offline_results/exp_ga_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
10.1007/s12640-009-9063-8,365.0,Bercker,2009,rats,postnatal day 6,Y,propofol,sevoflurane,wistar,"10.1007/s12640-009-9063-8
Materials and Methods
The experiments were performed according to the guidelines of the German Animal Protection Law and were approved by the Berlin State authorities.

Wistar rat pups were purchased from the Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin BgVV, Berlin, Germany.

Experimental Protocol
Six-day-old Wistar rats received either intraperitoneal (i.p.) injections of propofol or underwent inhalational anesthesia with sevoflurane and were separated from their mother during the experimental phase. In every litter animals were randomized either for anesthesia or controls.

For propofol anesthesia doses of 30 mg/kg body weight were given every 90 min up to a cumulative dose of 90 mg/kg. For gas administration rats were placed into an incubation chamber (Billups Rothenberg Inc., Del Mar, USA), which was connected to an anesthesia system (F.Stephan GmBH, Gackenbach) for 6 h. Carbon dioxide and sevoflurane concentrations were monitored using a gas monitor (Datex Ohmeda, GE Healthcare, Munich, Germany). To avoid rebreathing of carbon dioxide, inspired CO2 concentration was continuously monitored and kept below 1 vol% by adjusting the fresh gas flow. Pilot studies established that sevoflurane concentrations between 3 and 5% maintained sufficient depth of anesthesia, as determined by lack of reaction to a painful stimulus. However, it was also observed that skin color changed and respiratory diminished in some animals, suggesting respiratory insufficiency. Accordingly, animals were closely monitored during the experiment and sevoflurane concentration adjusted between 3 and 5 vol% to maintain normal skin color and adequate respiratory efforts.

The animals were observed for another 90 min until they were awake and active to be returned to their mother after the last injection and 6 h of gas application respectively. To maintain body temperature and prevent hypothermia, animals were placed on a heating device. During anesthesia respiratory frequency and skin color were observed to detect apnea and hypoxia. If bradypnoe occurred, rats received a pain stimulus, if breathing did not restart or resuscitation efforts were necessary rats were excluded from further processing and analysis. Verum and control animals received injections of PÄD II cristalloid/glucose (50 g/l) solution (Fresenius-Kabi, Bad Homburg, Germany) to prevent hypoglycemia and hypovolemia. Control animals were separated from the mother for the same period as the anesthetized animals and received injections of the crystalloid/glucose solution as well. In order to reduce the amount of laboratory animals, the control animals of the propofol group were pooled with control animals from the sevoflurane group. Therefore, sham injections have not been performed. However, all experiments were performed using the same experimental settings and laboratories during the same time.

For perfusion fixation animals were killed with an injection of an overdose of chloral hydrate 24 h after starting anesthesia. A solution of PBS (phosphate buffered saline) mixed with heparine (Thrombophob 25,000, Heparin-Natrium; Nordmark Arzneimittel GmbH, Uetersen, Germany) was injected slowly into the heart and ascending aorta in order to wash out the blood from the vessels. Afterwards, rats were perfused with a solution containing paraformaldehyde 4% (Merck, Darmstadt, Germany) with cacodylate buffer (Sigma, Deisenhofen, Germany) for 10 min (De Olmos cupric silver staining).

Histology
To visualize degenerating cells, coronal sections (70 μm) of the whole brain were cut on a vibratome and stained with silver nitrate and cupric nitrate (De Olmos and Ingram 1971). This technique stains degenerating cells dying via an apoptotic or non-apoptotic mechanism. Degenerating cells were identified by their distinct dark appearance due to silver impregnation.

Quantification of Damage
Quantification of brain damage was assessed in the frontal, parietal, cingulate, retrosplenial cortex, caudate nucleus (mediodorsal part), thalamus (laterodorsal, mediodorsal, and ventral nuclei), septum, dentate gyrus, hypothalamus, cornu ammonis field CA1, and subiculum in silver stained sections by estimating mean numerical densities (Nv) of degenerating cells (Gundersen et al. 1988). An unbiased counting frame (0.05 mm × 0.05 mm: dissector height 0.07 mm) and a high aperture objective were used for sampling. The Nv for each brain region was determined with 8–10 dissectors. Regional Nv values from 17 brain regions were summed to give a total score for degenerating neurons for each brain. Figure 1 shows representative silver stained brain regions.

Fig. 1
figure 1
Light microscopic overviews of silver-stained transverse sections picturing neurodegenerative changes in 6-day-old rats. The images of the rat thalamus show examples 24 h after treatment a after propofol treatment, b sevoflurane treatment, and c for controls. Degenerated neurons are pictured as small dark dots

Full size image
Behavioral Task
For behavioral testing n = 13 propofol treated, n = 7 sevoflurane treated animals and n = 6 controls were anesthetized as described above. Only male pups were used. At the beginning of behavioral testing, animals were 7 weeks old. Animals were group-housed (4–5 animals per cage) under standard laboratory conditions (22 ± 2°C room temperature) with an artificial 12 h light-dark cycle (lights on 6.00–18.00 h). Rats had access to food (Altromin 1326, Lage, Germany) and water ad libitum. They were acclimated to the animal unit for at least 2 weeks before testing. One hour prior to testing rats were transferred to a quiet anteroom. All experiments were performed between 8.00 and 13.00 h and only male animals were tested. In order to avoid possible carry-over effects, a pause of 7 days, during which the animals were left undisturbed in their home cages, was introduced between the hole board and water maze task.

Morris Water Maze (MWM) Test
The water maze task was conducted by using the same experimental design as described previously (Bert et al. 2002). A blue circular tank (diameter 200 cm, 60 cm deep) was filled up with water (21 ± 1°C) to a height of 42 cm. The tank was surrounded by several visual cues and was indirectly illuminated (120 lx at the centre of the pool).

For a single adaptation trial (day 0, without platform), the rats were released into the pool for 90 s with no escape platform present. On the following 8 days (day 1–8, place version) a transparent platform (16 × 16 cm) was submerged 1.5 cm below the surface in the middle of one of four virtual quadrants which was according to adaptation trial neither preferred nor avoided by the rat. Each day the animals were lowered into the water facing the wall from three different starting points (left, opposite, and right from the platform quadrant). Animals that did not find the escape platform within 90 s were placed onto it by the experimenter. All rats were allowed to remain on the platform for 30 s for orientation and were afterwards removed to rest for 60 s in a heated cage until the next trial. For each trial the escape latency to reach the platform was measured by a computerized tracking system (TSE VideoMot, Version 1.43, Bad Homburg, Germany). For each animal the three daily trials were averaged. On day 9 the escape platform was removed (spatial probe) and the time spent in each quadrant during a single 90 s trial was registered. On day 10 (cued version) the platform was elevated 1 cm above water level, signaled by a white cylinder (diameter 3 cm and 4 cm high), and moved to the quadrant opposite to the initial quadrant. This test was performed to assess the motivation to escape from the water and sensor-motor integrity. The testing procedure and recorded parameter during the cued version were the same as for the hidden platform version of the task (Morris 1984).

Hole Board Test
The hole board apparatus consisted of a square box (50 × 50 cm) made of grey Perspex with 16 equally spaced holes (diameter 2.5 cm), and was situated in a sound-attenuated chamber. The behavior of rats was monitored by an overhead installed video camera which was linked to a computerized tracking system (TSE VideoMot2, Bad Homburg, Germany). The test was conducted on two consecutive days. On both days rats were placed in the centre of the apparatus and observed for 10 min. The numbers of nose pokes and rearings as well as the distance traveled were recorded. After each animal the box was cleaned with 2-propanol 30%. Habituation to the apparatus was defined as a significant reduction of nose pokes, rearings and locomotor activity from the 1st to the 2nd day (Voits et al. 1995).

Blood Gas Analysis
To exclude severe hypoxia, hypercapnia or lactic acidosis, a blood gas analysis was performed for example in one animal of each group by transcutaneous puncture of the left ventricle. The probe was analyzed by a blood gas analyzer (Radiometer ABL series, Radiometer, Copenhagen, Denmark).

Statistical Analysis
The Kolmogorov Smirnov test was used to test for normal distribution. The results of the sum scores were compared using the Mann–Whitney U test between controls and propofol as well as between controls and sevoflurane. The place version data of the water maze test were analyzed by two-way ANOVAs on repeated measures followed by Holm-Sidak method for post-hoc multiple pair-wise comparisons. The spatial probe and the cued version data were analyzed by one-way ANOVAs followed by Holm-Sidak post-hoc tests. The data of the hole board test were analyzed by paired t-tests. Differences were considered to be significant if P < 0.05.",rats,"['Wistar rat pups were purchased from the Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin BgVV, Berlin, Germany.']",postnatal day 6,['Six-day-old Wistar rats received either intraperitoneal (i.p.) injections of propofol or underwent inhalational anesthesia with sevoflurane and were separated from their mother during the experimental phase.'],Y,"['For behavioral testing n = 13 propofol treated, n = 7 sevoflurane treated animals and n = 6 controls were anesthetized as described above.', 'Morris Water Maze (MWM) Test', 'Hole Board Test']",propofol,['For propofol anesthesia doses of 30 mg/kg body weight were given every 90 min up to a cumulative dose of 90 mg/kg.'],sevoflurane,"['For gas administration rats were placed into an incubation chamber (Billups Rothenberg Inc., Del Mar, USA), which was connected to an anesthesia system (F.Stephan GmBH, Gackenbach) for 6 h.', 'Carbon dioxide and sevoflurane concentrations were monitored using a gas monitor (Datex Ohmeda, GE Healthcare, Munich, Germany).']",wistar,"['Wistar rat pups were purchased from the Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin BgVV, Berlin, Germany.']",True,True,True,True,True,True,[ Passage 1/25 ] 10.1007/s12640-009-9063-8
10.1002/cbin.10349,4738.0,Cao,2015,mice,postnatal day 14,Y,ketamine,none,c57bl/6,"PMID: 25052764 DOI: 10.1002/cbin.10349
Materials and methods
Animals
C57BL/6 mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). The in vivo induction of ketamine-related hippocampal neurotoxicity was done at 2 weeks. Quantitative real time PCR of miR-34 family was used at 3 weeks, as was hippocampal injection of lentivirual vector of miR-34c. For analyses of TUNEL staining and Western blotting, 2-month old mice were used. All experimental procedures were reviewed and approved by the Animal Care Committee at the first affiliated Hospital of XinXiang Medical College.

Induction of ketamine-related hippocampal neurotoxicity
The in vivo protocol to induce ketamine-related hippocampal neurotoxicity was done as before with slight modifications (Hayashi et al., 2002; Huang et al., 2012, Liu et al., 2012). Young C57BL/6 mice, postnatal 14 days, were intraperitoneally administrated with repeated dosage of 75 mg/kg ketamine per day for six consecutive days (n = 28). Normal saline was injected in the control group of mice (n = 25).

RNA isolation and reverse transcription
Hippocampal RNA was isolated with Trizol reagent (In Vitrogen, Carlsbad, CA, USA). Briefly, mice were anesthetized and decapitated. Hippocampal samples were retrieved and homogenized at 1 mL Trizol/0.1 g tissue. The quantity of RNA was assessed by spectrophotometry followed by 1% agarose gel electrophoresis. Total RNA was treated with 10 U of RNase free DNase I, and reverse transcription (RT) was done in a total volume of 20 μL with random hexamer primers using a High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). cDNA was stored at −20°C until further use.

Quantitative RT-PCR
Expression of miR-34a, miR-34b, miR-34c, and house-keeping gene GAPDH were measured by TaqMan microRNA RT-PCR on the ABI 7900 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Expression profiles of each gene were quantified using corresponding standard curves. End-point RT-PCR of miR-34a, miR-34b, miR-34c, and GAPDH used 50 ng of total RNA with a mirVana RT-PCR miRNA Detection Kit (Ambion, Austin, Texas, USA). PCR products were separated and visualized on a 4% agarose gel. Each sample was run in triplicate and a mean value of each Ct triplicate was used.

Lentivirus production and transduction
To downregulate miR-34c, the coding sequence for a 2’-O-methyl oligonucleotide of miR-34c inhibitor was UCCGUCACAUCAAUCGACUAACG, and the non-specific control antisense sequence was UACUCUUUCUAGGAGGUUGUUAUU (Yu et al., 2012). These two sequences were amplified and cloned into pCDH-CMV-MCS-EF1-coGFP for in vivo gene transfer, resulting in a miR-34c inhibitor vector (lenti-miR34c-I) and miR-34c non-specific control vector (lenti-miR34c-C) (System Biosciences, Mountain View, CA, USA). The lentivirual expression vectors and pPACK packaging vector were co-transfected into 293T cells, and viral particles were collected and concentrated to high titer.

Hippocampal injection
One day after the 6-day ketamine treatment, the injections of lent viruses were performed on the right side of the cortex. A tiny hole was drilled above hippocampus and a Hamilton syringe was used to inject 2 μL of lentivirus of miR-34c inhibitor (lenti-miR34c-I, 20 μM, n = 17) or non-specific control (lenti-miR34c-C, 20 μM, n = 14) at the coordinates assessed from bregma and skull surface: anteroposterior −2.0 mm, lateral +1.5 mm, and vertical −1.5 mm. After injection, the incision was quickly sealed with dental cement.

Western blotting
Western blotting analysis was conducted at 2 months. Four mice with Lenti-miR34c-I injection and four mice with Lenti-miR34c-C injection were included in this analysis. Forty micrograms of hippocampal protein were collected and separated on an 8% NuPage Gel with MES buffer (Invitrogen, Carlsbad, CA, USA) and transferred to a polyvinylidene difluoride membrane. Primary antibody dilutions included 1:500 BCL2 (Santa Cruz, USA), 1:100 phosphorylated-PKC (p-PKC) (Sant Cruz Biotechnologies, Santa Cruz, CA, USA), 1:100 phosphorylated-ERK (p-ERK) (Sant Cruz Biotechnologies, Santa Cruz, CA, USA), and 1:1,000 β-actin (Cell Signaling, Danvers, MA, USA). Membranes were then incubated in primary antibody in Odyssey Blocking Buffer at 4°C for 24 h, followed by three washes in 0.1% PBS-T and 1 h incubation at RT with 1:1,000 secondary antibodies. The films were visualized and quantified on the Odyssey Infrared Imaging Center (Li-Cor, Lincoln, NE, USA).

TUNEL staining for hippocampal apoptosis
Hippocampal slices (350 μm) were prepared for terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining to detect the apoptosis, using an In Situ Cell Death Detection Kit according to manufacturer's protocol (Roche, Branchburg, NJ, USA). Five mice with Lenti-miR34c-I injection and 5 mice with Lenti-miR34c-C injection were included in the analysis. Hippocampal CA1 region was examined under a fluorescent scope. The apoptotic CA1 neurons were identified based on their size, location and immuno-reaction to TUNEL staining. The average number of the apoptotic neurons per 0.01 mm2 was measured and compared between control hippocampi and hippocampi treated with miR-34c inhibitor.

Morris water maze (MWM) testing
The MWM testing was carried out 1 month after hippocampal transfection of miR-34c knockdown. Eight mice with Lenti-miR34c-I injection and 5 mice with Lenti-miR34c-C injection were included in this analysis. In a large circular tank with a transparent platform (10 cm × 10 cm), warm water at 26°C was added to submerge the platform 1 cm below the surface. Visual cues of color paints were used to aid mice in locating the platform. The mice were given training sessions four times per day for one week before final testing. In each training session, the mice were put in the maze to locate the platform in 2 min followed by resting on the platform for 30 s. If mice did not locate the platform in 2 min, they were aided with flashing lights to the platform with 30 s of rest on top of the platform. On the final day of examination, the average swimming time and swimming distance were compared between control mice and the mice with miR-34c knockdown.

Statistical analysis
Statistic analysis was conducted with SPSS software (version 11.0). The measured data were presented as mean ± standard deviations. The statistical differences were measured with a Student's t-test, and the significance set at P < 0.05.",mice,"['C57BL/6 mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China).']",postnatal day 14,"['Young C57BL/6 mice, postnatal 14 days, were intraperitoneally administrated with repeated dosage of 75\u2009mg/kg ketamine per day for six consecutive days (n\u2009=\u200928).']",Y,['The MWM testing was carried out 1 month after hippocampal transfection of miR-34c knockdown.'],ketamine,"['Young C57BL/6 mice, postnatal 14 days, were intraperitoneally administrated with repeated dosage of 75\u2009mg/kg ketamine per day for six consecutive days (n\u2009=\u200928).']",none,[],c57bl/6,"['C57BL/6 mice were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China).']",True,True,True,True,True,True,[ Passage 2/25 ] 10.1002/cbin.10349
10.1213/ANE.0000000000000030,903.0,Cheng,2014,mice,postnatal day 7,N,isoflurane,none,cd-1,"PMID: 24413549 PMCID: PMC4029883 DOI: 10.1213/ANE.0000000000000030
METHODS
Animal Exposures
The care of the animals in this study was in accordance with National Institutes of Health and Institutional Animal Care and Use Committee guidelines. Study approval was granted by the Children’s National Medical Center. Six- to 8-week-old CD-1 pregnant female mice (20–30 grams) were acquired (Charles River, Wilmington, MA) to yield newborn pups. CD-1 mice were chosen because pups have been shown to reliably demonstrate neuronal changes consistent with human neonatal injury in specific experimental models.23 On postnatal day 7 (P7), we exposed male CD-1 mouse pups to 0 ppm CO (air), 5 ppm CO in air, or 100 ppm CO in air with and without isoflurane (2%) for 1 hour in a 7-L Plexiglas chamber (25 × 20 × 14 cm). The 3 experimental CO cohorts represented: negative control (0 ppm CO), low concentration subclinical CO (5 ppm), and high concentration subclinical CO (100 ppm). The chamber had a port for fresh gas inlet and a port for gas outlet that was directed to a fume hood exhaust using standard suction tubing. Specific concentrations of CO in air (premixed gas H-cylinders, Air Products, Camden, NJ) were verified using an electrochemical sensing CO detector (Monoxor III, Bacharach, Anderson, CA). Designated CO mixtures were delivered through the variable bypass isoflurane vaporizer and exposure chamber at a flow rate of 8 to 12 L/min. Mice were kept warm with an infrared heating lamp (Cole-Parmer, Court Vernon Hills, IL). P7 was chosen because synaptogenesis peaks at day 7 in rodents and is completed by the second or third week of life.24,25 One hour exposure to 2% isoflurane has been shown to activate brain capsase-3 in 7-day-old mice and is a brief anesthetic exposure.26 After exposure, pups were placed with their respective dams. Eighty-four newborn mice were evaluated.

Carboxyhemoglobin (COHb) Levels
COHb levels were measured immediately after 1-hour exposure. At the time of euthanasia, after pentobarbital injection (150 mg/kg, intraperitoneal), 200 μL blood was sampled from the left ventricle and COHb measured via 6 wavelength co-oximetry (Radiometer Osm3 Hemoximeter, Copenhagen, Denmark, range 0–100 ± 0.2%). Five animals per cohort were evaluated.

Activated Caspase-3 Immunohistochemistry
Five hours after exposure, following euthanasia with pentobarbital injection (150 mg/kg, ip), the brain was perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) via left ventricle injection for 30 minutes and then postfixed in additional fixative solution for 24 hours at 4°C. Serial sections were cut at a thickness of 6 μm in the coronal plane through the cerebral hemispheres beginning at −1.7 mm from bregma and 2.1 mm from interaural, and individual sections were slide mounted. Immunohistochemistry was performed on 3 to 4 nonserial nonadjacent sections using polyclonal antirabbit activated caspase-3 (Cell Signaling Technology, Beverly, MA), biotinylated secondary antibody (goat antirabbit, Cell Signaling Technology), and developed with diaminobenzidine. Nuclei were counterstained with hematoxylin. The number of activated caspase-3 positive cells per square millimeter was quantified at ×10 magnification in neocortex (primary and secondary somatosensory and auditory neocortices), hippocampus (dentate gyrus, CA1, CA2, and CA3 regions), and hypothalamic/thalamic region (laterodorsal, mediodorsal, ventromedial, ventrolateral, ventroposteromedial, ventroposterolateral thalamic nuclei, ventromedial hypothalamic nucleus, peduncular part of the lateral hypothalamus, and the central anterior hypothalamic area) of both hemispheres in 3 to 4 animals per group. Brain regions were defined in accordance with Mouse Brain Atlas.27,28

Terminal Deoxynucleotidyl Transferase-Mediated UTP Nick End-Labeling Staining
Five hours after exposure, following euthanasia, the brain was perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) via left ventricle injection for 30 minutes and then postfixed in additional fixative solution for 24 hours at 4°C. Paraffin-embedded brain sections were cut into 6-μm sections in the coronal plane through the cerebral hemispheres beginning at −1.7 mm from bregma, 2.1 mm from interaural, slide mounted, and stained for terminal deoxynucleotidyl transferase-mediated UTP nick end-labeling (TUNEL). Sections were incubated in 0.5% Triton at room temperature, followed by proteinase K at 37°C, then immersed in terminal deoxynucleotidyl transferase (TdT) buffer (30 mmol/L Tris-HCl buffer, pH 7.2, 140 mmol/L sodium cacodylate, and 1 mmol/L cobalt chloride) at room temperature. This was followed by incubation with TdT and biotin-16-dUTP for 60 minutes at 37°C. The reaction was terminated with TB buffer (300 mmol/L sodium chloride with 30 mmol/L sodium citrate) at room temperature, followed by immersion in 3% hydrogen peroxide and 2% fetal bovine serum at room temperature. The sections were then covered with an Avidin Biotin Complex (1:200 dilution) for 30 minutes at room temperature, incubated with FITC-Avidin D for detection, and counterstained with DAPI. The numbers of TUNEL positive nuclei in neocortex, hippocampus, and hypothalamic/thalamic region (identical regions as for activated caspase-3) were quantified at ×10 magnification in 3 to 4 nonserial sections per mouse, and 3 to 4 mice per cohort were evaluated. Brain regions were defined in accordance with Mouse Brain Atlas.27,28

Cytochrome C Peroxidase Activity
Immediately after 1-hour exposure, cytochrome c was extracted from fresh mitochondria as previously described.29 Isolated forebrain mitochondria (20 mg/mL) were suspended in a hypotonic 0.015 M KCl solution for 10 minutes on ice and then centrifuged at 105,000g for 15 minutes at 4°C. The pellet was resuspended in 0.15 M KCl solution for 10 minutes on ice and then centrifuged again at 105,000g for 15 minutes at 4°C. The supernatant was collected and cytochrome c content quantified with spectrophotometry. The peroxidase activity of 0.5 to 1 μM cytochrome c was determined by measuring the rate of oxidation of 50 μM 2,2′-azinobis-(2-ethylbenzthiazoline-6-sulfonate) (ABTS) in 10 mM potassium phosphate buffer (pH 7.4) at 415 nm (ε415 = 3.6 × 104 M−1 cm−1) after the addition of hydrogen peroxide.30 Five animals per cohort were evaluated.

Heme C Determination
Immediately after 1-hour exposure, forebrain mitochondria and cytosol were isolated by differential centrifugation.31 As previously described, forebrain was harvested and homogenized in ice-cold H medium (70 mM sucrose, 220 mM mannitol, 2.5 mM Hepes, pH 7.4 and 2 mM EDTA).31 The homogenate was spun at 1500g for 10 minutes at 4°C. Supernatant was removed and centrifuged at 10,000g for 10 minutes at 4°C. Cytosolic supernatant was collected, and pellet was resuspended in H medium and centrifuged again at 10,000g for 10 minutes at 4°C. Pellet was again resuspended in H medium, and mitochondrial and cytosolic protein concentrations subsequently determined using the method of Lowry.31

Mitochondrial and cytosolic heme c content were calculated from the difference in spectra (dithionate/ascorbate reduced minus air-oxidized) of mitochondria or cytosolic protein (0.5–1 mg) solubilized in 10% lauryl maltoside using an absorption coefficient of 20.5 mM−1 cm−1 at 550 to 535 nm.32,33 Five animals per cohort were evaluated.

Statistical Analysis
Sample sizes for each end point were chosen based on previous work.19 Our previous study used 8 animals per cohort for COHb and heme c determination, 3 to 4 animals per cohort for activated caspase-3 and TUNEL assessment, and 5 animals per cohort for measurement of cytochrome c peroxidase activity, and data followed normal probability distribution.19 For this work, sample sizes were based on the number of animals needed to detect a 30% difference from air-exposed control values with a power of 80 based on an α of 0.01. Data are presented as mean ± SE. To assess statistical significance, we performed pairwise comparisons in an analysis of variance design using Tukey test.",mice,"['Six- to 8-week-old CD-1 pregnant female mice (20–30 grams) were acquired (Charles River, Wilmington, MA) to yield newborn pups.']",postnatal day 7,"['On postnatal day 7 (P7), we exposed male CD-1 mouse pups to 0 ppm CO (air), 5 ppm CO in air, or 100 ppm CO in air with and without isoflurane (2%) for 1 hour in a 7-L Plexiglas chamber (25 × 20 × 14 cm).']",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,"['On postnatal day 7 (P7), we exposed male CD-1 mouse pups to 0 ppm CO (air), 5 ppm CO in air, or 100 ppm CO in air with and without isoflurane (2%) for 1 hour in a 7-L Plexiglas chamber (25 × 20 × 14 cm).']",none,[],cd-1,"['Six- to 8-week-old CD-1 pregnant female mice (20–30 grams) were acquired (Charles River, Wilmington, MA) to yield newborn pups.']",True,True,True,True,True,True,[ Passage 3/25 ] 10.1213/ANE.0000000000000030
10.1007/s12640-018-9877-3,437.0,Chen,2018,rats,postnatal day 7,Y,sevoflurane,none,sprague dawley,"PMID: 29427282 DOI: 10.1007/s12640-018-9877-3
Methods
Ethical Approval
The use of rats in this study was approved by the Institutional Animal Care and Use Committee at Sun Yat-sen University (Guangzhou, China). All experiments were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and ARRIVE guidelines. Sprague-Dawley multiparous dams (n = 31) with litters containing male pups (n = 135) were purchased from Experimental Animal Center of Sun Yat-sen University, China. We only used male offspring to exclude the influence of estrogen on the biochemical data and neurocognitive functions. The pups from postnatal day 0 (P0) to P20 were housed with the dams in a 12-h:12-h light:dark cycle (light from 07:00 to 19:00), and room temperature (RT) was maintained at 21 ± 1 °C. On P21, the pups were weaned and housed 4–6 per cage in a standard environment.

