Visualizing ocular lens fluid dynamics using MRI: manipulation of steady state water content and water fluxes

Studies using various MRI techniques have shown that a water-protein concentration gradient exists in the ocular lens. Because this concentration is higher in the core relative to the lens periphery, a gradient in refractive index is established in the lens. To investigate how the water-protein concentration profile is maintained, bovine lenses were incubated in different solutions, and changes in water-protein concentration ratio monitored using proton density weighted (PD-weighted) imaging in the absence and presence of heavy water (D(2)O). Lenses incubated in artificial aqueous humor (AAH) maintained the steady state water-protein concentration gradient, but incubating lenses in high extracellular potassium (KCl-AAH) or low temperature (Low T-AAH) caused a collapse of the gradient due to a rise in water content in the core of the lens. To visualize water fluxes, lenses were incubated in D(2)O, which acts as a contrast agent. Incubation in KCl-AAH and low T-AAH dramatically slowed the movement of D(2)O into the core but did not affect the movement of D(2)O into the outer cortex. D(2)O seemed to preferentially enter the lens cortex at the anterior and posterior poles before moving circumferentially toward the equatorial regions. This directionality of D(2)O influx into the lens cortex was abolished by incubating lenses in high KCl-AAH or low T-AAH, and resulted in homogenous influx of D(2)O into the outer cortex. Taken together, our results show that the water-protein concentration ratio is actively maintained in the core of the lens and that water fluxes preferentially enter the lens at the poles.     

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.     

Crystallin distribution patterns in concentric layers from toad eye lenses

Protein distribution patterns across eye lenses from the Asiatic toad Bufo gargarizans were investigated and individual crystallin classes characterised. Special fractionation that follows the growth mode of the lens was used to yield nine fractions corresponding to layers laid down at different chronological (developmental) stages. Proportions of soluble and insoluble crystallins within each fraction were measured by Bradford assay. Water‐soluble proteins in all fractions were separated by size‐exclusion HPLC and constituents of each class further characterised by electrophoresis, RP‐HPLC and MS analysis. In outer lens layers, α‐crystallin is the most abundant soluble protein but is not found in soluble proteins in the lens centre. Water‐soluble β‐crystallins also decrease from their highest level in the outer lens to negligible mounts in the central lens. The proportion of soluble γ‐crystallin increases significantly towards the lens centre where this is the only soluble protein present. Insoluble protein levels increase significantly towards the lens centre. In B. gargarizans lenses, as with other anurans, the predominant water‐soluble protein class is γ‐crystallin. No taxon‐specific crystallins were found. The relationship between the protein distribution patterns and the functional properties of the lens this species is discussed.     

Dioptrics of the facet lenses of male blowflies Calliphora and Chrysomyia

1. The dioptrics of the facet lenses of two blowfly species, Calliphora erythrocephala and Chrysomyia megacephala, was investigated. Measurements were performed on facet lenses ranging in diameter from 20 to 80 microns. 2. The radius of curvature of the front surface of the facet lenses, measured by microreflectometry, increases approximately linearly with the facet lens diameter. 3. The optical path difference of the facet lens and water, measured by interference microscopy, depends on the distance from the optical axis according to a parabolic function. Average refractive index values, calculated from the optical path difference profile together with estimates of the thickness profile, are between 1.40 and 1.43, with the lowest values in the largest lenses. 4. The F-number calculated from the experimental data ranges from 1.5 to 2.2. It is argued that the range of effective F-numbers is 2.1-2.4.     

Cell growth patterns and lens geometry: a quantitative study from three-dimensional reconstructions

The growth of the lens of the sea lamprey, Petromyzon marinus, was studied over the 5 years of larval development. Whole lenses (25) and Golgi-impregnated cells (393) were reconstructed with computer-assisted microscopy. Several cellular geometric parameters (length, width, curvature, surface, volume, shape) were correlated with the position of the cell's base on the lens capsular perimeter. Based on these correlations, the cells formed four groups that correspond to the central anterior, germinative, transitional and cortical fiber zones. A fifth zone, containing nuclear fiber cells, never stained. Lens growth is exponential during the 5 years. The anterior epithelium increases in size and in cell number by cell growth and division. The posterior mass increases in cell number by recruitment and increases in size by cell growth. A model is proposed to account for the size and shape of the lens based upon the coupling of anterior and posterior growth patterns. Four zonal boundaries are defined by changes in cell growth patterns. With growth, cells are subsumed into adjacent zones and zonal boundaries move away from the lens center. We find no support for the suggestion that cells migrate centrally.     

