Preclinical animal modals that have been used for neuroprotection studies relevant to glaucoma include cutting or crushing of the optic nerve, acute retinal ischemia-reperfusion, inducing elevated IOP, chronic optic nerve ischemia, and intravitreal injection of an excitotoxic neurotransmitter or analogue (e.g., glutamate, N-methyl-D-aspartate [NMDA], and kainic acid). Justification for the optic nerve crush, ischemia, and excitotoxic models is based on theories for the initiation and development of retinal pathology in glaucoma. Each model is thought to mimic at least one component of glaucomatous optic neuropathy but may be very different from human glaucoma. Considerable progress has been made toward the development of a spontaneous glaucoma model. In rodents, a strain of mice (DBA/2)^1,2 and rats³ with spontaneous glaucoma now exist. A wide range of drug classes shows neuroprotective activity in RGC culture systems, but fewer have been tested and found active in the acute in vivo models of RGC death, and fewer still in chronic high-IOP models, which arguably have the closest relevance to glaucoma. For the purposes of this volume, this chapter emphasizes the mechanisms leading to RGC death and neuroprotective studies done on in vivo model systems and excludes studies on regeneration.

Glaucomatous neuropathy is likely a response to abnormal stressors (primary risk factors) interacting with several mostly undefined secondary factors, such as in some cases a genetic predisposition. It is abundantly clear from numerous clinical and experimental studies that the level of IOP is a primary risk factor for optic neuropathy. A consistent observation is that a high IOP, however caused in humans, results in retinal damage. Human studies have confirmed that the lower the IOP, the slower the progression of glaucomatous damage will be, even for IOPs considerably below the currently accepted range of “normal.” Pressure may act as a direct primary stimulus for apoptosis in neural cells,⁴ but it is the prevailing hypothesis that secondary signals cause RGCs to become apoptotic. In addition to elevated IOP there is now also sufficient evidence from studies on patients with glaucoma with IOPs in the normal range (normal tension glaucoma) to indicate that chronic hemodynamic changes, such as those associated with sleep apnea syndrome, and/or localized episodic vasospastic dysfunction in the ophthalmic microcirculation may be another primary risk factor.⁵ This would be particularly damaging to RGCs and their axons located in areas of perfusion such as the retinal and optic nerve head/lamina cribrosa microvasculatures. Focal dysfunction in the regulation of blood flow by intrinsic (autoregulatory) or extrinsic mechanisms at these locations could cause, respectively, ischemic stress to RGC somata or to their unmyelinated axons in the optic nerve head. There is some experimental evidence that excessive vasoconstriction by endothelin may be a contributor to the optic nerve head vascular pathophysiology in glaucoma.⁶

There may be an important interaction between vascular dysfunction and elevated IOP.⁷ One may hypothesize a defective regulation of optic nerve head or retinal blood flow in response to IOP compression of the microcirculation to be the predisposing factor for glaucomatous RGC loss. Subjects with normal microvascular regulation may be able to counteract moderate increases in IOP and thus avoid ischemic damage and glaucomatous retinal pathology, as may be the case with ocular hypertensive patients who do not develop injury. Some degree of vascular dysregulation may not be pathologic by itself but may become so with the added stress when IOPs are elevated above normal, as in the case of primary open-angle glaucoma, whereas a greater degree of vascular dysregulation could be pathologic even with normal IOP levels, as in the case of normal-tension glaucoma. Thus, since the optic nerve vascular supply is under direct IOP-induced strain, the vascular and mechanical factors involved are intrinsically intertwined, and it is likely that both mechanical and vascular phenomena play a role in varying degree in all clinical manifestations of glaucomatous optic neuropathy.

Any hypothesis for the initiation and progression of glaucomatous neuropathy needs to account for the focal and chronic nature of RGC and nerve fiber layer (nfl) loss. In vascular dysfunction, inability of retinal microvessels in a localized area to autoregulate against IOP compression could lead to hypoperfusion and a focal ischemia that in turn triggers overriding autoregulatory mechanisms that temporarily restore perfusion (e.g., mediated by the localized rise in pCO₂ level). This response cycle could result in repetitive transient episodes of ischemia-reperfusion over an extended period of time, causing a gradual focal loss of RGCs and thinning of the nfl leading to that location. A similar postulate can be offered for ischemic-reperfusion episodes occurring in the optic nerve head/lamina cribrosa region when subjected to compression by elevated IOP. Localization of the microvascular pathology to subregions of the optic nerve head could account for the observed focal pattern of RGC death and nfl thinning. These conjectures for a vascular pathology initiating glaucomatous optic neuropathy provide some justification for using ischemia-reperfusion models to develop neuroprotective agents for glaucoma. This vascular hypothesis includes both current general theories about where retinal glaucoma pathology actually begins, namely, axon pathology leading to RGC loss (termed primary, or retrograde cell death) or damage to or death of the ganglion cell body first, followed by axon degeneration (termed secondary, or anterograde death). As discussed subsequently, it is likely that both types of death processes occur in glaucoma.

