the total number of caspase-3 positive cells in the hippocampal CA1 region and retrosplenial cortex. The areas of entire hippocampal CA1 region and retrosplenial cortex were defined according to the Atlas of the Developing Rat Brain, Figure 96 (Paxinos et al., 1990) and the area measured using IPLab Suite v3.7 imaging processing and analysis software (Biovision Technologies, Exton, PA, http://www.BioVis.com). The density of caspase-3 positive cells in a particular brain region was calculated by dividing the number of caspase-3 positive cells by the area of that brain region.
+
+2.6. TUNEL for DNA fragmentation
+Three brain sections (10 μm) adjacent to the sections used for caspase-3 detection were used for TUNEL staining using the DeadEnd™ Colorimetric TUNEL System Kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocol (Gavrieli et al., 1992). Briefly, sections were permeabilized by proteinase K solution (20 μg/ml) for 8 min, incubated in equilibration buffer for 10 min and the terminal deoxynucleotidyl transferase (TdT) and biotinylated nucleotide were added to the section and incubated in a humidified chamber at 37 °C for 1 h. The reaction was then stopped, followed by incubation with horseradish peroxidase-labeled streptavidin, colorization with DAB/ H2O2 and counterstained with modified hematoxylin. For positive-controls, the tissue sections were first treated with DNase I (1000 U/ml, pH 7.6) for 10 min at room temperature to initiate breakdown of DNA. Incubation of sections in reaction buffer without TdT provided negative controls. Images were acquired, and TUNEL quantitation performed as described above for caspase-3.
+
+2.7. Spatial reference memory and learning performance
+2.7.1. Morris Water Maze (MWM)
+Pregnant rats were allowed to deliver after the isoflurane treatment and 4 pups per litter (2 females and 2 males) were raised. The body weights of the rat pups were recorded at P0, P3, P5, P11, P17 and P28 to determine growth rate. We determined spatial reference memory and learning with the MWM as reported previously with some modification (Jevtovic-Todorovic et al., 2003). A schematic of the experimental paradigm is shown in Fig. 2. A round, fiberglass pool, 150 cm in diameter and 60 cm in height, was filled with water to a height of 1.5 cm above the top of the movable clear 15 cm diameter platform. The pool was located in a room with numerous visual cues (including computers, posters and desks) that remained constant during the studies. Water was kept at 20 °C and opacified with titanium dioxide throughout all training and testing. A video tracking system recorded the swimming motions of animals and the data were analyzed using motion-detection software for the MWM (Actimetrics Software, Evanston, IL, USA). After every trial, each rat was placed in a holding cage, under an infrared heat lamp, before returning to its regular cage.
+2.7.2. Cued trials
+The cued trials were performed only for postnatal rats at P28 and P29 (28 rats in control group and 31 rats in treatment group) to determine whether any non-cognitive performance impairments (e.g. visual impairments and/or swimming difficulties) were present, which might affect performance on the place or probe trials. A white curtain surrounded the pool to prevent confounding visual cues. All rats received 4 trials per day. On each trial, rats were placed in a fixed position in the swimming pool facing the wall and were allowed to swim to a platform with a rod (cue) 20 cm above water level randomly placed in any of the 4 quadrants of the swimming pool. They were allotted 60 s to find the platform upon which they sat for 30 s before being removed from the pool. If a rat did not find the platform within 60 s, the rat was gently guided to the platform and allowed to remain there for 30 s. The time for each rat to reach the cued platform and the swim speed was recorded and the data at P28/29 were analyzed.
+
+2.7.3. Place trials
+After completion of cued trials, we used the same rats to perform the place trials to determine the rat's ability to learn the spatial relationship between distant cues and the escape platform (submerged, no cue rod), which remained in the same location for all place trials. The starting points were random for each rat. The time to reach the platform was recorded for each trial. The less time it took a rat to reach the platform, the better the learning ability. The juvenile rats (P32) received two blocks of trials (two trials per block, 30 s apart, 60 s maximum for each trial and 2 h rest between blocks) each day for 5 days. The adult rats (P115) received only one block of trials each day for 5 days using a new platform location in an effort to increase task difficulty and improve test sensitivity.
