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Behavioral recovery and anatomical plasticity in adult rats after cortical lesion and treatment with monoclonal antibody IN-1
Behavioural Brain Research 152 (2004) 315–325
Research report
Behavioral recovery and anatomical plasticity in adult rats after cortical lesion and treatment with monoclonal antibody IN-1 April J. Emerick a,b,∗ , Gwendolyn L. Kartje a,b b
a Research and Neurology Service, Edward Hines Veterans Affairs Hospital, Hines, IL 60141, USA Neuroscience Program, Departments of Neurology and Cell Biology, Neurobiology and Anatomy, Loyola University Medical Center, Maywood, IL, USA
Received 30 June 2003; received in revised form 8 September 2003; accepted 10 October 2003 Available online 9 January 2004
Abstract We have previously reported that monoclonal antibody (mAb) IN-1 treatment after ischemic infarct in adult rats results in significant recovery of skilled forelimb use. Such recovery was correlated with axonal outgrowth from the intact, opposite motor cortex into deafferented subcortical motor areas. In the present study, we investigated the effects of mAb IN-1 treatment after adult sensorimotor cortex (SMC) aspiration lesion on behavioral recovery and neuroanatomical plasticity in the corticospinal tract. Adult rats underwent unilateral SMC aspiration lesion and treatment with either mAb IN-1 or a control Ab, or no treatment. Animals were then tested over a 6-week period in the skilled forelimb use task and the skilled ladder rung walking task. We found that animals treated with mAb IN-1 after SMC lesion fully recovered the use of forelimb reaching, but showed no improvement in digit grasping as tested in the skilled forelimb use task. The mAb IN-1 treatment group was also significantly improved as compared to control groups in the skilled ladder rung walking test. Furthermore, neuroanatomical tracing revealed a significant increase in the corticospinal projections into the deafferented motor areas of the spinal cord after mAb IN-1 treatment. These results indicate that treatment with mAb IN-1 after cortical aspiration lesion induces remodeling of motor pathways resulting in recovery in only certain behavioral tasks, suggesting that the cause of brain damage influences behavioral recovery after mAb IN-1 treatment. © 2003 Elsevier B.V. All rights reserved. Keywords: Motor cortex; Nogo-A; Corticospinal tract; Cortical injury; Plasticity; Behavioral recovery
1. Introduction Functional recovery after adult CNS lesions is often incomplete and is limited in part by the failure of axons to regenerate after injury. Barriers to axonal outgrowth include Nogo-A [10,13,17,37,40], an inhibitory protein expressed in oligodendrocyte myelin sheaths and compact myelin [18]. The application of the monoclonal antibody (mAb) IN-1 neutralizes inhibitory effects of Nogo-A [10] and induces transient axonal outgrowth in normal adult Purkinje cells [5] and corticospinal neurons [1]. Immunochemical assays confirm that Nogo-A is the antigen for mAb IN-1 [12]. Treatment with mAb IN-1 and other antibodies raised against Nogo-A promotes behavioral recovery in adult rats after ischemic stroke [35,47,48], pyramidotomy [38,40,41] and spinal cord injury [3,4,29]. Furthermore, pharmacological blockade of a receptor to Nogo-66, one of the inhibitory domains of Nogo-A, enhances locomotor recovery after spinal hemisection [14,26]. Behavioral recovery in these ∗
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studies was observed in parallel with the remodeling of neuroanatomical pathways and the reinnervation of subcortical motor areas affected by the lesion. Neuronal plasticity has often been suggested as an underlying substrate for the spontaneous recovery observed after CNS lesions in the young (for review, see [21]). Studies have shown that after unilateral cortical ablation in the neonate, the intact, opposite motor cortex projects in a target-specific pattern to the deafferented striatum [22,23], red nucleus [25,32,33], basilar pontine nuclei [9,20,25] and spinal cord gray [2,8,16,24,25,39,46]. While such growth is limited in the adult, we have reported that rats treated with mAb IN-1 after adult SMC aspiration lesions form new cortico-efferent connections from the intact, opposite motor cortex to deafferented subcortical targets including the striatum [19], red nucleus and basilar pontine nuclei [44]. However, the effects of mAb IN-1 on recovery of motor function after adult SMC aspiration lesions have not yet been examined. In using the aspiration lesion model, damage can be restricted to areas of the cortex concerned with skilled motor behaviors. Specifically, functional recovery was assessed by the utilization of proximal muscle groups during the skilled reach-
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ing task and distal muscle groups during the digit grasping test, while overall limb coordination was measured with the skilled ladder rung-walking test. We further investigated the corticospinal connections from the non-injured cortex to the ␣-motoneurons that innervate such muscle groups and whether new growth facilitates behavioral recovery. Based on previous reports of improved behavioral outcome and anatomical plasticity, we hypothesized that mAb IN-1 treatment would result in behavioral recovery in adults with cortical aspiration lesions and also promote neurite outgrowth in the corticospinal tract. Interestingly, we found that treatment with mAb IN-1 after adult cortical lesion resulted in variable amounts of recovery, depending upon the task, i.e., recovery in both skilled forelimb reaching and skilled ladder rung walking, and no recovery in skilled digit grasping. Anatomical analysis demonstrated a significant increase in the number of corticospinal axons projecting into the deafferented motor areas of the spinal cord, indicating a probable role for the intact motor cortex in behavioral recovery after mAb IN-1 therapy.
2. Materials and methods 2.1. Animals All animal procedures were approved by the Joint Institutional Animal Care and Use Committee of Loyola University and Hines Veterans Affairs Hospital and conform to the National Institutes of Health guidelines. Animals were housed three to a cage and maintained in a temperature and humidity controlled room on a 12-h light:12-h dark cycle. All testing was performed during the light portion of the cycle. Forty male Long Evans black-hooded rats at 8–10 weeks of age were divided into three experimental groups: (1) SMC lesion-only (n = 16); (2) SMC lesion + control Ab (n = 10); (3) SMC lesion + mAb IN-1 (n = 14). Animals were number-coded and investigators were blind to treatment groups. 2.2. Behavioral tests 2.2.1. Skilled forelimb use task Animals were reduced to ∼95% of their original body weight for motivational purposes and maintained on a foodrestricted diet throughout the training and testing period. As previously described [35], animals were trained and tested in a transparent Plexiglas chamber (30 cm × 36 cm× 30 cm) with a small window (1.5 cm × 3 cm) opening on the left or right side of the front wall at the level of the chamber floor. A shelf was attached outside the box and beneath the window for placement of small sucrose pellets (45 mg; Bilaney Consultants, Frenchtown, NJ) at a distance of 1.5 cm from the window. Animals were trained daily, Monday through Friday, at the same time of day over a 2-week period. During the training period, animals learned
to use the forelimb to reach through the window and grasp the pellets that were placed in succession onto the shelf (see Fig. 1A). Upon grasping the pellets, animals would then have to retract the forelimb into the box and bring the pellet to the mouth. When animals began to show a limb preference (greater than 70% of pellets obtained using either the left or right forelimb), the training was conducted through the window that favored the use of the preferred forelimb. Animals were then trained for 2 weeks to reach and grasp 20 pellets using the preferred forelimb within 5 min. The average scores and time from the last three training sessions before surgery were calculated for each animal and recorded as the preoperative baseline scores. For consistency in data analysis, only animals achieving baseline scores of 16 or greater in grasping were included in the study (animals that did not meet this criterion were still included in the skilled ladder rung walking test). All animals were then randomly assigned to experimental groups. Beginning on the first day after cortical lesion surgery as described below, animals were tested Monday through Friday for 6 weeks in a 20-pellet use task. Motor recovery of the lesion-impaired forelimb was measured in three ways: forelimb reach, digit grasp, and time. The forelimb reach score indicates the number of times an animal successfully extended its forelimb through the window in an attempt to grasp a pellet (only one reach per pellet was measured, yielding a maximum score of 20). The digit grasp score indicates the number of pellets an animal picked up, carried into the box, and placed into its mouth. The time taken to retrieve 20 pellets was recorded in each testing session. Testing sessions were video recorded for further analysis. 2.2.2. Skilled ladder rung walking The ladder walk apparatus consisted of a 1-m long wooden-rung walk way with a varying distance of 1–2 cm between rungs (described in [30]) (see Fig. 1B). Animals were acclimated to the testing apparatus during three separate sessions within a 2-week period before cortical lesion surgery. Animals traversed the walkway freely and without reinforcement. During the third session, animals were video-recorded as they traversed the walkway three times. Tapes were later analyzed to assess preoperative baseline scores. Animals were tested on the first day after surgery and then once a week for the next 6 weeks. In a single testing session, the animals crossed the walkway three times and received a score for the number of forelimb foot faults per 10 steps. Only the lesion-affected forelimb was analyzed and a foot fault was characterized as a total miss or slip from the rung or a misplacement of the paw on the rung, i.e., using the wrist or digits instead of the plantar surface. 2.2.3. Statistical analysis of behavioral data The behavioral data was analyzed using GraphPad Prism 2.01 (GraphPad Software, Inc.). A one-way ANOVA was used to compare the mean values across all groups in a single testing session. Differences in reach, grasp, and time were
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Fig. 1. Behavioral tests. Deficits in reaching and digit grasping were measured in the skilled forelimb use test (A). Successful reaching requires aiming and extension (as demonstrated by a representative animal during training in the film sequence above) of the forelimb through a small window at the front of the testing box. Successful grasping is accomplished when the digits close around the pellet and the animal carries the pellet in its paw to the mouth. The target pellet can be seen on the shelf beneath the paw. Deficits in forelimb placement and accuracy were measured using the skilled ladder rung walking test (B). The number of lesion-impaired forelimb footfaults was measured per 10 steps across the runway. Footfaults include a slip from the rung (arrow) as depicted in a representative animal.
further analyzed with the Bonferroni’s multiple comparison post-test. For comparison within an experimental group, i.e., postoperative and week 6 scores, an unpaired Student’s t-test was used. To measure the percent recovery in the skilled ladder rung walking task, weekly postoperative scores were normalized to the day one postoperative score in each animal. A P value less than or equal to 0.05 was considered significant. All data are presented as mean values ± S.E.M.
taxic frame, and the scalp was incised along the midline. With bregma as a landmark, the skull was opened to expose either the left or right sensorimotor cortex (SMC), and using suction the forelimb motor cortex was gently aspirated within a specific boundary: 4.0 mm rostral; 2.0 mm caudal; 1.0 mm medial; 4.0 mm lateral to bregma [34]. Cortical aspiration lesions were made in the SMC contralateral to the preferred forelimb as determined in the earlier training sessions.
