| Studies have indicated that reperfusion may trigger a series of injury responses,and cause the corresponding organs lethality damag e.Lung reperfusion injury could occur after lung transplant ation,extraco rporeal circulation surgery,pulmonary artery thromboendarter ectomy,pulmona ry embolism thrombolysis and so on,but few therapies have been demonstrated for preventing such injury.The mechanism of lung reperfusion injury is still not clearly elucidated,which might possibly be involved with many kinds of factors such as the inflammation medium,the oxygen free radicals,the alveolar epithelium and the lung blood vessel endothelium damage.At present,some problems are still existed and waiting for further studies and solutions.In recent years,people are unceasingly exploring and seeking interventional measures in order to reduce reperfusion injury.Some researches found that ischemic postconditioning was one of the most powerful protective mechanism,which has become a new research focus.Reperfusion is a process which can be foreseen and controlled.Some scholar's studies have shown that "controlled reperfusion"could reduce myocardial infarction size,lessen myocardial edema and no reflow response,which promoted the concept of ischemic postconditioning.Therefore,we established the animal model to interpret the mechanism of lung ischemia/reperfusion from the pulmonary parenchyma injury,changes in lung vascular endothelial permeability,interstitial substance and ischemic postconditioning. During surgery usinging some drug is an important element, some researches have found that the lidocaine, dexamethasone, anisodamin preconditioning have protective effects for L1RI. But there are some different opinions, and lidocaine, dexamethasone, anisodamin postconditioning researeh is rare in the literature.So it is important to performing more researches on the effects of lidocaine, dexamethasone, anisodamin to LIRI.By setting the rabbit model of LIRI and admililstrating lidocaine, dexamethasone, anisodamin before and after ischemia, we try to compare the protective effeets of them, and further reveal the possible mechanisms of these methods, so as to provide theoretical evidences for the corrective selection of clinical drug and surgery, and develop a new routine for organ protection.The current study includes the following two parts as felows:Chapter I ALI result from Lung Ischemia-Reperfu sion Injury in Rabbit:Immunologic MechanismObjective:To investigate the mechanism of the lung ischemia/reperfusion injury result in ALIMethods:The New Zealand big ear white rabbits were used in this experiment. By Eppinger Methods to restore blood flow to allow reperfusion,in order to establish the acute rabbit lung ischemia/reperfusion injury model.The morphological changes,lung wet/dry ratio(W/D),and the dynamic changes of the TNF-a, IL-1, IL-6, IL-8 in lung tissues were observed.Immunohistochemistry and in situ hybridization test was used to detect expression of TNF-a, IL-1, IL-6, IL-8 in the lung tissue during the pulmonary ischemia and reperfusion,Results:(1)Compared with control group,white blood cell count in the BALF significantly increased 60min,120min after lung ischemia,further increased at 60min after reperfusion,and reached its peak value 120min after reperfusion(22.36±1.65,24.17±1.28,54.93±3.65, P<0.01)。The white blood cell counts in BALF during reperfusion were significantly higher than those during ischemia(all P<0.01).(2)The BALF neutrophil percentage was significantly 60min,120min after ischemia, further increased 60min after reperfusion,and reached the peak value at reperfusion 120min (0.88±0.24,1.26±0.84,8.42±0.68, P<0.01,all P<0.01).(3) The lung W/D ratio at lung ischemia 60min,120min was significantly higher than control group and sham group.The lung W/D ratio at reperfusion 60min,120min (3.90±0.10,4.19±0.12,5.42±0.66, P<0.01)was significantly higher than the first two groups.(4)Changes of the TNF-a:Compared with control group,there is no obvious changes in the lung tisse homogenate TNF-a, at ischemia 60min (7.90±1.89, P>0.05),it was increased at lung ischemia 120min,and was continuously increased at reperfusion 60min,and reached the peak value at reperfusion 120min (6.03±1.24,7.20±1.48,11.56±2.55, P<0.05).The lung tissue homogenate TNF-a in reperfusion 120min group was higher than in reperfusion 60min group(P<0.05),it was significantly higher than ischemia 60min,120min group(P<0.05).The TNF-a changes in BALF was similar to the changes in the lung tisse homogenate.(5)Changes of IL-1, IL-6, IL-8:Compared with control group,there is no obvious changes in the lung tissue homogenate IL-1, IL-6, IL-8 at ischemia 60min (0.38±0.09, P>0.05),it was decreased at lung ischemia 120min,further decreased at reperfusion 60min,and the decrease was most obvious at reperfusion 120min (0.29±0.09,0.39±0.12,1.45±0.33, P<0.05).The lung tissue homogenate IL-1, IL-6, IL-8 in reperfusion 120min group was higher than in reperfusion 60min group(P<0.05).The IL-1, IL-6, IL-8 change tendency in BALF was also similar to that in the lung tissue homogenate.