Fulton Wong

Apoptosis: Final common pathway of photoreceptor death in rd, rds, and mutant mice

Mutations in the retinal degeneration, retinal degeneration slow(/peripherin) and rhodopsin genes cause photoreceptor degeneration in humans and mice. Although the phenotypes arising from these mutations are different, suggesting different mechanisms of pathogenesis, we present evidence that apoptosis may be the final common pathway of the disease process linking genotype to phenotype. We observed internucleosomal cleavage of retinal DNA by gel electrophoresis and fragmented DNA at the single cell level by labeling the nicked DNA ends with biotinylated poly(dU). In retinal degeneration mice, DNA fragmentation occurred during the period of photoreceptor degeneration. In retinal degeneration slow mice and in transgenic mice expressing a mutant (Pro347Ser) rhodopsin gene, DNA fragmentation occurred after normal histogenetic cell death (also apoptosis) had ceased. Since DNA fragmentation by internucleosomal cleavage is a cardinal feature of apoptosis, our data suggest that all three of these genetic mutations lead to apoptosis.

Oxidative damage is a potential cause of cone cell death in retinitis pigmentosa

Retinitis pigmentosa (RP) is a prevalent cause of blindness caused by a large number of different mutations in many different genes. The mutations result in rod photoreceptor cell death, but it is unknown why cones die. In this study, we tested the hypothesis that cones die from oxidative damage by performing immunohistochemical staining for biomarkers of oxidative damage in a transgenic pig model of RP. The presence of acrolein‐ and 4‐hydroxynonenal‐adducts on proteins is a specific indicator that lipid peroxidation has occurred, and there was strong immunofluorescent staining for both in cone inner segments (IS) of two 10‐month‐old transgenic pigs in which almost all rods had died, compared to faint staining in two 10‐month‐old control pig retinas. In 22‐ and 24‐month‐old transgenic pigs in which all rods and many cones had died, staining was strong in cone axons and some cell bodies as well as IS indicating progression in oxidative damage between 10 and 22 months. Biomarkers for oxidative damage to proteins and DNA also showed progressive oxidative damage to those macromolecules in cones during the course of RP. These data support the hypothesis that the death of rods results in decreased oxygen consumption and hyperoxia in the outer retina resulting in gradual cone cell death from oxidative damage. This hypothesis has important therapeutic implications and deserves rapid evaluation.

Ectopic synaptogenesis in the mammalian retina caused by rod photoreceptor-specific mutations

In addition to rod photoreceptor loss, many mutations in rod photoreceptor-specific genes cause degeneration of other neuronal types. Identifying mechanisms of cell–cell interactions initiated by rod-specific mutations and affecting other retinal cells is important for understanding the pathogenesis and progression of retinal degeneration. Here we show in animals with rod and cone degeneration due to mutations in the genes encoding rhodopsin and cGMP phosphodiesterase β-subunit (PDE-β) respectively, that rod bipolar cells received ectopic synapses from cones in the absence of rods. Thus, synaptic plasticity links certain rod-specific mutations to retina-wide structural alterations that involve different types of neurons.

Proper function of the Drosophila trp gene product during pupal development is important for normal visual transduction in the adult.

The response of invertebrate photoreceptors consists of the summation of quantum bumps, each representing the response to a single photon. The bumps adapt depending on the intensity of the stimulus: their average size is relatively large in dim light and small in bright light. The rate of occurrence of the bumps varies proportionally with light intensity. In the Drosophila mutant trp, unlike in the wild type, the rate does not increase with increasing light intensity and the bumps do not adapt. Here we report an analysis of the trp gene and its expression in normal and mutant flies. Our results suggest that the trp protein is a novel photoreceptor membrane-associated protein, that this protein is not required for the occurrence of bumps but is necessary for adaptation, and that proper function of the trp gene product during pupal development is important for normal visual transduction in the adult.

Retinal Rod Photoreceptor-Specific Gene Mutation Perturbs Cone Pathway Development

Rod-specific photoreceptor dystrophies are complicated by the delayed death of genetically normal neighboring cones. In transgenic (Tg) swine with a rod-specific (rhodopsin) gene mutation, cone photoreceptor physiology was normal for months but later declined, consistent with delayed cone cell death. Surprisingly, cone postreceptoral function was markedly abnormal when cone photoreceptor physiology was still normal. The defect was localized to hyperpolarizing cells postsynaptic to the middle wavelength-sensitive cones. Recordings throughout postnatal development indicated a failure of cone circuitry maturation, a novel mechanism of secondary cone abnormality in rod dystrophy. The results have implications for therapy for human retinal dystrophies and raise the possibility that rod afferent activity plays a role in the postnatal maturation of cone retinal circuitry.

