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From ocular hypertension to ganglion cell death: a theoretical sequence of events leading to glaucoma
From ocular hypertension to ganglion cell death: a theoretical sequence of events leading to glaucoma Robert W. Nickells, PhD ABSTRACT • RÉSUMÉ
There is substantial evidence that elevated intraocular pressure (IOP) is a critical risk factor for glaucoma.Even with exhaustive investigation, however, there is still little understanding of the pathologic events that translate increased IOP into the process of retinal ganglion cell death. New studies, particularly in rat and mouse models of glaucoma, have helped elucidate some of the important events associated with the initiation of glaucoma. This review summarizes a 5-stage series hypothesizing that elevated IOP causes deleterious changes to glia in the optic nerve head (stage 1), which activate the autonomous self-destruction of ganglion cell axons (stage 2), leading to the loss of neurotrophic support and apoptotic death of ganglion cell somas in the retina (stage 3). In the initial wave of ganglion cell death, dying cells may adversely affect their neighbouring cells in a wave of secondary degeneration involving glutamate exposure (stage 4). As ganglion cell structures disappear through the processes of cell death, glia are again involved, but this time to replace the lost neural tissue with a glial scar (stage 5). Il est solidement établi que la pression intraoculaire (PIO) élevée est un facteur de risque crucial du glaucome. Toutefois, même après des recherches exhaustives, on comprend toujours peu l’activité pathologique par laquelle la PIO élevée se traduit par la mort des cellules ganglionnaires de la rétine. De nouvelles études, effectuées notamment sur des modèles de glaucome chez des rats et des souris, ont aidé à élucider certaines activités importantes associées avec le déclenchement du glaucome. Cette revue résume en 5 étapes l’hypothèse selon laquelle la PIO élevée cause des modifications nocives à la névroglie de la tête du nerf optique (1re étape). Cela déclenche l’autodestruction autonome des axones des cellules ganglionnaires (2e étape) et mène à la perte du soutien neurotrophique et à la mort apoptotique des somas de cellules ganglionnaires de la rétine (3e étape). Dans la vague initiale de décès des cellules ganglionnaires, les cellules mourantes peuvent affecter les cellules adjacentes entraînant une deuxième vague de dégénérescence impliquant l’exposition au glutamate (4e étape). À mesure que disparaissent les structures des cellules ganglionnaires dans le processus de mortalité des cellules, la névroglie est de nouveau impliquée, mais cette fois pour combler la perte de tissu nerveux par une cicatrice gliale (5e étape).
G
laucoma is an optic neuropathy characterized by the progressive degeneration of both the retinal ganglion cell axons in the nerve and cell bodies (somas) in the retina proper. Although glaucoma is considered a multivariate and complex genetic disease, the unifying and most important risk factor is elevated intraocular pressure (IOP). Nearly all individuals who have glaucoma exhibit higher than normal IOPs. Large, multicentre clinical trials, along with some controlled laboratory studies, have now verified that lowering IOP is associated with an attenuation of progressive optic nerve damage.1–6 Similarly, even persons with normal-tension glaucoma benefit from IOP-lowering therapy.7 In this assessment, it is reasonable to speculate that IOP, at any
level, applies some critical stress to the ganglion cells and their axons. The nature of the stress applied to ganglion cells is still not well characterized. Studies conducted by biomechanical engineers using finite element modeling of the sclera and lamina cribrosa have suggested a striking relation between the level of IOP, the mechanical properties of the sclera, and the sheer force applied to the laminar region.8–11 This force can have an impact on all the “soft” tissues occupying the lamina cribrosa, including the ganglion cell axons, capillaries supplying blood to the region, and the glial cellular component, including astrocytes and microglia. A theory, encompassing 5 different stages, is emerging that ties together the relation between elevated
From the Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisc.
Correspondence to: Robert W. Nickells, PhD, Ophthalmology and Visual Sciences, 6640 MSC, 1300 University Ave., University of Wisconsin, Madison WI 53706; fax 608-262-0479; [email protected]
Originally received Jan. 16, 2007 Accepted for publication Feb. 2, 2007
This article has been peer-reviewed. Cet article a été évalué par les pairs. Can J Ophthalmol 2007;42:278–87 doi: 10.3129/can.j.ophthalmol.i07-036
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Ganglion cell death in glaucoma—Nickells
IOP and the sequence of events that lead to ganglion cell death (Fig. 1). Evaluation of these stages, along with supporting experimental data, shows how this simple model may explain many of the conflicting hypotheses of glaucoma pathogenesis that have arisen over the years. STAGE 1: ELEVATION
OF
IOP AND THE ACTIVATION
OF
OPTIC NERVE GLIA IN THE LAMINA CRIBROSA
Several years ago, seminal studies by Quigley and colleagues suggested that the initial site of damage in glaucoma was at the lamina cribrosa.12–14 An important aspect of this observation was that both retrograde and
anterograde axonal transport was blocked at this level in glaucomatous eyes.15–17 More recent studies have shown that there is disruption of the transport of specific molecules, including neurotrophins and their receptors18,19 and dynein motor proteins.20 The majority of this work relied on nonhuman primate and rat models of experimental glaucoma. Within the last 10 years, however, a model of chronic inherited disease has been characterized in the DBA/2J inbred mouse.21–26 These mice carry genetic defects that cause iris atrophy and iris pigment dispersion, which lead to the obstruction and scarification of the trabecular meshwork. By 8–9 months of age, they have developed elevated IOP, and by 10–12 months they exhibit an optic neuropathy with similarities to human glaucoma. Anatomic studies of glaucoma in these mice support the concept that initial damage occurs at the lamina. Independent examinations of the pattern of retinal degeneration in these animals has revealed that loss of Fig. 1—The 5 stages of disease progression in glaucoma: from elevated intraocular pressure (IOP) to retinal ganglion cell death. A series of 7 cartoons depicting the 5 stages of glaucoma progression. A mouse eye is drawn because many of the new details emerging about the pathology of glaucoma are coming from studies using rodent models. In the top panel, the basic features of the eye are shown. Ganglion cell structures are depicted in light blue and are made up of axons in the optic nerve, and cell somas and dendritic trees in the retina. In stage 1 of glaucoma (second panel from the top), elevated intraocular pressure exerts force around the globe (dark arrows).This force is distributed along the sclera toward the weakest point in the globe, at the scleral canal (red arrows).The force exerts some unknown stress at the level of the scleral canal, which affects the structures and tissues making up the lamina cribrosa (yellow shading). In this theoretical model, the stress causes changes in the glia of the optic nerve head. In stage 2, the optic nerve head glia become activated and probably cause damage to the axons in the region (red shading).The damage likely both affects axonal transport, which is blocked in each direction at this level, and initiates an autonomous axonal self-destruct program (yellow shading). In stage 3, the axons are undergoing disassembly in a retrograde fashion (red–yellow gradient shading), while there is complete blockage of the transport of neurotrophins to the retina at the level of the lamina (red shading). In the retina, ganglion cell somas most affected by this begin their own self-destruct program called apoptosis (yellow circles in the retina). In stage 4, ganglion cells affected in stage 3 are dead (red circles in the retina) and have affected their neighbouring cells (yellow circles in the retina) by releasing excess glutamate into the extracellular space. These ganglion cells die by apoptosis in a process termed secondary degeneration. During the period of ganglion cell soma death, there has been significant loss of neuronal tissue in the optic nerve. Glial cells respond by generating a scar (grey shading). In stage 5, a glial scar has replaced all remnants of ganglion cell structures in both the retina and optic nerve (grey shading).
