Pulsed focused ultrasound treatment of muscle mitigates paralysis-induced bone loss in the adjacent bone: a study in a mouse model

Abstract

Bone loss can result from bed rest, space flight, spinal cord injury or age-related hormonal changes. Current bone loss mitigation techniques include pharmaceutical interventions, exercise, pulsed ultrasound targeted to bone and whole body vibration. In this study, we attempted to mitigate paralysis-induced bone loss by applying focused ultrasound to the midbelly of a paralyzed muscle. We employed a mouse model of disuse that uses onabotulinumtoxinA-induced paralysis, which causes rapid bone loss in 5 d. A focused 2 MHz transducer applied pulsed exposures with pulse repetition frequency mimicking that of motor neuron firing during walking (80 Hz), standing (20 Hz), or the standard pulsed ultrasound frequency used in fracture healing (1 kHz). Exposures were applied daily to calf muscle for 4 consecutive d. Trabecular bone changes were characterized using micro-computed tomography. Our results indicated that application of certain focused pulsed ultrasound parameters was able to mitigate some of the paralysis-induced bone loss.

Keywords: Micro-computed tomography; Mitigation of trabecular bone loss; Mouse model of disuse; Musculoskeletal; Paralysis; Pulsed focused ultrasound.

Fig. 1

(a) Experimental setup for pulsed focused ultrasound (pFUS) treatment of the mouse calf muscle, as described in the text. Treatments were performed in a heated water bath to ensure good acoustic coupling, (b) The leg is rotated 90°, and the positioning of the ultrasound focus is indicated. The long axis of the transducer focus lies completely within the muscle midbelly without approaching the tibia. The region of micro-computed tomography analysis is noted in the proximal tibia.

Fig. 2

Ultrasound fields produced by the 2 MHz transducer, as measured by the fiberoptic probe hydrophone in water. (a) Non-linearly distorted ultrasound pulses, measured at the focus, used in the exposures with 20 and 80 Hz pulse repetition frequencies (top), and detailed view of the highest amplitude section of the pulse produced with “High” (thin line) and “Low” (thick line) power output settings (bottom). Dashed arrows mark the shock front that forms in the case of the “High” setting. (b) Lower amplitude, linear pulses used in the exposures with 1 kHz pulse repetition frequency (top), and detailed view of the highest amplitude section of the pulse (bottom). (c, d) Peak pressure distributions in the focal region across (c) and along (d) the transducer axis, in linear (thick line) and non-linear regimes (thin line for peak positive pressure and dotted line for peak negative pressure). The vertical lines mark the relative position of the tibia (c) and the skin-water boundaries (d). As seen, the focus was placed so that the calf would be affected directly by the main lobe. The second side lobe was incident on the tibia, whereas the first side lobe could potentially affect it in some cases because of small inaccuracies in positioning of the limb (±0.25 mm). However, the side lobes were unlikely to produce any bioeffect inside the bone because of the very short pulse duration and low amplitude. The maximum peak negative pressure at the bone surface (High80 exposure) was 20% of the peak negative pressure in the main lobe, or 1.8 MPa (corresponding mechanical index = 1.2), and the maximum peak positive pressure was 2.1 MPa.

Fig. 3

Passive cavitation detection (PCD) in ex vivo murine calf muscles (n = 6) was performed during each pulsed focus ultrasound (pFUS) exposure listed in Table 1. (a) Measurement arrangement: The 5 MHz PCD transducer was positioned confocally with the pFUS transducer. (b) Cavitation occurrence, as measured by the level of broadband noise emissions and the amplitude of ultraharmonics, was not observed in any of the pFUS exposures, even at the highest focal pressure level (High80). To induce measurable cavitation activity, the pulse duration had to be increased from 5.3 µs up to 23–46 µs at the High80 setting. Examples are provided of the spectral amplitude of PCD signals recorded during a 53-µs pulse (black line) and a 46-µs pulse (gray line), in which the presence of broadband noise and ultraharmonics is clear.

Fig. 4

Trabecular bone loss in mouse proximal tibia 5 d after onabotulinumtoxinA (Obtx)-induced paralysis of the calf muscle and pulsed focused ultrasound (pFUS) treatment of the muscle, evaluated with ex vivo micro-computed tomography. The results are expressed as percentage change relative to the contralateral, non-experimental limb. (a) BV/TV (bone volume/total volume) = Trabecular bone volume: higher loss indicates bone degradation. (b) BS/BV = specific bone surface: higher change indicates bone loss. (c) Tb.Th = trabecular thickness: higher loss indicates bone degradation; (d) Tb.N = trabecular number: higher loss indicates bone degradation. (e) Tb.Pf = trabecular pattern factor: higher change indicates bone loss. Treatment group abbreviations are described in Table 1. Animals per group: Obtx sham, n = 7; Saline sham, n = 6; Low20, n = 6, High20, n = 6; Low80, n = 7; High80, n = 5; Low1k, n = 5; High1k, n = 6. Data are presented as averages ± standard errors. *Significant difference from the Obtx sham group at p < 0.05. ^†Significant difference from the Saline sham group at p < 0.05.

Fig. 5

Example images of 3D volume renderings of micro-computed tomography data reveal the proximal tibia with the trabecular bone extracted in both the experimental (right) and contralateral (left) limbs. Top: Animal injected with ona-botulinumtoxinA (Obtx) and treated with pulsed focused ultrasound (pFUS) using the Low20 treatment protocol. Bottom: Animal from the Obtx sham group. This image qualitatively indicates the ability of pFUS treatment of the paralyzed muscle to mitigate paralysis-induced trabecular bone loss (Obtx + Low20 pFUS). Mean values for the different parameters characterizing bone loss for all experimental and sham groups are given in Figure 4.

Fig. 6

Representative histologic images of formalin-fixed calf muscle stained with H&E. Images reveal longitudinal muscle fibers (pink) with nuclei (purple). No damage was detected in the (a) Obtx sham, (b) Low20-treated, (c) High80-treated, or Saline sham (image not shown) limb compared with (d) an uninjected contralateral limb. There was some evidence of localized mild perifascicular inflammation in many of the injected tissue muscles, whether sham treated or ultrasound treated. Examples of inflammation {arrowhead) in Obtx sham (e) and High80-treated (f) samples are presented. Bar = 100 µm (a–d) and 40 µm (e–f).