1. Art and Diseño

Journal of Cerebral Blood Flow & Metabolism
23:154–165 © 2003 The International Society for Cerebral Blood Flow and Metabolism
Published by Lippincott Williams & Wilkins, Inc., Philadelphia
Anti–Nogo-A Antibody Infusion 24 Hours After Experimental
Stroke Improved Behavioral Outcome and Corticospinal
Plasticity in Normotensive and Spontaneously Hypertensive Rats
*Christoph Wiessner, †Florence M. Bareyre, ‡Peter R. Allegrini, *Anis K. Mir, *Stefan Frentzel,
‡Mauro Zurini, †Lisa Schnell, †Thomas Oertle, and †Martin E. Schwab
*Nervous System Research, ‡Core Technology Area, Novartis Pharma AG, Basel; †Brain Research Institute,
University of Zurich, and Department of Biology, Swiss Federal Institute of Technology Zurich, Zurich, Switzerland
Summary: Nogo-A is a myelin-associated neurite outgrowth
inhibitory protein limiting recovery and plasticity after central
nervous system injury. In this study, a purified monoclonal
anti–Nogo-A antibody (7B12) was evaluated in two rat stroke
models with a time-to-treatment of 24 hours after injury. After
photothrombotic cortical injury (PCI) and intraventricular infusion of a control mouse immunoglobulin G for 2 weeks,
long-term contralateral forepaw function was reduced to about
55% of prelesion performance until the latest time point investigated (9 weeks). Forepaw function was significantly better in
the 7B12-treated group 6 to 9 weeks after PCI, and reached
about 70% of prelesion levels. Cortical infarcts were also produced in spontaneously hypertensive rats (SHR) by permanent
middle cerebral artery occlusion (MCAO). In the control group,
forepaw function remained between 40% and 50% of prelesion
levels 4 to 12 weeks after MCAO. In contrast, 7B12-treated
groups showed significant improvement between 4 and 7
weeks after MCAO from around 40% of prelesion levels at
week 4 to about 60% to 70% at 7 to 12 weeks after MCAO.
Treatment in both models was efficacious without influencing
infarct volume or brain atrophy. Neuroanatomically in the spinal cord, a significant increase of midline crossing corticospinal
fibers originating in the unlesioned sensorimotor cortex was
found in 7B12-treated groups, reaching 2.3 ± 1.5% after PCI
(control group: 1.1 ± 0.5%) and 4.5 ± 2.2% after MCAO in
SHR rats (control group: 1.8 ± 0.8%). Behavioral outcome and
the presence of midline crossing fibers in the cervical spinal
cord correlated significantly, suggesting a possible contribution
of the crossing fibers for forepaw function after PCI and
MCAO. The results suggest that specific anti–Nogo-A antibodies bear potential as a new rehabilitative treatment approach
for ischemic stroke with a prolonged time-to-treatment window. Key Words: Nogo—Neurite outgrowth inhibitor—Focal
cerebral ischemia—Sensorimotor function—Plasticity.
Injuries to the adult human cerebral motor cortex often
result in persisting limb movement deficits (Nudo et al.,
2001). The leading cause of cortical injury and serious
long-term disability is ischemic stroke (Stapf and Mohr,
2002). Epidemiologic data suggest that after a long period of decline, stroke incidence is now rising again (Gillum and Sempos, 1997). Because treatment options are
limited to thrombolysis with a time-to-treatment window
of only 3 hours (The NINDS study group, 1997; del
Zoppo, 2000), and a large number of neuroprotective
drugs failed in clinical trials (Martinez-Vila and Sieira,
2001), an urgent medical need for new treatment strategies for stroke exists.
In comparison to the adult brain, recovery of motor
function after injury is much better in the neonatal brain
(Milner, 1974; Whishaw and Kolb, 1988), and neuroanatomic plasticity is thought to be the underlying mechanism. Studies in newborn rats have shown that, after
sensorimotor cortical lesion, the uninjured contralateral
cortex is able to form bilateral connections with the striatum (Kolb et al., 1992), thalamus (Yu et al., 1995), red
nucleus (Naus et al., 1985), tectum (Leong and Lund,
1973), basilar pontine gray (Kartje-Tillotson et al.,
1986), and spinal cord (Rouiller et al., 1991). The capacity to form such new connections declines with progressing myelination (Huber and Schwab, 2000), which led to
the hypothesis that plasticity in the adult brain is restricted by neurite outgrowth inhibitors residing in myelin (Kapfhammer and Schwab, 1994). One of the most
Received July 4, 2002; final version received September 18, 2002;
accepted September 19, 2002.
Supported by the NCCR on Neural Plasticity and Repair.
Address correspondence and reprint requests to Dr. Christoph
Wiessner, Novartis Pharma AG, Nervous System Research, Neurodegeneration Unit WKl125.5.15, CH-4002 Basel, Switzerland; e-mail:
[email protected]
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DOI: 10.1097/01.WCB.0000040400.30600.AF
ANTI–NOGO-A ANTIBODY TREATMENT AFTER STROKE
potent neurite growth inhibitory activities associated
with oligodendrocyte membranes and CNS myelin is the
membrane protein Nogo-A, which has recently been
cloned (Chen et al., 2000; Grand Pre´ et al., 2000; Prinhja
et al., 2000). A neutralizing antibody (mAb IN-1), raised
against NI-250 (Caroni and Schwab, 1988), improved
regeneration of lesioned axons in the adult rat spinal
cord, optic nerve, or brain (Schnell and Schwab, 1990;
Huber and Schwab, 2000), and increased compensatory
fiber growth of unlesioned tract systems (Thallmair
et al., 1998; Z’Graggen et al., 2000; Wenk et al., 1999).
In behavioral assays in rats, functional recovery was observed after injury and IN-1 hybridoma treatment (Thallmair et al., 1998; Z’Graggen et al., 1998). In all of these
studies, IN-1 was delivered by implanted antibodysecreting hybridoma cells with concomitant immunosuppression. As an important step towards transfer of
Nogo-A neutralization to the level of clinical trials, we
generated a new monoclonal anti–Nogo-A antibody of
the immunoglobulin G (IgG)1-subtype (7B12), which
was purified and delivered in a highly controlled manner
by minipumps. In the present study, we showed that
7B12 infusion initiated 24 hours after stroke improved
long-term behavioral recovery without affecting lesion
volumes. Behavioral outcome correlated with sprouting
of corticospinal fibers of the unlesioned sensorimotor
cortex, which was stimulated in 7B12-treated animals.
The results support the concept that Nogo-A neutralization augments the inherent and functionally meaningful
plastic capacity of the adult CNS, which is restricted by
outgrowth inhibitors.
