A Novel Retinal Nerve Fiber Layer Biomarker of Amyotrophic Lateral Sclerosis (ALS) Identified Using Longitudinal in vivo Ocular Imaging

Abstract

Purpose: Like motor neurons, retinal ganglion cells (RGCs) have long axons and high metabolic demands, making them vulnerable to disruption of axonal transport. Unlike motor neurons, the RGC axons are accessible to high-resolution non-invasive optical imaging in their intraocular portion. A non-invasive in vivo retinal imaging biomarker can be valuable for amyotrophic lateral sclerosis (ALS) diagnosis and monitoring. We aim to assess the presence of inner retinal pathology in a mouse model of ALS and its possible progression with age.

Methods: Transgenic SOD1G93A mice (n=8, 4M/4F) and age-matched controls (n=8, 4M/4F) underwent in vivo retinal imaging with confocal scanning laser ophthalmoscopy (cSLO) coupled with optical coherence tomography (OCT) at 20 weeks of age. Another group of SOD1G93A mice (n=20, 6M/14F) and age-matched controls (n=20, 6M/14F) underwent longitudinal in vivo retinal imaging with the same device. Each retinal imaging session included infrared reflectance (IR) and blue reflectance (BR) cSLO coupled with OCT. Hyperreflective puncta located in the retinal nerve fiber layer (RNFL) were counted in a blinded fashion in ALS and control mice. The number of puncta at 20 weeks of age in ALS mice was compared with controls using Wilcoxon test. The rates of increase of puncta number were analyzed using a Generalized Linear Mixed-Effect Model (GLMM) for genotype, time, and sex.

Results: IR-cSLO coupled with OCT revealed hyperreflective puncta located in the RNFL of ALS mice. IR-cSLO fundus imaging at the age of 20 weeks showed ALS mice had significantly higher number of puncta compared to controls (2.1±2.3 vs 0.5±0.8; (mean±SD), respectively, p=0.036). GLMM analysis showed both ALS mutation and age were significantly associated with the rate of increase of puncta number (p=0.000232 and p=0.000366, respectively). In addition, female ALS mice had a steeper increase of puncta compared to male ALS mice (0.21±0.04 log number puncta/week vs 0.16±0.04, respectively; p=0.037).

Conclusion: Our findings demonstrate distinct inner retinal nerve fiber layer pathology, detected using cSLO coupled with OCT, which worsens over time. These findings support the potential of retinal imaging as a translationally relevant, non-invasive biomarker for ALS diagnosis or disease monitoring in humans.

Keywords: amyotrophic lateral sclerosis; eye imaging; hyperreflective; mouse; optical coherence tomography; pathology; puncta; retinal nerve fiber layer; sex; spheroids; superoxide dismutase.

Figure 1

Retinal Pathology in ALS. IR-cSLO imaging of the right fundus of a female mouse at 9 weeks (A) showed hyperreflective puncta (B). OCT B-scan (green arrow) localized puncta (white arrows) to the retinal nerve fiber layer. Puncta were distinguishable from blood vessels by being more hyperreflective (C) Calibration bars, 200 µm.

Figure 2

Number of IR-Puncta at 20 weeks. Boxplot of number of puncta at 20 weeks. In the first set of experiments, ALS mice (n = 8, 4M/4F, red) showed significantly more puncta compared to controls (n=8, 4M/4F, green) at 20 weeks (2.1 ± 2.3 vs 0.5 ± 0.8 puncta, respectively, mean ± SD, p = 0.036, Wilcoxon Rank Sum Test). *p < 0.05. The horizontal line within each box represents the median; the box shows the interquartile range (IQR); whiskers indicate the range excluding outliers.

Figure 3

Retinal Pathology with Age. (A) Longitudinal IR-cSLO imaging of the left fundus of a female control and ALS mouse at 9, 13, and 17 weeks showed hyperreflective puncta increase in number. (B) Plot showing the puncta number of an ALS (red) and control mouse (blue) over time. Calibration bars, 200 µm.

Figure 4

Retinal Pathology by Sex. In the second set of experiments, the rate of puncta increase over time was significant in female ALS (0.21 ± 0.04 vs 0.08 ± 0.04 log number puncta/week, respectively, p = 0.006, left panel) and male ALS mice (0.16 ± 0.04 vs 0.09 ± 0.05 log number puncta/week, respectively, p = 0.039, right panel). Analysis with GLMM revealed that the interaction between genotype and sex over time was significant (p = 0.029). **p < 0.01, *p < 0.05.

Figure 5

Axonal spheroids in ALS retinal ganglion axons. Immunofluorescence staining for phosphorylated neurofilament (P-NF) of the wholemount retina of a female control mouse at the age of 20 weeks (A) showed retinal ganglion cell axons. Wholemount retina of a female ALS mouse at 20 weeks (B) showed P-NF+ axonal spheroids (white arrows) in retinal ganglion cell axons in RNFL. P-NF, phosphorylated neurofilament; Calibration bars, 20 µm.