This work was supported by NIH R01 EY029130 (S.B.) and P30 EY002687 (S.B.), R01 HL53325 and the Ines Mandl Research Foundation (R.P.M.), the Marfan Foundation, and an unrestricted grant to the Department of Ophthalmology and Visual Sciences at Washington University from Research to Prevent Blindness. J.R. also received a grant from the University of Health Sciences and Pharmacy in support of this project.

All animal experiments were approved by the Washington University Animal Studies Committee and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
1. Fabrication of specialized parts and construction of apparatus
<ol>
	<li>Fabrication of specialized parts
	<ol>
		<li>Probe fabrication. Hold a glass capillary at an angle as shown in the left panel of Figure 2A. Place a flame from a cigarette lighter about 2&#160;cm from one end and keep it there until the end bends by 90&#176;, as shown in the right panel of Figure 2A.</li>
		<li>Sample platform fabrication. Using 3D drawing software, design a platform measuring 30 x 30 x 5 mm and containing hemispherical indentations of 2.0, 2.5, and 3.0 mm in diameter, as shown in Figure 2B.</li>
		<li>Probe holder fabrication. Using the 3D drawing software, design a mount that holds the capillary probe and attach it to a micromanipulator (see Figure 2C). 
		NOTE: A sample 3D file for platform fabrication and probe holder fabrication in STL format is available on request from the corresponding author.</li>
		<li>Negative lens assembly. Place a negative cylindrical lens (-75 mm in focal length and approximately 50 mm in height and length) as shown in Figure 1C and Figure 1D to correct the distortion caused by the addition of fluid to the Petri dish (addition of fluid distorts the view of the dissected eye when imaged from the side).</li>
		<li>Glue the negative lens to one of the 2-slotted bases (see Figure 2D for positioning of the lens on the base).</li>
		<li>Assemble the remaining parts as shown in Figure 2D.</li>
		<li>Adjust the height of the post so that the lens barely hovers over the scale and tighten the screw in the post-holder.</li>
	</ol>
	</li>
	<li>Construction of apparatus
	<ol>
		<li>Install on a computer the logging program supplied with the scale, the microscope camera software, and the motorized micrometer controller application.</li>
		<li>Connect the motorized micrometer to the servo motor controller and the latter to the computer. Start the&#160;motor controller application and edit the motor settings. 
		NOTE: The motor settings, which are listed below, were chosen following preliminary experiments that revealed that stresses relaxed on a time scale of 10-20 s. Based on this determination, we selected a speed and acceleration that allowed the motor to complete a 50 &#181;m displacement in a time smaller than the relaxation time, but not too short to avoid jolting&#160;the sample. Here we chose a displacement time of about 5-10 s.</li>
		<li>Set the maximum velocity to 0.01 mm/s and the acceleration to 0.005 mm/s2.</li>
		<li>Install the camera in the inspection microscope and test the camera imaging software.</li>
		<li>Place the scale on the bench space devoted to the apparatus.</li>
		<li>Glue a 3D-printed platform (from step 1.1.2) to a Petri dish and add a 2-3 mm glass bead to one of the wells. Place the Petri dish on the scale so that the bead is located near the center of the pan.</li>
		<li>Replace the manual micrometer from the micromanipulator with the motorized one.</li>
		<li>Screw the two 4-40 screws into the probe holder. Attach the probe holder to the manipulator as shown in Figure 1C.</li>
		<li>Prepare a probe as illustrated in Figure 2A, place it inside the probe holder with the bent portion facing down, and tighten the screws.</li>
		<li>Position the micromanipulator on the table such that the tip of the probe is over the bead on the platform. Affix the micromanipulator to the table to prevent accidental movement during the experiment.</li>
