Neuromuscular dysfunction, independent of gait dysfunction, modulates trabecular bone homeostasis in mice

Abstract

Objectives: To clarify the effects of neuromuscular dysfunction on hindlimb loading, muscle atrophy, and bone homeostasis.

Methods: We quantified changes to hindlimb loading, muscle atrophy, and bone morphology following either Botulinum toxin A (BTxA) induced muscle paralysis or peripheral nerve injury (PNI) in mice; two in vivo models that we anticipated would differently alter gait and mechanical loading patterns due to their distinct effects on neuromuscular signaling. To confirm the expected behavioral effects of PNI, we assessed mechanical allodynia of the ipsilateral hindlimb using von Frey testing and activity (distance traveled and speed) was monitored in both groups using open field testing. Peak vertical ground reaction forces (GRF) and ankle and knee kinematics during normal locomotion were quantified and used to estimate peak mid-diaphyseal normal strains. Muscle atrophy and trabecular and cortical bone morphology were assessed via high-resolution microCT imaging.

Results: BTxA-induced calf paralysis caused severe muscle atrophy and altered gait kinetics and kinematics and reduced gait-induced normal strains. PNI increased mechanical allodynia but did not alter gait, nor did it cause muscle atrophy. We observed that muscle paralysis and PNI both led to severe trabecular bone loss but only BTxA-induced paralysis increased cortical bone resorption.

Conclusions: While mechanical stimuli clearly have essential functions in bone development and adaptation, these data emphasize that neuromuscular signaling, independent of load-induced mechanical strains, may modulate trabecular bone homeostasis in normal and disease states.

Figure 1

Mechanical allodynia induced by PNI. Mechanical allodynia reached significance (represented by decreased withdrawal threshold) by d12 and was sustained through d 28. No changes in mechanical allodynia were observed in the contralateral limb (*, p<0.01 vs d 0; ^, p <0.01 vs PNI).

Figure 2

Locomotor activity following BTxA or PNI. The total distance traveled during baseline habituation (d0) was higher in the PNI group vs d 5, d 12, and d 28 (A; (*, p<0.05 vs d 0). There were no significant differences in the BTxA treatment or between groups. There were no differences in average speed at any time point (B).

Figure 3

Hind limb ground reaction forces during free ambulation. GRF deficits were observed only following calf paralysis and were confined to the ipsilateral limb (A; *, p<0.01 vs d 0; ^, p <0.01 vs PNI). Contralateral peak vertical GRFs during free locomotion were not altered by either intervention (B).

Figure 4

Ankle and knee kinematics during free ambulation. Compared to d 0, calf paralysis decreased ankle flexion at d 5 (A) and increased knee extension at d 5 and d12 during free locomotion (B). Ankle and knee kinematics were not altered vs d 0 in PNI mice (*, p<0.01 vs d 0; ^, p<0.01 vs PNI; #, p<0.05 vs PNI).

Figure 5

Tibia mid-shaft normal strains at time of peak GRF during free ambulation. Following calf paralysis, peak tibia mid-shaft normal strain was rapidly diminished compared to d 0, but not altered by PNI (*, p<0.01 vs d 0; ^, p <0.01 vs PNI).

Figure 6

Muscle volume alterations due to BTxA induced paralysis and PNI. Lower limb musculature demonstrated profound atrophy due to calf paralysis (A). Muscle volume was significantly diminished within d 5 of calf paralysis and continued to atrophy through d 28. PNI did not alter muscle volume at any time point (*, p<0.01 vs d 0; ^, p <0.01 vs PNI).

Figure 7

Proximal tibia trabecular bone alterations due to BTxA induced paralysis and PNI. Both calf paralysis and PNI diminished metaphyseal trabecular bone (A, red). Trabecular BV/TV of the proximal tibia metaphysis was significantly diminished by calf paralysis by d 5, while PNI induced bone loss was significant by d 12 (*, p<0.01 vs d 0).

Figure 8

Tibia mid-shaft cortical bone alterations due to BTxA induced paralysis and PNI. Neither intervention altered the tibia mid-shaft P.Vol (A). Calf paralysis resulted in a significant increase in EC.Vol by d 12 that reached 12% by d 28. PNI did not induce significant EC expansion at any time point (*, p<0.01 vs d 0, ^, p <0.01 vs PNI).

Figure 9

Regression of gait induced normal strain and lower limb muscle volume vs trabecular BV/TV across time points. While normal strain was significantly correlated proximal tibia BV/TV, it only predicted a small portion of the variance of the data (7%; A). In contrast, altered muscle volume predicted 51% of the variability of the BV/TV data (B).