Muscle fiber and satellite cell apoptosis in the aging human thyroarytenoid muscle: a stereological study with confocal laser scanning microscopy.

OBJECTIVE: This study determines the role of changes in numerical densities of apoptotic myonuclei and satellite cells in age-related remodeling of the thyroarytenoid muscle (TA). DESIGN: Changes in numerical densities of apoptotic myonuclei and satellite cells were estimated for the entire TA by using stereological techniques. RESULTS: There was an increase in N(V apoptotic myonucleus, fiber type) (P < 0.05) and in the percentage of apoptotic myonuclei (P < 0.05) in type 1 but not in type 2 muscle fibers. There was also an increase in N(V apoptotic satellite cell, fiber type) (P < 0.05) and in the percentage of apoptotic satellite cells (P < 0.05) in type 1 fibers but not in type 2 fibers. CONCLUSION: Apoptosis may contribute to age-related fiber loss and atrophy in the TA. SIGNIFICANCE: Therapeutic techniques based on decreasing the frequency of apoptosis may block age-related fiber loss and atrophy in the TA.

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-related changes in muscle fiber regeneration in the human thyroarytenoid muscle.

BACKGROUND: Muscle fiber regeneration is essential to maintain normal muscle fiber populations and muscle mass by continuous replacement of fibers lost to acute muscle injury or overuse. However, the extent of ongoing muscle fiber regeneration in the laryngeal muscles is unknown. OBJECTIVE: The present study provides statistically unbiased, quantitative estimates of the content of regenerating fibers in the human thyroarytenoid muscle over the adult lifespan. DESIGN: In the adult, only regenerating muscle fibers express the developmental myosin isoform. Therefore, regenerating fibers were identified using immunohistochemical techniques. The content of regenerating muscle fibers in the entire muscle volume was then estimated using stereological techniques. Through the use of a computer-automated sampling protocol, stereological data were collected from sets of isotropic uniform random cryostat sections. Overprojection error was minimized by using a confocal laser-scanning microscope to image thin optical sections for use as sample fields. SUBJECTS: Eight autopsy cases, subjects ranging in age from 19 to 81 years. RESULTS: The summed length of fibers expressing developmental myosin increased significantly (P=.02) with age when compared with the overall muscle fiber length. CONCLUSIONS: This finding indicates that muscle fibers maintain the capability for spontaneous regeneration, and that the proportion of regenerating fibers increases as the thyroarytenoid muscle ages. This increase is possibly a compensatory response to an age-related increase in muscle fiber injury or death.

Age-related changes in muscle fiber types in the human thyroarytenoid muscle: an immunohistochemical and stereological study using confocal laser scanning microscopy.

A decline in motor performance contributes to laryngeal dysfunction in the elderly, but the pathogenetic mechanisms are unknown. Quantitative 3-dimensional, age-related changes in the muscle fiber content of the human thyroarytenoid muscle were estimated from geometric probability (stereology) by use of a technique that provided a statistically unbiased sample of all possible section orientations and locations in the entire muscle volume. There was a preferential 27% age-related loss in the length density (L(V type, muscle)) of type 1 (slow) fibers in contrast to the selective type 2 (fast) fiber loss typical of aging limb muscles. In type 2 fibers there was no significant loss in the L(V), but there was an age-related decrease (P < 0.05) in the surface density (S(V type, muscle)) and an increase (P < 0.05) in the atrophy factor, an index of the content of very small, atrophic fibers. There was also an age-related increase in the length fraction (L(L type, all fibers)) of muscle fibers that coexpress both fast and slow myosin heavy-chain isoforms (P < 0.05). These findings demonstrate a type-specific fiber loss and atrophy that differs from that in aging limb muscles and an age-related increase in motor unit remodeling.

Quantitative histochemical studies on the cat infrahyoid muscles.

The intrinsic laryngeal muscles have been reported to be composed of muscle fibers that have unusual physiologic, morphologic, and biochemical characteristics, and it has been suggested that the relatively unique differentiation of these muscle fibers is the result of their specific activity patterns. Because the infrahyoid muscles are recruited for some of the same laryngeal functions, it was of interest to determine if they also included unusual fiber types. In order to examine this possibility, microdensitometry was carried out on type I, type IIA, and type IIB fibers in the cat sternohyoid, sternothyroid, and thyrohyoid muscles using histochemical techniques for a variety of enzymatic markers of oxidative and glycolytic capacity and for fiber size. It was found that the infrahyoid muscles were composed of muscle fibers having enzyme profiles generally similar to those of fiber types in the limb muscles.

