Fine Structure of Tracheo-Bronchial Epithelial Nerves of the Cat KUEN-SHAN HUNG Depurtment of Anatomy, University of Kansas Medical Center, Kansas City, Kansas 66103
ABSTRACT The nerves in and immediately under the epithelial layer of the trachea and primary bronchi of the cat are studied with the electron microscope. The axons are unmyelinated and contain microtubules and mitochondria. Epithelial axons are observed throughout the entire epithelial layer, but, are usually concentrated in the basal area immediately above the basal lamina. Most axons are near the basal cells; others are in the vicinity of granulated cells or occur in the intercellular spaces between other columnar cells. A few of the epithelial axons are large and crowded with axoplasmic mitochondria. The possibility that some epithelial axons may have sensory functions is discussed.
Previous electron microscopy has demonstrated unmyelinated epithelial axons in the human trachea (Rhodin, '66), at the tracheal bifurcations of rabbits and guinea pigs (Fillenz and Woods, ' 7 0 ) , in the rat trachea and bronchi (Jeffery and Reid, ' 7 3 ) , and the bronchi of birds (King et al., '74) and mice (Hung and Loosli, '75). While physiological studies have revealed evidence of the presence of sensory receptors and related reflexes initiated near the surface of the tracheo-bronchial tree of the cat (Widdicombe, '54a,b), the precise anatomical locations and structures of these receptors are not known. A recent ultrastructural study on the tracheo-bronchial tree of the cat demonstrated autonomic motor innervation, but did not show the presence of sensory nerves (Silva and Ross, '74). In this paper, the fine structure of the nerves in the surface epithelium and subepithelial areas of the trachea and primary bronchi of the cat will be described. MATERIAL AND METHODS
Seven adult mongrel cats were used in this study. Each cat was anesthetized by intravenous injection of pentobarbital sodium. The trachea was exposed and phosphate buffered 2% glutaraldehyde (pH 7 . 2 ) was immediately injected into the lumen of the trachea, primary bronchi and lungs. The lungs with the bronchi and trachea attached were removed and placed ANAT. REC., 185: 85-92.
in glutaraldehyde fixative overnight. Portions of the lower one third of the trachea and the primary bronchi were dissected, removed, cut into longitudinal strips, left in fresh glutaraldehyde fixative for one hour, and post-fixed in phosphate buffered 1% osmium tetroxide for another hour. The tissues were then dehydrated and embedded in Araldite in flat molds. Thin sections were double stained with uranyl acetate and lead citrate and examined with an electron microscope. During sectioning, the tissue blocks were oriented so that the airways were always cut in cross section. OBSERVATIONS
The epithelium of the trachea and primary bronchi consisted of basal cells, ciliated cells, goblet cells and a few granulated cells. The granulated cells were characterized by numerous cytoplasmic dense-core granules, 100 n m to 160 n m in diameter (figs. 1, 2 ) , and some perinuclear cytoplasmic microfilaments. These cells often appeared singly (fig. 1 ) although a few were seen to form small clusters. Unmyelinated axons were located in the immediate subepithelial area (fig. 1) and throughout the entire layer of the epithelium. These axons were identified by their oval shape and the presence of characteristic microtubules and mitochondria. Received June 20, '75. Accepted NQV. 28, '75.
