Search

Rat lenses incubated in dimethyl sulfoxide (DMSO)‐water binary mixtures of different compositions became turbid. A slight haziness developed up to 0.062 mole fraction of DMSO; at higher concentrations dense turbidity developed. Microscopic examination of the incubated rat lenses showed damage restricted to the epithelium and outer cortex at low DMSO concentrations; maximum damage occurred, in terms of fiber cell swelling, extracellular fluid (lake) formation and disintegration of epithelium, at 0.25 mole ratio of DMSO. Scanning electron microscopic observation showed that at high DMSO concentrations, especially at 0.25 mole fraction, the contents of the fiber cells were greatly damaged. Apparently a large part of the crystallins had coalesced around the cytoskeletal bodies. Polarized light-scattering intensity measurements and their analyses indicated that when dense opacities developed at high DMSO concentrations, the major contribution to the turbidity came from the optical anisotropy fluctuations. The change in the organization of the components within the fiber cells disturbs the balance between intrinsic birefringence and form birefringence necessary for transparency. Thus, the DMSO-caused opacification can be described as a good model for the involvement of optical anisotropy fluctuations in cataractogenesis. © 1996 Society of Photo-Optical Instrumentation Engineers.

Previous evidence has shown clearly that sugar cataract formation results from unusually high intracellular levels of polyol. Documentation of polyol-related histological changes in the cortical fiber cells of the equatorial zone has been extensive. However, little attention has been given to the early changes in the lens epithelial cells, in spite of the fact that the highest level of aldose reductase is found in this layer of the lens. Also, cultured lens epithelial cells exposed to high sugar levels exhibit rapid accumulation of polyol and show ultrastructural alterations. Therefore, a study was designed to evaluate the role of the lens epithelium in sugar cataract formation. Specifically, an attempt was made to localize the earliest fine structural lesions in intact lenses of galactose-fed rats and to test their relation to aldose reductase. Rats were fed either a normal diet or a 50% galactose diet with or without sorbinil, an aldose reductase inhibitor. Rats were killed at varying periods of time ranging from 6 to 96 hr, and the eyes were processed for light and electron microscopy. The first detectable abnormalities occurred after 36 hr of galactose feeding, and were limited to the central lens epithelium. Cell edema, apparent dilution of cytoplasm, rounding of nuclei, aberrant intracellular vacuoles, and loss of normal tortuosity of cell boundaries were the salient lesions. No changes were detectable in the equatorial zone until 48 hr, and no deviation from the control structure was found in any of the rats treated with an aldose reductase inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

While many of the diverse crystallins of the transparent lens of vertebrates are related or identical to metabolic enzymes, much less is known about the lens crystallins of invertebrates. Here we investigate the complex eye of scallops. Electron microscopic inspection revealed that the anterior, single layered corneal epithelium overlying the cellular lens contains a regular array of microvilli that we propose might contribute to its optical properties. The sole crystallin of the scallop eye lens was found to be homologous to Omega-crystallin, a minor crystallin in cephalopods related to aldehyde dehydrogenase (ALDH) class 1/2. Scallop Omega-crystallin (officially designated ALDH1A9) is 55-56% identical to its cephalopod homologues, while it is 67 and 64% identical to human ALDH 2 and 1, respectively, and 61% identical to retinaldehyde dehydrogenase/eta-crystallin of elephant shrews. Like other enzyme-crystallins, scallop Omega-crystallin appears to be present in low amounts in non-ocular tissues. Within the scallop eye, immunofluorescence tests indicated that Omega-crystallin expression is confined to the lens and cornea. Although it has conserved the critical residues required for activity in other ALDHs and appears by homology modeling to have a structure very similar to human ALDH2, scallop Omega-crystallin was enzymatically inactive with diverse substrates and did not bind NAD or NADP. In contrast to mammalian ALDH1 and -2 and other cephalopod Omega-crystallins, which are tetrameric proteins, scallop Omega-crystallin is a dimeric protein. Thus, ALDH is the most diverse lens enzyme-crystallin identified so far, having been used as a lens crystallin in at least two classes of molluscs as well as elephant shrews.

Previous studies have suggested that disturbances in plasminogen activator inhibitor (PAI)-1 may be relevant to the development of diabetic microvascular complications. To determine whether overexpression of PAI-1 in cells of retinal microvasculature would result in a disease similar to that observed in diabetes, ocular tissue from transgenic mice that overexpress human PAI-1 were examined.Transgenic mice were administered ZnSO4 (25 mM) in their water for up to 49 weeks to activate the metallothionein promoter and stimulate human PAI-1. Colloidal gold immunocytochemistry was used to quantify the human PAI-1 antigen at 7, 20, 34, and 49 weeks of ZnSO4 administration. Cross sections of retinal microvessels were examined by electron microscopy for changes in basement membrane (BM) thickness. Retinal digest preparations were examined by light microscopy for possible microangiopathy, including changes in endothelial cell-to-pericyte ratios.Human PAI-1 immunoreactivity was detected throughout the retinal capillaries of transgenic mice receiving zinc and increased significantly (P0.001) after 20 to 49 weeks of ZnSO4 administration compared with age-matched transgenic control mice. At 20 and 49 weeks, retinal capillaries of transgenic mice that received zinc showed significantly thickened BMs compared with control animals (P0.001). Moreover, wholemounts of the retinal vasculature from PAI-1 transgenic mice demonstrated an increased endothelial cell-to-pericyte ratio.PAI-1 overexpression in retinal microvasculature leads to retinal disease similar to that observed in diabetic retinopathy.

