Pulmonary Pharmacology (1992) 5, 191-198 PULMONARY PHARMACOLOGY
Relationship of Airway Responsiveness to Agents Causing Bronchoconstriction and Cough in Sensitized Guinea-pigs J.-A . Karlsson*, C . Zackrisson, J . Erjefalt, K . Forsberg Department of Exploratory Pharmacology, AB Draco, Box 34, S-221 00 Lund, Sweden
SUMMARY : The relationship between airway responsiveness to bronchoconstrictor- and cough-inducing stimuli has been examined in Ascaris suum-sensitized conscious guinea-pigs . Guinea-pigs were sensitized to Ascaris swum 14000 PNU and 100 mg Al(OH) 3 i .p. on days 1 and 71 and then challenged with aerosolized antigen on days 21, 28 and 35 . At day 35, antigen-exposure produced an early bronchoconstrictor response (EBR) and in about 50% of the animals also a late bronchoconstrictor response (LBR) commencing 4-8 h later . The bronchial responsiveness to inhaled histamine was increased in sensitized guinea-pigs and increased further 20-24 h after acute antigen challenge. Guinea-pigs developing only EBR were equally sensitive to histamine as those having both EBR and LBR . In contrast, the cough and reflex bronchoconstriction produced by inhaled citric acid (0.40 M, acting on capsaicinsensitive sensory neurons) and cigarette smoke (3 min exposure ; exciting both capsaicin-sensitive neurons and rapidly adapting stretch receptors) were not altered by sensitization . Furthermore, acute antigen challenge did not alter the effect of citric acid as measured 24 h later . The antigen-induced airway hyperresponsiveness to histamine was not accompanied by an altered sensitivity of airway sensory nerves mediating cough (and reflex bronchoconstriction), demonstrating that bronchial- (airway obstruction) and sensory- (cough) hyperresponsiveness involve separate and independent mechanisms .
However, contrary to these findings other investigators have been unable to detect differences between the effects of inhaled citric acid 10 and capsaicin" in asthmatic and healthy subjects . It therefore remains unclear whether airway hyperresponsiveness is accompanied by an increased responsiveness to cough stimuli . Airway hyperresponsiveness in asthma may be related to the degree of inflammation and the occurrence of a late bronchoconstrictor response after antigen challenge . Since we were interested in airway responsiveness to different types of mediators and irritants, we hypothesized that it would be advantageous to use an animal model comprising both EBR and LBR . Only a few guinea-pig models of allergeninduced early and late bronchoconstrictor responses have been described" , " and unfortunately there appear to be considerable variability in the occurrence of the LBR even in these models . More consistent early and late bronchoconstrictor responses were recently reported in Ascaris suum-sensitized guinea-pigs," and we therefore developed a model based on sensitization to this particular antigen . The purpose of the present study was to examine the relationship between airway responsiveness to bronchoconstrictor and cough stimuli in Ascaris
INTRODUCTION Exposure of the respiratory tract of allergic asthmatics to antigen causes an immediate, but reversible bronchoconstrictor response (EBR) . In many asthmatics a second period of airflow obstruction also develops 4-12 h after challenge . This late bronchoconstrictor response (LBR) is associated with an airways inflammatory reaction and often a further increase in airway hyperresponsiveness ." It has been speculated that the events involved in the LBR may be particularly relevant to clinical asthma .' ,' Airway hyperresponsiveness to non-specific stimuli is a characteristic feature of asthma . The observation that cough may be an early, and sometimes the only, symptom of asthma 7,8 suggests that airway hyperresponsiveness to bronchoconstrictor stimuli is accompanied by an increased sensitivity of sensory neurons in the airways . Indeed, Mitsuhashi and coworkers' have found that some asthmatic children are significantly more responsive to the tussive effect of nebulized acetic acid than adult asthmatics or healthy subjects. * For correspondence at : Rhone Poulenc Rorer Ltd ., Dagenham Research Centre, Rainham Road South, Dagenham, Essex RM 10 7XS, UK . 0952-0600/92/030191 + 08 $08 .00/0
