Hild Kjeldbye

Retinal Degeneration in Mice Lacking the γ Subunit of the Rod cGMP Phosphodiesterase

The retinal cyclic guanosine 3′,5′-monophosphate (cGMP) phosphodiesterase (PDE) is a key regulator of phototransduction in the vertebrate visual system. PDE consists of a catalytic core of α and β subunits associated with two inhibitory γ subunits. A gene-targeting approach was used to disrupt the mouse PDEγ gene. This mutation resulted in a rapid retinal degeneration resembling human retinitis pigmentosa. In homozygous mutant mice, reduced rather than increased PDE activity was apparent; the PDEαβ dimer was formed but lacked hydrolytic activity. Thus, the inhibitory γ subunit appears to be necessary for integrity of the photoreceptors and expression of PDE activity in vivo.

Transplantation of cultured human retinal epithelium to Bruch's membrane of the owl monkey's eye

Human retinal epithelial cells dividing in culture have been radiolabelled with tritiated thymidine and subsequently transplanted and identified on Bruchs membrane of owl monkey by autoradiography. Such cells have been followed from 2 hours to 7 days after transplantation. The transplant cells reattach within 2 hours to the basement membrane left on Bruchs membrane after the host epithelial cells have been removed by suction after trypsinization. Within 6 to 24 hours they form a layer on Bruchs membrane with junctional complexes between cells and an apical-basal polarity characteristic of such cells in culture or after retinal detachment. After 2 to 7 days multilayers form by continued mitosis but no transplant cells invade the choroid as long as Bruch membrane is intact. Within this time macrophages begin to appear in the choriocapillaris under the transplant. These cells migrate through Bruchs membrane and between the transplant cells but have not been seen phagocytizing transplant cells. Reattachment of the neural retina to the transplant layer has not been attempted but seems technically possible.

The effects of bFGF on RCS rat eyes

Basic fibroblast growth factor (bFGF) has been implicated as a factor in retinal differentiation and disease. Recent studies have shown that subretinal or intravitreal injections of bFGF delay the retinal degeneration of the RCS rat but the global nature of this effect has been quantified for few test animals and the mechanism underlying this effect is not understood. In order to determine more accurately the global effects of intravitreal bFGF and to further elucidate the mechanism of bFGF promoted photoreceptor cell saving, we injected one of three bFGF doses into the vitreal cavities of young RCS rats. Using measurements from several eyes, we confirmed that a single intravitreal bFGF injection globally delays the RCS dystrophy. Test eyes contained fewer debris zone macrophages and more inner retinal macrophages than did control eyes at 1 month post injection. As bFGFs saving effect waned, the number of inner retinal macrophages decreased and the number of debris zone macrophages increased toward control levels. Dose-dependent cataract formation occurred in 100% of test eyes. Eyes that received the highest bFGF dose showed increased retinal vascularization at 1, 2 and 3 months post injection. The possible relationships between bFGF promoted photoreceptor survival and our findings are discussed.

Tolerance of human fetal retinal pigment epithelium xenografts in monkey retina

Abstract• Background: RPE transplantation offers the possibility of treating certain forms of retinal degeneration. Understanding how to optimize the surgical technique for performing RPE transplantation, especially in primates, is therefore of considerable interest. • Methods: Fifteen patch RPE transplants were performed in six monkeys. The transplant sites were examined at follow-up by ophthalmoscopy, biomicroscopy, fluorescein angiography and histology. Foveal and peripheral retinal transplants were compared. • Results: Human fetal RPE xenografts can survive without rejection for at least 6 months after transplantation in monkey retina. Such grafts form a basal lamina and make intimate contacts with the outer segments of the host. Both rods and cones retain a normal appearance when in contact with unrejected transplants. Rejection occurred in only 30% (3/10) of the peripheral but in 60% (3/5) of the foveal transplants. • Conclusions: Cultured human fetal RPE patch transplants can survive and maintain local photoreceptor integrity for relatively long periods of time in monkey subretinal space without immunosuppression. Rejection, when it occurs, is more frequent near the fovea.

Patch culturing and transfer of human fetal retinal epithelium

Human retinal pigmented epithelium (RPE) can be cultured by removing small patches of this layer from the choroid of the fetal eye. Such RPE patches give rise to healthy, epithelioid monolayers in vitro within 1–2 weeks without contamination from retinal or choroidal cells. The viability and proliferative capacity of these cultures is independent of the initial polarity of the patch. These RPE monolayers develop apical/basal polarity and a basal lamina and rest on a field of collagen fibers; they are capable of phagocytizing outer segments. A patch can be lifted off a confluent monolayer and transferred to another culture dish without risking the viability of either the old or the new culture. This provides a means of transplanting an organized, polarized patch of human RPE from one place to another.

