Journal of Applied Physiology
Vol. 83, No. 3, pp. 723-730, September 1997
CONTROL OF BREATHING, CIRCULATION, AND TEMPERATURE

Reflex apneic response evoked by laryngeal exposure to wood smoke in rats: neural and chemical mechanisms

Y. S. Lin and Y. R. Kou

Institute of Physiology, School of Medicine and Life Science, National Yang-Ming University, Taipei, Taiwan 11221, Republic of China

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Lin, Y. S., and Y. R. Kou. Reflex apneic response evoked by laryngeal exposure to wood smoke in rats: neural and chemical mechanisms. J. Appl. Physiol. 83(3): 723-730, 1997.We investigated the neural and chemical mechanisms contributing to the immediate ventilatory responses to laryngeal exposure to wood smoke in anesthetized Sprague-Dawley rats. Five milliliters of wood smoke were delivered into a functionally isolated larynx at a constant flow rate of 1.4 ml/s while the animals breathed spontaneously. Within 1 s after exposure, laryngeal wood smoke consistently triggered an apnea in each of the 42 rats tested. The apneic duration reached 1,636.4 ± 105.4 (SE) % (n = 42) of the baseline expiratory duration. This apneic response was not affected by denervation of recurrent laryngeal nerves (n = 6) or by removal of smoke particulates (n = 14), but it was totally eliminated by topical application of an anesthetic (n = 8; lidocaine hydrochloride, 8%) to the laryngeal mucosa or by sectioning of the superior laryngeal nerves (n = 42). Furthermore, laryngeal application of a hydroxyl radical scavenger (dimethylthiourea; 500 mg/ml; n = 8) greatly diminished or abolished the smoke-induced apneic response, but it did not affect the apneic response evoked by laryngeal exposure to air saturated with 6% ammonia. These results suggest that the immediate apneic response to laryngeal wood smoke is a reflex resulting from the stimulation of the superior laryngeal afferents by the gas phase of wood smoke and that the stimulation is mediated through a hydroxyl radical mechanism.

laryngeal irritation; gas phase; particulates; hydroxyl radicals; dimethylthiourea; ammonia

INTRODUCTION

INHALATION OF WOOD SMOKE generated by a house fire or a campfire immediately causes airway irritation in humans. Wood smoke has been recognized as a potent inhaled irritant to the respiratory tracts (1, 9, 18). However, the role of respiratory reflexes in protecting the lungs against the laryngeal assault by wood smoke has not been fully elucidated.

The laryngeal sensory innervation is provided by the superior (SLNs) and recurrent laryngeal nerves (RLNs) (22, 28). Previous studies have demonstrated that airway reflexes are elicited when irritants such as ammonia (4, 17, 19), capsaicin (19, 26), cigarette smoke (4, 14), or distilled water (2) are administered into the larynx. The airway reflexes elicited by laryngeal irritants may include apnea, bradypnea, cough, and expiration reflex (22, 28). It is known that the SLNs are the common afferent pathway for eliciting these airway reflexes (22, 28). The chemical factors involved in these airway reflexes, however, vary according to the nature of laryngeal irritants. For example, a lack of chloride or other permeant anions contributes to the reflex apneic and laryngeal sensory receptors to water instilled in the laryngeal lumen (2, 22). The ability of laryngeal ammonia (4, 17, 19) or capsaicin (19, 26) to evoke airway reflexes presumably arises from their chemical effects on the laryngeal afferent nerve endings. The constituent of cigarette smoke responsible for evoking the reflex bradypneic and laryngeal afferent responses is not known (14, 15).

It has been shown that, during several minutes of inhalation of wood smoke from an exposure chamber, conscious mice (1) or guinea pigs (29) exhibit a sustained decrease in respiratory rate. Stimulation of trigeminal sensory receptors located in the nasopharynx has been postulated to be responsible for the smoke-induced bradypnea observed in these studies (1, 29). Conversely, when wood smoke is inhaled via tracheostomy (bypassing the larynx) into the lower airways in anesthetized rats, it immediately evokes a slowing of respiration or an augmented breath, both of which are mediated through vagal afferents (9-11). Accordingly, the respiratory reflexes evoked by laryngeal exposure to wood smoke have not been well defined, and the afferent pathways involved in triggering these reflex responses remain to be investigated.

