INTRODUCTION

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WOOD SMOKE generated from a fireplace or a house fire is a potent inhaled irritant (17). Inhalation of wood smoke immediately causes airway irritation in humans and laboratory animals (2, 14, 17, 27). However, the neural and chemical mechanisms contributing to the smoke-induced sensory irritation of the airways are not fully understood.

We recently reported that inhalation of wood smoke via tracheostomy immediately affects breathing in anesthetized rats (14-16). A slowing of respiration is the major immediate ventilatory response occurring within the first two breaths of smoke inhalation. This ventilatory response is preserved when the conduction of myelinated fibers is differentially blocked by cooling of both vagi to 6.7°C but is totally abolished when the conduction of unmyelinated C fibers is selectively blocked by perineural capsaicin treatment of cervical vagus nerves (16). Additionally, this immediate ventilatory response is not affected by removal of smoke particulates (16) but is largely attenuated or eliminated by pretreatment with antioxidants of hydroxyl radical (· OH) (15). · OH is an extremely reactive oxygen metabolite formed in the lung tissues after inhalation of wood smoke (22, 27). Lung vagal C-fiber afferents are known to be sensitive to a variety of inhaled irritants and chemical mediators (6). Taken together, the results obtained from our reflex studies suggest that lung vagal C fibers are stimulated by the gas phase of wood smoke and that · OH is primarily involved in this afferent stimulation. However, direct evidence provided by electrophysiological studies remains to be established.

In addition to oxygen-reactive metabolites, inhalation of toxic smoke generated from combustion of materials is known to cause an increase in the release of other chemical mediators, including cyclooxygenase products (10, 13, 25). When the cyclooxygenase pathway is activated, arachidonic acid is metabolized to various types of prostaglandins and thromboxane (1). Cyclooxygenase products, either produced endogenously or administered exogenously, have been shown to stimulate lung vagal C fibers (5, 6, 11). However, whether these arachidonate metabolites are involved in the activation of lung vagal C fibers after smoke inhalation is not yet known.

In this study, we recorded afferent activity arising from vagal pulmonary C fibers in anesthetized rats to determine 1) whether these sensory nerve endings are stimulated by delivery of wood smoke into the lungs, 2) whether the gas phase of wood smoke is responsible for this afferent stimulation, and 3) whether · OH and cyclooxygenase products are involved in this afferent stimulation.

	METHODS

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Sprague-Dawley rats (weight 334 ± 6 g) of either sex were anesthetized with intraperitoneal injection of chloralose (100 mg/kg) and urethan (500 mg/kg). A polyethylene catheter was inserted into the jugular vein and advanced until the tip was close to the right atrium for intravenous administration of pharmacological agents. The right femoral artery was cannulated for measuring arterial blood pressure. During the course of the experiments, supplemental doses of chloralose (20 mg · kg¹ · h¹) and urethan (100 mg · kg¹ · h¹) were administered to maintain abolition of pain reflexes induced by pinching the animal's tail. During the recording of vagal action potentials, the rats were paralyzed with pancuronium bromide (0.05 mg/kg iv; Orgnon Teknika). Periodically, the effect of pancuronium was allowed to wear off so that the depth of anesthesia could be checked.

The rats were tethered in a supine position, and the trachea was cannulated below the larynx with a short tracheal tube via a tracheotomy. A midline thoracotomy was performed, and the edges of the rib cage were retracted. The lungs were ventilated by a rodent respirator (model 683, Harvard) at a constant volume of 2 ml. The frequency of the respirator was set at 65-75 breaths/min and was kept constant in each experiment. The expiratory outlet of the respirator was placed under 3-4 cm of water to maintain a near-normal functional residual capacity. Tracheal pressure (Ptr; transpulmonary pressure in an open-chest preparation) was monitored by a pressure transducer (MP45-28, Validyne) via a side tap of the tracheal cannula. Body temperature was maintained at ~36°C throughout the experiment by means of a servo-controlled heating blanket.

