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The possible role of ultraviolet light in the formation of cataract is not well understood. In this study, guinea pigs were exposed to a chronic, low level of UVA light (0.5 mWcm(-2), 340-410 nm wavelength, peak at 365 nm) for 4-5 months. It is known that the lens of the guinea pig possesses unusually high levels of the UVA chromophore NADPH. In a preliminary analysis, it was found that isolated guinea pig corneas transmitted 70-90% of 340-400 nm light, and that UVA radiation was able to penetrate deep into the nucleus of the guinea pig lens, where it was absorbed. Exposure of guinea pigs to UVA in vivo produced a 60% inactivation of lens epithelial catalase; however, analysis by transmission electron microscopy (TEM) showed no apparent morphological effects on either the lens epithelium or the cortex. A number of UVA-induced effects were found in the nucleus of the guinea pig lens, but were observed either not at all or to a lesser extent in the cortex. The effects included an increase in light scattering (two-fold; slit-lamp examination), distention of intercellular spaces (TEM), an increase in lipid peroxidation (30-35%; infrared spectroscopy), a decrease in GSH level (30%), an increase in protein-thiol mixed disulfide levels (80%), loss of water-soluble protein (20%), an increase in the amount of protein disulfide (two-fold; two-dimensional diagonal electrophoresis), degradation of MIP26 (15%) and loss of cytoskeletal proteins including actin, alpha- and beta- tubulin, vimentin and alpha-actinin (60-100%). The results indicate that a 4-5 month exposure of guinea pigs to a biologically relevant level of UVA light produces deleterious effects on the central region of the lenses of the animals. UVA radiation, coupled presumably with the photoreactive UVA chromophore NADPH and trace amounts of O(2) present in the lens nucleus, produced significant levels of oxidized products in the nuclear region over a five month period. The data demonstrate the potentially harmful nature of UVA light with respect to the lens, and highlight the importance of investigating a possible role for this type of radiation in the formation of human cataract.

Purpose: Previous in vitro studies with transgenic and gene-knockout mice have shown that lenses with elevated levels of glutathione peroxidase (GPX)-1 activity are able to resist the cytotoxic effect of H(2)O(2), compared with normal lenses and lenses from GPX-1-deficient animals. The purpose of this study was to investigate the functional role of this enzyme in antioxidant mechanisms of lens in vivo by comparing lens changes of gene-knockout mice with age-matched control animals., Methods: In vivo lens changes were monitored by slit lamp biomicroscopy, and enucleated lenses were examined under a stereomicroscope in gene-knockout animals and age-matched control animals ranging in age from 3 weeks to 18 months. Transmission (TEM) and confocal microscopy were performed on different regions of lenses after the mice were killed at various times., Results: Slit lamp images showed an increase in nuclear light scattering (NLS) in gene-knockout mice compared with control animals. TEM revealed changes in the nucleus as early as 3 weeks of age by the appearance of waviness of fiber membranes. With increasing age, there was greater distortion of fiber membranes and distension of interfiber space at the apex of fiber cells compared with control mice. The changes in nuclear fiber membranes were even more dramatic, as observed by confocal microscopy, which was performed on thicker sections. In contrast to the changes in the lens nucleus, the morphology of the epithelium and superficial cortex remained unchanged in knockout animals during the same experimental period, consistent with slit lamp observations. Stereomicroscopy of ex vivo lenses demonstrated a significant increase in opacification in gene-knockout mice relative to control animals of the same age. This effect became evident in mice aged 5 to 9.9 months and persisted thereafter in older animals, resulting in mature cataracts after 15 months., Conclusions: The results demonstrate the critical role of GPX-1 in antioxidant defense mechanisms of the lens nucleus. The increased NLS appears to be associated with damage to fiber membranes in the nucleus, which is particularly susceptible to oxidative challenge because of the deficiency of GPX-1. It is suggested that the lens membrane changes in the knockout animals may be due to the formation of lipid peroxides, which serve as substrates for GPX-1. Cataract development in gene-knockout mice appeared to progress from focal opacities, apparent at an earlier age, to lamellar cataracts between 6 and 10 months, and finally to complete opacification in animals older than 15 months. This is the first reported phenotype in GPX-1-knockout mice.

Purpose: Recently, we reported that topical administration of 2-carboxyethyl germanium sesquioxide (Ge-132) concurrently with 50% galactose feeding delayed the establishment of mature cataracts and reduced advance glycation product. This study was to determine the effect of pretreatment of Ge-132 on galactose associated morphological changes and Na(+)-K(+)-ATPase activity., Methods: Young Sprague Dawley rats received topical eye drops four times a day of either saline or Ge-132 seven days prior to the 50% galactose diet and during galactose feeding. At desired intervals the lenses were extracted, photographed and processed for either light microscopy, scanning electron microscopy or the determination of Na(+)-K(+)-ATPase activity., Results: In Ge-132 pretreated lenses as compared to saline pretreated lenses the following results were observed: (a) the galactose-induced morphological alterations in the majority of lenses were delayed and (b) Na(+)-K(+)-ATPase activity was protected., Conclusions: Our previous and current studies show that in addition to osmotic stress post-translational protein modification, such as glycation, including enzymes may play a role in initiating changes that lead to cataract development. The inhibition of protein glycation by antiglycating compounds, such as Ge-132, delays sugar cataract formation. Currently, we are investigating the status of protein glycation and advanced glycation end products following pretreatment with Ge-132 and the role of Ge-132 on the activities of enzymes such as aldose reductase and Na(+)-K(+)-ATPase.

