5-azacytidine enhances proliferation in transplanted rat fetal epiglottis.

Fetal rat epiglottis and its developmental potential in ectopic transplants under the influence of the epigenetic drug was investigated. Epiglottises from 17-days-old rat embryos were transplanted under kidney capsules of adult rats for 14 days. 5-azacytidine (5 mg/kg) was injected intraperitoneally during first three days and controls were sham treated. TEM, immunohistochemical detection and quantitative stereological analysis of the Proliferating Cell Nuclear Antigen (PCNA) expression (numerical density N(v)) were performed. Typical chondroblasts with long surface processes and sparse lipid droplets were found in fetal epiglottis and chondrocytes with shorter processes, numerous lipid droplets and elastic fibers in adult. PCNA was expressed in almost all cells of the fetal epiglottis while in the adult it was expressed in minority of cells. In transplants, differentiation progressed towards the differentiation found in the adult. Application of 5-azacytidine increased the capacity for proliferation (N(v PCNA)) in comparison to controls but no difference in differentiation was observed. Data about the developmental potential and induction of proliferation in mammalian epiglottis by epigenetic modulation is of importance for regenerative medicine.

Development of the movement of the epiglottis in infant and juvenile pigs.

Although backward folding of the epiglottis is one of the signal events of the mammalian adult swallow, the epiglottis does not fold during the infant swallow. How this functional change occurs is unknown, but we hypothesize that a change in swallow mechanism occurs with maturation, prior to weaning. Using videofluoroscopy, we found three characteristic patterns of swallowing movement at different ages in the pig: an infant swallow, a transitional swallow and a post-weaning (juvenile or adult) swallow. In animals of all ages, the dorsal region of the epiglottis and larynx was held in an intranarial position by a muscular sphincter formed by the palatopharyngeal arch. In the infant swallow, increasing pressure in the oropharynx forced a liquid bolus through the piriform recesses on either side of a relatively stationary epiglottis into the esophagus. As the infant matured, the palatopharyngeal arch and the soft palate elevated at the beginning of the swallow, so exposing a larger area of the epiglottis to bolus pressure. In transitional swallows, the epiglottis was tilted backward relatively slowly by a combination of bolus pressure and squeezing of the epiglottis by closure of the palatopharyngeal sphincter. The bolus, however, traveled alongside but never over the tip of the epiglottis. In the juvenile swallow, the bolus always passed over the tip of the epiglottis. The tilting of the epiglottis resulted from several factors, including the action of the palatopharyngeal sphincter, higher bolus pressure exerted on the epiglottis and the allometry of increased size. In both transitional and juvenile swallows, the subsequent relaxation of the palatopharyngeal sphincter released the epiglottis, which sprang back to its original intranarial position.

Differentiation of epiglottal epithelia during prenatal and postnatal human development.

Differentiation of epiglottal epithelia during human development was for the first time investigated by the light microscopy and documented in celoidine collection of human embryos from the Archive of the Department of Histology and Embryology, School of Medicine University of Zagreb, Croatia. At 6 weeks epiglottal swelling was found to be covered by a simple squamous epithelium consisting of a single layer of cells. At 8 weeks epithelium changed to a two-layered cuboidal epithelium which at the end of the 8th week transformed to multilayered columnar epithelium without cilia and goblet cells. In the one-day-old newborn, the majority of epiglottis was found to be covered by the mature ciliated columnar pseudostratified epithelium with goblet cells while only a minor part of the oral surface next to the tongue by the stratified squamous epithelium. This unexpected finding is in contrast to the domination of the stratified squamous epithelium found at the age of 13 years and in 35-years-old adult. Reversal of proportion covered by different types of epithelia between birth and puberty /adulthood is probably connected to the establishment of the air-flow which could be stimulating for differentiation of stratified squamous epithelium.

Development of the supralaryngeal vocal tract in Japanese macaques: implications for the evolution of the descent of the larynx.

