Published ahead of print on November 8, 2002, doi:10.1164/rccm.200207-768BC
© 2003 American Thoracic Society
Hypersensitivity of Laryngeal C-Fibers Induced by Volatile Anesthetics in Young Guinea PigsDepartments of Veterinary Surgery and Comparative Pathophysiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo; and Division of Molecular Brain Science, Department of Brain Sciences, Kobe University Graduate School of Medicine, Kobe, Japan Correspondence and requests for reprints should be addressed to Hirokazu Tsubone, D.V.M., Ph.D., Department of Comparative Pathophysiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail: aahiro{at}mail.ecc.u-tokyo.ac.jp
Inhalation induction of anesthesia with a single volatile anesthetic is commonly used in children but is sometimes associated with increased cough, secretion, and airway obstruction, which may result in part from stimulation of laryngeal C-fibers. We examined the effects of two popular volatile anesthetics, halothane and sevoflurane, on laryngeal C-fiber responsiveness in urethane-anesthetized guinea pigs (from age 4–5 weeks). After administration of halothane or sevoflurane to the functionally isolated upper airway, laryngeal C-fiber afferents recorded from the internal branch of the superior laryngeal nerve and identified by a conduction velocity of less than 2.0 m/second were tested for responsiveness to chemical and mechanical stimuli. Halothane doubled C-fiber responsiveness to capsaicin injected into the left atrium or nebulized to the larynx and to laryngeal hyperinflation, compared with sevoflurane, but it had no effect on baseline activity. The data indicate that, compared with sevoflurane, halothane more markedly enhances laryngeal C-fiber sensitivity to chemical and mechanical stimuli in young guinea pigs, which would explain the greater number of respiratory-related complications in children during induction of anesthesia with this agent.
Key Words: airway irritation • laryngeal C-fibers • volatile anesthetics The induction of anesthesia using a facemask with a single volatile agent is common in pediatric patients but is occasionally associated with upper airway reflexes such as cough, breath-holding, laryngospasm, and hypersecretion (1). Clinically, these undesirable responses are thought to be the result of an irritation of the upper airway mucosa (2), the effects of which place patients at risk for exposure to hypoxia or hypercapnea and inhibit smooth induction with certain volatile anesthetics. The degree of the upper airway irritation is known to vary with the type and concentration of inhalant. Isoflurane is not tolerated by patients because of its pungency and a relatively high incidence of coughing, airway obstruction, and oxygen desaturation during induction (3). Halothane is less irritating to airways than isoflurane (2) and has been widely used for mask induction in children. Recently, the induction technique has engendered renewed interest with the introduction of a new halogenated volatile anesthetic, sevoflurane, into clinical practice. Studies in children and adults have shown that high inspired concentrations of sevoflurane are well tolerated because sevoflurane not only acts more rapidly but also facilitates induction with a lower incidence of respiratory complications and "better patient acceptance" (4, 5). Defense and protection of the lower airway has frequently been viewed as the main role of the larynx. The epithelium of the larynx contains sensory afferent fibers that are responsible for eliciting various airway reflexes (e.g., cough, apnea, bradypnea, expiration reflex, and bronchospasm) (6–8). The internal branch of the superior laryngeal nerves (SLNs) is the common afferent pathway for eliciting these airway reflexes. Morphologic evidence has shown that approximately 50% of afferent fibers in the SLN are nonmyelinated C-fibers (9). Physiological studies using indirect measures, such as preventing the reflex responses by blocking C-fiber conduction or measuring the local release of tachykinins, indicate that the laryngeal C-fibers may play an important role in triggering the airway reflexes evoked by exposure to irritants such as ammonia, capsaicin, and bradykinin (8, 10). These observations have led to the suggestion that some of the same respiratory symptoms associated with anesthetic induction in children are also elicited by stimulation of the laryngeal C-fibers. Recently, Mutoh and coworkers (11) have provided more direct evidence for this proposal by showing that exposure to high concentrations (3–5%) of halothane and isoflurane, but not sevoflurane, sensitized the baseline activity of the laryngeal "capsaicin-sensitive" receptors. Consistent with these findings, several other studies have also documented an increased excitability of primary lung sensory C-fibers (12, 13) or somatic nociceptor afferents (i.e., the counterpart of C-fiber endings in the peripheral tissue) (14) after exposure to halothane or isoflurane. However, it remains unknown whether the potentiating effect of volatile anesthetics is also present in the responses of laryngeal C-fibers of the developing organism to chemical and mechanical stimulation because all other similar studies have been performed in adult animals, and the electrophysiologic data on the newer inhalant sevoflurane is limited. Hence, the main objective of the current study was to examine the effects of acute exposure to two popular volatile anesthetics, halothane and sevoflurane, in young guinea pigs during the age-equivalent period of human childhood on the baseline and evoked increases in impulse activity of laryngeal C-fibers.
