We have developed a quantitative method of characterizing mechanical stability of the cornea and sclera in vitro that minimizes tissue damage during specimen preparation and mounting and retains the loading geometry that occurs in vivo. Digital imaging enables intact globe expansion measurements22
to be performed without mechanically constraining the globe20
or attaching a strain gauge.23
The present method incorporates desirable features of prior experimental methods, using image analysis to achieve sensitive, noncontact measurement of globe dimensions22
and controlling the environment around the eye to minimize changes in hydration during measurement.20
The measured parameters have correlates in vivo (corneal and scleral length are directly related to axial length)28
and in vitro (corneal and scleral perimeters are related to extension measured in tensile tests). The intact globe expansion method enables sensitive discrimination between specimens at slight strains and low strain rates. Application of this method reveals that rabbit kit eyes can serve as a model tissue mimicking the poor mechanical integrity characteristic of keratoconus and degenerative myopia. Treating this weak tissue with riboflavin/UVA or with glyceraldehyde strengthens it beyond the level of normal adult tissue.
We suggest that GEM with immature tissue could be used to optimize treatment parameters in vitro (e.g., to meet or exceed a benchmark in strength, such as the change produced by riboflavin/UVA treatment or the strength of normal tissue). The ability of GEM to discriminate between different treatments in small samples makes it an attractive tool for comparative studies of efficacy of collagen cross-linking.
Such in vitro studies could guide the selection of drug and irradiation combinations to advance to in vivo experiments. Furthermore, GEM may prove useful in conjunction with in vivo studies, providing a method of evaluating differences in mechanical stability between treated and fellow globes that were treated in vivo, then characterized postmortem. It is not intended to replace in vivo studies.
One challenge facing those conducting in vitro studies is the change in the hydration state of the tissue during transportation, dissection, and experimental measurement. Limited pachymetry readings on untreated kit eyes with intact epithelium showed that the corneas did swell during shipment in saline (received corneal thickness was 530 ± 50 μm), as expected based on prior literature.29
During shape restoration, the corneal thickness of untreated eyes decreased to 390 ± 20 μm (in vivo thickness is ~300 μm).30
During expansion at high pressure, thickness measurements were not obtained; future experiments can be designed to facilitate thickness measurements without disturbing the eyes. However, there was no indication of swelling during the intact globe expansion (e.g., corneal transparency did not significantly change over the 23 hours of the experiment). Nor was there any indication that swelling markedly changed the tissue mechanical response (e.g., creep rates were indistinguishable for untreated kit eyes with intact epithelium and dextran controls that were de-epithelialized and given drops of 20% dextran for 1 hour). Therefore, we attribute the observed changes in mechanical stability to collagen cross-linking. Future experiments could evaluate the possible effects of swelling by modifying tissue handling and the environment (e.g., examine eyes immediately after enucleation and replace DPBS with corneal storage medium).
Precise characterization of tissue mechanics requires repeatable techniques that measure changes from a well-defined initial state. The biomechanics literature describes a plethora of techniques for establishing the initial conditions (e.g., applying a cyclic load or deformation, deforming the tissue until a selected stress is recorded or applying a small stress until deformation ceases). Although any of these methods could be implemented with GEM, we chose to restore in vivo shape in a simple manner. When the eye is enucleated, there is a drop in IOP, and handling the eyes without the stabilization provided by the IOP causes each specimen to have a perturbed shape, different from its in vivo shape. Using GEM with an intact globe allows its natural constraints to be used in restoring a shape analogous to that in vivo. The protocol we used is based on physiological data (we applied an IOP that is slightly above the physiological range). The results showed very good reproducibility, perhaps as a consequence of using an IOP that was high enough to eliminate any variations between eyes that had different IOPs in vivo, while not producing a gross continual creep. The time necessary for each eye to reach a stable shape was found to be less than 30 minutes for all 11 eyes examined (). After reaching a stable shape, the sclera did not undergo statistically significant creep (, left column) and the cornea exhibited very gradual creep (strain at <0.02%/min; , left column). If the shape restoration period is too short, then the initial creep measurements will show artificially high creep rates with larger variability. Therefore, we chose to allow more than 30 minutes, to ensure minimal variability in the initial condition while keeping the shape restoration time short enough that negligible creep had time to occur (hence, a 1-hour shape-restoration period was chosen).
