Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.
The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the lens, leading to an increase in the lens' optical power to focus on nearby objects, a process known as accommodation. Age-related changes in lens stiffness have been linked to presbyopia, a reduction in the lens' ability to accommodate, and, by extension, the need for reading glasses. Even though mouse lenses do not accommodate or develop presbyopia, mouse models can provide an invaluable genetic tool for understanding lens pathologies, and the accelerated aging observed in mice enables the study of age-related changes in the lens. This protocol demonstrates a simple, precise, and cost-effective method for determining mouse lens stiffness, using glass coverslips to apply sequentially increasing compressive loads onto the lens. Representative data confirm that mouse lenses become stiffer with age, like human lenses. This method is highly reproducible and can potentially be scaled up to mechanically test lenses from larger animals.
The lens is a transparent and avascular organ in the anterior chamber of the eye that is responsible for fine focusing of light onto the retina. A clear basement membrane, called the lens capsule, surrounds a bulk of elongated fiber cells covered by an anterior monolayer of epithelial cells1,2. Life-long growth of the lens depends on the continuous proliferation and differentiation of epithelial cells at the lens equator into new fiber cells that are added onto previous generations of fiber cells in a concentric manner2. Over time, lens fiber cells undergo compaction, resulting in a rigid center in the middle of the lens called the nucleus1. Accommodation, defined as a dioptric change in the optical power of the eye, occurs in humans when the ciliary muscles contract to deform the lens and allow a true increase in optical power to focus on near objects3-5. In the unaccommodated eye, the lens is held in a relatively flattened state due to tension from zonular fibers. When the ciliary muscles contract, the tension on the lens is released, leading to decreased lens equatorial diameter and increased axial thickness. Age-related changes in the lens cause presbyopia, a progressive loss of lens accommodation, which leads to the need for reading glasses.
Several studies have linked presbyopia to age-related increase in the intrinsic stiffness of the lens6-11. Stiffness is defined as the resistance of an elastic object to deform under applied load. A variety of methods have been used to examine stiffness of human lenses, including spin compression12-14, actuator compression15, probe indentation16, dynamic mechanical analysis 6,10 and bubble-based acoustic radiation force17. While mouse lenses do not accommodate or develop presbyopia, mouse models for lens pathologies are valuable tools because mice are less expensive than larger animals, well characterized genetically and undergo accelerated age-related changes due to rapid aging. A handful of studies have examined mouse lens stiffness with compression methods and demonstrated changes in lens stiffness due to aging or targeted genetic disruptions18-21. Thus, mouse lenses are good models for studying age-related changes in lens stiffness.
This protocol describes a simple and inexpensive, yet precise and reproducible, compression method for determining mouse lens stiffness based on application of glass coverslips onto the lens in conjunction with photographing the lens through a dissection microscope and mirror. This method yields robust strain and morphometric data with an easily fabricated and assembled apparatus. The representative results confirm that mouse lenses increase in stiffness with age.
All animal procedures were performed in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health and under an approved protocol by the Institutional Animal Care and Use Committee at The Scripps Research Institute.
1. Lens Dissection
2. Stiffness Measurements
Figure 1: Stiffness Measurement Chamber. A photo showing the dimensions of the custom-made stiffness measurement chamber with a variety of divots of different depths and shapes. The round divots that are 200 µm or 300 µm deep (yellow arrowheads) are used for the measurements on mouse lenses. Divots are 2 mm in diameter and ~13 – 14 mm from the edge of the chamber. Please click here to view a larger version of this figure.
