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61 

Neuroprotection and Neurorepair LEONARD A LEVIN*

Summary Glaucoma is a distinctive optic nerve disease in which the primary damage occurs to the retinal ganglion cell axon. The loss of either the retinal ganglion cell or its axon is sufficient to cause visual loss. Therapies that prevent death of the retinal ganglion cell (neuroprotection) or its axon (axoprotection), or that regenerate retinal ganglion cells and their axons, theoretically should be useful in treating glaucoma. At present most data are derived from animal studies, with only one randomized clinical trial that had results that could be consistent with neuroprotection. More extensive randomized clinical trials are crucial for assessing whether these strategies are useful for treating patients with glaucoma, and eventually, for assessing neurorepair in patients who have already lost significant amounts of vision.

Introduction Glaucoma is the most common optic neuropathy. It is also an optic neuropathy distinguished by a highly characteristic morphology of the optic disk, namely cupping with a relative absence of pallor. The distinctive appearance of the glaucomatous disk reflects the fact that the primary pathophysiology of the disease takes place at that location. This locus of injury is supported by a wealth of clinical, experimental, and explanatory data.1–7 The concept of glaucoma as an axonopathy was comprehensively reviewed in 2012.8 As with other optic neuropathies, glaucoma is manifested by loss of retinal ganglion cell axons, death of retinal ganglion cells, and loss of vision. For each of these neurobiological consequences there is a corresponding clinical manifestation. The loss of retinal ganglion cell axons results in focal or generalized thinning of the retinal nerve fiber layer, optic nerve atrophy, and a decrease in the size of the optic nerve on neuroimaging. The thinning of the retinal nerve fiber layer is most easily demonstrated by optical coherence tomography or polarization-sensitive imaging, but can also be observed clinically, especially with red-free illumination. The optic atrophy of glaucoma is virtually pathognomonic, being a morphological excavation of the disk without significant pallor, and is not seen in other optic neuropathies. The loss of retinal ganglion cell bodies is not seen on clinical examination, but can be quantitatively *Grant Support: Canadian Institutes of Health Research, National Institutes of Health, Research to Prevent Blindness, Retina Research Foundation, American Health Assistance Foundation, Glaucoma Research Foundation.

demonstrated by a decrease in the pattern electroretinogram (pattern ERG), a decrease in the retinal ganglion cell components of the multifocal ERG, and thinning of the ganglion cells and inner plexiform layer on macular optical coherence tomography. The loss of visual function is manifested by a variety of clinical measures, predominantly visual field loss in a nerve fiber bundle defect pattern. Decreased visual acuity and color vision only late in the course of glaucomatous optic neuropathy, and signify the terminal loss of axons responsible for vision. The recognition that glaucoma is an optic neuropathy (albeit often associated with an elevated intraocular pressure or a response to intraocular pressure lowering) led to suggestions that therapies directed at (1) preventing or (2) repairing the neuronal loss may be efficacious for patients with the disease. The first concept is called neuroprotection, and parallels neuron-centered therapies in a variety of acute and chronic neurological diseases. The second concept is called neurorepair, and is necessary because mammalian neurons are almost always post-mitotic and cannot be replaced. Given that the permanent loss of vision in glaucoma reflects the permanent loss of retinal ganglion cells and their axons, visual restoration requires a restoration of retinal ganglion cells and their axons. This chapter reviews neuroprotection and regeneration with respect to glaucomatous optic neuropathy.

Mechanisms of Retinal Ganglion Cell Death and Neuroprotection The damage in glaucoma primarily occurs at the optic disk. It is through the disk that the axons of the retinal ganglion cells course. Therefore, the death of retinal ganglion cells is an irreversible consequence of axonal injury, the likely mechanism for injury in glaucomatous optic neuropathy.1–7 Historically, much is known about the effect of axonal injury on retinal ganglion cells because of several years of research studying acute transection of the axons in experimental animals. Cutting the optic nerve within the orbit causes the retinal ganglion cells to die over the course of days to weeks.9 The process of glaucomatous cell death involves apoptosis, a suicide-like process in which axonal injury signals the retinal ganglion cell to die.10 Blocking apoptosis (e.g. by blocking the expression of apoptosisrelated proteins or using chemical inhibitors of apoptosis) increases cell survival. Similarly, there are survival factors that can be injected into the eye and maintain retinal ganglion cell survival, despite a severe axonal injury. For example, researchers crushed the optic nerve of rats, injecting a survival factor called brain-derived neurotrophic 625

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factor into the vitreous.9 In the control eyes, approximately 50% of the retinal ganglion cells died after seven days, while in the BDNF-injected eyes, almost all of the cells survived. Combinations of survival factors, and expressing the receptors for the survival factors on retinal ganglion cells, can increase survival, so that a great number of retinal ganglion cells can be preserved.11 Since these early studies, there has been a tremendous increase in knowledge relating to RGC death after axonal injury. Some of the mechanisms include the previously mentioned neurotrophin deprivation, but also include excitotoxicity, increases in intra-axonal Ca++, accumulation of excess retrogradely transported macromolecules, induction of various kinases, and induction of superoxide and other redox signaling molecules.9,12–20 Mechanisms of RGC death in glaucoma were comprehensively reviewed in 2012.21 Correspondingly, drugs or other therapies that interfere with these processes can protect RGCs from dying in axonal injury.22–26 Beginning more than a decade ago there has been a realization that glia play a critical role in glaucoma. These are non-neuronal nervous system cells, and are categorized as either macroglia (astrocytes and oligodendrocytes) or microglia (macrophage-like cells). The contribution of glia to glaucomatous pathophysiology occurs at several levels, and includes the role of activation of astrocytes within the optic nerve head, response of oligodendrocytes within the retrolaminar optic nerve, and microglia within the retina and elsewhere. The role of glia in glaucoma was recently reviewed.27