Germanium compounds have been shown to be effective in preventing the formation of advanced glycation end-products and for reversible solubilization of glycated proteins. As protein glycation has been proposed to play a role in lens opacification, we initiated studies to evaluate the effects of 2-carboxyethyl germanium sesquioxide (germanium compound 132 or Ge-132) on galactose-induced cataractogenesis. For this study young Sprague-Dawley rats were fed a 50% galactose diet. One group of rats received topical saline and another group was administered Ge-132 in saline four times a day. The lenses were periodically examined with an ophthalmoscope and at desired intervals processed for light and scanning electron microscopy. Our observations, beginning at 3 days and continuing to 21 days of galactose feeding, exhibited the characteristic galactose-induced morphological alterations, which include the formation of vacuoles, cysts, membrane disruption and swelling of fibers and epithelial cells as well as disorganization of the bow in lenses of rats in both groups. However, in the majority of rats administered Ge-132 these alterations were delayed as compared to the lenses of rats administered saline. Our findings show that, although the initiation, progression and pattern of lens opacification in rats receiving saline and Ge-132 were similar, in the majority of lenses the progression and establishment of mature cataracts in the Ge-132 group of rats were delayed. Analysis of the water-soluble and water-insoluble lens-protein fractions for glycated proteins showed increased levels of the Amadori products and advanced glycation related fluorescent products in galactosemic rats treated with saline eye drops. In rats receiving the topical Ge-132 treatment the levels of these glycation products were substantially reduced to levels lower than control values. Prevention of glycation seems to be a mechanism by which cataract progression is delayed.

Nuclear cataract, a major cause of loss of lens transparency in the aging human, has long been thought to be associated with oxidative damage, particularly at the site of the nuclear plasma membrane. However, few animal models have been available to study the mechanism of the opacity. Hyperbaric oxygen (HBO) has been shown to produce increased nuclear light scattering (NLS) and nuclear cataract in lenses of mice and human patients. In the present study, older guinea pigs (Initially 17-18 months of age) were treated with 2.5 atmospheres of 100% O2 for 2-2.5-hr periods, three times per week, for up to 100 times. Examination by slit-lamp biomicroscopy showed that exposure to HBO led to increased NLS in the lenses of the animals after as few as 19 treatments, compared to lenses of age-matched untreated and hyperbaric air-treated controls. The degree of NLS and enlargement of the lens nucleus continued to increase until 65 O2-treatments, and then remained constant until the end of the study. Exposure to O2 for 2.5 instead of 2 hr accelerated the increase in NLS; however, distinct nuclear cataract was not observed in the animals during the period of investigation. A number of morphological changes in the experimental lens nuclei, as analysed by transmission electron microscopy, were similar to those recently reported for human immature nuclear cataracts (Costello, Oliver and Cobo, 1992). O2-induced damage to membranes probably acted as scattering centers and caused the observed increased NLS. A general state of oxidative stress existed in the lens nucleus of the O2-treated animals, prior to the first appearance of increased NLS, as evidenced by increased levels of protein-thiol mixed disulfides and protein disulfide. The levels of mixed disulfides in the experimental nucleus were remarkably high, nearly equal to the normal level of nuclear GSH. The level of GSH in the normal guinea pig lens decreased with age in the nucleus but not in the cortex; at 30 months of age the nuclear level of GSH was only 4% of the cortical value. HBO-induced changes in the lens nucleus included loss of soluble protein, increase in urea-insoluble protein and slight decreases in levels of GSH and ascorbate; however, there was no accumulation of oxidized glutathione. Intermolecular protein disulfide in the experimental nucleus consisted mainly of gamma-crystallin, but crosslinked alpha-, beta- and zeta-crystallins were also present.(ABSTRACT TRUNCATED AT 400 WORDS)

Cataract development has been reported in Dahl salt-sensitive (DS) hypertensive rats with high-salt intake. An alteration in ionic transport and electrolyte balance has been reported to be associated with hypertension in these animals. A morphological evaluation of the lenses of salt-sensitive 'cataract-prone' (DS) and salt-resistant (DR) control animals is lacking. We undertook this investigation to evaluate changes in lenticular morphology and in systolic blood pressure (SBP) following administration of high-salt. Both DS and DR rats were given high-salt for up to 33 weeks. Lenses were extracted at desired intervals following the initiation of the high-salt diet and processed for light and transmission electron microscopy. Except for some variations between animals, our study showed that DR rats, in general, exhibited normal SBP and relatively normal morphology. In comparison, the lenses of DS hypertensive rats demonstrated the following major changes: (1) some epithelial cells showed multilayering, nodule formation, pyknotic nuclei, swelling of cell organelles, presence of a large number of lysosomes, and distension of intercellular spaces, (2) disorganization of the bow and (3) swelling of fiber cells in the equatorial region. These changes were similar to those observed in precataractous stages during the development of several types of experimentally induced cataracts and indicate an alteration in normal electrolyte balance as well as hyperosmolarity. Changes in ion transport and electrolyte imbalance have been reported in the lenses of DS rats on high-salt diet. These alterations and a defect in the sodium-potassium pump may be responsible for cataract development in these hypertensive rats.

We reported that in utero galactose-induced cataracts could be inhibited if aldose reductase inhibitors (ARIs) were included in the galactose diet of pregnant rats. These studies involved morphological and cytochemical approaches. We undertook this investigation to evaluate the effects of ARIs in preventing the formation, accumulation and depletion of dulcitol in lenses of in utero galactose exposed neonates and in mothers during and following pregnancy. Sprague Dawley rats were fed Purina Rat Chow with 50% galactose either with or without 15mg Sorbinil or 1mg Eisai compound E-0722/day/Kg body weight during and following pregnancy. The lenses of neonates and mothers were processed to determine dulcitol concentrations. At parturition there was a significant amount of dulcitol in the lenses of pups and their mothers, which reduced rapidly in the lenses of pups regardless of the diet fed to the nursing mother. While galactose had a cross-placental but not a milk-mediated effect, the ARIs had both cross-placental and milk-mediated effects on dulcitol accumulation and depletion, respectively. The galactose feeding of mothers post-parturition maintained the high lenticular dulcitol concentration and the absence of galactose led to a reduction in lenticular dulcitol. The correlation between dulcitol accumulation and cataract development is discussed.

Alterations in elemental composition of the normal lens have been reported to accompany galactose cataract development in rats. In this report we present the changes in regional distribution of Na, K, Cl, P, S and Ca during the reversal of galactose-induced cataracts. Elemental X-ray maps of lenses from young female Sprague Dawley rats fed 50% galactose for 20 days were examined at 0, 20, 40 and 90 days following the transfer of galactose fed rats to Purina Rat Chow diet. Reinstatement of normal elemental distribution accompanied the progression of lens transparency. By 90 days on the rat chow diet, K had increased and Na, Cl and Ca had decreased so that a near normal lenticular distribution of these elements was established. The reinstatement of elemental distribution during cataract reversal followed a pattern similar to that observed for alterations during cataract development, initiating near the equatorial surface and expanding centrally. The correlation between the alterations in the distribution of the elements studied and our previously reported morphological investigation of lenses during galactose cataract reversal is discussed in this report.

