GLAUCOMA – CURRENT CLINICAL AND RESEARCH ASPECTS

Edited by Pinakin Gunvant

Glaucoma – Current Clinical and Research Aspects

Edited by Pinakin Gunvant

Published by InTech

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Contents

VI Contents

Preface

It is difficult to imagine how intellectually gifted individuals, like Leonardo da Vinci, could demonstrate such mastery of so many subjects: art, mathematics and medicine. Such comprehensive academic proficiency was uncommon in the 15th century, and perhaps is even more rare today. It would be an understatement to say that we live in an era where information is exploding at a rate faster than what can be assimilated. This means that it is nearly impossible to be specialists in all areas and has, in turn, produced “super specialists” in all fields––including medicine.

This book features a collection of articles authored by various researchers who are super specialists in the pathogenesis, diagnosis and treatment of glaucoma. With such a heterogeneous nature, you may even argue that glaucoma is not one disease, but a group of diseases. A lot is already known about glaucoma, and a lot more has yet to be determined. Likewise, what information we deem to be "fact" today may be questioned and deconstructed by the thought leaders of tomorrow. But, that is the beauty of science and research.

This book summarizes current literature about research and clinical science in glaucoma. By no means is this a comprehensive guide to the subject; rather, it is more of a synopsis and translation of the research conducted by individuals who are known in each of their respective areas.

The book can be divided into two broad sections: basic science and clinical science. The basic science section examines benchand animal-modeling research in an attempt to understand the pathogenesis of glaucoma. The clinical science section addresses various diagnostic issues and the medical, laser and surgical techniques used in glaucoma management. We hope that both clinicians and scientists find this work useful and stimulating.

The e-book and open-access model provided by the publishers ensure that the information in this book will be widely circulated and available to everyone who wants to learn. This model echoes the words of Nobel laureate Rabindranath Tagore who wrote the poem given below in his book Gitanjali about 100 years ago:

“Where the mind is without fear and the head is held high; Where knowledge is free;

XPreface

Where the world has not been broken up into fragments by narrow domestic walls; Where words come out from the depth of truth;

Where tireless striving stretches its arms towards perfection;

Where the clear stream of reason has not lost its way into the dreary desert sand of dead habit; Where the mind is led forward by thee into ever-widening thought and action---

Into that heaven of freedom, my Father, let my country awake.”

My sincere gratitude goes to Subba Gollamudi MD, Eye Specialty Group, Memphis Tennessee; Shelly Gupta MD, faculty The Ohio State University, Columbus, Ohio; and Jasmine Yumori OD, Western University of Health Sciences, Pomona, California. Their expert opinion and assistance in reviewing parts of the book was invaluable. Additionally, I would like to thank the managing editor, Ms. Petra Zobic and editorial team manager Ms. Anna Nikolic for their assistance throughout the duration of this project. Most importantly, thanks to all the contributing authors whose dedication to research and the art of scientific knowledge dissemination has made this book a reality.

This book is dedicated to

My dear parents Minaben and Gunvant Davey for all that they have done so I am here today. My darling wife Payal for her care and continued support to my professional career. My beloved Professors Daniel O’Leary and Edward Essock, whose teaching laid the foundation of my research and understanding glaucoma

Dr. Pinakin Gunvant

Department of Research, Southern College of Optometry,

Adjunct Faculty University of Louisville,

Adjunct Faculty University of Memphis,

USA

Part 1

Basic Science Research in

Understanding Pathogenesis in Glaucoma

1

Oxidative Stress in Anterior Segment

of Primary Open Angle Glaucoma

Sergio C. Saccà1 and Alberto Izzotti2

1Department of Head/Neck Pathologies, St Martino Hospital, Ophthalmology Unit, Genoa, 2Department of Health Sciences, Faculty of Medicine, University of Genoa, Italy

1. Introduction

The glaucomas is a group of complex and heterogeneous ocular diseases representing the second leading cause of blindness, and almost 75 million people are affected worldwide (Quigley 1999) being a major issue for public health. The prevalence of glaucoma increases with age (Friedman et al. 2006). The reported prevalence among whites in their 80s varies widely across these studies, with estimates as low as 1.9%3 to as high as 8.8%. Glaucoma is a syndrome characterized by a progressive optical atrophy resulting from the apoptosis of the retinal ganglion cells (RGCs). Growing evidence obtained from clinical and experimental studies over the past decade strongly suggests the involvement of the reactive oxygen species (ROS) in glaucoma. Free radicals can directly induce neuronal death by a protease and phosphatase-gated mechanism distinct from apoptosis (Sée and Loeffler 2001). In glaucoma free radicals may damage the trabecular meshwork (TM) (Saccà et al. 2005) while in the posterior segment of the eye the process of apoptotic retinal ganglion cell death starts with exposure of glial cells to elevated concentrations of free radicals (Nakazawa et al. 2006). The final neurological damage results in progressive RGCs death, axon atrophy and degeneration also extending to the brain cortex (visual areas) finally leading to the characteristic optical-cup neuropathy and irreversible visual loss (Weber and Harman 2005) (Yucel et al. 2000). In addition to the loss of the ganglion cells, the most of glaucoma types is characterized by having a high intraocular pressure (IOP). This is the most important risk factor for this disease, even if it is not yet clear what are the pathogenic events connecting IOP to glaucoma phenotype. In any case the TM damage has a key role in the increasing of IOP.

1.1 Trabecular meshwork: Functional anatomy

The chambers of the eye are filled with aqueous humour, a fluid with an ionic composition very similar to the blood plasma and with two main functions: to provide nutrients to the structures of the eye: cornea, iris and lens and to maintain intraocular pressure. Therefore the anterior chamber of the eye can be regarded as a highly specialized vascular compartment whose inner walls are composed of the endothelia of iris, cornea, and trabecular meshwork (Brandt and O’Donnell 1999). Aqueous humor is secreted by the ciliary body into the posterior chamber of the eye. Aqueous humor cannot traverse the intact iris and thus it passes through the pupil to reach the anterior chamber of the eye. At the iris-

corneal angle, the main part of this flow enters a pathway composed of the trabecular meshwork (TM), the juxtacanalicular connective tissue (JCT), the endothelial lining of the inner wall of Schlemm’s canal, Schlemm’s canal itself, and the collecting channels that lead to the episcleral veins and episcleral vessels. This outflow pathway is called the “conventional way” to distinguish it from the non-conventional outflow called the uveoscleral way. The posterior way or uveoscleral outflow pass through the iris root and the anterior face of ciliary muscle, passing in the connective tissue interposed between the bundles of ciliary muscle to sovracoroideal space. This pathway carries less than 10% of the total flow in the older adult human eye (Gabelt and Kaufman 1989). The TM resides in the ocular limbus between the cornea and the sclera and comprises perforated, interlacing collagenous lamellae, called the TM beams. These have a core of collagenous and elastic fibers, and are covered by flat cells which rest on a basal lamina. The space between the beam is filled with extracellular matrix where the AH filters through (Chen and Kadlubar 2003). The beams are encapsulated by a single layer of endothelial-like cells (Polansky and Alvarado 1994) (Figure 1). The outermost juxtacanalicular or cribriform region has no collagenous beams, but rather several cell layers which some authors claim to be immersed in loose extracellular material/matrix (Tian et al., 2000). Histologic studies of POAG do not find a specific ‘‘plug’’ of the outflow pathways, suggesting instead that derangement of a cellular physiologic function may be involved (Johnson 2005). The functional aspects and morphology of the aqueous outflow pathways is still not clearly understood (Epstein and Rohen 1991). Some authors think that aqueous humor (AH) flow through TM structures in a passive way (Freddo and Johnson 2008; Tamm 2009) relegating the role of TM to a passive filter. Still others believe the TM is a tissue that is actively crossed from an active flow (Saccà et al. 2005; Alvarado et al. 2005a and b). Anyway, the locus of aqueous humor outflow resistance in the normal eye has not yet been unequivocally determined. Nevertheless experimental evidence supports the conclusion that the source of normal outflow resistance as well as the source of increased outflow resistance in glaucoma is attributable to the inner wall endothelium, its basement membrane, JCT, or some combination of all three of these tissues.

1.2 The juxtacanalicular tissue

The JCT, is the region of the meshwork positioned between the beams of the corneoscleral meshwork and the basal lamina of the inner wall of Schlemm’s canal. Its small flow pathways would suggest a significant outflow resistance, but it is not supported by hydrodynamic studies (Ethier et al. 1986; Seiler and Wollensak 1985). Rather it manifests a decrease in extracellular matrix (ECM) components in hyaluronan (Knepper et al. 1996a) or an increase in outflow resistance to excess accumulation of glycosaminoglycans (Knepper et al. 1996b). It is possible that other extracellular matrix components have a major role in contributing to outflow resistance in human eyes. Several ECM proteins may contribute to homeostatic modifications of AH outflow resistance, being upor downregulated (Vittal et al., 2005) and lower concentrations of oxidized low-density lipids stimulate ECM remodeling (Bachem et al., 1999). Interestingly, an increased fibronectin synthesis could result in concomitant increase of IOP (Fleenor et al., 2006). Transforming growth factors (TGFs) are a family of cytokines that control the production of a wide variety of ECM genes, including elastin, collagens, fibrillin, laminin, and fibulin. One of its isofom the TGF-b2 levels are elevated in glaucomatous human AH (Tripathi et al., 1994) and alter ECM

Fig. 1. Scanning electron microscope photograph of the human sclerocorneal trabecular meshwork (magnification 2000 X).The conventional outflow pathway consists of trabecular lamellae covered with human trabecular meshwork (HTM) cells, in front of a resistor consisting of juxtacanalicular HTM cells and the inner wall of Schlemm’s canal. This tissue has unique morphologic and functional properties involved in the regulation of AH outflow. Endothelial cells of TM seem to have a leading role in outflow: probably, their tridimensional architecture and allocation on the trabecular beams considerably increases the filtration surface whose degeneration, resulting in the decay of HTM cellularity, causes IOP increase and triggers glaucoma (Saccà and Izzotti 2008).

metabolism (Wordinger et al., 2007). TGF in the AH is also responsible for anterior chamberassociated immune deviation, a mechanism that protects the eye from inflammation and immune-related tissue damage (Wilbanks et al., 1992). Indeed, TGF-b2 is one of the most important immunosuppressive cytokines in the anterior chamber of the eye and has a fibrogenic effect in trabecular cells (Alexander et al., 1998). Finally ECM production in the TM may be mediated by vitamin C (Epstein et al., 1990; Sawaguchi et al., 1992). Ascorbic acid is reported to stimulate increased hyaluronic acid synthesis in glaucomatous TM cells compared with normal human TM cells (Schachtschabel and Binninger, 1993). Also, ascorbate reduces the viscosity of hyaluronic acid, thus increasing outflow through the trabeculum (McCarty, 1998). Indeed, Virno already in 1966 discovered that high doses of vitamin C decreases IOP (Virno 1966). Other molecules that seem to play a very important role on collagen remodeling are the metalloproteinases (MMPs). MMPs are a family of calciumand zinc-dependent extracellular endoproteases that degrade ECM proteins (Nagase and Woessner, 1999). Matrix metalloproteinases (MMPs) comprise a family of at least 25 secreted zinc proteases, which are of eminent importance not only for the ECM turnover, but also for interactions between cells and their surrounding structures (Sternlicht and Werb 2001). Indeed, increased MMP activity decreases collagen deposition, and AH outflow facility is increased by stimulating MMP activity (Saccà and Izzotti 2008). Anyway, it remains unclear what fraction of total resistance is attributable to the JCT and how ECM or specific ECM molecules might be involved in generation of this resistance (Overby et al. 2009). Anyway, ECM turnover is required to maintain the appropriate outflow resistance. (Bradley et al. 1998).

