THE LARYNGEAL MUCOSA AND THE SUPERIOR LARYNGEAL NERVE OF THE RAT An immunohistochemical and electron microscopic study AKADEMISK AVHANDLING som med vederbörligt tillstånd av rektorsämbetet vid Umeå universitet för avläggande av doktorsexamen i medicinsk vetenskap kommer att offentligen försvaras i Institutionens för Histologi med Cellbiologi föreläsningssal fredagen den 19 oktober 1990, kl 09.00 av Siw Domeij D i ™ t ^ ■ ■ ■ ^ O A lA<<o UMEÅ 1990 UMEÅ UNIVERSITY MEDICAL DISSERTATIONS NEW SERIES No 287-ISSN 0346-6612 From the Department of Histology and Cell Biology University of Umeå, Umeå, Sweden THE LARYNGEAL MUCOSA AND THE SUPERIOR LARYNGEAL NERVE OF THE RAT. AN IMMUNOHISTOCHEMICAL AND ELECTRON MICROSCOPIC STUDY SIW D O M EIJ ABSTRACT Neuropeptides are present in nerve fibers of the upper and lower airways. Local release of these substances may be of importance for the pathophysiology of airway disorders and may play a role in responses to different stimuli. However, little is known about the distribution of neuropeptides in the larynx. The superior laryngeal nerve is one of the vagal branches supplying the larynx. The aim of the present study was to investigate the fiber composition of this nerve and to analyse the distribution of different neuropeptides and mast cells in the larynx. The internal and the external branches of the superior laryngeal nerve had a similar number and size of the nerve fibers. Numerous unmyelinated fibers were evenly distributed in the branches. A large majority of the fibers were sensory myelinated and unmyelinated fibers; only a few of the myelinated fibers of the external branch ( 2-10 %) were motor. About a quarter of the unmyelinated fibers of the internal and the external branches had their cell bodies in the brainstem, and single myelinated and unmyelinated fibers emanated from the superior cervical ganglion. In every superior laryngeal nerve examined one to three spherical paraganglia were observed. These paraganglia contained cells which were similar to the type I and type II cells found in the carotid body and the paraganglia of the recurrent laryngeal nerve. Thin-walled sinusoidal blood vessels which were sometimes fenestrated were also present The laryngeal mucosa was supplied with nerve fibers exhibiting substance P- and calcitonin gene-related peptide-like immunoreactivity with regional differences in the distribution. The laryngeal side of the epiglottis and the ventral recess were richly supplied, and the vocal cords showed no evidence of immunoreactive nerve fibers. The distribution of connective tissue mast cells and mucosal mast cells/globular leucocytes was similar to that of nerve fibers displaying substance P- and calcitonin gene-related peptide-like immunoreactivity. These cells were found in close approximation to nerve fibers. Acetylcholinesterase-positive ganglionic cells in the larynx showed vasoactive intestinal polypeptide-, neuro­ peptide Y-and enkephalin-like immunoreactivity. Neuropeptide Y-like immunoreactivity was co-localized with tyrosine-hydroxylase/dopamine beta-hydroxylase-like immunoreactivity in nerve fibers in some blood vessel walls. Enkephalin-like immunoreactivity was rarely found in this location and co-localization with tyrosine- hydroxylase-like immunoreactivity was not detected. In glands and some blood vessel walls neuropeptide Y- and enkephalin-like immunoreactivity were localized in nerve fibers showing a positive acetylcholinesterase reaction and vasoactive intestinal polypeptide-like immunoreactivity. Thus, this indicates that neuropeptide Y is present in both the sympathetic and parasympathetic nervous systems, while enkephalin and vasoactive intestinal polypeptide are confined to the parasympathetic nervous system in the rat larynx. The present study shows that the superior laryngeal nerve is mainly sensory, and the study also provides a morphological basis for neuropeptide effects in laryngeal physiology/pathophysiology. Key words: Superior laryngeal nerve, paraganglia, substance P, calcitonin gene-related peptide, neuropeptide Y, vasoactive intestinal polypeptide, enkephalin, mast cells, electron microscopy, immunohistochemistry. UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No 287 — ISSN 0346-6612 From the Department of Histology and Cell Biology University of Umeå, Umeå, Sweden THE LARYNGEAL MUCOSA AND THE SUPERIOR LARYNGEAL NERVE OF THE RAT An immunohistochemical and electron microscopic study Siw Domeij AX'' University of Umeå Umeå 1990 Copyright © 1990 by Siw Domeij ISBN 91-7174-552-X CONTENTS REPORTS CONSTITUTING THE THESIS 4 ABSTRACT 5 ABBREVIATIONS 6 INTRODUCTION 7 BACKGROUND 8 General description 8 The superior laryngeal nerve 8 The larynx 8 Epithélia 9 Receptors 9 Neurotransmitters 9 Mast cells 10 AIMS OF