All animal experiments were performed in accordance with guidelines established by the Animal Care and Use Committee of Nanfang Hospital, Southern Medical University (Guangzhou, China). Male Sprague-Dawley rats (350-400 g) were purchased from the Experimental Animal Center of Southern Medical University. We used male Trpm4−/− mice on C57BL/6 background (8-10 weeks old, 20-25 g, Model Organisms Center, Shanghai, China) and wild type littermates. Wild type and Trpm4−/− mice were both confirmed by extracting and separating tail DNA in agarose gel. All animals in current study were housed in a specific pathogen-free facility under a strict 12-hour light/dark cycle with free access to food and water.
Cell Lines Culture and Treatments
Murine BV2 microglial cells and RAW264.7 macrophage cells were purchased from American Type Culture Collection (VA, USA) and 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, under a humidified atmosphere of 5% CO2 at 37°C.
When BV2 or RAW264.7 cells reached 80 to 90% confluence, they were primed via radiation, oxygen-glucose deprivation/reperfusion (OGD/R) or 3-hour LPS (1µg/mL; Sigma-Aldrich, MO, USA) incubation. LPS was dissolved in sterile water, while the equal volume of sterile water was added as control. The 0.5-hour incubation with NaN3 (1 mmol/L; Sigma-Aldrich), nigericin (10µg/mL; MCE, NJ, USA), diazoxide (100 µmol/L; Sigma-Aldrich) or ATP (5 mmol/L; Roche, Basel, Switzerland) was conducted 6 hours after the radiation, OGD, or LPS stimulation. Additionally, the following reagents were added into the medium 0.5 hour preceding the intervention with ATP, NaN3, nigericin or diazoxide: MCC950 (10 µmol/L; MCE) against NLRP3, PAP-1 (10 nmol/L; MCE) inhibiting Kv1.3, EGTA (4 mmol/L; Sigma-Aldrich) chelating extracellular Ca2+, Brilliant Blue G (BBG) (100 µmol/L; Sigma-Aldrich) against P2X7R, 9-phenanthrol (9-Ph) (50 µmol/L; Sigma-Aldrich) against TRPM4, quinine (100 µmol/L; MCE) inhibiting K2P family, and sulfonylureas (100 µmol/L; Sigma-Aldrich) against SUR1 including GLB, gliclazide (GLZ) and glimepiride (GLM). Particularly, cells were pre-incubated with transcription factor inhibitors such as Mithramycin A (2 µmol/L; MCE) blocking specificity protein 1 (Sp-1) and JSH23 (30 µmol/L; MCE) against nuclear factor kappa-B (NF-κB) for 1 hour before challenged by LPS, radiation, or OGD.
Solutions Used in Ion-replacement Experiments
The preparation of solutions was in accordance with the previous study (23). Nominally K+ free solution contains (in mM): 150 NaCl, 2 CaCl2, 1 MgCl2, 10 Hepes; Nominally K+, Na+, Ca2+ free solution contains (in mM): 150 NMDG-HCl, 1 MgCl2, 10 Hepes.
Asphyxial Cardiac Arrest Model in Rats and Treatments
Rats were randomly assigned to appropriate groups, and the 10-minute asphyxial cardiac arrest/cardiopulmonary resuscitation (CA/CPR) model was performed as per our previous study (28). In brief, the rats were anesthetized with isoflurane (4% for induction and 2% for maintenance; RWD, Shenzhen, China), followed by the orotracheal intubation with a 14G cannula (BD, Suzhou, China) and connection to a ventilator (RWD). Intravascular catheters (PE50; Smiths Medical, Ashford, UK) were inserted into the right femoral artery and vein for dynamic blood pressure monitoring and drug delivery, respectively. After 10-minute stabilization, rats were chemically paralyzed by IV Vecuronium (2 mg/kg), and then the ventilator was disconnected for 10 minutes, typically leading to circulatory arrest in 5 minutes, which was characterized by the cessation of arterial pulse and a decrease of mean arterial pressure (MAP) to 20 mmHg below. At the end of 10-minute asphyxia, cardiopulmonary resuscitation was initiated by effective ventilation with 100% oxygen and intravenous administration of epinephrine (0.01 mg/kg), simultaneously accompanied by the chest compressions (200 compressions/minute). Additional doses of epinephrine (0.02 mg/kg) were given at 2-minute intervals until the return of spontaneous circulation (ROSC), as defined by an increase of MAP to 60 mmHg above for at least 10 minutes. After spontaneous respiration recovery, rats were weaned from ventilator and extubated, but the rats that failed to ROSC within 5 minutes or were unable to be weaned from ventilator after 1-hour observation were excluded from the continuing experiments.
