November 2009
Volume 50, Issue 11
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Glaucoma  |   November 2009
Sensitivity of Ocular Anterior Chamber Tissues to Oxidative Damage and Its Relevance to the Pathogenesis of Glaucoma
Author Affiliations & Notes
  • Alberto Izzotti
    From the Department of Health Sciences, Faculty of Medicine, University of Genoa, Genoa, Italy; and
  • Sergio C. Saccà
    the Division of Ophthalmology, St. Martino Hospital, Genoa, Italy.
  • Mariagrazia Longobardi
    From the Department of Health Sciences, Faculty of Medicine, University of Genoa, Genoa, Italy; and
  • Cristina Cartiglia
    From the Department of Health Sciences, Faculty of Medicine, University of Genoa, Genoa, Italy; and
  • Corresponding author: Alberto Izzotti, Department of Health Sciences, University of Genoa, Via A. Pastore 1, Genoa I-16132, Italy; izzotti@unige.it
Investigative Ophthalmology & Visual Science November 2009, Vol.50, 5251-5258. doi:10.1167/iovs.09-3871
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      Alberto Izzotti, Sergio C. Saccà, Mariagrazia Longobardi, Cristina Cartiglia; Sensitivity of Ocular Anterior Chamber Tissues to Oxidative Damage and Its Relevance to the Pathogenesis of Glaucoma. Invest. Ophthalmol. Vis. Sci. 2009;50(11):5251-5258. doi: 10.1167/iovs.09-3871.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: The anterior chamber of the eye is a highly specialized structure delimited by the cornea, lens, and iris. It contains the aqueous humor, secreted by the ciliary body and drained by the trabecular meshwork. Alteration of aqueous humor homeostasis plays a major role in the pathogenesis of glaucoma. The trabecular meshwork is the target tissue of glaucoma in the anterior chamber, and the development and progression of glaucoma are accompanied by accumulation of oxidative damage in this tissue. This study was conducted to comparatively evaluate the sensitivity to oxidative stress of anterior chamber tissues including the cornea, iris, and trabecular meshwork.

Methods.: Cornea, iris, and trabecular meshwork fragments collected from six cornea donors were either left untreated or treated with hydrogen peroxide. Oxidative damage was determined by evaluating nucleotide oxidative modifications (8-hydroxy-2′-deoxyguanosine) and apurinic alkali–fragile sites by capillary electrophoresis.

Results.: The results indicated that the basal level of oxidative nucleotide modifications was higher in the cornea than in the iris and trabecular meshwork. The trabecular meshwork was the most sensitive tissue to oxidative damage, as after exposure to hydrogen peroxide both markers of oxidative damage dramatically increased in the trabecular meshwork but not in the cornea and iris.

Conclusions.: Because the cornea and iris are directly exposed to light, they possess antioxidant defense mechanisms that are not activated in the trabecular meshwork. The peculiar sensitivity of the trabecular meshwork to oxidative stress is consistent with the damage selectively induced in it, triggering glaucoma's pathogenic cascade.

