offline_results/exp_test_old.csv

DOI,Citation ID,First author,Year,animal type,exposure age,behavior test: Y/N,intervention1,intervention2 （anesthetics only),genetic chain,content,Question 1,Question 1_original_sentences,Question 2,Question 2_original_sentences,Question 3,Question 3_original_sentences,intervention_1,Question 4intervention_1_original_sentences,intervention_2,Question 4intervention_2_original_sentences,Question 5,Question 5_original_sentences,correct_1,correct_2,correct_3,correct_4,correct_5,correct_6,fn
10.1016/j.biopha.2018.09.111,279.0,Bi,2018,rats,postnatal day 7,Y,sevoflurane,none,sprague dawley,"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
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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.",rats,"['A total of 30 Sprague –Dawley (SD) rats(10 males, 20 females), weighting 220–250\u2009g, were purchased from Liaoning Changsheng Bio-Technology Co., Ltd.']",postnatal day 7,"['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\u2009g, were used in this study.']",Y,"['To assess neurodevelopmental outcomes in adolescence, particularly learning and memory functions, twenty-four rats from four groups including control, control\u2009+\u2009SP600125, sevoflurane and sevoflurane\u2009+\u2009SP600125, were subjected to the MWM after 28 d (six rats in each group), as previously described.']",sevoflurane,"['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\u2009h.']",none,[],sprague dawley,"['A total of 30 Sprague –Dawley (SD) rats(10 males, 20 females), weighting 220–250\u2009g, were purchased from Liaoning Changsheng Bio-Technology Co., Ltd.']",True,True,True,True,True,True,[ Passage 1/12 ] 10.1016/j.biopha.2018.09.111
10.1093/toxsci/kfn152,1107.0,Boctor,2008,rats,postnatal day 7,Y,ketamine,none,sprague dawley,"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.",rats,"['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.']",postnatal day 7,"['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. ... 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.']",Y,"['Home Cage Pup Behavior To determine the immediate effects of treatment, home cage behavior was assessed on PNDs 7–11.', '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).', 'Forelimb Hang Behavior Muscle strength/coordination was examined using a forelimb hang test as previously described (Cada et al., 2000).']",ketamine,"['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.']",none,[],sprague dawley,"['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.']",True,True,True,True,True,True,[ Passage 2/12 ] 10.1093/toxsci/kfn152
10.1093/bja/aet073,794.0,Boscolo,2013,rats,postnatal day 7,Y,"midazolam, isoflurane, nitrous oxide",none,sprague dawley,"PMID: 23616588 PMCID: PMC3732064 DOI: 10.1093/bja/aet073
Methods
We exposed postnatal day 7 (P7) Sprague-Dawley rats of both sexes to one of four treatment protocols: (i) sham controls (mock GA-vehicle, 0.1% dimethyl sulfoxide+21% oxygen for 6 h); (ii) GA-treated (midazolam, 9 mg kg−1, i.p.; single injection immediately before administration of 0.75% isoflurane+75% nitrous oxide+24% oxygen for 6 h); (iii) GA+PPX-treated (PPX, 1 mg kg−1 i.p.; four doses—at 9 h before, immediately before, immediately after, and 9 h after 6 h of GA); and (iv) PPX alone (same dosing regimen but mock GA). At the end of the treatments, rat pups were reunited with their mothers. Rats were housed using standard housing on a 12 h light/dark cycle with ad libitum access to food and water. All experiments were approved by the Animal Care and Use Committee of the University of Virginia Health System and were done in accordance with the Public Health Service's Policy on Human Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used.
R(+)PPX doses and the dosing regimen were selected based on results from our prior work7 and the half-life of R(+)PPX which is estimated to be 8–12 h9 [i.e. R(+)PPX was dosed ∼every half-life around the time of anaesthesia exposure]. An adult rodent dose of 1 mg kg−1 is equivalent to a human dose of 0.2 mg kg−1.9 10 Thus, these doses are very small and clinically feasible. Based on high-pressure liquid chromatography assays, R(+)PPX was >99.9% chemically and >99% enantiomerically pure.11
