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Purpose: To determine the safety, sensitivity, and specificity of a novel flash photorecovery timing instrument with response verification in differentiating normal from abnormal maculae, and in detecting worsening macular disease., Methods: Right and left eye photorecovery times were determined at baseline and after 5 min using a xenon arc, flash filtered for infrared, ultraviolet, and visible short wavelengths, delivered through an aperture in a hand-held tube. A push-button actuated timer and flash and stopped timer when lighted numbers became visible post-flash. A numeric keypad verified responses. Normal subjects (two eyes tested, n = 144; one eye tested, n = 108) ranged in age from 15 to 84. Photorecovery times were measured in one eye of subjects with small drusen and 20/20 acuity (53-55 correct ETDRS letters; n = 57); in both eyes of subjects with dry age-related macular degeneration (AMD; n = 118); wet AMD with (n = 19) or without (n = 17) macular fluid; and eyes of diabetics with background retinopathy with (n = 19) or without (n = 17) macular retinal thickening. Once-weekly photorecovery measurements for 6 months in each eye of 10 dry AMD subjects and 10 dry diabetic maculopathy subjects provided longitudinal data., Results: Normal subjects' mean right eye recovery time was 9.6 sec (+/- 1.9 SD); left 10.8 sec (+/- 1.0 SD). Photorecovery lengthened after age 55, nearly doubling that of young subjects by age 80. Macular edema, serous macular detachment, or worsened dry AMD were accompanied by prolonged photorecovery (p < .01). When abnormal new vessels or retinal thickening appeared in three serially followed patients, photorecovery at least doubled (p < .01). In all three, photorecovery prolongation occurred without clinical symptoms. None of the 499 tested subjects reported adverse events due to the flash testing., Conclusions: These findings support the usefulness of a reproducible light flash macular vision recovery measurement as an indicator of macular pathology and worsening disease.

Purpose: To test the hypothesis that daily use of zinc-monocysteine (ZMC) supplement will be well tolerated and result in improved macular function in persons with dry age-related macular degeneration (AMD)., Methods: Eligible, consenting subjects were randomized to either ZMC 25 mg or placebo twice daily for 6 months. Both ZMC and placebo groups enrolled 40 participants, with best corrected visual acuity 20/25 to 20/70, macular drusen, and pigment changes. Masked personnel determined baseline, 3- and 6-month best-corrected visual acuity, contrast sensitivity, and light flash recovery time. Differences between ZMC and placebo were analyzed by a one-sided unpaired t-test of the paired differences between baseline and 3- and 6-month timepoints for right and left eyes separately., Results: By 6 months the ZMC group showed improved visual acuity (p < 0.0001) and contrast sensitivity (p < 0.0001). Macular light flash recovery time shortened in the ZMC group at 3 months by 2.1 sec (left eye, p = 0.0001) to 3.6 sec (right eye, p < 0.0001), and at 6 months by 7.2 sec (left eye, p < 0.0001) to 7.4 sec (right eye, p < 0.0001). This variable had no improvement in the placebo group. ZMC had a gastrointestinal irritation rate of under 2%., Conclusion: ZMC 25 mg twice daily was well tolerated and was associated with improved macular function in comparison to a placebo in persons with dry AMD.

Purpose: The aim of this study was to document the effect of a novel zinc amino acid combination on the concentrations of important antioxidants in cultured human retinal pigment epithelial (hRPE) cells., Methods: Primary confluent hRPE cells were treated with 30 microM of zinc acetate, zinc chloride, zinc cysteine, and zinc sulfate. The antioxidants catalase, glutathione, glutathione peroxidase, and metallothionein were measured. MTT assays were performed to determine the relative protection of the zinc compounds from the cytotoxic effects of H202 and t-butyl hydroperoxide., Results: Catalase and glutathione peroxidase activities were increased by the zinc formulations compared with the untreated control. Glutathione and metallothionein content were also increased. The greatest increases occurred with zinc conjugated to the amino acid cysteine. The MTT assays showed that zinc monocysteine protected cultured RPE cells from the toxicity of H2O2 and t-butyl hydroperoxide., Conclusions: Our results demonstrate that zinc treatment of RPE cells increases antioxidants and protects cultured RPE cells from the cytotoxic effects of H2O2 and t-butyl hydroperoxide. The results show that zinc conjugated to cysteine offers greater benefits than either zinc salts or cysteine alone.