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ganglion cell somas occurred in distinctive, fan-shaped sectors extending from the optic nerve head.27,28 The most likely reason for this characteristic pattern of soma death is focal damage to bundles of axons originating from these cells. In the mouse, such discrete bundling of axons occurs only in the optic nerve head,28 implicating this region as the most likely area for a focal insult and site of initial damage. The question still remaining is: What is the nature of this localized damage? An early hypothesis, often referred to as the “mechanical damage model,” predicted that the increased IOP distorted the arrangement of the collagen plates of the lamina; this caused them to deform, resulting in the compression or binding of the axons as they worked their way through the pores in this structure. This model has been challenged by evidence obtained from rodent models of chronic and experimental glaucoma. The animals studied can develop glaucoma even though they have a lamina made up of columns of cells rather than the collagen plates found in primates,29,30 suggesting that physical compression of the axons is unlikely. In addition, axonal degeneration in mice with chronic inherited glaucoma occurs according to a pattern suggesting only mild damage in the laminar region as apposed to the severe crush or axotomy expected from physical compression of the axons (for a more complete discussion of this, see stage 2, next section). If not direct physical damage to the axons, then what is the nature of the damaging insult elicited by elevated IOP? Several hypotheses have been brought forward and are in the process of being rigorously tested. The most important of these may be local involvement of glial cells residing in the optic nerve head and lamina cribrosa. For decades, glia have not been considered anything more than “brain glue” or neuronal support cells, but more evidence is pointing to their essential role in neuronal homeostasis and disease.31 Under normal conditions, these cells provide a variety of functions in support of neurons, including regulation of extracellular K+ levels, removal of glutamate and GABA neurotransmitters (particularly at synapses), metabolic renewal of precursors used in the synthesis of glutamate, ammonium ion detoxification, regulation of extracellular pH levels and osmolarity, and energy support to neurons by the provision of lactate and (or) glucose from the breakdown of intracellular glycogen stores.31 In the damaged CNS, glia change their behaviour and dramatically alter their gene expression profile. Collectively, these changes are termed the “activation” response. Several marker genes have been described that characterize an activated glial cell, but the most extensively examined have been two
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intermediate filament proteins: glial fibrillary acidic protein (GFAP) and vimentin. Researchers have known for many years that astrocytes and microglia in the optic nerve head of a glaucomatous eye become activated and begin to express GFAP. Microarray analysis of astrocytes isolated from glaucomatous optic nerve heads indicates that they upregulate the expression of more genes, at least 150 different ones, as compared with astrocytes from control optic nerves.32 Gene cluster analysis of the data from this study shows that the majority of these genes are involved in proliferation, cell adhesion, and the synthesis of new extracellular matrix, suggesting that most of the gene expression changes associated with glial activation are part of the formation of a glial scar (see stage 5). Some of the genes, however, are clustered into a group associated with changes in signal transduction that can arguably alter the nurturing role of astrocytes (see later). A critical element to understanding the role of glia in the process of damage to the optic nerve head is deciphering when specific changes occur in these cells relative to the overall pathology of glaucoma. The reason for this is that glial cells likely have at least two distinct roles, one early in the disease process and one late in the process.33,34 To date, the existing studies examining the changes in astrocytes and other optic nerve glia during glaucoma have not reached a high enough resolution to distinguish the early from the late events. The exact interaction between activated glial cells and the neurons they associate with is not known, but several theories have been proposed on the basis of the available information. One possibility is that these cells, particularly microglia, become directly pathogenic to axons. Studies using a rat model of experimental glaucoma have suggested that microglia synthesize and release toxic compounds such as nitric oxide,35,36 a highly reactive molecule that can cause damaging covalent modifications of amino acids in neighbouring cells and adjacent axons.37,38 Although this concept is compelling, it has not been verified in other models of experimental glaucoma.39 Different theories suggest a more passive debilitating role for activated glia. One hypothesis is that activated astrocytes may induce mini-strokes in the optic nerve head by stimulating increased vasoconstriction of regional small capillaries. New evidence has demonstrated the release of vasoactive peptides from astrocytic end-feet (which typically surround small capillaries) during times of stress, at least under conditions that cause increased intracellular Ca2+ in these cells.40 Increased vasoconstriction, in tandem with the antagonizing increase in IOP, may lead to reduced blood flow in the
Ganglion cell death in glaucoma—Nickells
capillaries of the optic nerve head and ischemic conditions. In this scenario, axonal damage could be mediated by changes in ion exchangers found in the axon membrane. Ischemia, associated with reduced energy stores, can negatively affect an axonal Na+/K+ ATPase, leading to an increase in intracellular Na+. Elevated Na+ then reverses the activity of a Na+/Ca2+ exchanger causing an overload of intracellular Ca2+.41,42 This latter event is commonly considered a potent stimulus of autonomous axonal degeneration (reviewed by Whitmore et al43). Associated with the effects of ischemia, axons may also become starved of energy sources, especially in conditions of blocked axonal transport from the cell soma. Again, a normal activity of astrocytes is to provide fuel for neurons, either in the form of lactate or glucose from the breakdown of glycogen. Although highly speculative, one possible process is that stress-induced activation of these cells may alter or impede their role as a fuel supplier to neurons, thus exacerbating or mimicking the energy demands caused by localized ischemia. STAGE 2: DAMAGE TO THE AXON AND THE
PROCESS OF
DEGENERATION
An important concept when considering the sequence of events leading to ganglion cell death is that neuronal degeneration can be compartmentalized (Whitmore et al43). According to this concept, a neuron is able to execute autonomous self-destruct pathways to eliminate different parts of itself, including synapses, axon, dendritic tree, and cell body (soma). The molecular pathways involved in each compartment are not necessarily the same. For example, studies of mice carrying mutant copies of specific genes have shown that molecules essential for soma death are not required for axonal degeneration.44 The compartmentalized degeneration of retinal ganglion cells also appears to occur in glaucoma. Detailed labelling studies by Weber et al45 showed that ganglion cells undergo dendritic tree shrinkage before soma loss in experimental glaucoma. In addition, DBA/2J mice carrying knock-out alleles for the proapoptotic gene Bax exhibited complete axonal loss but no degeneration of the retinal ganglion cell bodies.46 Results like this suggest that a discrete, damaging stimulus occurs to the axon without first requiring the death of the soma. Compartmentalized neuronal degeneration is an essential component of the theory of ganglion cell death presented here, because it relies on the ability of the axon to be independently damaged and undergo degeneration before the apoptotic program is activated in the ganglion cell soma. As discussed in stage 1, it is unclear what stimulates
the axons of retinal ganglion cells to begin to degenerate in glaucoma, but the process may be initiated by an increase in the levels of intracellular Ca2+. Once the axon is damaged, it will execute a self-destruction program that involves the systematic dismantling of its cytoskeleton and other organelles. This includes the breakdown of microfilaments and microtubules, and the deregulation and swelling of mitochondria.43,47 There are 2 basic patterns of degeneration that are activated, depending on the severity of the axonal lesion.48 Severely damaged axons, such as those present after axotomy, undergo a process called Wallerian degeneration, characterized by the rapid degeneration of the axon along the entire length of the severed process. Less severe insult to the axon results in a different pattern of degeneration, termed “die-back,” which is characterized by the slower degeneration of the axon, usually beginning at the synaptic end and progressing in a retrograde fashion toward the neuronal soma. Morphologically, axons undergoing Wallerian degeneration or die-back have many features in common. They are distinct by virtue of the extent of the lesion to the axon, the speed at which the axon degenerates, and the direction or pattern of degeneration. Currently, it is not known whether ganglion cell axons in glaucoma die by Wallerian degeneration or by die-back, but fluorescent dye labelling and histopathologic studies of the optic nerves in the DBA/2J mouse suggest that axons die slowly in a retrograde fashion, consistent with the latter process.28 These results also suggest that the damage to the axon is relatively mild, (e.g., somewhat less severe than complete axotomy). An important aspect of the 5-stage theory presented here is that axonal damage and degeneration precede ganglion cell soma death. Although there have been no comprehensive studies of the temporal progression of each process in glaucoma, functional studies with monkeys trained to undergo visual field examinations have shown that the development of visual field defects initially does not correlate well with ganglion cell soma loss.49 In general, this variability is higher for field defects in the central retina than in the periphery.50,51 After about 40% cell loss, defects progress linearly with cell loss. One explanation for this lack of correlation is that ocular hypertension and glaucoma cause outer retinal dysfunction, particularly in the red and green cone photoreceptors, which are prevalent in this region.52 An alternative hypothesis, however, is that in the early stages of glaucoma, axon function is compromised before the loss of the ganglion cell body. Combined retrograde and cell soma labelling studies in the DBA/2J mouse suggest