It has been proposed that oxidative tissue damage is involved in the development of diabetic angiopathies. To evaluate this hypothesis, experiments were conducted to identify the retinal vessel changes induced by the oxidative stress related to alpha-tocopherol deficiency and examine possible similarities with the lesions characteristic of diabetic retinopathy. Twenty-one-day-old male Fisher 344 albino rats were divided randomly to receive a basal, chemically defined diet either with (adequate group) or without (deficient group) alpha-tocopherol. After 6 and 8 months, some rats (n = 3 per group) were killed and the eyes removed. In order to evaluate cell integrity and localization of lipofuscin-specific autofluorescence by light and fluorescence microscopy, some of the retinas were prepared for cryostat-sections while others were digested by elastase to isolate intact retinal vasculatures. After 8 and 14 months, the central retina of one eye per rat (n = 6 to 8 per group) was examined by electron microscopy for retinal capillary basement membrane (RCBM) thickening and other ultrastructural changes. At 6 and 8 months, the deficient rats exhibited extensive shortening and disarray of rod outer segments (ROS), marked loss of photoreceptor cells, and pronounced increases in the numbers of granules with lipofuscin-specific autofluorescence in the retinal pigment epithelium (RPE) and retinal vessels. At 14 months, the ultrastructure revealed that the damage to ROS involved disruption of membranes and that the capillary lipofuscin was contained mainly within the endothelial cells. Membrane remnants were found in the lipofuscin granules of both the RPE and retinal vessels. In addition, there was an increase in RCBM thickness (98.7 +/- 2.6 nm vs. 86.9 +/- 2.9 nm). RCBM thickening was the only finding common with diabetic retinopathy, and the thickening was 13.6%, significantly less than that reported in diabetic rat models with 8 and 14 months durations (34% and 53.1%, respectively). Capillary lipofuscin accumulation, which was prominent in the deficient rats, is not notable in diabetes. Both the moderate RCBM thickening and marked lipofuscin accumulations seen in alpha-tocopherol-deficient rats were similar to changes occurring in the aging process, though more pronounced. The spectrum of microangiopathies characteristic of diabetic retinopathy did not develop in alpha-tocopherol-deficient rats. These findings suggest that oxidative damage, though probably involved, is unlikely to play a predominant role in the development of diabetic retinal microangiopathies.

To determine whether the diabetic-like thickening of retinal capillary basement membrane (RCBM) that develops in the galactose-fed rat model of diabetic ocular complications could be halted or ameliorated after 4 or 8 months of galactosemia by treatment with ARI-509, a potent new aldose reductase inhibitor (ARI), or by withdrawal of the galactose diet.Weanling female Sprague-Dawley rats were randomized into eight groups and fed laboratory chow plus 50% starch, control group (CON); 50% D-galactose, galactose-fed group (GAL); 50% D-galactose with ARI-509 at 25 mg/kg or 10 mg/kg body wt per day, high-dose prevention group (HDP) and low-dose prevention group (LDP), respectively; 50% D-galactose for 4 or 8 months and then intervention by addition of ARI-509 (25 mg/kg body wt per day), 4-month intervention group (4IN) and 8-month intervention group (8IN), respectively; or 50% D-galactose for 4 or 8 months and then intervention by withdrawing galactose and replacing it with the 50% starch diet, 4-month galactose withdrawal group (4GW) and 8-month galactose withdrawal group (8GW), respectively. After 4, 8, 16, and 24 months of the experimental diets, the levels of carbohydrates in tissues and the extent of RCBM thickening in capillaries of the outer plexiform layer were determined in all groups.Retinal polyol was reduced by 95% in all ARI-treated groups and by 100% in the 4GW and 8GW groups after withdrawal of the galactose. The mean RCBM thickness increased rapidly in GAL rats, becoming almost two times greater (189 +/- 9.4 nm) than in CON rats (103 +/- 3.4 nm) by 24 months. Treatment with ARI-509 in high and low doses (HDP, LDP) initiated with the introduction of the galactose diet significantly prevented RCBM thickening at all time points (P0.05). In contrast, intervention by withdrawing galactose from the diet or by adding the high dose of ARI-509 had no significant effect (P0.05) on RCBM thickening until the 24-month time point (4IN, 166 +/- 10.3 nm; 8IN, 161 +/- 8.2 nm; 4GW, 136 +/- 5.1 nm; 8GW, 163 +/- 9.6 nm).Both early and late interventions decreased RCBM thickening compared with that in untreated GAL rats. The decreased thickening, however, was not evident until 16 to 20 months after the intervention. Because RCBM thickening is one of the earliest changes in diabetic and galactosemic retinopathy, the findings suggest that RCBM thickening and possibly subsequent retinal lesions are caused by early biochemical alterations induced by the galactose diet that are not readily reversed. The delayed response to therapy is consistent with that observed in the Diabetes Control and Complications Trial. The cumulative evidence indicates that intervention should begin as early after onset of diabetes as possible, and long follow-up periods should be used to evaluate efficacy.

This study investigated whether diabetic-like corneal sensory deficits occur in the galactose-fed rat model of diabetic ocular complications and if such deficits could be prevented using either of two structurally different aldose reductase (AR) inhibitors, CT-112 or AL-1576. S-D rats were randomly grouped to receive a diet of Purina chow with either 50% starch (n=25) or 50% D-galactose (n=65). Some of the galactosemic rats received either 0.25% CT-112 topically 3x daily (n=15) or 28 mg/kg body wt/day AL-1576 systemically (n=10). The control and untreated galactosemic rats in the CT-112 portion of the study received equivalent topical doses of the vehicle. Sensitivity measurements were made with a Cochet-Bonnet Aesthesiometer mounted on a micromanipulator. The filament was applied to the central corneal surface (mean pressure of 0.96 g/mm2) and viewed using a slit-lamp biomicroscope. Ten consecutive stimuli were conducted on each cornea and the average number of blink-responses was expressed as a percent of total stimuli effected. Mean initial corneal sensitivities were similar in all groups. Corneal sensitivity in the galactosemic rat was reduced (p

