Ascorbate and glutathione levels in the developing normal and dystrophic rat retina: effect of intense light exposure

Ascorbic acid and glutathione were measured in retinas excised from normal rats reared in a cyclic light or dark environment and in dystrophic rats from the dark environment. Similar measurements were made on retinas from age matched rats exposed to intense visible light for periods of up to 24 hours. In other rats, ascorbic acid was given for various periods before exposure to intense light and the degree of photoreceptor cell death determined subsequently by rhodopsin measurements. In non-intense light treated rats ascorbate and glutathione were 12.1 nmol/retina at 20 days of age and 13.3 - 15.9 nmol/retina in 60 day old animals. In dystrophic rat retinas glutathione was 4-8% higher and ascorbate 10-20% higher than in normal dark reared rats. Although the levels of ascorbate and glutathione per retina increased during development, the molar ratios of the antioxidant materials to rhodopsin decreased by 36% and 60% in normal and dystrophic rats respectively. The levels of glutathione in young cyclic light or dark reared normals were unaffected by intense light exposure of either short (2-4 hrs) or long (24 hrs) duration. However, in both 20 and 40 day old dystrophic rats, intense light exposure resulted in a significant increase in retinal glutathione. In contrast to glutathione, retinal ascorbate decreased in normal rats exposed to intense light for 24 hrs, in an age and prior light environment dependent fashion. At ages greater than 20 days, normal rats exposed to light had significantly lower retinal ascorbate levels than their non-light exposed counterparts. The levels of ascorbate in 21-40 and 41-60 day old dark reared rat retinas were also significantly lower than in comparable intense light treated-cyclic light reared rats. In the youngest dystrophic rats whole eye ascorbate (retina, RPE, choroid and sclera) was 20-30% lower than in non-light treated rats, but in older mutant rats (41-60 day) light had no effect on the level of ascorbate in the retina. As determined by the level of rhodopsin remaining in the eye two weeks after 24 hrs light exposure, cyclic light reared rats lost 50-55% of their visual cells. However, cyclic light rats supplemented with ascorbic acid before intense light exposure lost only 30-35% of their visual cells.     

Protection by dimethylthiourea against retinal light damage in rats.

The protective effect of dimethylthiourea (DMTU) against retinal light damage was determined in albino rats reared in darkness or in weak cyclic light. Rats maintained under these conditions were treated with DMTU at different concentrations and dosing schedules and then exposed for various times to intense visible light, either intermittently (1 hr light and 2 hr dark) or continuously. The extent of retinal light damage was determined 2 weeks after light exposure by comparing rhodopsin levels in experimental rats with those in unexposed control animals. To determine the effect of DMTU on rod outer segment (ROS) membrane fatty acids, ROS were isolated immediately after intermittent light exposure, and fatty acid compositions were measured. The time course for DMTU uptake and its distribution in serum, retina, and the retinal pigment epithelium (RPE)/choroid complex was determined in other rats not exposed to intense light. After intraperitoneal injection of the drug (500 mg/kg body weight), DMTU appeared rapidly in the serum, retina, and the RPE and choroid. In the ocular tissues, it was distributed 70-80% in the retina and 20-30% in the RPE and choroid. This antioxidant appears to have a long half-life because it was present in these same tissues 72 hr after a second intraperitoneal injection. For rats reared in the weak cyclic light environment, DMTU (two injections) provided complete protection against rhodopsin loss after intense light exposures of up to 16 hr. Only 15% rhodopsin loss was found in cyclic-light DMTU-treated rats after 24 hr of intermittent or continuous light. For rats reared in darkness, DMTU treatment resulted in a rhodopsin loss of less than 20% after 8-16 hr of continuous light and approximately 40% after similar exposure to intermittent light. Irrespective of the type of light exposure, rhodopsin loss in the dark-reared DMTU-treated rats was nearly identical to that found in uninjected cyclic light-reared animals. In rats from both light-rearing environments, DMTU treatment prevented the light-induced loss of docosahexaenoic acid from ROS membranes. As measured by rhodopsin levels 2 weeks later, DMTU was most effective when given as two doses administered 24 hr before and just before intense light exposure. As a single dose given during continuous light exposure, DMTU protected cyclic light-reared rats for at least 4 hr after the start of exposure but was ineffective in dark-reared animals if injected 1 hr after the start of light. It was also ineffective in both types of rats when given after light exposure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Melatonin-binding in the frog retina: autoradiographic and biochemical analysis