Aldose reductase (AR), a major enzyme in the polyol pathway, is thought to be responsible for accumulation of polyols in lenses exposed to high doses of galactose or glucose, and it may be linked to some of the complications found in diabetes. In this report we examined the level of expression of AR mRNA in lens epithelia undergoing development of galactose cataracts in vivo. The AR mRNA was quantitated by Northern blot hybridization with a [35S]-RNA transcript from a previously described AR cDNA clone. This was done on normal lens epithelia and on epithelia from lens of rats fed a diet of Purina Chow containing 50% galactose for periods of from 6 hr to 20 days. We found AR mRNA to elevate to about 5-fold the control levels by 12-24 hr on galactose, then decrease to the control levels by day 4. The increase in AR mRNA appears to be transitory. The high abundance in AR mRNA by 24 hr on galactose was confirmed by in situ hybridization. At later periods, from 8 to 20 days on galactose, a slow increase in AR mRNA took effect, as we have previously reported. Changes in the levels of galactose and dulcitol between 0 and 96 hr were also quantitated by gas chromatography, showing that there was a significant increase in both galactose and dulcitol occurring throughout the experimental period.(ABSTRACT TRUNCATED AT 250 WORDS)

We have shown that galactose cataract development is delayed or inhibited with the administration of aldose reductase inhibitors (ARIs). Dulcitol forms and accumulates in the lens of rats fed galactose. We undertook investigations to study the effectiveness of ARIs in preventing the formation and accumulation of dulcitol in the lens. Young Sprague Dawley rats were fed Purina Rat Chow with 50% galactose either with or without 15 mg sorbinil, 0.15, 0.5, or 1.0 mg E-0722/day/Kg body weight. At desired intervals following the initiation of diets, the lenses were processed for the determination of galactose and dulcitol levels. The lenticular dulcitol increased significantly in all animals fed galactose reaching a maximum level by approximately 15 days with comparatively lower levels in the groups fed ARIs with galactose; this increase was dose dependent in the groups fed E-0722. There was a subsequent, rapid drop in lenticular dulcitol by 18 days in all dietary groups. Interestingly, a second peak of increased lenticular dulcitol was observed in all groups. The correlation between dulcitol accumulation and cataract development is discussed.

It is well established that normal patterns of epithelial cell proliferation and metabolism, and of fiber cell differentiation and maturation are essential for the maintenance of transparency in the ocular lens. Several factors, including exposure to high levels of sugars, have been known to result in the compromise of lens transparency. For example, initiation of lens cell damage by galactose induces lens epithelial cells to proliferate. Elevated levels of c-myc mRNA have usually been correlated with rapid cell growth and increased entry of cells into the S phase. Therefore, changes in c-myc mRNA levels may provide an early indication of the stimulation of lens epithelial cells to proliferate and differentiate, which has been postulated to be an early and important event in response to lens cell injury by galactose. By Northern blot hybridization analysis we quantitated c-myc mRNA levels in the lens capsule epithelia of rats (1) exposed to galactose, and (2) undergoing a partial recovery from the galactose-induced cell damage. At the onset of lens cell damage, we find c-myc mRNA to elevate to 6-fold by 24 hr, and by 48 hr decreases to about 3-fold the normal levels. During recovery, c-myc mRNA continues to be expressed at high levels approaching a 10-fold increase by day 12, then decreasing to levels of about 8-fold the control by day 30. The 24 h transitory elevation in c-myc mRNA in lens epithelial cells is in accord with our previous observations on the 24 h increase in MP26, gamma crystallin and aldose reductase mRNAs following a high influx of galactose. Therefore, the elevation in c-myc mRNA as well suggest that galactose appears to cause lens cells to undergo an early transitory period of gene induction following the exposure of lens cells to galactose.

Lens permeability has been shown to be compromised during galactose-induced cataract development in rats. Recent studies have demonstrated permeability and diffusion pathways as well as endocytotic activity in normal lenses of different species using tracers of different molecular weights. We investigated the permeability and diffusion of tracers in normal rat lenses and in lenses during cataract development using three different molecular weight tracers, lanthanum nitrate (LN, MW 430), ruthenium red (RR, MW 858.5) and horseradish peroxidase (HRP, MW 40.000) for this study. Sprague Dawley rats weighing 50gms were fed Purina Rat Chow with or without galactose. Our results, based on transmission electron microscopy, show that all tracers penetrated through the capsule and the basal portion of the intercellular spaces. While the diffusion of RR was restricted to the epithelial cell layer, LN and HRP were observed in intercellular spaces in cortical fiber cells. In the HRP "washout" studies, HRP could be readily removed from the intercellular spaces in the basal region in the epithelial cell layer. This and other observations suggest the presence of a barrier (tight-junction) in the apical region. Our studies also suggest an influx of tracers in the lens, specifically LN and HRP, through the equatorial region. The permeability of the tracers increased and endocytotic vesicles with tracers were often found associated with the lateral and basal membranes of epithelial cells in the galactose-fed rats at the precataractous and mature cataractous stages. This study provides further support for the presence of a tight-junction between epithelial cells and indicates the movement of tracers through the equatorial region. Moreover, it confirms previous observations that indicated alterations in lens permeability during galactose cataractogenesis.

