This work was approved by Ethics Committee of Hubei Provincial Hospital of Traditional Chinese Medicine (approval no. HBZY2020-C47-01) and all experimental procedures were performed according to the Chinese Animal Welfare Legislation for protection of animals used for scientific purposes. Every effort was made to reduce animal suffering and to minimize the number of animals used in the present work. A total of 40 male adult C57BL/6 (6-week-old, 24-30 g) mice were purchased from Experimental Animal Research Center of Hubei Province. All mice were housed on a constant temperature (21 ± 3 ℃), moisture (60 ± 5%), and photoperiod (12-h light/dark cycle) condition. All mice were allowed to free access to food and water before and after surgery.
Bilateral Carotid Artery Stenosis (BCAS)
Bilateral carotid artery stenosis (BCAS) surgery was performed as previously described [7,26]. Mice were anesthetized using isoflurane/air mixture (2 l/min). All surgical procedures were conducted under aseptic condition and body temperature was maintained at 37 ± 0.3 °C using a feedback-controlled heating system (Zhongshi, inc., Beijing, China) during surgery. In brief, a small cervical midline incision was introduced, and the bilateral carotid arteries were exposed under a stereomicroscope (SZM45, Sunny, NingBo, China). A 2.5 mm-long microcoil (0.18-mm internal diameter, 0.08-mm diameter, 0.5 mm thread pitch) was placed around one-side carotid artery. Subsequently, the mouse was allowed to recover for 30 minutes. The second microcoil was placed around the other carotid artery using the same method. In groups of Sham and Quercetin (Que), surgery was carried out in the same approach except for the placement of microcoils around bilateral carotid arteiries in mice. After surgery, mice were randomly assigned into the following groups: Sham group (without BCAS/CRS, without Que administration), Que group (without BCAS/CRS, with Que administration), BCAS/CRS group (with BCAS/CRS, without Que administration), BCAS/CRS + Que group (with BCAS/CRS, with Que administration).
Chronic Restraint Stress (CRS)
Chronic restraint stress (CRS) is a common approach to introduce anxious and depressive symptoms in animal models with cerebral hypoperfusion, as previously described . Hence, the CRS was imposed to mice after BCAS surgery, and the effect of treatment on neuropsychiatric symptoms was investigated. CRS was performed to introduce cerebral hypoperfusion as previously described . A mouse restraint system was daily applied for CRS from 9:00 to 15:00 lasting for consecutive 14 days. Briefly, each mouse was put into a 50 mL plastic tube with some breathing holes, which was well ventilated. There is a hopper at the front of the tube, which allowed restrained mice free access to food and water. Restrained mice were horizontally maintained in cages during the restraint sessions. Mice in the groups of Sham and Quercetin were kept in the cages without stress.
Quercetin (Que) was purchased from Sigma-Aldrich (cat. no. Q4951, St. Louis, MO, USA). It was firstly dissolved in DMSO, then diluted in 0.9% saline. Que was intraperitoneally injected at the dose of 60 mg/kg body weight. In the Que group, mice daily received Que for 14 days. In the BCAS/CRS + Que group, mice received Que from the first day of CRS 1 hour before restraint stress once a day for 14 days. Mice in groups of Sham and BCAS/CRS received the same volume of DMSO and 0.9% saline as Que-treated mice.
For behavioral tests, all experiments and analyses were performed by an individual investigator blinded to every designed group, and detailed procedures were demonstrated as follows.
For Morris Water Maze (MWM) test, all procedures were performed 2 weeks after surgery, as described previously [28,29]. Mice were trained 5 times each day at a 20-minute interval for 5 days. In each test, mice were given 90 seconds to find the platform. Swimming was video-tracked, and the mean escape latency was recorded as a major outcome. If the latency was more than 90 seconds, mice were randomly divided into group BCAS/CRS or group BCAS/CRS + Quercetin.
For Elevated Plus Maze (EPM) test, the instrument and procedures were stated in our previous report . In brief, mice were placed in the central platform facing the open arm. Then, mice moving trace was recorded by a camera (Zhongshi, inc., Beijing, China) and analyzed using an automatic animal behavior analysis software (Yihong Technology, Wuhan, China).
