Pattern-specific loss of aquaporin-4 immunoreactivity
distinguishes neuromyelitis optica from multiple
Shanu F. Roemer,1Joseph E. Parisi,2Vanda A. Lennon,1^3Eduardo E. Benarroch,1Hans Lassmann,4
Wolfgang Bruck,5Raul N. Mandler,6Brian G.Weinshenker,1Sean J. Pittock,1,2Dean M.Wingerchuk7and
Claudia F. Lucchinetti1
Departments of1Neurology,2Laboratory Medicine and Pathology and3Immunology, Mayo Clinic College of Medicine,
Rochester, MN,USA,4Center for the Clinical Trials Network, National Institutes of Health, Bethesda, MD,5Department of
Neurology, Mayo Clinic College of Medicine, Scottsdale, AZ,USA,6Center for Brain Research, Medical University of Vienna,
Vienna, Austria and7Department of Neuropathology, Institute for Multiple Sclerosis Research,Georg-August University,
Correspondence to: Claudia F. Lucchinetti, MD, Neurology, Mayo Clinic, College of Medicine, 200 First St. SW, Rochester,
Neuromyelitis optica (NMO) is an inflammatory demyelinating disease that typically affects optic nerves and
spinalcord.Its pathogenic relationship to multiple sclerosis (MS) is uncertain.Unlike MS,NMOlesions are char-
acterized by deposits of IgG and IgM co-localizing with products of complement activation in a vasculocentric
pattern around thickened hyalinizedblood vessels, suggesting a pathogenic role for humoralimmunity targeting
an antigen in the perivascular space. A recently identified specific serum autoantibody biomarker, NMO-IgG,
targets aquaporin-4 (AQP4), the most abundant waterchannelprotein in the CNS, which is highlyconcentrated
in astrocytic foot processes.We analysed and compared patterns of AQP4 immunoreactivity in CNS tissues of
nine patients with NMO, 13 with MS, nine with infarcts and five normal controls. In normal brain, optic nerve
and spinal cord, the distribution of AQP4 expression resembles the vasculocentric pattern of immune complex
deposition observed in NMOlesions.In contrast to MS lesions, which exhibit stage-dependent loss of AQP4, all
NMO lesions demonstrate a striking loss of AQP4 regardless of the stage of demyelinating activity, extent of
tissue necrosis, or site of CNS involvement.We identified a novel NMO lesion in the spinal cord and medullary
tegmentum extending into the area postrema, characterized by AQP4 loss in foci that were inflammatory and
oedematous, but neither demyelinated nor necrotic. Foci of AQP4 loss coincided with sites of intense
vasculocentric immune complex deposition.These findings strongly support a role for a complement activating
AQP4-specific autoantibody as the initiator of the NMOlesion, and further distinguish NMO from MS.
Keywords: neuromyelitis optica; aquaporin-4; multiple sclerosis; demyelination; astrocyte; blood^brain barrier
Abbreviations: AQP4¼aquaporin-4; HIVE¼human immunodeficiency virus encephalitis; GFAP¼ glial fibrillary acidic
protein; NMO¼Neuromyelitis optica; PML¼progressive multifocal leucoencephalopathy
Received August 25, 2006. Revised November 3, 2006. Accepted December12, 2006
Neuromyelitis optica (NMO) is an idiopathic and usually
relapsing inflammatory demyelinating disease of the CNS
characterized by severe attacks of optic neuritis and
myelitis. It can be distinguished from classical multiple
sclerosis(MS) byclinical, neuroimaging,CSF and
In contrast to classical MS, which is thought to be
mediated by effector T cells, increasing evidence supports
a role for an autoantibody-mediated pathogenesis in NMO
(Lucchinetti et al., 2002). NMO is often associated with
criteria(Wingerchuk et al.,1999,2006).
doi:10.1093/brain/awl371Brain (2007), Page1of12
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Brain Advance Access published February 4, 2007
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other organ-specific or non-organ-specific autoimmune
(O’Riordan et al., 1996; Wingerchuk et al., 2006). In
addition, plasmapheresis is therapeutically effective in NMO
patients with severe steroid-unresponsive relapses (Keegan
et al., 2002). NMO lesions are characterized by extensive
demyelination with partial necrosis in the spinal cord,
involvingboth grey and
extending over multiple segments (Lucchinetti et al.,
2005). The lesions are associated with acute axonal injury,
and inflammatory cell infiltrates containing eosinophils and
granulocytes. Deposits of IgG and IgM co-localize with
products of complement activation in a vasculocentric pattern
around thickened hyalinized blood vessels, suggesting a
pathogenic role for humoral immunity targeting an antigen
in the perivascular space (Lucchinetti et al., 2002). This
hypothesis is strengthened by the recent identification of a
specific serum autoantibody biomarker, NMO-IgG (Lennon
et al., 2004), which targets the most abundant water channel
protein in the CNS aquaporin-4 (AQP4), (Lennon et al., 2005).
