Dental pulp-derived stem cell conditioned medium to regenerate peripheral nerves in a novel animal model of dysphagia.

In nerve regeneration studies, various animal models are used to assess nerve regeneration. However, because of the difficulties in functional nerve assessment, a visceral nerve injury model is yet to be established. The superior laryngeal nerve (SLN) plays an essential role in swallowing. Although a treatment for SLN injury following trauma and surgery is desirable, no such treatment is reported in the literature. We recently reported that stem cells derived from human exfoliated deciduous teeth (SHED) have a therapeutic effect on various tissues via macrophage polarization. Here, we established a novel animal model of SLN injury. Our model was characterized as having weight loss and drinking behavior changes. In addition, the SLN lesion caused a delay in the onset of the swallowing reflex and gain of laryngeal residue in the pharynx. Systemic administration of SHED-conditioned media (SHED-CM) promoted functional recovery of the SLN and significantly promoted axonal regeneration by converting of macrophages to the anti-inflammatory M2 phenotype. In addition, SHED-CM enhanced new blood vessel formation at the injury site. Our data suggest that the administration of SHED-CM may provide therapeutic benefits for SLN injury.

Development of topography within song control circuitry of zebra finches during the sensitive period for song learning.

Refinement of topographic maps during sensitive periods of development is a characteristic feature of diverse sensory and motor circuits in the nervous system. Within the neural system that controls vocal learning and behavior in zebra finches, axonal connections of the cortical nucleus lMAN demonstrate striking functional and morphological changes during vocal development in juvenile males. These circuits are uniquely important for song production during the sensitive period for vocal learning, and the overall size of these brain regions and their patterns of axonal connectivity undergo dramatic growth and regression during this time. Axonal connections to and from lMAN are topographically organized in adult males that have already learned song. We wondered whether the large-scale changes seen in lMAN circuitry during the time that vocal behavior is being learned and refined could be accompanied by the emergence of topographic mapping. However, results presented herein demonstrate that most of these song-control circuits show the same broad patterns of axonal connectivity between subregions of individual nuclei at the onset of song learning as seen in adult birds. Thus, coarse topographic organization is not dependent on the types of experience that are crucial for vocal learning. Furthermore, this maintenance of topographic organization throughout the period of song learning is clearly not achieved by maintenance of static axonal arbors. In fact, because the volumes of song-control nuclei are growing (or regressing), topography must be maintained by active remodeling of axonal arbors to adapt to the changes in overall size of postsynaptic targets. A salient exception to this pattern of conserved topography is the projection from lMAN to the motor cortical region RA: this pathway is diffusely organized at the onset of song learning but undergoes substantial refinement during early stages of song learning, suggesting that remodeling of axonal connections within this projection during the period of vocal learning may signify the production of increasingly refined vocal utterances.

Thyroid hormone controls the onset of androgen sensitivity in the developing larynx of Xenopus laevis.

Gonadal differentiation, the onset of androgen-stimulated laryngeal growth and the genesis of a sex difference in laryngeal innervation, all temporally coincide with thyroid hormone (TH)-induced metamorphosis in Xenopus laevis. To explore the role TH plays in the ontogeny of the Xenopus androgen-sensitive vocal neuromuscular system, we examined gonadal and laryngeal development in tadpoles in which metamorphosis had been blocked by treatment with the thyroxine synthesis inhibitor propylthiouracil (PTU). PTU treatment did not arrest gonadal differentiation. Testes from PTU-treated male tadpoles had seminiferous tubules and advanced stage male germ cells, while in females stage 1 oocytes were present. In contrast to the gonads, PTU did block morphological development of the larynx. Tadpoles treated with PTU for 50 or 100 days had larynges which structurally resembled those of stage 54 control tadpoles. PTU-treated animals did not exhibit the extensive development of the laryngeal cartilage seen in untreated animals. Laryngeal cartilages of hypothyroid tadpoles exhibited low density and minimal patterning of chondrocytes; the complex lumen and marked expansion of the dilator muscles characteristic of 50- and 100-day untreated animals were absent. Laryngeal growth evoked by exposure to exogenous androgen (dihydrotestosterone) was entirely prevented by PTU treatment. Hypothyroid tadpoles did not exhibit the decline in laryngeal nerve axon number characteristic of age-matched controls, nor were laryngeal nerve axon numbers sexually dimorphic. PTU treatment also interfered with the myelination of laryngeal axons. We conclude that while gonadal differentiation is independent of TH, androgen sensitive laryngeal development and sexually dimorphic laryngeal innervation require exposure to secreted TH.

