diff --git "a/offline_results/exp_gb.csv" "b/offline_results/exp_gb.csv" --- "a/offline_results/exp_gb.csv" +++ "b/offline_results/exp_gb.csv" @@ -30,7 +30,7 @@ Spectrophotometric Detection of ROS Control and experimental groups of rats were killed immediately postanesthesia, and the subicular and thalamic brain tissues were quickly removed. ROS were measured as hydrogen peroxide using the horseradish peroxidase-linked spectrophotometric assay kit according to the manufacturer’s instructions (Amplex Red, Invitrogen). Briefly, extracted brain mitochondria samples (120 μg) were added to a 96-well plate containing 100 μl of reaction buffer consisting of 0.1 U/ml of the horseradish peroxidase, 50 μm Amplex UltraRed, and 1 μl of dimethyl sulfoxide. Reactions were incubated at room temperature for 30 min and protected from light. Resorufin absorptions were followed at 560 nm using a VersaMax tunable microplate reader (Molecular Devices, Chicago, IL). Hydrogen peroxide levels are expressed in arbitrary units (per milligram protein). The group sizes for each experimental condition are indicated in the figure legends. Statistical Analysis -Single comparisons among groups were made using an unpaired two-tailed t test. When ANOVA with repeated measures was needed, the Bonferroni correction was used to help maintain prescribed alpha levels (e.g., 0.05). Histograms in cumulative frequency analysis were compared with chi-square-test. Using the standard version of GraphPad Prism 5.01 software (Media Cybernetics, Inc., Bethesda, MD), we considered P < 0.05 to be statistically significant. All the data are presented as mean + SEM. No experimental data were missing or lost to statistical analysis.",rats,"['Sprague–Dawley rat pups (Harlan Laboratories, Indianapolis, IN) at P7 were used for all experiments.']",postnatal day 7,"['Sprague–Dawley rat pups (Harlan Laboratories, Indianapolis, IN) at P7 were used 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'.""]",isoflurane,['Isoflurane was administered using an agent-specific vaporizer that delivers a set percentage of anesthetic into the anesthesia chamber.'],midazolam,"['Midazolam (Sigma–Aldrich Chemical, St. Louis, MO) was dissolved in 0.1% dimethyl sulfoxide just before administration.']",sprague dawley,"['Sprague–Dawley rat pups (Harlan Laboratories, Indianapolis, IN) at P7 were used for all experiments.']",True,True,True,False,False,True,10.1097/ALN.0b013e318289bc9b +Single comparisons among groups were made using an unpaired two-tailed t test. When ANOVA with repeated measures was needed, the Bonferroni correction was used to help maintain prescribed alpha levels (e.g., 0.05). Histograms in cumulative frequency analysis were compared with chi-square-test. Using the standard version of GraphPad Prism 5.01 software (Media Cybernetics, Inc., Bethesda, MD), we considered P < 0.05 to be statistically significant. All the data are presented as mean + SEM. No experimental data were missing or lost to statistical analysis.",rats,"['Sprague–Dawley rat pups (Harlan Laboratories, Indianapolis, IN) at P7 were used for all experiments.']",postnatal day 7,"['Sprague–Dawley rat pups (Harlan Laboratories, Indianapolis, IN) at P7 were used 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'.""]",isoflurane,['Isoflurane was administered using an agent-specific vaporizer that delivers a set percentage of anesthetic into the anesthesia chamber.'],midazolam,"['Midazolam (Sigma–Aldrich Chemical, St. Louis, MO) was dissolved in 0.1% dimethyl sulfoxide just before administration.']",sprague dawley,"['Sprague–Dawley rat pups (Harlan Laboratories, Indianapolis, IN) at P7 were used for all experiments.']",True,True,True,False,False,True,[ Passage 1/25 ] 10.1097/ALN.0b013e318289bc9b 10.1016/S1995-7645(14)60066-3,408.0,Cao,2014,rats,postnatal day 7,Y,ketamine,propofol,sprague dawley,"PMID: 25063071 DOI: 10.1016/S1995-7645(14)60066-3 2. Materials and methods 2.1. Animals @@ -126,7 +126,7 @@ SPSS 13.0 software. After the variance test, the difference between two groups was compared with single factor analysis of variance. P<0.05 was considered as statistical significant difference. -",rats,"['A total of 80 healthy 7-day-old SD rats, male or female, weighing 12-18 g were selected.']",postnatal day 7,"['A total of 80 healthy 7-day-old SD rats, male or female, weighing 12-18 g were selected.']",Y,['Behavior of rats was observed by Morris water maze[3].'],ketamine,"['The animals in experiment group A received 80 mg/kg ketamine l mL by intraperitoneal injection every 2 h, continuous for 3 times.']",propofol,"['The animals in experiment group B received 80 mg/kg propofol 1 mL by intraperitoneal injection every 2 h, continuous for 3 times.']",sprague dawley,"['A total of 80 healthy 7-day-old SD rats, male or female, weighing 12-18 g were selected.']",True,True,True,True,True,True,10.1016/S1995-7645(14)60066-3 +",rats,"['A total of 80 healthy 7-day-old SD rats, male or female, weighing 12-18 g were selected.']",postnatal day 7,"['A total of 80 healthy 7-day-old SD rats, male or female, weighing 12-18 g were selected.']",Y,['Behavior of rats was observed by Morris water maze[3].'],ketamine,"['The animals in experiment group A received 80 mg/kg ketamine l mL by intraperitoneal injection every 2 h, continuous for 3 times.']",propofol,"['The animals in experiment group B received 80 mg/kg propofol 1 mL by intraperitoneal injection every 2 h, continuous for 3 times.']",sprague dawley,"['A total of 80 healthy 7-day-old SD rats, male or female, weighing 12-18 g were selected.']",True,True,True,True,True,True,[ Passage 2/25 ] 10.1016/S1995-7645(14)60066-3 10.1016/j.bbrc.2021.03.063,234.0,Chen,2021,mice,gestational day 14,Y,sevoflurane,none,c57bl/6,"PMID: 33756347 DOI: 10.1016/j.bbrc.2021.03.063 2. Methods and materials 2.1. Animals @@ -143,7 +143,7 @@ The three-chambered social box (40L × 60W × 22H cm) with two enclosures (7D × The offspring mice (N = 17 Control, 9 males and 8 females; N = 14 Sevoflurane, 9 males and 5 females) were tested at one- and two-month-old (i.e., the juvenile and early-adult age). In advance, the testing mouse was housed single for 1-h isolation in the behavioral room. The stranger mice were the identical background, same gender and similar age as the testing mouse, and they had exactly no contact before. Social interaction test is composed of three 10-min sessions of habituation, sociability, and preference for social novelty. Firstly, the testing mouse was allowed to freely explore in social box with two doorways opening. Next, an unfamiliar conspecific (Stranger 1) was introduced into one enclosure, and the testing mouse was allowed to sniff the stranger 1 or explore the empty enclosure (Fig. 1E, G). After that, another unfamiliar conspecific (Stranger 2) was introduced into the other enclosure, and the testing mouse was allowed to sniff the stranger 1 and stranger 2 (Fig. 1F, H). Placement of the stranger 1 on the left or right side was systematically altered between trials, and social apparatus was cleaned after each trial to minimize olfactory disturbance. 2.5. Statistical analysis -Data were shown as Mean ± SD. Graphpad Prism 5.0 software (San Diego, USA) was used for statistical analyses. Social data obtained from the left and right side were mutually exclusive in each 10-min session and they were normally distributed by Kolmogorov-Smirnov tests. Therefore, two-tailed paired t-test was used to compare the side preferences (stranger 1 vs. the opposite). Two-way repeated measures (RM) ANOVAs were used to analyze the interaction effects of treatment (control or sevoflurane) × side (stranger 1 or the opposite). Based on the preliminary study, a sample size of more than 5 (sociability) and 13 (preference for social novelty) could lead to a 90% power to detect a difference in side preference with 5% type I error. P values less than 0.05 (∗), 0.01 (∗∗) and 0.001 (∗∗∗) were considered statistically significant.",mice,"['Adult C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Suzhou, China).']",gestational day 14,['Six pregnant mice on gestational day 14 were randomly assigned to receive either 2.5% sevoflurane in 100% oxygen or just 100% oxygen as the control.'],Y,['The three-chambered social box (40L × 60W × 22H cm) with two enclosures (7D × 15H cm) was used for social interaction test (Fig. 1A–C).'],sevoflurane,['Six pregnant mice on gestational day 14 were randomly assigned to receive either 2.5% sevoflurane in 100% oxygen or just 100% oxygen as the control.'],none,[],c57bl/6,"['Adult C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Suzhou, China).']",True,True,True,True,True,True,10.1016/j.bbrc.2021.03.063 +Data were shown as Mean ± SD. Graphpad Prism 5.0 software (San Diego, USA) was used for statistical analyses. Social data obtained from the left and right side were mutually exclusive in each 10-min session and they were normally distributed by Kolmogorov-Smirnov tests. Therefore, two-tailed paired t-test was used to compare the side preferences (stranger 1 vs. the opposite). Two-way repeated measures (RM) ANOVAs were used to analyze the interaction effects of treatment (control or sevoflurane) × side (stranger 1 or the opposite). Based on the preliminary study, a sample size of more than 5 (sociability) and 13 (preference for social novelty) could lead to a 90% power to detect a difference in side preference with 5% type I error. P values less than 0.05 (∗), 0.01 (∗∗) and 0.001 (∗∗∗) were considered statistically significant.",mice,"['Adult C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Suzhou, China).']",gestational day 14,['Six pregnant mice on gestational day 14 were randomly assigned to receive either 2.5% sevoflurane in 100% oxygen or just 100% oxygen as the control.'],Y,['The three-chambered social box (40L × 60W × 22H cm) with two enclosures (7D × 15H cm) was used for social interaction test (Fig. 1A–C).'],sevoflurane,['Six pregnant mice on gestational day 14 were randomly assigned to receive either 2.5% sevoflurane in 100% oxygen or just 100% oxygen as the control.'],none,[],c57bl/6,"['Adult C57BL/6 mice in breeding age were purchased from Zhaoyan Laboratory (Taicang, Suzhou, China).']",True,True,True,True,True,True,[ Passage 3/25 ] 10.1016/j.bbrc.2021.03.063 10.1002/brb3.2556,878.0,Chen,2022,rats,postnatal day 7,Y,sevoflurane,none,sprague dawley,"PMID: 35726359 PMCID: PMC9304839 DOI: 10.1002/brb3.2556 2 MATERIALS AND METHODS Animals and treatment @@ -187,7 +187,7 @@ Cell viability assay Cell viability was detected by the cell counting kit-8 (CCK8) assay. Transfected hippocampal neurons were seeded onto 96-well plates at approximately 103 cells/well (100 μl/well). Then, the neurons were cultured for 1 h and mixed with 10 μl CCK8 reagent (Dojindo, Kumamoto, Japan) for 2 h. Next, the optical density was measured at 450 nm by utilizing a Bio-EL340 automatic microplate reader (Tek Instruments, Hopkinton, USA). Statistical analysis -All data are presented as the mean ± standard deviation (SD) of three independent experiments. Unpaired Student's t test and one-way ANOVA were used to test the mean difference between groups. Statistical analysis was carried out using GraphPad Prism 7 (GraphPad Inc., San Diego, CA, USA). A p-value < .05 was considered statistically significant.",rats,['Seven-day-old Sprague–Dawley rats were used in this study.'],postnatal day 7,"['The hippocampus was dissected from neonatal rats (7 days old), triturated, and dissociated through trypsin.']",Y,['The Morris water maze (MWM) test was used to evaluate the learning and memory abilities of rats at the age of 2 months.'],sevoflurane,"['Sevoflurane was used to anesthetize rats as previously described (Zhou et al., 2017).']",none,[],sprague dawley,['Seven-day-old Sprague–Dawley rats were used in this study.'],True,True,True,True,True,True,10.1002/brb3.2556 +All data are presented as the mean ± standard deviation (SD) of three independent experiments. Unpaired Student's t test and one-way ANOVA were used to test the mean difference between groups. Statistical analysis was carried out using GraphPad Prism 7 (GraphPad Inc., San Diego, CA, USA). A p-value < .05 was considered statistically significant.",rats,['Seven-day-old Sprague–Dawley rats were used in this study.'],postnatal day 7,"['The hippocampus was dissected from neonatal rats (7 days old), triturated, and dissociated through trypsin.']",Y,['The Morris water maze (MWM) test was used to evaluate the learning and memory abilities of rats at the age of 2 months.'],sevoflurane,"['Sevoflurane was used to anesthetize rats as previously described (Zhou et al., 2017).']",none,[],sprague dawley,['Seven-day-old Sprague–Dawley rats were used in this study.'],True,True,True,True,True,True,[ Passage 4/25 ] 10.1002/brb3.2556 10.31083/j.jin2003065,1043.0,Gao,2021,rats,gestational day 14.5,N,sevoflurane,none,sprague dawley,"PMID: 34645094 DOI: 10.31083/j.jin2003065 2. Methods 2.1 Animals @@ -236,7 +236,7 @@ All the data was expressed as mean ± standard deviation. JMP software version 16.0 (SAS Institute, Cary, NC, USA) was used for statistical processing. All parameters were tested for normal distribution using the Kolmogorov-Smirnov test. Two independent-sample t tests were conducted used to compare the parameters differences between the control and sevoflurane groups, including NRG1, ErbB4, PV, GAD67, NR2B and NR2A. Dendrites were analyzed with Kruskal-Wallis test (Sholl analysis) and Steel Dwass post hoc test using JMP software version 16.0 (SAS Institute, Cary, NC, USA) [28]. It was considered that a difference was statistically significant when P < - 0.05. GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) software was used to make drawings.",rats,"['Six adult female Sprague-Dawley (SD) rats, weighing 180–220 g, were raised with free diet and water intake in polypropylene cages for 7 days.']",gestational day 14.5,"['The six female SD rats were raised to G14.5 (middle pregnancy).', 'On pregnancy day 14.5, the rats allocated to sevoflurane exposure were put inside a 30 cm × 20 cm × 120 cm box.']",N,[],sevoflurane,['A mixture of oxygen and sevoflurane (2 L/min with 4% sevoflurane) was delivered through an inlet port connected to a vaporizer.'],none,[],sprague dawley,"['Six adult female Sprague-Dawley (SD) rats, weighing 180–220 g, were raised with free diet and water intake in polypropylene cages for 7 days.']",True,True,True,True,True,True,10.31083/j.jin2003065 + 0.05. GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) software was used to make drawings.",rats,"['Six adult female Sprague-Dawley (SD) rats, weighing 180–220 g, were raised with free diet and water intake in polypropylene cages for 7 days.']",gestational day 14.5,"['The six female SD rats were raised to G14.5 (middle pregnancy).', 'On pregnancy day 14.5, the rats allocated to sevoflurane exposure were put inside a 30 cm × 20 cm × 120 cm box.']",N,[],sevoflurane,['A mixture of oxygen and sevoflurane (2 L/min with 4% sevoflurane) was delivered through an inlet port connected to a vaporizer.'],none,[],sprague dawley,"['Six adult female Sprague-Dawley (SD) rats, weighing 180–220 g, were raised with free diet and water intake in polypropylene cages for 7 days.']",True,True,True,True,True,True,[ Passage 5/25 ] 10.31083/j.jin2003065 10.3892/mmr.2019.10397,1134.0,Guan,2019,rats,postnatal day 7,N,propofol,none,sprague dawley,"PMID: 31257533 PMCID: PMC6625379 DOI: 10.3892/mmr.2019.10397 Materials and methods Rat HPC model All animal procedures were conducted with the approval of the Animal Care and Use Committee of Guangxi Medical University (Nanning, China). Seven-day-old (P7) male Sprague-Dawley pups (average body weight, 10–15 g, n=70) were identified and numbered using picric acid, which were revealed to the investigator only after the completion of experiments and analyses. All pups were housed in a temperature-controlled room (22±1°C) with a 12-h light/dark schedule. H89 (Selleck Chemicals) and Sp-cAMP (Sigma-Aldrich; Merck KGaA) were prepared in 5 µl double-distilled water. The experimental set-up is illustrated in Fig. 2 (n=10) and the following experimental groupings were used: i) Normal saline group (NS group) received intraperitoneal injections of an equal volume of normal saline; ii) propofol group (P group) received intraperitoneal injections of 100 mg/kg propofol; iii) following the propofol treatment as in the P group, the propofol + Sp-cAMP group (P+Sp-cAMP group) received intracerebroventricular injections of 20 nmol/5 µl Sp-cAMP (a cAMP-dependent protein kinase agonist); iv) HPC+P group rats were placed in a chamber containing 8% oxygen and 92% nitrogen for 10 min, and the pups were subsequently exposed to room air for a further 10 min, and following five HPC cycles, the rats received an intraperitoneal injection of 100 mg/kg propofol; v) HPC+P +H89 group was exposed to 5 µmol/5 µl H89 [a protein kinase A (PKA) inhibitor] by intracerebroventricular injections, followed by the same protocol as in the HPC+P group; vi) the remaining pups in the two blank test groups received intracerebroventricular injections of dimethyl sulfoxide (D-ICV group) or normal saline (NS-ICV group). All pups were sacrificed according to standard protocols (100 mg/kg intraperitoneal sodium pentobarbital). Brain tissue slices were prepared for immunohistochemistry and the levels of PKA, CREB, phosopho (p)-CREB, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 were evaluated by western blotting. Morphological and structural changes were evaluated by haematoxylin and eosin (H&E) staining and transmission electron microscopy. @@ -245,7 +245,7 @@ ELISA The intracellular concentrations of adenylyl cyclase in the pups was deter Western blot analysis All pups were sacrificed to harvest the brain tissue. The protein was extracted by RIPA Lysis Buffer (Beijing Solarbio Science & Technology Co., Ltd.) and protein concentration measured using a bicinchoninic acid protein assay (Biotype Biotech Co.). The mass of protein loaded per lane was 20 µl. Equal amounts of proteins were loaded onto 12% SDS-polyacrylamide gels. Electrophoresed proteins were transferred to polyvinylidene difluoride membranes (0.22-µm pore size; EMD Millipore). The membranes were blocked using 5% bovine serum albumin (blocking buffer) for 2 h at room temperature and incubated with the following primary antibodies overnight at 4°C: β-tubulin (1:2,000; cat. no. 48885), caspase-3 (1:1,000; cat. no. 48658) and cleaved caspase-3 (1:1,000; cat. no. 29034; all from Signalway Antibody) Bcl-2 (1:1,000; cat no. ab196495; Abcam), Bax (cat. no. 27727) PKA (cat. no. 5842S), CREB (cat. no. 9197S) and p-CREB (cat. no. 9198S) (all 1:1,000; from Cell Signaling Technology, Inc.), and GAPDH (1:10,000; cat. no. 10494-1-AP; Proteintech, Inc.). The membranes were washed three times with Tris-buffered saline 1% Tween-20 (TBST; pH 7.4) and then incubated in horseradish peroxidase-conjugated secondary antibody (1:10,000; cat. no. 134658; LI-COR Biosciences) for 2 h at room temperature (23–25°C) and washed three times with TBST. The bands were developed using an Odyssey infrared imaging system (LI-COR Biosciences) and evaluated using densitometric analysis (ImageJ 1.52 h, National Institutes of Health). H&E and immunohistochemical staining Morphological and structural changes were observed by H&E staining. Tissues were fixed in 4% ice-cold paraformaldehyde at 4°C for 2 h and paraffin-embedded sections were obtained. The paraffin sections were dewaxed in xylene for 15 min and rehydrated using graded ethanol. The sections were immersed in haematoxylin for 30 sec and then subjected to antigen retrieval using 0.01 mol/l sodium citrate and incubated with 10% normal goat serum at room temperature for 30 min to block nonspecific binding, followed by incubation with the primary antibodies against PKA C and p-CREB (cat. nos. 5842S and 9198S, 1:1,000; Cell Signaling Technology, Inc.) at 4°C overnight. The sections were incubated with streptavidin-horseradish peroxidase at room temperature for 30 min and then stained with 0.05% 3,3-diaminobenzidine substrate, followed by counterstaining with 1% haematoxylin at 37°C for 30 sec. The sections were observed using a microscope (Olympus BX53; Olympus Corporation) and four fields of the hippocampus were randomly selected in every section which represented the areas of interest and the positive cells were counted using Image-Pro Plus version 6.0 software (Media Cybernetics Inc.). Electron microscopy The ultrastructures of neurocytes were observed by transmission electron microscopy (HITACHI H-7650; Hitachi, Ltd.). Briefly, 2.5% glutaraldehyde solution was perfused into the rats, and the tissues were fixed in 1% OsO4 at 4°C for 1 h, dehydrated in increasing concentrations of ethanol and embedded in Epon. Then, the samples were sectioned into semi-thin slices (1 µm) and stained with 1% uranyl acetate and 5% uranyl acetate at 37°C for 20 min. The ultrastructures of the entire mitochondria were measured by manually measuring length using Image Pro Plus (version 6.0.0.260, Media Cybernetics, Inc.). -Statistical analysis Data are presented as the mean ± standard error, and were analysed using SPSS version 17.0 (SPSS, Inc.) and GraphPad Prism 5 software (GraphPad Software Inc.). Multiple comparisons were performed using one-way analysis of variance (ANOVA), followed by Dunnett's post hoc test, as appropriate. P<0.05 was considered to indicate a statistically significant difference.",rats,"['Seven-day-old (P7) male Sprague-Dawley pups (average body weight, 10–15 g, n=70) were identified and numbered using picric acid, which were revealed to the investigator only after the completion of experiments and analyses.']",postnatal day 7,"['Seven-day-old (P7) male Sprague-Dawley pups (average body weight, 10–15 g, n=70) were identified and numbered using picric acid, which were revealed to the investigator only after the completion of experiments and analyses.']",N,"['Brain tissue slices were prepared for immunohistochemistry and the levels of PKA, CREB, phosopho (p)-CREB, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 were evaluated by western blotting.', 'Morphological and structural changes were evaluated by haematoxylin and eosin (H&E) staining and transmission electron microscopy.']",propofol,['ii) propofol group (P group) received intraperitoneal injections of 100 mg/kg propofol;'],none,[],sprague dawley,"['Seven-day-old (P7) male Sprague-Dawley pups (average body weight, 10–15 g, n=70) were identified and numbered using picric acid, which were revealed to the investigator only after the completion of experiments and analyses.']",True,True,True,True,True,True,10.3892/mmr.2019.10397 +Statistical analysis Data are presented as the mean ± standard error, and were analysed using SPSS version 17.0 (SPSS, Inc.) and GraphPad Prism 5 software (GraphPad Software Inc.). Multiple comparisons were performed using one-way analysis of variance (ANOVA), followed by Dunnett's post hoc test, as appropriate. P<0.05 was considered to indicate a statistically significant difference.",rats,"['Seven-day-old (P7) male Sprague-Dawley pups (average body weight, 10–15 g, n=70) were identified and numbered using picric acid, which were revealed to the investigator only after the completion of experiments and analyses.']",postnatal day 7,"['Seven-day-old (P7) male Sprague-Dawley pups (average body weight, 10–15 g, n=70) were identified and numbered using picric acid, which were revealed to the investigator only after the completion of experiments and analyses.']",N,"['Brain tissue slices were prepared for immunohistochemistry and the levels of PKA, CREB, phosopho (p)-CREB, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 were evaluated by western blotting.', 'Morphological and structural changes were evaluated by haematoxylin and eosin (H&E) staining and transmission electron microscopy.']",propofol,['ii) propofol group (P group) received intraperitoneal injections of 100 mg/kg propofol;'],none,[],sprague dawley,"['Seven-day-old (P7) male Sprague-Dawley pups (average body weight, 10–15 g, n=70) were identified and numbered using picric acid, which were revealed to the investigator only after the completion of experiments and analyses.']",True,True,True,True,True,True,[ Passage 6/25 ] 10.3892/mmr.2019.10397 10.3892/mmr.2014.2751,623.0,Han,2015,mice,postnatal day 7,N,sevoflurane,none,c57bl/6,"PMID: 25338822 DOI: 10.3892/mmr.2014.2751 Materials and methods Animals and sevoflurane exposure @@ -258,7 +258,7 @@ Protein extraction and western blot analysis Resected hippocampi were placed into 1.5-ml centrifuge tubes and preserved in liquid nitrogen. All methods were conducted on ice. An NE-PER Nuclear and Cytoplasmic Extraction kit (cat no.78835; Thermo Fisher Scientific, Waltham, MA, USA) was used to extract protein samples. All steps were conducted according to the manufacturer’s instructions. Sodium dodecyl sulphate (SDS) was added to the samples prior to boiling for 10 min at 100°C. Equal quantities of protein (15 μg) were used to detect the expression of the proteins of interest. Samples were electrophoresed on 10 or 15% SDS polyacrylamide gel, blotted onto polyvinylidine fluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA) and then incubated with the following antibodies overnight at 4°C: Anti-phospho-CREB (ser133), (cat no. 06-519, EMD Millipore, Billerica, MA, USA) 1:4,000 dilution in 5% non-fat milk; anti-CREB (cat no. MAB5432, Millipore) 1:5,000 dilution in 5% non-fat milk; anti-BDNF (cat no. AB1779SP, Millipore) 1:1,000 dilution in 5% non-fat milk; anti-MeCP2 (cat no. 3456P, Cell Signaling Technology, Danvers, MA, USA) 1:4,000 dilution in 5% non-fat milk; anti-phospho-MeCP2-S421 (cat no. AP3693a, Abgent Biotech, Suzhou, China) 1:2,000 dilution in 5% non-fat milk); and anti-actin (cat no. A5441, Sigma-Aldrich, St. Louis, MO, USA) 1:10,000 dilution in 5% non-fat milk. The following day, the blots were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary goat anti-rabbit or goat anti-mouse immunoglobulin G (Kangchen, Shanghai, China), 1:5,000 dilution in 5% non-fat milk. Immunoreactive bands were visualized using Amersham ECL Prime Western Blotting Detection kit (cat NO.RPN2232; GE Healthcare, Chalfont St. Giles, UK). The protein signals were quantified using Quantity One software and a GS-800 Calibrated Imaging Densitometer (Bio-Rad Laboratories) and normalized to a corresponding internal reference: CREB for the exression of p-CREB-S133, MeCP2 for P-MeCP2-S421 and actin for BDNF. Statistical analysis -All data are presented as the mean ± standard error. Data were analyzed using the unpaired Student’s t-test in Origin software, version 7.5 (OriginLab, Northampton, MA, USA). P<0.05 was considered to represent a statistically significant difference.",mice,"['At postnatal day 7 (P7), male C57BL/6 mice (weight, 3–5 g; Shanghai Laboratory Animal Center, Shanghai, China) were randomly divided into a sevoflurane-treated group (n=6) and an air-treated control group (n=6) for analysis of the effects of sevoflurane on CREB phosphorylation, BDNF expression and MeCP2 phosphorylation levels.']",postnatal day 7,"['At postnatal day 7 (P7), male C57BL/6 mice (weight, 3–5 g; Shanghai Laboratory Animal Center, Shanghai, China) were randomly divided into a sevoflurane-treated group (n=6) and an air-treated control group (n=6) for analysis of the effects of sevoflurane on CREB phosphorylation, BDNF expression and MeCP2 phosphorylation levels.']",N,"[""The document does not mention any behavior tests such as 'Open field test', 'Morris water task', 'fear conditioning test', 'Dark/light avoidance'; 'passive/active avoidance test'; 'elevated maze', 'Forced swim test', 'Object recognition test', 'Social interaction/preference'.""]",sevoflurane,"['Mice were placed in a plastic container and continuously exposed to 1.5% sevoflurane (Maruishi Pharmaceutical Co., Osaka, Japan) in air, or to air alone for 2 h, with a gas flow of 2 l/min.']",memantine,['Mice received 1 mg/kg saline or memantine intraperitoneally prior to sevoflurane or air treatment.'],c57bl/6,"['At postnatal day 7 (P7), male C57BL/6 mice (weight, 3–5 g; Shanghai Laboratory Animal Center, Shanghai, China) were randomly divided into a sevoflurane-treated group (n=6) and an air-treated control group (n=6) for analysis of the effects of sevoflurane on CREB phosphorylation, BDNF expression and MeCP2 phosphorylation levels.']",True,True,True,True,False,True,10.3892/mmr.2014.2751 +All data are presented as the mean ± standard error. Data were analyzed using the unpaired Student’s t-test in Origin software, version 7.5 (OriginLab, Northampton, MA, USA). P<0.05 was considered to represent a statistically significant difference.",mice,"['At postnatal day 7 (P7), male C57BL/6 mice (weight, 3–5 g; Shanghai Laboratory Animal Center, Shanghai, China) were randomly divided into a sevoflurane-treated group (n=6) and an air-treated control group (n=6) for analysis of the effects of sevoflurane on CREB phosphorylation, BDNF expression and MeCP2 phosphorylation levels.']",postnatal day 7,"['At postnatal day 7 (P7), male C57BL/6 mice (weight, 3–5 g; Shanghai Laboratory Animal Center, Shanghai, China) were randomly divided into a sevoflurane-treated group (n=6) and an air-treated control group (n=6) for analysis of the effects of sevoflurane on CREB phosphorylation, BDNF expression and MeCP2 phosphorylation levels.']",N,"[""The document does not mention any behavior tests such as 'Open field test', 'Morris water task', 'fear conditioning test', 'Dark/light avoidance'; 'passive/active avoidance test'; 'elevated maze', 'Forced swim test', 'Object recognition test', 'Social interaction/preference'.""]",sevoflurane,"['Mice were placed in a plastic container and continuously exposed to 1.5% sevoflurane (Maruishi Pharmaceutical Co., Osaka, Japan) in air, or to air alone for 2 h, with a gas flow of 2 l/min.']",memantine,['Mice received 1 mg/kg saline or memantine intraperitoneally prior to sevoflurane or air treatment.'],c57bl/6,"['At postnatal day 7 (P7), male C57BL/6 mice (weight, 3–5 g; Shanghai Laboratory Animal Center, Shanghai, China) were randomly divided into a sevoflurane-treated group (n=6) and an air-treated control group (n=6) for analysis of the effects of sevoflurane on CREB phosphorylation, BDNF expression and MeCP2 phosphorylation levels.']",True,True,True,True,False,True,[ Passage 7/25 ] 10.3892/mmr.2014.2751 10.1016/j.ntt.2020.106890,520.0,Burks,2020,rats,postnatal day 7,N,sevoflurane,none,sprague dawley,"PMID: 32413489 DOI: 10.1016/j.ntt.2020.106890 2. Methods 2.1. Animals @@ -285,7 +285,7 @@ Thionine staining was performed to verify brain regions. Sections from regions w Imaging of brain tissue was conducted using a Nikon Eclipse Ni microscope equipped with digital cameras (Photometrics, USA; Nikon, USA). FJC, Mki67, NeuN, and thionine labeling were quantified in the somatosensory cortex, motor cortex, CPu, thalamus, CA1 region of the hippocampus, septum and amygdala at 10× magnification using NIS Elements AR automated software (Nikon, USA). Brain regions were defined in accordance with brain atlases for adult and neonatal rats (Paxinos and Watson, 2014; Ramachandra and Subramanian, 2011). 