The anatomical distribution and cellular sites of AQP4
expression in normal mammalian tissues, including brain
and spinal cord, have been investigated extensively. AQP4 is
a homotetrameric integral plasma membrane protein
anchored in the astrocytic foot process membrane by the
dystroglycan complex (Amiry-Moghaddam and Ottersen,
2003). Prior studies demonstrate intense AQP4 immuno-
reactivity in the plasma membrane of the astrocytic endfeet
that abut capillaries and pia in the brain and spinal cord,
particularly in the subpial and subependymal zones, as well
as in the glial lamellae of the supraoptic nucleus in the
hypothalamus (Jung et al., 1994; Frigeri et al., 1995; Nielsen
et al., 1997; Venero et al., 1999; Amiry-Moghaddam and
The localization of AQP4 in the astrocytic foot processes
surrounding endothelial cells is consistent with the role of
astrocytes in the development, function and integrity of the
interface between brain parenchyma and perivascular space,
and between brain and cerebrospinal fluid, and serves to
mediate water flux (Nicchia et al., 2004). Experimental rodent
models of ischaemia, trauma and hyponatraemia implicate
AQP4 in the development of brain oedema regardless of
cause (Manley et al., 2000; Taniguchi et al., 2000; Saadoun
et al., 2002; Vajda et al., 2002; Warth et al., 2005).
Enhanced AQP4 expression has been reported in a
wide spectrum of human neuropathological conditions,
including ischaemia, trauma, brain tumours, bacterial
(HIVE), MS and Creutzfeldt–Jakob
et al., 2002; Aoki et al., 2003, 2005; Misu et al., 2006). A
recent report (Aoki et al., 2005), suggests that the
distribution of intensely AQP4-positive astrocytes differs
among disease states. In PML and HIVE, abundant AQP4-
expressing astrocytes were noted at the boundary between
greyand white matter
whitematter, and usually
inflammatory foci. The absence of a comparative analysis
of AQP4 expression in normal control brain tissue in this
study, however, may have led to erroneous attribution of
AQP4 expression patterns to disease states. Additionally,
rostral–caudal regional differences in AQP4 mRNA expres-
sion levels have been described (Venero et al., 1999, 2001).
Thus, it is essential to compare similar regions in diseased
and normal control brain, before inferring a ‘pathological
Upregulated AQP4 was reported in astrocytes located at the
periphery of MS plaques, but the plaque centre and
unaffected normal appearing white matter areas were
essentially devoid of AQP4 (Aoki et al., 2005; Misu et al.,
2006). However, the stage of demyelinating activity within the
MS lesions was not specified, precluding determination of
any association of lesional stage with AQP4 expression.
The distribution of AQP4 at glial–fluid interfaces in the
mouse spinal cord coincides with sites to which NMO-IgG
binds, and is similar to the deposition pattern of Ig and
demyelinating NMO lesions (Lucchinetti et al., 2002;
Lennon et al., 2004). These observations make a compelling
but circumstantial case for AQP4-IgG being a primary
effector of NMO lesions. A recent immunohistochemical
study of a single NMO case reported perivascular loss of
AQP4 immunoreactivity in spinal cord lesions (Misu et al.,
2006). The authors contrasted this observation with
high AQP4 expression in a section of normal cervical
spinal cord grey matter, and with an apparent increase of
AQP4 expression in reactive astrocytes of MS lesions.
This study, however, did not specify the number of MS
cases, the number of lesions analysed, or the stage of
demyelinating activity in any of the NMO or MS lesions.
lesions corresponded to regions of tissue necrosis and
cavitation. Although the authors suggested that functional
may underlie NMO lesion pathogenesis, they did not address
the potential influence of other biological factors on AQP4
expression, such as demyelinating or remyelinating activity,
the extent of tissue necrosis, or the degree of astrocyte loss.
The present study describes AQP4 expression in a large
series of NMO cases that are well characterized clinically
and pathologically (Lucchinetti et al., 2002). We compare
expression patterns of AQP4 immunoreactivity in NMO
lesions with patterns observed in normal brain, optic nerve
and spinal cord; in acute, subacute and chronic infarcts of
brain, optic nerve and spinal cord; and in acute and
chronic MS lesions. We observed that the pattern of AQP4
expression in normal tissues is similar to the rim and
rosette pattern of Ig deposits and products of complement
activation observed in NMO lesions. In contrast to a stage-
dependent loss of AQP4 in MS lesions, we observed in all
NMO lesions a striking loss of AQP4 regardless of the stage
of demyelinating activity, extent of tissue necrosis, or
CNS region involved. We further observed AQP4 loss at
loss of AQP4 in some
Page 2 of12Brain (2007)S.F. Roemer et al.
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sites of vasculocentric Ig deposition and complement activa-
tion in NMO lesions that lacked demyelination, but were
inflammatory. These lesions were identified in both the spinal
cord and medulla at the floor of the fourth ventricle,
particularly involving the area postrema, a region lacking
a blood–brain barrier (BBB) and rich in osmoreceptors. The
pathogenic implications of these observations are discussed.
Material and methods
The study was performed on archival brain, optic nerve and
spinal cord material from nine patients with NMO, 13 with
MS, nine withinfarcts and
patients withoutCNS histopathological
study was approved by the Mayo Clinic Institutional Review
Board (2067–99). Causes of death in the control cases were
acute myocardial infarct (3), pneumonia (1) and unknown (1).
All materials were obtained at autopsy except for two acute MS
cases that were obtained surgically (diagnostic brain biopsy).
