| Background and ObjectiveAs very severe cases, thromboembolic diseases, which often appeare in patients urgently and dangerously, have a high morbidity, mortality and high mousee of deformity. At present the diagnosis of thromboembolic diseases depends on clinical feature of patients and it depends on the invasive contrast venography, expensive computed tomography or magnetic resonance imaging. But clinical feature of patients is not so easy to find out and the examinations will miss acute thrombi when angiostenosis doesn't happen. Because of the poor sensitivity and specificity in detection of acute thrombi, the patients always miss the suitable treatment method, which make mortality and disability mousee high. Therefore, if we can develop a technology capable of early non-invasive, sensitive and specific diagnosis of thrombosis and thus early treatment of thrombosis, which is critical to reducing patient mortality and the incidence of complications. It will have great clinical value.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 technology is emerging as a novel imaging modality that shows a greater perspective for sensitively and specifically detecting thrombi. Arg-Gly-Asp (RGD) peptide-modified microbubbles, which can selectively bind to GPⅡb/Ⅲa, have been successfully applied to evaluate venous, atrial and arteriolar thrombus, but have so far failed in the detection of arterial thrombus. Ultrasound molecular imaging of arterial thrombus currently remains a difficult problem due to the high-shear arterial flow resulting in limited binding of site-targeted microbubbles to thrombus. And arterial thrombosis are mainly composed of activated platelets, platelet activation and aggregation at sites of vascular endothelial disruption are key events in arterial thrombus formation. When platelet aggregation, the GPⅡb-Ⅲa-fibrinogen complexes can be internalized or moved towards the canalicular system gradually, so the RGD-modified microbubbles can't bind to GPⅡb/Ⅲa. To make ultrasound molecular imaging of arterial thrombosis successful, we need to find new target for arterial thrombus.As P-selectin is expressed in a high density on activated platelets for at least one hour, the greatest clinical use of P-selectin is as a marker of platelet activation in vivo. P-selectin expression in platelets has been shown to be elevated in disorders associated with arterial thrombosis such as coronary artery disease, acute myocardial infarction, stroke, and peripheral artery disease. Merten et al. have shown that the GPⅡb-Ⅲa-fibrinogen complexes can be internalized or moved towards the canalicular system gradually, leaving P-selectin as the only bridging molecule on activated platelets in later phases of platelet aggregation. P-selectin binds to sulfatides on adjacent platelets by means of its lectin domain, stabilizings interactions between already bridged platelets, thereby allowing the formation of large stable platelet aggregates. In fact, Isenberg et al. have shown that P-selectin was the only glycoprotein present in the contact zone between platelet aggregates 15 minutes after activation, suggesting we can chose P-selectin as the target for arterial thrombus.Therefore, in this study, 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 contrast enhanced ultrasound (CEU), the ex-vivo thrombus were evaluated in an agarose flow chamber model and in vivo mouse model of abdominal aortic thrombosis were evaluated. Our aim is to explore the feasibility of targeted microbubble to evaluate aterial thrombosis. P-selectin may be likely one of optimal targeted molecular probes for ultrasound molecular imaging of arterial thrombus.Methods1. Prepamouseion of microbubbles and determine their biological propertiesLlipid microbubbles with biotin were prepared by sonication of perfluorocarbon gas (C3H8) with aqueous dispersion of several lipids in determinate mouseio. After being washed (4×) to remove excess free unincorpomouseed lipid, streptavidin in determinate mouseio were added to the lipid microbubbles with biotin, then washed (2×) to removed excess free unincorpomouseed streptavidin and the biotin conjugated mouse anti-mouse P-selectin monoclone antibodies in determinate mouseio were added to complete the prepamouseion of MBp. At last, the MBp were washed (2×) to remove excess free unincorpomouseed antibodies. Microbubbles biotinylated isotype control antibody (MB) were made by the same method. Both MB and MBp were storaged in refrigemouseor at 4℃. The mean diameter and density in both MB and MBp were measured by coulter counter.2. 