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MS 9923 Received 30 July 1999; accepted after revision 14 November 1999.
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ABSTRACT |
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 TopAbstract IntroductionMethodsResultsDiscussionReferences |
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INTRODUCTION |
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Environmental tobacco smoke (passive smoke), the product of the smoldering end of cigarettes mixed with exhaled mainstream smoke, harms the respiratory health of children. Children living in homes where they are exposed to passive smoke have more coughs (Dodge, 1982; Ekwo et al. 1983), wheeze (Dodge, 1982) and airway obstruction (Wang et al. 1994), increased airway reactivity (Ekwo et al. 1983; Frischer et al. 1992) and more sputum production (Dodge, 1982). In addition, these children have an increased risk of lower respiratory tract illnesses (Strachan & Cook, 1997), an increased rate and earlier onset of asthma (Weitzman et al. 1990), and an increased incidence of sudden infant death syndrome (Klonoff-Coher et al. 1996).
Some of the same respiratory symptoms associated with environmental tobacco smoke exposure are also elicited by stimulation of the vagal sensory bronchopulmonary C-fibres. In this regard, studies using direct measures of bronchopulmonary C-fibre impulse activity or indirect measures such as preventing the reflex responses by blocking C-fibre conduction or measuring the local release of tachykinins, indicate that the bronchopulmonary C-fibres are stimulated by acute exposures to components of environmental tobacco smoke including nicotine receptor agonists (Saria et al. 1988), acrolein (Lee et al. 1992) and oxidants (Coleridge et al. 1993), as well as by acute exposures to mainstream tobacco smoke (Lee et al. 1989, 1990; Delay-Goyet & Lundberg, 1991; Pisarri et al. 1991). The stimulated C-fibres initiate both a central and a local reflex (Coleridge & Coleridge, 1984; Saria et al. 1988). The central reflex responses, thought to protect the lung from further injury from inhaled agents, include rapid shallow breathing, expiratory apnoea, cough, bronchoconstriction, increased mucous secretion, hypotension and bradycardia (Coleridge & Coleridge, 1984). The reflex respiratory responses of cough, bronchoconstriction and increased mucous secretion are also hallmark symptoms of chronic environmental tobacco smoke exposure (Dodge, 1982; Ekwo et al. 1983; Frischer et al. 1992; Wang et al. 1994). These observations have led to the suggestion that hyperresponsiveness of the bronchopulmonary C-fibre reflex causes some of the respiratory symptoms evoked or exacerbated by environmental tobacco smoke exposure. Recently, electrophysiological recordings of single bronchopulmonary C-fibre afferent activity in young guinea-pigs have provided more direct evidence for this proposal by showing that chronic exposure to environmental tobacco smoke sensitizes bronchopulmonary C-fibres to both chemical and mechanical stimuli (Mutoh et al. 1999). A number of other studies have also documented an increased excitability of primary lung sensory fibres or sensory fibre somata in the nodose ganglia following exposure to environmental pollutants, including allergen (Undem et al. 1993, 1999; Fischer et al. 1996), sidestream tobacco smoke (Bonham et al. 1996) and ozone (Ho & Lee, 1998; Joad et al. 1998). To our knowledge, however, there are no data on whether the sensitization of primary lung sensory afferent fibres by chronic exposure to an environmental pollutant is sustained in the central circuitry of the reflex or has any physiological consequences on the reflex control of respiratory function. The nucleus tractus solitarii (NTS) is the first site in the central circuitry where afferent signals from the primary bronchopulmonary C-fibres are transmitted and susceptible to modulation. The principal neurotransmitter and a number of neuromodulators at these synapses have been identified (Seifert & Trippenbach, 1995; Sevoz et al. 1996; Wilson et al. 1996; Wang et al. 1997). Signal conditioning at these first central synapses may be pivotal, in that the sensory information may be unfailingly transmitted or further modulated, e.g. amplified, blunted or extinguished.
We hypothesized that if an increased responsiveness of the bronchopulmonary C-fibres contributes to the exaggerated respiratory symptoms associated with environmental tobacco smoke via the central reflex pathway, then the increase should be manifest at NTS neurones in the central network and ultimately lead to an increase in at least some component of the reflex output. If, on the other hand, the increased excitability of the sensory fibres is extinguished by signal modulation in the central circuitry, the reflex output may be unchanged. Thus, the purpose of this study was to examine the effects of 5 weeks of exposure to sidestream smoke (the surrogate for environmental tobacco smoke) in young guinea-pigs during the age-equivalent period to human childhood on (1) the baseline and evoked increases in impulse activity of bronchopulmonary C-fibre-activated NTS neurones and (2) the associated reflex changes in phrenic nerve discharge and tracheal pressure measured in the whole animal. As a secondary objective, we also examined the exposure effects on baseline and evoked changes in arterial blood pressure and heart rate. We used the potent C-fibre stimulant capsaicin (Coleridge & Coleridge, 1984).
