| Objective: The aim of this study was to investigate the effects of LIP to spinal cord ischemia-reperfusion injury on the expression of ERK pathway in rats, and to explore the spinal cord protection mechanism by LIP, which could provide us new ideas to reduce spinal cord secondary injury in clinical.Method: Ninety male Sprague-Dawley rats, weighting250-300 g, were randomly divided into 5groups with 18 each.(1)group with sham operation served as negative control(2)group with SCII(3)group with LIP(Limb ischemia precondition)(4)group with LIP+PD98059(5)group with LIP+DMSO,And then randomly divided into three sub-groups according to different sampling time point(2h,6h, 12 h,n=6,respectively). The sham group open the abdomen to expose the abdominal aorta below the kidney without blocking;The SCII group blocking the abdominal aorta at the level below the renal artery for 25 minutes, then restore blood flow; The LIP group consisted of three cycles of 5-minute ischemia followed by 5-minute reperfusion in right hind limb by rubber band before establishing SCII model for 24 hours; The PD group and DMSO group respectively were injected intravenously with ERK inhibitor PD98059(0.2mg/kg) or DMSO(0.2mg/kg), then were treated the same as group LIP after 24 hours. All animals were neurologically assessed at 6h, 8h, 10 h, 12 h after reperfusion and then sacrificed for histopathological evaluation, stored at-80℃. Three representative sections taken from L3 to L5 segments were stained with Hematoxylin and Eosin(HE) for histopathologic evaluation. Neuronal injury was evaluated at a magnification of ×200 in the ischemic ventral spinal cord.Neuron apoptosis and the influence of LIP on apoptosis was detected by terminal deoxynucleotidyl transferase-mediated d UTP-biotin nick end labeling(TUNEL) staining. The expressions of p-erk and total erk were detected by immunohistochemical method and western blot.Result: BBB results show: The LIP group and the DMSO group were significantly higher score than the SCII and PD group(p<0.001). For the detection of neuronal cell damage the tissue slices were analyzed using the TUNEL method,the number of TUNEL positive cells in SCII group was significantly higher than other groups(P<0.05),on the other hand, the PD group and the DMSO group were significantly lower than the SCII group(P<0.05), while the PD group was significantly higher than the group LIP(P<0.05). There were significant differences in expression of p-erk protein between each sub-group of the five major groups, p-erk protein were significantly expression at 2 hour, and then it reached a peak at 6 hour and a trough at 12 h. The expression of p-erk have correlation with the neurological outcome(r=0.657, p<0.05).Conclusion: Limb ischemia preconditioning can reduce deformation and necrotic neurons after spinal cord ischemia-reperfusion injury, improved neurological function after reperfusion; it can improve the spinal cord reperfusion injury tolerance by upgrading the expression of ERK pathway. |
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