Intrathecal lipid-specific oligoclonal IgM synthesis associates with retinal axonal loss in multiple sclerosis

Abstract

Objective:
It has been suggested that autoantibodies may induce axonal damage in multiple sclerosis (MS). Optical coherence tomography (OCT) showed that thinning of peripapillary retinal nerve fiber layer (RNFL) and ganglion cell layer/inner plexiform (GCIPL) measurements reflect axonal loss in the disease. We investigated whether the intrathecal synthesis of lipid-specific oligoclonal IgM bands (LS-OCMB) associates with thinning of these structures in MS patients.

Methods:
58 consecutive MS patients and 70 age-matched healthy controls were assessed. LS-OCMB was studied in cerebrospinal fluid by isoelectric focusing and immunoblotting. RNFL and GCIPL imaging were quantified by spectral domain OCT.

Results:
RNFL and GCIPL were significantly reduced in MS patients compared to controls (p<0.01). RNFL thickness was further reduced in LS-OCMB positive MS patients compared to LS-OCMB negative MS subjects mainly in papillomacular bundle (p<0.05), temporal and inferior quadrants (p<0.05) and inferotemporal sector (p=0.01).

Conclusions:
The presence of LS-OCMB associates with increased retinal axonal loss in MS. This reinforces the relationship found between the intrathecal synthesis of IgM and the axonal damage observed in immunological and pathological studies even in normal-appearing white matter. OCT seems an optimal tool to monitor axonal damage in LS-OCMB positive patients, relevant for therapeutic decisions and quantification of the effects of new neuroprotective treatments.

Full text

Intrathecal lipid-specific oligoclonal IgM synthesis associates with retinal
axonal loss in multiple sclerosis
José C. Álvarez-Cermeño
a,
⁎, Francisco J. Muñoz-Negrete
b
, Lucienne Costa-Frossard
a
, Susana Sainz de la Maza
a
,
Luisa M. Villar
c,1
, Gema Rebolleda
a,1
a
Department of Neurology, Multiple Sclerosis Unit, Ramón y Cajal University Hospital, IRYCIS, University of Alcalá de Henares, Madrid, Spain
b
Department of Ophthalmology, Multiple Sclerosis Unit, Ramón y Cajal University Hospital, IRYCIS, University of Alcaláde Henares, Madrid, Spain
c
Department of Immunology, Multiple Sclerosis Unit, Ramón y Cajal University Hospital, IRYCIS, University of Alcalá de Henares, Madrid, Spain
abstractarticle info
Article history:
Received 19 August 2015
Received in revised form 3 November 2015
Accepted 16 November 2015
Available online 18 November 2015
Objective: It has been suggested that autoantibodies may induce axonal damage in multiple sclerosis (MS).
Optical coherence tomography (OCT) showed that thinning of peripapillary retinal nerve fiber layer (RNFL)
and ganglion cell layer/inner plexiform (GCIPL) measurements reflect axonal loss in the disease. We investigated
whether the intrathecal synthesis of lipid-specific oligoclonal IgM bands (LS-OCMB) associates with thinning of
these structures in MS patients.
Methods: 58 consecutive MS patients and 70 age-matched healthy controls were assessed. LS-OCMB was studied
in cerebrospinal fluid by isoelectric focusing and immunoblotting. RNFL and GCIPL imaging were quantified by
spectral domain OCT.
Results: RNFL and GCIPL were significantly reduced in MS patients compared to controls (p b0.01). RNFL
thickness was further reduced in LS-OCMB positive MS patients compared to LS-OCMB negative MS subjects
mainly in papillomacularbundle (p b0.05), temporal and inferior quadrants(p b0.05) and inferotemporal sector
(p = 0.01).
Conclusions: The presence of LS-OCMB associates with increased retinal axonal loss in MS. This reinforces the
relationship found between the intrathecal synthesis of IgM and the axonal damage observed in immunological
and pathological studies even in normal-appearing white matter. OCT seems an optimal tool to monitor axonal
damage in LS-OCMB positive patients, relevant for therapeutic decisions and quantification of the effects of new
neuroprotective treatments
© 2015 Elsevier B.V. All rights reserved.
