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Cough reflex responses during pulmonary C-fibre receptor activation in anesthetized rabbits
Neuroscience Letters 448 (2008) 200–203
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Cough reflex responses during pulmonary C-fibre receptor activation in anesthetized rabbits Donatella Mutolo a , Fulvia Bongianni a , Elenia Cinelli a , Giovanni A. Fontana b , Tito Pantaleo a,∗ a b
Dipartimento di Scienze Fisiologiche, Unità Funzionale di Medicina Respiratoria, Università degli Studi di Firenze, Viale G.B. Morgagni 63, 50134 Firenze, Italy Dipartimento di Area Critica Medico Chirurgica, Unità Funzionale di Medicina Respiratoria, Università degli Studi di Firenze, Viale G.B. Morgagni, 50134 Firenze, Italy
a r t i c l e
i n f o
Article history: Received 3 September 2008 Received in revised form 16 October 2008 Accepted 17 October 2008 Keywords: Cough Phenylbiguanide Pulmonary chemoreflex Pulmonary C-fibres Rabbit Respiratory reflexes
a b s t r a c t We investigated the changes induced by pulmonary C-fibre receptor activation in the cough reflex evoked by mechanical stimulation of the tracheobronchial tree in pentobarbitone anesthetized, spontaneously breathing rabbits. Phrenic nerve and abdominal muscle activities were monitored along with tracheal and arterial blood pressures. The activation of pulmonary C-fibre receptors by means of right atrial injection of phenylbiguanide (PBG) caused the pulmonary chemoreflex characterized by tachypnea, bradycardia and hypotension. During the pulmonary chemoreflex, the time components (total cycle duration, inspiratory and expiratory times) of the cough motor pattern significantly decreased, whereas no consistent changes in peak phrenic and abdominal activity, peak tracheal pressure and number of coughs evoked by each stimulation trial were observed. At variance with previous findings in cats and dogs, the results show that tracheobronchial cough is not significantly reduced in the rabbit during PBG-induced chemoreflex. This study is the first to provide evidence supporting the hypothesis that the time components of the cough motor pattern are, to some extent, dependent upon the timing characteristics of the ongoing respiratory activity and suggests a novel mechanism leading to cough depression. © 2008 Elsevier Ireland Ltd. All rights reserved.
Cough is a very important defensive reflex aimed at removing mucus and foreign particles from the respiratory tract ([9]; see also [13] for further Refs.). During the activation of pulmonary C-fibre receptors by intravenous injections of capsaicin or phenylbiguanide (PBG) in anesthetized cats and dogs the cough reflex from the larynx and the tracheobronchial tree is inhibited or abolished [6,21,22,25]. The same is true for stimulation of bronchial C-fibres in dogs [21,25]. In particular, it has been reported that cough is abolished during apnea, i.e. the initial phase of the pulmonary chemoreflex due to C-fibre receptor stimulation, and significantly reduced during the rapid shallow breathing that immediately follows apnea. These respiratory effects were accompanied by marked bradycardia and hypotension. In the present study we investigated the influence of pulmonary C-fibre receptor activation on the cough reflex in the spontaneously breathing anesthetized rabbit, i.e. a different animal species that recently we have shown to be suitable for studies on the physiology and pharmacology of cough [5,12,13]. In more detail, we have shown that ionotropic glutamate receptors located in the caudal portion of the nucleus tractus solitarii mediate the cough reflex evoked by the mechanical stimulation of the tracheobronchial tree
∗ Corresponding author. Tel.: +39 055 4237311/316; fax: +39 055 4379506. E-mail address: tito.pantaleo@unifi.it (T. Pantaleo). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.10.057
