The Protection Of Remote Limbs Ischemic Perconditioning Against Lung Ischemia-reperfusion Injury On The Model Of Acute Pulmonary Thromboembolism In Rabbits

Acute pulmonary thromboembolism is a group of disease that various embolus from the venous system or right ventricular obstructed pulmonary artery or its branch-es.It’s main symptom is a pathological syndrome that pulmonary circulaton and respi-ratory dysfunction. At present,it’s incidence is increased because of the ageing popula-tion and the proportion of elderly patients hospitalized.European society of Cardiolo-gy(ESC) in 2008 and United States Heart Association(AHA) in 2011 recommend that based on clinical features,right heart failure and myocardial damage markers as a hierarchical index and risk of acute pulmonary embolism can be divided into high, medium and low risk types.Rapid thrombolytic or embolectomy was recommended in patients with high-risk.Hospitalization was recommended in intermediate risk patients.Discharge from hospital or outpatient treatment was recommended in patients with low risk. Althou-ght thrombolytic therapy is the primary method of clinical treatmen of acute pulmon-ary embolism, we found that the mortality and death rate did not reach the expected therapeutic effect after thrombolysis in patients with high-risk of actue pulmonary em-holism in clinical works.The result may be related to lung ischemia/reperfusion injury after thrombolysis.In recent years,the protective effects of remote limbs ische-mic perconditioning against ischemia-reperfusion of target organs was explored in domestic and foreign medical institutions.At first,the main research was on the heart and brain.At present,the studies generally involve the liver,kidneys, lungs and other vital organs.Remote limbs ischemic perconditioning was that multiple brief,repetitive ischemia/reperfusion intervention were refer to the limbs during the phase of targer organ ischemia,what can significantly reduce ischemia-reperfusion injury of target organs.However,the research of remote limb ischemic perconditioning on the lung ischemia/reperfusion injury of acute pulmonary embolism after thrombolytic therapy was rare.We established the rabbit model of acute pulmonary thromboembolism,when the model was established 20 mins later we used remote ischemic perconditioning intervention.The aim was to explore the protective effects and mechanism of remote limbs ischemic perconditioning against lung ischemia-reperfusion injury in the model of acute pulmonary thromboembolism in rabbit.The research was divided into three parts as the followed.Part I. The establish of acute pulmonary thromboembolism modelsObjective:To establish the acute pulmonary thromboembolism models of rabbits.Methods:1.The animal group:12 Big-eared Japanese rabbits(SPF,no limit of male or female)weighting 2.0±0.5kg are divided into 2 groups randomly(six in each group): Sham group(Sham-operated group) and PE group(pulmonary embolism group). Sham group:The rabbit did not receive thrombus injection,which only injection of 5ml saline.PE group:The rabbit receive five thrombus injection,and followed by bolus injection of 5ml saline to prevent local venous thrombosis.2.The establish of acute pulmonary thromboembolism models in rabbit:Preope-rative fasting 8h,water 4h.The 10% chloralic hydras 4ml/kg intraperitoneal injection, we tracheotomy and ventilation with small animal breathing machine when succeed at anesthesia,breathing rate 50 times per min,tidal volume set up to l0ml/kg with an inspiratory/expiratory ratio 1:2,the vital signs was monitor by Powerlab electro card-scope monitor.Right ventricular intubation was connected with bar-receptor after inserting the right ventricular through right jugular vein,which was used as right ventricular systolic pressure monitoring.2ml arterial blood put in the disposable sterilized syringe,stand for natural solidification,mothball.Then,all animals received 3mg/kg of heparin intravenously in saline. Thrombus at 2mm diameter and 4mm in length were prepared from the clot.The thrombus was injected through the right ventricular intubation,which was followed by bolus injection of 5ml saline to prevent local venous thrombosis.Each rabbit received the injection of five emboli.3.The marker for the detection:(1)Right ventricular systolic pressure:Record the different time points of right ventricular systolic pressure such as before embolization and 1h,2h,4h after embolization.