The Research Of The Micro Vessel Effect On Diabetes Aggravated Cerebral Ischemia-reperfusion Injury

Objective To investigate the change of number and structure of microvascular andendothelial cell ultrastructure changes of diabetes/hyperglycemia after cerebral ischemia.Toevaluate the damage the effects and its possible mechanism of the change of microvascular onfocal ischemia-reperfusion injury in SD rat with hyperglycemia.Further the mechanism ofhyperglycemia increase damage of cerebral ischemia.Methods The male SD rats,260-300g,were randomly divided into4groups:the diabeticsham operation group(DS),the normal sham operation group(NS),the normal cerebralischemia-reperfusion group(NM),and diabetic cerebral ischemia-reperfusion group(DM). Todevelop a rat model of typeⅠdiabetes mellitus in SD rats,and those in diabetic group werereceived intraperitoneal injection of STZ,those in normal group were received intraperitonealinjection of physiological saline.After blood glucose is being constant higher than16.7mol/ml to established the model of Middle cerebral artery occlusion(MCAO) that wastransient focal ischemia by placement of an intraluminal filament at the origin of right middlecerebral artery of surgical group.Neuronogical behavior was evaluated by Longa’s scoringmethod. we collect the brain of rats in different time point. Take Serial coronal planesections from the place before anterio fontanelle2mm to after3mm,section3mm thick andkept the brain tissue section fixed into4%paraformaldehyde for HE andimmunohistochemical staining.Take the infarction area surrounding the cortex of brain keptinto-80℃for Western-blotting and take the fresh brain tissue by2.5%Glutaraldehyde fixation. HE and PAS staining was applied to observe blood vessels injury and regeneration indiabetic model group and the normal groups cerebral ischemia in MCAO. vWFimmunohistochemical staining were used to detect the degree of damage vascularendothelial; Immunohistochemistry and Western-blotting were used to observe the expressionof VEGF,VEGFR2positive cells in the ischemic brain tissue in theat different time ofischemia and reperfusion. The location of VEGF was observed by fluorescent double-stainingimmunofluorescence.Transmission electron microscopy（TEM） was used to observe themorphological change of vascular endothelial cells cerebral ischemia reperfusion1d,3d,7d.Further the influences to microvessel and their possible molecular mechanisms and provides anew method for the therapy of hyperglycemia increase damage of cerebral ischemia.Results The volume of cerebral infarct in DM group is obviously bigger than the NMgroup.Histochemical results suggest that control group no significant change.After1d ofischemia,brain tissue slightly edema, lumen of blood vessel broaden,neurons swelling inischemic area,part of the nucleus karyopyknosis.Clearly edema also appeared at contralateralbrain at3d of reperfusion,vascular lumen is irregular,the quantity of micro vessels wereincreases.At7d,a great number of microglia were activated,vascular injury better thanbefore.When to28d,blood vessels is no no significant difference between the normal state.Thelesions degree of DM was serious than NM,and the heaviest injury in3d.PAS showsthat:After reperfusion1d,in DM and NM group,the lumen of blood vessel broaden,basementmembrane thinner, the stain was lighter than normal control group.At3d, vascularendothelial were further aggravate than the1d group,the basement membrane is thin, andform cavitations and different thickness phenomenon is obvious.Some areas even existedfracture phenomenon. Vessels were irregular and dyeing bleached.Reperfusion7d basementmembrane hyperplasia,some parts became thickening and dyeing were thicken than1，3d.At14d, the vessel return to normal,basement membrane were thickened than normal bloodvessels slightly and are complete,deeper PAS stain.Transmission electron microscopy（TEM） results show the normal cerebral ischemia-reperfusion group,and diabetic cerebralischemia-reperfusion group vascular endothelial cells appear different degree of damage,thehigh glucose group showed higher damage than normal glucose.Compared with the NM, DMvessel wall surrounding vacuoles obviously increase, larger, vessel wall thickening andluminal stenosis.In DM1d, endothelial cell apoptosis body formation, the severe edema,almost all basal membrane stripping, appear a large number of vacuoles.In3d, basementmembrane thickness differ phenomenon obviously, some places even break, basementmembrane nucleus pycnosis, visible neovascularization, irregular lumen.7d, vessel wall wasobvious thickening than NM, thickening like hyperplasia, vascular were decreased than1,3dgroup significantly, the vessel lumen narrowing.vWF immunohistochemical results show:at1d of reperfusion,the NM group, a small amount of damage to the vascular endothelium.At3dof reperfusion,damaged blood vessels increased in quantity, declined in7d,returned to normalin28d. Immunohistochemical results show：in sham groups, there were scattered VEGF,VEGFR2positive cells (mainly in the cortex).VEGF mainly localized in the ischemicpenumbra.It significantly increased in diabetic and euglycemic ischemic rats(P<0.01).At thesame reperfusion time, VEGF expression in euglycemic ischemic rats is more than diabeticischemic rats(P<0.05). In ischemic groups VEGF, VEGFR2positive cells expressiondecreased at1d, peaked at3d, with the prolongation of reperfusion, the expression of VEGF,VEGFR2decreased, to28d was still expression. Western Blot results show that VEGF,VEGFR2protein the normal cerebral ischemia-reperfusion group and sham group was muchless than model group. In model group, there was a certain amount at1d after reperfusion,reached to peak at3d, then reduced gradually, to28d was still expression.(P<0.05）Fluorescent double-staining immunofluorescence results show that VEGF immune positivecell expressed in neuron and astroglia cell.Conclusion1.Diabetes can aggravate the brain damage，increase the infarction area and micro vascular damage. Microvascular damage including: endothelial cell, basement membraneand astrocytes damage, participated in the diabetes/hyperglycemia is aggravating cerebral ischemia injury.2. Diabetes can promote microvessel in the process of ischemia-reperfusion, microvesselof hyperplasia were growth in clusters, structure is not complete. May be related to diabetescaused poor prognosis of cerebral ischemia.3. Under the condition of diabetes,cerebral ischemia reperfusion injury induced theexpression of VEGF,VEGFR2in the early time. It is possible that the early expression ofVEGF, VEGFR2were involved to promote the angiogenesis and remodeling of peripheral ofischemia,with promoting the recovery of neurological function.4. In diabetes cerebral ischemia-reperfusion injury the changes of VEGF and VEGFR-2protein expression may be the key factor of aggravating of microvessel.