| ObjectiveIntracerebral hemorrhage(ICH) is a common and often fatal stroke subtype that produces severe neurological deficits in survivors with no effective treatment. Erythropoietin(EPO) is a cell factor of regulating haematopoiesis, recently study discover that EPO and EPOR widespreadly exist in central nervous system,both boinding possessed nerve growth,neurotropy and neuroprotective effect,whose mechanism was complicate and not clear. We produe model of ICH according to method of autoblood injection,observe apoptosis of nerve cells and expression of Bcl-2,Bax and explore EPO interventional effct and mechanism.Materials and methods1 .Experimentation groups110 health adult male Wistar rats,each weighing 220-270g from Animal Center China Medical Uinversity,were randomly divided into four group,i.e.normal group(n=5),sham operation group,ICH group,EPO treatment group. Last three groups were divided into 6h,12h,24h,48h,72h,120h and168h sub-group(n=5).2.Animal modelsAccording to method of Xue and Yang,the rats of different groups were anesthetized with 10% hydration chloral(300mg/kg) after weighing. The animals were positioned in a stereotaxic frame,and a cranial burr hole (0.5mm) was drilled on the localization. Autologous blood was withdrawn from the tail artery and infused (50uL)immediately into the left caudate nucleus through a 26-gauge needle at a rate of 20mL/min with a micro infusion pump.The coordinates were 0.5mm anterior,6mm ventral,and3.0mm lateral to the bregma.After intracerebral infusion,the needle was removed,and the skin incision was closed with suture.The rats of sham operation group did not infuse blood.The rats of treatment group were injected rhEPO(3000UI/Kg) after operation within 5 minutes,the rats of sham operation group and ICH group were injected 0.5ml saline,the rats of normal group did not operate and cure.3.Get the brain tissuesThe rats of different groups were anesthetized with 10% hydration chloral(300mg/kg).At once,we cut open thoracic cavity;eposure heart;shears open pericardium,left venticle;insert catheter and fixation,at the same time,we use surgical scissors to right auricle,to put blood,then fill saline 4℃300ml and 4% paraformaldehyde 400ml,via systemic circulation. After liver color turn bright into grey white,we stop filling,get brain tissue after decapitation,and fix with 4% paraformaldehyde. After dehydration transparence and embedment,we get section of brain with 5um thickness and stain with HE,take picture with digital camera.4.ApoptosisApoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling technique(TUNEL).We treat sample by 3%H2O2 and wash it by distilled water;use ProteinaseK to digest the sample,wash it by distilled water;add tagging buffer to the sample,wash it by TBS;add blocking fluid and biotic anti-DIG-antibody,reach for 30 minutes,wash it by TBS.We add SABC for 30 minutes reaction;wash it by TBS;deal it by DAB;nuclei were counterstained with hematoxylin,dehydration mounting,observating with light microscope.The cases with brown nuclei staining were positive,we count positive cells of 5 random high time field each section and calculate the mean value. 5.Bcl-2,Bax ImmunohistochemistryBcl-2,Bax was detected by immunohistochemistry. We treat sample by and wash it by distilled water;use microwave to repair antigen,block non-specificity antigen with normal goat serum incubating 20 minutes;add Bcl-2,Bax polyclonal antibody 4℃to stay overnight;add biological tagging antibody and incubate 20 minutes;deal it by DAB;cells were counterstained with hematoxylin,dehydration mounting,observating with light microscope.The cases with brown kytoplasm and cell membrane staining were positive,we count positive cells of 5 random high time field each section and calculate the mean value.6.Statistical AnalysisSPSS for windows 13.0 statistical was used to solve the data.All data in this study are presented as mean±SD.Data multiplicity sample compare were used one-way ANOVA followed by LSD-t tests (comparisons between 2 groups);Pearson linear correlation was performed to examine the relationship between Bax protein expression and apoptosis,Bax/Bcl-2 and apoptosis,Significance levels were measured at P<0.05.Results1 .HE stainingThere were no necrosis cells,quantity of neuron,abundant kytoplasm,micro caryoplasm,distnct nucleolus in cortex of normal group.The necrosis cells were few in surrounding of pin hole in sham operation group.There were no neuron in center area of hemorrhage,a few colloid cells whose intercellular substances were vacuolar alteration.In perifocal tissue intracerebral hemorrhage there were rarefaction neuron,cell spaces augmentation,cell diminution,distinct demarcation of cell membrane and surrounding,and we discovered a lot of degeneration and necrosis nerve cells,cell body collapsed,pycnosis anachromasis,nucleoli disappeared. In EPO group we discovered that center area of hemorrhage shinked,nerve cells of degeneration and necrosis decreased in perifocal tissue,majority cells morphous were normal and pathological changes were light.The apoptotic cells and necrosis nerve cells of hemorrhage group,erythropoietin treated group obviously increased after 6h,reached peak 72h and decreased after 120h,the apoptotic cells on different time points significantly decreased compared with that of in hemorrhage grou.2.Apotosis of perifocal tissue intracerebral hemorrhageTUNEL positive cells were not discovered in cerebral cortex of normal rats,there were few positive cells in surrounding of pin hole in cerebral cortex of sham operation. The apoptotic cells of hemorrhage group,erythropoietin treated group obviously increased after 6h,reached peak 72h and decreased after 120h,the apoptotic cells on different time points significantly decreased compared with that of in sham operation group(P<0.01).The positive cells in EPO group on different time points significantly decreased compared with that of in ICH group(P<0.01).3.Compare of Bcl-2 positive cellsBcl-2 positive cells were not discovered in cerebral cortex of normal rats,there were few positive cells in surrounding of pin hole in cerebral cortex of sham operation. The Bcl-2 positive cells of hemorrhage group,erythropoietin treated group obviously increased after 6h,reached peak 72h and decreased after 120h,the Bcl-2 positive cells on different time points significantly increased compared with that of in sham operation group (P<0.01).The positive cells in EPO group on different time points significantly increased compared with that of in ICH group (P<0.01).4.Compare of Bax positive cellsBax positive cells were not discovered in cerebral cortex of normal rats,there were few positive cells in surrounding of pin hole in cerebral cortex of sham operation.The Bax positive cells of hemorrhage group,erythropoietin treated group obviously increased after 6h,reached peak 72h and decreased after 120h,the Bax positive cells on different time points significantly increased compared with that of in sham operation group (P<0.01).The positive cells in EPO group on different time points significantly decreased compared with that of in ICH group (P<0.01).5.Pearson linear corellation analysisBax protein expression and apoptosis presented positive correlation (r=0.889, P<0.01),Bax/Bcl-2 and apoptosis presented positive correlation (r=0.423,P<0.01).ConclusionApoptosis mechanisms may mediate some of the brain injury after ICH, erythropoietin can decrease the number of apoptotic cells after ICH by up-regulating Bcl-2 and down-regulating Bax. |
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