The Laryngeal Mucosa and the Superior Laryngeal Nerve of the Rat

Home , Submandibular ganglion, Superior laryngeal nerve

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

Siw Domeij

D i ™ t ^ ■ ■ ■ ^ O A lA <

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

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

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.

5 Abbreviations

ACh acetylcholine AChE acetylcholinesterase CGRP calcitonin gene-related peptide DBH dopamine ß-hydroxylase ENK enkephalin 5-HT 5-hydroxytryptamine -LI -like immunoreactivity NPY neuropeptide Y SP substance P TH tyrosine hydroxylase VIP vasoactive intestinal polypeptide

6 INTRODUCTION

The main functions of the human larynx, The laryngeal mucosa is provided with nerve protection, respiration and phonation, are controlled by endings which have the ability to respond to complicated interrelationships of diverse polysynaptic mechanical, chemical and thermal stimuli (Koizumi, brainstem reflexes (Widdicombe, 1977). The protective 1953; Ogura & Lam, 1953; Suzuki & Kirchner, 1968; function is entirely reflexive, while respiration and Widdicombe, 1977). Taste buds are also found in the phonation can be initiated voluntarily, but are regulated larynx; the function of these receptors still remains involuntarily through an array of feedback reflexes unclear (Palmieri et al., 1983). (Sasaki & Isaacson, 1988). The reflex mechanisms are Neuropeptides, a new group of neurotransmitters in mediated through two vagal branches; the recurrent addition to the "classical” transmitters, are present in laryngeal nerve and the superior laryngeal nerve. The nerve fibers in the body, including the airways. The larynx participates in the modulation of respiration by neuropeptides play important roles. For example, a changing the airway resistance, and respiration is participation of different neuropeptides in locally influenced by chemoreceptor organs (paraganglia), such induced neurogenic inflammation in the lower airways as, the carotid body (Dixon et al.,1974; Bartlett, 1980). and in the nose has been described (Lundberg & Saria, Paraganglia are also found close to and in the superior 1982; 1983; Lundberg et al., 1983 a, b; Lundblad et and recurrent laryngeal nerves of both the rat and man al., 1983 a). There is, however, limited information on (Watzka, 1963; Kleinsasser, 1964; McDonald & the distribution of neuro-peptides in the larynx. Blewett, 1981; Dahlqvist et al., 1986).

7 BACKGROUND

General description contains myelinated fibers of all sizes, but both the internal and external branches have a majority of small The anatomy of the larynx and the ability of the fibers, 1.5 to 4 pm, (DuBois & Foley, 1936; Miller & mucosal lining to secrete mucous was first described in Loizzi, 1974). Only a few unmyelinated fibers have the writings of Galen (131-210 A. D.). The main been found in the internal and external branches by laryngeal nerves, the recurrent laryngeal nerve and the using light (DuBois & Foley, 1936) and electron superior laryngeal nerve, are vagal branches. The microscopy (Miller & Loizzi, 1974). The laryngeal sensory fibers of these nerves have their cell bodies in nerves also contain small-diameter vasomotor and the jugular and nodose ganglia (Molhant, 1913) and secromotor fibers of sympathetic (superior and middle terminate in the tractus nucleus solitarius. Motor fibers cervical ganglia) (Hisa, 1982) and parasympathetic emanate from the nucleus ambiguus, and the dorsal (dorsal motor nucleus of the vagus nerve) (Mitchell, motor nucleus of the vagal nerve is a source of 1954; Hinrischen et al.,1981; Wallach et al.,1983) parasympathetic secromotor fibers (Mitchell, 1954; origin. Studies on the human superior laryngeal nerve Hinrischen et al.,1981; Wallach et al., 1983). The have revealed that the fibers range from 1 to 18 pm in recurrent laryngeal nerve supplies the subglottic diameter; two-thirds of the fibers range from 1 to 10 mucosa with sensory fibers, and all intrinsic laryngeal pm and the majority from 1 to 3 pm (Ogura & Lam, muscles except the cricothyroid muscle with motor 1953; Scheuer, 1964). fibers (Suzuki & Kirchner, 1969). The superior The proportion of sensory and motor fibers, as laryngeal nerve divides into two branches: The internal well as the proportion of sympathetic and para branch innervates the supraglottic mucosal lining of sympathetic fibers, in the superior laryngeal nerve, is the larynx, and the external branch carries motor fibers to our knowledge undetermined. to the cricothyroid muscle (Onodi, 1902; Lemere, 1932; Galen, 1962). The internal branch is believed to be purely a sensory nerve (Ogura & Lam, 1953; The larynx Andrew, 1956; Suzuki & Kirchner, 1968; Rueger 1972), and the external branch mainly a motor nerve The larynx can be divided into the supraglottic, (DuBois & Foley, 1936). However, afferent discharge glottic and subglottic regions. A fold in the has been recorded from the external branch (Andrew, supraglottic region between the epiglottis and the 1956; Suzuki & Kirchner, 1968). Morphologically, the arytenoid cartilages is termed the aryepiglottic fold recurrent laryngeal nerve has been studied more (Fig. 1). extensively than the superior laryngeal nerve.

The superior laryngeal nerve

Anatomical and physiological studies in cat, dog and man have revealed that the laryngeal motor fibers vary widely in diameter. These fibers are in general somewhat smaller than those innervating striated muscle elsewhere in the body; most of the fibers being between 6 and 10 pm in diameter (Ogura & Lam, 1953; Scheuer, 1964). In an electrophysiological study of the main trunk of the rat superior laryngeal nerve estimation of the fiber size was based on recordings of a b the impulse size. It was found that the majority of the nerve fibers were less than 4 pm in diameter and that fibers above 10 pm were rare (Andrew, 1956). DuBois Fig. 1. A schematic drawing showing two & Foley (1936) showed that in the cat superior sectioned larynxes, (a) a lateral view, (b) a view from laryngeal nerve, small fibers (1.5 to 5 pm) were more behind. 1 = the supraglottic region, 2 = the frequent than large ones (5 to 8 pm). The internal aryepiglottic fold, 3 = the glottic region, 4 = the branch of the superior laryngeal nerve of the cat subglottic region.

