Establishment And Improvement Of The Non-heart-beating-donor Model In Rat Orthotopic Left Allograft Lung Transplantation

Establishment and improvement of the non-heart-beating-donor model in rat orthotopic left allograft lung transplantationObjectives: To establish the non-heart-beating-donor (NHBD) model in rat orthotopic left single allograft lung transplantation, to improve and simplify the operative procedures, to compare the injury of the allograft with different warm ischemia time (WIT), and to provide animal experimental model for the research in expanding the donor pool, improving the techniques of lung preservation, decreasing the lung injury, predicting the function of the allograft and so on.Methods: Thirty-two male SD rats were randomized into 16 pairs for donors and recipients, divided into 4 groups: group 1, heartbeating donor (HBD, control group); group 2, NHBD with 1 hour of WIT (NHBD1h, experimental group); group 3, NHBD with 2 hours of WIT (NHBD2h, experimental group); group 4, NHBD with 3 hours of WIT (NHBD3h, experimental group). There were four pairs in each group. The donor lungs in group 1 were flushed with low potassium dextran (LPD) solution at 4℃after asystolia, while the lungs in group 2, group 3 and group 4 remained ventilated at the room temperature for 1 hour, 2 hours and 3 hours respectively after asystolia and then were flushed with LPD solution. The donor lungs were preserved in LPD solution at 4℃for four hours after harvesting. The recipient rat underwent left thoracotomy. The left pulmonary hilum with the structure of pulmonary artery, pulmonary vein and left bronchus were clamped and blocked together. The orthotopic left single lung transplantations were performed in 16 rats using cuff technique. Analysis of the blood gas from the recipient's right cervical artery and the whole pulmonary compliance were tested before the operation and 10 minutes, 1 hour and 2 hours after reperfusion. The blood gas from the left pulmonary vein of the allograft, wet to dry weight ratio and histomorphology findings after 2 hours reperfusion were analyzed.Results: After 10 minutes, 1 hour and 2 hours reperfusion, the PaO₂ from right cervical artery in NHBD1h group (177.8±15.5, 162.0±19.3 and 195.5±26.4) was no significant difference with which in control group (187.3±34.4, 191.8±36.6 and 198.8±36.6). After 10 minutes and 2 hours reperfusion, the PaO₂ from right cervical artery in NHBD2h group (154.8±25.2 and 116.5±32.1), and in NHBD3h group (121.0±28.2 and 88.3±23.6) was significant difference with which in control group (P<0.05). After 2 hours reperfusion, PaO₂ from left pulmonary artery was no significant difference with which in control group (132.3±33.8 vs. 143.8±17.1), but in NHBD2h group and NHBD3h group, it was significant difference with which in control group (78.8±16.1 vs. 143.8±17.1, 70.8±15.5 vs. 143.8±17.1, P<0.05). The wet to dry weight ratio and histomorphology score in NHBD3h group were significant difference with which in control group (P<0.05).Conclusions: We have successfully established the NHBD model in rat orthotopic left single allograft lung transplantation. We improved and simplified the operative procedures to make the model more reliable and easy to work. It could be applied in the research of NHBD lung preservation, ischemia-reperfusion injury, etc. One hour of WIT could be suitable for lung transplantation in our model. The protective effect and its mechanism of ischemia preconditioning on lung ischemia-reperfusion injury in rats undergoing non-heart-beating-donor lung transplantationObjectives: To investigate the protective effect and its possible mechanism of ischemia preconditioning (IP) on lung ischemia-reperfusion injury (IRI) in rats undergoing non-heart-beating-donor (NHBD) lung transplantation. To seek the strategy and approach of treatments for lung IRI following NHBD lung transplantation.Methods: The NHBD model in rat orthotopic left single allograft lung transplantation was established in the same way of Part I. The warm ischemia time (WIT) was 60 min. Thirty-two male SD rats were randomized into 16 pairs for donors and recipients, divided into 4 groups: group 1, NHBD with 1 hour of WIT (NHBD1h, control group); group 2, IP by one cycle of blocking the left pulmonary artery for 5 min and then release for 10 min before killing the donor rats (IP, experimental group); group 3, the same IP was performed, and the recipients received 5mg/kg of 5-hydroxydecanoate (5-HD) 30 min preoperatively (IP + 5-HD, experimental group); group 4, the same IP was performed, and the recipients received 0.5 ml DMSO 30 min preoperatively (IP + DMSO, DMSO control group). All the donor lungs remained ventilated at the room temperature for 1 hour after asystolia and then were flushed with low patassium dextran (LPD) solution. The donor lungs were preserved in LPD solution at 4℃for four hours after harvesting. Then the recipient rats underwent orthotopic left single lung transplantations. The blood gas from the recipient's right cervical artery and the whole pulmonary compliance were tested before the operation and 10 minutes, 1 hour and 2 hours after reperfusion. The blood gas from the allograft left pulmonary vein, TNF-αand IL-8 level, the activity of malondialdehyde (MDA) and total antioxidative capabilities (T-AOC) of the the serum and the lung tissue, wet to dry weight ratio, activity of myeloperoxidase (MPO) of the lung tissue, apoptosis of alveolar cells, and histomorphology findings of the allograft by microscopy and electro-microscope were analyzed after 2 hours reperfusion.Results: There was no significant difference in PaO₂ from right cervical artery between the experimental groups and the control groups