Greatly improved neurological outcome after spinal Samira Saadoun, B. Anthony Bell,

doi:10.1093/brain/awn014
Brain (2008), 131, 1087^1098
Greatly improved neurological outcome after spinal
cord compression injury in AQP4-deficient mice
Samira Saadoun,1 B. Anthony Bell,1 A. S. Verkman2 and Marios C. Papadopoulos1
1
Academic Neurosurgery Unit, St George’s, University of London, London SW17 0RE, UK and 2Department of Medicine
and Department of Physiology, University of California, San Francisco, CA 94143- 0521 USA
Correspondence to: Marios C. Papadopoulos, MD FRCS(SN), Academic Neurosurgery Unit, Room 1.122 Jenner Wing,
St George’s, University of London, Cranmer Terrace, Tooting, London SW17 0RE, UK
E-mail: [email protected]
Keywords: aquaporin- 4; astrocyte; oedema; spinal cord injury; water channel
Abbreviations: AQP4 = aquaporin- 4; AQP4+/+ = wildtype; AQP4 / = AQP4 -null; BMS = Basso Mouse Scale; EBD = Evans
blue dye; ECS = extracellular space; FJC = Fluoro Jade C; GFAP = glial fibrillary acidic protein; LFB = Luxol Fast Blue;
NeuN = neuronal nuclear protein; SCI = spinal cord injury; SSER = spinal somatosensory evoked response
Received October 9, 2007. Revised December 21, 2007. Accepted January 18, 2008. Advance Access publication February 11, 2008
Introduction
Traumatic spinal cord injury (SCI) is a devastating condition
primarily affecting young males with an annual incidence of
15–40 cases per million (Sekhon and Fehlings, 2001;
McDonald and Sadowsky, 2002; Wyndaele and Wyndaele,
2006). Care for individuals with SCI in the USA is estimated
to cost more than $7 billion per year. Most SCIs are
incomplete and even in patients whose motor and sensory
paralysis appears complete, post-mortem studies show spared
neural tissue (Kakulas, 1999). These observations raise the
possibility that early drug treatments may preserve these
neurons, thus improving outcome. Studies in animals suggest
that even small anatomical gains can produce disproportionate functional benefits. For example, fewer than 10% of
functional long-tract connections are necessary for locomotion (Blight, 1983). Such limited restoration might not enable
people with severe SCI to walk, but it might improve bowel
and bladder control, hand grasp, limb mobility and breathing.
Currently, there is no drug treatment that effectively
improves outcome after SCI.
Pathophysiological mechanisms that produce secondary
injury following the initial impact include spinal cord
oedema, ischaemia, free radical damage, electrolyte imbalance, excitotoxicity, inflammatory injury and apoptosis
ß 2008 The Author(s)
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Aquaporin- 4 (AQP4) is a water channel protein expressed in astrocytes throughout the CNS. In brain, AQP4
facilitates water balance and glial scar formation, which are important determinants of outcome after injury.
Here, we provide evidence for AQP4 -dependent spinal cord swelling following compression injury, resulting in
remarkably improved outcome in AQP4 -null mice. Two days after transient T6 spinal cord compression injury,
wild-type mice developed more severe hindlimb weakness than AQP4 -null mice, as assayed by the Basso openfield motor score, inclined plane method and footprint analysis. Basso motor scores were 1.3 0.5 (wild-type)
versus 4.9 0.6 (AQP4 -null) (SE, P _ 0.001). Improved motor outcome in AQP4 -null mice was independent of
mouse strain and persisted at least 4 weeks. AQP4 -null mice also had improved sensory outcome at 2 days, as
assessed by spinal somatosensory evoked responses, with signal amplitudes »10 kV (uninjured), 1.7 0.7 kV (wildtype) and 6.4 1.3 kV (AQP4 -null) (P _ 0.01).The improved motor and sensory indices in AQP4 -null mice corresponded to remarkably less neuronal death and myelin vacuolation, as well as reduced spinal cord swelling and
intraparenchymal spinal cord pressure measured at T6 at 2 days after injury. AQP4 immunoreactivity at the
injury site was increased in grey and white matter at 48 h. Taken together, our findings indicate that AQP4 provides a major route for excess water entry into the injured spinal cord, which in turn causes spinal cord swelling
and elevated spinal cord pressure. Our data suggest AQP4 inhibition or downregulation as novel early neuroprotective manoeuvres in spinal cord injury.
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Brain (2008), 131, 1087^1098
Methods
Mice
AQP4 / mice in CD1 and C57/BL/6 genetic backgrounds were
generated as described (Ma et al., 1997). Experiments were done
at the University of California, San Francisco and St George’s,
University of London using weight-matched mice (25–35 g),
typically 8–12 weeks old. Protocols were approved by the
University of California, San Francisco Committee on Animal
Research and the British Home Office, as appropriate. Investigators
were unaware of mouse genotype during the experiments.