8 Epithélia participate not only in the co-ordinated control of the laryngeal musculature in respiration and phonation, but Depending on the function of the different regions also have a proprioceptive function (Andrew, 1956; of the larynx, different types of epithelia are found. Suzuki & Kirchner, 1968; Sasaki & Isaacson, 1988). Surfaces subject to wear and tear are covered with stratified squamous epithelium. This epithelium is Muscular receptors found on the lingual and laryngeal side of the A paucity of muscle spindles is reported for the epiglottis, on the upper part of the aryepiglottic folds intrinsic laryngeal muscles of man and the cat and on the vocal cords. In the rat, a thin keratinized (Gracheva, 1963). However, the laryngeal muscles layer is apparent on the lingual side of the epiglottis seem to have an abundant supply of spiral nerve and the epithelium on the vocal cords is only two cell endings coiled around individual muscle fibers layers thick (Smith, 1977; Lewis & Prentice, 1980). (Gracheva, 1963). These receptors are likely to Also in the rat, a pouch is located ventral to the vocal participate in reflex contractions and relaxations of the cords (the ventral recess). The pouch is covered with a laryngeal muscles (Gracheva, 1963). cuboidal epithelium (Smith, 1977; Lewis & Prentice, 1980). Posteriorly, at the level of the vocal cords and Paraganglia (peripheral chemoreceptors) in the subglottic region, a pseudostratified ciliated columnar epithelium is present. In the zones between Paraganglia are clusters of cells with the same the stratified squamous and pseudostratified columnar embryological origin as the cells of the adrenal epithelia (i. e. the lower parts of the epiglottis and medulla. These cells show a chromaffin reaction. The aryepiglottic folds and the caudal part of the glottic cell clusters form small organs and are thought to area), a cuboidal pseudostratified epithelium covered belong to the sympathetic nervous system (Kohn, with microvilli is present (Lewis & Prentice, 1980). 1900; Pearse et al., 1973). These organs are found throughout the body; not only in the head and neck region, but also in the kidney, the ovary, the testis, Receptors and the liver. The largest and most well-studied paraganglion is the carotid body, which is located in Mucosal receptors the bifurcation of the carotid arteries (Adams, 1958). By using silver impregnation techniques, a regional The cells of the carotid body react to changes of pH, difference in the distribution of glomerular pC>2 and pCC>2 tension of the blood (Heymans et al., terminations and a widespread network of free nerve 1930) and participate in regulation of breathing (de endings can be observed in the mucosa (Koizumi, Burgh Daly & Scott, 1958; de Burgh Daly & Angell- 1953; König & von Leden, 1961). This receptor James, 1979). Paraganglia are highly vascularized and system is thought to be supplied by small and medium consist of type I and type II cells. Type I cells contain sized myelinated and unmyelinated afferent fibers numerous dense-cored vesicles and type II cells lack (Ogura & Lam, 1953; Suzuki & Kirchner, 1968). The dense-cored vesicles and are sustentacular cells that receptors can be stimulated by chemical, mechanical envelope the type I cells. and thermal stimuli (Ogura & Lam, 1953; Suzuki & In the adult human larynx, paraganglia are found in Kirchner, 1968; Boushey et al., 1973), causing reflexes the vestibular folds (superior paraganglia) (Watzka, like coughing, expiratory efforts, apnea and 1963) and at the dorsal aspect of the cricoid cartilage swallowing. Furthermore, sensory reflex effects like (inferior paraganglia) (Kleinsasser, 1964). More airway mucous secretion, laryngeal constriction, recently, paraganglia have also been described in the bronchoconstriction, changes in the breathing pattern, main trunk of the superior and recurrent laryngeal hypertension and bradycardia can also be triggered nerves of the rat (endoneurial paraganglia) (McDonald (Widdicombe, 1977). Reflexes can easily be elicited, & Blewett, 1981; Carlsöö et al., 1983) and in the and can sometimes even be fatal due to glottic closure human recurrent laryngeal nerve (Dahlqvist et al., accompanied by apnea (Angell-James & de Burgh 1986). Endoneurial paraganglia were first described in Daly, 1975; de Burgh Daly & Angell-James, 1979). the vagal trunk by Aschoff & Goodhart (1909). The Another serious condition is laryngeal edema, which functions of the laryngeal paraganglia are not known at can be induced by many agents or factors such as present. bacteria, virus, and thermal, chemical or mechanical stimuli. The underlying pathophysiological mechanisms of the condition are not completely Neurotransmitters understood. Acetylcholine (ACh) and noradrenaline (NA) are Articular receptors "classical" neurotransmitters in the nervous system. With the isolation and characterization of different Nerve endings have been found in the neuronal peptides, a new group of transmitter intercartilaginous joints of the larynx, i. e. the crico molecules has emerged. The peptides often coexist arytenoid, the cricothyroid, the thyro-epiglottic and with the "classical" transmitters (Hökfelt, et al., 1980, thyrohyoid joints (Gracheva, 1963). These nerve fibers 1986; Lundberg & Hökfelt , 1983). "Classical"

