Chronic Antioxidant Capacity Loss in Anterior Chamber Environment After Iridectomy

Abstract

Purpose: To compare the ascorbic acid concentration and total antioxidant capacity in the aqueous humor of pigmented Rex rabbits after sham operation (control), iridectomy, and trabeculectomy.

Methods: Pigmented Rex rabbits were divided into control, iridectomy, and trabeculectomy groups and followed up for 12 months after surgery. Ascorbic acid concentration and total antioxidant capacity in the aqueous humor, intraocular pressure, and the occurrence of cataracts were examined in each group.

Results: The ascorbic acid concentration and total antioxidant capacity after iridectomy and trabeculectomy were significantly lower at one week and at one, six, and 12 months after operation than those in the control group (P ≤ 0.03). Ascorbic acid concentration was positively and significantly correlated with total antioxidant capacity in the aqueous humor (P < 0.01). Compared to the control and the iridectomy groups, intraocular pressure in the trabeculectomy group was significantly lower at one week and at one and six months after surgery (one week: P < 0.01 and P < 0.01, respectively; one month: P < 0.01 and P = 0.03, respectively; six months: P = 0.03). Histological findings in the iridectomy and trabeculectomy groups included the appearance of vacuoles in the lens at six and 12 months after surgery.

Conclusions: Iridectomy causes a sustained decrease in ascorbic acid concentration, followed by a long-term decrease in the total antioxidant capacity within the aqueous humor.

Translational Relevance: The animal model possibly predicts the vulnerability focusing on the antioxidant level in the anterior chamber environment after trabeculectomy and iridectomy per se in clinical settings.

Introduction

Glaucoma and cataracts are the leading causes of blindness worldwide^1,2 and often coexist because both are age related. Treatment of cataracts is surgical, whereas treatment for glaucoma can be performed by medical, laser, surgical, or combination therapy. Trabeculectomy is a typical filtration surgery for glaucoma that is effective in lowering intraocular pressure (IOP).^3–5 However, one of the disadvantages of trabeculectomy is postoperative cataract progression.^6–8 The cause of cataract progression after trabeculectomy is unclear. It has been postulated that cataract progression might be caused by postoperative corticosteroid use,⁹ hypotony, and the collapse of the anterior chamber,^10,11 or it might be the result of changes in the dynamics of the aqueous humor.¹² 

An alternative filtration surgery, Ex-PRESS filtration surgery, uses a unique device designed to stabilize the filtered aqueous volume to prevent postoperative complications derived from overfiltration. Previously, we compared postoperative complications after Ex-PRESS filtration surgery with those after trabeculectomy during a two-year follow-up.¹³ Interestingly, cataract progression was significantly slower in the Ex-PRESS filtration surgery group than in the trabeculectomy group. Ex-PRESS filtration surgery does not require iridectomy during the surgical procedure. We hypothesized that iridectomy might change the content of aqueous humor nourishing the lens, resulting in cataract progression. 

Factors that disrupt homeostasis in the anterior chamber such as inflammation,¹⁴ lipid abnormalities,¹⁵ glycation,¹⁶ and oxidative stress^17–19 are closely linked to cataract progression. Oxidative stress, including free radicals and reactive oxygen species (ROS), is known to cause cellular damage²⁰ and protein degeneration.²¹ Ascorbic acid in the aqueous humor is present at much higher concentrations than in plasma^22,23 to prevent cataract progression^24,25 as an antioxidant. Total antioxidant capacity (TAC) has been measured as an indicator of oxidative resistance of antioxidants in the human body.²⁶ Ascorbic acid is expected to account for a high proportion of TAC in aqueous humor.²⁷ Therefore we aimed to determine whether iridectomy per se would alter the antioxidant environment in the aqueous humor by measuring ascorbic acid concentration and total antioxidant capacity. 

Methods

Pigmented male Rex rabbits weighing 2.0 to 2.5 kg (Japan SLC Co. Ltd, Shizuoka, Japan) were acclimated to their surroundings for at least one week. All rabbits were kept under pathogen-free conditions at 23°C ± 1°C, 60% ± 10% humidity, and 12 hours of light with 12 hours of darkness. Animals were housed with free access to water and food (RC4; Oriental Yeast Co. Ltd., Tokyo, Japan) throughout the day. The maintenance and experimentation of animals conformed to the guidelines of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research during all procedures. A total of 96 right eyes of 96 rabbits were divided 1:1:1 into the sham operation (control), trabeculectomy, and iridectomy groups. Each group included 32 eyes. The postoperative measurement points were at one week and at one, six, and 12 months. Eight eyes were assigned to each postoperative measurement point in each group for sample collection. General anesthesia was administered by intramuscular injection of a mixture of ketamine (35 mg/kg) and xylazine (5 mg/kg) during surgery in the surgery room of the Department of Biological Resources at the University of Fukui. 

