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w hen a photoreceptor cannot perform its function because of a structural or biochemical defect, it may degenerate. If the disease affects rods primarily, visual field loss progresses from the mid-periphery towards the middle of the retina, leaving the patient with night blindnes and 'tunnel vision'. Conversely, diseases that primarily affect cones have the opposite effect, destroying the central region of the retina (the macula), where cones are most abundant. The steps leading to retinal degeneration, of either rods or cones, are not well understood, although several scenarios have been proposed. A current theory is that apoptosis, a form of programmed cell death, is ultimately responsible for retinal degeneration (LoUey et al., 1994).

1.6.5.1 Pathogenesis

Apoptosis is an active and highly regulated cellular activity that occurs normally in physiological situations in which the elimination of particular cells is necessary for the well being of an organism, for example during embryonic development. It has been well recorded in many tissues of both vertebrates and invertebrates as a cause of cell loss (Portera-Cailliau et al., 1994). A cell committed to apoptosis withdraws from its

neighbours and is characterised morphologically by disintegration of the nucleolus and generalised condensation of the chromatin resulting from incision of most of the nuclear DNA into short chains of nucleosomes by an endogenous non-lysosomal nuclease (Wyllie et al., 1981). Inability to repair multiple breaks in the DNA strands is the lethal event which probably kills the cell (Schwartzman and Cidlowski, 1993). The ceU breaks down into membrane bound apoptotic bodies which are engulfed by neighbouring cells or sometimes by circulating macrophages, neatly removing them without leakage of the cell contents which may be detrimental to surrounding cells (as shown by inflammation in necrosis). In contrast to necrosis, the process affects individual cells within a tissue, with its neighbours remaining healthy, and takes place in the absence of inflammation (Gregory and Bird, 1995). In the normal developing retina approximately 50% of ganglion, amacrine and bipolar cells and around 5% of photoreceptor cells die by apoptosis (Cepko, 1996). Apoptosis has been shown to be the cause of cell death in all animals with genetically determined retinal degeneration examined to date, whether naturally occurring or induced by transfection by mutant genes (Chang et ah, 1993; Tso

et ah, 1994). Thus, it appears that cell loss is not a direct consequence of the intrinsic metabolic environment of the tissue.

1.6.5.2 Animal models of retinal degeneration

Apoptosis has been found to be the final common pathway of cell death due to mutations in three genes whose primary site of action is in the photoreceptor (Chang

et ah, 1993). Mutations in three photoreceptor-spedfic genes have been identified as the cause of disease in individuals affected by retinitis pigmentosa (RP). These are the P subunit of cyclic GMP (cGMP) phosphodiesterase (McLaughlin et ah, 1993), rhodopsin (Dryja et ah, 1990), and the retinal degeneration slow (rds)/ peripherin genes (Farrar et ah,

1991). Mouse models for RP involving mutations in these three genes are available, and the major feature of the phenotypes is photoreceptor degeneration.

For the p subunit of cGMP phosphodiesterase gene, the retinal degeneration (rd)

mouse is an animal model that has been extensively studied (Chader et ah, 1988). The mutation in the rd mouse is recessive and results in no enzyme production (Pittler and Baehr, 1991). The photoreceptors develop normally up to the early part of the second week of life at which time photoreceptor degeneration starts. Degeneration is rapid and by postnatal day 17, virtually all the rod photoreceptors have disapperared (Carter- Dawson et ah, 1978). No spontaneous mutations of the rhodopsin gene have been identified in animals. Transgenic technology has been used however to create several lines of mice expressing mutant rhodopsin (Humphries et ah, 1997), simulating a form of autosomal dominant RP seen in humans (Dryja et ah, 1990a). In one line of transgenic mice, 50% of the nuclei in the outer nuclear layer (ONL) had degenerated by 7 weeks.

Degeneration was virtually complete by 12 months, with very few photoreceptor nuclei remaining in the retina (Chang et fl/., 1993). In another line of homozygous rhodopsin deficient mice, rod outer segments lose their photoreceptors over 3 months (Humphries

et al., 1997). In heterozygotes outer segments are present but shortened and disorganised in older mice. The rds/peripherin gene was first studied in the rds mouse (Sanyal et al.,

1980). In homozygous rds mice, the outer segment of the photoreceptor appears normal. Beginning at about 3 weeks of age, the photoreceptor nuclei in the ONL start to disappear, and by about 12 weeks, 50% of the nuclei have been lost. The degeneration is virtually complete by 12 months (Sanyal et al, 1980). Heterozygous rds mice also demonstrate abnormal photoreceptor morphology. However, the rate of degeneration is much slower than in homozygotes, and the process is never complete (Hawkins et al,

1985).

Although these three animal models represented different basic mutations, subsequent cell death was remarkably similar, with a lack of inflammatory response despite the large number of degenerated photoreceptors. Even in the Royal College of Surgeons (RCS) rat model, where the expression of the primary genetic defect is in the retinal pigment epithelium, apoptois was observed in the photoreceptors (Tso et al,

1994). In every case, photoreceptors were shown to die via the apoptotic pathway, as evidenced by the histological picture, by terminal deoxynucelotidyl transferase- mediated biotin-dUTP nick end-labelling assays (TUNEL technique) (Gavrieli et al,

1992), and/or by direct demonstration of retinal-DNA nucleosomal laddering by gel electrophoresis.