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similar early damage to the ganglion cell axons and (or) axonal transport.27 In these experiments, ganglion cell somas could not be labelled by retrograde transport of dyes applied to the superior colliculus, even though they were clearly still viable in the retina. More recently, in a temporal study of both optic nerve and retinal degeneration, also in DBA/2J mice, it was observed that a majority of mice showed optic nerve disease at a younger age than when they typically exhibit retinal disease. These data suggested that optic nerve degeneration did, in fact, precede ganglion cell loss.28 Although compelling, this latter study was conducted on 2 separate cohorts of mice, and a more comprehensive evaluation of retinal and optic nerve degeneration in the same eyes is still lacking. STAGE 3: SIGNALLING
GANGLION CELL APOPTOSIS—
PRIMARY DEGENERATION
Apoptosis is broadly defined as a molecular process by which cells kill themselves. Neurons, including retinal ganglion cells, can execute a complex series of molecular events that culminate in the activation of a cascade of proteases (caspases or, in some cases, calpains) that systematically digest the internal contents of the dying cells.53 The process is designed to leave very little cellular debris remaining as extracellular “garbage” that could lead to an inflammatory response causing damage to the surrounding tissue. Apoptosis, which is characterized by a complex series of interacting events involving dozens of molecules, can be divided into 2 basic pathways. The “intrinsic” pathway leads to mitochondrial dysfunction, which causes the generation of reactive oxygen species, a loss of ATP production, and the release of cytochrome c.54 This latter protein is normally involved in the electron transport chain, but during apoptosis it forms part of a cytoplasmic complex called the apoptosome, which initiates the caspase protease cascade. The “extrinsic” pathway relies on cell surface signalling between a ligand and a cellular receptor containing a death domain. Activation of the death domain directly activates caspases, bypassing the mitochondria. A complex side pathway of extrinsic apoptosis, however, is an amplification step that activates critical molecules, which then recruit the intrinsic pathway to accelerate the cell death process. Extrinsic apoptosis is a fundamental process of the immune system, involving activator molecules like tumor necrosis factor and the Fas ligand. Neurons often do not undergo apoptosis using the extrinsic pathway and instead rely on intrinsic apoptosis. Retinal ganglion cells are not unlike most neurons in the CNS in that they die by intrinsic apoptosis. The
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most compelling evidence for this statement comes from studies using mice that lack a functional Bax gene. BAX is a proapoptotic protein that is critical in causing mitochondrial dysfunction in neurons. Bax knock-out mice have ganglion cells that are resistant to programmed cell death during development,55 experimental lesion of the optic nerve,56 and chronic inherited glaucoma.46 Similarly, antagonists of Bax, including the antiapoptotic gene Bcl2, provide a similar protective effect on ganglion cells.57–60 What activates the intrinsic apoptotic program in ganglion cells during glaucoma? In our theoretical model of the events leading to glaucoma, we speculate that the primary cause of ganglion cell death is neurotrophin deprivation. Neurotrophins are a class of growth factors that act on neurons. All neurons require support of these factors, and ganglion cells acquire them from target neurons in the superior colliculus and lateral geniculate nucleus in the brain. Ganglion cells respond to a variety of neurotrophins, but principally to brain-derived neurotrophic factor, neurotrophin-4, ciliary nerve trophic factor, and glial-cell derived neurotrophic factor.61–64 In the sequence of events leading to ganglion cell death in glaucoma, there is a blockade of both retrograde and anterograde axonal transport at the level of the lamina cribrosa, and it is likely that the process of axonal degeneration has already been initiated (stages 1 and 2). Because of this, critical neurotrophins from the brain are not supplied to the ganglion cell somas, affecting important signalling cascades within the cells. Consistent with the activation of intrinsic apoptosis, neurotrophin deprivation activates c-Jun N-terminal kinases, which stimulate both the de novo expression and post-translational modification of so-called BH-3-containing proteins. These proteins facilitate the actions of BAX, leading to mitochondrial dysfunction.53,65 In many respects, glaucoma recapitulates developmental programmed cell death, during which ganglion cells that do not correctly innervate target neurons in the brain are activated to die. The activation signal for this early wave of programmed cell death is the lack of neurotrophin support from the brain.66,67 In support of this theory, there have been numerous studies showing that the replacement of neurotrophins in animal models of ganglion cell death dramatically attenuate the cell death process.63,64,68–73 STAGE 4: SIGNALLING
GANGLION CELL APOPTOSIS—
SECONDARY DEGENERATION
A popular but controversial theory of how ganglion cells die in glaucoma is the concept of secondary degen-
Ganglion cell death in glaucoma—Nickells
eration. This theory was popularized by evidence that glaucoma was associated with increased levels of the neurotransmitter glutamate in the vitreous.74,75 Glutamate at high doses is damaging to neurons because it can hyperstimulate the ionotropic N-methyl-D-aspartate (NMDA) receptor, leading to a toxic influx of extracellular Ca2+. A classical assumption is that this mode of toxicity is operating after ischemia in stroke, leading to the penumbra of neuronal cell death outside the region of the original infarction.76,77 In this widely accepted hypothesis, neurons initially damaged by ischemia release their glutamate stores into the extracellular space, where the glutamate then affects their nonischemic neighbours. Ganglion cells contain a high concentration of NMDA receptors, making them very sensitive to elevated concentrations of glutamate. Several studies have suggested that there is a second phase of ganglion cell death after partial lesion of the optic nerve,78–80 in which cells initially damaged by axonal lesion presumably expel their glutamate and kill surrounding ganglion cells. Supporting this model is evidence from experiments in rat eyes after partial optic nerve crush. These eyes exhibit elevated glutamate in the vitreous,81 and drugs that act as open channel blockers of the NMDA receptor (thereby preventing the influx of Ca2+) prevent secondary ganglion cell loss.82 It is not certain that a similar phase of secondary ganglion cell loss is common in glaucoma. There is compelling evidence that the glutamate–glutamine cycle is disrupted in eyes with ocular hypertension.83 In this cycle, Müller glial cells normally take up excess extracellular glutamate and convert it to glutamine, which is benign compared with its neurotransmitter counterpart. The glutamine is then transported from the Müller cells back to neurons in the retina. A disruption in normal Müller cell function that affects this cycle would be predicted to lead to elevated extracellular glutamate levels, and this has been confirmed in experimental paradigms in which glutamate uptake by these cells is artificially disrupted.84 Thus, in glaucoma, there may be additive effects of glutamate being released from damaged neurons (stage 3) and impairment of the glial function designed to scavenge extracellular glutamate. Confounding this theory of glutamate toxicity, however, is that the initial reports of elevated glutamate levels in the vitreous have not been confirmed in studies by independent laboratories.85,86 In addition, a study using memantine, an open channel blocker of the NMDA receptor, did not provide compelling evidence of protection of ganglion cells in a monkey model of experimental glaucoma.87 Nevertheless, a large-scale
human clinical trial has been ongoing to test the effects of memantine in glaucoma. When the results of this trial are finally released, it is hoped that the importance of glutamate-mediated secondary degeneration will be clearer. STAGE 5: GLIAL ACTIVATION
IN RESPONSE TO
NEURODEGENERATION
Stage 5 of this process is not limited to an ordered sequential pathway. Several studies have documented glial activation in the optic nerve and retina, but none of them has established when glial changes occur relative to the pathology of the ganglion cells. This lack of careful temporal evaluation of the activation response has been one of the greatest problems in trying to assess the role of glial activation in glaucoma. Glaucoma is associated with the activation of glia in both the optic nerve and retina, although microarray studies suggest that these cells are activated in the nerve first. What distinguishes the changes in glia in stage 1 from the changes in stage 5 of this model is that, in the latter, cells appear to be responding to the progressive degeneration of the retinal ganglion cells and their axons rather than to the effects of elevated IOP. This response may be quite complex and appears to be directed at a variety of factors. In the retina, Müller cells initially respond by synthesizing and secreting neurotrophic factors, possibly in an effort to provide a local neuroprotective environment to damaged ganglion cells.88,89 Later, as ganglion cell somas die and the nerve fibre layer thins, both Müller cells and astrocytes may become more active in laying down new connective tissue as part of the process of generating a glial scar. Similarly, in the optic nerve, astrocytes likely initiate mechanisms designed to protect the ganglion cell axons. These mechanisms may involve sequestration of excess K+ and Ca2+ ions and the release of glucose and lactate to axons as neuronal energy stores become depleted. During later stages, the loss of axons may alter the response of activated astrocytes to lay down new connective tissue.32,90–92 In both the optic nerve and retina, these different phases of glial activation probably occur continuously throughout the stages of progressive neuronal degeneration. WHAT
IS THE IMPACT OF THE
5
STAGES ON THE
CLINICAL MANAGEMENT OF GLAUCOMA?
