INTRODUCTION

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WOOD SMOKE GENERATED from a house fire or a fireplace has been recognized as a potent inhaled irritant to the respiratory tract (9, 15). The nasal cavity is the first site in the airways that can trigger powerful airway reflexes in response to inhaled irritants (3, 19, 30). However, the role of respiratory reflexes in protecting the lungs against nasal irritation by wood smoke has not been elucidated and the airway responses to nasal wood smoke have not been well defined.

Previous studies have demonstrated that protective or defensive airway reflexes, including apnea, sniff, and sneeze, are elicited when chemical irritants such as cigarette smoke, ammonia, or capsaicin are administered into the nasal cavity (3, 12, 14, 19, 30). It is known that the ophthalmic and maxillary divisions of the trigeminal nerves are the common afferent pathway for eliciting these nonolfactory airway reflexes (19, 30). Indeed, electrophysiological studies in various animal species reveal that many types of chemical irritants stimulate nasal sensory receptors, the afferent activity of which is conduced by trigeminal nerve fibers (19-22, 29, 30). Although the physiological properties of these irritant-sensitive nasal sensory receptors are not completely understood, several investigators have suggested the important role of trigeminal C-fiber sensory nerve endings in eliciting the reflex and receptor responses to nasal chemical irritants (12, 19, 20, 30). Nevertheless, the role of the trigeminal sensory pathway, especially unmyelinated C-fiber afferents, in triggering the airway reflexes during nasal exposure to wood smoke remains to be investigated.

In a closed space, incomplete combustion of wood yields smoke containing particulates and numerous irritant gases, which have been suggested to be potential irritants to the airways (9, 15). Furthermore, wood smoke contains high concentrations of free radicals and radical precursors, which may cause an increase in the oxygen radical burden in the airways after smoke exposure (17, 24). Among the major types of oxygen radicals, hydroxyl radical is an extremely reactive oxygen species (16) and is actively involved in eliciting airway reflexes when wood smoke is inhaled into the lower airways (5) or delivered through a functionally isolated larynx (10). Despite the possibility that various smoke constituents may be involved, the chemical factors contributing to the sensory irritation induced by nasal wood smoke are still unclear.

The objectives of the present study were 1) to characterize the airway reflexes evoked by nasal exposure to wood smoke in anesthetized rats and 2) to investigate the neural and chemical mechanisms underlying these smoke-induced responses. To study the neural mechanisms, we focused on the importance of the trigeminal sensory pathway and the role of unmyelinated C-fiber afferents. To study the chemical mechanisms, we concentrated on the relative contributions of the gas and particulate phases of wood smoke and the possible involvement of hydroxyl radical.

	METHODS

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Animals. A total of 58 adult male Sprague-Dawley rats were used in this study; 42 rats (wt 356 ± 9 g) were normal animals, and the remaining 16 (wt 338 ± 14 g) were animals that received neonatal treatments. For neonatal treatments, male rat pups newly born from seven mothers were evenly divided into two groups. On day 2 postpartum, one group of pups received a subcutaneous injection of capsaicin (50 mg/kg, Sigma), whereas the other group received the same amount of vehicle (10% ethanol, 10% Tween 80, and 80% saline). This capsaicin treatment has been shown to chronically and preferentially ablate unmyelinated C fibers and some A fibers (18). At the age of 3-4 mo, these neonatal treatment animals were subjected to a wiping test. For this purpose, a drop of 0.01% capsaicin solution was instilled into the right eye of these animals. The effectiveness of neonatal capsaicin treatment was confirmed by the result that the blepharospastic response to this local application displayed by the vehicle-treated rats was absent in the capsaicin-treated rats. All animals were housed in and purchased from the Animal Center of National Yang-Ming University, Taipei, Taiwan. All protocols were approved by the Committee of the National Science Council, Taipei, Taiwan.

General preparations. Animals were anesthetized with intraperitoneal injection of chloralose (100 mg/kg) and urethan (500 mg/kg). The femoral artery and the jugular vein were cannulated for recording arterial blood pressure and for 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 superior and recurrent laryngeal nerves were sectioned to minimize possible mechanical irritation on the upper airway during the experiment. Body temperature was maintained at ~36°C throughout the experiment by means of a servo heating blanket.