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© 1992 Academic Press Limited
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suum-sensitized guinea-pigs and, thus, whether the development of bronchial and sensory-hyperresponsiveness involve separate mechanisms . The term 'sensory responsiveness' is used for the sensitivity of the respiratory tract to agents evoking a CNS-mediated protective reflex like cough . Conscious animals had to be used in the present experiments since cough and airway obstruction were measured simultaneously and anaesthetics suppress these airway reflexes to different degrees . Effects of inhaled histamine, citric acid and cigarette smoke were examined in naive, sensitized or sensitized and antigen-challenged animals . Nebulized citric acid, like capsaicin, produces cough and reflex bronchoconstriction through an action on capsaicin-sensitive sensory neurons which seems to involve an axon reflex within the airway wall ." , " Cigarette smoke-induced bronchospasm is also inhibited by capsaicin treatment whereas the cough reflex triggered by this irritant is mediated by other pathways ." In conscious guinea-pigs, histamine is resistant to capsaicin desensitization ." The present data demonstrate that airway- and sensory-hyperresponsiveness can occur independently and involve different mediators and mechanisms .
MATERIALS AND METHODS Sensitization procedure Guinea-pigs (Dunkin Hartley, Sahlins Malmo, Sweden) of either sex were used in this study . Guinea-pigs weighing 150-200 g were sensitized by i .p . administration of an Ascaris suum extract (Artu Biologicals, The Netherlands) . Two i .p . injections of a mixture of 4000 PNU Ascaris suum and 100 mg Al(OH) 3 were given 7 days apart . To increase the sensitivity, the guinea-pigs were repeatedly exposed to nebulized Ascaris suum (4000 PNU/ml in saline) at weekly intervals from day 21 onwards . The guinea-pigs were placed individually in a transparent perspex box and exposed to aerosol (generated by an MAD2 jet nebulizer driven by compressed air ; flow 51/min, output 0 .2 ml/min, 3 .0 µm MMAD) of Ascaris extract until bronchoconstriction developed (usually between 40 and 180s ; defined as the development of a slow laboured breathing with exaggerated abdominal movements, see below), or for a maximum period of 10 min .
Recording of
sGAW
in conscious guinea-pigs
Specific airway conductance (sGAw, 1 /kPa s) was determined in a constant-volume, whole-body plethysmograph which was constructed according to the principles described by Agrawal ." sGAw is calculated from air flow at the snout and an estimate of the
driving pressure . Conscious animals were placed in a restrainer that kept the head fixed . Air flow at the snout was measured with a Fleisch no . 0 pneumotachograph connected to a Validyne DP45-14 differential pressure transducer . Box pressure changes were recorded by a differential pressure transducer (Validyne DP45-14) . The pneumotachograph and the box pressure were calibrated daily with constant flows of air and rapid injections of boluses of air from a syringe, respectively . Air flow and box pressure signals were simultaneously delivered to an X-Y oscilloscope (Nicolet, 3091) and a flow pressure loop generated . The slope of the rising limb of this loop provided the ratio of airflow change, in the transition period from exhalation to inhalation, to the corresponding change in box pressure . During the transition period there is only a small change in lung volume, and therefore the temperature-humidity artefact in this ratio is negligible ." sGAW was calculated by dividing the slope of the rising limb of this loop by the barometric pressure minus water vapour pressure (at body temperature) ." Recording of
GAW
in anaesthetized guinea-pigs