+
+2.7.4. Probe trials
+Probe trials were conducted after the last place trials for the juveniles (P36) and adults (P119) to evaluate memory retention capabilities. After all rats completed the last place trial on the fifth day, the platform was removed from the water maze and rat was started to swim in the quadrant opposite to one the platform was placed before. The rats were allowed to swim for 60 s during each probe trial and the time the rats spent in each quadrant was recorded. The percentage of the swimming time spent in the target (probe) quadrant where the platform was placed before was calculated. The time spent in the target quadrant compared to other quadrants was an indication of memory retention.
+
+2.7.5. Learning to reach criterion test
+After the last probe test for the adult rats, the animals performed the learning to reach criterion test during the next 9 days as described previously (Chen et al., 2000). The experimental procedure was similar to the place trial except that the platform location was changed. For each rat, the platform was moved between nine different locations set up by the computer. Each rat received up to eight trials per day. In order to advance to the next platform location, each rat had to reach the criterion of three successive trials with escape latency of 20 s or less. If a rat reached a criterion in 8 or less trials, a new platform location would be selected the following day. The numbers of learned platforms and the number of trials used to reach the criteria were recorded and compared. The number of platforms learned and the number of trials to reach a criterion indicated the learning ability of the rats.
+
+2.8. Statistical analysis
+To reduce variance from different size litters, we averaged the data from all fetal or postnatal rats from the same mother and considered them as a single sample. Results of weight gain of postnatal rat pups, ABG and MABP of pregnant rats and place trials of postnatal rats were analyzed using 2-way ANOVA for repeated measurements. Data for immunohistochemistry, TUNEL and other behavioral studies were analyzed using Student’s t-test for comparison of two groups or by ANOVA followed by Fisher's post hoc multiple comparison tests for those with more than two groups. In all experiments, difference were considered statistically significant at P < 0.05.

20230808-AI coding-1st round/564 – Lei 2013.txt

@@ -0,0 +1,33 @@
+PMID: 23967080 PMCID: PMC3742769 DOI: 10.1371/journal.pone.0070645
+Materials and Methods
+Animal/Anesthesia treatment
+The rats used in the present study were obtained from the Animal Care Center of Fudan University. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee, Fudan University. One-day pregnant female Sprague Dawley rats (weight 220–250 g) were randomly assigned to one of the three groups: control, sevoflurane, or sevoflurane with n-3 PUFAs (n = 3 per group). Fish oil, the main source of n-3 PUFAs (Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA)), was extracted from the capsule (1000 mg/capsule that containing 180 mg EPA and 120 mg DHA, Puritan's Pride, Bohemia, NY, USA) and added to food. Pregnant dams in the sevoflurane and control groups were fed a regular laboratory rodent diet with a low n-3 PUFAs concentration (0.5% of total fatty acid), whereas the sevoflurane with n-3 PUFAs group were fed the same diet, but supplemented with n-3 PUFAs (15 mg fish oil/g regular diet) from day 2 of pregnancy to 14 days after parturition. Dams were given free access to food and water, and dams for all groups were kept under identical housing conditions with a 12-h light cycle. On postnatal day 7 (P7), the rat pups in the sevoflurane and sevoflurane with n-3 PUFAs groups received sevoflurane anesthesia.
+
+P7 rats were placed in a sealed box ventilated with 3% sevoflurane in 60% oxygen and treated for 6 h. The temperature in the sealed box was maintained at 33–35°C. The total survival percentage of P7 rats after 6-h anesthesia was 88.4%; the likely cause of death was respiration depression. After anesthesia, the pups were returned to the dams. Control rat pups were placed in the same box without sevoflurane exposure and under identical experimental conditions. The flow chart for the experimental protocol is summarized in Figure 1.
+Blood gas analysis
+Twelve naïve P7 rats that that did not participate in other experiments were used to assess the effect of sevoflurane on blood gases. Blood was percutaneously aspirated from the left cardiac ventricle after 0, 2, 4, and 6 h of anesthesia (n = 3 per time point). From these samples, we measured partial pressures of carbon dioxide and oxygen, pH, and blood lactate and glucose levels with a Radiometer ABL 800 blood gas analyzer (Radiometer, Copenhagen, Denmark).