By analogy to other basement membranes in the body, the inner wall basement membrane has the potential to generate a significant portion of outflow resistance. This is discontinuous (Gong et al. 1996) and this characteristic may be related to the flow of aqueous humor into Schlemm’s canal (Buller and Johnson, 1994). The resistance by this tissue seems to be substantially limited (Overby et al. 2009).

On the basis of electron microscopy studies, it has been proposed that aqueous humor mainly crosses the inner endothelium wall of Schlemm’s canal by two different mechanisms: a paracellular route through the junctions formed between the endothelial cells (Epstein and Rohen 1991) and a transcellular pathway through intracellular pores of the same cells (Johnson and Erickson 2000).

Nevertheless trabecular meshwork pores contribute only 10% of the aqueous outflow resistance (Sit et al. 1997). Furthermore characteristics of inner wall pores depend on fixation conditions. In particular, the density of inner wall pores increases with the volume of fixative perfused through the outflow pathway (Johnson et al. 2002). Scott et al. (2009) provided by confirmation that the inner wall and underlying juxtacanalicular connective tissue work together to regulate outflow resistance .

1.3 Meshwork endothelial cells

According to Alvarado, we know that in conventional aqueous outflow pathway there are two endothelial cell barriers separating the venous circulation from the aqueous humor, which are specialized and positioned in series: the trabecular meshwork endothelial (TME) cells and then, subsequently, the endothelial cells that line the lumen of Schlemm’s canal (SCE) cells. Between these two barriers, there is the juxtacanalicular tissue, which contains a loose extracellular matrix through which the AH flows (Alvarado et al. 2004). The TME cells release factors into the AH, and these ligands flow downstream from TMEs to bind and

actively regulate the permeability properties of the SCEs. These factors, upon binding to SCE cells, increase the permeability of the SCE barrier (Alvarado et al. 2005a) inducing a 400% enhance in SCE conductivity by means of the activation of specific TME genes (Alvarado et al. 2005b). In particular interleukin-1α and 1β and tumor necrosis factor-α released by TME cells induce cell division and migration (Bradley et al. 2000) in those cells near Schwalbe’s line, while inducing the release of matrix metalloproteinases (Kelley et al. 2007) and an increase of fluid flow across extracellular matrix tissues near JCT (Alvarado et al. 2005b). In a recent research (Izzotti et al. 2010a) for the first time we have provided evidence that aqueous humour molecular alterations reflect glaucoma pathogenesis. The expression of 1,264 proteins was analysed detecting remarkable changing in the aqueous humour proteins of glaucomatous patients as compared to matched controls. Among the others AH proteins we have observed that in patients with glaucoma those cytokines referred by Alvarado are expressed in significantly greater amount compared with controls. This finding is likely related to the fact that these cytokines are produced to improve the TM working but in the case of glaucoma TM does not respond properly because malfunctioning and therefore we are seeing an over-expression of these cytokines. Therefore, the cytokines released by TME cells regulate the permeability of the SCE barrier in active way (Alvarado et al. 2005b) . Regulatory volume responses of TM cells influence the tissue permeability too; indeed, hyperosmotic solutions increased and hyposmotic solutions decreased outflow facility, respectively (Al-Aswad et al. 1999; Gual et al. 1997 ). The molecular mechanisms for regulating water balance in many tissues are unknown, but TM cells express aquaporin-1, a multiple water channel protein transporting water through membranes that can modulate cell volume (Stamer et al. 2001). Aquaporins also facilitate cell migration, (Verkman 2005 ), cell proliferation, neuroexcitation, fat metabolism, hydration, and others cell functions (Tradtrantip et al. 2009 ). Aquaporin may be implicated in the pathogenesis of glaucomatous optical neuropathy, indeed in animal model elevated IOP reduce its expression (Naka et al. 2010). Anyway, chronic sublethal injury due to cellular stress is a common theme in the pathogenesis of diverse diseases including atherosclerosis, glomerulonephritis and pulmonary fibrosis (Dunn 1991; Ross 1995). During glaucoma course, sublethal damage to the outflow pathways is developing, being the result of accumulated oxidative stress arising from the environment, vascular dysregulation, aging and/or the pathogenic processes (Flammer et al. 1999). Molecular changes in the surviving cells determine the expression of new genes (Dunn 1991; Ross 1995) dependent on the nature of the damaging stimulus and on tissue type (Mercurio and Manning 1999; Itoh and Nakao 1999). Glaucomatous eyes exhibit a high level of TM cell loss, above and beyond that of age-matched controls (Alvarado et al. 1984). The decline of human TM cellularity is linearly related to age (Alvarado et al., 1984). Grierson has calculated that at 20 years of age the estimated TM cell number for the whole meshwork is 763,000 (Grierson and Howes, 1987) and the cells number decreases to 403,000 by the age of 80 years, with a loss rate of 6000 per year (Grierson et al., 1982).

The mechanism of cell loss and the environmental factors contributing to it are not yet known. However, this phenomenon may be brought about by cell death caused by noxious insult, such as free radical attack (Yan et al., 1991; Padgaonkar et al., 1994).

In the anterior chamber (AC), oxidized lipoproteins and free radicals are considered to be major causes of tissue stress and serve as local triggers for tissue inflammation (Xu et al. 2009). The up-regulation of a large number of inflammatory genes, including genes involved

in complement activation and inflammatory cytokine/chemokine production, which in turn cause abnormal leukocyte-endothelial interactions and ultimately vascular damage (Xu et al. 2009). Furthermore, the innate immune system in general and monocytes in particular play an important role in aqueous outflow homeostasis: presumably under the influence of chemotactic signals, the monocytes circulate through the trabecular meshwork in the normal state and cytokines regulate the permeability of Schlemm's canal endothelial cells (Shifera et al.2010) and monocytes increase aqueous outflow (Alvarado et al. 2010).

This last mechanism is most easily understood if we think AC as a vase and its endothelium as that of specialized vase in which flows AH; the endothelia of this vase is the TM whose complex structure represent a system to increase the area of contact between the TME cells and the AH. Indeed, the TM has been shown to be composed of contractile elements, which helps to regulate the outflow facility (Wiederholt et al. 2000). Therefore, opening or fastening its slots, TM can change the quantity of cells involved in the passage of AH from AC to SC. Its malfunction thus leads to the intraocular pressure increase. Finally, it is to remember that fluid flow is not equal within the trabecular meshwork and that preferential pathways or flow to areas of lower resistance exist in the trabecular meshwork. (De Kater et al. 1989; Tripathi 1971). One factor contributing to preferential flow may be changes in extracellular matrix interactions with Schlemm's canal cells in collector channel regions. As collector channels become altered with age or disease, other collector channels are available to assume the functional burden (Hann and Fautsch 2009).

2. Oxidative stress and Trabecular Meshwork

The interaction of many risk factors converge on intracellular signaling pathways, affecting the balance between protein synthesis and breakdown, inducing mitochondrial damage and apoptosis, which cause the primary glaucoma pathology through a significant loss of TM endothelial cells. It is not known the exact sequence in which this disease starts, but it is a fact that glaucoma is not a condition relating to IOP alone. Oxidative stress (Izzotti et al. 2006), vascular abnormalities (vasospasm, systemic hypotension, reduced vascular perfusion in the optic nerve head and/or retina) (Flammer et al. 2002), glial activity (Kirwan et al. 2005), immune system (Tezel 2007), inflammatory stimulus (Rönkkö et al. 2007) are involved in glaucoma injury.

The “trait de union” of all these components is the oxidative stress (Kumar and Agarwal 2007). Oxidative DNA damage is an inevitable consequence of cellular metabolism and it is secondary to free-radical formation (Cooke et al., 2003).

The oxidative stress and related molecular damages occur in a cell, tissue, and organ in response to the exposure to oxidizing agents. The mayor types of free radicals are reactive oxygen species (ROS) and reactive nitrogen species. The free radicals are molecule fragments equipped with an unpaired electron (odd number of electrons in the last orbital, when normally the electrons are coupled). Under normal physiological conditions, a small fraction of the oxygen consumed by mitochondria is constantly converted to superoxide anions, hydrogen peroxide, hydroxyl radicals, and other ROS. To cope with the ROS, human cells express an array of antioxidant enzymes, including Mn2+-dependent superoxide dismutase (SOD), copper/zinc SOD, glutathione peroxidase (GP), glutathione reductase (GR), and catalase (CAT). SOD convert superoxide anions to hydrogen peroxide, which is then transformed to water by CAT. The NO radical is produced in organisms by the oxidation of one of the terminal guanidonitrogen atoms of L-arginine (Palmer et al. 1988).

This process is catalyzed by the enzyme NOS. Depending on the microenvironment, superoxide and NO are readily converted by enzymes or nonenzymic chemical reactions into reactive non radical species such as singlet oxygen, hydrogen peroxide, or peroxynitrite , i.e., species which can in turn give rise to new radicals. At moderate concentrations, however, nitric oxide (NO), superoxide anion, and related reactive oxygen species (ROS) play an important role as regulatory mediators in signaling processes. Many of the ROSmediated responses actually protect the cells against oxidative stress and reestablish “redox homeostasis.” (Dröge 2002 ). Anyway, there is an age dependent increase in the fraction of ROS and free radicals that may escape these cellular defense mechanisms and exert damage to cellular constituents, including DNA, RNA, lipid, and proteins. Any signal or stimulus that triggers overproduction of ROS may induce the opening of the membrane permeability transition pore in mitochondria and release of cytochrome c and other apoptogenic factors, which ultimately lead the cell into apoptosis (Tatton and Olanow 1999).

The anterior chamber (AC) is a highly specialized structure of the eye. It is composed of several tissues and structures, including the posterior surface of the cornea, the anterior surface of the iris, the pupil, the pupillary portion of the lens, and peripherally, the sclerocorneal angle, where the trabecular meshwork (TM), the scleral spur, the ciliary body, and the iris root are located.

Both vitamin C and glutathione operate in fluid outside the cell and within the cell (Cardoso et al., 1998); the ascorbate content is higher in diurnal than in nocturnal aqueous humor (Reiss et al. 1986). Aqueous humor could act as a liquid ultraviolet-light filter for the lens by virtue of the ascorbic acid in the anterior chamber (Ringvold 1980). Also in vitreous vitamin C has a very important role; indeed human vitreous gel consumes oxygen by an ascorbate dependent mechanism. Anyway, the concentration of ascorbate in human vitreous is remarkably high (Shui et al. 2009). Hence, the vitamin might protect against oxidative or photo-oxidative damage (Garland 1991; Rose et al. 1998) in both the central corneal epithelium and aqueous humour (Giblin et al., 1984) and reacts with O2 to form H2O2, which in turn is neutralized by SOD and CAT. Continuous exposure of HTM cells to oxidative stress via H2O2 results in ROS generation in mitochondria. This, in turn, stimulated NF-kB activation and subsequent production of interleukins and the induction of inflammatory mediators. (Li et al., 2007). A high level of ascorbic acid is necessary to maintain oxidative balance in the AH (Izzotti et al. 2009). A synergism between vitamin E and C has been envisaged, because vitamin C reduces oxidized vitamin E, which is crucial for protecting cell membranes from lipid peroxidation; thus, this synergism may have a role in the pathogenesis of glaucoma (Varma 1991; Kang et al. 2003). Indeed, resistance to AH outflow increases in the presence of high levels of H2O2 in eyes with a glutathione (GSH)-depleted TM (Kahn et al. 1983). GSH plays a critical role as an intracellular defense system providing detoxification of a broad spectrum of reactive species and allowing their excretion as watersoluble conjugates (Meister, 1989). Glaucomatous patients exhibit low levels of circulating glutathione (Gherghel et al. 2005) and glutathione participates directly in the neutralization of free radicals and reactive oxygen species, and maintains exogenous antioxidants such as vitamins C and E in their reduced (active) forms (Saccà et al. 2007). Insufficient glutathione combined with exogenous H2O2 may induce collagen matrix remodeling and TM cell apoptosis independently of mitochondria (Veach 2004). Therefore in glaucoma course occurs a decline in total antioxidant defenses and in particular in AH (Ferreira et al. 2004) that have a great impact on the TM endothelium: because this is not more protected

properly and because TM is the most sensitive tissue to oxidative radicals in the AC (Izzotti et al. 2009). This leads to the reduction in the TM cellularity, to TM failure and subsequently to IOP increase. Of course, oxidative DNA damage in the TM of patients with primary openangle glaucoma is significantly higher than in controls (Izzotti et al. 2003). Furthermore, oxidative DNA damage in patients with glaucoma correlate significantly with intraocular pressure and with visual field defects (Saccà et al. 2005, Izzotti et al. 2003; FernándezDurango et al. 2008). Therefore it is possible that the process that occurs in AC and at the level of the optic nerve head is the same, and that in both districts the oxidative stress play an important role. Indeed, in glaucoma animal model, through the induction of oxidative damage, mechanical and vascular factors working synergistically lead to the same final pathologic consequence, i.e. glaucoma (Prasanna et al., 2005).