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METHODS |
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All experimental protocols in this work were reviewed and approved by the Institutional Animal Care and Use Committee in compliance with the Animal Welfare Act and in accordance with Public Health Service Policy on Humane Care and Use of Laboratory Animals.
Chronic exposure to sidestream tobacco smoke
Male Dunkin-Hartley guinea-pigs (Charles River Laboratories, Raleigh, NC, USA) were randomly assigned to a group exposed to either sidestream smoke, the surrogate for environmental tobacco smoke (n = 12), or filtered air (n = 10) for 6 h a day, 5 days a week, from age 7-9 days to age 41-45 days of life. During exposures, the guinea-pigs were housed in polycarbonate cages (41 cm × 20 cm cross-sectional area) with wire lids and autoclaved wood shavings for bedding. They were fed guinea-pig chow and water ad libitum, including during the exposure periods. Sidestream smoke was generated by a modified ADL/II smoke-exposed system (Little, Cambridge, MA, USA) from conditioned 1R4F cigarettes from the University of Kentucky Tobacco and Health Research Institute (Lexington, KY, USA). Two cigarettes at a time were smoked under Federal Trade Commission conditions in a staggered fashion at rate of 1 puff min-1 (35 ml, 2 s duration). The sidestream smoke was diluted with filtered air in a mixing chamber, then passed into a stainless steel and glass Hinners-type exposure chamber, 0·44 m3 in size. The exposure chamber was characterized by the following parameters expressed as means ± S.D.: relative humidity of 37·5 ± 10·8 %, temperature of 22·8 ± 0·4°C, total suspended particulate concentration of 1·00 ± 0·07 mg m-3, carbon monoxide concentration of 6·2 ± 0·8 p.p.m., and nicotine concentration of 224·4 ± 99·9 µg m-3. Relative humidity and temperature were sampled continuously. Nicotine was sampled daily for 15 min during each 6 h exposure period. The total suspended particle concentration was sampled with the piezobalance technique for 2 min every 0·5 h.
General animal preparation
Sixteen hours after the final exposure, each guinea-pig was anaesthetized with an injection of urethane (1·6 g kg-1 I.P.) and then given supplemental doses of pentobarbital sodium (4 mg kg-1 I.V.) about every hour as needed. Before neuromuscular blockade, the adequacy of anaesthesia was determined every half an hour by pinching the hindlimb paw and monitoring for hindlimb flinch or withdrawal or sudden fluctuation of arterial blood pressure (ABP; > 5 mmHg) or heart rate (HR; > 10 %). During neuromuscular blockade, the adequacy of anaesthesia was tested every half an hour by determining whether there was a spontaneous or paw pinch-evoked fluctuation or increase (> 5 mmHg) in ABP or increase (> 10 %) in HR. Each guinea-pig was placed on a servo-controlled water blanket and body temperature was monitored via a rectal temperature probe and maintained within 37 ± 1°C. Catheters were introduced into the jugular vein for administering fluids and drugs and into the carotid artery for monitoring ABP and withdrawing samples for arterial blood gas determination. The trachea was cannulated and a catheter was connected to a side port of the endotracheal tube to monitor tracheal pressure (TP). TP, ABP and HR were recorded through a Modular Instruments data-acquisition system (model M100, Malvern, PA, USA). Each guinea-pig was prepared with bilateral pneumothoraces by incisions made in the chest wall and mechanically ventilated with oxygen-enriched humidified air with a tidal volume of 8 ml kg-1. The ventilator rate was set initially at 35-40 breaths min-1, and the positive end-expiratory pressure was set at 2 cmH2O. At these settings, neural respiratory activity was entrained 1:1 to mechanical lung inflation in < 10 % of the guinea-pigs. Arterial blood gases and pH were maintained so that the pH was between 7·30 and 7·40 and the arterial PCO2 was between 35 and 45 Torr, by adjusting ventilator rate and by infusing sodium bicarbonate. After tracheal intubation each animal was paralysed with gallamine (3 mg kg-1 I.V.) every hour as needed. The pericardium was opened and a cannula (0·58 mm i.d.) prefilled with capsaicin (2·5 µg ml-1) was inserted into the left atrium through the left atrial appendage. Both cervical vagus nerves were separated from the carotid artery and sectioned below the diaphragm to eliminate afferent traffic from vagally innervated viscera below the diaphragm. The aortic depressor nerves were carefully separated from the vagus nerves and cut bilaterally to eliminate aortic baroreceptor input. The fourth cervical (C4) branch of the left phrenic nerve was isolated in the neck and cut distally.
The guinea-pigs were placed in a stereotaxic head frame. The vagus nerve ipsilateral to the recording site was placed on a bipolar silver hook electrode, covered with a mixture of warm petroleum jelly and mineral oil and connected to a stimulus isolation unit driven by a Grass S48 stimulator. This was generally the left vagus nerve, but if a blood vessel on the surface of the left side of the brainstem interfered with proper placement of the electrode, then the right vagus nerve was isolated and the right side of the brainstem was searched. For recording neural respiratory rate and expiratory time (TE), the central end of the phrenic nerve was placed on a bipolar silver hook electrode and covered with a mixture of warm petroleum jelly and mineral oil. A vertebral clamp was placed on the T2 spinal process and an occipital craniotomy was performed. For exposing the brainstem the caudal portion of the fourth ventricle was exposed by removing the dura mater and arachnoid membranes and then covered with warm mineral oil. At the end of the experiments, the guinea-pigs were killed with injection of a lethal dose of pentobarbital.
