Experimental Study Of Ultrasound Microbubble-Mediated Gene Therapy For Lower Limb Vascular Occlusion

PartⅠ PREPARATION OF ALBUMIN ULTRASOUND CONTRAST AGENT AND ENHANCEMENT OF ULTRASOUND IMAGING ON RABBIT KIDNEY Objective: To develop the technique of preparation of albumin ultrasound contrast agent, and to investigate the efficiency and safety of this contrast agent for rabbit kidney imaging. Methods : Effects of different solutions, sonication time and ultrasound intensity on microbubbles were evaluated by orthogonal method. The concentration and diameter of the microbubbles were measured. Before and after intravenous injection of the microbubbles(0.05 ml/kg), The kidneys of ten rabbits were imaged with two-dimensional grey scale, color Doppler and power Doppler imaging system. Results: Concentration and diameter of microbubbles were significantly different among different intensities, time and solutions. 5% albumin mixed with 5% dextrose (1:4), and 80s of sonication time(Sonicator XL2020,output grade 9) were the ideal condition for preparation of the contrast agent. In vivo experiment showed that grey ultrasound contrast agent; albumin; microbubble;kidney ultrasound; microbubble;plasmid; skeletal muscleultrasound; microbubble; β-galactosidaseMethods:Thirty rabbits with left femoral artery ligation were randomly divided into 3 groups: the plasmid pcDNA3.1/VEGF165 local intramuscular injection group(group A), plasmid and microbubble mixture local intramuscular injection plus 1MHz, 2.0W/cm2 ultrasound local irradiation group(group B), and the control group (group C). Changes of blood flow in the internal iliac artery (IIA) and changes of Doppler power of the skeletal muscles after contrast were measured by ultrasonography 1 and 28 days later. In order to evaluate the collateral circulation and angiogenesis in treated regions, Digital subtraction angiography(DSA), immunohistochemical staining of VEGF and factor Ⅷ were performed and microvascular density(MVD) was counted 28 days later. Results:One day later, no significant difference in the blood flow of the internal iliac artery (P>0.05) and power Doppler imaging among the groups was observed,while twenty-eight days later, there was significant difference in the blood flow of the internal iliac artery (P<0.05) and power Doppler imaging between groups. DSA showed that collateral circulation in group B was more than that in group A and C. Immunohistochemical staining showed that the expression of VEGFand factor Ⅷ in group B was higher than that in group A and C. The MVD was significant different between groups(P<0.05). It was the highest in ultrasound-mediated microbubble destruction group. Conclusion: Microbubble-mediated VEGF165 plasmid therapy could promote new vessels growth and collateral circulation development in ischemic skeletal muscles. This provides a new method for the treatment of limb ischemia diseases.