2.8. Stereological analysis -The brain regions in which the highest levels of FJC positive neurons were identified with the aid of adult and neonatal atlases as guides. Given the absence of a complete neonatal atlas, these locations were verified using thionine stained sections from the same regions that the FJC sections were taken to determine neurodegeneration from the pups sacrificed at PND 7, 8 and 10 [see Fig. 1]. Subsequent to identifying these regions, unbiased stereological estimates of positively immunolabeled cells/structures were performed from images that were captured with Photometrics (fluorescent) or Nikon (brightfield) digital cameras using NIS elements AR software for analysis.",rats,"['Pregnant Sprague-Dawley rats (Charles Rivers, USA) arrived on gestational day 5.']",postnatal day 7,"['On PND 7, rats were exposed to vehicle gas alone (75% oxygen/25% nitrogen) or 2.5% Sevo (in vehicle gas) for 6 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'.""]",sevoflurane,"['On PND 7, rats were exposed to vehicle gas alone (75% oxygen/25% nitrogen) or 2.5% Sevo (in vehicle gas) for 6 h.']",none,[],sprague dawley,"['Pregnant Sprague-Dawley rats (Charles Rivers, USA) arrived on gestational day 5.']",True,True,True,True,True,True,10.1016/j.ntt.2020.106890 +The brain regions in which the highest levels of FJC positive neurons were identified with the aid of adult and neonatal atlases as guides. Given the absence of a complete neonatal atlas, these locations were verified using thionine stained sections from the same regions that the FJC sections were taken to determine neurodegeneration from the pups sacrificed at PND 7, 8 and 10 [see Fig. 1]. Subsequent to identifying these regions, unbiased stereological estimates of positively immunolabeled cells/structures were performed from images that were captured with Photometrics (fluorescent) or Nikon (brightfield) digital cameras using NIS elements AR software for analysis.",rats,"['Pregnant Sprague-Dawley rats (Charles Rivers, USA) arrived on gestational day 5.']",postnatal day 7,"['On PND 7, rats were exposed to vehicle gas alone (75% oxygen/25% nitrogen) or 2.5% Sevo (in vehicle gas) for 6 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'.""]",sevoflurane,"['On PND 7, rats were exposed to vehicle gas alone (75% oxygen/25% nitrogen) or 2.5% Sevo (in vehicle gas) for 6 h.']",none,[],sprague dawley,"['Pregnant Sprague-Dawley rats (Charles Rivers, USA) arrived on gestational day 5.']",True,True,True,True,True,True,[ Passage 8/25 ] 10.1016/j.ntt.2020.106890 10.1111/pan.12263,801.0,Hu,2013,rats,postnatal day 7,N,sevoflurane,none,wistar,"PMID: 24102683 DOI: 10.1111/pan.12263 Materials and methods Animals @@ -306,7 +306,7 @@ For the Western blot analysis, samples (80 μg protein) were prepared using neon Tau assay and quantitative real-time PCR Total RNA was isolated from sevoflurane group and control group. Hippocampus neurons using the RNAeasy mini kit (Takara, Dalian, China) according to the manufacture's instruction. First-strand cDNA was synthesized from 5 μg of total RNA using the Super Script III first-strand synthesis kit (Takara) and random hexamer system (Roche, Shanghai, China). Quantification of the target genes was performed with Power SYBR Green PCR master mix kit (ABI, Carlsbad, CA, USA) in Bio-Rad MX3000P real-time PCR system according to the manufacturer's instructions. Triplicate quantitative reverse transcription PCRs were carried out for each sample. The PCR amplification cycles were as follows: initial denaturation at 95°C for 15 min, followed by 40 cycles with denaturation at 95°C for 20 s, and annealing-extension at 60°C for 35s. The specificity of the SYBR Green PCR signal was confirmed by melting curve analysis. Acquired data were analyzed by lightcycle 2000 software 3.5 (Roche). The Ct value of each gene was normalized against that of GAPDH. Tau primer sequences were as follows: tau-sense 5′ACC CCG CCA GGA GTT TGA C-3′, tau-antisense 5′-GAT CTT CGC CCC CGT TTG-3′ 244 bp, GAPDH-sense 5′-CTA CAA TGA GCT GCG TGT GGC-3′, GAPDH-antisense 5′-CAG GTC CAG ACG CAG GAT GGC-3′ 207 bp. -All data are expressed as mean ± sd. spss 12.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Numerical data including Oxygen saturation, PaO2, PaCO2, pH, and the levels of tau phosphorylation at phosphor-Ser396/404 and tau mRNA between groups were analyzed by the Student's t-test, and intragroup numerical data were analyzed by repeated measures anova. Statistical significance was accepted as P < 0.05.",rats,"['Thirty-six neonatal male Wistar rats aged 7 days were purchased from Zhejiang Academy of Medical Science (Hangzhou, China) (SYXK(zhe)2005-0072).']",postnatal day 7,['Thirty-six 7-day-old male rats were allocated by computer-generated random numbers to a 6 h exposure in an anesthesia chamber with either 3% sevoflurane plus 60% oxygen (group S) or air as a normal control (group NC).'],N,"[""The document does not mention any behavior tests such as 'Open field test', 'Morris water task', 'fear conditioning test', 'Dark/light avoidance'; 'passive/active avoidance test'; 'elevated maze', 'Forced swim test', 'Object recognition test', 'Social interaction/preference'.""]",sevoflurane,"['Thirty-six 7-day-old male rats were allocated by computer-generated random numbers to a 6 h exposure in an anesthesia chamber with either 3% sevoflurane (H20100586; Abbott, Chicago, IL, USA) plus 60% oxygen (group S) or air as a normal control (group NC).']",none,['The document only mentions the use of sevoflurane as an intervention.'],wistar,"['Thirty-six neonatal male Wistar rats aged 7 days were purchased from Zhejiang Academy of Medical Science (Hangzhou, China) (SYXK(zhe)2005-0072).']",True,True,True,True,True,True,10.1111/pan.12263 +All data are expressed as mean ± sd. spss 12.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. Numerical data including Oxygen saturation, PaO2, PaCO2, pH, and the levels of tau phosphorylation at phosphor-Ser396/404 and tau mRNA between groups were analyzed by the Student's t-test, and intragroup numerical data were analyzed by repeated measures anova. Statistical significance was accepted as P < 0.05.",rats,"['Thirty-six neonatal male Wistar rats aged 7 days were purchased from Zhejiang Academy of Medical Science (Hangzhou, China) (SYXK(zhe)2005-0072).']",postnatal day 7,['Thirty-six 7-day-old male rats were allocated by computer-generated random numbers to a 6 h exposure in an anesthesia chamber with either 3% sevoflurane plus 60% oxygen (group S) or air as a normal control (group NC).'],N,"[""The document does not mention any behavior tests such as 'Open field test', 'Morris water task', 'fear conditioning test', 'Dark/light avoidance'; 'passive/active avoidance test'; 'elevated maze', 'Forced swim test', 'Object recognition test', 'Social interaction/preference'.""]",sevoflurane,"['Thirty-six 7-day-old male rats were allocated by computer-generated random numbers to a 6 h exposure in an anesthesia chamber with either 3% sevoflurane (H20100586; Abbott, Chicago, IL, USA) plus 60% oxygen (group S) or air as a normal control (group NC).']",none,['The document only mentions the use of sevoflurane as an intervention.'],wistar,"['Thirty-six neonatal male Wistar rats aged 7 days were purchased from Zhejiang Academy of Medical Science (Hangzhou, China) (SYXK(zhe)2005-0072).']",True,True,True,True,True,True,[ Passage 9/25 ] 10.1111/pan.12263 10.4196/kjpp.2017.21.6.579,827.0,Jiang,2017,rats,postnatal day 7,Y,isoflurane,none,sprague dawley,"PMID: 29200900 PMCID: PMC5709474 DOI: 10.4196/kjpp.2017.21.6.579 METHODS Chemicals and reagents @@ -335,7 +335,7 @@ For the place trials, the cloth surrounding the pool and the cue rod attached to Probe trials were conducted to assess memory retention. The test was performed 24 h after the place trials. The platform was placed in a different quadrant than where the submerged platform had been during the cued and place trials (target quadrant). The time spent by the rats in the target quadrant searching for the submerged platform was recorded. Statistics -The results are presented as means±standard deviation (SD) of six independent experiments. Data were analyzed for statistical significance by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test as a post hoc analysis using SPSS (version 22.0; SPSS, Chicago, IL, USA). In all analyses, p<0.05 was taken to indicate statistical significance.",rats,['Pregnant Sprague–Dawley rats were housed in individual sterile plastic cages under standard animal house conditions.'],postnatal day 7,"['On P7, the pups were exposed to 0.75% isoflurane (6 h) in 30% oxygen or air.']",Y,"['Behavioral analysis: open field test P35 rats exposed to anesthesia on P7 were subjected to open field tests to evaluate their anxiety behavior and general locomotory activity.', 'Fear conditioning test Hippocampal-dependent and -independent responses were assessed by a fear conditioning test performed as described previously.', 'Learning and memory analysis: Morris water maze test The Morris water maze test was used to assess learning ability and memory retention.']",isoflurane,"['On P7, the pups were exposed to 0.75% isoflurane (6 h) in 30% oxygen or air.']",none,[],sprague dawley,['Pregnant Sprague–Dawley rats were housed in individual sterile plastic cages under standard animal house conditions.'],True,True,True,True,True,True,10.4196/kjpp.2017.21.6.579 +The results are presented as means±standard deviation (SD) of six independent experiments. Data were analyzed for statistical significance by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test as a post hoc analysis using SPSS (version 22.0; SPSS, Chicago, IL, USA). In all analyses, p<0.05 was taken to indicate statistical significance.",rats,['Pregnant Sprague–Dawley rats were housed in individual sterile plastic cages under standard animal house conditions.'],postnatal day 7,"['On P7, the pups were exposed to 0.75% isoflurane (6 h) in 30% oxygen or air.']",Y,"['Behavioral analysis: open field test P35 rats exposed to anesthesia on P7 were subjected to open field tests to evaluate their anxiety behavior and general locomotory activity.', 'Fear conditioning test Hippocampal-dependent and -independent responses were assessed by a fear conditioning test performed as described previously.', 'Learning and memory analysis: Morris water maze test The Morris water maze test was used to assess learning ability and memory retention.']",isoflurane,"['On P7, the pups were exposed to 0.75% isoflurane (6 h) in 30% oxygen or air.']",none,[],sprague dawley,['Pregnant Sprague–Dawley rats were housed in individual sterile plastic cages under standard animal house conditions.'],True,True,True,True,True,True,[ Passage 10/25 ] 10.4196/kjpp.2017.21.6.579 10.3389/fncel.2020.00004,415.0,Ju,2020,mice,postnatal day 16,N,sevoflurane,none,c57bl/6,"PMID: 32047423 PMCID: PMC6997293 DOI: 10.3389/fncel.2020.00004 Materials and Methods Animals @@ -357,7 +357,7 @@ Electrophysiology Whole-cell voltage-clamp recordings of pyramidal neurons in the CA1 region of the hippocampus were obtained as described (Chung et al., 2015a). Twenty-four hours after exposure to sevoflurane or fresh gas, sagittal slices of the hippocampus (300 μm) were prepared in ice-cold dissection buffer (212 mM sucrose, 25 mM NaHCO3, 5 mM KCl, 1.25 mM NaH2PO4, 10 mM d-glucose, 2 mM sodium pyruvate, 1.2 mM sodium ascorbate, 3.5 mM MgCl2, and 0.5 mM CaCl2) aerated with 95% O2/5% CO2, using a VT1200S vibratome (Leica, Arrau, Switzerland). Slices were transferred immediately to a 32°C chamber containing artificial cerebrospinal fluid (aCSF: 125 mM NaCl, 25 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 10 mM d-glucose, 1.3 mM MgCl2, and 2.5 mM CaCl2, continuously aerated with 95% O2/5% CO2) and incubated for 30 min. Glass capillaries were filled with two kinds of internal solutions. For miniature excitatory postsynaptic current (mEPSC) recordings, the glass capillaries were filled with an internal solution containing 117 mM CsMeSO4, 10 mM tetraethylammonium chloride, 8 mM NaCl, 10 mM HEPES, 5 mM QX-314-Cl, 4 mM Mg-adenosine triphosphate (ATP), 0.3 mM Na-guanosine triphosphate, and 10 mM EGTA; for miniature inhibitory postsynaptic current (mIPSC) recordings, the glass capillaries were filled with an internal solution containing 115 mM CsCl, 10 mM tetraethylammonium chloride, 8 mM NaCl, 10 mM HEPES, 5 mM QX-314-Cl, 4 mM Mg-ATP, 0.3 mM Na-guanosine triphosphate, and 10 mM EGTA. Whole-cell recordings were performed under visual control (BX50WI; Olympus, Tokyo, Japan) with a multi clamp 700A amplifier (Molecular Devices, San Jose, CA, USA). Data were acquired with Clampex 9.2 (Molecular Devices, San Jose, CA, USA) and analyzed using Clampfit 9 software (Molecular Devices, San Jose, CA, USA). Statistical Analysis -The sample size was determined based on previous experience or as previously described (Chung et al., 2015b, 2017b). All statistical analyses were performed using R statistical software (3.1.2: R Core Team, Austria). All continuous variables were tested to determine whether they met conditions of normality and homogeneity of variance. One-way ANOVA with post hoc Tukey HSD test was performed when both conditions were met, Welch’s ANOVA with post hoc Tukey HSD test was performed when homogeneity of variance was unmet, and the Kruskal–Wallis test with post hoc Dunn’s test was performed if normality was unmet. P < 0.05 was considered statistically significant. Statistical results are presented as Supplementary Statistics.",mice,"['C57BL/6J mice were maintained in a specific pathogen-free (SPF) room maintained at 22°C, with a 12 h light/dark cycle, and fed ad libitum.']",postnatal day 16/17,"['PND 16/17 mice were randomly divided into three groups: control, sevoflurane, and sevoflurane plus rapamycin groups.']",N,"[""The document does not mention any behavior tests such as 'Open field test', 'Morris water task', 'fear conditioning test', 'Dark/light avoidance'; 'passive/active avoidance test'; 'elevated maze', 'Forced swim test', 'Object recognition test', 'Social interaction/preference'.""]",sevoflurane,"['Mice in the sevoflurane and sevoflurane plus rapamycin groups were placed in a 1-l plastic chamber and exposed to a constant flow of fresh gas [fraction of inspired oxygen (FiO2) 0.4, 4 L/min] containing 2.5% sevoflurane for 2 h.']",none,[],c57bl/6,"['C57BL/6J mice were maintained in a specific pathogen-free (SPF) room maintained at 22°C, with a 12 h light/dark cycle, and fed ad libitum.']",True,False,True,True,True,True,10.3389/fncel.2020.00004 +The sample size was determined based on previous experience or as previously described (Chung et al., 2015b, 2017b). All statistical analyses were performed using R statistical software (3.1.2: R Core Team, Austria). All continuous variables were tested to determine whether they met conditions of normality and homogeneity of variance. One-way ANOVA with post hoc Tukey HSD test was performed when both conditions were met, Welch’s ANOVA with post hoc Tukey HSD test was performed when homogeneity of variance was unmet, and the Kruskal–Wallis test with post hoc Dunn’s test was performed if normality was unmet. P < 0.05 was considered statistically significant. Statistical results are presented as Supplementary Statistics.",mice,"['C57BL/6J mice were maintained in a specific pathogen-free (SPF) room maintained at 22°C, with a 12 h light/dark cycle, and fed ad libitum.']",postnatal day 16/17,"['PND 16/17 mice were randomly divided into three groups: control, sevoflurane, and sevoflurane plus rapamycin groups.']",N,"[""The document does not mention any behavior tests such as 'Open field test', 'Morris water task', 'fear conditioning test', 'Dark/light avoidance'; 'passive/active avoidance test'; 'elevated maze', 'Forced swim test', 'Object recognition test', 'Social interaction/preference'.""]",sevoflurane,"['Mice in the sevoflurane and sevoflurane plus rapamycin groups were placed in a 1-l plastic chamber and exposed to a constant flow of fresh gas [fraction of inspired oxygen (FiO2) 0.4, 4 L/min] containing 2.5% sevoflurane for 2 h.']",none,[],c57bl/6,"['C57BL/6J mice were maintained in a specific pathogen-free (SPF) room maintained at 22°C, with a 12 h light/dark cycle, and fed ad libitum.']",True,False,True,True,True,True,[ Passage 11/25 ] 10.3389/fncel.2020.00004 10.1213/ANE.0b013e3182a8c709,815.0,Kato,2013,rats,postnatal day 7,N,sevoflurane,none,wistar/st,"PMID: 24132013 DOI: 10.1213/ANE.0b013e3182a8c709 METHODS Animals @@ -372,7 +372,7 @@ To assess respiratory disturbances caused by sevoflurane exposure, arterial bloo Electrophysiological Study Between postnatal days 63 and 70, an electrophysiological study was performed following our reported method.21 Rats were anesthetized with 1% halothane in a mixture of 21% O2 and 79% nitrogen through a tracheal catheter, and their lungs mechanically ventilated (SN-480–7, Shinano, Tokyo, Japan). They were placed in a stereotaxic apparatus with the bregma and lambda in the same horizontal plane, and their body temperatures were maintained at 37°C ± 0.5°C with a heating pad throughout the recording period. The concentration of halothane and expired CO2 tension was continuously monitored through a tracheal catheter using an anesthetic gas monitor (5250RGM, Datex-Ohmeda GE Healthcare), and the expired CO2 tension was maintained between 35 and 45 mm Hg. A monopolar recording electrode was inserted into the pyramidal cell body layer of the hippocampal CA1 region (5.0 mm posterior and 3.0 mm lateral to the bregma and approximately 2.2 mm ventral to the dura), to record extracellular population spike amplitude (PSA) (Fig. 1A). A bipolar stimulating electrode was inserted into the ipsilateral Schaffer collaterals (3.0 mm posterior and 1.5 mm lateral to the bregma, and 2.8 mm ventral to the dura) to deliver cathodal stimulus (frequency 0.1 Hz, pulse duration 250 μs) (Fig. 1A). A single electrical stimulus evoked action potentials in the Schaffer collaterals, resulting in activation of pyramidal cells of the CA1 region, and extracellular PSA was recorded (MacLab, ADInstruments, Sydney, Australia). We measured the PSA following our previous method.21 Briefly, the PSA was defined as the absolute voltage of a vertical line running from the population spike minimum to its intersection with a line tangential to the population spike onset and population