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W. Stamer, D.W. Chan, C.R. Ethier; Targeted Viral Delivery to Schlemm’s Canal by Retroperfusion in Organ Culture . Invest. Ophthalmol. Vis. Sci. 2006;47(13):220.
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The ability to deliver agents directly to the lumen of Schlemm’s canal (SC) provides a useful experimental tool to study conventional drainage function. In enucleated human and bovine eyes this was previously accomplished using retroperfusion. Our goal was to extend this technique to the perfused human anterior segment model, thereby enabling targeted delivery in long–term experiments such as those involving virally mediated transgene expression.
Anterior segments from fresh post mortem human eyes (n=9) were prepared for organ culture using standard techniques and were perfused at constant flow (2.5 µl/min) while recording facility. After reaching a stable facility within physiological limits, a small plastic strip sealed with grease on its lower edge was placed into the clamping ring of the perfusion dish to make a fluid–tight fence encircling the limbus. Media containing an adenoviral construct expressing the lacZ reporter gene under control of the CMV promoter (6x106 pfu/ml) was loaded into this fence to submerge the limbus and IOP was lowered to –1 mmHg for 30–60 min, then maintained at 0 mmHg for 40–60 min. During the zero time pressure interval, IOP was occasionally varied ±1 mmHg for 15 sec intervals to promote mixing in SC. In some eyes, the eye was also placed on a small inclined plane and rotated through 90 degrees every 15 min. After retroperfusion, conventional (forward) perfusion was restarted and continued for 5–7 days, after which anterior segments were fixed and processed for visualization of lacZ activity.
Retroperfusion with adenovirus containing reporter gene did not affect outflow facility. Gross examination of outflow tissues showed patchy, fragmented distribution of lacZ activity in SC, except in segments that were put on an inclined plane and rotated, where the distribution of lacZ activity was more uniform. Sagittal histologic sections showed lacZ activity in SC endothelial cells as well as the outermost juxtacanalicular cells.
Retroperfusion of adenovirus into cultured human anterior segments successfully delivered a reporter gene to the lumen of Schlemm’s canal. This technique enables the delivery of agents that are removed from the perfusion media by uveal and corneoscleral meshwork cells during conventional (forward) perfusion, or for any approach where targeting of Schlemm’s canal endothelial cells in organ culture is desired.
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