Age-related changes in Schwann cells of the internal branch of the rat superior laryngeal nerve.

While previous studies have examined the role of neurons in age-related changes of laryngeal sensory nerve function, investigators have not focused on the significant role of Schwann cells in the aging process. This investigation used an electron microscopic morphometric technique to systematically study Schwann cells of the internal branch of the superior laryngeal nerve of three Wistar rat age groups: young = 3 to 5 months; old = 25 months; very old = 29 to 31 months. In Schwann cells of myelinated axons, a substantial and significant reduction (p = 0.0127) in mitochondrial volume fraction was found between the young and old groups, and also between the young and very old groups (young = 0.0034, old = 0.00175, very old = 0.0019). The volume fraction of compact myelin also showed a trend (p = 0.0683) toward decreasing with age. The volume fractions of cytoplasm, nuclei, and lipofuscin granules showed no significant age-related changes (lipofuscin in myelinating Schwann cells: young = 0.0025, old = 0.0020, very old = 0.0051; lipofuscin in non-myelinating Schwann cells: young = 0.012, old = 0.0061, very old = 0.0051). Our observations on mitochondria lead to two possible conclusions regarding energy availability in aged Schwann cells. One possibility is that aged cells have a decreased energy requirement, which is reflected in the decreased mitochondrial densities in old and very old specimens. The other possibility is the reduced mitochondrial volume fractions are independent of cellular energy requirements and are indicative of some aging or pathologic process.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural changes with age in the human superior laryngeal nerve.

Electron-microscopic morphometric techniques were used to examine age-related changes in postmortem human superior laryngeal nerves obtained at autopsy (young adult, aged 20 to 30 years; old, 60 years and over). A statistically significant age-related loss of myelinated nerve fibers was detected (old, 7032 +/- 1572; young, 10,179 +/- 1969), representing a 31% decrease. Much of this loss occurred selectively in small myelinated fibers (1 to 2 microns) where there was an extensive (67%) and significant age-related fiber loss. A corresponding analysis of axonal diameter of myelinated nerve fibers similarly showed an extensive (67%) and statistically significant decrease in myelinated fibers with small axonal diameters (0 to 0.5 microns) in the old age group compared with the young adult age group. These findings may represent a significant histomorphologic correlate to the observed age-related sensorimotor dysfunction of the upper aerodigestive tract common in the elderly.

Age-related changes in the neuromuscular junctions in the human posterior cricoarytenoid muscles: a quantitative study.

The posterior cricoarytenoid muscle is unique histochemically, ultrastructurally, and functionally. It has been suggested that the posterior cricoarytenoid muscle undergoes early aging. Aging in peripheral nerves has been reported to resemble a "dying back" neuropathy in that the most severe and earliest age-related changes occur at the most distal levels of the nerve fibers. The lengths and terminal axonal branching of neuromuscular junctions in 17 human posterior cricoarytenoid muscles aged 4 days to 95 years were determined. Both neuromuscular junction lengths and the numbers of axonal terminal branches in the very young group differed significantly from values in two older groups. In contrast to results reported for some other muscles, there was no significant age-related change in these parameters in the posterior cricoarytenoid muscle during adult life. This difference may be related to the repetitive contraction of the posterior cricoarytenoid muscle.

Actomyosin adenosine triphosphatase activities of the cat infrahyoid muscles.

By use of actomyosin ATPase histochemistry, it was found that there were large differences among the three cat infrahyoid muscles (sternohyoid, sternothyroid, and thyrohyoid) with respect to their percentages of different muscle fiber types. It has been established that the individual activity patterns of the component motor units in each muscle drive the biochemical and physiologic differentiation of the muscle fibers associated with each motor unit. Therefore, the data obtained in the present investigation provide an indication of the characteristics of long-term use of each of the various types of motor units, as well as the associated differences in the physiologic capacities of the different motor unit types composing each of these infrahyoid muscles.

Age-related changes in the internal branch of the rat superior laryngeal nerve.