Binding of melatonin was examined in the retina of Rana pipiens. When intact frog retinas were incubated with 3H-melatonin and processed for autoradiography, most of the radioactivity was localized to the melanosomes of the retinal pigment epithelium-choroid (RPE-choroid) and to the outer plexiform layer of the retina. Melanosome-enriched fractions of the RPE-choroid and membrane-enriched fractions of the neural retina demonstrated saturable melatonin binding when incubated with increasing melatonin concentration. Thin-layer chromatography showed that greater than 98% of the bound radioactivity was authentic melatonin. Scatchard analysis revealed a single population of binding sites with apparent Kd values of 6 X 10(-7) M for both the RPE-choroid and neural retina. When various indole analogs were tested for their ability to inhibit 3H-melatonin binding to the neural retina, both 5-methoxytryptophol and 6-chloromelatonin demonstrated complete displacement of melatonin binding. Endogenous retinal melatonin levels were measured by radioimmunoassay. A twofold increase in melatonin levels was observed during the dark period with peak levels at 384.5 +/- 28.8 pgms melatonin/pair retinas. Melatonin levels persisted in constant darkness, but were suppressed in constant light. Our data suggest that in the frog, the sites of action of retinal melatonin are the melanosomes of the RPE-choroid and the outer plexiform layer of the neural retina.     

Adaptive changes in visual cell transduction protein levels: effect of light.

Long-term environmental light-mediated changes in visual cell transduction proteins were studied to assess the influence of rearing environment on their levels and their potential effects on intense light-induced retinal damage. The levels of rhodopsin, S-antigen and the alpha subunit of transducin were measured in whole eye detergent extracts, retinal homogenates or rod outer segments isolated from rats reared in weak cyclic light or darkness, and following a change in rearing environment. Rats changed from weak cyclic light to darkness had 22% more rhodopsin per eye than cyclic-light rats after 12-14 days in the new environment. Western trans-blot analysis of retinal proteins from these dark-maintained animals contained 65% higher levels of immunologically detectable alpha transducin; S-antigen levels were approximately 45% lower than in cyclic-light rats. In rats changed from the dark environment to weak cyclic light, rhodopsin levels decreased by 18% during a comparable period; retinal alpha transducin was 35% lower, S-antigen was 30% higher. At various times after the change in rearing environment, some rats were exposed to intense visible light to determine their susceptibility to retinal damage. Two weeks after an 8-hr exposure, cyclic-light reared rats had rhodopsin levels only 10% lower than control (2.1 nmol per eye). However, rhodopsin was 75% lower when cyclic-light rats were maintained in darkness for 2 weeks before intense light. For animals originally reared in darkness, rhodopsin was 78% lower following 8 hr of intense light, whereas only 30% rhodopsin loss occurred in dark-reared rats after previous maintenance for 2 weeks in weak cyclic-light.(ABSTRACT TRUNCATED AT 250 WORDS)     

地塞米松对光损伤大鼠视细胞凋亡的防治作用

目的 :研究地塞米松对光损伤及视细胞凋亡的防治作用 ,以探讨光损伤视细胞凋亡的发生机制。方法 :所有SD大鼠经循环光环境适应 7d ,实验前暗适应 36h。实验A组的大鼠只光照 ,实验B组的大鼠光照 6h后在暗箱中喂养 ,实验a组的大鼠在暗适应后腹腔注射地塞米松再光照 ,实验b组的大鼠光照 6h后在暗箱中喂养 ,且每天应用地塞米松。对经以上处理过的大鼠行灌流固定 ,摘除眼球。光镜标本在常规脱水、透明、石蜡包埋切片后 ,行HE、TUNEL法染色 ,光镜观察。应用CIAS 10 0 0图像分析系统定量检测外核层面积和视细胞凋亡指数 ,所得数据做统计学分析。结果 :在实验A组中 ,随着光照时间的增加 ,视网膜光损伤逐渐加重 ,视细胞凋亡逐渐增多 ,外核层面积逐渐减少。而在实验a组中 ,也出现如实验A组的规律性变化 ,但应用地塞米松后 ,视网膜光损伤程度减轻 ,发生视细胞凋亡的时间延迟 3h。两实验组定量检测结果经统计学分析表明 ,地塞米松对视网膜光损伤及视细胞凋亡有明显的预防作用。实验B组和实验b组的定量检测结果经统计学分析表明 ,地塞米松对视网膜光损伤及视细胞凋亡有明显的治疗作用。结论 :地塞米松在实验性大鼠视网膜光损伤及视细胞凋亡过程中发挥较好的防治作用     

Light-sensitive melatonin synthesis by Xenopus photoreceptors after destruction of the inner retina.

