2.3. Inflation testing

Stiffening agents were evaluated by comparing mechanical strain measurements (stiffened versus control regions) during whole globe inflation tests. We modulated the IOP of each eye while submerged in a PBS bath at physiological temperature. Calibrated stereo cameras (including compensation for refraction through PBS) imaged a speckle pattern on the surface of the eye throughout the inflation test, and three-dimensional digital image correlation (DIC) was used to quantify surface strain (Q-400 DIC; Dantec Dynamics, Holtsville, NY).

The eye was submerged in a temperature-controlled, PBS-filled plastic chamber (Kritter Keeper; Lee's Aquarium & Pets, San Marcos, CA) during experimentation. To model physiological conditions ex vivo, the temperature of the PBS in the chamber was maintained at 37°C ± 2°C throughout the experiment by pumping saline through a thermoelectric heater assembly (LA-045-24-02-00-00; Laird Technologies, London, UK; temperature controller TC-XX-PR-59; measured by thermistor TC-NTC-1 immersed next to the eye) using a peristaltic pump (BT300 L; Golander LLC, Duluth, GA; pump head DT15-44; tubing no. 25 (ID 4.8 mm, OD 8 mm)) at 60 ml min⁻¹. This low flow rate was selected so as not to produce any turbulence and resultant optical distortion in the PBS around the eye.

To avoid evaporation of PBS during experimentation, a 1/8″ thick borosilicate glass sheet was placed over the chamber and warmed to 70°C to prevent condensation (3682K25; McMaster Carr, Douglasville, GA; PID controller 36815K71). The mounted eye was then illuminated from above with dual gooseneck lighting (Mi-LED-US-DG; Dolan-Jenner Industries, Boxborough, MA).

An adjustable-height pressure reservoir [36] was connected to the base of the chamber through silicone tubing connected to a bulkhead fitting. This presented a female luer connection on the inside surface of the chamber where we could attach mounted eyes and modulate their IOP using hydrostatic pressure.

Prior to experimentation, custom-made mounting blocks (figure 2) were manufactured from acrylic sheets (8560K369; McMaster Carr, Douglasville, GA). A 1/4″ diameter ball end mill created a hemispherical cradle for rat eyes, and a thin channel was drilled through the block with a 1/16″ drill bit. This hole was widened opposite the indentation for the eye using a 3/16″ drill bit that could accept a luer fitting adaptor.

Side view of the acrylic mounting block. Eyes are placed in the hemisphere at the top, and a threaded luer fitting mates with the hole in the bottom.

Following overnight (16 h) incubation, the orientation of the eye relative to the solution was recorded. The cornea was blotted dry with a Kimwipe, and a small, continuous bead of gel superglue was applied along the inner rim of the mounting block hemisphere. The eye was then placed onto the hemisphere, cornea-side down, with the optic nerve centred upwards and excess glue was scraped away. The mounting block was marked with a waterproof marker to record the region of the eye that was incubated in stiffening solution.

In order for DIC to evaluate displacements, a speckle pattern must be applied to the tissue. For this study, the speckle pattern was applied to the posterior sclera with graphite powder (no. 970 PG; General Pencil Company, Inc., Redwood City, CA). Graphite was poured onto a fine mesh sieve (tensile bolting cloth no. 60; Amazon), and an airbrush was used to blow the powder through the sieve onto the external surface of the eye and allowed to dry briefly. This method was repeated until the graphite powder formed a speckle pattern that did not detach from the surface of the eye when submerged in PBS. Eyes were immersed in ice-cold PBS until testing began.

Prior to testing each day, the PBS chamber was filled and heated to temperature, and the intrinsic stereo-calibration parameters of the cameras were determined using a standardized chessboard calibration target. To inflate the eye, the cornea was punctured by inserting a 1 mm biopsy punch through the 3/16″ hole in the mounting block and twisting gently until slight collapse of the eye was observed. Care was taken to ensure that the eye did not detach from the mounting block, nor that the biopsy punch deeply penetrated the eye. A threaded male luer fitting (EW-45505-84; Cole-Parmer, Vernon Hills, IL) was then glued into the 3/16″ hole.