Alterations in physical parameters and proteins of lens from Rana catesbeiana during development

Lens proteins and lens gross morphology were examined during tadpole and adult development of the bullfrog, Rana catesbeiana. Significant increases in the lens physical parameters of diameter, wet weight, dry weight (94–97% protein), and percent water were observed to accompany both natural and thyroxine-induced metamorphosis. These increases in lens parameters were not accompanied by growth of tadpoles during metamorphic change. Lens proteins were isolated from whole lenses and also from specified layers within whole lenses by a new fractionation method. Electrophoretic examination of whole lenses showed that the lens proteins did not change rapidly, one for another, prior to or during metamorphosis. However, changes became apparent during post metamorphic development. These changes included an increase in the relative concentration and mobility of alpha crystallin, a decrease in the relative concentration of gamma crystallin and an increase in the relative concentration of beta crystallin. Examination of specified layers within tadpole and frog lenses demonstrated that changes in the patterns of lens protein synthesis and modification may occur during development. Rapid and reproducible methods for quantitating changes in lens gross morphology and lens proteins, and for fractionating both tadpole and frog lenses into a number of definable layers were devised in the course of this study.     

The role of macromolecular crowding in the evolution of lens crystallins with high molecular refractive index

Crystallins are present in the lens at extremely high concentrations in order to provide transparency and generate a high refractive power of the lens. The crystallin families prevalent in the highest density lens tissues are γ-crystallins in vertebrates and S-crystallins in cephalopods. As shown elsewhere, in parallel evolution, both have evolved molecular refractive index increments 5-10% above those of most proteins. Although this is a small increase, it is statistically very significant and can be achieved only by very unusual amino acid compositions. In contrast, such a molecular adaptation to aid in the refractive function of the lens did not occur in crystallins that are preferentially located in lower density lens tissues, such as vertebrate α-crystallin and taxon-specific crystallins. In the current work, we apply a model of non-interacting hard spheres to examine the thermodynamic contributions of volume exclusion at lenticular protein concentrations. We show that the small concentration decrease afforded by the higher molecular refractive index increment of crystallins can amplify nonlinearly to produce order of magnitude differences in chemical activities, and lead to reduced osmotic pressure and the reduced propensity for protein aggregation. Quantitatively, this amplification sets in only at protein concentrations as high as those found in hard lenses or the nucleus of soft lenses, in good correspondence to the observed crystallin properties in different tissues and different species. This suggests that volume exclusion effects provide the evolutionary driving force for the unusual refractive properties and the unusual amino acid compositions of γ-crystallins and S-crystallins.     

Mechanism of insolubilization by a single-point mutation in alphaA-crystallin linked with hereditary human cataracts

AlphaA-crystallin is a small heat shock protein that functions as a molecular chaperone and a lens structural protein. The R49C single-point mutation in alphaA-crystallin causes hereditary human cataracts. We have previously investigated the in vivo properties of this mutant in a gene knock-in mouse model. Remarkably, homozygous mice carrying the alphaA-R49C mutant exhibit nearly complete lens opacity concurrent with small lenses and small eyes. Here we have investigated the 90 degrees light scattering, viscosity, refractive index, and bis-ANS fluorescence of lens proteins isolated from the alphaA-R49C mouse lenses and found that the concentration of total water-soluble proteins showed a pronounced decrease in alphaA-R49C homozygous lenses. Light scattering measurements on proteins separated by gel permeation chromatography showed a small amount of high-molecular mass aggregated material in the void volume which still remains soluble in alphaA-R49C homozygous lens homogenates. An increased level of binding of beta- and gamma-crystallin to the alpha-crystallin fraction was observed in alphaA-R49C heterozygous and homozygous lenses but not in wild-type lenses. Quantitative analysis with the hydrophobic fluorescence probe bis-ANS showed a pronounced increase in fluorescence yield upon binding to alpha-crystallin from mutant as compared with the wild-type lenses. These results suggest that the decrease in the solubility of the alphaA-R49C mutant protein was due to an increase in its hydrophobicity and supra-aggregation of alphaA-crystallin that leads to cataract formation. Our study further shows that analysis of mutant proteins from the mouse model is an effective way to understand the mechanism of protein insolubilization in hereditary cataracts.     

