Abstract
Purpose :
In healthy sclera, actin and collagen fibrils are highly aligned. We hypothesized that elevated intraocular pressure cause a loss of alignment. To test this hypothesis, we conducted a quantitative and non-biased analysis of actin-collagen alignment and nuclear morphology in mouse peripapillary (PPS) and peripheral sclera 7 days and 6 weeks after IOP elevation.
Methods :
Serial cross-sections from the PPS and peripheral regions of control, 7-day, and 6-week microbead-induced (BI) ocular hypertension (OHT) mouse eyes were fluorescently labelled and imaged using confocal microscopy (for nuclei and fibrillar actin) and second harmonic generation (SHG) imaging (for collagen fibers). For each image, regional fibrillar actin (FA) alignment with collagen fibers was quantitatively assessed using a vector field-based approach to analyze vector orientation in 4096 x 64 µm2 regions - zero degrees being complete alignment and 90 degrees being perpendicular/poor alignment. Nuclear morphology was assessed using VAMPIRE – an unsupervised machine learning image analysis program that divides input nuclei into different shape modes.
Results :
In control eyes, mean FA collagen alignment was greater in PPS (7.1°) than peripheral sclera (10.7°, p=0.02). At 7 days, median FA-collagen alignment in the peripheral sclera (15.0°, n=8) and PPS (14.9°, n=8) decreased compared to control peripheral (p=0.05) and PPS (p=0.03). At 6 weeks, alignment in the peripheral sclera returned to control levels (11.6°; p=0.12, n=6), however, alignment in PPS remained reduced (11.5°, p=0.004). Poor alignment (>40° misalignment) in the PPS at 6 weeks was seen in 14% of vector regions per image versus only 6% of vectors in control PPS (p=0.003). VAMPIRE analysis of nuclear structure identified two PPS regions with the inner layer having a greater proportion of high aspect ratio nuclei which was reflected in the lower Shannon Entropy of the inner (2.07) versus outer (2.14) PPS. Nuclear morphology remained stable following IOP elevation for 7 days; however, there was a significant decrease in one high aspect ratio shape mode’s frequency in glaucomatous (10%) versus control (15.1%) PPS.
Conclusions :
Ocular hypertension induced a temporary reduction of FA-collagen alignment in the peripheral sclera with a sustained reduction in alignment in PPS. Subtle alterations in nuclear morphology were detected in the PPS but not in the peripheral sclera.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.