+
+BrdU injection
+To determine whether sevoflurane affects progenitor cell proliferation in the S-phase of the cell cycle, bromodeoxyuridine ((+)-5′-bromo-2′-deoxyuridine [BrdU]; 97%; Sigma-Aldrich, St. Louis, MO, USA) in 0.9% sterile saline solution was injected intraperitoneally using the procedure described by Wojtowicz [16]. The first dose (150 mg/kg) was administered immediately before sevoflurane treatment, and the three subsequent injections (50 mg/kg BrdU) were given at 24-h intervals following sevoflurane anesthesia.
+
+Tissue preparation and immunohistochemistry
+Animals were deeply anesthetized with chloral hydrate and then transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4. The brains were removed, postfixed overnight in 4% paraformaldehyde/PBS, and placed in 30% sucrose until they sank in the solution. Coronal sections (30 µm) were cut on a microtome (Leica CM1900 UV, Wetzlar, Germany) and every sixth section was stored in 30% sucrose containing 30% ethylene glycol to stain BrdU or cleaved caspase-3.
+
+For immunocytochemical detection of BrdU-labeled nuclei, DNA was denatured to expose the antigen for incubation with 2 N hydrochloric acid for 30 min at 37°C, followed by neutralization with two 10-min incubation periods in 0.5 M boric acid (pH 8.5) at room temperature (RT). Sections were subjected to three 10-min washes in PBS with 0.3% Triton-X with 10 min between each wash. Nonspecific epitopes were blocked with 1% serum for 30 min at RT, and were incubated overnight at 4°C with either BrdU (1∶100; BD Pharmingen, Franklin Lakes, NJ, USA) or cleaved caspase-3 (1∶1,000; Cell Signaling, Danvers, MA, USA) antibody in PBS and 1% serum. On day 2, the sections were incubated with the appropriate secondary fluorescent antibodies (Alexa Fluor 488, 1∶200; Invitrogen, Carlsbad, CA, USA) for 2 h at RT, followed by three 5-min washes in PBS. Nuclear counterstaining was performed with 4′,6-diamidino-2-phenylindole (1∶500; Beyotime Institute of Biotechnology, Haimen, China), which was followed by mounting and coverslipping with an aqueous mounting medium. Images were acquired with a microscope (Leica DM2500). BrdU- or cleaved caspase-3-positive cells were counted in a blinded manner at ×20 magnification [17]. Questionable structures were excluded from the count if their identification remained uncertain under ×40 magnification.
+
+Western blot analysis
+The cerebral cortex, thalamus, and hippocampus were harvested 18 h after sevoflurane treatment. The brain tissues were homogenized in RIPA buffer (Millipore, Temecula, CA, USA) containing complete protease inhibitor cocktail and 2 mM phenylmethylsulfonyl fluoride. The lysates were collected and centrifuged at 12,000 rpm for 30 min at 4°C. After the protein samples were quantified using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA), 60 µg of each sample was electrophoresed through a 14% sodium dodecyl sulfate-polyacrylamide gel and wet electrotransferred to 0.45-µm nitrocellulose membranes (Millipore). The blots were incubated overnight at 4°C with a polyclonal anti-cleaved caspase-3 antibody, and then incubated with a rabbit anti-mouse polyclonal horseradish peroxidase-conjugated secondary antibody (1∶5,000; Epitomics, Hangzhou, Zhejiang Province, China) at RT for 1 h. Protein signals were detected using an enhanced chemiluminescence detection system (Pierce Biotechnology). A β-actin antibody (1∶1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to normalize sample loading and transfer. Band intensities were densitometrically quantified using Gel-Pro Analyzer (Media Cybernetics, Bethesda, MD, USA).
+
+Neurobehavioral tests
+We used only male offspring (n = 9 per group) in the neurobehavioral tests to exclude estrogen influences on neurocognitive evaluations. The water maze setup in spatial reference memory task and memory consolidation task was shown in Figure 2.
