The aim of this study was to compare the kinematic patterning of patients diagnosed with VPD and IPD during a reach-to-grasp task. The results indicate that patients with VPD showed similar movement durations and hand-grip conformation to patients with IPD, but longer movement duration and smaller hand opening than controls. Furthermore, for patients with VPD the onset of the grasping component was delayed with respect to the onset of the transport component when compared to the performance of controls. Although this pattern has been retrieved also for the patients with IPD, the VPD group showed a significantly shorter

delay between the beginning of the arm movement towards the target object and the opening of the fingers to grasp it. VPD, vascular parkinsonism; IPD, idiopathic Parkinson's disease; HC, healthy controls.

delay. Interestingly, the standard prehension task provides a simple and natural opportunity to examine whether the organization of upper-limb movements is somewhat dysfunctional in patients with VPD. The nature of this task, composed of a proximal transport component and a distinct but inter-related distal manipulation constituent, makes it a potentially good candidate for the exploration of the motor consequences of the disorder. This view is also supported by empirical evidence suggesting that subthalamic nucleus and internal pallidum overactivity is responsible for motor-related deficit in VPD (42) and that in primates the pallidal output of the basal ganglia is directed toward the ventrolateral thalamus, which selectively innervates the hand representation in the primary motor cortex (43, 44). Nevertheless this assumption should be taken with a certain degree of caution, given that the putative pathophysiology of VPD varies according to the type of evidence found and the behavioral manifestations observed can be linked to lesions at any level of the cortico-subcortical motor loops (45).

Zooming on the results of the kinematic analysis, significantly different patterns were found for the two target sizes in all the groups studied. The movement time was longer and the maximum grip aperture was reduced for smaller as compared to larger targets in both groups of patients (19, 24) as well as in the neurologically healthy participants (8–10). Thus, patients with VPD, as for the other groups, were able to modify the spatiotemporal characteristics of the grasping pattern in response to experimentally imposed changes in the size of the object. Patients with VPD showed longer movement duration for actions requiring greater accuracy such as when reaching for smaller objects (8–10). And they were able to scale hand opening in relation to the size of the object to be grasped (8–10). It appears, therefore, that VPD does not necessarily lead to any significant impairment of the central processes involved in organizing the reach-to-grasp movement.

Patients with VPD took longer to complete the movement and reached a smaller peak aperture than age-matched control participants. Similarly, and as previously demonstrated, patients with IPD demonstrated that their reach-to-grasp movements were slower (19, 24) and their maximum grip aperture smaller (41) with respect to control participants. Thus, VPD patients do show bradykinesia and hand hypometria, which limits the speed of movement execution and affects the modulation of hand aperture, respectively. This suggests that, as reported for patients with IPD, patients with VPD might have problems modulating movement speed and the command related to the opening/closing phases of the hand.

The kinematic analysis of the reach-to-grasp task allows examining the hypothesis that Parkinson's disease leads to a problem with concurrent execution of functionally independent motor programs with the same limb (19, 46). In this respect, significant grasp-transport coordination impairments have been observed (19, 47). On average, IPD patients tended to start distancing the index finger and the thumb later than control subjects, relative to the onset of the transport movement (i.e., delay). It appears, therefore, that IPD does lead to a significant impairment of the central processes involved in organizing the concurrent execution of functionally independent motor programs, which are executed by the same effector system. It is possible that the disease affects the well-established motor programs controlling the coordination of subcomponents in the performance of everyday actions such as reaching and grasping.

Here, we found that also patients with VPD started to open the hand later than controls. A point worth noting, however, is that the extent of the delay between the transport and the manipulation components was less for the patients with VPD than for patients with IPD, resembling the delay exhibited by the control participants. Nevertheless, this effect but might be the result of the same mechanism, namely the difficult coordination of movements with a motor output system – disrupted by pathological descending signals – which significantly limit the ability to assemble movement components. Tentatively, we suggest that lesions linked to VPD motor outcomes may affect the responsiveness of cortical areas to activation – defined as the readiness to the elaboration of triggers not originating from the basal ganglia – and result in an inadequate cortical preparation of the movement. If this lack of cortical responsiveness was confined to a specific neural channel (e.g., reach or grasping), this would explain why a movement shows a delay of activation. The different pattern of results might indicate that the more focal pathophysiology resulting in VPD less affects this cortical readiness phenomenon. The ultimate reason why this is so, still remains to be determined.

We are fully aware that the present study has some limitations. Indeed, VPD encompasses a heterogeneous set of conditions and the extent of the spectrum of VPD remains quite imprecise. However, given the promising results in finding markers differentiating VPD and IPD kinematical profiles, further work should address a full characterization of the unfolding of the reach-to-grasp movement in this population.

In conclusion, the present study provides the first attempt to compare the kinematic patterning of reach-to-grasp movements in VPD with respect to the better characterized IPD, in the effort of unveiling possible upper-limb dysfunctions in this population. The results indicate that the basic pattern of performance is similar across the two groups of patients. They both show bradykinesia, hypometria, and loss of coordination between the reach and the grasp components. However, the dysfunction in the concurrent execution of the coordinated motor plans, patients with VPD appear to be much less compromised than patients with IPD. With a certain degree of caution, we contend that this kinematical landmark might be a useful tool for distinguishing across different parkinsonian syndromes.

## **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 27 March 2014; paper pending published: 10 April 2014; accepted: 02 May 2014; published online: 16 May 2014.*

*Citation: Parma V, Zanatto D, Straulino E, Scaravilli T and Castiello U (2014) Kinematics of the reach-to-grasp movement in vascular parkinsonism: a comparison with idiopathic Parkinson's disease patients. Front. Neurol. 5:75. doi: 10.3389/fneur.2014.00075*

*This article was submitted to Movement Disorders, a section of the journal Frontiers in Neurology.*

*Copyright © 2014 Parma, Zanatto, Straulino, Scaravilli and Castiello. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## The Irvine, Beatties, and Bresnahan (IBB) forelimb recovery scale: an assessment of reliability and validity

#### **Karen-Amanda Irvine† , Adam R. Ferguson, Kathleen D. Mitchell, Stephanie B. Beattie, Amity Lin, Ellen D. Stuck, J. Russell Huie, Jessica L. Nielson, Jason F. Talbott,Tomoo Inoue† , Michael S. Beattie and Jacqueline C. Bresnahan\***

Brain and Spinal Cord Injury Center, Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, USA

#### **Edited by:**

Renée Morris, The University of New South Wales, Australia

#### **Reviewed by:**

David Magnuson, University of Louisville, USA Ian Q. Whishaw, University of Lethbridge, Canada

#### **\*Correspondence:**

Jacqueline C. Bresnahan, Brain and Spinal Injury Center, Department of Neurological Surgery, University of California San Francisco, 1001 Potrero Avenue, Building 1, Room 101, San Francisco, CA 94110, USA e-mail: jacqueline.bresnahan@ ucsf.edu

#### **†Present address:**

Karen-Amanda Irvine, Department of Neurology, San Francisco VA Medical Center, University of California San Francisco, San Francisco, CA, USA; Tomoo Inoue, Department of Neurological Surgery, Tohoku University, Sendai, Japan

#### **INTRODUCTION**

Motor function loss is a major consequence of spinal cord injury (SCI) and has been the focus of experimental studies for over a century. Most studies have used thoracic injury models and assessed locomotor function as the primary outcome measure. A number of cervical injury models have been developed (3–9), and are being used more frequently due to the understanding that the majority of SCI occurs at this level in the human population (10). Individuals with cervical injuries are reported to be most interested in the reinstatement of hand function (11), and hence outcome measures focused on recovery of forelimb use are becoming more commonplace.

In our attempts to model cervical SCI, we chose to use unilateral injuries to reduce the burden of neurological deficits, including bladder dysfunction and quadriplegia. Prior work (4) had shown the feasibility of this approach. We used the well-established MAS-CIS injury device for the early studies (6), but are now using the IH device (2, 12) due to its currently widespread use in the SCI research community. We selected outcome measures that evaluated spontaneously expressed behaviors, thus reducing training requirements and food deprivation since weight loss is a consistent consequence of SCI. In our initial studies (6), we measured paw placement during vertical exploration as originally described

The IBB scale is a recently developed forelimb scale for the assessment of fine control of the forelimb and digits after cervical spinal cord injury [SCI; (1)]. The present paper describes the assessment of inter-rater reliability and face, concurrent and construct validity of this scale following SCI. It demonstrates that the IBB is a reliable and valid scale that is sensitive to severity of SCI and to recovery over time. In addition, the IBB correlates with other outcome measures and is highly predictive of biological measures of tissue pathology. Multivariate analysis using principal component analysis (PCA) demonstrates that the IBB is highly predictive of the syndromic outcome after SCI (2), and is among the best predictors of bio-behavioral function, based on strong construct validity. Altogether, the data suggest that the IBB, especially in concert with other measures, is a reliable and valid tool for assessing neurological deficits in fine motor control of the distal forelimb, and represents a powerful addition to multivariate outcome batteries aimed at documenting recovery of function after cervical SCI in rats.

**Keywords: spinal cord injury, recovery of function, forelimb functional task, reliability, validity**

by Schallert et al. (13) for assessing forebrain injuries, grooming as originally described by Bertelli and Mira (14) for assessing brachial plexus injuries, over-ground locomotion in an open field and on the Catwalk apparatus (Noldus Information Technology, Sterling, VA, USA), and locomotion on a horizontal ladder (4, 15, 16). Performance on most of these measures reflected graded injury effects, and using principle components analysis (PCA), these behavioral outcomes were seen to co-vary with biomechanical and anatomical descriptors of the lesion (2). However, what was missing in this battery of tests was an assessment of distal forelimb and digit function.

Food retrieval and manipulation for consumption is a critical behavior that is spontaneously expressed in all individuals across mammalian species, and requires involvement of both proximal and distal forelimb. A novel task involving food manipulation was described by Allred et al. (17) and was based on the observations of Whishaw and Coles (18). In this task, pasta is presented to rats for eating and forelimb use is assessed during consumption. This test was sensitive to a number of forebrain injuries. In our initial attempts to use this test with spinal cord injured animals, we discovered that our rats were not particularly interested in eating pasta but would readily consume sugared cereal, which is available in a variety of shapes of consistent size. The manipulation of these cereal pieces was observed to involve detailed movements of the forelimbs and digits as the rats rotated the cereal pieces and somewhat systematically bit off small chunks to eat. Therefore, we attempted to evaluate the movements that were used to manipulate these food items while recovering from unilateral cervical contusion injuries. The first attempt to establish a recovery scale was presented in a video and manuscript (1) describing the methods, and termed the "IBB." The scale was generated by characterizing the movements made during cereal eating over the post-SCI recovery period, and assigning an ascending series of numbers for each functional set, and adjusting the scale until it reflected a sequential representation of the recovery (1). This procedure was based on our prior experience in developing and testing the Basso–Beattie– Bresnahan (BBB) locomotor rating scale (19). In that effort, we used an iterative process to construct an ordinal scale that withstood the test of inter-rater reliability (IRR) and construct validity (20, 21). The usefulness and metric properties of motor outcome scales are not always tested or considered in the SCI literature. But in response to suggestions made as more and more laboratories adopted the BBB and more data became available, this scale was modified in light of a growing body of data that suggested the metric properties were not optimized (22). A similar approach has been taken in the construction of scales for walking in human SCI patients (23). Similarly, in the present paper, we describe modifications to the original IBB scale based on our iterative evaluation of its usefulness and attempt to establish its validity and reliability. In addition, using the syndromics approach described recently for cervical SCI (2), we are now able to evaluate the relationship of this new outcome scale to other forelimb functional tests currently in use in our laboratory and in the field.

We first provide a brief history of the scale and metric properties analysis that guided its initial development. We then present results of IRR testing across a group of 9–10 novice and expert raters, and propose some minor revisions that improve reliability. Finally, we address the issue of validity (face, concurrent, predictive, external, and construct validity) for the IBB scale.

The results demonstrate that the IBB is a reliable and valid scale that is sensitive to injury severity and recovery over time. In addition, the IBB correlates with other outcome measures and is highly predictive of biological measures of tissue pathology. Multivariate analysis using PCA demonstrates that the IBB is highly predictive of the syndromic outcome after SCI, and is among the best predictors of bio-behavioral function, that is, there is good evidence of construct validity. Altogether, the data suggest that the IBB, especially in concert with other measures, is a reliable and valid tool for assessing neurological deficits in fine motor control of the distal forelimb, and represents a powerful addition to multivariate outcome batteries aimed at documenting recovery of function after cervical SCI in rats. Further, the similarities of "hand function" across rodents and primates may make such measures as this especially important in translating therapeutic strategies from rodent studies to clinical studies in man.