Recording of afferent activity of vagal pulmonary C fibers. Afferent activity arising from pulmonary C fibers was recorded by using techniques described elsewhere (19). Briefly, a fine afferent filament was split from the desheathed nerve trunk of the right vagus and placed on a platinum-iridium recording electrode. Action potentials were amplified (P511K, Grass), monitored by an audio monitor (AM8, Grass), and displayed on an oscilloscope (model 420, Gould). The fine nerve filament was subdivided until activity from only one or two units was obtained. All physiological signals were recorded simultaneously by a thermal array recorder (TA11, Gould) and also recorded on tape (DR-890, Neurocorder) for later analysis.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Afferent responses of a vagal pulmonary C fiber to lung inflation (A), capsaicin (B), unfiltered wood smoke (C), and gas-phase smoke (D). A: lungs were hyperinflated to 4 times tidal volume. B: capsaicin (1 µg/kg) was injected into catheter (1st arrowhead) and flushed into vein (2nd arrowhead). C and D: 6 ml of unfiltered smoke or gas-phase smoke were delivered into lungs in 3 ventilatory cycles as indicated by horizontal bars. Twenty minutes elapsed between capsaicin injection and smoke delivery, and 30 min elapsed between 2 smoke deliveries. C fiber had a conduction velocity of 1.5 m/s. Ptr, tracheal pressure; AP, action potential; ABP, arterial blood pressure.

Generation and delivery of smoke. The electric furnace and the methods for generating wood smoke are described in detail in our previous study (14). In brief, 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 the wood smoke through a standard grass-fiber Cambridge filter, which removed >99% of the smoke particulates (16). The smoke was sampled and analyzed for its O₂ (OM-11, Beckman), CO₂ (LB-2, Beckman), CO (model 961, Neotronics), and particulate (P-5H2, Sibata) concentrations. Unfiltered smoke generated by this method contains ~1.5% O₂-15% CO₂-24% CO, and 25 mg/l particulates (14, 16). The gas-phase smoke contains similar concentrations of these gases but is free of particulates (16). Immediately after its generation, 6 ml of unfiltered smoke or gas-phase smoke at a temperature of ~25°C were delivered by the respirator in three ventilatory cycles by using a circuit similar to that described previously (19).

Pharmacological agents. Dimethylthiourea (DMTU, an · OH scavenger; Sigma Chemical) was dissolved in isotonic saline to a concentration of 500 mg/ml. Indomethacin (Indo; a cyclooxygenase inhibitor; Sigma Chemical) was first dissolved in polyethylene glycol and then diluted at 1:1 ratio in isotonic saline to a concentration of 5 mg/ml. Capsaicin solution (1-2 µg/ml) was made from a refrigerated stock solution (5 mg/ml), which was prepared by dissolving capsaicin in 10% ethanol, 10% polyoxyethylene sorbitan monooleate, and 80% saline.

Experimental procedures. A total of 58 rats was used in this investigation. Sixty-eight pulmonary C fibers were recorded and studied for their control responses to unfiltered wood smoke. In 15 pulmonary C fibers recorded from 15 rats, afferent responses to gas-phase smoke were compared with those to unfiltered smoke. In another 36 pulmonary C fibers recorded from 36 rats, challenges of unfiltered smoke were repeated after pretreatment with vehicle for DMTU (n = 4), vehicle for Indo (n = 4), DMTU (500 mg/kg; n = 12), Indo (5 mg/kg; n = 8), or a combination of the same doses of DMTU and Indo (DMTU+Indo; n = 8). A solution of these chemicals at a volume of 0.7 ml was slowly injected into the vein over a period of 20 s. The doses of DMTU and Indo noted above have been previously used in the study of pulmonary responses to toxic smoke (10, 15, 18). To exclude the possible deleterious effects of these pharmacological agents, afferent responses of pulmonary C fibers to an injection of the same dose of capsaicin (1 or 2 mg/kg iv) before and after administration of a vehicle or these chemicals were also studied. Before each test of smoke delivery or capsaicin injection, the animal's lungs were hyperinflated (4 × VT) to maintain a constant volume history. Challenges of unfiltered smoke and gas-phase smoke were alternated among the animals to achieve a balanced design. An interval of at least 30 min elapsed between the two deliveries of smoke to avoid tachyphylaxis; our preliminary study indicated that the C-fiber responses to smoke were reproducible when this period of recovery time was allowed. A 20-min period elapsed before the study was resumed after the administration of DMTU, Indo, or DMTU+Indo.

Data analysis and statistics. Neural activity of pulmonary C fibers, mean arterial blood pressure, and heart rate was measured at 1-s intervals. Ptr was measured on a breath-by-breath basis. Baseline data of these physiological parameters were calculated as the average values over the 10-s or 10-breath period immediately preceding the smoke challenge. Pulmonary C fibers were judged to be stimulated by the smoke when the peak evoked discharge exceeded the baseline activity by at least 1 impulse/s. These physiological parameters were analyzed by using 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 for accuracy with those calculated manually. Results were analyzed by a paired t-test or a two-way repeated-measures analysis of variance 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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The baseline activity of the C fibers studied was irregular and sparse (0.5 ± 0.1 impulses/s, n = 68). The nerve endings were stimulated by hyperinflating the lungs up to 3 or 4 VT (Fig. 1A) and by bolus intravenous injection of capsaicin (Fig. 1B). The mean conduction velocity of the afferent fibers conducting impulses from 51 of the C fibers was 1.1 ± 0.1 m/s (range 0.5-2.1 m/s); the conduction velocity of the remaining 17 fibers was not measured. All C fibers studied were localized within the lung structure, and their physiological properties were consistent with those reported in rats (3, 20) and in other species (6).