		<li>Position the side microscope on the table so that the bead is at the center of its field of view and in focus.</li>
	</ol>
	</li>
</ol>
2. Sample preparation and data acquisition
<ol>
	<li>Eye fixation and dissection
	<ol>
		<li>Maintain wild-type mice and Magp1-null animals on an identical C57/BL6J background. Euthanize 1-month-old or 1-year-old mice by CO2 inhalation.</li>
		<li>Remove the eyes with fine forceps and fix the enucleated globes at 4 &#176;C overnight in 4% paraformaldehyde/phosphate-buffered saline (PBS, pH 7.4). Maintain a positive pressure of 15-20 mmHg in the eye during the fixation process, as described6. 
		NOTE: Experiments are conducted on male mice, to control for possible sex-related differences in the size of the ocular globe. The positive pressure ensures that the globe remains inflated, preserving the gap between the lens and the wall of the eye spanned by the zonular fibers.</li>
		<li>Wash the eyes for 10 min in PBS. Using ophthalmic surgical scissors and working under a stereomicroscope, make a full-thickness incision in the wall of the eye near the optic nerve head.</li>
		<li>Extend the cut forward to the equator, and then around the equatorial circumference of the eye. Take care to spare the delicate ciliary processes and associated zonular fibers.</li>
		<li>Remove the back of the globe, exposing the posterior surface of the lens.</li>
		<li>Use the forceps to remove a dissected eye from the buffer solution and place it on a dry task wipe with the cornea facing down. Gently drag the cornea over the surface of the wipe to dry it.</li>
		<li>Add 3 &#181;L of instant glue to the platform wells that will accommodate the eye in the Petri dish.</li>
		<li>Place the dish on the stage plate of the stereomicroscope so that the well with the glue is in view.</li>
		<li>Transfer the eye from the wipe to the edge of the well that contains glue. Then, carefully drag the eye into the well and quickly adjust its orientation so that the back of the lens is uppermost.</li>
		<li>Dry the exposed side of the lens by gently blotting it with the corner of a dry wipe.</li>
		<li>Apply a dab of instant glue to the bottom of a 50 mm Petri dish and cement the platform to it.</li>
	</ol>
	</li>
	<li>Measurement of zonular viscoelastic response
	<ol>
		<li>Turn on the scale, start the scale logging program and the camera software. Ensure that the logging program can acquire data for 30 min, as some trials can last that long.</li>
		<li>Switch on the servo motor controller and start the controller application on the computer. Make sure the controller is set to move in 50 &#181;m increments using motion parameters similar to those outlined in the NOTE in step 1.2.2.</li>
		<li>Create a 90&#176; bend in a capillary rod as described in step 1.1.1.</li>
		<li>Slip the bent capillary into the capillary probe holder and tighten the securing screws. 
		NOTE: To minimize sample dehydration, we recommend that steps 1-4 be completed prior to, or during, eye dissection.</li>
		<li>Add a small (~1 mm) bead of UV-curing glue to the tip of the capillary.</li>
		<li>Using the manual adjustments on the manipulator, move the tip of the capillary probe so that it is directly over the center of the lens. Check whether the bottom portion of the UV glue appears centered over the top of the lens when viewed from the front (by visual inspection) and the side (through the microscope camera).</li>
		<li>While looking through the camera, lower the probe tip until the UV glue makes contact with the lens and covers one-third to one-half of its upper surface.</li>
		<li>Use a low-intensity (~ 1 mW), directional, near-visible UV (380-400 nm) light source to cure the glue. 
		NOTE: These specifications suffice to cure the glue in a few seconds while minimizing the potential for inducing protein crosslinking. The UV light sources supplied with commercial UV glue pens meet these specifications.</li>
		<li>Add PBS solution to the dish until the eye is covered by fluid to a depth of at least 2 mm.</li>