## **MATERIALS AND METHODS**

#### **ANIMALS**

Long Evans and Sprague Dawley rats aged 77–87 days at the time of injury were used in the initial scale development and validity testing (*N* = 70). All experiments adhered to the National Institutes of Health Guide for the Care and Use of Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California San Francisco (UCSF). For many of the subjects, the primary data on non-IBB outcomes have been presented elsewhere as part of recently published papers (2, 24). These data are re-plotted here (with permission) for the purposes of comparative (concurrent) validity testing of the IBB.

#### **SURGICAL PROCEDURES FOR CERVICAL SCI**

All surgical procedures were performed aseptically as described previously (6). Briefly, animals were anesthetized with Ketamine HCL (80 mg/kg, Abbott Laboratories, North Chicago, IL, USA) and Xylazine (20 mg/kg, TraquidVed, Vedco Inc., St Joseph, MO, USA) intraperitoneally (ip) or with isoflurane before surgery. A dorsal, midline skin incision was made, the skin dissected, and the trapezius muscle was cut just lateral to the midline from C2 to T2. Spinous processes from C4 to T1 were exposed and a C5 dorsal laminectomy was performed to expose the entire right side and most of the left side of the underlying spinal cord. Contusion injuries were produced using the Infinite Horizon Impactor (Precision Systems and Instrumentation LLC, Fairfax, VA, USA) with a modified impactor tip 2 mm in diameter, with a force of 75 (mild) or 100 (moderate) kdynes. Cord hemisections were performed in a separate group of animals at the same vertebral level by inserting the tip of a #11 blade at the midline and sweeping laterally to cut all fibers of the hemi-cord. The sham group of animals underwent the laminectomy without SCI. The wound was closed in anatomical layers. The analgesic, buprenorphine (0.05 mg/kg, Buprenex, Hospira, IL, USA), and the antibiotic, Cefazolin (50 mg/kg, Henry Schein, Melville, NY, USA) were administered, and the animal recovered overnight in an incubator (Thermocare®, Intensive Care Unit with Dome Cover; Thermocare, Incline Village, NV, USA). All animals were inspected daily for wound healing, weight loss, dehydration, autophagia, and discomfort. Appropriate veterinary care was provided when needed.

#### **SURGICAL PROCEDURES FOR TRAUMATIC BRAIN INJURY**

A controlled cortical contusion injury (CCI) was produced using a device that has been described in detail elsewhere (25). Briefly, rats were mounted in a Kopf stereotaxic frame under isoflurane anesthesia. A unilateral craniectomy (6.0 mm diameter) between 3.0 mm posterior and 3.0 mm anterior to bregma, and between 1.0 and 7.0 mm lateral to bregma was produced using a high-speed drill. CCI was produced using a 5.0 mm diameter impactor with a convex tip (Custom Design & Fabrication, Inc., Sandston, VA, USA), oriented perpendicular to the cortical surface. The cortex was compressed to a depth of 2.0 mm at 4.0 m/s velocity with a dwell time of 150 ms. Sham animals received the craniectomy only. During the surgical procedure, heart rate and blood oxygenation were monitored with a Mouse Ox™ pulse-oximeter (Torrington, CT, USA); temperature was monitored and maintained at 37.5°C. The injury sites were closed and the animals were recovered in an incubator (Thermocare®, Intensive Care Unit with Dome Cover; Thermocare, Incline Village, NV, USA).

#### **COMBINED SCI** + **TBI**

In animals with both traumatic brain injury (TBI) and SCI, both surgical sites were prepared and then the TBI was performed followed by the SCI. All other aspects of the procedure were as described above and previously (24).

## **BEHAVIORAL TESTING**

All behavioral testing for the IRR and validity testing was performed by raters who were blind to the experimental condition. Testing was typically performed pre-operatively and on post-operative days 2, 7, 14, 21, 28, 35, and 42 after injury.

## **Forelimb testing using the Irvine, Beattie, and Bresnahan (IBB) Scale**

Rats were given pieces of cereal in their home cage twice daily beginning as soon as they entered the lab. Forelimb function was assessed while rats were eating cereal as described previously (1). Briefly, rats were individually placed in a Plexiglas cylinder (diameter = 20 cm; height = 46 cm) or in their home cage and given spherical- and donut-shaped pieces of cereal ("Reeses Puffs™," The Hershey Co., and"Froot Loops™,"Kellogg's Co.) that were of a consistent size and shape prior to the initiation of eating. Rats were not scored when eating cereal pieces that were broken prior to the initiation of testing. Each trial was recorded to allow slow motion HD playback and evaluation of forelimb use. Videos of animals eating the cereal were evaluated using a standardized scoring sheet (**Figure 1**) to record observations of forelimb behaviors, including joint position, object support, wrist and digit movement, and grasping method used while consuming both cereal shapes. An IBB score was assigned using the 10-point (0–9) ordinal scale for each shape, and the highest score reflecting the greatest amount of forelimb recovery, was assigned.

#### **Grooming test**

Forelimb grooming function was assessed using a scoring system described previously (6). Cool tap water was applied to the animal's head and back with soft gauze, and the animal was placed in a clear plastic cylinder (diameter = 20 cm; height = 46 cm) or in their home cage. Grooming activity was recorded with a video camera from the onset of grooming through at least two stereotypical grooming sequences (~2 min). A score was assigned depending on the highest region touched by the hand as follows: 0, no contact with the head; 1, contact with the mouth only; 2 contact with the snout below the eyes; 3, contact with the face from the eye level to below the ears; 4, contact with the ears; 5, contact with the head behind the ears. Slow motion video playback was used to score each forelimb independently by the maximal contact made while initiating any part of the grooming sequence. The animals were tested on day 2 post-operatively, and then at least weekly until sacrifice.

## **Forelimb use during vertical exploration: forelimb asymmetry or cylinder test**

Animals were placed in a clear plastic cylinder and spontaneous exploratory behavior was recorded for 5 min. Slow motion video playback was used to determine the number of times the animal placed its left, right, or both hands against the side of the

cylinder during weight-supported movements according to previously published criteria (26). Individual placements were scored as either "left" or "right" when 0.5 s or more passed without the other limb contacting the side of the cylinder. If both hands were used for weight-supported movements within 0.5 s of each other, a score of "both" was given. Results are reported as a percentage of contralateral limb use versus total placements and reported as the "paw preference" outcome.

#### **Over-ground locomotion**

Forelimb use during over-ground locomotion was assessed in an open field. Limb use for stepping was assessed using a simple four-point scale: 0, no use of the forelimb; 1, stepping on the dorsal surface of the paw; 2, stepping on both the dorsal and plantar surface of the paw; 3, stepping on the plantar surface only.

## **CatWalk**

The walkway and CatWalk analysis program was used to measure forelimb function during gait as described previously (27). Briefly, animals were trained to cross a glass walkway (120 cm long) with black Plexiglass walls and ceiling. Light transmitted through the walkway floor revealed foot contacts which were captured and collected by a digital video camera placed underneath the runway (for details, see **Figure 9**). A digital file for each run across the middle 90 cm of the walkway was analyzed using the CatWalk program (version 7). Measurements for locomotion included stride length, print area during maximal contact, and the distribution of total

steps among the four limbs. During training, animals were gently guided to make complete passes across the walkway and were reinforced with sugared cereal or access to the home cage. Data were gathered pre-operatively (baseline), and then at 2–3 weekintervals post-operatively. Data were averaged across five runs in which the animal maintained a constant speed across the middle 90 cm of the CatWalk runway.

#### **Inter-rater reliability testing protocol**

Inter-rater reliability was assessed by measuring means and standard deviations of ratings of the same 10 rat videos chosen to represent all parts of the IBB scale, across multiple raters similar to that described for the BBB (21). In the first IRR, nine participants were given an initial IBB training session in which videos of the pattern of recovery in rats with cervical unilateral SCI were shown and the method of scoring using the IBB was explained. The rating of individual rats was then practiced with concurrent discussions, followed by individuals silently rating, and then comparing and discussing scores with those of the trainers. Then each participant was given a CD with ten videos of rats performing at all levels of recovery; each CD presented the videos in a different, randomized order. Also provided to each rater were a set of data recording sheets (**Figure 1**), a copy of the originally published IBB manuscript and video instructions (1), a set of frequently asked questions with answers, and a score determination guide for ease of assigning scores (**Figure 2** shows the revised version). All participants then independently

**FIGURE 2 |The score determination guide**. This guide can be used to aid in the selection of the correct IBB score after viewing the video and filling out the IBB score sheet.

evaluated the 10 videos and assigned IBB scores based on the descriptions provided in Ref. (1). Data sheets were then collected, analyzed, and compared to a consensus score for each rat, arrived at by the original scale developers viewing, discussing, and arriving at a consensus score for each video. This consensus score was determined after all raters (including the experienced raters) had completed and submitted their independent ratings of the videos. The initial IRR test results then were discussed with the participants and problems in recognizing behavioral elements and in assigning scores were identified. Choices, definitions, and the score sheet were then revised to overcome the identified issues for the purpose of improving clarity and consistency in score assignment. Subsequently, a second IRR test was performed approximately 3 months later, with 10 raters most of whom participated in the first IRR test described above, and using the newly revised definitions and the modified score sheet. Consensus scores were determined as in test 1 and individual scores were again assessed for variation from the consensus score as in the first IRR test.

#### **HISTOLOGICAL PREPARATION AND MORPHOLOGICAL ANALYSIS**

Animals were perfused through the left ventricle of the heart with 4% paraformaldehyde under deep anesthesia with pentobarbital or ketamine–xylazine. The cords were removed and post-fixed in 4% paraformaldehyde for 2 h and then cryoprotected in PBS containing 30% sucrose. A 2 mm block containing the lesion epicenter was then incubated in 100% OCT for 1 h and then mounted in a cryomold (filled with OCT) in coronal orientation and rapidly frozen using dry ice. The blocks were stored at −80°C until sectioning. The cords were cut coronally at 10µm and every section was retained and mounted. Sections were stained with Luxol fast blue or eriochrome cyanine for myelin/white matter integrity and counterstained with Cresyl violet or neutral red for cell body assessment.

#### **Sparing at lesion epicenter**

A camera lucida drawing of the section with the largest lesion extent (i.e., the lesion epicenter) was made outlining intact gray and white matter, and the lesion. Pixel counts from digitized drawings in Adobe Photoshop 5.5 (Adobe Systems Inc., San Jose, CA, USA) were used to determine the area of spared tissue for both hemi-cords at the lesion epicenter. The percent sparing for the ipsilateral hemi-cord was determined by dividing the total spared ipsilateral tissue area, spared white matter tissue area, or spared gray matter tissue area, by the same measure from the contralateral hemi-cord [(ipsilateral spared tissue area/contralateral spared tissue area) × 100]. Quantifying pathology in this manner normalized tissue sparing within subjects and corrected for any biological differences in spinal cord size or tissue preparation. Motor neuron counts through the lesion region were performed as in Ferguson et al. (28).

#### **STATISTICAL ANALYSIS**

All analyses were performed using SPSS v.19 (IBM) using base, regression, advanced models, and missing values packages. All graphs were generated in Graphpad Prism.

#### **Inter-rater reliability assessment**

Comparisons across raters were analyzed by assessing individual rater deviations from the "gold standard" or experienced raterderived consensus scores on the same set of behavioral videos, using the formulas

$$Difference = \sum\_{i,j} |X\_j - \mu\_j| \tag{1}$$

and the mean difference score (MDS) is represented by

$$\text{MDDS} = \frac{\text{Difference}}{n\_{i,j}} \tag{2}$$

where *i* = individual rater, *j* = individual rat, *Xij* = observed score on rat *j* by rater *i*,µ*<sup>j</sup>* = consensus score on rat *j*, *nij* = total number of observations by all raters for all rats.

Separate *MDS* values were calculated for expert and novice raters. In addition, MDS values for the novice and expert raters were regressed onto the consensus scores to assess the degree of linear correlation of assessments across raters.

#### **Validity assessment**

Internal and face validity were examined by testing whether the IBB responded to the impact of graded injury and recovery over time using two-way mixed analysis of variance (ANOVA). In addition, we assessed sensitivity/propriety of applying parametric statistics (e.g., ANOVA) to the IBB by assessing variance-explained (eta squared). Concurrent validity was assessed by correlating the IBB with other more established behavioral measures used by the SCI research community. Predictive validity was assessed by correlating IBB scores with terminal histology. Construct validity was assessed at a multivariate level using exploratory factor analysis using the principal component analysis (PCA) extraction method (2, 29, 30).