Extracellular single unit recording
Extracellular recordings of single unit activity were made through a single-barrel glass electrode filled with 2 % Pontamine Sky Blue dye (BDH Chemicals Ltd, Poole, UK) in 0·5 M sodium acetate. Unit activity was fed via high-impedance source followers to second-stage amplifiers, filtered (0·3-3 kHz), and fed in parallel to an oscilloscope, thermal chart recorder, audio monitor, and a digital tape-recorder with a sampling rate of 11 kHz per channel for off-line analysis.
Based on our previous experience (Bonham & Joad, 1991; Wilson et al. 1996) we focused the search for bronchopulmonary C-fibre-activated neurones in the region of the caudomedial NTS extending from 700 µm rostral to 500 µm caudal to the calamus scriptorius, from midline to 500 µm lateral to midline, and from 0 µm to 1800 µm ventral to the dorsal surface. The NTS was searched for potential bronchopulmonary C-fibre-activated neurones by continually stimulating the vagus nerve (100-700 µA, 0·5-0·7 ms square wave cathodal pulses) at 0·5 Hz as the recording electrode was lowered slowly through the NTS. Vagally activated neurones were suspected to be bronchopulmonary C-fibre-activated neurones if they were silent or irregularly, sparsely firing neurones that did not discharge robustly with ventilatory cycles (eliminating slowly adapting receptor-activated neurones) (Knowlton & Larrabee, 1946).
Bronchopulmonary C-fibre-activated NTS neurones
The impulse activity (action potentials s-1) of a suspected vagal bronchopulmonary C-fibre-activated neurone, TE (the interval between phrenic nerve activity bursts), TP, ABP and HR were tested in response to left atrial injections of capsaicin (0·5 and 2·0 µg kg-1). Vagal C-fibres in the lungs and airways have been categorized as bronchial or pulmonary based on their blood supply, with pulmonary C-fibres being distinguished from bronchial C-fibres by their rapid onset response to right atrial or pulmonary circulation injections of stimulants (0·3-4·0 s) and a slower-onset, smaller or no response to left atrial or bronchial circulation injections in dogs, cats and rats (Coleridge & Coleridge, 1977; Coleridge et al. 1984). The criteria for distinguishing bronchial from pulmonary C-fibres in small animals are somewhat tenuous because of the fast circulation times, the presence of anastomoses (Sant'Ambrogio & Sant'Ambrogio, 1982) and the finding that some C-fibres are accessible from both circulations with similar onset latencies (Delpierre et al. 1980). Because of these uncertainties regarding the criteria for segregating bronchial and pulmonary C-fibres, we categorized the C-fibre-activated neurones as bronchopulmonary.
Capsaicin was administered in doses of 0·5 µg kg-1 in order to compare the responses of the bronchopulmonary C-fibre NTS neurones to the responses of the primary bronchopulmonary C-fibres studied previously (Mutoh et al. 1999) and in doses of 2·0 µg kg-1 to provide dose-response curves. The time intervals separating the doses were determined in pilot studies. From the pilot studies, NTS responses to capsaicin doses of 0·5 µg kg-1 separated by 30 min intervals showed no tachyphylaxis, whereas the responses to 2·0 µg kg-1 frequently showed tachyphylaxis. Thus, if a neurone was tested with 0·5 µg kg-1 capsaicin first and the higher dose could not subsequently be tested, for example if the neurone was lost, then a second neurone in the same animal was not tested for at least 30 min. Once a neurone was tested with 2·0 µg kg-1 capsaicin, the protocol was terminated. Three neurones (two from filtered air-exposed and one from a sidestream smoke-exposed animal) were tested with a capsaicin dose of 2·0 µg kg-1 at 2 min after an injection of 0·5 µg kg-1. The responses of these three neurones were not statistically significantly different from the responses evoked by a single 2·0 µg kg-1 dose of capsaicin that was not preceded by an injection of 0·5 µg kg-1 (P = 0·42, Student's unpaired t test) and thus were included in the analysis.
Experimental protocol
For a suspected vagal bronchopulmonary C-fibre-activated neurone, we measured the onset latency of the response by averaging the time between the stimulus artifact and the onset of the action potential for ten successive vagal stimuli. All neurones were subjected to the presumptive criterion for classification as being monosynaptically activated if they discharged an action potential to each of two stimuli separated by 5 ms (Miles, 1986; Scheuer et al. 1996). NTS neuronal activity was monitored in conjunction with TE (as an index of the respiratory component of the C-fibre reflex output), TP (as a global index of airway tone) and the cardiovascular indices of ABP and HR. After 1 min of recording baseline activity, capsaicin (0·5 or 2·0 µg kg-1) was injected into the left atrium and NTS unit impulse activity, TE, TP, ABP and HR were measured.