Mechanical stability is of obvious clinical importance. In vivo, the eye sustains an IOP while maintaining its shape. Creep tests permit the study of how well the tissue can resist deformation under a constant IOP. In disease states associated with elevated pressure or with weakened tissue, the eye can become susceptible to distension. This inspired our decision to examine an intact globe using a creep test at a constant IOP instead of injecting specified volumes of liquid22
or ramping up the pressure.19,23
Rather than forcing the tissue to undergo a specific extent of deformation (e.g., a step strain imposed by injection of a known volume of liquid), we probed the ability of the tissue to resist deformation. Instead of imposing a brief window of time at each incrementally higher pressure in a continuous ramp, we allowed many hours of observation at a fixed IOP, to discover whether the tissue deforms continuously or reaches a steady shape. Treatments aimed at making the tissue stronger could be evaluated by using other mechanical methods, but creep tests are particularly appropriate for revealing whether a specific treatment can actually prevent expansion of the tissue when challenged at a particular IOP.
The IOP that the cornea can withstand without ongoing deformation is lower than that which the sclera can withstand (, ), in accordance with current knowledge of corneal and scleral mechanical properties.19,22
Although the hydration state of the tissue may shift these thresholds, a clear trend is evident: At low IOP only the cornea continues to creep, whereas at high IOP, both the cornea and the sclera undergo creep, with expansion increasing linearly with time. For the purpose of discriminating between different proposed collagen cross-linking conditions, it is useful to find an imposed IOP that produces nominal strain that is substantially greater than the experimental uncertainty. Simply stated, if the control eyes do not deform, then the treated tissues cannot be distinguished from the control. Comparison of low and high IOP illustrates the criteria for selecting a suitable IOP to use for comparative evaluation of treatments: Low IOP did not induce adequate deformation of the control specimens (, , left, open symbols), whereas high IOP applied to the kit cornea induced a deformation that was approximately five times greater than the SD of the data (, filled squares).
At the same time, the magnitude of the strain at which the experiment achieves this level of confidence should be small enough to be physiologically relevant. Therefore, methods for comparative evaluation of corneal cross-linking protocols must have small experimental uncertainty. Examination of the results in kit corneas subjected to high IOP showed that a nominal strain of 6% was sufficient (SD less than one fifth of the mean) and that consistent results were obtained over the range from 6% to 20% nominal strain. Further reduction in the uncertainty of GEM may be possible by reducing animal-to-animal variability. The most obvious direction to pursue is pair-wise comparison of eyes from the same animal. Analysis of the instrumental uncertainty shows that little can be gained by improved image acquisition or processing until other sources of variability are reduced by an order of magnitude.
A final consideration illustrated by the response to high IOP is that it generates a significant degree of expansion within 24 hours without rupturing the eyes. The experiment is short enough that the tissue does not significantly deteriorate, yet long enough that the deformation occurs gradually (~0.01%/min). It is hoped that choosing the in vitro IOP in this way will allow the experiment to reveal information pertinent to prevention of progressive changes in corneal and scleral shape that occur at normal IOP over much longer periods in diseases such as keratoconus and degenerative myopia.
In addition to selection of the appropriate IOP to impose in vitro, we considered the choice of tissue to use as a model. Andreassen et al.15
have shown that the cornea and sclera in keratoconus and myopia have reduced biomechanical stability compared with healthy tissue. In in vitro studies of the mechanical changes induced by prospective treatments for these ocular diseases, normal adult porcine eyes have been used primarily, with human donor tissue used in a few (Spoerl E et al. IOVS
1999;40:ARVO Abstract 1800).24,25
In a study of in vivo treatments and evaluation of their effects, postmortem mature rabbits with healthy ocular tissue were used.31
It is well known that mature, healthy tissue undergoes considerable enzymatic cross-linking of collagen, which increases the strength of the cornea and sclera. Collagen in young animals does not have the full complement of cross-links present in mature collagen.32
There is clinical evidence that immature ocular tissue is more susceptible to deformation: Marked axial enlargement of the globe occurs in infantile glaucoma; for example, although no such changes are noted in adult forms.33
Therefore, we propose that tissues in which this maturation process is incomplete serve as a model that is more representative of weaker, diseased tissue. The reasoning is distinct from in vivo young animal models of myopia that exploit the neurophysiological feedback system to remodel the tissue during development and emmetropization.16,17,34
Rather, the low mechanical strength of the young tissue mimics the weakness found in diseased tissue that is addressed by therapeutic collagen cross-linking.