In a closed space, incomplete combustion of wood yields smoke containing particulates and numerous irritant gases (18), which have been suggested to be potential irritants to the airways (6, 11, 17, 28). Furthermore, wood smoke contains high concentrations of free radicals and radical precursors, which may cause an increase in oxygen radical burden in the airways after smoke inhalation (21). Among the major types of oxygen radicals, hydroxyl radical (· OH) is an extremely reactive oxygen species (20) and is actively involved in eliciting respiratory reflexes when wood smoke (10) or cigarette smoke (13) is inhaled into the lower airways. Despite the possibility that various smoke constituents may be involved, the chemical factors contributing to the respiratory reflexes evoked by laryngeal wood smoke are still unclear.

The purposes of the present study were 1) to determine the immediate ventilatory responses to laryngeal exposure to wood smoke in anesthetized rats, 2) to assess the importance of the SLNs and RLNs in evoking these responses, 3) to study the relative contribution of the gas-phase smoke and smoke particulates to these responses, and 4) to investigate the involvement of · OH in eliciting these responses.

METHODS

Adult Sprague-Dawley rats (weight 310 ± 7 g) of either sex were anesthetized with -chloralose (100 mg/kg ip) and urethan (500 mg/kg ip). The femoral artery and vein were cannulated for recording arterial blood pressure and for intravenous administration of pharmacological agents, respectively. During the experiment, the depth of anesthesia was regularly monitored at fixed intervals; supplemental doses of the anesthetics were administered intravenously whenever necessary to maintain abolition of the pain reflex induced by pinching the animal's tail. The animal was tethered in a supine position, the neck was opened in the midline, and the esophagus was ligated as rostrally as possible. The SLNs and/or RLNs were isolated carefully for later sectioning. Body temperature was maintained at ~36°C throughout the experiment by means of a servo heating blanket.

RESULTS

Within 1 s after laryngeal exposure to wood smoke, an apneic response was elicited in each of the 42 rats studied (Fig. 1A). The apneic breath had a prolongation of TE, reaching 8.9 ± 0.7 s or 1,636.4 ± 105.4% (n = 42) of the baseline TE and resulting in a marked decrease in f (Fig. 2). For this apneic breath, the accompanied VT also decreased (Fig. 2). After the immediate apneic response, it took 6-18 breaths for both f and VT to return to their baseline values (Fig. 2). During this ensuing bradypneic period or just a few breaths afterward, a delayed augmented breath took place in 33 of the animals tested (Fig. 1A). The augmented breath was characterized by a two-step inspiratory flow, leading to an exceedingly large VT (Fig. 1A). In contrast to the smoke effect, delivery of air or a gas mixture containing 2% O₂-15% CO₂-24% CO-balance N₂ at the same flow rate into the isolated larynx did not cause any detectable change in breathing pattern (Fig. 2).

In one group of six rats, two additional challenges of laryngeal wood smoke were repeated to study the reproducibility of the responses. On average, the immediate apneic response (apneic ratio, 1,951.7 ± 321.5 and 1,475 ± 339.1%; n = 6) evoked by each of the additional wood smoke challenges was not significantly different from that (apneic ratio, 1,774.7 ± 378.9%; n = 6) evoked by the first challenge. In a second group of six rats, the RLNs were cut before challenges of laryngeal wood smoke were repeated to assess their involvement. Denervation of RLNs generally did not affect the baseline f and VT (Fig. 3A, Table 1) and did not alter either the amplitude or the time course of the ventilatory responses to laryngeal wood smoke (Fig. 3A). On average, the immediate apneic response (apneic ratio, 1,608.3 ± 240.9%; n = 6) evoked by laryngeal wood smoke after denervation of RLNs was not significantly different from that (apneic ratio, 1,718.3 ± 224.7%; n = 6) before denervation.

