Retinal Transplantation Research Articles

Visual information processing by intracerebral retinal transplants in rats.

We have developed a simple system involving the implantation of retinae over the midbrain of rodents to examine whether, in a clearly defined system such as the primary optic pathway, it is possible to re-create circuits lost as a result of injury or developmental disorder. For much of the work, immature rat hosts have been used, in part to maximise optimal conditions and to provide a baseline for similar transplants in adults. In this review we summarise the sequence of studies that has led us to the conclusion that transplanted retinae are capable not only of differentiating and responding to light but also of relaying luminance information to visual centres of the host brain where appropriate behavioural responses are elaborated.

Ultrastructural circuitry in retinal cell transplants to rat retina.

The development of five transplants of fetal retinal tissue to adult rat eyes was examined with the electron microscope. The transplants were of 9 to 10 weeks total age after conception in four cases and 20 weeks in one case. They were at stage E15 when transplanted. Transplants developed in both the epiretinal and subretinal spaces. The transplants were heterogeneously developed with some parts showing almost normal differentiation and others little. Subretinal transplants examined in this study were more developed than epiretinal grafts. Photoreceptor cells developed both inner and outer segments. Their synaptic terminals possessed output ribbon synapses with postsynaptic processes similar to those seen in normal retinas. In regions corresponding to the inner plexiform layer, the adult complement of synapses was seen, including advanced features such as serial synapses as well as reciprocal synapses at bipolar cell dyads. Incompletely differentiated synapses of both the amacrine and bipolar cell types were often observed, especially in the rat epiretinal transplants. Ganglion cell processes could not be identified with certainty. Although transplant cells were adjacent to host photoreceptor cells and pigment epithelium, obvious specializations or interactions were not observed. The experiments suggest that embryonic rat retinal cell transplants develop most or perhaps all of the structural components and neuronal circuitry necessary to transduce light and process some visual information.

Function and structure in retinal transplants.

Embryonic mammalian donor retina transplanted into the subretinal space of a mature host develops into a graft with wellorganized, but atypical retinal structure. We tested the effect of this organization on rabbitto-rabbit graft functional properties, isolating the graft to avoid contamination of graft responses by host retinal activity. Transient ON or ON-OFF spike-like responses and local electroretinograms (L-ERGs) were recorded simultaneously via a single electrode on the graft surface. These response components depended on stimulus diameter, sometimes in a way indicating antagonistic center-surround receptive field organization and spatial tuning (43%). Other times, the responses were an increasing function of stimulus diameter which saturated for large spots (57%). Response amplitudes were transplantation surgery is to be done with therapeutic aims.

Anatomical and functional consequences of induced rejection of intracranial retinal transplants

Retinae from embryonic rats transplanted over the midbrain of newborn host rats establish connections with visual centres of the host brain, which mediate a pupilloconstrictor response in the host eye when the transplant is stimulated by light. The changes in the size of the host pupil can be measured accurately with a pupillometry system. We have taken advantage of the additional observation that while grafts between rat strains, as between Long Evans and Sprague-Dawley strains, may survive indefinitely, they can be induced to reject by skin grafting from the strain providing the donor retinal tissue. Combining pupillometry with skin grafting provides a useful way of examining correlated anatomical and behavioural changes associated with graft rejection from its earliest stage to the point of overt destruction. Even within three days of skin grafting, the amplitude and speed of constriction as well as the response latency all showed significant enhancement from normal, and this was sustained for a further week or more. Response deterioration followed during the second week post-skin grafting, but the exact timing varied considerably among animals. Anatomical observations of the process of retinal rejection showed the first invasion of lymphocytes to occur between days 5 and 7 and total degeneration of the retinal transplant and its projections to occur by two to three weeks post-skin grafting. The lymphocytic infiltration was preceded by upregulation of microglia, which expressed both class I and II major histocompatibility antigens and by activation of astrocytes identified by their expression of glial fibrillary acidic protein. Within the target region of retinal transplant axons, major histocompatibility antigen expression and astrocytic responses preceded degeneration of transplant derived axons (demonstrated by the Fink-Heimer stain) and there was no evidence for any lymphocytic infiltration during transplant rejection. These observations show that the earliest stages of microglial activation are accompanied by an enhancement of response parameters, but that the functional failure finally occurs only at an advanced stage of graft destruction. The absence of lymphocytic infiltration into areas receiving terminals from axons of transplant origin, even though these contain significant numbers of reactive microglia, suggests that the terminal axonal processes are not a primary target for the immune response.