9 transmitters are usually detected indirectly by Hara et al., 1985). Vasoactive intestinal polypeptide- demonstration of the presence of enzymes participating containing fibers are present around blood vessels and in the synthesis or degradation of the neuro in glands in the nose and lower airways (Uddman et al., transmitters. Neuropeptides are usually visualized by 1978; Lundberg et al., 1980; Luts et al., 1989). immunohistochemical techniques. Recently it was reported that VIP and NPY coexist in nerve fibers in the lower airways (Martling et Catecholamine-synthesizing enzymes al., 1990). Whether this also is the case in the larynx is not known. Tyrosine hydroxylase (TH) and dopamine-ß- hydroxylase (DBH), the first and second enzymes in the catecholamine synthetic pathway, are often used as Substance P and calcitonin gene-related peptide markers for sympathetic nerve fibers. Substance P (SP) (von Euler & Gaddum, 1931) and calcitonin gene-related peptide (CGRP) (Amara et a l., Neuropeptide Y 1982; Rosenfeld et al., 1983) are two peptides found in Neuropeptide Y (NPY) (Tatemoto et al., 1982), has sensory nerve fibers (Hökfelt et al., 1975; Lundberg & been found in the sympathetic nervous system Saria, 1982; Lundberg et al., 1983 b, 1985). These (Lundberg et al., 1982; Ekblad et al., 1984 a) and to be peptides are present in nerve fibers in epithelium, a potent vasoconstrictor (Edvinsson et al., 1983). In around blood vessels and in glands in the airways (Nilsson et al., 1977; Wharton et al.l979; Uddman et the mucosa of the nose and lower airways, NPY- al., 1981; Lundblad et al., 1983 a; Lundberg et al., containing nerve fibers are present around blood vessels and in glands (Lundberg et al., 1983 c; Luts & 1984; Hisa et al., 1985; Cadieux et al., 1986; Martling Sundler, 1989). Neuropeptide Y has been found in et al., 1988). A high degree of coexistence of SP and nonadrenergic nerve fibers in the gut (Ekblad et CGRP is often found in nerve fibers in the airways (Martling et al., 1988; Luts & Sundler, 1989). A al.,1984 b), but it is not known whether this peptide is detailed study of the distribution of nerve fibers present in such fibers in the larynx . showing SP- and CGRP-LI in the different regions of Enkephalin the larynx is lacking. Sensory nerves mediate afferent impulses to the The opioid penta-peptide enkephalin (ENK) central nervous system, but may also play an "efferent" (Hughes et al., 1975) is located in the sympathetic role, mediating a local response to irritant substances. nervous system (Elde et al., 1976; Schultzberg et al., The local tissue response is known as neurogenic 1978 ), including sympathetic acetylcholine-containing inflammation or neurogenic edema. In the central preganglionic neurons (Schultzberg, 1983). Recently it nervous system different sensations like discomfort has also been shown that ENK is present transiently and/or pain are elicited. Substance P participates in the during postnatal development in the rat neurogenic inflammatory reaction, which is parasympathetic submandibular ganglion (Kanagawa- characterized by an increased vascular permeability and Terayama et al., 1989). In the lower airways of the rat, vasodilation (Jansco et al., 1967). Neurogenic ENK has been found in nerve fibers in glands and inflammation can be induced in the airways by around a few blood vessels (Shimosegawa et al., antidromic stimulation of the vagus nerve, by injection 1989). It is , however, not known if ENK-containing of SP, or exposure to capsaicin, the pungent agent of nerve fibers are present in the larynx and if the ENK hot peppers, which causes release of SP and CGRP found in the airways is confined to the sympathetic (Lundberg & Saria, 1982; Lundberg et al., 1983 a, b; and/or parasympathetic nervous system(s). Lundblad et al., 1983 b; Martling et al.,1988; McDonald, 1988 a, b; McDonald et al., 1988;). Acetylcholinesterase Acetylcholinesterase (AChE) degrades acetylcholine Mast cells (ACh) and staining for this enzyme is often used morphologically to identify cholinergic nerve fibers. Mast cells are large, granulated cells found in However, since this enzyme is not only restricted to connective tissue. The knowledge of mast cells is cholinergic nerve fibers, caution must be taken when based mainly on studies of dermal, peritoneal and interpreting AChE-positive nerve structures (Eränkö et intestinal cells. Mast cells constitute a heterogeneous al., 1970; Papka et al., 1981). The distribution of cell population. The existence of two distinct types nerve fibers showing a positive AChE -reaction in the have been demonstrated, the connective tissue mast cell larynx has to the best of our knowledge not been and the mucosal mast cell (Enerbäck, 1966). The two described in detail. cell populations differ structurally, biochemically and functionally, but also in localization and staining Vasoactive intestinal polypeptide properties. The connective tissue mast cell contains Vasoactive intestinal polypeptide (VIP) (Said & heparin, a high histamine content, 5- Mutt, 1970) is present in postganglionic hydroxytryptamine (5-HT), does not respond to parasympathetic nerve fibers (Lundberg et al., 1980; corticosteroids and the anionic sites are not blocked by

10 aldehyde fixation. In contrast to the connective tissue inflammatory reactions as both SP and CGRP are mast cell, the mucosal mast cell contains condroitin known to induce degranulation of mast cells (Johnson sulfate instead of heparin, has a low histamine and 5- & Erdös, 1973; Lembeck & Holzer, 1979; Piotrowski HT content, responds to corticosteroids and the anionic & Foreman, 1986). However, a close spatial sites are blocked by aldehyde fixation (Enerbäck, association between mast cells and nerve fibers is 1986). required for a physiological interaction (Newson et Mast cells participate in hypersensitivity reactions al., 1983). Another type of granulated cell present in by releasing preformed or newly synthesized the laryngeal epithelium is termed globular leucocyte biologically active substances, e. g. chemotactic (Kent, 1966). -This type of cell is thought to be related peptides, proteoglycans, proteases, biogenic amines to or even identical to the mucosal mast cell (Kent, such as histamine, lipids such as leucotrienes, 1966; Tam et al., 1988). The occurrence and prostaglandins, and a platelet activating factor (Raud, distribution of mast cells in the larynx has to our 1989). Mast cells may also participate in neurogenic knowledge not been studied.

11 AIMS OF THE STUDY

Against the background outlined above, 4. the To examine the distribution of mast cells aims of the present study were: and if there is a spatial relationship between the cells and substance P- and calcitonin 1. To determine the composition of motor gene-related peptide-containing nerve fibers and sensory fibers in the superior laryngeal in the larynx. nerve quantitatively and qualitatively. 5. To investigate the distribution of nerve 2. To morphometrically analyse and fibers showing neuropeptide Y-, vasoactive characterize the cells of the endoneurial intestinal polypeptide, - and enkephalin-like paraganglia of the superior laryngeal nerve.immunoreactivity in relation to fibers exhibiting tyrosine hydroxylase/dopamine beta hydroxylase-like immunoreactivity and 3. To study the distribution of nerve fibers a positive acetylcholinesterase reaction in showing substance P- and calcitonin gene- the larynx, and to investigate whether the related peptide-like immunoreactivity in different immunoreactivities/enzyme reac different regions of larynx. tions coexist.

