The content of rhodopsin in the eyes of 15 donors (30 eyes) was determined. Both retinal and pigment epithelial fractions were collected from each globe, extracted using 1% CTAB, and the rhodopsin difference spectrum of each fraction was obtained separately. The total amount of rhodopsin, obtained by summing the amounts recovered from the retinal and PE fractions, ranged from 2.00 to 11.94 (median: 6.40) nmoles/eye. Previously reported mean values of about 3.5 to 4.0 nmoles per retina have been obtained using a variety of methods. The present higher values, perhaps largely dependent on procedural details described herein, appear plausible given the known concentrations of rhodopsin in rod outer segments, rod outer segment volumes, and number of rods in the human retina.
BackgroundThe current method of delivering gene replacement to the posterior segment of the eye involves a three-port pars plana vitrectomy followed by injection of the agent through a 37-gauge cannula, which is potentially wrought with retinal complications. In this paper we investigate the safety and efficacy of delivering adeno-associated viral (AAV) vector to the suprachoroidal space using an ab externo approach that utilizes an illuminated microcatheter.Methods6 New Zealand White rabbits and 2 Dutch Belted rabbits were used to evaluate the ab externo delivery method. sc-AAV5-smCBA-hGFP vector was delivered into the suprachoroidal space using an illuminated iTrackTM 250A microcatheter. Six weeks after surgery, the rabbits were sacrificed and their eyes evaluated for AAV transfection using immunofluorescent antibody staining of GFP.ResultsImmunostaining of sectioned and whole-mounted eyes demonstrated robust transfection in all treated eyes, with no fluorescence in untreated control eyes. Transfection occurred diffusely and involved both the choroid and the retina. No apparent adverse effects caused by either the viral vector or the procedure itself could be seen either clinically or histologically.ConclusionsThe ab externo method of delivery using a microcatheter was successful in safely and effectively delivering a gene therapy agent to the suprachoroidal space. This method presents a less invasive alternative to the current method of virally vectored gene delivery.
The use of detomidine hydrochloride sedation and the DTL microfiber electrode allowed the recording of good quality ERGs. This protocol should permit the detection of functional problems in the retina without the risk involved with general anesthesia.
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