Detailed clinical histories were available for all cases. The NMO
cohort comprised eight women and one man, with an average age
of 50 years (range 16–80 years). The clinical course was relapsing
in eight patients, and monophasic in a single case. Mean disease
duration was 2.4 years (SEM?0.8 years). All patients died from
respiratory compromise directly attributable to attacks of NMO.
The presenting syndrome was optic neuritis in four patients, and
myelitis in five patients, with a mean interval of 19months (range
4–41months) between optic neuropathy and myelopathy. The MS
cohort comprised: (i) 11 acute MS cases (nine women and two
men) with an average age of 43 years (range 26–70 years). Mean
disease duration was 4.1months (SEM?13.6months). Death was
attributable to an acute MS attack associated with herniation in
five cases, sepsis due to pneumonia in three and unknown in one.
Two patients are still living. (ii) Two chronic cases, both women
(aged 36 and 71 years) who had a secondary progressive
course and mean disease duration of 18 years (6 and 30 years).
Both patients died of respiratory compromise associated with
Neuropathological evaluation and
Specimens were fixed in 10–15% formalin and embedded in
paraffin. Sections were stained with haematoxylin and eosin (HE),
Luxol-fast blue-periodic acid-Schiff (LFB/PAS) and Bielschowsky
silver impregnation. Immunohistochemistry
without modification using an avidin–biotin or an alkaline-
described previously (Vass et al., 1986). The primary antibodies
were specific for myelin proteins (myelin basic protein [MBP;
Boehringer Mannheim, Germany], proteolipid protein [PLP,
protein [MOG; Dr S Piddlesden, Department of Biochemistry,
[CNPase; Sternberger, USA], myelin associated glycoprotein
[MAG; Serotec]), glial fibrillary acidic protein (GFAP; Dako),
neurofilament protein (NF; Dako), T lymphocytes (all T cells;
CD3 and cytotoxic CD8 T cells; [Dako]), B lymphocytes (CD20;
Dako), plasma cells (CD138; Dako), macrophages/microglial
cells (KiM1P; Dr Radzun, University of Go ¨ttingen, Germany),
Department of Biochemistry, Cardiff, UK), immunoglobulin G
(IgG; Dako), immunoglobulin M (IgM; Dako) and AQP4
Sigma-Aldrich). Table 1 lists the antibodies and specific conditions
used for immunohistochemistry. The primary antibodies were
omitted in controls. All antibodies were incubated at 4?C
Staging of demyelinating activityin NMOand MS
Lesions were classified with respect to demyelinating activity, as
previously described(Brucket al., 1995).‘Earlyactive
T able1 Antibodies used for immunohistochemistry
AntibodyCloneDilution Antigen retrievalCompany/source
Boehringer, Mannheim; Roche,Germany
Dr Sarah Piddlesden; Cardiff,UK
Dr Radzun; Goettingen,Germany
Dr Paul Morgan; Cardiff,UK
Dr Paul Morgan; Cardiff,UK
All monoclonal antibodies are from mouse and all polyclonals from rabbit. EDTA¼ethylenediaminetetraacetic acid; NA¼not applicable.
Aquaporin-4 immunoreactivity, neuromyelitis optica and MS Brain (2007) Page 3 of12
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demyelinating lesions’ were diffusely infiltrated by macrophages
containing immunoreactive products for all myelin proteins. ‘Late
active demyelinating lesions’ were more advanced with respect to
myelin degradation, and were immunoreactive for major myelin
proteins (MBP and PLP), but not for MOG or CNPase.
‘Remyelinating lesions’ were characterized by uniformly thin and
irregularly arranged myelin sheaths. ‘Inactive demyelinated lesions’
were completely demyelinated without evidence of active demye-
lination. We examined 77 lesions from 9 autopsy cases of clinically
confirmed NMO. Demyelinating activity in the NMO lesions was
classified immunohistochemically as ‘early active’ in 22, ‘late
active’in 18, ‘inactive’in 37
Demyelinating activity in 57 lesions from the 11 acute MS cases
was classified as ‘early active’ in 45, ‘early remyelinating’ in 6 and
‘inactive’ in 6. The 2 chronic MS cases had no ‘active’ lesion, 24
‘inactive’, and 3 ‘late remyelinated’ lesions (i.e. shadow plaques).
Early active MS lesions in the acute MS cohort were further
classified immunopathologically based on previously published
criteria (Lucchinetti et al., 2000), into patterns I (T cell/
macrophage associated) (n¼2 lesions from two MS biopsy
cases), II (antibody/complement associated) (n¼30 lesions from
six cases), III (distal oligodendrogliopathy) (n¼11 lesions from
two cases) or IV (primary oligodendrogliopathy) (n¼2 lesions
from one case). We also evaluated AQP4 expression in nine NMO
lesions and seven MS lesions with cavitation.
and ‘remyelinating’in 0.
Table 2 summarizes AQP4 immunoreactivity patterns in
infarct, MS and NMO tissues relative to baseline expression
in normal CNS control tissues. Specific characteristics of
AQP4 immunoreactivity are discussed subsequently.