Several lines of evidence indicate that retinal photoreceptors produce melatonin. However, there are other potential melatonin sources in the retina, and melatonin synthesis can be regulated by feedback from the inner retina. To analyze cellular mechanisms of melatonin regulation in retinal photoreceptors, we have developed an in vitro method for destruction of the inner retina that preserves functional photoreceptors in contact with the pigment epithelium. Eyecups, which include the neural retina, retinal pigment epithelium, choriod, and sclera were prepared. The vitreal surface of the retina in each eyecup was washed sequentially with 1% Triton X-100, water, and culture medium. This lysed the ganglion cells and neurons and glia of the inner nuclear layer, causing the retina to split apart within the inner nuclear layer. The damaged inner retina was peeled away, leaving photoreceptors attached to the pigment epithelium. The cell density of the inner nuclear layer was reduced 94% by this method, but there was little apparent damage to the photoreceptors. Lesioned eyecups produced normal melatonin levels in darkness at night, and melatonin production was inhibited by light. These results indicate that the inner retina is not necessary for melatonin production nor for regulation of photoreceptor melatonin synthesis by light. The lesion method used in this study may be useful for other physiological and biochemical studies of photoreceptors.     

Light damage in the developing retina of the albino rat: an electroretinographic study

The albino rat retina is severely damaged by exposure to bright light. The degree of damage depends upon the intensity of the exposure and its duration. In the present study, electroretinographic (ERG) responses were measured in rats exposed at different ages during the period of retinal development to 24 hr of bright light and then transferred to darkness for about 2 months. The ERG data indicated that if the bright-light exposure was done prior to 20 days of age, the retina was resistant to the light damage, and the dark-adapted ERG responses measured later were normal. In rats older than 20 days, light damage increased with age until, in rats exposed to light at age 30 days, the ERG was unrecordable. Further experiments showed that light exposure did damage the 15-day-old rat retina; however, complete recovery was attained within 15 days postexposure.     

The protective effect of ascorbate in retinal light damage of rats

Cyclic light and dark-reared rats were exposed to intense visible light for various periods and then rhodopsin-measured following recovery in darkness for up to 14 days. Animals were injected with ascorbic acid or ascorbate derivatives at various doses prior to light exposure in green Plexiglas chambers. The results show that ascorbic acid administration elevates retinal ascorbate and reduces the loss of rhodopsin and photoreceptor cell nuclei resulting from intense light. When given in comparable doses, L-ascorbic acid, sodium ascorbate, and dehydroascorbate were equally effective in preserving rhodopsin. The ascorbate protective effect in the retina is also dose dependent in both cyclic light and dark-reared rats and exhibits a requirement for the L-stereoisomer of the vitamin. Ascorbic acid is effective when administered before, but not after, light exposure, suggesting that protection from light damage in the retina occurs during the light period. In some experiments, rod outer segments were isolated from rats immediately after light exposure, lipids extracted, and fatty acid composition determined. As judged by the preservation of rod outer segment docosahexaenoic acid in rats given ascorbate, the vitamin may act in an antioxidative fashion by inhibiting oxidation of membrane lipids during intense light.     

The dopaminergic amacrine system and its response to light stimulation in rats with inherited retinal dystrophy

In both the RCS mutant strain of rats with hereditary progressive retinal dystrophy and in controls, concentrations of dopamine (DA) and its metabolic DOPAC increased steeply in retina from 14 to 60 days postnatally with no further elevations in older animals indicating normal dopaminergic system evolution in the RCS rats. In 60-, 90-, and 135-day-old but not in younger (14- and 30-day-old) RCS rats killed in the light phase of the light-dark cycle, retinal DA, and more markedly DOPAC levels, were lower than those in age-matched controls. In normal rats aged 24, 35 and 70 days that were dark-adapted for 24 hr, 2-hr light exposure increased DA and mainly DOPAC levels in retina. Light stimulation after dark adaptation elevated retinal DA and DOPAC only in 24- but not in 35- or 70-day-old RCS rats. In RCS rats with advanced retinal dystrophy, decreases in retinal DA and DOPAC levels and lack of response of DA amacrine cells to light exposure are probably secondary to degeneration or impairment of photoreceptors which are no longer capable of transmitting light stimuli onto DA neurons in retina.     

