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Vaghefi E, Pontre BP, Jacobs MD, Donaldson PJ. Visualizing ocular lens fluid dynamics using MRI: manipulation of steady state water content and water fluxes. Am J Physiol Regul Integr Comp Physiol 301: R335-R342, 2011. First published May 18, 2011 doi:10.1152/ajpregu.00173.2011.-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 (D2O). 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 2O, which acts as a contrast agent. Incubation in KCl-AAH and low T-AAH dramatically slowed the movement of D 2O into the core but did not affect the movement of D 2O into the outer cortex. D2O seemed to preferentially enter the lens cortex at the anterior and posterior poles before moving circumferentially toward the equatorial regions. This directionality of D 2O 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 2O 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. homeostasis; D 2O; water-protein gradient OUR VISION IS CRITICALLY DEPENDENT on our ability to focus light onto the retina. This refractive power is the combined result of the optical properties of the cornea and lens. While the cornea contributes most of the eye's refractive power, its focus is fixed (24). Young lenses, however, can adjust their focal power through the process of accommodation to elongate or shorten the focal length. As an optical element, the cornea normally contributes a positive spherical aberration to the light path, which is later corrected by the inherent negative spherical aberration of the ocular lens (2, 34). This negative spherical aberration is the result of a gradient in refractive index (21) that is produced by changes in the water-protein concentration ratio of the lens, such that water content is highest in the outside of the lens and lowest in the core (4). While this water-protein concentration ratio is known to create a smooth refractive index gradient that corrects for spherical aberration (26), how this gradient is maintained is unknown.The existence of a gradient in water content would be expected to generate a...

Citation: Vaghefi E, Kim A, Donaldson PJ. Active maintenance of the gradient of refractive index is required to sustain the optical properties of the lens. Invest Ophthalmol Vis Sci. 2015;56:7195-7208. DOI:10.1167/ iovs.15-17861 PURPOSE. To determine whether the cellular physiology of the lens actively maintains the optical properties of the lens and whether inhibition of lens transport affects overall visual quality. METHODS.One lens from a pair of bovine lenses was cultured in artificial aqueous humor (AAH), while the other was cultured in either AAH-High-K þ or AAH þ 0.1 mM ouabain for 4 hours. Lens pairs or whole enucleated eyes were then imaged in 4.7 Tesla (T) high-field small animal magnet. Lens surface curvatures, T1 measurements of water content, and T2 measurements of water/protein ratios were extracted from cultured lenses, while the geometrical parameters that define the optical pathway were obtained from whole eyes. Gradients of refractive index (GRIN), calculated from T2 measurements, and the extracted geometric parameters were inputted into optical models of the isolated lens and the whole bovine eye. RESULTS.Inhibiting circulating fluxes by inhibiting the Na/K-ATPase with ouabain or depolarization of the lens potential by High K þ caused changes to lens water content, the water/protein ratio (GRIN) and surface geometry that manifested as an increase in optical power and a decrease in negative spherical aberration in cultured lenses. Changes to optical properties of the lens resulted in a myopic shift that impaired vision quality in the optical model of the bovine eye.CONCLUSIONS. The cellular physiology of the lens actively maintains its optical properties and inhibiting the Na/K/ATPase induces a myopic shift in vision similar to that observed clinically in patients who go on to develop cataract.Keywords: lens physiology, magnetic resonance imaging, gradient of refractive index, physiological optics, optical modeling, cataract O ur sense of sight is critically dependent on the optical properties of the ocular lens that enables light to be focused onto the retina.1 Like a glass window, the lens allows light rays that enter the eye to pass through it with minimal scattering. However, the lens is more than a simple pane of glass, because its curved surfaces enable it to focus light. In addition, the lens needs to correct for positive spherical aberration introduced to the optical pathway via the cornea. This spherical aberration is an optical error caused by the increased refraction of light rays that strike the periphery of the cornea relative to those that strike its center.2 The lens compensates for this optical error by imposing and maintaining a compensating negative spherical aberration through the establishment an inherent gradient of refractive index (GRIN). 3Being a biological tissue, this gradient is generated by over expressing different subtypes of crystallin proteins with varying refractive indices, 4 thereby ensuring that incoming light is accurately focussed on the retina. While ...

PurposeSignal transduction pathways influence lens growth, but little is known about the role(s) of the class 1A phosphoinositide 3-kinases (PI3Ks). To further investigate how signaling regulates lens growth, we generated and characterized mice in which the p110α and p110β catalytic subunits of PI3K were conditionally deleted in the mouse lens.MethodsFloxed alleles of the catalytic subunits of PI3K were conditionally deleted in the lens by using MLR10-cre transgenic mice. Lenses of age-matched animals were dissected and photographed. Postnatal lenses were fixed, paraffin embedded, sectioned, and stained with hematoxylin-eosin. Cell proliferation was quantified by labeling S-phase cells in intact lenses with 5-ethynyl-2′-deoxyuridine. Protein kinase B (AKT) activation was examined by Western blotting.ResultsLens-specific deletion of p110α resulted in a significant reduction of eye and lens size, without compromising lens clarity. Conditional knockout of p110β had no effect on lens size or clarity, and deletion of both the p110α and p110β subunits resulted in a phenotype that resembled the p110α single-knockout phenotype. Levels of activated AKT were decreased more in p110α- than in p110β-deficient lenses. A significant reduction in proliferating cells in the germinative zone was observed on postnatal day 0 in p110α knockout mice, which was temporally correlated with decreased lens volume.ConclusionsThese data suggest that the class 1A PI3K signaling pathway plays an important role in the regulation of lens size by influencing the extent and spatial location of cell proliferation in the perinatal period.