Spinal cord injury
We used an established compression model previously used to
study spinal cord injury in mice (Faulkner et al., 2004; Terayama
et al., 2007), rats (Guth et al., 1985; Fujiki et al., 2005), guinea
pigs (Blight, 1991; Borgens and Shi, 2000; Luo et al., 2002b), and
isolated guinea pig spinal cords (Luo et al., 2002a; McBride et al.,
2007). Briefly, mice were anaesthetized using 2,2,2-tribromoethanol (125 mg/kg i.p., Avertin, Sigma, Poole, UK). A longitudinal
skin incision was made to expose the spine between T5–T7
vertebral body levels. The spine was then immobilized using two
pairs of forceps attached to a metal frame. A Foredom
micromotor drill with a 0.7 mm stainless steel burr
(MyNeuroLab, St Louis, MO) was used to remove the spinous
process, lamina and pedicles of T6 in order to expose the
underlying intact dura and spinal cord. The skin wound was then
closed in sham-operated mice. The cord at T6 was injured by
bilateral compression for 2 min using forceps that were 1 mm wide
with a 1 mm spacer (Fig. 1). Rectal temperature was kept within
37–38 C with an infrared lamp. Mice were returned to their cage
once they regained the righting reflex. After the injury, urinary
retention was relieved by twice-daily bladder expressions, as
described (Wrathall et al., 1985; Joshi and Fehlings, 2002; Basso
et al., 2006).
Neurological score
The open-field Basso Mouse Scale (BMS) was used to assess
hindlimb locomotion (Basso et al., 2006). Mice were evaluated by
two observers, and the final score was given by consensus.
Fig. 1 Spinal cord injury model. (A) Intraoperative photograph
after T6 laminectomy showing the spine clamped with forceps with
the dura/spinal cord exposed. (B) Schematic showing forceps with
spacer compressing the cord at T6. (C, D) Exposed theca in sham
(C) and injured (D) mice at 2 days after surgery. Evans blue dye
was injected intracisternally. Blue arrows mark the cranial end. Red
arrows indicate obstruction preventing the flow of dye.
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(McDonald and Sadowsky, 2002; Kwon et al., 2004). Water
accumulation in spinal cord parenchyma occurs in the
acute phase after contusion SCI in humans (Flanders et al.,
1999; Boldin et al., 2006; Miyanji et al., 2007), and several
animal species (Wagner and Stewart, 1981; Fujii et al., 1993;
Sharma et al., 2005), with substantial evidence indicating
that the amount of oedema correlates with the severity of
trauma and subsequent motor dysfunction (Wagner and
Stewart, 1981; Flanders et al., 1999; Sharma et al., 2005;
Boldin et al., 2006; Miyanji et al., 2007). We hypothesized
that the water channel protein aquaporin-4 (AQP4) may be
an important determinant of spinal cord oedema early after
injury and thus of ultimate neurological outcome. AQP4 is
expressed in glial cells throughout the central nervous
system, including brain and spinal cord (Frigeri et al., 1995;
Nielsen et al., 1997; Rash et al., 1998; Oshio et al., 2004).
In the brain, a considerable body of evidence implicates
AQP4 as a major route for brain water accumulation in
cytotoxic (cell swelling) oedema in water intoxication,
ischaemic stroke and bacterial meningitis (Manley et al.,
2000; Amiry-Moghaddam et al., 2003; Papadopoulos and
Verkman, 2005), and in clearance of excess brain water in
vasogenic (vessel leak) oedema in tumour and brain abscess
(Papadopoulos et al., 2004; Bloch et al., 2005), as well as in
obstructive hydrocephalus (Bloch et al., 2006).
AQP4 protein is very strongly expressed in glial cells of
spinal cord (Rash et al., 1998; Oshio et al., 2004). A recent
study reported that changes in AQP4 expression in injured
spinal cords parallel changes in spinal cord water content
(Nesic et al., 2006), providing indirect evidence for
involvement of AQP4 in SCI-related oedema. Here we
demonstrate a major role of AQP4 in spinal cord oedema
and neuronal function using a well-established experimental
crush injury model involving transient compression of the
mid-thoracic spinal cord. Rodent models producing blunt
injury to the cord mimic several features of human SCI and
are widely used for studying acute pathophysiological
processes (Guth et al., 1985; Wrathall et al., 1985; Blight,
1991; Borgens and Shi, 2000; Joshi and Fehlings, 2002; Luo
et al., 2002a, 2002b; Faulkner et al., 2004; Fujiki et al., 2005;
Huang et al., 2007; McBride et al., 2007; Nishi et al., 2007;
Terayama et al., 2007). A series of clinical and histological
outcome measures were compared in wild-type (AQP4+/+)
versus AQP4-null (AQP4 / ) mice. AQP4 / mice have
normal physiological parameters (heart rate, blood pressure,
rectal temperature), blood chemistries and hematology, and
do not show upregulation of other aquaporins (Ma et al.,
1997; Manley et al., 2000; Papadopoulos et al., 2004).
Furthermore, they are indistinguishable from AQP4+/+ mice
in their brain and cerebrovascular anatomy, blood-brain
barrier integrity, intracranial pressure and bulk intracranial
compliance (Manley et al., 2000; Papadopoulos et al.,
2004). We found a remarkably improved clinical and
histological outcome in AQP4 / mice following spinal
cord compression injury, which was explicable by reduced
spinal cord oedema.
S. Saadoun et al.
Role of AQP4 in spinal cord injury
Inclined plane test
Mice were placed on an adjustable inclined plane constructed as
described (Rivlin and Tator, 1977). The slope was progressively
increased every 20 s noting the angle at which the mouse could
not maintain its position for 5 s. The test was repeated twice for
each mouse and the average angle was recorded.
Myelography
Mice were anaesthetized and immobilized in the spinal frame.
Evans blue dye (EBD, 10 ml, 4% in saline, Sigma, Poole, UK) was
injected into the cisterna magna. Five minutes after injection,
a digital image of the dorsal theca exposed at T4–T7 was taken
to determine whether intrathecal dye could flow past the injury
level (T6).
