Evaluation of Total Reactive Antioxidant Potential of the Vitreous Humor in Dogs with Primary Glaucoma
N. Weichsler; S. Ferreira; S. Llesuy; H.D. Herrera
Glaucoma is the final common pathway of a group of diseases with decreased retinal ganglion cells (RGC) sensitivity and function, RGC death and optic nerve head (ONH) cup enlargement. The neurodegenerative glaucomatous damage is due to ischemia, (Gelatt et al 2007) obstruction of axoplasmic flow (Anderson & Hendrickson 1974) and oxidative damage produced by both increase of reactive oxygen species (ROS) or diminution of the antioxidant ability of the retina and vitreous (Gelatt et al 2007). The purpose of this study was to evaluate the status of the total reactive antioxidant potential (TRAP) of the vitreous of dogs with primary glaucoma.
Materials and Methods
Seven dogs with primary glaucoma, 5 females and 2 males were included. Age ranged from 5 to 14 year-old (mean 9.71 ± 3.14 years). Glaucoma was diagnosed on these dogs by using biomicroscopy, direct and indirect ophthalmoscopy, gonioscopy and applanation tonometry. At the time of inclusion, mean of intraocular pressure (IOP) in the glaucomatous eyes was 52.57 mmHg (SD 14.76 range 23 to 70 mmHg). Four cadaverous dogs were also included as control group. These dogs died due to traumatic causes in an Emergency Center, had no ocular diseases and less than two hours death. One milliliter of vitreous was collected from the vitreous chamber from each glaucomatous eye prior to ciliary body ablation procedure, and from the right eye of control dogs using a 21-gauge needle. All samples were stored at -15°C until assays were performed. The TRAP in the vitreous humor was considered as the capacity to inhibit ROS generation by a system composed of 2.2'-azobisamidinopropano (ABAP) 20 mM and luminol 40 μM. Chemiluminescence emitted by Peroxyl radicals produced by ABAP and scavenged by antioxidants of 10μl of vitreous, was measured using a scintillation counter. Trolox 150mM, a hydrosoluble antioxidant analogous of vitamin E, was used for calibration.
TRAP evaluation is based on the measurement of the induction time elicited by the additive incorporation to the reaction medium. This induction time is a measure of the amount of antioxidants present in the sample that are able to scavenge the radicals. The TRAP value of vitreous from glaucomatous eyes was 21 ± 3μM whereas for control dogs was 156 ± 8 μM .The average TRAP values from glaucoma patients was significantly lower than that obtained from normal dogs (p < 0.001), showing a significant decrease of antioxidant defenses of the vitreous due to an increase of prooxidant substances.
Discussion and Conclusions
In order to assess the occurrence of oxidative stress in the vitreous of glaucoma patients, we evaluated the antioxidant status through the determination of TRAP levels and antioxidant enzymes activities. Antioxidant status of biological samples is regarded as an indicator of oxidative stress. A decrease in the antioxidant capacity of tissues and body fluids may be the consequence of increased oxidative processes. Among the methods that determine the antioxidant capacity of a biological sample, TRAP is one of the most common used procedures to evaluate the antioxidant status of biological fluids. (Ferreira et al 2008) These results indicate a significant reduction in the level of water-soluble antioxidants in the vitreous, mainly represented by glutathione and ascorbate. This decrease may be due to the occurrence of oxidative stress in a glaucomatous eye that makes the organ more susceptible to damage associated with ROS production. The vitreous plays an important role in ocular metabolism. It serves as a storage site for retinal metabolites, including glycogen, amino acids and potassium. Retinal and lenticular waste products, including lactic acid and free radicals, are also taken up by the vitreous, which thus serves to protect the lens and retina from toxic compounds. (Gum et al 2007) Prooxidant substances plays an important role in the glaucomatous damage of the ONH, because of this, the study of prooxidant and antioxidant substances in the healthy eye and its modification in a disease is an interesting investigation field since many years. Chemiluminescence is one of the most used methods for measuring antioxidant potencial in many substances in different organs (Hirayama et al 1997) including the eye (Barros et al 2003). Measure of levels of enzymatic antioxidants and molecules with antioxidant potencial are very important for the diagnosis and following of conditions associated with oxidative damage (Repetto et al 1996) Neuroprotective therapy in glaucoma is an interesting complement to hypotensive drugs since the neuronal damage remains even if the IOP returns to normal levels (Yoles & Schwartz 1998). The results obtained here indicate that the concentration of antioxidants in the vitreous of glaucomatous eyes was lower than vitreous from non glaucomatous dogs. In other words, high and sustained levels of IOP in glaucomatous dogs were associated to a significant decrease of the antioxidant defenses in the vitreous. This could be useful to evaluate antioxidant therapies as associate treatment in glaucoma.
1. Gelatt KN, Brooks DE, Koollberg ME. The canine glaucomas. In: Gelatt, K.N. (Ed): Veterinary Ophthalmology (fourth edition), Blackwell Publishing, 2007; 753-811.
2. Anderson DR, Hendrickson A. Effect of intraocular pressure on rapid axoplasmic transport in monkey optic nerve. Invest Ophthalmol Vis Sci. 1974; 13: 77183.
3. Ferreira SM, Lerner SF, Brunzini R, Evelson PA, Llesuy SF. Antioxidant status in the aqueous humour of patients with glaucoma associated with exfoliation syndrome. Eye 2008; 21: 352.
4. Gum GG, Gelatt KN, Esson DW. Physiology of the eye. In: Gelatt, K.N. (Ed): Veterinary Ophthalmology (fourth edition), Blackwell Publishing, 2007; 753-811.
5. Hirayama O, Takagi M, Hukumoto K, Katoh S. Evaluation of antioxidant activity by chemiluminescence. Anal Biochem. 1997, 247 (2): 237-41.
6. Barros PS, Padovani CF, Silva VV, Queiroz L, Barros SB. Antioxidant status of dog aqueous humor after extracapsular lens extraction. Braz J Med Biol Res. 2003; 36 (11): 14911494.
7. Repetto M, Reides C, Gómez M, Costa M, Griemberg G, Llesuy S. Oxidative stress in blood of HIV infected patients. Clin Chim Acta. 1996; 255 (2): 107-17.
8. Yoles E, Schwartz M. potential neuroprotective therapy for glaucomatous optic neuropathy. Surv Ophthalmol. 1998; 42 (4): 367-72.