MATERIAL AND METHODS
Characterization of monoclonal antibody 7B12
Binding of purified antirat monoclonal antibody (mAb)
7B12 (IgG1) to rat NiG (the protein domain specific to
Nogo-A) and rat NiG delta-6 fragment (Oertle et al., 2000) was
determined by enzyme-linked immunosorbent assay. NiG and
NiG delta-6 were coated on enzyme-linked immunosorbent assay plates at a concentration of 2 ␮g/mL and incubated with
different concentrations of 7B12 mAb. The binding of 7B12 to
the two proteins was quantified by using a goat antimouse
horseradish peroxidase–coupled antibody and peroxidase substrate (Roche Diagnostics, Rotkreuz, Switzerland) and reading
the optical density of the color reaction in an enzyme-linked
immunosorbent assay plate reader at 450 nm (Spectracount;
Packard, Mississauga, Ontario, Canada). Results are means ±
SD of samples in at least duplicate wells.
Animals. Animal maintenance conditions and all surgical
procedures were approved by the veterinary authorities of the
Kanton Basel-Stadt, Switzerland.
Photothrombotic cortical injury. A modification of a
method described by Watson et al. (1985) was used. Male
Fischer F344 rats (Iffa Credo, L’Arbresele, France) weighing
300 g were anesthetized with 2% isoflurane in a 70:30 (by
volume) nitrous oxide–oxygen mixture and 80 mg/kg RoseBengal in saline was infused into the femoral vein (0.5 mL/kg
at 95 ␮L/min). Immediately afterwards, a fiberglass probe with
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10-mm diameter was positioned directly over the exposed right
skull. The skull was illuminated (Volpi Intralux 6000, 150 W;
Volpi AG, Schlieven, Switzerland) for 7 minutes at maximum
output. Body temperature was maintained at 37°C by means of
a heating pad (CMA 150; Carnegie Medicine, Stockholm,
Sweden) connected to a rectal probe during surgery and until
animals regained consciousness. Thereafter, animals were
returned to their home cages and allowed free access to food
and water.
Permanent middle cerebral artery occlusion in spontaneously hypertensive rats. Irreversible right middle cerebral
artery occlusion (MCAO) was performed in male spontaneously hypertensive rats (SHR) rats, 220 to 300 g (Iffa Credo),
as described previously (Barone et al., 1993), with minor modifications. Briefly, animals were anesthetized (see preceding
paragraph) and the right middle cerebral artery was exposed by
a subtemporal craniectomy and occluded by bipolar electrocoagulation. Afterwards, retracted soft tissue was replaced,
wounds were sutured, and anesthesia was discontinued. Body
temperature was maintained at 37°C during surgery and until
animals regained consciousness (see preceding paragraph).
Thereafter, animals were returned to their home cages and were
allowed free access to food and water.
Magnetic resonance imaging. Infarct volume was determined 24 hours after PCI or MCAO by means of quantitative
in vivo magnetic resonance imaging (MRI). Measurements
were performed on a 4.7-T 30-cm-bore Spectrospin DBX
(Bruker, Karlsruhe, Germany) equipped with a 20-cm gradient
coil as described in detail elsewhere (Allegrini and Sauer, 1992;
Wiessner et al., 2000). Briefly, rats were anesthetized with
isoflurane (see preceding paragraphs) and the head was positioned in a 35-mm resonator. Thirteen contiguous T2-weighted
coronal slices with a thickness of 1.2 mm were taken using
a RARE sequence (repetition time, 3,000 milliseconds; effective echo time, 87 milliseconds; spatial resolution in plane, 156
␮m2; measure time, 5 minutes). For morphometric evaluation,
damaged tissue was segmented by setting an intensity threshold
using a semiautomatic image analysis software (Analyze, Biomedical Imaging Resource; Mayo Foundation, Rochester, MN,
U.S.A.) on a Silicon Graphics (Mountain View, CA, U.S.A.)
O2 computer. Infarct volume was calculated based on the damaged area in each slice and the distance between slices. Nine
weeks after PCI, volumes of the infarcted and contralateral
hemispheres were measured again by MRI and the atrophy of
the infarcted hemisphere was calculated as their difference.
Statistical comparison between control and treatment groups
was made using t-test or analysis of variance (ANOVA) followed by Bonferroni t-test. A P value < 0.05 was regarded as
being statistically significant.
Treatment with 7B12. A brain infusion cannula (Alzet brain
infusion kit; Alza Corp., Palo Alto, CA, U.S.A.) was stereotaxically implanted under anesthesia (2% isoflurane in 70:30
nitrous oxide–oxygen mixture) in the left lateral ventricle (coordinates: lateral 1.3; anterior–posterior −0.8; dorsoventral
−3.8, relative to bregma). Control antibody (mouse IgG;
Chemicon, Temecula, CA, U.S.A.) or 7B12 were applied intraventricularly in phosphate-buffered saline (PBS) by continuous infusion for 2 weeks using Alzet osmotic minipumps
(5 ␮L/h; model 2ML2; Alza Corp.). After PCI, total antibody
doses were 5 mg control mouse IgG (n ⳱ 13) or 5 mg 7B12
(n ⳱ 14) infused at 15 ␮g/h. After MCAO in SHR rats, total
antibody doses were 1.68 mg 7B12 (5 ␮g/h; n ⳱ 9), or 5 mg
7B12 (15 ␮g/h; n ⳱ 9). In this experiment, the control antibody
was given at a dose of 1.68 mg 7B12 (5 ␮g/h; n ⳱ 9), to match
the lower dose 7B12 group. The pumps were removed after
2 weeks and quantitative delivery was controlled. The surgeons
J Cereb Blood Flow Metab, Vol. 23, No. 2, 2003
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C. WIESSNER ET AL.
implanting osmotic minipumps were masked with respect to
treatment. In addition, brains from animals receiving 7B12,
control antibody, or PBS were sampled 2 weeks or 4 weeks
after pump implantation (n ⳱ 3 to 5) and frozen at −70°C
in Tissue-Tek O.C.T. compound (Sakura, Zoeterwoude, Netherlands). Coronal sections (10 ␮m) of these brains were cut
on a cryostat, and 7B12 distribution was visualized using a
biotinylated antimouse antibody and the ABC kit (Vector
Laboratories, Burlingame, CA, U.S.A.) as recommended by
the supplier.
Evaluation of sensorimotor performance. Montoya’s
staircase test was carried out as described in detail previously
(Montoya et al., 1991) and by investigators masked with regard
to treatment. Rats were placed in a Plexiglas box with a baited
double staircase. Food pellets were placed on the staircase and
presented bilaterally at seven graded stages of reaching difficulty to provide objective measures of maximum forelimb extension and grasping skill. Rats were food deprived for at least
24 hours before and throughout the whole weekly test session,
being fed once a day with approximately 10 g standard rat
chow. A complete test series was done on 4 consecutive days.
