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

Summary

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.

Abstract

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's and the role of external factors such as ionic concentration, temperature, and interactions with signaling molecules.

Introduction

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 µm 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 and have similar dimensions to zonular fibers⁶. Other proteins, such as latent-transforming growth factor β-binding proteins (LTBPs) and microfibril-associated glycoprotein-1 (MAGP-1), are found in association with both types of fibers^7,8,9,10,11. The elastic modulus of zonular fibers is in the range of 0.18-1.50 MPa^{12,13,14,15,16}, 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 animals^{12,13,14,15,16}. 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 assay^4,18, 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.

Protocol

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

1. Fabrication of specialized parts
   1. 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 cm from one end and keep it there until the end bends by 90°, as shown in the right panel of Figure 2A.
   2. 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.
   3. 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.
   4. 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).
   5. Glue the negative lens to one of the 2-slotted bases (see Figure 2D for positioning of the lens on the base).
   6. Assemble the remaining parts as shown in Figure 2D.
   7. Adjust the height of the post so that the lens barely hovers over the scale and tighten the screw in the post-holder.
2. Construction of apparatus
   1. Install on a computer the logging program supplied with the scale, the microscope camera software, and the motorized micrometer controller application.
   2. Connect the motorized micrometer to the servo motor controller and the latter to the computer. Start the 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 µm displacement in a time smaller than the relaxation time, but not too short to avoid jolting the sample. Here we chose a displacement time of about 5-10 s.
   3. Set the maximum velocity to 0.01 mm/s and the acceleration to 0.005 mm/s².
   4. Install the camera in the inspection microscope and test the camera imaging software.
   5. Place the scale on the bench space devoted to the apparatus.
   6. 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.
   7. Replace the manual micrometer from the micromanipulator with the motorized one.
   8. Screw the two 4-40 screws into the probe holder. Attach the probe holder to the manipulator as shown in Figure 1C.
   9. Prepare a probe as illustrated in Figure 2A, place it inside the probe holder with the bent portion facing down, and tighten the screws.
   10. 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.
   11. Position the side microscope on the table so that the bead is at the center of its field of view and in focus.

2. Sample preparation and data acquisition

1. Eye fixation and dissection
   1. 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.
   2. Remove the eyes with fine forceps and fix the enucleated globes at 4 °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.
   3. 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.
   4. 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.
   5. Remove the back of the globe, exposing the posterior surface of the lens.
   6. 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.
   7. Add 3 µL of instant glue to the platform wells that will accommodate the eye in the Petri dish.
   8. Place the dish on the stage plate of the stereomicroscope so that the well with the glue is in view.
   9. 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.
   10. Dry the exposed side of the lens by gently blotting it with the corner of a dry wipe.
   11. Apply a dab of instant glue to the bottom of a 50 mm Petri dish and cement the platform to it.
2. Measurement of zonular viscoelastic response
   1. 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.
   2. Switch on the servo motor controller and start the controller application on the computer. Make sure the controller is set to move in 50 µm increments using motion parameters similar to those outlined in the NOTE in step 1.2.2.
   3. Create a 90° bend in a capillary rod as described in step 1.1.1.
   4. 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.
   5. Add a small (~1 mm) bead of UV-curing glue to the tip of the capillary.
   6. 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).
   7. 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.
   8. 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.
   9. Add PBS solution to the dish until the eye is covered by fluid to a depth of at least 2 mm.
   10. Place the cylindrical lens in front of the inspection microscope and as close as possible to the Petri dish without touching it.
   11. Simultaneously start the logging program and a timer program. Take a picture of the eye/probe using the camera.
   12. After 60 s, initiate another 50 µm 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.
   13. 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.
   14. 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 each time point.
       NOTE: If using a spreadsheet, the interpolation can be performed automatically by entering the formula = B2 - $B$2 + ($B$2 - @INDIRECT("B"&COUNTA(B:B)))/(COUNTA(B:B)-2) * A2 in the cell to the right of the first scale reading, then moving 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1/4-20 hex screws 3/4 inch long	Thorlabs	SH25S075
1/4-20 nut	Hardware store
3D SLA printer	Anycubic	Photon
4-40 screws 3/8 inch long, 2	Hardware store
Capillaries, OD 1.2 mm and 3 inches long, no filament	WPI	1B120-3
Cyanoacrylate (super) glue	Loctite
Digital Scale accurate to 0.01 g	Vernier	OHAUS Scout 220
Excel	Microsoft		Spreadsheet
Gas cigarette lighter
Inspection/dissection microscope	Amscope	SKU: SM-4NTP	Working distance ~ 15 cm
Micromanipulator, Economy 4-axis	WPI	Kite-L
Motorized micrometer	Thorlabs	Z812B
Negative cylindrical lens	Thorlabs	LK1431L1	-75 mm focal length
Petri dishes, 50 mm
Post holder, 3 inches	Thorlabs	PH3
Post, 4 inches	Thorlabs	TR4
Scale logging software	Vernier	LoggePro
Servo motor controller	Thorlabs	KDC101
Servo motor controller software	Thorlabs	APT
Slotted base, 1	Thorlabs	BA1S
Slotted bases, 2	Thorlabs	BA2
Stand for micromanipular	WPI	M-10
USB-camera for microscope	Amscope	SKU: MD500
UV activated glue with UV source	Amazon