thiourea and Retinal-Degeneration

Retinal degeneration (RD) such as retinitis pigmentosa and age-related macular degeneration are major causes of blindness in adulthood. As one of the model for RD, intraperitoneal injection of N-methyl-N-nitrosourea (MNU) is widely used because of its selective photoreceptor cell death. It has been reported that MNU increases intracellular calcium ions in the retina and induces photoreceptor cell death. Although calcium ion influx triggers the neuronal nitric oxide synthase (nNOS) activation, the role of nNOS on photoreceptor cell death by MNU has not been reported yet. In this study, we investigated the contribution of nNOS on photoreceptor cell death induced by MNU in mice. MNU significantly increased NOS activation at 3 day after treatment. Then, we evaluated the effect of nNOS specific inhibitor, ethyl[4-(trifluoromethyl) phenyl]carbamimidothioate (ETPI) on the MNU-induced photoreceptor cell death. At 3 days, ETPI clearly inhibited the MNU-induced cell death in the ONL. These data indicate that nNOS is a key molecule for pathogenesis of MNU-induced photoreceptor cell death.

Topics: Alkylating Agents; Animals; Apoptosis; Enzyme Activation; Enzyme Inhibitors; Immunohistochemistry; In Situ Nick-End Labeling; Injections, Intraperitoneal; Male; Methylnitrosourea; Mice, Inbred C57BL; NADPH Dehydrogenase; Nitric Oxide Synthase Type I; Photoreceptor Cells, Vertebrate; Retina; Retinal Degeneration; Retinal Photoreceptor Cell Inner Segment; Thiourea

N-methyl-N-nitrosourea (MNU) is widely used to study the mechanism of retinal degenerative diseases (RDs) because of its selectivity of photoreceptor cell death. Many reports suggest that excessive nitric oxide (NO) plays a crucial role in neuronal cell death. We hypothesized that nitric oxide synthase (NOS)/NO are involved in photoreceptor cell death by MNU. We found that the levels of NO increased after MNU treatment. Furthermore, we demonstrated that neuronal NOS specific inhibitor attenuated photoreceptor cell death by MNU in mice. We believe that our findings might be a new target for the treatment of RDs.

Topics: Animals; Cell Death; Enzyme Inhibitors; Male; Methylnitrosourea; Mice, Inbred C57BL; Molecular Targeted Therapy; Nitric Oxide; Nitric Oxide Synthase Type I; Photoreceptor Cells; Retinal Degeneration; Thiourea

Potentiation of retinal degeneration by intense light exposure, and its amelioration by an antioxidant, were studied in a rat model of Smith-Lemli-Opitz syndrome (SLOS), in comparison with normal (control) Sprague-Dawley rats. The SLOS model is created by treating rats with AY9944, a selective inhibitor of cholesterol synthesis at the level of 3beta-hydroxysterol-Delta7-reductase. A subset of rats was treated with dimethylthiourea (DMTU), a synthetic antioxidant, 24 and 1 hr prior to light exposure. Half of the animals (+/-DMTU) were exposed to intense, constant, green light (24hr, 1700lx, 490-580 nm), while the others were maintained in darkness. Subsequently all animals were returned to dim cyclic light (20-40 lx, 12 hr light-12 hr dark) for 2 weeks, after which electroretinograms were recorded. One eye from each rat was taken for histological and quantitative morphometric analyses; sterol analysis was performed on retinas from contralateral eyes. HPLC analysis confirmed the accumulation of 7-dehydrocholesterol (7DHC) in retinas of AY9944-treated rats; cholesterol represented >99% of the sterol in control retinas. Histology of retinas from unexposed, AY9944-treated rats (6-week-old) was normal. In contrast, age-matched, light-exposed rats exhibited massive photoreceptor cell loss in both the superior and inferior hemispheres, and concomitant rod and cone dysfunction. The severity and geographic extent of the damage was far greater than that observed in normal albino rats exposed to the same conditions. DMTU pre-treatment largely prevented these degenerative changes. These findings indicate that the AY9944-induced rat SLOS model is hypersensitive to intense light-induced retinal damage, relative to normal rats. DMTU protection against light-induced damage implicates free radical-based oxidation in the retinal degeneration process. Furthermore, the use of green light (corresponding to the absorption maxima of rhodopsin) implicates rhodopsin in the initiation of the pathobiological mechanism. We propose that generation of cytotoxic oxysterols (by-products of 7DHC oxidation) is an integral part of retinal cell death in the SLOS rat model, which is exacerbated by intense light. Furthermore, the results predict light-dependent potentiation of retinal degeneration in SLOS patients, and the possible ameliorative effects of antioxidant therapy.

