Quantitative assessment of forelimb motor function after cervical spinal cord injury in rats: Relationship to the corticospinal tract

Experimental Neurology 194 (2005) 161 – 174 www.elsevier.com/locate/yexnr

Quantitative assessment of forelimb motor function after cervical spinal cord injury in rats: Relationship to the corticospinal tract Kim D. Anderson, Ardi Gunawan, Oswald StewardT Departments of Anatomy and Neurobiology, Neurobiology and Behavior, Neurosurgery, Reeve-Irvine Research Center, University of California at Irvine College of Medicine, 1105 Gillespie Neuroscience Research Facility, Irvine, CA 92697-4292, USA Received 15 August 2004; revised 30 January 2005; accepted 14 February 2005 Available online 30 March 2005

Abstract Approximately 50% of human spinal cord injuries (SCI) are at the cervical level, resulting in impairments in motor function of the upper extremity. Even modest recovery of upper extremity function could have an enormous impact on quality of life for quadriplegics. Thus, there is a critical need to develop experimental models for cervical SCI and techniques to assess deficits and recovery of forelimb motor function. Here, we analyze forelimb and forepaw motor function in rats after a lateral hemisection at C5 and assessed the relationship between the functional impairments and the extent of damage to one descending motor system, the corticospinal tract (CST). Female Sprague–Dawley rats were trained on various behavioral tasks that require the forelimb, including a task that measures gripping ability by the hand (as measured by a grip strength meter, GSM), a food reaching task, and horizontal rope walking. After 8 weeks of post-injury testing, the distribution of the CST was evaluated by injecting BDA into the sensorimotor cortex either ipsi- or contralateral to the cervical lesion. Complete unilateral hemisection injuries eliminated the ability to grip and caused severe impairments in food retrieval by the forepaw ipsilateral to the lesion. There was no indication of recovery in either task. In cases in which hemisections spared white matter near the midline, there was some recovery of forelimb motor function over time. Assessment of rope climbing ability revealed permanent impairments in forelimb use and deficits in hindlimb use and trunk stability. Sensory testing using a dynamic plantar aesthesiometer revealed that there was no increase in touch sensitivity in the affected forelimb. For the cases in which both histological and behavioral data were available, spared forelimb motor function was greatest in rats in which there was sparing of the dorsal CST. D 2005 Elsevier Inc. All rights reserved. Keywords: Cervical injury; Forelimb behavior; Corticospinal tract; Digital flexors; Grip strength

Introduction Currently, there are an estimated 400,000 individuals in the United States living with spinal cord injury (SCI) and, of these, approximately 50% are quadriplegic as a result of injury at the cervical level. The average lifetime cost for a quadriplegic is estimated at over $1,000,000, due largely to the lack of independence resulting from impaired function of the upper extremities, forcing the need for health care attendants to assist with essential activities of daily living. A recent survey demonstrated that regaining arm and hand function is considered the highest priority for improving the T Corresponding author. Fax: +1 949 824 2625. E-mail address: [email protected] (O. Steward). 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.02.006

quality of life of quadriplegics (Anderson, 2004). These statistics document the imperative need to study deficits and recovery of upper extremity function following cervical SCI in animal models. Traditionally, the thoracic spinal cord has been the preferred site of injury for animal studies using models that include complete transection, crush, compression, and contusion. The most obvious functional consequence of injuries at the thoracic level is the loss of hindlimb motor function, and considerable effort has been devoted to developing functional assessment techniques that measure hindlimb locomotor ability. Recently, however, there has been an increasing use of cervical SCI models ranging from graded unilateral contusions (Schrimsher and Reier, 1992; Soblosky et al., 2001), select dorsal column or other partial lesions

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Table 1 Experimental grouping Experiment

Number of animals

Behavioral assessment

Histological verification of lesion

Inferred lesion based on GSM

06-30-03 (Exp. 1)

15

GSM, food pellet, bladder

10-06-03 (Exp. 2)

16

GSM, food pellet, DPA

9 (5 complete hx, 3 sham, 1 incomplete hx) 1 (1 incomplete hx)

01-12-04 (Exp. 3)

18

GSM, DPA, horizontal rope

Total

49

6 (3 complete hx, 2 sham, 1 incomplete hx) 15 (6 complete hx, 6 sham, 2 incomplete hx, 1 over hx) 16 (5 complete hx, 4 sham, 5 incomplete hx, 2 over hx) 37

(Ballermann et al., 2001; Nash et al., 2002; Schrimsher and Reier, 1993; Webb and Muir, 2003), unilateral crush injuries (Schallert et al., 2000), or unilateral hemisections (Fujiki et al., 2004; Jin et al., 2002; Webb and Muir, 2002). In addition, these models have been used from cervical level 1 (C1) through C5 with varying degrees of gray and white matter damage. With the increasing focus on injuries at the cervical level, there is a need to develop assessment techniques to measure impairments and recovery of forelimb motor function. Tasks that measure paw function are especially desirable, because rats use their forepaws for highly skilled motor tasks, and in some skilled tasks like forelimb reaching and grasping, exhibit behavioral capabilities that are analogous with humans (Whishaw et al., 1992). In a previous study, we described the use of a grip strength meter to assess paw function after cervical injuries in mice (Anderson et al., 2004). We found that hemisection injuries lead to a complete loss of gripping ability for about 2 weeks and then a sudden partial recovery in grip strength at about 14 days post lesion. This task offers considerable advantages as an assessment tool because it is easy to administer, is unlearned (and thus does not require specific training), provides a quantitative measure of digital flexor strength, and can be done repeatedly for many days with highly consistent results. Despite its advantages, however, the grip strength task does not measure highly skilled digit movements, and it is not known whether recovery of flexor strength is correlated with recovery of dexterity and ability to carry out skilled movements. It is also not known whether the recovery of digital flexor strength reliably indicates improved function in tasks that actually require gripping ability (for example, walking along a rope). The goal of the present study was to compare the usefulness of several assessment tasks for studies of forelimb motor deficits and recovery after cervical level injuries in rats. Tasks include the assessment of grip strength using the grip strength meter, a forelimb reaching task, and ability to walk along a horizontal rope. The forelimb reaching task especially involves the type of fine, voluntary movement that is thought to be controlled by the corticospinal tract (CST). We chose to evaluate the consequences of hemisection lesions at C5 (the most common level for cervical injuries in humans). These lesions eliminate descending inputs to motor neuron pools supplying digital flexors of the forelimb ipsilateral to the

