Robot-assisted mechanical therapy attenuates stroke-induced limb skeletal muscle injury

Abstract

The efficacy and optimization of poststroke physical therapy paradigms is challenged in part by a lack of objective tools available to researchers for systematic preclinical testing. This work represents a maiden effort to develop a robot-assisted mechanical therapy (RAMT) device to objectively address the significance of mechanical physiotherapy on poststroke outcomes. Wistar rats were subjected to right hemisphere middle-cerebral artery occlusion and reperfusion. After 24 h, rats were split into control (RAMT^-) or RAMT⁺ groups (30 min daily RAMT over the stroke-affected gastrocnemius) and were followed up to poststroke d 14. RAMT⁺ increased perfusion 1.5-fold in stroke-affected gastrocnemius as compared to RAMT^- controls. Furthermore, RAMT⁺ rats demonstrated improved poststroke track width (11% wider), stride length (21% longer), and travel distance (61% greater), as objectively measured using software-automated testing platforms. Stroke injury acutely increased myostatin (3-fold) and lowered brain-derived neurotrophic factor (BDNF) expression (0.6-fold) in the stroke-affected gastrocnemius, as compared to the contralateral one. RAMT attenuated the stroke-induced increase in myostatin and increased BDNF expression in skeletal muscle. Additional RAMT-sensitive myokine targets in skeletal muscle (IL-1ra and IP-10/CXCL10) were identified from a cytokine array. Taken together, outcomes suggest stroke acutely influences signal transduction in hindlimb skeletal muscle. Regimens based on mechanical therapy have the clear potential to protect hindlimb function from such adverse influence.-Sen, C. K., Khanna, S., Harris, H., Stewart, R., Balch, M., Heigel, M., Teplitsky, S., Gnyawali, S., Rink, C. Robot-assisted mechanical therapy attenuates stroke-induced limb skeletal muscle injury.

Keywords: cerebrovascular accident; ischemia; physical therapy; rehabilitation.

Figure 1.

Robot-assisted mechanical therapy (RAMT) hardware. A) The RAMT apparatus is controlled by a customized version of software fabricated to our design (1) and connected to a controller (2). A pair of servo amplifiers (3) drive the x- and y-axis motorized linear stages (5, 6). A servo amplifier integrated to the controller drives the z-axis voicecoil stage (7). The controller monitors (4) stage and contact head (9) positions using optical sensors (not shown) mounted on each of the stages. A load cell (8) mounted on the z-axis stage (7) provides the force feedback signal used to monitor and control the load applied by the stage. The bottom stage is mounted on rails (11) that allow the stack to be positioned on either side of a breadboard-type base plate (10). B) The customized software platform enables programmable control of RAMT load (force), pattern (linear, orbital, vibratory), and duration. The RAMT paradigm employed for the current study was 30 min linear motion (10 mm) at a frequency of 1 Hz and a constant load of 0.5 N over the stroke-affected hindlimb. C, D) The RAMT hardware was conceptualized and prototyped using computer-aided design (C) before delivery of final hardware (D).

Figure 2.

RAMT improves perfusion in stroke-affected gastrocnemius muscle. A) Perfusion maps covering the stroke-affected and contralateral saphenous artery were acquired immediately after 30 min of RAMT on poststroke d 7 and 14 (poststroke d 14 shown). Top: rat in supine position with a software-defined bounding box covering the stroke-affected gastrocnemius. Perfusion (relative perfusion units, PU) was quantified from a software-defined region of interest (10 × 1.25 mm) placed over the RAMT site that is inclusive of the femoral artery that supplies the gastrocnemius muscle. B) Mean PU in stroke-affected hindlimb of RAMT⁺ rats was significantly higher than that of the pair-matched contralateral hindlimb on poststroke d 14. Furthermore, mean PU was significantly higher in stroke-affected hindlimb of RAMT⁺ rats as compared with stroke-affected hindlimb of RAMT⁻ rats on poststroke d 14. Data are means ± sd (n = 4). Black bars, contralateral limbs; white bars, stroke-affected limbs. *P < 0.05, RAMT⁺ stroke affected vs. contralateral; ^†P < 0.05, RAMT⁺ stroke affected vs. RAMT⁻ stroke affected.

