Experimental animal studies were performed according to the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines. Animal experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University, Shanghai, China. A total of 78 adult male ICR mice weighing 25-30 grams were purchased from Jie Si Jie Laboratory Animal Co., Ltd. (Shanghai, China) and housed in a standard facility with 12-hour light-dark cycle, free access to the food and water, ambient humidity of 20∼50%, and temperature of 21∼25°C. Mice were randomly assigned to 3 groups, sham group (n=12), transient middle cerebral artery occlusion (tMCAO) group (n=40) and tMCAO with semaglutide treated group (n=26). Experimental designs were summarized in Fig. 1a.
Transient middle cerebral artery occlusion (tMCAO)
The mouse model of tMCAO was established using a method described previously . Briefly, mice were anesthetized with 1.5-2% isoflurane in a mixture of oxygen/nitrous oxide (30%/70%). First, the common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were carefully separated. Then, a 6-0 nylon suture (Dermalon, 1756-31, Covidien) coated with silica gel was inserted through an incision in the ECA, into the ICA, and advanced until the suture tip reached the bifurcation of the middle cerebral artery (MCA). The inserted suture length was about 0.95 0.05 cm. The success of occlusion was confirmed by the decrease of surface cerebral blood flow (CBF) in the MCA territory to 20% of baseline CBF using a laser Doppler flowmetry (Moor Instruments, Devon, UK). Reperfusion was performed by withdrawing the suture 90 minutes after occlusion. The success of reperfusion was confirmed by more than 70% CBF recovered, compared to the baseline. Animals failed in occlusion or reperfusion were excluded from the study.
Semaglutide was purchased from China peptides Ltd. Company (Shanghai, China). The amino acid sequence of semaglutide is HXEGTFTSDVSSYLEGQAAKN6-(N-(17-carboxy-1-oxoheptadecyl)-L-gammaglutamyl-2-(2-(2-aminoethoxy)ethoxy)acetyl-2-(2-(2-aminoethoxy) ethoxy)acetyl)EFIAWLVRGRG-OH . The semaglutide treated mice received 200 μl of semaglutide solution at a dose of 30 mg/kg at 2 hours after reperfusion via intraperitoneal (i.p.) injection, followed by the same dose injection every 5 days. The control group received 200 μl of PBS i.p. injection at 2 hours after reperfusion, followed by the same dose injection every 5 days.
Neurobehavioral tests were carried out by an investigator blinded to the experimental design using the modified neurological severity score (mNSS), hanging wire test, and rotarod test. mNSS was performed at 1, 3, 7, 14, 21 and 28 days after tMCAO. Hanging wire test and rotarod test were carried out at 3, 7, 14 21 and 28 days after tMCAO. mNSS was a composite score of motor, reflex, and balance tests. The severity score was graded at a scale from 0 to 14, where 0 represents normal and 14 indicates the most severe injury .
Hanging wire test was used to assess muscle function and motor coordination . In the hanging test, mice were hung on a horizontal wire. The wire was 1.6 mm in diameter, 50 cm in length, and elevated at 30 cm above the floor. Each mouse was given a score of 10 at the beginning of the test. The mice were then scored based on the number of times they reached the terminal (earn one point) and the number of falls (loss one point) in 180 seconds. If the mouse reached the terminal without falling in one trial, no score was given or taken. Therefore, the highest score an animal could receive is 10. The average score of three 180-second tests was used for analysis. A Kaplan-Meier-like curve was created using the scores, in which 10 indicates best muscle function and motor coordination, while a lower score indicated worse muscle function and motor coordination. Holding impulse (s*g), which was calculated by multiplying body mass (g) and hanging time (s), was used as an outcome measure of overall limb strength.
Rotarod test was used to evaluate motor coordination and balance . Mice were trained for 3 consecutive days before tMCAO. On the first and second day of training, the mice were placed on the non-rotating rod to adapt for 1 minute, then the rod was accelerated to 20 revolutions per minute (rpm) and maintained for 5 minutes. On the third day of training, baseline test after two training sessions was carried out. The rotating rod was accelerated to 40 rpm while the mice were monitored and the fell off rate within 5 minutes was recorded and analyzed.