To determine whether the diabeticlike retinal microangiopathies of the galactose-fed rat model could be ameliorated if intervention by withdrawal of the galactose diet or treatment with the aldose reductase inhibitor AL-3152 was initiated after quantifiable microangiopathies had occurred.Weanling male Sprague-Dawley rats were randomized into five groups and fed for up to 24 months Purina laboratory chow (#5001) plus 50% starch (control [CON]), 50% D-galactose (galactose [GAL]), 50% D-galactose with AL-3152 (approximately 14 mg/kg per day) (prevention [PRV]), 50% D-galactose for 6 months followed by intervention with the inhibitor (intervention [INT]), or 50% D-galactose for 6 months followed by replacement with the 50% starch diet (withdrawal [GWD]). In rats on experimental diets and killed after 6, 18, and 24 months, one retina was prepared for transmission electron microscopy; the other was used for vessel wholemounts using elastase digestion. Capillary images were analyzed by computer morphometry.At 6 months, the GAL rats exhibited statistically significant (P0.05) increases over CON rats in mean capillary basement membrane thickness, capillary density, and dilated channels. These parameters tended to increase with time in most groups, and the differences between GAL and age-matched CON rats were maintained at the 18- and 24-month endpoints. Although the microangiopathies were ameliorated by AL-3152 treatment from the onset (PRV), intervention after 6 months of galactosemia with either galactose withdrawal (GWD) or addition of inhibitor (INT) showed amelioration in only some parameters at 18 months and no statistically significant benefit at the 24-month endpoint.Amelioration of galactose-induced retinal microangiopathies with AL-3152 in the prevention group suggests an efficacious application of aldose reductase inhibitors in treating diabetic retinopathy, provided treatment can begin soon after the onset of diabetes. Intervention after some of the earliest microscopic lesions neither halted progression of the angiopathy nor provided appreciable benefit at the 24-month follow-up.

The purpose of the present study was to determine if prolonged systemic arterial administration of basic fibroblast growth factor (bFGF) at a dose sufficient to enhance collateral vessel formation in the ischaemic hearts of dogs would produce retinal neovascularisation in these same animals. Adult dogs (15-25 kg) were subjected to gradual occlusion of a coronary artery and randomised to receive 1 of 3 treatments via an indwelling left atrial catheter: (1) bFGF 1.74 mg/d, 5 d/wk for 63 d (n = 7); (2) bFGF 1.74 mg/d, 5 d/wk, for 35 d followed by physiological saline, 5 d/wk, for 28 d (n = 10); or (3) physiological saline, 5 d/wk, for 63 d (n = 10). After 63 d the retinal vasculatures from these dogs were isolated and examined for capillary varicosity, neovascularisation and other histopathological signs of angiopathy. All data were collected under masked conditions. The results suggest that chronic, systemic arterial administration of bFGF stimulates neovascularisation in the ischemic myocardium, but has no significant structural or vasoproliferative effect on the nonischaemic retina of the same animal.

To determine if the retinal microangiopathies of the galactose-fed rat model of diabetic retinopathy can be prevented with the aldose reductase inhibitor sorbinil.Sprague-Dawley rats were fed 50% d-galactose with or without sorbinil (0.05% wt/wt), mixed biweekly with fresh diet. Rats in each group were examined frequently by slit lamp and were killed after 8, 16, and 24 months. Computer-assisted morphometry was performed on wholemounts of elastase retinal digest preparations.Cataracts developed in all galactose-fed untreated rats within 3 weeks but not in the sorbinil-treated rats even after 24 months. At 8 months, the galactose-fed untreated rats exhibited statistically significant increases in the mean capillary width, the percent of retinal area occupied by capillaries (capillary density), and the percent of microvascular area with capillaries20 microns wide (dilated channels), compared to controls. At 16 months, the galactose-fed untreated rats showed statistically significant increases over controls in both total mean capillary length and density, and two of the four rats examined had microaneurysms. At 24 months, all the galactose-fed untreated rats had microaneurysms and extensive areas with hypercellular meshworks composed of dilated channels characteristic of intraretinal microvascular abnormalities (IRMA). By contrast, galactose-fed, sorbinil-treated rats, at 24 months, had no IRMA and showed no statistically significant differences from control rats in any of the parameters measured morphometrically.All the galactose-induced retinal microangiopathies were prevented with sorbinil. Aldose reductase inhibitors may be beneficial in ameliorating the similar vascular lesions characteristic of human diabetic retinopathy, though the mechanism remains obscure.

The purpose of this study was to modify the gold chloride procedure for studies of total innervation in corneal whole mounts to provide a decrease in nonspecific background staining and to eliminate the progressively deteriorating stain quality of standard gold chloride techniques. Modifications included use of cryoprotective agents, mechanical removal of Descemet's membrane-endothelium complex prior to fixation, treatment with alpha amylase, and halting the reduction of gold chloride to metallic gold using Kodak rapid fixer with hardener. Rat corneas were stored at -70 C in O.C.T. compound. The Descemet's membrane-endothelium complex was removed after thawing, and corneas were fixed in 4% NaPO4-buffered paraformaldehyde with 8% sucrose. Fixed corneas were incubated in NaPO4-buffered saline containing alpha amylase, placed in 100% lemon juice, then in 1% gold chloride solution, transferred to glacial acidic acid, placed in rapid fixer, rinsed in NaPO4-buffered saline, and dehydrated in graded alcohols. Flat mounts of whole corneas were examined using contralateral corneas as controls. Freezing corneas in O.C.T. compound, removal of the Descemet's membrane-endothelium complex, and treatment with alpha amylase reduced non-specific background staining compared to controls. Treatment with Kodak rapid fixer prevented the deterioration of staining quality for at least 8 months. These improvements allow the gold chloride technique to be used with immunohistochemical procedures where the reaction products would be obscured by background staining.

