anti–nogo-a antibody infusion 24 hours after … · the hypothesis that plasticity in the adult...

Anti–Nogo-A Antibody Infusion 24 Hours After ExperimentalStroke 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 outgrowthinhibitory protein limiting recovery and plasticity after centralnervous system injury. In this study, a purified monoclonalanti–Nogo-A antibody (7B12) was evaluated in two rat strokemodels with a time-to-treatment of 24 hours after injury. Afterphotothrombotic cortical injury (PCI) and intraventricular in-fusion of a control mouse immunoglobulin G for 2 weeks,long-term contralateral forepaw function was reduced to about55% of prelesion performance until the latest time point inves-tigated (9 weeks). Forepaw function was significantly better inthe 7B12-treated group 6 to 9 weeks after PCI, and reachedabout 70% of prelesion levels. Cortical infarcts were also pro-duced in spontaneously hypertensive rats (SHR) by permanentmiddle cerebral artery occlusion (MCAO). In the control group,forepaw function remained between 40% and 50% of prelesionlevels 4 to 12 weeks after MCAO. In contrast, 7B12-treatedgroups showed significant improvement between 4 and 7

weeks after MCAO from around 40% of prelesion levels atweek 4 to about 60% to 70% at 7 to 12 weeks after MCAO.Treatment in both models was efficacious without influencinginfarct volume or brain atrophy. Neuroanatomically in the spi-nal cord, a significant increase of midline crossing corticospinalfibers originating in the unlesioned sensorimotor cortex wasfound in 7B12-treated groups, reaching 2.3 ± 1.5% after PCI(control group: 1.1 ± 0.5%) and 4.5 ± 2.2% after MCAO inSHR rats (control group: 1.8 ± 0.8%). Behavioral outcome andthe presence of midline crossing fibers in the cervical spinalcord correlated significantly, suggesting a possible contributionof the crossing fibers for forepaw function after PCI andMCAO. The results suggest that specific anti–Nogo-A anti-bodies bear potential as a new rehabilitative treatment approachfor ischemic stroke with a prolonged time-to-treatment win-dow. Key Words: Nogo—Neurite outgrowth inhibitor—Focalcerebral ischemia—Sensorimotor function—Plasticity.

Injuries to the adult human cerebral motor cortex oftenresult in persisting limb movement deficits (Nudo et al.,2001). The leading cause of cortical injury and seriouslong-term disability is ischemic stroke (Stapf and Mohr,2002). Epidemiologic data suggest that after a long pe-riod of decline, stroke incidence is now rising again (Gil-lum and Sempos, 1997). Because treatment options arelimited to thrombolysis with a time-to-treatment windowof only 3 hours (The NINDS study group, 1997; delZoppo, 2000), and a large number of neuroprotectivedrugs failed in clinical trials (Martinez-Vila and Sieira,

2001), an urgent medical need for new treatment strate-gies for stroke exists.

In comparison to the adult brain, recovery of motorfunction after injury is much better in the neonatal brain(Milner, 1974; Whishaw and Kolb, 1988), and neuroana-tomic plasticity is thought to be the underlying mecha-nism. Studies in newborn rats have shown that, aftersensorimotor cortical lesion, the uninjured contralateralcortex is able to form bilateral connections with the stria-tum (Kolb et al., 1992), thalamus (Yu et al., 1995), rednucleus (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 capac-ity to form such new connections declines with progress-ing myelination (Huber and Schwab, 2000), which led tothe hypothesis that plasticity in the adult brain is re-stricted by neurite outgrowth inhibitors residing in my-elin (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, Neurode-generation Unit WKl125.5.15, CH-4002 Basel, Switzerland; e-mail:[email protected]

Journal of Cerebral Blood Flow & Metabolism23:154–165 © 2003 The International Society for Cerebral Blood Flow and MetabolismPublished by Lippincott Williams & Wilkins, Inc., Philadelphia

154 DOI: 10.1097/01.WCB.0000040400.30600.AF

potent neurite growth inhibitory activities associatedwith oligodendrocyte membranes and CNS myelin is themembrane protein Nogo-A, which has recently beencloned (Chen et al., 2000; Grand Pre et al., 2000; Prinhjaet al., 2000). A neutralizing antibody (mAb IN-1), raisedagainst NI-250 (Caroni and Schwab, 1988), improvedregeneration of lesioned axons in the adult rat spinalcord, optic nerve, or brain (Schnell and Schwab, 1990;Huber and Schwab, 2000), and increased compensatoryfiber growth of unlesioned tract systems (Thallmairet al., 1998; Z’Graggen et al., 2000; Wenk et al., 1999).In behavioral assays in rats, functional recovery was ob-served after injury and IN-1 hybridoma treatment (Thall-mair et al., 1998; Z’Graggen et al., 1998). In all of thesestudies, IN-1 was delivered by implanted antibody-secreting hybridoma cells with concomitant immunosup-pression. As an important step towards transfer ofNogo-A neutralization to the level of clinical trials, wegenerated a new monoclonal anti–Nogo-A antibody ofthe immunoglobulin G (IgG)1-subtype (7B12), whichwas purified and delivered in a highly controlled mannerby minipumps. In the present study, we showed that7B12 infusion initiated 24 hours after stroke improvedlong-term behavioral recovery without affecting lesionvolumes. Behavioral outcome correlated with sproutingof corticospinal fibers of the unlesioned sensorimotorcortex, which was stimulated in 7B12-treated animals.The results support the concept that Nogo-A neutraliza-tion augments the inherent and functionally meaningfulplastic capacity of the adult CNS, which is restricted byoutgrowth inhibitors.

