Retinal Segmentation as Noninvasive Technique to Demonstrate Hyperplasia in Ataxia of Charlevoix-Saguenay

Abstract

Purpose.: To present a new retinal layer segmentation technique for evaluation of nerve fiber hyperplasia in patients with autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS).

Methods.: Five patients with a molecular diagnosis of ARSACS and five age- and sex-matched healthy controls underwent a full ophthalmologic examination, which included a new technique to segment the retinal layers using Spectralis optical coherence tomography (OCT). Images and data were correlated with diffusion tensor color-encoded magnetic resonance imaging maps, diffusion tensor tractographies, and retinal anatomopathologic analysis.

Results.: Optical coherence tomography evaluation revealed increased thickness in the internal layers of the retina (inner limiting membrane, nerve fiber layer, and ganglion cell layer) in each patient with ARSACS compared with controls. These findings suggest that the presence of neurofilamentous hyperplasia in the retinas of patients with ARSACS correlates with the anatomopathologic findings.

Conclusions.: We found evidence of ganglion cell and nerve fiber hyperplasia in the retinas of ARSACS patients. The etiopathogenic mechanisms of this disease thus require reconsideration.

Introduction

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a condition that affects muscle movement due to atrophy of the superior vermis, cervical spinal cord, cerebellum, and cerebral cortex. The signs and symptoms worsen over the years, and spasticity and ataxia of the arms and legs increase. Patients with ARSACS typically experience early-onset (age 12–18 months) difficulties in walking, and gait unsteadiness. Ataxia, dysarthria, spasticity with extensor plantar reflexes, distal muscle wasting and sensory loss, and horizontal gaze-evoked nystagmus are the most frequent neurologic signs. ^1,2  

ARSACS is a highly prevalent neurodegenerative disease in the Charlevoix-Saguenay-Lac-Saint-Jean region of the Province of Quebec (carrier frequency 1/22) and is also diagnosed in various other countries; and the two initial founder mutations identified in Canada have increased to more than 70. ³ Patients from the province of Quebec become wheelchair bound at an average age of 41 years; cognitive skills are preserved over the long term, however, and individuals remain able to perform daily living tasks late into adulthood. Death commonly occurs in the sixth decade. SACS is the gene associated with ARSACS (cytogenetic location: 13q12; molecular location on chromosome 13: base pairs 23,902,964–24,007,840). ⁴ Canadian patients are usually homozygotes or compound heterozygotes for the founder mutations. ^5,6 Although initial descriptions of the disease were confined to Quebec, genetically confirmed ARSACS has now been reported in individuals from France, Tunisia, Italy, Spain, Japan, and Turkey. The actual worldwide incidence of ARSACS remains unknown, however, as underdiagnosis is likely. ^5,7,8  

A recent article about ARSACS described atrophy of the cerebellar vermis, cervical spinal cord, and cerebral cortex in the central nervous system, as well as yellow streaks of hypermyelinated fibers that focally covered the retinal vessels. ⁹ Previous studies have suggested that the yellow streaks around the optic nerve are due to an increased density of the retinal nerve fiber layer (RNFL) around the head of the optic nerve, ^10–13 and that hyperplasic pontocerebellar fibers compress the pyramidal tracts at the pons and cause cerebellar atrophy by glutamate-induced excitotoxicity. ^12,13 In this case, the spasticity would be mediated by compression of the pyramidal tracts; ataxia, by cerebellar degeneration; and weakness and sensory loss, by axonal degeneration superimposed on peripheral dysmyelinopathy, pointing to a developmental cause of ARSACS rather than to a degenerative cause. ^12,13  

In the present study, the peripapillary areas of five patients with ARSACS ^10–13 and those of five age- and sex-matched healthy subjects were analyzed using a new optical coherence tomography (OCT) technique to identify the individual retinal layers and quantify their thickness in cross-sectional foveal scans and determine whether the etiopathogenic mechanism of the retinal abnormalities in ARSACS is fiber hyperplasia, hypertrophy, or myelination. A normal retina was also analyzed pathologically, and an attempt to establish a correlation between the imaging and anatomopathological findings was made. 

