The experimental protocol was approved by the Animal Ethics Committee of Zhongnan Hospital of Wuhan University, and all experiments were performed following the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Female C57BL/6 mice (Changsha Tianqin Biotechnology CO., LTD., Changsha, China), 18 months old, and weighing 30–40 g were group-housed 4–5 per cage on a 12-hour light/dark cycle in a temperature-controlled (25 ± 2˚C) room with free access to standard rodent water and food.
The mice were randomly divided into the control group, surgery group, NPD1 group, or NPD1 + surgery group. NPD1 (Cayman Chemical, Ann Arbor, MI, USA) was given at 2 µg/ml in saline with 1.4% ethanol, i.p. 600 ng (300 µl) per mouse in NPD1 group and NPD1 + surgery group, while the equal volume of 1.4% ethanol in saline was given in control group and surgery group. One hour after administration of NPD1 or vehicle, mice in the surgery group and NPD1 + surgery group were subjected to a simple laparotomy under isoflurane anesthesia, while the mice in the control group and NPD1 group were placed in their home cages with 100% oxygen for two hours without surgery treatments. The mice had multiple behavioral tests at 24 hours before the surgery (baseline), and at 6, 9, and 24 hours after the surgery/anesthesia. Within each group, separate cohorts were subjected to assessment at each time point (n = 8–10 per cohort). To measure BBB permeability by immunohistochemistry and spectrophotometric quantification, mice were given with 10-kDa dextran i.v. at 6 hours after surgery/anesthesia, and decapitated 15 min later to harvest the brain (n = 5 per cohort). To detect inflammatory cytokine, mice were sacrificed at 6, 9, and 24 hours after the surgery/anesthesia to collect blood, hippocampus, and prefrontal cortex for Western blotting and ELISA (n = 5 per cohort). At 24 hours postoperatively, mice were anesthetized and transcardially perfused with ice-cold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. Then, hippocampal and prefrontal cortex tissues were collected for immunostaining (n = 3–5 per cohort).
A simple laparotomy was performed under isoflurane anesthesia using the methods as described in our previous studies [26, 27]. Specifically, each mouse was induced with 1.4% isoflurane in 100% oxygen in a transparent acrylic chamber. Fifteen minutes after induction, the mouse was moved out of the chamber and placed on a heating pad to maintain body temperature between 36 ℃ to 37 ℃ during the surgery. Isoflurane anesthesia was maintained via a cone device with a 16-gauge needle sensor monitoring the concentration of isoflurane. A longitudinal midline incision was made from the xiphoid to the 0.5 cm proximal pubic symphysis on the skin, abdominal muscles and peritoneum. Abdominal organs were partially exposed for 2 min, then the incision was sutured layer by layer with 5 − 0 Vicryl thread. The procedure for each mouse lasted about 10 minutes, then the mouse was put back into the anesthesia chamber for up to 2 hours to receive the rest of the anesthesia. Blood pressure was monitored with the mouse-tail blood pressure cuff (Softron BP-2010A, Softron Beijing Biotechnology Co., Ltd., Beijing, China), and blood gas and blood glucose levels were tested by a blood gas analyzer (i-STAT, Abbott Point of Care Inc., Princeton, NJ, USA). Analgesia was given 5 min before skin incision, at the end of the procedure and every 8 hours for one day postoperatively with EMLA cream (2.5% lidocaine and 2.5% prilocaine).
The behavioral changes were detected by multiple behavioral tests in the order of buried food test, open field test and Y maze test at 24 hours before the surgery, and at 6, 9, and 24 hours after the surgery as described in our previous studies . In all the tests each apparatus was cleaned with 75% ethanol after each mouse to remove odors.
Firstly, for buried food test, each mouse was given 2 pieces of sweetened cereal two days before the test in their home cage. Mice were habituated for one hour before the test by placing the home cage with mice in the testing room. The test cage was contained 3 cm deep of clean bedding and a piece of sweetened cereal pellet buried underneath 0.5 cm below the surface. The location of the food pellet was randomly changed every time. The mouse was placed in the center of the test cage for 5 minutes, and the latency to eat the food was measured as the time interval from the mouse placed in the test cage to when it uncovered the food pellet and grasped it in the forepaws and/or teeth. If the mouse failed to find the pellet within 5 minutes, the latency was defined as 300 seconds.
