Materials
The I3C (#I-7256, Sigma-Aldrich, Munich,Germany) was dissolved in a solution containing dimethyl sulfoxide (DMSO), PEG300, Tween-80, and saline31.
Animals
Six- to eight-week-old male SD rats (180–220 g) were obtained from the Experimental Animal Center of Nantong University. Rats were housed under standard laboratory conditions (5 rats per cage, 12h light-dark cycle, 08:00 ~ 20:00 light on, 24 ± 1°C ambient temperature, 55 ± 10% relative humidity) for 2 weeks with free access to water and food. Animal experiments were approved by the University Animal Ethics Committee of Nantong University (Permit Number: 2110836) and were conducted in accordance with the internationally accepted guidelines for the use of animals in toxicology adopted by the Society of Toxicology in 1999.
Cell culture
Rat microglia (HAPI) were cultured in vitro in an incubator (5% CO2, 37°C) containing 10% Australian origin fetal bovine serum (10100147, Gibco, USA), 1% penicillin/streptomycin and high sugar medium (10-013-CV, DMEM, Corning, NewYork, USA).
Experimental Design and Drug Treatment
After 2 weeks of habituation, 60 rats were randomly divided into sham-operated group, MCAO group and MCAO + I3C group, 20 rats in each group. I3C was administered intraperitoneally to rats at a dose of 150 mg/kg per day on the day of surgery and on the third day of the second day after surgery.
HAPI cells of appropriate density were inoculated in 6-well plates containing complete culture medium and divided into blank group, OGD/R group and OGD/R + I3C group. There were at least 3 replicates in each group.
Establishment of the CIRI Model and OGD/R model
The procedure of the middle cerebral artery occlusion-reperfusion (MCAO/R) model is described below32. Rats were anesthetized by inhalation of isoflurane via the isoflurane delivery system. The rats were fixed in the supine position and the hair on the neck was shaved off with a razor. The rat's neck was disinfected with a cotton ball containing iodophor. Then a small incision of about 1 cm was made in the midline of the neck with surgical scissors to bluntly separate the subcutaneous tissue and muscle and expose the right common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA). The distal common carotid artery (CCA) was ligated, and the internal carotid artery (ICA) was temporarily blocked with an arterial clip. The mice in the sham-operated group were subjected to the same procedure except for obstruction. The body temperature of the rats was maintained at 37℃±0.5℃, and the basic vital signs of the rats were closely monitored.
The OGD/R model was established as described below33. HAPI microglia were inoculated in sugar-free DMEM, divided into two groups, one of which was supplemented with I3C and transferred to an incubator at 37°C, 94% N_2, 1% O_2 and 5% CO_2 for 4 hours. The sugar-free DMEM was then replaced with normal medium (10% Australian-derived fetal bovine serum, 1% penicillin/streptomycin and high sugar medium) and incubated in an aerobic incubator for 24 hours.
Neurological assessment
The modified Neurological Severity Score(mNSS) consisted of motor, sensory, reflex and balance assessments with a normal score of 0 and a maximum score of 18. Each mouse was assessed on a score of 0 to 18 on days 1, 3, 7 and 14 after MCAO surgery.
Measurement of Infarct Size
After 3 days of continuous supplementation with I3C, the rats were executed after deep anesthesia. The whole brain was quickly removed and placed in a Petri dish and dried after washing the remaining blood clots from the brain. The brain was placed in a brain tank, frozen at -20◦ C for 30 min, and cut into 2-mm coronal sections with a razor blade. The sections were immersed in 2% TTC dye for 30 min at 37°C under dark conditions. then fixed with 4% paraformaldehyde overnight. Uninjured brain tissues were stained red and brain infarct tissues were stained white. Finally, the percentage of total brain infarct area was calculated using Image J software.
Hematoxylin and Eosin (H&E) staining
Paraffin sections were dewaxed and washed with different concentrations of ethanol and distilled water, then transparent with xylene. Then they were stained with H&E and sealed with neutral resin. Finally, they were observed under a microscope and photographed. The tissue sections are also scored. Scores are judged on the morphological structure of the cells and the condition of the pericellular area. 0 points: normal; 1 point: small amount of cell morphological alterations, no vacuoles around the cells; 2 point: Significant morphological changes with a few vacuoles around the cells; 3 points: significant morphological changes with significant vacuoles around the cells; 4 points: substantial morphological changes with a large number of vacuoles around the cells.
