Involvement of carbon monoxide in the innervation of the canine cervical esophagus and trachea.

We investigated the involvement of carbon monoxide (CO) in the innervation of the canine cervical esophagus and trachea by means of immunohistochemistry using an antiserum against heme oxygenase-2 (HO-2). We detected HO-2-immunoreactive nerve fibers around the blood vessels and submucosal glands of the esophagus and trachea. We found HO-2-immunoreactive neurons in ganglia in the trachea and in the myenteric plexus of the esophagus. These results suggest that CO is involved as a neurotransmitter in the innervation of the canine esophagus and trachea.

Neurotransmitters and neuromodulators involved in laryngeal innervation.

The distribution and role of neurotransmitters and neuromodulators in laryngeal innervation are reviewed, and our recent findings regarding the nitrergic innervation of the larynx are demonstrated for the better understanding of the complexity of the laryngeal innervation system. Noradrenergic innervation of the larynx was studied with fluorescence histochemistry and electron microscopy after application of 5-hydroxydopamine. These studies confirmed the existence of noradrenergic innervation for the submucosal glands and blood vessels, and the origin and course of noradrenergic nerve fibers contained in the laryngeal nerves and their destinations in the larynx. Cholinergic innervation of the larynx has not been clarified in detail. Many kinds of neuropeptides have been demonstrated to be involved in laryngeal innervation. Vasoactive intestinal polypeptide originating from intralaryngeal ganglionic neurons participates in laryngeal vasodilation and reduction of laryngeal seromucous secretion. Neuropeptide Y nerve fibers are few in the larynx, and most originate from the superior cervical ganglion. They are distributed around the large or medium-sized blood vessels, especially arteries. They are also associated with excretory structures. Substance P was the first neuropeptide found to be a sensory neurotransmitter in the laryngeal afferent system. It is also involved in regulation of laryngeal blood flow and secretion. Calcitonin gene-related peptide is associated with the sensory, autonomic, and motor innervation of the larynx. The majority of enkephalin nerve fibers are located close to excretory structures, although no information on the physiological significance of enkephalin is available. In addition to the above neuropeptides, the peptides histidine isoleucine, histidine methionine, and helospectin have been shown to exist in the larynx. The nitrergic innervation of the larynx has been recently studied with NADPH-diaphorase histochemistry and immunohistochemistry using antiserum against nitric oxide synthase. Nitric oxide originates from the neurons in the intralaryngeal ganglia and is believed to modulate blood flow and secretion of the larynx. It controls the laryngeal exocrine secretion in cooperation with intrinsic vasoactive intestinal polypeptide and/or extrinsic calcitonin gene-related peptide. Nitric oxide from the nodose ganglion may modulate nociception of the larynx. The existence of nitrergic neurons located in the intrinsic laryngeal muscles has been demonstrated. Many of them are bipolar or pseudounipolar, so they might be sensory in nature. The effect of injury of the recurrent laryngeal nerve on the induction of nitric oxide synthase in the laryngeal motoneurons is also discussed.

Calcitonin gene-related peptide-like immunoreactive motoneurons innervating the canine intrinsic laryngeal muscles.

The percentage of calcitonin gene-related peptide (CGRP)-immunoreactive motoneurons in the nucleus ambiguus innervating the intrinsic laryngeal muscles of colchicine-treated dogs was examined by using cholera toxin B subunit-gold (CTBG) as a retrograde tracer and by immunohistochemistry. Neurons that were labeled with CTBG from the cricothyroid muscle were located in the ventromedial division of the rostral part of the nucleus ambiguus, and the ratio of CGRP-positive neurons was 93.0%. Neurons labeled with CTBG from the thyroarytenoid muscle were located in the dorsal division of the medial part of the nucleus ambiguus, and the percentage of neurons with CGRP was 71.4%. Neurons labeled with CTBG from the posterior cricoarytenoid muscle were located in the ventral division of the medial part of the nucleus ambiguus, and the percentage of CGRP-positive neurons was 85.5%. These findings suggest that the innervation and/or the neurotrophic mechanism involving CGRP for each intrinsic laryngeal muscle is different.

Coexistence of calcitonin gene-related peptide and NADPH-diaphorase in the canine superior cervical ganglion.