To improve the 30-year-old "trypsin digestion" procedure for isolation of the complete retinal vasculature, which, in its time, was a revolutionary advance that allowed important discoveries about diabetic retinopathy; to provide a method that will yield more consistent results when applied to retinas representing a wide range of ages, species, and severity of vascular disease, such as that occurring in diabetes.Because the Difco trypsin preparation (Difco Laboratories, Detroit, MI) is a crude pancreatic extract, containing variable amounts of chymotrypsin, elastase, amylase, lipase, ribonuclease, collagenase, and other contaminants, an attempt was made to determine which of the major enzymes alone (using purified preparations), or what combination of enzymes, might be most effective in providing consistently clean yet intact retinal vasculatures from eyes of different origins.Purified elastase alone (40 U/ml) in 100 mmol/l sodium phosphate buffer with 150 mmol/l sodium chloride and 5 mmol/l EDTA at pH 6.5 and 37 degrees C gave better results than various concentrations of purified trypsin or chymotrypsin alone, or mixtures of trypsin/chymotrypsin, trypsin/elastase, chymotrypsin/elastase, or the crude trypsin preparation.Elastase, which exhibits broad protease activity, and not trypsin, is the most important enzyme of the standard, crude trypsin digestion procedure for removal of the nonvascular tissues of the retina.

The progressive increase in urinary albumin excretion, which precedes the development of diabetic nephropathy, can be prevented in diabetic rats if the aldose reductase inhibitor, tolrestat, is administered at the initiation and throughout the duration of hyperglycaemia. We therefore determined the ability of tolrestat to intervene in the further progression of already established urinary albumin excretion of streptozotocin-diabetic female Wistar rats. Two months after streptozotocin injection, diabetic rats were grouped as low-urinary albumin excretion (0.2–1.0 mg albumin/day) or high-urinary albumin excretion (1.9–5.9 mg albumin/day), at which time tolrestat intervention (25 mg/kg per day) was begun for half of the diabetic rats in each urinary albumin excretion group. After six months of treatment tolrestat caused a significant reduction in the urinary albumin excretion rate of the low-urinary albumin excretion group only. The diabetes-induced rise of total urinary protein in both groups was significantly reduced by tolrestat. Furthermore, the diabetes-induced increase (49%) in the thickness of the basement membranes of retinal capillaries from the outer plexiform layer was significantly diminished by tolrestat administration. In conclusion, intervention therapy with the aldose reductase inhibitor, tolrestat, can reduce the progression of urinary albumin excretion and retinal basement membrane thickening in long-term diabetic rats.

In order to determine whether neuronal cell death in the retina accompanies senescence, retinas from pigmented ACI rats of various ages were analysed for changes in dimensions, cell densities, DNA content, and rhodopsin levels. Between the ages of 4 and 30 months, there were significant declines in the densities of nuclei in all three nuclear layers of the retina. Over this age span, nuclear density in the outer nuclear layer (ONL) declined by 32–41 %, depending on the region of the retina examined, while inner nuclear layer (INL) nuclear density decreased by 25–34 %. Over the same age range, the nuclear density in the ganglion cell layer (GCL) declined by 25–28 %. The eye increased in size over the 4–30 month age span, particularly between 4 and 12 months. The retinal stretching that resulted from this growth could only partially account for the age-related decline in retinal cell densities. Depending on the region of the retina examined, decreases in nuclear density of 17–26 % for the ONL, 9–17 % for the INL, and 16–20 % for the GCL were apparently due to cell death. Measurements of retinal DNA content support the conclusion that significant cell loss from the retina occurs during senescence; between 4 and 30 months of age retinal DNA content decreased by about 13 %. Age-related changes in retinal rhodopsin content did not parallel apparent changes in photoreceptor number. Between 4 and 11 months of age, rhodopsin levels rose by about 10 %, and then remained constant up to 26 months. Over this same age span, there was an apparent decrease in photoreceptor number of about 15 %. These findings suggest that the amount of rhodopsin per photoreceptor cell increased by up to 25 % between 4 and 26 months of age. Beyond 26 months, rhodopsin levels fell in parallel with photoreceptor cell numbers.

Relatively high proportions of long-chain, polyunsaturated fatty acids seem to be required in rod photoreceptor membranes in order to provide the precise microenvironment for the proper function of the visual pigment rhodopsin. At the same time, such high levels of lipid unsaturation put the photoreceptor membranes at a high risk for autoxidation. The antioxidant vitamin E which can minimize autoxidation of polyunsaturated fatty acids is found in rather high concentrations in the outer segment membranes. Dietary deficiency in vitamin E induces disintegration of rod outer segment membranes, probably by increasing autoxidation. Also, it greatly accelerates the accumulation of aging pigments in the retinal pigment epithelium, probably because these lipofuscin granules do indeed represent the end products of lipid peroxidation. Vitamin E supplements, up to threefold normal levels, appear to provide no significant protection of the retina from light damage produced either by short but acute or by long-term, low level exposures to light. This is not consistent with current theories which implicate lipid peroxidation in the destruction of rod outer segments in light damaged retinas; more work is needed before any relation between retinal light damage and vitamin E levels can be assessed. Surprisingly, the amount of lipofuscin granule accumulation in the retinal pigment epithelium is influenced dramatically by dietary levels of vitamin A. Even retinas lacking a source of polyunsaturated fatty acids from rod outer segments still may accumulate massive lipofuscin if dietary vitamin A is provided. Perhaps vitamin A, which has such a dynamic relationship with the retinal pigment epithelium, becomes oxidized, and then contributes to the formation of a lipofuscin-like pigment. Centrophenoxine, a drug claimed to be effective in reversing the accumulation of age-related lipofuscin in the central nervous system, has no obvious effect in the eye or uterus in removing the lipofuscin granules induced by vitamin E deficiency. Microperoxisomes are abundant in the retinal pigment epithelium, and may be associated with rapid lipid turnover and/or utilization of lipid soluble vitamins. Their potential roles, however, need further documentation and clarification. Recently developed techniques and new discoveries in lipid research open the way for many fruitful studies on the interactions and precise roles of lipids and lipid-soluble vitamins in vision.