Keywords:
Axonal loss
OCT
IgM
Oligoclonal bands
Neurodegeneration
Multiple sclerosis
1. Introduction
Multiple sclerosis (MS) is a chronic inflammatory disease of the
central nervous system (CNS) where a broad immune attack leads to
demyelination and axonal loss, the major source of disability. In recent
years, the function of the cellular response in tissue damage including
myelin-specific auto-reactive lymphocytes (mainly CD8+ T cells) and
microglia has been extensively studied [1–3]. Nevertheless, the exact
role of antibodies in this issue remains elusive. They have been implicat-
ed in plaque formation [4] and found in a major percentage of MS brains
[5]. Even, they have been proposed as a common finalstepindemyelin-
ation [6]. Moreover, the presence of oligoclonal bands of IgG is a
hallmark of the disease, and a very useful tool in its diagnosis [7].Cere-
brospinal fluid (CSF) proteome mirrors the IgG-transcriptome of
inflamed CNS tissue in MS [8]. In addition, most of the antibodies
detected in CSFbelong to the IgG or IgM isotypes, are activatingcomple-
ment, and contain somatic hypermutations. All these features indicate
sustained exposure to specific antigens within CNS and a direct role of
these molecules in MS pathogenesis [8,9].
The intrathecal synthesis of lipid-specific oligoclonal IgM bands (LS-
OCMB) in MS associates with disability [10,11], brain atrophy and great-
er lesion load from the initial phase of the disease [12].Likewise,these
oligoclonal antibodies are associated with increased CSF levels of
neurofilament light protein (NFL), a marker of neurodegeneration and
axonal damage [13].Allthesefindings suggest that LS-OCMB are associ-
ated with axonal loss, the source of permanent disability in MS.
Optical coherence tomography (OCT) is a non-invasive technique
that permits the study of retinal axonal loss in vivo. Thinning of RNFL,
typically in the temporal sector using OCT, is a well-documented
structural marker of axonal loss in the eyes of patients with multiple
sclerosis. It occurs with and without a previous history of optic neuritis
(ON) and reflects global neurodegeneration in MS [14].
Therefore, we considered of interest to study whether MS patients
with LS-OCMB show reduced RNFL thickness as a result of the axonal
damage associated with these autoantibodies [13].Itwouldoffera
Journal of the Neurological Sciences 360 (2016) 41–44
⁎Corresponding author at: Department of Neurology, Multiple Sclerosis Unit, Hospital
Universitario Ramón y Cajal, Ctra de Colmenar Km. 9,100, 28034 Madrid, Spain.
E-mail address: josecarlos.alvarez@salud.madrid.org (J.C. Álvarez-Cermeño).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.jns.2015.11.030
0022-510X/© 2015 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Journal of the Neurological Sciences
journal homepage: www.elsevier.com/locate/jns

reliable marker of the noxious effect of these molecules in the everyday
clinical setting.
2. Methods
2.1. Subjects
This is a single-centre cross-sectional study approved by the Ramón
y Cajal ethics committee. After informed consent, we assessed 58
consecutive patients with RRMS. They all fulfilled the 2010 McDonald
criteria for MS diagnosis [15]; the clinical work-up followed for their
study and diagnosis has been specified elsewhere [16]. Patients with
acute ON or evidence of optic disc swelling on fundoscopy within 3
months of inclusion, or during study follow-up, were excluded. 70
age-matched healthy controls without any previous ophthalmological
or neurological history were also studied and recruited from the hospi-
tal staff.
2.2. Cerebrospinal fluid (CSF) study
Oligoclonal IgG bands and lipid-specific oligoclonal IgM bands were
studied by isoelectric focusing and immunoblotting as previously
described [10,16].