Cellular organization in retinal transplants using cell suspensions or fragments of embryonic retinal tissue.

We have investigated the cellular organization in two different types of retinal transplants using cell type-specific monoclonal antibodies. Both fragments and cell suspensions of E17-E19 Sprague-Dawley rat retina were transplanted to a subretinal site in congenic adult rat hosts. After a survival time of 28 days, the transplants were stained by immunocytochemistry with antibodies against rhodopsin, which stained rods; with antibodies against HPC-1, which stained amacrine cells and outer and inner plexiform layers; and with antibodies against vimentin, which stained Müller cell fibers and horizontal cells. In the host retina, the distribution of immunocytochemical staining was similar, irrespective of transplantation technique. In the transplants, the anti-rhodopsin staining showed that fragment transplants developed photoreceptors in rosettes, whereas in cell suspension transplants, this staining showed a scattered distribution of photoreceptors. The HPC-1 staining showed that regions corresponding to the inner nuclear layer surrounded both types of transplants and made large invaginations into them. In one case, using the cell suspension technique, fibres were found to run from the inner plexiform layer of the transplant to the outer plexiform layer of the host. The vimentin staining revealed a disorganized array of Müller cell fibres in both types of transplants, but with some concentration to the regions corresponding to the inner plexiform layer.

Horizontal Cells of the Normal and Dystrophic Rat Retina: A Wholemount Study Using Immunolabelling for the 28-kDa Calcium-binding Protein

Immunocytochemistry with a monoclonal antibody against the 28-kDa calcium-binding protein (28-kDa CaBP) was used to study horizontal cells in both congenic control (RCS- rdy +) and dystrophic (RCS) 3-month-old rat retina. We found that horizontal cells in the RCS- rdy + rat retinas were distributed evenly from the centre to the peripheral retina with a mean density of 783 cells mm -2. Cell size ranged between 6·5-12·5 μm in diameter with a mean of 8·87 μm. Individual cell processes could not be followed readily, however they formed a narrow plexus within the outer plexiform layer with the primary dendrites extending radially from each cell soma in an organized manner. In the RCS rat, by contrast, the horizontal cell processes were more disorganized than in the congenic controls. The degree of disorganization varied across the retina. In the posterior pole, where all outer layers had been lost, many processes were grossly swollen along their length and at the tips. The cell somas were also distributed more widely in the depth of the retina. In the peripheral retina, where a debris layer was still present, the processes were less swollen but they extended abnormally widely in depth. Despite the disorganized structure the mean and range of cell size (9·23 μm, 5·5-15·5 μm), and cell density (796 cells mm -2) were similar to that of the control RCS- rdy + animals. These findings indicate that in dystrophic retinas beyond a certain stage of degeneration the horizontal cells become abnormal in their structure but are present at a normal density and the somas are not grossly swollen or shrunken. This tendency for horizontal cell processes to reorganise is important for retinal pathology and transplantation studies.