(2)The ratio of lung tissue wet weight/dry weight: Executed rabbits after embolization for 4h,and take about 1g lung tissue,mearsured the wet weight with electronic analytical balance and dry weight after baking at 80 ℃ in the electric thermostatic drier over 48h,calculated the wet/dry weight.(3) Determi-nation the lung tissue Superoxide Dismutase activity and Malondialdehyde content: Mixed the lung tissue and 0.9% NS then homogenate the lung,3000r/min centrifugal 20min at the place of 4 ℃.Measured the concentration changes of MDA and SOD level by colorimetric method,take on the operation strictly according to the operation instructions.(4) Pathological changes of lung tissue:The lung tissue, bleeding points, was fixed in 4% buffered formalin and embedded in paraffin.Thin sections(4um)cut from each paraffin block were stained with hematoxylin and eosin and used for histo-pathologic examination under a light microscrope, and lung injury was scored.4.Analysis was performed using SPSS version 20.0(SPSS Inc,Chicago,USA). The statistical description through Mean±SD(x±s),and comparisons between groups were analyze with independent-samples T test. P<0.05 is considered signigicant.Results:(1)Right ventricular systolic pressure.Analyze with repeated measures test we found there were significant different between different groups (F=245.516,P=0.000), and there were significant different at different time point(F=42.734,P=0.000). Analy-ze with independent-samples T test we found there were no difference between groups before embolization(t=0.681,P=0.511);But there were significant difference between different groups at different time point after embolization(t1h=-15.544、 t2h=-12.9566、4h=-12.807,P=0.00). Analyze with independent-samples T test we found the right ventricular systolic pressure of PE group increased more significantly than S group after embolization(P<0.01).(2)The ratio of lung tissue wet weight/dry weight:Analyze with independent-samples T test we found there were significant difference between PE group and S group(t=:-6.340,P=0.00),and the ratio of lung tissue wet/dry weight of PE group increased more significantly than S group(4.79±0.23 vs 4.01±0.19^P=0.00).(3)Determination the lung tissue Superoxide Dismutase activity and Malondia-ldehyde content:Analyze with independent-samples T test we found there were signi-ficant difference between PE group and S group(t=-10.550,.P=0.00), and the MDA content of PE group increased more significantly than S group(3.49±0.29nmol/mg vs 2.12±0.12nmol/mg,P=0.000). Analyze with independent-samples T test we found there were significant difference between PE group and S group(t=10.377, P=0.00), and the SOD activity of PE group increased more significantly than S group (127.04 ±13.52U/mg vs 216.30±16.16U/mg,P=0.00).(4)Pathological changes of lung tissue:There was no thrombosis in S group when we anatomized lung,but the thrombosis can be found in the pulmonary artery system in PE group.We can found that the structure of mitochondria and epithelial cells are normal in S group.Mitochondria dissolved into vacuole in PE group under an electron microscope.Conlusion:We successfully establish a acute pulmonary thromboembolism model which is similar with clinical APTE patient. We confirmed the APTE model by monitor the risen of right ventricular systolic blood pressure after embolization,anatomical see there are thrombus formation in the pulmonary artery. The model is simple,repeat-able and with high success rate.Part II. The research of lung ischemia-reperfusion injury after thromboly-tic in the acute pulmonary thromboembolismObjective:To observe the phenomenon of lung ischemia/reperfusion injury after thrombolytic in the acute pulmonary thromboembolism.Methods:1.The animal group:18 Big-eared Japanese rabbits(SPF,no limit of male or female)weighting 2.0±0.5kg are divided into 3 groups randomly(six in each group),that is Sham group(Sham-operated group),PE group(pulmonary embolism group),UK group(thrombolytic group).Sham group,PE group are same as part 1.UK group:After the APTE model established,the urokinase 50000 U/kg was injected through right ventricular intubation with micro perfusion pump at 1h after embolism, all the urokinase should be injected within 2h.The other operation is same as part 1.2.The establish of acute pulmonary thromboembolism models in rabbit:The establish of acute pulmonary thromboembolism models is same as the part 1.3.The marker for the detection:(1)Right ventricular systolic pressure:It’s same as the part 1.