We're trying to understand how people lose their near vision with age. We know that this isn't due in part to a change in lens stiffness, but we still don't know why the lens stiffens as we age. Current experimental methods rely on crude methods for loading the lens, such as placing microscope cover slips on the lens.
These methods are tedious and lack appropriate controls for the rate of loading. We have determined that the lens shape is the primary driver of presbyopia, and we know that the shape is primarily driven by biomechanical parameters. This protocol offers a completely objective reproducible method for measuring lens stiffness.
Automation allows us to control the rate of loading, which is a key parameter when characterizing a viscoelastic material like the lens. We now have an objective repeatable protocol for measuring lens biomechanical properties. This will allow us to rigorously characterize how the lens stiffens with age, as well as how specific proteins modify the lens biomechanical properties.
To begin, place the locally sourced pig eye on a petri dish. Remove the peripheral tissue from the pig eyes. Then use curved forceps and small dissection scissors to remove the excess flesh from the sclera until the optic nerve is visible.
Next, with a scalpel, make a shortcut at the limbus. Make another cut at the equator. Now insert a pair of micro scissors into the cut at the limbus, and use a pair of fine blunt tip forceps to lift the cornea.
Cut around the circumference of the cornea to remove it. With the blunt tip forceps, lift the iris and cut it with micro scissors to excise it. Now insert the dissection scissors into the cut at the equator and cut around the circumference to bisect the sclera.
Remove the posterior portion of the sclera. Then gently remove the vitreous humor with forceps. If required, cut the vitreous humor coronally to allow the posterior to pull away from the lens.
Use micro scissors to make a Meridiano cut through the sclera from the anterior end to the posterior end. Starting from the cut, gently stretch the zonules while pulling the lens and sclera apart, and cut between the lens and ciliary body through the zonules and around the lens circumference to isolate the lens. If desired, use forceps to puncture the capsule at the equator and peel it away to remove the capsule.
Place the lens in PBS solution, and visually inspect the lens for any damage before further testing. To begin, construct a parallel plate compression apparatus with a 50 gram force capacity loading cell enough to measure a displacement of one micrometer order. After programming the motorized stage, load the cell to perform the loading regimen.
Now fill a square box with PBS solution and place it on the compression platform. To determine the lower limit of motion and absolute gap height, lower the upper plate until it meets the lower plate. Then raise the upper plate by approximately 15 millimeters.
Now position the isolated poor sign ocular lens centrally in the box. Lower the upper plate until it is close to the upper surface of the lens. Initiate motion to move the upper plate into contact with the lens.
Commence data recording upon determination of contact, recording time, position of the upper plate relative to the lower plate and force at 500 hertz. Then apply a preconditioning loading where the lens is progressively compressed by 2.5%then 5%and 7.5%of its initial height, each occurring three times at a rate of 1%per second. Hold the position of the upper plate constant for a minute after preconditioning.
Then apply 15%compression at the rate of 1%per second before unloading at the same rate. Continue to unload until the upper plate has traveled in additional 2%of the unloaded lens thickness away from the bottom plate. To estimate the lens modulus, estimate the thickness of the lens based on the instrument's gap at the point of contact.
Alternatively, measure the thickness from a photograph taken before testing. Compute the elastic modulus using the Hertz model for compression of a sphere between parallel plates. The forced displacement curves of both encapsulated and decapsulated lenses were well-fitted by the Hertz model.
Decapsulation resulted in a significant decrease in effective elastic modulus.
