Age-related compaction of lens fibers affects the structure and optical properties of rabbit lenses.

BACKGROUND: The goal of this investigation was to correlate particular age-related structural changes (compaction) to the amount of scatter in rabbit lenses and to determine if significant fiber compaction occurred in the nuclear and inner cortical regions. METHODS: New Zealand White rabbits at 16-20 months old (adult; n = 10) and at 3.5-4 years old (aged; n = 10) were utilized for this study. Immediately after euthanising, scatter was assessed in fresh lenses by low power helium-neon laser scan analysis. Scatter data was analyzed both for whole lenses and regionally, to facilitate correlation with morphometric data. After functional analysis, lenses were fixed and processed for scanning electron microcopy (SEM; right eyes) and light microscopy (LM; left eyes). Morphometric analysis of SEM images was utilized to evaluate compaction of nuclear fibers. Similarly, measurements from LM images were used to assess compaction of inner cortical fibers. RESULTS: Scatter was significantly greater in aged lenses as compared to adult lenses in all regions analyzed, however the difference in the mean was slightly more pronounced in the inner cortical region. The anterior and posterior elliptical angles at 1 mm (inner fetal nucleus) were significantly decreased in aged vs. adult lenses (anterior, p = 0.040; posterior, p = 0.036). However, the average elliptical angles at 2.5 mm (outer fetal nucleus) were not significantly different in adult and aged lenses since all lenses examined had comparable angles to inner fetal fibers of aged lenses, i.e. they were all compacted. In cortical fibers, measures of average cross-sectional fiber area were significantly different at diameters of both 6 and 7 mm as a function of age (p = 0.011 and p = 0.005, respectively). Accordingly, the estimated fiber volume was significantly decreased in aged as compared to adult lenses at both 6 mm diameter (p = 0.016) and 7 mm diameter (p = 0.010). CONCLUSION: Morphometric data indicates that inner cortical fibers undergo a greater degree of age-related compaction than nuclear fibers. Increased scatter appears to be only tentatively correlated with regions of fiber compaction, suggesting that it is simply one of an array of factors that contribute to the overall decreased transparency in aged rabbit lenses.

Gap junctions contain different amounts of cholesterol which undergo unique sequestering processes during fiber cell differentiation in the embryonic chicken lens.

PURPOSE: To determine the possible changes in the distribution of cholesterol in gap junction plaques during fiber cell differentiation and maturation in the embryonic chicken lens. The possible mechanism by which cholesterol is removed from gap junction plaques is also investigated. METHODS: Filipin cytochemistry in conjunction with freeze-fracture TEM was used to visualize cholesterol, as represented by filipin-cholesterol complexes (FCCs) in gap junction plaques. Quantitative analysis on the heterogeneous distribution of cholesterol in gap junction plaques was conducted from outer and inner cortical regions. A novel technique combining filipin cytochemistry with freeze-fracture replica immunogold labeling (FRIL) was used to label Cx45.6 and Cx56 antibodies in cholesterol-containing gap junctions. Filipin cytochemistry and freeze-fracture TEM and thin-section TEM were used to examine the appearance and nature of the cholesterol-containing vesicular structures associated with gap junction plaques. RESULTS: Chicken lens fibers contain cholesterol-rich, cholesterol-intermediate and cholesterol-free gap junction populations in both outer and inner cortical regions. Filipin cytochemistry and FRIL studies confirmed that cholesterol-containing junctions were gap junctions. Quantitative analysis showed that approximately 86% of gap junctions in the outer cortical zone were cholesterol-rich gap junctions, whereas approximately 81% of gap junctions in the inner cortical zone were cholesterol-free gap junctions. A number of pleiomorphic cholesterol-rich vesicles of varying sizes were often observed in the gap junction plaques. They appear to be involved in the removal of cholesterol from gap junction plaques through endocytosis. CONCLUSIONS: Gap junctions in the young fibers are enriched with cholesterol because they are assembled in the unique cholesterol-rich cell membranes in the lens. A majority of cholesterol-rich gap junctions in the outer young fibers are transformed into cholesterol-free ones in the inner mature fibers during fiber cell maturation. A distinct endocytotic process appears to be involved in removing cholesterol from the cholesterol-containing gap junctions, and it may play a major role in the transformation of cholesterol-rich gap junctions into cholesterol-free ones during fiber cell maturation.

The role of Src family kinases in cortical cataract formation.