+
+
+Morris water maze spatial reference memory Probe training: Rats trained for 4 consecutive days (postnatal days 35–38, P35–38) in the Morris water maze following treatment with a vehicle or 3% sevoflurane for 6 h. A platform (10.3-cm diameter) was submerged in a circular pool (180-cm diameter, 50-cm depth) filled with warm (23–25°C) opaque water. Rats performed two training sessions each day. In each session, rats performed four trials in which they were released from one of four pseudorandomly assigned release points while facing the tank wall. This provided two short and two medium swims per session. Animals were allowed 60 s to locate the hidden platform, and if they failed to find the hidden platform in the allotted time, the investigator guided the animal to the platform. In either case, the rats were removed from the platform after 15 s. Training sessions were conducted until the rats could locate the hidden platform in less than 15 s in at least five sessions (average time per session). All trials were videotaped, and rat swim paths were recorded with ANY-maze video tracking system (Stoelting Co., Wood Dale, IL, USA), which allowed us to measure the time taken (latency) to find the platform(s), as well as other behavioral information obtained during the spatial reference memory test. The animals were dried and placed beneath a heating lamp after completing each test.
+Probe test: A probe trial was performed with the platform removed from the tank to assess memory retention for the hidden platform location. Probe trials were administered 1 day after the last training session (P38). During the 60-s probe trial, we determined the number of entries into the platform quadrant zone, the swimming speed (cm/s), the total distance (cm), and the time spent in the target quadrant relative versus the other quadrants.
+
+Fear conditioning test Rats underwent fear conditioning tests on postnatal days 63–64 (P63–64). Every time four rats randomly chosen from three groups were trained in each session. Rats were placed in plastic chambers with a grid floor constructed from 19 stainless steel bars (4-mm diameter, spaced every 16 mm). The floors were connected to a shock delivery system (Coulbourn, Whitehall, PA, USA), and electrical shocks were delivered through the stainless steel bars. The chamber was illuminated with overhead fluorescent bulbs, and a ventilation fan provided background noise (65 db). The training context was considered the appearance, odor, and texture of the environment (chamber and room) in which the rats were trained. After a 3-min baseline exploratory period, rats were presented with three auditory tones (2,000 Hz, 90 db) that were followed 1 min later by an electric shock (1 mA, 2 s). We quantified the rats' fear response with freezing, which is an innate defensive fear response in rodents and a reliable measure of learned fear. Freezing was defined as the lack of movement, except for respiration. We examined rats in the fear condition test the day after they first received the electrical shock to determine whether they showed fear to the training context or the auditory tone. For the context test, rats were placed in the chamber where they were trained on the previous day. The rats remained in the chamber for 8 min, without an auditory tone or shock. For the tone test, rats were transported in groups to a context chamber with black boards covering the walls. Rats were allowed a 3-min exploratory period before three 30-s tones were played (2,000 Hz, 90 db, separated by 60 s). Rats were removed from the chamber 30 s after the tone presentation. The order of the context and tone tests was counterbalanced so that half of each treatment group first was tested for context and then for tone, whereas the other half of the treatment group was tested in the reverse order. FreezeView software (Coulbourn) was used to score each animal's freezing behavior separately for the training period and the context and tone tests, which were expressed as a percentage.
+Morris water maze memory consolidation Working memory (WM): On postnatal day 70 (P70), the testing room was rearranged by repositioning the water tank and adding new spatial cues. The platform was submerged 1.5 cm below the water surface in one of four designated platform positions. From P70 onward, one session was conducted per day. Each session began with a 60-s free swim (performance not scored) in which rats explored the maze, and was followed by a 1-min rest interval and three subsequent scored trials. Rats that found the platform during the free swim were allowed to rest on the platform for 15 s. Rats that failed to find the platform during the free swim were guided to the platform and remained there for 15 s. After the free swim, three trials were administered in which the rat was released from one of six pseudorandomly chosen locations that faced the tank wall. The platform location was identical for all animals in a session, but the drop location was pseudorandomly varied to incorporate one short, one medium, and one long swim. Training sessions were administered until the session average for finding the hidden platform was less than 15 s. The latency for reaching the platform was recorded by the ANY-maze video tracking system.
