Electrophysiological and clinical tests in dry age-related macular degeneration follow-up: differences between mfERG and OCT.

BACKGROUND: Age-related macular degeneration (AMD) is one of the major causes of progressive and debilitating visual impairment in developed countries and has become a growing health and social issue that needs to be addressed. Imaging techniques and functional tests are useful to assess the degree of macular dysfunction and AMD progression. However, given the slow progression of the disease, it is necessary to identify which techniques are more sensitive for the diagnosis and monitoring of patients with AMD. PURPOSE: To study changes observed with both imaging techniques and electrophysiological tests in dry AMD-diagnosed patients during 2 years in order to identify the most sensitive technique. METHODS: Fundus photography, OCT (macular thickness and number of drusen), Pattern VEP (P100 wave), Pattern ERG (P50 wave) and multifocal ERG (central rings) were carried out in 30 patients that were diagnosed with dry AMD in both eyes. The tests were repeated 1 and 2 years later. RESULTS: No statistically significant changes were observed in visual acuity or in the severity of the disease throughout the study. OCT showed an increase in the number of drusen, as well as in macular thickness. As for the electrophysiological techniques, no significant changes were observed throughout the study in Pattern VEP or Pattern ERG. mfERG showed significant alterations. Statistical analysis showed that mfERG is more efficient in detecting changes throughout the experimental period. CONCLUSIONS: OCT and mfERG are useful in the diagnosis and monitoring of dry AMD patients, whilst mfERG is the most sensitive technique to study the progression of this disease in short periods of time.

Oxidative stress gradient in a medium during human corneal organ culture.

PURPOSE: Lipid peroxidation content was measured in an organ culture medium after one-week storage of human donor corneas. Moreover, the effects of the medium on oxidative stress, antioxidant capacity, and the proliferation of cultured human corneal cells were studied. METHODS: The medium was sampled from the upper and lower halves of storage vials and from controls (n=42). Malondialdehyde (MDA) was measured by high pressure liquid chromatography (HPLC). Cultured human corneal epithelium (CRL-11515) was exposed to different medium samples and monitored for changes in MDA (enzyme-linked immunosorbent assay [ELISA]), total antioxidant capacity (antioxidant assay kit), and proliferation (Ki-67). RESULTS: A significant increase in MDA was observed in the organ culture medium in the lower level of storage vials. The addition of this fraction to cultured cells increased MDA significantly after 3 days, and the medium from both levels significantly increased MDA after 7 days. The medium from both levels significantly decreased the total antioxidant capacity of the cells but did not affect proliferative activity. CONCLUSIONS: An oxidative gradient with an evident biologic effect is established in the medium in vials during organ culture of human donor corneas. Donor tissue stored at the bottom or in lower levels of such vials is exposed to a significant amount of oxidative stress.

Protection by DHA of early hippocampal changes in diabetes: possible role of CREB and NF-κB.

The mechanisms underlying diabetic encephalopathy, are only partially understood. In this study, we try to address the mechanisms of diabetes induced damage and whether docosahexaenoic acid (DHA) could attenuate the degenerative changes in diabetic hippocampus in a rodent model of diabetes. Diabetes was induced in rats by an intraperitoneal injection of streptozotocin. Animals were divided into the following experimental groups: control rats; control animals treated with DHA; untreated diabetic rats; diabetic rats treated with insulin; diabetic rats treated with DHA; diabetic rats treated with insulin and DHA. At the end of week 12, rats were killed and one of the hemispheres was cryosectioned and the other was dissected and hippocampi homogenized. The number of bromodeoxyuridine positive cells in the hippocampus of diabetic rats was decreased, and the latency time to find the platform in the Morris Water maze was significantly increased in the diabetic rats when compared to controls. No changes where observed in the expression of p21 in the hippocampus of control and diabetic rats. Biochemical markers of oxidative stress were altered in hippocampus of diabetic rats, and NFκB-positive cells were increased in the hippocampus of diabetic rats when compared to controls. Treatment with DHA, or the combination of DHA with insulin, significantly restored to control levels all the values mentioned above. Our findings confirm a pivotal role for oxidative stress as well as NF-κB, but not p21, in diabetes-induced hippocampal impairments. Administration of DHA as well as insulin prevented the changes induced by diabetes in hippocampus.