Histology
Recording sites were marked by passing current (10 µA for 7 s every 14 s for 15 min; electrode negative) through the recording electrode to deposit the Pontamine Sky Blue dye. At the end of an experiment, the brainstem was removed and fixed in 4 % paraformaldehyde and 10 % sucrose. The brainstems were cut in 40 µm coronal sections and counterstained with Neutral Red. Recording sites were reconstructed from dye spots with the aid of a drawing tube.
Pharmacological agents
A stock solution of capsaicin (1 × 10-2 M; Sigma Chemical Co.) was prepared in a vehicle of 10 % Tween 80, 10 % ethanol and 80 % saline. All drugs were kept frozen and the desired concentration was prepared from concentrated stock solutions on the day of the experiment by dilution in saline.
Data analysis
Data collected included the latency and variability of the onset of the action potentials evoked by vagus nerve stimuli, whether the responses were evoked in second- or higher-order NTS neurones, and capsaicin-evoked changes in NTS neurone impulse activity, phrenic nerve activity, TP, ABP and HR. The onset latency of the vagally evoked responses was determined by using peristimulus time histograms (1·6 ms bins) generated using EGAA/Computerscope software (RC Electronics, Goleta, CA, USA). The onset variability was defined as the difference between the shortest and longest latency of the evoked action potentials.
For capsaicin-evoked responses, the impulse activity of NTS neurones was analysed for each 1 s interval by using EGAA/Computerscope software. The baseline impulse activity was determined over a 1 min period. The peak NTS response was defined as the mean number of action potentials per second during the most active 5 s out of the initial 10 s after left atrial capsaicin injection. NTS neurones that were activated by left atrial capsaicin injection after the occurrence of the phrenic nerve apnoea were excluded because of a lack of temporal relationship between neuronal and reflex responses. The onset latency for the increase in NTS unit activity was defined as the time between the injection of capsaicin and the first detectable increase in NTS unit activity. Phrenic nerve response to capsaicin was determined by the ratio of the peak TE after capsaicin injection to the mean TE during the control period.
The peak increase in TP and the peak decrease in ABP or HR were defined as the 5 s bin with the biggest change within the initial 30 s after left atrial capsaicin injection. To determine whether the evoked peak response of unit activity, TE, TP, ABP and HR were significantly different from the 1 min baseline values within either the sidestream smoke- or filtered air-exposed groups, a paired t test was used.
To determine whether the capsaicin-evoked changes (
) from the baseline value to the peak response for neuronal activity, TE, TP, ABP and HR were significantly different in animals in the filtered air-exposed versus sidestream smoke-exposed group, a two-way ANOVA was used with exposure and dose as between-subject effects. The duration of the neuronal responses was taken as the time from the onset of the increase in activity to the time when the activity returned to within 20 % of the baseline. The duration of the capsaicin-evoked changes in neuronal activity was compared using a two-way ANOVA with exposure and dose as between-subject effects.
To determine whether capsaicin-evoked peak changes in neuronal activity, TE, TP, ABP and HR were different from baseline, we used a paired t test. Body weights, blood gases and conduction velocities were compared with an unpaired t test.
Statistical significance was claimed when the probability of a type I error was < 0·05. All values are expressed as means ± S.E.M., unless otherwise indicated.
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RESULTS |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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At the commencement of the experiments, the weights and arterial blood gases were not different between the two groups (P > 0·05). In the filtered air- and sidestream smoke-exposed groups, the weights were 502 ± 14 and 486 ± 19 g, the PO2 was 384 ± 14 and 382 ± 19 Torr, the PCO2 was 42·2 ± 1·9 and 42·4 ± 1·3 Torr, and the pH was 7·34 ± 0·02 and 7·34 ± 0·01, respectively.
Characteristics and location of bronchopulmonary C-fibre NTS neurones
Of the 44 neurones activated by sequential vagus nerve stimuli delivered at 0·5 Hz, 28 neurones were subsequently stimulated by left atrial capsaicin injections. These 28 neurones were recorded in 22 guinea-pigs (13 neurones in 10 filtered air-exposed animals and 15 neurones in 12 sidestream smoke-exposed animals). Sidestream smoke exposure had no effect on either the onset latency or the variability of the onset latency of the vagal-evoked action potentials (P > 0·05, exposure effect). The onset latency was 27 ± 2·5 ms with a variability of 1·44 ± 0·19 ms in the filtered air-exposed animals, and 30·7 ± 2·6 ms with a variability of 1·38 ± 0·20 ms in the sidestream smoke-exposed animals. Based on the mean onset latencies and a measured distance of 25 mm between the cervical vagus nerve stimulating electrode and the NTS recording electrode, the conduction velocity of the primary bronchopulmonary C-fibres was estimated to be 0·99 ± 0·09 m s-1 in the filtered air-exposed animals and 0·91 ± 0·08 m s-1 in the sidestream smoke-exposed animals. These values were not different from each other (P > 0·05).