Aqueous humor samples were collected to analyze whether there were significant differences in ascorbic acid concentration and TAC among the groups at one week and at one, six, and 12 months after surgery. In addition, the lenses of rabbit were enucleated for histological examination among the groups at one week and at one, six, and 12 months after surgery. The presence of cataracts was visually assessed using a portable slit lamp before enucleation. The enucleated lenses were cut to a thickness of 3 µm using a microtome for pathological specimen preparation with paraffin embedding. The specimens were observed under a microscope (IX70; Olympus, Tokyo, Japan). Conventional hematoxylin and eosin staining was used to examine the postoperative lens pathology. IOP was measured one day before surgery and at one week and at one, six, and 12 months after surgery using TONOVET (Icare, Helsinki, Finland) in conscious rabbits. 

Surgical Techniques

Conjunctival sacs were disinfected prior to all the surgeries with 10% polyvinyl alcohol iodine (Nitten Pharmaceutical, Nagoya, Japan) diluted ×6. 

Trabeculectomy

A 7-mm conjunctival incision was made along the corneal limbus. A square scleral flap with a side length of 3 mm was created along the corneal limbus of the upper sclera. Three sponges soaked in mitomycin C (0.4 mg/mL; Kyowa Kirin, Tokyo, Japan) were applied under the conjunctival and scleral flaps for four minutes. The subconjunctival tissue and scleral flap were then washed with 10 mL normal saline solution (Otsuka, Tokyo, Japan). The trabecular meshwork below the scleral flap was excised. The iris was pulled from the excisional wound, and peripheral iridectomy was performed. The scleral flap was sutured using 10-0 nylon, maintaining aqueous humor filtration. The conjunctival wound was tightly closed using 10-0 nylon. 

Iridectomy

A 7-mm conjunctival incision was made along the corneal limbus. A slit knife was inserted into the sclera near the corneal limbus, and the iris was removed using forceps. The incision was sutured using 10-0 nylon. 

Sham Operation

A 7-mm conjunctival incision was made along the corneal limbus. A slit knife was inserted into the sclera near the corneal limbus, and the iris was touched with forceps without cutting the iris. 

Eye drops of 1.5% levofloxacin hydrate as an antibacterial drug were administered three times a day after the surgeries in the sham control, the iridectomy, and the trabeculectomy groups for a week. Corticosteroid eye drops were not used after the surgeries because corticosteroid administration affects cataract progression and postoperative inflammation. 

Samples

Before sample collection, rabbits were anesthetized with an intramuscular injection of a mixture of ketamine (35 mg/kg) and xylazine (5 mg/kg). The aqueous humor was collected with a 30 G needle connected to an insulin syringe and stored in a −80°C freezer until measurement. After sample collection, the rabbits were euthanized with an intravenous injection of 10 mL sodium pentobarbital. The lens was enucleated immediately after euthanasia, then soaked in 4% PFA, and stored in a −4°C freezer until tissue staining. A total of eight eyes were collected at each measurement point in each group for the purpose of measuring ascorbic acid concentration and TAC in the aqueous humor, and examining histological changes of the lens. 

Ascorbic Acid Measurement

The samples were diluted 50 times and then measured using an assay kit (Ascorbic Acid Quantification Kit; Bio Vision Inc., Milpitas, CA, USA). The concentration of ascorbic acid was determined by fluorometric methods (Ex/Em = 535/587 nm). The assay could detect the amount of 0.01 to 10 nmol of ascorbic acid per assay. 

Total Antioxidant Capacity Measurement

TAC in the aqueous humor was measured using a commercially available kit (Total Antioxidant Status Assay Kit; MEGA TIP San. Tic. Ltd, Mücahitler, Turkey). Antioxidants in the kit reduce dark blue-green ABTS radicals to a colorless reduced ABTS form. The change in the absorbance at 660 nm was related to the total antioxidant level of the sample. The assay is calibrated with a stable antioxidant standard solution, traditionally called Trolox equivalent, which is a vitamin E analog. 

