Harold J. Sheedlo

Functional and structural characteristics of photoreceptor cells rescued in RPE-cell grafted retinas of RCS dystrophic rats

In this study retinas of pink-eyed 26 day-old RCS dystrophic rats were injected with normal, melanotic retinal pigment epithelial (RPE) cells into the subretinal space. The areas containing underlying photoreceptor cells were subsequently investigated by electron microscopy at 60 days and light microscopy immunocytochemistry at 60 and 120 days. Retinal regions containing viable RPE-cell grafts exhibited melanin-contained RPE cell transplants attached to Bruchs membrane and containing shed rod outer segments (ROS). Also, the ROS of these photoreceptor cells exhibited a normal-appearing structural relationship with apical membrane projections of these RPE cells. The debris zone, prominent in retinas of 60 day-old RCS dystrophic rats, was much reduced in RPE-cell grafted regions. By at least 3 months after RPE transplantation, immunostaining for Na+,K+-ATPase was demonstrated along inner segments (IS) of photoreceptor cells in grafted regions, as was also shown in control retinas, but not in non-grafted regions. Opsin immunostaining in RPE-cell grafted regions and in control retinas was detected along ROS and rod inner segments (RIS) and at the periphery of cell bodies in the outer nuclear layer. This study has shown that transplanted normal RPE cells in retinas of RCS dystrophic rats appear to have normal functional and structural characteristics at least three months after transplantation. Furthermore, the rescued PRCs exhibited a normal distribution of and immunostaining density for the membrane-bound enzyme Na+,K+-ATPase and the photo-pigment opsin.

Chapter 20 Long-term rescue of photoreceptor cells in the retinas of RCS dystrophic rats by RPE transplants

Publisher Summary This chapter discusses the effects of retinal pigment epithelial (RPE)-cell transplants in the retinas of Royal College of Surgeon (RCS) dystrophic rats that are stable for at least 6 months after transplantation. Studies are currently in progress that will determine if these transplants continue to affect photoreceptor cells (PRC) survival 1 year after transplantation. A long-term stability of RPE-cell transplants is very important, because ideally this surgical technique and subsequent RPE-cell injection should be performed once, thus limiting trauma and injury to the host eye. Grafted RPE cells have been frequently found interposed between host RPE, along Bruchs membrane, and also attached to the apical surface of host RPE cells. It is also important that rescued PRCs continue to synthesize and transport proteins that are essential in the proper functioning of this cell. In this regard, detecting immunocytochemically the membrane-bound enzyme (Na + + K+)—ATPase—and the photopigment opsin in their normal cellular sites provide evidence that rescued PRCs properly expressed these two proteins. The RPE-cell transplants in dystrophic retinas may stabilize cells of the intermediate nepheloid layer (INL), which is indicated by the increased INL thickness when compared to nongrafted dystrophic retinas. The transplanted RPE cells in the retinas of 6-month-old RCS dystrophic rats appear to be functioning normally as these cells actively phagocytize shed ROS and their microvilli are interposed between and contact rod outer segments (ROS).

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.

Effects of RPE-cell factors secreted from permselective fibers on retinal cells in vitro

This study was undertaken to determine if retinal pigment epithelial (RPE) cells encased in permselective hollow fibers survive in a tissue culture environment and secrete a diffusible trophic factor(s) that may affect retinal cell survival in vitro. In this study, RPE cells were isolated from 6- to 8-day-old Long-Evans rats, then loaded into hollow fibers. The RPE-cell fibers were then cultured for at least one week in serum-containing medium. These RPE-cell fibers were subsequently co-cultured with cells isolated from retinas of day 2 Long-Evans rats in a defined medium. For at least 6 days in culture, opsin-positive cells were observed on the surface of larger flat cells. Over 80% of the small, round cells immunostained for opsin. However, opsin-immunostained cells were seldom seen in cultures with control fibers, that lacked RPE cells. In addition, conditioned medium collected from either the RPE-cell fibers or cultured RPE cells affected survival of opsin-positive retinal cells in culture in a manner similar to that of the RPE-cell fibers. Furthermore, selected growth factors such as epidermal, nerve and fibroblast growth factors, were unable to sustain retinal cell survival and affect morphological development as seen in RPE-CM supplemented cultures. In vivo companion developmental studies demonstrated that few opsin-positive cell bodies were observed in retinas of day 2 Long-Evans rats, the age corresponding to the stage of retinal cell isolation. In retinas of day 5 Long-Evans rats, the age corresponding to the end point of the in vitro assay, a dramatic increase in the number of opsin-immunostained cell bodies was noted, which corresponds to the developmental sequence also seen in culture. Light and electron microscopic examination revealed that the RPE cells cultured in the hollow tubes maintained an RPE-like structure for several months, in that these cells contained melanosomes and extended microvilli from their apical border and formed junctional complexes with adjacent cells. Results of this study confirm our earlier findings that RPE cells secrete an apparent novel factor(s) that affects retinal cell survival in vitro and, most significantly, the described encapsulation/secretion mechanism may provide a convenient method to deliver such factors for further in vivo testing of this phenomenon.

