Acrylamide inhibits nerve sprouting induced by botulinum toxin type A

Abstract

Botulinum toxin type A is a potent muscle relaxant that blocks the transmission and release of acetylcholine at the neuromuscular junction. Intramuscular injection of botulinum toxin type A has served as an effective and safe therapy for strabismus and focal dystonia. However, muscular weakness is temporary and after 3-4 months, muscle strength usually recovers because functional recovery is mediated by nerve sprouting and reconstruction of the neuromuscular junction. Acrylamide may produce neurotoxic substances that cause retrograde necrotizing neuropathy and inhibit nerve sprouting caused by botulinum toxin type A. This study investigated whether acrylamide inhibits nerve sprouting after intramuscular injection of botulinum toxin type A. A tibial nerve sprouting model was established through local injection of botulinum toxin type A into the right gastrocnemius muscle of Sprague-Dawley rats. Following intramuscular injection, rats were given intraperitoneal injection of 3% acrylamide every 3 days for 21 days. Nerve sprouting appeared 2 weeks after intramuscular injection of botulinum toxin type A and single-fiber electromyography revealed abnormal conduction at the neuromuscular junction 1 week after intramuscular injection of botulinum toxin type A. Following intraperitoneal injection of acrylamide, the peak muscle fiber density decreased. Electromyography jitter value were restored to normal levels 6 weeks after injection. This indicates that the maximal decrease in fiber density and the time at which functional conduction of neuromuscular junction was restored were delayed. Additionally, the increase in tibial nerve fibers was reduced. Acrylamide inhibits nerve sprouting caused by botulinum toxin type A and may be used to prolong the clinical dosage of botulinum toxin type A.

Keywords: acrylamide; action potential mean consecutive difference; botulinum toxin type A; dysmyotonia; electromyography; fiber density; nerve fibers; nerve regeneration; nerve sprouting; neural regeneration; neuromuscular junction; peripheral nerve regeneration; single-fiber EMG.

Figure 1

Single-fiber action potential of gastrocnemius muscle of rats during nerve sprouting. (A) Fiber density in acrylamide group; (B–D) fiber density in botulinum toxin type A group at 2, 3 and 4 weeks after injection with botulinum toxin type A. Fiber density was increased and mean consecutive difference was significantly prolonged, suggesting neuromuscular blockage occurred during nerve terminal sprouting. Electrical stimulation parameters: 4.0 mA, 10 Hz, 0.1 ms, 1 ms/D, 200 μV/D.

Figure 2

Fiber density of gastrocnemius muscle of rats during nerve sprouting. *P < 0.05, vs. normal group; #P < 0.05, vs. BTX-A group. Data are expressed as mean ± SD of five rats for each group (one-way analysis of variance and the least significant difference test). BTX-A: Botulinum toxin type A; Acr: acrylamide; FD: fiber density.

Figure 3

Changes of mean consecutive difference (MCD) of rat gastrocnemius muscle action potentials during nerve sprouting process. *P < 0.05, vs. normal group; #P < 0.05, vs. BTX-A group. Data are expressed as mean ± SD of five rats for each group (one-way analysis of variance and the least significant difference test). BTX-A: Botulinum toxin type A; Acr: acrylamide.

Figure 4

Nerve sprouting after intramuscular injection of botulinum toxin type A in the right side of rat gastrocnemius muscles (Gros-Bielschowsky silver staining, × 200). (A) At 2–3 weeks post-injection, several short and thin sprouting nerves grew from the terminal of tibial nerve. (B) At 4 weeks post-injection, the sprouts were longer and extended into adjacent muscle. (C) The normal control group showed no nerve fiber spouting. Sprouts are indicated by arrows.

Figure 5

Effect of botulinum toxin type A (BTX-A) and acrylamide (Acr) on tibial nerve fibers count in rats. *P < 0.05, vs. normal group; #P < 0.05, vs. BTX-A group. Data are expressed as mean ± SD of five rats for each group (one-way analysis of variance and the least significant difference test).

Figure 6

Effect of botulinum toxin type A (BTX-A) plus acrylamide (Acr) on muscle strength of rats. *P < 0.05, vs. normal group; #P < 0.05, vs. BTX-A group. Data are expressed as mean ± SD of five rats for each group (one-way analysis of variance and the least significant difference test). Muscle power: 0 is normal, 4 is the worst; the lower the score is, the poorer the muscle power is.

Figure 7

The wet weight of right gastrocnemius muscle of rats after BTX-A injection. *P < 0.05, vs. normal group. Data are expressed as mean ± SD in five rats for each group (one-way analysis of variance and the least significant difference test). BTX-A: Botulinum toxin type A; Acr: acrylamide.