Table  1.   Effects of various experimental interventions on baseline respiratory parameters n Respiratory Frequency, breaths/min Tidal Volume, ml RLN denervation 6   Before RLN Cut 69.9 ± 4.4 1.4 ± 0.1   After RLN Cut 72.8 ± 2.9 1.6 ± 0.2 Lidocaine application 8   Before lidocaine 76.5 ± 5.9 1.8 ± 0.2   After lidocaine 79.3 ± 4.6 1.7 ± 0.1 DMTU application 8   Before DMTU 64.1 ± 7.3 1.4 ± 0.1   After DMTU 67.9 ± 8.3 1.4 ± 0.1 SLN denervation 42   Before SLN Cut 72.8 ± 3.3 1.8 ± 0.1   After SLN Cut 70.6 ± 2.9 1.8 ± 0.1 Values are means ± SE over 10 breaths immediately before smoke exposure; n, no. of animals. RLN, recurrent laryngeal nerve; Cut, denervation; DMTU, dimethylthiourea; SLN, superior laryngeal nerve. No statistical significance (P > 0.05) was found in any comparison of values before and after intervention.

In a third group of eight rats, lidocaine was topically applied to the laryngeal mucosa to elucidate the role of laryngeal sensory nerve endings. Twenty minutes after lidocaine application, baseline f and VT did not change significantly (Fig. 1B, Table 1). However, a repeated challenge of laryngeal wood smoke no longer evoked any change in breathing pattern in each of the rats tested (Fig. 1B) (apneic ratio before vs. 20 min after lidocaine, 1,881.1 ± 272.8 vs. 99.0 ± 4.4%; n = 8). Two hours after lidocaine application, the smoke-induced ventilatory responses reappeared (Fig. 1C), and the immediate apneic response partially recovered to 58.6% of the control response. In a fourth group of 14 rats, delivery of the gas phase of wood smoke into the isolated larynx evoked ventilatory responses of very similar amplitude and time course compared with the responses evoked by unfiltered wood smoke in the same animals (Fig. 3B). On average, the immediate apneic response (apneic ratio, 1,289.1 ± 157.1%; n = 14) to laryngeal gas-phase smoke was not significantly different from that (apneic ratio, 1,398.0 ± 112.6%; n = 14) to laryngeal unfiltered smoke.

In a fifth group of eight rats, DMTU was topically applied to the laryngeal mucosa to assess the involvement of · OH. Before DMTU application, laryngeal exposure to 5 ml of air saturated with 6% ammonia immediately evoked ventilatory responses similar to those evoked by laryngeal wood smoke in the same animals (Fig. 4). Thirty minutes after DMTU application, baseline f and VT did not change significantly (Fig. 4, Table 1). However, the ventilatory responses to laryngeal wood smoke were totally eliminated in four rats (Fig. 4A) and largely attenuated in the other four. As a result, the average apneic response induced by laryngeal wood smoke was markedly diminished by DMTU application (Fig. 5A). In contrast, the ventilatory responses to laryngeal ammonia were not affected by DMTU application (Fig. 4B), and the average apneic response evoked after DMTU was not significantly different from that before DMTU (Fig. 5B).

Before the end of the experiment, all 42 rats underwent denervation of SLNs. Thirty minutes after denervation of SLNs, baseline f and VT did not change significantly (Fig. 2; Table 1). The immediate apneic response and the following changes in breathing pattern evoked by laryngeal exposure to wood smoke (Figs. 1D, 2, 4A, and 5A), gas-phase smoke, or ammonia vapor (Figs. 4B and 5B) were completely abolished by sectioning the SLNs.

During control, laryngeal wood smoke generally caused a transient (<9 s) and mild hypertension and bradycardia, which began 1-4 s after smoke exposure (Figs. 1, 3, and 4A). For the group (n = 42), mean arterial blood pressure increased from a baseline of 102.9 ± 3.1 to a peak of 119.5 ± 3.6 mmHg, whereas heart rate decreased from a baseline of 359.7 ± 8.0 to a peak reduction of 294.0 ± 13.1 beats/min. These smoke-induced immediate cardiovascular responses were totally blunted by denervation of SLNs; the changes in mean arterial blood pressure and heart rate after smoke exposure reached only 101.9 ± 0.6 and 99.1 ± 0.1%, respectively, of their baseline values.