12 MATERIAL AND METHODS sections, 0.5 J i m , were cut using an LKB Ultrotome. The sections were stained with toluidine blue and examined in a Zeiss light microscope. Ultrathin Animals sections, about 70 nm thick, were cut, collected on gold grids and contrasted with uranyl acetate and lead Adult male Sprague-Dawley rats (250-400 g), kept under standard conditions with free access to water and citrate and examined and photographed using a Philips EM 300 or a Jeol 100 CX electron microscope. a standard pellet diet, were used. Immunohistochemistry (III-VI) Denervation procedures (I, III, V, VI) The larynx together with the cranial part of the All experimental procedures were carried out under trachea, the superior cervical ganglion and the nodose anaesthesia with pentobarbital (Mebumal^) ganglion were excised and fixed by immersion intraperitoneally or with methohexital (Brietal^) overnight in a solution of 4 % formaldehyde. The injected into a tail vein. The surgical denervation laryngeal blocks were cut transversely at specific procedures were carried out on either the left or the levels: the epiglottis, the glottic region including the right side of the animals. aryepiglottic folds, the subglottic region and the cranial part of the trachea. The specimens were mounted Surgical denervation separately and frozen in propane chilled in liquid nitrogen. Series of sections, 10 pm, were cut using a Sympathectomy was performed by excising the cryostat. The sections were incubated in Triton X-100, superior cervical ganglion including 0.5 cm of the rinsed in phosphate buffered saline (PBS), incubated in cervical sympathetic trunk. Extracranial vagotomy was normal swine serum, followed by an incubation for 60 performed by an excision of a 1.5 mm segment of the min at 37° C with primary antibodies, raised in rabbits vagus nerve close to the nodose ganglion above the against SP, CGRP, VIP, NPY, ENK, TH, DBH and origin of the superior laryngeal nerve. Intracranial 5-HT. After a wash in PBS, the sections were vagotomy was performed by cutting the rootlets of immersed for 30 min at 37° C in fluorescein- cranial nerves IX, X, and XI. Sympathectomy was also isothiocyanate-(FITC-) conjugated swine anti-rabbit combined with either extracranial vagotomy or IgG, followed by a wash in PBS. A Leitz Ortoplane intracranial vagotomy. Seven or fourteen days after photomicroscope equipped with epifluorescence optics surgery the animals were sacrificed. was used to examine the sections. For comparison, adjacent sections were stained for demonstration of Capsaicin treatment (HI) tissue morphology and activity of acetylcholinesterase (AChE) (Forsgren, 1987). Capsaicin treatment was performed by In sequential double-staining experiments, the subcutaneous injections of capsaicin (10 mg/ml) three antibodies were eluted with acid potassium times daily for two days, giving a total dose of 150 permanganate (Tramu et al., 1978). After photography mg/kg body weight (Lundberg et a l., 1983 a). and after the completeness of the antibody elution had been tested, by application of FITC-conjugated anti Histological methods (I-VI) rabbit IgG, the sections were restained with another antibody, re-examined and re-photographed. Transmission electron microscopy (I, II, IV) In immunohistochemistry, positive and negative Fixation was performed by perfusing a solution of results need to be interpreted carefully. Crossreactivity 1.25% glutaraldehyde and 1% paraformaldehyde in 0.1 with known and unknown substances present in the M cacodylate buffer, pH 7.2, for 1 min, followed by 5 tissue cannot be excluded. The sensitivity of the % glutaraldehyde and 4% paraformaldehyde in 0.1 M staining reaction is highly dependent on the antiserum cacodylate buffer, pH 7.2, through the left cardiac used, the purity of the antigen, the fixation technique ventricle for 4 min. In IV, the animals were perfused and the choice of the immunohistochemical procedure. with 3% glutaraldehyde in 0.1 M cacodylate buffer for Thus, the term "-like immunoreactivity" (-LI) is used 5 min. The excised specimens were then postfixed in to describe the immunohistochemically stained 3% glutaraldehyde in 0.1 M cacodylate buffer for 2 h. structures. In I and II, prior to fixation, in order to detect paraganglia 0.7 ml/100 mg body weight (flow rate 1.2 Demonstration of mast cells (IV) ml/min) of an Evans blue solution (100 mg dye/ ml, To detect connective tissue mast cells and mucosal in saline, pH 7.4) was injected in a tail vein mast cells, different fixation techniques and staining (McDonald & Blewett, 1981). methods were used. The avidine-peroxidase staining All specimens were rinsed in 0.2 M cacodylate method reveals the connective tissue mast cells as buffer overnight, postfixed in 1 % OSO4 in the same avidine reacts with heparin; mucosal mast cells contain buffer, dehydrated in a graded series of ethanol condroitin sulfate instead of heparin (Enerbäck, 1966; solutions and embedded in Epon 812. Semithin Fritz et al., 1986). Since the connective tissue mast

13 cells of the rat contain higher amounts of 5-HT than The number and diameter of the myelinated fibers were the mucosal mast cell (Enerbäck, 1986), mainly the determined using a computer-aided image analysis connective tissue mast cells are stained when system (MOP-Videoplane). The irregular fibers were immunolabelling for 5-HT is performed. Both the approximated to a circle and the corresponding diameter connective tissue mast cells and mucosal mast cells was calculated. The unmyelinated fibers were measured can be detected after fixation with lead acetate and on electron micrographs, magnification x 6 000 and x Camoy’s fixative (Enerbäck, 1966) and staining with 13 000, using a Zeiss TZG-3 particle size analyser. 1% toluidine blue and 1% Alcian blue. Also in glutaraldehyde fixed, toluidine blue stained ( 0.1 %) Paraganglia semithin sections mast cells and intraepithelial granulated cells are observed. Volume densities of the cell nuclei, mitochondria, and dense-cored vesicles were calculated by using a point-counting method (Weibel, 1979) on paper prints, Morphometrie methods (I, II) magnification 24 000. The diameter of the dense-cored vesicles was measured on micrographs, magnification Nerve fibers x 48 000, using a Zeiss TZG-3 particle size analyser. Photomicrographs, magnification x 1 500, of The true vesicle diameter was estimated according to semithin sections showing the internal and external the methods of Bach (1967) and Giger & Riedwyl branches of the superior laryngeal nerve were analysed. (1970).