3. Lens Nucleus Measurement
Figure 2: A Mouse Lenses Compressed by Coverslips. (A) Schematic and (B) photograph of the experimental setup showing a 2-month-old mouse lens in a 200-µm-deep divot in the measurement chamber filled with 1x PBS. A right angle mirror and a digital camera mounted on a dissection microscope were used to collect images of the lens during compression by coverslips. (C) Photos of sagittal views of a 2-month-old wild-type lens compressed by successively increasing numbers of coverslips provided the raw data for measuring axial and equatorial diameters and calculating axial and equatorial strains during coverslip-based compression testing. A reflection of the lens can sometimes be seen in the coverslips (most clearly visible in the 1 coverslip image). When making measurements, ignore the reflection and measure to the apex of the lens. (D) Photos of sagittal views of the 2-month-old wild-type lens post-compression and the isolated lens nucleus. The post-compression lens and isolated nucleus are sitting outside of the divot. Scale bars, 1 mm. This figure is modified from Gokhin, et al. PloS one, 201219. Please click here to view a larger version of this figure.
4. Image Analysis
The stiffness and dimensions of 2-, 4- and 8-month-old mouse lenses were measured. Mice were all wild-type animals on a pure C57BL6 strain background obtained from the TSRI Animal Breeding Facility, and each lens was loaded with 1 to 10 coverslips. The axial and equatorial strains were calculated as a function of applied load by measuring the axial and equatorial diameters of the lens after the addition of each coverslip, and then normalizing each change in diameter to the corresponding unloaded diameter. Eight lenses from each age were tested, and the results are expressed as the average ± standard error. As shown previously19, axial strain is a logarithmic function of applied load (Figure 3A). There was a statistically significant age-dependent decrease in axial and equatorial strains under the maximum applied load (Figure 3), indicating that the mouse lens stiffens with age. Strain measurements were highly reproducible across lenses of the same age, as demonstrated by the small standard errors.
The image data collected during this experiment were also used to determine several other lens morphological characteristics (Figure 4). As expected, axial and equatorial diameters and lens volume increased with age (Figure 4A, 4B and 4D). The aspect ratio indicates that the lens has a slightly larger equatorial diameter than axial diameter, and this parameter did not change with age (Figure 4C). The diameter, volume and fraction of the lens nucleus increased with age (Figure 4E, 4F and 4G). These results suggest that the lens nucleus remodels to increase in relative size as the lens ages.
This data show that mouse lenses increase in stiffness with age, similar to changes in aging human lenses9,15. These data also agree with previous observations made using a similar method18 and by Brillouin optical microscopy23 that mouse lenses increase in stiffness with age. Two other studies have used the described method to show that tropomodulin-1, an actin pointed-end capping protein, CP49, a beaded intermediate filament protein, and aquaporin 0 are needed to maintain lens stiffness19,20. With this method, the plethora of mouse models for lens pathologies and the accelerated aging of mice can be used to understand lens stiffness changes due to genetic variation and/or aging. This method can also be adapted for lenses from other species. The dimensions of the chamber used for these experiments are optimized for mouse lenses, but can easily be scaled up for lenses from larger species. In the future, it would be interesting to determine whether lens size scales with lens stiffness across species.
At the lowest load (1 coverslip, 129.3 mg), there was no obvious difference in axial and equatorial strains between 2-, 4- and 8-month-old lenses. The increase in lens stiffness with age became more apparent at higher loads. It is possible that the outermost cortex of the lens (~100 µm) that is compressed by first coverslip does not change in stiffness with age, or that this method is unable to distinguish small changes in strain at low loads. The morphometrics data indicate that increases in both the whole lens and nuclear volume correlate with increased lens stiffness as a function of age.
Figure 3: Axial and Equatorial Strain-load Curves for 2-, 4- and 8-month-old (2M, 4M and 8M) Wild-type Mouse Lenses. (A) Axial compressive strain plotted as a function of the applied load (mg). (B) Equatorial compressive strain plotted as a function of the applied load (mg). Four- and 8-month-old lenses exhibited less strain than 2-month-old lenses at equivalent maximum loads, indicating an increase in lens stiffness with age. **, p <0.01. Note that the Y-axis is different between (A) and (B). Please click here to view a larger version of this figure.
