Protein profiles in cortical and nuclear regions of aged human donor lenses: A confocal Raman microspectroscopic and imaging study.

A combination of Raman spectroscopy, imaging, hierarchical cluster analysis (HCA) and peak ratio analysis was used to analyze protein profiles in the superficial cortex (SC), deep cortex (DC) and nucleus of old human lenses with cortical, nuclear and mixed cataracts. No consistent differences were observed in protein spectra and after cluster analysis between the three locations irrespective of the presence or absence of cortical opacities and/or coloration. A sharp increase (∼15%-∼33%) in protein content from SC to DC, normal for human lenses, was found in 7 lenses. In 4 lenses, characterized by the absence of cortical opacities, the SC has a protein content of ∼35%. A significant increase in the disulfide-to-protein ratio is found only in the SC of the 7 cortical cataracts. No changes were found in sulfhydryl-to-protein ratio. The relative contents of α-helices and ß-sheets increase from SC to nucleus. ß-Sheets are more common in the SC of lenses with cortical cataract. The absence of significant and consistent changes in protein profiles between nucleus and cortex even in cases of severe coloration is not favoring the prevailing concept that ubiquitous protein oxidation is a key factor for age related nuclear (ARN) cataracts. The observations favor the idea that multilamellar bodies or protein aggregates at very low volume densities are responsible for the rise in Mie light scatter as a main cause of ARN cataracts leaving the short-range-order of the fiber cytoplasm largely intact. The absence of significant changes in the protein spectra of the deep cortical opacities, milky white as a result of the presence of vesicle-like features, indicate they are packed with relatively undisturbed crystallins.

Absence of amyloid-beta in lenses of Alzheimer patients: a confocal Raman microspectroscopic study.

We have compared the protein profiles in plaques and tangles in the hippocampus of post-mortem Alzheimer brains and in opaque and clear regions in the deep cortex of eye lenses of the same donors. From the 7 Alzheimer donors studied, 1 had pronounced bilateral cortical lens opacities, 1 moderate and 5 only minor or no cortical opacities. We focused on beta-sheet levels, a hallmarking property of amyloid-beta, the major protein of plaques and tau protein, the major protein of tangles in Alzheimer brains. Confocal Raman microspectroscopy and imaging was used in combination with hierarchical cluster analysis. Plaques and tangles show high levels of beta-sheets with a beta-sheet to protein ratio of 1.67. This ratio is 1.12 in unaffected brain tissue surrounding the plaques and tangles. In the lenses this ratio is 1.17 independently of the presence or absence of opacities. This major difference in beta-sheet conformation between hippocampus and lens is supported by Congo red and immunostaining of amyloid-beta and tau which were positive for plaques and tangles in the hippocampus but fully negative for the lens irrespective of the presence or absence of opacities. In line with a previous study (Michael et al., 2013) we conclude that cortical lens opacities are not typical for Alzheimer patients and are not hallmarked by accumulation of amyloid-beta, and can thus not be considered as predictors or indicators of Alzheimer disease as claimed by Goldstein et al. (2003).

Absence of beta-amyloid in cortical cataracts of donors with and without Alzheimer's disease.

Eye lenses from human donors with and without Alzheimer's disease (AD) were studied to evaluate the presence of amyloid in cortical cataract. We obtained 39 lenses from 21 postmortem donors with AD and 15 lenses from age-matched controls provided by the Banco de Ojos para Tratamientos de la Ceguera (Barcelona, Spain). For 17 donors, AD was clinically diagnosed by general physicians and for 4 donors the AD diagnosis was neuropathologically confirmed. Of the 21 donors with AD, 6 had pronounced bilateral cortical lens opacities and 15 only minor or no cortical opacities. As controls, 7 donors with pronounced cortical opacities and 8 donors with almost transparent lenses were selected. All lenses were photographed in a dark field stereomicroscope. Histological sections were analyzed using a standard and a more sensitive Congo red protocol, thioflavin staining and beta-amyloid immunohistochemistry. Brain tissue from two donors, one with cerebral amyloid angiopathy and another with advanced AD-related changes and one cornea with lattice dystrophy were used as positive controls for the staining techniques. Thioflavin, standard and modified Congo red staining were positive in the control brain tissues and in the dystrophic cornea. Beta-amyloid immunohistochemistry was positive in the brain tissues but not in the cornea sample. Lenses from control and AD donors were, without exception, negative after Congo red, thioflavin, and beta-amyloid immunohistochemical staining. The results of the positive control tissues correspond well with known observations in AD, amyloid angiopathy and corneas with lattice dystrophy. The absence of staining in AD and control lenses with the techniques employed lead us to conclude that there is no beta-amyloid in lenses from donors with AD or in control cortical cataracts. The inconsistency with previous studies of Goldstein et al. (2003) and Moncaster et al. (2010), both of which demonstrated positive Congo red, thioflavin, and beta-amyloid immunohistochemical staining in AD and Down syndrome lenses, is discussed.

