Single, high-dose local injection of bFGF improves thyroarytenoid muscle atrophy after paralysis.

OBJECTIVES/HYPOTHESIS: Unilateral vocal fold paralysis (UVFP) induces hoarseness due to progressive atrophy of the denervated thyroarytenoid (TA) muscle. Therefore, treatments aimed at regenerating the atrophied TA muscle are required. Basic fibroblast growth factor (bFGF) is involved in muscle development and regeneration. This study aimed to elucidate the effects of bFGF injection on atrophied TA muscle. STUDY DESIGN: Animal research. METHODS: A recurrent laryngeal nerve-paralysis rat model was established, and low- (200 ng) or high-dose (2,000 ng) bFGF or saline (control) was injected into the TA muscle 28 days later. The larynges were excised on day 1, 3, 7, 14, and 28 after treatment. The cross-sectional area of the TA muscle in normal and paralyzed sides was compared, and the Ki67-positive (Ki67+ ) dividing cells, paired box 7-positive (Pax7+ ) satellite cells (SCs), and myogenic differentiation-positive (MyoD+ ) myoblasts were counted. RESULTS: The TA muscle area of animals administered high-dose bFGF increased with time and was significantly larger than that of the saline-injected controls 28 days after treatment (P < .05). The counts of Ki67+ and Pax7+ cells were the highest on day 1, whereas the MyoD+ myoblast count was highest on day 7. These results suggest that bFGF administration into the denervated TA muscles compensated for the atrophied TA muscles by inducing proliferation of SCs and their differentiation to myoblasts. CONCLUSIONS: A single injection of high-dose bFGF augmented regeneration and differentiation of the atrophied TA muscle by enhancing proliferation and differentiation of muscle SCs, suggesting its possible clinical application in humans with UVFP. LEVEL OF EVIDENCE: NA Laryngoscope, 130:159-165, 2020.

Creation of Laryngeal Grafts from Primary Human Cells and Decellularized Laryngeal Scaffolds.

Current reconstruction methods of the laryngotracheal segment fail to replace the complex functions of the human larynx. Bioengineering approaches to reconstruction have been limited by the complex tissue compartmentation of the larynx. We attempted to overcome this limitation by bioengineering laryngeal grafts from decellularized canine laryngeal scaffolds recellularized with human primary cells under one uniform culture medium condition. First, we developed laryngeal scaffolds which were generated by detergent perfusion-decellularization over 9 days and preserved their glycosaminoglycan content and biomechanical properties of a native larynx. After subcutaneous implantations in rats for 14 days, the scaffolds did not elicit a CD3 lymphocyte response. We then developed a uniform culture medium that strengthened the endothelial barrier over 5 days after an initial growth phase. Simultaneously, this culture medium supported airway epithelial cell and skeletal myoblast growth while maintaining their full differentiation and maturation potential. We then applied the uniform culture medium composition to whole laryngeal scaffolds seeded with endothelial cells from both carotid arteries and external jugular veins and generated reendothelialized arterial and venous vascular beds. Under the same culture medium, we bioengineered epithelial monolayers onto laryngeal mucosa and repopulated intrinsic laryngeal muscle. We were then able to demonstrate early muscle formation in an intramuscular transplantation model in immunodeficient mice. We supported formation of three humanized laryngeal tissue compartments under one uniform culture condition, possibly a key factor in developing complex, multicellular, ready-to-transplant tissue grafts. Impact Statement For patients undergoing laryngectomy, no reconstruction methods are available to restore the complex functions of the human larynx. The first promising preclinical results have been achieved with the use of biological scaffolds fabricated from decellularized tissue. However, the complexity of laryngeal tissue composition remains a hurdle to create functional viable grafts, since previously each cell type requires tailored culture conditions. In this study, we report the de novo formation of three humanized laryngeal tissue compartments under one uniform culture condition, a possible keystone in creating vital composite tissue grafts for laryngeal regeneration.

Characteristics of single large-conductance Ca2+-activated K+ channels and their regulation of action potentials and excitability in parasympathetic cardiac motoneurons in the nucleus ambiguus.

