Adult male C57BL/6J mice (weight, 25–28 g) were anesthetized with 1% pentobarbital sodium (50 mg/kg, i.p.) and subjected to MCAO using nylon monofilament（Jia Ling ,China）. The nylon monofilament was slowly inserted into the lumen of the internal carotid artery through the common carotid artery and advanced from the bifurcation until it blocked the origin of the right middle cerebral artery[23, 24]. In the sham group, the nylon monofilament was not advanced, although it was introduced into the common carotid artery. The nylon monofilament was left in the artery for 60 min and then withdrawn for blood reperfusion. All animals were randomly divided into four groups: sham group, ischemia-reperfusion group, ischemia-reperfusion+TAT-W61 group, and ischemia-reperfusion+TAT-Scr group. The peptide was injected into the cortical M1 and hippocampal CA1 regions. The MNSS score was used to assess neurological deficits.
The interaction of endogenous S100b with the Rage protein was detected by Co-IP. Anti-S100b ( Santa, USA) or anti-Rage ( Santa, USA) antibody and protein A/G (Proteintech, USA) magnetic bead (30 μL; added ferrite beads to each sample) were treated with 1000 μg of total protein and incubated overnight at 4°C in a rotary mixer. Subsequently, these samples were placed on a magnetic rack for several minutes until the ferrite beads were fully absorbed. Each sample was mixed with 2× protein loading buffer and incubated in a metal bath at 100°C for 10 min; it was then analyzed by western blotting.
Analysis of brain infarct volume
The brain was removed at 24 h after MCAO, and the infarct volume was calculated according to a previously described method. Briefly, the brain was sliced into 3-mm-thick coronal sections. The sections were stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC) for 15 min and then fixed in 4% buffered formaldehyde solution. These brain sections were photographed, and the infarct part of each section was measured using ImageJ.
Western blot analysis
Proteins were extracted with the RIPA lysis buffer containing PMSF. Protein concentration was determined using the BCA kit (Beyotime Biotechnology , China ). Post-preparation was performed using the RIPA lysis buffer and 5× protein loading buffer. Electrophoresis and electrotransfer were performed sequentially. After electrotransfer, protein samples were loaded onto SDS-PAGE gels (Beyotime Biotechnology , China ) and transferred to PVDF ( Pall , USA ) membranes. After blocking with 5% skimmed milk, the samples were incubated with primary antibodies against S100b (Abcam , UK ), Rage (Abcam , UK ) , IL-1β ( Cell Signaling Technology , USA ), TNF-α ( Cell Signaling Technology , USA ) , IL-6 ( Santa , USA), Bcl2 ( Cell Signaling Technology , USA ) , Bax ( Cell Signaling Technology , USA ) , cleaved caspase-3 (1:1000 dilutions; Abclonal), caspase-3 ( Cell Signaling Technology , USA ) , and NF-κB ( Cell Signaling Technology , USA ) at 4°C overnight. Subsequently, the membranes were incubated with mouse or rabbit secondary antibodies for 2 h at room temperature and analyzed with ImageJ.
At 24 h after surgery, the mice were euthanized with pentobarbital sodium. The mice were treated with 0.9% saline followed by 4% paraformaldehyde. Mice brains were fixed with 10% paraformaldehyde. Paraffin-embedded brains were cut into 5-μm sections. The slices were stained with HE Kit (Beyotime Biotechnology , China ) and observed and photographed under an ordinary microscope.
To assess mice memory and spatial learning ability, we performed the Morris water maze experiment 7 days after MCAO. Before a formal experiment, the mice were subjected to water maze training for 4 consecutive days, including 60 s of being submerged to ascertain mice ability to find an underwater platform. The mice were randomly assigned a starting position. Mice that failed to find the platform within the specified time were guided to the platform, where they remained for 20 s. The fourth day was the testing day, and mice performance was recorded.
In the open field test, the mice were placed in a box sized 45 cm × 45 cm × 45 cm. The box was divided into central and surrounding areas, and the mice were placed at the center of the site. Video tracking software was used to record the action route of the mice in the specified time, and ImageJ was used for statistical analysis.
Contextual fear conditioning test was performed based on a previously reported method. On the first day of training, the mice were allowed to freely explore the chamber without any stimulation for 5 min. The mice were placed in the box and observed for 6 min. After 3 min, the mice were subjected to three rounds of auditory and electric stimuli of 10 s each (80 dB, 5 kHZ), and an additional electric stimulus for 2 s (0.7 mA) at the eighth second of stimulation. After the end of each mouse, the excreta in the box were wiped with alcohol. Subsequently, the mice were placed back in the box for 8 min without being subject to any stimuli. Finally, the sound fear experiment was conducted; the mice were put into the white box for 5 min. After 3 min, they were exposed to three successive auditory stimuli of 10 s each. The freezing time of each mouse was recorded and used as an index to evaluate the memory of situational fear in mice.
The rotarod test was performed to measure coordination and balance. Before the test, the mice were placed on a stick for 4 min, then the rotation speed was increased from 5 to 40 r/min within 5 min. The time on the rod was recorded until the mouse fell off the rotarod. Each mouse underwent three trials, and the mean of the observed values was recorded[15, 26-28].
Flow cytometry analysis for cell apoptosis
Experiments were performed using Annexin-V-kFluor647/PI (Keygen BioTECH , China ), and cells were collected after treatment and gently washed and blown twice with PBS. The collected cells were resuspended in 20 µl binding buffer, 5 µl Annexin-V FITC, and incubated for 5–10 min at 22-25℃. The samples were evaluated on a computer, and the results are presented.