Footprint analysis
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incubated in 0.06% potassium permanganate solution for 10 min,
water-rinsed and transferred for 10 min to a 0.0001% solution of
Fluoro-Jade C (Chemicon Europe, Hampshire, UK) dissolved in
0.1% acetic acid. Sections were also immunostained for the
neuronal nuclear marker NeuN using a mouse polyclonal antibody
(1:500, Chemicon Europe, Hampshire, UK), which confirmed that
the FJC positive cells are neurons. Slides were then rinsed with
distilled water, air-dried at 50 C, coverslipped with VectaMount
mounting medium (Vector Labs, Peterborough, UK), and
visualized using an Olympus BX51 microscope system with a
CC12 CCD camera. FJC and NeuN positive cells were then
counted in six high power fields within grey matter.
Luxol fast blue staining
Luxol Fast Blue (LFB) selectively stains myelin. Formalin-fixed,
paraffin-embedded axial tissue sections through the injury site at
T6 were deparaffinized, rehydrated and incubated in 0.1% LFB (in
95% alcohol and 0.5% acetic acid) (Sigma, Poole, UK) solution at
40 C overnight. After rinsing excess stain with 95% ethanol and
washing in distilled water, slides were immersed in 0.05% lithium
carbonate solution for 30 s. Sections were then dehydrated in serial
alcohols, and mounted in VectaMount mounting medium (Vector
Labs, Peterborough, UK). A digital image (8-bit jpeg format) was
taken of the dorsal, lateral and ventral funiculus from each side
(six images in total). We used Image J (v. 1.33u, NIH) to
arbitrarily set pixels with intensities 175–255 as white, and the
remaining pixels (intensities 0–174) blue, and calculated the
percentage of white pixels in each image. For each cord, myelin
vacuolation was defined as the average percentage of white pixels
in the six images.
Spinal somatosensory evoked responses
Evoked responses were obtained using stainless steel needle
electrodes and a Biopac MP35 System equipped with BSLPro
software and a BSLSTM Stimulator Box (Linton Instrumentation,
Norfolk, UK). Mice were anaesthetized using avertin (Sigma,
Poole, UK) and rectal temperature was maintained at 37–38 C.
Two stimulating electrodes were inserted into the calf to stimulate
the tibial nerve. After a longitudinal skin incision, recording
electrodes were positioned in the interspinous ligaments at T3/4
and T4/5. A reference electrode was inserted subcutaneously, halfway between stimulating and recording electrodes. A supramaximal
(typically 5 V) rectangular stimulus (0.2 ms duration) was applied at
a frequency of 3 Hz. Data were recorded at 100 kHz without filtering
and 300–500 recordings were averaged. Recordings were made from
each hindlimb and averaged.
In control experiments (data not shown), we found that SSER
amplitude was sensitive to the duration of SCI (crush at 1 mm
for 0.5–2.0 min), but insensitive to the amount of anaesthetic
(62.5–375 mg/kg avertin i.p.), rectal temperature (32–38 C) and
mouse age (8–12 weeks), whereas SSER latency was sensitive to
each of these parameters. Also, because latency could not be
determined accurately in many injured mice because of the low
amplitudes, we analysed only SSER amplitudes.
Immunostaining
Axial tissue sections taken through the injury site at T6 were
deparaffinized, rehydrated and incubated with polyclonal antiAQP4 antibody (1:200) or polyclonal anti-GFAP antibody (1:200)
for 1 h at room temperature (Chemicon Europe, Hampshire,
UK), followed by biotinylated secondary antibody, and avidinhorse-radish peroxidase (Vector Labs, Peterborough, UK).
Immunolabelling was visualized brown using diaminobenzidine,
with haematoxylin counterstain. Two observers unaware of the
experimental groups graded AQP4 immunorectivity in grey
and white matter of each section as follows: 0, no staining;
+, pericapillary staining; ++ marked pericapillary and diffuse
parenchymal staining.
Spinal cord pressure
Intraparenchymal cord pressure was measured in anaesthetized
mice that were immobilized using the spinal frame. A parenchymal pressure microcatheter (SPR-1000) was inserted through the
dura within spinal cord parenchyma at T6 and was connected to a
pressure control unit (TC510) from PMS Instruments, Berkshire,
UK. Parenchymal pressure was recorded for 1 min using a
BIOPAC MP35 system (Linton Instrumentation, Norfolk, UK).
Fluoro Jade C staining
Fluoro Jade C (FJC) selectively stains degenerating neuronal cell
bodies and processes, regardless of the mechanism of cell death; it
is more specific and sensitive than Fluoro Jade A and B (Schmued
et al., 2005; Brown and Sawchenko, 2007). Axial tissue sections
through the injury site at T6 were deparaffinized, rehydrated,
Spinal cord water content
Spinal cords were removed from T4–T8 vertebral body levels,
weighed, heated at 98 C for 48 h and re-weighed. Percent
water content was calculated as [wet weight – dry weight)/wet
weight 100].
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The plantar surface of each hindpaw was coloured blue and the
dorsal surface red using non-toxic dyes, and mice were allowed to
run on white paper towards a dark tunnel (Ma et al., 2001;
Faulkner et al., 2004). From each set of footprints the blue
(normal plantar paw placing) and red (abnormal dorsal paw
placing) areas were determined using Adobe Photoshop (Adobe
Systems Inc., Seattle, WA). The percentage of red pixels defined as
[red/(blue + red) 100] was computed using Image-J (NIH freeware), to quantify toe dragging. We also measured stride length
(distance between ipsilateral adjacent footprints) and stride width
(distance between left and right foot tracks) averaged using a
minimum of 10 steps in each case.