UPSTREAM VS. DOWNSTREAM THERAPIES Inhibiting RGC death after axonal injury in glaucoma is an example of neuroprotection. Neuroprotection can either be prophylactic or instituted after the injury has commenced. The loss of the cell is targeted, not the disease process by which the loss occurs. In glaucomatous optic neuropathy, the retinal ganglion cell is treated, and not the elevated intraocular pressure or other etiology that indirectly causes the death of the retinal ganglion cell. Although intraocular pressure lowering and other such therapies can be considered indirectly neuroprotective, by strict definition and by comparison with other cytoprotective therapies, a neuroprotective therapy is directed at the neuron itself. Strategies for neuroprotection are diverse (Box 61-1).

Assessment of Neuroprotective Therapies – Theory Current therapies of glaucoma are directed at lowering the intraocular pressure. The assessment of these therapies only requires demonstration that the intraocular pressure is lowered, and to what degree. With neuroprotection, the therapy is directed at neuronal death, and therefore the assessment of efficacy must reflect this endpoint. There are at least two different ways of looking at retinal ganglion cell endpoints, namely functional and structural. Preclinical studies should use models that are useful for predicting the response when the therapy is applied to patients. Furthermore, if a drug is used, there must be evidence that it, or an

Box 61-1  Methods for Neuroprotection Pharmacological Neurotrophic factors Calcium channel blockade Glutamate receptor antagonists NMDA receptors AMPA/kainate antagonists α2 Adrenergic agonists Nitric oxide synthase inhibitors Inhibition of death signaling cascades Activation of survival signaling cascades Reactive oxygen species scavengers Redox modulators Apoptosis inhibitors Inhibition of cytochrome C release Caspase inhibitors Immune Modulation Preconditioning Stem Cells

Similarity to human glaucoma

Primate OHT/chronic ischemia

Mechanisms and screening

Rodent OHT/Ischemia Non-primate optic nerve injury Retinal cell cultures

Neuronal cultures

Retinal injury

Figure 61-1  Glaucoma models. OHT, ocular hypertension.

indirect effector, is active at the retinal ganglion cell or its axon.

DOES THE MODEL IN WHICH THE THERAPY IS BEING TESTED MIMIC ASPECTS OF CLINICAL GLAUCOMA? Glaucoma models can be represented in a pyramid with the most applicable models at the top, and the relatively less applicable models at the bottom (Fig. 61-1). This does not mean that models at the top of the pyramid are intrinsically superior to those at the bottom, as there is a role for each in terms of the similarity to human glaucoma with respect to morphology, time-course, cell biology, and how the model is carried out. A retinal ganglion cell culture cannot reflect the complicated pathophysiology that is far more closely reproduced in a non-human primate model. On the other hand, thousands of drugs cannot be screened in monkeys or even rodents, but can be in cell culture models. Experimental paradigms of neuroprotective strategies in glaucoma include in vitro cell models, in vivo models of optic nerve injury and glaucoma in animals, and clinical trials in humans. Each has its advantages and disadvantages. The most important aspects to consider are the similarity to glaucoma and the utility for testing neuroprotection (Table 61-1).

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Table 61-1  Neuroprotection Models Model

Similarity to Glaucoma

Utility for Testing Neuroprotection

Neuronal culture models other than retinal ganglion cells Retinal ganglion cell culture models

Low

Retinal photic injury

Not an optic neuropathy

Retinal ischemia

Causes retinal ganglion cell and axonal ischemia, and therefore indirectly causes an optic neuropathy, but not cupping Causes optic neuropathy with cupping Causes axonal damage with consequent effects on the retinal ganglion cell. No cupping Causes axonal damage and cupping. Also can cause cell body injury if intraocular pressure is too high

Good for understanding mechanisms of neuronal death Good for understanding mechanisms of retinal ganglion cell death May be good test of whether a drug is neuroprotective for retinal cells (e.g. photoreceptors) other than retinal ganglion cells Good for testing neuroprotection of cell body injuries

Optic nerve head ischemia Crush or other mechanical insult to the optic nerve Animal ocular hypertension

Common feature is retinal ganglion cell death

Cultures of retinal ganglion cells are useful for understanding mechanisms of how axonal injuries cause cell death, but the culture itself as a disease model is very different from glaucoma. Given that the act of producing the cell culture causes an axonal injury at the cell body, it can be difficult to simulate the axonal injury of glaucoma, where the injury occurs further away. Attempts to use hydrostatic pressure within cultures do not simulate the effects on the axon from pressure gradients across the lamina cribrosa in glaucoma.28 There are many animal models of central nervous system injury and disease or cultures of other neuronal cell types, but these are pathophysiologically distant from the glaucomatous process itself, and thus cannot be used to provide good evidence of therapeutic efficacy for neuroprotection in glaucoma. Other helpful, but less extrapolative models include those where the IOP is raised to higher than the systolic blood pressure, causing retinal ischemia, or models where NMDA or other excitotoxic chemicals are injected into the eye, killing retinal ganglion cells.29,30 Further up the pyramid are optic nerve injuries (e.g., optic nerve transection or optic nerve crush). This is an acute injury of the optic nerve, different from glaucoma, but sharing the characteristics of optic neuropathy. The model’s usefulness is the ability to study signaling mechanisms of retinal ganglion cell death, in this case after axonal injury. The next higher level on the pyramid are experimental glaucoma models, particularly where the IOP is chronically raised,31 simulating the effect of higher IOP in humans. Rodent models of ocular hypertension have become well characterized, but the primate model of ocular hypertension after argon laser damage to the trabecular meshwork represents the closest model akin to human glaucoma. Often, the morphological functional and histological features of these animal models closely match those of the human disease, although taking place over a shorter time period. A parallel model is that of chronic disk ischemia, induced by infusing vasoconstrictive endothelin-132–35 into the subarachnoid space, but this may be confounded by nonglaucomatous optic atrophy. Finally, the randomized

Good for testing neuroprotection of axonal injuries Good for testing neuroprotection of axonal injuries Good for testing neuroprotection of glaucoma

controlled clinical trial in patients remains the gold standard for assessing efficacy.

