There were several limitations in our study. First, the sample size (
n = 4) for each group was small. However, the biomechanical contrast between the two groups was consistent with the biochemical contrast, and both comparisons reached statistical significance with the present sample size. The current results, thus, serve as a starting point for future mechanical studies on this canine glaucoma model. Second, IOP was measured only once in the eight young dogs. Previous reports have shown that the affected animals of this selected age range (5–7 months) have not yet developed ocular hypertension.
6,28 Our monthly diurnal IOP data collected from a representative subgroup of the animals in this colony also validated that IOP was not significantly different between the affected dogs and controls of this age range. Future studies should characterize the detailed diurnal IOP curves of the studied animals to capture fully the dynamic IOP information. Third, the use of uniaxial tensile testing on excised strips has multiple drawbacks as noted in previous studies with excised corneoscleral tissue, including the deviation from the natural loading condition, which is multiaxial; lack of incorporation of natural curvature; severed collagen fibrils; and alignment of collagen in the loading direction, which all may alter the mechanical properties of the tissue.
26,54,55 Thus, the mechanical properties reported in our study are most relevant for relative comparisons between the two groups. We recently have developed an inflation testing method based on high-resolution ultrasound,
56 which will enable future mechanical testing on whole globes under physiologic loadings. Fourth, the hydroxyproline assay and histology staining were performed on tissue following mechanical testing and flash freezing. These steps most likely introduced a certain degree of collagen swelling in addition to degradation. However, careful analysis of the H&E stains for all specimens revealed that the collagen appeared to be intact, consistent between specimens, and comparable to previously reported H&E staining of sclera.
57 Previous studies also found only mild changes in collagen content due to short freezing times of soft tissues.
44,58 The hydration level of the sclera strips also was unknown, and could be greater than that of fresh, intact sclera due to the potential swelling during transport, mechanical testing, and freeze/thaw. This most likely decreased the magnitudes of μg collagen/mg wet weight for each strip, potentially making these data less comparable to the collagen content of fresh scleral tissue. However, all strips were put through the same mechanical testing, flash freezing, and weighing protocols, allowing for the relative comparison between groups and correlations with mechanical properties, as was the objective of our study. Fifth, the amount of insoluble collagen was a convenient but indirect way of measuring collagen cross-linking. Future analysis with methods, such as HPLC, will be necessary to measure directly the amount and types of cross-linking within the tissue.