Name: sequential application of glass coverslips to assess the compressive stiffness of the mouse lens: strain and morphometric analyses
Uploaded: 2016-05-03T12:00:00
Description: Watch this Scientific Journal Video about Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses at JoVE.com

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).

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
<ol>
	<li>Euthanize mice according to recommendations in the National Institutes of Health &#34;Guide for the Care and Use of Laboratory Animals&#34; and approved institution animal use protocols...

<ol>
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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&#176;) with respect to the bottom of the chamber (&#952;). This will apply a very small component of the load equatorially rather than axially. However, this equatorial load is considered negligible because sin &#952; &#8776; 0.119. If this method is adapted for larger lenses, the angle of the coverslips to the bottom of the chamber would need...

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.

<table border="0" cellpadding="0" cellspacing="0" width="811">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:361px;">Fine tip straight forceps</td>
			<td style="width:139px;">Fine Scientific Tools</td>
			<td style="width:119px;">11252-40</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microdissection scissors, straight edge</td>
			<td style="width:139px;">Fine Scientific Tools</td>
			<td style="width:119px;">15000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved forceps</td>
			<td style="width:139px;">Fine Scientific Tools</td>
			<td style="width:119px;">11272-40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Seizing forceps</td>
			<td style="width:139px;">Hammacher</td>
			<td style="width:119px;">HSC 702-93</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection dish</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">12565154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60mm petri dish</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">0875713A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X phosphate buffered saline (PBS)</td>
			<td style="width:139px;">Life Technologies</td>
			<td style="width:119px;">14190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18mm x 18mm glass coverslips</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">12-542A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Measurement chamber with divots to hold lenses</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td>Custom-made (see Figure 1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Right-angle mirror</td>
			<td style="width:139px;">Edmund Optics</td>
			<td style="width:119px;">45-591</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light source</td>
			<td style="width:139px;">Schott/Fostec</td>
			<td style="width:119px;">8375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Illuminated dissecting microscope</td>
			<td style="width:139px;">Olympus</td>
			<td style="width:119px;">SZX-ILLD100</td>
			<td>With SZ-PT phototube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital camera</td>
			<td style="width:139px;">Nikon</td>
			<td style="width:119px;">Coolpix 990</td>
			<td></td>
		</tr>
	</tbody>
</table>

sequential application of glass coverslips to assess the compressive stiffness of the mouse lens: strain and morphometric analyses

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&#39; 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&#39; 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 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...

Watch this Scientific Journal Video about Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses at JoVE.com

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

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 ...

The overall goal of this procedure is to evaluate the compressive stiffness and geometry of mouse lenses using a simple and inexpensive approach. This method can answer key questions in the lens field. Such as how different genetic and experimental perturbations affect lens biomechanical properties.The main advantages of this technique are that it is precise, reproducible, and does not require expensive or specialized equipment. This method provides insight into the properties on mouse lenses, with potential application to larger model organisms such as rats and rabbits. Visualization of the method is critical because lens dissection and coverslip application steps are difficult to learn without demonstration.Demonstrating the procedure will be Catherine Cheng, a post doc from my laboratory. Begin the dissection using curved forceps to depress the tissue around the eye of a euthanized mouse bringing the eye out of the socket. Then, remove the eye and place it in a dissection dish containing fresh PBS under a dissecting microscope.Cut off the optic nerve as close to the eyeball as possible. And gently and carefully insert fine, straight tweezers into the eyeball through the hole where the optic nerve exits the posterior of the organ. Carefully make an incision with scissors from the posterior to the edge of the cornea, taking care not to damage the lens.Continue the incision along the junction between the cornea and the sclera, at least halfway around the eyeball. And gently push on the cornea to remove the lens from the eye through the opening. Then, use fine tipped straight forceps to carefully remove any large debris that is still attached to the lens.And visually confirm that the lens is free of damage. At least two hours before starting the stiffness measurements, use an analytical balance to weigh at least 10 cover slips from the same box. Determine the average weight of the cover slips.Then, pre wet the cover slips and a right angle mirror in room temperature PBS. While the materials are being hydrated, fill the measurement chamber with 65 to 75 milliliters of PBS. And place the chamber under a dissecting microscope set to a 30 X magnification, with illumination from the bottom and a fiber optic light source on the left and right sides.When the cover slips are ready, place the right angle mirror into the chamber at a constant distance from the divot that will be used to hold the lens. Next, using curved forceps, carefully transfer the dissected lenses to the measurement chamber. Set the power optic power supply to 80%of the maximum light intensity, and adjust the power supply output based on the ambient lighting, user preference, and picture quality as needed.Obtain top view images of the unloaded lenses from directly overhead of the chamber. Then, take a picture of the mirror edge in focus, followed by a side view image of the unloaded lens through the mirror outside of the divot. Then, place a lens into the divot, and confirm that the lens is seated securely and straight within the divot on it's anterior or posterior pole.Take a photo of the lens in the mirror. Then, gently place a cover slip onto the lens. After two minutes, take another side view image of the loaded lens.Continue adding cover slips, obtaining side view images two minutes after the addition of each cover slip, until the total of 10 cover slips have been applied. Then, remove all of the cover slips, wait a final two minutes, and obtain side view images of the lens inside and outside of the divot. And a top view image of the lens outside the divot.To determine the lens nucleus size, place a lens into a clean petri dish filled with PBS and use fine, straight forceps to gently decapsulate the lens. To off the cortical fiber cells, roll the lens between gloved fingers. Next, gently rinse the lens nucleus in the PBS and place the nucleus back into the measurement chamber.Acquire an image of the lens nucleus through the right angle mirror. Finally, to analyze the images, use the appropriate image analysis software to measure the equatorial and axial diameters of the lenses before and after each loading step. Measure the diameter of each lens nucleus as well.In this representative experiment, the stiffness and dimensions of two, four, and eight month old mouse lenses were measured and the axial and equatorial strains were calculated. The axial strain is a logarithmic function of applied load with a statistically significant, age dependent decrease in the axial and equatorial strains under the maximum applied load, indicating that the mouse lens stiffens with age. The image data collected during this experiment were also used to determine several other lens morphological characteristics.As expected, the axial and equatorial diameters, and the lens volume, increased with age. The aspect ratio indicates that the lens has a slightly larger equatorial diameter than axial diameter. And this parameter did not change with age.The diameter, volume, and fraction of the lens nucleus increase with age, suggesting that the lens nucleus remodels to increase in relative size as the lens ages. Once mastered, this technique can be completed in 30 to 45 minutes per lens, if performed properly. While attempting this procedure, it's important to position the lens evenly inside the divot, and to allow the lens to stress relax for two minutes between each additional cover slip.After watching this video, you should have a good understanding of how to assess the compressive stiffness, and morphometrics of mouse lenses using the sequential application of cover slips. This precise, simple, and inexpensive technique paves the way for lens researchers to explore the biomechanical effects of gene mutations in mice.

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