Large-conductance Ca2(+)-activated K+ channels (BK) regulate action potential (AP) properties and excitability in many central neurons. However, the properties and functional roles of BK channels in parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus (NA) have not yet been well characterized. In this study, the tracer X-rhodamine-5 (and 6)-isothiocyanate (XRITC) was injected into the pericardial sac to retrogradely label PCMNs in FVB mice at postnatal 7-9 days. Two days later, XRITC-labeled PCMNs in brain stem slices were identified. Using excised patch single-channel recordings, we identified voltage-gated and Ca(2+)-dependent BK channels in PCMNs. The majority of BK channels exhibited persistent channel opening during voltage holding. These BK channels had a conductance of 237 pS and a 50% opening probability at +27.9 mV, the channel open time constant was 3.37 ms at +20 mV, and dwell time increased exponentially as the membrane potential depolarized. At the +20-mV holding potential, the [Ca2+]50 was 15.2 µM with a P0.5 of 0.4. Occasionally, some BK channels showed a transient channel opening and fast inactivation. Using whole cell voltage clamp, we found that BK channel mediated outward currents and afterhyperpolarization currents (IAHP). Using whole cell current clamp, we found that application of BK channel blocker iberiotoxin (IBTX) increased spike half-width and suppressed fast afterhyperpolarization (fAHP) amplitude following single APs. In addition, IBTX application increased spike half-width and reduced the spike frequency-dependent AP broadening in trains and spike frequency adaption (SFA). Furthermore, BK channel blockade decreased spike frequency. Collectively, these results demonstrate that PCMNs have BK channels that significantly regulate AP repolarization, fAHP, SFA, and spike frequency. We conclude that activation of BK channels underlies one of the mechanisms for facilitation of PCMN excitability.

Morphologic evaluation of the fetal recurrent laryngeal nerve and motor units in the thyroarytenoid muscle.

This study is a morphologic description of the recurrent laryngeal nerve (RLN) and of the number and size of motor units (MUs) in the thyroarytenoid (TA) muscle bilaterally of a human fetus aged 25 weeks. A quantitative analysis of RLN and MUs is presented to investigate similarities with equivalent structures in adults. In the fetus used in our study, the morphologic organization of the RLN was similar to that commonly described in the adult RLN. Moreover, as is observed in adult TA, the TA of the analyzed fetus, particularly the right TA, showed MUs typical of muscles with great motor accuracy. These results may be used to increase our knowledge of the features of the voice in adults and newborns.

Development of the rat larynx: a histological study.

OBJECTIVES/HYPOTHESIS: To evaluate and describe the cartilaginous and muscular development of the rat larynx. STUDY DESIGN: Histologic evaluation. METHODS: The larynges of Sprague Dawley rats of embryonic day (E) 13, 15, 17, 19, 21, postnatal day 0, 14, and adult of 250 gm were collected. Four larynges of each age were harvested, cut into 15-µm serial sections, stained with hematoxylin and eosin, and evaluated under light microscopy. Representative digital images were recorded and evaluated at the preglottic (supraglottic in humans), glottic, and postglottic (subglottic in humans) levels. RESULTS: Brachial arches were observed at E13. At E17, immature structures of the larynx, including skeletal muscle, cartilage, and the lumen were identifiable. Chondrification and muscle formation were clearly seen by E19. The muscular and cartilagenous components of the larynx were well established by E21. During the span between birth and adult maturation, the size of the larynx increased from a height of 1.10 mm to 2.90 mm, and from a width of 1.80 mm to 5.40 mm, and from a length of 1.38 mm to 4.77 mm in the stained section. Although developed at E21, the laryngeal structures continued to grow by approximately 30%. CONCLUSION: Rat laryngeal development parallels that in mice and humans. In the rat, at E17 immature structures of the larynx are identifiable, they are well developed at birth and grow by approximately 30% into adulthood. Understanding the chronology and morphology of the embryogenesis of the rat laryngeal musculature is essential and will allow for further evaluation of the embryologic innervation of these muscles.

Age-related changes in rat intrinsic laryngeal muscles: analysis of muscle fibers, muscle fiber proteins, and subneural apparatuses.

