.
Abstract
Life-long eye lens function requires an appropriate gradient refractive index, biomechanical integrity and transparency. We conducted an extensive study of wild-type mouse lenses 1-30 months of age to define common age-related changes. Biomechanical testing and morphometrics revealed an increase in lens volume and stiffness with age. Lens capsule thickness and peripheral fiber cell widths increased between 2 to 4 months of age but not further, and thus, cannot account for significant age-dependent increases in lens stiffness after 4 months. In lenses from mice older than 12 months, we routinely observed cataracts due to changes in cell structure, with anterior cataracts due to incomplete suture closure and a cortical ring cataract corresponding to a zone of compaction in cortical lens fiber cells. Refractive index measurements showed a rapid growth in peak refractive index between 1 to 6 months of age, and the area of highest refractive index is correlated with increases in lens nucleus size with age. These data provide a comprehensive overview of age-related changes in murine lenses, including lens size, stiffness, nuclear fraction, refractive index, transparency, capsule thickness and cell structure. Our results suggest similarities between murine and primate lenses and provide a baseline for future lens aging studies.
Introduction
The eye lens is required for fine focusing of light onto the retina to form a clear image, and the function of the lens is intimately tied to its shape, biomechanical properties, transparency and refractive index. It has long been known that age-related changes in these lens properties lead to two major lens pathologies, cataracts and presbyopia [1]. Cataracts, defined as any lens opacity, are the leading cause of blindness in the world [2], and almost all mammalian and avian species develop age-related cataracts [3–15]. Changes in lens transparency occur in several locations, cortical, subcapsular and nuclear, and there are many hypothesized causes for opacities, including UV light exposure, reactive oxygen species, nutrition and genetic variations [16–18]. Though previous studies have demonstrated that wild-type mouse lenses develop age-related cataracts [10, 12–15, 19, 20], the age at which defects appear and the mechanisms for opacities in different locations in lenses from aged mice has not been thoroughly studied. Presbyopia is caused by a reduction in the lens’ ability to change shape during focusing (accommodation), and, by extension, the need for reading glasses [21–23]. Studies have linked age-related increases in lens stiffness to presbyopia [21–26], and increased lens stiffness with age has been reported in humans [21, 26–34] and animal models [34–36], including mice [37–41].
The lens is composed of two cell types, a monolayer of epithelial cells covering the anterior hemisphere and a bulk mass of differentiated lens fiber cells (Supplementary Figure 1). A basement membrane, called the capsule, encapsulates the lens [42]. Life-long lens growth occurs through the proliferation, elongation and differentiation of equatorial epithelial cells into new fiber cells that are added in concentric layers surrounding previous generations of fibers [43–45]. Hexagonal packing of lens fibers is established by cell reorganization and shape changes in equatorial epithelial cells [46, 47]. High refractive index in the lens is established by the high concentration and short range order of crystallin proteins [48, 49]. Previous studies, including our work, suggest that cytoskeletal structures are important to maintain lens mechanical integrity [38, 40, 50, 51]. The lens is a unique organ where all the cells are retained since the formation of the tissue during embryonic development, and there is little or no protein turnover in cells at the center of the lens [42]. This presents a rare opportunity to study cellular aging by allowing comparison of cells made in the embryo vs. cells added during in adulthood vs. cells formed in old age.
Little is known about the morphological, mechanical, refractive and cellular changes that occur with advanced age in the lens. Mice offer an opportunity to investigate changes in lens morphometrics, stiffness, transparency and refractive properties with age in a relatively shortened period of time. We carried out a comprehensive study of the properties of mouse lenses from young adult mice (1–2 months old) to very old mice (24–30 months old), measuring size, Gradient Refractive Index (GRIN) and stiffness. We also determined that anterior subcapsular and cortical ring cataracts appear in wild-type mouse lenses around 12 months of age. Confocal and electron microscopy revealed morphological changes in lens fiber cells that are correlated with these changes in lens transparency, suggesting a cellular basis for these age-related cataracts. In all, we demonstrate that age-related changes in mouse lenses mimic some aspects of aging in human lenses.
Results
Mouse lenses have increased size, nuclear fraction, stiffness and resilience with age
We first examined the morphometrics of mouse lenses between 2–30 months of age (Figure 1). We measured the axial (Figure 1, red double-headed arrows) and equatorial diameters (Figure 2, green double-headed) arrows for each lens to calculate lens volume, lens aspect ratio, nuclear volume and biomechanical properties. We observed that lenses from very old mice (24–30 months) have an optical discontinuity in the lens cortex (Figure 1, yellow arrowheads). For morphometric measurements, we used dot plots with lines to show the average and standard deviation, and also included a graph for each parameter of the Tukey 95% confidence interval from the statistical analysis, to assess the significance of the multiple comparisons between the age groups. The horizontal lines representing each comparison are statistically significant if they do not cross the vertical dotted line (95% confidence), which is similar in concept to a p-value less than 0.05. We found that lenses steadily increased in volume between 2 to 8 months of age, with growth slowing down between 8 to 18 months of age (Figures 1 and 2A). Mouse lenses do not increase significantly in volume after 18 months of age. The equatorial diameter of lenses does not increase significantly after 8 months of age while axial diameter increases slightly until 18 months of age (data not shown). Mouse lenses are nearly spherical in shape, becoming slightly more spherical between 2 and 4 months of age (Figure 2B). After 4 months, the lens aspect ratio does not change significantly with age. Lens fiber cells at the center, or nucleus, of the lens become compacted [21, 52], and in mouse lenses, the nucleus is a hard and spherical structure that can be isolated by removing the softer cells of the lens cortex [38, 41]. In contrast to the constant lens volume after 18 months of age, the size of the nucleus continues to increase with age up to 30 months of age (Figures 1 and 3). While the rate of increase is slow (no change from 8 to 12 months, or from 18 to 24 months), the size of the nucleus increases significantly between 24 to 30 months of age. These data suggest that while overall lens growth slows down, the remodelling and compaction of the nucleus continues in the mouse lens with age.
Figure 1. Side view pictures of mouse lenses between 2-30 months of age pre-compression, during coverslip compression (1, 5 and 10 coverslips) and post-compression, and the isolated lens nucleus. With age, the application of the same load compressed the older lenses less than young lenses. There is an overall increase in lens size and nucleus size with age. The axial diameter (red double-headed arrows) and the equatorial diameter (green double-headed arrows) for each lens were measured to calculate lens volume, lens aspect ratio, axial compressive strain, equatorial expansion strain, resilience and nuclear volume. In very old lenses (24-30 months), there is an area of optical discontinuity in the lens cortex (yellow arrowheads). Scale bar, 1mm.
.../...
F O R T H E R E S T O F T H E S T U D Y, P L E A S E V I S I T T H E S O U R C E .
.