in the rabbit [13] and that this medullary region is a site of action of some centrally acting antitussive drugs [12]. The pulmonary chemoreflex can be induced in several animal species, including the rabbit [7,8,10,24]. In general agreement with previous findings, we have recently shown that in the anesthetized rabbit this reflex response, evoked by right atrial injections of PBG, lacks the initial apneic phase and mainly consists of tachypnea, bradycardia and hypotension [13]. Experiments were performed on five male New Zealand white rabbits (2.7–3.2 kg) anesthetized with sodium pentobarbital (40 mg/kg i.v., supplemented by 2–4 mg/kg every 30 min; Sigma–Aldrich, St. Louis, MO). The adequacy of anesthesia was assessed by the absence of reflex withdrawal of the hindlimb in response to noxious pinching of the hindpaw. All animal care and experimental procedures were conducted in accordance with the Italian legislation and the official regulations of the European Community Council on the use of laboratory animals (Directive 86/609/EEC). The study was approved by the Animal Care and Use Committee of the University of Florence. Experimental procedures and details about the methods employed have been previously described [5,12,13,15]. After cannulation of the trachea, polyethylene catheters were inserted into the right femoral artery and vein for the measurement of arterial blood pressure and drug administration, respectively. The tip of the polyethylene cannula inserted into the femoral vein was
D. Mutolo et al. / Neuroscience Letters 448 (2008) 200–203
advanced to the right atrium to evoke the pulmonary chemoreflex by right atrial injections of PBG (Sigma–Aldrich). The position of the cannula was confirmed post-mortem. The animal was placed in a prone position and fixed by a stereotaxic head holder and vertebral clamps. Rectal temperature was maintained at 38.5–39 ◦ C. The efferent phrenic nerve activity was recorded using bipolar platinum electrodes from the central stump of a cut and desheathed C3 or C5 phrenic root. The electromyographic (EMG) activity of abdominal muscles was recorded by wire electrodes (Nichrome wires, insulated except for 1 mm at the tips, diameter 0.1 mm) inserted into the external or the internal oblique abdominal muscles. Phrenic and abdominal activities were amplified, full-wave rectified, and “integrated” (low-pass RC filter, time constant 100 ms). Intratracheal and arterial blood pressure as well as end-tidal CO2 partial pressure were monitored. The signals of all studied variables were acquired and analyzed using a personal computer, equipped with an analogto-digital interface (Digidata 1200, Axon Instruments, Union City, CA) and appropriate software (Axoscope, Axon Instruments). Cough was induced by means of a 0.5-mm diameter nylon fibre inserted through a lateral port of the tracheal cannula until the tip was judged to be near the carina and main bronchi [4,5,12,13,15,23]. Back and forth movements of the fibre aimed at touching repeatedly (approximately 1 time/s) the carina or nearby airway walls were made over periods of 4–5 s. These manoeuvres were executed by the same experimenter in order to maintain stimulation intensity and frequency as far as possible at similar levels (see e.g. Refs. [5,12,13]). PBG was dissolved in 0.9% NaCl solution and administered in dose of 80 g/kg in 0.3 ml volumes over about 2 s (see e.g. Ref. [8]). The volume of PBG utilized was less than the dead space of the catheter. The PBG was loaded into the catheter, and the drug was infused with a 0.5-ml saline flush. Three right atrial injections of PBG were performed in each animal at an interval of about 60 min. Mechanical stimulation of the tracheobronchial tree was applied in the control period as well as within 10 and 20 s from the PBG injection, respectively. The tussigenic stimulation was also repeated after complete recovery of control breathing. Cardiorespiratory variables were measured according to the procedures described in previous reports [12,13] during eupneic breathing and cough efforts. To evaluate the intensity of the pulmonary chemoreflex, respiratory frequency and mean arterial blood pressure were measured and averaged both during the period (10 s) immediately preceding PBG injections and during the time period (3–4 s) immediately following the beginning of cardiorespiratory responses. Average values for each period were taken as single measurements for the purpose of analysis (Student’s paired t-tests). Peak amplitude of phrenic nerve and abdominal EMG activities were measured on integrated traces and normalized by expressing them in relative units (RUs), i.e. as a fraction of the highest amplitude observed in each animal (see e.g. Refs. [2,13]). The highest peak
201
