Oxidants in Corneal Diseases

Figure 1 Histological section of the cornea, showing the epithelium (top), the stroma (middle) and the monolayer endothelium (bottom). See text for details.

most of its nutrients are derived from the aqueous humor. On the contrary, the major part of the cornea’s oxygen supply is directly derived from the air. This rather odd route of oxygenation is more or less unique to the cornea, and is naturally associated with the avascularity of the cornea. As a practical consequence of its oxygenation route, the oxygen tension in the superficial cornea is higher when the eye is open, but is significantly reduced when the eye is closed during sleep (the local oxygen tension of the superficial cornea may in fact vary as much as three-fold over a 24-hour period).4 The cornea consists of three layers, separated by basal laminae and acellular layers: an epithelium, a stroma, and a monolayer endothelium (Fig. 1). These layers are separately described below.

The Corneal Epithelium

The epithelium of the cornea is a squamos epithelium with 4–6 cell layers, which makes up about 10% of the total corneal thickness. The germinative capacity of the epithelium is found in the columnar basal cells. Bowman’s layer is a 10mm thick amorphous layer, which separates the epithelium from the stroma. The epithelial cells continuously regenerate, and they move gradually from Bowman’s layer towards the surface, while undergoing a transition to squamous superficial cells, which in turn undergo continuous apoptosis, cellular disintegration and desquamation, not unlike the superficial cells of the skin. Simultaneously, the epithelial cells move from the periphery of the cornea towards the corneal center.5,6 The stem cells of the corneal epithelium are located in deep crypts at the corneoscleral transition (the limbus),7 and the regenerative capacity of the corneal epithelium is virtually unlimited under normal conditions. These cells are capable

of creating a whole new corneal epithelium after larger injuries or surgery.8 The flat superficial epithelial cells form a watertight layer that prevents water from entering the stroma, which is essential for corneal transparency.2 Also, it provides a smooth, even refractive surface, and a mechanical protection against invading microorganisms. The epithelial thickness and morphology may be influenced by environmental factors, and interestingly from an oxidative perspective, the epithelial thickness increases in response to light exposure.9

ROS can be generated within the corneal epithelium by multiple mechanisms, including photochemical and inflammatory processes.10 Also, xanthine oxidase, an enzyme known to generate ROS, is present in the corneal epithelium.11 There are many examples of situations where the influence of ROS affects the integrity and normal function of the corneal epithelium. For example, the process of epithelial wound healing is slower when oxidative processes are involved, such as in diabetes mellitus. Healing of corneal epithelial wounds can be accelerated by addition of antioxidants, such as

trolox,12 Vitamin E,13,14 Vitamin C (ascorbic acid)15 or superoxide dismutase derivates.10,16,17

The Corneal Stroma

The corneal stroma constitutes 90% of the corneal thickness in humans and is mainly made of stacked lamellae of collagen fibrils. Especially in the posterior stroma, these lamellae are arranged in a highly precise and regular manner. Between the lamellae are the keratocytes, cells which maintain the stoma by synthesizing collagen and an extracellular matrix of glycosaminoglycans (GAGs), mainly keratan sulphate (KS) and chondroitin sulphate/dermatan sulphate (DS).2 Oxygen tension apparently has a role in regulating the synthesis of the GAGs.18 Accordingly, the keratocytes synthesize more DS and less KS in the anterior stroma, where the oxygen tension is higher.4,19 The polyanionic GAGs are essential to keep the lamellae in the regular arrangement with a constant distance between them, which, in turn, is essential for corneal transparency. The

alterations in the composition of GAGs with a decreased KS/DS ratio and appearance of other GAGs like heparan sulphate in corneal scar tissue20–22 and in

corneal healing processes23 may contribute to the reduced transparency of a corneal scar. Also in deeper wounds, involving the corneal stroma, beneficial

effects on the healing can be seen with antioxidants13 and superoxide dismutase derivates.10,16,17

The Corneal Endothelium

The corneal endothelium is a 5 mm thick monolayer of flat, uniform, hexagonal cells covering the entire inside of the cornea,2,24–26 the endothelium rests on a

5–10 mm thick basement membrane, the Descemet’s membrane, which in turn is loosely attached to the stroma. The hexagon is the ‘‘roundest’’ of the three

geometrical figures that can cover a plane, which explains the hexagonal shape of the endothelial cells. Uniform hexagonal cells means that the endothelium is

‘‘at rest’’, and in a state of minimal stress.27 The endothelial cells dehydrate the corneal stroma by pumping fluid continuously from the stroma.2,28,29 Keeping the

stroma dehydrated is essential for preservation of the stromal lamellar geometry, and thereby for the visual function; a loss of corneal endothelial pump function will immediately cause corneal swelling, opacification of the stroma and loss of visual function.

The corneal endothelial cells increase in size, in humans from 200–250 mm2 at birth to 400–700 mm2 in adulthood. From a few years of age this is mainly explained by a continuous loss of corneal endothelial cells.2,24 Loss of endothelial cells in

humans is exclusively compensated for by sliding and thinning of adjacent cells to cover the defect.2,30–32 Mitosis may also play a role in lower mammals,25,31,33,34 but

corneal endothelial cells are essentially amitotic under resting conditions.35 Therefore, a gradual enlargement of cells is seen with age in many species.36

The normal enlargement of cells can accelerate under different stress conditions, such as intraocular surgery,25,37,38 endothelial wounds,29,39 ocular40–42 and

systemic diseases such as diabetes.43 Oxidative stress has been shown to cause corneal endothelial cell death by apoptosis or necrosis,44,45 and ROS generated from ultrasonic energy may be a major mechanism behind corneal endothelial cell damage in phacoemulsification cataract surgery46 (see below).

EXAMPLES OF IMPORTANT CORNEAL ANTIOXIDANTS

The Superoxide Dismutases

Superoxide dismutases (SOD) generally catalyze the reaction 2 O2 + 2 H+ O2 + H2O2.

