REGIONAL AND TOPOGRAPHIC VARIATION OF PERIPAPILLARY SCLERAL FIBROBLAST MORPHOLOGY AND ALPHA SMOOTH MUSCLE ACTIN EXPRESSION

Abstract

Purpose : Peripapillary scleral (PPS) collagen fibers change with aging and glaucoma. We examine how structure relates to cellular behavior by comparing scleral fibroblast nuclear and cytoskeletal orientation to that of PPS matrix fibrils.

Methods : We imaged fibrillar actin (FA) and α-smooth muscle actin (αSMA), their nuclear shape, and collagen of the peripapillary region from 5 normal adult human eyes. Immunofluorescence for FA, αSMA, and nuclei was performed using phalloidin, a monoclonal antibody, and SYTOX^TM Green, respectively. Second harmonic generation (SHG) imaging was used to visualize collagen fibril structure/orientation in PPS and adjacent juxtaperipapillary (JPP) regions. Nuclear aspect ratio and density were determined using ImageJ. Vector fields were created of corresponding SHG and immunofluorescent images to quantify vector alignment of collagen and fibrillar actin (0° is perfect alignment and 90° is perpendicular).

Results : PPS had a densely cellular inner (i) region and a less cellular outer (o) region. Compared to JPP, the nuclear density/µm² was 3.6 ± 1.6 times greater in iPPS and 2.6 ± 1.1 greater in oPPS (mean ± SD, p <0.0001 and p=0.02, respectively). Nuclear aspect ratio in the iPPS was rounder than JPP (2.0 ± 0.3 vs 2.7 ± 0.5, p = 0.002), but oPPS (2.4 ± 0.4, p=0.5) did not significantly differ from JPP. FA was present in all scleral zones, with actin spacing and direction similar to that of collagen structures. Circumferentially arranged PPS FA and collagen were aligned (14.7 ± 13 °, p <0.0001). In the typical basket-weave orientation of JPP scleral lamellae, actin and collagen were less well aligned, (24.7 ± 22.4 °). Individual bundles of collagen and FA within the JPP, however, were highly aligned (7.8 ± 9.4 °). In both PPS and JPP, αSMA fibers were found associated with penetrating blood vessels and in strongly αSMA expressing cells in within intersections of non-aligned collagen lamella in the JPP.

Conclusions : Cells of normal human PPS is distinct from adjacent sclera. Cytoskeletal organization is aligned with surrounding collagen fibrils and αSMA expressing cell bodies are located within lamellar intersections in the JPP.

This is a 2020 ARVO Annual Meeting abstract.

 

SHG (a) and FA (b) of a single section of JPP shows overlap of fiber orientation (c). Heat map of a vector field comparison of SHG and FA (d) shows alignment (light squares) along collagen lamellae.