		<li>Place the cylindrical lens in front of the inspection microscope and as close as possible to the Petri dish without touching it.</li>
		<li>Simultaneously start the logging program and a timer program. Take a picture of the eye/probe using the camera.</li>
		<li>After 60 s, initiate another 50 &#181;m displacement, and thereafter every 60 s until the experiment is complete, i.e., until all the fibers have been broken. Note that the signal will not return to baseline levels due to buffer evaporation during the experiment. Correct the ensuing drift in the readings during the data analysis, as exemplified in step 2.2.14.</li>
		<li>Upon completion of a run, save the scale logging data and export it into a format compatible with the spreadsheet, e.g., a .csv format. Save the lens pictures that were collected during the run.</li>
		<li>Import data into a spreadsheet. Use the first and the last scale reading to interpolate the drift in the background reading over time due to evaporation (see Figure 3). Subtract the interpolated reading from the reading at&#160;each time point. 
		NOTE: If using a spreadsheet, the interpolation can be performed automatically by entering the formula = B2 - $B$2 + ($B$2 - @INDIRECT(&#34;B&#34;&#38;COUNTA(B:B)))/(COUNTA(B:B)-2) * A2 in the cell to the right of the first scale reading, then moving&#160;the cursor to the right-lower corner of the cell and dragging it down to the last data value. The formula assumes that the data is organized in a column with the first data point appearing in cell B2. If desired, the data processed in step 2.2.14 may be analyzed with the quasi-elastic viscoelastic model developed by one of the co-authors, Dr. Matthew Riley4.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Bassnett, S., Shi, Y., Vrensen, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+glass:+structural+determinants+of+eye+lens+transparency.">Biological glass: structural determinants of eye lens transparency.</a> Philosophical Transactions of the Royal Society B Biological Sciences. 366 (1568), 1250-1264 (2011).</li><li>Bassnett, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinn's+zonule.">Zinn's zonule.</a> Progress in Retinal and Eye Research. 82, 100902 (2021).</li><li>Dureau, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+zonular+diseases.">Pathophysiology of zonular diseases.</a> Current Opinion in Ophthalmology. 19 (1), 27-30 (2008).</li><li>Shi, Y., 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=Latent-transforming+growth+factor+beta-binding+protein-2+(LTBP-2)+is+required+for+longevity+but+not+for+development+of+zonular+fibers.">Latent-transforming growth factor beta-binding protein-2 (LTBP-2) is required for longevity but not for development of zonular fibers.</a> Matrix Biology. 95, 15-31 (2021).</li><li>Ushiki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fibers,+reticular+fibers+and+elastic+fibers.+A+comprehensive+understanding+from+a+morphological+viewpoint.">Collagen fibers, reticular fibers and elastic fibers. A comprehensive understanding from a morphological viewpoint.</a> Archives of Histology and Cytology. 65 (2), 109-126 (2002).</li><li>Bassnett, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+preserving+and+visualizing+the+three-dimensional+structure+of+the+mouse+zonule.">A method for preserving and visualizing the three-dimensional structure of the mouse zonule.</a> Experimental Eye Research. 185, 107685 (2019).</li><li>Todorovic, V., Rifkin, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LTBPs,+more+than+just+an+escort+service.">LTBPs, more than just an escort service.</a> Journal of Cellular Biochemistry. 113 (2), 410-418 (2012).</li><li>Mecham, R. P., Gibson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microfibril-associated+glycoproteins+(MAGPs)+and+the+microfibrillar+niche.">The microfibril-associated glycoproteins (MAGPs) and the microfibrillar niche.</a> Matrix Biology. 47, 13-33 (2015).