Secondly, for open field test, mouse was placed in the center of an open field chamber (40 × 40 × 40 centimeters) in a quiet and illuminated room and allowed to freely explore for 5 minutes. The movement parameters of the mouse were monitored and analyzed via a video camera connected to the Any-Maze animal tracking system software (Xinruan Information Technology Co. Ltd., Shanghai, China). Parameters of total distance moved, freezing time and time spent in the center were recorded and analyzed.
At last, the Y maze test was executed in a two trials task in a quiet and illuminated room, with the apparatus consists of three arms (width 8 cm × length 30 cm × height 15 cm) at 120° angles extending from a central space, and each wall of the arms were pasted with cardboards in different patterns as spatial cues. Three arms of Y maze were randomly distributed into the novel arm, which is blocked at the first trial but opened at the second trial; start arm, in which the mouse starts to explore; and another arm, which is always open. The first trial is the training trial which allowed the mouse to explore the start arm and the other arm for 10 minutes, with the novel arm being blocked. After 2 h (for the tests of 6 and 24 hours after surgery) or 4 hours (for the tests of 9 hours after surgery) time interval, the second trial as the retention trial was conducted. The mouse was placed back in the maze in the same start arm with free access to all the 3 arms for 5 minutes. A video camera, which was linked to the Any-Maze animal tracking system software, was installed 200 centimeters above the chamber to monitor and analyze the number of entries and the time spent in each arm.
Bbb Permeability Assay
The method is based on the established BBB dye-injection assay with slight modification [28, 29]. Specifically, 6 h after surgery, each mouse was injected intravenously through tail veins with 100 µl 10-kDa dextran Texas Red (Invitrogen, D1863). Fifty minutes after injection, each mouse was anesthetized and decapitated. The brains were harvested and fixed by immersion in 4% paraformaldehyde overnight at 4 ℃, then cryopreserved in 30% sucrose and frozen in TissueTek OCT (Sakura). Frozen sections of 20 µm were collected and post-fixed in 4% PFA at room temperature (20–25 ℃) for 15 min, washed in PBS and were blocked with 10% goat serum (Boster Biologic Technology, China) for 2 hours, permeabilized with 0.5% Triton X-100, then incubated with isolectin B4 (20 µg/ml, I21411, Molecular Probes, San Francisco, CA, USA) for immunostaining to visualize blood vessels. The fluorescence images of the injected tracer and isolectin were detected under 40⋅ objective lens.
Spectrophotometric quantification of extravascular 10-kDa dextran Texas Red was carried out with extracts of the hippocampus and prefrontal cortex at 6 hours after surgery. Specifically, anesthetized animals were perfused transcardially for 5 min with 50 ml PBS, then the brains were removed and homogenized in 1% Triton X-100 in PBS (100 µl/100 mg brain tissue). Brain lysates were centrifuged at 16,000 r.p.m. for 20 min and the relative fluorescence of the supernatant was measured on a fluorometer POLARstar Omega (BMG Labtech) (ex/em 595/615 nm).
Western Blot Analysis
The total protein samples from hippocampal and prefrontal tissues were homogenized using RIPA lysis buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris, 1% Triton, 0.1% sodium dodecyl sulfate, and 0.5% deoxycholate) containing protease and phosphatase inhibitors. The lysate was centrifuged at 12,000 rpm for 5 minutes at 4 ℃ to remove the sediment. The supernatants were collected, and the protein concentration was determined by a bicinchoninic acid (BCA) protein assay kit (Aspen, Wuhan, China). After the determination of the contents, the proteins were separated by SDS-PAGE (8–12%) and then transferred to PVDF membranes (Aspen, Wuhan, China). After being blocked with 5% skim milk for 1 hour at room temperature, the membranes were incubated overnight at 4 ℃ with the following primary antibodies: anti-ZO-1 (1:500, Abcam, ab96587), anti-occludin (1:2000, Abcam, ab167161), anti-claudin-5 (1:500, Biorbyt, orb214680), anti-β-actin (1:10,000, TDY Biotech, ab37168). The membranes were washed three times with TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween-20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Aspen, AS1107) for 30 minutes at room temperature. Specific immunoreactivity was detected using enhanced chemiluminescence (Aspen, Wuhan, China). The bands were measured using image analysis software (AlphaEaseFC software).
