Ординатура / Офтальмология / Английские материалы / Macular Degeneration_Penfold, Provis_2005

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Contents

9.1Introduction 149

9.2Approach 150

9.3Stages in Photoreceptor Degeneration

at the Edge of the Retina 150

9.4.2Why Is the Protection

of Photoreceptors Stress-Inducible? 162

9.4.3Vulnerability to Oxidative Stress

in Mice and Humans 163 9.4.4 The Link to AMD 163

References 164

9.1 Introduction

Surveys of the human retina have reported the degeneration of the anterior edge of the retina to be a common feature (Vrabec 1967; Gartner

in Mammalian Retina: Lessons from the Edge

Jonathan Stone, Kyle Mervin, Natalie Walsh, Krisztina Valter, Jan M. Provis, Philip L. Penfold

1975; Gartner and Henkind 1981; Ahnelt 1998), sufficiently common in older humans to be regarded as “normal”. Detailed observations of mouse and rat retina have suggested that these degenerative phenomena begin very early in postnatal life (Mervin and Stone 2002a, 2002b), with a period of localized photoreceptor death. Conversely, in experimental studies of photoreceptor degeneration in adult rodents, the edge of the retina has been described as highly resistant to degeneration, whether lightor muta- tion-induced (LaVail 1981; Valter et al. 1998; Bowers et al. 2001).

This paradox, that a chronically degenerative region of retina is resistant to acute stress, has been reproduced in studies of “pre-condi- tioning” of the retina, in which limited damage to the retina (including some photoreceptor death) upregulates protective mechanisms which make the surviving photoreceptors resistant to subsequent stress (Liu et al. 1998; Nir et al. 1999; Cao et al. 2001). The same paradox is evident in the impact of ambient light levels on photoreceptor structure and vulnerability (Penn and Anderson 1991). Bright ambient light kills some photoreceptors and makes the survivors resistant to acute stress. Penn and Anderson’s data make clear that degenerative and stabilizing properties appeared in the same cell, photoreceptors exposed to stress showing degenerative membrane changes, yet proving highly resistant to light challenge.

This chapter summarises results of studies of degenerative phenomena at the edge of the retina. Taken together, these observations suggest that the edge of the retina is subject throughout life to a localized stress, which in-

duces a progressive degenerative process. These edge-specific changes are part of the life history of the normal retina, and form part of the baseline against which the retinal degenerations take place.

9.2 Approach

It was of interest to study the edge of the retina from neonatal development to senescence. For practical reasons, these observations were collected from different species. Neonatal development was studied in rodents, while the most striking phenomena of senescence were apparently only in adult human retina, after several decades of normal function. Segments of human retina were obtained through the Eye Bank of the Save Sight Institute, University of Sydney. Material was successfully obtained from individuals aged 62, 47 and 29 years. The eyes had been immersion-fixed in paraformaldehyde for up to several years. Medical records in each case contained no evidence of ocular pathology. Segments of two marmoset retinas were obtained by courtesy of Dr. Paul Martin, from animals studied in unrelated physiological experiments. One baboon eye was obtained, from an individual aged 20 years killed because of multiple organ failure which did not involve the eyes. Rodent material is summarized from recent published studies (Maslim et al. 1997; Bowers et al. 2001; Walsh et al. 2001; Mervin and Stone 2002a).

9.3

Stages in Photoreceptor Degeneration at the Edge of the Retina

9.3.1

Postnatal Development of the Edge: Site of Early Stress and Degeneration

The most detailed evidence available of the onset of stressed conditions at the edge of the retina comes from the mouse retina (Mervin and Stone 2002a, 2002b), in which a clustering of photoreceptor death at the edge of the retina

has been observed in early postnatal material (Fig. 9.1A–C). Evidence of the cause of this death comes from the observation that the site of the death (the extreme edge) is also the site of an upregulation of two stress-inducible proteins, the trophic factor FGF-2 (green in Fig. 9.1D, E) and the structural protein GFAP in the processes of Müller cells (red in Fig. 9.1D). The locus of this early episode of photoreceptor death is a narrow (<100 µm; Fig. 9.1H) strip at the extreme edge of the retina. The episode is also transient; by adulthood, the rate of photoreceptor death at the edge is not measurably higher than in the midperiphery. Nevertheless, the edge-specific upregulation of GFAP and FGF-2 persists into adulthood (Fig. 9.1F, G), suggesting that the stress persists after the rate of death slows. Finally, the upregulation of both FGF-2 and GFAP is maximal at the edge and falls towards the center of the retina (Fig. 9.1I), suggesting that the stress acts at the extreme edge, and reduces with distance from the edge.