value was consistently obtained during coughing. Cough-related variables included cough-related total duration of the respiratory cycle (TT ), cough-related inspiratory (TI ) and expiratory (TE ) times, peak phrenic amplitude, peak abdominal activity, peak tracheal pressure and the cough number, i.e. the number of coughs evoked by each stimulation trial. The time components of the cough motor pattern were measured on recordings of raw respiratory activity. In each animal, cough-related variables were measured and averaged before PBG injections, at each of the two scheduled times during the pulmonary chemoreflex and after the complete recovery of control respiratory pattern. The average values of cough-related variables for each period in each preparation (three stimulation trials, during three PBG-induced pulmonary chemoreflexes) were taken as single measurements for subsequent analysis by means of one-way repeated measures analysis of variance followed by Student–Newman–Keuls tests. All reported values are means ± S.E.; P < 0.05 was taken as significant. In agreement with our previous findings [13], the rapid bolus injections of PBG into the right atrium consistently caused tachypnea, bradycardia and hypotension often accompanied by the development of some tonic inspiratory activity and decreases in expiratory muscle discharges. In the period immediately following (3–4 s) the beginning of PBG-induced responses, respiratory frequency raised from 50.0 ± 2.7 to 126.2 ± 6.2 breaths/min, while mean arterial blood pressure decreased from 95.0 ± 1.4 to 58.0 ± 2.9 mmHg. A complete recovery was achieved within 20 min. Cough responses to mechanical stimulation of the tracheobronchial tree were characterized by repeated coughs consisting of coordinated bursts of inspiratory and expiratory activity [12]. On few occasions, the first obvious event in a cough epoch was a small-amplitude expiratory effort not preceded by an augmented inspiration and beginning during the expiratory phase. These expiratory bursts fit more appropriately the definition of expiration reflex. We have extensively reported information and discussion on this reflex in previous papers ([12,13]; see also Ref. [19]). These expiratory responses were not considered for data analysis. Changes in cough-related variables observed during the pulmonary chemoreflex are reported in Table 1 and illustrated by original recordings in Fig. 1. Noticeably, significant changes were observed in the time components of the cough motor pattern consisting of significant reductions in cough-related TT due to decreases in both TI and TE . No consistent changes were observed in the other variables, whose values turned out to be slightly but not significantly smaller during the pulmonary chemoreflex. The present results show that the cough motor pattern during PBG-induced pulmonary chemoreflexes displays marked changes in its time components, without consistent changes in the other variables. Therefore, they are at variance with previous findings obtained in cats [6,22,25] and dogs [21]. Tatar et al. [21,22] gave
Table 1 Changes in cough-related variables during the pulmonary chemoreflex evoked by right atrial injections of phenylbiguanide (n = 5).
CN TT (s) TI (s) TE (s) PPA (RUs) PAA (RUs) TP (cmH2 O)
Control
First cough challenge within 10 s after PBG
Second cough challenge within 20 s after PBG
Cough challenge 25 min after PBG
3.8 ± 0.20 0.60 ± 0.03 0.41 ± 0.02 0.18 ± 0.01 0.59 ± 0.02 0.52 ± 0.02 7.22 ± 0.12
3.4 ± 0.22 0.47 ± 0.01** 0.35 ± 0.005* 0.12 ± 0.004* 0.58 ± 0.02 0.49 ± 0.02 7.20 ± 0.17
3.4 ± 0.21 0.52 ± 0.01** 0.37 ± 0.005* 0.14 ± 0.005* 0.57 ± 0.02 0.50 ± 0.02 7.20 ± 0.15
3.6 ± 0.24 0.62 ± 0.02 0.42 ± 0.02 0.18 ± 0.01 0.58 ± 0.01 0.50 ± 0.01 7.22 ± 0.18
Values are means ± S.E.; CN, cough number; TT , cycle duration; TI , inspiratory time; TE , expiratory time; PPA, peak phrenic activity in relative units (RUs); PAA, peak abdominal activity; TP, tracheal pressure (cmH2 O). * P < 0.05 compared with controls. ** P < 0.01 compared with controls.
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D. Mutolo et al. / Neuroscience Letters 448 (2008) 200–203
Fig. 1. Changes in the cough reflex induced by the mechanical stimulation of the trachebronchial tree at different times during the pulmonary chemoreflex and after recovery of control breathing in one anesthetized rabbit. The pulmonary chemoreflex was evoked by a right atrial injection of 240 g PBG (arrow). Traces are: Phr IN, phrenic integrated neurogram; Phr N, phrenic neurogram; Abd IEMG, abdominal integrated electromyographic activity; Abd EMG, abdominal electromyographic activity; BP, arterial blood pressure; TP, tracheal pressure. Tussigenic stimulation marked by filled bars.