SOD comprises the main enzymatic system for O2 scavenging, and is present in all higher organisms and most aerobic bacteria. There are three specific superoxide dismutases in higher organisms, each confined to its own compartment in cells and tissues: the cytosolic Copper-Zinc-containing SOD (SOD1),47 the mitochondrial Manganese-containing SOD (SOD2),48 and the Extracellular SOD (SOD3).49

The cornea contains unusually large amounts of SOD3, among the highest levels measured in the human body, and close to that of SOD1. The cornea also has a relatively high SOD2 activity, just below that of the other two isoenzymes. SOD3 shows an uneven distribution within the human cornea, with significantly lower contents in the central cornea than in the periphery, and immunohistochemically lower contents in the anterior, than in the posterior stroma (Fig. 2A). The corneal epithelium is rich in SOD3, which indicates a high synthesis of SOD3 in the epithelial cells, given the high turnover rate of these cells (Fig. 2B). The epithelium is also rich in SOD1, localized in the cytosol of the epithelial cells (Fig. 2C).

Figure 2 (See color insert.) Immunohistochemical staining for SOD3 in the human cornea. A: Note a pronounced staining of the cell borders in the epithelium, and a stromal staining which is interleaved between the stromal collagen lamellae. The stromal staining is slightly weaker in the anterior, than in the posterior stroma. B: Detail of immunohistochemical staining for SOD3 in the human corneal epithelium. Note intense staining of the cell borders and intercellular space. C: Staining for SOD1 in the human corneal epithelium. Note the staining of the cytosol and nuclei.

Ascorbic Acid

Ascorbic acid (Vitamin C) is hydrophilic and acts as a reducing agent, which may sometimes be of benefit and sometimes not. It reacts rapidly with ROS, such as O2 and OH , to give the less reactive semidehydroascorbic acid, but oxidation of ascorbic acid in the presence of certain transition metal ions, especially Cu2+ can also produce both H2O2 and OH .50 Ascorbic acid has long been known to exert special protective functions in certain tissues and fluids. It is accumulated in very high concentrations (10–100x the concentrations in serum9) in, for example, the lens, the aqueous humor, and the cornea of the eye. There is at least indirect evidence to support that the role of ascorbate in the anterior part of the

eye has to do with protection from light-induced damage, and that ascorbate acts as a filter for ultraviolet light in the eye.9,51,52 In diurnal species, including

humans, which are exposed to high levels of light, there are high concentrations of ascorbic acid present in these tissues, as opposed to in nocturnal species.52–54 The distribution of ascorbic acid in the cornea is just as interesting as that of SOD3. The concentrations in the corneal endothelium and stroma approximately

equal those in the aqueous humor and the lens, but the concentrations in the corneal epithelium are about 6-fold higher.9,52,55 The epithelial concentrations

also vary with the degree of light exposure in the same species,9 and within the epithelium in the same individual, with higher concentrations in the centre, over the pupil area.55

When evaluating the radical protective properties of ascorbate it is important to remember that they may be situation-dependent (as mentioned, ascorbate can have opposite effects under certain conditions), and also that ascorbate is consumed (oxidized) when scavenging radicals, and needs to be regenerated. In the anterior part of the eye, however, this should be a minor problem, since the aqueous humor has a rather high turnover rate, with an exchange of several percent of its volume each minute.

among family members of KC patients. The exact genetic mechanisms underlying

the disease are current under investigation,61 but may vary among cases.58

KC corneas show a decreased KS/DS ratio (see above),22,62,63 but this may be secondary to the thinning or the scarring of advanced forms of the disease. There is also a degradation of the extracellular matrix of the superficial stroma with elevated degradative enzymes64,65 and wound-healing and stress-related proteins66,67 as well as altered proteinase inhibitors67–69 in KC. These biochemical changes may be initiated by keratocyte apoptosis mediated by the interleukin-1 system.70–72 Indeed, apoptosis of anterior stromal keratocytes and basal epithelial cells is found in KC.71

Various types of stress, including mechanical and oxidative stress, to the superficial cornea can induce apoptosis of these cells.71,73,74 Kenney et al. have suggested a

working hypothesis for KC pathogenesis, with formation of peroxynitrite (ONOO ) from O2 and nitric oxide (NO)72 as an initiating factor causing the keratocytes to

undergo apoptosis, and subsequent studies have provided further support for oxidative stress as a causative factor behind KC.1,75,76 KC corneas show immunohis-

tochemical staining for both nitrotyrosine and malondialdehyde, markers of ONOO and lipid peroxidation, respectively,1 an up-regulation of catalase,76 and increased degradative enzymes.76 Indeed, a spatial relationship is seen between nitrotyrosine, the nitric oxide synthetase NOS III, and fibrosis, which is interpreted

as an insufficient superoxide radical processing capacity, resulting in ONOO formation, in the KC cornea .1,72 NO has a variety of functions in the eye,77 and is

synthesized by keratocytes under stress conditions.78,79 The chain reaction thereafter, eventually resulting in resorption of collagen with stromal thinning, may reflect an unspecific corneal reaction pattern under such circumstances, analogous to the local resorption of corneal stroma seen clinically after, for example, corneal trauma and infectious processes.

Our group has demonstrated that the levels of Extracellular Superoxide Dismutase (SOD3) in KC are significantly reduced, to about half of the levels

in normal central cornea, whereas the other two SOD isoenzymes, SOD1 and SOD2, are unaltered.80,81 It is striking that the earliest changes in KC occur in

the anterior stroma, where the levels of SOD3 are the lowest, also in the normal cornea (Fig. 2A). Subsequently, Kenney’s group have demonstrated that the basal expression of SOD3 on the mRNA level in the KC cornea does not differ from that in the normal cornea,76 but alterations in the corneal SOD3 expression pattern globally or locally, or in response to cytokines, oxidative stress or trauma, may still be altered in the KC cornea. In conclusion, there is growing evidence that the KC cornea is unable to handle superoxide radicals in a normal manner,76,81 and that oxidative stress is an important factor in KC pathogenesis.

