Leslie T. Malmgren

Localization of adductor and abductor motor nerve fibers to the larynx.

Knowledge of the location of motor nerve fibers to the adductor and abductor muscles of the larynx may be useful in the diagnosis and treatment of innervation disorders in this organ. Anterograde degeneration and retrograde tracer anatomical techniques have demonstrated the central and peripheral positions of these two groups of motor nerve fibers in the cat. Traditional nerve fiber degeneration methods applied following intracranial transection of the vagus nerve rootlets indicated that: 1) Most of the fibers in the recurrent laryngeal nerve (RLN) are motor; 2) Almost all of these motor fibers leave the brain stem in the most rostral rootlet(s) of the vagus nerve; and 3) Motor fibers to the larynx form a discrete bundle within the trunk of the vagus nerve before forming the RLN. A tracer (horseradish peroxidase) of retrograde axoplasmic flow in motor neurons has been employed to demonstrate: 1) Dorsoventral division of the adductor and abductor neurons in the nucleus ambiguus; and 2) Diffuse arrangement of both adductor and abductor nerve fibers in the vagus nerve but collection of these fibers into abductor and adductor halves of the RLN prior to entering the larynx. These findings dispel theories of differential cord paralysis (Semons law) based on a vulnerable position of abductor fibers at the periphery of the RLN. Furthermore, the diffuse arrangement of these fiber groups explains the usually mixed functional results obtained following reimplantation of the RLN into a laryngeal muscle.

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 (LV 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 LV, but there was an age-related decrease (P < 0.05) in the surface density (SV 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 (LL 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.

Histochemical Characteristics of Muscle Fiber Types in the Posterior Cricoarytenoid Muscle

The muscle fiber type composition of the human posterior cricoarytenoid muscle (PCA) was examined using a large battery of histochemical techniques. Staining for myosin ATPase (pH 9.9) indicated that the muscles were composed of 52% ± 11.8 SD type 1 (slowly contracting) fibers and 48% ± 11.9 SD type 2 (rapidly contracting) fibers. In order to obtain information concerning the probable fatigue resistance of the type 2 fibers, serial sections were processed to determine the relative extent of ATPase inactivation at various pH levels in the acid range and to obtain data concerning the relative activities of oxidative and glycolytic enzymes and their substrates. The great majority of the type 2 fibers were of the 2A (fatigue resistant) fiber type. This indication of a capacity for prolonged activity was substantiated by the presence of high activities of succinic dehydrogenase, a mitochondrial enzyme which is involved in oxidative metabolism. Type 2C fibers (generally considered to be an undifferentiated fiber type) were also present but relatively rare. The overall enzyme profiles of many of the muscle fibers in the human PCA differed from those typical of fibers having the same alkaline ATPase and acid ATPase characteristics in most other mammalian muscles. Since muscle fiber biochemistry reflects the activity pattern of the motor unit, these unusual enzyme profiles may be the result of activity patterns that are associated with the inspiratory cycle and/or patterns of activity that are relatively specific to the PCA. Four of the ten muscles examined had unequivocal evidence of muscle fiber type grouping, a manifestation of partial denervation followed by reinnervation. This is interesting since most of the cases were in the fifth decade. Muscle fiber type grouping has been shown to occur selectively in certain other human muscles and to increase with age, eventually resulting in muscle atrophy. This suggests the possibility that the human PCA is for some reason selectively vulnerable to partial denervation and indicates the need for more extensive data concerning the relationship of muscle fiber type grouping in the PCA and other laryngeal muscles to age.

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.

Age-related changes in the neuromuscular junctions in the human posterior cricoarytenoid muscles: A quantitative study†

The posterior cricoarytenoid muscle is unique histo‐chemically, 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.

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-supphed 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.

Localization of abductor and adductor fibers in the kitten recurrent laryngeal nerve: Use of a variation of the horseradish peroxidase tracer technique

Abstract Horseradish peroxidase was injected into either the posterior cricoarytenoid or thyroarytenoid muscle of seven newborn kittens. After allowing 3 to 5 h for retrograde axoplasmic transport in motor axons, the animals were killed and the recurrent laryngeal nerves were stained histochemically for the peroxidase. Transmission electron microscopy was used to examine serial sections of both nerves for axons containing horseradish peroxidase reaction product. The results demonstrated that the motor axons supplying an abductor muscle p. cricoarytenoid and those to an adductor muscle thyroarytenoid are diffusely arranged in the recurrent laryngeal nerve.

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.

ACETYLCHOLINESTERASE STAINING OF FIBER COMPONENTS IN FELINE AND HUMAN RECURRENT LARYNGEAL NERVE Topography of Laryngeal Motor Fiber Regions

The amount of epineurial connective tissue in the human recurrent laryngeal nerve (RLN) (87.65.8% S.D. of x.s. area; 0-3 cm inferior to cricoid) was greater than in most other human nerves (generally 30-75 % of x.s. area). This probably makes the RLN resistant (viscoelastic properties) to stretch and other mechanical trauma. Two types of nerve fiber population groups were found in the human RLN. One type of grouping (group 1) included primarily nerve fibers (4-12 urn diameter) that stained moderately or intensely for axonal acetylcholinesterase (AChE) with very few unstained axons. Experimental studies using the cat indicated that this group provides mainly motor innervation to the human intrinsic laryngeal muscles. in other regions of the human RLN the populations of nerve fibers (group 2) differed from group 1 in that myelinated axons of the same size (4-12 fim diameter) were almost entirely unstained for AChE while smaller myelinated axons (1-5 p.m diameter) were either darkly or moderately stained. Gr...

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 ceils 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, veryold = 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. In view of the “giant” mitochondria and other degenerative Schwann cell features noted in this and earlier studies, the latter possibility seems to be a more reasonable explanation of our findings.