PBG or capsaicin intravenously in a bolus, probably targeting pulmonary C-fibres, resulting in profound depression of respiration, i.e. apnea, followed by rapid and shallow breathing. Given that the neural network implicated in the generation of the eupneic pattern of breathing is also involved in the production of the cough motor pattern [1,3,4,15–18], it is not surprising that coughing is abolished under conditions where central respiratory drive is profoundly depressed. However, in agreement with Widdicombe and Singh [25], it seems unlikely that the suppression of cough observed in cats and dogs is related to the apnea per se. In fact, Tatar et al. [22] have reported that the strength of cough was significantly reduced during the phase of rapid and shallow breathing and that cough was decreased, but not abolished during apnea induced by intravenous injections of veratrine in the cat [20]. In keeping with this interpretation, we have observed that the cough reflex evoked by mechanical stimulation of the tracheobronchial tree is still present, although greatly attenuated during apnea induced by severe hyperventilatory hypocapnia (Bongianni F, Mutolo D, Fontana GA, Pantaleo T, unpublished observations). We do not know the reasons of the discrepancies between present and previous results. However, we believe that differences in the animal species employed may have played the major role. Present results do not allow us to establish whether cough-related changes in timing are directly dependent upon the activation of pulmonary C-fibres or are secondary to the increases in respiratory frequency. Nevertheless, the observed changes in the time components of the cough motor pattern support for the first time the hypothesis that the neural mechanisms generating cough are dependent upon the characteristics of the ongoing respiratory activity. In particular, it appears that the timing of the cough motor pattern is, to some extent, related to the timing of baseline respiratory activity, thus implying that the reconfigured respiratory pattern generator during coughing (see e.g. Refs. [1,4,16,18]) retains memory of the preceding respiratory pattern. Furthermore, it seems reasonable to hypothesize that decreases in duration of cough-related TI and TE beyond a certain limit may lead to reductions in the peak amplitude of inspiratory and expiratory cough efforts due to the lack of sufficient time for their development. As an extreme consequence of this phenomenon at very high respiratory frequency or even during tonic activity [11,14], reductions in the cough number or cough suppression could be produced without any involvement of an inhibitory action of pulmonary C-fibres. Interestingly, the results also suggest that further studies may increase our knowledge on the distinctive features of the pulmonary chemoreflex in different animal species. In addition, comparative studies performed by using not only mechanical but also other tussigenic stimuli, such as inhalation of citric acid or