+Short-term memory (STM) and early long-term memory (ELTM): When the WM latencies of rats in task were plateaued on postnatal day 77 (P77), we increased the delay between the free swim and the subsequent trials. The delay was extended from 1 min on P77 to 1 h on postnatal day 78 (P78) to test STM, and then to 4 h on P79 to test ELTM. Performances on the last trial after the free swim on P77 (1-min delay), P78 (1-h delay), and P79 (4-h delay) were used as measures of WM, STM, and ELTM, respectively.
+
+Statistical methods
+All data are presented as mean ± standard deviation. We performed two-tailed t tests (assuming equal variances) to determine differences in cleaved caspase-3 immunohistochemistry and blood gas parameters between the control and sevoflurane groups. We used a one-way analysis of variance followed by Newman-Keuls post hoc tests to determine differences among groups for interactions between n-3 PUFAs or sevoflurane and cleaved caspase-3 activation, BrdU quantification, or neurobehavioral tests. For all tests, p<0.05 was considered statistically significant.

20230808-AI coding-1st round/570 – Huang 2015.txt

@@ -0,0 +1,18 @@
+PMID: 25591773 DOI: 10.1159/000369698
+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/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.
+
+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.

20230808-AI coding-1st round/579 – Lu 2016.txt

@@ -0,0 +1,27 @@
+PMID: 26898457 DOI: 10.1016/j.biopha.2016.01.034
+2. Materials and methods
+2.1. Animals
+Because peak anesthesia-induced neurodegeneration in rodents occurs on postnatal day (PND) 7 [22], Sprague-Dawley (SD) PND7 rats weighing 14–18 g, provided by the Animal Center of Shanghai Jiao Tong University School of Medicine (Shanghai, China) were used in this study. The housing and treatment of the animals were in accordance with the National Institutes of Health guidelines for animal experimentation and approved by the institutional animal care and use committee. The animals were kept on a 12-h light/dark cycle (light from 7 am to 7 pm) with room temperature (23 ± 1 °C).
+
+2.2. Sevoflurane exposure
+Rat pups were separated from their mothers for acclimatization prior to sevoflurane exposure. Pups from the same litter were randomly allocated to three different groups. Totally, ninety PND7 rats were included in this study (n = 30 for each group). Rats in the control group received 100% oxygen for 6 h in a chamber at 37 °C. Rats in the other two groups were exposed to either 2% sevoflurane (SEVOFRANE®, Osaka, Japan) for 3 h (Sevo1 group) or 3% sevoflurane for 6 h (Sevo2 group) under 100% oxygen in the same chamber at 37 °C as described previously [13]. The concentration of sevoflurane in the chamber was monitored and maintained by a vaporizer as we described previously [23]. The gas flow to the chamber was 2 l/min. We chose these treatments because 3 h exposure to 2% seveflurane more closely approximates typical general pediatric anesthetic episodes for anesthesia maintenance [16] and 6 h exposure to 3% sevoflurane can cause neuronal apoptosis in developing animals [11], [12], [14], [15].
+
+2.3. Arterial blood gas analysis
+To determine adequacy of ventilation and oxygenation，arterial blood samples (n = 6) were obtained from the left cardiac ventricle in each group at the end of anesthesia, and the samples were immediately analyzed by a blood gas analyzer (Radiometer, ABL800, Denmark). We compared the pH, pO2, pCO2, oxygen saturation (sO2), and the concentrations of blood glucose (Glu), lactic acid (Lac) and bicarbonate (HCO3−) among the groups. Animals were killed by lethal injection of pentobarbital at the time of blood sampling.
+
+2.4. Analysis of apoptotic levels
+2.4.1. TUNEL assay of brain
+Twenty-four hours after sevoflurane exposure, six rats from each group (n = 6) were anesthetized with sodium pentobarbital and the brains were perfused, fixed, dehydrated and made into paraffin sections (5 μm), as described previously [24]. Apoptotic cells in the brain sections were detected by TUNEL Assay using the FragEL™ DNA Fragmentation Detection Kit (Merck, Darmstadt, Germany), according to the manufacturer’s protocol. Briefly, brain sections were permeabilized with proteinase K (20 μg/ml) at room temperature for 20 min. Endogenous peroxidase was inactivated by 3% H2O2. Specimens were incubated for 1.5 h with terminal deoxynucleotidyl transferase (TdT) labelling reaction mixture, and apoptotic cells were visualized with 3,3′-diaminobenzidine (DAB), and normal nuclei were counterstained with methyl green. Because the cerebral cortex reaches peak vulnerability to anesthetics at PND7 and the hippocampus is closely related to learning and memory [25], the number of apoptotic neurons in the frontal cortex and the CA1 region of the hippocampus was quantified. We selected two random viewing fields (400×) per region (frontal cortex and CA1) from one brain section per animal for analysis in a double blinded manner.