For Open Field Test (OFT), mice were placed into the corner of an open arena (100 × 100 × 40 cm), and allowed to freely explore for 5 minutes. The arena was divided into 16 (4 × 4) equal-size squares and the middle nine grids (2 × 2) were considered as the central zone. Moving distance, time spent, and grooming times in the center of the arena were recorded by a camera (Yihong Technology, Wuhan, China). Thereafter, the behavioral records were analyzed by an AVTAS version 4.0 system (Yihong Technology, Wuhan, China).
For Tail Suspension Test (TST), the tail of mouse was fixed on a metal wire hook which was about 1 meter away from the ground. The immobility time of each mouse was recorded within 5 min, including the period when the whole body was shaking, the limb and head were relatively motionless. Mice were not allowed to climb up their tails and fall off the end of their tails during suspension. The total amount of immobility time was measured for each mouse, and considered as an index of “depression-like” behavior.
For Forced Swim Test (FST), mice were placed in a large graduated cylinder filled halfway with water. Primarily, mice exhibited a period of vigorous activity when the mice tried to escape. Eventually, mice gave up vigorous activity and represented a typical immobility in which they only moved to maintain its head above water. This physical immobility was considered as an indicator of behavioral despair. Investigators measured the amount of time between when mice were placed in the cylinder and the onset of immobility. Mice with depression exhibited a decrease in the time spent trying to escape.
For Sucrose Preference Test (SPT), mice were placed in their home cage for 3 days with presence of two drinking bottles (one containing plain drinking water and the other one containing 2% sucrose). Next, mice were permitted to free access to either drinking the plain water or 2% sucrose solution for a period of 4 days. Water and sucrose solution consumption was measured daily, and the position of two bottles was changed daily to reduce any confound produced by a side bias. Sucrose preference was represented as a percentage of the volume of sucrose intake over the total volume of fluid intake and averaged over the 4 days of testing.
Primary Microglia Culture
Primary microglia were harvested from postnatal day 0-2 C57BL/6 mice as previously described [30,31]. Briefly, after removal of the meninges and cortical tissues under a dissecting microscope, hippocampi were incubated in enzymatic solution (1.2 M glucose, 100 mM MgSO4, 0.25 M EGTA, 200 U or 0.47 mg/ml DNase I, 0.48 mM Papain) for digestion at 37 °C for 30minutes. Then, the samples were triturated using a ﬁre-polished Pasteur pipette and passed through a 70-µm Nylon cell strainer (Nest, Wuxi, Jiangsu, China) after they were rinsed twice with Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12; Gibco, Grand Island, NY, USA). Thereafter, the mixed cell suspension was seeded on a T75 cell culture flask precoated with poly-L-lysine (PLL, Sigma) in DMEM/F12 containing 10% FBS (Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin after being centrifuged at 1,500 rpm for 5 minutes. The cells were cultured in a constant-temperature incubator (Sanyo, Osaka, Japan) at 37 °C under 5% CO2 humidified condition. Microglia were isolated from primary mixed glial cell cultures on day 8 by shaking the flasks 1 hour at 150 rpm on a rotary shaker at 37°C. Afterwards, the detached microglial cells were reseeded into cell culture plates at a density of 5 × 105 cells/ml in complete cell culture medium. The purity of the microglia was more than 90% that were confirmed by immunostaining with the microglial marker Iba1.
For microglia treatment, Que (cat. no. S2391, Selleck, Houston, TX, USA) was dissolved in DMSO and diluted to the working concentration with culture medium. The microglia were assigned into the following 6 groups: Control, Que 30 μM, Que 60 μM, OGD, OGD + Que 30 μM; (6) OGD + Que 60 μM. The same amount of DMSO was added in each group as the groups of Que 60 μM and OGD + Que 60 μM.
