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Robotic mapping of motor cortex in children with perinatal stroke and hemiparesis

Abstract Brain stimulation combined with intensive therapy may improve hand function in children with perinatal stroke‐induced unilateral cerebral palsy (UCP). However, response to therapy varies and underlying neuroplasticity mechanisms remain unclear. Here, we aimed to characterize robotic motor mapping outcomes in children with UCP. Twenty‐nine children with perinatal stroke and UCP (median age 11 ± 2 years) were compared to 24 typically developing controls (TDC). Robotic, neuronavigated transcranial magnetic stimulation was employed to define bilateral motor maps including area, volume, and peak motor evoked potential (MEP). Map outcomes were compared to the primary clinical outcome of the Jebsen–Taylor Test of Hand Function (JTT). Maps were reliably obtained in the contralesional motor cortex (24/29) but challenging in the lesioned hemisphere (5/29). Within the contralesional M1 of participants with UCP, area and peak MEP amplitude of the unaffected map were larger than the affected map. When comparing bilateral maps within the contralesional M1 in children with UCP to that of TDC, only peak MEP amplitudes were different, being smaller for the affected hand as compared to TDC. We observed correlations between the unaffected map when stimulating the contralesional M1 and function of the unaffected hand. Robotic motor mapping can characterize motor cortex neurophysiology in children with perinatal stroke. Map area and peak MEP amplitude may represent discrete biomarkers of developmental plasticity in the contralesional M1. Correlations between map metrics and hand function suggest clinical relevance and utility in studies of interventional plasticity.