2.3. Ophthalmological studies
2.3.1. Visual field and evoked potentials
The visual field (VF) was tested only in the eyes of patients with
RRMS, using a Humphrey Field Analyser (Carl Zeiss Meditec AG, Jena,
Germany) and the SITA Standard protocol (program 24-2). VF test
was considered reliable when fixation losses were less than 20% and
false-positive and false-negative errors were less than 15%. The VEP
(visual evoked potential) testing was performed in strict accordance
with the ISCEV (International Society for Clinical Electrophysiology of
Vision) standard and using the device RETIscan 21 4.13.1.6 (Roland
Consult, Brandenburg, Germany).
2.3.2. Optical coherence tomography measurements
A single, well-trained optometrist performed all OCT examinations
in random order to prevent any fatigue bias. Methodology for peri-
papillary RNFL Spectralis and ganglion cell layer/inner plexiform
(GCIPL) imaging in Cirrus have been reported previously [17]. Spectralis
OCT (software version 5.2.0.3) simultaneously captures infrared fundus
and SD-OCT images at 40,000 Ascans per second. Peripapillary RNFL
measurements were obtained using the N-site axonal protocol, which
differs from the standard RNFL scan because it starts and terminates in
the nasal side of optic nerve. Scans were obtained using the high resolu-
tion (HR) mode. The RNFL Spectralis protocol generates a map showing
the average thickness, maps with 4 quadrants (superior, inferior, nasal,
and temporal), and maps with 6 sector thicknesses (superonasal, nasal,
inferonasal, inferotemporal, temporal, and superotemporal).
Imaging of the macular area was performed using Cirrus OCT
macular cube (512 × 128). Cirrus OCT provides quantitative assessment
of the ganglion cell and inner plexiform layers (GCIPL) in 6 circular
sectors centered in the fovea (superonasal, superior, inferonasal,
inferotemporal, inferior, and superotemporal).
All poor-quality scans were rejected, defined as those with signal
strength of ≤6 by Cirrus. For Spectralis OCT only those images with a
signal-to-noise score higher than 25 dB were analysed. Scans with
misalignment, segmentation failure, decentration of the measurement
circle were excluded from the analysis.
2.4. Statistical analysis
Results were analysed with the Prism 5.0 statistical package
(GraphPadPrism, San Diego, CA, USA). The Mann–Whitney U test was
used for comparisons between groups and the Dunn's Test procedure
was used to correct significance tests for multiple comparisons.
The Fisher exact test was used for categorical variable comparisons
between groups.
The Spearman test was used for correlations. P values below 0.05
were considered significant.
3. Results
All 58 MS patients studied showed oligoclonal bands of IgG. Patients
were divided in two groups: LS-OCMB + (n = 19) and LS-OCMB −
(n = 39) according to the presence or not of lipid-specific oligoclonal
bands of IgM respectively. Age, time elapsed since the diagnosis,disease
duration, percentage of eyes with previous ON and of patients with
immunomodulatory treatment were similar in both groups of patients.
There were no differences in EDSS score: 2 (1.5–2) in LS-OCMB+ and
1.5 (1.5–2) in LS-OCMB −patients (median [95% C.I.])(p = 0.16)
However, MSSS score was higher in LS-OCMB+ patients (p = 0.0004)
(Table 1).
Visual acuity (VA), latency and cup/disk ratio were similar in
controls and MS patients (data not shown). Visual evoked potential
latencies were similar in LS-OCMB+ and LS-OCMB−patients: 117.2
(15.10) ms vs 114.1 (15.9), [Mean (SD) (p = 0.18)] respectively. Similar
results were also observed when only patients without previous ON
were studied: 115.8 (16.43) vs 112.7 (13.75) [Mean (SD) (P = 0.32)].
RNFL and GCIPL were significantly reduced in most quadrants and
sectors measured in MS eyes in both groups of patients compared to
controls. Only superonasal sector in MS was similar to that of controls
and that was also the case for inferonasal sector in LS-OCMB- patients.
