Cytochrome c oxidase deficiency in human posterior cricoarytenoid muscle.

BACKGROUND: Mitochondrial alterations occur in skeletal muscle fibers throughout the normal aging process, resulting from increased accumulation of reactive oxide species (ROS). These result in respiratory chain abnormalities, which decrease the oxidative capacity of muscle fibers, leading to decreased contractile force, sarcopenia, or fiber necrosis. Intrinsic laryngeal muscles are a cranial muscle group that possesses some distinctive genotypic, phenotypic, and physiologic properties. Their susceptibility to mitochondrial alterations resulting from biological processes that increase levels of oxidative stress may be one of these distinctive characteristics. OBJECTIVES: The incidence of cytochrome c oxidase (COX) deficiency (COX(-)) was determined in human posterior cricoarytenoid (PCA) muscle when compared with the human thyrohyoid (TH) muscle, an extrinsic laryngeal muscle that served as "control" muscle. Ten PCA and 10 TH muscles were harvested postlaryngectomy from 10 subjects ranging in age from 55 to 86 years. Differences in COX(-) were compared within and between muscle types using tissue section staining and standard morphometric analysis. RESULTS AND CONCLUSIONS: COX(-) fibers were identified in both the PCA and TH muscles. The PCA muscle had 10 times as may affected fibers as the TH muscle, with significant differences in COX(-) found between muscle type and fiber type (P=0.003). Almost all of this effect was the result of elevated levels of COX(-) in type I fibers from the PCA muscle (P=0.002) that showed a strong positive correlation with increased age. These results suggest that increased mitochondrial alterations may occur in the PCA muscle during normal aging.

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 muscle fibre types.

The internal laryngeal muscles have evolved to subserve the highly specialized functions of airways protection, respiration, and phonation. Their contractile properties, histochemistry, biochemical properties, myosin heavy chain (MyHC) expression and their regulation by nerves and hormones are reviewed and compared with limb muscle fibres. Cricothyroid, the vocal cord tensor, is limb-like in MyHC composition and fibre type properties, while the vocal fold abductor and adductors are allotypically different, with capacity for expressing an isoform of MyHC that is kinetically faster than the fastest limb MyHC. In rats and rabbits the faster isoform is the extraocular (EO) MyHC, while in carnivores, it is the IIB MyHC. These adaptations enable the abductor and adductor muscles to remain always faster than the cricothyroid as the latter changes in speed during evolution to match changing metabolic and respiratory rates in relation to scaling with body mass. Such phylogenetic plasticity is vital to the airways protection and respiratory functions of these muscles. The posterior cricoarythenoid, the abductor muscle, is tonically driven during expiration, and consequently has a slower fibre type profile than the principal adductor, the thyroarythenoid. The human thyroarythenoid appears not to express EO or IIB MyHC significantly, but is unique in expressing the slow-tonic MyHC. The concepts of allotype and phylogenetic plasticity help to explain differences in fibre type between limb and laryngeal muscles and between homologous laryngeal muscles in different species. Laryngeal muscle fibres exhibit physiological plasticity as do limb muscles, being subject to neural and hormonal modulation.

[The study of the enzyme histochemistry changes of thyroarytenoid when one side of RLN was excised].

OBJECTIVE: To chronologically evaluate the changes in both sides of thyroarytenoid (TA) muscles. when one side of recurrent laryngeal nerve (RLN) was cut off. METHOD: In 7 adult mongrel dogs, 2 dogs for control and the other 5 dogs for the following experiment: a 2 cm segment of right RLN was excised under intraperitoneal sedation. The animals were killed at 2, 4, 6, 8, 10 week respectively and the thyroarytenoid were processed for histochemical reaction and electromicroscope. The mean muscle fiber, standard deviation and muscle fiber type composition were determined. RESULT: For the injured sides, denervation resulted in shrinking of all the fibers at four weeks. At ten weeks, however, all the muscles were fibrosed. For the uninjured sides (left side), TA diameter appeared compensatory changes at four weeks, this changes were obvious at ten weeks. Variability of the fiber composition was observed throughout the study; however, there was no specific trend in changes before ten weeks. CONCLUSION: When the RLN was excised, the fibers in the uninjured side of thyroarytenoid appeared compensatory changes in fiber diameter, which could give rise to the recovery of the laryngeal function.