On the first 2 days, the platform was baited with 4 chow pellets
(Dustless Precision Pellets [45 mg]; Bio-Serv, Frenchtown, NJ,
U.S.A.) at the far end of the central platform, and in addition
stairs 1 and 2 were baited bilaterally with 2 pellets each. The
rats were tested twice daily (morning/afternoon) for 10 minutes
each. On days 3 to 4, stairs 2 to 6 were bilaterally baited with
three pellets each to avoid rats successfully retrieving pellets
with their tongue or with the “unaffected” paw. The rats were
tested again twice daily for 10 minutes each. After each test
series, the total number of pellets grasped and eaten on days 3
to 4 with each forepaw was counted. In addition, the number of
displaced pellets was determined. Performance in the staircase
test was expressed in percent of the last test session before
lesioning (eaten pellets) or in success rate (eaten pellets ×
100/[eaten pellets + displaced pellets]). For statistical analysis
between groups, two-way repeated-measures ANOVA was
used followed by a pairwise multiple comparison procedure
(i.e., Bonferroni t-test). For analysis of recovery within the
groups, the first test session after lesioning was used as a reference value, and statistical analysis was done using one-way
repeated-measures ANOVA.
Anterograde tracing with biotinylated dextran amine.
Tracing and analysis was performed by an investigator masked
with regard to treatment. Location of the forelimb sensorimotor
cortex was determined by measuring positions on the skull
relative to Bregma (Neafsey et al., 1986). Nine weeks after PCI
or 13 weeks after MCAO, pressure injections of a 10% solution
of biotinylated dextran amine (10,000 d [Molecular Probes,
Eugene, OR, U.S.A.], in 0.01 mol/L phosphate buffer, pH 7.4)
were made into the left forelimb sensorimotor cortex (0.5 mm
posterior, 2.5 mm lateral; 1.5-mm depth, relative to bregma)
through a glass capillary that remained in its position for 2
minutes after the end of the injection. Two weeks after biotinylated dextran amine injection, animals were deeply anesthetized with pentobarbital (450 mg/kg intraperitoneally; Abbott
Laboratories, Cham, Switzerland) and perfused transcardially
with 100 mL of Ringer solution containing 100,000 IU/L heparin (Liquemin; Roche, Basel, Switzerland) and 0.25% NaNO2
followed by 300 mL of 4% paraformaldehyde in 0.1 mol/L
phosphate buffer with 5% sucrose. Brains and spinal cords
were dissected and postfixed overnight at 4°C in the same
fixative. Meninges were removed and the pons and cervical
enlargement (C4–C6) was embedded in a gelatine–chicken albumin solution polymerized with 2.5% glutaraldehyde. Fiftymicrometer coronal sections were cut using a vibratome (Leica
J Cereb Blood Flow Metab, Vol. 23, No. 2, 2003
vt 1000S). All sections were collected in 50 mmol/L Trisbuffered 0.9% saline, pH 8, and 0.5% Triton X-100 (TBS-Tx).
They were serially mounted on slides (Superfrost/Plus; Menzel-Gla¨ ser, Braunschweig, Germany) according to the semifree floating technique (Herzog and Brosamle, 1997). Sections
were washed three times for 30 minutes in TBS-Tx before
overnight incubation in avidin-peroxidase in TBS-Tx (ABC
Elite; Vector Laboratories). The following day, the slides were
washed three times for 30 minutes in TBS-Tx. After an additional wash in 50 mmol/L Tris-HCl, pH 8, a preincubation for
10 minutes in 0.4% ammonium nickel sulfate (Sigma, St.
Louis, MO, U.S.A.) was performed, followed by a second preincubation in 0.4% ammonium nickel sulfate and 0.015% 3,3⬘diaminobenzidine (DAB; Sigma, Buchs, Switzerland) for 10
minutes. The tissue was then reacted in 0.4% ammonium nickel
sulfate, 0.015% DAB, and 0.004% H2O2 in 50 mmol/L Tris
buffer, pH 8, for another 10 minutes. The process was stopped
by washing with Tris-HCl buffer. The sections were air dried,
slightly counterstained with cresyl violet, and coverslipped
with Eukitt (Kindler, Freiburg, Germany). Corticospinal projections were quantified by counting the number of axons
sprouting across the midline in the pons and in the cervical
enlargement. The bilateral innervation of the pons was determined using an image analysis software routine (MCID/M4;
Imaging Research, St. Catherines, Ontario, Canada). The pixels
per area were automatically quantified in the ipsilateral and
contralateral sides after defining a threshold for the background
that was kept identical all through the analysis. A ratio between
the contralateral and ipsilateral sides was then computed. For
the cervical enlargement, the fibers sprouting across the midline were counted manually. Quantification of the total number
of fibers labeled in the main corticospinal tract (CST) was also
performed to generate a ratio of crossing fibers to account for
differences in biotinylated dextran amine labeling. After PCI,
successful tracing was achieved in 12 of 13 control-Ab–treated
animals and in all 7B12-treated animals (n ⳱ 14). After
MCAO, success rates for tracing were 8 of 9 in controlAb–treated animals, and 7 of 9 animals in both groups treated
with 7B12. All data were analyzed using a nonparametric
Kruskal-Wallis test in case of multiple comparisons followed
by nonparametric Mann-Whitney test in case of paired comparisons. Significance was taken to be P < 0.05.
RESULTS
Generation and characterization of the monoclonal
anti–Nogo-A antibody 7B12
The antirat Nogo-A antibody mAb 7B12 has subnanomolar binding for the Nogo-A specific domain NiG
(amino acids 174 to 979 of Nogo-A; Chen et al., 2000)
(Fig. 1) and within NiG for a part called NiGdelta-6
comprising amino acids 763 to 975 of Nogo-A (Fig. 1;
Chen et al., 2000). Details of antibody generation and
further characterization have been described elsewhere
(Oertle et al., 2000). Briefly, female mice, 5 to 6 weeks
old, (C3H- and C57BI6/J-strains) were immunized subcutaneously with recombinant prokaryotically produced
rat NiR-G, corresponding to amino acids 1 to 979 of
Nogo-A, that is, Nogo-A lacking the C-terminal reticulon domain (Chen et al., 2000). Positive clones were
subcloned twice, and the supernatants were collected to
ANTI–NOGO-A ANTIBODY TREATMENT AFTER STROKE
FIG. 1. Binding of purified antirat monoclonal antibody (mAb)
7B12 (immunoglobulin G1) to rat NiG (the protein domain specific
to Nogo-A) and rat NiG delta-6 fragment (Oertle et al., 2002)
using enzyme-linked immunosorbent assay (ELISA). Both proteins were coated in the ELISA plates at a concentration of
2 µg/mL and incubated with different concentrations of 7B12
mAb. The binding of 7B12 to the two proteins was quantified by
using a goat antimouse horseradish peroxidase–coupled antibody and peroxidase substrate and reading the optical density
(OD) of the color reaction in an ELISA plate reader at 450 nm.
