Overview of the study design. Twenty-nine healthy adults completed 4 wk of wrist-hand immobilization of the nondominant limb, and 15 adults served as a control group. A subset of study participants in the immobilization group (n = 14) were also assigned to perform MI training 5 days/wk. Descriptive statistics are provided in Table 1. The Ohio University Institutional Review Board approved this study, and subjects provided written consent. Potential participants were excluded if they were taking any medications or supplements, had any major medical issues, or had any known neurological or musculoskeletal limitations of the upper limbs. The nondominant arm was assessed for isometric muscle strength, VA, and SP duration during a 15% maximal voluntary isometric contraction (MVC) at baseline, 4 wk later (during which the immobilization groups were immobilized), and 5 wk after baseline (1 wk after cast removal and the restoration of normal activity for participants in the immobilization groups). Subjects abstained from alcohol (24 h) and caffeine (4 h) prior to the sessions. Testing sessions were performed at the same time of day for each subject. Individuals involved in assessments were blinded to experimental group assignment. Subjects were not randomly assigned to treatment group per se, but rather were assigned based on whether they were willing to undergo the immobilization procedures as well as the investigators opinion on whether subjects would comply with the imagery training (e.g., feasibility of their schedule availability for permitting them to report to the facilities 5 days/wk for imagery training). Table 1. Descriptive statistics of the study participants Group N (%female) Age, yr Height, cm Weight, kg BMI, kg/m2 Immobilization 15 (46) 21.2 ± 3.5 170.8 ± 10.9 70.1 ± 10.8 24.2 ± 4.2 Immobilization + MI 14 (40) 20.9 ± 3.6 179.4 ± 9.1 78.4 ± 16.1 24.1 ± 3.0 Control 15 (47) 21.5 ± 3.4 170.0 ± 10.2 67.4 ± 13.7 23.3 ± 3.8

Cast immobilization. Subjects in the immobilization groups were fitted with a rigid wrist-hand cast on the nondominant forearm (model 1101–1103, Orthomerica, Orlando, FL), as previously described (Clark et al. 2008, 2010). In brief, lightweight polyethylene casts were applied, which extend from just below the elbow past the fingers (eliminates wrist flexion/extension movements and finger usage). Casts were removed 3–4 times/wk under supervision to wash the arm and inspect for complications. During the recovery period, subjects in the immobilization groups were instructed to return to their normal daily activities, but not begin rehabilitation or a strengthening protocol.

MI training. MI training was performed 5 times/wk. For each session, subjects performed 52 imagined maximal contractions of the casted wrist flexor muscles in a quiet room. The duration of each imagined contraction was 5 s, followed by 5 s of rest. Training was performed in four blocks of 13 imagined contractions each with 1 min of rest between the blocks. During the imagery sessions, subjects were instructed to relax their arm muscles, and to maximally activate the brain, but not the muscles. The electromyogram (EMG) was recorded from the flexor carpi radialis (FCR) muscle to ensure that muscle activation did not occur and real-time feedback was provided. Quantitative analyses of these EMG signals were not performed, as we did not visually observe any voluntary interference EMG activity beyond nominal levels that occasionally occurred during the first session. More specifically, an unblinded scientist supervised these sessions. The imagery script was digitized such that this person did not have to actually read the script. Rather, they were charged with monitoring the EMG recordings in real time on a computer monitor and to provide feedback to the subject if any interference EMG was subjectively noted (i.e., activity is observable above baseline noise with the y-axis scale such that very small increases in activity were noticeable). On a verbal signal to begin, subjects were instructed to “imagine that you are maximally contracting the muscles in your left (or right) forearm and imagine that you are making your wrist flex and push maximally against a hand grip with your hand. We will ask you to do this for 5 s at a time followed by a 5-s rest period for a total time of around 2 min. When we tell you to start, we want you to imagine that you are pushing in against a handgrip as hard as you can and continue to do so until we tell you to stop. After a 5-s rest we will ask you to repeat this. Ready, and begin imagining that you are pushing in as hard as you can with your left wrist, push, push, push… and stop (5 s of silence). Start imagining that you are pushing in again as hard as you can, keep pushing, keep pushing… and stop (5 s of silence).” This verbal cuing and imagery continued for 2 min, at which time the study participant was instructed that they would have a short break (1 min), and then the next blocks would subsequently begin. It should be noted that this mental exercise was not simply a visualization of oneself performing the task; rather, the performers were instructed to adopt a kinesthetic imagery approach, in which they urged the muscles to contract maximally (Ranganathan et al. 2004).

Muscle strength and VA. To quantify wrist flexion forces, subjects were seated with the elbow at 90°, the hand pronated and the forearm supported and restricted while the head rested on a pad (Fig. 1A) (Biodex System 4, Biodex Medical Systems, Shirley, NY). The wrist joint was aligned to the rotational axis of a torque motor to which a constant-length lever arm was attached. The signal was scaled to maximize its resolution (208.7 mV/Nm; Biodex Researchers Tool Kit Software), smoothed over a 10-point running average, and sampled at 625 Hz (MP150 Biopac Systems). Subjects received visual feedback of all exerted forces on a computer monitor located 1 m directly in front of them. Fig. 1.A: setup for assessing wrist flexion strength, voluntary activation (VA), and the cortical silent period (SP). TMS, transcranial magnetic stimulation; EMG, electromyogram. B: immobilization (open circles; n = 15) resulted in a 45% reduction in strength. Mental imagery training (open triangles; n = 14), however, attenuated the loss of muscle strength by ∼50% (strength loss of 24%). No changes were observed in the control group (solid circles; n = 15). Values are means ± SE. Significant differences vs. *baseline, **baseline and recovery, §control group value. Download figureDownload PowerPoint

