Lipopolysaccharide (LPS) was purchased from Solarbio (Peking, China). Glibenclamide (GLB, an inhibitor of SUR1) and 9-phenanthrol (9-Phe, an inhibitor of TRMP4) were purchased from Sigma-Aldrich (St.Louis, MO, USA). TGFα recombinant proteins were purchased from Abcam (Cambridge, UK). CaMKII-IM-1 (an inhibitor of CaMKII) and KG-501 (an inhibitor of CREB) were purchased from MedChemExpress (NJ, USA). NCGC00067819 (an agonist of CREB) was purchased from Probechem (IL, USA). STAT3 inhibitor VI was purchased from Santa Cruz (CA, USA).
All animal experiments in this study were approved by the Animal Care and Use Committee of the Nanfang Hospital, Southern Medical University (Guangzhou, China), and were adhered to the National Institute of Health Guide for the care and use of laboratory animals. In this research, male Trpm4-/- mice on C57BL/6J background (6-8 weeks, 22-24g, Shanghai Model Organisms, China) and wild-type (WT) littermates propagated by homozygous mating were adopted. In RNA-sequencing, 10 mice (5 Trpm4-/- mice, 5 WT mice) were selected. In other experiments, 108 mice (36 Trpm4-/- mice, 36 WT mice, 36 sham mice) were adopted. A total of 118 mice were used in all experiments.
Temporary Middle Artery Occlusion (tMCAO)
The intraluminal filament model of focal ischemia was adopted. Briefly, right common carotid artery, external and internal carotid arteries were isolated from surrounding nerves and fascia through a midline incision under anesthesia of isoflurane (induced with 3%, maintained with 1%). A monofilament nylon suture with a silicone-coated tip (silicone diameter: 0.22 ± 0.02 mm) was then inserted through an arteriotomy of the external carotid artery and gently advanced into the internal carotid artery to the opening of the middle cerebral artery. After 1 h of occlusion, the suture was removed to allow reperfusion for 24 h. Rectal temperature was maintained at 37 ± 0.2 °C during surgery using a heating pad (RWD Life Science, Shenzhen, China). Successful modeling of tMCAO was defined as a reduction of focal cerebral blood flow by more than 75% of baseline and complete recovery of blood flow after reperfusion, which were monitored by a laser Doppler flowmetry (Moor Instruments, Wilmington, USA). After 24 h of reperfusion, the brain tissues of the PIA were isolated and reserved for subsequent experiments. Sham surgery included the exposure of the common, external, and internal carotid arteries with all ligations and transections; no occlusion occurred in the sham group.
Treatment and Drug Administration
In the first part, GLB and Trpm4-/- are the main interventions inhibiting SUR1-TRPM4 channels. In the tMCAO model, mice were randomly allocated to receive vehicle or GLB at 1 h after ischemia. Based on previous studies, GLB (Sigma-Aldrich, St Louis, United States) was administrated with an initial dose of 10 μg/kg and additional doses of 0.2 μg every 8 hours. Trpm4-/- mice undergone tMCAO received vehicle injection at the same time point with GLB injection. To elucidate the influence of infarction volume, delayed GLB injection at 10 h after ischemia and reperfusion were set as a control group. GLB was prepared for intraperitoneal injection by dissolving in dimethyl sulfoxide (DMSO) and clarifying the solution as needed using a minimum amount of NaOH to a pH approximately 8 to 8.5 and 7.8. Mice in different groups randomly received equivalent volumes of vehicle (normal saline containing 0.05% DMSO) and GLB (vehicle and 2.5 μg/ml GLB).
In the second part, intracerebroventricular injection of recombinant TGFα was performed using stereotaxic surgery. Mice after ischemia for 1 h received 50 ng recombinant TGFα (n = 6) in the lateral ventricle according to Paxinos atlas. Phosphate buffered saline (PBS) treated vehicle group (n = 6) underwent stereotaxic surgery and received 2 μl PBS in the right lateral ventricle set as a control group.
Agarose gel electrophoresis of tail DNA
Genotyping of each mouse was identified by using the Mighty Amp Genotyping kit (TAKARA), according to the manufacturer’s protocol. Firstly, DNA was extracted from tail tissue and amplified. Then, by mixing DNA with primers and 5x loading dye for agarose gel electrophoresis, genotypes were identified.
Different primer sequences were listed as follows:
Trpm4-/-: Forward primer 5’-3’: GGAGCAACACCTGAGCTTATGAC;
Reverse primer 5’-3’: AGGCTGAGCTGGAACTCAGAG.
Trpm4+/+: Forward primer 5’-3’: CTATCTGGAGCTGCATCCCTG;
Reverse primer 5’-3’: GTTCCACCTATCCTTAGGACCTG
RNA isolation and cDNA synthesis
Total RNA from the cerebral PIA was isolated using the Animal Total RNA Isolation Kit (Foregene, China), according to the manufacturer’s protocol. cDNA synthesis was conducted in 20 μl of reaction mixture containing 2 mg of RNA by using PrimeScript RT reagent Kit with gDNA Eraser (TAKARA).