14 RESULTS AND DISCUSSION

The superior laryngeal nerve (I, II) superior cervical ganglion was performed, the results were similar to those for intracranial vagotomy alone, The superior laryngeal nerve divides into the except for the occurrence of single degenerated internal and external branches. The internal branch myelinated fibers in the internal branch. Removal of divides just before penetrating the thyrohyoid the superior cervical ganglion caused degeneration of membrane and the external branch divides just before occasional myelinated and a small number of entering the cricothyroid muscle. The internal and the unmyelinated fibers in the superior laryngeal nerve (I; external branches had myelinated fibers of similar Fig. 4). number and size (200 - 450 fibers) (I; Figs. 1 a, b, There has been diverging opinions as to whether Table I). The diameters of the myelinated fibers in both both sensory and motor activities are present in the branches showed a unimodal distribution pattern with a laryngeal nerves (Onodi, 1902; DuBois & Foley, fiber range from 0.5 to 12 pm and with peaks at 2 - 4 1936; Rueger, 1972). Lemere (1932) reported that the |im (I; Fig. 2). These results are in line with internal branch was sensory and the external branch physiological studies, in which the majority of the mixed; findings which were later confirmed by fibers are smaller than 4 jj.m, with a few fibers being electrophysiological studies (Andrew, 1956; Suzuki & above 10 jum in diameter; the laryngeal sensory and Kirchner, 1968). However, in spite of these findings, motor fibers have equal conduction velocities (Ogura & Widdicombe and co workers stated as recently as 1988 Lam, 1953; Andrew, 1956). In light and electron that the internal branch is to be regarded as a purely microscopic studies of the superior laryngeal nerve of sensory nerve and the external branch mainly as a the cat and man, the nerve was reported to contain only motor nerve. few unmyelinated fibers (DuBois & Foley, 1936; Our denervation studies showed that the majority of Ogura & Lam, 1953; Miller & Loizzi, 1974). the fibers of the internal and external branches of However, in the rat superior laryngeal nerve, we found the rat superrior laryngeal nerve were sensory, only a numerous unmyelinated fibers evenly distributed all small number of the myelinated fibers were motor and over the nerve and its branches. The differences in continued in the external branch. A minority of the number of unmyelinated fibers may reflect species unmyelinated fibers were motor fibers and these may differences, but it can also partly be explained by the be parasympathetic fibers originating in the dorsal fact that electron microscopy was not used in the two motor nucleus. The superior cervical ganglion was the earliest studies. source of single sympathetic myelinated and After denervation, the number of motor myelinated unmyelinated fibers. Thus, a large number of sensory fibers of the superior laryngeal nerve was determined. nerve fibers are provided to the rat larynx via the After extra- and intracranial vagotomy the myelin internal and external branches. sheets of the affected axons were folded and myelin For every superior laryngeal nerve examined, one to debris and lipid droplets were seen (I; Fig. 3). The three spherical paraganglia (II; Fig. 1) were found. The affected unmyelinated fibers displayed numerous largest paraganglion was always detected at the irregular or rounded processes (I; Fig. 4). Interpretation bifurcation of the nerve into the internal and external of the nature of these processes is difficult since both branch (II; Fig. 2), and smaller paraganglia were regenerating axons and Schwann cell processes contain detected in the internal and external branches. The microtubuli, filaments and mitochondria (Payer, 1979). volume of the paraganglion located at the bifurcation The processes can thus correspond either to was estimated to vary between 0.18 and 3.31 x 10 "6 cytoplasmic Schwann cell processes or to degenerating pm3 (n = 4). or regenerating affected unmyelinated axons. The paraganglia were surrounded by a thin One or two weeks after extracranial vagotomy, in connective tissue capsule separating the paraganglionic five of six nerves a total degeneration of myelinated cells from the nerve fibers. However, myelinated and fibers was observed. After extracranial vagotomy unmyelinated fibers were found within the paraganglia combined with excision of the superior cervical (II; Fig. 1, 2), as has been found for endoneurial ganglion, degeneration of all myelinated and paraganglia elsewhere (McDonald & Blewett, 1981; unmyelinated fibers was found in four of six nerves. Dahlqvist et al., 1984). The superior laryngeal nerve Intracranial vagotomy caused degeneration of 10 - 20 paraganglion was composed of clusters of cells myelinated fibers in each superior laryngeal nerve resembling the type I and type II cells of the carotid studied (I; Fig. 5), and all the degenerated myelinated body. The type I cells were characterized by numerous fibers were traced to continue in the external branch. dense-cored vesicles which were often located close to About a quarter of the unmyelinated fibers were the cell membrane (II; Fig. 3). The type II cells affected in the internal and the external branches. When enclosed the cell clusters with slender cytoplasmic intracranial vagotomy combined with removal of the processes. Sinusoidal thin-walled blood vessels,