Results are means ± SD of samples in at least duplicate wells.
Note the binding of the mAb 7B12 to the Nogo-A fragment used
for immunization (rat NiG delta-6) and the full-length Nogo-A–
specific fragment (rat NiG) at low nanomolar concentrations.
be tested on a standard fibroblast spreading test in vitro
as described (Spillmann et al., 1998). Like the extensively characterized antibodies IN-1 and AS-472 (Chen
et al., 2000), mAb 7B12 recognizes a single 190-kd band
in Western blots of rat and mouse oligodendrocyte lysates corresponding to Nogo-A (T. Oertle, unpublished
observations, 2002). In addition, in living cultures of
oligodendrocytes incubated with 7B12, immunostaining
of cell bodies and radial processes was observed, showing that the Nogo-A domain recognized by 7B12 is at the
cell surface (T. Oertle, unpublished observations, 2002).
Infarct morphology and volumes after
photothrombotic cortical injury and 7B12 treatment
Lesions were measured noninvasively by T2-weighted
MRI 24 hours after PCI, and before the implantation of
minipumps (Fig. 2). At this time, hyperintensive regions
denoting cytotoxic edema were sharply demarcated in
the MRI scan in the right hemisphere (Fig. 3A). The
lesions extended from approximately bregma 5.0 mm to
bregma −6.0 mm. Affected cortical areas included the
frontal cortex (area 1, 2, and 3), parietal cortex, the forelimb and hind-limb area of the cortex, and partly the
occipital cortex (according to Paxinos and Watson, 1986)
in all animals. Previous experiments had shown that lesions of this type resulted in persisting deficits of grasping performance with the contralateral forepaw, whereas
smaller lesions were followed by complete recovery (C.
Wiessner, unpublished observations, 2001). The total infarct volume was 308.1 ± 26.7 ␮L (mean ± SD) and
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313.0 ± 24.3 ␮L in the control-Ab group and the 7B12
group, respectively (Table 1). Thus, both groups had
very similar lesion volumes 24 hours after PCI and before Nogo-A antibody treatment was initiated. Magnetic
resonance imaging measurements were repeated 9 weeks
after PCI, that is, 7 weeks after termination of antibody
infusion (Fig. 3A). Loss of brain tissue (brain atrophy)
was determined by subtracting the volumes of the lesioned and unlesioned hemisphere. Brain atrophy was
very similar in the control-Ab–treated and the 7B12treated groups (Table 1), indicating that 7B12 infusion
did not influence the development of brain atrophy. In
addition, body weight—a sensitive general indicator for
the severity of the brain lesion and of the animal’s health
status—was not significantly different among the groups
before or at any time point after surgery (Table 1). These
results excluded the possibility that any observed differences in sensorimotor performance or fiber sprouting between both groups could be attributed to variations of
lesion size, or differences in the general health status of
the animals.
Behavioral improvement after photothrombotic
cortical injury and delayed treatment with 7B12
A skilled forepaw-reaching test was used to evaluate
sensorimotor function after PCI by counting food pellets
successfully grasped with the left or the right forepaw
and subsequently eaten (Montoya’s staircase test, Fig.
3B). Before PCI, rats were trained daily for 2 weeks to
establish the baseline performance, which was very similar in the control-Ab and the 7B12 groups (Table 1). In
the first week after PCI, grasping performance with the
(left) forepaw contralateral to the lesioned (right) hemisphere was reduced to about 10% of prelesion values
FIG. 2. Experimental protocol. The time flow of experiments is
shown on the left. On the right, the ischemia-induced cortical
lesion in the right hemisphere is shown as a shaded area. The
site of the brain infusion cannula for pump-controlled antibody
infusion was in the left lateral ventricle. The biotinylated dextran
amine tracer injection site was in the forelimb region of the left
sensorimotor cortex (i.e., contralateral to the lesion). MRI, magnetic resonance imaging; icv, intracerebroventricular.
J Cereb Blood Flow Metab, Vol. 23, No. 2, 2003
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C. WIESSNER ET AL.
FIG. 3. After photothrombotic cortical injury (PCI), treatment with the anti–Nogo-A antibody 7B12 improved regaining of contralateral
forepaw function. (A) Representative example for lesion morphology (bright areas) in the right hemisphere of the same rat 24 hours and
9 weeks after PCI as determined by noninvasive T2-weighted magnetic resonance imaging. (B) As a behavioral readout, Montoya’s
staircase test (Montoya et al., 1991), which is an integrative sensorimotor task for the forelimbs, was used. Rats enter the compartment
containing the baited staircase voluntarily from an anteroom partially visible on the right. (C) Grasped and eaten pellets with the left
forepaw were counted and expressed in percent of baseline (prelesion) performance determined in the last test session before lesioning
(Table 1). Performance was significantly better in the 7B12-treated group from 6 weeks after lesion onward. The control group received
unspecific mouse immunoglobulin Gs. Two-way repeated-measures analysis of variance: treatment, P = 0.031; time, P = 0.001; time ×
treatment; P = 0.43; *0.05 versus control group, t-test. (D) Successful attempts (eaten pellets × 100/[eaten pellets + displaced pellets])
with the left forepaw. This measure excluded the possibility that differences resulted from less motivation to use the impaired forepaw in
the control group. *0.05 versus control group, t-test. Error bars indicate standard deviation.
without significant differences between groups (Fig. 3C).
In the control group, grasping ability with the left paw
recovered between week 1 and week 6 after lesion,
reaching about 55% of prelesion performance without
further improvement at 7 and 9 weeks (Fig. 3C). Thus,
PCI resulted in a persistent sensorimotor deficit. This
result was similar to a previous pilot study, where rats
received an intracerebroventricular infusion of PBS after
PCI (n ⳱ 8; lesion volume: 310.6 ± 15.5 ␮L). In these
animals, grasping ability with the left forepaw was reduced to 12.5% and 56% in week 1 and week 8 after
lesion, respectively.
In the 7B12-treated group, behavioral improvement
also occurred between week 1 and week 6, however, was
better than in the control group and reached a plateau at
about 70% of prelesion performance, which was significantly different from the control group 6 to 9 weeks after
PCI (P < 0.05). Similar results were obtained when the
percentage of eaten pellets in relation to the total number
of grasping attempts (⳱ successful attempts) was determined (Fig. 3D). This finding excluded differences in
motivation to perform the sensorimotor task between
the groups.