MATERIAL AND METHODS

Characterization of monoclonal antibody 7B12Binding of purified antirat monoclonal antibody (mAb)

7B12 (IgG1) to rat NiG (the protein domain specific toNogo-A) and rat NiG delta-6 fragment (Oertle et al., 2000) wasdetermined by enzyme-linked immunosorbent assay. NiG andNiG delta-6 were coated on enzyme-linked immunosorbent as-say plates at a concentration of 2 �g/mL and incubated withdifferent concentrations of 7B12 mAb. The binding of 7B12 tothe two proteins was quantified by using a goat antimousehorseradish peroxidase–coupled antibody and peroxidase sub-strate (Roche Diagnostics, Rotkreuz, Switzerland) and readingthe optical density of the color reaction in an enzyme-linkedimmunosorbent 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 surgicalprocedures were approved by the veterinary authorities of theKanton Basel-Stadt, Switzerland.

Photothrombotic cortical injury. A modification of amethod described by Watson et al. (1985) was used. MaleFischer F344 rats (Iffa Credo, L’Arbresele, France) weighing300 g were anesthetized with 2% isoflurane in a 70:30 (byvolume) nitrous oxide–oxygen mixture and 80 mg/kg Rose-Bengal in saline was infused into the femoral vein (0.5 mL/kgat 95 �L/min). Immediately afterwards, a fiberglass probe with

10-mm diameter was positioned directly over the exposed rightskull. The skull was illuminated (Volpi Intralux 6000, 150 W;Volpi AG, Schlieven, Switzerland) for 7 minutes at maximumoutput. Body temperature was maintained at 37°C by means ofa heating pad (CMA 150; Carnegie Medicine, Stockholm,Sweden) connected to a rectal probe during surgery and untilanimals regained consciousness. Thereafter, animals werereturned to their home cages and allowed free access to foodand water.

Permanent middle cerebral artery occlusion in sponta-neously hypertensive rats. Irreversible right middle cerebralartery occlusion (MCAO) was performed in male spontane-ously hypertensive rats (SHR) rats, 220 to 300 g (Iffa Credo),as described previously (Barone et al., 1993), with minor modi-fications. Briefly, animals were anesthetized (see precedingparagraph) and the right middle cerebral artery was exposed bya subtemporal craniectomy and occluded by bipolar electroco-agulation. Afterwards, retracted soft tissue was replaced,wounds were sutured, and anesthesia was discontinued. Bodytemperature was maintained at 37°C during surgery and untilanimals regained consciousness (see preceding paragraph).Thereafter, animals were returned to their home cages and wereallowed free access to food and water.

Magnetic resonance imaging. Infarct volume was deter-mined 24 hours after PCI or MCAO by means of quantitativein vivo magnetic resonance imaging (MRI). Measurementswere performed on a 4.7-T 30-cm-bore Spectrospin DBX(Bruker, Karlsruhe, Germany) equipped with a 20-cm gradientcoil as described in detail elsewhere (Allegrini and Sauer, 1992;Wiessner et al., 2000). Briefly, rats were anesthetized withisoflurane (see preceding paragraphs) and the head was posi-tioned in a 35-mm resonator. Thirteen contiguous T2-weightedcoronal slices with a thickness of 1.2 mm were taken usinga RARE sequence (repetition time, 3,000 milliseconds; effec-tive 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 thresholdusing a semiautomatic image analysis software (Analyze, Bio-medical 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 dam-aged area in each slice and the distance between slices. Nineweeks after PCI, volumes of the infarcted and contralateralhemispheres were measured again by MRI and the atrophy ofthe infarcted hemisphere was calculated as their difference.Statistical comparison between control and treatment groupswas made using t-test or analysis of variance (ANOVA) fol-lowed by Bonferroni t-test. A P value < 0.05 was regarded asbeing statistically significant.

Treatment with 7B12. A brain infusion cannula (Alzet braininfusion kit; Alza Corp., Palo Alto, CA, U.S.A.) was stereo-taxically implanted under anesthesia (2% isoflurane in 70:30nitrous oxide–oxygen mixture) in the left lateral ventricle (co-ordinates: 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 in-traventricularly in phosphate-buffered saline (PBS) by continu-ous infusion for 2 weeks using Alzet osmotic minipumps(5 �L/h; model 2ML2; Alza Corp.). After PCI, total antibodydoses were 5 mg control mouse IgG (n � 13) or 5 mg 7B12(n � 14) infused at 15 �g/h. After MCAO in SHR rats, totalantibody doses were 1.68 mg 7B12 (5 �g/h; n � 9), or 5 mg7B12 (15 �g/h; n � 9). In this experiment, the control antibodywas given at a dose of 1.68 mg 7B12 (5 �g/h; n � 9), to matchthe lower dose 7B12 group. The pumps were removed after2 weeks and quantitative delivery was controlled. The surgeons

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implanting osmotic minipumps were masked with respect totreatment. In addition, brains from animals receiving 7B12,control antibody, or PBS were sampled 2 weeks or 4 weeksafter pump implantation (n � 3 to 5) and frozen at −70°Cin Tissue-Tek O.C.T. compound (Sakura, Zoeterwoude, Neth-erlands). Coronal sections (10 �m) of these brains were cuton a cryostat, and 7B12 distribution was visualized using abiotinylated antimouse antibody and the ABC kit (VectorLaboratories, Burlingame, CA, U.S.A.) as recommended bythe supplier.

Evaluation of sensorimotor performance. Montoya’sstaircase test was carried out as described in detail previously(Montoya et al., 1991) and by investigators masked with regardto treatment. Rats were placed in a Plexiglas box with a baiteddouble staircase. Food pellets were placed on the staircase andpresented bilaterally at seven graded stages of reaching diffi-culty to provide objective measures of maximum forelimb ex-tension and grasping skill. Rats were food deprived for at least24 hours before and throughout the whole weekly test session,being fed once a day with approximately 10 g standard ratchow. 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 additionstairs 1 and 2 were baited bilaterally with 2 pellets each. Therats were tested twice daily (morning/afternoon) for 10 minuteseach. On days 3 to 4, stairs 2 to 6 were bilaterally baited withthree pellets each to avoid rats successfully retrieving pelletswith their tongue or with the “unaffected” paw. The rats weretested again twice daily for 10 minutes each. After each testseries, the total number of pellets grasped and eaten on days 3to 4 with each forepaw was counted. In addition, the number ofdisplaced pellets was determined. Performance in the staircasetest was expressed in percent of the last test session beforelesioning (eaten pellets) or in success rate (eaten pellets ×100/[eaten pellets + displaced pellets]). For statistical analysisbetween groups, two-way repeated-measures ANOVA wasused followed by a pairwise multiple comparison procedure(i.e., Bonferroni t-test). For analysis of recovery within thegroups, the first test session after lesioning was used as a ref-erence value, and statistical analysis was done using one-wayrepeated-measures ANOVA.

