The Effects Of The Nitric Oxide Synthase On Ischemia-reperfusion Injury Following Rat Lung Transplantation

This research was composed of three parts:1.Establishment of the model of rat orthotopic left lung allografl transplantation;2.The alterations of nitric oxide synthase expression durning rat lung transplantation;3.The effects of L-Arg and aminoguanidine on ischemia-reperfusion injury following rat lung transplantation.1.Establishment of the model of rat orthotopie left lung allograft transplantationObjective To approach rat orthotopic left lung allograft transplantation base on the "cuff-like vessel anastomosis technique",which will provide a base model for further research of lung ischemia-reperfusion injury(IRI).Methods We established rat orthotopic left lung allograft transplantation in which the anastomosis of pulmonary artery,vein and bronchus were performed by using the cuff technique under microscope and evaluated the possibility of the model by achievement ratio of operation,time of operation,graft function, pathologic change and imageology examination.Results In 20 consecutive cases,17 have been performed successfully.The achievement ratio of operation was 85%.The time of donor lung perfusion-picking,donor lung vessel anastomosis and recipient vessel anastomosis was 13.1min±0.9min,9.3min±0.7min and 33.7min±1.7min respectively.The occlusion test showed the donor lung could maintain over 20 min breath alone.2h after reperfusion,pathologic study found the typical pathologic change of IRI.Conclusions By using the improved "cuff-like vessel anastomosis technique" and analyzing the model of rat lung transplantation domestic and abroad,we tried to establish the best model of rat orthotopic left lung allograft transplantation which shortened the operation time,simplified the operative difficulty and improved the achievement ratio of operation by a single surgeon with microscope.The pathologic study showed the typical pathologic change of IRI,which can be applied to study donor lung IRI.2.The alterations of nitric oxide synthase expression durning rat lung transplantationObjective To investigate the alterations of nitric oxide synthase expression durning rat lung transplantation.Methods Twelve health adult male Sprague-Dawley rats were underwent the left lung transplantation.At the time point of open the chest(Ⅰgroup),after flush(Ⅱgroup),after cold preserved(Ⅲgroup),we take the samples of the donor right lung.After 2h reperfusion,we take the samples of transplantated left lung(Ⅳgroup) and the recipient right lung(Ⅴgroup).Then total RNA was extracted from this samples and reverse transcription of total RNA from each sample was done according to the manufacturers instructions.The expression of eNOS and iNOS was determined by using Real Time PCR system.Results Compared withⅠgroup,there was no significant change of mRNA of eNOS and iNOS inⅡgroup,Ⅲgroup andⅤgroup.But the mRNA of eNOS increased significantly inⅣgroup,the mRNA of iNOS decreased significantly.Conclusions It was improved that the reperfusion is the most important reason that leading to alteration of nitric oxide synthase expression,through investigating the expression of mRNA in each stage of lung transplantation ischemia-reperfusion procession.3.The effects of L-Arg and aminoguanidine on ischemia-reperfusion injury following rat lung transplantationObjective To investigate the effects of L-Arg and aminoguanidine on ischemia-reperfusion injury following rat lung transplantation and to explore its mechanisms.Methods Fourty-eight health adult male Sprague-Dawley rats were randomly allocated to 4 groups with 12 rats in each group:lung transplantation group(A group), L-Arg treatment group(B group),aminoguanidine treatment group(C group) and L-Arg + aminoguanidine treatment group(D group).Single left lung transplantation with allografts were underwent and the Sodium chloride,L-Arg,aminoguanidine and L-Arg + aminoguanidine were respectively administered to the recipient rats through the abdominal cavity.After 2h reperfusion,the above of the lung graft was harvested to measure the activity of myeloperoxidase(MPO),the content of malondialdehyde(MDA) and the activity of super-oxide dismutase(SOD),the activity of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase(iNOS);measure the content of plasma nitric oxide(NO).The lower for wet/dry weight ratio(W/D);The 4 groups were respectively for studying pathomorphology.Results After 2h reperfusion,compared with A group,the activity of eNOS and iNOS of B group increased significantly(P＜0.05),the activity of erythrocuprein(SOD) and the content of plasma nitric oxide(NO) were higher than A group(P＜0.05),the wet/dry weight ratio(W/D),the activity of myeloperoxidase(MPO) and the content of malondialdehyde(MDA) were lower than A group(P＜0.05).the activity of iNOS and the content of plasma nitric oxide(NO) of C group was lower than A group (P＜0.05),there was no change in the activity of eNOS,MPO,MDA and SOD.the activity of eNOS of D group increased,the content of plasma nitric oxide(NO) and the activity of erythrocuprein(SOD) of D group were higher than A group(P＜0.05),the activity of iNOS,the wet/dry weight ratio(W/D),the activity of myeloperoxidase(MPO) and the content of malondialdehyde(MDA) were lower than A group(P＜0.05).the activity of eNOS and the activity of erythrocuprein(SOD) of D group were higher than B group (P＜0.05),the activity of iNOS,the content of plasma nitric oxide(NO),the wet/dry weight ratio(W/D),the activity of myeloperoxidase(MPO) and the content of malondialdehyde (MDA) were lower than B group(P＜0.05).The result of pathomorphology showed that the inflammatory cell infiltration of D group was the lowest,the B group was lower,the A group and D group was the highest.Conclusions The L-Arg protects the lung function against ischemia-reperfusion injury after transplantation,it was better than L-Arg that administered L-Arg + aminoguanidine to the recipient in protects the lung function against ischemia-reperfusion injury after transplantation,but the aminoguanidine can not be against ischemia-reperfusion injury after transplantation.The protection of L-Arg is related to the increase of the production of NO,which was produced by the endothelial nitric oxide synthase(eNOS).