2.3. SMC aspiration lesion
2.4. Antibody application
Animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) supplemented with ketamine (40 mg/kg, i.m.). Anesthetized animals were then secured in a stereo-
Immediately after cortical aspiration lesion and while animals were still anesthetized, mouse hybridoma cells (1 × 105 cells in 1 l) secreting either mAb IN-1 or control
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Ab (anti-horseradish peroxidase) were delivered by Hamilton syringe into the hippocampus posterior to the lesion site (4.0 mm caudal, 5.0 mm lateral and 5.0 mm ventral to bregma). Animals were monitored after surgery until they were mobile and then returned to standard housing conditions. All animals, including controls, received daily injections of Cyclosporin-A (10 mg/kg, i.p.) 1 day before and 7 days after surgery to suppress host rejection of the mouse hybridoma cells. 2.5. Biotin dextran amine neuroanatomical tracing After 6 weeks of behavioral testing, animals were re-anesthetized as described above and placed in a stereotaxic frame. The bone overlaying the motor cortex opposite the SMC lesion was removed, and a 10% biotinylated dextran amine (BDA) solution in 0.01 M phosphate buffer was pressure injected (1 l per injection at a rate of 0.2 l per 1 min) using a Hamilton syringe at four sites within the motor cortex. Animals were returned to standard housing conditions for 2 weeks and then were sacrificed by sodium pentobarbital overdose (200 mg/kg, i.p.) and transcardial perfusion with saline supplemented with 10,000 IU heparin and 0.25% NaNO2 , followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The brains and spinal cords were removed and postfixed in 4% paraformaldehyde at 4 ◦ C overnight followed by 30% sucrose for cryoprotection at 4 ◦ C overnight. Tissue was then embedded in an albumin/sucrose compound (as described in [15]) and frozen in −50 ◦ C isopentane. Tissue was cut in the coronal plane on a cryostat in 50 m serial sections and collected on glass slides for semi-free floating processing with avidin– biotin–peroxidase complex (ABC kit elite, Vector Labs) for visualization of BDA. Alternate brain sections were collected on gelatin-coated glass slides and counterstained with toluidene blue for visualization of cell bodies.
2.6. Neuroanatomical tracing analysis The CST projections from the opposite, intact hemisphere to the spinal cord were evaluated at levels C6 through C8 . Using standard anatomical landmarks, five comparable sections per animal were analyzed under light microscopy using NIH Image Software 5.1 to evaluate BDA-positive labeled axons in the ventral motor horn. Within the motor horn, we evaluated the medial and lateral ␣-motoneuron cell columns (see Figs. 2 and 3). Using a square counting frame, BDA-positive fibers that intersected a total line length of 0.26 mm were counted in each cell column, both ipsilateral and contralateral to the injection site. In order to normalize the results and to take into account tracer variability between animals, the number of fibers on the ipsilateral (lesion-affected) was divided by the contralateral (normal) count on each section to provide a ratio as previously described [1,19,41,47]. The mean ratio for each animal was averaged for each group and was expressed as a percent of the contralateral fibers and compared between experimental groups. These data were statistically analyzed using GraphPad Prism 2.01 (GraphPad Software, Inc.). The Mann– Whitney test was used to compare mean values between groups. A P value less than or equal to 0.05 was considered significant. All data are presented as mean values ± S.E.M. 3. Results 3.1. SMC lesion cavity Gross inspection of brains from all animals showed that SMC lesion size and location was comparable among groups. The area of cortical damage as identified using Paxinos and Watson coordinates [36] included +4.2 mm rostral through −3.3 mm caudal and extended from the midline
Fig. 2. A schematic diagram illustrating the experimental procedures. The placement of the SMC aspiration lesion (shaded) and the implantation site of antibody-secreting hybridoma cells is shown in the left hemisphere. The biotinylated dextran amine (BDA) tracer injection site is contralateral to the aspiration lesion in the non-injured cortex. The pathway of the CST is depicted arising from the right forelimb sensorimotor cortex (SMC), crossing the midline (arrow) and projecting into the spinal cord. (Modified from Papadopoulos et al. [35].)
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Fig. 3. Neuroanatomical plasticity was examined in the cervical spinal cord. The BDA-positive labeled CST fibers projecting from the non-injured cortex were counted in the lateral α-motoneuron cell column (box a, a ) and medial α-motoneuron cell column (box b, b ) on both ipsi- and contralateral sides.
through 4.5 mm lateral, and did not differ in animals treated with mAb IN-1 as compared to lesion-only and control Ab treatment (Fig. 4). 3.2. Behavioral recovery in animals treated with mAb IN-1 3.2.1. Skilled forelimb use Functional recovery in the impaired forelimb was evaluated using the skilled forelimb reach test, and performance
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was measured in forelimb reach, time, and digit grasp. In the forelimb reach test (Fig. 5A), preoperative baseline measurements showed that animals in all experimental groups scored between 19 and 20 in forelimb reach (i.e., extension of the forelimb through the window), indicating no significant difference between groups (P > 0.05, one-way ANOVA). At postoperative day 1, animals from all treatment groups showed limited mobility in the impaired forelimb and most failed to use the limb in the reaching task. All groups showed minimal improvement at the end of postoperative week 1 (data not shown). By postoperative week 2, animals from all treatment groups were increasingly using the impaired forelimb and all groups were significantly improved as compared to the corresponding postoperative deficits measured at day 1 (P < 0.05, unpaired Student’s t-test). However, we observed that most animals continued to have difficulty with aiming and extending the forelimb through the window. By week 6, the SMC lesion-only and lesion plus control antibody groups did not show further improvement from weeks 2 or 3 (data not shown) and their average score remained significantly less than their preoperative baseline scores (P < 0.001, unpaired Student’s t-test). Furthermore, there was no significant difference between the SMC lesion-only group and the SMC lesion plus control Ab treatment group at 6 weeks postoperatively (P > 0.05, one way ANOVA). In marked contrast, animals treated with mAb IN-1 following SMC lesion showed continued improvement through the testing period and a remarkable recovery of skilled forelimb reach. Beginning at week 4, animals in the SMC plus mAb IN-1 treatment group showed significant improvement in forelimb reaching as compared to SMC lesion-only (P < 0.001, one-way ANOVA) and SMC lesion plus control Ab (P < 0.05, one-way ANOVA) treatment groups. This improvement was sustained in the mAb IN-1 treatment group through week 5 (data not shown) and at 6 weeks the forelimb reach scores were not statistically different from the
Fig. 4. Cross-sectional reconstructions of the maximum (black) and minimum (gray) extent of the lesion cavities did not appear to differ among the experimental groups. Only the lesion-side hemisphere is displayed. Coordinates represent distance in millimeters from bregma.
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Fig. 5. Treatment with mAb IN-1 leads to behavioral recovery in the skilled forelimb use test as measured by the number of successful forelimb reaches using the impaired forelimb in a 20-pellet test (A). At 4 weeks following surgery, animals treated with mAb IN-1 were significantly improved compared to control treatment animals. This recovery was sustained through postoperative week 6 at which time point animals treated with mAb IN-1 showed no statistical difference from preoperative baseline scores (P > 0.05, unpaired t-test). SMC lesion-only and SMC lesion + control AB were not significantly different throughout the course of the study (P > 0.05). Treatment with mAb IN-1 improves the time it takes animals to complete the 20-pellet test (B) Time (seconds) to reach was significantly faster in the mAb IN-1 treatment group as compared to the SMC lesion-only and SMC lesion plus control Ab groups at postoperative week 6 (P < 0.001). Digit grasping abilities showed little or no recovery in all treatment groups (C). At postoperative week 6, animals treated with mAb IN-1 were not significantly different from SMC lesion-only or SMC lesion plus control Ab animals (P > 0.05). Data shown are mean ± S.E.M. Asterisks indicate significance compared to SMC lesion-only and SMC lesion + control Ab treatment groups: ∗∗ P < 0.01; ∗∗∗ P < 0.001, one-way ANOVA, followed by Bonferroni’s Multiple Comparison for post hoc evaluation of group differences (dpo: days postoperative; wpo: weeks postoperative).
preoperative baseline scores (P > 0.05, unpaired Student’s t-test). The time to retrieve 20 pellets in the skilled forelimb use task showed no difference in preoperative baseline measurements among the experimental groups (P > 0.05, one-way ANOVA), with the average time to complete the task less than 69 s (Fig. 5B). On postoperative day 1, animals from all experimental groups were unable to complete the task within the 5 min allotted, with no significant difference between groups (P > 0.05, one-way ANOVA). By week 6, the control treatment groups were not statistically different from one another (P > 0.05, one-way ANOVA) and remained significantly slower at performing the task as compared to preoperative baseline scores (P < 0.001, unpaired Student’s t-test). In comparison, animals treated with mAb IN-1 after SMC lesion showed a considerable improvement in the time required to complete the task. At postoperative week 6, animals treated with mAb IN-1 were significantly faster than animals from the control groups (P < 0.001, one-way ANOVA). Recovery was limited, however, and animals treated with mAb IN-1 after SMC lesion remained slower than their preoperative baseline measurements (P < 0.001, unpaired Student’s t-test). Preoperative baseline scores for digit grasp indicate animals were highly successful in grasping the sucrose pellets (Fig. 5C). On postoperative day 1, digit grasp scores were extremely low in all groups, reflective of the motor deficits in forelimb reaching. By week 2, animals began to show some improvement, but clearly at week 6 there was just a modest change in the digit grasp scores. Most animals showed a lack of fine digit control and could not flex the digits around the small pellets. Some animals modified their retrieval tactics and would swat at or scoop up a pellet, but in most cases, would fumble the pellet out of the paw before bringing it to the mouth. Animals treated with mAb IN-1 after SMC lesion were not statistically different from control treatment groups (P > 0.05) nor did they recover to their preoperative baseline scores. 3.2.2. Skilled ladder rung walking Accurate placement of the impaired forelimb was examined on the skilled ladder rung walking test. Similar to our earlier findings in normal animals performing this test [49], preoperative baseline measurements showed that animals from all experimental groups were able to cross the ladder with fewer than 1.5 foot faults per 10 steps (data not shown). Following SMC lesion, the average number of foot faults had tripled from preoperative baseline values in all experimental groups. We assessed recovery as a percent of preoperative baseline scores in each individual animal (Fig. 6). By week 2, all experimental groups showed slight improvement, but were not significantly different from postoperative day 1 (P > 0.05, unpaired Student’s t-test). There was no statistical difference in the percent recovery between the control groups throughout the experiment (P > 0.05, one-way ANOVA) and within each control group there was no signif-
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Fig. 6. Treatment with mAb IN-1 improves performance in the skilled ladder rung walking test. The percent recovery was significantly higher in the SMC lesion + mAb IN-1 treatment group compared to both SMC lesion-only and SMC lesion + control Ab groups at postoperative weeks 4 and 6 (∗ P < 0.05, unpaired Student’s t-test). Animals in the SMC lesion+mAb IN-1 treatment group showed significant recovery at 6 weeks compared to day 1 postoperative values (∗∗ P < 0.05, unpaired Student’s t-test), whereas the percent recovery did not improve over the 6 weeks in either of the control experimental groups.
