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.&#160;</li>
	<li>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 pluck the eye from the socket with the forceps. Transfer eyes to fresh 1x phosphate buffered saline (PBS) in dissection dish.</li>
	<li>Cut off the optic nerve as close to the eyeball as possible. Gently and carefully insert fine straight tweezers into the eyeball through the hole where the optic nerve exits the posterior.</li>
	<li>Carefully make an incision with scissors into the eyeball from the posterior to the edge of the cornea. Rodent lenses occupy ~30% of the eye. Make these incisions carefully, and do not insert tweezers or scissors too deep into the eye to avoid damaging the lens.</li>
	<li>Cut along the junction between the cornea and sclera at least half way around the eyeball.</li>
	<li>Gently push on the cornea to remove the lens from the eye through the opening made in steps 1.4 and 1.5.</li>
	<li>Use fine tip straight forceps to carefully remove any large debris that is still attached to the lens. Visually inspect the lens for any damage before proceeding to the stiffness measurements.</li>
</ol>
2. Stiffness Measurements
<ol>
	<li>Weigh at least 10 coverslips from the same box using an analytical balance. Find the average weight of the coverslips. For consistency, use the same box of coverslips for all experiments. Pre-wet the coverslips and right-angle mirror in 1x PBS at room temperature for at least 2 hr before starting experiments.</li>
	<li>Fill the measurement chamber (see Figure 1) with 65 - 75 ml of 1x PBS. The measurement chamber was made out of Plexiglas by an in-house machine shop, and divots in the chamber were made by a drill press set to the desired depth with an appropriate drill bit. &#160;Lenses remain transparent in 1x PBS at room temperature for the duration of mechanical testing.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53986/53986fig1.jpg" /> 
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 &#181;m or 300 &#181;m 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. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53986/53986fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Place right-angle mirror into the chamber at a constant distance from the divot that will be used to hold the lens. Make sure the mirror does not move during the experiment.</li>
	<li>Transfer dissected lenses to measurement chamber carefully with seizing forceps or curved forceps.</li>
	<li>Take a top-view picture of the unloaded lens from directly overhead. Take mouse lens photos at 30X magnification with illumination from dissecting microscope (bottom) and a fiber optic light source on the left and right sides. Set the fiber optic power supply to 80% of the maximum light intensity. Adjust the power supply output based on ambient lighting, user preference and picture quality as needed.</li>
	<li>Take a side-view picture of the unloaded lens, which can be seen through the right-angle mirror. If camera is not calibrated, take a picture of the mirror edge in focus. The mirror edge is 5 mm long, and this measurement can later be used to determine the pixels/mm and serve as a scale bar in the images.</li>
	<li>Place lens into the divot, and confirm that the lens is seated securely and straight in the divot. Take a picture of the lens before loading. The lens should be resting in the divot on its anterior or posterior pole.</li>
	<li>Place 1 coverslip gently onto the lens. Wait 2 min to permit creep, and take another side-view picture of the loaded lens.</li>
	<li>Continue adding coverslips as in step 2.8 and taking side-view pictures after the addition of each coverslip as in step 2.8 until a total of 10 coverslips are applied.</li>
	<li>Remove all coverslips. Wait 2 min, and take a side-view picture of the lens (inside and outside of the divot) after removing all coverslips.</li>
</ol>
3. Lens Nucleus Measurement
<ol>
	<li>To determine the lens nucleus size, move the lens to a clean Petri dish filled with 1x PBS.</li>
	<li>Gently decapsulate the lens using fine straight forceps.</li>
	<li>Slough off the cortical fiber cells by rolling the lens between gloved fingers. The remaining lens nucleus will feel like a hard marble. Use this procedure to isolate the nucleus on adult lenses starting at 1 month of age. Since the isolated nucleus is a rigid body, further mechanical testing of the lens nucleus cannot be performed using this described method.</li>
	<li>Gently rinse the lens nucleus in 1x PBS in the Petri dish.</li>
	<li>Place the lens nucleus back into the measurement chamber (not in the divot), and take an image of the lens nucleus through the right-angle mirror.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53986/53986fig2.jpg" /> 
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-&#181;m-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.&#160;(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. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53986/53986fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Image Analysis
<ol>
	<li>Measure equatorial and axial diameters of lenses before loading and after each loading step using ImageJ or similar software. Measure the diameter of each lens nucleus. The lens nucleus is nearly spherical so a measurement at any orientation will suffice19,21.</li>
	<li>Correct the lens axial diameters by adding the depth of the divot used. In the measurement chamber, the divot obscured 200 &#181;m (2-month-old mouse lenses) or 300 &#181;m (4-month-old and 8-month-old mouse lenses) of the axial thickness of the lens.</li>
	<li>Calculate the axial and equatorial strains from the lens diameter measurements using the equation, &#949; = (d - d0) / d0, where &#949; is strain, d is the axial or equatorial diameter at a given load, and d0 is the corresponding axial or equatorial diameter at zero load.</li>
	<li>Plot the axial and equatorial strains as functions of the imposed load (in mg).</li>
	<li>Plot the axial, equatorial and nuclear diameters. Calculate and plot the lens aspect ratio by dividing the axial diameter by the equatorial diameter.</li>
	<li>Calculate and plot the lens volume using the equation, volume = 4/3 &#215; &#960; &#215; rE2&#160;&#215; rA, where rE is the equatorial radius and rA is the axial radius measured from the picture taken in step 2.6. This equation assumes the lens is an oblate spheroid (ellipsoid)1,22.</li>
	<li>Calculate and plot the nuclear volume using the equation, volume = 4/3 &#215; &#960; &#215; rN3, where rN is the radius of the lens nucleus as measured from the picture taken in step 3.5. This equation assumes the lens nucleus is a sphere19,21.</li>
	<li>Calculate and plot the nuclear fraction as the ratio of the nuclear volume to the lens volume.</li>
</ol>