Regional distribution of the Na+ and K+ currents around the crystalline lens of rabbit

Early studies described asymmetricalelectrical properties across the ocular lens in theanterior-to-posterior direction. More recent results obtained with avibrating probe indicated that currents around the lens surface are notuniform by showing an outwardly directed K⁺ efflux at thelens equator and Na⁺ influx at the poles. The latterstudies have been used to support theoretical models for fluidrecirculation within the avascular lens. However, the existence of anonuniform current distribution in the lens epithelium from theanterior pole to the equator has never been confirmed. The present workdeveloped a modified short-circuiting technique to examine the netflows of Na⁺ and K⁺ across arbitrarily definedlens surface regions. Results indicate that passive inflows ofNa⁺ occur at both the anterior polar region and posteriorlens surface, consistent with suggestions derived from the vibratingprobe data, whereas K⁺ efflux plus theNa⁺-K⁺ pump-generated current comprise thecurrents at the equatorial surface and an area anterior to it.Furthermore, Na⁺-K⁺ pump activity was absent atthe posterior surface and its polar region in all lenses examined, aswell as from the anterior polar region in most lenses. The latterunexpected observation suggests that the monolayered epithelium, whichis confined to the anterior surface of the lens, does not express anactive Na⁺-K⁺ pump at its anterior-most aspect.Nevertheless, this report represents the first independent confirmationthat positive currents leave the lens around the equator and reenteracross the polar and posterior surfaces.

     

Fine structure and optical properties of an ostracode (Crustacea) nauplius eye

Summary InNotodromas monachus, the three cups of the nauplius eye are formed by four pigment cells. The insides of the cups are lined with tapetal cells, which produce several layers of reflecting crystals. The reflecting crystals form a concave mirror in each cup upon which the retinular cells rest. The two-celled rhabdoms are few and perpendicular to the tapetal layer. The axons from the tripartite eye leave the retinular cells distally in three separate groups. The eye is thus of the inverse type. Large lens cells, with a low refractive index, are present in the open part of each cup. Distal to the lens cells, highly refractive lenses are formed in the cuticle. These lenses serve to decrease the effective curvature of the mirrors, thus enabling the reflectors to produce a focused image on the retina. The ventral cup differs by the lack of a cuticular lens and has degenerated-appearing cellular elements. The investigated nauplius eye is the only one known with both a mirror and a highly refractive lens in the dioptric apparatus.This investigation has been supported by grants from the Swedish Natural Science Research Council (grant no. 2760-009) and the Royal Physiographic Society of Lund.     

Spherical aberration of the fish lens: interspecies variation and age

Summary Spherical aberration of the eyes of a spectrum of freshwater fishes was determined by photographing the refractive effects of excised crystalline lenses on multiple parallel split laser beams. In general, spherical aberration is minimized by the developmentally related variation in lens refractive index. However, spherical aberration is marked and non-monotonic in a non-visual species such as the bullhead. Furthermore, the size and variability of the aberration appears to be related to visual need as indicated by diet and feeding habits. For example, the lenses of predatory sight feeders such as the pike (Esox lucius) or rock bass (Ambloplites rupestris) are optically superior to that of an omnivorous feeder as the carp (Cyprinus carpio).The effect of age was tested by examining rock bass lenses from fish two to seven years of age. Lens quality, as indicated by the amount of change in posterior focal length for beams of varying eccentricity from the optic axis, is optimum in lenses from five year old fish. The significance of this variation in lens quality is uncertain and requires further study with greater attention to specimens of advanced age.     

A Focus on the Optical Properties of the Regenerated Newt Lens

Lens regeneration studies in the adult newt suggest that molecular aspects of lens regeneration are complete within 5 weeks of lentectomy. However, very little is known about the optical properties of the regenerated lens. In an aquatic environment, the lens accounts for almost all of the refractive power of the eye, and thus, a fully functional lens is critical. We compared the optical properties of 9- and 26-week regenerated lenses in the red spotted newt, Notophthalmus viridescens, with the original lenses removed from the same eyes. At 9 weeks, the regenerated lenses are smaller than the original lenses and are histologically immature, with a lower density of lens proteins. The 9 week lenses have greater light transmission, but significantly reduced focal length and refractive index than the original lenses. This suggests that following 9 weeks of regeneration, the lenses have not recovered the functionality of the original lens. By 26 weeks, the transmission of light in the more mature lens is reduced, but the optical parameters of the lens have recovered enough to allow functional vision.     