Amelioration of photic injury in rat retina by ascorbic acid: a histopathologic study

It has been postulated that ascorbic acid may help to protect the retina from oxidative insult by light. To confirm this hypothesis, the authors compared light-damaged retinas of rats with or without ascorbate supplement by morphologic and morphometric studies at different time periods after light exposure. No dramatic morphologic differences were observed in the photoreceptor-retinal pigment epithelium complex between the two groups six hr after light exposure to 200 to 250-foot candles of visible light. Six to 13 days after 24 hr of exposure, the retina of rats that received ascorbate supplement showed significantly less severe damage than the retina of unsupplemented rats. The superior and temporal quadrants of the retina appeared to be most susceptible to the light damage when comparing rats with or without ascorbate supplement. These findings suggested that ascorbate ameliorates the photic injury in rat retina.     

Retinal protein synthesis in relationship to environmental lighting

A series of in vivo and in vitro experiments using Xenopus laevis juvenile toads was conducted to probe the relationship between environmental lighting and protein synthesis in the retina. Autoradiographic and biochemical analyses indicated that measurable changes in protein synthesis did not occur during a normal diurnal cycle when animals were conditioned to 12 hr light followed by 12 hr darkness each day (LD). However, when retinas from animals maintained in continuous darkness (DD) for 3 days were incubated with 3H-leucine, there was a 40% reduction in the specific radioactivity of total retinal proteins compared with retinas from animals maintained in continuous light (LL) for 3 days or on the LD cycle. Retinas from DD animals injected with 3H-leucine showed a 48% reduction in protein synthesis compared with retinas of LL animals. In autoradiographs of retinas from in vivo or in vitro experiments, grain counts were 40% lower in the total retinas of the DD animals compared with retinas of LL animals. This reduction occurred throughout the entire retina and was not restricted to any specific cell type. There was also a 35% reduction in the rate of radioactive band displacement in the rod outer segments of DD animals, although the percent of 3H-leucine incorporated into opsin relative to total retinal protein was the same for both groups. We conclude from these studies that fluctuations in the rate of protein synthesis during the normal light-dark cycle are not detectable. However, major differences in protein synthesis are evident when animals are stressed with continuous darkness for several days. This effect is not restricted to any particular retinal layer but occurs throughout the entire retina. Moreover, prolonged darkness affects protein synthesis in extraocular tissues as well.U     

Retinal light damage in rats exposed to intermittent light. Comparison with continuous light exposure

Visible light-induced photoreceptor cell damage resulting from exposure to multiple intermittent light-dark periods was compared with damage resulting from continuous light in albino rats maintained in a weak cyclic-light environment or in darkness before light treatment. The time course of retinal damage was determined by correlative measurements of rhodopsin and visual cell DNA at various times after light exposure, and by histopathological evaluation. The effect of intense light exposures on rhodopsin regeneration and on the level of rod outer segment docosahexaenoic acid was also determined. For rats previously maintained in weak cyclic light, 50% visual cell loss was measured 2 weeks after 12 1 hr light/2 hr dark periods, or following 24 hr of continuous light. A comparable 50% loss of visual cells was found in dark-reared rats after only 5 hr of continuous illumination or 2-3 hr of intermittent light. As judged by histology, cyclic-light-reared rats incurred less retinal pigment epithelial cell damage than dark-reared animals. In both experimental rat models intermittent light exposure resulted in greater visual cell damage than continuous exposure. Visual cell damage from intermittent light was found to depend on the duration of light exposure and on the number of light doses administered. Measurements of rhodopsin and DNA 2 hr and 2 weeks after light exposure of up to 8 hr duration revealed that visual cell loss occurs largely during the 2 week dark period following light treatment. The loss of docosahexaenoic acid from rod outer segments was also greater in rats exposed to intermittent light than in animals treated with continuous light. It is concluded that intermittent light exposure exacerbates Type I light damage in rats (involving the retina and retinal pigment epithelium) and the schedule of intense light exposure is a determinant of visual cell death.     