<ol>
	<li>Lovicu, F. J., Robinson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Development of the ocular lens. , (2004).</li><li>Piatigorsky, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lens+differentiation+in+vertebrates.+A+review+of+cellular+and+molecular+features.">Lens differentiation in vertebrates. A review of cellular and molecular features.</a> Differentiation. 19 (3), 134-153 (1981).</li><li>Glasser, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+accommodation:+surgical+options+for+correction+of+presbyopia.">Restoration of accommodation: surgical options for correction of presbyopia.</a> Clin Exp Optom. 91 (3), 279-295 (2008).</li><li>Keeney, A. H., Hagman, R. E., Fratello, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dictionary of ophthalmic optics. , (1995).</li><li>Millodot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dictionary of optometry and visual science. 7, (2009).</li><li>Heys, K. R., Cram, S. L., Truscott, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massive+increase+in+the+stiffness+of+the+human+lens+nucleus+with+age:+the+basis+for+presbyopia.">Massive increase in the stiffness of the human lens nucleus with age: the basis for presbyopia.</a> Mol Vis. 10, 956-963 (2004).</li><li>Heys, K. R., Friedrich, M. G., Truscott, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presbyopia+and+heat:+changes+associated+with+aging+of+the+human+lens+suggest+a+functional+role+for+the+small+heat+shock+protein,+alpha-crystallin,+in+maintaining+lens+flexibility.">Presbyopia and heat: changes associated with aging of the human lens suggest a functional role for the small heat shock protein, alpha-crystallin, in maintaining lens flexibility.</a> Aging Cell. 6 (6), 807-815 (2007).</li><li>Pierscionek, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+response+of+human+lenses+to+stretching+forces.">Age-related response of human lenses to stretching forces.</a> Exp Eye Res. 60 (3), 325-332 (1995).</li><li>Glasser, A., Biometric Campbell, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=optical+and+physical+changes+in+the+isolated+human+crystalline+lens+with+age+in+relation+to+presbyopia.">optical and physical changes in the isolated human crystalline lens with age in relation to presbyopia.</a> Vision Res. 39 (11), 1991-2015 (1999).</li><li>Weeber, H. A., van der Heijde, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+relationship+between+lens+stiffness+and+accommodative+amplitude.">On the relationship between lens stiffness and accommodative amplitude.</a> Exp Eye Res. 85 (5), 602-607 (2007).</li><li>Weeber, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+mechanical+properties+of+human+lenses.">Dynamic mechanical properties of human lenses.</a> Exp Eye Res. 80 (3), 425-434 (2005).</li><li>Fisher, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+properties+of+the+human+lens.">Elastic properties of the human lens.</a> Exp Eye Res. 11 (1), 143 (1971).