3. Mitochondrial dysfunction

The most important and frequent ocular degenerative diseases including cataract, glaucoma and age-related macular degeneration are caused by multiple interacting factors. A large number of environmental and genetic factors play a role in common eye pathologies. Many of these risk factors are genotoxic therefore being emerging evidence that DNA damage play a role in the development of the degenerative diseases of the eye (Saccà et al. 2009). ROS production has been principally implicated in the pathogenesis of eye disease. In particular, it is possible to believe that the ROS are responsible for the decline of cells that occurs in TM during aging and glaucoma that is the basis of its bad functioning and failure (Alvarado et al. 1984 and 2005a; Saccà et al. 2005).

Nowadays it is established that POAG patients bear a spectrum of mitochondrial abnormalities (Abu-Amero et al. 2006). Mitochondrial theory of ageing, a variant of free radical theory of ageing, proposes that the accumulation of damage in mitochondria, and in particular in mitochondrial DNA (mtDNA), leads to human and animal ageing. Oxidative modification and mutation of mtDNA easily and frequently occur, and the extent of such alterations of mitochondrial DNA increases exponentially with age. Oxidative modification in mtDNA is much more extensive than that in nuclear DNA (Ames et al.1993; Yakes and Van Houten 1997). Age-related alterations in the respiratory enzymes not only decrease ATP synthesis but also enhance the production of reactive oxygen species (ROS) through increasing electron leakage in the respiratory chain. With the accumulation of genetic defects in mechanisms of mitochondrial energy production, the issue of neuronal susceptibility to damage as a function of ageing becomes important (Parihar and Brewer 2007). Damage to mtDNA induces alterations to the polypeptides encoded by mtDNA in the respiratory complexes, with a consequent decrease in electron transfer efficiency, leading to further production of ROS, thus establishing a vicious circle of oxidative stress and energy decline. This deficiency in mitochondrial energy capacity is regarded as the cause of ageing and age-related degenerative diseases (Genova et al. 2004). On the basis of the fact that mitochondria are the major intracellular source and vulnerable target of ROS (Linnane et al. 1989) accumulation of somatic mutations in mtDNA is a major contributor to human aging and degenerative diseases. Hence, a vicious cycle contributes to the progression of degenerative process. In this cycle, first a primary mitochondrial mutations induces a mitochondrial respiratory defect, which increases the leakage of ROS from the respiratory chain. Then the ROS would trigger accumulation of secondary mtDNA mutations in

postmitotic cells, leading to further aggravation of mitochondrial respiratory defects and increased production of ROS and lipid peroxides from mitochondria, and thus resulting in degeneration of cellular components (Tanaka et al. 1996). This is the basis of mitochondrial dysfunction that occurs in glaucoma. In the anterior chamber of POAG patients the relationship between mitochondria and TM is still rather obscure. From a morphological point of view, we know that in the cribriform layer often contained small mitochondria (Rohen et al.1993). Besides using an in vitro culture system of bovine trabecular meshwork cells Dexamethasone-treatment developed an increased number of mitochondria (Sibayan et al. 1998). TM cells from patients with POAG cells have high endogenous reactive oxygen species, low ATP, and that mitochondrial complex I defect is associated with the degeneration of TM cells (He et al. 2008). Therefore other information is taken not “in vivo”, but only from the study of cell cultures. Recently, it has been demonstrated in vitro that expression of mutant myocilin, a mitochondrial protein whose role in the arising of early glaucoma is established, sensitizes Cells to Oxidative Stress-Induced Apoptosis (Myung Kuket al., 2010).

Recently we have demonstrated that mtDNA damage occurs in the target tissue of POAG: the TM, but not in other anterior chamber districts, e.g, the iris (Izzotti et al. 2010b). Furthermore genetic polymorphisms for antioxidant and apoptosis related genes affect the amount of mtDNA damage in TM (Izzotti et al. 2010b). The remarkable interindividual variability observed in this study could be due to differences in diseases status because all patients were carriers of advanced unbalanced POAG or related to mitochondrial haplotypes, which are characterized by different sensitivity to oxidative damage (Kofler et al.2009). The lack of mtDNA damage in tissues different from TM observed in this study in iris is in agreement with the negative findings reported by other studies in blood lymphocytes of patients with POAG (Abu-Amero et al.: 2007). These findings indicate that glaucoma may be envisaged as a mitochondriopathy specifically occurring in TM.

Mitochondrial damage and loss occurring in TM trigger both degenerative and apoptotic phenomena resulting in cell loss, as specifically occurring only in primary open-angle glaucoma and in pseudoexfoliative glaucoma and not in other glaucoma types ( Izzotti et al. 2011). Decreased cellularity of the TM appears to be a particular characteristic of POAG, but other authors reported that it does not seem to play a role in the pathogenesis of PEX glaucoma (Schlotzer-Schrehardt and Naumann 1995). Anyway, in all types of glaucoma in which IOP is high, TM malfunction resulting from cellularity decrease is the key to the development of the disease, in acute and chronic angle closure glaucoma too (Sihota et al. 2001).

Furthermore, glaucoma itself could also produce apoptosis of TM cells through mechanical stress (Grierson 1987) or through trabecular hypoperfusion (Rohen et al. 1993). An increase in oxidative stress may also contribute to cell loss or alterations in the functioning of TM cells (Izzotti et al. 2003; Saccà et al. 2005; Dela Paz et al. 1996). Mitochondrial damage has been detected only in primary open angle and pseudo-exfoliation glaucoma, as the outflow dysfunction in the other glaucomas studied may have a different underlying bases ( Izzotti et al. 2011). A further confirmation of this type of pathogenesis is given by the study of AH.

4. Aqueous Humour proteome reflects glaucoma pathogenesis

The AC endothelium (ACE) is not only a group of cells that act as a barrier between AH and the surrounding tissues, but like in vessels, is a real organ with the function of modulating

12 Glaucoma – Current Clinical and Research Aspects

the tone and the flow rate in response to humoral, nervous and mechanics stimuli. In physiological conditions the ACE plays an active role cellular interchange, being able to adapt functionally and structurally to changes in the environment (Verma et al. 2003). The normal endothelial function depends on both the continuity of cellular anatomical monolayer and by its functional integrity (Furchgott and Zawadzki 1980). In vessels, the endothelial dysfunction is characterized by vasoconstriction, platelet aggregation, proliferation of smooth muscle cells, and is related to a reduced bioavailability of nitric oxide (NO), to an excess oxidative burden, and to an increased action of endothelin ET-1 (Monnink et al. 2002; Landmesser et al. 2002).

NO is one of the more important substances produced by endothelium; it is a powerful vasodilatator, an inhibitor of endothelial cell growth and inflammation. NO is a major intracellular and extracellular effective agent against oxidative stress, and it has a beneficial antioxidant effects against reactive oxygen species, such as H2O2, whose detrimental effects on aqueous humor outflow are established (Lutjen-Drecoll 2000). A prominent role in endothelial dysfunction must be assigned to NO inactivation by ROS. ROS react with NO producing peroxy-nitrites, which are cytotoxic agents, thus decreasing NO bioavailability or directly inactivating NO (Aslan et al. 2008).

The Endothelin-1(ET-1), a potent vasoconstricting peptide of endothelial production acts on specific receptors present only on smooth muscle cells and cause vasoconstriction and cell growth. ET-1 determine stimulates the NO production, which acts as negative feedback by inhibiting further ET-1production (Heitzer et al. 2001). In case of reduced NO bioavailability this negative feedback mechanism is compromised, and consequently ET-1 vasoconstricting effect is increased (Haynes and Webb 1998).

A balance between vasoconstrictors and vasodilators is necessary for the maintenance of the physiological structure and function of endothelia (Gibbons 1997). Whenever the balance between vasoconstriction and vasodilatation is disrupted, as in glaucoma, the outcome is endothelial dysfunction and injury that has as consequence cellular proteins loss in AH.

Anterior chamber (AC) is a lumen of a vessel constituted by the cornea and iris and joint together by means of TM. The AC contains the aqueous humor (AH). The volume of the AC is approximately 0.25 mL, whereas the volume of the posterior chamber is 0.06 mL. AH is needed to guarantee optical transparency, structural integrity, and nutrition in the absence of blood vessels (Izzotti et al. 2009). Furthermore, this liquid has the task of protecting and supplying nutrients to the cornea, lens, and TM (Izzotti et al. 2006; Fuchshofer and Tamm 2009). Other functions ascribed to AH inflow have been less clearly defined (Krupin and Civan 1996) and include the delivery of antioxidant, such as ascorbate, and participation in local immune responses. The ciliary epithelium concentrates ascorbate in AH rendering its concentrations 40-fold higher in AH than in blood plasma (Krupin and Civan 1996). It is possible that ascorbate is not only a scavenger of ROS, but may be also a regulator of ion channel activity functioning as an endogenous modulator of neuronal excitability (Nelson et al. 2007). In any case, cells in the outflow pathway are subjected to chronic oxidative stress and go towards an impaired proteasome activity in TM (Govindarajan et al. 2008). Therefore, any alteration in proteasome function due to oxidative stress or aging would be also expected to increase the rate of accumulation of misfolded mutant myocilin in the endoplasmic reticulum and contribute to the pathogenic effect of this mutant protein in the mitochondrial functions in human TM cells (He et al. 2009). AH represents a proteincontaining biological fluid fundamental for eye pathophysiology (Civan 2008). However,

the relationship between TM and AH proteins as related to POAG pathogenesis has not yet been explored. Many proteins expressed at high levels in healthy patients are reduced in POAG patients, while other proteins detected at low levels in normal subjects are increased in POAG patients.

Recently we have discovered 6 classes of protein specifically present in the AH of advanced glaucomatous patients (Izzotti et al. 2010a).

5. Proteins in glaucomatous Aqueous Humour

The first class of proteins were mitochondrial proteins involved in the electron transport chain, trans-membrane transport, protein repair, and mitochondrial integrity maintenance. The second class were proteins involved in apoptosis induction, through the intrinsic, i.e., mitochondrial-dependent, pathway.

The third class were proteins connecting cells. These include catenins, junctional plaque protein, dynein, and cadherins.

The fourth class is composed by neuronal proteins like optineurin or growth and differentiation factors involved in neurogenesis and neuron survival.

