Isolation of novel biflavonoids from Cardiocrinum giganteum seeds and characterization of their antitussive activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Seeds of Cardiocrinum giganteum var. yunnanense (Leichtlin ex Elwes) Stearn (Liliaceae), also known as Doulingzi, have been used as a folk substitute for conventional antitussive herb "Madouling" (Aristolochia species) to treat chronic bronchitis and pertussis. The active antitussive phytochemicals in C. giganteum seeds are not known. AIM OF THE STUDY: The present work aims at isolating the active phytochemicals in C. giganteum seeds and confirming their antitussive effects. MATERIALS AND METHODS: Active chemicals were isolated from C. giganteum seeds ethanol extract and identified their structures. Antitussive effects were evaluated with the cough frequency of guinea pigs exposed to citric acid. Electrical stimulation of the superior laryngeal nerve in guinea pigs was performed to differentiate the acting site of potential antitussives. RESULTS: Two racemic biflavonoids (CGY-1 and CGY-2) were isolated from C. giganteum seeds. CGY-1 was identified as (S)-2″R,3″R- and (R)-2″S,3″S-dihydro-3″-hydroxyamentoflavone-7- methyl ether, which are new compounds and firstly isolated from C. giganteum seeds. Racemic CGY-2 was identified as (S)-2″R,3″R- and (R)-2″S,3″S-dihydro-3″-hydroxyamentoflavone. Both CGY-1 and CGY-2 could significantly inhibit coughs induced by inhalation of citric acid. Further, they acted on the peripheral reflex pathway to inhibit cough after electrical stimulation of the superior laryngeal nerve in guinea pigs. CONCLUSIONS: These chemicals isolated from C. giganteum seeds showed good antitussive effects. The data provide scientific evidence to support the traditional use of C. giganteum seeds as an antitussive herbal medicine.

Of mice, birds, and men: the mouse ultrasonic song system has some features similar to humans and song-learning birds.

Humans and song-learning birds communicate acoustically using learned vocalizations. The characteristic features of this social communication behavior include vocal control by forebrain motor areas, a direct cortical projection to brainstem vocal motor neurons, and dependence on auditory feedback to develop and maintain learned vocalizations. These features have so far not been found in closely related primate and avian species that do not learn vocalizations. Male mice produce courtship ultrasonic vocalizations with acoustic features similar to songs of song-learning birds. However, it is assumed that mice lack a forebrain system for vocal modification and that their ultrasonic vocalizations are innate. Here we investigated the mouse song system and discovered that it includes a motor cortex region active during singing, that projects directly to brainstem vocal motor neurons and is necessary for keeping song more stereotyped and on pitch. We also discovered that male mice depend on auditory feedback to maintain some ultrasonic song features, and that sub-strains with differences in their songs can match each other's pitch when cross-housed under competitive social conditions. We conclude that male mice have some limited vocal modification abilities with at least some neuroanatomical features thought to be unique to humans and song-learning birds. To explain our findings, we propose a continuum hypothesis of vocal learning.

Modifications of Miccoli minimally invasive thyroidectomy for the low-volume surgeon.

OBJECTIVE: The objective of the study was to describe our experience with modifications of the Miccoli minimally invasive thyroidectomy. DESIGN: Planned analysis of a prospectively maintained database was undertaken after Institutional Review Board approval. METHODS: Demographic and surgical data were obtained and analyzed with attention to age, sex, pathology, incision lengths, and complications. RESULTS: From a single-surgeon series of 785 consecutive thyroidectomies, 178 patients were identified who underwent an endoscopic minimally invasive thyroidectomy. A series of modifications of the classic Miccoli technique evolved over a period of 4 years and include presurgical factors (patient marking in holding area, intubation with laryngeal EMG tube using videolaryngoscope, rotation of operating table away from anesthesia), intraoperative principles (use of operative loupes, slave monitor, laryngeal nerve monitoring, and novel instrumentation; identification of the medial cleft and ligation of superior pedicle bundle using ultrasonic technology; avoidance of clips), and postoperative techniques (deep extubation, laryngeal endoscopy, outpatient management, and oral calcium supplementation). CONCLUSIONS: A minimally invasive endoscopic thyroidectomy is possible even in a practice with moderate surgical volumes by using several techniques that facilitate the performance of this procedure. A high success rate and low complication rate can be achieved, resulting in improved patient satisfaction.