Figure 3.

RAMT improves poststroke gait function. A) Representative still frames matched for stride position in control (RAMT⁻) rat at baseline and on poststroke d 7 demonstrate hunched posture (1), reduced rear track width (2), and shorter stride length (3). B) Compared to RAMT⁻ controls (black bars), RAMT⁺ rats (white bars) had significantly increased track width, spent less time in quad support, and had a longer stride at poststroke d 7. Data are means ± sd (n = 7). *P < 0.05 vs. baseline within group; ^†P < 0.05 vs. RAMT⁻ at the same timepoint.

Figure 4.

RAMT improves poststroke sensorimotor function. A) Representative screen capture of baseline RAMT⁻ (control) rat with software overlay tracking head (green dot), middle body (orange dot), tail (yellow dot), and motion vector (orange line) in real time. Software was used to divide the open field into 4 quadrants (orange boxes Q1–Q4) for tracking the amount of time spent in each zone during the test. B) Average heat map of RAMT⁻ and RAMT⁺ rats during field test on poststroke d 14. A video tracking system calculated mean time spent in each zone (RAMT⁻, black bars; RAMT⁺, white bars). C) Compared to RAMT⁻ rats, RAMT⁺ rats traveled greater distance and spent more time mobile and less time freezing on poststroke d 14. Data are means ± sd (n = 7). *P < 0.05 vs. RAMT⁻ at same timepoint.

Figure 5.

Poststroke myostatin expression is down-regulated in stroke-affected gastrocnemius muscle after RAMT treatment. A) Myostatin mRNA expression in stroke-affected gastrocnemius muscle was significantly lower in RAMT⁺ vs. RAMT⁻ rats on poststroke d 14. *P < 0.05 vs. RAMT⁻. B) Representative myostatin (red) in contralateral and stroke-affected gastrocnemius muscle on poststroke d 14. Sections counterstained with DAPI (blue). Scale bar, 50 μm. C) Quantification (% area) of myostatin expression in contralateral and stroke-affected gastrocnemius muscle of RAMT⁻ (black bars) and RAMT⁺ (white bars) rats. Data are means ± sd (n = 7). *P < 0.05 vs. contralateral within group; ^†P < 0.05 vs. stroke-affected RAMT⁻.

Figure 6.

RAMT protects against stroke-induced loss of BDNF in skeletal muscle. A) BDNF mRNA expression in stroke-affected gastrocnemius muscle was significantly higher in RAMT⁺ vs. RAMT⁻ rats on poststroke d 14. *P < 0.05 vs. RAMT⁻. B) BDNF (green) immunostaining in contralateral and stroke-affected gastrocnemius muscle on poststroke d 14. Sections counterstained with DAPI (blue). Scale bar, 50 μm. C) Quantification (% area) of BDNF expression in contralateral and stroke-affected gastrocnemius muscle of RAMT⁻ (black bars) and RAMT⁺ (white bars) rats. Data are means ± sd (n = 7). *P < 0.05 vs. contralateral within group; ^†P < 0. 05 vs. stroke-affected RAMT⁻.

Figure 7.

Cytokine profile of stroke-affected skeletal muscle in response to RAMT. A) Images of chemiluminescent intensity from the cytokine array. Rat cytokine array coordinates indicate the position of cytokines measured in Table 1. Optical density of each signal was measured by ImageJ, and values were calculated based on the signal intensity of the positive controls from the same membrane. B, C) Immunohistology was used to validate targets with the greatest fold-change up (IL-1ra, B) and down (IP-10/CXCL10, C). Immunohistology was quantified as % area in contralateral and stroke-affected gastrocnemius muscle of RAMT⁻ (black bars) and RAMT⁺ (white bars) rats. Data are means ± sd (n = 4). *P < 0.05 vs. contralateral within group, ^†P < 0.05 vs. stroke-affected RAMT⁻.