2.1 Animals and experimental design
Male Sprague-Dawley (SD) rats weighting 200-250 g were obtained from the Experimental Animal Center of Southwest Medical University and placed in a thermostatic specific pathogen-free laboratory animal room with free access to feeds and water. In this experiment, all animals were starved for one week prior to the experimental work, then randomly divided into six groups (n=10): NC, CO, BMSC-ex, GW4869, DAPT, and BMSC-ex +DAPT group. DEACMP models were induced as previously described. Briefly rats excluding the NC group were exposed to CO (1,000 ppm) for 40 min followed by a second exposure to CO (3,000 ppm) for 20 min in a hyperbaric oxygen chamber. Rats in the BMSC-ex, GW4869, and BMSC-ex + DAPT groups were injected with exosomes (100 µg/mouse) by the tail vein 1 h after DEACMP induction. Next, GW4869 (2.5µg/g, Sigma, USA) was diluted in dimethyl sulfoxide and intraperitoneally administered in the GW4869 group. The DAPT was applied to the DAPT and BMSC-ex +DAPT group at doses of 1 mg /kg. Fourteen days post-CO exposure, rats were sacrificed for brain tissue collection under deep anesthesia by 10% pentobarbital sodium (40 mg/kg).
2.2 Isolation and culture of BMSCs
Isolation and culturing of BMSCs was performed as previously reported protocol. The rats were anesthetized using pentobarbital and immersed in ethanol (70%) for disinfection. The femurs and tibia of rats were removed from tissues. Bones were sterilized in a tube with 70% ethyl alcohol for 2 min and then steeped in phosphate-buffered saline (PBS) for 30 min under ultraviolet disinfection. The marrow cavity is exposed by cutting off the epiphysis at each end of the bone. A syringe was used to draw the cell growth fluid, with the needle was alternately inserted into their ends to rinse, until the marrow cavity turned white. The cell growth fluid consisted of Dulbecco’s modified Eagle medium (90%; Gibco, USA), fetal bovine serum (10%; HYCLONE, USA), PEN-STREP (1%; Lonza, USA) and L-VC (1%). Collected samples were placed in 60 mm culture dishes and routinely cultured in a 5% CO2 incubator at 37 ° C. After 48 h, half of the cell growth fluid was discarded and the medium was replaced completely every three days. BMSCs at approximately 80% confluency were subcultured.
2.3 Extraction and identification of exosomes
The BMSCs culture supernatant was collected to extract exosomes by ultracentrifugation as described previously. After incubating BMSCs for 72 h in an exosome-depleted medium to avoid the interference of exosomes from media, the spent medium was collected and alternately centrifuged at low and high speeds. Centrifugation was carried out at 300 x g for 10 min to remove cells, and then at 2000 × g for 10 min to remove dead cells. The supernatant was centrifuged at 100,000 x g for 30 min to discard cells debris and centrifuged again at 100,000 x g for 70 min to discard the supernatant. The pellet was washed in PBS and recentrifuged at 100,000 x g for 70 min. Finally, the pellet was resuspended in PBS and stored at -80 ℃. Further procedures were carried out at 4 ℃ to authenticate exosomes at three levels: surface markers authenticated by western blot, morphology visualized by transmission electron microscopy, and size by nanoparticle tracking analysis.
2.4 Morris water maze test
The cognitive impairment of the rats was evaluated by the Morris water maze test as reported earlier. In order to adapt to the new environment, the rats were put into the water to swim for 2 min before the formal training. In the place navigation test, rats were placed into each of four quadrants toward the wall of the pool under water, and the time required for the rats to find the underwater platform and stand on it was recorded as escape latency. If the rats could not find the platform after 2 min, they were pulled onto the platform and the time was recorded as 120 s. When the rats learned to find the platform, we removed the platform and placed rats into water from the same position. The number of times of crossing the platform was recorded.
2.5 Luxol fast blue (LFB)
Paraffin slices of brain tissue were sequentially immersed in toluene for 20 min, toluene - anhydrous alcohol for 20 min, anhydrous alcohol for 10 min, 95% alcohol for 5 min, 90% alcohol for 5 minutes, 80% alcohol for 5 min, and 70% alcohol for 5 min. The processed sections were incubated overnight in 0.1% LFB solution, and successively immersed in 95% HCI and 70% ethanol until white matter stained blue and gray matter appeared colorless.
2.6 Enzyme-linked immunosorbent assay
Brain tissues were centrifuged at 5000 x g for 10 min, and the supernatant retained. Rat tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-10 and tumor necrosis factor (TGF)-β kits (eBioscience, USA) were used to estimated cytokine levels according to the manufacturer’s instruction. A microplate reader (Biotek, USA) was used to measure the absorbance at 450 nm; the data obtained were analyzed using the Gen5 software.
2.7 Immunofluorescence analysis
Paraffin slices of the brain tissue were dewaxed with water in a 65 ℃ oven for 2h and were washed with PBS. The non-specific binding sites were blocked with 5% BSA for 20 min. After removing the BSA solution, primary antibodies for anti-Foxp3 (1:50; Ab22510; Abcam, USA), anti-CD4 (1:50; A0362; Abclonal, Wuhan, China), anti-MBP (1:200; BA0094; Boster, USA), anti-Notch1 (1:150; 10062-2-Ap, Ptg) were added accordingly to each section and incubated overnight at 4 ℃. After washing thrice with PBS, sections were incubated with a secondary antibody, such as goat-anti-rabbit (1:50; AS-1110; Aspen, USA) or goat-anti-mouse (1:50; AS-1112; Aspen, USA) at 37 ℃ for 50 min. Sections were incubated with 50-100µL DAPI solution (1:1000, AS1075; Aspen, USA) at room temperature for 5 min, in darkness. An appropriate amount of anti-fluorescence quenching agent was added to the tissue, the cover glass sealed, and observed under a fluorescence microscope.
2.8 Western blotting analysis
Proteins samples were separated by 12% SDS-PAGE (AS1012; ASPEN, USA) from exosomes or brain tissues, then transferred onto polyvinylidene difluoride membranes (IPVH00010; Millipore, USA) and blocked with TBST for 1 h. Primary antibodies foe anti-CD9 (1:1000; bs23032R; BIOSS, USA), CD63 (1:1000; Ab109201; Abcam, USA), anti-TSG101 (1:1000, 14497-1-AP), anti-GAPDH (1:10000; Ab37168; Abcam, USA), anti-Foxp3 (1:1000; Ab215206; Abcam, USA), anti-CD4 (1:1000, 19068-1-AP), anti-MBP (1:1000; Ab11159; Abcam, USA), anti-Notch1 (1:500; sc-376403; Santa, USA), and anti-Hess-1 (1:10000; Ab108937; Abcam, USA) were added to the membranes and incubated together overnight at 4°C. After washing thrice with TBST, secondary antibodies (all diluted 1:10000; ASPEN, AS1106 for mouse-antibody and AS1107 for rabbit-antibody) were added and incubated at room temperature for 1 h. Finally, the band was detected by chemiluminescence and the intensity was analyzed by ImageJ software.
2.9 Statistical analysis
GraphPad Prism v 7.01. was used for all statistical analyses in this experiment. Data was analyzed using one-way or two-way repeated measures analysis of variance with post-hoc Tukey’s multiple comparisons. All data presented as mean ± SEM (n=3) were statistically significant when P <0.05.