Brain infarct volume and atrophy volume measurement
Six mice from each group were euthanized with an excess of 10% chloral hydrate at 3 days and 14 days after tMCAO. Mouse brain samples were collected after intracardial perfusion with 0.1 ml/L PBS followed by 4% paraformaldehyde (PFA, Sinopharm Chemical Reagent, Shanghai, China). Brains were immediately placed in 4% PFA for 6 hours at 4°C and then transferred to 30% sucrose in PBS until the brain sank to the bottom. Processed mouse brains were placed in −42°C pre-chilled isopentane for 10 min and then stored at −80°C. The pre-OCT-embedded brains were cut into floating coronal sections at a thickness of 30 μm from the anterior commissure to the hippocampus and stored in a 24-well plate containing antifreeze (Meilunbio, Dalian, China). Cresyl violet staining was carried out by sampling 16 sections spaced 300 μm apart that collectively spans the entire injury region for each mouse. The selected brain slices were mounted on glass slides and air dried. The sections were then stained in 0.1% Cresyl violet solution (Meilunbio, Dalian, China) followed by de-staining in ethyl alcohol. The ratio of staining in the ipsilateral and contralateral hemispheres was calculated using ImageJ (National Institutes of Health, Bethesda, MD).
Infarct volume were calculated by the following formula: V =∑h/3 * [ΔSn + (ΔSn * ΔSn+1)1/2 + ΔSn+1, V represents volume. ΔS were calculated by subtracting the normal area of the ipsilateral hemisphere from the contralateral hemisphere area. ΔSn and ΔSn+1 represent the infarct areas of two adjacent sections. h represents the thickness between two adjacent brain slices sampled (h=300 μm) .
Immunostaining and quantification
Brain slices or cell slides were fixed with 4% PFA for 10 minutes and incubated in 0.3% Triton X-100 solution for 10 minutes followed by blocking with 10% BSA for 1 hour at room temperature. Astrocytes were incubated with antibodies against C3d (1:100, AF2655, R&D system, Minneapolis, MN), S100A10 (1:100, AF2377, R&D system), and glial ﬁbrillary acidic protein (GFAP, 1:200, AB5804, Millipore, Burlington, MA). Microglia were incubated with antibodies against Iba-1 (1:200, NB100-1028, Novusbio, CO), CD16/32 (1:200, 553141, Thermo fisher scientific, MA), and Arginase (1:200, SC-271430, Santa Cruz Biotechnology, CA). Brain sections were incubated with antibodies against occludin (1:100, 33-1500, Invitrogen, Carlsbad, CA), ZO-1 (1:100, 61-7300, Invitrogen), claudin-5 (1:100, 35-2500, Invitrogen), CD31 (1:200, AF806, R&D Systems) overnight at 4°C. After being washed three times in PBS, brain sections and cell slides were incubated with fluorescent conjugated secondary antibodies for 1 hour at 37°C. The fluorescent images were collected with a TCS SP5 Confocal Scanning System (Leica, Solms, Germany).
Brain sections stained for occludin, ZO-1, or claudin-5 were analyzed for tight junction gap length, which was presented as a percentage (%) of whole tight junction staining. GFAP staining results were analyzed by comparing the GFAP integral optical density (IOD). The number of C3d-positive, S100A10-positive, CD16/32-positive, GFAP-labelled, and IBA-1 labeled cells was counted from randomly selected fields in the peri-focal striatum of the ipsilateral hemisphere in three mice from each group. For each mouse, four-section were evaluated and three fields per section were sampled. The data were analyzed using ImageJ (National Institutes of Health, MD) and Prism Graphpad 8 (GraphPad Software, San Diego).
