Cellular remodeling in mammalian retina: results from studies of experimental retinal detachment

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Abstract

Retinal detachment, the separation of the neural retina from the retinal pigmented epithelium, starts a cascade of events that results in cellular changes throughout the retina. While the degeneration of the light sensitive photoreceptor outer segments is clearly an important event, there are many other cellular changes that have the potential to significantly effect the return of vision after successful reattachment. Using animal models of detachment and reattachment we have identified many cellular changes that result in significant remodeling of the retinal tissue. These changes range from the retraction of axons by rod photoreceptors to the growth of neurites into the subretinal space and vitreous by horizontal and ganglion cells. Some neurite outgrowths, as in the case of rod bipolar cells, appear to be directed towards their normal presynaptic target. Horizontal cells may produce some directed neurites as well as extensive outgrowths that have no apparent target. A subset of reactive ganglion cells all fall into the latter category. Müller cells, the radial glia of the retina, undergo numerous changes ranging from proliferation to a wholesale structural reorganization as they grow into the subretinal space (after detachment) or vitreous after reattachment. In a few cases have we been able to identify molecular changes that correlate with the structural remodeling. Similar changes to those observed in the animal models have now been observed in human tissue samples, leading us to conclude that this research may help us understand the imperfect return of vision occurring after successful reattachment surgery. The mammalian retina clearly has a vast repertoire of cellular responses to injury, understanding these may help us improve upon current therapies or devise new therapies for blinding conditions.

Introduction

Two decades of studying experimental retinal detachment (and reattachment) have led us into new avenues of thinking about the responsiveness of mammalian retina to injury, especially with respect to the degree of cellular remodeling that occurs among the retinal neurons and glia. In general, the neural circuitry of the adult vertebrate retina has not been considered as a highly “plastic” or malleable component of the central nervous system. Its task, transducing light energy into electrical impulses and then encoding parts of a visual image for reconstruction by the visual centers of the brain, may seem best served by a relatively hard-wired system. For example, the fact that it is possible to identify specific neural circuits in the retina (Dowling, 1970; Kolb and Famiglieti, 1976; Linberg et al., 2001b; Kolb et al., 2001) and that many of these circuits are preserved between species could be interpreted to support this concept. Indeed, there are references dating back to the late 1800s describing the reactivity of neurons in the brain and spinal cord to injury. However, only recently have similar changes in the retina received much attention, especially from those who study photoreceptor degenerative diseases.

In the last few years descriptions of cellular remodeling in the vertebrate retina have begun to appear with some regularity in the published literature. Prior to this, the few descriptions of structural remodeling came from studies of fish retinas where Wagner and colleagues (Wagner, 1975, Wagner, 1980; Wagner and Ali, 1977) described the remodeling of cone photoreceptor synapses and horizontal cell processes in response to transitions of the light/dark cycle. Peichl and Bolz (1984) described structural remodeling of retinal neurons in response to severe retinal degeneration induced by kainic acid. It was nearly a decade later that reports of cellular remodeling in mammalian retina began to appear with some regularity (Chu et al., 1993; Li et al., 1995; Lewis et al., 1998; Fariss et al., 2000). Thus, the published literature reinforced the impression of the retina as relatively static structurally. Even total photoreceptor cell loss was not regarded as causing significant changes to the neuronal components of the inner retina, probably due in part to the difficulties of visualizing the often subtle changes in organization that accompany such alterations. Now it seems likely that the retina shares with the rest of the CNS a significant capacity for remodeling of its cellular architecture. The functional consequences of such remodeling largely remain unknown, although various informal and anecdotal clinical reports along with a few systematic studies of human vision after retinal reattachment surgery suggest that there are functional consequences because visual recovery occurs over a much longer time-period than would be expected based on outer segment regeneration alone.

Many of the recent descriptions of structural remodeling in mammalian retina are from observations in humans or rodent species in which massive photoreceptor cell death is induced by light damage or genetic mutations. The literature on this topic has recently been reviewed extensively by Marc et al. (2003), and data from the detachment models compliment data from those systems because in most species there is not massive photoreceptor cell death after detachment. Another important distinction is that the earliest, and most obvious damage induced by detachment, outer segment degeneration, is reversible by reattaching the retina. The reversibility of inner retinal changes has, for the most part not been studied in detail.

