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

PMID: 22535834 PMCID: PMC3393078 DOI: 10.1093/bja/aes121
Methods
Animals
The use of animals in this study was approved by the Institutional Animal Care and Use Committee at Sun Yat-sen University (Guangzhou, Guangdong, China). All efforts were made to minimize the number of animals used and their suffering. Male Sprague–Dawley (sd) rats were obtained from the Experimental Animal Centre of Sun Yat-sen University. The rats were housed under a 12 h light–dark cycle (light from 07:00 to 19:00) at 20–22°C. In addition, the rats were given ad libitum access to water and food. A total of 19 litters consisting of 99 male pups were used in this study. Each experimental condition had its own group of littermate controls to minimize variability in the rate of apoptosis.21

Sevoflurane exposure
Rats at postnatal day 7 (P7, 16–17 g) were randomly divided into a sevoflurane-treated group (51 rats) and an air-treated control group (48 rats). Rats in the sevoflurane-treated group were placed in a plastic container and continuously exposed to 2.3% sevoflurane for 6 h using air as a carrier with a gas flow of 2 litre min−1. During sevoflurane exposure, the container was heated to 38°C (NPS-A3 heated device, Midea, Co., Guangdong, China). Sevoflurane, oxygen, and carbon dioxide in the chamber were monitored using a gas monitor (Detex-Ohmeda, Louisville, KY, USA). After 6 h, the rats were exposed to air only and, when able to move freely, were placed back into their maternal cages. During sevoflurane exposure, an investigator monitored respiratory frequency and skin colour; if signs of apnoea or hypoxaemia were detected, the rat was immediately exposed to air and excluded from the experiment. Rats in the control group were placed into the container and were exposed to air only for 6 h.

Arterial blood gas analysis
Arterial blood analysis was performed on P7–8 rats (16–17 g) from the sevoflurane- and air-treated groups.1,22 Arterial blood samples were obtained from the left cardiac ventricle immediately after removal from the maternal cage (0 h, n=3 in each subgroup) or after anaesthesia (6 h, n=3 in each subgroup). Samples were transferred into heparinized glass capillary tubes and analysed immediately by a blood gas analyser (Gem premier 3000). The pups were killed by decapitation at the time of blood sampling and the analysis of each sample was repeated at least three times.