It has been proposed that in the absence of blood supply, the ocular lens operates an internal microcirculation system that delivers nutrients to internalized fiber cells faster and more efficiently than would occur by passive diffusion alone. To visualize the extracellular space solute fluxes potentially generated by this system, bovine lenses were organ cultured in artificial aqueous humor (AAH) for 4 h in the presence or absence of two gadolinium-based contrast agents, ionic Gd(3+), or a chelated form of Gd(3+), Gd-diethylenetriamine penta-acetic acid (Gd-DTPA; mol mass = 590 Da). Contrast reagent penetration into the lens core was monitored in real time using inversion recovery-spin echo (IR-SE) magnetic resonance imaging (MRI), while steady-state accumulation of [Gd-DTPA](-2) was also determined by calculating T1 values. After incubation, lenses were fixed and cryosectioned, and sections were labeled with the membrane marker wheat germ agglutinin (WGA). Sections were imaged by confocal microscopy using standard and reflectance imaging modalities to visualize the fluorescent WGA label and gadolinium reagents, respectively. Real-time IR-SE MRI showed rapid penetration of Gd(3+) into the outer cortex of the lens and a subsequent bloom of signal in the core. These two areas of signal were separated by an area in the inner cortex that limited entry of Gd(3+). Similar results were obtained for Gd-DTPA, but the penetration of the larger negatively charged molecule into the core could only be detected by calculating T1 values. The presence of Gd-DTPA in the extracellular space of the outer cortex and core, but its apparent absence from the inner cortex was confirmed using reflectance imaging of equatorial sections. In axial sections, Gd-DTPA was associated with the sutures, suggesting these structures provide a pathway from the surface, across the inner cortex barrier to the lens core. Our studies have revealed inner and outer boundaries of a zone within which a narrowing of the extracellular space restricts solute diffusion and acts to direct fluxes into the lens core via the sutures.

Meta-analyses showed that flavonoids have a promising role in improving visual function in patients with glaucoma and ocular hypertension (OHT), and appear to play a part in both improving and slowing the progression of visual field loss.

It has been proposed that optical properties of the lens are actively maintained by an internal microcirculation system that utilizes ionic and fluid fluxes to deliver nutrients to deeper regions of the lens tissue via the extracellular space, faster than would occur by passive diffusion alone. To test this hypothesis, we utilized a range of commercially available MRI reagents of varying molecular sizes that served as tracers of extracellular solute delivery. The penetration of these tracers into bovine lenses incubated in the absence and presence of solutions that inhibit the microcirculation, was monitored in real time, over a period of 4 hours, using T1-weighted MRI. We found that only the smaller contrast agents were delivered to the core of the lens and that the rate of solute penetration was significantly faster than that calculated simple diffusion. Next, the lenses were first incubated in either high extracellular K to depolarize the lens potential, or ouabain to inhibit the Na pump. These two perturbations are known to inhibit the circulating ionic and fluid fluxes that are proposed to drive solute delivery into the lens core. Both perturbations inhibited the delivery of the extracellular tracer molecules to the lens core. Our findings suggest that the microcirculation system can be potentially harnessed to deliver exogenous antioxidants to the lens core to afford mature fiber cells protection against oxidative damage that ultimately manifests as age related nuclear cataract.

Although the functionality of the lens water channels aquaporin 1 (AQP1; epithelium) and AQP0 (fiber cells) is well established, less is known about the role of AQP5 in the lens. Since in other tissues AQP5 functions as a regulated water channel with a water permeability (P) some 20 times higher than AQP0, AQP5 could function to modulate P in lens fiber cells. To test this possibility, a fluorescence dye dilution assay was used to calculate the relative P of epithelial cells and fiber membrane vesicles isolated from either the mouse or rat lens, in the absence and presence of HgCl, an inhibitor of AQP1 and AQP5. Immunolabeling of lens sections and fiber membrane vesicles from mouse and rat lenses revealed differences in the subcellular distributions of AQP5 in the outer cortex between species, with AQP5 being predominantly membranous in the mouse but predominantly cytoplasmic in the rat. In contrast, AQP0 labeling was always membranous in both species. This species-specific heterogeneity in AQP5 membrane localization was mirrored in measurements of P, with only fiber membrane vesicles isolated from the mouse lens, exhibiting a significant Hg-sensitive contribution to P. When rat lenses were first organ cultured, immunolabeling revealed an insertion of AQP5 into cortical fiber cells, and a significant increase in Hg-sensitive P was detected in membrane vesicles. Our results show that AQP5 forms functional water channels in the rodent lens, and they suggest that dynamic membrane insertion of AQP5 may regulate water fluxes in the lens by modulating P in the outer cortex.