Establishment of animal model
A total of 348 C57BL/6J male mice (12 to 16-week-old; 25-28g; Jackson Laboratory, stock no. 000664) were purchased from the Animal Center of the Third Military Medical University. Mice were housed in a pathogen-free environment and had free access to food and water with the light/dark cycle of 12:12 hours.
As previously described, the MCAO model was used to generate focal unilateral cerebral ischemia . Briefly, C57BL/6 mice were anesthetized using a 2% isoflurane/air mixture (1-2 L/min). A vertical midline incision exposed the submandibular glands on the neck. The carotid sheath was removed, and the common carotid artery was separated to avoid accidental injury to the vagus nerve. A 2.0-cm silicone-coated nylon suture with a diameter of 0.23mm (Sunbio Co., Ltd., Beijing, China) was gently inserted from the external carotid artery stump internal carotid artery, which terminated at the beginning of the middle cerebral artery. The cerebral blood flow was blocked for 60 minutes. The same procedures were performed on sham group mice apart from occlusion. The temperature of experimental animals was monitored during the surgery and the recovery from anesthesia. All surgeries and tests were carried out randomly by blinded researchers.
Experimental grouping and design
To evaluate the recovery of motor function in MCAO animals treated with different ART concentrations, mice were randomly sorted into five groups: Sham (n=10), MCAO+Veh (saline, n=10); MCAO+50mg/kg ART (n=10); MCAO+150mg/kg ART (n=10) and MCAO+250mg/kg ART (n=10). The ART (Holley-Wulingshan pharmaceuticals Ltd., Chongqing, China) was given intraperitoneally once per day, and the first dose was given immediately after reperfusion. Furthermore, each group's recovery of forelimb motor function was assessed using cylinder task, grid-walking task pasta handing, and single-pellet retrieval task. Animals were tested once on these tasks 1 week before surgery to establish baseline performance levels and were then tested at 3 and 7 days post-MCAO. Behaviors were scored by observers blinded to the grouping (Fig S1A of Supplemental Material). Detailed descriptions were presented in supplementary materials.
To further examine the efficacy of ART and investigate the underlying mechanisms of its neuroprotective roles, the established MCAO model mice were randomly grouped: Sham, MCAO+Veh (saline; Ad-Flag,), MCAO+ART (150mg/kg; Ad-Flag) and MCAO+ART +FOXO3aOE (150mg/kg/day; Ad-TM-FOXO3a). T2-weight MRI was used to measure the cerebral infarcted volume. Diffusion tensor imaging (DTI), transmission electron microscopy, and immunofluorescence assay were performed to examine white matter injury. Immunofluorescence staining and western blotting (WB) were carried out to assess the proliferation of NSPCs in the SVZ and the perilesional cortex, which revealed the proliferation and migration of NSPCs after MCAO. FOXO3a/p27kip1-associated molecules' expression levels were determined at three days post-MCAO (Fig S1B of Supplemental Material).
To further investigate ART's effects on the stroke model, the three experimental groups: sham, MCAO+Veh, and MCAO+ART, were assessed by comparing by neuro-function, infracted volume, and white matter injury. Additionally, MCAO+ART and MCAO+ART+FOXO3aOE groups were also examined to elucidate the underlying mechanisms of ART-induced neuroprotective roles.
Magnetic resonance imaging (MRI) and diffusion tensor imaging (DTI）
Mice were anesthetized using a 2% isoflurane/air mixture before MRI. Serial and T2-weighed imaging was carried out on 16 coronal slices (thickness= 1.0mm) using a 7.0-T Varian MR scanner (field of view = 35x25mm; Bruker Analytik GmbH, Karlsruhe, Germany) at 72h after MCAO.
DTI assay enabled the visualization and characterization of white matter fibers in three dimensions and was performed as previously described . Diffusion Toolkit software (Harvard Medical School, Boston, US) was used to track white matter fiber bundles and calculate apparent diffusion coefficient (ADC) and the fractional anisotropy (FA) values in the ipsilateral internal capsule. The FA value (Ipsil/contral) was calculated as (FA value at ipsilateral IC)/ (FA value at contralateral internal capsule) x100%.
