Regional changes of AQP0-dependent square array junction and gap junction associated with cortical cataract formation in the Emory mutant mouse.

The Emory mutant mouse has been widely used as an animal model for human senile cataract since it develops late-onset hereditary cataract. Here, we focus on the regional changes of aquaporin-0 (AQP0) and connexins that are associated with the cortical cataract formation in the Emory mutant mice. Emory mutant and CFW wild-type mice at age 1-16 months were used in this study. By using an established photography system with dissecting microscopy, the opacities were first detected at the anterior or posterior lens center surface in Emory mice at age 7 months, and gradually extended toward the equator during the 16 months examined. Scanning EM verified that disorganized and fragmented fiber cells were associated with the areas of opacities within approximately 200 µm from the lens surface, indicating that Emory mouse cataracts belong to the cortical cataracts. Freeze-fracture TEM further confirmed that cortical cataracts exhibited extensive wavy square array junctions, small gap junctions and globules. Immunofluorescence analysis showed that in contrast to the high labeling intensity of AQP0-loop antibody, the labeling of AQP0 C-terminus antibody was decreased considerably in superficial fibers in Emory cataracts. Similarly, a significant decrease in the labeling of the antibody against Cx50 C-terminus, but not Cx46 C-terminus, occurred in superficial and outer cortical fibers in Emory cataracts. Western blotting further revealed that the C-termini of both AQP0 and Cx50 in Emory cataracts were decreased to over 50% to that of the wild-type. Thus, this systematic study concludes that the Emory mouse cataract belongs to the cortical cataract which is due to regional breakdown of superficial fibers associated with formation of AQP0-dependent wavy square array junctions, small gap junctions and globules. The marked decreases of the C-termini of both AQP0 and Cx50 in the superficial fibers may disturb the needed interaction between these two proteins during fiber cell differentiation and thus play a role in the cortical cataract formation in Emory mutant mice.

Simple fixation and storage protocol for preserving the internal structure of intact human donor lenses and extracted human nuclear cataract specimens.

PURPOSE: Increased use of phacoemulsification procedures for cataract surgeries has resulted in a dramatic decrease in the availability of cataractous nuclear specimens for basic research into the mechanism of human cataract formation. To overcome such difficulties, a fixation protocol was developed to provide good initial fixation of human donor lenses and extracted nuclei, when available, and is suitable for storing or shipping cataracts to laboratories where structural studies could be completed. METHODS: Cataractous lens nuclei (n=19, ages 12 to 74 years) were obtained from operating suites after extracapsular extraction. Transparent human donor lenses (n=27, ages 22 to 92 years) were obtained from the Ramayamma International Eye Bank. After the dimensions were measured with a digital caliper, samples were preserved in 10% formalin (neutral buffered) for 24 h and followed by fixation in 4% paraformaldehyde (pH 7.2) for 48 h. Samples were stored cold (4 °C) in buffer until shipped. Samples were photographed and measured before further processing for transmission electron microscopy. RESULTS: The dimensions of the samples varied slightly after short fixation followed by 1 to 5 months' storage before transmission electron microscopy processing. The mean change in the axial thickness of the donor lenses was 0.15±0.21 mm or 3.0±5.4%, while that of the extracted nuclei was 0.05±0.24 mm or 1.8±7.6%. Because the initial concern was whether the nuclear core was preserved, thin sections were examined from the embryonic and fetal nuclear regions. All cellular structures were preserved, including the cytoplasm, complex edge processes, membranes, and junctions. The preservation quality was excellent and nearly equivalent to preservation of fresh lenses even for the lens cortex. Cell damage characteristic of specific nuclear cataract types was easily recognized. CONCLUSIONS: The novel fixation protocol appears effective in preserving whole donor lenses and cataractous nuclei over a wide age range. Dimensions varied only 2%-3%, and fiber cell damage correlated well with standard fixation. These methods enable researchers and clinicians in remote settings to preserve donor lenses and rare examples of extracapsular extractions for detailed examination at later times.