Developmental changes in histochemical properties of intrinsic laryngeal muscles in rats.

OBJECTIVE: Using neonatal rats, the developmental changes in muscle fiber type of the intrinsic laryngeal muscles were analyzed. The potential influence of two factors were also studied, that were predicted would influence developmental changes in muscle fiber type, denervation and hypothyroidism. METHODS: Using the histochemical technique of myosin ATPase staining, postnatal changes in the ratio of muscle fiber types of each intrinsic laryngeal muscle were determined. In addition, to clarify factors influencing the development of the intrinsic laryngeal muscles, the same technique was employed to study recurrent laryngeal nerve-denervated rats and rats with experimentally-induced hypothyroidism. RESULTS: In normal pups, type 2C fibers had almost disappeared by postnatal day (PND) 14. In denervated pups, differentiation to type 1 and 2A muscle fibers was not observed. In contrast, differentiation to type 2B muscle fibers was impaired in the hypothyroid pups. CONCLUSION: The differentiation of intrinsic laryngeal muscles occur earlier than that of hindlimb muscles. Each intrinsic laryngeal muscle exhibits a particular pattern of developmental changes in normal pups. The developmental changes in the intrinsic laryngeal muscles are affected by recurrent laryngeal nerve innervation and thyroid hormonal control. The findings suggest that both recurrent laryngeal nerve innervation and thyroid hormone play important roles in the differentiation of the intrinsic laryngeal muscles.

Mechanism of the differential sensitivity in the rat adductor and abductor laryngeal muscles to a non-depolarizing neuromuscular blocker.

We have compared the neuromuscular blocking effects of tubocurarine at pre- and postsynaptic sites in the lateral cricoarytenoid muscle (LCA) (one of the adductor muscles of the vocal cords) and in the posterior cricoarytenoid muscle (PCA) (sole abductor muscle of the vocal cords) of the rat. Fine wire electrodes were inserted into both muscles and evoked compound electromyographic (EMG) responses measured by supramaximal stimulation of the recurrent laryngeal nerves. End-plate potentials (EPP), mean quantal content and carbachol sensitivity to tubocurarine in both muscles were measured using intracellular microelectrodes. Tubocurarine produced a concentration-dependent reduction in the EMG responses, EPP amplitude, mean quantal content and carbachol sensitivity. The LCA muscle was more resistant to tubocurarine than the PCA muscle in EPP amplitude, mean quantal content and carbachol sensitivity, suggesting unequal pre- and postsynaptic sensitivity for inhibition of elicited acetylcholine release, reduction in EPP amplitude and loss of evoked muscle action potentials. Examination of muscle fibre composition demonstrated that the LCA muscle contained a significantly higher fraction of slow twitch muscle fibres than PCA muscle. However, the sizes of the fibres were similar in both muscles. We conclude that the mechanism of unequal sensitivity to a non-depolarizing neuromuscular blocker in the LCA and PCA muscles may be explained by differential sensitivities at the pre- and postsynaptic sites of the neuromuscular junction.

Histochemical and morphometric study of the cricoarytenoideus lateralis muscle in the horse.

Histochemical and morphometric parameters of the cricoarytenoideus lateralis muscle of the horse are presented. Using myosin ATPase staining after acid preincubation, 3 fibre types (I, IIA and IIC) were identified. Using NADH-TR staining, type I fibres showed high oxidative capacity, whereas type II fibres had high or low oxidative capacity. The type I to type II ratio was of 35:65. This ratio remained constant in the age range examined. Statistically significant (p < 0.01) differences were found in values for fibre size between groups of horses weighing more than 500 kg and less than 400 kg. Mean area of type II fibres was greater (p < 0.001) than that of type I fibres. There were no significant differences in mean area between left and right muscles in the group of animals with less weight. In contrast, significant differences (p < 0.05) in mean area between left and right muscles were found for type I fibres in the group of animals exhibiting a higher weight. The histographical distribution of fibre type areas was unimodal. Most adult horses showed muscle fibre type grouping in the left muscle.

