Study Of The Mechanism About The Effect Of Dexamethasone On Brain Glioma Edema And The Permeability Of Blood Tumor Barrier

ObjectiveBrain glioma edema is an important phathophsiological change in the development of brain glioma.It is associated with many clinical symptoms;meanwhile, the rise of intracranial pressure caused by is a major reason for morbility and mortality. The application of dexamethasone(DEX)in clinic can reduce the mortality during and after operation dramatically,which close relate with its physiological function,for instance,rapid reduction of brain edema and repairmen of damaged blood-brain barrier. However,the molecular mechanism is still not clear.The Aquaporins(AQPs)are a family of channel-forming transmembrane proteins that facilitate the movement of water across the plasma membrane of cells.AQP4,the Predominant aquaporin in the endfeet of astrocytes surrounding blood vessels and also in the vascular endothelium of brain,is the key part in this biological process. AQP4-mediated transcellular water movement is crucial for fluid clearance in vasogenic brain edema,which suggested the activation and/or up-regulation of this channel could be a novel therapeutic option in vasogenic brain edema.However,little is known about the molecular mechanism about AQP4 regulation,which is necessary for the new therapy discovery. Increasing evidence suggests that there is an obvious decrease in both surgical morbidity and mortality following dexamethasone(DEX)treatment,which is routinely used in the management of patients with brain tumors and peritumoral edema.Recently, we found that DEX could reduce the brain hemorrhage edema by down regulation of AQP4 expression which was around the edema area.However,it's still need to investigate whether DEX decrease brain glioma edema by regulating the expression of AQP4.AQP4 was found located in blood-brain barrier and took part in the formation of it.According to literature,it was not found in 9 days of chicken brain,little expressed in 14 days and reached mature level in 20 days.This was correspondance with blood-brain barrier(BBB)mature.Another group reported that AQP4 was up-regulated after BBB damage which induced by LPS;it was up-regulated dramatically inbrain injury disease with BBB damage,but not in the brain without BBB damage.This indicated that the change of AQP4 expression which was around brain tumor may associate with brain edema and BBB permeability change.Recently some studies reported that AQP4 could act as osmosensor and be expressed in osmosensory regions of the brain,where it played a role in water accumulation.Up-regulation of AQP4 expression in the hypothalamus surrounding the third ventricle could contribute to mediating water drainage and CSF production.Taken together,these studies implicate that AQP4 is involved in the modulation of osmotic balance and BBB integrity. Previous study demonstrated that DEX could help to restore BBB integrity and reduce inflammatory cytoldnes thereby decrease osmotic change.AQP4 facilitates glial water uptake at sites of brain injury and water efflux at distant sites.Therefore,we hypothesizes that DEX,by regulating AQP4 expression in different brain regions, attributes to water clearance from the tumor site to distal areas and maintains osmotic balance between proximal and distal sites to the zone of tumor.The permeability of BTB was increased in patients with brain glioma edema and DEX could decrease the permeability of BTB.However,the mechanism about the effect of DEX on blood-brain tumor barrier is still not clear.The blood-brain barrier consists of capillary endothelial cells,basement membranes and astrocyte foot processes.The normal brain capillary endothelial cells lack fenestrations and are connected together with continuous tight junctions,which forms a major part of BBB. Patients with brain gliomas,whose capillary endotheliums are destroyed by tumor cells, the endothelial cells of tumor vessels ultrastructurally showed increased fenestrations, swelling and disrupted junctions.Simultaneous BTB-analysis was obtained in 4 patients showing correlation between oedema resorption in vivo and reduction of BTB transport rate constant Ki after 7 days of steroid treatment.Thus,the pathophysiological mechanisms of DEX-mediated action on the BTB permeability should be further investigated.In cultured endothelial cells,dexamethasone caused up-regulation and dephosphorylation of tight junction proteins,including occludin and ZO-1,which directly implicated DEX as one of the physiological modulators of tight junctions in vivo.Thus,we hypothesize that DEX could