HOW IS THE EFFECT OF NEUROPROTECTION ASSESSED? Functional Assessment The functional assessment of the retinal ganglion cell and its axon is reflected in the: (1) ability to generate action potentials based on incoming information generated from its synapses with bipolar and amacrine cells; (2) conduct these impulses down the optic nerve toward cells in the lateral geniculate nucleus and other targets within the central nervous system; and (3) be relayed to processing neurons of the visual cortex within the occipital lobe. At the highest level, this pathway can be assessed with visual function measures, particularly the visual field. A wide variety of measurements of visual fields have been developed, with the most common being white-on-white automated perimetry, as well as short-wave (blue on yellow) automated perimetry, frequency-doubling perimetry, highpass perimetry, and several other techniques. There are also several methods for assessing whether or not there is progression in the visual field. Some are based on looking at single points, others on clusters of points, and others on summation of the entire visual field. These range from techniques as simple as looking at the mean deviation of the visual field compared with an age-matched normal control group to complex techniques assessing the border of the increasing scotoma or other techniques.36 The techniques for visual field progression are not unique to neuroprotection studies, but as is discussed below, sensitive and low-variability methodologies are useful for testing the efficacy of putative neuroprotective therapies. Some visual parameters are less sensitive for assessing neuroprotection in glaucoma because they are only affected late in the course of the disease. These include visual acuity and color vision measures. Mass measures of optic nerve function such as the afferent papillary defect

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are useful only when there is significant asymmetrical optic neuropathy. The summation of the electrical generation of action potentials within the retinal ganglion cell can be determined with the pattern ERG and late components of the multifocal ERG to detect retinal ganglion cell outputs.37,38 The latter has the advantage of generating a retinotopic mapping, with the focal loss of retinal ganglion cell function in a specific area depicted in a manner similar to that used for visual fields. The photopic negative response (PhNR)39–41 and scotopic threshold response (STR)42,43 can similarly be used to detect changes in glaucoma, with varying degrees of sensitivity. Conduction down the optic nerve can be measured with the visually evoked response to a contrast-reversing checkerboard pattern, or as a retinotopic map from a multifocal visual evoked response. These techniques, which usually rely on measurements made at the cortex, actually reflect not just the conduction down the optic nerve, but also the geniculocortical connection. In animals, electrodes can be placed at the superior colliculus (the target for most retinal ganglion cell axons in lower animals), thereby isolating the function of the anterior visual pathways. The analysis of retinal ganglion cell and axonal function on a single cell basis is important for preclinical study and evaluation of neuroprotective techniques, even if it is currently inapplicable to routine clinical use. Animal retinas can be dissociated and retinal ganglion cells, cultured. This allows study of multiple retinal ganglion cell functions, e.g. electrical activity and calcium influx.44 Retinal ganglion cells within the living animal can also be imaged and studied. For example, Cordeiro and colleagues described the use of annexin V, which binds exposed phosphatidylserine on apoptotic cells, as a measure of an early step in apoptosis.45,46 Similar techniques can be used to study early signaling events in retinal ganglion cell bodies and their axons.19,47,48 Axonal transport along the RGC axon can be assessed in animals using fluorescently labeled tracers.49

Structural Assessment The retinal nerve fiber layer thins in step with the disappearance of retinal ganglion cells and their axons in glaucoma. This can be detected on the fundus examination, especially when there is focal thinning. Examination of the nerve fiber layer is aided by red-free photography. The nerve fiber layer can be assessed more precisely with optical coherence tomography (OCT), which directly measures the thickness, or with polarization-sensitive imaging, which measures the amount of birefringence from microtubules within the axons contained in the nerve fiber layer. If there is microtubule dysfunction or destruction independent of loss of retinal ganglion cell axons, then there may be a discrepancy between the OCT and polarimetry measurements.50 In glaucoma, changes in the morphology of the optic nerve head also reflect the number of remaining retinal ganglion cell axons. As glaucoma progresses, the size of the cup increases as the size of the neuroretinal rim decreases. The change in the disk can be assessed by observation, with stereophotography, or with specialized instruments, e.g. Heidelberg retinal tomography or OCT. Changes within the orbital optic nerve also reflect the decreased number of retinal ganglion cell axons in