other appropriate irritants, would be of importance for a better understanding of the role played by pulmonary C-fibres in the control of the cough reflex. Acknowledgements This study was supported by grants from the University of Florence and the Ministero dell’Istruzione, dell’Università e della Ricerca of Italy. References [1] D.C. Bolser, P.W. Davenport, F.J. Golder, D. Baekey, K. Morris, B. Lindsey, R. Shannon, Neurogenesis of cough, in: F. Chung, J. Widdicombe, H. Boushey (Eds.), Cough: Causes, Mechanisms and Therapy, Blackwell Publishing, Oxford, UK, 2003, pp. 173–180. [2] D.C. Bolser, J.A. Hey, R.W. Chapman, Influence of central antitussive drugs on the cough motor pattern, J. Appl. Physiol. 86 (1999) 1017–1024. [3] D.C. Bolser, I. Poliacek, J. Jakus, D.D. Fuller, P.W. Davenport, Neurogenesis of cough, other airway defensive behaviors and breathing: A holarchical system? Respir. Physiol. Neurobiol. 152 (2006) 255–265. [4] F. Bongianni, D. Mutolo, G.A. Fontana, T. Pantaleo, Discharge patterns of Bötzinger complex neurons during cough in the cat, Am. J. Physiol. 274 (1998) R1015–R1024. [5] F. Bongianni, D. Mutolo, F. Nardone, T. Pantaleo, Ionotropic glutamate receptors mediate excitatory drive to caudal medullary expiratory neurons in the rabbit, Brain Res. 1056 (2005) 145–157. [6] J. Hanacek, M. Tatar, J. Widdicombe, Regulation of cough by secondary sensory inputs, Respir. Physiol. Neurobiol. 152 (2006) 282–297. [7] W. Karczewski, J.G. Widdicombe, The role of the vagus nerves in the respiratory and circulatory responses to intravenous histamine and phenyl diguanide in rabbits, J. Physiol. 201 (1969) 271–291. [8] I.S. Kay, D.J. Armstrong, Phenylbiguanide not phenyldiguanide is used to evoke the pulmonary chemoreflex in anaesthetized rabbits, Exp. Physiol. 75 (1990) 383–389. [9] J. Korpáˇs, Z. Tomori, Cough and Other Respiratory Reflexes, Karger, Basel, Switzerland, 1979, 1–356 pp. [10] L.Y. Lee, T.E. Pisarri, Afferent properties and reflex functions of bronchopulmonary C-fibers, Respir. Physiol. 125 (2001) 47–65. [11] D. Mutolo, F. Bongianni, M. Carfi, T. Pantaleo, Respiratory changes induced by kainic acid lesions in rostral ventral respiratory group of rabbits, Am. J. Physiol. 283 (2002) R227–R242. [12] D. Mutolo, F. Bongianni, E. Cinelli, G.A. Fontana, T. Pantaleo, Modulation of the cough reflex by antitussive agents within the caudal aspect of the nucleus tractus solitarii in the rabbit, Am. J. Physiol. Regul. Integr. Comp. Physiol. 295 (2008) R243–R251. [13] D. Mutolo, F. Bongianni, G.A. Fontana, T. Pantaleo, The role of excitatory amino acids and substance P in the mediation of the cough reflex within the nucleus tractus solitarii of the rabbit, Brain Res. Bull. 74 (2007) 284–293. [14] D. Mutolo, F. Bongianni, F. Nardone, T. Pantaleo, Respiratory responses evoked by blockades of ionotropic glutamate receptors within the Bötzinger complex and the pre-Bötzinger complex of the rabbit, Eur. J. Neurosci. 21 (2005) 122–134. [15] D. Mutolo, F. Bongianni, T. Pantaleo, Effects of lignocaine blockades and kainic acid lesions in the Bötzinger complex on spontaneous expiratory activity and cough reflex responses in the rabbit, Neurosci. Lett. 332 (2002) 175– 179.
D. Mutolo et al. / Neuroscience Letters 448 (2008) 200–203 [16] T. Pantaleo, F. Bongianni, D. Mutolo, Central nervous mechanisms of cough, Pulm. Pharmacol. Ther. 15 (2002) 227–233. [17] R. Shannon, D.M. Baekey, K.F. Morris, Z. Li, B.G. Lindsey, Functional connectivity among ventrolateral medullary respiratory neurones and responses during fictive cough in the cat, J. Physiol. 525 (2000) 207–224. [18] R. Shannon, D.M. Baekey, K.F. Morris, S.C. Nuding, L.S. Segers, B.G. Lindsey, Production of reflex cough by brainstem respiratory networks, Pulm. Pharmacol. Ther. 17 (2004) 369–376. [19] M. Tatar, J. Hanacek, J. Widdicombe, The expiration reflex from the trachea and bronchi, Eur. Respir. J. 31 (2007) 385–390. [20] M. Tatar, B. Nagyova, J.G. Widdicombe, Veratrine-induced reflexes and cough, Respir. Med. 85 (Suppl. A) (1991) 51–55.