Presbyopia and heat: changes associated with aging of the human lens suggest a functional role for the small heat shock protein, alpha-crystallin, in maintaining lens flexibility

Presbyopia, the inability to focus up close, affects everyone by age 50 and is the most common eye condition. It is thought to result from changes to the lens over time making it less flexible. We present evidence that presbyopia may be the result of age-related changes to the proteins of the lens fibre cells. Specifically, we show that there is a progressive decrease in the concentration of the chaperone, α-crystallin, in human lens nuclei with age, as it becomes incorporated into high molecular weight aggregates and insoluble protein. This is accompanied by a large increase in lens stiffness. Stiffness increases even more dramatically after middle age following the disappearance of free soluble α-crystallin from the centre of the lens. These alterations in α-crystallin and aggregated protein in human lenses can be reproduced simply by exposing intact pig lenses to elevated temperatures, for example, 50 °C. In this model system, the same protein changes are also associated with a progressive increase in lens stiffness. These data suggest a functional role for α-crystallin in the human lens acting as a small heat shock protein and helping to maintain lens flexibility. Presbyopia may be the result of a loss of α-crystallin coupled with progressive heat-induced denaturation of structural proteins in the lens during the first five decades of life.     

Chick eye optics: zero to fourteen days

Ocular dimensions and refractive state data for chicks 0 to 14 days of age were obtained from 234 untreated control eyes of birds treated unilaterally in previous work involving various defocussing lenses and/or translucent goggles. Refractive state and corneal curvatures were measured in vivo by retinoscopy and ophthalmometry respectively. Intraocular dimensions were measured by A-scan ultrasonography, after which the eyes were removed, weighed and measured. In some cases (n=52) intraocular dimensions and lens curvatures were obtained from frozen sections of enucleated eyes. The hyperopia of hatchling chicks (+6.5+4.0 D) initially decreases rapidly and then more gradually to + 2.0 ± 0.5 D by 16 days. The distribution of refractive errors is very broad at Day 0, but becomes leptokurtotic, with a slight myopic skew, by Day 14. Corneal radius is constant for the first four days, possible as a result of pre-hatching lid pressure, and then increases linearly, as do all lens dimensions, axial diameter and equatorial diameter. Schematic eyes were developed for Days 0, 7, and 14.     

A biochemical characterization of the photophore lenses of the midshipman fish,Porichthys notatus Girard

The present study is a biochemical characterization of the photophore lenses of the midshipman fish, Porichthys notatus, a species that bears 800 photophores distributed over the body surface. The biochemical properties of the photophore lenses were compared with those of the eye lens with which they share a similar developmental origin and analogous function. To achieve a high refractive index, the vertebrate eye lens has a relatively high concentration of structural proteins (20–50%, depending on species) and a simple protein composition, that is, relatively few proteins are synthesized in comparison to other tissues. Similarly, the photophore lenses of P. notatus had a relatively high protein concentration (average = 29%, n = 5) and approximately 60% of the total soluble protein was represented by two subunit species of 33 kD and 35 kD on denaturing polyacrylamide gels. The structural proteins of the eye lens are of two principle types: 1) and polypeptides which belong to vertebrate lens-specific crystallin families, and, 2) enzymes recruited into the lens which take on the function of structural proteins. Here, we report that the two major photophore lens subunits of 33 kD and 35 kD are biochemically similar to each other, but are clearly distinct from any of the previously characterized crystallins. Therefore, we propose that photophore lenses appear to recruit a novel protein.     

Surface change of the mammalian lens during accommodation

Classical theories suggest that the surface area of the crystalline lens changes during accommodation while the lens volume remains constant. Our recent work challenged this view by showing that the lens volume decreases as the lens flattens during unaccommodation. In this paper we investigate 1) the magnitude of changes in the surface of the in vitro isolated cow lens during simulated accommodation, as well as that of human lens models, determined from lateral photographs and the application of the first theorem of Pappus; and 2) the velocity of the equatorial diameter recovery of prestretched cow and rabbit lenses by using a custom-built software-controlled stretching apparatus synchronized to a digital camera. Our results showed that the in vitro cow lens surface changed in an unexpected manner during accommodation depending on how much tension was applied to flatten the lens. In this case, the anterior surface initially collapsed with a reduction in surface followed by an increase in surface, when the stretching was applied. In the human lens model, the surface increased when the lens unaccommodated. The lens volume always decreases as the lens flattens. An explanation for the unexpected surface change is presented and discussed. Furthermore, we determined that the changes in lens volume, as reflected by the speed of the equatorial diameter recovery in in vitro cow and rabbit lenses during simulated accommodation, occurred within a physiologically relevant time frame (200 ms), implying a rapid movement of fluid to and from the lens during accommodation.     

