Magnetic implants in the tongue for assistive technologies: Tests of migration; oromotor function; and tissue response in miniature pigs.

OBJECTIVE: Uncertain biological consequences of titanium-magnet (Ti-mag) tongue implants constrain application of the Tongue Drive System (TDS), a brain-tongue-computer interface for individuals with severe physical impairment. Here we describe oromotor function and tongue tissue response following Ti-Mag implantation and explantation in the miniature pig, an animal model with a tongue similar in size to humans. DESIGN: A 1.8×6.2mm Ti-mag tracer was implanted into the anterior tongue in five Yucatan minipigs. X-rays were taken immediately and >six days after implantation to evaluate tracer migration. In three minipigs, the tracer was explanted >16days after implantation. Twenty-five days post-explantation, tongue tissue was harvested and processed for histological and immunohistochemical (IHC) markers of healing. In two minipigs tissue markers of healing were evaluated post-mortem following >12days implantation. Drink cycle rate (DCR) was characterized to determine the impact of procedures on oromotor function. RESULTS: Neither implantation (N=5) nor explantation (N=3) changed DCR. X-rays revealed minimal tracer migration (N=4, 0-4mm). By histology and IHC a robust capsule was present two weeks post-implantation with limited fibrosis. Explantation produced localized fibrosis and limited muscle remodeling. CONCLUSIONS: These findings suggest the safety of Ti-mag anterior tongue implants for assistive technologies in humans.

Absence of morphological and molecular correlates of sarcopenia in the macaque tongue muscle styloglossus.

INTRODUCTION: Equivocal decline of tongue muscle performance with age is compatible with resistance of the tongue to sarcopenia, the loss of muscle volume and function that typically occurs with aging. To test this possibility we characterized anatomical and molecular indices of sarcopenia in the macaque tongue muscle styloglossus (SG). METHODS: We quantified myosin heavy chain (MHC), muscle fiber MHC phenotype and size and total and phosphorylated growth- and atrophy-related proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblot and immunohistochemistry (IHC) in the SG in twenty-four macaque monkeys (Macaca rhesus, age range 9months to 31years) categorized into Young (<8years of age), Middle-aged (15-21years of age) and Old (>22years of age) groups. RESULTS: In Young, Middle and Old age groups, by SDS-PAGE MHCI comprised ~1/3 and MHCII ~2/3 of total MHC. MHCI relative frequency was lower and MHCII higher in Middle versus Young (p=0.0099) and Middle versus Old (p=0.052). Relative frequencies of MHC fiber phenotype were not different by age but were different by phenotype (rates 233, 641 and 111 per 1000 fibers for MHCI, MHCII and MHCI-II respectively, p=0.03). Few or no fibers were positive for developmental MHC. Mean cross-sectional area (CSA) was not different among the three age groups for MHCII and MHCI-II; however MHCI fibers tended to be larger in Middle versus Old and Young (mean=2257µm2,1917µm2 (p=0.05) and 1704µm2 (p=0.06), respectively). For each age group, mean CSA increased across MHC phenotype (lowest mean CSA for MHCI and highest mean CSA for MHCII). Spearman analysis demonstrated age-related increases in total p70 ribosomal protein S6 kinase (P70), phosphorylated P70421/424, phosphorylated P38 mitogen-activated protein kinase and muscle atrophy F-Box, a trend to age-related decrease in total extracellular signal-regulated kinase (ERK), and no age-related change in total protein kinase B (Akt/PKB), phosphorylated Akt, phosphorylated ERK, phosphorylated c-Jun N-terminal kinase (JNK46) and phosphorylated P70389. CONCLUSION: Common anatomical and molecular indices of sarcopenia are absent in our sample of macaque SG. Relative frequencies of MHCII protein and phenotype are preserved with age. Although MAFbx expression increases with age, this is not associated with fiber atrophy, perhaps reflecting compensatory growth signaling by p70. The resistant nature of the styloglossus muscle to sarcopenia may be related to routine activation of tongue muscles in respiration and swallowing and the preservation of hypoglossal motoneuron number with age.

Absence of developmental and unconventional myosin heavy chain in human suprahyoid muscles.