Ectopic synaptogenesis during retinal degeneration in the Royal College of Surgeons rat

Rod photoreceptor-specific mutations cause ectopic synapses to form between cone photoreceptor terminals and rod bipolar cell dendrites in degenerating retinas of rhodopsin transgenic (P347L) pigs and retinal degeneration mice. Since the mutations occur in rod photoreceptor-specific genes in these two models, it is not known if ectopic synaptogenesis occurs specifically due to some rod photoreceptor cell-autonomous properties of a mutation or as a general consequence of photoreceptor degeneration. In the Royal College of Surgeons (RCS) rat, a mutation in the receptor tyrosine kinase gene, Mertk, causes failure of the retinal pigment epithelial (RPE) cells to phagocytose shed photoreceptor outer segments; subsequently, both rod and cone photoreceptors die. The non-phagocytic phenotype of the RCS rat is RPE cell-autonomous and the photoreceptors degenerate secondarily. Here we show that in 35-day-old RCS rats, where a majority of rod and cone photoreceptors remained, rod bipolar cell dendrites had abnormal (flat-contact type) synaptic contacts with rod and cone terminals. Demonstration of ectopic synapses in the RCS rat suggested that ectopic synaptogenesis could occur as a result of photoreceptor degeneration, even when the rods and cones were developmentally normal. This further supported the hypothesis that ectopic synaptogenesis may be a common step in the disease progression of different forms of retinal degeneration that include photoreceptor death as a feature, such as retinitis pigmentosa.

Effects of indocyanine green on the retina and retinal pigment epithelium in a porcine model of retinal hole.

Purpose: This study was designed to emulate human macular hole surgery and to test the effects of indocyanine green (ICG) on the retina and retinal pigment epithelium (RPE). Methods: Yorkshire Cross pigs (n = 23) underwent vitrectomy, separation of the posterior cortical vitreous, and creation of a single retinal hole. In three study groups (n = 6, each group), air–fluid exchange was performed, following which balanced salt solution (BSS), 1.0% ICG, or 0.5% ICG was applied over the retinal hole. In one additional group (n = 5), 0.5% ICG was injected into the fluid-filled eye. At 4 weeks, the eyes were examined clinically, and fundus photographs were obtained before enucleation and light microscopic examination. Results: Clinical evaluations documented a statistically significant difference between study groups (P = 0.036). There was a higher rate of moderate or severe RPE atrophy among animals where 1% or 0.5% ICG was applied in air-filled eyes (83% and 67%, respectively) compared with BSS controls (17%) and fluid-filled eyes receiving 0.5% ICG (40%). Histologic evaluation demonstrated a statistically significant difference between groups (P = 0.044), with extensive outer retinal degeneration observed in air-filled eyes receiving 1% or 0.5% ICG (66% and 60%, respectively) compared with BSS controls or fluid-filled eyes receiving 0.5% ICG (none of the eyes in either group). None of the study groups had any changes in the inner retina except at the retinal hole site. Conclusions: Retina exposed to ICG concentrations used in human vitreoretinal surgery had greater RPE atrophy and outer retinal degeneration than control eyes undergoing the same surgery without ICG. Eyes filled with infusion fluid during ICG injection had less damage to the RPE and outer retina than did air-filled eyes receiving ICG.

Porcine global flash multifocal electroretinogram: possible mechanisms for the glaucomatous changes in contrast response function.

