Juliani Maslim

Limiting photoreceptor death and deconstruction during experimental retinal detachment: the value of oxygen supplementation ☆

PURPOSE To assess the role of hypoxia in causing the death and deconstruction of photoreceptors in detached retinas and the effectiveness of supplemental oxygen in limiting such damage. METHODS Retinal detachment was induced surgically in the right eye of each of 10 cats. The cats were allowed to survive surgery for 3 days. Two were kept for these 3 days in normoxia (room air, 21% oxygen) and eight in hyperoxia (70% oxygen). The retinas were examined for cell death by use of labels for normal and fragmenting DNA, with antibodies and a cone sheath-specific lectin to demonstrate the status of their inner and outer segments, the synaptic structures of the outer plexiform layer, and the distribution of basic fibroblast growth factor (bFGF) and with in situ hybridization to demonstrate bFGF mRNA. RESULTS Retinal detachment without oxygen supplementation caused the death of some photoreceptors; the loss of cytochrome oxidase from the inner segments and the collapse of the outer segments of surviving photoreceptors; the loss of synaptophysin profiles from the outer plexiform layer; and the loss of bFGF protein from retinal neurons and neuroglia but not from retinal vessels. Oxygen supplementation (hyperoxia) during detachment mitigated all these changes, reducing photoreceptor death, maintaining the specialized structures of surviving photoreceptors, and stabilizing the bFGF within the retina. CONCLUSIONS In experimental retinal detachment, hypoxia caused by the separation of outer retina from its normal source of nutrients is a factor in inducing the death and deconstruction of photoreceptors as well as in the loss of bFGF from the detached retina. Hyperoxia offered to human patients between diagnosis of retinal detachment and surgery may enhance the function of the reattached retina.

Limiting the proliferation and reactivity of retinal Müller cells during experimental retinal detachment : The value of oxygen supplementation

PURPOSE To assess the role of hypoxia in inducing the proliferation, hypertrophy, and dysfunction of Muller cells in detached retina and the effectiveness of supplemental oxygen in limiting these reactions. METHODS Retinal detachments were produced in the right eye of each of 13 cats; the cats survived surgery for 3 days, during which six were kept in normoxia (room air, 21%) and seven in hyperoxia (70% oxygen). Retinas were labeled for proliferation with an antibody (MIB-1) to a cell cycle protein (Ki-67), for evidence of hypertrophy employing antibodies to the intermediate filament protein glial fibrillary acidic protein (GFAP) and to beta-tubulin and for disturbance of glutamate neurochemistry employing antibodies to glutamate to a glutamate receptor (GluR-2) and to glutamine synthetase. RESULTS Results from the two animals kept in normoxia after retinal detachment confirmed previous reports that detachment caused the proliferation of Muller cells, the hypertrophy of Muller cell processes, and the disruption of glutamate recycling by Muller cells. Oxygen supplementation during detachment reduced Muller cell proliferation and hypertrophy and reduced the abnormalities in the distributions of glutamate, GluR-2, and glutamine synthetase. CONCLUSIONS Oxygen supplementation reduced the reaction of retinal Muller cells to retinal detachment, limiting their proliferation and helping to maintain their normal structure and function. In the clinical setting, oxygen supplementation between diagnosis and reattachment surgery may reduce the incidence and severity of glial-based complications, such as proliferative vitreoretinopathy.

Fate of DNA from retinal cells dying during development: uptake by microglia and macroglia (Müller cells).

The tunel technique of labelling fragmenting dna was used to examine cell death in the developing retina of the rabbit, rat and cat. TUNEL-labelled structures included the still-intact nuclei of retinal cells and smaller, strongly labelled bodies interpreted as fragments of disintegrating nuclei (apoptotic or pyknotic bodies). With confocal microscopy, the cytoplasm around labelled nuclei was observed to be labelled, suggesting that DNA fragments spread into the cytoplasm of the dying cell. Also observed were cells whose nuclei were TUNEL-but whose cytoplasm was TUNEL+, so that their morphology could be discerned. Evidence is presented that these are phagocytes, their cytoplasmic labelling resulting from the ingestion of the fragmenting DNA of a dying neighbour. Results suggest that in developing retina fragmenting DNA is phagocytosed principally by microglia and Müller cells, with a few neurones and no astrocytes active as phagocytes. In the postnatal material studied, microglia are the predominant phagocytes for cells dying in the ganglion cell and inner nuclear layers. Müller cells appear able to phagocytose cells dying in any retinal layer and, since microglia do not normally enter the outer nuclear layer, may be important for the phagocytosis of dying photoreceptors.

