Experimental animals
A total of 54 aged SD male rats (Ji’nan Peng Yue Experimental Animal Breeding Co., Ltd.; production license no. SCXK (LU) 20140007; age, 18 months; weight, 540 ± 50g) were used in the current study. All animals were quarantined as required. During the quarantine period, animal activities, including diet, were observed. Animals were required to pass quarantine prior to participation in the subsequent experiment. The environmental conditions for laboratory animal feeding and management were as follows: Room temperature 20-26˚C, daily temperature difference ≤ 4°C, relative humidity 40-70%, alternation between light and dark 12/12 h. During the quarantine and experimental period, the rats had access to food and water ad libitum. During housing, animals were monitored twice daily for health status. No adverse events were observed. All experimental protocols were conducted according to the Nursing and Use Guidance for Animal Experiment Operation guidelines of the National Institutes of Health (NIH Pub. no. 85-23; revised in 1996) and were approved by the Animal Protection and Use Committee of the Affiliated Yantai Yuhuangding Hospital of Qingdao University. All sections of this report adhere to the ARRIVE Guidelines for reporting animal research [13]. A completed ARRIVE guidelines checklist is included in Checklist S1.
Animal grouping, anesthesia and administration
A total of 54 sprague dawley rats were randomly divided into 6 groups: Blank control group (Control), sevoflurane (NMPN-H20070172; Shanghai Hengrui Pharmaceutical Co., Ltd.) group (Model), DHA group (Rongcheng Baihe Biotechnology Co., Ltd.; 3 g/kg), Sev + DHA (0.3g/kg) group, Sev + DHA (1g/kg) group, Sev + DHA (3g/kg) group with 9 rats in each group.
Rats in the Model, Sev + DHA (0.3g/kg) group, Sev + DHA (1g/kg) group, Sev + DHA (3g/kg) groups were placed in a self-made transparent anesthesia box (50x40x40cm). One side hole of the box was connected to a Drager anesthesia machine (Drager Company, Germany), which administered the rats’ with 2.5% sevoflurane anesthetic for 3 h, meanwhile the heart rate (HR), respiratory frequency (RF) and blood oxygen saturation (BOS) were continuously monitored using an electrocardiogram monitor (NORDEP LTD) to eliminate brain damage caused by hypoxia. When the rats were induced into a coma, they were immediately exposed to air to wake them. After their righting reflex was recovered and they were able to move freely, the rats were then placed in the box again for another dose of anesthetic. The control group received no treatment.
Rats in DHA, Sev + DHA (0.3g/kg), Sev + DHA (1g/kg), Sev + DHA (3g/kg) groups received daily intragastric administration of DHA on days 1-10 of the experiment (10 days prior to the administration of sevoflurane), while the Control and Model group received no treatment. On days 11 - 15 rats in the Model received daily inhalation of sevoflurane for repeated anesthesia, Sev + DHA (0.3g/kg), Sev + DHA (1g/kg), Sev + DHA (3g/kg) groups received DHA administration and daily inhalation of sevoflurane for repeated anesthesia, DHA group received DHA administration, while the Control group received no treatment.
Rats in the Model, Sev + DHA (0.3g/kg), Sev + DHA (1g/kg), Sev + DHA (3g/kg) groups received sevoflurane inhalation for anesthesia or DHA administration with the Morris water maze (MWM) test was carried out from 16 - 20 days, and no drugs was administered on day 21 before the test. The body weight of rats in each group was recorded on day 1, day 7, day 14 and day 21. The experimental flow chart was shown in Figure 1.
Morris water maze experiment
Learning capability test (positioning navigation experiment)
Rat navigational abilities were assessed using Morris water maze experiments. The MWM (Institute of Materia Medica; Chinese Academy of Sciences) consisted of two parts: One round pool (120 cm in diameter; 50 cm in height; stainless steel) and one movable platform. The platform was located 2 cm below the water. The water temperature of the pool was ~25°C. The pools surface was divided into four parts and was set as four quadrants (quadrants 1, 2, 3 and 4). Within a specified period of time or when the rats begin to swim until they climbed onto the platform, the computer automatically tracked the rats' swimming trajectories and calculated the incubation period (the time when the rats located the platform in the pool) automatically using software (Etho Vision XT, Noldus Information Technology BV). The positioning navigation experiment recorded the time taken for the rats to locate the platform hidden under the water surface, and tested the spatial orientation learning ability of the rats.