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[21] M. Tatar, G. Sant’Ambrogio, F.B. Sant’Ambrogio, Laryngeal and tracheobronchial cough in anesthetized dogs, J. Appl. Physiol. 76 (1994) 2672–2679. [22] M. Tatar, S.E. Webber, J.G. Widdicombe, Lung C-fibre receptor activation and defensive reflexes in anaesthetized cats, J. Physiol. 402 (1988) 411–420. [23] Z. Tomori, J.G. Widdicombe, Muscular, bronchomotor and cardiovascular reflexes elicited by mechanical stimulation of the respiratory tract, J. Physiol. 200 (1969) 25–49. [24] D. Trenchard, N.J. Russell, H.E. Raybould, Non-myelinated vagal lung receptors and their reflex effects on respiration in rabbits, Respir. Physiol. 55 (1984) 63–79. [25] J. Widdicombe, V. Singh, Physiological and pathophysiological down-regulation of cough, Respir. Physiol. Neurobiol. 150 (2006) 105–117.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Cough reflex responses during pulmonary C-fibre receptor activation in anesthetized rabbits Donatella Mutolo a , Fulvia Bongianni a , Elenia Cinelli a , Giovanni A. Fontana b , Tito Pantaleo a,∗ a b
Dipartimento di Scienze Fisiologiche, Unità Funzionale di Medicina Respiratoria, Università degli Studi di Firenze, Viale G.B. Morgagni 63, 50134 Firenze, Italy Dipartimento di Area Critica Medico Chirurgica, Unità Funzionale di Medicina Respiratoria, Università degli Studi di Firenze, Viale G.B. Morgagni, 50134 Firenze, Italy
a r t i c l e
i n f o
Article history: Received 3 September 2008 Received in revised form 16 October 2008 Accepted 17 October 2008 Keywords: Cough Phenylbiguanide Pulmonary chemoreflex Pulmonary C-fibres Rabbit Respiratory reflexes
a b s t r a c t We investigated the changes induced by pulmonary C-fibre receptor activation in the cough reflex evoked by mechanical stimulation of the tracheobronchial tree in pentobarbitone anesthetized, spontaneously breathing rabbits. Phrenic nerve and abdominal muscle activities were monitored along with tracheal and arterial blood pressures. The activation of pulmonary C-fibre receptors by means of right atrial injection of phenylbiguanide (PBG) caused the pulmonary chemoreflex characterized by tachypnea, bradycardia and hypotension. During the pulmonary chemoreflex, the time components (total cycle duration, inspiratory and expiratory times) of the cough motor pattern significantly decreased, whereas no consistent changes in peak phrenic and abdominal activity, peak tracheal pressure and number of coughs evoked by each stimulation trial were observed. At variance with previous findings in cats and dogs, the results show that tracheobronchial cough is not significantly reduced in the rabbit during PBG-induced chemoreflex. This study is the first to provide evidence supporting the hypothesis that the time components of the cough motor pattern are, to some extent, dependent upon the timing characteristics of the ongoing respiratory activity and suggests a novel mechanism leading to cough depression. © 2008 Elsevier Ireland Ltd. All rights reserved.
Cough is a very important defensive reflex aimed at removing mucus and foreign particles from the respiratory tract ([9]; see also [13] for further Refs.). During the activation of pulmonary C-fibre receptors by intravenous injections of capsaicin or phenylbiguanide (PBG) in anesthetized cats and dogs the cough reflex from the larynx and the tracheobronchial tree is inhibited or abolished [6,21,22,25]. The same is true for stimulation of bronchial C-fibres in dogs [21,25]. In particular, it has been reported that cough is abolished during apnea, i.e. the initial phase of the pulmonary chemoreflex due to C-fibre receptor stimulation, and significantly reduced during the rapid shallow breathing that immediately follows apnea. These respiratory effects were accompanied by marked bradycardia and hypotension. In the present study we investigated the influence of pulmonary C-fibre receptor activation on the cough reflex in the spontaneously breathing anesthetized rabbit, i.e. a different animal species that recently we have shown to be suitable for studies on the physiology and pharmacology of cough [5,12,13]. In more detail, we have shown that ionotropic glutamate receptors located in the caudal portion of the nucleus tractus solitarii mediate the cough reflex evoked by the mechanical stimulation of the tracheobronchial tree
∗ Corresponding author. Tel.: +39 055 4237311/316; fax: +39 055 4379506. E-mail address: tito.pantaleo@unifi.it (T. Pantaleo). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.10.057
