| Objective:To establish a model of rat spinal cord ischemia reperfusion injury(SCII),and investigate the expression regularity of matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase(MMP-9/TIMP-l)in spinal cord ischemia reperfusion injury,and observe the neural protection effect of Neuregulin-1?(NRG-1?)in spinal cord injury.Methods:Seventy-eight healthy special pathogen-free male Sprague-Dawley rats,6-8 weeks old,weighing 200g-250 g,were selected.The experiment is divided into two parts:Part I: 48 rats were randomly divided into 3 groups: control group(n=16),ischemia reperfusion group(model group,n=16),NRG-1? treatment group(n=16),spinal cord ischemia reperfusion injury was induced by using the modified Zivin method,ischemia time 30min;The NRG-1?(10?g/kg)was injected through tail vein immediately after blood perfusion in the treatment group,and the ischemia reperfusion model group was injected with an equal amount of 0.1mol/L PBS buffer solution after blood perfusion.Neurological function was assessed by using the modified Tarlov standard.Sampling test was performed at 3h,6h,12 h,24h respectively after injury(4 rats at each time point).The pathological changes were observed by HE staining,Protein and mRNA expressions of MMP-9,TIMP-1,TNF-? and ICAM-1 were assessed by immunohistochemistry and real-time PCR.Part II: 30 rats were randomly divided into 3 groups: Schwann cells with high expression of NRG-1? transplantation group(A group),Schwann cells with reduced expression of NRG-1? transplantation group(B group),and normal Schwann cells transplantation group(C group).The animal model was established by modified Zivin method,Rats in A group,B group and C group were injected different expression of NRG-1? Schwann cells through intervertebral space by microinjector.Neurological function was assessed by using the modified Tarlov standard.Sampling test was performed at 24 h after Schwann cells transplantation.The pathological changes were observed by HE staining,Protein and mRNA expressions of MMP-9,TIMP-1,TNF-? and ICAM-1 were assessed by immunohistochemistry and real-time PCR.Result:Part I:1.Neurological function score: The Tarlov score of rats in model group was lower than that in control group,and the Tarlov score of rats in treatment group was higher than that in model group,the difference was statistically significant(P<0.05).2.Pathological manifestations of spinal cord: Spinal cord in control group had no obvious damage,while they showed significant pathological damage in the model group and treatment group,but the degree of spinal cord injury in treatment group reduced significantly compared with that in model group.3.Immunohistochemical results: Compared with model group,the number of positive cells of MMP-9 and TNF-? in the treatment group reduced at reperfusion 6h,12 h,24h,and the number of TIMP-1 positive cells increased at reperfusion 12 h,24h,and the number of ICAM-1 positive cells reduced at reperfusion 12 h,24h,the difference was statistically significant(P<0.05).4.Real-time PCR test results: Compared with model group,the mRNA expression of MMP-9 and TNF-? in the treatment group decreased at reperfusion 6h,12 h,24h,and the expression of TIMP-1 mRNA increased at reperfusion 12 h,24h,and the expression of ICAM-1 mRNA decreased at reperfusion 12 h,24h,the difference was statistically significant(P<0.05).Part II:1.Neurological function score: Rats in the B group and C group had obvious motor dysfunction,while that in the A group had only mild motor dysfunction.Tarlov score of rats in B group compared with that in C group,the difference was not statistically significant,Tarlov score of rats in A group compared with that in C group,the difference was statistically significant(P<0.05).2.Pathological manifestations of spinal cord: Spinal cord in A group had only slight pathological changes,while they showed obvious pathological damage in B group and C group.3.Immunohistochemical results: Compared with C group,the number of MMP-9,TNF-? and ICAM-1 positive cells reduced significantly,and the number of TIMP-1 positive cells increased significantly,and the difference was statistically significant(P<0.05).4.Real-time PCR test results: MMP-9,TIMP-1,TNF-? and ICAM-1 mRNA expression in A group compared with C group was statistically significant difference(P<0.05).While the m RNA expression showed no significant difference between B group and C group.Conclusion:1.The rat spinal cord ischemia reperfusion injury model was established successfully,and that can cause rats dysfunction,pathological damage of spinal cord and the expression change of MMP-9,TIMP-1,TNF-? and ICAM-1.2.Neuregulin plays a neural protective role in spinal cord ischemia reperfusion injury,which may be related to down-regulation of MMP-9 expression and up-regulation of TIMP-1 expression.3.The experiment confirmed that the high expression of NRG-1? Schwann cells transplantation can down-regulate the expression of MMP-9 and up-regulate the expression of TIMP-1,and inhibit the expression of inflammatory factor TNF-? and ICAM-1,which may be the activation mechanism of neural protection in spinal cord ischemia reperfusion injury. |
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