Oxidative stress in keratoconus?

PURPOSE: The purpose of this study was to establish the alterations of oxidative stress-related markers in keratoconus (KC) corneas. METHODS: A total of 6 healthy and 11 ectatic corneas (7 KC and 4 post-LASIK) were studied. Different oxidative stress-related markers were determined to assess their implication in the KC pathophysiology. Total antioxidant capacity and total nitrites present in the samples were assayed. Furthermore, lipid peroxidation products and the glutathione contents were determined, together with 4-hydroxynonenal (4-HNE) immunohistochemistry, to establish the relationship between KC and oxidative stress. RESULTS: The antioxidant capacity and glutathione content in KC corneas were decreased significantly when compared with healthy corneas. Moreover, the total nitrites and lipid peroxidation were significantly elevated in the corneas with KC when compared with the controls. There was a statistically significant difference in the amount of HNE-positive cells in KC corneas when compared with healthy corneas by immunohistochemistry. Post-LASIK ectatic corneas and KC corneas showed similar results. CONCLUSIONS: The increased levels of oxidative stress markers and the decreased antioxidant capacity and antioxidant defenses in KC corneas, as well as in the post-LASIK ectatic corneas, indicate that oxidative stress might be involved in the development of this disease and may provide new insights for its prevention and treatment in the future.

Intravitreal injection of bevacizumab induces inflammatory alterations in a uveitis experimental model.

PURPOSE: Bevacizumab is currently used as an intravitreal agent in the treatment of inflammatory-associated eye diseases. The aim of the current study is to explore the effects of the intravitreal injection of bevacizumab on aqueous humour cytokines and chemokines in an experimental uveitis model. METHODS: Endotoxin-induced uveitis was induced in rats by footpad injections. Bevacizumab was administered by intravitreal injection (75 µg in 3-µL samples) and different chemokine and cytokine proteins were quantified in aqueous humor. RESULTS: Intravitreal administration of bevacizumab led to a several-fold increase of RANTES, MCP-1, and IFN-γ concentrations in aqueous humor of endotoxin-treated rats. CONCLUSIONS: Given the exacerbating effect of bevacizumab on inflammation agents and considering the increasing use of bevacizumab as an off-label intravitreal agent, care should be taken if an underlying inflammatory disease is present.

Comparison of the acute effects of anti-TNF-alpha drugs on a uveitis experimental model.

PURPOSE: To compare histopathological and biochemical effects of the anti-TNF-alpha drugs adalimumab and infliximab in a uveitis experimental model. METHODS: Histopathological evaluation was performed 24 h after endotoxin (200 microg into the footpad) and drug administration, as well as biochemical analysis of oxidative stress-related markers in the aqueous humor. RESULTS: Severe inflammation was found in rat anterior chamber of the eye 24 h after endotoxin. Only infliximab administration partially prevented the endotoxin-induced disruption of the blood-aqueous barrier, as well as the increase in Rantes and MCP-1 concentration in aqueous humor. Both drugs ameliorated the histopathological score after endotoxin. Biochemical analysis revealed that both drugs protected against endotoxin-induced oxidative stress, restoring all markers to control levels, except infliximab, which failed to restore GSH concentration. CONCLUSIONS: Both anti-TNF-alpha drugs were effective in reducing histopathological inflammation but their mechanism of action appears to be different.

IL-2 and IFN-gamma in the retina of diabetic rats.