+
+2.4.2. Western blot
+Apoptosis was also assessed using western blot to quantify cleaved caspase-3 (Cl-Csp3) in all groups (n = 6). Briefly, tissue samples of the frontal cortex and CA1 region were collected from three groups twenty-four hours after sevoflurane exposure. Tissues were lysed in a buffer containing a protease inhibitor cocktail (Calbiochem, San Diego, CA, USA) and homogenated. The homogenate was centrifuged and the supernatant was collected for further analysis. Protein concentrations were measured by BCA Protein Assay Kit (Novagen, San Diego, CA, USA). Equal amounts of protein were boiled in loading buffer (Beyotime, Beijing, China) and separated by 10% polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose, and the blots were probed overnight with anti-cleaved caspase-3 (1:200, Millipore, Darmstadt, Germany) and β-actin antibodies (1:500, internal standard, Santa Cruz, San Diego, CA, USA) at 4 °C. Primary antibodies were visualized using secondary antibodies conjugated to horseradish peroxidase (Santa Cruz, San Diego, CA, USA) and ECL reagent (Pierce, Rockford, IL, USA). Quantitative analysis of Cl-Csp3 was normalized to β-actin using the Quantity One software.
+
+2.5. Neurologic assessment
+2.5.1. Morris water maze
+To assess neurodevelopmental outcomes, particularly the learning and memory functions of juveniles, rats from all groups were subjected to Morris water maze after reaching 6 weeks of age (n = 12), as previously described [24]. Briefly, a circular pool (1.6 m diameter, 60 cm height) was used for the water maze, and a submerged platform (10 cm diameter, 2 cm below the surface of the water) was located at a fixed position in the pool. The water temperature was set at 23 ± 1 °C. Probe trials were conducted twice a day for five consecutive days. In the trials, rats were trained to swim to and locate the hidden platform. The time spent in finding the hidden platform and the swimming distance before reaching the platform were recorded. After the probe trials, the platform was removed, and the rats were allowed to swim freely for 120 s: the number of times that the former platform was crossed and the percentage of time spent in the target quadrant were determined. The entire behavioral test was recorded and analyzed using a MS-type Water Maze Video analysis system (Chengdu Instrument Ltd., Chengdu, China). Finally, to investigate cognitive function during development, the passive avoidance test was performed at 3 months.
+
+2.5.2. Passive avoidance test
+The passive avoidance test was performed as previously described [26]. The apparatus used for the passive avoidance test included a behavioral stimulation controller and a video shuttle box (Chengdu Instrument Ltd., Chengdu, China). The test relies the natural preference of rats for darkness. Briefly, on the first trial day, the rats were placed in the illuminated compartment after 2 min of habituation to the dark compartment and allowed to re-enter the dark compartment. On the following day, an electric foot shock was delivered through the grid floor of the dark compartment after the rats entered. Twenty-four hours later, the retention of passive avoidance was determined by comparing the time elapsed prior to re-entry into the dark compartment with the arbitrary maximum time of 180 s.
+
+2.6. Statistical analysis
+All data are expressed as the mean ± SEM. SAS 9.2 (SAS Institute Inc., Cary, North Carolina, USA) was used for statistical analysis. One-way ANOVA was used to determine statistically significant differences between the three groups, and Tukey’s post hoc analysis was performed to correct for multiple comparisons when applicable. Statistical significance was accepted as P < 0.05.

20230808-AI coding-1st round/588 – Sun 2016.txt

@@ -0,0 +1,29 @@
+PMID: 27147542 PMCID: PMC4913390 DOI: 10.1093/bja/aew064
+Methods
+Ethical approval
+The Animal Care and Use Committee of Tokyo Medical and Dental University approved the protocol and the study was performed in accordance with relevant aspects of ARRIVE guidelines.
