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Colleen Cowan, Beatrix Kovas, Steven Feinstein, Matthew MacCumber, Shunbin Xu; Non-invasive, Targeted Gene Delivery to Retina and Retinal Endothelial Cells and Enhanced Live Retinal Imaging Using Contrast-enhanced Ultrasound Delivery System. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4980.
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Ultrasound-mediated microbubble destruction (UMD) is a new approach of non-invasive gene delivery. Microbubbles carrying DNA fragments will be injected to the blood; ultrasound application to the eyes may release DNA and help enter retinal endothelial cells and the retina. Additionally, microbubbles are also ideal ultrasound contrast agent, which enhances the contrast of ultrasound imaging of the retina. Therefore, this approach may improve non-invasive, site-specific delivery, and improve live imaging of the retina and ocular tissues. The purpose of this research is to test these hypotheses.
We performed tail-vein injection of microbubbles (OPTISON™, GE Medical Diagnostics) loaded with plasmid DNA of pCS2-Venus GFP (100 ug/200 ul) in young adult male Sprague-Dawley rats (n=6). 10 seconds after injection, diagnostic imaging ultrasound was performed using the Vevo2100 imaging system (VisualSonic). Subsequently, therapeutic ultrasound pulses were applied for ~1s duration 5 second intervals over the course of the two-minute infusion period. 24 hours later, we harvested the eyes to prepare cryosections for fluorescent microscopy.
1. Contrast-enhanced ultrasound increased the imaging intensity of the eye. Structures of eyes, including the retina, can be readily visualized.2. Contrast-enhanced ultrasound can detect ocular blood vessels, distinguishing arteries and veins and measuring the width of the vessels, and evaluate the status of blood flow, including direction and speed of the blood flow.3. Ocular therapeutic pulse did not increase gene delivery in the eyes.
1. Contrast-enhanced Ultrasound is a promising new method to evaluate the blood vessels and flow in different regions of the eyes. Therefore, this procedure may have important applications to studies of retinal diseases with vascular damages or dysfunctions, e.g. diabetic retinopathy, glaucoma and age-related macular degeneration (AMD).2. The Contrast-enhanced Ultrasound Delivery System did not help introduce plasmid DNA into retinal cells. However, because of the limited funding, we couldn’t afford to repeat and make improvement on the methodology, or to verify this negative result. Therefore, we do not exclude the possibility that modification of this protocol may help gene delivery, including small RNA molecules to the retina, as showed in other system.
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