2 (2 sham) 12

lesion, which are located within the C6 through T1 spinal levels in the rat (McKenna et al., 2000). Because the lesions are unilateral, the contralateral forelimb can be assessed as an internal control. We show here that, in contrast to mice, complete hemisection injuries at the C5 level in rats lead to a permanent loss of gripping ability as measured by the grip strength meter. There was also a complete loss of ability to use the affected limb to retrieve a food pellet and a loss of ability to use the limb to grasp a rope. Analyses of animals with partial lesions that spared part of the CST revealed that residual gripping ability was related to sparing of parts of the CST. Together, these analyses provide a very useful tool for assessing recovery of forelimb motor function after partial cervical injuries.

Methods SCI surgery All studies were performed on female Sprague–Dawley rats (from Harlan, Inc., San Diego, CA) that were 200–230 g at the beginning of each experiment and between 3–4 months of age. In three separate experiments, a total of 32 rats received a hemisection surgery and 17 received sham surgery (n = 49). Post-hoc histological assessments were performed on 37 of the 49 spinal cords (see Tables 1 and 2) and revealed 14 bcompleteQ unilateral hemisections and 12 shams with no damage. In 3 rats, the lesions extended across the midline and were thus bover-hemisectionsQ. Hemisections were incomplete in 8 other rats. Of the 12 remaining spinal cords in which

Table 2 Total number of animals used for the GSM

Complete hx Sham Incomplete hx Over hx Total

Grouped based on histological verification of lesion (Fig. 1)

Grouped based on inferred lesion (Fig. 2)

14 12 8 3 37

5 5 2 0 12

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histology could not be obtained, the lesions were inferred based on the outcome of the GSM behavior (5 complete, 5 sham, and 2 incomplete). For surgery, rats were anesthetized with an intraperitoneal injection of Ketamine and Xylazine (100 mg/ kg and 10 mg/kg, respectively; Western Medical Supply, Inc., Arcadia, CA). Hair overlying the cervical vertebrae was removed by shaving, the skin was treated with betadine and incised, and the three layers of muscle overlying the vertebral column were bluntly dissected. A dorsal laminectomy was then performed on the fifth cervical vertebra (C5). A complete lateral hemisection was performed using a Moria micro knife, (FST catalog #7040A, Fine Science Tools, Foster City, CA) on the right- or left-hand side of the spinal cord. The microknife was inserted into the spinal cord at the midline, with the blunt back-side of the knife abutting the dorsal blood vessel and the sharp-side facing laterally, until the ventral surface of the vertebra column was touched. The knife was then pulled across the spinal cord until it came out the lateral side. This incision was repeated once and the completeness of the lesion was verified microscopically. Sham-operated controls received a C5 dorsal laminectomy only. The muscle was sutured in layers, and the skin was closed with wound clips. Post-operatively, animals received 1 ml of 0.9% saline, 1 ml of 5% dextrose, 0.5 mg/kg Baytril, and 0.01 mg/kg Buprenorphine subcutaneously and were placed on a warming blanket at 378C overnight. Bladders were manually expressed for the first week; however, there were never any noticeable impairments in voiding ability (that is, bladders were empty or contained minimal urine when expressed). Behavioral testing A 3 week handling and pre-training procedure was developed in order to calm the rats and enhance reliability when testing, during which the animals were trained for all the tasks. Behavioral testing was then conducted for 8 weeks post-injury, as described below for the individual tasks. Grip strength meter test Reliable assessment of gripping ability requires that animals are used to handling. Accordingly, the first 5 sessions of training were limited to handling each animal for 5 min. Then, there were 10 training sessions during which animals were held around the midsection, facing the handle of the grip strength meter (GSM, designed by TSE-Systems and distributed by SciPro, Inc.), and one forearm was manually restrained by the experimenter. When the unrestrained forepaw is brought into contact with the handle, the animals reliably grasp the bar, and the animal is then gently pulled away from the device. The GSM then measured the maximal force before the animal released the bar. Each testing session assessed

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each forepaw separately four times. The handling and training took 3 weeks to complete, after which surgery was performed as described above and GSM testing (4 trials/paw/session) was carried out 3 times per week for 8 weeks post-injury. Horizontal rope walking As described above for the GSM training, animals were handled for the first 5 sessions. Following that, each animal received 8 training sessions during which they learned to walk across a horizontal rope for a distance of 80 cm. The starting point was a small platform 2 in.  4 in. and the ending point was a large, black counter-top. Each training session consisted of 5 trials and the number of sets of footsteps with the forelimbs was counted for each trial. The rope was 1 m above the ground and animals traversed from the small platform to the large platform. A descriptive log was maintained in order to record deficits in hindlimbs and trunk stability. Surgery was performed after the 10 training sessions, and then testing on the rope task was carried out 2 times per week for 8 weeks post-injury. Food pellet reaching task The food pellet reaching task was performed in a similar manner to previously published work (see for example Whishaw and Pellis, 1990) and the apparatus was designed following the detailed description by Metz and Whishaw (2000). The only major difference is that our rats were not food-deprived. This was taken as a precautionary measure to prevent weight loss following the injury. To motivate animals, chocolate-flavored food pellets were used (20 mg, Bio-Serv, Inc., Frenchtown, NJ). During the first week of handling for other behavioral tests, the rats were also placed in the food pellet reaching box to allow acclimation. Food pellets were first placed inside the box, then gradually on the shelf outside the box, and animals learned to reach through the slot, grasp the pellets, and pull them back into the box. During the second and third week of training, each rat spent 7 min in the reaching box for a total of 10 sessions (5 sessions per week). The number of successful pellet retrievals was recorded as well as the percentage of successful retrievals with 1 to 2 reaches (a measure of accuracy) and the hand used to retrieve each pellet. The right- or left-handedness was determined for each animal, and the hemisection injuries were made on the preferred side. Some animals were ambidextrous during the entire training time and for those animals the side of the spinal cord to be lesioned was chosen randomly. After receiving a hemisection or sham operation, animals were tested on the reaching task 2 times per week (7 min per session) for 8 weeks. None of the animals began reaching with the contralateral bun-impairedQ limb following SCI. Beginning on day 15 post-injury, animals were videotaped once per week for 3 of the 7 min of a session and this was