Experimental and Surgical Aspects of Retinal Pigment Epithelial Cell Transplantation

OBJECTIVES: To discuss the current status of experimental studies on transplantation of the retinal pigment epithelium (RPE), and surgical results on the excision of subfoveal choroidal new vessel membranes in age-related macular degeneration (AMD) and presumed ocular histoplasmosis (POHS). STUDY DESIGN: Experimental study on transplantation of the RPE in organ culture and in vivo, and results of a prospective series on surgical excision of choroidal neovascularization. SETTING: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine. PATIENTS: Patients with subfoveal neovascular membranes secondary to AMD and POHS. MAIN OUTCOME MEASURES: Histological results on transplantation of the RPE onto Bruch's membrane in experimental animal models, and visual results on surgical excision of subfoveal choroidal neovascularization in patients with AMD and POHS. RESULTS: We have demonstrated that the RPE monolayer can heal after surgical debridement in the experimental animal, and retinal recovery occurs after healing of the RPE monolayer. We have demonstrated that RPE can reattach to healthy Bruch's membrane in vitro, and have performed RPE transplantation into the subretinal space in vivo. CONCLUSIONS: We are currently involved in a multifaceted approach aimed at RPE transplantation and surgical reconstruction of the photoreceptor-RPE-Bruch's membrane interface. We are hopeful that our approach will be applicable to the clinical problem of subfoveal choroidal new vessel membranes in AMD and POHS, in addition to the broad applicability of RPE transplantation to patients with diseases arising from primary RPE dysfunction.

Retinal transplants in congenitally blind mice: patterns of projection and synaptic connectivity.

Embryonic retinae were transplanted onto the midbrain of neonatal congenitally anophthalmic mice and neonatal mice from which both eyes had been removed. When donor mice of the AKR strain were used, the detailed patterns of the transplant projections to the host brain were demonstrated with an antibody to Thy-1.1, which specifically stains neural tissue derived from AKR donors. Many of the subcortical visual centers were innervated, and only small differences were encountered between anophthalmic and eye-enucleated mice. The terminal arbors of transplant-derived axons could not be classified as in normal animals, although several distinct arbor types were seen. In the superior colliculus, the laminar arrangements that characterize normal retinal arbors were disrupted. Despite this, the synaptic patterns formed by transplant-derived axons in the superior colliculus of anophthalmic mice compared very closely with those of retinal axons in normal, sighted animals. These observations indicate that the ability of a retinal transplant to innervate the host brain and to form the synaptic arrays characteristic of optic terminals are not dependent on prior innervation, nor do they appear to be influenced by the events that follow eye removal.

Retinal pigment epithelial cell transplantation in RCS rats: Normal metabolism in rescued photoreceptors

Photoreceptor cells in Royal College of Surgeons (RCS) rats with inherited retinal dystrophy can be rescued by the transplantation of normal, wild-type retinal pigment epithelial (RPE) cells, if done before photoreceptor cell death. In the present study, we have examined several metabolic features of rescued photoreceptors and transplanted RPE cells at 2.3-3.3 months after transplantation. Rescued photoreceptors with a structurally normal RPE interface showed a rod outer segment renewal rate similar to that of normal control rats of 2.2 microns day-1, as measured in autoradiograms. Rod outer segment disc shedding had values indistinguishable from those in normal controls, as measured by the number of phagosomes in the transplanted RPE cells both during the burst of disc shedding soon after the onset of light in the morning and during the middle of the light cycle when disc shedding is low. The interphotoreceptor matrix, which is synthesized by both photoreceptors and the RPE, was distributed normally in the regions where normal-appearing photoreceptors were present underlying normal, transplanted RPE cells. Thus, the rescued photoreceptors show normal metabolic rates and normal interactions with the RPE in each of the parameters examined. These findings, combined with the previous demonstration of opsin and Na+,K(+)-ATPase expression by the rescued photoreceptors, support our interpretation that the surviving, normal-appearing photoreceptors may function normally. Moreover, transplantation of normal RPE cells reversed pathological changes in the photoreceptors that had already occurred by the time of transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

CFos labeling in rat superior colliculus: Activation by normal retinal pathways and pathways from intracranial retinal transplants