GLB was dissolved in dimethyl sulfoxide (DMSO) and diluted in saline (2.5 µg/mL). After ROSC, GLB was administrated intraperitoneally with an initial dose of 10 µg/kg at 15 minutes and 4 maintenance doses of 1.2 µg per 6 hours (28), whereas rats in the vehicle group received equivalent volume of DMSO and saline (Fig. S1A). Rats that underwent all procedures except asphyxial CA/CPR were used as sham control.
The Trpm4 in vivo Ready siRNA and the universal in vivo negative control siRNA were purchased from Ambion (MA, USA). The sequences of Trpm4 siRNA were as follows: sense 5′-CGCUAGUAGCAGCAAAUCUtt-3′ and antisense 5′-AGAUUUGCUGCUACUAGCGtg-3′, which downregulated the expression of TRPM4 in vivo as per our previous study (29). 500 pmol of Trpm4 or negative control siRNA was injected into the right lateral ventricle of the rats at a rate of 0.5 µl/min prior to cardiac arrest.
Radiation-Induced Brain Injury (RBI) Model in Mice and Treatments
Male C57BL/6 mice weighing 20–25 g were randomly divided into different groups. The RBI model was conducted as described in the previous study (30). After anesthetization, the head of a prone mouse was placed in a radiation ଁeld (1×1 cm2) from the post-canthus line to the post-aurem line. Afterward, radiation was administered with a single dose of 30 Gy (3 Gy/min) employing the small animal/cell X-ray MultiRad 225 irradiator (Faxitron, AZ, USA), characterized by a source-to-skin distance of 58 cm.
According to the previous study (31), the irradiated mice were administrated 10 µg of GLB (100 µg/mL) intraperitoneally posterior to the whole-brain radiation, followed by the maintenance dose of 10 µg once daily until euthanized, whereas irradiated mice in the vehicle group received equivalent volume of DMSO and saline (Fig. S1A). The mice in the sham group received anesthetic procedures at the same time as those irradiated but not the whole-brain radiation.
Cell Irradiation Model
The BV2 cells were seeded into a 6-well plate and irradiated with a single dose of 10 Gy (1 Gy/min) using the X-ray MultiRad 225 irradiator, since earlier studies reported that 10 Gy was the optimal radiation dose to activate microglia (32). After irradiation, cells were returned to the 5% CO2 incubator. The control cells received sham-irradiation. Cells or culture supernatants were collected at indicated time points post-irradiation.
Cell OGD/R Model
OGD/R on BV2 cells was performed as described previously (33). The cells were cultured in the glucose-free DMEM (Gibco), and placed in a hypoxic chamber with 1% O2, 5% CO2, and 94% N2 at 37°C for 3 hours to mimic the hypoxic-ischemic injury, followed by the restoration with high-glucose DMEM at the normoxic condition to mimic the reperfusion. Control cells were maintained in high-glucose DMEM without oxygen deprivation.
Western blotting was routinely performed as previously reported (34). Rodent brain tissues, BV2 cells, and RAW264.7 cells were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail. After denatured in loading buffer, the samples were subjected to SDS-PAGE and then transferred to PVDF membranes (Millipore, MA, USA). After blocked by 5% non-fat milk, the membranes were incubated overnight at 4°C with the primary antibodies as below: Mouse anti-β-actin (Proteintech, IL, USA), rabbit anti-GAPDH (Proteintech), rabbit anti-TRPM4 (Sigma-Aldrich), mouse anti-SUR1 (Abcam, Cambridge, UK), rabbit anti-NLRP3 (Novus, CO, USA), rabbit anti-pro-caspase-1 (Abcam), rabbit anti-caspase-1 p20 (Bioss, Beijing, China), rabbit anti-precursor of IL-1β (pro-IL-1β) (Proteintech), rabbit anti-IL-1β p17 (Novus), rabbit anti-Kir6.1 (Abcam), and rabbit anti-Kir6.2 (Abcam), followed by the detection via the secondary antibodies (CST, MA, USA). The densities of protein blots were quantified by ImageJ software (NIH, MD, USA) and normalized to the level of β-actin or GAPDH.
Measurement of Gene Expression
The mRNA levels of SUR1, TRPM4, NLRP3, caspase-1, IL-1β, Kir6.1, Kir6.2, K2P family, Kv family, and GADPH were routinely measured by quantitative real-time polymerase chain reaction (qRT-PCR) (35). Briefly, total RNA was isolated using Trizol Reagent (Thermo Fisher Scientiଁc, MA, USA) and reverse transcribed to cDNA with the PrimeScriptTM RT Master Mix Kit (Takara, Dalian, China) according to the manufacturer’s instructions. qRT-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.
