Quantification of Retinal Ganglion Cell Morphology in Human Glaucomatous Eyes

Abstract

Purpose: To characterize retinal ganglion cell morphological changes in patients with primary open-angle glaucoma associated with hemifield defect (HD) using adaptive optics-optical coherence tomography (AO-OCT).

Methods: Six patients with early to moderate primary open-angle glaucoma with an average age of 58 years associated with HD and six age-matched healthy controls with an average age of 61 years were included. All participants underwent in vivo retinal ganglion cell (RGC) imaging at six primary locations across the macula with AO-OCT. Ganglion cell layer (GCL) somas were manually counted, and morphological parameters of GCL soma density, size, and symmetry were calculated. RGC cellular characteristics were correlated with functional visual field measurements.

Results: GCL soma density was 12,799 ± 7747 cells/mm2, 9370 ± 5572 cells/mm2, and 2134 ± 1494 cells/mm2 at 3°, 6°, and 12°, respectively, in glaucoma patients compared with 25,058 ± 4649 cells/mm2, 15,551 ± 2301 cells/mm2, and 3891 ± 1105 cells/mm2 (P < 0.05 for all locations) at the corresponding retinal locations in healthy participants. Mean soma diameter was significantly larger in glaucoma patients (14.20 ± 2.30 µm) compared with the health controls (12.32 ± 1.94 µm, P < 0.05 for all locations); symmetry was 0.36 ± 0.32 and 0.86 ± 0.13 in glaucoma and control cohorts, respectively.

Conclusions: Glaucoma patients had lower GCL soma density and symmetry, greater soma size, and increased variation of GCL soma reflectance compared with age-matched control subjects. The morphological changes corresponded with HD, and the cellular level structural loss correlated with visual function loss in glaucoma. AO-based morphological parameters could be potential sensitive biomarkers for glaucoma.

Figure 1.

Images of the right eye of a 54-year-old control subject (6289). Clinical data includes (A) fundus photograph, (B) Spectralis scanning laser ophthalmoscopy, and (C) optical coherence tomography B-scan at 2.5° superior retina, location denoted by blue arrow line in (B). There are no signs of glaucoma, such as disc-rim thinning or RNFL defects. The white rectangular box in (A) corresponds to the same region in (B) and the labeled seven white boxes in (B) are the locations where our adaptive optics–optical coherence tomography (AO-OCT) data were acquired. The white arrows in (C) corresponded with the three retinal locations (L1, L3, and L5) in (B). (D) Representative AO-OCT B-scans of the inner retina (from ILM to INL) on the left oriented vertically for comparison of hemifield differences and en face views of a single plane at the six corresponding retinal eccentricities (L1–L6). ILM, inner limiting membrane; RNFL, retinal nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer.

Figure 2.

Images of the left eye of a 51-year-old glaucoma patient (1733) with superior hemifield defect. Clinical data include (A) optical coherence tomography (OCT) macula ganglion cell layer (GCL) thickness measurement, (B) hemisphere asymmetry map, (C) peripapillary retinal nerve fiber layer thickness classification, (D) 10-2, and (E) 24-2 VF PD maps, (F) Spectralis scanning laser ophthalmoscopy macula scan, and (G) OCT B-scan at 2.5° superior retina (denoted by blue arrow line in F). The light blue dots shown in the VF maps (D and E) correspond to the AO-OCT imaged locations at 3°, 6°, and 12°, respectively. OCT B-scan showed thinning of inner retina. The three white arrowheads associate with the three superior locations (L1, L3, and L5) in F. (H) AO-OCT B-scan and en face projection (single plane) at six retinal locations (L1–L6) showed a significant decrease in GCL soma density in the superior retina correlated with the HD. Representative three-dimensional volumetric visualization of 12°(L5) data set showed in Supplemental Video S2. B-scans at the paired locations were approximately aligned to the IPL for comparison. RNFL, retinal nerve fiber layer; optic nerve head (ONH), ILM, inner limiting membrane; RNFL, retinal nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer.

