2.1 Animals
Adult male Sprague Dawley rats were used in this study (6–8 weeks old, 250 ± 20 g, Shanghai SLRC Laboratory Animal Co., Ltd., Shanghai, China). The rats were housed in a controlled condition with a 12 h light/dark cycle (6 am–6 pm, day; 6 pm–6 am,night), a controlled temperature (20 to 24˚C) and humidity (40–70%), and free access to food and water for three day to adapt to the environment. After three-days acclimatisation, all rats were randomly divided for either middle cerebral artery occlusion(MCAO) or sham surgery. The experimental protocol of this study were approved by the Animal Ethical Committee of Shanghai University of Traditional Chinese Medicine.
2.2 Establishment of PSCI model
All SD rats were fed adaptively for 3 days before surgery. During the operation, the rats were injected with 5% isoflurane(Art No. T8877, Sigma, USA) and supplemented with 1-1.5% isoflurane. The rectal temperature was maintained at 37.0 ± 0.5℃ during the operation. MCAO model was achieved by inserting a monofilament(0.32 ± 0.02 mm)(Rayward Life Technology Co., Ltd., Shenzhen, China) into the left common carotid artery of the rat via internal carotid artery. And cerebral blood flow in MCA area was monitored by laser Doppler flowmeter(Moor Instruments, Devon, USA) (Fig. 1). Effective blockade was confirmed by cerebral blood flow by ༞70%. After 90 minutes, the monofilaments were removed, skin incisions were sutured, and the rats were put back into the cage after heat preservation. In the sham operation group, the other surgical methods were the same as above except the insertion of nylon filament. After recovering from anesthesia, neurobehavioral were scored using the Longa scoring system, and rats with scores of 0 or 4 were excluded from the study. Rats with a score of 1–3 were considered as the models of success and were placed in standard conditions. After 24 hours, the rats were randomly assigned to each group.
2.3 EE paradigm and animal groups
Animals were initially assigned into two main groups, sham and MCAO. Three days after the surgery, rats were randomly divided to four subgroups: (1) sham + standard environment(SE)(n = 7); (2) sham + environment enrichment(EE) (n = 7); (3) MCAO + standard environment(SE) (n = 7) and (4) MCAO + environment environment(EE) (n = 7).
The home cage in EE group was 39cm wide×54.5cm long×20cm high and contained a ladder, plastic pipes and tunnels, running wheels and small boxes. Meanwhile, in order to simulate the rehabilitation environment of the clinical ward, the MCAO group and the sham operation group were mixed and randomly divided into the EE group (7 rats in each enriched environment cage), because stroke patients may also come into contact with healthy people, who may give them different social stimuli. Standard environment (15cm wide x 29.5cm long x 12.5cm high) is normal living conditions, no objects, 3 rats per cage.
2.4 Quantification of infarct volume
The rats were sacrificed 72 hours after the completion of cerebral ischemia surgery, and the fresh brain tissue was quickly frozen and cut into coronal sections of about 1mm. Using 2,3, 5-Triphenyltetrazolium Chloride (T8877, Sigma), TTC was dehydrogenase in living tissue Reduced to red methoxymidine products and pale staining corresponding to infarct areas. The measurement of infarct volume is available. The infarcted tissue was analyzed using digital image analysis software (Sigmascan Pro, Jandel, San Rafael, CA, USA). The infarct size of each section was measured (1mm) by subtracting the total area of the contralateral hemisphere from the non-infarct area of the ipsilateral hemisphere, and then the final infarct volume was calculated by adding up the infarct area of each section and multiplying it by section thickness.
2.5 Neurobehavioral assessment
Neurobehavioral function tests were assessed 7, 14, 28 days after MCAO by two researchers who were blinded to the test groups. The Longa scoring system was used to detect neurological impairment: the score was 0, and there was no neurological impairment. 1 point (full extension of right forepaw) mild deficiency; 2 points (rotated right), 3 points (totward right), moderate inadequacy; 4 points (inability to walk independently, decreased consciousness), serious deficiency.
2.6 Morris Water Maze (MWM) Test
The MWM test was performed 28 days after MCAO to evaluate the ability of spatial learning and memory of the rats. The test was performed as described in the protocols(15). The experiment consists of two evaluations: the space acquisition test and the detection test. All the experiments were carried out in a quiet environment. The pool is a circular pool, 122 cm in diameter, 50 cm high and 30 cm deep, divided almost into four equal quadrants. The water temperature is about 20 ~ 22℃. The 10 cm2 concealed circular platform is located in the NE quadrant and submerged 0.5 ~ 1.0 cm below the surface of the water. The water maze test will take seven days. The first day of the test was an adaptive swim, followed by five consecutive days of spatial acquisition trials. There are four trials a day. The starting position is SE, S, NW, W, as shown in Table 1. If the mouse does not reach the platform within 90 seconds, guide the rats to the platform. After the animal reached the escape table, it was allowed to remain there for 15 seconds before another experiment was conducted. The escape latency is defined as the time it takes for the animal to reach the escape platform. The probe test was carried out on the seventh day. When the escape platform was removed from the pool, the rats could swim in the pool for 90 seconds. The time the animal stayed in the target quadrant and the distance it traveled were recorded, as well as the number of times the animal crossed the previous platform area. All swim routes were monitored using image detectors and analyzed using ANY-maze software (ANY-maze, Stoelting Co., IL, USA).