Failure of ascorbate to protect against broadband blue light-induced retinal damage in rat

· Background: Excessive generation of free radicals due to light absorption is proposed as the most likely mechanism for photochemical retinal damage. The observed reduction of green light-induced retinal injury after ascorbate treatment is believed to be an antioxidative effect. The aim of the present study was to evaluate the possible protection of ascorbate against blue light-induced photoreceptor damage. · Methods: Cyclic light-reared albino rats were injected intraperitoneally with either ascorbate (1 mg/g body weight) or, as placebo, physiological saline 24 h before and just prior to exposure to blue light. After 20–22 h of dark adaptation, two groups of the rats were exposed in pairs to the blue light (400–480 nm) for 6 h at an average irradiance of 0.7 W/m² in the cage. Six days after light exposure, all rats were killed and retinal samples were analyzed. · Results: Diffuse blue light irradiation resulted in an uneven distribution of damage in the retina. As judged from the pathological changes in the retina irradiated, no microscopic difference was observed between the two groups. The preserved thickness of the outer nuclear layer was on average 61.3% in the ascorbate-treated and 66.4% in the placebo-treated group. The photoreceptor loss was not significantly different between the two groups. · Conclusion: The ascorbate did not protect the retina from blue-light induced damage. This favors the assumption that the mechanisms for blue light-induced retinal damage might differ from that for green light. Received: 13 October 1998 Revised version received: 22 January 1999 Accepted: 18 February 1999     

Ascorbate peroxidase in bovine retinal pigment epithelium and choroid

Peroxidase, catalyzing hydrogen peroxide reduction concurrent with ascorbate oxidation, was demonstrated in the extract of retinal pigment epithelium and choroid. The peroxidase in the choroid, RPE, and retina are 236.1, 25.1, and 0.5 units/mg protein respectively. Ammonium sulfate fractionation and high pressure liquid chromatography showed that the peroxidase in the RPE-choroid is associated with a group of heme proteins with absorption maxima at 410 nm, and optimal activity at pH 4.5. The high peroxidase activity in the RPE-choroid explains the observation of dehydroascorbate in these tissues and indicates a possible role of this enzyme in the removal of H2O2.     

Zinc deficiency and oxidative stress in the retina of pigmented rats

PURPOSE: To determine the effect of moderate zinc deficiency on antioxidant defenses and measures of oxidative stress in the retina and retinal pigment epithelium (RPE) of Brown Norway Rats. METHODS: Twenty-four rats were housed individually and divided into three groups of 8 rats each. Group 1 was fed ad libitum a semipurified control diet formulated to contain 50 parts per million [ppm] total zinc; group 2 was fed ad libitum an identical diet but containing 5 ppm total zinc; and group 3 was pair-fed the control diet but restricted in amount to that consumed by group 2. Food intake was measured daily and the rats weighed weekly. After 6 weeks, the rats were killed and the following measurements were made: serum zinc, serum alkaline phosphatase, retinal zinc, RPE-choroid zinc, RPE-choroid catalase, liver metallothionein (MT), retinal MT, RPE-choroid MT, retinal catalase, and retinal thiobarbituric reactive substances (TBARS). RESULTS: The following showed statistically significant differences between groups 2 and 3, respectively: serum Zn (1216 micro/l versus 1555 microg/l, P < or = 0.01), serum alkaline phosphatase (3.75 U/mg versus 5.10 U/mg, P < or = 0.05), liver MT (4.3 microg/mg protein versus 16.7 microg/mg, P < or = 0.0001), RPE-choroid MT (1.3 microg/mg protein versus 2.2 microg/mg, P < or = 0.02), retinal MT (0.85 microg/mg protein versus 2.8 microg/mg, P < or = 0.05), and retinal TBARS (6.2 nM/mg protein versus 2.2 nM/mg, P < or = 0.05). CONCLUSIONS: The results show that retinal MT and RPE MT concentrations are very sensitive to intake of dietary zinc. The increase in retinal TBARS in group 2 indicates that moderate zinc deficiency increases oxidative stress to the retina. The results also suggest that MT is protective against lipid peroxidation of retinal membranes.     

