Research On Contrast-enhanced Ultrasound To Evaluate Testicular Ischemia-reperfusion Injury And The Protective Effects Of P-selectin Inhibitor

Objective To discuss the protective effects and the correlative mechanism of P-selectin inhibitor on testicular completely moderate ischemia-reperfusion injury(IRI).Research on the contrast-enhance ultrasound performance of lipid microbubbles with anti-P-selectin monoclonal antibody(TMB-P)before and after the intervention by low molecular weight heparin.Explore an effective and noninvasive method to evaluate the testicular microvascular injury.Methods To prepare the general lipid microbubbles(MB)and ipid microbubbles with antiP-selectin monoclonal antibody(TMB-P)and detect the physical property and correlative performance of both contrast agent.A total of 36 rabbits were randomly divided into 6 groups including control groups(Group S1,S2),ischemia reperfusion groups(Group R1,R2)and low molecular weight heparin intervention groups(Group D1,D2)with 6 in each group.Group S1,S2 were randomly dissected to expose one side of rabbits' permatic cord and threaded without ligation.Group R1,R2 and Group D1,D2 were randomly dissected to expose one side of rabbits' permatic cord with ligation for building a completely testicular ischemia model of operation side.After 4h complete ischemia the reperfusion was performed on the operation side of the testis with 1h for group R1,D1 and 2h for group R2,D2.In addition,group S1,R1,R2 were given ear vein injection with equivalent normal saline after operation,group S2,D1,D2 were given ear vein injection with low molecular weight heparin 300IU/kg.Rabbits in each group were performed on contrast-enhanced ultrasound(CEUS)by contrast agent MB and TMB-P before the operation and after the successful build of each model,followed by analyzing the changes of contrast parameters of each group with the ultrasound contrast quantitative analysis technologies including PI,TP,Slope,MTT,DT/2 and AUC and comparing both results of evaluation.Meanwhile,rabbits in each group were performed on the heart puncture before and after the operation to obtain 3ml blood for the laboratory testing of the plasma level of v WF,ET-1 and NO and coagulation indexes including PT,APTT.HE staining and immunohistochemistry staining were performed on the testis of the operation side after ultrasonic contrast for observation of the pathological changes and comparison of changes about P-selectin expression level of each group.Results Correlative detection of lipid microbubble: MB and TMB-P both are milky white turbid liquid by naked-eye observing and roundness under light microscope,the distribution and particle size of the microbubble is uniform.After 30 minutes' Standing at room temperature the number of microbubbles reduced and the particle size of the microbubble became nonuniform.In addition,after one day's Standing at room temperature the number of microbubbles became sparse and the particle size of the microbubble was small.The concentration of the microbubble is(5.66±0.92)×108/ml approximately.Lipid microbubble of TMB-P is surrounded by one layer of green fluorescent under fluorescent microscopy.Expression of CEUS: There was no significant difference in preoperative MB and TMB-P parameters of CEUS among each group(P>0.05).There was also no significant difference in postoperative MB and TMB-P parameters of CEUS between group S1 and S2(P>0.05),which parameters of CEUS had no significant difference compared with preoperative parameters(P>0.05).To compare postoperative group R1 and group S1 showed that MB parameters of CEUS including PI,Slope,AUC increased obviously(P<0.01),DT/2 extended obviously(P<0.01),TP reduced(P<0.05),and MTT extended(P<0.05),TMB-P parameters of CEUS compared with MB changed more obviously,PI,Slope,AUC increased(P<0.01),TP reduced(P<0.01),MTT and DT/2 extended(P<0.01).To compare postoperative group R2 and group R1 showed that MB parameters of CEUS including PI,Slope,AUC further increased(P<0.01),DT/2 extended obviously(P<0.01),TP reduced(P<0.05),and MTT extended(P<0.05),TMB-P parameters of CEUS compared with MB changed more significantly,PI,Slope,AUC increased(P<0.01),TP reduced(P<0.01),MTT and DT/2 extended(P<0.01).To compare postoperative group D1 and group R1 showed that MB parameters of CEUS including PI,AUC decreased(P<0.05),whereas there was no statistically significant difference in changes among TP,Slope,MTT and DT/2(P<0.05),TMB-P parameters of CEUS compared with MB embodied changes more sensitively,PI,AUC decreased obviously(P<0.01),Slope decreased(P<0.05),TP extended(P<0.05),MTT reduced(P<0.05),DT/2 reduced(P<0.01).To compare postoperative group D2 and group R2 showed that MB parameters of CEUS including PI,Slope,AUC decreased obviously(P<0.01),TP extended(P<0.05),MTT and DT/2 reduced(P<0.01),TMB-P parameters of CEUS compared with MB changed more significantly,PI,Slope,AUC decreased(P<0.01),TP extended(P<0.01),MTT and DT/2 reduced(P<0.01).Detection of plasma: There was no significant difference in postoperative plasma level of v WF,ET-1 and NO and coagulation indexes including PT,APTT among each group(P>0.05).There was also no significant difference in postoperative plasma level of v WF,ET-1 and NO and PT,APTT between group S1 and S2(P>0.05),which various indexes had no significant difference compared with preoperative indexes(P>0.05).To compare postoperative group R1 and group S1 showed that the plasma level of v WF,ET-1 and NO increased obviously(P<0.01).To compare postoperative group R2 and group R1 showed that the plasma level of v WF,ET-1 and NO further increased(P<0.01).In addition,to compare postoperative group D1 and group R1 showed that the plasma level of v WF,ET-1 and NO decreased(P<0.05),whereas the difference of PT,APTT was no statistical significance(P>0.05).To compare postoperative group D2 and group R2 showed that the plasma level of v WF,ET-1 and NO decreased obviously(P<0.01),whereas the difference of PT,APTT was no statistical significance similarly(P>0.05).Detection of pathological tissue: the testicular microvascular structure of interstitial substance of Group S1,S2 was clear without expansion and congestion of lumen,endothelial cells lined up tightly and the basement membrane is still intact.The microvascular endothelial cells of testicular interstitial substance of Group R1,R2 developed turgidly with expansion and congestion of lumen,many leukocytes aggregated and adhered to the surface of endothelial cells and surrounding interstitial edema.Immunohistochemical staining showed that P-selectin has high expression in the surface of microvascular endothelial cells of testicular interstitial substance.To compare group D1,D2 and group R1,R2 showed that the expression of P-selectin reduced obviously in the surface of microvascular endothelial cells,and tumidness of microvascular endothelial cells reduced,the number of aggregated and adhesive leukocytes also reduced.Conclusions 1.Low molecular weight heparin can effectively inhibit the expression and effect of P-selectin of microvascular endothelial cells in testicular moderate completely ischemia-reperfusion injury,which contributes to reduce microvascular inflammatory response and injury of endothelial cells and maintains microvascular integrity.2.TMB-P evaluates the degree of injury of microvascular endothelial cells in testicular ischemia-reperfusion injury more effectively and sensitively than MB.