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used to document reaching strategies using a computerbased video analysis program (MicrosoftR Windows Movie Maker). Dynamic plantar aesthesiometer To assess for changes in sensation or the development of mechanical allodynia, sensitivity to tactile stimulation was assessed using the Dynamic Plantar Aesthesiometer (DPA, UGO BASILE, catalog #37400, Italy), which is an automated version of the von Frey hair assessment. Plantar von Frey hairs are used to detect changes in touch sensitivity, in response to mechanical stimulation, resulting from neural damage. The standard von Frey hairs are a set of 20 monofilaments in a linear scale of physical force. The different von Frey hairs are pressed against the skin on the plantar surface of the foot until the animal withdraws its paw. Animals are placed individually in a small enclosed testing arena (20 cm  18.5 cm  13 cm, length  width  height) with a wire mesh floor for 5 min. The DPA device is positioned beneath the animal so that the filament is directly under the plantar surface of the foot to be tested. When a trial is initiated, the device raises the filament to touch the foot and progressively increases force until the animal withdraws its foot, or until it reaches a maximum of 40 g of force. The DPA automatically records the force at which the foot is withdrawn and the withdrawal latency (actually, latency and maximum force are directly related because the device progressively increases force until withdrawal occurs). Each paw is tested twice per session. This test does not require any special pretraining, just an acclimation period to the environment and testing procedure, although as will be seen, there is a training effect in which latency to withdraw decreases over time in normal rats. Testing was performed once per week for 2 weeks prior to surgery and 8 weeks after surgery.

bregma, 1.0 mm posterior, and 2.0 mm posterior to bregma at a depth of 1 mm from the cortical surface, and 0.5 Al of tracer was injected at each site. After completing the injections, the scalp was sutured, and rats were placed on soft bedding on a warming blanket held at 378C for 4 h after surgery. Tissue preparation Animals survived for 18 days after BDA injections, after which they were killed humanely with an overdose of EuthasolR (195 mg/ml pentobarbital sodium and 25 mg/ml phenytoin sodium; Delmarva Laboratories, Inc., Richmond, VA) and perfused transcardially with cold saline followed by cold 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (Na2HPO4), pH 7.4. The bladders from 8 rats that had received complete hemisections and 4 sham rats were removed and blotted dry for weighing. The length and width were measured while the bladder was lying flat. The spinal cords were carefully dissected out, keeping the dorsal roots attached, and the tissues were post-fixed for 4 h at 48C in 4% PFA, rinsed for 1 h in 0.1 M Na2HPO4, and then stored overnight at 48C in 30% sucrose buffer. Equilibrated tissues were immersed in TissueTekR (VWR International, West Chester, PA) and frozen using liquid nitrogen. The cervical dorsal roots were identified and used as reference points when preparing the tissue blocks for freezing. The injury site was identified visually and a block of tissue was cut that included two segments above and below the injury. Brains and spinal cords were embedded separately. Frozen tissue blocks were stored at 808C until ready to be sectioned with a cryostat. BDA staining

Mini-ruby BDA tracing of corticospinal tract (CST) projections CST projections were traced in 28 of the 49 total rats by injecting mini-ruby/biotinylated dextran amine (BDA) into the sensorimotor cortex at 8 weeks post-injury. Half of the rats received injections into the cortex on the side contralateral to the lesion to assess whether the CST was completely transected; the remaining rats received injections into the cortex ipsilateral to the hemisection injury to assess the integrity of the CST contralateral to the injury. For the BDA injections, animals were anesthetized as described above; the hair on the scalp was shaved and swabbed with betadine, and small holes were drilled in the skull over the sensorimotor cortex. A 10 Al Hamilton microsyringe fitted with a pulled glass micropipette was used to inject tetramethylrhodamine and biotin conjugated dextran amine, 10,000 MW, lysine fixable (mini-rubyBDA, Molecular Probes, Eugene, OR) at a total of 8 sites. Injection coordinates were 1.5 mm and 2.5 mm lateral to the midline at 1.0 mm anterior, centered on

Injections of mini-ruby BDA allowed tract tracing based on either fluorescence (for the mini-ruby) or histochemistry (for BDA). For all of the present experiments, histochemical staining for BDA was used. Hence, throughout the text, we refer to bBDA labelingQ. For BDA staining, the spinal cord was sectioned in the longitudinal, horizontal plane at 25 Am thickness using a cryostat. Serial sections were thaw-mounted onto polylysine treated slides (2 mg/ml; Sigma-Aldrich, St. Louis, MO) and stored at 808C until ready to be stained. For the staining procedure, sections were warmed for 1.5 h at 378C to promote adherence to slides. The tissue was rinsed for 5 min in 1 PBS (phosphate buffered saline) and 30 min in PBS with 0.3% H2O2 then incubated overnight at 48C with avidin and biotinylated horseradish peroxidase (Vectastain ABC Kit, Vector Labs, Burlingame, CA). The following day, the tissue was rinsed for 1 h in 1 PBS (on a shaker), 5 min in 0.1 M acetate buffer, and then reacted for 30 min in a 70 ml solution of 0.1 M acetate buffer containing 1.875 g nickel

