2.1 Middle cerebral artery occlusion (MCAO)
Male C57BL/6 J mice (2-3 months old, 20-25 g) were maintained in temperature-controlled rooms with 12 h light/dark cycles where they received food and water ad libitum. A total of 238 male mice (Vital River Laboratory Animal, Co) were used in the experiment. Only male mice were included in the study design due to potential effects of sex hormones. All experiments with animals were performed following protocols approved by the Animal Care and Use Committee of Jinzhou Medical University in accordance with the Care and Use of Laboratory Animals guidelines from the National Institutes of Health. All mice were randomized for stroke studies and treatments.
The MCAO model of acute ischemic stroke was prepared as previously reported [23-25]. Briefly, anesthesia was induced with 2% isoflurane in N2O:O2 (70%:30%). The right common carotid artery and ipsilateral internal carotid artery were exposed, and focal cerebral ischemia was produced by MCAO with a surgical nylon monofilament. After 60 min, reperfusion was allowed by withdrawal of the filament. In the sham group, the mice underwent an identical procedure without inserting the nylon monofilament. Body temperature during surgery was regularly maintained at 37.0±0.5°C by a homeothermic heating pad. The mice displayed typical neurologic deficit of MCAO, circling to the non-ischemic side (left), were included in this study. A total of 28 mice were excluded in this study.
2.2 Experimental design
For the time course study, mice were assigned to five groups: (1) Sham; (2) MCAO (6 h sacrifice (sac)); (3) MCAO (12 h sac); (4) MCAO (24 h sac); (5) MCAO (72 h sac).
For the dose and outcome study, mice were assigned to seven groups: (1) Sham (72 h sac); (2) MCAO + PBS (72 h sac); (3) MCAO + rh-NMNAT1 (3μg/kg, 72 h sac); (4) MCAO + rh-NMNAT1 (10μg/kg, 72 h sac); (5) MCAO + rh-NMNAT1 (30μg/kg, 72 h sac); (6) MCAO + PBS (24 h sac); (7) MCAO + rh-NMNAT1 (30μg/kg, 24 h sac).
For the mechanism study, mice were assigned to six groups (24 h sac or 72 h sac): (1) Sham; (2) MCAO + PBS; (3) MCAO + rh-NMNAT1 (30μg/kg); (4) MCAO + NAD; (5) MCAO + Negative siRNA + rh-NMNAT1 (30μg/kg); (6) MCAO + SIRT1 siRNA + rh-NMNAT1 (30μg/kg).
2.3 Drug administration
Intranasal administration was performed as previously described [26, 27]. Mice were placed on their backs and three doses (3 μg/kg, 10 μg/kg, 30 μg/kg) of recombinant human NMNAT1 (rh-NMNAT1, Sino Biological) were given. A total 10 μl of rh-NMNAT1 dissolved in PBS was delivered 1 h post MCAO within 10 min. Mice were then further kept on their backs for an additional 5 min to allow for absorption of drug before being returned to their cage. NAD+ (Sigma-Aldrich) dissolved in PBS was administered intraperitoneally at a dosage of 100 mg/kg according to previous reports [28, 29].
2.4 Intracerebroventricular injection
Pre-designed small interfering RNA (siRNA) targeting SIRT1 (RiboBio, Guangzhou, China) were stereotaxically injected into the lateral ventricle of mice. Mice were anesthetized with pentobarbital sodium and then fixed in a stereotaxic apparatus. 24 h before MCAO, 2 μl of SIRT1 siRNA (300 pmol/μl) or a non-targeting negative control siRNA was injected into the right lateral ventricle at the point of 0.6 mm posterior to bregma, 1.5 mm lateral to bregma, and 1.7 mm ventral to the brain surface at the rate of 0.3 μl/min and the needle was kept in the place for 10 min.
