The Specific Molecular Composition and Structural Arrangement of Eleutherodactylus Coqui Gular Skin Tissue Provide Its High Mechanical Compliance.

A male Eleutherodactylus Coqui (EC, a frog) expands and contracts its gular skin to a great extent during mating calls, displaying its extraordinarily compliant organ. There are striking similarities between frog gular skin and the human bladder as both organs expand and contract significantly. While the high extensibility of the urinary bladder is attributed to the unique helical ultrastructure of collagen type III, the mechanism behind the gular skin of EC is unknown. We therefore aim to understand the structure-property relationship of gular skin tissues of EC. Our findings demonstrate that the male EC gular tissue can elongate up to 400%, with an ultimate tensile strength (UTS) of 1.7 MPa. Species without vocal sacs, Xenopus Laevis (XL) and Xenopus Muelleri (XM), elongate only up to 80% and 350% with UTS~6.3 MPa and ~4.5 MPa, respectively. Transmission electron microscopy (TEM) and histological staining further show that EC tissues' collagen fibers exhibit a layer-by-layer arrangement with an uninterrupted, knot-free, and continuous structure. The collagen bundles alternate between a circular and longitudinal shape, suggesting an out-of-plane zig-zag structure, which likely provides the tissue with greater extensibility. In contrast, control species contain a nearly linear collagen structure interrupted by thicker muscle bundles and mucous glands. Meanwhile, in the rat bladder, the collagen is arranged in a helical structure. The bladder-like high extensibility of EC gular skin tissue arises despite it having eight-fold lesser elastin and five times more collagen than the rat bladder. To our knowledge, this is the first study to report the structural and molecular mechanisms behind the high compliance of EC gular skin. We believe that these findings can lead us to develop more compliant biomaterials for applications in regenerative medicine.

A mutation in the Warburg syndrome gene, RAB3GAP1, causes a similar syndrome with polyneuropathy and neuronal vacuolation in Black Russian Terrier dogs.

An autosomal recessive disease of Black Russian Terriers was previously described as a juvenile-onset, laryngeal paralysis and polyneuropathy similar to Charcot Marie Tooth disease in humans. We found that in addition to an axonal neuropathy, affected dogs exhibit microphthalmia, cataracts, and miotic pupils. On histopathology, affected dogs exhibit a spongiform encephalopathy characterized by accumulations of abnormal, membrane-bound vacuoles of various sizes in neuronal cell bodies, axons and adrenal cells. DNA from an individual dog with this polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV) was used to generate a whole genome sequence which contained a homozygous RAB3GAP1:c.743delC mutation that was absent from 73 control canine whole genome sequences. An additional 12 Black Russian Terriers with POANV were RAB3GAP1:c.743delC homozygotes. DNA samples from 249 Black Russian Terriers with no known signs of POANV were either heterozygotes or homozygous for the reference allele. Mutations in human RAB3GAP1 cause Warburg micro syndrome (WARBM), a severe developmental disorder characterized by abnormalities of the eye, genitals and nervous system including a predominantly axonal peripheral neuropathy. RAB3GAP1 encodes the catalytic subunit of a GTPase activator protein and guanine exchange factor for Rab3 and Rab18 respectively. Rab proteins are involved in membrane trafficking in the endoplasmic reticulum, axonal transport, autophagy and synaptic transmission. The neuronal vacuolation and membranous inclusions and vacuoles in axons seen in this canine disorder likely reflect alterations of these processes. Thus, this canine disease could serve as a model for WARBM and provide insight into its pathogenesis and treatment.

Evolution of Courtship Songs in Xenopus : Vocal Pattern Generation and Sound Production.

The extant species of African clawed frogs (Xenopus and Silurana) provide an opportunity to link the evolution of vocal characters to changes in the responsible cellular and molecular mechanisms. In this review, we integrate several robust lines of research: evolutionary trajectories of Xenopus vocalizations, cellular and circuit-level mechanisms of vocalization in selected Xenopus model species, and Xenopus evolutionary history and speciation mechanisms. Integrating recent findings allows us to generate and test specific hypotheses about the evolution of Xenopus vocal circuits. We propose that reduced vocal sex differences in some Xenopus species result from species-specific losses of sexually differentiated neural and neuromuscular features. Modification of sex-hormone-regulated developmental mechanisms is a strong candidate mechanism for reduced vocal sex differences.

