The relationship between retinal ganglion cell axon constituents and retinal nerve fiber layer birefringence in the primate

Abstract

Purpose: To determine the degree of correlation between spatial characteristics of the retinal nerve fiber layer (RNFL) birefringence (Delta n(RNFL)) surrounding the optic nerve head (ONH) with the corresponding anatomy of retinal ganglion cell (RGC) axons and their respective organelles.

Methods: RNFL phase retardation per unit depth (PR/UD, proportional to Delta n(RNFL)) was measured in two cynomolgus monkeys by enhanced polarization-sensitive optical coherence tomography (EPS-OCT). The monkeys were perfused with glutaraldehyde and the eyes were enucleated and prepared for transmission electron microscopy (TEM) histologic analysis. Morphologic measurements from TEM images were used to estimate neurotubule density (rho(RNFL)), axoplasmic area (A(x)) mode, axon area (A(a)) mode, slope (u) of the number of neurotubules versus axoplasmic area (neurotubule packing density), fractional area of axoplasm in the nerve fiber bundle (f), mitochondrial fractional area in the nerve fiber bundle (x(m)), mitochondria-containing axon profile fraction (m(p)), and length of axonal membrane profiles per unit of nerve fiber bundle area (L(am)/A(b)). Registered PR/UD and morphologic parameters from corresponding angular sections were then correlated by using Pearson's correlation and multilevel models.

Results: In one eye there was a statistically significant correlation between PR/UD and rho(RNFL) (r = 0.67, P = 0.005) and between PR/UD and neurotubule packing density (r = 0.70, P = 0.002). Correlation coefficients of r = 0.81 (P = 0.01) and r = 0.50 (P = 0.05) were observed between the PR/UD and A(x) modes for each respective subject.

Conclusions: Neurotubules are the primary source of birefringence in the RNFL of the primate retina.

Figures

Figure 1

The peripapillary RNFL was divided into 8 and 16 angular sections. Each angular section is labeled by the symbols superimposed on the area surrounding the ONH (white circle) for subjects (A)1853 OS and (B)57204 OD, The uncertainty in RNFL bundle positions are ± 22.5° and ± 11.25° for subjects 1853 and 57204, respectively. Modified with permission from Histology of the Human Eye, Hogan MJ, Alvarado JA, Weddell JE, Copyright Elsevier 1971.

Figure 2

Low magnification image (2200X) of a retinal nerve fiber layer bundle from subject 57204 in angular section NSI. Scale bar, 2 μm.

Figure 3

(A) A montage of a nerve fiber bundle from subject 57204 in angular section ITL. Montages were transferred to a tablet PC, where cellular structures were traced, counted, and measured. The montage was used to keep track of photographed high magnification images of RGC axons using the Image J’s Region of Interest (ROI) Manager to ensure that there were not any repeat or missed images of any RGC axons within the nerve fiber bundle. Scale bar, 2 μm (B) Medium magnification (11000X to 14000X) TEM image used to create montage in (A). Scale bar, 1 μm.

Figure 4

High magnification images (28000X to 44000X) were used to measure RGC axon neurotubules and organelles. Sparse axons were rare in nerve fiber bundles except in the temporal macular region of the eye in both subjects. Axons that had a neurotubule density that was less than 25% percent of the average neurotubule density of the axon population were classified as sparse axons. (A) Sparse axons (S) appear pale and have fewer nerurotubules. Scale bar, 0.5 μm. (B) A RGC axon from nasal region of subject 57204. Scale bar, 0.5 μm.

Figure 5

(A) Original positions of the mean ρRNFL values and PR/UD radial area and ring scan measurements used for correlation with ρRNFL for subject 57204. The yellow and blue bar highlight the original position of angular section TIS and the lowest PR/UD measurements within an angular section, respectively. (B) Radial area and ring scans of PR/UD falling within an angular section were averaged together resulting in an equal number of PR/UD measurements for every ρRNFL value.The green bar marks the optimally registered ρRNFL value for angular section TIS and PR/UD measurement. N, nasal; T, temporal; S, superior; I, inferior

Figure 6

Phase retardation per unit depth (PR/UD, proportional to birefringence) measurements along a 360° circular sweep around the ONH. PR/UD measurements (ring or radial) in each eye are plotted with a unique symbol. The black squares in both figures represent the average PR/UD of combined ring and radial scans for each angular section region that was used to determine neurotubule density. (A)Two different radial area and single ring scan for subject 52704. (B) Area scan and ring scan of 1853 OS. N, nasal; T, temporal; S, superior; I, inferior

Figure 7

(A) Mean retinal nerve fiber layer neurotubule density (ρRNFL) estimates averaged over 3 nerve fiber bundles are presented for both subjects. (B) ρRNFL estimates for subject 57204 within angular section octants analogous to subject 1853 were further averaged to produce eight ρRNFL values for comparison with subject 1853. N, nasal; T, temporal; S, superior; I, inferior

Figure 8

(A) Least-squares linear fits of neurotubule number (k) versus axoplasmic area (Ax) and (B) axoplasmic area probability density function from angular section NSS of 57204 OD used in the statistical algorithm to estimate ρRNFL

Figure 9

Average birefringence measurements calculated from EPS-OCT PR/UD radial area and ring scans plotted with respective regional neurotubule density (ρRNFL) for subjects (A) 57204 (B) and 1853. Area and ring scans of PR/UD measured within angular regions that were sampled for neurotubule density surrounding the ONH were averaged together within the respective angular section region resulting in an equal number of PR/UD measurements for every ρRNFL value. N, nasal; T, temporal; S, superior; I, inferior

Figure 10

Phase retardation per unit depth (PR/UD, proportional to birefringence) measurements from radial area and ring scans for subject 57204 were averaged together within its respective angular section around the (ONH) and superimposed on (A) mean (Ax) modes for subject 57204 and (B) mean (Aa) and (Ax) modes for subject 1853 measured in corresponding regions of the same eye. N, nasal; T, temporal; S, superior; I, inferior

Figure 11

Phase retardation per unit depth (PR/UD, proportional to birefringence) measurements from radial area and ring scans for subject 57204 were averaged together within its respective angular section around the (ONH) and superimposed on mean (u) [neurotubule packing density] data measured in corresponding regions of the same eye. N, nasal; T, temporal; S, superior; I, inferior