Chapter 29 Cellular Effects of Detachment and Reattachment on the Neural Retina and the Retinal Pigment Epithelium
In retinal detachment the separation of the neural retinal from the retinal pigment epithelium (RPE) initiates a complex series of cellular and molecular changes.1 Left untreated, retinal detachment results in permanent visual loss; however, early intervention may be associated with good visual outcomes, suggesting that some of these molecular changes may be arrested or reversed.2,3
By studying the cellular and molecular changes that occur after detachment and/or reattachment clinicians may gain a more precise understanding of the degenerative processes within the retina that lead to visual impairment and the mechanisms underlying the serious complications of detachment, such as proliferative vitreoretinopathy (PVR). In addition, these insights may aid the development of future treatment strategies and adjunctive therapies aimed at improving visual outcomes.
This chapter reviews the many changes that occur in retinal cells following retinal detachment and the ensuing process of morphologic recovery following reattachment, as revealed by human case series and studies of experimental retinal detachment and reattachment in animal models.
Human studies of cellular changes following acute retinal detachment are limited to isolated case reports as surgical management does not routinely involve removal of retinal tissue. More recently data have become available from patients undergoing macular translocation surgery in which the retina is detached as part of the procedure, allowing sampling of the retina as early as 1 hour following detachment.4 In patients with advanced stages of retinal detachment and PVR, surgical management may involve excising areas of scarred retinal tissue allowing histopathological analysis. However, the data from human studies is still limited by small numbers, the challenges of sampling and analyzing small retinal specimens, and an inability to study cellular recovery following reattachment.4–7
This difficulty in obtaining retinal tissue from patients with retinal detachments has led to the use of animal models. Animal models have been developed in a variety of mammalian species from rodents to primates, most commonly in rabbits and cats, and more recently in mice. The feline retina is rod-dominant, and has an intraretinal circulation that is excluded from the photoreceptor layer and a choroidal circulation that supplies the photoreceptor layer. The rabbit retina is also rod-dominant but has no intraretinal vasculature, with the inner retina being supplied by vessels that lie on the vitreal surface. The rabbit retina has proved to be a more difficult animal model in long-term experiments because the retina tends to degenerate very rapidly following detachment; for short-term studies, however, (i.e., 3–7 days) rabbits continue to be a very useful model.
Ideally the characteristics of an experimental detachment should closely mimic those found in humans while allowing for precise control over the extent of separation between the two layers (detachment height), the location of the detachment, its surface area, and the onset of detachment (or reattachment). A number of methods have been used to simulate human retinal detachment in animal models. These range from creating large retinal tears to subretinal injections of fluid or viscous substances. Experiments where retinal detachment induction is standardized with a micropipette provide a very controlled environment for analysis; however they differ from the clinical pattern of events in which acute retinal tears of variable size are induced by vitreoretinal traction at the time of posterior vitreous detachment. It is possible that retinal tearing may act as a more potent stimulus for cellular disorganization, loss, and remodeling, leading more rapidly to the advanced pathology usually seen following longer periods of retinal detachment in animal models. Although experiments involving animal models may differ in methodology, species used, and outcome measures, they have yielded similar results to give a relatively detailed profile of the changes that occur after detachment. Retinal tissue removed from human postmortem specimens and from patients undergoing retinal detachment surgery has demonstrated changes similar to those seen in animal models.6–8
Animal models have proved invaluable in providing insight into the regenerative capacity of photoreceptor cells and the ability of reattachment to slow, stop, or reverse changes induced by detachment. They also continue to provide opportunities to test adjunctive agents targeting neuroprotection and wound healing before progressing to human surgical trials. Further, in mouse models the large variety of genetic mutations that exist provides additional scope to study retinal detachment and potential gene therapies.
Descriptions of the cellular response to retinal detachment are frequently divided into those observed in the early stages of retinal detachment and those observed in more chronic cases and in PVR. There is, however, a continuous progression of pathological change.
A rapid response to retinal detachment has been shown to occur within 15 minutes, including phosphorylation of fibroblast growth factor receptor (FGFR-1) and increased expression by RPE and Müller cells of extracellular signal-regulated kinase and activator protein transcription factor.9 This initiates a cascade of events that leads to a number of molecular and cellular changes within the retina and RPE.
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 position10,14 (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 retinas10,16 (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.)
Retinal pigment epithelial (RPE) proliferation can be seen as subretinal pigment deposition in chronic retinal detachments. It is likely that the demarcation lines noted in human retinal detachments represent zones of proliferated RPE occurring at transitions between detached and attached regions of the eye (Fig. 29.4).
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
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
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.