Experimental Study On Evaluating Testicular Ischemia/Reperfusion Injury With Novel Lipid Microbubbles With Matrix Metalloproteinase-9Targetin In Rabbit

Object Investigate the change of MMP-9on moderate ischemia/reperfusion injury inrabbit testicles; Triturate the novel lipid microbubbles with matrix metalloproteinase-9targeting and detect the physical parameter of targeted microbubbles; valuate the valueof targeted microbubbles on moderate ischemia/reperfusion injury in rabbit testicles.Method Part I:12healthy adult white rabbits were randomly divided into: controlgroup (sham-operated group/S group), ischemia/reperfusion group(ISI group),6pergroup. S group exposed randomly either side of the spermatic cord,6h later the exposedside of testes were resected and fixed in10%formaldehyde prepared for HE andimmunohistochemical staining. IRI group selected one side randomly to have testicularcomplete torsion surgery,4h after ligation, released toreperfusion2h respectively, theoperated testes were resected and fixed in10%formaldehyde prepared for HE andimmunohistochemical staining.Observe the distribution of MMP-9in the testis underthe light microscope.Analyse the mean optical density of MMP-9with image-proplus6.0ver.Part Ⅱ: Preparation of general lipid microbubbles(MB) and lipid microbubblestarget. Microbubbles with biotin were prepared by sonication of sulfur hexafluoridegas(SF6) with aqueous dispersion of several lipids in determinate ratio.After washed toremove excess free indispersed lipid, avidin in determinate ratio were added to thelipid microbubbles with biotin，then washed to removed excess free indispersed avidinand the biotin conjugated anti MMP-9monoclone antibodies were added to completethe preparation of TMB9.At last,the TMB9was washed to remove excess freeindispersed antibodies．Both MB and TMB9were storaged in refrigerator at4℃．Themean diameter and density in both MB and TMB9were measured byhemocytometer.FITC-labeled secondary antibody was used to observe the combinationbetween MMP-9antibody and microbubbles by fluorescence microscopy. Part Ⅲ:12healthy adult white rabbits were randomly divided into: control group(sham-operated group/S group), ischemia/reperfusion group(ISI-1group), schemia/reperfusion and blasting group(ISI-2group),6per group. S group exposed randomlyeither side of the spermatic cord,6h later the testes were performed with CEU by usingMB and TMB9. IRI-1group selected one side randomly to have testicular completetorsion surgery,4h after ligation, released to reperfusion2h respectively. Then CEUwas performed as S group. The rabbits were killed after the end of the experiment, theoperated testes were resected and fixed in10%formaldehyde prepared for HE andimmunohistochemical staining, Observe the distribution of MMP-9in the testis underthe light microscope.Analyse the mean optical density of MMP-9with image-proplus6.0ver. IRI-1group selected one side randomly to have testicular complete torsionsurgery,4h after ligation, released to reperfusion2h respectively.Then bolus thecontrast media and blast them to increase microvascular permeability,20m later thetestes were performed with CEU by using TMB9. All images were stored in themachine to prepare for off-line analysis. Using professional CEU software to analysethe contrast images. Evaluate the contrast curve of rabbits’ testis (include slope, peaktime, peak to peak, half time, the average time and area). Statistical analysis wasperformed using SPSS19.0software, mean was output as χ±s,and t-test was picked toanalyse the differences between groups at the level of p <0.05.Conclusions1. In a testicular I/R remodeling rabbits model,MMP-9can be found invascular basement membrane and interstitial,,and expressed in the cytoplasm andnucleus showed no expression;2.Microbubbles targeted to MMP-9can besuccessfully constructed by combinding anti MMP-9antibodies to lipid microbubblesvia ‘avidin-biotin’ bridging chemistry;3.Base on the temporary MMP-9increasing,MMP9-targeted contrast-enhanced ultrasound imaging may provide a novel andnon-invasive approach to localize and study MMP9activation in a post I/R remodelingrabbits model;4."Acoustic cavitation effect” did not enhance the capabilities ofTMB9targeted imaging.