Developmental and functional considerations of masseter muscle partitioning.

The masseter muscle participates in a wide variety of activities including mastication, swallowing and speech. The functional demands for accurate mandibular positioning and generation of forces during incising or a power stroke require a diverse set of forces that are determined by the innate muscle form. The complex internal tendon architecture subdivides the masseter into multiple partitions that can be further subdivided into neuromuscular compartments representing small motor unit territories. Individual masseter compartments have unique biomechanical properties that, when activated individually or in groups, can generate a wide range of sagittal and off-sagittal torques about the temporomandibular joint. The myosin heavy chain (MyHC) fibre-type distribution in the adult masseter is sexually dimorphic and is influenced by hormones such as testosterone. These testosterone-dependent changes cause a phenotype switch from slower to faster fibre-types in the male. The development of the complex organization of the masseter muscle, the MyHC fibre-type message and protein expression, and the formation of endplates appear to be pre-programmed and not under control of the muscle nerve. However, secondary myotube generation and endplate maturation are nerve dependent. The delayed development of the masseter muscle compared with the facial, tongue and jaw-opening muscles may be related to the delayed functional requirements for chewing. In summary, masseter muscle form is pre-programmed prior to birth while muscle fibre contractile characteristics are refined postnatally in response to functional requirements. The motor control mechanisms that are required to coordinate the activation of discrete functional elements of this muscle remain to be determined.

Differential activation of neuromuscular compartments in the rabbit masseter muscle during different oral behaviors.

The rabbit masseter muscle is composed of multiple anatomical partitions that produce different mechanical actions. The purpose of this study was to test the hypothesis that these compartments are differentially activated during the performance of different oral behaviors. Rhythmic activation of the masticatory muscles was elicited by stimulating the cortical masticatory area (CMA) while recording forces generated at the incisors in three dimensions with the mandible immobilized. Torques about the right temporomandibular joint (TMJ) were calculated using these forces recorded during isometric function. A set of 1-15 unique rhythmic behaviors was identified for each rabbit using torque phase plot patterns. Electromyographic recordings were made at nine different compartments in the right masseter, two compartments in the left masseter, two regions in the right digastric, and single locations in the left digastric and right and left medial pterygoid muscles. In activation cycles producing similar mechanical actions, activity patterns at the 16 recording sites were clustered into three to six groups using principal component analysis (PCA). To test for similarities in the activation of masseter compartments, pair-wise comparisons of the PCA assignment for the nine masseter compartments were conducted and frequencies of common assignment were compiled for each unique rhythmic behavior for each rabbit. Masseter muscle compartments were found to vary significantly in their PCA from the expected distribution of 100% common principal component (PC) assignment (i.e., similar activation pattern). This finding is consistent with the independent activation of masseter compartments.

Modeling rabbit temporomandibular joint torques during a power stroke.

Little information exists regarding the effects of changes in mandibular form as a result of orthognathic surgery on torques produced about the temporomandibular joint (TMJ). In this study, we have modeled torques produced about the working side TMJ by selected compartments of the rabbit masseter muscle based on published electromyographic activity. The masseter muscle is composed of multiple subregions or compartments that have unique mechanical actions. In a previous study, forces were elicited by electrical stimulation of each compartment and were recorded by a multiaxis force transducer attached to the anterior mandible. Torques were calculated using mandibular lever arms measured from the center of the TMJ. We have extended this modeling to include variations in mandibular width, length, or height to determine the torques that would be generated with variations in mandibular form. Three superficial masseter compartments on the working side and one posterior deep compartment from the balancing side masseter were examined using data collected from a companion study. It was found that the working and balancing side compartments were synergists for pitch torque components but were antagonists for roll and yaw. In modeling an increase of each mandibular dimension by 20%, nonuniform changes in compartment-generated torques were found. The largest increase was found for the posterior superficial masseter yaw torque component. The effects of changing mandibular form on torques produced about the TMJ may be greater than predicted by previous models that assumed a single line of force produced by each jaw muscle.

Functional activation of the extensor carpi radialis muscles in humans.

OBJECTIVES: To assess activity of radial wrist extensors caused by isometric radial deviation and extension by using magnetic resonance imaging (MRI) and to assess measures that might be used to normalize T2-weighted data. DESIGN: Two-way analysis of variance (ANOVA) design. SETTING: Laboratory and children's hospital. PARTICIPANTS: Three healthy volunteers. INTERVENTIONS: Ten repetitions of 10-second randomly ordered 30% or 60% of maximum voluntary isometric contractions toward wrist extension or radial deviation. MAIN OUTCOME MEASURES: Average T2 values from T2-weighted MR images of the extensor carpi radialis brevis (ECRB) and the extensor carpi radialis longus (ECRL), flexor digitorum profundus (FDP), and radius marrow were determined across 7 sections and 4 exercise bouts and a preexercise condition. RESULTS: Significant differences across task and across sections were determined. Post hoc analysis revealed differences in activity between proximal and distal ECRB and ECRL during an exercise and differential activation of the same muscle across the 2 exercise tasks. Bone marrow and FDP did not show task-related changes. The range of average T2 values of bone marrow across sections was greater than a muscle (FDP) that was not the target of the exercise protocol. However, FDP did show small but significant differences across sections. CONCLUSIONS: T2-weighted MR images can be used to study muscle activation at 30% and 60% of maximum voluntary contractions. The use of inactive muscle and bone marrow for normalizing data requires further investigation.