INTRODUCTION: Contradictory reports of the myosin heavy chain (MHC) composition of adult human suprahyoid muscles leave unresolved the extent to which these muscles express developmental and unconventional MHC. METHODS: By immunohistochemistry, separation sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-Coomassie, separation SDS-PAGE-Western blot, and mRNA PCR, we tested for conventional MHCI, MHCIIA, MHCIIX, developmental MHC embryonic and MHC neonatal, and unconventional MHC alpha-cardiac, MHC extraocular, and MHC slow tonic in adult human anterior digastric (AD), geniohyoid (GH), and mylohyoid (MH) muscles. RESULTS: By separation SDS-PAGE-Coomassie and Western blot, only conventional MHC are present. By immunohistochemistry all muscle fibers are positive for MHCI, MHCIIA, or MHCIIX, and fewer than 4 fibers/mm(2) are positive for developmental or unconventional MHC. By PCR, mRNA of MHCI and MHCIIA dominate, with sporadically detectable MHC alpha-cardiac and without detectable mRNA of other developmental and unconventional MHC. CONCLUSIONS: We conclude that human suprahyoid muscles AD, GH, and MH are composed almost exclusively of conventional MHC isoforms.

Myosin heavy chain composition of the human genioglossus muscle.

BACKGROUND: The human tongue muscle genioglossus (GG) is active in speech, swallowing, respiration, and oral transport, behaviors encompassing a wide range of tongue shapes and movement speeds. Studies demonstrate substantial diversity in patterns of human GG motor unit activation, but whether this is accompanied by complex expression of muscle contractile proteins is not known. PURPOSE: The authors tested for conventional myosin heavy chain (MHC) MHCI, MHCIIA, MHCIIX, developmental MHCembryonic and MHCneonatal and unconventional MHCαcardiac, MHCextraocular, and MHCslow tonic in antero-superior (GG-A) and posterior (GG-P) adult human GG. METHOD: SDS-PAGE, Western blot, and immunohistochemistry were used to describe MHC composition of GG-A and GG-P and the prevalence of muscle fiber MHC phenotypes in GG-A. RESULTS: By SDS-PAGE, only conventional MHC are present with ranking from most to least prevalent MHCIIA > MHCI > MHCIIX in GG-A and MHCI > MHCIIA > MHCIIX in GG-P. By immunohistochemistry, many muscle fibers contain MHCI, MHCIIA, and MHCIIX, but few contain developmental or unconventional MHC. GG-A is composed of 5 phenotypes (MHCIIA > MHCI-IIX > MHCI > MHCI-IIA > MHCIIX). Phenotypes MHCI, MHCIIA, and MHCI-IIX account for 96% of muscle fibers. CONCLUSIONS: Despite activation of GG during kinematically diverse behaviors and complex patterns of GG motor unit activity, the human GG is composed of conventional MHC isoforms and 3 primary MHC phenotypes.

Changes in growth-related kinases in head, neck and limb muscles with age.

Sarcopenia coincides with declines in several systemic processes that signal through the MAP kinase and Akt-mTOR-p70S6k cascades typically associated with muscle growth. Effects of aging on these pathways have primarily been examined in limb muscles, which experience substantial activity and neural changes in addition to systemic hormonal and metabolic changes. Head and neck muscles are reported to undergo reduced sarcopenia and disuse with age relative to limb muscles, suggesting muscle activity may contribute to maintaining mass with age. However many head and neck muscles derive from embryonic branchial arches, rather than the somites from which limb muscles originate, suggesting that developmental origin may be important. This study compares the expression and phosphorylation of MAP kinase and mTOR networks in head, neck, tongue, and limb muscles from 8- and 26-month old F344 rats to test the hypothesis that physical activity and developmental origin contribute to preservation of muscle mass with age. Phosphorylation of p38 was exaggerated in aged branchial arch muscles. Phosphorylation of ERK and p70S6k T421/S424 declined with age only in the biceps brachii. Expression of p70S6k declined in all head and neck, tongue and limb muscles although no change in phosphorylation of p70S6k on T389 could be resolved. A systemic change that results in a loss of p70S6k protein expression may reduce the capacity to respond to acute hypertrophic stimuli, while the exaggerated p38 signaling in branchial arch muscles may reflect more active muscle remodeling.

