Acute and long-term effects of botulinum neurotoxin on the function and structure of developing extraocular muscles

Abstract

Strabismus is a misalignment of the visual axes, due to an imbalance in extraocular muscle (EOM) function. Botulinum neurotoxin (BoNT) treatment can correct the misalignment with permanent therapeutic effects in infants, possibly because the toxin causes structural alterations in developing EOM. To determine whether BoNT indeed permanently weakens developing EOMs, we examined the chicken oculomotor system. Following injections of BoNT in hatchling chicks, we quantified physiological parameters (contractile force measurements) and morphological parameters (myofiber morphometry, innervation, quantitative transmission electron microscopy of mitochondria/fiber types). Treatment of developing EOM with BoNT caused acute reductions of muscle strength and mitochondrial densities, but minimal changes in muscle fiber diameter and neuromuscular junction structures. Contrary to expectations, contractile force was fully recovered by 3-4 months after treatment. Thus, permanent therapeutic effects of BoNT most likely do not cause permanent changes at the level of the peripheral effector organ, but rather involve central (CNS) adaptive responses.

Measurement of contractile force of skeletal and extraocular muscles: Effects of blood supply, muscle size and in situ or in vitro preparation

“…In adult Wistar rats, the in vitro stimulation of the inferior rectus muscle was found to be 0.66 g (Close and Luff, 1974), which was significantly less than the 51 mN (~5.2 g) reported for the in situ stimulation of the superior rectus muscle from Sprague-Dawley rats (Frueh et al, 2001). Extraocular muscle force in two-day old hatchling chicks, stimulated in vitro, was reported to be 1.43 mN (~146 mg) for the superior oblique muscle (Chen and von Bartheld, 2004), which was significantly less than the 391 mg reported for the in situ stimulation of the same muscle with blood supply intact (Croes et al, 2007). …”

“…In a previous comparison, Frueh and colleagues (2001) found that muscle force (force per cross-sectional area, kN/m 2 ) measured in situ were about six times greater than the highest of those reported previously for extraocular muscles in vitro (Close and Luff, 1974;Asmussen and Gaunitz, 1981;Luff, 1981;Asmussen et al, 1994;Frueh et al, 1994). Our own lab has reported force measurements of the superior oblique muscle obtained in situ to be 3 to 6 times greater than that of the same muscle in vitro (Chen and von Bartheld, 2004;Croes et al, 2007).…”

“…The superior oblique muscle is innervated by the trochlear nerve, which is unique in that it has a sole muscle target. This method enabled us to effectively measure the contractile forces generated by the superior oblique muscle in chickens at 2 days (P2) and 3 weeks (P21) of age in the current study and up to 4 months of age in a previous study (Croes et al, 2007). Representative traces of twitch contractions of the superior oblique muscle via indirect (nerve) stimulation are shown in Fig.…”

“…The stimulation rates that elicit maximum tetanic tension were determined previously (Croes et al, 2007). Three single-tetanic tension contractions were elicited in 5 s intervals to determine the average P o at each time point.…”

“…Muscle mass, optimal muscle length L o , muscle fiber length L f , and P o were used to calculate maximum specific isometric tetanic force (sP o ) or maximum P o normalized per total muscle CSA (kN/m 2 ), as described in Frueh et al (2001). The CSA was estimated in four ways: (i) by the conventional formula that divides muscle mass (mg) by the product of the measured optimal muscle length, L o , and 1.06 mg/mm3 (the density of mammalian skeletal muscle, Close, 1972); (ii) by correcting for the actual muscle fiber length = mean myofiber length, L f (estimated to be 45%, Frueh et al, 2001); (iii) by correcting to exclude extracellular spaces in muscle cross sections (Close, 1972; based on data from Baryshnikova et al, 2007); and (iv) by correcting for actual myofibrillar area (Taylor and Kandarian, 1994; based on data from Baryshnikova et al, 2007). Absolute P o values were normalized for muscle CSA using the formula sP o (kN/m 2 ) = P o (mN)/CSA (mm 2 ).…”