Differentially expressed genes analysis
After ischemia for 1 h and reperfusion for 4 h, the total RNA was extracted in the PIA. In this analysis, two groups (WT versus Trpm4-/-) were compared for differential gene expression. Transcriptomic analysis was performed using the HISAT2-Stringtie-DESeq pipeline. Genes that exhibited changes in expression > 2-fold and had a P-value adjusted using the Benjamini–Hochberg procedure lower than 0.05 (Padj < 0.05) were identified as differentially expressed genes (DEGs). Enrichment Analysis based on Gene Ontology Database annotates gene functions of DEGs to obtain the related pathways that the genes participate in, and then calculates the significance level (P-value) and misjudgment rate (FDR) of each pathway by using hypergeometric test (or Fisher's exact test) and multiple comparison test.
Mice were euthanized after reperfusion for 24 h and transcardially perfused with saline. Following fixation with 4% PFA overnight, brains were immersed in 15% and 30% sucrose at 4°C for cryoprotection. Sequential 6 μm-thick coronal sections of the brain were prepared by cryoutramicrotomy (CM1950, Leica, Germany). The slices were simultaneously incubated at 4°C overnight with two types of primary antibody from different species for the co-localization staining: primary antibody from mouse: anti-SUR1 (1:100, Sigma-Aldrich), anti-Iba1 (1:100, Sigma-Aldrich), anti-GFAP (1:100, Abcam), anti-NeuN (1:100, Abcam) with rabbit anti-TRPM4 (1:100, Sigma-Aldrich), respectively; mouse anti-Iba1 (1:100) with rabbit anti-TGFα (1:100, Santa Cruz, CA, USA). Afterwards, the slices were washed and detected with secondary antibodies: Goat anti-mouse Cy3 and Goat anti-rabbit FITC (Beyotime, China).
Immunofluorescence staining for neuronal apoptosis
Double staining of Terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) and neuronal nuclei (NeuN) was conducted to determine the co-localization of apoptotic cells and neurons. In brief, frozen sections were immunostained with mouse anti-NeuN antibody (1:100, Abcam) at 4 °C overnight and subsequently subjected to TUNEL staining using a One Step TUNEL Apoptosis Assay Kit (Beyotime, China) according to the manufacturer’s protocol. Afterwards, the slices were washed and detected with Goat anti-mouse Cy3 at 37 °C for 2 h. Finally, the sections were covered with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). Positive cells around the injured cortex were calculated per square millimeter from six random microscopic fields of each section (three sections per animal) under a fluorescence microscope (Olympus).
Measure of Infarct Volume and the selection of the PIA
At 24 hours after ischemia, the whole mice brains were extracted after euthanasia and sectioned into 7 contiguous coronal slices from the frontal pole with placement in a brain matrix. Then, all slices were incubated in 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma Aldrich) for 30 minutes at 37 °C. After correction for edema, corrected infarct volume (%) was calculated by a researcher blinded to group allocation with Image J (NIH, Bethesda, United States) as follow: corrected infarct volume (%) = [contralateral hemisphere volume − (ipsilateral hemisphere volume − infarct volume)] / contralateral hemisphere volume × 100%. The peri-infarct territory, which is found in the 2 mm around the infarct core, was dissected for protein analysis.
Morris water maze
Spatial learning and memory were evaluated in animals 8 days post-tMCAO using the Morris water maze as described previously [40, 41]. Firstly, mice were trained to reach for the platform for 5 consecutive days with 4 trials per day. Movements of the mice were tracked by TSE VideoMot2 tracking system (Bad Homburg, Germany) to record the path and time taken to escape from 4 randomly assigned locations. The latency time required to locate the hidden platform was assessed among groups. After acquisition trial, the probe trial was performed on the following day, when mice were allowed 60 seconds to explore the platform which had been removed. The percentage of total time that mice spent in the target quadrant and the number of platform location crossing were recorded and analyzed.
The global neurological and motor function were evaluated with longa test and string test by a researcher blinded to the group allocation. The longa test is a 5-point scale with a minimum score of 0 for no neurological deficit and a maximum score of 5 for no spontaneous walking and depressed. The string test was performed by placing the mouse on a 50 cm string at a point between the supports and rated by the following system:0, falls off; 1, hangs onto string by two forepaws; 2, as for 1, but attempt to climb onto string; 3, hangs onto string by two forepaws plus one tail wrapped around string; 4, hangs onto string by all four paws plus one tail wrapped around string 5, escape.
After successful tMCAO modeling, mice were perfused with the use of 4% paraformaldehyde in PBS via the heart, followed by the extraction of the brains. After being fixed with 4% paraformaldehyde and dehydrated using 15% and 30% sucrose solution, the brain tissues were sliced into 10-μm sections, followed by 1-h staining with 0.04% cresyl violet dissolved in acetate buffer. Next, the sections were observed under a light microscope. Cell counting was performed in the peri-infarct area of tMCAO mice using five sections of each brain tissue.