2.7 Histological Analysis of WM Injury of hippocampal area
The animals were deeply anesthetized with isoflurane, followed by perfusion with saline and then perfusion with 4% paraformaldehyde in 0.1-M phosphate-buffer saline (PBS; PH 7.4). The brains were removed and placed overnight in the same fixative at 4°C, then stored in PBS with 15% and 30% sucrose until the tissue sank. Coronal sections (about 8 mm thick), including the hippocampus and cortex, were placed in an optimal cutting temperature (OCT) compound, frozen in liquid nitrogen, and then cut into 8µm sections using a microtomy-cryo thermostat. Luxol-Fast Blue (LFB) staining was used to assess the degree of demyelination or regeneration. We took 3 slices from each rat and randomly selected 3 regions of hippocampal CA3 for evaluation. Imagej is used to quantify LFB staining. The image was taken using a bright field microscope (Leitz Dialux 20, Leica) with a 5 objective lens and a Lumenera Infinity digital camera (Lumenera Corporation, Canada). The areas covered by the LFB stain were expressed as a percentage of the area of the entire field of view assessed.
2.8 Immunofluorescence Staining
For immunofluorescence, frozen sections of brain tissue were fixed in 4% paraformaldehyde (PFA) overnight. After fixing the brain tissue on the microtome with OTC, cut multiple sets of frozen sections (8µm thick, hippocampus) in a cryostat (Leica CM1950) at 24°C, and collect every 3 microtome on each Super Frost Plus slide, then store at 80°C for further dyeing. In order to perform immunofluorescence staining, the brain sections were fixed with 4% paraformaldehyde for 10 minutes at room temperature (25 ~ 26°C) and blocked with 10% BSA for 60 minutes. The slides were incubated with primary antibodies of IBA-1 (1:1000, Abcam, ab178847), MBP (1:3000, Servicebio, GB12226), NG2 (1:1000, Servicebio, DF12589) at 4°C overnight. After rinsing 3 times with PBS, we incubate the brain slices with fluorescently coupled secondary antibody (anti-mouse, 1:3000) at room temperature for 60 minutes. Images were taken using a confocal microscope (LeicaTCS SP2). For immunofluorescence staining analysis, four fields of view (200×) of each area were photographed under a confocal microscope (Leica TCS SP2), and Image-Pro Plus 6.0 (Media Cyber Netics Inc., MD, USA) aims to evaluate the proportion of positive areas.
2.9 Western Blot Analysis
We collect bilateral intact hippocampal tissue samples and sonicate them in homogeneous buffer (RIPA with protease cocktail inhibitor, phosphatase inhibitor, and phenylmethanesulfonyl fluoride). Perform SDS-PAGE on an equal amount of sample on a 10% gel, and transfer the protein to a PVDF membrane (Millipore, Massachusetts, USA). Then the membrane was incubated overnight at 4°C with the following primary antibodies: IBA1 (1:1000, Abcam, ab178847), MBP (1:3000, Servicebio, GB12226), PDGFR-α (1:750, Servicebio, GB111342) and GAPDH (1:1000, Servicebio, DF12589). After washing 3 times with TBST, the membrane was incubated with horseradish peroxide (HRP)-conjugated secondary antibody for 60 minutes at room temperature and visualized by chemiluminescence (Servicebio, G2014). The results were recorded with an imaging system (Adobe, CA, USA). The optical density ratio of the target band to GAPDH is used as the relative expression of the target protein.
2.10 Enzyme-linked immunosorbent assay (ELISA).
Blood samples were taken at 28 days after MCAO induction to separate serum. An ELISA was
performed to measure IL-1β (cat. no., RLB00, R&D Systems Inc., Minneapolis, MN, USA) levels in the serum and hippocampus by measuring the optical density (OD) at 450 nm. The experiment was repeated at least three times to calculate the average value.
2.11 Statistical analysis
Data are expressed as mean SD. Using IBM SPSS statistics 25 software, the t-test was used to compare the western blot immunofluorescence staining, and ELISA data in each group. The Mann-Whitney U test or Kruskal-WallisH test was performed for non-parametric analysis, and repeated measures one-way analysis of variance (ANOVA) followed by Scheffe's post hoc comparison was used to evaluate the differences in Longa scores, escape latency and platform crossing time among groups. In all analyses, P < 0.05 was defined as statistically significant.
Table 1
Morris water maze spatial(hidden platform)start positions
Acquistion
|
Day
|
Trial 1
|
Trial 2
|
Trial 3
|
Trial 4
|
1
|
S
|
W
|
NW
|
SE
|
2
|
NW
|
S
|
SE
|
W
|
3
|
SE
|
NW
|
W
|
S
|
4
|
W
|
SE
|
S
|
NW
|
5
|
S
|
NW
|
W
|
SE
|
6(Probe)
|
SW
|
NW, Northwest; S, south; SE,southeast; W, west. |