Brain (2008), 131, 1087^1098
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S. Saadoun et al.
Behavioural outcome measures
Data are expressed as mean SEM. Groups were compared with
Student’s t-test, Mann–Whitney U-test, or ANOVA with post hoc
Student–Newman–Keuls test.
Hindlimb motor performance was evaluated using three
behavioural tests, including the BMS open-field assessment
of motor function, the balancing of mice on an inclined
plane, and footprint analysis.
Figure 3A summarizes the BMS hindlimb motor scores
for AQP4+/+ and AQP4 / mice in CD1 and C57/Bl/6
genetic backgrounds. Before surgery all mice scored 9/9.
Injury produced marked hindlimb weakness in AQP4+/+
and AQP4 / mice. In AQP4+/+ mice, motor scores fell
within 6 h of surgery and did not improve significantly for
48 h. Motor scores also fell after SCI in AQP4 / mice, but
in CD1 mice there was marked improvement between
6 and 48 h. For both mouse strains, significantly lower
BMS scores were found at 1 and 2 days in AQP4+/+ than
AQP4 / mice. BMS scores improved over several days,
reaching a plateau by 3 weeks. The motor scores of CD1
AQP4+/+ and C57/Bl/6 AQP4+/+ mice remained remarkably
lower than those of corresponding AQP4 / mice even at
long time-points (4 weeks for CD1 and 3 weeks for C57Bl/6).
We also measured hindlimb motor function using the
well-established inclined plane method (Rivlin and Tator,
1977; Wrathall et al., 1985). This test is rapid, non-invasive
and quite reproducible. Slope data are summarized in
Fig. 3B. Before surgery, AQP4+/+ and AQP4 / mice could
maintain their positions on slopes of about 36 (CD1 mice)
and 41 (C57/Bl/6). The slopes were reduced after SCI,
though they remained remarkably greater in AQP4 /
versus AQP4+/+ mice at short (1 and 2 days) and long (3–4
weeks) time-points after injury.
After colouring the hindpaws (dorsal—red, plantar—
blue), we compared the stepping patterns of non-injured
AQP4+/+ versus AQP4 / CD1 mice (Fig. 4). Before injury,
running parameters were identical in 10 AQP4+/+ versus
10 AQP4 / mice, including hindlimb stride length
(4.7 0.2 cm versus 4.2 0.3 cm, SEM), stride width
(2.8 0.1 cm versus 2.6 0.1 cm), and normal plantar
stepping with clearly defined blue sole and toe hind-paw
prints. SCI produced toe dragging, increasing the percentage of red (dorsal) versus blue (plantar) footprints. Data
from a large set of experiments, summarized in Figs 4B–D,
show significantly increased toe dragging and reduced step
length in AQP4+/+ than in AQP4 / mice between 2 days
and 4 weeks. Stride width was also reduced in AQP4 /
mice at 2 days and 1 week but not at later time points. The
three assessments (motor score, inclined plane, footprint
analysis) indicate remarkably improved hindlimb motor
performance in AQP4 / versus AQP4+/+ mice after SCI.
Results
Spinal cord compression injury model
Spinal cords from non-injured AQP4 / mice appeared
normal by gross and microscopic examination. Several
parameters (measured at the T6 level) were similar as
measured in 3 AQP4+/+ versus 3 AQP4 / CD1 mice,
including the shapes and cross sectional areas of the ventrolateral (56.0 9 versus 51.6 9 mm2, SEM) and dorsal
(8.9 3 versus 7.9 2 mm2) funiculi, as well as grey matter
(81.8 4 versus 76.7 4 mm2). Vascular anatomy was
also indistinguishable in spinal cords of AQP4+/+ versus
AQP4 / mice, with supply by one anterior and two
posterior spinal arteries, as well as several segmental
arteries.
In preliminary experiments (data not shown), we graded
the extent (0.5, 1.0, 1.5 mm) and duration (0.25, 1, 2 min)
of spinal cord compression in AQP4+/+ mice. Compression
of 1 mm for 2 min produced moderately severe hindlimb
weakness that did not recover fully over 3 weeks.
These conditions were chosen because they could detect
improved or worse outcome in AQP4 / compared with
AQP4+/+ mice. Crush injury produced in this way caused
marked spinal cord swelling at the injury site, evidenced
by obstructed myelograms at 2 days (Fig. 1C and D).
EBD injected intracisternally flowed freely intrathecally
across T6 in sham mice, but did not flow past T5 in
injured mice.
Histological appearances after compression injury to the
spinal cord have been described before in several species
(Guth et al., 1985; Blight, 1991; Borgens and Shi, 2000;
Luo et al., 2002a, 2002b; Faulkner et al., 2004; Fujiki et al.,
2005; McBride et al., 2007; Terayama et al., 2007).
We found widespread damage to the spinal cord in
H&E-stained axial sections taken through the centre of
injury at T6 (Fig. 2A–I). At 2 days after surgery, intraparenchymal haemorrhages and vacuolation were visible
throughout the white matter of injured cords (Fig. 2E and
inset), but not in sham-operated mice (Fig. 2D and inset).