</li><li>Hubmacher, D., Reinhardt, D. P., Plesec, T., Schenke-Layland, K., Apte, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+eye+development+is+characterized+by+coordinated+expression+of+fibrillin+isoforms.">Human eye development is characterized by coordinated expression of fibrillin isoforms.</a> Investigative Ophthalmology &amp; Visual Science. 55 (12), 7934-7944 (2014).</li><li>Inoue, T., 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=Latent+TGF-&#946;+binding+protein-2+is+essential+for+the+development+of+ciliary+zonule+microfibrils.">Latent TGF-&#946; binding protein-2 is essential for the development of ciliary zonule microfibrils.</a> Human Molecular Genetics. 23 (21), 5672-5682 (2014).</li><li>De Maria, A., Wilmarth, P. A., David, L. L., Bassnett, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+the+bovine+and+human+ciliary+zonule.">Proteomic analysis of the bovine and human ciliary zonule.</a> Investigative Ophthalmology &amp; Visual Science. 58 (1), 573-585 (2017).</li><li>Wright, D. M., Duance, V. C., Wess, T. J., Kielty, C. M., Purslow, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+supramolecular+organization+of+fibrillin-rich+microfibrils+determines+the+mechanical+properties+of+bovine+zonular+filaments.">The supramolecular organization of fibrillin-rich microfibrils determines the mechanical properties of bovine zonular filaments.</a> Journal of Experimental Biology. 202 (21), 3011-3020 (1999).</li><li>Bocskai, Z. I., Sandor, G. L., Kiss, Z., Bojtar, I., Nagy, Z. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+mechanical+behaviour+and+estimation+of+the+elastic+properties+of+porcine+zonular+fibres.">Evaluation of the mechanical behaviour and estimation of the elastic properties of porcine zonular fibres.</a> Journal of Biomechanics. 47 (13), 3264-3271 (2014).</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=The+ciliary+body+in+accommodation.">The ciliary body in accommodation.</a> Transactions of the Ophthalmological Societies of the United Kingdom. 105, 208-219 (1986).</li><li>Michael, R., 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=Elastic+properties+of+human+lens+zonules+as+a+function+of+age+in+presbyopes.">Elastic properties of human lens zonules as a function of age in presbyopes.</a> Investigative Ophthalmology &amp; Visual Science. 53 (10), 6109-6114 (2012).</li><li>van Alphen, G. W., Graebel, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elasticity+of+tissues+involved+in+accommodation.">Elasticity of tissues involved in accommodation.</a> Vision Research. 31 (7-8), 1417-1438 (1991).</li><li>Green, E. M., Mansfield, J. C., Bell, J. S., Winlove, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+and+micromechanics+of+elastic+tissue.">The structure and micromechanics of elastic tissue.</a> Interface Focus. 4 (2), 20130058 (2014).</li><li>Jones, W., Rodriguez, J., Bassnett, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+deletion+of+fibrillin-1+in+the+mouse+eye+results+in+ectopia+lentis+and+other+ocular+phenotypes+associated+with+Marfan+syndrome.">Targeted deletion of fibrillin-1 in the mouse eye results in ectopia lentis and other ocular phenotypes associated with Marfan syndrome.</a> Disease Models &amp; Mechanisms. 12 (1), 037283 (2019).</li><li>Weinbaum, J. 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=Deficiency+in+microfibril-associated+glycoprotein-1+leads+to+complex+phenotypes+in+multiple+organ+systems.">Deficiency in microfibril-associated glycoprotein-1 leads to complex phenotypes in multiple organ systems.</a> Journal of Biological Chemistry. 283 (37), 25533-25543 (2008).</li><li>Comeglio, P., Evans, A. L., Brice, G., Cooling, R. J., Child, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+FBN1+gene+mutations+in+patients+with+ectopia+lentis+and+marfanoid+habitus.">Identification of FBN1 gene mutations in patients with ectopia lentis and marfanoid habitus.</a> British Journal of Ophthalmology. 86 (12), 1359-1362 (2002).</li></ol>

