Axonal and Non-axonal Contributions to Circumpapillary Retinal Nerve Fiber Layer in Experimental Glaucoma

Abstract

Purpose : Circumpapillary retinal nerve fiber layer (RNFL) thickness is commonly used to assess glaucoma-associated retinal ganglion cell (RGC) loss. In addition to RGC axons, this layer also includes glial and vasculature components. Each of these constituents is known to change with glaucoma progression, but their contribution to RNFL thickness is not fully understood. This study aimed to determine the axonal and non-axonal contributions to circumpapillary RNFL thickness in the non-human primate (NHP) experimental glaucoma model.

Methods : Unilateral experimental glaucoma was induced in five animals (Macaca mulatta, age range: 4.8-8.9 yrs) by lasering the trabecular meshwork. Animals were monitored with optic nerve head imaging to varying endpoints. Following final optical coherence tomography imaging, animals were euthanized and perfusion fixed. After 24hrs in fixative, six circumpapillary NFL regions, approximately 300-1000µm from the optic nerve head rim margin, were carefully dissected and prepared for serial block face scanning electron microscopy. A single high-resolution image of the block face was captured for analysis. Each axon in a 60µm region of the NFL was manually outlined and quantified.

Results : Circumpapillary RNFL thickness of the experimental eyes ranged from 49 to 99 µm (control eyes: 103 to 111 µm). Experimental eyes had reduced axon counts in each sector compared to controls (5531±1695 vs. 10301±4173, P<0.05). Each of the sectors analyzed, other than the superior nasal, had reduced axon density in experimental eyes, compared to the control eyes (P<0.05). Axons on the scleral third of the RNFL were smaller compared to the vitreal third in both control and experimental eyes (controls: 0.39 vs. 0.52 µm², P<0.05; experimental eyes: 0.39 vs. 0.46 µm², P<0.05). Though the proportion of non-axonal components of the RNFL was greater in experimental eyes compared to controls (34% vs. 24%, P<0.05), the non-axonal area was not significantly different between the two eyes (1310±206 vs. 1495±278 µm², P=0.19).

Conclusions : Our findings suggest that while there is a reduction in axon number and density in eyes with experimental glaucoma, the total area occupied by non-axonal tissue does not significantly change. When assessing in vivo circumpapillary RNFL thickness, the ratio of axonal and non-axonal tissue should be considered.

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