icant improvement as compared to postoperative day 1 (P > 0.05, unpaired Student’s t-test). Conversely, animals treated with mAb IN-1 showed significant improvement at week 4 when compared to the control groups (P < 0.05, one-way ANOVA). This improvement was sustained through week 6, at which point animals treated with mAb IN-1 also showed significant recovery when compared to postoperative day 1 (P < 0.05, unpaired Student’s t-test). 3.3. Neuroanatomical tracing of the CST The intact, opposite SMC was injected with BDA tracer to identify the CST and its projections into the cervical spinal cord gray matter. The dorsal funiculus contralateral to the BDA-injected SMC showed dense labeling of the CST through all spinal cord levels in animals from all experimental groups. The pattern of CST axons terminating contralaterally in Rexed’s lamina I through X was consistent with previous neuroanatomical studies [1,24,27]. We analyzed the percent of CST fibers projecting into the lateral and medial ␣-motoneuron columns of the ventral motor horn ipsilateral to the injection side. The ␣-motoneurons of these two regions typically innervate the distal and proximal muscles of the forearm, respectively [28]. Since there was no apparent difference between SMC lesion-only and SMC lesion + control Ab, these data were pooled together as SMC lesion control. The percent of CST fibers projecting into the lateral ␣-motoneuron cell column was not significantly different between the mAb IN-1 treated animals and the lesion control group (Fig. 7A) (P > 0.05, Mann–Whitney). When we assessed the medial ␣-motoneuron cell column, however, we found a significant increase in the percentage of CST fibers as compared to the SMC lesion control group (Fig. 7B and C) (P < 0.05, Mann–Whitney). Thus, the mAb IN-1 treatment group had more CST connections to ␣-motoneurons that facilitate
movements in the impaired forelimb related to proximal (i.e., shoulder) muscles but not distal (i.e., digit) muscles.
4. Discussion This study demonstrates that intracerebral application of mAb IN-1 following SMC aspiration lesion results in recovery of specific skilled forelimb movements. Animals treated with mAb IN-1 achieved preoperative baseline scores in skilled forelimb reaching and were significantly better than control groups. However, these animals showed no recovery in digit grasping and were no different from control animals. In addition, treatment with mAb IN-1 resulted in significant improvement in the skilled ladder rung walking test. The recovery of animals treated with mAb IN-1 after SMC aspiration lesion correlates with increased plasticity of corticospinal projections in the spinal cord to areas that facilitate proximal forelimb movement. 4.1. Treatment with mAb IN-1 results in significant motor recovery after SMC lesion Skilled forelimb reaching is a highly conserved action pattern that is organized into a series of forelimb movements mediated by the CST among other motor pathways [30]. Accordingly, damage to the motor cortex results in functional deficits from which animals may never completely recover [7,45,46]. In agreement with these previous studies, we found that skilled reaching movements were severely impaired in the forelimb contralateral to the SMC aspiration lesion. Animals from the SMC lesion-only and lesion+control Ab groups often retained severe motor deficits in precise forelimb motor control and were thus unsuccessful in forelimb reaching and grasping. Animals treated with mAb IN-1, on the other hand, displayed a remarkable degree of recov-
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Fig. 7. Treatment with mAb IN-1 after SMC lesion results in an increase in CST fibers projecting to the ␣-motoneurons that innervate proximal muscles of the impaired forelimb. The percentage of CST fibers projecting to the ipsilateral (lesion-affected) lateral ␣-motoneuron cell column did not change after SMC lesion and treatment with mAb IN-1 as compared to SMC lesion control (pooled data from lesion-only and lesion + control Ab) (P > 0.05, Mann–Whitney) (A). However, there was a significant increase in the percentage of CST fibers projecting to the medial ␣-motoneuron cell column (B) in animals treated with mAb IN-1 as compared to lesion-control animals. Error bars indicate mean ± S.E.M. Asterisk (∗) indicates P < 0.05, Mann–Whitney. (C) Photomicrographs of representative cross-sections through the medial ␣-motoneuron cell column in the lesion-affected side of the spinal cord. BDA-positive CST fibers (black) were identified in all groups, yet the mAb IN-1 treatment group was characterized by increased CST fibers that were often located adjacent to ␣-motoneurons (∗).
ery in the use of proximal limb muscles as indicated by the successful performance in forelimb reaching and the time to reach for 20 pellets. Rodents depend on the ascending and descending pathways required for locomotion during the skilled forelimb ladder rung walking test. Balance, fine-adjusted muscle control, and limb coordination are all necessary for the animal to cross the rungs accurately [31]. We found that following SMC aspiration lesion animals had forelimb placement deficits, and that most errors resulted from incorrect aiming of the lesion-impaired forelimb or improper positioning of the forelimb on the rung with either the wrist or digits. The SMC aspiration lesions resulted in chronic impairments in the control of limb and muscle movements in the lesion-only and lesion + control Ab treatment groups, which is consistent with previous reports of forelimb placing deficits observed 3–6 months after unilateral motor cortex lesion or pyramidotomy [31]. The mAb IN-1 treatment
group, however, showed significant recovery from postoperative deficits, and, although they did not reach preoperative baseline values by postoperative week 6, there was a trend for continued improvement. Animals from all experimental groups showed chronic impairments in fine digit motor control and had limited success in digit grasping after SMC lesions. Treatment with mAb IN-1 alone did not ameliorate this lesion effect. The failure of animals treated with mAb IN-1 to recover in digit grasping suggests that this task is inherently more difficult than the reaching task, and a longer survival period or additional training might be necessary for improvement. Likewise, skilled digit use might require more CST involvement than skilled reaching, and the extensive damage to the motor cortex could contribute to enduring deficits. Interestingly, the lack of recovery in digit grasping conflicts with previous reports in animals treated with mAb IN-1 following unilateral ischemic stroke [35] or CST le-
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sion [41,49]. In those studies, mAb IN-1 treatment led to significant recovery in fine digit motor control, and animals were highly successful in grasping and retrieving the small food pellets. Similarly, intraventricular infusion of a purified monoclonal anti-Nogo-A antibody following unilateral ischemic stroke also improved digit-grasping ability during the staircase task [47,48]. In our study, animals treated with mAb IN-1 after SMC aspiration lesion did not even show a trend for improvement at postoperative week 6. This discrepancy in results indicates that behavioral outcome after CNS injury and mAb IN-1 treatment may depend in part on the lesion type (i.e., ischemic versus aspiration). In our experience there is a much greater area of damage specifically to the motor cortex after aspiration lesion than after ischemic insult. Furthermore, the cellular response varies according to the lesion type; for instance, ischemic insult to the cerebral cortex induces spontaneous axonal growth [42] concomitantly with synchronized neuronal EEG patterns [6]. On the contrary, aspiration lesion alone shows limited axonal outgrowth and lacks any EEG activity related to axonal sprouting. Likewise, following cortical aspiration lesion there is an absence of dendritic plasticity in layer V pyramidal neurons [43], which is also suggested to contribute to enduring forelimb impairments.
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ment with either mAb IN-1 or other anti-Nogo-A antibodies appears to sustain such neuronal outgrowth or plasticity into denervated motor areas and concomitantly improves behavioral outcome after ischemic stroke [35,47,48], pyramidotomy [41,49], and spinal cord injury [3,29]. While the availability of antibody is limited to 7–10 days, we are still investigating the down-stream molecular effects of blocking Nogo-A. Indeed, the application of mAb IN-1 after CST injury significantly enhances the expression of growth-cone guidance molecules essential for target pathfinding [1]. These changes in gene expression might also contribute to the long-lasting anatomical reorganization and behavioral recovery that is observed after damage to the motor cortex. Here we show that after a focal aspiration lesion to the motor cortex and mAb IN-1 treatment corticospinal plasticity does occur and appears to correspond to behavioral recovery on specific tasks related to the re-organized pathway. However, mAb IN-1 treatment does not ameliorate all motor deficits, and in particular, digit grasping was not improved. In conclusion, lesion type should be an important consideration when evaluating treatment paradigms in recovery after CNS injury.
Acknowledgements 4.2. Neuroanatomical remodeling of the CST parallels behavioral recovery After SMC lesion, motoneurons in the spinal cord as well as several brain stem targets have lost their connection to the motor cortex. Here we show anatomical evidence that following aspiration lesion and treatment with mAb IN-1, the motor cortex opposite to the damaged hemisphere connects to this deafferented spinal cord through the intact CST. Treatment with mAb IN-1 resulted in an increase in CST projections into the medial ␣-motoneuron cell column but not to the lateral cell column. Given the proximity of the nuclei, it is doubtful that the distance was too great to allow for sprouting into the lateral cell column. Needless to say, the lack of recovery in digit grasping might be explained in part by the limited plasticity observed in this area of the spinal cord, which tends to innervate the distal muscles of the forelimb. On the other hand, the ␣-motoneurons of the medial cell column innervate the proximal muscles such as shoulder and elbow [30] that function in forelimb extension, and plasticity in this region was correlated with recovery in forelimb reaching. In agreement with these findings, electrophysiological studies in animals treated with mAb IN-1 after aspiration lesion confirmed that the opposite, non-injured cortex increases motor output to the lesion-affected forelimb resulting primarily in shoulder and elbow movements [11]. In the absence of injury, the application of mAb IN-1 induces transient neuronal outgrowth in adult cerebellar Purkinje cells [5] and corticospinal neurons in parallel with the upregulation of growth factors and growth-related proteins necessary for axonal outgrowth [1]. After injury, treat-
This work was supported by the Department of Veterans Affairs and a DVA Career Development Award (G.L.K.), the Swiss NSF and NIH Grant NS 40960. We thank Dr. Martin Schwab for his critical comments and Melanie Bollnow for her excellent technical support.