This investigation was initiated to provide data on the ultrastructural basis for neurologic age-related changes in laryngeal sensory function. In the present study, an animal model (female Wistar rats: age range: young [Y], 3 to 5 months; old [O], 25 months; and very old [VO], 29 to 30 months) was used to examine systematically changes in the internal branch of the superior laryngeal nerve with age using electron microscopic morphometric techniques. Total fiber counts, fiber populations (size categories), and mean fiber size for myelinated and unmyelinated fibers did not change with age. Qualitative changes were consistent with segmental demyelination and axonal degeneration in the older animals. There was also a significant age-related increase in the volume fraction of adaxonal Schwann cell cytoplasm (Y, 0.019; O, 0.041; and VO, 0.042). Ultrastructural correlates of intracellular support and axonal transport showed a significant decrease in the numerical density of neurofilaments (Y, 0.126/micron2; O, 0.073/micron2; and VO, 0.078/micron2) in the older animals, but no change in the numerical density of microtubules. Energy metabolism correlates in the form of mitochondrial volume fraction did not change with age. There was a significant increase in the volume fraction of the intrafascicular extracellular space (Y, 0.224; O, 0.271; and VO, 0.301), indicating a late, age-related change in the extracellular environment. These changes could lead to decreased conduction velocity or complete fiber dysfunction. A number of these changes resembled those of aged human peripheral nerves already examined.

Muscle fiber types in the cat middle ear muscles. II. Tensor tympani.

Using quantitative histochemical techniques, it was determined that the tensor tympani muscle of the cat consists of three muscle fiber types: type 1, type 2A (staining characteristics similar to the type 1 and type 2A muscle fibers found in the control tibialis anterior muscles), and a third unclassified fiber type (type 3) similar to the 2A fiber type except that it had extremely dense alkaline actomyosin adenosine triphosphatase staining (mean transmittance, type 2A = 33.6%; type 3 = 17.3%), as well as dense staining for periodic acid-Schiff, menadione-linked alpha-glycerolphosphate dehydrogenase, nicotinamide-adenine dinucleotide tetrazolium reductase, and succinic dehydrogenase. The type 1 fiber population was smaller in diameter (mean +/- SD, 14 +/- 4 microns) than the type 2A fiber (mean +/- SD, 21 +/- 5 microns) and the type 3 fiber (mean +/- SD, 22 +/- 6 microns) populations. In all muscles, intrafascicular and extrafascicular fat accumulations were found, with the majority being extrafascicular. Calculations indicate that the tendon occupies approximately 41% of the total muscle volume, while the muscle fibers constitute 59% of the volume.

Histochemical study of posterior cricoarytenoid muscle reinnervation by a nerve-muscle pedicle in the cat.

Reinnervation of the posterior cricoarytenoid (PCA) muscle with a nerve-muscle pedicle (NMP) has been proposed for patients with bilateral abductor vocal cord paralysis. Since its success has been controversial, a glycogen depletion histochemical technique was used to examine reinnervation. An ansa cervicalis NMP was implanted into the denervated PCA in nine cats. Eight months later, vocal cord activity was evaluated. The NMP nerve was stimulated extensively in seven cats (experimental group). Optical densities of NMP-supplied PCA muscle fibers from experimental and control groups were compared to detect differences in glycogen content. The results demonstrated quantitative evidence of reinnervation in two experimental animals. Electrical stimulation of the NMP produced abduction in one of these two animals, but was never observed during spontaneous respiration or airway occlusion. These observations indicate that reinnervation can occur but abduction requires electrical stimulation. The NMP technique may be more successful with an electrical pacer.

Motor end-plate distribution in the human interarytenoid muscle.

Histochemical and computer graphic techniques were used to determine the distribution of neuromuscular junctions in the human interarytenoid (IA) muscle. The motor end-plates of four IA specimens obtained from normal postmortem larynges were visualized using an acetylcholinesterase stain. The three-dimensional distribution of motor end-plates was later reconstructed using computer graphics. The motor end-plates in the IA were found to be distributed in an inverted "Y" configuration. A broad band of end-plates was found at the midpoint of the muscle fibers in the superior and middle aspect of the muscle. This band divided obliquely in a lateral direction toward the inferior border of the muscle. This pattern of motor end-plate distribution is possibly consistent with the bilateral innervation of the IA by the recurrent laryngeal nerves.

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.

Motor end-plate distribution in the human lateral cricoarytenoid muscle.

Knowledge of the neuromuscular junction distribution in the intrinsic laryngeal muscles has potential clinical application in directing procedures that affect the function of these muscles through their neuromuscular junctions. We used histochemical techniques and computer graphics to determine the three-dimensional distribution of the motor end-plates in the human lateral cricoarytenoid muscle. In contrast to the results that have been reported for the human thyroarytenoid, cricothyroid, and posterior cricoarytenoid muscles, where end-plates are more diffusely distributed, the results of our study indicate that in the human lateral cricoarytenoid muscle, the neuromuscular junctions are generally found within a broad band at the midlength of the muscle. This more focused distribution should be advantageous clinically in facilitating the manipulation of the motor end-plates in this muscle.