The BV-2 mouse microglial cells (obtained from Bnbio, Peking, China) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco, NY, USA) containing 10% fetal bovine serum (FBS; Gibco), 100 IU/mL penicillin, and 100 mg/mL streptomycin. SH-SY5Y cells (obtained from Bnbio, Peking, China) were cultured in DMEM/F12 containing 15% fetal bovine serum, 100 IU/mL penicillin, and 100 mg/mL streptomycin, under a humidified atmosphere of 5% CO2 at 37 °C.
Oxygen and glucose deprivation/reoxygenation (OGD) of BV-2
The cultured BV-2 cells were washed two times with PBS and incubated with DMEM without glucose in a chamber (Thermofisher, USA) that was filled with 95% N2 and 5% CO2 at 37°C. Control group cells were incubated under normal culture conditions for the same time period. After 3 h of OGD, the medium of both OGD and control group was replaced with the normal DMEM medium and cultured under normal conditions for 24 h.
Cell proliferation assay
SH-SY5Y were seeded in a 96-well culture plate at 1× 104 cells/well in 100 μl for 24h to adhere. The medium was then replaced with a 100 μl conditioned medium from BV-2 cells. After 24 h incubation, 10 μl CCK-8 were added per well and then absorbance value at 450 nm was tested to examine the survival of SH-SY5Y.
The concentration of TGFα in the cell-free supernatants was detected by means of a mouse TGFα Elisa detection kit (Boshen, Jiangsu, China) according to its manufacturer’s protocol.
Measurement of Gene Expression
The mRNA levels of SUR1, TRPM4, TGFα, and GADPH were routinely measured by quantitative real-time polymerase chain reaction (q-PCR). Briefly, total RNA was isolated using a Total RNA/DNA isolation kit (Tiangen, Peking, China) and reverse transcribed to cDNA with PrimeScriptTM RT Master Mix Kit (TAKARA) according to the manufacturer’s instructions. q-PCR was performed using the SYBR Green master mixes (TAKARA) and Roche LightCycler480 System. Relative changes of mRNA expression were normalized to the level of GADPH.
Tested genes and primer sequences were listed as follows:
Abcc8: Forward primer 5’-3’: CATCCGGGTGAGGAGATACG;
Reverse primer 5’-3’: CAGGTTAACGAAGGGCTGCA.
Trpm4: Forward primer 5’-3’: TGATGAGCACACCACGGAGA;
Reverse primer 5’-3’: ATCCGTGCGATCAGACAGC.
Tgfα: Forward primer 5’-3’: CACTCTGGGTACGTGGGTG;
Reverse primer 5’-3’: CACAGGTGATAATGAGGACAGC.
Gapdh: Forward primer 5’-3’: AGGTCGGTGTGAACGGATTTG;
Reverse primer 5’-3’: TGTAGACCATGTAGTTGAGGTCA.
Ischemic tissues in PIA were resolved in RIPA solution (Beyotime Biotechnology, Shanghai, China) and restored at -80 °C until used. Denatured protein was separated by polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane (Millipore, Billerica, United States). The membrane was incubated overnight at 4 °C with antibodies involving anti-SUR1 (1:1000; Bioss, Beijing, China; Rabbit), anti-TRPM4 (1:1000; Sigma-Aldrich; Rabbit), anti-TGFα (1:1000; Santa Cruz; Rabbit), and anti-GAPDH (1:10000; Proteintech; Mouse). Primary antibodies were detected with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies. All signals were detected by enhanced chemiluminescence detection method followed by quantification with Image J and normalizing to GAPDH levels.
Preparation of microglia conditioned medium (MCM)
Cultured BV-2 microglia were plated in DMEM serum-free media at a density of 1x106 cells/well, in six-well tissue culture-treated plates (Corning. Lowell, MA) and maintained in a 37 °C incubator under conditions of 5 % CO2 and 95 % humidity for 24 h. After 24 h, media was aspirated out and 1 mL of fresh DMEM serum-free media was added. After modeling for 24 h, the medium was collected after centrifugation at 1000 g for 10 minutes with sterile filtration (0.22 μM filter) of the supernatant. The microglia conditioned medium after OGD or LPS were named OGD-MCM or LPS-MCM, respectively.
siRNA duplex sequences were designed by RIB BIO (Guangzhou, China) and transfected using ribo FECTTM CP (Guangzhou, China) according to its protocol.
The number of positive cells in immunofluorescence staining was compared using two independent sample t-test. Statistical differences in multiple groups were compared using one-way analysis of variance followed by the Tukey's multiple comparisons test, while two groups were analyzed by unpaired t-tests. All statistics were completed by SPSS 25.0, and GraphPad Prism 8.0 was used for image drawing and processing. P < 0.05 was statistically significant.