glaucoma. This causes an anatomically smaller optic nerve, that can be quantitated by neuroimaging. Magnetic resonance imaging (MRI), computed tomography, and ultrasonography have all been used for this purpose. For MRI, the appropriate pulse sequences are necessary in order to avoid problems in discriminating the optic nerve from orbital fat, e.g. the use of fat suppression in T1-weighted MRI. The inherent contrast between fat and the optic nerve makes computed tomography particularly straightforward for assessing the optic nerve, although the discrimination between a large amount of subarachnoid cerebrospinal fluid in an atrophic nerve and a large number of axons in a normal nerve may be difficult. Specific MRI sequences for analyzing optic nerve axons have been developed.51–53 Ultrasonography also can be used, with the advantage of not requiring complex instrumentation but being highly operator-dependent. Neuroprotective strategies addressed at maintaining ganglion cell viability can be assessed by counting the number of living retinal ganglion cells after an experimental intervention. Most commonly these are done on either retinal whole-mounts or optic nerve cross-sections. In a wholemount the entire retina is flattened out onto a microscope slide. Retinal ganglion cells are identified by retrograde labeling with a fluorescent dye that had previously been injected into one of the targets of retinal ganglion cells, e.g. the superior colliculus, by immunohistochemistry, with ganglion-cell-specific promoters driving a fluorescent dye,54 or with antibodies to ganglion-cell-specific antigens. The optic nerve can also be sectioned and the number of retinal ganglion cell axons counted. Each axon corresponds to a living retinal ganglion cell. These techniques are obviously inappropriate for clinical testing. However, adaptive optics can be used to increase the resolution of conventional or fluorescent imaging of cells within the retina,55–57 allowing noninvasive quantitation of retinal ganglion cell survival. The techniques for localizing dying retinal ganglion cells with fluorescent dyes (mentioned above) are also suitable for structural imaging of single cells,45,46 and this is applicable to humans. This is an example of the convergence of structure and function. Finally, transgenic mice expressing a fluorescent protein such as yellow fluorescent protein under the control of a neuron-specific promoter can be used to longitudinally follow RGC numbers over time.58–60 The number of neurons within the targets of the retinal ganglion cell are also important for studying neuroprotective therapies. There is loss or dysfunction of lateral geniculate nucleus neurons in glaucoma.61–64 Even if a neuroprotective therapy rescues a retinal ganglion cell and its axon, if the process which causes the lateral geniculate neuron (or its target in the cortex) to die is not halted, then visual function may not be spared. The techniques for counting lateral geniculate neurons are usually stereological, i.e. require corrections for the number and thickness of the tissue slices and other factors. Therefore, lateral geniculate nucleus assessment is not as simple as counting retinal whole-mounts or optic nerve cross-sections. If the endpoint is instead the size of the lateral geniculate nucleus or its layers, then high-resolution MRI can be used.65 Preservation of a retinal ganglion cell and its axon is ineffective if the cell is not connected to those cells in the

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retina that excite or inhibit it. These connections occur via the dendritic tree and the cell body. The nature of the dendritic tree of retinal ganglion cells, which retracts in glaucoma,66 was initially studied by electrophysiologically identifying retinal ganglion cells and directly injecting Lucifer yellow or other dyes into the ganglion cell body.66 Such techniques necessitated postmortem analysis of tissue, and thus were not suitable for longitudinal imaging. The use of transgenic animals fluorescently expressing fluorescent proteins in the RGC dendritic tree allows longitudinal imaging with confocal scanning laser ophthalmoscopy,67 but like all techniques can be limited by the toxic effect of the fluorescent protein itself.68

DOES THE CONCENTRATION OF THE DRUG AT ITS TARGET MATCH THE CONCENTRATION NEEDED TO BE NEUROPROTECTIVE? There have been many attempts at producing comparable concentrations of ocular neuroprotectants in both in vivo and in vitro models. Although it is often difficult to extrapolate drug dosing and intraocular concentrations from the in vivo model to the human model, this helps in determining whether a neuroprotective therapy is likely to be effective. Chronic use of some drugs that bind to melanin results in deposition in ocular depots such as the retinal pigment epithelium, which can increase bioavailability over time.69

Assessment of Neuroprotective Therapies – Practice PRECLINICAL STUDIES OF NEUROPROTECTION FOR GLAUCOMA In the past, the evidence for neuroprotection was mostly generated by preclinical data from the bottom two layers of the pyramid (Fig. 61-1). Models of ocular hypertension provide more evidence, one layer higher in the pyramid. However, the randomized control trial is the gold standard for truly deciding whether a therapy is efficacious. The variability in disease progression and the inherent variability in measuring visual function or optic nerve structure has implications for the study of glaucoma, necessitating that good randomized control trials require somewhere in the region of hundreds of patients. In the early years of studying neuroprotection in retinal ganglion cells, most studies focused either on survival of retinal ganglion cells in culture or survival after severe injuries like optic nerve crush. Subsequently, the focus shifted to animal models of glaucoma that are closer to the human disease. These are usually intraocular hypertension models. Rodents are most commonly used, but non-human primates are also employed. The main interventions for neuroprotection that have been studied in preclinical research are pharmacological, preconditioning, and immune-based therapies. The vast majority are pharmacological. The following section discusses some seminal studies of neuroprotection in animal models of glaucoma.