(6)After the lung ischemia/reperfusion,immunohistoch emistry and in situ hybridization test demonstrated that the massive expressions of IL-1, IL-6, IL-8positive cell in rabbit lung tissue were mainly located in pulmonary alveolusepithelial cells,bronchial epithelium and inflammatory cells(mononuclear cell,granular leukocytes).Compared with control group, the positive area of TNF-a, IL-1, IL-6, IL-8 in rabbit lung tissue was remarkably increased at ischemia 60min,120min,and reperfusion 60min,120min (P<0.01). TNF-a IL-1, IL-6, IL-8 expression in reperfusion 60min,120min groups were higher than ischemia 60min,120min groups, TNF-a IL-1, IL-6, IL-8 expression in reperfusion 120min group was higher than reperfusion 60min group(P<0.01). (7)Linear regression analysis showed that the TNF-a expression level was significantly correlated with lung W/D,lung tissue TNF-a content,BALF PMN proportion(r=-0.95, r=-0.93, r=-0.97, r=-0.91,all P<0.01). TNF-a expression level was also significantly correlated with IL-1, IL-6, IL-8content in lung tissue andBALF,(r=0.91 and r=0.94,both P<0.05).(9)Transmission electron microscope showed cell swelling,vacu olization,partial plasmolysis,and dense nuclearchromatin in capillary endothelial.Conclusion:(1)In our experiment,we successfully established lung ischemia/reperfusion animal model by Eppinger Methods.(2)The lung injury existed in both the processes of lung ischemia and reperfusion,and more obvious during reperfusion.(3)Our experiment indicated that the possible mechanism for acute lung ischemia/reperfusion was the accumulationof the PMN in lung tissue,rapid generation of TNF-a,which resulted in the activation of TNF-a,followed by IL-1, IL-6, IL-8 overexpression.ChapterⅡprotective effects of drug for ALI result from Lung Ischemia-Reperfusion Injury in RabbitObjective:With the developed cardiopulmonary surgical techniques, lung ischemia reperfusion injury(L1RI) result in ALI still is a hard work.While clinical preventing researches haven't got ideal progressions now. During surgery usinging some drug is an important element, some researches have found that the lidocaine, dexamethasone, anisodamin preconditioning have protective effects for L1RI. But there are some different opinions, and lidocaine, dexamethasone, anisodamin postconditioning research is rare in the literature.So it is important to performing more researches on the effects of lidocaine, dexamethasone, anisodamin to LIRI.By setting the rabbit model of LIRI and admililstrating lidocaine, dexamethasone, anisodamin before and after ischemia, we try to compare the protective effects of them, and further reveal the possible mechanisms of these methods, so as to provide theoretical evidences for the corrective selection of clinical drug and develop a new routine for organ protection.Method:32 New Zealand big ear white rabbits were randomly divided into 4 groups(n=8). (1) Isehemia reperfusion group (I/R, group):the left pulmonary hilum was oceluded for 60min and reperfused: (2) lidocaine-preconditioning group(lido-Pregroup):giving lidocaine 3o min before occlusion, others similar to I/R group; (3) anisodamin-postconditioning group(anisodamin—posgroup):admiisting anisodamin 3o min accompanied by reperfusion; (4) dexamethasone-preconditioning and postconditioning group:administing dexamethasone 30 min before the occlusion; administing dexamethasone 30 min accompanied by reperfusion.At the end of experiment, each group sacrifice 1 rabbits at reperfused 0min,30min,60min,120min,240min respectively then get arterial blood, lung tissue and bronchoalveolarlavagefluid(BALF)samples.The parameters include artery blood oxygen pressure, amount of protein, white blood cell differential counts, expression of tumor neerosis factorsa(TNF-a), interleukin(IL-1), IL-6 and IL-8 in BALF, and the expression of TNF-a, IL-1, IL-6 and IL-8 in lung tissures were detected..Using light microscope and electron microscope investigate pathological changes of lung, immunohistochemistry and in situ hybridization test..Results:With the increasing of reperfusion stage, all test groups show apparent lung injury changes, arterial blood oxygen pressure decreasing, the expression of TNF-a, IL-1, IL-6, IL-8 and amount of protein, white blood cell differential count increasing apparenily. The expression of TNF-a, IL-1, IL-6, and IL-8 in BALF increasing also.Comparing with I/R group there are apparent statistic variances in lidocaine, dexamethasone and anisodamin groups.In 1/R group, significant membrane damages of typeⅡalveolar epithelial cell, vacuolization of amellar body, and disorganzation of mitoehondrion were revealed by electron microscope examination. While pathological changes were slight in lidocaine, dexamethasone and anisodamin groups.There are integrated cell membrane and novacuolization of lamellar body.However, there are no differences between preconditioning and postconditioning groups.Conclusion:As commonly used drug, lidocaine, dexamethasone and anisodamin preconditioning or postconditioning all show protective effects for the LIRI in rabbit。The possible protective mechanisms include inhibiting inflammatory reaction, decreasing PMN infiltration and down-regulating expression of inflammatory factors TNF-a, IL-1, IL-6, IL-8.so as to alleviating systemic and local inflammatory reaction, decreasing PMN aggregation and permeability of lung.There are similar protective effects between lidocaine, dexamethasone and anisodamin groups.So we interfere that they may share the same mechanisms to develop the protective effects.
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