Bullous Keratopathy/Fuchs Endothelial Dystrophy

These two conditions have resemblances, but distinctly different pathogenetic backgrounds. The common feature of these two corneal diseases is the corneal

Figure 4 Specular microscopy photographs of the human corneal endothelium in beginning bullous keratopathy (A). Note the enlarged endothelial cells. In B, a normal endothelium is shown, for comparison. C: Fuchs endothelial dystrophy. Note the fibrous patches, appearing dark in the picture, clinically known as guttae. Between the guttae, the endothelial morphology may appear rather normal.

edema, which occurs because of a failure of the corneal endothelial cells to remove fluid from the stroma. Corneal edema is painful and sight threatening, and together, these two diagnoses comprise the majority of corneal transplantation cases. In bullous keratopathy, the endothelium is damaged by external forces (mainly surgical procedures), whereas in Fuchs endothelial dystrophy, the etiology is largely unknown. Sometimes, there may have been a mild, subclinical form of Fuchs prior to a surgical procedure, which has contributed to the subsequent development of edema. In other words, mixed forms of the two diseases may occur, and there is likely some uncertainty regarding the clinical diagnosis in a portion of the cases. In typical cases, however, the endothelial morphology differs between the two diseases (Fig. 4A–B), which indicates that the conditions may also differ in pathogenesis and/or biochemical changes.

The density of corneal endothelial cells decreases continuously with time due to cell loss.2,24 In man, the cell densities decrease from 3500–4000 cells

mm 2 at birth to 1400–2500 cells mm 2 in adulthood. In man, and other higher

mammals, the loss of endothelial cells is compensated for only by sliding and thinning of adjacent cells to cover the defect,2,30–32 but mitosis may also play a role in lower mammals25,31,33–35 Even so, a gradual enlargement of cells is seen

with age is seen also in lower mammals.36 The normal enlargement of corneal endothelial cells seen with time can be accelerated in various stress conditions, and then often in combination with a deviation from the uniform hexagonal cellular pattern normally seen.24–26 Examples of such stress conditions include

endothelial wounds,29,39 systemic43 or ocular diseases,40 including uveitis40–42 and intraocular surgery.25,37,38 There is much evidence to support that oxidative

stress is a major factor behind corneal endothelial cell loss.

Figure 5 The corneal endothelium of SOD3 null mice shows an accelerated spontaneous cell loss with age (A), which is further accentuated after an LPS-induced uveitis (B).

Corneal endothelial cell death can be induced by hydrogen peroxide perfusion of the anterior chamber in rabbits.45 In Fuchs’ endothelial dystrophy, an accelerated age-dependent endothelial cell loss with increased apoptosis of the cells is seen.82 Photooxidative injury with formation of ROS has been shown to induce corneal endothelial cell apoptosis in animal models,83,84 and may also be a mechanism underlying cell loss in Fuchs’dystrophy. Our group has demonstrated that mice lacking SOD3 have an accelerated loss of corneal endothelial cells with an otherwise largely preserved morphology in normal ageing (Fig. 5A), a finding which strongly indicates that superoxide radicals and oxidative stress contributes to the age-dependent corneal endothelial cell loss. Reduced scavenging of O2 generated by photooxidation with subsequently increased apoptosis of endothelial cells may be a mechanism behind the increased cell loss seen in the SOD3 null mouse strain.

Interestingly, recent investigations have demonstrated that the formation of ROS and the oxidative tissue injury differs between Fuchs’ endothelial dystrophy and bullous keratopathy. Bullous keratopathy corneas predominantly accumulate byproducts of lipid peroxidation, whereas in Fuchs’ dystrophy corneas, signs of peroxynitrite formation dominate.1 These findings strongly suggest that these two diseases differ from a pathogenetic and oxidative point of view.

Loss of Corneal Endothelial Cells in Inflammatory Eye Diseases

In an acute or chronic inflammation of the anterior segment of the eye, the corneal endothelium always suffers some degree of injury. As opposed to in

normal ageing, altered cell morphology, cell elongation and pleomorphism are more pronounced features in inflammations.25,29,31,37,39 A prominent feature of a

uveitis is the invasion of polymorphonuclear leucocytes, known to generate both O2 and NO.85,86 Endotoxin-induced uveitis, with administration of lipopolysaccaride (LPS) systemically87 or intravitreally88 is often employed in models to study endothelial viability and regenerative capacity in vivo in inflammatory

loss is almost invariably seen after phacoemulsification, and many investigations indicate that ROS generation is involved in this process, as opposed to in the previous extracapsular technique, where the endothelial damage may have been more mechanical.92 With the phacoemulsification technique, there is a strong correlation between the reversible decrease in corneal endothelial cell function the day after surgery, and the irreversible cell loss seen,93 a finding which aligns well with an oxidative mode of endothelial injury (Fig. 6). The endothelial damage induced by ultrasonic energy in phacoemulsification cataract surgery can be diminished by the addition of SOD91 or hyaluronate, acting in this concept as a ROS scavenger.46 In addition, Rubowitz et al have elegantly demonstrated that addition of ascorbic acid to the irrigation solution can reduce the corneal endothelial cell loss in a rabbit model of phacoemulsification surgery with as much as 70%,94 a finding which indicates that oxidative mechanisms likely play the main role in this particular cell damage.

REFERENCES

1.Buddi R, Lin B, Atilano SR, et al. Evidence of oxidative stress in human corneal diseases. J Histochem Cytochem 2002; 50:341–351.

2.Klyce S, Beuerman R. Structure and function of the cornea. In: Kaufman H, Barron B, McDonald M, eds. The Cornea. 2nd ed. Boston: Butterworth-Heinemann, 1998:3–50.

3.Ringvold A. Cornea and ultraviolet radiation. Acta Ophthalmol (Copenh) 1980; 58:63–68.

4.Fatt I, Bieber MT. The steady-state distribution of oxygen and carbon dioxide in the in vivo cornea. I. The open eye in air and the closed eye. Exp Eye Res 1968; 7:103–112.

5.Thoft RA, Friend J. The X, Y, Z hypothesis of corneal epithelial maintenance [letter]. Invest Ophthalmol Vis Sci 1983; 24:1442–1443.