85
86
KUEN-SHAN HUNG
A majority of the axons were cut in cross section, although a few longitudinal sections were found either parallel to the surface of the epithelium or extending perpendicularly from the base of the epithelium toward the lumen. None of the axons seemed to be exposed directly to the lumen of the airways. In the subepithelial connective tissue. the axons either aggregated into small bundles covered by Schwann cells (figs. 1, 2 ) or appeared as isolated single axons, each of which was surrounded by a Schwann cell. The sizes of the axons in this area measured from 0.3 pm to 1.0 pm in diameter. In the epithelial layer, a majority of the axons were just above the basal lamina oE the epithelium (figs. 1, 3 ) . These were observed with and without Schwann cell coverings (figs. 2, 3). Most of them were wrapped by the basal cells (figs. 1, 3 ) ; a few others were located immediately under the granulated cells or along their lateral surfaces (figs. 1, 2 ) . The axons in the basal area apparently extended toward the luminal surface of the epithelium as some of them appeared above the basal cells and others were within 0.1 pm from the lumen of the airways. These axons were often seen between or surrounded by the ciliated cells (figs. 4, 5), with a few near the goblet cells. Some of these axons contained occasional large dense-core vesicles in addition to microtubules and mitochondria (fig. 4). A few large axons near the ciliated cells were crowded with numerous small mitochondria (fig. 5). The axonal membrane and the surface membrane of the epithelial cell maintained a space of 11 nm to 14 n m in width. Most axons in the epithelium ranged from 0.2 pm to 0.9 pm in diameter, with a few unusually large ones reaching 2.2 pm in diameter. DISCUSSION
The present electron microscopic studies demonstrate the presence of unmyelinated epithelial axons in the trachea and primary bronchi of the cat. Most axons were surrounded by the basal cells as shown by Jeffery and Reid ( ' 7 3 ) in their studies of the rat. It is likely that some of the epithelial axons merely pass between the epithelial cells, and are not functionally
related to them. However, it is interesting to note that the association of granulated cells with the nerves appears to be common findings in the distal intrapulmonary airways (Lauweryns et al., ' 7 2 ; Lauweryns and Cokelaere, ' 7 3 ; Hung et al,,' 7 3 ; Hung and Loosli, '74) and in the rat, bird and mouse trachea and bronchi (Lucian0 and Reale, '70; Jeffery and Reid, '73; King et al., '74; Ericson et al., '72). Previously, the axons associated with the granulated cells have been identified as sensory (Lauweryns and Cokelaere, '73; Hung et al.. '73; Hung and Loosli, "74; King et al., '74), cholinergic (Lauweryns and Cokelaere, ' 7 3 ) and adrcnergic (Ericson et al., ' 7 2 ) . Functionally, many investigators feel that these axon-granulated cell complexes form chemo-receptors (Cook and King, '69; Hung et al., ' 7 3 ; King et al., '74; Lauweryns et al., '72). Unusually large axons packed with numerous mitochondria are observed near the lumen of the airways. Similar mitochondria-rich axons have been reported to appear in many sensory organs (Cauna and Ross, '60; Farbman, '65; Iggo and Muir, '69; Chiba, '72). It is therefore possible that such axons in the tracheo-bronchial epithelium of the cat have sensory functions. ACKNOWLEDGMENTS
This work was supported by a grant from the American Lung Association. The author wishes to thank Dr. A. L. Chapman for the use of his electron microscope facilities, and for suggestions and comments on the manuscript. Technical assistance was given by Mary Mejia. LITERATURE CITED Cauna, N., and L. L. Ross 1960 The fine structure of Meissner's touch corpuscles of h u m a n fingers. J. Biophys. Biochem. Cytol.. 8: 467-482. Chiba, T. 1972 Fine structure of baroreceptor nerve terminals in thc carotid sinus of the dog. J . Electr. Microsc., 2 1 : 139-148. Cook, R. D., and A . S. King 1969 A neuritereceptor complex in t h e avian lung. Electron microscopical observations. Experientia, 25: 1162-1 164. Ericson, L. E., R. Hakanson, B. Larson, Ch. Owm a n a n d F. Sundler 1972 Fluorescence and electron microscopy of amine-storing enterochromaffin-like cells in tracheal epithelium of mouse. Z. Zellforsch., 124: 532-545. Farbman, A. I. 1965 Fine structure of the taste bud. J. Ultrastruct. Res.. 1 2 : 328-350.