Mechanisms of retinal angiogenesis

Studies of vessel growth in the retina have contributed to the understanding of retinal development and disease, and also to the establishment of principles of angiogenesis. The earliest formulation of the concept of a vasoformative factor, produced by a tissue to control its vascularisation, came from studies of vessel growth in developing retina, prompted by the clinical importance of neovascularising retinal disease. As in vitro techniques developed, a large number of factors—growth factor proteins, peptides, adhesion molecules and cytokines—were shown to be capable of promoting, facilitating or inhibiting the growth of vessels. This range of possible angiogenic factors was narrowed by subsequent in vivo studies of the retina, in particular studies of the pathogenesis of retinopathy or prematurity, a proliferative vasculopathy of developing retina. From these studies came the understanding that the level of oxygen in retinal tissue drives both normal development and neovascularising disease. This made clear that if there is a principal angiogenic factor which controls the growth of retinal vessels it must be hypoxia-inducible and led to investigation of the role of vascular endothelial growth factor (VEGF) in retinal angiogenesis. VEGF was first identified as a potent and specific angiogenic factor less than a decade ago. When its expression was shown in tumour models to be hypoxia-inducible, studies followed which showed that VEGF has the properties and is expressed in the retina in spatial and temporal patterns which indicate that it plays key role in the induction of normal vessel growth, in the maintenance of vessels once formed and in the induction of neovascularising disease of the retina. Future work will explore the interaction of VEGF with other angiogenic molecules and will explore the therapeutic potential of controlling the expression and secretion of VEGF by the retina, and of enhancing or blocking the binding of VEGF to its receptors.

Receptor Degeneration is a Normal Part of Retinal Development

The degeneration of photoreceptors is regarded as pathological, for the tangible reason that it causes blindness. The causes of such degeneration include genetic defects specific to photoreceptors, many specific to the rhodopsin molecule, and genetic defects in the retinal pigment epithelium (reviewed in ref. 5 and this volume). The death of affected photoreceptors involves DNA fragmentation (2, 7, 8,18) characteristic of apoptosis or programmed cell death (3). This paper presents evidence from the rat and rabbit that DNA fragmentation occurs in committed photoreceptors in normally developing retina; that the affected cells undergo death; that this physiological death of photoreceptors occurs during a discrete period in retinal development, which coincides with the growth of inner and outer segments; and that, in the res strain of rat in which photoreceptors degenerate pathologically, the onset of their degeneration coincides with the onset of physiological degeneration.

The Influence of Oxygen on the Survival and Death of Photoreceptors

We have studied the influence of oxygen on the rates of death of photoreceptors in the normogenetic mouse and rat, and in the genetically dystrophic RCS rat. Hyperoxia delays photoreceptor death during development of the normogenetic and RCS rats. Hypoxia accelerated photoreceptor death in the adult normogenetic rat and mouse. Hypoxia also accelerates photoreceptor death during development of the normogenetic rat and mouse, and of the RCS rat. The effects of hypoxia were greater in the juvenile, confirming the concept of a critical period in photoreceptor development, during which photoreceptors are particularly vulnerable to oxygen levels. In the RCS rat, evidence was obtained that the edges of the retina are resistant to hypoxic damage, explaining earlier observations of long-term photoreceptor survival at the edges. It is suggested that nutrient supply is a limiting factor in photoreceptor survival, in both normal and genetically damaged RCS retinas. If these ideas prove relevant also to the human effective therapy for some photoreceptor dystrophies may be achieved by nutrient supplementation.