We found no differences among the groups in general appearance or body weight over the next 7 months (data not shown). Cognitive abilities were assessed between 5 and 7 months of age using the Morris water maze test with the adult size pool (180 cm inner diameter).1 To examine their ability to swim, animals were tested in cued trials using a visible platform that was switched to a new location for each trial. During the place trials, rats were tested on their ability to learn the location of a platform (submerged, not visible), which remained in the same location during all trials. Two acquisition place trials were performed, each four blocks long (2 days per block). Probe trials were performed after each acquisition place trial (after blocks four and nine). During these trials, the platform was removed and times and patterns of swimming were analysed with special attention focused on time spent in the target quadrant.
Data were analysed by analysis of variance using treatment and sex as between-subject variables and blocks of trials as within-subject variables. Pairwise comparisons were done after analysis of significant treatment effects and P-values exceeding Bonferroni corrected levels were noted. We considered P<0.05 to be statistically significant.",rats,['We exposed postnatal day 7 (P7) Sprague-Dawley rats of both sexes to one of four treatment protocols:'],postnatal day 7,['We exposed postnatal day 7 (P7) Sprague-Dawley rats of both sexes to one of four treatment protocols:'],Y,['Cognitive abilities were assessed between 5 and 7 months of age using the Morris water maze test with the adult size pool (180 cm inner diameter).'],midazolam,"['GA-treated (midazolam, 9 mg kg−1, i.p.; single injection immediately before administration of 0.75% isoflurane+75% nitrous oxide+24% oxygen for 6 h);']",isoflurane,"['GA-treated (midazolam, 9 mg kg−1, i.p.; single injection immediately before administration of 0.75% isoflurane+75% nitrous oxide+24% oxygen for 6 h);']",sprague dawley,['We exposed postnatal day 7 (P7) Sprague-Dawley rats of both sexes to one of four treatment protocols:'],True,True,True,False,False,True,[ Passage 3/12 ] 10.1093/bja/aet073
10.1093/bja/aes121,505.0,Feng,2012,rats,postnatal day 7,N,sevoflurane,none,sprague dawley,"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.",rats,['Male Sprague–Dawley (sd) rats were obtained from the Experimental Animal Centre of Sun Yat-sen University.'],postnatal day 7,"['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).']",Y,"['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.']",sevoflurane,['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.'],none,[],sprague dawley,['Male Sprague–Dawley (sd) rats were obtained from the Experimental Animal Centre of Sun Yat-sen University.'],True,True,False,True,True,True,[ Passage 4/12 ] 10.1093/bja/aes121
10.1038/s41598-021-85125-5,966.0,Hogarth,2021,rats,postnatal day 7,N,sevoflurane,none,sprague dawley,"PMID: 33707598 PMCID: PMC7952562 DOI: 10.1038/s41598-021-85125-5
Materials and methods
Experimental animals
Experiments were performed using male Sprague Dawley rats (total N = 12), randomly assigned to experimental (sevoflurane exposed) and control groups. Experimental and control animals were equally co-populated and maintained in identical environments, including food and water, temperature and light/dark cycles, to the best of our ability. There was no animal mortality during the entire time of observation. All experiments were approved by the Institutional Animal Care and Use Committee of the University of California (San Francisco, California) and performed in accordance with national and institutional guidelines for animal care. All animal experiments were carried out according to the ARRIVE 2.0 guidelines, as described in the relevant section.

