Nitric oxide levels in the anterior chamber of vitrectomized eyes with silicon oil Níveis de óxido nítrico na câmara anterior em olhos vitrectomizados com óleo de silicone

Purpose: To investigate the nitric oxide levels in the anterior chamber of eyes who underwent pars plana vitrectomy (PPV) with silicone oil. Methods: Patients who underwent PPV with silicon oil injection, from february 2005 to august 2007, were selected. Nine patients (nine eyes) participated in the study (five women and four men). Nitric oxide concentration was quantified after the aspiration of aqueous humor samples during the procedure of silicon oil removal. Data such as: oil emulsification; presence of oil in the anterior chamber; intraocular pressure and time with silicone oil were evaluated. Values of p <0.05 were considered to be statistically significant. Results: A positive correlation between nitric oxide concentration and time with silicon oil in the vitreous cavity (r=0.799) was observed. The nitric oxide concentration was significantly higher (p=0.02) in patients with silicon oil more than 24 months (0.90μmol/ml + 0.59, n=3) in the vitreous cavity comparing to patients with less than 24 months (0.19μmol/ml + 0.10, n=6). Conclusion: A positive correlation linking silicone oil time in the vitreous cavity with the nitric oxide concentration in the anterior chamber was observed.


G
laucoma is a possible complication in patients who underwent posterior pars plana vitrectomy (PPV) with silicon oil injection.Its frequency has been reported between 5.9 and 56% (1) .Aphakia, steroids use and previous vitreoretinal surgery have been described as risk factors for glaucoma in patients who underwent PPV with silicon oil injection (2) .After removal of the silicon oil, the intraocular pressure can continue high due to possible structural damage to the trabecular meshwork (1)(2)(3)(4)(5)(6)(7)(8)(9) .
Experimental studies demonstrated that silicon oil acts as a barrier to the diffusion of oxygen, maintaining a high oxygen concentration in the anterior chamber (10)(11)(12)(13)(14)(15)(16) .The oxygen tension in the anterior chamber is higher in eyes treated with lensectomy/ vitrectomy using silicon oil than in eyes just treated with lensectomy/vitrectomy (10) .There are no prior studies in humans that reveal the relation of free oxygen radicals in the anterior chamber of vitrectomized eyes with silicon oil.
The purpose of this study was to evaluate the nitric oxide levels, related to oxidative stress, in the anterior chamber of patients who underwent PPV with silicon oil injection.

METHODS
A prospective and analytical study was developed in the Department of Retina and Vitreous of the Altino Ventura Foundation and in the Laboratory of Immunopathology Keizo Asami (LIKA) -Pernambuco Federal University.
Patients who underwent PPV with silicon oil injection, from february 2005 to august 2007 were selected.Nine patients (nine eyes) participated in the study, five women and four men.The oil emulsification, presence of oil in the anterior chamber, intraocular pressure elevation (above 21mmHg) and time of permanence of the oil in the eye were evaluated.
The diagnosis requiring silicon oil injection was retinal detachment with proliferative vitreoretinopathy in eight cases and proliferative diabetic retinopathy in one case.Six patients were pseudophakic, two aphakic and one phakic.
During the surgery of silicon oil removal and before the approach to the posterior segment, 0.2ml of aqueous humor was aspirated with a paracentesis using an insulin syringe (1ml).The material was transferred to a sterile recipient, and then the nitric oxide concentration was quantified with the Griess reagent, that

Table 1 Patient's data who have undergone PPV with silicon oil injection
provides a well characterized assay for nitrites and nitrates.Nitric oxide is readily oxidized to nitrite and nitrate.Thus, the Griess reagent can indirect measure the nitric oxide concentration, quantifying the amount of nitrite.
The study was conducted after approval by the Ethics Committee of the Altino Ventura Foundation.All patients signed a consent form.The statistical tests used for continuous variables data were the non-parametric Mann-Whitney test and the Pearson correlation coefficient, to measure the linear dependence of two variables: nitric oxide concentration and time with silicon oil.The level of statistical significance was p < 0.05.

RESULTS
The data such as subject age, lens status, previous pathology, emulsification, presence of oil in the anterior chamber, ocular hypertension (above 21mmHg) and time permanence of the silicon oil are described in table 1.
A positive correlation was observed between the nitric oxide concentration and the permanence time of silicone oil in the vitreous cavity (r=0.799,p=0.01).
To compare nitric oxide concentration and emulsification (table 2), oil in the anterior chamber (table 3), intraocular pressure and silicon oil time (table 4), the Mann-Whitney test was used.
There is a statistically significant difference (p <0.05) between the mean values of the nitric oxide concentration in patients with less than 24 months duration of silicone oil and those over 24 months (table 4).