Brief exposure to testosterone is sufficient to induce sex differences in the rabbit masseter muscle.

In order to evaluate whether testosterone is responsible for inducing long-lasting sex differences in myosin heavy chain gene expression in the rabbit masseter muscle, we castrated young adult animals and administered supra-physiologic doses of testosterone for 3 or 6 weeks duration. Biopsies were taken from the masseter muscles of these animals at the time of castration and 3, 6 and 9 weeks later. Both immunoblot and immunohistochemical analyses of the myosin heavy chain (MyHC) isoform content of these muscle samples were performed. Exposure to testosterone for either duration resulted in a dramatic decrease in the content of the cardiac alpha MyHC isoform and a comparable increase in the content of the IIa MyHC isoform. The decrease in the cardiac alpha MyHC isoform content persisted for as long as 6 weeks after the end of treatment, but the increase in the content of the IIa MyHC isoform declined to normal during this time. Significant numbers of fibers were found containing both the cardiac alpha (and slow) and the IIa isoforms. Several fibers were encountered that contained both IIa and, presumably, the IIx isoform. Thus, a brief exposure to testosterone during postnatal maturation is able to produce a long lasting myosin heavy chain isoform switch that is similar in magnitude to that found during normal development.

Post-translational phosphorylation of the slow/beta myosin heavy chain isoform in adult rabbit masseter muscle.

Four different phenotypes of slow muscle fibers, characterized by differential epitope expression in the slow/beta myosin heavy chain (MyHC) isoform, have been identified in adult rabbit masseter muscle. We investigated the role of post-translational phosphorylation in the expression of these four phenotypes. Serial cryostat sections were treated either with alkaline phosphatase to dephosphorylate proteins in the tissue, or with a brain kinase solution and ATP to phosphorylate them, and then stained, using four antibodies that bind specifically to the slow/beta MyHC isoform. In sections pre-treated with phosphatase, immunoreactivity to antibody A4.840 was abolished, but it could be restored by subsequent kinase/ATP treatment or ATP alone, indicating that the expression of its epitope requires phosphorylation. Phosphatase treatment resulted in an exposure of the epitope for antibody A4.951 in cells that normally bind this antibody only weakly or not at all, but since heat treatment alone produced similar effects, the role of phosphorylation in this enhancement is less certain. Immunoreactivity to antibodies S58 and BA-D5 were not influenced by phosphatase pre-treatment. Kinase/ATP treatment was only effective in changing antibody binding when tissues already had been phosphatase treated. We interpret these results to mean that sites of potential phosphorylation may already be occupied by O-linked glycosylation.

Ciliary neurotrophic factor is required for motoneuron sprouting.

We used mutant mice that lack the gene for ciliary neurotrophic factor (CNTF) to test the hypothesis that it is an endogenous sprouting factor. Fibers in the lateral gastrocnemius muscle were either partially denervated by transection of one of the branches of its nerve or paralyzed by intramuscular injection of botulinum toxin. This results in a significant sprouting response at the terminals of intact motoneurons in normal animals. We did not detect sprouting produced by either stimulus in mice lacking CNTF. When exogenous CNTF was administered to CNTF knockout mice following partial muscle denervation, they mounted a typical sprouting response. Thus CNTF is a critical factor in the process of sprout formation after both partial denervation injury and neuromuscular paralysis. It may function as part of a cellular compensatory mechanism after neuronal injury.

Sexually dimorphic expression of myosin heavy chains in the adult mouse masseter.

Little is known regarding the role of androgenic hormones in the maintenance of myosin heavy chain (MHC) composition of rodent masticatory muscles. Because the masseter is the principal jaw closer in rodents, we felt it was important to characterize the influence of androgenic hormones on the MHC composition of the masseter. To determine the extent of sexual dimorphism in the phenotype of masseter muscle fibers of adult (10-mo-old) C57 mice, we stained tissue sections with antibodies specific to type IIa and IIb MHC isoforms. Females contain twice as many fibers containing the IIa MHC as males, and males contain twice as many fibers containing the IIb MHC as females. There is a modest amount of regionalization of MHC phenotypes in the mouse masseter. The rostral portions of the masseter are composed mostly of type IIa fibers, whereas the midsuperficial and caudal regions contain mostly type IIb fibers. Using immunoblots, we showed that castration results in an increase in the expression of type IIa MHC fibers in males. Ovariectomy has no effect on the fiber type composition in females. We conclude that testosterone plays a role in the maintenance of MHC expression in the adult male mouse masseter.