A commonly held notion suggests that initiation and progression of retinal damage in IOP-induced primary open-angle glaucoma, and most secondary glaucomas as well, occur on the unmyelinated axons, particularly at the optic nerve head/lamina cribrosa. Elevated IOP is thought to cause structural distortion of the optic nerve head, which alone, or in conjunction with compression of the microvasculature, results in localized ischemia. Structural distortion of the cribriform plates, ischemia, or just the increased pressure itself may be directly deleterious to unmyelinated nerve fibers and disrupt axoplasmic flow of neurotrophins to RGCs. These stresses could also cause an activation response in astrocytes or oligodendrocytes of the optic nerve head and in the nfl of the retina. The activation of astrocytes results in physical damage to axons by erosion or remodeling of the extracellular matrix (ecm),⁸ clinically seen as cupping of the optic nerve head, and additional axon damage caused by the release of toxic mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and excess nitric oxide.⁹ The end result of all these postulated cellular pathologic events is disruption of a functional axonal connection between RGCs and their target neurons in the lateral geniculate nucleus of the brain. Interruption of the anterograde flow of information and materials, but more particularly of the retrograde axoplasmic flow of neurotrophic proteins to RGC somata, is thought to be a critical signal for RGC death. However, as noted previously, the injured axon or its surrounding glial cells may also signal RGC death. On the basis of these hypotheses, potential neuroprotective agents acting at the level of the optic nerve head or the nfl can be targeted either to axons or to retinal glial cells (astrocytes, oligodendrocytes, Müller cells, microglia). A better understanding of the pathophysiology of unmyelinated RGC axons and of retinal glial cells is needed to develop such protective agents.

Axon degeneration can be triggered by various insults, and it appears to be an autodestructive process with similarity to apoptosis. Axonal injury causes the release of cytochrome C from mitochondria but does not appear to follow subsequent apoptotic pathway steps involving caspases.¹⁰ In normal mice transected axons distal to their soma fail to conduct an action potential within 1 day of the injury, but in the Wallerian degeneration-Slow (WldS) mouse mutant¹¹ such axons remain structurally intact and can conduct action potentials for up to 3 weeks. It is likely that axon preservation in the WldS mouse applies also to the proximal part of a damaged optic nerve axon and may increase survival of the RGC.

The WldS mutation causes overexpression of a chimeric protein composed of a component of the ubiquitin proteosome system (UPS) and the nicotinamide adenine dinucleotide (NAD) synthesizing enzyme nicotinamide mononucleotide adenyltransferase-1. The mechanism for axonal protection by this chimeric protein remains under investigation. Proposed mechanisms include increased NAD synthesis,¹² the UPS effect on Wallerian degeneration,¹³ or modification of the cell cycle pathway.¹⁴ This finding supports neuroprotective strategies aimed at increasing the cellular NAD supply, and it may explain earlier reports on the beneficial effect of nicotinamide treatment in several neurodegeneration models.

The damaging initial effect of ischemia on axons that has been most studied is a persistent depolarization that occurs through opening of Na⁺ and/or Ca²⁺ entry channels. Sodium channel blockade by some anesthetics, anticonvulsant drugs, and ββadrenergic receptor blockers can significantly reduce axon injury in such models. These agents include, for example, lidocaine, riluzole, flunarizine, betaxolol,¹⁵ diazepam, carbamazepine, and phenytoin. In an in vivo study of a rat model of glaucoma, orally administered phenytoin was found to have a protective effect on RGCs.¹⁶ The wide chronic therapeutic use of this agent provides an opportunity for a retrospective clinical study to assess whether Na⁺ channel blockade is of value in glaucoma therapy. Ca²⁺ channel blockers, such as nifedipine and nimodipine, which are primarily used as vasodilators, gave marginally positive results when evaluated in a clinical study.¹⁷ The present status in glaucoma therapy of established channel blocking drugs that affect axons, alone or combined with a vascular effect, is that they may have limited value in specific instances,¹⁸ but their general use is probably unwarranted. Because of the multiplicity of neural Na⁺ and Ca²⁺ channel subtypes, and the state-dependent binding at multiple sites of potential blockers, we may not yet have identified the appropriate channel type or the kind of blocking agent needed for a more effective glaucoma therapeutic agent of this drug class.

In normal conditions, glial cells help with homeostasis of glucose, metabolites, pH, ions, and neurotransmitters (e.g., glutamate and gamma-aminobutyric acid) of the optic nerve head and retina.¹⁹ In response to stress/injury, glial cell activation occurs and can result in cellular hypertrophy and hyperplasia as well as increased expression of intermediate filament proteins such as glial fibrillary acidic protein and vimentin. With glaucomatous progression these ecm materials fill areas previously occupied by RGC axons. Ecm organization and composition changes may alter the biochemical/structural stresses in the optic nerve head, thereby increasing the vulnerability of the remaining axons.²⁰ Changes of ecm proteins, their metabolizing enzymes, and many other proteins are also noted to occur during activation.²¹ Activated astrocytes may damage the ecm by increased nitric oxide (see later discussion of nitric oxide). Activated astrocytes show induction of the ecm modulating enzyme matrix metalloproteases (MMP) in response to elevated IOP.²² A combination of ischemia and trauma has been shown to result in a loss of the ecm component laminin in the nfl/RGC layers.²³ The importance of this cell–matrix interaction for cell survival is indicated by the finding that neurons grown on a laminin substrate exhibit sustained antiapoptotic signaling through the phosphoinositide 3-kinase (PI3-k)/Akt pathway. Loss of laminin and RGCs was associated with increased MMP-9 activity²³ or when ecm modification was blocked by inhibition of plasminogen activators.²⁴ These findings indicate that, as a consequence of glial activation, the loss of appropriate interaction with the ecm might be an important signal within the retina for initiating axon degeneration and apoptosis in RGC. In contrast with causing damage, reactive glial cells in the retina could potentially have some beneficial effects on the basis of up-regulation of various survival/neurotrophic/growth factors, such as insulin-like growth factor (IGF)-1, IL-10, ciliary neurotrophic factor (CNTF), and members of the chaperone/heat stress proteins (HSPs), such as HSP-27.²⁵