Functionally isolated nasal cavity. After the trachea was exposed, a tracheal cannula (polyethylene tubing; Clay Adams, PE-260) was inserted caudally just above the thoracic inlet, while an upper airway catheter (polyethylene tubing; Clay Adams, PE-206) was inserted cranially with its tip placed inside the nasopharynx. The outlet of the nostrils was covered by a plastic funnel, which allowed the air or smoke to flow out. The oral cavity was stuffed with small cotton balls and sealed to prevent any air leak. The position of the catheter tip at the nasopharynx was confirmed by autopsy after animals had been killed at the end of the experiment. During the experiment, rats breathed spontaneously via the lower tracheal cannula. Respiratory flow () was measured with a pneumotachograph (Fleisch 4/0) coupled with a differential pressure transducer (MP45-14, Validyne). The flow signal was integrated to give tidal volume (VT). Tracheal pressure (Ptr) was monitored by another differential pressure transducer (MP45-28, Validyne) via a side port of the lower tracheal cannula. The pneumotachograph was disconnected from the lower tracheal cannula after each test. All physiological signals were recorded on a chart recorder (model TA11, Gould) and a tape recorder (model DR-890, Neurocorder) for later analysis.

Generation of smoke. The electric furnace and the methods for generating wood smoke are described in detail in our previous study (4). Briefly, 100 g of dry wood dust (lauan wood) were thermally decomposed by the furnace at a core temperature maintained at 500 ± 8°C for 5 min, and the effluent smoke was collected in a 25-liter plastic balloon attached to the furnace outlet. Gas-phase smoke was generated by passing wood smoke through a standard glass-fiber Cambridge filter that removed >99% of the smoke particulates (6). Unfiltered smoke generated from this method contained ~2% O₂, 15% CO₂, 24% CO, and 25 mg/l particulates (4, 6). Gas-phase smoke contains similar concentrations of these gases but is free of particulates (6).

Nasal exposure to smoke. Immediately after thermal decomposition, fresh wood smoke or gas-phase smoke was withdrawn into a 20-ml syringe. The smoke, at a temperature of ~25°C, was continuously delivered at a constant flow rate of 1.4 ml/s by a syringe pump (model 367, Sage) into a section of 6-ml Teflon tubing (8 mm ID) connected to the proximal end of the upper airway catheter. The communication between the Teflon tubing and the upper airway catheter was quickly blocked by a three-way stopcock at the end of the smoke delivery. In each smoke challenge, the total amount of smoke delivered via the upper airway catheter was 11 ml; 5 ml of smoke passed through the isolated nasal cavity and flowed out to the environment via the nostrils, whereas the rest remained in the luminal space of the Teflon tubing for the rest of the experiment. To avoid contamination, the smoke that flowed out the nostrils was drawn into a fume hood via a suction line, and the syringe and its connecting Teflon tubing were replaced after each smoke delivery. Gas samples were drawn continuously (1.2 ml/s, transportation lag 0.5 s) via a sampling tubing placed near the nostrils and analyzed by a capnograph (model 9000, Biochem) for CO₂ concentration. Because the smoke contained a high CO₂ concentration (9, 11), the time when the smoke passed through the isolated nasal cavity was signaled by an abrupt increase in the CO₂ concentration (Fig. 1).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Experimental records illustrating immediate responses evoked by nasal exposure to wood smoke before (left) and 20 min after nasal application of dimethylthiourea (right; 500 mg/ml, 0.1 ml) in two anesthetized rats. A: animal responded to smoke challenge with apneic reflex at control. B: animal responded to smoke challenge with sniff-like reflex at control. Time between 2 smoke challenges was 30 min. CO2, CO2 concentration of gas sampled continuously from outlet of nostrils; , respiratory flow; VT, tidal volume; ABP, arterial blood pressure. Wood smoke (5 ml) was delivered through a functionally isolated nasal cavity at a constant flow rate of 1.4 ml/s. Time when smoke passed though nasal cavity was signaled by an abrupt increase in CO2 concentration. See text for further explanation.