DISCUSSION

The results of this study demonstrate that delivery of a small amount (5 ml) of wood smoke through a functionally isolated larynx immediately caused a prominent inhibition of respiration. The immediate apneic response occurred abruptly after laryngeal exposure to wood smoke in each rat tested and was reproducible. Additionally, the apneic response was totally abolished by denervation of SLNs but was unaffected by denervation of RLNs. Furthermore, the apneic response was completely prevented by topical application of lidocaine confined to the laryngeal mucosa but reappeared when the effects of lidocaine gradually wore off. Taken together, these observations suggest that the immediate apnea is a reflex response resulting from stimulation of laryngeal sensory receptors whose activity is conducted by SLNs. Because changes in transmural pressure and flow can stimulate laryngeal sensory receptors and produce an inhibition of respiration (22, 28), it is possible that the reflex apneic response we observed was due to the flow delivered into the laryngeal segment. This possibility is excluded by the present finding that delivery of air into the larynx at the same flow rate did not cause any detectable changes in breathing pattern. Thus it is clear that wood smoke was the causative stimulus responsible for evoking the reflex apnea after laryngeal smoke exposure.

In this study, no attempt was made to identify which type(s) of laryngeal sensory receptors was responsible for eliciting the smoke-induced apneic response. The reflex apnea evoked by laryngeal cigarette smoke (4), capsaicin (19, 26), or ammonia (4, 17, 19) is thought to be a result originating from the stimulation of laryngeal irritant-sensitive nerve endings, laryngeal C-fiber nerve endings, or both. Because the pattern of the immediate apnea evoked by laryngeal wood smoke is very similar to that evoked by these laryngeal irritants (4, 17, 19, 26), it is not known whether the wood smoke-induced apneic response results from the stimulation of one or both types of laryngeal sensory receptors. When wood smoke is inhaled directly into the lower airways via tracheostomy, it stimulates both lung irritant receptors and vagal C fibers, thus triggering the corresponding respiratory reflexes (11, 12).

We found that removal of smoke particulates did not affect the reflex apneic response to laryngeal wood smoke, suggesting that the gas-phase smoke is responsible for triggering this response. Because of combustion, the gas-phase smoke contained a low concentration of O₂ and high concentrations of CO and CO₂ (9, 11, 18); the latter has been reported to reflexly produce an inhibition of respiration in cats (3). However, the role of these gases in eliciting the apneic response after smoke exposure is doubtful because delivery into the isolated larynx of a gas mixture containing gas concentrations matching those in the wood smoke generated in this study did not cause any measurable change in breathing pattern. The discrepancy between the effect of laryngeal CO₂ in rats and in cats may be, at least in part, because of the species differences.

We demonstrate that topical application of DMTU, an effective · OH scavenger (8), to the laryngeal mucosa greatly diminished or abolished the reflex apneic response to laryngeal wood smoke, suggesting that · OH is actively involved in eliciting this response. This observation is consistent with previous findings from this laboratory that the respiratory responses originating from the lower airways after inhalation of wood smoke can be largely attenuated by pretreatment with antioxidants for · OH (10). The source of the · OH remains unclear, but wood smoke may be one possible origin. The gas phase of wood smoke is known to contain high concentrations of free radicals and radical precursors that are formed during combustion (21). These free radicals and their precursors may continuously generate · OH either in the smoke or on reaching the airways (21, 23). The other possible origin is that · OH may be formed and released endogenously by certain lung cells when they are activated by smoke inhalation (25). On the other hand, the particulate phase of wood smoke also contains free radicals (21) but did not contribute to the reflex apnea observed in this study. It is not clear whether the difference in the contribution of gas and particulate phases to the apneic response is because of the dissimilarity in the type, concentration, and lifetime of · OH formed by these two smoke components. However, it is known that the radical chemistry of the gas-phase smoke is quite different from that of the particulate phase (21).