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ammonium sulfate, 40 mg diaminobenzidine, 150 mg hd-glucose, 30 mg ammonium chloride, and 0.6 mg glucose oxidase (all chemicals from Sigma-Aldrich, St. Louis, MO). Slides were then rinsed 2 times in 0.1 M acetate buffer, dehydrated, and coverslipped. An advantage of this staining procedure was that axons were darkly stained and there was also light overall staining of gray matter, which allowed visualization of cytoarchitectural organization. For each labeled spinal cord (n = 28), all the sections were analyzed, from dorsal to ventral, and the completeness of the lesion (dorsally, laterally, and ventrally) was visually assessed, as well as the completeness of the lesioning of the dorsal CST. For those spinal cords in which CST tracing was not available (n = 9), the lesion was verified by cresyl violet staining. Thus, the lesions were histologically verified in a total of 37 animals. Statistical analysis On each testing day, the maximal average force (in grams) exerted on the GSM by each forepaw at the point just before grip was released was calculated from four trials per paw per time point for each rat. Then, for each time point, the average force was determined per paw for the animals in each group (hemisection and sham). A repeated measures two-way ANOVA was performed to identify differences between paws and time post-injury. The Bonferroni test was used for posthoc analysis to correct for multiple comparisons. A repeated measures one-way ANOVA was used to analyze changes in the contralateral forepaw over time. Post-hoc analysis was performed with the Tukey test. For the food pellet reaching task, a two-way ANOVA comparing hemisection versus sham-operated animals over time was performed to analyze differences in the percentage of successful retrievals in 1–2 reach attempts. The Bonferroni test was used for post-hoc analysis to correct for multiple comparisons. The mean number of pairs of footsteps taken with the forelimbs during the horizontal rope walking task was calculated from five trials per time point for each rat. Then, at each time point, the average was determined for each group. To analyze the change in number of sets of footsteps taken by sham animals over time, a one-way repeated measures ANOVA was performed followed by post-hoc analysis with the Tukey test. For analysis of DPA data, the average force needed to induce paw withdrawal (in grams) was calculated from two trials per paw per time point for each animal. For each group (hemisection and sham), the mean force was determined for each paw at each of the time points. As with the GSM, a repeated measures two-way ANOVA was performed to identify differences between paws and time post-injury for each of the groups and the Bonferroni test was used for post-hoc analysis to correct for multiple comparisons. To analyze each paw individually across the multiple time

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points, a repeated measures one-way ANOVA was used along with the Tukey test for post-hoc analysis.

Results Summary of animals As described in Table 1, a total of 49 animals were used for these studies (32 injured and 17 shams). Because the different behavioral tasks required different time investments, not all animals were tested on every task. Rather, the experiment was performed 3 separate times with the first experimental group being tested on the food pellet reaching task and the GSM, the second experimental group being tested on the food pellet reaching task, the GSM, and the DPA, and the third experimental group being tested on the GSM, the DPA, and the horizontal rope climbing (Table 1). Lesions were verified by histological analysis after the completion of testing for a total of 37 rats (12 sham and 25 SCI animals; see Table 2). Of these 37 histology cases, 14 of the lesions were complete hemisections that did not extend significantly across the midline and 12 sham cases showed no damage at all. In a few animals, however, lesions were incomplete (n = 8) or were over-hemisections in which the lesion extended across the midline (n = 3), and data from animals with incomplete lesions or over-hemisections were analyzed separately. The remaining 12 animals were not analyzed histologically and so the extent of the lesion was not verified. For these cases, we inferred the extent of the lesion based on performance in the grip strength task (whether individual animals exhibited functional recovery characteristic of partial lesions or the permanent deficits in function in the contralateral forelimb that were characteristic of overhemisections, see below). These cases were grouped separately based on the behavioral assessments (Table 2). Gripping strength in the digital flexors To assess function in the digital flexors, grip strength was measured using a grip strength meter as described previously for mice with cervical hemisections (Anderson et al., 2004). The data presented in Fig. 1 are from animals that were grouped post-hoc according to histological lesion verification. A baseline measure of grip strength was established during a 2 week training period prior to spinal cord injury. This training time is referred to as days 14 through 1. On day 3, the mean force exerted by either forepaw for all rats was 133.62 F 2.31 g (mean F standard deviation, n = 37). After receiving a C5 unilateral hemisection (n = 14), all observable voluntary gripping ability in the impaired forepaw was lost for the 8 week duration of the experiment (Fig. 1A). The forepaw ipsilateral to the lesion often became stiff, and the wrist was held in a flexed

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position, but no rhythmic spastic movements were observed. Thus, when the animal’s forepaw was drawn over the bar, the forepaw would passively hook onto the bar and the only force generated was the result of the stiffness of the forepaw as the experimenter pulled the animal away from the bar. Although a measurable force was recorded under these circumstances, trials in which the recorded response was due to paw stiffness were scored as b0Q force for the data in Fig. 1A because the animal did not grasp and release the bar

(active grasping was easily detected through careful observation during the task). This decision was made because the force recorded by the GSM was generated by the experimenter and is not an accurate reflection of hand function. In addition to the complete loss of gripping ability by the ipsilateral forepaw, there was a transient decrease in the strength of the forepaw contralateral to the injury during the first week post-injury. Statistical analysis of the force exerted by the contralateral forepaw of animals that received a hemisection starting with day 3 revealed a significant decrease in grip strength at days 1, 3, and 5 ( P b 0.05). Sham-operated animals (n = 12) did not exhibit any significant impairment in gripping ability (Fig. 1B). Fig. 1C shows the behavioral data from 8 rats with incomplete hemisections. Beginning during the third week post-injury, there was a gradual recovery of gripping ability in the forepaw ipsilateral to the lesion, but this plateaued and strength never fully returned to pre-injury or contralateral levels. Fig. 1D demonstrates the gripping ability of 3 rats with over-hemisections, in which the CST on the contralateral side was damaged. Grasping was permanently lost in the ipsilateral forepaw, as with bcompleteQ hemisections. In addition, grip strength was also impaired in the contralateral forepaw, although there was some recovery over time. The grip strength of the contralateral forepaw never fully recovered to pre-injury levels, however. Animals in which the lesion could not be verified by histology (n = 12) were analyzed separately. The extent of the lesion in these animals was inferred based on the performance on the gripping task. There were 5 animals that displayed a complete loss of gripping ability in the ipsilateral forepaw and a transient weakness in the contralateral forepaw (Fig. 2A). Additionally, 5 animals exhibited no loss of gripping ability in either forepaw, similar to shams (Fig. 2B). The final 2 animals displayed behavior patterns much like an incomplete hemisection (Fig. 2C). These results suggest that the gripping ability measured by the grip strength meter is predictive of the extent of unilateral hemisection, at least for lesions at the fifth cervical level. For the analyses of the remaining behavioral tests, these 12 animals (grouped as just described) were included with the animals in which the lesion was verified with histology. Fig. 1. Grip strength meter graphs based on verified lesion. Each graph represents the force (in grams) pulled by the ipsilateral and contralateral forepaw. (A) C5 unilateral hemisection (n = 14) results in permanent loss of grip strength in the ipsilateral forepaw. There is a temporary impairment in strength in the contralateral forepaw (*P b 0.05), which returns to preinjury levels after the first week post-injury. Error bars represent standard deviation. (B) Sham-operated controls (n = 12) show no behavioral impairments. Error bars represent standard deviation. (C) Incomplete lesion of the ipsilateral CST as indicated by behavioral improvement in grip strength of the ipsilateral forepaw (n = 8). Error bars represent standard deviation. (D) Over-hemisection of the contralateral CST as exemplified by behavioral impairment of grip strength in the contralateral forepaw (n = 3). Error bars represent standard deviation.

