Restoration of diaphragmatic function after diaphragm reinnervation by inferior laryngeal nerve; experimental study in rabbits

OBJECTIVES: To assess the possibilities of reinnervation in a paralyzed hemidiaphragm via an anastomosis between phrenic nerve and inferior laryngeal nerve in rabbits. Reinnervation of a paralyzed diaphragm could be an alternative to treat patients with ventilatory insufficiency due to upper cervical spine injuries. MATERIAL AND METHOD: Rabbits were divided into five groups of seven rabbits each. Groups I and II were respectively the healthy and the denervated control groups. The 3 other groups were all reinnervated using three different surgical procedures. In groups III and IV, phrenic nerve was respectively anastomosed with the abductor branch of the inferior laryngeal nerve and with the trunk of the inferior laryngeal nerve. In group V, the fifth and fourth cervical roots were respectively anastomosed with the abductor branch of the inferior laryngeal nerve and with the nerve of the sternothyroid muscle (originating from the hypoglossal nerve). Animals were evaluated 4 months later using electromyography, transdiaphragmatic pressure measurements, sonomicrometry and histological examination. RESULTS: A poor inspiratory activity was found in quiet breathing in the reinnervated groups, with an increasing pattern of activity during effort. In the reinnervated groups, transdiaphragmatic pressure measurements and sonomicrometry were higher in group III with no significant differencewith groups IV and V. CONCLUSION: Inspiratory contractility of an hemidiaphragm could be restored with immediate anastomosis after phrenic nerve section between phrenic nerve and inferior laryngeal nerve

Upper Airway Control in Airway Defense

Upper airways (UA) are an organic component of the respiratory tract, they serve to respiration, respiratory tract protection and defense, phonation, deglutition, etc. The functions of UA are regulated by motor control of the oral, pharyngeal, and laryngeal muscles

Influence of microinjections of D,L-homocysteic acid into the Bötzinger complex area on the cough reflex in the cat.

Microinjections of D,L-homocysteic acid (DLH) were used to test the hypothesis that neuronal activation within the Botzinger complex area can modify the spatiotemporal characteristics of the cough reflex in 17 spontaneously breathing pentobarbitone anesthetized cats. DLH (50 mM, 1.25-1.75 nmol, 9 cats) reduced the number (P<0.01) of coughs and expiratory amplitude of abdominal electromyographic activity (P<0.01), and also esophageal pressure (P<0.001) during mechanically induced tracheobronchial cough. The duration of cough abdominal activity was shortened by 48% (P<0.05). DLH microinjections also temporarily reduced the respiratory rate (P<0.01) and increased the mean arterial blood pressure (P<0.001), baseline of esophageal pressure (P<0.01), and end tidal CO 2 concentrations (P<0.01). Lower doses of 8 cats) induced few alterations in cardiorespiratory or cough characteristics. The results support predominantly inhibitory effects of neurons in the region of the Bötzinger complex on cough abdominal activity and cough number

Molecular Mechanisms of Dysautonomia During Heart Failure. Focus On “Heart Failure-Induced Changes of Voltage-Gated CA2+ Channels and Cell Excitability in Rat Cardiac Postganglionic Neurons”

