Effects of Subdural Monopolar Cortical Stimulation Paired With Rehabilitative Training on Behavioral and Neurophysiological Recovery After Cortical Ischemic Stroke in Adult Squirrel Monkeys

Subjects

Fifteen adult squirrel monkeys (genus Saimiri, 10 male, 5 female; birth dates unknown, veterinary estimates ranged between 3 and 10 years at start of experiment) were used. Monkeys were experimentally naïve and free of obvious physical and neurological problems at study initiation. Five squirrel monkeys (2 from the cohort above along with 3 others) participated in additional experiments to evaluate technical aspects of the monopolar CS method; results of these studies informed the final methods design. All procedures were conducted in accordance with federal guidelines for the care and use of laboratory animals and with approval of the Institutional Animal Care and Use Committee at the University of Kansas Medical Center.

Experimental Design

Figure 1 illustrates the experimental timeline. After determination of hand preference on a pellet reach-and-retrieval task (Kluver board),^21,22 monkeys were fitted with a short-sleeved, torso-covering, nylon mesh primate jacket. Monkeys underwent training on the pellet retrieval task for two weeks, followed by a surgical procedure during which functional regions of M1, PMD, and ventral premotor cortex (PMV) were identified via intracortical microstimulation (ICMS) mapping.^14,22,23 In the same procedure, an ischemic infarct was produced in the M1 DFL representation.^14,24,25 After infarct, in either the same procedure (n = 2) or in a separate procedure 2 to 3 weeks later (n = 13), a monopolar electrode was implanted over spared DFL areas in premotor cortex. This variation in timing of electrode implantation was guided by veterinary consultation regarding the impact of the multiple survival surgery schedule on the overall health and well-being of the monkeys.

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Figure 1.

Summary of experiment timeline highlighting key procedural elements. The middle series of boxes delineate major intervals (preinfarct, postinfarct, therapy, follow-up). The top boxes, arrows, and brackets indicate the timing of surgical procedures (motor mapping, ischemic infarct, electrode implantation); brackets reflect timing variations for electrode implantation and posttherapy motor mapping (see Methods section). The bottom box and arrows indicate the timing of behavior assessments reported in the article.

Postinfarct motor deficits were monitored during a 6-week recovery period. This timing was chosen to model the expected clinical treatment paradigm in which human stroke survivors would enroll into CS/RT treatment programs secondary to an earlier poststroke recovery period^17,18,26; prior results suggested monkeys would have sufficient residual deficits on the pellet retrieval task at 6 weeks postinfarct to enable meaningful comparisons between treatment groups. ²⁷ At 7 weeks postinfarct, monkeys were randomly assigned to either the RT therapy group (rehabilitative training for 4-6 weeks; n = 8; 4 male, 4 female) or the CS/RT therapy group (cortical electrical stimulation concurrent with 4-6 weeks of RT; n = 7; 6 male, 1 female). At the conclusion of the therapy period, task performance was monitored for 12 weeks to assess retention of performance gains. Within this follow-up period, monkeys underwent a surgical procedure at 1 week (n = 5; RT = 3, CS/RT = 2) or 7 weeks (n = 9; RT = 4, CS/RT = 5) posttherapy to remove the electrode and remap DFL representations in M1, PMD, and PMV. Since only limited data was available when the present study was designed to suggest that posttherapy retention of performance gains would not be dependent on ongoing CS treatment (retention for 2 days post-CS/RT in rats ¹² ; up to 4 months in monkeys but in only 3 subjects ¹⁴ ), the variation in posttherapy surgical timing was planned in anticipation of investigating neurophysiological changes associated with a potential relapse of behavioral performance in either group. One RT monkey died prior to the second mapping procedure (missing posttherapy map data and 12-week follow-up behavior data). A technology failure caused behavior video from 2 RT monkeys to be lost after experiment completion but prior to analysis of behavioral performance (behavior data missing, but mapping data present). For the 15 monkeys described here, complete behavior results were available for 12 monkeys (5 RT, 7 CS/RT) and ICMS mapping results were available for 14 monkeys (7 RT, 7 CS/RT).

