Optimization of the rat digit abduction score (DAS) assay: Evaluation of botulinum neurotoxin activity in the gastrocnemius lateralis, peronei, and extensor digitorum longus

Abstract

The mouse digit abduction score (DAS) assay is commonly used to measure muscle flaccidity-inducing effects of botulinum neurotoxin (BoNT) in vivo. Adapting the assay to rats has been challenging, as injection of onabotulinumtoxinA (onaBoNT-A) into the gastrocnemius muscle, as performed in mice, or into the tibialis anterior leads to sub-optimal sensitivity of the test (Broide et al., 2013). To optimize the experimental design of the rat DAS assay, we evaluated the effects of research-grade, purified, native BoNT serotype A1 (BoNT-A) in three muscles: the gastrocnemius lateralis, peronei, and extensor digitorum longus using female animals. Following injection, animals were tested daily for the digit abduction and body weight. BoNT-A caused dose-dependent inhibition of digit abduction when injected into the gastrocnemius lateralis or peronei. BoNT-A was six-fold more potent when injected into the peronei in comparison to the gastrocnemius lateralis. As injection of BoNT-A into the extensor digitorum longus muscle resulted in an all-or-none digit abduction response and therefore prevented calculation of the ED₅₀, it was considered unsuitable for the rat DAS assay. At equipotent doses, peronei- and extensor digitorum longus-injected animals showed normal body weight gain, while those injected with BoNT-A into the gastrocnemius lateralis gained less weight in comparison to vehicle-treated controls. Thus, injecting the peronei muscles of female rats offers optimized conditions for evaluating the biological properties of BoNTs in the rat DAS assay; for assessing the potency, onset, and duration of action across natural and recombinant BoNT in a robust and reproducible manner.

Keywords: Botulinum neurotoxin type A; Digit abduction score; Extensor digitorum longus muscle; Gastrocnemius muscle; Peronei muscle.

Fig. 1

BoNT-A dose-response in the mean peak digit abduction score (DAS) following injection into the peronei or gastrocnemius lateralis. The curves correspond to mean peak DAS responses observed 1 or 2 days post-administration. All values are means ± standard error of the mean from two independent experiments (n = 6–8 animals dose/experiment).

Fig. 2

The time course of DAS following injection of BoNT-A at the closest dose to ED₅₀ (diamond) and the first dose reaching DAS 4 (square) for the peronei and the gastrocnemius lateralis in comparison to that following injection of vehicle, gelatin phosphate buffer (GPB; circle) in either of the two muscles. All values are means ± standard error of the mean from two independent experiments (n = 6–8 dose/experiment).

Fig. 3

Effects of BoNT-A and vehicle (gelatin phosphate buffer; GPB) on body weight gain in the rat digit abduction score (DAS) assay. The effects were assessed at the half-maximal (DAS 2) and maximal (DAS 4) doses injected into the peronei muscles (A) or the gastrocnemius lateralis (B). All values are means ± standard error of the mean from two independent experiments with 6–8 animals per dose. *p < 0.05, **p < 0.01 and ***p < 0.001 significantly different from the GPB-injected control group by one-way ANOVA followed by Dunnett's post-hoc analysis.