6.Buck RC. Measurement of centripetal migration of normal corneal epithelial cells in the mouse. Invest Ophthalmol Vis Sci 1985; 26:1296–1299.

7.Tseng SC. Regulation and clinical implications of corneal epithelial stem cells. Mol Biol Rep 1996; 23:47–58.

8.Coster D. Surgical procedures to restore the corneal epithelium. Kaufman HE, Baron BA, McDonald MB, eds. The Cornea. 2nd ed. Boston: Butterworth-Heinemann, 1998:715–726.

9.Ringvold A, Anderssen E, Kjonniksen I. Impact of the environment on the mammalian corneal epithelium. Invest Ophthalmol Vis Sci 2003; 44:10–15.

10.Ando E, Ando Y, Inoue M, et al. Inhibition of corneal inflammation by an acylated superoxide dismutase derivative. Invest Ophthalmol Vis Sci 1990; 31:1963–1967.

11.Cejkova J, Ardan T, Filipec M, et al. Xanthine oxidoreductase and xanthine oxidase in human cornea. Histol Histopathol 2002; 17:755–760.

12.Hallberg CK, Trocme SD, Ansari NH. Acceleration of corneal wound healing in diabetic rats by the antioxidant trolox. Res Commun Mol Pathol Pharmacol 1996; 93:3–12.

13.Vetrugno M, Maino A, Cardia G, et al. A randomised, double masked, clinical trial of high dose vitamin A and vitamin E supplementation after photorefractive keratectomy. Br J Ophthalmol 2001; 85:537–539.

14.Bilgihan K, Ozdek S, Ozogul C, et al. Topical vitamin E and hydrocortisone acetate treatment after photorefractive keratectomy. Eye 2000; 14(Pt 2):231–237.

15.Saika S, Uenoyama K, Hiroi K, et al. Ascorbic acid phosphate ester and wound healing in rabbit corneal alkali burns: epithelial basement membrane and stroma. Graefes Arch Clin Exp Ophthalmol 1993; 231:221–227.

16.Alio JL, Artola A, Serra A, et al. Effect of topical antioxidant therapy on experimental infectious keratitis. Cornea 1995; 14:175–179.

17.Matsumoto K, Shimmura S, Goto E, et al. Lecithin-bound superoxide dismutase in the prevention of neutrophil-induced damage of corneal tissue. Invest Ophthalmol Vis Sci 1998; 39:30–35.

18.Scott JE, Haigh M. Keratan sulphate and the ultrastructure of cornea and cartilage: a ‘stand-in’ for chondroitin sulphate in conditions of oxygen lack? J Anat 1988; 158:95–108.

19.Kwan M, Niinikoski J, Hunt TK. In vivo measurements of oxygen tension in the cornea, aqueous humor, and anterior lens of the open eye. Invest Ophthalmol 1972; 11:108–114.

20.Cintron C, Covington HI, Kublin CL. Morphologic analyses of proteoglycans in rabbit corneal scars. Invest Ophthalmol Vis Sci 1990; 31:1789–1798.

21.Funderburgh JL, Cintron C, Covington HI, et al. Immunoanalysis of keratan sulfate proteoglycan from corneal scars. Invest Ophthalmol Vis Sci 1988; 29:1116–1124.

22.Funderburgh JL, Chandler JW. Proteoglycans of rabbit corneas with nonperforating wounds. Invest Ophthalmol Vis Sci 1989; 30:435–442.

23.Goodman WM, SundarRaj N, Garone M, et al. Unique parameters in the healing of linear partial thickness penetrating corneal incisions in rabbit: immunohistochemical evaluation. Curr Eye Res 1989; 8:305–316.

24.Laing RA, Sanstrom MM, Berrospi AR, et al. Changes in the corneal endothelium as a function of age. Exp Eye Res 1976; 22:587–594.

25.Glasser DB, Matsuda M, Gager WE, et al. Corneal endothelial morphology after anterior chamber lens implantation. Arch Ophthalmol 1985; 103:1347–1349.

26.Yee RW, Edelhauser HF, Stern ME. Specular microscopy of vertebrate corneal endothelium: a comparative study. Exp Eye Res 1987; 44:703–714.

27.Collin HB, Grabsch BE. The effect of ophthalmic preservatives on the shape of corneal endothelial cells. Acta Ophthalmol (Copenh) 1982; 60:93–105.

28.Stiemke MM, Edelhauser HF, Geroski DH. The developing corneal endothelium: correlation of morphology, hydration and Na/K ATPase pump site density. Curr Eye Res 1991; 10:145–156.

29.Yee RW, Geroski DH, Matsuda M, et al. Correlation of corneal endothelial pump site density, barrier function, and morphology in wound repair. Invest Ophthalmol Vis Sci 1985; 26:1191–1201.

30.Chung JH, Fagerholm P. Corneal alkali wound healing in the monkey. Acta Ophthalmol (Copenh) 1989; 67:685–693.

31.Van Horn DL, Sendele DD, Seideman S, et al. Regenerative capacity of the corneal endothelium in rabbit and cat. Invest Ophthalmol Vis Sci 1977; 16:597–613.

32.Gwin RM, Lerner I, Warren JK, et al. Decrease in canine corneal endothelial cell density and increase in corneal thickness as functions of age. Invest Ophthalmol Vis Sci 1982; 22:267–271.

33.Chung JH, Fagerholm P. Endothelial healing in rabbit corneal alkali wounds. Acta Ophthalmol (Copenh) 1987; 65:648–656.

34.Olsen EG, Davanger M. The healing of rabbit corneal endothelium. Acta Ophthalmol (Copenh) 1984; 62:796–807.

35.Gordon SR, Rothstein H, Harding CV. Studies on corneal endothelial growth and repair. IV. Changes in the surface during cell division as revealed by scanning electron microscopy. Eur J Cell Biol 1983; 31:26–33.

36.Fitch KL, Nadakavukaren MJ. Age-related changes in the corneal endothelium of the mouse. Exp Gerontol 1986; 21:31–35.