PURPOSE The aim of this study was to obtain a better understanding of the cellular contributions to the porcine global flash mfERG by using a pharmacologic dissection method, together with the method using variation of stimulus contrast which has been used to demonstrate mfERG changes in human glaucoma. METHODS Global flash mfERGs with different stimulus-contrast settings (99%, 65%, 49% or 29%) were recorded from 14 eyes of ten 6-week-old Yorkshire pigs in control conditions and after suppression of inner retinal responses with inhalation of isoflurance (ISO), and injections of tetrodotoxin (TTX) and N-methyl-d-aspartic acid (NMDA). ON- and OFF-pathway responses were isolated by injection of 2-amino-4-phosphonobutyric acid (APB) and cis-2,3-piperidinedicarboylic acid (PDA). RESULTS The porcine global flash mfERG consisted of an early direct component (DC) and a late induced component (IC). ISO and TTX removed inner retinal contributions to the IC; NMDA application further abolished the oscillatory wavelets in the DC and removed the residual IC waveform. The inner retina contributed regular oscillation-like wavelets (W1, W2 and W3) to the DC and shaped the IC. After removing the inner retinal contributions, the porcine global flash mfERG waveform becomes comparable to that obtained with conventional mfERG stimulation. The remaining waveform (smoothed DC) was mainly contributed by the ON- and OFF-bipolar cells as revealed after APB or PDA injection. Photoreceptors contributed a small signal to the leading edge of N1. The characteristic of contrast response function of DC was demonstrated to be contributed by the inner retinal oscillation-like wavelets. CONCLUSION We believe that the DC of the porcine global flash mfERG is mainly composed of contributions from photoreceptors, and ON- and OFF-bipolar cells, where inner retinal activity partially shaped the DC with superimposed regular wavelets. However, the IC is dominated by inner retinal activity. The contrast response functions of DC consisted of both outer retinal response and oscillation-like wavelets of the inner retinal response. Both contain different characteristics during contrast modulation of the stimulus, where the changes of W2 of the inner retinal response seem independent of contrast modulation. The DC contrast response feature depends mainly on the relative contribution of inner retinal activities; the loss of inner retinal cells may alter the DC contrast response function, making it tend toward linearity.

Dispase facilitates posterior vitreous detachment during vitrectomy in young pigs.

Purpose To evaluate the role of intravitreal dispase in conjunction with pars plana vitrectomy to facilitate the creation of a posterior vitreous detachment (PVD) in young pig eyes. Methods Twenty-four eyes of 24 animals were randomized to receive an intravitreal injection of dispase (50 &mgr;g/0.05 mL) or phosphate buffered saline (PBS) immediately after core vitrectomy and before attempted creation of a posterior cortical vitreous detachment. Following a 15-minute waiting period, surgical creation of a posterior vitreous separation was attempted by aspiration of the posterior vitreous immediately adjacent to the optic disk. Eyes were evaluated postoperatively by clinical examination (1, 4, and 8 weeks) and electroretinography (4 and 8 weeks), after which they were enucleated for light, scanning, and transmission electron microscopy. Results Based on intraoperative findings and postoperative scanning electron microscopy, eyes receiving intravitreal dispase exhibited a higher incidence of PVD compared to eyes receiving PBS (P = 0.029). Electroretinographic responses recorded at postoperative weeks 4 and 8 were similar in both dispase and PBS eyes compared to the unoperated fellow eyes. Clinical examinations, including indirect ophthalmoscopy, were indistinguishable between the PBS eyes and 11 of 12 eyes in the dispase group. Light and transmission electron microscopy demonstrated no differences in the retina between the dispase eyes and the PBS operated controls. Conclusion Dispase is a useful adjunct in facilitating surgical creation of a PVD in young pig eyes.

Transplantation of full-thickness retina in the rhodopsin transgenic pig.

Purpose To establish the morphology of full-thickness neuroretinal grafts transplanted to hosts with degenerative photoreceptor disease. Methods Twenty rhodopsin transgenic pigs received a neuroretinal sheet from a neonatal normal pig in one eye. Following vitrectomy and retinotomy with bleb formation, the grafts were positioned inside the bleb between the host neuroretina and retinal pigment epithelium. After a survival time of 4 months, eye specimens were studied by light and electron microscopy as well as with immunohistochemical markers. Results One eye developed endophthalmitis in the immediate postoperative period and was terminated. Laminated grafts with correct polarity were found in 13 of the remaining 19 eyes. In most cases, these grafts had well-developed organized photoreceptors with outer segments apposed to the host retinal pigment epithelium. The inner layers of the graft contained mostly Müller cells. Both eyes of the hosts had a reduction of photoreceptor cells in most of the retina, while inner layers remained relatively intact. Conclusions Full-thickness neuroretinal grafts can be transplanted to a large animal host with photoreceptor degeneration. The transplantation procedure is relatively atraumatic to both graft and host tissue, and the grafts survive well for at least 4 months. The graft and host retina does not seem to form extensive neuronal contacts, and future work must be directed at stimulating such activity without disrupting the retinal neuronal organization.