Ultrasound Assessment Of Ischemia-reperfusion Injury In Skeletal Muscle Of Mice With Microbubbles Targeted To P-selectin And Preparation Of The Thrombus Model For Experiments In Vivo

Backgroundsl:Ischemia-reperfusion injury(I-R) is a common pathophysiology phenomenon,which are often following coronary artery disease,acute myocardial infarction and stroke.Endothelium inflammation of blood vessel usually happened with I-R.In the developing course of the endothelium inflammation,inflammation medium and adhesion molecule were excreted much more than normal level.P-selectin excreted more than before in acute inflammation,too.It has an important clinical value undoubtly if we can use a non-invasive method of imaging to judge the changes in this process.Targeted ultrasound molecular imaging was a new examination method of disease in recent years,microbubbles could be detained in the tissue or the organs through the special treated to the microbubbles.Targeted ultrasound molecular imaging were more widely used than the examination of ultrasound by using microbubbles.One is the development of the molecular images,a non-invasive examination method of various tissues and organs,such as vascular endothelial injury, thrombosis,tumor angiogenesis,the other is the targeted release of genes and drugs.It is come true of ultrasound assessment of ischemia-reperfusion injury in heart and renal with microbubbles targdted to P-selectin on abroad,there will be enormous development in the research of targeted ultrasound molecular imaging.However,the study of targeted ultrasound molecular imaging was no development at home.Peripheral atherosclerotic disease was concerned a dangerous disease whose risk similar with the coronary artery disease,which can lead to claudication and gangrene.With the increasing of morbility of peripheral atherosclerotic diseases,it is important to detect the I-R injury of limbs,which can induce the complication of atherosclerotic disease obviously.There were enormous significance to set up a non-invasion examnation method to detect the limbs I-R injury early.There were a lot of studies of targeted microbubble and CEU evaluating I-R injury of heart and renal.Volume of blood flow of limbs were more less than heart and renal,so it was uncertain of ultrasound assessment of ischemia-reperfusion injury in skeletal muscle of mice with microbubbles targdted to P-selectin,there have no relative report at home.Therefor,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),theâ…¥(video intensity) of limbs undergoing IR or SH were determined by CEU were measured.We hypothesized that the microvascular inflammation in limbs IR could be accurately evaluated with microbubbles targeted to the endothelial cell adhesion molecule P-selectin and CEU imaging.Baekgrounds2:The morbility of thrombotic diseases have increased obviously in recent years,according to the statistic in our country,the mortality of stroke,coronary artery disease and acute mycrodial infraction have always located from the first to the fourth place of all the fatality disease since 2001.About 17,000 thousands people died for heart and brain vascular diseases every year according to the statistic of WTO in 2002.The formation of thrombus or thromboembolism were the lastest key of the emergency events of cardic,brain and peripheral vescular,which were direct reasons of death or disability,no thrombus,no events.So it is very important of early diagnosis and therapy of thrombotic diseases,it can reduce the mortality and improve the prognosis of them obviously.However,examination methods of thrombotic diseases such as CT and MRI had some defects.Ultrasound was only used as a diagonosis method first,the effects of ultrasound in thrombolysis had been confirmed in recent years,it would be manifest enhanced with the existence of microbubbles.With the invention of the thrombus targeted microbubbles,the effects of it in the diagnose and therapy in thrombus diseases were more and more thought highly of the people.Now the thrombolysis by ultrasound and thrombus targeted microbubbles were in stages of elementary research and animal experiments.Objectives:The first part of this study Assessment of ischemia-reperfusion injury in skeletal muscle of mice with microbubbles targeted to P-selectin which prepared by our laboratory with ultrasound.The second part of this study To set up a new rat crester microcirculation thrombotic model which was quite similar to the clinical features by using photochemistry method,providing a more practical animal model for microcirculation experiments of targeted microbubbles and experiments of evaluating drug effects of thrombolysis.Methods:The first part of this study Microbubbles preparation:General lipid microbubbles and biotinylation lipid microbubbles were prepared by sonication of perfluorocarbon gas(C₃H₈) with aqueous dispersion of several lipids in determinate ratio.After being washed(4×) to remove exess free unincorporated lipid,streptavidin in determinate ratio were added to the biotinylation lipid microbubbles,then washed (2×) to removed exess free unincorporated streptavidin and the biotin conjugated rat anti-mouse P-selectin monoclone antibodies in determinate ratio were added.At last, the targeted microbubbles combinded anti-mouse P-selectin monoclone antibodies via "avidin-biotin" bridging chemistry were washed(2×) to remove exess free unincorporated antibodies and storaged in refrigerator at 4℃.The mean diameter and density in both MB and MBp were measured by coulter counter.Using green fluorescent-labeled antiantibody for anti P-selectin monoclone antibody to identify the linking antibodies on the MBp.Mice were anesthetize with(0.6ml/100g) and fixed them on the board,trochar were putted into the vena caudalis,prepared the skin of inguinal groove and hindlimbs, the skin of the right side