The ultrastructure of transplanted rabbit retinal epithelium

Retinal epithelial cells from pigmented rabbits have been cultured and radiolabelled during division with 3H-thymidine and transplanted to the subretinal space of albino rabbits using a pars plana transvitreal approach. The host rabbits were maintained on cyclosporine immunosuppression after transplantation surgery. The transplant cells survive and maintain a morphologically normal appearance for at least 5 months. The pigmented epithelial cells retain their pigmentation and become integrated with the host retinal epithelium forming junctional complexes with both the host and other transplant cells. The transplant cells contact host photoreceptors with their apical processes and phagocytize outer segments. There appears to be no significant cell division among the transplant cells.

Monitoring photoreceptor transplants with nuclear and cytoplasmic markers

Two methods are described for identifying transplanted photoreceptors in a foreign host retina. One involves the use of [3H]thymidine to label the nuclei of photoreceptors which are dividing for 1 week after birth in myomorphic retina. These photoreceptors can be identified by autoradiography. The second involves the use of a transgenic mouse carrying a bovine rhodopsin promoter in tandem with the bacterial LacZ gene. These mice express beta-galactosidase in their rods. X-gal reaction allows these rods to be identified by routine light and electron microscopy. These methods have been used to follow photoreceptor transplants in adult Royal College of Surgeons strain rat and C3H mouse mutants which have lost virtually all their photoreceptors. Dissociated photoreceptors transplanted to the subretinal space of these animals survive for at least 3 months. The inner segment, cell body, and synaptic terminal of these transplanted photoreceptors remain morphologically normal; the outer segment, however, becomes rudimentary.

Reporter gene expression in cones in transgenic mice carrying bovine rhodopsin promoter/lacZ transgenes

Rhodopsin gene expression has been used as a model system to study the mechanisms regulating photoreceptor gene expression. Previous transgenic experiments using rhodopsin promoter/lacZ fusion constructs identified some of the cis-acting DNA elements responsible for photoreceptor cell-specific expression. However, the issue of rod specificity vs. photoreceptor (rod and cone) specificity of the elements was not resolved. To address this issue, the specificity of reporter gene expression in the retinas of transgenic mice carrying bovine rhodopsin promoter/lacZ (beta-galactosidase) fusion genes was assessed using X-gal staining and electron microscopy. Two independent transgenic lines, one carrying a rhodopsin promoter fragment extending from -2174 to +70 base pairs (bp) relative to the messenger RNA start site and another line carrying a fragment from -222 to +70 bp, both showed reporter gene expression in cones as well as rods, although the level of staining appeared to be less in the cones than in the rods. These results demonstrate that the -2174 to +70 bp and -222 to +70 bp bovine rhodopsin promoter fragments are not rod-specific in transgenic mice and indicate that the existence of rod promoter mediated-expression in cones must be considered when interpreting results from transgenic experiments utilizing the rhodopsin promoter.

Transplantation of retinal cells

This paper traces the history of transplantation of dissociated retinal epithelial and/or photoreceptor cells. It describes our most recent results on the long term allotransplantation of cultured, tritiated thymidine labeled pigmented retinal epithelial cells into the retina of albino rabbits immunosuppressed with cyclosporine. Cultured rabbit retinal epithelial cells survive for months after transplantation, resume phagocytosis of host outer segments and form junctional complexes with each other and with host retinal epithelium. It also describes long term transplantation of photoreceptors (rods) from normal rats into the almost receptorless retina of a congenic dystrophic rat. These rods maintain primitive outer segments and appear to form synapses on host retinal neurons. The results suggest that cellular reconstruction of the degenerated retina may become a feasible undertaking.

Isolation of human fetal cones

Purpose. To describe a method for isolating a monolayer of human fetal cone photoreceptors and to compare their structure and ultrastructure before and after preparation. Methods. Eyes from human fetuses (fetal week 20 to 24) were dissected and the neural retina of the developing fovea identified, cut out and placed on 10% gelatin. A VISX Star TM excimer laser was used to remove the inner retinal layers. The isolated cone monolayers were cultured for 18 hours and compared with untreated retinas by light microscopy and transmission electron microscopy. Results. Excimer laser ablation removed the inner nuclear and ganglion cell layers leaving a monolayer of fetal cones. These cones survive in culture for at least 18 hours. The laser ablation disorganized the ultrastructure of the synaptic pedicles of these cones, left their plasma membranes intact. Conclusions. The developing central retina of human fetal eyes provides a source of fetal cones, which can be isolated from inner retinal cells using the excimer laser. Such a monolayer of human fetal cones may be useful for transplantation or biochemical studies.