15 sometimes fenestrated, intermingled with the clusters al., 1977). These results are somewhat astonishing (II; Fig. 3). The number of the type I cells per cross- since the vocal cord is regarded to be a very sensitive section in the superior laryngeal nerve paraganglion area in the larynx (Jeffery et al., 1977). varied between 40 and 80, and the type II cells roughly Immunohistochemical double-labelling revealed a represented 10 - 25 % of the total cell population. The high degree of co-existence of SP- and CGRP-LI in the mean cell profile area of the type I cells was calculated nerve fibers (III; Fig. 16 a, b). Similar observations to be 54. 3 ± 14. 6 pm2 (II; Table 1), which is about have also been made for the lower respiratory tract of the same as the value for the type I cells of the several species including man (Martling et al., 1988). paraganglia of the recurrent laryngeal nerve and the The SP- and CGRP-LI observed is probably located in carotid body (Laidler & Kay, 1978; Dahlqvist et al., afferent C-fibers, since capsaicin, known to deplete 1984). SP- and CGRP-LI from primary sensory fibers The volume density of the dense-cored vesicles in (Lundberg et al., 1983 a), induced a substantial the superior laryngeal nerve paraganglionic cells was reduction in the number of nerve fibers exhibiting SP- calculated to be 3.5 ± 0.5 %, which is higher than that and CGRP-LI. of the carotid body, but lower than the values reported No nerve fibers showing a positive AChE reaction for the paraganglia of the recurrent laryngeal nerve or TH/DBH-, VIP-, NPY- and ENK-LI were seen in (Hellström, 1975; Laidler & Kay, 1978; Dahlqvist et the epithelium in any part of the larynx. However, in al., 1984, 1987). The distribution pattern was strictly the lamina propria nerve fibers showed VIP- and NPY- unimodal with regard to the profile diameter of the LI and a positive AChE reaction and sometimes ENK- dense-cored vesicles, range 45 - 195 nm, mean 113.8 ± LI but not TH/DBH-LI. 4.9 nm (II; Fig. 4, Table 1). On the basis of the Regional differences in the distribution of the profile diameter of the vesicles, only one type of type I connective tissue mast cells were seen in the lamina cell was found. This is also the case in the paraganglia propria of the larynx (IV; Figs. 1 - 4, Table 1). A large of the recurrent laryngeal nerve (Dahlqvist et al., number of cells was found on the laryngeal side of the 1984). In the carotid body of the rat, two types of type epiglottis, a moderate number in the ventral recess, but I cells have been observed based on the vesicle profile only a few cells were seen on the lingual side of the diameter: Large vesicle cells and small vesicle cells epiglottis and at the subglottic level. No cells at all (Hellström, 1975; McDonald & Mitchell, 1975). were observed in the vocal cords (IV; Figs. 1, 2,4). Nerve endings representing afferent and efferent nerves Besides the connective tissue mast cells, a were closely associated with the type I cells (II; Fig. population of intraepithelial granulated cells was 5). found. These cells had roughly a similar regional distribution as the connective tissue mast cells (IV; The larynx (III-VI) Fig. 7 a). In a previous study of the rat trachea, it has been found that mast cells are present in the lamina Ganglionic cells propria, and that another population of granulated cells is present in the epithelium (Tam et al., 1988). These The local ganglionic cells in the larynx showed a cells were termed globular leucocytes, and have been positive AChE reaction. These cells are presumably suggested to be related to or even identical to mucosal parasympathetic postganglionic cells. A large number mast cells (Kent, 1966; Enerbäck, 1986; Miller et al., of the cells expressed VIP- and NPY- like 1986; Tam et al., 1988). Mucosal mast cells and immunoreactivity (LI) and some of the cells also ENK- globular leucocytes show a similar response to LI. Neither TH-LI nor SP/CGRP-LI were detected in corticosteroids, exhibit granules of variable size and these cells. These observations indicate that VIP and contain low levels of histamine in contrast to NPY and sometimes also ENK are co-expressed in connective tissue mast cells (Miller et al., 1986; Tam parasympathetic ganglionic cells. et al., 1988). The connective tissue mast cells and the mucosal mast cells/globular leucocytes showed a The mucosa (epithelium and lamina propria)similar distribution as the nerve fibers showing SP- Analysis of the rat larynx revealed great regional and CGRP-LI. Parallel sections stained for mast cells differences in the distribution of nerve fibers showing and SP or CGRP revealed that mast cells sometimes SP- and CGRP-LI in the mucosa. Numerous nerve were in the vicinity of peptide-containing nerve fibers fibers displayed SP-and CGRP-LI in the mucosa on (IV; Figs. 6 a, b). Also at an ultrastructural level, the laryngeal side of the epiglottis (III; Fig. 1) and in nerve fibers were found close (within 100 nm) to the ventral recess (III; Fig. 4). A moderate number of granulated cells in the epithelium (IV; Fig. 10). nerve fibers was found at the subglottic level and in Our study suggests that the intraepithelial the trachea, (III: Fig. 3). No immunoreactive nerve granulated cells in the rat larynx may differ with fibers were seen in the vocal cords (III; Fig. 2), which respect to 5-HT-content. In the subglottic epithelium a correspond to studies using silver impregnation large number of granulated cells exhibited 5-HT-LI, techniques, in which the vocal cord of the cat, man and whereas the granulated cells within the supraglottic dog have been found to be devoid of nerve fibers epithelium did not display 5-HT-LI (IV; Figs. 3, 4 b, 5 (Koizumi, 1953; König & von Leden, 1961, Jeffery et a). Further studies are needed to elucidate whether these

16 two cell populations play different functional roles and Joints and cartilages whether they are involved in laryngeal diseases. Nerve fibers displaying SP- and CGRP-LI were apparent outside the laryngeal joints and cartilages (III; Blood vessels and glands Figs. 10 a, b). Nerve fibers located close to laryngeal cartilages and joints have previously been detected Nerve fibers displaying SP- and CGRP-LI, with a using silver impregnation techniques (Koizumi, 1953; high degree of co-existence of the two Gracheva, 1963). immunoreactivities, were frequently observed around Both NPY- and VIP-LI were detected in a blood vessels and in seromucous glands in all the subpopulation of the AChE-reactive nerve fibers in the different regions of the larynx (III; Figs. 8 b, 9). perichondrium (V; Figs. 16 a, b, 17 a, b). Some nerve Similar observations have been made for the mucosa of fibers showing ENK-LI were also seen close to the nose, larynx and lower respiratory tract of the rat, cartilage (VI; Figs. 13 b, 18 a). The ENK-LI was cat, guinea pig and man (Uddman et al., 1981, 1985; colocalized with NPY- or VIP-LI and a positive AChE Lundberg et al.,1984 a; Hisa et al., 1985; Cadieux et reaction (VI; Figs. 13 a, b, 14 a, b, 18 a, b). Thus, not al., 1986; Martling et al., 1988; Luts & Sundler, 1989 only SP/CGRP-innervation is associated with )• cartilages, but also presumably parasympathetic fibers Nerve fibers displaying TH/DBH-LI, presumably showing VIP-, NPY- and ENK-LI are present in the sympathetic nerve fibers, were restricted to the walls of perichondrium. arteries and arterioles (V; Figs. 6 a, 7 a) and a co existence with NPY-LI was found in some of these Effects of denervation peirvascular fibers (V; Fig. 6 b). Enkephalin-LI was very rarely found in fibers located around blood vessels Vagotomy (VI; Figs. 13 b, 15 a, b). Only a few blood vessels Some cells of the nodose and jugular ganglia show have been found to be supplied with nerve fibers SP- and CGRP-LI (Lundberg et al., 1978; Cadieux et exhibiting ENK-LI even in the lower airways of the rat al., 1986). These ganglia supply the larynx with nerve (Shimosegawa et al., 1989). Glands in the nasal fibers via the superior and recurrent laryngeal nerves. mucosa and the peripheral airways are supplied by Extracranial vagotomy induced an almost total nerve fibers displaying TH/DBF1-LI (Partanen et al., disappearance of nerve fibers displaying SP- and 1982; Pack & Richardson, 1988; Uddman et al., 1984; CGRP-LI in the ipsilateral side in the upper parts of Luts & Sundler, 1989), but fibers showing these two the epiglottis and the aryepiglottic folds (III; Figs. 13 immunoreactivities were not found to supply the acini a, b). In the caudal parts of the larynx such nerve fibers and ducts of the glands in the larynx (V; Figs. 14 b, 15 were seen bilaterally, but the number of fibers on the b). intact side was somewhat larger than on the operated Arteries, arterioles and glands were supplied by side (III; Figs. 14, 15). Thus, results indicate that the nerve fibers exhibiting a positive AChE reaction (V; sensory innervation is ipsilateral in the upper parts of Fig. 8 b), NPY- and VIP-LI (V; Figs. 1 a, 8 a, 1 c). In the epiglottis and the aryepiglottic folds and bilateral the glands nerve fibers showing ENK-LI were also below this level. In a tracer study (horseradish observed. In perivascular nerve fibers a co-localization peroxidase) in the cat, it was also found that the upper of VIP- and NPY-LI and a positive AChE reaction was part of the larynx is ipsilaterally innervated while the sometimes detected (V; Figs. 5 a, b, 1 a, c, 8 a, b). In more caudal part is bilaterally innervated - but with an the glands, a co-localization of VIP- and NPY-LI and ipsilateral predominance (Yoshida et al., 1986). In of ENK-LI and VIP- or NPY-LI was detected in some dissection and physiological studies, only ipsilateral nerve fibers showing a positive AChE reaction (V; innervation of the whole larynx has been reported Figs. 11 a, b, 12 a, b, 13 a, b, VI; Figs. 12 a, b, 17 a, (Ogura & Lam, 1953; Suzuki & Kirchner, 1969; b). In the nasal mucosa and the lower respiratory tract, Rueger, 1972). nerve fibers showing VIP- NPY- and ENK-LI are Extracranial vagotomy did not affect the number present in nerve fibers around blood vessels and in and distribution of nerve fibers displaying VIP-, NPY- glands (Uddman et al., 1978, 1984; Lundberg et al., and ENK-LI and a positive AChE reaction. The source 1980, 1981, 1983 c, 1984 b; Luts & Sundler, 1989; of these nerve fibers may be the local presumably Shimosegawa et al., 1989), and recently, in line with parasympathetic ganglionic cells, as some of these our results, a co-localization of VIP- and NPY-LI was cells as discussed above, displayed VIP-, NPY- and observed around arteries and glandular structures in the ENK-LI. lower airways (Martling et al., 1990). Also in airway smooth muscle, NPY-LI has been detected in nerve Removal of the superior cervical ganglion fibers showing VIP-LI and in a subpopulation of fibers exhibiting DBH-LI (Luts & Sundler, 1989). However, Some of the ganglionic cells of the superior a co-localization of ENK-LI and VIP- or NPY- LI in cervical ganglion show NPY-LI (Lundberg et al., nerve fibers has previously not been documented for 1982). When this ganglion was excised unilaterally, the airways. nerve fibers showing TH/DBH-LI and some of those

