Purchase this article with an account.
C Nathaniel Roybal, Cindy X. Ruan, Kendal Kernstein, Chinatsu Tosha, Dean Bok, Steven Nusinowitz, Gabriel H. Travis; A Novel Approach To RPE Transplant In Mouse Models. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4022. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Atrophic age-related macular degeneration (AMD) is thought to involve the retinal pigment epithelium (RPE) initially with secondary involvement of photoreceptors. Replacement of degenerating RPE cells is a potentially promising therapeutic strategy for this disease. We and others are working to develop this strategy using mouse models of AMD. Requirements for testing this strategy in mice include the ability to deliver cells into the subretinal space with minimal damage to the retina, to quantitate survival of the RPE graft, and to measure RPE-cell function. Here we report our progress toward transplantation and functional analysis of RPE cells in mice.
We employed a high-definition camera to visualize the fundus during trans-scleral injection of cells into the subretinal space. Wild-type BALB/c and rpe65-/- mice, in some cases following administration of sodium iodate, were used as transplant recipients. Placement of cells into the subretinal space and the subsequent return to normal retinal anatomy were ascertained in vivo by optical-coherence tomography (OCT) and fundus photography. RPE-cell survival and integration were measured by quantitative genomic PCR (qPCR) and retinal histology. RPE function was assessed in vivo by electroretinography and in isolated retinas by liquid chromatographic analysis of visual chromophore.
The visualized-injection approach allowed for reproducible subretinal RPE cell transplant in 100/138 trials (72%). Light damage experiments confirmed minimal photolytic damage by the imaging system. Return to normal retinal anatomy occurred within three days after transplantation by OCT. Light microscopy documented integration of mouse RPE cells into the native RPE monolayer. Electroretinography and chromophore analysis have demonstrated a post-transplant increase in b-wave amplitudes with correlating increases in 11-cis-retinaldehyde in rpe65-/- mice. Utilizing differences in the rpe65 gene between host and graft, we quantitated survival of the transplanted RPE cells by qPCR with sensitivities to 200 transplant cells per eye.
The above-described approach yields reproducible transplantation of RPE cells into the mouse subretinal space with minimal trauma to the retina. We have also presented methods to quantitate RPE integration and evaluate transplanted RPE function. These methods will be useful in developing an RPE transplantation approach to treat AMD.
This PDF is available to Subscribers Only