#### **RESULTS**

#### **INITIAL SCALING**

Based on general observations of rats with SCI while consuming cereal, we first divided the behaviors into different categories (posture, proximal forelimb joint movement, contact with the food object, digital clubbing, wrist movements, digital movements, and grasping method). These categories were further subdivided into ranks (e.g., no, yes but abnormal, yes but normal) and operational definitions were developed to describe the categories and attributes. Categories were loosely arranged to reflect the sequence of recovery, and scores were assigned (0, 1, 2) to reflect the rank-ordered attributes. Initial scaling involved summation of these ranked features and then the resulting 55-point scale was subjected to evaluation of the metric properties such as score frequency distribution, ordinality, discontinuities, and interval properties (22). This analysis revealed that certain features did not progress in an ordered sequence and further reanalysis revealed problems with reliability and sensitivity that increased measurement error and reduced ordinality. Through this process, we improved the operational definitions of observed behaviors and switched from a summation-based scale to an ordinal scale with fixed definitions of each point. Ultimately, scores were winnowed down to a 10-point (0–9) scale that was published in video format (1). In the present paper, further modifications to the operational definitions are reported to correct for inconsistencies and interpretational difficulties identified during the formal IRR testing analysis as presented below.

#### **DATA RECORD SHEET**

An initial scoring sheet was developed to use with the IBB for ease of recording observations while viewing subjects eating cereal, and was provided in the original IBB manuscript and video (1). The data sheet was organized from left to right to reflect the course of recovery after SCI, with the earliest behaviors to recover being positioned on the left and the later behaviors on the right. The individual subcategories were organized from top to bottom to reflect less to more recovery. This data sheet was revised to reflect changes resulting from the current analysis as described below; the revised data sheet is now shown in **Figure 1**.

#### **INTER-RATER RELIABILITY**

#### **Inter-rater reliability test 1**

The results of the first IRR test (nine raters; three experienced, six novice) are shown in **Figure 3** and present the MDS (i.e.,

**FIGURE 3 | Results of inter-rater reliability testing using a standardized set of rat behavioral videos before and after revision of the IBB operational definitions and score sheet**. **(A)** Three experienced raters and six novice raters participated in the first round of inter-rater reliability testing. Mean difference scores (MDS) from a "gold-standard" consensus score were calculated as described in the methods. Following score-sheet revisions, a second round of inter-rater reliability testing was performed by three experienced and seven novice raters. Note that the MDS values as well as their standard errors (SE) were reduced after the revisions, indicating an increase in inter-rater reliability. **(B)** Pearson correlations between the mean IBB score and the consensus score suggest a high degree of agreement with consensus in both novice and experienced raters, providing strong evidence that the IBB has high inter-rater reliability that improves with practice.

the absolute value of the difference between the assigned score and the consensus or "gold standard" score) for ratings of performance shown in the 10 videos. Experienced raters scored within <1 point of the consensus score (0.8 ± 0.36) while novice raters scored within an average of 1.5 ± 0.5 points of the consensus score. This suggests that experienced raters independently assigning scores for the 10 videos are more accurate than novice raters, but novice raters could clearly get in the range of experienced raters with only a one-day training session. Correlational analysis of the separate expert inter-rater scores revealed significant reliability (all *r* values >0.9, *p* < 0.0001)**.**

On review of the results by the group, a number of issues were identified that caused problems for the raters. These were:

The revised IBB scale and definitions are shown in **Table 1**; the changes from that provided in Irvine et al. (1), are indicated by *italics* and underlining.

**FIGURE 4 | Amendment: predominant elbow position**. The rat is assessed for the most common position (more than 50% of the time) assumed by the elbow during eating. Extended is when the elbow is held straight with an

angle of more than 160°. Flexed – The elbow is flexed with an angle of less than 160°. (Revisions of the IBB scale from the JoVE 2010 version are highlighted in italics.)

**FIGURE 5 | Amendment: proximal forelimb movements**. The rat is assessed for movements made by the shoulder and/or elbow of the impaired forelimb that may or may not result in contact of the forelimb with the cereal. These proximal forelimb movements are defined as either: none – there are no shoulder and/or elbow movements of the impaired forelimb. Slight **(A,B)** is defined as infrequent movements (<5% of the time) through less than third the range of the shoulder and/or elbow joint; twitches and shrugs fall into this category. Extensive is defined as frequent movements (>5% of the time) by the impaired forelimb OR movements **(C,D)** that are more than third the range of the shoulder and/or elbow joint. In early recovery, these movements can be numerous and erratic. (Revisions of the IBB scale from the JoVE 2010 version are highlighted in italics.)

#### **Inter-rater reliability test 2**

After the changes were made, a second IRR test (three experienced, seven novice raters) was performed to determine if the changes increased clarity and thus accuracy. As shown in **Figure 3**, following the revisions, experienced raters had a mean difference from consensus score of 0.16 ± 0.15 points and novice raters had

a MDS of 1.23 ± 0.05. Experienced observers continued to show more accurate ratings, but all raters increased accuracy. The revisions not only increased accuracy, but also reduced variability in score assignment and improved IRR as reflected by a reduction in the overall variability in score assignments. Improved accuracy is revealed by the reduction in deviation from the consensus score. In addition, Pearson correlations between each rater and the gold standard were consistently high (**Figure 3B**).

#### **VALIDITY**

#### **Internal and face validity**

To assess internal and face validity of the IBB, we tested its sensitivity to a well-established experimental manipulation: graded SCI. We assessed sensitivity using a mixed repeated measures ANOVA (*F*-test) as well as effect size calculations (eta squared,η 2 ). To assess the IBB's sensitivity to recovery we performed repeated IBB testing over the post-injury interval. As shown in **Figure 8A**, the IBB was highly sensitive to the main effect of injury [sham, 75, 100 kdynes, or hemisection; *F*(3,24) = 120.89, *p* < 0.00001]. Effect size calculations indicated a very large effect of injury on IBB (η <sup>2</sup> = 0.94), over six times higher than the classical definition of "large" effect size (0.14) (31). This indicates that the IBB was highly sensitive to the effect of SCI. The IBB also performed very well as a measure of recovery over time, *F*(3,72) = 27.52, *p* < 0.00001, η <sup>2</sup> = 0.53. In addition, the IBB was highly sensitive to the injury × time interaction,*F*(9, 72) = 7.20, *p* < 0.00001,η <sup>2</sup> = 0.47. The interaction term, in particular, indicates that the IBB is highly sensitive to the variable patterns of recovery produced by different SCI gradations. In addition, as shown in **Figure 8A** (inset), the IBB correlated very highly with the observed ("actual") injury force biomechanical read-out from the IH device force transducer (*r* = −0.96; *r* <sup>2</sup> = 0.93), providing strong evidence of face validity. Altogether these findings indicate that the IBB is an internally valid measure for assessment of recovery after SCI.

#### **Concurrent validity: relationship to other functional tests**

To assess concurrent validity, we compared the IBB to other established tests of outcome after SCI performed within the same

**FIGURE 6 | Amendment: predominant forepaw position**. The rat is assessed for the most common position (more than 50% of the time) assumed by the digits. Scored as either **(A)** clubbed flexed fixed – the digits are flexed and held in a fist with joint angles of about 90°. **(B)** Extended, non-adaptable – One or more of the digits are partially extended with joint angles between 180° and 160°; in addition, these digits DO NOT CONFORM to the shape of the cereal. **(C)** Partially extended, adaptable – digits are partially extended with joint angles between 160° and 90°; in addition, these digits CONFORM to the shape of the cereal. Diagrams within the squares are observing the impaired forepaw, depicting digits 1 and 3 (\*), from above. (Revisions of the IBB scale from the JoVE 2010 version are highlighted in italics.)

subjects, i.e., the grooming task, paw placement in a cylinder, CatWalk, and forelimb use for over-ground locomotion in the open field (**Figures 8B-D**; **Figure 9**). The IBB demonstrated a similar overall pattern of recovery as other measures, however, with mild injuries (75 kdynes) it appeared to show less of an asymptotic performance ceiling in later recovery stages, suggesting that it may have greater sensitivity to continued recovery in high-functioning individuals. In addition, the IBB significantly correlated with paw preference asymmetry in the cylinder (**Figure 8B**, *r* = −0.87; *r* <sup>2</sup> = 0.75), forelimb grooming test (**Figure 8C**, *r* = 0.85; *r* <sup>2</sup> = 0.73), and forelimb open-field (**Figure 8D**, *r* = 0.66; *r* <sup>2</sup> = 0.43). Comparisons to the CatWalk yielded less robust correlations (**Figure 9**), with significance reached (*r* crit = 0.317) for the correlation with left (contralateral) forelimb print area (*r* = 0.32; *r* <sup>2</sup> = 0.10), right (ipsilateral) forelimb step distribution (*r* = 0.55; *r* <sup>2</sup> = 0.31), and right forelimb

stride length (*r* = 0.37; *r* <sup>2</sup> = 0.14). This reinforces prior work suggesting that only a subset of CatWalk measures are sensitive to the effects of unilateral cervical contusion injuries (2, 6). Altogether, the analytics reveal that the IBB has high concurrent validity.

#### **Predictive validity: relationship to terminal histology**

To assess the predictive validity of the IBB test, we assessed its ability to predict postmortem histology (**Figure 10**). The IBB scores were averaged over the 42-day recovery interval and the binned IBB scores were correlated with postmortem histopathological assessment of total tissue sparing, white matter sparing, and gray matter sparing and motor neuron counts. The results revealed significant correlations for each of these measures (*r* = 0.93, *r* <sup>2</sup> = 0.87; *r* = 0.89, *r* <sup>2</sup> = 0.79; *r* = 0.88, *r* <sup>2</sup> = 0.77; *r* = 0.68, *r* <sup>2</sup> = 0.46, respectively; **Figure 10**, insets). Together, these results suggest that the IBB is highly predictive

#### **Table 1 | Revised IBB Forelimb Recovery Scale**.

#### **REVISED IBB DEFINITIONS**

#### **Predominant elbow joint position:**

The rat is assessed for the most common position (more than 50% of the time).

EXTENDED: The elbow is held straight with an angle of >160°.

FLEXED: The elbow is flexed with an angle of <160°.

#### **Proximal forelimb movements:**

The rat is assessed for movements made by the shoulder and/or elbow of the impaired forelimb that may or may not result in contact of the forelimb with the cereal.

NONE: There are no shoulder and/or elbow movements of the impaired forelimb.

SLIGHT: Infrequent movements (<5% of the time) by the impaired forelimb through less than a third of the range of the shoulder and/or elbow. (Twitches and shrugs fall into this category.)

EXTENSIVE: Frequent movements (>5% of the time) by the impaired forelimb OR movements that are greater than one-third of the range of the shoulder and/or elbow. In early recovery, these movements can be numerous and erratic.

Note: If animals show both slight and extensive proximal forelimb movements during eating they are scored as having extensive movements.

(Continued)

#### **Table 1 | Continued**

#### **Contact non-volar support:**

The rat is assessed for its ability to use the non-volar surface of the impaired forelimb to stabilize the cereal piece and in doing so, maintaining it in a position to aid eating. (Areas of the forelimb that may act as supports are the forearm above the wrist, the wrist or the back of digits.)

NONE: No non-volar support by the forelimb during eating (<5% of the time).

SOME: Non-volar support of the object does occur during eating but not always.

ALMOST ALWAYS: Non-volar support of the object occurs nearly always or always during eating (>95% of the time).

#### **Predominant forepaw position:**

The rat is assessed for the most common position (more than 50% of the time) assumed by the digits, from flexed to extended, during eating.

CLUBBED, FLEXED, AND FIXED: Digits are flexed with joint angles greater than 90° and are held in a fist.

EXTENDED, NON-ADAPTABLE: One or more of the digits are partially extended with joint angles between 180° and 160°; in addition, these digits do not conform to the shape of the cereal.

PARTIALLY EXTENDED, ADAPTABLE: Digits are partially extended with joint angles between 160° and 90°; in addition, these digits conform to the shape of the cereal.

#### **Contact volar support:**

The rat is assessed for its ability to use the volar (palmar) surface of the impaired forepaw to stabilize the cereal and, in doing so, maintains a position to aid eating.

NONE: No volar support by the forelimb during eating (<5% of the time).

SOME: Volar support of the object does occur during eating but not always.

ALMOST ALWAYS: Volar support of the object occurs nearly always or always during eating (>95% of the time).

#### **Cereal adjustments (Control):**

The rat is assessed for movements made by the shoulder and/or elbow and or/wrist of the impaired forelimb that are synchronized (in time) with successful manipulatory movements of the unimpaired forelimb, and that contribute to the proper adjustment (control) of the cereal position by the volar surface of both forepaws.

NONE: There are NO manipulatory movements made by the volar surface of the impaired forepaw.

EXAGGERATED: Hypermetric movements of the shoulder and/or elbow and/or wrist of the impaired forelimb that:

Cause a loss of contact between the volar surface of the impaired forepaw and the cereal, **and** DO NOT adjust (control) the cereal position or DO NOT contribute to the proper manipulation of the cereal by the volar surface of the forepaws.

SUBTLE: Tiny movements of the shoulder and/or elbow and/or wrist of the impaired forelimb that:

May or may not momentarily cause a loss of contact between the volar surface of the impaired forepaw and the cereal, **and**

DO adjust (control) the cereal position or DO contribute to the proper manipulation of the cereal by the volar surface of the forepaws.