17 showing NPY-LI around the arteries and arterioles AChE reaction. Thus, NPY appears to be present in disappeared ipsilaterally in the epiglottis and the both the sympathetic and the parasympathetic neurons aryepiglottic folds (V; Figs. 18 a, b, 19 a) but not of the larynx. Furthermore, the number and below that level. This indicates that sympathetic fibers distribution of nerve fibers showing SP- and CGRP-LI in the upper part of rat larynx emanate from the did not change either. ipsilateral superior cervical ganglion. The lower part of Enkephalin-LI is present in the sympathetic the larynx may be provided with sympathetic fibers nervous system in cattle, coexisting with noradrenaline from the superior cervical ganglion and/or lower in the large dense-cored vesicles (Elde et al., 1976; sympathetic ganglia. In a catecholamine histofluo- Schultzberg et al., 1978; Bastiaensen et al., 1988). In rescence study of the innervation of the dog larynx, the present study, however, removal of the superior Hisa (1982) found that the superior cervical ganglion cervical ganglion neither affected the number nor the was the source of the sympathetic fibers of the superior distribution of fibers showing ENK-LI in the larynx. laryngeal nerve, and that the recurrent laryngeal nerve This finding taken together with the findings that contained sympathetic fibers from the superior cervical ENK-LI co-existed with VIP- and NPY -LI in AChE- ganglion and the middle cervical ganglion. positive nerve fibers indicate that the source of fibers Removal of the superior cervical ganglion did not exhibiting ENK-LI in the larynx are the laryngeal affect the distribution of nerve fibers showing a ganglionic cells. colocalization of VIP- and NPY-LI and a positive