The fifth class includes the protein kinase involved in apotosis activation and signal transduction.

Finally, the sixth class concerns the oxidative stress and includes: nitric oxide synthetase, superoxide dismutase and microsomal glutathione S-transferase 1.

All these proteins testify that the AH composition is affected and reflects the mechanisms of glaucoma pathogenesis. Mitochondrial proteins presence, segregated into intact mitochondrial under normal conditions, reflects that TM endothelium cells is affected by mitochondria loss and dysfunction, as specifically occurring only in TM and not other AC tissues during glaucoma. The presence of mitochondrial proteins in AH indicates that TM undergoes structural alterations and that in particular its endothelial cells loss mitochondrial proteins as a consequence of mitochondrial DNA deletion that leads to mitochondrial TM malfunction and destruction leading to apoptosis. Cellular loss is determined not only by proapoptotic proteins but also by other mechanisms involved and revealed by the presence of other protein: inflammation, vascular dysregulation, and hypoxia (Choi and Benveniste 2004; Li et al. 2006). Indeed, proteins of these functional groups are expressed in the AH of glaucomatous patients. This is the situation for BIK protein, normally located inside mitochondria, which activates apoptosis process through the intrinsic pathway, and for FAS protein, that is responsible for the activation of apoptosis through the extrinsic pathway in response to inflammation and/or oxidative stress. Furthermore, FAS has been demonstrated to provoke apoptosis increasing myocilin release from mitochondria to cytosolic compartments of TM cells (Sakai et al. 2007).

The presence of “proteins connecting cells” in glaucomatous AH, reflects the impairment of cytoskeleton organization, cell-cell adhesion and migration (Lee and Tomarev 2007). Calnexin presence in AH can be linked with the presence of mutant myocilin. Myocilin, the first protein genetically associated with the development of glaucoma, is a constituent of human AH and is expressed in many ocular tissues, with highest expression observed in cells of the trabecular meshwork (Adam et al., 1997;). While calnexin is a calcium-binding protein playing a major role in the quality control apparatus of the endoplasmic reticulum by the retention of incorrectly folded proteins. Normally, located in melanosomes, calnexin

presence in AH is likely to be caused by the death of pigmented HTM cells induced by mutant and wild-type myocilin depending upon the presence of misfolded protein in the ER (Liu Y, Vollrath 2004).

Myocilin is a signal secretory protein (Hebert and, Molinari 2007). It has both intracellular and intercellular functions (Ueda et al.2002) and can be found in various organelles such as the endoplasmic reticulum (Liu Y, Vollrath 2004), Golgi apparatus (O’Brien et al. 2000), and moreover mitochondria (Wentz-Hunter et al. 2002). Myocilin increases both calcium concentration in cytoplasm and in mitochondria, possibly through the dysregulation of calcium channels (He et al. 2009). Excessive cytoplasmic Ca2+ leads to mitochondrial Ca2+ overload, which triggers ROS overproduction, mitochondrial membrane depolarization, and ATP production inhibition, all hallmark events of mitochondrial dysfunction and eventual apoptosis (Jackson JG, Thayer 2006; Dahlem et al. 2006) . Hence mutant myocilin impairs mitochondrial functions in human trabecular meshwork cells (He et al. 2009) and may confer different sensitivity to oxidative stress depending on the mutation of this protein (Joe and Tomarev 2010) as induced on genetic basis in juvenile glaucoma or on degenerative ROS-mediated basis in POAG.

The presence of neuronal proteins in AH is not surprising, because TM cells have a neuroectodermic origin, expressing, at least in part, a neural-like phenotype (Steely et al. 2000) TM cells deriving from mesenchymal cells of the neural crest (Cvekl and Tamm 2004). The role of neural proteins in glaucoma pathogenesis is established. Optineurin plays a neuroprotective role in the eye and optic nerve. Optineurin protects the cell from oxidative damage and blocks the release of cytochrome c from mitochondria (De Marco et al. 2006). Its presence in glaucomatous AH suggests an antiapoptotic attempt by TM cells through NFk-B pathway regulation (Ray and Mookherjee 2009). Conversely, the presence of ankyrin bears witness to the TM cells degenerative process (Scotland et al. 1998).

Of great interest seems to be the presence of Kinase proteins in AH. Indeed, protein kinases C (PKC) could influence AH outflow affecting cellular relaxation, contraction, and morphological changes in TM and sclerocorneal cells (Khurana et al. 2003) and leading to secretion of matrix metalloproteinase (Husain et al. 2007 ). Cyclic mechanical stress induces changes in a large number of genes that are known to affect the AH outflow facility altering extracellular matrix composition, cellular cytoskeleton, and cell adhesion (Luna et al. 2009) . The finding that AH proteome alterations reflects glaucoma pathogenesis confirms the importance of TM motility. TM malfunction is multifactorial being related not only to mitochondrial dysfunction leading to TM endothelial cell loss but also to the alteration of extracellular cell matrix and to the altered expression of genes governing TM functions and motility.

It is necessary the action of many factors for developing glaucoma, i.e. ageing, genetic predisposition, exogenous environmental and endogenous factors. Environmental factors can interact with genetic predisposition. In some families, the disease has a clearly dominant inheritance, but it is very rare cases. In a greater number of cases there is a certain genetic predisposition, witnessed by the presence of another member of a family, even though having a far relationship, affected by the disease. In particular, a concerning aspect is the individual sensitivity to light: in fact, this could encourage the radical free production that could induce damage. Indeed, reactive oxygen species are increased in glaucomatous AH determining decrease of the total antioxidant potential (Ferreira et al. 2004). In particular, we demonstrated that in the AH of glaucomatous patients as compared to normal subjects the antioxidant enzymes superoxide dismutases 1/2 and glutathione S transferase 1 are

significantly lower in POAG patients than in controls while the pro-oxidant enzyme nitric oxide synthetase 2 and glutamate ammonia ligase are significantly higher in POAG patients than in controls (Figure 1). This unbalance results in a pro-oxidative status mainly affecting in the AC of the eye TM, which indeed is particularly sensitive to oxidative damage thus triggering the glaucoma’s pathogenic cascade (Izzotti et al. 2009).

The importance of oxidative stress in glaucoma pathogenesis is further highlighted by the recent discovery that some active antiglaucomatous drugs like Timolol (Izzotti et al. 2008) and Dorzolamide (Saccà et al. 2011), commonly used in glaucoma treatment, have antioxidant properties and counteracts adverse consequences of oxidative damage as occurring in whole TM and in specifically in its endothelial component. Timolol has an antioxidant effect on the whole cell while Dorzolamide exerts protective activity towards oxidative stress only in presence of intact mitochondria. Therefore, drugs targeting basic mitochondrial processes such as energy production and free radical generation, or specific interactions of disease-related protein with mitochondria, hold great promise for glaucoma therapy.

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2

Manipulating Glia to Protect Retinal

Ganglion Cells in Glaucoma

Denise M. Inman, Caroline B. Lupien and Philip J. Horner

University of Washington, Seattle WA United States of America

1. Introduction

Increasing evidence supports both direct and indirect roles for retinal glia in the pathogenesis of glaucoma. To complicate these roles is the realization that glial activity can be both beneficial and detrimental to the survival of retinal ganglion cells (RGCs) and their axons. The contribution of glia to glaucoma pathogenesis also varies by compartment; glia in retina react differently to disease-induced stressors than glia in the optic nerve head or in the optic nerve. We will describe the evidence to date for the various roles of glia in each of these compartments. From this foundation, we have explored two hypotheses: whether manipulating gliosis can protect RGCs or their axons; and whether manipulating the antioxidant supportive role of retinal glia could prevent RGC degeneration and preserve vision. Encouragingly, we have observed that retinal gliosis can be altered to positive effect for RGCs. Improving glial support of RGCs has also increased RGC and optic nerve axon survival.

Glia greatly outnumber neurons in the CNS, but due to their reputation as secondary support cells, their study has lagged that of neurons. Within the retina, there are three types of glia: Astrocytes and Müller glia (the macroglia), and microglia. The Müller glia form the structural scaffolding of the retina, with endfeet that comprise both the inner and outer limiting membranes. The astrocytes reside among the retinal ganglion cells and their axons, while the microglia exist in non-overlapping tiled arrangements throughout the neuronal and synaptic layers of the inner retina (Bosco et al., 2011). Astrocytes and Müller glia provide homeostatic support to retinal neurons, including neurotransmitter and ion buffering, and anti-oxidant, nutrient and growth factor provision. Microglia, the resident immune cells, survey the retinal environment and respond to changes or threats. Müller glia, a macroglia subtype specific to the retina, serve all of the functions of parenchymal astrocytes, but with additional unique qualities such as transdifferentiation after specific kinds of injury (Bringmann & Reichenbach, 2001).

Glia have garnered attention in the visual system through their emergence as fascinating arbiters of health and disease. Glia respond quickly to even the slightest homeostatic alterations, including pressure, electrical activity, infection, degeneration, and pH changes. Astrocytes and Müller glia undergo gliosis in response to many of these stimuli, a cellular hypertrophy that includes, but is not limited to, upregulating the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. GFAP expression is always apparent in astrocytes, but Müller glia only express this intermediate filament in times of

stress (Kim et al., 1998). In some contexts, gliosis is accompanied by proliferation, but not in glaucoma. Of the glia subject to review here, only microglia proliferate in the DBA/2J murine model of glaucoma (Inman & Horner, 2007). Gliosis and the accompanying hypertrophy rearranges glial processes which can change glial connectivity and position. Microglia responding to environmental change often increase their secretion of matrix metalloproteinases (MMPs) and become motile, retracting processes and upregulating their expression of Iba1, a Ca2+-binding protein (Ito et al., 1998; Bosco et al., 2008). Gliosis and microglial response are the earliest signs of pathology in glaucoma models that include intraocular pressure (IOP) increase (Inman & Horner, 2007; Bosco et al., 2011). Increased IOP, like age, is a major risk-factor in developing glaucoma (Flanagan, 1998). Glia possess mechanoreceptors that could transduce the pressure signal for the retina (Gottlieb et al., 2004). Some mechanoreceptors flux ions and likely initiate signal transduction that can lead to changes in glial production of intermediate filaments (GFAP, vimentin) and proteoglycans of extracellular matrix.

Both astrocytes and Müller glia manage glucose metabolism, maintain the blood-retinal- barrier, control ion and water homeostasis (Bringmann et al., 2006), and contribute to signal processing by recycling neurotransmitters and modulating neuron excitability (Stevens et al., 2003). Fundamental to the role of retinal glia is their exchange of substrates (pyruvate, glutamine) and their uptake of byproducts (glutamate, CO2) from neurons. Retinal glia often rely on anaerobic glycolysis which generates lactate; conversion of lactate to pyruvate then release from the glia via a monocarboxylate transporter, MCT2 (Lin et al.,1998) supplies neurons that take up the pyruvate and use it as a substrate in their own Krebs cycle. Like astrocytes, Müller glia also have glycogen deposits (Kuwabara & Cogan, 1961) that could provide a ready substrate during ischemia or glucose shortage.