We tested the hypothesis, motivated in part by a coordinated computational cough network model, that alterations of mean systemic arterial blood pressure (BP) influence the excitability and motor pattern of cough. Model simulations predicted suppression of coughing by stimulation of arterial baroreceptors. In vivo experiments were conducted on anesthetized spontaneously breathing cats. Cough was elicited by mechanical stimulation of the intrathoracic airways. Electromyograms (EMG) of inspiratory parasternal, expiratory abdominal, laryngeal posterior cricoarytenoid (PCA), and thyroarytenoid muscles along with esophageal pressure (EP) and BP were recorded. Transiently elevated BP significantly reduced cough number, cough-related inspiratory, and expiratory amplitudes of EP, peak parasternal and abdominal EMG, and maximum of PCA EMG during the expulsive phase of cough, and prolonged the cough inspiratory and expiratory phases as well as cough cycle duration compared with control coughs. Latencies from the beginning of stimulation to the onset of cough-related diaphragm and abdominal activities were increased. Increases in BP also elicited bradycardia and isocapnic bradypnea. Reductions in BP increased cough number; elevated inspiratory EP amplitude and parasternal, abdominal, and inspiratory PCA EMG amplitudes; decreased total cough cycle duration; shortened the durations of the cough expiratory phase and cough-related abdominal discharge; and shortened cough latency compared with control coughs. Reduced BP also produced tachycardia, tachypnea, and hypocapnic hyperventilation. These effects of BP on coughing likely originate from interactions between barosensitive and respiratory brainstem neuronal networks, particularly by modulation of respiratory neurons within multiple respiration/cough-related brainstem areas by baroreceptor input. cough is initiated by stimulation of mechano- and chemosensitive sensory endings of cough receptors and also may be influenced by a cough-related subgroup of rapidly adapting “irritant” receptors and C fibers within tracheobronchial and laryngeal mucosa (23, 24, 148). However, the pattern of coughing (the number of coughs and their strength and timing; Refs. 67, 149) is profoundly affected by other peripheral and central afferent inputs (17, 49), particularly during pathological processes such as infection, inflammation, and allergic reactions (28, 118). Stimuli within the larynx (141) and nose (109, 110) enhance cough induced from the tracheal-bronchial region. Stimulation of cardiac receptors (140), chemoreceptors (138), and pulmonary as well as bronchial C fibers in anesthetized animals (139) reduces coughing. Afferent signaling from muscles, joints, skin, and possibly the viscera may also alter the expression of cough (66, 67, 118). Coughing induces vigorous intrathoracic and intra-abdominal pressure oscillations and changes of sympathetic and parasympathetic nervous activities (27, 67, 145) that significantly affect the cardiovascular system including dynamic changes of blood pressure (BP) and regional blood flow (60, 67). Coughing is associated with peaks in systemic arterial blood pressure during systole and cough expulsions followed by post-tussive hypotension (unpublished observations; Refs. 67, 127). This relationship involves central reflex mechanisms (27, 127, 145); it is observed also in neuromuscular-blocked decerebrate animals (unpublished observations). However, very little is known about the effects of systemic BP and baroreceptor afferent input on the excitability and patterning of cough. Available data were mostly obtained with stimulation of multiple sensory afferents resulting in expression of the chemoreflex (96, 140), and the results suggested either no changes (139, 140) or only transient alterations of the cough reflex (96) during reduced BP. The respiratory neuronal network is a crucial component in the generation of cough and the transmission of its central motor pattern to the respiratory muscles (54, 119, 123–125). There is a close relationship between the control of the respiratory and cardiovascular systems. It is well established that an increase in blood pressure resulting in the baroreflex (83, 98, 135) can prolong expiration, significantly reduce breathing frequency, and reduce inspiratory drive by an action on selected populations of brainstem respiratory neurons (2, 35, 72, 76, 107) sensitive to afferent impulses originating from baroreceptors. Thus baroreceptor reflex feedback mechanisms that modulate breathing may also limit cough intensity and/or number. Hence, changes in cough excitability and/or the pattern of coughing due to the stimulation of baroreceptors (and alternatively by their unloading) are consistent with the multifunctional role of the respiratory pattern generator in controlling cough and breathing. Motivated by this consideration, we undertook a computational modeling study of the respiratory/cough neuronal network and in vivo experiments to test the role of blood pressure changes in modulation of cough motor pattern. The study also allowed us to address a more general hypothesis that this model could be used to predict, not just motor patterns and neuronal responses of the brainstem respiratory network, but regulation of this system as well. Models simulating concurrent cough and baroreceptor perturbations of breathing predicted that an increase of mean systemic arterial BP would alter the motor pattern and excitability of the cough reflex