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Fig. 2. Grip strength meter graphs based on inferred lesion. (A) Complete hemisection (n = 5). (B) Sham-operated controls (n = 5). (C) Incomplete hemisection (n = 2). Error bars represent standard deviation.

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successfully retrieve pellets at all times post-injury ( P b 0.001). Moreover, rats that were able to retrieve pellets used adaptive strategies that were quite different than the welldefined strategies used by uninjured or sham-operated rats, as documented by the slow motion video analysis (below). Slow motion video analysis provides a sensitive and informative measure of the strategies employed by rats when retrieving pellets in which the different components of a reach and grasp can be analyzed. For example, Metz and Whishaw (2000) identify 4 main components of the task (Pronation, Grasp, Supination I, and Supination II) that can be further separated into 11 sub-components. The reach begins as the arm is extended through the slot in a pronated position with the paw open. The digits then contact the surface of the shelf in an arpeggio motion, in which digit 5 is the first to contact followed by 4, 3, and 2 (Whishaw and Gorny, 1994). The pellet is typically grasped between digits 3 and 4. After the pellet is grasped, the paw is then supinated and withdrawn from the slot then supinated further as the pellet is brought to the mouth. In the present experiments, sham-operated animals always extended their arm through the slot in a pronated fashion with the paw open, as described by Metz and Whishaw (2000). The digits contacted the surface of the shelf either in the arpeggio movement or appeared to make contact all at once. The apparently simultaneous contact may be due to the fact that our video recording was not as high-speed as that used by the Whishaw laboratory. The majority of the time (57%) the pellet was located between digits 3 and 4, but other times it was located either between digits 2 and 3 (22%) or 4 and 5 (21%). Finally, in 75% of the reaches scored, the paw was supinated and the pellet was brought to the mouth and released. Animals with complete hemisection injuries, however, displayed different strategies while reaching that were

Food pellet reaching task The food pellet reaching task is a more complex and highly functional task involving forelimb reaching and grasping with the digital flexors. This task requires an animal to reach through a slot with its forearm, grasp a food pellet that is sitting on a shelf in front of the slot with its paw, and bring the food pellet to its mouth. A simple way to assess an animals’ performance on this task is to determine the percentage of successful pellet retrievals with 1 or 2 reach attempts (this is also a measure of accuracy, as multiple reach attempts are usually required to successfully obtain a pellet when animals are first learning this task; over time, the ratio of successful retrievals to attempts increases to a plateau of about 60%). Fig. 3 demonstrates that shamoperated animals (n = 11) maintained a steady rate of accuracy on the task both before and after surgery (~60%). Animals that received a hemisection injury (n = 15), however, experienced severe impairments in the ability to

Fig. 3. Food pellet reaching. A representation of the percentage of successful retrievals of a food pellet within 1 or 2 reach attempts for both sham-operated and hemisection-injured animals. Sham-operated animals maintain a steady rate of retrievals following surgery (~60%) while hemisection-operated animals exhibit a significant decrease in pellet retrieval at all post-injury time intervals tested ( P b 0.001). Error bars represent standard error of the mean.

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consistent over time, indicating a lack of improvement. First to note, injured animals demonstrated far fewer reaches compared to sham-operated controls (93 attempts versus 556 for sham controls). Usually, the arm was extended through the slot in a pronated manner, but sometimes the arm was held in a half supinated position. In addition, the paw was often open, but 43% of the time, the paw was held in a closed fist. Another behavior sometimes used by animals with C5 hemisections was to set the paw down on the surface of the shelf in a half supinated position with the side of digit 5 being the only digit touching the surface, so that the pellet was cupped within the paw. The most obvious difference between injured and sham-operated animals was in the manner in which the paw/pellet was removed from the slot. On most reaches, injured animals held the paw in a pronated or partially supinated position, and the pellet was drawn across the surface of the shelf rather than being grasped and raised. As a result, the pellet often fell into the box at the edge of the shelf. Animals with complete hemisections exhibited these impairments for the duration of the post-injury testing period, as was also the case with gripping ability as measured with the grip strength meter. The reaching patterns adopted by animals with incomplete hemisections were similar to sham-operated animals in that the arm was extended through the slot in a pronated manner with the paw open. Similarly, the digits contacted the surface of the shelf in the arpeggio movement or all at once and the pellet was typically between digits 3 and 4. The major difference was that the animals still used adaptive methods to retrieve the pellet and remove the arm from the slot; however, 20% of the retrievals fit the bnormalQ criteria of grasping the pellet, supinating the paw, and bringing the pellet to the mouth. This behavior was not exhibited by any of the animals in the complete hemisection group. It is important to consider that animals that received a lesion were videotaped performing the reaching task during weeks 3 through 8 post-injury, but not during the first 2 weeks following injury. Therefore, it could not be determined whether animals with incomplete hemisections recovered reaching ability immediately following the lesion or if there was a period of initial impairment followed by gradual recovery. However, during the assessments between 3 to 8 weeks post-injury, there were no further changes in the reaching patterns employed by the animals with incomplete hemisections. Only 1 animal was analyzed that had an over-hemisection and it only reached twice and did not successfully bring the pellet to the mouth.

