Hideki Bando

Molecular Clocks in Mouse Skin

Clock genes in the skin exhibit day-night changes in expression; however, whether these changes are brought by external light or intrinsic mechanisms is unclear. In this study, we demonstrated that expression of the clock and clock-controlled genes showed robust rhythms in mouse skin under constant dark conditions, whereas these rhythms were completely lost in Cry1/Cry2 knockout mice lacking a molecular clock. At the cellular level, the main oscillatory protein in the mammalian molecular clock, PER2, was expressed in the nuclei of keratinocytes in the epidermis and hair follicles, with expression peaking at CT16 (subjective dusk), 4-8 hours after expression of its mRNA. These expression patterns in the skin stopped after the ablation of the central clock in the suprachiasmatic nucleus (SCN), which was not recovered even in animals housed in 12 hour-light/12 hour-dark conditions. These findings demonstrate that the intrinsic oscillating molecular clock exists in the epidermis, and that signaling from the SCN is essential for the maintenance of the epidermal clock, and cannot be compensated by external light.

Vagal Regulation of Respiratory Clocks in Mice

The present study addresses the role of the circadian system in day–night changes of respiratory functions in the mouse. In all airway tissues investigated (i.e., larynx, trachea, bronchus, and lung), we observed clear rhythmic expression of the Per1, Per2, Bmal1, and Clock core oscillator genes (the latter two genes oscillating in antiphase with the Per genes), as well as the clock-regulated Dbp gene. Oscillations were abolished in arrhythmic Cry1−/−Cry2−/− knock-out mice and after lesioning of the master clock in the suprachiasmatic nucleus (SCN) in wild-type animals. These findings indicate that respiratory system cells contain a functional peripheral oscillator that is controlled by the SCN. Furthermore, we found that the muscarinic acetylcholine receptor genes Chm2, Chm3, and Chm4 are expressed in a circadian manner, and that mucin secretion (rather than synthesis) by the airway submucosal glands is under circadian control. Signals from the SCN are mainly transmitted by the vagal nerve because unilateral vagotomy completely abolished rhythms in mucin and PER2 protein levels in the (operated) ipsilateral side of the submucosal glands, but not in the (intact) contralateral side. Thus, peripheral clock mediated circadian expression of muscarinic acetylcholine receptor proteins, and parasympathetic signaling between SCN and respiratory tissues are essential gears in conferring circadian “time” information to airway glands.

Vocal Fold Paralysis as a Sign of Chest Diseases: A 15-year Retrospective Study

BackgroundVocal fold paralysis (VFP) is sometimes the only sign of chest diseases. However, some patients with VFP due to chest diseases are not diagnosed correctly at the first examination, which may leave the patients untreated for a long time. Depending on the situation, chest x-ray is not enough for detecting the primary lesion. The objective of this study was to discuss the diagnostic procedure for VFP based on the retrospective analysis of the cases.MethodsA total of 42 patients (29 males and 13 females) with VFP due to chest disease examined at the Department of Otolaryngology of Kyoto Prefectural University of Medicine between 1988 and 2002 were reviewed retrospectively.ResultsOf the primary chest diseases, lung cancer (15 cases) was the most common, followed by thoracic aortic aneurysm (TAA) (9 cases), metastatic tumor from other regions (6 cases), pulmonary and mediastinal tuberculosis (TB) (5 cases), and esophageal cancer (4 cases). While the primary lesions were easily detected with chest x-ray in most of the cases, some lesions in the aortopulmonary window were difficult to detect. Contrast-enhanced computed tomography (CT) was useful to detect any mass in this region.ConclusionsIn the diagnosis of VFP due to chest diseases, chest x-ray was useful but not always enough for detecting the primary lesion. Necessity of further examinations including contrast-enhanced chest CT must be kept in mind for the cases with negative chest radiographs.