TRACHEO-BRONCHIAL EPITHELIAL NERVES Fillenz, M., and R. I. Woods 1970 Sensory i n nervation of the airways. In: Breathing, HeringBreuer Centenary Symposium, Ciba Foundation. R. Porter, ed. J . and A. Churchill, London, pp. 101-107. Hung, K.-S., M. S. Hertweck, J. D. Hardy and C. G. Loosli 1973 Ultrastructure of nerves and associated cells i n bronchiolar epithelium of the mouse lung. J. Ultrastruct. Res., 43: 426-437. Hung, K.-S., and C. G. Loosli 1974 Bronchiolar neuro-epithelial bodies i n the neonatal mouse lungs. Am. J. Anat., 140: 191-200. 1975 Ultrastructural studies of tracheal and bronchial epithelial nerves. Anat. Rec., 181: 300 (abstract). Iggo, A , , and A. R. Muir 1969 The structure and function of a slowly adapting touch corpuscle in hairy skin. J. Physiol., 200: 763-769. Jeffrey, P., and L. Reid 1973 Intra-epithelial nerves in normal rat airways. A quantitative electron microscopic study. J. Anat., 114: 35-45 King, A . S . , J. McLelland, R. D. Cook, D. A . King and C. Walsh 1974 The ultrastructure of afferent nerve endings in the avian lung. Respirat. Physiol., 22: 21-40. I
87
Lauweryns, J. M., and M. Cokelaere 1973 Hypoxia-sensitive neuro-epithelial bodies, intrapulmonary secretory neuroreceptors modulated by the CNS. Z. Zellforsch., 145: 521-540. Lauweryns, J. M., M. Cokelaere and P. Theunynck 1972 Neuroepithelial bodies i n respiratory mucosa of various animals. A light, histochemical and ultrastructural investigation. Z. Zellforsch., 135: 569-592. Luciano, L., and E. Reale 1970 Die Innervation des Bronchialepithels. In: Septeme Conges International de Microscopie Electronique. Vol. 3. P. Favard, ed. Societe Francaise de Microscopie Electronique, Paris, pp. 719-720. Rhodin, J. A. G. 1966 Ultrastructure and function of the h u m a n tracheal mucosa. Am. Rev. Resp. Dis., 93: 1-15. Silva, D. G., and G . Ross 1974 Ultrastructural and fluorescence histochemical studies on the innervation of the tracheo-bronchial muscle of normal cats and cats treated with 6-hydroxydopamine. J. Ultrastruct. Res., 47: 310-328. Widdicombe, J. G . 1954a Respiratory reflexes from the trachea and bronchi of the cat. J . Physiol., 123: 55-70. 1954b Receptors in the trachea and bronchi of the cat. J. Physiol., 123: 71-104.
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PLATE 1 EXPLANATION OF FIGURES
88
1
A granulated cell ( A ) is located in the basal area of the bronchial epithelium. One nerve (N,) is directly under this cell: and another (N2) is under a basal cell. A large nerve bundle (Na) is i n the immediate sub-epithelial area. B, basal cells. x 7590.
2
This is a higher magnification of the basal area of the granulated cell shown i n figure 1. Note the dense granules ( D ) ; and a n axon ( N ) closely related to this cell. The sub-epithelial nerve bundle contains several axons. S, Schwann cell. x 25,070.
3
An axon ( N ) i n the basal area of the epithelium is first covered by the Schwann cell ( S ) , and then by the basal epithelial cell ( B ) . L, basal lamina of the epithelium. x 35,850.
TRACHEO-BRONCHIAL EPITHELIAL NERVES Kuen-Shan Hung
PLATE 1
89
PLATE 2 EXPLANATION OF FIGURES
90
4
This axon ( N ) near the lumen of the bronchus appears in the intercellular space between two ciliated cells. The axoplasm contains many microtubules, some mitochondria and occasional large dense-core vesicles. x 36,970.
5
This large axon ( N ) is covered by a ciliated cell. It is filled with mitochondria and a few microtubules. x 24,960.
