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

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.