Of the several glutamate transporters identified in the CNS, retinal astrocytes and Müller glia primarily express GLAST (glutamate-aspartate transporter). The importance of glutamate transport in retinal glia extends beyond managing neurotransmitter levels in the extracellular milieu. Glutamate is an important stimulator of glycolysis in glia, via its cotransport of Na+. In addition, glutamate, through the glial enzyme glutamine synthetase, gets converted to glutamine in glia, which then provides the glutamine to neurons for their production of glutamate and GABA (Pow & Crook, 1996). More important, however is the use of glutamate in the production of glutathione (GSH). This ubiquitous and quickly metabolized anti-oxidant is present in high concentrations in the astrocyte (up to 20mM) and released to the extracellular space. Once there, a glia membrane-bound ectoenzyme, γ- GT (γ-glutamyltranspeptidase), breaks GSH into the cysteine-glycine dipeptide that can be taken up by neurons. This reaction is key because neurons maintain GSH at low levels in the cytoplasm (<1mM) and they cannot import it directly. Neurons require GSH to reduce sideproducts of oxidative phosphorylation and detoxification pathways. The cysteine-glycine precursors for GSH are provided to neurons solely by astrocytes or Müller glia. Mechanisms of neurodegeneration related to glutamate handling and oxidative stress have been implicated in glaucoma, discussed in greater detail below.

In this chapter, we review the role of glia by compartment of the visual system— retina, optic nerve head (ONH) and optic nerve (ON)— which encompass the potential sites of glaucoma initiation and progression. Astrocytes and microglia appear in each compartment while Müller glia are confined to the retina. The overlapping function of astrocytes and Müller glia reinforces common mechanisms for disease pathogenesis across visual system compartments; however, the unique environment in each compartment allows for distinct

causes and consequences of disease-related pathology. The various environments and stresses experienced by glia demand that we understand the time line of glial activation and find ways to manipulate that activation for RGC neuroprotection. Once we have discussed the mechanisms of glaucoma involving glia by compartment, we review the methods we have used glia to limit glaucoma pathology and protect RGCs and visual function.

2. Retina

Glaucomatous changes that result in RGC death occur in the retina. This general hypothesis for the pathogenesis of glaucoma draws strength from the fact that the retina contains the RGC somas and the glia that support them. The intraocular pressure (IOP) increases that pose a risk factor for glaucoma translate directly into key concerns for the RGCs in the retinal compartment.

2.1 Astrocytes

Astrocytes in the retina migrate in from the optic nerve as the retina develops (Huxlin et al., 1992). Likely a reflection of their role in the blood-retinal-barrier, astrocytes are only present in the retinas of mammals with vascularized retinas (Stone & Dreher, 1987). They form a syncytium across the nerve fiber layer with processes that extend into the ganglion cell layer. Astrocytes ensheath RGC axons and have processes that converge on the initial segments of RGCs (Hollander et al., 1991). In places where vessels and axon bundles prevent Müller glia endfeet from forming the glia limitans of the inner limiting membrane, astrocytes extend the glial coverage over both vessels and axon bundles. Adherent junctions form between astrocytes and Müller glia, but gap junctions form only between astrocytes, never between astrocytes and Müller glia. The glial sheaths around RGCs are primarily comprised of Müller glia processes, though the occasional astrocyte process has been observed. Astrocytes (but not Müller glia) have processes that contact node-like specializations of RGC axons (Hollander et al., 1991). In human retina, there is limited astrocyte support in the macula, the region of tightly-packed multi-layered RGCs responsible for central high resolution vision. Vasculature also avoids this region; these facts have implications for glaucoma pathogenesis since the arcuate pattern of RGC loss in human glaucoma follows the arc of vessels and astrocytes beyond the macula (Araie, 1995).

Astrocytes respond to even subtle changes in retinal homeostasis in ways that can be protective or detrimental. The most overt response of astrocytes to glaucoma in the retina is gliosis, which includes hypertrophy of the astrocytic cytoskeleton along with various context dependent changes in gene expression and astrocyte function. In some regions of the CNS, gliosis includes astrocyte proliferation and migration, but gliosis in glaucomatous retina is simply reactive (Inman & Horner, 2007). As RGCs die in glaucoma, hypertrophied astrocyte and Müller glia processes fill in the empty spaces. Increased proteoglycan production concomitant with GFAP and vimentin over-expression occurs in the DBA/2J mouse retina with early glaucoma (Figure 1). These proteoglycans, important to retinal development for neural patterning (Brittis et al., 1992), significantly inhibit axon growth or cellular migration when re-expressed (Inatani et al., 2000; Inman & Horner, 2006). Proteoglycan deposition restricts the ability of the retina to engage in any endogenous repair. Two signal transduction pathways, the JAK/STAT and NFκB, have been implicated in the reactive gliosis observed in retinal astrocytes and Müller glia. Astrocytes in the DBA/2J mouse retina express STAT3 and phosphorylated STAT3 protein at higher levels as glaucoma progresses (Lupien & Horner,

2007). Retinal astrocytes and Müller glia in vitro upregulate GFAP expression through the STAT3 pathway, as determined by increased GFAP expression with the addition of the STAT3 activators ciliary neurotrophic factor (CNTF) or leukemia inhibitory factor (LIF) (Lupien et al., 2008). GFAP expression in retinal glia decreased when inhibitors of NFκB were introduced four weeks after glaucoma induction (Lupien et al., 2009). We have manipulated these pathways to understand the role of gliosis in glaucoma (see below).

Fig. 1. A. Western blot of whole retina from DBA/2J mice from 2 to 6 months of age shows a significant increase in GFAP and CSPG expression concomitant with early increases in IOP (5-6 months of age, p<0.05). B & C. Immunolabeling of retina with antibodies against CSPG (green) and GFAP (red) show significant increases in GFAP expression in astrocytes of the nerve fiber layer (NFL) and significant upregulation of CSPG in Müller glia endfeet when comparing 4 month (B) and 8 month (C) DBA/2J retinal sections. Arrowheads point to Müller glia somas expressing CSPG in the inner nuclear layer (INL) in c’ and arrows point to GFAP expression in Müller glia processes (c’’) Scale bar=50 microns. repair.

Retinal astrocytes release cytokines and chemokines in response to various stimuli, and changes in many of these signaling molecules have been observed in glaucoma. Astrocytes decrease their release of IL-6 when cultured under hydrostatic pressure, a condition designed to recapitulate the increased IOP observed in glaucoma (Sappington & Calkins, 2006). Lower IL-6 expression may be detrimental to cell survival in retina because astrocytederived IL-6 has been shown to regulate expression of metallothionein I and II, potent antioxidants in the CNS (Penkowa et al., 2003). RGCs import astrocyte-derived metallothioneins through their megalin receptors; their import has been associated with subsequent axon regeneration (Chung et al., 2008). Regardless, IL-6 activates astrocyte gliosis through the activity of the JAK/STAT pathway, a potentially autocrine mechanism that may be helpful or detrimental depending on the context and timing of activation.

Analysis of human glaucoma retina shows mRNA and intense immunolabel for TNFα in glia—likely both astrocytes and Müller cells, while the TNF-R1 was observed primarily on RGCs (Tezel et al., 2001). TNFα is a pro-inflammatory cytokine that can be released by microglia, astrocytes or Müller glia, and its effect depends upon the intracellular signals induced after binding to the TNFα-R1 or 2. For example, the TNFα-R1 receptor has a cell

death domain that promotes apoptosis, but only if NFκB anti-apoptotic mechanisms are also inhibited (Kraft et al., 2009). TNFα binding that leads to NFκB nuclear translocation enables transcription of inhibitors of apoptosis, thereby promoting cell survival (Beg & Baltimore, 1996). Robust and sustained activation of JNK, which occurs when NFκB is suppressed, would indicate the TNFα-R1 directed apoptosis as opposed to survival. One model of experimental glaucoma found no evidence of JNK activation in RGCs or other cells of the retina (Levkovitch-Verbin et al., 2005), while JNK expression was observed in RGCs from glaucoma donor eyes, though the timing or persistence of expression is unknown (Tezel et al., 2003). TNFα-R1 can also activate cell protective heat shock proteins such as hsp70 in response to cellular stress (Heimbach et al., 2001), and this activity involves cross-talk with NFκB. TNFα represents just one signaling system between glia and neurons with finelytuned, context-dependent effects on RGC survival. TNFα undoubtedly can cause RGC apoptosis, but for which cells and when, and in which glaucoma-related contexts, remains a topic of active investigation.

Astrocytes that surround synapses actively modulate synaptic activity in a Ca2+-dependent, glutamate mediated way (Jourdain et al., 2007). Early studies showed how the amplitude of Ca2+ waves moving through retinal astrocytes and Müller glia modulated the spike activity of RGCs (Newman & Zahs, 1998). Since glutamate antagonists reduce the inhibition of neuronal activity associated with glial Ca2+ waves, inhibition is likely mediated by inhibitory interneurons stimulated by glutamate release from glial cells. In the hippocampus, TNFα has been shown to regulate the glial release of glutamate that can escape glial reuptake and bind to neuronal NMDA receptors (Santello et al., 2011). TNFα improvement of glutamate exocytosis from astrocytes could be quite detrimental if the transport mechanisms to prevent glutamate RGC overstimulation are compromised. On the other hand, we have already outlined how loss of TNFα could limit the ability of astrocytes to support activity-dependent RGC survival. Well-timed glutamate release from glia activates glutamate transporters, potentially extending the interval of extracellular glutamate signaling. TNFα also works through the TNFα-R1 to increase AMPA-R insertion in the post-synaptic membrane. This multi-pronged modulation of glutamate signaling through TNFα significantly complicates our understanding of the role of TNFα in glaucoma. Müller glia, through their more intimate association with retinal synapses in which TNFα modulation of glutamate release would occur, will be the object of intense study of these phenomena.

TNFα contributing to glutamate dysregulation implicates glutamate-induced excitotoxicity as a glial-related mechanism of glaucoma. Evidence for neurotoxic levels of glutamate has been observed in the vitreous of canines (Brooks et al., 1997) but not in monkeys (CarterDawson et al., 2002) with induced glaucoma. However, focal depletion of glutamate in glaucomatous canine retina occurred concomitant with significantly lower levels of glutamine and glutamine synthetase (GS) (Madl et al., 2005; Chen et al., 2008). Reduced glutamate disrupted important anti-oxidant systems, making oxidative stress secondary to glutamate dysregulation a potential mechanism of RGC loss in glaucoma. In support of this concept, anti-oxidant treatment α-luminol restored glutamate, glutamine and GS levels and protected RGCs in the DBA/2J mouse model of glaucoma (Gionfriddo et al., 2009). Further detail regarding the role of oxidative stress appears in the Müller glia section below.

In a microarray study that compared 3 and 8-month retinas from DBA/2J mice prior to outright glaucoma, a few of the mRNAs that underwent the greatest decrease were members of the crystallin family (Steele et al., 2005), a group of proteins with diverse

function but primary expression in the lens. The βA3/A1crystallin subtype, which was down 2-fold in 8-month DBA/2J, is also expressed in retinal astrocytes. Elimination of βA3/A1crystallins results in structural and functional defects in astrocytes that lead to abnormalities in vascular development and maturation (Sinha et al., 2008). Though these observations were made in young knockout mice, the implications of eliminating an astrocyte-derived vascular modulator in the DBA/2J glaucoma mice could have a significant effect on retinal function and RGC survival. Autoantibodies against α-crystallin have been observed in normal-tension glaucoma patients, and these antibodies could cause apoptosis of RGCs in vitro (Tezel et al., 1998). These data as well as data described in the microglia section raises the possibility of an auto-immune component to glaucoma. Evidence for pathogenic glial interactions in glaucoma continues to mount.

2.2 Müller glia

Müller glia have overlapping roles with astrocytes, including redox homeostasis, maintenance of extracellular milieu through ion buffering and growth factor provision. However, by virtue of their role in retina structure, their contact of all retinal neurons, and their anatomical link between the vitreous of the inner retina and the pigment epithelium of the outer retina, Müller glia form a functional unit with a much more critical role to play in retina than astrocytes. Müller glia contribute more to the glia limitans and to wrapping cell somas in the inner retina than do astrocytes (Hollander et al., 1991). Through their expression of myriad receptors, ion channels, and transporters, Müller glia engage in extensive interplay (energetic and signaling) with their neuronal neighbors (Bringmann et al., 2006).