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.

Hyperspectral Raman imaging of neuritic plaques and neurofibrillary tangles in brain tissue from Alzheimer's disease patients.

Neuritic plaques and neurofibrillary tangles are crucial morphological criteria for the definite diagnosis of Alzheimer's disease. We evaluated 12 unstained frontal cortex and hippocampus samples from 3 brain donors with Alzheimer's disease and 1 control with hyperspectral Raman microscopy on samples of 30 × 30 µm. Data matrices of 64 × 64 pixels were used to quantify different tissue components including proteins, lipids, water and beta-sheets for imaging at 0.47 µm spatial resolution. Hierarchical cluster analysis was performed to visualize regions with high Raman spectral similarities. The Raman images of proteins, lipids, water and beta-sheets matched with classical brain morphology. Protein content was 2.0 times, the beta-sheet content 5.6 times and Raman broad-band autofluorescence was 2.4 times higher inside the plaques and tangles than in the surrounding tissue. The lipid content was practically equal inside and outside. Broad-band autofluorescence showed some correlation with protein content and a better correlation with beta-sheet content. Hyperspectral Raman imaging combined with hierarchical cluster analysis allows for the identification of neuritic plaques and neurofibrillary tangles in unstained, label-free slices of human Alzheimer's disease brain tissue. It permits simultaneous quantification and distinction of several tissue components such as proteins, lipids, water and beta-sheets.

Effect of bag-in-the-lens implantation on posterior capsule opacification in human donor eyes and rabbit eyes.

PURPOSE: To evaluate bag-in-the-lens implantation by studying the feasibility of implanting a new type of intraocular lens (IOL) and the occurrence of posterior capsule opacification (PCO) in human postmortem eyes and in eyes of living rabbits. SETTING: Department of Ophthalmology, University of Antwerp, Belgium, and Netherlands Research Institute of Amsterdam, Amsterdam, The Netherlands. METHODS: The IOL was implanted in 10 postmortem human donor eyes (in vitro study) and in 17 eyes of 10 rabbits (in vivo study). The postmortem capsular bags were cultured for 4 to 6 weeks, and the rabbits were killed 1 to 5 months after implantation. All capsular bags with the bag-in-the-lens were examined by light microscopy and scanning electron microscopy. RESULTS: The IOL design was highly effective in restricting lens epithelial cell (LEC) proliferation in the remaining lens bag in human donor eyes and in rabbit eyes. In eyes in which the capsules were not positioned well within the groove of the IOL, LEC proliferation and PCO occurred. CONCLUSION: Bag-in-the-lens implantation was highly effective in preventing PCO in vitro and in vivo provided the anterior and posterior capsules were secured properly in the peripheral groove of the IOL.

Expression of vascular endothelial growth factor receptors 1, 2, and 3 in quiescent endothelia.