Efficacy and safety of blue-light scleral cross-linking

PURPOSE: To evaluate the efficacy of blue-light scleral cross-linking as well as its safety in preventing retinal damage beneath the treated sclera. METHODS: Six rabbits were unilaterally treated with topical riboflavin (0.5%) and blue light (465 nm) on the equatorial sclera using a light emitting diode source with an exposure area of 9 mm in diameter. Four weeks after the treatment, the animals were euthanized and the exposed sclera and contralateral eye sclera excised for comparative testing of biomechanical rigidity and histologic retinal cellular damage. Extensiometry was performed to evaluate the stress-strain curve of treated versus untreated sclera, and light microscopy of the treated sclera and underlying retina were also comparatively evaluated. RESULTS: Blue-light scleral cross-linking showed a three-fold increased stiffening in all tested animals in the stress-strain curve. Histological investigation revealed no retinal damage in any of the treated eyes. CONCLUSIONS: Scleral cross-linking with riboflavin and blue light (465 nm) has a stiffening effect on the sclera without histological tissue damage to the retina.     

Upregulation of VEGF in murine retina via monocyte recruitment after retinal scatter laser photocoagulation

PURPOSE: This study was conducted to determine changes in the expression of vascular endothelial growth factor (VEGF) in murine retina after retinal scatter laser photocoagulation. METHODS: Photocoagulation (PHC) was performed on wild-type C57BL/6J mice using a diode laser, and the eyes were enucleated 1, 2, 3, 4, 7, and 14 days after laser treatment. VEGF and monocyte chemoattractant protein (MCP)-1 levels in the sensory retina and retinal pigmented epithelial (RPE) cells in both tissues were measured by ELISA. The VEGF mRNA was measured by real-time RT-PCR. Leukocyte infiltration into the RPE-choroid was determined by flow cytometry. VEGF comparisons between mice subjected to PHC and those treated with monocyte recruitment inhibitor (anti-MCP-1) were performed and statistically analyzed. The expression of VEGF and MCP-1 in the retina was determined by immunohistochemistry. RESULTS: VEGF protein levels significantly increased 1 day after PHC in both the RPE-choroid and the sensory retina. VEGF concentrations were maximum at day 3 after photocoagulation and stayed elevated until day 7. The number of choroid-infiltrating macrophages was markedly increased in mice with laser treatment compared with those without laser treatment. VEGF expression decreased after treatment with neutralized antibody to monocyte recruitment. We demonstrate that MCP-1 expression in the retina increased markedly after scatter laser photocoagulation by immunohistochemistry and ELISA. CONCLUSIONS: Retinal scatter laser photocoagulation induced upregulation of VEGF in the sensory retina and RPE-choroid at an early period. The authors speculate that the major source of VEGF in the retina after retinal scatter laser photocoagulation is the recruited monocytes.     

Expression pattern of erythropoietin and erythropoietin receptor in experimental model of retinal detachment

PURPOSE: Erythropoietin (EPO), known for its role in erythroid cell differentiation, has been suggested to have a neuroprotective effect on retinal neurons. The aim of the current study was to investigate the expression pattern of EPO and erythropoietin receptor (EPOR) in the detached retina of a rat model of retinal detachment (RD). METHODS: Forty-eight albino Sprague-Dawley rats were randomized into normal control group (n = 6) and RD model group (n = 42). The rat RD model was established by slow injection of 1.4% sodium hyaluronate into the upper half of the subretinal space, in which the upper half of the neurosensory retina was detached from the underlying retinal pigment epithelium (RPE). The expression of EPO and EPOR mRNA/protein was determined at 1, 3, 6, 12, 24, 48, and 72 hr after sodium hyaluronate injection by semiquantitative RT-PCR and Western blot analysis, respectively. Meanwhile, the distributions of EPO and EPOR were examined by immunohistochemistry. RESULTS: EPO and EPOR mRNA levels increased gradually after sodium hyaluronate injection and peaked 48 hr later. EPO mRNA level became significantly higher than that in the normal control group at 12 hr after injection, and EPOR mRNA level became significantly higher at 6 hr (p < 0.05). Moreover, the protein levels of EPO and EPOR presented a similar tendency to the mRNA level and became significantly higher than those in normal control group at 3 hr after injection (p < 0.05). Immunohistochemical results showed that both EPO and EPOR were strongly expressed all over the neurosensory retina 48 hr after injection, but EPOR was not found in the rod outer segment. Only weak expression of EPO and EPOR was noticed in the retina of normal control group. CONCLUSIONS: The levels of EPO and EPOR in detached retina increase with the duration of retinal detachment and reach their peaks at 48 hr; most layers of detached neurosensory retina can express EPO and EPOR. It is indicated that the EPO/EPOR system might play an important role in protection of retinal neurons during RD; supplementation with exogenous EPO might promote the survival of retinal neurons.     