2.5 Evaluation of BBB permeability in ischemic mice by Evan's Blue leakage
Evan's Blue (EB, 2% wt/vol in PBS; Sigma) was intravenously administered via tail vein. After 1 h circulation, the mouse was transcardially perfused with saline. The brain was removed and then cut into five 1-mm-thick coronal slices. Brain slices were photographed to visualize EB leakage. Quantitative assessment of BBB disruption was achieved by measuring EB content in the ischemic hemispheric tissue as we previously reported . In brief, ischemic hemispheric tissue was weighed and homogenized in 50% wt/vol trichloroacetic acid. The fluorescence intensity of the supernatant was measured on a microplate fluorescence reader. The EB content was calculated using a standard curve and expressed as ng per g of brain tissue.
2.6 Measurements of neurological deficits
Neurological deficits of mice were assessed according to neurological disability status scale (NDSS) reported by Rodriguez et al . NDSS has 10 progressive steps beyond 0 (normal), extending to status 10 (death). Motor performance was determined by the RotaRod test. Mice were trained to remain on the rod for 1.5 min at 50 revolutions/min for 3 days prior to MCAO. These mice were placed on the rotating rod after MCAO and the latency to fall was measured. Neurological deficit measurements were performed in a blinded way.
2.7 Measurements of infarct volume
After the evaluation of neurological deficits, the mouse was transcardially perfused with saline, and the brain was quickly removed and sliced into 1.0-mm-thick coronal sections. The brain sections were incubated for 15 min in a solution of 0.5% 2, 3, 5-triphenyltetrazolium chloride (TTC) at 37°C, and then the brain sections were scanned into a computer. The digital images of stained sections were recorded and analyzed using ImageJ software.
Frozen 20-µm-thick brain sections were blocked by 5% normal goat serum for 1 h and then incubated with primary antibodies overnights. The primary antibodies were: NMNAT1 (1:300, Santa Cruz Biotechnology USA), occludin (1:200, Invitrogen), claudin-5 (1:200, Invitrogen), SIRT1 (1:500, Invitrogen), and CD31 (an endothelial cell marker, 1:100, Santa Cruz Biotechnology), followed by Alexa Fluor 488 or Cy3-conjugated secondary antibodies (1:4000, Invitrogen) for 2 h at room temperature. The sections were then counterstained with 4', 6-diamidino-2-phenylindole (DAPI) for nuclear labeling. Fluorescence images were obtained by Leica DMI 4000B microscope (Leica, Germany).
2.9 Isolation of cerebral microvessels from the brain after ischemia/reperfusion
To examine the impacts of NMNAT1 on cerebral microvessels, we extracted cerebral microvessels from brains of ischemic mice as previously reported [32, 33]. Briefly, at the end of reperfusion, the hemispheric cerebral tissue was minced and homogenized in ice-cold PBS. Then the homogenate was filtered through a 41 μm nylon mesh. Cerebral microvessels retained on the mesh were then purified with Dextran T-500 and stored at −80 °C until further analysis.
2.10 NAD+/NADH Assay
The NAD+ and NADH levels were measured using the NAD+/NADH Assay Kit (BioVision), according to the manufacturer’s instructions.
2.11 Western blot analysis
Antibodies used in this study were NMNAT1 (1:500, Santa Cruz Biotechnology), occludin (1:500, Invitrogen), claudin-5 (1:500, Invitrogen), SIRT1 (1:1000, Invitrogen), acetyl-NF-κB p65 (1:1000, Cell Signaling Technology), acetyl-p53 (1:1000, Cell Signaling Technology), cleaved Caspase-3 (1:500, ABclonal), MMP-9 (1:500, Invitrogen), β-actin (1:4000, Sigma) and corresponding secondary antibodies (1:5000, Invitrogen). The membranes were incubated with the primary antibody overnight and secondary antibodies for 1 h. The Enhanced Chemiluminescence (ECL) kit (GE Healthcare, UK) was employed to detect the signals. Protein levels were quantified by densitometry and normalized to β-actin, an internal standard.
2.12 Statistical analysis
Statistical analyses were performed using GraphPad Prism version 7. Data reported are presented as mean±SD. Statistical differences were evaluated with Student’s t-test between two groups or by one-way analysis of variance (ANOVA) followed by all pairwise multiple comparison procedures using Bonferroni post-hoc test. P < 0.05 was defined as statistically significant.