37.Matsuda M, Suda T, Manabe R. Serial alterations in endothelial cell shape and pattern after intraocular surgery. Am J Ophthalmol 1984; 98:313–319.

38.Olsen T. Variations in endothelial morphology of normal corneas and after cataract extraction. A specular microscopic study. Acta Ophthalmol (Copenh) 1979; 57:1014–1019.

39.Landshman N, Solomon A, Belkin M. Cell division in the healing of the corneal endothelium of cats. Arch Ophthalmo 1989; 107:1804–1808.

40.Olsen T. Changes in the corneal endothelium after acute anterior uveitis as seen with the specular microscope. Acta Ophthalmol (Copenh) 1980; 58:250–256.

41.Setala K. Corneal endothelial cell density in iridocyclitis. Acta Ophthalmol (Copenh) 1979; 57:277–286.

42.Brooks AM, Gillies WE. Fluorescein angiography of the iris and specular microscopy of the corneal endothelium in some cases of glaucoma secondary to chronic cyclitis. Ophthalmology 1988; 95:1624–1630.

43.Schultz RO, Matsuda M, Yee RW, et al. Corneal endothelial changes in type I and type II diabetes mellitus. Am J Ophthalmol 1984; 98:401–410.

44.Cho KS, Lee EH, Choi JS, et al. Reactive oxygen species-induced apoptosis and necrosis in bovine corneal endothelial cells. Invest Ophthalmol Vis Sci 1999; 40:911–919.

45.Hull DS, Green K. Oxygen free radicals and corneal endothelium. Lens Eye Toxic Res 1989; 6:87–91.

46.Takahashi H. Free radical development in phacoemulsification cataract surgery. J Nippon Med Sch 2005; 72:4–12.

47.McCord JM, Fridovich I. Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J Biol Chem 1969; 244:6049–6055.

48.Weisiger RA, Fridovich I. Mitochondrial superoxide simutase: site of synthesis and intramitochondrial localization. J Biol Chem 1973; 248:4793–4796.

49.Marklund SL. Human copper-containing superoxide dismutase of high molecular weight. Proc Natl Acad Sci U S A 1982; 79:7634–7638.

50.Wolff SP, Wang GM, Spector A. Pro-oxidant activation of ocular reductants. 1. Copper and riboflavin stimulate ascorbate oxidation causing lens epithelial cytotoxicity in vitro. Exp Eye Res 1987; 45:777–789.

51.Ringvold A. Corneal epithelium and UV-protection of the eye. Acta Ophthalmol Scand 1998; 76:149–153.

52.Ringvold A, Anderssen E, Kjonniksen I. Ascorbate in the corneal epithelium of diurnal and nocturnal species. Invest Ophthalmol Vis Sci 1998; 39: 2774–2777.

53.Reddy VN, Giblin FJ, Lin LR, et al. The effect of aqueous humor ascorbate on ultraviolet-B-induced DNA damage in lens epithelium. Invest Ophthalmol Vis Sci 1998; 39:344–350.

54.Reiss GR, Werness PG, Zollman PE, et al. Ascorbic acid levels in the aqueous humor of nocturnal and diurnal mammals. Arch Ophthalmol 1986; 104: 753–755.

55.Ringvold A, Anderssen E, Kjonniksen I. Distribution of ascorbate in the anterior bovine eye. Invest Ophthalmol Vis Sci 2000; 41:20–23.

56.Taylor A, Jacques PF, Nadler D, et al. Relationship in humans between ascorbic acid consumption and levels of total and reduced ascorbic acid in lens, aqueous humor, and plasma. Curr Eye Res 1991; 10:751–759.

57.Taylor A, Jahngen-Hodge J, Smith DE, et al. Dietary restriction delays cataract and reduces ascorbate levels in Emory mice. Exp Eye Res 1995; 61:55–62.

58.Marguire L. Ectatic corneal degenerations. In: Kaufman H, Barron BA, McDonald MB, eds. The Cornea. 2nd ed. Boston: Butterworth-Heinemann, 1998:525–538.

59.Rao SN, Raviv T, Majmudar PA, et al. Role of Orbscan II in screening keratoconus suspects before refractive corneal surgery. Ophthalmology 2002; 109: 1642–1646.

60.Auffarth GU, Wang L, Volcker HE. Keratoconus evaluation using the Orbscan Topography System. J Cataract Refract Surg 2000; 26:222–228.

61.Rabinowitz YS, Dong L, Wistow G. Gene expression profile studies of human keratoconus cornea for NEIBank: a novel cornea-expressed gene and the absence of transcripts for aquaporin 5. Invest Ophthalmol Vis Sci 2005; 46:1239–1246.

62.Funderburgh JL, Funderburgh ML, Rodrigues MM, et al. Altered antigenicity of keratan sulfate proteoglycan in selected corneal diseases. Invest Ophthalmol Vis Sci 1990; 31:419–428.

63.Sawaguchi S, Yue BY, Chang I, et al. Proteoglycan molecules in keratoconus corneas. Invest Ophthalmol Vis Sci 1991; 32:1846–1853.

64.Kenney MC, Nesburn AB, Burgeson RE, et al. Abnormalities of the extracellular matrix in keratoconus corneas. Cornea 1997; 16:345–351.

65.Sawaguchi S, Yue BY, Sugar J, et al. Lysosomal enzyme abnormalities in keratoconus. Arch Ophthalmol 1989; 107:1507–1510.

66.Zhou L, Yue BY, Twining SS, et al. Expression of wound healing and stress-related proteins in keratoconus corneas. Curr Eye Res 1996; 15:1124–1131.

67.Kenney MC, Chwa M, Alba A, et al. Localization of TIMP-1, TIMP-2, TIMP-3, gelatinase A and gelatinase B in pathological human corneas. Curr Eye Res 1998; 17:238–246.

68.Sawaguchi S, Twining SS, Yue BY, et al. Alpha-1 proteinase inhibitor levels in keratoconus. Exp Eye Res 1990; 50:549–554.

69.Whitelock RB, Fukuchi T, Zhou L, et al. Cathepsin G, acid phosphatase, and alpha 1-proteinase inhibitor messenger RNA levels in keratoconus corneas. Invest Ophthalmol Vis Sci 1997; 38:529–534.