The configuration of the supralaryngeal vocal tract depends on the nonuniform growth of the oral and pharyngeal portion. The human pharynx develops to form a unique configuration, with the epiglottis losing contact with the velum. This configuration develops from the great descent of the larynx relative to the palate, which is accomplished through both the descent of the laryngeal skeleton relative to the hyoid and the descent of the hyoid relative to the palate. Chimpanzees show both processes of laryngeal descent, as in humans, but the evolutionary path before the divergence of the human and chimpanzee lineages is unclear. The development of laryngeal descent in six living Japanese macaque monkeys, Macaca fuscata, was examined monthly during the first three years of life using magnetic resonance imaging, to delineate the present or absence of these two processes and their contributions to the development of the pharyngeal topology. The macaque shows descent of the hyoid relative to the palate, but lacks the descent of the laryngeal skeleton relative to the hyoid and that of the EG from the VL. We argue that the former descent is simply a morphological consequence of mandibular growth and that the latter pair of descents arose in a common ancestor of extant hominoids. Thus, the evolutionary path of the great descent of the larynx is likely to be explained by a model comprising multiple and mosaic evolutionary pathways, wherein these developmental phenomena may have contributed secondarily to the faculty of speech in the human lineage.

A morphometric study of age-related changes in adult human epiglottis using quantitative digital analysis of cartilage calcification.

The epiglottis plays an important role in deglutition in humans. The present study investigated age-related changes in the epiglottis using macroscopic and microscopic measurements. Epiglottic specimens from 281 Japanese adult cadavers (177 males, 104 females) were obtained. Specimens were divided into three groups according to age: group I: 20-39 years old (32 males, 26 females), group II: 50-69 years old (82 males, 36 females), and group III: 80-98 years old (63 males, 42 females). Width, height, and thickness were measured macroscopically. To evaluate the degree of calcium deposition, the calcium volume in digitalized von Kossa-stained sections was assessed using a quantitative analysis. An elemental analysis of the area detected with von Kossa staining was done using energy-dispersive X-ray fluorescence spectrometer (EDX). Measurements of the thickness and cell density in the superficial and deep layers of epiglottic cartilage were performed in horizontal histological sections. No significant differences in macroscopic width or height were found across the age groups in either sex. A series of three measurements in males was significantly larger than in females (p<0.05). The volume of the calcium deposit area was greater in males than in females (p<0.05) and was significantly increased in group III in males (p<0.05). The lower level of the epiglottic cartilage showed a greater calcium deposit area than the upper level. In the scanning image by line and surface analysis using EDX, the calcium deposit areas detected with von Kossa staining indicated a close association of calcium and phosphorus ions. The mean Ca/P molar ratio in the calcium deposit area was 1.32+/-0.12. Microscopic cartilage thickness increased significantly with age (p<0.05), and was greater in males than in females (p<0.05). Cartilage cell density in the superficial cartilage layer was higher than in the deep layer and was decreased in group III (p<0.05). Cartilage cell density was lower in males compared to females. Diameter of chondrocytes significantly increased in group III (p<0.05) and was larger in males than in females in group III (p<0.05). Epiglottic cartilage exhibited marked sex-related differences and progression of calcification with age. Calcification of epiglottic cartilage in elderly individuals may affect movement patterns in deglutition.

Magnetic resonance imaging procedures to study the concurrent anatomic development of vocal tract structures: preliminary results.

The vocal tract structures undergo drastic anatomic restructuring during the course of development from infancy to adulthood. This study demonstrates the feasibility of using MRI to examine the growth processes of the vocal tract. This method affords precise and detailed visualization of the soft tissues in the oro-pharyngeal region, while also providing images of related bony and cartilaginous structures. Information on anatomic restructuring contributes to the understanding of how speech emerges and develops, and it also establishes normative information that can be used in the assessment of developmental anomalies. This paper describes the method used to measure and examine the concurrent anatomic development of the various vocal tract structures during early childhood. Preliminary results from two pediatric subjects indicate that there is synchrony of growth in the different structures-both soft and hard tissues-, and that such synchronous growth appears to persist during periods of growth spurts.

Myxoid tissue: its morphology, histochemistry, and relationship with other supporting tissues.

