Photoreceptor Cell Rescue at Early and Late RPE-Cell Transplantation Periods During Retinal Disease in RCS Dystrophic Rats

Maximal PRC rescue was affected by RPE-cell transplantation in retinas of RCS dystrophic rats at early stages of the retinal disease, while little or no rescue was detected when transplantation was performed at late time periods.

months the outer nuclear layer (ONL) was [8][9][10] cells in thickness as shown in retinas of agematched control rats. Of these transplantation times, day 17 appeared to affect the best rescue of photoreceptor cells. Nongrafted retinas of 4 month-old RCS dystrophic rats exhibited scattered PRC's, most prevalent in the peripheral retina. In addition, a small, but significant increase in the ONL thickness was detected in vehicle-injected retinas (sham control) of 17 day-old RCS dystrophic rats at 2 months; however, at 3 months, the ONL thickness was reduced to control levels. A normal distribution of (Na + + K/)-ATPase immunostain was demonstrated beneath grafted RPE cells in retinas of 4 month-old RCS dystrophic rats. Dense immunostaining was shown along rescued photoreceptor cell inner segments (IS), within the inner (IPL) and outer (OPL) plexiform layers and on plasmalemma of cell bodies in the inner nuclear layer (INL). In nongrafted retinas of age-matched RCS dystrophic rats, immunostaining for (Na + + K+)-ATPase was observed only in the INL and IPL.
Under RPE-cell transplants in retinas of 4 month-old RCS dystrophic rats, opsin immunostaining was detected along both rescued photoreceptor cell inner and outer (OS) segments and on plasmalemma of ONL cell bodies. However, immunostaining for opsin was restricted to a debris zone in nongrafted retinas of age-matched RCS dystrophic rats. In contrast to PRC rescue resulting from early stage transplantation at  The purpose of this study was to determine by microscopic and immunocytochemical methods the effects of RPE-cell transplantation in retinas of RCS dystrophic rats at early (prior to PRC degeneration) and late (during the period of advanced PRC degeneration) stages of the retinal disease process. Specifically, we wanted to know how far the disease process would progress before normal RPE-cell transplantation had little or no therapeutic value in regard to photoreceptor cell rescue.

Animals
Eyes of Long Evans rat pups between 8-10 days of age were the source of normal RPE cells. Isolated pigmented RPE cells were injected into the retinas of pink-eyed RCS dystrophic rats at 10, 17, 26, 39, 43, 61 and 68 days and retinas were examined when these rats were 2 to 6 months of age. Control animals were agematched nongrafted RCS dystrophic, pigmented Long Evans, nonpigmented Sprague-Dawley rats and vehicle-injected (sham control) RCS dystrophic rats.

RPE Cell Isolation and Transplantation
RPE cells were isolated from Long Evans rat pups using a previously described procedure /6,11L The cells were concentrated to 60,000 cells//zl for transplantation. The isolated RPE cells were injected into the subretinal space of RCS dystrophic rats using a dorsal lesion method /6,15/. Briefly, the dorsal surface of the eye was exposed, then a small lesion was made through the sclera, choroid and Bruch's membrane between the two vorticose veins. The cells were injected into the retina with a 32 gauge bluntedged needle at the lateral edge of the lesion. As sham controls, retinas of age-matched dystrophic rats were injected with  ONL thicknesses and total rescued ONL area were measured using JAVA software and an IBM computer. Any statistical differences of corresponding ONL measurements were determined by the students t-test.

Immunocytochemistry
The immunocytochemical method followed in this study has been described previously/13,14/. Briefly, deparaffinized sections were treated with 20% normal goat or rabbit serum prior to incubation with primary rabbit antibovine brain (Na + + K+)-ATPase /4/ or sheep antibovine opsin /12/antisera, respectively. Sections were then incubated in the appropriate secondary antibody conjugated to horseradish peroxidase (F(ab')2-HRP), then treated with the chromogen diaminobenzidine in Tris buffer. Finally, sections were rehydrated, cleared in xylene, mounted in Permount, then examined with a Zeiss Universal light microscope using Nomarski optics.