+
+Experimental mice
+Postnatal day five C57BL/6 mice (average body weight: 2.7 g), as a litter with their mother, were purchased from SLC (SLC Japan Inc., Shizuoka, Japan). Mice were housed under a 12-h light-dark cycle (lights on from 08:00 to 20:00), and room temperature was maintained at 21 (1°) C. The same number of pups from each litter was used for the experiments to reduce variability related to the use of different litters (total number of pups used=110). For the behavioural study, male mice were weaned at three or four weeks of age and housed four mice per cage for each experimental group. All mice had ad libitum access to water and food. Behavioural testing was carried out only on male mice to avoid potential variability caused by the oestrous cycle.16
+
+Anaesthesia
+We used sevoflurane anaesthesia protocol as previously published.3 On postnatal day six, mice were placed in a humidified chamber (180×180×200 mm) heated to 38 (1)°C, and were exposed to 3% sevoflurane in 40% oxygen for 6 h, which was delivered via a calibrated flowmeter (Shinano, Tokyo, Japan), with a total gas flow of 1 L min−1. During sevoflurane anaesthesia, all mice maintained spontaneous breathing and eventually exhibited the loss of the righting reflex.
+
+For the experimental treatments, the pups from the same litter were randomly divided into four groups; (1) non-anaesthesia (NA group, control mice), (2) NADPH oxidase inhibitor treatment (apocynin) group where mice received intraperitoneal apocynin, 50 mg kg−1,17,18 in a total injection volume of 10 µl, (3) 3% sevoflurane exposure (SEVO group, where mice received 3% sevoflurane for 6 h), and (4) apocynin in combination with sevoflurane (apocynin+SEVO group, where mice received 3% sevoflurane for 6 h in addition to intraperitoneal apocynin, 50 mg kg−1). Apocynin powder (Abcam, Cambridge, MA, USA) was dissolved in ethanol to make a stock solution, which was then diluted with phosphate buffered saline (PBS) to the appropriate concentration for administration. As pups are very small, injection volumes were limited to 10 µl and so the percentage of alcohol in the solution needed to be sufficient to fully dissolve the apocynin powder. However, during perfusion, the abdominal tissue did not show any sign of damage 24 h after anaesthesia induction, suggesting that local ethanol toxicity was negligible. The preparation of apocynin i.p. injection was done according to previously published studies.17,18 Mice in the NA and apocynin groups received carrier gas. Mice were returned to the original litter after treatment.
+
+Detection of superoxide
+Mice were anaesthetized using i.p. pentobarbital (50 mg kg−1) and adequate anaesthesia was ascertained by lack of response to tail pinch. They were then rapidly transcardially perfused with 10 ml cold 0.1 M PBS and the brain was quickly removed and immediately frozen at −80°C. Slices, 10-μm-thick, were cut on a cryostat and mounted onto microscope slides. Concentrations of superoxide in the brain were assessed using dihydroethidium (DHE) (Sigma-Aldrich, St. Louis, MO, USA) fluorescence.19 Each slice was incubated with 10 μM DHE for 30 min at 37°C in the dark. After incubation, the sections were washed with PBS (pH 7.4) three times and analysed by fluorescence microscopy using the Zeiss LSM Image Browser (Carl Zeiss MicroImaging, Jena, Germany). Mean DHE fluorescence values of five regions of interest in the cortex layer (two for each slice) were quantified.
+
+Histopathological evaluation
+For histopathology, mice were anaesthetized as before, 18 h after treatment, and perfusion was performed. The brain was removed and immersed in 4% paraformaldehyde in PBS overnight at 4°C. Subsequently, 50-μm-thick coronal sections were cut, and then washed with PBS in 0.3% Triton X-100 for 10 min, and endogenous peroxidase activity was blocked with 1% H2O2 for 30 min. Thereafter, sections were incubated with rabbit anti-cleaved caspase-3 antibody (Cell Signaling Technology; 1:300) in PBS/Tween-20 overnight at 4°C for detecting apoptosis. Sections were incubated with peroxidase-conjugated secondary antibody (EnVision+System, Dako, Tokyo, Japan) for 2 h and washed with PBS. Then, the sections were reacted with 3, 3′-diaminobenzidine (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions. The sections were mounted on slides and counterstained with 0.5% neutral red solution. Our previous investigation3 showed that the apoptotic response to sevoflurane was robust over the whole brain.