THE STUDY 12 MATERIAL AND METHODS 13 Animals 13 Denervation procedures 13 Capsaicin treatment 13 Histological methods 13 Morphometrie methods 14 RESULTS AND DISCUSSION 15 The superior laryngeal nerve 15 The larynx 16 GENERAL DISCUSSION 19 SUMMARY 21 ACKNOWLEDGEMENTS 22 REFERENCES 23 PAPERI 31 PAPER II 43 PAPER III 51 PAPER IV 63 PAPER V 73 PAPER VI 87 REPORTS CONSTITUTING THE THESIS This thesis is based of the following reports. They will be referred to in the text by their Roman numerals: I Domeij, S. , Carlsöö, B. , Dahlqvist, Å. , Hellström, S. & Kourtopoulos, H. Motor and sensory fibers of the superior laryngeal nerve in the rat. A light and electron microscopic study. Acta Otolaryngol (Stockh) 108: 469-477, 1989. II Domeij, S. , Carlsöö, B. , Dahlqvist, Å. & Hellström, S. Paraganglia of the superior laryngeal nerve of the rat. Acta Anat. 130: 219-223, 1987. III Domeij, S ., Dahlqvist, Å. & Forsgren, S. Regional differences in the distribution of nerve fibers showing substance P- and calcitonin gene-related peptide-like immunoreactivity in the rat larynx. Manuscript. IV Domeij, S ., Carlsöö, B. , Dahlqvist, Å. & Forsgren, S. Occurrence mast cells in relation to nerve fibers in the rat larynx. Manuscript. V Domeij, S. , Dahlqvist, Å. & Forsgren, S. Studies on colocalizatoin of neuropepitde Y, vasoactive intestinal polypeptide, catecholamine-synthesizing enzymes and acetylcholinesterase in the larynx of the rat. Cell Tissue Res. In press. VI Domeij, S. , Dahlqvist, Å. & Forsgren, S. Enkephalin-like immunoreactivity in the rat larynx and the superior cervical ganglion. Manuscript. 4 UMEÅ UNIVERSITY MEDICAL DISSERTATIONS NEW SERIES No 287-ISSN 0346-6612 From the Department of Histology and Cell Biology University of Umeå, Umeå, Sweden THE LARYNGEAL MUCOSA AND THE SUPERIOR LARYNGEAL NERVE OF THE RAT. AN IMMUNOHISTOCHEMICAL AND ELECTRON MICROSCOPIC STUDY SIW DOM EIJ ABSTRACT Neuropeptides are present in nerve fibers of the upper and lower airways. Local release of these substances may be of importance for the pathophysiology of airway disorders and may play a role in responses to different stimuli. However, little is known about the distribution of neuropeptides in the larynx. The superior laryngeal nerve is one of the vagal branches supplying the larynx. The aim of the present study was to investigate the fiber composition of this nerve and to analyse the distribution of different neuropeptides and mast cells in the larynx. The internal and the external branches of the superior laryngeal nerve had a similar number and size of the nerve fibers. Numerous unmyelinated fibers were evenly distributed in the branches. A large majority of the fibers were sensory myelinated and unmyelinated fibers; only a few of the myelinated fibers of the external branch ( 2-10 %) were motor. About a quarter of the unmyelinated fibers of the internal and the external branches had their cell bodies in the brainstem, and single myelinated and unmyelinated fibers emanated from the superior cervical ganglion. In every superior laryngeal nerve examined one to three spherical paraganglia were observed. These paraganglia contained cells which were similar to the type I and type II cells found in the carotid body and the paraganglia of the recurrent laryngeal nerve. Thin-walled sinusoidal blood vessels which were sometimes fenestrated were also present. The laryngeal mucosa was supplied with nerve fibers exhibiting substance P- and calcitonin gene-related peptide-like immunoreactivity with regional differences in the distribution. The laryngeal side of the epiglottis and the ventral recess were richly supplied, and the vocal cords showed no evidence of immunoreactive nerve fibers. The distribution of connective tissue mast cells and mucosal mast cells/globular leucocytes was similar to that of nerve fibers displaying substance P- and calcitonin gene-related peptide-like immunoreactivity. These cells were found in close approximation to nerve fibers. Acetylcholinesterase-positive ganglionic cells in the larynx showed vasoactive intestinal polypeptide-, neuro­ peptide Y-and enkephalin-like immunoreactivity. Neuropeptide Y-like immunoreactivity was co-localized with tyrosine-hydroxylase/dopamine beta-hydroxylase-like immunoreactivity in nerve fibers in some blood vessel walls. Enkephalin-like immunoreactivity was rarely found in this location and co-localization with tyrosine- hydroxylase-like immunoreactivity was not detected. In glands and some blood vessel walls neuropeptide Y- and enkephalin-like immunoreactivity were localized in nerve fibers showing a positive acetylcholinesterase reaction and vasoactive intestinal polypeptide-like immunoreactivity. Thus, this indicates that neuropeptide Y is present in both the sympathetic and parasympathetic