AQP4 immunoreactivity in normal CNS
AQP4 immunoreactivity in normal tissues, at all levels of
the CNS, was most intense at the glia limitans externa and
the subependyma (Fig. 1A–C). Within the cerebral cortex,
AQP4 immunoreactivity was concentrated in astrocytic foot
processes extending to the abluminal surface of blood
vessels (Fig. 1D). As is typical of astrocytes, GFAP
immunoreactivity was concentrated in the cell body and
soma, and did not extend to the astrocytic endfeet
associated with either small or medium-sized blood vessels
(Simard et al., 2003), (Fig. 1E). Cerebral white matter
restricted to the abluminal surface of occasional penetrating
blood vessels (Fig. 1F). Within the brainstem (midbrain to
caudal medulla), AQP4 immunoreactivity was most intense
in subependymal regions at the floor of the fourth ventricle
(Fig. 1G). Brainstem white matter exhibited a mesh-like
pattern ofAQP4 immunoreactivity,
perivascular staining in a rim and rosette pattern. In the
spinal cord, AQP4 immunoreactivity was prominent within
both grey and white matter (Fig. 1H). AQP4 immunor-
eactivity in the optic nerve exhibited meshwork and rim
and rosette vasculocentric patterns (Fig. 1I), similar to
those seen in brainstem and spinal cord white matter.
AQP4 immunoreactivity in other
The intensity and distribution of AQP4 immunoreactivity
in brain, optic nerve and spinal cord infarcts were
compared with the baseline expression patterns observed
in regionally matched normal control tissues. In acute
infarcts (512 h; one cerebral and two spinal cord) and
subacute infarcts (57 days; one cerebral and two spinal
cord), AQP4 was lost in the necrotic centre and diffusely
increased at the periphery of the infarct (Fig. 2A and E).
A similar pattern of GFAP immunoreactivity was seen in
the necrotic centre and ischaemic periphery (Fig. 2B).
Higher magnification demonstrated increased AQP4 out-
(Fig. 2C), whereas GFAP was more concentrated in the
cell body (Fig. 2D). AQP4 was lost in the necrotic zone
(Fig. 2A, E and F). Chronic infarcts (42 years; two cerebral
of reactive astrocytes
T able 2 Aquaporin-4 immunoreactivity in infarct, MS and
NMO relative to baseline expression in normal controls
Chronic multiple sclerosis
Late remyelinating (n¼3)
Acute multiple sclerosis?
Early remyelination (n¼6)
N ¼ number of cases; n, number of lesion areas; GM ¼ grey
matter; WM ¼ white matter; NA ¼ not applicable; þ/?, þ, þþ,
þþþ¼intensity scale of AQP4 immunoreactivity in CNS control
tissue; ", "", """ ¼ degree of increasein AQP4 immunoreactivity
relative to baseline expression in regionally matched CNS
control tissue; $ ¼ no change in AQP4 immunoreactivity relative
to baseline expression in regionally matched CNS control tissue;
0 ¼ complete loss of AQP4 immunoreactivity.
difference in AQP4 immunoreactivity pattern between immuno-
pathologically classified acute MS cases (patterns I¼2, II¼6,
inflammatory NMO foci lacking demyelination associated with
vasculocentric immune complex deposition.
?There is no
yAQP4 immunoreactivity is also absent in
Page 4 of12 Brain (2007)S.F. Roemer et al.
by guest on June 9, 2013
and one optic nerve) demonstrated no increase in AQP4
immunoreactivity at the periphery, while cavitary regions
were devoid of AQP4.
AQP4 immunoreactivity in MS lesions is
AQP4 expression in MS lesions correlated with the
stageof demyelinating activity.
immunopathologically classified as patterns I, II, III or
IV, the pattern of AQP4 immunoreactivity was identical.
AQP4 was diffusely increased in the periplaque white
matter of early (Fig. 3A) and late active lesions, and
outlined the cytoplasmic surface of enlarged reactive
astrocytes dispersed throughout the active lesion centre
(Fig. 3B). Early remyelinating lesions demonstrated a
In acuteMS cases
diffuse increase in AQP4 immunoreactivity in both the
periplaque white matter and lesion centre (Fig. 3C), and
also outlined the cytoplasmic surface of reactive astrocytes
(Fig. 3D). In contrast, inactive lesions identified within the
acute MS cohort were devoid of AQP4 (Fig. 3E), despite
the continued presence in some lesions of PAS-positive
macrophages that lacked myelin degradation products.
In both chronic MS cases, AQP4 was absent in inactive
long-standing plaques (Fig. 3F), but was diffusely increased
within late remyelinated shadow plaques. AQP4 was absent
in all seven cavitary acute MS lesions.
In active pattern II MS lesions (Fig. 4A), Ig and
complement deposits were distributed in a pattern quite
distinct from the striking perivascular rosette and rim
pattern of Ig and complement activation product deposi-
tion described in NMO lesions (Lucchinetti et al., 2002).
