Vinod P. Gaur

RPE conditioned medium stimulates photoreceptor cell survival, neurite outgrowth and differentiation in vitro.

In the present study we have investigated retinal pigment epithelium-photoreceptor cell interactions in vitro, and their contributions to photoreceptor cell survival and differentiation. Preparations enriched for intact photoreceptor cells from neonatal rat retina were grown in either serum-free medium supplemented with RPE-conditioned medium (RPE-CM) or in serum-free medium alone. A variety of substrate conditions were tested for the best neurite outgrowth. Cultures were monitored for 7 days by light and electron microscopy, as well as by opsin, vimentin and carbonic anhydrase-C immunocytochemistry. RPE-CM was found to stimulate both proliferation of flat cells and photoreceptor differentiation. The number of photoreceptors bearing neurites and their neurite length measurements showed significant differences between the RPE-CM group and the control group within 20 hr in culture. Elimination of contaminating flat cells by the addition of an antimitotic drug prevented photoreceptor cell morphological maturation; however, these cells survived as round cell bodies without processes for at least 10 days in the presence of RPE-CM and expressed opsin during this period. Conditioned medium from the flat-cell monolayers did not support photoreceptor differentiation or their survival. However, the presence of flat cells was a requisite to achieve any neurite outgrowth even in the presence of RPE-CM. In the absence of RPE-CM, neither photoreceptors nor flat cells survived or proliferated. Heat and trypsin treatment of the RPE-CM abolished all its growth-supporting activities which indicates its proteinaceous nature. This represents the first time in vitro that an RPE-derived factor(s) has been shown to be responsible for photoreceptor cell survival and differentiation.

Transplantation to the diseased and damaged retina

Retinas of Royal College of Surgeons (RCS) dystrophic rats undergo a dramatic loss of photoreceptor cells as a result of defective retinal pigment epithelial (RPE) cells. These retinas are therefore a valuable model in the investigation of the role of the RPE on photoreceptor-cell survival and development. Also, rat retinas damaged by excessive light serve as a suitable environment to study survival of transplanted photoreceptor cells. Even though photoreceptor cells are lost in these retinas, a normal inner retinal structure is retained. Both models have recently been used in successful RPE-cell and/or photoreceptor-cell transplantation studies designed to replace defective or lost cells due to retinal disease or damage. These new approaches in the field of retinal transplantation offer unique and novel opportunities for the development of possible therapeutic strategies in human eye disease, and for improving our understanding of the normal relationships between retinal cells.

Effects of macrophage and retinal pigment epithelial cell transplants on photoreceptor cell rescue in RCS rats.

The effects of macrophage transplants on photoreceptor cell survival in retinas of Royal College of Surgeons (RCS) dystrophic rats were contrasted with RPE-cell transplants, sham-injection and surgical controls. The effects of these different treatments on the thickness and total area of the outer nuclear layer (ONL) were evaluated by light and electron microscopy at 1, 2 and 5 months after transplantation or surgical manipulations. Macrophage transplants into dystrophic retinas, although significantly reducing the debris zone thickness (p less than 0.01), had little effect on photoreceptor cell survival (2-3 cells thick ONL) after two months. In contrast, two months after RPE-cell transplantation, retinas exhibited an 8-10 cell thick ONL. Also, inner and outer segments of rescued photoreceptor cells were present, especially in areas directly beneath RPE-cell transplants. At the same time period, retinas injected with saline had a 2-3 cell thick ONL with no organized inner or outer segments. Furthermore, the affected ONL area in macrophage-transplanted or saline-injected retinas was significantly smaller than that seen in RPE-cell transplanted retinas (p less than 0.0001). Surviving photoreceptor cells were found only in the RPE-cell transplanted retinas five months after treatment. No effect on photoreceptor cell survival was seen in saline-injected, needle-inserted or incision-only retinas. Thus, transplantation of healthy RPE cells is an effective long-term therapeutic approach to correct the genetic defect in retinas of RCS dystrophic rats.

Photoreceptor rescue in the dystrophic retina by transplantation of retinal pigment epithelium

Publisher Summary This chapter describes the effects of retinal pigment epithelial (RPE) cell transplants in the retinas of Royal College of Surgeons (RCS) dystrophic rats. The chapter describes the mechanism by which RPE cell transplants affect the survival of photoreceptor cells in dystrophic retinas and in vitro. Retinal cell transplantation and techniques are also presented in the chapter. Prior to and during the early stages of the study of RPE cell transplantation, retinas as whole or partial segments or as cell suspensions had been grafted into mammalian eyes. Various methods were employed to transplant donor retinal tissue—intact or as a cell suspension—into a host eye. Often the donor tissue was of embryonic or neonatal origin, while the host animals were adults. The anterior chamber in rats was used as a suitable environment for retinal tissue transplantation. Small pieces of retina from newborn rats were transplanted into a retinal lesion bordered by the vitreous using a transscleral approach. Photoreceptor cells as an intact layer survived for several weeks when transplanted into light-damaged and dystrophic retinas. Exposing adult rats to excess light destroyed most photoreceptor cells within a few weeks, while the inner retina remained relatively unchanged.