Myosin heavy chain-2b transcripts and isoform are expressed in human laryngeal muscles.

Three fast myosin heavy chain (MyHC) isoforms, i.e. MyHC-2a, -2x and -2b, are expressed in skeletal muscles of smaller mammals. In contrast, only MyHC-2a and -2x have been revealed in humans so far. The expression of MyHC isoforms is known to be wider in the functionally more specialized laryngeal muscles. Though mRNA transcripts of the MyHC-2b gene were found to be expressed in certain human skeletal and laryngeal muscles, the corresponding isoform has not been demonstrated in these muscles. To our knowledge, we are the first to demonstrate not only the expression of MyHC-2b transcripts using an in situ hybridization technique but also the corresponding protein, i.e. the MyHC-2b isoform, in some human laryngeal muscles by immunohistochemistry but not by polyacrylamide gel electrophoresis. Using a set of antibodies specific to MyHC isoforms, we demonstrated that MyHC-2b was always co-expressed with the major MyHC isoforms, not only with the fast ones (MyHC-2a and -2x) but with the slow isoform (MyHC-1) as well.

Decellularized rabbit cricoarytenoid dorsalis muscle for laryngeal regeneration.

OBJECTIVES: Although considerable progress has been made in regenerative medicine, a quantum step would be the replacement and/or regeneration of functional muscle tissue. For example, although patients' airways can now be successfully replaced with stem cell-based techniques, a much greater patient need would be addressed by regeneration of the muscles required for engineering a functional larynx, in which active movement is critical. The rabbit cricoarytenoid dorsalis muscle was chosen for the present study because it is equivalent to the posterior cricoarytenoid muscle, the only significant abductor muscle in human larynges. METHODS: Rabbit cricoarytenoid dorsalis muscles were harvested, and different decellularization methods were compared by use of a combination of histologic, immunohistochemical, and molecular techniques. Decellularized scaffolds were implanted into Sprague-Dawley rats as part of a 2-week biocompatibility study to assess immunogenicity. RESULTS: Decellularization with a combination of latrunculin B, potassium iodide, potassium chloride, and deoxyribonuclease resulted in total DNA clearance and reduced levels of major histocompatibility complex class II expression, with relative preservation of the scaffold's structural integrity (collagen, elastin, and glycosaminoglycan content). The scaffolds showed minimal signs of rejection at 2 weeks in a cross-species (xenotransplantation) study. CONCLUSIONS: Decellularized laryngeal muscles, which are nonimmunogenic, may provide the optimal scaffold source for the generation of a fully functional tissue-engineered larynx.

Estimation of the number and size of motor units in intrinsic laryngeal muscles using morphometric methods.

The number and size of motor units in the intrinsic laryngeal muscles were estimated by morphometric methods. Laryngeal muscles with their respective nerve branches were obtained from 64 fresh cadavers (32 older than 60 years, mean age 74 +/- 9 years and 32 younger than 60 years, mean age 51 +/- 8 years). Myelinated nerve fibers and the total number of muscle fibers were counted. Motor unit size was estimated by dividing the total number of muscle fibers by the total number of motor units in each case. The mean number of motor units ranged from 268 +/- 1.3 (interarytenoid muscle) to 431 +/- 1.6 (cricothyroid muscle). Thyroarytenoid and cricothyroid muscle presented the smallest (9.8 +/- 0.2) and largest (20.5 +/- 0.9) motor unit size, respectively, suggesting that thyroarytenoid muscle has a greater capacity to fine-tune its total force compared with the other intrinsic laryngeal muscles. No differences in motor unit number or size were observed between the right and left sides or between younger and older subjects. It is suggested that synaptic rearrangements may occur at the level of the neuromuscular junction in the human larynx that may explain the age-related changes in motor units reported by clinical methods.