Death of edge photoreceptors is evident in mouse retina from P14 (Fig. 9.1A). Upregulation of FGF-2 in edge photoreceptors is not evident at P14 (Fig. 9.2A), but is detected at P16 (Fig. 9.2B) and increased thereafter (Fig. 9.2C–F). It thus seems likely that the edge-specific stress causes photoreceptor death and then, with a 1- to 2-day delay, the upregulation of stress-indu- cible factors.

Insight into the nature of the edge-specific stress come from an analysis (Mervin and Stone 2002b) of the influence of hypoxia on photoreceptor death. In most of the retina, hypoxia induces photoreceptor-specific death. At the edge at P14–18 (when the episode of edgespecific photoreceptor death is maximal), hypoxia has the opposite effect, reducing death. Mervin and Stone have suggested that hyperoxic toxicity is a factor in edge-specific stress, the hyperoxia arising because of oxygen flowing to the edge from the choriocapillaris just beyond the edge of the retina.

In the rat, as in the mouse, the adult retina shows evidence of edge of stress, and is structurally and neurotrophically distinct. At the edge of the Sprague-Dawley rat retina for example, stress-inducible trophic factors CNTF and FGF-2 are upregulated, the CNTF princi-

Fig. 9.1A–I. Evidence of stress at the edge of the neonatal C57BL/6J mouse retina. This strain is non-degenera- tive. A–C At the edge of the retina, TUNEL labelling demonstrates clustering of dying cells (red) at the extreme edge of the retina, and in the ONL. D–G By P26 (D, E) and in the adult (F, G),FGF-2 (green) is upregulated in the ONL at the edge (D, F), but not in mid-periph- eral retina (E, G). GFAP (red) is present in all regions in astrocytes at the inner surface of the retina. At the edge, GFAP-labelling (red) is also seen in the radial processes of Müller cells. H Averaged over multiple sections of tis-

sue, the cluster of dying cells is confined to the extreme edge of the retina, to a region <80 µm wide, at P14, P16 and P18. I Quantitation of the immunolabel signal in F confirms the visual impression that FGF-2-labelling is maximal at the edge and decreases towards the centre of the retina, following to low levels 200 µm from the edge. The red trace shows GFAP-labelling along a transect along the INL, crossing successive processes of Müller cells. GFAP-positive Müller cell processes show as peaks in the red line; they are confined to the most peripheral 150 µm of retina. (Data from Mervin and Stone 2002a)

Fig. 9.2A–F. The development of the upregulation of FGF-2 in the ONL, in the developing C57BL/6J mouse retina. The upregulation can be detected at P16 and progresses over the following 10 days. The upregulation of

FGF-2 thus seems to follow the occurrence of photoreceptor death at the edge, which is well developed at P14 (Fig. 9.1). (Data from Mervin and Stone 2002a)

Fig. 9.3A–F. Edge/midperiphery comparison for a rat retina (non-degenerative, Sprague-Dawley albino): A–D are from control retinas, raised in dim cyclic light; F, G are from a retina after exposure to bright continuous light. A, B FGF-2-immunolabelling in mid-peripheral retina is prominent only in Müller cell somas in the INL and in the nuclei of astrocytes at the inner surface (A). CNTF-labelling (red) is prominent only in astrocytes processes at the inner surface (A). At the edge (B), FGF- 2 is prominent in the ONL and CNTF in Müller cells, cell processes extending across the retina. Note the overall thinning of the retina, including the ONL. C, D Immunolabelling (red) for CO (cytochrome oxidase) shows that in mid-peripheral retina (C) the enzyme is prominent in the inner segments (lower two asterisks) and in the OPL (top asterisk). Opsin (green) is prominent in outer segments (arrow in C). At the edge (D), the ONL

has thinned to <50% of its normal thickness, and COlabelling is less intense in both the outer segments (low- er asterisk) and in the OPL (upper asterisk). The outer segments remain strongly opsin-positive but are markedly shortened (arrow). E, F Labelling for DNA (blue) and fragmenting DNA (red, using the TUNEL technique) in mid-peripheral and edge regions of a rat retina suffering light damage after exposure to bright continuous light (Bowers et al. 2001). The photoreceptors in the ONL undergo massive DNA fragmentation (red) in mid-peripheral retina (E), with some of the fragmented DNA appearing in Müller cells in the INL. The neat stacking of somas in the ONL, apparent in C, is broken down. At the edge (F), by contrast, the photoreceptors are light-damage resistant, only a few showing evidence of DNA fragmentation. The neat packing of ONL cells is maintained