J Cereb Blood Flow Metab, Vol. 23, No. 2, 2003
A moderate initial impairment of grasping ability was
observed for the (right) forepaw ipsilateral to the lesion,
amounting to 65% ± 22% and 67% ± 20% of prelesion
performance in the control group and the 7B12 group,
respectively. Five weeks after PCI performance had recovered to 92% ± 11% and 91% ± 29% in the control
group and 7B12 group, respectively, and remained in
the range of 90% to 100% of prelesion performance up to
TABLE 1. Body weight and lesion volumes after
photothrombotic cortical injury
PCI
Control-Ab (n ⳱ 13)
7B12 (n ⳱ 14)
Baseline performance (%)
Body weight, prelesion (g)
Body weight, 9 wk (g)
Infarct volume, 24 h (␮L)
Brain atrophy, 9 wk (␮L)
70.2 ± 7.3
235.4 ± 4.6
310.6 ± 14.3
308.1 ± 26.7
91.8 ± 35.6
71.7 ± 6.0
237.0 ± 8.8
310.0 ± 11.6
313.0 ± 24.3
90.5 ± 31.5
Baseline performance is expressed as the rate (%) of successful
grasping attempts in the week before photothrombotic cortical injury
(PCI); no significant differences were found for any of the parameters
shown.
Control-Ab, group treated with mouse immunoglobulin G; 7B12,
groups treated with monoclonal antibody anti–Nogo-A.
ANTI–NOGO-A ANTIBODY TREATMENT AFTER STROKE
9 weeks after PCI, the latest time point investigated.
Differences between the groups were not significant at
any time after PCI.
Brain distribution of intraventricularly infused
antibodies after photothrombotic cortical injury
The brain distribution of 7B12 and control antibody
was investigated by antimouse IgG immunohistochemistry in brains 2 weeks and 4 weeks after PCI. Two
weeks after PCI (i.e., at the end of the infusion period
into the left lateral ventricle), a staining gradient from the
ventricles and surface staining was found (Fig. 4A).
Staining intensity was highest in the left hemisphere,
where the antibody was infused, but was also prominent
in the lesioned hemisphere. The antibody distribution
was widespread and extended far into the caudal direction, including the hippocampus and gray matter of the
cervical spinal cord (Fig. 4A). Higher magnification
showed predominantly diffuse parenchymal staining for
both 7B12 (Fig. 4B) and the control antibody (not
shown). Both antibodies were still detectable 4 weeks
after PCI (i.e., 2 weeks after removal of the pumps),
again showing a very similar distribution pattern (Figs.
4C and E). Instead of the diffuse parenchymal distribu-
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tion, a more localized pattern was observed, including
the lesion border, corpus callosum, dorsolateral striatum
(Figs. 4C and E), cerebral peduncle, and the fascicles of
the capsula interna (Fig. 4C). In addition, moderate staining of the gray matter in the cervical spinal cord was still
observable (Fig. 4C). Higher magnification showed
staining of punctate and also elongated structures in the
mentioned areas, exemplified for the corpus callosum in
Fig. 4D. The precise nature of these structures remains to
be determined using techniques such as electron microscopy and double-staining immunohistochemistry. The
specificity of detection of mouse IgGs in brain tissue was
demonstrated by using brain sections from rats subjected
to PCI followed by minipump infusion of the carrier
solution (PBS). In this case no staining was observed
(Fig. 4F). In antibody-infused but unlesioned animals,
2 weeks after termination of infusion only marginal
staining around the infusion site (visible only at highpower magnification) was found (Fig. 4G), indicating an
involvement of the brain lesion in the prolonged presence of the infused mouse antibodies in the brain. In
summary, these results demonstrated that the mouse
IgG1 antibodies were widely distributed in the brain and
spinal cord after intraventricular infusion and were still
FIG. 4. After photothrombotic cortical injury (PCI) and 2 weeks of intraventricular infusion into the left lateral ventricle, antibodies
displayed widespread brain distribution and were still detectable 2 weeks after termination of intracerebroventricular infusion. Mouse
immunoglobulin G (IgG), 7B12 or control-Ab groups, was shown by immunostaining with an antimouse IgG antibody. (A) Typical antibody
distribution exemplified for 7B12 after 2-week infusion in two brain sections (bregma −0.8 mm to 3.2 mm) and one section from the cervical
spinal cord (C5). (Space bar = 1.5 mm.) (B) At higher magnification, mostly diffuse parenchymal distribution of the infused antibodies was
observed after 2 weeks of infusion. (Scale bar = 20 µm.) (C) Brain distribution of 7B12 4 weeks after PCI (i.e., 2 weeks after termination
of intraventricular pump infusion). Note the clearly detectable but more restricted distribution pattern as compared with A. (D) Higher
magnification 4 weeks after PCI showed staining of elongated and punctate structures. As an example, staining in the corpus callosum
of a 7B12-infused animal is shown. (Scale bar = 20 µm.) (E) Distribution patterns 4 weeks after PCI were similar for 7B12 and the mouse
control–antibody, as exemplified in this image. (F) Only weak background staining was observed in brains of animals receiving infusions
of phosphate-buffered saline into the lateral ventricle after PCI, showing the specificity of mouse IgG detection. (G) After intraventricular
infusion of antibodies into unlesioned brains, no widespread distribution was found 2 weeks after the end of infusion. (Scale bars in A,
C, E, F, G = 1.5 mm.)
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C. WIESSNER ET AL.
present in the brain at a time when control-Ab–treated
and 7B12-treated animals were beginning to diverge in
grasping performance.
Anterograde tracing of the pyramidal tract after
photothrombotic cortical injury
We hypothesized that recovery could be related to
neuroanatomic changes of fiber tracts originating in the
unlesioned cortex contralateral to the lesion. The anterograde tracer biotinylated dextran amine was, therefore,
injected into the forelimb region of the left sensorimotor
cortex 9 weeks after PCI. Two weeks later, the cortical
projection to the ipsilateral pons showed the typical topographical forelimb-specific innervation pattern as described earlier (Z’Graggen et al., 1998). Labeled fibers
running in the cerebral peduncle over the basilar pons
left the peduncle ventrally and formed typical termination fields (Figs. 5A and B). We have previously observed that in normal Lewis rats or after pyramidotomy,
only very few fibers cross the midline and terminate
within the contralateral pontine gray (Wenk et al., 1999;
Z’Graggen et al., 1998). In the present study, photothrombotic-lesioned Fischer rats from the control-Ab
group showed a prominent bilateral innervation of the
pons (Fig. 5A) that, however, was not significantly different in the 7B12-treated group (Figs. 5B and C). The
crossed projection formed a termination pattern specific
for fibers originating in the forelimb area, mirroring the
projections of the ipsilateral side. No correlation between
crossed fibers in the pons and grasping performance in
the staircase test was found.
