Exclusion Criteria for Patients and Healthy Volunteers

NEUROLOGY72012/490680 Liuzzi et al.

Appendix e-1

Exclusion criteria for patients and healthy volunteers

Exclusion criteria encompassed a history of neurological or psychiatric diseases, severe head traumas, drug abuse, neuroactive medication, metal implants in the head and neck region, cardiac pacemaker, pregnancy and cognitive or language deficits interfering with appropriate participation in the study.

Behavioral evaluation

For each grip strength assessment, the mean value (in kg) of three consecutive measurements separated by approximately 30 s of rest was calculated. For the assessment of FT, patients were seated in front of an electronic keyboard in an upright position with the forearm lying on a table. They were instructed to press a specific key with the paretic index finger as quickly as possible for a total of 10 s (1).

This task was repeated three times with approximately thirty seconds rest in between repetitions. The keyboard was connected to a laboratory computer that recorded the timing of the taps (in ms). FT was defined as the mean number of taps in the three repetitions.

Assessment of movement-related intracortical inhibition

Prior to TMS measurements, patients were familiarized with a simple reaction time task. They were instructed to abduct the paretic index finger as quickly as possible after presentation of a visual Go-signal on a computer screen. Electromyographic

(EMG) activity was recorded from the affected FDI. In the absence of TMS, each individual’s reaction time (RT) was calculated in 25 trials as the mean latency in ms

1 NEUROLOGY72012/490680 Liuzzi et al.

between the Go-signal and EMG onset (fig. 1A). The measurements with TMS during movement preparation were then adjusted to the individual RT: after 20% (t1), 50%

(t2), 80% (t3) and 95% (t4) of RT comparable to previous work (fig. 1B).

TMS was delivered by two Magstim 200 stimulators (Magstim Co., Withland, Dyfed

UK) connected through a Bistim module to a figure-of-eight shaped coil (70 mm in outer diameter). Before testing ICI, the optimal location for stimulating the M1 representation of the affected FDI (hot spot: M1FDI) and the individual resting motor threshold (rMT) was determined, as described previously (2, 3).

In order to assess ICI, dpTMS requires single-pulse (only suprathreshold pulse) and double-pulse trials (subthreshold conditioning pulse + suprathreshold pulse; fig. 1C).

In a single pulse trial, one suprathreshold pulse was given over the M1 FDI of the ipsilesional hemisphere, and the resulting motor-evoked-potential (MEP) was recorded from the affected FDI. In a double-pulse trial, a conditioning stimulus (CS) followed by a suprathreshold pulse was given. ICI was defined as the ratio (in %) of

MEP peak-to-peak amplitudes in double-pulse trials divided by MEPs in single-pulse trials (fig. 1C). Values below 100 indicate inhibition that is smaller MEPs in double- pulse trials compared with unconditioned MEPs in single-pulse trials. In order to determine movement-related ICI, single pulses and double pulses were randomly applied at four time points during the preparatory period of a voluntary movement with the affected index finger (fig. 1C). For each time point, 18 single- and 18 double- pulse MEPs were collected. The inter-trial interval randomly varied between 6, 7 or 8 s to avoid anticipation of the Go-signal.

Short-interval ICI can be observed at inter-pulse intervals of 1–6 ms and reflects the activation of local GABAergic circuits (4-6). We chose an inter-pulse interval of 3 ms

and a stimulus intensity for the subthreshold conditioning pulse of 80% of the rMT, a reliable procedure to assess movement-related ICI in stroke patients (7). The stimulation intensity of the suprathreshold pulse was adjusted to obtain MEPs of approximately 1 mV during movement preparation (50 % of RT) (8), because the amplitudes of MEPs substantially differ between rest and when cued for a reaction

(9). MEPs of approximately 1 mV reflect an optimal excitability level to induce ICI as demonstrated in a previous study that tested ICI at different intensities of the suprathreshold pulse (10).

Using the same dpTMS setup, ICI was determined during the preparation of a voluntary movement with the right hand in the age-matched healthy control group.