Note: If animals show both exaggerated and subtle proximal forelimb movements during eating, they are scored as having exaggerated movements, as these disappear with further recovery.

#### **Wrist movements:**

The rat is assessed for the presence of wrist movements of the impaired forepaw during eating, once volar support has been established. Movements of the wrist that occur in the absence of contact between the impaired forepaw and the cereal are not scored. These movements can occur in any direction, e.g., a dorsal (towards the back) to ventral (down towards the stomach) direction or medial (in towards the body midline) to lateral (away from the body midline) direction:

YES

NO

#### **Presence of digit movements:**

The rat is assessed for the presence of movements made by the individual digits during eating.

NON-CONTACT, YES or NO: Movements of the digits occur but these movements do not result in volar contact with the cereal.

CONTACT MANIPULATORY, YES or NO: Movements of the digits occur that do result in volar contact of the digit with the object and, in doing so, contribute to manipulation of the cereal.

(Continued)

#### **Table 1 | Continued**

#### **Grasping method:**

The rat is assessed for the most common (more than 50% of the time) grasping technique used during the eating phase. Several grasping methods exist but the most common are the "pincer," the "hook," and the "whole" grasp. The grasping techniques used by the rat are stereotypical depending on the size and shape of the cereal piece.

ABNORMAL: Consistent use of an alternative method of grasping to the method used prior to injury to support and control the cereal piece during the eating phase.

SOMETIMES NORMAL: Inconsistent use of the grasping method used prior to injury to support and control the cereal piece during the eating phase.

ALMOST ALWAYS NORMAL: Consistent use of the grasping method used prior to injury to support and control the cereal piece during the eating phase.

The changes from that provided in Ref. (1), are indicated by italics and underlining.

of histological changes after SCI, providing strong support for its use as a behavioral biomarker for SCI outcome assessment.

Correlations of individual variables with the IBB score were done using all animals including the shams. The reason for this was that we wanted the entire range of behavior and anatomy to be represented (i.e., from most injured with no function to no injury and normal function). An alternative approach is to ask if the scale is sensitive within the range of injury and partial function, i.e., without the shams. **Table 2** presents the correlations figured both ways. Pearson correlations (*r*) and shared variance (*r* 2 ) deflated without shams, indicating a smaller but often still significant dynamic range within different injury conditions. This suggests that the IBB has sensitivity across a wide dynamic range of injury conditions. Note that *r* crit = 0.31 for *p* < 0.05.

#### **External validity: responsiveness to other types of neurological injuries**

To assess whether the IBB has external validity, we tested a new population of subjects and also assessed its sensitivity to alternative forms of neurological injury in the context of a modeldevelopment effort for central nervous system (CNS) polytrauma (SCI + TBI; (24)). IBB was assessed in subjects receiving either a unilateral cervical SCI alone (75 kdynes), TBI alone, or SCI + TBI combined injuries (with the TBI either ipsilateral or contralateral to the SCI). If the IBB has high external validity then it should show graded sensitivity in this new population of subjects. The results are shown in **Figure 11**, and demonstrate that IBB was highly sensitive to the impact of injury condition, *F*(4,37) = 15.74, *p* < 0.00001. The sensitivity of the IBB to CNS injury was reinforced with a very large effect size η <sup>2</sup> = 0.63, over four times higher than the classical cut off for "large" effect size [η <sup>2</sup> = 0.14; (31)]. Together, the results indicate that the IBB has high external validity for the combinatorial effect of SCI + TBI. Note, that the IBB was selectively sensitive to the impact of TBI contralateral to the SCI, but little impacted by TBI alone. This suggests that the IBB, like the grooming test, is somewhat selective for the effects of SCI, and perhaps, selectively sensitive to anatomical substrates through which contralateral cortical contusion impacts SCI recovery [see Ref. (24), and "Discussion" section for further review).

#### **Construct validity: multidimensional syndromic assessment**

Spinal cord injury is an intrinsically multifaceted syndrome that can be conceptualized within a multivariate, big-data analytic framework (2, 32–37). In this context, we can assess construct validity of SCI outcome batteries by borrowing well-established methods from the educational and neuropsychiatric testing fields. Namely, we can apply multivariate exploratory factor analysis on the full set of multi-trait multi-method outcomes to derive the underlying latent structure of the SCI syndromic space (2, 29, 38, 39). This approach is a realization of classical arguments about strong inference and the need to leverage full-information to deal with complexity in biology and neuroscience (40).

To assess the relationship of the IBB to multidimensional SCI, we performed exploratory factor analysis using the extraction method of PCA. PCA integrates the full bivariate cross-correlation matrix of all biological and functional outcomes through multivariate pattern detection coupled with dimension-reduction ((2, 29); **Figure 12**). In essence, PCA reduces the total number of observed variables down to a small number of principal components (PCs; or "latent variables") that concisely summarize the overall set of observations within the dataset. We performed PCA on the full set of outcome variables presented (in univariate form) in **Figures 8**–**11**. PCA revealed three latent multivariables (PC 1–3) that together accounted for 81.4% of the variance in outcome (**Figures 12A–C**). To understand how individual outcome metrics relate to the PC syndromic patterns, we plotted the correlation (so called "loadings") of each outcome metric on the PC patterns. Significant loadings above 0.45 are represented as arrows where arrow size indicates magnitude and heat represents valence (positive vs. inverse relationships). Note that IBB loads very highly on PC1, indicating that it is a highly de-noised measure of the latent construct represented by PC1. As in prior work (2), the PC1 loading pattern suggests that it represents the relationship between tissue sparing and recovery of function – the multidimensional target for neuroprotective therapies. The fact that the IBB is the highest loading variable on PC1, suggests that it is a powerful surrogate biomarker for the set of variables represented by PC1. In addition, note that IBB does not load on PC2 or PC3, which are both devoid of histological loadings. This suggests that the IBB is a highly selective detector of the histopathology–behavior

**FIGURE 8 | Face, internal, and concurrent validity of the IBB score**. **(A)** Face and internal validity of the IBB score is provided by responsiveness to experimentally graded spinal cord injuries as well as the correlation (inset) with a biomechanical measurement of tissue displacement at the time of contusion injury. Concurrent validity is provided by comparisons with other

established outcomes including **(B)** paw placement, **(C)** grooming score, and **(D)** forelimb open field. Insets reflect the scatterplot and regression line between the IBB and each of the established tests. The Pearson correlation (r) and the shared variance (r 2 ) for each appear above the scatterplot; group identity for each point is color coded.

relationship. Combined with the univariate validity testing, the multivariate results provide strong validation of the IBB as a measure of recovery of function following cervical SCI.

#### **DISCUSSION**

#### **DEVELOPMENT OF THE IBB**

A major goal of preclinical modeling for SCI is to identify methods that can be used to evaluate treatments for translation to clinical trials. Our prior work on cervical SCI (6) used a variety of tasks to measure forelimb function including the grooming task, paw placement in a cylinder, CatWalk, and forelimb open-field locomotion. It is noteworthy that these tasks largely assessed proximal forelimb movements with some limited information about hand use. None of these tests focused on digit function, which we consider to be important to assess for the translational relevance of our preclinical outcome testing. A number of tasks that assess distal forelimb movements in rodents have been described especially by Whishaw and colleagues, and many have focused on the "reachto-grasp task" [reviewed in Ref. (41)]. This task however, requires extensive training and food deprivation. We also considered an alternative task, pasta eating, that required hand use to accommodate a variety of food shapes (17, 18) and was sensitive to forebrain injuries. However, during the process of trying to acclimate rats to a variety of food items, we noticed that acutely injured subjects demonstrated movements of the affected limb during eating that did not contribute to food manipulation. The hand was fixed in a fisted position preventing the digits from grasping the food, and the forelimb was only used to support the food item. In contrast, the contralateral limb showed fine digital movements. Allred and colleagues (17) had made similar observations in their description of the "Vermicelli handling task," in which rats are filmed eating pieces of thin pasta and manipulation of the pasta was compared to pre-injury handling methods. However, the juxtaposition of the digits during pasta eating made it difficult to discern movement of individual digits, and only movements with physical contact with the pasta were described and assessed. We considered that this strategy would ignore the rats' attempts to use the forepaw ipsilateral to the SCI, and its continued improvement over time.

We therefore explored developing a formal observational scale to rate recovery of both proximal and distal forelimb movements in the affected limb during food manipulation, including fine digital control. Using a high-definition camera, we filmed subjects eating consistently sized cereal pieces in a Plexiglas cylinder surrounded by mirrors to enable 360° viewing of the movements.

**analysis on the CatWalk**. **(A)** Left forelimb step distribution. **(B)** Left forelimb stride length. **(C)** Left forelimb print area. **(D)** Right forelimb step distribution. **(E)** Right forelimb stride length. **(F)** Right forelimb print area. Insets reflect the

outcomes. The Pearson correlation (r) and the shared variance (r ) appear above each scatterplot; group identity for each point is color coded. \* Indicates significant correlation above r crit = 0.317.

Both uninjured subjects and subjects with a range of unilateral cervical injuries produced by the IH device were examined over 6 weeks. Initial observations were unconstrained notes based loosely on the structured note-taking scheme of the BBB locomotor rating scale (19). Like the BBB, attention was first given to gross position of the joints in the affected limb and then to more refined features of movement. We also noted differences in the grasping techniques across different cereal shapes, largely inspired by work of Whishaw and colleagues. The result of this analysis, termed the "IBB," was described in Irvine et al. (1).

In the current paper, we have assessed this method for both reliability and validity. These are distinct but related issues in the field of testing theory. IRR deals with the issue of consistent scoring of observations whereas validity deals with the issue of whether a measurement assesses what it purports to assess. These issues will be discussed separately below.

## **INTER-RATER RELIABILITY**

Inter-rater reliability deals with whether an assessment tool is consistent from rater to rater. To assess IRR, we used an approach similar to that used during the development of the BBB Locomotor Rating Scale (21). This approach relied on assessing deviations from a gold-standard consensus score that is derived by expert raters working together as a team. The current study used a consistent set of videos to assess IRR. This provided some advantages over the live-rating strategies used to assess the BBB scale. First, it ensured that there was only one view of the behavior, providing a more direct assessment of inter-rater variability. Second, we could randomize the presentation of the exact same behavior allowing us to control for sequence effects in raters. We found that there was a high concurrence of score assignment for both experienced and novice raters, and that concurrence was improved after some minor adjustments to the scale definitions and procedures.

**FIGURE 10 | Predictive validity of the IBB score with respect to histological outcome after spinal cord injury**. **(A)** IBB score. **(B)** Total tissue sparing at lesion epicenter. **(C)** White matter sparing at lesion epicenter. **(D)** Gray matter sparing at lesion epicenter. Insets reflect the

scatterplot and regression line between the IBB (averaged over time) and each of the established tests. The Pearson correlation (r) and the shared variance (r 2 ) appear above each scatterplot; group identity for each point is color coded.

#### **Table 2 | Correlations of individual variables with IBB score**.

Note that separate correlations were calculated for all injury conditions (all subjects) and excluding shams (no shams). Note that Pearson correlations (r) and shared variance (r<sup>2</sup> ) deflated without shams, indicating a smaller but often still significant dynamic range within different injury conditions. This suggests that the IBB has sensitivity across a wide dynamic range of injury conditions. Note: rcrit = 0.31 for p < 0.05.

We also found that experience improves consistency and accuracy of score assignment [as was observed with the BBB; Ref. (21)]. Novice raters could be trained to identify the behavioral features for rating within a single day, and were able to identify definitional issues that, when changed, improved accuracy for both novice and experienced raters. The full set of IRR assessment videos and materials are available to qualified neurobiological researchers upon request. Given that the videos are identical, researchers should be able to match their results to those presented in the current paper.

#### **INTERNAL/FACE VALIDITY**

The internal or face validity of this measure is reflected in its ability to detect differences in the degree of injury to the nervous system. Performance in cohorts of animals with 75 and 100 kdyne unilateral contusion SCI, lateral hemisection, and combined SCI with TBI showed that the IBB was sensitive to varying damage to the spinal cord and cortex, both individually and in combination. Graded SCI produced differential recovery (**Figure 8A**). Interestingly, TBI alone produced a mild initial deficit which quickly

recovered (by 1 week post-TBI; **Figure 11**, green line). Whishaw et al. (42) showed that cortical lesions did not affect the ability of rats to pick up food with their mouth and transfer it to their hands for manipulation, but did observe that cortical injuries produced difficulty with pronation and supination. This type of deficit could be reflected in the early mild suppression of the IBB score after the cortical injury alone. Interestingly, the addition of a cortical injury contralateral to an SCI, produced a significant depression of IBB scores over the SCI alone, suggesting that the contralateral cortex was involved in the recovery from the SCI. A TBI placed ipsilaterally to the SCI, did not show the same effect as the contralaterally placed TBI, and in fact slightly, but not significantly, improved outcome on this measure. The dual lesions' effect on the circuitry supporting paw use is complex and a multivariate approach to determining the output shows that this is indeed the case (35) but is beyond the scope of the present discussion.