Statistical Analysis

In each group, normality was measured using the Shapiro-Wilk test. To compare means among the three groups, an analysis of variance (ANOVA) test was first performed, followed by a post-hoc test using the Tukey-Kramer method. The proportional correlation between ascorbic acid concentration and TAC in aqueous humor was presented as a scatter diagram analyzed by Spearman's correlation analysis. SPSS version 27.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. For all tests, a P value less than 0.05 was considered significant. Preoperative data are shown as the mean ± standard deviation. Postoperative data are shown as the mean ± standard error. 

Additional Experiment

We performed an additional experiment to clarify the mechanism of antioxidant capacity loss in the aqueous humor postoperatively. Eight right eyes of eight rabbits were divided 1:1 into the sham-surgery group and anterior lamellar excision of peripheral iris group, which was the left posterior stroma group (defined as “anterior iridectomy” group). Each group included four eyes. We measured and compared the concentrations of ascorbic acid and various cytokines including vascular endothelial growth factor-A, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in aqueous humor between the sham-surgery group and the anterior iridectomy group with assay and enzyme-linked immunosorbent assay kits. Mann-Whitney U test was performed to analyze the significance between the groups. The postoperative measurement points were at one week and at one and two months. The aqueous humor was collected repeatedly in the same eye at each measurement point. The surgical techniques for the sham-surgery group, anesthetic methods for sample collection, and sample storage were the same as described in this “Methods” section, in the “Surgical techniques” and “Samples” subsections. For the surgical technique in anterior iridectomy group, a 7-mm conjunctival incision was made along the corneal limbus. A slit knife was inserted into the sclera near the corneal limbus, and the anterior lamellar of peripheral iris was excised using forceps, which left the posterior stroma. The incision was sutured with 10-0 nylon. 

Results

Ascorbic Acid Concentration

The comparison of the means of the ascorbic acid concentrations among the three groups showed significant differences with ANOVA at all measurement points (P < 0.01). The post-hoc test showed that the ascorbic acid concentrations after iridectomy and trabeculectomy were significantly lower at all measurement points at one week and at one, six, and 12 months after surgery than in the control group (one week: P < 0.01 and P < 0.01, respectively; one month: P < 0.01 and P = 0.03, respectively; six months: P < 0.01 and P < 0.01, respectively; 12 months: P < 0.01 and P < 0.01, respectively) (Table 1). 

Table 1.

 

Comparison of Ascorbic Acid Concentration (mM) Among the Groups

 

Total Antioxidant Capacity

In the comparison of TAC among the three groups, there were significant differences with ANOVA at all measurement points (P < 0.01). The post-hoc test showed that the TAC after iridectomy and trabeculectomy was significantly lower at all measurement points at one week and at one, six, and 12 months after surgery compared to the control group (one week: P < 0.01 and P < 0.01, respectively; one month: P < 0.01 and P < 0.01, respectively; six months: P < 0.01 and P < 0.01, respectively; 12 months: P < 0.01 and P < 0.01, respectively) (Table 2). Spearman's correlation analysis revealed a significant positive correlation between the ascorbic acid concentration and TAC in all samples (n = 96, r = 0.66, P < 0.01) (Fig. 1). 

Table 2.

 

Comparison of TAC (mM) Among the Groups

 

Figure 1.

 

Correlation of ascorbic acid concentration and total antioxidant capacity. Spearman's correlation analysis revealed significantly positive correlation between AA concentration and TAC in all samples (n = 96, r = 0.66, P < 0.01). AA, ascorbic acid.

Postoperative IOPs

Table 3 shows the IOPs in the control, iridectomy, and trabeculectomy groups. In the comparison of IOPs among the three groups, there were significant differences with ANOVA at all measurement points (P < 0.01). The post-hoc test showed that IOPs after trabeculectomy were significantly lower at one week and at one and six months after surgery compared to the control group and the iridectomy group (one week: P < 0.01 and P < 0.01, respectively; one month: P < 0.01 and P = 0.03, respectively; six months: P = 0.03). 

Table 3.

 

The Comparison of IOPs (mm Hg) Among the Groups

 

Cataract Progression

Postoperative anterior chamber photographs of the iridectomy group were obtained using a portable slit lamp (Fig. 2). Cortical opacity in the lens was visually observed at 12 months after surgery. The results of hematoxylin and eosin staining in the control group, the iridectomy group, and the trabeculectomy group are shown Figure 3. There were no cataractous changes in the lens either at one week or one month in each group, but numerous vacuole changes between the cortex and nucleus were observed at six and 12 months in the iridectomy and the trabeculectomy group. 