Delivery of unfiltered wood smoke stimulated 60 of the 68 pulmonary C fibers studied. When the fibers were stimulated, an intense and nonphasic burst of discharge was evoked within 1 or 2 s after smoke delivery (Fig. 1C). This immediate increase in C-fiber activity quickly peaked in 1-2 s after smoke delivery and lasted for 4-8 s (Figs. 1C and 2A). After this immediate-phase stimulation, the evoked discharge of these pulmonary C fibers declined yet remained at a level higher than the baseline activity (Fig. 2A). This delayed and more sustained increase in C-fiber activity was not in phase with ventilatory cycles (Fig. 1C). The remaining eight pulmonary C fibers were not stimulated, either immediately or later, by delivery of unfiltered smoke. For the whole group of 68 pulmonary C fibers, the mean duration of the overall stimulation induced by unfiltered smoke was 25.5 ± 2.2 s (range: 0-75 s).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   A: effect of unfiltered wood smoke on vagal pulmonary C fibers. Data are means ± SE of all 68 C fibers studied. Vertical dashed line, onset of smoke challenge. FA, fiber activity. B: comparison of mean responses of vagal pulmonary C fibers to unfiltered wood smoke and to gas-phase smoke. Data are means ± SE of 15 C fibers studied. Mean fiber activity is plotted during control period (baseline) and in 10-s intervals after smoke challenge. No statistical significance (P > 0.05) was found in any paired comparison of values between 2 types of smoke within same time intervals.

In 15 pulmonary C fibers that were stimulated by unfiltered wood smoke, afferent responses were compared with those evoked by the gas phase of wood smoke. Unfiltered and gas-phase smoke essentially produced a similar pattern of stimulation in the same 15 C fibers (Fig. 1). Statistical analysis revealed that mean responses of pulmonary C fibers to unfiltered smoke measured during each 10-s interval after smoke delivery were not significantly different from those to gas-phase smoke (Fig. 2B). Furthermore, the average duration (22.0 ± 2.4 s) of the overall afferent stimulation produced by unfiltered smoke was not significantly different from that (19.7 ± 2.1 s) produced by gas-phase smoke.

In 36 pulmonary C fibers initially stimulated by unfiltered smoke, smoke challenges were repeated after the animals had received a pretreatment with vehicle, DMTU, Indo, or DMTU+Indo. Twenty minutes after pretreatment with vehicle or these chemicals, the baseline activity of pulmonary C fibers did not change significantly (Figs. 3 and 4). In the vehicle-treated group, a repeated smoke challenge evoked afferent responses of similar amplitude and time course in the same eight C fibers compared with their control responses. In contrast, in the DMTU-, Indo-, or DMTU+Indo-treated groups, a repeated smoke challenge evoked a milder afferent stimulation in each of the C fibers tested (Fig. 3). On average, the immediate responses of C fibers evoked during the first 10-s interval after smoke delivery were not altered by vehicle or Indo but were largely attenuated by DMTU or DMTU+Indo (Fig. 4). Furthermore, the delayed responses of C fibers evoked beyond that first 10-s interval after smoke delivery were not altered by vehicle, but were significantly diminished by DMTU or Indo, and were totally abolished by DMTU+Indo (Fig. 4). As a result, the average duration of the afferent stimulation produced by unfiltered smoke was not significantly affected by vehicle but was greatly shortened by DMTU, Indo, or DMTU+Indo (Fig. 5). At the end of the test period, all the pulmonary C fibers in the vehicle-, DMTU-, Indo-, and DMTU+Indo-treated groups still responded to intravenous injection of capsaicin, and their average responses were not significantly altered by pretreatment with vehicle or these chemicals (Table 1).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Afferent responses of three vagal pulmonary C fibers to unfiltered wood smoke before and after pretreatment with dimethylthiourea (DMTU; A), indomethacin (Indo; B), or both dimethylthiourea and indomethacin (DMTU+Indo; C). Left: control responses. Right: responses after pretreatment. Horizontal bars, delivery of 6 ml of wood smoke. Thirty minutes elapsed between 2 smoke deliveries. See legend for Fig. 1 for further explanation.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effects of pretreatment with vehicle (A; n = 8), DMTU (B; n = 12), Indo (C; n = 8), or DMTU+Indo (D; n = 8) on mean responses of vagal pulmonary C fibers to unfiltered wood smoke. Data are means ± SE. * Significantly different from values before pretreatment within same time intervals, P < 0.05. See legend for Fig. 2 for further explanation.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Effects of pretreatment with vehicle (A; n = 8), DMTU (B; n = 12), Indo (C; n = 8), or DMTU+Indo (D; n = 8) on mean duration of C-fiber stimulation induced by unfiltered wood smoke. Data are means ± SE. * Significantly different from values before pretreatment, P < 0.05.