[Histochemical study of intrinsic laryngeal muscles in neonates].

The motor end-plates and muscle fiber types in muscles of cricothyroid, posterior cricoarytenoid, thyroarytenoid were in five neonates and two adults studied by staining with acetylcholinesterase (AChE) and succinic dehydrogenase (SDH). The muscle fibers in neonate could be classified into three types: red, white and intermediate. The diameters of muscle fibers were different, with the white muscle fiber widest and the red ones narrowest. Each of the laryngeal muscles was composed of three muscle types in proportion. The motor end-plate showed a concentrated distribution band.

Androgen regulation of muscle fiber type in the sexually dimorphic larynx of Xenopus laevis.

We used histochemical techniques [assays for adenine triphosphatase (ATPase) and succinate dehydrogenase (SDHase) activity] to identify muscle fiber types in the larynx of Xenopus laevis. Male muscle is made up of one fiber type, medium-sized fibers (approximately 9 microns2) that stain lightly for acid-stable ATPase and intensely for SDHase activity. In contrast, the female has 3 fiber types: small fibers (approximately 6 microns2) that stain intensely for ATPase and SDHase, medium-sized fibers (approximately 13 microns2) with moderate staining for ATPase and dark staining for SDHase, and large fibers (approximately 15 microns2) with little SDHase or ATPase activity. Long-term castration (6 months) has no effect on histochemical staining of adult male fibers. Long-term testosterone treatment (5 months) increases the proportion of medium-sized, moderately staining fibers in adult females, and reduces the proportion of both the small, darkly staining fibers and the large, lightly staining fibers. At metamorphosis, both males and females have 3 fiber types whose ATPase activity is similar to that of the adult female. However, no SDHase activity is observed. Treatment of juveniles for 3 weeks with testosterone results in nearly complete masculinization of muscle fibers, as judged by increased cross-sectional area, homogeneous ATPase staining, and a marked increase in SDHase activity. Thus, juvenile muscle is considerably more responsive to testosterone than is adult female muscle. We propose that the uniform metabolic properties of male laryngeal muscle contribute to the production of the rapid (66 Hz) mate call vocalizations characteristic of this species. Further, our results suggest that androgens direct the masculinization of laryngeal muscle fibers during postmetamorphic development.

Experimental laryngeal reinnervation by phrenic nerve implantation into the posterior cricoarytenoid muscle.

Under general anaesthesia, 5 dogs underwent sectioning of the right recurrent nerve followed by implantation of the phrenic nerve into the posterior cricoarytenoid (PCA) muscle. Some 6-7 months later the dogs were sacrificed after registration of vocal cord motility. Still photographs and movie film of the larynx were taken during quiet and forced respiration and at electrical stimulation of the implanted phrenic nerve. The PCA and vocal muscles were removed for histochemical studies. We found practically no abductory movement of the vocal cord on the reinnervated side, either during quiet or forced respiration. During forced inspiration there was, however, a slight medial bowing of the right vocal cord. At electrical stimulation there was a sphincteric movement of the entire larynx. Histochemistry showed a reinnervation picture of both the PCA and the vocal muscles on the experimental side. The conclusion drawn from this study is that axonal escape, probably from the implantation site, results in an unwanted reinnervation of laryngeal adductor muscles, which neutralize the abducting effect of the PCA muscle during inspiration. This method therefore does not seem to be suitable as a treatment alternative for bilateral recurrent nerve paralysis.

The effect of tracheostomy on posterior cricoarytenoid muscle.

It has been reported previously that the amount of electromyographic (EMG) potential of the posterior cricoarytenoid (PCA) decreases after prolonged tracheostomy. It is, therefore, reasonable to assume that a significant alteration of the biochemical characteristics of this muscle would also occur. In addition to histochemical analysis, endoscopic and EMG data were recorded to give a direct comparison in each subject. Seven male beagles were used for this study. Four were tracheostomized and three served as controls. They were examined immediately before and after surgery and again after 4 weeks by EMG and endoscopic techniques. Histochemical staining was performed on each subject. All three modalities failed to demonstrate a substantial difference between the controls and the experimental dogs.

Neuromuscular junctions of the posterior cricoarytenoid muscle in the human adult, human fetus and cat. Histochemical and electron microscopic study.