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

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

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

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

ADP/ATP ratio assay
As an indicator of cellular energy status, ADP/ATP ratio was performed using the Abcam Bioluminescent ADP/ATP Ratio Assay Kit and performed according to manufacturer’s instructions (ab65313, Cambridge, MA, USA). In brief, 100 µL of ATP Reaction Mix (1× ATP Monitoring Enzyme in Nucleotide Releasing Buffer) was loaded on a 96-well plate, followed by a baseline plate reading. To measure ATP levels, 100 µg of cortical lysate was then added to each well, followed by 2 min incubation and plate read. Finally, to determine ADP levels, 100 µL ADP (1× ADP Converting Enzyme in Nucleotide Releasing Buffer) was then added to each well and incubated for 2 min, followed by a final plate reading. ADP and ATP content quantified using a standard curve. Bioluminescence measured on Varioskan LUX Plate Reader (Thermo Scientific, MA, USA).

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

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

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

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

Statistical analysis
Unless otherwise noted, all results are presented as means ± SEM. Following a Kolmogorov–Smirnov test for normalcy, statistical significance was determined using either un-paired Student’s t-test with Welsh correction or un-paired Mann–Whitney U-test, as appropriate. Where more than one comparison was performed, significance was determined using an ANOVA, using the Dunnett method for multiple comparison correction. Statistical significance was calculated using GraphPad Prism 6.0 using a p-value of < 0.05 as a cut-off for significance.",rats,"['Experiments were performed using male Sprague Dawley rats (total N\u2009=\u200912), randomly assigned to experimental (sevoflurane exposed) and control groups.']",postnatal day 7,"['Briefly, on postnatal day 7 (P7), rat pups were randomly assigned to either exposed to 1 minimum alveolar concentration of sevoflurane (corresponding to\u2009~\u20095% atm), or identical environment without anesthetic agent as sham controls (6 animals per group) for a total of 4 h (FiO2\u2009=\u20090.5), as we performed previously11,13,21,22,47.']",N,"[""The document does not mention any behavior tests such as 'Open field test', 'Morris water task', 'fear conditioning test', 'Dark/light avoidance'; 'passive/active avoidance test'; 'elevated maze', 'Forced swim test', 'Object recognition test', 'Social interaction/preference'.""]",sevoflurane,"['Experimental animals Experiments were performed using male Sprague Dawley rats (total N\u2009=\u200912), randomly assigned to experimental (sevoflurane exposed) and control groups.']",isoflurane,"['Ten months following anesthetic exposure (P300), all animals were euthanized following deep anesthetization with isoflurane (greater than 5% with loss of pedal pain reflex) by subsequent transcardial perfusions of 0.9% saline and 4% paraformaldehyde in 0.1 M phosphate buffered saline (pH 7.4).']",sprague dawley,"['Experiments were performed using male Sprague Dawley rats (total N\u2009=\u200912), randomly assigned to experimental (sevoflurane exposed) and control groups.']",True,True,True,True,False,True,[ Passage 5/12 ] 10.1038/s41598-021-85125-5
10.1159/000369698,570.0,Huang,2015,rats,postnatal day 7,N,ketamine,none,sprague dawley,"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.",rats,['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.'],postnatal day 7,['The PND-7 male rats (11-14g) selected from all the pups were used in the experiments.'],N,"[""The document does not mention any behavior tests such as 'Open field test', 'Morris water task', 'fear conditioning test', 'Dark/light avoidance'; 'passive/active avoidance test'; 'elevated maze', 'Forced swim test', 'Object recognition test', 'Social interaction/preference'.""]",ketamine,"['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.']",none,[],sprague dawley,['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.'],True,True,True,True,True,True,[ Passage 6/12 ] 10.1159/000369698
10.1016/j.neuropharm.2014.03.011,359.0,Lee,2014,rats,postnatal day 7,Y,isoflurane,none,sprague dawley,"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).",rats,"['Sprague Dawley dams with litters containing male-only and female-only pups were obtained from Charles River Laboratories (Gilroy, CA).']",postnatal day 7,"['On postnatal day (P)7, animals were randomly assigned to control or treatment groups (Fig. 1).']",Y,"['All behavioral testing occurred during the light cycle between 0800 and 1700 h.', 'Testing began at postnatal day 38 (P38) with novel object recognition (Fig. 3).', 'Social interaction and recognition were assessed using a discrimination paradigm.']",isoflurane,"['Briefly, isoflurane was delivered into the anesthetic chamber, and gas concentrations were continuously monitored.']",none,[],sprague dawley,"['Sprague Dawley dams with litters containing male-only and female-only pups were obtained from Charles River Laboratories (Gilroy, CA).']",True,True,True,True,True,True,[ Passage 7/12 ] 10.1016/j.neuropharm.2014.03.011
10.3892/etm.2017.5004,4315.0,Ling,2017,rats,postnatal day 7,Y,sevoflurane,none,sprague dawley,"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.",rats,"['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.']",postnatal day 7,"['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.']",Y,"['Behavioral experiments As described in our previous study (9), three tests were conducted in sequence, including the elevated plus-maze (EPM), O-maze and Y-maze.']",sevoflurane,"['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.']",isoflurane,"['Rats were anaesthetized by isoflurane with an induction dosage of 4%, maintained at 2% (RuiTaibio, Beijing, China) and decapitated to obtain the hippocampus.']",wistar,"['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.']",True,True,True,True,False,False,[ Passage 8/12 ] 10.3892/etm.2017.5004
10.1016/j.brainres.2014.02.008,618.0,Liu,2014,rats,postnatal day 7,N,sevoflurane,none,sprague dawley,"PMID: 24518287 DOI: 10.1016/j.brainres.2014.02.008
4. Experimental procedures
4.1. Animals
This experiment was approved by the Institutional Animal Care and Use Committee at Tianjin Medical University. Sprague-Dawley (SD) rats (both male and female) were obtained from the Experimental Animal Centre of Tianjin Medical University, Tianjin, China. Rats were housed under 12 h light-dark cycles with free access to food and water at room temperature (21–24 °C). 1-week-old SD rats were randomly assigned to control group, 2.1% sevoflurane group and 3% sevoflurane group to receive gas exposure. After gas exposure, electrophysiological recording were performed on rats of 5 different ages (from 1 week to 5 weeks).

