RPE–photoreceptor interface

The earliest structural effects of retinal detachment are seen at the interface of photoreceptor outer segments and the RPE.¹⁰ The mature RPE is a polarized monolayer of neuroepithelial cells that rests on Bruch’s membrane, between the choriocapillaris and the neural retina.¹¹ The relationship of the apical surface of the RPE to differentiated photoreceptors is anatomically complex. There are no actual cellular junctions between the two layers in the mature eye, but the two are adherent, with the degree of adhesion varying among species.¹² With the onset of retinal detachment changes to this interface include alterations in the RPE apical surface, proliferation of RPE cells, migration of cells into the subretinal space, degeneration of photoreceptor outer segments, and changes in photoreceptor outer-segment renewal.¹

Within a few hours of retinal detachment, the long and elaborate sheet-like and villous processes that normally ensheath the outer segments are lost and replaced by a “fringe” of short microvilli (Fig. 29.1).¹³ At the same time, the overall surface morphology of the RPE cells changes into a rounded contour, as cytoplasm protrudes past the normal limits of the apical surface into the subretinal space, and the nucleus becomes displaced to a more apical position^10,14 (Fig. 29.2).

Fig. 29.1 Electron micrograph of the retinal pigment epithelium (RPE) 1 day after production of a retinal detachment. Compared with normal RPE cells, the apical surface is mounded. The sheet-like apical projections that normally ensheath the outer segments have been replaced by a homogeneous fringe of short, microvillous processes (MV). In this particular cell, the nucleus (N) is displaced into the mounded region. The cells’ lateral junctions are indicated by arrows. SRS, subretinal space.

(Reproduced with permission from Anderson DH, Stern WH, Fisher SK, et al. Retinal detachment in the cat: The pigment epithelial–photoreceptor interface. Invest Ophthalmol Vis Sci 1983;24:909.)

Fig. 29.2 Scanning electron micrograph of the apical surface of the retinal pigment epithelium 6 weeks after production of an experimental detachment, demonstrating the pronounced mounding response of the epithelial cells.

(Reproduced with permission from Anderson DH, Stern WH, Fisher SK, et al. Retinal detachment in the cat: The pigment epithelial–photoreceptor interface. Invest Ophthalmol Vis Sci 1983;24:910.)

In the feline model, experiments using ³H-thymidine have shown that within 72 hours of retinal detachment the RPE has begun to proliferate and may be observed as areas of hyperplasia within the RPE monolayer.¹⁰ This proliferative response transforms the RPE’s uniform monolayer into a heterogeneous morphology in which strands of cells extend from the original monolayer into the subretinal space or result in the formation of multiple layers of cells whose polarity does not necessarily match that of the original monolayer (Fig. 29.3). This effect is limited to the region of detachment; in attached regions the RPE remains mitotically quiet, suggesting that attachment of the RPE to the neural retina acts to keep the RPE mitotically inactive and its apical surface highly differentiated.^15–17 The proliferative response of the RPE cells also appears to be self-limiting with only low levels of proliferation observed after long detachment intervals (e.g., 12–14 months) in owl, monkey, and cat retinas^10,16 (Box 29.1 and Fig. 29.4).

Fig. 29.3 Light micrograph of an area of retinal pigment epithelium (RPE) cell proliferation in a cat retina detached for 14 days and reattached for 30 days. Three layers of RPE cells are present (L1-L3), each displaying different surface polarity. The apical surfaces of L1 and L2 face each other, as do the basal surfaces of L2 and L3. The basal lamina of L2 is clearly evident (arrow). Only outer-segment fragments (asterisk) appear near the inner segment (IS) tips (×800). ONL, outer nuclear layer.

(Reproduced with permission from Anderson DH, Guerin CJ, Erickson PA, et al. Morphological recovery in the reattached retina. Invest Ophthalmol Vis Sci 1986;27:174.)

Fig. 29.4 Color fundus photograph of a chronic retinal detachment with a pigmented demarcation line indicating the interface between attached and detached retina.

The subretinal space is usually free of cells; however within 24 hours of retinal detachment a number of cell types (polymorphonuclear neutrophils, monocytes, and macrophages) migrate into this space from the choroidal and retinal capillaries.^10,18 Free RPE cells are also seen in the subretinal space within 72 hours of retinal detachment and frequently contain outer-segment fragments, indicating that they may play a role in phagocytosis of cellular debris.^10,18

Photoreceptors

Within 12 hours of experimental retinal detachment, photoreceptor outer segments show evidence of structural damage. Initially, the distal end of the outer segment becomes vacuolated or distorted and by 24–72 hours all rod and cone outer segments are significantly shorter and distorted with disoriented discs.¹⁹ The degeneration of outer segments may proceed until those in the zone of detachment appear only as empty sacs of membrane attached to the connecting cilium.¹⁰

Outer-segment debris is shed into the subretinal space where it is phagocytosed by RPE cells and macrophages which have migrated into the area.^10,18

Although retinal detachment interrupts the process of disc production and shedding, outer-segment specific proteins continue to be produced but localize to abnormal cellular locations. Opsin, normally concentrated in the outer segment, begins to accumulate in the plasma membrane vitread to the outer segment within a day following experimental retinal detachment (Fig. 29.5).¹⁹ Peripherin/rds, another outer-segment protein specific to the disc rims, is also redistributed and begins to appear in cytoplasmic vesicles.²⁰ Cone outer-segment proteins appear to be more susceptible to damage, with redistributed cone opsins persisting for just 1 week following retinal detachment, after which their expression is downregulated.²¹