A complex muscle fiber network in the cricothyroid muscle: a scanning electron microscopic study.

OBJECTIVES: To examine the three-dimensional ultrastructure of cricothyroid (CT) muscle fibers to elucidate their morphologic characteristics with regard to the specific functions of the muscle. STUDY DESIGN: An anatomic animal study. METHODS: The CT muscles of five adult rats were processed using the HCl-hydrolysis method to remove the peri- and intramuscular connective tissue components. The fine muscle fiber arrangements were observed by scanning electron microscopy. RESULTS: Complex muscle fiber interconnections (myomyous junctions) were identified in the muscles. Myomyous junctions were characterized by the connection of many villous processes that were 1 to 2 mum in diameter and 2 to 4 mum in length with a serrated appearance at the ends of a lateral branch or a bifurcating trunk of the muscle fibers. CONCLUSIONS: Myomyous junctions form a complex muscle fiber network, which is thought to synchronize the activity of the CT muscle fibers. This is the first three-dimensional demonstration of the muscle fiber network in the CT muscle; this network may play a major role in the functional specificity of the CT muscle.

Small clusters of granule-containing cells at the lateral side of the posterior cricoarytenoid muscle of the young adult rat.

Small clusters consisting of granule-containing cells, sustentacular cells and capillaries around them, similar in structure to the carotid body-like paraganglia, sometimes existed at the lateral side of the posterior cricoarytenoid (PCA) muscle of young adult (3 months) rats. Differing from the paraganglia, however, these cell clusters were discontinuously invested by slender cytoplasmic processes of fibroblasts. In individual granule-containing cells, granules varied in size and had a concentrically or eccentrically arranged, electron-dense material, resembling those of chromaffin cells of the adrenal medulla. A series of desmosome-like structures were frequently observed between adjacent granule-containing cells, but synapses between them were not necessarily clear. Nerve endings containing clear synaptic vesicles and occasional granulated vesicles, being possibly cholinergic in nature, sometimes formed synapses with the granule-containing cells, probably indicating that the granule-containing cells receive the efferent nerve innervation. On the other hand, the sustentacular cells lacked cytoplasmic granules and sent their cytoplasmic processes around the granule-containing cells. Capillaries in and around clustered cells were of the fenestrated type. From these findings, it is suggested that unlike the carotid body-like paraganglia, the noncapsulated cell clusters at the lateral side of the PCA muscle of the young adult rat may be identical to groups of extra-adrenal chromaffin tissues.

Effect of recurrent laryngeal nerve section on the laryngeal muscles of adult rabbits.

OBJECTIVE: To examine the effect of recurrent laryngeal nerve section on myonuclear addition and myofiber remodeling in adult rabbit laryngeal muscles. STUDY DESIGN AND SETTING: Unilateral section of the recurrent laryngeal nerve was performed on adult rabbits. Rabbits were labeled with bromodeoxyuridine, euthanized, and the laryngeal muscles were examined for bromodeoxyuridine (brdU) and neonatal myosin heavy chain (MyHC) expression. RESULTS: One week after nerve section, brdU-positive myonuclei were significantly increased on the denervated and contralateral sides. Neonatal MyHC expression significantly increased. By 24 weeks, the upregulation of myonuclear addition returned to control levels. CONCLUSION: Laryngeal muscle denervation results in significant upregulation of myonuclear addition. Denervation also resulted in a significant upregulation of neonatal MyHC expression. SIGNIFICANCE: Laryngeal muscles can be successfully reinnervated after long periods of denervation. This significant and maintained increase in myonuclear addition may contribute to the ability of laryngeal muscles to survive for extended periods following interruption of the recurrent laryngeal nerve.

Scanning electron microscopic study of the muscle fiber arrangement in the rat cricopharyngeal muscle.