70.Wilson SE, He YG, Weng J, et al. Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res 1996; 62:325–327.

71.Kim WJ, Helena MC, Mohan RR, et al. Changes in corneal morphology associated with chronic epithelial injury. Invest Ophthalmol Vis Sci 1999; 40:35–42.

72.Connon CJ, Meek KM, Newton RH, et al. Hyaluronidase treatment, collagen fibril packing, and normal transparency in rabbit corneas. J Refract Surg 2000; 16:448–455.

73.Trinkaus-Randall V, Leibowitz HM, Ryan WJ, et al. Quantification of stromal destruction in the inflamed cornea. Invest Ophthalmol Vis Sci 1991; 32:603–609.

74.Helena MC, Baerveldt F, Kim WJ, et al. Keratocyte apoptosis after corneal surgery. Invest Ophthalmol Vis Sci 1998; 39:276–283.

75.Brown DJ, Lin B, Chwa M, et al. Elements of the nitric oxide pathway can degrade TIMP-1 and increase gelatinase activity. Mol Vis 2004; 10:281–288.

76.Kenney MC, Chwa M, Atilano SR, et al. Increased levels of catalase and cathepsin V/L2 but decreased TIMP-1 in keratoconus corneas: evidence that oxidative stress plays a role in this disorder. Invest Ophthalmol Vis Sci 2005; 46:823–832.

77.Becquet F, Courtois Y, Goureau O. Nitric oxide in the eye: multifaceted roles and diverse outcomes. Surv Ophthalmol 1997; 42:71–82.

78.Dighiero P, Behar-Cohen F, Courtois Y, et al. Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation. Invest Ophthalmol Vis Sci 1997; 38:2045–2052.

79.Sennlaub F, Courtois Y, Goureau O. Nitric oxide synthase-II is expressed in severe corneal alkali burns and inhibits neovascularization. Invest Ophthalmol Vis Sci 1999; 40:2773–2779.

80.Behndig A, Svensson B, Marklund SL, et al. Superoxide dismutase isoenzymes in the human eye. Invest Ophthalmol Vis Sci 1998; 39:471–475.

81.Behndig A, Karlsson K, Johansson BO, et al. Superoxide dismutase isoenzymes in the normal and diseased human cornea. Invest Ophthalmol Vis Sci 2001; 42:2293–2296.

82.Borderie VM, Baudrimont M, Vallee A, et al. Corneal endothelial cell apoptosis in patients with Fuchs’ dystrophy. Invest Ophthalmol Vis Sci 2000; 41:2501–2505.

83.Ashok BT, Ali R. The aging paradox: free radical theory of aging. Exp Gerontol 1999; 34:293–303.

84.Podskochy A, Gan L, Fagerholm P. Apoptosis in UV-exposed rabbit corneas. Cornea 2000; 19:99–103.

85.Ishimoto S, Wu GS, Hayashi S, et al. Free radical tissue damages in the anterior segment of the eye in experimental autoimmune uveitis. Invest Ophthalmol Vis Sci 1996; 37:630–636.

86.Parks DJ, Cheung MK, Chan CC, et al. The role of nitric oxide in uveitis. Arch Ophthalmol 1994; 112:544–546.

87.Rosenbaum JT, McDevitt HO, Guss RB, et al. Endotoxin-induced uveitis in rats as a model for human disease. Nature 1980; 286:611–613.

88.Ohta K, Norose K, Wang XC, et al. Apoptosis-related fas antigen on memory T cells in aqueous humor of uveitis patients. Curr Eye Res 1996; 15:299–306.

89.Behar-Cohen FF, Savoldelli M, Parel JM, et al. Reduction of corneal edema in endotoxin-induced uveitis after application of L-NAME as nitric oxide synthase inhibitor in rats by iontophoresis. Invest Ophthalmol Vis Sci 1998; 39:897–904.

90.Behndig A, Karlsson K, Brannstrom T, et al. Corneal endothelial integrity in mice lacking extracellular superoxide dismutase. Invest Ophthalmol Vis Sci 2001; 42:2784–2788.

91.Holst A, Rolfsen W, Svensson B, et al. Formation of free radicals during phacoemulsification. Curr Eye Res 1993; 12:359–365.

92.Bourne RR, Minassian DC, Dart JK, et al. Effect of cataract surgery on the corneal endothelium: modern phacoemulsification compared with extracapsular cataract surgery. Ophthalmology 2004; 111:679–685.

93.Lundberg B, Jonsson M, Behndig A. Postoperative corneal swelling correlates strongly to corneal endothelial cell loss after phacoemulsification cataract surgery. Am J Ophthalmol 2005; 139:1035–1041.

94.Rubowitz A, Assia EI, Rosner M, et al. Antioxidant protection against corneal damage by free radicals during phacoemulsification. Invest Ophthalmol Vis Sci 2003; 44:1866–1870.

Figure 1 Oxidative stress is imposed on cells because of an increase in oxidant generation (i.e reactive oxygen species or ROS), a decrease in endogenous antioxidant protection or a failure to repair oxidant damage.

altered perfusion occurring when IOP is moderately elevated. Compelling evidence therefore exists to show that raised IOP can be the cause of visual loss in glaucoma. This is supported by the finding that the lowering IOP is often linked with the prevention of visual loss. On the other hand a substantial number of glaucoma patients do not have raised IOP and often lowering of elevated IOP does not result in the prevention of visual loss. Moreover, not all ocular hypertensive patients have glaucoma.

POSSIBLE CAUSES FOR GANGLION CELL DEATH IN GLAUCOMA

It is now clear that the cause of ganglion cell death in glaucoma is not solely due to raised IOP and it has been hypothesised that a number of risk factors (one of which includes raised IOP) induce glaucoma or loss of ganglion cell function.2 The common aspect associated with all the putative risk factors is that they are proposed to cause an inadequate blood delivery to the components in the optic nerve head region.3 The following have been suggested to be risk factors in glaucoma: fluctuation of IOP, ageing, family history, severe myopia, central cornea thickness, hypertension, hypotension, vasospasm, hemorheology, immune system, diabetes mellitus, sleep disturbances, family history and light (Figure 2). It is likely that a combination of these risk factors is necessary to cause glaucoma. This might also explain why not all ocular hypertensive patients have glaucoma.