The effects of dibutyryl cyclic 3',5'-adenosine monophosphate (BcAMP) and Sephadex G-25 fractions of chick embryo extract on the growth rate, morphology, and pigmentation of normal chick retinal pigmented epithelium (PE) were investigated. Seven cloned PE cell lines were each grown in modified Ham's F-12 medium alone (F-12), or in F-12 supplemented with either high molecular weight (H) or low molecular weight (L) fractions of chick embryo extract. Cells grown in F-12 alone or in L medium formed compact epithelial sheets, whereas cells grown in H had a fibrocytic appearance and formed poorly organized monolayers. In H plus BcAMP, cell morphology was more epithelioid than in H alone, and generally the monolayers appeared more differentiated. Under each of these three culture conditions, 2 x 10-4 M BCAMP retarded the increase in cell number and decreased the final number of cells per culture dish, but had little effect on plating efficiency. BcAMP also increased the rate of cell adhesion to a plastic substratum. Pigmentation was marked in cultures grown in F-12 or in L alone, but the addition of BcAMP dramatically reduced visible pigmentation. This effect was reversed when BcAMP was removed from the culture medium. Thus BcAMP modifies cell and colonial morphology, rate of cell accumulation, adhesive properties, and pigmentation of normal PE cells.

Apparent acceleration in the phagocytosis of rod outer segment tips by the retinal pigment epithelium was induced, using white light of 400 ft cd, in black, beige, and albino mice by varying the exposures from 5 days to a few hours. In black and albino mice the enhanced phagocytosis led to only minor modifications in the processing of ingested outer segment membranes. In beige mice, however, remnants of outer segment membranes accumulated in giant secondary lysosomes and in giant pigment granules, suggesting an impairment in intracellular digestion like that reported for leukocytes of beige mice and patients manifesting the Chediak-Higashi syndrome. Defective digestion by the retinal pigment epithelium could alter the turnover rates of rod outer segment membranes and decrease the reversibility from damage by light. Microperoxisomes, which are cell organelles known to contain oxidative enzymes and catalase, were closely associated with lipid droplets that accumulated at the lateral cell boundaries during light exposure.

Albino-beige mice were produced in order to combine two experimentally useful characteristics, albinism and lysosomal dysfunction, in the same animal. The retinal pigment epithelium of albino-beige mice formed giant intracellular granules. Exposure of albino-beige mice to white light of 150 foot-candles for 3 to 10 hr induced marked phagocytosis of rod outer segment fragments by the retinal pigment epithelium, resulting in intracellular accumulations of undigested disk membranes within the giant granules. Additional, incompletely processed membranes accumulated as the mice aged or were exposed to 150 foot-candle light for longer periods. Such accumulations of ingested membranes were not observed in the pigment epithelium of exposed or aging albino mice heterozygous for the beige gene. Because of its altered processing of ingested outer segment membranes, this new albino mouse should be useful for studying the possible roles of the retinal pigment epithelium in the maintenance of photoreceptor cells and in their recovery from light damage and other insults.

The origin of giant granules in the retinal pigment epithelium of the beige mouse was investigated with electron microscopy and ultrastructural histochemistry. These granules were found to contain melanin and acid phosphatase. Apparently they arise from fusions of primary lysosomes with melanin granules which are already enlarged from multiple fusions among melanosomes. Therefore, the giant granules are not primary lysosomes, nor are they simply enlarged melanin granules as suspected from light microscopic studies. A deficiency of primary lysosomes in the pigment epithelium results, suggesting a defect in intracellular digestion similar to that found in the leukocytes of Chediak-Higashi patients and several animal models. Affected humans probably have defective digestion in their retinal pigment epithelium also; which could impair the renewal process for rod outer segments. Thus, Chediak-Higashi patients may show an increased susceptibility to light damage due not only to hypopigmentation, but to defective intracellular digestion, as well.

Adult rhesus and pigtail monkeys were exposed to a uniform field of light from daylight fluorescent lamps to determine the initial site and characteristics of structural retinal damage induced by continuous exposure and the threshold intensity required to produce the effects. Electron microscopic examination revealed that the initial site of damage is the photoreceptor outer segments. Damage remained restricted to the outer segments at intensities as high as 24,700 lux and with 12 hr exposure periods repeated for up to 4 days. Rods were swollen at the distal tip and showed disc membrane separation. Come outer segments were affected at the proximal end with vesiculation and membrane rearrangement. The threshold intensity for morphological changes to comes in the macula for a single 12 hr exposure was between 195 and 361 mu W/cm2 at the retina (400 to 700 mm; uncorrected for ocular transmittance), whereas the threshold for changes in rods was higher, at between 361 and 615 mu W/cm2. These levels correspond to between 5900 and 10,800 lux (550 to 1000 ft-cd) for comes and 10,800 to 19,400 lux (1000 to 1800 ft-cd) for rods of monkeys with fully dilated pupils. The paramacular areas of the retina were less sensitive to damage than macular areas of the same animal. No paramacular changes were observed in animals exposed to 10,800 lux or less. The patched (control) eyes of each monkey remained structurally normal in both the macula and paramacula at all exposure levels. These results demonstrate the sensitivity of the adult primate retina to damage by relatively moderate levels of light.