Patients and Methods

Five patients with a molecular diagnosis of ARSACS ^12,13 and five age- and sex-matched healthy subjects were analyzed. The study was conducted in accordance with the Ethics Committee of Hospital Universitario Miguel Servet and with the principles of the Declaration of Helsinki. There were no significant differences in refractive error and axial length between ARSACS and healthy subjects. Each of the ARSACS patients showed lower limb spasticity, abnormal tendon reflexes, extensor plantar responses, ataxic gait, nystagmus, pes cavus, and hammertoes. 

All subjects underwent a full neuro-ophthalmologic examination that included visual acuity examination, Goldmann applanation tonometry, standard automatic perimetry, stereophotographs of the optic disc, red-free digital fundus photographs, topographic analysis of the optic disc using Heidelberg Retina Tomograph (HRT) 3, and the new segmentation application of the Spectralis OCT device (Heidelberg Engineering, Heidelberg, Germany). All procedures were evaluated by an experienced neuro-ophthalmologist (EG-M). Standard automatic perimetries were performed using a Humphrey field analyzer (model 750i; Carl Zeiss Meditec, Inc., Dublin, CA) with the SITA Standard 24-2 program, and near addition was added to the subject's refractive correction. Simultaneous stereophotographs of the optic discs were obtained after mydriasis (0.5% tropicamide; Alcon Laboratories, Inc., Fort Worth, TX) using a Canon CF-60UV fundus camera (Canon, Inc., Tokyo, Japan). A series of five red-free digital fundus photographs (Canon CF-60UVi, with a Canon EOS D60 digital camera and a filter with maximum transmission at 490 nm of each eye was acquired for RNFL evaluation. Topographic analysis of the optic disc was performed using an HRT 3 instrument that provides topographic measurements of the optic nerve head, derived from 16 to 64 optical sections to a depth of 4 mm depending on the longitudinal field of view. ¹¹ The new technique for retinal segmentation by the Spectralis OCT device (Heidelberg Engineering) was used to identify each retinal layer and quantify its thickness. Images were acquired using the image alignment eye tracking software (TruTrack; Heidelberg Engineering) to obtain perifoveal volumetric retinal scans comprising 25 single horizontal axial scans (scanning area: 666 square mm, centered at the fovea). Poor-quality scans (0.20 decibels [dB]) were excluded from the analysis. Segmentation of the retinal layers in single horizontal foveal scans was performed automatically by the new segmentation application (segmentation technology, Heidelberg Engineering) to divide the retina into 10 layers: 1, inner glial limiting membrane; 2, nerve fiber layer; 3, ganglion cell layer; 4, inner plexiform layer; 5, inner nuclear layer; 6, outer plexiform layer; 7, outer nuclear layer; 8, outer glial limiting membrane; 9, photoreceptors (rods and cones); and 10, retinal pigment epithelium (Fig. 1). The 768 measurements of the peripapillary thickness of the 10 layers were registered in a database for the 10 eyes of ARSACS patients and for those of the controls. These measurements were distributed in eight uniformly divided sectors for each of the 10 retinal layers evaluated. 

Figure 1

 

Representation of retinal layer division determined by the new segmentation application of the Spectralis OCT segmentation technology in a healthy subject (A) and in a patient with ARSACS (B). The software automatically marked the following layers in a single horizontal foveal scan: 1, inner limiting membrane; 2, nerve fiber layer; 3, ganglion cell layer; 4, inner plexiform layer; 5, inner nuclear layer; 6, outer plexiform layer; 7, outer nuclear layer; 8, outer limiting membrane; 9, photoreceptors (rods and cones); 10, retinal pigment epithelium.

Within this study, the five patients with ARSACS underwent a complete neurologic evaluation and an imaging study comprising cranial computed tomography, magnetic resonance imaging, and diffusion tensor tractographies. 

Serial sections (6 μm thick) of a normal retina were cut and stained with hematoxylin and eosin according to routine protocols and compared with images provided by the Spectralis OCT. 

Statistical analysis was performed using IBM SPSS software (version 20.0; SPSS, Inc., Chicago, IL). The dependent variable was ARSACS diagnosis (yes or no), and the predictive variables were the 768 thickness measurements of each retinal layer, mean RNFL thickness of each retina, and eight sector thicknesses of each retinal layer, measured with the new segmentation technique of the Spectralis OCT. Retinal thickness was compared between ARSCACS patients and healthy controls using the Mann Whitney U test, considering a P value of ≤0.05 to indicate statistical significance. 

Results

Patient and healthy control characteristics are shown in Table 1. 

