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Department of Biomedical Engineering, Centre for Neuroscience, University of Alberta, Edmonton, Alberta, Canada
Submitted 16 September 2004; accepted in final form 8 March 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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), and the benefits are retained for some time after training (de Leon et al. 1999; Wernig et al. 1998). Clinical studies in humans suggest that the timing of training onset may be important because a long delay period between injury and commencement of training appears to reduce the beneficial effects of training regimens (Wernig et al. 1995). However, due to the inability to accurately measure lesion profiles and control the training delay period in humans, clinical results are very difficult to interpret. Although the effects of training onset on motor recovery has not been studied systematically in animals, it appears that training-induced recovery of treadmill walking in completely spinalized cats is similar whether step training is implemented 1 wk (de Leon et al. 1998) or 1 mo (Lovely et al. 1986) after injury. However, as mentioned by the authors, the criterion for measuring locomotor improvement was more stringent for animals trained 1 wk after injury; thus the two studies cannot be directly compared. Therefore the goal of this study was to develop an animal model of motor training after spinal cord injury and to examine how delaying the onset time of training by an appreciable amount (i.e., 3 mo) affects functional motor recovery. If early training after spinal cord injury is in fact more beneficial, then determining mechanisms that underlie this phenomenon may help to identify therapies that make the spinal cord more responsive to training after a long delay.
The unique challenges of human bipedal locomotion are difficult to model with quadrupedal animals. For instance, spinal cord injuries that are analogous anatomically, such as destruction of the crossed corticospinal tracts alone do not produce noticeable deficits in overground locomotion in rodents (Little et al. 1988; Metz et al. 1998; Muir and Whishaw 1999), whereas in humans, they are enough to produce a complete loss in walking ability (Nathan 1994). The anatomical findings in the human have recently been supported by studies using transcranial magnetic stimulation and demonstrate that in noninjured subjects, corticospinal inputs provide part of the drive to activate muscles for walking (Capaday et al. 1999; Petersen et al. 2001). In quadrupedal animals, recordings of corticospinal tract neurons have shown that the corticospinal pathway is directly involved in skilled movements and gait modifications (Beloozerova and Sirota 1993; Drew et al. 1996). Likewise, after damage to the crossed corticospinal tract alone, rats have difficulty traversing a horizontal ladder (grid test), a task that requires accurate placement of the feet onto cylindrical ladder rungs (Little et al. 1988; Metz and Whishaw 2002; Z'Graggen et al. 1998). Thus for our training model, we chose to study accurate stepping in rats because this task involves control from descending pathways much in the same way that descending control is involved in bipedal walking in humans.
In rats, a unilateral transection of the crossed corticospinal tract at the level of the pyramids produces a 20% error in the affected leg when crossing a horizontal ladder (Metz and Whishaw 2002; Z'Graggen et al. 1998). Because we wanted a larger deficit that potentially could produce a greater range of training-induced recovery, a more extensive lesion was required. The main descending tracts that are involved in obstacle avoidance and accurate placement of the limbs in cats and rats are the corticospinal and rubrospinal tracts (Lavoie and Drew 2002; Metz and Whishaw 2002; Widajewicz et al. 1994). Thus we performed a dorsal quadrant lesion, aiming to unilaterally sever the entire dorsal corticospinal tract and the majority of the rubrospinal tract to produce initial errors of >20% (Schucht et al. 2002). The advantage of using rats versus cats for this type of lesion is that the amount of damage to each of the dorsal corticospinal and rubrospinal tracts can be quantified from histological examination of the spinal cord due to the separate anatomical location of these tracts in the dorsal and dorsolateral funiculi (Brosamle and Schwab 1997; Schucht et al. 2002).
In summary, this study investigates a rat model of locomotor training on a horizontal ladder to test how a 3-mo delay in the onset of training after a spinal cord injury affects the recovery of accurate stepping in comparison to animals trained immediately after an injury. A lesion of moderate severity was performed so that significant training-induced recovery of accurate stepping could take place. Importantly, the amount of training that the animals received was directly controlled because accurate stepping, unlike overground walking, could not take place in the rat's regular home cage. Finally, recovery of descending (tactile and proprioceptive) and segmental (ground support response) reflexes was also examined to determine if the time course of the recovery of these reflexes was correlated with functional recovery in accurate stepping.
Parts of this paper have been presented in abstract form (Norrie et al. 2002).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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All procedures were conducted according to guidelines of the Canadian Council on Animal Care and approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee. Forty-three female adult Sprague-Dawley rats were studied. Animals were trained and tested on a raised horizontal ladder that was 1 m in total length and with a testing field of 0.8 m that allowed space for the animal to maneuver on each end (Fig. 1A). The testing field contained 10 rungs of 
1 cm in diameter that were spaced 3–8 cm apart, randomly allocated and rearranged regularly so animals could not memorize a pattern. Animals crossed freely, or were gently coaxed by the trainer, and a food reward was placed at the end of the ladder. A training session consisted of 10 consecutive ladder crossings; all animals were trained three times daily, 5 days/week for 3 wk, which was found to be sufficient for notable motor improvement in preliminary studies with immediately trained animals (n = 6). In eight immediately trained animals and in four delay-trained animals (see Delay protocol) the training period was extended to 6 and 5 wk, respectively.
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Stepping performance was measured only in the hindlimb that was ipsilateral to the lesion, i.e., the affected hindlimb. Error rate in stepping of the affected hindlimb was recorded as a percentage of the number of misplaced steps divided by the total number of steps on the ladder [100 x (steps with error/total steps taken)]. Misplaced steps were counted as any steps in which the foot or toes of the affected hindlimb descended past the rung midpoint, thereby causing failure of the animal to achieve weight support because the toes and ventral metatarsal pad did not land on the center of the rung (Fig. 1B). These foot slips correspond approximately to values of 0–3 in the foot fault score outlined by Metz and Whishaw (2002). Prior to surgery, animals were trained for 1 wk on the ladder to become acquainted with the ladder and handler. Intact rats had little trouble navigating the ladder, and at the end of the first week of prelesion training, the error rate was 2.5 ± 0.5% for the immediately trained group and 2.6 ± 0.6% for the delay trained group (not significantly different, P = 0.87). After surgery, rats were videotaped daily once training began. Error measurements in accurate stepping of the affected hindlimb were calculated from video recordings taken every second weekday by a person not involved with training and data collection, but not blinded to the training group. The error for each training day was calculated from the second of three training sessions that day, with an average error obtained from the 10 runs across the ladder during that session. The percent improvement in stepping error from the first day of training (day 0) to the last day of training (day 18) was calculated as 100 x (initial error -final error)/initial error.
Surgery
A T8 partial laminectomy was performed under anesthesia with hypnorm/versed (0.635 ml/kg and 6 mg/kg) delivered subcutaneously. A small incision was made in the dura mater and lidocaine was applied to the spinal cord to prevent reflexive movements during cutting of the cord. A fine razor, broken and filed down to a 1.5-mm width, was inserted to a depth of 1 mm at the midline, and was moved to the lateral edge of the spinal cord to unilaterally sever the thoracic (T8) dorsal and dorsolateral funiculi. Animals were allowed to recover from anesthesia on a water circulation blanket maintained at 37°C, and 0.05 mg/kg buprenorphine was given for analgesia.
Delay protocol
After surgery, training began immediately (3–4 days postoperative, n = 14 animals) or after a 3-mo delay period (n = 17 animals). Age matched controls (n = 6) were also studied; these animals were kept in their cages for 3 mo before the surgery then trained immediately after injury to account for the possibility of an aging effect on training. The mean ages and body masses of rats at commencement of training were: immediate group: 10 wk old, 220 g; 3-mo delay group: 22 wk old, 330 g; age-matched immediate group: 22 wk old, 310 g. All animals were considered to be mature, adult rats.
Histology
After training, animals were anesthetized with sodium pentobarbital (300 mg/kg) and then transcardially perfused with saline followed by a 4% paraformaldehyde fixative solution. Spinal cords were dissected free, placed in a paraformaldehyde solution overnight and then cryoprotected in 30% sucrose solution for 24 h. The tissue was then frozen in dry iced-cooled isopentane (–40°C) and stored in a –80°C freezer. The spinal cord surrounding the T8 lesion site (approximately T6–T12) was mounted in Tissue Tek freezing medium and 25-µm sagittal sections were sliced in a cryostat (Fig. 2A). The spinal cord was cut sagittally rather than transversely because this typically resulted in less tissue loss at the lesion site as intact tissue rostral and caudal to the lesion helped to support the tissue during cutting. Every second section was serially mounted on slides, cresyl violet or silver-stained as described in Kiernan (1999) and cover slipped. Typically, 35–40 sagittal sections were obtained for each spinal cord.
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Serial reconstruction of the lesion was done as described in Schucht et al. (2002). First, the mediolateral location of each serially mounted sagittal section was located on a representative transverse schematic of the T8 spinal cord (Fig. 2B) by using landmarks such as the central canal, gray and white matter. The most mid-line section(s) containing the central canal (e.g., Fig. 2A3) and the sections containing only white matter (e.g., Fig. 2A1) were first identified (marked at locations 3 and 1, respectively, in Fig. 2B). Sections between these positions were then located based on the profile of the gray and white matter in addition to the serial order of the section. For example, sagittal section 2 in Fig. 2A, which was the fourth section down from the mid-line section, was positioned slightly off mid-line at location 2 in Fig. 2B because the amount of gray matter (noted by the lighter stained tissue) comprised approximately one-third of the entire dorsoventral cord and the white matter above the gray matter was thicker than the white matter below, similar to the gray-white profile at location 2 in Fig. 2B. When spinal cords were sectioned slightly out of alignment in the sagittal plane, as noted by tapering bands of gray and white matter, the location of the section and the maximum extent of ventral damage to the spinal cord were taken from a smaller but consistent rostrocaudal location around the lesion site.
After the mediolateral location was identified the amount of dorsoventral damage (observed as a disruption of white matter tracts or scar formation) was measured for each sagittal section as shown for the white arrows on the three representative sagittal sections in Fig. 2A. The maximum depth of ventral damage for each sagittal section was then drawn on the corresponding location on the transverse schematic (black arrows in Fig. 2B). The total lesion area for each animal (e.g., medium shaded area in Fig. 2B) was then measured by joining together all points of maximum ventral damage for each sagittal section (at end of arrows in Fig. 2B). To measure the amount of damage sustained to each of the corticospinal and rubrospinal tracts, the location of these tracts, as obtained from anterograde labeling studies (Brosamle and Schwab 1997; Schucht et al. 2002), was drawn on the transverse schematic (dark shaded areas in Fig. 2B). A grid was placed over the schematic, and the number of squares that were within the lesioned area for a given tract were counted and expressed as a percentage of the total number of squares in the entire tract. The combined area of damage to the ipsilateral rubro- and corticospinal tracts and contralateral corticospinal tract was used as an indication of "total tract damage." Thus the measurement of total tract damage did not take into account the relative proportion of damage from each of the individual tracts. The amount of damage for each individual tract or all three tracts combined (total tract damage) was then compared with the initial (day 0) error and eventual percent improvement achieved (on day 18). In addition to the rubro- and corticospinal tracts, the extent of damage sustained to the ventrolateral funiculus located below the rubrospinal tract, which contains tracts from the reticulo- and vestibulospinal tracts (Schucht et al. 2002), was also measured. Thus damage to this area represents combined damage to the reticulo- and vestibulospinal tracts given that both tracts are interspersed throughout the ventrolateral funiculus (Shamboul 1980). Formation of fluid-filled cysts sometimes complicated the estimation of tract damage because such cavitations can compress white matter without compromising tract function (Olby and Blakemore 1996). Of 43 animals, the formation of cysts appreciably displaced and compressed white matter in 4 animals. When this happened, the proportion of damaged fibers in these compressed tracts, rather than the absolute depth of damage to the spinal cord, was indicated on the representative T8 transverse schematic. Lesion reconstruction was performed by an individual blinded to the tissue's training group.
Reflexes
Recovery of the mainly descending (tactile and proprioceptive) and segmental (ground support response) reflexes was examined in both the affected and unaffected leg to determine if they contributed to the recovery of accurate stepping. Reflexes were recorded 3 days after the injury and then weekly for 4 wk in animals receiving training (immediately trained group) or not receiving training (delay-trained group). To examine the tactile placing response, an animal was held upright with the tested hindlimb pendant and all other limbs tucked into the hand of the examiner. The dorsum of the paw was lightly brushed against a force transducer plate without producing movement of the ankle joint (Goldberger and Murray 1982; Kunkel-Bagden et al. 1993). Typically, a contact force of 
10 g did not result in movement of the ankle joint. A positive tactile placing response was considered to have occurred when the leg made a stepping motion over the plate. For the proprioceptive placing response, a visible movement of the ankle joint (10°) was required to evoke the reflex. The threshold to evoke the proprioceptive placing response was measured as the minimum contact force required to move the ankle before a placing response occurred. To record ground support responses, the animal was held upright with the tested hindlimb pendant over a scale. The plantar surface of the paw was slowly lowered onto the scale and the maximum reaction force applied onto the scale before the limb buckled was recorded for each leg (Helgren and Goldberger 1993). Percentage changes in reflex amplitude (ground support) or threshold (proprioceptive) were calculated as 100 x (prelesion reflex -postlesion reflex)/prelesion reflex.
Statistics
Differences between initial error, percent improvement, lesion size, and reflex responses between immediately and delay-trained animals were compared using a two-tailed, unpaired Student's t-test and a Mann-Whitney U test with statistical significance set to P < 0.05. Mean values ± SE are quoted in the text. Correlation analysis between initial error or percent improvement and total tract damage was performed using SigmaPlot 8 software (SPSS) to calculate the correlation coefficient (r, Pearson's product) of the relationship. Initial error or percent improvement in stepping performance was plotted against total tract damage and a linear regression line was fit to the data. When the data were not well represented by a straight line, a four-parameter sigmoidal curve was fit to the data using SigmaPlot 8 software. Goodness of fit of linear lines or sigmoidal curves was assessed by calculating the variance accounted for by the model (R2), which is equivalent to r2 for linear relations [R2 = 
(ym –
)2/(yd –); ym = model outcome, yd = data].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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After unilateral lesions of the dorsal quadrant, rats quickly recovered the ability to walk on a flat surface given that damage to the ventrolateral white matter was not appreciable (Schucht et al. 2002). In animals with large lesions (>40% ventrolateral funiculus damage, see Comparison of accurate stepping performance and lesion size), foot drag was present during walking on a flat surface but disappeared within 1 wk after the lesion. In contrast to overground walking, accurate stepping on the ladder was impaired in all animals. A large range of error values was present prior to training (initial error) in both the immediately trained animals tested at 3–4 day postlesion and in the delay trained animals that were allowed to move freely in their cages and then tested at 3 mo postlesion (Fig. 3). The average initial error for the immediately trained group [69.4 ± 5.3% (n = 20, 
)] was similar to that of the delay-trained group [62.7 ± 4.1 (n = 17, 
), P = 0.33] suggesting that impairments in accurate stepping did not improve spontaneously over time. However, there were more animals in the immediately trained group having initial errors >80% compared with the delay trained animals, most likely due to the acute effects of surgery. The variability of the initial error within the groups reflects the variability in lesion size (see following text).
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The 3-mo delay period reduced the amount of functional recovery in accurate stepping that was produced by training the animals three times per day, 5 day/wk for 3 wk (Fig. 4). By the second week of training (day 7, Fig. 4), the immediately trained group had, on average, a statistically greater amount of improvement [defined as 100 x (initial error/final error)/initial error] in accurate stepping compared with the delay trained group (46.3 ± 6.8% immediate vs. 26.6 ± 6.1% delay; significantly different at P = 0.04). The spread between the two groups increased over the course of training so that by the end of the 3-wk training period (day 18, Fig. 4) improvements in accurate stepping achieved by the immediately trained animals (61.5 ± 6.2%) were even greater than that achieved by the delay-trained animals (35.9 ± 7.1%, significantly different at P = 0.009). Immediately trained animals that were aged in their cages for 3 mo before receiving a dorsal quadrant lesion (age-matched controls, n = 6) showed improvements (60.3 ± 7.2%) that were not significantly different from the nonage-matched animals (62.5 ± 6.5%; P = 0.78). Therefore the age-matched controls were included in the overall immediately trained group in Fig. 4 (n = 20).
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Comparison of anatomical damage between the two groups
To ensure that differences in stepping improvement between the immediately and delay trained groups were not due to differences in lesion profiles, i.e., that the greater improvement in the immediately trained animals was not due to smaller lesions, the distribution of lesion sizes between the two groups was compared. A two-tailed unpaired Student's t-test demonstrated no statistical difference in the average total tract damage (i.e., combined damage to the corticospinal and rubrospinal tracts, see METHODS) between the immediate (57.5 ± 5.2%) and delay (60.8 ± 6.0, P = 0.68; Fig. 5) -trained animals. Likewise, the nonparametric Mann-Whitney test also demonstrated no significant difference between the mean ranks of total tract damage for the two groups (immediate 17.2 vs. delay 18.9, P = 0.55), indicating that the distribution of lesion sizes was similar in the two groups. Moreover, when animals were divided into three groups having an equal range of lesion sizes with small (10–40%), medium (40–70%) and large (70% –100%) total tract damage, the proportion of immediately and delay-trained animals in each sub-group was similar. For example, 25% of immediately trained and 24% of delay-trained animals fell into the small lesion group, 40% immediate and 35% delay-trained animals into the medium lesion group and 35% immediate and 41% delay-trained animals into the large lesion group.
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The values of initial error were plotted against total tract damage to determine if there was a relationship between these two variables (Fig. 6A). The amount of combined tract damage was significantly correlated to the initial error rate for both the immediately (r = 0.89, P < 0.001) and delay-trained (r = 0.77, P < 0.001) animals (r = correlation coefficient). When fitting a linear regression line to the data, a large percentage of the variance in initial error was accounted for by the linear model for immediately trained animals (R2 = 0.79) and delay-trained animals (R2 = 0.59). Linear regression lines fit through data comparing initial error to percent damage of individual tracts (ipsilateral corticospinal/rubrospinal or contralateral corticospinal tracts) resulted in lower R2 values (by 0.1–0.24, data not shown) compared with combined damage of all three tracts, indicating that measurement of total tract damage was a better determinant of initial error than individual tracts. The slope of the regression line fit through the data comparing initial error and total tract damage was greater for the immediately trained group (0.92) compared with the delay-trained group (0.53) because the immediately trained group had higher initial errors for lesions that were 
70%, most likely due to the acute effects of the surgery as mentioned previously.
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Regardless of the detailed relationship between tract damage and percent improvement, the critical point is that, for all lesion sizes, the delay-trained group had less percent improvement compared with the immediately trained group (the sigmoidal curve for the delay-trained group fell below the sigmoidal curve for the immediate-trained group in Fig. 6B). Delay-trained animals with <60% total tract damage had, on average, significantly lower percent improvement compared with immediately trained animals [lower by 21.3% (76.9–55.6%), P = 0.007]. Likewise, the average percent improvement of delay-trained animals for lesions >60% was 30.9% lower compared with immediately trained animals (42.7–11.8%, P = 0.02, statistically significant).
The extent of damage to the ventrolateral funiculus below the level of the rubrospinal tract, which contains the reticulospinal and vestibulospinal tracts, was also assessed. The immediately trained group had a slightly lower amount of damage to this region (14.0 ± 3.3%, range: 0–43%) compared with the delay trained animals (22.9 ± 4.8, range: 0–55%, not statistically different). Similar to the corticospinal and rubrospinal tracts when combined damage was <60%, the relationship between the amount of damage sustained to the ventrolateral funiculus was not statistically correlated to the initial error rate for both immediately (r = 0.30) and delay (r = 0.35)-trained animals. Likewise, percent improvement was not statistically correlated to damage of the ventrolateral funiculus (r = 0.12 immediate; r = 0.20 delay) (see also Schucht et al. 2002).
Comparison of stepping pattern
We observed that there were two stepping patterns that could be used for traversing the ladder: the affected leg could be placed ahead of the unaffected leg on each step (i.e., leading step), or it could be placed on the same rung as the unaffected limb (i.e., trailing step). To ensure that group differences in stepping pattern did not account for the group differences in accurate stepping, we observed the stepping pattern in a subset of training sessions (n = 10) for three animals in each group. Stepping patterns were compared on the first day of training when there was no difference in accurate stepping between groups and after 3 wk of training when there was a difference in accurate stepping. At the start of training, the immediately trained group took 75.7 ± 0.11% leading steps while the delay-trained group took 70.7 ± 0.10% leading steps (P = 0.21). At the end of 3 wk of training, the values were 82.6 ± 0.09% leading steps for the immediately trained group and 79.9 ± 0.10% for the delay-trained group (P = 0.58). Thus the stepping pattern was similar between groups and did not cause changes in accurate stepping or the discrepancy in accuracy between groups.
Time course of recovery of descending and segmental reflexes
Although the data described in the preceding text suggest that functional recovery is mediated by alterations in the activation of spared descending systems controlling accurate stepping, we analyzed some basic descending and segmental stepping-like reflexes that may also have contributed to the improved stepping performance on the ladder. The ground support response (see METHODS) was tested before and immediately after spinal cord injury in both immediately (n = 9) and delay-trained (n = 15) animals. In both groups, the ground support response displayed similar trends in that it decreased in the affected leg (open circles in Fig. 7, A and B) 3 days after injury compared with prelesion values (day 0, significant difference in both), and afterward, slowly increased with time. Interestingly, in both the delayed and immediate groups, at 28 days after the injury the ground support reflex in both the affected and unaffected leg increased above prelesion values (day 0, significant difference in the delay trained group only), even though the delay-trained group was not being trained during this period. Three months later when the delay-trained group was being trained, the ground support response did not change during the training period (not shown in Fig. 7). On average, only an 8% bilateral increase in the magnitude of the ground support response occurred after the 3-wk training period (not significantly different from values measured on the 1st day of training).
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; Bradley et al. 1983), was abolished in 24/27 animals after injury in that a light brushing to the dorsum of the paw produced a stepping response before the lesion but no longer did so after the lesion. In those animals, the tactile placing response never returned despite the recovery of accurate stepping. A preserved contact placing response was only observed in animals with 35% damage to the ipsilateral CST (n = 3 animals, similar to Basso et al. 2002) and in these animals this response did not undergo any marked changes with training.
The proprioceptive response, which is believed to have both descending and segmental components (De Ryck et al. 1992), remained intact after injury. However, the threshold for this response in the affected leg (Fig. 8, ) increased soon after injury in both the immediately trained group (by 45% at day 3, significantly different, Fig. 8A) and the delay-trained group (by 65% at day 7; significantly different, Fig. 8B) compared with prelesion control values (day 0). In the following weeks, the threshold to elicit a proprioceptive response decreased and returned to approximately prelesion values after 3 wk for both groups (not significantly different from day 0), irrespective of whether the animals were being trained (immediate group) or not (delay group). When the delay group was trained, the improvement in accurate stepping, while not as great as that of the immediate group, occurred without any corresponding changes in proprioceptive threshold. For example, on completion of training at 104 days postlesion, threshold values in the delay trained group were 104% (affected leg) and 109% (unaffected leg) of values recorded at 28 days after injury (not significantly different).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Mechanisms of training-induced recovery
Accurate placement of the hindlimb during walking on a ladder is not directly guided by visual inputs. On the ladder, the rat is unable to see its hind feet and thus must predict where to place them based on sensory information from the forelimbs, the intact hindlimb and/or based on remembered visual information. The lesion seems to interrupt this information relay, preventing accurate placement of the hindpaw due to the inability of spared pathways to accurately act on that information. Specifically, some of the errors in ladder walking may be due to a loss of cutaneous inputs reaching supra-spinal centers given that removal of cutaneous innervation to the hindpaw has dramatic affects on ladder walking (Bouyer and Rossignol 2003). Recovery of ladder walking after cutaneous denervation is abolished when lesions are applied to the motor cortex (Bouyer and Rossignol 2000) and suggests that the improvements in stepping errors observed from the dorsal quadrant lesions in our study, which also disrupts ascending cutaneous information to the sensorimotor cortex, may be due to the functional reorganization of spared corticospinal pathways.
Sprouting and/or strengthening of existing synaptic connections (Bach-y-Rita 1990), rather than sprouting of lesioned axons, are probably the main mechanisms of the observed functional recovery. It is unlikely that functional sprouting from lesioned axons can occur quickly enough to mediate the rapid improvement seen in the first week of training in the immediate group because sprouting of severed axons usually does not occur until 3 wk after injury (Hill et al. 2001). Thus reorganization of existing (spared) descending pathways is most likely responsible for the observed improvement. The cortico- and rubrospinal tracts are likely candidate descending pathways because extensive damage (>70%) to these tracts resulted in poor-training induced recovery of accurate stepping. However, a poor correlation existed between percent improvement and total tract damage for lesions <60%. This may be due to the poor resolution of reconstructing the lesion site using serial sagittal sections. Alternatively, the constant percent improvement for lesions between 10 and 60% total tract damage may be due to the fact that only a set number of spared connections is needed to produce a substantial amount of training-induced recovery of accurate stepping. When the number of spared connections falls below a critical number, reorganization of these few spared pathways is not sufficient to produce noticeable changes in motor function. In addition, other spared descending tracts such as the reticulo- and vestibulospinal pathways, may have contributed to the training-induced improvements in accurate stepping. Although the amount of sparing of reticulo- and vestibulospinal fibers was poorly correlated to the amount of recovery of accurate stepping achieved, damage to these tracts was not extensive (<60%) so it is not feasible to discount a contribution from these pathways to the recovery of accurate stepping.
Physiological effects of delay
The reduced benefits of training after a time delay can be attributed to the changes that occur in the brain and spinal cord during the delay. Specifically, we suggest that the immediate group is being trained for a specific behavioral function when synapses are most plastic and before the development of aberrant reflex connections (Calancie 1991). Potentially, transient increases in neurotrophin production in the spinal cord that occur within the first week after injury (Ikeda et al. 2001; Nakamura and Bregman 2001), facilitate the formation of new—or increase the strength of existing—synapses involved in accurate stepping. In addition, exercise-induced increases in neurotrophin production (e.g., BDNF) (Cotman and Berchtold 2002; Neeper et al. 1995; Ying et al. 2003) within spared circuits of the brain, brain stem, and spinal cord may be more effective when introduced early after a spinal cord lesion. The delay group receives no such timely behavior-specific training; thus reorganization of synapses in the cortex, brain stem and spinal cord that is not task-specific for ladder walking takes place. By the end of the delay period, a large degree of reorganization has likely occurred, including the formation of synapses that may serve functions not related to accurate stepping and that are not easily reversed (Tillerson and Miller 2002). This hypothesis can be tested by treating animals with therapies that promote reorganization of spared pathways, such as administration of factors that neutralize neurite growth inhibitors (IN-1 antibodies) (Raineteau et al. 2002) or factors that promote neurite growth (neurotrophins) (Bregman et al. 2002). An increase in task-specific plasticity of spared descending pathways should correlate with improvements in functional recovery, especially for the delay trained group.
Recovery of descending and segmental reflexes
Alterations in descending and segmental reflexes did not correlate with functional recovery on the ladder. While the tactile and proprioceptive placing responses bear some resemblance to the stepping motion on the horizontal ladder, there was no relation of improvement in accurate stepping to the reflexes studied. This is clear because the timeline of reflex recovery after spinal cord injury was similar for the immediate and delay groups, despite the fact that one group was being trained, whereas the other was not. Further, recovery of accurate stepping in the immediately trained group occurred before the recovery in ground support and proprioceptive reflexes, indicating that training had little impact on these reflexes, and these reflexes had little impact on improvement in accurate stepping. However, it should be noted that for simplicity the reflexes were studied in a stationary position rather than during walking and that task-dependent modulation of reflexes may still have occurred (Stein 1995). In addition to active reflex properties, in preliminary studies of four anesthetized animals, changes in the passive stiffness of leg muscles did not contribute to the differences in motor performance between the two groups. Passive stiffness in muscles of the affected and unaffected leg were nearly identical when measured after training, both in the immediately and delay-trained animals (unpublished results).
Interestingly, in both groups of animals, the ground support response after injury recovered to values that were greater than those recorded prior to the lesion, for both the affected and unaffected legs (Fig. 7). A part of this increase may have been due to a progressive increase in the weight of the animals as they aged during the 3-wk recording session. However, in the delay-trained group, no appreciable increases in ground support response were observed during training which occurred 3 mo after injury, even though these animals were still gaining weight at this time. Thus the bilateral increases in ground support responses that developed during the 3 wk after injury may have been due to heightened extensor reflex activity associated with the development of the spastic syndrome produced by unilateral damage to the dorsal aspect of the spinal cord (Carter et al. 1991; Heckman 1994). Such lesions reduce, bilaterally, descending inhibitory monoaminergic inputs to dorsal horn afferents and interneurons while at the same time leaving excitatory monoaminergic inputs to the ventral horn intact to produce heightened reflex activation of motoneuron pools on both sides of the spinal cord (Heckman 1994; Jankowska and Hammar 2002). The heightened ground support responses and associated extensor activity in both hindlimbs took 2–3 wk to develop and is consistent with the time course of development of muscle spasticity after spinal cord injury in rats and cats (Bennett et al. 1999; Carter et al. 1991). Excessive extension of the affected hindlimb between the ladder rungs also occurred during the foot slips and supports the idea of heightened spasticity as a result of the dorsal quadrant lesion. Alternatively, it is also possible that the increases in force with which the rats pushed against the ground was a learned response that cannot be ruled out in this study.
Functional implications
Our results indicate that rehabilitative training should begin as early as possible after incomplete spinal cord injury. In contrast, studies of motor training after cerebral ischemia have shown that initiating forced overuse of the impaired limb within the first week after injury actually increases motor deficits and tissue damage around the infarct (Humm et al. 1998; Kozlowski et al. 1996). It has been suggested that activity-induced increases in excitatory neurotransmitter release (e.g., glutamate and catecholamines) and/or brain temperature within the first 7 days after injury contributes to the loss of vulnerable tissue surrounding the lesion (Humm et al. 1999; Risedal et al. 1999). In spinal cord injury, training-induced neuronal plasticity occurs at sites that are remote from the thoracic lesion (e.g., hindlimb sensorimotor cortex, brain stem, and lumbar spinal cord) and, thus these areas are not susceptible to damage from increases in neuronal and metabolic activity (Risedal et al. 1999). Early training may increase damage to cell bodies near the thoracic lesion, such as oliogodendrocytes that do undergo apoptosis (Beattie et al. 2002), but not to axons traversing past the lesion. Thus when feasible, improving functional motor recovery via training should be optimal when initiated immediately after spinal cord injury.
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Address for reprint requests and other correspondence: M. Gorassini, 513 HMRC, Centre for Neuroscience, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada (E-mail: monica.gorassini{at}ualberta.ca,)
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) was not significantly different from that of the delay trained animals (62.7 ± 4.1, 
, 2-tailed Student's t-test, P = 0.33). Error bars represent SE. The variability within groups reflects the variability of the lesion size (see 