Anterograde tracing with biotinylated dextran amine.Tracing and analysis was performed by an investigator maskedwith regard to treatment. Location of the forelimb sensorimotorcortex was determined by measuring positions on the skullrelative to Bregma (Neafsey et al., 1986). Nine weeks after PCIor 13 weeks after MCAO, pressure injections of a 10% solutionof 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 mmposterior, 2.5 mm lateral; 1.5-mm depth, relative to bregma)through a glass capillary that remained in its position for 2minutes after the end of the injection. Two weeks after bioti-nylated dextran amine injection, animals were deeply anesthe-tized with pentobarbital (450 mg/kg intraperitoneally; AbbottLaboratories, Cham, Switzerland) and perfused transcardiallywith 100 mL of Ringer solution containing 100,000 IU/L hep-arin (Liquemin; Roche, Basel, Switzerland) and 0.25% NaNO2

followed by 300 mL of 4% paraformaldehyde in 0.1 mol/Lphosphate buffer with 5% sucrose. Brains and spinal cordswere dissected and postfixed overnight at 4°C in the samefixative. Meninges were removed and the pons and cervicalenlargement (C4–C6) was embedded in a gelatine–chicken al-bumin solution polymerized with 2.5% glutaraldehyde. Fifty-micrometer coronal sections were cut using a vibratome (Leica

vt 1000S). All sections were collected in 50 mmol/L Tris-buffered 0.9% saline, pH 8, and 0.5% Triton X-100 (TBS-Tx).They were serially mounted on slides (Superfrost/Plus; Men-zel-Glaser, Braunschweig, Germany) according to the semi-free floating technique (Herzog and Brosamle, 1997). Sectionswere washed three times for 30 minutes in TBS-Tx beforeovernight incubation in avidin-peroxidase in TBS-Tx (ABCElite; Vector Laboratories). The following day, the slides werewashed three times for 30 minutes in TBS-Tx. After an addi-tional wash in 50 mmol/L Tris-HCl, pH 8, a preincubation for10 minutes in 0.4% ammonium nickel sulfate (Sigma, St.Louis, MO, U.S.A.) was performed, followed by a second pre-incubation in 0.4% ammonium nickel sulfate and 0.015% 3,3�-diaminobenzidine (DAB; Sigma, Buchs, Switzerland) for 10minutes. The tissue was then reacted in 0.4% ammonium nickelsulfate, 0.015% DAB, and 0.004% H2O2 in 50 mmol/L Trisbuffer, pH 8, for another 10 minutes. The process was stoppedby washing with Tris-HCl buffer. The sections were air dried,slightly counterstained with cresyl violet, and coverslippedwith Eukitt (Kindler, Freiburg, Germany). Corticospinal pro-jections were quantified by counting the number of axonssprouting across the midline in the pons and in the cervicalenlargement. The bilateral innervation of the pons was deter-mined using an image analysis software routine (MCID/M4;Imaging Research, St. Catherines, Ontario, Canada). The pixelsper area were automatically quantified in the ipsilateral andcontralateral sides after defining a threshold for the backgroundthat was kept identical all through the analysis. A ratio betweenthe contralateral and ipsilateral sides was then computed. Forthe cervical enlargement, the fibers sprouting across the mid-line were counted manually. Quantification of the total numberof fibers labeled in the main corticospinal tract (CST) was alsoperformed to generate a ratio of crossing fibers to account fordifferences in biotinylated dextran amine labeling. After PCI,successful tracing was achieved in 12 of 13 control-Ab–treatedanimals and in all 7B12-treated animals (n � 14). AfterMCAO, success rates for tracing were 8 of 9 in control-Ab–treated animals, and 7 of 9 animals in both groups treatedwith 7B12. All data were analyzed using a nonparametricKruskal-Wallis test in case of multiple comparisons followedby nonparametric Mann-Whitney test in case of paired com-parisons. Significance was taken to be P < 0.05.

RESULTS

Generation and characterization of the monoclonalanti–Nogo-A antibody 7B12

The antirat Nogo-A antibody mAb 7B12 has subnano-molar 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-6comprising amino acids 763 to 975 of Nogo-A (Fig. 1;Chen et al., 2000). Details of antibody generation andfurther characterization have been described elsewhere(Oertle et al., 2000). Briefly, female mice, 5 to 6 weeksold, (C3H- and C57BI6/J-strains) were immunized sub-cutaneously with recombinant prokaryotically producedrat NiR-G, corresponding to amino acids 1 to 979 ofNogo-A, that is, Nogo-A lacking the C-terminal reticu-lon domain (Chen et al., 2000). Positive clones weresubcloned twice, and the supernatants were collected to

C. WIESSNER ET AL.156


be tested on a standard fibroblast spreading test in vitroas described (Spillmann et al., 1998). Like the exten-sively characterized antibodies IN-1 and AS-472 (Chenet al., 2000), mAb 7B12 recognizes a single 190-kd bandin Western blots of rat and mouse oligodendrocyte ly-sates corresponding to Nogo-A (T. Oertle, unpublishedobservations, 2002). In addition, in living cultures ofoligodendrocytes incubated with 7B12, immunostainingof cell bodies and radial processes was observed, show-ing that the Nogo-A domain recognized by 7B12 is at thecell surface (T. Oertle, unpublished observations, 2002).

