The Experimental Study Of Neuroprotection By Erythropoietin Following Traumatic Brain Injury

On the basis of stable and reliable TBI models,we studied the expression oferythropoietin and its receptor around the injured brain tissue in the rats aftertraumatic brain injury;then,by the means of Western blot,immunohistochemisty andTUNEL,the apoptosis index and related apotosis gene expression were tested;at last,it was certified that effect of erythropoietin on the TBI rats' cognitive function.Part One:The expression of erythropoietin and its receptor in the rats followingtraumatic brain injury.Objective:The part was aimed to study the expression of erythropoietin and itsreceptor around the injured brain tissue in the rats after traumatic brain injury.Methods:78 SD rats were randomly divided into 3 groups including controlgroup(six rats),sham group(thirty-six rats) and fluid percussion injurygroup(thirty-six rats).According to the time of death,the latter two groups weredivided into six subgroups which were 6 hours,twenty-four hours,three days,fivedays,seven days and fourteen days after traumatic brain injury (TBI).Six rats weresentenced to death in the sham and TBI group at each time point.It test the messengerRNA and protein of erythropoietin and its receptor of the brain tissue surrounding theinjuries by the methods of real-time PCR and Western Blot respectively.Results:EPO and its receptor mRNA expressed in normal control and sham groupat each time point were just a small amount.Real-time PCR and Western Blotindicated that EPO mRNA and protein expressions increased at 6h,peaked at 3d,which sustained two days,and gradually declined to 7 d,returned to normal level at14d.EPOR mRNA and protein expressions in the TBI group began to increase at 6hafter injury,peaked at 24h,sustained to 14d.Conclusion:EPOR expression was significantly upregulated at least for 14 daysafter traumatic brain injury,while EPO mRNA and its protein were only shortlyelevated.The protective mechanism of EPO on central nervous system is possiblyrelated to the markedly increased expression of erythropoietin receptor after TBI.Part Two:The effect of erythropoietin on apoptosis of nerve cells in the TBI modelsin Vivo.Objective:The part was aimed to evaluate the effect of erythropoietin on apoptosis inthe TBI model,so as to conclude the neuroprotection mechanism of erythropoietin.Methods:76 SD rats was randomly divided into six groups including control,sham, TBI,EPO,EPO+DMSO and EPO+LY294002 group,each group had twelve rats.Thelatter three groups were injected with erythropoietin (5000u/Kg,Q8h) by the way ofintraperitoneal.Meanwhile,the latter two groups were injected with DMSO orLY294002 through intraventricule in order to block PI3K pathway.After 2 days,allrats were sentenced to death,in order to test the protein of EPO and pAKT byWestern Blot,and the protein of apoptosis by immunohistochemisty,and apoptosisindex by TUNEL.Results:it is indicated in Western Blot that the erythropoietin treatment can promotethe expression of its protein,while downregulated the expression of pAKT.It isindicated in immunohistochemisty that EPO can inhibit the expression of BAX andCaspase-3,thus reduce apoptosis,while LY294002 can block the PI3K pathway andupregulated BAX and Caspase-3,so the apoptosis index was more than the EPOtreatment group.Conclusion:EPO can inhibit the apoptosis in the TBI model,when it is blocked withthe PI-3K pathway,which increased the apoptosis index.Part Three:The effect on cognitive function of erythropoietin in the rats aftertraumatic brain injury.Objective:The part was aimed to evaluate the effect on cognitive function oferythropoietin.Methods:48 SD rats were randomly divided into four groups including control,sham,TBI and EPO+TBI group,each have twelve rats.The last group were injected witherythropoietin immediately after injury,the others were injected with saline at thesame time.After eight days,we used the Morris Water Maze to evaluate the referencememory and expression of BDNF by immunohistochemisty.Results:In the navigation experiment,the latent period that all the rats found theplatform was shorter and shorter,and there is no difference in speed,while the controland sham groups' latent period was the shortest,and the TBI group's was longest,EPO can improve the latent period.Meanwhile,it can promote the expression ofBDNF.Conclusion:TBI can damage the cognitive function of rats,while exogenous EPOcan improve the rats' cognitive function and upregulated the expression of BDNF.