The vascular endothelial growth factor (VEGF) family is involved in angiogenesis, and therefore VEGFs are considered as targets for anti-angiogenic therapeutic strategies against cancer. However, the physiological functions of VEGFs in quiescent tissues are unclear and may interfere with such systemic therapies. In pathological conditions, increased levels of expression of the VEGF receptors VEGFR-1, VEGFR-2, and VEGFR-3 accompany VEGF activity. In this study we investigated normal human and monkey tissues for expression patterns of these receptors. Immunohistochemical staining methods at the light and electron microscopic level were applied to normal human and monkey tissue samples, using monoclonal antibodies (MAbs) against the three VEGFRs and anti-endothelial MAbs PAL-E and anti-CD31 to identify blood and lymph vessels. In human and monkey, similar distribution patterns of the three VEGFRs were found. Co-expression of VEGFR-1, -2, and -3 was observed in microvessels adjacent to epithelia in the eye, gastrointestinal mucosa, liver, kidney, and hair follicles, which is in line with the reported preferential expression of VEGF-A in some of these epithelia. VEGFR-1, -2, and -3 expression was also observed in blood vessels and sinusoids of lymphoid tissues. Furthermore, VEGFR-1, but not VEGFR-2 and -3, was present in microvessels in brain and retina. Electron microscopy showed that VEGFR-1 expression was restricted to pericytes and VEGFR-2 to endothelial cells in normal vasculature of tonsils. These findings indicate that VEGFRs have specific distribution patterns in normal tissues, suggesting physiological functions of VEGFs that may be disturbed by systemic anti-VEGF therapy. One of these functions may be involvement of VEGF in paracrine relations between epithelia and adjacent capillaries.

In vivo angiogenic phenotype of endothelial cells and pericytes induced by vascular endothelial growth factor-A.

VEGF-A is a major angiogenesis and permeability factor. Its cellular effects, which can be used as targets in anti-angiogenesis therapy, have mainly been studied in vitro using endothelial cell cultures. The purpose of the present study was to further characterize these effects in vivo in vascular endothelial cells and pericytes, in an experimental monkey model of VEGF-A-induced iris neovascularization. Two cynomolgus monkeys (Macaca fascicularis) received four injections of 0.5 microg VEGF-A in the vitreous of one eye and PBS in the other eye. After sacrifice at day 9, eyes were enucleated and iris samples were snap-frozen for immunohistochemistry (IHC) and stained with a panel of antibodies recognizing endothelial and pericyte determinants related to angiogenesis and permeability. After VEGF-A treatment, the pre-existing iris vasculature showed increased permeability, hypertrophy, and activation, as demonstrated by increased staining of CD31, PAL-E, tPA, uPA, uPAR, Glut-1, and alphavbeta3 and alphavbeta5 integrins, VEGF receptors VEGFR-1, -2 and -3, and Tie-2 in endothelial cells, and of NG2 proteoglycan, uPA, uPAR, integrins and VEGFR-1 in pericytes. Vascular sprouts at the anterior surface of the iris were positive for the same antigens except for tPA, Glut-1, and Tie-2, which were notably absent. Moreover, in these sprouts VEGFR-2 and VEGFR-3 expression was very high in endothelial cells, whereas many pericytes were present that were positive for PDGFR-beta, VEGFR-1, and NG2 proteoglycan and negative for alpha-SMA. In conclusion, proteins that play a role in angiogenesis are upregulated in both pre-existing and newly formed iris vasculature after treatment with VEGF-A. VEGF-A induces hypertrophy and loss of barrier function in pre-existing vessels, and induces angiogenic sprouting, characterized by marked expression of VEGFR-3 and lack of expression of tPA and Tie-2 in endothelial cells, and lack of alpha-SMA in pericytes. Our in vivo study indicates a role for alpha-SMA-negative pericytes in early stages of angiogenesis. Therefore, our findings shed new light on the temporal and spatial role of several proteins in the angiogenic cascade in vivo.

Influence of age, diabetes, and cataract on calcium, lipid-calcium, and protein-calcium relationships in human lenses.

PURPOSE: Calcium is elevated in most cataractous human lenses and may contribute to cataractogenesis. In this study, age-related changes were examined in the total calcium content of clear human lenses and the binding of calcium to lens lipids and proteins. METHODS: Total lens calcium was determined by atomic absorption spectroscopy. Calcium binding was measured by light scattering and measurement of calcium by atomic absorption spectroscopy in bound and unbound fractions. RESULTS: The calcium content of clear human lenses decreased between 18 and 55 years of age and increased between 55 and 75 years, as well as in the presence of cataract. Total calcium levels in clear lenses from subjects with insulin-dependent diabetes did not differ from that in lenses of age-matched control subjects. In vitro binding studies have shown that lens lipids can bind nearly all the calcium present in the human lens. Age and cataract diminished the capacity of lens lipids to bind calcium. Calcium-induced light-scattering, measured in vitro for lens proteins, correlated with increasing age and cataract. CONCLUSIONS: The data support the hypothesis that increased intracellular calcium concentrations and a diminished capacity of lens lipids to bind to calcium initiate a cascade of events that culminates in increased light-scattering from lipids and especially proteins. Calcium binding to lipid membranes cannot directly contribute to light-scattering in cataractous lenses. It has been suggested that most of the diffusible calcium in the lens is in the intercellular spaces and that lens lipids in the outer leaflet of the bilayer bind to that calcium. If so, this could account for the 150-fold difference between free and bound calcium levels in the lens.