K+-Cl-Cotransporter 1 (KCC1) Negatively Regulates NGF-induced Neurite Outgrowth in PC12 Cells

Potassium chloride cotransporters (KCCs) mediate electroneutrally-coupled transport of K+ and Cl-, and play crucial roles in various cell functions including regulation of cell volume and homeostasis of cellular Cl-content. Four isoforms of KCCs (KCC1, 2, 3, and 4) have been identified. KCC1 is ubiquitously expressed, whereas KCC2 is mainly expressed in neuronal cells of central nervous system. KCC3 is highly expressed in heart, skeletal muscle, kidney, lung and placenta. KCC4 is mainly expressed in epithelial cells. In this study, we investigated roles of KCCs in NGF-induced neurite outgrowth of rat pheochromocytoma PC12 cells. The most abundantly expressed isoform in PC12 cells was KCC1. Inhibition of KCCs using [(dihydronindenyl)oxy] alkanoic acid (DIOA), an inhibitor of KCCs, enhanced the NGF-induced neurite outgrowth of PC12 cells in a dose-dependent manner. Treatment of PC12 cells with NGF significantly decreased mRNA expression of KCC1, whereas other isoforms, KCC2-4, showed no changes in their mRNA expression in response to NGF treatment. Knockdown of KCC1 using small interfering RNA (siRNA) enhanced the NGF-induced neurite outgrowth. These results suggest that KCC1 negatively regulates the NGF-induced neurite outgrowth of PC12 cells.

Differential Responses to Steroid Hormones in Fibroblasts From the Vocal Fold, Trachea, and Esophagus

There is accumulating evidence that fibroblasts are target cells for steroids such as sex hormones and corticoids. The characteristics of fibroblasts vary among tissues and organs. Our aim in this study is to examine differences in responses to steroid hormones among fibroblasts from different cervicothoracic regions. We compared the actions of steroid hormones on cultured fibroblasts from the vocal folds, which are considered to be the primary target of steroid hormones, and the trachea and esophagus in adult male rats. Expression of steroid hormone receptors (androgen receptor, estrogen receptor α, and glucocorticoid receptor) was identified by immunofluorescence histochemistry. Androgen receptor was much more frequently expressed in fibroblasts from the vocal fold than in those from the trachea and esophagus. Cell proliferation analysis showed that administration of testosterone, estradiol, or corticosterone suppressed growth of all 3 types of fibroblasts. However, mRNA expression for extracellular matrix-associated genes, including procollagen I and III and elastin, and hyaluronic acid synthase I was elevated only by addition of testosterone to fibroblasts from the vocal fold. These results indicate that each steroid hormone exerts region-specific effects on cervicothoracic fibroblasts with different properties through binding to specific receptors.

Endolaryngeal extension of thyroglossal duct cyst

Thyroglossal duct cysts are the most common congenital neck masses that develop during childhood, The masses develop from remnants of thyroglossal ducts, and typically appear as midline neck masses. Endolaryngeal extension of thyroglossal duct cysts has been reported mostly as midline neck swelling. We observed a case of extension of the thyroglossal duct cyst to the supraglottic area without neck swelling. A 50-year-old man presented with a 1-month history of foreign-body sensation in the throat. Fiberscopic and radiologic findings were similar to those associated with a saccular cyst, but its proximity to the hyoid bone raised the possibility of thyroglossal duct cyst. Operation was performed via an external incision to completely remove the cyst. Postoperative fiberscopy revealed that the aryepiglottic fold swelling had disappeared. Diagnosis of thyroglossal duct cyst was confirmed on the basis of pathological findings. In cases in which it is difficult to remove the cyst from the hyoid membrane, the hyoid bone midline portion should be dissected. Thyroglossal duct cysts should be considered in cases with a submucosal tumor in the supraglottic region, and radiological examinations should be performed.

