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extracellular ATP released through pressure sensitive pathways either stimulating lethal P2X7 receptors on ganglion cells, or being converted to adenosine and protecting them. Additional theories propose a role for reactive astrocytes, compressive forces and even increased age itself in weakening ganglion cells to the point where they eventually die. Many of the basic patterns may not be restricted to the posterior of the eye, but may hold lessons for the study on the anterior chamber.

II. INTRODUCTION

At first glance, a chapter on retinal ganglion cells may seem out of place in a book about aqueous humor. Ganglion cells have an unknown influence on the composition, production, or drainage of the humor, while only 5% of the aqueous humor flows towards the posterior of the eye, limiting even unidirectional communication (Maurice, 1987). However, a mismatch between rates of aqueous humor secretion and drainage is of clinical interest primarily because the resulting increase in intraocular pressure (IOP) is a predominant risk factor for glaucomatous optic neuropathy (Quigley, 1996; Gordon et al., 2002; Sigal et al., 2005). A key goal in balancing the inflow and outflow of aqueous humor in glaucoma is to maintain or restore ganglion health. As such, a basic understanding of ganglion cells and how they are injured by elevated IOP is beneficial.

This report will first summarize the general characteristics of ganglion cell injury in glaucoma, detailing the types of ganglion cells lost, the influence of pressure, the time course of their loss, and the loss of ganglion cell function that precedes cells death. The second section deals with selected theories to explain how increased IOP can lead to ganglion cell loss. It is becoming increasingly evident that glaucoma is a multifactorial disease with both genetic and environmental influences. It is also evident that the pathogenesis of glaucoma generates considerable controversy. This chapter will not attempt to address all the putative contributions to ganglion cell death in glaucoma; many of these have been detailed in extensive reviews and readers are encouraged to pursue them for more information (e.g., Hernandez, 2000; Morgan, 2000; Osborne et al., 2001, 2003; Levin and Gordon, 2002; Wax and Tezel, 2002; Neufeld and Liu, 2003; Votruba, 2004; Morrison et al., 2005; Tezel, 2006; Gupta and Yucel, 2007). An abridged discussion does provide a necessary structure to understand recent developments, and in some cases provides evidence for opposing viewpoints. Particular attention will be given to the emerging role of purines as a link between elevated ocular pressure, ganglion cell death, and neuroprotection.

III. INFLUENCES ON GLAUCOMATOUS DAMAGE TO GANGLION CELLS

A. Ganglion cell Type

There are 1.2–1.5 million ganglion cells in the human retina. They receive visual information from photoreceptors via the bipolar and amacrine cells, and deliver the visual signal through the axons of the optic nerve to the superior colliculus (SC), and the lateral geniculate nucleus (LGN). Morphologic criteria are used to classify ganglion cells into basic groups (Kolb et al., 1992). The midget cells (P cells) have relatively small cell bodies and dendritic trees, and project to parvocellular layers of LGN (Dacey, 1993). Parasol cells (M cells) have larger cell bodies and dendritic fields and project to magnocellular layers of the LGN. Bistratified retinal ganglion cells have the smallest cell bodies and project to the koniocellular layers of the LGN. While all cell types are present across the retina, larger cells are concentrated in the periphery, while the central retina contains a higher percentage of cells with smaller somata (Dacey, 1994).

The distribution of ganglion cell types to particular retinal regions has apparent relevance to the identification of susceptible populations, and it is thought that a correlation could provide insight into the causes of cell death. Although ganglion cells throughout the retina are lost in glaucoma (Desatnik et al., 1996), peripheral ganglion cells typically die at a higher rate (Laquis et al., 1998; Sawada and Neufeld, 1999). This agrees with clinical findings where peripheral vision is aVected first. The nasal field is usually compromised during early stages of glaucoma, with an arcuate pattern of loss surrounding the fovea leading to enhanced axonal loss in the superior and inferior regions (Quigley and Green, 1979).

Some psychophysical evidence indicates parasol cells may be particularly susceptible to glaucomatous damage (Anderson and O’Brien, 1997). However, evaluation of patients in the early stages of the disease found no preferential loss of sensitivity from the magnocellular pathway (Ansari et al., 2002). A detailed morphologic analysis of labeled ganglion cells in primates with ocular hypertension found no significant diVerence between the loss of parasol and of midget ganglion cells (Morgan et al., 2000). Other research has approached the problem through cell size, attempting to correlate likelihood of loss with soma or axon dimensions. The mean diameter of axons in the optic nerve of primates with experimental glaucoma was significantly smaller than in control eyes (Quigley et al., 1987). Likewise, there were fewer ganglion cells with larger cell bodies in the retina of glaucomatous primate eyes (Glovinsky et al., 1991). While larger cells do seem more susceptible to damage changes in cell size with the progression of glaucoma undermine this form of analysis. For example, the dendritic field, soma and axon of ganglion

cells from glaucomatous primates were reduced before cell death occurred (Weber et al., 1998). The extent of shrinkage correlated with evidence of optic nerve atrophy. This shrinkage may also contribute to reduced visual function, as discussed below.