We further compared RNFL values in LS-OCMB positive and negative
individuals. RNFL thickness was significantly reduced in LS-OCMB+ pa-
tients compared to LS-OCMB −ones in papillomacular bundle and
temporal and inferior quadrants and temporoinferior sector in positive
patients (Table 2). We found no correlation between these measure-
ments and EDSS, MSSS or AV in the whole group of MS individuals.
Similarly, no differences were found in visual evoked potentials ampli-
tude or latency between patients and controls. We next analysed the
same parameters in patients with previous ON (ON+)(eyes n = 27)
and without ON (ON−) (eyes n = 89) in relation to the presence of
LS-OCMB. LS-OCBM+ eyes with previous ON (n = 9) showed reduced
RNFL values compared to eyes from LS-OCMB −individuals with ON
(n = 18). Nevertheless, probably due to the small number of cases,
differences were not significant (data not shown).
We also studied patients without prior ON (ON−). They showed
also significant RNFL thinning in LS-OCMB + eyes (n = 29) vs LS-
OCMB−ones (n = 60) in papillomacular bundle [43.96 (10.01) vs
48.72 (10.43) (p = 0.04)], temporal [58.63 (13.26) vs 65.76 (15.30)
(p = 0.04)] and inferior quadrants [114.5 (20.66) vs 124.1 (20.84
(p = 0.04)]. LS-OCMB+ NO −patients also showed thinning of tempo-
ral inferior [124.3 (21.49) vs 139.3 (25.20) (p = 0.01)] sector.
GCPIL analysis showed also a significant thinning in MS patients
compared to controls in all sectors investigated in both LS-OCMB posi-
tive and negative patients (p b0.01) (Table 2).
Moreover, we found reduced average GCIPL thickness in LS-OCMB+
vs LS-OCMB−eyes in inferior quadrant [83.42 (12.25) vs 87.94 (11.79)
(p = 0.04)]. Diminished GCIPL thickness was also found in LS-OCMB
positive patients with previous ON compared with LS-OCMB negative
individuals with ON. Nevertheless, differences were not significant.
For instance, the average GCIPL thickness was 62.00 (13.81) vs 70.17
(8.60) respectively (p = 0.08) and the inferior thickness values were
59.00 (14.80) vs 69.28) (p = 0.07).
4. Discussion
The presence of antibodies withpossible deleterious effects on axons
in MS has been proposed [18,19].However,theirclinicalvalueremains
42 J.C. Álvarez-Cermeño et al. / Journal of the Neurological Sciences 360 (2016) 41–44

uncertain. This is due in part to the difficulty in finding a variable or sur-
rogate marker that could reflect their role in neurodegeneration to be
applied in the clinical setting.
The measurement of RNFL and GCIPL by OCT seems highly appropri-
ate for this aim [20]. Axons are unmyelinated within the retina. They
originate from the ganglion cell neurons forming the RNFL which can
be measured by means of OCT [14]. After transection and/or demyelin-
ation of the anterior optic pathways retinal axons may undergo retro-
grade degeneration causing RNFL and GCIPL atrophy [21,22].
Moreover, lesions in the posterior visual pathway may induce trans-
synaptic degeneration with both RNFL and GCIPL thinning [14,23].Im-
provements in OCT technology can measure the ganglion cell and
inner plexiform layer thickness of themacula. Up to 40% of the thickness
in the macular area is occupied by the ganglion celllayer. Therefore both
RNFL and GCIPL are ideal structures to visualize by means of OCT the
processes of neurodegeneration in MS [14,20,22].
The main goal of this study was to assess if RNFL and GCIPL measure-
ments by OCT reflect the potential association of LS-OCMB with axonal
loss in MS. For this purpose we studied 58 MS patients whose demo-
graphic data are depicted in Table 1. We divided our patients in two
groups, according to the presence or absence of LS-OCMB. The unequal
distribution of patients in those groups, a possible limitation of the
study, reflects the actual distribution of LS-OCMB in the MS population,
as observed in previous publications [10–13]. Therefore, we consider
that this fact does not distort our results.