Under controlled conditions, delivery of unfiltered wood smoke did not cause any detectable change in Ptr (Fig. 3). The peak response of Ptr after smoke delivery was 8.1 ± 0.1 cmH₂O, which was not significantly different from its baseline (8.0 ± 0.1 cmH₂O; P > 0.05; n = 58). In contrast, mean arterial blood pressure initially increased from a baseline of 84.7 ± 3.1 mmHg to a peak of 89.4 ± 3.5 mmHg (P < 0.05; n = 58) at 5-9 s and subsequently decreased to its lowest level of 63.6 ± 2.6 mmHg (P < 0.01; n = 58) at 15-26 s after delivery of unfiltered smoke (Fig. 3). During the hypertensive and ensuing hypotensive period, mean heart rate decreased from a baseline of 338.1 ± 8.8 to 324.9 ± 8.2 and 325.1 ± 8.8 beats/min (P < 0.01; n = 58), respectively. Overall, it took <4 min for these cardiovascular parameters to return to their normal baseline.

	DISCUSSION

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Results of this study demonstrate that vagal pulmonary C fibers were instantly stimulated when three tidal breaths of wood smoke were delivered into the lower airways and lungs. The afferent responses to wood smoke usually consisted of an immediate and a delayed phase: a vigorous and transient stimulation followed by a moderate and more sustained increase in C-fiber activity. Both the immediate- and delayed-phase stimulations seem to be linked to the gas phase of wood smoke because the overall afferent responses were not affected by removal of smoke particulates. The immediate-phase stimulation was largely attenuated by pretreatment with DMTU or DMTU+Indo but was unaffected by pretreatment with Indo. Conversely, the delayed-phase stimulation was partially diminished by pretreatment with DMTU or Indo but was totally abolished by pretreatment with DMTU+Indo. In contrast to these effects, pretreatment with vehicle did not affect the overall C-fiber stimulation. DMTU is an effective scavenger for · OH, whereas Indo is a cyclooxygenase inhibitor that inhibits the production of prostaglandins and thromboxane. Hence, these results suggest that · OH, but not cyclooxygenase products, is actively involved in the immediate-phase stimulation of pulmonary C fibers evoked by wood smoke, whereas both metabolites are responsible for eliciting the delayed-phase stimulation.

That · OH and cyclooxygenase products are involved in the stimulation of pulmonary C fibers is not surprising. These two metabolites have been demonstrated to be important activators for the stimulation of a variety of C-fiber nerve endings under other pathophysiological conditions, including pulmonary C-fiber responses to pulmonary air embolism (5), abdominal visceral C-fiber responses to ischemia and/or reperfusion (21, 26), and cardiac vagal C-fiber responses to ischemia and reperfusion (29). Nevertheless, the mechanisms by which · OH and cyclooxygenase products are associated with the stimulation of pulmonary C fibers by wood smoke remain unclear. Because a direct effect of · OH and cyclooxygenase products on C-fiber nerve endings has been postulated (5, 6), one possibility is that these two metabolites may act directly on pulmonary C fibers. On the other hand, it has been suggested that · OH (28) and cyclooxygenase products (20) have the ability to maintain or to potentiate the sensitivity of C-fiber nerve endings to chemical irritants such as capsaicin. Therefore, a second possibility is that lowering the levels of these two metabolites by using DMTU, Indo, or DMTU+Indo may make pulmonary C fiber less sensitive to a smoke-related stimulus. However, this possibility is doubtful, at least in the case of cyclooxygenase products, because Indo only depressed the delayed-phase stimulation evoked by wood smoke. Additionally, pretreatment with DMTU, Indo, or DMTU+Indo in this study did not affect the responses of pulmonary C fibers to capsaicin. The persistence of the C-fiber responses to capsaicin after pretreatment with these chemicals may suggest that the attenuation of the C-fiber responses to wood smoke observed after administration of these chemicals was not likely due to anesthetic or deleterious effects on these nerve endings. A third possibility is that · OH and cyclooxygenase products may indirectly stimulate pulmonary C fibers through their bronchoconstrictive effects (1, 12). However, the smoke-evoked discharges of pulmonary C fibers that we observed were not modulated by ventilatory cycles. Furthermore, delivery of wood smoke did not cause any detectable change in Ptr in this study. Accordingly, this possibility is questionable.