Myxoid tissue was studied in the supporting organ of the cat epiglottis ("epiglottic cartilage"). Under the light microscope, myxoid tissue was characterized by stellate cells placed into an avascular acidic extracellular matrix. This extracellular matrix was alcianophilic at pH = 2.5, reacting with the colloidal iron stain, and staining metachromatically with toluidine blue O at pH = 5.0. Treatment of sections with testicular hyaluronidase abolished these reactions. In addition, staining persisted after methylation/saponification pretreatment, indicating hyaluronic acid as the main acidic component of myxoid extracellular matrix. Under the electron microscope, myxoid extracellular matrix formed flocculent electron dense precipitates. Stellate myxoid cells were characterized by bundles of intermediate (8 nm) cytoplasmic filaments. Myxoid cells were devoid of a basal lamina, contained a few small lipid droplets, and stored some glycogen. Bundles of collagen fibrils, 80-120 nm in diameter, were seen in myxoid areas. Myxoid cells reacted to S-100 protein, glial fibrillary acidic protein, and neuron specific enolase. Moreover, in adult animals, myxoid cells stained for neurofilament protein 200. All these markers were also present in chondrocytes of elastic and fibrous cartilage, indicating a close relationship between myxoid cells and chondrocytes. This was supported by the observation of continuous transitional forms of myxoid tissue into elastic or fibrous cartilage. In 8-week-old kittens, the supporting organ of the epiglottis was found mainly to consist of myxoid tissue with only a few interspersed islets of chondrocytes. It is therefore concluded that myxoid tissue can serve as a precursor of cartilage.

Maturational descent of the epiglottis.

BACKGROUND: Otolaryngologists and anesthesiologists have described a maturational descent of the epiglottis that occurs in infancy and childhood. OBJECTIVE: To investigate the changing level of the epiglottis to confirm and characterize this phenomenon more completely. DESIGN: A survey of 500 images with 338 images selected for the study. SETTINGS: A tertiary care facility. PATIENTS: Asymptomatic children aged 1 day to 18 years. MAIN OUTCOME MEASURE: The position of the tip of the epiglottis was correlated with the cervical vertebral level. RESULTS: Data indicate that maturational descent of the epiglottis starts in infancy and continues into adolescence. These results are statistically significant (P < .01). CONCLUSION: Maturational descent of the epiglottis occurs in a predictable pattern. Understanding this phenomenon may facilitate laryngoscopic, as well as clinical and radiologic, evaluation of the airway in children.

Morphology, histochemistry, and differentiation of the cat's epiglottic cartilage: a supporting organ composed of elastic cartilage, fibrous cartilage, myxoid tissue, and fat tissue.

BACKGROUND: In carnivores, the supporting organ of the epiglottis is usually called "epiglottic cartilage" (EC) although it is composed of elastic cartilage and unilocular fat storing cells. We studied the cat's EC in order to decide whether these fat storing cells are true adipocytes or fat storing (dedifferentiated) chondrocytes. METHODS: ECs were studied in cat embryos at gestation days 40 and 60, in newborn, postnatal, and adult cats. We used classical staining methods, immunohistochemistry, and transmission electron microscopy to identify the different kinds of tissues contributing to the EC and to follow their differentiation. RESULTS: The cat's EC was defined by a layer of coarse collagen fibers representing a tunica albuginea. This tunica covered irregularly formed and irregularly sized areas of elastic cartilage, fibrous cartilage, myxoid tissue, and lobules of unilocular fat cells. All these tissue showed regular morphology. Adipocytes were provided with continuous basal laminae and fat lobules were well supplied with capillaries. Alcianophilia of ground substance was observed in all tissue components but was strongest in elastic cartilage. Most islets of elastic cartilage adhered to the tunica albuginea of the EC at one surface and were connected to the opposite surface by coarse strands of connective tissue traversing the organ. Intercalated areas of fibrous cartilage contained fuchsinophilic collagen bundles. Myxoid tissue was characterized by stellate cells in alcianophilic ground substance with intermingled fuchsinophilic bundles. All kinds of supporting tissues combined with each other without clear demarcation. Immunohistochemistry revealed strong reactivity for S-100 of chondrocytes, myxoid cells, and fat cells. Chondrocytes and myxoid cells also stained for glial fibrillary acidic protein, neurofilament protein 200, and neuron specific enolase. During development, condensation of mesenchymal cells indicated the blastema of the EC at gestation day 40. At day 60, delicate collagen fibrils indicated the future tunica albuginea, faint alcianophilia was noted in the ground substance, and multilocular fat cells were scattered throughout the blastema. At birth, alcianophilia was moderate and multilocular fat cells were numerous. Three weeks after birth, single and grouped unilocular fat cells were seen, alcianophilia of ground substance was prominent, and former blastema cells presented as ramified myxoid cells. Eight weeks after birth, the EC primarily consisted of myxoid tissue, but the first islets of cartilage were seen in the center of myxoid areas. Unilocular fat cells already formed lobules. CONCLUSIONS: These results show that in the cat EC a) differentiation of adipocytes precedes differentiation of all the other tissue components, and b) differentiation of myxoid tissue precedes differentiation of cartilage. It is concluded that myxoid tissue may serve as a precursor of fibrous and elastic cartilage.

