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&#16...

<ol>
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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. 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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 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>
</table>

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...

Watch this Scientific Journal Video about Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers at JoVE.com

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 ...

The importance of our method lies in its ability to answer the question, what are the viscoelastic properties of zonular fibers, the fibers that suspend the lens inside the eye? The main advantage of our technique is that it can quantify the viscoelastic properties of the small and delicate zonular fibers from mice with and without targeted gene modifications. Mutations in genes encoding microfibril proteins underlie several inherited syndromic conditions.Using our technique, we can investigate how specific mutations alter the biomechanical properties of the microfibrils. To begin, post-euthanizing the mouse, remove the eyes using fine forceps. Insert the tip of a fixation needle through the wall of the eye and position it in the center of the vitreous humor, taking care not to damage the lens.Carefully transfer the impaled eye to a tube filled with 4%paraformaldehyde and phosphate buffered saline. Open the valve connecting the needle to a fixated reservoir located 25 centimeters above the sample which maintains a positive pressure equivalent to 15 to 20 millimeters of mercury within the eye during the fixation period and leave the sample to fix overnight. The next day, remove the fixation needle and wash the eye for 10 minutes in phosphate buffered saline.By employing ophthalmic surgical scissors and a stereo microscope, make a full thickness incision in the wall of the eye near the optic nerve head. Extend the cut forward towards the equator and then around the equatorial circumference of the eye while carefully sparing the delicate ciliary processes and associated zonular fibers. Expose the posterior surface of the lens by removing the back of the globe.Remove the dissected eye from the buffer solution using forceps and place it on a dry task wipe with the cornea facing downwards. Gently drag the cornea over the surface of the wipe to dry it. Apply a drop of instant glue to the bottom of a 50 millimeter Petri dish and fix the platform to it.Place the dish on the stage plate of the stereo microscope so that the well with the glue is in view. To accommodate the eye in the platform wells of the Petri dish, add three microliters of instant glue. Carefully place it into the well and quickly adjust so that the back of the lens faces upwards.Dry the exposed side of the lens by gently blotting with the corner of a dry wipe. For measuring the zonular viscoelastic response, turn on the scale, place the Petri dish on the scale, then start the scale logging program and camera software. Switch on the servomotor controller.Start the controller application on the computer and set the motion parameters with 50 micrometer increments. Create 90 degrees bend in a capillary rod. Slip the bent capillary into the capillary probe holder and tighten the securing screws.To the capillary tip, add a small bead of UV curing glue. Move the tip of the capillary probe using the manual adjustments on the manipulator and place it directly over the top of the lens. Through front visual inspection and the side view using a microscope camera, examine that the bottom portion of the UV glue appears centered over the top of the lens.While looking through the camera, lower the probe tip until the UV glue makes contact with the lens and covers 1/3 to 1/2 of its upper surface. Cure the glue using a directional near-visible UV light source of 380 to 400 nanometer wavelength and an intensity of about one milliwatt. Add PBS solution to the Petri dish until the eye is covered to a depth of at least two millimeters.Place a cylindrical lens in front of the microscope and in a close way to the Petri dish without touching it. At the same time, start the logging in a timer program. Take a picture of the eye and the probe using the camera.After 60 seconds, begin a 50 micrometer displacement. Repeat every 60 seconds until the experiment is completed as indicated by the breaking of all zonular fibers. Save the scale logging data upon completion of a run and export it into a compatible spreadsheet format.Also, save the lens pictures obtained during the run. Import the data into a spreadsheet. Using the first and the last scale reading, interpolate the drift in the background reading over time due to evaporation, then subtract the interpolated background from the reading in each time point.After performing the assay and correcting for evaporation, the graph of the viscoelastic data is inverted. The magnitude of the instantaneous and relaxed forces increases with each step up to about 1, 000 seconds, then drops as the zonular fibers begin to break until the 1, 500 second time point is reached when all fibers are broken. For this demonstration, the viscoelastic response for wild type and MAGP-1 deficient mice were also compared.At the initial time of zero to 600 seconds, the graphs resemble each other suggesting that the viscoelastic properties of the zonular fibers were not significantly altered. However, in the MAGP-1 knockout mouse, the fibers break prematurely. Zonular fiber breaking forces were obtained with the pull-up method for MAGP-1 versus wild type mice at one month and a year old stages.The results indicated that the strength of the fibers increases with age for wild type mice. Any accidental contact with the Petri dish during these steps may cause the eye wall to shift relative to the eye lens, potentially damaging the zonular fibers. This pull-up assay helps identify proteins that contribute to the tensile strength of microfibrils.We'll use this information to build more realistic models of the interior of zonular fibers.

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1.9K visualizações.  University of Health Sciences and Pharmacy. A importância do nosso método reside na sua capacidade de responder à pergunta, quais são as propriedades viscoelásticas das fibras zonulares, as fibras que suspendem a lente dentro do olho? A principal vantagem de nossa técnica é que ela pode quantificar as propriedades viscoelásticas das pequenas e delicadas fibraszonulares de camundongos com e sem modificações genéticas direcionadas. Mutações em genes que codificam proteínas microfibrilas estão por trás de várias condiçõ...