Naturally occurring motoneuron cell death in rat upper respiratory tract motor nuclei: a histological, fast DiI and immunocytochemical study of the nucleus ambiguus.

The mammalian upper respiratory tract (URT) serves as the common modality for aspects of respiration, deglutition, and vocalization. Although these actions are dependent on coordinated and specific neuromuscular control, little is known about the development of URT control centers. As such, this study investigated the occurrence of naturally occurring motoneuron cell death (MCD) in the nucleus ambiguus (NA) of a developmental series of rats. Standard histological techniques were used to count motoneurons in the ventrolateral brainstem where the mature NA is found. In addition, the neural tracer, fast DiI, was used to determine whether motoneurons were still migrating into the region of the NA during the period that cell counts were first taken. Furthermore, to elucidate the potential effect of inadvertently counting large interneurons on the assessment of motoneuron numbers, an antibody to gamma-aminobutyric acid (GABA) was used. The results of this study have, for the first time, demonstrated that MCD occurs in a URT-related motor nucleus. Approximately a 50% cell death was observed during the prenatal development of NA, with no further loss seen postnatally. The fast DiI studies showed that by embryonic day 17, NA was fully formed, suggesting that motoneuron migration from the basal plate was complete. In addition, use of the GABA antibody showed a lack of inhibitory interneurons within the NA. The finding of MCD in the NA helps define a critical period in the formation of URT neuromuscular control. As the course of MCD is modifiable by epigenetic signals, insult to the organism during this prenatal period may compromise future URT control.

Androgen directs sexual differentiation of laryngeal innervation in developing Xenopus laevis.

In adult Xenopus laevis, innervation of the vocal organ is more robust in males than in females. This sex difference originates during tadpole development; at stage 56, when the gonads first differentiate, the number of axons entering the larynx is the same in the sexes, but by stage 62, innervation is greater in males. To determine if androgen secretion establishes sex differences in axon number, we treated tadpoles with antiandrogen or androgen beginning at stage 48 or 54 and counted laryngeal nerve axons at stage 62 using electron microscopy. When male tadpoles were treated with the antiandrogen hydroxyflutamide, axon numbers were reduced to female-typical values; axon numbers in females were unaffected by antiandrogen treatment. When female tadpoles were treated with the androgen DHT (dihydrotestosterone), axon numbers were increased to male-like values. These findings suggest that endogenous androgen secretion during late tadpole stages in males is required for the sexual differentiation of laryngeal innervation observed from stage 62 on. Because androgen treatment and laryngeal innervation affect myogenesis in postmetamorphic frogs, numbers of laryngeal dilator muscle fibers were determined for hormonally manipulated tadpoles. At stage 62, vehicle-treated males had more laryngeal axons than females; laryngeal muscle fiber numbers did not, however, differ in the sexes. Both male and female tadpoles, treated from stage 54 with DHT, had more muscle fibers at stage 62 than vehicle-treated controls. Thus, while endogenous androgen secretion during late tadpole stages is subthreshold for the establishment of masculinized muscle fiber numbers, laryngeal myogenesis is androgen sensitive at this time and can be increased by suprathreshold provision of exogenous DHT. A subgroup of tadpoles, DHT treated from stage 54 to 62, was allowed to survive, untreated, until postmetamorphic stage 2 (PM2: 5 months after metamorphosis is complete). Androgen treatment between tadpole stages 54 and 62 does not prevent the ontogenetic decrease in axon numbers characteristic of laryngeal development. In addition, the elevation in stage 62 axon numbers produced by DHT-treatment at late tadpole stages was not associated with elevated numbers of laryngeal muscle fibers at PM2. Juvenile males normally maintain elevated axon numbers (relative to final adult values) through PM2 and the presence of these additional axons may result from--rather than contribute directly to--laryngeal muscle fiber addition.