Oxygen-Glucose Deprivation and Reoxygenation (OGD/R)
A hypoxia chamber was performed according to the procedures of OGD as previously described [31,2]. Before the experiment, microglial cells were washed twice by PBS and immersed in serum-and glucose-free Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA). Then, they were placed in the hypoxia chamber with a premixed gas (1% O2, 94% N2, 5% CO2) at 37°C. Two hours later, the OGD was terminated by bringing the plates back to a normoxic chamber after culture medium were exchanged to prewarmed DMEM/F12 supplemented with 10% FBS (Gibco, Grand Island, NY, USA). To determine the effect of Que on microglial cells, microglial were pretreated with 30 µM or 60 µM Que for 2h prior to OGD and presented during the whole experiment of OGD/R. Microglia in Control group were incubated under normal condition during the procedures without exposure to OGD.
Myelin Fragments Isolation
The myelin fragments were isolated as previously described . In short, hippocampi were dissected from whole brains collected from five young adult C57BL/6 mice and myelin fragments was isolated using a discontinuous sucrose gradient. Hippocampi were homogenized using 20 ml 0.32 M sucrose and 10 ml of lysis was layered between 18.5 ml 0.85 M sucrose and 8.5 ml 0.32 M sucrose. Thereafter, the suspension was ultracentrifuged at 75000 g for 20 minutes without brake. Subsequently, the interface was collected, homogenized in 35 ml distilled water, and ultracentrifuged at 75000 g for a further 15 minutes. The supernatant was aspirated and the pellet re-suspended in 35 ml distilled water. Afterwards, the suspension was ultracentrifuged at 12000 g for 10 minutes. The pellet was re-suspended in 10 ml 0.32 M sucrose, layered over 20 ml 0.85 M sucrose in a clean tube and then ultracentrifuged at 75000 g for 30 minutes. Finally, the interface was collected and centrifuged at 16000 g for 15 minutes to collect the myelin fragments that was stored at -80 °C for future experiments under sterile condition.
Transmission Electron Microscopy (TEM)
TEM was carried out as previously described . The mouse ventral hippocampi (vHIP) tissues from each group were incubated in 2.5% glutaraldehyde for 4 hours at 4℃. Then, the samples were fixed in 1% citric acid for 2 hours at 20 °C. Afterwards, the specimens were dehydrated with gradient acetone after soaking in uranyl acetate. Thereafter, samples were embedded with epoxy resin and sliced into 70 nm, and counterstained with lead citrate after placing on the copper trough grid. Subsequently, the ultrastructure of axon caliber and myelinated axons was observed using a transmission electron microscope Tecnai G220 (FEI, Hillsboro, OR, USA). Myelinated axons were counted in all 5 images per vHIP, and the G-ratio and axon caliber of all myelinated axons perpendicular to the field of view were determined using ImageJ and the ImageJ distribution Fiji, as previously described . All data were analyzed and counted by two investigators blind to sample information.
Klüver-Barrera Luxol Fast Blue (LFB) Staining
The frozen brain sections from each group were soaked in 0.1% Luxol Fast Blue (LFB) staining solution overnight at 28 °C. Then, the surplus dye solution was rinsed with 95% ethanol on the next day. Next, the samples were differentiated in 0.05% Lithium Carbonate Solution for 30 seconds after being washed with distilled water. Thereafter, the specimens were re-differentiated with 70% ethanol and transparented by xylene. Afterwards, the slides were observed using a light microscopy after being mounted with resinous medium. The spared myelin in white matter was examined using Image pro-plus (Media Cybernetics, Silver Spring, USA). The classification score according to the degree of myelin morphology damage was as follows: grade 0 (0 points), the myelin fibers were arranged in an orderly manner; grade 1 (1 point), the myelin fibers were arranged disorderly; grade 2 (2 points), vacuoles were formed in the myelin sheath; grade 3 (3 points): Myelin fibers disappeared.