| INTRODUCTION
Perinatal stroke is a focal vascular brain injury near the beginning of life (Dunbar & Kirton, 2019;Raju et al., 2007). It is the most common etiology of unilateral cerebral palsy (UCP) (Wu et al., 2006), affecting approximately 1:1000 live births (Dunbar et al., 2020;Raju et al., 2007) and millions of people globally (Oskoui et al., 2013). Such early brain injury interrupts typical development. Depending on lesion type, size, location, and many other factors, functional limitations after perinatal stroke may include motor deficits, sensory dysfunction, cognitive delay, language and speech impairment, and epilepsy (Kirton & Deveber, 2013). Such limitations result in life-long morbidity and decrease the quality of life of children and families.
UCP is the most common form of cerebral palsy (CP), with motor and sensory impairment lateralized to half of the body. The focal, unilateral lesions of perinatal stroke offer an ideal model for studying developmental neuroplasticity of the human motor system, with strong evidence from both animal and human mapping literature Martin et al., 2007Martin et al., , 2011Staudt, 2007). In typical prenatal development, corticospinal projections from diffuse cortical areas connect to diffuse areas of spinal cord grey matter with approximately equal contralateral and ipsilateral connections. During early postnatal development, these connections are refined with most projecting from the primary motor cortex (M1) to the anterior horn of the contralateral spinal cord while ipsilateral projections are pruned.
Perinatal stroke may alter this process, whereby corticospinal projections from the lesioned hemisphere lose their ability to compete for contralateral spinal synapses. This in turn may allow contralesional M1 projections to preserve their ipsilateral corticospinal projections to the affected hand Zewdie et al., 2017). The relative relationship between these contra-and ipsi-lateral corticospinal arrangements have been variably associated with hand function in children with UCP though the relationship is incompletely understood Riddell et al., 2019;Simon-Martinez et al., 2019;Smorenburg et al., 2017), suggesting additional developmental alterations in motor neurophysiology are occurring.
Categorical characterization of corticospinal organization as ipsilateral or contralateral is crude and unable to account for most of the variance in function in children with UCP (Friel et al., 2016;Marneweck et al., 2018). Motor maps are topographic representations that may reflect motor control of specific body parts (Penfield & Rasmussen, 1950). Motor map metrics such as area and volume may be useful biomarkers of functional impairment and recovery in animal studies (Milliken et al., 2013; and in adult stroke (Grefkes & Ward, 2014). Specifically, animal studies suggested that motor map territory can be a valuable neurophysiological measure for quantifying the neural substrate topography and plasticity associated with functional change due to brain injury or motor skill training Nudo, Wise, et al., 1996). Important findings pertinent to rehabilitation include observations that skill training-induced improvements in motor function can be accompanied by expansion of map area and normalization of corticospinal neuron distribution in the spinal cord (Friel et al., 2000(Friel et al., , 2012Kleim et al., 1998).
While map territories have been measured with intracortical electrophysiology in animals, noninvasive and painless transcranial magnetic stimulation (TMS) provides similar opportunity in humans and has been used extensively in adult stroke patients to better understand recovery, training effects, and function (Thickbroom et al., 2004;Yarossi et al., 2019). TMS experience in the developing brain appears to be safe and well tolerated  with valuable applications in CP populations. Kesar et al. (2012) showed that the geometric location of peak motor activations may be displaced, the degree of which is associated with function in children with nonspecific CP. In a more homogeneous group of children with UCP, Friel et al. (2016) showed structured intensive bimanual training could expand motor map area and increase MEP amplitudes of the more-affected hand. These promising studies were not able to study specific disease-states such as perinatal stroke and were vulnerable to the challenges of performing manual TMS motor mapping in children.
Robotic TMS systems may mitigate such challenges, affording near real-time localization of coil placement, reducing human technical errors, and decreasing experiment time. We have demonstrated the reliability  and feasibility of robotic TMS to map M1 neurophysiology in typically developing children (TDC; Giuffre et al., 2019Giuffre et al., , 2021Grab et al., 2018). Robotic TMS has not been applied to children with perinatal brain injuries and CP.
Understanding the mechanisms of developmental plasticity that occur after perinatal stroke is germane to the development of novel therapies . Early, randomized, controlled clinical trials of noninvasive brain stimulation suggest possible efficacy (Gillick et al., 2018;Kirton et al., 2017). Most trials have targeted the contralesional hemisphere, but precise targets and mechanisms of interventional plasticity remain poorly defined. The importance of the entire contralesional hemisphere in determining clinical function in children with perinatal stroke is increasingly understood (Craig et al., 2020) but more refined models of the role of the primary motor cortex are required. Personalized maps of motor neurophysiology may also help identify optimal candidates for, and the interventional plasticity effects of, both traditional (e.g., constraint-induced) and neuromodulatory therapies. However, evidence to date suggests simple corticospinal tract organization (Friel et al., 2021) or more advanced imaging methods (Juenger et al., 2013) have a limited ability to do so, suggesting further possible utility of detailed, individualized motor maps.
Here, we aimed to employ robotic TMS motor mapping to define bilateral map characteristics and their association with clinical function in children with perinatal stroke-induced UCP. We hypothesized that motor map area of children with UCP would be smaller than that of TDC and that the motor map area would be positively correlated with hand function in children with UCP.

| Participants
Participants with perinatal stroke were recruited from the Alberta Perinatal Stroke Project, a population-based research cohort (Cole et al., 2017) to participate in a rehabilitation clinical trial called SPORT (Stimulation for Perinatal Stroke Optimizing Recovery Trajectories, clinicaltrials.gov/NCT03216837). SPORT is an on-going, randomized, sham-controlled, double-blinded multicenter trial examining the efficacy of camp-based, child-centered intensive rehabilitation paired with tDCS. Baseline (preintervention) neurophysiology assessments included the motor mapping described here.
Typically developing controls (TDC) were recruited to participate in the AMPED (accelerated motor learning in PEDiatrics) trial to examine the effects of high-definition and conventional tDCS on motor learning in children (Cole et al., 2018). AMPED was a randomized, double-blinded, sham-controlled clinical trial (clinicaltrials.gov/ NCT03193580). Briefly, children who were between 12 and 18 years of age, right-handed, and of typical neurodevelopment without major medical conditions or TMS/MRI contraindications were recruited. The baseline (preintervention) mapping results are used as the comparison group for the current study. The methods used for MRI and TMS mapping were identical for both children with perinatal stroke and TDC.