Neurologic Function Evaluation
The neurologic outcome of post-cardiac arrest rats was assessed at 24, 48, 72 hours and 7 days after ROSC, utilizing a scale of neurologic deficit scores (NDSs) in which 80 was considered normal, whereas 0 represented brain death (36). The total NDSs scale consisted of 7 components: general behavioral deficit, brain-stem function, motor assessment, sensory assessment, motor behavior, behavior, and seizures.
The Morris Water Maze was carried out to evaluate short-term spatial learning and memory as described previously (17, 37). The water maze apparatus is a circular tank filled with opaque water (divided into four quadrants, called Q1, Q2, Q3, Q4), and a hidden platform is submerged 2-3 cm for rats (1 cm for mice) below the water surface and not visible to the rodents. Firstly, the rodents were trained to search for the platform on day 9-12 post-cardiac arrest or for 5 consecutive days after 8 weeks post-radiation at a frequency of four trials/day, orienting by referencing 3 external cues surrounding the tank. If the rodents did not find the platform in 60 seconds, they were manually placed on it for 15 seconds. Rodents’ movements were tracked by TSE VideoMot2 tracking system (Bad Homburg, Germany) to record the path and latency time taken to escape from 4 randomly assigned locations. After the training termed as acquisition trial, the probe trial was performed on the following day, when the rodents were allowed 60 seconds to explore the platform which has been removed. The percentage of total time that rodents spent in the target quadrant and the number of platform location crossings were recorded and analyzed.
For detecting neuronal loss and the levels of certain markers, rats were deeply anesthetized and transcardially perfused with saline on day 14 post-surgery. Brains were fixed with 4% paraformaldehyde (PFA) overnight and embedded in paraffin, and the 4-µm-thick coronal slices located at 3.5 mm posterior to bregma were stained with cresyl violet (Beyotime) and then observed under optical microscope (Olympus, Tokyo, Japan). Viable neurons in the hippocampal CA1 region had the characteristics of visible nucleus and intact cytoplasm with discernable Nissl staining, while those shrunken cell bodies surrounded by empty space were regarded as dead neurons.
Immunohistochemistry of hippocampus CA1 region from rats was conducted on day 14 post-surgery by incubation with antibodies against neuronal nuclei (NeuN; CST) for neurons, microtubule-associated protein 2 (MAP-2; Sigma-Aldrich) for dendrites, glial fibrillary acidic protein (GFAP; Abcam) for astrocytes, ionized calcium-binding adapter molecule-1 (Iba-1; Novus) and CD68 (Novus) for microglia. After the immunoperoxidase staining via secondary antibodies, the slides were counterstained with hematoxylin. In each brain section, 3 fields were randomly examined. The relative intensity of MAP-2 staining and the number of NeuN-, GFAP-, Iba-1- or CD68-positive cells in hippocampus CA1 region, were quantified using ImageJ software.
For immunofluorescence, rats or mice were euthanized after 12 hours post-cardiac arrest, 1 week or 8 weeks post-radiation, respectively. Following fixation with 4% PFA, brains were immersed in 15% and 30% sucrose at 4°C for cryoprotection. With regard to the BV2 cells, they were fixed with 2% PFA for 10 minutes at room temperature. The slides of brain sections and cells were incubated with the following primary antibodies: rabbit anti-TRPM4, mouse anti-SUR1, mouse anti-CD68, goat anti-Iba-1, rabbit anti-GFAP (Proteintech), and rabbit anti-Doublecortin (DCX; Abcam) for immature neurons. Afterwards, the slides were washed and detected with appropriate Alexa Fluor dye of secondary antibodies preceding counterstaining with DAPI in the dark. When immunofluorescent co-localization was employed, the pairs of primary or secondary antibodies were incubated simultaneously. Finally, fluorescent signaling was observed under confocal microscope (Olympus) and analyzed using ImageJ software.
Caspase-1 Activity Detection
BV2 cells were seeded in 96-well plates, of which the activated caspase-1 was evaluated utilizing a FAM-FLICA detection kit (Immunochemistry Technologies, MN, USA) as described previously with some adjustments (38). The fluorescent probe FAM-YVAD-FMK (FLICA) was employed to irreversibly label in situ activated caspase-1 in living cells, and the green fluorescent signal directly reflected the caspase-1 activity at the time the reagent was added. Briefly, following the reconstitution with DMSO, FLICA was diluted with phosphate buffered saline (PBS), added to each sample and incubated for 1 hour prior to the nuclear staining with Hoechst. FLICA excited from 490 to 495 nm and emitted from 515 to 525 nm. The images were captured with a fluorescence microscope and the fluorescence intensity of cleaved caspase-1 was quantified by the multiscan spectrum (BMG, Offenburg, Germany).