4.2. Sevoflurane exposure
The rats were placed in a home-made anesthetic induction chamber using an electric blanket to maintain the temperature at 35–37 °C, and calcium lime was spread on the bottom of the chamber to absorb carbon dioxide. Rats in 3% sevoflurane group and 2.1% sevoflurane group were respectively exposed to 3% and 2.1% sevoflurane for 6 h using oxygen as gas carrier with a gas flow of 4 L/min, while the rats in control group breathed independently oxygen with a gas flow of 4 L/min for 6 h in the same induction chamber. During sevoflurane exposure, the gas concentration of carbon dioxide, oxygen and sevoflurane in the chamber were monitored by gas monitor (Detex-Ohmeda, Louisville, KY, USA). After anesthesia, rats were exposed to oxygen only until they recovered from anesthetic. Then, all of rats were placed back into the maternal cages.

4.3. Blood gas analysis
As described previously (Feng et al., 2012, Lu et al., 2006), we performed blood gas analysis on rats of the both 3 treated groups before anesthesia exposure (0 h) and at the end of 6 h of anesthesia (6 h). Three pups of each group were used for that analysis. Arterial blood samples were collected from the left cardiac ventricle and immediately transferred into the blood gas analyzer (GEM Premier 3000, Instrumentation laboratory, Bedford, MA) to measure the blood pH, PaCO2 (arterial carbon dioxide tension) and PaO2 (arterial oxygen tension). The values of blood pH, PaCO2 and PaO2 in the sevoflurane groups did not significantly differ from that values in control group (Table 4).
4.4. Acute dissociation of hippocampal CA1 pyramidal neurons
In this study, hippocampal pyramidal neurons were acutely isolated as described previously by Zhang et al. (2009) with some modifications. Briefly, rat was decapitated and the brain was rapidly removed, iced for 1–2 min in 0–4 °C artificial cerebrospinal fluid (ACSF) bubbled with 95%O2+5%CO2 gas. ACSF solution contains 120 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 and 10 mM Glucose, pH 7.4, with HCl and NaOH. The hippocampus was separated out and cut into 400–600 μm thick slices on home-made ACSF ice pillow. The slices were then placed into ACSF bubbled with 95%O2+5%CO2 to incubate for 1 h at 32 °C. The CA1 region of hippocampus was dissected out using 1 mL syringe needle under stereomicroscope and transferred into ACSF containing 0.67 mg/mL Pronase XIV (Sigma) saturated with 95%O2+5%CO2 gas to dissociate for 20–45 min at 32 °C (1 w:20 min; 2 w:30 min; 3 w/4 w:40 min; 5 w:45 min). After enzyme digestion, slices were washed three times in standard extracellular fluid bubbled with 100% O2 gas and dissociated by a graded series of fire-polished Pasteur pipettes. The standard extracellular fluid contains 130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM Glucose and 10 mM HEPES, pH 7.4, with HCl and NaOH. Then, the cell suspension was plated into a 35 mm Lux Petri dish containing oxygen saturated standard extracellular fluid. And the neurons were allowed to settle to the button of the dish before recording.

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

Under inverted microscope (BX51-WI, Olympus, Japan), we selected randomly pyramidal neurons with a smooth and bright appearance and no visible organelles for recording. During the recording, neurons were bathed in an extracellular solution, which contains 110 mM Choline chloride, 20 mM TEA-Cl, 10 mM 4-AP, 1 mM MgCl2, 10 mM BaCl2, 10 mM HEPES, 10 mM Glucose and 0.001 mM TTX, PH=7.4, with HCl. Voltage-clamp recordings were obtained with Axon patch 200B patch-clamp amplifier (Molecular Devices, Foster City, CA, USA) and Digidata 1440 interface (Molecular Devices, Foster City, CA, USA). After establishing a gigaseal (>2 GΩ), the membrane was broken to form whole-cell configuration. Series resistance and membrane capacitance were routinely compensated by 60–80%, leakage and capacity currents were subtracted on-line using a P/4 protocol. The data were acquired using pCLAMP 10.0 (Molecular Devices, Foster City, CA, USA) running on a computer. Recording signals were low-pass filtered at 2 kHz and digitized at 10 kHz. In this study, whole-cell recording was voltage-clamped at −50 mV. Data were obtained from 10 to 14 cells per group.

