[Evaluation of tumor angiogenesis using microbubbles conjugated with RGD peptides and contrast enhanced ultrasound].

OBJECTIVE: To assess the feasibility of usage of microbubbles conjugated with RGD peptides and contrast enhanced ultrasound (CEU) in detection of tumor angiogenesis. METHODS: Lipid microbubbles (MB) were prepared, and the RGD peptides were covalently conjugated to the lipid shell of MB (MB(RGD)). Six nude mice with tumor created by dorsal inoculation of HepG2 tumor cells were used as the test group. Six nude mice without tumor were served as the control group. 10 minutes after bolus injection of MB and MB(RGD) randomly (30 min interval) via a tail vein catheter, CEU was performed on the tumors of the test group and the thigh skeletal muscles of control group. The video intensity (VI) of tumors and the skeletal muscles were measured. The tumors and the skeletal muscles were harvested for immunohistochemical examination. RESULTS: Only a slight contrast enhancement of the tumor was seen with MB, and the VI was 5.33 ± 1.71. While a remarkable enhancement of the tumor was observed after injection of MB(RGD). The VI was up to 17.03 ± 3.58, 3.18 folds higher as compared with that obtained by injection of MB (P < 0.05). As expected, there were no obvious contrast enhancement of the skeletal muscles with both MB(RGD) and MB. There was a high expression of αvß3-integrin in tumor neovascular endothelium, however, no apparent expression of αvß3-integrin was observed in the skeletal muscle vascular endothelium. CONCLUSION: CEU with MB(RGD) can be used to effectively evaluate the angiogenesis of tumors, and it may greatly contribute to the early judgement of the nature of tumor.

[Evaluation of myocardial ischemia-reperfusion injury in mouse by molecular imaging of P-selectin with targeted contrast echocardiography].

OBJECTIVE: To assess the feasibility of evaluating myocardial ischemia-reperfusion injury in mouse with targeted myocardial contrast echocardiography (MCE). METHODS: Phospholipid microbubbles targeted to P-selectin (MBp) and control microbubbles (MBc) were created by conjugating monoclonal antibody against murine P-selectin or isotype control antibody with the lipid shell via "avidin-biotin" bridging. Ten mice with myocardial ischemia-reperfusion were injected intravenously of MBp and MBc in a random order with a 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. RESULTS: A significant ultrasonic enhancement was observed in ischemic region of MBp-group. Increment in VI value of ischemic region in MBp-group was great and it amounted to (26.0 +/- 6.2) U. However, increment in VI value of ischemic region in MBc-group was minor and it was merely (9.1 +/- 0.9) U. Difference was evident in ischemic region between of two groups (P < 0.05). In both MBp-group and MBc-group, the VI value of ischemic region was significantly greater than that of non-ischemic region (6.5 +/- 1.0) U vs (6.4 +/- 0.8) U (P < 0.05). There was no obvious difference in the VI of non-ischemic region between two groups. CONCLUSION: Molecular imaging of P-selectin with targeted contrast echocardiography can effectively evaluate myocardial ischemia-reperfusion injury.

[Impact of ultrasound-mediated microbubbles on myocardial vascular permeability in rats].

OBJECTIVE: To investigate the impact of high-dose microbubbles induced by high mechanical index myocardial contrast echocardiography (MCE) on vascular permeability and its recovery time in rats. METHODS: Thirty male Wistar rats were randomized into 4 MCE groups (groups A-D) and a control group. In the MCE groups, Evans blue was injected at 10 s before MCE (A), immediately after the end of MCE (B), and at 5 min (C) and 20 min after the end of MCE (D). In the control group, the microbubbles and Evans blue were injected at the end of a 5-min ultrasound exposure. All the rats were sacrificed 5 min after Evans blue injection, and the content of Evans blue in the myocardium and the percentage of Evans blue leakage area were determined. RESULTS: The percentage of Evans blue leakage area in groups A, B and C were significantly higher than that in the control group (P<0.05), while the percentage was similar between group D and the control group (P>0.05). Evans blue contents in groups A and B were significantly higher than that in the control group (P<0.05), but groups C and D showed comparable contents with the control group E (P>0.05). No significant changes of the heart rates and premature beat number were observed during and after MCE in these groups (P>0.05). CONCLUSION: High mechanical index MCE and a high contrast dose may induce increased microvascular leakage in rats, and the vascular permeability can recover in 20 min after MCE.

[Molecular imaging of thrombus with microbubbles targeted to alphavbeta3-integrin using an agarose flow chamber model].

