Oxidative stress and glaucoma: injury in the anterior segment of the eye

Introduction

The destructive action of free radicals is focused largely on cells, particularly on membrane lipids (due to the peroxidation process), sugars, phosphates and proteins, and DNA, which is one of the major factor contributing to cell aging. These

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cellular alterations in aged individuals and senescent cells are similar and they are related to cellular response to sublethal doses of oxidative stress. These alterations lead to a decline in mitochondrial respiratory functions and to a decrease in the capacities of degradation of proteins and other macromolecules, establishing an early cellular dysfunction (Lee and Wei, 2001).

Light, continually penetrating the eye through its tissues, produces a large number of free radicals that are often at the basis of the main diseases of

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the eye, such as age-related macular degeneration, cataract, and glaucoma.

Many findings lead us to believe that these diseases are the cytopathological consequence of an unfavorable state between the intracellular concentration of free radicals and the cells’ capacity to neutralize them through an increase in endogenous production of free radicals, a reduction of antioxidant molecules and/or a decrease of the capacity to repair the oxidative damage on cellular macromolecules.

The main eye diseases have a common pathogenetic mechanism: a long period of latency between induction and clinical manifestation with a multifactorial etiology resulting from the interaction between environmental risk factors and heightened individual genetic susceptibility.

In particular, in glaucoma, free radicals have different target tissues and, especially, in the anterior segment of the eye, they affect the trabecular meshwork (TM) and, more specifically, its endothelium. Contemporarily, in the posterior segment, free radicals affect/involve retinal ganglion cells (RGCs) and optic nerve head (ONH) in its extracellular component, leading to a series of events defined as ‘‘the glaucomatous cascade.’’

Oxidative stress

The loss of electrons from the outer orbit of atoms is defined as ‘‘oxidation’’ and leads to the formation of highly reactive molecules. A molecular species whose atoms contain one or more unpaired electrons in their outer orbits is defined as a free radical. This structure gives rise to the fundamental property of such molecules: instability. Indeed, in order to restore the equilibrium of their magnetic fields, atoms containing unpaired electrons tend to capture electrons from other atoms of nearby molecules, which in turn become free radicals, thus a chain reaction is triggered.

Organisms use oxygen to oxidize food; nutrients composed of carbohydrates, proteins, and fats are oxidized to carbon dioxide and water. The energy released during the oxidation process is stored in the form of adenosine triphosphate (ATP) and is subsequently used in numerous metabolic reactions.

While oxygen is essential to animal metabolism, it can also be harmful. Indeed, numerous uncontrolled oxidation reactions (auto-oxidation) may take place in the presence of oxygen and can cause cell damage. Free radicals are oxygen compounds resulting from the numerous metabolic reactions involving oxygen. They tend to readdress their imbalance by ‘‘attacking’’ nearby molecules in order to recover their missing electron, thereby making other molecules unstable. The ensuing chain reaction gives rise to the formation of new compounds, some of which may be toxic.

Although many atoms and molecules can form free radicals in vivo, the most important for biological systems seem to be the radical ions associated with oxygen reduction. The diatomic molecules of oxygen (O2) can be reduced to form the superoxide (O2 ) and hydroxyl (OH ) radicals. These oxygen free radicals have an enormous potential to harm living organisms. The superoxide is not particularly reactive in aqueous environments, while it is highly destructive in the lipophilic linings of biological membranes. The hydroxyl form is the most reactive and the most dangerous of all free radicals: it survives only momentarily before combining with one of the molecules nearby, such as DNA, proteins, and other macromolecules.

In physiological conditions, there is a balance between the endogenous production of free radicals and their neutralization by antioxidant defense mechanisms. When the production of radicals exceeds the organism’s capacity to neutralize them (‘‘scavenger’’ activity) or when antioxidant substances activity decreases, damage ensues.

Free radicals are neutralized both by a range of enzymes, such as superoxide dismutase (SOD), glutathione peroxidase, or catalase, and by numerous molecules that are either endogenously produced, such as glutathione (GSH), or dietary introduced, such as flavonoids, vitamins C, E, and others. These molecules are able to capture free radicals and accept the unpaired electron and ‘‘pass it on.’’ Molecules with the most effective action are those which have aromatic rings, particularly those with several hydroxyl groups, such as the polyphenols.

The most well-known free radical chain reaction is lipid peroxidation. In this process, a free radical removes a hydrogen atom from a lateral chain of a polyunsaturated fatty acid. Consequently, the carbon atom from which the hydrogen atom has been removed is left with an unpaired electron. In other words, the lateral chain of the fatty acid is transformed into a reactive free radical. These carbon atom radicals (lipids) normally end up combining with molecular oxygen (O2) in the membrane to produce a peroxyl radical lipid (O2). O2 is extremely reactive and triggers a chain reaction within the lateral chains of the polyunsaturated fatty acids. Over time, peroxidation produces enough peroxidated lipids to damage the structure and fluidity of the membrane. Through a similar mechanism, peroxidation can cause severe damage to the essential proteins of the membrane.

The lateral chains of the polyunsaturated fatty acids, which guarantee the necessary fluidity of the membrane lipids, are particularly sensitive to attack by free radicals. If the free radicals of the chains are not deactivated, their chemical reactivity can damage all types of cellular macromolecules. The main targets of peroxidation reactions are proteins, cell membranes, and nucleic acids (DNA and RNA), including mitochondrial DNA (mtDNA). Indeed, mtDNA is less protected than nuclear DNA, and is therefore more sensitive to free radical attack (De Grey, 1997). mtDNA damage is another possible mechanism involved in the etiopathogenesis of degenerative diseases.

