Astrocyte Morphology in the Collagenous Lamina Cribrosa Revealed by Multicolor DiOlistic Labeling

Abstract

Purpose : Astrocytes in the lamina cribrosa (LC) play important roles in cell signaling and extracellular matrix remodeling, impacting the physiology of neural tissues necessary for vision. Although LC astrocyte morphology is known to undergo substantial changes throughout the pathogenesis of glaucoma, techniques to evaluate LC astrocyte morphology are limited. We hypothesized that multicolor DiOlistic labeling allows visualization of LC astrocytes and evaluation of key morphological features.

Methods : Gold microparticles were coated with all combinations of three fluorescent lipophilic dyes (DiI, DiD, and DiO) to create 7 different groups of microcarriers. Coronal vibratome sections were obtained through the LC of pig, sheep, and goat eyes (N = 2 sections each) at 200µm thickness. Dye-coated microcarriers were ballistically delivered into sections with a Helios gene gun. Cells were imaged via confocal microscopy and collagen was imaged via second harmonic generation (SHG). 25 total dyed astrocytes were segmented and morphological features quantified in Imaris.

Results : Microcarriers embedded within cells delivered dye that distributed across their respective cell membranes. Somas and fine ramified processes of astrocytes labeled with all 7 different combinations of dyes were visualized. Label density allowed visualization of individual astrocytes within LC pores. Distinct dye combinations allowed discerning individual astrocytes with processes in close proximity to one another, sharing spatial domains. SHG imaging allowed for visualization of astrocytes within the context of surrounding collagen beams. Average astrocyte branch number, length, depth, and straightness were 147.4 ± 106.7, 11.5 ± 10.2µm, 9.1 ± 5.3, and 0.9 ± 0.1.

Conclusions : Multicolor DiOlistic labeling in vibratome sections of collagenous LC is a suitable technique for visualization and morphological analysis of astrocytes. LC astrocytes demonstrated diverse morphologies and physical interactions with surrounding structures. Healthy LC astrocyte morphologies and interactions can later be compared with those in glaucomatous LCs to better understand the role of astrocytes in pathogenesis.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

A: LC labeled with multicolor DiOlistics. B: Detail and SHG within solid box in A. C: Segmentation of example astrocyte (dashed box in B) showing quantified morphological features. DiX = DiI, DiD, DiO.