High Myopia and the Complement System: Factor H in Myopic Maculopathy.

High myopia (HM) is both a medical problem and refractive error of the eye owing to excessive eyeball length, which progressively makes eye tissue atrophic, and is one of the main causes for diminishing visual acuity in developed countries. Despite its high prevalence and many genetic and proteomic studies, no molecular pattern exists that explain the degenerative process underlying HM, which predisposes patients to other diseases like glaucoma, cataracts, retinal detachment and chorioretinal atrophy that affect the macular area. To determine the relation between complement Factors H (CFH) and D (CFD) and the maculopathy of patients with degenerative myopia, we studied aqueous humor samples that were collected by aspiration from 122 patients during cataract surgery. Eyes were classified according to eyeball axial length as high myopia (axial length > 26 mm), low myopia (axial length 23.5-25.9 mm) and control (axial length ˂ 23.4 mm). The degree of maculopathy was classified according to fundus oculi findings following IMI's classification. Subfoveal choroid thickness was measured by optical coherence tomography. CFH and CFD measurements were taken by ELISA. CFH levels were significantly high in the high myopia group vs. the low myopia and control groups (p ˂ 0.05). Significantly high CFH values were found in those eyes with choroid atrophy and neovascularization (p ˂ 0.05). In parallel, the CFH concentration correlated inversely with choroid thickness (R = -0.624). CFD levels did not correlate with maculopathy. All the obtained data seem to suggest that CFH plays a key role in myopic pathology.

Lipoic acid lessens Th1-mediated inflammation in lipopolysaccharide-induced uveitis reducing selectively Th1 lymphocytes-related cytokines release.

Inflammation results in the production of free radicals. We evaluated the anti-inflammatory and antioxidant capacity of lipoic acid in an experimental uveitis model upon a subcutaneous injection of endotoxin into Lewis rats. The role of oxidative stress in the endotoxin-induced uveitis model is well-known. Besides, the Th1 response classically performs a central part in the immunopathological process of experimental autoimmune uveitis. Exogenous sources of lipoic acid have been shown to exhibit antioxidant and anti-inflammatory properties. Our results show that lipoic acid treatment plays a preventive role in endotoxin-induced oxidative stress at 24 h post-administration and reduced Th1 lymphocytes-related cytokines by approximately 50-60%. Simultaneously, lipoic acid treatment caused a significant reduction in uveal histopathological grading and in the protein concentration in aqueous humors, but not in cellular infiltration.

Lutein and docosahexaenoic acid prevent cortex lipid peroxidation in streptozotocin-induced diabetic rat cerebral cortex.

The mechanisms underlying diabetic encephalopathy, are largely unknown. Here, we examined whether docosahexaenoic acid (DHA) and lutein could attenuate the oxidative changes of the diabetic cerebral cortex. The levels of malondialdehyde (MDA) were significantly increased and glutathione (GSH) and glutathione peroxidase activity (GPx) were decreased in diabetic rats. The number of 4-hydroxynonenal (4-HNE) positive cells was increased. Treatment with insulin, lutein or DHA and the combination of each antioxidant with insulin, significantly restored all markers concentrations mentioned above, and the increase in 4-HNE inmunofluorescence. We combined 4-HNE immunofluorescence with NeuN (Neuronal Nuclei) staining. The latter demonstrated extensive overlap with the 4-HNE staining in the cortex from diabetic rats. Our findings demonstrate a clear participation of glucose-induced oxidative stress in the diabetic encephalopathy, and that the cells suffering oxidative stress are neurons. Lowering oxidative stress through the administration of different antioxidants may be beneficial for the central nervous tissue in diabetes.

Lutein prevents the effect of high glucose levels on immune system cells in vivo and in vitro.