PURPOSE: The goal of this study was to determine the role of Src family kinases (SFKs) in the development of lens cataract. This question was particularly significant, because these tyrosine kinases mediate the stress pathways known to lead to cataract formation. The experiments were focused on whether the inhibition of SFK activity suppresses the formation of lens opacities. METHODS: A whole-lens culture system was developed, in which cortical opacities formed within 5 days, in embryonic day (E)10 lenses grown in medium containing 10% fetal bovine serum. SFK activity was blocked in the cultured lenses by growth in the presence of the SFK-specific inhibitor PP1. Control cultures were grown in medium without inhibitor or in the presence of PP3, the inactive analogue of PP1. Lenses were cultured for 10 days, observed, and photographed daily. Opacification was quantified with image-analysis software. Tissue architecture was determined after hematoxylin and eosin staining and cellular organization by fluorescent localization of filamentous actin with fluorescein-conjugated phalloidin. RESULTS: Almost all lenses in the control cultures developed cortical opacities covering approximately 50% of the lens area by day 10. Similar to control cultures, PP1-treated lenses showed mild posterior opacities during the first 5 days in culture, but then became strikingly transparent. Only 7% of the PP1-treated lenses showed development of cortical cataract, and the average area of opacity was just 0.5% by culture day 10. In all cultured lenses, even in the presence of the PP1 inhibitor, the bow region of the lens extended to the posterior pole, and distribution of nuclei from the posterior pole toward the anterior aspects of the lens suggested that newly added fiber cells were misdirected. However, neither this feature, nor the presence of vacuoles appeared to correlate with the development of opacity in the cultured lenses. Instead, the lens opacities appeared to result from gross abnormalities in the shape and organization of cells in the equatorial and cortical fiber zones, as observed by F-actin staining. Culturing the lenses in the presence of the SFK inhibitor prevented these lens cell aberrations as well as the development of lens opacity. CONCLUSIONS: The formation of cataract can involve activation of SFK-mediated pathway(s) leading to disorganization of developing lens fiber cells, and inhibiting these tyrosine kinases blocks cataract progression.

Conservation of gene expression during embryonic lens formation and cornea-lens transdifferentiation in Xenopus laevis.

Few molecular comparisons have been made between the processes of embryogenesis and regeneration or transdifferentiation that lead to the formation of the same structures. In the amphibian, Xenopus laevis, the cornea can undergo transdifferentiation to form a lens when the original lens is removed during tadpole larval stages. Unlike the process of embryonic lens induction, cornea-lens transdifferentiation is elicited via a single inductive interaction involving factors produced by the neural retina. In this study, we compared the expression of a number of genes known to be activated during various phases of embryonic lens formation, during the process of cornea-lens transdifferentiation. mRNA expression was monitored via in situ hybridization using digoxigenin-labeled riboprobes of pax-6, Xotx2, xSOX3, XProx1, and gamma6-cry. We found that all of the genes studied are expressed during both embryogenesis and cornea-lens transdifferentiation, though in some cases their relative temporal sequences are not maintained. The reiterated expression of these genes suggests that a large suite of genes activated during embryonic lens formation are also involved in cornea-lens transdifferentiation. Ultimately functional tests will be required to determine whether they actually play similar roles in these processes. It is significant that the single inductive event responsible for initiating cornea-lens transdifferentiation triggers the expression of genes activated during both the early and late phases of embryonic lens induction. These findings have significant implications in terms of our current understanding of the "multistep" process of lens induction. Dev Dyn 1999;215:308-318.

Expression of transforming growth factor beta in the embryonic avian lens coincides with the presence of mitochondria.

During their maturation, lens cells lose all membrane bound organelles, including mitochondria. In chicken embryos this process begins in the central lens fibers beginning around embryonic day 12 (E12). Transforming growth factor beta (TGF beta) is a multipotent growth modulator thought to play a role in numerous developmental processes. TGF beta 1 has been localized to mitochondria in rat liver cells and muscle cells. In the present study, we examined the expression of TGF beta isoform mRNAs and proteins during chicken embryonic lens development. PCR analysis demonstrated TGF beta 2 and TGF beta 3 transcripts in the lens epithelium and fibers throughout pre- and post-hatching development. TGF beta isoforms were detected throughout the lens epithelium and fibers early in development (E6). However by E19, the distribution of TGF beta 2 and TGF beta 3 transcripts and proteins coincided with regions of the lens that contained mitochondria. In addition, intense TGF beta staining was observed in the basal portions of the equatorial epithelial cells, a region with abundant mitochondria. Transcripts for TGF beta 1 and TGF beta 4 were not detected in any tissue or time frame examined. Similarly, no immunostaining for TGF beta 1 was observed.

Lens fiber differentiation correlated with activation of two different DNAases in lens embryonic cells.

In order to identify the different DNAases present in the lens differentiating tissue, we have used an assay which reveals their activity directly on DNA-containing gels after SDS polyacrylamide gel electrophoresis. DNAase renaturation from nuclear embryonic lens extracts does not occur after separation in 0.1% SDS polyacrylamide gel electrophoresis in contrast to that observed with purified micrococcal nuclease. When the SDS concentration in the running buffer and separating gel is decreased to 0.075%, renaturation of lens DNAase and enzyme activities are observed. Isoelectrofocusing was carried out in a polyacrylamide gel which was overlaid with an agarose gel containing DNA, permitting the visualization of the pI of DNAase activity. The presence of several DNAase isoenzymes was demonstrated in 11-day embryonic lenses. In epithelial lens nuclei, high molecular weight (MW) isoenzymes with basic pI were predominant. In post-mitotic fiber lens nuclei, two lower MW isoenzymes with acidic pI were detected as well as high MW activity with a basic pI.