In vitro study on the closure of posterior capsulorrhexis in the human eye.

PURPOSE: An unexplained clinical observation is the development of posterior capsular opacification (PCO), even when the central part of the posterior capsule has been removed. The purpose of this study was to investigate in vitro the mechanisms involved in the closure of the posterior capsulorrhexis in a capsular bag model. METHODS: A sham extracapsular cataract extraction was performed in 71 human donor eyes, followed by a central posterior capsulorrhexis 3 to 4 mm in diameter. Each capsular bag was pinned to a PMMA ring with a central hole of 5 mm and placed in a Petri dish. The capsular bags were cultured and monitored for 3 to 7 weeks by phase-contrast microscopy, after which they were prepared for light, transmission, and scanning electron microscopy. RESULTS: Proliferation of lens epithelial cells (LECs) within the posterior rhexis area was found in 22 cases (31%) of which 3 had a complete closure. In the absence of the posterior capsule, a monolayer of LECs was observed growing on a basal lamina, consisting of loosely arranged fibers. Further observations on noncultured capsular bags revealed that this basal lamina corresponds to the anterior hyaloid membrane. CONCLUSIONS: This study corroborates the clinical observation that LECs that remain after cataract extraction have the potential to proliferate, in the absence of their natural substrate, on a basal lamina of vitreous origin and are able to close the posterior capsulorrhexis partially or totally in approximately one third of cases.

Altered expression patterns of VEGF receptors in human diabetic retina and in experimental VEGF-induced retinopathy in monkey.

PURPOSE: The vascular endothelial growth factor (VEGF) family is involved in vascular leakage and angiogenesis in diabetic retinopathy (DR) in the eye, but may also have physiological functions. Based on the hypothesis that differential VEGF receptor (VEGFR) expression in the retina is an important determinant of effects of VEGF, this study was conducted to investigate VEGFR expression in the diabetic retina and in an experimental monkey model of VEGF-A-induced retinopathy. METHODS: In retinas of 27 eyes of diabetic donors, 18 eyes of nondiabetic control donors, and 4 monkey eyes injected with PBS or VEGF-A, expression patterns of VEGFR-1, -2, and -3 in relation to leaky microvessels, as identified by the marker pathologische anatomie Leiden-endothelium (PAL-E) were studied by immunohistochemistry. RESULTS. In control human retinas and retinas of PBS-injected monkey eyes, all three VEGFRs were expressed in nonvascular areas, but only VEGFR-1 was constitutively expressed in retinal microvessels. In diabetic eyes, increased microvascular VEGFR-2 expression was found in association with PAL-E expression, whereas microvascular VEGFR-3 was present in a subset of PAL-E-positive cases. In VEGF-A-injected monkey eyes, VEGFR-1, -2, and -3 and PAL-E were expressed in retinal microvessels. CONCLUSIONS: The VEGFR-1, -2, and -3 expression patterns in control retinas suggest physiological functions of VEGFs that do not involve the vasculature. Initial vascular VEGF signaling may act primarily through VEGFR-1. In diabetic eyes, expression of retinal VEGFR-2 and -3 is increased, mainly in leaky microvessels, and VEGF-A induces vascular expression of the VEGF-A receptor VEGFR-2 and the VEGF-C/D receptor VEGFR-3. These findings indicate a dual role of VEGFs in the physiology and pathophysiology of the retina and suggest that microvascular VEGFR-2 and -3 signaling by VEGFs occurs late in the pathogenesis of DR, possibly initiated by high levels of VEGF-A in established nonproliferative DR.

Blood-brain barrier integrity is unaltered in human brain cortex with diabetes mellitus.