At 3 weeks, white matter vacuolation disappeared from the
injured cords (Fig. 2F), but there was widespread myelin
degeneration, evident as loss of LFB staining (Fig. 2F, inset).
Haemorrhages were also found in the grey matter at 2 days
(Fig. 2H) and by 3 weeks there was loss of motor neurons
in the anterior horns and cavitation within the grey
matter (Fig. 2I). GFAP staining revealed widespread glial
scarring throughout the white and grey matter at 3 weeks
(Fig. 2L, O), with no apparent reactive gliosis at 2 days
in injured mice (Fig. 2K, N) compared with sham mice
(Fig. 2J and M).
Histological outcome measures
We quantified grey matter damage in axial cord sections
through the centre of injury at T6 by staining for dying
neurons. FJC-positive cells showed green fluorescence, had
neuronal morphology at high magnification, and their
nuclei stained positive for the neuronal marker NeuN.
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Statistics
Role of AQP4 in spinal cord injury
Brain (2008), 131, 1087^1098
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Fig. 2 Spinal cord histology. Transverse sections at T6 from AQP4+/+ mice. Whole sections (H&E) (A) 2 days after sham surgery, (B) 2 days
after crush injury and (C) 3 weeks after crush injury. White matter (H&E, dorsal funiculus) (D) 2 days after sham surgery, (E) 2 days after
crush injury and (F) 3 weeks after crush injury. Grey matter (H&E, anterior horn) (G) 2 days after sham surgery, (H) 2 days after crush
injury and (I) 3 weeks after crush injury. Blue arrows show haemorrhages, shows vacuolation, dn = degenerating neurons, dm = degenerating myelin, g = glial nuclei, n = neuronal nuclei. Whole sections (GFAP immunostaining) (J) 2 days after sham surgery (K) 2 days after crush
injury, and (L) 3 weeks after crush injury. Grey matter (GFAP immunostaining, anterior horn) (M) 2 days after sham surgery (N) 2 days
after crush injury, and (O) 3 weeks after crush injury. Bars: 0.5 mm (A^ C, J^L), 50 mm (D ^I, M^ O), 25 mm (Insets D ^F).
Non-injured neurons expressed NeuN, but did not stain
with FJC (Fig. 5A). At 2 days, injured mice had more FJC+
NeuN+ cells per high-power field than the corresponding
sham-operated mice, with more dying neurons in injured
AQP4+/+ versus AQP4 / mice (Fig. 5B).
White matter damage was evaluated in axial cord section
through the centre of injury at T6 using the myelin stain
LFB. At 2 days after surgery, widespread vacuolation was
seen in white matter of injured mice, representing oedema
and early myelin breakdown (Pantoni et al., 1996; Yam
et al., 1998; Gomes-Leal et al., 2004), compared with uniform
myelin staining in sham mice (Fig. 5C). The overall
percentage of white versus blue pixels in high-magnification
images of dorso-lateral and ventral white matter, an index
of vacuole formation, was markedly greater in AQP4+/+
versus AQP4 / mice 2 days after SCI (Fig. 5D).
Electrophysiological outcome measures
SSERs were measured to evaluate sensory pathways.
Figure 6A diagrams the positions of the two stimulating
electrodes (subcutaneous, adjacent to tibial nerve), the two
recording electrodes (in the T3/4 and T4/5 inter-spaces),
and the reference electrode. In non-injured mice, the SSER
was comprised of a complex of peaks and troughs. Some
peaks and troughs were not seen consistently in all mice;
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S. Saadoun et al.
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Fig. 3 Improved hindlimb motor function following SCI in AQP4 / mice. (A) Hindlimb BMS motor score, and (B) Inclined plane test. We
used CD1 (left, 11 AQP4+/+ versus 11 AQP4 / injured, 3 AQP4+/+ versus 3 AQP4 / sham preoperatively and at 2 days, followed by 5
AQP4+/+ versus 5 AQP4 / injured, 2 AQP4+/+ versus 2 AQP4 / sham for 1^ 4 weeks) and C57/BL/6 (right, 7 AQP4+/+ versus 7 AQP4 /
injured, 3 AQP4+/+ versus 3 AQP4 / sham) mice. Mean SEM, P50.05, P50.01, P50.001, #P55 10 5.
however, the most prominent trough was present in all mice,
occurring at 2.58 0.07 and 2.55 0.03 ms (SEM) after
stimulation in normal AQP4+/+ and AQP4 / mice, respectively. The amplitude () of this consistently seen trough was
measured. In validation studies (Fig. 6B, left), the recorded
signal amplitude increased with increased stimulating pulse
voltage from 1 to 4 V. Stimuli more intense than 4 V had no
effect on amplitude, suggesting supramaximal stimulation of
the tibial nerve. SSER amplitude was reduced to zero after
spinal cord transection at T6, indicating the recorded signal
originates from within the spinal cord. Figure 6B (right)
shows representative SSERs from two AQP4+/+ and two
AQP4 / mice, before and 2 days after SCI. There was no
apparent difference in the shape of SSERs from non-injured
AQP4+/+ versus AQP4 / mice. Before surgery, SSER
amplitude was about 10 mV in AQP4+/+ and AQP4 / mice
(Fig. 6C). Amplitudes were reduced at 2 days after injury,
with significantly greater reduction in AQP4+/+ mice.