The motor end plate of the posterior cricoarytenoid muscle (PCA muscle) in the human adult, human fetus and cat was examined by using electron microscopy and histochemical methods. In the present study, we observed the single-type motor end plate and en plaque type neuromuscular junction. At the neuromuscular junction of the fetal PCA muscle, the primary synaptic cleft, the basement membrane and the postsynaptic density could already be observed; however, there was no secondary synaptic cleft. Histochemically, the subneural apparatus was filled with electron-dense products, indicating acetylcholinesterase (AChE) activity. The primary and secondary synaptic clefts in the adult PCA muscle were well developed and intense AChE activity was present. The appearance of the neuromuscular junction and its localization of AChE activity was similar to that in the cat PCA muscle.

[Intrinsic muscles of the human larynx. Histoenzymological characteristics of the muscle fibers]. / Muscles intrinsèques du larynx de l'homme. Caractéristiques histoenzymologiques des fibres musculaires.

ATPasic, oxidative, and glycogenolytic activities were demonstrated in human laryngeal intrinsic muscles (a total of 15) employing a series of histoenzymatic investigational techniques. Staining methods applied to 10 of the muscles revealed the presence of motor plates on the fibers, either with or without associated ATPase activity. These findings enabled clarification of the different muscle fiber populations, enzymatic equipment, and type of innervation of the laryngeal muscles. Certain of these muscles (cricothyroid, thyroarytenoid, and posterior cricoarytenoid) possess remarkable particularities in relation to known skeletal muscles, and the significance of these features is discussed.

Morphologic and histochemical characteristics of laryngeal muscle.

Laryngeal muscle (LM) is highly specialized for phonation and sphincter activity. We queried whether this specialization is reflected in the structure of LM. We examined, using histochemical techniques, the structure of five LM from three men who died suddenly and who had no evidence of laryngeal disease. Compared with nonlaryngeal skeletal muscle, our specimens demonstrated moderate fibrosis, rounding of fibers, basophilia, and ragged red fibers that were shown to be mitochondria. In general, LM fibers are smaller, have more variability in size, and contain a greater percentage of histochemically type 1 fibers than limb skeletal muscles. These differences suggest that theories of motor control derived from studies of limb skeletal muscles may not apply to LM.

Androgen affects cholinergic enzymes in syringeal motor neurons and muscle.

We examined the role of testosterone (T) in regulating the weight of the songbird syrinx and the activity of two cholinergic enzymes, choline acetyltransferase (CAT) and acetylcholinesterase (AChE). Castration of adult male zebra finches or neonatal canaries results in a lowering of syringeal weight and CAT and AChE activity. Administration of T for 1-4 weeks restores syringeal weight and AChE to intact levels in male zebra finches. Activity of CAT in muscle is not fully restored. Ovariectomy of female zebra finches and canaries does not affect these syringeal parameters, but T administration to ovariectomized females for 1 month increases syringeal weight and AChE activity. In the zebra finch tracheosyringealis nerve, activity of CAT and AChE is decreased one month after castration. T administration to castrates maintains nerve AChE activity but not CAT. In contrast to changes in the syrinx, tongue muscles do not change in weight or enzyme activity when circulating T levels are altered. Effects of muscle use and disuse were found on syringeal weight and AChE activity, but an androgenic effect also operates in addition. Results suggest that one mechanism for T regulation of singing in passerine birds is through induction of specific enzymatic proteins in androgen target neurons and muscles.

[Myonic makeup of the muscles innervated by the vagus nerve]. / Mionnyi sostav myshts, innerviruemykh bluzhdaiushchim nervom.

Histochemical investigation on succinic dehydrogenase activity and morphometric studies have demonstrated certain differences in the dog sublingual group of muscles. The thyreohyoid and sternohyoid muscles innervated by spinal nerves possess three types of myons differing in succinic dehydrogenase activity and in the area of transversal section. The cricothyreoid muscle and the superior pharyngeal constrictor obtaining their motor innervation from the vagus nerve are composed of unitypical muscular fibres with nearly the same areas of transversal section and high enzymic activity. The differences noted should be explained by different sources of motor innervation.