Neuroprotection And Mechanisms Of Glibenclamide In A Rat Model Of Cerebral Edema After Status Epilepticus

BackgroundStatus epilepticus(SE)is a common neurological emergency with high morbidity and mortality.Cerebral edema during SE,together with the subsequent intracranial hypertension and brain herniation,are the major causes of early death.Sustained seizures can lead to blood-brain barrier(BBB)breakdown and vasogenic edema.More importantly,cerebral edema aggravates the progression of spontaneous recurrent seizures(SRS),while serum components penetrated to brain parenchyma after BBB disruption worsen exitotoxicity of neurons.A vicious circle between cerebral edema and sustained seizure is formed to aggravate cerebral edema,accelerate the progression of epilepsy,and deteriorate the outcome of SE patients.Cerebral edema is formed followed by three events in turn:cytotoxic edema,BBB disruption and vasogenic edema.During the sequential process of cerebral edema,cytotoxic edema formation is believed as the first and critical step to induce following severe cerebral edema and swelling.The opening of sulfonylurea receptor 1-regulated transient receptor potential M4(SUR1-TRPM4)channel can induce influx of Na+ and water to form cytotoxic edema.The SUR1 blocker glibenclamide(GBC),an FDA approved drug for the treatment of type-2 diabetes.In recent years,glibenclamide receives great concerns for its pluripotential neuroprotection in acute neurological injury through blocking the SUR1-TRPM4 channel.It has been proved to alleviate cerebral edema and protect neurons in animal models of cerebral infarction,cerebral hemorrhage,traumatic brain injury,and hepatic encephalopathy.Previously we also demonstrated that GBC markedly improved neurological outcome in a rat model of cardiac arrest and resuscitation,whose effectiveness was comparable to target temperature management.In this study,we aimed to evaluate whether GBC might reduce cerebral edema and improve outcomes in SE model.Methods1.The establishment of cerebral edema in a rat model of SEIn this study,SE was induced by li-pilocarpine.SE rats were randomly assigned to the SE 1.5h group(n=15),SE 2h group(n=15),SE 2.5h group(n=25)or SE 3h group(n=20),by using random number table.All of the rats were followed up for 3 days and assessed for survival,cerebral edema,histological injury and neuroinflammation.(1)The brain water contents in hemisphere,hippocampus and piriform cortext were assessed by dry/wet weight.(2)Neurons were stained with cresyl violet,and observed under microscope.Immunohistochemistry was performed with antibodies against rat IgG for BBB permeability,Iba1 for microglia,GFAP for astrocyte,according to the protocol suggested by the manufacture.2.Neuroprotection and mechanisms of GBC in a rat model of cerebral edema after SESE rats with behaviol seizures terminated at 2.5h after SE onset,were randomly assigned to the GBC group or the vehicle group,by using random number table.GBC was dissolved in dimethyl sulfoxide(DMSO)and diluted in saline.Rats in the GBC group were received i.p.administration of GBC with a loading dose of 10 μg/kg followed by 1.2 μg/6h for 3 days,while rats in the vehicle group received equal volume of DMSO and saline.The present study includes four parts.In part 1,the rats in the non-SE group(n=10),vehicle group(n=36)and GBC group(n=23)were followed up for 28 days and assessed for survival and neurological outcome and histological injury.Neurological outcome were assessed by recording of SRS and test of Morris water maze(MWM).In the recording of SRS,seizures were monitored 24 h per day by video recording with infrared light in the dark from day 8 to day 28 after SE.Outcome measures were the average number,duration and stage of SRS per day.MWM was performed at day 28 after SE using an animal behavior analysis system.Then,the rats were sarcrificed.Number of viable neuron was observed with Nissl staining in the hippocampal CA1 region and piriform cortext,as well as activated microglia with antibody against Iba1.In part 2,rats were euthanized at day 3 after SE,and the brains were harvested for examinations of brain water contents,histological injury,neuroinflammation and the expressions of SUR1 and TRPM4 in every experimental group(all n=5).