| Experiment 1 Effect of limb remote preconditioning on antioxidant enzymes activities in spinal cord ischemic rabbitsObjective To examine the hypothesis that spinal cord ischemic tolerance induced by limb remote preconditioning (RPC) is mediated by an increase of antioxidant enzyme activities. Methods Sixty male adult New Zealand white rabbits were randomly assigned into four groups: control group, RPC group, ischemia group and RPC+ischemia group (6 in control group, 18 in each of other three groups). The animals of RPC group were underwent two cycles of 10min hind-limb ischemia; the animals in ischemia group were underwent infrarenal aorta occlusion for 20min; control group was sham group, animals in which were neurologically assessed by using modified Tarlov criteria at 48h after reperfusion and then sacrificed. Other three groups were respectively divided into three subgroups according to the time of reperfusion (6h, 24h and 48h). Each subgroup had six animals which were sacrificed after neurological assessment. Arterial blood was sampled at pre-ischemia, 20min post-ischemia, reperfusion 20min and reperfusion 6h in each group for the determination of antioxidant enzyme activities and MDA content. Spinal cord tissue of L5-7 level was also sampled for determination of antioxidant enzyme activities and MDA content. Results The neurological outcomes in control, RPC and RPC+ischemia groups were better than that of ischemia group at 6h, 24h and 48h after reperfusion(P<0.05). At each time points, serum activities of SOD and CAT in RPC group were higher than those in the control group(P<0.05). The serum activities of SOD and CAT in RPC+ischemia group were higher than those in ischemia group(P<0.05). At 20min after ischemia, serum activities of SOD and CAT in ischemia group were lower than those in the control group; while RPC+ischemia group lower than RPC group(P<0.05). At 24h and 48h after reperfusion, activities of SOD and CAT of spinal cord tissue in control group were lower than those in RPC group; while ischemia group lower than RPC+ischemia group(P<0.01). MDA content revealed contrary results(P<0.05). Activities of SOD and CAT of spinal cord tissue have correlation with the neurological outcome. Conclusion RPC upregulated activities of antioxidant enzymes, which then scavenge the free radicals produced during ischemia-reperfusion and protect the spinal cord from ROS-mediated injury.Experiment 2 Effect of free radical scavenger dimethylthiourea (DMTU) on the ischemic tolerance against spinal cord ischemia induced by limb remote preconditioningObjective To investigate the effect of free radical scavenger DMTU on spinal cord ischemic tolerance induced by RPC and the relationship between the effect and the change of antioxidant enzymes activities. Methods Twenty-four rabbits were randomly divided into four groups (n=6 for each): the control, RPC, DMTU and RPC+DMTU groups. In DMTU and RPC+DMTU groups, 10% DMTU saline solution 5ml·kg-1 was administered intravenously to the rabbits 1h before pretreatment (2h before spinal cord ischemia). In control and RPC groups, the 5ml·kg-1 of saline was administered at the same time point. The method for spinal cord ischemia and RPC was the same as experiment 1. Arterial blood was sampled at 10min pre-ischemia, pre-ischemia instantly, 20min post-ischemia, 20min after reperfusion and 6h after reperfusion in each group for the determination of SOD and CAT activities. The hindlimb motor function of all animals was evaluated respectively at 6h, 24h, 48h after reperfusion. Then all animals were sacrificed at 48h after reperfusion for histopathological analysis in H&E-stained sections from lumbar spinal cord. Results At each time points, serum activities of SOD and CAT in RPC group were higher than that in other three groups(P<0.01). Free radical scavenger DMTU abolished the upregulation of CAT and SOD activities induced by RPC. The neurologic outcome in the RPC group was better than that of the control and RPC+DMTU groups at 6h, 24h after reperfusion, respectively(P<0.01); while RPC group better than other three groups at 48h after reperfusion(P<0.05). Compared to control group and RPC+DMTU group, there were more normal motor neurons in anterior horn of spinal cord in RPC group (P<0.05). The neurologic outcome at 48h after reperfusion has correlation with the histopathologic outcome. Conclusion DMTU administration before RPC could abolish the neuroprotection of RPC and the up-regulation of antioxidant enzymes activities. Neuroprotection of antioxidant enzymes might be induced by free radicals.PartⅡEffect of ganglion blocker hexamethonium (HEX) on the ischemic tolerance against spinal cord ischemia induced by limb remote preconditioningObjective To investigate the effect of ganglion blocker HEX on spinal cord ischemic tolerance induced by RPC. Methods Thirty rabbits were randomly assigned into five groups averagely (n=6 for each): control, RPC, HEX1, HEX2 and HEX3 groups. Control and RPC groups were the same as experiment 2. In HEX1, HEX2 and HEX3 groups, HEX 20mg·kg-1 were administered intravenously to rabbits 30min, 15min and instant before preconditioning respectively. All animals were neurologically assessed at 6h, 24h, 48h after reperfusion and then sacrificed for histopathological evaluation. The numbers of normal neurons per section were compared among five groups. Results At 6h, 24h,48h after reperfusion, animals in the RPC group showed higher hind-limb motor function scores than those in the control group(P<0.05). There were no significant differences in hind-limb motor function scores of animals in the RPC group in comparison with the HEX1, HEX2 and HEX3 groups . The histopathologic outcome in the RPC group was better than that in the control group. Also, no difference was observed among the HEX1, HEX2, HEX3 and RPC groups. Conclusion HEX had no effect on the neuroprotection of RPC. Thus neurogenic pathway may not be related to the neuroprotection of RPC.
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