The source of the origin of · OH and cyclooxygenase products cannot be determined in this study. Several investigators (22, 27, 30) have suggested that oxygen-reactive metabolites formed in lung tissues after inhalation of toxic smoke might originate from an exogenous source. The gas phase of wood smoke is known to contain high concentrations of free radicals and radical precursors (22), which may continuously generate · OH either in the smoke or when they reach lung tissues after smoke inhalation (22, 30). However, the particulate phase of wood smoke also contains free radicals (22) but did not appear to be a major contributor to the stimulation of pulmonary C fibers observed in this study. One plausible explanation for the difference in the contribution of gas and particulate phases of wood smoke is that these two smoke components might each have different accessibility to pulmonary C fibers, the nerve endings of which are believed to be located primarily in the alveolar region (6). Because of their gravitational settling and deposition in the central airways (9), smoke particulates presumably have less ability to reach the lung periphery compared with gas-phase smoke. The other plausible explanation is that the oxygen-reactive metabolites generated by these two smoke components may be different in their types, concentrations, and life spans. Although still not fully understood, the radical chemistry of the gas phase of wood smoke is known to be quite different from that of the particulate phase (22).

Both · OH and cyclooxygenase products may be formed and released endogenously in the lungs after delivery of wood smoke. Certain lung cells, such as polymorphonuclear leukocytes and alveolar macrophages, are primary oxygen radical releasers (8, 27) and were found to be activated after acute inhalation of toxic smoke (23, 27). Additionally, it is known that the lungs are a rich source of arachidonate products and the enzymes necessary for their metabolism (1). Previous studies indicated that significant amounts of oxygen-reactive metabolites are formed during the metabolism of arachidonic acid via cyclooxygenase (7) and that oxygen-reactive metabolites may increase the production of cyclooxygenase products (24). Therefore, these two metabolites may be interrelated in their biosynthesis. Because the involvement of cyclooxygenase products was observed only during the delayed-phase stimulation, it may suggest that a longer delay is required for the arachidonate metabolites to be produced and/or to act on C-fiber nerve endings after smoke delivery compared with · OH.

The time required to initiate the immediate-phase stimulation of pulmonary C fibers is very similar to that needed to evoke the slowing of respiration observed in our previous studies (14-16), thus confirming the importance of · OH in eliciting this respiratory reflex. In this study, pretreatment with DMTU did not totally abolish the immediate-phase stimulation, an observation that is in good agreement with the finding that a similar dose of DMTU could not completely prevent the C-fiber-mediated respiratory reflex evoked by wood smoke (15). It is speculated that oxygen-reactive metabolites other than · OH and/or smoke constituents that are not related to free radicals were also involved in this immediate-phase stimulation of C fibers. In our previous studies of the reflex responses (14-16), inhaled wood smoke generally evoked an immediate and prominent bradycardia and hypotension, coinciding with the slowing of respiration (occurred within 1 or 2 s). We have hypothesized that these immediate cardiovascular responses to wood smoke are also elicited by stimulation of lung vagal C fibers (14, 15). In this study, however, immediate cardiovascular responses to wood smoke were absent, probably because the right vagus nerve was sectioned. The mild hypertension and ensuing hypotension after smoke delivery observed in this study may be due to the activation of arterial chemoreceptors by the extremely low oxygen and high carbon dioxide concentrations in the smoke (14).

Inhalation of toxic smoke not only causes airway irritations (2, 14, 17, 27) but also produces lung injury (4, 10, 13, 17, 25, 27, 30). Although the information regarding the inhalation injury of the lungs is relatively well established (27, 30), the mechanisms underlying the airway irritation induced by toxic smoke have been largely overlooked. It is generally believed that vagal pulmonary C fibers function as an important sensory system in detecting the onset of pathophysiological changes in the airways (6). Although oxygen-reactive metabolites and cyclooxygenase products have been strongly implicated in the pathogenesis of inhalation injury (4, 13, 25, 27, 30), their involvements in eliciting the sensory irritation of the airways after smoke inhalation are obscure. The observations made in this study provide the first electrophysiological evidence that vagal pulmonary C fibers play an important role in detecting the airway assault by wood smoke and that their functional significance is mediated through mechanisms involving · OH and cyclooxygenase products.