Our previously reported investigations showed that cataracts could be induced in fetal lenses through the maternal feeding of galactose during pregnancy. We also reported that the lens opacity present at birth reverses completely by 30 days of age if there is no further post-natal exposure to galactose. This investigation was designed to investigate if an aldose reductase inhibitor (ARI) has any cross-placental effect in preventing galactose-induced cataracts in fetuses. We have also evaluated if there are any milk-mediated effects of galactose on cataract induction and of galactose and ARI on the maintenance or reversal of opacities induced in utero. Pregnant Sprague-Dawley rats were fed either 50% galactose or rat chow with or without the ARI, (2R,4S)-6-fluoro-2-methyl spirochroman-4,4'-imidazolidine-2',5'-dione (Eisai compound E-0722). Following parturition, pups of mothers from the different dietary groups were either fed by their own mothers or foster fed by lactating females fed either rat chow or 50% galactose with or without the ARI. Lenses of the pups were examined at desired intervals with light and scanning electron microscopes. We observed that: (a) both galactose and the ARI had a cross-placental effect on the fetal lenses in the development and inhibition of cataracts, respectively; (b) galactose had very little, if any, milk-mediated effect on either the induction of cataracts in newborn pups that were born with transparent lenses or the maintenance of cataracts induced in utero; (c) the ARI appeared to have a milk-mediated effect, which accelerates the reversal of cataract associated alterations in lenses of pups with cataracts induced in utero, leading to further reinstatement of lens transparency; and (d) the presence of ARI in the diet of rats during pregnancy and/or post-parturition provided continued protection to the lenses of pups that were exposed to a galactose diet following birth.

It is well established that high concentrations of sugar in the lens of the eye eventually lead to fiber cell destruction and cataracts. In these studies the decrease in crystallin mRNAs was quantified as a result of influx of high concentrations of galactose into the lens of rats. The alpha A-, alpha B1-, and gamma-crystallin mRNA concentrations were assessed in normal lens and in lens undergoing development of sugar cataracts by northern blot and in situ hybridization methods. In a normal, 28-day-old lens, alpha A-crystallin mRNA accumulated to high levels throughout the fiberplasm, and alpha B-crystallin mRNA was present at low levels in epithelial cells, with increased expression in elongating epithelial and fiber cells. The beta B1-crystallin mRNA was distributed to about the same grain density throughout the fiberplasm but at significantly lower levels than alpha A-crystallin mRNA. The gamma-crystallin mRNA first emerged in the terminally differentiated fiber cell, with insignificant amounts detected in the elongating epithelial and fiber cells at the bow. Measurements of hybridization levels on the same RNA population isolated from a single lens showed that in the controls, alpha A-crystallin mRNA comprised about ten times the level of alpha B-crystallin mRNA and twice the level of beta B1- and gamma-crystallin mRNAs. In the cataractous lens the rate of decrease in the concentrations of alpha A-, alpha B- and beta B1-crystallin mRNAs was the same; the decrease in gamma-crystallin mRNA was far more severe. By 20 days of feeding of galactose, at the age of 48 days, gamma-crystallin mRNA diminished to about 9% of the control levels, alpha A-crystallin mRNA to 49%, alpha B-crystallin mRNA to 55%, and beta B1-crystallin mRNA to 65%. In the normal lens, at 48 days of age, the levels of alpha A-, alpha B-, and beta B1-crystallin mRNAs showed no significant changes; the gamma-crystallin mRNA level decreased significantly, to about 70% of the day-28 level, the time at which galactose feeding began. Overall, these data suggest that the loss in crystallin mRNAs in response to the development of galactose cataracts follows this order of decline: gamma greater than alpha B greater than alpha A greater than beta B1.

It is well established that in response to feeding of galactose to four-week-old rats cataracts develop (Unakar, Genyea, Reddan & Reddy, Exp. Eye Res. 26, 123-33, 1978). Initiation of cataracts leads to epithelial cell proliferation. In the lens, the main intrinsic membrane protein, MP26, is recognized as a fiber cell-specific gene product, and gamma-crystallin is found in terminally differentiated fiber cells. By Northern blot analysis we quantitated the MP26 and gamma-crystallin mRNAs found in the lens at various times spanning from 0 to 96 hr on galactose. We find that by 24 hr, MP26 mRNA, as well as gamma-crystallin mRNA, increased significantly above the zero time levels. The data was also confirmed by in situ hybridization of various lens sections to both gamma-crystallin and MP26 35S-labeled antisense RNA probes. As expected, at later periods beyond 24 hr, the levels of gamma-crystallin and MP26 mRNAs dropped to below the control levels. The initial increase in MP26 and gamma-crystallin mRNAs lead us to conclude that initiation of galactose cataracts appears to support, although for a short time period, both epithelial cell elongation and fiber cell differentiation.

Lanthanum nitrate (LN) and horseradish peroxidase (HRP) were used as tracers to study intercellular permeability as well as the existence and location of tight junctions and changes in them, if any, in the lens epithelium of Sprague Dawley rats. Thin sections of lenses taken from animals at birth and at three day intervals until the neonates were weaned at approximately 22 days were studied at the electron microscope level. At every age both tracers permeated the intercellular spaces of the anterior region epithelium and between the fiber cells of the equatorial region. The LN precipitates terminated near the apical part of the epithelium in the anterior polar region and were not seen at the epithelial-fiber interface or between fibers in this region. At the site of the tracer termination the intercellular space was found to be considerably constricted or completely sealed. The HRP precipitates, however, were present in these intercellular spaces and by using the HRP "washout" procedure could be washed out of the basolateral site of the intercellular spaces of the epithelium up to the point of constriction if a tight junction were present. The lens area occupied by epithelium with tight junctions was age related being very small in lenses from animals less than 24 hours old and becoming increasingly enlarged as the newborn aged. These findings further confirm the existence of a barrier in the form of tight junctions between epithelial cells in the central anterior region of the maturing rat lens. Moreover, our observations also provide information regarding the pattern of tight junction development and distribution during lens maturation.

Aldose reductase (AR) is implicated in the development of sugar cataracts by its reduction of galactose or glucose to polyols. The authors' recent work suggested that AR mRNA is found to be expressed in high concentrations in rat-lens epithelial cells after exposure of the animal to a diet containing 50% galactose. They localized the AR mRNA in the lens cells by in situ hybridization with a previously described AR clone. The data establish that AR mRNA is apparently an epithelial and not a fiber-cell gene transcript. It accumulates in the epithelial cell, then it is carried into the newly differentiated fiber cell, and finally it concentrates in the posterior region of the matured fiber cell. The AR mRNA is found in all of the anterior epithelial cells including the equatorial and central epithelia. It is present at highest concentrations in the elongating epithelial cells, and it is distributed equally throughout the secondary fiber cells at the bow, with no indication of a preferential buildup of AR mRNA in any of the nucleated fiber cells in the cortex. This differs from what the authors reported to occur with MP26 mRNA, a fiber cell-specific gene transcript. They found that MP26 mRNA was absent from the epithelial cells but was preferentially found in the secondary fiber cells. Present data suggest that the increase in AR mRNA concentration observed to occur in cataractous lenses is a result of epithelial cell proliferation, where every cell appears to be competent in expressing AR mRNA. The results of this research imply that AR mRNA is a lens epithelial cell-specific gene transcript in both normal and cataractous lenses.

