Evaluation of cataract formation in fish exposed to environmental radiation at Chernobyl and Fukushima.

Recent studies apparently finding deleterious effects of radiation exposure on cataract formation in birds and voles living near Chernobyl represent a major challenge to current radiation protection regulations. This study conducted an integrated assessment of radiation exposure on cataractogenesis using the most advanced technologies available to assess the cataract status of lenses extracted from fish caught at both Chernobyl in Ukraine and Fukushima in Japan. It was hypothesised that these novel data would reveal positive correlations between radiation dose and early indicators of cataract formation. The structure, function and optical properties of lenses were analysed from atomic to millimetre length scales. We measured the short-range order of the lens crystallin proteins using Small Angle X-Ray Scattering (SAXS) at both the SPring-8 and DIAMOND synchrotrons, the profile of the graded refractive index generated by these proteins, the epithelial cell density and organisation and finally the focal length of each lens. The results showed no evidence of a difference between the focal length, the epithelial cell densities, the refractive indices, the interference functions and the short-range order of crystallin proteins (X-ray diffraction patterns) in lens from fish exposed to different radiation doses. It could be argued that animals in the natural environment which developed cataract would be more likely, for example, to suffer predation leading to survivor bias. But the cross-length scale study presented here, by evaluating small scale molecular and cellular changes in the lens (pre-cataract formation) significantly mitigates against this issue.

Cell compaction is not required for the development of gradient refractive index profiles in the embryonic chick lens.

The development of the eye requires the co-ordinated integration of optical and neural elements to create a system with requisite optics for the given animal. The eye lens has a lamellar structure with gradually varying protein concentrations that increase towards the centre, creating a gradient refractive index or GRIN. This provides enhanced image quality compared to a homogeneous refractive index lens. The development of the GRIN during ocular embryogenesis has not been investigated previously. This study presents measurements using synchrotron X-ray Talbot interferometry and scanning electron microscopy of chick eyes from embryonic day 10: midway through embryonic development to E18: a few days before hatching. The lens GRIN profile is evident from the youngest age measured and increases in magnitude of refractive index at all points as the lens grows. The profile is parabolic along the optic axis and has two distinct regions in the equatorial plane. We postulate that these may be fundamental for the independent central and peripheral processes that contribute to the optimisation of image quality and the development of an eye that is emmetropic. The spatial distributions of the distinct GRIN profile regions match with previous measurements on different fibre cell groups in chick lenses of similar developmental stages. Results suggest that tissue compaction may not be necessary for development of the GRIN in the chick eye lens.

Chondroitin Sulfate as a Potential Modulator of the Stem Cell Niche in Cornea.

Chondroitin sulfate (CS) is an important component of the extracellular matrix in multiple biological tissues. In cornea, the CS glycosaminoglycan (GAG) exists in hybrid form, whereby some of the repeating disaccharides are dermatan sulfate (DS). These CS/DS GAGs in cornea, through their presence on the proteoglycans, decorin and biglycan, help control collagen fibrillogenesis and organization. CS also acts as a regulatory ligand for a spectrum of signaling molecules, including morphogens, cytokines, chemokines, and enzymes during corneal growth and development. There is a growing body of evidence that precise expression of CS or CS/DS with specific sulfation motifs helps define the local extracellular compartment that contributes to maintenance of the stem cell phenotype. Indeed, recent evidence shows that CS sulfation motifs recognized by antibodies 4C3, 7D4, and 3B3 identify stem cell populations and their niches, along with activated progenitor cells and transitional areas of tissue development in the fetal human elbow. Various sulfation motifs identified by some CS antibodies are also specifically located in the limbal region at the edge of the mature cornea, which is widely accepted to represent the corneal epithelial stem cell niche. Emerging data also implicate developmental changes in the distribution of CS during corneal morphogenesis. This article will reflect upon the potential roles of CS and CS/DS in maintenance of the stem cell niche in cornea, and will contemplate the possible involvement of CS in the generation of eye-like tissues from human iPS (induced pluripotent stem) cells.

The eye lens: age-related trends and individual variations in refractive index and shape parameters.

The eye lens grows throughout life by cell accrual on its surface and can change shape to adjust the focussing power of the eye. Varying concentrations of proteins in successive cell layers create a refractive index gradient. The continued growth of the lens and age-related changes in proteins render it less able to alter shape with loss of capacity by the end of the sixth decade of life. Growth and protein ageing alter the refractive index but as accurate measurement of this parameter is difficult, the nature of such alterations remains uncertain. The most accurate method to date for measuring refractive index in intact lenses has been developed at the SPring-8 synchrotron. The technique, based on Talbot interferometry, has an X-ray source and was used to measure refractive index in sixty-six human lenses, aged from 16 to 91 years. Height and width were measured for forty-five lenses. Refractive index contours show decentration in some older lenses but individual variations mask age-related trends. Refractive index profiles along the optic axis have relatively flat central sections with distinct micro-fluctuations and a steep gradient in the cortex but do not exhibit an age-related trend. The refractive index profiles in the equatorial aspect show statistical significance with age, particularly for lenses below the age of sixty that had capacity to alter shape in vivo. The maximum refractive index in the lens centre decreases slightly with age with considerable scatter in the data and there are age-related variations in sagittal thickness and equatorial height.