Circadian gene expression in the murine larynx

OBJECTIVE Symptoms of airway diseases are often associated with specific times of the day. For example, midnight worsening of cough is a frequent complaint of patients with laryngitis and bronchitis. We speculate that these symptoms are under the control of the circadian clock, and the clock genes in the airway epithelium play some important roles. In the present study, we tried to prove the time specific expressions of clock oscillating genes in the murine larynx. MATERIALS AND METHODS Adult wild-type C57/Bl6 mice and mCry1(-/-)mCry2(-/-) mutant mice were used for this study. We employed immunohistochemistry and/or Northern blotting for examining the circadian expression of mPer1, mPer2, C/EBPbeta, HNF3beta, and MUC5b. RESULTS The expression of mPer2 mRNA showed a strong day-night expression difference, which was abolished after the lesion of the suprachiasmatic nucleus, and in mCry1(-/-)mCry2(-/-) mutant mice. mPER1 and mPER2 proteins both showed very similar expression profiles in the epithelium and submucosal glands with a peak in the evening and a trough in the early morning. Other nuclear proteins such as C/EBPbeta and HNF3beta did not show the rhythms. MUC5b protein showed circadian oscillation in the laryngeal submucosal gland. CONCLUSION In this study, we confirmed the existence of a local laryngeal clock which is controlled by the central clock in the suprachiasmatic nucleus. MUC5b protein in the submucosal mucous gland also showed circadian rhythm. We consider that these rhythmic expressions may cause the time specific symptoms among laryngeal diseases.

Expression of Sex Steroid Hormone Receptors in Vagal Motor Neurons Innervating the Trachea and Esophagus in Mouse

The medullary vagal motor nuclei, the nucleus ambiguus (NA) and dorsal motor nucleus of the vagus (DMV), innervate the respiratory and gastrointestinal tracts. We conducted immunohistochemical analysis of expression of the androgen receptor (AR) and estrogen receptor α (ERα), in relation to innervation of the trachea and esophagus via vagal motor nuclei in mice. AR and ERα were expressed in the rostral NA and in part of the DMV. Tracing experiments using cholera toxin B subunit demonstrated that neurons of vagal motor nuclei that innervate the trachea and esophagus express AR and ERα. There was no difference in expression of sex steroid hormone receptors between trachea- and esophagus-innervating neurons. These results suggest that sex steroid hormones may act on vagal motor nuclei via their receptors, thereby regulating functions of the trachea and esophagus.

Localization and regulation of aquaporins in the murine larynx

Abstract Conclusion: Expression of aquaporins (AQPs) in the vocal folds and the parasympathetic regulation of AQPs in the laryngeal glands may be important for controlling laryngeal secretion and vocal fold hydration. These findings are thought to be an initial step towards understanding potential mechanisms of water flow through the vocal fold surface. Objectives: Lubrication of the vocal folds is important for phonation and laryngeal defense. However, the system of water transport in the laryngeal epithelium has not yet been clarified. We investigated the expression of AQP water channnels (AQP1–9) in the murine larynx and examined the neural regulating mechanisms of these subtypes. Methods: The distribution of AQPs (AQP1–9) in the murine larynx was examined by immunohistochemistry. Next, the neural mechanism regulating AQPs in the larynx was investigated using unilaterally vagotomized mice. Results: AQP1, 4, and 5 were expressed in the laryngeal epithelium including the vocal folds. Vocal fold AQP expression was minimal at the vocal process. AQP2, 3, 5, 6, 7, and 8 were localized to the submucosal glands. Expression of AQPs in the vagotomized region was significantly decreased compared with the contralateral intact side. The expression in the laryngeal glands was also decreased in the aged mice.

Superior Cervical Ganglion

The sympathetic innervation provides regulations of the larynx including vasoconstriction of blood vessels and mucous secretion of laryngeal glands via cervical ganglions. As we analyzed the origin of the sympathetic fibers in the canine laryngeal nerves in the cervical sympathetic ganglions using cholera toxin B, most of the sympathetic nerve fibers were projected to the larynx via the superior laryngeal nerve. Recent studies have revealed that sympathetic ganglion cells express not only the classical neurotransmitters such as noradrenaline and acetylcholine but various neuropeptides which play roles as neurotransmitter or neuromodulator. Although several studies have identified CGRP- and nitric oxide (NO)-positive cells in sympathetic ganglions, the roles in sympathetic nervous system have not been illuminated. We conducted immunohistochemical study for CGRP in canine superior cervical ganglion (SCG), and CGRP-positive cells with a number of dendrites were distributed diffusely. In immunohistochemistry for CGRP and histochemistry for NADPH diaphorase (NADPHd), nitric oxide synthase was applied for a single specimen of canine SCG. The study revealed that there are a number of NADPHd-positive cells in SCG, and the major part of them expresses both NADPHd and CGRP. These results indicate that laryngeal NANC fibers with both NO and CGRP immunoreactivity are originated from SCG.