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.

Structural evidence of human nuclear fiber compaction as a function of ageing and cataractogenesis.

This study was conducted to quantify structural change associated with human nuclear fiber compaction as a function of ageing and nuclear cataract formation. Normal donor lenses in three age ranges, young (15--25 years), middle-aged (36--46 years) and aged (59--81 years) were compared to each other and to age-related nuclear cataracts (55--81 years) surgically removed by extracapsular extraction. Several structural modifications which occurred as a manifestation of fiber compaction were noted. In the fetal nucleus (FN), the average anterior and posterior fiber angles decreased approximately 20% with age. Additionally, there was a reduction in the thickness of both the anterior and posterior segments of fetal fibers with age. On average, the anterior--posterior (A--P) axis in the embryonic nucleus (EN) decreased 33% with age. The average length of EN fibers decreased significantly (37%) as a function of age. This change in EN fiber length was accomplished by effecting compaction folds along fiber length. By comparison, in nuclear cataracts the anterior and posterior angles of FN fibers were about 12% smaller than comparably aged normal lenses. Similarly, the A--P axis and the length of EN fibers were 13% smaller than age-matched normals. Nuclear fiber compaction in early adulthood was significant and may contribute to the lens hardening and loss of accommodative ability symptomatic of presbyopia. 3D-CAD reconstructions of fiber compaction show how the reduction in the spacing of lateral interdigitations along fiber length causes an increase in the fiber membrane complexity along the A--P axis in relation to fiber cytoplasm as light passes through lenses. These results may explain, at least in part, how an increase in large particle scatter occurs as light is transmitted through fiber membranes, resulting in reduced lens optical quality as a function of age. By extrapolation, the significantly increased compaction of nuclear fibers in age-related nuclear cataracts may be a contributing factor for excessive scatter in nuclear opacification.

The internalization of posterior subcapsular cataracts (PSCs) in Royal College of Surgeons (RCS) rats. I. Morphological characterization.

PURPOSE: To document lens ultrastructure during and after internalization of posterior subcapsular cataracts (PSCs) in Royal College of Surgeons (RCS) rats, a model for human autosomal retinal degenerative disease. METHODS: RCS rat lenses at 2, 2.5, 3, 4, 6, 9, 12, and 15 months old were enucleated and fixed. For light and transmission electron microscopy (TEM), lenses were embedded in epoxy and sectioned along the visual axis. For scanning electron microscopy, lenses were dissected to expose the posterior fibers in concentric growth shells down to the internalized PSC plaques. RESULTS: Overgrowth of the plaque began between 8 and 9 weeks postnatal and proceeded from the periphery to the posterior pole. This is in contrast to PSC formation which begins centrally and enlarges radially between 4-6 weeks postnatal. Peripheral-to-central overgrowth resulted in the formation of a convexo-concave, disk-shaped suture plane oriented parallel to the capsule. The initial fibers overlying the plaque were extremely flattened at their posterior ends. However, by 3 months postnatal, fiber ultrastructure was relatively normal and displayed only minor morphological irregularities. These temporal and structural changes were used to create 3-dimensional computer assisted-drawing (3D-CAD) reconstructions and animations. TEM examination of plaques revealed scattered fiber defects such as membrane whorls, globular aggregates and intracellular voids in both the internalized plaques and the initial overgrowth. The internalized PSC plaques had comparable morphology in all animals, regardless of age. Specifically, the posterior segments of fibers were enlarged and curved abnormally toward the capsule. CONCLUSIONS: PSC plaques are not internalized and broken down in the classical cell biological sense (i. e. via lysosomal degradation). Rather the plaques retain their structure indefinitely as lens growth proceeds (albeit not entirely normally). This demonstrates that the lens has a restricted ability to respond to growth defects and effect a limited recovery after PSC formation.

The internalization of posterior subcapsular cataracts (PSCs) in Royal College of Surgeons (RCS) rats. II. The inter-relationship of optical quality and structure as a function of age.