Western blotting analysis
The ischemic tissue from the ipsilateral hemisphere of the striatum was dissected and lysed in the protein lysis buffer (RIPA, protease cocktail inhibitor and phosphatase inhibitor) on ice immediately after brain sample collection . The protein concentration of each protein sample was determined by a BCA kit (Meilunbio, Dalian, China). An aliquot of 30 μg of total protein from each sample was loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and subjected to electrophoresis. The protein samples on the SDS-PAGE gel were transferred to polyvinylidene fluoride membrane (Merck KGaA, Darmstadt, Germany). The membrane was blocked with 5% non-fat milk for 1 hour at room temperature and incubated with the primary antibodies against GFAP (1:4000, AB5804, Millipore, MA), C3d (1:1000, AF2655, R&D system), and β-actin (1:1000, 66009, Invitrogen) overnight at 4°C. After being washed three times in TBST buffer (Meilunbio), the membranes were incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Immunoblots were developed by incubating with solutions from an enhanced chemiluminescence kit (ECL, Pierce). The results of ECL were analyzed using Image J software.
Real-Time PCR Analysis
The ischemic tissue extracted from the ipsilateral hemisphere of the striatum was used to isolate total RNA using TRIzol reagent (Invitrogen). The Extracted RNA was used for real-time PCR analysis to detect the expression levels of IL-1α, TNFα, and C1q. Single-strand cDNA was synthesized through a universal cDNA synthesis kit (Qiagen, Hilden, Germany) under the conditions of 42°C for 1 hour and then 95°C for 5 minutes. The expression of RNA was tested by a fast real-time PCR system (7900 HT, ABI, Foster City, CA) using an SYBR Green master mix (Qiagen) with the following cycling conditions: 95°C for 10 minutes followed by 40 cycles of 95°C for 10 seconds and 60°C for 1 minute. GAPDH was used as the control for tissues. The primers used for real-time PCR analysis are listed in Table 1.
Primary microglia and astrocyte cell cultures
Primary microglia and astrocytes were isolated from postnatal ICR mice (JieSiJie, Shanghai, China). Briefly, the cortex was isolated from the brain of mice and then trypsinized for 10 minutes. After centrifugation, the cell pellet was resuspended in glial cell culture medium with 10% inactivated fetal bovine serum, filtered with a 70 μm filter (Millipore), and then inoculated on the culture dish prepared in advance. The mixed glial cells were kept in an incubator at 37°C and cultured for 10 days under the conditions of 95% humidity and 5% CO2. When the astrocytes were confluent, the primary microglia were isolated from the culture by briefly shaking. To obtain primary astrocytes, mixed glial cells were seeded into the petri dish and the medium was replaced with fresh culture medium every 3 days. A1 astrocytes were obtained by treating primary astrocytes with IL-1α (3 ng/ml, I3901, Sigma, MO), TNF (30 ng/ml, 8902SF, Cell Signaling Technology, MA), and C1q (400 ng ml−1, MBS143105, MyBio Source, CA) for 24 hours in vitro.
Culture of bEnd.3 cells with different types of astrocyte conditioned medium
Mouse brain capillary endothelial cell line (bEnd.3) cells were cultured under normal culture conditions, and then were cultured with different types of astrocyte-conditional medium (ACM) for 24 hours to prepare for the subsequent experiments. The sources of ACM included medium from the resting A0 astrocytes (A0-CM), from the A1 astrocytes (A1-CM) and from astrocytes treated with medium derived from LPS stimulated microglia (LPS-MCM-AS-CM).
RNA sequencing and differentially expressed gene analysis
Total RNA was extracted from cells using Trizol (Invitrogen). RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The libraries were constructed using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA). The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). The genome-wide transcriptomic analysis was performed on 4 independent experiments in the A0 astrocytes and A1 astrocytes groups. Differential expression analysis was performed using the DESeq (2012) R package. p<0.05 and fold change>2 or fold change<0.5 were set as the threshold for significantly differential expression. Gene set enrichment analysis (GSEA; https://www.broadinstitute. org/gsea/index.jsp) was performed to find differential phenotypes between A0 astrocytes and A1 astrocytes groups.
The parametric data were analyzed using Prism Graphpad 8. Comparisons between two groups were carried out using Student's t-test. For comparison among multiple groups, statistical significance between each group were examined by one-way ANOVA followed by Bonferroni correction for multiple analyses. All data were expressed as mean±standard error of the mean (SEM), and two-tail p<0.05 was considered statistically significant .