Nasal application of pharmacological agents. To locally apply pharmacological agents to the nasal mucosa, 0.1 ml of solution containing either lidocaine hydrochloride (8%, Roxane Laboratories; a local anesthetic), dimethylthiourea (500 mg/ml dissolved in isotonic saline, Sigma; a hydroxyl radical scavenger), or capsaicin (400 µg/ml, Sigma) was introduced into the nasal cavity via the upper airway catheter. The effectiveness of the blocking effect of lidocaine was confirmed by the absence of any changes in breathing pattern to the mechanical probing of the nasal mucosa by a nylon thread (0.3 mm diameter). Conversely, the possible deleterious effects of dimethylthiourea were excluded by the persistence of changes in breathing pattern evoked by the same mechanical probing. Additionally, nasal application of capsaicin at this concentration has been reported to stimulate nasal C-fiber sensory nerve endings (20, 22).

Experimental procedures. In study 1, which used 42 normal rats, first the immediate ventilatory responses to delivery (1.4 ml/s) of air and then to delivery of 5 ml of wood smoke into the isolated nasal cavity were studied. Subsequently, animals were divided into five groups that received different experimental interventions. Nasal smoke challenges were repeated after denervation of trigeminal nerves (n = 11) or removal of smoke particulates (n = 10) and after nasal application of lidocaine (n = 6), dimethylthiourea (n = 9), or saline (n = 6; vehicle of dimethylthiourea). For trigeminal nerve denervation, the ophthalmic and maxillary divisions were identified within the orbits and sectioned after the eyeballs had been removed. Each animal received only two smoke challenges, and at least 30 min were allowed to elapse between the two smoke challenges to avoid possible tachyphylaxis. In these denervation tests, at least 30 min elapsed after nerve sectioning to obtain a stable breathing pattern for baseline before the second smoke challenge. In the nasal application tests, 20 min elapsed before the second smoke challenge. In study 2, eight neonatal capsaicin-treated rats and eight neonatal vehicle-treated rats were used. In both groups, first the apneic responses to intravenous injection of capsaicin (2 µg/kg) and to a maintained lung inflation (Ptr = 10 cmH₂0) were studied. The immediate ventilatory responses to delivery of air, delivery of 5 ml of wood smoke, and a subsequent nasal application of capsaicin were investigated. Between any two challenges, 30 min were allowed to elapse. In both studies 1 and 2, the animal's lungs were hyperinflated (Ptr > 25 cmH₂O) by a syringe for 2 s to establish a constant volume history before each test.

Data analysis and statistics. Inspiratory and expiratory duration (TE), respiratory frequency (f), VT, and were all analyzed on a breath-by-breath basis and were averaged in 1-s intervals. These respiratory parameters were measured at least 15 s before and 30 s after delivery of air or smoke. Mean arterial blood pressure and heart rate were measured at 1-s intervals. Baseline data for each respiratory and cardiovascular parameter were calculated as the mean over 10 s immediately before delivery. These physiological parameters were analyzed on a computer equipped with an analog-to-digital convertor (DASA 4600, Gould) and software (BioCybernatics 1.0). Results obtained from the computer analysis were routinely checked and compared with those obtained by manual calculation for accuracy. Between-group comparisons were evaluated by a Student's t-test. Within-group comparisons were evaluated by a paired t-test or a one-way repeated-measures ANOVA followed by Duncan's test when appropriate. P < 0.05 was considered significant. All data are presented as means ± SE.