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Non-Human Primate Glaucoma The non-human primate model of glaucoma,70 based on trabecular meshwork ablation with the argon laser, is the closest to the human disease in terms of disk morphology and the relationship of progression with intraocular pressure. This model has been used to study memantine, an NMDA antagonist. The original rationale for studying NMDA antagonists in glaucoma was the activity of NMDA receptor-mediated excitotoxicity of retinal ganglion cells,71 by analogy to how neurons die by excitotoxicity from glutamate in stroke, and early studies demonstrated elevated intraocular glutamate levels in experimental primate and human glaucoma72 and rodent optic nerve crush.73 Subsequently, large increases in levels of extracellular glutamate in glaucoma were not confirmed,74,75 and the nature of glutamate excitotoxicity in retinal ganglion cells was questioned.76,77 A solution to these discrepancies was suggested by findings that glutamate activates NMDA receptors on Müller cells, which secrete TNF-alpha and increase Ca++permeable AMPA receptors in RGCs.78 In a long-term monkey study, memantine partly reduced the functional loss associated with glaucoma, as measured by multifocal ERG and visual evoked responses,79 maintained disk structural integrity,80 and partly decreased retinal ganglion cell death.80 Significantly, memantine also reduced glaucoma-induced changes in the brain, i.e. cell shrinkage in the lateral geniculate nucleus.81 However, the number of animals used was small, and the power of the study to detect significant differences was low. Memantine, and probably other NMDA antagonists, could also preserve visual function and structure in glaucoma not by blocking excitotoxicity, but by another means, perhaps decreasing metabolic load or a similar mechanism. Drug concentrations of systemically delivered memantine appear to be similar to that necessary for inhibiting NMDA receptor activation.82 In monkeys treated with 4 mg/kg/day,83 the plasma levels of memantine reached the same concentration as that measured in human subjects treated for Parkinson’s disease. Rodent Ocular Hypertension Multiple drugs have been studied in rat and mouse models of ocular hypertension. Mechanisms include blockade of nitric oxide synthase with two antagonists, aminoguanidine and L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide84,85 (but this is controversial86), activation of the α2 adrenergic receptor with brimonidine,87 erythropoietin,88,89 the NMDA antagonist memantine,90 brain-derived neurotrophic factor,91 morphine,38 cannabinoid receptor activation,92 blocking LINGO-1,93 activating the unfolded protein response,94 and many others. These and other drugs are summarized in Table 61-2. A comprehensive list of drugs that have been studied in optic nerve injury paradigms can be found in a recent review.95 Other Mechanisms Focal activation of the immune system in the optic nerve and/or retina is a way of preserving retinal ganglion cells and their functions in the face of optic neuropathy. Activated T lymphocytes primed to optic nerve constituents, e.g. myelin basic protein, are home to sites of injury and

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Table 61-2  Drugs that are Neuroprotective in Animal Models of Glaucoma Drug or Other Therapy 84

Mechanism 86

2-Aminoguanidine (conflicting results ) Brain-derived neurotrophic factor (BDNF)/S-PBN91 Brimonidine87 Ciliary neurotrophic factor (CNTF)173 Electroacupuncture174 Erythropoietin88,89 Geranylgeranylacetone; heat stress175,176 Glatiramer acetate98 (conflicting results177) Glial cell line-derived neurotrophic factor (GDNF)178 L-N6-(1-iminoethyl)lysine 5-tetrazole amide85 LINGO-1 antibody93 Lycium barbarum Lynn179 Memantine90 Minocycline180 Morphine38 Phenytoin181 R(-)-1-(benzo [b] thiophen-5-yl)-2-[2-(N, N-diethylamino) ethoxy] ethanol hydrochloride (T-588)182 CHOP depletion or XBP-1 activation94 WIN55212-292

release factors which are neuroprotective.96 This was first demonstrated with optic nerve crush models, where the crush was partial, and thus there were several marginally injured axons.97 Similar findings were seen in a laser ocular hypertension model in the rat.98 Not only was generation of an immune response to myelin basic protein a mechanism for inducing this immune-mediated neuroprotection, but also to copolymer-1,99 a synthetic polypeptide which is also used for the treatment of multiple sclerosis. Another mechanism for neuroprotection is the secretion of neurotrophic or other salutary factors from some kinds of stem cells. For example, intravitreal oligodendrocyte precursor cells and mesenchymal stem cells survive in the eye, where they demonstrate a neuroprotective role for retinal ganglion cells.100,101 Finally, preconditioning with a sub­ lethal injury can protect against subsequent and more severe glaucomatous pathophysiology.102

CLINICAL TRIALS IN NEUROPROTECTION Despite the seductive premise of neuroprotection for glaucoma, there are barriers to this therapy being studied in future large-scale clinical trials. The biggest concern is the failure of two very large clinical trials of memantine in glaucoma (see below). Almost as discouraging is the long history of clinical trials in stroke that failed to show efficacy.103,104 No neuroprotective drug has been approved by the United States Food and Drug Administration for the treatment of stroke, despite dozens of drugs being successful in animal models of stroke and hundreds of millions of dollars being spent on randomized clinical trials.105 In one case a drug was effective in one large phase III study,106 but not in a second and larger study.107 Yet in every case the decision to go to clinical trial was based

iNOS inhibitor Neurotrophin/reactive oxygen species scavenger α2 agonist Neurotrophin Unknown PI3 kinase/Akt kinase activator Heat shock protein activator T-cell activator Neurotrophin Prodrug for iNOS inhibitor RhoA/JNK inhibition and Akt activation Medicinal herb NMDA antagonist Anti-apoptosis (blocks cytochrome c release) Opioid receptor activation Sodium channel blocker Upregulation of MAP kinase pathways Activation of unfolded protein response Cannabinoid receptor agonist

on preclinical studies showing that the drugs worked very well.108 The discrepancy between the preclinical and clinical results could result from several causes: (1) the model did not properly simulate the human disease; (2) the variability in patients was much higher than the variability of the disease in laboratory animals; or (3) the pathophysiology of the disease in humans is intrinsically different from animals. Most laboratory animals are small and their brains not as highly developed as humans. It is possible that the ratio of axonal damage to neuronal damage differs in human versus animal studies, and this may be one of the reasons why human neuroprotective studies of stroke have in general failed to show efficacy. Is the failure of neuroprotection in stroke relevant to neuroprotection in glaucoma? There is a wealth of studies demonstrating that various pharmacological agents and other agents are neuroprotective for retinal ganglion cells in optic nerve injuries, including ocular hypertension models that simulate human glaucoma. In stroke, drugs that were neuroprotective in the laboratory failed to show efficacy in patients. Analogously, animal data of neuroprotection in glaucoma is therefore insufficient for deciding whether a drug is effective in patients. The consequence of this line of reasoning is the necessity that well-designed randomized control trials of potential neuroprotective agents are the acid test, and not just preclinical data.