Impaired laryngeal mobility and entrapment neuropathies.

Entrapment neuropathies are rarely mentioned in phoniatrics because a definite diagnosis is very difficult to make. However, several clinical examples described here strongly support this etiology, which should now be considered in the diagnosis of both partial and complete laryngeal immobility as well as in minor neuromuscular disorders.

Multiple forebrain systems converge on motor neurons innervating the thyroarytenoid muscle.

The present study investigated the central connections of motor neurons innervating the thyroarytenoid laryngeal muscle that is active in swallowing, respiration and vocalization. In both intact and sympathectomized rats, the pseudorabies virus (PRV) was inoculated into the muscle. After initial infection of laryngomotor neurons in the ipsilateral loose division of the nucleus ambiguus (NA) by 3 days post-inoculation, PRV spread to the ipsilateral compact portion of the NA, the central and intermediate divisions of the nucleus tractus solitarii, the Botzinger complex, and the parvicellular reticular formation by 4 days. Infection was subsequently expanded to include the ipsilateral granular and dysgranular parietal insular cortex, the ipsilateral medial division of the central nucleus of the amygdala, the lateral, paraventricular, ventrolateral and medial preoptic nuclei of the hypothalamus (generally bilaterally), the lateral periaqueductal gray, the A7 and oral and caudal pontine nuclei. At the latest time points sampled post-inoculation (5 days), infected neurons were identified in the ipsilateral agranular insular cortex, the caudal parietal insular cortex, the anterior cingulate cortex, and the contralateral motor cortex. In the amygdala, infection had spread to the lateral central nucleus and the parvicellular portion of the basolateral nucleus. Hypothalamic infection was largely characterized by an increase in the number of infected cells in earlier infected regions though the posterior, dorsomedial, tuberomammillary and mammillary nuclei contained infected cells. Comparison with previous connectional data suggests PRV followed three interconnected systems originating in the forebrain; a bilateral system including the ventral anterior cingulate cortex, periaqueductal gray and ventral respiratory group; an ipsilateral system involving the parietal insular cortex, central nucleus of the amygdala and parvicellular reticular formation, and a minor contralateral system originating in motor cortex. Hypothalamic innervation involved several functionally specific nuclei. Overall, the data imply complex CNS control over the multi-functional thyroarytenoid muscle.

Electromyographic control of a hands-free electrolarynx using neck strap muscles.

UNLABELLED: Three individuals with total laryngectomy were studied for their ability to control a hands-free electrolarynx (EL) using neck surface electromyography (EMG) for on/off and pitch modulation. The laryngectomy surgery of participants was modified to preserve neck strap musculature for EMG-based EL control (EMG-EL), with muscles on one side maintaining natural innervation and those on the other side receiving a transferred recurrent laryngeal nerve (RLN). EMG from each side of the neck controlled the EMG-EL across a day of unstructured practice followed by a day of formal training, including EMG biofeedback. Using either control source, participants spoke intelligibly and fluently with the EMG-EL before formal training. This good initial performance did not consistently improve across testing for either control source in terms of voice timing, speech intelligibility, fluency, and intonation of interrogative versus declarative sentences. Neck strap muscles have activation patterns capable of simple alaryngeal voice control without requiring RLN transfer. LEARNING OUTCOMES: The reader will better understand (1) functionality of the hands-free electrolarynx (2) modification of laryngectomy surgery to preserve neck strap musculature and (3) performance of hands-free electrolarynx with different control sources.

BDNF-TrkB signaling interacts with the GABAergic system to inhibit rhythmic swallowing in the rat.