after 10 minutes, 1 hour and 2 hours reperfusion, but there was significant difference in PaO₂ from right cervical artery after 10 minutes and 1 hour reperfusion with preoperative own control between group 1, 3 and group 2,4 (P<0.05). There was significant difference in PaO₂ from left pulmonary artery after 2 hours reperfusion between group 1, 3 (132.3±33.8, 121.5±19.7) and group 2, 4 (175.5±14.3, 171.8±15.4), P<0.05. There was significant difference in pulmonary compliance after 1 hour and 2 hours reperfusion between group 1, 3 and group 2, 4 (P<0.05). After 2 hours reperfusion, there was significant difference in serum and pulmonary MDA, T-AOC, pulmonary MPO, TNF-α, IL-6 between group 1 and group 2, 4 (P<0.05), whereas no significant difference was found between group 1 and group 3, no significant difference was found in serum TNF-αand IL-6 among all the groups. There was significant difference in apoptotic index between group 1 and group 2, 4 (P<0.05), whereas no significant difference was observed between group 1 and group 3. The morphological changes of lung injury were less severe in group 2 and group 4 compared with them in group 1 and group 3. Conclusions: In our model, IP by one cycle of blocking the left pulmonary artery for 5 min and then release for 10 min, showed the early protective effects of IRI in NHBD donor lung with 1 hour WIT after 2 hours reperfusion. The oxygenation of the allograft was increased, the pulmonary compliance and the morphological changes of lung injury were improved, and the cell apoptosis was less. The protective effects were obtained by inhibiting the lipid peroxidation and polymorphonuclear leukocyte aggregation and decreasing the concentration of TNF-αand IL-6. There were more significant changes in pulmonary cytokines than in serum after 2 hours reperfusion, suggesting that paracrine and autocrine of cytokines might play important role in IRI of the lung. The opening of the mitochondria ATP-sensitive potassium channel might play important role in the protection of IRI by IP, since the protective effects could be completely eliminated by 5-HD. Protection of diazoxide preconditioning on lung ischemia-reperfusion injury in rats undergoing non-heart-beating-donor lung transplantationObjectives: To investigate the protective effect of diazoxide preconditioning on lung ischemia-reperfusion injury (IRI) in rats undergoing non-heart-beating-donor (NHBD) lung transplantation. To offer the experimental evidence and possible drug for treating lung IRI following NHBD lung transplantation.Methods: The NHBD model in rat orthotopic left single allograft lung transplantation was established in the same way of Part I. The warm ischemia time was 60 min. Thirty-two male SD rats were randomized into 16 pairs for donors and recipients, divided into 4 groups: group 1, NHBD with 1 hour of warm ischemia time (NHBD1h, control group); group 2, ischemia preconditioning (IP) by one cycle of blocking the left pulmonary artery for 5 min and then release for 10 min before killing the donor rats (IP, IP control group); group 3, the recipients received 5mg/kg of diazoxide (DA) 30 min preoperatively (DA, experimental group); group 4, the recipients received 0.5 ml DMSO 30 min preoperatively (DMSO, DMSO control group). All the donor lungs remained ventilated at the room temperature for 1 hour after asystolia and then were flushed with LPD solution. The donor lungs were preserved in LPD solution at 4°C for four hours after harvesting. Then the recipient rats underwent orthotopic left single lung transplantations. Analysis of the blood gas from the recipient's right cervical artery and the whole pulmonary compliance were tested before the operation and after 10 minutes, 1 hour and 2 hours reperfusion. The blood gas from the allograft left pulmonary vein, TNF-αand IL-6 level, the activity of malondialdehyde (MDA) and total antioxidative capabilities (T-AOC) of the serum and the lung tissue, wet to dry weight ratio, activity of myeloperoxidase (MPO) of the lung tissue, apoptosis of alveolar cells, and histomorphology findings of the allograft by microscopy and electro-microscope were analyzed after 2 hours reperfusion.Results: There were significant differences in PaO₂ from left pulmonary artery after 2 hours reperfusion between group 3 and group 1, 4 (176.5±14.5 vs. 132.3±33.8 / 130.3±18.4, P<0.05), and no significant difference between group 3 and group 2. There were significant differences in pulmonary compliance after 1 hour reperfusion between group 3 and group 1, 4 (P<0.05), also no significant difference between group 3 and group 2. After 2 hours reperfusion, there were significant differences in serum and pulmonary MDA, T-AOC, pulmonary MPO, TNF-α, IL-6 between group 3 and group 1, 4 (P<0.05), whereas no significant difference was found between group 3 and group 2. There were significant differences in apoptotic index between group 3 and group 1, 4 (P<0.05), no significant difference between group 3 and group 2. The morphological changes of lung injury were less severe in group 2 and group 3 compared with them in group 1 and group 4.Conclusions: In our model, the early protective effects of IRI were observed by diazoxide pretreatment in NHBD donor lung with 1 hour warm ischemia time after 2 hours reperfusion. The oxygenation of the allograft was increased, the pneumonedema and morphological changes of lung injury were improved and the cell apoptosis was less. The protective effects of diazoxide pretreatment were similar with IP. Both of them were obtained by inhibiting the lipid peroxidation and inflammatory reaction. The opening of the mitochondria ATP-sensitive potassium channel might play the same role in these two pretreatments.