Memantine in Open-Angle Glaucoma Memantine, an NMDA receptor antagonist, was studied in two industry-supported (Allergan, Irvine, CA) Phase 3 studies, each with more than 1000 patients and lasting multiple years. Patients received standard management of their glaucoma and were randomized to receive oral

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memantine or placebo. The primary outcome measure was visual function. As of early 2013 this was the largest study of neuroprotection in an ophthalmic disease. The results of the memantine studies have not been published or presented. However, two press releases from the sponsor revealed that neither of the two studies met their primary outcome measure. The first109 stated: ‘Two measures of visual function were selected in the statistical analysis plan to assess the efficacy of memantine in glaucoma. The functional measure chosen as the primary endpoint did not show a benefit of memantine in preserving visual function. In a number of analyses using the secondary functional measure, memantine demonstrated a statistically significant benefit of the high dose compared to placebo.’ The second110 stated: ‘Although the study showed that the progression of disease was significantly lower in patients receiving the higher dose of memantine compared to patients receiving the low dose of memantine, there was no significant benefit compared to patients receiving placebo. Therefore, the study failed to meet its primary endpoint and to sufficiently replicate the results of the first Phase 3 trial. While additional analyses are ongoing, the company does not believe that these analyses will support an approval of the drug.’

Low-Pressure Glaucoma Treatment Study The Low-Pressure Glaucoma Treatment Study (LoGTS) randomized patients with normal-tension glaucoma to topical brimonidine 0.2% or timolol 0.5%.111 These drugs were chosen because they have equivalent pressure-lowering effects, and therefore the hypothesized neuroprotective effects of brimonidine could be tested independent of intraocular pressure lowering. All subjects completed enrollment and were followed for a minimum of four years. Subjects had to have intraocular pressures less than or equal to 21 mmHg on a diurnal curve. Subjects, physicians, technicians and the reading centers for visual fields and optic disk photographs were masked. The primary outcome was progression at three or more points in the Humphrey 24-2 full-threshold automated visual field by pointwise linear regression and confirmed on three successive fields. The study showed that 9% of the brimonidine group and 39% of the timolol group progressed (p = 0.001), despite equivalent amount of IOP lowering. The decreased progression in the brimondine group was also seen when glaucoma change probability mapping or the 3-omitting method for pointwise linear regression were used to analyze the results. Although these results are consistent with a neuroprotective effect of brimonidine, given equivalent degrees of IOP lowering, there are other mechanisms that could account for the results seen or that should temper the interpretation of the data.112 (1) Differences in the diurnal effects of IOP lowering between brimonidine and timolol might have had consequences on progression at specific times of day or night. (2) The effects on blood flow to the optic nerve head might have differed between the groups, and this might have been amplified in a study looking at subjects who were progressing at low IOP. (3) The amount of IOP lowering was low overall, with only about 40% of subjects having IOP lowering of 20% or greater. Future studies of neuroprotection would likely not tolerate such a

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low degree of IOP lowering. (4) There was a significantly greater number of subjects in the brimonidine group who dropped out from the study because of ocular allergy, and it is theoretically possible that those drop-outs were also the ones who might have been at higher risk of progression.

Ongoing Studies As of April 2013, there are several studies listed in the clinical trials registry (clinicaltrials.gov) that include the word ‘neuroprotection’ or related terms and include a patient population with a disease of the optic nerve or retina. Therapies being examined include: intravitreal QPI1007, a caspase 2 siRNA, in nonarteritic anterior ischemic optic neuoropathy (Quark); a ciliary neurotrophic factor intraocular implant in glaucoma (Bascom Palmer Eye Institute); topic MRZ-99030, a beta-amyloid aggregation modulator, in glaucoma (Merz); and brimonidine implants in glaucoma and other ocular diseases (Allergan). Other studies are likely planned but not yet available in publicly accessible databases. Implications It is encouraging that at least one study showed an effect that could be interpreted as being consistent with neuroprotection. However, overall there have been many studies in the neurological literature that failed to show efficacy, and there are parallels with ophthalmology.113 Clearly, future trials will require careful and clever study design in the future, which do not require immense resources, as were used in the memantine studies.114 Neuroprotection is an example of a failure to translate preclinical results to clinical therapies. This problem is endemic to translational research, and a recent review identified three issues that cause the ‘Lost in Translation’ problem.115 The first is the ‘Butterfly in Brazil’ problem, a reflection of the chaotic nature of preclinical models, where a small difference in how the model is carried out may lead to a large difference in the result. If models are sensitive to small changes, then they are less likely to translate to human subjects. The second is the ‘Princess and the Pea’ problem, where the drug development process classically results in less effect and more variance along the chain from chemistry to cell biology to animals to humans. The third is the ‘Two Cultures’ problem, a reflection that how studies are carried out in the laboratory are not always as rigorous as the clinical trials used for approval by regulatory agencies. There are possible solutions for some of these problems,116 but in general these are general barriers to achieving successful translational research in general, and specifically, neuroprotection in glaucoma.