Purpose: To determine the relative expression of metallothionein isoforms and their differential induction by oxidative stress in cultured RPE cells and to localize the isoforms in the human chorioretinal complex., Methods: Total RNA was isolated from cultured human retinal pigment epithelial cells using TRI-Reagent. An "anchor-oligo-dT primer" was used for the synthesis of cDNA, reverse transcribed using avian reverse transcriptase and subsequently subjected to PCR analysis using oligonucleotides specific for metallothionein (MT) I, MT II, and MT III. The selected transcripts were then used to assess the expression of the above elements in fixed tissue sections by in situ hybridization. Cultured RPE cells were allowed to phagocytose bovine photoreceptor outer segments (ROS) or were treated with H(2)O(2) for 6 hours and then analyzed by RT-PCR or in situ hybridization to ascertain the effect of oxidative stress on metallothionein mRNA isoform expression., Results: Relative density analysis of amplified products demonstrate the presence of MT I, MT II and MT III in RPE cells, with an apparent relative expression MT II > MT I > MT III [corrected]. Expression of MT I and MT II mRNA was increased by both phagocytosis and hydrogen peroxide, however MT III was not induced by either stress. In situ hybridization corroborated the findings of the RT-PCR analysis and showed that MTs were mainly localized in the RPE and the photoreceptor layer of the retina., Conclusions: The localization of MT and the response of MT to oxidative stress are consistent with a role for MT as an antioxidant in the RPE and retina. Studies are ongoing to determine the specific mechanisms of action of these antioxidants in RPE cells.

Purpose: Due to a possible link in humans between atherosclerosis, a high-fat diet, and the development of age-related retinal degenerations, we investigated retinal changes with age and diet in high-fat-fed C57BL/6 mice, a mouse model of human atherosclerosis., Methods: We fed C57BL/6J mice either a normal chow diet or an atherogenic diet containing 15% total fat for either 15, 30 or 45 weeks. We sacrificed the animals and examined the eyes using fluorescence microscopy, light microscopy and transmission electron microscopy., Results: At 15 weeks, in high-fat-fed mice, there was an increase in the number of autofluorescent granules in the retinal pigment epithelium (RPE) compared to chow-fed mice. By light microscopy, we noted no remarkable differences in the inner retinal layers between mice fed either diet for 15 or 30 weeks. In contrast, we noted major changes in transmission electron micrographs from the 30 week high-fat group. In the RPE, these included: (1) an increase in the number and size of autophagocitic and empty cytoplasmic vacuoles; (2) accumulations of lipid-like droplets in the cytoplasm, and (3) RPE atrophy. Changes in Bruch's membrane included: (1) thickening; (2) fragmentation of the elastic lamina, and (3) the accumulation of electron dense particulate and vesicular structures within the inner and outer collagenous zones. These changes were not seen in mice fed normal diets even at 45 weeks., Conclusions: We suggest that this model may prove useful for investigations into mechanisms on the effects of diet on the RPE and Bruch's membrane.

Purpose: We have previously shown that an experimental, low-zinc environment decreased catalase activity in cultured human fetal retinal pigment epithelial (RPE) cells. The purpose of this study was to investigate the effect of zinc supplementation on catalase expression in cultured human fetal RPE cells., Methods: Confluent fetal RPE cells incubated in Coon's modified Ham's F12 (CMF-12) were treated (18 h) with zinc chloride (ZnCl2) (15, 30, or 100 microM) to assess changes in catalase enzyme activity or for 6 h to assess the induction of catalase mRNA by Northern analysis and in situ hybridization. RPE cells were also treated with 30 microM ZnCl2 for 2, 6, 24, 48 and 72 h to assess the time course of changes in catalase enzyme activity, changes in mRNA levels and status of the Sp1 transcription factor., Results: Catalase activity was increased above control by the addition of 15, 30 and 100 microM ZnCl2. Catalase gene expression was induced by 30 microM zinc in 6 h, but decreased to non-treated control levels by 24 h. The transcription factor Sp1 was also activated by zinc treatment (30 microM) which peaked at 2 h and declined to non-treated control levels by 24 h. Catalase enzyme activity peaked at 24 h and decreased to control levels by 72 h., Conclusions: Our results demonstrate that zinc treatment of RPE cells increases catalase expression and activates the transcription factor Sp1. The results suggest zinc may play a role in the transcriptional regulation of catalase in RPE cells.