Behavior Training

Skilled motor behavior was assessed using a modified Kluver board task.^22,23,28 In brief, food pellets (45 mg, Bioserve) were placed individually into 1 of 5 wells for the monkey to retrieve and eat. The wells ranged in diameter from 25 mm (larger than the width of the monkey’s hand) to 9.5 mm (wide enough to insert 1-2 fingers). Success on this task is defined as controlled (ie, in the grasp of the fingers) removal of the pellet from the food well. Since well depth was identical, retrieval difficulty increased with decreasing well diameter. Normal, uninjured monkeys with no prior task exposure can readily perform the largest wells with few errors, but require training to achieve proficiency with the smaller wells.^22,23

Preinfarct training was conducted in 30-minute sessions, twice per day for 10 days. For the first session, pellets were placed exclusively into the largest well. In subsequent sessions, placements were progressively shifted toward smaller wells, with all placements into the smallest well by the last several training days. Monkeys typically performed 400 to 500 training trials per day with this regimen. Prior to each training session, “probe” trials were conducted, which consisted of 25 to 50 trials distributed evenly between each well. Probe trials thus served as a consistent testing mechanism to assess overall performance on the retrieval task, and are the basis of the reported performance data in the present study.

Rehabilitative training sessions were similar to preinfarct training (twice daily probe trials followed by training trials). Well progression during RT was based on achieving criterion performance at successive stages: Monkeys had to achieve at least 80% of their preinfarct performance level for the tested well for 2 consecutive days. All monkeys met this criterion within 4 days for each well. During the 6-week postinfarct recovery period and the 12-week follow-up period, task performance was assessed 1 to 3 times per week with a probe test consisting of 50 probe trials (10/well).

In a prior study, ¹⁴ we demonstrated that the time to remove a pellet from the food wells (“dwell time”) exhibited more substantial and persistent deficits after M1 infarct than the number of finger flexions required to remove pellets from the well (“flexions per retrieval”). As such, dwell time values for each well were used as the primary performance data in the present study. To facilitate comparisons between monkeys that could have a range of task skill levels after the preinfarct training protocol, a motor performance index^14,27 was calculated as follows. The average dwell time values from each test well were calculated from the final 5 days of preinfarct training, then used to normalize performance for the experimental periods listed below, such that an index of 1.0 is equivalent to the 5-day preinfarct average, 2.0 is double the preinfarct average, and so on. It is possible for individual monkeys to have a performance index less than 1.0, indicating performance better than preinjury levels.

Five time points were preselected as the primary endpoints to assess changes in motor performance: (1) “preinfarct” (the final 3 days of probe trials prior to ischemic injury), (2) “postinfarct” (probe trials during the first week after injury), (3) “pretherapy” (probe trials during week 6 of the recovery period), (4) “posttherapy” (probe trials during the first week after the therapy period), and (5) “follow-up” (probe trials during the final week of the follow-up period).

Surgical Procedures

Surgical and neurophysiological mapping methods were similar to previous reports.^14,21-23,29 In brief, monkeys were sedated with ketamine (20 mg/kg), followed by isoflurane anesthesia (1% to 2%, with 70% nitrous oxide, 30% oxygen) during surgical procedures. Using aseptic techniques, a craniotomy was performed to expose motor cortex in the hemisphere opposite the monkey’s preferred forelimb on the pellet retrieval task. The dura was excised and a plastic cylinder was secured around the cranial opening with dental acrylic and filled with sterile silicone oil to prevent tissue desiccation. Gas anesthesia was discontinued and ketamine (15-20 mg/kg/h) supplemented with diazepam (~0.01 mg/kg/h) was administered during the neurophysiological mapping procedure. At the conclusion of mapping, gas anesthesia was reinstated, the plastic chamber removed, and the craniotomy was closed using a layered combination of silicone sheeting (as dura replacement), gelfoam (as space filler), and dental acrylic (as skull replacement).

After completion of the first mapping procedure, and prior to surgical closing, the ischemic infarct was created (see below). All monkeys in both groups also underwent implantation of the monopolar electrode system (see below).