(2)Determination the lung tissue TNF-a and IL-6 content:After the APTE model established,draw 2ml blood from right carotid artery into EDTA tube before embolization and at lh,2h,3h,4h after embolization,3000r/min centrifugal 20min at the place of 4 ℃,detected TNF-a,IL-6 of plasma concentration by ELISA. (3)The ratio of lung tissue wet weight/dry weight:It’s same as the part 1. (4) Path-ological changes of lung tissue:It’s same as the part 1.4.Analysis was performed using SPSS version 20.0(SPSS Inc,Chicago,USA). The statistical description through Mean±SD(x±s),and comparisons between groups were analyze with one-way ANVOA test.The comparisons between pair-wise with the LSD test, P<0.05 is considered signigicant.Results:(1)Right ventricular systolic pressure:Analyze with repeated measures test we found there were significant different between different groups (F=164.020,P=0.000), and there were significant different at different time point(F=106.643,P=0.000). Analyze with one-way ANVOA test we found there were no difference between groups before embolization(F=0.537,P=0.595).But there were significant difference between different groups at different time point after embolization (Fih=150.500、 F2h=120.93、F4h=83.868,P=0.00). Analyze with LSD test we found the right ventricular systolic pressure of UK group,PE group increased more significantly than S group at h after embolization(21.17±1.33mmHg、 20.67±1.21mmHg vs 10.50± 1.05mmHg,P=0.00).But there were no differenc between PE group and UK group (P=0.482). The right ventricular systolic pressure of UK group was decreased more significantly than PE group at 4h after embolization(17.50±1.52mmHg vs 20.33± 1.37mmHg,P=0.004).(2)Determination the lung tissue TNF-a and IL-6 content:Analyze with repeat-ed measures test we found there were significant different between different groups (F=921.233,P=0.000),and there were significant different at different time point (F=454.285,P=0.000).Analyze with one-way ANVOA test we found there were no difference between groups before embolization (FTNF-α=0.238, P=0.791). But analyza with LSD test we found the TNFa content of PE group increased more significantly than S group after embolism(P<0.05),and the TNFa content of UK group increased more significantly than PE group after embolism,too(P=0.000). Analyze with repeat-ed measures test we found there were significant different between different groups (F=181.119,P=0.000),and there were significant different at different time point (F=115.252,P=0.000).Analyze with one-way ANVOA test we found there were no difference between groups before embolization (FIL-6=0.367, P=0.699). But analyza with LSD test we found the IL-6 content of PE group increased more significantly than S group after embolism(P=0.000),and the IL-6 content of UK group increased more significantly than PE group after embolism, too(P<0.05).(3)The ratio of lung tissue wet weight/dry weight:Analyze with one-way ANVOA test we found there were significant difference between groups (F=48.913, P=0.00). Analyze with LSD test we found the ratio of lung tissue wet/dry weight of UK group increased more significantly than PE group, S group(P<0.05).(4)Pathological changes of lung tissue:There was no thrombosis in S group when we anatomized lung,but the thrombosis can be found in the pulmonary artery system in PE group. We can found that the structure of mitochondria and epithelial cells are normal in S group.Mitochondria dissolved into vacuole in PE group under an electron microscope.A lot of lamellar body formatted in lung cell in the UK group under an electron microscope.Conlusion:There are lung ischemia-reperfusion injury phenomenon after thrombolytic in the acute pulmonary thromboembolism.The TNF-a and IL-6 were increased after embolization.The TNF-a and IL-6 were significant increased after thrombolytic.Part III. The protection of remote limbs ischemic perconditioning against lung ischemia-reperfusion injury on the model of acute pulmonary thromboem-bolism in rabbitsObjective:To observe the protection of remote limbs ischemic perconditioning against lung ischemia-reperfusion injury on the model of acute pulmonary thromboembolism in rabbits.Methods:1.The animal group:24 Big-eared Japanese rabbits(SPF,no limit of male or female)weighting 2.0±0.5kg are divided into 4 groups randomly(six in each group),that is Sham group(Sham-operated group),PE group(pulmonary embolism group),UK group(thrombolytic group),UK+LIPerC group(thrombolytic and limb ischemic perconditioning).Sham group,PE group and UK group are same as part 2.UK+LIPerC group:After the APTE model established,the limb ischemic percon-ditioning induced by brief cycles of ischemia and reperfusion of bilateral former limb was applied during sustained lung ischemia at 20mins after embolism. Four cycles of limb ischemic perconditioning were performed by applying rubber band tourniquet high around each thigh for 5 mins followed by reperfusion for 5 mins to achieve effective intervention. The other operation is same as part 1.2.The establish of acute pulmonary thromboembolism models in rabbit:The establish of acute pulmonary thromboembolism models is same as the part 1.3.The marker for the detection:(1)Right ventricular systolic pressure:It’s same as the part 1. (2)The ratio of lung tissue wet weight/dry weight:It’s same as the part 1.