(1)The brain water contents in hemisphere,hippocampus and piriform cortext were assessed by dry/wet weight.(2)Neurons were stained with cresyl violet,and observed under microscope.Immunohistochemistry was performed with antibodies against rat IgG for BBB permeability,microtubule-associated protein 2 for dendrite,Iba1 for activated microglia according to the protocol suggested by the manufacture.For immunofluorescence,brain cryo-sections were incubated with primary antibodies directed against SUR1,TRPM4,NeuN or vWF.Bound primary antibodies were detected with Alexa Fluor(?)dye-conjugated donkey anti-mouse or anti-rabbit and observed with a confocal microscopy.The relative intensity and the region size of IgG leakage were determined with Image J.(3)Total protein were extracted from brain tissues separated as hippocampus and piriform cortext,and Western blotting was performed to assess the expression of SUR1 and TRPM4 at day 3 after SE in the vehicle group and GBC group.In part 3,RNA and total protein were extracted from brain tissues,separated as hippocampus and piriform cortext,at 6 h,12 h,24 h,day 3 and day 7 after SE in every experimental group.Quantitative PCR was performed to check the mRNA expression of Abcc8(encoding SUR1)and Trpm4;Western blotting was performed to assess the expression of SUR1 and TRPM4(all n=5).In part 4,TRPM4 expression,BBB disruption and neuronal degeneration were examined in rats under Trpm4 siRNA or scramble siRNA treatment(n=5,respectively).Results.1.Cerebral edema,neurological injury and neuroinflammation were obviously observed in a rat model of SE induced by Li-Pilocarpine,with behavioral seisures terminated at 2.5 h after SE onset.Firstly,as compared to the Non-SE group,brain water contents were not changed in the SE 1.5h group,but were much higher in the SE 2h,SE 2.5h,and SE 3h group at day 3 after SE,all in hemisphere,hippocampus and piriform cortex(all P<0.05).Secondly,BBB breakdown,an important marker of cerebral edema,was assessed using extravasated endogenous IgG immunohistochemistry in brain sections isolated from different group rats at day 3 after SE.Increased IgG extravasations were observed in hippocampus and piriform cortex all in SE 1.5h,SE 2h and SE 2.5h group.More importantly,IgG extravasations in SE 2.5h group were more than that in SE 2h group both in hippocampus and piriform cortex(P<0.001 and P<0.05,respectively).Thirdly,as compared to Non-SE group,the numbers of neurons in SE 1.5h group were not changed at day 3 after SE in hippocampal CA1 region and piriform cortex,but much less in SE 2h group and SE 2.5h group,expecially in SE 2.5h group.Fourthly,the number of activated microglias and astrocyte cells in SE rats were much more than that in the non-SE rats.And activated microglias and astrocyte cells in SE 2.5h group were more than that in SE 2h group.Fifthly,the 3-day survival was 93.33%(14/15)in SE 1.5h group,73.33%(11/15)in SE 2h group,40%(10/25)in SE 2.5h group and 20%(3/15)in SE 3h group.There are no significant difference of 3-day survival rates of SE 1.5h group,as well as that of SE 2h group,while survival rates of SE 2.5h group and SE 3h group were much lower,when compared to Non-SE group(100%,10/10;P=0.41,P=0.08,P<0.01 and P<0.001,respectively).As the survival rate in SE 3h group was too much lower for harvesting enough brain tissue specimens,it suggests that SE lasting for 2.5h can lead to early death with suitable survival rate.2.GBC alleviated cerebral edema,ameliorated neuronal injury,decressed neuroinflammation,inhibited SUR1-TRPM4 upregulation and improved outcomes of SE.Firstly,results showed water content in the non-SE group,vehicle group and GBC group was 78.48%,80.76%,79.89%in hemisphere,79.86%,81.63%,80.37%in hippocampus,and 79.84%,82.13%,80.52%in piriform cortex at day 3 after