References [1] Bareyre FM, Haudenschild B, Schwab ME. Long-lasting sprouting and gene expression changes induced by the monoclonal antibody IN-1 in the adult spinal cord. J Neurosci 2002;22:7097– 110. [2] Barth TM, Stanfield BB. The recovery of forelimb-placing behavior in rats with neonatal unilateral cortical damage involves the remaining hemisphere. J Neurosci 1990;10:3449–58. [3] Bregman BS, Kunkel-Bagden E, Schnell L, Dai HN, Gao D, Schwab ME. Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors. Nature 1995;378:497–501. [4] Brösamle C, Huber AB, Fiedler M, Skerra A, Schwab ME. Regeneration of lesioned corticospinal tract fibers in the adult rat induced by a recombinant, humanized IN-1 antibody fragment. J Neurosci 2000;20:8061–8. [5] Buffo A, Zagrebelsky M, Huber AB, Skerra A, Schwab ME, Strata P, et al. Application of neutralizing antibodies against NI-35/250 myelin-associated neurite outgrowth inhibitory proteins to the adult rat cerebellum induces sprouting of uninjured Purkinje cell axons. J Neurosci 2000;20:2275–86. [6] Carmichael ST, Chesselet M-F. Synchronous neuronal activity is a signal for axonal sprouting after cortical lesions in the adult. J Neurosci 2002;22:6042–70. [7] Castro AJ. The effects of cortical ablations on digital usage in the rat. Brain Res 1972;37:173–85.
324
A.J. Emerick, G.L. Kartje / Behavioural Brain Research 152 (2004) 315–325
[8] Castro AJ. Ipsilateral corticospinal projection after large cerebral lesion in neonatal rats. Exp Neurol 1975;46:1–8. [9] Castro AJ, Mihailoff GA. Corticopontine remodelling after cortical and/or cerebellar lesions in newborn rats. J Comp Neurol 1983;219:112–23. [10] Chen MS, Huber AB, van der Haar ME, Frank M, Schnell L, Spillmann AA, et al. Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 2000;403:434–9. [11] Emerick AJ, Neafsey EJ, Schwab ME, Kartje GL. Functional reorganization of the motor cortex in adult rats after cortical lesion and treatment with mAb IN-1. J Neurosci 2003;23:4826– 30. [12] Fiedler M, Horn C, Bandtlow C, Schwab ME, Skerra A. An engineered IN-1 F(ab) fragment with improved affinity for the Nogo-A axonal growth inhibitor permits immunochemical detection and shows enhanced neutralizing activity. Protein Eng 2002;15: 931–41. [13] Grand Pré T, Nakamura F, Vartanian T, Strittmatter SM. Identification of Nogo inhibitor of axon regeneration as a reticulon protein. Nature 2000;403:439–44. [14] Grand Pré T, Li S, Strittmatter SM. Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature 2002;417:547– 51. [15] Herzog A, Brösamle C. “Semi-free-floating” treatment: a simple and fast method to process consecutive sections for histochemistry and neuronal tracing. J Neurosci Methods 1997;72:57–63. [16] Hicks SP, D’Amato JC. Motor-sensory and visual behavior after hemispherectomy in newborn and mature rats. Exp Neurol 1970;29:416–38. [17] Huber AB, Schwab ME. Nogo-A, a potent inhibitor of neurite outgrowth and regeneration. Biol Chem 2000;381:407–19. [18] Huber AB, Weinmann O, Brösamle C, Oertle T, Schwab ME. Patterns of Nogo mRNA protein expression in the developing and adult rat and after CNS lesions. J Neurosci 2002;22:3553–67. [19] Kartje GL, Schulz MK, Lopez-Yunez A, Schnell L, Schwab ME. Corticostriatal plasticity is restricted by myelin-associated neurite growth inhibitors in the adult rat. Ann Neurol 1999;45:778– 86. [20] Kartje-Tillotson G, Neafsey EJ, Castro AJ. Topography of corticopontine remodeling after cortical lesions in newborn rats. J Comp Neurol 1986;250:206–14. [21] Kolb B, Whishaw IQ. Plasticity in the neocortex: mechanisms underlying recovery from early brain damage. Prog Neurobiol 1989;32:235–76. [22] Kolb B, Gibb R, van der Kooy D. Cortical and striatal structure and connectivity are altered by neonatal hemidecotication in rats. J Comp Neurol 1992;322:311–24. [23] Kolb B, Gibb R, van der Kooy D. Neonatal frontal cortical lesions in rats alter cortical structure and connectivity. Brain Res 1994;645:85– 97. [24] Kuang RZ, Kalil K. Specificity of corticospinal axon arbors sprouting into denervated contralateral spinal cord. J Comp Neurol 1990;244:163–73. [25] Leong SK, Lund RD. Anomalous bilateral corticofugal pathways in albino rats after neonatal lesions. Brain Res 1973;62:218–21. [26] Li S, Strittmatter SM. Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J Neurosci 2003;23:4219– 27. [27] Liang F, Moret V, Wiesendanger M, Rouiller EM. Corticomotoneuronal connections in the rat: evidence from double-labeling of motoneurons and corticospinal axons arborizations. J Comp Neurol 1991;311:356–66. [28] McKenna JE, Prusky GT, Whishaw IQ. Cervical motoneuron topography reflects the proximodistal organization of muscles and movements of the rat forelimb: a retrograde carbocyanine dye analysis. J Comp Neurol 2000;419:286–96.
[29] Merkler D, Metz GAS, Raineteau O, Dietz V, Schwab ME, Fouad K. Locomotor recovery in spinal cord injured rats treated with an antibody neutralizing the myelin-associated neurite growth inhibitor Nogo-A. J Neurosci 2001;21:3665–73. [30] Metz GAS, Whishaw IQ. Skilled reaching an action pattern: stability in rat (Rattus norvegicus) grasping movements as a function of changing food pellet size. Behav Brain Res 2000;116:111– 22. [31] Metz GA, Whishaw IQ. Cortical and subcortical lesions impair skilled walking in the ladder rung walking test: a new task to evaluate fore- and hindlimb stepping, placing, and co-ordination. J Neurosci Methods 2002;115:169–79. [32] Nah SH, Leong SK. Bilateral corticofugal projections to the red nucleus after neonatal lesions in the albino rat. Brain Res 1976;107:433– 6. [33] Naus CG, Flumerfelt BA, Hrycyshyn AW. Contralateral corticorubral fibers induced by neonatal lesions are not collaterals of the normal ipsilateral projections. Neurosci Lett 1986;70:52–8. [34] Neafsey EJ, Bold EL, Haas G, Hurley-Gius KM, Quirk G, Sievert CF, et al. The organization of the rat motor cortex: a microstimulation mapping study. Brain Res Rev 1986;11:77–96. [35] Papadopoulos CM, Tsai S-Y, Alsbiei T, O’Brien TE, Schwab ME, Kartje GL. Functional recovery and neuroanatomical plasticity following middle cerebral artery occlusion and IN-1 antibody treatment in the adult rat. Ann Neurol 2002;51:433–41. [36] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. New York: Academic Press; 1998. [37] Prinjha R, Moore SE, Vinson M, Blake S, Morrow R, Christie G, et al. Inhibitor of neurite outgrowth in humans. Nature 2000;403: 383–4. [38] Raineteau O, Fouad K, North P, Thallmair M, Schwab ME. Functional switch between motor tracts in the presence of the mAb IN-1 in the adult rat. Proc Natl Acad Sci USA 2001;98:6929–34. [39] Rouiller EM, Fengyi L, Moret V, Wiesendanger M. Trajectory of redirected corticospinal axons after unilateral lesion of the sensorimotor cortex in neonatal rat a Phaseolu vulgaris leucoagglutin (PHA-L) tracing study. Exp Neurol 1991;114:53–65. [40] Schwab ME. Repairing the injured spinal cord. Science 2002;295:1029–31. [41] Thallmair M, Metz GAS, Z’Graggen WJ, Raineteau O, Kartje GL, Schwab ME. Neurite growth inhibitors restrict structural plasticity and functional recovery following corticospinal tract lesions. Nat Neurosci 1998;1:124–31. [42] Uryu K, MacKenzie L, Chesselet M-F. Ultrastructural evidence for differential axonal sprouting in the striatum after thermocoagulatory and aspiration lesions of the cerebral cortex in adult rats. Neuroscience 2001;105:307–16. [43] Voorhies AC, Jones TA. The behavioral and dendritic growth effects of focal sensorimotor cortical damage depend on the method of lesion induction. Behav Brain Res 2002;133:237–46. [44] Wenk CA, Thallmair M, Kartje GL, Schwab ME. Increased corticofugal plasticity after unilateral cortical lesions combined with neutralization of the IN-1 antigen in adult rats. J Comp Neurol 1999;410:143–57. [45] Whishaw IQ. Loss of the innate cortical engram for action patterns used in skilled reaching and the development of behavioral compensation following motor cortex lesions in the rat. Neuropharmacy 2000;39:788–805. [46] Whishaw IQ, Kolb B. Sparing of skilled forelimb reaching and corticospinal projections after neonatal motor cortex removal or hemidecortication in the rat: support for the Kennard doctrine. Brain Res 1988;451:97–114. [47] Wiessner C, Baeyre FM, Allegrini PR, Mir AK, Fretzel S, Zurini M, et al. Anti Nogo-A antibody infusion 24 hours after experimental stroke improved behavioral outcome and corticospinal plasticity in normotensive and spontaneously hypertensive rats. J Cereb Blood Flow Metab 2003;23:154–65.
A.J. Emerick, G.L. Kartje / Behavioural Brain Research 152 (2004) 315–325 [48] Wiessner C, Kartje GL, Hillenbrand R, Mir AK, Schwab ME. Anti-Nogo-A antibody therapy after experimental stroke. In: Krieglstein J, Klumpp S, editors. Pharmacology of cerebral ischemia. Stuttgart: Medpharm Scientific Publishers; 2002. p. 343–54.
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[49] Z’Graggen WJ, Metz GAS, Kartje GL, Thallmair M, Schwab ME. Functional recovery and enhanced corticofugal plasticity after unilateral pyramidal tract lesion and blockade of myelin-associated neurite growth inhibitors in adult rats. J Neurosci 1998;18:4744–57.