forelimbs while traversing the 80 cm distance of rope for both the hemisection and sham-operated rats, before and after surgery. The mean number of sets of footsteps taken for all animals (n = 16) on day 3 was 7.3 F 1.1. A repeated measures one-way ANOVA revealed a significant difference ( F 4,5 = 25.35, P b 0.0001) in the sets of footsteps taken by sham animals (n = 6) at days 1 and 5 post-injury, and post-hoc analysis with the Tukey test demonstrated that this was a significant increase ( P b 0.01) in the number of sets of footsteps when compared to pre-injury numbers (Fig. 4A). All rats with complete hemisection injuries (n = 10) were unable to traverse the rope for the first 8 days postinjury. At later post-lesion intervals, rats exhibited extreme variability in the ability to traverse the rope (Fig. 4B). Three rats were either unable or unwilling to perform the task, 3 rats began to traverse the rope with the forepaws by the end of week 2 but always took a greater number of pairs of footsteps compared to sham animals. The remaining 4 rats displayed sporadic ability to perform the task. Of the 3 rats that reliably recovered the ability to cross the rope, 1 was verified to be histologically incomplete by CST tracing methods and 2 were histologically complete. Of the 4 rats displaying sporadic ability to traverse the rope, 1 was histologically incomplete, 2 were histologically complete, and the histology was not available for one animal because of a histological failure. Of the 3 rats unwilling/unable to

Horizontal rope walking

Fig. 4. Horizontal rope walking. (A) The number of pairs of footsteps taken with the forelimbs in sham-operated animals, before and after surgery. (B) The number of pairs of footsteps taken with the forelimbs in hemisectioninjured animals, before and after surgery. The number over each time point indicates the number of animals that successfully traversed the rope on that testing day. Note the variability as described in the text. Error bars represent standard error of the mean.

The horizontal rope walking task requires full use of the body, and thus revealed not only forepaw deficits, but also impairments in hindlimb use and trunk stability. Figs. 4A–B demonstrate the number of sets of footsteps taken with the

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ever cross the rope, 2 were histologically complete and 1 was histologically incomplete. Not included here were 2 other rats determined to have over-hemisections; neither of these animals ever crossed the rope. Although some rats regained the ability to cross the rope, many deficits were qualitatively apparent. First, rats were not able to grasp the rope with the digits of the forepaw ipsilateral to the lesion. Rather, the paw was held in a fist, although the entire forelimb was placed against the rope to assist in balance while traversing the rope. Second, impairments in the hindlimb were observed with this task; most notably, the hindlimb ipsilateral to the lesion often extended off the rope and the rat would attempt to walk with the other 3 limbs. Third, similar impairments were observed regarding trunk instability. Often, the animals adopted compensatory posturing behaviors to help maintain balance on the rope, such as curving the body or shifting their weight to one side. The trunk balance in animals with sham lesions was not altered. Dynamic plantar aesthesiometer In order to analyze changes in sensation in response to mechanical stimulation, an automated version of von Frey hair testing was used (Dynamic Plantar Aesthesiometer, DPA). The mean force necessary to induce withdrawal of either forepaw for all animals on day 4 was 12.5 F 2.6 g. In animals that received a complete C5 hemisection (n = 17), the force necessary to elicit withdrawal of the forepaw ipsilateral to the lesion remained steady throughout the duration of the experiment and at day 52 the mean was 13.9 F 8.3 g (Fig. 5A). Interestingly, the force needed to induce withdrawal of the forepaw on the contralateral side decreased over time and at day 52, the mean was 4.3 F 3.9 g. This difference between paws was statistically significant at days 31, 38, 45, and 52 ( P b 0.05) post-injury. To further analyze the change over time in the forepaw contralateral to the lesion, a repeated measures one-way ANOVA was performed and a significant difference was revealed ( F 7,16 = 4.162, P b 0.005). Post-hoc analysis with the Tukey test demonstrated a significant decrease in force required to induce withdrawal at days 31, 38, 45, and 52 compared to day 3 post-injury ( P b 0.05). Sham animals (n = 12) demonstrated a similar decrease in force needed to trigger forepaw withdrawal over time (Fig. 5B) and this was statistically significant at days 17, 24, 31, 38, 45, and 52 compared to day 3 post-injury ( P b 0.05). These results suggest that, with an intact spinal cord, the force necessary to elicit forepaw withdrawal decreases with repeated testing, perhaps due to learning, whereas this decrease is not seen in the case of the forepaw ipsilateral to the hemisection injury. There were no demonstrable differences in the force needed to induce withdrawal of the hindpaws in either hemisection or sham-operated rats (Figs. 5C, D). Three animals not included in the complete hemisection group had an over-hemisection (data not shown). These animals displayed extensive variability, but the

Fig. 5. Dynamic plantar aesthesiometer. Graphs represent the average force required to induce paw withdrawal in response to mechanical stimulation. (A) Forepaw withdrawal for hemisection-injured animals. Note that the force needed to induce withdrawal of the ipsilateral forepaw remains steady while the force needed to stimulate the contralateral forepaw decreases (* denotes significant decrease in contralateral forepaw compared to ipsilateral, P b 0.05). (B) Forepaw withdrawal for sham-operated animals. (C) Hindpaw withdrawal for hemisection-injured rats. (D) Hindpaw withdrawal for shamoperated rats. Error bars represent standard error of the mean.

contralateral forepaw did not show the decrease in force needed to stimulate withdrawal to the same degree as the contralateral forepaw of animals with a complete hemi-