Anesthesia
SD rats at P7 (weight 14–16 g) were randomly divided into the air-treated control (C group), the 1.2% sevoflurane-exposed (1.2% sevo group), and the 2.4% sevoflurane-exposed (2.4% sevo group). Rats in the 1.2% sevo group and the 2.4% sevo group were placed in a plastic container and exposed to 1.2 or 2.4% sevoflurane continuously for 6 h, using air as a carrier, with a total gas flow of 2 L min−1. A nasopharyngeal airway tube was put in their mouth to prevent apnea and hypoxia when the rats stopped moving in the container. During exposure, the temperature inside the container was maintained at 30 °C using an external heating device (NPS-A3 heating device, Midea Co., Guangdong, China) and a hot water bag on the bottom of the container with a constant temperature maintained between 30 and 35 °C. The concentrations of sevoflurane, oxygen, and carbon dioxide in the chamber were monitored by a gas monitor (Detex-Ohmeda, Louisville, CO, USA). During exposure, an investigator monitored the rats’ spontaneous respiratory frequency and skin color every 5 min to detect any apnea or hypoxia. The rats were immediately exposed to air and excluded from the experiment if these symptoms were detected. Sevoflurane administration was terminated 6 h later, and the rats were exposed only to air. When the rats were moving freely again, they were placed back into their maternal cages. Rats in the C group were exposed to the same container as the rats in the 1.2% sevo and 2.4% sevo group but were exposed to air alone for 6 h.

Arterial Blood Gas Analysis
We performed arterial blood analysis in order to exclude the influence of respiratory or metabolic disorder. The arterial blood samples from the C, 1.2% sevo, and 2.4% sevo groups were obtained from the left cardiac ventricle immediately after removal from the maternal cage (n = 5 in each group) at the end of anesthesia. They were analyzed immediately after collection using a blood gas analyzer (Gem Premier 3000, US). We analyzed the pH, arterial carbon dioxide tension (PaCO2), arterial oxygen tension (PaO2), and blood glucose levels of the arterial blood samples.

Morris Water Maze Test
On P35, the rats were tested for spatial learning and memory ability using the Morris water maze (MWM). Three groups of rats (n = 10 in each group, weight 90–100 g) were tested on the MWM, which consists of two different tests including hidden platform acquisition and a probe trial test, at P35–P40 using the Water Maze Tracking System (MT-200; Chengdu, China). A white platform (12 cm diameter) was submerged in a circular pool (160 cm in diameter, 50 cm in height) that was filled with warm water (23 ± 2 °C). The pool, located in a room with no windows, was virtually divided into four quadrants. A video camera connected to the computer running the tracking software was suspended above the pool and captured the rats’ movements for analysis. At P35, before the test, a single habituation trial was performed without the platform; in this trial, the rats were placed in the water for 120 s. In the hidden platform acquisition test, performed at P36–P39, each rat was placed, facing the wall of the pool, in one of the four quadrants and allowed to swim freely in search of the escape platform for a maximum of 120 s. The experiment was repeated with four trials per day for four consecutive days. The average escape latency time (latency to reach the platform) was measured to evaluate spatial learning ability. At P40, a probe trial test was performed by removing the platform and releasing the rats into the water for 120 s. We calculated the time spent in the quadrant that previously contained the target and the frequency of crossing the former location of the platform. The rats were dried and placed back into a heated cage after completing each test.

Western Blot Analysis
On P10 and 28, rats (n = 5 in each group at each sacrifice time point) were sacrificed by rapid decapitation, and the bilateral hippocampus areas were harvested and stored at − 80 °C until use. Protein was extracted using RIPA lysis buffer (Keygen Biotech, Nanjing, China). The amount of protein in each hippocampal tissues was measured using a protein assay kit (BCA, Pierce, Thermo, USA). Polyacrylamide-SDS gels with an equal amount of 50-μg load in each lane were electrophoresed, and the proteins transferred onto PVDF membranes (Millipore, Carrigtwohill, Ireland). The blots were blocked with 5% skim milk in Tris-buffered saline (150 mM NaCl, 0.1% TWEEN 20, 20 mM Tris, pH 7.4) for 1 h and then incubated overnight at 4 °C with anti-BDNF (1:1000, Novusbio, USA), anti-TrkB (1:800, Millipore, Ireland), anti-postsynaptic density (PSD-95) (1:2000, Abcam, England), and anti-synaptophysin (1:20,000, Abcam, England) primary antibodies. After rinsing, membranes were probed with corresponding secondary antibodies at RT for 2 h. Immunoreactive bands were detected with an enhanced chemiluminescence detection system (Bio-Rad, USA). A β-actin antibody (1:1000, ABclonal, China) was used to normalize for sample loading and transfer. The intensities of the bands were densitometrically quantified using ImageJ.

BrdU Injections and Immunofluorescence
For the 5′-bromo-2-deoxyuridine (BrdU) injections, we followed the methods as previously described (Chen et al. 2015; Tozuka et al. 2005). BrdU has been described as a marker of neurogenesis and can incorporate into DNA only during the S-phase of the mitotic process (Kee et al. 2002). BrdU (Sigma, America) was dissolved in normal saline (10 mg mL−1) and injected at a dosage of 300 mg/kg. To investigate the effects of 1.2% sevoflurane on cellular proliferation, we performed a single injection of BrdU i.p. 24 h after sevoflurane exposure. Three days later, the rats were perfused, and their brains were processed for immunofluorescence. To investigate the effects of 1.2% sevoflurane on the survival of newborn cells, we performed a single injection of BrdU i.p. 24 h before sevoflurane exposure. Four weeks (28 days) after the BrdU injection, the rats were perfused, and their brains were processed for immunofluorescence.

For morphological examination, rats were deeply anesthetized with chloral hydrate at P10 and P35 (n = 5 in each group at each sacrifice time point, 80–100 g) and then transcardially perfused with 0.9% normal saline at RT followed by a fixative solution of 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in 0.1 M PBS (pH 7.4) at 4 °C. The brains were harvested, postfixed in 4% paraformaldehyde for 8 h, and subsequently soaked in 30% sucrose until they sank. Consecutive frozen coronal sections of the hippocampus were cut at a thickness of 30 μm. Every fifth section of the consecutive sections was processed by BrdU staining. DNA was first denatured by incubation with 2 N HCl for 30 min at 37 °C followed by a 15-min wash in 0.1 M boric acid (pH 8.5), with three 10-min washes in 0.01 M PBS before each step. The sections were blocked in 3% BSA and 0.4% Triton X-100 for 2 h at RT before being incubated with primary antibody (rat anti-BrdU, 1:200, ab6326, Abcam, UK) in 1% BSA overnight at 4 °C. Then, the sections were incubated with secondary antibody (Cy3 goat anti-rat IgG, 1:200, KGAB018, Keygentee, China) for 2 h at RT. Fluorescence was detected with a fully automatic fluorescence microscope (Olympus BX63, Japan). An observer who was blinded to group assignment was responsible for counting the number of BrdU-positive cells at × 200 magnification. Total cell counts were divided by the total number of sections for analysis.

Transmission Electron Microscopy
TEM was used to assess synaptic plasticity in the hippocampus after exposure to treatment (n = 5 in each group) at P35. Twenty-eight days after exposure to treatment, the rats were perfused transcardially with 50 mL of 0.9% normal saline, followed by 50 mL of a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde (Sigma-Aldrich, G6257, USA) in 0.1 M PBS. Approximately 1 mm3 of tissue per rat was dissected from the hippocampus and fixed in 2% glutaraldehyde for 2 h at 4 °C. The tissues were rinsed in 0.1 M cacodylate buffer and postfixed with 1% osmium tetroxide for 2 h. Then, the tissue was rinsed with distilled water before undergoing dehydration in a graded ethanol series. Subsequently, the tissue was infiltrated overnight at 4 °C using a mixture of half acetone and half resin. The tissue was embedded in resin 24 h later and then cured fully as follows: 37 °C overnight, 45 °C for 12 h, and 60 °C for 24 h. After that, 70-nm sections were cut and stained with 3% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Ultrastructural changes in synapses in the hippocampus were observed under TEM. Five pictures of each subregion per ultrathin section (five rats in total per group) were taken at each of two magnifications: × 13,500 and × 37,000. All pictures taken at × 13,500 magnification were used to observe the number of synapses, and all pictures taken at × 37,000 magnification were used to measure the thickness of the postsynaptic density and the width of the synaptic cleft. The number of synapses was expressed as the average number of synapses in each picture taken at × 13,500. The thickness of the postsynaptic density and the width of the synaptic were expressed as the average values for all synapses in all pictures taken at × 37,000, as described. We measured the distances using the image analysis software ImageJ.

Statistical Analysis
The results were expressed as the mean ± standard deviation (SD) for each group. The statistical tests were conducted using the computerized statistical package SPSS 19.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism Software version 5.0 (GraphPad Software, Inc., San Diego, CA, USA). The arterial blood data were analyzed using Student’s t test. One-way ANOVA was used to evaluate differences in the quantities of hippocampal proteins, numbers of BrdU-positive cells and synapses, and ultrastructure parameters of synapses among groups. Unpaired t tests and two-way ANOVA were used to analyze the results of the MWM. Each experiment was performed at least three times. A value of P < 0.05 was considered statistically significant.",rats,"['The use of rats in this study was approved by the Institutional Animal Care and Use Committee at Sun Yat-sen University (Guangzhou, China).']",postnatal day 7,"['SD rats at P7 (weight 14–16 g) were randomly divided into the air-treated control (C group), the 1.2% sevoflurane-exposed (1.2% sevo group), and the 2.4% sevoflurane-exposed (2.4% sevo group).']",Y,"['On P35, the rats were tested for spatial learning and memory ability using the Morris water maze (MWM).']",sevoflurane,"['the 1.2% sevoflurane-exposed (1.2% sevo group), and the 2.4% sevoflurane-exposed (2.4% sevo group).']",none,[],sprague dawley,"['Sprague-Dawley multiparous dams (n =\u200931) with litters containing male pups (n =\u2009135) were purchased from Experimental Animal Center of Sun Yat-sen University, China.']",True,True,True,True,True,True,[ Passage 4/25 ] 10.1007/s12640-018-9877-3
10.1021/acschemneuro.0c00106,3879.0,Chen,2020,mice,postnatal day 7,Y,sevoflurane,none,c57bl/6,"PMID: 32271540 DOI: 10.1021/acschemneuro.0c00106
Materials and Methods
ARTICLE SECTIONSJump To
Animals and Treatments
Seven day old C57BL/6 male mice (Beijing Vital River Company, Beijing, China) were used in this study. The mice were bred and maintained in the animal care facility following the standard rearing conditions of 12 h light and 12 h dark. All mouse studies were performed following the guidelines established by the Institutional Animal Care and Use Committee in Quanzhou First Hospital Affiliated to Fujian Medical University (QFH2017jb43i).
BRL-50481 (Tocris Bioscience, Bristol, United Kingdom) was dissolved in 2.5% dimethyl sulfoxide (Sigma, St. Louis, MO) with 0.9% NaCl and injected intraperitoneally into pups before subjecting them to sevoflurane, with a vehicle injection as control. Thirty minutes later, the injected pups were put into a semiclosed chamber and exposed to 3% sevoflurane for 4 h. After exposure, pups were returned to the parents’ cages and monitored for health status until the following tests.
The pups were randomly divided into five groups as follows:
Sham: vehicle intraperitoneal injection;
Control: 5 mg/kg BRL-50481 intraperitoneal injection;
B0: Sevoflurane anesthesia, vehicle intraperitoneal injection;
B1: Sevoflurane anesthesia, 1 mg/kg BRL-50481 intraperitoneal injection;
B5: Sevoflurane anesthesia, 5 mg/kg BRL-50481 intraperitoneal injection.
Each group contained 10 pups.
Morris Water Maze Test and Analysis
The spatial memory ability of control and treated mice was determined using the Morris water maze test developed by Richard Morris. (31) In brief, a 160 cm diameter and 60 cm high circular tank was filled with water at 30 cm high. The water temperature was maintained at 22 °C. A 12 cm diameter circular platform was submerged 1 cm below the water surface in the center of one of the four virtual quadrants. (32)
The control and treated mice were trained four times per day for 6 days. The mouse was released into the water, and it navigated to reach the platform. The maximum swimming time of the tested mouse was 80 s. If the mouse could escape to the refuge within 60 s, the delay to find the platform time was recorded as 60 s. Mice were allowed to stay on the platform for 15 s, and then they were sent to their cages under a heat lamp to maintain their core temperature. The escape latency was recorded by a tracking system, and data were analyzed using ViewPoint video tracking system (ViewPoint Behavior Technology, Civrieux, France). Three daily trials were averaged for each animal. (32)
Immunohistochemistry (IHC) Analyses
Mice were euthanized and perfused with cold phosphate-buffered saline and 4% paraformaldehyde immediately. The brains were fixed with 4% paraformaldehyde overnight and then cryoprotected by immersion in 30% sucrose at 4 °C for 48 h. Coronal sections (25 μm) were cut using a manual rotary microtome (Leica, Wetzlar, Germany).
The caspase-3 IHC staining was performed as previously described. (33) The cleaved caspase-3 antibody (ab13847) was purchased from Abcam (Cambridge, MA).
Immunoblotting Analyses
Frozen hippocampus homogenates were lysed using radioimmunoprecipitation buffer (Bioequip, Shanghai, China). The samples were subjected to immunoblotting analysis as described previously. (33) The pCREB (Ser133, #9198, 1:1000 dilution) and CREB (#9197, 1:2000 dilution) primary antibodies were ordered from Cell Signaling Technology (Danvers, MA), and the internal control β-actin antibody was ordered from Abcam (ab8226, 1:2000 dilution).
cAMP Concentration Assay
cAMP levels were measured using the mouse cAMP ELISA kit (ab133051, Biocompare, South San Francisco, CA) following the manufacturer’s instructions.
Statistical Analysis
Statistical analyses were carried out by using the SPSS 11.0 package. Differences between groups were analyzed using analysis of variance (ANOVA) or two-sample t test with Bonferroni correction. All data represent mean ± standard deviation (SD). Statistical significance thresholds were set at *P < 0.05.",mice,"['Seven day old C57BL/6 male mice (Beijing Vital River Company, Beijing, China) were used in this study.']",postnatal day 7,"['Seven day old C57BL/6 male mice (Beijing Vital River Company, Beijing, China) were used in this study.']",Y,['The spatial memory ability of control and treated mice was determined using the Morris water maze test developed by Richard Morris.'],sevoflurane,"['Thirty minutes later, the injected pups were put into a semiclosed chamber and exposed to 3% sevoflurane for 4 h.']",none,[],c57bl/6,"['Seven day old C57BL/6 male mice (Beijing Vital River Company, Beijing, China) were used in this study.']",True,True,True,True,True,True,[ Passage 5/25 ] 10.1021/acschemneuro.0c00106
10.1016/j.ijdevneu.2019.04.002,248.0,Goyagi,2019,rats,postnatal day 7,Y,sevoflurane,none,wistar,"PMID: 30959098 DOI: 10.1016/j.ijdevneu.2019.04.002
2 Material and methods
All animal protocols were approved by the animal research committee of Akita University, Japan (Approval number: a-1-2625). Seven-day-old (P7) Wistar rats (male and female) rat pups (body weight, 12–15 g) were used in this study. Animals were housed under standard conditions (12 h light/12 h dark cycle at 22 °C) in the Animal Research Laboratory at Akita University. All efforts to reduce the number of animals and their suffering were made. The animals were randomly divided into 6 groups (n = 10 per group) as follows: no anesthesia and no injection (sham), no anesthesia and intraperitoneal 25 μg/kg DEX (control), intraperitoneal saline (DEX 0), intraperitoneal 6.6 μg/kg DEX (DEX 6.6), intraperitoneal 12.5 μg/kg DEX (DEX 12.5), and intraperitoneal 25 μg/kg DEX (DEX 25).

After 30 min intraperitoneal injection on P7, the pups were put into a plastic chamber, exposed to 3% sevoflurane with 2 L/min of 21% oxygen for 4 h, and returned to their mother's cage. The oxygen and sevoflurane concentration were measured using a gas analysis system (GE Healthcare BioSciences, Pittsburgh, PA). The chamber was maintained at 30 ± 1 °C using an infrared heat lamp during the exposure.

Cognitive tests
2.1.1 Morris water maze
Spatial memory retention was examined using the Morris water maze by blinded observer as described previously (Goyagi, 2018). At P27 – P29, acquisition trials were executed 4 times per day for 3 successive days. The latency and the swimming path length to reach the hidden platform were measured using a video image motion analyzer (DVTrack DVT-11; Muromachi Kikai Co. Ltd, Tokyo, Japan). If the rat could not reach the hidden platform within 90 s, it was placed on the platform for 30 s during an acquisition trial. At P47 – P49, retention trials were executed 4 times per day. If the rats failed to find the platform within 90 s, the latency was regarded as 90 s. In this study, a probe trial was not done during the acquisition trials.

2.1.2 Fear conditioning test
Fear conditioning was performed to evaluate contextual memory retention using the fear conditioning system (MK-450RSQ; Muromachi Kikai Co., Ltd, Tokyo, Japan) as described previously (Goyagi, 2018). The apparatus consisted of a clear rectangular Plexiglas box with a floor of for the delivery of electric currents. At P42, the rats were placed on the cleaned parallel metallic rods to be accustomed to new environment for 1 min, before they were presented with a 70-dB white noise for 30 s A mild foot shock (0.4-mA) was administered through the metallic rods during the last 1 s of the tone presentation. The tone-shock pairing was repeated once per minute for the next 2 min. The rats were left in the cage for an additional 60 s before returned to their cage. At P49, cued fear memory was tested by placing rats into an unrelated environment for 90 s without any tone and presenting the auditory cue for a further 60 s used for conditioning. Freezing time was measured by the percent of time during the tone presentation using a video image motion analyzer (DVTrack DVT-11; Muromachi Kikai Co. Ltd, Tokyo, Japan).

Histological analyses
2.2.1 Neuronal nuclei staining
After finished the water maze task and fear conditioning test at P49, the rats’ brains were removed and embedded in paraffin following the perfusion of heparinized saline then 150 mL of 4% paraformaldehyde in phosphate buffer (pH 7.4) to use further neuronal nuclei (NeuN) stain, as described previously (Goyagi, 2018). In brief, 3-μm-thick serial transverse sections were incubated with a mouse monoclonal antibody to NeuN antigen (NeuN; 1:100 diluted in blocking solution; Millipore Corporation, Temecula, CA) for 10 min at 37 °C. Immunodetection was performed using avidin-horse radish peroxidase complexes with biotinylated antibodies to rabbit and mouse IgG (MILLIPORE IHC SelectR Immunoperoxidase Secondary Detection System; Millipore Corporation), with diaminobenzidine. Then we counterstained those with hematoxylin. The NeuN-positive cells express as mature typical neurons after growth. We counted the number of NeuN-positive cells in bilateral 500 μm × 300 μm areas in the CA1 hippocampus, amygdala, and cerebral cortical layer 3, as described previously (Goyagi, 2018).

2.2.2 Positive cell density map (PCDM)
The PCDM was made as described previously (Goyagi, 2018; Wada et al., 2006). In brief, the composite image was FFT- bandpass-filtered using the Image J program (National Institute of Health, Bethesda, MD) to eliminate low-frequency drifts (>20 pixels [50 μm]) and high-frequency noises (<1 pixel [2.5 μm]). The PDCM was made with a custom-made program using MATLAB (MathWorks INC., Natick, MA) (Wada et al., 2006), then adjusted for each section automatically and enumeration of NeuN-positive cells in each 100 μm × 100 μm square section. Finally, the normalized PCDMs were seen as averaged for each group (Fig. 6 A). As mentioned our previous study (Goyagi, 2018), the PCDMs were analyzed whether the DEX-treated groups showed increased NeuN cell density compared with the DEX 0 group. The areas were mapped as colored to indicate significantly increased normal neurons in blocks where the P value was less than 0.05 (Fig. 6B).

Statistical analysis
The escape latency, the swimming speed, the swimming path length, the freezing time, and the number of NeuN-positive cells are expressed as means ± standard deviation (SD). Comparisons of these variables among the groups were performed using a one-way or two-way analysis of variance (ANOVA) for multiple comparisons followed by Bonferroni post hoc tests. Each PCDM using a Gaussian filter of the block size (SD = 100 μm) was analyzed using t-tests for each block. Differences with p-values less than 0.05 were considered statistically significant. We performed all analyses using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA).",rats,"['Seven-day-old (P7) Wistar rats (male and female) rat pups (body weight, 12–15\u2009g) were used in this study.']",postnatal day 7,"['Seven-day-old (P7) Wistar rats (male and female) rat pups (body weight, 12–15\u2009g) were used in this study.']",Y,"['Spatial memory retention was examined using the Morris water maze by blinded observer as described previously (Goyagi, 2018).', 'Fear conditioning was performed to evaluate contextual memory retention using the fear conditioning system (MK-450RSQ; Muromachi Kikai Co., Ltd, Tokyo, Japan) as described previously (Goyagi, 2018).']",sevoflurane,"['the pups were put into a plastic chamber, exposed to 3% sevoflurane with 2\u2009L/min of 21% oxygen for 4\u2009h']",none,[],wistar,"['Seven-day-old (P7) Wistar rats (male and female) rat pups (body weight, 12–15\u2009g) were used in this study.']",True,True,True,True,True,True,[ Passage 6/25 ] 10.1016/j.ijdevneu.2019.04.002
10.1097/EJA.0b013e328330d453,667.0,Han,2010,rats,postnatal day 7,N,ketamine,none,sprague dawley,"PMID: 19918184 DOI: 10.1097/EJA.0b013e328330d453
Materials and methods
Animals
Seven-day-old male and female Sprague Dawley rats (body weight 11.1–17.5 g) were housed in plastic cages with their mothers and maintained on a 12: 12 h light/dark cycle at 22–25°C ambient temperature with food and water available ad libitum for the mothers. All of the experimental procedures were approved by the Animal Use and Care Committee for Research and followed the ethical guidelines for investigation of experimental pain in conscious animals [7].

Rats (n = 40) were divided into two random groups. In one group (n = 20) the rats were injected with ketamine intraperitoneally (i.p.) at PND 7 [8] and sacrificed within 24 h. In the other group (n = 20) the rats were also injected with ketamine at PND 7 and were sacrificed at PND 28. Each group was divided into five random subgroups (n = 4 per subgroup). The control group received 0.9% physiological saline. The other four groups received i.p. injections of ketamine (K1–K4) [9] (see Table 1).
Semiquantitative reverse-transcriptase PCR and quantitative real-time reverse-transcriptase PCR
All animals from the different groups (n = 4) were killed by decapitation under ether anaesthesia. sqRT-PCR was used to qualitatively assess the effect of NMDA subtype receptor expression. qRT-PCR was then applied in order to further quantify the observed effects. Table 2 summarizes information about the oligonucleotide primers used in this study. All primer sequences were checked in GenBank (National Center for Biotechnology Information, Bethesda, Maryland, USA) to avoid inadvertent sequence homologies. β-actin was used as an internal control. Animals were decapitated under ether anaesthesia, and the hippocampus was quickly dissected out and frozen at −80°C until use. Total RNA was isolated using Trizol reagent (Invitrogen, Virginia, USA), according to the manufacturer's instructions, and then reverse transcribed with Moloney murine leukaemia virus (M-MLV) reverse transcriptase (Invitrogen) and oligo(dT)12–18 primers.
For sqRT-PCR, a PCR reaction mixture containing 10 mmol l−1 Tris (pH 8.3), 50 mmol l−1 KCI, 1.5 mmol l−1 MgCI2, 100 μl of deoxyribonucleotide triphosphate (dNTP), 2.5 units of Taq DNA polymerase (Takara, Kyoto, Japan), 0.5 μl of synthesized cDNA and 20 pmol of each sense and antisense primer pair. The PCR reaction was performed for 30 cycles using a PTC-100 Programmed Thermal Controller (MJ Research, Watertown, Massachusetts, USA) as follows: 1 min at 93°C, 30 s at appropriate annealing temperature (Table 2) and 1 min at 72°C, with 1 min of 93°C treatment before starting the thermal cycles, and, finally, an 8 min extension at 72°C was conducted. PCR was performed simultaneously on control and experimental rat samples, with the internal controls (β-actin) running in parallel with the examined mRNAs. In all reverse transcriptase PCR experiments including negative controls, in which template RNA or reverse transcriptase was omitted, no PCR product was detected. Ten microlitres of each PCR product was electrophoresed on a 3% agarose gel containing ethidium bromide. Resulting gel bands were visualized in an ultraviolet (UV) transilluminator, and images were captured with an eight-bit charge coupled device (CCD) camera (Ultra-Violet Products, Upland, California, USA).

Quantitative PCR was set up using SYBR Green-containing premix from Takara. The reverse transcriptase reaction product (100 ng) was amplified in a 25 μl reaction with 12.5 SYBR Premix EX Taq (Takara, Shiga, Japan). Samples were heated to 90°C for 30 s, and then amplified for 40 cycles consisting of 95°C for 15 s and 60°C for 15 s. Relative quantification of NMDA subtype receptors was performed by a comparative threshold cycle method. All data are expressed as mean ± SEM. Experimental groups were compared by analysis of variance. P values of less than 0.05 were considered to be statistically significant.