Infarct morphology and volumes afterphotothrombotic cortical injury and 7B12 treatment

Lesions were measured noninvasively by T2-weightedMRI 24 hours after PCI, and before the implantation ofminipumps (Fig. 2). At this time, hyperintensive regionsdenoting cytotoxic edema were sharply demarcated inthe MRI scan in the right hemisphere (Fig. 3A). Thelesions extended from approximately bregma 5.0 mm tobregma −6.0 mm. Affected cortical areas included thefrontal cortex (area 1, 2, and 3), parietal cortex, the fore-limb and hind-limb area of the cortex, and partly theoccipital cortex (according to Paxinos and Watson, 1986)in all animals. Previous experiments had shown that le-sions of this type resulted in persisting deficits of grasp-ing performance with the contralateral forepaw, whereassmaller lesions were followed by complete recovery (C.Wiessner, unpublished observations, 2001). The total in-farct volume was 308.1 ± 26.7 �L (mean ± SD) and

313.0 ± 24.3 �L in the control-Ab group and the 7B12group, respectively (Table 1). Thus, both groups hadvery similar lesion volumes 24 hours after PCI and be-fore Nogo-A antibody treatment was initiated. Magneticresonance imaging measurements were repeated 9 weeksafter PCI, that is, 7 weeks after termination of antibodyinfusion (Fig. 3A). Loss of brain tissue (brain atrophy)was determined by subtracting the volumes of the le-sioned and unlesioned hemisphere. Brain atrophy wasvery similar in the control-Ab–treated and the 7B12-treated groups (Table 1), indicating that 7B12 infusiondid not influence the development of brain atrophy. Inaddition, body weight—a sensitive general indicator forthe severity of the brain lesion and of the animal’s healthstatus—was not significantly different among the groupsbefore or at any time point after surgery (Table 1). Theseresults excluded the possibility that any observed differ-ences in sensorimotor performance or fiber sprouting be-tween both groups could be attributed to variations oflesion size, or differences in the general health status ofthe animals.

Behavioral improvement after photothromboticcortical injury and delayed treatment with 7B12

A skilled forepaw-reaching test was used to evaluatesensorimotor function after PCI by counting food pelletssuccessfully grasped with the left or the right forepawand subsequently eaten (Montoya’s staircase test, Fig.3B). Before PCI, rats were trained daily for 2 weeks toestablish the baseline performance, which was very simi-lar in the control-Ab and the 7B12 groups (Table 1). Inthe first week after PCI, grasping performance with the(left) forepaw contralateral to the lesioned (right) hemi-sphere was reduced to about 10% of prelesion values

FIG. 2. Experimental protocol. The time flow of experiments isshown on the left. On the right, the ischemia-induced corticallesion in the right hemisphere is shown as a shaded area. Thesite of the brain infusion cannula for pump-controlled antibodyinfusion was in the left lateral ventricle. The biotinylated dextranamine tracer injection site was in the forelimb region of the leftsensorimotor cortex (i.e., contralateral to the lesion). MRI, mag-netic resonance imaging; icv, intracerebroventricular.

FIG. 1. Binding of purified antirat monoclonal antibody (mAb)7B12 (immunoglobulin G1) to rat NiG (the protein domain specificto Nogo-A) and rat NiG delta-6 fragment (Oertle et al., 2002)using enzyme-linked immunosorbent assay (ELISA). Both pro-teins were coated in the ELISA plates at a concentration of2 µg/mL and incubated with different concentrations of 7B12mAb. The binding of 7B12 to the two proteins was quantified byusing a goat antimouse horseradish peroxidase–coupled anti-body 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 usedfor immunization (rat NiG delta-6) and the full-length Nogo-A–specific fragment (rat NiG) at low nanomolar concentrations.



without significant differences between groups (Fig. 3C).In the control group, grasping ability with the left pawrecovered between week 1 and week 6 after lesion,reaching about 55% of prelesion performance withoutfurther improvement at 7 and 9 weeks (Fig. 3C). Thus,PCI resulted in a persistent sensorimotor deficit. Thisresult was similar to a previous pilot study, where ratsreceived an intracerebroventricular infusion of PBS afterPCI (n � 8; lesion volume: 310.6 ± 15.5 �L). In theseanimals, grasping ability with the left forepaw was re-duced to 12.5% and 56% in week 1 and week 8 afterlesion, respectively.

In the 7B12-treated group, behavioral improvementalso occurred between week 1 and week 6, however, wasbetter than in the control group and reached a plateau atabout 70% of prelesion performance, which was signifi-cantly different from the control group 6 to 9 weeks afterPCI (P < 0.05). Similar results were obtained when thepercentage of eaten pellets in relation to the total numberof grasping attempts (� successful attempts) was deter-mined (Fig. 3D). This finding excluded differences inmotivation to perform the sensorimotor task betweenthe groups.

A moderate initial impairment of grasping ability wasobserved for the (right) forepaw ipsilateral to the lesion,amounting to 65% ± 22% and 67% ± 20% of prelesionperformance in the control group and the 7B12 group,respectively. Five weeks after PCI performance had re-covered to 92% ± 11% and 91% ± 29% in the controlgroup and 7B12 group, respectively, and remained inthe range of 90% to 100% of prelesion performance up to

TABLE 1. Body weight and lesion volumes afterphotothrombotic cortical injury

PCI Control-Ab (n � 13) 7B12 (n � 14)

Baseline performance (%) 70.2 ± 7.3 71.7 ± 6.0Body weight, prelesion (g) 235.4 ± 4.6 237.0 ± 8.8Body weight, 9 wk (g) 310.6 ± 14.3 310.0 ± 11.6Infarct volume, 24 h (�L) 308.1 ± 26.7 313.0 ± 24.3Brain atrophy, 9 wk (�L) 91.8 ± 35.6 90.5 ± 31.5

Baseline performance is expressed as the rate (%) of successfulgrasping attempts in the week before photothrombotic cortical injury(PCI); no significant differences were found for any of the parametersshown.

Control-Ab, group treated with mouse immunoglobulin G; 7B12,groups treated with monoclonal antibody anti–Nogo-A.