Tri-partite complex for axonal transport drug delivery achieves pharmacological effect.

BACKGROUND: Targeted delivery of pharmaceutical agents into selected populations of CNS (Central Nervous System) neurons is an extremely compelling goal. Currently, systemic methods are generally used for delivery of pain medications, anti-virals for treatment of dermatomal infections, anti-spasmodics, and neuroprotectants. Systemic side effects or undesirable effects on parts of the CNS that are not involved in the pathology limit efficacy and limit clinical utility for many classes of pharmaceuticals. Axonal transport from the periphery offers a possible selective route, but there has been little progress towards design of agents that can accomplish targeted delivery via this intraneural route. To achieve this goal, we developed a tripartite molecular construction concept involving an axonal transport facilitator molecule, a polymer linker, and a large number of drug molecules conjugated to the linker, then sought to evaluate its neurobiology and pharmacological behavior. RESULTS: We developed chemical synthesis methodologies for assembling these tripartite complexes using a variety of axonal transport facilitators including nerve growth factor, wheat germ agglutinin, and synthetic facilitators derived from phage display work. Loading of up to 100 drug molecules per complex was achieved. Conjugation methods were used that allowed the drugs to be released in active form inside the cell body after transport. Intramuscular and intradermal injection proved effective for introducing pharmacologically effective doses into selected populations of CNS neurons. Pharmacological efficacy with gabapentin in a paw withdrawal latency model revealed a ten fold increase in half life and a 300 fold decrease in necessary dose relative to systemic administration for gabapentin when the drug was delivered by axonal transport using the tripartite vehicle. CONCLUSION: Specific targeting of selected subpopulations of CNS neurons for drug delivery by axonal transport holds great promise. The data shown here provide a basic framework for the intraneural pharmacology of this tripartite complex. The pharmacologically efficacious drug delivery demonstrated here verify the fundamental feasibility of using axonal transport for targeted drug delivery.

Sarcomeric myosin expression in the tongue body of humans, macaques and rats.

Expression of developmental and unconventional myosin heavy chain (MHC) isoforms in some adult head and neck muscles is thought to reflect specific contractile demands of muscle fibers active during kinematically complex movements. Mammalian tongue muscles are active during oromotor behaviors that encompass a wide range of tongue movement speeds and tongue shape changes (e.g. respiration, oral transport, swallowing, rejection), but the extent to which tongue muscles express developmental and unconventional MHC is not known. Quantitative PCR was used to determine the mRNA content of conventional MHC-beta, MHC-2a, MHC-2b and MHC-2x, the developmental isoforms embryonic MHC and neonatal MHC and the unconventional isoforms atrial/cardiac-alpha MHC (MHC-alpha), extraocular MHC, masseter MHC and slow tonic MHC in tongue body muscles of the rat, macaque and human. In all species, conventional MHC isoforms predominate. MHC-2b and MHC-2x account for 98% of total MHC mRNA in the rat. MHC-2a, MHC-2x and MHC-beta account for 94% of total MHC mRNA in humans and 96% of total MHC mRNA in macaque. With the exception of MHC-alpha in humans (5%), developmental and unconventional MHC mRNA represents less than 0.3% of total MHC mRNA. We conclude that in these species, there is limited expression of developmental and unconventional MHC and that diversity of tongue body muscle fiber contractile properties is achieved primarily by MHC-beta, MHC-2a, MHC-2x and MHC-2b. Whether expression of MHC-alpha mRNA in tongue is unique to humans or present in other hominoids awaits further investigation.

Myosin heavy-chain composition of the human hyoglossus muscle.

The human tongue muscle hyoglossus (HG) is active in oromotor behaviors encompassing a wide range of tongue movement speeds. Here we test the hypothesis that the human HG is composed of "uncommon" myosin heavy-chain (MHC) isoforms MHCembryonic, MHCneonatal, and MHCslow tonic as has been reported for other head and neck muscles active during kinematically diverse behaviors. Following reaction of human HG with antibodies specific for MHCI, MHCIIA, MHCII, MHCembryonic, MHCextraocular, MHCneonatal, and MHCslow tonic, only antibodies to MHCI, MHCIIA, and MHCII label more than occasional muscle fibers. These antibodies describe five phenotypes with prevalence MHCIIA > MHCI > MHCI-IIX > MHCI-IIA > MHCIIX. In MHC composition, the human HG is thus similar to human appendicular muscles and many human head and neck muscles but different from human masseter and extraocular muscles which contain five or more MHC isoforms.

