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Extraocular muscle (EOM) myofibers do not fit the traditional fiber typing classifications normally used in noncranial skeletal muscle, in part, due to the complexity of their individual myofibers. With single skinned myofibers isolated from rectus muscles of normal adult rabbits, force and shortening velocity were determined for 220 fibers. Each fiber was examined for myosin heavy chain (MyHC) isoform composition by densitometric analysis of electrophoresis gels. Rectus muscle serial sections were examined for coexpression of eight MyHC isoforms. A continuum was seen in single myofiber shortening velocities as well as force generation, both in absolute force (g) and specific tension (kN/m(2)). Shortening velocity correlated with MyHCIIB, IIA, and I content, the more abundant MyHC isoforms expressed within individual myofibers. Importantly, single fibers with similar or identical shortening velocities expressed significantly different ratios of MyHC isoforms. The vast majority of myofibers in both the orbital and global layers expressed more than one MyHC isoform, with up to six isoforms in single fiber segments. MyHC expression varied significantly and unpredictably along the length of single myofibers. Thus EOM myofibers represent a continuum in their histological and physiological characteristics. This continuum would facilitate fine motor control of eye position, speed, and direction of movement in all positions of gaze and with all types of eye movements-from slow vergence movements to fast saccades. To fully understand how the brain controls eye position and movements, it is critical that this significant EOM myofiber heterogeneity be integrated into hypotheses of oculomotor control.

Purpose: Human extraocular muscles (EOM) are preferentially susceptible to thyroid eye disease. Although the specific cause of this autoimmune disorder is unknown, it is often associated with elevated thyroid hormone levels. Thus, the effect of elevated thyroid hormone levels on cross-sectional area, myofiber size, satellite cells, and myosin heavy chain (MyHC) isoform expression was examined in adult rabbit EOMs, to determine how elevated thyroid hormone alters EOM biology., Methods: After 1 month of elevated thyroid hormone levels, the EOMs were removed and prepared for histologic examination. Total muscle mass, myofiber size, patterns of MyHC isoform expression, and the number of satellite cells were determined., Results: Elevated thyroid hormone levels significantly decreased muscle mass, total number of myofibers, and mean cross-sectional area of the myofibers. Alterations in MyHC isoform expression were extremely complex, but several basic patterns emerged. The percentages of neonatal- and developmental-positive myofibers decreased in almost all EOM regions examined, and the percentages of slow-positive myofibers significantly increased. In contrast to normal EOMs, which retain a population of activated satellite cells throughout life, elevated thyroid hormone levels resulted in the virtual disappearance of MyoD-positive cells and a decrease in Pax7-positive cells., Conclusions: The reductions in EOM size, number of fibers expressing developmental and neonatal MyHC, and number of MyoD- and Pax7-positive satellite cells suggest that elevated thyroid hormone levels decrease the ongoing myofiber remodeling normally seen in the EOM. These catabolic changes have important implications for maintenance of function in the EOMs.

Purpose: The density and three-dimensional localization of neuromuscular junctions (NMJs) of normal and botulinum toxin-treated normal adult rabbit and monkey extraocular muscles (EOMs) were analyzed. To demonstrate average myofiber length, randomly selected individual myofibers were reconstructed and compared with total muscle length., Methods: Normal adult rabbit and monkey EOM and normal adult rabbit tibialis anterior were dissected in their entirety, frozen, sectioned longitudinally, and immunostained for NMJ localization. In addition, adult rabbit EOMs were injected with 5 U botulinum toxin, and NMJ density was determined after 2 weeks. NMJ locations for the three groups of EOM were reconstructed, and density of NMJ was determined. Individual myofibers were reconstructed from the orbital and global layers to determine mean fiber length., Results: NMJs were dispersed throughout the entire length of all EOMs examined from adult rabbits and monkeys and were visualized by alpha-bungarotoxin staining and three-dimensional reconstruction of serial sections. In leg muscle, two relatively tight bands of NMJs were seen. Botulinum toxin significantly increased total NMJ density. Mean fiber lengths were 1.9 and 4.83 mm in the orbital and global layers, respectively, approximately 10% and 24% of the total origin-to-insertion muscle lengths. In addition, individual myofibers continuously changed their intrafascicular relationships over their lengths., Conclusions: The density and distribution of NMJs in normal EOMs are more extensive than previously described. Individual myofibers are significantly shorter than the tendon-to-tendon muscle length in both muscle layers. Botulinum toxin results in a doubling of NMJ density. NMJ localization in normal EOMs has ramifications for understanding eye movement control, but it is also important when surgical or pharmacologic intervention is used for the treatment of strabismus, nystagmus, or other eye muscle disorders.

