Muscle type-specific effects of bilateral abobotulinumtoxinA injection on muscle growth and contractile function in spastic mice

Abstract

Intramuscular injection of botulinum neurotoxin type A (BoNT-A) is commonly used to improve or maintain the joint range of motion in young children with spasticity. However, the effectiveness of BoNT-A treatment is variable and movement limitations are recurrent. Here we show long-term effects of a single, bilateral abobotulinumtoxinA (aboBoNT-A) injection in the gastrocnemius medialis and soleus muscles of wild-type and spastic (B6.Cg-Glrbspa/J with a mutation in the glycine receptor) mice at a young age (6-7 days). Specifically, we evaluated the impact of aboBoNT-A-A on gait, physical performance, and spontaneous physical behavior, as well as on contractile force characteristics, morphology, and histological phenotype of soleus and gastrocnemius muscles by comparing their results to those of saline-injected controls up to 9 weeks after the injection. The detailed time course of the study specifies the timing of the aboBoNT-A injection at 1 week, the period of behavioral studies from 4-9 weeks, and the age of the mice (10 weeks) at the time of contractile force characteristics and histology assessments. In spastic mice, aboBoNT-A injection had a minor and very specific effect on physical performance, by only modestly increasing stride length as a function of age. aboBoNT-A injection caused a reduction in the force-generating capacity and a slightly smaller physiological cross-sectional area in gastrocnemius medialis, but not in soleus. Reduced physiological cross-sectional area in aboBoNT-A-injected muscles was due to a lower number of muscle fibers, rather than reduced muscle fiber cross-sectional area. The percentage of slow-type muscle fibers and mitochondrial succinate dehydrogenase activity were increased, which was associated with an improved muscle endurance capacity. In conclusion, aboBoNT-A injection reduced the number of muscle fibers, causing muscle hypertrophy in remaining fibers and a shift towards more oxidative fibers, resulting in an improved endurance capacity and gait. This study proposed potential cellular mechanisms for the therapeutic efficacy of aboBoNT-A in spasticity.

Keywords: chemical denervation; gait; muscle endurance; muscle fiber typing; muscle oxidative capacity; physiological cross‐sectional area; plantar flexor muscles; sarcomeres.

FIGURE 1

Effects of aboBoNT‐A on body growth. (A) Body weight of male mice during development. (B) Body weight of female mice during development. Note that for assessment of the number of sarcomeres was used for further assessment, but due to the time‐intensive nature of the muscle contraction measurements study, only one mouse could be measured per day, resulting in a lower number of mice being assessed. (C) Tibia length at 10 weeks of age. Results are reported as mean ± SEM. *Significant differences (p < .05) between saline and treated mice, ^#Significant differences between WT and spastic mice, and ^$Significant differences between male and female mice. AboBoNT‐A and saline‐injected mice had similar body mass, comparable rates of increase of body mass and tibial bone lengths during development.

FIGURE 2

Effects of aboBoNT‐A on motor function and spontaneous behavior. (A) Rotarod performance of male and female mice. (B) Grip strength of front and hind limbs together of male and female mice. (C) Activity time during light of male and female mice. (D) Activity time during dark of male and female mice. (E) Shelter time during dark of male and female mice. (F) Long distances displacement velocity of male and female mice. Results are reported as mean ± SEM. *Significant differences between saline and treated mice, ^#Significant differences (p < .05) between WT and spastic mice, and ^$Significant differences between male and female mice. AboBoNT‐A and saline‐injected mice showed similar motor function and activity levels, with no significant differences in rotarod performances, grip forces, or home cage activity parameters. The neurological scores remained unchanged by aboBoNT‐A injection.

FIGURE 3

Effects of aboBoNT‐A on gait. (A) Stride length of hind limbs of male and female mice. (B) Print length of hind limb of male and female mice. (C) Print width of hind limbs of male and female mice. (D) Contact time of single hind limb of male and female mice. (E) Dual stance time of hind limbs of male and female mice. (F) Step width of front limb of male and female mice. (G) Step width of hind limb of male and female mice. (H) Example of footprints as registered by the Catwalk. Results are reported as mean ± SEM. *Significant differences (p < .05) between saline and treated mice, ^#Significant differences between WT and spastic mice, and ^$Significant differences between male and female mice. AboBoNT‐A‐injected mice showed improved stride length in spastic mice between weeks 4 and 6 and had smaller hind paw step widths at all ages compared to saline‐injected mice. However, both groups exhibited similar increases in step width from week 4 to week 6.

FIGURE 4

Effects of aboBoNT‐A on muscle length‐force relationship, maximal rate of force development (MRFD) and force‐frequency relationship as well as determinants of muscle (−tendon complex) length (l_m and l_mtc) and muscle force (F_m and F_mtc). (A) Soleus (SO) relative length‐force relationship (l_m rel to l_o) of male mice. (B) Soleus relative length‐force relationship of female mice. (C) Gastrocnemius medialis (GM) relative length‐force relationship of male mice. (D) Gastrocnemius medialis relative length‐force relationship of female mice. (E) Absolute rate of maximal force development of soleus muscle (SO). (F) Rate of maximal force development of soleus normalized for maximal force. (G) Absolute rate of maximal force development of gastrocnemius medialis muscle (GM), (H) Rate of maximal force development of gastrocnemius medialis, (I) Force‐frequency relationship of soleus muscle, (J) Force‐frequency relationship of gastrocnemius medialis muscle. (K) Soleus muscle volume. (L) Gastrocnemius medialis muscle volume. (M) Physiological cross‐sectional area (PCSA) of soleus. (N) Physiological cross‐sectional area of gastrocnemius medialis. (O) Number of muscle fibers within a soleus cross‐section area. (P) Number of muscle fibers within a gastrocnemius medialis cross‐section area. Results are reported as mean ± SEM. *Significant differences (p < .05) between saline and iterated mice, ^#Significant differences between WT and spastic mice, and ^$Significant differences between male and female mice. In spastic mice, aboBoNT‐A injections led to lower optimal muscle force in both soleus and the gastrocnemius medialis muscles. The force‐frequency relationships and optimum muscle lengths were unaffected, but the volumes of the injected SO and gastrocnemius medialis were lower, and muscle fiber numbers were significantly reduced in both soleus and gastrocnemius medialis.