A diabetic-like thickening of retinal capillary basement membranes induced in rats fed for 207 consecutive days a diet containing 50% galactose was prevented by the addition to the diet of tolrestat, a potent, structurally novel inhibitor of aldose reductase. Analysis of electron micrographs (× 25,000) of capillaries from the outer plexiform layer of the retina by computer planimetry showed that the basement membranes were approximately twofold thicker in rats fed galactose than in those fed either a standard diet or a diet containing galactose and tolrestat in doses of 43 or 57 mg/kg/day. The thickening of basement membranes in galactosefed rats was accompanied by other ultrastructural alterations mimicking changes typical of diabetic microangiopathy, such as multilamination and the formation of vacuoles and dense inclusions. Therefore, the galactosemic rat represents a useful model for studying basement membrane-related complications of diabetes and their possible prevention by aldose reductase inhibitors.

Relatively high proportions of long-chain, polyunsaturated fatty acids seem to be required in rod photoreceptor membranes in order to provide the precise microenvironment for the proper function of the visual pigment rhodopsin. At the same time, such high levels of lipid unsaturation put the photoreceptor membranes at a high risk for autoxidation. The antioxidant vitamin E which can minimize autoxidation of polyunsaturated fatty acids is found in rather high concentrations in the outer segment membranes. Dietary deficiency in vitamin E induces disintegration of rod outer segment membranes, probably by increasing autoxidation. Also, it greatly accelerates the accumulation of aging pigments in the retinal pigment epithelium, probably because these lipofuscin granules do indeed represent the end products of lipid peroxidation. Vitamin E supplements, up to threefold normal levels, appear to provide no significant protection of the retina from light damage produced either by short but acute or by long-term, low level exposures to light. This is not consistent with current theories which implicate lipid peroxidation in the destruction of rod outer segments in light damaged retinas; more work is needed before any relation between retinal light damage and vitamin E levels can be assessed. Surprisingly, the amount of lipofuscin granule accumulation in the retinal pigment epithelium is influenced dramatically by dietary levels of vitamin A. Even retinas lacking a source of polyunsaturated fatty acids from rod outer segments still may accumulate massive lipofuscin if dietary vitamin A is provided. Perhaps vitamin A, which has such a dynamic relationship with the retinal pigment epithelium, becomes oxidized, and then contributes to the formation of a lipofuscin-like pigment. Centrophenoxine, a drug claimed to be effective in reversing the accumulation of age-related lipofuscin in the central nervous system, has no obvious effect in the eye or uterus in removing the lipofuscin granules induced by vitamin E deficiency. Microperoxisomes are abundant in the retinal pigment epithelium, and may be associated with rapid lipid turnover and/or utilization of lipid soluble vitamins. Their potential roles, however, need further documentation and clarification. Recently developed techniques and new discoveries in lipid research open the way for many fruitful studies on the interactions and precise roles of lipids and lipid-soluble vitamins in vision.

The earliest histopathologic signs of diabetic retinopathy include selective loss of intramural pericytes and thickening of capillary basement membranes. Previous evidence from animal models indicated that aldose reductase inhibitors could prevent these capillary wall lesions, but only recently have aldose reductase inhibitors been tested for prevention of the subsequent retinal complications of diabetes, such as microaneurysms. In the present study, Sprague-Dawley rats were fed diets containing 50% galactose with or without an aldose reductase inhibitor (tolrestat). After 28 months of galactose feeding, the retinal capillaries in whole mounts exhibited a marked increase in periodic acid-Schiff (PAS) staining, extensive pericyte loss, endothelial cell proliferation, acellularity, diffuse dilation, occluded lumens, microaneurysms, and complex microvascular abnormalities including gross dilation and formation of multiple shunt networks. The PAS hyperchromaticity of basement membrane material and pericyte loss occurred throughout the retinal vasculature, while while the microaneurysms and complex lesions were limited to the capillaries of the central and paracentral retina. The changes were associated with both the arterial and venous portions of the capillary plexus. Treatment with orally administered tolrestat prevented essentially all of the vessel abnormalities. Thus, long-term galactose feeding of rats induced microvascular lesions simulating those occurring in background diabetic retinopathy in humans, and these lesions were prevented by treatment with an aldose reductase inhibitor.

Retinas and livers were studied with histochemical methods for catalase combined with light and electron microscopy, following intramuscular injections of C57 black mice with retinyl acetate or retinyl palmitate in total doses of 0.9 to 6.7 X 10(6) I.U./kg. body weight. There were clear, dose-related increases in the numbers and sizes of vitamin A--storing lipid droplets in the stellate cells of the liver. Concomitantly, more conservative increases in similar lipid droplets occurred in the pigment epithelium but not in other cells of the retina. Such lipid droplets may represent physiological sites of vitamin A storage which are important for the maintenance of photoreceptor cells by the retinal pigment epithelium. No changes in lipids of the retina or liver were observed in mice injected with retinoic acid or with peanut oil. Both peroxisomes containing catalase and the putative vitamin A--storing lipid droplets were distributed along the basal and lateral cell surfaces of the pigment epithelium where receptors for plasma retinol-binding protein have been reported. Peroxisomes may play a role in the reactions related to the esterification and sequestering of vitamin A.