Here we will discuss remodeling events in the retinal cell types illustrated in Fig. 1, including: Retinal pigmented epithelium, Müller cells, photoreceptors, rod bipolar cells, horizontal cells, ganglion cells, and astrocytes.

Animal models of retinal detachment have been available since the late 1960s (Kroll and Machemer, 1968). In early light and EM studies (Erickson et al., 1983; Anderson et al., 1983) outer segment degeneration, photoreceptor cell loss, Müller cell hypertrophy, and changes in the retinal pigmented epithelium (RPE) apical surface were all recognized as significant cellular events induced by detachment. More recently as we have gained the technological advantages of immunocytochemistry and confocal imaging (along with other methods of advanced imaging), many studies have shown the events after detachment to be far more complex than previously expected, and to include a significant amount of neural and glial remodeling. Presumably each cellular change induced by detachment indicates a change that is ideally reversed by reattachment in order for the retina (and vision) to return to its pre-detachment condition. Clearly, changes in the retina created by cell death are not going to be reversed by any current therapy. Other events may be reversed by reattachment, often incompletely and usually slowly—over a time-course that can vary from days to years. Because experimental studies have, out of practical necessity, been limited in time we do not know what effects may continue for years after reattachment. Retinal circuitry may have a great deal of built in redundancy, in which case it may not be necessary to reverse all of the damage caused by detachment in order to have relatively normal vision. Likewise, photoreceptor outer segments may need only some fraction of their normal length in order to function with reasonable efficiency. The link between molecular expression, cellular architecture and recovery of normal vision remains relatively unexplored territory. The literature now clearly demonstrates that reactions to detachment occur across the retina, from photoreceptors to ganglion cells, and this research is still in an early stage. As a model system, retinal detachment has some advantages: (1) The ability to study events associated with both degeneration (after detachment) and recovery (after reattachment). (2) Control over the starting times for these two events, and the duration of each. (3) The effect of detachment height (the distance the neural retina and RPE are separated) can be controlled. (4) The extent (size) of a detachment can be controlled which also provides the opportunity to study precise transitions between detached and attached retinas.

The difficulties of describing cellular events in the CNS are well documented from historical arguments that occurred well into the mid-20th century questioning the application of cell theory to neural tissue. The vast organizational complexity of CNS tissue, the huge numbers of neurons and glial cells involved, the vast range of intricacy in neuronal architecture, the small size of neuronal cell bodies relative to other cells, and the small size of the neuronal processes that intertwine to make up the traditional synaptic neuropil (or plexiform layers in the case of the retina) all combine to make convincing and reliable observations of changes in cellular morphology difficult-to-nearly impossible by the methods of traditional light and electron microscopy. Historically, discovery of the Golgi impregnation method was the breakthrough that allowed for a detailed description of individual neurons and their morphologic diversity (Ramón y Cajal, 1892).

Similarly, immunocytochemistry and other current techniques that label individual cells or populations of cells have permitted the discovery of remodeling events in the retina in recent years. In many ways the Golgi method would be ideal for uncovering changes in neuronal morphology occurring as a result of injury or insult to the nervous system, except that such discovery requires a reliable method that will give reproducible results in a large population of cells (to account for individual variation among cells). This is not a strength of the Golgi technique; well-known for its capriciousness and apparent randomness in producing stained cells. Recent advances in biological imaging (i.e., confocal microscopy), image processing, and cytochemical labeling (usually but not always by antibodies) have allowed for consistent, reliable means of documenting changes in cell morphology and the expression pattern of specific molecules. Thus data that have lead us to view the retina as a highly plastic tissue, one capable of undergoing significant cellular remodeling in response to injury. With this technology whole populations of cells can be reliably studied in different tissue samples. Antibody labeling of tissue sections can provide precise quantitative information under the right conditions (see Marc et al., 2003), or at least semi-quantitative comparisons of molecular expression levels if one is careful to keep control of variables in specimen preparation (fixation, hydration, dehydration, embedding medium, etc.), section thickness, and image collection parameters.