pally in Müller cells, the FGF-2 in photoreceptor somas in the ONL (Walsh et al. 2001; Fig. 9.3A, B). The ONL at the edge thins to <50% of its thickness in central retina (compare Fig. 9.3A with B, C with D), suggesting significant cumulative loss of photoreceptors. The thinning of layers at the edge, and the upregulation of FGF-2 and CNTF, are shown quantitatively in Fig. 9.4. FGF-2 (green in Fig. 9.4A, B) is most prominently upregulated in the ONL (between the OPL and OLM in Fig. 9.4B), while CNTF (red in Fig. 9.4B) is most strongly upregulated at the inner surface of the retina, and in the OPL and OLM.

9.3.2

The Edge of the Retina

Is Functionally Degraded

It is a consistent feature in the rat (Fig. 9.3C, D) and other species (below) that, at its edge, the retina appears functionally degraded. This is

evident when functional molecules are labelled. In Fig. 9.3C (mid-peripheral retina of the rat),the red label is immunolabelling for cytochrome oxidase (CO), the mitochondrial enzyme which sequesters oxygen for oxidative metabolism. The green label is immunolabelling for opsin, the protein of rhodopsin, and the blue is a DNA-specific dye. The ONL is 10–12 cells thick. CO is highly prominent in the outer part of the inner segments (between the two lower asterisks in Fig. 9.3C), and prominent also in the OPL (top asterisk in Fig. 9.3C),as blobs which correspond to the large mitochondria in the axon terminals of photoreceptors. Opsin is prominent in the outer segments (arrow in Fig. 9.3C). At the edge of the retina (Fig. 9.3D), the ONL is reduced to two to four cells, the labelling of CO in the OPL (upper asterisk in Fig. 9.3D) and in inner segments (lower asterisk in Fig. 9.3D) is less intense and the inner segments appear shorter, while opsin labelling of the OS is intense (Fig. 9.3D) but the OSs are relatively short.

9.3.3

The Edge of the Retina Is Highly Stable in the Face of Acute Stress

The edge/mid-peripheral difference in trophic factor expression and functional morphology is complemented by a striking difference in the vulnerability of photoreceptors to stress. In the Sprague-Dawley rat, exposure of the retina to bright continuous light (e.g. 2,000 lux for 24 h or 48 h) induces a photoreceptor-specific death. The severity of the death depends on the brightness, wavelength and duration of the exposure, and on the light history of the retina (reviewed by Organisciak et al. 1998). However, within one retina (thus with all the above factors constant), the severity of damage also varies with retinal location, being least at the retinal edge (Fig. 9.3E, F).

In the RCS rat, an inherited abnormality of a transport enzyme of the retinal pigment epithelium (D’Cruz et al. 2000) causes a failure of phagocytosis of the shed membrane of photoreceptor outer segments (LaVail et al. 1992). The outer segment membrane accumulates in the subretinal space,lengthening the diffusion path for oxygen to reach the photoreceptors. During the critical period of photoreceptor degeneration, RCS photoreceptors die at a high rate, driven partly by hypoxia (Valter et al. 1998). In the face of this genetically induced stress, the photoreceptors at the edge of the retina are relatively resistant, confirming earlier observations (LaVail and Battelle 1975).

In brief, photoreceptors at the edge are degraded morphologically and functionally by a chronic edge-specific stress, but contain or are surrounded by protective factors, and are highly resistant to damage.

9.3.4

The Adult Retina Aged 2 Years: Observations in the Marmoset

The morphology of a 2-year-old marmoset retina near the anterior edge is shown in Fig. 9.5A–H. Centrally, the laminar structure of the retina seems well differentiated (Fig. 9.5D). Towards the edge (Fig. 9.5D-A), the retina thins,

GFAP becomes more prominent in the radial processes of Müller cells (Fig. 9.5B) and FGF-2, which centrally is prominent only in the INL (Fig. 9.5D), is found in most surviving neurons (Fig. 9.5A). Centrally, the inner and outer segments of photoreceptors are well differentiated (Fig. 9.5H),with CO prominent in the inner segments (red) and opsin in the outer segments (green). Towards the edge (Fig. 9.5G, F, H), both inner and outer segments become shorter and less well separated. At the edge (Fig. 9.5E), neither inner nor outer segments are apparent; instead a few cells, probably surviving photoreceptors, appear rounded, with opsin prominent in their cytoplasm.