We compared age-related changes in the intrinsic laryngeal muscles of aged and young adult rats by determining the number and diameter of muscle fibers, contractile muscle protein (myosin heavy chain isoforms, MHC) composition, and the morphology of the subneural apparatuses. In aged rats, both the numbers and the diameters of muscle fibers decreased in the cricothyroid (CT) muscle. The number of fibers, but not diameter, decreased in the thyroarytenoid (TA) muscle. In the posterior cricoarytenoid (PCA) muscle, neither the number nor the diameter of fibers changed significantly. Aging was associated with a decrease in type IIB and an increase in type IIA MHC isoform levels in CT muscle, but no such changes were observed in the TA or PCA muscles. Morphological examination of primary synaptic clefts of the subneural apparatus revealed that aging resulted in decreased labyrinthine and increased depression types in only the CT muscle. In the aged group, morphologically immature subneural apparatuses were found infrequently in the CT muscle, indicating continued tissue remodeling. We suggest, therefore, that age-related changes in the intrinsic laryngeal muscles primarily involve the CT muscle, whereas the structures of the TA and PCA muscles may better resist aging processes and therefore are less vulnerable to functional impairment. This may reflect differences in their roles; the CT muscle controls the tone of the vocal folds, while the TA and PCA muscles play an essential role in vital activities such as respiration and swallowing.

Histological organization is similar in human vocal muscle and tongue--a study of muscles and nerves.

One of the most exciting questions about the human voice is how the vocal fold produces and modulates different sounds. One hypothesis to explain the wide range of movements found in the vocal fold is based on the variety of muscle fiber orientations in the thyroarytenoid (TA) muscle. The tongue (TO) muscle is considered the most complex structure in the body in terms of muscle fiber orientation and movements. Thus, possible similarities between these two muscles and their innervations, the recurrent laryngeal nerve (RLN) and hypoglossal nerve (XII), could explain the complex movements executed by the focal fold. Moreover, such studies help us to understand some microanatomical aspects of vocal fold reinnervation, based on XII-to-RLN anastomosis. Therefore, this study investigates the histological organization of TA and TO muscles and their innervations (n=12 subjects). The muscle fibers were classified into three categories according to their orientation (transverse, undefined, and longitudinal). To quantify the percentage of fibers in each category in the TA and TO, the shape coefficient (shape Z) was estimated. Qualitative analysis and estimation of fiber area and shape Z show that the histological organization of TA and TO muscle is similar. Both muscles present the same percentage of transversal (~72%), undefined (~15%), and longitudinal fibers (~10%). By contrast, the authors' analysis of the morphometric parameters of the RLN and XII shows that there is no correlation between these nerves. In conclusion, in humans, TA and TO muscles present similar histological organization and this finding could help to explain interesting questions about human phonation.

CpG-DNA suppresses poly(I:C)-induced TSLP production in human laryngeal arytenoid fibroblasts.

Thymic stromal lymphopoietin (TSLP) exerts a marked influence on the polarization of dendritic cells to drive T helper (Th) 2 cytokine production, and has been linked to allergic airway diseases. Although TSLP is produced by airway epithelium, TSLP production in laryngeal arytenoid fibroblasts remains largely unexplored. We examined the effect of Toll-like receptor (TLR) ligands and the cross-talk that occurs among different TLR ligands on TSLP production in arytenoid fibroblasts. Since mRNA of TLR 2, 3, 4, and 9 has been found to be expressed in arytenoid fibroblasts, we examined the effect on its production of TLR ligands. TSLP production by arytenoid fibroblasts was strongly induced in the presence of polyinosinic-polycytidylic acid (poly(I:C)), a ligand of TLR3. Its production was synergistically induced in the presence of IL-4, to a level more than 100 times higher than that observed in the absence of poly(I:C) or IL-4. We also revealed that B type DNA containing CpG motifs (CpG-DNA) coding for a TLR9 ligand markedly suppressed both poly(I:C)-induced and poly(I:C)-plus-IL-4-induced TSLP production. B type CpG-DNA decreased the poly(I:C)-induced phosphorylation of c-Jun N-terminal kinase (JNK), and pre-incubation with SP600125 (inhibitor of JNK) reduced the poly(I:C)-induced TSLP-production. These results indicate that human arytenoid fibroblasts strongly induce TSLP production with stimulation by double-stranded RNA (dsRNA), which can be inhibited by CpG-DNA and participate in immune allergic responses.