Amplitude was not affected by sham surgery. We also
investigated whether reduced SSER amplitude correlates
with greater damage to the major afferent spinal cord white
matter tracts. Figure 6D shows significant inverse correlation
between SSER amplitude and dorsal column damage. White
matter damage was quantified as loss of LFB staining at the
site of injury.
AQP4 expression and spinal cord oedema
Motivated by altered AQP4 expression in central nervous
system in several pathologies (Saadoun et al., 2002; Sun et al.,
2003; Aoki-Yoshino et al., 2005; Papadopoulos and Verkman,
2005; Nesic et al., 2006; Ribeiro Mde et al., 2006), we
investigated AQP4 immunoreactivity in the SCI model used
here (Fig. 7A). In sham cords, AQP4 was strongly expressed in
grey and white matter, around capillaries, in radial astrocytes,
and glia limitans (Figs 7A, D, F), with no detectable
Role of AQP4 in spinal cord injury
Brain (2008), 131, 1087^1098
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expression in AQP4 deficiency (Fig. 7B). Overall, AQP4
expression in AQP4+/+ mice was markedly upregulated 2 days
after SCI in grey matter and white matter as shown in Figs 7C,
E, G, and summarized in Fig. 7H. In injured cords with
upregulated AQP4 expression, there were regions with
extensive tissue damage where reduced expression was seen
(data not shown).
In the brain, AQP4 deletion limits cytotoxic oedema (cell
swelling) during early ischaemia, water intoxication and
bacterial meningitis (Manley et al., 2000; Papadopoulos and
Verkman, 2005) by reducing the uptake of water from the
vasculature into brain parenchyma. AQP4 deletion increases
vasogenic oedema (extracellular water accumulation) in
brain tumour and brain abscess (Papadopoulos et al., 2004;
Bloch et al., 2005) by impeding water outflow from the
brain. We therefore investigated the effect of AQP4 deletion
on spinal cord oedema following SCI. Figure 8A shows that
non-injured AQP4 / mice have greater baseline spinal
cord water content than AQP4+/+ mice. However, 2 days
after injury water content increased considerably in
AQP4+/+ but not AQP4 / mice.
At 2 days after injury the spinal cord was significantly
swollen, evidenced by obstruction in the flow of intrathecal
EBD (Fig. 1C and D), and increased spinal cord water
content (Fig. 8A) at the injury site. We therefore
hypothesized that compression by the surrounding dura
produces increased intraparenchymal pressure within the
injured, swollen cord. The intraparenchymal pressure at T6
was thus measured using a micro-probe. Typical pressure
traces are shown in Fig. 8B (left), and the data are
summarized in Fig. 8B (right). Although T6 cord pressures
were comparable in sham-operated AQP4+/+ and AQP4 /
mice, markedly higher pressures were found in AQP4+/+
versus AQP4 / mice at 2 days after injury.
Discussion
Our data show substantially improved outcome after SCI
in AQP4 deficiency. Three behavioural assessments
(open-field, inclined plane, footprint analysis) revealed
better motor function in injured AQP4 null mice, and
electrophysiological analysis by SSERs indicated better
sensory preservation in hindlimbs. The improved functional
outcome in AQP4 deficiency was associated with reduced
neuronal death and myelin breakdown, and was a robust
finding seen in both outbred (CD1) and inbred (C57/BL)
mouse strains, evident within 24 h of injury and persisting
for at least 4 weeks.
The findings of significantly increased spinal cord water
content and intrathecal obstruction to the flow EBD at the
site of injury indicate marked circumferential cord swelling
within the intact theca at 2 days. Spinal cord oedema is a
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Fig. 4 Footprint analysis. (A) Hindlimb footprints (dorsumçred, plantarçblue) of (left) AQP4 / mouse and (right) AQP4+/+ mouse,
pre- and 2 days post- injury. (B^D) Summary of hindlimb footprint data including (B) Toe dragging, (C) Step length, and (D) Stride width.
We used 11 AQP4+/+ versus 11 AQP4 / injured and 3 AQP4+/+ versus 3 AQP4 / sham preoperatively and at 2 days, followed by 5
AQP4+/+ versus 5 AQP4 / injured and 2 AQP4+/+ versus 2 AQP4 / sham for 1^ 4 weeks. Mean SEM, P50.05, P50.01, P50.001.
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Brain (2008), 131, 1087^1098
S. Saadoun et al.
well-recognized cause of secondary neuronal damage after
SCI in humans and animals (Wagner and Stewart, 1981;
Fujii et al., 1993; Flanders et al., 1999; McDonald and
Sadowsky, 2002; Kwon et al., 2004; Sharma et al., 2005;
Boldin et al., 2006; Miyanji et al., 2007). Our data suggest
that the swollen spinal cord becomes compressed against
the surrounding theca, which causes the intraparenchymal
cord pressure to rise. In a few mice (not included in the
analysis) in which the dura was inadvertently torn whilst
re-opening the skin at 2 days, marked cord herniation
through the dural opening was seen consistent with
increased cord pressure. Increased spinal cord pressure
may limit venous outflow from the cord, producing a
further rise in intraparenchymal pressure, leading to a
vicious circle that eventually causes cord ischaemia.
If our finding of dural cord compression can be
extrapolated to humans, it may explain why early bony
decompression after SCI (without dural decompression) has
produced mixed results (Fehlings and Perrin, 2006). Bony
decompression alone would only benefit those SCI patients
who do not have dural cord compression, whereas bony
removal and dural opening is required for patients with
significant cord swelling. This is analogous to the situation
in brain-injured humans in whom brain oedema causes
a rise in intracranial pressure that is not relieved by bony
decompression alone (decompressive craniectomy) unless
the overlying dura is also opened (Hutchinson et al., 2006).