FIGURE 5

Effects of aboBoNT‐A on passive muscle force determinants. (A, B) Typical example of a cross‐section stained with sirius red of saline and an aboBoNT‐A‐injected muscle, connective tissue within a cross‐section is stained red, magnifications show different areas within cross‐sections. (C) Soleus (SO) muscle fiber cross‐sectional area (fCSA) per muscle fiber type. (D) Gastrocnemius medialis (GM) muscle fiber cross‐sectional area per muscle fiber type in the low oxidative muscle region (E) and high oxidative muscle region. (F) Soleus primary perimysium length (lp1). (G) secondary perimysium length (lp2). (H) Soleus tertiary perimysium length (lp3). (I) Soleus percentage of endomysium area per fiber area. (J) Gastrocnemius medialis primary perimysium length. (K) Gastrocnemius medialis secondary perimysium length. (L) Gastrocnemius medialis tertiary perimysium length. (M) Gastrocnemius medialis percentage of endomysium area per fiber area. Results are reported as mean ± SEM. *Significant differences (p < .05) between saline and treated mice, ^#Significant differences between WT and spastic mice, and ^$Significant differences between male and female mice. AboBoNT‐A injection resulted in larger mean muscle fiber cross‐sectional areas in the soleus compared to saline, and in larger cross‐sectional areas for type IIB fibers in the low‐oxidative region of the gastrocnemius medialis. Additionally, in the high‐oxidative region of the gastrocnemius medialis, type I, IIA, IIX, and IIB fibers were significantly larger in aboBoNT‐A‐injected muscles compared to saline‐injected muscles.

FIGURE 6

Effects of aboBoNT‐A on endurance capacity, as well as endurance determinants. (A) Endurance curve of soleus (SO). (B) Percentage of decreased muscle force at the end of the fatigue protocol of soleus. (C) Endurance curve of gastrocnemius medialis (GM). (D) Percentage of decreased muscle force at the end of the fatigue protocol of gastrocnemius medialis. (E–G) Typical example of muscle fiber type matching with SDH stained cross‐section of a saline‐injected muscle. (H–J) Typical example of muscle fiber type matching with SDH stained cross‐section of an aboBoNT‐A‐injected muscle. (K) Soleus MHC distribution of male mice. (L) Soleus MHC distribution of female mice. (M) Gastrocnemius medialis MHC distribution in the high oxidative muscle region. (N) Gastrocnemius medialis MHC distribution in the low oxidative muscle region. (O) SDH activity of soleus. (P) SDH activity in the high oxidative muscle region of gastrocnemius medialis muscle. (Q) SDH activity in the low oxidative muscle region of gastrocnemius medialis muscle. Results are reported as mean ± SEM. *Significant differences (p < .05) between saline and treated mice, ^#Significant differences between WT and spastic mice, and ^$Significant differences between male and female mice. Fmax: Maximal muscle force at optimum muscle length. AboBoNT‐A‐injected gastrocnemius medialis muscles exhibited improved fatigue resistance with lower force reductions after 4 min of stimulation compared to saline‐injected muscles. Additionally, aboBoNT‐A injection caused a shift towards slower muscle fiber types in both the soleus and the high oxidative region of the gastrocnemius medialis, with increased percentages of type I fibers and decreased percentages of type IIA, IIX, and IIB fibers.”

FIGURE 7

Effects of aboBoNT‐A on myotrophic determinants. (A) Number of satellite cells (SC) per cross‐section in soleus (SO). (B) Number of satellite cells per cross‐section (10 μm thick section) in soleus. (C) Number of satellite cells per cross‐section area in gastrocnemius medialis (GM). (D) Number of satellite cells per muscle fiber cross‐section (10 μm thick section) in gastrocnemius medialis. (E) Number of myonuclei per 1 mm of muscle fiber in soleus. 45 (F) Number of myonuclei per 1 mm of muscle fiber in the low oxidative muscle region of the gastrocnemius medialis. (G) Number of myonuclei per 1 mm of muscle fiber in the high oxidative muscle region of the gastrocnemius medialis. (H) Myonuclear domain in soleus. (I) Myonuclear domain in the low oxidative muscle region of the gastrocnemius medialis. (J) Myonuclear domain in the high oxidative muscle region of the gastrocnemius medialis. Results are reported as mean ± SEM. “*” indicates significant differences (p < .05) between saline and treated mice, “#” indicates significant differences between WT and spastic mice, and “$” indicates significant differences between male and female mice. In both soleus and gastrocnemius medialis muscles, the numbers of satellite cells per muscle cross‐section and per muscle fiber were similar between saline and aboBoNT‐A‐injected groups. However, in the low oxidative region of the gastrocnemius medialis, aboBoNT‐A injection resulted in a higher number of myonuclei per 1 mm of fiber length compared to saline injection, while myonuclear domains were similar across all conditions. A typical example of myonuclei and satellite cells (indicated by white arrows) in saline and aboBoNT‐A‐injected muscles can be found in this figure.