of the inguinal groove was opened and separated the femoral artery,then clamped it with bulldog clamp,released clamp after 1 hour.After reperfusion 1 hour,all experimental mice were performed with CEU respectively by using general lipid microbubbles and microbubbles targeted to P-selectin(intravenous injection,30 minutes interval).After ten minutes of intravenous injection, microbubbles in the circulation were eliminated,then the ultrasound molecule imaging from the targeted microbubbles and lipid microbubbles were got by CEU. After CEU imaging,all hindlimbs of experimental mice were harvested for the examination of pathology.All analysis were performed using the SPSS version 11.5, Data are expressed as mean±SD.Statistics of two groups comparisons were made with Independent-Samples Test.Differences were considered significant at a value of P＜0.05(2-sided).The second part of this study Fifteen male SD rats were devided into five groups at random,after i.m.injection of different dose(0.6ml/100g) urethane(13.3%) and chloralose(1%) in croup.Polythylene catheter(PE-50,inner diameter 0.58mm) was inserted into left femoral vein,the skin of the right side of the scrotum was opened and the cremaster was incised longitudinally,keeping the principal nerves and blood vessels to the muscle intact.The cremaster was spread with sutures over the glass slide,moistened with modified Krebs solution,then put the testis and epididymis into the abdominal.The animal and cremaster preparation were positioned on a modified stage of a microscope(Nikon,Tokyo,Japan) so that the cremaster,which is approximately 200 to 250 mm thick,could be observed by transmitted light or epi-illumination.After i.v.injection of different dose(0.2ml～0.6ml) of FITC-dextran through the catheter 5 minutes later,FITC-dextran,photochemical thrombus formation was induced by continuous blue light(450～490nm) from a 100-W mercury lamp exposure to the arteriole of the rat cremester,while the process was displayed on the computer monitor,the degree of stenosis in different time were calculated.All analysis were performed using the SPSS version 11.5,Data are expressed as mean±SD.Comparisons of stenosis degree of different exposed time were commited to One-way ANOVA.Stenosis degree in using different dose was compared by using Repeated Measures.Differences were considered significant at a value of P＜0.05(2-sided).Results:The first part of this study 1.Average size of general lipid microbubbles were 2.83 um,the concentration of it about 1.5×10⁹/ml.P-selectin targeted microbubbles distributed uniform,average size were 2.73 urn,concentration of it about 7.1×108/ml.2.The anti-mouse anti P-selectin monoclone antibodies linked well to the surface of microbubbles,which were observed with fluorescence microscopy.3. Significant enhanced ultrasound imaging was observed in CEU picture of the I-R group compared with the normal group with MBp,while there was significant CEU imaging enhancement of I-R group compared with normal group with MB in the CEU picture too.4.In I-R as compared to the contral group,there were significant of VI with MB and MBp(P＜0.05),but no significant difference ofâ…¥in contral group with MBp and MB(P＞0.05).However,compared with the MB group,there were significant increase ofâ…¥in I-R group with MBp(P＜0.05).5.Pathological examination showed that,skeletal muscle cells no ischemia were normal and no congestion and edema;skeletal muscle cells of IR group were edema,with a large number of neutrophil infiltration.The second part of this study 1.The relation of stenosis degree and dose of FITC-dextran and illumination time.There were significant increases of stenosis degree in 0.6ml group compared to 0.2ml and 0.3ml group when the exposure time was 3,6,9 minutes(P＜0.05),but between 0.2ml and 0.3ml group there have no significant differences(P＞0.05).The degree of stenosis was much higher in 0.4ml^. 0.6ml group than 0.2ml and 0.3ml group when the time was 6 and 9 minutes(P＜0.05).Except 0.2ml group,the microvessels of all the other five groups were all occluded in 30 minutes.Significantly less time of occlusion of the vessels were observed in 0.4ml～0.6ml group than in 0.3ml group,while there have no significant differences in 0.4ml～0.6ml group.After 30 minutes of occlusion,there have no thrombolysis in all obstructed vessels.2.The process of thrombotic in microscope.There were no occlusion before exposed to the fluorescent light in vessels,blood flow was very fast,being linear flow.After i.v.injection of 0.4ml FITC-dextran,first,endothelial cell were swelled,vessel wall were thickened,leukocyte in the blood flow moving to the vessel wall and rolling on it.then adhesion on it,being "white microthrombus";red blood cell agglutination in clumping, blood flow slower than before,with the prolonging exposed to the fluorescent light,volume of the thrombus was greater than before,embolus amoticed from the thrombus,flowed with the blood,then the vessel was occluded completely,blood flow stopped.However,the vessels hadn't been exposed to the light were normal.3.Study of pathology of microvascular show with thrombosis,which were red thrombus,and endothelial cells were swollen.Conclusions:The first part of this study 1.Microbubbles targeted to P-selectin can be successfully constructed by combinding anti P-selectin monoclone antibodies to lipid microbubbles via "avidin-biotin" bridging chemistry.2.Microbubbles targeted to P-selectin(MBp) and CEU that create "active targeted CEU imaging" can effectively evaluate the limbs ischemia-reperfusion injury in mouse,and may be used to evaluate the microvascular inflammation and other endothelial responses. The second part of this study It is a simple and reliable approach to develop an experimental rat cerester thrombus model,providing a new method for the experiment of thrombus targeted microbubbles.