Comparative analysis of rodent lens morphometrics and biomechanical properties

Abstract

Introduction: Proper ocular lens function requires biomechanical flexibility, which is reduced during aging. As increasing lens size has been shown to correlate with lens biomechanical stiffness in aging, we tested the hypothesis that whole lens size determines gross biomechanical stiffness by comparing lenses of varying sizes from three rodent species (mice, rats, and guinea pigs).

Methods: Coverslip compression assay was performed to measure whole lens biomechanics. Whole mount staining on fixed lenses, followed by confocal microscopy, was conducted to measure lens microstructures.

Results: Among the three species, guinea pig lenses are the largest, rat lenses are smaller than guinea pig lenses, and mouse lenses are the smallest of the three. We found that rat and guinea pig lenses are stiffer than the much smaller mouse lenses. However, despite guinea pig lenses being larger than rat lenses, whole lens stiffness between guinea pigs and rats is not different. This refutes our hypothesis and indicates that lens size does not solely determine lens stiffness. We next compared lens microstructures, including nuclear size, capsule thickness, epithelial cell area, fiber cell widths, and suture organization between mice, rats, and guinea pigs. The lens nucleus is the largest in guinea pigs, followed by rats, and mice. However, the rat nucleus occupies a larger fraction of the lens. Both lens capsule thickness and fiber cell widths are the largest in guinea pigs, followed by mice and then rats. Epithelial cells are the largest in guinea pigs, and there are no differences between mice and rats. In addition, the lens suture shape appears similar across all three species.

Discussion: Overall, our data indicates that whole lens size and microstructure morphometrics do not correlate with lens stiffness, indicating that factors contributing to lens biomechanics are complex and likely multifactorial.

Keywords: allometry; lens biomechanics; lens microstructures; lens stiffness; morphometrics.

Figure 1

Lens and nucleus size are significantly different among different rodent models. (A, B) Top view and side view images of lenses and (C) side view images of lens nucleus from mouse, rat, and guinea pig (GP). Scale, 1 mm. (D) Calculated gross lens volumes, (E) equatorial to axial diameter ratio of lenses (aspect ratio), (F) Calculated nuclear volume and (G) nucleus to lens fraction in mouse, rat, and GP. N= 6-14 lenses per species. **p <0.01: ***p <0.001 ****p < 0.0001.

Figure 2

Mouse lenses are significantly softer than rat and guinea pig lenses. (A) Side-view images of mouse, rat, and guinea pig (GP) lenses captured during the coverslip compression assay, under varying levels of compression: 0, 2, 4, 10, 14, 20, and the release of 20 coverslips (-20 CS). Scale bars, 1 mm. The calculated (B) axial and (C) equatorial strain of the lenses under compression indicate that rat and GP lenses exhibit similar biomechanical properties, whereas mouse lenses are notably softer in comparison to both. N= 10-13 lenses per species. (D) % of pre-compression axial diameter reveal that the rodent lenses can recover to near their original size after the removal of 20 coverslips. N=9-19 lenses per species. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Blue and red asterisks indicate that mouse lenses are significantly different from rat and GP lenses, respectively.

Figure 3

Capsule thickness is significantly different among different rodent models. Fixed lensed were labeled with WGA (green) for lens capsule and phalloidin (red) to visualize basal F-actin in epithelial cells at the epithelial-capsule interface. (A) representative (XZ reconstruction) confocal images of the mouse, rat, and guinea pig (GP) anterior lens capsule. Scale bar, 20 µm. (B) The anterior capsule is significantly thicker in GP compared to mice and rats, with rats having the thinnest anterior capsule among the three species. The plot represents the mean ± SD of 6 lenses per species. *p<0.05; **p<0.01; ****p < 0.0001.

Figure 4

Lens epithelial cell and fiber cell widths are greater in guinea pig lenses. Fixed whole lenses were labeled with phalloidin (gray scale) to visualize F-actin at cell boundaries, and Hoechst (blue) to visualize epithelial cell nuclei. Representative confocal images of (A) anterior epithelial cells and (B) equatorial fiber cells from mouse, rat, and guinea pig (GP) lenses. In B, the yellow letter indicates the number of fiber cells. (C) The average anterior epithelial cell area measurements reveal that GP cell area is significantly higher than both mouse and rat cell area. (D) Fiber cell width measurements reveal that GP lenses have wider fiber cells compared to mouse and rat lenses. Additionally, mouse fiber cell widths are significantly greater than those of rat lenses. N=4-6 lenses per species. ****p < 0.0001.

Figure 5

Suture organization in mouse rat and guinea pig lenses are similar. Lenses were stained with phalloidin (grayscale) to stain F-actin at cell boundaries and Hoechst (blue) to stain nuclei (only present at the anterior region of the lens). This revealed that Y-shaped sutures at the lens’s anterior (top panels) and posterior (bottom panels) regions are similar in mouse, rat, and guinea pig (GP) lenses. Scale bars, 100 µm.

Figure 6

Summary diagram of the lens morphometrics and microstructural features in mouse, rat, and guinea pig. Lens volume is smallest in the mouse, followed by the rat and guinea pig (GP), while the nuclear fraction is highest in the rat. The mouse lens exhibits the highest axial and equatorial strain under compressive load, indicating a softer lens compared to the rat and GP. The lens capsule is thickest in the GP and thinnest in the rat. Anterior epithelial cell area is largest in the GP, while fiber cell width is smallest in the rat, followed by the mouse and then the GP. Created with BioRender.com.