Aldose reductase (AR) mRNA concentration in rat lens was quantitated by hybridization of a RNA transcript from a previously described AR cDNA clone to mRNA found in epithelial and cortical cytosols. This was done on normal rat lens and on lens initially made cataractous by feeding of a diet of Purina Chow containing 50% galactose, followed by reversal of the cataracts due to the removal of the galactose from the diet. Recent data from this laboratory has shown that AR mRNA was increased in lens epithelial cells upon administration of galactose; while in the cortex it was reduced to insignificant levels when fiber cell damage became extensive by day 20 on galactose. Present data reveals that, upon removal of galactose from the diet, the lens epithelial AR mRNA was gradually reduced from the high levels found at day 20 of galactose feeding to low levels by day 30 of reversal. On the other hand, the cortex exhibited an initial sudden increase in AR mRNA at days 1 to 6 of reversal and by day 30 it was reduced to levels below those found in the untreated lens. DNA content in the epithelium also began to decrease to normal levels by day 16 following reversal of the cataracts. The data demonstrate that the concentration of AR mRNA in lens of reversed cataracts appears to faithfully reflect the loss of epithelial cellular need for AR mRNA in favor of enhanced differentiation of epithelial cells to secondary fiber cells.

Primary cultures of adult rat hepatocytes were prepared by releasing liver cells through fractional trypsinization and by a novel nonenzymatic procedure involving the imprinting of liver explants on the surface of the culture flask (explant imprinting). The comparative properties of these two primary cultures and their abilities to synthesize alpha 2u-globulin under multihormonal stimulation were investigated. The hepatocytes isolated from male rats by fractional trypsinization were found to divide rapidly and form a confluent monolayer within 5-7 days of culture. However, these cells failed to synthesize alpha 2u-globulin even after treatment with 5 alpha-dihydrotestosterone, GH, T3, and dexamethasone, hormones with known stimulatory effects on the synthesis of alpha 2u-globulin in vivo. On the contrary, hepatocytes prepared from male rats by the nonenzymatic procedure of explant imprinting were found to synthesize alpha 2u-globulin for more than 2 weeks and responded to GH, T3, and dexamethasone with enhanced production of this protein. Fortification of the culture medium with all of the above three hormones caused an approximately 9-fold enhancement of alpha 2u production within 4 days of culture compared to production in control culture without these hormones. The above results substantiate earlier findings concerning the multiple hormonal requirement for hepatic synthesis of alpha 2u-globulin in vivo and show that isolation and culture of hepatocytes by nonenzymatic explant imprinting may serve as a useful procedure for studying endocrine regulation of gene activity in vitro.

Biochemical studies have demonstrated the presence of ouabain-sensitive Na-K-ATPase in lens epithelium. The development of certain experimental cataracts has been associated with the decrease in the level of activity of this enzyme. Cytochemical investigations at the ultrastructural level to localize this important enzyme in the ocular lens are needed to exhibit the site of Na-K-ATPase action. In this investigation cytochemical methods originally described by Ernst for the localization of Na-k-atpase through K-NPPase reaction have been used for normal and cataractous ocular lens. The reaction product was observed to be present on the lateral and apical portion of the epithelial cell membranes. Mooreover, the membranes of the cortical fibers in the anterior polar and equatorial region of the lens also exhibited reaction product of Na-k-atpase. The presence of ouabain reduced the reaction product of Na-K-ATPase; however, continuous exposure to ouabain during preincubation fixation, and incubation demonstrated greater reduction in enzyme action in the lens. The Na-K-ATPase activity in the lenses of animals fed galactose decreased with increase in time on galactose diet. In this study, using ultrastructural cytochemistry, we have demonstrated gradual decrease in Na-K-ATPase activity in the lenses of galactose-fed animals.

Previous cytochemical and biochemical studies have shown an increase in the activity of acid phosphatase and arylsulfatase during the induction of galactose cataracts in rat lenses. It was postulated that these enzymes may be involved in lens fiber degradation observed during cataractogenesis, however, the role of these enzymes in the repair process was not ruled out. The present investigation has evaluated the level of acid phosphatase activity in lenses in which the induction of opacity is inhibited with the aldose reductase inhibitor sorbinil and during the recovery of galactose induced opacity. Sprague-Dawley rats received 50% galactose diet, or galactose diet with sorbinil, or laboratory chow diet. Following 20 days on this diet all rats received lab chow plus 50 mg kg-1 sorbinil (recovery diet). The lenses were removed at desired intervals following the initiation of the above three diets and following the transfer of animals to the recovery diet. Cytochemical localization and biochemical quantitation of acid phosphatase activity were performed with methods previously reported. Most of the enzyme activity was localized within the epithelial cells and superficial cortical fibers. In the epithelial cell layer, the enzyme activity was primarily localized in lysosomes and at extracellular sites near the epithelial cell membrane which abut each other and cortical fibers. In cortical fibers the enzyme activity was observed at various extracellular sites between the cell membranes of neighboring fibers. The effect of sorbinil, if any, and the possible role of acid hydrolases in the repair process during cataract reversal is discussed.