Previous studies in this laboratory have shown that intracranial retinal transplants can establish both anatomical and functional connections with the host brain. Embryonic rat retinae transplanted intracranially into neonatal host brains are capable of evoking appropriate physiological and behavioral responses when illuminated. The present study employs the specific expression of the cFos protein to identify brain regions in which immediate-early gene activation can be recognized in response to flash stimuli delivered to retinal transplants and to normal intact retinae. Stimulation of the intact eye induced significant cFos expression in various visual centers, including the stratum griseum superficiale of the superior colliculus and the pretectal area, but not in the dLGN, suprachiasmatic nucleus, or retina. Animals with functional transplants expressed cFos throughout the depth of the stratum griseum superficiale and stratum opticum of the superior colliculus, thus apparently activating an additional population of superior collicular cells. Like the eye-stimulated animals, animals with functional transplants failed to elicit significant cFos expression in the dLGN or transplanted retina. This study indicates that intracranial retinal transplants are capable of forming functional connections with the host superior colliculus which not only mediate transient changes in electrical activity but also may affect gene expression through the induction of the c- fos gene.

Sensorineural improvement in rats after retinal transplants: M. del Cerro, J. R. Ison, E. Lazar, D. A. Grover, P. Bowen, D. DiLoreto, & C. del Cerro. University of Rochester; Rochester, NY, U.S.A

Sensorineural improvement in rats after retinal transplants: M. del Cerro, J. R. Ison, E. Lazar, D. A. Grover, P. Bowen, D. DiLoreto, & C. del Cerro. University of Rochester; Rochester, NY, U.S.A

Development of parvalbumin immunoreactive neurons in normal and intracranially transplanted retinas in the rat.

Retinas from embryonic day 14 Sprague-Dawley rats were transplanted to the midbrain or cerebral cortex of newborn (P0) rats of which the right eye was enucleated at the time of transplantation. Parvalbumin immunoreactive (PV-I) neurons were studied in the developing retinal transplants, and in the remaining retina of the host, as well as in normal retinas. PV-I neurons were identifiable in retinas of normal and host rats from postnatal day 5 (P5) onward, with the PV-I somata primarily in the inner half of the inner nuclear layer and in the ganglion cell layer. An adult-like distribution of PV-I neurons was attained at P35, as judged by cell packing density, intensity of immunostaining, laminar distribution and soma size of subpopulations of PV-I cells. A similar time course of development and distribution of PV-I somata was observed in the retinal transplants, except for some minor differences such as a slight delay in PV-I cells achieving their final distribution. These findings provide evidence that PV-I neurons can survive, differentiate and mature according to predetermined programmes intrinsic to the retinal tissue following transplantation to a new and foreign environment.

Transplantation of embryonic retina to the subretinal space in rabbits

Embryonic rabbit retina can be transplanted to the subretinal space of adult rabbit with a new method, which gives a high rate of successful short-term transplants. Embryonic (stage E 15) neural retina cells were injected through an incision just behind the sclerocorneal border with a thin (inner diameter 0·15 – 0·4 mm, outer diameter 0·3 – 0·5 mm) plastic tube attached to a specially designed instrument, by which the length of the protruding plastic tip could be controlled. The retina was penetrated from the vitreous side and the donor tissue was injected into the subretinal space. The cells survived in the host for at least 5 months, although the long-term survival rate tended to decrease. The transplanted cells matured and differentiated, forming an approximation of the layered, retinal structure with some anomalies (e.g. rosettes). The subretinal location offers an interesting and convenient way of studying the development of retinal cell transplants in rabbits. Large transplants can be produced, and the risk for failures due to erroneous vitreous placement is small.

Development of Light-activated Pupilloconstriction in Rats as Mediated by Normal and Transplanted Retinae.