FIG. 3. After photothrombotic cortical injury (PCI), treatment with the anti–Nogo-A antibody 7B12 improved regaining of contralateralforepaw function. (A) Representative example for lesion morphology (bright areas) in the right hemisphere of the same rat 24 hours and9 weeks after PCI as determined by noninvasive T2-weighted magnetic resonance imaging. (B) As a behavioral readout, Montoya’sstaircase test (Montoya et al., 1991), which is an integrative sensorimotor task for the forelimbs, was used. Rats enter the compartmentcontaining the baited staircase voluntarily from an anteroom partially visible on the right. (C) Grasped and eaten pellets with the leftforepaw 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 receivedunspecific 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 inthe control group. *0.05 versus control group, t-test. Error bars indicate standard deviation.



9 weeks after PCI, the latest time point investigated.Differences between the groups were not significant atany time after PCI.

Brain distribution of intraventricularly infusedantibodies after photothrombotic cortical injury

The brain distribution of 7B12 and control antibodywas investigated by antimouse IgG immunohistochem-istry in brains 2 weeks and 4 weeks after PCI. Twoweeks after PCI (i.e., at the end of the infusion periodinto the left lateral ventricle), a staining gradient from theventricles and surface staining was found (Fig. 4A).Staining intensity was highest in the left hemisphere,where the antibody was infused, but was also prominentin the lesioned hemisphere. The antibody distributionwas widespread and extended far into the caudal direc-tion, including the hippocampus and gray matter of thecervical spinal cord (Fig. 4A). Higher magnificationshowed predominantly diffuse parenchymal staining forboth 7B12 (Fig. 4B) and the control antibody (notshown). Both antibodies were still detectable 4 weeksafter 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-

tion, a more localized pattern was observed, includingthe lesion border, corpus callosum, dorsolateral striatum(Figs. 4C and E), cerebral peduncle, and the fascicles ofthe capsula interna (Fig. 4C). In addition, moderate stain-ing of the gray matter in the cervical spinal cord was stillobservable (Fig. 4C). Higher magnification showedstaining of punctate and also elongated structures in thementioned areas, exemplified for the corpus callosum inFig. 4D. The precise nature of these structures remains tobe determined using techniques such as electron micros-copy and double-staining immunohistochemistry. Thespecificity of detection of mouse IgGs in brain tissue wasdemonstrated by using brain sections from rats subjectedto PCI followed by minipump infusion of the carriersolution (PBS). In this case no staining was observed(Fig. 4F). In antibody-infused but unlesioned animals,2 weeks after termination of infusion only marginalstaining around the infusion site (visible only at high-power magnification) was found (Fig. 4G), indicating aninvolvement of the brain lesion in the prolonged pres-ence of the infused mouse antibodies in the brain. Insummary, these results demonstrated that the mouseIgG1 antibodies were widely distributed in the brain andspinal 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, antibodiesdisplayed widespread brain distribution and were still detectable 2 weeks after termination of intracerebroventricular infusion. Mouseimmunoglobulin G (IgG), 7B12 or control-Ab groups, was shown by immunostaining with an antimouse IgG antibody. (A) Typical antibodydistribution exemplified for 7B12 after 2-week infusion in two brain sections (bregma −0.8 mm to 3.2 mm) and one section from the cervicalspinal cord (C5). (Space bar = 1.5 mm.) (B) At higher magnification, mostly diffuse parenchymal distribution of the infused antibodies wasobserved after 2 weeks of infusion. (Scale bar = 20 µm.) (C) Brain distribution of 7B12 4 weeks after PCI (i.e., 2 weeks after terminationof intraventricular pump infusion). Note the clearly detectable but more restricted distribution pattern as compared with A. (D) Highermagnification 4 weeks after PCI showed staining of elongated and punctate structures. As an example, staining in the corpus callosumof a 7B12-infused animal is shown. (Scale bar = 20 µm.) (E) Distribution patterns 4 weeks after PCI were similar for 7B12 and the mousecontrol–antibody, as exemplified in this image. (F) Only weak background staining was observed in brains of animals receiving infusionsof phosphate-buffered saline into the lateral ventricle after PCI, showing the specificity of mouse IgG detection. (G) After intraventricularinfusion 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.)



present in the brain at a time when control-Ab–treatedand 7B12-treated animals were beginning to diverge ingrasping performance.

Anterograde tracing of the pyramidal tract afterphotothrombotic cortical injury

We hypothesized that recovery could be related toneuroanatomic changes of fiber tracts originating in theunlesioned cortex contralateral to the lesion. The antero-grade tracer biotinylated dextran amine was, therefore,injected into the forelimb region of the left sensorimotorcortex 9 weeks after PCI. Two weeks later, the corticalprojection to the ipsilateral pons showed the typical to-pographical forelimb-specific innervation pattern as de-scribed earlier (Z’Graggen et al., 1998). Labeled fibersrunning in the cerebral peduncle over the basilar ponsleft the peduncle ventrally and formed typical termina-tion fields (Figs. 5A and B). We have previously ob-served that in normal Lewis rats or after pyramidotomy,only very few fibers cross the midline and terminatewithin the contralateral pontine gray (Wenk et al., 1999;Z’Graggen et al., 1998). In the present study, photo-thrombotic-lesioned Fischer rats from the control-Abgroup showed a prominent bilateral innervation of thepons (Fig. 5A) that, however, was not significantly dif-ferent in the 7B12-treated group (Figs. 5B and C). Thecrossed projection formed a termination pattern specificfor fibers originating in the forelimb area, mirroring theprojections of the ipsilateral side. No correlation betweencrossed fibers in the pons and grasping performance inthe staircase test was found.

When fiber outgrowth from the dorsal pyramidal tractin the cervical spinal cord was examined, animals receiv-ing the control antibody showed only few midline cross-ing fibers (Fig. 5D). Treatment with 7B12 prominentlyincreased the number of midline crossing fibers at thislevel (Fig. 5E). Quantification confirmed that in the7B12-treated group, midline crossing increased signifi-cantly in the cervical spinal cord from 1.1% ± 0.5% to2.3% ± 1.5% (P < 0.05) (Fig. 5F). The degree of fibercrossing in the cervical spinal cord correlated signifi-cantly with grasping performance 9 weeks after PCI(Fig. 5G).

