| Background and ObjectiveTargeted ultrasound molecular imaging research has become a frontline focus in international iconography technosphere.The classic targeted molecular imaging technologies are nuclide imaging and MRI.However,ultrasonic image research on molecular imaging with high space resolution and temporal resolution,extensive sphere of application,low cost,repeatable use,and without radiation pollution,is greatly fall behind to the other iconography technologies.For the past few years, with the presence of contrast media of targeted ultrasound microbubbles,targeted ultrasound molecular imaging gradually come true,which greatly expand the applied limit of general ultrasound microbubbles,make the early diagnosis of various kinds of diseases on molecular level by using ultrasound techniques become possible.With great research and application value,targeted ultrasound molecular imaging must play a very important role in molecular iconography technosphere.Vascular endothelium injury/inflammation generally resides in coronary artery disease,hypertension and diabetes mellitus,which are harmful to the health of human race.Moreover,the happening,progression and prognosis of aforesaid diseases are closely relevant to vascular endothelium injury/inflammation. Endothelial P-selectin expression increases in vascular endothelium injury/ inflammation.P-selectin can promote the recruitment and extravasation of platelets and neutrophilic granulocytes,which were activated and release lots of mediators of inflammation and chemotactic factors that aggravate the injuries and inflammation. Therefore,by using targeted ultrasound molecular imaging with microbubbles targeted to the related inflammatory molecules(P-selectin) on vascular endothelium, early and noninvasive evaluation of vascular endothelium injury/inflammation could be accomplished,which will have great clinical significance.For the past few years,with extensive perspective and great clinical significance,evaluation of microvascular inflammation and homologue endothelial responses with targeted ultrasound molecular imaging technology has increasingly become a frontline focus in ultrasound research.Now days,relevant researches on abroad are still in starting stage of empirical study and has no substantive progression at home,and there are many problems urgently to be solved.As one of the hypertransfusion organs,kidney is susceptible with ischemia-reperfusion(IR). Clinically,as a very important pathophysiology phenomenon,renal ischemia-reperfusion injury(IRI) can emerge in hypovolemic shock and renal transplantation.Renal IRI is the main element of ischemia renal failure and the important influential factor about functional rehabilitation and long-term survival time of transplanted kedneys.The expression of P-selectin on vascular endothelial cells(VEC) increases in kidneys undergoing IR,which can promote the adhesion and aggregation of platelets and neutrophilic granulocytes to the IR area.As a main cause of renal IRI,the neutrophilic granulocyte result in inflammatory reaction and release many mediators of inflammation or proinflammatory cytokine(TNF-a,IL-1, TXA2),cause the injury of renal capillary vessel and cells.Now,there is still no noninvasive and effective test facility on evaluation of renal IRI.So,by using targeted ultrasound molecular imaging with microbubbles targeted to the related inflammatory molecules(P-selectin) on vascular endothelium in kidney with IRI, early and noninvasive evaluation of vascular endothelium injury/inflammation in kidney with IRI could be accomplished,which will have active clinical significance.Therefore,we constructed the microbubbles targeted to P-selectin by combinding the anti-mouse P-selectin monoclone antibodies to the shell of general lipid microbubbles via "avidin-biotin" bridging chemistry.By using this microbubbles targeted to P-selectin(MBp) and the general lipid microbubbles without anti P-selectin monoclone antibodies(MB),theⅥ(video intensity) of kidneys undergoing IR or SH were determined by CEU were measured.We hypothesized that the microvascular inflammation in renal IR could be accurately evaluated with microbubbles targeted to the endothelial cell adhesion molecule P-selectin and CEU imaging.Methods1.Microbubbles preparation:General lipid microbubbles(MB) and lipid microbubbles with biotin were prepared by sonication of perfluorocarbon gas(C3H8) with aqueous dispersion of several lipids in determinate ratio.After being washed(4×) to remove excess free unincorporated lipid,streptavidin in determinate ratio were added to the lipid microbubbles