Fig.1 AQP4 immunoreactivity (IR) in normal CNS tissues. (A) AQP4 is concentrated at the glia limitans externa, throughout the cerebral
cortex and at the grey^white junction. (B) Higher magnification of boxed region in A demonstrates intense AQP4at the glia limitans
(arrows) and a rosette pattern of staining in the underlying cortex. (C) AQP4 is concentrated in the subependymal region. (D) AQP4 is
intense at the abluminal surface of penetrating cortical blood vessels in a rim pattern (arrow) and at astrocytic foot processes abutting
vessels in a rosette pattern (arrowhead). (E) GFAP is concentrated at the glia limitans and astrocyte cellbody, butdoes notlabel astrocytic
endfeet associated with small to medium sized vessels. (F) AQP4 is minimal in normal cerebral white matter, apart from occasional pene-
trating capillaries. (G) AQP4 is intense at the floor of the fourth ventricle. (H) Spinal cord white and grey matter show moderate diffuse
AQP4. (I) Optic nerve shows diffuse mesh-like and vasculocentric (arrow) AQP4 IR. A^D, F^I, AQP4 Immunohistochemistry (IHC); E,
Aquaporin-4 immunoreactivity, neuromyelitis optica and MSBrain (2007) Page 5 of12
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The inflammatory infiltrates in pattern II MS lesions
were lymphocytic and products of complement activation
were less pronounced in degree, and were detected on
degenerating myelin sheaths, within macrophages and on
oligodendrocytes along the active plaque edge (Fig. 4B)
(Lucchinetti et al., 2000). AQP4 immunoreactivity was
less intense in the plaque centre relative to its increased
expression in the periplaque white matter, and outlined
the cytoplasm of reactive astrocytes, as well as astrocytic
foot processes surrounding blood vessels (Fig. 4C).
AQP4 immunoreactivity in NMOlesions
The pattern of AQP4 expression in NMO lesions was
fundamentally different. No AQP4 immunoreactivity was
detectable in any lesion, irrespective of stage of demyelinat-
ing activity. Actively demyelinated NMO lesions (Fig. 4D)
contained granulocytes and eosinophils, and exhibited a
striking vasculocentric deposition of Ig and the C9neo
product of complement activation (Fig. 4E) in a rim and
rosette pattern (Fig. 4E, inset), in regions of AQP4 loss
(Fig. 4F). Periplaque white matter demonstrated a similar
degree of AQP4 immunoreactivity as normal regionally
matched control tissue (Fig. 4F). The identical pattern of
also observed in the optic nerve of a single NMO case
[early active (Fig. 5A and B), inactive, and cavitary lesion
areas], in contrast to the stage-dependent pattern of
periplaque and lesion AQP4 expression observed in early
active (Fig. 5C, D), remyelinated, inactive and cavitary optic
nerve lesion areas from a single acute MS case.
An unanticipated observation was loss of AQP4 in spinal
cord (Fig. 6C) and brainstem regions characterized by
eosinophil and plasma cell infiltrates and vasculocentric
deposits of IgM, IgG (Fig. 6A) and complement activation
products (Fig. 6B), but lacking evidence of demyelination
(n¼5 cases) (Fig. 6D and E). These regions appeared
rarefied, particularly around blood vessels (Fig. 6D),
and despite AQP4 loss, they retained normal staining of all
myelin proteins [MBP, PLP, CNPase, MAG, MOG (Fig. 6E)],
and lacked macrophages containing myelin degradation
products. Axons were structurally preserved with no
evidence of acute axonal pathology (Fig. 6F).
Of particular note, three of the NMO cases had
inflammatory foci lacking demyelination associated with
AQP4 loss situated below the floor of the fourth ventricle
extending laterally along the subependymal surface (Fig. 7).
The involved tissue appeared rarefied (Fig. 7A), and
CD138þ plasma cells (Fig. 7B) and eosinophils (Fig. 7C).
Myelin was preserved in the subependymal white matter
(Fig. 7D). IgG and IgM deposits were diffuse (Fig. 7E),
as well as vasculocentric (Fig. 7G), and serial sections
revealed they co-localized with products of complement
activation (Fig. 7H) and regions of AQP4 loss (Fig. 7F and
I). These inflammatory foci associated with AQP4 loss
extended into the adjacent area postrema (Fig. 7G–I), a site
known to lack a BBB.
Despite a complete loss of AQP4 immunoreactivity
(Fig. 8A and C), reactive
demyelinated NMO foci (Fig. 8B and D). This is in
contrast to active (Fig. 8E and F; Fig. 3B) and remyelinating
(Fig. 3C and D) MS lesions, as well as acute and subacute
infarcts (Fig. 2A, C and E), in which AQP4 immuno-
reactivity is diffusely increased, and outlines the cytoplasmic
surface of GFAP positive astrocytes.
GFAP positive astrocytes
The relationship between MS and NMO has long been
debated. We previously reported a unique pattern of tissue
destruction inactiveNMO lesions characterizedby
Fig. 2 AQP4 immunoreactivity (IR) in early acute (A^D) and
subacute (E^F) infarcts. (A) The early acute infarct demonstrates
loss of AQP4 in the necrotic centre (*), with increased IR in the
periphery. (B) With GFAP, there is a similar pattern and distribu-
tion of IR, absent in the necrotic centre (*), and increased at the
periphery. (C) Higher magnification of the infarct periphery in
A demonstrates increased AQP4 outlining the cytoplasmic surface
of reactive astrocytes (arrowheads). (D) Higher magnification of
the infarct periphery in B demonstrates GFAP is similarly
increased in reactive astrocytes, but concentrated in cell bodies
(arrowheads). (E) The subacute infarct also demonstrates
increased AQP4 IR in the periphery, with relative lack of AQP4
IR in macrophage-rich necrotic areas (*). (F) There is absence
of AQP4 in the necrotic zone. A, C, E, F, AQP4 IHC; B, D,
Page 6 of12Brain (2007)S.F. Roemer et al.