Inadequate blood delivery to the optic nerve head region will result in ischemic/hypoxic insults being delivered to the components in the region. These will include retinal ganglion cell axons, astrocytes, microglia and the lamina cribosa. Since oxidative stress is intricately associated with ischemia4 it follows that oxidative stress is likely to play a major role in the pathogenesis of glaucoma.

Figure 2 Likely causes for the initiation for loss of vision in different glaucoma patients. Any insult alone or in combination with other insults all result in a similar pathogenesis of ganglion cell death initiated by common influences on the optic nerve head components.

We have hypothesised5,6 that the initial ischemic/hypoxic insults to the ganglion cell axons does not result in the neurones dying but rather forces them to survive at a lower energetic status and in the process making them more susceptible to any additional insults. We have also suggested that this will ultimately occur because of altered glial function (astrocytes, Mu¨ller cells, microglia), originating from ischemia to the optic nerve head region. This is based partly on experimental studies which have shown that a variety of toxic substances (glutamate, TNF-a, serine, nitric oxide, potassium) become elevated in the extracellular retinal spaces when retinal glial cell function is affected. Elevation of such substances will particularly affect the survival of retinal ganglion cells because they are energetically compromised. Moreover, they will affect ganglion cells differentially depending partly on the nature of their receptors. As a consequence, ganglion cell death will occur at varying times as it does in glaucoma.

Recent observations have made it necessary to modify this hypothesis because of the realisation that ganglion cell axons within the globe contain many mitochondria7 and that light can interact with mitochondrial enzymes to generate ROS.8 It is established that light can act on the mitochondrial photosensitizers, cytochrome and flavin-containing oxidases to generate ROS.8 It is therefore proposed that the secondary insults to initiate apoptosis to energetically compromised ganglion cells in glaucoma can also be mediated by light effects upon their many axonal mitochondria (Figure 3). It should be emphasised that light is probably not a risk factor to healthy ganglion cells where their mitochondria are likely to be able to scavenge all ROS produced in metabolism or because of light. However, in glaucoma the ganglion cells are proposed to exist initially at a compromised energetic state and only at this stage become prone to elevation of ROS caused by light.

Figure 3 Hypothesis for differential ganglion cell apoptosis caused by variable or sustained changes in the normal blood supply to the optic nerve head.

RETINAL GANGLION CELL AXONS, MITOCHONDRIA, AND OXIDATIVE STRESS

Mitochondria are the seat of a number of important cellular functions, including essential pathways of intermediate metabolism, amino acid biosynthesis, fatty acid oxidation, steroid metabolism, and apoptosis. Of key importance is the role of mitochondria in energy metabolism. Oxidative phosphorylation generates most of the cell’s ATP, and any impairment of the organelle’s ability to produce energy can have catastrophic consequences, not only due to primary loss of ATP, but also due to impairment of ‘‘downstream’’ functions, such as maintenance of organelle and cellular calcium homeostasis. Moreover, deficient mitochondrial metabolism may generate ROS that can wreak havoc in the cell because of oxidative stress. It is for such reasons that it is believed that mitochondrial dysfunction leads to apoptosis.

Retinal ganglion cell axons within the globe are laden with mitochondria.7 The abundance of mitochondria is thought to satisfy the high energy requirements for nerve transmission within unmyelinated axons, compared with the lower amount required for salutatory conduction in the myelinated axons of the optic nerve, including the laminar and prelaminar portions of the optic nerve head.7 The abundance of mitochondria within the intraretinal retinal ganglion cell axons makes them particularly vulnerable to ischemic/hypoxic insults and to the light that constantly impinging upon them. It is now established that light can act on the mitochondrial photosensitizers, cytochrome and flavin-containing

oxidases to generate ROS.8 The probability for mitochondrial dysfunction and oxidative stress being a primary cause for retinal ganglion cell death in glaucoma is therefore extremely high.

ANALYSIS OF PLASMA FOR THE INVOLVEMENT

OF OXIDATIVE STRESS IN POAG

Yildirin et al9 compared the plasma level of the malondialdehyde and the enzymes myeloperoxidase and catalase in 40 POAG patients with 60 healthy controls, and concluded that malondialdehyde was elevated in the glaucoma subjects. Malondialdehyde is a product generated during oxidative stress. Such data suggest that there is a reduced systemic capacity for POAG patients to oxidative stress. This is supported by a recent study which demonstrated a reduced plasma level of glutathione (GSH) in newly diagnosed POAG patients when compared to age matched controls.10 Circulating GSH is a very important enzyme involved in counteracting oxidative stress and might be reduced either by reduction in synthesis or by increased consumption due to oxidative stress. Altered metabolism of GSH could also be the cause as indicated by the work of Izzotti and collaborators.1,11 These authors analysed the glutathione S-transferase isoenzymes (GSTM1 and GSTM2) involved in the synthesis of GSH and found that the GSTM1-null genotype was more common in POAG patients. These studies therefore suggest that there is an association between low systemic antioxidative capacity and POAG.

OXIDATIVE STRESS AND RAISED IOP

Aqueous humour contains several active oxidative agents such as hydrogen peroxide and superoxide anion12 and a rise in their levels could affect, for example trabecular cell function. Indeed, laboratory studies have shown that trabecular cells are susceptible to hydrogen peroxide which alters their adhesion properties and compromises their cellular integrity.13 Moreover, studies on the isolated perfused eye show that hydrogen peroxide affects the drainage of aqueous so causing a raise in IOP.14 These experimental studies are consistent

with the hypothesis that trabecular cell malfunction might be caused by oxidative stress in glaucoma patients.15,16 Further support for this idea comes from studies

which reveal that oxidative DNA damage17 and the expression of endothelialleukocyte adhesion molecule (ELAM-1)18 are significantly elevated in trabecular cells of glaucoma patients compared with unaffected controls.

Both trabecular cells and aqueous humour contain a number of oxidative stress markers and an alteration in any of these can cause oxidative stress to the cells. There is some evidence that these oxidative stress markers are altered in the aqueous humour of glaucoma patients. For example, Ferreira et al19 found that the total antioxidant potential value in the aqueous humour of glaucoma patients is 64% less than that of a cataract group of patients. Moreover, aqueous

Figure 5 (See color insert.) A rise in extracellular glutamate and overactivation of glutamate ionotropic receptors leads to generation of ROS and cell death.

of ROS.6 Ischemia/hypoxia to the astrocytes is also likely to generate ROS to potentially influence ganglion cell function.22

It would appear therefore that there are very good reasons to suggest that increased extracellular and intracellular levels of ROS initiated by ischemia/ hypoxia to the optic nerve head region causes ganglion cells to die at a differential rate (Figure 6). Increased retinal ROS also affects glial function and possibly activates immune responses. Detailed molecular mechanisms of the real impact of oxidative stress on the development and progression of glaucomatous neurodegeneration, however, remains to be elucidated. A greater understanding may offer unique opportunities for neuroprotective intervention with appropriate antioxidants.

CONCLUSION

Good evidence exists to support the tenant that oxidative stress to the trabecular cells and retinal cells are instrumental in the eventual cause for ganglion cells dying in glaucoma (Figure 6). Logic therefore suggests that adjunct treatment of glaucoma patients with IOP lowering agents and suitable antioxidants would be worthy of consideration. Oral intake of powerful antioxidants like a-lipoic acid and/or vitamin E, which are well tolerated and will reach the retina, are possible candidates.

Figure 6 Oxidative stress may be involved in a number of events that are associated with the pathogenesis of glaucoma?

REFERENCES

1.Izzotti A, Bagnis A, Sacca SC. The role of oxidative stress in glaucoma. Mutat Res 2006; 612:105–114.

2.Pache M, Flammer J. A sick eye in a sick body? Systemic findings in patients with primary open-angle glaucoma. Surv Ophthalmol 2006; 51:179–212.

3.Osborne NN, Ugarte M, Chao M, et al. Neuroprotection in relation to retinal ischemia and relevance to glaucoma. Surv Ophthalmol 1999; 43 (suppl 1):S102–S128.

4.Osborne NN, Casson RJ, Wood JP, et al. Retinal ischemia: mechanisms of damage and potential therapeutic strategies. Prog Retin Eye Res 2004; 23:91–147.

5.Osborne NN, Ugarte M, Chao M, et al. Neuroprotection in relation to retinal ischemia and relevance to glaucoma. Surv Ophthalmol 1999; 43 (Suppl 1):S102–S128.

6.Osborne NN, Lascaratos G, Bron AJ, et al. A hypothesis to suggest that light is a risk factor in glaucoma and the mitochondrial optic neuropathies. Br J Ophthalmol 2006; 90:237–241.

7.Carelli V, Ross-Cisneros FN, Sadun AA. Mitochondrial dysfunction as a cause of optic neuropathies. Prog Retin Eye Res 2004; 23:53–89.

8.Godley BF, Shamsi FA, Liang FQ, et al. Blue light induces mitochondrial DNA damage and free radical production in epithelial cells. J Biol Chem 2005; 280:21061–21066.

9.Yildirim O, Ates NA, Ercan B, et al. Role of oxidative stress enzymes in open-angle glaucoma. Eye 2005; 19:580–583.

10.Gherghel D, Griffiths H, Hilton E, et al. Systemic reduction in glutathione levels occurs in patients suffering from primary open-angle glaucoma. Invest Ophthalmol Vis Sci 2005; 46:877–883.

11.Izzotti A, Sacca SC, Cartiglia S, et al. Oxidative deoxyribonucleic acid damage in eyes of glaucoma patients. Am J Med 2003; 114:638–646.

12.Spector A, Garner WH. Hydrogen peroxide and human cataract. Exp Eye Res 1981; 33:673–681.

13.Zhou L, Li Y, Yue BY. Oxidative stress affects cytoskeletal structure and cellmatrix interactions in cells from an ocular tissue: the trabecular meshwork. J Cell Physiol 1999; 180:182–189.

14.Kahn MG, Giblin FJ, Epstein DL. Glutathione in calf trabecular meshwork and its relation to aqueous humor outflow facility. Invest Ophthalmol Vis Sci 1983; 24:1283–1287.

15.Alvarado J, Murphy C, Polansky J, et al. Age-related changes in trabecular meshwork cellularity. Invest Ophthalmol Vis Res 1981; 21:714–727.

16.Alvarado J, Murphy C, Juster R. Trabecular meshwork cellularity in primary openangle glaucoma and non-glaucomatous normals. Ophthalmology 1984; 91:564–579.

17.Sacca SC, Pascotto A, Camicione P, et al. Oxidative DNA damage in human trabecular meshwork: clinical correlation in patients with primary open-angle glaucoma. Arch Ophthalmol 2005; 123:458–463.

18.Wang N, Chintala SK, Fini ME, et al. Activation of a tissue specific stress response in the aqueous outflow pathway of the eye defines the glaucoma disease phenotype. Nat Med 2001; 7:304–309.

19.Ferreira SM, Lerner SF, Brunzini R, et al. Oxidative stress markers in aqueous humor of glaucoma patients. Am J Ophthalmol 2004; 137:62–69.

20.Xu YC, Wu RF, Gu Y, et al. Involvement of TRAF4 in oxidative activation of c-Jun N-terminal kinase. J Biol Chem 2002; 277:28051–28057.

21.Tezel G, Yang X, Yang J, et al. Role of tumor necrosis factor receptor-1 in the death of retinal ganglion cells following optic nerve crush injury in mice. Brain Res 2004; 996:202–212.

22.Liu B, Neufeld AH. Expression of nitric oxide synthase-2 (NOS-2) in reactive astrocytes of the human glaucomatous optic nerve head. Glia 2000; 30:178–186.