The vocal motor neurons of Xenopus laevis: development of sex differences in axon number.

Sex differences in the number of muscle fibers in the larynx of clawed frogs (Xenopus laevis) develop after metamorphosis. In order to examine possible contributions of neural innervation to this process, we prepared sections of the laryngeal nerve from tadpole stage 56, when the sexes can first be distinguished, through adulthood, and counted axons on electron micrographs. The adult number of axons is achieved by a sexually differentiated pattern of axonal addition and loss. Axon numbers are high at tadpole stage 56 and equal for males and females; thereafter, males have more axons. Sex differences are most pronounced at tadpole stage 62 because between stages 59 and 62 the number of laryngeal axons in males increases by an average of 119 per nerve. Ultrastructural evidence is congruent with the hypothesis that new axons are added to the laryngeal nerve between tadpole stages 56 and 62. The loss of axons from the laryngeal nerve is greater for females than for males. Between tadpole stages 56 and adulthood, overall axon number decreases by 47% in males and by 64% in females. Signs of axonal degeneration are present in both sexes before metamorphosis but are rare at juvenile or adult stages. The numbers of axons in juvenile frogs do not differ from those in adults and continue to be greater in males than in females. In contrast to the amount of axon addition and loss, the timing of axon loss and the percentage of myelinated axons is the same for males and females throughout development. Thus sex differences in the innervation of laryngeal muscle originate before metamorphosis and could contribute to the marked sex differences in muscle fiber addition that occur thereafter.

Development of laryngeal function: etiologic significance in the sudden infant death syndrome.

Recent clinical evidence implicates transient upper airway obstruction as a cause of potentially fatal cardiorespiratory disturbances. This investigation identifies age-related neurologic mechanisms which may be pertinent to the production of abnormal laryngeal closure as a possible cause of unexpected infant death. A period of transient laryngeal hyper-excitability is identified in pups 50--75 days post-natally. The mechanism of the hyper-excitable state resulting in increased risk of laryngeal spasm, appears related to: 1. the completion of central synaptic maturation: 2. transient reduction in central latency; and 3. a reduction in central inhibition. Such observations provide clues to neurologic vulnerability occurring not immediately after birth, but during a discrete time period thereafter, prior to complete neurologic maturation. As such, these observations fulfill a criterion of utmost importance in the search for etiologic significance in the Sudden Infant Death Syndrome, and, in a broad sense, support the concept of selective maturational failure as a possible cause for age-dependent, unexpected infant death.

Characterization of the postnatal development of superior laryngeal nerve fibers in the postnatal kitten.

A combined electron microscopic and electrophysiological study of the superior laryngeal nerve (SLN) was undertaken in postnatal kittens ranging in age from 1-63 days. The superior laryngeal nerve is predominantly a sensory nerve innervating the upper respiratory tract, and could play a potential role in the modulation of respiration, particularly in the infant animal. Distribution of fibers in the developing SLN indicates that within the first postnatal month, 75% of the fibers are unmyelinated, and by 42 days, the myelinated fibers increase in number to approximately 50%. Of the myelinated fibers present in the one day old kitten, 3-4% of those exceeded 4 mum in total diameter, which is the minimum diameter for normal conduction velocity of action potentials. The distribution of the diameter sizes of the myelinated fibers is bell-shaped within the first 45 days after which the curve becomes skewed to the right (43-61 days; mean 2.6 mum, range 0.5-8.0 mum) to resemble the adult distribution of myelinated fibers (mean 4.2 mum, range 1.6-13.0 mum). Two variable plots of myelin width to axon diameter suggest a steeper slope for developing fibers as compared to that of the adult fibers. Electrical stimulation of the sectioned SLN indicates that evoked potentials could be recorded from the recurrent laryngeal nerve innervating the laryngeal intrinsic muscles and from the hypoglossal nerve to the tongue musculature in the youngest kittens tested (i.e., age 9 days). Stimulation at selected frequencies of 3 and 30/sec readily evoked apnea in the youngest kitten studied (i.e., age 5 days), while swallowing was more readily evoked at 28-30 days when using electrical stimulation.