The goal of this study was to determine the effect of extracellular matrix components on the phagocytic function of the retinal pigment epithelial (RPE) cell. Cultured human fetal RPE cells were established in culture and plated on three commercially-prepared substrates: collagen IV, fibronectin and laminin and on three native matrices: bovine corneal endothelial cell matrix (BCEM) denuded bovine Bruch's membrane and denuded human Bruch's membrane. Cultured cells were allowed to become confluent and maintained for an additional two weeks before uptake of fluorescent bovine retinal outer segments (ROS) was measured by flow cytometry. Morphology by phase contrast microscopy and melanization was also determined as measures of differentiation. The results showed that morphology, melanization and ROS uptake by cells on collagen IV, laminin and fibronectin were not different from control cells plated on tissue culture plastic. However, ROS uptake by cells plated on BCEM was significantly less than that of cells cultured on plastic and melanization was greater. ROS uptake by cells plated on both types of Bruch's membrane was also significantly less than control cells. Treatment of cells plated on tissue culture plastic with 44 mM NaHCO3, which increased melanization, also reduced ROS uptake. We conclude that native matrices seem to contain components that significantly depress ROS uptake in culture. The inhibition is not mimicked by collagen, laminin or fibronectin coated wells. The ECM may play a significant role in controlling phagocytosis of ROS either by determining morphology, increasing differentiation or by directly influencing intracellular metabolism, and thus serve as another level of control for this RPE function which may not occur in cells plated on tissue culture plastic. These results may also have implications for the effects of aging or disease in which there are changes in the ECM.

Zinc is a necessary micronutrient, usually abundant in human RPE. Our study was undertaken to determine the effects of short-term, zinc deficiency on human retinal pigment epithelium (RPE) using a culture model of fetal human RPE cells. Human fetal RPE cells were isolated and cultured in Coon's modified Ham's F-12 medium. For zinc depletion studies, cells were cultured for 1 week in Chelex-treated Dulbecco's modified Eagle's medium containing low (0.25 microM) or physiologic (11 microM) total zinc concentrations as determined by flame atomic absorption spectroscopy. Protein synthesis was determined by incorporation of 35S-cysteine/methionine and labeled proteins analysed by polyacrylamide gel electrophoresis. Several cell parameters and enzymes were significantly reduced below control when cultured in low zinc: zinc content (40%), proliferation (63%), protein/well (50%), catalase activity (68%), alkaline phosphatase activity (61%), alpha-mannosidase activity (68%), and metallothionein (82%). No statistically significant decline was seen in acid phosphatase activity, superoxide dismutase activity, glutathione peroxidase activity and dexamethasone induction of metallothionein. Zinc repletion (100 microM, 1 h) increased catalase and alpha-mannosidase activities from 32% and 33% of control to 75% and 73%, respectively. Cycloheximide did not inhibit this short-term zinc-induced repletion of catalase or alpha-mannosidase. Protein synthesis in low zinc medium was depressed, but not significantly, as shown by incorporation of radiolabeled 35S-cysteine/methionine into newly synthesized proteins. The effects of zinc deficiency in cultured human RPE are selective. Adequate intracellular zinc was required for maximal activity of some enzymes. The dependence of catalase activity on zinc was not predicted and may help explain the observed decline in catalase activity seen with age in RPE. Our model of zinc deficiency should prove useful in elucidating the complex effects of zinc deficiency and repletion in human RPE.

