Serial Block-Face Scanning EM (SBEM) Provides Evidence for Retrolaminar Demyelination of Structurally Intact Axons in Non-Human Primate (NHP) Early Experimental Glaucoma (EG)

Abstract

Purpose : To test the hypothesis that structurally intact retrolaminar optic nerve (ON) axons are demyelinated in NHP early EG.

Methods : Unilateral early EG¹ NHPs (n=8) were perfusion fixed, EG and Control (C) eyes were enucleated, and ONs were axon counted²_. For each eye, the optic nerve head (ONH) was trephined, and serial 100µm vibratome sectioned parallel to the OCT fovea to Bruch’s membrane opening (FoBMO) axis (Fig.1). Each section was imaged and each image colocalized to a fundus photograph allowing sectoral post-mortem ON axon loss to be estimated.² To guide locations of 3D reconstruction data by SBEM, x-ray microscopy and tomographic reconstructions (μCT) were obtained from the same resin-embedded samples, and used to precisely select the x-y-z location of the 200 x 200 x 50 μm region of interest (ROI). The posterior-most laminar beams were segmented and best fit with a posterior laminar reference surface (PLRS). All axons were manually identified within a digital section-image parallel to the vitreous block surface. Axons were randomly selected and 3D traced, until they ended (non-intact) or left the block (intact). For each axon, myelin onset was marked, if present, and myelin onset distance (MOD) was measured along the axon from the PLRS (Fig. 2). Intact axons without myelin were labeled unmyelinated and the full retrolaminar distance was used. Axons were traced until 50 axons with a MOD were identified. Cox proportional hazards models were used to compare MOD between axons in each EG ROI and axons in a pooled C of 4 ROIs.

Results : To date, 6 ROIs have been studied: 2 ROIs from 2 sectors of a single early EG eye (NHP1-EG1 with 8% estimated axon loss, and NHP1-EG2 with 15% loss), 2 ROIs from the matched contralateral C eye sectors (NHP1-C1 and NHP1-C2) (Fig. 2) and 2 ROIs from a second NHP C eye (NHP2-C1 and NHP2-C2). Axons in both EG eye ROIs were unmyelinated further from the posterior lamina than in the pooled C eye ROIs (p<0.0001, Fig. 2). The median MODs from the survival analysis were 49.6μm for the pooled C eye ROIs; 165.6μm for NHP-EG1; and 125.5μm for NHP1-EG2.

Conclusions : We describe a novel SBEM colocalization technique and report preliminary findings that suggest structurally intact axons are demyelinated in the immediate retrolaminar ON in NHP early EG¹.

¹He et al. IOVS. 2014
²Reynaud, et al. IOVS. 2012

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.