BACKGROUND: The pathophysiology of the early events leading to diabetic retinopathy is not fully understood. It has been suggested that Inflammatory processes are involved in the development of the disease; however, the concentrations of tissue retinal inflammatory mediators and their possible alteration in diabetic retinopathy have not been described. The aim of this work was to study T-helper cell cytokine and chemokine profiles, and tyrosine nitration in retinal tissue of diabetic rats. METHODS: Cytokines (interleukin IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-10, TNFa, GM-CSF, IFN-g), chemokines (MIP-1a, MIP-2, MIP-3a, MCP-1, GRO/KC, RANTES, Fractalkine), and tyrosine nitration were measured in retinal homogenate obtained from Long-Evans rats after 5 months of experimental diabetes. RESULTS: The T-helper type 1 cytokines IL-2 and INF-gamma, in addition to NO production (measured as nitrotyrosine), were found to be significantly elevated in diabetic rat retina homogenates. None of the other cytokines and chemokines studied were affected by the diabetic condition. CONCLUSIONS: Immunoregulatory cytokines belonging to the Th-1 group (IL-2 and IFN-gamma) were increased in the retina of experimental diabetic rats. Moreover, the nitrotyrosine formation (as an expression of increased NO production) was significantly elevated in the diabetic retina, supporting the concept of an inflammatory element in the development of diabetic retinopathy.

Antioxidants rescue photoreceptors in rd1 mice: Relationship with thiol metabolism.

We have previously shown that the use of a combination of antioxidants delayed the degeneration process in rd1 mouse retina. In an effort to understand the mechanism of action of these substances (zeaxanthin, lutein, alpha-lipoic acid, glutathione, and Lycium barbarum extract) the changes in the levels of several proteins and oxidative stress markers in the rd1 retina have been studied. The treatment increased glutathione peroxidase activity and glutathione levels and decreased cystine concentrations in rd1 retinas. Considering all the results obtained from treated and untreated animals, a high correlation was present between glutathione concentration and glutathione peroxidase activity, and there was a negative correlation between glutathione retinal concentration and number of TUNEL-positive cells. No difference was observed between the numbers of nNOS- and NADPH-diaphorase-positive cells in treated and untreated rd1 mice. Thiol contents and thiol-dependent peroxide metabolism seem to be directly related to the survival of photoreceptors in rd1 mouse retina.

Beneficial effect of docosahexanoic acid and lutein on retinal structural, metabolic, and functional abnormalities in diabetic rats.

PURPOSE: To assess the effect of docosahexanoic acid (DHA) and lutein (both compounds with anti-inflammatory and antioxidant properties) on experimental diabetic retinopathy. METHODS: Male Wistar rats were studied: non-diabetic controls, untreated diabetic controls, and diabetic rats were treated with DHA and lutein or the combination of DHA + insulin and lutein + insulin for 12 weeks. Oxidative stress and inflammatory markers, apoptosis, and functional tests were studied to confirm biochemical and functional changes in the retina of diabetic rats. Malondialdehyde (MDA), glutathione concentrations (GSH), and glutathione peroxidase activity (GPx) were measured as oxidative stress markers. TUNEL assay and caspase-3 immunohistochemistry and electroretinogram were performed. RESULTS: Diabetes increases oxidative stress, nitrotyrosine concentrations, and apoptosis in the retina. At 12 weeks after onset of diabetes, total thickness of retinas of diabetic rats was significantly less than that in control rats. Specifically, the thickness of the outer and inner nuclear layers was reduced significantly in diabetic rats and demonstrated a loss of cells in the GCL. These retinal changes were avoided by the administration of insulin and DHA and lutein alone or in combination with insulin. Impairment of the electroretinogram (b-wave amplitude and latency time) was observed in diabetic rats. DHA and lutein prevented all these changes even under hyperglycemic conditions. CONCLUSIONS: Lutein and DHA are capable of normalizing all the diabetes-induced biochemical, histological, and functional modifications. Specifically, the cell death mechanisms involved deserve further studies to allow the proposal as potential adjuvant therapies to help prevent vision loss in diabetic patients.

Lutein prevents cataract development and progression in diabetic rats.