Previous morphological and biochemical studies indicate that a late appearing hereditary Emory mouse cataract may be a good model for certain human senile cataracts. The development of lenticular opacity in the Emory mouse is a slow process which provides an opportunity to conduct analysis of the progression of alterations that lead to cataract development. Biochemical investigations have not yet demonstrated any specific correlation between alterations in the lens and the extent of opacity. We have conducted studies to determine the role of Na+K+-ATPase in the development of cataract in the Emory mouse. In this report we present results obtained on the site and level of activity of Na+K+-ATPase in six- and twelve-month-old Emory mouse lenses in which visible cataractous changes are beginning to appear. CFW mice (the parent strain) were used for controls in this study. Ultrastructural cytochemistry for the localization of Na+-K+-ATPase exhibited the enzyme reaction product for this enzyme to be present mainly between the lateral epithelial cell membranes and between the apical epithelial cell membranes and superficial cortical fiber membranes. In cortical fibers the reaction product was localized between fiber membranes. Although there was very little or no significant differences in the extent of reaction product in epithelial cells, the reaction product in the cortical fibers of six-month-old Emory mouse was less extensively distributed as compared to lenses from control CFW mice of the same age.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphological changes preceding insulin-induced mitosis in cultured rabbit lenses were characterized. The fine structure of these lenses was compared with that of lenses exposed to culture conditions in which a mitotic response failed to materialize. Lenses were cultured in: (1) medium KEI-4, i.e., a completely defined medium, which retains the central epithelium in a nonproliferating state, (2) KEI-4 plus insulin, and (3) KEI-4 plus insulin, theophylline, and dibutyryl adenosine 3':5'-cyclic monophosphate (I-DBcAMP-T). The insulin-induced mitotic response was inhibited by dibutyryl cAMP and theophylline or papaverine or 8-bromoadenosine cAMP. At one hour, lenses cultured in KEI-4 alone or in KEI-4-I-DBcAMP-T showed a moderate increase in free ribosomes relative to zero hour controls. In contrast, lenses exposed to KEI-4-insulin exhibited a pronounced increase in the number of free ribosomes and had prominent nucleoli. An increase in the degree of folding of the cell membrane was detected in all lenses at three hours but was decidedly more prominent in lenses cultured in KEI-4-insulin. At seven hours, lenses exposed to KEI-4-insulin showed an increase in the size and apparent number of mitochondria. At all intervals, ribosomes were more numerous in lenses cultured in KEI-4-insulin. Lenses cultured in medium KEI-4 alone for 24 and 52 hours displayed a morphology ostensibly similar to that of zero hour controls. At 52 hours the epithelial cells of lenses exposed to insulin, DBcAMP and theophylline were multilayered and markedly elongated. The increase in ribosomes (one hour), changes in the cell membrane (three hours), and the heightened mitochondria profile all precede the initiation of DNA synthesis and mitosis which commenced at 24 and 40 hours, respectively.

A series of rat galactose lenses, from 1 to 20 days on the 50% galactose diet, were frozen in the whole eye, and fractured from pole to pole in the frozen state. Lyophilized half-lenses were prepared for analysis by energy dispersive spectrometry (EDS). Following elemental analysis, some specimens were embedded and sectioned for histological studies. Elemental X-ray maps, and/or profiles, were made for K, Na, Cl, Ca, P, and S. As early as two days on the galactose diet, a crescent-shaped region ("streak") of Cl, Na, and Ca gain, and K loss develops near the equatorial surface. Between this region and the equatorial surface are the nucleated differentiating fiber cells which maintain low Cl, Na, and Ca, and high K (viable equatorial zone, VEZ). With time the "streak" expands anteriorly, centrally and posteriorly, eventually (by 20 days) including most of the lens. The VEZ, including the epithelium, however, is non-reactive to the galactose diet, which is deleterious to the fully differentiated fiber cells. Eventually, the VEZ undergoes a characteristic morphological change, apparently due in part to changes in its physical environment.

Prolactin has been known to affect the water and electrolyte balance. Because increased lens hydration has been shown to be a common phenomenon in most, if not all types of cataracts, we have been interested in investigating a possible role of prolactin in sugar cataract induction and progression. For this study, we have used morphological and biochemical approaches. The prolactin delivery method involved intraperitoneal implantation of one or more pellets in Sprague-Dawley female rats. Following implantation of the desired number of prolactin or control (nonprolactin) pellets, animals were either fed galactose and lab chow, or lab chow diet. Gross morphological observations of whole lenses, slit-lamp examination of lenses and light microscopic analysis of lens sections showed that in the galactose-fed prolactin group, galactose associated alteration progressed faster and total opacification (mature cataract development) was achieved earlier than in the nonprolactin group. The levels of galactose and dulcitol were higher in the lenses of galactose-fed prolactin treated rats as compared to lenses from nonprolactin (control) rats. No significant difference in lens Na+-K+ ATPase activity between the prolactin and nonprolactin group was observed. Our results indicate that prolactin accelerates galactose-induced cataractogenesis in rats.

The diaminobenzidine (DAB) technique was used to investigate the localization of the peroxidatic activity of catalase in cultured lens epithelial cells at the ultrastructural level. Cultured rabbit, bovine and mouse lens epithelial cells incubated in an alkaline DAB reaction mixture contained catalase-positive microperoxisomes which were randomly distributed throughout the cytoplasm. The reaction product of catalase was abolished when cells were treated with 3-amino-1-H-1,2,4-triazole, a specific inhibitor of catalase, and was not detected when DAB or H2O2 was omitted from the incubation medium, or when the pH of the reaction mixture was lowered to 7.0. The fine structure of lens epithelial cells that were constantly exposed to 0.025 or 0.05 mM hydrogen peroxide was also determined. Lens epithelial cells that were constantly exposed to 0.05 mM H2O2 for 1-3 h exhibited damaged mitochondria, a phenomenon that did not occur when the cells were exposed to 0.025 mM peroxide.

The importance of enzyme Na-K-ATPase in the development of galactose-induced cataractogenesis is now well realized. In our recent studies we reported decreased level of activity of this enzyme with an increased duration of galactose feeding and the induced alterations in rat ocular lens. Our approach was to determine the level of Na-K-ATPase activity by ultrastructural cytochemical analysis of lenses and spectrophotometric analysis of the incubating media used for cytochemical localization as described by Ernst. Using these approaches, we have determined the activity level of this enzyme during the reversal phase of the galactose-induced injury to the lens. Our findings are presented in this report and show that the activity of Na-K-ATPase recovers rapidly and attains the normal level when the animals were transferred to Rat Chow diet after the establishment of mature cataracts resulting from galactose feeding. This study supports the previous biochemical and morphological findings that partial reversal of galactose-induced cataractous lens occurs upon discontinuation of feeding of cataractogenic agent galactose.