TRACIIEO-BRONCHIAL EI’ITHELIAL XERVES Kuen-Shan Hung
PLATE 2
91
ABSTRACT The nerves in and immediately under the epithelial layer of the trachea and primary bronchi of the cat are studied with the electron microscope. The axons are unmyelinated and contain microtubules and mitochondria. Epithelial axons are observed throughout the entire epithelial layer, but, are usually concentrated in the basal area immediately above the basal lamina. Most axons are near the basal cells; others are in the vicinity of granulated cells or occur in the intercellular spaces between other columnar cells. A few of the epithelial axons are large and crowded with axoplasmic mitochondria. The possibility that some epithelial axons may have sensory functions is discussed.
Previous electron microscopy has demonstrated unmyelinated epithelial axons in the human trachea (Rhodin, '66), at the tracheal bifurcations of rabbits and guinea pigs (Fillenz and Woods, ' 7 0 ) , in the rat trachea and bronchi (Jeffery and Reid, ' 7 3 ) , and the bronchi of birds (King et al., '74) and mice (Hung and Loosli, '75). While physiological studies have revealed evidence of the presence of sensory receptors and related reflexes initiated near the surface of the tracheo-bronchial tree of the cat (Widdicombe, '54a,b), the precise anatomical locations and structures of these receptors are not known. A recent ultrastructural study on the tracheo-bronchial tree of the cat demonstrated autonomic motor innervation, but did not show the presence of sensory nerves (Silva and Ross, '74). In this paper, the fine structure of the nerves in the surface epithelium and subepithelial areas of the trachea and primary bronchi of the cat will be described. MATERIAL AND METHODS
Seven adult mongrel cats were used in this study. Each cat was anesthetized by intravenous injection of pentobarbital sodium. The trachea was exposed and phosphate buffered 2% glutaraldehyde (pH 7 . 2 ) was immediately injected into the lumen of the trachea, primary bronchi and lungs. The lungs with the bronchi and trachea attached were removed and placed ANAT. REC., 185: 85-92.
in glutaraldehyde fixative overnight. Portions of the lower one third of the trachea and the primary bronchi were dissected, removed, cut into longitudinal strips, left in fresh glutaraldehyde fixative for one hour, and post-fixed in phosphate buffered 1% osmium tetroxide for another hour. The tissues were then dehydrated and embedded in Araldite in flat molds. Thin sections were double stained with uranyl acetate and lead citrate and examined with an electron microscope. During sectioning, the tissue blocks were oriented so that the airways were always cut in cross section. OBSERVATIONS
The epithelium of the trachea and primary bronchi consisted of basal cells, ciliated cells, goblet cells and a few granulated cells. The granulated cells were characterized by numerous cytoplasmic dense-core granules, 100 n m to 160 n m in diameter (figs. 1, 2 ) , and some perinuclear cytoplasmic microfilaments. These cells often appeared singly (fig. 1 ) although a few were seen to form small clusters. Unmyelinated axons were located in the immediate subepithelial area (fig. 1) and throughout the entire layer of the epithelium. These axons were identified by their oval shape and the presence of characteristic microtubules and mitochondria. Received June 20, '75. Accepted NQV. 28, '75.