Research report
Behavioral recovery and anatomical plasticity in adult rats after cortical lesion and treatment with monoclonal antibody IN-1 April J. Emerick a,b,∗ , Gwendolyn L. Kartje a,b b
a Research and Neurology Service, Edward Hines Veterans Affairs Hospital, Hines, IL 60141, USA Neuroscience Program, Departments of Neurology and Cell Biology, Neurobiology and Anatomy, Loyola University Medical Center, Maywood, IL, USA
Received 30 June 2003; received in revised form 8 September 2003; accepted 10 October 2003 Available online 9 January 2004
Abstract We have previously reported that monoclonal antibody (mAb) IN-1 treatment after ischemic infarct in adult rats results in significant recovery of skilled forelimb use. Such recovery was correlated with axonal outgrowth from the intact, opposite motor cortex into deafferented subcortical motor areas. In the present study, we investigated the effects of mAb IN-1 treatment after adult sensorimotor cortex (SMC) aspiration lesion on behavioral recovery and neuroanatomical plasticity in the corticospinal tract. Adult rats underwent unilateral SMC aspiration lesion and treatment with either mAb IN-1 or a control Ab, or no treatment. Animals were then tested over a 6-week period in the skilled forelimb use task and the skilled ladder rung walking task. We found that animals treated with mAb IN-1 after SMC lesion fully recovered the use of forelimb reaching, but showed no improvement in digit grasping as tested in the skilled forelimb use task. The mAb IN-1 treatment group was also significantly improved as compared to control groups in the skilled ladder rung walking test. Furthermore, neuroanatomical tracing revealed a significant increase in the corticospinal projections into the deafferented motor areas of the spinal cord after mAb IN-1 treatment. These results indicate that treatment with mAb IN-1 after cortical aspiration lesion induces remodeling of motor pathways resulting in recovery in only certain behavioral tasks, suggesting that the cause of brain damage influences behavioral recovery after mAb IN-1 treatment. © 2003 Elsevier B.V. All rights reserved. Keywords: Motor cortex; Nogo-A; Corticospinal tract; Cortical injury; Plasticity; Behavioral recovery
1. Introduction Functional recovery after adult CNS lesions is often incomplete and is limited in part by the failure of axons to regenerate after injury. Barriers to axonal outgrowth include Nogo-A [10,13,17,37,40], an inhibitory protein expressed in oligodendrocyte myelin sheaths and compact myelin [18]. The application of the monoclonal antibody (mAb) IN-1 neutralizes inhibitory effects of Nogo-A [10] and induces transient axonal outgrowth in normal adult Purkinje cells [5] and corticospinal neurons [1]. Immunochemical assays confirm that Nogo-A is the antigen for mAb IN-1 [12]. Treatment with mAb IN-1 and other antibodies raised against Nogo-A promotes behavioral recovery in adult rats after ischemic stroke [35,47,48], pyramidotomy [38,40,41] and spinal cord injury [3,4,29]. Furthermore, pharmacological blockade of a receptor to Nogo-66, one of the inhibitory domains of Nogo-A, enhances locomotor recovery after spinal hemisection [14,26]. Behavioral recovery in these ∗
Corresponding author. Tel.: +1-708-202-5992; fax: +1-708-202-2703. E-mail address: [email protected] (A.J. Emerick).
0166-4328/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2003.10.010
studies was observed in parallel with the remodeling of neuroanatomical pathways and the reinnervation of subcortical motor areas affected by the lesion. Neuronal plasticity has often been suggested as an underlying substrate for the spontaneous recovery observed after CNS lesions in the young (for review, see [21]). Studies have shown that after unilateral cortical ablation in the neonate, the intact, opposite motor cortex projects in a target-specific pattern to the deafferented striatum [22,23], red nucleus [25,32,33], basilar pontine nuclei [9,20,25] and spinal cord gray [2,8,16,24,25,39,46]. While such growth is limited in the adult, we have reported that rats treated with mAb IN-1 after adult SMC aspiration lesions form new cortico-efferent connections from the intact, opposite motor cortex to deafferented subcortical targets including the striatum [19], red nucleus and basilar pontine nuclei [44]. However, the effects of mAb IN-1 on recovery of motor function after adult SMC aspiration lesions have not yet been examined. In using the aspiration lesion model, damage can be restricted to areas of the cortex concerned with skilled motor behaviors. Specifically, functional recovery was assessed by the utilization of proximal muscle groups during the skilled reach-
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ing task and distal muscle groups during the digit grasping test, while overall limb coordination was measured with the skilled ladder rung-walking test. We further investigated the corticospinal connections from the non-injured cortex to the ␣-motoneurons that innervate such muscle groups and whether new growth facilitates behavioral recovery. Based on previous reports of improved behavioral outcome and anatomical plasticity, we hypothesized that mAb IN-1 treatment would result in behavioral recovery in adults with cortical aspiration lesions and also promote neurite outgrowth in the corticospinal tract. Interestingly, we found that treatment with mAb IN-1 after adult cortical lesion resulted in variable amounts of recovery, depending upon the task, i.e., recovery in both skilled forelimb reaching and skilled ladder rung walking, and no recovery in skilled digit grasping. Anatomical analysis demonstrated a significant increase in the number of corticospinal axons projecting into the deafferented motor areas of the spinal cord, indicating a probable role for the intact motor cortex in behavioral recovery after mAb IN-1 therapy.
2. Materials and methods 2.1. Animals All animal procedures were approved by the Joint Institutional Animal Care and Use Committee of Loyola University and Hines Veterans Affairs Hospital and conform to the National Institutes of Health guidelines. Animals were housed three to a cage and maintained in a temperature and humidity controlled room on a 12-h light:12-h dark cycle. All testing was performed during the light portion of the cycle. Forty male Long Evans black-hooded rats at 8–10 weeks of age were divided into three experimental groups: (1) SMC lesion-only (n = 16); (2) SMC lesion + control Ab (n = 10); (3) SMC lesion + mAb IN-1 (n = 14). Animals were number-coded and investigators were blind to treatment groups. 2.2. Behavioral tests 2.2.1. Skilled forelimb use task Animals were reduced to ∼95% of their original body weight for motivational purposes and maintained on a foodrestricted diet throughout the training and testing period. As previously described [35], animals were trained and tested in a transparent Plexiglas chamber (30 cm × 36 cm× 30 cm) with a small window (1.5 cm × 3 cm) opening on the left or right side of the front wall at the level of the chamber floor. A shelf was attached outside the box and beneath the window for placement of small sucrose pellets (45 mg; Bilaney Consultants, Frenchtown, NJ) at a distance of 1.5 cm from the window. Animals were trained daily, Monday through Friday, at the same time of day over a 2-week period. During the training period, animals learned
to use the forelimb to reach through the window and grasp the pellets that were placed in succession onto the shelf (see Fig. 1A). Upon grasping the pellets, animals would then have to retract the forelimb into the box and bring the pellet to the mouth. When animals began to show a limb preference (greater than 70% of pellets obtained using either the left or right forelimb), the training was conducted through the window that favored the use of the preferred forelimb. Animals were then trained for 2 weeks to reach and grasp 20 pellets using the preferred forelimb within 5 min. The average scores and time from the last three training sessions before surgery were calculated for each animal and recorded as the preoperative baseline scores. For consistency in data analysis, only animals achieving baseline scores of 16 or greater in grasping were included in the study (animals that did not meet this criterion were still included in the skilled ladder rung walking test). All animals were then randomly assigned to experimental groups. Beginning on the first day after cortical lesion surgery as described below, animals were tested Monday through Friday for 6 weeks in a 20-pellet use task. Motor recovery of the lesion-impaired forelimb was measured in three ways: forelimb reach, digit grasp, and time. The forelimb reach score indicates the number of times an animal successfully extended its forelimb through the window in an attempt to grasp a pellet (only one reach per pellet was measured, yielding a maximum score of 20). The digit grasp score indicates the number of pellets an animal picked up, carried into the box, and placed into its mouth. The time taken to retrieve 20 pellets was recorded in each testing session. Testing sessions were video recorded for further analysis. 2.2.2. Skilled ladder rung walking The ladder walk apparatus consisted of a 1-m long wooden-rung walk way with a varying distance of 1–2 cm between rungs (described in [30]) (see Fig. 1B). Animals were acclimated to the testing apparatus during three separate sessions within a 2-week period before cortical lesion surgery. Animals traversed the walkway freely and without reinforcement. During the third session, animals were video-recorded as they traversed the walkway three times. Tapes were later analyzed to assess preoperative baseline scores. Animals were tested on the first day after surgery and then once a week for the next 6 weeks. In a single testing session, the animals crossed the walkway three times and received a score for the number of forelimb foot faults per 10 steps. Only the lesion-affected forelimb was analyzed and a foot fault was characterized as a total miss or slip from the rung or a misplacement of the paw on the rung, i.e., using the wrist or digits instead of the plantar surface. 2.2.3. Statistical analysis of behavioral data The behavioral data was analyzed using GraphPad Prism 2.01 (GraphPad Software, Inc.). A one-way ANOVA was used to compare the mean values across all groups in a single testing session. Differences in reach, grasp, and time were
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Fig. 1. Behavioral tests. Deficits in reaching and digit grasping were measured in the skilled forelimb use test (A). Successful reaching requires aiming and extension (as demonstrated by a representative animal during training in the film sequence above) of the forelimb through a small window at the front of the testing box. Successful grasping is accomplished when the digits close around the pellet and the animal carries the pellet in its paw to the mouth. The target pellet can be seen on the shelf beneath the paw. Deficits in forelimb placement and accuracy were measured using the skilled ladder rung walking test (B). The number of lesion-impaired forelimb footfaults was measured per 10 steps across the runway. Footfaults include a slip from the rung (arrow) as depicted in a representative animal.
further analyzed with the Bonferroni’s multiple comparison post-test. For comparison within an experimental group, i.e., postoperative and week 6 scores, an unpaired Student’s t-test was used. To measure the percent recovery in the skilled ladder rung walking task, weekly postoperative scores were normalized to the day one postoperative score in each animal. A P value less than or equal to 0.05 was considered significant. All data are presented as mean values ± S.E.M.
taxic frame, and the scalp was incised along the midline. With bregma as a landmark, the skull was opened to expose either the left or right sensorimotor cortex (SMC), and using suction the forelimb motor cortex was gently aspirated within a specific boundary: 4.0 mm rostral; 2.0 mm caudal; 1.0 mm medial; 4.0 mm lateral to bregma [34]. Cortical aspiration lesions were made in the SMC contralateral to the preferred forelimb as determined in the earlier training sessions.