CONCLUSIONS: Myomyous junctions comprise a complex muscle fiber network, which is thought to synchronize the activity of the cricopharyngeal (CP) muscle fibers. Myomyous and myotendinous junctions explain the heterogeneity in muscle fiber length which contributes to the efficient behavior of the muscle. This scanning electron microscopy (SEM) study demonstrated the complex muscle fiber arrangement of the CP muscle and improved on the previous description of its morphological specificity. OBJECTIVE: To examine the 3D ultrastructure of the inferior pharyngeal constrictor muscle fibers to obtain further knowledge of their morphological characteristics with regard to the specific functions of the muscle in deglutition. MATERIAL AND METHODS: Six adult rats were used. Their CP and thyropharyngeal (TP) muscles were obtained and processed using the HCl hydrolysis method to remove i.m. connective tissue. The fine muscle fiber structure was observed by means of SEM. RESULTS: Multifaceted muscle fiber interconnections (myomyous junctions) were identified in the CP muscle. The myomyous junctions were characterized by the tight connection of many finger-like processes at the ends of a lateral branch or bifurcating trunk of the muscle fibers. In addition, muscle fibers occasionally tapered and ended within the muscle belly, forming myotendinous junctions. The TP muscle lacked these structures.

Motor innervation of the guinea pig interarytenoid muscle: reinnervation process following unilateral denervation.

The authors investigated the process of denervation and reinnervation of the interarytenoid (IA) muscle in the guinea pig using transmission electron microscopy and glycogen depletion technique after unilateral transection of the recurrent laryngeal nerve (RLN) and superior laryngeal nerve to clarify the innervation pattern of the unpaired IA muscle. Anastomosis between the bilateral arytenoid branches was confirmed in the belly of the IA muscle. Five weeks after transection, all of the IA muscle fibers appeared to have been reinnervated by the contralateral RLN. As the arytenoid branch of the RLN runs together with that of the contralateral RLN in a single intramuscular nerve funiculus, it is possible that collateral sprouting branches grow and extend into the adjacent denervated Schwann's sheaths. The authors conclude that the unpaired IA muscle, as a whole, receives specific motor nerve supply from the bilateral RLNs, although each muscle fiber is innervated unilaterally.

Human insulinlike growth factor 1 gene transfer into paralyzed rat larynx: single vs multiple injection.

OBJECTIVE: To compare the biological effects of single vs multiple treatment of rat denervated laryngeal muscle with human insulinlike growth factor 1 (hIGF1) gene therapy. EXPERIMENTAL METHODS OR DESIGN: A muscle-specific nonviral vector containing the alpha-actin promoter and hIGF1 gene formulated with polyvinyl polymers was injected into denervated adult rat thyroarytenoid muscle. The effects on animals given a single injection (n = 16) vs those given multiple injections (n = 14) vs control groups (n = 18) were evaluated. Twenty-eight days after the first injection, gene expression, muscle fiber size, motor endplate length, and nerve-to-motor endplate contact were evaluated. RESULTS: Gene expression, detected by reverse transcriptase polymerase chain reaction for hIGF1 messenger RNA, occurred in 13 (81%) of 16 animals receiving single injections and 14 (100%) of 14 animals receiving multiple injections. Compared with controls, hIGF1-transfected animals in both single- and multiple-injection groups had a significant increase in the lesser diameter of muscle fiber, a significant decrease in motor endplate length, and a significant increase in the percentage of endplates with nerve contact (P <.05 for all). There was no statistical difference between single- and multiple-injection groups. CONCLUSIONS: Applied to laryngeal paralysis, hIGF1 gene therapy provides an opportunity to augment surgical treatment modalities by the prevention or reversal of muscle atrophy, and enhancement of nerve sprouting and muscle reinnervation. Although the percentage of denervated muscles demonstrating hIGF1 expression was increased following multiple injections, no difference was observed in the biological response compared with that in the single-injection treatment groups. Further investigation will be conducted to assess long-term benefits and physiological responses and to define the limitations of this potentially valuable therapeutic strategy.

Numerical densities of myonuclei and satellite cells in muscle fiber types in the aging human thyroarytenoid muscle: an immunohistochemical and stereological study using confocal laser scanning microscopy.