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section. A two-way ANOVA of the over-hemisection group showed no significant difference between ipsilateral and contralateral forepaws over time. A comparison of the contralateral paw of the complete hemisection group versus the contralateral paw of the over-hemisection group demonstrated no significant separation. There were 2 other animals not included in the complete hemisection group that had incomplete lesions and a two-way ANOVA revealed no significant difference between paws over time (data not shown). Again, these animals displayed extensive variability. No observable changes in bladder Cervical hemisections did not result in obvious impairments in bladder function. Rats were able to void the day following spinal cord surgery (although bladders were manually expressed twice per day for the first 5 days postinjury as a precautionary measure). To assess whether hemisection caused any gross abnormalities in the bladder that were not evident behaviorally, bladders from 8 complete hemisection animals and 4 sham animals were weighed and measured at the end of the experiment, immediately following perfusion. Other laboratories have previously correlated increased bladder weight to the development of a neurogenic bladder (Kojima et al., 1996; Pikov et al., 1998). The average weight of bladders was 0.10 F 0.036 g from injured rats and 0.10 F 0.013 g from sham animals. The average size of bladders was 15.8  7.1 cm for injured rats and 17.3  8.5 cm for sham rats. There were no significant differences between the two groups. CST tracing Since the CST is generally thought to be involved in the control of fine, voluntary movement of the forepaw and the GSM has been demonstrated to be a sensitive measure of grasping function, it was of interest to assess the relationship between gripping ability as measured by the GSM behavioral data and the extent of CST damage in animals (n = 28) in which the CST had been labeled by BDA injections into the motor cortex. In 9 cases, BDA labeling revealed a complete interruption of the ipsilateral CST without damage to the contralateral CST; in all of these cases, there was a permanent loss of gripping ability in the ipsilateral forepaw and a temporary impairment in strength of the contralateral forepaw during the first week post-injury, which then returned to pre-injury levels. Fig. 6B is a representative example of an animal with a bcompleteQ hemisection as demonstrated by BDA labeling and GSM data. In 3 cases, BDA labeling revealed an over-hemisection of the contralateral CST and in each of these cases the GSM data reflected an impairment of the contralateral forepaw. A representative example of an animal with an over-hemisection is depicted in Fig. 6A. Of the 4 animals that showed incomplete behavioral impairment on the GSM, 2 showed

incomplete lesioning of the ipsilateral CST as revealed by surviving BDA labeled CST axons that passed the lesion site (exemplified in Fig. 6C). Two additional cases could not be examined histologically due to technical problems. There were, however, 4 cases in which BDA tracing revealed that the lesion of the ipsilateral CST was incomplete, but these animals did not show recovery of gripping ability by the ipsilateral forepaw with the GSM. Ten cases of shamoperated animals were examined, and all showed no CST damage and no behavioral impairment on the GSM. For the cases in which both histological and behavioral data were available, the integrity of the dorsal CST appeared to be a good indicator of skilled forelimb reaching and grasping function.

Discussion Our goal in the present study was to compare the usefulness of different assessment tasks for evaluating deficits and recovery of forelimb function in rats following cervical hemisection lesions. All tasks reveal impairments in forelimb motor function following complete hemisections, and surprisingly, given previous findings in mice, there was no evidence of recovery of any forelimb motor ability. There was some recovery, however, when hemisections were incomplete. Also surprisingly, assessment of tactile sensitivity revealed experience-dependent changes in function in control forelimbs that were not seen when testing the forelimb ipsilateral to the injury. The decreases in withdrawal latency may represent a learned behavior that depends on descending motor pathways. In what follows, we will consider the advantages and disadvantages of each of the assessment tasks that we used. In considering the results, it is important to consider the anatomical nature of the injury. A complete hemisection at C5 interrupts descending motor pathways including the dorsal CST in the dorsal column and the dorsolateral CST (Casale et al., 1988; Goodman et al., 1966; Rouiller et al., 1991; Schreyer and Jones, 1982) and rubrospinal tract (Antal et al., 1992; Brown, 1974; Muir and Whishaw, 2000; Whishaw and Gorny, 1996; Whishaw et al., 1998) in the lateral column, which are important for distal flexor function. The hemisection also interrupts the vestibulospinal and reticulospinal tracts in the ventral funiculus, which are especially important for proximal musculature and extensors (Alstermark et al., 1981; Lawrence and Kuypers, 1968; Schrimsher and Reier, 1993), and the coeruleospinal tract projections to the ventral horn that modulate motor function (Proudfit and Clark, 1991). Ascending sensory fibers in the dorsal column and the spinothalamic tracts are also severed (Giesler et al., 1979, 1981, 1988). The hemisection also damages local circuitry at the segmental level, including motoneurons, second order sensory neurons, and interneurons serving the C5 segment (Schrimsher and Reier, 1992). The lower motoneur-

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Fig. 6. Relationship between CST damage and grip strength behavior. (A) A representative example of an animal that received an over-hemisection (right side of spinal cord plus part of the left CST). The ipsilateral CST is labeled with BDA and it can be seen that both CSTs are lesioned. The GSM graph for this animal shows impairment in the contralateral paw. (B) A representative example of an animal that received a full unilateral hemisection (right side of spinal cord). The ipsilateral CST is labeled with BDA and is completely lesioned. The GSM graph for this animal. (C) A representative example of an animal that received an incomplete hemisection (left side of spinal cord). The ipsilateral CST is labeled with BDA and is not completely lesioned. The GSM graph for this animal shows behavioral recovery in the ipsilateral paw. In all images, the arrow is pointing to the midline between both CSTs. Scale bar = 500 Am.

ons that would be directly damaged at C5 include those supplying the spinodeltoidius (shoulder), biceps (upper forearm), extensor carpi radialis longus (dorsal surface of lower forearm), extensor carpi radialis brevis (dorsal surface of lower forearm), and some of the motoneurons supplying the flexor carpi radialis (ventral surface of lower forearm) that run in longitudinal columns between C4 and C6 (McKenna et al., 2000). Thus, in terms of CST control of the forelimb, C5 injuries would produce mixed upper and lower motoneuron symptoms involving the digital flexors (both flaccid and spastic paralysis) and mainly lower

motoneuron symptoms involving the proximal extensors (flaccid paralysis). Grip strength meter Previous studies in mice demonstrated that the grip strength meter can provide a quantitative, sensitive, reproducible measure of strength of the digits of the forepaw (Anderson et al., in press). The present results indicate that the task is also useful for assessing forelimb function following cervical injury in rats. One noteworthy difference