Nanomechanical characterization of the stiffness of eye lens cells: a pilot study.

PURPOSE: The purpose of this study is to probe the mechanical properties of individual eye lens cells isolated from nucleus and cortex of adult sheep eye lens, and to characterize the effect of cytoskeletal drugs. METHODS: We used atomic force microscopy (AFM), featuring a spherical tip at the end of a soft cantilever, to indent single lens cells, and measure the Young's modulus of isolated nuclear and cortical lens cells. Measurements were performed under basal conditions, and after addition of drugs that disrupt actin filaments and microtubules. RESULTS: We found that single lens cells were able to maintain their shape and mechanical properties after being isolated from the lens tissue. The median Young's modulus value for nuclear lens cells (4.83 kPa) was ~ 20-fold higher than for cortical lens cells (0.22 kPa). Surprisingly, disruption of actin filaments and microtubules did not affect the measured Young's moduli. CONCLUSIONS: We found that single cells from the lens nucleus and cortex can be distinguished unambiguously using the elastic modulus as a criterion. The uncommon maintenance of shape and elastic properties after cell isolation together with the null effect of actin filaments and microtubules targeting drugs suggest that the mechanical stability of fiber cells is provided by cellular elements other than the usual cytoskeletal proteins.

Inhibitory effects of chlorogenic acid on aldose reductase activity in vitro and cataractogenesis in galactose-fed rats.

Chlorogenic acid (5-O-caffeoylquinic acid, CA), a phenolic compound found ubiquitously in plants, has antidiabetic effect in diabetic animal models. In this study, we investigated the inhibitory effect of CA on diabetic cataractogenesis. We evaluated the aldose reductase (AR) activity during cataract development in 50% galactose-fed rats, an animal model of sugar cataract. Galactose-fed rats were treated orally with CA (10 and 50 mg/kg body weight) once a day for 2 weeks. In vehicle-treated galactose-fed rats, lens opacity was increased, and lens fiber swelling and membrane rupture were observed. In addition, AR protein was highly expressed in lens epithelial cells and lens cortical fibers of galactose-fed rats. However, CA inhibited the rat AR activity in vitro, and the administration of CA prevented the development of sugar cataract through the inhibition of AR activity. These observations suggest that CA is useful for the treatment of sugar cataract.

Evolution of damage in the lens after in vivo close to threshold exposure to UV-B radiation: cytomorphological study of apoptosis.

The purpose of the present study was to investigate cataractogenesis and recovery of lens damage after in vivo close to threshold ultraviolet (UV)-B radiation around 300 nm. Eighty six-week-old albino Sprague-Dawley rats were familiarized to a rat restrainer five days prior to exposure. Groups of non-anesthetized rats were exposed unilaterally to 8 kJ/m(2) UVR-300 nm. The animals were sacrificed at 1, 7, 48 and 336h following exposure. The lenses were extracted for imaging of dark-field lens macro anatomy and measurement of intensity of forward lens light scattering to quantify lens opacities. Three exposed lenses and one non-exposed lens from each time interval were examined with light and transmission electron microscopy (TEM). Macro anatomy and lens light scattering revealed that all contralateral non-exposed lenses were clear. The degree of lens opacity (difference in lens light scattering between exposed and non-exposed lenses) increased during the 336h, reaching a plateau towards the end of the observation period. Light microscopy and TEM demonstrated that apoptotic features appeared in the epithelium already 1h after UVR exposure, and small vacuoles were seen in the outer cortex. Epithelial damage occurs during the first 48h after exposure and is followed by regenerative repair at 336h post-exposure. Apoptotic epithelial cells were phagocytized by adjacent epithelial cells. Cortical fiber cells exhibited increasing damage throughout the observation period without any clear repair after 336h. In conclusion, acute UVR-induced cataract is partly a reversible. Lens epithelium is a primary target for UVR exposure. Damage to cortical fiber cells remained irreversible.

AlphaA-crystallin R49Cneo mutation influences the architecture of lens fiber cell membranes and causes posterior and nuclear cataracts in mice.