EDSS score was similarin the two groupsand relatively low despite
the disease duration. However, MSSS score, which provides a cross-
sectional measure of comparative disease severity for patients with
different disease duration [24] was significantly higher in LS-OCMB+
group (p = 0.0004). This finding reflects, as previously observed, that
the presence of lipid-specific oligoclonal IgM bands associates with a
more aggressive MS course [10] and disability progression [11].Al-
though only significant in the inferior quadrant measure (p = 0.04),
probably due to the relatively small cohort, LS-OCMB+ patients
showed a tendency to have diminished GCIPL thickness in most ex-
plored areas than patients without such bands. This is in agreement
with recent data showing that patients with active MS, asthose having
LS-OCMB [12,13], have also faster rates of GCIPL thinning. This seems to
reflect a greater availability of retinal ganglion cells for neurodegenera-
tion earlier in the disease course in these patients [25]. Further studies
with larger series of patients are needed to confirm these data.
Our results show that the presenceof LS-OCMB also associates with
retinal axonal loss being the temporal quadrant mostly damaged. These
findings are in line with a previous study [17] analysing the color-code
classification of RNFL in RRMS patients, that identified the temporal
quadrant to be the most abnormally color-coded by both Cirrus and
Spectralis. This may reflect the fact that small-diameter axons, which
are more abundant in the temporal quadrant [26] and are preferentially
affected in MS [14] are known to be more susceptible to noxious mech-
anisms in the disease [27]. It is tempting to speculate that these fibers
might be more sensitive to be damaged by the deposit of lipid-specific
IgM [28].
RFNL thinning in LS-OCMB positive patients is further reduced as
compared to MS patients without LS-OCMB, even in eyes without previ-
ous ON. This reinforces the association of intrathecal synthesis of lipid-
specific IgM antibodies with axonal damage in MS. This is consistent
Table 1
Demographic and clinical data of patients and controls studied.
Controls LS-OCMB+ LS-OCMB−p Cont vs LS-OCMB + p Cont vs LS-OCMB−p LS-OCMB + vs LS-OCMB−
N701939
Age m (SD) 36.93 (9.99) 35.79 (10.47) 38.82 (6.98) ns ns ns
Time since diagnosis years M (SD) na 6.83 (1.821) 5.84 (0.875) na na ns
Disease duration na 7.645 (1.78) 8.743 (1.25) na na ns
EDSS median (95% CI) na 2 (1.5–2) 1.5(1.5–2) na na ns
MSSS median (95% CI) na 4.30 (1.76–6.14) 1.98 (1.45–2.87) na na 0.0004
Eyes with previous ON yes/no 0 9/29 18/60 na na ns
IM treatment yes/no 0 14/5 29/10 na na ns
Values given as mean (SD) or median (95% CI). M: mean. LS-OCMB+: Patients with lipid-specific oligoclonal M bands. LS-OCMB−: Patients without lipid-specific oligoclonal M bands.
Cont: controls. na: not applicable.SD: standarddeviation.ON: Optic neuritis. IM: immunomodulatory. ns: not significant.
Table 2
OCT studies in MSpatients and controls according to the presence or absence of lipid-specific oligoclonal IgM antibodies (LS-OCMB).