The anterior chamber (AC) is a highly specialized structure of the eye. It is composed of several different 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. The cellular lining of this space includes the corneal and TM endothelium and the melanocytes and fibroblasts of the anterior border layer of the iris and the ciliary body. The narrowest portion of the anterior chamber is at the angle. 
The AC contains the aqueous humor (AH), and, being a space surrounded by endothelium that encloses a liquid, may be considered to be a highly specialized vessel. 1 AH is secreted in the posterior chamber by the ciliary body and leaves the AC via two routes: the conventional (anterior) route is the through the TM, and the unconventional (posterior) route is through the uveoscleral pathway. 2 Uveoscleral outflow contributes to approximately 30% of the total amount of AH drainage. 3  
The volume of the AC is approximately 0.25 mL, whereas the volume of the posterior chamber is 0.06 mL. The rate of production and elimination of AH contained in the AC is responsible for intraocular pressure. AH is needed to guarantee optical transparency, structural integrity, and nutrition in the absence of blood vessels. 
The AH contains a protein concentration approximately one hundredth that of the serum that enters it directly by diffusion through the root of the iris. 4 The aqueous fluid has 0.1% to 0.2% of the concentration of plasma protein and has higher concentrations of amino acids than does plasma. Ascorbate, lactate, and bicarbonate concentrations are also high in the AH. 57 The rate of AH production is approximately 2 to 2.5 mL/min. Approximately 1% of the AC and 3% of the posterior chamber volume of AH is replaced each minute. 7 The metabolism of all the many components of the AC depends on the AH. In addition, this fluid provides nutrients for the avascular lens and cornea and the egress for waste products from these structures. 8  
The most common human diseases affecting the eye are glaucoma, cataracts, and macular degeneration. The common denominator of these diseases is the involvement of free radicals. The antioxidant ascorbic acid is a primary substrate in ocular protection because of its high concentration in the eye. 9 A high level of ascorbic acid is necessary to maintain oxidative balance in the AH. Furthermore, the antioxidant vitamin E plays a major role in the protection of ocular structures. In fact, vitamin E deficiency increases H2O2 levels and the intracellular production of reactive oxygen species. 10 Vitamin E prevents apoptosis during hypoxia and oxygen reperfusion, protecting the lens and the retina from the damaging effects of ultraviolet radiation. 5,11 In addition, vitamin C exerts a filterlike function against UV radiation in both the central corneal epithelium and the AH and reacts with O2 to form H2O2. 11 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. 12,13 High doses of vitamin C decrease intraocular pressure, probably by depolymerizing the TM's hyaluronic acid component. 14,15  
Another antioxidant agent that plays a major role in eye defense is reduced glutathione, a tripeptide composed of the amino acids γ-glutamic acid, cysteine, and glycine. Glutathione participates directly in the neutralization of free radicals and reactive oxygen species (ROS) and maintains exogenous antioxidants, such as vitamins C and E. Thus, glutathione protects ocular tissues from the damage induced by low H2O2 concentrations, whereas catalase protects ocular tissues from the damage induced by high H2O2 concentrations. 16,17 When mitochondrial glutathione and vitamin E levels are reduced to 20% of the normal level, lipid peroxidation occurs. 18  
Numerous scientific investigations have confirmed the presence of oxidative stress in ocular diseases. Indeed, ROS are a cause of cataracts, are implicated in age-related macular degeneration, and also may play a significant role in glaucoma's pathophysiology. 1926 However, the exact effect of oxidative stress on AC tissues is still far from being completely understood. Light has a direct oxidizing effect that is exerted both locally and at a systemic level in exposed organisms. 27,28  
All AC endothelial cells from the cornea, TM, and iris are immersed in AH and accordingly are exposed to free radicals. Light may induce the formation of oxidative radicals, which could indirectly target the TM by altering the oxidant–antioxidant balance in the AH, thus contributing to the pathogenesis of glaucoma. 29 Ultraviolet (UV) light induces the formation of 8-hydroxy-2′-deoxyguanosine (8-oxo-dG) in DNA via a singlet oxygen mechanism, and it has been established that in age-related macular degeneration, retinal cells die because of apoptosis in response to photic injury. 27,30 In glaucoma, a decrease in total antioxidant potential in the AC occurs, and levels of 8-oxo-dG are significantly higher in the TM of glaucomatous eyes than in control eyes. 24,25 Nevertheless, in glaucoma, this structural damage occurs only in the TM and does not affect other structures of the AC, such as the cornea or iris. 
The purpose of the present study was to evaluate the susceptibility to oxidative stress in the various tissues of the AC, thus shedding light on the mechanism that contributes to the onset of the most common degenerative ocular diseases. 
Material and Methods
Patients and Sample Collection
Iridocorneal angular-region fragments (six) were made available by the Melvin Jones Eye Bank in Genoa, Italy. These samples were obtained from six organ donors from whom eyes were obtained for corneal transplant within 3 hours after death. The procurement of the human tissues conformed to the tenets of the Declaration of Helsinki. 
Tissue and cell viability were accurately checked in all collected samples before proceeding to cornea transplant. The absence of any eye disease in these donors was ascertained in agreement with Italian national law (no. 301 of 12 August 1993). 
Iridocorneal angular region fragments were transferred to Petri dishes and dissected into the iris, TM, and cornea components (Fig. 1). Two fragments were obtained from each tissue type: One was used as the control and one was exposed to oxidative stress. Thus, the total number of fragments analyzed was 36 (6 subjects × 2 treatments × 3 tissues). Tissue fragments were immersed in glucose-enriched phosphate-buffered saline isotonic solution, and oxidative stress was induced by adding H2O2 25 μM for 30 minutes. The reaction was stopped by removing the H2O2-containing medium and washing the tissue fragments in isotonic buffer phosphate. 
Figure 1.
 
AC tissues (cornea, TM, and iris) as collected from corneal donors. Molecular analyses were performed on the whole collected tissues, including (1) cornea: epithelium, basement membrane, Bowman's layer, stroma, Descemet's membrane, and endothelium; (2) TM: trabecular endothelium, juxtacanalicular tissue, and Schlemm's canal; and (3) iris: anterior and posterior layer, surface endothelium, stroma, basement membrane, and the pigmented epithelial layer.
Figure 1.
 