Diabetic patients present an increased susceptibility to frequent and protracted infections. The recognition of an impaired immune system has implications for the diagnosis, treatment and outcome of infections. Nuclear Factor kappa B (NF-kappaB) is a redox sensitive transcription factor involved in immune response, cell proliferation and apoptosis that has been associated to the development of diabetic complications. Herein we study the effects of high glucose on oxidative stress markers (malondialdeyde and glutathione contents) and NF-kappaB activity in U937 cells (a human promonocytic cell line). Furtheremore effects of lutein treatment in lymphocytes from diabetic rats was studied. The results show that high glucose induces oxidative stress in immune system cells, both in vitro and in vivo, as well as an increase in their NF-kappaB activity. It is also showed that lutein, a natural antioxidant without hypoglycemiant properties, is able to prevent all the alterations observed. Thus, this study confirms the role of oxidative stress in the immune system impairment described in diabetes, and allows the proposal of antioxidants for the clinical management of the diabetes-associated susceptibility to infections.

[Chronic ethanol feeding induces oxidative stress in the rat retina: treatment with the antioxidant ebselen]. / Estrés oxidativo en la retina de la rata inducido por la administración crónica de etanol: tratamiento con el antioxidante ebselen.

PURPOSE: To assess the involvement of biochemical and functional changes to the retina after chronic ethanol intake in adult rats, and the capacity of the antioxidant ebselen to prevent these changes. METHODS: Male Sprague-Dawley rats were used in the study. They were fed an ethanol-containing liquid diet, whereas a control group was given an ethanol-free isocaloric diet. After six weeks of experiment, the eyes were extracted and homogenized without the lens, and markers of oxidative stress were assayed, i.e., glutathione (GSH) and malondialdehyde (MDA) as an intracellular antioxidant and a lipid peroxidation product, respectively. Moreover, retinal function was assessed by electroretinogram (ERG). RESULTS: The retinal MDA concentration was significantly increased in the ethanol-fed animals compared to controls, whereas the GSH content was significantly reduced in the ethanol-fed group compared to controls. Ethanol also induced a decrease in ERG b-wave amplitude. Ebselen treatment restored the MDA and GSH concentrations and ERG b-wave amplitude to control values. CONCLUSION: These results indicate that chronic alcohol consumption alone and without the influence of nutritional factors alters the retinal redox status as well as its function (ERG). Further studies are required to better understand the protective mechanism of ebselen in this experimental model of chronic alcoholism.

Protective effect of viscoelastics against retina lipid peroxidation.

Viscoelastics or ophthalmic viscosurgical devices are routinely used during anterior segment surgery and also in posterior segment surgery. Studies of the harmful effects of phacoemulsification on corneal endothelial cells suggest that much of this damage is mediated by free radicals. In this study, we compare the possible effects against lipid peroxidation in the retina of three different viscoelastic substances: Viscoat, Healon and Visiol. Herein we demonstrate for the first time that viscoelastics are effective to protect the retina against lipid peroxidation, as can be seen by the slight increase of malondialdehyde in the homogenates incubated with viscoelastic exposed to light and to a temperature of 37 degrees C when compared with the control homogenates.

[Oxidative stress in a model of experimental diabetic retinopathy: the utility of peroxinytrite scavengers]. / Estrés oxidativo en un modelo de retinopatía diabética experimental II: utilidad de agentes secuestrantes de peroxinitritos.

PURPOSE: The retina is the neurosensorial tissue of the eye and is extremely rich in polyunsaturated lipid membranes. This feature makes it especially sensitive to oxygen and/or nitrogen activated species and lipid peroxidation. Several authors have postulated the importance of superoxide (O2-) and peroxynitrite production in the development of diabetic complications. In the present study, we have used two different antioxidants, ebselen and lutein, that present as a common feature their peroxynitrite scavenging capacity, to ameliorate the oxidative stress that exists in the retina in diabetic patients. METHODS: Hyperglycemia was accomplished by the intraperitoneal injection of Alloxan in a mouse model of diabetic retinopathy. Malondialdehyde (MDA) and glutathione (GSH) concentrations in eye homogenates (without the lens) were determined. We also recorded serial electroretinograms (ERG) and measured latency and implicit times. RESULTS: The MDA concentration increased and the GSH concentration decreased in the eyes of the diabetic animals. Treatment with ebselen and lutein restored the MDA and GSH concentrations to control values. Latency and implicit times were not affected by the diabetes. CONCLUSION: New studies are required to better understand the protective mechanism of ebselen and lutein in this model of experimental diabetic retinopathy.