Figure 2.

 

Anterior chamber examination by a slit lamp in the iridectomy group. Cortical opacity was observed 12 months postoperatively. The red arrow indicates the area of cortical opacity in the lens.

Figure 3.

 

Histological changes in the lens after the surgeries. The top row represents the control group. The middle row shows the results of the iridectomy group. The bottom row shows the results of the trabeculectomy group. There were vacuole changes between the cortex and nucleus in the lens at six and 12 months in the iridectomy and trabeculectomy groups after surgery. Vacuole changes are indicated in red.

Additional Experiment

The ascorbic acid and IL-6 concentrations of anterior iridectomy group were significantly lower at one week and at one and two months after surgery than those of the sham-surgery group (one week: P = 0.03; one month: P = 0.03; two months: P = 0.03). The MCP-1 concentration in anterior iridectomy group was significantly lower at one week and at two months after surgery than those in the sham-surgery group (one week: P = 0.03; two months: P = 0.03). There were no significant differences in vascular endothelial growth factor-A and TNF-α concentrations between the groups. The numerical data are presented in Supplementary Tables S1 to S5. 

Discussion

The present study showed that the concentration of ascorbic acid, a reductant agent in aqueous humor, and TAC were significantly lower in the iridectomy and the trabeculectomy group than in the control group. Additionally, regression analysis showed that the ascorbic acid concentration had a significant positive correlation with the TAC (P < 0.01). The antioxidant environment associated with ascorbic acid and TAC in the aqueous humor is altered after iridectomy and trabeculectomy. There have been reports that focused on oxidative stress and glaucoma, including serum oxidative stress level,^28,29 damage to retinal neurons,³⁰ retinal ganglion cell death,³¹ glaucoma severity,³² and supplements for glaucoma,³³ although no studies have examined oxidative stress levels after trabeculectomy. This study provides new insights related to the vulnerability in anterior chamber environment after iridectomy, focusing on antioxidant condition in aqueous humor. 

Ascorbic acid is a powerful reducing agent of ROS in the anterior chamber environment. ROS are generated as a result of oxygen consumption by air respiration,³⁴ exposure to radiation,³⁵ ultraviolet light,³⁶ smoking,³⁷ inflammatory reaction,³⁸ and surgery.³⁹ ROS include sequential one-electron reduction products of O₂, such as O₂−, H₂O₂, and OH. The OH group of ascorbic acid donates H+ to convert OH into H₂O. The immediate postoperative decline in ascorbic acid concentration and TAC in the iridectomy and the trabeculectomy groups may be due to the consumption of ascorbic acid to remove OH from postoperative transient inflammation. Ascorbic acid concentration and TAC in the iridectomy and the trabeculectomy groups remained significantly lower than those in the control group until 12 months after surgery. The long-term loss of ascorbic acid and TAC may be related to chronic inflammation due to iris damage. Aketa et al.⁴⁰ demonstrated that iris damage elevated proinflammatory cytokines such as IL-α, IL-6, IL-8, TNF-α, MCP-1, and interferon-γ in the collection of aqueous humor samples six months to 10 years after intraocular surgeries,⁴¹ including laser peripheral iridectomy and trabeculectomy. Our additional experiment showed that the anterior lamellar excision of the peripheral iris sustained an increase of inflammatory cytokine levels including MCP-1 and IL-6 and a decrease of ascorbic acid concentration in the aqueous humor for two months after surgery. Furthermore, previous studies have shown that the interaction between iris pigment epithelial cells and the components of the aqueous humor maintains the homeostasis of the immune system in the anterior chamber, because the iris pigment epithelium has immunosuppressive and immunomodulatory effects.^42–45 Inflammatory responses inevitably occur when cells and tissues are exposed to stress or injury, as when infection occurs, because some receptors on immune cells detect damage-associated molecular pattern molecules released from damaged cells and tissues.^46,47 Proteomic approaches⁴⁸ from human samples suggest that iris atrophy results in blood aqueous barrier disruption and elevated protein levels, with concurrent complement activation and oxidative stress in the aqueous humor. This study suggests that failure of immunomodulatory factors caused by iris damage disturbs the microenvironment in the aqueous humor, resulting in long-term elevation in the levels of aqueous inflammatory cytokines accompanied by ascorbic acid consumption for ROS removal. 