OBJECTIVE: To assess the binding ability of microbubbles targeted to alphavbeta3-integrin (MBp) for thrombus-targeted contrast-enhanced ultrasound. METHODS: Targeted microbubbles were prepared by conjugating the monoclonal antibody against alphavbeta3-integrin to lipid shell of the microbubble via the avidin-biotin bridges. Equivalent isotype control microbubbles (MB) or targeted ultrasound microbubbles (MBp) were randomly added into the flow chamber. After a 30-min incubation with the thrombus fixed in an agarose flow chamber model, the thrombus was washed with a continuous flow of PBS solution (15 cm/s) for 2, 4, 6, 8 and 10 min, followed by thrombus imaging using contrast-enhanced ultrasound and measurement of the video intensity (VI) values of the images. RESULTS: The VI of the thrombus in MBp group was reduced by 28%-66%, while that in control MB group was decreased by 87%-94%, and the VI values of the thrombus group were significantly greater in former group at each of the time points (P<0.05). CONCLUSION: MBP has good targeting ability to the thrombus with resistance to the shear stress after adhesion to the thrombus. In vitro evaluation of the thrombus-binding capability of the targeted microbubble (MBp) by simulating the shear stress in vivo can be helpful for predicting the in vivo effects of ultrasonic molecular imaging using MBp.

[Effect of polyethylene oxide on red blood cell velocity in rat cremaster microcirculation].

OBJECTIVE: To investigate the drag-reducing effect of polyethylene oxide (PEO) on the velocity of red blood cells in rat cremaster microcirculation. METHODS: Blood samples were collected from 6 Wistar male rats (100-110 g) via the post-orbital venous plexus. The red blood cells were separated by centrifugation and labeled by fluorescinisothiocyate (FITC). After successful establishment of cremaster model, the labeled red blood cells were injected into the jugular vein, and the microcirculation was observed and recorded under fluorescence microscope. The hemodynamic parameters and microcirculation video was recorded every 4 min since 4 min before PEO or normal saline injection. Both PEO (10 ppm) and normal saline was injected into the same rat in random sequence at a constant rate of 3.5 ml/h for 20 min followed by observation for another 20 min. The velocity of the labeled-red blood cells was determined by IPP 6.0 software. RESULTS: Compared with normal saline, PEO significantly increased the velocity of the red blood cells in the rat cremaster microcirculation (498.7-/+182.89 microm/s vs 773.54-/+308.27 microm/s, P=0.012). No significant changes in the heart rate and arterial blood pressure were observed during the experiment (P=0.836, P=0.420). CONCLUSION: PEO at an extremely low concentration can significantly increase the velocity of the red blood cells in rat cremaster microcirculation and produces no significant impact on heart rate and arterial blood pressure.

[Ultrasound-mediated microbubble destruction increases capillary permeability in rat skeletal muscles].

OBJECTIVE: To investigate the effects of ultrasound mediated microbubble destruction on capillary permeability in rat skeletal muscles. METHODS: Eighteen SD rats were randomized into 3 groups, namely the Evans blue (EB) group, EB+ultrasound (E+U) group and EB+microbubble+ultrasound (U+E+M) group with corresponding treatments, using EB injected into the carotid artery as the indicator for capillary permeability. The microbubbles were injected through the carotid artery with fixed ultrasound parameters. The spillover of EB was estimated under fluorescence microscope according to the visual staining scores. The contents of EB in the skeletal muscles were calculated according to the standard curve and spectrophotometry. RESULTS: EB spillover was observed around the capillaries in E+U+M group, which had a significantly higher visual score than EB group and E+U group (0 and 0-1, respectively, P<0.05). The EB content was 51.57-/+3.89 microg/g in E+U+M group, also significantly higher than those in EB group (28.99-/+4.67 microg/g) and E+U group (30.99-/+4.11 microg/g) (P<0.05). CONCLUSION: Exposure to both ultrasound and microbubble contrast agents results in increased capillary permeability of rat skeletal muscles, which might be an important mechanisms for gene delivery enhancement by ultrasound contrast agents.

[Effect of phospholipid- and albumin-coated microbubbles for myocardial opacification: a comparative study].

OBJECTIVE: To evaluate the effect of a phospholipid-coated microbubble contrast agent for myocardium opacification in comparison with a albumin-coated microbubble contrast agent (Quanfuxian). METHODS: In 10 dogs with single coronary artery stenosis involving the anterior descending branch or circumflex branch randomly received infusion of the two contrast agents through the femoral vein. The myocardial blood flow, heart rate and blood pressure were analyzed qualitatively and quantitatively. The concentration and the particle diameter of the two contrast agents were determined. RESULTS: The concentration of the phospholipid-coated microbubbles was (1.06-/+0.22) x10(9)/ml, with a diameter of 3.04-/+0.34 microm, similar to the concentration and diameter of Quanfuxian ((1.31-/+0.33)x10(9)/ml and 2.88-/+0.58 microm, respectively, P>0.05). Both of the agents achieved grade three myocardium opacification, and produced no obvious effect on the heart rate and blood pressure. Quantitative analysis of myocardial opacification in terms of myocardial blood volume (A), blood velocity (beta), and blood flow (A x beta) revealed no significant difference between the two agents (P>0.05), and the parameters derived from the two agents showed good correlations (P<0.05, rA=0.809, r beta=0.932, rA.beta=0.925). CONCLUSION: The phospholipid-coated microbubble contrast agent shows good effect for myocardial opacification without significant difference from Quanfuxian. Both of the agents are good ultrasound contrast agents for quantitative analysis of myocardium blood flow.