Figure 3.

AO-OCT quantification of (A) ganglion cell layer (GCL) soma density, (B) Soma diameter, (C) Symmetry, and correlations between (D) Adaptive optics–optical coherence tomography (AO-OCT) measured GCL soma density and OCT (Spectralis) measured GCL thickness, (E) Adaptive optics–optical coherence tomography (AO-OCT) measured GCL soma density percentage and pattern deviation (PD), and (F) clinical OCT measured GCL thickness percentage and PD. A statistically significant difference was revealed between the glaucoma (purple) and control (green) groups in terms of GCL soma density, diameter and symmetry. Glaucoma caused cell morphology changes correlated with the hemifield defect. Open purple symbols () in (A) and (B) denote more diseased hemifield and filled purple symbols () represent relatively healthier hemifield. For the control group, open green symbols were from () superior retina and filled green symbols () are from inferior retina. In each plot, different symbols represent different subjects. In (D) data is color-coded with respect to eccentricity: 3° data in darkest color symbol and 12° in lightest color symbol. Colors in (E) and (F) are coded with respect to retinal eccentricities, and the percentage was normalized to control eyes. The black dashed line denotes x = −y that indicates an equal rate of structural and functional loss. While the true structure-function relationship is non-linear and more complex, a coarse rule-of-thumb is that datapoints above the x = −y line (log-log scaling) represent structural losses preceding functional loss and datapoints below represent the opposite. The red dashed line denotes 3 dB PD loss. P-Value < 0.01 **, and P-Value < 0.05 * (Students’ t-test). r in (D-E) is the Pearson correlation coefficient.

Figure 4.

AO-OCT revealed subcellular reflectance changes in ganglion cell layer (GCL) somas in a 51-year-old early glaucoma patient (1733) at 12° (L5). (A) Representative AO-OCT en face image showed reflectance variations of three identified cells (arrows). (B–D) Magnified regions (65 µm × 65 µm) of the cells labeled in (A). (E) Magnified region of GCL somas from the same eccentricity of a 49-year-old healthy subject. (F) Radial profiles from the center of the cell reveal a distinctive pattern associated with subcellular hyporeflectivity in comparison to normal function in control subjects.

Figure 5.

AO-OCT reveals hyperreflective structures in the defective hemifield (inferior retina) in the right eye of a 62-year-old glaucoma patient (1541, also see Supplemental Video S3 for the three-dimensional volumetric visualization of Adaptive optics–optical coherence tomography (AO-OCT) data taken at L2). Adaptive optics–optical coherence tomography (AO-OCT) B-scan and en face projections at six retinal locations (L1–L6) show a significant soma density reduction in the ganglion cell layer (GCL) (green) in the inferior retina correlated with the HD. hyperreflective structures were seen from en face projections of the inner plexiform layer (IPL) (purple) in two inferior retinal locations (L2 and L4). B-scans at the paired locations were approximately aligned to the IPL for comparison purpose. RNFL, retinal nerve fiber layer.

Figure 6.

AO-OCT tracking of inner retinal changes at a 3° retinal location (L2) in the right eye of a 58-year-old patient (7365) with two visits. B-scan images taken: (A) first visit and (B) second visit. The Adaptive optics–optical coherence tomography (AO-OCT) volumes between the two visits were axially aligned to the top of IPL for comparison purpose. En face projection across 10 pixels (∼7 µm) of the ganglion cell layer (GCL) soma mosaic taken at (C) first visit and (D) second visit. (E) Magnified views of two labeled regions (blue boxes) from two visits. (F) Color merged en face projection across 20 pixels (∼14 µm) of IPL vessels between first (cyan) and second (magenta) visits demonstrated good registration and little vascular remodeling. Depth color maps showed superficial vessels in GCL in first visit (G) migrated posteriorly in the second visit in (H). The example branching vessel is labeled with white arrows. IPL, inner plexiform layer; NFL, nerve fiber layer.