#### **CONCURRENT VALIDITY**

Concurrent validity asks how performance on this test relates to performance on other tests used to assess recovery after unilateral SCI [e.g., Ref. (4, 6, 9)]. The current study found that IBB scores correlate very highly with paw placement and grooming scores, and less highly, but still significantly, with forelimb use for locomotion in the open field and on the Catwalk (although only on some of the Catwalk measures). These tests evaluate hand use during vertical exploration, during grooming of the face and head, and for locomotion respectively. Other tests which evaluate hand use during grasp and retrieval [e.g., Ref. (42–44)] were not tested. The IBB test focuses on a different aspect of forelimb use than the reach and grasp tasks. The IBB represents an assessment of hand use during food manipulation for consumption as opposed to reaching and grasping tasks, which involve forelimb use for retrieval of items distal to the animal (41, 45). During reaching tasks, animals are required to extend their arm through a slot to reach a food object. The hand is then brought over the food pellet using a stereotyped arpeggio movement and the pellet is grasped, followed by bringing the food to the mouth. For the IBB, animals first locate the food on the floor of the cage using at least olfaction and somatosensory input via the vibrissae, they pick the food up with their mouth and then bring the forelimbs to the mouth to support and manipulate the food, especially if the item is large. The food is then rotated and positioned for biting with both hands. The reach and grasp tasks do not focus on this proximal manipulation during consumption. In this sense the IBB is complementary to reach and grasp tasks.

Whishaw has pointed out that "reach and grasp" is a highly evolutionarily conserved function that is similar across the mammalian class, and thus is likely to be a useful tool for translational modeling (41). While the ability to use fine digital movements increases and individuates as one "ascends" the class from rodents to primates, the basic organization of the neural systems underling these behaviors are likely to be similar. Therefore, attempts to develop outcome measures with similar features across species that can be combined to develop batteries of tests evaluating different substrates for recovery, would seem to increase the probability of translation from rodent injury models to the human clinical situation. In this sense, the IBB represents an important addition to a complete battery of tests that can be used to assess recovery

of function after cervical SCI. By combining data from multiple tests, we will have a better, more holistic view of recovery after neurological injury.

#### **PREDICTIVE AND EXTERNAL VALIDITY**

To test the predictive validity of the IBB, we examined the relationship with the underlying tissue damage in the spinal cord. We found that the IBB scores were highly and significantly correlated with the amount of tissue sparing at the SCI lesion site. How the IBB predicts SCI severity in comparison to other tests is discussed in the multivariate section below. The IBB was minimally sensitive to the impact of TBI alone, but as mentioned above, showed a similar sensitivity to combined SCI + TBI as the paw placement test (24). In a recent report from Speck et al. (46), the IBB was also shown to be sensitive to recovery from peripheral nerve injuries in mice.

#### **CONSTRUCT VALIDITY: MULTIVARIATE ASSESSMENT OF FUNCTION**

Findings from multifaceted outcome batteries applied to the same subject ultimately need to be integrated in some manner to derive a complete picture of forelimb recovery. Multivariate statistical pattern detectors such as PCA and the related approach of exploratory factor analysis provide quantitative means to perform this integration across outcomes (29, 39). This approach has classically been applied in the human assessment literature as a tool to gauge construct validity: the degree to which an individual test measures or "taps into" an underlying trait of interest [e.g., intelligence, executive function, memory etc.; Ref. (39)]. Indeed, this application of multivariate statistics is the underlying basis for most modern, standardized human achievement and neuropsychological tests. However, PCA has rarely been applied in preclinical research studies to assess the validity of scales used in animal models of neurobiological disorders. In the present paper we applied PCA to, (1) integrate outcome across multiple assessment tools, and (2) to assess the construct validity of the IBB. Based on prior work, we knew that PCA has the capacity to detect specific neurobiological substrates for forelimb recovery after SCI, specifically tapping into the relationship between tissue sparing and multifaceted forelimb function on the first principal component (PC1) (2, 32, 33, 37). The question in the current paper was, "does the IBB predict (or "load onto") the established forelimb neurobehavioral recovery construct outcome set?" The results indicated that not only did the IBB predict the forelimb neurobehavioral recovery construct (PC1), but it actually had the highest loading of all of the outcome variables assessed, providing strong evidence of construct validity for the IBB.

It is noteworthy that the IBB did not correlate as well with CatWalk measures of gait during locomotion. This suggests that the CatWalk assesses different neurobiological substrates than the IBB. This is consistent with prior work showing that the CatWalk outcome metrics do not have high construct validity with respect to multivariate tissue sparing in contusive SCI (PC1) but do tap into orthogonal variance (PC2, PC3) related to hemisection injuries (2). This indicates that the CatWalk may reflect tissue changes not captured by crude measures of histological sparing after unilateral cervical SCI. This could account for the observation that hemisection injuries impact CatWalk, a model in which white matter and gray matter sparing at the lesion epicenter are relatively consistent. This dissociation between CatWalk and tissue sparing is reminiscent of the pattern observed in prior analyses that have included the horizontal ladder test after cervical SCI (6, 47). The horizontal ladder, the CatWalk and forelimb locomotor function clustered together as a coherent functional assessment construct (PC2); however, this outcome cluster did not correlate with histological sparing (47). We have argued that this indicates that CatWalk and horizontal ladder reflect finedetails of locomotor recovery that are organized by more subtle neurobiological changes (perhaps due to sprouting and plasticity), not reflected by gross gray and white matter sparing metrics *per se* (2, 37).

#### **FORELIMB OBJECT MANIPULATION AS A TRANSLATIONAL TOOL**

Our group has begun developing a primate analog to the IBB to facilitate cross-species translation of SCI research findings (34, 48, 49). Early work suggests that the IBB can be scaled up into an analogous object manipulation task in a non-human primate (NHP) model of cervical SCI in the rhesus macaque (48, 49). The primate version of the task shows strong sensitivity for loss and recovery of function after cervical lateral hemisection injuries. In addition, early cross-species testing of construct validity suggests that the rodent IBB and primate object manipulation task co-load along with tissue sparing on PC1, enabling consistent assessment of translational features of forelimb recovery (34, 48, 49).

Of course, the utility of object manipulation as a translational outcome measure may depend on the neurobiological substrates under study. It is often assumed that much of the loss and recovery of fine digital movement, and reach and grasp, in humans after CNS damage or degeneration is due to loss of cortico-spinal tract (CST) function. The classic work of Lawrence and Kuypers (50–52) indeed points to the pyramidal tract as a critical mediator of forelimb and especially fine digital control in primates. However, attempts to assign specific roles to the multitude of descending tracts and intra-spinal circuits in experimental models of SCI have proven to be difficult, and recent work suggests that there may be considerable redundancy in the organization of forelimb motor function. For example, Fouad and colleagues tested performance on a single pellet reaching task after various lesions of the dorsal and lateral funiculi, and found little correlation between lesion size and performance in the rat (53). In a related study, Morris et al. (54) found that lesions restricted to the dorsolateral funiculus where the rubrospinal tract is located, only affected the "arpeggio" movement, and not other aspects of reach and grasp.

It seems clear that more flexibility and individuation of movement might be supported by the development of the cortical system mediated through the CST as the primate CST developed, and that the ability of primates to produce highly accurate ballistic movements in space and to produce individual finger movements is extraordinary. However, recent work from several laboratories using NHPs suggests that recovery of fine digital control can be accomplished via reorganization of descending reticular systems impinging upon interneurons in the cervical cord. This raises the issue of how much of the forelimb control is mediated by cortical brainstem circuits versus those organized intrinsically within the cervical cord. In the case of the IBB scale, the results of our CCI studies suggest that the circuits in the sensorimotor cortex are involved in recovery of forelimb and fine digital movements, but that certainly much of this circuitry is organized at the spinal level, at least in the rodent.

Comparative studies of the neurobiology of forelimb recovery after rodent and primate SCI are a major focus of ongoing studies (55, 56). Object manipulation tasks such as the IBB will play an important role in making these cross-species comparisons to unravel the neurobiological substrates of forelimb recovery in the context of translational therapeutic testing.

### **CONCLUSION**

The IBB is a recently developed forelimb scale for the assessment of fine control of the digits after damage to the nervous system (1). The present paper suggests that the IBB has strong IRR and validity (face, concurrent, and construct). Thus, the IBB may be useful in conjunction with, and in comparison to, other measures of forelimb and fine digital control in other mammalian species including primates. And, it may be a valuable adjunct to the armamentarium of translational tools for assessing recovery after nervous system damage and degeneration.

#### **AUTHOR CONTRIBUTIONS**

Karen-Amanda Irvine, Stephanie B. Beattie, Jacqueline C. Bresnahan, Michael S. Beattie provided the testing concept, Karen-Amanda Irvine, Stephanie B. Beattie, Jacqueline C. Bresnahan, Adam R. Ferguson, Michael S. Beattie designed the studies; all authors participated in the data collection, IRR testing (except Stephanie B. Beattie), and the interpretation of the results; Adam R. Ferguson supervised the data analysis; Karen-Amanda Irvine implemented the figures with input from Adam R. Ferguson, Jacqueline C. Bresnahan, and Michael S. Beattie; Karen-Amanda Irvine, Adam R. Ferguson, Jacqueline C. Bresnahan, and Michael S. Beattie generated the manuscript draft, and all authors critically reviewed and revised the manuscript.

#### **ACKNOWLEDGMENTS**

Supporting grants: NIH R01-NS038079; NIH R21-AG032518, New York State CORE-SCI, NIH R01-NS067092, Wings for Life Foundation, Craig H. Nielson Foundation.

#### **REFERENCES**

skilled reaching. *Behav Brain Res* (2010) **206**:21–31. doi:10.1016/j.bbr.2009.08. 030

anatomical fractionation. *J Neurotrauma* (2011) **28**:2439–51. doi:10.1089/neu. 2010.1708

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 16 May 2014; paper pending published: 23 May 2014; accepted: 20 June 2014; published online: 07 July 2014.*

*Citation: Irvine K-A, Ferguson AR, Mitchell KD, Beattie SB, Lin A, Stuck ED, Huie JR, Nielson JL, Talbott JF, Inoue T, Beattie MS and Bresnahan JC (2014) The Irvine, Beatties, and Bresnahan (IBB) forelimb recovery scale: an assessment of reliability and validity. Front. Neurol. 5:116. doi: 10.3389/fneur.2014.00116*

*This article was submitted to Movement Disorders, a section of the journal Frontiers in Neurology.*

*Copyright © 2014 Irvine, Ferguson, Mitchell, Beattie, Lin, Stuck, Huie, Nielson, Talbott, Inoue, Beattie and Bresnahan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Time reproduction and numerosity interaction in the parietal cortex: some missing links

## *Carmelo M. Vicario1,2\**

*<sup>1</sup> School of Psychology, The University of Queensland, St Lucia, Brisbane, QLD, Australia*

*<sup>2</sup> Faculty of Motor Science, University of Palermo, Palermo, Italy*

*\*Correspondence<EMAIL_ADDRESS>
#### *Edited by:*

*Renée Morris, The University of New South Wales, Australia*

#### **A commentary on**

## **Interaction of numerosity and time in prefrontal and parietal cortex**

*by Hayashi, M. J., Kanai, R., Tanabe, H. C., Yoshida, Y., Carlson, S., Walsh, V., and Sadato, N. (2013). J. Neurosci. 33, 883–893.*

The interaction of time and numerical representation in the human brain is currently object of debate in the field of cognitive neuroscience. A wide amount of research based on "A theory of Magnitude (ATOM)" (Walsh, 2003), a model which proposes common cortical metric for the representation of space, time, and numbers, has provided extensive evidence in support of this suggestion (Dormal et al., 2006; Xuan et al., 2007; Oliveri et al., 2008; Vicario et al., 2008, 2012; Lu et al., 2009; Vicario, 2011). For instance, it has been shown that merely looking at numbers causes a bias in a timebisection task that depends on its magnitude (Vicario, 2011). Nevertheless, there are also works which contrast with such proposal as it seems that these two dimensions refer to at least partially separated neural segregations (for instance, see Dormal et al., 2008; Agrillo et al., 2010). Thus, we are only beginning to understand the complex neuronal mechanisms underlying the interaction of these magnitudes in the human brain.