Measurement of Intracellular Ca2+ and Membrane Potential
The procedure was established as described previously (39, 40). Experiments were typically conducted using BV-2 cells seeded into 96-well plates and allowed to adhere overnight. After treatment with LPS and indicated antagonists, the cultures were washed three times in buffer and loaded for 30 minutes in the dark at 37°C, with 10 µmol/L of either cell-permeable Ca2+ fluorescent indicator Fluo-4AM (Beyotime) or membrane potential indicator Dioc5 (Abbkine, CA, USA). Cells were then washed again in buffer and all experiments were performed at room temperature in the dark. Using the multiscan spectrum, emission intensity was measured up to 15 minutes, and the decline in Dioc5 fluorescent intensity at 430 nm represents the depolarization of membrane potential. Stimulating drugs including ATP, diazoxide and nigericin were added 30 seconds after the first measurement.
Measurement of Intracellular K+ and Na+
This procedure was routinely performed as previously reported (41). The BV-2 cells were seeded into 96-well plates to adhere overnight. After treatment with LPS and indicated antagonists, the cells were loaded with either potassium- or sodium-sensitive fluorescent dyes, PBFI-AM (5 µmol/L, Abcam) or SBFI-AM (5 µmol/L, Abcam), which were freshly prepared by combining with equal volume of 25% Pluronic F-127 (Solarbio, Beijing, China) for 40 minutes at 37℃. The change in 340 nm/380 nm ratio that represents the alteration of intracellular K+ or Na+ was analyzed with the multiscan spectrum. Stimulating drugs including ATP and diazoxide were added 30 seconds after the first measurement.
Measurement of Intracellular ATP
Cells were cultured in a 96-well plate and allowed to grow overnight. After treatment, the intracellular ATP level was measured using CellTiter-Glo luminescent cell viability assay kit (Promega, WI, USA) as previously reported (42). The luminescence was measured to calculate the ATP concentration using the standard curve.
This assay was performed in line with our previous study (17). BV2 and RAW264.7 cells were lysed in moderate lysis buffer (Genstar, Beijing, China) containing phenylmethylsulfonyl fluoride. After centrifuged at 14,000g for 15 minutes, the supernatant was obtained and quantified, of which the portion containing 500 µg protein was immunoprecipitated with 1µg rabbit anti-TRPM4 or mouse anti-SUR1 under rotation overnight at 4°C. Following the rotary incubation with 30 µl protein A/G magnetic beads (Bimake, TX, USA) for another 20 minutes at room temperature and subsequent magnetic separation, the complexes were washed 5 times, and resuspended in 40 µl loading buffer prior to denatured at 100°C. Finally, the protein complexes were subjected to Western blotting as described above, and the employed antibodies were as follows: rabbit anti-TRPM4, mouse anti-SUR1, rabbit anti-Kir6.1, and rabbit anti-Kir6.2. Whole cell lysates were used as an input control, and homophytic IgG was used as a negative control.
The 7.0T nuclear magnetic resonance scanner (Bruker Biospin, Ettlingen, Germany) was utilized to scan the consecutive 0.5-mm thick mice brain transverse sections. Mice were anesthetized with isoflurane (5% induction, 1-2% maintenance) before the prostration on a custom-made holder with strapping to minimize head motion. Transverse T2-weighted images were obtained using a 2-dimensional turbo spin echo sequence (repetition time/echo time, 1600 ms /125 ms; flip angle, 90°; field of view, 40 mm; matrix, 200/400 r; echo planar imaging factor, 1; turbo spin echo factor, 10; number of signal averages, 14; total scan time, 11 min) (30), ranging from the superior of the parietal lobe to the inferior of the temporal lobe. The size of lateral ventricles was analyzed via ImageJ software.
All data were presented as means ± SD or medians and 25th to 75th percentiles (NDSs). Continuous data were analyzed with one-way ANOVA followed by Tukey’s post hoc multiple comparison tests, or as indicated. NDSs were compared using Mann-Whitney U test. The data of escape latency in the water maze test were analyzed with repeated-measures ANOVA comprising treatment groups, time points, and groups×time interaction, followed by Tukey’s post hoc multiple comparison tests. SPSS 20.0 (IBM, NY, USA) and GraphPad Prism 8.0 (GraphPad, CA, USA) were used for statistical analyses. P < 0.05 was considered statistically significant.