Another reason for the long-term loss of ascorbic acid and TAC may be related to alterations in the aqueous humor flow after iridectomy. Razzak et al.⁴⁹ hypothesize that cataracts are caused by trauma to the lens due to the continuous flow of aqueous humor through a surgical fistula created by trabeculectomy. It is possible that chronic trauma to the lens because of the alteration of aqueous flow may have led to the sustained consumption of ascorbic acid and TAC because ascorbic acid is absorbed by the lens and protects it from oxidation.⁵⁰ However, in our additional experiment, there was a significant decrease of ascorbic acid concentration with a concurrent increase of inflammatory cytokines in the aqueous humor after surgery without direct aqueous humor flow from the posterior to anterior chamber for two months after surgery. Therefore we consider that the long-term antioxidant capacity loss is due to the prolonged inflammation rather than changes in the aqueous humor flow. 

In the current study, cataracts were associated with iridectomy and trabeculectomy in slit-lamp examination and pathological specimens. To the best of our knowledge, no study has focused on postoperative oxidative stress and cataract progression after iridectomy and trabeculectomy. Despite the relationship between oxidative stress levels and cataract progression,^17–19 studies to date have been limited to diet,⁵¹ epidemiology,⁵² and plasma oxidative stress levels.⁵³ Few reports have mentioned changes in the antioxidant capacity of aqueous humor, with which the lens is in direct contact. Tsao et al.²⁷ reported that ascorbic acid and TAC levels in the aqueous humor have a significant negative correlation with cataract severity, which is consistent with our results. 

In addition, vacuoles were histologically observed between the cortex and nucleus of the lens at six and 12 months after surgery in the present study, whereas observation using the portable slit showed cortical cataractous changes at 12 months only. The areas of lens opacity after glaucoma surgery are controversial, with some studies reporting nuclear opacity,¹³ others reporting posterior subcapsular cataract,⁵⁴ and still others stating that all types of cataracts progress.⁶ Our data indicate that cataract development after trabeculectomy begins with small vacuolar changes between the cortex and nucleus. Whether this change is specific to the long-term loss of antioxidant capacity requires further study. 

In this study, trabeculectomy significantly reduced IOPs compared to the control and iridectomy groups at one week and at one and six months after surgery. In contrast, IOPs were not significantly different between the iridectomy and control groups. Although a shallow anterior chamber and low IOP have been reported as risk factors for cataract progression after trabeculectomy,^10,11 the iridectomy group did not encounter hypotony in the present study. This suggests that postoperative antioxidant levels in the aqueous humor with concurrent cataract progression are independent of IOP and anterior chamber collapse. Our data indicate that peripheral iris defects cause the consumption of antioxidants in the aqueous humor. 

This study has several limitations. Other antioxidants, such as glutathione and uric acid, were not measured because of the limited amount of aqueous humor samples available. The sample size for the additional experiments was small, and other inflammatory and anti-inflammatory cytokines need to be investigated. A longer measurement period is necessary to confirm the long-term loss of ascorbic acid and the elevation levels of the cytokines in the aqueous humor after surgery. In addition, the TAC was not measured in the additional experiment because of the small volume of the aqueous humor that can be collected from one eye. We will have to conduct comprehensive studies to evaluate the effect of postoperative inflammatory cytokines on antioxidant levels in the aqueous humor by multivariate analysis. Although this study visually examined the appearance of cataracts, there were no objective data regarding lens opacity. Some reports have shown the progression of cataracts numerically using Image J with photographs taken under a microscope⁵⁵ or with an anterior segment imaging device.¹³ The correlation between lens opacity and antioxidant levels in the aqueous humor should be analyzed in the future. 

In conclusion, we observed a long-term loss in ascorbic acid concentration and TAC in the aqueous humor and cataract progression after iridectomy and trabeculectomy. This study provides a new information related to the vulnerability of anterior chamber environment after iridectomy, especially in antioxidant conditions. 

Acknowledgments

Supported in part by JSPS KAKENHI Grants 19K18875 and 22K19582. 

Disclosure: S. Arimura, None; K. Iwasaki, None; T. Neo, None; Y. Orii, None; T. Matsumura, None; Y. Takamura, None; M. Oki, None; M. Inatani, None