The present comparison of tissues from 2- to 3-week-old rabbit kits and adult rabbits (>6 mo) indicates significantly greater expansion of immature globes (, , middle columns). This difference cannot be explained simply by the difference in size of the globes. For a given imposed IOP, the stress in the tissue scaled linearly with a characteristic radial dimension and inversely with a characteristic tissue thickness. We found that the ratio of the globe radial size and the tissue thickness was very similar in 2- to 3-week-old and adult rabbits (within 5%–10%, depending on the position chosen for comparing the tissue thickness). The observed differences (300% for the cornea and up to 700% for the sclera) were much greater, indicating that the kit eyes are more distensible than the adult eyes. Therefore, we regard kit eyes as the better model of weak tissue susceptible to expansion.
Comparative Evaluation of Treatments
The clinical significance of riboflavin/UVA treatment is becoming increasingly evident, motivating the development of further refinements to reduce the treatment time (~1 hour per eye), to reduce toxicity to keratocytes,35
to enable treatment of thin corneas without damaging the endothelium,36
and to enable collagen cross-linking of the sclera without retinal toxicity.26
Therefore, researchers are modifying the established riboflavin/UVA protocol (e.g., reducing light intensity, irradiation time, and concentration of riboflavin) and examining alternative agents (e.g., glyceraldehyde).9,10
In the treatment optimization process, GEM can be used for in vitro evaluation of the relative efficacy of different treatments. The present experiments readily distinguished between control corneas and corneas treated using the riboflavin/UVA procedure (, right column). Groups of four samples were sufficient to achieve statistically significant differences between riboflavin/UVA-treated and control eyes, based on a t
-test tolerating a 5% chance of a false-affirmative and a 5% chance of a false-negative conclusion (statistical analysis program, G*Power).37
Clinically, interesting comparisons would be those between diseased tissue that has been strengthened and normal tissue that is stable. As discussed, we chose kit eyes as a model of weakened tissue compared with stable adult eyes. Riboflavin/UVA treatment strengthened the weak kit cornea well beyond the normal adult rabbit cornea (, middle, right columns). The clinical success of the riboflavin/UVA corneal collagen cross-linking technique as a treatment for keratoconus indicates that treatment achieves stability; however, a milder treatment might be sufficient based on our expansion results in rabbits, which indicate that riboflavin/UVA treatment may strengthen tissue beyond normal levels. It would be interesting to study the efficacy of milder treatments. Furthermore, the alternative treatment with glyceraldehyde demonstrated greater stability than the riboflavin/UVA treatment.
In evaluating small differences between optimized treatments the testing method must be subject to little variability and must be sensitive. The variability of GEM is minimized by eliminating the need to cut tissue and the variability due to clamping or securing the tissue and by providing a method of establishing the initial size of the eye. The uncertainty of the measurement technique is well below the animal-to-animal variability. Even with the observed animal-to-animal variability, we have demonstrated that the sensitivity of this method allows for discrimination between treatments that change the ultimate strain (24 hours) by as little as 2%. To further improve sensitivity, pairs of fellow eyes given different treatments can be compared. Alternatively, increasing the IOP may create larger distension of strengthened tissue, allowing greater discrimination among treatments. Together, these techniques may provide the capability of distinguishing between treatments of different concentration, exposure time, and light intensity, for example. Finally, rabbit kit tissue affords greater sensitivity to treatment than does adult tissue, which has been used in prior studies of corneal and scleral cross-linking. The inherent stability of normal adult tissue obscures the incremental change in strength due to treatment. Indeed, a weak tissue model such as kit eyes may prove useful in conjunction with other mechanical testing methods as well.
In relation to myopia treatments, we note that glyceraldehyde cross-linking prevents expansion of the entire eye (measured changes are indistinguishable from 0% expansion). The intact globe expansion apparatus is capable of measuring differences in scleral expansion (, right column), with sensitivity similar to that for corneal expansion. Further experiments and characterization of treatments on the sclera could provide insight for development of future treatments for degenerative myopia (e.g., glyceraldehyde and nitroalcohols)9–11