SE,respectively.Water contents were all significantly increased in the three regions in SE rats,compared with non-SE rats,which all were decreased by GBC treatment(all P<0.05).More importantly,GBC administration markedly decreased relative intensity of leaked IgG in hippocampus and piriform cortex(both P<0.001),as well as the region size of IgG leakage in piriform cortex(P<0.01).Secondly,The numbers of neurons in hippocampal CA1 region and piriform cortex at day 3 after SE were much less than that in non-SE group,but were significantly ameliorated in the GBC group when compared to the vehicle group(both P<0.05).In addition,dendritic injury revealed as decrease of MAP2-staining was apparent in hippocampal CA1 region in the vehicle group but partly prevented by GBC treatment(P=0.001).At day 28 after SE,The numbers of neurons in hippocampal CA1 region and piriform cortex were much more in GBC group than in Vehicle group(P<0.01 and P<0.05,respectively).Thirdly,the number of activated microglias in GBC group were much less than that in the Vehicle group both in hippocampus and piriform cortex at day 3 after SE(P<0.01,P<0.05,respectively),as well as the COX2、IL-6(P<0.05 and P<0.01,respectively).At day 28 after SE,the number of activated microglias in GBC group were decreased in hippocampus and piriform cortex,compared with that in Vehicle group(P<0.01 and P<0.001,respectively).Fourthly,GBC treated SE rats displayed less frequency and shorter duration of SRS than vehicle control.In addition,a tendency of lower incidence and grade of SRS were observed in the GBC group than in the vehicle control,although differences were not statistically significant.Meanwhile,data in MWM test showed that shorted time in latencies were observed in rats from the GBC group as compared to rats in the Vehicle group at the fourth day of acquiring training(P<0.01).In probe trial,time of staying in the target quarter and frequency of crossing the location of platform were much more in the GBC group as compared to the Vehicle group(both P<0.05).Fifthly,survival rate at day 28 after SE was 22.22%(8/36)in the vehicle group,which was much lower than in the GBC group(47.83%,11/23)(χ2=3.877,P=0.045).Finally,SUR1-TRPM4 complex has been proved to be the putative therapeutic target of GBC.We then examined the expressions of these channel subunits before and at several time points after SE.Both the mRNA and protein levels of SUR1 and TRPM4 were significantly upregulated at 6 h after SE,and last for at least three and seven days,respectively.Additionally,SUR1 was evident in neurons and endothelial cells,with paralleled upregulation of TRPM4.Furthermore,the elevations of SUR1 and TRPM4 protein levels at day 3 after SE were reduced by GBC treatment.Knockdown of TRPM4 reduced BBB disruption and neuronal degeneration,similar to that with GBC treatment.Therefore,the protective mechanism of GBC after SE was associated with the inhibition of upregulation of SUR1-TRPM4.Conclusions1.Cerebral edema,disruption of BBB,neuronal injury and neuroinflammation can be induced by SE lasted for 2.5 h.2.Low-dose GBC treatment alleviates cerebral edema and disruption of BBB induced by SE.3.Cerebral edema during SE can further lead to neuronal degeneration in hippocampus CA1 region and piriform cortext,as well as dendritic damage in hippocampus CA1 region.Low-dose GBC treatment ameliorated neuronal degeneration in hippocampal CA1 region and piriform cortex.4.Cerebral edema during SE can lead to neuroinflammation.Low-dose GBC treatment decreases neuroinflammation in hippocampal CA1 region and piriform cortex.5.Low-dose GBC treatment improves 28-day survival,as well as cognitive function,and decreases both the frequency and duration of SRS.6.SE leads to the upregulation of SUR1-TRPM4 channel,which are partly inhibited by GBC treatment.Knockdown of TRPM4 reduced BBB disruption and neuronal degeneration,similar to that with GBC treatment.The salutary effects of GBC on the outcome of SE were associated with the inhibition of upregulation of SUR1-TRPM4.