Adult neuron addition to the zebra finch song motor pathway correlates with the rate and extent of recovery from botox-induced paralysis of the vocal muscles.

In adult songbirds, neurons are continually incorporated into the telencephalic nucleus HVC (used as a proper name), a premotor region necessary for the production of learned vocalizations. Previous studies have demonstrated that neuron addition to HVC is highest when song is most variable: in juveniles during song learning, in seasonally singing adults during peaks in plasticity that precede the production of new song components, or during seasonal reestablishment of a previously learned song. These findings suggest that neuron addition provides motor flexibility for the transition from a variable song to a target song. Here we test the association between the quality of song structure and HVC neuron addition by experimentally manipulating syringeal muscle control with Botox, which produces a transient partial paralysis. We show that the quality of song structure covaries with new neuron addition to HVC. Both the magnitude of song distortion and the rate of song recovery after syringeal Botox injections were correlated with the number of new neurons incorporated into HVC. We suggest that the quality of song structure is either a cause or consequence of the number of new neurons added to HVC. Birds with naturally high rates of neuron addition may have had the greatest success in recovering song. Alternatively, or in addition, new neuron survival in the song motor pathway may be regulated by the quality of song-generated feedback as song regains its original stereotyped structure. Present results are the first to show a relationship between peripheral muscle control and adult neuron addition to cortical premotor circuits.

Posterior cricoarytenoid bellies: relationship between their function and histology.

OBJECTIVES/HYPOTHESIS: Complete physiological information about human posterior cricoarytenoid muscle (PCA) is essential and is not only of basic science interest but also could lead directly to understanding phonation and many clinical issues in neurolaryngology. The purpose of the study was to investigate and compare the histochemical and morphological properties to know contractile muscle fiber characteristics of two bellies of the PCA. STUDY DESIGN: Cross-sectional experimental study. METHODS: The PCAs were harvested from the total laryngectomy simples. Serial transverse sections of the two PCA bellies were performed and studied by immunohistochemical analysis. RESULTS: Two separate muscle bellies were always identified within 15 PCA. The following muscle fiber types were observed: I, I-IIA, and IIA. Comparisons of the vertical and horizontal bellies of the PCA reveled differences in the fiber-type composition. CONCLUSION: In our experience, the PCA should be considered as a combination of two functional subunits, which significantly differ in their muscle fiber-type composition.

Tissue-engineered larynx using perfusion-decellularized technique and mesenchymal stem cells in a rabbit model.

CONCLUSION: Reseeding mesenchymal stem cells (MSCs) into the decellularized laryngeal muscle matrix for construction of a tissue-engineered larynx is feasible. This in vivo maturation in the omentum could be the first step before in situ implantation of the construct. This construct could facilitate a tissue-engineered method for laryngeal reconstruction. OBJECTIVES: The extracellular matrix (ECM) and MSCs have been widely used for organ reconstruction. Our study aimed to prepare a soft tissue decellularized laryngeal scaffold with intact laryngeal cartilages utilizing a perfusion-decellularized technique, reseeding cells on it, and then construct a recellularized larynx. METHODS: Perfusion-decellularized larynges were obtained from 20 rabbits by perfusion of the common carotid arteries with detergents. Twelve perfused larynges were observed by macroscopic visualization, histological examination, scanning electron microscopy (SEM), and cartilage viability. The remaining eight perfusion constructs were reseeded with induced MSCs aspirated from eight receptor rabbits. Composites were transferred into greater omentums of receptor rabbits after adherence for 1 day in vitro. Rabbit larynges were harvested after 4 weeks and 8 weeks, respectively. Macroscopic visualization, histological examination, and immunohistochemistry were performed. RESULTS: Larynges perfused by sodium dodecyl sulfate became transparent after 2 h of perfusion. Histology and SEM indicated that the perfusion method showed a better decullularized effect. Almost no intact cells or nuclei were found, while more pores and collagen fibers were retained in the decellularized matrix. The chondrocyte vitality assay indicated that chondrocyte vitality was high. Vascularization was clearly seen by 4 weeks and relatively integrated cartilage frameworks remained by 8 weeks. Histological and immunohistochemical examinations clearly showed muscle bundles and vessels.

[A preliminary study on the preparation of perfusion-decellularized laryngeal scaffold and the feasibility of laryngeal muscle reconstruction].