decrease the permeability of BTB by up-regulating occluding,one of the tight junction proteins in BTB.Transcellular way is another important way for drug transportation,which could enhance the permeability of BTB by increase the vesicular transportation.However,the effect which DEX exerts on transcellular transportation is still not reported. ATP-sensitive potassium channel and calcium-activated potassium channel are two important targets for transcelluar transportation in BTB,which could increase the formation speed of fenestrations in brain tumor capillary endothelial cells and tumor cells.Studies suggest that potassium channels on vascular endothelial cells are important targets for glucocorticoids regulation.DEX could regulate K_ATPchannels activity in primary vascular smooth muscle cell;and also regulate K_Cachannels activity by activation of serine/threonine protein phosphatase and protein kinase A in pituitary CH3 and AtT-20 cells.We hypothesize that DEX could regulate transcellular transportation by targeting ATP-sensitive potassium channel and calcium-actived potassium channels.Is whether DEX decreases brain edema correlated with regulation of AQP4 expression in different brain area? What's the molecular mechanism of the DEX regulating BTB permeability? These problems are still not clear and need be investigated.Methods1.Establishment of rat brain C6 tumor model.1×10⁶/10μl C6 cells were injected into the right caudate nucleus using the stereotaxic apparatus in rats,according to Nicolau.Tumor bearing models of rat were successfully prepared after 14 days.2.Brain edema content was analyzed after DEX-administration by dry-wet weight method in C6 glioma rat model.3.Fluorescence microscopes analysis was used to detect the colocalization and expression of AQP4 protein in different brain regions in C6 glioma rat model.4.Western blot and RT-PCR analysis was performed to investigate the expression of AQP4 mRNA and protein after DEX or/and BK ttreatment in C6 glioma rat model.5.BTB permeability was quantitatively evaluated by extravasation of EB as a marker of albumin extravasation after DEX or/and BK administration in C6 glioma rat model.6.Western blot analysis was performed to investigate the expression of the Kir6.2 subunit of K_ATPchannel,αsubunit of K_Cachannels and occluding protein in different groups.7.Immonohischemistry method was performed to investigate the expression of the Kir6.2 subunit of K_ATPchannel andαsubunit of K_Cachannels in different groups.8.The whole cell-recording mode of the patch-clamp technique was used to analysis the change of IK_ATPand IK_Cain C6 cells with DEX or/and BK pretreatment.Results1.Model of C6 glioma-rat was successfully established.2.Brain edema content was decreased by DEX administration for 3 days. 3.AQP4 protein was located in the endfeet of astrocytes surrounding blood vessels in brain and was significantly increased in the peritumoral brain tissue.4.AQP4 mRNA was markedly down-regulated after DEX administration for 1 day and AQP4 protein was significantly down-regulated after DEX administration for 3 days in the peritumoral brain tissue.5.AQP4 mRNA was markedly up-regulated after DEX administration for 1 day and AQP4 protein was significantly up-regulated after DEX administration for 3 days in the hypothalamus surrounding the third ventricle.6.The permeability of BTB was significantly decreased after DEX administration.7.The expression of occluding protein in brain tumor tissue was increased by DEX administration for 3 days.8.The expression ofα-subunit of K_Caprotein was significantly increased by DEX administration for 3 days,but the density of I_Kcain C6 cells could not be enhanced.9.The expression ofα-subunit of Kir6.2 protein was significantly increased by DEX administration for 3 days,but the density of IK_ATPin C6 cells could not be enhanced.10.The density of IK_ATPand IK_cainduced by BK was increased by DEX administration for 3 days.Conclusion1.AQP4 mRNA and protein was markedly up-regulated in the peritumoral brain tissue and was not changed in the hypothalamus surrounding the third ventricle.2.DEX downregulated the expression of AQP4 protein in the peritumoral brain tissue and upregulated it in the hypothalamus surrounding the third ventricle.3.DEX up-regulated the expression of occluding protein in brain tissue of C6 glioma rats.4.DEX up-regulated the expression of K_ATPchannel and K_Cachannel protein in brain tissue of C6glioma rats. 5.DEX enhanced the increase of IK_ATPand IK_Cainduced by BK.6.DEX inhibited the permeability of BTB,but not inhibited the increase of BTB permeability induced by BK.