The pressure reservoir was set to the height corresponding to the baseline IOP of 3 mmHg (approximately the minimum necessary to prevent the eye globe from buckling under its own weight). PBS was injected through polyethylene tubing into the lumen of the mounting block to purge all air bubbles. The eye was then submerged in the PBS chamber 25 mm below the surface, imparting an external pressure of approximately 2 mmHg to the eye, and attached to the luer fitting at the base of the chamber connected to the pressure reservoir. Extrinsic camera calibration parameters were then determined after the eye was mounted to account for refraction through the borosilicate glass sheet and PBS [37].

Effective IOP was calculated by subtracting the external pressure on the eyes (2 mmHg from the tissue bath) from the internal hydrostatic pressure from the reservoir. Images were captured every 30 s at an exposure time of 20 ms for 30 min (see DIC system characterization results) at each of three pressures: 3 (low/hypotensive IOP), 13 (normal/normotensive IOP) and 28 mmHg (high/hypertensive IOP). The pressure reservoir was raised after each set of 60 images to the next height via a stepper motor at a speed of 5 mm s⁻¹. Eyes were not preconditioned prior to inflation testing.

Dantec's Istra 4D software (v. 4.4.1) was used to compute displacement and the resulting principal strains from the image dataset using DIC. Correlation settings were: 99-pixel facets, 45-pixel grid spacing, maximum permissible start point accuracy 0.2 pixels, residuum of 30 grey values and three-dimensional residuum of 1.1 pixels. All strain calculations were performed from smoothed displacement data using a two-dimensional bicubic spline function to the dataset. The grid reduction factor (minimizes the difference between the data point and the spline function) was set to 3 for displacement and 2 for contours, and the smoothness factor (straightens the filtered data) was set to 0 for both items.

Strain was computed relative to the reference state (3 mmHg after 30 min). Exported strain data for each image were then segmented (figure 3) in custom Matlab software (R2016a; MathWorks, Natick, MA) by manually tracing the experimental and control regions of the posterior sclera (excluding the optic nerve) based upon the markings made on the mounting block prior to testing. Relative stiffness as a per cent change between E_exp (elastic modulus in the experimental region) and E_con (elastic modulus in the control region; see appendix A for derivation) was defined as

where ɛ_con represents strain in the control region, and ɛ_exp represents strain in the stiffened region. The calculation was performed following outlier removal, as described in the Data analysis section.

DIC was used to spatially resolve the surface strains in individual eyes. (a) The speckle pattern on the posterior sclera is overlaid with manually traced masks (made prior to calculating strain) denoting the locations treated with cross-linking agent or PBS as a control, taking care not to include the optic nerve. (b) We have overlaid these same masks on the computed surface strains at an inflation pressure of 13 mmHg (normotensive). Regions of comparatively low and high strain match closely with the treatment and control zones.

DIC data are noisy, particularly when dealing with small strains, as tiny errors in displacements become amplified in strain computations. Although smoothing displacements helps minimize this type of error, we required outlier detection to remove spurious data points. Having verified that the data were normally distributed within both the experimental and control regions of each eye at each time point (Anderson–Darling normality test, p > 0.05), the median absolute deviation (MAD) was calculated according to 1.4826 times the median of the absolute values of the difference between each data point and the median [38]. Any values that were more than two MADs away from the median were considered to be outliers and removed from the dataset.

We then computed the mean and standard deviation of the first principal Lagrange strain, as this metric is sensitive to deformation in the direction of local stretching, for a given control or experimental region at each time point. The primary deformation mode of a spherical eye is expected to be a hoop deformation, which would result in in-plane extension of the sclera; thus, the principal Lagrange strains should capture this effect. Following outlier removal, we used a weighted linear fit of this strain metric (weighted by 1/σ²) using Matlab's lmfit function using strains from the final 10 min at normotensive and hypertensive IOPs each. If the slope of this fit was above 0.5 millistrain (mStrain) per minute, we assumed the eye was creeping and had not reached its steady state, and thus the eye was discarded from further analysis (2 of 73 total inflation tests were excluded under this criterion). We then recorded the intercept of fits that were not excluded as well as the 95% confidence interval (CI) of the intercept of this fit as an indication of the uncertainty of the test.

Finally, we used equation (2.1) to compute the relative stiffness at normotensive and hypertensive IOPs for both the control and experimental halves of the eye. Using a nested 2-factor ANOVA (relative stiffening as a function of pressure nested within concentration; R v. 3.3.1), we compared the relative stiffness of each ocular region as a function of treatment and inflation pressure.