Name: biological preparation and mechanical technique for determining viscoelastic properties of zonular fibers
Uploaded: 2021-12-16T13:30:00
Description: Watch this Scientific Journal Video about Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers at JoVE.com

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
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<ol>
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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.

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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...

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 ...

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