Animals. Thirty-six healthy SPF adult male SD rats(6–7 weeks old, 230-260g) were fed at room temperature (23 ± 1℃) and 12 hours of alternating light and dark cycles, and were free to obtain food and water. The rats were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd. Rats were randomized into four groups: the sham operation group, the ISO-1group, the cerebral I/R group, and the ISO-1 + I/R group. This experiment complies with the provisions of the Guide to Experimental Animals of the People's Republic of China and has been approved by the Animal Ethics Committee of the First Clinical College of Xinxiang Medical University (2020026). This study followed ARRIVE animal reporting guidelines.
Cerebral I/R models. The cerebral ischemia-reperfusion model was made by line bolt method, and the specific process was based on methods of Yuan Yang18. et al, and improved according to our case. Rats were anesthetized with 2% pentobarbital (2ml/kg), then the left common carotid, external carotid and internal carotid artery were isolated and ligated at the vascular bifurcation. A line bolt was removed after 2 hours ischemia and reperfusion lasted for 24 hours. No line bolt was inserted in the sham group, and no surgical procedures was performed in the ISO-1 group.
Pharmacological Treatment. When the line bolt was removed, ISO-1 dissolved with dinitrosulfoxide (DMSO) at a concentration of 1mg/kg was and intraperitoneal injected. Sham and I/R group were injected with the same dose of DMSO.
Behavioral tests. Neurological deficits was assessed using modified nerve severity score (mNSS)19 after 24 hours brain reperfusion. The score mainly detected motor ability, sensory, and reflex function. The highest score was 11, and the higher the score represented heavier degree of neurological deficit in the rats.
Infarct Volume Measurement. The brains were cut into six 2mm-thick coronal sections and incubated in 2%TTC (Shenzhen, Solarbio, China) at 37℃ for 30 minutes. After that, slices were fixed in 4% neutral formaldehyde and photographed on the next day. Pictures were analyzed using ImageJ software (NIH, USA).
Nissl's staining. Rat brains were fixed with 4% neutral formaldehyde for 48h, then embedded in paraffin and cut into 3µm-thick slices. Slices were then dewaxing in xylene and dehydrated in graded ethanol. The slices were stained with Nissl dye solution. Changes of Nissl bodies and pathomorphology of brain tissue were observed under light microscope (55i, Nikon, Japan).
Immunohistochemistry. Brain slices were dewaxed and rehydrated and put into a heat-resistant glass container containing sodium citrate buffer to a microwave oven for 10 minutes for antigen repair, and then were permeabilized in 3% hydrogen peroxide and 0.2%Triton buffer for 10 minutes. Between the two permeation process, slices were washed for 5 minutes in PBS, and the wash repeated for 3 times. 5% fetal bovine serum was used to block for 30 minutes and then slices were cultured overnight at 4℃ with Rabbit Anti-MIF Polyclonal Antibody (Abbkine, 1:200). Next day, HRP labeled Goat anti-rabbit IgG was used for incubation 1 hour. Neutral gum sealing and MIF expression in the infarct core and penumbra zone by light microscope.
Western Blotting. Cerebral ischemic penumbra tissues were collected from different groups according to the method of Stephen Ashwal. et al20. RIPA containing protease inhibitor was added to brain tissue and homogenized in a glass homogenizer. Brain protein samples were isolated by 15% polyacrylamide gel electrophoresis and then transferred to PVDF membrane. The membrane was blocked with 5% nonfat milk powder for 2 hours. Primary antibody Rabbit Anti-MIF Polyclonal Antibody (Abbkine, 1:1000), Rabbit Anti-Bax Antibody (Wanleibio, 1:1000), Rabbit Anti-Bcl2 Antibody (Wanleibio, 1:500), Rabbit Anti-Caspase3 Antibody (Wanleibio, 1:1000), Rabbit Anti-cytochrome coxidase-IV Antibody (Affinity Biosciences, 1:1000), Rabbit Anti-AIF Polyclonal Antibody (Bioss, 1:1000) were added, and stayed overnight at 4℃. Next day, the membrane was washed for 10 minutes in TBST for 3 times. Later, HRP labeled Goat anti-rabbit IgG or HRP labeled Goat anti-mouse IgG were used for incubation for 1 hour. The sample was washed with TBST again and added hypersensitive immunoblot luminescent solution. Images were photographed with a fluorescence imager (Amersham Imager600, USA), and subsequently analyzed with ImageJ software.
Terminal deoxynucleotide-transferase (TdT-)-mediated d-UTP nicked end labeling (TUNEL) staining. After brain slices were dewaxed and rewatered, 100ul of protease K working solution was added to each tissue slice, which was incubated at 37℃ oven for 30 minutes, and washed with PBS for 3 times and 5 minutes each time. TUNEL reaction mixture was added on tissue. Slices were put in a wet box to avoid light and incubated in oven at 37℃for 1 hour. Apoptotic cells were dyed red. Slices were sealed with DAPI-containing anti-fluorescence quenching tablets. Finally, brain tissue apoptosis was observed by fluorescence microscope (DM4B, Leica, Germany) and photographed for subsequent analysis.
Immunofluorescence. Brain slices were washed for 3 times with PBS for 5 minutes each after TUNEL reaction. Primary antibody Rabbit Anti-AIF Polyclonal Antibody (Bioss, 1:100), Rabbit Anti-ENDOG Polyclonal Antibody (Signalway Antibody, 1:25) were added, and slices were left overnight at 4℃. Next day, slices were incubated with FITC-labeled goat anti-rabbit IgG (Biosharp, 1:100) for 1 hour. Finally, tablets were sealed with anti-fluorescence quenching agent containing DAPI and brain tissue was visualized by fluorescence microscopy.
Statistical analysis. All data in this study were expressed with mean ± standard mean error (SEM) and analyzed using Graphpad Prism 8.0.1 software. Difference between multiple groups was analyzed using one-way analysis of variance (ANOVA), and data between two groups were analyzed using unpaired t-test. The difference was statistically significant when P < 0.05.