The whole lysate was extracted from microglia or hippocampi in different groups, and the concentration was determined by a BCA kit (Beyotime, Shanghai, China) according to the manufacturer’s instruction. Then, samples were separated by electrophoresis using 8% or 10% SDS-PAGE, and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA). Subsequently, membranes were incubated in primary antibodies at 4°C overnight after they were blocked with 5% skim milk or 5% BSA at room temperature for 2 hours. Thereafter, membranes were incubated in horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 2 hours after rinsed twice with TBST. The signals for every band were detected using ECL luminous fluid (Beyotime, Shanghai, China) by a ChemiDoc™ XRS+ imaging system (Bio-Rad, California, USA). Densitometric measurement of each membrane was performed using ImageJ. The primary antibodies used in the present study were as follows: MBP (1:1000, cat. no. 78896s, Cell Signaling Technology, Danvers, MA, USA), MAG (1:1000, cat. no. 114386-1-AP, Proteintech Group, Inc, Wuhan, China), MOG (1:1000, cat. no. 12690-1-AP, Proteintech Group, Inc, Wuhan, China), IL-4 (1:1000, cat. no. 66142-1-Ig, Proteintech Group, Inc, Wuhan, China), IL-10 (1:1000, cat. no. 60269-1-Ig, Proteintech Group, Inc, Wuhan, China), GAPDH (1:1000, cat. no. 60004-1-Ig, Proteintech Group, Inc, Wuhan, China), iNOS (1:1000, cat. no. 13120s, Cell Signaling Technology, Danvers, MA, USA), or Arg1 (1:1000, cat. no. 93668s, Cell Signaling Technology, Danvers, MA, USA), TNF-α (1:1000, cat. no. 11948, Cell Signaling Technology, Danvers, MA, USA), or IL-1β (1:1000, cat. no. 63124, Cell Signaling Technology, Danvers, MA, USA).
For immunofluorescence, coverslips or frozen brain sections from each group were post-ﬁxed using 4% paraformaldehyde (PFA) in 0.01 M phosphate buffer saline (PBS) for 30 minutes at room temperature after being twice washed with PBS. Then, the samples were incubated in 5% bovine serum album (BSA; Beyotime, Shanghai, China) solution supplemented with 0.3% Triton X-100 (Solarbio, Beijing, China) in PBS at room temperature for 2 hours. Thereafter, specimens were incubated in MBP (1:1000, cat. no. 78896s, Cell Signaling Technology, Danvers, MA, USA), iNOS (1:200, cat. no. 13120s, Cell Signaling Technology, Danvers, MA, USA), Iba1 (1:300, cat. no. 17198S, Cell Signaling Technology, Danvers, MA, USA), or Arg1 (1:200, cat. no. 93668s, Cell Signaling Technology, Danvers, MA, USA) overnight at 4˚C. On the next day, the specimens were incubated in Alexa Fluor® 555 or 488-conjugated secondary antibody (1:300; cat. nos. A0453 and A0423; Beyotime, Shanghai, China) at room temperature for 2 hours. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, Munich, Germany) at room temperature for 10 minutes. Subsequently, sections were mounted onto slides and images were captured using a fluorescence microscopy (Zeiss, Göttingen, Germany). Meanwhile, blocking buffer without the primary antibodies was used as the negative control. Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used for semi-quantitative analysis of immunostaining images. For each sample, at least twelve slices were analyzed, and the cross-sectional areas were calculated and reported as the average of four independent measurements. All measurements were performed by an individual investigator who was blinded to sample information.
Enzyme Linked Immunosorbent Assay (ELISA)
The secretion of cytokines of IL-1β, TNF-α, IL-10 and IL-4 was determined using ELISA. The same amount of 100 μl supernatant from each group were used to quantify the concentration of IL-1β (cat. no. DY401, R&D Systems, Minneapolis, MN, USA), IL-4 (cat. no. M4000B, R&D Systems, Minneapolis, MN, USA), TNF-α (cat. no. DY410, R&D Systems, Minneapolis, MN, USA), and IL-10 (cat. no. M1000B, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Each experiment was repeated six times.
Data were presented as mean ± SE, and analyzed using GraphPad Prism 6.0 software (GraphPad Software, Inc., San Diego, CA, USA) or SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). The normal distribution of data was tested a using a Shapiro–Wilk normality test, and the significance of difference was determined by two-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Statistical significance was represented as p < 0.05 (*/#), or p < 0.01 (**/##).