Botulinum toxin treatment of extraocular muscles in rabbits results in increased myofiber remodeling

Abstract

Purpose: Botulinum toxin A (Botox) is commonly used for strabismus treatment. Although other muscles atrophy after intramuscular injection with Botox, extraocular muscles (EOMs) do not. A continuous process of myonuclear addition in normal uninjured adult myofibers in rabbit EOMs was studied. In this study, the effect of Botox-induced muscle paralysis on myofiber remodeling in adult EOMs was examined.

Methods: The superior rectus muscles of adult rabbits were each injected with 5 units of Botox. The contralateral muscle received injections of saline only. Bromodeoxyuridine (BrdU) was administered for various periods after Botox treatment, followed by various BrdU-free periods. Myonuclear addition, the number of BrdU-positive satellite cells, and the number of MyoD-positive satellite cells were quantified, as were alterations in expression of immature myosins.

Results: Intramuscular injection of Botox resulted in a significant increase in both the number of BrdU-positive myonuclei and satellite cells. MyoD expression in both satellite cells and myonuclei was significantly increased after Botox injection in EOMs. In Botox-treated EOMs, an increased number of myofibers positive for the neonatal myosin heavy chain (MyHC) isoform was detected in the orbital layer.

Conclusions: Botox-induced EOM paralysis resulted in a significant short-term increase in satellite cell activation and myonuclear addition in single myofibers in adult rabbit EOMs compared with control muscles. The appearance of MyoD-positive myonuclei suggests that protein synthesis becomes upregulated after Botox injection, and this, in turn, may help explain the minimal effects on myofiber size in EOMs after Botox injection. Understanding the effect of Botox on satellite cell activation and myonuclear addition in existing myofibers may suggest new ways to maximize the clinical effectiveness of Botox in patients with strabismus.

Figure 1

Photomicrographs of a superior rectus muscle labeled with daily BrdU for 7 days 1 week after a single injection of Botox. BrdU is immunostained black and dystrophin, which demarcates the sarco-lemma, is immunostained brown. Horizontal arrow: a BrdU-positive myonucleus; vertical arrow: BrdU-positive cell in the satellite cell position. Bar, 50 μm.

Figure 2

Quantification of (A) myonuclear addition (B) and satellite cell activation, as measured by BrdU incorporation. Botox was administered on day 0, BrdU was administered daily from days 7 to 14 after the Botox injection, and the animal was euthanatized on day 15 (7,7,1) *Significant difference from the saline-injected control.

Figure 3

Quantification of (A) myonuclear addition (B) and satellite cell activation, as measured by BrdU incorporation. Botox was administered on day 0, BrdU was administered daily from days 3 to 8 after the Botox injection, and the animal was euthanatized on day 21 (3,8,10) *Significant difference from the saline-injected control.

Figure 4

Quantification of (A) myonuclear addition and (B) satellite cell activation, as measured by BrdU incorporation. Botox was administered on day 0, BrdU was administered daily from days 7 to 14 after the Botox injection, and the animal was euthanatized on day 21 (7,7,7) *Significant difference from the saline-injected control.

Figure 5

Injection of normal adult superior rectus with Botox resulted in a significant increase in the number of MyoD-positive satellite cells (arrowheads) compared with the normal EOM. Normally, the myonuclei in adult skeletal muscle are negative for MyoD expression. However, Botox treatment resulted in the appearance of MyoD-positive myonuclei, which were randomly located throughout the treated muscle (arrows).

Figure 6

The number of MyoD-positive satellite cells and myonuclei was quantified in the rectus muscles. *Significant increase compared with the noninjected controls. **Essentially no MyoD-positive myonuclei in control superior rectus muscle.