By means of double staining technique of NADPH-diaphorase (NADPH-d) histochemistry and calcitonin gene-related peptide (CGRP) immunohistochemistry, we investigated the coexistence of NADPH-d reactivity and CGRP immunoreactivity in the canine superior cervical ganglion (SCG). Most of NADPH-d reactivity and CGRP immunoreactivity were coexisted in the principal postganglionic neurons. These neurons were distributed throughout the ganglion without specific localization. The present findings suggest the intimate role of CGRP and nitric oxide in postganglionic neurons of the canine SCG.

Relationship of neuropeptides to nitrergic innervation of the canine laryngeal glands.

The participation of vasoactive intestinal polypeptide (VIP) or calcitonin gene-related peptide (CGRP) in the nitrergic innervation of the canine laryngeal glands was investigated using a double-staining technique of NADPH-diaphorase (NADPHd) histochemistry and VIP or CGRP immunohistochemistry. NADPHd-positive nerve fibers with varicosities appeared to terminate in some acinar cells. Double staining revealed that NADPHd reactivity and VIP- or CGRP-like immunoreactivity were colocalized in some nerve fibers distributed around the acini. A cluster of NADPHd-positive cells were occasionally found in the larynx. Many NADPHd-positive cells had VIP-like immunoreactivity and no NADPHd-positive cells were CGRP-like immunoreactive. These findings suggest that nitric oxide participates in the neural control of the laryngeal exocrine secretion in cooperation with intrinsic VIP and/or extrinsic CGRP.

Nitrergic innervation of the rat larynx measured by nitric oxide synthase immunohistochemistry and NADPH-diaphorase histochemistry.

We evaluated the involvement of nitric oxide (NO) in the laryngeal innervation of rats using NADPH-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. The findings obtained by NADPH-d histochemistry were identical with those obtained by nNOS immunohistochemistry, indicating that NADPH-d is nNOS in the laryngeal innervation system. We found NADPH-d-positive nerve fibers in every region of the larynx. In the epithelia of the mucosa, a small number of NADPH-d-positive nerve fibers were detected. The plexus of NADPH-d-positive nerve fibers was commonly found in the lamina propria, and some of these fibers were clearly associated with blood vessels. We also noted NADPH-d-positive nerve fibers in the region of laryngeal glands. Some of these fibers appeared to terminate in the glandular cells. We found NADPH-d-positive nerve fibers with varicosities in the intrinsic laryngeal muscle and free-ending nerve fibers on the muscle fiber. Motor end plate-like structures were positive for NADPH-d histochemistry. The NADPH-d-positive nerve fibers appeared to terminate at motor end plate-like structures in two of nine rats examined. A cluster of NADPH-d-positive neurons were occasionally present in the lamina propria of the laryngeal mucosa, in the connective tissue between the thyroid cartilage and intrinsic laryngeal muscle, and in the connective tissue near the cricoarytenoid joint. The present findings suggest that NO participates in the autonomic, sensory, and motor innervation of the larynx.

Tyrosine hydroxylase-immunoreactive cells in the nodose ganglion for the canine larynx.

Several substances have been reported as candidates for the neurotransmitter in the laryngeal afferent system. In the present study we demonstrated that catecholamine is also a candidate neurotransmitter in the canine laryngeal afferent system using tyrosine hydroxylase (TH) immunochemistry in combination with retrograde labelling with cholera toxin B in subunit-conjugated gold (CTBG). A few cells in the nodose ganglion labelled by application of CTBG to the internal branch of the superior laryngeal nerve were also TH-immunoreactive. These cells were also labelled following application of CTBG to the nucleus of the solitary tract. These results indicate that some of the TH-IR cells in the nodose ganglion could be primary afferent neurones for the canine larynx.

Nitrergic neurons in the canine intrinsic laryngeal muscle.

Nitrergic ganglionic cells located in the canine intrinsic laryngeal muscle were studied by NADPH-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. Cells intensely stained by NADPH-d histochemistry were found between the striated muscle fibers of the intrinsic laryngeal muscle. Most of these cells were bipolar or pseudounipolar in form. Some NADPH-d negative cells were observed to be enveloped in a mesh by varicose NADPH-d positive nerve fibers. The findings obtained by nNOS immunohistochemistry corresponded well with those obtained by NADPH-d histochemistry, indicating that NADPH-d activity in the ganglion in the intrinsic laryngeal muscle is nNOS. The present findings clearly indicate that some of the ganglion cells located in the canine intrinsic laryngeal muscle are nitrergic, and that the ganglionic cells synapse together with the participation of nitric oxide in integrating ganglionic cells.