</li><li>Krueger, R. R., Sun, X. K., Stroh, J., Myers, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+increase+in+accommodative+potential+after+neodymium:+yttrium-aluminum-garnet+laser+photodisruption+of+paired+cadaver+lenses.">Experimental increase in accommodative potential after neodymium: yttrium-aluminum-garnet laser photodisruption of paired cadaver lenses.</a> Ophthalmology. 108 (11), 2122-2129 (2001).</li><li>Burd, H. J., Wilde, G. S., Judge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+spinning+lens+test+to+determine+the+stiffness+of+the+human+lens.">An improved spinning lens test to determine the stiffness of the human lens.</a> Exp Eye Res. 92 (1), 28-39 (2011).</li><li>Glasser, A., Campbell, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presbyopia+and+the+optical+changes+in+the+human+crystalline+lens+with+age.">Presbyopia and the optical changes in the human crystalline lens with age.</a> Vision Res. 38 (2), 209-229 (1998).</li><li>Pau, H., Kranz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+increasing+sclerosis+of+the+human+lens+with+age+and+its+relevance+to+accommodation+and+presbyopia.">The increasing sclerosis of the human lens with age and its relevance to accommodation and presbyopia.</a> Graefes Arch Clin Exp Ophthalmol. 229 (3), 294-296 (1991).</li><li>Hollman, K. W., O'Donnell, M., Erpelding, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+elasticity+in+human+lenses+using+bubble-based+acoustic+radiation+force.">Mapping elasticity in human lenses using bubble-based acoustic radiation force.</a> Exp Eye Res. 85 (6), 890-893 (2007).</li><li>Baradia, H., Nikahd, N., Glasser, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+lens+stiffness+measurements.">Mouse lens stiffness measurements.</a> Exp Eye Res. 91 (2), 300-307 (2010).</li><li>Gokhin, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tmod1+and+CP49+synergize+to+control+the+fiber+cell+geometry,+transparency,+and+mechanical+stiffness+of+the+mouse+lens.">Tmod1 and CP49 synergize to control the fiber cell geometry, transparency, and mechanical stiffness of the mouse lens.</a> PLoS One. 7 (11), e48734 (2012).</li><li>Sindhu Kumari, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Aquaporin+0+in+lens+biomechanics.">Role of Aquaporin 0 in lens biomechanics.</a> Biochem Biophys Res Commun. , (2015).</li><li>Fudge, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermediate+filaments+regulate+tissue+size+and+stiffness+in+the+murine+lens.">Intermediate filaments regulate tissue size and stiffness in the murine lens.</a> Invest Ophthalmol Vis Sci. 52 (6), 3860-3867 (2011).</li><li>Kuszak, J. R., Mazurkiewicz, M., Zoltoski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+modeling+of+secondary+fiber+development+and+growth:+I.+Nonprimate+lenses.">Computer modeling of secondary fiber development and growth: I. Nonprimate lenses.</a> Mol Vis. 12, 251-270 (2006).</li><li>Scarcelli, G., Kim, P., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+age-related+stiffening+in+the+crystalline+lens+by+Brillouin+optical+microscopy.">In vivo measurement of age-related stiffening in the crystalline lens by Brillouin optical microscopy.</a> Biophys J. 101 (6), 1539-1545 (2011).</li></ol>

