K. J. Al-Ghoul

Disruption of lens fiber cell architecture in mice expressing a chimeric AQP0-LTR protein.

Aquaporin‐0 (AQP0) is the major intrinsic protein of lens fiber cells and the founder member of the water channel gene family. Here we show that disruption of the AQP0 gene by an early transposon (ETn) element results in expression of a chimeric protein, comprised of ∼75% AQP0 and ~25% ETn long terminal repeat (LTR) sequence, in the cataract Fraser (CatFr) mouse lens. Immunoblot analysis showed that mutant AQP0‐LTR was similar in mass to wild‐type AQP0. However, immunofluorescence microscopy revealed that AQP0‐LTR was localized to intracellular membranes rather than to plasma membranes of lens fiber cells. Heterozygous CatFr lenses were similar in size to wild‐type but displayed abnormal regions of translucence and light scattering. Scanning electron microscopy further revealed that mature fiber cells within the core of the heterozygous Cat Fr lens failed to stratify into uniform, concentric growth shells, suggesting that the AQP0 water channel facilitates the development of the unique cellular architecture of the crystalline lens.—Shiels, A., Mackay, D., Bassnett, S., Al‐Ghoul, K., Kuszak, J. Disruption of lens fiber cell architecture in mice expressing a chimeric AQP0‐LTR protein. FASEB J. 14, 2207–2212 (2000)

Fiber cell morphology and cytoplasmic texture in cataractous and normal human lens nuclei

PURPOSE The goal of this study was to compare the ultrastructure of the oldest cells in opaque and transparent human lenses. METHODS Age-related nuclear cataracts, late-onset diabetic nuclear cataracts and normal aged lenses were examined by transmission electron microscopy. Cross-sectional profiles of fiber cells in the embryonic, fetal and juvenile nuclear regions were obtained to facilitate direct comparisons between lens regions and between sample groups. Image analysis was performed to determine cross-sectional areas of fiber cells in each region. RESULTS The average cross-sectional area increased approximately sixfold from the outer to the inner nuclear regions in all lenses measured. In each nuclear region, fiber cells displayed a characteristic size, shape, arrangement and type of interdigitations which were consistently seen in all the lenses examined. Some lenses had more complex interdigitations than others. Gap junctions were identified as pentalamellar structures having 16 nm width and appeared identical throughout the nuclei of both normal and cataractous lenses. The cytoplasm of all lenses was smooth and free of large density variations. However, the cytoplasm of some cataractous lenses appeared more granular in texture than noncataractous lenses. Cellular degeneration, debris or large cellular defects were not seen in the cores of cataractous lens nuclei. CONCLUSIONS These results indicate that only minor ultrastructural differences exist between the oldest fiber cells in normal and cataractous lenses, and that the presence of extensive cellular damage and disruptions is not necessary for the generation of nuclear opacities in aged lenses. Our observations suggest that light scattering sufficient for vision impairment may involve structural alterations much smaller than previously proposed.

A quantitative analysis of sutural contributions to variability in back vertex distance and transmittance in rabbit lenses as a function of development, growth, and age

Purpose. To correlate specific parameters of lens structure (anterior and posterior suture branch length and planar area) with variability in back vertex distance (BVD) and scatter in rabbit lenses as a function of development, growth, and age. Methods. Lenses from juvenile (n = 9), adult (n = 9), and aged (n = 10) New Zealand White rabbits were utilized in this study. After sacrifice, lens suture patterns were photographed using a stereo surgical dissecting microscope. Within 5 min of sacrifice, average BVD, variability in BVD, and scatter were assessed with a Scantox In Vitro Assay System. Laser beams were passed incrementally along anterior and posterior suture planes through right eye (oculus dexter, OD) lenses, and between suture planes through left eye (oculus sinister, OS) lenses. After fixation, lens axial dimensions and suture branch lengths were assessed and used to create scaled, 3-dimensional computer assisted drawings (3-D CADs) depicting gross lens shape and sutural changes throughout life. Results. Whereas average BVD only increased significantly as a function of growth, variability in BVD only increased significantly as a function of aging. However, anterior sutures exerted a greater influence on variability of BVD than posterior sutures throughout growth and aging. This difference is consistent with anterior suture branches being longer, or extending farther peripherally, than posterior sutures. Scatter was essentially unchanged between juvenile and adult lenses but significantly increased in aged lenses. Notably, posterior sutures effected a greater age-related increase in scatter than anterior sutures. This difference was consistent with the formation of numerous small, posterior subbranches and subplanes later in life. Structural analysis also suggested that asymmetric age-related lens compaction had occurred, predominantly affecting posterior lens dimensions. Conclusions. Lens sutures significantly influence average BVD throughout development and growth, and variability in BVD throughout aging. In addition, even though the rabbit lenses appeared transparent throughout growth and aging, unequal length and area of anterior vs. posterior suture branches and planes respectively, as well as a greater degree of age-related posterior lens compaction, were factors contributing to increased scatter.