spike offset. To adjust the test stimulus, changes in PSA caused by varied stimulus intensity were recorded, and the intensity of the test stimulus was then fixed to produce a half-maximal response for each rat. After establishing a stable baseline for 30 minutes, LTP was induced by applying high-frequency stimulation (HFS; 10 trains at 1 Hz each, composed of 8 pulses at 400 Hz), at the same intensity as the test stimulation. Then relative ratios of PSAs before and after the HFS were then plotted every 5 minutes for 60 minutes after HFS. On completion of the experiment, small lesions were made using a direct electric current (5 µA for 30 seconds) at the tips of the recording and stimulating electrodes. The positions of the electrodes were examined histologically (Fig. 1) according to methods in similar previous reports.22,23 Histological confirmation of the locations of the electrodes showed misplacement of the electrodes in 2 rats in each group, so the data obtained from those electrodes were excluded from the following assessment. All data were collected by investigators who were blinded to treatment assignment. Statistical Methods -All statistical analyses were performed using GraphPad Prism version 5.04 (GraphPad Software, Inc., San Diego, CA). First, we confirmed normal distribution of the obtained data by the Shapiro-Wilk test and a normal-probability plot. Data obtained in arterial blood gas analysis were analyzed using 1-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test for each time point. In the comparisons of LTP data, we used 2-way repeated-measures ANOVA with treatment and time as factors to assess the interaction, and we used 1-way ANOVA followed by Bonferroni multiple comparison test to evaluate differences among the groups at each time point. All averaged data are presented as mean ± SEM. Probability values (P) of <5% were considered significant.",rats,"['Pregnant Wistar/ST rats were obtained from Shizuoka Laboratory Animal Center (Hamamatsu, Japan).']",postnatal day 7,"['At postnatal day 7, male pups were divided into 4 treatment groups as follows:']",N,"['After gas exposure, rat pups were brought up in an austere environment in standardized cages to avoid the influence of environmental enrichment on later neurocognitive functions.']",sevoflurane,"['animals exposed to (1) 1% sevoflurane with oxygen (O2), (2) 2% sevoflurane with O2']",none,[],wistar/st,"['Pregnant Wistar/ST rats were obtained from Shizuoka Laboratory Animal Center (Hamamatsu, Japan).']",True,True,True,True,True,True,10.1213/ANE.0b013e3182a8c709 +All statistical analyses were performed using GraphPad Prism version 5.04 (GraphPad Software, Inc., San Diego, CA). First, we confirmed normal distribution of the obtained data by the Shapiro-Wilk test and a normal-probability plot. Data obtained in arterial blood gas analysis were analyzed using 1-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test for each time point. In the comparisons of LTP data, we used 2-way repeated-measures ANOVA with treatment and time as factors to assess the interaction, and we used 1-way ANOVA followed by Bonferroni multiple comparison test to evaluate differences among the groups at each time point. All averaged data are presented as mean ± SEM. Probability values (P) of <5% were considered significant.",rats,"['Pregnant Wistar/ST rats were obtained from Shizuoka Laboratory Animal Center (Hamamatsu, Japan).']",postnatal day 7,"['At postnatal day 7, male pups were divided into 4 treatment groups as follows:']",N,"['After gas exposure, rat pups were brought up in an austere environment in standardized cages to avoid the influence of environmental enrichment on later neurocognitive functions.']",sevoflurane,"['animals exposed to (1) 1% sevoflurane with oxygen (O2), (2) 2% sevoflurane with O2']",none,[],wistar/st,"['Pregnant Wistar/ST rats were obtained from Shizuoka Laboratory Animal Center (Hamamatsu, Japan).']",True,True,True,True,True,True,[ Passage 12/25 ] 10.1213/ANE.0b013e3182a8c709 10.1016/j.ejphar.2011.08.050,307.0,Kong,2011,rats,gestational day 14,Y,isoflurane,none,none,"PMID: 21930122 DOI: 10.1016/j.ejphar.2011.08.050 2. Materials and methods 2.1. Animals @@ -403,7 +403,7 @@ The sections mentioned above were washed in 0.01 M PBS containing 0.3% Triton X- After Morris Water Maze test, two pups from each pregnant mother were anesthetized with a lethal dose of Nembutal. Then their thoracic cavities were opened and perfused intracardially with 100 mL of normal saline. Hippocampus, including CA1 and dentate gyrus field, of each rat was taken out immediately to obtain fresh tissue specimens. Protein concentration was determined by the BCA method using bovine serum albumin as the standard. Protein samples (50 μg) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membranes were blocked by nonfat dry milk buffer for 2 h and then incubated overnight at 4 °C with primary antibody against CHOP or caspase-12 (1:500, Santa Cruz Biotechnology, USA). The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies and developed with ECL kit. The optical densities of bands were quantitatively analyzed using Bio-Rad Quantity One 4.6.2 (Bio-Rad Laboratories, USA). The results were expressed as a relative density. Equal protein loading in each lane was confirmed by hybridization with a 1:2000 dilution of β-actin antibody (Santa Cruz Biotechnology, USA). 2.8. Statistical analysis -All data were presented as mean ± S.E.M. Results of weight of postnatal rat pups and place trials of postnatal rats were analyzed using 2-way ANOVA for repeated measurements. Other data were analyzed using Student's t-test for comparison of two groups. A P value of < 0.05 was considered statistically significant. All statistical tests and graphs were performed or generated, respectively, using GraphPad Prism Version 4.0 (GraphPad Prism Software, Inc. CA, USA).",rats,['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).'],gestational day 14,"['The dams at gestational day 14 were used for all experiments, because this time corresponds approximately to midgestation in humans (Clancy et al., 2001, Clancy et al., 2007), the period when most nonobstetric surgeries and fetal interventions are performed (Goodman, 2002, Tran, 2010).']",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 (Jevtovic-Todorovic et al., 2003).']",isoflurane,"['In these boxes, the dams were either exposed to 1.3% isoflurane (Lot 826005U, ABBOTT, USA) in a humidified 30% oxygen carrier gas or simply humidified 30% oxygen without any inhalational anesthetic for 4 h.']",none,[],not specified,[],True,True,True,True,True,False,10.1016/j.ejphar.2011.08.050 +All data were presented as mean ± S.E.M. Results of weight of postnatal rat pups and place trials of postnatal rats were analyzed using 2-way ANOVA for repeated measurements. Other data were analyzed using Student's t-test for comparison of two groups. A P value of < 0.05 was considered statistically significant. All statistical tests and graphs were performed or generated, respectively, using GraphPad Prism Version 4.0 (GraphPad Prism Software, Inc. CA, USA).",rats,['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).'],gestational day 14,"['The dams at gestational day 14 were used for all experiments, because this time corresponds approximately to midgestation in humans (Clancy et al., 2001, Clancy et al., 2007), the period when most nonobstetric surgeries and fetal interventions are performed (Goodman, 2002, Tran, 2010).']",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 (Jevtovic-Todorovic et al., 2003).']",isoflurane,"['In these boxes, the dams were either exposed to 1.3% isoflurane (Lot 826005U, ABBOTT, USA) in a humidified 30% oxygen carrier gas or simply humidified 30% oxygen without any inhalational anesthetic for 4 h.']",none,[],not specified,[],True,True,True,True,True,False,[ Passage 13/25 ] 10.1016/j.ejphar.2011.08.050 10.1371/journal.pone.0105340,269.0,Lee,2014,rats,postnatal day 7,Y,isoflurane,desflurane,sprague dawley,"PMID: 25165850 PMCID: PMC4148240 DOI: 10.1371/journal.pone.0105340 Methods Subjects @@ -436,7 +436,7 @@ Total FluoroJade-positive cells for each brain region were compared among the gr Recognition tasks were first assessed by comparing the investigation times of each target using paired tests for each group. Paired t-test was used for normally distributed data, and nonparametric data were analyzed with the Wilcoxon matched-pairs rank test. Also, to identify possible confounding effects of varying investigation times on subsequent object/animal recognition, the times during the exposure phase were compared between the groups using either one-way ANOVA with Bonferroni’s post-test or the Kruskal-Wallis test with Dunn’s post-test. -In addition, a “discrimination index” (DI) was calculated and represents the relative time spent exploring each target (eg. Familiar versus Novel). To calculate DI, the time spent investigating the familiar target was subtracted from the time spent on the novel target, and this was divided by the total time spent investigating the two (eg. DI = (Novel-Familiar)/(Total Time)). This value was compared to a theoretical value of zero using one sample t-test to assess whether a preference was shown for one of the objects, and a positive DI indicates preference for the novel aspect of the task. For each task, DI of control animals was compared against DI of all anesthetized animals. Also, within the group of anesthetized animals, the DI of desflurane-treated subjects was compared with that of isoflurane-treated subjects. These comparisons were made using either unpaired t-test for parametric data or the Mann Whitney test for nonparametric data.",rats,"['Five Sprague-Dawley dams with litters of postnatal day 6 (P6) pups from were obtained from Charles River Laboratories (Gilroy, CA).']",postnatal day 7,"['On P7, animals from each litter were randomly assigned to control and treatment groups.']",Y,"['Object recognition was assessed using similar arrangements as others [19], [28].', 'Social interaction and recognition were assessed using a discrimination paradigm one week after completing object recognition testing at P80.']",isoflurane,['animals in the treatment groups received either isoflurane or desflurane as a single agent in air and oxygen (FiO250%) at 1 Minimum Alveolar Concentration [27] for four hours.'],desflurane,['animals in the treatment groups received either isoflurane or desflurane as a single agent in air and oxygen (FiO250%) at 1 Minimum Alveolar Concentration [27] for four hours.'],sprague dawley,"['Five Sprague-Dawley dams with litters of postnatal day 6 (P6) pups from were obtained from Charles River Laboratories (Gilroy, CA).']",True,True,True,True,True,True,10.1371/journal.pone.0105340 +In addition, a “discrimination index” (DI) was calculated and represents the relative time spent exploring each target (eg. Familiar versus Novel). To calculate DI, the time spent investigating the familiar target was subtracted from the time spent on the novel target, and this was divided by the total time spent investigating the two (eg. DI = (Novel-Familiar)/(Total Time)). This value was compared to a theoretical value of zero using one sample t-test to assess whether a preference was shown for one of the objects, and a positive DI indicates preference for the novel aspect of the task. For each task, DI of control animals was compared against DI of all anesthetized animals. Also, within the group of anesthetized animals, the DI of desflurane-treated subjects was compared with that of isoflurane-treated subjects. These comparisons were made using either unpaired t-test for parametric data or the Mann Whitney test for nonparametric data.",rats,"['Five Sprague-Dawley dams with litters of postnatal day 6 (P6) pups from were obtained from Charles River Laboratories (Gilroy, CA).']",postnatal day 7,"['On P7, animals from each litter were randomly assigned to control and treatment groups.']",Y,"['Object recognition was assessed using similar arrangements as others [19], [28].', 'Social interaction and recognition were assessed using a discrimination paradigm one week after completing object recognition testing at P80.']",isoflurane,['animals in the treatment groups received either isoflurane or desflurane as a single agent in air and oxygen (FiO250%) at 1 Minimum Alveolar Concentration [27] for four hours.'],desflurane,['animals in the treatment groups received either isoflurane or desflurane as a single agent in air and oxygen (FiO250%) at 1 Minimum Alveolar Concentration [27] for four hours.'],sprague dawley,"['Five Sprague-Dawley dams with litters of postnatal day 6 (P6) pups from were obtained from Charles River Laboratories (Gilroy, CA).']",True,True,True,True,True,True,[ Passage 14/25 ] 10.1371/journal.pone.0105340 10.1002/brb3.514,299.0,Lin,2016,mice,postnatal day 7,Y,sevoflurane,none,c57bl/6,"PMID: 27688943 PMCID: PMC5036436 DOI: 10.1002/brb3.514 Materials and Methods Treatment with sevoflurane @@ -456,7 +456,7 @@ Three‐chamber interaction A three‐chamber apparatus made of clear plexiglass Communication Olfaction habituation/dishabituation The mouse was transferred to a new cage containing a thin layer of fresh bedding and a hole for inserting a cotton tipped swab. After a 10‐min habituation period in the new cage, the mouse was presented with nonsocial and social odors. Each odor was presented for three consecutive times; the order of presentation was water, almond extract (1:100, Spice Supreme), orange extract (1:100, McCormick), mouse socials 1 and 2. The mouse social odors were taken by wiping in a zigzag pattern across the bottom surface of different cages for odors 1 and 2; each cage housed unfamiliar mice of the same sex and strain. Each presentation of odor lasted 2 min. The amount of time that the mouse spent sniffing the cotton swab, including nose poking, chewing, sniffing, and close proximity (2 cm) of the nose to cotton swab was scored (Silverman et al. 2010). Statistical analysis -All statistical analysis was done using GraphPad Prism 5.0 (GraphPad, San Diego, CA). Data with one variable such as the open field and the reciprocal social interaction were analyzed by t test. Data with two variables such as the APA, the NOR, the three‐chamber interaction, and the olfaction habituation/dishabituation were analyzed by two‐way ANOVA, followed by Bonferroni posttests.",mice,"['C57/BL6 mice were used throughout the study, which was approved by the SUNY Downstate IACUC.']",postnatal day 7,"['A separate set of 11 litters of mice were used for treatment without tail clamp and mice from this group were used for behavioral tests later on in life. At P7, all male pups from each litter (ranging from 2 to 6 pups) were randomly assigned to either the sevo or the no sevo (control) group, while the female pups remained with the dam.']",Y,"['The behavior tests were given sequentially for the active place avoidance (APA), reciprocal social interaction, and olfaction habituation/dishabituation.', 'After completion of these tests, we then introduced three‐chamber interaction, open field, and novel object recognition (NOR).']",sevoflurane,"['Treatment with sevoflurane', 'During a 2‐h treatment period, pups from the sevo group were separated from the dam and exposed to sevo in a 40% oxygen (O2) and 60% nitrogen (N2) gas mixture.']",none,[],c57bl/6,"['C57/BL6 mice were used throughout the study, which was approved by the SUNY Downstate IACUC.']",True,True,True,True,True,True,10.1002/brb3.514 +All statistical analysis was done using GraphPad Prism 5.0 (GraphPad, San Diego, CA). Data with one variable such as the open field and the reciprocal social interaction were analyzed by t test. Data with two variables such as the APA, the NOR, the three‐chamber interaction, and the olfaction habituation/dishabituation were analyzed by two‐way ANOVA, followed by Bonferroni posttests.",mice,"['C57/BL6 mice were used throughout the study, which was approved by the SUNY Downstate IACUC.']",postnatal day 7,"['A separate set of 11 litters of mice were used for treatment without tail clamp and mice from this group were used for behavioral tests later on in life. At P7, all male pups from each litter (ranging from 2 to 6 pups) were randomly assigned to either the sevo or the no sevo (control) group, while the female pups remained with the dam.']",Y,"['The behavior tests were given sequentially for the active place avoidance (APA), reciprocal social interaction, and olfaction habituation/dishabituation.', 'After completion of