We studied zinc uptake by nonhuman primate retinal pigment epithelium (RPE) and choroid, using 65Zn as a probe. With intravenously administered 65ZnCl2, virtually all detectable tracer was lost from the plasma after 20 hours but the pigment epithelium-choroid showed prominent uptake and retention. Plasma concentrations of oral 65ZnO remained high 20 hours after feeding. Uptake and retention of orally administered 65Zn as 65ZnO from the bloodstream by the RPE/choroid was avid in both young and old animals. Excretion in urine and feces was minimal. All pigmented ocular tissues took up and retained 65Zn. A survey of total zinc content of human and nonhuman primate ocular tissues showed that the pigmented tissues had consistently higher concentrations of zinc. Our results demonstrate for the first time direct uptake and retention of zinc from the blood by primate RPE and other ocular tissues.

The retinal pigment epithelium (RPE) plays several important roles in the continual support and renewal of photoreceptor outer segments. In the present study, we have demonstrated that RPE cells contain a low molecular weight protein with a high capacity for zinc binding that is dependent on available sulfhydryl groups. This protein is inducible by a 24 hour incubation of cultured RPE in medium supplemented with zinc, cadmium, or dexamethasone. The induction of this protein is correlated with an increased capacity for zinc-65 uptake into cultured RPE. Analysis with a cDNA probe specific for the human metallothionein II gene corroborated the existence and induction of metallothionein gene products in RPE cells. Based on these properties, we have identified this protein as metallothionein. The induction of metallothionein likely has a critical influence on the zinc economy of the RPE.

The flash evoked ERG is used extensively as a measure of retinal function. To understand better the relationship between extent of retinal damage and the ERG we created multistage laser lesions in one eye each of three Macaca fascicularis monkeys. An argon-dye laser was used to deliver 630 nm energy to cumulative quarter sectors of the temporal hemiretina and the foveal region at two week intervals. Latencies of the a- and b-waves and amplitude from the trough of the a-wave to peak of the b-wave were measured preoperatively and one week after each new lesion. Following the last recording session the eyes were removed and prepared for histological examination. In general, but depending on stimulus condition, the maximum decrease in ERG amplitude (50-70% of normal) occurred following the third or fourth sector lesions. These lesions accounted for a loss of the outer nuclear layer in 18-25% of the entire retinal surface area extending from the edge of the fovea into the anterior equatorial region. Surprisingly, subsequent lesions of the remaining sector and fovea did not result in further reduction of ERG amplitudes. Significant increases in amplitudes were even observed for some stimulus conditions following these final lesions. Latencies of the a- and b-waves were not affected. The fact that in the present study ERG amplitudes recovered, suggest that such values may be tenuous evidence of amount of functional retina in laser-lesioned eyes.

Posterior subcapsular cataracts (PSC) are associated with hereditary retinal dystrophy in the Royal College of Surgeons (RCS) rat model and with human retinitis pigmentosa. The relationship of lens and retinal pathology has never been explained. Previous studies of pink-eyed RCS rats aged 2.5 to 11 months had shown an incidence of cataract of 24% when observed by the unaided eye and 60% by direct ophthalmoscopy, while 40% of rats were considered to have clear lenses. Unlike the retinal degeneration, which appeared in all homozygous animals, cataract seemed not to be predictably associated with the rdy mutation. To test this further, we studied the lenses of rats of different ages with a diagnostic slitlamp. We confirmed that by 8 to 15 months of age, rats fed a diet containing recommended concentrations of all known nutrients for rodents developed cataracts with an incidence of 23% when observed by unaided eye. In addition, opacities were seen in 74% with the indirect ophthalmoscope and 20 D lens; but 100% had at least a "sugar grain" type PSC by slitlamp. The slitlamp-detectable cataract was first seen in some animals by 49 days, and by 56 days all rats examined had bilateral PSC. This is an age at which the rod photoreceptors have degenerated. We concluded that slitlamp-detectable PSC are predictably associated with the retinal dystrophy of the rdy mutation. The RCS rat model may be relevant to a type of retinal degeneration having a constant association of cataract.