Maintenance of opsin and S-antigen gene expression in RCS dystrophic rats following RPE transplantation

The effects of retinal pigment epithelium (RPE) transplantation in rescuing photoreceptor cells (PRC) were studied in RCS rats bearing the rdy mutation. RPE preparations from Long-Evans rats were transplanted into the subretinal space of post-natal (P) 17-19-day-old RCS rats. Age-matched RCS animals in the control group received injections of the transplantation vehicle buffer alone. The animals were killed at various post-natal ages after RPE transplantation. Immunocytochemical studies using antibodies to opsin and S-antigen indicated intact photoreceptor cells in the transplanted retinas. In contrast age-matched sham-injected animals did not show any rescue of photoreceptor cells at P90. Single RPE transplantation of 60,000 cells in each eye resulted in restoration of near normal mRNA levels for opsin, and S-antigen proteins. Comparisons of mRNA levels of two visual proteins using cDNA probes demonstrated a therapeutic effect of RPE transplantation in preventing a progressive decline in mRNA levels due to retinal degeneration. In contrast, P109 sham-injected controls showed no detectable mRNA levels for these proteins. In vitro protein synthesis in RPE-transplanted retinas implied further competence of these retinas. These data suggest that RPE transplantation not only rescues photoreceptors from degeneration, but more importantly, it allows normal transcription and translation in these cells rendering them capable of participating in the visual and signal transduction cascade.

Ocular distribution of 70-kDa heat-shock protein in rats with normal and dystrophic retinas.

SummaryStress proteins are thought to play an important role in cellular development and in survival mechanisms. We compared the immunolocalization of the 70-kDa stress protein (SP70) in the ocular tissue of the normal Sprague-Dawley (SD) rat with that in the Royal College of Surgeons (RCS) rat with retinal dystrophy. SP70 was present in the maturing ocular tissues of both rat strains. However, once retinal degeneration began in the RCS rat, the retinal pigment epithelium and photoreceptor cells showed increased immunostaining for SP70 over that observed in age-matched SD rats. In late stages of retinal degeneration, immunostaining for SP70 was considerably reduced in the RCS retina, whereas normal distribution of immunostaining for SP70 in the SD retina was preserved, albeit decreased, through postnatal day 180. The optic nerve, ciliary body, and corneal epithelium were also influenced by the dystrophic disease condition, although the pattern of changes in SP70 immunostaining differed for each tissue. These results suggest that the genetic defect in the RCS rat produces a state of metabolic stress in all ocular tissues as the degeneration progresses, but that the subsequent rise in ocular SP70 is insufficient to prevent progression of the disease.

Cellular localization of somatostatin mRNA in rat retina

In an attempt to determine the localization of the messenger RNA (mRNA) encoding somatostatin in the rat retina, we studied Sprague-Dawley rats by in situ hybridization histochemistry using radiolabelled oligodeoxyribonucleotides complementary for rat somatostatin mRNA. Among the layers of retina, we found specific labelling in the soma of some cells in the innermost and outermost laminae of the inner nuclear layer and in the ganglion cell layer; no specific labelling was observed in the inner and outer plexiform layers or in the outer nuclear layer. These data indicate the major site of somatostain synthesis within the rat retina.

Development and distribution of opsin-like immunoreactivity in the dystrophic retinas of rdle mutant mice

The opsin-like immunoreactivity in the retinas of C57BL/6J rdle mutant mice has been studied by light-microscopic immunocytochemistry. Positively labeled cells were found in the normal heterozygous and homozygous mutant mouse outer nuclear layer (ONL) as early as postnatal day 3 (PN3). Beginning at PN10, in the retinas of the homozygous mutant mice, labeled photoreceptor cells rapidly decreased in number and disappeared after PN42. In the decreased ONL, remaining opsin-positive cells were labeled at higher density than those of controls. The retinal pigment epithelium was also moderately labeled during the loss of opsin-positive photoreceptor cells. In addition, sparse opsin-immunoreactive cells were demonstrated in the inner nuclear layer (INL) in the retinas of both the mutant and non-dystrophic mice as early as PN10 and are presumed to be ectopic photoreceptor cells. However, these displaced photoreceptor cells disappeared by PN28 in mutants along the same time course as those in the ONL but were still present in the PN28 retina of controls and seemed to be more abundant at later adult ages. There was no difference in the developmental regulation of opsin in the heterozygous and normal controls.