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

The Fox battery test Fox battery tests were conducted on 12 rats from P1 to P21 (4–55 g) daily between 08:30 and 23:00 which corresponded to the rats' active period, as described in previous studies.23,24 At P7, rats were randomly divided into sevoflurane-treated (n=6) and the air-treated groups (n=6) that were exposed to sevoflurane or to air for 6 h, respectively. The Fox battery test was performed after the rats had fully recovered from anaesthesia and were able to move freely as described.23,24 The time of the appearance (days) of the eye opening, incisor eruption, limb grasp, crossed extensor reflex, negative geotaxis reflex, and gait reflex was recorded for each rat. Additionally, the time needed to achieve the righting reflex, the negative geotaxis reflex, and the gait reflex was recorded. The maximum angle at which the animals could maintain the position on an inclined board test for 5 s was also documented.
MWM test Based on a previous study,25 we performed the MWM test on P28 (80–100 g) rats using the Water Maze Tracking System (TME; Chengdu, China) with minor modifications (1984).26 This test was conducted on both sevoflurane-treated (n=9) and air-treated groups (n=6). The MWM consisted of a grey circular tank (100 cm diameter, 50 cm in depth), which was surrounded by several visual cues. Immediately before the test, the tank was filled with water [22 (1)°C] to a height of 30 cm. The tank was equally divided into the target (T, where a plastic platform was submerged), right (R), opposite (O), and left (L) quadrants, with four starting locations that were equidistant from the rim. We conducted memory-acquisition trials (training) four times daily for 5 days. A single adaptation trial (without the platform) was performed and the rats were released into the pool for 60 s in the absence of any escape platform on day 0. On the following 4 days (days 1–4, Place Navigation), two blocks of tests (morning 08:30–11:00 and afternoon 14:30–15:00) were performed with four trials per block per day for each rat. In each trial, the rat was placed into the water facing the wall from one of the four starting points. The escape latency (time to find the submerged platform), the swimming route to reach the platform, and the swimming speed were measured by a computer-operated video tracking system. Once the rat had reached the platform, it was allowed to remain on the platform for 15 s for orientation purposes. Those rats that failed to independently find the escape platform within 60 s were placed on to the escape platform by the experimenter. The rats were removed afterwards to rest in a heated cage until the next trial. Four daily trials were averaged for each animal. On day 5, the memory retention tests (Spatial Probe) were performed in the absence of a submerged platform in the tank. The rat was placed into the water, facing the wall from one of the four starting points. Within 120 s, both the time spent in each quadrant and the swimming route were recorded. The rats were then removed from the tank and placed back into the heated cage.
Western blot analysis
Immunoblotting was performed on hippocampi obtained from 48 P7–8 rats (16–17 g) as previously described.27,28 Briefly, rats were killed by decapitation at 0, 2, 6, and 24 h after 6 h sevoflurane or air treatments, with six rats at each time point per treatment. The rat brain was quickly dissected, and the hippocampus was quickly removed and homogenized in 100 mg ml−1 RIPA Lysis Buffer (Shenergy Biocolor Co., China) with 1% (v/v) PMSF (Shenergy Biocolor Co., China). The homogenate was centrifuged at 13 000g for 20 min at 4°C, and the supernatant was separated and stored at −80°C until further use. The proteins extracted from the hippocampus were separated on a 10% gel by electrophoresis and transferred on to polyvinylidene fluoride membranes (Pall Co., USA). The blots were then incubated with anti-cleaved caspase-3 (1:1000, rabbit polyclonal, Asp175; Cell Signaling Technology, Inc., USA) or anti-β-actin (1:2000, mouse monoclonal; Santa Cruz Biotechnology, USA) antibodies. The changes in the protein expression levels of nNOS using an anti-nNOS antibody (1:500, mouse monoclonal; Santa Cruz Biotechnology, USA) were examined using the ECL-PLUS system (CWBIO, China) and imaged. The β-actin levels were used as a loading control. Optical density was measured by analysing scanned images using the Image J software (NIH, USA). Changes in protein expression ratio (compared with β-actin) were determined by optical density measurements (n=3 for each rat hippocampus sample).

Histopathological examination
Sevoflurane-treated (n=6) and air-treated (n=6) rats (P7–8, 16–17 g) were killed for the Nissl staining at 6 h after a 6 h exposure to either sevoflurane or air. Animals were anaesthetized with a lethal dose of 10% chloral hydrate and transcardially perfused with saline through the left cardiac ventricle until the liver and lungs were cleared of blood, followed by 4% paraformaldehyde in 0.1 M PB (NaH2PO4.2H2O 2.96 g, Na2HPO4.12H2O 29 g dissolved in 1000 ml water, PH 7.4). The perfusion lasted for 15–25 min. The brains were removed and incubated overnight in the same fixative. Paraffin blocks of brain tissue (0.5 mm thick) included sections of the hippocampus at different levels along the septotemporal axis and associated areas.29 Coronal hippocampal sections 5 μm in thickness were Nissl-stained, and examined under a light microscope (Nikon ECLIPSE, 50i, Japan) to study the morphological changes of pyramidal neurones in the CA1 and CA3 regions of the hippocampus. We counted cells imaged from three sections per animal (n=3 for each group). Nissl-positive cells were counted only if the structures were of the appropriate size and shape, possessed a Nissl-positive nucleus and cytoplasmic Nissl-positive particles. The number of Nissl-positive neurones in the pyramidal cell layers of the bilateral CA1 regions was counted at ×400 magnification by two individuals in a blinded manner.30 Questionable structures were examined under ×1000 magnification and were not counted if identification remained uncertain.

Statistical analysis
Values are presented as mean (sem). The SPSS 13.0 software was used for statistical analysis. We tested for normality using the Shapiro–Wilk test and homogeneity of variance by Levene's test. Comparisons of means between two groups were performed using Student's t-test or the Wilcoxon W-test. Statistical significance was assessed using multivariate analysis of variance followed by the Bonferroni multiple comparison testing. When appropriate, 2×2 comparisons were made using a least significant difference test. P-values of ≤0.05 were considered statistically significant.