Experimental Study Of Effect Of Tumor Necrosis Factor-α On Skeletal Muscle Ischemia-reperfusion Injury

Experimental study of effect of tumor necrosis factor-a on skeletal muscle ischemia-reperfusion injuryIschemia in skeletal muscle followed by reperfusion results in local as well as remote organ injury. The mechanisms is very complex. In the past decade, studies showed that aside from oxygen free radicals and calcium overload, cytokines also play an important role in skeletal muscle ischemia- reperfusion injury (SMIRI). This study investigated the effect of tumor necrosis factor-a on skeletal muscle ischemia-reperfusion injury. It not only would help for further expounding the pathogenesis of SMIRI, but also could be informative for new prevention and cure of SMIRI.A model of rat hind limb ischemia and reperfusion was established in this study. Anti-TNF-a monoclonal antibody was used to block the function of TNF-a to investigate the role of TNF-oc in SMIRI.In the first part of this study, monocyte mRNA transcription level of TNF-a was determined by reverse transcription-polymerase chain reaction (RT-PCR), standard assay was performed for plasma level of TNF-a. Results showed that skeletal muscle ischemia-reperfusion (SMIR) contributed to significant increase in the level of TNF-a mRNA in monocyte and the level of TNF-a in plasma, anti-TNF-a monoclonal antibody managed to block the increasing TNF-a in plasma. The hyperexpression of TNF-a mRNA was firstly determined by RT-PCR in this study, this will help for further understanding the pathogenesis of SMIRI.In the second part of this study, intercellular adhesion molecule-1 (ICAM-1) protein translation level of endothelium and CD 18 of neutrophil were assessed by immunohistochemistry and flow cytometry respectively. Results showed that ICAM-1 of vascular endothelium in skeletal muscle and lung was markedly upregulated by SMIR, so was CD 18 of neutrophil. The expression of these adhesion molecules was effectively blocked by treatment with anti-TNF-a monoclonal antibody.In the third part of this study, standard assays were performed for plasma levels of malondialdehyde (MDA), creatine kinase (CK) and nitric oxide (NO), skeletal muscle and lung myeloperoxidase (MPO) activity was scored as well. The oedema degree was quantified by calculating the wet/dry weight ratio of skeletal muscle. Skeletal muscle and lung were also observed histologically and ultrastructurally. Terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling (TUNEL) method was used to identify apoptosis in skeletal muscle. Results showed that skeletal muscle ischemia-reperfusion resulted in a significant increase in the level of MDA, NO, CK, MPO and wet/dry weight ratio. Treatment with anti-TNF-a monoclonal antibody significantly lowered the levels of MDA, NO, CK and MPO, but couldn't reduce the oedema degree of skeletal muscle. Intravenous administration of anti-TNF-a monoclonal antibody at the time of reperfusion provided significant protection against histological and ultrastructural injury in both skeletal muscle and lung. A great number of TUNEL-positive cells in skeletal muscle were detected at 4-hour reperfusion. Monoclonal antibody targeting TNF-a distinctly reduced the rate of apoptotic cells. Skeletal muscle ischemia-reperfusion initiates a systemic TNF-a response. Both local(skeletal muscle) and remote organ (lung) injury after ischemia-reperfusion requires participation of TNF-a, and the injury can be effectively attenuated by treatment with anti-TNF-a monoclonal antibody. This will be informative for new prevention and cure of SMIRI.