Experimental Study On Evaluating Testicular Ischemia/Reperfusion Injury With Targeted Microbubbles In Rabbit

Objective Detect the physical parameter of ordinary lipid microbubbles and targetedmicrobubbles, and evaluate the value of targeted microbubbles on completed ischemia/reperfusion injury in rabbit testicles.Method Part I:Microbubbles preparation General lipid microbubbles(MB)and lipidMicrobubbles with biotin were prepared by sonication of sulfur hexafluoride gas(SF6) withaqueous dispersion of several lipids in determinate ratio.After washed to remove excess freeindispersed lipid, avidin in determinate ratio were added to the lipid microbubbles withbiotin，then washed to removed excess free indispersed avidin and the biotin conjugated antiP-selectin polyclone antibodies were added to complete the preparation of MBp.At last,theMBp was washed to remove excess free indispersed antibodies．Both MB and MBp werestoraged in refrigerator at4℃．The mean diameter and density in both MB and MBp weremeasured by hemocytometer.FITC-labeled secondary antibody was used to observe thecombination between anti-P-selectin antibody and microbubbles by fluorescence microscopy.Part II:30healthy adult New Zealand white rabbits were randomly divided into: controlgroup (sham-operated group/S group), ischemia/reperfusion group (IR, IR group is dividedinto0.5h,1h,2h,4h group),6per group. S group exposed randomly either side of the spermaticcord,4h later the testes were performed with CEU by using MB and MBp. IR group selectedone side randomly to have testicular complete torsion surgery,4h after ligation, released toreperfusion0.5h,1h,2h,4h respectively. Then CEU was performed as S group. Each injectedMB and MBP approximately0.2ml/kg,(concentration of5.0×108/mL), dynamic observationwas recorded and after5~10min when the microbubbles in blood circulation pool fadedcompletely, the power(10E-5AU) of the first CEU frame was recorded.All images were storedin the machine to prepare for off-line analysis. The rabbits were killed after the end of theexperiment, the operated testes were resected and fixed in10%formaldehyde prepared forpathological examination.Using professional CEU software to analyse the contrast images.Statistical analysis was performed using SPSS13.0software, mean was output as χ±s,and one-way ANOVA was picked to analyse the differences between groups at the level of p <0.05.Results Part I: General microbubbles and targeted microbubbles were white suspension,uniformly distributed circular microbubble, particle size were about1.5~4.0μm (MB),1.0~3.0μm(MBp), the concentrations were about5.0×108/mL (MB),1.0×109/mL(MBp). TheMB showed no fluorescence while MBp showed bright green fluorescence under thefluorescence microscope.Part II: Significant enhanced ultrasound imaging was observed in CEU picture of1h、2hgroup，slight encanced imaging was observed in0.5h、4h group compared with SH group andnormal testes showed no enhancement by MBp;there were slight enhanced imaging in each I-Rgroup by MB.And power value of MB was less than MBp in operated testes(P <0.05) in eachI-R group,too. The Power value of MB and MBp had significant differences between operatedand normal testis (P <0.05),and had no significant difference in all group before operation(P>0.05). Pathological examination showed normal seminiferous tubules, no congestion,edema in mesenchymal, IR group showed varying degrees of necrosis, congestion, edema andneutrophil infiltration. Immunohistochemistry showed that only interstitial vascular endothelialcell of I-R group expressed P-select.Conclusions1.Microbubbles targeted to P-selectin can be successfully constructed bycombinding anti P-selectin antibodies to lipid microbubbles via ‘avidin-biotin’ bridgingchemistry．2.Microbubbles targeted to P-selectin(MBp)and CEU that create ‘active targetedCEU imaging’ can effectively evaluate the testis ischemia-reperfusion injury in rabbit，and maybe used to evaluate the microvascular inflammation and other endothelial responses.3.P-selectin secreted vascular endothelial cells mediating inflammatory response I-R injury couldbe detected at0.5h,2h to peak, then decreased gradually in rabbit testes.