There is no question that conventional therapy to lower IOP will still be 1 of the main lines of treatment for glaucoma. If the hypothesis that ganglion cell damage and loss occurs in progressive stages of glaucoma
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proves to be accurate, however, what impact will this new knowledge have on current treatments? One area of treatment may be directed at blocking or reversing the initial phase of glial activation (stage 1). A recent study, for example, tested an inhibitor of the receptor for epidermal growth factor (EGF) in a rat model of experimental glaucoma. EGF is thought to be one of the early signalling molecules that activate glia in the optic nerve head. In this model, rats treated with the inhibitor showed a significant attenuation of ganglion cell loss.93 Drugs that block the activation response may even enhance IOP-lowering therapy. Patients who exhibit glaucomatous progression even after successful lowering of IOP may still be suffering from a prolonged glial activation response that takes time to reverse when the IOPrelated stress is relieved. Although hypothetical, treatments that help to reverse the glial activation response would be expected to be synergistic with IOP reduction. A second area of treatment would be directed at the degenerative responses of the ganglion cell axons and somas (stages 2, 3, and 4). At present, the molecular pathways involved in axonal degeneration are only beginning to be elucidated. Nevertheless, drugs like timolol, which appear to have neuroprotective effects, may act by blocking Ca2+ influx into axons.94 Similarly, numerous studies have been conducted using treatments designed to block the death of retinal ganglion cell somas. Many of these studies have shown limited promise, but experiments aimed at reducing the function of the Bax gene are completely effective at blocking ganglion cell death in glaucomatous mice.46 A confounding problem with blocking soma death, however, is that these treatments do not prevent axonal degeneration. This leaves the clinician and the researcher with an interesting paradigm. Treatments may need to be developed that would include a regimen of approaches, such as therapies to lower IOP, prevent or reverse glial activation, block axonal degeneration, and block ganglion cell soma apoptosis. A word of caution is necessary, however, because treatments directed at preventing glial activation may be a double-edged sword. Studies have clearly shown that CNS injuries heal abnormally in mice that cannot activate glia, but these same mice are more permissive for regeneration.33,95–97 In this case, treatments that prevent late-stage glial activation (stage 5) may compromise the integrity of the damaged optic nerve, but this may be essential to promote regeneration of the surviving ganglion cell somas. In patients who have few options remaining for the recovery of some vision, this may be an option well worth taking. In this case, therapy directed at ganglion cells may then involve a treatment that prevents soma loss combined with a second treat-
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ment that stimulates axonal regeneration. This work was supported by a grant from the National Eye Institute of the United States to RWN (R01 EY12223) and by a grant from Research to Prevent Blindness, Inc., to the Department of Ophthalmology and Visual Sciences at the University of Wisconsin.
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eyes with glaucoma. Am J Ophthalmol 1989;107:453–64. 51. Harwerth RS, Carter-Dawson L, Smith III EL, Barnes G, Holt WF, Crawford ML. Neural lossess correlated with visual losses in clinical perimetry. Invest Ophthalmol Vis Sci 2004; 45:3152–60. 52. Pelzel HR, Schlamp CL, Poulsen GL, Ver Hoeve JN, Nork TM, Nickells RW. Decrease of cone opsin mRNA in experimetal ocular hypertension. Mol Vis 2006;12:1272–82. 53. Nickells RW. The molecular biology of retinal ganglion cell death: caveats and controversies. Brain Res Bull 2004;62:439–46. 54. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309–12. 55. Mosinger Ogilvie J, Deckwerth TL, Knudson CM, Korsmeyer SJ. Suppression of developmental retinal cell death but not photoreceptor degeneration in Bax-deficient mice. Invest Ophthalmol Vis Sci 1998;39:1713–20. 56. Li Y, Schlamp CL, Poulsen KP, Nickells RW. Bax-dependent and independent pathways of retinal ganglion cell death induced by different damaging stimuli. Exp Eye Res 2000;71:209–13. 57. Cenni MC, Bonfanti L, Martinou J-C, Ratto GM, Strettoi E, Maffei L. Long-term survival of retinal ganglion cells following optic nerve section in adult bcl-2 transgenic mice. Eur J Neurosci 1996;8:1735–45. 58. Bonfanti L, Strettoi E, Chierzi S, et al. Protection of retinal ganglion cells from natural and axotomy-induced cell death in neonatal transgenic mice overexpressing bcl-2. J Neurosci 1996;16:4186–94. 59. Qin Q, Patil K, Sharma SC. The role of Bax-inhibiting peptide in retinal ganglion cell apoptosis after optic nerve transection. Neurosci Lett 2004;372:17–21. 60. Isenmann S, Engel S, Gillardon F, Bähr M. Bax antisense oligonucleotides reduce axotomy-induced retinal ganglion cell death in vivo by reduction of Bax protein expression. Cell Death Differ 1999;6:673–82. 61. Mansour-Robaey S, Clarke DB, Wang Y-C, Bray GM, Aguayo AJ. Effects of ocular injury and administration of brainderived neurotrophic factor on survival and regrowth of axotomized retinal ganglion cells. Proc Natl Acad Sci U S A 1994;91:1632–36. 62. Sawai H, Clarke DB, Kittlerova P, Bray GM, Aguayo AJ. Brain-derived neurotrophic factor and neurotrophin-4/5 stimulate growth of axonal branches from regenerating retinal ganglion cells. J Neurosci 1996;16:3887–94. 63. Klocker N, Braunling F, Isenmann S, Bahr M. In vivo neurotrophic effects of GDNF on axotomized retina ganglion cells. NeuroReport 1997;8:3439–42. 64. Ji JZ, Elyaman W, Yip HK, et al. CNTF promotes survival of retinal ganglion cells after induction of ocular hypertension in rats: the possible involvement of STAT3 pathway. Eur J Neurosci 2004;19:265–72. 65. Putcha GV, Le S, Frank S, et al. JNK-mediated BIM phosphorylation potentiates BAX-dependent apoptosis. Neuron 2003;38:899–914. 66. Cohen-Cory S, Escandón E, Fraser SE. The cellular patterns of BDNF and trkB expression suggest multiple roles for
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82. Yoles E, Muller S, Schwartz M. NMDA-receptor antagonist protects neurons from secondary degeneration after partial optic nerve crush. J Neurotrauma 1997;14:665–75. 83. Martin KR, Levkovitch-Verbin H, Valenta D, Baumrind L, Pease ME, Quigley HA. Retinal glutamate transporter changes in experimental glaucoma and after optic nerve transection in the rat. Invest Ophthalmol Vis Sci 2002;43:2236–43. 84. Vorwerk CK, Naskar R, Schuettauf F, Quinto K, Zurakowski D, Gochenauer G, Robinson MB, Mackler SA, Dreyer EB. Depression of retinal glutamate transporter function leads to elevated intravitreal glutamate levels and ganglion cell death. Invest Ophthalmol Vis Sci 2000;41:3615–21. 85. Carter-Dawson L, Crawford MLJ, Harwerth RS, Smith III EL, Feldman R, Shen FF, Mitchell CK, Whitetree A. Vitreal glutamate concentration in monkeys with experimental glaucoma. Invest Ophthalmol Vis Sci 2002;43:2633–37. 86. Levkovitch-Verbin H, Martin KR, Quigley HA, Baumrind LA, Pease ME, Valenta D. Measurement of amino acid levels in the vitreous humor of rats after chronic intraocular pressure elevation or optic nerve transection. J Glaucoma 2002;11: 396–405. 87. Hare WA, WoldeMussie E, Weinreb RN, Ton H, Ruiz G, Wijono M, Feldmann B, Zangwill L, Wheeler LA. Efficacy and safety of memantine treatment for reduction of changes associated with experimental glaucoma in monkeys II: Structural measures. Invest Ophthalmol Vis Sci 2004;45:2640–51. 88. Kostyk SK, D’Amore PA, Herman IM, Wagner JA. Optic nerve injury alters basic fibroblast growth factor localization in the retina and optic nerve tract. J Neurosci 1994;14:1441–9. 89. Honjo M, Tanihara H, Kido N, Inatani M, Okazaki K, Honda Y. Expression of ciliary neurotrophic factor activated by retinal Muller cells in eyes with NMDA- and kainic acid-induced neuronal death. Invest Ophthalmol Vis Sci 2001;41:552–60.