Our laboratory is involved in studying the mechanism of repair in the ocular lens. As lysosomal enzymes have been shown to play an important role in tissue repair, we have been investigating the status of lysosomal enzymes, such as acid phosphatase and arylsulfatases, in the normal and injured lens. In the present investigation, we have examined the presence, distribution, and possible role of arylsulfatases (E.C. 3.1.6.1) in lenses of normal and galactose-fed rats. Arylsulfatases were localized in lenses using the cytochemical procedure described by Hopsu-Havu and Helminen (1974) using p-nitrocatechol sulfate as a substrate and then examined at the ultrastructural level. The reaction product resulting from arylsulfatase activity was mainly localized in the epithelial cells with very little activity in the cortical fibers. The intracellular activity was confined to lysosomes. Some extracellular activity was visible in the intercellular regions in both the epithelium and superficial cortex. With the progression of galactose-induced lesion in the epithelium the number of lysosomes exhibiting enzyme reaction product was found to have increased, and the lysosomes closely abutted the capsule. The biochemical assay indicated a considerable increase in the activity of arylsulfatases with the continuation of a galactose diet. The possible role of arylsulfatases in the normal and cataractous lens is discussed.

The present investigation was designed to assess whether lens membrane permeability is affected by changes in levels of intracellular calcium. Lanthanum, an inhibitor of Ca-ATPase, affected an increase in the concentration of intracellular calcium (Cai) measured in cortical fiber cells. Preculture of lenses in lanthanum (1.0mM) caused an accumulation of 36Cl during subsequent culture at a rate three-fold higher than control lenses. Changes in calcium levels, however, were not responsible for the observed flux changes because a 40mV depolarization was observed to occur prior to a significant increase in calcium levels. The non-specific effects of lanthanum and other potential inhibitors of calcium transport were avoided by preculturing lenses in an ion-HEPES medium containing 20mM calcium chloride. In lenses with a six-fold increase in calcium levels there resulted only a 10% increase in 36Cl uptake over a 3 hr period. 86Rb efflux was also measured and the rate constant was unchanged compared to control lenses. Calcium accumulation did lead to a small (8mV) depolarization which may account for the small increase in chloride accumulation. By light microscopy, morphology of cortical lens fibers and the epithelium appeared unchanged in the calcium-loaded lens. The results provide little evidence that an increase in Cai leads to acute changes in lens membrane permeability.

In the present investigation we have examined the presence, distribution and probable role of acid phosphatase in lenses of normal and galactose-fed rats. Acid phosphatase was localized in lenses using two separate cytochemical procedures (Gomori and Barka-Anderson methods) and examined at the ultrastructural level. Both procedures, in general, provided similar sites of acid phosphatase activity, although with the Barka-Anderson method finer and larger amounts of well-defined reaction product were observable at the site of reaction. The reaction product was observed in lenses of both rats fed on regular laboratory chow and galactose-fed rats. The intracellular location of the reaction of this enzyme was primarily in lysosomes and occasionally in the endoplasmic reticulum cisternae. At the extracellular sites, it was near the epithelial cell membranes which abut each other and cortical fibers. However, in the cortical fibers the extracellular localization was at various sites on the entire intercellular space between neighboring fibers. The possible role of hydrolases in the lens tissue is discussed.

Ultrastructural and cytochemical investigations of ocular lens have been difficult due to limited applicability of currently available techniques for the preparation of this tissue for such studies Several modifications in currently available techniques are being made in order to preserve the fine structure of the lens. In this report we present a modified method which provides good preservation of lens fine structure and allows cytochemical localization of enzymes in this tissue. Brief freezing permits preparation of thin slices of the lens which are subsequently incubated in the appropriate media for enzyme cytochemistry at an ultrastructural level. We have used this modified method for the localization of Na-K-dependent ATPase and the results are presented in this report.

Lenses of rats maintained on a 50% galactose diet displayed the development of a progressive cataract which was cortical at 3-11 days, and progressively internalized (nuclear as well) and mature at 16-20 days of feeding. Lens fiber plasma membranes were isolated from female rats subjected to the galactose diet and from controls at 11, 19, and 31 days of feeding, and analyzed by SDS-PAGE. Examination of the fiber plasma membranes from whole lenses of galactose-fed rats demonstrated the limited proteolysis of MP26 into MP23-24, in both the cortical and mature stages of the resultant cataracts. The limited proteolysis of MP26 was first evident in the lens cortex at 11 days of galactose feeding, and was evident as well, and more severe in proportion, in the lens nucleus at 19 days of feeding. The greatest proportion in MP26 limited proteolysis was observed in whole lenses at 31 days of galactose feeding. The regional progression of MP26 limited proteolysis closely paralleled the morphological progression of the galactose-induced cataract in the rat. The proportion of lens MP26 which underwent limited proteolysis into MP23-24 increased the longer the animals were kept on the galactose diet.

Lens ultrastructure and Na- K-ATPase activity in the lenses of rats fed galactose and a galactose + sorbinil diet (aldose reductase inhibitor) were studied. Lenses of rats on the galactose diet exhibited development of peripheral opacity within 3-4 days. This opacity progressed with the continuation of the galactose feeding, and by 20 days mature cataracts were observed in these animals. The formation of vacuoles, cysts, membrane disruption in the epithelium and fibers, and swelling of fibers accompanied the development of opacity. With the progression of opacity there was a considerable drop in lens Na- K-ATPase activity in the galactose-fed animals. However, the lenses of rats that were treated with sorbinil did not show any of the alterations in the ultrastructure of the epithelium and fibers that accompany galactose cataractogenesis. The level of Na-K-ATPase activity in the sorbinil-treated animals was similar to that found in lenses from the laboratory chow-fed group of rats. These observations further substantiate the role of aldose reductase in sugar-cataract development.