Brain-derived neurotrophic factor (BDNF) acts as an anorexigenic factor in the dorsal vagal complex (DVC) of the adult rat brain stem. The DVC contains the premotoneurons controlling swallowing, a motor component of feeding behavior. Although rats with transected midbrain do not seek out food, they are able to swallow and to ingest food. Because BDNF and tropomyosin-related kinase B (TrkB) receptors are expressed in the DVC, this study hypothesized that BDNF could modify the activity of premotoneurons involved in swallowing. Repetitive electrical stimulation of the superior laryngeal nerve (SLN) induces rhythmic swallowing that can be recorded with electromyographic electrodes inserted in sublingual muscles. We show that a microinjection of BDNF in the swallowing network induced a rapid, transient, and dose-dependant inhibition of rhythmic swallowing. This BDNF effect appeared to be mediated via TrkB activation, since it no longer occurred when TrkB receptors were antagonized by K-252a. Interestingly, swallowing was inhibited when subthreshold doses of BDNF and GABA were coinjected, suggesting a synergistic interaction between these two signaling substances. Moreover, BDNF no longer had an inhibitory effect on swallowing when coinjected with bicuculline, a GABA(A) receptor antagonist. This blockade of BDNF inhibitory effect on swallowing was reversible, since it reappeared when BDNF was injected 15 min after bicuculline. Finally, we show that stimulation of SLN induced a decrease in BDNF protein within the DVC. Together, our results strongly suggest that BDNF inhibits swallowing via modulation of the GABAergic signaling within the central pattern generator of swallowing.

The descending motorcortical pathway to the laryngeal motoneurons in the squirrel monkey.

The motor cortex of primates contains an area ("larynx area") which, when stimulated unilaterally, produces bilateral vocal fold adduction. In order to identify the pathway along which the cortical larynx area exerts its control on the laryngeal motoneurons, we have blocked excitatory neurotransmission in each of the main projection fields of the cortical larynx area and tested for the elicitability of vocal fold movements from this area in the squirrel monkey. Blocking was carried out by injection of the glutamate antagonist kynurenic acid. We found that injection into the dorsal reticular nucleus of the caudal medulla ipsilateral to the stimulation site blocked vocal fold movements bilaterally; injections invading major parts of the nucleus ambiguus blocked vocal fold movements exclusively ipsilateral to the injection site; and injections centered on the parvocellular reticular formation bordering the nucleus ambiguus blocked exclusively contralateral vocal fold movements. We conclude from this that the corticobulbar laryngeal control pathway synapses in the ipsilateral dorsal reticular nucleus and then divides into one component running directly to the ipsilateral nucleus ambiguus and a second component crossing to the contralateral nucleus ambiguus after having synapsed in the ipsilateral peri-ambigual reticular formation.

Sensory regulation of swallowing and airway protection: a role for the internal superior laryngeal nerve in humans.

During swallowing, the airway is protected from aspiration of ingested material by brief closure of the larynx and cessation of breathing. Mechanoreceptors innervated by the internal branch of the superior laryngeal nerve (ISLN) are activated by swallowing, and connect to central neurones that generate swallowing, laryngeal closure and respiratory rhythm. This study was designed to evaluate the hypothesis that the ISLN afferent signal is necessary for normal deglutition and airway protection in humans. In 21 healthy adults, we recorded submental electromyograms, videofluoroscopic images of the upper airway, oronasal airflow and respiratory inductance plethysmography. In six subjects we also recorded pressures in the hypopharynx and upper oesophagus. We analysed swallows that followed a brief infusion (4-5 ml) of liquid barium onto the tongue, or a sip (1-18 ml) from a cup. In 16 subjects, the ISLN was anaesthetised by transcutaneous injection of bupivacaine into the paraglottic compartment. Saline injections using the identical procedure were performed in six subjects. Endoscopy was used to evaluate upper airway anatomy, to confirm ISLN anaesthesia, and to visualise vocal cord movement and laryngeal closure. Comparisons of swallowing and breathing were made within subjects (anaesthetic or saline injection vs. control, i.e. no injection) and between subjects (anaesthetic injection vs. saline injection). In the non-anaesthetised condition (saline injection, 174 swallows in six subjects; no injection, 522 swallows in 20 subjects), laryngeal penetration during swallowing was rare (1.4 %) and tracheal aspiration was never observed. During ISLN anaesthesia (16 subjects, 396 swallows), all subjects experienced effortful swallowing and an illusory globus sensation in the throat, and 15 subjects exhibited penetration of fluid into the larynx during swallowing. The incidence of laryngeal penetration in the anaesthetised condition was 43 % (P < 0.01, compared with either saline or no injection) and of these penetrations, 56 % led to tracheal aspiration (without adverse effects). We further analysed the swallow cycle to evaluate the mechanism(s) by which fluid entered the larynx. Laryngeal penetration was not caused by premature spillage of oral fluid into the hypopharynx, delayed clearance of fluid from the hypopharynx, or excessive hypopharyngeal pressure generated by swallowing. Furthermore, there was no impairment in the ability of swallowing to halt respiratory airflow during the period of pharyngeal bolus flow. Rather, our observations suggest that loss of airway protection was due to incomplete closure of the larynx during the pharyngeal phase of swallowing. In contrast to the insufficient closure during swallowing, laryngeal closure was robust during voluntary challenges with the Valsalva, Müller and cough manoeuvres under ISLN anaesthesia. We suggest that an afferent signal arising from the ISLN receptor field is necessary for normal deglutition, especially for providing feedback to central neural circuits that facilitate laryngeal closure during swallowing. The ISLN afferent signal is not essential for initiating and sequencing the swallow cycle, for co-ordinating swallowing with breathing, or for closing the larynx during voluntary manoeuvres.

