Nalin J. Unakar

Nuclear light scattering, disulfide formation and membrane damage in lenses of older guinea pigs treated with hyperbaric oxygen

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)

The Control of Cell Division in the Ocular Lens

Publisher Summary This chapter discusses the control of cell division in the ocular lens and primarily deals with the nature of the effects of environmental factors on the control of cell division. In certain very important related areas, such as differentiation, regeneration, and aging, the lens has proved to be the most valuable model system for analysis. These active areas of investigation, including studies on cataract formation are addressed in the chapter. Moreover, emphasis is placed on concepts of the mechanisms that underlie the control of cell division, particularly as they apply to the ocular lens. It is assumed that the knowledge gained for one tissue on a fundamental mechanism such as the control of cell division, may apply, at least in part, to control mechanisms in other tissues.

Ultrastructural changes during the development and reversal of galactose cataracts

Abstract Ultrastructural alterations in rat lens that accompany the induction of galactose cataract and the reinstatement of tissue transparency that occurs subsequent to removal of the animals from galactose diet were documented. Lenses examined at different times after the initiation of galactose diet exhibited edematous, liquefied fibers and cellular cysts. Electron dense aggregates were initially found in sections obtained from the pre-equatorial region and subsequently in the tissue from the anterior polar region. The location of the aggregates in the galactose-fed animals followed a spatio-temporal pattern which paralleled cataract maturation at the macroscopic level. The aggregates were not present in either the epithelium of the cataractous lenses or in the epithelium or fibers of lenses from animals that were fed glucose or laboratory chow alone. Mature cataracts were observed in rats fed galactose for 20 days. However, a reinstatement of fiber morphology and lens transparency was realized if these animals were subsequently placed on a galactose free diet. Normalization of fiber morphology and alignment was noted after the animals had been on a galactose free diet for 28 days. However, a nuclear opacity persisted throughout the 3 month observation period. The extent to which the repair of existing fibers and/or the formation of newly formed fibers contribute to the reversal of lens opacity remains to be ascertained.

Inhibition of galactose-induced alterations in ocular lens with sorbinil

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.

Relative abundance of aldose reductase mRNA in rat lens undergoing development of osmotic cataracts

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.

Scanning Electron Microscopy

Our laboratory undertook extensive light, transmission (TEM) and scanning elctron microscope (SEM) studies of rat lenses during the development and reversal phase of galactose-induced cataracts. These studies were undertaken in order to gain insight into the morphological manifestation of known biochemical changes that accompany development and reversal of galactose cataracts. In a recent report we presented TEM studies describing ultrastructural alterations associated with induction and reversal of galactose cataracts in rat lens. This report presents SEM findings of lenses undergoing such processes. Lenses of galactose and Purina Lab Chow-fed 50 g Sprague-Dawley rats were removed at desired times after initiation of diet and processed for SEM. When examined with SEM, some of the alterations induced with galactose included intercellular cyst formation, decrease in inter-digitations between fibers, abnormal configurations, conformation, granulation roughening and fragmentation of the lens fibers. These alterations progressed from equatorial regions to lens nucleus. Upon removal of galactose from the diet after the establishment of mature cataracts, normal lens fiber morphology was reestablished and the progression of normalization followed similar equator to nucleus pattern. However, lens damage and transparency was not restored throughout the lens even after 90 days following cessation of galactose when small nuclear opacity and damage was still evident. These observations compliment TEM findings reported previously from our laboratory. A probable mechanism for the reestablishment of lens transparency is proposed.

Sodium-potassium-dependent-ATPase activity in Emory mouse lens

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)

Effect of germanium-132 on galactose cataracts and glycation in rats

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.

Separation of rat liver lysosomes and mitochondria in the A-XII zonal centrifuge☆

Abstract Rat liver lysosomes and mitochondria were isolated free of mutual cross-contamination on the basis of differences is sedimentation velocity during a single run in the type A-XII zonal centrifuge. Cytochrome oxidase was employed as the biochemical marker for the mitochondria, and acid phosphatase (nitrophenyl phosphate substrate, sodium acetate buffer, pH 5.0, in the presence of the non-ionic detergent Turgitol NPX) was employed as the biochemical marker for the lysosomes. Structure-linked latency and activation was demonstrated for the “lysosomal” phosphatase following exposure to Turgitol NPX, osmotic lysis by treatment with distilled water, and fragmentation in a Waring blender. Electron microscopy of the isolated fractions showed that the microsomes were contaminated by glycogen but free of mitochondria and lysosomes, the lysosomes were contaminated by microsomes and glycogen but were free of mitochondria, and the mitochondria were cross-contaminated only by a few microsomal vesicles.

An enzymatic assay for DNA in epoxy-tissue-sections by light microscope-autoradiography

Thick and thin sections of glutaraldehyde- and osmium-fixed and epoxy-embedded tissues were incubated with calf-thymus terminal deoxynucleotidyl transferase, and the resulting incorporation of 3H-dAMP was located by light microscope-autoradiography. This cyto-enzymatic method is rapid and permits detection of nuclear DNA in a large variety of cell types from the rat trachea, rabbit lens epithelium, and newt ovaries.