The Influence of Experimental Environment on the Mechanical Properties of Porcine Iris Stroma Using Micro-Indentation

Purpose: Iris mechanical properties are important in pathophysiology of angle-closure glaucoma. Previous experiments such as micro indentation have been used to quantify such properties ex vivo. Given the varying experimental environments used in previous studies, we aimed to identify how factors such as keeping samples in phosphate-buffered saline (PBS) solution or conducting the experiments in room temperature (RT), i.e., 21°C, versus physiologically relevant 37°C affects the iris stromal mechanical properties.

Methods: Porcine eyes (N=9) were harvested within a 24-h period from 6-month-old pigs (4 male, 5 female). The irides were excised and their posterior surfaces were attached to a flat surface with a biocompatible adhesive. Three irides were used for 3 experimental groups: 1) sprayed with PBS, 2) submerged in RT PBS, and 3) submerged in 37°C PBS. The iris from each group was then indented in 6 clockwise points starting at 1 o’clock then moving to 3, 5, 7, 9, and 11. A spherical indenter (Mach-1, Biomomentum, Canada) with 0.5mm diameter was used to indent the iris to 0.15mN at 0.005mm/s. The samples were then kept in the same environment for 20 minutes before being submerged in 37°C PBS. The indentation was then repeated. The load-displacement data were used to calculate the elastic modulus E using established mathematical methods. Two-way ANOVA and Tukey post-hoc test were used for statistical analysis.

Results: The mean moduli of initial experiments were 4.29, 3.73, 3.35kPa, for groups 1, 2, and 3, respectively. The respective mean moduli after all samples were submerged in 37°C PBS were 4.66, 2.98, and 2.13kPa. We found no statistical difference between the before and after conditions, but there was a significant difference between the group interactions. With 95% confidence, the post-hoc test revealed there was no significant difference between any of the groups except for group 1 and group 3 (p=0.02).

Conclusions: It is important that ex vivo experiments mimic physiological conditions. Our study indicates that mechanical properties of iris stroma may not change significantly if the samples are tested in room temperature as compared to more physiologically relevant 37°C in the given timeframe of our experiments.