In a recent issue of the *Journal of Neuroscience*, Hayashi et al. (2013) added new insight into the current debate on the interaction of time and numerosity by exploring their neural correlates. The use of transcranial magnetic stimulation (TMS) for the investigation of this issue in the human brain is not new in the literature, as an attempt was previously made by Dormal et al. (2008). However, in the current work Hayashi et al. (2013) have used a clever methodology for testing their hypothesis as they applied TMS upon the neural regions which resulted in a joint activation during the execution of time and quantity processing tasks. Moreover, they used two different timing tasks (time discrimination vs. time reproduction) with the purpose of exploring the time numerosity interaction in the presence and in the context of a reduced involvement of categorical magnitude judgment. Remarkably, this work shows that TMS upon the right inferior frontal gyrus (IFG) impairs categorical duration discrimination, but in contrast, it has no effect on time estimation in the duration reproduction task. On the other hand, the TMS upon the right intraparietal cortex (IPC) modulates the degree of influence of numerosity on time reproduction and impairs precise time estimation. This evidence is striking insofar as it provides clear evidence of a common neural origin for the processing of time and numerosity in a precise region of the parietal cortex. The provided results lead the authors to propose a two-stage model of numerosity–time interactions, in which it is stated that the categorical decision takes place in the frontal cortex, whereas the interaction of numerosity information on perception of time occurs within the parietal cortex. Nevertheless, in the context of the present discussion there are two aspects worthy of receiving attention: the "*motor*" nature of the time reproduction task and the involvement of hand/fingers body part for its execution.

Previous studies have shown neural activity in the parietal cortex for particular visuomotor actions involving right hand/ fingers representation such as grasping (Baumann et al., 2009), and the go/no go task (Sugawara et al., 2013). Interestingly, this parietal activation occurred after the Go stimulus, which can be considered the equivalent of the stimulus disappearance in the time reproduction task, of Hayashi et al. (2013). In fact, in this task participants were instructed to start the time reproduction action once the stimulus (digit or dot arrays) disappeared from the computer screen. It is also interesting to note that the parietal cortex of the right hemisphere is directly involved in motor tasks performed with the right hand. For instance, Hinkley et al. (2009) have recently reported that two regions of the right posterior parietal cortex were active in all the experimental subjects asked to perform a grasping task with their right hand. Thus, in consideration of this evidence, it is not possible to exclude that TMS upon the right IPC might affect performance by modulating the accuracy with which participants performed the required movement with their right hand, rather than their timing ability. In fact, by using a time reproduction it is not possible to disentangle the effect played by TMS on the motor and temporal dimensions of this task.

In strict connection with this argument is the close relationship between the representation of numerosity and hand/ fingers in the parietal cortex, as showed by the common parietal activation for finger movements (Sugawara et al., 2013) and numerical cognition (Dormal et al., 2008). Moreover, previous works have extensively shown that, in both forced (Dehaene et al., 1990) and free-response (Daar and Pratt, 2008; Vicario, 2012) paradigms, perceiving numbers affects the execution of fingers action. The relationship between fingers and numerosity is also supported by the study of Costa et al. (2011) showing that deficits in fingers gnosia were found in association to mathematical difficulties. Interestingly, the authors provide argument that the deficits in fingers gnosia could not be attributed to a shortage in working memory capacity but rather to a specific inability to use fingers to transiently represent magnitudes. All these works provide further support to the argument of a close relationship between hand/fingers and numerical magnitude through different experimental designs. Although the studies above mentioned have explored mechanisms qualitatively different from those investigated by Hayashi et al. (2013), the involvement of the hand/fingers at motor and/or representational level represents the *trait d'union* among all of these studies.

Taken together these results suggest that there may be a limit related with the use of the time reproduction task for testing the interaction of time and numerosity since it becomes difficult to establish: (i) if numerosity modulates performance by affecting the execution of the hand/fingers movements or the temporal representation of participants; (ii) if the effect played by TMS on the current timing task is due to its modulatory effect on hand/fingers motor planning rather than on timing performance. To overcome these concerns a good control would have been to subject participants to a non-timing motor task which involves the hand also (as in the case of the current time reproduction task), to ensure that the reported effect is not related to the execution of the action and/or to the hand/fingers involvement. Perspective works devoted to dissociate the interaction between time and numerosity might focus on experimental paradigms that are able to control the factors mentioned above (action and hand/fingers representation). The use of a *verbal time estimation* task (Hurks and Hendriksem, 2011) could represent a valid solution to clear these limits. In fact, this task does not require the involvement of hand/fingers movements.

## **References**

the effects of task modality, duration, and symptom dimensions. *Child Neuropsychol.* 17, 34–50.

#### *Received: 08 April 2013; accepted: 22 April 2013; published online: 06 May 2013.*

*Citation: Vicario CM (2013) Time reproduction and numerosity interaction in the parietal cortex: some missing links. Front. Neurol. 4:45. doi: 10.3389/fneur.2013.00045 This article was submitted to Frontiers in Movement Disorders, a specialty of Frontiers in Neurology.*

*Copyright © 2013 Vicario. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

## Effect of auditory constraints on motor performance depends on stage of recovery post-stroke

#### **Viswanath Aluru<sup>1</sup> ,Ying Lu<sup>2</sup> , Alan Leung<sup>3</sup> , Joe Verghese<sup>4</sup> and Preeti Raghavan1,5\***

<sup>1</sup> Department of Rehabilitation Medicine, New York University School of Medicine, New York, NY, USA

<sup>2</sup> Center for the Promotion of Research Involving Innovative Statistical Methodology, Steinhardt School of Culture, Education and Human Development, New York University, New York, NY, USA

<sup>5</sup> Department of Physical Therapy, Steinhardt School of Culture, Education and Human Development, New York University, New York, NY, USA

#### **Edited by:**

Renée Morris, University of New South Wales, Australia

#### **Reviewed by:**

William Hutchison, University Health Network, Canada Andrew Paul Tosolini, University of New South Wales, Australia

#### **\*Correspondence:**

Preeti Raghavan, Department of Rehabilitation Medicine, Rusk Institute of Rehabilitation Medicine, NYU Langone Ambulatory Care Center, 240 E 38th Street, 17th Floor, New York, NY 10016, USA e-mail<EMAIL_ADDRESS>
In order to develop evidence-based rehabilitation protocols post-stroke, one must first reconcile the vast heterogeneity in the post-stroke population and develop protocols to facilitate motor learning in the various subgroups. The main purpose of this study is to show that auditory constraints interact with the stage of recovery post-stroke to influence motor learning. We characterized the stages of upper limb recovery using task-based kinematic measures in 20 subjects with chronic hemiparesis. We used a bimanual wrist extension task, performed with a custom-made wrist trainer, to facilitate learning of wrist extension in the paretic hand under four auditory conditions: (1) without auditory cueing; (2) to non-musical happy sounds; (3) to self-selected music; and (4) to a metronome beat set at a comfortable tempo.Two bimanual trials (15 s each) were followed by one unimanual trial with the paretic hand over six cycles under each condition. Clinical metrics, wrist and arm kinematics, and electromyographic activity were recorded. Hierarchical cluster analysis with the Mahalanobis metric based on baseline speed and extent of wrist movement stratified subjects into three distinct groups, which reflected their stage of recovery: spastic paresis, spastic co-contraction, and minimal paresis. In spastic paresis, the metronome beat increased wrist extension, but also increased muscle co-activation across the wrist. In contrast, in spastic co-contraction, no auditory stimulation increased wrist extension and reduced co-activation. In minimal paresis, wrist extension did not improve under any condition.The results suggest that auditory task constraints interact with stage of recovery during motor learning after stroke, perhaps due to recruitment of distinct neural substrates over the course of recovery.The findings advance our understanding of the mechanisms of progression of motor recovery and lay the foundation for personalized treatment algorithms post-stroke.

**Keywords: bimanual movements, upper extremity, rehabilitation, motor learning/training, electromyography, task specificity, cerebrovascular disorders**

#### **INTRODUCTION**

Stroke strikes one in six people worldwide. It is the leading cause of disability in the United States (1) and Europe (2), and the second leading cause of disability in the world (3). Hemiparesis is the most common reason for stroke-related disability, and the majority of individuals with hemiparesis have persistent deficits in hand function (4). There has been a recent surge in the availability of new rehabilitation strategies post-stroke. However, several large randomized controlled trials have failed to show the benefit of any one intervention over conventional treatment (5), and there remains a lack of understanding about how to select an appropriate treatment strategy for a given individual. While it is now accepted that task-specific training is an important aspect of a rehabilitation intervention, the constraints under which the task(s) should be practiced to be optimally therapeutic are not known. A constraint may be defined as the specific conditions under which a task is performed, for example, with one hand or both, with auditory/visual/multi-sensory feedback or without, etc. Task constraints are important because they regulate the information that is processed and assimilated by the nervous system, and the selection of constraints for any specific task may depend on the integrity and/or capacity to recruit specific neural substrates that facilitate processing of the relevant movement-related information. The stage of motor recovery, as measured by the level of motor impairment, may provide an indication for the type of task-specific constraints that are useful during practice for a given individual.

Fortunately, recovery of motor function after a hemiplegic stroke has been shown to follow a predictable pattern. Twitchell (6), Brunnstrom (7), and Fugl-Meyer et al. (8) described a hierarchical progression of recovery of patients who initially present with flaccid paralysis on one side of the body with areflexia. The

<sup>3</sup> University of Pittsburgh Medical Center, Pittsburgh, PA, USA

<sup>4</sup> Department of Neurology, Albert Einstein College of Medicine, Bronx, NY, USA

reflex activity returns next and becomes heightened as spasticity emerges, and voluntary movements occur in stereotypical flexor and extensor synergy patterns. Spasticity then reaches its maximum level, producing characteristic patterns of stretch-sensitive responses such as spastic co-contraction. Eventually, the synergy patterns start to break up and spasticity begins to reduce as normal patterns of voluntary movement are restored. The emergence and disappearance of spasticity are thus important milestones in motor recovery (9, 10), although the severity of spasticity may vary considerably and temporary arrests in recovery or "plateaus" can occur at any stage (6).

Recent imaging studies further show how recovery processes unfold after a stroke [see Ref. (11) for review]. Early in recovery, the undamaged contralesional hemisphere shows increased activation (12–15), but eventually normal sensorimotor lateralization is restored in the stroke-affected hemisphere (16–18). Importantly, increases in neural activity in the contralesional motor areas in the first weeks after stroke correlate with better motor recovery in humans (19, 20) and monkeys (15), although persistent activation of the motor and non-motor areas in the contralesional hemisphere is noted in patients with poor motor outcome (18, 21). A recent longitudinal case study of a patient's recovery over 21 months revealed continuous change in activation in the contralesional hemisphere with concomitant improvement in motor performance, whereas the ipsilesional hemisphere demonstrated significant change only toward the end of the study period (22). Taken together, these studies suggest that (1) redundant homologous pathways in the intact hemisphere can facilitate re-organization of the central nervous system, particularly in the earlier stages of recovery, and (2) that motor recovery occurs over a protracted and variable time period post-stroke. Hence the time since stroke may not reflect where an individual is in his or her recovery process.

Two kinds of bimanual training protocols have been developed to capitalize on contralesional cortical activity post-stroke. In active bimanual training, both arms move independently and simultaneously, requiring that individuals have at least some active movement on the paretic side. Active bimanual arm training combined with rhythmic auditory stimulation (BATRAC protocol) led to increased recruitment in the contralesional and ipsilesional hemispheres with concomitant improvement in performance of the paretic hand (23, 24). These data suggest that there may be a synergistic effect of bimanual and auditory constraints, but their individual contribution to performance improvement has not been ascertained. Rhythmic auditory stimulation by itself has also been found to be a useful adjunct to post-stroke rehabilitation (25–28). In active–passive bimanual training, the non-paretic arm drives movements of the paretic arm and leads to simultaneous mirror movements of both arms. Here, bimanual training occurred without auditory stimulation, was used to prime the ipsilesional motor cortex for subsequent training with the paretic arm, and also led to significant gains in arm function (29–32). An advantage of the active–passive approach is that it requires little active movement in the paretic arm and can therefore be used in individuals with significant paresis. Furthermore, the active–passive approach may be used to probe subsequent motor learning with the paretic arm. We have previously shown that motor learning is often impaired with the paretic hand, but may be temporarily restored after prior practice with the non-paretic hand (33).

In this study, we sought to determine the effect of various auditory constraints on bimanual-to-unimanual (paretic hand) learning in individuals at different stages of motor recovery poststroke. Rhythmic stimulation with a metronome has been shown to improve spatiotemporal control of arm movements, perhaps via activation of brainstem–cerebellar networks (34, 35). However, several lines of evidence suggest that emotional drive via activation of limbic networks may also be an important predictor of motor performance (36) and post-stroke motor recovery (37). Music has been shown to activate a bilateral network of mesolimbic structures involved in processing emotions and reward information (38), and affective vocalizations have been shown to modulate attention via activation of pre-frontal–limbic networks (39). It is not clear when over the course of recovery one type of auditory stimulation versus another or no auditory stimulation will be beneficial. Hence, the objectives of this study were to: (1) characterize the stage of recovery in a disparate group of subjects with post-stroke hemiparesis using task-based kinematic measures, and (2) to examine how various types of auditory constraints interact with stage of recovery to facilitate learning with the paretic limb on a bimanual-to-unimanual learning task. Since voluntary wrist extension is frequently compromised post-stroke (40) and active wrist extension ability is predictive of hand function (41), we focused our task on training of wrist extension in the paretic hand. We hypothesized that auditory constraints that enhance emotional drive would facilitate learning of wrist extension with the paretic arm particularly in the early stages of recovery post-stroke.

