Neuroprotection Of TRPM4 Cation Channel In A Mouse Model Of Cerebral Edema After Status Epilepticus

BackgroundTRPM4 is a Calcium-activated,phosphatidylinositol-4,5-bisphosphate(PtdIns(4,5)P2)-modulated,non-selective Cation channel that belongs to the family of melastatin-related transient receptor potential(TRPM)channels.TRPM4 allows Na+entry into the cell upon activation,but is completely impermeable to Ca2+.In Trpm4-/mice,secondary hemorrhages after spinal cord injury are reduced,and neurobehavioral performance was improved compared to wild-type animals.The hypothesis is that sustained opening of TRPM4 channels,as would arise with the severe ATP depletion associated with SCI,could lead to continuous influx of Na+,which,if unchecked,could induce oncotic swelling and oncotic death of endothelial cells,resulting in capillary fragmentation.Asimilar hypothesis was put forward when the clinical development of experimental autoimmune encephalomyelitis was analyzed in WT and Trpm4-/-mice.In Trpm4-/-mice,reduced axonal and neuronal degeneration was observed,which correlated with improved clinical disease scores in Trpm4-/-mice.The same effect was obtained by treating mice with a nonspecific blocker of TRPM4,glibenclamide,which had no additional effect in Trpm4-/animals.Status epilepticus(SE)is broadly defined as a prolonged seizure or multiple seizures with incomplete return to baseline and remains a common neurological emergency with an annual incidence of 10-41 per 100,000 population.The overall mortality associated with status epilepticus approaches 20%.Generalized convulsive status epilepticus(GCSE)is the most dramatic,readily recognized,and dangerous form of status epilepticus.SE can lead to permanent pathological damage and altered physiological function in certain brain regions and induces major changes in membrane phospholipids,massive increases in arachidonic acid concentrations,diacylglycerol-mediated activation,of protein kinase C,calcium-mediated changes in calmodulin kinase Ⅱ and possibly generation of free radicals that could play an essential role in the mechanism of oxidative stress involved in neural damage.Our previous studies of SE in rats have demonstrated that the 28-day survival and neurological outcomes after SE can be significantly improved,by alleviating cerebral edema and neurological injury via inhibiting the upregulation of TRPM4 channel.The relevance of the Trpm4-/-mice and SE is however still unclear.ObjectiveIn this study,we aimed to evaluate whether Trpm4-/-might reduce cerebral edema and improve outcomes in SE model.Methods1.Breeding of Trpm4 gene knockout miceA total of 31 hybrid mice of Trpm4 gene were purchased from the Nanjing Biomedical Research Institute(NBRI).Aged 6-8 week,male and female mice were 1:2 mating in the same cage,housed on a 12-h light/dark cycle with an average temperature of 23℃,humidity of 56%and free access to water and food.The offspring of mice were identificated their genotype by their tails respectively.The obtained hybrids were used for propagation and replacement of breeding cages,and the Trpm4-/-homozygous and wild type were used in the experimental group.2.The establishment of cerebral edema in a C57BL/6J mouse model of SEIn this study,SE was induced by different doses of Li-pilocarpine.C57BL/6J mice were randomly assigned to the Li 3mEq/kg+pilocarpine 300-350mg/kg(Li 3)group(n=26);Li 10mEq/kg+pilocarpine 30-40mg/kg(Li 10)group(n=21),and sham-operated group(n=6),by using random number table.All of the mice were followed up for 3 days and assessed for survival,cerebral edema and histological injury.(1)MRI examination at 24h after SE to assess brain edema.(2)Neurons were stained with cresyl violet,and observed under microscope.Immunohistochemistry was performed with antibodies against mouse IgG for BBB permeability,Ibal for microglia,GFAP for astrocyte,according to the protocol suggested by the manufacture.3.Neuroprotection of Trpm4-/-mice model of cerebral edema after SEWT mice(n=61)and Trpm4-/-mice(n=61)with behaviol seizures terminated at 2.5h after SE onset,were induced by Li 10mEq/kg+pilocarpine 30-40mg/kg.After SE,WT mice(n=28)and Trpm4-/-mice(n=27)were followed up for 28 days and assessed for survival,the others were evaluated for cerebral edema,histological injury and neurological outcomes.Results1.Cerebral edema and neurological injury were obviously observed in a mouse model of SE induced by Li 10mEq/kg+pilocarpine 30-40mg/kg,with behavioral seisures terminated at 2.5 h after SE onset.Firstly,as compared to Li 3 group,Li 10 group was in shorter duration from pilocarpine injection to SE,and had higher incidence of SE.Secondly,Brain edema was much serious in Li 10 group compared to Li 3 group by MRI examination at 24h after SE.Thirdly,BBB breakdown was improved by increased IgG extravasations in hippocampus and piriform cortex in SE group.Fourthly,the numbers of neurons in Li 10 group were much less than that in Li 3 group(P<0.001)after SE Fifthly,the number of activated microglias and astrocytes in Li 10 group were much more than that in Li 3 group(P<0.001)after SE.2.Trpm4-/-alleviated cerebral edema,ameliorated neuronal injury,and improved outcomes of SE.Firstly,28-day survival rate in the Trpm4-/-SE group(77.78%)was much higher than that in the WT-SE group(50%)(x2=3.888,p=0.0486,Log-rank[Mantel-Cox]test).Secondly,at 72h after SE,water content in the Trpm4-/-SE group was much less than WT-SE group in hippocampus,piriform cortex and cortex,respectively(all p<0.001).More importantly,a much more increased signal was observed on the T2-weighted images(T2WI)and diffusion weighted images(DWI)at 48h after SE in WT-SE group than that in Trpm4-/--SE group.Apparent diffusion coefficient(ADC)were lower in Trpm4-/--SE group than that in WT-SE group,which indicated that cerebral edema was alleviated in Trpm4-/-mice.Thirdly,the number of neurons in hippocampal CA1 and CA3 region and piriform cortex at day 7 after SE were significantly ameliorated in the Trpm4-/--SE group when compared to the WT group(both p<0.001).There were less PI-positive cells in the Trpm4-/--SE group than the WT-SE group in hippocampus and piriform cortex(all p<0.001).In addition,much more cleaved caspase 3-positive cells were detected in the WT-SE group than Trpm4-/--SE group(p<0.01),which suggested that Trpm4 deficiency inhibited cellular apoptosis induced by SE.Fourthly,Evans blue(EB)extravasation was decreased in Trpm4-/--SE group than that in WT-SE group.Moreover,relative intensity of leaked IgG was markedly decreased in hippocampus and piriform cortex in Trpm4-/--SE group than that in WT-SE group(both p<0.01).Fifthly,the number of activated microglias and astrocytes in Trpm4-/--SE group were much less than that in WT-SE group(both p<0.01).Sixthly,Trpm4-/--SE group displayed less frequency and shorter duration of SRS than WT-SE group(both p<0.05).Finally,Trpm4-/--SE mice showed a significant decrease in the latency to the platform during acquisition compared with WT-SE mice during the 5 consecutive training days(p=0.0014,two-way ANOVA).Conclusions1.Cerebral edema and neurological injury can be induced by Li 10mEq/kg+pilocarpine 30-40mg/kg in a C57BL/6J mouse model of SE.2.Trpm4 deficiency improved survival rate of SE mice.3.Trpm4 deficiency alleviates cerebral edema,also ameliorates neuronal injury and BBB breakdown after SE.4.Trpm4 deficiency reduces the development of SRS and improves neurological outcomes of SE mice.