Because the immediate apneic response evoked by laryngeal wood smoke may be a respiratory reflex resulting from stimulation of laryngeal sensory receptors, it is plausible that · OH may participate in the process of excitation of sensory nerve endings. This hypothesis is supported by the preliminary results from our electrophysiological study in rats (12), wherein it was demonstrated that pretreatment with DMTU markedly attenuated the stimulation of both lung irritant receptors and vagal pulmonary C fibers induced by inhaled wood smoke. A similar hypothesis has also been proposed recently by several investigators who demonstrate that pretreatment with antioxidants for · OH diminished or abolished the stimulation of sensory nerve endings located in the gastrointestinal tracts (24), heart (27), and the lower airways and lungs (5) under various experimental conditions. The mechanisms by which · OH is associated with activation of laryngeal sensory receptors by wood smoke are not known. It is possible that · OH may directly stimulate these sensory nerve endings, but their direct effects on neural functions are still obscure. · OH may also be involved in the local release of chemical mediators (16) that, in turn, may stimulate laryngeal sensory receptors. Whatever the mechanisms, lowering the · OH burden by DMTU does not seem to affect the reflex responses evoked by other chemical stimulation of laryngeal sensory receptors. As demonstrated in this study, the reflex apneic response evoked by laryngeal ammonia was not altered by pretreatment with DMTU. This observation suggests that the suppressive effect of DMTU on the reflex apneic response to laryngeal wood smoke is not likely because of anesthetic or deleterious effects on laryngeal sensory receptors and that the mechanism of sensory activation induced by laryngeal wood smoke may be different from that induced by laryngeal ammonia.

The immediate apneic response evoked by laryngeal wood smoke was always followed by a bradypnea. Additionally, during this bradypneic period or just a few breaths afterward, a delayed augmented breath was evoked in 78% of the animals studied. These changes in breathing pattern were also reduced or prevented by denervation of SLNs and by laryngeal application of lidocaine or DMTU, suggesting that they are mediated through neural and chemical mechanisms similar to those responsible for triggering the immediate apnea. However, although the bradypnea has been well documented as a reflex response to laryngeal irritants (17, 19, 22, 28), the augmented breath seems to be uncommon. Rather, the augmented breath is known as an airway reflex resulting from activation of irritant receptors located in the lower airways (6, 11). Stimulation of laryngeal sensory receptors has been shown to produce reflex bronchoconstriction (22, 28), which can activate lung-irritant receptors (6). Accordingly, it is plausible that the delayed augmented breath may be a vagally mediated response secondary to the stimulation of laryngeal sensory receptors by wood smoke. After smoke exposure, laryngeal wood smoke generally induces hypertension and bradycardia. The fact that these cardiovascular responses are totally eliminated by laryngeal application of lidocaine or by denervation of SLNs indicates that they are also part of the reflex responses evoked by laryngeal wood smoke.

In summary, the immediate apneic response evoked by laryngeal wood smoke is a reflex resulting from the stimulation of superior laryngeal afferents by the gas phase of wood smoke, and an increase in · OH burden in the larynx is responsible for evoking this reflex. In our animal model, the smoke-induced apneic response may possibly be potentiated by anesthesia, a result that is presumably due to depression of the arousal system (7, 28). The apnea elicited by laryngeal irritants has been suggested as an airway-protective reflex (17, 22, 28). In addition to its irritant effects, inhaled wood smoke has been shown to produce lung injury (25, 30). Although · OH has been strongly implicated in the pathogenesis of inhalation injury (25, 30), the observations made in this study provide evidence to support the notion that laryngeal sensory receptors are capable of detecting an increase in · OH burden in the upper airway immediately after smoke exposure, which in turn elicits the resultant protective airway reflexes.

ACKNOWLEDGEMENTS

We are grateful to Dr. L.-Y. Lee for valuable comments on the manuscript and to Al Vendooris for English language consultancy.

FOOTNOTES

Address for reprint requests: Y. R. Kou, Institute of Physiology, School of Medicine and Life Science, National Yang-Ming Univ., Shih-Pai, Taipei, Taiwan 11221, Republic of China.

Received 13 February 1997; accepted in final form 7 May 1997.

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