#### **MATERIALS AND METHODS**

#### **SUBJECTS**

Twenty subjects with chronic post-stroke hemiparesis (at least 6 months prior to enrollment) were recruited through referrals from physicians at the Rusk Institute of Rehabilitation Medicine and through public advertisement. Subjects provided informed consent in accordance with the Institutional Review Board of the New York University School of Medicine. All subjects had at least 15° of passive and 5° of active wrist extension on the paretic side to perform unimanual movements, and they were screened to rule out hearing deficits prior to participation.

#### **PROTOCOL**

The clinical assessments and experimental protocols were administered by well-trained research staff at the Motor Recovery Research Laboratory in the Rusk Institute of Rehabilitation Medicine. At the first visit, the Fugl-Meyer Scale (8) was used to assess upper extremity motor impairment; the Modified Ashworth Scale (42) assessed spasticity in the affected shoulder, elbow, wrist, and finger joints; active and passive range-of-motion at shoulder, elbow, wrist, and finger joints were measured using a goniometer (43), and the threshold for joint proprioception was also assessed. Depression and mood were assessed using the 15 item Geriatric Depression Scale, which has been recommended for

the assessment of post-stroke depression in adults of all ages (44, 45), and the Brunel Mood Scale (BRUMS) (36), respectively. An appropriate tempo for the metronome beat was then determined by asking subjects to flex and extend their paretic wrist at a comfortable pace using a custom-made wrist trainer (**Figure 1**) for 15 s. Subjects then selected three familiar songs from public media to increase their feeling of vigor, happiness, and calmness. An uptempo major key song that matched their metronome speed, or was in multiples of their metronome speed, was chosen to induce a positive mood-state. The BRUMS Scale was repeated after the subjects listened to their self-selected song to verify improvement in mood-states (**Figure 2**).

At the second visit, subjects performed repeated bimanual and unimanual (with the paretic hand) wrist flexion-extension movements using a custom-made wrist trainer. The device was designed to constrain movement of the wrist in the sagittal plane, and limit compensatory movement of the forearm and arm. The height of the chair was adjusted to keep the shoulders level and maintain proper alignment of the trunk. Table height and the position of the wrist trainer were maintained across all task conditions for each subject. Before the start of the experiment, subjects were informed that the goal of training was to facilitate wrist extension. Electromagnetic motion sensors (trakSTAR, Ascension Technology Corporation, Shelburne, VT, USA) affixed to the limb segments on each side measured wrist kinematics. Bipolar surface electrodes (DE 2.1, Delsys Inc., Natick, MA, USA) affixed over the flexor carpi ulnaris (FCU) and extensor carpi radialis longus (ECRL) muscles on each limb recorded electromyographic (EMG) signals. Video, kinematic, and EMG data were captured synchronously using The Motion Monitor (Innovative Sports Training Inc., Chicago, IL, USA), and analysis was performed offline using Spike 2 (Cambridge Electronic Design, Cambridge, England).

Wrist movements were performed under four different auditory conditions: (1) at baseline without auditory cueing; (2) to positively valenced affective "happy" sounds (baby's laughter) recorded for 11 s and looped continuously, providing non-musical and non-rhythmic auditory stimulation; (3) to the self-selected up-tempo major key song chosen during visit 1; and (4) to a metronome beat set at each individual's comfortable tempo. The subject was required to complete one cycle of wrist extension and flexion to each beat. Each condition consisted of 18 15-s trials of wrist flexion and extension, where subjects performed two bimanual trials followed by one unimanual trial with the paretic hand. A 20-s rest break was provided between each trial to prevent fatigue. The order of the conditions was counterbalanced across subjects, and subjects rated their fatigue levels after the completion of all trials for each condition.

#### **DATA ANALYSIS**

Kinematic data were sampled at 120 Hz and EMG data were sampled at 1206 Hz. The kinematic data were low pass filtered at 6 Hz and up-scaled to 1206 Hz using linear interpolation. The EMG data were filtered using a dual band pass filter (10–52.5 and 67.5–500 Hz) and the root mean square (RMS) of the signal was obtained for wrist flexion and extension phases of the movement separately. The EMG signals were normalized to the maximum amplitude recorded for each muscle across all trials and conditions (46, 47) to facilitate within- and between-subject comparisons. This method was chosen after extensive reliability testing of different methods of normalization (by Ying Lu). Movement speed, amplitude of wrist extension, wrist extensor activation (RMS of agonist, ECRL), wrist flexor activation (RMS of antagonist, FCU) during extension, and co-activation (defined as RMS of antagonist, FCU/RMS of agonist, ECRL) were the variables used in the analyses. Recognizing that the subjects may present at various stages of recovery at the time of the study, we used hierarchical cluster analysis with the Mahalanobis metric (48) based on baseline wrist kinematics to stratify subjects into groups. The stratification scheme corresponded well with recovery characterized by the Fugl-Meyer Scale as shown in the results below. We then fit linear mixed effect models with group interactions and individual random effects to assess: (1) differences among subject clusters, and (2) learning rates across repeated unimanual trials with the paretic hand after bimanual priming with the four auditory conditions. Learning rate on unimanual trials was defined as the slope of the linear trend fit over the six unimanual trials. All the statistical analyses were conducted using R (v. 2.15.1). The R package "lme 4" was used for the mixed effect model estimation. To control for multiple comparisons but preserve statistical power (due to low sample size in the subgroups), we present all results but choose to interpret results with marginal statistical significance (0.01 < *p* < 0.05) with caution.

## **RESULTS**

Our first objective was to characterize the stages of recovery across a disparate group of patients with post-stroke hemiparesis. Since wrist kinematics provide direct, objective, and reliable measures of movement ability in the paretic hand, we used the movement speed and extent of wrist extension from the first trial with the paretic hand under the baseline condition (no auditory cueing) to perform hierarchical cluster analysis (48), which stratified subjects into three distinct groups (**Figure 3**).

Clinical metrics (**Table 1**) showed clear differences across the three groups. The Fugl-Meyer scores were lowest in group 1, followed by group 2, and then group 3 (*p* = 0.047). Active wrist motion, measured using goniometry separately from the wrist extension task, showed that both wrist flexion and extension were surprisingly lowest in group 2, intermediate in group 1, and highest in group 3 (*p* < 0.001). Spasticity at the wrist flexors was, however,

**the first trial with the paretic hand at baseline (without auditory stimulation)**. Three distinct groups emerged.

highest in group 1, and similar in groups 2 and 3 (*p* = 0.066). There were no significant differences among the three groups in joint proprioception at the wrist, depression scores, mood scores, tempo of the metronome beat or selected song, or fatigue levels. Note that the mean time since stroke was also not different across the three groups (*p* = 0.89).

Baseline performance metrics on the wrist extension task also showed clear differences across the three groups. Movement speed was higher in group 3 compared to groups 1 and 2 (*p* < 0.001, **Figure 4A**). Extent of wrist extension was lowest in group 2 (where attempted wrist extension produced paradoxical flexion), intermediate in group 1, and highest in group 3 (*p* < 0.001, **Figure 4B**). Wrist extensor muscle (ECRL) activation was also lowest in group 2, intermediate in group 1, and highest in group 3 (*p* = 0.047, **Figure 4C**), whereas wrist flexor muscle (FCU) activation was not differentiated in the three groups (*p* = 0.877, **Figure 4D**). Co-activation between wrist extensor and flexor muscles was highest in group 2, intermediate in group

Bolded variables showed statistically and/or clinically important differences across the three groups.

1, and lowest in group 3 (*p* = 0.07, **Figure 4E**). Taken together, the baseline performance and clinical metrics enabled characterization of recovery patterns into the three descriptive groups below.

#### **GROUP 1 – SPASTIC PARESIS**

In this group, performance on the paretic side (**Figure 4**, shown in blue) relative to the non-paretic side showed low movement speed (~10%), moderate wrist extension (~20%), moderate activation in the wrist extensor (~30%), and five times greater coactivation. Clinically, these subjects had the lowest Fugl-Meyer scores (range 21–51), but had 15–33° of active wrist flexion and 10–35° of active wrist extension. Spasticity was observed predominantly in the wrist flexors. Lesion location and stroke subtype (**Table 1**) suggest that these subjects had very severe strokes that were caused predominantly by intracerebral hemorrhage (subject #s 2, 3, 5, 18) or hemorrhagic transformation of ischemic infarcts (subject # 7).

#### **GROUP 2 – SPASTIC CO-CONTRACTION**

In this group, performance on the paretic side (**Figure 4**, shown in red) relative to the non-paretic side showed very slow movement speed (~7%), paradoxical wrist flexion on attempted wrist extension (−27%), minimal activation of the wrist extensor muscle (~10%), and ~10 times greater co-activation. Clinically, these subjects had higher Fugl-Meyer scores (range 25–62) than those in group 1. However, they had only 10–20° of active wrist flexion and 5–15 of active wrist extension. Spasticity was distributed equally in both wrist flexors and extensors for the most part. Lesion location and stroke subtype (**Table 1**) suggest that these subjects had moderately severe strokes caused predominantly by infarcts in the MCA territory (subject #s 1, 9, 12, 21).

#### **GROUP 3 – MINIMAL PARESIS**

In this group, performance on the paretic side (**Figure 4**, shown in green) relative to the non-paretic side showed relatively high movement speed (~60%), substantial wrist extension (~77%), and wrist extensor activation (~75%), and twice the co-activation as the non-paretic side. Clinically, these subjects had the highest Fugl-Meyer scores (range 35–65) and the greatest range of active wrist flexion (30–75°) and extension (25–70°) of the three groups. Lesion location and stroke subtype (**Table 1**) suggest that these subjects had a mixed variety of strokes predominantly in the MCA territory.

Our second objective was to examine how different types of auditory stimuli interact with bimanual training to facilitate subsequent learning with the paretic limb in the three groups. Subjects performed six cycles of two bimanual trials followed by one unimanual trial with the paretic hand, where each trial consisted of multiple repeats of wrist flexion-extension over 15 s. We were interested in the changes in wrist extension, wrist extensor activation, wrist flexor activation, and co-activation over the six unimanual trials for each of the auditory conditions (represented by the different line patterns, see **Figure 5**). The mean level of the trend lines provides an indication of the amplitude of overall performance, whereas the slope of the trend lines quantifies the rate of learning on the paretic side. A positive slope suggests sustained improvement whereas a negative slope suggests reduced performance under that constraint. Subjects in the spastic paresis (**Figure 5A**) and spastic co-contraction (**Figure 5B**) groups started with low or negative wrist extension, but showed sustained improvements under certain auditory constraints. Subjects in the minimal paresis group (**Figure 5C**), showed good wrist extension at first, but did not improve much over the repeated trials.

The slope (unit change per trial) succinctly summarizes which auditory conditions couple with bimanual training for sustained improvement on the paretic side in the three groups (**Figure 6**). In the spastic paresis group (**Figure 6A**), wrist extension improved most with the metronome beat (slope *b* = 0.86, *p* = 0.03), even though it also increased wrist flexor activity (*b* = 0.0021, *p* < 0.0001) and co-activation (*b* = 0.07, *p* = 0.004).

Self-selected music did not increase wrist extension, but marginally increased flexor muscle activity (*b* = 0.0010, *p* = 0.04). Thus rhythmic auditory constraints improved motor control in subjects with spastic paresis who were at an earlier stage in motor recovery post-stroke. In the spastic co-contraction group (**Figure 6B**), wrist extension improved most *without* any auditory cueing (*b* = 1.83, *p* < 0.0001), which increased wrist extensor muscle activation (*b* = 0.004, *p* = 0.0002) and decreased co-activation across the wrist joint (*b* = −0.1, *p* = 0.0006). In contrast, self-selected music increased co-activation (*b* = 0.059, *p* = 0.04) in this group. Thus practice without auditory constraints was most beneficial in subjects with spastic co-contraction. In the minimal paresis group (**Figure 6C**), there was no improvement in wrist extension across the auditory conditions. The slope for wrist extension was most negative with happy sounds (*b* = −0.86, *p* = 0.03), wrist extensor activation decreased with the metronome beat (*b* = −0.0022, *p* = 0.02), and wrist flexor activation increased without auditory stimulation (*b* = 0.0012, *p* = 0.015).