[Oxidative stress in a model for experimental diabetic retinopathy: treatment with antioxidants]. / Estrés oxidativo en un modelo de retinopatía diabética experimental: tratamiento con antioxidantes.

PURPOSE: Diabetic retinopathy is the primary cause of blindness in developed countries, and though strict glycemic control is desirable to prevent complications, this is not always achievable. Thus, adjunctive therapies are needed to help in preventing or delaying the onset of diabetic complications. We have studied the biochemical and functional changes in the retina of diabetic mice, and the ability of ebselen and lutein, two antioxidants, to reverse these effects, using as a comparison the effect of insulin therapy. METHODS: Alloxan injection was used to achieve hyperglycemia. Malondialdehyde (MDA) concentration in blood and glutathione peroxidase (GPx) activity in eye homogenate were measured. Serial electroretinograms (ERG) were recorded. RESULTS: MDA concentration in the blood was high in diabetic animals. GPx activity in eye homogenate decreased in the diabetic conditions. Maximal electroretinogram amplitude decreased in diabetic animals with respect to controls. Ebselen and lutein restored MDA levels, GPx activity and ERG amplitude, and had no effect on glycemia. CONCLUSION: These results call for further studies on ebselen or lutein as adequate adjunctive therapies for diabetes.

[Chronic ethanol feeding induces oxidative stress in the optic nerve of rats]. / Estrés oxidativo en el nervio óptico de la rata inducido por la administración crónica de etanol.

PURPOSE: To assess the involvement of oxidative stress in optic nerves after chronic intake of ethanol in adult rats, when compared to animals fed with an ethanol-free isocaloric diet. METHODS: Male Sprague-Dawley rats were used throughout the study. They were fed an ethanol-containing liquid diet, whereas the pair-fed group was given an ethanol-free isocaloric diet. After six weeks of the experiment, optic nerves were extracted and markers of oxidative stress were assayed, i.e., antioxidants such as glutathione and lipid peroxidation products such as malondialdehyde (MDA). RESULTS: The GSH content in the optic nerves of ethanol-fed animals was significantly reduced, and the concentration of MDA was significantly higher in this group when compared with the pair-fed group. Time-course of body weight of animals in both groups varied identically throughout the six weeks of the experiment. CONCLUSION: The increase in lipid peroxidation products (MDA), together with the decrease in cellular antioxidants (GSH) confirm, in this experimental model, the involvement of oxidative stress in ethanol-induced toxicity of the optic nerves of rats. In view of the body weight time course, the influence of nutritional status on the parameters studied could also be discarded (Arch Soc Esp Oftalmol 2002; 77: 263-268).

Beta-amyloid-induced activation of caspase-3 in primary cultures of rat neurons.

It is known that beta-amyloid peptide (Abeta) contributes to the neurodegeneration in Alzheimer's disease (AD) and operates through activation of an apoptotic pathway. Apoptotic signal is driven by a family of cysteine proteases called caspases. The beta-amyloid precursor protein (APP) is directly and efficiently cleaved by caspases during apoptosis, resulting in elevated beta-amyloid peptide formation. Cerebellar neurons from rat pups were treated with the aged Abeta(25-35) at 1 and 5 microM and fluorescence assays of caspase activity performed over 4 days. We observed an increase in caspase activity after 48 h treatment in both 1 and 5 microM treated cells, then (72-96 h) caspase activity decreased to control values. The data presented support the hypothesis that Abeta(25-35)-induced apoptosis is mediated by the activation of Caspase-3 and that this is a transient effect.