AC tissues (cornea, TM, and iris) as collected from corneal donors. Molecular analyses were performed on the whole collected tissues, including (1) cornea: epithelium, basement membrane, Bowman's layer, stroma, Descemet's membrane, and endothelium; (2) TM: trabecular endothelium, juxtacanalicular tissue, and Schlemm's canal; and (3) iris: anterior and posterior layer, surface endothelium, stroma, basement membrane, and the pigmented epithelial layer.
The level of oxidative damage was tested by various methods that analyzed different endpoints including: oxidative nucleotide modifications and DNA fragmentation and formation of alkali fragile sites. 
Oxidative Nucleotide Modifications
Oxidative DNA damage was determined by quantifying 8-hydroxy-2′-deoxyguanosine (8-oxo-dG), the most important and abundant indicator of oxidative lesions of DNA in chronic degenerative diseases. 8-oxo-dG has also been demonstrated to be present at high levels in the TM of glaucomatous eyes. 25 This molecule results from the interaction between the hydroxyl radical OH° and the C2 of guanine, resulting in a hydroxylated guanine that, if unrepaired by specific glycosylases, may cause G→A transversions and formation of apurinic sites. The analytical method used for 8-oxo-dG detection was 32P postlabeling and thin-layer chromatography. 25,31 DNA (1–3 μg) was depolymerized to 3′-monophosphate nucleotides by incubation with micrococcal nuclease (0.14 U/μg DNA) and spleen phosphodiesterase (1 mU/μg DNA) at 37°C for 3.5 hours. Unmodified dGp nucleotides were selectively removed by incubation with 80% vol/vol trifluoroacetic acid (30 μL) for 10 minutes at room temperature. Samples were dried by vacuum centrifugation, and 3′-phosphate-8-oxo-dG was labeled by incubation with T4 plasmid polynucleotide kinase (8 U) dissolved in 200 mM bicine, 100 mM dithiothreitol, and 10 mM spermidine. This process was conducted in the presence of AT-γ-32P (64 μCi, specific activity ≥ 750 Ci/mmol; ICN, Irvine, CA), which acted as the 32P donor. The reaction was performed at 24°C for 40 minutes. The mixture underwent nuclease P1 digestion (2.7 U at 37°C for 60 minutes) to selectively separate 32P from the normal nucleotides. 32P-labeled 8-oxo-dG was purified from the reaction mixture by monodirectional thin-layer chromatography on an 18 × 3-cm cellulose sheet coated with the anion exchanger polyethylenimine (Macherey and Nagel, Düren, Germany). The chromatographic development was performed in unbuffered 1.5 M formic acid. Under these conditions, 32P-labeled 8-oxo-dG slowly migrated to the central part of the chromatographic area, whereas normal nucleotides accumulated on the upper edge of the chromatographic sheet that was cut away. 32P-labeled 8-oxo-dG was identified by electronic autoradiography and quantified by measuring the emitted β radiation using a 32P imager (Instant Inmager; Packard, Meriden, CT). Positive reference standards were obtained by incubating calf thymus DNA with 1 mM CuSO4 and 50 mM H2O2 or using an authentic 8-oxo-dG reference standard (National Cancer Institute Chemical Carcinogen Reference Standard Repository; Midwest Research Institute, Kansas City, MO). DNA-free samples were used as a negative control. Untreated bacterial DNA extracted from Salmonella typhimurium was used as the reference DNA because it has low levels of 8-oxo-dG. 
DNA Fragmentation by Capillary Electrophoresis
Oxidative damage to DNA was also evaluated in terms of formation of apurinic sites, which are a result of 8-oxo-dG formation and are particularly labile to alkali fragmentation. Apurinic sites were evaluated by digestion with endonuclease IV, an endonuclease that specifically catalyzes the formation of single-strand breaks at abasic sites. Fragmentation of digested DNA was determined by alkaline capillary electrophoresis. 
The treatment with endonuclease IV was performed as follows. DNA (100 ng) was added to a mixture composed of 0.1 U endonuclease IV (Fermentas International Inc, Burlington, ON, Canada), 1 μL of 10× reaction buffer, and sterile water to a final volume of 10 μL, then incubated for 30 minutes at 37°C. Reactions were terminated by incubation for 15 minutes at 80°C. Digested samples were lyophilized and resuspended in 4 μL EDTA 20 mM (pH 12.25), then incubated for 30 minutes at room temperature. 
Samples underwent capillary electrophoresis in a bioanalyzer (model 2100; Agilent Technology, Waldbronn, Germany) using a DNA assay (12000 Chip; Agilent). The chip was prepared by filling each well with 9 μL of gel-dye mix (1 μL DNA dye concentrate in 60 μL filtered gel matrix; Agilent Technology) and 5 μL of marker. DNA (25 ng in 1 μL) was added to each sample well, and ladder (1 μL) was added to a separate well. The chip was then vortexed for 1 minute at 2400 rpm and run in the bioanalyzer. An electric field was applied between each well and the microchannels so that the DNA was electrophoretically driven by a voltage gradient and the molecules were separated by size. The molecular sizes that can be analyzed with the chip range between 100 and 12,000 bp. The dye intercalated with the DNA was detected by laser-induced fluorescence. Data were translated into gel-like images and electropherograms. 
Analyses of the gel-like images were performed with the ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). For each lane, a rectangular area between 2,000 and 10,380 bp, corresponding to an elution time of 75 to 90 seconds for the reference ladder, was analyzed, and the average intensity signal was recorded. The intensity of each control sample was compared to that of the corresponding treated sample. 
Standardization of Results Obtained from Different Tissues
Since the cellularity of tested tissues varies widely and because of differences in their structural elements attention was focused to quantify the examined end points on the basis of an equal amount of cellular DNA or of unmodified nucleotides to allow comparison of data obtained among cornea, iris, and TM. In particular, for each one of the two main end points, analyzed data have been standardized and reported as follows: (1) 8-oxo-dG was expressed as 8-oxo-dG/105 normal nucleotides; unmodified normal nucleotides were quantified in each sample in parallel to 8-oxo-dG by relative adduct 32P labeling, as detailed in other reports 31,32 ; (2) DNA fragmentation; in all electropherograms performed the amount of total DNA was accurately quantified by fiber optic spectrophotometry (Nanodrop Technology, Wilmington, DE) and standardized at 50 ng DNA per chip. 
GSTM1 Gene Polymorphism
The GSTM1 polymorphism was analyzed by qPCR. The GSTM1 gene had three possible allelic attributes, including homozygous positivity, heterozygous positivity; and homozygous deletion referred to as a GSTM1 null polymorphism. Only the homozygous deletion, usually occurring in approximately 40% of the population, results in a lack of function of the encoded activity. The qPCR reaction was performed as previously described. 32  
Statistical Analyses
Comparisons among quantitative variables in different groups of patients were executed by ANOVA and Student's t-test for unpaired data. The accepted level of significance was P < 0.05 (Statview software; Abacus Concept, Berkeley, CA). 
Results
Basal Oxidative Damage in Different Ocular Tissues
An example of the results obtained by analyzing 8-oxo-dG in parallel under the same experimental conditions in the iris, cornea, and TM of the same subject, in the absence or presence of oxidative stress, is shown in Figure 2. As reported in Table 1, the greatest amount of nucleotide oxidative damage in the AC tissues examined under basal conditions was observed in the cornea, followed by the TM and the iris. Observed differences were statistically significant between the cornea and both the TM and the iris (P < 0.01), whereas the difference between the TM and the iris was not statistically significant. Accordingly, the basal level of 8-oxo-dG in the cornea was 1.7- and 1.4-fold higher than that in the iris and the TM, respectively. 
Figure 2.
 