Refractive index degeneration in older lenses: A potential functional correlate to structural changes that underlie cataract formation.

A major structure/function relationship in the eye lens is that between the constituent proteins, the crystallins and the optical property of refractive index. Structural breakdown that leads to cataract has been investigated in a number of studies; the concomitant changes in the optics, namely increases in light attenuation have also been well documented. Specific changes in the refractive index gradient that cause such attenuation, however, are not well studied because previous methods of measuring refractive index require transparent samples. The X-ray Talbot interferometric method using synchrotron radiation allows for measurement of fine changes in refractive index through lenses with opacities. The findings of this study on older human lenses show disruptions to the refractive index gradient and in the refractive index contours. These disruptions are linked to location in the lens and occur in polar regions, along or close to the equatorial plane or in lamellar-like formations. The disruptions that are seen in the polar regions manifest branching formations that alter with progression through the lens with some similarity to lens sutures. This study shows how the refractive index gradient, which is needed to maintain image quality of the eye, may be disturbed and that this can occur in a number of distinct ways. These findings offer insight into functional changes to a major optical parameter in older lenses. Further studies are needed to elicit how these may be related to structural degenerations reported in the literature.

Optical properties of the lens: an explanation for the zones of discontinuity.

The structural basis of zones of discontinuity in the living human eye lens has not been elucidated, and there is no conclusive explanation for what relevance they may have to the structure and function of the lens. Newly developed synchrotron radiation based X-ray Talbot interferometry has enabled the detection of subtle fluctuations in the human eye lens which, when used in mathematical modelling to simulate reflected and scattered light, can recreate the image of the lens seen in the living human eye. The results of this study show that the zones of discontinuity may be caused by subtle fluctuations in the refractive index gradient as well as from random scattering in the central regions. As the refractive index contours are created by cell layers with progressively varying protein concentrations, the zones are linked to growth and will contain information about ageing and development. The index gradient is important for image quality and fluctuations in this gradient may add to quality optimisation and serve as models for designs of new generation implant lenses.

The gradient index lens of the eye: an opto-biological synchrony.

The refractive power of a lens is determined largely by its surface curvatures and the refractive index of its medium. These properties can also be used to control the sharpness of focus and hence the image quality. One of the most effective ways of doing this is with a gradient index. Eye lenses of all species, thus far, measured, are gradient index (GRIN) structures. The index gradation is one that increases from the periphery of the lens to its centre but the steepness of the gradient and the magnitudes of the refractive index vary so that the optics of the lens accords with visual demands. The structural proteins, the crystallins, which create the index gradient, also vary from species to species, in type and relative distribution across the tissue. The crystallin classes do not contribute equally to the refractive index, and this may be related to their structure and amino acid content. This article compares GRIN forms in eye lenses of varying species, the relevance of these forms to visual requirements, and the relationship between refractive index and the structural proteins. Consideration is given to the dynamics of a living lens, potential variations in the GRIN form with physiological changes and the possible link between discontinuities in the gradient and growth. Finally, the property of birefringence and the characteristic polarisation patterns seen in highly ordered crystals that have also been observed in specially prepared eye lenses are described and discussed.

Optical properties of in situ eye lenses measured with X-ray Talbot interferometry: a novel measure of growth processes.

The lens, a major optical component of the eye, has a gradient refractive index, which is required to provide sufficient refractive power and image quality. The refractive index variations across the lens are dependent on the distributions and concentrations of the varying protein classes. In this study, we present the first measurements of the refractive index in the in situ eye lens from five species using a specially constructed X-ray Talbot grating interferometer. The measurements have been conducted in two planes: the one containing the optic axis (the sagittal plane) and the plane orthogonal to this (the equatorial plane). The results show previously undetected discontinuities and fluctuations in the refractive index profile that vary in different species. These may be linked to growth processes and may be the first optical evidence of discrete developmental stages.

The interaction of unfolding α-lactalbumin and malate dehydrogenase with the molecular chaperone αB-crystallin: a light and X-ray scattering investigation.

