| Spinal cord injury is a kind of serious injury , whose mutilation rate is high,itoften bring incubus to family and society, the treatment of spinal cord injury isstill a cosmopolitical puzzle. The exact mechanism of spinal cord injury is unclear,while it is recognized that spinal cord injury contain primary injury and secondinjury. Moreover, the vascular mechanism and neurobiochemistry mechanism arethe important mechanism of second injury, including lipid peroxidation,excitedamino acid, nitrogen monoxideium,endogenous opium peptide,etc.Erythropoietin(EPO), 34kd, is asialoglycoprotein. EPO can take action whenit bind to its receptor.one molecular EPO can bind to two molecular EPOR, theend is the receptor-binding area.when the intracellular zone tyrosin take phos-phorylation, induce the conduction of intercellular signal, modulate theintranuclear gene expression, finally control the survive, proliferation,differentiation of cell.EPO was considered to be excreted only by the kidney andresponsible for the proliferation, maturation, and differentiation of the precursorsof the erythroid cell line blood factor. Recent studies have demonstrated that EPOand its receptors(EPOR) are widely expressed in the central nervous system(CNS).Moreover,some research has confirmed that EPO is a multifunctional nutritionalfactor and neuroprotective factor, which can modulate the neurodevelopment, andhave neurotrophy and neuroprotective function in different conditions of neuronaldamage. The exact mechanism of EPO's neuroprotective effect after the CNSinjury are still not well understood. However, it was not documented that theeffect and mechanism of EPO on spinal cord injury. The study aimed at to explorethe effect and mechanism of EPO on spinal cord injury.I EPO and EPOR expression after injured spinal cord in rats.Objective: To investigate the change rule of EPO and EPOR expression afterspinal cord injury in rats.Methods:.By the modified Allen's method, the model of SCI is established,24 SD rats were divided into two groups: experimental group(n=12) subject tolaminectomy and bounce of spinal cord, control group(n=12) only underwentlaminectomy. EPO and EPOR expression in cytoplasm of spinal cord wasdetermined by immunohistochemical analysis(SABC method). EPO and EPORpositive cells were counted by Leicaquantitation570 image analysis system, all thedata was expressed as±S, difference analysis was made by t test .Results:①In normal spinal cord: weak immunoexpression of EPO and EPORobserved in neurons and glial cells, the mean number was individually16±2.5,28.6±6.2.Weak EPO and EPOR staining was also observed in capillaries andependymal cells. These findings were unchanged during subsequent observations.②at 8 hours and 2 days after injury: it was no significant difference that the meannumber of EPO immunostained cell and EPOR-positive cell between experimentalgroup and control group(P>0.05)③In experiment group, a marked increase ofexpression of EPO and EPOR was observed in neurons, vascular endothelium,and glial cells, peaking at 8 days after injury, the mean number was individually290.7±7.3,370.8±9.2,after which it gradually decreased. Two weeks after injury,EPO immunoreactivity was scarcely detected in neurons, whereas glial cells andvascular endothelium expressed strong EPOR immunoreactivity. the mean numberwas individually 43.8±5.4,200.6±8.1.Conclusions: EPO and EPOR expression assumed time-related after spinalcord injury. The increase in EPOR staining strongly support the possibleusefulness of a therapeutic approach based on exogenous EPO administration.II The effect of recombinant human erythropoietin on the lipidperoxidation after spinal cord injury in ratsObjective: To explore the effect of recombinant human erythropoietin on thelipid peroxidation and ultrastructure after spinal cord injury in rats.Methods:By the modified Allen's method, the model of SCI is established, 27SD rats were divided into three groups: experimental group(n=9) subject tolaminectomy and bounce of spinal cord, then rHuEPO intraperitonealadministration control group(n=9) subject to laminectomy and bounce of spinalcord, then equal volume saline intraperitoneal administration, normal group(n=9)underwent no disposal. The content of malondialdehyde at every time-point afterspinal cord injury was measured by thiobarbituric acid method. The ultrastructurechange of injured spinal cord tissue at every time-point after spinal cord injurywas observed by transmission electron microscope, meanwhile, ultrastructurescore was carried out by blind method. All the data was expressed as X ±S,difference analysis was made by t test .Results: The content of malondialdehyde of injured spinal cord increasedwith time shift in treatment group and control group,The content ofmalondialdehyde of injured spinal cord increased significantly in control groupthan that in normal group at every