Previous work from this laboratory has shown that in cataractous lens from rats fed a 50% galactose diet up to 32 days, synthesis of crystallin mRNAs was reduced while synthesis of mRNAs for the non-crystallin proteins appeared to be maintained at equivalent or higher levels than found in the controls. In this study, we find that the population of mRNA from the 45-day cataractous lens included all of the crystallin mRNAs and substantial amounts of mRNAs for the non-crystallin proteins, in particular those proteins of molecular weights ranging from 45,000 to 92,000, and pIs from 5.5 to 7.0. At the 45-day cataractous stage, the observed mRNA products totaled between 100 and 120, while for the control they numbered between 40 and 50. In addition, hybrid-select measurements with a lambda gt11 MP26 clone established that MP26 mRNA persisted in the fiber cells of 11-, 20-, 32- and 45-day galactosemic lens. The data, therefore, suggest that continuous exposure of the lens to galactose apparently leads to significant fluctuations in mRNA synthesis and survival. Indirect immunofluorescence (with a monospecific polyclonal rabbit anti-MP26 antibody) and light-microscopy studies demonstrated that although the cortical fiber cells were swollen and structurally disoriented, they retained their nuclei, while the enucleated fiber cells were absent. The microscopy data also suggest that differentiation of the epithelial cells to fiber cells continued in the cataractous lens. Fiber-cell migration was backward as compared with the control, and the surviving cells were localized within the cortex and appeared to be surrounded by damaged cells or cell debris. It is suggested that persistent fiber-cell survival, and continued synthesis of various classes of mRNA by the nucleated cells in the cataractous lens could explain why reversal of cataracts results in recovery in both cell morphology and transparency in the non-nuclear portion of the lens.

We have demonstrated an increase in activity of arylsulfatase A and B during galactose induced cataract development in rats. Our recent investigation shows that acid phosphatase activity, which increases substantially during galactose cataract development in rats, could be contained to near normal level if Sorbinil, an aldose reductase inhibitor, was fed along with galactose to the rat. We have observed that the activity of other lysosomal enzymes, arylsulfatase A and/or B, also increases during galactose cataractogenesis. In the present report, we provide information with regards to the effect of Sorbinil on the activity of these enzymes during cataractogenesis. A modified Hopsu-Havu and Helminen method (1974) with p-nitrocatecholsulfate as substrate was used for localization of both arylsulfatase A and B; and the method of Hara et al. (1979) was utilized to obtain quantitative data on the level of arylsulfatase A and B activity. Ultrastructural cytochemistry shows that arylsulfatase activity in all lenses was primarily localized in epithelial cells in lysosomes with very little or no activity in cortical fibers. The number of arylsulfatase positive lysosomes and the activity level of these enzymes increased with the progression of cataract development. Galactose induced damage to lens morphology and increase in activity of arylsulfatase A and B was inhibited by inclusion of 50mg/Kg (diet) Sorbinil in the galactose containing cataractogenic diet. However, Sorbinil had no significant effect on the enzyme activity following the establishment of mature cataracts.

Our recent investigations have shown that the Eisai compound, E-0722, (2R-4S-6-fluoro-1-2-methylspirochroman 4,4'-imidazolidine 2,5'-dione) is a more potent aldose reductase inhibitor than Sorbinil (D-6-fluorospirochroman 4,4'-imidazolidine 2,5'-dione). In the previous studies these aldose reductase inhibitors were added to the 50% galactose diet fed to rats to determine their effect on galactose-induced alterations in the lens and the development of cataract. In this report we present our results on the effect of prefeeding the aldose reductase inhibitor, E-0722, on the alterations in rat lens following subsequent feeding of galactose. For this study, young Sprague Dawley rats were prefed either rat chow or rat chow plus 50% galactose containing 1mg/day/Kg body weight of E-0722 for 1 or 2 weeks. After this dietary regimen, the animals were transferred to diets containing 50% galactose for different periods. For controls, rats were fed either rat chow or 50% galactose without the prefeeding of E-0722. Our results obtained through gross observation of the lenses, light microscopic studies of lens sections and assay of Na+-K+-ATPase (NPPase) activity show that the prefeeding of E-0722 prior to galactose feeding delays galactose-induced alterations and the development of mature cataract.

In our previous work, two-dimensional gel electrophoretic analysis of the translational products from mRNA of lens from rats maintained on 50% galactose up to 45 days has suggested that synthesis of mRNA was not arrested by the disease process, but it decreased significantly relative to the control. The loss in mRNA number was due mainly to loss in cell population. Specifically, the gamma-crystallin mRNA product decreased to very low levels at onset of the disease. However, this mRNA was resynthesized in the surviving cells as the cataracts matured. Therefore, it became of interest to explore whether reversal or inhibition of cataracts would lead to some measurable changes in mRNA population in the experimental lens. The results show that the percentage (relative to the total mRNA population) of the in vitro [35S]-labeled translational products from alpha-, beta- and gamma-crystallin mRNAs combined, remained unchanged irrespective of the state of the lens. The severity of the cataracts was examined by indirect immunofluorescence with polyclonal MP26 antibody. Reversal of cataracts led to partial recovery of normal fiber cell morphology. Treatment with sorbinil in combination with galactose led to inhibition of cataracts with indication of appearance of vacuoles at longer periods of exposure to the drug. The translational products profile reflected the expected variation in non-crystallin and crystallin mRNA synthesis. It is concluded that there appears to be a combined fixed level of synthesis for the crystallins, such that inhibition in synthesis of gamma-crystallin mRNAs appears to lead to an increase in synthesis of alpha-crystallin mRNAs, while synthesis of beta-crystallin mRNAs showed insignificant fluctuation. A similar conclusion may also be drawn relative to the combined synthesis for the non-crystallin mRNAs.

Aldose reductase (AR) messenger RNA concentration was determined in normal rat lens and in lens from rats fed a 50% galactose diet over a period of 20 days. The AR mRNA was detected by using a previously described AR cDNA clone. The relative concentration of the AR mRNA was estimated by cpm of 35S-UTP labeled antisense RNA hybridized to dot-blots prepared from cytosols isolated from single lens, decapsulated lens (cortex) and its respective capsule (epithelia). The results demonstrated that the concentration of the AR mRNA in the epithelium doubled over the 20 day period. Correspondingly, an increase in the concentration of the DNA was also observed, suggesting that the increase in epithelial cytosolic mRNA might be partially due to the increase in the number of epithelial cells occurring in lens undergoing cataractogenesis. The increase in AR mRNA in the epithelia was gradual, and it doubled by day 12 on galactose, while the increase in DNA was rapid and reached an optimum level by about day 4. By day 4 the cortex AR mRNA concentration increased, then rapidly decreased to insignificant levels by day 20. Changes in AR mRNA and in DNA following a high influx of galactose in the lens might suggest a heightened gene response to changes in the cellular environment for the lens epithelium.