| Magnetic resonance imaging
MRI was performed at Alberta Children's Hospital Diagnostic Imaging Suite using a 3T General Electric MR750w scanner (GE Healthcare, Chicago, IL). High resolution T1-weighted anatomical images were obtained using fast spoiled gradient echo sequence (FSPGR BRAVO, 226 contiguous axial slices, repetition time = 8.5 ms, echo time = 3.2 ms, voxels = 1 mm isotropic, duration $5 min). Individual T1 structural images were utilized in the TMS neuronavigation software, described below.

| TMS motor mapping
TMS motor mapping was conducted at the Pediatric Non-invasive Neurostimulation Laboratory at the Alberta Children's Hospital in Calgary Canada. Robotic TMS (Axilum Robotics, France) was utilized to perform all motor mapping procedures, the details of which are described elsewhere (Giuffre et al., 2019;Grab et al., 2018) and below. The Axilum TMS robot accommodates a 70-mm Air-Film coil (Magstim, UK), allows precise neuronavigation, near real-time motion correction, and compensates for human operation errors associated with manual mapping (Goetz et al., 2019).
Neuronavigation (Brainsight2, Rogue Research, Montréal) was used to reconstruct skin and curvilinear brain, to generate stimulation grids and targets (details below), and to record stimulation trials. The neuronavigation software enables maintenance of stimulation target alignment tangentially to the reconstructed scalp and stimulation trajectories projected perpendicular to the cortical target. Adjustments of individual trajectories were sometimes required, especially in participants with large brain lesions. Coil orientation was maintained by the robotic system at a 45 angle to the interhemispheric fissure to produce posterior-anterior current.
Following participant orientation, a pair of Ag-AgCl electrodes (Kendall, Chicopee, MA) were applied to record surface electromyogram (EMG) from the first dorsal interosseous (FDI) muscles bilaterally with a ground electrode on the wrist styloid process. EMG signals were amplified (gain = 1000, Bortec Biomedical, Calgary), band-pass filtered (20-2500 Hz) and then digitized at a rate of 5000 Hz using CED 1401 hardware and Signal 6.0 software (Cambridge Electronic Design Limited). Raw EMG data were displayed on a computer screen to allow online monitoring by investigators. Participants were seated comfortably in the robot chair with both arms resting on a cushioned support, watched a movie among a selection in our laboratory, and encouraged to take breaks when needed.
Experiments began by mapping the contralesional (intact) motor cortex of children with UCP and the right hemisphere in TDC. Stimulations were first delivered in proximity to the anatomical hand knob to identify the "hotspot" of the contralateral FDI; the location producing the largest and most consistent MEP. This location was subsequently marked using the neuronavigation software and used for determining the resting motor threshold (RMT). Details for determining RMT were described in details in Giuffre and colleagues . Briefly, RMT was estimated from a stimulus response curve (SRC) which was produced by delivering four stimuli at 100% maximum stimulator output (%MSO) and then decreasing in 5% increments until no MEPs >50 μV were observed. For children with UCP whose input-output data did not fit a sigmoidal input-output curve, a conventional RMT thresholding method was performed. Intensity was adjusted with increments of 2%MSO based on the MEP responses to determine the minimal intensity inducing MEP >50 μV in >5/10 trials.
The same thresholding procedure was then performed for the ipsilateral, affected FDI within the contralesional hemisphere.
Modified procedures to localize the contralateral FDI hotspot were performed for the lesioned hemisphere in children with UCP.
For participants with arterial stroke, the anatomical hand knob may have been absent or altered. In such cases, task-based functional MRI peak activation location (during performance of a squeeze ball task as described elsewhere (Baker et al., 2020) was marked and used as a reference in the neuronavigation software. The perilesional area was probed for any location producing the largest and most consistent MEP.
A customized rectangular grid (13 Â 13 grid-points, 0.7-cm spacing) was then created and centered at the hand knob area (or the task-based fMRI activation location if the anatomy was altered) of individual participant's MRI. Four single-pulses were delivered at 120% of the RMT at 1-s inter-stimulus intervals to each grid point that allows acceptance or rejection of individual MEPs using the robotic TMS system (contralesional M1, n = 20, lesioned M1, n = 7). For children who were not able to tolerate such frequency of pulses, the inter-stimulus interval was adjusted to 5-10 s. A serial progression across the grid was employed to define each map as described previously (Giuffre et al., 2019Grab et al., 2018). Beginning with the hotspot location, the coil was then moved anteriorly or posteriorly until no MEP responses were observed. The same approach was then completed in the medial to lateral plane in the neighboring grid row and repeated until a border of nonresponsive sites (<2/4 stimulations producing a MEP >50 μV) was established. If the RMT of the affected FDI differed from the unaffected FDI by >5%MSO, the contralesional M1 was mapped at two stimulator intensities, with each intensity corresponding to 120% of RMT of the affected or unaffected FDI.
Similar procedures were utilized for mapping in TDC, except for that the robotic TMS system always delivered four pulses at 120% RMT at 1 Hz to each grid point.