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eosinophil and neutrophil infiltration, vascular fibrosis and
intense vasculocentric deposition of Ig and products of
complement activation in a characteristic ‘rim’ and ‘rosette’
pattern (Lucchinetti et al., 2002). Based on those findings,
we suggested that humoral autoimmunity targeting an
antigen in the perivascular space may play a role in the
pathogenesis of NMO. Lennon et al. subsequently identified
a specific marker autoantibody (NMO-IgG), which interacts
selectively with the AQP4 water channel (Lennon et al.,
2005). AQP4 is concentrated at the astrocytic endfeet,
which are typically GFAP-negative (Frigeri et al., 1995;
Bushong et al., 2002; Simard et al., 2003). Whereas GFAP-
positive processes do not systematically cover smaller
vessels (516mm) and capillaries, AQP4 expression outlines
Fig. 4 AQP4 immunoreactivity (IR) in acute pattern II MS (A^C) and NMO (D^F) lesions. (A) Numerous macrophages containing myelin
debris are dispersed throughout the active lesion (arrowheads and inset; LFB/PAS). (B) C9neo antigen is present within macrophages
(arrowheads), but absent around blood vessels (arrow). (C) Higher magnification reveals AQP4 IR is prominent in a rosette pattern
surrounding a penetrating blood vessel in the lesion. (D) In NMO, there is extensive demyelination involving both grey and white matter
(LFB/PAS); *indicates preserved myelin in the PPWM. (E) C9neo is deposited in a vasculocentric rim and rosette pattern (inset) within the
active lesions, but not in the PPWM. (F) The lesions lack AQP4, which is retained in the PPWM (*) and grey matter. A, D, LFB/PAS; B, E,
C9neo IHC; C, F, AQP4 IHC.
Fig. 3 Stage-dependent pattern of AQP4 immunoreactivity (IR) in active (A, B), early remyelinated (C, D) and inactive (E, F) MS lesions.
(A) An active MS lesion demonstrates increased AQP4 IR that is marked in the adjacent cortical grey matter (*) and periplaque white
matter (PPWM), and moderate in the lesion centre (þ). (B) Higher magnification of the active lesion centre demonstrates AQP4 outlining
the cytoplasmic surface of reactive astrocytes (arrowheads) and their processes. (C) An early remyelinated MS lesion (circle) shows diffuse
increase in AQP4 extending throughout the lesion, and surrounding PPWM. (D) AQP4 stains the cytoplasmic surface of reactive astro-
cytes (arrowheads) in an early remyelinatedlesion. (E^F).Inactive MS lesions from an acute case (E) and a chronic case (F) show complete
loss of AQP4; (*) indicates normal grey matter AQP4 IR. A^F, AQP4 IHC.
Aquaporin-4 immunoreactivity, neuromyelitis optica and MSBrain (2007)Page 7 of12
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the entire network of vessels (Simard et al., 2003; Lennon
et al., 2005). A pathogenic role for NMO-IgG remains to be
proven, but the strategic location of AQP4at the BBB
strengthens our original hypothesis of a perivascular target
The present study describes a unique pattern of AQP4
loss in nine of nine NMO cases. This pattern is unrelated to
stage of demyelinating activity and is distinct from the
patterns of AQP4 expression observed in MS, infarcts and
normal controls. Changes in the intensity of AQP4
immunoreactivity in MS lesions were dependent on the
stage of demyelination. The increase in AQP4 immunor-
eactivity observed in the periplaque white matter and
within reactive astrocytes of active MS lesions is consistent
with published studies (Aoki et al., 2005; Misu et al., 2006).
However, we also observed complete loss of AQP4
immunoreactivity in inactive MS lesions sampled from
both the acute and chronic phases of the disease.
This contradicts the findings of Misu et al. (2006) whose
study concluded that AQP4 is not lost in MS lesions, but
in which the stage of demyelinating activity was not
It is not unexpected to find AQP4 expression increased
in actively demyelinating and remyelinating MS lesions,
since astrocytic proliferation is a physiological host response
to inflammation. Similarly, it is not unexpected for AQP4
expression to be reduced or undetectable in inactive gliotic
and quiescent non-inflammatory MS lesions. Increased
AQP4 immunoreactivity outlining GFAP positive reactive
periplaque and lesion centre of active and remyelinating
MS lesions, and in the periphery of infarcts. However, both
active MS and NMO lesions are characterized by a
proliferative astrocytic response, yet AQP4 immunoreactiv-
ity was not observed in either the periplaque or lesion
centre of any NMO lesion, regardless of location, or stage
of demyelinating activity. These findings suggest a targeted
attack against AQP4. The presence of GFAP positive
was observedin the
Fig. 6 Spinal cord AQP4 loss in inflammatory NMO foci lacking demyelination. (A^B) Immune complexes (IgG, A; C9neo, B) are depos-
itedin a rosette pattern surrounding thickened, hyalinized vessels (arrows). (C^F) The lesion is characterizedby complete loss of AQP4 IR
(C; note residual AQP4 in grey matter at lower left), with preservation of myelin (D, LFB/PAS; E, MOG) and axons (F, Bielschowsky).