Guinea-pigs were anaesthetized by i .v . pentobarbitone (30 mg/kg) . An incision was made in the neck, the trachea exposed and a tracheal cannula inserted . The animal was ventilated (Ugo Basile type 7025 Rodent ventilator) with a volume of 10 ml/kg at a rate of 80 breaths/min . Airflow was measured with a Fleisch pneumotachograph (model 000) connected to a Validyne (model DP 45) differential pressure transducer and recorded on a Grass model 7D polygraph . Transpulmonary pressure was recorded with a Validyne (model DP 45-14) differential pressure transducer and recorded on the Grass polygraph . Flow and pressure signals were fed into a Victor computer and airway conductance (GAW, l/kPa s) and tidal volume calculated on a breath per breath basis according to Amdur and Mead ." Each GAW value reported is the mean of five to ten consecutive breaths . Polyethylene catheters (PE 50) were inserted into a cartoid artery for recording of blood pressure and heart rate (Statham P23AC pressure transducer connected to a Grass polygraph) and into a jugular vein for injection of additional pentobarbitone and bolus doses of histamine . Measurement of cough and bronchoconstriction in conscious guinea-pigs To be able to study cough and bronchoconstrictor responses simultaneously in individual guinea-pigs, conscious unrestrained animals had to be used . Unfortunately, we were not able to measure cough frequency while the animals were restrained in the
Airway Responsiveness in Sensitized Guinea-pigs 193
plethysmograph used for recording of sGAw and therefore these experiments were performed separately . Thus, guinea-pigs were placed individually in a transparent perspex,chamber and exposed to nebulized aqueous solutions of either histamine (0 .70 mm) or citric acid (0 .40 M) for up to 7 min. Exposure to saline (vehicle) caused no respiratory effects . The aerosols were generated by an ultrasonic nebulizer (Omron NEUIO) and had an aerodynamic mass median diameter of 0 .9 gm. About 0.5 ml solution was nebulized per min . Conscious guinea-pigs were also acutely exposed to cigarette smoke (Kentucky 2R1 research cigarettes, Tobacco and Health Research Institute, Lexington, KT, USA) . In these experiments smoke from one cigarette was drawn continuously during 3 min and blown into a perspex box . The animals were continuously watched by a trained observer, who counted the number of coughs and noted the time to onset of bronchoconstriction (defined below) without knowing the previous treatment of the animal . The sounds of cough (which could readily be distinguished from sneezes) were recorded by a microphone inside the exposure chamber and amplified by use of a tape-recorder and loudspeaker . The number of coughs produced during the initial 3 min of exposure to citric acid or cigarette smoke was counted . Bronchoconstriction was defined as the abrupt onset
of a slow laboured breathing with exaggerated abdominal movements which correlated closely in time with the onset of a pronounced wheezing from the chest and with a significantly altered air flow (recorded on a Grass Polygraph, Fig . 1) . 16 This method measures changes in airways tone and with increasing concentrations of histamine 19 and citric acid20 the time to onset of bronchoconstriction is progressively shortened. Furthermore, inhaled citric acid has been shown to increase lung resistance in anaesthetized guinea-pigs ." ," The wheezing and altered breathing pattern could be rapidly and completely reversed by terbutaline (0 .4 gmol/kg i .p .) . Statistics and drugs Data are given as mean ± SEM . A positive LBR was defined as a secondary fall in sGAw/GAw amounting to > 25% and occurring after recovery from the immediate bronchospasm to antigen inhalation . Airway responses to antigen, histamine and citric acid were compared using analysis of variance (ANOVA) . If the overall F-value was significant, differences between group mean values were evaluated by use of Student's t-test for unpaired observations using the estimated standard deviation from the ANOVA . Parametric statistical tests were used to evaluate cough since we have previously shown that citric acid-
2 ml Ascaris
I
4000 PNU/ml, 40s
Cough
I Visible dyspnea
I-I Citric acid 0 .40 M, I min ft Coughs --------------------Visible dyspnea
-------
5s
Fig. 1 The tracings show tidal volume, electronically integrated from airflow, before and after exposure of conscious sensitized guinea-pigs to aerosols of either Ascaris suum or citric acid . Changes in breathing pattern (slow, laboured breathing) could be observed at the same time as changes in tidal volume were recorded . Note that coughing did not start until well after the onset of bronchoconstriction .