The authors have nothing to disclose.

The zonule is an unusual ECM system where fibers are arranged symmetrically and can be manipulated identically by displacing the eye lens along the optical axis. The space can also be readily accessed without cellular disruption, allowing the fibers to be studied in an environment close to their native state. The pull-up technique takes advantage of this ECM presentation to manipulate the delicate fibers from mice, a genetically tractable system, and accurately quantify their mechanical properties. This has allowed us to...

The eye of a vertebrate contains a living optical lens that helps focus images on the retina1. The lens is suspended on the optical axis by a system of delicate, radially-oriented fibers, as illustrated in Figure 1A. At one end, the fibers attach to the lens equator and, at the other, to the surface of the ciliary body. Their lengths span distances ranging from 150 &#181;m in mice to 1 mm in humans. Collectively, these fibers are known as the zonule of Zinn2, the ciliary zonule, or simply the zonule. Ocular trauma, disease, and certain genetic disorders can affect the integrity of the zonular fibers3, resulting in their eventual failure and accompanying loss of vision. In mice, the fibers have a core comprised mostly of the protein fibrillin-2, surrounded by a mantle rich in fibrillin-14. Although zonular fibers are unique to the eye, they bear many similarities to elastin-based ECM fibers found elsewhere in the body. The latter are covered by a fibrillin-1 mantle5&#160;and have similar dimensions to&#160;zonular fibers6. Other proteins, such as latent-transforming growth factor &#946;-binding proteins (LTBPs) and microfibril-associated glycoprotein-1 (MAGP-1), are found in association with both types of fibers7,8,9,10,11. The elastic modulus of zonular fibers is in the range of 0.18-1.50 MPa12,13,14,15,16, comparable to that of elastin-based fibers (0.3-1.2 MPa)17. These architectural and mechanical similarities lead us to believe that any insight into the roles of zonule-associated proteins may help elucidate their roles in other ECM elastic fibers.
The main purpose of developing the method described here is to gain insights into the role of specific zonular proteins in the progression of inherited eye disease. The general approach is to compare the viscoelastic properties of zonular fibers in wild-type mice with those of mice carrying targeted mutations in genes encoding zonular proteins. While several methods have been used previously to measure the elasto-mechanical properties of zonular fibers, all were designed for the eyes of much larger animals12,13,14,15,16. As such models are not genetically tractable; we sought to develop an experimental method that was better suited to the small and delicate eyes of mice.
The method we developed for assessing the viscoelasticity of mouse zonular fibers is a technique we refer to as the pull-up assay4,18, which is summarized visually in Figure 1. A detailed description of the pull-up method and the analysis of the results is provided below. We begin by describing the construction of the apparatus, including the three-dimensional (3D)-printed parts used in the project. Next, we detail the protocol used for obtaining and preparing the eyes for the experiment. Lastly, we provide step-by-step instructions on how to obtain data for the determination of the viscoelastic properties of zonular fibers. In the Representative Results section, we share previously unpublished data obtained with our method on the viscoelastic properties of zonular fibers from mice lacking MAGP-119 as well as a control set obtained from age-matched wild-type animals. Finally, we conclude with general remarks on the advantages and limitations of the method, and suggestions for potential experiments that may elucidate how environmental and biochemical factors affect the viscoelastic properties of ECM fibers.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1/4-20 hex screws 3/4 inch long</td>
			<td>Thorlabs</td>
			<td>SH25S075</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4-20 nut</td>
			<td>Hardware store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D SLA printer</td>
			<td>Anycubic</td>
			<td>Photon</td>
			<td></td>
		</tr>
		<tr>
			<td>4-40 screws 3/8 inch long, 2</td>
			<td>Hardware store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Capillaries, OD 1.2 mm and 3 inches long, no filament</td>
			<td>WPI</td>
			<td>1B120-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate (super) glue</td>
			<td>Loctite</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Scale accurate to 0.01 g</td>
			<td>Vernier</td>
			<td>OHAUS Scout 220</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>Spreadsheet</td>
		</tr>
		<tr>
			<td>Gas cigarette lighter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inspection/dissection microscope</td>
			<td>Amscope</td>
			<td>SKU: SM-4NTP</td>
			<td>Working distance ~ 15 cm</td>
		</tr>
		<tr>
			<td>Micromanipulator, Economy 4-axis</td>
			<td>WPI</td>
			<td>Kite-L</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized micrometer</td>
			<td>Thorlabs</td>
			<td>Z812B</td>
			<td></td>
		</tr>
		<tr>
			<td>Negative cylindrical lens</td>
			<td>Thorlabs</td>
			<td>LK1431L1</td>
			<td>-75 mm focal length</td>
		</tr>
		<tr>
			<td>Petri dishes, 50 mm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Post holder, 3 inches</td>
			<td>Thorlabs</td>
			<td>PH3</td>
			<td></td>
		</tr>
		<tr>
			<td>Post, 4 inches</td>
			<td>Thorlabs</td>
			<td>TR4</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale logging software</td>
			<td>Vernier</td>
			<td>LoggePro</td>
			<td></td>
		</tr>
		<tr>
			<td>Servo motor controller</td>
			<td>Thorlabs</td>
			<td>KDC101</td>
			<td></td>
		</tr>
		<tr>
			<td>Servo motor controller software</td>
			<td>Thorlabs</td>
			<td>APT</td>
			<td></td>
		</tr>
		<tr>
			<td>Slotted base, 1</td>
			<td>Thorlabs</td>
			<td>BA1S</td>
			<td></td>
		</tr>
		<tr>
			<td>Slotted bases, 2</td>
			<td>Thorlabs</td>
			<td>BA2</td>
			<td></td>
		</tr>
		<tr>
			<td>Stand for micromanipular</td>
			<td>WPI</td>
			<td>M-10</td>
			<td></td>
		</tr>
		<tr>
			<td>USB-camera for microscope</td>
			<td>Amscope</td>
			<td>SKU: MD500</td>
			<td></td>
		</tr>
		<tr>
			<td>UV activated glue with UV source</td>
			<td>Amazon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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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 pull-up technique described here provides a straightforward approach for determining viscoelastic properties of zonular fibers in mice. In brief, the mouse eye is first preserved by injection of a fixative at physiological intraocular pressure. This approach maintains the natural inflation of the eye and keeps the fibers properly pre-tensioned (fixation was deemed acceptable after preliminary experiments demonstrated it did not alter the elasticity or strength of the fibers significantly). The back of the mouse eye i...