In the brain, AQP4 deletion limits cytotoxic oedema
(Manley et al., 2000; Amiry-Moghaddam et al., 2003;
Papadopoulos and Verkman, 2005), but increases vasogenic
oedema (Papadopoulos et al., 2004; Bloch et al., 2005). The
concepts of cytotoxic versus vasogenic oedema are not well
defined in the spinal cord. Our finding of increased spinal
cord swelling in AQP4+/+ versus AQP4 / mice supports a
major role for cytotoxic oedema (cell swelling) in SCI with
the excess water entering the site of injury primarily
through an AQP4-dependent route. Reports of astrocyte
foot process enlargement (Verdu et al., 2003) and reduced
water diffusion detected by MRI (Schwartz and Hackney,
2003; Krzyzak et al., 2005; Deo et al., 2006; Gaviria et al.,
2006) in animal SCI models are consistent with marked
cytotoxic oedema. Excess water (vasogenic oedema) may
also enter the extracellular space (ECS) of the injured
cord through a damaged blood–spinal cord barrier (Joshi
and Fehlings, 2002), which is an AQP4-inependent route.
The pathway of oedema fluid elimination from the site
of injury is unknown, but is probably independent of AQP4
involving diffusion of excess water away from the injury
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Fig. 5 Histological assessment of spinal cord damage. (A) Spinal cord grey matter at T6, 2 days after surgery stained with FJC. Insets:
magnified view of neurons stained with FJC (green) and NeuN (red). Non-injured neurons are NeuN+ FJC-, whereas injured neurons
are NeuN+ FJC+. (B) Summary of FJC data (average of 6 high power fields). (C) Spinal cords 2 days after surgery stained with LFB.
Panels show whole section through the injury site at T6 (left) and magnified view of white matter in the dorsal funiculus (right).
(D) Summary of LFB data (average of ventral, lateral, and dorsal funiculi). Bars: 50 mm (A), 10 mm (A, insets) 0.5 mm (C, left), 50 mm
(C, right). Mean SEM, P50.05, P50.01.
Role of AQP4 in spinal cord injury
Brain (2008), 131, 1087^1098
1095
site up and down the cord. We suggest that oedema
formation (cytotoxic component) is slowed in AQP4 /
mice, whereas oedema elimination is unaffected by AQP4.
Reduced oedema formation may thus explain why
AQP4 / mice develop less cord swelling after SCI.
Within 2 days of SCI, there is neuronal death (FJC
staining) and demyelination (loss of LFB staining). The
number of FJC+ cells and the extent of demyelination
positively correlate with the Basso Motor Score (data not
shown). It is thus impossible to determine with certainty
whether the neurological deficit (hindlimb weakness) is
primarily caused by neuronal injury versus demyelination.
Another contributor to improved outcome after SCI in
AQP4 / mice may be delayed glial scarring (Saadoun
et al., 2005; Auguste et al., 2007), which enhances neuronal
regeneration. The inhibitory effects of glial scarring on
axonal sprouting and synaptogenesis after SCI are well
documented (Silver and Miller, 2004). In brain, AQP4
deletion remarkably impairs astrocyte migration and
reduces glial scarring after a stab injury (Saadoun et al.,
2005; Auguste et al., 2007). Glial scarring, however, was not
detectable at 2 days after acute SCI and so cannot account
for the improved outcome in AQP4 / mice observed
within 24 h of injury.
We chose an acute blunt SCI model, which is technically
simple, reproducible and well-established (Guth et al., 1985;
Blight, 1991; Borgens and Shi, 2000; Luo et al., 2002a,
2002b; Faulkner et al., 2004, Fujiki et al., 2005; McBride
et al., 2007; Terayama et al., 2007). Blunt SCI rodent
models involve cord contusion or compression. Contusion
is often caused using an impactor (Nishi et al., 2007) or
weight drop (Wrathall et al., 1985), whereas cord
compression can be done at a constant pressure [by
applying an aneurysm clip across the cord (Joshi and
Fehlings, 2002) or placing a constant weight on the cord
(Huang et al., 2007)] or at a constant compression distance
[produced by applying forceps across the cord (Guth et al.,
1985; Blight, 1991; Borgens and Shi, 2000; Luo et al., 2002a,
2002b; Faulkner et al., 2004; Fujiki et al., 2005; McBride
et al., 2007; Terayama et al., 2007)]. Blunt models produce
damage by mechanical disruption of cells as well as spinal
cord ischaemia and reperfusion arising from transient
vascular occlusion. Unlike rat and human SCI, previously
described mouse SCI models do not cause cavitation
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Fig. 6 Improved sensory outcome in AQP4 / mice after SCI assessed by spinal somatosensory evoked responses (SSERs). (A) Schematic
showing position of electrodes for stimulation, recording and reference. A supramaximal 0.2 ms square pulse stimulus was used and the
amplitude () of the most prominent trough was determined. (B) Recordings obtained by increasing stimulus amplitude (1^ 8 V) and after
complete cord transection at T6. Arrows mark beginning and end of stimulus. (C) (left) Representative SSERs from two AQP4+/+ and two
AQP4 / mice before (black trace) and 2 days after (grey trace) injury. (C) SSER amplitudes () in AQP4+/+ versus AQP4 / mice recorded
before injury (13 versus 13 mice) and 2 days after injury (11 versus 12 mice). (D) Correlation between SSER and dorsal column vacuolation
(filled circle AQP4+/+, open circle AQP4 / , r = 0.6, P50.01). Mean SEM, P50.01.