The authors have nothing to disclose.

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><tbody><tr><td>Fine tip straight forceps</td><td>Fine Scientific Tools</td><td>11252-40</td><td></td></tr><tr><td>Microdissection scissors, straight edge</td><td>Fine Scientific Tools</td><td>15000-00</td><td></td></tr><tr><td>Curved forceps</td><td>Fine Scientific Tools</td><td>11272-40</td><td></td></tr><tr><td>Seizing forceps</td><td>Hammacher</td><td>HSC 702-93</td><td>Optional</td></tr><tr><td>Dissection dish</td><td>Fisher Scientific</td><td>12565154</td><td></td></tr><tr><td>60 mm Petri dish</td><td>Fisher Scientific</td><td>0875713A</td><td></td></tr><tr><td>1x phosphate buffered saline (PBS)</td><td>Life Technologies</td><td>14190</td><td></td></tr><tr><td>18 x 18 mm glass coverslips</td><td>Fisher Scientific</td><td>12-542A</td><td></td></tr><tr><td>Measurement chamber with divots to hold lenses</td><td></td><td></td><td>Custom-made (see &lt;strong&gt;Figure 1&lt;/strong&gt;)</td></tr><tr><td>Right-angle mirror</td><td>Edmund Optics</td><td>45-591</td><td></td></tr><tr><td>Light source</td><td>Schott/Fostec</td><td>8375</td><td></td></tr><tr><td>Illuminated dissecting microscope</td><td>Olympus</td><td>SZX-ILLD100</td><td>With SZ-PT phototube</td></tr><tr><td>Digital camera</td><td>Nikon</td><td>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 ...

sequential-application-glass-coverslips-to-assess-compressive

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

7.2K Views.  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.

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

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

Cancer Research

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved through the contraction of the ciliary body and relaxation of zonule fibers, which results in the thickening of the lens necessary for near vision. Here, we present a novel, simple method in which accommodation is replicated by tensing the zonules connected to the lens capsule via a manual lens stretcher (MLS). This method monitors the radial stretching achieved by a lens when subjected to a consistent force and allows for a comparison of accommodating lenses, which can be stretched, to non-accommodating lenses, which cannot be stretched. Importantly, the stretcher couples to the zonules directly, and not to the sclera of the eye, thus only requiring the lens, zonules, and ciliary body rather than the entire globe sample. This difference can significantly decrease the cost of acquiring donor cadaver lenses by about 62% compared to acquiring an entire globe.

We present an efficient method of studying lens accommodation by using a manual lens stretcher. The protocol mimics physiological accommodation by pulling the zonules connected around the lens capsule, thereby, stretching the lens.

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

Bioengineering

Immunofluorescence Staining of Membrane Interdigitations in Lens Fiber Cells

Preparation and Immunofluorescence Staining of Bundles and Single Fiber Cells from the Cortex and Nucleus of the Eye Lens

The lens is a transparent and ellipsoid organ in the anterior chamber of the eye that changes shape to finely focus light onto the retina to form a clear image. The bulk of this tissue comprises specialized, differentiated fiber cells that have a hexagonal cross section and extend from the anterior to the posterior poles of the lens. These long and skinny cells are tightly opposed to neighboring cells and have complex interdigitations along the length of the cell. The specialized interlocking structures are required for normal biomechanical properties of the lens and have been extensively described using electron microscopy techniques. This protocol demonstrates the first method to preserve and immunostain singular as well as bundles of mouse lens fiber cells to allow the detailed localization of proteins within these complexly shaped cells. The representative data show staining of the peripheral, differentiating, mature, and nuclear fiber cells across all regions of the lens. This method can potentially be used on fiber cells isolated from lenses of other species.

Author Spotlight: Advancing Lens Biomechanics Research Through a Novel Protocol for Imaging Complex Interdigitations and Protein Staining 

This protocol describes methods to prepare peripheral, mature, and nuclear eye lens fiber cells for immunofluorescence staining to study complex cell-to-cell interdigitations and the membrane architecture.

The lens is a transparent and ellipsoid organ in the anterior chamber of the eye that changes shape to finely focus light onto the retina to form a clear ...

