Variability in Posterior Laminar Surface segmentation and colocalization does not affect the detection of significant increases in retrolaminar Myelin Onset Distance in Non-Human Primate Early Experimental Glaucoma

Abstract

Purpose : We previously reported that retrolaminar myelin onset distance (MOD) in structurally intact axons was further behind the lamina in the experimental glaucoma (EG) eye of n=4 EG vs Control (C) eye comparisons from n=2 non-human primates (NHPs) with unilateral early EG.¹ Here we assess the effect of three independent colocalizations of the 4X microcomputed tomography (µCT) posterior laminar surface (PLS) segmentation (see methods) to the scanning block-face electron microscopy reconstruction (SBEMR) on our ability to detect significant increases in EG eye MOD within the single EG vs C eye comparison that demonstrates the smallest EG vs C eye increase in MOD.

Methods : In both the EG and C eye SBEMRs, (each 200 x 200 x 50 microns), axons had previously been randomly selected and traced through the block so that the myelin onset point in each axon could be identified and MOD measured relative to the PLS. To create a PLS in the SBEMR, we first segmented and surfaced the posterior-most laminar beams within the µCT Digital Reconstruction of its Source Vibratome Tissue Section (µCTDRSVTS) because its 100 µm thickness increased the number of identifiable beams. To transfer the µCTDRSVTS PLS into the SBEM, we segmented all vessels and their junction points within the SBEMR, and then sought to identify and segment the same vessels and junction points within the µCTDRSVTS. Using the fiducial junction points in each volume, the SBEMR was 3D colocalized into the µCTDRSVTS using a “Closest Point Algorithm” allowing the PLS surface to be transferred into the SBEMR as facet points from which an SBEMR PLS surface was created.

Results : While some differences in the tilt of the PLS is evident in both the EG and C eye SBEMRs, among the Original, Reproducibility1 and Reproducibility2 versions of the PLS (Figure 1), survival curve comparisons demonstrate EG vs C eye increases in MOD (p<0.001) within each version of the reproducibility data (Figure 2).

Conclusions : Our data suggest that variability in our use of the µCTDRSVTS to generate a PLS that is then colocalized into the SBEM, did not prevent us from consistently detecting EG eye increases in the retrolaminar MOD of structurally intact axons in NHP early EG.
¹Stowell_IOVS_2019 ARVO Abstract; 60(9):5658

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.