When fiber outgrowth from the dorsal pyramidal tract
in the cervical spinal cord was examined, animals receiving the control antibody showed only few midline crossing fibers (Fig. 5D). Treatment with 7B12 prominently
increased the number of midline crossing fibers at this
level (Fig. 5E). Quantification confirmed that in the
7B12-treated group, midline crossing increased significantly in the cervical spinal cord from 1.1% ± 0.5% to
2.3% ± 1.5% (P < 0.05) (Fig. 5F). The degree of fiber
crossing in the cervical spinal cord correlated significantly with grasping performance 9 weeks after PCI
(Fig. 5G).
Delayed anti–Nogo-A antibody treatment after
middle cerebral artery occlusion in spontaneously
hypertensive rats
Because hypertension is the single most important risk
factor for stroke, we sought to investigate effects of 7B12
in a stroke model in SHR rats—an animal model of
genetically determined chronic hypertension resulting in
vascular changes and increased susceptibility to brain
ischemia (Barone et al., 1992). Permanent focal cerebral
ischemia was induced by distal MCAO, resulting in infarcts extending from approximately bregma +3 mm to
bregma −5 mm and including the sensorimotor cortex
J Cereb Blood Flow Metab, Vol. 23, No. 2, 2003
FIG. 5. Midline crossing of corticospinal fibers originating in the
unlesioned left sensorimotor cortex after photothrombotic cortical
injury (PCI) increased in 7B12-treated animals and correlated
with left forepaw function. The anterograde tracer biotinylated
dextran amine (BDA) was injected into the left sensorimotor cortex. (A) Cross section at the midpontine level of a control-Ab–
treated animal. Note that numerous BDA-labeled fibers end in the
contralateral pons (arrows). (B) Cross section at the midpontine
level of a 7B12-treated animal showing a similar BDA staining
pattern compared with control animals. (C) Quantification of bilateral innervation of the pons at the midpontine level did not
disclose significant differences between the groups. (D) Pyramidal tract labeling at the level of the cervical spinal cord in a control
animal receiving mouse immunoglobulin G. Substantial midline
crossing of fibers is not obvious. (E) In an animal treated with
7B12, numerous pyramidal tract fibers cross the midline and terminate in the gray matter of the left cervical spinal cord (arrows).
The inlet shows the fibers marked by the left arrow at higher
magnification. (F) For quantification, fibers sprouting across the
midline in the cervical spinal cord and the total number of fibers
labeled in the right pyramidal tract (originating in the left sensorimotor cortex) were counted and used to generate the ratio of
crossing fibers in percent. Error bars indicate standard error.
Data were analyzed using nonparametric Mann-Whitney test.
Significance was taken at P < 0.05. (G) Percentage of midline
crossing fibers in the cervical spinal cord was plotted against
performance (success rate [see Fig. 2D] in the last staircase
session before tracing for all animals, that is, including 7B12treated and control-Ab–treated animals]. Linear regression analysis showed a significant correlation of fiber crossing the cervical
spinal cord and grasping ability. (Scale bar in A, B, D, E =
0.25 mm.)
(Fig. 6A). The infarct volume at 24 hours after MCAO
amounted to 238.8 ± 51.8 ␮L, 216.5 ± 36.9 ␮L, and
223.8 ± 49.2 ␮L in the control group, the low-dose 7B12
group (1.68 mg), and the high-dose 7B12 group (5 mg),
respectively (Table 2). Differences in infarct volume between the groups before treatment were not significant.
ANTI–NOGO-A ANTIBODY TREATMENT AFTER STROKE
161
FIG. 6. Treatment with the anti–Nogo-A antibody 7B12 improved recovery of grasping
ability and midline crossing of corticospinal
fibers after middle cerebral artery occlusion
(MCAO) in spontaneously hypertensive
rats. (A) Representative example for the lesion morphology (bright areas) in the right
hemisphere 24 hours after MCAO as determined by T2-weighted magnetic resonance
imaging. (B) Grasped and eaten pellets with
the left forepaw in the staircase test were
counted and expressed in percent of baseline performance determined in the last test
session before lesioning. Two-way repeated-measures analysis of variance
showed a significant time effect (P < 0.001).
The significance of time effect within treatment groups was isolated by the StudentNewman-Keuls method (#P < 0.05 versus
4-week test). (C) After anterograde tracing
of fibers from the left unlesioned sensorimotor cortex, more pyramidal tract fibers crossing the midline and terminating in the gray
matter of the left cervical spinal cord (left
part of figure) are obvious in a 7B12-treated
animal (right) as compared with a controlAb–infused animal (middle). (D) Quantification of midline crossing fibers in the cervical
spinal cord was done exactly as described
for the photothrombotic cortical injury experiment (see Fig. 4). Error bars indicate
standard deviation. Data were analyzed using nonparametric Kruskal-Wallis test. (E)
Midline crossing of fibers in the cervical spinal cord was plotted against performance in
the last staircase session (week 12 after
MCAO) before tracing for all animals, that is,
including both 7B12-treated groups and
control-Ab–treated animals. Linear regression analysis showed a significant correlation between fiber crossing in the cervical
spinal cord and grasping ability.
The same was true for the baseline performance in the
staircase test before MCAO (Table 2). Of note, the baseline performance (in absolute terms) of the SHR rats in
the staircase test after the 2-week training period was
inferior compared with normotensive Fischer rats, and
the interindividual variability was higher (Table 2). Because of the invasive nature of MCAO, the first assessment of sensorimotor function could be done 4 weeks
after MCAO. At this time, all three groups showed
grasping deficits with the paw contralateral to the lesion
in the range of about 40% of prelesion performance (Fig.
6B). In the control group, no significant subsequent improvement was observed up to 12 weeks after MCAO,
and the deficit remained in the range of 50% of baseline
performance, which is in good agreement with previously published data (Grabowski et al., 1993). In contrast, for both 7B12-treated groups, prominent improvement in the staircase test was observed 7 to 12 weeks
after MCAO, approaching 70% of prelesion performance. Because of high interindividual variability of
TABLE 2. Body weight and lesion volumes after permanent middle cerebral artery occlusion
pMCAO (SHR)
Control-Ab (n ⳱ 9)
7B12, 1.68 mg (n ⳱ 9)
7B12, 5 mg (n ⳱ 9)
Baseline performance (%)
Body weight, prelesion (g)
Body weight, 12 wk (g)
Infarct volume, 24 h (␮L)
50.4 ± 10.7
212.8 ± 9.4
310.9 ± 9.4
238.8 ± 51.8
48.2 ± 8.8
213.9 ± 6.0
323.7 ± 14.6
216.5 ± 36.9
45.5 ± 8.2
215.0 ± 8.3
313.1 ± 14.1
223.8 ± 49.2
Baseline performance is expressed as the rate (%) of successful grasping attempts in the week before
permanent middle cerebral artery occlusion (pMCAO); no significant differences were found for any of the
parameters shown.