The power of the newer technology can be illustrated by an example from data published from one of our earlier studies of retinal detachment. Erickson et al. (1983) published ultrastructural evidence suggesting that some rod photoreceptors retract their synaptic terminals after detachment. This conclusion was based on the observation that synaptic ribbons and vesicles came to lie in the cell body, adjacent to the nucleus instead of at the end of a rod axon (or synaptic spherule) in the outer plexiform layer (OPL). The limited sampling provided by electron microscopy made visualizing the extent of this event cumbersome and thus a number of questions were left unanswered by the study: was this a rare event, or did it occur widely throughout the photoreceptor population? Did it only occur in photoreceptor cells on the path to cell death? If it is a widespread phenomenon, then what are the effects on cells connected to the retracted rod terminals (i.e., rod bipolar dendrites and horizontal cell axon terminals)? These questions were answered only as antibodies became available to label the specific players: photoreceptor synaptic terminals, rod bipolar, and horizontal cells (Lewis et al., 1998). However, the ability to correlate changes in antibody labeling patterns with the ultrastructural images was critical, because protein expression profiles (e.g., amount, labeling pattern) can change dramatically in a cell responding to injury (as occurs in cone photoreceptors). Specimen preparation techniques can also lead to different results. Detached feline retinal tissue embedded in wax using traditional dehydration techniques, provided an image of rod bipolar cells without dendrites, i.e. victims of extreme dendritic “pruning”, while the current technology (Matsumoto and Hale, 1993) in which specimens are not dehydrated, demonstrated prominent neurite outgrowth (with some significant pruning as well) from this same cell type (Lewis et al., 1998). Thus, advances in bioimaging technology have lead to some unexpected insight into the range and variety of the retina's responses to injury. Speculation based on EM data in 1983 resulted in the following conclusion: “In addition to the effects of retinal detachment in the outer retina, we strongly suspect that the inner nuclear layer, inner plexiform layer, ganglion cell layer and, perhaps, more central areas of the visual system may be affected as well” (Erickson et al., 1983). Remodeling has now been firmly established in the inner retina, and leads one to believe more strongly that central changes, for example ganglion cell axonal arborizations and synaptic contacts, will be eventually identified as well. It is clear that the adult retina is not a static structure. Like the rest of the CNS, it has a wide repertoire of inherent responses to injury and many of these result in significant cellular remodeling.

In general, we are using the term “remodeling” to refer to structural changes in retinal cells, although these structural events will always be built on a foundation of molecular changes, and ultimately it is these that we will need to identify, understand and manipulate the mechanisms involved. Some of the changes that we describe can occur generally across a whole population of cells within the affected retinal area, although there is always inter-cell variation. For example, photoreceptor cell death changes the photoreceptor population and alters overall thickness of the outer nuclear layer (ONL). Outer segments are greatly shortened in all photoreceptors in a region of detachment, but the exact amount of outer segment loss may vary slightly from cell to cell. All inner segments appear to lose mitochondria and to show a “mixing” of organelles segregated normally to the ellipsoid (mitochondria) or myoid (ER, RER, Golgi). The apical surface of the RPE in a region of detachment is remodeled as the cells become “mounded” and their distinctive and highly specialized apical processes are replaced by a uniform fringe of microvilli (Figs. 2A–C, asterisk). Indeed, the latter may be the most invariant cellular remodeling that occurs in response to detachment. It appears to be constant from cell-to-cell and species-to-species. This surface must remodel again in response to reattachment as the apical processes are reformed, and here the remodeling is not so homogeneous (Figs. 2D and E); a good indicator that regeneration is not a perfect recapitulation of developmental events. In many cases, it is difficult to determine the number of cells within a population that are responding to detachment, and quantifying the responses may be critical to understanding the reaction to detachment and the recovery process. Cellular proliferation is stimulated in all non-neuronal cell types (Müller cells, RPE, astrocytes, vascular endothelium, pericytes) by detachment (Fisher et al., 1991), but the proportion of the different cell populations that proliferate is unknown. While all Müller cells appear to upregulate intermediate filament proteins, only some extend processes into the subretinal space or onto the vitreal surface, and both of these events result in serious ophthalmic diseases. Only a subpopulation of ganglion cells appear to remodel in response to detachment (Coblentz et al., 2003), and identifying which types may lead to a better understanding of some visual disturbances that occur after successful reattachment surgery.