these tests, we then introduced three‐chamber interaction, open field, and novel object recognition (NOR).']",sevoflurane,"['Treatment with sevoflurane', 'During a 2‐h treatment period, pups from the sevo group were separated from the dam and exposed to sevo in a 40% oxygen (O2) and 60% nitrogen (N2) gas mixture.']",none,[],c57bl/6,"['C57/BL6 mice were used throughout the study, which was approved by the SUNY Downstate IACUC.']",True,True,True,True,True,True,[ Passage 15/25 ] 10.1002/brb3.514 10.1097/ALN.0000000000002904,263.0,Li,2019,mice,postnatal day 7,Y,isoflurane,none,c57bl/6,"PMID: 31436548 PMCID: PMC6800770 DOI: 10.1097/ALN.0000000000002904 Materials and Methods Animal paradigm and experimental timeline. @@ -496,14 +496,14 @@ Electron microscopy. Two animals from each group were perfused with 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) plus 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in PBS at postnatal day 63 (after behavior tests) and post-fixed at 4°C for 1 week. Brains containing fimbria were dissected into small blocks (2mm × 2mm × 2mm). The blocks were placed into 1% OsO4 (Electron Microscopy Sciences, Hatfield, PA, USA) for 1 hour, stained in 0.5% uranyl acetate (Electron Microscopy Sciences, Hatfield, PA, USA) overnight, and dehydrated in a series of alcohols followed by propylene oxide for 3 hours. After being infiltrated with a 1:1 mixture of propylene oxide and EMBed-812 embedding resin (Electron Microscopy Sciences, Hatfield, PA, USA) for 3 hours, the blocks were embedded with the same resin in the plastic templates at 60ºC overnight.21 Parasagittal semi-thin sections (1 μm) were cut and stained with 1% Toluidine blue for preliminary light microscopy observation. Then, 90 nm ultrathin sections were cut, picked up on Forvar-coated slotted grids, and stained with 0.5% uranyl acetate and 0.5% lead citrate (Electron Microscopy Sciences, Hatfield, PA, USA). Thin sections were observed and imaged with a Hitachi 7600 transmission electron microscope (Chiyoda, Tokyo, Japan). For each case, 10 photos were randomly photographed at 20,000×. The thickness of myelin was quantitatively measured by determining g-ratio, which was calculated by dividing the diameter of the axon by the diameter of the entire myelinated fiber as previously described. ImageJ (NIH, Bethesda, MD, USA) was used by first opening ultrastructural images. The scale was set according to the scale bar in the images by selecting “Analyze>Set Scale”. The “straight line tool” was selected to measure axonal caliber and diameter of myelinated axons. One hundred axons per group (two animals, fifty from each) were randomly selected and quantitatively analyzed (n=100).16 Statistical analysis. -The statistics were performed with GraphPad Prism 6 (La Jolla, CA, USA) program. The sample size was based on our previous experience with this design. No a priori statistical power calculation was conducted. Normal distribution was verified using the D’Agostino Pearson test. Data for immunohistochemistry, Western blotting, and electron microscopy were analyzed using one-way analysis of variance (ANOVA). The factor of variable was comparisons among groups (control vs. isoflurane plus vehicle vs. isoflurane plus rapamycin). The behavior tests were analyzed with two-way ANOVA. For this analysis, the second factor was animal’s choice between old vs. novel positions (or arms) and only the values for this variable in each individual group were compared. The Tukey post hoc test was employed for intergroup comparisons. The two-tailed test was set according to convention. The criteria for significant difference was set a priori at p<0.05. In this study, all results were expressed as mean ± standard deviation (SD). The sample size “n” represents the number of animals for each group. Only exception is g-ratio analysis with electron microscopy in which “n” indicates the number of randomly selected axons from two mice per group (n=100). This analysis way is extensively applied for g-ratio study.16 Because all animals survived tests, there were no missing data in this study. No exclusions for outliers were made in this study. In some experiments, the sample size was increased in response to peer review.",mice,['A total of 120 (61 male and 59 female) immature C57BL/6 mice (body weight = 4.4±0.9 g. at postnatal day 7) were used in this study.'],postnatal day 7,"['At postnatal day 7, animals were exposed to isoflurane or room air for 4 hours.']",Y,['The novel object position recognition test and Y-maze test were performed at the last week of the survival period (postnatal days 56-62).'],isoflurane,"['At postnatal day 7, two-thirds of the mice were evenly distributed across littermate groups and were randomly selected for isoflurane exposure.']",none,[],c57bl/6,['A total of 120 (61 male and 59 female) immature C57BL/6 mice (body weight = 4.4±0.9 g. at postnatal day 7) were used in this study.'],True,True,True,True,True,True,10.1097/ALN.0000000000002904 +The statistics were performed with GraphPad Prism 6 (La Jolla, CA, USA) program. The sample size was based on our previous experience with this design. No a priori statistical power calculation was conducted. Normal distribution was verified using the D’Agostino Pearson test. Data for immunohistochemistry, Western blotting, and electron microscopy were analyzed using one-way analysis of variance (ANOVA). The factor of variable was comparisons among groups (control vs. isoflurane plus vehicle vs. isoflurane plus rapamycin). The behavior tests were analyzed with two-way ANOVA. For this analysis, the second factor was animal’s choice between old vs. novel positions (or arms) and only the values for this variable in each individual group were compared. The Tukey post hoc test was employed for intergroup comparisons. The two-tailed test was set according to convention. The criteria for significant difference was set a priori at p<0.05. In this study, all results were expressed as mean ± standard deviation (SD). The sample size “n” represents the number of animals for each group. Only exception is g-ratio analysis with electron microscopy in which “n” indicates the number of randomly selected axons from two mice per group (n=100). This analysis way is extensively applied for g-ratio study.16 Because all animals survived tests, there were no missing data in this study. No exclusions for outliers were made in this study. In some experiments, the sample size was increased in response to peer review.",mice,['A total of 120 (61 male and 59 female) immature C57BL/6 mice (body weight = 4.4±0.9 g. at postnatal day 7) were used in this study.'],postnatal day 7,"['At postnatal day 7, animals were exposed to isoflurane or room air for 4 hours.']",Y,['The novel object position recognition test and Y-maze test were performed at the last week of the survival period (postnatal days 56-62).'],isoflurane,"['At postnatal day 7, two-thirds of the mice were evenly distributed across littermate groups and were randomly selected for isoflurane exposure.']",none,[],c57bl/6,['A total of 120 (61 male and 59 female) immature C57BL/6 mice (body weight = 4.4±0.9 g. at postnatal day 7) were used in this study.'],True,True,True,True,True,True,[ Passage 16/25 ] 10.1097/ALN.0000000000002904 10.3892/etm.2017.5651,1395.0,Liu,2018,mice,postnatal day 7,Y,sevoflurane,none,c57bl/6,"PMID: 29434807 PMCID: PMC5776508 DOI: 10.3892/etm.2017.5651 Materials and methods Animals The current study was approved by the Animal Care Committee at Sun Yat-sen University (Guangzhou, China) and performed in accordance with the National Institutes of Health Guide for the Use of Laboratory Animals (27). A total of 24 C57BL/6 male mouse pups, aged 7 days (P7) and weighing 3.5–4.5 g were obtained from Guangdong Medical Laboratory Animal Center (Guangdong, China; permission no. SCXK2011-0029). The pups were housed in the same cage as their mothers and were kept under temperature-controlled environmental conditions (26°C) on a 14:10 constant light-dark cycle until P7. The mother mice had free access to food and water. The mouse pups at P7 were exposed to 2.6% sevoflurane (Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) for 6 h [~1.0 minimal alveolar concentration (MAC) in P7 mice] in 50% oxygen in a temperature-controlled chamber, following a previously described protocol (n=12) (17). The control mice were exposed to normal air for 6 h under the same condition (n=12). The concentrations of anesthetic gas, oxygen and carbon dioxide in the chamber were measured using a gas analyzer (Datex-Ohmeda; GE Healthcare, Chicago, IL, USA). All animals were sacrificed 2 h following termination of sevoflurane/oxygen exposure and their cortices were used for western blotting (sevoflurane group, n=6; control group, n=6) or TdT-mediated dUTP nick end labeling (TUNEL) with fluorescent dye (sevoflurane group, n=6; control group, n=6). Tissue preparation Half of the mice in each group were used for western blotting and half of the mice for TUNEL studies. For western blotting, mouse pups were anaesthetized by inhaling 3% of sevoflurane until loss of the righting reflex (LORR), which indicated the mice had lost consciousness. Then the mice were sacrificed by decapitation. Cortices were isolated immediately on ice and then stored at −80°C until use. For TUNEL studies, mouse pups were sacrificed by inhaling 3% of sevoflurane until LORR and perfused transcardially with ice-cold normal saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min at 4°C. Their brains were post-fixed in the same fixative for 48 h at 4°C, and then paraffin embedded and sectioned into 6-µm-thick sections. As described in previous studies (15,16,25), at least three sections in the same plane of the hippocampus for each animal were selected to detect cells that exhibited positive TUNEL staining; all sections used in TUNEL were 100 µm apart and the sections were according to Figures 129–131 in the Atlas of the Developing Mouse Brain (28). Western blotting Western blotting was performed as previously described (15,16,25). Briefly, the protein concentration in each sample was determined using a BCA protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Sample proteins (40 µg/lane) were separated on 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% bovine serum albumin (Beyotime Institute of Biotechnology, Shanghai, China) in Tris-buffered saline with Tween-20 (TBST) at room temperature for 1 h. Membranes were subsequently incubated at 4°C overnight with the following primary antibodies: Anti-cleaved caspase-3 (cat no. 9664) at 1:2,000 dilution, anti-α-fodrin (which contain SBDP145 and SBDP120 fragments; cat no. 2122) at 1:2,000 dilution, anti-phosphorylated-(p)-JNK (cat no. 4668) at 1:2,000 dilution, anti-JNK (cat no. 9252) at 1:2,000 dilution, anti-p-ERK1/2 (cat no. 4376) at 1:1,000 dilution, anti-ERK1/2 (cat no. 4695) at 1:1,000 dilution, anti-p-P38 (cat no. 4631) at 1:1,000 dilution, anti-P38 (cat no. 9212) at 1:1,000 dilution, anti-p-CREB (cat no. 9198) at 1:1,000 dilution, anti-p-nuclear factor-κB (NF-κB) (cat no. 3033) at 1:1,000 dilution, anti-p-Akt (Ser 473) (cat no. 4060) at 1:2,000 dilution, anti-Akt (cat no. 4685) at 1:5,000 dilution, anti-p-GSK-3β (Ser 9) (cat no. 5558) at 1:2,000 dilution, anti-GSK-3β (cat no. 9315) at 1:2,000 dilution, anti-p-CRMP-2 (Thr 514) (cat no. 9397) at 1:2,000 dilution, anti-CRMP-2 (cat no. 9393) at 1:2,000 dilution and anti-β-actin (cat no. 3700) at 1:2,000 dilution (all Cell Signaling Technology, Inc., Danvers, MA, USA) and anti-p-GSK-3β (Ty 216) (cat no. ab75745; Abcam, Cambridge, USA) at 1:2,000 dilution. The membranes were washed with TBST three times and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse IgG, cat no. A0216; goat anti-rabbit IgG, cat no. A0208; 1:2,000; Beyotime Institute of Biotechnology) at room temperature for 1 h. The membranes were washed with TBST three times and visualized using an enhanced chemiluminescence detection system (cat no. 34580; Thermo Fisher Scientific, Inc.). Images were scanned using an Image Master II scanner (GE Healthcare) and were analyzed using Image Quant TL software (v2003.03, GE Healthcare). The band signals of p-ERK1/2, p-JNK, p-p38, p-Akt, p-GSK-3 and p-CRMP-2 were normalized to the bands of total ERK1/2, JNK, p38, Akt, GSK-3β and CRMP-2 from the same samples. The band signals of the other proteins were normalized to those of β-actin and the results in each group were normalized to that of the corresponding control group. TUNEL assay TUNEL was performed following a previously described protocol (15,16). A Dead End™ fluorometric TUNEL system (Promega Corporation, Madison, WI, USA) was used and staining following the manufacturer's protocol. Briefly, TUNEL labeling was conducted with a mix of 45 µl equilibration buffer, 5 µl nucleotide mix and 1 µl recombinant terminal deoxynucleotidyl transferase (rTdT) enzyme in a humidified, lucifugal chamber for 1 h at 37°C, and then Hoechst 33258 (H-33258; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to stain nuclei for 10 min at room temperature. The sections were protected by anti-Fade solution and mounted on glass coverslips with clear nail polish sealing the edges. Slides were protected from direct light during the experiment. The images of TUNEL positive cells in the retrosplenial cortex (RS), frontal cortex (FC) and parietal association cortex (PtA) areas were acquired by Ti-S inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan) and analyzed using NIS-Elements Basic Research imaging processing and analysis software (version 3.0; Nikon Corporation). The density of TUNEL positive cells in the three cortical regions was calculated by dividing the number of TUNEL positive cells by the area of that brain region. -Statistical analysis Sample size was calculated using PASS 11 software (NCSS, LLC, Kaysville, UT, USA) to achieve 80% power at a significance level of P<0.05. All data were determined to be normally distributed using the Shapiro-Wilk test and had no significant heterogeneity of variance as detected by Levene's test. GraphPad Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA) was used to conduct all statistical analyses. Data were presented as mean ± standard deviation and were analyzed by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.",mice,"['A total of 24 C57BL/6 male mouse pups, aged 7 days (P7) and weighing 3.5–4.5 g were obtained from Guangdong Medical Laboratory Animal Center (Guangdong, China; permission no. SCXK2011-0029).']",postnatal day 7,"['A total of 24 C57BL/6 male mouse pups, aged 7 days (P7) and weighing 3.5–4.5 g were obtained from Guangdong Medical Laboratory Animal Center (Guangdong, China; permission no. SCXK2011-0029).']",N,"['The mouse pups at P7 were exposed to 2.6% sevoflurane (Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) for 6 h [~1.0 minimal alveolar concentration (MAC) in P7 mice] in 50% oxygen in a temperature-controlled chamber, following a previously described protocol (n=12) (17).']",sevoflurane,"['The mouse pups at P7 were exposed to 2.6% sevoflurane (Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) for 6 h [~1.0 minimal alveolar concentration (MAC) in P7 mice] in 50% oxygen in a temperature-controlled chamber, following a previously described protocol (n=12) (17).']",none,[],c57bl/6,"['A total of 24 C57BL/6 male mouse pups, aged 7 days (P7) and weighing 3.5–4.5 g were obtained from Guangdong Medical Laboratory Animal Center (Guangdong, China; permission no. SCXK2011-0029).']",True,True,False,True,True,True,10.3892/etm.2017.5651 +Statistical analysis Sample size was calculated using PASS 11 software (NCSS, LLC, Kaysville, UT, USA) to achieve 80% power at a significance level of P<0.05. All data were determined to be normally distributed using the Shapiro-Wilk test and had no significant heterogeneity of variance as detected by Levene's test. GraphPad Prism 6.0 software (GraphPad Software Inc., La Jolla, CA, USA) was used to conduct all statistical analyses. Data were presented as mean ± standard deviation and were analyzed by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.",mice,"['A total of 24 C57BL/6 male mouse pups, aged 7 days (P7) and weighing 3.5–4.5 g were obtained from Guangdong Medical Laboratory Animal Center (Guangdong, China; permission no. SCXK2011-0029).']",postnatal day 7,"['A total of 24 C57BL/6 male mouse pups, aged 7 days (P7) and weighing 3.5–4.5 g were obtained from Guangdong Medical Laboratory Animal Center (Guangdong, China; permission no. SCXK2011-0029).']",N,"['The