Neurophysiological Mapping of Motor Cortex Using ICMS

Functional maps of DFL movement representations in M1, PMD, and PMV were derived prior to infarct (baseline map) and after the intervention period (posttherapy map) using ICMS.^21,22 In brief, guided by a scaled digital image of the cortical surface, a sharp beveled glass micropipette (10-25 µm outer diameter, filled with 3.5 M saline) was used to deliver high-frequency low-amplitude electrical stimuli to layer V neurons (thirteen 200-µs pulses, 3.3-ms interval, 1-Hz repeat, 0-30 µA, 1750 µm depth). The objective was to identify the extent of digit, wrist, and forearm representations (collectively, the DFL representation) in each of the 3 cortical regions (Figure 2). Cortical territory containing representations of other parts of the body, such as the proximal forelimb, torso, face, and hindlimb, were extensively explored to ensure all DFL locations were identified at both mapping timepoints. The total extent of cortical mapping area was typically ~95 to 105 mm², based on 250 to 350 ICMS sites/map, using a 500-µm interpenetration distance in M1 and PMD and a 350-µm interpenetration distance in PMV.

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Figure 2.

(Left) Representative baseline motor map from 1 cortical stimulation/rehabilitative training (CS/RT) monkey illustrating distal forelimb (DFL) and surrounding non-DFL representations in primary motor (M1), dorsal premotor (PMD), and ventral premotor (PMV) cortices, overlaid onto an image of the surgically exposed cortical surface. Colors represent different movement types evoked by ICMS mapping: DFL movements (red = finger or thumb, green = wrist or forearm, yellow = digit + wrist/forearm, light blue = digit + elbow/shoulder (labeled “Prox” as abbreviation for “proximal forelimb”), magenta = wrist/forearm + elbow/shoulder; the latter 3 colors are dual-response sites at which two movements were evoked within 2 µA of each other^22,23), non-DFL movements (dark blue; eg, elbow, shoulder, torso, face, hindlimb), and nonresponsive (black). The white dashed line indicates the infarct target in this monkey, based on the location of M1 DFL responses and constraints imposed by vascular patterns. (Top right) Image of the cortical surface (from left panel) immediately after infarct creation. Surface arteries and veins within the target region have been occluded by bipolar electrocoagulation, producing a blanched appearance indicative of the absence of blood flow. Note the difference between blanched tissue and white areas due to reflective glare (dashed arrows). (Bottom right) The monopolar electrode developed by Northstar Neuroscience. The exposed portion of the cortical contact was placed on the pial surface overlying the spared PMD DFL area (black circle in left and top right panels) and secured in place during surgical closing. The return electrode was subcutaneously implanted in the mid-torso to complete the electrical circuit.

Ischemic Infarct

Bipolar electrocoagulation of surface vasculature was used to produce the ischemic infarct.^14,24,30 The baseline M1 DFL map was used to determine the cortical territory subjected to a permanent ischemic infarct, with the goal of targeting all of the M1 DFL area while limiting the extent of injury to other representations, notably adjacent proximal forelimb areas and nearby PMD DFL areas. All arterial and venous vessels and vessel branches within the target region were occluded, with an exception for the main “trunk” of large pass-through vessels that supplied other cortical territories. Vessels were monitored for 10 minutes for signs of reperfusion, and reoccluded as necessary. A digital image of the infarct tissue was captured to document and verify the extent of the target region (Figure 2). This infarct technique produces an injury with sharp borders that extends through all cortical layers but spares the underlying white matter^24,29; as such, it produces a selective injury that is ideal for examining functional plasticity in spared tissue. Since the duration between infarct creation and final follow-up behavior testing was 22 to 24 weeks in each monkey, calculation of lesion volume based on histological data is unreliable. ²⁹ Instead, the size of the infarct (in mm²) was calculated from the scaled image of the preinfarct blood vessels compared with the postinfarct blood vessels (Figure 2). By superimposing ICMS maps of motor representations, this allows us to describe the infarct size in terms of the amount of loss of the M1 DFL representation.^{14,24,25,27,31,32}

Electrode Implantation and Cortical Stimulation

All monkeys were implanted with a monopolar electrode system (developed by Northstar Neuroscience, Seattle, WA) consisting of a cortical surface electrode with a single (monopolar) 4-mm diameter circular contact and a separate “return” electrode with three 4.5-mm diameter circular contacts. Wire leads connected the electrodes to a portable battery-powered stimulator unit.