Reduction of brain antioxidant defense upon treatment with butylated hydroxyanisole (BHA) and Sudan III in Syrian golden hamster.

Treatment with the antioxidant butylated hydroxyanisole (BHA) or the azo dye Sudan III during two weeks led to changes in the brain enzymatic antioxidant defense of Syrian golden hamsters. BHA was able to induce liver superoxide dismutase (SOD) 2-fold but had no effect on the brain SOD activity, whereas SOD activity was reduced to 50% in brain and remained unchanged in liver with Sudan III. These two substances are known inducers of DT-diaphorase and in fact this enzymatic activity was induced 4- and 6-fold in liver with BHA and Sudan III, respectively. However, BHA promoted a significant 40% reduction, whereas no change was observed with Sudan III in brain DT-diaphorase activity. Glutathione(GSH)-related enzymatic activities were also assayed in brain and liver. No induction was observed with BHA or Sudan III for any of the activities tested in hamster brain: GSH S-transferase (GST), GSH peroxidase (GSH-Px) and glutathione disulfide (GSSG) reductase (GR). Only 1.3- and 1.4-fold increases of GST and GR activities were observed in liver and no change in any of these enzymatic activities in brain with BHA; a partial limitation of permeability to BHA of the blood-brain barrier may explain this results. Furthermore, Sudan III promoted reductions in all these GSH-related enzymatic activities in brain and liver. The possible explanations for these results are discussed.

Efficacy of the antioxidant ebselen in experimental uveitis.

Inflammation results in the production of free radicals. In a model of experimental uveitis upon subcutaneous injection of endotoxin to Lewis rats, i.e., endotoxin-induced experimental uveitis (EIU), we have evaluated the status of the antioxidant capacity of ocular tissues. EIU results in a decrease of glutathione (GSH) content and glutathione peroxidase (GPx) activity in whole eye homogenates 24-h after endotoxin administration. Furthermore, an increase in malondialdehyde (MDA) content was observed in these same samples, thus confirming the involvement of oxidative stress in the pathophysiology of the process. In view of the ability of the antioxidant ebselen as GPx enzyme mimic, we tested the effect of the oral treatment with two doses of 100 mg/kg body weight of ebselen (first dose administered at the same time of endotoxin, and the second after 12 h). Ebselen administration normalized the GSH and MDA contents and protected the GPx activity of the EIU rat eyes. The GPx activity in the eye homogenate of the treated rats could be completely acounted for by the ebselen-dependent GPx-like activity, i.e., GPx activity measured in the acidic supernatant of the homogenate after neutralization. Unmodified ebselen was detected in whole eye homogenates, thus it shows for the first time the penetration of ebselen through the blood-aqueous and blood-retina barrier. The results herein may allow the proposal of ebselen as a suitable antiinflammatory agent in ocular tissues.

4-Hydroxynonenal inhibits glutathione peroxidase: protection by glutathione.

4-Hydroxy-2,3-trans-nonenal, a lipid peroxidation product, inhibits glutathione peroxidase in a concentration-dependent manner. The concentration providing 50% inhibition is 0.12 mM. This inhibition can be almost completely (89%) prevented by 1 mM glutathione added to the incubation mixture 30 min before 4-hydroxy-2,3-trans-nonenal or 2,3-trans-nonenal, but not by other thiol-containing antioxidants such as 0.5 mM dithiothreitol or beta-mercaptoethanol. Again the addition of 1 mM glutathione, and not of 0.5 mM dithiothreitol or beta-mercaptoethanol, to the enzyme 30 min after incubation with 4-hydroxy-2,3-trans-nonenal restores activity to the same extent as does the preincubation with GSH. In view of the known reactivity of 4-hydroxy-2,3-trans-nonenal with lysine residues and the reversibility of the inhibition, the involvement of a lysine residue in GSH binding to glutathione peroxidase is proposed. The potential relevance of the inhibition of glutathione peroxidase by 4-hydroxy-nonenal to oxidative tissue damage is discussed with particular emphasis on neurological disorders.

