The Inflammation Injury In Lung Tissue Of Rat Model With Perpheral Arterrial Occlusive Diseasis

Objection: Peripheral Arterial Occlusive Disease (PAOD) is a common disease with high incidence, including thromboangiitis obliterans (TAO) and arteriosclerosis obliterans(ASO). Bogen Zhang suspected there are twenty millions ASO patients in china, and increace 0.6 millions per years, recent research manifest 14% patients with POAD have combined pulmonary diseases and after bronchial sleeve resection for malignancy these patient have more complications such as pneumonia, empyema, bronchopleural fistula. In different phase of PAOD there are ischemia, gangrene and infection. These disorders could induce systemic inflammation and inflammation medium expression in lung, inflammation cells invade and lead to lung injury. TNF-a is an important factor in acute lung injury, it is believed that TNF-α has action in lung injury as followed: first, TNF-α could directly injure endothelial cell by enhancing the adhesion ability of endothelial cell of vessel for neutrophil cell. Second , activate neutrophil cell. As neutrophil was activated ,they can construct a series of frame work leading to lung injury, inflammation medium released from distant place such as TNF-α can Activated neutrophil and other inflammation cell release a mass of injury Medium. Third, interaction with other cytokine. TNF-α can induce the release of interleukin-1 and interleukin-8. Meanwhile interleukin-4, interleukin-6 and transforming growth factor-β can prevent the transcription of TNF-α and interleukin-1 induced by endotoxin. Fourth, prevent synthesis of surfactant in lung type Ⅱ cell. Fifth , Activate blood coagulation system. Sixth, Activate complement system. Recent research certificate that the concentration of TNF-α in blood was elevated in some chronic hypoxia and ischemia disease such as ischemic heart disease or cerebral infarction. Ischemia reperfusion injury can increase concentration of TNF-α in blood significantly. Our research built the PAODrat model by injecting lauric acid sodium into femoral artery.The experiment rats were divided into sham-operation group and three trial groups based on the degree of the disease. Observing the proportion of neutrophil in BALF by Wright's staining , pathological alteration by hematoxylin and eosin Staining, detecting concentration of TNF-a in Homogenate of lung tissue, plasma and BALF by Enzyme-linked immunoadsordent assay technique, Observing the ratio of TNF-a positive cells in lung tissue by immunohistochemistry Technique. To study that if there are release of inflammation medium and increasing of inflammation cells in lung tissue of rat model with PAOD. Methods:1 Creation of rat model with PAODClean grade Wistar male rats were chosen with weight ranging from 250g to 350g. All of rats were bred for a week to adapt to environment, experiment rat model was built on left hindlimb. Anesthesia by injection in abdominal cavity. Cutdown skin and separate hypodermis to expose femoral artery blood tube sheath. Then dissect and free femoral artery under microscope followed by injecting 0.2ml lOmg/ml solutions of lauric acid sodium. Finally Block the puncture point with medical anastomosis glue. Artery clamp was taken away after 1 minute, after operation 0.2 million unit penicillin was injected into abdominal cavity for infection prevention. Suture operative incision. 0.9% Saline replace solutions of lauric acid sodium in sham-operation group (control group) o2 obtain stuffInjecting ketamine hydrochloride into abdominal cavity by O.lg/kg for anesthesia. Then open abdominal cavity by median incision, Sacrifice rats by Withdrawal blood from inferior vena cava. blood was put in test tube with anticoagulant, then Centrifugation 3000met/min for 3 minutes. Obtained plasma was stored in -20°C refigerabo. BALF from left lung: open the thoracic cavity and separate skin and muscle of neck to expose the trachea and hilum of lung o Tie right hilum of lung, then make a lateropulsion Incisions on trachea . A blunt hard hollow tube was inserted by the incision. Turn left into left principal bronchus after feeling Resistance. 2 ml Salinewas administrated into left lung by the tube, wait for 30 second,then retrieve. Replicate this procedure three times. Other 2ml Saline replicate. In sum 6ml saline was administrated and 3-5 ml was retrieved. The retrieved saline was stored in silic-plastics tube which can prevent cell from attaching for observation of cell smear. Cut right lobe of lung down and cut it open. A half was dipped in neutrality Formaldehyde solution for HE Staining and immunohistochemistry. The other half was collected in sterile EP tube in -20 °C refigerabo for ELISA.3 PMN proportion in total cell count of BALFThe depositor obtained from 1 ml BALF by centrifugation was made smear for Wright's staining. Observe the PMN proportion in total cell count of BALF in 20 random sights.4 HE StainingHE Staining was performed by routine method to observe morphology alteration in lung tissues.5 ELISAConcentration of TNF-a of lung tissue Homogenate, BALF and plasma was detected with Test Kit made by Wuhan Boster company.6 immunohistochemistryTNF-a positive cell proportion in lung tissue was detected with kit made by Wuhan Boster company.7 Pathology observation: routine HE staining8 Statistics analysisThe difference of value of observing marker was analysised by analysis of variance and multiple comparisons with SPSS 10.0 software and P value less than 0.05 was considered significant. Correlation among these marker value was check by correlation analysis.ResultsConcentration of TNF-a in lung tissue increased in three model group compared with control group, especially in gently gangrene group. Severe gangrene group is lower than gently gangrene group, and have not significant difference with limpinggroup.The difference of concentration of TNF-a in plasma among four groups have not significant difference.In gently gangrene group and severe gangrene group, Concentration of TNF-a in BALF increased significantly. There was no significant difference of concentration of TNF-a between gently gangrene group and severe gangrene group, limping group and control group.PMN proportion in total cell count of BALF in gently gangrene group are higher than those of limping group and control group. There was no significant difference of PMN proportion in total cell count of BALF between gently gangrene group and severe gangrene group, limping group and control group. There was no significant difference between severe gangrene group and limping group and control group.TNF-a positive cell proportion in lung tissue in gently gangrene and severe gangrene group are higher than that of limping and control group. There was no significant difference of TNF-a positive cell proportion in lung tissue between gently group and Severe gangrene group,limping group and control group.pathologic alteration: inflammation cell in the blood vessel of the lung increased. Polymorphonuclear leukocyte attached to the wall of the vessel. There was infiltrating of the inflammation cell around the vessel and edema in the interval of the alveolus. All of these alteration increased according with degree of the gangrene. In Severe gangrene group, polymorphonuclear leukocyte aggregated and formatted micro-abscess in the interval of the alveolus.Correlation AnalysisCorrelation Analysis Certificate that there are significant correlation among TNF-a positive cell proportion in lung tissue, PMN proportion in total cell count of BALF, Concentration of TNF-a in BALF and Concentration of TNF-a in lung tissue, but these Observing marker are not relative with Concentration of TNF-a in plasma.9 Conclusion1) The level of all marker including TNF-a in BALF, lung tissue, TNF-a positive cell proportion in lung tissue ,PMN proportion in total cell count of BALF are elevated. There was no significant difference between gently gangrene group and severe gangrene group, inflammation cell in the blood vessel of the lung increased. Polymorphonuclear leukocyte attached to the wall of the vessel. There was infiltrating of the inflammation cell around the vessel and edema in the interval of the alveolus. All of these alteration increased according with degree of the gangrene. In Severe gangrene group, polymorphonuclear leukocyte aggregated and formatted micro-abscess in the interval of the alveolus.2) Concentration of TNF-a in BALF and lung tissue, TNF-a positive cell proportion in lung tissue ? PMN proportion in total cell count of BALF are relative. But there was no significant correlation between these marker and concentration of TNF-a in plasma.