BACKGROUND: AlphaA-crystallin (CRYAA/HSPB4), a major component of all vertebrate eye lenses, is a small heat shock protein responsible for maintaining lens transparency. The R49C mutation in the alphaA-crystallin protein is linked with non-syndromic, hereditary human cataracts in a four-generation Caucasian family. METHODS: This study describes a mouse cataract model generated by insertion of a neomycin-resistant (neor) gene into an intron of the gene encoding mutant R49C alphaA-crystallin. Mice carrying the neor gene and wild-type Cryaa were also generated as controls. Heterozygous knock-in mice containing one wild type gene and one mutated gene for alphaA-crystallin (WT/R49Cneo) and homozygous knock-in mice containing two mutated genes (R49Cneo/R49Cneo) were compared. RESULTS: By 3 weeks, WT/R49Cneo mice exhibited large vacuoles in the cortical region 100 mum from the lens surface, and by 3 months posterior and nuclear cataracts had developed. WT/R49Cneo mice demonstrated severe posterior cataracts at 9 months of age, with considerable posterior nuclear migration evident in histological sections. R49Cneo/R49Cneo mice demonstrated nearly complete lens opacities by 5 months of age. In contrast, R49C mice in which the neor gene was deleted by breeding with CreEIIa mice developed lens abnormalities at birth, suggesting that the neor gene may suppress expression of mutant R49C alphaA-crystallin protein. CONCLUSION: It is apparent that modification of membrane and cell-cell interactions occurs in the presence of the alphaA-crystallin mutation and rapidly leads to lens cell pathology in vivo.

Morphology of age-related cuneiform cortical cataracts: the case for mechanical stress.

We evaluated the gross morphology, location, and fiber cell architecture of equatorial cortical opacities in the aging human lens. Using dark-field stereomicroscopy, we photographed donor lenses in toto and as thick slices. In addition, we investigated the details of the fiber cell architecture using fluorescent staining for membranes and by scanning electron microscopy. We then combined our data with data from recent studies on lens viscoelasticity. We found that small cortical and cuneiform opacities are accompanied by changes in fiber structure and architecture mainly in the equatorial border zone between the lens nucleus and cortex. Because the lens cortex and nucleus have different viscoelastic properties in young and old lenses, we hypothesize that external forces during accommodation cause shear stress predominantly in this border zone. The location of the described changes suggests that these mechanical forces may cause fiber disorganization, small cortical opacities, and ultimately, cuneiform cataracts.

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.

Up-regulation of tau, a brain microtubule-associated protein, in lens cortical fractions of aged alphaA-, alphaB-, and alphaA/B-crystallin knockout mice.

PURPOSE: Alpha-crystallin is expressed at high levels in the lens in a complex of alphaA- and alphaB-crystallin subunits in 3:1 molar ratios, and is known to maintain the solubility of unpolymerized tubulin and enhance the resistance of microtubules to depolymerization, but its effect on proteins classically associated with microtubule stability (microtubule associated proteins) in the lens is unknown. In the present study we examined the expression of the brain microtubule associated protein tau in lenses of alpha-crystallin gene knockout mice. METHODS: Quantitative RT-PCR, immunoblotting, cryo-immunoelectron microscopic and immunohistochemical methods were used to characterize the expression of tau in the lenses of alphaA(-/-)-, alphaB(-/-)-, and alphaA/B(-/-)-crystallin mice. RESULTS: Immunoreactivity to tau, a 45-66 kDa brain microtubule associated protein that has been best characterized in neurons and neuronal pathologies, was uniquely upregulated in lens cortical fiber cells with aging and was associated with the microtubule fraction of alphaA(-/-)-, alphaB(-/-)-, and alphaA/B(-/-)-crystallin mouse lenses, but was undetectable in wild type lenses. Quantitative RT-PCR analysis further showed an upregulation of tau transcripts in alphaA(-/-)- and alphaA/B(-/-)-crystallin lenses. Brain microtubule fractions served as a positive control for tau in these experiments. An increase in phosphorylation of tau was detected in alphaA(-/-)- and alphaB(-/-)-crystallin brain proteins. CONCLUSIONS: Although tau aggregation and alphaB-crystallin expression have been shown to increase in neurodegenerative diseases, surprisingly tau expression increases in the alpha-crystallin knockout lenses, suggesting that alphaA- and alphaB-crystallins are potentially important regulators of tau expression in lens.