The effects of vitamin E and A deficiencies on the formation of lipofuscin in the melanocytes and fibroblasts of the choroidal stroma and in the endothelial cells of the choriocapillaris were studied. Weanling female albino rats (Sprague-Dawley) were divided into three groups and fed purified diets adequate or deficient in vitamins E and A: +E, +A; -E, -A. After 35 weeks, vitamin E deficient rats (-E, +A) exhibited increased lipofuscin-specific autofluorescence in the choroid compared to the controls (+E, +A). By electron microscopy and morphometric methods the choroidal stroma of vitamin E deficient rats displayed an increase in lipofuscin content as measured by the number of lipofuscin granules and their size, if compared with the controls. However, in vitamin E deficiency, only animals with a supply of vitamin A (-E, +A) showed higher amounts of lipofuscin in the choroidal stroma; animals deficient in both vitamins (-E, -A) stayed on the same level as the controls (+E, +A). In the endothelial cells of the choriocapillaris, on the other hand, no significant increases in lipofuscin content were observed in either group of vitamin E deficient animals (-E, +A), (-E, -A). Apparently vitamin E deficiency affects the choroid by increasing lipofuscin formation only in the melanocytes and fibroblasts. Vitamin A appears to play a role in lipofuscin formation.

Capillary basement membrane thickness is known to increase in a variety of tissues during aging and as a consequence of certain systemic disease states. In order to determine whether capillary basement membrane thickness increases during senescence in the outer plexiform (OPL) and ganglion cell (GCL) layers of the retina, eyes of pigmented ACI rats aged 4, 11, 17, and 32 mo were examined. Basement membrane thickness in the OPL and GCL were determined at each of these ages using computer-assisted ultrastructural morphometry. Capillary basement membranes in both the OPL and GCL showed a linear increase in thickness over the 4 to 32 mo age span. The increase in thickness averaged 54% for the OPL and 65% for the GCL over this age range. At all ages examined, the GCL basement membranes were 80 to 90% thicker than those in the OPL. Age-related changes in the substructure of the capillary basement membranes differed between the OPL and GCL. It is possible that the observed age-related changes in retinal capillary basement membranes may play a role in the development of senile retinopathies.

Microperoxisomes were found to be abundant in the retinal pigment epithelium of the human, rhesus monkey, mice, rats, domestic fowl, and frog by ultrastructural histochemistry. They were rare in other cells of the retina and choroid. These organelles had a granular matrix, ranged in diameter from 0.15 mum to 0.30 mum, and were bound by a single tripartite membrane which often maintained slender connections with the smooth endoplasmic reticulum and other microperoxisomes. They exhibited a positive reaction (electron opaque product) following incubation in diaminobenzidine and H2O2 for the demonstration of the peroxidatic activity of catalase (Novikoff et al., J. Histochem. Cytochem. 20: 1006, 1972). The reaction was inhibited by: (1) aminotriazole; (2) dichlorophenol-indophenol; (3) preheating at 95 degrees C.; or (4) elimination of H2O2. Microperoxisomes, like the well-known peroxisomes (microbodies) of liver cells have been inplicated in various aspects of lipid metabolism and the detoxification of H2O2. We demonstrated for the first time that microperoxisomes respond to drug-induced changes in lipid metabolism, as previously shown for peroxisomes. Nafenopin is a recently utilized drug which greatly decreases serum lipids, increases hepatic catalase activity, and induces an increased size and number of hepatic peroxisomes. Black, beige, albino, and obese mutant mice of the C57BL/6J strain treated with nafenopin for several weeks showed a two- to threefold increase in the number of microperoxisomes in the retinal pigment epithelium. Microperoxisomes of the retinal pigment epithelium may be involved in the transport, storage, and rapid turnover of lipids associated with the maintenance of photoreceptor outer segment disc membranes.

Before any clinical signs of diabetic retinopathy can be detected, two major histopathological lesions occur in the walls of retinal capillaries: basement membrane thickening; and loss of intramural pericytes (mural cells). A decrease in the number of pericyte to endothelial cell junctions also occurs under diabetic conditions and may result in subsequent proliferation of endothelial cells and microaneurysm formation. Aldose reductase inhibitors prevent all these diabetic microangiopathies of the retina in animal models of diabetes and are presently being tested in clinical trials on diabetic retinopathy. Utilization of aldose reductase inhibitors is a novel approach which would potentially provide a very effective diabetic therapy to be used in addition to insulin.

Capillary basement membrane thickening is typical of the diabetic retina, and aldose reductase appears to be involved since a diabetic-like thickening can be induced by galactose feeding and prevented with aldose reductase inhibitors. Because aldose reductase is present in the Mueller's cells, studies were undertaken to determine if thickening of the retinal inner limiting membrane, which is the basement membrane of these cells, can be induced by long-term galactose feeding and be prevented with an aldose reductase inhibitor. Weanling male, Sprague-Dawley rats were given a 50% galactose diet with or without an aldose reductase inhibitor (0.04% tolrestat, Ayerst). Quantitative computer planimetry on electron micrographs demonstrated a significant galactose-induced thickening of the inner limiting membrane which was prevented by the aldose reductase inhibitor. The results were consistent with the notion that basement membrane thickening is involved in diabetic retinopathy and can be delayed or prevented with aldose reductase inhibitors.

The interrelationships of vitamins E and A in maintaining various structural components of the retina were studied in four groups of weanling female rats fed purified diets adequate or deficient in each vitamin: +E, +A; -E, +A; +E, -A; and -E, -A. Groups deficient in retinol (-A) were supplemented with retinoic acid. After 14, 21, and 35 weeks, the retinas were examined histologically and ultrastructurally. At 35 weeks, the doubly deficient rats (-E, -A) had lost 92% of their rod nuclei, whereas rats deficient in vitamins A (+E, -A) or E (-E, +A) alone had lost only 34% and 20%, respectively. Vitamin E deficiency resulted in extensive lipofuscin deposits in the retinal pigment epithelium as early as 21 weeks, but the presence of vitamin A doubled the number of lipofuscin granules (-E, +A vs. -E, -A) and induced an even greater increase in their autofluorescence. Another clear influence of vitamin A was seen when +E, +A retinas autofluoresced not only much more than +E, -A retinas, which had similar numbers of granules, but also more than -E, -A retinas, which had about twice as many lipofuscin granules. In the retina, unlike the uterus, the lipofuscin-specific autofluorescence and lipofuscin granule number were not proportional. Moreover, the numbers of granules were influenced by both vitamins E and A, whereas the intensity of lipofuscin-specific autofluorescence was determined almost exclusively by vitamin A. Probably the accelerated loss of photoreceptor cells in -E, -A retinas resulted from both oxidation of membranes and oxidation of retinal vitamin A stores in the absence of vitamin E protection.