B. Elevated IOP

The normal range of IOP in adult humans has traditionally been defined as between 10 and 21 mmHg, with pressure above this considered a major risk factor for the establishment of glaucoma. Multiple studies have demonstrated a correlation between elevated IOP and cell death. Pharmacological treatment that decreases IOP reduces the likelihood of disease progression (AGIS, 2000). Even in so called ‘‘normal tension’’ glaucoma, a decrease in IOP is beneficial (CNTG, 1998), suggesting that the set point for damage could be lower in some patients. Interestingly, an improvement in the patterned electroretinogram (PERG) response in patients with normotensive glaucoma accompanied a decrease in IOP, implying that reduced cellular function can be reversed (Ventura and Porciatti, 2005). The correlation between elevated pressure and ganglion cell loss is far from perfect however. A substantial proportion of patients with primary open angle glaucoma (POAG) experience an increase in optic disc cupping even after pressure is reduced below 17 mmHg (Tezel et al., 2001). POAG patients whose loss of visual field progressed could not be distinguished from those whose fields remained the same on the basis of pressure alone (Martinez Bello et al., 2000).

Large diurnal variations in pressure present an increased risk of glaucomatous damage even in patients whose IOP is normal during examination (Asrani et al., 2000) and diurnal variation in absolute IOP can be larger in patients with untreated POAG than in controls (Sehi et al., 2005). Laboratory work indicates that fluctuations in pressure may indeed lead to pathological changes. For example, cyclical stretch of glial cells from the lamina cribrosa produced a clear change in the expression of many genes that could aVect the structure of the lamina cribrosa, as well as genes aVecting axonal signaling (Kirwan et al., 2005). These observations indicate changes in pressure, in addition to absolute pressure levels, contribute to the pathology.

Recent analysis has provided some interesting insight into the mechanical eVects of increased pressure on the eye, and there is currently some debate as to whether the moderate increases in hydrostatic pressure associated with glaucoma are suYcient to induce changes on a molecular level (Knepper et al., 2005; Ethier, 2006). DiVerences in IOP capable of inducing considerable strain on ocular tissues are thought to produce a negligible eVect on molecules

present on a rigid surface. This suggests that diVerential rates of compression between various cellular or ocular structures could transduce pressure increases into structural damage. Stress and strain on optic nerve head tissues were strongly associated with scleral thickness and stiVness of the lamina cribrosa, supporting the importance of relative compression (Sigal et al., 2005). Nevertheless, the reproducible eVects of hydrostatic pressure on cells in numerous in vitro studies suggest hydrostatic pressure can itself initiate responses (Hernandez, 2000; Knepper et al., 2005).

There is typically a long delay between the initial detection of an elevated IOP and a noticeable loss of vision. This delay reflects both the slow nature of the pathological processes and the relatively insensitive tools available to detect ganglion cell loss. The death of ganglion cell axons can be detected structurally as a thinning of the retinal nerve fiber layer and changes to the optic disk, including a loss of the neuroretinal rim and an increase in the cup to disk (C/D) measurements. However, the predominant method of detection remains standardized automated field assessment. The rate of progressive field loss in glaucomatous patients can be diYcult to measure (Katz et al., 1997). Assessment over eight years found an average decrease of only 1.3% per year across the entire visual field (Pereira et al., 2002). This small a degree of annual change is less than the variability of the test itself. Alternative techniques for detection are being developed. For example, the PERG has been known to detect changes in the glaucomatous eye for some time (Weinstein et al., 1988), and recent reports indicate it can detect relatively small changes at the early stages of glaucoma (Hood et al., 2005; Bach et al., 2006; Porciatti et al., 2007). This brings hope that a more sensitive assessment of the progression may become available in the near future.

Novel preliminary work suggests that the acceleration of loss of ganglion cells with age may reflect a general inability of aged eyes to endure the eVects of pressure, rather than just the cumulative response to a chronic elevation in IOP (Cepurna et al., 2006). When the IOP of 8 and 28 month old rats was elevated following injection of hypertonic saline into the episcleral veins, the optic nerves of the older rats displayed a significantly greater degree of degeneration than that of the younger rats for a given pressure elevation. This is consistent with increased susceptibility of ganglion cells in older animals to ischemic damage (Kawai et al., 2001), and implies that older tissues are either more susceptible to injury, and/or less able to repair the damage.