Eighteen of the bronchopulmonary C-fibre-activated neurones could be histologically verified. All were located in the intermediate and caudal NTS and medial to the tractus. Figure 1 shows an example of a dye spot marking the recording site in the NTS in a coronal slice (Fig. 1A) and a composite of the reconstructed recording sites (Fig. 1B). There were no detectable differences between the distribution of recordings obtained from the filtered air- (
) and sidestream smoke-exposed (
) animals.
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A, photomicrograph of a dye spot indicating an NTS recording site (arrow). B, composite of histologically verified recording sites in filtered air-exposed ( | ||
Effect of sidestream tobacco smoke on bronchopulmonary C-fibre-activated neurones
A total of 31 capsaicin injections were made (10 of 0·5 µg kg-1 and 5 of 2·0 µg kg-1 in filtered air-exposed guinea-pigs and 6 of 0·5 µg kg-1 and 10 of 2·0 µg kg-1 in sidestream smoke-exposed guinea-pigs).
Following chronic exposure to sidestream tobacco smoke, the responses of NTS neurones to bronchopulmonary C-fibre input were greater compared to those recorded in the filtered air-exposed animals. Figure 2 shows an example of the activity of a bronchopulmonary C-fibre-activated neurone and phrenic nerve in response to left atrial injections of capsaicin (2·0 µg kg-1) from a filtered air- (Fig. 2A) and sidestream smoke-exposed guinea-pig (Fig. 2B). Each neurone met the presumptive criterion for monosynaptic activation by discharging an action potential in response to each of two stimuli separated by 5 ms (insets in Fig. 2A and B). In the sidestream smoke-exposed guinea-pig, the NTS neurone response was more robust and the increase in TE was longer compared to the filtered air-exposed control animal.
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Left atrial capsaicin injection (2·0 µg kg-1) evoked responses of NTS neurones, phrenic nerve activity (PNA) and ABP from a guinea-pig exposed to filtered air (FA, A) or sidestream smoke (SS, B). The SS-exposed guinea-pig had a greater NTS response and longer apnoea compared to the FA-exposed guinea-pig. Insets, vagus nerve stimulus artifact ( | ||
As shown in the group data (Fig. 3), left atrial capsaicin injections (0·5 and 2·0 µg kg-1) produced dose-dependent increases in NTS neuronal activity in both the filtered air- and sidestream smoke-exposed groups (P = 0·012, dose effect, ANOVA). In the sidestream smoke-exposed group the peak increase in impulse activity was statistically different from baseline at both doses of capsaicin (P < 0·05); in the filtered air-exposed animals, the difference reached statistical significance with the 0·5 µg kg-1 capsaicin dose and showed a trend with the 2·0 µg kg-1 dose (P = 0·058). The peak increase in NTS activity in the sidestream smoke-exposed animals was significantly greater than that in the filtered air-exposed animals with the 2·0 µg kg-1 dose of capsaicin (Fig. 3, P = 0·030, exposure effect; P = 0·02, Fisher's LSD), but not with the 0·5 µg kg-1 dose (P > 0·05, Fisher's LSD). Sidestream smoke exposure had no effect on the baseline activity of the NTS neurones (P > 0·05, exposure effect). In addition, there was no difference in the extent to which sidestream smoke exposure enhanced the responsiveness of the NTS neurones to either dose of capsaicin based on their presumptive classification as second-order (9 in each group) or higher-order (4 in the filtered air-exposed and 6 in the sidestream smoke-exposed group; P < 0·05, exposure effect; P > 0·05, neurone order effect).
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Left atrial capsaicin (LA CAP) injections produced dose-dependent increases in unit activity (action potentials s-1; P = 0·012, dose effect, ANOVA) that were significantly greater following sidestream smoke (SS) exposure compared to filtered air (FA) exposure for the 2·0 µg kg-1 dose (* P = 0·030, exposure effect, ANOVA; P = 0·02, Fisher's LSD). FA: 0·5 µg kg-1, n = 10; 2·0 µg kg-1, n = 5; SS: 0·5 µg kg-1, n = 6; 2·0 µg kg-1, n = 10). | ||
Figure 4 shows the entire time course of the capsaicin-evoked changes in impulse activity of the NTS neurones in the sidestream smoke- and filtered air-exposed guinea-pigs presented on a second-by-second basis. As illustrated by the time course, the onset latency ranged from 1 to 5 s in all groups. Statistical analysis of the grouped data confirmed that the mean onset latencies did not differ based on the dose of capsaicin or the exposure (P > 0·05, dose effect; P > 0·05, exposure effect). The overall mean onset latency was 2·3 ± 0·4 s. Following the 0·5 µg kg-1 dose of capsaicin, the increased activity returned to within 20 % of the baseline within 20 s in both the filtered air- and the sidestream smoke-exposed groups. As illustrated in the right panel of Fig. 4, with the 2·0 µg kg-1 capsaicin dose the increase in activity was prolonged in the sidestream smoke-exposed animals. The analysis of the grouped data confirmed a significant overall effect of sidestream smoke exposure on the duration of the neuronal response (P = 0·008, exposure effect). The duration was significantly prolonged following the high dose of capsaicin (30·2 ± 6·2 s in the sidestream smoke-exposed animals vs. 10·4 ± 3·4 s in the filtered air-exposed control animals; P = 0·01, Fisher's LSD), but not the low dose (P > 0·05, Fisher's LSD).