1 94
J .-A . Karlsson et al
(P
Relationship of Airway Responsiveness to Agents Causing Bronchoconstriction and Cough in Sensitized Guinea-pigs J.-A . Karlsson*, C . Zackrisson, J . Erjefalt, K . Forsberg Department of Exploratory Pharmacology, AB Draco, Box 34, S-221 00 Lund, Sweden
SUMMARY : The relationship between airway responsiveness to bronchoconstrictor- and cough-inducing stimuli has been examined in Ascaris suum-sensitized conscious guinea-pigs . Guinea-pigs were sensitized to Ascaris swum 14000 PNU and 100 mg Al(OH) 3 i .p. on days 1 and 71 and then challenged with aerosolized antigen on days 21, 28 and 35 . At day 35, antigen-exposure produced an early bronchoconstrictor response (EBR) and in about 50% of the animals also a late bronchoconstrictor response (LBR) commencing 4-8 h later . The bronchial responsiveness to inhaled histamine was increased in sensitized guinea-pigs and increased further 20-24 h after acute antigen challenge. Guinea-pigs developing only EBR were equally sensitive to histamine as those having both EBR and LBR . In contrast, the cough and reflex bronchoconstriction produced by inhaled citric acid (0.40 M, acting on capsaicinsensitive sensory neurons) and cigarette smoke (3 min exposure ; exciting both capsaicin-sensitive neurons and rapidly adapting stretch receptors) were not altered by sensitization . Furthermore, acute antigen challenge did not alter the effect of citric acid as measured 24 h later . The antigen-induced airway hyperresponsiveness to histamine was not accompanied by an altered sensitivity of airway sensory nerves mediating cough (and reflex bronchoconstriction), demonstrating that bronchial- (airway obstruction) and sensory- (cough) hyperresponsiveness involve separate and independent mechanisms .
However, contrary to these findings other investigators have been unable to detect differences between the effects of inhaled citric acid 10 and capsaicin" in asthmatic and healthy subjects . It therefore remains unclear whether airway hyperresponsiveness is accompanied by an increased responsiveness to cough stimuli . Airway hyperresponsiveness in asthma may be related to the degree of inflammation and the occurrence of a late bronchoconstrictor response after antigen challenge . Since we were interested in airway responsiveness to different types of mediators and irritants, we hypothesized that it would be advantageous to use an animal model comprising both EBR and LBR . Only a few guinea-pig models of allergeninduced early and late bronchoconstrictor responses have been described" , " and unfortunately there appear to be considerable variability in the occurrence of the LBR even in these models . More consistent early and late bronchoconstrictor responses were recently reported in Ascaris suum-sensitized guinea-pigs," and we therefore developed a model based on sensitization to this particular antigen . The purpose of the present study was to examine the relationship between airway responsiveness to bronchoconstrictor and cough stimuli in Ascaris
INTRODUCTION Exposure of the respiratory tract of allergic asthmatics to antigen causes an immediate, but reversible bronchoconstrictor response (EBR) . In many asthmatics a second period of airflow obstruction also develops 4-12 h after challenge . This late bronchoconstrictor response (LBR) is associated with an airways inflammatory reaction and often a further increase in airway hyperresponsiveness ." It has been speculated that the events involved in the LBR may be particularly relevant to clinical asthma .' ,' Airway hyperresponsiveness to non-specific stimuli is a characteristic feature of asthma . The observation that cough may be an early, and sometimes the only, symptom of asthma 7,8 suggests that airway hyperresponsiveness to bronchoconstrictor stimuli is accompanied by an increased sensitivity of sensory neurons in the airways . Indeed, Mitsuhashi and coworkers' have found that some asthmatic children are significantly more responsive to the tussive effect of nebulized acetic acid than adult asthmatics or healthy subjects. * For correspondence at : Rhone Poulenc Rorer Ltd ., Dagenham Research Centre, Rainham Road South, Dagenham, Essex RM 10 7XS, UK . 0952-0600/92/030191 + 08 $08 .00/0