Enzyme-linked Immune-sorbent Assay (elisa)
The concentrations of TNF-α, IL-6, IL-10, IL-12 in the plasma and brain tissue of mice or the primary bone marrow-derived macrophages for in vitro experiments were determined using ELISA kits (eBioscience) according to the manufacturer’s instructions.
At 24 hours after surgery, mice were deeply anesthesia with isoflurane and perfused transcardially with ice-cold 0.1 M PBS followed by 4% PFA in 0.1 M PBS at pH 7.4. Brains were harvested and post-fixed in 4% PFA in 0.1M PBS at 4 ℃, and cryoprotected in 30% sucrose for 72 hours. Brains were freeze-mounted in TissueTek OCT (Sakura), and were cut sequentially to 20 µm. After washed in PBS and permeabilized in 0.5% Triton X-100, the coronal sections were blocked with 10% goat serum in PBS for 2 hours at room temperature to block non-specific binding, then the following primary antibodies were used: mouse anti-glial fibrillary acidic protein (GFAP) (1:500, Abcam, I21411), rabbit anti-Iba-1 (1:200, Abcam, ab178847) at 4 ℃ overnight. For secondary detection, (goat anti-mouse, goat anti-rabbit) conjugated with Alexa Fluor dyes (405 and 488) from Invitrogen (1:500) were used. Immunolabelled sections were coverslipped with 40,6-diamidino-2-phenylindole (DAPI; Invitrogen) and visualized by microscopy (Olympus, Tokyo, Japan). Five high magnifications were chosen in three non-overlapping fields randomly acquired in hippocampal subregions using a counting frame size of 0.4 mm2. Images were processed and the area of the astrocytes and microglia quantified using ImageJ software (NIH). The area of the selected cells was converted into a binary image using the dilation method and the cell outline measured. Total immunoreactivity was calculated as percentage area density defined as the number of pixels (positively stained areas) divided by the total number of pixels (sum of positively and negatively stained area) in the imaged field.
Primary Cell Culture And Grouping
Bone marrow-derived macrophages (BMDMs) were purchased from iCell Bioscience Inc (MIC-iCELL-i017, Shanghai, China), and were cultured in DMEM/F-12 containing 10% fetal bovine serum (FBS), 100 µg/mL streptomycin, and 100 U/mL penicillin and supplementary factor (iCell Bioscience Inc, PriMed-iCell- 011) 37˚C under 5% CO2 and 95% air. Cells were then stimulated with 10 ng/ml lipopolysaccharide (LPS, Sigma, L2880), with or without NPD1 (80 ng/ml) for 24 h, respectively. The same batches of BMDMs were untreated as control.
Flow Cytometry Analysis
For cell staining, anti-mouse CD 68-PE, CD16/CD32-PE-Cy7, CD206-PE (Invitrogen) were used. The cells were detached and suspended in flow cytometry staining buffer and treated with Fc-receptor blocker antibody for 20 min at 4 °C to eliminate nonspecific binding. Then the cells were stained by these antibodies for 30 min at 4 ℃ in dark. After washed twice in flow cytometry staining buffer and resuspended in the staining buffer, samples were detected using a BD FACSCalibur system was used for analysis.
The statistical analyses were performed with SPSS 19.0 (IBM, New York, USA) or GraphPad Prism 6 (GraphPad, New York, USA). Quantitative data are expressed as the means ± standard error of the mean (SEM). Statistical significance was determined using 1-way or 2-way analysis of variance, followed by the Bonferroni post hoc test. A p value less than 0.05 was considered statistically significant.