in the rabbit [13] and that this medullary region is a site of action of some centrally acting antitussive drugs [12]. The pulmonary chemoreflex can be induced in several animal species, including the rabbit [7,8,10,24]. In general agreement with previous findings, we have recently shown that in the anesthetized rabbit this reflex response, evoked by right atrial injections of PBG, lacks the initial apneic phase and mainly consists of tachypnea, bradycardia and hypotension [13]. Experiments were performed on five male New Zealand white rabbits (2.7–3.2 kg) anesthetized with sodium pentobarbital (40 mg/kg i.v., supplemented by 2–4 mg/kg every 30 min; Sigma–Aldrich, St. Louis, MO). The adequacy of anesthesia was assessed by the absence of reflex withdrawal of the hindlimb in response to noxious pinching of the hindpaw. All animal care and experimental procedures were conducted in accordance with the Italian legislation and the official regulations of the European Community Council on the use of laboratory animals (Directive 86/609/EEC). The study was approved by the Animal Care and Use Committee of the University of Florence. Experimental procedures and details about the methods employed have been previously described [5,12,13,15]. After cannulation of the trachea, polyethylene catheters were inserted into the right femoral artery and vein for the measurement of arterial blood pressure and drug administration, respectively. The tip of the polyethylene cannula inserted into the femoral vein was
D. Mutolo et al. / Neuroscience Letters 448 (2008) 200–203
advanced to the right atrium to evoke the pulmonary chemoreflex by right atrial injections of PBG (Sigma–Aldrich). The position of the cannula was confirmed post-mortem. The animal was placed in a prone position and fixed by a stereotaxic head holder and vertebral clamps. Rectal temperature was maintained at 38.5–39 ◦ C. The efferent phrenic nerve activity was recorded using bipolar platinum electrodes from the central stump of a cut and desheathed C3 or C5 phrenic root. The electromyographic (EMG) activity of abdominal muscles was recorded by wire electrodes (Nichrome wires, insulated except for 1 mm at the tips, diameter 0.1 mm) inserted into the external or the internal oblique abdominal muscles. Phrenic and abdominal activities were amplified, full-wave rectified, and “integrated” (low-pass RC filter, time constant 100 ms). Intratracheal and arterial blood pressure as well as end-tidal CO2 partial pressure were monitored. The signals of all studied variables were acquired and analyzed using a personal computer, equipped with an analogto-digital interface (Digidata 1200, Axon Instruments, Union City, CA) and appropriate software (Axoscope, Axon Instruments). Cough was induced by means of a 0.5-mm diameter nylon fibre inserted through a lateral port of the tracheal cannula until the tip was judged to be near the carina and main bronchi [4,5,12,13,15,23]. Back and forth movements of the fibre aimed at touching repeatedly (approximately 1 time/s) the carina or nearby airway walls were made over periods of 4–5 s. These manoeuvres were executed by the same experimenter in order to maintain stimulation intensity and frequency as far as possible at similar levels (see e.g. Refs. [5,12,13]). PBG was dissolved in 0.9% NaCl solution and administered in dose of 80 g/kg in 0.3 ml volumes over about 2 s (see e.g. Ref. [8]). The volume of PBG utilized was less than the dead space of the catheter. The PBG was loaded into the catheter, and the drug was infused with a 0.5-ml saline flush. Three right atrial injections of PBG were performed in each animal at an interval of about 60 min. Mechanical stimulation of the tracheobronchial tree was applied in the control period as well as within 10 and 20 s from the PBG injection, respectively. The tussigenic stimulation was also repeated after complete recovery of control breathing. Cardiorespiratory variables were measured according to the procedures described in previous reports [12,13] during eupneic breathing and cough efforts. To evaluate the intensity of the pulmonary chemoreflex, respiratory frequency and mean arterial blood pressure were measured and averaged both during the period (10 s) immediately preceding PBG injections and during the time period (3–4 s) immediately following the beginning of cardiorespiratory responses. Average values for each period were taken as single measurements for the purpose of analysis (Student’s paired t-tests). Peak amplitude of phrenic nerve and abdominal EMG activities were measured on integrated traces and normalized by expressing them in relative units (RUs), i.e. as a fraction of the highest amplitude observed in each animal (see e.g. Refs. [2,13]). The highest peak