Immunohistochemistry
Rats in the control group and those in the K1 and K3 subgroups (n = 4), which were sacrificed on PND 7 or PND 28, were perfused transcardially with 100 ml of 0.01 mol l−1 PBS (pH 7.4), followed by 100 ml of 4% (w/v) paraformaldehyde and 75% (v/v) saturated picric acid in 0.1 mol l−1 phosphate buffer (pH 7.4). The brains were then removed immediately and placed into the same fresh fixative for an additional 2 h at 4°C. Subsequently, the brains were placed into 30% (w/v) sucrose solution in 0.1 mol l−1 phosphate buffer (pH 7.4) overnight at 4°C (the sucrose solution contained 0.02% NaN3), and then cut serially into 30 μm thick coronal sections by the use of a freezing microtome (Kryostat 1720; Leitz, Mannheim, Germany). The sections were placed into five different dishes according to their numerical order while cutting (e.g. sections 1 and 7 in dish 1; sections 2 and 8 in dish 2; sections 3 and 9 in dish 3; sections 4 and 10 in dish 4; and sections 5 and 11 in dish 5). Each dish usually contained 28–32 sections. All sections were washed carefully with 0.01 mol l−1 PBS. The sections in the first three dishes were used for immunohistochemistry for NR2A–2C. Briefly, the sections were incubated at 4°C sequentially with: a mixture of rabbit anti-NR2A, 2B and 2C serum (1: 200 dilution; Elek molnar) for 24 h; biotinylated goat antirabbit immunoglobulin G (1: 200 dilution; Vector) for 2 h; and avidin-labelled horseradish peroxidase compound (1: 100 dilution; Vector) for 1 h. The diluent used for all antibodies was 0.05 mol l−1 PBS containing 5% (v/v) normal donkey serum, 0.5% (v/v) Triton X-100, 0.05% (w/v) sodium azide (NaN3) and 0.25% (w/v) carrageenan (pH 7.3). In the fourth dish, normal rabbit serum was used instead of rabbit anti-NR2A, 2B and 2C serum, and the following steps were the same as mentioned above. The fifth dish was used for Nissl staining in order to locate a positive construction. The sections were rinsed at least three times in 0.01 mol l−1 PBS (pH 7.4) after each incubation, for at least 10 min. The sections were coloured with diaminobenzidine (DAB) and H2O2, then sections were mounted onto clean glass slides, air dried and cover-slipped with a mixture of 50% (v/v) glycerin and 2.5% (w/v) triethylene diamine (antifading agent) in 0.01 mol l−1 PBS. Finally, the sections were studied under a microscope.
",rats,['Seven-day-old male and female Sprague Dawley rats (body weight 11.1–17.5 g) were housed in plastic cages with their mothers and maintained on a 12: 12 h light/dark cycle at 22–25°C ambient temperature with food and water available ad libitum for the mothers.'],postnatal day 7,['Rats (n = 40) were divided into two random groups. In one group (n = 20) the rats were injected with ketamine intraperitoneally (i.p.) at PND 7 [8] and sacrificed within 24 h.'],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,['In one group (n = 20) the rats were injected with ketamine intraperitoneally (i.p.) at PND 7 [8] and sacrificed within 24 h.'],none,[],sprague dawley,['Seven-day-old male and female Sprague Dawley rats (body weight 11.1–17.5 g) were housed in plastic cages with their mothers and maintained on a 12: 12 h light/dark cycle at 22–25°C ambient temperature with food and water available ad libitum for the mothers.'],True,True,True,True,True,True,[ Passage 7/25 ] 10.1097/EJA.0b013e328330d453
10.1007/s12640-016-9615-7,341.0,Huang,2016,rats,postnatal day 7,Y,ketamine,none,sprague dawley,"PMID: 26966008 DOI: 10.1007/s12640-016-9615-7
Materials and Methods
Animal Treatment
All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. The timed-pregnant Sprague–Dawley rats were housed in a temperature-controlled (22–23 °C) room on a 12 h:12 h light:dark cycle (light on at 8:00 AM) with free access to food and water. The PND-7 male rat pups (11–14 g) were randomly assigned to ketamine-treated and control groups. In the treated group, ketamine was diluted in 0.9 % normal saline, and PND-7 rats were intraperitoneally administered with 40 mg/kg doses of ketamine in four injections at 1 h intervals (40 mg/kg × 4 injections). Control rats received an equal volume of normal saline. Temperature probes were used to facilitate control of temperature at 36.5 ± 1 °C using computer-controlled heater/cooler plates integrated into the floor of the chamber. Between each injection, animals were returned to their chamber to help maintain body temperature and reduce stress.

BrdU Injections
All animals received an intraperitoneal injection of BrdU (5-bromo-2-deoxyuridine; Sigma) at a dosage of 100 mg/kg after ketamine anesthesia according to the following experimental schedule.

Experiment 1: To evaluate the effect of ketamine on the proliferation and differentiation of NSCs in the DG during the BGS, the PND-7 rats received a single intraperitoneal injection of BrdU on PND-7, 13, and 20 after ketamine treatment. The animals were then anesthetized and fixed by perfusion at 24 h after each BrdU injection. The experimental protocol is described in Tables 1a and 2.
Experiment 2: To exclude the GFAP/BrdU double-positive cells that were proliferative astrocytes, the PND-7 rats received a single intraperitoneal injection of BrdU on PND-7, 13, and 20 after exposure to treatment. The animals were then perfused at 3 h after each BrdU injection. The experimental protocol is detailed in Tables 1b and 2.

Experiment 3: To determine the effect of ketamine on the migration of newborn granule neurons in the DG, the PND-7 rats received three consecutive BrdU injections on PND-7, 8, and 9 after exposure to treatment. At 28 and 35 days after the last BrdU injection, the animals were anesthetized and fixed by perfusion. The experimental protocol is described in Table 1c and 2.

Cell Apoptotic Assays
Nestin/caspase-3 and GFAP/caspase-3 double-immunofluorescence staining was utilized to detect whether ketamine could induce the apoptosis of NSCs or astrocytes. At 12 h after the end of control and ketamine-anesthesia treatment, the neonatal rats were anesthetized and fixed by perfusion (n = 5 per group).

Tissue Preparation and Immunofluorescence
At the indicated time point, animals were deeply anesthetized and then transcardially perfused with 0.9 % normal saline followed by 4 % paraformaldehyde. The brains were removed, postfixed overnight in 4 % paraformaldehyde, and placed in 30 % sucrose until sunk. The coronal sections of brain were cut consecutively at a thickness of 30 μm when the hippocampus was initially exposed. The fifteenth section was taken and stored in PBS. According to the Atlas of the Developing Rat Brain and previous reports (Ashwell and Paxinos 2008; Paxinos and Watson 1986), the positions of hippocampus coronal sections selected in our study were about 2.20–2.25 mm posterior to the bregma at PND-8 rats, about 2.35–2.40 mm posterior to the bregma at PND-14 rats, about 2.50–2.55 mm posterior to the bregma at PND-21 rats, and about 2.75–2.85 mm posterior to the bregma at PND-37 and PND-44 rats, respectively.

For Nestin/BrdU, β-tubulin III/BrdU, GFAP/BrdU, and NeuN/BrdU double-immunofluorescence staining, the BrdU antigen was exposed by incubating the sections in 2-normal hydrochloric acid for 30 min at 37 °C and then washed three times with PBS for 5 min between each of these steps. Blocking of nonspecific epitopes with 10 % donkey serum in PBS with 0.3 % Triton-X for 2 h at room temperature preceded incubation overnight at 4 °C with the primary antibodies listed in Table 3 in PBS with 0.3 % Triton-X. On the next day, the sections were incubated with the appropriate secondary fluorescent antibodies (Invitrogen Carlsbad, CA) for 2 h at room temperature.
Astrocytic development was detected by using GFAP single-labeled staining. The sections were incubated overnight at 4 °C with a fluorescent antibody for the GFAP (Table 3). After three washes in PBS, sections were incubated with secondary fluorescent antibody (Invitrogen) for 2 h at room temperature.

To characterize the phenotype of cell apoptosis, brain sections were analyzed by double-labeled staining. The sections were incubated overnight at 4 °C with the appropriate primary antibodies listed in Table 3. After three washes with PBS, the sections were incubated with the suitable secondary fluorescent antibodies (Invitrogen) for 2 h at room temperature.

A skilled pathologist blinded to the study conditions examined the labeled sections using a laser scanning confocal microscope (Fluoview 1000, Olympus). The number of single- or double-positive cells in the hippocampal DG was quantified using Image-Pro Plus software.

Western Blot Analysis
Thirty and thirty-seven days after the control or ketamine-anesthesia treatment, the animals were decapitated, and the hippocampal DG tissue was dissected carefully with anatomic microscope (leica EZ4HD). The harvested hippocampal tissues were homogenized on ice using lysate buffer plus protease inhibitors. The lysates were centrifuged at 14,000 rpm for 15 min at 4 °C and were resolved by 12 % polyacrylamide gel electrophoresis, and the target proteins were transferred to nitrocellulose membranes. The blots were incubated with blocking buffer for 2 h at room temperature and then incubated for 24 h at 4 °C with the primary antibodies: rabbit anti-GFAP antibody (1:1000, Millipore) and GAPDH. The membranes were then incubated with appropriate secondary alkaline phosphatase-conjugated donkey anti-rabbit antibody (1:10,000, Abcam) for 1 h. The band intensity was quantified using Image J software (n = 5 per group).

Morris Water Maze Test
The hippocampal-dependent spatial memory abilities were tested by using the Morris water maze (MWM). Different set of rats were tested 2 months after administration of ketamine on PND-7. A circular, black painted pool (180 cm diameter, 50 cm deep) was filled with water to a depth of 30 cm. The water temperature was maintained at 25 ± 1 °C. An invisible platform (10 cm diameter) was submerged 1 cm below the water surface and placed in the center of the III quadrant which was determined with four starting locations called I, II, III, and IV at equal distance on the edge of the pool. During five consecutive days, the experiments were conducted in a dark and quiet laboratory, all the rats were trained four times per day, the starting positions were random for each rat. When the rat found the platform, the rat was allowed to stay on it for 30 s. If a rat did not find the platform within 120 s, the rat would be guided gently to the place and allowed to stay on it for 30 s, and the latency time to find the hidden platform was recorded as 120 s. The average time from four trials represented as the daily result for the rat. On the sixth day, the hidden platform was removed, and the rat was placed in the opposite quadrant. Rats were allowed to swim freely for 120 s. The numbers the rat swam to cross the previous platform area, and the times the rat stayed in the target quadrant within 120 s were recorded. Each animal’s path was tracked by a computerizing video system. After every trial, each rat was placed in a heater plates for 1 to 2 min until dry before being returned to its chamber. The data were analyzed using software for the MWM (Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, China).

Statistical Analysis
The statistical analysis was conducted using SPSS 13.0, and the graphs were created using GraphPad Prism 5. The data were analyzed using Mann-Whitney U test. The interaction between time and group factors in a two-way ANOVA was used to analyze the difference of escape latency between rats in the control group and rats treated with ketamine in the MWM. The data are presented as the mean ± SD, and p < 0.05 was considered statistically significant.",rats,['The timed-pregnant Sprague–Dawley rats were housed in a temperature-controlled (22–23 °C) room on a 12 h:12 h light:dark cycle (light on at 8:00 AM) with free access to food and water.'],postnatal day 7,['The PND-7 male rat pups (11–14 g) were randomly assigned to ketamine-treated and control groups.'],Y,['The hippocampal-dependent spatial memory abilities were tested by using the Morris water maze (MWM).'],ketamine,"['In the treated group, ketamine was diluted in 0.9 % normal saline, and PND-7 rats were intraperitoneally administered with 40 mg/kg doses of ketamine in four injections at 1 h intervals (40 mg/kg × 4 injections).']",none,[],sprague dawley,['The timed-pregnant Sprague–Dawley rats were housed in a temperature-controlled (22–23 °C) room on a 12 h:12 h light:dark cycle (light on at 8:00 AM) with free access to food and water.'],True,True,True,True,True,True,[ Passage 8/25 ] 10.1007/s12640-016-9615-7
10.1186/s12871-018-0471-2,1187.0,Huang,2018,rats,gestational day 21,Y,isoflurane,none,sprague dawley,"PMID: 29325538 PMCID: PMC5765622 DOI: 10.1186/s12871-018-0471-2
Methods
This study was approved by the Ethics Committee of Affiliated Shengjing Hospital of China Medical University, and specific pathogen free SD pregnant rats weighing 380–420 g were purchased from the Experimental Animal Center of Affiliated Shengjing Hospital of China Medical University. Animals were housed at 22–24 °C, 40–60% humidity with a 12-h light /dark cycle and had free access to food and water. Rats at the gestational age of 21 days (E21) were used in subsequent experiments. According to the isoflurane dose, rats were divided into 3 groups: the Iso1 group (1.3% isoflurane), the Iso2 group (2.0% isoflurane) and the control group (0% isoflurane; O2).

In the absence of anesthesia, intratracheal intubation was difficult in the control group. Thus, all the rats retained spontaneous breathing and did not receive intratracheal intubation. Inhalation of isoflurane at a high concentration may inhibit respiration and cause hypoxia. Thus, in our pilot study, pregnant rats at the gestational age of 20 days (E20) were anesthetized intraperitoneally with pentobarbital sodium and catheter indwelling was done in the right carotid artery; rats were then allowed to recover at room temperature. At E21, rats were placed in a box filled with prefilled gas according to the following groups: 50% O2 was administered in the control group; 1.3% isoflurane was administered in the Iso1 group (50% oxygen, balanced with nitrogen); 2.0% isoflurane was administered in the Iso2 group (50% oxygen, balanced with nitrogen). All rats were retained spontaneous breathing and exposed in the box for 3 h (the concentrations of isoflurane and oxygen were monitored). The mean arterial blood pressure was continuously monitored via a catheter in the carotid artery, and arterial gas analysis was performed hourly. The results showed that inhalation of isoflurane at 1.3% or 2.0% had no influence on the arterial gas and mean arterial blood pressure. Rats used in pilot study will not be used for formal study.

In this study, a total of 48 rats at E21 were randomly assigned into 3 groups and exposed to isoflurane at the predesigned concentration for 3 h. Animals were allowed to recover at room temperature and housed until they delivered. The number of fetuses was recorded, and healthy male neonatal rats were used in the experiments. At day 28 after birth (P28), the male offsprings were randomly assigned into two groups: one for Morris water maze (MWM) test to evaluate memory and learning and the other one were housed until day 90 after birth (P90) to receive the same MWM test.

MWM test used a round swimming pool sized 150 cm in diameter and 60 cm in height with a platform sized 10 cm in diameter in the maze. The removable platform was 1.5 cm lower than the water surface. The visual cues (a variety of figures) on the maze’s inner wall remained unchanged during the study. Training and examination were performed in the water at 20 °C. After each examination, rats were dried under a lamp and returned to the cages.

Place navigation test was performed for consecutive 5 days. In brief, platform were placed in a quadrant (the 4th quadrant in this study). At predesigned time point, rats were placed in a random quadrant (once for each quadrant). If the rat found the platform within 90s, it was allowed to stay on the platform for 15 s and then placed out of the pool. The spatial navigation test was performed on the 6th day to evaluate memory. In brief, the platform was removed, rats were placed in a random quadrant and the swimming trajectory was recorded within 90s. In the test, the proportion of swimming distance in the platform quadrant to the total swimming distance and the times of crossing the platform were calculated. The swimming distance in the platform quadrant reflects spatial localization and the times of crossing the platform reflects the accuracy of spatial memory. Before training, the platform was visible above the water surface, which may exclude rats with visual defects that were unable to find the platform. In addition, rats with poor performance in the test, such as those could not find the hidden platform and swam along the wall, were also excluded from this study.

Two hours after the spatial navigation test, rats were intraperitoneally anesthetized with pentobarbital sodium. Half of each group of the rats were used to collect brain and followed by the separation of hippocampus. The hippocampus was weighed and lysed for total protein extraction. Samples were then stored at −80 °C for later use. Western blotting was performed to detect the protein expression of CREB and p-CREB in the hippocampus. The half of the rats were transcardially perfused with 4% paraformaldehyde and the brain was collected and fixed in 4% paraformaldehyde. Immunohistochemistry was performed to detect CREB and p-CREB expression. (Fig. 1).
The neonatal rats were randomly assigned into different groups to reduce variation. We normalized CREB and p-CREB protein expression in control group as 1. CREB and p-CREB expression in the Iso1 and Iso2 group was compared with the controls. All data are expressed as mean ± standard deviation. Statistical analyses were performed by using SPSS software (version 21.0; IBM, Corp., Armonk, NY, USA). One-Way ANOVA was used to compare the means between groups. A value of P < 0.05 indicated significance.",rats,['specific pathogen free SD pregnant rats weighing 380–420 g were purchased from the Experimental Animal Center of Affiliated Shengjing Hospital of China Medical University.'],gestational day 21,['Rats at the gestational age of 21 days (E21) were used in subsequent experiments.'],Y,"['At day 28 after birth (P28), the male offsprings were randomly assigned into two groups: one for Morris water maze (MWM) test to evaluate memory and learning and the other one were housed until day 90 after birth (P90) to receive the same MWM test.']",isoflurane,"['According to the isoflurane dose, rats were divided into 3 groups: the Iso1 group (1.3% isoflurane), the Iso2 group (2.0% isoflurane) and the control group (0% isoflurane; O2).']",none,[],sprague dawley,['specific pathogen free SD pregnant rats weighing 380–420 g were purchased from the Experimental Animal Center of Affiliated Shengjing Hospital of China Medical University.'],True,True,True,True,True,True,[ Passage 9/25 ] 10.1186/s12871-018-0471-2
10.1213/ANE.0b013e318281e988,407.0,Istaphanous,2013,mice,postnatal day 7,N,isoflurane,none,none,"PMID: 23460572 DOI: 10.1213/ANE.0b013e318281e988
METHODS
All procedures were approved by the Institutional Animal Care and Use Committee and conformed to the guidelines for ethical treatment of animals. Efforts were made to minimize the number of animals used. Breeding pairs of male CD1 and female C57BL/6 mice were housed in a 12/12-hour light-dark cycle at 22°C with free access to food and water. This hybrid was selected because they exhibit robust anesthesia-induced apoptosis with acceptable survival.3

Isoflurane Treatment
For caspase 3 immunohistochemistry, 7-day-old CD1 and C57BL/6 hybrid littermates (n = 14) were randomly assigned to a 6-hour exposure to 1.5% isoflurane (approximately 0.6 minimum alveolar concentration in these mice) in 30% oxygen (anesthesia, n = 8) or to 6 hours in room air (control, n = 6). Immediately after treatment, animals were euthanized with an overdose of ketamine, acepromazine, and xylazine. Brains were immersion-fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4), postfixed overnight at 4°C, and cryopreserved in 25% sucrose. Brains were snap frozen and 40-μm coronal sections were cut on a cryostat (Thermo Electronics, Kalamazoo, MI). Sections were mounted to charged slides and stored at −80°C until use.

For protein analyses, a separate set of animals (n = 22) was treated and euthanized as described above. The left hemispheres were cut into 4 coronal sections, frozen in liquid nitrogen, and stored at −80°C until use. At a later date, sections of neocortex around bregma −3 mm were separated with a razor blade on dry ice and then homogenized twice in cell lysis buffer solution for approximately 10 seconds each time at 4°C. The homogenate was then centrifuged at 13,000 rpm using a refrigerated microcentrifuge (Fresco centrifuge, Sorvall, Buckinghamshire, UK). The supernatant was removed and used for testing for the specific proteins.

Immunohistochemistry
Slide-mounted brain sections were blocked for 1 hour in normal goat serum, followed by incubation in rabbit antiactivated caspase 3 polyclonal antibodies (1:100, 9661L; Cell Signaling, Danvers, MA) for 18 hours at −4°C, combined with one of the following antibodies: (1) mouse anti–Neuronal Nuclei (NeuN) monoclonal antibodies (NeuN, 1:500, Chemicon, MAB377; Millipore, Billerica, MA), (2) mouse antiglutamate decarboxylase isoform 67 (antiglutamic acid decarboxylase [GAD]67, 1:2000, MAB5406; Chemicon), (3) mouse anti-S100β (1:500, CB1040; Millipore), or (4) chicken antiglial fibrillary acidic protein (GFAP) (1:500, AB5541; Chemicon). Sections were then rinsed in blocker and incubated in Alexa Fluor 488 goat antirabbit secondary antibodies (1:200, A11034, Molecular Probes Inc.; Invitrogen, Carlsbad, CA) for 4 hours at 20°C, combined with either Alexa Fluor 594 goat antimouse (1:250, A11032, Molecular Probes) or Alexa Fluor 594 goat antichicken (1:250, A11042, Molecular Probes) secondary antibodies, as appropriate for the primary antibody species. After immunostaining, sections were dehydrated in an ascending ethanol series, cleared in xylenes, and mounted with Krystalon (EMD, Gibbstown, NJ).

Identification of Cellular Phenotype
To determine the phenotype of degenerating cells, brain sections from anesthesia-treated and control animals, corresponding to Bregma −2.46 to −2.70 (figures 51–53 in the mouse brain atlas by Paxinos and Franklin11) and double-immunostained for caspase 3 and NeuN or triple stained for caspase 3, S100β, and GFAP, were examined by an observer unaware of group assignment. NeuN and S100β stains cannot be combined in the same section, because both secondary antibodies are raised in the same species.

Caspase 3 immunostaining was excited using the 488-nm laser line, and emission wavelengths between 510 and 540 nm were collected to identify caspase-positive cells in layers II/III from retrosplenial cortex to piriform cortex using an SP5 confocal microscope set up on a DMI6000 stand (Leica Microsystems, Wetzlar, Germany) equipped with a 63× objective (NA 1.4). This region was selected because it has repeatedly demonstrated increased numbers of apoptotic cells in immature rodents.2,3 Immunostaining for NeuN, S100β, or GFAP was excited using the 543-nm laser line, and emission wavelengths between 600 and 650 nm were collected. Confocal optical sections were collected through the midpoint of the caspase 3–positive cell (pinhole = 1 Airy unit). Data are expressed as the percentage of caspase 3–immunoreactive cells that were also NeuN- or GFAP-positive, respectively.

Quantification of Apoptotic Cells Using the Optical Dissector Method
Further quantification of the effects of isoflurane exposure on cortical neurons and on GABAergic interneurons was performed as previously described.3,8 Briefly, confocal image stacks of caspase 3/GAD67 double labeling were collected at 1-µm increments through the entire Z-depth of the tissue (40 μm) using 1× optical zoom. Six image stacks were collected from layers II/III of visual cortex, corresponding to figures 51 to 53 in the mouse brain atlas by Paxinos and Franklin,11 from each animal, as follows: for each hemisphere, 3 adjacent confocal image stack frames were collected beginning 750 μm from the midline and moving laterally (Leica SP5, 63× 1.4 NA objective, 1-μm steps). Image stacks, which were 120 × 120 µm in dimension for NeuN and 240 × 240 µm for GAD67, because of the significantly lower cellular density for the latter stain compared with NeuN, were transferred to Neurolucida software (v7.50.4; MBF Bioscience, Williston, VT) for analysis. Using the optical dissector method, an observer unaware of group assignment quantified the respective numbers of NeuN-positive or GAD67-positive cells, the corresponding number of caspase 3–positive cells, and the number of caspase 3/GAD67 or caspase 3/NeuN double-positive cells in each field.12,13 Cells were considered positive if their fluorescence intensity was 2 times or greater than the background intensity. Counts from all 6 respective image stacks were averaged for each animal.

Quantification of GAD67 and GAD65 Expression Using Competitive Enzyme-Linked Immunosorbent Assay
We used a competitive enzyme-linked immunosorbent assay to quantify the expression of the two γ-aminobutyric acid A (GABAA) synthesizing enzymes, GAD67 and GAD65. Rat antiglutamate decarboxylase isoform 67 (Anti GAD67, 1:5000, 671-C; Alpha Diagnostics Inc., San Antonio, TX) and goat antiglutamate decarboxylase isoform 65 (Anti GAD65, 1:32,000, Ab67725; Abcam, Cambridge, MA) antibodies were incubated overnight with the homogenized cortical tissue samples. These bound antibody/antigen complexes were then added to a GAD67 or GAD65 antigen-coated well blocked with 5% bovine serum albumin. Rabbit antirat and rabbit antigoat secondary antibodies were added to GAD67 and GAD65 complexes, respectively. The secondary antibodies were covalently bound to horseradish peroxidase, an enzyme that cleaves the peroxide in the chromophore 3,3′,5,5′-tetramethylbenzidine. This enzyme activation turned on the chromophore and emitted a blue signal, which when treated with 2 M sulfuric acid turned to a yellow color, which was measured at 450 nm using a spectrophotometer (Jenway Genova Life Science Spectrophotometer; Bibby Scientific Limited, Staffordshire, UK). Absorbancy was then compared with a standard curve allowing for the determination of the isoforms’ concentrations.

Statistical Analysis
All sample sizes for group assignment were made a priori. For each animal, the total NeuN-positive cells were counted over the 6 fields. The number of caspase 3/NeuN double-positive cells was defined as an event. The data were normalized to events (caspase 3/NeuN double-positive cells) per 400 NeuN-positive cells counted, the lower end of cells encountered in each animal, to avoid extrapolation. Gross inspection of the raw data revealed that caspase 3 activation in NeuN-positive cells was a rare event with a mean incidence of 2.4% and a maximal incidence of 3.6% in the anesthesia-treated animals. This event rate met the criteria for analysis using the Poisson distribution.

The Poisson mean event rate, λ, and its 95% CI were determined using the MATLAB® function [lambdahat, lambdaci] = poissfit(data, alpha). The vector “data” represented the number of events per 400 counted NeuN cells for each animal in the group of interest and α = (1 − CI). The mean event rates, λ, derived from the MATLAB function, were used to construct probability distribution function curves for the 2 groups (see Appendix).

The raw event counts were used to compute the ratio of events in the anesthesia-treated group to the control group using equations 6 and 7 in Graham et al.14 This method was used as an independent means to assess the mean event ratio and to determine the 95% CI for the event ratio.