Diabetes-related cognitive dysfunction has been recognized for many years in humans, but the pathogenesis of this condition is poorly understood. Evidence from animal studies suggests that altered function of the blood-brain barrier (BBB) could be a potential cause contributing to this disease. This study aimed to investigate whether the permeability of the BBB is affected in the brains of persons with diabetes mellitus (DM). On postmortem prefrontal and temporal cortex of diabetic patients and controls, immunohistochemical stainings were carried out using specific antibodies against three proteins (PAL-E, IgG and albumin), which are considered as markers for the vascular permeability status of the BBB. Rare or no PAL-E staining was found in the capillaries of the prefrontal and temporal cortex parenchyma, in both DM and control materials. IgG and albumin were localized in and directly around blood vessel walls in the prefrontal and temporal cortex. No obvious differences in the staining pattern of IgG and albumin were observed between brain samples of persons with DM and controls. This study suggests that the BBB in diabetic patients is well maintained.

Morphological differences between lens fibers in albino and pigmented rats.

The purpose of this study was to investigate the morphological characteristics of lens fibers in albino and pigmented rats by scanning electron microscopy. In addition to the ubiquitous interdigitating edge protrusions many ball-and-socket junctions were found on the lateral surfaces of lens fibers in pigmented rats. Notable differences in density, shape and size between superficial and deep cortical layers were observed. Especially, in the intermediate equatorial cortex large ball-and-socket junctions were found. In contrast, only few and small ball-and-socket junctions were observed in albino rats and many ruptures of lens fiber membranes were present in the anterior, superficial and intermediate equatorial cortex. The present observations show that different strains of rats have a different morphology of lens fibers. In view of a postulated role of ball-and-socket junctions in calcium homeostasis in the lens this may account for differences in cataractogenesis between albino and pigmented rats.

Bag-in-the-lens implantation of intraocular lenses.

PURPOSE: To report a new intraocular lens (IOL) and an IOL implantation concept, the bag-in-the-lens implantation technique, designed to prevent posterior capsule opacification (PCO). SETTING: The University of Antwerp, Department of Ophthalmology, Edegem, Belgium, and the Netherlands Ophthalmic Research Institute, Department of Morphology, Amsterdam, The Netherlands. METHODS: After identical curvilinear capsulorhexes are created in both the anterior and posterior capsules, the capsules are inserted in a flange of the IOL, thus the term bag-in-the-lensas opposed to the currently used lens-in-the-bagtechnique. The IOL was implanted in an in vitro human capsular bag model and in 10 eyes of 9 patients with cataract. Lens epithelial cell (LEC) outgrowth and PCO formation were observed. RESULTS: When both capsular blades were well stretched around the IOL optic, the in vitro capsular bag model showed LEC proliferation only within the space of the remaining lens bag. The LEC proliferation was limited, and there was no tendency toward proliferation approaching the visual axis. In all 10 eyes, the optical axis remained clear during a follow-up between 4 and 15 months. CONCLUSIONS: This new IOL prevented LEC proliferation in vitro and seems promising in vivo. Target patients are those at risk of PCO including those with congenital cataract, uveitis, diabetes, or cataract extraction combined with vitrectomy.

Biological glass: structural determinants of eye lens transparency.

The purpose of the lens is to project a sharply focused, undistorted image of the visual surround onto the neural retina. The first pre-requisite, therefore, is that the tissue should be transparent. Despite the presence of remarkably high levels of protein, the lens cytosol remains transparent as a result of short-range-order interactions between the proteins. At a cellular level, the programmed elimination of nuclei and other light-scattering organelles from cells located within the pupillary space contributes directly to tissue transparency. Scattering at the cell borders is minimized by the close apposition of lens fibre cells facilitated by a plethora of adhesive proteins, some expressed only in the lens. Similarly, refractive index matching between lens membranes and cytosol is believed to minimize scatter. Refractive index matching between the cytoplasm of adjacent cells is achieved through the formation of cellular fusions that allow the intermingling of proteins. Together, these structural adaptations serve to minimize light scatter and enable this living, cellular structure to function as 'biological glass'.

Homeostasis in the vertebrate lens: mechanisms of solute exchange.