The foregoing discussion indicates several potential mechanisms that could be targeted for developing neuroprotective agents that interfere with destructive actions by activated astrocytes or other glial cells, namely, inhibitors of nitrous oxide synthase (NOS)-2 or enzymes that modify the ecm, and agents or gene therapy that promote local neurotrophin/survival factor synthesis and release to make up for the interruption of axonal supply of such factors from the lateral geniculate nucleus.

Neurotrophic factors play an important role in RGC survival. RGCs not receiving the appropriate chemical factors from the brain and lateral geniculate nucleus can undergo apoptosis. Elevated IOP has been shown to interrupt axoplasmic flow of RGCs at the level of the optic nerve head.²⁶ This blockade and interruption of neurotrophic factors at the optic nerve head has been postulated as a theory for glaucoma.²⁷ Neurotrophins such as brain-derived neurotrophic factor (BDNF), nerve growth factor, neurotrophins 3 and 4, as well as their receptors (TrkA, TrkB, p75) are examples of potential targets for neuroprotective strategies.²⁸

In ischemic eyes, BDNF and its receptor TrkB have been shown to play a role in preventing apoptosis.²⁹ Although BDNF/TrkB signaling is not essential for RGC survival during development, it is required for maintenance of RGC in adulthood and appears to be essential for the immediate survival of RGCs after injury.³⁰ Intravitreal injection of BDNF was found to significantly delay the death of axotomized RGCs.³¹ These findings on BDNF led to the recent promising study using retinal BDNF gene therapy in a rat model of chronic elevated IOP.³²

There are some experimental indications that activation of receptors mediating neurotransmission can affect signaling by BDNF and other neurotrophins. Elevated levels of cyclic adenosine monophosphate (cAMP) increase the survival effect of neurotrophins in RGC cultures. BDNF receptor activation leads to an intracellular antiapoptosis signal pathway involving the activation of PI3-k and the serine-threonine kinase, Akt, resulting in inactivation of proapoptotic proteins, such as BAX or BCL-2-associated death protein (BAD), by phosphorylation. cAMP can potentiate the BDNF signal cascade either by blocking BAX/BAD proapoptotic signals or up-regulating antiapoptotic BCL-2 expression.³³ On this basis one might expect cAMP activators to be beneficial in optic nerve damage models, but there is as yet no evidence for this in vivo. Instead, contrary to this expectation, it has been found that a β-adrenergic antagonist, betaxolol, is beneficial in the ischemia model and also up-regulates mRNA for BDNF³⁴ and other neurotrophic/growth factors.³⁵ This response, and other mechanisms of some β-adrenergic antagonists, such as ion-channel blockade, suggests that their neuroprotective effect involves signal systems other than the β-adrenergic receptor/adenylyl cyclase system.

Adult RGC survival and regeneration in vivo after optic nerve damage or ischemia can also be promoted by CNTF,³⁶ ββFGF,³⁷ and IGF-1.³⁸ FGF can also be up-regulated in the retina by activation of other receptors, for example, by αα-adrenergic agonists,³⁹ and this may account in part for the neuroprotective effect of this class of drug.^40,41,42 These findings on ββantagonists and ααagonists affecting growth factors hold out the possibility that conventional pharmaceutical agents acting through well-established receptor systems (such as those linked to cAMP), or those that have novel drug mechanisms that modulate neurotrophin/growth factor expression, may be a useful therapeutic approach to minimize loss of trophic support caused by damaged axons. For the near term, this approach deserves further investigation because it seems more clinically feasible than the promising but long-term approach of direct replacement of growth factors and neurotrophins by gene therapy.

The normal rat RGC layer has some resident cells that express RNA for BDNF, which becomes up-regulated and expressed in additional cells for approximately 2 weeks after an optic nerve crush.⁴³ Up-regulation in the retina itself of BDNF, other neurotrophins/growth factors, and heat shock proteins thus appear to be components of a general local protective response against intraocular injury. This important endogenous protective response occurs with various preconditioning insults and is not yet understood for all types of insult. For example, even a saline injection into the vitreous can increase local neurotrophin/growth factor levels, and an injury to the lens is effective in promoting survival of RGCs and even axon regeneration.^44,45