Analysis of nuclear fiber cell compaction in transparent and cataractous diabetic human lenses by scanning electron microscopy

BackgroundCompaction of human ocular lens fiber cells as a function of both aging and cataractogenesis has been demonstrated previously using scanning electron microscopy. The purpose of this investigation is to quantify morphological differences in the inner nuclear regions of cataractous and non-cataractous human lenses from individuals with diabetes. The hypothesis is that, even in the presence of the osmotic stress caused by diabetes, compaction rather than swelling occurs in the nucleus of diabetic lenses.MethodsTransparent and nuclear cataractous lenses from diabetic patients were examined by scanning electron microscopy (SEM). Measurements of the fetal nuclear (FN) elliptical angles (anterior and posterior), embryonic nuclear (EN) anterior-posterior (A-P) axial thickness, and the number of EN fiber cell membrane folds over 20 μm were compared.ResultsDiabetic lenses with nuclear cataract exhibited smaller FN elliptical angles, smaller EN axial thicknesses, and larger numbers of EN compaction folds than their non-cataractous diabetic counterparts.ConclusionAs in non-diabetic lenses, the inner nuclei of cataractous lenses from diabetics were significantly more compacted than those of non-cataractous diabetics. Little difference between diabetic and non-diabetic compaction levels was found, suggesting that diabetes does not affect the degree of compaction. However, consistent with previous proposals, diabetes does appear to accelerate the formation of cataracts that are similar to age-related nuclear cataracts in non-diabetics. We conclude that as scattering increases in the diabetic lens with cataract formation, fiber cell compaction is significant.

Discordant expression of the sterol pathway in lens underlies simvastatin-induced cataracts in Chbb Thom rats

Simvastatin rapidly induced cataracts in young Chbb:Thom (CT) but not Sprague Dawley (SD) or Hilltop Wistar (HW) rats. Oral treatment for 14 but not 7 days committed CT rat lenses to cataract formation. The cholesterol to phospholipid molar ratio in lenses of treated CT rats was unchanged. Differences between strains in serum and ocular humor levels of simvastatin acid poorly correlated with susceptibility to cataracts. No significant differences were found between rat strains in the capacity of simvastatin acid to inhibit lens-basal sterol synthesis. Prolonged treatment with simvastatin comparably elevated HMG-CoA reductase protein and enzyme activity in lenses of both cataract resistant and sensitive strains. However, in contrast to SD and HW rats, where sterol synthesis was markedly increased, sterol synthesis in CT rat lenses remained at baseline. Discordant expression of sterol synthesis in CT rats may be due to inadequate upregulation of lens HMG-CoA synthase. HMG-CoA synthase protein levels, and to a much lesser extent mRNA levels, increased in lens cortex of SD but not CT rats. Because upregulation of the sterol pathway may result in increased formation of isoprene-derived anti-inflammatory substances, failure to upregulate the pathway in CT rat lenses may reflect an attenuated compensatory response to injury that resulted in cataracts.

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

BackgroundThe 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.MethodsNew 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.ResultsScatter 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).ConclusionMorphometric 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.