90. Hernandez MR, Andrzejewska W, Neufeld A. Changes in the extracellular matrix of the human optic nerve head in primary open-angle glaucoma. Am J Ophthalmol 1990;102:180–8. 91. Johnson EC, Morrison JC, Farrell S, Deppmeier L, Moore CG, McGinty MR. The effect of chronically elevated intraocular pressure on the rat optic nerve head extracellular matrix. Exp Eye Res 1996;62:663–74. 92. Hernandez MR. The optic nerve head in glaucoma: role of astrocytes in tissue remodeling. Prog Retin Eye Res 2000;19:297–321. 93. Liu B, Chen H, Johns TG, Neufeld AH. Epidermal growth factor receptor activation: an upstream signal for transition of quiescent astrocytes into reactive astrocytes after neural injury. J Neurosci 2006;26:7532–40. 94. Osborne NN, Wood JP, Chidlow G, Casson R, DeSantis L, Schmidt KG. Effectiveness of levobetaxolol and timolol at blunting retinal ischemia is related to their calcium and sodium blocking activities: relevance to glaucoma. Brain Res Bull 2004;62:525–8. 95. Wilhelmsson U, Li L, Pekna M, et al. Absence of glial fibrillary acidic protein and vimentin prevents hypertrophy of astrocytic processes and improves post-traumatic regeneration. J Neurosci 2004;24:5016–21. 96. Kinouchi R, Takeda M, Yang L, et al. Robust neural integration from retinal transplants in mice deficient in GFAP and vimentin. Nat Neurosci 2003;6:863–8. 97. Menet V, Prieto M, Privat A, y Ribotta MG. Axonal plasticity and functional recovery after spinal cord injury in mice deficient in both glial fibrillary acidic protein and vimentin genes. Proc Natl Acad Sci U S A 2003;100:8999–9004. Key words: glaucoma, ganglion cell death, glial activation, compartmentalized neuronal self-destruct pathways
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There is substantial evidence that elevated intraocular pressure (IOP) is a critical risk factor for glaucoma.Even with exhaustive investigation, however, there is still little understanding of the pathologic events that translate increased IOP into the process of retinal ganglion cell death. New studies, particularly in rat and mouse models of glaucoma, have helped elucidate some of the important events associated with the initiation of glaucoma. This review summarizes a 5-stage series hypothesizing that elevated IOP causes deleterious changes to glia in the optic nerve head (stage 1), which activate the autonomous self-destruction of ganglion cell axons (stage 2), leading to the loss of neurotrophic support and apoptotic death of ganglion cell somas in the retina (stage 3). In the initial wave of ganglion cell death, dying cells may adversely affect their neighbouring cells in a wave of secondary degeneration involving glutamate exposure (stage 4). As ganglion cell structures disappear through the processes of cell death, glia are again involved, but this time to replace the lost neural tissue with a glial scar (stage 5). Il est solidement établi que la pression intraoculaire (PIO) élevée est un facteur de risque crucial du glaucome. Toutefois, même après des recherches exhaustives, on comprend toujours peu l’activité pathologique par laquelle la PIO élevée se traduit par la mort des cellules ganglionnaires de la rétine. De nouvelles études, effectuées notamment sur des modèles de glaucome chez des rats et des souris, ont aidé à élucider certaines activités importantes associées avec le déclenchement du glaucome. Cette revue résume en 5 étapes l’hypothèse selon laquelle la PIO élevée cause des modifications nocives à la névroglie de la tête du nerf optique (1re étape). Cela déclenche l’autodestruction autonome des axones des cellules ganglionnaires (2e étape) et mène à la perte du soutien neurotrophique et à la mort apoptotique des somas de cellules ganglionnaires de la rétine (3e étape). Dans la vague initiale de décès des cellules ganglionnaires, les cellules mourantes peuvent affecter les cellules adjacentes entraînant une deuxième vague de dégénérescence impliquant l’exposition au glutamate (4e étape). À mesure que disparaissent les structures des cellules ganglionnaires dans le processus de mortalité des cellules, la névroglie est de nouveau impliquée, mais cette fois pour combler la perte de tissu nerveux par une cicatrice gliale (5e étape).
G
laucoma is an optic neuropathy characterized by the progressive degeneration of both the retinal ganglion cell axons in the nerve and cell bodies (somas) in the retina proper. Although glaucoma is considered a multivariate and complex genetic disease, the unifying and most important risk factor is elevated intraocular pressure (IOP). Nearly all individuals who have glaucoma exhibit higher than normal IOPs. Large, multicentre clinical trials, along with some controlled laboratory studies, have now verified that lowering IOP is associated with an attenuation of progressive optic nerve damage.1–6 Similarly, even persons with normal-tension glaucoma benefit from IOP-lowering therapy.7 In this assessment, it is reasonable to speculate that IOP, at any
level, applies some critical stress to the ganglion cells and their axons. The nature of the stress applied to ganglion cells is still not well characterized. Studies conducted by biomechanical engineers using finite element modeling of the sclera and lamina cribrosa have suggested a striking relation between the level of IOP, the mechanical properties of the sclera, and the sheer force applied to the laminar region.8–11 This force can have an impact on all the “soft” tissues occupying the lamina cribrosa, including the ganglion cell axons, capillaries supplying blood to the region, and the glial cellular component, including astrocytes and microglia. A theory, encompassing 5 different stages, is emerging that ties together the relation between elevated
From the Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisc.