In the CS/RT group, the monopolar electrode was placed over the PMD DFL, with overlap onto adjacent proximal forelimb (elbow and shoulder) representations due to the size of the contact area (Figure 2). In the RT group, similar electrode placement was conducted over either PMD DFL (n = 6) or PMV DFL (n = 2), but no therapy stimulation was conducted in these animals. In all monkeys, the return electrode was implanted subcutaneously in the mid-to-lower back, 1 to 2 cm lateral to the spine, on the same side of the body as the implanted cortical hemisphere, with the contacts facing the underside of the skin. Wire leads from both the cortical and return electrodes were tunneled subcutaneously to a common exit incision in the mid-back and connected to the stimulator unit worn in a backpack-like pocket of the primate jacket. The lead connections were arranged to deliver cathodal current via the cortical electrode.

The CS-evoked movement threshold (MT) for each monkey in the CS/RT group was determined prior to RT initiation. Individual stimuli were triggered remotely (via computer and wireless controller) and evoked movements were observed while monkeys were at rest in their home cages. MT was determined by delivering a burst of cathodal pulses at 100 Hz for 1 second. Each pulse was charge balanced but asymmetrically biphasic, with an active-driven 100-µs square phase followed by a passive 9900-µs decaying exponential phase. Starting at a level below the expected MT, the amplitude of MT test bursts was gradually increased in 0.12-mA steps until a motor response was detected visually, then reduced in 0.06-mA steps until the response was unobservable (0.06 mA was the minimum step level possible for this stimulation system). The interval between each MT test was a minimum of 10 seconds. The lowest current that produced an observed movement was defined as the MT and an amplitude of 50% MT was used during CS therapy.^12-15 MT was defined twice per week during the RT period and the therapy amplitude was adjusted accordingly.

During CS/RT therapy sessions, continuous 100 Hz pulses (at 50% MT) were delivered for a total duration of 35 minutes; CS was initiated 5 minutes prior to the start of the RT session and remained on for the remainder of the session (30 minutes).

Data Analysis and Study Outcomes

Statistical analysis was performed using Prism 6 (GraphPad Software), with 2-tailed hypotheses and α = .05, and reported/graphed as mean ± standard error of the mean. Behavioral performance and neurophysiological mapping data were analyzed with 2-way repeated-measures analysis of variance and Bonferroni post hoc tests. Relationships between behavioral recovery and DFL map areas were examined with correlation analysis. CS movement thresholds were analyzed with 1-way repeated-measures analysis of variance and Bonferroni post hoc tests. All behavior and mapping data were normally distributed (Kolmogorov-Smirnov test).

The primary outcome measure was motor performance at 1 week posttherapy. Secondary outcome measures included motor performance at follow-up, within-group changes in behavior over the course of the experiment, and changes in DFL representations in M1, PMD, and PMV after therapy. Subgroup statistical analysis revealed no effect on outcomes related to the timing of postinfarct electrode implantation, location of cortical electrode, or posttherapy timing of ICMS mapping; thus, data were pooled and analyzed based only on RT or CS/RT group assignment.

Infarct Size in Baseline M1

For the RT group (n = 8), the infarct affected 98.1% of the baseline M1 DFL map, intentionally sparing 0.25 ± 0.09 mm² of M1 DFL area (Figure 2). For the CS/RT group (n = 7), the infarct affected 97.2% of the baseline M1 DFL map, sparing 0.43 ± 0.14 mm² of M1 DFL area. Additional non-DFL territory included in the infarct region was 8.20 ± 0.86 mm² in the RT group and 8.35 ± 1.32 mm² in the CS/RT group. There were no statistical differences between groups for any of these measurements.