Controls n = 70 LS-OCMB+ n = 19 LS-OCMB −n = 39 p Controls vs LS-OCMB+ p Controls vs LS-OCMB−p LS-OCMB + vs LS-OCMB−
Spectralis retinal nerve fiber layer (RNFL) μm
Papillomacular bundle
(PMB) 54.94 (7.64) 42.39 (10.77) 47.11 (11.36) b0.01 b0.01 0.03
Temporal 71.38 (10.56) 55.69 (14.55) 63.42 (16.35) b0.01 b0.01 0.02
Inferior 131.10 (16.28) 112.6 (21.23) 122.4 (20.68) b0.01 b0.01 0.02
Central 99.31 (8.74) 86.42 (12.32) 91.14 (13.11) b0.01 b0.01 ns
Inferotemporal 147.6 (17.58) 122.2 (24.74) 136.72 (26.56) b0.01 b0.01 0.01
Superotemporal 136.3 (16.02) 123.2 (21.0) 125.9 (27.9) b0.01 b0.01 ns
Superior 119.0 (14.75) 108.6 (18.44) 112.3 (21.20) b0.01 b0.05 ns
Nasal 75.35 (13.56) 68.36 (13.91) 68.30 (13.03) b0.05 b0.01 ns
Superonasal 101.8 (20.62) 93.75 (20.19) 98.64 (20.40) ns ns ns
Inferonasal 114.7 (23.87) 102.81 (25.5) 108.13(24.9) b0.05 ns ns
Cirrus ganglion cell/inner plexiform (GCIPL) analysis μm
Average 83.84 (5.86) 71.47 (13.15) 75.14 (8.12) b0.01 b0.01 ns
Minimum 81.86 (6.14) 67.08 (15.26) 70.79 (11.09) b0.01 b0.01 ns
Superior 84.85 (6.23) 71.97 (15.63) 75.84 (8.42) b0.01 b0.01 ns
Superonasal 85.47 (6.30) 72.56 (12.44) 75.81 (9.29) b0.01 b0.01 ns
Inferonasal 83.97 (6.26) 71.64 (12.20) 74.35 (10.69) b0.01 b0.01 ns
Inferior 82.65 (6.22) 69.22 (14.48) 74.40 (9.90) b0.01 b0.01 0.04
Superotemporal 82.41 (6.46) 71,64 (14.95) 74,73 (7.93) b0.01 b0.01 ns
Inferotemporal 83.82 (6.14) 71,89 (14.69) 75,84 (7.86) b0.01 b0.01 ns
Values expressed as mean (SD). LS-OCMB+: Patients with lipid-specific oligoclonal M bands. LS-OCMB−: Patients without such bands.
43J.C. Álvarez-Cermeño et al. / Journal of the Neurological Sciences 360 (2016) 41–44

with the axonal loss observed in LS-OCMB individuals in pathological
studies, within lesions and normal appearing white matter [28],andin
immunological assessments [13]. It also offers an explanation of the
relationship of LS-OCMB with disability progression and brain atrophy
[10–12]. Due to its high sensitivity to detect axonal loss from the very
early phases of MS [29] and disease activity [30], OCT seems an optimal
tool to detect and monitor axonal damage in LS-OCMB positive patients.
This may be relevant for therapeutic decision and quantification of the
effects of new neuroprotective treatments.
In summary, our results show that the intrathecal synthesis of lipid-
specific IgM oligoclonal autoantibodies associates with retinal axonal
loss in MS. This is in line with previous reports showing that these anti-
bodies correlate with disability progression, brain atrophy and
increased CSF neurofilaments, a marker of neurodegeneration.
Conflicts of interest
Drs Villar and Álvarez-Cermeño received compensations due to
board membership or payment for lectures from Biogen, Merck-
Serono, Bayer HealthCare, Novartis, Teva, Roche Farma and Genzyme.
Dr. Costa-Frossard obtained compensation for lectures from Biogen-
Idec, Roche and Novartis. The other authors declare no disclosures.
Acknowledgements
This work was supported by grants from Plan Estatal de I + D + I
2013-2016, PI12-00239 from FIS, Instituto de Salud Carlos III
and FEDER, and SAF 2012-34670 from Ministerio de Economıay
Competitividad.
References
[1] S.L. Hauser, J.R. Oksenberg, Theneurobiology of multiple sclerosis: genes,inflamma-
tion, and neurodegeneration, Neuron 52 (2006) 61–76.
[2] A. Bitsch,J. Schuchardt, S. Bunkowski, T. Kuhlmann, W. Brück, Acute axonal injury in
multiple sclerosis. Correlation with demyelination and inflammation, Brain 123
(2000) 1174–1183.
[3] M.A. Michell-Robinson, H. Touil, L.M. Healy, D.R. Owen, B.A. Durafourt, A. Bar-Or,
et al., Roles of microglia in brain development, tissue maintenance and repair,
Brain 138 (2015) 1138–1159.