Correspondence to: Robert W. Nickells, PhD, Ophthalmology and Visual Sciences, 6640 MSC, 1300 University Ave., University of Wisconsin, Madison WI 53706; fax 608-262-0479; [email protected]
Originally received Jan. 16, 2007 Accepted for publication Feb. 2, 2007
This article has been peer-reviewed. Cet article a été évalué par les pairs. Can J Ophthalmol 2007;42:278–87 doi: 10.3129/can.j.ophthalmol.i07-036
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IOP and the sequence of events that lead to ganglion cell death (Fig. 1). Evaluation of these stages, along with supporting experimental data, shows how this simple model may explain many of the conflicting hypotheses of glaucoma pathogenesis that have arisen over the years. STAGE 1: ELEVATION
OF
IOP AND THE ACTIVATION
OF
OPTIC NERVE GLIA IN THE LAMINA CRIBROSA
Several years ago, seminal studies by Quigley and colleagues suggested that the initial site of damage in glaucoma was at the lamina cribrosa.12–14 An important aspect of this observation was that both retrograde and
anterograde axonal transport was blocked at this level in glaucomatous eyes.15–17 More recent studies have shown that there is disruption of the transport of specific molecules, including neurotrophins and their receptors18,19 and dynein motor proteins.20 The majority of this work relied on nonhuman primate and rat models of experimental glaucoma. Within the last 10 years, however, a model of chronic inherited disease has been characterized in the DBA/2J inbred mouse.21–26 These mice carry genetic defects that cause iris atrophy and iris pigment dispersion, which lead to the obstruction and scarification of the trabecular meshwork. By 8–9 months of age, they have developed elevated IOP, and by 10–12 months they exhibit an optic neuropathy with similarities to human glaucoma. Anatomic studies of glaucoma in these mice support the concept that initial damage occurs at the lamina. Independent examinations of the pattern of retinal degeneration in these animals has revealed that loss of Fig. 1—The 5 stages of disease progression in glaucoma: from elevated intraocular pressure (IOP) to retinal ganglion cell death. A series of 7 cartoons depicting the 5 stages of glaucoma progression. A mouse eye is drawn because many of the new details emerging about the pathology of glaucoma are coming from studies using rodent models. In the top panel, the basic features of the eye are shown. Ganglion cell structures are depicted in light blue and are made up of axons in the optic nerve, and cell somas and dendritic trees in the retina. In stage 1 of glaucoma (second panel from the top), elevated intraocular pressure exerts force around the globe (dark arrows).This force is distributed along the sclera toward the weakest point in the globe, at the scleral canal (red arrows).The force exerts some unknown stress at the level of the scleral canal, which affects the structures and tissues making up the lamina cribrosa (yellow shading). In this theoretical model, the stress causes changes in the glia of the optic nerve head. In stage 2, the optic nerve head glia become activated and probably cause damage to the axons in the region (red shading).The damage likely both affects axonal transport, which is blocked in each direction at this level, and initiates an autonomous axonal self-destruct program (yellow shading). In stage 3, the axons are undergoing disassembly in a retrograde fashion (red–yellow gradient shading), while there is complete blockage of the transport of neurotrophins to the retina at the level of the lamina (red shading). In the retina, ganglion cell somas most affected by this begin their own self-destruct program called apoptosis (yellow circles in the retina). In stage 4, ganglion cells affected in stage 3 are dead (red circles in the retina) and have affected their neighbouring cells (yellow circles in the retina) by releasing excess glutamate into the extracellular space. These ganglion cells die by apoptosis in a process termed secondary degeneration. During the period of ganglion cell soma death, there has been significant loss of neuronal tissue in the optic nerve. Glial cells respond by generating a scar (grey shading). In stage 5, a glial scar has replaced all remnants of ganglion cell structures in both the retina and optic nerve (grey shading).
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ganglion cell somas occurred in distinctive, fan-shaped sectors extending from the optic nerve head.27,28 The most likely reason for this characteristic pattern of soma death is focal damage to bundles of axons originating from these cells. In the mouse, such discrete bundling of axons occurs only in the optic nerve head,28 implicating this region as the most likely area for a focal insult and site of initial damage. The question still remaining is: What is the nature of this localized damage? An early hypothesis, often referred to as the “mechanical damage model,” predicted that the increased IOP distorted the arrangement of the collagen plates of the lamina; this caused them to deform, resulting in the compression or binding of the axons as they worked their way through the pores in this structure. This model has been challenged by evidence obtained from rodent models of chronic and experimental glaucoma. The animals studied can develop glaucoma even though they have a lamina made up of columns of cells rather than the collagen plates found in primates,29,30 suggesting that physical compression of the axons is unlikely. In addition, axonal degeneration in mice with chronic inherited glaucoma occurs according to a pattern suggesting only mild damage in the laminar region as apposed to the severe crush or axotomy expected from physical compression of the axons (for a more complete discussion of this, see stage 2, next section). If not direct physical damage to the axons, then what is the nature of the damaging insult elicited by elevated IOP? Several hypotheses have been brought forward and are in the process of being rigorously tested. The most important of these may be local involvement of glial cells residing in the optic nerve head and lamina cribrosa. For decades, glia have not been considered anything more than “brain glue” or neuronal support cells, but more evidence is pointing to their essential role in neuronal homeostasis and disease.31 Under normal conditions, these cells provide a variety of functions in support of neurons, including regulation of extracellular K+ levels, removal of glutamate and GABA neurotransmitters (particularly at synapses), metabolic renewal of precursors used in the synthesis of glutamate, ammonium ion detoxification, regulation of extracellular pH levels and osmolarity, and energy support to neurons by the provision of lactate and (or) glucose from the breakdown of intracellular glycogen stores.31 In the damaged CNS, glia change their behaviour and dramatically alter their gene expression profile. Collectively, these changes are termed the “activation” response. Several marker genes have been described that characterize an activated glial cell, but the most extensively examined have been two
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intermediate filament proteins: glial fibrillary acidic protein (GFAP) and vimentin. Researchers have known for many years that astrocytes and microglia in the optic nerve head of a glaucomatous eye become activated and begin to express GFAP. Microarray analysis of astrocytes isolated from glaucomatous optic nerve heads indicates that they upregulate the expression of more genes, at least 150 different ones, as compared with astrocytes from control optic nerves.32 Gene cluster analysis of the data from this study shows that the majority of these genes are involved in proliferation, cell adhesion, and the synthesis of new extracellular matrix, suggesting that most of the gene expression changes associated with glial activation are part of the formation of a glial scar (see stage 5). Some of the genes, however, are clustered into a group associated with changes in signal transduction that can arguably alter the nurturing role of astrocytes (see later). A critical element to understanding the role of glia in the process of damage to the optic nerve head is deciphering when specific changes occur in these cells relative to the overall pathology of glaucoma. The reason for this is that glial cells likely have at least two distinct roles, one early in the disease process and one late in the process.33,34 To date, the existing studies examining the changes in astrocytes and other optic nerve glia during glaucoma have not reached a high enough resolution to distinguish the early from the late events. The exact interaction between activated glial cells and the neurons they associate with is not known, but several theories have been proposed on the basis of the available information. One possibility is that these cells, particularly microglia, become directly pathogenic to axons. Studies using a rat model of experimental glaucoma have suggested that microglia synthesize and release toxic compounds such as nitric oxide,35,36 a highly reactive molecule that can cause damaging covalent modifications of amino acids in neighbouring cells and adjacent axons.37,38 Although this concept is compelling, it has not been verified in other models of experimental glaucoma.39 Different theories suggest a more passive debilitating role for activated glia. One hypothesis is that activated astrocytes may induce mini-strokes in the optic nerve head by stimulating increased vasoconstriction of regional small capillaries. New evidence has demonstrated the release of vasoactive peptides from astrocytic end-feet (which typically surround small capillaries) during times of stress, at least under conditions that cause increased intracellular Ca2+ in these cells.40 Increased vasoconstriction, in tandem with the antagonizing increase in IOP, may lead to reduced blood flow in the