Nissl staining
After dewaxing, the paraffin sections were washed three times with PBS buffer and then immersed in nylon staining solution at 60◦ C for 30 min. The sections were again washed three times with PBS and dried at 60◦ C. Then after xylene treatment, the sections were sealed with neutral glue. Finally, the pathology of neurons in the rat brain tissue was observed under a microscope and photographed. The number of neurites in each section was counted independently by three individuals in a triple-blind situation and the average was taken.
Survival analysis
Sixty SD rats divided into three groups were used for a 7-day survival study. After surgery, the rats recovered in separate cages and were fed standard chow. Survival was assessed every 24 hours after surgery until 7 days post-surgery. All animals in the treatment group were treated with the same dose of I3C (150 mg/kg/d).
TUNEL Assay
Apoptosis was detected using the TUNEL one-step apoptosis assay kit (Meilunbio, Dalian, China). Brain tissues were taken and fixed in 4% paraformaldehyde (PFA), dehydrated, and OCT-embedded. The OCT-embedded sections were cut into 10 µm-thick sections, then treated with paraformaldehyde, permeabilized with 0.5% Triton X-100, and incubated in the prepared working solution for 60 min at 37°C according to the manufacturer's instructions. cells in six-well plates were fixed with 4% paraformaldehyde (PFA) and washed three times in PBS. Finally, the cells were analyzed under a fluorescence microscope (Leica, Wetzlar, Germany) and photographed.
Cell Viability/Cytotoxicity Detec
Three groups of cultured cells (blank group, OGD/R group OGD/R + I3C group) were inoculated into six-well plates, and the cells were washed two to three times with PBS according to the instructions (Meilunbio, China) to ensure that the active esterase contained in the culture medium was removed. 100 µl of dye solution was added to each well (working solution was added to 10 ml of PBS along with 5 µl of PI solution and 5 µl of calcein Am solution). cells were observed and photographed by fluorescence microscopy after incubation at 37°C for 30 to 60 min.
RNA Extraction and Real-Time PCR
Total RNA was extracted using the Trizol kit according to the manufacturer's instructions and the RNA concentration was determined.
The reverse transcription reaction conditions were 42°C, 15 min and 85°C, 5 min. The mixed samples were added to wells of a 96-well plate and the polymerase chain reaction was carried out at 95°C, 10 min denaturation, 95°C, 15s, 60°C, 60s, 40 cycles. The following primers were used in the experiment: IL-1β : 5'-tgccaccttttgacagtgatg-3'(f), 5'-tgatgtgctgcagatt-3'(r).
il-6 : 5'-tctggagcccaccaagaacgatag-3'(f), 5'-gtcaccagcatcagtcccaagaag'-(r).
IL-4: 5 '-aaaactttgaacagcctacag-'(f),5'-ggtttccttctcagttgtgttc-3'(r).
IL-10:5'-gttgttaaaggagtccttgctg-3'(f), 5'-ttcacagggaagaaatcgatga-3'-(r).
Casp3: 5'-CTGGACTGCGGTATTGAGACA-3'(f), 5'-CGGGTGCGGTAGAGAGTAAGC-3'(r)
CASP9: 5'-CCTTGTGTCCTACTCCACCTTCC-3'(f), 5'-GGAAGTTAAAACAGCCAGGAATC-3'(r)
Bax: 5'-GGGTGGTTGCCCTTTTCTACTT-3'(f), 5'- GAAGTCCAGTGTCCAGCCCAT-3'(r)
BCL2: 5'-TTGTGGCCTTCTTTGAGTTCG-3'(f), 5'-GCATCCCAGCCTCCGTTAT-3'(r)
INOS: 5'-CTACTACTACCAGATCGAGCCCTG-3'(f), 5'-CTAGCGCTTCCGACTTTCCT-3'(r)
Immunofluorescence staining
After the brain tissue samples were frozen and sectioned, the frozen sections were dried in an oven at 37 ◦ C for 1 h. The sections were washed three times with PBS solution for 10 min each time. Then, the sections were soaked in antigen repair solution for 30 min and washed three times with PBS solution. Sections were blocked with goat serum for 60 min for antigen, and the blocking solution was removed. The appropriate amount of primary antibody was added and incubated overnight at 4◦ C before removing the blocking solution, adding the appropriate amount of primary antibody and incubating at 4◦ C for 16 h. Then, the secondary antibody was added and incubated for 2 h. The nuclei were stained with DAPI. Finally, the cells were observed under fluorescence microscope and photographed.
Statistical analysis
One-way analysis of variance (ANOVA) or two-way ANOVA was used for multiple comparisons, and all data were statistically analyzed using GraphPad Prism9 software. Data were expressed as mean ± standard deviation of the mean (SEM). values of P < 0.05 were considered statistically significant.