In a 7-year-old retina (Fig. 9.5I), the same trends are apparent, shown here at low power. Centrally (to the right in Fig. 9.5I), GFAP (red) is prominent in astrocytes at the inner surface of the retina. Towards the edge (arrow in Fig. 9.5I) the intensity of GFAP-labelling increases, both at the inner edge of the retina, and across its thickness. Centrally, FGF-2 (green in Fig. 9.5I) is prominent in Müller cells in the INL; towards the edge (arrow), FGF-2 is prominent in all cells present. The DNA label (blue in Fig. 9.5I) shows the cellular layers of the retina; two of the layers (INL and ONL) are seen at this magnification. At the edge (arrow in Fig. 9.5I) the separation between these two layers becomes indistinct.

9.3.5

The Adult Retina Aged 20 Years: Observations in the Baboon

In this retina, again, there is a gradual breakdown of retinal structure over the peripheralmost few millimeters of retina. For example, the inner and outer segments of photoreceptors are neatly arranged in mid-peripheral retina (>10 mm from the edge, Fig. 9.6A), with opsin (green in Fig. 9.6A) detectable in the membranes of somas, showing a limited concentration in the inner parts of the inner segments and prominent in the outer segments. CO (red) is prominent in the outer parts of the inner segments. At 1 mm and 0.6 mm from the edge (Fig. 9.6A–C), this neat lamination progressive-

Fig. 9.5A–I. Degeneration at the edge of the marmoset retina. A–D From a section labelled for GFAP (red), FGF-2 (green) and DNA (blue), from a retina aged 2 - years. At the extreme edge (A) and 1 mm away (B), the retina is thin, and GFAP is prominent in astrocytes and Müller cells. FGF-2 is prominent in cells in the INL. More centrally (C, D) the retina is thicker, and GFAP less prominent in Müller cells. E–H From a section labelled for CO (red), opsin (green) and DNA (blue), from the retina aged 2 years. Centrally (H) CO is prominent in the inner segments of photoreceptors, and opsin in outer segments. Towards the edge (G–E) the separation of in-

ner and outer segments becomes less clear. At the edge (E) inner and outer segments cannot be distinguished, and opsin is found in the cytoplasm of a few cells, presumably surviving photoreceptors. I Section at the edge of marmoset retina aged 7 years,labelled for GFAP,FGF- 2 and DNA, with the fluophores shown separately. At lower power the upregulation of GFAP (red) is apparent at the edge (arrow in top image). The upregulation of FGF-2 (green) in photoreceptors is also apparent (arrow in middle image) and the breakdown of laminar structure is also seen (arrow in lower image)

ly degrades, and opsin-positive neurites grow abnormally into inner retina (Fig. 9.6C). At the extreme edge (Fig. 9.6D), inner and outer segment structure are totally lost, although a few opsin-positive structures remain. Some of these structures resemble cells, with opsin-pos- itive cytoplasm. There is evidence of pigmentary invasion (arrow in Fig. 9.6G).

At lower power (Fig. 9.6E, F, H), it is apparent that the retina thins markedly towards the edge, to <50% of its central thickness (compare Fig. 9.6E, H). Centrally (Fig. 9.6H), GFAP (red) is confined to astrocytes at the inner surface of the retina, while, towards the edge, GFAP is expressed increasingly in the radial processes of Müller cells (Fig. 9.6E, F). The green label in

Fig. 9.6A–K. Degeneration at the edge of the baboon retina aged 20 years. A–D, G From a section labelled for CO (red) and opsin (green). Centrally (A), CO is prominent in the outer part of inner segments of rods and cones, and opsin in outer segments and, less markedly, in the inner parts of inner segments of rods. Towards the edge this separation of inner and outer segments breaks down (B). Some cells grow opsin-positive processes which extend into inner retina (C). At the edge (D), opsin is found only in the cytoplasm of isolated cells, presumably surviving photoreceptors. Pigmentary inclusions were seen in some cells, at the edge (arrow in G). E, F, H From a section labelled for GFAP (red), and

for blood vessels (green), with the lectin G. simplicifolia. Centrally (H) the retina is thick, GFAP is confined to astrocytes at the inner edge of the retina and capillaries (green) extend to the outer aspect of the INL. Near the edge (E, F), the retina is markedly thinned, blood vessels are absent and the lectin labels occasional cone sheaths and cells in the ganglion cell layer. GFAP is present in Müller cells processes, becoming particularly prominent at the edge (F). I–K From a section labelled for GFAP (red), FGF-2 (green and DNA (blue) Again, towards the edge, the thinning of the retina is clear, FGF-2 becomes prominent in cells of the outer nuclear layer (arrows in K) and GFAP in the processes of Müller cells