Effects of perineural capsaicin treatment on cardiopulmonary reflexes elicited by laryngeal instillations of capsaicin and distilled water in sevoflurane-anesthetized dogs.

The aim of this study was to determine the effect of perineural capsaicin (CAPS) treatment on cardiopulmonary reflexes elicited by topical laryngeal instillation of CAPS and distilled water (DW) in sevoflurane-anesthetized dogs. Cardiopulmonary reflexes elicited by CAPS (10 microg/ml, 10 ml) were attenuated by perineural CAPS treatment to the superior laryngeal nerves (SLNs) (P<0.05), whereas those by DW (10 ml) remained unaffected (P>0.05). The reflex responses to DW that remained even after the perineural CAPS treatment were eliminated by laryngeal anesthesia with lidocaine. These results suggest that cardiopulmonary reflexes from the laryngeal mucosa elicited by CAPS instillation can be blocked by perineural CAPS treatment to the SLNs, which may result from inhibition of the laryngeal CAPS-sensitive C-fiber afferents.

Effects of perineural capsaicin treatment on compound action potentials of superior laryngeal nerve afferents in sevoflurane-anesthetized dogs.

Effects of perineural capsaicin (CAPS) treatment on compound action potentials of the superior laryngeal nerve (SLN) afferents were studied in 6 sevoflurane-anesthetized dogs. Perineural CAPS (100 microg/ml) to the bilateral SLNs reduced (P<0.01) the peak and integral amplitudes of the C-wave of the compound action potential. By contrast, the perineural CAPS had no effect on the A-wave component (P>0.05). Removal of the perineural CAPS recovered the C-wave to pretreatment level. The perineural CAPS treatment selectively blocks C-wave compound action potentials of the SLN afferents, providing a useful tool for studies of laryngeal C-fibers in respiratory physiology.

Anesthesia for the superior laryngeal nerves and tartaric acid-induced cough.

OBJECTIVE: The internal branch of the superior laryngeal nerve (ibSLN) conveys impulses for the laryngeal cough reflex, which protects the laryngeal aditus and prevents the development of aspiration pneumonia. The purpose of this study was to determine the effect of bilateral anesthesia of the ibSLN on the cough reflex after inhalation of a nebulized chemoirritant solution of tartaric acid. DESIGN: Prospective, clinical investigation. SETTING: Outpatient. PARTICIPANTS: Nine healthy volunteers. INTERVENTIONS: Bilateral injections of 2% lidocaine solution without epinephrine into the paraglottic space containing the ibSLN. MAIN OUTCOME MEASURES: The tidal volume after inhalation of a nebulized 20% tartaric acid solution and forced vital capacity (FVC) were measured before and after injection. Data were analyzed using the Wilcoxon signed ranks, Mann-Whitney, and sign tests. RESULTS: Complete anesthesia of the ibSLN abolished the laryngeal cough reflex. Postinjection tidal volumes were significantly lower than preinjection volumes (p<.01). The decrease in tidal volumes for six subjects with complete bilateral anesthesia was significantly larger than the decrease in tidal volumes for three subjects with partial anesthesia (p<.05). FVC in both the six subjects with complete bilateral anesthesia and the three subjects with partial anesthesia did not significantly change from preinjection to postinjection. None of the subjects in this study had complications or adverse respiratory sequelae. CONCLUSION: Tartaric acid-induced cough may be useful in assessing the integrity of the laryngeal cough reflex after anesthesia or in patients with neurologic injury who are at risk of developing aspiration pneumonia. It may also be useful in making the decision whether to resume oral feeding.

