All animals used in this experiment were in compliance with guidelines from Animal Experiment Ethical Inspection of Laboratory Animal Centre at Wenzhou Medical University. Eight-week-old male and female mice were housed in standard cages with 12h dark/light cycle at 23±1℃ and 50±5% humidity and supplied with food and water ad libitum.
Laser-induced ischemia model in the brain and TTC staining
The laser-induced ischemia model in mice was described in detail from a previous publication . Briefly, C57BL/6 mice were first injected with rose bengal stain intraperitoneally. Then, mouse head was fixed on a three-dimensional brain locator and opened at the sagittal line after general anesthesia (50mg/kg) by intraperitoneal injection of 1% Pentobarbital sodium. A hole was created in the calvarium (2.0 mm left of the sagittal line and 1.0 mm in front of the coronal line), and an optic fiber was inserted to a depth of 3.0 mm where the brain was irradiated with the 473nm blue laser for 15min. Mice were then sutured up and monitored on a heating plate. After 24 hours, the mice were sacrificed and the brains were stripped off the cerebellum, olfactory bulb and lower brainstem, and placed in 4℃ PBS solution. Then, the brain was left frozen at -20℃ for 30 min, and cut into slices with a thickness of 1 mm. The slices were immersed in 2% 2,3,5-triphenyl tetrazolium chloride (TTC) solution in a 37℃ water bath shaker for 30 min. Afterwards, the slices were washed in PBS solution for 5 min and photographed.
Primary cell culture and treatments
Microglia were isolated from 1-day-old postnatal mouse based on established protocol. Briefly, cortical cells were acquired from the pups sacrificed by cervical dislocation. Cells were cultured in DMEM/F12 supplemented with 10% fetal bovine serum and 0.5 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF). At the end of 10 days, microglia were sub-cultured into 6-well or 24-well plates at a density of 5×104 cells/cm2 for experiments. Low glucose condition describes cells cultured in medium with 5mM glucose. Pyrollidine dithiocarbamate, an NF-κB inhibitor, was used to inhibit NF-kB signaling at various concentrations.
BV2 microglia were fixed with 4% paraformaldehyde at room temperature, permeabilized for 5min with 0.1% Triton-X (Sigma) and blocked with 1% BSA in PBST for 1h. Then, cells were incubated with primary antibodies in 1% BSA overnight at 4°C, 1h the next day with secondary antibodies (Jackeson, 1:1000) and then with DAPI for 30 min at room temperature. The primary antibodies included mouse anti-MCT1 ( ab10582, abcom, 1:250)，rabbit anti-argnase-1(sc-166902, Santa Cruz, 1:200)，goat anti-Iba1(MABN92, Novus, 1:2000), rabbit anti-iNOS (#13120,CST, 1:1000). Cells were evaluated under 40× magnification using a Vision microscope (Olympus, Japan). All imaging conditions, including exposure times and image acquisition, were identical.
Brain tissue samples were harvested and 10 μm-thick cryosections were generated using a microtome (Leica CM1950). Sections were incubated in citrate buffer for antigen recovery, then in 0.3% hydrogen peroxide for 20 min, and blocked with blocking buffer for 1 h. Similarly, sections were incubated with primary and secondary antibodies as described above and processed using the fluorescence microscope (Olympus, Japan).
20 ng of protein lysates were loaded on 10% SDS polyacrylamide gel for protein separation and transferred to a polyvinylidene difluoride membrane. After blocking with 5% nonfat milk for 1 h, membranes were incubated with primary antibodies at 4℃ overnight and rinsed with tris buffered saline tween (TBST) buffer before incubation with secondary antibodies for 1hour. Membranes were imaged using Tanon-4500 after addition of peroxidase reagents.
All small interfering RNAs were purchased from MICRO-G BIOTECK company (Shanghai, China). According to the manufacture’s protocol, BV2 microglia were seeded at 105 cells/well. 20 μM of siRNA was added to serum-free DMEM and then mixed with 5 μl of Lipo3000 transfection reagent. After incubation at room temperature for 15 min, the mixture was added to cells and cultured for 48 h. The sequences of siRNAs were as follows (5’-3’): forward UUCUCCGAACGUGUCACGUTT, reverse ACGUGACACGUUCGGAGAATT for siRNA-control; forward CCAAAUCCAUCACUGUCUUTT, reverse AAGACAGUGAUGGAUUUGG for siRNA-MCT1.
MCT1-containing plasmid transfection
The GV230 eukaryotic plasmids were purchased from TSINGKE BIOTECK (Tianjin, China). First, XhoI/KpnI double enzyme digestion was performed on plasmids for MCT1 gene insertion. For transfection, microglia were grown in Dulbecco’s Modified Eagle’s Medium(Gibco), supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) in a 5% CO2 incubator at 37℃. Appropriate plasmids (5μg per well) were transfected into the cells using Lipofectamine™ 3000 Transfection Reagent (L3000-015, Invitrogen) according to the manufacturer’s instructions.
Transmission electron microscopy
BV2 microglia were harvested with cell scrapper and fixed with 2.5% glutaric dialdehyde for 2h at 4℃, rinsed with 0.1 M of phosphoric acid, and fixed again with osmic acid for 3 h. Cells were further incubated in 1% uranyl acetate for 2 h. After dehydration in acetone, cells were dried at 65℃ for 48 h. Cells were analyzed with transmission electron microscope (h7500, HITACHI).
For western blotting, triplicates were performed for three independent experiments. For immunofluorescence imaging, 3 random views of each group were selected and at least 100 cells were counted. All data were analyzed by one-way ANOVA and Tukey post hoc test. p<0.05 was viewed as statistically significant.