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between rats and mice is that following a complete C5 hemisection in mice, grip strength recovers to about 50% of control levels at about 14 days post-injury, whereas in rats, complete hemisections cause a permanent loss of gripping ability in the affected forepaw. When hemisections were incomplete, however, there were profound deficits in gripping ability that recovered over time. Thus, in rats, the GSM is useful for measuring recovery only if the lesions are incomplete. In both mice and rats, CST projections to cervical segments are largely unilateral, but there are some recrossing projections. Thus, one possible explanation for the recovery that is seen in mice is that there could be sprouting of re-crossing projections or some functional change leading to enhanced efficacy of re-crossing CST pathways. If this is the mechanism, then the re-crossing projections may not be sufficient to mediate the recovery of gripping ability in rats. In this regard, it will be of interest to determine whether the extent of CST recrossing is greater in mice than in rats. The recovery that is seen after partial hemisections may be mediated by sprouting from surviving CST projections ipsilateral to the injury. Of course, it is also possible that gripping is not mediated by the CST alone or even predominantly and that other pathways are primarily responsible. Although gripping does require distal flexors, which are controlled by the CST, gripping is not a highly skilled task and may be determined in part by brainstem motor pathways like the rubrospinal tract or even the vestibulospinal or reticulospinal tract (because digital flexion is important for posture and balance). In considering the pathways that might be involved, it is noteworthy that, following partial injuries, there was a recovery of gripping ability. In all cases, the lesions were considered partial because they did not extend to the midline, and in some cases, there was evidence from BDA tracing for surviving CST projections. Nevertheless, in all animals, the lesions were complete laterally, and so descending rubrospinal axons would have been completely interrupted. Thus, if pathways other than the CST are involved in the recovery, the most likely candidates are the vestibulospinal or reticulospinal axons that descend in the medial part of the ventral column. It may also be that recovery is due to relays via propriospinal pathways. Further studies will be required to explore these issues. Food pellet reaching The food pellet reaching task is skilled and goaldirected and is the type of behavior that is mediated by the CST. The present results indicate that the ability to reach is severely impaired following complete hemisection injuries in rats. The food pellet reaching task provides a wealth of information about how rats use their forelimb and detailed analyses revealed substantial abnor-

malities in the fine aspects of the reach that did not recover over the post-lesion testing interval. In this regard, the results are consistent with the results of the grip strength task. Nevertheless, although the data are rich in detail, the food pellet reaching task has significant disadvantages because it does not provide direct quantitative measures (except in terms of percent success) and requires detailed off-line slow-motion video analysis to parse out the changes in reach strategy. Based on the data from the small number of animals that received incomplete lesions and displayed reaching strategies similar to sham-operated animals, with the exception of the final phase of grasping the pellet and supination, the reaching task did not appear to be as useful as the grip strength meter for measuring gradual recovery of function of the digits of the forepaw. Rope walking All animals learned the rope walking task, but many animals were unable or unwilling to cross the rope after cervical SCI. It might be possible to modify the task so as to overcome this shortcoming by increasing motivation, but without this, many animals cannot be assessed after injury. The rats that were able/willing to cross the rope exhibited profound deficits in forelimb function (they were unable to use the paw to grip the rope) although the forelimb was used to help maintain balance. There were also obvious deficits in hindlimb use and trunk stability. It would be feasible to develop a quantitative measure of these deficits, but the fact that many animals cannot be assessed detracts from the usability of the task. Tactile sensitivity We measured tactile sensitivity following C5 hemisections to determine whether animals exhibited evidence of mechanical allodynia and found no evidence of increased sensitivity in the affected forelimb. At the same time, the von Frey hair (DPA) assessments did reveal an interesting change in the response of the control forelimbs to applied mechanical stimuli. Specifically, with repeated testing, there was a progressive decrease in force required to induce withdrawal and a decrease in latency to withdraw. We report the force measurements, but the force required to induce withdrawal and the latency to withdraw are directly related because the DPA progressively increases force until withdrawal occurs. Thus, the changes could indicate either increased tactile sensitivity or a faster withdrawal to an applied mechanical stimulus. One interesting possibility is that the decrease in latency/force necessary to stimulate withdrawal is a learned response and that this learning is not seen when descending projections are interrupted. If this is the case, the change seen with repeated von Frey hair testing might provide another useful tool for assessing the integrity of descending pathways.

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Conclusions Hemisection injuries destroy all descending and ascending pathways on one side, and so it is not possible to relate the functional deficits seen here with damage to particular pathways. Nevertheless, previous studies provide some hints in this regard. Schrimsher and Reier (1992) assessed food pellet reaching following bilateral cervical contusion injuries at C4/5, which damaged the main CST in the dorsal columns as well as the dorsolateral and ventrolateral funiculi and most of the gray matter at the level of the lesion. They found that reaching success was correlated with sparing of the ascending tracts in the dorsal columns and sparing of the ventral white matter tracts, but when the main CST in the dorsal column was damaged, there was no recovery of function in the digits. Thus, reaching is dependent upon multiple ascending and descending systems. In this regard, the task may be useful as a general assessment for forelimb deficits, in the same way that the BBB is useful as a general measure of hindlimb locomotor function. In a subsequent study, Schrimsher and Reier (1993) demonstrated that focal lesions of the dorsal columns or the dorsolateral columns result in permanent impairments of grasping function in the digital flexors but did not impair overall reaching ability of the proximal forelimb. The dorsal columns contain the main component of descending CST axons, whereas the dorsolateral column contains the dorsolateral CST and rubrospinal tract. In contrast, lesions of the ventrolateral funiculi (which contains the ventral CST, reticulospinal, and vestibulospinal tracts) resulted in a mild forelimb reaching hypometria. Taken together, these studies suggest that gripping ability, which involves digital flexors, depends on the integrity of the dorsal and dorsolateral CST. Although the reaching task can reveal these deficits, the grip strength meter may provide a more convenient and quantitative measure of strength in the musculature of the digits. Of course in the present study, there was no detectable recovery of either dexterity (as measured by the reaching task) or grip strength following complete hemisections, and so it remains to be seen whether recovery of grip strength is correlated with recovery of gripping ability in the reaching task and whether recovery of these abilities, when it occurs (for example following incomplete hemisections), depends on plasticity of descending CST pathways.

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