Developmental Biology

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Biomechanical Characterization of Porcine Ocular Lens with Automated Compression

Automated Compression Testing of the Ocular Lens

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically with age in the human lens, resulting in a loss of near vision called presbyopia. However, the mechanisms of these changes remain unknown. Lens compression offers a relatively simple method for assessing the lens' biomechanical stiffness in a qualitative sense and, when coupled with appropriate analytical techniques, can help quantify biomechanical properties. A variety of lens compression tests have been performed to date, including both manual and automated, but these methods inconsistently apply key aspects of biomechanical testing such as preconditioning, loading rates, and time between measurements. This paper describes a fully automated lens compression test wherein a motorized stage is synchronized with a camera to capture the force, displacement, and shape of the lens throughout a preprogrammed loading protocol. A characteristic elastic modulus may then be calculated from these data. While demonstrated here using porcine lenses, the approach is appropriate for the compression of lenses of any species.

Author Spotlight: Advancing Understanding of Age-Related Lens Stiffness Changes 

We present an automated method for characterizing the effective elastic modulus of an ocular lens using a compression test.

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...

Neuroscience

Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular matrix (ECM), the protein meshwork that binds cells and tissues together. How the elastic properties of an ECM network relate to its composition, and whether the relaxation properties of the ECM play a physiological role, are questions that have yet to be fully addressed. Part of the challenge lies in the complex architecture of most ECM systems and the difficulty in isolating ECM components without compromising their structure. One exception is the zonule, an ECM system found in the eye of vertebrates. The zonule comprises fibers hundreds to thousands of micrometers in length that span the cell-free space between the lens and the eyewall. In this report, we describe a mechanical technique that takes advantage of the highly organized structure of the zonule to quantify its viscoelastic properties and to determine the contribution of individual protein components. The method involves dissection of a fixed eye to expose the lens and the zonule and employs a pull-up technique that stretches the zonular fibers equally while their tension is monitored. The technique is relatively inexpensive yet sensitive enough to detect alterations in viscoelastic properties of zonular fibers in mice lacking specific zonular proteins or with aging. Although the method presented here is designed primarily for studying ocular development and disease, it could also serve as an experimental model for exploring broader questions regarding the viscoelastic properties of elastic ECM&#39;s and the role of external factors such as ionic concentration, temperature, and interactions with signaling molecules.

The protocol describes a method for the study of extracellular matrix viscoelasticity and its dependence on protein composition or environmental factors. The matrix system targeted is the mouse zonule. The performance of the method is demonstrated by comparing the viscoelastic behavior of wild-type zonular fibers with those lacking microfibril-associated glycoprotein-1.

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular ...

Medicine

Replicating Cataract Surgical Techniques in a Mouse Model Through LEC Extraction

Modeling Cataract Surgery in Mice

Cataract surgery (CS) is an effective treatment for cataracts, a major cause of visual disability worldwide. However, CS leads to ocular inflammation, and in the long term, it can result in posterior capsular opacification (PCO) and/or lens dislocation driven by the post-surgical overgrowth of lens epithelial cells (LECs) and their conversion to myofibroblasts and/or aberrant fiber cells. However, the molecular mechanisms by which CS results in inflammation and PCO are still obscure because most in vitro models do not recapitulate the wound healing response of LECs seen in vivo, while traditional animal models of cataract surgery, such as rabbits, do not allow the genetic manipulation of gene expression to test mechanisms. Recently, our laboratory and others have successfully used genetically modified mice to study the molecular mechanisms that drive the induction of proinflammatory signaling and LEC epithelial to mesenchymal transition, leading to new insight into PCO pathogenesis. Here, we report the established protocol for modeling cataract surgery in mice, which allows for robust transcriptional profiling of the response of LECs to lens fiber cell removal via RNAseq, the evaluation of protein expression by semi-quantitative immunofluorescence, and the use of modern mouse genetics tools to test the function of genes that are hypothesized to participate in the pathogenesis of acute sequelae like inflammation as well as the later conversion of LECs to myofibroblasts and/or aberrant lens fiber cells.