Delayed anti–Nogo-A antibody treatment aftermiddle cerebral artery occlusion in spontaneouslyhypertensive rats

Because hypertension is the single most important riskfactor for stroke, we sought to investigate effects of 7B12in a stroke model in SHR rats—an animal model ofgenetically determined chronic hypertension resulting invascular changes and increased susceptibility to brainischemia (Barone et al., 1992). Permanent focal cerebralischemia was induced by distal MCAO, resulting in in-farcts extending from approximately bregma +3 mm tobregma −5 mm and including the sensorimotor cortex

(Fig. 6A). The infarct volume at 24 hours after MCAOamounted to 238.8 ± 51.8 �L, 216.5 ± 36.9 �L, and223.8 ± 49.2 �L in the control group, the low-dose 7B12group (1.68 mg), and the high-dose 7B12 group (5 mg),respectively (Table 2). Differences in infarct volume be-tween the groups before treatment were not significant.

FIG. 5. Midline crossing of corticospinal fibers originating in theunlesioned left sensorimotor cortex after photothrombotic corticalinjury (PCI) increased in 7B12-treated animals and correlatedwith left forepaw function. The anterograde tracer biotinylateddextran amine (BDA) was injected into the left sensorimotor cor-tex. (A) Cross section at the midpontine level of a control-Ab–treated animal. Note that numerous BDA-labeled fibers end in thecontralateral pons (arrows). (B) Cross section at the midpontinelevel of a 7B12-treated animal showing a similar BDA stainingpattern compared with control animals. (C) Quantification of bi-lateral innervation of the pons at the midpontine level did notdisclose significant differences between the groups. (D) Pyrami-dal tract labeling at the level of the cervical spinal cord in a controlanimal receiving mouse immunoglobulin G. Substantial midlinecrossing of fibers is not obvious. (E) In an animal treated with7B12, numerous pyramidal tract fibers cross the midline and ter-minate in the gray matter of the left cervical spinal cord (arrows).The inlet shows the fibers marked by the left arrow at highermagnification. (F) For quantification, fibers sprouting across themidline in the cervical spinal cord and the total number of fiberslabeled in the right pyramidal tract (originating in the left senso-rimotor cortex) were counted and used to generate the ratio ofcrossing 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 midlinecrossing fibers in the cervical spinal cord was plotted againstperformance (success rate [see Fig. 2D] in the last staircasesession before tracing for all animals, that is, including 7B12-treated and control-Ab–treated animals]. Linear regression analy-sis showed a significant correlation of fiber crossing the cervicalspinal cord and grasping ability. (Scale bar in A, B, D, E =0.25 mm.)



The same was true for the baseline performance in thestaircase test before MCAO (Table 2). Of note, the base-line performance (in absolute terms) of the SHR rats inthe staircase test after the 2-week training period wasinferior compared with normotensive Fischer rats, andthe interindividual variability was higher (Table 2). Be-cause of the invasive nature of MCAO, the first assess-ment of sensorimotor function could be done 4 weeksafter MCAO. At this time, all three groups showedgrasping 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 im-provement was observed up to 12 weeks after MCAO,and the deficit remained in the range of 50% of baselineperformance, which is in good agreement with previ-ously published data (Grabowski et al., 1993). In con-trast, for both 7B12-treated groups, prominent improve-ment in the staircase test was observed 7 to 12 weeksafter MCAO, approaching 70% of prelesion perfor-mance. 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 (%) 50.4 ± 10.7 48.2 ± 8.8 45.5 ± 8.2Body weight, prelesion (g) 212.8 ± 9.4 213.9 ± 6.0 215.0 ± 8.3Body weight, 12 wk (g) 310.9 ± 9.4 323.7 ± 14.6 313.1 ± 14.1Infarct volume, 24 h (�L) 238.8 ± 51.8 216.5 ± 36.9 223.8 ± 49.2

Baseline performance is expressed as the rate (%) of successful grasping attempts in the week beforepermanent middle cerebral artery occlusion (pMCAO); no significant differences were found for any of theparameters shown.

Control-Ab, group treated with mouse immunoglobulin G; 7B12, groups treated with monoclonal antibodyanti–Nogo-A; SHR, spontaneously hypertensive rats.

FIG. 6. Treatment with the anti–Nogo-A an-tibody 7B12 improved recovery of graspingability and midline crossing of corticospinalfibers after middle cerebral artery occlusion(MCAO) in spontaneously hypertensiverats. (A) Representative example for the le-sion morphology (bright areas) in the righthemisphere 24 hours after MCAO as deter-mined by T2-weighted magnetic resonanceimaging. (B) Grasped and eaten pellets withthe left forepaw in the staircase test werecounted and expressed in percent of base-line performance determined in the last testsession before lesioning. Two-way re-peated-measures analysis of varianceshowed a significant time effect (P < 0.001).The significance of time effect within treat-ment groups was isolated by the Student-Newman-Keuls method (#P < 0.05 versus4-week test). (C) After anterograde tracingof fibers from the left unlesioned sensorimo-tor cortex, more pyramidal tract fibers cross-ing the midline and terminating in the graymatter of the left cervical spinal cord (leftpart of figure) are obvious in a 7B12-treatedanimal (right) as compared with a control-Ab–infused animal (middle). (D) Quantifica-tion of midline crossing fibers in the cervicalspinal cord was done exactly as describedfor the photothrombotic cortical injury ex-periment (see Fig. 4). Error bars indicatestandard deviation. Data were analyzed us-ing nonparametric Kruskal-Wallis test. (E)Midline crossing of fibers in the cervical spi-nal cord was plotted against performance inthe last staircase session (week 12 afterMCAO) before tracing for all animals, that is,including both 7B12-treated groups andcontrol-Ab–treated animals. Linear regres-sion analysis showed a significant correla-tion between fiber crossing in the cervicalspinal cord and grasping ability.