| TMS data analysis and motor maps
EMG sweeps were inspected visually during online data acquisition; trials were excluded when background signals were above 50 μV or when children were moving and repeated until a clean recording was obtained. EMG data were exported offline and subsequently analyzed using in-house scripts (MATLAB R2016b, The MathWorks, Inc., Natick, MA). Recordings with background EMG signal (10-30 ms prior to the TMS pulse) >50 μV were considered as facilitated and were discarded. MEPs >50 μV occurring 20-80 ms after the TMS pulse were included. Peak-to-peak MEP amplitude was averaged across valid trials at each responsive site.
Individualized two-dimensional (2D) heatmaps and threedimensional (3D, x-y-MEP; where x-axis is parallel to the central sulcus and y-axis is parallel to the interhemispheric fissure) motor maps were generated and overlaid on individual MRIs (Figures 1 and 2).
Based on previous experience (Grab et al., 2018), a minimally acceptable number of responsive sites was determined to be ≥9.
The primary motor mapping outcome was motor map area, calculated as the number of responsive sites (grids) multiplied by one grid area (0.7-cm Â 0.7-cm = 0.49 cm 2 ). Secondary mapping outcomes included the following: 1. Peak MEP amplitude of the 3D map, defined as the maximum averaged peak-to-peak MEP of the entire map.
2. Map volume, calculated as the cumulative sum of the peak-to-peak MEP amplitude at each grid-location multiplied by map area (0.49 cm 2 ).  Jebsen et al., 1969). To avoid frustration, when participants had difficulty completing the task within a given 2-min duration, the duration for that task was marked as 120 s (Duncan et al., 1998). Higher scores on the JTT reflect poorer performance. The Box and Blocks Test (BBT) evaluated unimanual dexterity by measuring how many blocks a participant can pick up, move over a F I G U R E 1 A typical example of a three-dimensional (3D) motor map. X and y axes are the coordinates from the mapping grid and the z axis represents the peak-to-peak averaged motor evoked potential (MEP) amplitude at the corresponding grid location. Color bar on the right represents the size of the MEP amplitude barrier, and drop off with each hand within 1 min (Mathiowetz et al., 1985). Both JTT and BBT assessments are standardized and reliable assessment protocols in TDC and children with UCP (Araneda et al., 2019;Jongbloed-Pereboom et al., 2013;Reedman et al., 2016;Taylor et al., 1973). The Assisting Hand Assessment (AHA) evaluated upper extremity hand function in various bimanual play activities in children with hemiparesis (Krumlinde-Sundholm et al., 2007). Scores are reported in AHA logit units (0-100). Higher scores on both BBT and AHA reflect better performance. The JTT was also administered to TDC (Cole et al., 2018;Giuffre et al., 2021). Note that we acknowledge the unaffected upper extremity should ideally be termed "lessaffected" and the affected side "more-affected." We use the terms "unaffected" and "affected" to keep the terms consistent in the manuscript.