2.3. SMC aspiration lesion
2.4. Antibody application
Animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) supplemented with ketamine (40 mg/kg, i.m.). Anesthetized animals were then secured in a stereo-
Immediately after cortical aspiration lesion and while animals were still anesthetized, mouse hybridoma cells (1 × 105 cells in 1 l) secreting either mAb IN-1 or control
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Ab (anti-horseradish peroxidase) were delivered by Hamilton syringe into the hippocampus posterior to the lesion site (4.0 mm caudal, 5.0 mm lateral and 5.0 mm ventral to bregma). Animals were monitored after surgery until they were mobile and then returned to standard housing conditions. All animals, including controls, received daily injections of Cyclosporin-A (10 mg/kg, i.p.) 1 day before and 7 days after surgery to suppress host rejection of the mouse hybridoma cells. 2.5. Biotin dextran amine neuroanatomical tracing After 6 weeks of behavioral testing, animals were re-anesthetized as described above and placed in a stereotaxic frame. The bone overlaying the motor cortex opposite the SMC lesion was removed, and a 10% biotinylated dextran amine (BDA) solution in 0.01 M phosphate buffer was pressure injected (1 l per injection at a rate of 0.2 l per 1 min) using a Hamilton syringe at four sites within the motor cortex. Animals were returned to standard housing conditions for 2 weeks and then were sacrificed by sodium pentobarbital overdose (200 mg/kg, i.p.) and transcardial perfusion with saline supplemented with 10,000 IU heparin and 0.25% NaNO2 , followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The brains and spinal cords were removed and postfixed in 4% paraformaldehyde at 4 ◦ C overnight followed by 30% sucrose for cryoprotection at 4 ◦ C overnight. Tissue was then embedded in an albumin/sucrose compound (as described in [15]) and frozen in −50 ◦ C isopentane. Tissue was cut in the coronal plane on a cryostat in 50 m serial sections and collected on glass slides for semi-free floating processing with avidin– biotin–peroxidase complex (ABC kit elite, Vector Labs) for visualization of BDA. Alternate brain sections were collected on gelatin-coated glass slides and counterstained with toluidene blue for visualization of cell bodies.
2.6. Neuroanatomical tracing analysis The CST projections from the opposite, intact hemisphere to the spinal cord were evaluated at levels C6 through C8 . Using standard anatomical landmarks, five comparable sections per animal were analyzed under light microscopy using NIH Image Software 5.1 to evaluate BDA-positive labeled axons in the ventral motor horn. Within the motor horn, we evaluated the medial and lateral ␣-motoneuron cell columns (see Figs. 2 and 3). Using a square counting frame, BDA-positive fibers that intersected a total line length of 0.26 mm were counted in each cell column, both ipsilateral and contralateral to the injection site. In order to normalize the results and to take into account tracer variability between animals, the number of fibers on the ipsilateral (lesion-affected) was divided by the contralateral (normal) count on each section to provide a ratio as previously described [1,19,41,47]. The mean ratio for each animal was averaged for each group and was expressed as a percent of the contralateral fibers and compared between experimental groups. These data were statistically analyzed using GraphPad Prism 2.01 (GraphPad Software, Inc.). The Mann– Whitney test was used to compare mean values between groups. A P value less than or equal to 0.05 was considered significant. All data are presented as mean values ± S.E.M. 3. Results 3.1. SMC lesion cavity Gross inspection of brains from all animals showed that SMC lesion size and location was comparable among groups. The area of cortical damage as identified using Paxinos and Watson coordinates [36] included +4.2 mm rostral through −3.3 mm caudal and extended from the midline
Fig. 2. A schematic diagram illustrating the experimental procedures. The placement of the SMC aspiration lesion (shaded) and the implantation site of antibody-secreting hybridoma cells is shown in the left hemisphere. The biotinylated dextran amine (BDA) tracer injection site is contralateral to the aspiration lesion in the non-injured cortex. The pathway of the CST is depicted arising from the right forelimb sensorimotor cortex (SMC), crossing the midline (arrow) and projecting into the spinal cord. (Modified from Papadopoulos et al. [35].)
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Fig. 3. Neuroanatomical plasticity was examined in the cervical spinal cord. The BDA-positive labeled CST fibers projecting from the non-injured cortex were counted in the lateral α-motoneuron cell column (box a, a ) and medial α-motoneuron cell column (box b, b ) on both ipsi- and contralateral sides.
through 4.5 mm lateral, and did not differ in animals treated with mAb IN-1 as compared to lesion-only and control Ab treatment (Fig. 4). 3.2. Behavioral recovery in animals treated with mAb IN-1 3.2.1. Skilled forelimb use Functional recovery in the impaired forelimb was evaluated using the skilled forelimb reach test, and performance
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was measured in forelimb reach, time, and digit grasp. In the forelimb reach test (Fig. 5A), preoperative baseline measurements showed that animals in all experimental groups scored between 19 and 20 in forelimb reach (i.e., extension of the forelimb through the window), indicating no significant difference between groups (P > 0.05, one-way ANOVA). At postoperative day 1, animals from all treatment groups showed limited mobility in the impaired forelimb and most failed to use the limb in the reaching task. All groups showed minimal improvement at the end of postoperative week 1 (data not shown). By postoperative week 2, animals from all treatment groups were increasingly using the impaired forelimb and all groups were significantly improved as compared to the corresponding postoperative deficits measured at day 1 (P < 0.05, unpaired Student’s t-test). However, we observed that most animals continued to have difficulty with aiming and extending the forelimb through the window. By week 6, the SMC lesion-only and lesion plus control antibody groups did not show further improvement from weeks 2 or 3 (data not shown) and their average score remained significantly less than their preoperative baseline scores (P < 0.001, unpaired Student’s t-test). Furthermore, there was no significant difference between the SMC lesion-only group and the SMC lesion plus control Ab treatment group at 6 weeks postoperatively (P > 0.05, one way ANOVA). In marked contrast, animals treated with mAb IN-1 following SMC lesion showed continued improvement through the testing period and a remarkable recovery of skilled forelimb reach. Beginning at week 4, animals in the SMC plus mAb IN-1 treatment group showed significant improvement in forelimb reaching as compared to SMC lesion-only (P < 0.001, one-way ANOVA) and SMC lesion plus control Ab (P < 0.05, one-way ANOVA) treatment groups. This improvement was sustained in the mAb IN-1 treatment group through week 5 (data not shown) and at 6 weeks the forelimb reach scores were not statistically different from the
Fig. 4. Cross-sectional reconstructions of the maximum (black) and minimum (gray) extent of the lesion cavities did not appear to differ among the experimental groups. Only the lesion-side hemisphere is displayed. Coordinates represent distance in millimeters from bregma.
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Fig. 5. Treatment with mAb IN-1 leads to behavioral recovery in the skilled forelimb use test as measured by the number of successful forelimb reaches using the impaired forelimb in a 20-pellet test (A). At 4 weeks following surgery, animals treated with mAb IN-1 were significantly improved compared to control treatment animals. This recovery was sustained through postoperative week 6 at which time point animals treated with mAb IN-1 showed no statistical difference from preoperative baseline scores (P > 0.05, unpaired t-test). SMC lesion-only and SMC lesion + control AB were not significantly different throughout the course of the study (P > 0.05). Treatment with mAb IN-1 improves the time it takes animals to complete the 20-pellet test (B) Time (seconds) to reach was significantly faster in the mAb IN-1 treatment group as compared to the SMC lesion-only and SMC lesion plus control Ab groups at postoperative week 6 (P < 0.001). Digit grasping abilities showed little or no recovery in all treatment groups (C). At postoperative week 6, animals treated with mAb IN-1 were not significantly different from SMC lesion-only or SMC lesion plus control Ab animals (P > 0.05). Data shown are mean ± S.E.M. Asterisks indicate significance compared to SMC lesion-only and SMC lesion + control Ab treatment groups: ∗∗ P < 0.01; ∗∗∗ P < 0.001, one-way ANOVA, followed by Bonferroni’s Multiple Comparison for post hoc evaluation of group differences (dpo: days postoperative; wpo: weeks postoperative).
preoperative baseline scores (P > 0.05, unpaired Student’s t-test). The time to retrieve 20 pellets in the skilled forelimb use task showed no difference in preoperative baseline measurements among the experimental groups (P > 0.05, one-way ANOVA), with the average time to complete the task less than 69 s (Fig. 5B). On postoperative day 1, animals from all experimental groups were unable to complete the task within the 5 min allotted, with no significant difference between groups (P > 0.05, one-way ANOVA). By week 6, the control treatment groups were not statistically different from one another (P > 0.05, one-way ANOVA) and remained significantly slower at performing the task as compared to preoperative baseline scores (P < 0.001, unpaired Student’s t-test). In comparison, animals treated with mAb IN-1 after SMC lesion showed a considerable improvement in the time required to complete the task. At postoperative week 6, animals treated with mAb IN-1 were significantly faster than animals from the control groups (P < 0.001, one-way ANOVA). Recovery was limited, however, and animals treated with mAb IN-1 after SMC lesion remained slower than their preoperative baseline measurements (P < 0.001, unpaired Student’s t-test). Preoperative baseline scores for digit grasp indicate animals were highly successful in grasping the sucrose pellets (Fig. 5C). On postoperative day 1, digit grasp scores were extremely low in all groups, reflective of the motor deficits in forelimb reaching. By week 2, animals began to show some improvement, but clearly at week 6 there was just a modest change in the digit grasp scores. Most animals showed a lack of fine digit control and could not flex the digits around the small pellets. Some animals modified their retrieval tactics and would swat at or scoop up a pellet, but in most cases, would fumble the pellet out of the paw before bringing it to the mouth. Animals treated with mAb IN-1 after SMC lesion were not statistically different from control treatment groups (P > 0.05) nor did they recover to their preoperative baseline scores. 3.2.2. Skilled ladder rung walking Accurate placement of the impaired forelimb was examined on the skilled ladder rung walking test. Similar to our earlier findings in normal animals performing this test [49], preoperative baseline measurements showed that animals from all experimental groups were able to cross the ladder with fewer than 1.5 foot faults per 10 steps (data not shown). Following SMC lesion, the average number of foot faults had tripled from preoperative baseline values in all experimental groups. We assessed recovery as a percent of preoperative baseline scores in each individual animal (Fig. 6). By week 2, all experimental groups showed slight improvement, but were not significantly different from postoperative day 1 (P > 0.05, unpaired Student’s t-test). There was no statistical difference in the percent recovery between the control groups throughout the experiment (P > 0.05, one-way ANOVA) and within each control group there was no signif-
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Fig. 6. Treatment with mAb IN-1 improves performance in the skilled ladder rung walking test. The percent recovery was significantly higher in the SMC lesion + mAb IN-1 treatment group compared to both SMC lesion-only and SMC lesion + control Ab groups at postoperative weeks 4 and 6 (∗ P < 0.05, unpaired Student’s t-test). Animals in the SMC lesion+mAb IN-1 treatment group showed significant recovery at 6 weeks compared to day 1 postoperative values (∗∗ P < 0.05, unpaired Student’s t-test), whereas the percent recovery did not improve over the 6 weeks in either of the control experimental groups.
