Combined effects of botulinum toxin injection and hind limb unloading on bone and muscle

Abstract

Bone receives mechanical stimulation from two primary sources, muscle contractions and external gravitational loading; but the relative contribution of each source to skeletal health is not fully understood. Understanding the most effective loading for maintaining bone health has important clinical implications for prescribing physical activity for the treatment or prevention of osteoporosis. Therefore, we investigated the relative effects of muscle paralysis and reduced gravitational loading on changes in muscle mass, bone mineral density, and microarchitecture. Adult female C57Bl/6J mice (n = 10/group) underwent one of the following: unilateral botulinum toxin (BTX) injection of the hind limb, hind limb unloading (HLU), both unilateral BTX injection and HLU, or no intervention. BTX and HLU each led to significant muscle and bone loss. The effect of BTX was diminished when combined with HLU, though generally the leg that received the combined intervention (HLU+BTX) had the most detrimental changes in bone and muscle. We found an indirect effect of BTX affecting the uninjected (contralateral) leg that led to significant decreases in bone mineral density and deficits in muscle mass and bone architecture relative to the untreated controls; the magnitude of this indirect BTX effect was comparable to the direct effect of BTX treatment and HLU. Thus, while it was difficult to definitively conclude whether muscle force or external gravitational loading contributes more to bone maintenance, it appears that BTX-induced muscle paralysis is more detrimental to muscle and bone than HLU.

Fig. 1

Schematic of experimental design of four groups: CON, untreated and cage dwelling; HLU, untreated and hindlimb unloaded; CON+BTX, injected with BTX in left leg and cage dwelling; HLU+BTX, injected with BTX in left leg and hindlimb unloaded.

Fig. 2

Body mass changes following injection of BTX on day −3 and initiation of HLU on day 0 (mean ± SE) *Difference between day 21 and initial measurement On a given day: ^adifference between CON and CON+BTX, ^bdifference between HLU and HLU+BTX, ^cdifference between CON and HLU, ^ddifference between CON+BTX and HLU+BTX

Fig. 3

Scores on a) digit abduction and b) wire hang assessments of muscle paralysis (mean ± SD) ^*Difference between groups by day

Fig. 4

Effect of BTX and HLU on a) soleus and b) gastrocnemius muscle mass (mean ± SD) Brackets indicate significant differences (p<0.05) from respective control group *p<0.05, BTX-injected vs. uninjected leg within loading group

Fig. 5

Effect of BTX and HLU on hindlimb BMD (percent change from baseline, mean ± SD) Brackets indicate significant differences (p<0.05) from respective control group * p<0.05, BTX-injected vs. uninjected leg within loading group

Fig. 6

Effect of BTX and HLU on trabecular bone volume fraction of the a) proximal tibia and b) distal femur (mean ± SD) Brackets indicate significant differences (p<0.05) from respective control group * p<0.05, BTX-injected vs. uninjected leg within loading group

Fig. 7

Relative effect of unloading, direct BTX treatment, and indirect BTX treatment on a) hindlimb bone mineral density and b) proximal tibia trabecular bone volume fraction. The magnitude of the effect of unloading (black bars) was calculated as the difference between average values of HLU and CON groups. The direct effect of BTX alone (shaded bars) and BTX combined with unloading (unshaded bars) was calculated as the difference between the injected and contralateral limbs of the CON+BTX and HLU+BTX groups, respectively. The indirect effect of BTX alone and combined with unloading was calculated as the difference between the contralateral leg of the CON+BTX or HLU+BTX group and the untreated CON or HLU group, respectively