1096
Brain (2008), 131, 1087^1098
(Guth et al., 1985; Wrathall et al., 1985; Blight, 1991;
Borgens and Shi, 2000; Joshi and Fehlings, 2002; Luo et al.,
2002a, 2002b; Faulkner et al., 2004; Fujiki et al., 2005;
McBride et al., 2007; Nishi et al., 2007; Terayama et al.,
2007). The SCI model described here produces cavitation,
although not as prominent as in rat or human spinal cords.
The presence of cavitation suggests that the mouse
compression model described in our article may be a
good model of human spinal cord injury. Another
advantage of the compression model used here, unlike
models that result in sectioning of neuronal tracts
(Wrathall, 1992), is that it mimics the blunt, traumatic
human SCI in which bone fragments and haematoma
damage the spinal cord by compression.
Increased AQP4 expression was detected at 2 days after
SCI in contrast to Nesic et al. (2006) who reported AQP4
downregulation at 3 days in a rat model. The difference
between the results of Nesic et al. and our’s most likely
represents species and model differences. For example, a
very severe SCI causing extensive necrosis is likely to
produce AQP4 downregulation, but a milder injury with
little astrocyte necrosis is likely to stimulate astrocytes to
upregulate AQP4 expression. In general, AQP4 becomes
upregulated after central nervous system (CNS) injury
including focal cerebral ischaemia in mice (Ribeiro Mde
et al., 2006), brain inflammatory diseases in humans
(Aoki-Yoshino et al., 2005), meningitis in mice
(Papadopoulos and Verkman, 2005), traumatic brain
injury in rats (Sun et al., 2003), and human brain tumours
(Saadoun et al., 2002). AQP4 upregulation is expected to
amplify differences in outcome between wildtype and AQP4
null mice, enhancing water flow from the vasculature into
spinal cord parenchyma, thus increasing cord swelling after
injury. AQP4 upregulation following SCI thus represents a
maladaptive response, as was found in multiple other brain
pathologies.
AQP4 deletion in mice did not produce baseline
structural changes in the spinal cord. The shape, size and
myelination of AQP4 / spinal cords as well as their
vascular supply were indistinguishable from those in
AQP4+/+ mice. Sensorimotor function was also normal as
shown by neurological functional assessment and SSERs.
These findings are consistent with the normal brain
appearance, cerebrovascular supply, intracranial pressure
and intracranial compliance in AQP4 null mice (Manley
et al., 2000; Papadopoulos et al., 2004). AQP4 deficiency,
however, was associated with increased baseline spinal cord
water content, consistent with our findings in AQP4 /
mouse brain (Manley et al., 2000; Papadopoulos et al.,
2004; Bloch et al., 2005, 2006). Elevated CNS water content
in AQP4 / mice may result from expansion of CNS
extracellular space (ECS) volume (Binder et al., 2004; Zador
et al., in press). After injury, cell swelling constricts the ECS
thus impairing the diffusion of nutrients, metabolites and
neuro-transmitters, and increasing the extracellular concentration of excitotoxic amino acids. Therefore, enhanced
ECS diffusion in AQP4 deficiency is potentially beneficial
during SCI.
Our demonstration of improved outcome after SCI in
AQP4-deficient mice has potential therapeutic implications
in human SCI, which is associated with high morbidity and
mortality. SCI survivors have permanent neurological
deficits including paralysis and anaesthesia below the level
of injury, and urinary and fecal incontinence. About 13% of
those who reach the hospital alive, die within the first year
(Sekhon and Fehlings, 2001). We suggest that AQP4
inhibitors and downregulators may be novel neuroprotective agents that may be beneficial by reducing spinal cord
oedema when administered early after SCI.
Acknowledgements
This work was funded by the Wellcome Trust,
Neurosciences Research Foundation, and St George’s
Charitable Trust (MCP), the Harrison Fund (SS), and
the National Institutes of Health grant R37 DK35124
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Fig. 7 Increased AQP4 expression 2 days after SCI. Spinal cords
from (A) Sham AQP4+/+ mouse, (B) Sham AQP4 / mouse, and,
(C) Injured AQP4+/+ mouse. Magnified view of grey matter from
(D) Sham and (E) Injured AQP4+/+ mouse. Magnified view of white
matter from (F) Sham, and (G) Injured AQP4+/+ mouse. AQP4
expression around capillaries (black arrows) and glia limitans (red
arrows). (H) Numbers of mice expressing different levels of AQP4
protein (O, +, ++). For details see Methods. Bars: 0.5 mm (A^C),
50 mm (D^G).
S. Saadoun et al.
Role of AQP4 in spinal cord injury
Brain (2008), 131, 1087^1098
1097
Fig. 8 Reduced spinal cord oedema in AQP4 / mice after SCI. (A) Spinal cord water content, and (B) (left) Representative spinal cord
pressure recordings (arrow shows insertion of probe into the cord. (Right) Summary of T6 cord pressures, measured at 2 days after surgery.
Mean SEM, P50.05, P50.01.
(to A.S.V.). Funding to pay the Open Access publication
charges for this article was provided by the Wellcome Trust.
Downloaded from by guest on October 15, 2014
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