All experimental protocols in this work were reviewed and approved by the Institutional Animal Care and Use Committee. Male Dunkin-Hartley guinea pigs (age, 4–5 weeks; body weight, 300–380 g) were anesthetized with an injection of urethane (1.8 g/kg intraperitoneally). Full details of the methods can be accessed in the online supplement. The trachea was cannulated caudally just above the thoracic inlet and a catheter was connected to a side port of the tracheal tube to monitor intratracheal pressure, whereas a short catheter (PE-60) was inserted cranially with its tip placed slightly below the cricoid cartilage (15). A latex cuffed tube (3 mm internal diameter) was introduced orally with its tip placed at the pharynx with the aid of a bronchoscope. The oral tube and the upper tracheal catheter were connected to a semiclosed anesthesia circuit through which either hyperinflation or negative pressure, as well as administration of a constant airflow, could be applied to the isolated upper airway. Each guinea pig was mechanically ventilated via the lower tracheal tube with oxygen-enriched humidified air with a tidal volume of 8 ml/kg. The pericardium was opened, and a cannula (0.58 mm internal diameter) prefilled with capsaicin (2.5 µg/ml) was inserted into the left atrium through the left atrial appendage. The internal branches of both SLNs were isolated and cut to avoid reflexes that could interfere with the activity of the receptors. The peripheral cut end of the right SLN was placed on a dissecting platform in a pool of mineral oil. A nerve bundle containing a suspected C-fiber was split so that the fiber was the only active fiber discernible or the only fiber in which the signal-to-noise ratio was sufficient to differentiate its activity from the noise as determined by a window discriminator.
Suspected C-fibers were preliminarily identified as irregularly, sparsely firing fibers that did not discharge faithfully and robustly with laryngeal muscle contraction or passive transmissions through the trachea by chest wall respiratory muscles (tracheal tug; eliminating "drive" receptors) and that did not adapt, either rapidly or slowly, to a sharply increasing and then steadily maintained (22 cm H2O) hyperinflation applied to the larynx (this criterion eliminated rapidly adapting "irritant" receptors and slowly adapting drive or "pressure" receptors) (11, 16). Fibers that were silent with normal ventilation but activated with hyperinflation maneuvers without rapid adaptation and that did not adapt slowly to maintained (-10 cm H2O) negative pressure (eliminating "negative pressure" receptors) or to a constant flow ( Once a C-fiber was identified, it was tested for its responses to (1) laryngeal hyperinflation (22 cm H2O), (2) left atrial injection of capsaicin (0.5 µg/kg, bolus), (3) laryngeal bradykinin nebulization (3 x 10-3 M, 21 seconds), and (4) laryngeal capsaicin nebulization (1.6 x 10-4 M, 21 seconds). Drugs were nebulized with an ultrasonic nebulizer (NE-U22; Omron Healthcare, Kyoto, Japan), which was connected in series with the inspiratory line from the anesthetic gas vaporizer so that air was drawn through the nebulizer during inhalation. The time lag of the nebulizer from the onset to initial appearance of a bubble indicating the presence of a drug was 9 seconds. The dose of capsaicin and bradykinin, time intervals, and sequence of trials were determined both in pilot and previous studies (17). Because it is difficult to maintain the recording of single-unit activity for an extended period of time, some of the receptors were tested for no more than two of these stimuli. To avoid any tachyphylaxis, a lapse of at least 10 minutes was allowed between injection or nebulization challenges. At the beginning of the protocol, data were collected over a 2-minute baseline period, then the upper airway was hyperinflated for 3 to 5 seconds. After another 2-minute baseline period, capsaicin was injected into the left atrium. After a 10-minute interval, warm (35–37°C), saturated airflow (30 ml/second) was passed through the upper airway in the expiratory direction. Data were collected over a 2-minute baseline period, then one or two times the minimum alveolar concentration (MAC) of halothane or sevoflurane was added to the warm airflow for at least 2 minutes through a precalibrated agent-specific vaporizer to prime the laryngeal lumen with vaporized air. Each C-fiber was challenged with a single dose (either 1 or 2 MAC) administration of both anesthetic gases throughout the experiment. Concentrations of anesthetic gases were continuously monitored with a Datex gas analyzer and were normalized to guinea pig MAC values of approximately 1% for halothane and 2% for sevoflurane (18). Priming was considered to be complete when vapor concentrations were identical to the challenge dose and remained constant for at least 1 minute. After laryngeal anesthetic exposure of the animal, the responses to hyperinflation and capsaicin injection were tested in the same sequence as before the exposure. Laryngeal application of the anesthetic was continued for at least 5 minutes after the capsaicin injection. After a 10-minute interval, data were collected over a 2-minute baseline period, and bradykinin or capsaicin was nebulized for 21 seconds. After a 10-minute interval, the nebulization was repeated after laryngeal application of the anesthetics, using the same administration protocol. Animals were killed after the experiment by a lethal dose injection of pentobarbital.