| Safety and tolerability
We used a modified pediatric noninvasive stimulation safety and tolerability questionnaire to evaluate children's experiences of the robotic TMS session (Garvey et al., 2001;Zewdie et al., 2020). Children were asked to rank the experience of their TMS session against other common childhood experiences (e.g., play a video game). Additionally, possible side effects associated with TMS (e.g., headache, neck pain) were also screened for the duration and intensity (mild, moderate, or severe) immediately following the session.

| Statistical analysis
Shapiro-Wilk test was used to test data normality. First, motor map variables were compared between bilateral FDI of the contralesional hemisphere in children with UCP using paired or independent t-tests and Wilcoxon signed-rank or Mann-Whitney U tests, where appropriate. Next, motor map variables were compared between groups using

| Relationships between motor maps and clinical outcomes
For UCP participants with a valid contralesional M1 map (n = 23), the mean (SD) JTT of the affected hand was 225(206) s, the average BBT score of the affected hand was 25(13) blocks/min, and the average AHA score was 55 (16)  arman's r = À.47, p = .02). These relationships trended toward significance when corrected for age (partial correlation = À.33, p = .07; partial correlation = À.35, p = .05, respectively). In all the above correlations, higher values of mapping outcomes were associated with better hand function (lower score in the JTT). There were no other significant correlations between mapping and clinical outcomes.