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The mean onset latency of the responses to left atrial capsaicin (LA CAP) injections was the same in guinea-pigs exposed to both filtered air (FA; 0·5 µg kg-1, n = 10; 2·0 µg kg-1, n = 5) and sidestream smoke (SS; 0·5 µg kg-1, n = 6; 2·0 µg kg-1, n = 10) at both doses. The duration of the response was significantly prolonged with the high dose. Action potential responses are plotted in 1 s bins. | ||
Effect of sidestream tobacco smoke on reflex responses
Left atrial capsaicin produced a dose-dependent increase in the peak TE vs. control TE (TE,peak/TE,control) in both the filtered air- and sidestream smoke-exposed guinea-pigs (P = 0·0001, dose effect; Fig. 5). TE,peak was statistically different from TE,control at both capsaicin doses in the sidestream smoke-exposed group and in the filtered air-exposed group (P < 0·05). The prolongation of TE was significantly greater in the sidestream smoke-exposed animals compared to that in the filtered air-exposed control group (P = 0·030, exposure effect); the augmented increase in TE was only apparent at the larger dose of capsaicin (2·0 µg kg-1) (P = 0·016, interaction between dose and exposure; P = 0·003, Fisher's LSD). Sidestream smoke exposure had no effect on baseline TE (P > 0·05).
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Left atrial capsaicin (LA CAP) injections produced dose-dependent increases in TE,peak/TE,control (P = 0·0001, dose effect) that were significantly larger following sidestream smoke (SS) exposure compared to filtered air (FA) exposure (* P = 0·030, exposure effect) only with the 2·0 µg kg-1 dose (P = 0·016, interaction between dose and exposure). FA: 0·5 µg kg-1, n = 10; 2·0 µg kg-1, n = 5; SS: 0·5 µg kg-1, n = 5; 2·0 µg kg-1, n = 9). | ||
Sidestream smoke exposure also did not alter the baseline values or the capsaicin-evoked increases in TP, decreases in ABP or decreases in HR (Table 1). While these capsaicin-evoked reflex responses were small, most of responses to both doses of capsaicin were significantly different from baseline (P < 0·05) and all the rest showed a trend (P 
0·09) (Table 1).
Table 1. Changes in TP, ABP and HR for animals in filtered air (FA)- and sidestream smoke (SS)-exposed groups
| FA | SS | |||
| 0·5 µg kg-1 capsaicin |
2·0 µg kg-1 capsaicin |
0·5 µg kg-1 capsaicin |
2·0 µg kg-1 capsaicin |
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| TP (cmH2O) |
0·06 ± 0·02 * | 0·54 ± 0·33 * | 0·06 ± 0·03 * | 0·71 ± 0·32 * |
| ABP (mmHg) |
-1·09 ± 0·72 | -5·44 ± 1·90 | -1·14 ± 0·38 * | -3·35 ± 1·23 |
| HR (min-1) |
-0·44 ± 0·31 | -3·92 ± 1·16 * | -0·85 ± 0·21 * | -2·08 ± 0·70 |
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DISCUSSION |
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The new finding of this study is that the sensitization of primary afferent bronchopulmonary C-fibres by chronic exposure of guinea-pigs to sidestream tobacco smoke during the equivalent period to human childhood is sustained at the first central synapses in the NTS and is associated with a prolonged reflexively evoked expiratory apnoea. To our knowledge the current findings are the first to show that an increased excitability of lung sensory afferent fibres by chronic exposure to an environmental pollutant is preserved by signal processing in the central circuitry leading to functional changes in reflex output.
