| Background and ObjectiveAs a very important pathophysiology phenomenon,Myocardial ischemia-reperfusion injury(IRI) often appeares in patients after percutaneous coronary intervention(PCI) or acute coronary syndrome(ACS).Many relevant researches have proved that the electrocardiogram and initial serologic markers for injury are frequently non-diagnostic,which leads to a delayed or even missed diagnosis in 38%patients with acute myocardial ischemia.Imaging of wall motion and perfusion using ultrasound has been shown to provide incremental diagnostic information to established practice.Yet even this strategy is of limited value if performed late after symptoms have resolved or in those with pre-existing abnormalities from remote events.Moreover,in patients with known severe chronic coronary artery disease,often the most important question is not if ischaemia is present,but rather how much muscle is in jeopardy.Until now,there is still no noninvasive and effective test facility on evaluation of myocardial IRI.So if we could detect the recent ischemic "molecular memory" of myocardium successfully and it may be helpful to assess region and extent of myocardial ischemia-reperfusion injury, which will have great clinical significance.When myocardial ischemia-reperfusion injury happens,Endothelial P-selectin expression increases in vascular endothelium injury/ inflammation within a few minutes.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(TNF-α,IL-1,TXA2)that aggravate the injuries and inflammation in myocardial capillary vessel and cells.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.Molecular imaging researches have succeed in many areas such as kidney,tumor, brain,etc.However,the molecular imaging researches in heart are still in early stage and still have many problems needed to be solved.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 play an important role in prevention and treatment of myocardial ischemia-reperfusion injury.So 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 control microbubbles(MBc),theⅥ(video intensity) of region of ischemia-reperfusion and non ischemia-reperfusion were measured separately by MCE.We hypothesized that the microvascular inflammation in myocardial IR could be accurately evaluated with microbubbles targeted to the endothelial cell adhesion molecule P-selectin and MCE 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(1 ×) to remove excess free unincorporated lipid,streptavidin in determinate ratio were added to the lipid microbubbles with biotin,then washed(1×) to removed excess free unincorporated streptavidin and the biotin conjugated rat anti-mouse P-selectin monoclone antibodies and isotype control antibodies in determinate ratio were added to complete the preparation of MBp and MBc.At last,the MBp and MBc were washed(1×) to remove excess free unincorporated antibodies.Both MBc and MBp were storaged in refrigerator at 4℃.The mean diameter and density in both MBc and MBp were measured by coulter counter.2.Microbubble with FITC preparation:Biotin lipid microbubbles with were prepared by sonication of perfluorocarbon gas(C3H8) with aqueous dispersion of several lipids in determinate ratio.FITC-streptavidin in determinate ratio were added to the lipid microbubbles with biotin,then washed(1×) 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 with FITC.At last,the MBp were washed(1×) to remove excess free unincorporated antibodies.Both MB and MBp wih FITC were storaged in refrigerator at 4℃.3.Evaluation of microbubble in vitro:Using green fluorescent-labeled antiantibody for anti P-selectin monoclone antibody to identify the linking antibodies on the MBp.4.Evaluation of microbubble in vivo:To evaluate the adhesion pathways and performances of microbubbles targeted to P-selectin on the inflammatory endothelium using Fluorescence microscope.5.Animal preparation:Inflammation of the cremaster muscle was produced by intrascrotal injections of 0.5μg murine tumor necrosis factor(TNF)-α2 hours before surgery in 10 wild-type mice Ten untreated wild-type mice were served as controls.A cremaster muscle was prepared and was superfused continuously with isothermic bicarbonate-buffered saline.Intravital microscopy was performed in twenty mice to evaluate P-selectin-mediated microbubble adhesion Video recordings were made with a highresolutio camera. Ten mice with myocardial ischemia-reperfusion