The time course of ICI in healthy controls during movement preparation is illustrated in fig. 1D indicating a decrease of inhibition towards EMG onset. To determine the modulation of ICI during movement preparation, we calculated a ratio of t4 by t1

(t4/t1: ICImod), as previously described (7). In healthy subjects, ICImod is usually above

1, because inhibition typically decreases from the early to the late phase of movement preparation. In stroke patients, it could be demonstrated that ICImod is reduced after stroke (below or around 1) (7). However, the importance of movement- related intracortical inhibition (ICImod) for motor function and recovery after stroke is still not known. To evaluate the association of movement-related intracortical inhibition with motor recovery, ICImod was chosen as the primary predictor in the present study (please see also “Statistical analysis” below).

e-Figure Legends

Online Figure e-1: Individual MRI scans.

Images of individual patients in the acute phase of stroke are shown. Except for patient 10 (FLAIR image), diffusion-weighted images were chosen for illustration of individual lesions.

Online Figure e-2: Time plots of motor tests.

A Grip strength, B finger tapping speed (taps per 10s), C upper limb section of Fugl-

Meyer score (FMS) and D Action Research Arm Test (ARAT) of paretic arm/hand during the course after stroke. The x-axis indicates time points after stroke. On the y- axis, the group mean values of each test +/- standard errors (vertical bars) are given.

Online Figure e-3: Movement-related short-interval intracortical inhibition in stroke patients and healthy subjects.

The time course of movement-related short-interval intracortical inhibition (ICI% on y- axis) is shown during movement preparation (x-axis; t1: 20% of reaction time, t2:

50% of reaction time, t3: 80% of reaction time; t4: 95% of reaction time).

Group mean values +/- standard errors of stroke subjects in the A acute (5.8 ± 0.41 days), B subacute7w (50.72 ± 3.01 days), C subacute3m (91.91 ± 2.56 days) and D chronic phase (351.5 ± 15.92 days) compared with healthy subjects are given.

e-References

1. Werhahn KJ, Conforto AB, Kadom N, Hallett M, Cohen LG. Contribution of the ipsilateral motor cortex to recovery after chronic stroke. Annals of Neurology 2003;54:464-472.

2. Chen R, Cros D, Curra A, et al. The clinical diagnostic utility of transcranial magnetic stimulation: report of an IFCN committee. Clin Neurophysiol 2008;119:504- 532.

3. Rossini PM, Barker AT, Berardelli A, et al. Non-invasive electrical and magnetic stimulation of the brain, spinal cord and roots: basic principles and procedures for routine clinical application. Report of an IFCN committee. Electroencephalogr Clin Neurophysiol 1994;91:79-92.

4. Kujirai T, Caramia MD, Rothwell JC, et al. Corticocortical inhibition in human motor cortex. J Physiol (Lond) 1993;471:501-519.

5. Ziemann U, Lonnecker S, Steinhoff BJ, Paulus W. Effects of antiepileptic drugs on motor cortex excitability in humans: a transcranial magnetic stimulation study. Annals of Neurology 1996;40:367-378.

6. Ziemann U, Lonnecker S, Steinhoff BJ, Paulus W. The effect of lorazepam on the motor cortical excitability in man. Experimental Brain Research 1996;109:127- 135.

7. Hummel FC, Steven B, Hoppe J, et al. Deficient intracortical inhibition (SICI) during movement preparation after chronic stroke. Neurology 2009;72:1766-1772.

8. Liuzzi G, Hörniß V, Hoppe J, et al. Distinct temporospatial interhemispheric interactions in the human primary and premotor cortex during movement preparation. Cerebral Cortex 2009;in press.

9. Chen R, Yaseen Z, Cohen LG, Hallett M. Time course of corticospinal excitability in reaction time and self-paced movements. Annals of Neurology 1998;44:317-325.

10. Sanger TD, Garg RR, Chen R. Interactions between two different inhibitory systems in the human motor cortex. J Physiol 2001;530:307-317.