Serum vitamin E levels negatively correlate with severity of age-related macular degeneration.

Age-related macular degeneration (AMD) pathogenesis has been related to UV radiation and other factors that may promote increased oxidative damage to the retina. Patients with different AMD grading (n = 25) were compared with an age-matched group of AMD-free subjects (n = 15), both groups older than 60 years. A modification of the AMD grading system is proposed that allows patient grading and not single eye grading. AMD patients showed statistically significant lower serum levels of vitamin E and Zn than AMD-free subjects. Moreover, a negative correlation (Spearman's correlation coefficient r = -0.815, P < 0.001) could be established between AMD grading of both the patients' eyes and serum vitamin E levels. Sun exposure index (SEI) was also compared and found to be significantly higher in the AMD group. The results presented establish the importance of antioxidants in AMD, and set the basis for further studies on adjuvant therapies with antioxidants for AMD. Finally, the results also confirm the pathogenic role of UV radiation in AMD.

Glycemic control and not protein kinase C inhibition prevents the early decrease of glutathione peroxidase activity in peripheral nerve of diabetic mice.

Experimental diabetes promotes changes in biochemical activities of peripheral nervous tissue. Glutathione peroxidase activity decreases in sciatic nerve of diabetic mice very early after onset of experimental diabetes. Effective glycemic control with insulin restores the early lost glutathione peroxidase activity in peripheral nerve of diabetic mice to control values. Data are also presented demonstrating that glutathione peroxidase activity in diabetic mouse peripheral nerve is not modified by the constant delivery of calphostin C, a protein kinase C inhibitor, therefore this decrease seems to be independent on a protein kinase C mediated mechanism. Thus, the early glutathione peroxidase activity decrease in peripheral nerve of diabetic mice is closely related to hyperglycemia, and a tight glycemic control is rather effective in restoring the control levels of this enzymatic activity. The results herein do not rule out the benefits of antioxidant adjuvant therapies in diabetes to help recover the overall decrease in antioxidant defense in peripheral nerve elicited by the decrease of glutathione peroxidase activity.

Serum malondialdehyde: possible use for the clinical management of chronic hepatitis C patients.

Serum lipid peroxidation products are increased in inflammatory liver disease and, as we previously reported, also in chronic hepatitis C. We have performed a specific assay of malondialdehyde, the reported most abundant product of lipid peroxidation, in serum of twenty four chronic hepatitis C patients, before, during, and after interferon treatment. Liver biopsies were performed in each patient before and after interferon treatment. The results show higher serum malondialdehyde values in chronic hepatitis C patients than healthy subjects (n = 68) before interferon treatment (p < .001). Mean value of serum malondialdehyde levels after interferon treatment was significantly lower than before it (p < .002). Associating the histopathological findings in each of the 48 biopsies performed, with serum malondialdehyde and alanine aminotransferase activity levels, of the sample obtained the same day of biopsy, a much better correspondence with the histopathological severity was observed for malondialdehyde concentration than for alanine aminotransferase activity. These levels decreased significantly after interferon treatment. However, when the patients were grouped in responding (group I; n = 9) and non-responding (group II; n = 15) to interferon treatment, according to the histopathological findings before and after interferon, the values of group I before interferon treatment were significantly higher than group II (p < .03). Thus, a potential predictive value could be ascribed to the serum malondialdehyde levels before interferon treatment in these patients. We propose the utility of the specific assay of malondialdehyde for the clinical management of chronic hepatitis C patients.

Lipid peroxidation products and antioxidants in human disease.