PURPOSE: The Royal College of Surgeons (RCS) rat is an animal model for human retinal degenerative disease and posterior subcapsular cataracts (PSCs). The purpose of this study was to correlate the structure and optical quality of RCS lenses with PSCs as a function of their internalization, with normal, non-cataractous, age-matched control lenses. METHODS: Correlative light (LM), scanning electron microscopic (SEM), three-dimensional computer assisted drawings (3D-CADs) and low power helium-neon laser scan analysis were used to examine the structure and function of lenses. RESULTS: The optical properties (average focal length variability; sharpness of focus) of RCS rat lenses are quantitatively compromised by PSCs. Correlative LM and SEM analysis of RCS lenses at various stages of PSC internalization (1.5, 3, 6, 9, 12 and 15 months of age), revealed that the sutures formed by additional fiber growth were progressively more abnormal. During PSC internalization, two to nine small suture branches were formed and arranged in modified line to multiple y configurations rather than the normal three branch y sutures. These temporal changes were also chronicled in animated 3D-CAD videos derived from lens reconstructions based on LM and SEM micrographs from the selected time points stated above. However, laser scan analysis also revealed that as the PSCs of RCS rat lenses were progressively internalized, there was a steady improvement in total sharpness of focus that reached normal levels by 12 months of age. The correlation of laser scan and structural data from specific regions of lenses revealed the following: 1. The abnormal posterior sutures of RCS rats with internalized PSCs effect a greater reduction in optical quality than normal posterior sutures of age-matched controls; 2. However, the resulting abnormal suture plane area was cumulatively similar to that of age-matched controls; 3. Thus, total optical quality was similar between RCS lenses with internalized PSCs and age-matched controls by 12 months of age. CONCLUSIONS: The results of this study show that RCS lenses with internalized PSCs can appear grossly, and indeed optically perform, at levels comparable to aged lenses. These findings are consistent with clinical observations of spontaneous recovery from PSC. The results suggest that human PSCs that occur as a consequence of retinal degenerative disease could also be the result of abnormal posterior suture growth. If this is proven to be the case, such PSCs may have some capacity for repair or recovery thereby obviating their surgical removal.

Anterior polar cataracts in CS rats: a predictor of mature cataract formation.

PURPOSE: The objective of this study was to characterize the morphology of the anterior opacities formed during recovery from posterior subcapsular cataract (PSC) in Royal College of Surgeons (RCS) rats. METHODS: Lenses from RCS rats at 8 and 12 weeks postnatal (n = 14 and 12, respectively) were examined under a dissecting microscope for the presence of anterior opacities. Lenses with anterior opacities were fixed, embedded in epoxy resin, and sectioned along the optic axis for light microscopy (LM) and transmission electron microscopy (TEM). RESULTS: At eight weeks postnatal, 21.5% of animals (3/14) had anterior cataracts. Light microscopy of 1- to 2-microm-thick sections revealed an anomalous layer of material located at the epithelium-fiber interface, which was identified as a zone of liquefaction by TEM. Epithelial cells had minor structural defects but were not necrotic. Anterior portions of elongating and cortical fibers under the zone of liquefaction were undisrupted, whereas their posterior portions had numerous vacuoles. The anterior opacities were classified as anterior polar cataracts (APCs) based on the location and type of morphologic damage in the affected lenses. At twelve weeks postnatal, 25% of animals (3/12) had APCs that involved prominent vesiculation of the anterior cortex. Ultrastructural examination showed that large vesicles were located between and inside anterior fibers and that most extracellular spaces were abnormally widened. Posteriorly, internalization of the PSC by new fiber growth was disordered and displayed vesiculation and density variations. In the bow region, LM revealed minor structural irregularities that were identified as groups of apparently degenerating fibers by TEM. CONCLUSIONS: APCs in RCS rats are caused by degeneration of elongating fibers in the bow region and subsequent damage in the superficial anterior cortex. The percentage of animals with APCs (25%) was consistent with the percentage of animals in which mature cataracts eventually develop. The morphologic changes, time of onset, and percentage of animals affected suggest that APC is the initial manifestation of mature cataract formation in RCS rats.

The structure of posterior subcapsular cataracts in the Royal College of Surgeons (RCS) rats.

The Royal College of Surgeons (RCS) rat is an animal model for human autosomal recessive retinitis pigmentosa. As the retinas of these animals degenerate from two to six weeks after birth, posterior subcapsular cataracts (PSCs) develop, presumably in response to toxic lipid peroxides formed by degenerating rod outer segments. Morphologically, these PSCs are thought to be characterized by a proliferation of dysplastic bladder-like fibers, or Wedl cells, in the meridional region of the lens, that subsequently migrate to, and aggregate at, the posterior pole as the PSC. This report presents the results of correlative scanning (SEM) and transmission (TEM) electron microscopic as well as light microscopic (LM) analysis of the ultrastructure of RCS PSCs. SEM analysis of two, four and six week old lenses (n=6-10 specimens per age group) demonstrated that the PSCs of RCS rats resulted from a growth malformation of the posterior fiber ends from four to six weeks. The PSC is composed of markedly enlarged and irregular posterior fiber ends aberrantly curved away from the polar axis toward the vitreous rather than overlapping and abutting to form suture branches within and between concentric growth shells. LM analysis revealed evidence of progressively more numerous, enlarged, and irregular, ovate cellular profiles at the posterior pole from four to six weeks. However, there was no evidence of Wedl cells either within the meridional row region or along a migratory path from the equator to the posterior pole at any age. TEM analysis confirmed that the size and abnormal shapes of cellular profiles were consistent with SEM analysis and that nuclei were never observed within the plaque. In addition, there was considerable variation in cytoplasmic densities between cells. Also, dense deposits were frequently noted between cells and beneath the capsule. The orientation of posterior fiber end profiles to the posterior capsule was 45, 70 and 90 degrees at respectively two, four and six weeks of age. These results show that RCS PSCs are a consequence of abnormal posterior fiber end growth culminating in a posterior opacity.

