Classification and development of myofiber types in the superior oblique extraocular muscle of chicken.

Precise control of contractile force of extraocular muscles is required for appropriate movements and alignment of the eyes. It is unclear how such precise regulation of contractile force is achieved during development and maturation. By using the posthatch chicken as a model, we describe and quantify critical parameters of the developing superior oblique extraocular muscle from hatching to 16 weeks of age, including contractile force, muscle mass, myofiber diameters, classification of fiber types, and distribution and quantification of mitochondria. Analysis at the light- and electron microscopic levels shows that chicken myofiber types largely correspond to their mammalian counterparts, with four fiber types in the orbital and four types in the global layer. Twitch tension muscle force and muscle mass gradually increase and stabilize at approximately 11 weeks. Tetanic tension continues to increase between 11 and 16 weeks. Myofiber diameters in both the orbital and global layer increase from hatching to six weeks, and then stabilize, whereas the myofiber number is constant after hatching. This finding suggests that muscle mass increases during late maturation due to increasing fiber length rather than fiber diameter. Quantitative ultrastructural analysis reveals continuing changes in the composition of the four muscle fiber types, suggesting ongoing fiber type conversion or differential replacement of myofiber types. Muscle fiber composition continues to change into late juvenile and adult age. Our study provides evidence for gradual, incremental, and continuing changes in avian myofiber composition and function that is similar to postnatal oculomotor maturation in visually oriented mammals such as kitten.

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

Contractile forces can be measured in situ and in vitro. To maintain metabolic viability with sufficient diffusion of oxygen, established guidelines for in vitro skeletal muscle preparations recommend use of relatively thin muscles (< or =1.25 mm thick). Nevertheless, forces of thin extraocular muscles vary substantially between studies. Here, we examined parameters that affect force measurements of in situ and in vitro preparations, including blood supply, nerve stimulation, direct muscle stimulation, muscle size, oxygenated or non-oxygenated buffer solutions and the time after interruption of vascular circulation. We found that the absolute forces of extraocular muscle are substantially lower when examined in vitro. In vitro preparation of 0.58 mm thick extraocular muscle from 3-week-old birds underestimated contractile function, but not of thinner (0.33 mm) muscle from 2-day-old birds. Our study shows that the effective criteria for functional viability, tested in vitro, differ between extraocular and other skeletal muscle. We conclude that contractile force of extraocular muscles will be underestimated by between 10 and 80%, when measurements are made after cessation of blood supply (at 5-40 min). The mechanisms responsible for the declining values for force measurements are discussed, and we make specific recommendations for obtaining valid measurements of contractile force.

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

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.

Development of the neuromuscular junction in extraocular muscles of white Leghorn chicks.

Relatively little is known about the development of the neuromuscular junction of extraocular muscles (EOMs). In recent years, chicks have been increasingly used as a developmental model in ophthalmological research. To utilize this model system for understanding the development and plasticity of the extraocular motor system, we investigated the structural changes that occur at the developing neuromuscular junction in the chick between embryonic day 14 (E14) and posthatch day 2 (P2). Axons and nerve terminals were visualized with fluorescent neurofilament antibodies and motor endplates with rhodamine-conjugated alpha-bungarotoxin. Nerve fibers and endplates were colabeled within the same tissue samples. Motor endplates (density, length, width, and area) were measured and numbers of axons per neuromuscular junction were counted using confocal and conventional microscopy. In P2 chicks, densities of motor endplates were significantly greater in the superior oblique muscle when compared with the superior rectus and lateral gastrocnemius muscle. EOMs showed a two- to threefold larger area of motor endplate size as compared to gastrocnemius muscle. Motor endplate size also differed among EOMs with the superior oblique muscle having endplates with a larger area than those of the superior rectus muscle. The period of synapse elimination was similar between EOM and gastrocnemius muscle. Synapse elimination began at about E18 and was completed by P2. By describing the normal morphological changes in developing EOMs, this study provides a baseline for future work to elucidate underlying molecular mechanisms that regulate EOM innervation and strength.