Purpose. To determine whether RPE transplantation is successful, it is necessary to distinguish donor RPE from host RPE unequivocally after surgery. We investigated whether a modified Barr body staining of plastic-embedded sections can be used to identify donor RPE transplanted into the subretinal space of opposite-gender pigs. Methods. Primary porcine RPE sheets were harvested from 27 adult domestic pigs using Dispase and transplanted to the subretinal space of opposite-gender pigs using pars plana vitrectomy techniques. Eyes were enucleated 1-30 days later, fixed in 2.5% gluteraldehyde & 2% paraformaldehyde, postfixed in osmium, embedded in plastic (EM-Bed 812) and serially sectioned until the graft was visualized. Unstained sections were deplasticized and stained using a modification of Guard's sex chromatin staining method- Histologie sections of retina, RPE and choroid from female and male pigs served as positive and negative controls, respectively. Result. Barr bodies, visible as a red inclusions in the periphery of the RPE nucleus, were visible in 58.0 ± 11.3 % and 0 % of RPE cells seen in 1 micron sections harvested from female and male pigs, respectively. Ban body staining was sufficient to distinguish transplanted RPE from native RPE in all specimens. Conclusions. Barr body staining of deplasticized specimens is a simple, accurate and inexpensive technique to distinguish donor RPE from host RPE after RPE transplantation. This technique is preferable to the use of melanin and other cytoplasmtc or membrane markers to identify transplanted cells, since these markers may be unreliable if donor cell fragments are incorporated into host RPE by phagocytosis. Supported in part by grants from the Foundation Fighting Blindness, NIH grants EY10311 and EY02687, and unrestricted funds from Research to Prevent Blindness. None.
|Original language||English (US)|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Dec 1 1997|
All Science Journal Classification (ASJC) codes
- Sensory Systems
- Cellular and Molecular Neuroscience