Light microscopic variation of fiber cell size, shape and ordering in the equatorial plane of bovine and human lenses.

PURPOSE: A rapid means was sought to visualize and quantify the cross-sectional areas of fiber cells, the variations of cell area, and the regularity of packing in the equatorial plane of normal adult bovine and normal aged human lenses. METHODS: Vibratome sections of bovine and human lenses were fixed, embedded in LR White resin, and sectioned for light microscopic observation. Image analysis was performed to determine the cross-sectional areas of fiber cells in selected nuclear regions. RESULTS: Examination of bovine lenses revealed a pattern of cell size and shape in each region that was similar to that recently reported for normal human lenses (1). In the equatorial plane of bovine lenses, average cross-sectional areas were 20 +/- 6 micron2 in the adult nucleus, 43 +/- 19 micron2 in the fetal nucleus, and 63 +/- 61 micron2 in the embryonic nucleus. Light microscopy of human lenses was consistent with our previous electron microscopic observations. Moreover, in both bovine and human lenses, the distribution of cell sizes and the number of cell layers was readily available for each region. Overviews of the equatorial plane demonstrated a gradual improvement in the regular packing of radial cell columns proceeding from the relatively disordered embryonic and fetal nuclei through the well-ordered adult nucleus to the highly regular cortical region. CONCLUSIONS: Light microscopy revealed the highly irregular packing and large average size of cells in the embryonic nucleus and the gradual reduction in size and progressive improvements in regularity of packing in the outer layers. The methods used here have the advantage of rapidly giving a continuous view of the fiber cell structure and arrangement which is not available using electron microscopy.

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.

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.

Distribution and type of morphological damage in human nuclear age-related cataracts.

The distribution and type of fiber cell damage was evaluated in human age-related nuclear cataracts and in aged normal (non-cataractous) lenses. Ten age-related nuclear cataracts (53 to 89 years old) and four normal lenses (59 to 67 years old) were examined by electron microscopy of fixed Vibratome sections. Images from the adult, juvenile, fetal and embryonic nuclear regions were compared. Each cataractous lens contained a central region of increased light scattering which involved the embryonic and fetal regions with progressively less involvement in the juvenile and adult nuclear regions. Some damaged fiber cells were observed in all specimens, although damage was minor and infrequent in the normal lenses. Degeneration of single or groups of fiber cells was noted in all the adult nuclei of the cataractous lenses, becoming less frequent in the juvenile nuclei. The types of damage included localized voids, multilamellar membrane aggregates, globular bodies, enlarged cells and regions of highly convoluted membranes. The fetal and embryonic nuclei of the cataractous lenses exhibited rare and minor morphological defects, and were virtually identical to the equivalent regions of the normal aged lenses. Examination of cell interfaces in opaque regions of cataractous lenses revealed that the oldest fiber cells sustained apparent membrane loss. Extracellular spaces in the embryonic, fetal and juvenile regions of the cataractous lenses often contained dense deposits, presumably cytoplasmic material lost from adjacent fibers. The results indicate that the region of greatest nuclear opacity, located in the lens center, does not contain any significant cellular damage. This suggests that older fiber cells respond differently to pathological and senescent changes than younger cells made after fetal development. The observed loss of membranes and cytoplasmic material from the oldest fiber cells may be a contributory mechanism in the formation of age-related human nuclear cataracts.

Morphological changes in human nuclear cataracts of late-onset diabetics.

The ultrastructure of human diabetic lens nuclei is described for the first time. Two cataractous lenses from late-onset diabetics were examined using transmission electron microscopy to determine the type and distribution of cellular disruptions. The diabetic lens nuclei were compared to a transparent nucleus from a normal human lens. Cellular damage to the exterior region of the diabetic lens nuclei was extensive, especially at the cortical-nuclear interface. Areas of lens fiber condensation as well as areas of cytoplasmic loss were observed in the outer nucleus. Morphological defects commonly seen in this region included: multilamellar membrane aggregates, voids where cytoplasmic material was lost, deposits in the extracellular spaces, density variations between adjacent fiber cells, and heterogeneously staining globules. The opaque central regions of the nuclei displayed relatively little cell damage, but fiber cells were very irregular in shape and packing. The ultrastructure of inner nuclear fiber cells was comparable to that seen in the normal lens and in age-related nuclear cataracts in non-diabetics. It appears that the effect of hyperglycemia on lens fiber cells is dependent on their age and stage of differentiation.