Lipid peroxidation (LPO) is a free radical-related process that in biologic systems may occur under enzymatic control, e.g., for the generation of lipid-derived inflammatory mediators, or nonenzymatically. This latter form is associated mostly with cellular damage as a result of oxidative stress, which also involves cellular antioxidants in this process. This article focuses on the relevance of two LPO products, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), to the pathophysiology of human disease. The former has been studied in human serum samples of hepatitis C virus-infected adults and human immunodeficiency virus-infected children. In these two cases it is shown that the specific assay of serum MDA is useful for the clinical management of these patients. The presence of MDA in subretinal fluid of patients with retinal detachment suggests the involvement of oxidative stress in this process. Moreover, we were able to report the dependence of this involvement on the degree of myopia in these patients. The assay of MDA contents in the peripheral nerves of rats fed a chronic alcohol-containing diet or diabetic mice also confirms the pathophysiologic role of oxidative stress in these experimental models. In these two cases, associated with an increase in tissue LPO products content, we detected a decrease of glutathione peroxidase (GSHPx) activity in peripheral nerve, among other modifications. We have demonstrated that in vitro HNE is able to inhibit GSHPx activity in an apparent competitive manner, and that glutathione may partially protect and/or prevent this inactivation. The accumulation of LPO products in the brain of patients with Alzheimer's disease has also been described, and it is on the basis of this observation that we have tried to elucidate the role of oxidative stress and cellular antioxidants in beta-amyloid-induced apoptotic cell death of rat embryo neurons. Finally, we discuss the possible role of the observed vascular effects of HNE on human arteries.

Experimental diabetic neuropathy: role of oxidative stress and mechanisms involved.

Oxidative stress has been related to the development of diabetic neuropathy. Experimental diabetes (alloxan injection of mice) promotes early biochemical changes in peripheral nervous tissue, e.g. decrease in Na,K-ATPase activity and glutathione (GSH) peroxidase (GSHPx) activity. The former decrease can be reverted by inhibiting protein kinase C (PKC), since it has been reported that PKC is activated in these experimental conditions. Here we present data demonstrating that the inhibition of PKC, as early as 4 days after alloxan administration, is not able to return to normal values GSHPx activity in sciatic nerve of diabetes mice. Thus, it would fit with our previous proposal of the possible glycation of this protein as an early event in experimental diabetes, and apparently rules out the control of GSHPx activity by PKC in this tissue.

Serum malondialdehyde in HIV-seropositive children negatively correlates with CD4+ lymphocytes count.

Human immunodeficiency virus (HIV) infection is associated with oxidative stress as it has been demonstrated in adult-seropositive individuals. We show in this study that serum malondialdehyde (MDA) concentration of HIV-infected children was significantly higher than in control children. A negative correlation (r = -0.515) was found in HIV-infected children between their CD4+ lymphocyte count, and MDA concentration but not with serum antioxidant status. The increase of MDA concentration in HIV-seropositive children confirms the involvement of oxidative stress in the pathophysiology of this infection also in childhood. Because of the importance of oxidative stress and antioxidants for HIV viral replication, the adequacy of an adjuvant therapy with antioxidants should be considered; an adequate candidate for it could be N-acetylcysteine.

Chronic ethanol feeding induces cellular antioxidants decrease and oxidative stress in rat peripheral nerves. Effect of S-adenosyl-L-methionine and N-acetyl-L-cysteine.

Chronic ethanol feeding promotes oxidative stress in rat peripheral nerve. Malondialdehyde, a lipid peroxidation product, content increases in sciatic nerves of rats fed an ethanol-containing diet, when compared with pair-fed animals. Moreover, glutathione content and glutathione peroxidase activity in this same tissue decrease in ethanol-fed vs. pair-fed rats. S-Adenosyl-L-methionine and N-acetyl-L-cysteine, both with possible therapeutic action on alcoholism, were tested in this animal model. Only N-acetyl-L-cysteine was able to normalize malondialdehyde content and to restore glutathione content and glutathione peroxidase activity, to values not significantly different from those of sciatic nerves from pair-fed animals. The reasons for the different effect of both substances tested is also discussed.