Role Of Glibenclamide In Brain Injury After Intracerebral Hemorrhage

IntroductionBrain edema is a key factor in secondary brain injury following intracerebral hemorrhage(ICH),in which the destruction of the blood-brain barrier(BBB)is one of most primary factors causing brain edema.Our previous research and correlative reports have indicated that metalloproteinases(MMPs)play an important role in BBB integrity damage after ICH.Therefore,to attenuate the secondary brain injury of clinical ICH,it may be important to develop new therapies against BBB disruption.Glibenclamide(GLC)is a member of the sulfonylurea class of drugs and has been used as an oral hypoglycemic medicine for many years.All classes of sulfonylurea agents work via inhibition of Sur1.Sur1 is a subunit that regulates the activity of two distinct ion channels: the Sur1-Kir6.2 channel and Sur1-Trpm4 channel.Recently,GLC attracted significant attention for its pleiotropic protection in CNS diseases,such as ischemic stroke,spinal cord injury,cerebral metastases,and subarachnoid hemorrhage(SAH)by inhibiting the Sur1-Trpm4 channel.Previous studies have demonstrated that GLC effectively protected the BBB and reduced brain edema via blocking Sur1-Trpm4.Recently published study reported that GLC,selective Sur1 inhibitor,reduced MMP expression or activation.However,to date,the role of GLC in ICH remains unclear.Thus,we hypothesize that GLC treatment may have the potential to protect BBB integrity and alleviate secondary brain injury after ICH by suppressing MMP expression and enhancing BBB tight-junction proteins by inhibiting the Sur1-Trpm4 channel.The present study was designed to examine this hypothesis using a rat model of ICH.Part ? The expression of sulfonylurea receptor 1 around the hemorrhage after intracerebral hemorrhage.ObjectiveTo investigate the expression of sulfonylurea receptor1 after intracerebral hemorrhage.MethodsThirty six Male Sprague Dawley(SD)rats were randomized into sham group and ICH group.ICH was induced via the stereotaxic basal ganglia injection of autogenous blood.The expression of Sur1 and Kir6.2 was measured using immunofluorescence,western blot analysis and Real-Time-PCR.ResultsSur1 but Not Kir6.2 is Up-regulation after ICHSur1 was previously reported to be up-regulated in various rat models of CNS injury,but its expression has not been measured in the ICH model.Immunofluorescence revealed that Sur1 is upregulated and localized in neurons and endothelial cells surrounding the hematoma 24 h after ICH.We observed that the expression of Sur1 was significantly upregulated around the hemorrhage after ICH,and Sur1 was observed expressed in endothelial cells and neurons 24 h after ICH.Sur1 expression was not observed in microglial cells at 24 h and 72 h after ICH.The expression of KIR6.2 exhibited no difference between the sham group and ICH group.ConclusionOur data demonstrated that the expression of the Sur1-Trpm4 channel but not the Sur1-Kir6.2 channel is up-regulated following ICH.Part II The effect of glibenclamide on Brain Injury after Experimental intracerebral hemorrhageObjectiveTo investigate whether glibenclamide have neuroprotective effect after ICH.MethodsThe potent SUR1 blocker GLC was purchased from Tocris Bioscience.Stock so lutions of GLC were prepared in dimethylsulfoxide(DMSO)(50mg/ml),and the injection solution was made at the concentration of 200 ng/?l or at 1 ?g/ml by dilution with unbuffered saline(0.9% Na Cl)and clarifying the solution using a few microliters of 0.1N Na OH(final p H~8.5).GLC treatment was initiated administering a single loading dose of GLC(10?g/kg)intraperitoneally plus subcutaneous continuous infusion at 200ng/h via a miniosmotic subcutaneous pump(Alzet 2001,1.0?l/h;Alzet Corp,Cupertino,CA)beginning at the end of surgery.Solutions of vehicle control were made with DMSO,NS and Na OH,and were administered in the same way.Neurological scores,brain water content,Evans blue extravasation,Morris Water Maze Test,western blots,and immunofluo rescence were used to study the effects of glibenclamide.Results1.Treatment with GLC Decreased Brain Water Content and Improved Neurological Deficits2.Treatment with GLC Decreased Evans Blue Extravasation 72 h after ICH.3.Treatment with GLC Improved Rats Performance in the Morris Water Maze Test.4.GLC Prevents Endothelium,ZO-1 and Occludin Attenuation after ICH.5.GLC Reduce ICH-Induced MMPs Expression.ConclusionThe neuroprotective effects of the Sur1 inhibitor GLC on brain edema,BBB integrity,and neurological deficits were observed in a rat ICH model.We also note that the suppression of MMPs,such as MMP-9 and MMP-2,is likely involved in the process.These data suggest that GLC is a promising therapeutic option for neuroprotection following ICH.Further studies are needed to understand the molecular pathways underlying how Sur1 and MMP are correlated.