Detection of oxidative nucleotide alterations (8-hydroxy-2′-deoxyguanosine [8-oxo-dG/105 normal nucleotides]) in the cornea, TM, and iris of the same subject, either in the absence of any treatment (control) or after exposure to H2O2. 32P-postlabeled 8-oxo-dG, at variance with normal nucleotides, slowly migrated in formic acid chromatographic medium and was resistant to nuclease P1 digestion, thus being the only radioactive spot retained in the chromatographic area above the origin. 8-oxo-dG was significantly higher (P < 0.01) under basal conditions (Control) in the cornea than in the iris and TM. After oxidative stress (H2O2) 8-oxo-dG significantly (P < 0.001) increased only in the TM.
Figure 2.
 
Detection of oxidative nucleotide alterations (8-hydroxy-2′-deoxyguanosine [8-oxo-dG/105 normal nucleotides]) in the cornea, TM, and iris of the same subject, either in the absence of any treatment (control) or after exposure to H2O2. 32P-postlabeled 8-oxo-dG, at variance with normal nucleotides, slowly migrated in formic acid chromatographic medium and was resistant to nuclease P1 digestion, thus being the only radioactive spot retained in the chromatographic area above the origin. 8-oxo-dG was significantly higher (P < 0.01) under basal conditions (Control) in the cornea than in the iris and TM. After oxidative stress (H2O2) 8-oxo-dG significantly (P < 0.001) increased only in the TM.
Table 1.
 