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 arterial physiologic flow conditions2.1. Attachment study:MBp and MB (5×106/ml) were drawn through the parallel plate flow chamber that coated with PSfc (1000ng/ml) at the shear stress of 4 dyn/cm2, respectively. Quantitative analysis of microbubble accumulation was performed by counting the number of microbubbles adhered in the observed area under the microscope when the emergence of microbubble for 6 min; Control experiments on a plate that lack of PSFc (each n=3).2.2. Detachment study:The parallel plate flow chamber were coated with PSFc (1000ng/ml).0.2 ml MBp and MB were drawn into the flow chamber respectively and allowed to interact with the target surface by flotation at zero flow for 5 min. After 5 min, PBS was drawn through the flow chamber at a shear stress of 0.2 dyn/cm2, that would remove the microbubbles which did not adhere to PSFc.The total number of microbubbles adhered was recorded and microbubble detachment was then assessed by drawing PBS through the flow chamber with incremental shear stress increases every 30s until the microbubbles were completely detached. Recorded the full video and took photographys at a fixed field of vision (the objective lens was 20 times).3. Evaluation the efficacy of MBp targeted to thrombus using agarose flow chamber model in vitro studyAn agarose flow-chamber model was homely made as an imitation of the system of arterial blood flow. Thrombus was developed in vitro and fixed in flow-chamber model. After 30 min incubation with equivalent MB or MBp, the thrombus was washed with the continuous flow of PBS solution in high speed(15 cm/s). Contrast-enhanced ultrasound imaging of thrombi was performed at 2,4,6,8 and 10 min of the washing and the video intensity (Ⅵ) of thrombi was measured.4. Prepartion of mouse model of abdominal Aortic thrombosis and CEU examinationAnesthetized open-abdomen mice, non-occlusive thrombus resulted with injury in mouse abdominal aorta tunica intima was formatted. Two-dimensional ultrasound was used to determine the location of thrombus. Ten mice with non-occlusive thrombus were performed with CEU respectively by using MB and MBp, the intravenous injection of 10×106 microbubbles were made in random order with 30 minutes interval. After six minutes of intravenous injection, microbubbles in the circulation were nearly 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 0.2, transmission frequency of 7.0 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 thrombus were measured.5. Examination of pathologyAfter CEU examination, all ex-vivo thrombi and abdominal aortic were harvested for the examination of pathology and immunohistochemisty.Results1. Results for microbubble prepamouseion:The density of MBp and MB were about (2.8～4.2)×108/ml and (5.5～6.8)×108/ml sepamouseely, the sizes for MBp and MB were about 2.6±0.4μm,2.4±0.3μm respectively.2. The efficacy of MBp targeted to PSFc in the parallel plate flow chamber study:There was good targeted combination between MBp and PSFc with a shearing force (4 dyn/cm2) under arterial physiologic flow conditions, which was observed in the parallel plate flow chamber. However, it could not attach on the parallel plate in the group lack of PSFc-As expected, MB could not attach on the parallel plate in the test groups or control groups. Detachment study showed that the MBp could resist a certain shear stress, half-maximal detachment was achieved at a shear stress of (28.13±4.24) dyn/cm2. when the MBp were completely detached, the shear stress was up to (96.51±2.71) dyn/cm2, while MB can not resist low shear stress, its half-maximal detachment and maximal detachment were only achieved at a shear stress of (7.18±1.44) dyn/cm2 and (25.52±2.21) dyn/cm2.3. Results for ex-vivo thrombi CEU imaging:Before the washing, there was not significant difference in theⅥbetween both MBp and MB group in vitro (P>0.05). At each time points of the washing, the VI in the MBp were bigger than that in the MB (P=0.000). The visual enhancement of thrombus was still noted in the MBp with the washing of 10 min, while it was not seen even at 2 min in the MB.4. Results for arterial thrombi in mice abdominal aorta CEU imaging:As expected, all the arterial thrombi in mice abdominal aorta were clearly visualized after intravenous application of specific MBp. Obvious contrast enhancement of thrombi in comparison to the abdominal aortas lumen was showed in all thrombi.However, no obvious contrast enhancement of all thrombi can be observed when utilizing the nonspecific MB. Unlike the targeted microbubbles, the nonspecific MB in conbination with CEU failed to increase thrombi detectability after intravenous application. Mean VI of the abdominal aortic thrombi with MBp (20.91±2.17) was about 2.56 folds higher than that of the thrombi with MB (8.18±2.25) (P=0.000).5. Pathological and immunohistochemisty examination:In the thrombi we made in vitro and in vivo, formation of a large number of platelets trabecular were observed, and the surface of thrombi was positive for P-selectin.Conclusions1. MBp has a good targeting performance to thrombus and can resist to a certain degree of shear stress after adhesion to thrombus. In vitro evaluation of binding thrombus capability of targeted microbubble (MBp) by simulating shear stress in vivo environment could be helpful for predicting the effects of ultrasonic molecular imaging when MBp were used in vivo environment.2. The "active targeted CEU imaging" created by MBp and CEU can effectively evaluate the aterial thrombosis in mouse abdominal aortic, which may be used to diagnose the thrombosis in early stage, study targeting thrombolysis targeted therapy and monitoring the efficacy.3. MBp may be one of optimal targeted molecular probes for ultrasound molecular imaging of arterial thrombus. |
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