Neurotransmitters for the canine inferior pharyngeal constrictor muscle.

The inferior pharyngeal constrictor muscle plays an important role at the pharyngeal phase of deglutition and is anatomically composed of the thyropharyngeal muscle and cricopharyngeal muscle. In this study we investigated the distribution pattern of neuropeptidergic and catecholaminergic nerve fibers in the thyropharyngeal muscle and cricopharyngeal muscle of seven puppies by immunohistochemistry. Some of the calcitonin gene-related peptide-, substance P-, vasoactive intestinal polypeptide-, and tyrosine hydroxylase-immunoreactive nerve fibers were found to lie parallel to the muscle fibers in both the thyropharyngeal muscle and cricopharyngeal muscle. Nerve fibers with immunoreactivity to all substances examined were found to be associated with blood vessels in both the thyropharyngeal muscle and cricopharyngeal muscle, and the number of calcitonin gene-related peptide, neuropeptide Y, and tyrosine hydroxylase nerve fibers was higher than the number of substance P, vasoactive intestinal polypeptide, and galanin nerve fibers. Motor end plate-like structures with calcitonin gene-related peptide immunoreactivity were found in both the thyropharyngeal muscle and cricopharyngeal muscle. These structures in the cricopharyngeal muscle were clearly less than those in the thyropharyngeal muscle. Some clusters of neurons were detected only in the cricopharyngeal muscle of all dogs examined. Substance P-, vasoactive intestinal polypeptide-, galanin-, and neuropeptide Y-immunoreactive neurons were found in this ganglion, and the vasoactive intestinal polypeptide-immunoreactive neurons were the most abundant. Abundant calcitonin gene-related peptide- and vasoactive intestinal polypeptide-immunoreactive nerve fibers, and some substance P- and galanin-immunoreactive nerve fibers were distributed in the ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)

NADPH-diaphorase and nitric oxide synthase in the canine superior cervical ganglion.

By means of NADPH-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, we demonstrate that considerable numbers of NADPH-d-positive neurons are distributed throughout the canine superior cervical ganglion (SCG). These neurons also show NOS immunoreactivity. This finding indicates that NADPH-d histochemistry, a simple and reliable technique, can be used as a reliable marker of NOS activity in the sympathetic innervation of canine head and neck. The present findings suggest that the participation of nitric oxide in the SCG differs greatly between species.

Sympathetic preganglionic neurons contain nitric oxide synthase and project to the superior cervical ganglion: combined application of retrograde neuronal tracer and NADPH-diaphorase histochemistry.

Nitric Oxide (NO), which was initially identified as an endothelium-derived relaxing factor, has recently been demonstrated to be a neuronal messenger in central and peripheral nervous systems. In the present study, we examined the possibility of NO producing neurons in teh intermediolateral (IML) cell collum of the thoracic spinal cord (Th) project to the superior cervical ganglion (SCG). First, we observed the NADPH-diaphorase-positive/nitric oxide synthase (NOS)-immunoreactive neurons of the IML and the dorsal part of the central canal at the level of Th1-Th3, and numerous fiber-stainings in the superior cervical ganglion. Second, after injecting WGA-HRP (wheat germ agglutinin-horse radish peroxidase complex), a retrograde neuronal tracer, into the SCG, and developing WGA-immunohistochemistry and the NADPH-diaphorase histochemistry in the same sections, we detected double-labeled neurons in the IML. These findings provide evidence that sympathetic preganglionic NO producing neurons directly innervate to the SCG.

Neuropeptide participation in canine laryngeal sensory innervation. Immunohistochemistry and retrograde labeling.

We investigated the quantitative participation of calcitonin gene-related peptide (CGRP), substance P (SP), and leu-enkephalin (ENK) in canine laryngeal sensory innervation by immunohistochemistry in combination with retrograde labeling using the recently introduced retrograde tracer cholera toxin subunit B-conjugated gold (CTBG). In the nodose ganglion, neurons labeled from the internal branch of the superior laryngeal nerve with CTBG were investigated immunohistochemically by means of antisera against CGRP, SP, and ENK. The percentages of neurons immunoreactive to each neuropeptide were as follows: CGRP 81.5%, SP 24.5%, and ENK 7.0%. These results suggest that CGRP is the main sensory neurotransmitter in canine laryngeal sensory innervation.

