Tongue exercise and ageing effects on morphological and biochemical properties of the posterior digastric and temporalis muscles in a Fischer 344 Brown Norway rat model.

OBJECTIVE: This study sought to examin effects of age and tongue exercise on the posterior digastric (opener) and the temporalis (closer). We hypothesized 1) age would result in differing morphological (cross sectional area) and biochemical (myosin heavy chain isoform) components of these muscles; 2) tongue exercise would result in coactivation of these muscles inducing a decrease in age-related differences between age groups. DESIGN: Young adult (9 months) and old (32 months) Fischer 344 Brown Norway rats were randomized into a tongue exercise or control group. Post-training, posterior digastric and temporalis muscles were harvested and analyzed using: 1) Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to assess percent myosin heavy chain (MyHC) content; 2) Immunohistochemical staining to determine cross sectional area (CSA). RESULTS: A larger proportion of slowly contracting MyHC isoforms in the posterior digastric and temporalis muscles were found in old. No significant main effects for age or exercise in fiber size were found in posterior digastric muscle. An interaction between age and exercise for temporalis cross sectional area indicated the old exercise group had smaller average cross sectional area than all other groups. CONCLUSIONS FINDINGS: suggest that: 1) Increasing age induces biochemical changes in muscles of the jaw, specifically showing an increase the proportion of slower contracting MyHC isoforms; 2) Increasing age and tongue exercise induce a reduction in muscle fiber cross sectional area in the temporalis muscle only. However, continued study of these cranial muscle systems is warranted to better understand these changes that occur with age and exercise.

Detectability and medico-legal value of the gunshot residues in the intracorporeal channel.

The application of the histochemical stain of sodium rhodizonate to the entrance wound for the detection of the lead (Pb) residues coming from the gunshot may be affected by false positive cases due to the contamination of the environmental Pb. The aim of the Authors is to histochemically search the Pb of GSR in a region which should be more protected by the contamination: the intracorporeal channel. Two hundreds and eighteen serial histological specimens of the intracorporeal channels coming from 25 subjects (dead due to gunshots and being autopsied at the Section of Legal Medicine of the Milan University, in the years 2013-2014) were stained with the sodium rhodizonate and sodium rhodizonate in acid environment (HCl 5%), and then observed by the microscope. The sodium rhodizonate showed a positivity for the Pb residues in the intracorporeal channel, with the detection of the particles within the first 2 cm beyond the entrance wound in 6 cases over the total number of 25 (24%). Victims were characterized by common features: short-barreled weapon; contact shots or short-distance shots; involvement of regions that were not covered by clothing; preservation of the microscopic structure of organs interested by the intracorporeal channel. The searching of GSR in the intracorporeal channel, even in conditions securing a high sensitivity, could represent an important test for the discrimination between an environmental contamination of Pb and the presence of Pb residues by GSR: once confirmed the presence of GSR in the intracorporeal channel by the histochemical analysis, the diagnostic process should require the application of the SEM-EDX for the confirmation of the results. Although not yet studied, this combination could be applied to cadavers exposed to the environment, with advanced post-mortal phenomena permitting at least the suspects of the existence of gunshot wounds at the macroscopic autopsy evaluation. Indeed, in some cases, the putrefaction is so advanced that no suspect of gunshot injuries could be derived from the soft tissue.

Regional variation in IIM myosin heavy chain expression in the temporalis muscle of female and male baboons (Papio anubis).

OBJECTIVE: The purpose of this study was to determine whether high amounts of fast/type II myosin heavy chain (MyHC) in the superficial as compared to the deep temporalis muscle of adult female and male baboons (Papio anubis) correlates with published data on muscle function during chewing. Electromyographic (EMG) data show a regional specialization in activation from low to high amplitude activity during hard/tough object chewing cycles in the baboon superficial temporalis.(48,49) A positive correlation between fast/type II MyHC amount and EMG activity will support the high occlusal force hypothesis. DESIGN: Deep anterior temporalis (DAT), superficial anterior temporalis (SAT), and superficial posterior temporalis (SPT) muscle samples were analyzed using SDS-PAGE gel electrophoresis to test the prediction that SAT and SPT will show high amounts of fast/type II MyHC compared to DAT. Serial muscle sections were incubated against NOQ7.5.4D and MY32 antibodies to determine the breadth of slow/type I versus fast/type II expression within each section. RESULTS: Type I and type IIM MyHCs comprise nearly 100% of the MyHCs in the temporalis muscle. IIM MyHC was the overwhelmingly predominant fast MyHC, though there was a small amount of type IIA MyHC (≤5%) in DAT in two individuals. SAT and SPT exhibited a fast/type II phenotype and contained large amounts of IIM MyHC whereas DAT exhibited a type I/type II (hybrid) phenotype and contained a significantly greater proportion of MyHC-I. MyHC-I expression in DAT was sexually dimorphic as it was more abundant in females. CONCLUSIONS: The link between the distribution of IIM MyHC and high relative EMG amplitudes in SAT and SPT during hard/tough object chewing cycles is evidence of regional specialization in fibre type to generate high occlusal forces during chewing. The high proportion of MyHC-I in DAT of females may be related to a high frequency of individual fibre recruitment in comparison to males.