18 GENERAL DISCUSSION

The regional differences in the distribution of nerve pig have been found to be equipped with specific SP- fibers showing SP- and CGRP-LI indicate that the binding sites (Hoover & Hancock, 1987), and SP has larynx cannot be regarded as a uniformly innervated also been found to be a potent stimulant of mucous organ when interpreting the significance of the secretion in isolated canine trachea and human mucosal innervation. The large number of fibers in the bronchioles (Coles et al., 1986; Barnes et al., 1986). inlet of the larynx is probably related to the protective Furthermore, VIP has been found to potentiate ACh- function of this organ (Sasaki & Isaacson, 1988). induced glucoconjugate secretion in tracheal These fibers are most likely sensory fibers and may submucosal glands (Lundberg & Hökfelt, 1983; mediate responses to different stimuli. Shimura, 1988). The great regional differences in the number of A central finding in our studies was that VIP- and nerve fibers showing SP- and CGRP-LI and in the NPY-LI frequently co-existed in the AChE-positive number of connective tissue mast cells and mucosal ganglionic cells and nerve fibers. The effects of VIP mast cells/globular leucocytes may be of importance in and NPY have been reported to be antagonistic both in the understanding of the pathogenesis of laryngeal glandular tissues and in blood vessels. Thus, in the edema. Both SP and CGRP can induce histamine pancreas, whereas NPY inhibits secretion (Tatemoto et release from mast cells (Johnson & Erdös, 1973; al., 1982), VIP stimulates secretion (Holst et al., Piotrowski & Foreman, 1986; Pauwels et al., 1989) 1987). As also observed for other parts of the body, and vasodilation and an increased vascular permeability VIP causes vasodilation (Malm et al., 1980; Barnes, (von Euler & Gaddum, 1931; Lundberg & Saria, 1982; 1987) and NPY vasoconstriction (Wahlestedt et al., Lundberg et al., 1983 a; Lundblad et al., 1983 b; Brain 1985; Lundblad et al., 1987) in the nose, the trachea et al., 1985; Martling et al., 1988). A neurogenic and the lungs. All above mentioned effects are observed inflammatory response, mediated by the SP- and when the effects of VIP and NPY were studied CGRP-containing nerve fibers, has been shown to separately. In future, the combined effects of NPY and occur in the lower airways in response to mechanical VIP in the larynx should be studied. and chemical stimuli (Lundberg & Saria, 1982, 1983; Enkephalin-LI has previously not been detected in Lundberg et al., 1983 b; McDonald, et al., 1988). It laryngeal ganglionic cells and nerve fibers. The effects has also been shown that vagal stimulation causes an of ENK in the larynx are to our knowledge not known. increased airway epithelial permeability (McDonald, Very little is known about the effects of ENK in 1987). Furthermore, McDonald (1988 a) has shown peripheral tissue. It has been found that ENK acts on that tracheal epithelium of the rat infected by presynaptic opioid receptors, and inhibits the release of Mycoplasma pulmonis, Sendai virus or corona virus is noradrenaline (Gaddis & Dixon, 1982) and susceptible to neurogenic inflammation. acetylcholine (Mulder et al., 1984; Russel & Simons, The nerve fibers showing SP- and CGRP-LI close 1985). to the laryngeal cartilages and joints probably The physiological significance of the endoneurial participate in relaying sensory information required for paraganglia of the laryngeal nerves is unknown, but it the control of the musclar and cartilaginous movement is possible that they participate (via afferent and /or including movement and tension of the vocal cords in efferent nerve endings) in the regulation of breathing phonation. Movements of laryngeal joints result in via reflex effects on the breathing pattern and via reflex afferent discharge in the external branch of the superior adjustment of the vocal cords. The laryngeal mucosa is laryngeal nerve (Andrew, 1956; Suzuki & Kirchner, sensitive to the gaseous composition (p 0 2, pCOj), and 1968), affecting the laryngeal motor neuron pools in the lower brain stem (Wyke & Kirchner, 1976). From reflex changes of the breathing pattern has been these laryngeal motor neurons, co-ordinated reflexes are observed (Boushey & Richardson, 1973; Wyke & mediated (Wyke, 1983). Kirchner, 1976). It is still not known whether these In the present study, immunolabelling for all the changes are regulated directly by the paraganglia of the peptides examined were observed in the laryngeal laryngeal nerves or whether blood 0 2 and C 0 2 glands. Mucus secretion is an important defensive concentrations can affect the paraganglionic cells. The mechanism of epithelia providing a protective barrier carotid body plays a role in regulation of the breathing against foreign bodies. Several neuropeptides affect this responding to p 0 2, pC 0 2 and pH levels of the blood. secretion in different ways, and the individual effects of A rise in the content of dopamine under hypoxic neuropeptides has been studied. Mucus secretion into conditions has been reported (Dahlqvist et al., 1987) the airway lumen is thought to involve non- indicating a chemoreceptive role for the type I cells. cholinergic vagal axons since there is a secretion when An understanding of the complexity of the muscarinic cholinergic effects of vagal stimulation are laryngeal innervation and of its role in different blocked by atropine (McDonald , 1988, b). Sero- physiological events is fundamental. The present study mucous glands in the lower airways of the guinea has mainly focused on the ultrastructural appearance of

19 the superior laryngeal nerve and on the distribution of conditions is yet poorly understood but is probably different neurotransmitters in the larynx. The very complex. Further experimental work is required importance and role of these transmitters in normal to elucidate the roles of these neurotransmitters in the physiological processes and in pathophysiological larynx.

20 SUMMARY

4. The distribution of connective tissue mast 1. The majority of the fibers in the internal and cells and mucosal mast cells/globular the external branches of the superior leucocytes corresponded to the distribution laryngeal nerve were sensory myelinated of nerve fibers showing SP- and CGRP- and unmyelinated fibers. Single myelinated LI. A spatial relationship between nerve and a few unmyelinated fibers originated in fibers and these cells was observed, the superior cervical ganglion. About 25 % providing a structural basis for a functional of the unmyelinated fibers in each branch interaction between the cells and were parasympathetic. A small population SP/CGRP. of the fibers in the external branch were motor fibers; motor fibers were not observed in the internal branch. 5. A co-localization of VIP- and NPY-LI was observed in AChE-positive laryngeal ganglionic cells and nerve fibers. NPY-LI 2. The endoneurial paraganglia of the superior was co-localized with TH/DBH-LI in some laryngeal nerve exhibited clusters of type I perivascular nerve fibers. Thus, it is likely cells, type II cells and fenestrated blood that NPY is present in both the sympathetic vessels. One type of dense-cored vesicle and parasympathetic nervous systems in cell was found. the larynx.

3. There was a large regional variation in the 6. Enkephalin-LI co-existed with VIP- and distribution of nerve fibers showing SP- NPY-LI in some of the AChE-positive and CGRP-like immunoreactivity (LI). The ganglionic cells and nerve fibers. A co epiglottis and the ventral recess were most existence of TH/DBH- and ENK-LI was densely innervated, while there were no not detected. It is likely that enkephalin in fibers in the vocal cords. the larynx is present in parasympathetic nerve fibers.

21 ACKNOWLEDGEMENTS

The present study was performed at the Sten Hellström, coauthor, for his never failing Departments of Histology and Cell Biology, and enthusiasm, generosity and deep scientific knowledge. Anatomy, University of Umeå. I want to thank everyone, friends and colleagues, for the Kristina Forsgren, Gudrun Furthenbach, Berith encouragement and support I have received during Lundström and Lena Söderberg for excellent technical these years. I am especially indebted to: assistance and collaboration.

Bengt Carlsöö and Åke Dahlqvist, for introducing Per-Olof Fredriksson and Erik Öhlund for me to scientific work and also initiating this work. invaluable help whenever technical problems occurred. Their personal commitment, guidance, patience and great knowledge has been extremely important. Gunnar Engström, Barbara Frankel, Lars-Åke Idahl, Per Lindström, Lars Ohlsson, Pål Roth, Per-Erik Sture Forsgren, coauthor, for introducing me to the Sandström and Staffan Sundström, my friends at the world of neuropeptides and for fruitful discussions and Department who were always willing to help and/or scrutinizing manuscripts and for helping me to avoid explain things to me. pitfalls. Ann-Christine Edlund and Anita Dreyer for Janove Sehlin and Inge-Bert Täljdal, present and secretarial help. former Heads of the Department, for valuable advice, support and continuous interest throughout the study. Last but not least my family and close friends.

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