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I. Perlman, L. Bilgoray, B. Miller, R. Heinrich; Testing the Spread of Degeneration From Affected Photoreceptors to Non-Affected Ones. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5575.
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In retinitis pigmentosa (RP), there is a spread of degeneration from affected rods to healthy cones. We created a rat model of RP test the mechanisms underlying the spread of degeneration from affected photoreceptors to non-affected ones.
Two types of adeno-associated viral vector (AAVs) were constructed. (1) The plasmid pAAV-IRES-hrGFP used as a transfer vector to create an AAV with no transgene for control studies. (2) Mutated (P23H) rhodopsin gene was cloned into the pAAV-IRES-hrGFP plasmid for the experimental group. Albino rats were injected subretinally in one eye with one of the above viral vectors. The electroretinogram (ERG) was recorded monthly to assess deterioration of retinal function. Rats were sacrificed at different time intervals after the injection (one to nine months). Fluorescence analysis using confocal microscopy was used to detect GFP and localize the infected region. TUNEL was used to identify cells undergoing apoptosis. Labeling with anti GFAP, which usually expressed by Muller cells only in response to stress, was performed.
With subretinal injection only a limited retinal area is exposed to the injected AAVs. Both a-wave and b-wave in pAAV-P23H-IRES-hrGFP injected eyes deteriorated gradually as degeneration spreads across the retina and reached a stable level four months post injections. At the pAAV-IRES-hrGFP (control) injected eyes there was no significant reduction in the ERG recordings. TUNEL assay showed a positive staining from three months post injection at the pAAV-P23H-IRES-hrGFP injected eyes. There was no positive staining in the control infected eyes. At the pAAV-P23H-IRES-hrGFP infected eyes there was an extended staining of GFAP far beyond the infected area, comparing to control injected eyes in which there was a positive staining only at the injected area.
These findings indicate that we succeeded in creating a rat model for studying the spread of degeneration from affected photoreceptors to non-affected ones.
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