Therapeutic Treatment Of Spinal Cord Ischemia-Reperfusion Injury And Its Related Mechanism

Spinal cord ischemia reperfusion?I/R?injury?SCIR?is a disastrous complication in many pathophysiological conditions,which may result in devastating paraplegia and paraparesis.SCIR has been reported to occur in 3%–18%of patients undergoing descending thoracic and thoracoabdominal aneurysms surgery and endovascular aortic repair surgery.SCIR should be regarded not only as a medical problem,but also as a socioeconomic burden.Considerable therapeutic interventions have been attempted to mitigate this problem,including cerebrospinal fluid drainage,reattachment of segmental arteries,intercostal vessel reimplantation,and administering pharmaceutical?steroids,oxygenderived free radical scavengers,and vasodilators?drugs.However,the results were not that satisfactory.Objective:1.To verify hypothesis that NAD⁺could ameliorate oxidative stress-induced neuronal apoptosis.2.To explore the role of autophagy in SCIR.In addition,we also studied neuroprotective mechanisms of nicotinamide adenine dinucleotide?NAD⁺?,and their relationship to spinal cord ischemia-reperfusion injury?SCIR?was assessed as well.3.To explore the role of H₂S,which is the same as that of NAD⁺,in SCIR and its related mechanism.Methods:The model of spinal cord ischemia-reperfusion injury was established by using adult male SD rats?180-250g?,and different treatment and treatment methods were used as different groups.BBB score was used to evaluate the neurological function.SOD Activity and MDA content were evaluated by oxidative stress.Western blot,immunofluorescence and co-fluorescence staining and immunohistochemical staining were used to detect the level of oxidative stress,autophagy and apoptosis.The effect of pathway was tested by the corresponding pathway inhibitor.Results:1.The results showed that NAD⁺at 50 or 100 mg/kg significantly decreased the oxidative stress level?P<0.05?and neuronal apoptosis?P<0.05?in the spinal cord of ischemia–reperfusion rats compared with saline,as accompanied with the decreased oxidative stress,NAD⁺administration significantly restrained the neuronal apoptosis after ischemia injury while improved the neurological and motor function?P<0.05?.But 10mg/kg NAD⁺did not have significant therapeutic effect?P>0.05?2.Expression levels of Beclin-1,Atg12-Atg5,LC3B-II,Cleaved caspase 3,and Bax were upregulated in the I/R group and downregulated in the 75mg/kg NAD⁺group;p-mTOR,p-AKT,p62,and Bcl-2 were downregulated in the I/R group and upregulated in the 75mg/kg NAD⁺group.Numbers of LC3B-positive,Caspase 3-positive,Bax-positive,and TUNEL-positive cells were significantly increased in the I/R group and decreased in the 75mg/kg NAD⁺group?P<0.05?.The mean integrated option density of Bax increased and that of Nissl decreased in the I/R group,and it decreased and increased,respectively,in the 75mg/kg NAD⁺group?P<0.05?.BBB scores significantly increased in the 75mg/kg NAD⁺group relative to the I/R group?P<0.05?.No difference was observed between I/R and 10mg/kg NAD⁺groups for these indicators?P>0.05?.3.Compared with normal controls,autophagic cell death was upregulated in neuronal after SCIR?P<0.05?.Furthermore,we found autophagy promoted neuronal cells death during SCIR,as we found a significant number of TUNEL-positive cells was co-labeled with the autophagy marker LC3?P<0.05?.In additon,we demonstrated that oxidative stress was the main inducer of autophagic neuronal cell death after SCIR,as accompanied with the decreased malondialdehyde?MDA?concentration and increased superoxide dismutase?SOD?activity?P<0.05?.Finally,we identified hydrogensulfide?H₂S?,which alleviated autophagic cell death?P<0.05?and ameliorated neurological and motor function significantly by limiting oxidative stress in SCIR?P<0.05?,moreover,H₂S mediated autophagic neuronal cell death via AKT-mTOR pathway.Conclusion:1.NAD⁺might protect against spinal cord ischemia–reperfusion via reducing oxidative stress induced neuronal apoptosis.2.Excessive and sustained autophagy aggravates SCIR;administration of NAD⁺alleviates injury.3.Oxidative stress induced autophagic cell death,and H₂Salso acts as a neuroprotective effect by inhibiting the oxidative stress levels in SCIR to inhibit neuronal apoptosis caused by autophagy overactivation.