SHR rats in the staircase test, two-way repeated-measures ANOVA showed that the differences in per-formance between the groups did not reach significance(P > 0.05); statistical analysis, however, indicated ahighly significant time effect. The significance of timeeffect for the individual groups was isolated by the Stu-dent-Newman-Keuls method and showed that, comparedwith the 4-week measurement, only both 7B12-treatedgroups significantly (P < 0.05) improved 7 to 12 weeksafter MCAO (Fig. 6B). In the group treated with a lowerdose of 7B12 (1.68 mg), performance continued to im-prove until the latest time point measured (12 weeks).From these results, it appeared that the higher doseof 7B12 accelerated the speed of recovery, whereasthe maximal level of recovery was similar with both7B12 doses.

Anterograde tracing of the pyramidal tract aftermiddle cerebral artery occlusion

Tracing was done as described for PCI, and a similarstaining pattern was obtained. At the level of the pons,the mean values for midline crossing fibers were in asimilar 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, and7B12 (5 mg) group, respectively. Although there ap-peared to be a dose-dependent increase of crossing fibersin the pons in this experiment, the differences betweenthe groups were not statistically significant. In line withthe findings after PCI, no correlation between midlinecrossing in the pons and grasping performance with theleft paw was found (not shown). In the cervical spinalcord, 1.8% ± 0.8% midline crossing of CST fibers wasfound in the control-Ab group. Treatment with 7B12triggered a dose-dependent increased of midline crossingfibers (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 of7B12 more than doubled the number of midline crossingfibers, and this increase was statistically significant(P < 0.05). As for the PCI experiment, a significantcorrelation between midline crossing fibers in the cervi-cal enlargement and grasping performance determined12 weeks after MCAO was observed (Fig. 6E).

DISCUSSION

This study showed that pump-controlled delivery of aNogo-A–specific antibody (7B12) initiated 24 hoursafter experimental stroke promoted improvement oflong-term neurologic outcome in normotensive and SHRrats. Treated animals showed an increase of midlinecrossing in the cervical spinal cord of corticospinal fibersoriginating from the unlesioned sensorimotor cortex(Fig. 7). Midline crossing correlated with grasping abil-

ity, suggesting a possible contribution of the crossingfibers for behavioral improvement.

Pump infusion of 7B12 after experimental strokeimproved neurologic outcome

The experimental protocol in this study (Fig. 2) wasdesigned to evaluate postacute treatment effects onendpoint behavioral outcome without confounding influ-ences of infarct size. To achieve this goal, lesion vol-umes were determined 24 hours after stroke by T2-weighted MRI before treatment was initiated. It haspreviously been shown that lesion volumes derived fromnoninvasive T2-weighted MRI highly correlate with vol-umes as determined by conventional histologic methods(Rudin et al., 1999).

After focal cerebral ischemia in rodents, few sensori-motor tasks uncover permanent deficits (Virley et al.,2000; Hunter et al., 2000). One of these is Montoya’sstaircase test (Montoya et al., 1991; Grabowski et al.,1993), which is an integrative sensorimotor task for theforelimbs implicated with cortical as well as subcorticalcircuitries (Whishaw et al., 1986). Combining Montoya’sstaircase test and MRI-controlled cortical ischemia, weshowed that grasping ability of the forepaw contralateralto the lesion was permanently depressed in control rats,reaching a plateau at about 50% of baseline performanceup to the latest time points investigated (9 to 12 weeks).When rats with infarct volumes MRI-matched to the con-trol group were treated with 7B12 in the postacute phaseafter stroke (24 hours), grasping ability improved signifi-cantly and reached about 70% of baseline levels. These

FIG. 7. Diagram of projections from the left forelimb motor cortexto the right cervical spinal cord. A significant increase of midlinesprouting to the left side of the cervical spinal cord was observedafter treatment with the anti–Nogo-A antibody 7B12 both afterphotothrombotic cortical injury in normotensive Fischer rats andafter middle cerebral artery occlusion in spontaneously hyperten-sive rats. BDA, biotinylated dextran amine.



findings agree with a recent study showing improvementof skilled forelimb movements after MCAO by IN-1 hy-bridoma treatment initiated at the time of MCAO andunder immunosuppression (Papadopoulos et al., 2002).

Beneficial effects with 7B12 in the present study wereobserved both in healthy normotensive rats and in SHR,which develop vascular abnormalities, have reduced col-lateral cerebral blood flow, and show premature mortal-ity (Barone et al., 1993). Thus, these results indicate thatanti–Nogo-A antibodies could be a promising rehabili-tative treatment approach for human stroke with a pro-longed time-to-treatment window. Intraventricular ad-ministration of antibodies, however, seems not to besuitable for patients who have had stroke. Consideringdisturbances of the blood–brain barrier after stroke(Huber et al., 2001), it will be important to explorewhether peripheral administration can be achieved.

For spinal cord injury and head trauma, the demon-stration of efficacy after pump infusion of 7B12 has im-mediate clinical relevance. In previous studies showingbehavioral recovery after CNS injury, the antibody IN-1was secreted by implanted hybridoma cells under con-comitant immunosuppressive treatment with cyclosporin(Z’Graggen et al., 1998; Papadopoulos et al., 2002;Merkler et al., 2001; Raineteau et al., 2001), whichwould not be applicable to patients. Pump-controlled in-trathecal drug administration, however, is an establishedclinical method that is routinely used after spinal cordinjury (Schwab, 2002) and head trauma (Stempien andTsai, 2000). Intraventricularly infused antibodies of theIgG isotype are cleared rapidly from the cerebrospinalfluid via an Fc-receptor-mediated mechanism and with ahalf-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 werefound at high levels throughout the brain including thecervical spinal cord, indicating a steady-state concentra-tion gradient sufficient to drive penetration. The antibod-ies were still detectable 2 weeks after termination ofinfusion in brain structures for which plastic changeshave been previously reported after cortical injury, suchas the perilesion cortex (Carmichael et al., 2001), thedorsolateral striatum (Kartje et al., 1999; Papadopouloset al., 2002), and the cerebral peduncle (Wenk et al.,1999). Therefore, it seems that intraventricular infusionof anti–Nogo-A antibodies could be utilized for clinicalindications where this route of administration is feasible.