Behavior Performance

Five time points were selected to assess the hypothesis that CS/RT would enhance behavioral performance over RT alone (Figure 3). To facilitate comparisons between monkeys, a motor performance index score was calculated by normalizing each monkey’s preinfarct performance scores, with preinfarct equal to 1.0 and larger scores indicating impaired performance (see Methods section and Plautz et al ¹⁴ ).

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Figure 3.

Behavior performance on the pellet retrieval task for rehabilitative training (RT; dark gray) and cortical stimulation (CS)/RT (light gray) groups. Both groups exhibited postinfarct impairments (3-fold increase in motor performance index score; postinfarct bars), partial spontaneous recovery by 6 weeks postinfarct (pretherapy bars), and additional recovery during the intervention period (posttherapy bars). The CS/RT group continued to improve while the RT group regressed slightly during the 12-week follow-up period (follow-up bars). There was a significant effect of time (analysis of variance, double asterisk), and significant within-group effects (single asterisks) for postinfarct impairment (vs preinfarct) and posttherapy recovery (vs postinfarct). There were no significant differences between RT and CS/RT groups at any of the 5 time points.

There was a significant effect of Time on the motor performance index (F_{4, 44} = 22.66, P < .0001), demonstrating an effect of the infarct on behavioral performance. However, there were no significant group or group × time interaction effects. Importantly, there was no significant difference in the primary outcome measure comparing behavior recovery in the CS/RT group versus the RT group at 1 week posttherapy.

Each of the 15 monkeys exhibited postinfarct impairments on the retrieval task, with index scores of individual monkeys ranging from 2.05 to 5.35 during the first week postinfarct. Postinfarct performance for both groups was significantly impaired compared with preinfarct (RT index score = 3.18 ± 0.48, P < .0001; CS/RT index score = 3.16 ± 0.21, P < .0001; Figure 3). Index scores improved for both groups during the 6 weeks prior to therapy initiation (pretherapy time point, RT, 2.23 ± 0.41; CS/RT, 1.98 ± 0.32), consistent with the phenomenon of “spontaneous” recovery. After therapy (posttherapy time point), index scores were significantly improved in both groups compared with their postinfarct scores (RT, 1.70 ± 0.16, P < .002; CS/RT, 1.57 ± 0.24, P < .0001). At the end of the follow-up period, the RT group had regressed slightly (index score 1.79 ± 0.21) while the CS/RT group had improved slightly (index score 1.46 ± 0.26), though this difference was not significant (within-group vs posttherapy or between-group for RT vs CS/RT).

Changes in Cortical Motor Representations

Each cortical region (M1, PMD, PMV) was compared to evaluate differential effects of RT and CS/RT on functional representations in postinfarct surviving tissue (Figures 4 and 5). Compared with the baseline map, the M1 DFL representation in the posttherapy map was significantly reduced in size in both groups (RT, −95.0%, P < .0001; CS/RT, −95.3%, P < .0001) (Figure 4, top row, left graph), indicating that the infarct successfully destroyed the M1 DFL map as intended, and that behavior improvements were unlikely to be related to functional recovery in M1. ICMS stimulation thresholds for DFL responses in M1 were higher in both groups between baseline and posttherapy maps (RT, 14.6 ± 0.9 µA baseline, 21.7 ± 3.3 µA posttherapy; CS/RT, 12.9 ± 0.9 µA baseline, 17.0 ± 3.8 µA posttherapy). These increases were significant overall (effect of time, P < .036) though neither within-group change reached significance.

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Figure 4.