85
86
KUEN-SHAN HUNG
A majority of the axons were cut in cross section, although a few longitudinal sections were found either parallel to the surface of the epithelium or extending perpendicularly from the base of the epithelium toward the lumen. None of the axons seemed to be exposed directly to the lumen of the airways. In the subepithelial connective tissue. the axons either aggregated into small bundles covered by Schwann cells (figs. 1, 2 ) or appeared as isolated single axons, each of which was surrounded by a Schwann cell. The sizes of the axons in this area measured from 0.3 pm to 1.0 pm in diameter. In the epithelial layer, a majority of the axons were just above the basal lamina oE the epithelium (figs. 1, 3 ) . These were observed with and without Schwann cell coverings (figs. 2, 3). Most of them were wrapped by the basal cells (figs. 1, 3 ) ; a few others were located immediately under the granulated cells or along their lateral surfaces (figs. 1, 2 ) . The axons in the basal area apparently extended toward the luminal surface of the epithelium as some of them appeared above the basal cells and others were within 0.1 pm from the lumen of the airways. These axons were often seen between or surrounded by the ciliated cells (figs. 4, 5), with a few near the goblet cells. Some of these axons contained occasional large dense-core vesicles in addition to microtubules and mitochondria (fig. 4). A few large axons near the ciliated cells were crowded with numerous small mitochondria (fig. 5). The axonal membrane and the surface membrane of the epithelial cell maintained a space of 11 nm to 14 n m in width. Most axons in the epithelium ranged from 0.2 pm to 0.9 pm in diameter, with a few unusually large ones reaching 2.2 pm in diameter. DISCUSSION
The present electron microscopic studies demonstrate the presence of unmyelinated epithelial axons in the trachea and primary bronchi of the cat. Most axons were surrounded by the basal cells as shown by Jeffery and Reid ( ' 7 3 ) in their studies of the rat. It is likely that some of the epithelial axons merely pass between the epithelial cells, and are not functionally
related to them. However, it is interesting to note that the association of granulated cells with the nerves appears to be common findings in the distal intrapulmonary airways (Lauweryns et al., ' 7 2 ; Lauweryns and Cokelaere, ' 7 3 ; Hung et al,,' 7 3 ; Hung and Loosli, '74) and in the rat, bird and mouse trachea and bronchi (Lucian0 and Reale, '70; Jeffery and Reid, '73; King et al., '74; Ericson et al., '72). Previously, the axons associated with the granulated cells have been identified as sensory (Lauweryns and Cokelaere, '73; Hung et al.. '73; Hung and Loosli, "74; King et al., '74), cholinergic (Lauweryns and Cokelaere, ' 7 3 ) and adrcnergic (Ericson et al., ' 7 2 ) . Functionally, many investigators feel that these axon-granulated cell complexes form chemo-receptors (Cook and King, '69; Hung et al., ' 7 3 ; King et al., '74; Lauweryns et al., '72). Unusually large axons packed with numerous mitochondria are observed near the lumen of the airways. Similar mitochondria-rich axons have been reported to appear in many sensory organs (Cauna and Ross, '60; Farbman, '65; Iggo and Muir, '69; Chiba, '72). It is therefore possible that such axons in the tracheo-bronchial epithelium of the cat have sensory functions. ACKNOWLEDGMENTS
This work was supported by a grant from the American Lung Association. The author wishes to thank Dr. A. L. Chapman for the use of his electron microscope facilities, and for suggestions and comments on the manuscript. Technical assistance was given by Mary Mejia. LITERATURE CITED Cauna, N., and L. L. Ross 1960 The fine structure of Meissner's touch corpuscles of h u m a n fingers. J. Biophys. Biochem. Cytol.. 8: 467-482. Chiba, T. 1972 Fine structure of baroreceptor nerve terminals in thc carotid sinus of the dog. J . Electr. Microsc., 2 1 : 139-148. Cook, R. D., and A . S. King 1969 A neuritereceptor complex in t h e avian lung. Electron microscopical observations. Experientia, 25: 1162-1 164. Ericson, L. E., R. Hakanson, B. Larson, Ch. Owm a n a n d F. Sundler 1972 Fluorescence and electron microscopy of amine-storing enterochromaffin-like cells in tracheal epithelium of mouse. Z. Zellforsch., 124: 532-545. Farbman, A. I. 1965 Fine structure of the taste bud. J. Ultrastruct. Res.. 1 2 : 328-350.