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.

Analysis of the healthy rabbit lens surface using MAC Mode atomic force microscopy.

In this investigation healthy rabbit crystalline lenses were characterized by atomic force microscopy (AFM). The lenses were cut in slices with thickness with 1mm and thus, put after cortex distinct regions of nucleus and cortex for AFM examination. AFM analysis were carried out using a PicoSPM I operating in Mac Mode. We obtained topographic images of rabbit lenses and a quantitative analysis of the width and height of fibers for nucleus and cortex regions. The longitudinal section analysis of fibers in the nucleus region indicated structures with an average width of 200nm and average height of 200nm. The intershells distance was determined as 4microm. Fiber cell cross-section dimensions, longitudinal and transverse widths, could be estimated in these regions from the AFM images. Structures with average widths as small as 1.0microm are observed in the nucleus; the intershell distance is 4.0microm. In cortical regions, hexagonal structures with average longitudinal and transverse widths of 5.0mum and 3.0mum, respectively, were identified. Three-dimensional images of tissue sections with resolution on a nanometer scale were obtained. The potential of AFM analysis for characterizing healthy and pathologic lens tissues is discussed.

The influence of hypodynamic stress on ultrastructure of the cortical layer cells of eye lens.

The aim of the study was to determine the influence of 48-day hypodynamic stress on the ultrastructure of the cortical layer cells of eye lens. Hypodynamic stress was induced in Chinchilla male rabbits (weight 2.5-3.0 kg; n=8) by placing them in metal hutches for 48 days. Rabbits (n=8) of the control group (non-stressed) were kept under normal vivarium conditions. Following the hypodynamic regime (after 48 days) the rabbits (stressed and control) were anaesthetized using thiopental sodium (35 mg/kg). On removing their eyes, the cortical partes of lenses were prepared and immersed into a fixative solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for more than 4 hours at room temperature or overnight at 4 degrees C. The specimens were then postfixed, dehydrated and embedded in a mixture of Epon 812 and Araldite. Ultrathin sections stained with uranyl acetate and lead citrate were evaluated by electron microscopy (Philips-300). Microscopical examination of the cortical layer cells of lenses showed extensive cellular vacuolization, calcification, and formation of a light scattering center. Cortical fibers were found to be compacted along their length, and elongated with decreased interdigitations.

A distinct membrane current in rat lens fiber cells isolated under calcium-free conditions.

PURPOSE: To investigate whether lens fiber cells isolated and maintained under calcium-free conditions exhibit distinct membrane currents. METHODS: Fiber cells were isolated from the cortical portion of neonate rat lenses using a trypsin digestion protocol and were maintained in EDTA-buffered Ringer's solutions. Membrane currents were recorded from fiber bundles using the whole-cell patch-clamp technique. RESULTS: Cortical fiber cells of up to 600-microm length were viable and amenable to whole-cell patch-clamp recording. The major current recorded under these conditions was a slowly activating, voltage-dependent current that was markedly increased on membrane depolarization. This current appeared to be fiber cell specific and had similar properties to currents elicited by gap junction hemichannels previously recorded by others in Xenopus oocytes. CONCLUSIONS: This is the first report of whole-cell patch-clamp recordings from intact elongated fiber cells. Fiber cells kept in calcium-free bath medium appear to be electrically "leaky" and exhibit a distinct membrane current that has not been described previously for lens cells. This current is unlikely to be active in the normal lens but may play a role in the depolarized cataractogenic lens.