The response of Müller glia to glaucomatous changes resembles that of astrocytes in that many activities are neuroprotective, but these macroglia can also severely damage the retina through the release of cytokines, nitric oxide, and proteoglycans or by failing to release growth factors. Beyond the non-specific responses to retinal injury which include upregulation of GFAP, vimentin (Bringmann & Reichenbach, 2001) and activation of the extracellular signal-regulated kinases (ERKs) (Tezel et al., 2003), Müller glia have specific responses like upregulation of glutamine synthetase (GS) by mRNA and protein, which we have observed in the DBA/2J mouse retina at ages of accelerating RGC loss (10 months; see Figure 2). A similar upregulation of GS expression occurs in hepatic retinopathy where the GS detoxifies the retina of accumulated ammonia (Reichenbach et al., 1995). Upregulation of GS in Müller glia is neuroprotective against glutamate neurotoxicity (Gorovits et al., 1997), suggesting that the increased expression in DBA/2J retina may be a protective mechanism. As noted in the retinal astrocyte section, changes in GS have implications for managing oxidative stress and maintaining appropriate glutamate levels in retina.

A strong neuroprotective role for Müller glia emerges in their management of the neurotransmitter glutamate. Müller glia use GLAST to efficiently move glutamate from the extracellular space in the course of normal retinal function but also in pathological conditions such as ischemia, a proposed contributor to the mechanism of glaucomatous damage. Patients with primary open angle glaucoma had glial activation that correlated well with vascular dysregulation (Grieshaber et al., 2007), a potential source of retina ischemia. Blocking GLAST or GLT-1 in the retina of normal rats led to glutamate increases and RGC death (Vorwerk et al., 2000), establishing that RGCs are sensitive to excitotoxicity. While Müller glia endeavor to maintain signaling homeostasis, RGCs do not necessarily sit

passively by: RGCs expressed one isoform of the GLT-1 transporter (GLT-1c) upon induction of glaucoma in the rat eye (Sullivan et al., 2006). This unusual expression might be a protective response to increases in extracellular glutamate. GLAST can reverse glutamate and aspartate transport when extracellular K+ concentration is high, as would be the case with high neuronal activity or Müller glia depolarization (Marcaggi et al., 2005). However, an electrophysiological analysis in the DBA/2J mouse model of glaucoma determined that neither membrane currents nor membrane potentials were altered in the Müller glia in mice with clear glaucoma pathology (Bolz et al., 2008). Evidently, concrete evidence for excitotoxicity in the glaucomatous retina is elusive; the evidence for oxidative stress as a driver in glaucoma, which can be secondary to glutamate dysregulation, is much stronger.

Fig. 2. A. Glutamine synthetase (GS) mRNA in whole retina from DBA/2J mice from 3 to 10 months of age, as percent of control (3 month levels). GS mRNA levels peak at 8 months, coincident with significant gliosis but prior to significant RGC loss. B. GS protein levels in whole retina from DBA/2J mice from 3 to 10 months of age. The protein levels were normalized to β-actin. GS peaks at 10 months (p<0.05) when RGC loss is accelerating.

Retinal glia have a fundamental role in maintaining redox homeostasis so are equipped with the tools to resolve redox imbalance. Oxidative stress occurs when reactive oxygen species (ROS) creation outpaces its clearance by anti-oxidant enzymes and compounds in a cell. Oxidative stress can result from exposure to stimuli that range from environmental toxins, infection and energy dysregulation to ischemia; it has been implicated in several neurodegenerative diseases, including glaucoma (Gmitterova et al., 2009). Resolution of oxidative stress can halt neurodegeneration (De Luca et al., 2008). As will be discussed at length in the section on optic nerve head, increased IOP can affect blood flow to the retina. Lack of nutrient delivery and waste removal via the circulation leads to ischemia. Müller glia upregulate iNOS in times of ischemia and the subsequent NO can dilate blood vessels (Goldstein et al., 1996), but NO can also be quickly converted to peroxynitrite that can attack proteins. Protein thus nitrated can disrupt normal cellular function and lead to increased energy demand. ATP energy in the CNS comes from glycolysis or oxidative phosphorylation. Non-reduced diatomic oxygen (O2·), known as superoxide anion, is the primary mitochondrial reactive oxygen species (ROS) generated from oxidative phosphorylation (Skulachev, 2006). Other ROS can be derived from superoxide production. ROS attack glutamate transporters on redox-sensing cysteine residues (Aschner, 2000), leading to higher extracellular glutamate, lower glutamine (and therefore, glutathione)

production as well as compromised energy availability, a dangerous feedback loop. The loss of the anti-oxidant glutathione hinders the cell's ability to limit ROS damage. Cellular components accumulate ROS-related changes (oxidation, nitration, lipidation) and cease to function. As a sign of extreme distress, Müller glia exposed to ROS express major histocompatibility class II molecules, allowing these glia to present antigen and T cells to create antibodies against the presented protein (Tezel et al., 2007). Autoantibodies appear in the serum of glaucoma patients (see microglia section below). Oxidative stress can be initiated in several ways; it is certainly a secondary degenerative process in glaucoma, but it has the ability to amplify and extend damage at every level of cellular regulation, making it a formidable foe.

Müller glia from human glaucoma eyes showed increased levels of advanced glycation end products (AGE), an inflammatory stimulant, as well as the receptor for advanced glycation end products (RAGE) (Tezel et al., 2007). Müller glia express proinflammatory cytokines IL- 6 and TNFα (Tezel & Wax, 2000) in response to AGE exposure. TNFα release has been shown to impact the blood-retinal-barrier through increased vesicular transport of serum proteins by vascular endothelial cells. Serum proteins accumulate in Müller glia, perivascular microglia and pericytes after TNFα exposure, suggesting that these glia protect the retina by acting as secondary barriers (Claudio et al., 1994). However, Müller glia are just as likely as retinal microglia and astrocytes to be a source of the TNFα. Similarly to astrocyte-derived TNFα, Müller glial TNFα can dictate cell death or survival based on the complement of receptors and second messenger systems available to it.

2.3 Microglia

Microglia function in environment surveillance, synapse elimination, cytokine/chemokine release, innate immunity and debris clean-up. The release of specific cytokines and synapse elimination are two mechanisms of glaucoma in which microglia would play a significant role, possibly driving pathogenesis. In order to implicate microglia in the development of glaucoma, one research group used minocycline to reduce microglial activation and document changes in glaucoma disease progression. A significant decrease in microglial activation occurred with minocycline treatment, as shown through morphology and gene expression, and there was significant improvement of retrograde transport in RGC axons (Bosco et al., 2008). Minocycline inhibits the expression of NOS (Amin et al., 1996) and exerts a stimulus-dependent decrease or increase the production of IL-6 through TNF-α (Kloppenburg et al., 1996). Microglia in DBA/2J mouse retina have been shown to release the cytokine IL-6. These observations suggest protection of RGCs by decreasing microglial activation in glaucomatous retina, but it does not rule out a contribution of astrocytes, Müller glia or even neurons due to the systemic delivery of the minocycline treatment.

Microglia increase their expression and release of IL-6 when cultured under hydrostatic pressure, conditions meant to mimic the retina at increased IOP (Sappington & Calkins, 2006). Microglia-derived IL-6 rescued RGCs cultured under hydrostatic pressure from death (Sappington et al., 2006). Interestingly, retinal microglia express TRPV channels, cationselective transient receptor potential vanilloid-1 receptors sensitive to mechanical stimuli like pressure (Sappington & Calkins, 2008). IL-6 release from microglia under pressure required Ca2+ increase, mediated by both intracellular ryanodine receptors and TRPV channels (Sappington & Calkins, 2008). The same group also demonstrated that IL-6 increases were preceded by sustained NFκB nuclear translocation, suggesting that NFκB

drives IL-6 changes in retinal microglia (Sappington & Calkins, 2006). Antagonism of Ca2+ release reduced IL-6 release and NFκB activation in retinal microglia. These findings reflect the complexity of glial response during disease circumstances; not only is IOP translated by glia in myriad ways, but those ways can be in opposition, depending on the glial subtype.

Additional studies have underscored the complexity of glial-associated pathogenesis by showing the give and take of astrocytes and microglia. Astrocyte-derived signals (possibly ROS) initiated Nrf2 activity and expression of heme-oxygenase in microglia (Min et al., 2006). Conditioned media from those astrocytes suppressed interferon-γ (IFN-γ)-induced ROS production, leading to reduced iNOS expression and NO release. These data illustrate the feedback loop of ROS production that exists between these glia. Feedback loops regulated by astrocytes in microglia work in reverse too: Microglia can activate the Nrf2ARE (anti-oxidant response element) in astrocytes and change the redox level there.

An autoimmune component to glaucomatous optic neuropathy has emerged from observations of serum autoantibodies against proteoglycans in open-angle glaucoma patients (Tezel et al., 1999), and against heat shock proteins (hsp) hsp27 and hsp60 (Wax et al., 2008) in patients with normal tension glaucoma (NTG). Microglia have a complex, context-dependent role in autoimmunity. They can express MHC class II proteins which allow them to present antigen. Their expression of Fas ligand (FasL), a cytokine in the TNF superfamily, leads to apoptosis in cells expressing the FasL receptor. Immunization of rats with hsp27 or hsp60 activated microglia and upregulated FasL receptor expression in RGCs and cells in the INL (Wax et al., 2008). Heat shock proteins are upregulated in times of stress and function as protein chaperones or assembly of protein complexes (Young & Elliott, 1989). In NTG patients, antibody against hsp27 may reduce the ability of native hsp27 to stabilize the cytoskeleton, thereby initiating apoptosis in hsp27-expressing cells (Tezel & Wax, 2000). These data suggest that activated microglia could initiate apoptosis in cells targeted by autoantibodies. Conversely, the expression of FasL proteins by retinal microglia may be a mechanism of maintaining ocular immune privilege and avoiding damaging inflammatory reactions in the eye (Griffith et al., 1995) by inducing apoptosis of invading T- cells. Ocular immune privilege certainly limits T-cell travel in the retina unless coincident with blood-retinal-barrier breach, a circumstance that has been demonstrated only in canine glaucoma that is secondary to uveitis (Reilly et al., 2005; Mangan et al., 2007). Glaucoma experimental models that document persistent microglial activation do not also find T-cells (Ebneter et al., 2011). Glaucoma T-cell entry to the retina is receptor-mediated even in cases of clear autoimmune retinal pathological mechanisms such as in glaucomas secondary to sarcoidosis (Hamanaka et al., 2002) or B-cell lymphoma (Cockerham et al., 2000), in which T- cell or B-cells were observed in Schlemm's canal and trabecular meshwork. Therefore in retina, these studies suggest microglia would be the primary agents of autoimmune-related FasL-induced apoptosis capable of dispatching significant numbers of RGCs. Further investigation into which cells present antigen, what comprises that antigen and which cells express the FasL receptor would improve our understanding of potential autoimmune processes in glaucoma.

Based on evidence from innate immunity research, microglia and astrocytes coordinate management of synapses in the inner retina. The initiating member of the classical complement cascade, complement component 1q (C1q), coats dead cells, debris or pathogens and initiates a protease cascade that results in complement C3 deposition. C3 can activate receptors on microglia that signal the cells to phagocytose any C3-coated elements. Synapse elimination during CNS development proceeds by this mechanism (Stevens et al.,

2007), with immature astrocytes signaling to RGCs to express C1q. Not normally observed in the adult retina, C1q is nevertheless upregulated in glaucomatous eyes (Steele et al., 2005; Stasi et al., 2006), suggesting that activated astrocytes have overcome their developmental limitations and released a factor to which the RGCs respond by making C1q. Immunolabeling of the inner plexiform layer where RGCs synapse with amacrine and bipolar cells shows C1q upregulation coincident with synapse loss (Stevens et al., 2007). An unknown stimulus triggers the astrocytic release of the factor that leads to C1q upregulation in the glaucomatous retina. Microglia respond by eliminating the targeted synapses, reducing RGC connectivity. Evidence for dendritic arbor shrinkage and loss (Weber et al., 1998; Pavlidis et al., 2003; Shou et al., 2003) in glaucoma supports this mechanism of degeneration.