Fig. 5 Comparison of AQP4 immunoreactivity (IR) in active NMO
(A, B) and MS (C, D) optic nerve lesions. (A) Active demyelination
with macrophages containing MOG-immunoreactive myelin debris
(arrowheads), adjacent to PPWM (*). (B) AQP4 is lost in the active
lesion, but retained in the PPWM (*). (C) Active demyelination
with macrophages containing PLP-immunoreactive myelin debris
(arrowheads), adjacent to PPWM (*). (D) AQP4 IR is increased in
both the active lesion and PPWM (*). A, MOG IHC; B, D, AQP4
IHC; C, PLP IHC.
Page 8 of12Brain (2007)S.F. Roemer et al.
by guest on June 9, 2013
reactive astrocytes within inflammatory non-demyelinated
NMO lesions, despite the complete absence of AQP4
immunoreactivity, argues against AQP4 loss in NMO
lesions being secondary to astrocyte loss. Our finding of
stage-dependent differences in AQP4 expression in MS
lesions, in contrast to the absence of AQP4 in all NMO
lesions regardless of demyelinating stage, suggests that
different pathogenic mechanisms underlie disease initiation
and evolution in these two disorders.
This study also provides a plausible explanation for the
rim and rosette pattern of Ig and complement activation
product deposits that we reported previously as a distinctive
characteristic of NMO lesions (Lucchinetti et al., 2002).
The finding of an identical staining pattern of AQP4 in
normal brain, optic nerve and spinal cord localizing to
astrocytic endfeet in the perivascular glia limitans, suggests
that the rim and rosette pattern of Ig and terminal
complement deposition reflects the regional density of
AQP4 molecules (Nielsen et al., 1997; Venero et al., 1999).
The report of Misu et al. (2006) emphasized increased
AQP4 immunoreactivity within normal central spinal cord
grey matter. We additionally noted prominent staining of
white matter within normal optic nerve, brainstem and
spinal cord tissues, in contrast to limited expression of
AQP4 in supratentorial white matter. The widespread
expression of AQP4 in the brain is paradoxical in face of
the typically optic–spinal predominant locations of NMO
lesions and predilection for brainstem. Regional differences
in AQP4 concentration could contribute to this paradox, as
well as regional differences in the spatial distribution or
molecular orientation of AQP4 epitopes on astrocytic
endfeet that might preclude efficient complement activation
or intermolecular cross-linking at some sites (Lennon, 1978;
Lennon et al., 1984, 2005).
pathology, it is important to determine whether AQP4
loss in NMO is truly disease-specific, or rather reflects
tissue injury or loss of cellular elements and antigen
Fig. 7 Medullary floor of the fourth ventricle AQP4 loss in inflammatory NMO foci lacking demyelination. (A^C) Patchy perivascular
inflammatory infiltrate andparenchymalrarefaction are noted at low power (A,H & E).Major components of the inflammatory infiltrates
include plasma cells (B, CD138), and eosinophils (C,H & E), but macrophages are scarce. (D^F) There is myelin preservation (D, MOG),
while IgM is prominent in subventricular zones. (E, IgM). Note that AQP4 loss is most profound in region of IgM deposition (F, AQP4).
(G^I) In the area postrema: subependymal vessels (arrowheads) show a rosette pattern of staining for IgM (G), C9neo (H) and loss of
Aquaporin-4 immunoreactivity, neuromyelitis optica and MSBrain (2007) Page 9 of12
by guest on June 9, 2013
degradation associated with necrosis. Therefore, we exam-
ined AQP4 immunoreactivity in non-necrotic and necrotic
regions sampled from acute, subacute and chronic infarcts.
Our observations of enhanced AQP4 immunoreactivity in
the periphery of acute and subacute non-necrotic ischaemic
foci are consistent with published studies (Aoki et al.,
2003). In a rat ischaemia model, AQP4 mRNA expression
after middle cerebral artery occlusion was increased in the
region surrounding infarcted cortex during the observation
period (1–7 days, maximal at day 3), and the change was
related to the generation and resolution of brain oedema.
The increase of AQP4 immunoreactivity in the periphery of
acute and subacute infarcts may therefore reflect the
participation of AQP4 in the development of oedema.
In contrast, all necrotic ischaemic lesions lacked AQP4
immunoreactivity, regardless of infarct stage. AQP4 loss was
also observed in cavitated demyelinated regions analysed
from both NMO and acute fulminant MS lesions. Loss of
AQP4 immunoreactivity in these circumstances is not
surprising, because astrocytes are destroyed during necrosis,
regardless of the initiating disease. It is, therefore, critical to
evaluate non-necrotic NMO lesions in order to confirm
Our findings in spinal and brainstem NMO lesions of
AQP4 loss in foci that were inflammatory but neither
demyelinated nor necrotic, and co-localizing with intense
vasculocentric deposition of immune complexes, strongly
implicate a complement-activating AQP4-specific autoanti-
body as the initiator of the NMO lesion. Small amounts of
circulating IgG normally gain access to the CNS because the
BBB is not absolutely impenetrable (Brimijoin et al., 1990).
NMO may indeed be an antibody-mediated disease.