Table 1

 

Epidemiologic Characteristics, Visual Functional Parameters, and Topographic Analysis of the Optic Discs of Patients With ARSACS and Control Subjects

	ARSACS Patients, n = 10	Healthy Controls, n = 10	P
Age, y, mean (range)	49.36 (39–58)	49.68 (39–58)	0.850
Men:women	1:4	1:4	0.965
Intraocular pressure, mm Hg, mean (SD)	14.26 (2.44)	14.60 (1.98)	0.663
BCVA, Snellen scale, mean (SD)	0.65 (0.14)	0.89 (0.09)	0.005
MD of visual field, dB, mean (SD)	−7.26 (1.03)	−0.25 (0.28)	0.001
HRT measurements:
Rim area	2.15 (0.26)	2.06 (0.19)	0.369
Rim volume	0.39 (0.05)	0.37 (0.06)	0.451
Linear cup/disc ratio	0.48 (0.10)	0.45 (0.08)	0.758
Cup shape measure	−0.23 (0.03)	−0.22 (0.04)	0.469
Height variation contour	0.36 (0.06)	0.37 (0.04)	0.801
Mean RNFL thickness	0.16 (0.02)	0.19 (0.03)	0.227

 BCVA, best-corrected visual acuity; MD, mean deviation; SD, standard deviation. Bold values indicate statistically significant differences.

Neuro-ophthalmologic analysis revealed abnormal visual fields in ARSACS patients, with mild to severe nonspecific localized defects or general reduction of sensitivity, moderate to strikingly increased visibility of the RNFL in optic disc color stereophotographs and RNFL monochromatic photographs, and RNFL thickening using Spectralis OCT (Table 1, Fig. 2). Examination with the HRT 3 topographer by sectors was within normal limits in all ARSACS patients (Table 1). 

Figure 2

 

Neuro-ophthalmologic exploration of one eye of a patient with ARSACS and that of a healthy subject. Representation of optic disc color stereophotographs (A, F), RNFL monochromatic photographs (B, G), visual field (C, H), HRT (D, I), and optical coherence tomography assessments (E, J). Results reflect mild nonspecific defects in the visual field, increased density of the retinal nerve fiber layer around the optic nerve, and an increase in global RNFL thickness in the ARSACS patient compared with the healthy subject.

Segmentation of retinal layers in the single horizontal foveal scans by the new segmentation application revealed a statistically significant increase in the thickness of the three inner retinal layers: the inner glial limiting membrane (P = 0.001), the nerve fiber layer (P = 0.016), and the ganglion cell layer (P = 0.042; Table 2, Fig. 3). 

Figure 3

 

Comparison of segmentation of retinal layer analysis determined by the new segmentation application of the Spectralis OCT segmentation technology in a patient with ARSACS and a healthy subject. The ARSACS patient showed an increased thickness of the nerve fiber and ganglion cell layers.

Table 2

 

Retinal Layer Thicknesses, Mean (SD), Determined by the Segmentation Application of the Spectralis OCT in ARSACS Patients and Healthy Controls

Retinal Layer	ARSACS Patients	Healthy Controls	P
Inner glial limiting membrane	8.68 (2.36)	4.19 (0.93)	0.001
Nerve fiber layer	5.86 (1.88)	4.61 (0.91)	0.016
Ganglion cell layer	6.16 (1.91)	5.18 (1.11)	0.042
Inner plexiform layer	6.51 (1.89)	5.93 (1.20)	0.196
Inner nuclear layer	6.86 (1.91)	6.23 (1.19)	0.387
Outer plexiform layer	7.17 (1.89)	6.45 (1.21)	0.324
Outer nuclear layer	7.44 (1.91)	7.04 (1.26)	0.494
Outer glial limiting membrane	8.14 (1.88)	7.38 (1.25)	0.268
Photoreceptors	8.44 (1.88)	7.78 (1.36)	0.371
Retinal pigment epithelium	8.70 (1.85)	7.81 (1.26)	0.196
Mean thickness, all layers	73.96 (17.33)	62.6 (15.61)	0.001

 Bold values indicate statistically significant differences.

The magnetic resonance imaging study of ARSACS patients showed atrophy of the superior cerebellar vermix and upper cervical cord with a normal-appearing pons. Diffusion tensor imaging showed a large amount of pontocerebellar fibers that produced thick middle cerebellar peduncles and compressed the pyramidal tracts, which were thin or interrupted in same cases (Fig. 4). 