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.

Fiber type shifts in aging skeletal muscle have been studied with myofibrillar ATPase histochemistry and gel electrophoresis, but less commonly with immunohistochemistry. Immunohistochemical study of myosin heavy chains (MHCs) in single myofibers yields additional information about aged skeletal muscle. Furthermore, many studies of aging rodent skeletal muscle have been performed on fast-MHC-predominant muscle and in several different strains. The aim of this study was to evaluate immunohistochemically MHC characteristics in the slow-MHC-predominant soleus muscle in the Fischer Brown Norway F1 hybrid aging rat (FBN). Three age groups of FBN rats were studied: 12 months, 30 months, and 36 months. Soleus muscles were excised, quick-frozen, and stained immunohistochemically for slow, fast, developmental, and neonatal MHC isoforms. Cross-sections were evaluated for the number and cross-sectional areas of fibers expressing each isoform. Single myofibers in soleus muscles of the aged rats showed significantly greater amounts of coexpression of slow and fast MHC than did younger animals. This change began by 30 months of age, but did not reach statistical significance until 36 months of age. The soleus from 36-month-old rats also expressed greater amounts of developmental MHC than did the other groups. These developmental MHC-positive myofibers also coexpressed either slow or slow and fast MHC. The age-related increase in MHC coexpression of slow with fast isoforms may indicate a fiber type shift suggestive of denervation that outpaces reinnervation. The developmental MHC-positive fibers provide evidence of ongoing myofiber remodeling in the oldest rats in the midst of the fiber degeneration of aging., (Copyright 2005 Wiley-Liss, Inc)

Objectives: In normal mature limb skeletal muscle, satellite cells are quiescent and myonuclei do not divide after formation of their associated myofibers in the absence of injury. The possibility of myonuclear addition in uninjured laryngeal myofibers of adult rabbits was investigated in an immunohistochemical pilot study., Methods: Bromodeoxyuridine (brdU), a marker for cell division, was administered by intraperitoneal injection over a 12-hour period in rabbits. The number of brdU-positive myonuclei per myofiber was determined on cross sections through the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles., Results: In the TA muscle, 0.13% +/- 0.03% (mean +/- SEM) of the myofibers counted had a brdU-positive nucleus. In the PCA muscle, 0.13% +/- 0.01% of the myofibers counted had a brdU-positive nucleus. Approximately 0.2% and 0.3% of the myofibers of the TA and PCA muscles, respectively, had brdU-positive satellite cells associated with them. Tibialis anterior and pectoralis major muscle controls were negative for brdU-positive myonuclei., Conclusions: These data support the possibility of continuous remodeling in uninjured adult laryngeal myofibers and accentuate the distinct nature of laryngeal muscle relative to limb skeletal muscle in the rabbit model.

Unlike normal mature limb skeletal muscles, in which satellite cells are quiescent unless the muscle is injured, satellite cells in mammalian adult extraocular muscles (EOM) are chronically activated. This is evidenced by hepatocyte growth factor, the myogenic regulatory factor, Pax-7, and the cell-cycle marker, Ki-67, localized to the satellite cell position using serial sections and the positional markers laminin and dystrophin. Bromodeoxyuridine (brdU) labeling combined with dystrophin immunostaining showed brdU-positive myonuclei, presumably the result of fusion of activated satellite cells into existing myofibers. One new myonucleus was added to every 1000 myofibers in cross-section using a 12-hour brdU-labeling paradigm. The EOM thus appear to retain a stable nuclear population by an opposing process of apoptosis that results in myonuclear removal as visualized by terminal deoxynucleotidyltransferase-mediated nick end labeling (TUNEL). Activated caspase-3 was present in localized cytoplasmic domains extending from 10 to 210 microm within individual myofibers, suggesting segmental cytoplasmic reorganization. Understanding the cellular mechanisms that maintain this process of continuous myonuclear addition and removal in normal adult EOM may suggest new hypotheses to explain the preferential involvement or sparing of these muscles in skeletal muscle disease., (Copyright 2004 Wiley Periodicals, Inc.)

