Heme Oxygenase-1 Overexpression Protects Rat Lung Allografts From Ischemia/reperfusion Injury

Part1. The improvements in establishment of rat orthotopic left lung transplantation model.Objective To establish rat orthotopic left lung allograft transplantation model by improved techniques. Methods This study improved the traditional cuff technique in many aspects, including graft retrieval, cuff self-making, recipient pneumonoresection,"muff-like"vessel and trachea anastomosis techniques. In a group with 30 pairs of clean-grade SD rats, the left lung allograft was stored at 4 oC LPDG solution for 3 h, and then the left lung was transplanted into the recipient rat without microscope. When the transplanted lung had been reperfused for 4 h. Airway pressure, blood gas analysis were detected before and after the right hilus of lung was block up, then left lung was removed, and W/D, histological changes under microscopy were determined. The reproducible ability of this model about ischemical reperfusion injury of donor lung was evaluated. Results 30 pairs of rats receiving transplantation were performed the anastomosis of pulmonary artery, pulmonary vein and bronchus by single person without microscope. The time consuming was (4±1)min, (8±3)min(,2±0.5)minutes respectively. The total operational time consuming was (55±10)min. The operation successful rate was 90%, survival rate arrived 100%. The experimental outcome demonstrated that the model could duplicated the change of the ischemia-reperfusion injury. Conclusion The merit of this model corresponds with the construction of rat lung transplantation model. All the manipulations were performed by single person without microscope. The harvesting of donor lung was so fast that ischemic time was nearly 0 min. The cuff structure and the anastomosis technique were simpler than before. The improved"muff-like"technique increased the successful rate and shortened the total operational time consuming. This model duplicated the changes of ischemia reperfusion injury on lung allograft, and was demonstrated to be an ideal and suitable model for some researches just like IRI of donor lung .Part2. Endogenous heme oxygenase-1 overexpression protects rat lung allografts from ischemia reperfusion injury.Objective To investigate the effect of endogenous heme oxygenase-1 overexpression by using CoPP as the HO-1 inducer on ischemia reperfusion injury of rat left lung allograft and explore the mechanisms. Methods A left lung transplantation formwork was constructed by using SD rat both donor and recipient by improved muff-like technique. Three groups of rats were examined. At 24 hours before the harvest,the donors in group 1,which is the control group,received 0.9%saline,the donors in group 2 were pretreated with CoPP+ ZnPP,the donors in group 3 received CoPP . The donor lungs were all ex vivo reperfused by 4oC LPDG through pulmonary vein . After the left lung allograft was reperfused for 4 hours, we blocked the hilum of right lung, then measured airway pressure and made arterial blood gas analysis to measure PaO2 and PaCO2 .At last, rat was executed. Lung allograft tissue was harvested to be detected. W/D ratio, superoxide dismutase(SOD) activity, malondialdehyde (MDA) contents and myeloperoxidase(MPO) activity were detected. Graft pathologic histology was examined under light microscope. The message of expression of HO-1 in donor lung tissue was measured by immunohistochemical staining and reverse transcription-polymerase chain raction (RT-PCR). The content of TNF- a was quantified by ELISA. The expression of TNF-a,IL-10 and bcl-2 were detected by immunohistochemical staining. Apoptotic cell death was determined by TUNEL. Results The contents of HO-1 in group C was obviously higher than A and B (P<0.01), which demonstrated the successful expression of HO-1 in group C pulmonary allograft. Compared with A and B group, the airway pressure, PaO2 and the activity of SOD increased. W/D ratios, content of MDA and activity of MPO decreased(P<0.01). In group C, tissue edema, interstitial inflammation and exudation alleviated under light microscope. Lower expression of TNF- a and higher expression of IL-10 and bcl-2 were detected by immunohistochemistry(P<0.01). Less apoptotic cells were found by TUNEL(P<0.01). Conclusion The efficient expression of endogenous heme oxygenase-1 overexpression by using CoPP was demonstrated. HO-1 has the characters of anti-oxidative, anti-inflammatory and anti- apoptosis, which down-regulate the expression of TNF- a and up-regulate IL-10, can reduce interstitial inflammation, graft inflammatory reaction and cellular apoptosis, reduce ischemical reperfusion injury of lung allograft. This study demonstrated that the expression of HO-1 in lung grafts may become a strategy, which can reduce the IRI of lung allografts.Part3. The impact of adenovirus mediated gene transfer of HO-1 on the ischemical reperfusion injury of rat left lung allograftObjective To investigate the effect of adenovirus-mediated gene transfer of HO-1 on ischemia reperfusion injury of rat left lung allograft and explore the mechanisms. Methods A left lung transplantation formwork was constructed by using SD rat both donor and recipient with improved muff-like techniques. The experiment was divided into three groups: In groupⅠ, the donor lung was ex vivo retroperfused by 4oC LPDG through pulmonary vein .In groupⅡ, the donor lung was ex vivo retroperfused by empty adenovec which was diluted by 4oC LPDG fluid. In groupⅢ, the donor lung was retroperfused by Ad- HO-1 diluted by 40C LPDG fluid too. The donor lung which had been perfused was preserved for 3 hours in 4oC LPDG fluid. Then the left lung orthotopic transplantation was performed. After the left lung allograft was reperfused for 4 hours, we blocked the hilum of right lung, then measured airway pressure and made arterial blood gas analysis to measure PaO2 and PaCO2 . At last, rat was executed. Lung allograft tissue was harvested to be detected W/D ratio, superoxide dismutase (SOD) activity, malondialdehyde (MDA) contents and myeloperoxidase(MPO) activity. Graft pathologic histology was examined under light microscope. The message of expression of HO-1 in donor lung tissue was measured by immunohistochemical staining and reverse transcription-polymerase chain raction (RT-PCR). The content of TNF- a was quantified by ELISA. The expression of TNF-a,IL-10 and bcl-2 were detected by immunohistochemical staining. Apoptotic cell death was determined by TUNEL. Results The content of HO-1 in groupⅢwas obviously higher thanⅠandⅡ(P<0.01), which demonstrated the successful expression of HO-1 in pulmonary allograft of groupⅢ. Compared withⅠandⅡgroup, the airway pressure decreased. PaO2 and activity of SOD increased. W/D ratios content of MDA and activity of MPO decreased(P<0.01). In groupⅢ, tissue edema, interstitial inflammation and exudation alleviated under light microscope. Lower expression of TNF- a and higher expression of IL-10 and bcl-2 were detected by immunohistochemistry(P<0.01). Less apoptotic cells were found by TUNEL(P<0.01). Conclusion The efficient expression of HO-1 through adenovirus-mediated gene transfer of Ad-HO-1 was demonstrated. HO-1 has the characters of anti-oxidative, anti-inflammatory and anti- apoptosis, which down-regulate the expression of TNF- a and up-regulate IL-10, can reduce interstitial inflammation, graft inflammatory reaction and cellular apoptosis, reduce ischemical reperfusion injury of lung allograft. This study demonstrated that the expression of HO-1 in lung grafts may become a new strategy, which can reduce the IRI of lung allografts.