| Background: Ischemia-reperfusion injury was the most important cause of paraplegia after thoracic aorta and spine surgery. The incidence of paraplegia was over10% as reported in thoracoabdominal aneurysm repairs surgery. Although a number of studies have shown that the risk of postoperative paraplegia was decreased with the application of new techniques, preventing spinal cord ischemic injury is still a big concern in the thoracic aorta and spine surgery. Recent studies also found that the IPC induced ischemic tolerance was occurred not only within the same piece of tissue, but also between different regions as well as different organs. This phenomenon was called remote ischemic preconditioning (RIPC). RIPC has greater potential for clinical application than classic ischemic preconditioning, since it can be performed in a non-vital organ, avoiding the high risk of preconditioning in the vital organ, such as the brain or the spinal cord. Numerous studies have suggested that the endocannabinoids were implicated in the protective effects of IPC through cannabinoid (CB) receptor-dependent mechanisms. Vulnerability of motor neurons in the spinal cord against ischemia is considered to play an important role in the development of delayed paraplegia after surgery of the thoracic aorta. Recently, the ubiquitin system has been reported to participate in neuronal cell death. This study was designed to explore the protective effects and mechanism of RIPC, providing evidence of clinical application for RIPC.Experiment 1 Effect of repeated limb ischemic preconditioning on spinal cord ischemia-reperfusion injuryObjective To invest the effect of repeated limb ischemic preconditioning on spinal cord ischemia-reperfusion injury in rat. Methods The rat spinal cord ischemia model was used. A total of 40 male Sprague-Dawley rats were randomly divided into five groups (n=8 in each group):①. Sham group: Rats were operated in the same way as in other groups but without limb ischemia preconditioning and spinal cord ischemia.②Control group: After rats were anesthetized with 1-2% isoflurane for 30min, the spinal cord ischemia was induced.③RIPC2min-3c group: Three cycles of 2 minutes right femoral artery occlusion and reperfusion were induced prior to spinal cord ischemia-reperfusion.④RIPC3min-3c group: Three cycles of 3 minutes right femoral artery occlusion and reperfusion were induced prior to spinal cord ischemia-reperfusion.⑤RIPC5min-3c group: Three cycles of 5 minutes right femoral artery occlusion and reperfusion were induced prior to spinal cord ischemia-reperfusion. The animals were neurologically assessed at 24 and 48h after reperfusion by an observer who was unaware of the grouping. To evaluate the neuronal damage, motor function deficits in the hind limbs were evaluated according to the criteria as reported. Motor deficit scores were graded according to the following scale: a) walking/use of hind limbs: 0 scored as normal, 1 for toes flat under the body when walking, 2 for knuckle walking, 3 for movement of the hind limbs but unable to walk; and 4 for no movement, dragging hind limbs; b) placing/stepping reflex: 0 scored for normal, 1 for weak, and 2 for not stepping. Each motor deficit score was obtained by adding the scores for scales a and b. A histopathologic evaluation was performed in the spinal cord for 48h after reperfusion. Three representative sections taken from L-4 to L-6 segments were stained with Haematoxylin and Eosin (HE) for histopathologic evaluation. Neuronal injury was evaluated at a magnification of×200 by an observer who was unaware of the grouping. The remaining normal neurons in the ischemic ventral spinal cord were counted. Results 1. Physiologic variables. Except for sham group, the rectal temperature, arterial pH, PaCO2, PaO2, and blood glucose concentrations were similar in other four groups. In groups that spinal cord ischemia was induced, blood glucose was elevated and pH was dropped after reperfusion, but there is no difference among groups. Five minutes after the reperfusion, the value of the distal blood pressure was recovered to nearly preischemic level. 2. Neurologic outcome.All animals had survived until the final neurologic behavior assessment at 48 h after reperfusion. There was no neurologic change in sham group, and all of other groups of rats showed motor deficit in varying degrees. The neurologic outcome in the RIPC3min-3c group was better than that of control, RIPC2min-3c, and RIPC5min-3c groups, respectively (P < 0.01). Three rats in RIPC3min-3c group showed complete normal motor function (motor deficit index,MDI = 0). The hind-limb MDI of five groups at 48h after reperfusion were shown in. 3. Histopathologic evaluation.The number of normal neurons in the RIPC3min-3c group was significantly more than that in the RIPC2min-3c, RIPC5min-3c, and control group (P<0.01). No difference was found in the number of normal neurons at the anterior spinal cord among the control, RIPC2min-3c and RIPC5min-3c groups. Conclusion Brief episode of femoral artery occlusion and reperfusion can induce spinal cord ischemia tolerance in a rat model, and RIPC for 3min and 3 cycles is the best choice to prevent against spinal cord ischemia for 12min.Experiment 2 Effect of cannabinoid receptor antagonist on the ischemic tolerance induced by RIPCObjective To invest the effect of cannabinoid receptor antagonist on the ischemic tolerance induced by RIPC. Methods 64 male Sprague-Dawley rats were randomly divided into 8 groups averagely (n=8 in each group):①Control group: Right femoral artery was isolated without occlusion, followed by spinal ischemia/reperfusion protocol.