01_tomori

K e y w o r d s : auto-resuscitation, brainstem, cat, eupnoea, gasping, respiratory rhythmogenesis, aspiration reflex, expiration reflex 1) a short-lasting high-frequency activity in the phrenic nerve or the inspiratory muscles, indicating a rapid, strong, spasmodic sniff-and gasp-like inspiration; 2) no subsequent active expiration, but even a reflex interruption of any spontaneous activity of the abdominal muscles, suggesting an inhibition of active expiration and an exhalation performed passively; 3) a marked inspiratory pleural pressure decrease (-4 kPa), followed by a passive return of intrathoracic pressure to values slightly above zero, promoting an increased venous return of blood to the heart and supporting blood supply to various organs and tissues; 4) a marked positive increase in transmural pressure, promoting; 5) a passive inspiratory dilation of the intrathoracic airways; 6) a rapid short-lasting inspiratory reflex dilation of the pharyngeal lumen and reflex opening of the glottis, allowing lung inflation, followed by a glottal narrowing during the post-inspiratory phase which inhibits subsequent lung deflation; 7) a sniff-like sound; 8) reflex inhibition of spontaneous or any increased bronchoconstrictor fibre activity, resulting in transient bronchodilation; 9) a rapid inspiratory airflow (280-420 ml . s -1 ); 10) a moderately increased inspiratory tidal volume (7). The ten main components of ExpR are: 1) no preceding inspiration or even a reflex interruption of an occasional inspiration; 2) a solitary, short-lasting activity in the abdominal muscles, indicating a prompt expiratory effort (30-150 ms); 3) a sudden marked expiratory increase in the intrathoracic (pleural) pressure (+2.2 kPa), contributing to the systolic ejection of the blood from the heart; 4) a negative transmural pressure; causing 5) an expiratory compression of the intrathoracic airways; 6) a reflex glottal closure, promoting a pressure increase during the compressive phase, followed by reflex glottal opening allowing a rapid expulsion of airflow together with any irritants or fluids during the expulsive phase; 7) a brisk explosive sound; 8) a reflex bronchoconstriction indicated by ~2-fold increase of baseline bronchoconstrictor fibre activity; 9) a rapid and marked expiratory airflow (~130 ml . s -1 ); 10) a moderately increased expiratory tidal volume. The ten main components of the tracheo-bronchial cough reflex, are: 1) a strong activity of the phrenic nerve, the diaphragm and other inspiratory muscles, causing deep longer-lasting initial inspiration; 2) a very strong activation of the abdominal and other expiratory muscles, resulting in a powerful expiratory effort; 3) a considerable decrease followed by a large increase in intrathoracic (pleural) pressure (+4 kPa), promoting an increased venous return to the heart, followed by marked ejection of blood to various tissues; 4) a highly negative transmural pressure; promoting 5) a dynamic expiratory compression of the intrathoracic airways; 6) after inspiratory dilation a reflex glottal closure in the compressive phase, causing an intrathoracic pressure increase, followed by prompt glottal opening, allowing a powerful expulsion of air with any irritants; 7) an explosive cough sound; 8) reflex bronchoconstriction indicated by about a 3-fold increase in bronchoconstrictor fibre activity; 9) very rapid expiratory airflow (500 ml . s -1 ); 10) a large (about 5-fold) increase in expired tidal volume. DISTINCT LOCALIZATION OF BRAINSTEM STRUCTURES FOR THE ASPR AND EXPR Our previous results, obtained by the c-fos immuno-reactive method, were intricately re-evaluated to allow quantitative assessment and comparison of early c-fos gene production, indicating the level of activation of neurones involved in the two distinct airway reflexes. In separate experiments, 450±30 AspRs or 296±9 ExpRs were elicited during ~30 min, with recording of electrophysiological and cardio-respiratory parameters in pentobarbitone-anaesthetized spontaneouslybreathing cats. The results indicated that the AspR induced by Nph stimulation provoked much stronger activation of inspiratory modulated neurones in 16 of 35 investigated brainstem nuclei than did quiet breathing (8). These regions included the nucleus tractus solitarius, representing the first central station of the AspR, the caudal to middle ventrolateral medulla containing the pre-Botzinger complex (preBotC), known to be the site of the inspiratory rhythm generator (1, 9), and also the lateral tegmental field (10, 11), as well as the caudal medullary raphe and the ventrolateral pons (8), participating in the generation of gasping during severe brain hypoxia in decerebrate cats. In experiments on another cats, stimulation of the glottal area provoking the ExpR induced strong immuno-reactivity, indicating powerful activation of expiratory modulated neurones in the middle to rostral ventrolateral medulla, including the retrotrapezoid-parafacial region, postulated to serve as the expiratory rhythm generator (1, 12), and the rostral midline midbrain, compared with eupnoea Re-evaluation of results from successive rostral-to-caudal brainstem transection experiments RESPIRATORY RHYTHM AND PATTERN GENERATION AND ITS REFLEX MODIFICATIONS The persistence of spontaneous breathing during severe brain hypoxia is strongly supported by stimulation of peripheral and central chemoreceptors, as indicated by