	RESULTS

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Ventilatory responses in normal rats. Within 1 s after nasal exposure to wood smoke, either an apneic response (Fig. 1A, left) or a sniff-like response (Fig. 1B, left) was elicited in each of the 42 normal rats studied; 26 animals displayed the former response, whereas the other 16 exhibited the latter. The apneic breath had a prolongation of TE (3.71 ± 0.29 s) reaching 756 ± 60% (n = 26) of the baseline TE (0.49 ± 0.04 s) and resulting in a marked decrease in f (Fig. 2A). For this apneic breath, however, the accompanied VT was not significantly affected (Figs. 1A, left, and 2A). The sniff-like response was characterized by a single or several sets of one to three rapid and convulsive inspiratory efforts not followed by forced expirations (3) (Fig. 1B, left). Additionally, these large inspiratory efforts were usually preceded and/or followed by a rapid series of small inspirations (Fig. 1B, left). Consequently, both f and VT were vigorously affected (Figs. 1B, left, and 2B) and the average duration of the irregular breathing pattern reached 3.6 ± 0.5 s (n = 16). Subsequent to these immediate ventilatory responses, the breathing pattern became more regular, but it still took an additional 2-7 s before the breathing pattern returned to its baseline.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Mean ventilatory responses evoked by nasal exposure to air and to wood smoke in 42 animals studied. A: animals (n = 26) responded to smoke challenge with apneic reflex. B: animals (n = 16) responded to smoke challenge with sniff-like reflex. Wood smoke was delivered into the isolated nasal cavity during the time period between 2 vertical dotted lines. Data are means ± SE.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Average apneic responses evoked by nasal exposure to wood smoke before and after trigeminal nerve denervation (A; trig. cut), nasal application of lidocaine (B), removal of smoke particulates (C), nasal application of dimethylthiourea (D; DMTU), or nasal application of saline (E; vehicle of DMTU). Dotted lines indicate a ratio of 1. * Significantly different from control response, P < 0.05. Data are means ± SE; n, no. of animals tested. TE, expiratory duration.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Average durations of irregular breathing pattern in sniff-like response evoked by nasal exposure to wood smoke before and after trigeminal nerve denervation (A), nasal application of lidocaine (B), removal of smoke particulates (C), nasal application of DMTU (D), or nasal application of saline (E). * Significantly different from control response, P < 0.05. Data are means ± SE; n, no. of animals tested.

Ventilatory responses in neonatal treatment rats. In each of the neonatal vehicle-treated rats, an intravenous capsaicin injection instantly evoked a reflex apneic response that was essentially absent in every neonatal capsaicin-treated rat tested. The apneic ratio produced by this capsaicin injection in the neonatal vehicle-treated rats (5.50 ± 1.88; n = 8) was significantly greater than that in the neonatal capsaicin-treated rats (0.99 ± 0.02; n = 8, P < 0.05). Additionally, a maintained lung inflation (Ptr = 10 cmH₂O) evoked a reflex apneic response in both groups of the animals and the apneic ratio (18.85 ± 2.24; n = 8) measured in vehicle-treated rats was not significantly different from that (21.13 ± 2.15; n = 8, P > 0.05) measured in capsaicin-treated rats. Delivery of air into the isolated nasal cavity did not cause any detectable changes in breathing pattern in both groups of the neonatal treatment animals. In response to nasal challenges of wood smoke, six neonatal vehicle-treated rats immediately displayed an apneic response (Fig. 5A), whereas the other two instantly exhibited a sniff-like response. In the vehicle-treated rats displaying the apneic response, their f decreased from a baseline of 57 ± 2 breaths/min to a peak reduction of 22 ± 2 breaths/min (n = 6) and their VT was 2.1 ± 0.2 ml at baseline and 2.2 ± 0.2 ml after the smoke challenge. Contrastingly, nasal exposure to wood smoke only evoked a mild apneic response in two capsaicin-treated rats and did not affect the breathing pattern in the other six (Fig. 5B). In these capsaicin-treated rats, their f was 65 ± 8 breaths/min at baseline and 58 ± 10 breaths/min (n = 8) after the smoke challenge and their VT was 2.0 ± 0.1 ml at baseline and 2.0 ± 0.2 ml after the smoke challenge. As a result, the average apneic ratio (1.25 ± 0.17; n = 8) measured in the capsaicin-treated rats was significantly smaller (P < 0.05) than that measured in the vehicle-treated rats (4.20 ± 0.52; n = 6). Finally, in the vehicle-treated rats, nasal application of capsaicin evoked an apneic response (apneic ratio = 8.40 ± 2.98; n = 8) that was significantly greater (P < 0.05) than that (apneic ratio = 2.26 ± 0.32; n = 8) displayed by the capsaicin-treated rats (Fig. 5).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Experimental records illustrating immediate responses to nasal exposure to wood smoke and to nasal application of capsaicin in 1 neonatal vehicle-treated rat (A) and in 1 neonatal capsaicin-treated rat (B). At top, arrows indicate time when capsaicin (400 µg/ml, 0.1 ml) was introduced into nasal cavity. Thirty minutes elapsed between smoke and capsaicin challenges. Note that smoke challenge provoked apneic response in vehicle-treated rat but had no effect on breathing in capsaicin-treated rat. See legend of Fig. 1 for further explanation.