All other data are presented as means ± SEM. Group comparisons were made using the Mann-Whitney U test. Statistical calculations were analyzed using Stata/IC 10.1 for Mac OS X (Stata Corp., College Station, TX). Statistical significance was accepted at P < 0.05.",mice,['Breeding pairs of male CD1 and female C57BL/6 mice were housed in a 12/12-hour light-dark cycle at 22°C with free access to food and water.'],postnatal day 7,"['For caspase 3 immunohistochemistry, 7-day-old CD1 and C57BL/6 hybrid littermates (n = 14) were randomly assigned to a 6-hour exposure to 1.5% isoflurane...']",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,"['For caspase 3 immunohistochemistry, 7-day-old CD1 and C57BL/6 hybrid littermates (n = 14) were randomly assigned to a 6-hour exposure to 1.5% isoflurane...']",none,[],c57bl/6,['Breeding pairs of male CD1 and female C57BL/6 mice were housed in a 12/12-hour light-dark cycle at 22°C with free access to food and water.'],True,True,True,True,True,False,[ Passage 10/25 ] 10.1213/ANE.0b013e318281e988
10.1016/j.bja.2018.04.034,740.0,Ju,2018,rats,postnatal day 5,Y,sevoflurane,none,sprague dawley,"PMID: 30032879 PMCID: PMC6200111 DOI: 10.1016/j.bja.2018.04.034

Methods
Animals
All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee. Sprague-Dawley rats were housed under controlled illumination (12-h light/dark, lights on at 7:00AM) and temperature (23–24°C) with free access to food and water. Within 24 h of delivery, litters were culled to 12 pups. At 21 postnatal days (P21), pups were weaned and housed in sex-matched groups of two for the rest of the study.
Treatment groups
The P5 male and female rat pups were kept in a temperature-controlled chamber (37ºC) with a continuous supply of 30% oxygen in air (1.5 L min−1) during anaesthesia with 6 vol% sevoflurane for 3 min for induction and 2.1 vol% sevoflurane for 357 min as maintenance (sevoflurane group). Previously, we have shown that blood glucose and gas levels after 2.1% sevoflurane for 6 h were in the normal range.4 Control F0 animals were subjected to animal facility rearing only (control group).
The F0 male and female rats were sequentially evaluated on the elevated plus maze (EPM) starting on P60, for prepulse inhibition (PPI) of the acoustic startle response on P70, and for corticosterone responses to physical restraint for 30 min on ≥P160 followed by isolation of brain and gamete tissue samples for further analyses (Fig. 1). Twenty-four F0 males and 24 females were mated on ∼P90 to produce the F1 generation. F0 breeders were randomised into one of the following four groups for mating: 1) control males+control females (con-M*con-F); 2) exposed males+control females (sevo-M*con-F); 3) control males+exposed females (con-M*sevo-F); and 4) exposed males+exposed females (sevo-M*sevo-F). The female was kept alone throughout the entire gestation and postpartum rearing periods. The F1 rats, 144 in total [n=18 per sex (two) per group (four)], which were subjected to facility rearing only, were evaluated in the EPM starting on P60, PPI of startle on P70, Morris water maze (MWM) testing starting on P79, and for the corticosterone responses to restraint for 30 min on ≥P90, followed by isolation of brain tissue samples for further analyses. A separate cohort of F1 rats was sacrificed on P5 to collect brain tissue for bisulphite sequencing.
Basal and stress-induced activity of the HPA axis
Blood samples (∼300 μL) were collected at rest and 10, 60, and 120 min after the restraint, as previously described.7 Serum corticosterone was measured using commercial ELISA kits (Cayman Chemical Company, Ann Arbor, MI, USA) following the manufacturer's instructions.7,8
Behavioural tests
The EPM, acoustic startle response, PPI of startle, and MWM tests were performed as previously described.4,  5,  6,  7,  8
Tissue collection
Adult rats were anaesthetised with sevoflurane and decapitated. Whole brains were removed and immediately put in a stainless steel adult rat brain slicer matrix with 0.5 mm coronal section slice intervals (Zivic Instruments, Pittsburgh, PA, USA). Hypothalamic paraventricular nucleus (PVN) tissue was punched out with a 1-mm ID glass capillary tube. The hippocampus was isolated from the respective slices. Tissues were placed in vials filled with RNAlater solution (Invitrogen, Carlsbad, CA, USA) and stored at −80°C. Sperm were isolated from the caudal epididymis of adult males and stored at −80°C. After separation from the adipose tissues, ovaries were stored at −80°C.
Analyses of mRNA levels for Nkcc1, Kcc2, and glucocorticoid receptors (Gr)
The mRNA levels for Nkcc1, Kcc2 in the PVN of the hypothalamus and hippocampus, and for Gr in the hippocampus were analysed via qRT–PCR as previously described.7,8
Bisulphite sequencing
Genomic DNA was extracted from the sperm pellet and ovaries of adult F0 rats and from hippocampal and hypothalamic tissues of P5 F1 rats using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). The sodium bisulfite conversion was performed with EZ DNA Methylation kits (Zymo Research, Irvine, CA, USA) following the manufacturer's instructions. The primers (Nkcc1: forward: GAGAGGAGTTTATAGGGTT; reverse: AACCCTAC(A/G)CTAACCAACCTC; Kcc2: forward: GATTGTAAGTGTTTTTATTATTGAGTTGTATATT; reverse: AATAAACTTTTCCCCTTTTATACCC) were designed for the bisulfite-converted DNA sequences, using previously published sequences.23,24 PCR amplification was performed with HotStar Taq (Qiagen). Amplicons were cloned into pCR4-TOPO vector with the TOPO TA cloning kit for sequencing (Life Technologies, Carlsbad, CA, USA). Miniprep was performed on each positive clone using ZR Plasmid Miniprep kit (Zymo Research). Sanger sequencing was done by Genewiz (South Plainfield, NJ, USA) using M13R primers. The DNA methylation status of all CpG sites was analysed using Benchling Molecular Biology 2.0 Software (Benchling, San Francisco, CA, USA).
Statistical analysis
Values are reported as mean (standard deviation). Statistical analyses were carried out on raw data using SigmaPlot 13.0 software (Systat Software, Inc., San Jose, CA, USA). To assess differences in total corticosterone concentration, EPM behaviour and gene expression for Nkcc1, Kcc2, and Gr, t-test and one way analysis of variance (ANOVA) were used for F0 and F1 generations, respectively. Two way ANOVA with experimental groups and time as the independent variables was run to analyse changes in serum corticosterone concentrations at rest and at three time points after the restraint. Two way ANOVA was used to analyse the PPI data, with the treatment and prepulse intensity as independent variables, and the MWM latencies to escape data, with experimental groups and days of training as the independent variables. One-way ANOVA was used to analyse time spent in the target quadrant and numbers of crossings during the MWM probe test. Two way measures ANOVA with treatment as ‘between’-subject factor and CpG site as ‘within’-subject factor was used to analyse the frequency methylation of CpG sites. Multiple pairwise comparisons were done with the Holm-Sidak method. All comparisons were run as two-tailed tests. A P value <0.05 was considered significant. The sample sizes in this study were based on previous experience with the same experimental techniques.6,  7,  8",rats,"['Sprague-Dawley rats were housed under controlled illumination (12-h light/dark, lights on at 7:00AM) and temperature (23–24°C) with free access to food and water.']",postnatal day 5,['The P5 male and female rat pups were kept in a temperature-controlled chamber (37ºC) with a continuous supply of 30% oxygen in air (1.5 L min−1) during anaesthesia with 6 vol% sevoflurane for 3 min for induction and 2.1 vol% sevoflurane for 357 min as maintenance (sevoflurane group).'],Y,"['The F0 male and female rats were sequentially evaluated on the elevated plus maze (EPM) starting on P60, for prepulse inhibition (PPI) of the acoustic startle response on P70, and for corticosterone responses to physical restraint for 30 min on ≥P160 followed by isolation of brain and gamete tissue samples for further analyses (Fig. 1).', 'The F1 rats, 144 in total [n=18 per sex (two) per group (four)], which were subjected to facility rearing only, were evaluated in the EPM starting on P60, PPI of startle on P70, Morris water maze (MWM) testing starting on P79, and for the corticosterone responses to restraint for 30 min on ≥P90, followed by isolation of brain tissue samples for further analyses.']",sevoflurane,['The P5 male and female rat pups were kept in a temperature-controlled chamber (37ºC) with a continuous supply of 30% oxygen in air (1.5 L min−1) during anaesthesia with 6 vol% sevoflurane for 3 min for induction and 2.1 vol% sevoflurane for 357 min as maintenance (sevoflurane group).'],none,[],sprague dawley,"['Sprague-Dawley rats were housed under controlled illumination (12-h light/dark, lights on at 7:00AM) and temperature (23–24°C) with free access to food and water.']",True,True,True,True,True,True,[ Passage 11/25 ] 10.1016/j.bja.2018.04.034
10.1371/journal.pbio.2001246,384.0,Kang,2017,mice,postnatal day 18,Y,isoflurane,none,c57bl/6,"PMID: 28683067 PMCID: PMC5500005 DOI: 10.1371/journal.pbio.2001246
Methods
Ethics
All study protocols involving mice were approved by the Animal Care and Use Committee at the Johns Hopkins University (protocol MO14M315) and conducted in accordance with the NIH guidelines for care and use of animals.

Animals
C57BL/6 mice were housed in a temperature- and humidity-controlled room with a 12:12 hour light:dark cycle, and provided with ad libitum access to water and food. Both sexes were equally represented in all experiments. No animals were excluded.

Isoflurane treatment and physiologic monitoring of sentinel animals
P18 mouse littermates were randomly assigned to 2 groups. In Group 1 (isoflurane), mice were exposed to 1.5% isoflurane carried in 100% oxygen for 4 hours. A calibrated flowmeter was used to deliver oxygen at a flow rate of 5 L/min and an agent-specific vaporizer was used to deliver isoflurane. In Group 2 (control), mice were exposed to room air for 4 hours. Animals were returned to their cages together with their littermates upon regaining righting reflex. Mice were continually monitored and recorded for skin temperature, heart rate, and oxygen saturation during the 4-hour isoflurane treatment (PhysioSuite; Kent Scientific, Torrington, CT). Intracardiac puncture was used to collect left ventricular blood samples from selected sentinel animals, and those confirmed to be arterial are reported.

Production and stereotaxic injection of engineered retroviruses
Engineered self-inactivating murine retroviruses were used to express GFP under Ubiquitin promotor (pSUbGW vector) specifically in proliferating cells and their progeny [55,56]. High titers of engineered retroviruses (1 x 109 unit/ml) were produced by cotransfection of retroviral vectors and VSVG into HEK293gp cells followed by ultracentrifugation of viral supernatant as previously described [24,49,55–57]. After induction with a single ketamine injection (50mg/kg), high titers of GFP-expressing retroviruses were stereotaxically injected into the P15 mice dentate gyrus through a 32-gauge microsyringe (Hamilton Robotics, Reno, NV) at 2 sites of the following coordinates relative to the bregma (mm): AP: −2.2, ML: ±2.2, DV: −2.4. The retrovirus-containing solution was injected at a rate of 0.025 μl/min for a total of 0.5 μl per site. After infusion, the microsyringe was left in place for an additional 5 minutes to ensure full virus diffusion and to minimize backflow. After surgery, mice were monitored for general health every day until full recovery. In order to test for a possible confound related to the use of ketamine anesthesia, pS6 immunoreactivity in the dentate gyrus was quantified at P30 in naïve control animals and compared to pS6 immunoreactivity in animals doses with ketamine as above. No significant difference is seen in pS6 levels between these groups (S6 Fig).

Immunostaining
After transcardial perfusion fixation with 4% paraformaldehyde/PBS, brains were sliced transversely (50 μm thick) with microtome and processed for immunohistochemistry. Primary antibodies, including goat anti-GFP (Rockland, 1:1000) and chicken anti-GFP (Millipore, 1:1000) were used. Immunofluorescence was performed with a combination of Alexa Fluor 488- or Alexa Fluor 594-labeled anti-goat, anti-chicken, or anti-rabbit secondary antibodies (1:250) and 4ʹ,6ʹ-diaminodino-2-phenylindole (DAPI, 1:5000). For analysis of pS6 levels, primary antibodies against pS6-Ser235/236 (rabbit, 1:1000, Cell Signaling) were used. Effective immunostaining of pS6 required an antigen retrieval protocol as previously described [58]. Briefly, sections were incubated in target retrieval solution (DAKO) in 85°C for 20 minutes followed by washing with PBS for t3 times before the incubation with primary antibody.

Imaging and analyses
Images were acquired on a confocal system (Zeiss LSM 710 or Leica SPE) and morphological analyses were carried out as previously described [24,49,55,56,58,59]. Images for dendritic and spine morphology were deconvoluted with Auto Quant X (Media Cybernetics, Rockville, MD) using the blind algorithm, which employs an iteratively refined theoretical PSF. No further processing was performed prior to image analysis. For visualization, brightness, and contrast levels were adjusted using Image J (NIH). For analysis of dendritic development, three-dimensional (3D) reconstructions of entire dendritic processes of each GFP+ neuron were obtained from Z-series stacks of confocal images using excitation wavelength of 488 nm at high magnification (x 40 lens with 0.7x optical zoom). The two-dimensional (2D) projection images were traced with NIH Image J plugin, NeuronJ. All GFP+ DGCs with largely intact, clearly identifiable dendritic trees were analyzed for total dendritic length. The measurements did not include corrections for inclinations of dendritic process and therefore represented projected lengths. Sholl analysis for dendritic complexity was carried out by counting the number of dendrites that crossed a series of concentric circles at 10 μm intervals from the cell soma using ImageJ (NIH). For complete 3D reconstruction of spines, consecutive stacks of images were acquired using an excitation wavelength of 488 nm at high magnification (x 63 lens with 5x optical zoom) to capture the full depth of dendritic fragments (20–35 μm long, 40~70 dendritic fragments in each condition analyzed) and spines using a confocal microscope (Zeiss, Oberkochen. Germany). Confocal image stacks were deconvoluted using a blind deconvolution method (Autoquant X; Media Cybernetics, Rockville, MD). The structure of dendritic fragments and spines was traced using 3D Imaris software using a “fire” heatmap and a 2D x–y orthoslice plane to aid visualization (Bitplane, Belfast, UK). Dendritic fragments were traced using automatic filament tracer, whereas dendritic spines were traced by means of an autopath method with the semiautomatic filament tracer (diameter; min: 0.1, max: 2.0, contrast: 0.8). For spine classification, a custom MatLab (MathWorks, Natick, MA) script was used based on the algorithm; stubby: length (spine) <1.5 and max width (head)<mean_width (neck) *1.2; mushroom: max width (head) >mean width (neck) *1.2 and max_width (head) >0.3; if the spine was not classified as mushroom or stubby, it was defined as long-thin. Axonal bouton volume from axonal fragments was measured by using 3D Imaris software and using a magic wand menu (Bitplane, Belfast, UK) after deconvolution. For analysis of pS6 levels, the sections were processed in parallel and images were acquired using the identical settings, (Zeiss LSM 710, 20X lens). Fluorescence intensity was measured within the granular cell layer using ImageJ (NIH) and the value was normalized to background signal in the same image. These data were then subsequently normalized to the area of the dentate gyrus granule layer as defined by DAPI staining. All experiments were carried out in a blind fashion to experimental conditions.

Behavioral tests
Sixty-day-old mice housed in groups (5 mice per cage) were handled for at least 2 minutes per day for 3 days before the start of the behavioral experiments. All behavioral tests were performed during the light phase of the cycle between 8:00am and 6:00pm. Experimenters were blind to the samples when behavioral tests were carried out and quantified. The numbers of mice per condition are indicated in the figure legends.

Object-place recognition test Object-place recognition was performed as previously described [37]. Briefly, the test was assessed in a 27.5 cm × 27.5 cm × 25 cm opaque chamber with a prominent cue on 1 of the walls. Each mouse was habituated to the chamber for 15 minutes daily for 2 days. During the training phrase, each mouse was allowed to explore 2 identical objects (glass bottle, 2.7 cm diameter, 12 cm height, and colored paper inside) for 10 minutes. The mouse was then returned to its home cage for a retention period of 24 hours. The mouse was reintroduced to the training context and presented with 1 object that stayed in the same position as during training while the other object was moved to a new position. Movement and interaction with the objects was recorded with a video camera that was mounted above the chamber and exploratory behavior was measured by a blinded observer. Exploratory behavior was defined as sniffing, licking, or touching the object while facing the object.
Y-maze test In the Y-maze test, mice were released from the start arm (no visual cue) and allowed to habituate to only 1 out of 2 possible choice arms (overt visual cue) for 15 minutes. This was followed at 24 hours later by the recognition phrase in which the animal could choose between the 2 choice arms after being released from the start arm. The timed trials (5 minutes) were video recorded as well as graded by an observer blind to condition for total exploration time in each choice arm.
Rapamycin treatment
P21 mouse littermates were given IP injections of rapamycin (Sigma-Aldrich, St. Louis, MO) prepared from a stock solution (25 mg/ml in 100% ethanol, stored at -20°C) diluted to a final concentration of 4% (v/v) ethanol in the vehicle. Vehicle consisted of 5% Tween 80 (Sigma-Aldrich, St. Louis, MO) and 10% polyethylene glycol 400 (Sigma-Aldrich, St. Louis, MO) as previously described [58,60,61]. Both rapamycin- and vehicle-treated mice received the same volume for each injection (200 μl). Mice received treatments at 48 hour intervals from P21 to P29.

Statistics
Results are expressed as mean ± SEM. A one-tailed Student t test or ANOVA with Bonferroni test for intergroup comparisons were used for most statistical comparisons between groups as described in the figure legends using Prism Software (Graphpad Software Inc, La Jolla, CA). For Sholl analysis ANOVA was used at each point to test for differences between distributions. All data examined with parametric tests were determined to be normally distributed, and the criteria for statistical significance was set a priori at p < 0.05. Sample sizes were predicted based on experience from previous similar work [24]. All relevant data are available from the authors.",mice,['All study protocols involving mice were approved by the Animal Care and Use Committee at the Johns Hopkins University (protocol MO14M315) and conducted in accordance with the NIH guidelines for care and use of animals.'],postnatal day 18,['Isoflurane treatment and physiologic monitoring of sentinel animals P18 mouse littermates were randomly assigned to 2 groups.'],Y,"['Behavioral tests Sixty-day-old mice housed in groups (5 mice per cage) were handled for at least 2 minutes per day for 3 days before the start of the behavioral experiments.', 'Object-place recognition test Object-place recognition was performed as previously described [37].', 'Y-maze test In the Y-maze test, mice were released from the start arm (no visual cue) and allowed to habituate to only 1 out of 2 possible choice arms (overt visual cue) for 15 minutes.']",isoflurane,"['In Group 1 (isoflurane), mice were exposed to 1.5% isoflurane carried in 100% oxygen for 4 hours.']",ketamine,"['After induction with a single ketamine injection (50mg/kg), high titers of GFP-expressing retroviruses were stereotaxically injected into the P15 mice dentate gyrus.']",c57bl/6,"['C57BL/6 mice were housed in a temperature- and humidity-controlled room with a 12:12 hour light:dark cycle, and provided with ad libitum access to water and food.']",True,True,True,True,False,True,[ Passage 12/25 ] 10.1371/journal.pbio.2001246
10.1016/j.bcp.2012.06.001,470.0,Kong,2012,rats,gestational day 14,Y,isoflurane,none,none,"PMID: 22705347 DOI: 10.1016/j.bcp.2012.06.001
2. Materials and methods
2.1. Animals
All of the animals were treated according to the guidelines of the Guide for the Care and Use of Laboratory Animals (China Ministry of Health). The Laboratory Animal Care Committee of Zhejiang University approved all experimental procedures and protocols. All efforts were made to minimize the number of animals used and their suffering. The dams were housed in polypropylene cages, and the room temperature was maintained at 22 °C, with a 12-h light–dark cycle. The dams at gestational day 14 were used for all experiments, because this time corresponds approximately to mid-gestation in humans [15], [16], the period when most non-obstetric surgeries and fetal interventions are performed [9], [10].

2.2. Anesthesia exposure
The dams were randomly divided into three groups: control, low concentration of isoflurane (1.3%), and high concentration of isoflurane (3%) treatment groups (n = 8). The dams were placed in plastic containers resting in water baths with a constant temperature of 38 °C. In these boxes, pregnant rats in isoflurane treatment groups were exposed to 1.3 or 3% isoflurane (Lot 826005U, Abbott Laboratories Limited, USA) in a humidified 30% oxygen carrier gas for 1 h; the control group was exposed to simply humidified 30% oxygen without any inhalational anesthetic for 1 h. We chose 1.3% because it represents 1 MAC in the pregnant rats [17], and 3% is equal to ∼2 MAC. The determination of anesthetic duration based on our preliminary study which indicated that maternal physiological states remained stable throughout a 1-h isoflurane exposure. The isoflurane concentration, oxygen and carbon dioxide levels in the box were monitored with an agent gas monitor (Vamos, Drager Medical AG & Co. KgaA, Germany). Otherwise, control and experimental animals were under the same treatment and environment. Arterial blood gases (ABG) and blood glucose were measured at the end of the 1-h anesthetic exposure. The rectal temperature was maintained at 37 ± 0.5 °C. After exposure, all the dams were returned to their cages and allowed to deliver naturally. The postnatal body weights of the rat pups were monitored.

2.3. Memory and learning studies
Four rat pups (2 females and 2 males) from each dam were selected to determine cognitive function at postnatal day 28 with a Morris Water Maze test with minor modifications [1]. A round pool (diameter, 150 cm; depth, 50 cm) was filled with warm (24 °C) opaque water to a height of 1.5 cm above the top of the movable clear 15-cm-diameter platform in the third quadrant. A video tracking system recorded the swimming motions of animals, and the data were analyzed using motion-detection software for the Morris Water Maze (Actimetrics Software, Evanston, IL, USA). After every trial, each rat was wiped before returning to its regular cage, kept warm and allowed free access to food.

2.3.1. Place trials
The place trials were performed at postnatal day 29 for 4 days to determine the rats’ ability to obtain spatial information. At postnatal day 28, rats were tested for their ability to swim to a visible platform through a 30-s swimming training. A dark black curtain surrounded the pool to prevent confounding visual cues. All rats received 4 trials per day in each of the four quadrants of the swimming pool. On each trial, rats were placed in a fixed position into the swimming pool facing the wall. They were allotted 120 s to find the platform upon which they sat for 20 s before being removed from the pool. If a rat did not find the platform within 120 s, the rat was gently guided to the platform and allowed to remain there for 20 s. For all training trials, swim speed and the time to reach the platform (escape latency) were recorded. The less time it took a rat to reach the platform, the better the learning ability. We took the average of four trials as the escape latency each day.

2.3.2. Probe trials
Probe trials were conducted immediately after the four-day period to evaluate memory retention capabilities. The probe trials involved the submerged platform of the third quadrant from the pool and allowing the rats to swim for 120 s in any of the four quadrants of the swimming pool. Time spent in the third quadrant and the number of original platform crossing in the third quadrant was recorded.

2.4. Transmission electron microscopy
After the Morris Water Maze test, six pups per group were anesthetized with a lethal dose of Nembutal. The thoracic cavities were opened and perfused intracardially with 100 mL of normal saline. Then the hippocampus, including CA1 and dentate gyrus area, of each rat was taken out immediately. Immersion fixation was completed on tissues about 1 mm3 from the hippocampus. Samples were rinsed in cold phosphate-buffered saline (PBS) and placed in 2.5% glutaraldehyde at 4 °C for 4 h. The tissue was rinsed in buffer and post-fixed with 1% osmium tetroxide for 1 h. Then, the tissue was rinsed with distilled water before undergoing a graded ethanol dehydration series and was infiltrated using a mixture of half propylene oxide and half resin overnight. Twenty-four hours later, the tissue was embedded in resin. 120 nm sections were cut and stained with 4% uranyl acetate for 20 min and 0.5% lead citrate for 5 min. Ultrastructure changes of synapse in the hippocampus were observed under a transmission electron microscope (Philips Tecnai 10, Holland).

2.5. Tissue section preparation
After the Morris Water Maze test, two pups from each dam were anesthetized by intraperitoneal injection of a lethal dose of Nembutal. The aorta was cannulated and the animal was firstly perfused with 200 mL of normal saline, then with 250 mL of 4% formaldehyde (freshly made from paraformaldehyde) for 20–30 min. The fixed brain was then removed from the cranial cavity and post-fixed overnight in the same fixative at 4 °C. The tissues were embedded in paraffin, and transverse paraffin sections containing the hippocampal area were mounted on silanecoated slides. Sections were deparaffinaged and rehydrated. Then the sections were treated for antigen retrieval with 10.2 mmol/L sodium citrate buffer, pH 6.1, for 20 min at 95 °C for immunohistochemistry.

2.6. Immunohistochemistry for caspase-3
Caspase-3 positive cells were measured in the hippocampal CA1 region, using immunohistochemical methods described previously [7], [8]. The brain region was chosen because it is particularly vulnerable to anesthesia-induced neurodegeneration [1] and is important to memory and learning. Briefly, the sections mentioned above were washed in 0.01 M PBS containing 0.3% Triton X-100 (pH 7.4, PBS-T), followed by blocking in 5% normal goat serum in 0.01 M PBS. The sections were then incubated in the primary antibodies rabbit polyclonal against anti-caspase-3 (1:200, Santa Cruz Biotechnology, USA) overnight at 4 °C. After a thorough wash in PBS, sections were incubated with biotinylated goat anti-rabbit IgG antibody (1:200, Wuhan Boster Biological Technology, Ltd., China) for 2 h at room temperature, followed by avidin–biotin–peroxidase complex solution (ABC, 1:100, Wuhan Boster Biological Technology, Ltd., China) for 2 h at room temperature. Immunolabeling was visualized with 0.05% diaminobenzdine (DAB, Wuhan Boster Biological Technology, Ltd., China) plus 0.3% H2O2 in PBS and the reaction was stopped by rinsing the slides with 0.2 M Tris–HCl. Sections were mounted onto 0.02% poly-l-lysinecoated slides and allowed to dry at room temperature. Then the sections were dehydrated through a graded series of alcohols, cleared in xylene and finally coverslipped. Rat Immunoglobulin IgG (1:200, Biomeda Corporation, USA) was used instead of primary antibody as a negative control. Other chemicals used in this study were provided by Cell Signaling Technology (Beverly, MA). Three sections from hippocampal CA1 region of each animal were randomly selected and images were photographed under 400× magnification in 3 visual fields/per section, the caspase-3 positive neurons were counted in the same area. The optical densities of caspase-3 positive neurons were measured quantitatively using Image-Pro Plus version 6.0 (Media Cybernetics, Inc., Silver Spring, USA). The optical density of caspase-3 positive cells in a particular brain region was calculated by dividing the integrated optical density of caspase-3 positive cells by the area of that brain region.