Central to the conclusion drawn from this study is that the NTS neurones were, indeed, in the bronchopulmonary C-fibre reflex pathway; that is, they were synaptically driven by primary afferent vagal C-fibres which originated in the lungs and airways. The following data suggest that this was the case. First, the responses of the neurones, recorded in a specific NTS region where bronchopulmonary C-fibres have been shown to terminate (Bonham & Joad, 1991; Kubin et al. 1991; Wilson et al. 1996), were similar to the responses of the primary bronchopulmonary afferent C-fibres studied previously; the two studies used the same exposure protocol and the same dose (0·5 µg kg-1) and administration route of the potent C-fibre stimulant capsaicin. Moreover, the fibres in the previous study were unequivocally localized to the lungs by probing the receptive fields and had conduction velocities of C-fibres (Mutoh et al. 1999). The estimated conduction velocity of the vagus nerve fibres activating the NTS neurones in the current study was also consistent with the conduction velocity of C-fibres (Coleridge & Coleridge, 1984). In addition, capsaicin evoked rapid-onset, dose-dependent increases in spiking activity of the NTS neurones in conjunction with a prolonged TE and decrease in ABP and HR, classic reflex responses evoked by C-fibre stimulation (Coleridge & Coleridge, 1984). While capsaicin applied to tracheal mucosa in in vitro preparations has also been shown to stimulate some A
-fibres with cell bodies in the jugular ganglia (Riccio et al. 1996), in the current study the onset latency of the responses to vagal stimulation was consistent with activation of C-fibres. Finally, since both vagus nerves were transected below the diaphragm the potential contribution of sub-diaphragmatic C-fibres was eliminated. On the other hand, we cannot rule out the possibility that stimulation of supra-diaphragmatic C-fibres other than those originating in the lungs and airways (e.g. those originating in the oesophagus, larynx, pharynx or myocardium) could have contributed to the increases in NTS neuronal activity (Thoren, 1979; Dawid Milner et al. 1995). Given that the sidestream smoke was inhaled it seems unlikely that myocardial C-fibres would have been susceptible to plastic changes that resulted in an increased responsiveness. Finally, although we only tested NTS neurones that were activated by vagus nerve stimulation and while the majority of peripheral afferent fibres that terminate in the NTS are conveyed in the vagus nerve, it is possible that the NTS neuronal responses to capsaicin were mediated by stimulation of sympathetic afferent fibres.
We did not attempt to isolate selective inputs from bronchial vs. pulmonary C-fibres by comparing neuronal responses to injections made in the left vs. right heart. Interpretation of the data would have been complicated by the fast peripheral circulation times, the presence of anastomoses (Sant'Ambrogio & Sant'Ambrogio, 1982) and the finding that some primary afferent C-fibres are accessible from both circulations with similar onset latencies (Delpierre et al. 1980). Moreover, in previous recordings of NTS neurones, we found that some vagal C-fibre-activated NTS neurones were stimulated by injections in both the left ventricle and the right atrium, raising the possibility of convergent inputs to the NTS neurones from C-fibres supplied by the bronchial and pulmonary circulations (Wilson et al. 1996). Convergent inputs from cardiac and pulmonary C-fibre afferent fibres have been confirmed (Silva-Carvalho et al. 1998).
For the increase in excitability of the primary afferent bronchopulmonary C-fibres to affect the reflex output, the increased afferent traffic must be transmitted to the NTS and transformed into a comparable increase in NTS neuronal output. The sidestream smoke-induced augmentation of the peak NTS responses was significant only at the higher dose of capsaicin. However, there was a trend towards an augmentation of the peak response of the NTS neurones with the low dose of capsaicin which was similar to the twofold augmentation of the peak response of the primary afferent fibres previously recorded in the same age guinea-pigs subjected to the same sidestream smoke exposure protocol (Mutoh et al. 1999). Moreover, the short onset latencies of the NTS responses corresponded to the onset latencies of the early responding bronchopulmonary C-fibres in our previous study. The comparison suggests that the increased excitability of the afferent fibres was preserved without extensive amplification or blunting in the NTS. Sidestream smoke exposure had no significant effect on the duration of the responses of either the primary afferent fibres or the NTS neurones with the low dose of capsaicin. However, it significantly prolonged (about threefold) the NTS neuronal responses to the higher capsaicin dose. This finding coupled with the observation that the augmentation of the reflex apnoea was only apparent at the high capsaicin dose suggests that a sustained increase in NTS activity may be required for an enhancement of the reflex output.
Sidestream smoke exposure enhanced the responsiveness of neurones classified as either second- or higher-order to the same extent. This may suggest that the transmission of the increased input from the sensitized primary afferent fibres was not significantly altered by processing at successive synapses within the NTS. Nonetheless, the classification relied on the presumptive criterion (Miles, 1986) that monosynaptically activated neurones discharge an action potential to each of two vagal stimuli separated by 5 ms, while polysynaptically activated neurones do not. The criterion has been validated in vivo, insofar as NTS neurones identified independently as second-order (by anterograde tracing of the vagus nerve) reliably followed the two stimuli (Scheuer & Mifflin, 1998). Still, conclusions drawn using the criterion are tentative, given that other factors, including dendritic filtering at the postsynaptic neurones (Rose & Call, 1992) or a decreasing conduction safety factor of the vagal afferent C-fibres at this high stimulation rate, could also contribute to the failure of the neurones to follow the two stimuli.
There was no sidestream smoke-induced augmentation of the very small airway or cardiovascular reflex responses at either dose of capsaicin. It may be that with higher doses of capsaicin or with longer exposures to sidestream smoke, differences in these reflex responses could have been detected. There is an alternative explanation, however, based on the central circuitry of the reflex. For the complex output of the bronchopulmonary C-fibre reflex, the sensory signals after being processed at a number of synapses within the NTS must ultimately be transmitted out of the NTS over divergent pathways to neurones in the central respiratory network to change breathing pattern (cough, apnoea, rapid shallow breathing), to vagal pre-motoneurones to increase mucous secretion and airway tone, and to sympathetic and/or vagal pre-motoneurones to decrease heart rate and blood pressure. Thus, it could be the case that while the smoke-induced increase in NTS activity was preserved over projections to the respiratory network, the same increases in NTS activity were dampened or offset at synapses in the pathway between the NTS and the vagal and sympathetic pre-motoneurones.