Three-dimensional computer reconstruction of the neuromuscular junction distribution in the human posterior cricoarytenoid muscle.

Recent studies have indicated that in a denervated muscle highly specific interactions occur between regenerating axons and the basal lamina of the myofiber precisely at the original synaptic sites. Since these findings suggest that synapse regeneration is facilitated at the original synaptic sites, a knowledge of the distribution of neuromuscular junctions in the human posterior cricoarytenoid (PCA) muscle may guide reinnervation procedures in this muscle. We, therefore, have used histochemical and computer graphics techniques to reconstruct the three-dimensional distribution of neuromuscular junctions in the human PCA taken fresh at autopsy. The results indicated a more localized pattern of neuromuscular junctions as compared to the human thyroarytenoid muscle. The results will be discussed with respect to their implications for procedures for reinnervation of the posterior cricoarytenoid muscle.

Central projection of the sensory component of the rat recurrent laryngeal nerve.

The central projection from the sensory components in the rat recurrent laryngeal nerve was studied using WGA-HRP. Sensory terminals were found bilaterally in the nuclei of the tractus solitarius, although they were very sparse contralaterally. In the ipsilateral nucleus, most of these terminals were located in the interstitial subnucleus extending, from the most rostral area, near the obex, caudally for a distance of 1.5 mm.

Three-dimensional distribution of neuromuscular junctions in human cricothyroid.

Microinjections of myoneural blocking agents are effective in the treatment of strabismus and have been proposed as a possible technique to treat spastic dysphonia. The success of such a technique would rely on a precise knowledge of the neuromuscular junction distribution in the laryngeal muscle to be injected. In view of the possibility of an involvement of the cricothyroid muscle in spastic dysphonia or other neuromuscular disorders, we determined the three-dimensional distribution of neuromuscular junctions in the human cricothyroid muscle. Cricothyroid muscles obtained from autopsy cases were fixed, sectioned, and processed for the histochemical localization of neuromuscular junction acetylcholinesterase. Using serial sections and a computer interfaced X-Y digitizer, the neuromuscular junctions were referenced to various anatomic landmarks and the neuromuscular junction distribution and reconstructed in three dimension using computer graphics.

Three-dimensional computer reconstruction of the distribution of neuromuscular junctions in the thyroarytenoid muscle.

Microinjections of botulinum toxin have recently been shown to be effective in the treatment of strabismus, and it has also been suggested that microinjections of this myoneural blocking agent might be of value in the treatment of spastic dysphonia. The success of such a microinjection technique would rely on a precise knowledge of the distribution of myoneural junctions in the thyroarytenoid muscle. In view of this potential application as well as the need for such information in reinnervation procedures, we have used computer graphics to reconstruct the three-dimensional distribution of motor end-plates in the thyroarytenoid muscle. Three cat and one human (fresh autopsy specimen) larynges were frozen and sectioned on a cryostat. Serial sections were then processed for the histochemical localization of acetylcholinesterase activity to demarcate the neuromuscular junctions. An X-Y digitizer was used to reference the position of the motor end-plates in each serial section, and the three-dimensional distribution of the neuromuscular junctions was reconstructed on a computer graphics terminal. The results are discussed in regard to their applicability to clinical treatment of spastic dysphonia and other disorders of phonation.

A histochemical characterization of muscle fiber types in the middle ear muscles of the cat. 1. The stapedius muscle.

The muscle fiber content of the stapedius muscle of the cat was determined histochemically using a combination of oxidative enzymes and glycolytic markers. The major fiber type present was determined to be the fast oxidative glycolytic type (FOG; 77%). Two other types of fiber were found that could not be placed into any of the classical muscle fiber categories. One of these fibers had little staining for actomyosin ATPase (1', 13%) while the other stained densely for this enzyme (2', 10%). These fibers could also be separated using fiber diameter as a criterion. The mean diameters of these different fibers were 22.8 Mm+/-6.3 (FOG fiber type), 14.8 micrometers+/-3.7 (1' fiber) and 14.9 micrometers+/-5.5 (2' fiber). Since the predominant fiber type (FOG) is adapted for fast contraction and fatigue resistance, the stapedius muscle of the cat is probably capable of fast repetitive contractions, a conclusion that fits well with much of the physiological data. Due to the fact that the 2' fibers were always paired with the 1' fibers, it is conceivable that these pairs may represent some specialized sensory structures (i.e. unencapsulated muscle spindles).