Cardiovascular responses. In control rats, nasal wood smoke did not significantly affect the mean arterial blood pressure and heart rate. In normal rats (n = 42), mean arterial blood pressures before and after the nasal smoke challenge were 116 ± 2 and 118 ± 3 mmHg (P > 0.05), respectively, and mean heart rates were 347 ± 9 and 337 ± 9 beats/min (P > 0.05), respectively. In neonatal treatment rats (n = 16), mean arterial blood pressures before and after the nasal smoke challenge were 88 ± 10 and 89 ± 10 mmHg (P > 0.05), respectively, and mean heart rates were 331 ± 14 and 328 ± 13 beats/min (P > 0.05), respectively.

	DISCUSSION

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The results of this study demonstrate that delivery of a small amount (5 ml) of wood smoke through a functionally isolated nasal cavity of anesthetized rats immediately evoked either an apnea or a sniff-like response. These two airway responses had distinct characteristics, occurred abruptly after nasal exposure to wood smoke, and were reproducible. Additionally, both airway responses were totally abolished by denervation of the ophthalmic and maxillary divisions of the trigeminal nerves or by local application of lidocaine confined to the nasal mucosa. Together, our observations suggest that the apneic and sniff-like responses are reflexes resulting from the stimulation of nasal sensory receptors whose activity is conducted by trigeminal afferents. The former response is thought to be a protective reflex that may prevent the subsequent irritant insult, whereas the latter is believed to be a defensive reflex that may flush irritants away from the site of irritation (3). Because changes in transmural pressure and flow can stimulate nasal sensory receptors and affect breathing (19, 21, 25, 30), it is possible that the reflex responses we observed were due to the flow delivered. This possibility is excluded by the present finding that delivery of air into the nasal cavity 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 these airway reflexes following nasal smoke exposure.

It is known that the nasal mucosa is richly innervated with trigeminal sensory receptors, many of which are sensitive to chemical irritants (19, 30). Although the specific types of nasal sensory receptors that initiated the smoke-induced airway reflexes remain to be identified, an attempt using neonatal capsaicin treatment has been made in this study to assess the importance of unmyelinated C-fiber afferents. This is because treatment of neonatal rats with capsaicin, a chemical that has preferential actions on C fibers, causes an irreversible degeneration of unmyelinated C fibers, yet it produces very little damage on small myelinated A fibers in adulthood (18). In this study, neonatal capsaicin treatment selectively impaired the reflex apneic response to intravenous injection of capsaicin and to nasal application of capsaicin but had no effects on the reflex apneic response to a maintained lung inflation. The former two apneic responses are known to result from stimulation of pulmonary and nasal C-fiber afferents (2, 19, 30), whereas the latter is thought to be mediated by pulmonary stretch receptors (myelinated afferents) (2). In these same neonatal capsaicin-treated animals, nasal wood smoke evoked a very mild apneic response in only 25% of the animals studied and had no effects on breathing in the remaining 75%. These contrast to the findings that nasal wood smoke evoked an apneic response in 75% of the neonatal vehicle-treated animals and triggered a sniff-like response in the remaining 25%. Hence, the inability of the neonatal capsaicin-treated rats to exhibit an apneic reflex or a sniff-like reflex may be attributable to the effects of capsaicin treatment. Because neonatal capsaicin treatment preferentially ablates C and A fibers (18), these results suggest that trigeminal C-fiber and A-fiber afferents play an important role in eliciting both the apneic and sniff-like reflexes during nasal exposure to wood smoke. Indeed, an electrophysiological study in guinea pigs (20) demonstrated that 53% of the 36 ethmoidal sensory receptors recorded are activated by nasal applications of low doses of capsaicin that stimulate C-fiber afferents. A reflex study in guinea pigs (12) reported that sneezing, another powerful airway reflex evoked by nasal irritants, can be prevented by nasal applications of, and by systemic pretreatment with, high doses of capsaicin, which impair the functions of C-fiber nerve endings. Histological investigations (13, 26) indicated an abundant distribution of substance P-containing C-fiber nerve endings in the nasal mucosa of various species, including rats. A quantitative electron microscopic study of the anterior ethmoidal nerve, a branch of the ophthalmic division in cats (28), showed that the number of unmyelinated fibers is about six times greater than that of myelinated fibers. Thus our results are in good agreement with these observations that trigeminal C-fiber sensory nerve endings are important in eliciting the reflex and receptor responses to nasal chemical irritants.