Superior laryngeal nerve response patterns to chemical stimulation of sheep epiglottis.

Responses were recorded from single fibers of the sheep superior laryngeal nerve during stimulation of the epiglottis with 0.5 M KCl, NH4Cl, NaCl and LiCl, distilled water, 0.005 M citric acid, and 0.01 N HCl. Recordings were made from both lambs and ewes. KCl elicited a response from 99% of fibers followed in order of effective stimulation by NH4Cl, HCl, distilled water, citric acid, NaCl and LiCl. Analysis of the variation in response frequency with time demonstrated differences in the response patterns for these stimuli. The pattern of frequency over time is sufficient to discriminate among the salts, between some of the salts and acids, and between some of the salts and water. Therefore the response pattern may be significant in initiating the various reflex activities that occur during chemical stimulation of the larynx.

A quantitative study of cat epiglottal taste buds during development.

To quantify the development of upper airway chemoreceptors, epiglottal taste buds were counted in cat fetuses, kittens and adults. Taste buds with structural characteristics similar to those in the adult were observed in all fetuses close to term and in kittens from birth. During the first postnatal week a mean of 76 taste buds was present on the kitten epiglottis and by adulthood 800 were observed. The number of taste buds increased as a logarithmic function of both age and weight, but the correlation with weight is better than that with age. The presence of epiglottal taste buds from very early developmental stages suggests that these receptors may mediate the reflex apnoea and swallowing that occur in response to chemical stimulation of the larynx in newborns.

[Morphometrically definable aging velocities of cells and tissues]. / Morphometrisch fassbare Alternsgeschwindigkeiten von Zellen und Geweben.

We determined aging changes of the nucleus-cytoplasm-relation in the epithel of the human epiglottis and intestine of rats. The results were compared with data about the proliferation of connective tissue and the decrease of muscle cells in the wall of the intestine and in the ciliary muscle. We adapted exponential functions to the measured results. For comparing the several equations and the derivable aging velocities, the nucleus-cytoplasm-relation must be transformed into nucleus volume rate of the total cell volume. Similarities in the decrease of the aging speed in the course of life and in the value of the exponent of the aging function by W. Beier (approximately 0,03) were demonstrated.

Quantitative analysis of developing epiglottal taste buds in sheep.

Epiglottal taste buds of the sheep increase in number during development, and continue to increase until the epiglottis has reached its adult size. However, since the increase in taste bud numbers is paralleled by increase in the surface area of the epiglottis, the density of taste buds decreases progressively in the fetus and newborn. After birth the density remains relatively constant. From examination of the morphological stages of epiglottal taste bud development, we conclude that taste buds are originally produced by de novo formation, but that further increase in numbers is effected by division of existing, developing taste buds. Because of the large number of taste buds, and the maintenance of their density on the epiglottis, it is concluded that they have an important functional role in upper airway protective reflexes.

Survey of the development of laryngeal epithelium.

An overview of the developing laryngeal epithelium has been presented to establish the norm. The sequential relationships of the formation of the protective laryngeal covering, cellular and glandular, have been reviewed. The topographical distribution of the epithelial elements in the human larynx are illustrated in the adult and contrasted in the child. Unique and previously undescribed cellular ultrastructure and transition areas have been identified. Biological integrationof form and function is again well illustrated in the epithelium of the human larynx.