2.7. Statistical analysis
All data were presented as mean ± S.E.M. Results of weight of postnatal rat pups and place trials of postnatal rats were analyzed using 2-way ANOVA for repeated measurements. Other data were analyzed using one-way ANOVA, followed by Tukey post hoc multiple comparison tests. A P value of <0.05 was considered statistically significant. All statistical tests and graphs were performed or generated, respectively, using Graph-Pad Prism Version 4.0 (GraphPad Prism Software, Inc., CA, USA).",rats,"['The dams were housed in polypropylene cages, and the room temperature was maintained at 22 °C, with a 12-h light–dark cycle.']",gestational day 14,"['The dams at gestational day 14 were used for all experiments, because this time corresponds approximately to mid-gestation in humans [15], [16], the period when most non-obstetric surgeries and fetal interventions are performed [9], [10].']",Y,['Four rat pups (2 females and 2 males) from each dam were selected to determine cognitive function at postnatal day 28 with a Morris Water Maze test with minor modifications [1].'],isoflurane,"['The dams were randomly divided into three groups: control, low concentration of isoflurane (1.3%), and high concentration of isoflurane (3%) treatment groups (n = 8).']",none,[],not specified,[],True,True,True,True,True,False,[ Passage 13/25 ] 10.1016/j.bcp.2012.06.001
10.1016/j.brainres.2015.10.050,1730.0,Lai,2016,rats,postnatal day 7,Y,sevoflurane,none,sprague dawley,"PMID: 26541582 DOI: 10.1016/j.brainres.2015.10.050
4. Experimental procedures
4.1. Animals
A total of 240 male and female clean Sprague-Dawley rats, 7 days of age and weighting 12–16 g, (Shanghai Slac Laboratory Animal Co., Ltd., China) were used in this study. They were housed and treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 80-23, revised in 2011). All rats were maintained under standard laboratory temperature and humidity and a 12 day/night cycle (8 am/8 pm), and were allowed free access to food and water. The study was approved by the Experimental Animal Care Committee of the Fujian Medical University Union Hospital, and efforts were made to minimize the number of animals used and their suffering.

4.2. Experimental protocol
The animals were randomly allocated into 10 groups (n=24 per group; Fig. 1): (1) Sham, without hypoxia-ischemia; (2) HI/Control, received cerebral hypoxia-ischemia; (3) HI+Atractyloside (Atr), (4) HI+Cyclosporin A (CsA), treated like the control and respectively injected with Atr (10 mg/kg) and CsA (5 mg/kg); (5) HI+sevoflurane (Sev), treated like the control and received sevoflurane postconditioning; (6) HI+Sev+LY, (7) HI+Sev+L-N, (8) HI+Sev+SB, (9) HI+Sev+Atr, (10) HI+Sev+CsA, treated like the HI+Sev group and respectively injected with LY294002 (0.3 mg/kg), L-NAME (10 mg/kg), SB216763 (0.2 mg/kg), Atr (10 mg/kg), and CsA (5 mg/kg). LY294002, L-NAME, and SB216763 are specific blockers of Akt, eNOS, and GSK-3β, respectively. Atr and CsA open and close, respectively, mPTPs. In each group, the brains of the rats that received behavioral testing (from 32-days-old to 42-days-old; n=12 per group) were harvested for determination of hippocampal neuron count and morphology study, and the brains of the other 12 rats were harvested 24 h after the intervention for Western blot analysis, and study of mitochondrial permeability transition pore opening.

4.3. Cerebral HI model and sevoflurane postconditioning
The cerebral HI model was adapted from a procedure described previously (Ren et al., 2014). Briefly, the rats were anesthetized with pentobarbital sodium (0.5–1%, 40–50 mg/kg, intraperitoneal), and their left common carotid arteries were permanently ligated with a double 7–0 surgical silk; the arteries in the Sham group, however, were not ligated. A dose of 5 µL of 0.1% DMSO or drug (LY294002, L-NAME, SB216763, Atr, CsA) with 0.1% DMSO was injected into the left lateral ventricle immediately after the surgery as previously described (Satoh and Onoue, 2005). After waking, the rats were returned to their cages with the mothers for 1.5–2.5 h, and then placed in a chamber containing humidified 8% O2–92% N2 for 2 h. The air temperature in the chamber was maintained at 36.5±1 °C. The chamber was then exposed to room air for 15–20 min. For sevoflurane postconditioning, the animals were placed in a chamber containing 2.5% sevoflurane in 30% O2–70% N2 for 30 min after cerebral HI injury. After waking, the neonates were cleaned with 75% alcohol and returned to their mothers.

4.4. Novel object recognition test
The rats were evaluated with a nonspatial object recognition memory task 25 days after the intervention as described by Ennaceur and Delacour (1988) and Bruel-Jungerman et al. (2005). Briefly, for the first 3 days, after being comforted and stroked, each animal was put into an open chamber made of black plexiglas (80×80×60 cm3) for a 5 min acclimation and the test was conducted on the fourth day. Before the test, the animals received a 5 min training in the chamber containing 2 different objects (a white cube and a red cylinder) fixed at adjacent angles with a spacing of 10 cm from the field wall. Rats were put into the chamber with their backs turned towards the objects and allowed to explore the chamber freely for 5 minutes. Exploratory behavior can be identified when rats touch the objects with their noses or put their noses at places within 2 cm of the objects. To test memory storage, the white cube was kept in the chamber and the red cylinder was replaced by a blue semisphere. Exploratory time of new (T2) and old (T1) objects within 5 min was recorded and memorization ability of the rats was assessed by discrimination index: DI=T2/(T1+T2). The blue semisphere was replaced by a green prism 3 h after training, and the green prism was replaced by a yellow irregular shape 24 h after the training. The time each rat used to explore new and old objects was recorded for calculating DI. The DIs at 5 min, 3 h, and 24 h after the training (DI0 h, DI3 h, DI24 h) represent the instant, short-term, and long-term memory, respectively. Data with total exploration time less than 20 s were excluded from statistic analysis. The field was always provided with even light, and the objects and fields were cleaned with 75% ethanol after each testing.

4.5. Morris water maze test
After the novel object recognition test, the Morris water maze was used to test spatial learning and memory (Peng et al., 2012, Jiang et al., 2004). Briefly, a black circular pool (120 cm in diameter, 50 cm in height) was filled with water (25±1 °C) to a depth of 25 cm and located in a quiet room. Chinese ink was added to make the water opaque. The water maze was conceptually divided into 4 quadrants, and a hyaline platform (10 cm in diameter) was submerged 1 cm below the surface of the water at the midpoint of the third quadrant. In the place navigation trial, each rat underwent 4 successive trials a day for 5 days for memory acquisition training, with a 15 min interval between trials for the rat to recover physically. The sequence of water-entry points differed each day, but the location of the platform was constant. Escape latency (EL) to find the platform was measured up to a maximum of 120 s. On locating the platform, the rat was left there for 15 s before the next trial. If the rats failed to locate the platform within 120 s, it was guided to the platform and allowed to stay there for 15 s. Latency and the search strategies, including straight strategy, tendency strategy, marginal strategy, and random strategy, were recorded for each trial. Twenty-four hours after the last training session, a space exploration trial was performed. The platform was removed from the pool and rats were allowed to swim freely for 60 s. Four indexes were calculated: (1) the time spent by the rats in the third quadrant in which the platform was hidden during acquisition trials; (2) the number of rats crossing exactly over the original position of the platform; (3) the search path in the target quadrant; (4) the total movement distance. Search speed was calculated by total movement distance divided by 120 cm/s. All trials were videotaped by a camera located 2 m above the water surface and computer analyzed.

4.6. Histology of left hippocampal neurons
After the behavioral studies, rats were anesthetized with pentobarbital, transcardialy perfused with 200 mL of 4 °C heparin saline solution and then with 300 mL of 4% paraformaldehyde. Left hippocampus was made into a wax block according to Paxinos–Waston methods. Continual coronal sections (4 µm in thickness) at approximately 3.3 mm caudal to bregma were obtained, and subjected to hematoxylin–eosin (HE) staining. The sections were examined by an observer blinded to the rat group assignment. Neurons microscopically showed a clear boundary, a round or an oval shape, a smooth cell membrane, basophilic cytoplasm (Nissl body), a large and round nucleus, a clear nuclear membrane and a large and round nucleolus will be defined as surviving neurons. Apoptotic neurons will not be regarded as surviving ones. Surviving neurons in pyramidal cell layer of the CA1 and CA3 regions were counted (n/mm) by two investigators blind to experimental conditions, and a count was determined by averaging the total of 5 sections.

4.7. Western blot analysis
Proteins were separated on a 12% SDS-PAGE gel, and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, USA). The membrane was blocked using 5% nonfat milk and incubated with a mouse anti-p-Akt, t-Akt, p-eNOS, t-eNOS, p-GSK-3β, and t-GSK-3β monoclonal antibody (mAb) (Cell Signaling Technology, Beverly, MA, USA) or a mouse anti-β-actin mAb (Sigma, USA). The proteins were visualized and quantified using ECL reagents (Pierce, IL, USA).

4.8. mPTP opening assay
Preparation of mitochondria was adapted from a procedure described previously (Wu et al., 2006). All procedures were carried out in the cold (0–4 °C). Hippocampal pieces were placed in isolation buffer (250 mmol/L sucrose, 210 mmol/L mannitol, 1 mmol/L K-EDTA, 10 mmol/L Tris–HCl, pH 7.4) and homogenized (10 mL buffer/g). The homogenate was immediately centrifuged at 2000g for 3 min. The supernatant was centrifuged again at 2000g for 3 min, the second supernatant was decanted and centrifuged at 12,000g for 8 min, and the resulting supernatant was decanted and resuspended in isolation buffer without K-EDTA. The suspension was centrifuged at 12,000g for 10 min and the resulting mitochondrial pellet was resuspended in the same buffer. Mitochondrial protein concentration was quantified according to the Bradford׳s method using 1 g/mL bovine serum albumin (BSA) as standard. Purity and integrity of isolated mitochondria were confirmed by neutral red-Janus green B staining (Sigma, USA). Isolated mitochondria from the hippocampus (0.5 mg protein) was resuspended in swelling buffer (71 mmol/L sucrose, 215 mmol/L mannitol, and 10 mmol/L sodium succinate in 5 mmol/L HEPES, pH 7.4) to a final volume of 2 mL, and incubated at 25 °C for 2 min. mPTP-induced mitochondrial swelling was confirmed by 5 min incubation with the strong mPTP inhibitor CsA before addition of CaCl2, and was measured with a spectrophotometer (Beckman DU800, USA) as a reduction in optical density at 540 nm (OD540) (Kristal and Brown, 1999, Baines et al., 2003).

4.9. Statistical analysis
All data were presented as mean±standard deviation (SD). For comparison between multiple groups, data were analyzed by one-way ANOVA. When a statistical difference was determined by ANOVA, the least significant difference (LSD) procedure was applied. The percentage of search strategies were examined by the Mann–Whitney method, and repetitive measure ANOVA was used to measure mean EL at different time points. Spatial probe trial data were analyzed by one-way ANOVA and principal components analysis (PCA). All analyses were performed with SPSS 13.0 for Windows, and a value of P<0.05 was considered significant.",rats,"['A total of 240 male and female clean Sprague-Dawley rats, 7 days of age and weighting 12–16 g, (Shanghai Slac Laboratory Animal Co., Ltd., China) were used in this study.']",postnatal day 7,"['A total of 240 male and female clean Sprague-Dawley rats, 7 days of age and weighting 12–16 g, (Shanghai Slac Laboratory Animal Co., Ltd., China) were used in this study.']",Y,"['The rats were evaluated with a nonspatial object recognition memory task 25 days after the intervention as described by Ennaceur and Delacour (1988) and Bruel-Jungerman et al. (2005).', 'After the novel object recognition test, the Morris water maze was used to test spatial learning and memory (Peng et al., 2012, Jiang et al., 2004).']",sevoflurane,"['For sevoflurane postconditioning, the animals were placed in a chamber containing 2.5% sevoflurane in 30% O2–70% N2 for 30 min after cerebral HI injury.']",none,[],sprague dawley,"['A total of 240 male and female clean Sprague-Dawley rats, 7 days of age and weighting 12–16 g, (Shanghai Slac Laboratory Animal Co., Ltd., China) were used in this study.']",True,True,True,True,True,True,[ Passage 14/25 ] 10.1016/j.brainres.2015.10.050
10.1371/journal.pone.0070645,564.0,Lei,2013,rats,postnatal day 7,Y,sevoflurane,none,sprague dawley,"PMID: 23967080 PMCID: PMC3742769 DOI: 10.1371/journal.pone.0070645
Materials and Methods
Animal/Anesthesia treatment
The rats used in the present study were obtained from the Animal Care Center of Fudan University. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee, Fudan University. One-day pregnant female Sprague Dawley rats (weight 220–250 g) were randomly assigned to one of the three groups: control, sevoflurane, or sevoflurane with n-3 PUFAs (n = 3 per group). Fish oil, the main source of n-3 PUFAs (Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA)), was extracted from the capsule (1000 mg/capsule that containing 180 mg EPA and 120 mg DHA, Puritan's Pride, Bohemia, NY, USA) and added to food. Pregnant dams in the sevoflurane and control groups were fed a regular laboratory rodent diet with a low n-3 PUFAs concentration (0.5% of total fatty acid), whereas the sevoflurane with n-3 PUFAs group were fed the same diet, but supplemented with n-3 PUFAs (15 mg fish oil/g regular diet) from day 2 of pregnancy to 14 days after parturition. Dams were given free access to food and water, and dams for all groups were kept under identical housing conditions with a 12-h light cycle. On postnatal day 7 (P7), the rat pups in the sevoflurane and sevoflurane with n-3 PUFAs groups received sevoflurane anesthesia.

P7 rats were placed in a sealed box ventilated with 3% sevoflurane in 60% oxygen and treated for 6 h. The temperature in the sealed box was maintained at 33–35°C. The total survival percentage of P7 rats after 6-h anesthesia was 88.4%; the likely cause of death was respiration depression. After anesthesia, the pups were returned to the dams. Control rat pups were placed in the same box without sevoflurane exposure and under identical experimental conditions. The flow chart for the experimental protocol is summarized in Figure 1.
Blood gas analysis
Twelve naïve P7 rats that that did not participate in other experiments were used to assess the effect of sevoflurane on blood gases. Blood was percutaneously aspirated from the left cardiac ventricle after 0, 2, 4, and 6 h of anesthesia (n = 3 per time point). From these samples, we measured partial pressures of carbon dioxide and oxygen, pH, and blood lactate and glucose levels with a Radiometer ABL 800 blood gas analyzer (Radiometer, Copenhagen, Denmark).

BrdU injection
To determine whether sevoflurane affects progenitor cell proliferation in the S-phase of the cell cycle, bromodeoxyuridine ((+)-5′-bromo-2′-deoxyuridine [BrdU]; 97%; Sigma-Aldrich, St. Louis, MO, USA) in 0.9% sterile saline solution was injected intraperitoneally using the procedure described by Wojtowicz [16]. The first dose (150 mg/kg) was administered immediately before sevoflurane treatment, and the three subsequent injections (50 mg/kg BrdU) were given at 24-h intervals following sevoflurane anesthesia.

Tissue preparation and immunohistochemistry
Animals were deeply anesthetized with chloral hydrate and then transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4. The brains were removed, postfixed overnight in 4% paraformaldehyde/PBS, and placed in 30% sucrose until they sank in the solution. Coronal sections (30 µm) were cut on a microtome (Leica CM1900 UV, Wetzlar, Germany) and every sixth section was stored in 30% sucrose containing 30% ethylene glycol to stain BrdU or cleaved caspase-3.

For immunocytochemical detection of BrdU-labeled nuclei, DNA was denatured to expose the antigen for incubation with 2 N hydrochloric acid for 30 min at 37°C, followed by neutralization with two 10-min incubation periods in 0.5 M boric acid (pH 8.5) at room temperature (RT). Sections were subjected to three 10-min washes in PBS with 0.3% Triton-X with 10 min between each wash. Nonspecific epitopes were blocked with 1% serum for 30 min at RT, and were incubated overnight at 4°C with either BrdU (1∶100; BD Pharmingen, Franklin Lakes, NJ, USA) or cleaved caspase-3 (1∶1,000; Cell Signaling, Danvers, MA, USA) antibody in PBS and 1% serum. On day 2, the sections were incubated with the appropriate secondary fluorescent antibodies (Alexa Fluor 488, 1∶200; Invitrogen, Carlsbad, CA, USA) for 2 h at RT, followed by three 5-min washes in PBS. Nuclear counterstaining was performed with 4′,6-diamidino-2-phenylindole (1∶500; Beyotime Institute of Biotechnology, Haimen, China), which was followed by mounting and coverslipping with an aqueous mounting medium. Images were acquired with a microscope (Leica DM2500). BrdU- or cleaved caspase-3-positive cells were counted in a blinded manner at ×20 magnification [17]. Questionable structures were excluded from the count if their identification remained uncertain under ×40 magnification.

Western blot analysis
The cerebral cortex, thalamus, and hippocampus were harvested 18 h after sevoflurane treatment. The brain tissues were homogenized in RIPA buffer (Millipore, Temecula, CA, USA) containing complete protease inhibitor cocktail and 2 mM phenylmethylsulfonyl fluoride. The lysates were collected and centrifuged at 12,000 rpm for 30 min at 4°C. After the protein samples were quantified using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA), 60 µg of each sample was electrophoresed through a 14% sodium dodecyl sulfate-polyacrylamide gel and wet electrotransferred to 0.45-µm nitrocellulose membranes (Millipore). The blots were incubated overnight at 4°C with a polyclonal anti-cleaved caspase-3 antibody, and then incubated with a rabbit anti-mouse polyclonal horseradish peroxidase-conjugated secondary antibody (1∶5,000; Epitomics, Hangzhou, Zhejiang Province, China) at RT for 1 h. Protein signals were detected using an enhanced chemiluminescence detection system (Pierce Biotechnology). A β-actin antibody (1∶1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to normalize sample loading and transfer. Band intensities were densitometrically quantified using Gel-Pro Analyzer (Media Cybernetics, Bethesda, MD, USA).

Neurobehavioral tests
We used only male offspring (n = 9 per group) in the neurobehavioral tests to exclude estrogen influences on neurocognitive evaluations. The water maze setup in spatial reference memory task and memory consolidation task was shown in Figure 2.


Morris water maze spatial reference memory Probe training: Rats trained for 4 consecutive days (postnatal days 35–38, P35–38) in the Morris water maze following treatment with a vehicle or 3% sevoflurane for 6 h. A platform (10.3-cm diameter) was submerged in a circular pool (180-cm diameter, 50-cm depth) filled with warm (23–25°C) opaque water. Rats performed two training sessions each day. In each session, rats performed four trials in which they were released from one of four pseudorandomly assigned release points while facing the tank wall. This provided two short and two medium swims per session. Animals were allowed 60 s to locate the hidden platform, and if they failed to find the hidden platform in the allotted time, the investigator guided the animal to the platform. In either case, the rats were removed from the platform after 15 s. Training sessions were conducted until the rats could locate the hidden platform in less than 15 s in at least five sessions (average time per session). All trials were videotaped, and rat swim paths were recorded with ANY-maze video tracking system (Stoelting Co., Wood Dale, IL, USA), which allowed us to measure the time taken (latency) to find the platform(s), as well as other behavioral information obtained during the spatial reference memory test. The animals were dried and placed beneath a heating lamp after completing each test.
Probe test: A probe trial was performed with the platform removed from the tank to assess memory retention for the hidden platform location. Probe trials were administered 1 day after the last training session (P38). During the 60-s probe trial, we determined the number of entries into the platform quadrant zone, the swimming speed (cm/s), the total distance (cm), and the time spent in the target quadrant relative versus the other quadrants.

Fear conditioning test Rats underwent fear conditioning tests on postnatal days 63–64 (P63–64). Every time four rats randomly chosen from three groups were trained in each session. Rats were placed in plastic chambers with a grid floor constructed from 19 stainless steel bars (4-mm diameter, spaced every 16 mm). The floors were connected to a shock delivery system (Coulbourn, Whitehall, PA, USA), and electrical shocks were delivered through the stainless steel bars. The chamber was illuminated with overhead fluorescent bulbs, and a ventilation fan provided background noise (65 db). The training context was considered the appearance, odor, and texture of the environment (chamber and room) in which the rats were trained. After a 3-min baseline exploratory period, rats were presented with three auditory tones (2,000 Hz, 90 db) that were followed 1 min later by an electric shock (1 mA, 2 s). We quantified the rats' fear response with freezing, which is an innate defensive fear response in rodents and a reliable measure of learned fear. Freezing was defined as the lack of movement, except for respiration. We examined rats in the fear condition test the day after they first received the electrical shock to determine whether they showed fear to the training context or the auditory tone. For the context test, rats were placed in the chamber where they were trained on the previous day. The rats remained in the chamber for 8 min, without an auditory tone or shock. For the tone test, rats were transported in groups to a context chamber with black boards covering the walls. Rats were allowed a 3-min exploratory period before three 30-s tones were played (2,000 Hz, 90 db, separated by 60 s). Rats were removed from the chamber 30 s after the tone presentation. The order of the context and tone tests was counterbalanced so that half of each treatment group first was tested for context and then for tone, whereas the other half of the treatment group was tested in the reverse order. FreezeView software (Coulbourn) was used to score each animal's freezing behavior separately for the training period and the context and tone tests, which were expressed as a percentage.
Morris water maze memory consolidation Working memory (WM): On postnatal day 70 (P70), the testing room was rearranged by repositioning the water tank and adding new spatial cues. The platform was submerged 1.5 cm below the water surface in one of four designated platform positions. From P70 onward, one session was conducted per day. Each session began with a 60-s free swim (performance not scored) in which rats explored the maze, and was followed by a 1-min rest interval and three subsequent scored trials. Rats that found the platform during the free swim were allowed to rest on the platform for 15 s. Rats that failed to find the platform during the free swim were guided to the platform and remained there for 15 s. After the free swim, three trials were administered in which the rat was released from one of six pseudorandomly chosen locations that faced the tank wall. The platform location was identical for all animals in a session, but the drop location was pseudorandomly varied to incorporate one short, one medium, and one long swim. Training sessions were administered until the session average for finding the hidden platform was less than 15 s. The latency for reaching the platform was recorded by the ANY-maze video tracking system.
Short-term memory (STM) and early long-term memory (ELTM): When the WM latencies of rats in task were plateaued on postnatal day 77 (P77), we increased the delay between the free swim and the subsequent trials. The delay was extended from 1 min on P77 to 1 h on postnatal day 78 (P78) to test STM, and then to 4 h on P79 to test ELTM. Performances on the last trial after the free swim on P77 (1-min delay), P78 (1-h delay), and P79 (4-h delay) were used as measures of WM, STM, and ELTM, respectively.

Statistical methods
All data are presented as mean ± standard deviation. We performed two-tailed t tests (assuming equal variances) to determine differences in cleaved caspase-3 immunohistochemistry and blood gas parameters between the control and sevoflurane groups. We used a one-way analysis of variance followed by Newman-Keuls post hoc tests to determine differences among groups for interactions between n-3 PUFAs or sevoflurane and cleaved caspase-3 activation, BrdU quantification, or neurobehavioral tests. For all tests, p<0.05 was considered statistically significant.",rats,['The rats used in the present study were obtained from the Animal Care Center of Fudan University.'],postnatal day 7,"['On postnatal day 7 (P7), the rat pups in the sevoflurane and sevoflurane with n-3 PUFAs groups received sevoflurane anesthesia.']",Y,"['We used only male offspring (n\u200a=\u200a9 per group) in the neurobehavioral tests to exclude estrogen influences on neurocognitive evaluations.', 'Morris water maze spatial reference memory Probe training: Rats trained for 4 consecutive days (postnatal days 35–38, P35–38) in the Morris water maze following treatment with a vehicle or 3% sevoflurane for 6 h.', 'Fear conditioning test Rats underwent fear conditioning tests on postnatal days 63–64 (P63–64).']",sevoflurane,['P7 rats were placed in a sealed box ventilated with 3% sevoflurane in 60% oxygen and treated for 6 h.'],none,[],sprague dawley,"['One-day pregnant female Sprague Dawley rats (weight 220–250 g) were randomly assigned to one of the three groups: control, sevoflurane, or sevoflurane with n-3 PUFAs (n\u200a=\u200a3 per group).']",True,True,True,True,True,True,[ Passage 15/25 ] 10.1371/journal.pone.0070645
10.1111/jcmm.13524,1256.0,Lin,2018,rats,e7,Y,propofol,none,sprague dawley,"PMID: 29461008 PMCID: PMC5908131 DOI: 10.1111/jcmm.13524
2. MATERIALS AND METHODS
2.1. Drugs
All drugs were prepared just before use: propofol (Diprivan; AstraZeneca UK limited, Italy: jc393, 20 mL: 200 mg); 20% intralipid (2B6061; Baxter, Deerfield, IL, USA); SAHA (Selleck Chemicals LLC, Houston, TX, USA). HGN antisense was synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. Senegenin (purity ≥ 98%) was purchased from Nanjing SenBeiJia Biological Technology Co., Ltd. (Jiangsu province, China).