| DISCUSSION
We demonstrate that robotic TMS motor mapping is feasible and safe in children with perinatal stroke and UCP. We were able to successfully generate unaffected FDI motor maps in 83% and affected FDI motor maps in 52% of children with UCP when stimulating the contralesional M1. Generating affected FDI maps from stimulation of the lesioned M1 was challenging, accomplished in only 17% of the sample.
Our results show how cortical motor maps might integrate into such "pathway" oriented classifications of developmental plasticity after perinatal stroke (Hilderley et al., 2019;Kirton et al., 2021). Furthermore, addition of motor map characteristics to such models may provide new opportunities to study interventional plasticity in this population. Most existing noninvasive brain stimulation trials in children with UCP to date have targeted the contralesional M1 (Gillick et al., 2015(Gillick et al., , 2018Kirton et al., 2016Kirton et al., , 2017. Our findings of differences between the affected and unaffected FDI map characteristics add to recent studies exploring the underlying neurophysiology between the affected and the unaffected hands in children with UCP (Hawe et al., 2020;Rich et al., 2017). To date, most studies in this population have focused on the affected hand. However, the unaffected hand has also been shown to be impaired, including detailed robotic sensorimotor examinations in the perinatal stroke population (Kuczynski et al., 2018). Given that many children with UCP are severely impaired and rely heavily on the unaffected limb for daily function, understanding and even targeting this extremity for therapeutic intervention is considered but remains poorly studied.
While we found differences in map area and peak MEP amplitude and correlations to motor outcomes, it is unclear how these neurophysio- wide range of differences in such "laterality" and only modest correlations with clinical function (Friel et al., 2021;Kuo et al., 2021;Smorenburg et al., 2017). While the degree to which an ipsilateral map can be "installed" in the unintended, contralesional hemisphere is also not well defined and likely varies across individuals, that areas appear to fall within the normal range may indirectly support the occurrence of such developmental plasticity (Baker et al., 2020). This may be further supported by a sub-analysis showing that the unaffected FDI area from stimulating the contralesional M1 was larger than that of the left FDI when stimulating the right M1 in TDC.
In contrast, our findings of differences in the peak MEP amplitude between hands and the two groups are interesting. Friel et al. (2016) showed an increase in the overall MEP amplitude after motor learning in the structured group in children with UCP. Similarly, Nemanich et al. (2019) observed differential changes in the MEP amplitude in two groups children after receiving different therapies (active tDCS combined with constraint therapy vs. sham tDCS combined with constraint therapy). Recently, our group reported better correlations between subsets of mapping outcome (area and volume) and JTT in healthy adults . Briefly, this study showed decreased variabilities when using smaller subsets of mapping outcome as it contains larger MEPs of a motor map, when compared to using the entire map. Collectively, we speculate our findings here may suggest the peak of "3D-map mountain" to be an important biomarker and future studies may use such a simple metric to probe the underlying neurophysiology in children with UCP and TDC.
As the first study utilizing robotic TMS mapping in children with UCP, we found favorable safety and tolerability in support of future studies. Side effects reported were generally mild, self-limiting, and in keeping with what is now a large body of TMS safety data in children . Common experiences including tingling sensation, headache, and neck pain are expected but should be fully disclosed to participants and caregivers when obtaining informed consent. While rates appear similar to previously described CP and TDC populations, challenges for some children with UCP such as difficulty with attention or sitting still should be considered. While we had volunteers help support and protect children's head and provided breaks when necessary, the robotic coil requires a certain level of force feedback to ensure the contact between the coil and participants. Proper introduction, education, and orientation prior to initiating robotic TMS in children is recommended to optimize participant experience, compliance, and data collection.
Future directions include examining the application of robotic mapping in children with more severely affected CP as technology advances, and to understand the neurobiology of the motor maps in children with UCP and TDC as the goal of motor mapping research is to understand the neurophysiology and mechanisms associated with early brain injury and functional improvements. As robotic TMS has shown promising utility in providing details of neurophysiology, future studies would benefit from automatic mapping procedures to enhance experiment efficiency with established criteria. This technology may become a tool to obtain outcome as biomarkers to advance personalized rehabilitation in children with UCP.
Important limitations are acknowledged. First, the Axilum robotic TMS system only accommodates a 70-mm diameter biphasic Air-Film coil. The peak magnetic field strength (0.8T) is lower than that of other coils designed for neurophysiological evaluations (e.g., peak magnetic field of the Alpha Flat coil is >1.44T). This unmodifiable factor decreased our ability to obtain suprathreshold (and likely some rest threshold) MEP responses in our study, particularly given the known higher RMT in children and those with UCP, particularly in the lesioned hemisphere (Eyre et al., 2007;Rich et al., 2017;Smorenburg et al., 2017). Second, we may have underestimated motor map outcomes for the eight participants where stimulation of the contralesional M1 was unable to match the mapping intensity at 120% RMT (i.e., their RMT was >85%MSO). Similar phenomenon likely contributed to the challenges of mapping the lesioned M1. It is worthwhile considering this challenge of teasing apart whether the absence of MEPs when stimulating the lesioned hemisphere is due to true lack of cortical motor representation, insufficient stimulator intensity, or a combination of both. Other modalities, such as task-based fMRI, might be utilized to localize motor activation areas in the lesioned hemisphere when TMS fails to achieve localization of the hotspot.
Third, we allowed children to watch movies as in other studies (Friel et al., 2021;Smorenburg et al., 2017) to ensure participants remained awake, steady, and interested throughout the experiment. While the movies were child-friendly, we would cautiously note that we cannot entirely exclude the effects of action observation on mapping physiology. Fourth, while the JTT, BBT, and AHA are standardized assessments, it is possible that we were not able to capture individual differences in discrete fine-motor movement quality using these outcomes. Finally, our sample size is relatively small compared to some adult studies though it represents the largest perinatal stroke study to date.

ACKNOWLEDGMENT
We thank children and families participating in this study, the staff support of following: the N3 network, the ACH Pediatric TMS laboratory, the PONI laboratory, and Drs. McMaster and Kiss laboratories.
Hsing-Ching Kuo is funded by postdoctoral awards from the CPSR and ACHRI.

CONFLICT OF INTEREST
The authors declare that they have no conflicts of interest.