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

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 ...

biological-preparation-mechanical-technique-for-determining

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JoVE Journal

Medicine

2.0K Views.  University of Health Sciences and Pharmacy. 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.

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

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Cancer therapies which are less toxic and invasive than their existing counterparts are highly desirable. The use of RF electric-fields that penetrate deep into the body, causing minimal toxicity, are currently being studied as a viable means of non-invasive cancer therapy. It is envisioned that the interactions of RF energy with internalized nanoparticles (NPs) can liberate heat which can then cause overheating (hyperthermia) of the cell, ultimately ending in cell necrosis.
In the case of non-biological systems, we present detailed protocols relating to quantifying the heat liberated by highly-concentrated NP colloids. For biological systems, in the case of in vitro experiments, we describe the techniques and conditions which must be adhered to in order to effectively expose cancer cells to RF energy without bulk media heating artifacts significantly obscuring the data. Finally, we give a detailed methodology for in vivo mouse models with ectopic hepatic cancer tumors.

We describe the protocols used to investigate the interactions of 13.56 MHz radiofrequency (RF) electric-fields with gold nanoparticle colloids in both non-biological and biological systems (in vitro/vivo). These interactions are being investigated for applications in cancer therapy.

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength. Because ex vivo biological tissues are prone to lose their mechanical properties over time, specimen preparation for experimental testing must be performed carefully to obtain reliable results that represent in vivo conditions. For that reason, we designed a protocol and a set of fixtures to prepare the femoral specimens such that their mechanical properties experienced minimal changes. The femora were kept in a frozen state except during preparation steps and mechanical testing. The relevant clinical measures of total hip and femoral neck bone mineral density (BMD) were obtained with a clinical dual X-ray absorptiometry (DXA) bone densitometer, and the 3D geometry and distribution of bone mineral were obtained using CT with a calibration phantom for quantitative estimations based on the greyscale values. Any possible bone disease, fracture, or the presence of implants or artifacts affecting the bone structure, was ruled out with X-ray scans. For preparation, all bones were carefully cleaned of excess soft tissue, and were cut and potted at the internal rotation angle of interest. A cutting fixture allowed the distal end of the bone to be cut off leaving the proximal femur at a desired length. To allow positioning of the femoral neck at prescribed angles during later CT scanning and mechanical testing, the proximal femoral shafts were potted in polymethylmethacrylate (PMMA) using a fixture designed specifically for desired orientations. The data collected from our experiments were then used for validation of quantitative computed tomography (QCT)-based finite element analysis (FEA), as described in a different protocol. In this manuscript, we present the protocol for the precise bone preparation for mechanical testing and subsequent QCT/FEA modeling. The current protocol was successfully applied to prepare about 200 cadaveric femora over a 6-year time period.

