Dramatic changes in muscle contractile and structural properties after 2 botulinum toxin injections

Abstract

Introduction: Botulinum toxin is frequently administered serially to maintain therapeutic muscle paralysis, but the effect of repeated doses on muscle function are largely unknown. This study characterized the muscle response to 2 onabotulinum toxin (BoNT) injections separated by 3 months.

Methods: Animal subjects received a single toxin injection (n = 8), 2 BoNT injections separated by 3 months (n = 14), or 1 BoNT and 1 saline injection separated by 3 months (n = 8).

Results: The functional effect of 2 serial injections was exponentially greater than the effect of a single injection. While both groups treated with a single BoNT injection had decreased torque in the injected leg by approximately 50% relative to contralateral legs, the double BoNT injected group had decreased torque by over 95% relative to the preinjection level. Both single and double BoNT injections produced clear signs of fiber-type grouping.

Conclusions: These experiments demonstrate a disproportionately greater effect of repeated BoNT injections.

Keywords: Botox®; denervation; fiber type; muscle physiology; neurotoxin.

Figure 1

Schematic diagram of experimental procedures. Animal subjects were divided randomly into 3 groups: a single toxin injection (n=8), 2 botulinum toxin injections separated by 3 months (n= 14), or 1 toxin and 1 saline injection separated by 3 months (n=8). The latter group was used to insure that a time and age-matched comparison could be made to the BoNT+BoNT group.

Figure 2

(A) Torque measured from injected and contralateral hindlimbs. Note a reduction in torque in all BoNT injected limbs compared to contralateral uninjected muscle (P<0.00001). In the injected limbs, torque was reduced significantly in the double BoNT injected group compared to other groups (P<0.0001). (B) Average muscle fiber CSA measured in injected BoNT hindlimb and contralateral uninjected side. There was a significant decrease in fiber CSA in double BoNT injected muscle compared to single injected muscle, in BoNT followed by saline injected muscle (P<0.0001), and in all injected muscles when compared to contralateral uninjected muscles (P ≤ 0.05). (C) Percentage of lipid accumulation area in injected and contralateral uninjected TA muscles. Note a higher percentage of lipid accumulation in double BoNT injected muscles compared to contralateral (P<0.0001). (D) Collagen content of the experimental groups measured by hydroxyproline content. Collagen content increases with both double injection groups. This appears to be more of an effect of age than neurotoxin injection, since the second saline injection elicited the same response.

Figure 3

TA cross-section from a BoNT injected muscle. (A) Traditional hematoxyln and eosin reflects general morphology. (B) Oil Red O staining reveals red droplets that represent fat accumulation, which was not observed in any control muscles.

Figure 4

Histogram of percentage of type 1 fibers surrounded by type 2 fibers. Note that in control skeletal muscles, 85±1% of type 1 fibers were completely surrounded by type 2 fibers (i.e., number of fibers = 1) while in all 3 of the BoNT groups, less than 70% of type 1 fibers were completely surrounded by type 2 fibers (P<0.001). Data are shown only for “groups” up to 6 fibers, which was the limit of control muscles. Several BoNT injected muscles contained fiber groups of over 20 fibers.

Figure 5

Representative immunohistochemical images from (A) contralateral control of BoNT +Saline injected, (B) BoNT injected, (C) BoNT + BoNT, or (D) BoNT +Saline groups. All sections were labeled with a primary antibody against type 1 myosin heavy chain. Fiber type grouping was observed in all BoNT injected muscles, with groups ranging from 16–24 fibers. Scale bar: 100μm.