4.6. Data analysis
Current density was calculated by dividing current at membrane potential by the cell-membrane capacitance (i.e. pA/pF). All data were analyzed by pCLAMP Clampfit 10.0 (Molecular Devices, Foster City, CA, USA), Origin 6.0 (OriginLab Corp, Northampton, MA, USA). The data were presented as means±standard error of the mean (S.E.M.), one way ANOVA followed by SNK post hoc test (SPSS, Chicago, IL) were used for statistical analysis. Significant level was set to 0.05.",rats,"['Sprague-Dawley (SD) rats (both male and female) were obtained from the Experimental Animal Centre of Tianjin Medical University, Tianjin, China.']",postnatal day 7,"['1-week-old SD rats were randomly assigned to control group, 2.1% sevoflurane group and 3% sevoflurane group to receive gas exposure.']",N,"['After gas exposure, electrophysiological recording were performed on rats of 5 different ages (from 1 week to 5 weeks).', 'During sevoflurane exposure, the gas concentration of carbon dioxide, oxygen and sevoflurane in the chamber were monitored by gas monitor (Detex-Ohmeda, Louisville, KY, USA).', 'After anesthesia, rats were exposed to oxygen only until they recovered from anesthetic.', 'In this study, hippocampal pyramidal neurons were acutely isolated as described previously by Zhang et al. (2009) with some modifications.', 'In current experiment, as described in detail (Taketo and Yoshioka, 2000), whole-cell patch clamp recordings were performed to record voltage-gated calcium channel currents at room temperature (21–24 °C).']",sevoflurane,"['Rats in 3% sevoflurane group and 2.1% sevoflurane group were respectively exposed to 3% and 2.1% sevoflurane for 6 h using oxygen as gas carrier with a gas flow of 4 L/min, while the rats in control group breathed independently oxygen with a gas flow of 4 L/min for 6 h in the same induction chamber.']",none,[],sprague dawley,"['Sprague-Dawley (SD) rats (both male and female) were obtained from the Experimental Animal Centre of Tianjin Medical University, Tianjin, China.']",True,True,True,True,True,True,[ Passage 9/12 ] 10.1016/j.brainres.2014.02.008
10.1007/s11064-015-1529-x,460.0,Liu,2015,rats,postnatal day 7,N,sevoflurane,none,sprague dawley,"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.",rats,['Sprague–Dawley (SD) rats used in the present study were obtained from the Animal Care Center of Fudan University.'],postnatal day 7,"['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).']",N,"[""The document does not mention any behavior tests such as 'Open field test', 'Morris water task', 'fear conditioning test', 'Dark/light avoidance'; 'passive/active avoidance test'; 'elevated maze', 'Forced swim test', 'Object recognition test', 'Social interaction/preference'.""]",sevoflurane,"['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.']",none,['The document only mentions the use of sevoflurane as an intervention.'],sprague dawley,['Sprague–Dawley (SD) rats used in the present study were obtained from the Animal Care Center of Fudan University.'],True,True,True,True,True,True,[ Passage 10/12 ] 10.1007/s11064-015-1529-x
10.1213/ANE.0000000000000380,276.0,Peng,2014,rats,postnatal day 7,N,isoflurane,none,sprague dawley,"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.",rats,"['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.']",postnatal day 7,"['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.']",N,"[""The document does not mention any behavior tests such as 'Open field test', 'Morris water task', 'fear conditioning test', 'Dark/light avoidance'; 'passive/active avoidance test'; 'elevated maze', 'Forced swim test', 'Object recognition test', 'Social interaction/preference'.""]",isoflurane,"['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).', '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.']",none,[],sprague dawley,"['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.']",True,True,True,True,True,True,[ Passage 11/12 ] 10.1213/ANE.0000000000000380
10.1007/s11064-021-03301-5,214.0,Wen,2021,mice,postnatal day 7,N,isoflurane,none,fmr1-ko,"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.",mice,['The C57BL/6 WT mice and heterozygous Fmr1 KO (HET) mice were purchased from the Jackson Laboratory.'],postnatal day 7,['P7 mice were randomly divided into two experimental groups: an isoflurane exposure group and a control group.'],N,[],isoflurane,"['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.', '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).', '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.']",none,[],c57bl/6,['The C57BL/6 WT mice and heterozygous Fmr1 KO (HET) mice were purchased from the Jackson Laboratory.'],True,True,True,True,True,False,[ Passage 12/12 ] 10.1007/s11064-021-03301-5