mouse pups at P7 were exposed to 2.6% sevoflurane (Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) for 6 h [~1.0 minimal alveolar concentration (MAC) in P7 mice] in 50% oxygen in a temperature-controlled chamber, following a previously described protocol (n=12) (17).']",sevoflurane,"['The mouse pups at P7 were exposed to 2.6% sevoflurane (Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) for 6 h [~1.0 minimal alveolar concentration (MAC) in P7 mice] in 50% oxygen in a temperature-controlled chamber, following a previously described protocol (n=12) (17).']",none,[],c57bl/6,"['A total of 24 C57BL/6 male mouse pups, aged 7 days (P7) and weighing 3.5–4.5 g were obtained from Guangdong Medical Laboratory Animal Center (Guangdong, China; permission no. SCXK2011-0029).']",True,True,False,True,True,True,[ Passage 17/25 ] 10.3892/etm.2017.5651 10.3389/fncel.2019.00251,435.0,Li,2019,rats,postnatal day 7,N,ketamine,none,sprague dawley,"PMID: 31263401 PMCID: PMC6585163 DOI: 10.3389/fncel.2019.00251 Materials and Methods Animal Protocols @@ -533,7 +533,7 @@ Western Blot Analysis Brain tissues from the SVZ and SGZ of rats (n = 6 per group) at 12 h after anesthesia and cell cultures at 24 h following drug exposure were subjected to Western blot analyses as described in our previous studies (Lu et al., 2017). Briefly, the tissues were lysed by RIPA lysis buffer with protease and phosphatase inhibitors. The lysates were homogenized with an electric homogenizer and maintained on ice for 15 min. After being centrifuged for 15 min at 14000 rpm at 4°C, the supernatant was aspirated and the resulting lysates were placed in a new tube. We used the BCA protein assay kit to examine the protein concentrations. Bovine serum albumin (BSA) was used as a standard. An equal amount of the resulting lysate was resolved by sodium dodecyl sulfate-polyacrylamide gel and the separated proteins were transferred to polyvinylidene fluoride membranes. After being blocked for 1 h at room temperature, the membranes were then incubated with appropriate dilutions of primary antibodies at 4°C overnight. The used antibodies included anti-caspase-3 (cleaved, 17 KDa, 1:1000, Cell Signal Technology Inc. Beverly, MA, United States), anti-phosophorylated GSK-3β (p-GSK-3β, 1:1000, Cell Signal Technology Inc. Beverly, MA, United States), anti-GSK-3β (1:1000, Cell Signal Technology Inc. Beverly, MA, United States), and anti-β-actin (1:1000, Cell Signal Technology Inc., Beverly, MA, United States). The following day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit or anti-mouse) for 2 h at room temperature. After being enhanced by chemiluminescence (ECL), the signals were then exposed to X-ray films. Each band in the Western blot represented an independent experiment and at least three independent experiments were conducted. Data were expressed as the ratio to optical density (OD) values of the corresponding controls. The Western blots were quantified as described in our previous study. Statistical Analysis -Data obtained from the study were presented as mean ± SEM. Every data point represented a mean for each animal in a single case. SigmaPlot 12.0 was used for all statistical analysis. Data were tested and then confirmed with normality and equal variance criteria. A one-way analysis of variance (ANOVA) following the post hoc Holm-Sidak method was used to analyze the differences among different groups. A two-tailed probability value P < 0.05 was considered statistically significant.",rats,['PND 7 and embryonic day 18–19 Sprague-Dawley rats were obtained from Laboratory Animal Centre of Xi’an Jiaotong University.'],postnatal day 7,"['The PND 7 rats, weighing 13–18 g, were housed with their mother and maintained at a temperature of 24°C in a 12 h/12 h light/dark cycle with free access to food and water.']",Y,"['The spatial learning and memory function of rats after ketamine exposure were tested by MWM experiments as described in a previous study (Shen et al., 2013).']",ketamine,"['the rats in ketamine group received 40 mg/kg ketamine, diluted in normal saline and administrated by intraperitoneal injection (ketamine, Sigma–Aldrich Inc. St. Louis, MO, United States)']",17β-estradiol,"['the rats in the 17β-estradiol group received 17β-estradiol (17β-estradiol, Tocris, Minneapolis, MN, United States; DMSO, Sigma–Aldrich, St Louis, MO, United States) dissolved in dimethylsulfoxide (DMSO) at a concentration of 100 ug/ml, 100 ug/kg 17β-estradiol administered intraperitoneally 8 h, 1 h prior to and 3 h after ketamine’s initial injection (Liu et al., 2007).']",sprague dawley,['PND 7 and embryonic day 18–19 Sprague-Dawley rats were obtained from Laboratory Animal Centre of Xi’an Jiaotong University.'],True,True,False,True,False,True,10.3389/fncel.2019.00251 +Data obtained from the study were presented as mean ± SEM. Every data point represented a mean for each animal in a single case. SigmaPlot 12.0 was used for all statistical analysis. Data were tested and then confirmed with normality and equal variance criteria. A one-way analysis of variance (ANOVA) following the post hoc Holm-Sidak method was used to analyze the differences among different groups. A two-tailed probability value P < 0.05 was considered statistically significant.",rats,['PND 7 and embryonic day 18–19 Sprague-Dawley rats were obtained from Laboratory Animal Centre of Xi’an Jiaotong University.'],postnatal day 7,"['The PND 7 rats, weighing 13–18 g, were housed with their mother and maintained at a temperature of 24°C in a 12 h/12 h light/dark cycle with free access to food and water.']",Y,"['The spatial learning and memory function of rats after ketamine exposure were tested by MWM experiments as described in a previous study (Shen et al., 2013).']",ketamine,"['the rats in ketamine group received 40 mg/kg ketamine, diluted in normal saline and administrated by intraperitoneal injection (ketamine, Sigma–Aldrich Inc. St. Louis, MO, United States)']",17β-estradiol,"['the rats in the 17β-estradiol group received 17β-estradiol (17β-estradiol, Tocris, Minneapolis, MN, United States; DMSO, Sigma–Aldrich, St Louis, MO, United States) dissolved in dimethylsulfoxide (DMSO) at a concentration of 100 ug/ml, 100 ug/kg 17β-estradiol administered intraperitoneally 8 h, 1 h prior to and 3 h after ketamine’s initial injection (Liu et al., 2007).']",sprague dawley,['PND 7 and embryonic day 18–19 Sprague-Dawley rats were obtained from Laboratory Animal Centre of Xi’an Jiaotong University.'],True,True,False,True,False,True,[ Passage 18/25 ] 10.3389/fncel.2019.00251 10.1016/j.neuropharm.2007.09.005,531.0,Li,2007,rats,gestational day 21,Y,isoflurane,none,sprague dawley,"PMID: 17959201 PMCID: PMC2170454 DOI: 10.1016/j.neuropharm.2007.09.005 2. Materials and methods 2.1. Animals @@ -574,7 +574,7 @@ Probe trials were conducted after the last place trials for the juveniles (P36) After the last probe test for the adult rats, the animals performed the learning to reach criterion test during the next 9 days as described previously (Chen et al., 2000). The experimental procedure was similar to the place trial except that the platform location was changed. For each rat, the platform was moved between nine different locations set up by the computer. Each rat received up to eight trials per day. In order to advance to the next platform location, each rat had to reach the criterion of three successive trials with escape latency of 20 s or less. If a rat reached a criterion in 8 or less trials, a new platform location would be selected the following day. The numbers of learned platforms and the number of trials used to reach the criteria were recorded and compared. The number of platforms learned and the number of trials to reach a criterion indicated the learning ability of the rats. 2.8. Statistical analysis -To reduce variance from different size litters, we averaged the data from all fetal or postnatal rats from the same mother and considered them as a single sample. Results of weight gain of postnatal rat pups, ABG and MABP of pregnant rats and place trials of postnatal rats were analyzed using 2-way ANOVA for repeated measurements. Data for immunohistochemistry, TUNEL and other behavioral studies were analyzed using Student’s t-test for comparison of two groups or by ANOVA followed by Fisher's post hoc multiple comparison tests for those with more than two groups. In all experiments, difference were considered statistically significant at P < 0.05.",rats,"['Sprague–Dawley pregnant rats (Charles River Laboratories, Inc Wilmington, MA) were housed in polypropylene cages and the room temperature was maintained at 22 °C, with a 12 h light–dark cycle.']",gestational day 21,"['Pregnant rats at gestation day 21 (E21) were used for all experiments because it approximately corresponds to mid-gestation in human beings according to the theory of brain growth spurt (Dobbing and Sands, 1979, Jevtovic-Todorovic et al., 2003), and is a common time for most fetal surgeries (18–25 weeks) (Myers et al., 2002).']",Y,"['The behavioral study was performed to investigate the effects of fetal exposure to isoflurane on postnatal memory and learning.', 'In the behavioral study, pregnant rats were treated with 1.3% isoflurane (n = 8) or carrier gas (sham controls, n = 7) for 6 h.', 'We determined spatial reference memory and learning with the MWM as reported previously with some modification (Jevtovic-Todorovic et al., 2003).']",isoflurane,"['A pilot study was first conducted to find the highest concentration of isoflurane not accompanied by significant arterial blood gas (ABG) and mean arterial blood pressure (MABP) changes in the mothers.', 'Isoflurane is used clinically at a wide range of concentrations (about 0.2–3%), depending on the presence of other kinds of anesthetics or narcotics and the type and duration of surgery.']",none,[],sprague dawley,"['Sprague–Dawley pregnant rats (Charles River Laboratories, Inc Wilmington, MA) were housed in polypropylene cages and the room temperature was maintained at 22 °C, with a 12 h light–dark cycle.']",True,True,True,True,True,True,10.1016/j.neuropharm.2007.09.005 +To reduce variance from different size litters, we averaged the data from all fetal or postnatal rats from the same mother and considered them as a single sample. Results of weight gain of postnatal rat pups, ABG and MABP of pregnant rats and place trials of postnatal rats were analyzed using 2-way ANOVA for repeated measurements. Data for immunohistochemistry, TUNEL and other behavioral studies were analyzed using Student’s t-test for comparison of two groups or by ANOVA followed by Fisher's post hoc multiple comparison tests for those with more than two groups. In all experiments, difference were considered statistically significant at P < 0.05.",rats,"['Sprague–Dawley pregnant rats (Charles River Laboratories, Inc Wilmington, MA) were housed in polypropylene cages and the room temperature was maintained at 22 °C, with a 12 h light–dark cycle.']",gestational day 21,"['Pregnant rats at gestation day 21 (E21) were used for all experiments because it approximately corresponds to mid-gestation in human beings according to the theory of brain growth spurt (Dobbing and Sands, 1979, Jevtovic-Todorovic et al., 2003), and is a common time for most fetal surgeries (18–25 weeks) (Myers et al., 2002).']",Y,"['The behavioral study was performed to investigate the effects of fetal exposure to isoflurane on postnatal memory and learning.', 'In the behavioral study, pregnant rats were treated with 1.3% isoflurane (n = 8) or carrier gas (sham controls, n = 7) for 6 h.', 'We determined spatial reference memory and learning with the MWM as reported previously with some modification (Jevtovic-Todorovic et al., 2003).']",isoflurane,"['A pilot study was first conducted to find the highest concentration of isoflurane not accompanied by significant arterial blood gas (ABG) and mean arterial blood pressure (MABP) changes in the mothers.', 'Isoflurane is used clinically at a wide range of concentrations (about 0.2–3%), depending on the presence of other kinds of anesthetics or narcotics and the type and duration of surgery.']",none,[],sprague dawley,"['Sprague–Dawley pregnant rats (Charles River Laboratories, Inc Wilmington, MA) were housed in polypropylene cages and the room temperature was maintained at 22 °C, with a 12 h light–dark cycle.']",True,True,True,True,True,True,[ Passage 19/25 ] 10.1016/j.neuropharm.2007.09.005 10.1371/journal.pone.0160826,935.0,Luo,2016,rats,gestational day 18,N,isoflurane,none,sprague dawley,"PMID: 27536989 PMCID: PMC4990207 DOI: 10.1371/journal.pone.0160826 Experimental Procedures Subjects @@ -586,7 +586,7 @@ On E18, gravid rats in the I2, I4 and I8 groups were exposed to 1.5% isoflurane Morris Water Maze (MWM) Test The age of P30 in rat corresponds to preschool age in human [31]. Therefore, we evaluated the spatial learning and memory of the offspring begining on P30 with MWM according previous report [32]. All of the offspring were acclimated to the experimental environment for 30 min before testing. The Morris water maze is a black circular steel pool with a diameter of 150 cm and a height of 60 cm, filled with 24 ± 1°C water to a depth of 20 cm. A circular escape platform of 10 cm in diameter was submerged 1 cm below the water surface in the second quadrant. The swimming trail and speed of the rats was automatically recorded by the SLY-WMS Morris water maze test system (Beijing Sunny Instruments Co. Ltd., Beijing, China). The escape latency (time needed to find the platform), platform crossing times (number of times the rat swam across the submerged platform), and the target quadrant traveling time (time spent in the platform-hidden quadrant) were recorded automatically by the test system. The tests were begun at 9:00 am, one time per day for seven consecutive days. Each offspring rat was put into the pool to search for the platform one time per day for six days (training trial). The starting point was in the third quadrant, the farthest quadrant from the platform-hidden quadrant (the second quadrant in the present study, named the target quadrant). The rats were placed in the water facing the wall of the pool. The same starting point was used for each rat (with a colour marker on the pool wall). The animals were allowed to stay on the platform for 30 seconds when they found the platform. If an animal could not find the platform within 120 s, the escape latency was recorded as 120 s for that trial. The animal was then guided to the platform and allowed to stay on it for 30 s. On the seventh day, the platform was removed. Rats were allowed to swim for 120s to test their memory (platform-crossing times and target quadrant traveling time). The mean of the latencies, platform-crossing times and target quadrant traveling time of the offspring rats born by the same mother rat were calculated as the final results. -The offspring born to the same dam in each group were subdivided into the SAHA subgroup (I2S, I4S, I8S and CS subgroup) and the non-SAHA subgroup (I2N, I4N, I8N and CN subgroup) (Fig 1). Two hours before each MWM test, 90 mg/kg SAHA (Selleck Chemicals, Houston, TX, USA), at a concentration of 0.6 μM in dimethyl sulfoxide (DMSO) was given intraperitoneally to the offspring in the SAHA subgroups. An equal volume of DMSO was given to the rats in the non-SAHA subgroups. We selected 2 h before each MWM trial as the administration time point for SAHA based on the fact that 2 h after SAHA administration, the expression of NR2B increased in the hippocampus of Sprague-Dawley rats by enhancing histone acetylation, thus facilitating fear extinction [17].",rats,['Seventy-day-old female Sprague-Dawley (SD) rats (maternal rats) were supplied by the animal science research department of the Jiangxi Traditional Chinese Medicine College (JZDWNO: 2011–0030).'],gestational day 18,"['On E18, gravid rats in the I2, I4 and I8 groups were exposed to 1.5% isoflurane...']",Y,"['The learning and memory functions of the parental rats were assessed with the MWM before mating.', 'Therefore, we evaluated the spatial learning and memory of the offspring begining on P30 with MWM according previous report [32].']",isoflurane,"['gravid rats in the I2, I4 and I8 groups were exposed to 1.5% isoflurane (Abbott laboratories Ltd, Worcester, MA, USA) in 100% oxygen for 2, 4 and 8 hours, respectively']",none,[],sprague dawley,['Seventy-day-old female Sprague-Dawley (SD) rats (maternal rats) were supplied by the animal science research department of the Jiangxi