PURPOSE: The molecular chaperone αB-crystallin is found in high concentrations in the lens and is present in all major body tissues. Its structure and the mechanism by which it protects its target protein from aggregating and precipitating are not known. METHODS: Dynamic light scattering and X-ray solution scattering techniques were used to investigate structural features of the αB-crystallin oligomer when complexed with target proteins under mild stress conditions, i.e., reduction of α-lactalbumin at 37 °C and malate dehydrogenase when heated at 42 °C. In this investigation, the size, shape and particle distribution of the complexes were determined in real-time following the induction of stress. RESULTS: Overall, it is observed that the mass distribution, hydrodynamic radius, and spherical shape of the αB-crystallin oligomer do not alter significantly when it complexes with its target protein. CONCLUSIONS: The data are consistent with the target protein being located in the outer protein shell of the αB-crystallin oligomer where it is readily accessible for possible refolding via the action of other molecular chaperones.

Changes in the X-ray diffraction pattern from lens during a solid-to-liquid phase transition.

PURPOSE: The purpose of this study is to compare the structural integrity of bovine lenses using small-angle X-ray diffraction techniques, before and after freezing, using both liquid nitrogen and a -20 degrees C freezer to understand the molecular changes that occur and to see if any permanent structural changes result from the freezing and thawing process. MATERIALS AND METHODS: We used small-angle X-ray scattering to investigate the effects of freezing whole bovine eye lenses (i) in liquid nitrogen and (ii) at -20 degrees C, to better understand the structural basis of the phase transitions. RESULTS: Lenses frozen in liquid nitrogen thawed more rapidly than those placed at -20 degrees C. With both freezing methods, X-ray patterns taken during the thawing process indicated less protein order than before or after freezing. After both freezing methods, the X-ray reflection returned to its original spacing and close to its original intensity values before freezing. CONCLUSIONS: We explain these phenomena in terms of a simple model based on the melting of ice crystals. We also suggest that the liquid nitrogen method of freezing is probably the better method of cryo-preservation for maintaining lens crystallin order.

X-ray- and neutron-scattering studies of alpha-crystallin and evidence that the target protein sits in the fenestrations of the alpha-crystallin shell.

PURPOSE: Alpha-crystallin, a ubiquitous molecular chaperone, is found in high concentrations in the lens. Its structure and precise mechanism of action, however, are unknown. The purpose of these experiments was to further the understanding of the chaperone function of alpha-crystallin. METHODS: X-ray- and neutron-solution-scattering studies were used to measure the radius of gyration of bovine lens alpha-crystallin when complexed with its target protein beta-crystallin in both normal and heavy-water-based solutions. Spectrophotometry was used as a chaperone assay. RESULTS: The radius of gyration of alpha-crystallin on its own and when mixed with beta-crystallin was 69 +/- 1 A at 35 degrees C and increased with the temperature. In contrast to H2O-buffered solutions, the radius of gyration did not increase significantly in D2O-buffered solutions up to 55 degrees C, and at 70 degrees C was, on average, some 15 to 20 A smaller. CONCLUSIONS: Bovine lens alpha-crystallin in solution can be modeled as a fenestrated spherical shell of diameter 169 A. At physiological temperatures, a weak interaction between alpha- and beta-crystallin occurs, and beta-crystallin is located in the fenestrations. Deuterium substitution indicates that the superaggregation process is controlled by hydrogen bonding. However, the chaperone process and superaggregation appear not to be linked.

Transparency of the bovine corneal stroma at physiological hydration and its dependence on concentration of the ambient anion.

De-epithelialised and de-endothelialised bovine corneal stromas with a hydration of 3.2 equilibrated at 154 mM NaCl and buffered at pH 7.4 had their optical density (400-750 nm) measured. Stromas equilibrated against 10, 20, 30, 50 or 100 mM NaCl made isotonic to 154 mM NaCl by supplementing with sorbitol were progressively more transparent as NaCl increased. Hypertonic equilibration against 300, 600 or 1000 mM NaCl resulted in a progressive loss of transparency compared with 154 mM NaCl. Light scattering as a function of wavelength fitted a lambda(-3) function well for 10, 30, 50, 100 and 154 mM NaCl preparations between 450 and 650 nm, but not at higher wavelengths. However, hypertonic 300, 600 and 1000 mM NaCl preparations showed a lambda(-2) dependence in the 450-750 nm range. Experiments with 154 mM NaCl and either 0 or 300 mM sorbitol suggested that the changes in light scattering in hypertonic preparations are unlikely to be caused by osmotic alterations to the stromal keratocytes. Psychophysical studies of the optical transmission function of preparations indicated that corneal stromas dialysed against 154 mM NaCl had usable optical properties, but preparations dialysed against 10 mM NaCl were effectively unable to transmit an image. The results are related to the known increase of fixed negative charge in the corneal matrix when chloride ions are adsorbed onto the matrix. It is suggested that the ordering force between corneal collagen fibrils, generated in part by anion binding, may be crucial to the physiological functioning of the visual system.