Area of distal forelimb (DFL) representations in primary motor (M1), dorsal premotor (PMD), and ventral premotor (PMV) cortices prior to infarct (“baseline”, dark gray) and after rehabilitative training (RT) or cortical stimulation (CS)/RT treatment (“posttherapy,” light gray). (Top row) In M1, the ischemic infarct produced a significant and equivalent reduction in DFL area in both groups (P < .001, left graph). PMD DFL area significantly increased in the RT group (P < .029) but not in the CS/RT group (middle graph). There were no changes in total DFL area in PMV for either group (right graph). There were no between-group differences in baseline area in PMD or PMV for any of the data shown in this figure (all 9 graphs). (Middle row) Comparison of 3 movement types evoked by intracortical microstimulation (ICMS) in PMD. There were no significant within-group differences in digit (left graph), wrist/forearm (middle graph), or dual-response (right graph) representations in PMD. The increase in overall DFL area in the RT group (top row) was driven by the separate nonsignificant increases in wrist/forearm and dual-response representations (69% of these dual-responses included wrist/forearm movements). (Bottom row) Comparison of 3 movement types evoked by ICMS in PMV. There was a significant decrease in digit area (P < .009, left graph) and a significant increase in wrist/forearm area (P < .021, middle graph) for the CS/RT group in PMV. These changes offset each other in the overall size of the PMV DFL map in this group (top row). There were no significant changes in the RT group in PMV.

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Figure 5.

Representational maps from 2 monkeys in the cortical stimulation/rehabilitative training (CS/RT) group (panels A and B) and 1 monkey in the RT group (panel C). Black dots indicate the location of intracortical microstimulation (ICMS) sites in each map, and distal forelimb (DFL) sites are indicated by 1 of 5 colors (as in Figure 2). Non-DFL sites (proximal forelimb, face, etc) are shaded light gray (rather than dark blue, as in Figure 2) to improve visibility of the DFL regions. Borders between primary motor (M1), dorsal premotor (PMD), and ventral premotor (PMV) cortices (dashed black lines) were defined in preinfarct (baseline) maps based on ICMS data and vascular landmarks. Notably, some PMD DFL sites are often adjacent to the M1 DFL area, with as little as 500 µm separation. In posttherapy maps, the vascular landmarks from the baseline map were used to delineate the borders. Tissue necrosis of the infarct caused deformation of intact cortical tissue and surface vasculature, which is reflected by the shift in border positions in the posttherapy maps. In monkey C, the 2 infarct outlines indicate that a large pass-through artery was spared from occlusion (see Methods section). All 3 subjects had near-complete loss of DFL area in M1, with a limited number of ICMS-responsive sites at the rostral edge of the infarct (consistent with prior reports and likely reflecting uneven tissue distortion between the cortical surface and deeper layers ¹⁴ ). Movements were evoked by ICMS throughout PMD and PMV after therapy, indicating that CS did not adversely affect the physiological responsiveness of the cortical tissue. For all three subjects, the monopolar electrode was implanted 2 weeks postinfarct (shown on the preinfarct map only for reference), and posttherapy maps were derived 7 weeks posttherapy. Image scaling for figure display is responsible for the apparent change in size of the electrode between maps (all electrode circles are 4 mm in diameter).

In PMD (the target of CS in the CS/RT group), the posttherapy DFL representation increased significantly in the RT group (+100.2%, P < .029) but not in the CS/RT group (+14.5%) (Figure 4, top row, middle graph). ICMS thresholds for PMD DFL responses were not significantly different for either group (RT, 18.1 ± 3.1 µA baseline, 15.9 ± 2.6 µA posttherapy; CS/RT, 17.9 ± 1.6 µA baseline, 18.4 ± 2.4 µA posttherapy). In PMV, the posttherapy DFL representation increased in both groups (RT, +2.4%; CS/RT, +14.3%) (Figure 4, top row, right graph) but neither increase was significant. PMV DFL ICMS thresholds were not significantly different for either group (RT, 19.1 ± 2.0 µA baseline, 19.5 ± 1.9 µA posttherapy; CS/RT, 16.9 ± 0.6 µA baseline, 16.9 ± 0.9 µA posttherapy). Combining both premotor regions (PMD + PMV), DFL area increased in both groups (RT, +29.8%; CS/RT, +14.4%) but again neither increase was significant.