Permeability of retinal pigment epithelium: effects of permeant molecular weight and lipophilicity

PURPOSE: To determine the effects of solute molecular weight and lipophilicity on the permeability of a retinal pigment epithelium (RPE)-choroid preparation. METHODS: Fresh RPE-choroid specimens from bovine eyes were placed in diffusion chambers for permeability experiments with carboxyfluorescein, fluorescein isothiocyanate (FITC)-labeled dextrans with molecular masses from 4 to 80 kDa, and beta-blockers exhibiting a wide range of lipophilicity (atenolol, nadolol, pindolol, timolol, metoprolol, and betaxolol). Permeability experiments were performed both in the choroid-to-retina (inward) direction and in the retina-to-choroid (outward) direction. Carboxyfluorescein and FITC-dextrans were determined by fluorometry, and beta-blockers by HPLC. The transepithelial electrical resistance and potential difference were monitored during the experiments. RESULTS: Permeability of the fluorescent FITC-dextran probes through RPE-choroid decreased significantly with the increasing size of the probe. RPE-choroid was 35 times more permeable to carboxyfluorescein (376 Da) than to FITC-dextran 80 kDa. The permeabilities of lipophilic beta-blockers were up to 8 and 20 times higher than that of hydrophilic atenolol and carboxyfluorescein, respectively. The lag time of solute flux across the RPE-choroid increased with the molecular weight and lipophilicity. Compared with published data on isolated sclera, bovine RPE-choroid was 10 to 100 times less permeable to hydrophilic compounds and macromolecules. The permeability of lipophilic molecules in RPE-choroid was in the same range as in the sclera. CONCLUSIONS: RPE is a major barrier and may be the rate-limiting factor in the retinal delivery of hydrophilic drugs and macromolecules through the transscleral route. For lipophilic molecules, RPE-choroid, and sclera are approximately equal barriers.     

Light damage in the rat retina: the effect of dietary deprivation of N-3 fatty acids on acute structural alterations.

We investigated the effect of depleting membrane docosahexaenoic acid (DHA, 22:6n-3) content through dietary deprivation of n-3 fatty acids on the susceptibility of the photoreceptors and pigment epithelium cells to acute light-induced changes. Male Sprague-Dawley rats were raised throughout gestation, lactation and up to the age of 8 weeks on semi-purified diets containing either safflower oil (SFO, n-3 deficient diet) or soybean oil (SO) as the sole source of lipids. A third group was switched at weaning from safflower oil to soybean oil (SFO/SO). Rats were maintained on a 12 hr/12 hr light/dark cycle in which the light level at the front of the cages was 5-10 lx. Light damage was produced by exposing dark-adapted animals to diffuse white fluorescent light of 700-800 lx for 30 min followed by 90 min of darkness. In order to study recovery from light damage, additional groups of SFO and SO rats were returned to dim cyclic light for 27 hr following bright light exposure. DHA content in retinal phosphatidylethanolamine and phosphatidylcholine was 65-75% lower in rats fed SFO than in rats fed SO. The decrease was compensated for by an increase in 22:5n-6, the total content of polyunsaturated fatty acids (PUFA) being similar in both the SFO and SO groups. The SFO/SO rats had DHA levels similar to SO animals, but 22:5n-6 remained elevated resulting in a slightly higher level of total PUFA. Severe rod outer segment (ROS) membrane disruptions were seen following bright light exposure in rats on the SO and SFO/SO diets. The appearance of these disruptions did not change significantly during more than 24 hr in dim cyclic light. In contrast, there were virtually no acute ROS lesions in the SFO group. Furthermore, there was a strong light-elicited disk-shedding response in the SO rats but not in the other two groups. The pigment epithelium of the DHA deficient retinas showed a significantly greater accumulation of large lipid droplets in the dark-adapted state. Notably, whole retina rhodopsin levels were 15% higher in the SFO than in the SO group. These results indicate that depletion of retinal DHA reduces the susceptibility of the rod outer segments to acute light damage and at the same time may alter visual pigment photochemistry and other photoreceptor and pigment epithelium functions.