A Novel Terminal Web‐Like Structure in Cortical Lens Fibers: Architecture and Functional Assessment

This study describes a novel cytoskeletal array in fiber cells of the ocular lens of the rat and shows its relationship to the classical terminal web of other epithelial tissues. Naive adult Sprague‐Dawley rats (n = 28) were utilized. F‐actin, fodrin, myosin IIA, and CP49 distribution was assessed in anterior and posterior polar sections. For functional analysis, lenses were cultured with or without cytochalasin‐D for 3 hr, then processed for confocal microscopy or assessed by laser scan analysis along sutures. Phalloidin labeling demonstrated a dense mesh of F‐actin adjacent to posterior sutural domains to a subcapsular depth of 400 μm. Anterior polar sections revealed a comparable actin structure adjacent to anterior suture branches however, it was not developed in superficial fibers. Fodrin and myosin were localized within the web‐like actin apparatus. The data was used to construct a model showing that the cytoskeletal array is located within the blunt, variable‐width fiber ends that abut at sutures such that the “terminal web” flanks the suture on either side. Treatment with cytochalasin‐D resulted in partial disassembly of the “terminal web” and perturbed cellular organization. Laser scan analysis revealed that cytochalasin‐D treated lenses had significantly greater focal variability than control lenses (P = 0.020). We conclude that cortical fibers of rat lenses contain a bipolar structure that is structurally and compositionally analogous to classical terminal webs. The results indicate that the lens “terminal web” functions to stabilize lens fiber ends at sutures thus minimizing structural disorder, which in turn, promotes the establishment and maintenance of lens transparency. Anat Rec, 2010.

Aberrant basal fiber end migration underlies structural malformations in a streptozotocin-induced diabetic rat model

This study characterized early structural changes at posterior fiber ends in the crystalline lens after diabetic induction. Wistar rats (n = 49), randomized into one naïve control group and four experimental groups, were rendered diabetic via streptozotocin injection. Animals were euthanized at 1 week intervals, blood glucose levels recorded and lenses were evaluated grossly, by SEM and by confocal microscopy. Scoring Indices were developed to assess structural alterations and for statistical correlations between the scores and the duration of hyperglycemic exposure as well as blood glucose levels. Average blood glucose levels increased progressively from 98.5 mg/dL (controls) to 331.4 mg/dL (4 weeks). Diabetic lenses displayed abnormal suture sub-branches and opacity formation beginning late in the first week post-injection and rapidly progressing in severity through four weeks. SEM analyses showed gradual elongation of fiber ends and filopodia which comprised a disorganized configuration and a loss of recognizable migration patterns. Structural alterations culminated in foci of fiber degeneration by the third to fourth weeks. The F-actin distribution at basal fiber ends was significantly altered as compared to naïve controls. Cadherin distribution was altered as compared to controls, but largely at later time points. The grading system clearly shows increased structural compromise with elevated blood glucose levels in streptozotocin-induced diabetes. Further, although the initial rise in blood glucose levels was associated with pathological changes, their progression depended to a larger extent on continued hyperglycemic exposure. The data suggests that hyperglycemia initially disrupts fiber end migration, resulting in structural alterations and eventual fiber degeneration.

Morphology of the normal human lens

PURPOSE To provide a quantitative, morphologic description of differentiated lens fiber cells in all regions of aged normal human lenses. METHODS Transparent normal human lenses (age range, 44 to 71 years) were examined with correlative transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Vibratome sections allowed examination of internal structures, whereas dissected whole lenses revealed surface characteristics. Additionally, image analysis was used to measure cross-sectional areas of fiber cells. RESULTS Approximate regional dimensions (percentage of diameter and thickness, respectively) were determined for whole lenses: cortex 16%, 17%; adult nucleus 24%, 21%; juvenile nucleus 12%, 9%; fetal nucleus 45%, 49%; and embryonic nucleus 3%, 4%. Cortical cells were irregularly hexagonal, and the average cross-sectional area measured 24 +/- 9 microns2. Adult nuclear cells were flattened with intricate membranous interdigitations and an area of 7 +/- 2 microns2. Juvenile nuclear cells had an area of 14 +/- 5 microns2. Fetal nuclear cells were rounded with an area of 35 +/- 22 microns2. Embryonic nuclear cells also were rounded and had a variable area of 80 +/- 68 microns2. Fiber cell cytoplasm in all lens regions appeared smooth in texture and homogeneous in staining density. CONCLUSIONS Both TEM and SEM are necessary to obtain a complete description of fiber cells. Cross-sections of fibers give new insights into the lamellar organization of the lens, indicating that each region has characteristic cell shapes and sizes. Furthermore, average dimensions were used to demonstrate that the number of cells and approximate growth rates vary significantly between adjacent regions.