However, our findings to date have not excluded a role
for effector T cells in NMO pathogenesis.
Our study revealed another novel pathological finding in
NMO, namely lesions in the medullary tegmentum at the
floor of the fourth ventricle involving the subependymal
region and the area postrema. Recent MRI studies have
established conclusively that lesions in NMO may target the
brain, even relatively early in the course of the disease, and
that certain brain lesions are far more common in NMO
than in MS (Pittock et al., 2006a,b). In 10% of the cases,
NMO brain lesions affect the hypothalamus and brainstem,
especially the periventricular and subependymal regions.
Clinical reports have described endocrine dysfunction in
NMO (Vernant et al., 1997), as well as intractable hiccups
and nausea in 8 out of 47 cases of relapsing NMO (Misu
et al., 2005). In six of the latter report’s cases, MRI revealed
medullary lesions involving the ventricular and spinal canal
regions,the nucleus tractus
postrema. Our observations likely reflect the pathological
substrate underlying these clinical and imaging correlates
The area postrema, like other circumventricular organs,
shows intense AQP4 expression and has characteristic
‘hypendymal’ features, namely a neurovascular plexus with
a loose glial bed, a thin ependymal cover and lack of BBB
(Goren et al., 2006). Expression of AQP4 in astrocytic
endfeet and the glia limitans is critical for normal
CSF–brain interfaces. Neurons and astrocytes of the area
postrema are an important target for circulating signals
regulating blood pressure, cerebral blood flow and osmo-
larity, including angiotensin II, arginine, vasopressin and
atriopeptins (Simard and Nedergaard, 2004). This region
also serves as an interface between the immune system and
the brain. It contains receptors for circulating cytokines and
following peripheral challenges with immunostimulants,
may harbour immunocytes expressing cytokine immuno-
reactivity, such as IL-1b. In the area postrema, mast cells
are located subependymally and in close proximity to blood
vessels, suggesting that their products may regulate local
blood flow and blood vessel permeability. The area
postrema has robust connections with other CNS areas
involved in osmoregulation and brain volume homeostasis,
including the magnocellular hypothalamic nuclei, and
with areas involved in immunomodulation, such as the
paraventricular nucleus. Thus autoimmune targeting of
this region in NMO may disrupt several homeostatic
mechanismswhich may result
solitarius and the area
Fig. 8 AQP4 and astrocytes in NMO and MS. (A^D) There is loss
of AQP4 in the medullary subependyma (A) and raphe (C), despite
astrogliosis associated with the presence of GFAP immunoreactive
astrocytes (B, D). (E^F) Early MS lesion shows increased AQP4
(E) in the PPWM (*), the expanding macrophage rich border
and lesion centre. With GFAP (F), a similar increased distribution
associated with multiple reactive astrocytes is noted. *PPWM. A,
C, E, AQP4 IHC; B, D, F,GFAP IHC.
Page10 of12 Brain (2007) S.F. Roemer et al.
by guest on June 9, 2013
blood flow autoregulation, cerebral oedema and immune
In summary, we have documented two basic pathologies
in NMO, both associated with loss of AQP4 immuno-
reactivity. The most prevalent lesion type involved the
spinal cord and optic nerves, and AQP4 loss was in the
context of vasculocentric immune complex deposition,
active demyelination and vascular hyperplasia with hyalini-
zation. These lesions were often cavitary, and involved both
grey and white matter in the spinal cord. The less frequent
lesion type was found in the spinal cord and medulla
extendinginto thearea postrema,
inflammatory. AQP4 loss was associated with vasculocentric
IgG and IgM deposits and complement activation, and
tissue rarefaction, but there was no evidence of demyelina-
tion. Whether these inflammatory non-demyelinated brain-
stem lesions progress to demyelinated cavitary NMO lesions
is uncertain, but unlikely in light of recent MRI reports
describing reversible T2-weighted non-enhancing signal
abnormalities corresponding to the area postrema in
patients who recovered from intractable hiccups and
vomiting (Misu et al., 2005). Furthermore, in contrast to
the severe clinical manifestations of lesions involving the
optic nerves and spinal cord in NMO patients, brainstem
lesions in NMO are often asymptomatic, and have been
observed by imaging to resolve rapidly in some patients
(Pittock et al., 2006a,b). It is plausible, therefore, that these
medullary inflammatory non-demyelinated NMO lesions
reflect a transient functional impairment of the astrocyte’s
capacity to mediate water flux on initial binding of IgG to
AQP4 that may be rapidly compensated in regions richly
endowed with AQP4. It remains to be determined whether
these two NMO lesion types reflect different pathogenic
mechanisms in lesion formation, or alternatively represent
different anatomical region-specific responses to the same
pathogenic mechanism. Proof of the pathogenicity of
AQP4-specific IgG will require the induction of the
characteristic vasculocentric lesions in the spinal cord and
optic nerve of animals by passive transfer of AQP4-IgG or
by active immunization with AQP4.
The authors thank Patricia Ziemer for her expert technical
assistance and Peggy Chihak for photographic assistance.
Study supported in part by MSIF Du Pre Grant (SFR),
NIH RO1-NS049577-01-A2 (CFL), NMSS RG 3185-B-3
(CFL) and theRalphC.
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