Light scattering and morphology of cataract formation in transgenic mice containing the HIV-1 protease linked to the lens alpha A-crystallin promoter.

Two constructs of transgenic mice, TG61 and TG72, containing the HIV-1 protease linked to the lens alpha A-crystallin promoter develop cataract. The TG61 construct develop cataract in utero, while the TG72 construct exhibit frank opacities on the 24th day (homozygotes) and 26th day (hemizygotes) post natum. Polarized light scattering measurements were performed on cortical and nuclear sections of TG72 lenses from day 19 to day 26 as a function of scattering angle. The parallel components of the scattered light intensity increase gradually during opacification, the perpendicular components show very low values from day 19 to day 22 after which they increase exponentially. Analysis of the scattered light intensities yielded parameters describing the size of the protein aggregates, the size of the domains exhibiting optical anisotropy/birefringence, the difference in refractive index between (a) the protein aggregates and their surroundings and (b) the birefringent units and their surroundings. The last parameter accounts for the major portion of the increase in lens turbidity. The TG72 construct shows normal lens development on day 16. By day 21 the posterior cortex shows some disintegration, while the lens is still clear. By day 26 the lens nucleus migrates toward the posterior pole and there is a major alteration in the cortical fibers. Scanning electron microscopic studies reveal normal fiber cell organizations in control animals. In the TG72 construct the fiber cells are well organized at 16 days after birth but already develop some slight separation in the posterior cortical part of the lens. By post-natal day 21, the nucleus and the anterior cortex still exhibit well aligned fiber cell organization, but the posterior cortex shows disalignment. By day 26 in the TG72 construct, all areas of the lens show complete disintegration of the fiber cells and amorphous masses are present throughout. The light scattering parameters describing changes on the nanometer scale can be correlated with the changes in lens morphology during cataractogenesis that occur on the micrometer scale. In comparison, the light and scanning electron microscopic examinations of the postnatal TG61 construct show that the lens is severely disrupted and contains completely disintegrated fiber cell remnants at an early age.

Actin filament bundles in cortical fiber cells of the rat lens.

The distribution and organization of actin filament bundles were studied in cortical fiber cells of rat lenses at various ages (3 days to 2.5 months old), using thin-section electron microscopy, immunocytochemistry and immunoblotting. Electron microscopy showed that actin bundles were regularly found along cortical fiber cell membranes of the lens at all ages studied. The actin bundles were commonly arranged in three distinct units, one bundle in each fiber cell, located at the intersections where three hexagonal fiber cells meet as seen in cross sections. These actin bundles were approximately 150 nm in diameter and were composed of 7-nm small filaments. They were aligned parallel to the long axis of fiber cells as judged by both cross and longitudinal sections. The outside border of each bundle was always surrounded by a zone of 10-nm intermediate filaments which have the same orientation as that of the actin bundles. In longitudinal sections, elongated actin bundles were always parallel to the cell membranes. A number of individual actin bundles sometimes were found to form a chain with periodic short intervals. In addition, actin bundles were frequently associated with adherens junctions near the intersections and other regions of fiber cell membranes. By immunoelectron microscopy, we demonstrated that these filament bundles indeed contained actins. By rhodamine-phalloidin labelling, we found that labeled actin bundles appeared as large, distinct dots at the corners of hexagonal fiber cells in all ages studied. In addition, non-bundle F-actins were labeled preferentially along the cell membranes of the short sides of hexagonal fiber cells. This resulted in a unique zigzag pattern of actin labeling commonly seen in the cortical fiber cells of a mature rat lens. Finally, we showed that alpha-actinin was associated with the actin bundles in the fiber cells by immunofluorescent double labeling and immunoblotting. It is suggested that this unique arrangement of actin bundles in fiber cells may provide a stabilizing structure for forming a sharp angle at each corner of fiber cells, thereby the hexagonal shape of the cells can be maintained.

Liquefaction of cortical tissue in diabetic and galactosemic rat lenses defined by confocal laser scanning microscopy.

