Animals
C57BL/6J male mice (8-10 weeks, 23-25g), purchased from SLAC Company (Shanghai, China), were housed 5 per cage under the vivarium with constant temperature (23±1°C) and controlled light (12-h light/12-h dark cycle). All animals had free access to food and water. The Soochow University Committee on Animal Care approved the animal procedures which were performed following the NIH Guide for the Care and Use of Laboratory Animals. Details of animal use could be found in Figure legends.
Focal Cerebral Ischemia and Reperfusion model in mice
During surgical procedures, C57BL/6 mice were anesthetized with isoflurane (4% for induction, 1.75% for maintenance) in a mixture of N2 and O2 (70% N2: 30% O2), with the body temperature kept constant at 37.5±0.5°C by a heating pad. To mimic cerebral ischemic stroke, middle cerebral artery (MCA) occlusion (MCAO) was induced using an intraluminal monofilament as described previously (Gu et al. 2012). To summarize, the right common and internal carotid arteries were isolated and ligated through a midline incision of the neck under a microscope. A 6-0 nylon monofilament thread with silicon-coated tip was inserted into the right internal carotid artery through the common carotid artery, blocking the blood flow to MCA. After 90 min of occlusion, the thread was removed to allow reperfusion for 24 h. After completion of the surgical procedures, the incision was sutured and the mice were placed in a controlled temperature condition (24-25°C) to recover from anesthesia.
Drug administration and experimental groups
After MCAO, mice were randomly assigned into five different treatment groups: 1,“Saline”(containing 2% DMSO) group; 2, PNU282987 (a potent agonist of α7 nAChR,10 mg/kg) group; 3, Melatonin (15 mg/kg) group; 4, Melatonin+methyllycaconitine (MLA, an antagonist of α7 nAChR, 6 mg/kg) group; 5, MLA (6 mg/kg) group. The mice were intraperitoneally administered with either saline or one of the four treatments at the onset of reperfusion.
Assessment of Evan’s blue (EB) dye leakage
Leakage of EB dye from brain is a marker of BBB disruption (Sun et al. 2017). Hence, BBB disruption was determined by assessing the extravasation of EB dye (Sigma). EB (2%) was injected (3 ml/kg) through the tail vein after 22 h of reperfusion. After 2 h of the EB injection, mice were perfused transcardially with ice-cold PBS, then the brain was quickly removed and sliced into 1 mm coronal sections with a brain matrix. EB leakage appeared as blue on brain sections and quantitative assessment was done by detecting the EB contents (Liu et al. 2017). In brief, ischemic and non-ischemic brain hemispheres were weighed and homogenized in 50% wt/vol trichloroacetic acid (Sigma). After centrifugation (14000 g for 15 min) at 4°C, the supernatant was collected, and the OD values of supernatants were measured at 620 nm on a microplate fluorescence reader (Infinite M200 Pro; TECAN, Grodig, Austria). The quantity of EB was calculated according to the gradient concentrations of EB standard curve. The dye content (μg) detected in each sample was quantified as EB leakage and expressed as per gram of brain tissue (μg/g). The EB leakage was analyzed by investigators that were blind to treatment group designation.
Evaluation of BBB permeability by detection of immunoglobulin G leakage
Immunoglobulin G (IgG) leakage is another method to evaluate BBB permeability. As we described previously (Wang et al. 2016), the 20-μm-thick cryosection was fixed with 4 % paraformaldehyde (PFA) for 20 min, then incubated with Cy3-conjugated Affinity Pure Goat anti-Mouse IgG (1:500, Jackson, 112-165-167, USA) for 2 h. Immunostaining was visualized in an LSM 700 microscope (Carl Zeiss) and images were obtained.
Immunostaining for CRTC1, HMGB1, Iba-1, NeuN, occludin, p-CREB
The 20-μm-thick frozen slices were fixed with 4 % PFA for further analysis as we described previously (Zhang et al. 2019). In brief, the sections were blocked with 5 % goat serum for 2 h to inhibit nonspecific binding and incubated with primary antibody against HMGB1 (1:800; Abcam, ab79823, UK), CRTC1 (1:100; Cell Signaling Technology, #2587, USA), occludin (1:100; Invitrogen, 711500, USA), Iba-1 (1:2000; Wako, 019-19741, Japan), NeuN (1:800; Merck Millipore, MAB337, USA), p-CREB (1:100; Cell Signaling Technology, #9198, USA) overnight at 4°C. After being washed with PBS 3 times, sections were incubated with 488- or Cy3-conjugated secondary antibody (anti-rabbit, 1:800, Life Technology, A11008, USA; anti-mouse, 1:800, KPL, 072-01-18-06, USA) for 2 h in dark at room temperature, followed by DAPI staining for 5 min. Color images were snapped by investigators that were blind to treatment group designation using a confocal microscope (Zeiss LSM 700, Carl Zeiss). Neuron number was blindly measured in images captured from non-ischemic and ischemic hemispheres using Image J.
Western blot analysis for HMGB1, CRTC1, p-CREB, and occludin
Tissues in ischemic (I) and non-ischemic (NI) hemispheres were achieved at 24 h after MCAO (Yang et al. 2018). Proteins (30 μg of total protein) were boiled and electrophoresed in 10 % or 12 % SDS-PAGE acrylamide gels. Then the proteins were transferred onto PVDF membrane (Millipore, Billerica, MA, USA), and blocked for 2 h in TBS-T containing 5% nonfat milk. The membranes were incubated with primary antibodies against HMGB1 (1:20000; Abcam, ab79823, UK), CRTC1 (1:1000; Cell Signaling Technology, #2587, USA), p-CREB (1:1000; Cell Signaling Technology, #9198, USA), or occludin (1:300; Invitrogen, 711500, USA) overnight at 4°C, washed in TBS-T, and then followed by incubation with corresponding HRP conjugated anti-rabbit (1:3000; Boster, BA1054, China) or anti-mouse antibodies (1:3000; Boster, BA1050, China) for 2 h at room temperature. The protein bands were developed with an enhanced luminescence reagent (Millipore) and photographed with ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA). The intensities of protein band were quantitated via normalization to β-actin as the expression of the ratios of target proteins/β-actin.
TUNEL assay
The TUNEL Apoptosis Detection Kit (Yeasen, 40307ES20, China) was used to detect apoptosis cells according to the instructions from the manufacturer. Briefly, at first, brain sections were re-hydrated and nuclear was stripped with proteinase K. A mixture of 488-labeled nucleotides and terminal deoxynucleotidyl transferase was applied onto brain sections for 60 min at 37°C in a dark humidified incubator and followed by DAPI staining for 5 min. Incubation with labeling solutions without the enzyme served as a negative control (Yang et al. 2011). Microvessels labeled with RECA-1 were counted in images captured from ischemic hemispheres by using National Institutes of Health Image J. Indicators of animal identity on slides were blinded to the investigator. The number of TUNEL cells was calculated as the mean of the numbers per mm2 obtained from the imaged sections.
Statistical analysis
The data were expressed as mean ± SEM. The theoretical normal distribution of values was calculated. T-test was used for within-group or two-group comparison, and one-way ANOVA with Newman-keuls comparison post hoc test was used to evaluate the difference between groups. All statistical analyses were performed with the SPSS 17.0 software, and plotting was carried out by GraphPad Prism software version 5.0. Differences were considered to be significant when P<0.05.