RESULTS
The outer nuclear layer (ONL) in retinas of 4 month-old nonpigmented Sprague-Dawley rats was about 10 cells in thickness (Fig. 1A). Retinas of 4 month-old RCS dystrophic rats transplanted with RPE cells at 17 days exhibited an ONL of 8-10 cells in thickness. Also, outer (OS) and inner (IS) segments were observed beneath grafted pigmented RPE cells (Fig. 1B). However, in nongrafted retinas of age-matched dystrophic rats, few photoreceptors were detected at 4 months (Fig. 1C). Immunostaining for (Na / + K/)-ATPase under RPE-cell transplants was dense along IS, in the inner (IPL) and outer (OPL) plexiform layers and on plasmalemma of rescued photoreceptor cells and inner nuclear layer (INL) cell bodies (Fig. 1D). Immunostaining in respective regions of nongrafted retinas of 4 month-old dystrophic rats was still detected within the IPL and INL (Fig. 1E). Also, beneath RPE-cell grafts, opsin immunostaining was observed along OS, IS and on the plasmalemma of rescued photoreceptor cells (Fig. 1F), while only the debris zone immunostained for opsin in corresponding regions of nongrafted retinas (Fig. 1G).
Examination of retinas of 4 month-old RCS dystrophic rats transplanted with RPE cells at 10 days revealed somewhat similar PRC rescue as shown for the 17 day transplantations. The distribution of (Na / + K/)-ATPase beneath the RPE-cell graft was shown along IS, in the plexiform layers and surrounding_ INL cell bodies. Also, the immunostained ONL was almost as thick as observed at the 17 day grafting period and (Na / + K /)-ATPase immunostained IS were dearly demonstrated ( Fig. 2A) Photoreceptor cell rescue was clearly shown lateral to RPE-eell transplants and beneath membrane debris in 4 month-old RCS dystrophic rats grafted at 26 days (Fig. 4A). EM examination of these retinas revealed grafted pigmented RPE cells attached to Bruch's membrane (Fig. 4B). Also, rescued segmentbearing photoreceptor cells under the RPE-cell graft showed a normal-appearing ultrastructure (Fig. 4C). As shown in Figure 5, RPE-cell transplantation in retinas of RCS dystrophic rats at 17 days caused rescue of photoreceptor cells, such that at 4 months the ONL thicknesses approximated that of control retinas of agematched Sprague-Dawley rats. ONL thicknesses in RPE-cell transplanted dystrophic retinas at postnatal days 10 and 26 when examined at 4 months were slightly, but significantly, decreased from retinas of both age-matched control retinas and 17 day transplanted dystrophic retinas. RPEcell transplantation at 39 days resulted in no significant photoreceptor cell rescue when retinas were examined about 3 months after grafting (at 4 months). Injection of vehicle (sham control) into retinas of 17 day-old RCS dystrophic rats appeared to have a slight beneficial effect on photoreceptor cells about one month after treatment, but 2 months after the injection, the ONL thicknesses were reduced to control dystrophic retina levels and was confined only to the area of the needle track. The reason for this transitory effect on PRC's by vehicle-injection in retinas of RCS dystrophic rats remains unknown. This phenomenon is currently under investigation in this laboratory.
In conclusion, RPE cell transplantation into retinas of RCS dystrophic rats at early, but not late, stages of retinal degeneration caused extensive photoreceptor cell rescue. From these studies it appears that the optimum period for RPE-cell transplantation in the retinas of RCS dystrophic rats to achieve maximal photoreceptor cell rescue is at approximately postnatal day 17. RPE-cell transplantation in retinas of 10 day-old RCS dystrophic rats may have caused some trauma to the small eye, thus resulting in a lessened effect on photoreceptor cell survival than was shown at 17 days. Also, photoreceptor cells in retinas of 26 day-old RCS dystrophic rats have already begun to degenerate, which may explain the reduced effect of RPE-cell transplantation when compared to the 17 day transplants. These and earlier studies/7,9,15/are the first demonstrations of the rescue of neurons by a transplantation procedure that would normally die due to an inherited disease process.