Central nervous system control of cardiovascular function: neural mechanisms and novel modulators.

1. Studies are described that indicate that hypothalamic stimulation, at sites eliciting the defence reaction, results in an increase in adenosine levels in both the nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM) of the rat. 2. Adenosine receptor antagonists applied to these sites attenuate the pressor response elicited by hypothalamic stimulation. Adenosine appears to be produced extracellularly from ATP, which is released from axon terminals in both the NTS and RVLM. 3. The implications of these observations for the pharmacology of hypothalamic actions on reflex inputs that modify cardiorespiratory function are discussed.

Vocal function following vertical hemilaryngectomy: comparison of four reconstruction techniques in the canine.

The goals of laryngeal reconstruction have been prevention of aspiration, production of a functional voice, and maintenance of an adequate airway for decannulation. A number of procedures for partial laryngeal reconstruction have accomplished these objectives. However, few studies have attempted to compare patients' vocal characteristics following different reconstruction procedures. In this study, an in vivo canine model was used to compare acoustic and aerodynamic measures of vocal function for the following vertical hemilaryngectomy reconstruction techniques: 1) a superiorly based sternohyoid muscle flap, 2) a modified epiglottic laryngoplasty, 3) a new procedure using a layered vascularized buccal mucosal flap and a transversely oriented sternohyoid muscle flap, and 4) hemilaryngeal transplantation combined with arytenoid adduction. Hemitransplantation provided the most efficient phonation of the four techniques. The vascularized buccal mucosa flap produced the best phonation of the autologous tissue techniques examined. Both vascularized buccal mucosa flap and hemilaryngeal transplantation subjects demonstrated a mucosal wave on stroboscopy. The results indicate that vocal function will improve as the layered structure of the vocal fold is more accurately replicated in a reconstructed hemilarynx. Endoscopic findings and whole organ sections are presented.

Hypothalamic modulation of laryngeal reflexes in the anaesthetized cat: role of the nucleus tractus solitarii.

1. This investigation was initiated because activation of laryngeal afferents, either by electrical stimulation of the superior laryngeal nerve (SLN) or by natural stimulation of receptors in the laryngeal mucosa, results in a cardiorespiratory response comprising bradycardia, hypotension and apnoea (phrenic nerve activity was suppressed). This pattern of response is qualitatively equivalent to the response that is evoked on activation of the arterial baroreceptors. 2. Preliminary studies indicated that the effects of activating the SLN were suppressed during stimulation in the hypothalamic defence area (HDA) at points that also blocked the effects of baroreceptor stimulation. 3. Recordings were taken from seventy-two neurones localized within the ipsilateral nucleus tractus solitarii (NTS) whose activity was modified by SLN stimulation. Sixty neurones responded with an EPSP on SLN stimulation; nine of these had an inspiratory firing pattern. Five neurones were seen to receive an IPSP on SLN stimulation. 4. Five respiratory SLN-activated neurones were unresponsive to stimulation of the other nerve inputs, whilst four received convergent EPSP inputs on sinus nerve (SN) stimulation. One cell of these four also received inputs from the aortic and the vagus nerves. Sixty-one non-respiratory SLN-activated neurones also received convergent inputs from the sinus nerve. Of these, fifty displayed an EPSP, four an IPSP and seven an EPSP-IPSP. Fifteen neurones also received inputs from the aortic nerve and seventeen from the vagus. 5. From the population of neurones affected by SLN stimulation, twenty-four of seventy were also influenced by HDA stimulation (3 were respiratory cells). Sixteen of these responses consisted of an EPSP (2 respiratory cells), five of an IPSP (1 respiratory cell) and three of an EPSP-IPSP. 6. In neurones receiving an IPSP on HDA stimulation, the SLN-evoked excitatory response was reduced throughout the period of HDA-evoked inhibition. These neurones were all shown to receive excitatory inputs from the arterial baroreceptors and laryngeal mechanoreceptors. 7. Additionally, in the thirty-seven neurones that were excited by SLN stimulation but received no direct synaptic input on HDA stimulation, a conditioning stimulus to the HDA evoked a block of SLN-evoked responses without an accompanying change in membrane potential. Several of these neurones were also affected by both baroreceptor and laryngeal mechanoreceptor stimulation. 8. These observations are discussed in the context of the role of the NTS in cardiorespiratory control. The potential importance of these interactions in respiratory distress are highlighted and the implications for the organization of central pathways for the control of autonomic and respiratory function are discussed.