Ganglion cell death in glaucoma—Nickells
capillaries of the optic nerve head and ischemic conditions. In this scenario, axonal damage could be mediated by changes in ion exchangers found in the axon membrane. Ischemia, associated with reduced energy stores, can negatively affect an axonal Na+/K+ ATPase, leading to an increase in intracellular Na+. Elevated Na+ then reverses the activity of a Na+/Ca2+ exchanger causing an overload of intracellular Ca2+.41,42 This latter event is commonly considered a potent stimulus of autonomous axonal degeneration (reviewed by Whitmore et al43). Associated with the effects of ischemia, axons may also become starved of energy sources, especially in conditions of blocked axonal transport from the cell soma. Again, a normal activity of astrocytes is to provide fuel for neurons, either in the form of lactate or glucose from the breakdown of glycogen. Although highly speculative, one possible process is that stress-induced activation of these cells may alter or impede their role as a fuel supplier to neurons, thus exacerbating or mimicking the energy demands caused by localized ischemia. STAGE 2: DAMAGE TO THE AXON AND THE
PROCESS OF
DEGENERATION
An important concept when considering the sequence of events leading to ganglion cell death is that neuronal degeneration can be compartmentalized (Whitmore et al43). According to this concept, a neuron is able to execute autonomous self-destruct pathways to eliminate different parts of itself, including synapses, axon, dendritic tree, and cell body (soma). The molecular pathways involved in each compartment are not necessarily the same. For example, studies of mice carrying mutant copies of specific genes have shown that molecules essential for soma death are not required for axonal degeneration.44 The compartmentalized degeneration of retinal ganglion cells also appears to occur in glaucoma. Detailed labelling studies by Weber et al45 showed that ganglion cells undergo dendritic tree shrinkage before soma loss in experimental glaucoma. In addition, DBA/2J mice carrying knock-out alleles for the proapoptotic gene Bax exhibited complete axonal loss but no degeneration of the retinal ganglion cell bodies.46 Results like this suggest that a discrete, damaging stimulus occurs to the axon without first requiring the death of the soma. Compartmentalized neuronal degeneration is an essential component of the theory of ganglion cell death presented here, because it relies on the ability of the axon to be independently damaged and undergo degeneration before the apoptotic program is activated in the ganglion cell soma. As discussed in stage 1, it is unclear what stimulates
the axons of retinal ganglion cells to begin to degenerate in glaucoma, but the process may be initiated by an increase in the levels of intracellular Ca2+. Once the axon is damaged, it will execute a self-destruction program that involves the systematic dismantling of its cytoskeleton and other organelles. This includes the breakdown of microfilaments and microtubules, and the deregulation and swelling of mitochondria.43,47 There are 2 basic patterns of degeneration that are activated, depending on the severity of the axonal lesion.48 Severely damaged axons, such as those present after axotomy, undergo a process called Wallerian degeneration, characterized by the rapid degeneration of the axon along the entire length of the severed process. Less severe insult to the axon results in a different pattern of degeneration, termed “die-back,” which is characterized by the slower degeneration of the axon, usually beginning at the synaptic end and progressing in a retrograde fashion toward the neuronal soma. Morphologically, axons undergoing Wallerian degeneration or die-back have many features in common. They are distinct by virtue of the extent of the lesion to the axon, the speed at which the axon degenerates, and the direction or pattern of degeneration. Currently, it is not known whether ganglion cell axons in glaucoma die by Wallerian degeneration or by die-back, but fluorescent dye labelling and histopathologic studies of the optic nerves in the DBA/2J mouse suggest that axons die slowly in a retrograde fashion, consistent with the latter process.28 These results also suggest that the damage to the axon is relatively mild, (e.g., somewhat less severe than complete axotomy). An important aspect of the 5-stage theory presented here is that axonal damage and degeneration precede ganglion cell soma death. Although there have been no comprehensive studies of the temporal progression of each process in glaucoma, functional studies with monkeys trained to undergo visual field examinations have shown that the development of visual field defects initially does not correlate well with ganglion cell soma loss.49 In general, this variability is higher for field defects in the central retina than in the periphery.50,51 After about 40% cell loss, defects progress linearly with cell loss. One explanation for this lack of correlation is that ocular hypertension and glaucoma cause outer retinal dysfunction, particularly in the red and green cone photoreceptors, which are prevalent in this region.52 An alternative hypothesis, however, is that in the early stages of glaucoma, axon function is compromised before the loss of the ganglion cell body. Combined retrograde and cell soma labelling studies in the DBA/2J mouse suggest
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similar early damage to the ganglion cell axons and (or) axonal transport.27 In these experiments, ganglion cell somas could not be labelled by retrograde transport of dyes applied to the superior colliculus, even though they were clearly still viable in the retina. More recently, in a temporal study of both optic nerve and retinal degeneration, also in DBA/2J mice, it was observed that a majority of mice showed optic nerve disease at a younger age than when they typically exhibit retinal disease. These data suggested that optic nerve degeneration did, in fact, precede ganglion cell loss.28 Although compelling, this latter study was conducted on 2 separate cohorts of mice, and a more comprehensive evaluation of retinal and optic nerve degeneration in the same eyes is still lacking. STAGE 3: SIGNALLING
GANGLION CELL APOPTOSIS—
PRIMARY DEGENERATION
Apoptosis is broadly defined as a molecular process by which cells kill themselves. Neurons, including retinal ganglion cells, can execute a complex series of molecular events that culminate in the activation of a cascade of proteases (caspases or, in some cases, calpains) that systematically digest the internal contents of the dying cells.53 The process is designed to leave very little cellular debris remaining as extracellular “garbage” that could lead to an inflammatory response causing damage to the surrounding tissue. Apoptosis, which is characterized by a complex series of interacting events involving dozens of molecules, can be divided into 2 basic pathways. The “intrinsic” pathway leads to mitochondrial dysfunction, which causes the generation of reactive oxygen species, a loss of ATP production, and the release of cytochrome c.54 This latter protein is normally involved in the electron transport chain, but during apoptosis it forms part of a cytoplasmic complex called the apoptosome, which initiates the caspase protease cascade. The “extrinsic” pathway relies on cell surface signalling between a ligand and a cellular receptor containing a death domain. Activation of the death domain directly activates caspases, bypassing the mitochondria. A complex side pathway of extrinsic apoptosis, however, is an amplification step that activates critical molecules, which then recruit the intrinsic pathway to accelerate the cell death process. Extrinsic apoptosis is a fundamental process of the immune system, involving activator molecules like tumor necrosis factor and the Fas ligand. Neurons often do not undergo apoptosis using the extrinsic pathway and instead rely on intrinsic apoptosis. Retinal ganglion cells are not unlike most neurons in the CNS in that they die by intrinsic apoptosis. The
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most compelling evidence for this statement comes from studies using mice that lack a functional Bax gene. BAX is a proapoptotic protein that is critical in causing mitochondrial dysfunction in neurons. Bax knock-out mice have ganglion cells that are resistant to programmed cell death during development,55 experimental lesion of the optic nerve,56 and chronic inherited glaucoma.46 Similarly, antagonists of Bax, including the antiapoptotic gene Bcl2, provide a similar protective effect on ganglion cells.57–60 What activates the intrinsic apoptotic program in ganglion cells during glaucoma? In our theoretical model of the events leading to glaucoma, we speculate that the primary cause of ganglion cell death is neurotrophin deprivation. Neurotrophins are a class of growth factors that act on neurons. All neurons require support of these factors, and ganglion cells acquire them from target neurons in the superior colliculus and lateral geniculate nucleus in the brain. Ganglion cells respond to a variety of neurotrophins, but principally to brain-derived neurotrophic factor, neurotrophin-4, ciliary nerve trophic factor, and glial-cell derived neurotrophic factor.61–64 In the sequence of events leading to ganglion cell death in glaucoma, there is a blockade of both retrograde and anterograde axonal transport at the level of the lamina cribrosa, and it is likely that the process of axonal degeneration has already been initiated (stages 1 and 2). Because of this, critical neurotrophins from the brain are not supplied to the ganglion cell somas, affecting important signalling cascades within the cells. Consistent with the activation of intrinsic apoptosis, neurotrophin deprivation activates c-Jun N-terminal kinases, which stimulate both the de novo expression and post-translational modification of so-called BH-3-containing proteins. These proteins facilitate the actions of BAX, leading to mitochondrial dysfunction.53,65 In many respects, glaucoma recapitulates developmental programmed cell death, during which ganglion cells that do not correctly innervate target neurons in the brain are activated to die. The activation signal for this early wave of programmed cell death is the lack of neurotrophin support from the brain.66,67 In support of this theory, there have been numerous studies showing that the replacement of neurotrophins in animal models of ganglion cell death dramatically attenuate the cell death process.63,64,68–73 STAGE 4: SIGNALLING