DNA Oxidative Damage in Anterior Chamber Tissues
Table 1.
 
DNA Oxidative Damage in Anterior Chamber Tissues
8-oxo-dG/105 Nucleotides DNA Fragmentation (High-Molecular-Weight DNA %)
Control H2O2 Control H2O2
Iris 1.20 ± 0.27 1.54 ± 0.05 69.8 ± 27.22 58.2 ± 24.04
Cornea 2.08 ± 0.25* 2.42 ± 0.38 70.7 ± 31.40 64.1 ± 17.01
Trabecular meshwork 1.50 ± 0.29 2.78 ± 0.23† 79.1 ± 17.68 56.7 ± 6.43‡
No statistically significant differences were observed for DNA fragmentation among the different tissues under basal conditions. 
Different Sensitivity of Various Ocular Tissues to Oxidative Stress
Sensitivity to oxidative stress varied dramatically among the three different tissues examined, in both nucleotide oxidative alterations and DNA fragmentation. 
After treatment with H2O2, 8-oxo-dG increased slightly but not to a statistically significant extent in the iris (1.3-fold) and the cornea (1.16-fold), whereas the increase observed in the TM was substantial (1.9-fold) and statistically significant (P < 0.001; Fig. 2; Table 1). 
Regarding DNA fragmentation, the TM was more sensitive than the iris and cornea to the H2O2-induced damage. In fact, the increase in DNA fragmentation was statistically significant (P < 0.05) in the TM (1.4-fold) but not in the iris (1.2-fold) or cornea (1.1-fold; Figs. 3, 4; Table 1). 
Figure 3.
 
Analysis of DNA fragmentation in the cornea, TM, and iris of the same subject either in absence of any treatment or after exposure to H2O2. Pseudocolor scale indicates the DNA amount (50 ng for each lane) as distributed along the elution time (vertical axis). Undamaged high-molecular-weight DNA remained in the black rectangles, whereas damaged low-molecular-weight DNA migrated outside these areas. Vertical axis reports the elution time of biochip capillary electrophoresis. Digestion with endonuclease and use of alkaline buffer reveal the occurrence of H2O2-induced DNA fragmentation above the statistical significance threshold (P < 0.05) in the TM only.
Figure 3.
 
Analysis of DNA fragmentation in the cornea, TM, and iris of the same subject either in absence of any treatment or after exposure to H2O2. Pseudocolor scale indicates the DNA amount (50 ng for each lane) as distributed along the elution time (vertical axis). Undamaged high-molecular-weight DNA remained in the black rectangles, whereas damaged low-molecular-weight DNA migrated outside these areas. Vertical axis reports the elution time of biochip capillary electrophoresis. Digestion with endonuclease and use of alkaline buffer reveal the occurrence of H2O2-induced DNA fragmentation above the statistical significance threshold (P < 0.05) in the TM only.
Figure 4.
 
Electropherogram comparing the alkaline electrophoresis of the TM (top), cornea (middle), and iris (bottom) of the same subject, either untreated (gray line) or treated with H2O2 (black line). The horizontal axis reports the elution time (in seconds); the vertical axis reports DNA amount as inferred from the fluorescent signal. Undamaged high-molecular-weight DNA (black rectangle) remained in the area around the 10,380-bp peak (elution time interval 75 to 90 seconds of biochip capillary electrophoresis). H2O2-induced DNA fragmentation was revealed by endonuclease digestion and alkaline buffer as a flattening of the electropherogram in this area. TM was the most sensitive tissue to oxidative DNA damage.
Figure 4.
 