Figure 4: Morphological Characteristics of 2-, 4- and 8-month-old (2M, 4M and 8M) Wild-type Mouse Lenses. (A) Axial diameter and (B) equatorial diameter progressively increase with age. The lens aspect ratio (C) shows that mouse lenses are slightly wider at the lens equator. Lens volume (D), nuclear diameter (E), nuclear volume (F) and nuclear fraction (G) progressively increase with age. **, p <0.01. Please click here to view a larger version of this figure.
There are several key considerations when using this method to measure lens stiffness. First, the coverslips are applied to the lens at a slightly oblique angle (8 – 8.5°) with respect to the bottom of the chamber (θ). This will apply a very small component of the load equatorially rather than axially. However, this equatorial load is considered negligible because sin θ ≈ 0.119. If this method is adapted for larger lenses, the angle of the coverslips to the bottom of the chamber would need to be measured to determine whether equatorial load should be factored into strain calculations. Second, it is crucial to allow the lens to equilibrate after the addition of each coverslip. The waiting period of 2 minutes allows time-dependent deformation (i.e., creep) to occur, such that pictures are only taken when the lens is at an equilibrium shape19. Third, this protocol is optimized for measuring compressive strain on mouse lenses across a wide dynamic range of loads. In pilot studies, an applied load of 1,293 mg (i.e., ten 18 x 18 mm coverslips) compressed the mouse lens to a maximum strain, beyond which increased loads do not cause appreciable further deformation. This is due to the presence of the rigid lens nucleus that does not deform appreciably under compression19. Fourth, this protocol avoids irreversible tissue damage. In previously published experiments, no changes in the mechanical properties of mouse lenses were observed upon repeat loading, suggesting that this method does not damage the lens19. When mechanically testing lenses of a different species or mutant lenses, pilot tests should be done to determine the maximum load needed for maximum strain by repeating steps 2.8 – 2.10 and comparing the strain-load curves, lens diameters and lens volumes between the first and second loading. Finally, this method provides an empirical measurement of the stiffness of the whole lens and cannot differentiate the contributions of different cell types (epithelial cells, cortical fibers, nuclear fibers) and the lens capsule to whole-lens mechanical properties.
The axial and equatorial strains are plotted here as functions of the imposed load. Previous studies have quantified mouse lens stiffness19,21, resilience21 or the change in diameter18 upon applied load. Strain is a dimensionless quantity that allows direct comparison between lenses of different sizes. Note that equatorial extension (positive strain) occurs concurrently with the applied axial compression (negative strain) due to conservation of lens volume (i.e., Poisson effect). However, the observed equatorial strains were much smaller in absolute magnitude than the axial strains, indicating that this method has less resolution to detect small changes in equatorial strain as compared to axial strain.
In summary, this simple method with an easily assembled device for measuring mouse lens stiffness can be applied generally and widely in lens research to better understand how mutations in proteins, pathologies and/or aging affect lens stiffness. While mouse lenses do not accommodate, this method can still elucidate the proteins and age-related modifications that contribute to increased lens stiffness, and potentially contribute new knowledge to developing novel treatments for presbyopia.
The authors have nothing to disclose.
This work was supported by National Eye Institute Grant R01 EY017724 (VMF) and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant K99 AR066534 (DSG).
Fine tip straight forceps | Fine Scientific Tools | 11252-40 | |
Microdissection scissors, straight edge | Fine Scientific Tools | 15000-00 | |
Curved forceps | Fine Scientific Tools | 11272-40 | |
Seizing forceps | Hammacher | HSC 702-93 | Optional |
Dissection dish | Fisher Scientific | 12565154 | |
60mm petri dish | Fisher Scientific | 0875713A | |
1X phosphate buffered saline (PBS) | Life Technologies | 14190 | |
18mm x 18mm glass coverslips | Fisher Scientific | 12-542A | |
Measurement chamber with divots to hold lenses | Custom-made (see Figure 1) | ||
Right-angle mirror | Edmund Optics | 45-591 | |
Light source | Schott/Fostec | 8375 | |
Illuminated dissecting microscope | Olympus | SZX-ILLD100 | With SZ-PT phototube |
Digital camera | Nikon | Coolpix 990 |