TRACHEO-BRONCHIAL EPITHELIAL NERVES Fillenz, M., and R. I. Woods 1970 Sensory i n nervation of the airways. In: Breathing, HeringBreuer Centenary Symposium, Ciba Foundation. R. Porter, ed. J . and A. Churchill, London, pp. 101-107. Hung, K.-S., M. S. Hertweck, J. D. Hardy and C. G. Loosli 1973 Ultrastructure of nerves and associated cells i n bronchiolar epithelium of the mouse lung. J. Ultrastruct. Res., 43: 426-437. Hung, K.-S., and C. G. Loosli 1974 Bronchiolar neuro-epithelial bodies i n the neonatal mouse lungs. Am. J. Anat., 140: 191-200. 1975 Ultrastructural studies of tracheal and bronchial epithelial nerves. Anat. Rec., 181: 300 (abstract). Iggo, A , , and A. R. Muir 1969 The structure and function of a slowly adapting touch corpuscle in hairy skin. J. Physiol., 200: 763-769. Jeffrey, P., and L. Reid 1973 Intra-epithelial nerves in normal rat airways. A quantitative electron microscopic study. J. Anat., 114: 35-45 King, A . S . , J. McLelland, R. D. Cook, D. A . King and C. Walsh 1974 The ultrastructure of afferent nerve endings in the avian lung. Respirat. Physiol., 22: 21-40. I
87
Lauweryns, J. M., and M. Cokelaere 1973 Hypoxia-sensitive neuro-epithelial bodies, intrapulmonary secretory neuroreceptors modulated by the CNS. Z. Zellforsch., 145: 521-540. Lauweryns, J. M., M. Cokelaere and P. Theunynck 1972 Neuroepithelial bodies i n respiratory mucosa of various animals. A light, histochemical and ultrastructural investigation. Z. Zellforsch., 135: 569-592. Luciano, L., and E. Reale 1970 Die Innervation des Bronchialepithels. In: Septeme Conges International de Microscopie Electronique. Vol. 3. P. Favard, ed. Societe Francaise de Microscopie Electronique, Paris, pp. 719-720. Rhodin, J. A. G. 1966 Ultrastructure and function of the h u m a n tracheal mucosa. Am. Rev. Resp. Dis., 93: 1-15. Silva, D. G., and G . Ross 1974 Ultrastructural and fluorescence histochemical studies on the innervation of the tracheo-bronchial muscle of normal cats and cats treated with 6-hydroxydopamine. J. Ultrastruct. Res., 47: 310-328. Widdicombe, J. G . 1954a Respiratory reflexes from the trachea and bronchi of the cat. J . Physiol., 123: 55-70. 1954b Receptors in the trachea and bronchi of the cat. J. Physiol., 123: 71-104.
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PLATE 1 EXPLANATION OF FIGURES
88
1
A granulated cell ( A ) is located in the basal area of the bronchial epithelium. One nerve (N,) is directly under this cell: and another (N2) is under a basal cell. A large nerve bundle (Na) is i n the immediate sub-epithelial area. B, basal cells. x 7590.
2
This is a higher magnification of the basal area of the granulated cell shown i n figure 1. Note the dense granules ( D ) ; and a n axon ( N ) closely related to this cell. The sub-epithelial nerve bundle contains several axons. S, Schwann cell. x 25,070.
3
An axon ( N ) i n the basal area of the epithelium is first covered by the Schwann cell ( S ) , and then by the basal epithelial cell ( B ) . L, basal lamina of the epithelium. x 35,850.
TRACHEO-BRONCHIAL EPITHELIAL NERVES Kuen-Shan Hung
PLATE 1
89
PLATE 2 EXPLANATION OF FIGURES
90
4
This axon ( N ) near the lumen of the bronchus appears in the intercellular space between two ciliated cells. The axoplasm contains many microtubules, some mitochondria and occasional large dense-core vesicles. x 36,970.
5
This large axon ( N ) is covered by a ciliated cell. It is filled with mitochondria and a few microtubules. x 24,960.
TRACIIEO-BRONCHIAL EI’ITHELIAL XERVES Kuen-Shan Hung
PLATE 2
91