Muscle spindle reinnervation following phenol block.

Infusion of phenol into peripheral nerves is used clinically to manage spasticity. It produces relief of symptoms by chemical denervation. We simulated the clinical procedure by bathing the lateral plantar nerve of rats in 7% phenol solution for 20 min. We studied the innervation of muscle spindles in the plantar lumbrical muscles of untreated rats and in rats 4 and 6 weeks after a single phenol block. Spindles were identified by the immunoreactivity of nuclear bag(1) fibers to slow tonic myosin (antibody ALD 19). The integrity of the sensory and motor reinnervation of spindles was evaluated using a monoclonal antibody specific for a high molecular weight neurofilament protein. Four weeks after phenol block, muscle spindles were difficult to find, as their immunoreactivity to antibody ALD 19 was reduced. In those spindles studied, most (>80%) were completely denervated. The remainder of which were innervated by afferents only. None received efferent (gamma) innervation. After 6 weeks, spindles were readily identified and nearly all (>90%) received recognizable afferent innervation. A much smaller number (38%) received gamma innervation. Phenol block thus results in a complete denervation of muscle spindles, followed by a fairly rapid sensory reinnervation. Reinnervation by gamma motor neurons is either incomplete or significantly delayed.

Investigation of sexual dimorphism in the rabbit masseter muscle showing different effects of androgen deprivation in adult and young adult animals.

To evaluate the role played by androgens in the development and maintenance of sex differences in the proportion of muscle fibres of different phenotypes, the effects of castration in adult (>6 months old) and in young adult (2-3 months old) male rabbits was examined. Immunohistochemical methods were used to evaluate the proportion of muscle fibres containing different myosin heavy-chain isoforms in 10 different neuromuscular compartments of the masseter. In young adult animals of both sexes, the proportion of fibres of different phenotypes in different compartments was not significantly different from that of normal adult females. In animals castrated as young adults, the development of adult male phenotype proportions was completely blocked in most compartments. In animals castrated as adults, proportions were not significantly different from those of the intact males. For most masseter compartments, androgens produced permanent changes in muscle fibre phenotype during a critical period of postnatal development. However, in the posterior deep compartment, androgen deprivation in young adults had no effect on phenotype proportions, but castration of adults resulted in a striking increase in the proportion of fibres containing the IIa myosin heavy-chain isoform.

Neuromuscular compartments of cat lateral gastrocnemius produce different torques about the ankle joint.

The primary purpose of this study was to establish whether the neuromuscular compartments of cat lateral gastrocnemius produce different mechanical actions on the skeletal system, by determining the contributions made by these compartments to the torques produced about the ankle joint. It was postulated that neuromuscular compartments might represent output elements of the spinal circuits. If so, they should produce unique mechanical actions. Isometric torques about the center of the ankle joint produced by the neuromuscular compartments of the cat lateral gastrocnemius were measured with a multiaxis force-moment sensor connected to the plantar surface of the foot. Individual compartment torques were elicited by activation of the primary compartment branches of the lateral gastrocnemius nerve. The magnitude of the individual torque components, and thus of the resultant torque, was significantly different between compartments. In three of the four lateral gastrocnemius compartments, significantly different torque trajectories were noted. The results, together with those from previous studies demonstrating that compartments can be activated in a task-dependent manner, suggest that neuromuscular compartments represent anatomical substrates that can be used by the nervous system for regulating movement.

Sexual dimorphism in the rabbit masseter muscle: myosin heavy chain composition of neuromuscular compartments.

The myosin heavy chain (MyHC) isoform composition of six adult (>7 months old) male and female rabbit masseter muscles was studied using seven monoclonal antibodies. In matched serial tissue sections, muscle fibers in 10 different neuromuscular compartments were analyzed. Nearly all fibers were found to express one of five phenotypes. They either contained one of four different slow/beta MyHC phenotypes (I(1)-I(4)), nearly all of which co-express cardiac alpha MyHC, or they contained type IIa MyHC. Very few fibers contained slow/beta or cardiac alpha MyHC only or both the alpha/slow/beta and IIa isoforms. Most, but not all, of the compartments studied contained similar proportions of fibers of the five major phenotypes, at least within sex. For 7 of the 10 compartments studied, significant sex differences in the proportion of I(1) and IIa fibers were found. Males contained more IIa fibers and fewer I(1) fibers than females. Fibers of the IIa phenotype were significantly larger than fibers of all of the other phenotypes and larger in males than females.