Corticospinal plasticity was enhanced after 7B12treatment and correlated with behavioral outcome

Neuroanatomically, 7B12 treatment more thandoubled the number of midline crossing CST fibers inthe cervical spinal cord originating in the unlesioned sen-sorimotor cortex in normotensive and SHR rats (Fig. 6).A similar extent of compensatory fiber growth from the

CST was found after unilateral CST transection caudalfrom the lesion after IN-1 hybridoma treatment (Thall-mair et al., 1998). Corticospinal projections across themidline have been also described after sensorimotor cor-tex lesion in neonatal rats (Rouiller et al., 1991), wherethe degree of spontaneous behavioral recovery aftercomparable cortical lesions is much better comparedwith adult animals. In previous studies, plasticity aftercortical injury and IN-1 hybridoma treatment has notbeen investigated in the spinal cord, but interhemisphericfiber outgrowth in corticostriatal (Kartje et al., 1999),corticorubral (Wenk et al., 1999), and corticopontine(Wenk et al., 1999) pathways is well documented. Cor-ticostriatal plasticity was found also after MCAO inblack-hooded rats (Papadopoulos et al., 2002). In thepresent study, the number of corticopontine fibers cross-ing the midline was relatively high even in control-Ab–treated animals, and there was no increase in the 7B12-treated rats. It is important to note that even untreatedrats displayed corticospinal fiber crossing in the spinalcord (1% to 2%), although to a significantly lesser degreethan 7B12-treated animals. Similar observations weremade for interhemispheric corticostriatal fiber outgrowthafter cortical ablation (Kartje et al., 1999) or thermoco-agulation (Napieralski et al., 1996). The correlationanalysis of fiber crossing with endpoint forepaw functionfor all animals irrespective of treatment showed a sig-nificant correlation for both normotensive and SHR rats.It therefore seems that lesion-induced fiber outgrowth inthe cervical spinal cord possibly contributed to the be-havioral improvement after PCI and MCAO in SHR rats.Importantly, treatment with 7B12 did not induce a quali-tatively new fiber outgrowth pattern, but augmented anendogenous lesion-induced plastic capacity of the adultCNS. Without lesion, it has been shown that anti–Nogo-A antibodies induced only short-lasting transientsprouting of Purkinje axons in the adult cerebellum(Buffo et al., 2000).

The finding that the correlation coefficients for corti-cospinal collateral sprouting and behavioral improve-ment were—although significant—in a moderate rangesuggests that other brain areas and systems also are in-volved in the sensorimotor task and that such areas andsystems may also be affected by the 7B12 antibody.From previous studies with the antibody IN-1 (seeabove), it appears that corticostriatal and corticorubraltracts could be involved (Kartje et al., 1999; Papadopou-los et al., 2002). Spontaneous although limited improve-ment of deficits after experimental and clinical stroke iswell recognized, and functional takeover in the unle-sioned hemisphere has been implicated as a mechanismfor recovery after stroke by functional MRI studies.These studies showed that electrical forelimb (Dijkhui-zen et al., 2001) or hind-limb stimulation (Abo et al.,2001) resulted in activation of normally silent brain



regions ipsilateral to stimulation. Noninvasive imagingtechniques such as positron emission tomography, func-tional MRI, transcranial magnetic simulation, and mag-netoencephalography have been used to investigate func-tional organization of the cortex after stroke in humans(Nudo et al., 2001; Pineiro et al., 2001). These studiesprovided evidence that recovery of hand function wasassociated with increased bilateral activation of brain ar-eas. Experimental and clinical studies also showed thatreorganization in the ipsilateral injured cortex is a veryimportant mechanism for recovery after stroke (Abo etal., 2001; Carmichael et al., 2001; Dijkhuizen et al.,2002; Nudo et al., 1996; Pineiro et al., 2001). Plasticchanges in the lesioned hemisphere are also likely to playan important role in behavioral improvement in ourexperimental paradigms. Whether and how anti–Nogo-A antibody treatment influences plasticity and sprout-ing in the injured hemisphere will be addressed in fu-ture studies.

Underlying mechanisms for compensatory sproutingin the spinal cord

The Nogo-A–specific domain inhibits neurite out-growth and causes growth cone collapse in neurons viaan elusive receptor (Oertle et al., 2002) that is distinctfrom the Nogo-66 receptor NgR (Fournier et al., 2001),and involves Rho-mediated downstream signaling to thecytoskeleton (De Marco et al., 2002). Creation of a per-missive environment facilitating fiber outgrowth there-fore requires the local presence of the neutralizing anti-body. In our study the presence of 7B12 in the spinalcord after intraventricular infusion can explain the effecton fiber outgrowth from the unlesioned pyramidal tract.In addition, tonic Nogo-A–dependent inhibition ofgrowth-related genes in the cell bodies of cortical neu-rons may be released by Nogo-A neutralization with7B12 in cortical parenchyma. At present, it cannot beexcluded that 7B12 (or other anti–Nogo-A antibodies) donot only neutralize Nogo-A by binding to extracellularepitopes, but that binding results in an internalization andthus downregulation of Nogo-A. In vitro and in vivostudies have shown that anti–Nogo-A antibody IN-1 in-duced the expression of growth-related genes, such asc-jun, junD, synaptophysin, and GAP-43 (Zagrebelsky etal., 1998; Huber and Schwab, 2000).

In summary, the results of this study suggest that anti–Nogo-A antibodies bear potential as a new rehabilitativetreatment for ischemic stroke with a time-to-treatmentwindow of at least 24 hours. Although the precisemechanisms of the beneficial effects of anti–Nogo-A an-tibodies in the presented experimental stroke modelsneed to be further explored, the results indicate thatcompensatory growth of corticofugal fibers could beinvolved.

Acknowledgments: The authors thank Pascale Brebbia,Francesco D’Amato, Thomas Hafner, and Willi Theilkas fortheir excellent technical help.

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