Electropherogram comparing the alkaline electrophoresis of the TM (top), cornea (middle), and iris (bottom) of the same subject, either untreated (gray line) or treated with H2O2 (black line). The horizontal axis reports the elution time (in seconds); the vertical axis reports DNA amount as inferred from the fluorescent signal. Undamaged high-molecular-weight DNA (black rectangle) remained in the area around the 10,380-bp peak (elution time interval 75 to 90 seconds of biochip capillary electrophoresis). H2O2-induced DNA fragmentation was revealed by endonuclease digestion and alkaline buffer as a flattening of the electropherogram in this area. TM was the most sensitive tissue to oxidative DNA damage.
Influence of Age and GSTM1 Genotype
Three subjects were found to be GSTM1 positive and three GSTM1 negative. The only end point significantly affected by the GSTM1-null genotype was the increase in 8-oxo-dG in the TM after H2O2 treatment. The difference in the 8-oxo-dG increase was 1.42 ± 0.20 in GSTM1-null subjects and 0.91 ± 0.13 in GSTM1-positive subjects (P = 0.05). Thus, the GSTM1 homozygous deletion affected sensitivity to oxidative DNA damage, but only in the TM and not in the iris or cornea. 
Age, per se, did not exert significant effects on any of the variables tested. 
Discussion
The results obtained provide evidence that the various tissues composing the AC have a differential susceptibility to oxidative damage. The TM shows the highest sensitivity to the consequences of oxidative stress, both in terms of 8-oxo-dG formation and DNA fragmentation. 
We evaluated the TM as a whole, and thus it was not possible to say which layer, (e.g., endothelia or matrix intercellular beams), was the most affected by oxidative damage. The iridocorneal angular region has three components aligned in series (i.e., the TM, the juxtacanalicular tissue, and Schlemm's canal). The TM consists of interconnected channels lined by endothelial cell that offer minimal resistance to the ingress of AH. Schlemm's canal is a channel like a vascular vase that has intercellular junctions making up a fluid barrier and represents the major site of resistance to outflow. Between these two barriers, there is the juxtacanalicular tissue, which contains a loose extracellular matrix through which the AH flows. 33,34 TM endothelial cells release factors that regulate the permeability of Schlemm's canal endothelial cells through a complex mechanism of cytokines. 35 TM endothelial cells, which are in direct contact with AH, may be susceptible to injury as induced by oxidative free radicals contained in this fluid. 36 The TM is a complex cellular system that increases the contact surface between the endothelium and AH. 1 The TM is responsible for the AH outflow from the AC and Schlemm's canal. 33,35 The TM undergoes a progressive time-related decline in the number of cells until a dysfunction of the entire region occurs. It has been calculated that at 20 years of age, the estimated number of cells in the whole meshwork is 763,000, which decreases to 403,000 cells by 80 years of age with a loss rate of 6,000 cells per year. 37 At the level of Schlemm's canal, the decrease in cell population was estimated from linear regression equations to be on the order of 430 cells per year for the whole canal and 320 cells per year for the canal's inner wall. 38 Previous studies have shown that the total outflow resistance increases with age in humans. 3941 Several factors have been noted that could contribute to this age-related increase in outflow resistance, such as the existence of a protein depot. 42 Furthermore, a loss of trabecular cells with age could result in a reduction in matrix metalloproteinase activity in the TM, causing a reduced capacity of the TM to break down extracellular material. 43 Blockage of the endogenous activity of the metalloproteinases (MMPs) reduces the facility of the outflow, probably because extracellular matrix turnover, initiated by one or more MMPs, is essential in maintaining intraocular pressure homeostasis. 44 Resistance to AH outflow increases in the presence of high levels of H2O2 in eyes with a glutathione (GSH)-depleted TM. 45 The H2O2 effect on the adhesion of TM cells to extracellular matrix proteins results in rearrangement of cytoskeletal structures that may induce a decrease in TM cell adhesion, cell loss, and compromise of the TM's integrity. 46 Our experimental data obtained from TM biopsy indicate that the peculiar sensitivity of this tissue to oxidative damage is a major mechanism contributing to age-related TM cell loss resulting in progressive AH outflow difficulties. This pathogenic mechanism plays a fundamental role in primary open-angle glaucoma, in which TM pathologic changes, mainly including oxidative DNA damage, trigger the “glaucomatous cascade.” 47  
In the AH, there are many factors that have a protective function for endothelial cells, such as GSH, which protects anterior segment tissues from high levels of H2O2. 16 Likewise, a 28-amino acid neurotrophic factor present in human AH protects corneal endothelial cells from H2O2 and from other oxidative insults. 48 Failure of these antioxidant defenses is likely to result first of all in damages induced in the TM due to the remarkable susceptibility of this tissue to oxidative injury demonstrated by the herein presented results. Probably, the protection against free radicals in the AC goes beyond the composition of the AH, which washes and protects all three structures examined in our study: the iris, cornea, and TM. Therefore, it is likely that the different sensitivity of these three tissues to oxidative damage depends on the different composition and physiological situation of each one. This view is also supported by the different level of oxidative damage we detected under basal conditions in the three tissues, with the cornea having a higher level compared with the TM and iris but being the least sensitive tissue to the damage induced by hydrogen peroxide. 