+
+Western blot analysis
+For protein analysis using western blotting, mice were anaesthetized as before and 18 h after treatment, whole brains were quickly removed, and immediately homogenized on ice in 100 μl homogenization buffer containing 20 µM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM Na4P2O7, and protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). After centrifugation at 21,000×g for 30 min at 4°C, the supernatant was removed and stored at −80°C until use. The amount of protein in each sample was measured using a protein assay kit (BCA; Pierce, Rockford, IL, USA).
+
+For sodium dodecyl sulfate polyacrylamide gel electrophoresis, brain protein homogenates were mixed with lane marker sample buffer (Pierce) and denatured for 5 min at 95°C. The samples were cooled, separated using a 10% Mini-PROTEAN TGX™ Gel (Bio-Rad, Hercules, CA, USA), and transferred onto a nitrocellulose membrane (Bio-Rad). Rabbit anti-4-hydroxynonenal (4-HNE) antibody (Abcam; 1:1000) was used to detect concentrations of the oxidative stress marker. Rabbit anti-p22phox and rabbit anti-gp91phox antibodies (Santa Cruz Biotechnology, Dallas, TX, USA; both 1:1000) were used to detect NADPH oxidase subunits. For detecting apoptosis, rabbit anti-cleaved caspase-3 and rabbit anti-cleaved poly [adenosine diphosphate-ribose] polymerase (PARP) antibodies (Cell Signaling Technology, Beverly, MA, USA; both 1:1000) were used. Rabbit anti-cytochrome c antibody (Abcam; 1:5000) was used to evaluate mitochondrial function. After blocking in 5% non-fat milk for 30 min, the membrane was incubated with primary antibody in TBS containing 0.1% Tween-20 overnight at 4°C. The membrane was then incubated with secondary antibody (Abcam; 1:10,000) for 1 h at room temperature after washing with Tris-Buffered Saline and Tween-20. Mouse β-actin antibody (Sigma-Aldrich; 1:5000) was used as the loading control. The protein bands were visualized using a chemiluminescence detection system (SuperSignal West Pico; Pierce). For quantification of the oxidative stress marker 4-HNE, the total intensity of the 4-HNE lane was compared between groups.20,21
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+Fear conditioning test
+To assess long-term cognitive impairment induced by sevoflurane exposure, the fear conditioning test was performed.3,8 Male mice in each experimental group underwent behavioural testing in adulthood (11–13 weeks of age). The fear conditioning test was carried out in the daytime (between 09:00 and 12:00). The movement of mice was monitored using a computer-operated video tracking system. The apparatus used in this study were made by O'Hara & Co., Ltd. (Tokyo, Japan). The conditioning trial for contextual and cued fear conditioning consisted of a 6 min period followed by three conditioned stimulus–unconditioned stimulus pairings, each separated by 1 min. Each pairing was as follows: unconditioned stimulus, 0.5 mA foot shock intensity, 1 s duration; conditioned stimulus, 60 dB white noise, 20 s duration. The unconditioned stimulus was delivered during the last few seconds of the conditioned stimulus presentation. The contextual test was performed in the conditioning chamber for a five min period in the absence of white noise 24 h after conditioning. A cued test (on the same set of mice) was performed by presentation of a cue (60 dB white noise, 3 min duration) in an alternative context with distinct visual and tactile cues 48 h after conditioning. The duration of freezing time was recorded to assess fear memory. The freezing percentage during each test was compared between each group.
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+Statistical analysis
+Data are shown as individual raw data points. Data from behavioural testing are expressed as mean (sd). DHE fluorescence and protein expression are expressed as a percentage of the control (NA group) value. Statistical analysis was performed using SPSS statistical software package (Version 18, SPSS Inc. Chicago, IL). Kruskal-Wallis omnibus test was used for comparisons of non-parametric data and behavioural data were analysed using anova and post hoc Neuman-Keuls multiple comparison test. A P-value<0.05 was considered statistically significant.