To assess maximal wrist flexion strength, subjects performed a minimum of three MVCs with a 1- to 2-min rest period between each contraction. If subjects continually recorded more force with increasing trials, or if the two highest trials were not within 5% of each other, additional trials were performed until a plateau was reached. Verbal encouragement was provided during testing. The highest value was considered the MVC. To determine what percentage of the total force-generating capacity of the wrist flexors can be produced voluntarily, a combination of voluntary and electrically stimulated contractions was performed (Fig. 2A). Electrical stimulation (0.2-ms pulse duration) was delivered to the median nerve in the cubital fossa groove via stimulating electrodes (Ag-AgCl, 35 × 45 mm, no. 2015; Nikomed, Doylestown, PA). Stimuli were administered at increasing stimulation intensities until the FCR peak-to-peak (p-p) EMG amplitude reached a plateau (M max ), and for VA testing the intensity was subsequently increased 20% above that eliciting M max (DS7AH; Digitimer, Hertfordshire, UK). To assess VA, a supramaximal 100-Hz electrical doublet was delivered while the subject performed a 4- to 5-s MVC. The increase in force immediately following the stimulation was expressed relative to a potentiated response evoked 1–2 s after the MVC, and VA was calculated as follows. % V A = [ 1 − ( e v o k e d f o r c e d u r i n g M V C / e v o k e d f o r c e f o l l o w i n g M V C ) ] × 100 Fig. 2.A: example of a force trace assessing VA. Arrows represent the delivery of a 100-Hz electrical doublet to the peripheral nerve while an individual is maximally contracting (first arrow) and ∼2 s after the completion of the contraction (second arrow). MVC, maximum voluntary contraction. B: immobilization (open circles; n = 15) reduced VA ∼25%. Mental imagery training (open triangles; n = 14), however, attenuated the impairment in VA by ∼50%. No changes were observed in the control group (solid circles; n = 15). Values are means ± SE. Significant differences vs. *baseline, §control group value, †imagery group value. Download figureDownload PowerPoint



TMS. EMG was recorded from the nondominant FCR muscle using bipolar surface electrodes located longitudinally over the muscle on shaved and abraded skin with a reference electrode just distal to the medial epicondyle (Ag/AgCl electrodes with a 25-mm interelectrode distance). The EMG signals were amplified ×1,000, band-pass filtered (10–500 Hz), and sampled at 5,000 Hz (MP150, BioPac Systems, Goleta, CA). Single-pulse, monophasic waveform magnetic stimuli were delivered using a Magstim 2002 (The Magstim, Whitland, UK) magnetic stimulator with a 70-mm figure-of-eight focal coil positioned tangential to the scalp with the handle pointing backwards and laterally at 45° from midline. The stimulation location that elicited the largest p-p amplitude of the FCR motor-evoked potential (MEP) was identified and marked on a lycra cap for coil placement. This procedure was repeated for each testing session. Next, resting motor threshold (MT) was determined while study participants were seated in the dynamometer by delivering single pulses at gradually increasing stimulation intensities, as our laboratory previously described (Clark et al. 2008; Damron et al. 2008). Resting MT was determined and expressed as a percentage of the maximal stimulator output (SO). MT was determined by delivering TMS pulses at a low stimulus intensity and gradually increasing the intensity in 2% increments until MEPs were observed. Resting MT was defined as the stimulation intensity that elicited MEPs with a p-p amplitude of ≥50 μV in at least four of eight trials. During this assessment, the muscle was completely relaxed as monitored by the EMG signal. We should note that our laboratory has previously reported that the resting MT does not change following immobilization (Clark et al. 2008), which is consistent with what we observed in the present study. SP duration was quantified during brief 15% contractions (Fig. 3A). Here, eight single pulses were delivered at 130% of resting MT, and the SP was quantified and averaged. A single, blinded investigator visually defined the return of the interference EMG signal, and the duration between this TMS pulse and this event was quantified to represent the SP. We have previously reported that this quantification method displays high interrater reliability (r = 0.97) (Damron et al. 2008). Fig. 3.A: the TMS coil induces a magnetic field and a subsequent Eddy current that stimulates neurons within the motor cortex. B: example of an EMG trace illustrating a motor evoked potential (MEP) and SP. In this study, single TMS pulses were delivered to the primary motor cortex during 15% of maximum contraction to quantify the SP duration as an index of GABA B -mediated inhibition. C: immobilization resulted in a 12% prolongation in the SP (n = 15). Mental imagery training (n = 14), however, eliminated prolongation of the SP. No changes were observed in the control group (n = 15). Values are presented as a %change for clarity, but it should be noted that no baseline differences in groups were observed (baseline measures for control group: 108.5 ± 4.3 ms, immobilization group was 107.5 ± 4.4 ms, and immobilization + imagery group was 110.5 ± 4.9 ms). Values are means ± SE. Significant differences vs. *baseline, §immobilization group value. Download figureDownload PowerPoint



Sample size justification. Our sample size was calculated based on our observed effect size (ES) for imagery training to minimize disuse-induced strength loss (η2 = 0.11) (Clark et al. 2006b). The power calculation was based on the assumption of a mixed-model, within-between interaction ANOVA with α at 0.05 and power at 0.95. Based on this calculation our estimated sample size to detect significant changes in strength from preimmobilization to postimmobilization between the immobilization and the immobilization plus imagery groups was 15 subjects/group (G*Power 3.0.3, Universität Kiel, Kiel, Germany). We chose to set power to 0.95 because the success of this project was vitally dependent upon imagery training maintaining strength.