Extraocular muscles (EOM) are unique among mammalian skeletal muscles in that they normally express molecules associated with muscle development and regeneration. In this study we show that satellite cells of EOM, unlike those of other skeletal muscles, continually divide in the normal, uninjured adult. Adult EOM contained activated satellite cells positive for the myogenic regulatory factor MyoD. EOM satellite cells did not require a prolonged activation period prior to onset of cell division and differentiation in vitro. EOM satellite cells incorporated bromodeoxyuridine (brdU), a marker for cell division, and with longer postlabeling survival, brdU-labeled nuclei populated EOM myofibers. This was not seen with leg muscle. These findings suggest the possibility that continual division of satellite cells and fusion of their daughter myocytes with existing adult EOM myofibers contribute to the unique sparing or susceptibility of EOM to certain muscle diseases., (Copyright 2002 Wiley Periodicals, Inc.)

The sternocleidomastoid muscle is located in the neck and is both a neck rotator and flexor. Cervical dystonia, a focal dystonia disorder, is characterized by forceful involuntary contraction of a group of neck muscles, usually including the sternocleidomastoid. Little is known about the fiber type composition, fiber type compartmentalization and innervation patterns in this muscle in rabbit and primates. Sternocleidomastoid muscles from rabbit and monkey were analyzed for muscle fiber type composition and number, muscle fiber cross-sectional area and patterns of innervation. The sternocleidomastoid muscle was composed of two distinct regions, or compartments, with different fiber type compositions: an outer or superficial region composed of mostly type 2 myofibers and an inner deep region composed of both type 2 and type 1 myofibers. Neonatal myosin heavy chain isoform was detected in approximately 25% of the myofibers in both regions of the muscle. Neuromuscular junctions were located in seven endplate bands approximately 1-3 cm apart throughout the length of the muscle. There is clear evidence of anatomical subdivisions within this muscle. Not only is there variation in fiber type composition between superficial and deep regions of the muscle, but unlike most other mature skeletal muscles, it continues to express neonatal myosin heavy chain isoform in the adult. The motor program for neck movements is extremely complex, and the histological complexity plays a role in allowing for a continuum of movements of the head and neck, from maintenance of posture to rapid head movements.

Purpose: The anatomy of individual myofibers within the orbicularis oculi muscle was examined to determine individual myofiber lengths in the different regions of the muscle. A wide variety of eyelid conditions require eyelid surgery or drug injections directly into the eyelid. Knowledge of regional myofiber anatomy and physiology is important for accurate treatment of these conditions., Methods: Eyelid specimens from rabbits were treated with collagenase, fixed, and stained for neuromuscular junction location. Individual myofibers were dissected from these muscle specimens and were measured to determine individual myofiber length and neuromuscular junction position. Additional eyelid specimens of rabbits and humans were stained en bloc to visualize neuromuscular junction location in the pretarsal and preseptal regions of the orbicularis oculi muscle., Results: The myofibers showed variable lengths, shorter in the pretarsal region of the muscle and longer in the preseptal region. The average individual myofiber length in the pretarsal region was 36% as long as the entire length of the pretarsal muscle region. In the preseptal region, the myofibers were slightly longer, covering 54% of the entire length of this region of the muscle. In both the pretarsal and preseptal regions of the rabbit and human orbicularis oculi muscle, there were many clusters of neuromuscular junctions throughout the medial to lateral length of the muscle, with the majority of the neuromuscular junctions in the medial and lateral canthal regions of the preseptal portion of the lid. This indicates that the muscle is composed of relatively short, overlapping myofibers, and that the shortest myofibers reside in the medial and lateral canthal regions of the eyelid. Multiple innervation of one rabbit myofiber was observed as a rare occurrence., Conclusions: Individual myofibers of the orbicularis oculi muscle are relatively short, end intrafascicularly, and are of heterogeneous lengths varying regionally within the muscle. Thus, for drug injections into the eyelid, optimal drug effectiveness may require treatment of the entire lid from medial to lateral canthus to overcome the tissue barriers to diffusion. The existence of muscle fibers of heterogeneous lengths suggests that the complex organization of muscle fibers may play previously unappreciated but important roles in normal function, pathophysiology, and age-related changes in the eyelid.