The impulse activity of C-fibers was analyzed by using NOTOCORD-HEM software (Primetech Ltd., Tokyo, Japan) for each 1-second interval. The peak C-fiber response was defined as the average number of impulses/second during the most active 6 seconds out of the initial 15 seconds after left atrial capsaicin or 21 seconds during laryngeal nebulization of bradykinin or capsaicin (17). The onset latency for the increase in C-fiber activity was defined as the first detectable increase after the injection. To determine whether the evoked changes (
Characteristics of Laryngeal C-fibers A total of 45 C-fibers were studied in 39 guinea pigs. Conduction velocity was obtained for every C-fiber; the average conduction velocity was 1.26 ± 0.09 m/second (n = 24) for fibers in the halothane-administered group and 1.30 ± 0.12 m/second (n = 21) for fibers in the sevoflurane-administered group (p = 0.38). We identified all C-fibers (0.48 ± 0.08 impulses/second of baseline activity) were capsaicin-sensitive type of sensory afferents located in the laryngeal lumen from below the glottis to the epiglottis with a response onset of 5 seconds or less to left atrial capsaicin (0.5 µg/kg). The increase in activity (  ) of the C-fiber responses was 4.18 ± 0.52 impulses/second, with a mean onset latency of 3.35 ± 0.14 seconds.
Effects of Volatile Anesthetics on Laryngeal C-fiber Responses Both halothane and sevoflurane produced increases in C-fiber activity in response to left atrial capsaicin in a dose-dependent manner (p = 0.0001, dose effect, ANOVA). In the halothane-administered animals, the peak increase in impulse activity in response to left atrial capsaicin was statistically different from baseline at both doses (p < 0.05); in the sevoflurane-administered animals, the difference reached statistical significance only with the 2 MAC dose. The peak increase in C-fiber activity in the halothane-administered animals was significantly greater than that in the sevoflurane-administered animals with both doses (Figure 1 ; p = 0.004, drug effect; p < 0.01, Tukey's test).
For all C-fibers responsive to left atrial capsaicin, hyperinflation- and nebulized capsaicin–induced increases in activity over baseline ( ) in the halothane-administered group were higher than that in the sevoflurane-administered group with the 2 MAC dose (Figures 2 and 3
; p = 0.01, drug effect; p < 0.05, Tukey's test). Dose-dependent increases in the C-fiber activity were observed for the halothane-administered group (p = 0.001, dose effect, ANOVA). By contrast, the nebulized bradykinin–induced increase in C-fiber activity () was the same in the sevoflurane- and halothane-administered animals (Figure 4
; p > 0.05).