rapid replacement of spontaneous breathing by gasping in mice with transected carotid sinus nerves during 90 s inhalation of 10% O 2 in N 2 (17). During the gasping stage (SG) in these transection experiments, practically every mechanical stimulus, applied to the nasopharynx in 1 s intervals, elicited nearly 2-times stronger gasp-like AspRs, than before. These results in adult cats confirm the elimination of expiratory activity after similar brainstem transection observed in juvenile rats In addition, the AspR in the gasping stage frequently consisted of 2 to 4-phasic inspirations without interposed active expirations or total lung deflation. During severe hypoxia and/or transitory brain ischemia in cats, spontaneous inspiratory oscillatory discharges of a frequency of 10.7±0.6 Hz were observed, demonstrating transient inspiratory rhythm generation. These spontaneous discharges occurred either at a transition from eupnoea to gasping, or from gasping to eupnoea even after 13-17 min of hypoxic coma at a BP of only 75±8 mm Hg, or from gasping to terminal apnoea after cardiac arrest by early renewal of the air supply Higher animals, including cats and pigs, are more suitable for clinically applicable research. In adult cats in vivo, chemical interruption of synaptic activity within the preBotC also abolished the respiratory rhythm, but not the gasping (23). Stimulation of both the laryngeal and pharyngeal mucous membranes by a nylon fibre through a tracheotomy in cats with intact vagal afferentation increased the intensity of the cough reaction by evoking interposed spasmodic inspiratory efforts, mediated by stimulation of pharyngeal RARs with subsequent stronger expiratory efforts (3, 7). The intensity of these interposed inspirations gradually decreased during the I-phase of the cough cycle relative to the increasing momentary lung volume, activating the slowly-adapting receptors that mediate the Hering-Breuer inspiration inhibitory reflex (HBIIR). During the compressive phase of cough, the lung deflation is hindered, which evokes an increase of expiratory effort, termed the HeringBreuer expiration facilitating reflex, gradually decreasing during the lung deflation (24). Interaction of the inspiratory facilitation caused by rapid lung inflation with the HBIIR and expiration facilitatory reflexes may contribute to frequent repetition of cough efforts, manifesting in attacks of coughing, particularly in cases of airway inflammation, hyper-reactivity, etc. Lesioning of >70% of preBotC in awake goats eliminated diaphragm activity and spontaneous breathing, preserving the activity of the expiratory rhythm generator (25). The propriobulbar I neurones of preBotC, co-expressing neurokinin 1 and µ-opioid receptors, important for generation of the inspiratory rhythm, can be inhibited to sub-threshold level by opioids, and the signal transmission from the pre-inspiratory neurones, arising from higher levels, is blocked. This results in so-called quantal breathing, transient apnoea or arrhythmic polypnoeic breathing MECHANISMS OF THE ASPR AND EXPR Stimulation of polymodal RARs of the airways by irritants of various types elicits a prompt short-lasting solitary reaction in anaesthetized cats. Mechanical stimulation by a short contact, pressure pulse or airflow applied to the pharynx evokes a highfrequency response in the afferent single fibre of the glossopharyngeal nerve of A very stabile AspR can be evoked by electrical stimulation of the Nph in the region of the tuba auditiva in cats by a 0.5 ms pulse of 4-9 V which, after a latency of 25±2 ms, provokes diaphragmal activity lasting 70±11 ms, causing peak inspiratory airflow of 280-420 ml . s -1 followed by a successive inhibition for 64±11 ms The AspR and ExpR can be induced by various methods of stimulation (mechanical contact, pressure pulses, air-flow, and electrical, chemical and thermal stimulations) and from several areas of the body, which allows their relatively easy testing. The AspR can be induced from the naso-and oro-pharynx and similar gasp-like spasmodic inspirations from various acupuncture points EFFECTS OF THE ASPR AND EXPR IN CATS AND THEIR APPLICABILITY There are three main conclusions of this study, demonstrating the great importance of these two distinct airway reflexes for various applications in experimental and clinicophysiological practice: 1) Re-evaluation of the brainstem localization and mechanisms of the AspR and the ExpR in adult cats indicated that these reflexes separately activate the neurones of distinct rhythm generators for I and E, respectively, and provide a 9 support for the dual theory of respiratory rhythmogenesis. Therefore, they may be useful models for study of respiratory rhythm and pattern generation, including testing of various drugs, nervous sensors and membrane channels, even in genetically modified animals. 