[4] F.W. Gay, T.J. Drye,G.W. Dick, M.M. Esiri,1997. The application of multifactorialclus-
ter analysis in the staging of plaques in early multiple sclerosis. Identification and
characterization of the primary demyelinating lesion, Brain 120 (1997) 1461–1483.
[5] I. Metz,S.D. Weigand, B.F.G. Popescu,J.M. Frischer, J.E.Parisi, Y. Guo, et al.,Pathologic
heterogeneity persists in early active multiple sclerosis lesions, Ann. Neurol. 728-
738 (2014).
[6] E.C. Breij, B.P. Brink, R. Veerhuis, C. van denBerg, R. Vloet, R. Yan,et al., Homogeneity
of active demyelinating lesions in established multiple sclerosis, Ann. Neurol. 63
(2008) 16–25.
[7] J.C. Álvarez-Cermeño, L.M. Villar, Oligoclonal bands—ausefultooltoavoidMS
misdiagnosis, Nat. Rev. Neurol. 9 (2013) 303–304.
[8] B. Obermeier, L. Lovato, R. Mentele, W. Brück, I.Fome, A. Imhof, et al., Related B cell
clones that populate the CSF and CNS of patients with multiple sclerosis produce
CSF immunoglobulin, J. Neuroimmunol. 233 (2011) 245–248.
[9] E. Beltrán, B. Obermeier, M. Moser, F. Coret, M. Simó-Castelló, I. Boscá, et al.,
Intrathecal somatic hypermutation of IgM in multiplesclerosis and neuroinflamma-
tion, Brain 137 (2014) 2703–2714.
[10] L.M. Villar, M.C. Sádaba, E. Roldán, J. Masjuan, P. González-Porqué, N. Villarrubia,
et al., Intrathecal synthesis of oligoclonal IgM against myelin lipids predicts an
aggressive disease course in MS, J. Clin. Invest. 115 (2005) 187–194.
[11] M. Thangarajh, J. Gomez-Rial, A.K. Hedström, J. Hillert, J.C. Álvarez-Cermeño, T.
Masterman, et al., Lipid-specific immunoglobulin M in CSF predicts adverse long-
term outcome in multiple sclerosis, Mult. Scler. 14 (2008) 1208–1213.
[12] M.J. Magraner, I. Bosca, M. Simó-Castelló, G. García-Martí, A. Alberich-Bayarri, F.
Coret, et al., Brain atrophy and lesion load are related to CSF lipid-specificIgM
oligoclonal bands in clinically isolated syndromes, Neuroradiology 54 (2012) 5–12.
[13] L.M.Villar, C. Picón, L. Costa-Frossard, R. Alenda, J. García-Caldentey, M. Espiño, et al.,
Cerebrospinal fluid immunological biomarkers associated with axonal damage in
multiple sclerosis, Eur. J. Neurol. 22 (2015) 1169–1175.
[14] A. Petzold, J.F. deBoer, S. Schippling,P. Vermersch, R. Kardon, A. Green, et al., Optical
coherence tomographyin multiple sclerosis: a systematicreview and meta-analysis,
Lancet Neurol. 9 (2010) 921–932.
[15] C.H. Polman, S.C. Reingold, B. Banwell, M. Clanet, J.A. Cohen, M. Filippi, et al.,
Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria,
Ann. Neurol. 69 (2011) 292–302.
[16] J. Masjuan, J.C. Álvarez-Cermeño, N. García-Barragán, M. Díaz-Sánchez, M. Espiño,
M.C. Sádaba,et al., Clinically isolated syndromes: a new oligoclonal band test accu-
rately predicts conversion to MS, Neurology 66 (2006) 576–578.
[17] G. Rebolleda, J.J. González-López, F.J. Muñoz-Negrete, N. Oblanca, L. Costa-Frossard,
J.C. Álvarez-Cermeño, Color-code agreement among stratus, cirrus, and spectralis
optical coherence tomography in relapsing-remitting multiple sclerosis with and
without prior optic neuritis, Am J. Ophthalmol. 155 (2013) 890–897.
