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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

Published: May 3rd, 2016

DOI:

10.3791/53986

1Department of Cell and Molecular Biology, The Scripps Research Institute

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<....

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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

  1. Euthanize mice according to recommendations in the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" and approved institution animal use protocols.......

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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 u.......

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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.......

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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).

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Name Company Catalog Number Comments
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

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