2) Reassessment of the similarity of the brainstem structures (8, 16) and mechanisms of the gasp-like AspR in cats with hypoxic gasps 3) Such resetting of central control mechanisms of various vital functions provides a unique possibility to normalize various dysfunctions "on demand", when appropriate functional disorders occur, mostly based on reverberation of pathological excitation or inhibition, but without irreparable morphological changes: e.g. recent myocardial infarction or stroke. Such a mechanism might explain the unexpectedly successful treatment of longer-lasting attacks of hiccough in patients (47), as well as the repeated interruption of paroxysmal supraventricular tachycardia (48), by simple introduction of a nasogastric catheter. In premature infants, introduction of a nasogastric catheter for feeding occasionally induced short but marked inspiratory efforts, accompanied by a transient tachycardia and hypertension, resembling the AspR of cats (49). Similar unexpected reflex broncho-protective and broncho-spasmolytic effects have been observed after voluntary deep inspirations through the nose, but not through the mouth in healthy subjects and mild asthmatics (50). The occurrence of sigh and gasp in practically all mammals allows the study of their powerful restorative and "autoresuscitative" potential in animal models. Intensive studies have been performed in pigs and other animals with potentially fatal ventricular fibrillation, induced by intracardial stimulation and its reversal by autoresuscitation effect of spontaneously developing gasping during postponed defibrillation; these stimuli have been tested in animal experiments and clinicophysiological studies using also technical models (51). In humans, upper airway pressure more negative than -1.2 kPa induces a "pharyngeal dilatory reflex", reversing upper airway obstruction, which is less effective in patients with sleep disordered breathing (52). Negative pharyngeal pressure, induced by inspiratory resistive loading, provokes genioglossal muscle activation in awake humans. Both central outputs to the genioglossal muscle and pharyngeal reflex-mediated activation are important in maintaining upper airway patency (53). REVITALISATION EFFECTS OF THE ASPHYXIC GASPING AND THE ASPR In infants dying of SIDS, asphyxic gasping was regularly detected before death (28). The reaction of normal and SIDS infants to excessive stress is schematically illustrated in Nonspecific tactile stimulation of the foot of healthy infants elicits an arousal sequence commencing with a spinal withdrawal reflex, sometimes followed by respiratory and startle responses, occasionally ending in cortical arousal (54). Respiratory related stimulation, such as transient airway occlusion or a lack of O 2 and accumulation of CO 2 during sleep, evokes a sigh or gasp and a subsequent startle, associated with a neck extension and pharyngeal dilation. These reactions operate frequently as brainstem reflexes and may account for recovery of airway patency without evidence of cortical arousal. The magnitude of sigh, the intensity of startle with accompanying upper airway dilation and the subsequent increase in heart rate, correlated with the peak airway negative pressure, indicating graded intensity of the arousal reaction. For the shortness of upper airway closure, lung inflation and chemoreceptor stimulation are not prerequisites for the occurrence of these reflexes (55). Therefore, just as stimulation of upper airway receptors that elicit the AspR in cats Spontaneous gasps are regularly observed in various animals and in infants to prevent lung atelectasis (56). Continuous monitoring of infants with suspected SIDS in clinicophysiological trials indicated, that spontaneously developing agonal gasping, or a gasp reflex provoked after an alarm, resuscitated and saved some of the infants, as it does in animals (51, 55-60). Auto-resuscitation by gasping in infants usually starts with an increase in heart rate and BP, which may fail for various reasons (underdeveloped nervous control in premature infants, co-morbidities, exhaustion of functional reserves, etc.). Ineffective auto-resuscitation by gasping is assumed to contribute to the development of SIDS (28, 61) and sudden cardiac death. Asphyxic gasping may auto-resuscitate 10-15% of both animals and infants, depending on their onto-and phylogenetic development and functional reserves of their vital systems. The brainstem-mediated cardiovascular reflexes are mainly responsible for survival in acutely hypoxic infants and adults, due to auto-resuscitation by gasping. FUNCTIONS OF THE EXPR The ExpR without a preceding inspiration serves as a component of the laryngeal chemoreflex, evoked by chemical or mechanical irritants from the laryngo-pharyngeal region, with its 10 CONCLUSIONS Comparison of central neuronal structures, mechanisms and the effects of the two distinct airway reflexes with the generators for inspiration and expiration, indicated their similarity and supported the dual theory of respiratory neurogenesis. The AspR, characterized by solitary spasmodic inspiration without successive active expiration, and the ExpR manifesting as prompt expiration without preceding inspiration, may provide a unique model for study of various topics of respiratory rhythm and pattern generation. Both airway defensive reflexes have distinct characteristics, including reciprocal inhibition, and can contribute to prevention and reversal of severe life-threatening events. The AspR, like gasping, may prevent imminent death and even auto-resuscitate the moribund subject, while the ExpR as a component of laryngeal chemoreflex, can expel the irritants and inhibit their penetration into the lungs to prevent development of lung diseases. In affectionate memory of our friendship and collaboration with Juraj (George) Korpas

Correction: Suppression of Abdominal Motor Activity during Swallowing in Cats and Humans.

[This corrects the article DOI: 10.1371/journal.pone.0128245.]