A. Orts

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.

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.

Inhibition of adrenomedullary catecholamine release by propranolol isomers and clonidine involving mechanisms unrelated to adrenoceptors

1 Transmural electrical stimulation (10 Hz, 40 V, 1 ms for 60 s) increased total catecholamine secretion from perfused cat adrenal glands; this respnse was enhanced by neostigmine and inhibited by mecamylamine, suggesting that release of acetylcholine from splanchnic nerve terminals was stimulating nicotinic receptors and enhancing catecholamine secretion. 2 Isoprenaline, (‐)‐propranolol and (+)‐propranolol (10−7‐10−5 m) inhibited the electrically‐evoked secretory response by 40–70%; similar reductions were obtained with clonidine and yohimbine. Neither, (+)‐propranolol nor (‐)‐propranolol inhibited K‐evoked secretion from cat adrenals; in contrast, nimodipine potently inhibited it (IC50 = 24 nm). 3 Either, racemic propranolol or the (+)‐ or (‐)‐isomers (1–10 μm) equally inhibited [3H]‐noradrenaline release evoked by nicotine or acetylcholine from cultured bovine adrenal chromaffin cells; clonidine (10 μm) inhibited secretion by 50% and yohimbine or isoprenaline did not affect it. 4 The results indicate that adrenomedullary catecholamine release evoked by splanchnic nerve stimulation is not modulated by α‐or β‐adrenoceptors and suggest that propranolol may inhibit secretion by blocking ion fluxes through the acetylcholine receptor ionophore. Clonidine may inhibit secretion by this same mechanism, and/or by interfering with some intracellular event in the secretory mechanism.

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 µl 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 µg/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.

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.

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.

Effects of the Inhibition of Nitric Oxide Synthase and Lipoxygenase on the Development of Endotoxin-Induced Uveitis

The anti-inflammatory effect of the inhibition of nitric oxide synthase (NOS) or 5lipoxygenase (5-LO), and a concomitant inhibition of both pathways were investigated on the endotoxin-induced uveitis (EIU) model in the rabbit. EIU was induced in albino rabbits by intravitreal injection of 100 ng lipopolisaccharide (LPS) in both eyes. Four groups of rabbits were treated intraperitoneally with phosphate buffered saline (PBS) and 50% DMSO (control group), 10 mg/kg nordihydroguayaretic acid (NDGA) in 50% DMSO, 50 mg/kg NG-nitro-L-arginine methyl ester (L-NAME) in PBS, or with the combination NDGA+L-NAME. The severity of uveitis was graded under slit lamp examination. Six hours after the induction of EIU, animals were sacrificed, and protein (Bio-Rad assay), PGE2and LTB4(RIA), and nitrites were determined in aqueous humor. Leukocyte infiltration in iris/ciliary body was estimated by myeloperoxidase (MPO) activity. NDGA or L-NAME alone did not reduce the severity of EIU when compared to the control group. NDGA reduced MPO in iris/ciliary body correlating with a reduction in LTB4levels, but did not change protein, nitrite and PGE2levels. L-NAME produced a slight decrease in MPO activity and LTB4levels, and reduced protein, nitrite, and PGE2levels. The combination NDGA+L-NAME significantly reduced the severity of uveitis as well as all inflammatory parameters and mediators. We conclude that an additive effect between NO and LO-metabolites plays a role in the development of EIU, and we speculate that interactions between NO and oxygen free radicals could modulate the inflammatory response.

Effect of hypoxic stress in RPE cells : Influence of antioxidant treatments

Retinal pigmented epithelium (RPE) is an ocular tissue that performs several important functions such as light perception and phagocytosis. This study investigated the effects of hypoxia and serum and glucose deprivation of the RPE tissue. The possible protective effects of antioxidants were also studied by determining cell viability, ATP content, and DNA fragmentation. The results demonstrate that antioxidants can protect RPE tissue from death through the inhibition of some mechanisms that lead to cellular apoptosis.

An in vitro model of oxidative stress and hypoxia in retinal pigmented epithelial cells

Retinal pigmented epithelium (RPE) is an ocular tissue that performs a variety of functions including metabolism of photoreceptors. It constitutes an electrical and chemical barrier regulating the movement of solutes and ions between neural retina and the choriocapillary network. Reactive oxygen intermediate (ROI) generation has been implicated in the etiology of certain neurodegenerative diseases of the eye, which may be positively altered by treatment with antioxidant drugs. Cell viability under oxidative stress, hypoxia, and ischemic conditions in RPE were studied in vascular epithelium (VE) cells in culture to explore the possible role of vitamin C and N-acetyl-cysteine to prevent cell injury. The results of this study show that RPE cells may be protected against free radicals and products of peroxidation by endogenous scavengers by treatment with antioxidant drugs.