The Role Of ASK1-MAPK Signaling Cascades In Reperfusion Of Ischemic Spinal Cord

Objective:To determine the role and the signaling mechanism of ASK1-MAPKsignaling cascades induction during reperfusion of ischemic spinal cord.Methods:20 New Zealand white rabbits were divided into control group,ischemia 30 minutes / reperfusion 15 minutes group, ischemia 30 minutes /reperfusion 1 hour group and ischemia 30 minutes / reperfusion 24 hours group.The model of spinal cord ischemia and reperfusion injury was produced byoccluding the abdominal aorta with an arterial clamp. Changes in spinal cordmorphology were observed with hematoxylin and eosin (H&E) staining andelectron microscopy. The expressions and activations of apoptosissignal-regulating kinase 1(ASK1), c-Jun N-terminal kinase (JNK) and p38were assessed by immunoblotting. The interaction of ASK1 and 14-3-3 wasperformed by immunoprecipitation analyses. Immunohistochemical studies were performed to determine the cellular localization of phosphorylated ASK1(pASK1) and 14-3-3.Results:For hematoxylin and eosin (HE) stain, the shape of neuronal cell isregular and no hemorrhagic focus was found in the intercellular substance inthe control group and the ischemia 30 minutes / reperfusion 15 minutes group.However, in the ischemia 30 minutes / reperfusion 1 hour and 24 hours groups,large patchy hemorrhages were observed and neuronal cells were swollen.Electron microscopic examination of spinal cords revealed that these changeswere apoptotic in nature, characterized by the demyelination, coarse chromatincondensation, and breakdown of the nucleus into discrete fragments in theischemia 30 minutes / reperfusion 1 hour and 24 hours groups. However,ultrastructure of the spinal cord was normal in the control group and theischemia 30 minutes / reperfusion 15 minutes group. The phosphorylationlevels of ASK1, JNK and p38 were assessed by immunoblotting proteins fromanimals that received 30 minutes ofischemia followed by 1 hour or 24 hours ofreperfusion. Immunoprecipitation analyses suggested that 14-3-3 dissociatedfrom ASK1 during the activation of ASK1. The localization of phosphorylatedASK1 (pASK1) and 14-3-3 in cytoplasm were changed during reperfusion ofischemic spinal cords revealed by immunohistochemistry which also suggestedthe dissociation between ASK1 and 14-3-3.Conclusions: These findings suggest the possible involvement of the MAPKpathways through ASK1 in the transmission of apoptosis signals in reperfusedspinal cord injuries. The dissociation of 14-3-3 from ASK1 may contribute tothe activation of JNK and p38, which lead to increased apoptosis in reperfusedischemic spinal tissue.