Marc et al. (2003) discuss the remodeling of the inner retina as occurring largely in response to “deafferentation”, that is, the massive loss (usually approaching 100% but at varying rates) of photoreceptors in the retinas of animals and humans with inherited degenerations. They conclude that it is this deafferentation that leads to dramatic changes in the organization of the remaining retinal tissue; reorganization that is so extensive during the latter stages as to make it conceptually difficult to think of “recovery”, even if a new population of photoreceptor cells could be introduced into the tissue. Many of the remodeling events that we will describe here are similar to those occurring in the retinal degenerations, but they are often more subtle, may appear more quickly, and do not usually lead to gross rearrangements of the tissue. In the model system we have used for most of our studies (the feline retina), they are not occurring in response to a massive loss of photoreceptors. Indeed, more recent evidence (Claes et al., 2004) indicates that in mice in which both rod and cone degeneration is caused by knock-out mutations, early neuronal remodeling is very similar to what occurs in detachment, but with more dramatic, and drastic structural changes occurring in late stages when massive photoreceptor loss has occurred. Total photoreceptor cell death does occur after detachment in some species such as rabbit and ground squirrel. Interestingly, despite loss of virtually all of its photoreceptors after detachment, the ground squirrel retina shows little remodeling of inner retinal neurons and essentially none of the typical reactivity associated with Müller cells (Linberg et al., 2002a). Available evidence suggests that photoreceptor loss in human detachment is similar to that in the feline model (Chang et al., 1995). The changes that we have focused on are those that occur within hours, or days of detachment, because it is these changes that may be successfully manipulated by therapeutic intervention and thus lead to better visual recovery. Except for the loss of photoreceptors, the hypertrophy of Müller cells, and the remodeling of the RPE, the neuronal remodeling that is associated with detachment cannot be appreciated by ordinary histological observation. While photoreceptor cell death may play a role in inducing these events, it is probably not the sole cause. Indeed molecular events that may lead to remodeling are induced very rapidly in Müller cells (Geller et al., 2001) before TUNEL positive photoreceptors (a measure of apoptotic cell death) appear in the ONL. Furthermore, remodeling is not just associated with the detachment and the subsequent degeneration of photoreceptors, but with the recovery phase after retinal reattachment. Until some method for replacing lost photoreceptors is found, it is not possible to study recovery in the models that lead to deafferentation of the inner retina.

Section snippets

A description of the model system

The retina as a developmental outgrowth of the brain is highly specialized for the transduction and primary processing of visual information. It is well-known that neuronal regeneration is extremely limited in the brain and spinal cord, but at the same time, these tissues react vigorously to injury in many ways: astrocytes migrate, proliferate, hypertrophy and form glial scars (Pekny et al., 1999), and oligodendrocytes migrate and express molecules on their surface that appear to inhibit axonal

Retinal remodeling, detachment and reattachment

Studies of detached (and reattached) retina have shown us that the mammalian retina has remarkable remodeling capabilities. Fig. 27 shows in summary form the remodeling of the neural retina described here. The only change of this type for which we may have some underlying mechanistic explanation exists is outer segment regeneration. One of the most important discoveries in retinal cell biology was that of the phenomenon of outer segment renewal (Young, 1967). Although the details of the

Future directions

Although there are many unanswered questions, data from the detachment model, as well as data from a variety of other studies of retinal degeneration now suggests that retinal neurons remain capable of significant structural remodeling in adult mammals. This in turn may provide increased optimism for a variety of therapies for blinding diseases in which photoreceptor degeneration is the primary cause of visual loss. Although preventing photoreceptor cell death is the optimum therapy in these

Acknowledgments

We wish to acknowledge the contributions of many current and former members of our laboratory who have provided invaluable technical assistance, often on a volunteer basis, and data used in this discussion, these include: Edward Barawid, Kellen Betts, Meghan Brown, Katrina Carter, Amy Cline, Francie Coblentz, Ph.D., Scott Geller, Ph.D., Tonia Rex, Ph.D., Patrick Johnson, Ph.D., William Leitner, Linda Martin, Bryce Pulliam, Melvin Rabena, Tsotomu Sakai, M.D., Kevin Talaga, Bastel Wardak, Peter

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