Topics: Animals; Biomarkers; Cholesterol; Dark Adaptation; Docosahexaenoic Acids; Electroretinography; Free Radical Scavengers; Male; Models, Animal; Photic Stimulation; Photoreceptor Cells; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Smith-Lemli-Opitz Syndrome; Thiourea; trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride

To determine the relative susceptibility of rats to retinal light damage at different times of the day or night.. Rats maintained in a dim cyclic light or dark environment were exposed to a single dose of intense green light beginning at various times. Normally, light exposures were for 8 or 3 hours, respectively, although longer and shorter periods were also used. Some animals were treated with the synthetic antioxidant dimethylthiourea (DMTU) before or after the onset of light. The extent of visual cell loss was estimated from measurements of rhodopsin and retinal DNA levels 2 weeks after light treatment. The time course of retinal DNA fragmentation, and the expression profiles of heme oxygenase-1 (HO-1) and interphotoreceptor retinol binding protein (IRBP) were determined 1 to 2 days after exposure.. When dark-adapted, cyclic light-reared or dark-reared rats were exposed to intense light during normal nighttime hours (2000-0800) the loss of rhodopsin or photoreceptor cell DNA was approximately twofold greater than that found in rats exposed to light during the day (0800-2000). The relative degree of light damage susceptibility persisted in cyclic light-reared rats after dark adaptation for up to 3 additional days. For rats reared in a reversed light cycle, the light-induced loss of rhodopsin was also reversed. Longer duration light treatments revealed that dim cyclic light-reared rats were three- to fourfold more susceptible to light damage at 0100 than at 1700 and that dark-reared animals were approximately twofold more susceptible. Intense light exposure at 0100 resulted in greater retinal DNA fragmentation and the earlier appearance of apoptotic DNA ladders than at 1700. The extent of retinal DNA damage also correlated with an induction of retinal HO-1 mRNA and with a reduction in IRBP transcription. Antioxidant treatment with DMTU was effective in preventing retinal light damage when given before but not after the onset of light.. These results confirm earlier work showing greater retinal light damage in rats exposed at night rather than during the day and extend those findings by demonstrating that a single, relatively short, intense light exposure causes a circadian-dependent, oxidatively induced loss of photoreceptor cells. The light-induced loss of photoreceptor cells is preceded by DNA fragmentation and by alterations in the normal transcriptional events in the retina and within the photoreceptors. The expression profile of an intrinsic retinal factor(s) at the onset of light exposure appears to be important in determining light damage susceptibility.

Topics: Animals; Blotting, Northern; Circadian Rhythm; Dark Adaptation; DNA Damage; DNA Fragmentation; Electrophoresis, Agar Gel; Eye Proteins; Free Radical Scavengers; Gene Expression Profiling; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Light; Male; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Retinol-Binding Proteins; Rhodopsin; Thiourea

To investigate the functional protective effect of a synthetic (dimethylthiourea, DMTU) and a natural antioxidant (Ginkgo biloba extract, EGb 761) against light-induced retinal degeneration.. Wistar rats were exposed for 24 hours to 1700-lux light after treatment with DMTU or EGb 761. Electroretinograms were recorded before and on day (D)1, D3, D8, D15, D22, and D29 after light exposure. The b-wave amplitude was plotted against log L (ganzfeld luminance), providing the b-wave sensitivity curve. The Naka-Rushton function fitted to the sensitivity curve enabled derivation of the parameters Bmax (saturated amplitude) and K (luminance-inducing Bmax/2). In addition, rats from each group were killed for retinal morphometric analyses.. In the untreated group, light exposure caused collapse of the b-wave sensitivity curves. Bmax was reduced by 51% at D1 without subsequent recovery. K increased temporarily, reverting to normal values 8 days later. The outer nuclear layer thicknesses decreased markedly in the superior retina. In the treated groups, light exposure had a weaker effect on sensitivity curves. The values of Bmax were not significantly different from those in the unexposed-untreated group, although K increased temporarily. Retinal morphometry was preserved.. Dimethylthiourea and EGb 761 afford functional protection against light-induced retinal damage.

Topics: Animals; Antioxidants; Electroretinography; Flavonoids; Free Radical Scavengers; Ginkgo biloba; Male; Photoreceptor Cells, Vertebrate; Plant Extracts; Rats; Rats, Wistar; Reference Values; Retina; Retinal Degeneration; Thiourea

The neuronal redox status influences the expression of genes involved in neuronal survival. We previously showed that antioxidants may reduce the number of dying ganglion cells following axotomy in chick embryos. In the present study, we show that various antioxidants, including the new spin trap azulenyl nitrone and 1,3-dimethyl-2-thiourea, protect axotomized ganglion cells, confirming that neuronal death involves an imbalance of the cellular redox status towards oxidation. However, high concentrations of antioxidants did not protect ganglion cells, suggesting that excessive reduction is detrimental for neurons. Simultaneous injections of two different antioxidants gave results only partly supporting this view. Combinations of azulenyl nitrone and N-acetyl cysteine in fact gave greater protection than either antioxidant alone, whereas N-acetyl cysteine lost its neuroprotective effects and diminished those of alpha-phenyl-N-tert-butyl nitrone when the two compounds were injected simultaneously. The results of the combined treatments suggest that azulenyl nitrone and alpha-phenyl-N-tert-butyl nitrone do not have the same chemical effects within the ganglion cells. Moreover, N-acetyl cysteine's own antioxidant properties enhance the spin trapping effects of azulenyl nitrone but potentiate the toxicity of alpha-phenyl-N-tert-butyl nitrone. Our main conclusion is that neuronal survival requires the maintenance of the redox status near an optimal set-point. "Reductive stress" may be as dangerous as oxidative stress.