Reproductive cycles of farmed female chital deer (Axis axis).

Studies were conducted on chital deer hinds (Axis axis) living in a temperate region to advance the understanding of the patterns of reproduction of a tropical cervid species. The hinds exhibited regular patterns of oestrus cyclicity throughout the year as evidenced by concentrations of serum progesterone monitored over a 14-month period, and detection of behavioural oestrus by vasectomized stags. The mean length of the oestrous cycle was 18.0+/-0.7 days (range, 12-23 days). Profiles of serum progesterone showed concentrations of <0.5 ng mL(-1) at the time of oestrus, which rose to a peak (range 1.5-5.0 ng mL(-1)) about Day 13, and then declined to low concentrations at the next oestrus. Observations following parturition showed that the first detected oestrus occurred at a mean (+/- SEM) time of 26.9+/-3.0 days later for seven of nine hinds. The mean length of the oestrous cycle after the first post-partum oestrus was 16.6+/-1.0 days (range 7-20 days). The presence of a stag may influence the length of the post-partum period in chital deer hinds, and hinds in contact with a stag in this study had a significantly shorter interval from parturition to first ovulation (P<0.01) compared with hinds not in contact with a stag. By 7 weeks post partum a corpus luteum was detected in 93% of hinds. In comparison only 43% of hinds with no stag contact had a corpus luteum by 7 weeks post partum. It is suggested that the tendency towards seasonal calving in the study population may be related more to male than female factors.

Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities.

Skeletal muscle has a remarkable capacity to regenerate after injury, although studies of muscle regeneration have heretofore been limited almost exclusively to limb musculature. Muscle precursor cells in skeletal muscle are responsible for the repair of damaged muscle. Heterogeneity exists in the growth and differentiation properties of muscle precursor cell (myoblast) populations throughout limb development but whether the muscle precursor cells differ among adult skeletal muscles is unknown. Such heterogeneity among myoblasts in the adult may give rise to skeletal muscles with different regenerative capacities. Here we compare the regenerative response of a masticatory muscle, the masseter, to that of limb muscles. After exogenous trauma (freeze or crush injuries), masseter muscle regenerated much less effectively than limb muscle. In limb muscle, normal architecture was restored 12 days after injury, whereas in masseter muscle, minimal regeneration occurred during the same time period. Indeed, at late time points, masseter muscles exhibited increased fibrous connective tissue in the region of damage, evidence of ineffective muscle regeneration. Similarly, in response to endogenous muscle injury due to a muscular dystrophy, widespread evidence of impaired regeneration was present in masseter muscle but not in limb muscle. To explore the cellular basis of these different regenerative capacities, we analyzed the myoblast populations of limb and masseter muscles both in vivo and in vitro. From in vivo analyses, the number of myoblasts in regenerating muscle was less in masseter compared with limb muscle. Assessment of population growth in vitro indicated that masseter myoblasts grow more slowly than limb myoblasts under identical conditions. We conclude that the impaired regeneration in masseter muscles is due to differences in the intrinsic myoblast populations compared to limb muscles.

Different phenotypes among slow/beta myosin heavy chain-containing fibres of rabbit masseter muscle: a novel type of diversity in adult muscle.

Difference in the phenotype of different mammalian muscle fibres are usually attributed to differences in the expression of the product of different myosin heavy chain (MyHC) genes, which are known as isoforms. We studied differences in phenotype among fibres containing a single MyHC isoform (slow/beta) of the masseter muscle of adult rabbits. Four different monoclonal antibodies to slow/beta MyHC were used to stain serial sections from muscles in males and females. All antibodies recognize a single band on immunoblots and stain the same set of fibres in rabbit postcranial muscles. However, differential staining was observed in the masseter muscles. Antibody BA-D5 reacts with the most fibres, antibody A4.951 reacts with a subset of these fibres, and antibody A4.840 reacts with a subset of these fibres, and antibody A4.840 reacts with a subset of A4.951-positive fibres. Antibody S58 reacts only with an even smaller subset of fibres. Even though differential staining using four antibodies might allow for the expression of as many as 15 different staining patterns, or patterns, or phenotypes, only four were observed on > 99% of over 30 000 fibres studied. In females, nearly 40% of the fibres stain exclusively with antibody BA-D5, while in males, fewer than 8% of the fibres express this phenotype. The proportions of fibres of the other phenotypes do not differ so strikingly with gender. We conclude that an epitope diversity exists among muscle fibres in the adult rabbit masseter and that it is not necessarily a consequence of differences in gene expression. We feel that it is a regulated process and that, at least for some phenotypes, this regulation may be hormonally influenced.