Calcitonin gene-related peptide-like immunoreactive motoneurons innervating the canine inferior pharyngeal constrictor muscle.

The percentage of the calcitonin gene-related peptide-immunoreactive motoneurons (Mn) in the nucleus ambiguus (NA) innervating the canine inferior pharyngeal constrictor muscle was examined using the cholera toxin B subunit-gold (CTBG) as a retrograde tracer and by immunohistochemistry. Labeled neurons with CTBG (CTBG MNs) from the thyropharyngeal muscle (TP) were located in the dorsomedial division of the rostral part of the NA and the average percentage of neurons with CGRP immunoreactivity was 70.3%. CTBG MNs from the cricopharyngeal muscle (CP) were located in the dorsal division of the rostral part of the NA and the average percentage of neurons with CGRP immunoreactivity was 21.2%. This low percentage of CGRP immunoreactivity is noteworthy and supports the idea that the CP is a particular striated muscle in function and innervation manner.

Distribution of the afferent neurons from the canine recurrent laryngeal nerve.

Localization of the afferent neurons from the canine recurrent laryngeal nerve in the nodose ganglia (NG) and jugular ganglia was studied using the horseradish peroxidase technique. Labeled cells were detected in the ipsilateral NG and presented no distinct pattern of distribution, but rather were scattered throughout the ganglion. No labeled cells were found in the contralateral NG or bilateral jugular ganglia. The size of labeled cells varied from 15 microns to 60 microns. Two to six labeled cells larger than 45 microns were detected in each dog. Most of the labeled cells (62%) had a diameter between 30 microns and 45 microns. The percentage of labeled cells with a diameter between 15 microns and 30 microns was 30% and between 45 microns and 60 microns was 8%. This difference in size of labeled cells in the NG may be related to functional differences among the receptors mediated.

[Adenoid cystic carcinoma of the head and neck: a clinical review of 29 cases].

Pathological data and the outcomes of 29 patients with adenoid cystic carcinoma of the head and neck treated between 1975 and 1990 were reviewed. Patients consisted of 21 females and 8 males, with a median age of 59 years. The overall 5-year and 10-year survival rates determined by Kaplan-Meier analysis were 84.2% and 52.9%, respectively. Patients in whom pathological data indicated a frequent solid pattern of carcinoma had high incidences of metastasis and poor prognoses. The effectiveness of local control was found to have no relation to the pathological data of the tumor, but rather depended upon whether anatomical limitations restricted complete resection of the tumor. All 7 patients who had cervical lymph node metastasis underwent neck dissection, which was useful in controlling metastatic spread. Distant metastases were present in 6 patients, and the lung was the most frequent site of metastasis.

Distribution of calcitonin gene-related peptide nerve fibers in the canine larynx.

Immunohistochemistry was used to investigate the distribution pattern of calcitonin gene-related peptide (CGRP) nerve fibers in the laryngeal mucosa, glands and intrinsic muscles of the dog. CGRP immunoreactive nerve fibers were found more frequently than substance P immunoreactive nerve fibers in every region of the larynx. In the epithelia, CGRP nerve fibers were mainly found in the epiglottis, arytenoid region and subglottis. Many taste buds were observed in the arytenoid region and were densely innervated by the CGRP nerve fibers. In the lamina propria, the plexus of CGRP nerve fibers was present, with some of these fibers associated with blood vessels. Laryngeal glands were also innervated by a few CGRP nerve fibers. In the intrinsic laryngeal muscles, abundant immunoreactivity was observed and many motor end-plate-like structures were found with CGRP immunoreactivity. These findings strongly suggest that CGRP plays an important role in all of the sensory, motor and autonomic nervous systems of the larynx.

Pedunculated intraluminal osteochrondromatous hamartoma of the esophagus.

Pedunculated intraluminal hamartoma of the cervical esophagus of an adult female was treated by surgical resection. The patient presented with bloody sputum. A barium swallow revealed a pedunculated intraluminal tumor just below the conus of the thyroid cartilage. The tumor had a stalk of 6 cm in length and was attached to the cricopharyngeal muscle. Histopathologic examination showed a predominance of osseous and cartilaginous tissue. The tumor also contained fibrous tissue, adipose tissue and glandular structure. The embryology of such a cartilaginous tumor at the proximal esophagus was interesting.