(3) Determination the lung tissue Superoxide Dismutase activity and Malondialde-hyde content:It’s same as the part 1. (4) Determination the lung tissue IL-6 content: After the experiment,lung tissue was homogenized with isotonic chloride,3000r/min centrifugal 20min at the place of 4℃,detected IL-6 of plasma concentration by ELISA.(5)Pathological changes of lung tissue:It’s same as the part 1.4.Analysis was performed using SPSS version 20.0(SPSS Inc,Chicago,USA). The statistical description through Mean±SD(x±s),and comparisons between groups were analyze with one-way ANVOA test.The comparisons between pair-wise with the LSD test, P<0.05 is considered signigicant.Results:(1)Right ventricular systolic pressure:Analyze with repeated measures test we found there were significant different between different groups (F=112.316,P=0.000), and there were significant different at different time point(F=149.563,P=0.000). Analyze with one-way ANVOA test we found there were no difference between groups before embolization(F=0.686,P=0.571).But there were significant difference between different groups at different time point after embolization (F1h=79.733、 F2h=76.93、F4h=63.658,P=0.00). Analyze with LSD test we found the right ventri-cular systolic pressure of UK group,PE group and UK+LIPerC group increased more significantly than S group at 1h after embolization(10.67±1.21mmHg、21.17±1.33 mmHg、19.83±1.83mmHg vs 10.50±1.05mmHg,P=0.000).But there were no differenc between PE group,UK group and UK+LIPerC group(P>0.05). The right ventricular systolic pressure of UK group and UK+LIPerC group were decreased more significantly than PE group at 4h after embolization(17.50±1.52mmHg、 17.00±1.10mmHg vs 20.33±1.37mmHg,P< 0.002). But there were no differenc between UK group and UK+LIPerC group(P=0.535).(2)The ratio of lung tissue wet weight/dry weight:Analyze with one-way ANVOA test we found there were significant difference between groups (F=40.311, P=0.00). Analyze with LSD test we found the ratio of lung tissue wet/dry weight of UK group increased more significantly than PE group, S group(P< 0.001). UK+ LIPerC group decreased more significantly than UK group(4.96±0.15 vs 5.29± 0.25,P=0.015).(3)Determination the lung tissue Superoxide Dismutase activity and Malondia-ldehyde content:Analyze with one-way ANVOA test we found there were significan-tly difference between groups (FMDA=188.523, P=0.000; FSOD=110.774, P=0.000). Analyza with LSD test we found the MDA content of UK group increased more signi-ficantly than PE group(P=0.000), UK+LIPerC group decreased more significantly than UK group(P<0.01).Analyza with LSD test we found the SOD activity of UK group increased more significantly than PE group(6.31±0.42nmol/mg vs 8.75± 0.91nmol/mg,P=0.000), UK+LIPerC group decreased more significantly than UK group(148.93±14.53U/mg vs 78.69±7.25U/mg,P<0.01).(4)Determination the lung tissue IL-6 content:Analyze with one-way ANVOA test we found there were significantly difference between groups (F=546.231, P=0.000). Analyza with LSD test we found the IL-6 content of UK group increased more significantly than PE group(.P=0.000), UK+LIPerC group decreased more significantly than UK group(0.11±0.04ng/mg vs 0.14±0.04ng/mg, P=0.000).(5)Pathological changes of lung tissue:There was no thrombosis in S group when we anatomized lung,but the thrombosis can be found in the pulmonary artery system in PE group. We can found that the structure of mitochondria and epithelial cells are normal in S group.Mitochondria dissolved into vacuole in PE group under an electron microscope.A lot of lamellar body formatted in lung cell in the UK group under an electron microscope.UK+LIPerC group significantly attenuated these histologic changes in comparison with the UK group.Conlusion:The limbs ischemic perconditioning can decrease the lung ischemia-reperfusion injury on the model of acute pulmonary thromboembolism after thrombolytic in rabbits.