The Effect Study Of Melatonin Against Crush Syndrome Model-induced Ischemia Reperfusion Injury

Purpose.Melatonin(melatonin,MEL)and its metabolites have strong ability of radical scavenging,and the potential therapeutic effect in mammalian tissues and organs in the process of ischemia-reperfusion injury.Traumatic rhabdomyolysis,or the crush syndrome,is the consequence of prolonged continuous pressure on the limbs.It reflects the disintegration of muscle tissue and the influx of myoglobin,potassium,and phosphorus into the circulation.The syndrome is characterized by hypovolemic shock and hyperkalemia,followed by acute renal failure in the absence of treatment.Crush syndrome tend to occur in the traumatic events such as earthquakes,landslides,and vehicle accidents.The pathophysiologic features of derangement associated with the crush syndromen begin with the ischemia reperfusion injury.During the ischemia reperfusion period,the formation of oxygen free radicals and the release of proinflammatory factors induce the change of local tissue activity,lead to systemic inflammatory response syndrome.So it will induce secondary remote organ injury,such as heart,kidney,and lung.There has been no report evaluating the therapeutic effects of melatolin on CS and induced heart injury.We applied a tourniquet to rat hind limbs to creat a CS model in this study.This study is divided into two parts.On the one hand,we explore the role of melatonin to the change in acid-base balance disorders,oxidative stress damage,the influence of rhabdomyolysis and survival rate,on the other hand,we explore the protective effects of myocardial enzymes and myocardial oxidative stress injury,inflammatory cytokines and myocardial injury pathological changes on the animal model.Methods.In the first part.To compress bilateral hind limbs,we prepared a pair of Hemostatic clamp by modifying the ones used by Isamu Murata for mice,using 5% pentobarbital sodium solution(50 mg/kg)to abdominal cavity anesthesia in rats.The Hemostatic clamp is equal to 3 kg heavy pressure,five hours after,compression was released.The wistar male rats were randomly divided into four groups : blank group(SHAM group),model group(CS),the expansion group(NS group)and group of melatonin(MEL group)according to the principle of ensuring the survival of 10 rats after 6 hours of reperfusion.The level of potassium(K+),creatine kinase isoenzyme(CK),lactate dehydrogenase(LDH),glutathione peroxidase(GSH-px)and superoxide dismutase(SOD),catalase(CAT)and malondialdehyde(MDA)in serum were measured at predetermined time.Arterial blood gas was examined by i-STAT300.Hematoxylin eosin(Hematoxylin eosin,HE)staining pathological changes of skeletal muscle was observed.In a separate experiment.Four groups of rats(SHAM group:n =10;CS group:n =30,NS group:n =25,and MEL group:n =16)were prepared and observed for 48 hours after reperfusion to determine survival rates.The second part: according to the principle of ensuring the survival of 10 rats after 12 hours of reperfusion,wistar rats were randomly divided into four groups: SHAM group,the CS group,NS group and MEL.After 5h compression and 12 h reperfusion,the level of troponin I(Tn I)creatine kinase isoenzyme(CK-MB)and SOD,MDA,TNF-?,IL-1? level in serum and myocardial tissue were detected.HE staining pathological changes of myocardia tissue was observed.Results After 6h reperfusion,arterial blood gas of CS group was obviously metabolic acidosis,and the acid-base balance disorders of MEL group was improved significantly than CS group.Compared with the SHAM group,the content of K+ CK,LDH and the activity of GSH-px,SOD and CAT were significantly increased.But in MEL group,those were decreased significantly.CS group content of MDA in serum in the SHAM group increased significantly.MDA content in MEL group decreased significantly compared with CS group.SHAM group,calf muscle fibers did not see edema and inflammatory cell infiltration,muscle fiber normal distribution;The CS group,NS group gastrocnemius muscle fiber edema,obviously serious damage,muscle fiber structure horizontal stripes of the skeletal muscle disorders.MEL group of muscle fibers visible mild edema,fibrous tissue slightly disorder,its morphological structure was much better than CS group and NS group.CS group rat survival rate was 20%,the NS group rat survival rate was 29.2%,MEL rat survival rate was 56.3%.After 12 h reperfusion,the serum Tn I and CK-MB content of CS group had a significant rise.The serum levels of Tn I,CK-MB a in MEL group were significant decrease.The serum and myocardial tissue levels of SOD activity decreased significantly higher in the CS group,the tissue levels of MDA,TNF-??IL-1?increased significantly in CS group.Those in MEL group had opposite changing.We use HE staining to evaluate damage to the myocardial tissue.In the sham group,the cardiomyocytes were intact and there was no evidence of necrosis or inflammatory cell infiltration.The cardiac muscle cross striations were clearly visible.In the CS and NE group,necrosis and inflammatory cell infiltration were evident and the myocardial muscle cross striations were no longer visible.Conclusion Using tabbed hemostatic clamp can successful build crush syndrome rat model.Before the lower extremity to restore blood flow perfusion.The early intervention of melatonin can play protective effect to skeletal muscle ischemia-reperfusion injury.Melatonin can effectively inhibit oxidative stress damage during ischemia reperfusion,reduce rhabdomyolysis,can increase the survival rate of the crush syndrome model rats,improve pathological damage of the gastrocnemius muscle induced by ischemia reperfusion.Crush syndrome model can cause myocardial injury.Melatonin can influence myocardial enzyme level,endogenous antioxidant enzyme activity,inflammatory cytokines and myocardial tissue pathological damage.So melatonin can play the protective role to myocardial injury-induced by crush syndrome model.