OBJECTIVE: This study determines the role of changes in numerical densities of myonuclei and satellite cells in age-related remodeling of the thyroarytenoid muscle (TA). DESIGN: Changes in numerical densities (N(V)) and ratios (N(N)) of myonuclei and satellite cells were estimated for the entire TA by use of stereological techniques. RESULTS: There was no age-related change or difference between fiber types for N(V myonucleus, fiber), but N(V myonucleus, fiber) increased with decreasing fiber diameter. There was a trend toward a decrease in N(V satellite cell, fiber) and a decrease in N(N satellite cell, myonucleus). N(V satellite cell, fiber) was higher for type 1 than for type 2 fibers, and type 1 satellite cells increased disproportionately with increasing total satellite cell numerical density. CONCLUSION: Decreased satellite cell proliferation may contribute to age-related fiber loss and atrophy in the TA. SIGNIFICANCE: Therapeutic techniques based on activation of satellite cells may block age-related fiber loss and atrophy in the TA.

Age- and gender-related elastin distribution changes in human vocal folds.

The composition of the lamina propria in human vocal folds has been shown to affect vocal performance. Elastin plays a significant role in the biomechanical effects of the lamina propria. We obtained 19 larynges from the state medical examiner from subjects whose cause of death was unrelated to the trachea and laryngeal regions. The sample contained male and female subjects in the infant, adult, and geriatric age groups. We stained the vocal folds for elastin with Verhoeff's elastic tissue stain and studied them with use of an image analysis system configured for light microscopy. Distributions of elastin were measured from superficial to deep within the lamina propria (from epithelium to vocal muscle). These elastin distributions were then compared with the use of statistical software. The data showed that there was an increase in elastin content from the infant through geriatric stages. No gender-related differences were found. Infant folds had about 23% of the elastin found in adults, and geriatric subjects had about 879% of the elastin found in adults. Both of these results were statistically significant (p < 0.05). The distributions were consistent with previous observations that the lamina propria is a layered structure with most of the elastin present in the intermediate layer. This layer was larger in geriatric subjects than in adult and pediatric subjects. We observed that the fiber diameter appeared to be larger in geriatric subjects (this observation is currently being verified with electron microscopy) whereas smaller, spiraled fibers appeared in pediatric subjects.

Botulinum toxin for relief of bilateral abductor paralysis of the larynx: histologic study in an animal model.

We previously reported the effectiveness of botulinum toxin injections in the cricothyroid muscle under electromyographic guidance for lateralization of the true vocal cord in the mongrel dog. These additional experiments were performed to substantiate the effectiveness of botulinum toxin injections in laryngeal muscle to overcome airway obstruction produced by bilateral abductor vocal cord paralysis. A predetermined aliquot of the toxin was injected into the cricothyroid muscle of ten dogs, the duration of its effectiveness was noted, and repeated injections were given the animals. Biopsies of the injected muscles were obtained in three of the animals for routine histologic and electron microscopic studies. The effects of the toxin were recorded by cinelaryngoscopy and videotape documentation. In all the dogs, the true vocal cord was lateralized effectively and there was no morbidity, dysphagia, aspiration, or deaths. This study also confirms that the effects of the toxin in the canine larynx are spontaneously reversed and that multiple injections do not cause irreparable damage to the laryngeal muscles.

Thyroarytenoid muscle: functional subunits based on morphology and muscle fiber typing in cats.

Using parvalbumin immunohistochemistry to determine the distribution of muscle fiber types in the feline thyroarytenoid muscle (TA), we clearly distinguished the vocalis (with predominance of "slow" type 1 fibers) from the external TA (in which "fast" type 2 fibers predominated, especially in its rostral part). Reconstruction of serial frontal sections of the TA allowed the stereoscopic study of each division. The existence of a rudimentary laryngeal ventricle separating the true and false vocal folds in cats was demonstrated anatomically and histologically, and its relationships to each division of the TA were established. Our results suggest that the vocalis, fitted for enduring activities, is suited for voice control. The fast, rostral part of the external TA seems suited to laryngeal sphincteric demands, while its caudal counterpart may act in both functions. The anatomic individualization of the divisions of the TA may suggest that they play distinct physiological roles and may imply that they should not be considered a single functional unit.