Packing interactions in the eye-lens. Structural analysis, internal symmetry and lattice interactions of bovine gamma IVa-crystallin

gamma-Crystallins are a family of low molecular weight proteins found in high concentration in the densely packed regions of high refractive index in vertebrate lenses. Certain members have the characteristic property of a high critical temperature (tc) for phase separation. We report the three-dimensional structure determination of such a protein, bovine lens gamma IVa-crystallin, which has been refined to give an X-ray R-factor of 0.143. Its high tc contrasts with the low tc gamma II-crystallin, whose structure we have already published. The root mean square difference between the alpha-carbon atoms of these two proteins is 0.70 A and gamma IVa has an internal symmetry even higher than that of gamma II. The presence of a protein that exhibits the phenomenon of phase separation at body temperature renders the lens very susceptible to a transformation from transparent to an opaque state due to irregularities in the refractive index. Protein interactions of gamma IVa-crystallin have implications for the mechanism of cataract formation. Modes of self-association behaviour of gamma IVa-crystallin have been inferred from an analysis of the lattice interactions in the crystalline state, where the protein packing density is similar to that of the intact lens. It appears that the point mutation at position 103 from a serine residue in gamma II to a valine in gamma IVa gives rise to a lattice contact formed by two four-stranded beta-sheets in gamma IVa. A group-specific mutation at position 118 from leucine to phenylalanine induces subtle differences in core packing, leading to a reorganization around residue 103. However, the final phase separation determinant may be a complex combination of many side-chain functions.     

Optical quality of the ocular lens of the sea lamprey (<Emphasis Type="Italic">Petromyzon marinus</Emphasis>) during the mature and transformer periods of life

While larval sea lampreys exist as eyeless filter feeders for several years, they transform into free-swimming juveniles (transformers) that attach parasitically to prey fish as they develop sexual maturity. This study examines lamprey lens development and optics and, since the lens is often the only refractive component of an aquatic eye, the data also provide an indication of visual ability during transformer and adult periods of life. Seven adult sea lampreys (0.40–0.55 m) and eight transformers (0.15–0.18 m) were sacrificed, the eyes removed and lenses dissected, measured, and placed in an automated laser scanning instrument. Back vertex focal length (spherical aberration) was measured for 14 beam positions across each lens by using a digital camera to record the position of the refracted beam. Transformer lenses exhibit positive spherical aberration, with average focal lengths varying from about 2.40 mm near the lens center and 1.06 mm at the lens periphery. On the other hand, the lenses from adults are largely corrected for spherical aberration, with average focal lengths varying from 2.19 mm to 2.44 mm. This result indicates that the younger lenses do not have a gradient refractive index necessary to mitigate the aberration and that further study of this model may reveal the relation between lens embryology and the development of such a gradient.     

GROWTH OF LENS AND OCULAR ENVIRONMENT: ROLE OF NEURAL RETINA IN THE GROWTH OF MOUSE LENS AS REVEALED BY AN IMPLANTATION EXPERIMENT

The lens of 6-day-old normal mouse was implanted into the lentectomized eye of adult mouse to examine the effect of retina upon the growth of the implanted lens in vivo. The implanted lens grew normally and its transparency was kept for more than 5 months after implantation. The connection between the implanted lens and the ciliary part of the recipient iris was well established with the regeneration of zonular fibers from the recipient. In young lenses implanted reversely into adult eyes, the epithelial cells facing the retina elongated and a new epithelium was formed on the corneal side of the lens within 5 days. Young lenses implanted either in normal or reverse orientation into eyes from which the retina was previously removed did not grow. The cells of the original lens epithelium of these lenses were randomly accumulated beneath the posterior lens capsule, while the anterior portion of the implanted lenses became an epithelial structure without cell elongation. These results suggest that the growth of the implanted lens may be dependent on the retina of the adult eye.