The cornea is the primary physical barrier against environmental assault on the eye and functions as a dominant filter of UV radiation. Maintenance of corneal structure is crucial for the physiological functions of this tissue in refraction and biodefense. In the healthy cornea several defense mechanisms are present to minimize and reduce damage from ROS. Indeed, 20% to 40% of the soluble protein content of the cornea is an isoenzyme of aldehyde dehydrogenase 3, which directly absorbs UV radiation and removes cytotoxic aldehydes produced by UV-induced lipid peroxidation. 49,50 In addition, aldehyde dehydrogenase 3A1-induced reduction in cell growth may contribute to protection against oxidative stress by extending time for DNA and cell repair. 51  
The cornea is also rich in antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, all of which help in the removal of free radicals and ROS generated by constant absorption of UV light. 52,53 In normal corneas, there is little evidence of malondialdehyde (MDA), which is formed as a result of lipid peroxidation from UV-induced oxidative destruction of cell membranes. 54  
In normal corneas, constitutive nitric oxide synthase was expressed only in the epithelium and endothelium, though human corneas were found to adequately process the free radicals/ROS so that the cytotoxic peroxynitrite byproduct was not formed. 55,56 Moreover, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage. 57  
The corneal endothelium has also been shown to produce an autocrine factor against oxidative stress, vasoactive intestinal peptide, which may have some interest. 58 Finally, crystallins are abundant corneal proteins that display antioxidant properties. 59  
On the whole, these antioxidant defenses explain why the cornea, despite being the only tissue among the three tested in our study to be directly exposed to environmental stress, and thus having the highest basal level of oxidative damage, is the least sensitive tissue to hydrogen peroxide. 
The iris structure supports both unidirectional secretion of AH and its reabsorption. 60 The protective antioxidant system of the iris includes low-molecular-weight antioxidants that are present in this pigmented tissue at relatively high concentrations. 61 In particular, in the iris glutathione levels and glutathione redox cycling play an important role in cellular defense against oxidative stress jointly with good iron homeostasis in the anterior segment of the eye. 62,63 Furthermore, melanin, which colors the iris in nonalbino eyes, is a potent antioxidant that is likely to be a major contributor to the poor sensitivity of the iris to hydrogen peroxide observed in our study. 64 Together with melanin, the spectral properties of the extracellular matrix also contribute to the antioxidant activity defending the iris. 65 The iris in the human eye is exposed to relatively high-intensity UV radiation and visible light transmitted by the cornea. Melanin absorbs UV radiation and blue light more efficiently than visible light of longer wavelengths. 66  
The multiple antioxidant defensive mechanisms existing in the cornea and iris are not present to a similar extent in the TM. In fact, this tissue, in contrast with the cornea and iris, is not directly exposed to environmental light, which is likely to be a major determinant for inducing antioxidant defensive mechanisms in directly exposed tissues. The lack of effective antioxidant mechanisms in the TM explains why, as demonstrated in the present study, this is the most sensitive tissue of the AC to oxidative damage. Progressive accumulation of oxidative damage in the TM typically occurs in primary open angle glaucoma and is significantly related to an increase in intraocular pressure and visual field defects. 25,26 The peculiar sensitivity of the TM to oxidative damage should be taken into account in case of therapeutic intervention targeting the AC and having as a collateral effect the production of ROS. For example, cataract extraction by phacoemulsification produces free radicals, which could contribute to the accumulation of oxidative damage in previously altered TMs, thus aggravating a pre-existent glaucoma or leading to ocular hypertension. 67  
In conclusion, our study provides evidence that the TM is the most sensitive tissue to oxidative radicals in the AC. This issue contributes to explaining the pathogenic mechanism by which oxidative damage is involved in glaucoma's pathogenic cascade. 
Footnotes
 Supported by the U.S. Glaucoma Foundation.
Footnotes
 Disclosure: A. Izzotti, None; S.C. Saccà, None; M. Longobardi, None; C. Cartiglia, None
Footnotes
 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The authors thank Paola Pagani (Melvin Jones Eye Bank, Genoa, Italy) for collaboration. 
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Figure 1.
 