icant improvement as compared to postoperative day 1 (P > 0.05, unpaired Student’s t-test). Conversely, animals treated with mAb IN-1 showed significant improvement at week 4 when compared to the control groups (P < 0.05, one-way ANOVA). This improvement was sustained through week 6, at which point animals treated with mAb IN-1 also showed significant recovery when compared to postoperative day 1 (P < 0.05, unpaired Student’s t-test). 3.3. Neuroanatomical tracing of the CST The intact, opposite SMC was injected with BDA tracer to identify the CST and its projections into the cervical spinal cord gray matter. The dorsal funiculus contralateral to the BDA-injected SMC showed dense labeling of the CST through all spinal cord levels in animals from all experimental groups. The pattern of CST axons terminating contralaterally in Rexed’s lamina I through X was consistent with previous neuroanatomical studies [1,24,27]. We analyzed the percent of CST fibers projecting into the lateral and medial ␣-motoneuron columns of the ventral motor horn ipsilateral to the injection side. The ␣-motoneurons of these two regions typically innervate the distal and proximal muscles of the forearm, respectively [28]. Since there was no apparent difference between SMC lesion-only and SMC lesion + control Ab, these data were pooled together as SMC lesion control. The percent of CST fibers projecting into the lateral ␣-motoneuron cell column was not significantly different between the mAb IN-1 treated animals and the lesion control group (Fig. 7A) (P > 0.05, Mann–Whitney). When we assessed the medial ␣-motoneuron cell column, however, we found a significant increase in the percentage of CST fibers as compared to the SMC lesion control group (Fig. 7B and C) (P < 0.05, Mann–Whitney). Thus, the mAb IN-1 treatment group had more CST connections to ␣-motoneurons that facilitate
movements in the impaired forelimb related to proximal (i.e., shoulder) muscles but not distal (i.e., digit) muscles.
4. Discussion This study demonstrates that intracerebral application of mAb IN-1 following SMC aspiration lesion results in recovery of specific skilled forelimb movements. Animals treated with mAb IN-1 achieved preoperative baseline scores in skilled forelimb reaching and were significantly better than control groups. However, these animals showed no recovery in digit grasping and were no different from control animals. In addition, treatment with mAb IN-1 resulted in significant improvement in the skilled ladder rung walking test. The recovery of animals treated with mAb IN-1 after SMC aspiration lesion correlates with increased plasticity of corticospinal projections in the spinal cord to areas that facilitate proximal forelimb movement. 4.1. Treatment with mAb IN-1 results in significant motor recovery after SMC lesion Skilled forelimb reaching is a highly conserved action pattern that is organized into a series of forelimb movements mediated by the CST among other motor pathways [30]. Accordingly, damage to the motor cortex results in functional deficits from which animals may never completely recover [7,45,46]. In agreement with these previous studies, we found that skilled reaching movements were severely impaired in the forelimb contralateral to the SMC aspiration lesion. Animals from the SMC lesion-only and lesion+control Ab groups often retained severe motor deficits in precise forelimb motor control and were thus unsuccessful in forelimb reaching and grasping. Animals treated with mAb IN-1, on the other hand, displayed a remarkable degree of recov-
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Fig. 7. Treatment with mAb IN-1 after SMC lesion results in an increase in CST fibers projecting to the ␣-motoneurons that innervate proximal muscles of the impaired forelimb. The percentage of CST fibers projecting to the ipsilateral (lesion-affected) lateral ␣-motoneuron cell column did not change after SMC lesion and treatment with mAb IN-1 as compared to SMC lesion control (pooled data from lesion-only and lesion + control Ab) (P > 0.05, Mann–Whitney) (A). However, there was a significant increase in the percentage of CST fibers projecting to the medial ␣-motoneuron cell column (B) in animals treated with mAb IN-1 as compared to lesion-control animals. Error bars indicate mean ± S.E.M. Asterisk (∗) indicates P < 0.05, Mann–Whitney. (C) Photomicrographs of representative cross-sections through the medial ␣-motoneuron cell column in the lesion-affected side of the spinal cord. BDA-positive CST fibers (black) were identified in all groups, yet the mAb IN-1 treatment group was characterized by increased CST fibers that were often located adjacent to ␣-motoneurons (∗).
ery in the use of proximal limb muscles as indicated by the successful performance in forelimb reaching and the time to reach for 20 pellets. Rodents depend on the ascending and descending pathways required for locomotion during the skilled forelimb ladder rung walking test. Balance, fine-adjusted muscle control, and limb coordination are all necessary for the animal to cross the rungs accurately [31]. We found that following SMC aspiration lesion animals had forelimb placement deficits, and that most errors resulted from incorrect aiming of the lesion-impaired forelimb or improper positioning of the forelimb on the rung with either the wrist or digits. The SMC aspiration lesions resulted in chronic impairments in the control of limb and muscle movements in the lesion-only and lesion + control Ab treatment groups, which is consistent with previous reports of forelimb placing deficits observed 3–6 months after unilateral motor cortex lesion or pyramidotomy [31]. The mAb IN-1 treatment
group, however, showed significant recovery from postoperative deficits, and, although they did not reach preoperative baseline values by postoperative week 6, there was a trend for continued improvement. Animals from all experimental groups showed chronic impairments in fine digit motor control and had limited success in digit grasping after SMC lesions. Treatment with mAb IN-1 alone did not ameliorate this lesion effect. The failure of animals treated with mAb IN-1 to recover in digit grasping suggests that this task is inherently more difficult than the reaching task, and a longer survival period or additional training might be necessary for improvement. Likewise, skilled digit use might require more CST involvement than skilled reaching, and the extensive damage to the motor cortex could contribute to enduring deficits. Interestingly, the lack of recovery in digit grasping conflicts with previous reports in animals treated with mAb IN-1 following unilateral ischemic stroke [35] or CST le-
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sion [41,49]. In those studies, mAb IN-1 treatment led to significant recovery in fine digit motor control, and animals were highly successful in grasping and retrieving the small food pellets. Similarly, intraventricular infusion of a purified monoclonal anti-Nogo-A antibody following unilateral ischemic stroke also improved digit-grasping ability during the staircase task [47,48]. In our study, animals treated with mAb IN-1 after SMC aspiration lesion did not even show a trend for improvement at postoperative week 6. This discrepancy in results indicates that behavioral outcome after CNS injury and mAb IN-1 treatment may depend in part on the lesion type (i.e., ischemic versus aspiration). In our experience there is a much greater area of damage specifically to the motor cortex after aspiration lesion than after ischemic insult. Furthermore, the cellular response varies according to the lesion type; for instance, ischemic insult to the cerebral cortex induces spontaneous axonal growth [42] concomitantly with synchronized neuronal EEG patterns [6]. On the contrary, aspiration lesion alone shows limited axonal outgrowth and lacks any EEG activity related to axonal sprouting. Likewise, following cortical aspiration lesion there is an absence of dendritic plasticity in layer V pyramidal neurons [43], which is also suggested to contribute to enduring forelimb impairments.
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ment with either mAb IN-1 or other anti-Nogo-A antibodies appears to sustain such neuronal outgrowth or plasticity into denervated motor areas and concomitantly improves behavioral outcome after ischemic stroke [35,47,48], pyramidotomy [41,49], and spinal cord injury [3,29]. While the availability of antibody is limited to 7–10 days, we are still investigating the down-stream molecular effects of blocking Nogo-A. Indeed, the application of mAb IN-1 after CST injury significantly enhances the expression of growth-cone guidance molecules essential for target pathfinding [1]. These changes in gene expression might also contribute to the long-lasting anatomical reorganization and behavioral recovery that is observed after damage to the motor cortex. Here we show that after a focal aspiration lesion to the motor cortex and mAb IN-1 treatment corticospinal plasticity does occur and appears to correspond to behavioral recovery on specific tasks related to the re-organized pathway. However, mAb IN-1 treatment does not ameliorate all motor deficits, and in particular, digit grasping was not improved. In conclusion, lesion type should be an important consideration when evaluating treatment paradigms in recovery after CNS injury.
Acknowledgements 4.2. Neuroanatomical remodeling of the CST parallels behavioral recovery After SMC lesion, motoneurons in the spinal cord as well as several brain stem targets have lost their connection to the motor cortex. Here we show anatomical evidence that following aspiration lesion and treatment with mAb IN-1, the motor cortex opposite to the damaged hemisphere connects to this deafferented spinal cord through the intact CST. Treatment with mAb IN-1 resulted in an increase in CST projections into the medial ␣-motoneuron cell column but not to the lateral cell column. Given the proximity of the nuclei, it is doubtful that the distance was too great to allow for sprouting into the lateral cell column. Needless to say, the lack of recovery in digit grasping might be explained in part by the limited plasticity observed in this area of the spinal cord, which tends to innervate the distal muscles of the forelimb. On the other hand, the ␣-motoneurons of the medial cell column innervate the proximal muscles such as shoulder and elbow [30] that function in forelimb extension, and plasticity in this region was correlated with recovery in forelimb reaching. In agreement with these findings, electrophysiological studies in animals treated with mAb IN-1 after aspiration lesion confirmed that the opposite, non-injured cortex increases motor output to the lesion-affected forelimb resulting primarily in shoulder and elbow movements [11]. In the absence of injury, the application of mAb IN-1 induces transient neuronal outgrowth in adult cerebellar Purkinje cells [5] and corticospinal neurons in parallel with the upregulation of growth factors and growth-related proteins necessary for axonal outgrowth [1]. After injury, treat-
This work was supported by the Department of Veterans Affairs and a DVA Career Development Award (G.L.K.), the Swiss NSF and NIH Grant NS 40960. We thank Dr. Martin Schwab for his critical comments and Melanie Bollnow for her excellent technical support.
