Evidence of axonopathy during early-stage experimental glaucoma: relationship between in vivo imaging and histological findings

Abstract

 

Purpose

 

To compare results of longitudinal in vivo retinal ganglion cell (RGC) function testing and retinal nerve fiber layer (RNFL) imaging with post mortem histology in a non-human primate (NHP) model of experimental glaucoma (EG).

 

Methods

 

Longitudinal measurements of RNFL thickness (RNFLT, Spectralis SDOCT), RNFL retardance (GDxVCC) and multifocal electroretinography (mfERG, VERIS) were obtained in N=7 NHPs with unilateral EG. SDOCT scans centered on the optic nerve head (ONH) included single circular B-scans and 15x15 deg cube scans with 290 raster lines each. RGC function was measured specifically as the amplitude of mfERG high-frequency components (HFC, >75 Hz). Once the EG eye of each NHP demonstrated reliable loss of RNFLT (≥7% below baseline, BL, confirmed ≥1x), both eyes were enucleated after pentobarbital overdose and perfusion fixation for histology. Each retinal wholemount was evaluated by immunohistochemstry and confocal microscopy using antibodies against the axonal cytoskeletal components neurofilament (NF) and beta-III-tubulin (TUB) and the glial fibrillar acidic protein (GFAP). 100% axon counts were obtained from each optic nerve (ON).

 

Results

 

In EG eyes, final in vivo measurements of RNFLT were 20.0 ± 5.8% below fellow control eyes (CTL). Loss of RNFL retardance (30.0 ± 9.5% below CTL) and mfERG HFC (31.7 ± 10.6% below CTL) were both significantly worse than RNFLT (p=0.03 each). ON axon loss was also substantially worse than RNFLT ranging from 20-60% (mean 42 ± 19%) below CTL. The relationship between in vivo measures and ON axon counts differed significantly (p=0.03) such that retardance (p=0.03) and RGC functional loss (p=0.02) were worse than RNFLT changes at any level of axon loss. Axonal cytoskeletal disruption was profound, worse for NF than TUB and greatest close to the ONH, with little evidence of GFAP alteration (Fig 1). Areas of reduced peripapillary RNFL retardance as well as reduced RNFL reflectance imaged by SDOCT corresponded with areas of greatest axonal cytoskeletal disruption (Fig 2).

 

Conclusions

 

RNFL retardance abnormalities, RGC functional changes, RNFL axonal cytoskeletal disruption and ON axon loss all exceed early loss of RNFLT in NHP EG. Axonal cytoskeletal changes manifest in vivo as reduced RNFL retardance and reflectance and are greatest near (and within) the ONH.

 

 

Fig1. NHP 25997 retinal histology

 

 

Fig2. NHP 25997 SDOCT scans

 

Keywords: 531 ganglion cells • 493 cytoskeleton • 552 imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound)