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Plasticity of the human motor system following muscle reconstruction: a magnetic stimulation and functional magnetic resonance imaging study
Clinical Neurophysiology 114 (2003) 2434–2446 www.elsevier.com/locate/clinph
Plasticity of the human motor system following muscle reconstruction: a magnetic stimulation and functional magnetic resonance imaging study Robert Chena,*, Dimitri J. Anastakisb, Catherine T. Haywoodb, David J. Mikulisc, Ralph T. Manktelowb a
Division of Neurology, Toronto Western Research Institute, University Health Network, University of Toronto, Toronto, Ont., Canada Division of Plastic Surgery, Toronto Western Research Institute, University Health Network, University of Toronto, Toronto, Ont., Canada c Division of Medical Imaging, Toronto Western Research Institute, University Health Network, University of Toronto, Toronto, Ont., Canada b
Accepted 30 July 2003
Abstract Objective: Although motor system plasticity in response to neuromuscular injury has been documented, few studies have examined recovered and functioning muscles in the human. We examined brain changes in a group of patients who had a muscle transfer. Methods: Transcranial magnetic stimulation (TMS) was used to study a unique group of 9 patients who had upper extremity motor function restored using microneurovascular transfer of the gracilis muscle. The findings from the reconstructed muscle were compared to the homologous muscle of the intact arm. One patient was also studied with functional magnetic resonance imaging (fMRI). Results: TMS showed that the motor threshold and short interval intracortical inhibition was reduced on the transplanted side while at rest but not during muscle activation. The difference in motor threshold decreased with the time since surgery. TMS mapping showed no significant difference in the location and size of the representation of the reconstructed muscle in the motor cortex compared to the intact side. In one patient with reconstructed biceps muscle innervated by the intercostal nerves, both TMS mapping and fMRI showed that the upper limb area rather than the trunk area of the motor cortex controlled the reconstructed muscle. Conclusions: Plasticity occurs in cortical areas projecting to functionally relevant muscles. Changes in the neuronal level are not necessarily accompanied by changes in motor representation. Brain reorganization may involve multiple processes mediated by different mechanisms and continues to evolve long after the initial injury. Significance: Central nervous system plasticity following neuromuscular injury may have functional relevance. q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Motor cortex; Plasticity; Injury; Repair; Magnetic stimulation; Functional magnetic resonance imaging
1. Introduction The adult human brain maintains the ability to reorganize, adapt, and compensate for injury or changes in the environment (Sanes and Donoghue, 2000). Following transient deafferentation of the forearm, there are changes in the excitability (Brasil-Neto et al., 1993; Ridding and Rothwell, 1997), intracortical inhibition (Ziemann et al., 1998b,c) and blood flow (Sadato et al., 1995) in the contralateral motor cortex. Similarly, nerve injury or amputation leads to expansion of the cortical representation * Corresponding author. Toronto Western Hospital, 5W445, 399 Bathurst Street, Toronto, Ont. M5T 2S8, Canada. Tel.: þ1-416-603-5927; fax: þ 1416-603-5768. E-mail address: [email protected] (R. Chen).
of the muscle just proximal to the injury in rats (Donoghue et al., 1990; Sanes et al., 1990), primates (Schieber and Deuel, 1997; Wu and Kaas, 1999) and humans (Cohen et al., 1991; Kew et al., 1994; Pascual-Leone et al., 1996; Ridding and Rothwell, 1997; Roricht et al., 1999, 2001; Dettmers et al., 1999). Transcranial magnetic stimulation (TMS) studies in humans showed that the motor cortex contralateral to the amputation becomes more excitable (Cohen et al., 1991; Kew et al., 1994; Ridding and Rothwell, 1997; Chen et al., 1998a; Roricht et al., 1999; Dettmers et al., 1999) with reduced intracortical inhibition (Chen et al., 1998a). While motor system reorganization following peripheral injury is well documented, its functional significance remains unclear. Transient deafferentation of the forearm may increase the peak acceleration for elbow flexion
1388-2457/$30.00 q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S1388-2457(03)00283-9
R. Chen et al. / Clinical Neurophysiology 114 (2003) 2434–2446
movement (Ziemann et al., 2001). However, the muscle just proximal to amputation or transient deafferentation does not normally perform any useful function. Cutaneous anaesthesia of the hand was found to improve tactile spatial acuity of the contralateral hand (Werhahn et al., 2002). However, cortical reorganization following amputation can be associated with phantom pain (Flor et al., 1995) and may be maladaptive. Moreover, there was little evidence for functional changes in the deafferented somatosensory cortex leading some authors to question the functional relevance of brain plasticity after peripheral injury (Moore and Schady, 2000). At our institution, we have used a procedure known as free functioning muscle transfer (FFMT) to restore motor function in selected patients with severe neuromuscular injury (Manktelow and Zuker, 1989; Manktelow and Anastakis, 1999). FFMT involves microneurovascular transplantation of the gracilis muscle (a thigh adductor muscle) to replace a damaged upper limb muscle. The movements restored include shoulder flexion, elbow flexion, elbow extension, finger flexion and extension. In most cases, the gracilis muscle is reinnervated using a motor nerve of the upper extremity. In cases of complex brachial plexus avulsion with no available nerves in the paralyzed upper extremity, the transferred muscle is innervated by motor nerves outside the brachial plexus. The most common nerve used is the intercostal nerve (Manktelow and Anastakis, 1999). As with many surgical procedures, the final functional results vary from patient to patient despite a consistent technical approach. FFMT patients present a unique opportunity to examine motor system plasticity and recovery in functioning muscles. We investigated motor system reorganization with two complementary methods: TMS and functional magnetic resonance imaging (fMRI). TMS can measure different inhibitory and excitatory circuits in the motor cortex (Hallett,
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2000) while fMRI has high spatial resolution for assessment of motor representations. We hypothesize that adaptive changes in the motor system occur after FFMT and these changes are related to time after surgery and motor outcome.
2. Methods 2.1. Subjects Thirty-two patients had reconstruction of their upper extremity using FFMT at our institution between 1976 and 1999. Twelve patients were available for follow-up and 9 agreed to participate in the present study. The clinical information for the 9 patients (mean age 34.3 ^ 3.9 years) is summarized in Table 1. Each patient had suffered a devastating injury to the upper extremity that resulted in a significant functional loss. All subjects gave their written informed consent according to the declaration of Helsinki and the protocol was approved by the University Health Network Research Ethics Board. The gracilis muscle was the donor muscle in each transfer. The gracilis muscle was transferred to the arm and attached to the origin and insertion of the damaged muscle it was replacing. The new vascular and nerve supply was established by microneurovascular re-anastamosis with donor blood vessels and nerve (Manktelow and Zuker, 1989; Manktelow and Anastakis, 1999). 2.2. Clinical assessment The patients were examined by two investigators (DJA and CTH) independent of the physiological and fMRI studies. Muscle strength was assessed by the Medical Research Council scale (Medical Research Council, 1954). The motor outcome was graded as successful when
Table 1 Clinical information for the study patients Patient
Age/sex
Hand dominance
Injury
Time since surgery
Target muscle
Donor nerve
Muscle power and outcome
1 2 3 4 5 6 7
17M 42M 47M 41M 50M 30F 27F
Right Right Left Right Right Left Right
4 months 17 years 15 years 23 years 15 years 8 years 8 years
R biceps L biceps R finger flexors R finger flexors L finger flexors R finger flexors R deltoid
Intercostal nerves 2, 3 Musculo-cutaneousb Anterior interosseous Anterior interosseous Anterior interosseous Anterior interosseous Axillary
M3 fair M4 successful M4 successful M4 successful M4 successful M4 successful M4 successful
8 9
21F 32M
Right Right
C5,6,7 root avulsion Crush injurya Crush injury Crush injury Crush injury Crush injury Tumor resection (sarcoma) Atrophic deltoidc Laceration/trauma
1.25 years 6 years
R deltoid R triceps
Musculo-cutaneousd Axillary
M3 fair M4 successful
a
Crush injury involves significant trauma to the upper extremity with functional muscle loss. Awake nerve stimulation was used to differentiate sensory from motor nerve fibers within the musculocutaneous nerve to provide motor supply for the gracilis muscle transfer. c Complete deltoid paralysis – etiology idiopathic. d Motor branches to the brachialis were sacrificed and dissected proximally to provide motor supply for the gracilis muscle transfer. b
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the postoperative muscle strength was M4 or M5, as fair when the muscle strength was M3 and as poor with muscle strength of M2 or less. 2.3. Nerve conduction and transcranial magnetic stimulation studies Surface electromyogram (EMG) was recorded from the transferred gracilis muscle and from the analogous muscle (Table 1) on the intact side with disposable silver-silver chloride electrodes. The signal was amplified (Intronix Technologies Corporation Model 2024F, Bolton, Ont., Canada), filtered (bandpass 2 Hz– 5 kHz), digitized at 5 kHz (Micro 1401, Cambridge Electronics Design, Cambridge, UK) and stored in a laboratory computer for offline analysis. The compound muscle action potential (MMax) was determined by supramaximal electrical nerve stimulation. For deltoid, biceps and triceps muscles, brachial plexus stimulation at Erb’s point was used and for forearm flexors median nerve stimulation at the elbow was used. TMS was performed with a 7 cm figure-of-8 coil and two Magstim 200 stimulators (The Magstim Company, Whitland, UK) connected via a Bistim module. The coil was placed at the optimal position for eliciting motorevoked potentials (MEPs) from the target muscle. The optimal position was marked on the scalp to ensure identical placement of the coil throughout the experiment. The handle of the coil pointed backward and the induced current in the brain flowed from posterior to anterior. The FFMT and intact sides were studied in random order. The following TMS parameters were measured: Resting motor threshold (MT): Defined as the minimum stimulator output that produced MEPs of $ 50 mV in at least 5 out of 10 trials and was determined to the nearest 1% of the maximum stimulator power output. EMG silence was monitored on a computer screen and via speakers at high gain. Active MT: Defined as the minimum stimulator output that produced MEPs of $ 100 mV in at least 5 out of 10 trials. The EMG passed through a leaky integrator and the EMG level was displayed on an oscilloscope. With visual and auditory feedback, the subjects maintained a constant background contraction of 10% of the maximum integrated EMG. MEP recruitment curve and silent period (SP) duration: Stimulus intensities of 100, 110, 120, 130, 140 and 150% of the rest (for rest condition) or active (for active condition) MT were studied. Eight pulses at each stimulus intensity were delivered 6 s apart and the order of the stimulus intensities was random. Subjects were studied both at rest (rest condition) and during 10% maximum background contraction (active condition) in separate runs. The trials in the active condition were also used measurement of the SP. Short interval intracortical facilitation (SICF): The first wave SICF (also known as I-wave facilitation) was studied
using a paired pulse protocol similar to previous reports (Tokimura et al., 1996; Ziemann et al., 1998d; Chen and Garg, 2000). The first pulse was set to produce MEPs of about 1 mV for forearm flexors and 0.3 mV for deltoid, biceps or triceps muscle at rest and the second pulse was at rest MT. The first pulse alone (16 trials) and paired pulses at interstimulus intervals (ISIs) of 0.5, 0.7, 1.3, 1.5, 2.1, 2.3 ms (8 trials for each ISI) were delivered in random order. The rest and active conditions were studied in separate runs. Short interval intracortical inhibition (SICI) and intracortical facilitation (ICF): A paired-pulse protocol similar to that described by Kujirai et al. (1993) was used. The conditioning stimulus was set at 95% of active MT and the test stimulus evoked MEPs of about 1 mV for forearm flexors and 0.3 mV for deltoid, biceps or triceps muscle at rest. Single test pulse and paired pulses at ISI of 2 and 10 ms (10 trials for each condition) were delivered in random order. The rest and active conditions were studied in separate runs. Long interval intracortical inhibition (LICI): Suprathreshold conditioning and test pulses at ISIs of 50 –200 ms were used (Valls-Sole´ et al., 1992). The conditioning and test pulses were both set to produce 1 mV MEPs at rest. Single test pulse (20 trials) and paired pulses at ISIs of 50, 100, 150 and 200 ms (10 trials for each ISI) were delivered in random order. The rest and active conditions were studied in separate runs. Mapping of muscle representation: The cortical representation of the target muscle was studied by TMS mapping. A grid of 1 cm interval along the coronal axis and 2 cm interval along the saggital axis was drawn on the scalp. The coordinates of the scalp positions were expressed relative to the vertex (CZ, international 10 –20 system). Starting at the optimal position, 10 pulses at 120% rest MT were delivered 6 s apart. The coil was then moved to adjacent positions on the grid and the procedure repeated. Successive positions were stimulated until the area where MEPs were produced was surrounded by inactive positions. The rest and active conditions were studied in separate runs. Relationship between intercostal and biceps activation in Patient 1: Patient 1 differed from the other patients because the donor nerves were the intercostal nerves rather than a nerve from the injured arm. Therefore, the reconstructed muscle was innervated by thoracic rather than cervical spinal motoneurons. Additional studies were performed to examine the motor representations for the biceps and intercostal muscles. Physiological studies were performed 4 months after the FFMT operation. In addition to the nerve conduction and TMS studies described above, we determined whether the intercostal muscles and the biceps can be activated independent of each other. Surface EMG electrodes were placed on the reconstructed right biceps and the right fourth intercostal space at the mid-axillary line and 3 cm anterior. The effects of the patient at rest with normal respiration, performing volitional breathing at maximum
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effort and voluntary right elbow flexion at maximum effort were observed. 2.4. Data analysis The peak-to-peak MEP amplitude for each trial was measured offline. The MEP amplitude for MEP recruitment was also expressed as a percentage of the MMax to give an estimate of the percentage of motoneuron pool (%MMax) recruited. For SICF, SICI, ICF and LICI, the MEP amplitude for each conditioned trial was expressed as a ratio to the mean control (unconditioned) MEP amplitude for each subject. Ratios less than one indicate inhibition, and ratios greater than one indicate facilitation. The SP for each trial was measured offline from onset of the MEP to the first resumption of voluntary EMG activity. Values are expressed as mean ^ standard error. For the mapping study, the mean MEP amplitude of each active position was expressed a percentage of the site that elicited the maximum mean amplitude (normalized mean amplitude). The center of gravity (COG) and the number of top-half positions were calculated. The COG is an amplitude-weighted average and the lateral COG coordinate was computed as the sum of (lateral coordinate £ mean amplitude) for all active positions divided by the sum of mean amplitude for all active positions. The anterior-posterior COG coordinate was calculated similarly (Wassermann et al., 1992; Classen et al., 1998). The number of top-half positions was used as a measure of the map size and was the number of positions with mean amplitudes of at least 50% of the site with maximum mean amplitude (Classen et al., 1998). 2.5. Statistical analysis For MMax, rest MT, active MT, SICI and ICF, the FFMT and intact sides were compared by the paired t test. For MEP recruitment, the MEP amplitudes or %MMax recruited were analyzed with analysis of covariance (ANCOVA) with side (FFMT or intact) and stimulus intensity as factors in the ANCOVA model. For I-wave facilitation and LICI, the FFMT and intact sides were compared by analysis of variance (ANOVA) with repeated measures. The ISIs were the repeated measure. The relationship between time since FFMT, age at FFMT and the extent of reorganization was examined by ANCOVA. The factors in the ANCOVA model were time since FFMT and age at FFMT and the dependent variable was the ratio of the FFMT to intact sides for the measurements that were found to be significantly different between the two sides. For rest and active %MMax, the highest stimulus intensity in which both the FFMT and the intact sides were tested was used to calculate the FFMT/intact ratio. Variables that may affect the motor outcome were examined by logistic regression. The motor outcome was the dependent variable. Time
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since surgery and the ratio of the FFMT to intact sides for the measurements that were found to be significantly different between the two sides were the independent variables. Differences were considered significant if P # 0:05. 2.6. Functional magnetic resonance imaging studies fMRI studies were performed in Patient 1. Prior to the FFMT operation, the patient underwent a fMRI study performing volitional breathing consisting of rapid shallow breath at approximately two per second alternating with periods of normal, quiet breathing. A second fMRI study was performed 6 months after the FFMT operation. Self-paced, low amplitude elbow flexion (activates biceps) was carried out. Both the FFMT and intact sides were studied. The patient was trained to move at about 1 Hz and to make the movements as imperceptible as possible without ceasing the movement altogether. The movements were practiced by the patient before scanning and were observed during scanning to confirm that the amplitude of each movement was as low as possible. This ensured that image noise due to motion was minimized. Supports were placed under each subject’s upper extremity to further minimize unnecessary arm motions. Head motion was minimized by tightly cushioning the subject’s head within the head coil. 2.6.1. MR imaging The fMRI paradigm consisted of a block design alternating rest with movement every 15 s over 6 min for a total of 12 movement epochs and 12 rest epochs. Imaging was performed on a 1.5 Telsa scanner (Signa ‘Echospeed’ – GE Medical Systems, Milwaukee, WI) using a spiral gradient echo sequence. Twenty-eight contiguous slices of 4.5 mm thickness (no gaps) were obtained from the foramen magnum to the vertex. One spiral trajectory was collected per slice providing a 3.75 mm in-plane resolution. Other sequence parameters included: TR ¼ 2240 ms, TE ¼ 40 ms, flip angle ¼ 858 (set to the Ernst angle for maximum signal-to-noise within gray matter for TR ¼ 2240 ms and T1 of gray matter ¼ 850 ms). High resolution T1-weighted fast inversion recovery SPGR images were acquired at 1.5 mm slice thickness for co-registration with the functional images. 2.6.2. Data analysis Image analysis was performed offline using Stimulate (J.P. Strupp 1996, University of Minnesota Medical School) and AFNI (Analysis of Functional Neuroimage, R.W. Cox, The Medical College of Wisconsin) software. All images were corrected for head motion using automated image registration algorithms. Regions of activation were determined by cross-correlating the fMRI data to the predicted hemodynamic response (boxcar waveform) for each task using a pixel-by-pixel statistical analysis yielding r-values for all pixels in each image. Activation thresholds
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were set at two standard deviations above the mean r-value obtained from all pixels in each image.
3. Results 3.1. Clinical outcome The strength of the reconstructed muscle was greater than antigravity in all patients. Seven patients had successful outcomes (M4 or M5) while two patients had fair outcomes (M3). The motor outcome for each patient is shown in Table 1. 3.2. Nerve conduction studies Nerve conduction studies were performed in 8 patients and the MMax on the FFMT side (3.2 ^ 0.3 mV) was smaller than that of the intact side (13.1 ^ 0.8 mV, P ¼ 0:002) in all patients tested. 3.3. Motor thresholds and maximum MEP The rest and active MTs from TMS are shown in Fig. 1. There was a large between subject variation in part because we studied different muscles. As expected, forearm flexors tended to have lower MT than more proximal muscles (Fig. 1). In one patient the rest MT on the intact side was higher than maximum stimulator output. The rest MT was lower on the FFMT side (56 ^ 6.9%) than the intact side (68.4 ^ 8.6%) in all 9 patients (P ¼ 0:001, Fig. 1A).
However, the active MT was similar on the two sides (FFMT 51.9 ^ 5.9%, intact 53.1 ^ 4.1%, Fig. 1B). 3.4. MEP recruitment and silent period Rest MEP recruitment on the intact side cannot be tested in 3 patients and the higher stimulus intensities were omitted in some patients because of high motor threshold. Figs. 2A,B show the MEP amplitudes at different stimulus intensities. The effects of side (FFMT vs. intact) (P ¼ 0:04 rest, P ¼ 0:005 active) and stimulus intensity (P , 0:0001) were significant for both the rest and active conditions but their interaction was not significant. MEP amplitudes were higher on the intact than the FFMT side. Figs. 2C,D show the %MMax (MEP/MMax) recruited at different stimulus intensities. The %MMax recruited was higher for the FFMT side than the intact side. The effects of side (P ¼ 0:0004 rest, P ¼ 0:002 active) and stimulus intensity (P ¼ 0:005 rest, P ¼ 0:02 active) were significant for both the rest and active conditions and their interaction was not significant. The %MMax was higher on the FFMT than the intact side. The silent period was not significantly different between the FFMT and the intact sides (Fig. 2E). 3.5. SICF In 3 patients it was not possible to test SICF, SICI, ICF, LICI and TMS mapping at rest because the MEPs were too small even at maximum stimulator output. SICF was tested in 6 patients at rest and all 9 patients during voluntary contraction. The results are shown in Fig. 3. As expected, I-wave facilitation occurred at ISIs of 1.3 and 1.5 ms but not at 0.5, 0.7, 2.1 and 2.3 ms. The effect of ISI was significant but there was no significant difference between the FFMT and the intact sides. 3.6. SICI and ICF Resting SICI and ICF in the 6 patients tested is shown in Fig. 4A. The SICI was significantly reduced on the FFMT side (P ¼ 0:05, Figs. 4A,B) but the ICF did not differ significantly. SICI and ICF during voluntary contraction were tested in all 9 patients (Fig. 4C). SICI and ICF were reduced compared to rest but there was no significant difference between the FFMT and intact sides. 3.7. LICI
Fig. 1. Motor thresholds for magnetic stimulation. The rest (A); and active (B) motor thresholds for all 9 patients. Each line represents one subject and each error bar represents one standard error.
Rest LICI was tested in 6 patients and active LICI was tested all 9 patients. The effect of ISI was significant but there was no significant difference between the FFMT and the intact sides (Fig. 5).
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Fig. 2. MEP recruitment curve and silent period. (A, B) Show the MEP amplitude; and (C, D) show the %MMax (MEP amplitude divided by the MMax from peripheral nerve stimulation) for the rest (A, C); and active (B, D) conditions at different TMS intensities. (E) Shows the silent period. Each error bar represents one standard error.
3.8. TMS mapping TMS mapping at rest was performed in 6 patients and in all 9 patients during voluntary contraction. The location of the center of gravity was similar on the FFMT and the intact sides (Fig. 6). The number of
top-half positions (. 50% maximum) was also similar on the two sides (FFMT rest 3 ^ 0.4, active 3.9 ^ 0.4; intact rest 4.3 ^ 0.5, active 6 ^ 1.4). Patient 1 who had FFMT with the right biceps muscle innervated by the intercostal nerves to also showed similar map location and size on the two sides (Fig. 6B).
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Fig. 3. I wave facilitation for (A) rest; and (B) active conditions. The MEP amplitude for each interstimulus interval is expressed as a ratio to that of the first stimulus (S1) alone. There was no significant difference between the FFMT and intact sides. Each error bar represents one standard error.
3.9. Factors affecting extent of reorganization and motor outcome Since the MMax, rest %MMax, active %MMax, rest MT and rest SICI were found to be significantly different
between the FFMT and intact sides, the ratio of the FFMT to the intact sides for these measurements were used to examine factors that may affect the extent of reorganization. For the FFMT/intact ratio of MMax, rest %MMax and rest MT, the effects of time since FFMT was significant
Fig. 4. (A) Short interval intracortical inhibition (SICI, ISI 2 ms) and intracortical facilitation (ICF, ISI 10 ms) at rest for the FFMT and intact sides (6 patients). SICI was significantly reduced on the FFMT side compared to the intact side. (B) Rest SICI with each line representing one subject. (C) SICI and ICF in the active condition (9 patients). Each error bar represents one standard error.
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Fig. 5. Long interval intracortical inhibition (LICI) at rest (A) (6 patients); and active (B) (9 patients) conditions. There was no significant difference between the FFMT and intact sides. Each error bar represents one standard error.
(P ¼ 0:016 for MMax, P ¼ 0:026 for rest %MMax, P ¼ 0:008 for rest MT, ANCOVA) but the effect of age at the time of FFMT was not. The MMax on the FFMT side increased with time since surgery (Fig. 7A) and the difference in rest %MMax (Fig. 7B) and rest MT (Fig. 7C) between the FFMT and intact sides decreased with time since surgery. For active %MMax and rest SICI, the effects of time since FFMT and age at FFMT were not significant. Logistic regression for motor outcome showed a significant likelihood ratio for time since surgery (P , 0:0001), MMax (P ¼ 0:014) and rest MT (P ¼ 0:014) but there was no significant effect of rest and active %MMax and rest SICI.
3.10. Relationship between intercostal and biceps activation in Patient 1 Surface EMG recording showed rhythmic activity in the intercostal muscle and no activity in the reconstructed biceps during both normal breathing and voluntary breathing at maximum effort. With the biceps activated at maximum effort, there was no activity in the intercostal muscles.
3.11. Functional magnetic resonance imaging Volitional breathing prior to FFMT operation activated the primary motor cortex medial to the upper-limb area (Fig. 8A) but not the upper-limb area. Six months after FFMT the patient voluntarily contract the reconstructed right biceps innervated by the intercostal nerves and had anti-gravity strength. Right elbow flexion activated the upper limb area of the left motor cortex (Figs. 8B,C), similar to activation of the right motor cortex with movement of the intact left biceps (Figs. 8D,E).
Fig. 6. (A) Location of center of gravity (COG) of TMS maps for the rest (6 patients) and active conditions (9 patients). The bars in the medial-lateral column show the distance of the COG lateral to the vertex and the bars in the anterior-posterior column show the distance of the COG anterior to the vertex. There was no significant difference between the FFMT and intact sides. Error bars represent standard error. (B) TMS maps at rest for the patient 1 with FFMT involving intercostal nerves to the right biceps muscle. The vertex is the origin of the coordinates (0,0). The MEP amplitude of each point is expressed as a percentage of the position with maximum MEP amplitude on the same side. The size and location for the motor map for the right (FFMT) biceps (left hemisphere) is similar to that of the left (intact) biceps (right hemisphere).
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Fig. 7. The effects of time after FFMT. (A) The ratio of MMax for the FFMT to the intact side plotted against time after FFMT. Data from 8 patients who had nerve conduction studies. The ratio increased with time (P ¼ 0:016). (B) The ratio of rest % MMax for the FFMT to the intact side plotted against time after FFMT. Data from 6 patients. Three patients were excluded as rest MEP recruitment cannot be studied on the intact side due to high motor threshold. The ratio decreased with time (P ¼ 0:026). (C) The ratio of rest MT for the FFMT to the intact side plotted against time after FFMT. Data from 8 patients. One patient with threshold on the intact side higher than the maximum stimulator output was excluded. The ratio increased with time (P ¼ 0:008).
4. Discussion We used TMS and fMRI to study motor system reorganization following FFMT and found evidence of plasticity in the motor projection to functioning muscles. The reduced rest MT and SICI on the reconstructed side compared to the intact side is similar to the changes following lower limb amputation (Chen et al., 1998a). The rest MT may be related to membrane excitability in the motor cortex since it is increased by drugs that block voltage-gated sodium channels (Ziemann et al., 1996a; Chen et al., 1997) but is unaffected by drugs that alter GABA (Ziemann et al., 1996a) transmission. Our data do not allow us to distinguish whether the reduction in MT occurred predominately in cortical or subcortical sites. However, there is considerable evidence that SICI for the hand (Kujirai et al., 1993; Nakamura et al., 1997; Di Lazzaro et al., 1998) and proximal upper or lower limb representations (Chen et al., 1998a; Chen et al., 1998b) are mediated by cortical inhibitory mechanisms. SICI can be enhanced by drugs that increase GABAA activity (Ziemann
et al., 1996a) and by antiglutaminergic drugs (Liepert et al., 1997; Ziemann et al., 1998a). These finding are consistent with the hypothesis that motor cortex plasticity may involve adjustment of balance between inhibition and facilitation in the intrinsic horizontal connections mediated by GABAergic and glutaminergic mechanisms (Jacobs and Donoghue, 1991; Sanes and Donoghue, 2000). However, other mechanisms such as long-term potentiation (Hess and Donoghue, 1994) and axonal sprouting with formation of new synapses (Jones and Schallert, 1994) may also contribute to cortical plasticity (Sanes and Donoghue, 2000). The inhibitory circuits mediating LICI and SP and the excitatory circuits mediating ICF and SICF were not significantly altered by the muscle reconstruction procedure. The later part (. 50 ms) of the SP and LICI at ISIs of more than 50 ms (Fuhr et al., 1991; Chen et al., 1999) is due to cortical inhibition. SICI and LICI are mediated by different cortical inhibitory mechanisms (Sanger et al., 2001). LICI and SP may be mediated by GABAB receptors while SICI may predominately involve GABA A
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Fig. 8. fMRI color maps for Patient 1 showing areas of task related cortical activation. Threshold for activation was set to show pixels with r-values .2 standard deviations above the mean r-value for all pixels in the image. (A) Activation (blue arrows) in the medial aspect of both primary motor cortices during volitional breathing. (B, C) Activation maps in the same patient 6 months following surgery using a right biceps muscle transfer to restore elbow flexion. The patient was performing low amplitude elbow flexion using the intercostal nerves. The cortical areas activated are located in the primary motor cortex controlling upper extremity movement. They are clearly separate from the area activated by volitional breathing seen in (A) (blue arrows). (D, E) Cortical activation during flexion of the normal left elbow. The areas activated are in similar location (laterality) along the motor cortex for each extremity.
mechanisms (Siebner et al., 1998; Werhahn et al., 1999; Sanger et al., 2001). ICF (Ziemann et al., 1996b; Chen et al., 1998b) and SICF (Ziemann et al., 1998d) are also mediated by mechanisms distinct from SICI. Thus, our findings suggest that FFMT induce specific changes in the motor cortex affecting the circuits mediating SICI and MT but not the other excitatory and inhibitory circuits we tested. We found no change in the motor representation of the target muscle following FFMT with TMS mapping and fMRI. This indicates that alterations in the neuronal level, as demonstrated by changes SICI and MT, are not necessarily accompanied by changes in the motor representational level. The changes following FFMT are different from the expansion and shift of motor representation following nerve injury or amputation in animals (Donoghue et al., 1990; Sanes et al., 1990; Schieber and Deuel, 1997; Wu and Kaas, 1999) and humans (Cohen et al., 1991; Kew et al., 1994; Pascual-Leone et al., 1996; Ridding and Rothwell, 1997; Roricht et al., 1999; Dettmers et al., 1999) and following hand replantation in humans (Roricht et al., 2001). Following peripheral nerve injury, amputation and even hand replantation (Roricht et al., 2001), large areas of the body are deafferented. FFMT procedures do not lead to significant deafferentation because we strived to use a predominately motor donor nerve
although some patients had a small degree of deafferentation due to other nerve injuries. Deafferentation may be necessary for shift and expansion of motor representation to occur. The change in MT and SICI following FFMT was evident only with the muscle relaxed and not during voluntary contraction. It has been argued that changes observed in the resting state but not during muscle activation may be due to modulation in corticospinal excitability rather than change in cortical connectivity (Siebner and Rothwell, 2003). While decrease in rest MT may be related to increased corticospinal excitability, reduction in SICI likely involved changes in efficacy of inhibitory synapses. Moreover, we found no change in others measures of corticospinal excitability such as TMS map area (Siebner and Rothwell, 2003). The need to precisely control the excitability of cortical neurons to perform specific voluntary activity may account for the absence of significant change during voluntary activation. There are similar observations in other settings of cortical plasticity. Transient anesthesia of the forearm caused increased blood flow to the primary sensorimotor cortex at rest but not during biceps movement, implying that movement-related changes in cerebral blood flow was decreased (Sadato et al., 1995). In the muscle proximal to
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forearm ischemia or amputation, MEP amplitude and map area was increased at rest but not during muscle activation (Ridding and Rothwell, 1997) and the increase in MEP amplitude following immobilization occurred only with the target muscle tested at rest (Zanette et al., 1997). These observations suggest that reduced MT and SICI in the motor cortex may make it easier to activate corticospinal neurons to produce voluntary activity with less excitatory synaptic drive. This is supported by the progressive reduction in SICI just prior to voluntary movement (Reynolds and Ashby, 1999) and the finding that forearm deafferentation leads to faster elbow flexion movement (Ziemann et al., 2001). The lower MMax from peripheral nerve stimulation on the FFMT side than the normal side may indicate a lower degree of innervation for the reconstructed muscle. However, this may also reflect the greater muscle mass in intact upper extremity compared to the transferred gracilis muscle and in some cases difficulty in locating the reconstructed nerve for nerve conduction studies due to anatomical distortions from the operation. The increase in MMax with time suggests that the nerve regeneration continued long after the FFMT operation. While our small numbers need to be interpreted with caution, clinical experience with peripheral nerve injury has suggested that nerve regeneration occurred over a longer time than can be expected from an axonal regeneration rate of 1 – 3 mm per day (Sunderland, 1978; Kline and Hudson, 1995). Motor improvement may continue for 5– 7 years following nerve repair (Dagum, 1998). Training and increasing use of the muscle in activities of daily living may also contribute to the long-term changes in peripheral innervation and motor function. While the motor outcome of FFMT improved with time, the change in rest %MMax and rest MT diminished with time suggesting that the maximum change in these parameters probably occurred early. These measures may represent one step in motor reorganization that involves various mechanisms at different times. It seems likely that the motor reorganization continues to evolve and may be modified by training and experience long after the muscle reconstruction procedure. The increased %MMax and reduced MT may also be a compensatory mechanism for deficits in peripheral innervation which improved with time. Although we found no relationship between motor outcome and physiological changes, this may be related to the low sensitivity of the outcome measure. Patient 1 differed from the other patients because the donor intercostal nerve originally innervated truncal muscles rather than the upper-limb muscles. Our finding that volitional inspiration prior to the FFMT activated the motor cortex medial to the upper-limb area is similar to previous neuroimaging studies of volitional breathing that used passive mechanical ventilation in the rest period (Colebatch et al., 1991; Evans et al., 1999). Six months after the FFMT procedure, TMS mapping (Fig. 6) and fMRI
(Fig. 8) showed that the control of motoneurons that originally innervated the intercostal muscles (in the T2 and T3 spinal levels) had shifted to the upper-limb area of the motor cortex. Our results are similar to that of Mano et al. (1995) who reported that following coaptation of the musculocutaneous and intercostal nerves for treatment of cervical root avulsion, TMS mapping showed that cortical representation for the biceps was initially located in the areas of the intercostal muscles. It gradually moved laterally to the upper-limb area as the patients gained voluntary control of the biceps muscle (Mano et al., 1995). Thus, the adult human motor cortex is capable of relatively large scale reorganization. While changes in MT and SICI have been demonstrated in muscles proximal to transient deafferentation (Ziemann et al., 1998b) or amputation (Chen et al., 1998a), our results show that these changes occur in functioning muscles. Successful outcome following FFMT probably requires adaptive changes in the central nervous system because the movement mediated by the transferred muscle is often different from that mediated by the donor nerve and because of the random nature of axonal regeneration from the proximal stump into the distal pathways. Clinical experience suggested a period of rehabilitation and retraining is necessary for successful outcome (Manktelow and Anastakis, 1999) and there is evidence that cortical plasticity occurs with motor learning (Sanes and Donoghue, 2000). In conclusion, our results provided evidence for plasticity of the motor system projecting to functionally relevant muscles. Changes in the neuronal level are not necessarily accompanied by changes in motor representation and motor reorganization continued to evolve long after the initial injury.
Acknowledgements This work was supported by the Workplace Safety and Insurance Board of Ontario, the Canadian Institutes of Health Research, the Canada Foundation for Innovation, Ontario Innovation Trust and the University Health Network Krembil Family Chair in Neurology. RC is a Canadian Institutes of Health Research New Investigator.
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Plasticity of the human motor system following muscle reconstruction: a magnetic stimulation and functional magnetic resonance imaging study Robert Chena,*, Dimitri J. Anastakisb, Catherine T. Haywoodb, David J. Mikulisc, Ralph T. Manktelowb a
Division of Neurology, Toronto Western Research Institute, University Health Network, University of Toronto, Toronto, Ont., Canada Division of Plastic Surgery, Toronto Western Research Institute, University Health Network, University of Toronto, Toronto, Ont., Canada c Division of Medical Imaging, Toronto Western Research Institute, University Health Network, University of Toronto, Toronto, Ont., Canada b
Accepted 30 July 2003
Abstract Objective: Although motor system plasticity in response to neuromuscular injury has been documented, few studies have examined recovered and functioning muscles in the human. We examined brain changes in a group of patients who had a muscle transfer. Methods: Transcranial magnetic stimulation (TMS) was used to study a unique group of 9 patients who had upper extremity motor function restored using microneurovascular transfer of the gracilis muscle. The findings from the reconstructed muscle were compared to the homologous muscle of the intact arm. One patient was also studied with functional magnetic resonance imaging (fMRI). Results: TMS showed that the motor threshold and short interval intracortical inhibition was reduced on the transplanted side while at rest but not during muscle activation. The difference in motor threshold decreased with the time since surgery. TMS mapping showed no significant difference in the location and size of the representation of the reconstructed muscle in the motor cortex compared to the intact side. In one patient with reconstructed biceps muscle innervated by the intercostal nerves, both TMS mapping and fMRI showed that the upper limb area rather than the trunk area of the motor cortex controlled the reconstructed muscle. Conclusions: Plasticity occurs in cortical areas projecting to functionally relevant muscles. Changes in the neuronal level are not necessarily accompanied by changes in motor representation. Brain reorganization may involve multiple processes mediated by different mechanisms and continues to evolve long after the initial injury. Significance: Central nervous system plasticity following neuromuscular injury may have functional relevance. q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Motor cortex; Plasticity; Injury; Repair; Magnetic stimulation; Functional magnetic resonance imaging
1. Introduction The adult human brain maintains the ability to reorganize, adapt, and compensate for injury or changes in the environment (Sanes and Donoghue, 2000). Following transient deafferentation of the forearm, there are changes in the excitability (Brasil-Neto et al., 1993; Ridding and Rothwell, 1997), intracortical inhibition (Ziemann et al., 1998b,c) and blood flow (Sadato et al., 1995) in the contralateral motor cortex. Similarly, nerve injury or amputation leads to expansion of the cortical representation * Corresponding author. Toronto Western Hospital, 5W445, 399 Bathurst Street, Toronto, Ont. M5T 2S8, Canada. Tel.: þ1-416-603-5927; fax: þ 1416-603-5768. E-mail address: [email protected] (R. Chen).
of the muscle just proximal to the injury in rats (Donoghue et al., 1990; Sanes et al., 1990), primates (Schieber and Deuel, 1997; Wu and Kaas, 1999) and humans (Cohen et al., 1991; Kew et al., 1994; Pascual-Leone et al., 1996; Ridding and Rothwell, 1997; Roricht et al., 1999, 2001; Dettmers et al., 1999). Transcranial magnetic stimulation (TMS) studies in humans showed that the motor cortex contralateral to the amputation becomes more excitable (Cohen et al., 1991; Kew et al., 1994; Ridding and Rothwell, 1997; Chen et al., 1998a; Roricht et al., 1999; Dettmers et al., 1999) with reduced intracortical inhibition (Chen et al., 1998a). While motor system reorganization following peripheral injury is well documented, its functional significance remains unclear. Transient deafferentation of the forearm may increase the peak acceleration for elbow flexion
1388-2457/$30.00 q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S1388-2457(03)00283-9
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movement (Ziemann et al., 2001). However, the muscle just proximal to amputation or transient deafferentation does not normally perform any useful function. Cutaneous anaesthesia of the hand was found to improve tactile spatial acuity of the contralateral hand (Werhahn et al., 2002). However, cortical reorganization following amputation can be associated with phantom pain (Flor et al., 1995) and may be maladaptive. Moreover, there was little evidence for functional changes in the deafferented somatosensory cortex leading some authors to question the functional relevance of brain plasticity after peripheral injury (Moore and Schady, 2000). At our institution, we have used a procedure known as free functioning muscle transfer (FFMT) to restore motor function in selected patients with severe neuromuscular injury (Manktelow and Zuker, 1989; Manktelow and Anastakis, 1999). FFMT involves microneurovascular transplantation of the gracilis muscle (a thigh adductor muscle) to replace a damaged upper limb muscle. The movements restored include shoulder flexion, elbow flexion, elbow extension, finger flexion and extension. In most cases, the gracilis muscle is reinnervated using a motor nerve of the upper extremity. In cases of complex brachial plexus avulsion with no available nerves in the paralyzed upper extremity, the transferred muscle is innervated by motor nerves outside the brachial plexus. The most common nerve used is the intercostal nerve (Manktelow and Anastakis, 1999). As with many surgical procedures, the final functional results vary from patient to patient despite a consistent technical approach. FFMT patients present a unique opportunity to examine motor system plasticity and recovery in functioning muscles. We investigated motor system reorganization with two complementary methods: TMS and functional magnetic resonance imaging (fMRI). TMS can measure different inhibitory and excitatory circuits in the motor cortex (Hallett,
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2000) while fMRI has high spatial resolution for assessment of motor representations. We hypothesize that adaptive changes in the motor system occur after FFMT and these changes are related to time after surgery and motor outcome.
2. Methods 2.1. Subjects Thirty-two patients had reconstruction of their upper extremity using FFMT at our institution between 1976 and 1999. Twelve patients were available for follow-up and 9 agreed to participate in the present study. The clinical information for the 9 patients (mean age 34.3 ^ 3.9 years) is summarized in Table 1. Each patient had suffered a devastating injury to the upper extremity that resulted in a significant functional loss. All subjects gave their written informed consent according to the declaration of Helsinki and the protocol was approved by the University Health Network Research Ethics Board. The gracilis muscle was the donor muscle in each transfer. The gracilis muscle was transferred to the arm and attached to the origin and insertion of the damaged muscle it was replacing. The new vascular and nerve supply was established by microneurovascular re-anastamosis with donor blood vessels and nerve (Manktelow and Zuker, 1989; Manktelow and Anastakis, 1999). 2.2. Clinical assessment The patients were examined by two investigators (DJA and CTH) independent of the physiological and fMRI studies. Muscle strength was assessed by the Medical Research Council scale (Medical Research Council, 1954). The motor outcome was graded as successful when
Table 1 Clinical information for the study patients Patient
Age/sex
Hand dominance
Injury
Time since surgery
Target muscle
Donor nerve
Muscle power and outcome
1 2 3 4 5 6 7
17M 42M 47M 41M 50M 30F 27F
Right Right Left Right Right Left Right
4 months 17 years 15 years 23 years 15 years 8 years 8 years
R biceps L biceps R finger flexors R finger flexors L finger flexors R finger flexors R deltoid
Intercostal nerves 2, 3 Musculo-cutaneousb Anterior interosseous Anterior interosseous Anterior interosseous Anterior interosseous Axillary
M3 fair M4 successful M4 successful M4 successful M4 successful M4 successful M4 successful
8 9
21F 32M
Right Right
C5,6,7 root avulsion Crush injurya Crush injury Crush injury Crush injury Crush injury Tumor resection (sarcoma) Atrophic deltoidc Laceration/trauma
1.25 years 6 years
R deltoid R triceps
Musculo-cutaneousd Axillary
M3 fair M4 successful
a
Crush injury involves significant trauma to the upper extremity with functional muscle loss. Awake nerve stimulation was used to differentiate sensory from motor nerve fibers within the musculocutaneous nerve to provide motor supply for the gracilis muscle transfer. c Complete deltoid paralysis – etiology idiopathic. d Motor branches to the brachialis were sacrificed and dissected proximally to provide motor supply for the gracilis muscle transfer. b
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the postoperative muscle strength was M4 or M5, as fair when the muscle strength was M3 and as poor with muscle strength of M2 or less. 2.3. Nerve conduction and transcranial magnetic stimulation studies Surface electromyogram (EMG) was recorded from the transferred gracilis muscle and from the analogous muscle (Table 1) on the intact side with disposable silver-silver chloride electrodes. The signal was amplified (Intronix Technologies Corporation Model 2024F, Bolton, Ont., Canada), filtered (bandpass 2 Hz– 5 kHz), digitized at 5 kHz (Micro 1401, Cambridge Electronics Design, Cambridge, UK) and stored in a laboratory computer for offline analysis. The compound muscle action potential (MMax) was determined by supramaximal electrical nerve stimulation. For deltoid, biceps and triceps muscles, brachial plexus stimulation at Erb’s point was used and for forearm flexors median nerve stimulation at the elbow was used. TMS was performed with a 7 cm figure-of-8 coil and two Magstim 200 stimulators (The Magstim Company, Whitland, UK) connected via a Bistim module. The coil was placed at the optimal position for eliciting motorevoked potentials (MEPs) from the target muscle. The optimal position was marked on the scalp to ensure identical placement of the coil throughout the experiment. The handle of the coil pointed backward and the induced current in the brain flowed from posterior to anterior. The FFMT and intact sides were studied in random order. The following TMS parameters were measured: Resting motor threshold (MT): Defined as the minimum stimulator output that produced MEPs of $ 50 mV in at least 5 out of 10 trials and was determined to the nearest 1% of the maximum stimulator power output. EMG silence was monitored on a computer screen and via speakers at high gain. Active MT: Defined as the minimum stimulator output that produced MEPs of $ 100 mV in at least 5 out of 10 trials. The EMG passed through a leaky integrator and the EMG level was displayed on an oscilloscope. With visual and auditory feedback, the subjects maintained a constant background contraction of 10% of the maximum integrated EMG. MEP recruitment curve and silent period (SP) duration: Stimulus intensities of 100, 110, 120, 130, 140 and 150% of the rest (for rest condition) or active (for active condition) MT were studied. Eight pulses at each stimulus intensity were delivered 6 s apart and the order of the stimulus intensities was random. Subjects were studied both at rest (rest condition) and during 10% maximum background contraction (active condition) in separate runs. The trials in the active condition were also used measurement of the SP. Short interval intracortical facilitation (SICF): The first wave SICF (also known as I-wave facilitation) was studied
using a paired pulse protocol similar to previous reports (Tokimura et al., 1996; Ziemann et al., 1998d; Chen and Garg, 2000). The first pulse was set to produce MEPs of about 1 mV for forearm flexors and 0.3 mV for deltoid, biceps or triceps muscle at rest and the second pulse was at rest MT. The first pulse alone (16 trials) and paired pulses at interstimulus intervals (ISIs) of 0.5, 0.7, 1.3, 1.5, 2.1, 2.3 ms (8 trials for each ISI) were delivered in random order. The rest and active conditions were studied in separate runs. Short interval intracortical inhibition (SICI) and intracortical facilitation (ICF): A paired-pulse protocol similar to that described by Kujirai et al. (1993) was used. The conditioning stimulus was set at 95% of active MT and the test stimulus evoked MEPs of about 1 mV for forearm flexors and 0.3 mV for deltoid, biceps or triceps muscle at rest. Single test pulse and paired pulses at ISI of 2 and 10 ms (10 trials for each condition) were delivered in random order. The rest and active conditions were studied in separate runs. Long interval intracortical inhibition (LICI): Suprathreshold conditioning and test pulses at ISIs of 50 –200 ms were used (Valls-Sole´ et al., 1992). The conditioning and test pulses were both set to produce 1 mV MEPs at rest. Single test pulse (20 trials) and paired pulses at ISIs of 50, 100, 150 and 200 ms (10 trials for each ISI) were delivered in random order. The rest and active conditions were studied in separate runs. Mapping of muscle representation: The cortical representation of the target muscle was studied by TMS mapping. A grid of 1 cm interval along the coronal axis and 2 cm interval along the saggital axis was drawn on the scalp. The coordinates of the scalp positions were expressed relative to the vertex (CZ, international 10 –20 system). Starting at the optimal position, 10 pulses at 120% rest MT were delivered 6 s apart. The coil was then moved to adjacent positions on the grid and the procedure repeated. Successive positions were stimulated until the area where MEPs were produced was surrounded by inactive positions. The rest and active conditions were studied in separate runs. Relationship between intercostal and biceps activation in Patient 1: Patient 1 differed from the other patients because the donor nerves were the intercostal nerves rather than a nerve from the injured arm. Therefore, the reconstructed muscle was innervated by thoracic rather than cervical spinal motoneurons. Additional studies were performed to examine the motor representations for the biceps and intercostal muscles. Physiological studies were performed 4 months after the FFMT operation. In addition to the nerve conduction and TMS studies described above, we determined whether the intercostal muscles and the biceps can be activated independent of each other. Surface EMG electrodes were placed on the reconstructed right biceps and the right fourth intercostal space at the mid-axillary line and 3 cm anterior. The effects of the patient at rest with normal respiration, performing volitional breathing at maximum
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effort and voluntary right elbow flexion at maximum effort were observed. 2.4. Data analysis The peak-to-peak MEP amplitude for each trial was measured offline. The MEP amplitude for MEP recruitment was also expressed as a percentage of the MMax to give an estimate of the percentage of motoneuron pool (%MMax) recruited. For SICF, SICI, ICF and LICI, the MEP amplitude for each conditioned trial was expressed as a ratio to the mean control (unconditioned) MEP amplitude for each subject. Ratios less than one indicate inhibition, and ratios greater than one indicate facilitation. The SP for each trial was measured offline from onset of the MEP to the first resumption of voluntary EMG activity. Values are expressed as mean ^ standard error. For the mapping study, the mean MEP amplitude of each active position was expressed a percentage of the site that elicited the maximum mean amplitude (normalized mean amplitude). The center of gravity (COG) and the number of top-half positions were calculated. The COG is an amplitude-weighted average and the lateral COG coordinate was computed as the sum of (lateral coordinate £ mean amplitude) for all active positions divided by the sum of mean amplitude for all active positions. The anterior-posterior COG coordinate was calculated similarly (Wassermann et al., 1992; Classen et al., 1998). The number of top-half positions was used as a measure of the map size and was the number of positions with mean amplitudes of at least 50% of the site with maximum mean amplitude (Classen et al., 1998). 2.5. Statistical analysis For MMax, rest MT, active MT, SICI and ICF, the FFMT and intact sides were compared by the paired t test. For MEP recruitment, the MEP amplitudes or %MMax recruited were analyzed with analysis of covariance (ANCOVA) with side (FFMT or intact) and stimulus intensity as factors in the ANCOVA model. For I-wave facilitation and LICI, the FFMT and intact sides were compared by analysis of variance (ANOVA) with repeated measures. The ISIs were the repeated measure. The relationship between time since FFMT, age at FFMT and the extent of reorganization was examined by ANCOVA. The factors in the ANCOVA model were time since FFMT and age at FFMT and the dependent variable was the ratio of the FFMT to intact sides for the measurements that were found to be significantly different between the two sides. For rest and active %MMax, the highest stimulus intensity in which both the FFMT and the intact sides were tested was used to calculate the FFMT/intact ratio. Variables that may affect the motor outcome were examined by logistic regression. The motor outcome was the dependent variable. Time
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since surgery and the ratio of the FFMT to intact sides for the measurements that were found to be significantly different between the two sides were the independent variables. Differences were considered significant if P # 0:05. 2.6. Functional magnetic resonance imaging studies fMRI studies were performed in Patient 1. Prior to the FFMT operation, the patient underwent a fMRI study performing volitional breathing consisting of rapid shallow breath at approximately two per second alternating with periods of normal, quiet breathing. A second fMRI study was performed 6 months after the FFMT operation. Self-paced, low amplitude elbow flexion (activates biceps) was carried out. Both the FFMT and intact sides were studied. The patient was trained to move at about 1 Hz and to make the movements as imperceptible as possible without ceasing the movement altogether. The movements were practiced by the patient before scanning and were observed during scanning to confirm that the amplitude of each movement was as low as possible. This ensured that image noise due to motion was minimized. Supports were placed under each subject’s upper extremity to further minimize unnecessary arm motions. Head motion was minimized by tightly cushioning the subject’s head within the head coil. 2.6.1. MR imaging The fMRI paradigm consisted of a block design alternating rest with movement every 15 s over 6 min for a total of 12 movement epochs and 12 rest epochs. Imaging was performed on a 1.5 Telsa scanner (Signa ‘Echospeed’ – GE Medical Systems, Milwaukee, WI) using a spiral gradient echo sequence. Twenty-eight contiguous slices of 4.5 mm thickness (no gaps) were obtained from the foramen magnum to the vertex. One spiral trajectory was collected per slice providing a 3.75 mm in-plane resolution. Other sequence parameters included: TR ¼ 2240 ms, TE ¼ 40 ms, flip angle ¼ 858 (set to the Ernst angle for maximum signal-to-noise within gray matter for TR ¼ 2240 ms and T1 of gray matter ¼ 850 ms). High resolution T1-weighted fast inversion recovery SPGR images were acquired at 1.5 mm slice thickness for co-registration with the functional images. 2.6.2. Data analysis Image analysis was performed offline using Stimulate (J.P. Strupp 1996, University of Minnesota Medical School) and AFNI (Analysis of Functional Neuroimage, R.W. Cox, The Medical College of Wisconsin) software. All images were corrected for head motion using automated image registration algorithms. Regions of activation were determined by cross-correlating the fMRI data to the predicted hemodynamic response (boxcar waveform) for each task using a pixel-by-pixel statistical analysis yielding r-values for all pixels in each image. Activation thresholds
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were set at two standard deviations above the mean r-value obtained from all pixels in each image.
3. Results 3.1. Clinical outcome The strength of the reconstructed muscle was greater than antigravity in all patients. Seven patients had successful outcomes (M4 or M5) while two patients had fair outcomes (M3). The motor outcome for each patient is shown in Table 1. 3.2. Nerve conduction studies Nerve conduction studies were performed in 8 patients and the MMax on the FFMT side (3.2 ^ 0.3 mV) was smaller than that of the intact side (13.1 ^ 0.8 mV, P ¼ 0:002) in all patients tested. 3.3. Motor thresholds and maximum MEP The rest and active MTs from TMS are shown in Fig. 1. There was a large between subject variation in part because we studied different muscles. As expected, forearm flexors tended to have lower MT than more proximal muscles (Fig. 1). In one patient the rest MT on the intact side was higher than maximum stimulator output. The rest MT was lower on the FFMT side (56 ^ 6.9%) than the intact side (68.4 ^ 8.6%) in all 9 patients (P ¼ 0:001, Fig. 1A).
However, the active MT was similar on the two sides (FFMT 51.9 ^ 5.9%, intact 53.1 ^ 4.1%, Fig. 1B). 3.4. MEP recruitment and silent period Rest MEP recruitment on the intact side cannot be tested in 3 patients and the higher stimulus intensities were omitted in some patients because of high motor threshold. Figs. 2A,B show the MEP amplitudes at different stimulus intensities. The effects of side (FFMT vs. intact) (P ¼ 0:04 rest, P ¼ 0:005 active) and stimulus intensity (P , 0:0001) were significant for both the rest and active conditions but their interaction was not significant. MEP amplitudes were higher on the intact than the FFMT side. Figs. 2C,D show the %MMax (MEP/MMax) recruited at different stimulus intensities. The %MMax recruited was higher for the FFMT side than the intact side. The effects of side (P ¼ 0:0004 rest, P ¼ 0:002 active) and stimulus intensity (P ¼ 0:005 rest, P ¼ 0:02 active) were significant for both the rest and active conditions and their interaction was not significant. The %MMax was higher on the FFMT than the intact side. The silent period was not significantly different between the FFMT and the intact sides (Fig. 2E). 3.5. SICF In 3 patients it was not possible to test SICF, SICI, ICF, LICI and TMS mapping at rest because the MEPs were too small even at maximum stimulator output. SICF was tested in 6 patients at rest and all 9 patients during voluntary contraction. The results are shown in Fig. 3. As expected, I-wave facilitation occurred at ISIs of 1.3 and 1.5 ms but not at 0.5, 0.7, 2.1 and 2.3 ms. The effect of ISI was significant but there was no significant difference between the FFMT and the intact sides. 3.6. SICI and ICF Resting SICI and ICF in the 6 patients tested is shown in Fig. 4A. The SICI was significantly reduced on the FFMT side (P ¼ 0:05, Figs. 4A,B) but the ICF did not differ significantly. SICI and ICF during voluntary contraction were tested in all 9 patients (Fig. 4C). SICI and ICF were reduced compared to rest but there was no significant difference between the FFMT and intact sides. 3.7. LICI
Fig. 1. Motor thresholds for magnetic stimulation. The rest (A); and active (B) motor thresholds for all 9 patients. Each line represents one subject and each error bar represents one standard error.
Rest LICI was tested in 6 patients and active LICI was tested all 9 patients. The effect of ISI was significant but there was no significant difference between the FFMT and the intact sides (Fig. 5).
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Fig. 2. MEP recruitment curve and silent period. (A, B) Show the MEP amplitude; and (C, D) show the %MMax (MEP amplitude divided by the MMax from peripheral nerve stimulation) for the rest (A, C); and active (B, D) conditions at different TMS intensities. (E) Shows the silent period. Each error bar represents one standard error.
3.8. TMS mapping TMS mapping at rest was performed in 6 patients and in all 9 patients during voluntary contraction. The location of the center of gravity was similar on the FFMT and the intact sides (Fig. 6). The number of
top-half positions (. 50% maximum) was also similar on the two sides (FFMT rest 3 ^ 0.4, active 3.9 ^ 0.4; intact rest 4.3 ^ 0.5, active 6 ^ 1.4). Patient 1 who had FFMT with the right biceps muscle innervated by the intercostal nerves to also showed similar map location and size on the two sides (Fig. 6B).
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Fig. 3. I wave facilitation for (A) rest; and (B) active conditions. The MEP amplitude for each interstimulus interval is expressed as a ratio to that of the first stimulus (S1) alone. There was no significant difference between the FFMT and intact sides. Each error bar represents one standard error.
3.9. Factors affecting extent of reorganization and motor outcome Since the MMax, rest %MMax, active %MMax, rest MT and rest SICI were found to be significantly different
between the FFMT and intact sides, the ratio of the FFMT to the intact sides for these measurements were used to examine factors that may affect the extent of reorganization. For the FFMT/intact ratio of MMax, rest %MMax and rest MT, the effects of time since FFMT was significant
Fig. 4. (A) Short interval intracortical inhibition (SICI, ISI 2 ms) and intracortical facilitation (ICF, ISI 10 ms) at rest for the FFMT and intact sides (6 patients). SICI was significantly reduced on the FFMT side compared to the intact side. (B) Rest SICI with each line representing one subject. (C) SICI and ICF in the active condition (9 patients). Each error bar represents one standard error.
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Fig. 5. Long interval intracortical inhibition (LICI) at rest (A) (6 patients); and active (B) (9 patients) conditions. There was no significant difference between the FFMT and intact sides. Each error bar represents one standard error.
(P ¼ 0:016 for MMax, P ¼ 0:026 for rest %MMax, P ¼ 0:008 for rest MT, ANCOVA) but the effect of age at the time of FFMT was not. The MMax on the FFMT side increased with time since surgery (Fig. 7A) and the difference in rest %MMax (Fig. 7B) and rest MT (Fig. 7C) between the FFMT and intact sides decreased with time since surgery. For active %MMax and rest SICI, the effects of time since FFMT and age at FFMT were not significant. Logistic regression for motor outcome showed a significant likelihood ratio for time since surgery (P , 0:0001), MMax (P ¼ 0:014) and rest MT (P ¼ 0:014) but there was no significant effect of rest and active %MMax and rest SICI.
3.10. Relationship between intercostal and biceps activation in Patient 1 Surface EMG recording showed rhythmic activity in the intercostal muscle and no activity in the reconstructed biceps during both normal breathing and voluntary breathing at maximum effort. With the biceps activated at maximum effort, there was no activity in the intercostal muscles.
3.11. Functional magnetic resonance imaging Volitional breathing prior to FFMT operation activated the primary motor cortex medial to the upper-limb area (Fig. 8A) but not the upper-limb area. Six months after FFMT the patient voluntarily contract the reconstructed right biceps innervated by the intercostal nerves and had anti-gravity strength. Right elbow flexion activated the upper limb area of the left motor cortex (Figs. 8B,C), similar to activation of the right motor cortex with movement of the intact left biceps (Figs. 8D,E).
Fig. 6. (A) Location of center of gravity (COG) of TMS maps for the rest (6 patients) and active conditions (9 patients). The bars in the medial-lateral column show the distance of the COG lateral to the vertex and the bars in the anterior-posterior column show the distance of the COG anterior to the vertex. There was no significant difference between the FFMT and intact sides. Error bars represent standard error. (B) TMS maps at rest for the patient 1 with FFMT involving intercostal nerves to the right biceps muscle. The vertex is the origin of the coordinates (0,0). The MEP amplitude of each point is expressed as a percentage of the position with maximum MEP amplitude on the same side. The size and location for the motor map for the right (FFMT) biceps (left hemisphere) is similar to that of the left (intact) biceps (right hemisphere).
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Fig. 7. The effects of time after FFMT. (A) The ratio of MMax for the FFMT to the intact side plotted against time after FFMT. Data from 8 patients who had nerve conduction studies. The ratio increased with time (P ¼ 0:016). (B) The ratio of rest % MMax for the FFMT to the intact side plotted against time after FFMT. Data from 6 patients. Three patients were excluded as rest MEP recruitment cannot be studied on the intact side due to high motor threshold. The ratio decreased with time (P ¼ 0:026). (C) The ratio of rest MT for the FFMT to the intact side plotted against time after FFMT. Data from 8 patients. One patient with threshold on the intact side higher than the maximum stimulator output was excluded. The ratio increased with time (P ¼ 0:008).
4. Discussion We used TMS and fMRI to study motor system reorganization following FFMT and found evidence of plasticity in the motor projection to functioning muscles. The reduced rest MT and SICI on the reconstructed side compared to the intact side is similar to the changes following lower limb amputation (Chen et al., 1998a). The rest MT may be related to membrane excitability in the motor cortex since it is increased by drugs that block voltage-gated sodium channels (Ziemann et al., 1996a; Chen et al., 1997) but is unaffected by drugs that alter GABA (Ziemann et al., 1996a) transmission. Our data do not allow us to distinguish whether the reduction in MT occurred predominately in cortical or subcortical sites. However, there is considerable evidence that SICI for the hand (Kujirai et al., 1993; Nakamura et al., 1997; Di Lazzaro et al., 1998) and proximal upper or lower limb representations (Chen et al., 1998a; Chen et al., 1998b) are mediated by cortical inhibitory mechanisms. SICI can be enhanced by drugs that increase GABAA activity (Ziemann
et al., 1996a) and by antiglutaminergic drugs (Liepert et al., 1997; Ziemann et al., 1998a). These finding are consistent with the hypothesis that motor cortex plasticity may involve adjustment of balance between inhibition and facilitation in the intrinsic horizontal connections mediated by GABAergic and glutaminergic mechanisms (Jacobs and Donoghue, 1991; Sanes and Donoghue, 2000). However, other mechanisms such as long-term potentiation (Hess and Donoghue, 1994) and axonal sprouting with formation of new synapses (Jones and Schallert, 1994) may also contribute to cortical plasticity (Sanes and Donoghue, 2000). The inhibitory circuits mediating LICI and SP and the excitatory circuits mediating ICF and SICF were not significantly altered by the muscle reconstruction procedure. The later part (. 50 ms) of the SP and LICI at ISIs of more than 50 ms (Fuhr et al., 1991; Chen et al., 1999) is due to cortical inhibition. SICI and LICI are mediated by different cortical inhibitory mechanisms (Sanger et al., 2001). LICI and SP may be mediated by GABAB receptors while SICI may predominately involve GABA A
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Fig. 8. fMRI color maps for Patient 1 showing areas of task related cortical activation. Threshold for activation was set to show pixels with r-values .2 standard deviations above the mean r-value for all pixels in the image. (A) Activation (blue arrows) in the medial aspect of both primary motor cortices during volitional breathing. (B, C) Activation maps in the same patient 6 months following surgery using a right biceps muscle transfer to restore elbow flexion. The patient was performing low amplitude elbow flexion using the intercostal nerves. The cortical areas activated are located in the primary motor cortex controlling upper extremity movement. They are clearly separate from the area activated by volitional breathing seen in (A) (blue arrows). (D, E) Cortical activation during flexion of the normal left elbow. The areas activated are in similar location (laterality) along the motor cortex for each extremity.
mechanisms (Siebner et al., 1998; Werhahn et al., 1999; Sanger et al., 2001). ICF (Ziemann et al., 1996b; Chen et al., 1998b) and SICF (Ziemann et al., 1998d) are also mediated by mechanisms distinct from SICI. Thus, our findings suggest that FFMT induce specific changes in the motor cortex affecting the circuits mediating SICI and MT but not the other excitatory and inhibitory circuits we tested. We found no change in the motor representation of the target muscle following FFMT with TMS mapping and fMRI. This indicates that alterations in the neuronal level, as demonstrated by changes SICI and MT, are not necessarily accompanied by changes in the motor representational level. The changes following FFMT are different from the expansion and shift of motor representation following nerve injury or amputation in animals (Donoghue et al., 1990; Sanes et al., 1990; Schieber and Deuel, 1997; Wu and Kaas, 1999) and humans (Cohen et al., 1991; Kew et al., 1994; Pascual-Leone et al., 1996; Ridding and Rothwell, 1997; Roricht et al., 1999; Dettmers et al., 1999) and following hand replantation in humans (Roricht et al., 2001). Following peripheral nerve injury, amputation and even hand replantation (Roricht et al., 2001), large areas of the body are deafferented. FFMT procedures do not lead to significant deafferentation because we strived to use a predominately motor donor nerve
although some patients had a small degree of deafferentation due to other nerve injuries. Deafferentation may be necessary for shift and expansion of motor representation to occur. The change in MT and SICI following FFMT was evident only with the muscle relaxed and not during voluntary contraction. It has been argued that changes observed in the resting state but not during muscle activation may be due to modulation in corticospinal excitability rather than change in cortical connectivity (Siebner and Rothwell, 2003). While decrease in rest MT may be related to increased corticospinal excitability, reduction in SICI likely involved changes in efficacy of inhibitory synapses. Moreover, we found no change in others measures of corticospinal excitability such as TMS map area (Siebner and Rothwell, 2003). The need to precisely control the excitability of cortical neurons to perform specific voluntary activity may account for the absence of significant change during voluntary activation. There are similar observations in other settings of cortical plasticity. Transient anesthesia of the forearm caused increased blood flow to the primary sensorimotor cortex at rest but not during biceps movement, implying that movement-related changes in cerebral blood flow was decreased (Sadato et al., 1995). In the muscle proximal to
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forearm ischemia or amputation, MEP amplitude and map area was increased at rest but not during muscle activation (Ridding and Rothwell, 1997) and the increase in MEP amplitude following immobilization occurred only with the target muscle tested at rest (Zanette et al., 1997). These observations suggest that reduced MT and SICI in the motor cortex may make it easier to activate corticospinal neurons to produce voluntary activity with less excitatory synaptic drive. This is supported by the progressive reduction in SICI just prior to voluntary movement (Reynolds and Ashby, 1999) and the finding that forearm deafferentation leads to faster elbow flexion movement (Ziemann et al., 2001). The lower MMax from peripheral nerve stimulation on the FFMT side than the normal side may indicate a lower degree of innervation for the reconstructed muscle. However, this may also reflect the greater muscle mass in intact upper extremity compared to the transferred gracilis muscle and in some cases difficulty in locating the reconstructed nerve for nerve conduction studies due to anatomical distortions from the operation. The increase in MMax with time suggests that the nerve regeneration continued long after the FFMT operation. While our small numbers need to be interpreted with caution, clinical experience with peripheral nerve injury has suggested that nerve regeneration occurred over a longer time than can be expected from an axonal regeneration rate of 1 – 3 mm per day (Sunderland, 1978; Kline and Hudson, 1995). Motor improvement may continue for 5– 7 years following nerve repair (Dagum, 1998). Training and increasing use of the muscle in activities of daily living may also contribute to the long-term changes in peripheral innervation and motor function. While the motor outcome of FFMT improved with time, the change in rest %MMax and rest MT diminished with time suggesting that the maximum change in these parameters probably occurred early. These measures may represent one step in motor reorganization that involves various mechanisms at different times. It seems likely that the motor reorganization continues to evolve and may be modified by training and experience long after the muscle reconstruction procedure. The increased %MMax and reduced MT may also be a compensatory mechanism for deficits in peripheral innervation which improved with time. Although we found no relationship between motor outcome and physiological changes, this may be related to the low sensitivity of the outcome measure. Patient 1 differed from the other patients because the donor intercostal nerve originally innervated truncal muscles rather than the upper-limb muscles. Our finding that volitional inspiration prior to the FFMT activated the motor cortex medial to the upper-limb area is similar to previous neuroimaging studies of volitional breathing that used passive mechanical ventilation in the rest period (Colebatch et al., 1991; Evans et al., 1999). Six months after the FFMT procedure, TMS mapping (Fig. 6) and fMRI
(Fig. 8) showed that the control of motoneurons that originally innervated the intercostal muscles (in the T2 and T3 spinal levels) had shifted to the upper-limb area of the motor cortex. Our results are similar to that of Mano et al. (1995) who reported that following coaptation of the musculocutaneous and intercostal nerves for treatment of cervical root avulsion, TMS mapping showed that cortical representation for the biceps was initially located in the areas of the intercostal muscles. It gradually moved laterally to the upper-limb area as the patients gained voluntary control of the biceps muscle (Mano et al., 1995). Thus, the adult human motor cortex is capable of relatively large scale reorganization. While changes in MT and SICI have been demonstrated in muscles proximal to transient deafferentation (Ziemann et al., 1998b) or amputation (Chen et al., 1998a), our results show that these changes occur in functioning muscles. Successful outcome following FFMT probably requires adaptive changes in the central nervous system because the movement mediated by the transferred muscle is often different from that mediated by the donor nerve and because of the random nature of axonal regeneration from the proximal stump into the distal pathways. Clinical experience suggested a period of rehabilitation and retraining is necessary for successful outcome (Manktelow and Anastakis, 1999) and there is evidence that cortical plasticity occurs with motor learning (Sanes and Donoghue, 2000). In conclusion, our results provided evidence for plasticity of the motor system projecting to functionally relevant muscles. Changes in the neuronal level are not necessarily accompanied by changes in motor representation and motor reorganization continued to evolve long after the initial injury.
Acknowledgements This work was supported by the Workplace Safety and Insurance Board of Ontario, the Canadian Institutes of Health Research, the Canada Foundation for Innovation, Ontario Innovation Trust and the University Health Network Krembil Family Chair in Neurology. RC is a Canadian Institutes of Health Research New Investigator.
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