Polyol accumulation and myo-inositol depletion were accompanied by extensive vacuole formation in cultured canine lens epithelial cells that were incubated for up to 96 hr in growth medium supplemented with 30 mM D-galactose or 30 mM D-glucose. These changes did not occur in cells incubated in a hypergalactosemic or hyperglycemic medium which also contained an aldose reductase inhibitor (20 microM sorbinil). In addition, these changes were not observed in lens cells incubated in growth medium supplemented with either 30 mM mannitol, which is known to enter cells only slowly, or in 30 mM L-galactose, which is not a substrate for aldose reductase. The vacuoles were visible at the ultrastructural level after 6 hr of incubation in 30 mM D-galactose and increased in both number and size with time. These vacuoles had a unique fine structure. They did not result from swelling of mitochondria or other cell organelles. As demonstrated cytochemically, they did not represent either lysosomes or Golgi saccules. The proliferation pattern of cells incubated with 30 mM D-galactose was clearly different from that of control cells, but approached normal when an aldose reductase inhibitor was added to the incubation medium. Together these findings suggest that vacuole formation and altered cell proliferation were caused by polyol accumulation and/or myo-inositol loss, both of which result from aldose reductase activity.

To investigate the role of normal vitamin E levels and the interrelationships between vitamin E and A in maintaining the visual cells of the retina, weanling female Sprague-Dawley rats were fed vitamin E-free diets differing tenfold in their vitamin A content (0.8 and 8.0 mg of retinol per kilogram of diet). Rats on vitamin E-free diets with the higher vitamin A level exhibited marked disruption of photoreceptor outer segment membranes and a fivefold increase in the number of lipofuscin granules in the pigment epithelial cells which ingest these membranes. Rats on vitamin E-free diets with the lower vitamin A level showed the same retinal damages plus significant loss of photoreceptor cells compared to age-matched rats on control diets. Rods and cones were involved equally, and their pattern of loss was not like that found in vitamin A deficiency. Normal levels of vitamin E probably protect photoreceptor membranes from oxidative damage and retard the accumulation of their remnants and other products of lipid breakdown in the pigment epithelium. The vitamin A status of rats has a significant influence on the extent of damage induced by vitamin E deficiency.

Cluck neural retinal cells are relatively undifferentiated in appearance for several weeks in culture. Cells with long, thin processes are subsequently observed as well as foci of small, pigmented cells and large semi-transparent masses of cells or other material. Retinal cells thus appear to be able to differentiate into several divergent cell types in vitro. Chick pigment epithelial (PE) cells rapidly differentiate in culture, forming microvilli, pigment granules and other specialized subcellular organelles. 5-Bromodeoxyuridine greatly inhibits cellular differentiation of cultured PE cells as assessed by decreased visible pigmentation and by colonial disorganization. In contrast, several prostaglandins and dibutyryl cyclic-AMP plus isobutylmethylxanthine increase pigmentation and induce a more mature type of cell morphology and colonial development. By electron microscopy, increased numbers of pigment granules are apparent as well as an increase in the length of microvilli. Prostaglandins also increase glutathione levels in the cells. Prostaglandins and cyclic-AMP thus promote the general state of differentiation of PE cells in culture.

Normally, direct contacts between pericytes and endothelial cells occur frequently in both retinal and optic nerve capillaries. The contacts are made through fenestrae in the thick basement membranes which separate the plasma membranes of the two cell types over most of their juxtaposed surfaces. This study was initiated to determine if there was any change in the number of pericyte-endothelial cell contact regions in retinal or optic nerve capillaries of the galactose-fed rat model for polyol-related complications of diabetes. Sprague-Dawley rats were fed for 28 months a 50% galactose diet, with or without an aldose reductase inhibitor (tolrestat), or a normal diet. Electron micrographs of transected capillaries were taken from defined regions of the central retina and nearby portions of the optic nerve. The optic nerve capillaries showed no differences in the numbers of pericyte-endothelial cell contact regions nor in basement membrane thickness in the galactose-fed rats. In contrast, the retinal capillaries exhibited a 70% decrease in the numbers of contact regions and a 2.4-fold increase in basement membrane thickness in galactosemic rats. Both loss of cell contact regions and basement membrane thickening were prevented with tolrestat. The results are consistent with the proposed involvement of the polyol pathway in diabetic retinopathy and with the greater involvement of retinal versus optic nerve vessels in diabetic microangiopathy.

Oxygen can participate in a number of reactions that are potentially damaging to cells and tissues. Reactive forms of oxygen are generated during normal metabolic processes, and a variety of biologic mechanisms have evolved to protect organisms from damage by these compounds. Apparently these protective mechanisms are not fully developed in the fetus, which exists in a hypoxic environment in utero. Premature birth exposes the infant to a sudden increase in environmental oxygen content, for which it is not physiologically prepared. It is likely that this premature exposure to atmospheric oxygen triggers an elevation in the rate of autoxidative reactions in tissues and that the termination of normal retinal vascularization seen in ROP is the result of an attempt to minimize such reactions. This interpretation is supported by the finding that vitamin E, an antioxidant in vivo, reduces the severity of ROP when given at an early enough stage.