Effects of hypoxia on retinal pigmented epithelium cells: protection by antioxidants.

Age-related macular degeneration and other eye diseases, such as diabetic retinopathy, are probably linked to the effects of oxygen radicals derived from light or metabolic reactions. We have investigated the effects of hypoxia on bovine retinal pigmented epithelial cells (RPE) and the response of these cells to two antioxidants that have previously shown a beneficial action against free radical-linked senescent involution. The main results of the study were as follows: (i) Hypoxia induced apoptotic damage on RPE cells, with LDH leakage and ATP reduction; (ii) both vitamin C (VC) and N-acetyl-cysteine (NAC) treatment protected against hypoxia-induced apoptosis, with less DNA fragmentation. In our opinion, these findings justify further experimental and clinical work to investigate the role of hypoxia in the mechanisms of age-related RPE injury and death as well as the potential of antioxidant administration to prevent or delay retinal degenerative processes caused by oxygen-dependent pathophysiological conditions.

Inhibition of COX in ocular tissues: an in vitro model to identify selective COX-2 inhibitors.

The aim of this work was to study the regulation of LPS-stimulated PGE 2 synthesis by traditional NSAIDs (piroxicam and diclofenac) and a selective COX-2 inhibitor (NS-398), in cultured bovine corneal endothelial cells and retinal pigmentary epithelial cells. The IC50 values of piroxicam and diclofenac were compared with IC50 values of NS-398, diclofenac, in both types of cells, showed higher potency than piroxicam. Diclofenac seemed to be a COX-2 inhibitor because its IC50 values were similar to the IC50 values of NS-398. We suggest that this in vitro cell assay system could be useful for identifying compounds that selectively inhibit COX-2 in ocular tissues.

An in vitro model of ischemic-like stress in retinal pigmented epithelium cells: protective effects of antioxidants.

We have developed a model of in vitro cell oxidative stress in bovine retinal pigment epithelium cells exposed to a ischemia-like condition obtained by interference with glucose utilization through both oxidative phosphorylation and glycolysis. This resulted in a statistically significant decrease of the intracellular ATP levels, which reflects a bioenergetic decline similar to that associated with mitochondrial damage or loss in normal post-mitotic cells aging in vivo. This new model of cellular oxygen stress seems adequate for investigation of the protective action of antioxidants, in agreement with our finding of a statistically significant increase in the ATP levels over the values of the non-treated samples in retinal pigment epithelium cells exposed to the above oxygen stress in medium supplemented with 300 microM vitamin C or 10 mM N-acetylcysteine.

The ocular pharmacokinetics of topical diclofenac is affected by ocular inflammation.

The ocular pharmacokinetics of topical diclofenac sodium was studied in two experimental models of ocular inflammation and compared to physiological conditions. Keratitis or uveitis were induced by intrastromal injection of clove oil or by intravitreal lipopolysaccharide in rabbits. The control eyes were not inflamed. Simultaneously to the induction of inflammation, 30 microl of 0.1% diclofenac were applied topically in the right eye. Diclofenac levels were measured by HPLC in the cornea, aqueous humor (AH), iris/ciliary body (ICB) and plasma 30 min, 1, 3, 6 and 12 h after application. In physiological conditions, diclofenac reached a peak level in the cornea and ICB at 30 min slowly decreasing afterwards. Low levels of diclofenac were found in AH. In keratitic eyes, two peak levels which were significantly higher than in the controls were found in the cornea 30 min and 3 h after application. Diclofenac concentrations in keratitic AH and ICB were lower than in controls. In uveitic eyes, corneal and ICB levels peaked at 30 min, being significantly higher than in controls, and decreased quickly to very low levels at 1 h after application. In uveitic AH, diclofenac levels were lower than in controls. Plasma levels were very low (less than 0.1 microg/ml) in all experimental groups. It is concluded that the ocular pharmacokinetics of topical diclofenac is affected by inflammatory processes in the eye, reaching higher levels in the target tissues.

PGE2 synthesis by corneal endothelial cells: effect of glucocorticoids and NSAIDs.

The aim of this work was to study the effect of various anti-inflammatory drugs on PGE2 synthesis in cultured bovine corneal endothelial cells (BCECs) stimulated with calcium ionophore A23187 or lipopolysaccharide (LPS) of Salmonella typhimurium. NSAIDs were more potent in inhibiting LPS-stimulated PGE2 synthesis. Diclofenac was more potent than indomethacin, although both drugs showed a 98% maximal inhibitory effect. Dexamethasone inhibited 80% of the A23187-stimulated PGE2 synthesis and only 53% of the LPS-stimulated PGE2 synthesis. Prednisolone did not show an inhibitory effect. The results demonstrate the inhibitory effect of NSAIDs and show differences between the activity of glucocorticoids on PGE2 synthesis in BCECs. Prednisolone could not inhibit PGE2 synthesis in these cells in our experimental conditions.