Protruding masticatory (superfast) myosin heads from staggered thick filaments of dog jaw muscle revealed by X-ray diffraction.

To characterize the structure of jaw muscle fibres expressing masticatory (superfast) myosin, X-ray diffraction patterns of glycerinated fibres of dog masseter were compared with those of dog tibialis anterior in the relaxed state. Meridional reflections of masseter fibres were laterally broad, indicating that myosin filaments are staggered along the filament axis. Compared with tibialis anterior fibres, the peak of the first myosin layer line of masseter fibres was lower in intensity and shifted towards the meridian, while lattice spacings were larger at a similar sarcomere length. These suggest that the myosin heads of masticatory fibres are mobile, and tend to protrude from the filament shaft towards actin filaments. Lowering temperature or treating with N-phenylmaleimide shifted the peak of the first myosin layer line of tibialis anterior fibres towards the meridian and the resulting profile resembled that of masseter fibres. This suggests that the protruding mobile heads in the non-treated masticatory fibres are in the ATP-bound state. The increased population of weakly binding cross-bridges may contribute towards the high specific force of masticatory fibres during contraction. Electron micrographs confirmed the staggered alignment of thick filaments along the filament axis within sarcomeres of masticatory fibres, a feature that may confer efficient force development over a wide range of the sarcomere lengths.

Masticatory myosin unveiled: first determination of contractile parameters of muscle fibers from carnivore jaw muscles.

Masticatory myosin heavy chain (M MyHC) is a myosin subunit isoform with expression restricted to muscles derived from the first branchial arch, such as jaw-closer muscles, with pronounced interspecies variability. Only sparse information is available on the contractile properties of muscle fibers expressing M MyHC (M fibers). In this study, we characterized M fibers isolated from the jaw-closer muscles (temporalis and masseter) of two species of domestic carnivores, the cat and the dog, compared with fibers expressing slow or fast (2A, 2X, and 2B) isoforms. In each fiber, during maximally calcium-activated contractions at 12 degrees C, we determined isometric-specific tension (P(o)), unloaded shortening velocity (v(o)) with the slack test protocol, and the rate constant of tension redevelopment (K(TR)) after a fast shortening-relengthening cycle. At the end of the mechanical experiment, we identified MyHC isoform composition of each fiber with gel electrophoresis. Electrophoretic migration rate of M MyHC was similar in both species. We found that in both species the kinetic parameters v(o) and K(TR) of M fibers were similar to those of 2A fibers, whereas P(o) values were significantly greater than in any other fiber types. The similarity between 2A and M fibers and the greater tension development of M fibers were confirmed also in mechanical experiments performed at 24 degrees C. Myosin concentration was determined in single fibers and found not different in M fibers compared with slow and fast fibers, suggesting that the higher tension developed by M fibers does not find an explanation in a greater number of force generators. The specific mechanical characteristics of M fibers might be attributed to a diversity in cross-bridge kinetics.

Changes in the muscle fibre properties of the mouse temporal muscle after weaning.

To clarify changes in the muscle fibre properties of the temporal muscle related to the start of masticatory movement, we immunohistochemically investigated myosin heavy chain (MyHC) isoform protein expression using pre-weaning and post-weaning mice. In addition, we examined the expression of a gene coding for those MyHC proteins. Immediately after weaning, isoforms with fast and potent contractility were frequent. This suggests that the temporal muscle plays an important role in a marked functional change in the oral cavity from lactation to mastication, contributing to oral function in cooperation with other masticatory muscles.

Heterogeneous postnatal transitions in myosin heavy chain isoforms within the rabbit temporalis muscle.

Postnatal changes in the fiber type composition and fiber cross-sectional area were investigated in the superficial (TEM1) and deep (TEM23) temporalis of male rabbits. It was hypothesized that, due to the transition from suckling to chewing during early postnatal development, the proportion of fast fiber types would decrease, while the proportion of fibers positive for myosin heavy chain (MyHC) cardiac alpha would increase, and that, due to the influence of testosterone during late postnatal development, the proportion of these alpha fibers would decrease again. Classification of the fibers types was performed by immunohistochemistry according to their MyHC content. The proportion of alpha fiber types significantly increased in both muscle portions from 2% and 8% for TEM1 and TEM23 at week 1 to 29% and 54% at week 8, respectively,. While in TEM1 the proportion of this fiber type did not change thereafter, it decreased again to 27% in TEM23 at week 20. The change for the fast fiber types was opposite to that of the alpha fiber types. Significantly more MyHC IIX fibers were found in TEM1 than in TEM23 in adult rabbits. In the first 8 weeks, the cross-sectional areas of all fibers increased. After this period, only MyHC cardiac alpha + I fibers continued to increase significantly. It was concluded that there are developmental differences in the myosin heavy chain transitions of the two portions of the temporalis muscle.