were injected intravenous of MBp and MBc in random order with 30 min interval.After 5 min of intravenous injection of microbubble,targeted MCE imaging was performed in all mice.And then the video intensity(Ⅵ) was determined.According to the variety of microbubble injected,we have two groups:MBp group and MBc group.6.MCE imaging:Ten mice with myocardial ischemia-reperfusion were performed with MCE respectively by using MBp and MBc,the intravenous injection of 1×106 microbubbles were made in random order with 30 minutes interval.After five minutes of intravenous injection,microbubbles in the circulation were eliminated, the ultrasound signal(video intensity,Ⅵ) from MBc and MBp were measured by second harmonic MCE imaging with pulsing interval time(PI) of ten seconds and a mechanical index(MI) of 0.2,transmission frequency of 7.0 MHz.After the first picture of MCE 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 myocardial were measured.7.Examination of pathology and immunohistochemisty:After MCE imaging,all hearts of experimental mice were harvested for the examination of pathology and immunohistochemisty.8.Statistical analysis:Data are expressed as mean±SD.Experiment one: Comparisons between different groups were made with one-way-ANOVA.Interval comparisons were made with Dunnet T3.Experiment two:Comparisons between different groups were made with two stages cross-over design.Interval comparisons were made with paired t-test.Differences were considered significant at a value of P＜0.05(2-sided).Results1.Results for microbubble preparation:The density of MBp and MBc is about1.06×109个/ml and 1.03×109个/ml separately,the mean sizes for MBp and MBc were about 2.30±1.06μm and 2.07±1.13μm respectively.2.Results for evaluation of microbubble in vitro:The anti-mouse anti P-selectin monoclone antibodies linked well to the surface of microbubbles,which were observed with fluorescence microscopy.3.Results for evaluation of microbubble in vivo:MB could adhere to to the surface of an activated leukocyte.And fluorescent MBp were attached directly to the venular endothelium efficiently and specifically.Retention of MBp was significantly greater than that of MB in TNF-α-stimulated-group(P=0.002.The number of MBp amounted to 12.0±2.6/view and the number of MB was just 3.0±1.2/view.Adhesion quantity of MBp in TNF-α-stimulated mice were 6.4±1.7 and 9.9±2.1 fold greater separately than MBp and MB of wild-type mice without any treatments.4.Results for MCE imaging:A significant enhancement in ultrasound was observed in ischemic region of MBp-group.Increase inⅥvalue of ischemic region in MBp-group was great and it amounted to 25.98±6.23.However,increase inⅥvalue of ischemic region in MBc-group was minor,it was just 9.12±0.91.Difference was evident in ischemic region between of the two groups(P=0.000).In both MBp-group and MBc-group,theⅥvalue of ischemic region was significantly greater than non-ischemic region(P=0.000).TheⅥin ischemic region of group MBp is 4.03±1.08 times to the non-ischemic region(6.53±0.95).But TheⅥin ischemic region of group MBc is only 1.44±0.18 times to the non-ischemic region(6.41±0.78). There was no obvious difference in theⅥof non-ischemic region between the two groups(P=0.522).5.Results for examination of pathology and immunohistochemisty:Pathological examination showed that slightly swelling of myocardial cells,interstitial edema and Loose connective tissue were observed in ischemia region,while non-ischemia region remained unchanged.It was indicated by immunohistochemisty that the expression of endothelial P-selectin increased in ischemia region of myocardial tissues compared to non-ischemia region.Conclusions1.Microbubbles targeted to P-selectin can be successfully constructed by combinding anti P-selectin monoclone antibodies to lipid microbubbles via "avidinbiotin" bridging chemistry.2.The Phospholipid microbubbles targeted to P-selectin we constructed could adhere directly to vascular endothelial efficiently and specifically.These results suggest MBp may be used to assess inflammation of vascular endothelial or other tissue injury effectively.3.Microbubbles targeted to P-selectin(MBp) and MCE that create "active targeted MCE imaging" can effectively evaluate the myocardial ischemia-reperfusion injury in mouse,and may be used to evaluate the microvascular inflammation and other endothelial responses.
|
PDF Full Text Request