Anti‐β‐actin and anti‐rabbit IgG secondary antibody were obtained from Cell Signaling Technology (Cell Signaling Tech, MA, USA). Anti‐CREB (Phospho S133), anti‐NMDAR2B, anti‐HDAC2, antisynaptophysin, anti‐Ac‐H4K12 and anti‐Ac‐H3K14 antibodies were purchased from Abcam (Abcam, Cambridge, MA, USA). Anti‐HGN antibody was synthesized by Kitgen Bio‐tech Co., Ltd.(Zhejiang province, China).

2.2. Animals
The protocol in this study was approved by the institutional review board of the First Affiliated Hospital of Nanchang University on the Use of Animals in Research and Teaching. All the methods in this study were performed in co‐ordination with relevant guidelines and regulations. Sprague Dawley (SD) rats were purchased from the animal science research department of the Jiangxi Traditional Chinese Medicine College (JZDWNO: 2011‐0030; Nanchang, Jiangxi,China). The learning and memory functions of the parental rats were assessed using the Morris water maze (MWM) system before mating, so that to minimize the hereditary difference. Animals were housed separately under standard laboratory conditions with 12:12 light/dark cycle, 24 ± 1°C and had free access to tap water. Two female rats in cages with one male rat per cage for mating. Pregnancy was diagnosed by the sign of vaginal plug.

2.3. Drug treatment
On E7, pregnant rats received intravenous infusion of propofol (n = 10 dams) with the rate of 20 mg kg−1 h−1 for 4 hours, equal volume of saline (n = 10 dams) or intralipid (n = 5 dams), respectively.

Electrocardiograms, saturation of pulse oximetry (SpO2) and tail non‐invasive blood pressure were continuously monitored during maternal propofol exposure. Using heating lamp and temperature controller to monitor the rectal temperature and keep it at 37 ± 0.5°C. Arterial blood sampling from lateral caudal artery for blood gas analysis at the end of propofol anaesthesia. If the total time of SpO2 <95% and/or the systolic blood pressure <80% of the baseline in excess of 5 minutes, the pregnant rat was got rid of the study, and other pregnant rats were chosen to supply the sample size, so as to exclude the interfering effect of maternal hypotension or hypoxia on cognitive function in the pup rats.

After delivery, the offspring rats born to the same pregnant rat were randomly subdivided into the SAHA, SEN, HGNA group and their relative control groups (DMSO, NS(1) and NS(2) group, respectively; Figure ​Figure1).1). It has been proved that the acetylation level of histone in hippocampus obviously increased 2 hour after the administration of HDAC inhibitor.27 Therefore, 90 mg kg−1 SAHA (HDAC inhibitor), at a concentration of 0.6 μmol L−1 dissolved into dimethyl sulphoxide (DMSO) was injected to the offspring in SAHA group by the intraperitoneal route at 2 hours before each MWM trial. The same volume of DMSO was given to the DMSO group. Senegenin, a kind of Chinese medicine, was proved to up‐regulate the expression of NR2B mRNA and protein, thus to mitigate cognitive dysfunction.28 So, 15 mg kg−1 Senegenin and equal volume of saline were given intraperitoneally at 2 hours before each MWM trial to SEN or NS(1) groups, respectively. HGN antisense oligonucleotide (0.25 nmol μL−1, 1.5 μL) or normal saline (1.5 μL) was injected to offspring's hippocampus in HGNA or NS(2) group as previously described,18, 29 once daily for seven consecutive days before MWM trial.
2.4. Morris water maze test
Spatial learning and memory were assessed by the MWM test from post‐natal day 30 (P30) to P36 according to previously described5, 30 with SLY‐WMS Morris water maze test system (Beijing Sunny Instruments Co. Ltd., Beijing, China). Briefly, the trials start at 9 o'clock in the morning in the MWM system with the pool was filled with water to a height of 1.0 cm above the top of a 15‐cm‐diameter platform, in the second quadrant (target quadrant), and the water maintained at 24 ± 1°C. The training trial was performed once a day for six consecutive days. In each training trial, offspring rats were placed in the water facing the wall of the pool in the third quadrant, the farthest one from the target quadrant. The animals were allowed to search for the hidden platform or for 120 seconds. They were allowed to remain on the platform for 30 seconds when they found the platform and the time for the animal to find the platform was recorded as escape latency (indicating learning ability). For those who did not find the platform within 120 seconds, the animals were gently guided to the platform and allowed to stay there for 30 seconds, and their escape latency was recorded as 120 seconds. At the end of the reference training (P37), the platform was removed. The offspring rats were allowed to perform spatial probe test (memory function test) for 120 seconds. Times across the platform (platform crossing times, indicate memory function), the swimming trail and speed were automatically recorded by the system. The mean value of the platform crossing times, escape latency and speed of the offspring born to the same pregnant rats was taken as the final results.

2.5. Brain hippocampus harvest
The day after the MWM test, rats were anaesthetized with isoflurane and killed by cervical dislocation. Hippocampus tissues were harvested and stored in Eppendorf tubes that had been treated with 1% DEPC and were stored at −80°C (for Western blot analyses) or immersed in 4% paraformaldehyde (for immunofluorescence assay).

2.6. Western blot analysis
The hippocampus (n = 6, with three male and three female offspring rats from each group) were homogenized on ice in lysis buffer containing a protease inhibitors cocktail. Protein concentration was determined by the bicinchoninic acid protein assay kit. Protein samples (20 μg) were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a PVDF membrane. The membranes were blocked by non‐fat dry milk buffer for 1.5 h and then incubated overnight at 4°C with antihistone H3 (acetyl K14) (1:2000), antihistone H4 (acetyl K12) (1:10000), anti‐NMDAR2B (1:1000), anti‐HGN (1:1000), antisynaptophysin (1:10000) and anti‐β‐actin (1:2000), respectively. Thereafter, the membranes were washed three times with TBS‐T buffer for 15 minutes and incubated with the horseradish peroxidase (HRP)‐conjugated secondary antibody for 2 hours at room temperature. The immune complexes were washed three times with TBS‐T buffer and detected using the ECL system (Millipore Corporation, MA, USA). The images of Western blot products were collected and analysed by ImageJ 1.50i (Wayne Rasband, National Institutes of Health, USA). The density of observed protein band was normalized to that of β‐actin in the same sample. The results of offspring from all the other group were then normalized to the average values of normal saline control offspring (control+NS group) in the same Western blot. The mean expression level of all of the offspring born to the same mother rat in the same group was calculated as the final expression level of the observed proteins.

2.7. Immunofluorescence staining
Immunofluorescence staining was used to assess HDAC2 and phospho‐CREB in the hippocampus of offspring rats after the MWM test. Hippocampus from offspring rats (n = 6, with three male and three female offspring rats from each group) were fixated in paraformaldehyde. Five‐μm frozen sections of the hippocampus were used for the immunofluorescence staining. The sections were incubated with anti‐HDAC2 (1:300) and anti‐CREB (1:100) dissolved in 1% bovine serum albumin in phosphate‐buffered saline at 4°C overnight. Then, the sections were incubated with fluorescent‐conjugated anti‐rabbit secondary antibody (1:300) for 1 hour in the dark at room temperature. Negative control sections were incubated with PBS as a substitute for primary antibody. Finally, the sections were wet mounted and viewed immediately using a inverted fluorescence microscope (200×) (Olympus, Japan). The target protein was red, and nuclei were blue. The proteins of HDAC2 and p‐CREB were excited by the green light, while the DAPI was performed by UV blue light. All images were recorded at 10 × 20× (Exp Acq‐700mmm, Offset Acq‐1, Gain Acq‐1, Gamma Acq‐300). The density of HDCA2 and p‐CREB staining was conducted on the images using Image‐Pro Plus 6.0 (Media Cybernetics Inc., USA). The images were converted it into black and white pictures. After intensity calibration, hippocampal CA1 area was chosen to analyse and the integrated optical density (IOD) was measured. IOD/Area was calculated as the protein expression level.

2.8. Statistical analysis
All analyses were performed with SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Data from escape latency in the MWM test were subjected to a repeated measures two‐way analysis of variance (RM two‐way ANOVA) and were followed by least significant difference t (LSD‐t) analysis when a significant overall between‐subject factor was found (P < 0.05). Data from Western blot and immunofluorescence staining results were subjected to one‐way ANOVA analysis. All data well provided for any of the variables. The LSD t test was used to determine the difference between groups. Statistical significance was declared at P < .05.",rats,"['Sprague Dawley (SD) rats were purchased from the animal science research department of the Jiangxi Traditional Chinese Medicine College (JZDWNO: 2011-0030; Nanchang, Jiangxi,China).']",gestational day 7,"['On E7, pregnant rats received intravenous infusion of propofol (n = 10 dams) with the rate of 20 mg kg−1 h−1 for 4 hours, equal volume of saline (n = 10 dams) or intralipid (n = 5 dams), respectively.']",Y,"['Spatial learning and memory were assessed by the MWM test from post‐natal day 30 (P30) to P36 according to previously described5, 30 with SLY‐WMS Morris water maze test system (Beijing Sunny Instruments Co. Ltd., Beijing, China).']",propofol,"['On E7, pregnant rats received intravenous infusion of propofol (n = 10 dams) with the rate of 20 mg kg−1 h−1 for 4 hours, equal volume of saline (n = 10 dams) or intralipid (n = 5 dams), respectively.']",isoflurane,"['The day after the MWM test, rats were anaesthetized with isoflurane and killed by cervical dislocation.']",sprague dawley,"['Sprague Dawley (SD) rats were purchased from the animal science research department of the Jiangxi Traditional Chinese Medicine College (JZDWNO: 2011-0030; Nanchang, Jiangxi,China).']",True,False,True,True,False,True,[ Passage 16/25 ] 10.1111/jcmm.13524
10.1016/j.bbrc.2022.01.022,462.0,Liu,2022,mice,postnatal day 6,Y,sevoflurane,none,c57bl/6,"PMID: 35063768 DOI: 10.1016/j.bbrc.2022.01.022
2. Methods
2.1. Animals
This study was approved by the Institutional Animal Care and Use Committee at Soochow University (Suzhou, Jiangsu, China). Twenty-four female and six male C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Jiangsu, China) for producing next generation of mice. On postnatal day (PND) 21, the offspring mice were separated from dams and housed 4–5 per cage by gender. Four male and four female mice were specifically used as the stranger mice, which were trained to stay calmly in the enclosure before social interaction test. All the mice were raised with free access to food and water in a controlled environment (room temperature 21–22 °C, 12/12 h light/dark cycle, and light on at 7 a.m.).

2.2. Anesthesia
A retired anesthesia machine (Datex-Ohmeda, Inc.) was employed to supply a consistent concentration of anesthetic gas. A sealed plastic box (20 L × 20 W × 6 H cm) was used as the anesthetizing chamber, which was drilled with three holes for gas inflow, gas outflow and gas monitoring. An electric heater was placed underneath the anesthetizing chamber to keep neonatal mice warm during anesthesia. A gas analyzer (Datex-Ohmeda, Inc.) was applied to adjust gas concentrations. On postnatal day 6, the neonatal mice were randomly assigned into two groups. Twenty-eight mice (17 males and 11 female) received 3.0% sevoflurane in 60% oxygen for 2 h (Sevo), and thirty-one mice (11 males and 20 female) inhaled merely 60% oxygen for 2 h (Oxyg). Sex of each mouse and amount of each group were not identified until weaning on PND 21. These subject mice were tested for social interaction behavior at one- and two-month-old.

Another battery of neonatal mice were treated with the air condition (control) or 60% oxygen (oxyg) or 3.0% sevoflurane in 60% oxygen (sevo) for 2 h on PND 6, and then killed for harvesting brain tissues 24 h after treatment. Sevoflurane anesthesia was strictly performed by the protocols of previous studies [11,12], in which all neonatal mice could spontaneously breath during general anesthesia, and their arterial blood pressure and blood gas analysis showed within normal limits. The vapor for releasing sevoflurane was turned off at the end of anesthesia, and the residual anesthetic was washed out with 60% oxygen for 15 min. Finally, these pups were smeared with own bedding and sent back to their dams.

2.3. Social interaction paradigm
Social interaction test is performed with the three-chambered social box, with three chambers (40 L × 20 W × 22 H cm) and two enclosures (7 ID × 15 H cm). The floor is painted grey to provide a high contrast with the testing mice. Grid bars of the enclosure allow direct contacting between the subject and stranger mice. A novel video-tracking system was developed by hanging two video-cameras right above two enclosures. Thereby, two video-images were integrated into one with the montage effect in ANY-maze program (Stoelting Co., USA). The subject mouse initiates social interaction with the stranger mouse by nose-to-nose or nose-to-tail sniffing, thus the animal's head is tracked by the ANY-maze program.

2.4. Social interaction test
First of all, the stranger mice were transferred into behavioral room and hidden 2 m away from social apparatus. Each subject mouse was taken into behavioral room about 45 min before social interaction test. In the first session (Habituation, 10-min), the subject mouse was gently placed into the middle chamber, and allowed to freely explore in three chambers. In the second session (Sociability, 10-min), the subject mouse was guided into the middle chamber and transiently confined there. An unfamiliar conspecific (Stranger 1) was introduced into one enclosure, the subject mouse was allowed to explore in three chambers and sniff at two enclosures containing Stranger 1 or not. In the third session (Preference for social novelty 10-min), the subject mouse was again confined into the middle chamber. Another unfamiliar conspecific (Stranger 2) was introduced into the other enclosure, and the subject mouse was allowed to explore in three chambers and sniff at two enclosures containing Stranger 2 or Stranger 1. Placement of Stranger 1 on left and right side were balanced between trials, and two stranger mice were the same gender as the subject mice.

Sociability is characteristic of the mouse taking more time sniffing its conspecific mouse compared with an inanimate object. Preference for social novelty is characteristic of the mouse taking more time sniffing an unfamiliar mouse compared with a familiar one. Four parameters were measured for judging social choice, including 1) time sniffing at the enclosure, 2) number of sniffs, 3) time exploring in the chamber, and 4) number of entries. Sniffing time at the enclosure was primary outcome, number of sniffs at the enclosure and time exploring in the chamber were secondary outcomes. In social interaction test, “at the enclosure” is defined as the head of mouse entering an area about 3 cm around the enclosure, as described in similar social study [13]. And “in the chamber” is defined as the head of mouse entering into the chamber.

2.5. Immunoblotting analysis
The brain tissues of neonatal mice were harvested on dry ice at 24 h after treatment. Next, the cortex and hippocampus were homogenized on ice using the immunoprecipitation buffer plus protease inhibitor. And then, the lysates were centrifuged at 15,000 rpm for 30 min at 4 °C. After that, the lysates were quantified for total protein by the bicinchoninic acid (BCA) protein assay kit (MultiSciences Biotech Co., Ltd. Cat: PQ0012, Lot: A91041). Finally, western blot was performed by the protocols to analyze protein levels in cortex and hippocampus. Neuroligin-1 antibody (1:1000; Santa Cruz Biotechnology, Inc.) was used to detect neuroligin-1 (101 kDa). PSD-95 antibody (1:1000; Cell Signaling Technology, Inc.) was used to detect PSD-95 (95 kDa). Anti–β-actin (1:5000; Sigma) was used to detect β-actin (42 kDa).

2.6. Statistical analysis
Data were expressed as Mean ± SD. Statistical analyses were performed by using GraphPad Prism 5.0 (San Diego, USA). Data representing social behavior of testing mice were normally distributed by Kolmogorov-Smirnov test. Data of each mouse from the left or right side were mutually exclusive, and two-tailed paired t-test was used to determine side preference, which was supported by other social studies [14,15]. Student's t-test was used to assess differences in the levels of Neuroligin-1 and PSD95 expression in cortex and hippocampus of mice. P values less than 0.05 (∗), 0.01 (∗∗) and 0.001 (∗∗∗) were considered statistically significant.",mice,"['Twenty-four female and six male C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Jiangsu, China) for producing next generation of mice.']",postnatal day 6,"['On postnatal day 6, the neonatal mice were randomly assigned into two groups.']",Y,"['These subject mice were tested for social interaction behavior at one- and two-month-old.', 'Social interaction test is performed with the three-chambered social box, with three chambers (40 L × 20 W × 22 H cm) and two enclosures (7 ID × 15 H cm).']",sevoflurane,"['Twenty-eight mice (17 males and 11 female) received 3.0% sevoflurane in 60% oxygen for 2 h (Sevo), and thirty-one mice (11 males and 20 female) inhaled merely 60% oxygen for 2 h (Oxyg).']",none,[],c57bl/6,"['Twenty-four female and six male C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Jiangsu, China) for producing next generation of mice.']",True,True,True,True,True,True,[ Passage 17/25 ] 10.1016/j.bbrc.2022.01.022
10.1097/ALN.0b013e31819daedd,5173.0,Liu,2012,rats,postnatal day 7,N,isoflurane,none,sprague dawley,"PMID: 19352168 DOI: 10.1097/ALN.0b013e31819daedd
Materials and Methods
The study protocol was approved by the Home Office (London, United Kingdom) and conforms to the United Kingdom Animals (Scientific Procedures) Act of 1986.

In Vitro  Experiments
Organotypic hippocampal slices were derived from postnatal day 8 or 9 C57Bl/6 mice pups (Harlan Laboratories, Huntingdon, United Kingdom) and cultured by the interface method21,22with some modifications. In brief, the brain was quickly dissected and placed in ice-cooled (4°C) dissection solution. All stages of slice preparation were performed under sterile and ice-cooled conditions. Excess tissue (including the cerebellum, olfactory bulbs, and meninges) was removed, and the brain was cut into 400-μm sagittal slices using a McIllwain Tissue Chopper (Mickle Laboratory, Cambridge, United Kingdom). Under a dissecting microscope and avoiding contact with the hippocampus, the slices were separated using fine forceps. Slices containing the intact hippocampus were selected and positioned onto 30-mm-diameter semiporous cell culture inserts (five slices per insert) (Falcon; Becton Dickinson Labware, Millipore, Bedford, MA) and placed in a six-well tissue culture tray (Multiwell; Falcon, Becton Dickinson Labware). Eagle minimum essential medium enhanced with heat-inactivated horse serum (1.5 ml) was then transferred to each well.

The slices were incubated for 24 h in humidified air at 37°C, enriched with 5% carbon dioxide. The culture medium was replaced the next day with fresh, temperature-equilibrated medium before exposure to gas treatments. The groups of slices (n = 15 per group) were assigned to control (air + 5% carbon dioxide), dexmedetomidine 1 μm, gabazine 50 μm, 0.75% isoflurane, 0.75% isoflurane + dexmedetomidine 1 μm, and 0.75% isoflurane + gabazine 50 μm.

All subsequent gas exposure occurred in a specially constructed exposure chamber as previously described.23The gases, warmed by a water bath, were delivered in the headspace above the slices by a standard anesthetic machine at 2–3 l/min, and concentrations were monitored with an S/5 spirometry module (Datex-Ohmeda, Bradford, United Kingdom). After 3–4 min of gas flow, the chambers were sealed and placed in a 37°C incubator for 6 h (Galaxy R Carbon Dioxide Chamber; Wolf Laboratories, Pocklington, York, United Kingdom). After exposure, the slices were returned to the incubator for a further 12 h of culture to allow for suitable caspase-3 expression and then fixed overnight in 4% paraformaldehyde and subsequently immersed in 30% sucrose for a further 24 h at 4°C before slicing with a cryostat.

In Vivo  Experiments
Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane in 25% oxygen or air in a temperature-controlled chamber (n = 6 per group). Three doses of saline or dexmedetomidine (1, 10, or 25 μg/kg) were administered by intraperitoneal injection over the 6-h exposure (at 0, 2, and 4 h). One group received 0.75% isoflurane, 25 μg/kg dexmedetomidine, and 500 μg/kg nonselective α2adrenoceptor antagonist atipamezole in 3 doses over the 6-h exposure (n = 4 per group). An additional three doses of 75 μg/kg dexmedetomidine in air were given to establish at extreme doses of dexmedetomidine whether apoptosis could be induced (n = 6 per group).

The animals were sacrificed (with 100 mg/kg sodium pentobarbital by intraperitoneal injection) at the end of gas exposure and perfused transcardially with heparinized saline followed by 4% paraformaldehyde in 0.1 m buffer. After removal of the brain and storage overnight at 4°C in paraformaldehyde, it was transferred to 30% sucrose solution with phosphate buffer and 1% sodium azide and kept at 4°C until the brains were sectioned and stained immunohistochemically for caspase-3.

Immunohistochemistry
For the in vitro  experiments, the slices were sectioned at 25-μm intervals using a cryostat, and the inner sections were mounted onto Super Plus-coated glass slides (VWR International, Lutterworth, United Kingdom). The sections were allowed to dry at 37°C for 24 h and then immunostained while adherent to the slides. Concerning the in vivo  experiments, the brain was sliced at 30-μm intervals beginning at −3.6 mm from the bregma, the sections were then transferred to a six-well plate containing phosphate-buffered saline. Sections were dried at 37°C for 24 h and then immunostained while adherent to the slides, before preincubation with hydrogen 0.3% peroxidase in methanol for 30 min and then rinsed in phosphate-buffered saline. The sections were then incubated overnight at 4°C with rabbit anti-cleaved caspase-3 (1:2,500; New England Biolab, Hitchin, United Kingdom) and then washed three times in phosphate-buffered saline with 3% Triton at room temperature. Biotinylated secondary antibodies (1:200; Sigma, St. Louis, MO) and the avidin-biotin-peroxidase complex (Vector Laboratories, Orton Southgate, Peterborough, United Kingdom) were applied. The sections were again washed in phosphate-buffered saline before incubating with 0.02% 3,3′-diaminobenzidine with nickel ammonium sulfate in 0.003% hydrogen peroxide (DAB kit, Vector Laboratories). The sections were dehydrated through a gradient of ethanol solutions (70–100%) and then mounted (floating section) and covered with a cover slip.

Neurocognitive Evaluation
Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane in 25% oxygen or air in a temperature-controlled chamber (n = 6 per group). Three doses of saline or 25 μg/kg dexmedetomidine were administered by intraperitoneal injection over the 6-h exposure (at 0, 2, and 4 h). The animals were allowed to mature until postnatal day 40 and then tested for hippocampal-dependent memory and learning function in a previously reported contextual fear-conditioning behavioral paradigm24in which the rats were taken from the vivarium in the behavioral room on the first test day and allowed to sit undisturbed in their homecage for 10 min. Once placed in the conditioning chamber, the rats were allowed 198 s of exploration.

The conditioning chamber was cubic (30 cm × 24 cm × 21 cm; Med Associates, Inc., St. Albans, VT) and had a white opaque back wall, aluminum sidewalls, and a clear polycarbonate front door. The conditioning box had a removable grid floor and waste pan. Between each rat, the box was cleaned with an almond-scented solution and dried thoroughly. The grid floor contained 36 stainless steel rods (diameter, 3 mm) spaced 8 mm center to center. When placed in the chamber, the grid floor made contact with a circuit board through which a scrambled shock was delivered. During training and context testing, a standard high efficiency particulate air filter (HEPA) filter provided background white noise of 65 db.

Afterwards, all animals received 6 cycles of 214 s of trace fear conditioning. The tone was presented for 16 s (2 kHz) followed by a trace interval of 18 s and subsequent foot shock (2 s, 0.85 mA). The rats were removed from the conditioning chamber 198 s after the last shock and returned to their home cage. The total time of the acquisition phase was 26 min. Acquisition time was defined as the time spent immobile after a shock divided by the intertrial interval. On the next day, trained rats were exposed to the same acquisition environment but received neither tone nor shock for 8 min (context test). The percentage of time an animal froze during the 8-min observation periods was calculated as the number of observations judged to be freezing divided by the total number of observations in 8 min (i.e. , 60 observations). Freezing time was assessed using VideoFreeze software (Med Associates Inc., Burlington, VT); therefore, the assessment can be considered objective. The percentage of freezing time (context results) and the area under curve were derived from plots between the percentage freezing time and trial time in the tone test and were used for statistical comparison (mean ± SD, n = 6 per group).

Statistical Analyses
The number of caspase-3–positive neurons in the cortex, thalamus, and hippocampus in each brain slice were counted by an observer blinded to the experimental protocol. Four brain slices were counted per animal. The immunohistochemical and behavioral data are presented as mean ± SD. Statistical analyses was performed by ANOVA followed by post hoc  Newman Keuls testing using the INSTAT (London, United Kingdom) program. P < 0.05 was set as significant.",both,"['Organotypic hippocampal slices were derived from postnatal day 8 or 9 C57Bl/6 mice pups (Harlan Laboratories, Huntingdon, United Kingdom)', 'Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane in 25% oxygen or air in a temperature-controlled chamber']","postnatal day 7, postnatal day 8","['Organotypic hippocampal slices were derived from postnatal day 8 or 9 C57Bl/6 mice pups', 'Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane']",Y,['The animals were allowed to mature until postnatal day 40 and then tested for hippocampal-dependent memory and learning function in a previously reported contextual fear-conditioning behavioral paradigm'],isoflurane,['Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane in 25% oxygen or air in a temperature-controlled chamber'],none,[],sprague dawley,"['Organotypic hippocampal slices were derived from postnatal day 8 or 9 C57Bl/6 mice pups', 'Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane']",False,False,False,True,True,True,[ Passage 18/25 ] 10.1097/ALN.0b013e31819daedd
10.18632/oncotarget.15405,1027.0,Li,2017,rats,gestational day 14,Y,ketamine,none,wistar,"PMID: 28430606 PMCID: PMC5464800 DOI: 10.18632/oncotarget.15405
MATERIALS AND METHODS
Animals
Male and female Wistar rats, three months of age, weighing 200 ± 20 g, were purchased from the Animal Experimental Center of the Second Affiliated Hospital of the Harbin Medical University (Harbin, China). Prior to the experiment, rats were quarantined for two weeks at the Northeast Agricultural University (Harbin, China). All experiments were performed in accordance with the guidelines outlined by the Ethical Committee for Animal Experiments (Northeast Agricultural University, Harbin, China).