Several human diseases are accompanied by keratinization of the ocular surface and loss of corneal transparency. We describe a mouse strain in which 2/3 of the viable offspring manifest a corneal disease characterized initially by poor wetting followed by keratinization of the ocular surface, and progressive loss of corneal transparency. The corneal epithelium becomes vacuolated and the basement membrane thickened. Ulceration and perforation were seen in some cases. Standard bacterial cultures yielded no culpable pathogens. Blood antiviral titers and cultures of orbital and ocular tissues for herpes virus were negative. Double masked treatment trials with topical gentamicin, and topical and parenteral vitamin A had no significant effect on the course of the disease. The mouse strain exhibits corneal keratinization unresponsive to vitamin A therapy, and may serve as an excellent model of certain serious human ocular surface diseases.

Aging changes known to occur in Bruch's membrane may be associated with drusen formation and retinal pigment epithelial mottling, which often have a peripheral as well as a macular distribution. Few details of peripheral Bruch's membrane aging changes have been reported. We conducted a histochemical investigation of Bruch's membrane in 31 postmortem donor eyes and an ultrastructural morphometric investigation of these regional changes in a subgroup of 22 eyes. The age of our donors ranged from 12 days to 80 years. When we reacted 5 micron paraffin embedded sections of chorio-retinal complex fixed in 2% glutaraldehyde-1% paraformaldehyde, we observed increased PAS positivity, and staining with Weigert's elastin and alcian blue at pH 2.5 in Bruch's membranes from donors over 46 years. In this older group, the macula showed increased histochemical reactivity for glycoconjugates, glycosaminoglycans, collagen and elastin as compared with the equator and periphery. Thickening of Bruch's membrane was first detected in the periphery in tissue from donors aged 10 to 45. Thickening of macular Bruch's membrane was first detected in tissue from donors older than 45. The major proportion of thickening, which appears to be due to the deposition of fibrillar and amorphous material, occurred earlier in the inner than the outer collagenous zone. The equatorial region was relatively spared. Our findings confirm and extend the observations of the timing, regional predeliction, and extent of Bruch's membrane aging changes.

Retinal pigment epithelium (RPE) was isolated from the globe of a donor positive for human immunodeficiency virus (HIV) who had cytomegalovirus (CMV) retinitis secondary to acquired immunodeficiency syndrome (AIDS). In culture, the cells exhibited normal epithelioid morphology by phase contrast microscopy. After two weeks the cells developed cytomegaly and dense intranuclear and cytoplasmic inclusions and, eventually, died. Transmission electron microscopy (EM) demonstrated intranuclear nonenveloped virus particles 80-120 nm in diameter consistent with a herpes type infection. Immunofluorescence staining demonstrated the presence of CMV antigens. Conditioned medium from the infected cells caused infection in RPE cells isolated from normal donors. Hybridization assay demonstrated the presence of CMV DNA and indicated that the time course of the infection was similar, but not identical to infection in MRC-5 and HEL cells. We conclude that cultured human RPE is a permissive host for CMV.

Epiretinal membrane formation in the posterior pole is an almost constant feature of eyes with retinitis pigmentosa. Using a modified direct immunofluorescence reaction, we demonstrated a strong reaction with antilaminin in a band-like pattern on the inner retinal surface of 3 eyes with retinitis pigmentosa. There were also strongly-laminin-positive pigmented and unpigmented cells scattered throughout the degenerated retinas. This pattern was in sharp contrast to that seen in normal retinas, where the linear band of reaction at the internal limiting membrane was faint and accompanied by a zone of reaction at the level of the outer limiting membrane. Quantitation of laminin by ELISA revealed a six- to ten-fold increase (per mg protein) in retinitis pigmentosa retinas, as compared with normal. Since both glial and retinal pigmented epithelial cells can synthesize and deposit laminin, both may participate in the gliosis that occurs during the course of retinitis pigmentosa.

Periretinal membranes obtained at vitrectomy from three patients with massive periretinal proliferation were examined by immunofluorescence and electron microscopy. Immunofluorescent staining on fresh frozen sections showed positive stain with glial fibrillary acidic protein and a weaker stain with antibodies to actin, PBM-1 and laminin. Staining was negative for antibodies to actin, PBM-1 and laminin. Staining was negative for antibodies of collagen types I, III and IV. Electron microscopy revealed abundant glial cells arranged in a tubulo-acinar configuration with junctional complexes, apical microvillous processes and 9-10 nm cytoplasmic filaments.