Concomitant treatment with a 5-lipoxygenase inhibitor improves the anti-inflammatory effect of the inhibition of nitric oxide synthase during the early phase of endotoxin-induced uveitis in the rabbit.

Nitric oxide (NO) synthase inhibitors, such as NG-nitro-L-arginine methyl ester (L-NAME), have been shown to attenuate endotoxin-induced uveitis (EIU) but they could increase leukocyte adhesion to the vascular endothelium. We hypothesize that a concomitant treatment with the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) in 50% dimethylsulfoxide (DMSO, a hydroxyl radical scavenger) could improve the anti-inflammatory activity of L-NAME. EIU was induced in albino rabbits by intravitreal injection of 100 ng lipopolysaccharide. Animals were treated with multiple intraperitoneal injections of 50% DMSO in phosphate-buffered saline (PBS), NDGA (10 mg/kg) in 50% DMSO, L-NAME (50 mg/ kg) in PBS, or the combination NDGA+L-NAME. Uveitis was assessed by slit lamp examination, protein levels in aqueous humor, and myeloperoxidase (MPO) activity in the iris/ciliary body 6 h after induction. Nitrite, leukotriene B4 (LTB4), prostaglandin E2 (PGE2), platelet-activating factor (PAF) and interleukin-1 beta (IL-1 beta) levels in aqueous humor were also determined. NDGA or L-NAME alone did not show a significant reduction of uveitis intensity, although a significant decrease in MPO or in proteins was found, respectively. The combination NDGA+L-NAME significantly reduced the uveitis intensity, MPO in the iris/ciliary body, and the levels of nitrites, LTB4, PGE2, and PAF in aqueous humor. IL-1 beta levels were lower than the detection limit of the radioimmunoassay in all treatment groups. We conclude that concomitant treatment with NDGA in DMSO improves the anti-inflammatory activity of L-NAME during the early phase of EIU, suggesting that the inhibition of NO synthesis could enhance leukocyte infiltration and the release of oxygen free radicals.

Additive effect of nitric oxide and prostaglandin-E2 synthesis inhibitors in endotoxin-induced uveitis in the rabbit.

The involvement of nitric oxide (NO) and prostaglandin E2 (PGE2) was investigated in a model of intraocular inflammation induced by intravitreal injection of endotoxin (lipopolysaccharide, LPS, 10 ng) in rabbits. The severity of uveitis, the myeloperoxidase (MPO) activity in iris-ciliary body, and the protein concentration in aqueous humor were determined. Nitric oxide synthase (NOS) and cyclooxygenase (COX) activities were assessed respectively by nitrite and PGE2 levels in aqueous humor. Treatment with inhibitors of NOS (NG-nitro-L-arginine methyl ester, L-NAME, 50 mg/kp i.p.) or COX (diclofenac, 30 micrograms, topically), alone or in combination, were compared to a saline-treated group. Diclofenac or L-NAME alone reduced or delayed the intensity of uveitis, and partially decreased the protein concentration in aqueous humor; diclofenac, but not L-NAME, partially reduced the polymorphonuclear leukocyte infiltration in the iris ciliary body as indicated by the MPO activity. Treatment with both inhibitors in combination diminished the clinical uveitis, the disruption of the blood-aqueous barrier and the MPO activity in the iris-ciliary body. We conclude that NO and PGE2 have additive effects in endotoxin-induced uveitis in rabbits, and that the inhibition of both pathways would improve the therapeutical management of uveitis.

Protective properties of viscoelastic substances (sodium hyaluronate and 2% hydroxymethylcellulose) against experimental free radical damage to the corneal endothelium.

Hydrogen peroxide (H2O2) has potent oxidant properties due to the action of free radicals (OH.) induced from its degradation. The free radicals specie derived from H2O2 are extremely toxic to the corneal endothelium and quickly induce corneal edema. In the present work, in order to ascertain the endothelial cell protection from viscoelastic substances, we have studied experimental corneal endothelial cell damage caused in the rabbit eye after intracameral injection of different H2O2 concentrations, with and without previous filling and washing out of two widely used viscoelastic substances from the anterior chamber such as 1% sodium hyaluronate (Healon) and hydroxypropylmethylcellulose (HPMC). We observed a dose-dependent endothelial damage in the controls. The experimental groups protected with Healon or HPMC showed statistically fewer corneal endothelial cell lesions than the control group (p < 0.001) for all of the concentrations used. Healon showed superior protective properties than HPMC at higher H2O2 concentrations (100 mM). However, HPMC was superior with 1 and 10 mM peroxide. From this experimental evidence, we conclude that Healon and HPMC are effective as protectors against the corneal endothelial lesions caused by free radicals. This finding may explain some of the beneficial effects of these viscoelastic substances.