Neither the apneic response nor the sniff-like response to nasal wood smoke was altered by nasal application of a saline vehicle. This observation suggests that the airway reflexes in response to nasal wood smoke were consistent in each rat but varied among the animals studied. Similar variation has been described in the immediate reflex responses to inhalation of wood smoke into the lower airways (4). When wood smoke is inhaled via a tracheostomy into the rat's lung, it reflexly triggers either an apnea or an augmented inspiration (4). However, the airway responses to nasal wood smoke are mainly mediated through trigeminal C-fiber sensory nerve endings, whereas the airway responses to inhalation of wood smoke into the lower airways are mediated through both lung C-fiber sensory nerve endings and irritant receptors (6-8). The mechanisms underlying the variation of the airway responses observed in this study are not clear. The predisposition of the evoked response could be due to a preferential selection by the central respiratory controller.

We found that removal of smoke particulates did not affect either the apneic responses or sniff-like responses to nasal wood smoke, suggesting that the gas-phase smoke is responsible for triggering these reflexes. Additionally, local application of dimethylthiourea, an effective scavenger for hydroxyl radical, to the nasal mucosa totally abolished these two smoke-induced immediate responses, indicating that hydroxyl radical is actively involved in eliciting these airway reflexes. The source of the hydroxyl radical remains unclear, but wood smoke may be one possible origin (24). The gas phase of wood smoke is known to contain high concentrations of free radicals and radical precursors that are formed during combustion (17, 24). These free radicals and their precursors may continuously generate oxygen radicals either in the smoke or on reaching the airways (17). However, the particulate phase of wood smoke also contains free radicals (17), but it did not contribute to the airway reflexes observed in this study. The difference in the contributions of gas and particulate phases is not clear but may be due to the fact that the radical chemistry of the gas-phase smoke is quite different from that of the particulate phase (17). After its generation, the hydroxyl radical may possibly participate in the process of stimulation of nasal sensory receptors and trigger the resultant airway reflexes observed in this study. This notion is supported by the results from our electrophysiological studies in rats (7, 8), wherein we demonstrated that pretreatment with dimethylthiourea markedly attenuated the stimulation of both lung irritant receptors and vagal pulmonary C fibers induced by inhaled wood smoke. Indeed, several investigators have proposed that hydroxyl radical can stimulate sensory nerve endings located in the gastrointestinal tracts (23), the heart (27), and the lower airways and lungs (1) under various experimental conditions.

It is interesting to compare the above-mentioned smoke effects on the nasal cavity with those on other parts of the airways. Laryngeal exposure to wood smoke triggers an apnea that results from the stimulation of superior laryngeal afferents by the gas-phase smoke (10). In contrast to the situation in the nasal cavity, this apneic reflex does not depend on laryngeal C-fiber afferents (11). Lower airway exposure to wood smoke evokes airway reflexes that originate from the stimulation of both lung vagal C-fiber nerve endings and irritant receptors (myelinated afferents) by the gas-phase smoke and/or smoke particulates (4, 6-8). It seems that wood smoke irritates different levels of the airways and provokes airway reflexes involving different types of afferents (unmyelinated or myelinated) and smoke components (gas phase or particulates). Furthermore, it is amazing that hydroxyl radical serves as the common chemical mechanism that underlies the sensory irritation at various airway levels by the insult of wood smoke.

In summary, the apneic and sniff-like responses evoked by nasal exposure to wood smoke are reflexes resulting mainly from the stimulation of trigeminal nasal C-fiber and A-fiber afferents by the gas phase of wood smoke, and hydroxyl radical is the chemical factor responsible for evoking these airway reflexes.