Muscle transposition in the rehabilitation of the paralyzed larynx.

Muscle transposition of the innervated omohyoid muscle into the tendon of the paralyzed posterior cricoarytenoid muscle was studied in a small series of subhuman primates. A similar procedure was described previously by King for treatment of bilateral recurrent nerve injuries. Our studies confirm that this procedure adequately enlarges the airway to allow normal activity without a tracheostomy. Histologic studies appear to show nerve ingrowth occurring at 6 months as in other neuromuscular pedicle procedures. The procedure is easily performed and causes no other muscle denervation.

Electron microscopic study of muscle spindle in human interarytenoid muscle.

Using transmission electron microscopy, the fine structure of the muscle spindle in the interarytenoid muscle was studied. The specimen was made from a fresh adult male larynx which had been surgically removed. The muscle spindle showed the following characteristic features: (1) the capsule was thin, with only a small number of layers of capsular sheet cells; (2) the periaxial space was narrow and there were abundant collagenous fibrils; (3) the sensory endings consisted of irregularly coiled terminal axons with varicose swellings, and some endings penetrated the sarcoplasm; (4) one nuclear chain fiber terminated on the capsule, while the other branched into two bundles; and (5) special sarcoplasmic inclusions, possibly related to the sensory endings, were also observed. These findings suggest that the muscle spindle in the interarytenoid muscle is not only a very sensitive stretch receptor, but also a pressure receptor.

Scanning electron microscopic study of the muscle fiber ends at the myotendinous junction in the posterior cricoarytenoid and cricothyroid muscles in rats.

The fine structural organization of muscle fiber ends at the myotendinous junction in the posterior cricoarytenoid (PCA) and cricothyroid (CT) muscles in adult rats was studied by scanning electron microscopy, after removal of tendon connective tissues using the HCI-hydrolysis method. The muscle fiber ends in the PCA muscle had a relatively simple conical appearance and contained a great number of longitudinal slits on the surface. In the CT muscle, the muscle fiber ends were classified into two types. One type had a conical appearance similar to the PCA muscle with many slits on the surface, while the other type was characterized by a complex arrangement of cylindrical cytoplasmic processes and deep clefts, as seen in common skeletal muscles. These findings suggest that the PCA muscle is evolutionarily primitive, and that the CT muscles represent a transitional form between primitive and evolved muscles.

Scanning electron microscopic study of the neuromuscular junctions of the cricothyroid and thyroarytenoid muscles in rats.

Neuromuscular junctions were observed in the cricothyroid (CT) and thyroarytenoid (TA) muscles of adult rats by scanning electron microscopy after removing the intramuscular connective tissue components using the HCI hydrolysis method. Morphologically, the junctions were classified into three types in the CT muscle and two types in the TA muscle, based on the structural characteristics of the subneural apparatuses, including junctional folds. In the CT muscle, type 1 junctions (32%) consisted of more than 15 cup-like depressions with slit-like junctional folds. Type 2 junctions (20%) were characterized by approximately 10 cup-like depressions with a small number of pit- or slit-like junctional folds. Type 3 junctions (48%) had irregular labyrinthine gutters with slit-like junctional folds. In the TA muscle, type 1 (82%) and 2 (18%) junctions had similar structures to type 1 and 2 junctions in the CT muscle, respectively. Histochemical studies using myosin adenosine triphosphatase staining showed that both CT and TA muscles predominantly consisted of type II muscle fibers (78% and 82%, respectively), and that the diameter of type II fibers was larger than that of type I fibers. These findings suggest that the type 2 junction belongs to type I muscle fibers, while both type 1 and type 3 junctions belong to type II fibers, and that the type 3 junction is a structural variation of the type 1 junction. The significance of the structural differences of the subneural apparatuses in the intrinsic laryngeal muscles is discussed briefly.