Rats entered the swimming test from the center of any quadrant facing the pool wall. If the animal failed to locate the platform in the pool or climbed up the platform within 120 sec, then time taken was calculated as 120 sec. The rat was then guided and placed on the platform for 30 sec. The rats were subsequently removed from the platform and wiped dry. After the rats rested for 60 sec, they were trained for the next round. Rats were trained four times a day for five consecutive days. The average of the rats’ four training incubation periods was taken as the rat’s daily learning achievement.
Memory test (space exploration experiment)
A period of 24 h after the end of the positioning navigation experiment, and on day 21 of the experiment, the platform was withdrawn and the rats were placed in water at any of the same points of entry. The point of entry was the same as the position in the positioning navigation experiment. The rats were allowed to swim for 120 sec, and the time of target quadrant stay and the number of attempts to cross the platform were recorded.
H&E staining
After the rats' behavioral tests were completed, all animals were sacrificed by cervical spine dislocation after anesthetized by intraperitoneal injection of 1% pentobarbital sodium 40 mg/kg. Brain tissues were dissected out and placed in an ice bath, with the hippocampus being isolated from the brain. Histopathological examination was then performed using formalin fixation. The samples were embedded in paraffin and cut into 4 μm sections. The paraffin sections were dewaxed using xylene, dehydrated with serial dilutions of ethanol, stained with hematoxylin and eosin (H&E) (Beijing Solarbio Science & Technology Co., Ltd.), and rinsed in distilled water for 30 sec. The sections were washed with 95% anhydrous ethanol, soaked twice for 1 min, placed in a xylene soak three times for 5 min, and permount mounting medium (Thermo Fisher Scientific, Inc.) was used to place the samples on a coverslip, and pathological changes were observed using an optical microscope (magnification, ×100; Olympus Corporation).
Enzyme linked immunosorbent assay (ELISA)
The hippocampal tissue was collected from each group of rats and separated via centrifugation at 12,000 × g for 30 min, at 4°C. The supernatant was collected and the contents SOD, MDA and GSH-Px were detected by ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's protocol.
Immunohistochemistry
The hippocampal tissues were embedded in paraffin and sectioned (5 µm). The sections were deparaffinized with xylene twice, and rehydrated in a descending ethanol series. Endogenous peroxidase was inhibited by incubating the sections for 30 min using 3% H2O2. Antigen retrieval was performed using a citrate buffer at a high temperature for 10 min. The sections were subsequently blocked for 20 min in 5% BSA, and incubated using a primary anti‑HO-1 antibody (1:100; cat. no. ab13243; Abcam), and an anti-Nrf2 antibody (1:100; cat. no. ab31163; Abcam) overnight at 4°C. After rewarming, sections were incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:1000; cat. no. ab6721; Abcam) at 37°C for 1 h. Sections were visualized with 3, 3-diaminobenzidine tetrahydrochloride (DAB) as the chromogen (Beijing Solarbio Science & Technology Co., Ltd.). The sections were dehydrated and placed onto a coverslip with Permount mounting medium (Thermo Fisher Scientific, Inc.). The slides were observed under an optical microscope (Olympus Corporation; magnification, ×200). The data were expressed as the percentage of positive cells out of the total number of cells counted.
Western blot analysis
Total protein of the hippocampal tissues was extracted using RIPA lysis buffer containing proteinase inhibitor cocktail, and the concentration of protein was determined using a BCA assay. Proteins (50 µg) were separated using 10% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). The membranes were blocked using 5% skim milk at 4°C overnight, then incubated with primary anti Nrf2 antibody (1:1,000; cat. no. ab137550; Abcam) and anti HO-1 antibody (1:2,000; cat. no. ab13243, Abcam) at 4˚C overnight. After incubating with secondary antibody sheep anti‑rabbit IgG (1:5,000; cat. no. ab97095; Abcam) at 37°C for 1 h, protein bands were visualized using the ECL chemiluminescence system (Thermo Fisher Scientific, Inc.). Protein expression levels were normalized to β-actin (1:2,000; cat. no. ab8227; Abcam) and quantified using Image J software version 1.46 (National Institutes of Health).
Statistical analysis
All experimental data are expressed as mean ± standard deviation, and processed using SPSS 20.0 (IBM Corp.). Multiple comparisons were evaluated using a repeated measures ANOVA. A one-way analysis of variance (ANOVA) was used to compare the mean of multiple groups with a Dunnett’s post-hoc test. P<0.05 was considered to indicate a statistically significant difference.