### **DISCUSSION**

Neurological and behavioral differences between patients and within each patient over the course of post-stroke recovery can influence how learning occurs during task-specific interactions. Hence, it is necessary to reconcile the vast clinical and movement heterogeneity in the post-stroke population to develop evidencebased rehabilitation protocols directed toward more homogenous groups of patients. Toward this end, the purpose of this study was to: (1) stratify subjects with post-stroke hemiparesis according to their stage of recovery using task-based kinematic measures, and (2) to examine how various types of auditory constraints interact with stage of recovery to facilitate learning of a wrist extension task with the paretic limb. The subjects were stratified into three distinct groups based on their speed and extent of wrist extension. Differences in clinical metrics and task performance led to the characterization of stage of recovery into three groups: (1) the spastic paresis group showed weak extensor drive with flexor spasticity and moderate co-activation of the flexors and extensors, and higher level of motor impairment; (2) the

spastic co-contraction group showed higher flexor activation relative to the extensor and excessive co-activation of the flexors and extensors, with moderate level of motor impairment; and (3) the minimal paresis group showed restored extensor drive, low levels of co-activation, and minimal level of motor impairment. The effect of auditory constraints on rate of learning with the paretic hand after bimanual training was measured by the slope of wrist extension, and wrist extensor and flexor muscle activation patterns. Auditory stimulation with a metronome beat increased the rate of learning of wrist extension in subjects with spastic paresis, even though it increased flexor activation and co-activation across the flexor and extensor muscles. In contrast, bimanual training *without* auditory stimulation produced the greatest improvement in subjects with spastic co-contraction, increased wrist extensor activation, and reduced co-activation. Auditory stimulation in subjects with minimal paresis did not improve wrist extension, but performance was sensitive to the effects of auditory stimulation in this group. These results suggest that altering auditory task constraints during the same task can have different and even opposite effects on motor performance and learning in individuals at different stages of recovery post-stroke. These results

cannot be explained by differences in proprioceptive sensation, task difficulty, or fatigue across the groups or conditions. The results further our understanding of possible mechanisms underlying progression of recovery from one stage to the next after stroke.

## **STRATIFICATION OF SUBJECTS REFLECTS TEMPORAL STAGES IN POST-STROKE RECOVERY**

Subjects with stroke have traditionally been classified based on the time elapsed since their stroke into acute (0–3 months), subacute (3–6 months), and chronic (6 months onward) categories. Recovery has been found to be most rapid in the acute and subacute periods (49), but recently compiled evidence shows that it continues well into the chronic period (50), although the trajectory of recovery may be punctuated by "plateaus" or temporary arrests in recovery. All the subjects in our study were in the chronic phase and may be considered to have plateaued. In longitudinal studies, increases in Fugl-Meyer scores suggest progression toward recovery. The Fugl-Meyer Scale is based on the observation of sequential recovery of motor function by Twitchell and Brunnstrom (6, 9, 10). It is the most widely used quantitative measure of motor recovery

post-stroke (51, 52), and the scores have been shown to correlate with the extent of corticospinal tract damage (53). Hence, one can consider subjects with lower Fugl-Meyer scores as being more impaired or at an earlier stage in the recovery process compared to those with higher scores. In this study, subjects in the spastic paresis group had the lowest average Fugl-Meyer scores (both total and for the wrist and hand), which progressively increased in the spastic co-contraction and minimal paresis groups.

Fugl-Meyer scores have also been used to stratify subjects into groups (54, 55), but the cut-offs have been variable. Furthermore, the Fugl-Meyer Scale was constructed on the assumptions that recovery proceeds in a proximal-to-distal fashion and from synergistic-to-isolated movements (8, 51); however, both these assumptions have been contested recently (56–58). To circumvent the shortcomings of the Fugl-Meyer Scale in stratifying subjects, we used task-based kinematic measures, that is, speed and extent of wrist extension during the task, as direct, objective, and reliable

measures of movement ability to stratify subjects into groups. Note that wrist movement amplitudes recorded during the task were lower than those recorded with goniometry prior to the task as would be expected due to the repetitive nature of the task.

We found that the spastic paresis group showed higher speed and amplitude of movement than the spastic co-contraction group, even though the Fugl-Meyer scores were higher in the spastic co-contraction group. This may seem surprising and contradictory to the notion of a linear improvement in movement ability over the course of post-stroke recovery. However, Twitchell observed that spasticity or tone continues to increase and reaches a peak before it starts to decrease (6). In this study, we measured spasticity clinically using the Modified Ashworth Scale, and by the extent of co-activation across the flexors and extensors during the task. We found that the spastic co-contraction group had equally increased tone in both the flexors and extensors, and 10 times greater co-activation on the paretic side than on the non-paretic side. While some degree of co-activation between the agonist and antagonist muscles is normal during movement, excessive coactivation leads to reduced movement speed and amplitude (59). Therefore, it follows that a progression of recovery from spastic paresis would lead to a dip in movement ability due to increases in co-activation before it begins to improve again as seen across our three groups. Our results suggest that the processes underlying progression of recovery are non-linear, and predict that movement kinematics and muscle activation patterns may worsen as recovery progresses and then get better. These predictions should be confirmed by future longitudinal studies that measure kinematics and EMG over time.

Furthermore, our results show that auditory constraints increase movement amplitude but also increase muscle coactivation in subjects with spastic paresis, suggesting that individuals at earlier stages of motor recovery benefit from an excitatory drive. In contrast, in subjects with spastic co-contraction, who were at a later stage in recovery and showed excessive co-activation from excitatory overdrive, auditory constraints were not helpful. Instead, bimanual-to-unimanual training *without* auditory stimulation led to reduced muscle co-activation and increased agonist muscle activity, suggesting that an inhibitory drive may be more beneficial to transition from spastic co-contraction. These findings are discussed further in the sections below. Thus, we propose that stratification of subjects based on relatively simple kinematic parameters of speed and extent of movement into the groups: (1) spastic paresis, (2) spastic co-contraction, and (3) minimal paresis reflects temporal stages in the course of post-stroke recovery, and transition from each of these stages may be triggered by specific constraints imposed during training.

#### **RHYTHMIC AUDITORY STIMULATION IMPROVES PERFORMANCE IN INDIVIDUALS WITH SPASTIC PARESIS**

At baseline, subjects with spastic paresis had both weakness and spasticity, defined as velocity-dependent increase in muscle tone at rest (60), as measured by the Modified Ashworth Scale (61). The emergence of spasticity is thought to reflect re-organization of the descending brainstem pathways leading to diffuse and synergistic patterns of movement. Weakness predominates in the early stages of spasticity (62), hence, while subjects in the spastic paresis group could activate their wrist extensor muscle, their range of wrist extension was limited. Spasticity was greater in the flexor muscles, consistent with the emergence of a flexor synergy pattern (10). Co-activation across the flexors and extensors was increased, but not disabling, as it did not hinder wrist extension (63, 64). In this group, auditory stimulation with a metronome beat in conjunction with bimanual training led to increased wrist extension, while that with self-selected music and happy sounds did not. However, both the metronome beat and self-selected music increased wrist flexor activation.

Both the metronome beat and self-selected music have rhythmic components; the rhythm was even and constant with a metronome, but uneven and changing with music. Both even and uneven rhythmic stimulation have been shown to increase muscle co-activation (65). The underlying mechanism is thought to be increased excitability of spinal motor neurons via the reticulospinal pathway, with facilitation of the H-reflex response (66, 67). Using functional MRI and effective connectivity analyzes, it has been shown that listening to music relative to scrambled musical sounds, activates a bilateral network of mesolimbic structures including the nucleus accumbens and the ventral tegmental area (38) leading to dopamine release and arousal. The ventral tegmental area in turn forms part of the midbrain reticular formation where the reticulospinal tracts originate. Excitation of the reticular formation is known to increase spasticity via the reticulospinal projections to the spinal cord (68). Thus, both the metronome beat and stimulating music can increase muscle tone and co-activation that may be helpful in earlier stages of recovery from flaccid paralysis. Non-musical and non-rhythmic auditory stimulation, as in our happy sounds condition, does not produce this effect. Furthermore the type of music, whether stimulating or relaxing, can modulate the extent of arousal and may produce a different effect on muscle tone.

However, only auditory stimulation with a metronome beat in conjunction with bimanual training led to increased unimanual wrist extension, while that with self-selected music and happy sounds did not. Even rhythms have been shown to reduce the variability in EMG responses, whereas uneven rhythms increase the variability in healthy individuals (65). Patients with stroke show disordered motor unit recruitment on EMG (69–72), but training to even metronome beats has been shown to decrease EMG variability (73) and improve motor outcomes post-stroke (23, 25, 27, 73–75). More efficient motor unit recruitment and sensorimotor synchronization (28) to the even metronome beat can explain the increased wrist extension without a notable increase in extensor activation as seen in our subjects with spastic paresis. In contrast, the variable rhythms in music and subtle differences in the type of music chosen, the tempo of the song and its match to the individual's physical abilities may have influenced attention to the rhythmic component of music leading to a reduced peripheral synchronizing effect on wrist extension.

In healthy individuals, sensorimotor coupling to temporally structured auditory input has been shown to recruit a striatothalamo-cortical-system involving basal ganglia, thalamus, premotor cortex (PMC), supplementary motor area (SMA), and dorsolateral prefrontal cortex [see Ref. (76) for review]. Simultaneous bimanual rhythmic movements involve interhemispheric coupling primarily in the PMC, posterior parietal cortex, and cerebellum (77), and switching from simultaneous bimanual synchronized movements to unimanual movements leads to a higher degree of interhemispheric connectivity involving the PMC, SMA, and sensorimotor areas (78). Furthermore, studying acallosal patients has shown that temporal coupling during rhythmic movements arises in large part from interactions between the two hemispheres (79). Taken together with these data, our results suggest that bimanual-to-unimanual movements synchronized to rhythmic auditory stimulation excites a bilateral distributed sensorimotor network, which may facilitate the progression of motor recovery in individuals with spastic paresis.

## **AUDITORY STIMULATION DOES NOT IMPROVE INHIBITORY CONTROL IN INDIVIDUALS WITH SPASTIC CO-CONTRACTION**

When the threshold for reflex activity continues to reduce due to progressive re-organization of the supraspinal descending drive to the spinal cord, peripheral structures of the muscle, muscle spindles, and fascia are further shortened and spasticity evolves into stretch-sensitive forms such as spastic co-contraction (63). Spastic co-contraction refers to inappropriate antagonist recruitment triggered by volitional command (64). Clinically, spastic co-contraction opposes voluntary movement and contributes to impairment in active function, which was seen clearly in our subjects in this group where attempted wrist extension produced paradoxical wrist flexion. While some degree of co-activation between the agonist and antagonist muscles is normal during movement and necessary for joint stability, better movement accuracy and energy efficiency during functional activities, it has been shown to decrease with skill training (80–82). However, its persistence post-stroke signals disrupted reciprocal inhibition of antagonist muscles (83). Sensory feedback from muscle afferents mediates reciprocal inhibition through both spinal and cortical mechanisms (84, 85). Cortical suppression of the antagonist muscle is initiated centrally during preparation of agonist muscle contraction (86,87) and the degree of suppression is proportional to the amplitude of stretch of the muscle (88).

Bilateral synchronous mirror symmetric flexion-extension movements have been shown to modulate cortical inhibition in neurologically intact individuals (89) and subjects with stroke (30). Somatosensory and visual information from each side of the body is processed bilaterally (90–92), and interlimb coordination is mediated by motor representations in the parietal and premotor areas shared by both limbs (93). Transcallosal pathways between homotopic regions of the two hemispheres (94–96) may also facilitate transmission of accurate sensory information from the intact hemisphere (33). Passive wrist extension on the affected side (which was facilitated by linked movements with the unaffected hand in this study) in severely impaired patients has been shown to produce fMRI changes in contralesional secondary sensorimotor areas in the ventral premotor and parietal cortices (97), which play a crucial role in re-organization of motor output. Thus, in patients with spastic co-contraction, bimanual training *without* auditory stimulation may restore sensory feedback, and reinstate reciprocal control in the paretic hand, aiding progression to the next stage of post-stroke recovery. In contrast, self-selected music may have continued to potentiate the stretch reflex through facilitation of descending spinal pathways in this group as discussed above.

#### **INDIVIDUALS WITH MINIMAL PARESIS SHOW VARIED RESPONSES TO AUDITORY STIMULATION**

In subjects with minimal paresis, there was little change in wrist extension across the auditory conditions perhaps due to a ceiling effect. Later stages of recovery have been shown to be mediated by re-organization in the ipsilesional cortex (16–18). Thus, it is not surprising that subjects in this group, who were farther along in their recovery, did not benefit substantially from either bimanual-to-unimanual training or auditory stimulation at the wrist. These strategies would perhaps still be applicable for training of hand and finger control. Subjects with minimal paresis no longer had significant spasticity or co-contraction, but were clearly still impaired compared to the unaffected side. The challenge in these subjects is fine-tuning of muscular control

and restoration of dexterity, which may require different types of task constraint.

### **CONCLUSION**