Limited expression of slow tonic myosin heavy chain in human cranial muscles.

Recent reports of slow tonic myosin heavy chain (MHCst) in human masticatory and laryngeal muscles suggest that MHCst may have a wider distribution in humans than previously thought. Because of the novelty of this finding, we sought to confirm the presence of MHCst in human masticatory and laryngeal muscles by reacting tissue from these muscles and controls from extraocular, intrafusal, cardiac, appendicular, and developmental muscle with antibodies (Abs) ALD-58 and S46, considered highly specific for MHCst. At Ab dilutions producing minimal reaction to muscle fibers positive for MHCI, only extraocular, intrafusal, and fetal tongue tissue reacted with Ab S46 had strong immunoreaction in an appreciable number of muscle fibers. In immunoblots, Ab S46, but not Ab ALD-58, labeled adult extraocular muscles; no other muscles were labeled with either Ab. We conclude that, in humans, Ab S46 has greater specificity for MHCst than does Ab ALD-58. We suggest that reports of MHCst in human masticatory and laryngeal muscles reflect false-positive identification of MHCst due to cross-reactivity of Ab ALD-58 with another MHC isoform.

Immunohistochemical characterization of slow and fast myosin heavy chain composition of muscle fibres in the styloglossus muscle of the human and macaque (Macaca rhesus).

OBJECTIVE: Muscle fibre contractile diversity is thought to be increased by the hybridization of multiple myosin heavy chain (MHC) isoforms in single muscle fibres. Reports of hybrid fibres composed of MHCI and MHCII isoforms in human, but not macaque, tongue muscles, suggest a human adaptation for increased tongue muscle contractile diversity. Here we test whether hybrid fibres composed of MHCI and MHCII are unique to human tongue muscles or are present as well in the macaque. METHODS: MHC composition of the macaque and human styloglossus was characterized with antibodies that allowed identification of three muscle fibre phenotypes, a slow phenotype composed of MHCI, a fast phenotype composed of MHCII and a hybrid phenotype composed of MHCI and MHCII. RESULTS: The fast phenotype constitutes 68.5% of fibres in the macaque and 43.4% of fibres in the human (P<0.0001). The slow phenotype constitutes 20.2% of fibres in the macaque and 39.3% of fibres in the human (P<0.0001). The hybrid phenotype constitutes 11.2% of fibres in the macaque and 17.3% of fibres in the human (P=0.0002). Macaques and humans do not differ in fiber size (cross-sectional area, diameter). However, measures of fibre size differ by phenotype such that fast>hybrid>slow (P<0.05). CONCLUSION: These data demonstrate differences in the relative percent of muscle fibre phenotypes in the macaque and human styloglossus but also demonstrate that all three phenotypes are present in both species. These data suggest a similar range of mechanical properties in styloglossus muscle fibres of the macaque and human.

Neuromuscular organization of the superior longitudinalis muscle in the human tongue. 1. Motor endplate morphology and muscle fiber architecture.

Proper tongue function is essential for respiration and mastication, yet we lack basic information on the anatomical organization underlying human tongue movement. Here we use microdissection, acetylcholinesterase histochemistry, silver staining of nerves, alpha bungarotoxin binding and immunohistochemistry to describe muscle fiber architecture and motor endplate (MEP) distribution of the human superior longitudinalis muscle (SL). The human SL extends from tongue base to tongue tip and is composed of fiber bundles that range from 2.8 to 15.7 mm in length. Individual muscle fibers of the SL range from 1.2 to 17.3 mm in length (1.3-18.2% of muscle length). Seventy-one percent of SL fibers have blunt-blunt terminations; the remainder have blunt-taper terminations. Multiple MEPs are present along SL length and dual MEPs are present on some muscle fibers. These data demonstrate that the human SL is a muscle of "in-series" design. We suggest that SL motor units are organized to innervate specific regions of the tongue body and that activation of SL motor units according to anteroposterior location is one strategy employed by the nervous system to control tongue shape and tongue movement.