The relationship between the development of the pupilloconstriction response to changes in light levels and retinal maturation was studied in normal rats and rats that had received intracranial retinal transplants at birth. A pupillary response to light was first observed between postnatal days 7 and 9 in normal rats, and was typically of small amplitude and sluggish. By the time the eyelids first open, 2 weeks after birth, the pupillary response had improved to near adult levels. The inception of the pupillary response correlates with the first appearance of conventional synaptic contacts in the inner and outer plexiform layers of the retina, while improved responses correlate with maturation of photoreceptor outer segments and formation of synaptic ribbons in the inner plexiform layer. When embryonic retinae were transplanted to intracranial locations in newborn hosts and the transplants later illuminated as the host matured, the onset of a pupillary response to transplant illumination was delayed in proportion to the developmental disparity between the transplant and the host. The pattern of anatomical development in transplanted retinae was also similar, but delayed in time, compared to normal retinae. This indicates that the limiting factors for expression of light-activated pupilloconstriction exist within the retina, rather than being intrinsic to the central nuclei or to the output pathway subserving the response.

Intraretinal xenografts of differentiated human retinoblastoma cells integrate with the host retina

We report on the successful use of chemically modified Y79 human retinoblastoma cells for intraretinal xenografting into damaged adult mammalian eyes. Y79 cells were exposed in vitro to retinoic acid/butyrate to induce differentiation. Using a multisite transplantation method, the suspension was injected into the subretinal space of Fischer 344 rats. The survival, integration, and differentiation potential of these cells was studied, following their return to the intraocular milieu from which the progenitor cells originated. The grafted cells survived and differentiated into immature photoreceptor elements in the subretinal and intraretinal locations, as multiple clusters of rosette-forming cells intimately attached to the host neuroretina. The differentiation process included development of synaptic connectivity of the ribbon type with the surrounding neuropil. No signs of renewed cell division were found within grafts performed on 42 rat eyes, and there was no indication of cell-mediated host reaction against the transplants. This study indicates that tumorigenicity can be suppressed in mitotically arrested Y79 cells, and that these cells are capable of undergoing differentiation in vivo. This provides evidence of the remarkable differentiation properties of human retinoblastomas while indicating that Y79 cells may ultimately be able to substitute for fetal cells in experimental retinal transplantation.

Development of gamma-aminobutyric acid-immunoreactive neurons in normal and intracranially transplanted retinas in rats

Retinas from embryonic day 14 Sprague-Dawley rats were transplanted intracranially to the midbrain or cortex of newborn (P0) rats with right eyes enucleated at the time of transplantation and the gamma-aminobutyric acid (GABA) immunoreactivity in developing retinal transplants, host as well as normal retinas, was studied. The results showed that GABA-immunoreactive neurons were identified in retinas of normal and host rats from the day of birth (P0) onward and that their somata were distributed primarily in the inner half of the internal nuclear layer and in the ganglion cell layer. The adult pattern of GABA immunoreactivity was first observed at P16 when several immunoreactive sublaminae were clearly identifiable in the inner plexiform layer. In contrast, gamma-aminobutyric acid-immunoreactive somata could not be identified in retinal transplants until P4, with a significant reduction in the density and number of GABAergic neurons detected by P12. Moreover, only two immunoreactive sublaminae were observed in the inner plexiform layer in all transplants at P12, as well as in more mature stages. These results suggest that significant changes occurred in the GABA system of the transplanted retina, despite the fact that the overall pattern of organization of the GABAergic neurons and their processes in the retinal transplants was comparable to that of the normal retina.

A role for microglia in the maintenance of photoreceptors in retinal transplants lacking pigment epithelium.

Studies on intact retina have pointed to a necessary role for retinal pigment epithelium in the maintenance of photoreceptor outer segments and for regeneration of visual pigment. However, it has been shown that when embryonic retinae are separated from the pigment epithelium and transplanted into the brain of neonatal rats, the transplanted photoreceptors develop outer segments and the retina responds to light in the apparent absence of pigment epithelial cells. We confirm that there are no retinal pigment epithelium cells associated with transplanted retinae in the present series of experiments and show that a row of cells, composed predominantly of microglia of host origin, border the graft. These cells can be seen to contain engulfed outer segments when they are apposed to the outer retina, suggesting that the microglia have assumed, at the least, the phagocytic function normally associated with retinal pigment epithelium. Microglial cells and their processes are also found within the transplant, but these cells are typically devoid of phagosomes, indicating an absence of phagocytic activity. The close physical association of these resting microglia with the transplant may facilitate their role in antigen presentation under specific conditions of immune provocation.