Control-Ab, group treated with mouse immunoglobulin G; 7B12, groups treated with monoclonal antibody
anti–Nogo-A; SHR, spontaneously hypertensive rats.
J Cereb Blood Flow Metab, Vol. 23, No. 2, 2003
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C. WIESSNER ET AL.
SHR rats in the staircase test, two-way repeatedmeasures ANOVA showed that the differences in performance between the groups did not reach significance
(P > 0.05); statistical analysis, however, indicated a
highly significant time effect. The significance of time
effect for the individual groups was isolated by the Student-Newman-Keuls method and showed that, compared
with the 4-week measurement, only both 7B12-treated
groups significantly (P < 0.05) improved 7 to 12 weeks
after MCAO (Fig. 6B). In the group treated with a lower
dose of 7B12 (1.68 mg), performance continued to improve until the latest time point measured (12 weeks).
From these results, it appeared that the higher dose
of 7B12 accelerated the speed of recovery, whereas
the maximal level of recovery was similar with both
7B12 doses.
Anterograde tracing of the pyramidal tract after
middle cerebral artery occlusion
Tracing was done as described for PCI, and a similar
staining pattern was obtained. At the level of the pons,
the mean values for midline crossing fibers were in a
similar range as observed for the PCI-lesion experiment,
amounting to 7.5% ± 6.5%, 10.1% ± 4.5%, and 15.6% ±
12.5% in the control group, 7B12 (1.68 mg) group, and
7B12 (5 mg) group, respectively. Although there appeared to be a dose-dependent increase of crossing fibers
in the pons in this experiment, the differences between
the groups were not statistically significant. In line with
the findings after PCI, no correlation between midline
crossing in the pons and grasping performance with the
left paw was found (not shown). In the cervical spinal
cord, 1.8% ± 0.8% midline crossing of CST fibers was
found in the control-Ab group. Treatment with 7B12
triggered a dose-dependent increased of midline crossing
fibers (Fig. 6C), amounting to 2.9% ± 1.6%, and 4.5% ±
2.2% when given at 1.68 mg and 5 mg, respectively (Fig.
6D). Therefore, similar to the PCI experiment, 5 mg of
7B12 more than doubled the number of midline crossing
fibers, and this increase was statistically significant
(P < 0.05). As for the PCI experiment, a significant
correlation between midline crossing fibers in the cervical enlargement and grasping performance determined
12 weeks after MCAO was observed (Fig. 6E).
DISCUSSION
This study showed that pump-controlled delivery of a
Nogo-A–specific antibody (7B12) initiated 24 hours
after experimental stroke promoted improvement of
long-term neurologic outcome in normotensive and SHR
rats. Treated animals showed an increase of midline
crossing in the cervical spinal cord of corticospinal fibers
originating from the unlesioned sensorimotor cortex
(Fig. 7). Midline crossing correlated with grasping abilJ Cereb Blood Flow Metab, Vol. 23, No. 2, 2003
FIG. 7. Diagram of projections from the left forelimb motor cortex
to the right cervical spinal cord. A significant increase of midline
sprouting to the left side of the cervical spinal cord was observed
after treatment with the anti–Nogo-A antibody 7B12 both after
photothrombotic cortical injury in normotensive Fischer rats and
after middle cerebral artery occlusion in spontaneously hypertensive rats. BDA, biotinylated dextran amine.
ity, suggesting a possible contribution of the crossing
fibers for behavioral improvement.
Pump infusion of 7B12 after experimental stroke
improved neurologic outcome
The experimental protocol in this study (Fig. 2) was
designed to evaluate postacute treatment effects on
endpoint behavioral outcome without confounding influences of infarct size. To achieve this goal, lesion volumes were determined 24 hours after stroke by T2weighted MRI before treatment was initiated. It has
previously been shown that lesion volumes derived from
noninvasive T2-weighted MRI highly correlate with volumes as determined by conventional histologic methods
(Rudin et al., 1999).
After focal cerebral ischemia in rodents, few sensorimotor tasks uncover permanent deficits (Virley et al.,
2000; Hunter et al., 2000). One of these is Montoya’s
staircase test (Montoya et al., 1991; Grabowski et al.,
1993), which is an integrative sensorimotor task for the
forelimbs implicated with cortical as well as subcortical
circuitries (Whishaw et al., 1986). Combining Montoya’s
staircase test and MRI-controlled cortical ischemia, we
showed that grasping ability of the forepaw contralateral
to the lesion was permanently depressed in control rats,
reaching a plateau at about 50% of baseline performance
up to the latest time points investigated (9 to 12 weeks).
When rats with infarct volumes MRI-matched to the control group were treated with 7B12 in the postacute phase
after stroke (24 hours), grasping ability improved significantly and reached about 70% of baseline levels. These
ANTI–NOGO-A ANTIBODY TREATMENT AFTER STROKE
findings agree with a recent study showing improvement
of skilled forelimb movements after MCAO by IN-1 hybridoma treatment initiated at the time of MCAO and
under immunosuppression (Papadopoulos et al., 2002).
Beneficial effects with 7B12 in the present study were
observed both in healthy normotensive rats and in SHR,
which develop vascular abnormalities, have reduced collateral cerebral blood flow, and show premature mortality (Barone et al., 1993). Thus, these results indicate that
anti–Nogo-A antibodies could be a promising rehabilitative treatment approach for human stroke with a prolonged time-to-treatment window. Intraventricular administration of antibodies, however, seems not to be
suitable for patients who have had stroke. Considering
disturbances of the blood–brain barrier after stroke
(Huber et al., 2001), it will be important to explore
whether peripheral administration can be achieved.
For spinal cord injury and head trauma, the demonstration of efficacy after pump infusion of 7B12 has immediate clinical relevance. In previous studies showing
behavioral recovery after CNS injury, the antibody IN-1
was secreted by implanted hybridoma cells under concomitant immunosuppressive treatment with cyclosporin
(Z’Graggen et al., 1998; Papadopoulos et al., 2002;
Merkler et al., 2001; Raineteau et al., 2001), which
would not be applicable to patients. Pump-controlled intrathecal drug administration, however, is an established
clinical method that is routinely used after spinal cord
injury (Schwab, 2002) and head trauma (Stempien and
Tsai, 2000). Intraventricularly infused antibodies of the
IgG isotype are cleared rapidly from the cerebrospinal
fluid via an Fc-receptor-mediated mechanism and with a
half-life of less than an hour (Zhang and Pardridge,
2001). After continuous pump infusion for 2 weeks,
however, the IgG1 antibodies in the present study were
found at high levels throughout the brain including the
cervical spinal cord, indicating a steady-state concentration gradient sufficient to drive penetration. The antibodies were still detectable 2 weeks after termination of
infusion in brain structures for which plastic changes
have been previously reported after cortical injury, such
as the perilesion cortex (Carmichael et al., 2001), the
dorsolateral striatum (Kartje et al., 1999; Papadopoulos
et al., 2002), and the cerebral peduncle (Wenk et al.,
1999). Therefore, it seems that intraventricular infusion
of anti–Nogo-A antibodies could be utilized for clinical
indications where this route of administration is feasible.
