Ischemic preconditioning suppresses apoptosis of rabbit spinal neurocytes by inhibiting ASK1–14-3-3 dissociation

Abstract: The mechanism by which a brief episode of sublethal ischemia followed by reperfusion (ischemic preconditioning, IPC) prevents the lethal effects of subsequent periods of prolonged ischemia, are poorly understood. A completely randomized, controlled study was designed to study the effect of IPC using a rabbit model of ischemic spinal cord injury. Twenty-four white adult New England rabbits were randomly assigned to one of 3 groups (n =8 per group); the groups were assigned as follows: Group I: sham-operation group, Group II: ischemic reperfusion (I/R) group, and Group III: ischemic preconditioning group. Spinal cord ischemia was induced by introducing an infra renal aortic cross-clamp for 30min. Following injury, rabbits were subjected to 30min, 2h, or 8h of reperfusion in Group II. In Group III, subjects underwent three cycles, 5min each, of ischemia followed by 5min of reperfusion, before receiving 30min of ischemia. We previously reported that the association between ASK1 (apoptosis signal-regulating kinase 1) and 14-3-3 played an important role in regulating ischemia/reperfusion spinal cord injuries. To evaluate the effect of ischemic preconditioning in injured spinal cords, we examined alterations in spinal tissue morphology, activation of key members of the ASK1-mediated signaling pathway, and the association between ASK1 and 14-3-3. Changes in spinal cord morphology were observed with hematoxylin and eosin (H&E) staining and electron microscopy. The phosphorylation levels of ASK1, JNK, and p38 were assessed by immunoblot analysis. The association between ASK1 and 14-3-3 was analyzed by co-immunoprecipitation experiments. We observed that swelling of the neurocyte bodies and hemorrhage of the spinal cord were dramatically decreased in Group III compared to Group II. In addition, the degree of apoptosis among neurocytes was reduced in Group III compared to Group II. Finally, the phosphorylation of ASK1, JNK, p38 and the dissociation of ASK1 from 14-3-3 were dramatically decreased in Group III compared with Group II. These results indicate that ischemic preconditioning may have a protective affect against ASK1/14-3-3 dissociation-induced spinal cord injuries. [Copyright &y& Elsevier]