This study provides new evidence from electrophysiologic recordings of laryngeal C-fiber impulse activities that inhalation of halothane into the isolated upper airway of young guinea pigs augments the responsiveness of C-fibers to three stimuli—the chemical stimulus of capsaicin (through both intravascular and aerosolized administration routes to the larynx) and the mechanical stimulus of laryngeal hyperinflation—to a greater degree than does inhalation of sevoflurane. By contrast, the exposure had no effect on C-fiber responsiveness to inhaled bradykinin or on the baseline activity. The question is why volatile anesthetics, particularly halothane, augmented the laryngeal C-fiber responsiveness specifically to left atrial and aerosolized capsaicin and to laryngeal hyperinflation but not to aerosolized bradykinin. First, why did the exposure augment the responsiveness to both intravascular and aerosolized capsaicin? One hypothesis is that halothane causes all C-fibers to undergo the same changes in excitability, making them generally more responsive to inhaled stimulants. In fact, in the halothane-administered animals, for the same C-fibers that were excited by both left atrial capsaicin and nebulized capsaicin at the 2 MAC dose (n = 8), the responses to left atrial capsaicin (7.1 ± 0.4 impulses/second) and nebulized capsaicin (8.2 ± 0.9 impulses/second; p > 0.05) were not different. The changes in excitability may have been due to changes in membrane properties or in transduction processes or indirectly caused by increases in microvascular leak. The internal branch of the SLN is the main source of laryngeal afferent activity, and the majority of the cell bodies of laryngeal afferents are located in the nodose ganglion (7). Regarding changes in membrane properties, in primary sensory neuron cell bodies in the nodose ganglia, allergen exposure has been shown to cause membrane depolarization, changes in resting membrane conductance, inhibition of inward rectifying currents, and blockade of a slow afterhyperpolarization current, all of which can render the nerve more sensitive to stimuli (19). That halothane did not cause any significant changes in bradykinin-induced C-fiber stimulation may not be surprising considering the fact that these two different stimuli activate C-fibers via different receptor signal transduction mechanisms. The bradykinin-induced stimulation of nodose C-fiber afferents is secondary to activation of bradykinin B2 type receptors (20), whereas capsaicin activates C-fibers by directly binding to the vanilloid Type 1 receptor, a ligand-gated nonselective cation channel (8, 21). An alternative possibility is that the effectiveness of aerosolized bradykinin was simply reduced by epithelium-derived peptidases or by diffusion barriers in the epithelial layer. This possibility seems less likely because the exposure also increased the responsiveness of C-fibers to the mechanical stimulus of laryngeal hyperinflation as well as to the chemical stimulus of capsaicin. The mechanism whereby halothane augmented C-fiber responsiveness to laryngeal hyperinflation is not known. However, the finding that the exposure to halothane increases the responsiveness to three different stimuli, one chemical (with two different administration routes) and one mechanical, suggests the induction of some neuroplastic changes in the nerve endings or in transduction processes that resulted in an overall increase in excitability. Finally, consideration should be given to airway reflexes after laryngeal exposure to volatile anesthetics as a cause of the enhanced C-fiber responsiveness. Evidence has been accumulated to suggest that a type of myelinated afferents from the larynx classified into rapidly adapting irritant receptors is thought to be responsible for cough and related reflexes (6), whereas nonmyelinated laryngeal C-fibers probably mediate apnea and bronchoconstriction (7, 8). Analysis of the laryngeal rapidly adapting irritant receptors were excluded from this study because our early results clearly indicated their lack of sensitivity to volatile anesthetics similar to the nature of the airway irritation for individual anesthetics (11; T. Mutoh and H. Tsubone, unpublished data). However in healthy guinea pigs, C-fibers are responsive to capsaicin and bradykinin, express vanilloid Type 1 receptor, and are the only nerve fibers expressing neurokinins both in the airways and in the central nervous system (20, 22, 23). Given that the neurokinin-containing C-fibers are carried primarily by the SLNs (24), we also speculate that the interactions between rapidly adapting irritant receptors and C-fiber afferents both at the receptor level and in the centrally mediated reflex pathway are involved in mediating the reflexes initiated from the larynx.
Clinical Implications
Supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Received in original form July 30, 2002; accepted in final form November 5, 2002
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ABSTRACT
METHODS
25°C) of air (eliminating "cold" receptors) applied to the isolated upper airway were also suspected to be C-fibers. Once a suspected C-fiber was identified, its conduction velocity was measured. To measure the conduction velocity, a stimulating electrode was positioned on the internal branch of the SLN at its exit from the laryngeal cartridge and a field stimulus was applied by delivering rectangular constant-current pulses (duration: 1 millisecond; intensity: 0.3–3.0 mA) generated by a pulse generator and a stimulus-isolation unit. At the end of the experiment, we identified intralaryngeal localization of the C-fibers by gently probing the laryngeal mucosa with a nylon thread or a thin polyethylene catheter inserted through the tracheal tube (
5%) of the inhalants with a single deep breath. It should be noted here that volatile anesthetics, in particular halothane, are likely to cause an increase in the baseline excitability of C-fibers (