191
© 1992 Academic Press Limited
1 92
J .-A . Karlsson et al
suum-sensitized guinea-pigs and, thus, whether the development of bronchial and sensory-hyperresponsiveness involve separate mechanisms . The term 'sensory responsiveness' is used for the sensitivity of the respiratory tract to agents evoking a CNS-mediated protective reflex like cough . Conscious animals had to be used in the present experiments since cough and airway obstruction were measured simultaneously and anaesthetics suppress these airway reflexes to different degrees . Effects of inhaled histamine, citric acid and cigarette smoke were examined in naive, sensitized or sensitized and antigen-challenged animals . Nebulized citric acid, like capsaicin, produces cough and reflex bronchoconstriction through an action on capsaicin-sensitive sensory neurons which seems to involve an axon reflex within the airway wall ." , " Cigarette smoke-induced bronchospasm is also inhibited by capsaicin treatment whereas the cough reflex triggered by this irritant is mediated by other pathways ." In conscious guinea-pigs, histamine is resistant to capsaicin desensitization ." The present data demonstrate that airway- and sensory-hyperresponsiveness can occur independently and involve different mediators and mechanisms .
MATERIALS AND METHODS Sensitization procedure Guinea-pigs (Dunkin Hartley, Sahlins Malmo, Sweden) of either sex were used in this study . Guinea-pigs weighing 150-200 g were sensitized by i .p . administration of an Ascaris suum extract (Artu Biologicals, The Netherlands) . Two i .p . injections of a mixture of 4000 PNU Ascaris suum and 100 mg Al(OH) 3 were given 7 days apart . To increase the sensitivity, the guinea-pigs were repeatedly exposed to nebulized Ascaris suum (4000 PNU/ml in saline) at weekly intervals from day 21 onwards . The guinea-pigs were placed individually in a transparent perspex box and exposed to aerosol (generated by an MAD2 jet nebulizer driven by compressed air ; flow 51/min, output 0 .2 ml/min, 3 .0 µm MMAD) of Ascaris extract until bronchoconstriction developed (usually between 40 and 180s ; defined as the development of a slow laboured breathing with exaggerated abdominal movements, see below), or for a maximum period of 10 min .
Recording of
sGAW
in conscious guinea-pigs
Specific airway conductance (sGAw, 1 /kPa s) was determined in a constant-volume, whole-body plethysmograph which was constructed according to the principles described by Agrawal ." sGAw is calculated from air flow at the snout and an estimate of the
driving pressure . Conscious animals were placed in a restrainer that kept the head fixed . Air flow at the snout was measured with a Fleisch no . 0 pneumotachograph connected to a Validyne DP45-14 differential pressure transducer . Box pressure changes were recorded by a differential pressure transducer (Validyne DP45-14) . The pneumotachograph and the box pressure were calibrated daily with constant flows of air and rapid injections of boluses of air from a syringe, respectively . Air flow and box pressure signals were simultaneously delivered to an X-Y oscilloscope (Nicolet, 3091) and a flow pressure loop generated . The slope of the rising limb of this loop provided the ratio of airflow change, in the transition period from exhalation to inhalation, to the corresponding change in box pressure . During the transition period there is only a small change in lung volume, and therefore the temperature-humidity artefact in this ratio is negligible ." sGAW was calculated by dividing the slope of the rising limb of this loop by the barometric pressure minus water vapour pressure (at body temperature) ." Recording of
GAW
in anaesthetized guinea-pigs