X-ray microanalysis of elements in the masticatory muscle after paresis of the right masseter.

Muscle activity and function appear to be related to ionic concentrations in the muscle. We investigated whether muscle paresis induced by injection of Botulinum toxin A (Botox) in 16-week-old pigs over a 56-day period is associated with ionic changes in the affected muscles. Tissue samples were taken from the masseter, temporalis, medial pterygoid, and geniohyoid muscles by a standardized method and used for energy-dispersive x-ray microanalysis in an environmental scanning electron microscope. The largest increase in Na(+) was measured in the right and left sides of the masseter muscle in treated animals. Additionally, a significant elevation of Na(+) was measured in the anterior part of the temporalis muscle and in the pterygoid muscle (P < 0.05). In temporalis and pterygoid muscles, an increase in sulfur in both sides of treated pigs' heads was observed. Botox((R)) has an indirect impact on ion concentrations, resulting in changes in muscle functional capacity and adaptive compensation of paretic muscle function by other muscles.

Myosin heavy-chain isoform composition of human single jaw-muscle fibers.

Diversity in muscle contractile properties is based on the variability of contractile properties of single muscle fibers which in turn is related to the presence of different myosin heavy-chain (MyHC) isoforms. Human jaw muscles are featured by many hybrid fibers expressing more than one MyHC isoform. The purpose of this study was to determine the proportion of each isoform within these fibers for evaluation of the fiber's capacity of producing a large diversity in contractile properties. Electrophoretic separation of MyHC isoforms was performed on 218 single fibers of the temporalis and digastric muscles. Of these fibers, 100 were classified as hybrid fibers. Most hybrid fibers co-expressed MyHC-IIA and -IIX (n = 62); a smaller number co-expressed MyHC-I and -IIA (n = 14), MyHC-I and -IIX (n = 12), and MyHC-I, -IIA, and -IIX (n = 12). The proportions of the individual MyHC isoforms in the hybrid fibers varied highly, suggesting a large range of contractile properties among these fibers.

Histochemical studies of the masseter, the temporal and small zygomaticomandibular, and the temporomandibular masticatory muscles from aged male and female humans. Fiber types and myosin isoforms.

The purpose of this study was to investigate the histology of two small masticatory muscles from females and males of more than 70 years of age. By using immuno- and enzyme histochemistry the muscles were characterized by their fiber types and myosin heavy chain pattern. The observations were compared with similar studies of the masseter and temporalis muscles. Previously the two small muscles have been described based solely upon their gross anatomy. One muscle originates from the anterior, deep surface of the temporal fascia and inserts in the temporal tendon: the temporo-mandibular muscle (TM). The other muscle originates from the upper part of the temporal surface of the frontal process of the zygomatic bone and the adjacent part of the frontal bone and inserts in the temporal tendon: the zygomaticomandibular muscle (ZM). In the masseter, TM, and ZM, most of the autopsy samples contained an abundant number of fibers containing neonatal myosin heavy chains while in the temporal muscle specimens, such fibers were sparse and scattered. Electrophoresis followed by immuno-staining of Western blots supported the histochemical findings. There was no obvious correspondence between fiber typing based upon ATPase activity and the neonatal myosin heavy chain content in the muscle fibers. Neither did the fibers show accordance in their content of adult slow and fast myosin heavy chains and in their content of neonatal myosin heavy chain.

Regional differences in fibre type composition in the human temporalis muscle.

Anatomical and electromyographic studies point to regional differences in function in the human temporalis muscle. During chewing and biting the anterior portions of the muscle are in general more intensively activated and they are capable of producing larger forces than the posterior portions. It was hypothetised that this heterogeneity in function is reflected in the fibre type composition of the muscle. The composition and surface area of different fibre types in various anteroposterior portions of the temporalis muscle were investigated in 7 cadavers employing immunohistochemistry with a panel of monoclonal antibodies against different isoforms of myosin heavy chain. Pure slow muscle fibres, type I, differed strongly in number across the muscle. In the most posterior portion of the muscle there were 24% type I fibres, in the intermediate portion 57%, and in the most anterior portion 46%. The mean fibre cross-sectional area (m-fcsa) of type I fibres was 1849 microm2, which did not differ significantly across the muscle. The proportion of pure fast muscle fibres, type IIA and IIX, remained more or less constant throughout the muscle at 13% and 11% respectively; their m-fcsa was 1309 microm2 and 1206 microm2, respectively, which did not differ significantly throughout the muscle. Pure type IIB fibres were not found. The relative proportion of hybrid fibres was 31% and did not differ significantly among the muscle portions. Fibre types I + IIA and cardiac alpha + I + IIA were the most abundant hybrid fibre types. In addition, 5% of the type I fibres had an additional myosin isoform which has only recently been described by means of electrophoresis and was named Ia. In the present study they were denoted as hybrid type I + Ia muscle fibres. It is concluded that intramuscular differences in type I fibre distribution are in accordance with regional differences in muscle function.