References [1] Bareyre FM, Haudenschild B, Schwab ME. Long-lasting sprouting and gene expression changes induced by the monoclonal antibody IN-1 in the adult spinal cord. J Neurosci 2002;22:7097– 110. [2] Barth TM, Stanfield BB. The recovery of forelimb-placing behavior in rats with neonatal unilateral cortical damage involves the remaining hemisphere. J Neurosci 1990;10:3449–58. [3] Bregman BS, Kunkel-Bagden E, Schnell L, Dai HN, Gao D, Schwab ME. Recovery from spinal cord injury mediated by antibodies to neurite growth inhibitors. Nature 1995;378:497–501. [4] Brösamle C, Huber AB, Fiedler M, Skerra A, Schwab ME. Regeneration of lesioned corticospinal tract fibers in the adult rat induced by a recombinant, humanized IN-1 antibody fragment. J Neurosci 2000;20:8061–8. [5] Buffo A, Zagrebelsky M, Huber AB, Skerra A, Schwab ME, Strata P, et al. Application of neutralizing antibodies against NI-35/250 myelin-associated neurite outgrowth inhibitory proteins to the adult rat cerebellum induces sprouting of uninjured Purkinje cell axons. J Neurosci 2000;20:2275–86. [6] Carmichael ST, Chesselet M-F. Synchronous neuronal activity is a signal for axonal sprouting after cortical lesions in the adult. J Neurosci 2002;22:6042–70. [7] Castro AJ. The effects of cortical ablations on digital usage in the rat. Brain Res 1972;37:173–85.
324
A.J. Emerick, G.L. Kartje / Behavioural Brain Research 152 (2004) 315–325
[8] Castro AJ. Ipsilateral corticospinal projection after large cerebral lesion in neonatal rats. Exp Neurol 1975;46:1–8. [9] Castro AJ, Mihailoff GA. Corticopontine remodelling after cortical and/or cerebellar lesions in newborn rats. J Comp Neurol 1983;219:112–23. [10] Chen MS, Huber AB, van der Haar ME, Frank M, Schnell L, Spillmann AA, et al. Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 2000;403:434–9. [11] Emerick AJ, Neafsey EJ, Schwab ME, Kartje GL. Functional reorganization of the motor cortex in adult rats after cortical lesion and treatment with mAb IN-1. J Neurosci 2003;23:4826– 30. [12] Fiedler M, Horn C, Bandtlow C, Schwab ME, Skerra A. An engineered IN-1 F(ab) fragment with improved affinity for the Nogo-A axonal growth inhibitor permits immunochemical detection and shows enhanced neutralizing activity. Protein Eng 2002;15: 931–41. [13] Grand Pré T, Nakamura F, Vartanian T, Strittmatter SM. Identification of Nogo inhibitor of axon regeneration as a reticulon protein. Nature 2000;403:439–44. [14] Grand Pré T, Li S, Strittmatter SM. Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature 2002;417:547– 51. [15] Herzog A, Brösamle C. “Semi-free-floating” treatment: a simple and fast method to process consecutive sections for histochemistry and neuronal tracing. J Neurosci Methods 1997;72:57–63. [16] Hicks SP, D’Amato JC. Motor-sensory and visual behavior after hemispherectomy in newborn and mature rats. Exp Neurol 1970;29:416–38. [17] Huber AB, Schwab ME. Nogo-A, a potent inhibitor of neurite outgrowth and regeneration. Biol Chem 2000;381:407–19. [18] Huber AB, Weinmann O, Brösamle C, Oertle T, Schwab ME. Patterns of Nogo mRNA protein expression in the developing and adult rat and after CNS lesions. J Neurosci 2002;22:3553–67. [19] Kartje GL, Schulz MK, Lopez-Yunez A, Schnell L, Schwab ME. Corticostriatal plasticity is restricted by myelin-associated neurite growth inhibitors in the adult rat. Ann Neurol 1999;45:778– 86. [20] Kartje-Tillotson G, Neafsey EJ, Castro AJ. Topography of corticopontine remodeling after cortical lesions in newborn rats. J Comp Neurol 1986;250:206–14. [21] Kolb B, Whishaw IQ. Plasticity in the neocortex: mechanisms underlying recovery from early brain damage. Prog Neurobiol 1989;32:235–76. [22] Kolb B, Gibb R, van der Kooy D. Cortical and striatal structure and connectivity are altered by neonatal hemidecotication in rats. J Comp Neurol 1992;322:311–24. [23] Kolb B, Gibb R, van der Kooy D. Neonatal frontal cortical lesions in rats alter cortical structure and connectivity. Brain Res 1994;645:85– 97. [24] Kuang RZ, Kalil K. Specificity of corticospinal axon arbors sprouting into denervated contralateral spinal cord. J Comp Neurol 1990;244:163–73. [25] Leong SK, Lund RD. Anomalous bilateral corticofugal pathways in albino rats after neonatal lesions. Brain Res 1973;62:218–21. [26] Li S, Strittmatter SM. Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J Neurosci 2003;23:4219– 27. [27] Liang F, Moret V, Wiesendanger M, Rouiller EM. Corticomotoneuronal connections in the rat: evidence from double-labeling of motoneurons and corticospinal axons arborizations. J Comp Neurol 1991;311:356–66. [28] McKenna JE, Prusky GT, Whishaw IQ. Cervical motoneuron topography reflects the proximodistal organization of muscles and movements of the rat forelimb: a retrograde carbocyanine dye analysis. J Comp Neurol 2000;419:286–96.
[29] Merkler D, Metz GAS, Raineteau O, Dietz V, Schwab ME, Fouad K. Locomotor recovery in spinal cord injured rats treated with an antibody neutralizing the myelin-associated neurite growth inhibitor Nogo-A. J Neurosci 2001;21:3665–73. [30] Metz GAS, Whishaw IQ. Skilled reaching an action pattern: stability in rat (Rattus norvegicus) grasping movements as a function of changing food pellet size. Behav Brain Res 2000;116:111– 22. [31] Metz GA, Whishaw IQ. Cortical and subcortical lesions impair skilled walking in the ladder rung walking test: a new task to evaluate fore- and hindlimb stepping, placing, and co-ordination. J Neurosci Methods 2002;115:169–79. [32] Nah SH, Leong SK. Bilateral corticofugal projections to the red nucleus after neonatal lesions in the albino rat. Brain Res 1976;107:433– 6. [33] Naus CG, Flumerfelt BA, Hrycyshyn AW. Contralateral corticorubral fibers induced by neonatal lesions are not collaterals of the normal ipsilateral projections. Neurosci Lett 1986;70:52–8. [34] Neafsey EJ, Bold EL, Haas G, Hurley-Gius KM, Quirk G, Sievert CF, et al. The organization of the rat motor cortex: a microstimulation mapping study. Brain Res Rev 1986;11:77–96. [35] Papadopoulos CM, Tsai S-Y, Alsbiei T, O’Brien TE, Schwab ME, Kartje GL. Functional recovery and neuroanatomical plasticity following middle cerebral artery occlusion and IN-1 antibody treatment in the adult rat. Ann Neurol 2002;51:433–41. [36] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. New York: Academic Press; 1998. [37] Prinjha R, Moore SE, Vinson M, Blake S, Morrow R, Christie G, et al. Inhibitor of neurite outgrowth in humans. Nature 2000;403: 383–4. [38] Raineteau O, Fouad K, North P, Thallmair M, Schwab ME. Functional switch between motor tracts in the presence of the mAb IN-1 in the adult rat. Proc Natl Acad Sci USA 2001;98:6929–34. [39] Rouiller EM, Fengyi L, Moret V, Wiesendanger M. Trajectory of redirected corticospinal axons after unilateral lesion of the sensorimotor cortex in neonatal rat a Phaseolu vulgaris leucoagglutin (PHA-L) tracing study. Exp Neurol 1991;114:53–65. [40] Schwab ME. Repairing the injured spinal cord. Science 2002;295:1029–31. [41] Thallmair M, Metz GAS, Z’Graggen WJ, Raineteau O, Kartje GL, Schwab ME. Neurite growth inhibitors restrict structural plasticity and functional recovery following corticospinal tract lesions. Nat Neurosci 1998;1:124–31. [42] Uryu K, MacKenzie L, Chesselet M-F. Ultrastructural evidence for differential axonal sprouting in the striatum after thermocoagulatory and aspiration lesions of the cerebral cortex in adult rats. Neuroscience 2001;105:307–16. [43] Voorhies AC, Jones TA. The behavioral and dendritic growth effects of focal sensorimotor cortical damage depend on the method of lesion induction. Behav Brain Res 2002;133:237–46. [44] Wenk CA, Thallmair M, Kartje GL, Schwab ME. Increased corticofugal plasticity after unilateral cortical lesions combined with neutralization of the IN-1 antigen in adult rats. J Comp Neurol 1999;410:143–57. [45] Whishaw IQ. Loss of the innate cortical engram for action patterns used in skilled reaching and the development of behavioral compensation following motor cortex lesions in the rat. Neuropharmacy 2000;39:788–805. [46] Whishaw IQ, Kolb B. Sparing of skilled forelimb reaching and corticospinal projections after neonatal motor cortex removal or hemidecortication in the rat: support for the Kennard doctrine. Brain Res 1988;451:97–114. [47] Wiessner C, Baeyre FM, Allegrini PR, Mir AK, Fretzel S, Zurini M, et al. Anti Nogo-A antibody infusion 24 hours after experimental stroke improved behavioral outcome and corticospinal plasticity in normotensive and spontaneously hypertensive rats. J Cereb Blood Flow Metab 2003;23:154–65.
A.J. Emerick, G.L. Kartje / Behavioural Brain Research 152 (2004) 315–325 [48] Wiessner C, Kartje GL, Hillenbrand R, Mir AK, Schwab ME. Anti-Nogo-A antibody therapy after experimental stroke. In: Krieglstein J, Klumpp S, editors. Pharmacology of cerebral ischemia. Stuttgart: Medpharm Scientific Publishers; 2002. p. 343–54.
325
[49] Z’Graggen WJ, Metz GAS, Kartje GL, Thallmair M, Schwab ME. Functional recovery and enhanced corticofugal plasticity after unilateral pyramidal tract lesion and blockade of myelin-associated neurite growth inhibitors in adult rats. J Neurosci 1998;18:4744–57.