While this is the first simultaneous monitoring of the bronchopulmonary C-fibre-activated neuronal activity in the NTS and phrenic nerve activity in guinea-pigs chronically exposed to sidestream smoke, there are related data on the behavioural reflex responses with similar exposure periods to mainstream tobacco smoke. Karlsson et al. (1991) found that chronic mainstream smoke exposure (1 h twice daily for 2 weeks) increased the frequency of capsaicin- or citric acid-induced cough but not of capsaicin- or citric acid-induced bronchoconstrictor responses. One interpretation of these data is that while cough (elicited via the central reflex) was augmented, the bronchoconstrictor response (involving both central and local axon reflexes) was not. Using an isolated perfused lung preparation to focus on the axon reflex, we found that sidestream smoke exposure (the same exposure protocol in the same age guinea-pigs as in the present study) resulted in smaller capsaicin-induced increases in lung resistance compared to the filtered air-exposed lungs (Joad et al. 1995), suggesting that the local axon reflex-mediated bronchoconstrictor response was diminished. These previous findings coupled with the current findings raise the possibility that chronic tobacco smoke exposure may upregulate central reflex components (augmenting changes in cough frequency and TE as defensive mechanisms) and downregulate local axon reflex components (perhaps minimizing the deleterious effects of local inflammation).
Of consideration is the physiological relevance of the prolonged TE. Numerous epidemiological studies have documented the adverse pulmonary effects in humans, particularly in children, exposed to environmental tobacco smoke (Dodge, 1982; Delay-Goyet & Lundberg, 1991; Frischer et al. 1992; Wang et al. 1994; Klonoff-Coher et al. 1996). These studies focused on wheeze, cough, pulmonary function and airway reactivity without examining corresponding changes in breathing pattern. Thus, we have no direct information on whether there are increases in TE in children during exposure to environmental tobacco smoke. A prolonged TE is a key element of the airway defensive reflex, temporarily delaying inhalation to curb further access of the injurious agent. Thus, augmented episodic increases in TE may be beneficial, serving as a protective mechanism in children chronically exposed to environmental tobacco smoke. Moreover, if, as certain evidence suggests, bronchopulmonary C-fibre stimulation reflexively elicits cough (Forsberg et al. 1988; Karlsson et al. 1988, 1991), then cough may also be augmented and this may help to explain the increased cough associated with chronic passive smoke exposure. On the other hand, the episodic increases in TE may be detrimental. Tatar et al. (1988) have proposed that the reflex cough associated with stimulation of the lung rapidly adapting receptors (RAR) may be suppressed during pulmonary C-fibre-evoked apnoea. If that is the case, the prolonged TE (by suppressing the RAR-induced cough reflex) may be detrimental, leading to further airway irritation and injury from acute exposures to irritants. This may in turn help to explain some of the respiratory symptoms, such as wheezing, mucous hypersecretion and increased frequency of asthma exacerbations associated with chronic passive smoke exposure. Moreover, given the strong link between passive smoke exposure and sudden infant death, the exaggerated lengthening of TE may be detrimental if apnoea is a prominent feature of the syndrome (Klonoff-Coher et al. 1996).
Because of the increased prevalence of respiratory symptoms in children chronically exposed to environmental tobacco smoke, we performed these studies in young guinea-pigs. Guinea-pigs are like humans in that they have advanced development of lung function and morphology at birth (Sosenko & Frank, 1987). They reach puberty at 35-70 days of life and have a maximum life span of 
7 years (Witschi, 1973). Thus, these guinea-pigs were exposed during the equivalent of human childhood and were tested during the period equivalent to human adolescence.
In conclusion, the increased responsiveness of NTS neurones in the bronchopulmonary C-fibre reflex pathway and prolonged apnoea suggest that chronic environmental tobacco smoke exposure changes respiratory function via a central reflex. The results may provide a mechanism to explain some of the respiratory symptoms and the increased incidence of sudden infant death syndrome in children exposed to passive smoke.
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This research was supported by funds from the California Tobacco-Related Disease Research Program, grant 6RT-0024. T.M. is supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. The authors gratefully acknowledge the excellent technical support provided by Judy Stewart and Dr Kent Pinkerton.
Corresponding author
A. C. Bonham: University of California, Davis, Division of Cardiovascular Medicine, TB 172 One Shields Avenue, Davis, CA 95616, USA.
Email: acbonham{at}ucdavis.edu
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, medial aspect of the nucleus. AP, area postrema; ts, tractus solitarius; C, central canal; X, dorsal motor nucleus of the vagus. Calibration bar is 500 µm for A and 1000 µm for B.
, time of injection of capsaicin.