Reciprocal retinal transplantation: A tool for the study of an inherited retinal degeneration

The pathogenesis of retinal degeneration in rd mutant mice has been extensively studied, the gene responsible for the defect has been cloned, and the neural retina has been identified as the primary site for the degeneration. However, the possible contributory role of the ocular environment in this form of retinal degeneration remains undetermined. Retinal transplantation, which provides the opportunity to implant the neural retinal into a genetically defined intraocular environment, was used to examine this possibility. A reciprocal retinal transplantation paradigm was designed based on three experimental groups: (1) normal immature retina transplanted into rd rd mutatn eyes, (2) rd rd immature retina transplanted into normal eye, and (3) normal immature retina transplanted into normal eyes. The rates of survival and histological characteristics of the grafts were compared between the three groups. At post-transplantation Day 3 (PTD 3), there were no differences between the three groups. Between PTD 10 and 15, the retinal grafts in group 1 showed degeneration. In contrast, the retinal grafts in groups 2 and 3 survived and developed well. At PTD 30, the retinal grafts in both groups 1 and 2 showed degeneration, but the retinal grafts in group 3 survived and remained differentiated well. These results suggest that the retinal degeneration of rd mice may be caused by both a deficit of the neural retina and intraocular environmental changes which are elicited either as a result of mutation or as a sequel to retinal degeneration.

Photoreceptor differentiation in retinal xenografts of fetal monkey retina

Although the potential of retinal grafts to provide the host eye with rod cells is presently well established, the possibility of grafting cone photoreceptors has not been documented. In this study, the neural retinas of two Cebus apella monkey fetuses were xenografted into immunosuppressed Fischer 344 adult rats. Histological analysis showed intimate apposition between the grafted donor cells and the neighboring host rat retina. The transplanted cells survived well and often overgrew the boundaries of the host retina, expanding into the host vitreous cavity. These cells formed histogenetically differentiated structures predominantly populated by the photoreceptors. When transplanted into a foreign environment, donor cells formed inner segments which exhibited the basic morphology of cells developed in situ. This study demonstrates that embryonic monkey neural retina is a viable source of xenograft material. It also indicates that an advanced embryonic stage is not a deterrent to survival and differentiation of grafted primate neuroretinal cells. The successful transplantation of these cells, especially under the relatively adverse conditions of a xenograft, raises the hope that retinal transplantation may in fact be a useful technique for repairing both rod and cone function in damaged retinas of higher animals including humans.

Tyrosine-hydroxylase-immunoreactive neurons in retinal transplants in the rat.

Embryonic rat retinae were transplanted to the brains of newborn rats, and the distribution of catecholaminergic neurons in the retinal tissue was studied 1-2 months after transplantation, using the tyrosine hydroxylase (TH) immunohistochemical method. The results showed that distinct TH-positive cells were identified in all retinal transplants examined. The somata of the majority of these TH-immunoreactive cells were located along the inner margin of the inner nuclear layer in the retinal transplants; the processes of these cells were distributed mainly in the outer portion of the inner plexiform layer. This pattern is comparable to that observed in retinae of normal and host rats, suggesting that the organization of the catecholaminergic neurons in the transplant is largely similar to that in the normal retina. However, a reduction of the immunoreactivity in the plexiform layers and subpopulations of TH-positive cells with somatic diameter smaller than 8 microns or larger than 18 microns was observed in most of the retinal transplants studied. This implies that the organization of the catecholaminergic system in the transplant may not be as intact as in the normal retina.