The eye lens is avascular, deriving nutrients from the aqueous and vitreous humours. It is, however, unclear which mechanisms mediate the transfer of solutes between these humours and the lens' fibre cells (FCs). In this review, we integrate the published data with the previously unpublished ultrastructural, dye loading and magnetic resonance imaging results. The picture emerging is that solute transfer between the humours and the fibre mass is determined by four processes: (i) paracellular transport of ions, water and small molecules along the intercellular spaces between epithelial and FCs, driven by Na(+)-leak conductance; (ii) membrane transport of such solutes from the intercellular spaces into the fibre cytoplasm by specific carriers and transporters; (iii) gap-junctional coupling mediating solute flux between superficial and deeper fibres, Na(+)/K(+)-ATPase-driven efflux of waste products in the equator, and electrical coupling of fibres; and (iv) transcellular transfer via caveoli and coated vesicles for the uptake of macromolecules and cholesterol. There is evidence that the Na(+)-driven influx of solutes occurs via paracellular and membrane transport and the Na(+)/K(+)-ATPase-driven efflux of waste products via gap junctions. This micro-circulation is likely restricted to the superficial cortex and nearly absent beyond the zone of organelle loss, forming a solute exchange barrier in the lens.

Early cortical lens opacities: a short overview.

Cataract is still the dominant cause of blindness worldwide. Cortical cataract is the most prevalent of the age-related changes in the human lenses that require surgical intervention to restore vision. The absence of adequate cataract surgery in most developing countries is the main cause of the high prevalence of cataract blindness worldwide. Lens ageing is accompanied by dramatic increases in stiffness, light scattering and coloration of the lens nucleus. These changes start to become manifest as early as the fourth or fifth decade of life and lead to nuclear cataract in old age. In the same period the equatorial deep lens cortex starts to show small opaque shades, which eventually grow out to segmental and annular opacities. These opaque shades are filled with small vesicles and contain abnormal amounts of cross-linked proteins, cholesterol and phospholipids. They are bordered by membranes that are rich in square arrays, have 'degenerate' gap junctions and have few intramembranous particles. It has been shown that the opaque shades represent cohorts of locally affected fibres segregated from unaffected neighbouring fibres by 'non-leaky' membranes. This segregation is an effective mechanism delaying the outgrowth of these opacities to cuneiform cataracts entering the pupillary space and thus leading to blinding cortical cataracts. Although cataract formation is mostly considered to be a multi-factorial disease, oxidative stress might be one of the leading causes for both nuclear and cortical cataract. In cortical cataracts shear stress between cortex and nucleus during accommodation may also play a significant role.

Development and adult morphology of the eye lens in the zebrafish.

The zebrafish has become an important vertebrate model organism to study the development of the visual system. Mutagenesis projects have resulted in the identification of hundreds of eye mutants. Analysis of the phenotypes of these mutants relies on in depth knowledge of the embryogenesis in wild-type animals. While the morphological events leading to the formation of the retina and its connections to the central nervous system have been described in great detail, the characterization of the development of the eye lens is still incomplete. In the present study, we provide a morphological description of embryonic and larval lens development as well as adult lens morphology in the zebrafish. Our analyses show that, in contrast to other vertebrate species, the zebrafish lens delaminates from the surface ectoderm as a solid cluster of cells. Detachment of the prospective lens from the surface ectoderm is facilitated by apoptosis. Primary fibre cell elongation occurs in a circular fashion resulting in an embryonic lens nucleus with concentric shells of fibres. After formation of a monolayer of lens epithelial cells, differentiation and elongation of secondary lens fibres result in a final lens morphology similar to that of other vertebrate species. As in other vertebrates, secondary fibre cell differentiation includes the programmed degradation of nuclei, the interconnection of adjacent fibres via protrusions at the fibre cells' edges and the establishment of gap junctions between lens fibre cells. The very close spacing of the nuclei of the differentiating secondary fibres in a narrow zone close to the equatorial epithelium, however, suggests that secondary fibre cell differentiation deviates from that described for mammalian or avian lenses. In summary, while there are similarities in the development and final morphology of the zebrafish lens with mammalian and avian lenses, there are also significant differences, suggesting caution when extrapolating findings on the zebrafish to, for example, human lens development or function.

A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma.

