Abstract
Purpose :
Trabecular meshwork (TM) biomechanics is important for influencing aqueous humor outflow dynamics, and TM stiffness is elevated in primary open angle glaucoma. Notably, outflow through the TM is not uniform, and previous work in human donor eyes has shown that low flow (LF) regions of the TM are stiffer than high flow (HF) regions, as measured by atomic force microscopy (AFM) (Raghunathan et al., 2018); however, differences in stiffness between HF and LF regions in animal models are unknown. Here we investigate differences in stiffness between HF and LF regions of the mouse TM.
Methods :
Eyes from 4-month-old wild-type C57BL/6 mice (n=1 male, 2 females) were perfused in vivo with carboxylate-modified FluoSpheres (100 nm) at 15 mmHg for 1 hour to assess flow patterns. Each eye was dissected into quadrants and imaged by fluorescence microscopy (Fig A). A custom MATLAB script was used to quantify fluorescence distribution and identify HF and LF regions (Fig B). 16 µm thick sagittal cryosections were collected from both flow regions, and, with the observer masked to the region, an MFD-3D AFM (Asylum Research) was used to measure tissue stiffness via force mapping with a 10 µm spherical probe (Fig C). Force maps were repeated to quantify agreement between measurements at the same location, and the Hertz model was used to fit force-displacement curves and calculate Young’s modulus. Outliers were removed based on model fit, test-retest agreement, and indentation depth. Modulus values were log-transformed to obtain a normal distribution, and a paired t-test was used to compare the geometric means from HF and LF regions of each eye.
Results :
Test-retest agreement in Young’s modulus values was excellent, with linear regression of the repeated measurements showing average R2 = 0.97. LF regions were stiffer than HF regions (p=0.21), with average geometric mean = 23.8±14.0 kPa in LF regions vs. 13.9±4.59 kPa in HF regions; however, results between mice were highly variable. In the two females, the LF regions were consistently stiffer than HF, but in the male mouse, the HF region was stiffer (p < 0.001).
Conclusions :
Consistent with results in human eyes, LF regions of the TM tended to be stiffer than HF regions in healthy mice, but more animals will be tested to verify this trend and identify sources of variability. These data provide a baseline for studying differences in stiffness between HF and LF regions in a mouse model of glaucoma.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.