It is now evident that absolute level of ocular pressure is just one of many factors that influence the occurrence and progression of glaucoma. Although lowering IOP is helpful, ganglion cell loss continues in many cases, and additional treatments which directly preserve retinal ganglion cell viability oVer new potential for preventing visual loss (Hartwick, 2001; Levin and

Gordon, 2002). A more detailed understanding of the multiple mechanisms that injure them may aid in determining how pressure contributes, and perhaps more importantly, how pressure interacts with other factors to damage ganglion cells.

C. Chronic Dysfunction and Secondary Death

When discussing glaucomatous damage to ganglion cells, it is important to understand that with moderate increases in IOP, cells can perform at reduced levels for long intervals before finally dying. The characteristics of ganglion cell dysfunction, along with the spread of injury, may provide insight into the causes of eventual cell loss.

Psychophysical analysis indicates ganglion cell function is lost before significant thinning of the nerve fiber layer is detected, consistent with a defective transmission of the visual signal occurring independently from cell death (Ventura et al., 2006). Microelectrode recordings found the activity of ganglion cells was modified by short term changes in pressure, with even a moderate increased in IOP aVecting the flicker evoked responses (Grehn et al., 1984). The ability to restore function by reducing IOP also strengthens the theory that functional loss is distinct from death (Ventura and Porciatti, 2005). As mentioned, a shrinkage of the dendritic tree and soma of ganglion cells can precede death in chronic glaucoma, consistent with a loss in the eVectiveness of processing the visual message as a stage in disease progression (Morgan, 2002).

The susceptibility of ganglion cells to a secondary death indicates that signals emanating from injured cells are themselves detrimental. For example, partial transection of the optic nerve leads to the death of ganglion cells in quadrants corresponding to the severed axons. However, loss also extended beyond the regions of cut axons to encompass other cells not originally aVected (Levkovitch Verbin et al., 2001). The mechanisms involved in this secondary death, possibly including immunological or neurochemical signals, may involve processes shared with chronically injured ganglion cells. Secondary degeneration may also explain the continued ganglion cell loss after adequate IOP control is obtained.

Together, these observations suggest ganglion cells can be injured in glaucoma long before they die, and that injured cells aVect their neighbors. At least some of the functional changes in ganglion cells triggered by increased IOP are reversible, resulting in impairment that need not necessarily lead to death. Although the theories linking pressure and glaucoma described below were primarily based on assessment of cell mortality, the ability of these mechanisms to compromise function as well as survival

should be considered. The development of assays to monitor sick cells as well as dead ones will aid our understanding of both disease progression and neuroprotective approaches.

IV. MECHANISMS OF GANGLION CELL DEATH

The ability to preserve ganglion cells in glaucoma is hampered by our inability to fully explain why elevated ocular pressure leads to cell loss in the first place. As discussed above, the disease is multifactorial, with several mechanisms contributing to death, and it is likely that none are as mutually exclusive as their main proponents would like to believe. A compression of the lamina cribrosa, decreased vascular supply, reduction in availability of neurotropic factors, autoimmune and neurotransmitter imbalances, and parallels to other neurodegenerative all contribute. This survey has not attempted to cover all the possible factors that may damage ganglion cells, and readers are directed to the previously mentioned reviews for more comprehensive information. Instead, the focus here is on traditional themes and the novel role of purines. The emerging ability of purines to integrate increased pressure with neurochemical imbalances may have general relevance for both neurotoxic explanations and neuroprotective strategies.

A. Distention of the Lamina Cribrosa

The lamina cribrosa lies even with the sclera and serves as a scaVold to support axons of the optic nerve as they exit the eye. Structurally it is formed by a series of beams composed of extracellular matrix material and covered with cellular material, with glial cells of particular relevance to the pressurized eye (Morgan, 2000). Beams are arranged around pores through which the unmyelinated axons of ganglion cells pass. Myelination occurs posterior to the lamina cribrosa.

The lamina cribrosa is a major site of glaucomatous damage. The inferior/ superior pattern of ganglion cell loss in the retina correlates well with the topography of the lamina cribrosa, with the injured axons more likely to pass through laminar regions containing larger pores made of fewer beams (Quigley and Addicks, 1981). The lamina cribrosa of glaucomatous eyes shows sign of compression, with posterior bowing evident (Quigley et al., 1983). Swelling and an accumulation of organelles are found in axons around the lamina cribrosa in glaucomatous eyes, suggesting impaired axonal transport (Quigley et al., 1981).