OBJECTIVE: To prepare a decellularized whole laryngeal scaffold by utilizing a perfusion-decellularized technique, reseed cells on it, and construct decellularized laryngeal muscles. METHODS: Perfusion decellularized larynxes were obtained by common carotid arterious perfusion with detergents. Then they were performed by macroscopic view, histological examination, scanning electron microscopy (SEM) and cartilage viability. Decellularized laryngeal scaffold were then reseeded with inducted mesenchymal stem cells (MSCs). Composites were transferred into greater omentums of rabbits after one day's adherence and harvested after eight weeks. Macroscopic view, histological examination and immunohistochemistry were performed. RESULTS: Perfusion larynxes became transparent after two hours. Histology and SEM indicated that perfusion method showed better decullularized effect. More vintages and collagen fibers but no intact cell or nuclei were retained in the decellularized matrix. Porosity measured by Image pro plus 6.0 was 80.4% +/- 3.2% (x +/- s). Chondrocyte vitality assay indicated chondrocyte vitality rate in the perfusion group was 86.9% +/- 1.5%. After eight weeks, vascularization formed and integrated cartilage frameworks still remained. Histological examination could clearly show the presence of muscle bundles and vessels. Immunohistochemical examination indicated that sarcomeric-alpha actin expressed positively in corresponding areas. CONCLUSIONS: It is feasible to reseed MSCs into the decellularized laryngeal muscle matrix for constructing tissue-engineered laryngeal muscles. This in vivo maturation into the omentum could be the first step before in situ implantation of the construct.

Immunohistochemical analysis of myosin heavy chain expression in laryngeal muscles of the rabbit, cat, and baboon.

We studied myosin heavy chain (MyHC) expression and fiber type distribution in laryngeal muscles in the rabbit, cat, and baboon using immunohistochemistry with highly MyHC-specific antibodies. Two types of variation in MyHC expression were found: between muscles of different function within species and within specific muscles between species. Within species, thyroarytenoid (Ta), an adductor, had faster MyHCs and fiber type profiles than the abductor, posterior cricoarytenoid (PCA), which expressed faster MyHCs than the vocal fold tensor, cricothyroid (CT). Between species, laryngeal muscles generally expressed faster MyHCs in small animals than in larger ones: extraocular (EO) MyHC was expressed in the Ta and PCA of the rabbit but not in the cat and baboon, whereas 2B MyHC was expressed in these muscles of the cat but not of the baboon. The CT expressed only MyHC isoforms and fiber types found in the limb muscles of the same species. These results are discussed in light of the hypothesis that the between-species variations in laryngeal muscle fiber types are evolutionary adaptations in response to changes in body mass and respiratory frequency. Within-species variations in fiber types ensure that protective closure of the glottis is always faster than movements regulating airflow during respiration.

Characterization of laryngeal muscle stem cell survival and proliferation.

OBJECTIVES/HYPOTHESIS: Laryngeal muscle and skeletal muscle stem cells (MSC) have been shown to differ in physiological basal activity and responsiveness to stimuli. Given these differences, it is the purpose of this investigation to characterize the in vitro proliferation and survival of laryngeal and skeletal MSC to determine whether intrinsic differences exist that may account for differences noted in vivo. STUDY DESIGN: Basic science experiment utilizing rat MSC. METHODS: Cultures of both laryngeal and skeletal MSC were harvested and equal numbers from both groups were expanded under similar conditions, quantifying cellular population to determine proliferation rate for each population. Increased proliferative potential was confirmed using Western blot analysis of extracellular signal-regulated kinase phosphorylation. As per standard survival assay protocol, cultures were placed in serum-deprived medium and cell survival was assessed by terminal uridine deoxynucleotidyl transferase-mediated dUTP nick end labeling assay at 72 hours. RESULTS: Our results demonstrated increased proliferation of laryngeal MSC relative to the skeletal MSC when cultured under similar conditions. Western blot demonstrated increased activation of the proliferation pathway, extracellular signal-regulated kinase, in the laryngeal group. There was no detectable difference in the MSC survival between the two groups. CONCLUSIONS: Compared with skeletal MSC, laryngeal MSC demonstrate increased proliferation and regenerative capacity. This may explain some of the differences in physiological role and responses involved in each cell population's tissue of origin.