Peroxidation phenomena in the organism are countered by antioxidant compounds, which inhibit the formation of free radicals. These compounds include water-soluble antioxidants (e.g., ascorbic acid, cysteine, GSH), lipid-insoluble antioxidants (e.g., tocopherols and retinols), and enzymes such as SOD, which catalyzes the transformation of free radicals into hydrogen peroxide. Although hydrogen peroxide is also an active oxygen compound, it can be further transformed into oxygen and water by catalase. Also, several metal-binding proteins (e.g., transferrin) (Babizhayev and Costa, 1994; Rose et al., 1998) and flavonoids (e.g., genistein, diazine, glycyrrhizin, etc.) (Kapiotis et al., 1997; Wang et al., 1998; Tang et al., 2007) have an antioxidant activity.

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Table 1. Many studies underscore the role of endogenous oxidative damage in the pathogenesis of glaucoma

ROS effects in the eye

Note: This interpretation is in agreement with both vascular and mechanical pathogenic theories. The set of processes that make up the glaucomatous cascade triggered by free radicals results in the progressive apoptotic degeneration of trabecular meshwork, retina, and optic nerve.

The formation of metal-mediated free radicals causes various modifications of DNA nucleotides, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Lipid peroxides, formed by the attack of free radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals, finally producing mutagenic and other exocyclic DNA adducts (Valko et al., 2005), such as 8-hydroxy-2u- deoxyguanosine (8-OH-dG), which is an indicator of oxidative DNA damage.

Concerning eye diseases, all this phenomena are particularly important, above all in the pathogenesis of glaucoma (Table 1), where age and oxidative stress appear to play a fundamental pathogenic role (Sacca`et al., 2007).

Trabecular meshwork

Glaucoma is a group of optic neuropathies characterized by a progressive degeneration of RGCs and visual-field damage, which represents the final common pathway resulting from a number of different conditions that can affect the

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eye. Even if this disease has been known from the time of Hippocrates (Nathan, 2000), its pathogenesis is still misunderstood.

The chambers of the eye are filled with aqueous humor (AH), a fluid with an ionic composition very similar to blood plasma, with two main functions: to provide nutrients to eye tissues (e.g., cornea, iris, and lens) and to maintain intraocular pressure (IOP). 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 TM (Brandt and O’Donnell, 1999).

In most cases, glaucoma is accompanied by an increase of the IOP. Ocular hypertension is one of the major risk factors for the development and progression of primary open-angle glaucoma (POAG), a leading cause of blindness. TM is a tissue located in the anterior chamber angle of the eye, and it is a crucial determinant of IOP even if, at the moment, many aspects of the regulation of AH outflow remain unclear. Nevertheless, it is known that the region of maximal resistance to AH outflow resides at the peripheral juxtacanalicular TM, which connects the TM to the Schlemm’s canal (Johnson and Johnson, 2001). TM is directly involved in the regulation of AH outflow (Wiederholt et al., 1995). The subendothelial region of Schlemm’s canal does not form a continuous fluid system, and the pathways through the connective tissue of the cribriform region are responsible for outflow facility and determine the filtration area of the inner wall of Schlemm’s canal (Lutjen-Drecoll, 1973). This tissue has unique morphologic and functional properties involved in the regulation of AH outflow. The conventional outflow pathway is organized with a plumbing arrangement for maintaining a fluid barrier to prevent the passage of AH, consisting of trabecular lamellae covered with HTM cells, in front of a resistor, consisting of juxtacanalicular HTM cells and the inner wall of Schlemm’s canal. 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). TM cells also regulate the formation and turnover of

extracellular matrix (ECM) (Yue, 1996). A disproportionate accretion of ECM occurs in the TM region of POAG eyes, and this buildup is responsible for the development of greater resistance to AH outflow, resulting in increased IOP (Rohen and Witmer, 1972; Lee and Grierson, 1974). The main resistance to the AH outflow is located in the TM directly underneath the inner wall of Schlemm’s canal (Maepea and Bill, 1992). The inner wall of Schlemm’s canal is unique, sharing extraordinary characteristics with both types of specialized endothelia: lymphatic and blood capillary endothelia (Ramos et al., 2007). In this layer, there are the ‘‘giant vacuoles.’’ They are really outpouchings of the endothelium into Schlemm’s canal, caused by the pressure drop across inner wall endothelial cells (Brilakis and Johnson, 2001). The distal openings, or pores, in these vacuoles are a second feature of the inner wall endothelium (Johnson, 2006). The majority of these pores are transcellular. The transcellular pores do not connect the extracellular fluid with the cellular cytoplasm. These pores usually form on giant vacuoles because it is in this region in which the cell is greatly attenuated and the cytoplasm becomes thin (Johnson, 2006). When cell thickness is reduced below a critical value, vascular endothelium can form transcellular pores involved in transport processes (Neal and Michel, 1996; Savla et al., 2002). The region immediately underlying the inner wall and basement membrane is the juxtacanalicular connective tissue having many large spaces filled with an ECM gel (Ethier et al., 1986; Ten Hulzen and Johnson, 1996). Furthermore, the ECM may generate significant aqueous outflow resistance (Bradley et al., 1998). Studies found that some inner wall pores may be artifacts of the fixation process (Sit et al., 1997; Ethier et al., 1998). Actually, TM pores contribute only 10% of the aqueous outflow resistance (Sit et al., 1997). As stated by Johnson, ‘‘a fundamental reassessment of the mechanism by which AH crosses the inner wall endothelium is necessary’’ (Johnson et al., 2002). The concept of the outflow system as a passive filter has been surpassed; the structures through which AH leaves the anterior chamber may be objects of physical or pharmacologic manipulation for therapeutic