②Remote preconditioning(RIPC) group: Three cycles of 3 minutes right femoral artery occlusion and reperfusion were induced prior to spinal cord ischemia/reperfusion.③AM251+RIPC: CB1 receptor antagonist AM251 (1 mg/kg) was intravenously administered 15 min before right femoral artery occlusion.④AM630+RIPC: Cannabinoid CB2 receptor antagonist AM630 (1 mg/kg) was intravenously administered 15 min before right femoral artery occlusion.⑤Vehicle+RIPC: Vehicle was intravenously administered 15 min before right femoral artery occlusion (3 min) and reperfusion (3 min, three cycles) prior to spinal cord ischemia/reperfusion protocol.⑥AM251: AM251 (1 mg/kg) was intravenously administered 15 min before sham operation of RIPC.⑦AM630: AM630 (1mg/kg) was intravenously administered 15 min before sham operation of RIPC.⑧Vehicle: Vehicle was intravenously administered 15 min before sham operation of RIPC. Thirty minutes after preconditioning or 45 minutes after chemical pretreatment, the rats were subjected to spinal cord ischemia for 12 mins. The time of total anesthesia was identical in 8 groups. The animals were neurologically assessed at 24 and 48h after reperfusion. A histopathologic evaluation was performed in the spinal cord for 48h after reperfusion. Three representative sections taken from L-4 to L-6 segments were stained with Haematoxylin and Eosin (HE) for histopathologic evaluation. Neuronal injury was evaluated at a magnification of×200 and the remaining normal neurons in the ischemic ventral spinal cord were counted. Results 1. Physiologic variables. The hemodynamics, rectal temperature, arterial pH, PaCO2, PaO2, and blood glucose concentrations changes were similar with those in Experiment 1. 2. Neurologic outcome.All animals had survived until the final neurologic behavior assessment at 48 h after reperfusion. The neurologic outcome in the RIPC group was better than that of the Control (p<0.05) and AM251+RIPC group (p<0.01). The MDI in RIPC, AM630+RIPC, and Vehicle+ RIPC3 groups did not show significant difference. There was no significant difference among Control, AM251+RIPC, AM251, AM630, and Vehicle groups.3. Histopathologic evaluation.The number of normal motor neurons in the RIPC group was significantly more than that in the Control (p<0.05), AM251+RIPC groups (p<0.05). There was no significance among RIPC, AM630+RIPC, and Vehicle +RIPC groups. The ventral neurons in AM251, AM630, and Vehicle groups were damaged seriously, and the number of normal neurons among these groups did not show significant difference. Conclusion Cannabinoid 1 receptor has mediated the protective mechanism of RIPC. Experiment 3 The contents of AEA and 2-AG in lumbar spinal cord samples pre- and post- spinal cord ischemia.Objective To in vest the contents of two kind of endocannabinoids ,AEA and 2-AG, in spinal cord samples pre- and post- spinal cord ischemia. Methods 24 rats were divided into 2 groups averagely: Control group and RIPC group (n=12 in each group). Animals were sacrificed at prior (30 minutes after remote preconditioning or sham operation) or 1h after spinal cord ischemia. The spinal cord was removed within 1 minute (-20°C). A sample of 1 cm from L-4 to L-6 segments of spinal cord was frozen in dry ice and then stored in -80°C refrigerator. For endocannabinoid measurement, spinal cord from the rats of control or remote preconditioning groups (n=6 in each time point) were sampled. The endocannabinoid was detected by HPLC-MS. Results The preischemic AEA level in RIPC group was higher than that in Control group (p<0.05). The results indicated that RIPC (3 cycles of 3 min limb ischemia/reperfusion) stimulated the formation of AEA in the lumbar spinal cord. The postischemic AEA level did not show significant difference between two groups. There was no significant difference on 2-AG content in lumber spinal cord at corresponding time between two groups. Conclusion RIPC induces AEA increasing before ischemia in spinal cord which play a key role in the mechanism of spinal cord ischemia tolerance induced by RIPC.Experiment 4 The expression of ubiquitin in lumbar spinal cord after RIPC and the role of endocannabinoids.Objective To invest expression of ubiquitin, and the relationship among the ubiquitin molecular, cell apoptosis and endocnnabinoids after ischemia tolerance in the spinal cord induced by RIPC. Methods 24 rats were divided into 3 groups averagely: Control group and RIPC group (n=8 in each group). Animals were sacrificed at 3h and 24h after spinal cord ischemia. The section of L-4 to L-6 spinal cord segments was removed after fixation with routine method. The freezing section of 14μm were kept in -20°C refrigerator for immunohistochemical studies and apoptosis detection. Results Mean optical density value of ubiquitin in RIPC group is lower than the value in other two groups after reperfusion for 3h and 24h (p<0.05).The cell apoptosis in all three groups is not found after reperfusion for 3h in the sections. Conclusion RIPC suppressed the expression in spinal cord after ischemia, which is intercepted by AM251. Ubiquitin maybe a earlier marker of neuron stress when the cell apoptosis has not been detected.Conclusions1. Brief episode of femoral artery occlusion and reperfusion can induce spinal cord ischemia tolerance in a rat model.2. Cannabinoid 1 receptor has mediated the protective mechanism.3. RIPC induces AEA increasing before ischemia in spinal cord which play a key role in the mechanism.4. RIPC suppressed the expression in spinal cord after ischemia, which is intercepted by AM251. Ubiquitin maybe a earlier marker of neuron stress when the cell apoptosis has not been detected.
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