Traditional Chinese Medicine College (JZDWNO: 2011–0030).'],True,True,False,True,True,True,10.1371/journal.pone.0160826 +The offspring born to the same dam in each group were subdivided into the SAHA subgroup (I2S, I4S, I8S and CS subgroup) and the non-SAHA subgroup (I2N, I4N, I8N and CN subgroup) (Fig 1). Two hours before each MWM test, 90 mg/kg SAHA (Selleck Chemicals, Houston, TX, USA), at a concentration of 0.6 μM in dimethyl sulfoxide (DMSO) was given intraperitoneally to the offspring in the SAHA subgroups. An equal volume of DMSO was given to the rats in the non-SAHA subgroups. We selected 2 h before each MWM trial as the administration time point for SAHA based on the fact that 2 h after SAHA administration, the expression of NR2B increased in the hippocampus of Sprague-Dawley rats by enhancing histone acetylation, thus facilitating fear extinction [17].",rats,['Seventy-day-old female Sprague-Dawley (SD) rats (maternal rats) were supplied by the animal science research department of the Jiangxi Traditional Chinese Medicine College (JZDWNO: 2011–0030).'],gestational day 18,"['On E18, gravid rats in the I2, I4 and I8 groups were exposed to 1.5% isoflurane...']",Y,"['The learning and memory functions of the parental rats were assessed with the MWM before mating.', 'Therefore, we evaluated the spatial learning and memory of the offspring begining on P30 with MWM according previous report [32].']",isoflurane,"['gravid rats in the I2, I4 and I8 groups were exposed to 1.5% isoflurane (Abbott laboratories Ltd, Worcester, MA, USA) in 100% oxygen for 2, 4 and 8 hours, respectively']",none,[],sprague dawley,['Seventy-day-old female Sprague-Dawley (SD) rats (maternal rats) were supplied by the animal science research department of the Jiangxi Traditional Chinese Medicine College (JZDWNO: 2011–0030).'],True,True,False,True,True,True,[ Passage 20/25 ] 10.1371/journal.pone.0160826 10.3892/etm.2018.5950,1455.0,Lu,2018,mice,postnatal day 7,Y,sevoflurane,isoflurane,c57bl/6,"PMID: 29731813 PMCID: PMC5920718 DOI: 10.3892/etm.2018.5950 Materials and methods Animal model All experiments were performed according to the guidelines of the Guide for the Care and Use of Laboratory Animals (22) and were approved by the Institutional Animal Care and Use Committee of Ruijin Hospital Affiliated to Shanghai Jiaotong University (Shanghai, China). A total of 174 C57BL/6 mice (sex ratio, 1:1), were provided by the Model Animal Research Center of Nanjing University (Nanjing, China). They were housed in polypropylene cages (5 or 6 animals per cage) and kept at a 12 h light-dark cycle at room temperature (21–24°C) in 55% humidity for 7 days prior to testing. All animals had free access to food and water. @@ -600,10 +600,10 @@ Immunohistochemistry The hippocampal tissues were fixed overnight in 4% paraform Western blotting The preparation of hippocampus protein extraction was performed as previously described (28,29). Total proteins were extracted with radioimmunoprecipitation assay buffer [1% Triton X-100, 50 mM Tris, (pH 7.4), 150 mM NaCl, M, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA and 1% sodium deoxycholate]. Following 13,000 × g centrifugation at 4°C for 20 min, the supernatant was used for western blotting (30,31). The BCA method was used to assay protein concentrations. In brief, hippocampal tissue proteins were separated by 10% SDS polyacrylamide gel electrophoresis and then electrotransferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat powdered milk for 1 h at 25°C. The proteins were probed with rabbit anti-PARP antibodies (1:200, cat. no. AP102) or rat anti-GAPDH antibodies (1:5,000, cat. no. AG019) overnight at 4°C. Then, goat anti-rabbit (1:4,000; cat. no. A0208) or goat anti-rat (1:4,000; cat. no. A0192) horseradish peroxidase-conjugated secondary antibodies were used for 2 h incubation at room temperature (all from Beyotime Institute of Biotechnology). Proteins were visualized by an enhanced chemiluminescence method and analyzed with the Dolphin-Doc Plus Gel Documentation system (version 1141002; Wealtec Corp., Sparks, NV, USA). This procedure was repeated twice for all 3 groups. The relative level of PARP was presented as the band intensity and normalized to the corresponding band intensities of GAPDH. ELISA The method of hippocampus protein extraction mentioned above was also used for ELISA. The levels of BDNF, Ntrk2, pro-BDNF, p75NTR and PKB/Akt were measured using an ELISA kit (cat. no. EK0312; Wuhan Boster Bio-Engineering Co., Ltd., Wuhan, China) according to the manufacturer's instructions. Briefly, protein samples were added to the enzyme label plate and incubated for 1.5 h at 37°C. Next, the biotin-labeled antibodies were added for 1 h incubation at 37°C. Following washing, 30 min incubation with avidin peroxidase complex was conducted at 37°C. Color was developed using 3,3′,5,5′-tetramethylbenzidine following 20 min incubation at 37°C. Following reaction termination with a ‘stop’ solution, the products were measured at 450 nm using a microplate spectrophotometer (Spectramax 190; Molecular Devices LLC, Sunnyvale, CA, USA). All samples were assayed in duplicate and the readings were normalized to the amount of standard protein. Behavioral studies Prior to the MWM test, mice received 2 min of touch for 5 days to avoid the fear to touch during the test. The MWM test was performed as previously described (32,33), with minor modifications. The round pool (diameter, 122 cm) was filled with warm water, made opaque by the addition of titanium dioxide and an escape platform was placed in the northwest quadrant and hidden 0.5 cm below the surface of the water. The MWM test was performed on 7 consecutive days (6 days for training and 1 day for the probe test). Briefly, mice received 4 training sessions daily for 6 consecutive days. Each trial began from a different point and ended when the mice found the platform. The time from beginning to end was considered to be the time of escape latency. If mice could not find the platform within 90 sec, the time of escape latency was recorded as 90 sec. If mice found the platform within 90 sec, the real time from beginning to end was recorded as the time of escape latency. The swim rate during training was also recorded. On day 7, the probe test was performed by allowing the mice to swim for 60 sec in the absence of the platform. During 60 sec swimming, the time spent in the northwest quadrant and platform site crossovers was recorded and analyzed using the MWM JLBehv-FCS video analysis system (DigBehv-MG; Shanghai Jiliang Software Technology Co., Ltd., Shanghai, China). -Statistical analysis All data are presented as the mean ± standard error of the mean. A repeated measures analysis of variance (ANOVA) was used to measure the differences within groups over time. Meanwhile, one-way ANOVA was applied for comparison among groups (2.6, 1.3% sevoflurane and control groups), followed by Student Newman-Keuls post hoc test. The correlation between the swim rate and time of escape latency was identified using the Pearson Correlation coefficient. For all the analysis, P<0.05 was used to indicate a statistically significant difference. Additionally, SPSS 11.5 (SPSS, Inc., Chicago, IL, USA) was used for the analysis of the present study.",mice,"['A total of 174 C57BL/6 mice (sex ratio, 1:1), were provided by the Model Animal Research Center of Nanjing University (Nanjing, China).']",postnatal day ,[],Y,"['The MWM test was performed in half of the mice in each group.', 'The MWM test was conducted on the remaining mice at week 12.']",sevoflurane,"['For protocol one, 36 mice were randomly assigned into 3 groups with 12 mice in each group: The 2.6 and 1.3% sevoflurane groups and the control group (exposed to 30% O2).']",none,[],c57bl/6,"['A total of 174 C57BL/6 mice (sex ratio, 1:1), were provided by the Model Animal Research Center of Nanjing University (Nanjing, China).']",True,False,True,True,False,True,10.3892/etm.2018.5950 +Statistical analysis All data are presented as the mean ± standard error of the mean. A repeated measures analysis of variance (ANOVA) was used to measure the differences within groups over time. Meanwhile, one-way ANOVA was applied for comparison among groups (2.6, 1.3% sevoflurane and control groups), followed by Student Newman-Keuls post hoc test. The correlation between the swim rate and time of escape latency was identified using the Pearson Correlation coefficient. For all the analysis, P<0.05 was used to indicate a statistically significant difference. Additionally, SPSS 11.5 (SPSS, Inc., Chicago, IL, USA) was used for the analysis of the present study.",mice,"['A total of 174 C57BL/6 mice (sex ratio, 1:1), were provided by the Model Animal Research Center of Nanjing University (Nanjing, China).']",postnatal day ,[],Y,"['The MWM test was performed in half of the mice in each group.', 'The MWM test was conducted on the remaining mice at week 12.']",sevoflurane,"['For protocol one, 36 mice were randomly assigned into 3 groups with 12 mice in each group: The 2.6 and 1.3% sevoflurane groups and the control group (exposed to 30% O2).']",none,[],c57bl/6,"['A total of 174 C57BL/6 mice (sex ratio, 1:1), were provided by the Model Animal Research Center of Nanjing University (Nanjing, China).']",True,False,True,True,False,True,[ Passage 21/25 ] 10.3892/etm.2018.5950 10.4149/BLL_2017_017,282.0,Ozer,2017,rats,postnatal day 7,N,sevoflurane,none,wistar-albino,"PMID: 28814087 DOI: 10.4149/BLL_2017_017 Materials and methods The preclinical animal study was conducted at the Firat University Experimental Research Centre in December 2012. After receiving approval from the institutional Animal Experimentation Ethics Committee, seven-day-old male Wistar-Albino rats were obtained from the Experimental Research Centre. The Helsinki Universal Declaration of Animal Rights was followed at every stage of the study. The rats were kept in the rooms with ambient temperature of 22–24 °C and with 12/12 hour day/night cycle. Except for the time it took for the experimental tests, the subjects were kept in the same cage with their mothers until postnatal day 21 (PN21). After the 21st day, the rats were put in separate cages and fed with standard rat chow and tap water. Using permuted block randomisation methods, the subjects were divided into the two groups: Group C acted as the control and did not receive any anaesthesia, and in Group S, anaesthesia was achieved with 2.3 % sevofl urane in 50 % oxygen (O2 )-air mixture. The concentration of sevofl urane was adjusted according to the tail test. The tail test was applied every 15 minutes. The middle 1/3 of the tail was clamped, and if there was response, sevofl urane concentration was increased 15 %. All subjects were put in a plastic, transparent anaesthesia chamber that was connected to the anaesthesia device and was ventilated with 4 L/min fl ow and 50 % O2-air mixture. Immediately after the six-hour administration of anaesthesia, oxygen arterial blood gases were evaluated in 3 subjects from each group. At the end of the application period, half of the subjects were sacrifi ced to determine the early effects of sevofl urane (Group SE and Group CE), while the rest of the subjects were sacrifi ced 6 weeks after the application to determine the late effects of sevofl urane (Group SL and Group CL). In addition, the levels of serum BDNF, brain tissue BDNF and caspase 3 were evaluated for all subjects. The anaesthesia chamber was heated from the outside during the entire experiment to prevent hypothermia. Glucose and saline were administered intradermally to prevent hypoglycaemia and hypovolaemia. Subjects that experienced discolouration of the skin (cyanosis) or a decrease in respiratory rate that did not improve with stimuli, were excluded from the study. -The study’s primary outcomes were serum BDNF levels, cortex and hippocampal BDNF levels and neurocognitive status. Secondary outcomes were cortex and hippocampal caspase 3 levels. Blood samples were taken at decapitation phase into serum separator tube to evaluate serum BDNF. Blood samples were centrifuged at 1000 x g for 15 minute and stored at –20 °C. Serum BDNF levels were measured by the enzyme-linked immunosorbent assay (ELISA) method (EK0308, Boster Biological Technology, Ltd.). Brain tissue samples were kept for 24 hours in 10 % formaldehyde prepared with phosphate buffer saline. Brain tissue samples were taken for routine tissue processing for immunohistochemical (IHC) examination procedure following the sagittal reduction process. Brain tissue was divided at the midline on the sagittal plane. The BDNF levels (Abcam, ab108319, Cambridge, UK) and caspase 3 levels (Abcam, ab13847, Cambridge, UK) were assessed with IHC in brain hemispheres (0 – no staining, 1 – mild, 2 – moderate, 3 – severe). The behaviour, anxiety states and spatial learning abilities of the subjects during the long-term period (6 weeks later) were evaluated by using the plus arm test and the Morris water test, respectively. Open arm avoidance index was calculated according to the formula 100 – ((% of the time spent in the open arms + % of the entrance to the open arms)/2), while the total locomotor activity was calculated based on line crossings + rearing. Swimming tests were done 4 times a day for a period of 4 days. The test was repeated 2 days after training and the time it took to reach the platform (latency) and the time spent in the platform quadrant after the platform was removed were recorded. The subjects that completed the swimming test were put back into their heated cages. Those conducting the experiment knew, which group of subjects they were dealing with, but those evaluating biochemical, IHC and neurocognitive tests did not know, which samples and subjects belonged to which group. SPSS 15 was used for a statistical evaluation. Nonparametric methods were used for all variables because the sample size was small. Kruskal–Wallis test was used for one-way analysis of variance (ANOVA) of nonparametric data, therefore median values were calculated instead of mean. When it was determined not to the equal of medians with Kruskal–Wallis test, Mann–Whitney U test was used for post-hoc multiple comparisons. The escape latency within the group was evaluated by Wilcoxon test. The correlation between parameters was assessed by Spearman correlation test and p < 0.05 was considered signifi cant.",rats,"['After receiving approval from the institutional Animal Experimentation Ethics Committee, seven-day-old male Wistar-Albino rats were obtained from the Experimental Research Centre.']",postnatal day 7,"['After receiving approval from the institutional Animal Experimentation Ethics Committee, seven-day-old male Wistar-Albino rats were obtained from the Experimental Research Centre.']",Y,"['The behaviour, anxiety states and spatial learning abilities of the subjects during the long-term period (6 weeks later) were evaluated by using the plus arm test and the Morris water test, respectively.']",sevoflurane,"['and in Group S, anaesthesia was achieved with 2.3 % sevoflurane in 50 % oxygen (O2 )-air mixture.']",none,[],wistar-albino,"['After receiving approval from the institutional Animal Experimentation Ethics Committee, seven-day-old male Wistar-Albino rats were obtained from the Experimental Research Centre.']",True,True,False,True,True,True,10.4149/BLL_2017_017 +The study’s primary outcomes were serum BDNF levels, cortex and hippocampal BDNF levels and neurocognitive status. Secondary outcomes were cortex and hippocampal caspase 3 levels. Blood samples were taken at decapitation phase into serum separator tube to evaluate serum BDNF. Blood samples were centrifuged at 1000 x g for 15 minute and stored at –20 °C. Serum BDNF levels were measured by the enzyme-linked immunosorbent assay (ELISA) method (EK0308, Boster Biological Technology, Ltd.). Brain tissue samples were kept for 24 hours in 10 % formaldehyde prepared with phosphate buffer saline. Brain tissue samples were taken for routine tissue processing for immunohistochemical (IHC) examination procedure following the sagittal reduction process. Brain tissue was divided at the midline on the sagittal plane. The BDNF levels (Abcam, ab108319, Cambridge, UK) and caspase 3 levels (Abcam, ab13847, Cambridge, UK) were assessed with IHC in brain hemispheres (0 – no staining, 1 – mild, 2 – moderate, 3 – severe). The behaviour, anxiety states and spatial learning abilities of the subjects during the long-term period (6 weeks later) were evaluated by using the plus arm test