PURPOSE: To investigate whether a histologic link exists between osmotic fiber cell swelling and cortical tissue liquefaction in experimentally induced diabetic and galactosemic cataractogenesis of the rat lens. METHODS: Confocal laser scanning microscopy, in conjunction with specific membrane labels and correlative transmission electron microscopy, was used to image large cortical areas with precise definition of the individual cells. RESULTS: In both cataract models, tissue liquefaction--defined as the disintegration of tissue and the appearance of large fluid-filled spaces--typically was limited to a discrete zone in the lens cortex. The borders of the liquefaction zone were characterized by transitions between normal-appearing cells and swollen cells, which gained in size as plasma membranes ruptured and cytoplasmic contents fused and ultimately burst, thereby contributing to the formation of large fluid-filled spaces. During cataractogenesis, before tissue liquefaction became evident, selected fiber cells appeared swollen and accumulated specifically in the zone destined for tissue liquefaction. With increasing duration of diabetes or galactosemia, swollen fiber cells in this zone became more frequent and enlarged, resulting first in tissue disorder and then in tissue disintegration and the formation of large fluid-filled spaces. CONCLUSIONS: New imaging protocols strongly support a direct involvement of lens fiber cell swelling in the liquefaction of cortical tissue. The appearance of swollen fiber cells in the lens cortex, therefore, can be used as an early indicator of the histopathology of sugar cataractogenesis.

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.

[Ultrastructural changes in various cataractous lenses in rats].

OBJECTIVE: To study the ultrastructural changes of lenses aged 50 days from normal rats and rats with three kinds of cataract. METHOD: Transmission electron microscope (TEM) was used to investigate the ultrastructural changes of normal lens, selenium, galactosemic and congenital cataracts in the rat. RESULTS: In the normal group, the structure of cell membrane in cortex is not changed obviously, but in the lens nucleus there are slight cellular degeneration, low cytoplasmic density and the destruction of intercellular junctional complexes. In the selenium cataratous lens, in the lens cortex and nucleus the cellular junctional complexes are damaged severely, the cystic degeneration of mitochondria in the cytoplasm is obvious, the cytoplasmic density becomes lower and slight liquefaction can be observed in some parts of cytoplasm. In the galactosemic cataractous lens, in the cortex obvious liquefaction, vesicles and globular structures can be found within the cytoplasm, intercellular spaces are enlarged and liquified markedly, and in the lens nuclear area the cytoplasm appears as fine sands. In congenital cataractous lens, the cellular structure in cortex is essentially normal, but there are marked destruction of junctional complexes, obvious cystic degeneration of mitochondria and cytoplasmic aggregation with uneven distribution in the lens nuclear area. CONCLUSION: The ultrastructural changes in different kinds of cataractous lenses depend on the extent and location of opacification in lenses.

Calcium-mediated disintegrative globulization of isolated ocular lens fibers mimics cataractogenesis.

Single viable fiber cells have been isolated from the cortex of rat ocular lens by proteolytic digestion of the intact lens in calcium-free media. In isomolar sucrose, the isolated cells maintain their fiber-shaped morphology and exclude trypan blue. The surface morphology of the isolated fiber cells appears to be largely unaffected by the isolation procedure. The concentrations of adenine nucleotides, GSH, GSSG and the rate of glycolysis in the isolated fiber cells were comparable to those in the cortex. Upon perfusion of the tissue chamber with Ringer's solution, the fiber cells undergo a series of transformations, beginning with cell swelling, periodic blebbing along the longitudinal cellular axis, and eventual disintegration of the fiber into a number of resealed globules or round cells which resemble light-scattering areas in human cortical and supranuclear cataract. This disintegrative globulization of the fiber cells appears to be mediated by calcium influx, as it was prevented or delayed by a reduction in extracellular calcium concentration, verapamil or lanthanum. Since disturbances in calcium homeostasis are associated with various forms of cataract, such Ca(2+)-mediated disintegrative globulization of the fiber cells may be responsible for the formation of light scattering centers during cataractogenesis.