2, 3, 5-triphenyl tetrazolium hydrochloride (TTC) staining
At 72h after MCAO, TTC staining was performed as previously described. Brains were immediately removed following anesthetization, sectioned into coronal slices with 1mm intervals. The slides were further incubated with 2% 2,3,5-triphenyl tetrazolium hydrochloride (Sigma-Aldrich, St. Louis, MO) at 37˚C for 30 min in the dark.
Transmission electron microscopy
At 72h after MCAO, mice were anesthetized and perfused with pre-cooled 0.01M PBS and 4% formaldehyde. After the perfusion, brain tissues were obtained and immersed in glutaraldehyde. The per-infracted cerebral cortex was immediately removed from the solution. Subsequently, the white matter around the cerebral infarction was homogenized into small pieces (~1mm3) and then stored in 2% formaldehyde and 2% at glutaraldehyde at 4°C overnight. After dehydration, samples were impregnated with epoxy resin, sectioned, and then double-stained using lead citrate and uranyl acetate. Images were captured with an H-7100 transmission electron microscope (Hitachi, Tokyo, Japan) as previous described .
Bromodeoxyuridine (BrdU) injection and incorporation assay
100 mg/kg of BrdU was administered intraperitoneally every 24 h, and the first dose was given immediately after MCAO. The coronal sections (thickness=25 μm) were stained. Staining of BrdU and DCX double-positive cells in the peri-infarct and SVZ regions was visualized on the confocal images. Cell counting was performed on four slices for each brain to calculate the relative percentage of stained cells in the two areas.
Treated specimens of the peri-infarct brain region and SVZ were incubated with corresponding antibodies against BrdU (1:500, Millipore, Germany), DCX (1:500, Abcam, UK), Tuj-1 (1:1000, Abcam, UK) and Nestin (1:500, Abcam, UK) at 4°C overnight. The images were captured using a confocal microscope (Carl Zeiss, LSM780, Weimar, Germany)
Protein samples were extracted from the peri-infarct cortex of MCAO mice, separated on a 10% Bis-tris selective denaturing gel, and then transferred onto a PVDF membrane (Millipore Inc., MA, US). Bovine serum albumin (3%) in TBST was used to block the membranes incubated at room temperature for 2 hours. The primary antibodies were then incubated: rabbit polyclonal antibody against Foxo-3a (R&D, 1:1000), mouse monoclonal antibody against p27kip1 (CST, 1:2000), rabbit polyclonal antibody against Cyclin E (CST, 1:1000), rabbit polyclonal antibody against CDK2 (CST, 1:1000), rabbit polyclonal antibody against Nestin (Abcam, 1:2000) or mouse monoclonal antibody against GAPDH (Zsgb-bio, 1:1000) at 4˚C overnight. The following day, the membrane was rinsed three times with TBST to remove excessive primary antibodies. Subsequently, the membrane was further incubated with HRP-labeled secondary antibody (Zsgb-bio, 1:5000) at room temperature for 2 hours. The signals were detected using an ECL reagent (Thermo Scientific, US) and visualized on the ChemiDoc™ XRS+ imaging system. Protein bands were quantified by densitometry using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, US).
An adenoviral vector expressing constitutively active FOXO3a(AD-TM-FOXO3a) was adopted; thus, TM-FOXO3a could not be phosphorylated by p-Akt. Adenovirus Ad-TM (triple mutant)-FOXO3a (FOXO3aOE) and negative control Ad-Flag were purchased from Shanghai Heyuan Biological Co., Ltd. In the FOXO3a overexpression vector, three conserved Akt phosphorylation sites (Thr-32, Ser-253, and Ser-315) of FOXO3a were replaced by alanine residues to prevent phosphorylating by p-Akt. Ad-TM-FOXO3a and Ad-Flag were injected into the lateral ventricles (0.33 μl/min) using a specific coordinate (0.2mm posterior to the bregma, 2mm ventral) the skull, and 1mm lateral to the sagittal line) with stereotaxic frame 48 hours before MCAO surgery.
All data were presented as the mean ± standard error of the mean (SEM) and passed a normality test. Comparisons between two groups were analyzed using 2-tailed Student t-tests. Behavioral data collected at repeating time points were analyzed using two-way repeated-measures ANOVA, followed by the Bonferroni post hoc test. Other data were analyzed using one-way ANOVA followed by Tukey's post hoc test. The statistical analyses were conducted using SPSS v19.0 (SPSS Inc., Chicago, IL). P<0.05 was considered statistically significant.