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

PMID: 23967080 PMCID: PMC3742769 DOI: 10.1371/journal.pone.0070645
Materials and Methods
Animal/Anesthesia treatment
The rats used in the present study were obtained from the Animal Care Center of Fudan University. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee, Fudan University. One-day pregnant female Sprague Dawley rats (weight 220–250 g) were randomly assigned to one of the three groups: control, sevoflurane, or sevoflurane with n-3 PUFAs (n = 3 per group). Fish oil, the main source of n-3 PUFAs (Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA)), was extracted from the capsule (1000 mg/capsule that containing 180 mg EPA and 120 mg DHA, Puritan's Pride, Bohemia, NY, USA) and added to food. Pregnant dams in the sevoflurane and control groups were fed a regular laboratory rodent diet with a low n-3 PUFAs concentration (0.5% of total fatty acid), whereas the sevoflurane with n-3 PUFAs group were fed the same diet, but supplemented with n-3 PUFAs (15 mg fish oil/g regular diet) from day 2 of pregnancy to 14 days after parturition. Dams were given free access to food and water, and dams for all groups were kept under identical housing conditions with a 12-h light cycle. On postnatal day 7 (P7), the rat pups in the sevoflurane and sevoflurane with n-3 PUFAs groups received sevoflurane anesthesia.

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

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

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

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

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

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


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

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

Statistical methods
All data are presented as mean ± standard deviation. We performed two-tailed t tests (assuming equal variances) to determine differences in cleaved caspase-3 immunohistochemistry and blood gas parameters between the control and sevoflurane groups. We used a one-way analysis of variance followed by Newman-Keuls post hoc tests to determine differences among groups for interactions between n-3 PUFAs or sevoflurane and cleaved caspase-3 activation, BrdU quantification, or neurobehavioral tests. For all tests, p<0.05 was considered statistically significant.