[18] E.K. Mathey, T. Derfuss, M.K. Storch, K.R. Williams, K. Hales, D.R. Woolley, et al.,
Neurofascin as a novel target for autoantibody-mediated axonal injury, J. Exp.
Med. 204 (2007) 2363–2372.
[19] C. Elliott, M. Lindner, A. Arthur, K. Brennan,S. Jarius, J. Hussey,et al., Functionaliden-
tification of pathogenic autoantibody responses in patients with multiple sclerosis,
Brain 135 (2012) 1819–1833.
[20] B.M. Burkholder, B.Osborne, M.J. Loguidice, E. Bisker, T.C. Frohman, A. Conger, et al.,
Macular volume determined by optical coherence tomography as a measure of neu-
ronal loss in multiple sclerosis, Arch. Neurol. 66 (2009) 1366–1372.
[21] K.S. Shindler, E. Ventura, M. Dutt, A. Rostami, Inflammatory demyelination induces
axonal injury and retinal ganglion cell apoptosis in experimental optic neuritis,
Exp. Eye Res. 87 (2008) 208–213.
[22] S. Saidha, E.S. Sotirchos, J. Oh, S.B. Syc, M.A. Seigo, N. Shiee, et al., Relationships
between retinal axonal and neuronal measures and global central nervous system
pathology in multiple sclerosis, JAMA Neurol. 70 (2013) 34–43.
[23] J. Keller, B.F. Sánchez-Dalmau, P. Villoslada, Lesions in the posterior visual pathway
promote trans-synaptic degeneration of retinal ganglion cells, PloS One 9 (5) (2014)
e97444, http://dx.doi.org/10.1371/journal.pone.0097444 (Published online 2014
May 23).
[24] R.H.Roxburgh, S.R. Seaman, T. Masterman, A.E. Hensiek, S.J. Sawcer, S. Vukusic, et al.,
Multiple sclerosis severity score: using disability and disease duration to rate
disease severity, Neurology 64 (2005) 1144–1151.
[25] J.N. Ratchford, S. Saidha, E.S. Sotirchos, J.A. Oh, M.A. Seigo, C. Eckstein, et al., Active
MS is associated with accelerated retinal ganglion cell/inner plexiform layer thin-
ning, Neurology 80 (2013) 47–54.
[26] J.L. Bennett, M. de Seze, M. Lana-Peixoto, J. Palace, A. Waldman, S. Schippling, et al.,
Neuromyelitis optica and multiple sclerosis: seeing differences through optical co-
herence tomography, Mult. Scler. J. 21 (2015) 678–688.
[27] N. Evangelou, D. Konz, M.M. Esiri, S. Smith, J. Palace, P.M. Matthews, Size-selective
neuronal changes in the anterior optic pathways suggest a differential susceptibility
to injury in multiple sclerosis, Brain 124 (2001) 1813–1820.
[28] M.C. Sádaba, J. Tzartos, C. Paíno, M. García-Villanueva, J.C. Alvarez-Cermeño, L.M.
Villar, et al., Axonal and oligodendrocyte-localized IgM and IgG deposits in MS
lesions, J. Neuroimmunol. 247 (2012) 86–94.
[29] C. Oreja-Guevara, S. Noval, J. Álvarez-Linera, L. Gabaldón, B. Manzano, B. Chamorro,
et al., Clinically isolated syndromes suggestive of multiple sclerosis: an optical co-
herence tomography study, PLoS One 7 (3) (2012) e33907, http://dx.doi.org/10.
1371/journal.pone.0033907 (Published online 2012 Mar 0).
[30] J. Sepulcre, M. Murie-Fernández, A. Salinas-Alaman, A. García-Layana, B. Bejarano, P.
Villoslada, Diagnostic accuracy of retinal abnormalities in predicting disease activity
in MS, Neurology 68 (2007) 1488–1494.
44 J.C. Álvarez-Cermeño et al. / Journal of the Neurological Sciences 360 (2016) 41–44