Figure 4

 

Tractography in a healthy subject (left) and in a patient with ARSACS (right). Tractography in the healthy subject shows a normal thickness and location of the white matter fibers in the brainstem region. Tractography in the patient with ARSACS shows narrowing and displacement of the pyramidal tract as it passes through the pons; enlargement of the pontocerebellous fibers, which occupy most of the pons volume; and a normal-thickness medial meniscus, which is slightly posterior displaced.

We correlated the retinal layers marked by the segmentation application of the Spectralis OCT with the layers identified by pathologic analysis (Fig. 5) and found a good correlation between the results of the two techniques. 

Figure 5

 

Pathologic analysis of a normal retina and correlation with retinal layer segmentation determined by the new segmentation application of the Spectralis OCT (segmentation technology). Both techniques show nerve fiber and ganglion cell layers. These layers show increased thickness in the retina of ARSACS patients, suggesting hyperplasia of retinal nerve fibers in the optic disc of these patients.

Discussion

The presence of retinal hypermyelinated fibers has been considered a minor criterion for the diagnosis of ARSACS. In the present study, all ARSACS patients showed an abnormally thick RNFL without myelinated fibers radiating from the optic disc. Recent studies in ARSACS patients have described abnormal thickening of the RNFL as the cause of the anomalous fundus images, and the authors recommended performing exhaustive neuro-ophthalmologic examinations using stereophotographs, RNFL photographs, and digital image analysis devices. ^10,11  

Using the new segmentation application of the Spectralis OCT device in the present study, we observed increased thickness of the inner glial limiting membrane, nerve fiber layer, and ganglion cell layer in patients with ARSACS. This application has not been used before to evaluate eyes of patients with ataxia and might constitute a very useful tool to evaluate the nerve fiber structure and ganglion cell anomalies. The segmentation application should improve the delimitation of the retinal layers and the image resolution, because this application can overlook focal changes. In addition, some ARSACS patients showed a large increase in retinal layer thicknesses (see Figs. 1, 3) while others showed only a moderate increase (see mean values in Table 2). 

Neuro-ophthalmologic examinations preformed in ARSACS patients suggested the presence of retinal fiber hyperplasia, which could explain the increased thickness of the inner layers of the retina: An increase in the number of ganglion cell somata could cause thickening of the ganglion cell layer and inner glial limiting membrane, and an increase in the number of ganglion cell axons could cause thickening of the RNFL. There was a good correlation between the delimitation of the retinal layers provided by the Spectralis OCT and the pathologic analysis of these layers in a patient with ARSACS, as both techniques showed thickening of the retinal inner layers. 

Based on magnetic resonance and OCT findings, Gazulla et al. ^12,13 suggested that nerve fiber hyperplasia plays a role in the etiology of ARSACS; the neuro-ophthalmologic and pathologic results described in the present article support this proposal. 

The delimitation of the retinal layers provided by the Spectralis OCT was well correlated with the anatomopathologic analysis of these layers in a patient with ARSACS. Both techniques showed thicker inner layers. All of the neuro-ophthalmologic, radiologic, and neurologic tests registered support the notion that hyperplasia of nerve fibers could be the etiologic cause of ARSACS pathology. ^8,14 Our HRT measurements indicated normal optic disc morphology in ARSACS patients, although some patients seem to have smaller cup parameters and larger rim parameters (see Fig. 2). Studies evaluating a larger number of ARSACS patients may detect alterations in the optic disc morphology. Future studies with the segmentation application of the Spectralis OCT device should be performed to analyze the thickness data in different regions of the retinal layers and the correlation with volumetric measurements obtained using brain magnetic resonance imaging. 

SACS is hypothesized to be involved in nerve fiber development, which could cause hyperplasia of the nerve fibers in the retina and of pontocerebellar fibers in the central nervous system. Our results are consistent with this proposal and open new research possibilities to uncover the etiology of ARSACS to find therapies for this disease. In addition, the neuro-ophthalmologic examination is a useful, quick, innocuous, and comfortable diagnostic tool for patients with ARSACS that readily allows for the detection of RNFL hyperplasia. 

Acknowledgments

Disclosure: E. Garcia-Martin, None; L.E. Pablo, None; J. Gazulla, None; A. Vela, None; J.M. Larrosa, None; V. Polo, None; M.L. Marques, None; J. Alfaro, None