| Objective To investigate novel lipid microbubbles with matrix metalloproteinase-9 targeting in the evaluation of testicular different degree ischemia- reperfusion injury in rabbit, seeking for a more objective, more direct evaluation of testicular microvascular injury.Method Create general lipid microbubbles(MB) and novel lipid microbubbles with matrix metalloproteinase-9(TMB9).Detect their acoustic characteristics related.54 healthy adult male white rabbits were randomly divided into control group( group S), mild ischemia- reperfusion injury group(group R1,R2,R3,R4), moderate ischemia-reperfusion injury group(group R1’,R2’,R3’,R4’).A total of nine groups, each of 6. Randomly select one testis, the spermatic cords 2 cm above the testicle were ligated until testicular blood flow completely disappeared, with showing non-blood flow under color doppler ultrasound, to establish testicular completely ischemia model. Among them, group R1,R2,R3,R4 were re-established 2h, 6h, 12 h, 24 h respectively after ischemia reperfusion 2 h,to establish mild ischemia- reperfusion injury model.Group R1’,R2’,R3’,R4’ were re-established 2h, 6h, 12 h, 24 h respectively after ischemia re Perfusion 4 h, to establish moderate ischemia- reperfusion injury model. The S group was threaded but not ligated.Testes MB and TMB-9 CEUS were undertook preoperative and postoperative respectively. Ultrasound contrast angiographic Parameters of each group were analyzed with quantitative analysis techniques, including PI,TP,Slope,MTT,DT/2,Area and the difference between MB and TMB9. Laboratory test the levels of plasma, which were obtained by cardiac puncturevon guided by color doppler ultrasound, willebrand factor(v WF), nitric oxide(NO), endothelin-1(ET-1) before operation and after CEUS. Get the ipsilateral testicular to stain H.E. and immunohistochemical staining(MMP-9) after the operation and observe the pathological changes of each group. Statistical analysis was performed by SPSS13.0 software. All data are expressed in x ± s. Two sets of data were compared by two independent samples T test. Differences in test level is set to P <0.05(bilateral).Results Lipid microbubbles is milky white turbid liquid. the average particle was size of 3-5μm, evenly distributed round microbubbles by light microscope. After stored at room temperature for 30 minutes, liquid has a boundary, and microbubbles became larger while the number was decreased.After 24 h, liquid became clear and no obvious microbubble left.Preoperative, each parameter of testicular ultrasound contrast was no significant difference between the experimental group and the control group(P> 0.05). In mild ischemia-reperfusion injury group, the group R1’s TMB9 contrast parameter PI, Slope, Area increased significantly, while MTT,DT/2 was prolonged,TP decreased, which is a significant difference compared with the group S(P <0.01). With the prolongation of reperfusion, PI,Slope,Area decreased(P <0.05),while TP,MTT,DT/2 increased. Group TMB9 has higher in PI,MTT(P <0.05),greater in DT/2,Slope,Area,shorter in TP(P <0.01)compared with group MB.In moderate ischemia- reperfusion injury group, the group R1’’s TMB9 contrast parameter PI, Slope, Area increased significantly(P <0.01), while DT/2 was prolonged(P <0.05), TP decreased(P <0.05),which is a significant difference compared with the group S, With the prolongation of reperfusion, PI, Slope decreased(P <0.01),Area reduced(P <0.05)and TP,DT/2,MTT increased(P <0.01).Group TMB9 has greater in PI,MTT, DT/2,Slope,Area,shorter in TP(P <0.01)compared with group MB.Preoperative, the levels of plasma vWF, NO, ET-1 were not statistically different between the groups(P> 0.05). Each experimental group plasma v WF, NO, ET-1 levels were significantly higher than those in S group(P <0.01). In mild ischemia-reperfusion injury group,v WF, NO, ET-1 levels show a rising trend in group R1 and R2,group R3 has the highest content in ET-1. With the reperfusion time increasing, ET-1, NO levels shown a rising trend in moderate ischemia-reperfusion injury group(P<0.05).Testicular pathological findings: testicular interstitial vascular of group S were no expansion, no hemorrhage and thrombosis. In mild and moderate ischemia-reperfusion injury group,with reperfusion time increasing,testicular capillaries expansion obviously,thrombosis fomation in testicular capillaries, leakage, infiltration of inflammatory cells gradually increased and hyalinizationo of the lumen. MMP-9 expression in vascular basement membrane and interstitial edema gradually increased.Conclusions Novel lipid microbubbles with matrix metalloproteinase-9 targeting(TMB9) is better in evaluating ischemia- reperfusion injury of testicular microvascular compared with general lipid microbubbles(MB).Different ischemia time and different reperfusion time can establish different microvascular injury. |
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