Topics: Acetylcysteine; Animals; Antioxidants; Axotomy; Azulenes; Cell Count; Cell Survival; Chick Embryo; Cyclic N-Oxides; Eye; Neuroprotective Agents; Nitrogen Oxides; Oxidation-Reduction; Oxidative Stress; Retina; Retinal Degeneration; Retinal Ganglion Cells; Sesquiterpenes; Thiourea

To determine the effects of age and long-term light- or dark-rearing environments on acute, intense-light-mediated retinal degeneration.. Male albino rats were maintained in a dim cyclic light environment or in darkness for as long as 1 year. When aged 2, 4, 8, and 12 months, some rats were given the synthetic antioxidant dimethylthiourea (DMTU) by intraperitoneal injection and were exposed to intense visible light for as long as 24 hours. Uninjected control rats were exposed to light at the same time. Other rats were treated with light of lower intensity for various periods. Two weeks after intense-light treatment, photoreceptor cell degeneration was estimated by determining the level of rhodopsin and by measuring the content of photoreceptor cell DNA. Light-induced changes in retinal DNA were analyzed immediately after exposure by neutral gel electrophoresis and by 8-hydroxy-deoxyguanosine measurements. Expression of the antioxidative stress protein heme oxygenase-1 (HO-1) was determined by northern blot analysis of mRNA in retinal extracts.. At all ages, rats reared in cyclic dim-light conditions had lower rhodopsin levels than did rats reared in darkness; photoreceptor cell DNA levels were unaffected by the rearing environment. Senescent losses in rhodopsin and retinal DNA were significant after rats were 12 months old. Dim-light-reared rats exhibited an age-related increase in retinal light damage susceptibility, whereas dark-reared rats were equally susceptible to damage at all ages. In both types of rats, the mechanism of light-induced cell death involved an apoptotic process, visualized by the pattern of DNA fragments on electrophoretic gels. The process also induced the expression of HO-1 mRNA. Photoreceptor cell loss determined by biochemical measurement, DNA fragmentation, and HO-1 induction were dramatically reduced by the administration of DMTU.. The age-related increase in susceptibility to retinal light damage in rats is influenced by their long-term daily light history. Decreasing retinal irradiance by dark-rearing eliminates the age-related increase in light damage, suggesting a correlation between light environment and retinal gene expression associated with damage. In all rats, retinal light damage resulted in a pattern of DNA fragmentation consistent with apoptotic cell death and in an increased expression of HO-1 mRNA. Antioxidant treatment greatly reduced apoptosis and HO-1 expression. This indicates that light damage involves an oxidative process that may also trigger apoptosis in the retina. The rat aging model may provide useful insights into the role of light environment associated with retinal degeneration in an aging human population.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aging; Animals; Cell Death; Dark Adaptation; Deoxyguanosine; DNA Fragmentation; Electrophoresis, Polyacrylamide Gel; Free Radical Scavengers; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Light; Male; Radiation Injuries, Experimental; Rats; Rats, Sprague-Dawley; Retina; Retinal Degeneration; Rhodopsin; RNA, Messenger; Thiourea

Free radical scavengers and a calcium overload blocker have been demonstrated separately to ameliorate light-induced retinal degeneration, suggesting that both free radical formation and increased intracellular calcium levels are involved in the pathologic changes of this disease process. To understand the relationship between these two mechanisms, we studied the ameliorative effects of combined treatment with flunarizine and dimethylthiourea as well as individual treatment with either drug in a rat model of light-induced retinal degeneration. At 6 hr and 6 and 14 days after light exposure, morphologic and morphometric studies of the retinas from the rats receiving the combined treatment demonstrated better-preserved retinal pigment epithelial cells, photoreceptor elements, and nuclei than did retinas from rats receiving treatment with either flunarizine or dimethylthiourea alone. Rhodopsin level measurements at 6 and 14 days further substantiated the results of the protective effects on the photoreceptor outer segments. Because we used a saturating dose for dimethylthiourea, the enhanced ameliorative effect of the combination treatment suggested that free radical formation and elevated intracellular calcium levels were two separate mechanisms in light-induced retinal degeneration.

Topics: Animals; Drug Therapy, Combination; Flunarizine; Light; Male; Rats; Rats, Inbred Lew; Retina; Retinal Degeneration; Thiourea