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.

(Na+ + K+)-ATPase and opsin in retinas of RCS dystrophic rats: time course study

Retinas of Royal College of Surgeons (RCS) dystrophic rats were investigated immunocytochemically for the distribution of the membrane-bound enzyme (Na+ + K+)-ATPase and the photo-pigment opsin prior to and during the retinal disease process. Retinas of 11 day-old dystrophic and control Long Evans rats showed (Na+ + K+)-ATPase immunostain most dense in the outer nuclear layer (ONL) and inner plexiform layer (IPL). Also, in these retinas, immunostaining for opsin was dense along rod inner segments (RIS) and on plasmalemma of ONL cell bodies and several cell bodies in the inner nuclear layer (INL). In retinas of control rats at 30 days and later, less dense (Na+ + K+)-ATPase immunostain was detected in the ONL than at 11 days and opsin-immunostained ROS were also detected. However, (Na+ + K+)-ATPase immunostained RIS were shorter in retinas of 30 day-old dystrophic than in retinas of age-matched control rats and an opsin-immunostained debris zone was also observed in dystrophic retinas. In retinas of 60 day-old dystrophic rats, the opsin-immunostained debris zone was more prominent than at 30 days, while the few ONL cell bodies immunostained for both proteins. Also, the outer IPL of 60 day-old dystrophic rat retinas immunostained more densely for (Na+ + K+)-ATPase than the inner IPL. In 120 day-old dystrophic rat retinas, (Na+ + K+)-ATPase immunostain was detected in the INL and IPL, while opsin staining was demonstrated only in the debris zone. This opsin-immunostained debris disappeared in a central-to-peripheral gradient. (Na+ + K+)-ATPase immunostain was still present in the INL and IPL in retinas and the optic nerve of one year-old RCS dystrophic rats; however, opsin was restricted to a few surviving cell bodies in the peripheral retina.

Ciliary body degeneration in the royal college of surgeons dystrophic rat

We have studied the pathological changes of the ciliary body in Royal College of Surgeons (RCS) rats with an inherited retinal degeneration. Morphometric analyses were performed on sectioned ciliary bodies by a computerized morphometry system. Age-matched non-pigmented Sprague-Dawley (SD) rats were used as the control animals. The ciliary body of 26-day-old RCS dystrophic rats showed normal structure. However, the length and height of the pars plicata of the ciliary body became shorter and the area became smaller with increased age. Significant decreases in the values of these three parameters were observed between 26-day-old and 3-month-old RCS dystrophic rats. These parameters also showed significant differences when values of 3-month-old RCS dystrophic rats were compared to those of 3-month-old control SD rats. The same trends were observed in the ciliary body measurements in RCS dystrophic rats up to 1 year of age. Scanning electron microscopic examination demonstrated the progressive thinning of the pars plicata of the ciliary body with age in the RCS dystrophic rats. The total volume of the ciliary process of 6-month-old RCS dystrophic rats appeared to be one-half that of 26-day-old RCS dystrophic rats. Transmission electron microscopy revealed progressive cellular degenerative changes in the non-pigmented and pigmented ciliary epithelium of the RCS dystrophic rats. It was apparent that the pigmented ciliary epithelium had more severe degenerative changes than the non-pigmented ciliary epithelium. Immunostaining for Na+ + K+ ATPase of the ciliary epithelium was found to be less in the RCS dystrophic rats than in age-matched controls. This result suggests a possible dysfunction of ion transport in the ciliary body of the RCS dystrophic rats, which may account for their increased incidence of cataract formation. Although the mechanisms for the ciliary body degeneration in RCS dystrophic rats remain speculative, these findings add a new area of interest in this model of inherited retinal dystrophy.

Immunocytochemical localization of (Na+ + K+)-ATPase in the rat hippocampus

The adult rat hippocampus was investigated by light microscopic immunocytochemistry for (Na+ + K+)-ATPase. In the CA1, CA2 and CA3 hippocampal regions, dense immunostaining for (Na+ + K+)-ATPase, exhibiting a punctate appearance, was demonstrated along the soma plasmalemma of hippocampal pyramidal cells in the stratum pyramidale, thus outlining these cells distinctly, and along dendrites extending into the stratum radiatum. (Na+ + K+)-ATPase immunostaining was dense in the neuropil of the strata oriens and radiatum of the rat hippocampus, but much lighter in the corpus callosum. Immunostaining at the periphery of pyramidal cell soma may be associated with the plexus formed by axon terminals of hippocampal basket cells.