Guinea-pigs were anaesthetized by i .v . pentobarbitone (30 mg/kg) . An incision was made in the neck, the trachea exposed and a tracheal cannula inserted . The animal was ventilated (Ugo Basile type 7025 Rodent ventilator) with a volume of 10 ml/kg at a rate of 80 breaths/min . Airflow was measured with a Fleisch pneumotachograph (model 000) connected to a Validyne (model DP 45) differential pressure transducer and recorded on a Grass model 7D polygraph . Transpulmonary pressure was recorded with a Validyne (model DP 45-14) differential pressure transducer and recorded on the Grass polygraph . Flow and pressure signals were fed into a Victor computer and airway conductance (GAW, l/kPa s) and tidal volume calculated on a breath per breath basis according to Amdur and Mead ." Each GAW value reported is the mean of five to ten consecutive breaths . Polyethylene catheters (PE 50) were inserted into a cartoid artery for recording of blood pressure and heart rate (Statham P23AC pressure transducer connected to a Grass polygraph) and into a jugular vein for injection of additional pentobarbitone and bolus doses of histamine . Measurement of cough and bronchoconstriction in conscious guinea-pigs To be able to study cough and bronchoconstrictor responses simultaneously in individual guinea-pigs, conscious unrestrained animals had to be used . Unfortunately, we were not able to measure cough frequency while the animals were restrained in the
Airway Responsiveness in Sensitized Guinea-pigs 193
plethysmograph used for recording of sGAw and therefore these experiments were performed separately . Thus, guinea-pigs were placed individually in a transparent perspex,chamber and exposed to nebulized aqueous solutions of either histamine (0 .70 mm) or citric acid (0 .40 M) for up to 7 min. Exposure to saline (vehicle) caused no respiratory effects . The aerosols were generated by an ultrasonic nebulizer (Omron NEUIO) and had an aerodynamic mass median diameter of 0 .9 gm. About 0.5 ml solution was nebulized per min . Conscious guinea-pigs were also acutely exposed to cigarette smoke (Kentucky 2R1 research cigarettes, Tobacco and Health Research Institute, Lexington, KT, USA) . In these experiments smoke from one cigarette was drawn continuously during 3 min and blown into a perspex box . The animals were continuously watched by a trained observer, who counted the number of coughs and noted the time to onset of bronchoconstriction (defined below) without knowing the previous treatment of the animal . The sounds of cough (which could readily be distinguished from sneezes) were recorded by a microphone inside the exposure chamber and amplified by use of a tape-recorder and loudspeaker . The number of coughs produced during the initial 3 min of exposure to citric acid or cigarette smoke was counted . Bronchoconstriction was defined as the abrupt onset
of a slow laboured breathing with exaggerated abdominal movements which correlated closely in time with the onset of a pronounced wheezing from the chest and with a significantly altered air flow (recorded on a Grass Polygraph, Fig . 1) . 16 This method measures changes in airways tone and with increasing concentrations of histamine 19 and citric acid20 the time to onset of bronchoconstriction is progressively shortened. Furthermore, inhaled citric acid has been shown to increase lung resistance in anaesthetized guinea-pigs ." ," The wheezing and altered breathing pattern could be rapidly and completely reversed by terbutaline (0 .4 gmol/kg i .p .) . Statistics and drugs Data are given as mean ± SEM . A positive LBR was defined as a secondary fall in sGAw/GAw amounting to > 25% and occurring after recovery from the immediate bronchospasm to antigen inhalation . Airway responses to antigen, histamine and citric acid were compared using analysis of variance (ANOVA) . If the overall F-value was significant, differences between group mean values were evaluated by use of Student's t-test for unpaired observations using the estimated standard deviation from the ANOVA . Parametric statistical tests were used to evaluate cough since we have previously shown that citric acid-
2 ml Ascaris
I
4000 PNU/ml, 40s
Cough
I Visible dyspnea
I-I Citric acid 0 .40 M, I min ft Coughs --------------------Visible dyspnea
-------
5s
Fig. 1 The tracings show tidal volume, electronically integrated from airflow, before and after exposure of conscious sensitized guinea-pigs to aerosols of either Ascaris suum or citric acid . Changes in breathing pattern (slow, laboured breathing) could be observed at the same time as changes in tidal volume were recorded . Note that coughing did not start until well after the onset of bronchoconstriction .
1 94
J .-A . Karlsson et al
(P
