Modulating neuromuscular junction density changes in botulinum toxin-treated orbicularis oculi muscle

Abstract

Purpose: Botulinum toxin A is the most commonly used treatment for blepharospasm, hemifacial spasm, and other focal dystonias. Its main drawback is its relatively short duration of effect. The goal of this study was to examine the ability of corticotropin releasing factor (CRF) or antibody to insulin growth factor I-receptor (anti-IGFIR) to reduce the up-regulation of neuromuscular junctions that are associated with return of muscle function after botulinum toxin treatment.

Methods: Eyelids of adult rabbits were locally injected with either botulinum toxin alone or botulinum toxin treatment followed by injection of either CRF or anti-IGFIR. After one, two, or four weeks, the orbicularis oculi muscles within the treated eyelids were examined for density of neuromuscular junctions histologically.

Results: Injection of botulinum toxin into rabbit eyelids resulted in a significant increase in the density of neuromuscular junctions at one and two weeks, and an even greater increase in neuromuscular junction density by four weeks after treatment. Treatment with either CRF or anti-IGFIR completely prevented this increase in neuromuscular junction density.

Conclusions: The return of function after botulinum toxin-induced muscle paralysis is due to terminal sprouting and formation of new neuromuscular junctions within the paralyzed muscles. Injection with CRF or anti-IGFIR after botulinum toxin treatment prevents this sprouting, which in turn should increase the duration of effectiveness of single botulinum toxin treatments. Future physiology studies will address this. Prolonging botulinum toxin's clinical efficacy should decrease the number of injections needed for patient muscle spasm relief, decreasing the risk of negative side effects and changes in drug effectiveness that often occurs over a lifetime of botulinum toxin exposure.

Figure 1.

Photomicrographs of neuromuscular junctions stained with fluorescently labeled α-bungarotoxin. (A) Normal control rabbit orbicularis oculi muscle. (B) Two weeks after an injection of 5 units of botulinum toxin. (C) Two weeks after an injection of 5 units of botulinum toxin A followed by two injections of corticotrophin releasing factor. Scale bar, 20 μm. Inset magnification, 40×.

Figure 2.

Three-dimensional reconstruction of approximately 10% of the neuromuscular junctions in a representative eyelid specimen from (A) normal control orbicularis oculi muscle, (B) an eyelid treated two weeks earlier with botulinum toxin A, (C) an eyelid treated two weeks earlier with botulinum toxin A followed by CRF, and (D) an eyelid treated two weeks earlier with botulinum toxin A followed by anti-IGFIR. Each dot represents a single neuromuscular junction.

Figure 3.

Quantification of neuromuscular junction density of control orbicularis oculi muscles (white bar) as well as one (vertical hatched bar), two (black bar), and four (gray bar) weeks after an injection of 5 units botulinum toxin A (Botox). *Significantly different from control; **significantly different from control and from 1 and 2 weeks; #significantly different from pretarsal control.

Figure 4.

Quantification of neuromuscular junction density of control orbicularis oculi muscles (white bar) compared to two weeks after an injection of 5 units of botulinum toxin A only (Botox; black bar), and two weeks after botulinum toxin followed by corticotrophin releasing factor (gray bar) or an antibody to insulin growth factor-1 receptor (hatched bar). #Significantly different from control; *significantly different from Botox treated 2 weeks.