The purpose of the present study was to re-evaluate the three-dimensional organization of collagen fibrils and proteoglycans (PGs) in the human corneal stroma using an improved ultrastructural approach. After a short aldehyde prefixation, one half of seven fresh corneal buttons was stained for PGs with Quinolinic Phtalocyanin (QP) or Cupromeronic Blue (CB). Strips of 1 mm width were cut, subsequently treated with aqueous phosphotungstic acid (PTA) and further processed for light and electron microscopy. The other half of the corneas served as control and was routinely processed with OsO4. Embedding was as such that ultrathin sections could be cut precisely parallel (frontal sections) or perpendicular (cross sections) to the corneal surface. The mutual connections between collagen fibrils and PGs were studied and the length of PGs and their mutual distance were measured manually at a calibrated final magnification of 70,000 x. Prefixed fresh corneal tissue treated with QP and CB shows no signs of swelling and exhibits well contrasted PGs. In cross sections PGs form a repeating network of ring-like structures (approximately 45 nm) around the collagen fibrils. In frontal sections PGs are aligned orthogonal to the collagen fibrils, are equidistant (approximately 42 nm) attached to the collagen fibrils along their full length and have a thickness of approximately 11 nm and a length of approximately 54 nm. The observed maximal length of the PGs and the occurrence of ring-like structures enwrapping the collagen fibrils urged us to revisit the prevailing model of maurice (1962) on the organization of the corneal stroma. In the new model hexagonal arranged collagen fibrils are interconnected at regular distances with their next-nearest neighbours by groups of six PGs, attached orthogonal to the circumference of the fibrils. In this way a regular meshwork of ring-like structures enwrapping the collagen fibrils is formed. It is discussed that this new model more convincingly explains corneal resistance to compression and stretching and further rationalizes corneal transparency because of the low refractive index difference between the regularly arranged collagen fibrils and their inter-space filled with PGs.

Tryptophan deficiency arrests chromatin breakdown in secondary lens fibers of rats.

Tryptophan deficiency is known for long time to cause cataract in rats. However, up till now the underlying mechanism is still enigmatic. Histological studies showed an extended lens bow suggesting that the normal breakdown of nuclei in the lens fibres is arrested under these conditions. Using advanced ultrastructural techniques we aimed to clarify this aberrant final differentiation of lens fibres. Albino and pigmented rats were permanently or intermittently raised on a tryptophan deficient diet for 12 and 16 weeks, respectively. Rats of the same age raised on a normal diet served as controls. Lenses were treated for light and electron microscopy. For histology sections were stained for DNA and gamma-crystallins. In addition to routine transmission electron microscopy (TEM), ultrathin sections were subjected to electron tomography and energy dispersive X-ray microanalysis (EDX). Histology verified the extended lens bow for albino and pigmented rats and showed that in the intermittent period of normal diet the fibre nuclei are broken down as in controls. It was further shown that gamma-crystallins are co-localized with DNA in the nuclear domain. TEM revealed that during final differentiation nuclear chromatin becomes highly compacted in a chromosome-like manner and than rapidly evanesces in control rats. This compacted stage persists indefinitely in the tryptophan deficient rats. Electron tomography showed that during differentiation chromatin is first uncoiled to 30 nm solenoids, subsequently to highly compacted 10 nm beads-on-a-string fibrils and than is segregated from the nuclear proteins. EDX revealed that the late stage persisting nuclei consist of domains rich in DNA associated with histones and in domains with mainly proteins. This study corroborates previous findings on the final breakdown of nuclei of lens fibres. It further shows that the chromatin is ultimately uncoiled to beads-on-a-string fibrils and that as the last step chromatin is broken down at this unmasked stage. Except for this last step nuclear breakdown is identical in control and tryptophan deficient rats suggesting that it is not the availability of tryptophan for protein synthesis in general which causes the arrest. Two alternatives for this final arrest are discussed. A low tryptophan content, most pronounced in deeper cortical layers, may inhibit the late synthesis of the DNases and proteases necessary for chromatin breakdown. The radical scavenging by indoleamine 2,3-dioxygenase, which cleaves the pyrrole ring of tryptophan to form formylkynurenine using free oxygen radicals, is impaired by low levels of tryptophan. This decreased scavenging of oxygen radicals will expose the catalytic enzymes for chromatin breakdown, residing in the nucleus in an inactive form for quite a long period, to high levels of oxygen radicals and may affect the activity of these enzymes and therefore the execution of the chromatin breakdown.