DISCUSSION
The morphological and physiologic alterations in aqueous humor drainage, in glaucoma patients, have oxidative stress as a contributor factor.The trabecular meshwork is exposed to free oxygen radical species in the aqueous humor from the aerobic metabolism, and from the mechanical stress (17)(18)(19)(20) .The damage to the trabecular meshwork cells could lead to an intraocular pressure elevation and glaucoma (17)(18)(19) .
Reactive oxygen species generated by oxidative stress include: superoxide, hydrogen peroxide, hydroxyl and nitric oxide.The greater accessibility to quantify in the material studied is nitric oxide.mitocondrial oxidative phosforylation and production of highly reactive metabolites of oxygen as the radical superoxide, that when interacting with nitric oxide forms the peroxynitrite, which is a strong oxidant.Nitric oxide and its metabolites interact with reactive oxygen species leads to protein nitration, which is one of the chemical modifications that occur during oxidative stress (21) .The oxidative stress causes an injury to the trabecular meshwok through reduction of protease activity, a multicatalytic complex responsible for cellular protection, yielding reduction of cellular viability implicated in glaucoma physiopathology (19) .The damage to the trabecular meshwork due to free oxygen radicals is well established in previous studies (22)(23)(24) .Nitric oxide is also responsible to damage the ganglionar cells in the retina, and for the intraocular pressure elevation (25) .
Posterior vitrectomy can modify the gradient of oxygen concentration in the vitreous cavity (26,27) .When the vitreous gel is present and intact there is a diffusion of oxygen from the retinal arterioles into the vitreous and then, an uptake of this oxygen by the retinal tissue (27) .Before vitrectomy, the oxygen appears to be higher around the circumference of the vitreous body.After vitrectomy, the fluid in the vitreous cavity can mix freely and the oxygen concentration near the lens increases greatly (27) .So, the surgery increases oxygen concentration in the vitreous cavity, increasing the concentration of free oxygen radicals (26) .The lens exposure to these free radicals is a mechanism of cataract formation (27) .Pseudophakic or aphakic patients could have an increase concentration of free oxygen Rev Bras Oftalmol.2013; 72 (5): 326-30 Nitric oxide levels in the anterior chamber of vitrectomized eyes with silicon oil  radicals in the anterior chamber with possible structural damage to the trabecular meshwork cells and the development of glaucoma as considered by Chang (28) .The oxidative stress has been implicated as a possible cause of glaucoma in vitrectomized patients, due to injury to the trabecular meshwork cells and intraocular pressure elevation (28) .
The silicon oil has been used as a vitreous substitute in complex retinal detachment cases.It may lead to some complications as secondary open angle glaucoma, cataract and corneal decompensation.The silicon oil in the vitreous cavity acts as a barrier to the oxygen diffusion, increasing its concentration in the anterior chamber (10)(11)(12)(13)(14)(15)(16) .The presence of silicon oil in the eye could change the oxygen tension in the anterior chamber, leading to an increase of nitric oxide concentration and oxidative stress.Pseudophakic and aphakic patients would be more likely to have increased levels of oxygen free radicals in the anterior chamber due to the lens absence and its capacity to metabolize the excess oxygen.
Patients with proliferative diabetic retinopathy also have increased levels of nitric oxide when compared to diabetics without retinopathy (29) .These patients would be at increased risk to develop greater oxidative stress and possibly glaucoma when submitted to vitrectomy with silicon oil.
This study revealed a correlation between the duration of silicon oil in the eye and the oxide nitric concentration, suggesting an oxidative stress increase in the anterior chamber of these patients.This oxidative stress increase could lead, in a time period not well known, to damage to the trabecular meshwork and intraocular pressure elevation, thus a new mechanism of glaucoma in silicon oil patients.
The development of glaucoma was not observed in any patient of this study during the follow up time.This finding could be associated to the sample size and requirement of a larger follow up time.

CONCLUSION
The study observed a correlation between time with silicon oil in the vitreous cavity and nitric oxide levels in the anterior chamber.

Figure 2 :
Figure 2: Box plot (median, 25 th and 75 th percentiles) from two groups: less than 24 months duration and more than 24 months

Figure 1 :
Figure 1: Dispersion between the nitric oxide (NO) concentration and the silicon oil time; ( ___ ) best line of linear regression with the confidence interval ( ___ ); r=0.799 (p=0.010);r2=0.638 The achievement of aerobic metabolism occurs with RD: retinal detachment; PVR: proliferative vitreoretinopathy; DR: diabetic retinopathy Nitric oxide levels in the anterior chamber of vitrectomized eyes with silicon oil

Table 2 Comparison of nitric oxide concentration and emulsification (p = 0.176) Table 3 Comparison of nitric oxide concentration and presence of silicone oil in the anterior chamber (p = 0.195)
NO = nitric oxide