Lipid peroxidation in the iris and its protection by means of viscoelastic substances (sodium hyaluronate and hydroxypropylmethylcellulose).

Free radicals, especially the hydroxyl radical (OH.), are known to be toxic for several ocular structures including the cornea, lens, iris and retina through the initiation of lipid peroxidation of cell membranes. The oxidative damage to the iris epithelial-cell membranes, induced by the injection of hydrogen peroxide (H2O2) at different concentrations into the anterior chamber of the rabbit eye, was studied by means of the measurement of lipid peroxidation products. Thiobarbituric acid reactive (TBAR) products were significantly increased compared with normal control iris after the intracameral injection of H2O2 at concentrations of 0.1 (p < 0.02), 10 and 100 mM (p < 0.001). Viscoelastic substances, widely used in anterior ocular surgery, sodium hyaluronate (Healon) and hydroxypropylmethylcellulose, show a protective effect against rabbit iris lipid peroxidation. A statistically significant (p < 0.001) inhibition of the release of TBAR products occurred in both experimental groups that received an injection of these substances prior to a H2O2 injection. This is the first report of lipid peroxidation of the iris and the 'antioxidant'-protective effect of viscoelastic substances. This new technical approach could be used as a test of efficacy of the protective effect of viscoelastic substances.

Treatment of experimental acute corneal inflammation with inhibitors of the oxidative metabolism.

The authors studied the effect of topical 2% 6-aminonicotinamide and 0.1% adenosine on an experimental model of acute corneal inflammation. Luminol-enhanced chemiluminescence (LAC), as an indirect measurement of free-radical release, and computer-assisted planimetry of the corneal ulcer and its infiltrate were performed both ex vivo and in vivo on the fifth day following the induction of experimental alkali burn keratitis. The authors proved that both drugs significantly inhibited LAC both ex vivo and in vivo and that such treatments had also a significant beneficial effect on the evolution of the corneal ulcer and its infiltrate. To the best of the authors' knowledge, this finding had not been previously reported in experimental corneal inflammation and may indicate that treatment with inhibitors of the oxidative metabolism could offer a new approach in the pharmacological modulation of acute corneal inflammation.

The antiinflammatory effect of nordihydroguaiaretic acid in endotoxin induced uveitis in rabbits.

The authors evaluated the anti-inflammatory effect of nordihydroguaiaretic acid (NDGA) and dexamethasone on an endotoxin induced uveitis (EIU) model, in rabbits. Six groups of 12 rabbits were formed. In groups II to V a uveitis was induced by an intravitreal injection of 5 ul of saline, containing 10 ng of endotoxin of Salmonella typhi. In group I, which is considered as the control, an intravitreal injection of 5 ul of saline was given. Each group received a different treatment and the inflammatory reaction was evaluated after 24 hours, quantifying the following parameters: clinical scoring, cells, proteins, PGE2, LTB4 in the aqueous and histopathological scoring. Compared to group II (non treated), group VI (treated with intraperitoneal 2 mg/kg dexamethasone) showed a decrease of 61% of proteins and LTB4, and a decrease of more than 90% of the other parameters studied. All these differences are statistically significant (p < 0.001). In groups III (intraperitoneal NDGA 10 mg/kg), IV and V (NDGA 1% topically every two and four hours respectively), the proteins showed a change of less than 5.5% and the PGE2 was reduced to around 50% compared to group II; these changes are not statistically significant (p > 0.05). The authors observed an important and significant decrease of the other parameters when compared to group II (p < 0.001). It can be concluded that at the doses given here, NDGA shows an effective action on the lipoxygenase pathway without an increase of the production of PGE2.

Corneal concentration and systemic absorption of cyclosporin-A following its topical application in the rabbit eye.

To ascertain the corneal storage and the possible systemic absorption of 2% cyclosporin-A (CsA) eyedrops, the authors studied, by RIA with monoclonal specific antibodies, the corneal and blood levels of the drug following its topical administration on healthy albino rabbit eyes. When topical CsA was administered following a low-dose application schedule [such as a single dose of 100 microliters every 12 h for 5 days (group 1)], CsA corneal levels of 260.8 +/- 59.8 ng/ml and blood levels of 55.67 +/- 24.4 ng/ml (expressed as means +/- SD) were achieved. These levels significantly increased to 1,111.27 +/- 449.4 ng/ml (p > 0.0001) and 88.66 +/- 59.7 ng/ml (p > 0.05), respectively, when a higher dosage was used [100 microliters every 6 h for 10 days (group 2)]. It is shown that a dose-dependent corneal concentration of CsA, due to a cumulative effect, is achieved in the intact rabbit cornea following topical application of the drug. Systemic absorption of CsA, although irregular, does exist. Therefore, when using topical 2% CsA as in this study, a systemic effect of the drug (especially in low weight animals) should be considered when analyzing the results of topical application.