Mating and drug administration
Thirty-six Wistar rats were divided into 12 cages (one male and two females per cage) with an iron mesh at the bottom. On the next morning the vaginal suppository was investigated through the iron mesh. When sperm was detected, female rats were annotated as pregnant at day 0 (P0). The female rats were anesthetized via intravenous ketamine injection (200 mg/Kg) for 3 h on P14 [55]. The total volume of ketamine stayed below 2 mL/100 mg. Ketamine-treated offspring were recorded as K group, while individuals within the control group were recorded as C group. The first day after birth was recorded as B0. During B25-B30, Morris water maze task, contextual and cued fear conditioning, and olfactory tasks were used to test learning and memory capacity (n = 120, 5/dam, Figure ​Figure11).
Sample collections
Rat pups were sacrificed at B30 via cervical dislocation, and were recovered to collect brain tissue for Nissl staining (n = 24, 1/dam), Golgi staining (n = 24, 1/dam), and western blotting (n = 72, 3/dam). A subset of their hippocampuses were quickly dispensed on ice, put into a freezing tube, and frozen in liquid nitrogen, while other tissues were preserved in 10% formalin.

Nissl's staining
Coronal brain sections were cut in a vibratome (Leica VT1200S, Germany) after the brains were postfixed in the same fixative. To ensure matching of hippocampal sections between groups, we used anatomical landmarks provided by the brain atlas. The selected brain sections were stained with 0.5% cresyl violet and we selected three 104 μm2 areas for examination with a light microscope (Leica DFC420, Germany) to count neuron numbers in the CA1 and CA3 regions of the hippocampus.

Golgi staining
Golgi-Cox staining was utilized to obtain hippocampal dendritic spine density via the FD Rapid GolgiStainTM Kit (FD Neuro Technologies Inc), following the manufacturer's instructions. Coronal tissue sections of 150 μm thickness were cut at room temperature, using a vibratome (Leica VT1200S, Germany) and then, they were put on gelatin coated slides. Subsequently, slides were dehydrated with a gradient of 50%, 75%, 95%, to 100% ethanol and cleared in xylene, then the specimens were prepared with slide coverslips and sealed with Permount. The slides were then examined in detail with a light microscope (Leica DFC420, Germany). We analyzed the stained spine, using techniques similar to those described in previous study [56]. Five pyramidal neurons were analyzed that were well-impregnated and clearly distinguishable from others in each hippocampus (20 × objective lens). Five segments of 10 μm of apical and basal dendrites respectively, were randomly selected from each pyramidal neuron for inspection (via 200 × oil immersion lens) to quantify the density of spines. Spinal density of secondary apical and basal dendrites was analyzed at proximal segments emerging at more than 50 μm distance from the soma of the hippocampal CA1 neurons. All of these spines were required to exhibit a clearly distinguishable base or origin and were isolated from neighboring dendrites. Spine density was calculated per 10 μm of dendritic length. The open-source ImageJ 1.48 r Java image-viewing software and Adobe Photoshop CC 2015 were used to calibrate the scale and enlarge the segments of the spines. An investigator blinded to the experimental condition completed all analyses.

Morris water maze test
Place navigation trials
To test hippocampal-dependent spatial cognition, rats were trained in the standard morris water maze with a hidden platform [57]. A white escape platform (12 cm diameter) was submerged in a circular pool (160 cm diameter, at a 50 cm depth), filled with warm (23–25°C) opaque water. At B25-29, each rat pup underwent four trial sessions per day (60–70 min inter-trial interval) for five consecutive days. Each trial consisted of releasing the rat into the water, facing the outer edge of the pool at one of the quadrants (in random sequence) and permitting the animal to escape to the platform. They received four trials per day of training in search for the submerged and unmarked platform, with trial durations of 60 s on the platform at the end of trials. All trials were videotaped, and the swimming paths of rats were recorded with the ANY-maze video tracking system (Stoelting Co., IL, USA), which enabled us to measure the time taken (latency) to find the platform (s), as well as other behavioral information obtained during this spatial reference memory test. The animals were dried and placed beneath a heating lamp after completion of each test.

Spatial probe test
A probe trial was performed 1 d after the last trial at B30 where the platform was removed from the pool to assess memory retention for the location of the platform. During the 60 s test trial, we recorded and analyzed the swimming speed (cm/s), the swimming path tracks, and the number of entries into the platform quadrant zone.

Contextual and cued fear conditioning
Conditioning training on day one consisted of placing the rat pups in the chamber and exposing the animals to a mild footshock paired with an auditory cue. The rat pup was brought from the home cage to the testing room and placed into the conditioning chamber. It had 3 min to explore the novel environment. The auditory cue (a 90 dB tone) was sounded for approximately 30 sec. A stimulus light within the wall of the chamber may also be illuminated. During the last seconds of the auditory signal, an unconditioned aversive stimulus, a mild footshock in the range of 0.25 to 0.5 mA, was administered through the grid floor for 2 sec. The number of seconds spent freezing in the test chamber on the training day was considered the control measure of unconditioned fear. The rat pup was left in the conditioning chamber for 1 min after the last pairing, during which the association between the aversive stimulus and the properties of the conditioning chamber was further established. The rat pup was then returned to its home cage.

Testing on day 2 began approximately 24 hours after the conditioning session. The rat pup was returned to the same conditioning chamber and scored for bouts of freezing behavior. No footshock was administered on day two. The number of seconds spent freezing in the identical test chamber on day two was considered the measure of contextually conditioned fear, i.e., freezing within identical context. Freezing was defined as a lack of movement other than respiration. Presence or absence of freezing behavior was generally recorded by an investigator, who was blinded to the experimental condition, taking a note every 10 sec for 5 min, for a maximum total score of 30 freezing bouts. The rat pup was then returned to its home cage.

The second phase of testing began an hour later. A further testing chamber with very different properties provided the altered context. Changing the sensory cues as much as possible was essential so that the rat pup perceives the novel context as unrelated to the conditioning chamber. Such as triangle-shaped test chamber with different lighting was used and lemon juice was painted on the walls, while a different investigator wore gloves and a lab coat of different texture than on the training day. Freezing behavior was scored for 3 min. Contextual discrimination of fear conditioning was quantified by comparing the number of freezing bouts in the same contextual environment to the number of freezing bouts in the novel contextual environment.

At the end of the first 3 min, the tone that was presented on training day one (was well as the light stimulus cue if used on day one) was presented in the novel context environment. Freezing behavior was scored for the next 3 min in the presence of the sound (and light) cues. Cued conditioning was calculated via comparison of the number of freezing bouts in the novel context environment in the presence of the cue with the number of freezing bouts in the novel context environment in the absence of the cue (Figure ​(Figure5a5a).

Olfactory task
This task was designed to investigate the olfactory learning and memory abilities [58]. For this experiment, two holes (3 cm diameter and 4.5 cm deep) were used. A polypropylene swab, embedded in a fine plastic mesh and containing 20 μL of diluted odors (1:10) was placed at the bottom of each hole and covered with wood shavings. The acquisition test (one session) consisted of one odor (either limonene or carvone, Sigma-Aldrich) being presented in both holes for a 5 min period. In a preliminary experiment, with simultaneous presentation of the same pair of odors (one odor in each hole) in a one-trial test, rat pups spent the same amount of time exploring either hole, indicating no preference for one of the two odors. The recall test consisted of a 3 min session in which one hole was odorized with the previously presented odor, while the other hole was odorized with a new odor (Figure ​(Figure6a).6a). The delay between acquisition and recall tests was 60 min. During the recall test, the cumulated exploration time of each hole was converted as the percentage of the total exploration time of both holes. Rat pups were considered to have remembered the familiar odor when they spent less time exploring the hole containing it, in relation to the time spent exploring the hole containing the new odor. Equal exploration times for both holes during the recall test were considered to indicate that rat pup did not remember the familiar odor. Both odors were used alternatively during acquisition or recall and presented randomly in each of the two holes to avoid place preference bias (Figure ​(Figure6a6a).

Cell culture and drug treatment
PC12 cells were obtained from the Northeast Agricultural University, Harbin, China. The cells were cultured in DMEM medium (Gibco), supplemented with 10% (v/v) FBS, penicillin/streptomycin (100 U/mL; 100 μg/mL) at 37°C under an atmosphere of 5% CO2 and 95% air. The cells were seeded in 6-well plates with 2-9 × 105 cells/well or 96-well plates with 2-9 × 104 cells/well, and the culture medium was changed daily. Cells were pretreated for 3 h with Protein Kinase A (PKA) inhibitor (H89, 10 μM, H group), Extracellular Regulated Protein Kinases (ERK) inhibitor (SCH772984, 10 μM, S group), PKA inhibitor + ERK inhibitor (S+H group), DMSO (solvent of inhibitors, D group), and ketamine (K group).

Cell counting kit-8 (CCK-8) assay
Cell viability was detected via the CCK8 assay (Beyotime Institute of Biotechnology, Suzhou, Jiangsu, China). Following the indicated treatments, CCK8 solution (10 μl) was added to each well (96-well plates). Then, the cells were cultured at 37°C for one further hour. The optical density of each well was measured at 450 nm with a Bio-Tek microplate reader (Bio-Tek Instruments, Thermo Fisher Scientific, Winooski, VT).

WB
150 μg of protein were separated via 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (HybondTM-C Extra, GE Healthcare) via electroblotting. After washing, membranes were blocked with 3% (w/v) BSA (biotopped) for 4 h at room temperature and incubated overnight at 4°C in BSA with antibodies that are specific for Ca2+/Calmodulin-Dependent Protein Kinase II (CaMKII), p-CaMKII, CaMKIV, p-CaMKIV, ERK, p-ERK, PKA, CREB, p-CREB (1.5:1000, EnoGene), p-PKA, and Brain Derived Neurotrophic Factor (BDNF, 1:1000, abcam). Membranes were washed thrice with PBS containing 0.1% Tween and then incubated for 1 h at room temperature either with a horseradish peroxidase-conjugated secondary antibody (Goat anti-Rabbit IgG Antibody HRP (ABIN) or a goat anti-Mouse IgG Antibody HRP (Sigma)) in BSA.

Data analysis
All data were analyzed with GraphPad Prism 7.0 (GraphPad Software Inc., USA) via one-way ANOVA, followed by Turkey's Post Hoc test or unpaired two-tailed Student t-test. Values were considered to be statistically significant for P < 0.05. Data are presented as means ± standard deviation unless otherwise noted.",rats,"['Male and female Wistar rats, three months of age, weighing 200 ± 20 g, were purchased from the Animal Experimental Center of the Second Affiliated Hospital of the Harbin Medical University (Harbin, China).']",gestational day 14,['The female rats were anesthetized via intravenous ketamine injection (200 mg/Kg) for 3 h on P14 [55].'],Y,"['During B25-B30, Morris water maze task, contextual and cued fear conditioning, and olfactory tasks were used to test learning and memory capacity (n = 120, 5/dam, Figure \u200bFigure11).']",ketamine,['The female rats were anesthetized via intravenous ketamine injection (200 mg/Kg) for 3 h on P14 [55].'],none,[],wistar,"['Male and female Wistar rats, three months of age, weighing 200 ± 20 g, were purchased from the Animal Experimental Center of the Second Affiliated Hospital of the Harbin Medical University (Harbin, China).']",True,True,True,True,True,True,[ Passage 19/25 ] 10.18632/oncotarget.15405
10.1016/j.neulet.2013.04.008,1209.0,Li,2013,rats,postnatal day 7,N,isoflurane,none,sprague dawley,"PMID: 23603260 DOI: 10.1016/j.neulet.2013.04.008
2. Materials and methods
All animal procedures were in compliance with the NIH Guide for the Use of Laboratory Animals and approved by the Animal Care and Use Committee of Sun Yat-sen University. Seven-day-old (P7) Sprague-Dawley rat pups (Guangdong Medical Laboratory Animal Co, China) with body weight at 16 ± 3 g were exposed to 1.1% isoflurane (about 0.5 MAC in P7 rats [22]) for 4 h to induce neuronal apoptosis, or to air in a temperature-controlled chamber as we described before [21]. The concentrations of anesthetic gas, oxygen and carbon dioxide (CO2) in the chamber were measured by a gas analyzer (Datex-Ohmeda, Madison, WI).

Four doses of SP600125 (Selleck Chemicals LLC, Houston, TX, USA) (5, 10, 20 or 30 μg) or 12% dimethyl sulfoxide (DMSO) as the vehicle were administered by intracerebroventricular (i.c.v.) injection 15 min before isoflurane exposure. Some rats received 30 μg SP600125 or 12% DMSO only. The injection was performed as described before [6] under isoflurane anesthesia with a 5 μl microsyringe and 0.4 mm external diameter needle. The location of injection was 2.0 mm rostral, 1.5 mm lateral to the lambda and 2.0 mm deep to the skull surface of rats. The injection solution of 5 μl/rat was infused at a constant rate of 2.5 μl/min. The accuracy of i.c.v. injection was verified by methylene blue in our preliminary experiments. All animals were sacrificed 6 h after termination of gas exposure and their hippocampi were used for Western blotting (n = 6) or TdT-mediated dUTP nick end labeling (TUNEL) with fluorescent dye (n = 6).

For Western blotting studies, rat pups were anaesthetized with isoflurane and then sacrificed by decapitation. Hippocampi of rats were isolated immediately on ice and then stored at −80 °C until used. Western blotting was performed as we have described previously [20]. In brief, the protein concentrations of samples were determined using the BCA protein assay (Bio-Rad,Herts, UK). Sixty micrograms of each sample were subjected to Western blot analysis using the following primary antibodies: anti-cleaved caspase-3 at 1:2000 dilution, anti-phospho-JNK at 1:2000 dilution, anti-JNK at 1:2000 dilution, anti-phospho-c-Jun at 1:1000 dilution, anti-phospho-Akt (Ser 473) at 1:2000 dilution, anti-Akt at 1:5000 dilution, anti- phospho-GSK-3β (Ser 9) at 1:2000 dilution, anti-GSK-3β at 1:2000 dilution, anti-Bcl-xL at 1:2000 dilution and anti-β-actin at 1:2000 dilution. All antibodies were purchased from Cell Signaling Technology Company, USA. Images were scanned by an Image Master II scanner (GE Healthcare) and were analyzed using Image Quant TL software (v2003.03, GE Healthcare). The band signals of phospho-JNK, phospho-Akt and phospho-GSK-3β were normalized to their total JNK, Akt and GSK-3β from the same samples. The band signals of other interesting proteins were normalized to those of β-actin and the results in each group were normalized to that of corresponding control group.

For TUNEL studies, rat pups were anaesthetized with isoflurane and perfused transcardially with 4% paraformaldehyde. Their brains were paraffin embedded and sectioned at 6 μm thickness. As we described before [19], four or five sections (200 μm apart) for each animal at the same plane of the hippocampus were chosen for detecting apoptosis using TUNEL fluorescent method (Promega, Madision, WI, USA). The slides were protected from direct light during experiment. Hoechst was used to stain nuclei. The TUNEL positive cells in CA1, CA3 and dentate gyrus (DG) areas of hippocampus were analyzed immediately with NIS-Elements BR imaging processing and analysis software (Nikon Corporation, Japan). The densities of the TUNEL positive cells in CA1, CA3 and DG were calculated by dividing the number of TUNEL positive cells by the area of that brain region.

Data are presented in mean ± SEM. The Graphpad Prism 4.0 software was used to conduct the statistical analyses. A two-tailed P value of less than 0.05 was considered statistically significant. One way ANOVA with Newman–Keuls Multiple Comparison Test was used when data was normally distributed and had equal variances. Otherwise, non-parametric test with Dunn's Multiple Comparisons was used to compare the density of TUNEL positive cells as well as the relative protein abundance data among groups in Western blots.",rats,"['Seven-day-old (P7) Sprague-Dawley rat pups (Guangdong Medical Laboratory Animal Co, China) with body weight at 16 ± 3 g were exposed to 1.1% isoflurane']",postnatal day 7,['Seven-day-old (P7) Sprague-Dawley rat pups'],N,['All animal procedures were in compliance with the NIH Guide for the Use of Laboratory Animals and approved by the Animal Care and Use Committee of Sun Yat-sen University.'],isoflurane,['with body weight at 16 ± 3 g were exposed to 1.1% isoflurane'],none,[],sprague dawley,['Seven-day-old (P7) Sprague-Dawley rat pups'],True,True,True,True,True,True,[ Passage 20/25 ] 10.1016/j.neulet.2013.04.008
10.1016/j.biopha.2016.01.034,579.0,Lu,2016,rats,postnatal day 7,N,sevoflurane,none,sprague dawley,"PMID: 26898457 DOI: 10.1016/j.biopha.2016.01.034
2. Materials and methods
2.1. Animals
Because peak anesthesia-induced neurodegeneration in rodents occurs on postnatal day (PND) 7 [22], Sprague-Dawley (SD) PND7 rats weighing 14–18 g, provided by the Animal Center of Shanghai Jiao Tong University School of Medicine (Shanghai, China) were used in this study. The housing and treatment of the animals were in accordance with the National Institutes of Health guidelines for animal experimentation and approved by the institutional animal care and use committee. The animals were kept on a 12-h light/dark cycle (light from 7 am to 7 pm) with room temperature (23 ± 1 °C).

2.2. Sevoflurane exposure
Rat pups were separated from their mothers for acclimatization prior to sevoflurane exposure. Pups from the same litter were randomly allocated to three different groups. Totally, ninety PND7 rats were included in this study (n = 30 for each group). Rats in the control group received 100% oxygen for 6 h in a chamber at 37 °C. Rats in the other two groups were exposed to either 2% sevoflurane (SEVOFRANE®, Osaka, Japan) for 3 h (Sevo1 group) or 3% sevoflurane for 6 h (Sevo2 group) under 100% oxygen in the same chamber at 37 °C as described previously [13]. The concentration of sevoflurane in the chamber was monitored and maintained by a vaporizer as we described previously [23]. The gas flow to the chamber was 2 l/min. We chose these treatments because 3 h exposure to 2% seveflurane more closely approximates typical general pediatric anesthetic episodes for anesthesia maintenance [16] and 6 h exposure to 3% sevoflurane can cause neuronal apoptosis in developing animals [11], [12], [14], [15].

2.3. Arterial blood gas analysis
To determine adequacy of ventilation and oxygenation，arterial blood samples (n = 6) were obtained from the left cardiac ventricle in each group at the end of anesthesia, and the samples were immediately analyzed by a blood gas analyzer (Radiometer, ABL800, Denmark). We compared the pH, pO2, pCO2, oxygen saturation (sO2), and the concentrations of blood glucose (Glu), lactic acid (Lac) and bicarbonate (HCO3−) among the groups. Animals were killed by lethal injection of pentobarbital at the time of blood sampling.

2.4. Analysis of apoptotic levels
2.4.1. TUNEL assay of brain
Twenty-four hours after sevoflurane exposure, six rats from each group (n = 6) were anesthetized with sodium pentobarbital and the brains were perfused, fixed, dehydrated and made into paraffin sections (5 μm), as described previously [24]. Apoptotic cells in the brain sections were detected by TUNEL Assay using the FragEL™ DNA Fragmentation Detection Kit (Merck, Darmstadt, Germany), according to the manufacturer’s protocol. Briefly, brain sections were permeabilized with proteinase K (20 μg/ml) at room temperature for 20 min. Endogenous peroxidase was inactivated by 3% H2O2. Specimens were incubated for 1.5 h with terminal deoxynucleotidyl transferase (TdT) labelling reaction mixture, and apoptotic cells were visualized with 3,3′-diaminobenzidine (DAB), and normal nuclei were counterstained with methyl green. Because the cerebral cortex reaches peak vulnerability to anesthetics at PND7 and the hippocampus is closely related to learning and memory [25], the number of apoptotic neurons in the frontal cortex and the CA1 region of the hippocampus was quantified. We selected two random viewing fields (400×) per region (frontal cortex and CA1) from one brain section per animal for analysis in a double blinded manner.

2.4.2. Western blot
Apoptosis was also assessed using western blot to quantify cleaved caspase-3 (Cl-Csp3) in all groups (n = 6). Briefly, tissue samples of the frontal cortex and CA1 region were collected from three groups twenty-four hours after sevoflurane exposure. Tissues were lysed in a buffer containing a protease inhibitor cocktail (Calbiochem, San Diego, CA, USA) and homogenated. The homogenate was centrifuged and the supernatant was collected for further analysis. Protein concentrations were measured by BCA Protein Assay Kit (Novagen, San Diego, CA, USA). Equal amounts of protein were boiled in loading buffer (Beyotime, Beijing, China) and separated by 10% polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose, and the blots were probed overnight with anti-cleaved caspase-3 (1:200, Millipore, Darmstadt, Germany) and β-actin antibodies (1:500, internal standard, Santa Cruz, San Diego, CA, USA) at 4 °C. Primary antibodies were visualized using secondary antibodies conjugated to horseradish peroxidase (Santa Cruz, San Diego, CA, USA) and ECL reagent (Pierce, Rockford, IL, USA). Quantitative analysis of Cl-Csp3 was normalized to β-actin using the Quantity One software.

2.5. Neurologic assessment
2.5.1. Morris water maze
To assess neurodevelopmental outcomes, particularly the learning and memory functions of juveniles, rats from all groups were subjected to Morris water maze after reaching 6 weeks of age (n = 12), as previously described [24]. Briefly, a circular pool (1.6 m diameter, 60 cm height) was used for the water maze, and a submerged platform (10 cm diameter, 2 cm below the surface of the water) was located at a fixed position in the pool. The water temperature was set at 23 ± 1 °C. Probe trials were conducted twice a day for five consecutive days. In the trials, rats were trained to swim to and locate the hidden platform. The time spent in finding the hidden platform and the swimming distance before reaching the platform were recorded. After the probe trials, the platform was removed, and the rats were allowed to swim freely for 120 s: the number of times that the former platform was crossed and the percentage of time spent in the target quadrant were determined. The entire behavioral test was recorded and analyzed using a MS-type Water Maze Video analysis system (Chengdu Instrument Ltd., Chengdu, China). Finally, to investigate cognitive function during development, the passive avoidance test was performed at 3 months.

2.5.2. Passive avoidance test
The passive avoidance test was performed as previously described [26]. The apparatus used for the passive avoidance test included a behavioral stimulation controller and a video shuttle box (Chengdu Instrument Ltd., Chengdu, China). The test relies the natural preference of rats for darkness. Briefly, on the first trial day, the rats were placed in the illuminated compartment after 2 min of habituation to the dark compartment and allowed to re-enter the dark compartment. On the following day, an electric foot shock was delivered through the grid floor of the dark compartment after the rats entered. Twenty-four hours later, the retention of passive avoidance was determined by comparing the time elapsed prior to re-entry into the dark compartment with the arbitrary maximum time of 180 s.

2.6. Statistical analysis
All data are expressed as the mean ± SEM. SAS 9.2 (SAS Institute Inc., Cary, North Carolina, USA) was used for statistical analysis. One-way ANOVA was used to determine statistically significant differences between the three groups, and Tukey’s post hoc analysis was performed to correct for multiple comparisons when applicable. Statistical significance was accepted as P < 0.05.",rats,"['Because peak anesthesia-induced neurodegeneration in rodents occurs on postnatal day (PND) 7 [22], Sprague-Dawley (SD) PND7 rats weighing 14–18 g, provided by the Animal Center of Shanghai Jiao Tong University School of Medicine (Shanghai, China) were used in this study.']",postnatal day 7,"['Because peak anesthesia-induced neurodegeneration in rodents occurs on postnatal day (PND) 7 [22], Sprague-Dawley (SD) PND7 rats weighing 14–18 g, provided by the Animal Center of Shanghai Jiao Tong University School of Medicine (Shanghai, China) were used in this study.']",Y,"['To assess neurodevelopmental outcomes, particularly the learning and memory functions of juveniles, rats from all groups were subjected to Morris water maze after reaching 6 weeks of age (n = 12), as previously described [24].', 'Finally, to investigate cognitive function during development, the passive avoidance test was performed at 3 months.']",sevoflurane,"['Rats in the other two groups were exposed to either 2% sevoflurane (SEVOFRANE®, Osaka, Japan) for 3 h (Sevo1 group) or 3% sevoflurane for 6 h (Sevo2 group) under 100% oxygen in the same chamber at 37 °C as described previously [13].']",none,[],sprague dawley,"['Because peak anesthesia-induced neurodegeneration in rodents occurs on postnatal day (PND) 7 [22], Sprague-Dawley (SD) PND7 rats weighing 14–18 g, provided by the Animal Center of Shanghai Jiao Tong University School of Medicine (Shanghai, China) were used in this study.']",True,True,False,True,True,True,[ Passage 21/25 ] 10.1016/j.biopha.2016.01.034
10.1007/s12035-017-0730-0,230.0,Obradovic,2018,mice,postnatal day 7,N,ketamine,none,cd-1,"PMID: 28840469 PMCID: PMC5808855 DOI: 10.1007/s12035-017-0730-0
Materials and Methods
Animals
We used 7-day-old (PND7) CD-1 mice (Harlan Laboratories, Indianapolis, IN) for all experiments. We chose this age because 1) it is when rodents are most vulnerable to GA-induced developmental neurotoxicity [16] and, 2) it falls before developmental pruning of the IPB begins [15]. Our ketamine anesthesia protocol was as follows: experimental mouse pups were exposed to 6h of ketamine anesthesia and controls were exposed to 6h of mock anesthesia (vehicle) injected I.M. During anesthesia, pups were carefully monitored. After the administration of anesthesia, mice were reunited with their mothers until sacrifice (from P8 until P65). The weaning was done at P21 using the standard protocol. At the desired age mice were divided randomly into two groups: one group for assessing expression of pro- and the mature form of BDNF using the Western blotting technique and the second group for morphometric studies of IPB development. Our randomization process was designed to provide each group with roughly equal representation of pups from each dam.

The experiments were approved by the Animal Use and Care Committees at the University of Colorado the Office of Animal Resources (OLAR), Aurora, Colorado and the Animal Use and Care Committees of the University of Virginia, Charlottesville, Virginia. The experiments were done in accordance with the Public Health Service's Policy on Humane Care and Use of Laboratory Animals. Efforts were made to minimize the number of animals used while being able to conduct meaningful statistical analyses.

