Method to assess ganglion cell layer somas in non-human primates using multifunctional AO-OCT/fSLO for translational research in glaucoma

Abstract

Purpose : Glaucoma is a multifactorial disease characterized by progressive retinal ganglion cell (RGC) death. Histological studies can identify molecular mechanisms leading to RGC death. However, approaches are needed to observe and monitor compromised RGCs in life, to determine timing of pathological changes and assess interventions aiming to promote their recovery. We have developed and tested a multifunctional imaging approach to assess RGCs in non-human primates (NHP) for translational glaucoma research.

Methods : Somas within the RGC layer (GCL) were imaged in a living NHP eye using a newly developed adaptive optics optical coherence tomography/fluorescence scanning laser ophthalmoscopy (AO-OCT/fSLO) system. We acquired 300 AO-OCT volumes of 1.5°×1.5° area at 1 mm eccentricity, which were then registered and averaged to improve image contrast. Putative RGC somas were manually segmented to quantify their dimensions and density. Ex vivo, we imaged a whole mount retina of a different NHP using a confocal microscope (Leica TCS SPE, Leica) with RBPMS staining of RGCs and DAPI staining of nuclei. RGCs and nuclei were manually segmented in the confocal images 1 mm from the fovea. A peripheral region (5 mm eccentricity) was also imaged using both confocal microscopy and multifunctional AO-OCT/fSLO for comparison.

Results : AO-OCT revealed ~2000 multi-layered somas in the GCL 1 mm from the fovea the living NHP eye (Fig. 1). Their diameter was 10.1±3.3 µm (mean±SD), slightly smaller than RBPMS-positive RGCs observed ex vivo by confocal microscopy (11.9±2.20 µm) as shown in Fig. 2; although both values fall within the expected range (6-12 µm). GCL soma density was 15,200/mm², lower than histological estimates of 26,300/mm² [1] likely due to inter-subject variability. At 5mm eccentricity, RBPMS+ soma visible by confocal microscopy had an average size of 13.9±3.6 µm and density of 1,365 /mm2, matching the AO-fSLO images of the same patch (Fig. 2); however, no GCL somas were visible by AO-OCT image after the fixation [2]. GCL nuclei were 57% more numerous and 71% smaller (p<0.01) than RBPMS+ RGCs.

Conclusions : We developed a combined imaging approach to assess somas of the NHP GCL, both in vivo and ex vivo.
[1] Perry VH, Cowey A. Vis. Res. 1985;25(12):1795-1810.
[2] Grieve K et al., IOVS. 2016;57(9):OCT96-OCT104.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.