AC tissues (cornea, TM, and iris) as collected from corneal donors. Molecular analyses were performed on the whole collected tissues, including (1) cornea: epithelium, basement membrane, Bowman's layer, stroma, Descemet's membrane, and endothelium; (2) TM: trabecular endothelium, juxtacanalicular tissue, and Schlemm's canal; and (3) iris: anterior and posterior layer, surface endothelium, stroma, basement membrane, and the pigmented epithelial layer.
Figure 1.
 
AC tissues (cornea, TM, and iris) as collected from corneal donors. Molecular analyses were performed on the whole collected tissues, including (1) cornea: epithelium, basement membrane, Bowman's layer, stroma, Descemet's membrane, and endothelium; (2) TM: trabecular endothelium, juxtacanalicular tissue, and Schlemm's canal; and (3) iris: anterior and posterior layer, surface endothelium, stroma, basement membrane, and the pigmented epithelial layer.
Figure 2.
 
Detection of oxidative nucleotide alterations (8-hydroxy-2′-deoxyguanosine [8-oxo-dG/105 normal nucleotides]) in the cornea, TM, and iris of the same subject, either in the absence of any treatment (control) or after exposure to H2O2. 32P-postlabeled 8-oxo-dG, at variance with normal nucleotides, slowly migrated in formic acid chromatographic medium and was resistant to nuclease P1 digestion, thus being the only radioactive spot retained in the chromatographic area above the origin. 8-oxo-dG was significantly higher (P < 0.01) under basal conditions (Control) in the cornea than in the iris and TM. After oxidative stress (H2O2) 8-oxo-dG significantly (P < 0.001) increased only in the TM.
Figure 2.
 
Detection of oxidative nucleotide alterations (8-hydroxy-2′-deoxyguanosine [8-oxo-dG/105 normal nucleotides]) in the cornea, TM, and iris of the same subject, either in the absence of any treatment (control) or after exposure to H2O2. 32P-postlabeled 8-oxo-dG, at variance with normal nucleotides, slowly migrated in formic acid chromatographic medium and was resistant to nuclease P1 digestion, thus being the only radioactive spot retained in the chromatographic area above the origin. 8-oxo-dG was significantly higher (P < 0.01) under basal conditions (Control) in the cornea than in the iris and TM. After oxidative stress (H2O2) 8-oxo-dG significantly (P < 0.001) increased only in the TM.
Figure 3.
 
Analysis of DNA fragmentation in the cornea, TM, and iris of the same subject either in absence of any treatment or after exposure to H2O2. Pseudocolor scale indicates the DNA amount (50 ng for each lane) as distributed along the elution time (vertical axis). Undamaged high-molecular-weight DNA remained in the black rectangles, whereas damaged low-molecular-weight DNA migrated outside these areas. Vertical axis reports the elution time of biochip capillary electrophoresis. Digestion with endonuclease and use of alkaline buffer reveal the occurrence of H2O2-induced DNA fragmentation above the statistical significance threshold (P < 0.05) in the TM only.
Figure 3.
 
Analysis of DNA fragmentation in the cornea, TM, and iris of the same subject either in absence of any treatment or after exposure to H2O2. Pseudocolor scale indicates the DNA amount (50 ng for each lane) as distributed along the elution time (vertical axis). Undamaged high-molecular-weight DNA remained in the black rectangles, whereas damaged low-molecular-weight DNA migrated outside these areas. Vertical axis reports the elution time of biochip capillary electrophoresis. Digestion with endonuclease and use of alkaline buffer reveal the occurrence of H2O2-induced DNA fragmentation above the statistical significance threshold (P < 0.05) in the TM only.
Figure 4.
 
Electropherogram comparing the alkaline electrophoresis of the TM (top), cornea (middle), and iris (bottom) of the same subject, either untreated (gray line) or treated with H2O2 (black line). The horizontal axis reports the elution time (in seconds); the vertical axis reports DNA amount as inferred from the fluorescent signal. Undamaged high-molecular-weight DNA (black rectangle) remained in the area around the 10,380-bp peak (elution time interval 75 to 90 seconds of biochip capillary electrophoresis). H2O2-induced DNA fragmentation was revealed by endonuclease digestion and alkaline buffer as a flattening of the electropherogram in this area. TM was the most sensitive tissue to oxidative DNA damage.
Figure 4.
 
Electropherogram comparing the alkaline electrophoresis of the TM (top), cornea (middle), and iris (bottom) of the same subject, either untreated (gray line) or treated with H2O2 (black line). The horizontal axis reports the elution time (in seconds); the vertical axis reports DNA amount as inferred from the fluorescent signal. Undamaged high-molecular-weight DNA (black rectangle) remained in the area around the 10,380-bp peak (elution time interval 75 to 90 seconds of biochip capillary electrophoresis). H2O2-induced DNA fragmentation was revealed by endonuclease digestion and alkaline buffer as a flattening of the electropherogram in this area. TM was the most sensitive tissue to oxidative DNA damage.
Table 1.
 
DNA Oxidative Damage in Anterior Chamber Tissues
Table 1.
 
DNA Oxidative Damage in Anterior Chamber Tissues
8-oxo-dG/105 Nucleotides DNA Fragmentation (High-Molecular-Weight DNA %)
Control H2O2 Control H2O2
Iris 1.20 ± 0.27 1.54 ± 0.05 69.8 ± 27.22 58.2 ± 24.04
Cornea 2.08 ± 0.25* 2.42 ± 0.38 70.7 ± 31.40 64.1 ± 17.01
Trabecular meshwork 1.50 ± 0.29 2.78 ± 0.23† 79.1 ± 17.68 56.7 ± 6.43‡
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