GANGLION CELL APOPTOSIS—
SECONDARY DEGENERATION
A popular but controversial theory of how ganglion cells die in glaucoma is the concept of secondary degen-
Ganglion cell death in glaucoma—Nickells
eration. This theory was popularized by evidence that glaucoma was associated with increased levels of the neurotransmitter glutamate in the vitreous.74,75 Glutamate at high doses is damaging to neurons because it can hyperstimulate the ionotropic N-methyl-D-aspartate (NMDA) receptor, leading to a toxic influx of extracellular Ca2+. A classical assumption is that this mode of toxicity is operating after ischemia in stroke, leading to the penumbra of neuronal cell death outside the region of the original infarction.76,77 In this widely accepted hypothesis, neurons initially damaged by ischemia release their glutamate stores into the extracellular space, where the glutamate then affects their nonischemic neighbours. Ganglion cells contain a high concentration of NMDA receptors, making them very sensitive to elevated concentrations of glutamate. Several studies have suggested that there is a second phase of ganglion cell death after partial lesion of the optic nerve,78–80 in which cells initially damaged by axonal lesion presumably expel their glutamate and kill surrounding ganglion cells. Supporting this model is evidence from experiments in rat eyes after partial optic nerve crush. These eyes exhibit elevated glutamate in the vitreous,81 and drugs that act as open channel blockers of the NMDA receptor (thereby preventing the influx of Ca2+) prevent secondary ganglion cell loss.82 It is not certain that a similar phase of secondary ganglion cell loss is common in glaucoma. There is compelling evidence that the glutamate–glutamine cycle is disrupted in eyes with ocular hypertension.83 In this cycle, Müller glial cells normally take up excess extracellular glutamate and convert it to glutamine, which is benign compared with its neurotransmitter counterpart. The glutamine is then transported from the Müller cells back to neurons in the retina. A disruption in normal Müller cell function that affects this cycle would be predicted to lead to elevated extracellular glutamate levels, and this has been confirmed in experimental paradigms in which glutamate uptake by these cells is artificially disrupted.84 Thus, in glaucoma, there may be additive effects of glutamate being released from damaged neurons (stage 3) and impairment of the glial function designed to scavenge extracellular glutamate. Confounding this theory of glutamate toxicity, however, is that the initial reports of elevated glutamate levels in the vitreous have not been confirmed in studies by independent laboratories.85,86 In addition, a study using memantine, an open channel blocker of the NMDA receptor, did not provide compelling evidence of protection of ganglion cells in a monkey model of experimental glaucoma.87 Nevertheless, a large-scale
human clinical trial has been ongoing to test the effects of memantine in glaucoma. When the results of this trial are finally released, it is hoped that the importance of glutamate-mediated secondary degeneration will be clearer. STAGE 5: GLIAL ACTIVATION
IN RESPONSE TO
NEURODEGENERATION
Stage 5 of this process is not limited to an ordered sequential pathway. Several studies have documented glial activation in the optic nerve and retina, but none of them has established when glial changes occur relative to the pathology of the ganglion cells. This lack of careful temporal evaluation of the activation response has been one of the greatest problems in trying to assess the role of glial activation in glaucoma. Glaucoma is associated with the activation of glia in both the optic nerve and retina, although microarray studies suggest that these cells are activated in the nerve first. What distinguishes the changes in glia in stage 1 from the changes in stage 5 of this model is that, in the latter, cells appear to be responding to the progressive degeneration of the retinal ganglion cells and their axons rather than to the effects of elevated IOP. This response may be quite complex and appears to be directed at a variety of factors. In the retina, Müller cells initially respond by synthesizing and secreting neurotrophic factors, possibly in an effort to provide a local neuroprotective environment to damaged ganglion cells.88,89 Later, as ganglion cell somas die and the nerve fibre layer thins, both Müller cells and astrocytes may become more active in laying down new connective tissue as part of the process of generating a glial scar. Similarly, in the optic nerve, astrocytes likely initiate mechanisms designed to protect the ganglion cell axons. These mechanisms may involve sequestration of excess K+ and Ca2+ ions and the release of glucose and lactate to axons as neuronal energy stores become depleted. During later stages, the loss of axons may alter the response of activated astrocytes to lay down new connective tissue.32,90–92 In both the optic nerve and retina, these different phases of glial activation probably occur continuously throughout the stages of progressive neuronal degeneration. WHAT
IS THE IMPACT OF THE
5
STAGES ON THE
CLINICAL MANAGEMENT OF GLAUCOMA?
There is no question that conventional therapy to lower IOP will still be 1 of the main lines of treatment for glaucoma. If the hypothesis that ganglion cell damage and loss occurs in progressive stages of glaucoma
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proves to be accurate, however, what impact will this new knowledge have on current treatments? One area of treatment may be directed at blocking or reversing the initial phase of glial activation (stage 1). A recent study, for example, tested an inhibitor of the receptor for epidermal growth factor (EGF) in a rat model of experimental glaucoma. EGF is thought to be one of the early signalling molecules that activate glia in the optic nerve head. In this model, rats treated with the inhibitor showed a significant attenuation of ganglion cell loss.93 Drugs that block the activation response may even enhance IOP-lowering therapy. Patients who exhibit glaucomatous progression even after successful lowering of IOP may still be suffering from a prolonged glial activation response that takes time to reverse when the IOPrelated stress is relieved. Although hypothetical, treatments that help to reverse the glial activation response would be expected to be synergistic with IOP reduction. A second area of treatment would be directed at the degenerative responses of the ganglion cell axons and somas (stages 2, 3, and 4). At present, the molecular pathways involved in axonal degeneration are only beginning to be elucidated. Nevertheless, drugs like timolol, which appear to have neuroprotective effects, may act by blocking Ca2+ influx into axons.94 Similarly, numerous studies have been conducted using treatments designed to block the death of retinal ganglion cell somas. Many of these studies have shown limited promise, but experiments aimed at reducing the function of the Bax gene are completely effective at blocking ganglion cell death in glaucomatous mice.46 A confounding problem with blocking soma death, however, is that these treatments do not prevent axonal degeneration. This leaves the clinician and the researcher with an interesting paradigm. Treatments may need to be developed that would include a regimen of approaches, such as therapies to lower IOP, prevent or reverse glial activation, block axonal degeneration, and block ganglion cell soma apoptosis. A word of caution is necessary, however, because treatments directed at preventing glial activation may be a double-edged sword. Studies have clearly shown that CNS injuries heal abnormally in mice that cannot activate glia, but these same mice are more permissive for regeneration.33,95–97 In this case, treatments that prevent late-stage glial activation (stage 5) may compromise the integrity of the damaged optic nerve, but this may be essential to promote regeneration of the surviving ganglion cell somas. In patients who have few options remaining for the recovery of some vision, this may be an option well worth taking. In this case, therapy directed at ganglion cells may then involve a treatment that prevents soma loss combined with a second treat-
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ment that stimulates axonal regeneration. This work was supported by a grant from the National Eye Institute of the United States to RWN (R01 EY12223) and by a grant from Research to Prevent Blindness, Inc., to the Department of Ophthalmology and Visual Sciences at the University of Wisconsin.
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