Author Spotlight: Unraveling the Molecular Mechanisms in PCO and Fibrosis Following Cataract Surgery

This method models cataract surgery in vivo by removing lens fiber cells from adult mice and leaving behind the capsular bag with attached lens epithelial cells (LECs). The injury response is then assessed at various times post-surgery using molecular and morphological criteria.

Cataract surgery (CS) is an effective treatment for cataracts, a major cause of visual disability worldwide. However, CS leads to ocular inflammation, and ...

Preparation and Imaging of Murine Ocular Lens Whole Mounts

Whole Mount Imaging to Visualize and Quantify Peripheral Lens Structure, Cell Morphology, and Organization

The ocular lens is a transparent flexible tissue that alters its shape to focus light from different distances onto the retina. Aside from a basement membrane surrounding the organ, called the capsule, the lens is entirely cellular consisting of a monolayer of epithelial cells on the anterior hemisphere and a bulk mass of lens fiber cells. Throughout life, epithelial cells proliferate in the germinative zone at the lens equator, and equatorial epithelial cells migrate, elongate, and differentiate into newly formed fiber cells. Equatorial epithelial cells substantially alter morphology from randomly packed cobble-stone-shaped cells into aligned hexagon-shaped cells forming meridional rows. Newly formed lens fiber cells retain the hexagonal cell shape and elongate toward the anterior and posterior poles, forming a new shell of cells that are overlaid onto previous generations of fibers. Little is known about the mechanisms that drive the remarkable morphogenesis of lens epithelial cells to fiber cells. To better understand lens structure, development, and function, new imaging protocols have been developed to image peripheral structures using whole mounts of ocular lenses. Here, methods to quantify capsule thickness, epithelial cell area, cell nuclear area and shape, meridional row cell order and packing, and fiber cell widths are shown. These measurements are essential for elucidating the cellular changes that occur during lifelong lens growth and understanding the changes that occur with age or pathology.

Author Spotlight: In-Depth Morphometric Examination and Quantification of Native Lens Structure Using Whole Mount Imaging 

The present protocols describe novel whole mount imaging for the visualization of peripheral structures in the ocular lens with methods for image quantification. These protocols can be used in studies to better understand the relationship between lens microscale structures and lens development/function.

The ocular lens is a transparent flexible tissue that alters its shape to focus light from different distances onto the retina. Aside from a basement ...

Measurement of Stiffness in Mouse Retinal Capillaries and Subendothelial Matrix

Isolation of Mouse Retinal Capillaries and Subendothelial Matrix for Stiffness Measurement Using Atomic Force Microscopy

Retinal capillary degeneration is a clinical hallmark of the early stages of diabetic retinopathy (DR). Our recent studies have revealed that diabetes-induced retinal capillary stiffening plays a crucial and previously unrecognized causal role in inflammation-mediated degeneration of retinal capillaries. The increase in retinal capillary stiffness results from the overexpression of lysyl oxidase, an enzyme that crosslinks and stiffens the subendothelial matrix. Since tackling DR at the early stage is expected to prevent or slow down DR progression and associated vision loss, subendothelial matrix, and capillary stiffness represent relevant and novel therapeutic targets for early DR management. Further, direct measurement of retinal capillary stiffness can serve as a crucial preclinical validation step for the development of new imaging techniques for non-invasive assessment of retinal capillary stiffness in animal and human subjects. With this view in mind, we here provide a detailed protocol for the isolation and stiffness measurement of mouse retinal capillaries and subendothelial matrix using atomic force microscopy.

Author Spotlight: Investigating Vascular Stiffness and Inflammation in Early Diabetic Retinopathy

We recently identified retinal capillary stiffening as a new paradigm for retinal dysfunction associated with diabetes. This protocol elaborates the steps for isolation of mouse retinal capillaries and the subendothelial matrix from retinal endothelial cultures, followed by a description of the stiffness measurement technique using atomic force microscopy.