Corticospinal plasticity was enhanced after 7B12
treatment and correlated with behavioral outcome
Neuroanatomically, 7B12 treatment more than
doubled the number of midline crossing CST fibers in
the cervical spinal cord originating in the unlesioned sensorimotor cortex in normotensive and SHR rats (Fig. 6).
A similar extent of compensatory fiber growth from the
163
CST was found after unilateral CST transection caudal
from the lesion after IN-1 hybridoma treatment (Thallmair et al., 1998). Corticospinal projections across the
midline have been also described after sensorimotor cortex lesion in neonatal rats (Rouiller et al., 1991), where
the degree of spontaneous behavioral recovery after
comparable cortical lesions is much better compared
with adult animals. In previous studies, plasticity after
cortical injury and IN-1 hybridoma treatment has not
been investigated in the spinal cord, but interhemispheric
fiber outgrowth in corticostriatal (Kartje et al., 1999),
corticorubral (Wenk et al., 1999), and corticopontine
(Wenk et al., 1999) pathways is well documented. Corticostriatal plasticity was found also after MCAO in
black-hooded rats (Papadopoulos et al., 2002). In the
present study, the number of corticopontine fibers crossing the midline was relatively high even in control-Ab–
treated animals, and there was no increase in the 7B12treated rats. It is important to note that even untreated
rats displayed corticospinal fiber crossing in the spinal
cord (1% to 2%), although to a significantly lesser degree
than 7B12-treated animals. Similar observations were
made for interhemispheric corticostriatal fiber outgrowth
after cortical ablation (Kartje et al., 1999) or thermocoagulation (Napieralski et al., 1996). The correlation
analysis of fiber crossing with endpoint forepaw function
for all animals irrespective of treatment showed a significant correlation for both normotensive and SHR rats.
It therefore seems that lesion-induced fiber outgrowth in
the cervical spinal cord possibly contributed to the behavioral improvement after PCI and MCAO in SHR rats.
Importantly, treatment with 7B12 did not induce a qualitatively new fiber outgrowth pattern, but augmented an
endogenous lesion-induced plastic capacity of the adult
CNS. Without lesion, it has been shown that anti–
Nogo-A antibodies induced only short-lasting transient
sprouting of Purkinje axons in the adult cerebellum
(Buffo et al., 2000).
The finding that the correlation coefficients for corticospinal collateral sprouting and behavioral improvement were—although significant—in a moderate range
suggests that other brain areas and systems also are involved in the sensorimotor task and that such areas and
systems may also be affected by the 7B12 antibody.
From previous studies with the antibody IN-1 (see
above), it appears that corticostriatal and corticorubral
tracts could be involved (Kartje et al., 1999; Papadopoulos et al., 2002). Spontaneous although limited improvement of deficits after experimental and clinical stroke is
well recognized, and functional takeover in the unlesioned hemisphere has been implicated as a mechanism
for recovery after stroke by functional MRI studies.
These studies showed that electrical forelimb (Dijkhuizen et al., 2001) or hind-limb stimulation (Abo et al.,
2001) resulted in activation of normally silent brain
J Cereb Blood Flow Metab, Vol. 23, No. 2, 2003
164
C. WIESSNER ET AL.
regions ipsilateral to stimulation. Noninvasive imaging
techniques such as positron emission tomography, functional MRI, transcranial magnetic simulation, and magnetoencephalography have been used to investigate functional organization of the cortex after stroke in humans
(Nudo et al., 2001; Pineiro et al., 2001). These studies
provided evidence that recovery of hand function was
associated with increased bilateral activation of brain areas. Experimental and clinical studies also showed that
reorganization in the ipsilateral injured cortex is a very
important mechanism for recovery after stroke (Abo et
al., 2001; Carmichael et al., 2001; Dijkhuizen et al.,
2002; Nudo et al., 1996; Pineiro et al., 2001). Plastic
changes in the lesioned hemisphere are also likely to play
an important role in behavioral improvement in our
experimental paradigms. Whether and how anti–NogoA antibody treatment influences plasticity and sprouting in the injured hemisphere will be addressed in future studies.
Underlying mechanisms for compensatory sprouting
in the spinal cord
The Nogo-A–specific domain inhibits neurite outgrowth and causes growth cone collapse in neurons via
an elusive receptor (Oertle et al., 2002) that is distinct
from the Nogo-66 receptor NgR (Fournier et al., 2001),
and involves Rho-mediated downstream signaling to the
cytoskeleton (De Marco et al., 2002). Creation of a permissive environment facilitating fiber outgrowth therefore requires the local presence of the neutralizing antibody. In our study the presence of 7B12 in the spinal
cord after intraventricular infusion can explain the effect
on fiber outgrowth from the unlesioned pyramidal tract.
In addition, tonic Nogo-A–dependent inhibition of
growth-related genes in the cell bodies of cortical neurons may be released by Nogo-A neutralization with
7B12 in cortical parenchyma. At present, it cannot be
excluded that 7B12 (or other anti–Nogo-A antibodies) do
not only neutralize Nogo-A by binding to extracellular
epitopes, but that binding results in an internalization and
thus downregulation of Nogo-A. In vitro and in vivo
studies have shown that anti–Nogo-A antibody IN-1 induced the expression of growth-related genes, such as
c-jun, junD, synaptophysin, and GAP-43 (Zagrebelsky et
al., 1998; Huber and Schwab, 2000).
In summary, the results of this study suggest that anti–
Nogo-A antibodies bear potential as a new rehabilitative
treatment for ischemic stroke with a time-to-treatment
window of at least 24 hours. Although the precise
mechanisms of the beneficial effects of anti–Nogo-A antibodies in the presented experimental stroke models
need to be further explored, the results indicate that
compensatory growth of corticofugal fibers could be
involved.
J Cereb Blood Flow Metab, Vol. 23, No. 2, 2003
Acknowledgments: The authors thank Pascale Brebbia,
Francesco D’Amato, Thomas Hafner, and Willi Theilka¨ s for
their excellent technical help.
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