Fig. 29.5 Laser scanning confocal images of normal (A), 7-day (B), and 28-day (C) cat retinas, as well as a human detached retina (D), labeled with isolectin B4 (green), antiglial fibrillary acidic protein (anti-GFAP) (blue), and antirod opsin (red). In the normal retina, isolectin B4 labels microglia in the inner plexiform layer (IPL) (and blood vessels), anti-GFAP labels astrocytes and Müller cell endfeet, and antirod opsin labels rod outer segments (OS) (A). Following 7 days of detachment, isolectin B4 labeling illustrates an overall increase in the number of microglia and their presence in the outer retina, rod opsin becomes mislocalized to the photoreceptor cell bodies, and GFAP increases in Müller cells (B). At 28 days, numerous microglia can be observed throughout the retina, photoreceptor cell bodies are extruded out of the retina, and Müller cell processes extend into the subretinal space (C). A similar pattern of increased numbers of microglia, rod opsin mislocalization, and increased GFAP labeling is observed in human detached retinas (D). Note rod axons extending into the inner retina in (D), a common phenomenon in cat retinas following detachment and reattachment. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

During the first day of a detachment the inner segments appear essentially normal, but between the first and third days they begin to show signs of degeneration: most commonly swelling, disruption, and loss of mitochondria (and loss of anticytochrome oxidase labeling) in the ellipsoid region,^3,22 an overall disruption of the organized rough endoplasmic reticulum and Golgi apparatus in the myoid region, and, within a few days, an overall size reduction of the inner segment. It is interesting to note that the connecting cilium, which is essential for production of the outer segment, is retained even in severely affected inner segments in long-term detachments. This is crucial as its loss would prevent regeneration of outer segments following reattachment. Similarly, the loss of mitochondria also has the potential to affect the photoreceptors’ ability to regenerate significantly, because the metabolic rate in these cells is among the highest of any in the body.

The outer nuclear layer contains the cell bodies of the photoreceptor cells. These cell bodies extend a process toward the outer plexiform layer, where they form synapses with second-order neurons. Rods and cones have characteristic synaptic terminals called spherules and pedicles respectively.²³ The outer plexiform layer also contains the processes of second-order neurons, the cell bodies of which lie in the inner nuclear layer. These processes synapse with each other and with the photoreceptors. The photoreceptor cell bodies and synaptic terminals show a rapid response to detachment with extensive vacuolization, degeneration of mitochondria, and disorganization of the microtubules and actin filaments. Cell death via the apoptotic pathway peaks at day 3 following retinal detachment but continues at low levels for as long as the retina is detached, a process that appears to be mediated via caspases 3, 7, and 9.^24,25 Recent studies have shown that, when caspase pathways are blocked, receptor interacting protein (RIP) kinases promote necrosis and overcome apoptosis inhibition. Therefore, targeting of both caspase and RIP kinase pathways is required for effective photoreceptor protection²⁶ (Box 29.2).

Following cell death some photoreceptors are extruded into the subretinal space where they are phagocytosed by macrophages while others appear to undergo degeneration and phagocytosis within the outer nuclear layer.²⁸

Not all photoreceptors degenerate at the same rate; areas of extensive degeneration coexist with areas of relatively intact photoreceptors.¹⁹ It does appear that rod cell bodies appear to degenerate quicker than cones following retinal detachment.²¹ In a region in which nearly all of the rod cell bodies show signs of degeneration and even cell death, neighboring cone cell bodies may look relatively intact. Consistent with this observation, the rod spherules appear to be particularly susceptible to the effects of detachment. These synaptic terminals are normally filled with synaptic vesicles and contain one or two large presynaptic ribbons. When the retina has been detached for 3 days, many of these terminals appear depleted of vesicles, except for a few that remain as a halo around a greatly truncated ribbon.²⁹ Many terminals appear as if they have “retracted” into the cell body, and some synaptic structures generally associated with the outer plexiform layer now occur within the outer nuclear layer (Fig. 29.6).^29,30 As with the cone and rod photoreceptor cell bodies, the cone synaptic terminals seem to survive the early effects of detachment better than the rod terminals do. Although their shape can change fairly dramatically, they do not appear to retract and by electron microscopy they remain filled with synaptic vesicles.^30,31

Fig. 29.6 Laser scanning confocal images of normal (A) and 28-day detached (B) cat retinas labeled with antisynaptophysin (green), anti-protein kinase C (anti-PKC) (red), and antineurofilament (blue). In the normal retina, antisynaptophysin labels synaptic vesicles in the rod and cone terminals in the outer plexiform layer as well as synaptic terminals in the inner plexiform layer, anti-PKC labels rod bipolar cells, and antineurofilament labels ganglion cell axons and horizontal cell processes (A). After detachment, rod terminals retract into the outer nuclear layer (ONL), as evidenced by the synaptophysin labeling, the dendrites of rod bipolar cells extend into the ONL, while horizontal cell processes grow through the ONL into the subretinal space (B). Neurofilament labeling also increases in some ganglion cell bodies (B). OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

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