and the Morris water test, respectively. Open arm avoidance index was calculated according to the formula 100 – ((% of the time spent in the open arms + % of the entrance to the open arms)/2), while the total locomotor activity was calculated based on line crossings + rearing. Swimming tests were done 4 times a day for a period of 4 days. The test was repeated 2 days after training and the time it took to reach the platform (latency) and the time spent in the platform quadrant after the platform was removed were recorded. The subjects that completed the swimming test were put back into their heated cages. Those conducting the experiment knew, which group of subjects they were dealing with, but those evaluating biochemical, IHC and neurocognitive tests did not know, which samples and subjects belonged to which group. SPSS 15 was used for a statistical evaluation. Nonparametric methods were used for all variables because the sample size was small. Kruskal–Wallis test was used for one-way analysis of variance (ANOVA) of nonparametric data, therefore median values were calculated instead of mean. When it was determined not to the equal of medians with Kruskal–Wallis test, Mann–Whitney U test was used for post-hoc multiple comparisons. The escape latency within the group was evaluated by Wilcoxon test. The correlation between parameters was assessed by Spearman correlation test and p < 0.05 was considered signifi cant.",rats,"['After receiving approval from the institutional Animal Experimentation Ethics Committee, seven-day-old male Wistar-Albino rats were obtained from the Experimental Research Centre.']",postnatal day 7,"['After receiving approval from the institutional Animal Experimentation Ethics Committee, seven-day-old male Wistar-Albino rats were obtained from the Experimental Research Centre.']",Y,"['The behaviour, anxiety states and spatial learning abilities of the subjects during the long-term period (6 weeks later) were evaluated by using the plus arm test and the Morris water test, respectively.']",sevoflurane,"['and in Group S, anaesthesia was achieved with 2.3 % sevoflurane in 50 % oxygen (O2 )-air mixture.']",none,[],wistar-albino,"['After receiving approval from the institutional Animal Experimentation Ethics Committee, seven-day-old male Wistar-Albino rats were obtained from the Experimental Research Centre.']",True,True,False,True,True,True,[ Passage 22/25 ] 10.4149/BLL_2017_017 10.1097/ALN.0b013e3181974fa2,722.0,Satomoto,2009,mice,postnatal day 6,Y,sevoflurane,none,c57bl/6,"PMID: 19212262 DOI: 10.1097/ALN.0b013e3181974fa2 Materials and Methods The experiments were approved by the Committee for Animal Research at National Defense Medical College (Tokorozawa, Saitama, Japan). Pregnant C57BL/6 mice were purchased from SLC (SLC Japan Inc., Shizuoka, Japan). The animals were illuminated with a 12-h light–dark cycle (light from 07:00 to 19:00), and room temperature was maintained at 21°± 1°C. At the age of 3 weeks, the mice were weaned and housed in groups of 4 animals in a room. Mice had ad libitum access to water and food. @@ -660,7 +660,7 @@ Activity was measured as the total duration of interaction with an inanimate nov The same set of mice underwent social recognition (at 19 weeks of age), social interaction (at 14 weeks of age), olfactory (at 15 weeks of age), and novelty (at 15 weeks of age) tests. In other analyses, each test was conducted with a new set of animals. Statistical Analysis -Statistical analysis was performed using Statview software (SAS, Cary, NC). Comparisons of the means of two groups were performed using the Student t test. In the Y-maze task, comparisons of group performance relative to random levels were performed using the one-sample t test. Data of the social recognition task were analyzed by repeated-measures two-way analysis of variance. Values are presented as mean ± SEM.",mice,"['Pregnant C57BL/6 mice were purchased from SLC (SLC Japan Inc., Shizuoka, Japan).']",postnatal day 6,['Postnatal day 6 (P6) male mice were placed in an acrylic box and exposed to 3% sevoflurane or no anesthetics for 6 h.'],Y,"['Behavioral Studies', 'As described previously for CBF and histopathologic studies, some sets of mice for behavioral studies were exposed to 3% or 0% sevoflurane for 6 h at P6.', 'Open-field Test.', 'Elevated Plus-maze Test.', 'Spontaneous Alternation in the Y-maze Test.', 'Fear Conditioning Test.', 'Social Recognition Test.', 'Social Interaction Test.', 'Olfactory Test.', 'Novelty Test.']",sevoflurane,['Postnatal day 6 (P6) male mice were placed in an acrylic box and exposed to 3% sevoflurane or no anesthetics for 6 h.'],none,[],c57bl/6,"['Pregnant C57BL/6 mice were purchased from SLC (SLC Japan Inc., Shizuoka, Japan).']",True,True,True,True,True,True,10.1097/ALN.0b013e3181974fa2 +Statistical analysis was performed using Statview software (SAS, Cary, NC). Comparisons of the means of two groups were performed using the Student t test. In the Y-maze task, comparisons of group performance relative to random levels were performed using the one-sample t test. Data of the social recognition task were analyzed by repeated-measures two-way analysis of variance. Values are presented as mean ± SEM.",mice,"['Pregnant C57BL/6 mice were purchased from SLC (SLC Japan Inc., Shizuoka, Japan).']",postnatal day 6,['Postnatal day 6 (P6) male mice were placed in an acrylic box and exposed to 3% sevoflurane or no anesthetics for 6 h.'],Y,"['Behavioral Studies', 'As described previously for CBF and histopathologic studies, some sets of mice for behavioral studies were exposed to 3% or 0% sevoflurane for 6 h at P6.', 'Open-field Test.', 'Elevated Plus-maze Test.', 'Spontaneous Alternation in the Y-maze Test.', 'Fear Conditioning Test.', 'Social Recognition Test.', 'Social Interaction Test.', 'Olfactory Test.', 'Novelty Test.']",sevoflurane,['Postnatal day 6 (P6) male mice were placed in an acrylic box and exposed to 3% sevoflurane or no anesthetics for 6 h.'],none,[],c57bl/6,"['Pregnant C57BL/6 mice were purchased from SLC (SLC Japan Inc., Shizuoka, Japan).']",True,True,True,True,True,True,[ Passage 23/25 ] 10.1097/ALN.0b013e3181974fa2 10.3389/fncel.2017.00373,243.0,Xiao,2017,mice,postnatal day 7,N,propofol,none,c57bl/6,"PMID: 29249940 PMCID: PMC5715384 DOI: 10.3389/fncel.2017.00373 Materials and Methods Animals @@ -683,7 +683,7 @@ Quantification The quantification was obtained from regional analysis of lobe IX. All the sections were taken from similar medial-lateral position within the cerebellum, and each count area was chosen from the same field by the middle of the lobe IX. Calbindin-positive cells were analyzed along the long axis for 500 μm in the middle, and the dendrite length of Purkinje cells was evaluated by measuring the primary dendrite from the soma up to the surface of the ML (three Purkinje dendrites were measured per picture). Number of the NeuN-positive granule neurons were analyzed in the center region of the IGL in lobe IX along the long axis (unit area 2000 μm2). The number of BLBP- and GFAP-positive Bergmann fibers was counted from a 100-μm length in the middle area of lobe IX according to our previous methods (Yamada et al., 2000; Eiraku et al., 2005; Yang Y. et al., 2014). To analyze the astrocytes in the deep white matter, we compared the intensity of the GFAP-positive cells and fibers. Both the background integrated optical density (IOD) and surveyed area (same center area of the white matter from each group) were acquired, and the relative optical density (ROD) was calculated by subtracting the background from the IOD of the positive staining (Bao et al., 2017). Contact points between the calbindin-positive Purkinje cells and GFAP-positive Bergmann fibers were defined as where the tips of growing Purkinje cell dendrites were aligned parallel and attached directly to the rod-like domain of Bergmann fibers, entering the base of the overlying EGL, as previously reported (Yamada et al., 2000; Yamada and Watanabe, 2002; Lordkipanidze and Dunaevsky, 2005). Points were counted per image (212.5-μm length) at the interface between the EGL and ML. Only the yellow dots at the end of the dendrites in the direction of the Bergmann fiber were included, while the crossed ones were excluded in case of false positive. For quantifying granule neuron migration, BrdU-labeled cells were counted in a rectangular box (200 μm width and about 100 cells were counted) extending from the pial surface to the end of the IGL; this value was expressed as a percentage of the total number of BrdU-labeled cells. At least five sections were analyzed in each mouse and five mice from each group. All the quantitative statistics were performed blind to the experimental treatment. Statistical Analysis -All the data were presented as the mean ± standard deviation and analyzed using one-way analysis of variance (ANOVA) followed by Fisher’s protected least- significant difference post hoc test or a least-significant difference multiple-comparison. The differences were statistically significant when the P value was less than 0.05. Statistical analysis was performed using the SPSS 19.0 software (SPSS Inc., Chicago, IL, USA).",mice,['Male and female C57/BL6 mice were provided by the Third Military Medical University and housed under a 12 h light/dark cycle in a temperature-controlled room with free access to food and water.'],postnatal day 7,"['On P7, pups received a vehicle or propofol injection intraperitoneally (i.p.) at a subanesthetic dose of 30 or 60 mg/kg.']",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'.""]",propofol,"['On P7, pups received a vehicle or propofol injection intraperitoneally (i.p.) at a subanesthetic dose of 30 or 60 mg/kg.']",none,[],c57bl/6,['Male and female C57/BL6 mice were provided by the Third Military Medical University and housed under a 12 h light/dark cycle in a temperature-controlled room with free access to food and water.'],True,True,True,True,True,True,10.3389/fncel.2017.00373 +All the data were presented as the mean ± standard deviation and analyzed using one-way analysis of variance (ANOVA) followed by Fisher’s protected least- significant difference post hoc test or a least-significant difference multiple-comparison. The differences were statistically significant when the P value was less than 0.05. Statistical analysis was performed using the SPSS 19.0 software (SPSS Inc., Chicago, IL, USA).",mice,['Male and female C57/BL6 mice were provided by the Third Military Medical University and housed under a 12 h light/dark cycle in a temperature-controlled room with free access to food and water.'],postnatal day 7,"['On P7, pups received a vehicle or propofol injection intraperitoneally (i.p.) at a subanesthetic dose of 30 or 60 mg/kg.']",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'.""]",propofol,"['On P7, pups received a vehicle or propofol injection intraperitoneally (i.p.) at a subanesthetic dose of 30 or 60 mg/kg.']",none,[],c57bl/6,['Male and female C57/BL6 mice were provided by the Third Military Medical University and housed under a 12 h light/dark cycle in a temperature-controlled room with free access to food and water.'],True,True,True,True,True,True,[ Passage 24/25 ] 10.3389/fncel.2017.00373 10.1097/ALN.0b013e3182834d5d,268.0,Zheng,2013,mice,gestational day 14,Y,sevoflurane,none,c57bl/6,"PMID: 23314109 PMCID: PMC3580035 DOI: 10.1097/ALN.0b013e3182834d5d Materials and Methods Mice Anesthesia @@ -710,4 +710,4 @@ Environmental Enrichment The EE in the current experiment was created in a large cage (70 × 70 × 46 cm) that included five or six toys (e.g ., wheels, ladders, and small mazes) as described in previous studies, with modification.10,11The pregnant mice were put in the EE every day for 2 h before delivery. The pregnant mice delivered offspring mice at G21. Then, the mother and the babies were put in the EE again every day for 2 h from P4 to P30. The objects were changed two to three times per week to provide newness and challenge. Statistical Analysis -The nature of the hypothesis testing was two-tailed. Data were expressed as mean ± SD. The data for platform crossing time were not distributed normally and thus were expressed as median and interquartile range (IQR). The number of samples varied from 6–15, and the samples were distributed normally, with the exception of platform crossing time (tested by normality test, data not shown). Two-way ANOVA was used to determine the interaction of IL-6 antibody and sevoflurane treatment, and the interaction of EE and sevoflurane anesthesia. Interaction between time and group factors in a two-way ANOVA with repeated measurements was used to analyze the difference of learning curves (based on escape latency) between mice in the control group and mice treated with anesthesia in the MWM. Multiple comparisons in escape latency of MWM were adjusted using the Bonferroni method (with seven tests and a threshold of 0.05/7 = 0.0071). There were no missing data for the variables of MWM (escape latency and platform crossing time) during the data analysis. The Student two-sample t test was used to determine the difference between the sevoflurane and control conditions on levels of IL-6, PSD-95, and synaptophysin. Finally, the Mann–Whitney U test was used to determine the difference between the sevoflurane and control conditions on platform crossing times. Values of P < 0.05 were considered statistically significant. SAS software version 9.2 (SAS Institute Inc., Cary, NC) was used to analyze the data.",mice,"['Three-month-old C57BL/6J female mice (The Jackson Laboratory, Bar Harbor, ME) were mated with male mice.']",gestational day 14,"['At gestational day (G) 14, the pregnant mice were assigned randomly to an anesthesia group or a control group.']",Y,['The P31 offspring mice were tested in the Morris water maze (MWM) four times per day for 7 days.'],sevoflurane,"['Mice randomized to the anesthesia group received 2.5% sevoflurane in 100% oxygen for 2 h in an anesthetizing chamber.', 'The neurons were treated with 4.1% sevoflurane for 6 h as described in our previous studies.']",none,[],c57bl/6,"['Three-month-old C57BL/6J female mice (The Jackson Laboratory, Bar Harbor, ME) were mated with male mice.']",True,True,True,True,True,True,10.1097/ALN.0b013e3182834d5d +The nature of the hypothesis testing was two-tailed. Data were expressed as mean ± SD. The data for platform crossing time were not distributed normally and thus were expressed as median and interquartile range (IQR). The number of samples varied from 6–15, and the samples were distributed normally, with the exception of platform crossing time (tested by normality test, data not shown). Two-way ANOVA was used to determine the interaction of IL-6 antibody and sevoflurane treatment, and the interaction of EE and sevoflurane anesthesia. Interaction between time and group factors in a two-way ANOVA with repeated measurements was used to analyze the difference of learning curves (based on escape latency) between mice in the control group and mice treated with anesthesia in the MWM. Multiple comparisons in escape latency of MWM were adjusted using the Bonferroni method (with seven tests and a threshold of 0.05/7 = 0.0071). There were no missing data for the variables of MWM (escape latency and platform crossing time) during the data analysis. The Student two-sample t test was used to determine the difference between the sevoflurane and control conditions on levels of IL-6, PSD-95, and synaptophysin. Finally, the Mann–Whitney U test was used to determine the difference between the sevoflurane and control conditions on platform crossing times. Values of P < 0.05 were considered statistically significant. SAS software version 9.2 (SAS Institute Inc., Cary, NC) was used to analyze the data.",mice,"['Three-month-old C57BL/6J female mice (The Jackson Laboratory, Bar Harbor, ME) were mated with male mice.']",gestational day 14,"['At gestational day (G) 14, the pregnant mice were assigned randomly to an anesthesia group or a control group.']",Y,['The P31 offspring mice were tested in the Morris water maze (MWM) four times per day for 7 days.'],sevoflurane,"['Mice randomized to the anesthesia group received 2.5% sevoflurane in 100% oxygen for 2 h in an anesthetizing chamber.', 'The neurons were treated with 4.1% sevoflurane for 6 h as described in our previous studies.']",none,[],c57bl/6,"['Three-month-old C57BL/6J female mice (The Jackson Laboratory, Bar Harbor, ME) were mated with male mice.']",True,True,True,True,True,True,[ Passage 25/25 ] 10.1097/ALN.0b013e3182834d5d