A study in which a bone marrow transplant after irradiation rescued RGCs from cell death in the DBA/2J has intrigued many, and may be evidence for a vital role of microglia in glaucoma. Since microglia comprise the largest group of proliferating cells in the retina (Inman & Horner, 2007), the irradiation would have destroyed them and any endothelial cells. The bone marrow transplant repopulated the immune system and irradiation created a leaky blood-retinal-barrier, allowing peripheral immune cells to infiltrate. No one understands the mechanism of protection against optic neuropathy in this study, but the results highly suggest eliminating endogenous retinal microglia enabled RGC rescue (Anderson et al., 2005).

3. Optic nerve head

3.1 Locus of degeneration

A long-held hypothesis of the mechanism of RGC damage in glaucoma states that compression and rearrangement in the lamina cribosa cribriform plates, the connective tissue collagen plates through which the ganglion cell axons pass to form the optic nerve, impinge upon the axons, disrupting axon transport to a degree that leads to axon degeneration and RGC death (Quigley & Addicks, 1981). Supportive evidence for this hypothesis includes failure of retrograde delivery of target-derived growth factors in monkey and rat glaucoma models (Pease et al., 2000; Quigley et al., 2000) as well as the considerable extracellular remodeling that alters the lamina and deposes significant amounts of collagen and proteoglycans in humans (Tengroth & Ammitzboll, 1984; Tengroth & Ammitzboll, 1984) and in experimental glaucoma models (Johnson et al., 1996). The optic nerve head (ONH) undergoes considerable deformation in response to the diurnal fluctuation in IOP, and these conformational changes impact both glia and RGC axons. There are several mechanosensitive receptors on ONH astrocytes that could respond directly to changes in IOP (Oh, 1997).

Despite lacking a lamina cribosa, the DBA/2J mouse provides additional evidence for the optic nerve head as the site of initial damage in glaucoma. Fasciculated RGC axons travel through glial columns (glia lamina) in the mouse until myelination within the optic nerve. The glial columns are astrocytes arranged parallel to the axons and run caudal from the scleral boundary for about 200 microns (Howell et al., 2007), beyond which the ganglion cell axons become myelinated by optic nerve oligodendrocytes (May & Lutjen-Drecoll, 2002). Dystrophic neurites, with swollen and accumulated organelles and neurofilament breakdown, were observed in the glia lamina while the corresponding axons in the prelamina and in the retinal nerve fiber layer were intact (Howell et al., 2007). It appears that

axons underwent degeneration as entire fascicles, leaving sectors of ganglion cells axotomized and destined for degeneration within the retina (Jakobs et al., 2005; Howell et al., 2007). These data raise two interesting possibilities: for one, doubt is cast on the idea that the lamina cribosa pinches the axons to initiate glaucomatous degeneration; but secondly, the data implicates astrocytes in the lamina as prime drivers of pathology in this disease. The pattern of degeneration, that of ganglion cell loss in a pattern dictated by the position of axons lost, mirrors that observed in the human retina and proximal optic nerve. Astrocytes and microglia are the sole cell types in the lamina, so how might these cells initiate glaucoma? Recent evidence suggests mechanisms of action that include increased endothelin release, axon impingement from changes in extracellular matrix and astrocyte migration, oxidative stress from intermittent ischemia, and anterograde axon transport deficit of unknown etiology.

3.2 ONH astrocytes

As RGC axons undergo degeneration, the optic cup deepens, enabled by astrocyte remodeling and migration. The prelaminar portion of the optic nerve loses glial columns and astrocytes migrate between axon bundles (Hernandez & Pena, 1997). The ONH has both Type1A and Type1B astrocytes, distinguished primarily by the expression of NCAM in Type1B; both types express GFAP. Type1B astrocytes appear to be responsible for extracellular matrix production in ONH (Ye & Hernandez, 1995). Astrocytes depose new extracellular matrix (ECM) in the course of glaucoma development. Astrocytes isolated from the human optic nerve head upregulated their expression of neural cell adhesion molecule (NCAM) two-fold by 6h of exposure to 60mmHg hydrostatic pressure (Ricard et al., 2000). NCAM participates in cell anchoring, so documented changes in its expression in glaucoma (Ricard et al., 1999) suggest a potential point of intervention if astrocyte migration contributes to the incipient optic neuropathy. Integrins join GFAP and vimentin at focal adhesion complexes on astrocytes that connect the cells to ECM, giving them a role in cell migration and transmitting mechanical signals to the astrocyte (such as increased IOP). Integrins also undergo considerable changes in expression in glaucoma (Morrison, 2006). Altered ECM makeup means potential loss of compliance of this dynamic tissue.

As demonstrated in the retina (see Figure 1), glia overexpress proteoglycans during gliosis in glaucoma. Serum autoantibodies obtained from patients with normal tension glaucoma and those with increased IOP bind to chondroitin sulfate and heparan sulfate glycosaminoglycans, proteins upregulated in the optic nerve head of glaucoma patient tissue (Tezel et al., 1999). However, chondroitin sulfate proteoglycan 4 (CSPG4) and CSPG1 (aggrecan) mRNA stood out on one ONH astrocyte microarray for being downregulated 35fold and 29-fold, respectively in glaucoma patients (Hernandez et al., 2002). If there are feedback mechanisms that regulate proteoglycans, the mRNA downregulation might be a response to the persistence of the protein within the tissue. Optic nerve head astrocytes also degrade extracellular matrix as they migrate in vitro after exposure to hydrostatic pressure (Tezel et al., 2001).

Astrocytes in the optic nerve head (and retina) express G-protein-coupled endothelin receptors ETA and ETB, as do retinal vessels. Endothelin, a vasoactive and neuroactive peptide, has been shown to produce optic neuropathy when injected intravitreally. Patients with primary open-angle glaucoma have significantly higher levels of aqueous humor endothelin than age-matched normal patients (Tezel et al., 1997; Iwabe et al., 2010).

Endothelin leads to intracellular Ca+2 increases in astrocytes and can increase astrocyte hypertrophy and proliferation (Prasanna et al., 2003). This effect was mediated by the ETA receptor since it could be blocked by an ETA-specific antagonist. Ischemia and mechanical pressure can induce endothelin secretion from astrocytes. ETA has greatest affinity for ET-1 and ET-2 while ETB has equal affinity for all three isoforms of ET (1, 2 and 3). Endothelin can disrupt anterograde axon transport, making its upregulation in optic nerve head astrocytes (even retinal astrocytes) an important potential mechanism of glaucoma. Key to understanding the role of endothelin is recognizing that it had effects on specific transport types and times; early after intravitreal injection of 2nM endothelin, it increased fast anterograde axon transport of tubelovesicles, but by 28h after injection, it decreased transport of mitochondria and decreased slow anterograde transport of cytoskeletal proteins at 4 days (Stokely et al., 2002). A straightforward mechanism for optic neuropathy initiated by endothelin would combine its vasoconstriction (through the ETA) that leads to ischemia with the energy dysregulation in RGC axons, a result of the ETB-mediated deficit in mitochondrial anterograde transport. Not surprisingly, TNFα stimulates ET-1 release in ONH astrocytes exposed for 24h to hypoxia (Desai et al., 2004).

Some of the genes downregulated in ONH astrocytes cultured from human glaucoma patients have negative ramifications for cell survival. The loss of glucose transporter mRNA (GLUT5) and monocarboxylate transporter (MCT3) (Hernandez et al., 2002) would challenge astrocytes to maintain energy levels and exchange pyruvate or lactate with neurons. Fuel energy dysregulation would negatively impact membrane potentials and therefore action potential propagation in the neighboring RGC axons. Axonal Na+ overload occurs when energy substrates to decrease and Na+ initiates Ca2+ accumulation in the axon through reverse operation of the Na+/Ca2+ exchanger (Stys et al., 1992). Subsequent to Na+ overload, K+Cl- cotransporters, with subtypes present on both astrocytes and axons, release K+ and Cl- from the axons, disrupting the Cl- gradients that contribute to resting optic nerve membrane potential (Malek et al., 2003) Axons unable to conduct action potentials will fail to transmit the visual signal from RGCs to the target regions in brain. Energy depletion would be complete as ATPdependent processes such as axon transport would cease. This proposed mechanism supports many of the observations of generalized axon transport failure (Buckingham et al., 2008) or transport failure at the ONH (Minckler et al., 1977; Quigley et al., 2000). However, important observations of axon transport deficit in the DBA/2J mouse model of glaucoma showed that axon transport in glaucoma fails first in the target regions of the RGC axons, the superior colliculus (Crish et al., 2010). There are many activity-dependent regulatory mechanisms that maintain synapses and transmit sustaining growth factors back to the RGC soma. One might anticipate loss of synaptic connections with axon conduction loss, though the authors were able to demonstrate in the DBA/2J that collicular synapses were intact despite lack of transport to the target (Crish et al., 2010). Certainly distal transport failure highlights the logistical difficulties that RGCs face in getting cargo to remote synapses and highlights a potentially novel mechanism of glaucoma pathology.

Understanding the mechanism of optic neuropathy in glaucoma is complicated by the frequent overlap of expression and activation of potentially damaging intermediates by both astrocytes and microglia. Cytokines and chemokines expressed by glia have been implicated in neurodegeneration, and the feedback loops or escalation of inflammatory processes are difficult to tease apart. Astrocytes in human glaucomatous ONH express TNF-α and its receptor, TNF-R1 (Yuan & Neufeld, 2000). Upregulation of TNF-α and TNF-R1 occurred concomitant with increasing optic nerve degeneration; in severe glaucoma, TNF-α and TNF-

R1 were also expressed on activated microglia (Yuan & Neufeld, 2000). One consequence of TNF-R1 activation is NOS-2 induction, in both astrocytes and microglia (Neufeld, 1999). NOS-2 induction also occurs in reactive astrocytes from human ONH as a result of epidermal growth factor (EGF) receptor agonism (Liu & Neufeld, 2003). ONH astrocytes exposed to hydrostatic pressure showed increased EGF-R expression and phosphorylation, as did reactive, but not quiescent, astrocytes in human glaucoma ONH (Liu & Neufeld, 2003). A microarray analysis of EGF-R activation in ONH astrocytes showed upregulation of many genes associated with astrocyte activation, including various proteoglycans, ETA, leukemia inhibitory factor (LIF), insulin-like growth factor (IGF), fibroblast growth factor 2 (FGF-2), nerve growth factor (NGF), transforming growth factor- β (TGFβ) and tissue inhibitor of matrix metalloproteinase (TIMP) (Liu et al., 2006). Others have shown ONH astrocytes express TGFβ (Pena et al., 1999) which can upregulate ECM molecule expression, both possibly as a result of EGF-R activation. These data suggest that EGF is a potent astrocyte activator, but ironically, many of the upregulated genes are growth factors which generally support cell survival. Seven months delivery of an EGF-R tyrosine kinase inhibitor or NOS-2 inhibitor protected RGCs from cell death in a chronic rat model of glaucoma (Liu et al., 2006). The treatments sought to eliminate EGF-R activation and its target NOS-2 in astrocytes. Due to systemic delivery of the inhibitors, any cells with EGF-R would be antagonised, though the implication is that tempering glial reactivity protected RGCs from glaucoma-related cell death. However, RGC number and not function was the only outcome measure.