Anesthesia administration
To achieve general anesthesia state, we used a ketamine protocol known to cause significant developmental neurotoxicity in PND7 mice whereby mouse pups received a total of four doses of ketamine, at 75 mg/kg, IM every 90 minutes so that the loss of righting reflex and lack of response to tail pinch could be maintained for 6 hours [14]. For control animals, saline was administered using the same volume and administration schedule. During entire anesthesia procedure, animals were kept away from their mother and were housed in standard, tightly closed mice cages, with free air flow through air filters. Animals were kept in close proximity to each other in the cages, so they could preserve, even under anesthesia, important olfactory cues and stimuli, necessary to bust and sustain their metabolic output. During the experiment, we carefully monitored animals and measured environmental temperature in their breeding cages. We established that the ambient temperature in the breeding cage was around 37.0±1°C. Considering that animals at this age are very sensitive to change in body temperature, they were kept under constant ambient temperature maintained with heating pads conveniently set up around the cages. The ambient temperature was assessed at frequent time intervals using the thermometer.

Western blot studies
For BDNF protein quantification, we dissected the hippocampus immediately after the brains were removed from the individual pups using a dissecting scope (10× magnification). Tissue was collected on ice and was snap-frozen in liquid nitrogen immediately. The protein concentration of the lysates was determined with the Total Protein kit using the Bradford method (Cayman Chemical, Ann Arbor, MI). Approximately 10-25 μg of total protein was heat- denatured, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) through 4-20% Tris-glycine polyacrylamide gradient gels (BioRad, Hercules, CA). Separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA), blocked at room temperature for 1h in 3% bovine serum albumin (BSA) followed by incubation at 4°C overnight with primary antibody, anti-BDNF (1:500, Alomone Labs, Jerusalem, Israel), and anti-β-actin antibody (1:10000 Sigma Aldrich, USA) as a loading control.

Membranes were incubated for 1h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies - goat anti-rabbit or goat anti-mouse IgG (1:10000, Santa Cruz, Dallas, TX). Three washes with 0.3% Tween-20 in Tris-buffered saline were performed between all steps. Immunoreactivity was detected using enhanced chemiluminescence substrate (Super Signal west Femto; Thermo Scientific, UT). Images were captured using GBOX (Chemi XR 5, Syngene, MD) and gels were analyzed densitometrically with the computerized image analysis program ImageQuant 5.0 (GE Healthcare, Life Sciences, Piscataway, NJ).

Histological Preparation
Mice were deeply anesthetized with 2% isoflurane and immediately perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (at pH 7.4). Brains were extracted and immersed in fresh 4% paraformaldehyde and incubated at 4°C for additional 2-3 days before being embedded in agar. Briefly, brain coronal sections (50μm thickness) were cut using vibratome. Tissue sections were blocked with 1× TBS contains normal goat serum 5%, 1% BSA and 0.1% triton X-100 for 1h at room temperature before incubated with primary antibodies against calbindin (anti-calbindin D-28K antibody, 1:1000; Gene Tex, CA, USA) overnight at 4°C. Free floating sections were then washed three times with TBS, and then incubated with corresponding HRP-conjugated secondary antibodies (1:200) at room temperature for 2h. Tissue sections were mounted on glass slides and air dried. For detection, we used DAB Peroxidase substrate kit (Vector Laboratories) following manufacturer's instructions.

Histological Morphometric Assessment
The morphometric analyses of IBP developmental shortening (from PND10 until PND65 in both control and ketamine-treated mice) were performed using coronal hippocampal slices (50μm) cut from bregma -1.34mm to -2.30mm (as determined using a mouse brain atlas). The images were scanned at 20× magnification using an Aperio Scanscope XT digital slide scanner (Aperio Technologies Inc., Vista, CA) at University of Virginia, Charlottesville, VA and at University of Colorado, Aurora, CO. The hippocampal area in digital sections (.svs file) was extracted at 600μm scale to convert to a .tiff file and was spatially calibrated using 1000 μm2 grid prior to quantifying using Image-Pro Plus 7.0 software (Media Cybernetics, MD). The morphometric approach used to evaluate so called ‘normalized length of IPB’, which takes into consideration individual variability and developmental growth of hippocampus. The IPB length was approximated from the tip of the inferior blade of the dentate granule cell layer (“a”). The length of CA3 was approximated from the tip of the inferior blade to the apex of the curvature of the CA3 pyramidal cell layer (“b”). Normalized IPB length was taken as a ratio between “a” and “b”. The values from serial sections (n=3-6 serial sections per animal from 6-7 animals per age group) were averaged to provide a single data point and is presented as normalized IPB length. The results from different age groups were statistically analyzed by t test and between both groups by Two-way ANOVA using Graph Pad Prism 5.01 software (Graph Pad, CA). The experimenters were blinded to the experimental condition.

Statistical analysis
Comparisons among groups were made using one-way and two-way ANOVAs followed by Tukey's post hoc test. Using the standard version of GraphPad Prism 5.01 software (Media Cybernetics, Inc, Bethesda, MD), we considered p<0.05 to be statistically significant. All data are presented as mean ±SD or mean ±SEM. The sample sizes reported throughout the Results and in the Figure Legends were based on previous experience.",mice,"['We used 7-day-old (PND7) CD-1 mice (Harlan Laboratories, Indianapolis, IN) for all experiments.']",postnatal day 7,"['We used 7-day-old (PND7) CD-1 mice (Harlan Laboratories, Indianapolis, IN) for all 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,"['To achieve general anesthesia state, we used a ketamine protocol known to cause significant developmental neurotoxicity in PND7 mice whereby mouse pups received a total of four doses of ketamine, at 75 mg/kg, IM every 90 minutes so that the loss of righting reflex and lack of response to tail pinch could be maintained for 6 hours [14].']",isoflurane,['Mice were deeply anesthetized with 2% isoflurane and immediately perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (at pH 7.4).'],cd-1,"['We used 7-day-old (PND7) CD-1 mice (Harlan Laboratories, Indianapolis, IN) for all experiments.']",True,True,True,True,False,True,[ Passage 22/25 ] 10.1007/s12035-017-0730-0
10.1007/s10072-014-1726-4,4902.0,Ren,2014,rats,postnatal day 7,N,isoflurane,none,sprague dawley,"PMID: 24705859 DOI: 10.1007/s10072-014-1726-4
Methods
All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Virginia (Charlottesville, VA). All surgical and experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publications number 80-23) revised in 2011. Efforts were made to minimize the number of animals used and their suffering.

Neonatal brain hypoxia–ischemia modal
Brain HI was performed in 7-day-old male and female Sprague–Dawley rats as described previously [10, 11]. In brief, neonates were anesthetized by isoflurane and their left common carotid arteries were permanently ligated with a double 7-0 surgical silk. The procedure lasted <5 min. After surgery, neonates were returned to the cages with their mothers for 3 h. The neonates were then placed in a chamber filled with humidified 8 % oxygen–92 % nitrogen for 2 h at 37 °C. The oxygen concentration and temperature in the chamber were continuously monitored.

Drug application
The neonates were randomly divided into the following groups: (1) control, (2) brain HI, (3) brain HI and postconditioning with 1, 2 and 3 % sevoflurane, (4) brain HI and 5-HD treatment (10 mg/kg) and (5) brain HI, 5-HD treatment and postconditioning with 2 % sevoflurane. Sevoflurane postconditioning was performed by exposing neonates to various concentrations of sevoflurane in 30 % O2 for 1 h immediately after brain HI. Neonates of brain HI alone group were placed in a chamber flushed with 30 % O2 for 1 h. The mitochondrial KATP channel inhibitor 5-HD was dissolved in normal saline and administered intraperitoneally just before the start of brain HI. The dose of 5-HD was based on a previous study in which intraperitoneal injection of 10 mg/kg 5-HD blocked ischemic preconditioning-induced protection [12].

Brain injury/tissue loss quantification
After 7 days of the brain HI, rats were sacrificed under deep isoflurane anesthesia and then their brains were harvested as described previously [11, 13]. The hindbrain was removed from cerebral hemispheres and bilateral hemispheres were weighed separately. The weight ratio of left to right hemispheres was calculated.

Statistical analysis
The results are presented as mean ± SD (n ≥ 6). Statistical analysis was performed by one-way analysis of variance followed by the Tukey’s test. A P ≤ 0.05 was considered statistically significant.",rats,"['Brain HI was performed in 7-day-old male and female Sprague–Dawley rats as described previously [10, 11].']",postnatal day 7,"['Brain HI was performed in 7-day-old male and female Sprague–Dawley rats as described previously [10, 11].']",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,"['neonates were anesthetized by isoflurane', 'rats were sacrificed under deep isoflurane anesthesia']",sevoflurane,['Sevoflurane postconditioning was performed by exposing neonates to various concentrations of sevoflurane'],sprague dawley,"['Brain HI was performed in 7-day-old male and female Sprague–Dawley rats as described previously [10, 11].']",True,True,True,True,False,True,[ Passage 23/25 ] 10.1007/s10072-014-1726-4
10.1097/ALN.0000435846.28299.e7,1054.0,Takaenoki,2014,mice,postnatal day 6,Y,sevoflurane,none,c57bl/6,"PMID: 24061597 DOI: 10.1097/ALN.0000435846.28299.e7
Materials and Methods
Animals
All experiments were conducted according to the institutional ethical guidelines for animal experiments of the National Defense Medical College and were approved by the Committee for Animal Research at National Defense Medical College (Tokorozawa, Saitama, Japan). Inbred C57BL/6 mice were used in this study and maintained as described previously.5 

Anesthesia and Hydrogen Treatment
Sevoflurane anesthesia was carried out as described previously.5  In brief, on postnatal day 6 (P6), pups were placed in a humid chamber immediately after removal of mice from the maternal cage. A 3% concentration of sevoflurane was administered in 30% oxygen as the carrier gas. Control mice were exposed to 30% oxygen. Hydrogen gas (1.3%) was supplied as described previously.30  Total gas flow rate was 2 l/min.

Mouse Study Design
In each experiment, siblings from the same litter were randomly allocated into one of the following groups so that each group was balanced on littermate. No obvious differences (e.g., body size and weight) were observed within the litters, and there was no significant difference in mean body weight among the groups (data not shown).

Survival rate of delivered pups: control, sevoflurane, and sevoflurane + hydrogen groups (n = 17–19 dams for each group); a minimum biologically important difference was set at a 30% decrease from the baseline level in the control group.

Pup exchange test: control and sevoflurane groups (n = 6 dams for each group); a minimum biologically important difference was set at a 30% decrease from the baseline level in the control group.

Behavioral studies: control, sevoflurane, and sevoflurane + hydrogen groups (n = 10–11 dams for each group); the primary outcome measure was latencies for pup retrieval; in the pup retrieval test, a minimum biologically important difference was set at a 30% increase from the baseline level in the control group.

Hormonal assay: control, hydrogen, sevoflurane, and sevoflurane + hydrogen groups (n = 4–5 dams for each group); a minimum biologically important difference was set as 30% decrease from the baseline level in the control group.

Immunohistochemical study: control and sevoflurane groups (n = 5 dams for each group); a minimum biologically important difference was set at a 30% decrease from the baseline level in the control group.

In total, we prepared 160 female pups, which received anesthesia or hydrogen treatment at P6 (55 of control, 56 of sevoflurane, 40 of sevoflurane + hydrogen, and 9 of hydrogen groups). Among them, eight pups with sevoflurane and one pup with sevoflurane + hydrogen died during the treatment. Then, these siblings from the same litter were reunited and cohoused till the experiment (mice were similarly caged and housed in all groups). At 3 weeks of age, mice were weaned and allowed to further mature. At 7–9 weeks of age, female mice were mated with healthy males that had not been exposed to any anesthetic. Among them, 23 female mice did not get pregnant (eight of control, six of sevoflurane, five of sevoflurane + hydrogen, and four of hydrogen groups) and 1 control mouse died due to failure of delivery. These mice were excluded from the final analysis. Thus, for first delivery experiments, we used 46 control dams, 42 sevoflurane-treated dams, 34 sevoflurane + hydrogen–treated dams, and 5 hydrogen-treated dams. These mice were allocated as described above (1–5 in this section).

Among them, some dams were further analyzed for behavioral studies of parous dams: the same sets of mice for behavioral studies in first-time delivery were reused in behavioral studies in second-time (parous) delivery (control: 7 for survival rate and 11 for behavioral studies; sevoflurane-treated: 8 for survival rate and 10 for behavioral studies).

For paternal study experiments, 26 age-matched male mice were either subjected to anesthesia (n = 13) or control (n = 13) treatment at P6 (no mice died during the treatment). Siblings from the same litter were allocated into each group almost equally (i.e., groups were balanced on littermate).

Oxytocin and Vasopressin Assay
Plasma concentrations of oxytocin and vasopressin in dams at 10 weeks of age were examined by enzyme-linked immunosorbent assay using commercially available kits (Oxytocin enzyme-linked immunosorbent assay kit and arg8-Vasopressin enzyme-linked immunosorbent assay kit; Enzo Life Sciences, Farmingdale, NY). Assays were performed according to the manufacturer’s instructions. Blood samples were collected from the inferior vena cava within 6 h after parturition.

Immunohistochemical Study
Immunohistochemical studies using the anti-c-Fos antibody (rabbit polyclonal; sc-52; Santa Cruz Biotechnology, Santa Cruz, CA) were performed as previously described.30  Samples were obtained within 6 h after parturition. The numbers of immunoreactive cells were counted by an observer blinded to the groups.

Behavioral Studies
On the morning of parturition, maternal behaviors were examined. Maternal behavioral studies using first-time mothers were performed at 10–12 weeks of age. The same sets of female mice were reused in the maternal behavioral studies for second-time (parous) mothers: those mice were mated again at 19–25 weeks of age, and maternal behaviors were examined at 22–28 weeks of age. Paternal behavioral studies using male mice were performed at 11 weeks of age. Survival rate (percentage of the number of pups at the indicated day compared with that at birth) was recorded until P6. In each experiment, observation was made by the same observer who was blinded to the groups. All apparatus used in this study was made by O’Hara & CO., LTD. (Tokyo, Japan).

Evaluation of Maternal Behavior
Pregnant females were individually housed for a few days before parturition and examined for maternal behavior on the morning of parturition. The number of pups with milk in their digestive tract and that of poorly cleaned pups (with placenta, amniotic membrane, or umbilical cords) was recorded on that day. Nest quality was also evaluated at the same time using the score system described previously31  with some modifications: grade 3, shaped like a deep hollow surrounded by high banks; grade 2, a hollow with medium-height banks; grade 1, flat with low banks, but still discrete; grade 0, no depression in bedding with no banks. Each new dam was also evaluated for time spent crouching over pups and the percentage of newborns scattered for 20 min with minimal disturbance as described previously.32  The percentage of scattered pups was expressed as a percentage with respect to time. We calculated the percentage of scatter as follows for each pup: (duration of scatter/total time observed (20 min) × 100). We then calculated the average for each group. These evaluations were carried out before the pup retrieval test.

Pup Retrieval Test
The pup retrieval test was performed essentially as described previously.14  Before the test, pups were separated from dams for 30 min. At the beginning, each mouse was put in one corner of a cage and three of her pups were placed in different corners of the same cage. The cages were continuously observed for 10 min with minimal disturbance. Latencies to sniff a pup for the first time and to return each pup to the nest were evaluated.

Evaluation of Parental Behavior
Parental behavior of virgin male mice toward pups was evaluated for 20 min. At the beginning, each mouse was put in one corner of a cage and three new born pups were placed in different corners of the same cage as described in the pup retrieval test. Latencies to sniff a pup for the first time and the numbers of males which committed attacks toward pups were evaluated. If any of the pups was attacked during the test, all pups were removed immediately and this subject was considered as “attack.”

Pup Exchange Test
The pup exchange test was conducted as described previously with some modifications.14  Pups born to a female dam couple (a dam with sevoflurane exposure at P6 and a control), which were born on the same day, were exchanged within 12 h after delivery. The number of surviving pups was evaluated for 6 days after birth.

Olfactory Test
The olfactory test was conducted as described previously.3 

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA). Comparisons of the means of each group were performed using Student t test, one-way ANOVA followed by Bonferroni post hoc test, and two-way ANOVA followed by Bonferroni post hoc test. Comparisons of the survival rate until P6 were performed using a log-rank (Mantel-Cox) test. We did not exclude any data in this study. P values of less than 0.05 were considered statistically significant. Values are presented as the mean ± SEM in bar graphs.",mice,['Inbred C57BL/6 mice were used in this study and maintained as described previously.5'],postnatal day 6,"['Sevoflurane anesthesia was carried out as described previously.5  In brief, on postnatal day 6 (P6), pups were placed in a humid chamber immediately after removal of mice from the maternal cage.']",N,"['Behavioral studies: control, sevoflurane, and sevoflurane + hydrogen groups (n = 10–11 dams for each group); the primary outcome measure was latencies for pup retrieval; in the pup retrieval test, a minimum biologically important difference was set at a 30% increase from the baseline level in the control group.']",sevoflurane,['Sevoflurane anesthesia was carried out as described previously.5'],none,[],c57bl/6,['Inbred C57BL/6 mice were used in this study and maintained as described previously.5'],True,True,False,True,True,True,[ Passage 24/25 ] 10.1097/ALN.0000435846.28299.e7
10.1097/01.anes.0000291447.21046.4d,845.0,Zhao,2007,rats,postnatal day 6,Y,isoflurane,none,sprague dawley,"PMID: 18043065 DOI: 10.1097/01.anes.0000291447.21046.4d
Materials and Methods
The animal protocol was approved by the institutional Animal Care and Use Committee of the University of Virginia, Charlottesville, Virginia. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (  National Institutes of Health publication No. 80-23) revised in 1996. All reagents unless specified below were obtained from Sigma Chemical (St. Louis, MO).

Neonatal Cerebral Hypoxia–Ischemia Model
Cerebral hypoxia–ischemia was induced as we previously described.17Briefly, 7-day-old male and female Sprague-Dawley rats were anesthetized by isoflurane in 30% O2–70% N2, and their left common carotid arteries were permanently ligated with a double 7-0 surgical silk. The rats were allowed to awake and were returned to their cages with the mothers for 3 h. The neonates were then placed in a chamber containing humidified 8% O2–92% N2for 2 h at 37°C. The air temperature in the chamber was continuously monitored and maintained at 37°C. The chamber was then opened to room air for 15 min, and the animals were returned to their cages.

Isoflurane Preconditioning and Study Groups
Six-day-old rats were placed in a chamber containing 1.5% isoflurane carried by 30% O2–70% N2for 30 min at 24 h before the cerebral hypoxia–ischemia. The neonates usually started to feed within 30 min after the isoflurane application. In the first set of experiments, six groups of neonates were studied: (1) control, (2) 1.5% isoflurane treatment only, (3) cerebral hypoxia–ischemia, (4) 1.5% isoflurane pretreatment and then cerebral hypoxia–ischemia, (5) 1 mg/kg N -(3-(aminomethyl)benzyl)acetamidine (1400 W; BIOMOL Research Laboratories Inc., Plymouth Meeting, PA) injected intraperitoneally 24 h before cerebral hypoxia–ischemia, and (6) 1 mg/kg 1400 W injected intraperitoneally 30 min before the isoflurane pretreatment and then cerebral hypoxia–ischemia. Neonates from the same mother were assigned to these six experimental conditions. Neonates in groups 2, 4, and 6 were pretreated with isoflurane, whereas the others from the same mother were placed in a chamber containing 30% O2–70% N2but no isoflurane for 30 min and were assigned to groups 1, 3, and 5.

In the second set of experiments, four groups of rats were studied: (1) control, (2) 1.5% isoflurane treatment, (3) 200 mg/kg aminoguanidine administered intraperitoneally 30 min before the isoflurane treatment, and (4) 1 mg/kg 1400 W injected intraperitoneally 30 min before the isoflurane treatment. Aminoguanidine and 1400 W were dissolved in normal saline, and the injected volume was from 0.16 to 0.2 ml per rat. Rats in the control group and isoflurane treatment only group received 0.2 ml normal saline at the corresponding times. Aminoguanidine and 1400 W are inducible nitric oxide synthase (iNOS) inhibitors that have been shown to inhibit iNOS activity in rat brain18and iNOS-mediated neuroprotection induced by isoflurane and prenatal hypoxic preconditioning at the regimen used in this study.11,17The rat brains were harvested 24 h after isoflurane treatment for Western analysis.

Mortality and Body Weight Monitoring
Death during the period from the onset of cerebral hypoxia–ischemia to 1 month afterward was recorded, and the mortality rate was calculated. Rat body weights were measured just before and 1 month after the cerebral hypoxia–ischemia.

Brain Histopathology
Brain histopathologic evaluation was performed in rats in the first set of experiments. One month (30 days) after the cerebral hypoxia–ischemia, rats were euthanized by isoflurane and transcardially perfused with 30 ml saline. Brains were removed and stored in 4% phosphate-buffered paraformaldehyde for 4 h at room temperature. Eight-micrometer-thick cryostat coronal sections at approximately 3.3 mm caudal to bregma were obtained and subjected to Nissl staining. These sections were examined by an observer blinded to the group assignment of the sections. The cerebral cortical and hippocampal areas in each of the hemispheres were measured by using National Institutes of Health Image 1.60 (Bethesda, MD). The area ratio of the cerebral cortex and hippocampus in the left hemisphere to those in the right hemisphere was calculated and used to reflect brain tissue loss in the left hemisphere after brain hypoxia–ischemia. Neuronal density in the perirhinal cortex was determined as follows. A reticle (approximately 0.034 mm2) was used to count cells in the same size area. Nissl staining–positive cells were counted in the area. Three determinations, each on different locations in the left perirhinal cortex, were performed and averaged to yield a single number (density of the neurons) for the brain region of each individual rat. The neuronal density in the right perirhinal cortex was determined in the same way. The neuronal density ratio in the left/right perirhinal cortex was then calculated to measure the neuronal loss after brain hypoxia–ischemia.

Motor Coordination Evaluation
This evaluation was performed just before the rats were killed for brain histopathology. Rats were placed on a rotarod whose speed increased from 4 to 40 rpm in 5 min. The latency and the speed of rats’ falling off the rod were recorded. Each rat was tested three times, and the speed–latency index (latency in seconds × speed in rpm) for each trial was calculated. The mean index value of the three trials was used to reflect the motor coordination functions of each rat.

Y Maze and Social Recognition
The Y-maze and social recognition tests were performed as described previously19at 1 day before the rats were killed for brain histopathology. During Y-maze test, rats were placed in the center of a symmetrical Y maze and were allowed to explore freely in the maze for 8 min. The total number and sequence of arms entered were recorded. An arm was entered if the hind paws of the rat were completely in the arm. The percentage alternation that was the percentage of the number of entry into all three arms in the maximum possible alternations (the total number of arms entered divided by 3) was calculated for each rat.

The social recognition task was tested by placing a test rat in a clean acrylic cage. A male juvenile (3- to 4-week-old) rat was placed into the cage with the test rat for 2 min. The two rats were separated for 3 h and were placed together again for 2 min. The duration of social investigation of the juvenile rat by the test rat during the two 2-min periods was recorded. Social investigation behaviors include direct contact with the juvenile for inspection and close following (< 1 cm) of the juvenile. If there was any aggressive encounter between the rats, the experiments were terminated and the data were excluded from analysis. The ratio of duration of the social investigation during the second 2-min period in the duration of the first 2-min period was calculated to measure the social recognition memory.

Western Blot Analysis
Cerebral cortex and hippocampus were dissected from the rats in the second set of experiments and were sonicated in ice-cold 20 mm Tris-HCl (pH 7.5) containing 5 mm Mg Cl2, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 1 mm dl-dithiothreitol, and 2 mm sodium orthovanadate. The sample was centrifuged at 1,000g  at 4°C for 10 min. The protein concentrations in the supernatants were determined by the Lowry assay using a protein assay kit. Equal protein samples (50 μg per lane) were separated by 12% sodium dodecyl sulfate–polyacrylamide gels and then electrotransferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). The primary antibodies were rabbit polyclonal anti–heat shock protein 70 (HSP70) antibody (1:1,500 dilution, catalog No. SPA-812; Stressgen, Victoria, British Columbia, Canada), antisurvivin antibody (1:500 dilution, catalog No. S8191), antiactin antibody (1:2,000 dilution, catalog No. A2066), and mouse monoclonal anti–Bcl-2 antibody (1:2,000 dilution, catalog No. sc-509; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Protein bands were visualized by the enhanced chemiluminescence detection method with reagents from Amersham Pharmacia Biotech (Piscataway, NJ). The protein band volumes were quantified by a densitometry with ImageQuant 5.0 Windows NT software (Molecular Dynamics, Sunnyvale, CA). The volumes of Bcl-2, HSP70, and survivin protein bands were normalized to those of actin to control for errors in protein sample loading and transferring during the Western blot analysis. The results in the groups after isoflurane exposure were then normalized to those of control animals.

Statistical Analysis
Our previous study showed that a 2-h left hemisphere hypoxia–ischemia reduced the weight of left brain hemisphere by approximately 30% and isoflurane preconditioning decreased this brain loss to approximately 10% with an SD of approximately 12% when the brains were examined at 7 days after the brain hypoxia–ischemia.17Based on these results, it was estimated that 7 rats per group would be needed to detect the protective effects (brain loss reduction/brain pathology) of isoflurane preconditioning with a desired power of 80% at an α level of 0.05 by t  test. However, this sample estimate was used only as a reference in the experimental design of this study because of the obvious differences in the duration of observation after brain hypoxia–ischemia (1 week vs.  1 month) and outcome parameters between this study and our previous study.17 

Data are presented as mean ± SD. Results of hippocampal and cortical area ratio, neuronal density ratio, speed–latency index, percentage of alternation, and the ratio of the investigation times of the different study groups were compared by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls (SNK) method or by one-way ANOVA on ranks followed by the Dunn method as appropriate. The Western blot data were analyzed by one-way ANOVA on ranks followed by the Dunn method. The mortality rates among groups were analyzed by Z  test. The comparison of body weight among groups was performed by ANOVA for repeated measures followed by the SNK method. P < 0.05 was considered significant. All statistical analyses were performed with SigmaStat (Systat Software, Inc., Point Richmond, CA).

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