Jane Y. Tsui

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)

Limited proteolysis of MP26 in lens fiber plasma membranes of the galactose-induced cataract in the rat

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.

Scanning Electron Microscopy of Lens

Our previous transmission electron microscope and biochemical studies showed considerable regression of in utero induced galactose cataracts in rats following birth. The ultrastructural and biochemical alterations of the lens associated with such cataracts regressed considerably within a few days following birth. This report describes scanning electron microscope (SEM) observations made on lenses of offsprings of galactose-fed mothers. Lenses of rats delivered by galactose-fed mothers exhibited mature cataracts at birth. With SEM these lenses showed the presence of intercellular cysts, abnormal configuration, conformation and fragmentation of lens fibers. These changes were extensive at birth in the major portion of the lens, however, they became less extensive and gradually reduced in severity as the animal grew. By 28 days after parturition, lenses appeared normal and their morphology was comparable to that observed in lenses of offsprings from lab chow-fed animals. A probable mechanism(s) for the reestablishment of lens transparency is discussed.

Effect of Prolactin on Galactose Cataractogenesis

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.

Arylsulfatase-cytochemical localization in lenses of normal and galactose-fed rats

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.

Elemental studies in rat lens during galactose cataract reversal

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.

Effects of intracellular calcium on lens membrane permeability

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.