Distal forelimb representations in PMD and PMV were further explored by comparing changes in area for digit, wrist/forearm, and all dual-response movements evoked by ICMS. In PMD (Figure 4, middle row), there were no significant changes for any of the 3 movement types for either group. Although no individual change was significant, the increase in PMD DFL described earlier for the RT group was primarily driven by increases in wrist/forearm (+0.44 mm², +118%) and dual-response (+0.72 mm², +274%) movements, with 69% of the dual-response area including a wrist/forearm component. In PMV (Figure 4, bottom row), the CS/RT group had a significant decrease in digit area (−0.98 mm², −59%, P < .009) and a significant increase in wrist/forearm area (+1.19 mm², +96%, P < .021). These PMV changes in the CS/RT group offset each other in the overall DFL area analysis.

Baseline and posttherapy ICMS maps from 2 subjects in the CS/RT group and 1 subject in the RT group are illustrated in Figure 5. One CS/RT subject (Figure 5A) had large DFL expansion in both PMD (+1.33 mm², +177%) and PMV (+1.44 mm², +34%) while the other CS/RT subject (Figure 5B) had a reduction of DFL in PMD (−0.26 mm², −27%) and a smaller expansion in PMV (+0.56 mm², +18%). It is notable that CS (delivered to both subjects) did not affect tissue responsiveness to ICMS, indicating that group differences in PMD DFL were not due to a physiological dysfunction in the stimulated cortex. The RT subject (Figure 5C) had a large DFL expansion in PMD (+1.28 mm², +582%) and a smaller DFL expansion in PMV (+0.56 mm², +16%).

Relationship Between Behavior and Map Changes

Although significant recovery occurred in both groups, recovery levels of individual monkeys ranged from 10% to 104% of their postinfarct impairment level. Similarly, both groups had expanded premotor DFL maps, but changes in individual monkeys ranged from −47% to +66% (for the combined PMD + PMV area). Correlation analysis found that behavioral recovery was positively related to expanded DFL areas in PMD (r = 0.682, P < .015), PMV (r = 0.558, P < .06), and combined PMD + PMV (r = 0.693, P < .013) (Figure 6). This suggests that, independent of the application of CS during RT, changes in behavioral performance were associated with plasticity of motor representations in premotor cortex.

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Figure 6.

Scatterplot of behavior improvement (%Recovery) versus change in distal forelimb (DFL) representational area in dorsal premotor cortex (PMD, left graph), ventral premotor cortex (PMV, middle graph), and the combined premotor cortex area (PMD + PMV; right graph). There was a strong association between better behavioral recovery and larger DFL areas in the intact premotor cortex. Monkeys illustrated in Figure 5 are indicated on each of the scatterplots (“A,” “B,” and “C”); note that monkey A (cortical stimulation/rehabilitative training; CS/RT subject) had good recovery and larger DFL maps, monkey B (CS/RT subject) had poorer recovery and smaller DFL maps, and monkey C (RT subject) was intermediate between A and B. %Recovery (x-axis) is defined as the amount of performance improvement (postinfarct minus posttherapy) divided by the total deficit (postinfarct minus preinfarct). Change in area (y-axis) is defined as posttherapy DFL map area minus baseline DFL map area; thus negative values indicate a reduction in DFL area in posttherapy maps. Data are plotted for the 12 monkeys (5 RT, black circles; 7 CS/RT, gray circles) for which complete behavior and mapping results were available (see Methods section). Linear curve fit added to graphs for visual clarity after statistics were obtained by correlation analysis.

Cortical Stimulation–Evoked Movements and Movement Thresholds

Cortical stimulation reliably evoked movements in all 7 CS/RT subjects during each MT determination session. Evoked movements most often involved movements of the head, neck, and torso, typically characterized as “torso twitches” or “head ducking,” with very few instances of evoked forelimb movements. Movements did not appear to be visibly synchronized to the 100 Hz stimulation frequency, but rather were unitary events in response to each MT pulse burst.

The average MT in the CS/RT group was 2.98 ± 0.12 mA one week prior to therapy, 2.90 ± 0.15 mA during the first week of therapy, and 3.05 ± 0.05 mA during the final week of therapy. There were no significant differences in MT values over time; nor were there any significant correlations between MT and behavioral performance or between MT and DFL areas in motor maps.