Axoprotection Several treatments are successful for preventing the death of retinal ganglion cells in experimental models of glaucoma. However, treatments that maintain retinal ganglion cell viability without taking into account the functional consequences are unlikely to have a positive clinical outcome when translated to human use. For patients, visual function is the endpoint that is relevant to the patient’s

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quality of life, and therefore the value of neuroprotection directed at retinal ganglion cell soma survival alone has been questioned. If the optic nerve is transected, then maintaining the viability of a retinal ganglion cell despite a damaged axon will not prevent visual loss. Even if the retinal ganglion cell can send impulses through the nerve fiber layer towards the optic disk, if the axon is damaged or transected, then the impulse will never reach the brain, and the patient will not be able to see using that axon. We do not know whether axonal damage alone from glaucoma is irreversible, or whether it relies on the death of the retinal ganglion cell as well. However, from studies of other optic nerve injuries, it appears that once axonal injury has occurred that is sufficient to disrupt the structure of the axon, the axon will not be regenerated or maintained for the life of the animal. Therefore, a different strategy should be considered, one in which preservation of the axon is necessary. This strategy is called axonal protection or axoprotection, and represents the protection of axonal integrity and function in the face of damage. The following section is a summary from a recent comprehensive review on axon injury and optic neuropathy.117

AXONAL DEGENERATION Axonal degeneration after retinal ganglion cell injury takes place in two directions. The degeneration towards the cell is called retrograde degeneration, and the one away from the cell (and the site of injury) is called Wallerian or orthograde degeneration. It is important to realize that the processes of death of the cell body and degeneration of the axon are independent.118 Axonal responses can be divided into those associated with how the axon locally responds to injury; and those associated with degeneration of the proximal and distal healthy axon as a result of the injury. Mechanisms by which the axon degenerates after direct injury include excess accumulation of Ca++ ions, activation of calpains, loss of the membrane potential, and several other processes. In healthy fibers, ATP-dependent pumps support homeostasis of ionic gradients. When energy supply is limited, either due to inadequate delivery or excessive utilization, ion gradients break down, unleashing a variety of cascades leading to Ca++ overload, activation of destructive enzymes, and local axonal dissolution.119 Mechanisms for degeneration of the healthy remaining axon after injury are different, with most research focusing on Wallerian degeneration, i.e. loss of the distal segment. Neurons have a self-destruct program in their axon,118 parallel to the suicide program for apoptosis of the cell body. The program for Wallerian degeneration is initiated when the axon is injured. There is a naturally occurring mutation in mice, Wallerian degeneration slow (WldS), which blocks this axonal degeneration program.120 Interestingly, the WldS mutation does not itself prevent the calcium influx associated with early stages of axonal degeneration.121 Studies of experimental glaucoma in these mutant animals demonstrate loss of the RGC but preservation of the axon.122 This is the exact opposite of what occurs when the apoptosis program is blocked in glaucoma, where the cell body is preserved but the distal axons

are lost.6,123 Together, these data prove that the retinal ganglion cell body death and axonal degeneration programs are distinct.124

METHODS FOR AXOPROTECTION Much of our understanding of axonal protection relies on data from either ischemic injuries to the rodent optic nerve125 or understanding how the WldS mutation causes the distal part of the axon to maintain its integrity despite a transection proximal to the site of injury.126 In general, there is little known about how best to protect an axon in glaucoma. The most investigated area is ischemia of the isolated optic nerve, with work by Waxman and colleagues dating back to the 1990s. They and others showed that calcium and sodium influx mediate ischemic or anoxic damage to retinal ganglion cell axons,127 and that this can be ameliorated by drugs that block sodium channels.128 In recent years there has been accelerating interest in finding new methods for axoprotection.23,129–134 The WldS mouse, where Wallerian degeneration is slowed,135 and distal axons remain viable despite apoptosis of the cell body 122,136 is likely to be extraordinarily helpful in identifying novel methods for axoprotection. The molecular basis for WldS is a fusion of nicotinamide mononucleotide adenylyl transferase 1 (NMNAT-1) to ubiquitination factor e4b (UBE4B) by an 18-amino-acid linkage. The mechanism of WldS axoprotection involves increased axonal NMNAT-1 after axotomy.137 NMNAT-1 is critical to synthesis of NAD+, a key electron-accepting redox agent, and it is possible that one of the redox reactions by which elevated local NAD+ protects axons may be a target for future axoprotection research. It is unclear whether axoprotection related to a WldS mechanism would also be effective for retrograde degeneration. The WldS genotype protects against Wallerian but not retrograde axonal degeneration in the dopaminergic nigrostriatal pathway.138 This implies that there are targets for retrograde axo­ protection may be different from those for Wallerian degeneration.48 Although the concept of axoprotection is in its very early stages, it is likely that understanding axonal death and injury and how axons can be maintained will lead to advances in therapies for diseases such as glaucoma.

Neurorepair Neuroprotection is a strategy for preventing the death of neurons; however, once the neuron has died and the axon has disappeared, visual function cannot be restored unless a new neuron is delivered and connected to the appropriate afferent and efferent targets. Neurorepair of the optic nerve requires the production and differentiation of new retinal ganglion cells from stem cells, connection to bipolar and amacrine cells, and regeneration of their axons to their appropriate targets in the brain. In addition, the cells must maintain survival, connectivity, and function despite what is often an ongoing disease process. The multiplicity of barriers makes neurorepair one of the most difficult areas for restoring vision in glaucoma, and is discussed in several reviews.139