Decreased axonal transport likely contributes to ganglion cell dysfunction and death. The retrograde transport from the LGN to the retina is sensitive to pressure (Minckler et al., 1977), and the decreased transport of neurotrophic factors from the brain to the retina may contribute ganglion cell malaise (Pease et al., 2000). The transport of neurotrophic factors from the brain to the ganglion cell bodies in the retina is disrupted in eyes with increased IOP, with factors accumulating at the level of the lamina cribrosa (Quigley et al., 1980, 2000). The protective role of neurotrophic factors is indicated by the delayed loss of ganglion cells following transaction of the optic nerve in eyes given neurotrophic factors (Mey and Thanos, 1993). Bypassing the neurotrophic factor receptors by genetic upregulation of the eVector extracellular signal regulated kinase 1/2 (Erk1/2) is also eVective and increases neuronal survival in rats with ocular hypertension (Zhou et al., 2005).

Although neurotrophic factors can protect ganglion cells, their impaired transport may not be a primary cause of cell injury or death. Regions of maximal transport disruption do not correlate with areas of maximal nerve damage (Ogden et al., 1988). Particularly convincing was a careful study of the chronology of glaucomatous changes which found that ganglion cell death preceded the depletion of neurotrophic factors in the retina (Johnson et al., 2000). This implies that the death of retinal ganglion cells may involve additional processes that are exacerbated by the reduction in protective neurotrophic factors.

B. Vascular Compromise

It is likely that elevated IOP places a metabolic strain on ganglion cells in the optic nerve head (Osborne et al., 2001). These unmyelinated axons passing through the lamina cribrosa contain a high density of the mitochondrial enzymes cytochrome c oxidase and succinate dehydrogenase, consistent with a high energetic demand (Andrews et al., 1999). As such, the region could be particularly susceptible to a reduction in the eYciency of vascular supply to the optic nerve head. Under conditions where vascular delivery is less than optimal, energetically compromised cells may be less able to deal with environmental insults. It has been proposed that the reduction in energy production could compromise the function of the Naþ–Kþ ATPase pump and depolarize the membrane (Osborne et al., 2001). Transmission of this depolarization along the axon to the cell body could convey a metabolic strain initialized at the optic nerve head to the retina.

The theory that a general metabolic disorder leads to energetically challenged ganglion cells less able to withstand insults is supported experimentally. Rats with preexisting glaucoma lost many more ganglion cells than control animals after both were exposed to ischemia (Kawai et al., 2001). Whether this is because glaucomatous eyes have a higher metabolic need, or because the glaucomatous cells were already on the edge of survival is not clear. It is also not certain that a reduced vascular supply is directly caused by an increased IOP or reflects a secondary disorder. The pattern of ganglion cell loss accompanying occlusion of the carotid arteries diVers from that produced by elevating IOP (Osborne et al., 1999a), suggesting pressure may itself initiate additional pathologies independent of its eVects on blood flow.

Astrocytes make a major contribution to ganglion cell injury under conditions of vascular compromise, as well as to distortions of the laminar cribrosa discussed above. Hypoxic challenge elevates levels of intracellular calcium in astrocytes (Peers et al., 2006) and reduces their ability to remove glutamate from the extracellular space (Swanson et al., 1995). Astrocytes also become reactive after ischemia, triggering a number of pathological responses (Neufeld and Liu, 2003). Astrocytes in the glaucomatous optic nerve head show morphological changes and altered expression of certain proteins (Varela and Hernandez, 1997). In this respect an elevated pressure leads to increased secretion of elastin and to remodeling of the lamina cribrosa that may contribute to progressive optic atrophy (Hernandez, 2000). Anatomical connections imply astrocytes could also convey pathological signals from the optic nerve head to retinal regions, although such spreading remains to be demonstrated directly.

C. Neurochemical Imbalances

Altered levels of extracellular neurotransmitters lead to the death of cortical neurons in chronic neurodegenerative diseases, and can likewise disturb retinal ganglion cells in glaucoma. The distribution of excitatory and inhibitory receptors present on a particular ganglion cell is likely to aVect health and survival; ganglion cells with increased membrane expression of excitatory receptors capable of elevating intracellular calcium would be more vulnerable, while those cells with increased expression of inhibitory receptors that lower calcium levels would be relatively protected (Osborne et al., 1999b).

Although a general theory that altered neurochemical balance can alter ganglion cell function and survival has considerable merit, the precise identity of the receptors involved remains to be determined. For example, ganglion cells responsive to the inhibitory transmitter GABA had enhanced