Laryngeal muscles are spared in the dystrophin deficient mdx mouse.

PURPOSE: Duchenne muscular dystrophy (DMD) is caused by the loss of the cytoskeletal protein, dystrophin. The disease leads to severe and progressive skeletal muscle wasting. Interestingly, the disease spares some muscles. The purpose of the study was to determine the effects of dystrophin deficiency on 2 intrinsic laryngeal muscles, the posterior cricoarytenoid and the thyroarytenoid, in the mouse model. METHOD: Larynges from dystrophin-deficient mdx and normal mice were examined histologically. RESULTS: Results demonstrate that despite the absence of dystrophin in the mdx laryngeal muscles, membrane damage, inflammation, necrosis, and regeneration were not detected in the assays performed. CONCLUSIONS: The authors concluded that these muscles are 1 of only a few muscle groups spared in this model of dystrophin deficiency. The muscles may count on intrinsic and adaptive protective mechanisms to cope with the absence of dystrophin. Identifying these protective mechanisms may improve DMD management. The study also highlights the unique aspects of the selected laryngeal skeletal muscles and their dissimilarity to limb skeletal muscle.

Optimization of autologous muscle stem cell survival in the denervated hemilarynx.

OBJECTIVE: Current treatments for vocal fold paralysis are suboptimal in that they fail to restore dynamic function. Autologous muscle stem cell (MSC) therapy is a promising potential therapy for vocal fold paralysis in that it can attenuate denervation-induced muscle atrophy and provide a vehicle for delivery of neurotrophic factors, thereby potentially selectively guiding reinnervation. The goal of this project was to characterize optimal conditions for injected autologous MSC survival in the thyroarytenoid (TA) muscle following recurrent laryngeal nerve (RLN) injury by local administration of adjuvant factors. STUDY DESIGN: Animal experiment. METHODS: Unilateral RLN transection and sternocleidomastoid muscle (approximately 1 g) biopsies were performed in 20 male Wistar rats. One month later, 10 autologous MSCs labeled via retroviral-enhanced green fluorescent protein (EGFP) transduction were injected into the denervated hemilarynx of each animal with one of four adjuvant therapies: cardiotoxin [(CTX) 10 M], insulin-like growth factor-1 [(IGF- 1) 100 microg/mL], ciliary neurotrophic factor [(CNTF) 50 microg/mL], or saline. Animals were euthanized 1 month later and larynges harvested, sectioned, and analyzed for MSC survival. RESULTS: All specimens demonstrate extensive MSC survival, with fusion of the MSCs with the denervated myofibers. Based on mean fluorescent intensity of the laryngeal specimens, IGF-1 and CNTF had the greatest positive influence on MSC survival. Myofiber diameters demonstrated myofiber atrophy to be inversely related to MSC survival, with the least atrophy in the groups having the greatest MSC survival. CONCLUSIONS: Autologous MSC therapy may be a future treatment for vocal fold paralysis. These findings support a model whereby MSCs genetically engineered to secrete CNTF and/or IGF-1 may not only promote neural regeneration, but also enhance MSC survival in an autocrine fashion.

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.

Carotid body-like tissues around the rat posterior cricoarytenoid muscle.

We examined the carotid body-like tissues around the posterior cricoarytenoid (PCA) muscle of the rat by light and electron microscopy. One branch after bifurcation of the inferior (recurrent) laryngeal nerve frequently formed a small ganglion at the lateral side of this muscle and sometimes contained paraganglion cells (granule-containing cells). In addition, encapsulated structures (paraganglia) enveloped by a few layer of capsular cells were often observed on and near the muscle. Moreover, granule-containing cells resembling the encapsulated paraganglion cells were found in clusters outside the small nerve. These clustered cells were incompletely surrounded by fibroblastic processes. In addition to synapses between adjacent cells, afferent and efferent synapses were distinguished between nerve endings and these cells, possibly receiving both afferent and efferent innervation. These findings suggest that the clustered granule-containing cells outside the small nerve in the proximity of the PCA muscle may function as chemoreceptor cells as well as the paraganglion cells within the nerve bundles and the encapsulated paraganglia.

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.