61  •  Neuroprotection and Neurorepair

DIFFERENTIATION OF STEM CELLS INTO RETINAL GANGLION CELLS Differentiating neural stem cells into specific classes of retinal neurons is a first step in repopulating the neurons lost in glaucoma. Transplantation studies with stem cells have been increasingly used in attempts to repopulate neurons lost in degenerative or traumatic disease. A wide variety of stem cells have been studied for this purpose, e.g. embryonic stem cells,140 brain-derived precursor cells,141 hippocampal-derived neural stem cells,142 bone marrowderived stem cells,143 olfactory ensheathing cells,144 and inducible pluripotent stem cells.145 The eye itself contains cells capable of differentiation, including retinal pigment epithelium and Müller cells. Retinal stem cells have been demonstrated in the ciliary marginal zone of adult vertebrate eyes.146,147 However, the fact that visual loss from most optic nerve diseases (and particularly glaucoma) does not spontaneously improve implies that ciliary marginal zone stem cells and other endogenous stems cells do not normally divide, differentiate into RGCs, or repopulate the retina at clinically significant levels. The nature of these and other ocular stem cells was reviewed in 2012.148 A major goal is therefore to find ways of directing the differentiation of stem cells into specific retinal neurons, and thereby replace those lost due to pathology. The most complete data for retinal neuronal development exist for the RGC. It is known that several signals are involved in the developmental differentiation of progenitor cells into mature RGCs. This differentiation is paralleled by the expression of specific developmentally regulated genes.149 Although stem cell transplantation of brain-derived, hippocampal-derived, and bone marrow-derived stem cells into the retina leads to integration of these stem cells,140,141,150 the majority do not differentiate into RGCs, even after RGC depletion.151 Instead, they mostly develop into amacrine and horizontal cells.142 Müller glia can induce neural progenitor cells to differentiate into RGCs,152 and in vitro conditions can induce release of excitatory neurotransmitters from these newly produced RGCs.153 Conversely, Müller glia can be differentiated into RGCs. Singhal and colleagues differentiated human Müller glia and transplanted them into rat retinas deprived of retinal ganglion cells.154 The cells survived and were able to partly restore the negative scotopic threshold response, an electrophysiological measure of RGC cell body survival and connectivity to other retinal neurons. The axons of the transplanted cells did not reconnect through the optic nerve to targets in the rest of the central nervous system. Another group differentiated embryonic stem cells into neuronal precursor cells, that could be differentiated in vivo into RGC-like cells.155

AXONAL REGENERATION Regeneration of axons from retinal ganglion cells is a scientific problem that has been studied for many years. The mammalian optic nerve does not regenerate after it is transected, unlike lower animals such as the goldfish, which readily regenerate and form appropriate connections to targets in the brain. Nerves in the peripheral nervous system are also able to regenerate once transected, but mammalian central nerves cannot. Two decades ago,

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Aguayo and colleagues demonstrated that transection of the optic nerve, followed by replacement of part of the optic nerve with a sciatic (peripheral) nerve graft allowed limited regeneration.156 Regenerating axons are derived from retinal ganglion cells that express growth-associated protein-43 (GAP-43).157 Subsequently, studies of the factors which prevent regeneration in the central nervous system focused on the role of myelin-associated substances158 (e.g. myelin-associated glycoprotein159) and glial (astrocytederived) scarring (e.g. proteoglycans160) as inhibitory factors. Downregulation of the receptor for Nogo, a myelinassociated protein,161,162 enables regeneration of retinal ganglion cell axons when appropriately sensitized.163 Instead of targeting the receptors by which the inhibition of axonal extension is signaled, some groups have focused on the subcellular transduction pathways for inhibition. For example, up-regulation of cAMP allows regeneration of spinal cord164 and retinal ganglion cell165 axons, and mice lacking receptor protein tyrosine phosphatase sigma have increased retinal ganglion cell axon regeneration past a glial scar.166 Finally, there is a dramatic reduction in the axonal extension rate of retinal ganglion cells around the time of birth. This switch appears to be signaled by contact with amacrine cells,167 and the nature of the signal and how it is transduced is being studied. The field of optic nerve regeneration has grown dramatically in the past decade, and several comprehensive reviews have recently been written.168–170 Some of the more profound findings are the ability to regenerate RGC axons and induce connectivity. For example, deletion of both phosphatase and tensin homologue (PTEN) and suppressor of cytokine signaling 3 (SOCS3) allows sustained RGC axonal regeneration.171 Combining injections of zymosan and a cAMP analogue with deletion of the gene for PTEN not only allows full-length RGC regeneration, but allows some functional recovery, as measured by the optomotor response and circadian photoentrainment.172 Regeneration of optic nerve axons is one component of the challenge of regenerating the optic nerve. A greater problem is the guidance of extending axons along the complex path to the target. This trajectory begins at the retinal ganglion cell body within the inner retina to the optic nerve head, through the optic nerve, then crossing (of nasal fibers) at the chiasm, and continuing along the optic track to the retinotopic map within the lateral geniculate nucleus within the appropriate layer. In development, this complex pathway is determined by cell surface molecules and secreted chemotactic gradients. In many cases it is not known whether the same set of molecules is present in the adult organism. If not, those molecules would have to be recreated in order to allow an extending axon to find its appropriate target.

ASSESSMENT OF NEUROREGENERATION Assessment of replacement and regeneration of RGCs requires demonstration that its axon has extended out of the retina and ideally, to its target. This is usually done with orthograde labeling of axons, by injecting a dye or other marker into the eye, making sagittal or coronal sections of the optic nerve, and assessing the degree of axonal extension and connectivity. Assessment of RGC replacement in

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the retina, e.g. with stem cells, requires identification that the cells truly are RGCs, that they connect to neighboring neurons, and that they at least partially extend an axon towards the optic nerve. Functional assessment of stem cell replacement requires demonstration that the cells fire action potentials in an appropriate pattern corresponding to light input to photoreceptors in the immediate area of the RGC. Functional assessment of regeneration requires electrophysiological evidence of activation of target areas within the central nervous system, e.g. the superior colliculus for visual orienting, the lateral geniculate nucleus for depth perception, the suprachiasmatic nucleus for circadian photoentrainment, or the pretectal nuclei for the pupil. The complexity of the stem cell replacement and reconnection process for glaucoma is clearly immense. But even if replacement and regeneration could be achieved, without appropriate mapping to retinotopic targets, vision would be coarse and low resolution.

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