with biotin,then washed(2×) to removed excess free unincorporated streptavidin and the biotin conjugated rat anti-mouse P-selectin monoclone antibodies in determinate ratio were added to complete the preparation of MBp.At last,the MBp were washed(2×) to remove excess free unincorporated antibodies.Both MB and MBp were storaged in refrigerator at 4℃.The mean diameter and density in both MB and MBp were measured by coulter counter.2.Evaluation of MBp in vitro:Using green fluorescent-labeled antiantibody for anti P-selectin monoclone antibody to identifv the linking,antibodies on the MBo.To evaluate the combinding efficacy between MBp and PSFc(Recombinant Mouse P-Selectin/Fc Chimera) with a parallel plate flow chamber at a shearing force under physiologic flow conditions.3.Animal preparation:Sixteen experimental mice were divided into sham-operated (SH) group(six mice) and IR group(ten mice).One kidney of the postanesthetic mice in group IR was exposed and underwent 30 minutes of occlusive renal ischemia followed by 60 minutes of reperfusion,while kidneyes of the postanesthetic mice in group SH was exposed only without ischemia.4.Renal CEU imaging:All experimental mice were performed with CEU respectively by using MBp and MB,the intravenous injection of 4x106 microbubbles were made in random order with 30 minutes interval.After ten minutes of intravenous injection,microbubbles in the circulation were eliminated,the ultrasound signal(video intensity,Ⅵ) from MB and MBp were measured by second harmonic CEU imaging with pulsing interval time(PI) of ten seconds and a mechanical index(MI) of 0.18,transmission frequency of 7.0 MHz and receiving frequency of 14.0 MHz.After the first picture of CEU imaging being taken,the microbubbles were destroyed by two to three seconds of continuous imaging with a high MI of 1.9 and the background- subtractedⅥof kidneys were measured.5.Examination of pathology and immunohistochemisty:After CEU imaging, all kidneys of experimental mice were harvested for the examination of pathology and immunohistochemisty.Results1.Results for microbubble preparation:The density of MBp and MB is about 7.1x108/ml and 1.5x109/ml,the mean sizes for MBp and MB were about 2.83um and 2.73um respectively.2.Results for evaluation of MBp in vitro:The anti-mouse anti P-selectin monoclone antibodies linked well to the surface of microbubbles,which were observed with fluorescence microscopy.There was good targeted combination between MBp and PSFc with a shearing force(＜1.0dyn/cm2) under physiologic flow conditions,which was observed in the parallel plate flow chamber.3.Results for renal CEU imaging:Significant ultrasound imaging was observed in the fist CEU picture of the IR kidneys with MBp.while there was no significant CEU imaging in SH kidneys with MBp.Lower grade ultrasound imaging was observed in the fist CEU picture of the IR kidneys with MB,while no significant CEU imaging was observed in SH kidneys with MB in the fist CEU picture.4.Results for CEU-derivedⅣ:There was no significant increase ofⅥin kidneys in neither group SH-MBp(2.93±1.33) nor group SH-MB(2.73±1.29) (P=0.797).As compared to kidneys in group SH-MBp,a significant increase in CEU-derivedⅣwere noted in kidneys of group IR-MBp(30.99±9.83)(P=0.000), and the latter is 10.58 times to the former.TheⅣin kidneys in group IR-MB (9.87±3.68) slightly increased compared to kidneys in group SH-MB(P=0.000),and the former is 3.62 times to the latter.The averageⅥin group IR-MB was only 31.8%of averageⅥin group IR-MBp(P=0.001).5.Results for examination of pathology and immunohistochemisty: Pathological examination showed that,swelling and degeneration of renal tubular epithelial cells,cast and exfoliation of necrotic cell,interstitial edema and neutrophilic leukocytosis were observed in IR kidneys,while SH kidneys remained unchanged.It was indicated by immunohistochemisty that the expression of endothelial P-selectin increased in IR kidney tissues compared to SH kidney tissues.Conclusions1.Microbubbles targeted to P-selectin(MBp) and CEU that create "active targeted CEU imaging" can effectively evaluate the renal ischemia-repeffusion injury in mouse,and may be used to evaluate the microvascular inflammation and other endothelial responses.2.Microbubbles targeted to P-selectin can be successfully constructed by combinding anti P-selectin monoclone antibodies to lipid microbubbles via "avidin-biotin" bridging chemistry.3.The expression of endothelial P-selectin increase significantly in IR kidneys tissues with imflammation induced by IR.
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