Quantitative Microstructural Analysis of Cellular and Tissue Remodeling in Human Glaucoma Optic Nerve Head

Abstract

Purpose To measure quantitatively changes in lamina cribrosa (LC) cell and connective tissue structure in human glaucoma eyes. Methods We studied 27 glaucoma and 19 age-matched non-glaucoma postmortem eyes. In 25 eyes, LC cross-sections were examined by confocal and multiphoton microscopy to quantify structures identified by anti-glial fibrillary acidic protein (GFAP), phalloidin-labeled F-actin, nuclear 4′,6-diamidino-2-phenylindole (DAPI), and by second harmonic generation imaging of LC beams. Additional light and transmission electron microscopy were performed in 21 eyes to confirm features of LC remodeling, including immunolabeling by anti-SOX9 and anti-collagen IV. All glaucoma eyes had detailed clinical histories of open-angle glaucoma status, and degree of axon loss was quantified in retrolaminar optic nerve cross-sections. Results Within LC pores, the proportionate area of both GFAP and F-actin processes was significantly lower in glaucoma eyes than in controls ( P = 0.01). Nuclei were rounder (lower median aspect ratio) in glaucoma specimens ( P = 0.02). In models assessing degree of glaucoma damage, F-actin process width was significantly wider in glaucoma eyes with more damage ( P = 0.024), average LC beam width decreased with worse glaucoma damage ( P = 0.042), and nuclear count per square millimeter rose with worse damage ( P = 0.019). The greater cell count in LC pores represented 92.3% astrocytes by SOX9 labeling. The results are consistent with replacement of axons in LC pores by basement membrane labeled by anti-collagen IV and in-migrating astrocytes. Conclusions Alteration in LC structure in glaucoma involves migration of astrocytes into axonal bundles, change in astrocyte orientation and processes, production of basement membrane material, and thinning of connective tissue beams.

Comparison of the Biomechanics of the Mouse Astrocytic Lamina Cribrosa Between Glaucoma and Optic Nerve Crush Models

“…In human glaucoma eyes, by contrast, morphologic analysis of the LC beam and pore structure showed that greater glaucoma damage was associated with smaller pores, thinner connective tissue beams, and a greater number of cell nuclei in the LC pores, suggesting a migration of astrocytes into the former axonal bundle pores. 13 In contrast, morphologic analysis of the LC of early glaucoma monkey eyes reported both thinning and thickening of the connective tissue beams, where areas with thinner beams had greater pore diameters, and areas with thicker beams had smaller pore diameters. 64 We are presently comparing the differential gene expression between healthy, crush injury, and glaucoma mouse models 65 to help explain the differences in the ex vivo biomechanical strain behavior found in this study and the differences in the microanatomy found elsewhere.…”

“… 12 Reduction in connective beam thickness and decrease in astrocyte area coverage of the human glaucoma lamina cribrosa (LC) are features of more advanced disease. 13 Deformation of the ONH caused by a change in IOP in the living human eye has been estimated using optical coherence tomography (OCT). 14 – 19 The studies suggest from cross-sectional data that the more advanced the glaucoma, the greater the strains due to a change in the IOP, specifically the greater the maximum principal and maximum shear strains, 17 the greater the effective strain, 18 and the greater the anterior–posterior strain.…”

A Noninvasive Clinical Method to Measure in Vivo Mechanical Strains of the Lamina Cribrosa by OCT

“…Conceivably, the greater strains we have estimated in the LC with worse damage could result from greater glaucoma damage, causing remodeling in which LC beams become thinner. 23 Indeed, LC curvature in inflation testing was greater with greater damage, implying a more compliant response ( Invest Ophthalmol Vis Sci. 2021;62:1656).…”

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

“…In samples immunostained for GFAP, a common astrocyte marker, DiOlistically-labeled cells with astrocyte morphologies were GFAP + . Detailed structural analysis of GFAP, F-actin, nuclei, and collagen beams in the healthy and glaucomatous human LC (Guan et al, 2022) has provided important insights into the differences of these elements between health and advanced disease. However, detail of individual LC astrocyte morphologies cannot be determined through visualization of these elements alone.…”

“…DiOlistics relies on stochastic labeling of cell membranes and is not inherently discriminant of cell type. Astrocytes are abundant in the LC, accounting for over 90% of nuclei in the region, (Guan et al, 2022) with cell bodies largely oriented along the coronal plane. Therefore, many cells labeled in these coronal slices were astrocytes.…”