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

Summary

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.

Abstract

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.

Introduction

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 cells^1,2. 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 nucleus^1<....

Protocol

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. 
2. Enucleate the eye from mice using curved forceps. Depress the tissue around the eye with the forceps to bring the eye out of the socket, and then....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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
60 mm Petri dish	Fisher Scientific	0875713A
1x phosphate buffered saline (PBS)	Life Technologies	14190
18 x 18 mm 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