Overcoming laryngospasm by electrical stimulation of the posterior cricoarytenoid muscle.

The intent of this study was to demonstrate that the technique of transmucosal electrical stimulation of laryngeal muscles may be of clinical use in airway management. Specifically, its ability to overcome laryngospasm was evaluated. Laryngospasm was induced in eight tracheotomized dogs by hyperventilating each dog, and then applying 0.1 M ammonia to the laryngeal mucosa while administering continuous positive airway pressure (CPAP). Laryngospasm was defined by steady apposition of the vocal cords, massive electromyographic activity in the laryngeal adductor muscles, absence of such activity in the posterior cricoarytenoid muscle (PCA), and intraglottic pressure greater than 80 mm Hg. Upon transmucosal application of 10 mAmp current to the PCA bilaterally, the vocal cords abducted for the duration of the stimulus. We theorize that overcoming laryngospasm by electrostimulation involves a reflexive inhibition of the laryngeal adductors. This study provides an objective model for laryngospasm, and demonstrates that electrical manipulation of the vocal cords may have clinical relevance.

Bionic larynx: electronic control of the reimplanted organ in the dog.

Complete rehabilitation of paralyzed head and neck structures has been difficult, and attempts at reinnervation of striated muscles have not consistently succeeded. Recent studies have shown that disabled muscles can be electronically "paced." This, together with recent developments in microelectronics, has led to the concept of the Bionic Larynx. The current experiments involve transposed nerve-muscle pedicles used to selectively reinnervate larynges which were denervated, entirely detached from the animal except for their vascular supply, and then reimplanted. These pedicles were later stimulated using a potentially implantable "pacing unit." It was possible to selectively control the critical functions of the larynx, using as stimuli physiological events that are preserved even when the vocal folds are paralyzed or uncoordinated. Although laryngeal transplantation in humans remains theoretical, these studies show that it could be feasible in the future.

Transcutaneous electrical stimulation of the recurrent laryngeal nerve in monkeys.

The recurrent laryngeal nerve (RLN) of four anesthetized adult Macaca fascicularis monkeys was stimulated by applying current with blunt electrodes placed unilaterally and bilaterally on the intact neck skin along the tracheoesophageal groove. The stimulus consisted of 2.5- to 4.0-mA cathodal pulses, each of 1-ms duration, beginning at a frequency of 10 Hz and increasing by 10-Hz increments to 100 Hz. Unilateral stimulation from 10 to 30 Hz resulted in a graded vocal cord abduction, with the maximal glottic aperture occurring at 30 Hz. Stimulation above 30 Hz produced a graded cord adduction, with nearly complete glottic closure at 100 Hz; bilateral stimulation yielded similar results, with total glottic closure at 100 Hz. Confirmation of the RLN as mediator of this frequency-dependent cord motion was achieved by surgically isolating it and attaining identical results with direct stimulation. No cardiopulmonary alterations were observed in any trial. Transcutaneous electrical stimulation of the RLN seems to be a relatively safe, reliable, and noninvasive method of controlling vocal cord position and thereby the glottic airway in monkeys.