Intact globe expansion measurements were made in a transparent Plexiglas observation cell (
Fig. 1). The bottom of the observation cell had two holes sealed with rubber septa, used for the insertion of hypodermic needles (30-gauge) to regulate the IOP. The pressure imposed through the needles was achieved with a DPBS reservoir held at a corresponding height (
h) above the apparatus, imposing an IOP equal to the hydrostatic pressure (IOP = ρ
gh, where ρ is density and
g is gravitational acceleration). Before the globes were mounted, the integrity of the fluid path was confirmed by briefly opening the valve to observe fluid flow from the reservoir through the needle. To ensure that the hydrostatic pressure was indeed the IOP in the globe, it was necessary to minimize leakage of fluid from the eyes that could result in a pressure drop across the needle. Therefore, small-diameter needles were used to minimize the risk of a leak around the needle after the sclera was pierced. The apparatus makes it immediately obvious when a leak occurs: changes in fluid level in the sample chamber are readily observed and are recorded in the sequence of images. Of the experiments reported herein, three fourths had no detectable leak, and one fourth had detectable but very gradual leaks (bath fluid level rose <0.3 mm/h, corresponding to a volumetric flow rate of <0.9 mL/h, and perturbing the imposed IOP by <2%). There was no rupture of the eyes within the 24 hours allocated to this experiment.
After specimen preparation, two enucleated eyes were loaded onto transparent acrylic cylinders in the DPBS bath (
Fig. 1A). Because the tissues of interest are anisotropic and the eye is not symmetric, a consistent orientation was used; the eyes were aligned with the optical axis vertical, with the major axis of the equator parallel to the imaging plane, and with the optic nerve on the side closest to the camera. The eyes were nearly neutrally buoyant in the bath solution; therefore, loading was performed with the bath partially filled (fluid level slightly above the cylinder height), so that the weight of the eyes facilitated orienting the globes. The needles were then inserted through the posterior sclera. After insertion, friction between the needle and tissue was sufficient to hold the eyes in place without other methods of fixing the position. The bath was then filled with DPBS to maintain the state of hydration of the tissues. To minimize bacterial growth during the experiment, we added several antibiotic eye drops (neomycin, polymyxin B sulfate and gramicidin ophthalmic solution USP; Bausch & Lomb, Tampa, FL) to the solution in the observation cell. The eyes were allowed 15 minutes to reach bath temperature (∼22°C) before the valve was opened, and an IOP similar to the physiological state was reintroduced (see the Shape Restoration section). For creep experiments, the pressure was subsequently increased from low IOP to high IOP (see the Creep section).
Initially, experiments were performed by recording photographs from three orthogonal directions: along the optical axis of the globe and two projections orthogonal to the optical axis (in the plane of the major equatorial axis and in the plane of the minor equatorial axis). All three projections gave consistent results for the change of the perimeter of the sclera; and the two projections orthogonal to the optical axis gave consistent results for the change of the perimeter of the cornea. Therefore, a simplified method using a single projection orthogonal to the optical axis was adopted; specifically, the plane containing the optical axis and the major equatorial axis was chosen to characterize relative changes in eye shape for this article. Digital photographs of this projection were taken automatically every 15 minutes (PowerShot G3; Canon, Tokyo, Japan). A time series of images offers advantages over relying on initial and final photographs, such as permitting sensitive detection of leaks; tracking of anatomic features, such as the corneal–scleral intersection at the limbus; and evaluating changes in creep rates over the course of the experiment.
The images were analyzed to evaluate changes in ocular dimensions using a computer program written by one of the authors (MSM; MatLab; The MathWorks, Natick, MA). The code allows the user to select points along the perimeter of the eye defining the boundary of the cornea and sclera and uses cubic spline interpolation to trace the outline of the eye. Using the traces, we computed three measures of the sclera (scleral perimeter, SP; equatorial diameter, ED; and scleral length, SL) and three measures of the cornea: corneal perimeter, CP; corneal diameter, CD; and corneal length, CL (
Fig. 1B). The ratios of the scleral and corneal perimeters at time
t to their respective values at time 0, SP(
t)/SP(0) and CP(
t)/CP(0), are analogous to strain measurements in tensile tests. The corneal and scleral lengths are similar to the axial length measurements typical in biometry. The acrylic cylinders of known diameter (∼12.7 mm) provided a scale inside the bath for calibration of pixels to millimeters in each image sequence.
Uncertainty in the measured length ratios is small and predominantly due to animal-to-animal variability. Three potential sources of uncertainty are (1) inaccuracy of point placement during image analysis, (2) image distortion through the bath and the camera optics, and (3) errors in calibration. The first two were evaluated using images of grids with known line spacing placed in the DPBS bath. First, to characterize the uncertainty in point placement during image analysis, we performed repeat placement of points along a 1-mm line. Repeatability was good to the nearest pixel (SD ∼0.25 pixel); the resulting uncertainty in the measured length ratios was an order of magnitude less than the observed experimental uncertainty (e.g., for a 15% increase in CP(t)/CP(0), point placement accounts for <0.015% uncertainty, whereas the overall experimental uncertainty is ±2%). Second, the images of the uniform grid were used to characterize distortion resulting from refraction at the air–bath interface and from the optics of the low-cost cameras that were used for the experiments. The number of pixels per millimeter interval on the grid varied <3% from intervals near the center of a specimen to intervals near their perimeter. This image distortion has very little effect on the measured length ratio, since it affects the numerator and denominator almost identically (e.g., for a 15% increase in CP(t)/CP(0), the resulting error is <0.03%). Since the actual uncertainty due to animal-to-animal variability is approximately 10-fold greater, no benefit could be gained by improving the resolution of the camera or the quality of the lenses, or by implementing correction factors for refraction at the air–bath interface. Last, although uncertainty in the calibration of pixels per millimeter has no effect on the measured length ratios, it is characterized for completeness. From uncertainty in measuring the acrylic cylinder diameter, the number of micrometers/pixel was found to vary up to 0.3% (i.e., ±0.1 μm/pixel of 28 μm/pixel).