BACKGROUND: Diabetes mellitus is a heterogeneous metabolic disorder characterized by hyperglycemia. It is often associated with complications, such as cataracts. Cataract, characterized by cloudiness or opacity of the eye lens, is the leading cause of blindness worldwide. METHODS: The present study investigated the effect of lutein, alone or combined with insulin on the progression of eye lens opacities in streptozotocin-diabetic rats for a period of 12 weeks. Tissue markers of oxidative stress were also determined at the end of the experiment. RESULTS: Herein we demonstrate that lutein treatment prevents the development and progression of cataracts (0 eyes with mature cataract, and ten out of 16 eyes with clear lenses in the lutein-treated diabetic animals group, vs. seven and three eyes in the non-treated diabetic group, respectively). Lipid peroxidation is significantly increased in diabetic lens (up to three-fold); lutein and insulin, alone or in combination, are able to prevent this alteration. Only insulin and lutein together could prevent the diabetes-induced decrease of glutathione content. CONCLUSIONS: The combined treatment with lutein and insulin is useful in preventing the development of cataracts in streptozotocin-induced diabetic rats, supporting its utility in diabetes management, especially when a tight metabolic control is difficult to achieve.

Early lipoic acid intake protects retina of diabetic mice.

The aim of this study was to test the effect of lipoic acid treatment on the retina after a short diabetic insult. Diabetes was induced by alloxan and mice were divided into sub-groups; control, diabetic, diabetic+insulin and all groups received+/-lipoic acid (100 mg/kg body weight) for 3 weeks. GSH content, MDA concentration, GPx activity were measured and electroretinograms (ERG) were recorded. Early administration of lipoic acid to diabetic mice prevented the statistically significant decreases of GSH content and GPx activity and normalized MDA concentration. Moreover, lipoic acid restored electroretinogram b-wave amplitude of diabetic animals to control values. Lipoic acid has a protective effect on the diabetic retina.

CR-6 protects glutathione peroxidase activity in experimental diabetes.

Antioxidants can be useful as a supportive therapy in diabetes, and we try to elucidate some of the mechanisms by which these compounds are able to protect from diabetic complications. For this purpose we have assayed, in vitro and in vivo, the ability of CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran), an antioxidant able to scavenge nitrogen reactive species, to protect glutathione peroxidase (GPx) activity. Glucose, in vitro, inhibited GPx activity in a concentration-dependent manner; CR-6 was able to protect GPx activity from glucose-induced inactivation. Alloxan-induced experimental diabetes in mice promoted oxidative stress in the retina and hippocampus, after 3 weeks of hyperglycemia. CR-6 administration prevented not only the alterations of oxidative stress markers (tissue GSH and malondialdehyde (MDA) concentration and GPx activity) but also the impairment of retinal function (as assessed by the modifications in electroretinogram b-wave amplitude). The findings herein confirm the role of nitrogen reactive species in diabetes; therefore, antioxidants effectively quenching these species, such as CR-6, should be considered for the adjuvant treatment of diabetes.

Lutein effect on retina and hippocampus of diabetic mice.

Oxidative stress markers and functional tests were studied to confirm early biochemical and functional changes in retina and hippocampus of diabetic mice. The effects of lutein treatment were also tested. Mice were induced diabetic by alloxan injection and divided into subgroups: control, control+lutein, diabetic, diabetic+lutein, diabetic+insulin, and diabetic+insulin+lutein. Treatments started on Day 4 after alloxan injection and animals were sacrificed on Day 14. Malondialdehyde and glutathione concentrations and glutathione peroxidase activity were measured as oxidative stress markers. The following functional tests for retina and hippocampus were performed: electroretinogram and Morris water maze test. NFkappaB activity was also measured. Oxidative stress and NFkappaB activity increase in the retina and hippocampus after 15 days of diabetes. Impairment of the electroretinogram and a correlation between latencies of the water maze test and glycated hemoglobin (HbA1c) levels were observed. Lutein prevented all these changes even under hyperglycemic conditions. Retina appears to be affected earlier than hippocampus by diabetes-induced oxidative stress. Although a proper glycemic control is desirable in preventing the development of diabetic complications, it is not sufficient to prevent them completely. Lutein could be an appropriate coadjuvant treatment for the changes observed in this study.