We present a robust protocol on how to carefully preserve and prepare cadaveric femora for fracture testing and quantitative computed tomography imaging. The method provides precise control over input conditions for the purpose of determining relationships between bone mineral density, fracture strength, and defining finite element model geometry and properties.

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength.  Because ex vivo biological tissues are prone to ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

Mechanical Micronization of Lipoaspirates for Regenerative Therapy

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of cell products using collagenase. Here, we present a protocol to generate an injectable mixture of SVF cells and native extracellular matrix from adipose tissue by a purely mechanical process. Lipoaspirates are put into a centrifuge and spun at 1,200 x g for 3 min. The middle layer is collected and separated into two layers (high-density fat at the bottom and low-density fat on the top). The upper layer is directly emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance under the oil layer is collected and defined as the extracellular matrix (ECM)/SVF-gel. The oil on the top layer is collected. Approximately 5 mL of oil is added to 15 mL of high-density fat and emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance is also ECM/SVF-gel. After the transplantation of the ECM/SVF-gel into nude mice, the graft is harvested and assessed by histologic examination. The result shows that this product has the potential to regenerate into normal adipose tissue. This procedure is a simple, effective mechanical dissociation procedure to condense the SVF cells embedded in their natural supportive ECM for regenerative purposes.

Here, we present a protocol to obtain stromal vascular fraction from adipose tissue through a series of mechanical processes, which include emulsification and multiple centrifugations.

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of ...

Determining and Controlling External Power Output During Regular Handrim Wheelchair Propulsion

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which has led to improved research techniques becoming available over the last decades. To increase the understanding of wheeled mobility performance, monitoring, training, skill acquisition, and optimization of the wheelchair-user interface in rehabilitation, daily life, and sports, further standardization of measurement set-ups and analyses is required. A crucial stepping-stone is the accurate measurement and standardization of external power output (measured in Watts), which is pivotal for the interpretation and comparison of experiments aiming to improve rehabilitation practice, activities of daily living, and adaptive sports. The different methodologies and advantages of accurate power output determination during overground, treadmill, and ergometer-based testing are presented and discussed in detail. Overground propulsion provides the most externally valid mode for testing, but standardization can be troublesome. Treadmill propulsion is mechanically similar to overground propulsion, but turning and accelerating is not possible. An ergometer is the most constrained and standardization is relatively easy. The goal is to stimulate good practice and standardization to facilitate the further development of theory and its application among research facilities and applied clinical and sports sciences around the world.

Accurate and standardized assessment of external power output is crucial in the evaluation of physiological, biomechanical, and perceived stress, strain, and capacity in manual wheelchair propulsion. The current article presents various methods to determine and control power output during wheelchair propulsion studies in the laboratory and beyond.

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which ...

A Probing Device for Quantitatively Measuring the Mechanical Properties of Soft Tissues during Arthroscopy

Probing in arthroscopic surgery is performed by pulling or pushing the soft tissue, which provides feedback for understanding the condition of the soft tissue. However, the output is only qualitative based on the &#8220;surgeon&#8217;s feeling&#8221;. Herein is described a probing device developed to address this issue by measuring the resistance of soft tissues quantitatively with a tri-axial force sensor. Under both conditions (i.e., pull- and push-probing certain tissues mimicking the acetabular labrum and cartilage), this probing device is found to be useful for measuring some mechanical properties in joints during arthroscopy.

Probing during arthroscopy surgery is normally done to assess the condition of the soft tissue, but this approach has always been subjective and qualitative. This report describes a probing device that can measure the resistance of the soft tissue quantitatively with a tri-axial force sensor during arthroscopy.

Probing in arthroscopic surgery is performed by pulling or pushing the soft tissue, which provides feedback for understanding the condition of the soft ...