time point after SCI, There was statisticallysignificance between treatment group and control group(P<0.05). The content ofmalondialdehyde of injured spinal cord decreased significantly in treatment groupthan that in control group at every time point after SCI, There was statisticallysignificance between treatment group and control group at 2h,24h after SCI(P<0.05), There was statistically significance between treatment group and controlgroup at 48h after SCI (P<0.01).The ultrastructure score of injured spinal cordincreased gradually with time shift in control group. The ultrastructure score ofinjured spinal cord changed slightly with time shift in treatment group. Theultrastructure score decreased obviously in treatment group than that in controlgroup at every time point after SCI, There was statistically significance betweentreatment group and control group(P<0.05).Conclusions: Lipid peroxidation occurred after spinal cord injury, rHuEPOcan decrease the content of malondialdehyde after spinal cord injury, relieve thelipid peroxidation after spinal cord injury, increase the antioxidant capacity ofspinal cord, rHuEPO can decrease ultrastructure score of injured spinal cord,decrease ultrastructure change of injured spinal cord, protect spinal cord tissue,have neuroprotective effect.III The effect of recombinant human erythropoietin on the cell apoptosisafter spinal cord injury in rats.Objective: To explore the effect of recombinant human erythropoietin on thecell apoptosis after spinal cord injury in rats.Methods:By the modified Allen's method, the model of SCI is established, 60SD rats were divided into two groups: experimental group(n=30) subject tolaminectomy and bounce of spinal cord, then rHuEPO intraperitoneal administrationcontrol group(n=30) subject to laminectomy and bounce of spinal cord, then equalvolume saline intraperitoneal administration. The mRNA expression of bcl-2, bcl-xl,bax at every time-point after spinal cord injury was measured by reversetranscription polymerase chain reaction(RT-PCR) method. The cell apoptosischange of injured spinal cord tissue at every time-point after spinal cord injurywas detected by TUNEL method, meanwhile, apoptosis index was calculated byblind method. All the data was expressed as X ±S, difference analysis was madeby t test.Results: The level of bcl-2,bcl-xl,bax mRNA expression after spinal cordinjury.①The expression of bcl-2 mRNA can be seen at every time point afterspinal cord injury in control group, the level of bcl-2 mRNA increased graduallywith time shift, reach the peak at 24 h after SCI, then decrease gradually,Thelevels of bcl-2 mRNA expression in treatment group were increased obviouslythan that in control group at every time point after spinal cord injury. There wasstatistically significance between treatment group and control group (P<0.05) ②The expression of bcl-xl mRNA can be seen at every time point after spinal cordinjury in control group, the level of bcl-2 mRNA increased gradually with timeshift, reach the peak at 24 h after SCI, then decrease gradually, The levels of bcl-xlmRNA expression in treatment group were increased obviously than that incontrol group at every time point after spinal cord injury. There was statisticallysignificance between treatment group and control group(P<0.05) ③ Theexpression of bax mRNA can be seen at every time point after spinal cord injuryin control group, the level of bax mRNA increased gradually with time shift, reachthe peak at 24h after SCI, then decrease gradually, The levels of bax mRNAexpression in treatment group were not increased obviously than that in controlgroup at every time point after spinal cord injury. There was no statisticallysignificance between treatment group and control group(P>0.05) ④A smallquantity of TUNEL positive cells can be observed in gray matter and white matterof spinal cord at 6h,12h after spinal cord injury in treatment group and controlgroup.The cell apoptosis index decrease remarkably in treatment group than thatin control group, There was no statistically significance between treatment groupand control group(P>0.05) . At 24h after SCI, cell apoptosis index of treatmentgroup and control group increase gradually with time shift, the apoptosisincreased significantly in control group than that in treatment group, the apoptosisindex decrease obviously in treatment group than that in control group at everytime point(24h,48h,72h)after SCI, there was statistically significance betweentreatment group and control group( P<0.05).Conclusions: Cell apoptosis was observed after spinal cord injury, and it playan important role in second injury, rHuEPO can up-regulate the mRNA expressionof bcl-2,bcl-xl, decreased the apoptosis index of nerve cell after spinal cord injury,rescue injured spinal cord tissue, rHuEPO have neuroprotective effect. |
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