Second primary rhabdomyosarcomas in patients with bilateral retinoblastoma: a clinicopathologic and immunohistochemical study.

We reviewed six cases of rhabdomyosarcoma as a rare second primary malignancy in children with bilateral retinoblastoma after irradiation treatment. The patients comprised four females and two males (age range 1 year 4 months-7 years 11 months). Second tumors arose in the temporal muscle inside or close to the previously irradiated fields. All the children were alive and well 24-72 months after diagnosis. Microscopic examination showed proliferation of closely packed, small round cells with scanty cytoplasm, coarse nuclear chromatin, and increased mitotic activity without a myxoid background nor obvious alveolar architecture. The most characteristic feature was the presence of rosette-like structures in four tumors. Immunoreactivity for many skeletal muscle markers was evident, including desmin (six of six), muscle-specific actin (HHF35) (six of six), sarcomeric actin (six of six), myogenin (six of six), vimentin (six of six), and myoglobin (three of six). On reverse transcriptase-polymerase chain reaction examination, three second tumors lacked specific chimeric transcripts for alveolar rhabdomyosarcoma and Ewing's sarcoma. Unexpectedly, variable reactivity for neurofilament (150 kd) was identified in six of six second tumors as well as 15 of 20 sporadic primary rhabdomyosarcomas (75%) examined as controls, the result being confirmed by Western blot analysis. In addition, staining for retinoblastoma-susceptibility gene protein was negative in all second tumors, in contrast to positivity in 14 of 17 sporadic primary tumors (82%). This finding suggests that retinoblastoma-susceptibility gene abnormalities could be associated with the development of second primary rhabdomyosarcoma. We consider that knowledge of the occurrence of rhabdomyosarcoma and appropriate immunohistochemical study are helpful for avoiding a misdiagnosis of recurrent retinoblastoma or Ewing's sarcoma when encountering patients with a history of bilateral retinoblastoma who developed second small round cell neoplasms.

Adaptations of masticatory muscles to a hyperpropulsive appliance in the rat.

The plasticity of masticatory muscles was studied by comparing rats that were wearing a protrusive appliance and were kept on a liquid diet with two control groups: (1) pair-fed rats and (2) rats that had free access to ordinary pelleted food. The animals were 45 days old at the beginning of the experiment and were studied for a period of 20 days. Three jaw muscles with different functions were examined: masseter, temporalis, and digastric. Muscle fiber composition was determined (1) by fiber counting after staining with four monoclonal antibodies, which were able to recognize the four major myosin heavy chain (MHC) isoforms and therefore four fiber types (I, IIA, IIX, IIB) and (2) by electrophoresis on 6% polyacrylamide gels. The comparison between free-diet rats and pair-fed rats showed that the change from a hard pelleted diet to a liquid diet caused a shift in fiber type and MHC distribution, characterized by an increase of IIB MHC in temporalis and digastric muscles but not in the masseter muscle. The comparison between pair-fed rats and rats wearing appliances showed on the contrary a decrease in IIB MHC and an increase in IIA and IIX MHC. The results support the conclusions that (1) rat jaw muscles can quickly adapt to functional demand changing their fiber type composition, (2) the changes appear restricted inside the fast fiber population, and (3) fiber-type changes caused by dietary variation are not less than those caused by orthodontic intervention and must be taken into account to assess the effect of the appliance correctly.

Expression of superfast myosin in aneural regenerates of cat jaw muscle.

We investigated whether innervation is necessary for the expression of superfast myosin in regenerating cat jaw-closing muscle. Strips of jaw muscle were permitted to regenerate bilaterally in the beds of a fast limb muscle with innervation on one side being prevented surgically. Immunocytochemical analyses using anti-myosin heavy chain antibodies were done at various times postoperatively, the latest being after 78 days. We found little difference between innervated and uninnervated regenerates up to 27 days postoperatively. All regenerating myotubes expressed fetal myosin. In addition, some myotubes stained for superfast or slow myosin, while others stained for both superfast and slow myosins. Subsequently, uninnervated myotubes became atrophic but continued to express fetal, slow, and superfast myosins while innervated myofibers suppressed fetal and slow myosin expression. These results are consistent with the notion that satellite cells of jaw-closing muscles are committed to express superfast myosin during myogenesis, and that the expression of this program is independent of innervation.