3.3 ONH microglia

Microglia are activated in glaucomatous human ONH (Neufeld, 1999) and very early at the ONH in chronic glaucoma models, indicating either an unusual responsiveness to stressors there or an initiation of degenerative processes (Bosco et al., 2011). Microglia often appear activated and in high numbers near the peripapillary chorioretinal region, a possible site of tenuous blood-retinal-barrier, and in close proximity to retinal vessels (Neufeld, 1999). These cells' position and activation state suggest interaction with the periphery; perhaps the observed autoantibodies against proteins found in the ONH are generated with the assistance of ONH microglia. Microglia in ONH of humans with glaucoma (but not controls) express TNFα, TGFβ and shows signs of proliferation by being immunopositive for PCNA, the proliferating cell nuclear antigen (Yuan & Neufeld, 2001). Cytokine expression targets astrocytes, the primary cell type in the ONH besides microglia, and modulates many of the pathological processes already described. TNFα induces NOS-2 which leads to nitric oxide (NO) production in microglia. Microglia can affect axon transport of synaptic vesicle precursors by releasing nitric oxide (Stagi et al., 2005). This ability would be especially destructive in the ONH where the RGC axons do not have a protective myelin sheath and activated microglia have been observed very early in the DBA/2J mouse model of glaucoma (Bosco et al., 2011). Microglia constitutively express several matrix metalloproteinases (MMPs) that get upregulated with glaucoma progression. These enzymes can contribute to much of the documented tissue remodeling that occurs in the ONH.

4. Optic nerve

All glaucomas, regardless of etiology, share optic nerve (ON) degeneration. Despite this fact, the ON has not been a focus of research into the pathogenesis of glaucoma. This may change with recent evidence of phagocytic astrocytes and energy dysregulation in ON.

4.1 ON astrocytes

Unlike in the retina, astrocytes proliferate in the glaucomatous optic nerve, as well as increase their expression of vimentin (Son et al., 2010). There are age-related changes in optic nerve astrocytes that likely alter their ability to function. One recent glaucoma hypothesis posits that phagocytic astrocytes in a particular region of the optic nerve, an area just rostral to the beginning of myelination (the myelination transition zone, or MTZ), become dysregulated. These phagocytic MTZ astrocytes express Mac-2 and upregulate it with IOP increase; the Mac-2 levels correlated with the number of RGCs with damaged axons in the retina (Nguyen et al., 2011). Spheroids in the MTZ contained γ-synuclein; their numbers increased in DBA/2J mice with glaucoma. The MTZ astrocytes were observed with large, γ-synuclein-positive axon inclusions within their cytoplasm (Nguyen et al., 2011). These data suggest that glaucoma might be a synucleinopathy, with the γ-synuclein axon spheroids in the MTZ mimicking the α-synuclein accumulations in Lewy bodies that contribute to cell death in Parkinson's disease. Immunolabeling for γ-synuclein was observed in human optic nerve (on axons and GFAP+ astrocytes) from glaucoma patients (Surgucheva et al., 2002). In models of glaucoma, phagocytic astrocytes in the myelination transition zone (MTZ) may become hyperactive in a bid to control γ-synuclein accumulation, or they may be responding to pressure signals that encourage hyperphagocytosis and lead to premature axon destruction.

Also of note, one study of glaucomatous ON showed an inverse relationship between IOP and available ATP. Increased IOP correlated with significantly decreased ATP in ON (Baltan et al., 2010), suggesting a metabolic vulnerability in glaucoma. Given that astrocytes comprise 28 percent of the ON (Perge et al., 2009), it may be the case that ATP decrease occurs in ON astrocytes. This possibility, combined with the observation of Glut5 and MCT downregulation in ONH astrocytes (Hernandez et al., 2002) supports an astrocyte energy dysregulation mechanism of optic neuropathy. Efforts to resolve whether ATP decrease in extrinsic (astrocytes) or intrinsic (axons) to the ON in glaucoma continue.

4.2 ON microglia

Microglia also occupy non-overlapping areas in the optic nerve (ON). Microglia may have a role in the glaucomatous ON that resembles their function in the ONH. Microglia-derived TNFα recruited macrophages to the sciatic nerve during Wallerian degeneration (Liefner et al., 2000), in part by inducing MMP activity that allows migration of these cells to sites of debris. As discussed in the ONH section, TNFα figures prominently in the response of microglia to the increased IOP and intermittent ischemia that occurs at the ONH. In the glaucomatous optic nerve, microglia have been observed full of lipid droplets, the result of ingesting myelin debris (Figure 3), but only in ON undergoing significant degeneration. It has yet to be determined if ON microglia have a role to play in glaucoma pathogenesis.

5. Manipulating glia

5.1 Intracellular pathways

Manipulating gliosis is one strategy for diminishing any negatives, and improving upon any positive effects, of glial response on RGC survival and function in glaucoma. We have changed the course of gliosis in mice with glaucoma by manipulating two intracellular signaling pathways implicated in gliosis, the JAK/STAT and the NFκB pathways. In one

Fig. 3. Cross-sections of DBA/2J optic nerve at 3 months (left) and 12 months (right) shows green Iba1-positive microglia (arrows) filled with phagocytosed myelin debris (red) as labeled by a dye that stains lipid droplets (Oil Red O). Axons are labeled blue with an antibody against neurofilament-200 (NF-H) Scale bar=100 microns.

transgenic mouse, STAT3 is knocked out in GFAP+ cells (Herrmann et al., 2008); in the other, NFκB in GFAP+ cells is prevented from translocating to the nucleus (Brambilla et al., 2005).

STAT3, a critical regulator of astrocyte intermediate filament upregulation and hypertrophy (Herrmann et al., 2008), transduces the intracellular signal for IL-6, CNTF and LIF, cytokines released after CNS injury. STAT3 has been observed in the nuclei of Müller cells, RGCs and astrocytes (Thanos & Naskar, 2004; Wang et al., 2010). In general, STAT3 activation is present in neurons in the acute phase of neural tissue injury and in astrocytes during the chronic phase, though severe injury may disturb this distinction (Yi et al., 2007). Furthermore, contradictions about the protective nature of STAT3 activation abound. For example, Luo et al., found that inhibition of JAK/STAT promoted RGC survival and axon regeneration (Luo et al., 2007), while a separate study showed that activation of STAT3 protected RGCs (Ji et al., 2004). In the former, RGC survival accompanied inhibition of macrophage recruitment, suggesting that JAK/STAT inhibition protected RGCs by preventing immune cell infiltration. To further clarify the role of STAT3, we subjected the GFAP+-STAT3-KO mouse (Herrmann et al., 2008) to the microbead occlusion glaucoma model (Sappington et al., 2009). The STAT3-KO had decreased GFAP and proteoglycan expression in retinal glial cells and optic nerve when compared to control. Moreover, RGCs were significantly spared from degeneration in the STAT3-KO mice (Lupien et al., 2010).

NFκB is a transcription factor expressed in neuronal and glial cells (Kitaoka et al., 2006) that regulates the expression of genes involved in inflammation, cell survival and apoptosis (Hayden & Ghosh, 2004). NFκB is multi-talented, able to upregulate anti-apoptotic factors but also activate pro-apoptotic pathways. This dual role is reflected in retinal research; for example, deletion of the p50 subunit of NFκB led to accelerated age-related RGC death (Takahashi et al., 2007). Also, ONH astrocytes from glaucoma patients express higher levels of NFκB mRNA and show greater nuclear translocation than controls (Agapova et al., 2006). Conversely, GFAP-IκBα-dn mice given retinal ischemia achieved significant survival of RGCs and reduction of pro-inflammatory gene expression (Dvoriantchikova et al., 2009). Our research would echo the latter findings in that we observe decreased retinal gliosis in vitro and in vivo by using NFκB inhibitors in a glaucoma mouse model (Lupien et al, 2009). By subjecting the GFAP-IκBα-dn mice to acute glaucoma, we also observed a significant

decrease of GFAP expression in retinal glial cells that coincided with RGC survival and improved visual function (unpublished data).

5.2 Oxidative stress pathways

Manipulating essential functions of glia can augment glial support of retinal neurons. One major glial support function is the management of retinal redox homeostasis. Through delivery of exogenous anti-oxidants or the manipulation of anti-oxidant pathways within the retina, we have altered the neural-glial interaction in a way that improves RGC survival and function. In many circumstances, retinal glia provide protection from oxidative stress through the ROS-mediated liberation of a transcription factor, Nrf2, that binds to the antioxidant response element (ARE) (Itoh et al., 1999) and promotes the expression of antioxidants such as heme-oxygenase, ceruloplasmin (Miyahara et al., 2003; Lambert et al., 2006), peroxiredoxin 1, catalase, glutathione peroxidase, superoxide dismutase and thioredoxin (Lee et al., 2003; Kobayashi & Yamamoto, 2005). Nrf2 controls expression of genes encoding the catalytic (GCLC) and modifier (GCLM) subunits of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione (GSH) biosynthesis (Wild et al., 1999). Glutathione is such an important component of redox homeostasis that it comprises two percent of the total protein in the Müller glia, a level that decreases with age (Paasche et al., 1998). As oxidative stress increases in glaucoma (Tezel et al., 2005; Wang et al., 2005), endogenous antioxidants are likely insufficient to maintain redox homeostasis.

Our first efforts at decreasing oxidative stress in glaucoma delivered exogenous lipoic acid, an organosulfur compound also made by mitochondria, over several months to DBA/2J mice with glaucoma. Lipoic acid protected RGCs from cell death and improved retrograde transport; indices of anti-oxidant activity were also increased (Lambert et al., 2008). In researching the mechanism of lipoic acid neuroprotection, we determined that the compound was working through the transcription factor Nrf2. In other contexts, lipoic acid was able to restore GSH levels in the aged liver by increasing Nrf2 nuclear translocation and its binding to the anti-oxidant response element (Suh et al., 2004). Nrf2 overexpressing astrocytes, when cocultured with motor neurons expressing mutant hSOD1, protected the neurons from cell death through GSH-enabled reduction of ROS (Vargas et al., 2008). These data demonstrate that increasing Nrf2-ARE activity decreases ROS generation through upregulation of anti-oxidant enzymes. We hypothesized that Nrf2 activation in retinal glia could provide additional anti-oxidant capacity to the retina, thereby protecting RGCs from pressure and ischemia-induced cell death. First, we subjected GCLM knockout mice, which have just 10 to 15 percent of their normal production of glutathione (Yang et al., 2002), to acute glaucoma. GCLM-/- mice with glaucoma experienced a significant increase in RGC death and decreased retrograde axon transport in the remaining RGCs compared to wildtype littermates. This implicated glutathione as important to RGC survival in glaucoma. Our next experiments will target astrocytes and Müller glia to overexpress Nrf2. Glaucoma will provide the initial stimulus for Nrf2 activation, but the additional transcripts will enhance Nrf2 promotion of anti-oxidant genes.

6. Conclusions

Glaucoma is a complex disease. Diverse etiology has challenged researchers to determine disease mechanism, and several hypotheses have provided exciting ideas about how to

achieve neuroprotection. The latest research supporting the symbiotic relationship of neurons and glia suggest the pursuit of glial manipulation as a worthwhile means to protect RGCs in glaucoma.

7. Acknowledgements

We would like to thank Sahar Manavi for animal care and technical assistance. The Catalyst for a Cure initiative through the Glaucoma Research Foundation, the Melza M. & Frank Theodore Barr Foundation and R21 EY018203-01 supported this work.

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