201
value was consistently obtained during coughing. Cough-related variables included cough-related total duration of the respiratory cycle (TT ), cough-related inspiratory (TI ) and expiratory (TE ) times, peak phrenic amplitude, peak abdominal activity, peak tracheal pressure and the cough number, i.e. the number of coughs evoked by each stimulation trial. The time components of the cough motor pattern were measured on recordings of raw respiratory activity. In each animal, cough-related variables were measured and averaged before PBG injections, at each of the two scheduled times during the pulmonary chemoreflex and after the complete recovery of control respiratory pattern. The average values of cough-related variables for each period in each preparation (three stimulation trials, during three PBG-induced pulmonary chemoreflexes) were taken as single measurements for subsequent analysis by means of one-way repeated measures analysis of variance followed by Student–Newman–Keuls tests. All reported values are means ± S.E.; P < 0.05 was taken as significant. In agreement with our previous findings [13], the rapid bolus injections of PBG into the right atrium consistently caused tachypnea, bradycardia and hypotension often accompanied by the development of some tonic inspiratory activity and decreases in expiratory muscle discharges. In the period immediately following (3–4 s) the beginning of PBG-induced responses, respiratory frequency raised from 50.0 ± 2.7 to 126.2 ± 6.2 breaths/min, while mean arterial blood pressure decreased from 95.0 ± 1.4 to 58.0 ± 2.9 mmHg. A complete recovery was achieved within 20 min. Cough responses to mechanical stimulation of the tracheobronchial tree were characterized by repeated coughs consisting of coordinated bursts of inspiratory and expiratory activity [12]. On few occasions, the first obvious event in a cough epoch was a small-amplitude expiratory effort not preceded by an augmented inspiration and beginning during the expiratory phase. These expiratory bursts fit more appropriately the definition of expiration reflex. We have extensively reported information and discussion on this reflex in previous papers ([12,13]; see also Ref. [19]). These expiratory responses were not considered for data analysis. Changes in cough-related variables observed during the pulmonary chemoreflex are reported in Table 1 and illustrated by original recordings in Fig. 1. Noticeably, significant changes were observed in the time components of the cough motor pattern consisting of significant reductions in cough-related TT due to decreases in both TI and TE . No consistent changes were observed in the other variables, whose values turned out to be slightly but not significantly smaller during the pulmonary chemoreflex. The present results show that the cough motor pattern during PBG-induced pulmonary chemoreflexes displays marked changes in its time components, without consistent changes in the other variables. Therefore, they are at variance with previous findings obtained in cats [6,22,25] and dogs [21]. Tatar et al. [21,22] gave
Table 1 Changes in cough-related variables during the pulmonary chemoreflex evoked by right atrial injections of phenylbiguanide (n = 5).
CN TT (s) TI (s) TE (s) PPA (RUs) PAA (RUs) TP (cmH2 O)
Control
First cough challenge within 10 s after PBG
Second cough challenge within 20 s after PBG
Cough challenge 25 min after PBG
3.8 ± 0.20 0.60 ± 0.03 0.41 ± 0.02 0.18 ± 0.01 0.59 ± 0.02 0.52 ± 0.02 7.22 ± 0.12
3.4 ± 0.22 0.47 ± 0.01** 0.35 ± 0.005* 0.12 ± 0.004* 0.58 ± 0.02 0.49 ± 0.02 7.20 ± 0.17
3.4 ± 0.21 0.52 ± 0.01** 0.37 ± 0.005* 0.14 ± 0.005* 0.57 ± 0.02 0.50 ± 0.02 7.20 ± 0.15
3.6 ± 0.24 0.62 ± 0.02 0.42 ± 0.02 0.18 ± 0.01 0.58 ± 0.01 0.50 ± 0.01 7.22 ± 0.18
Values are means ± S.E.; CN, cough number; TT , cycle duration; TI , inspiratory time; TE , expiratory time; PPA, peak phrenic activity in relative units (RUs); PAA, peak abdominal activity; TP, tracheal pressure (cmH2 O). * P < 0.05 compared with controls. ** P < 0.01 compared with controls.
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Fig. 1. Changes in the cough reflex induced by the mechanical stimulation of the trachebronchial tree at different times during the pulmonary chemoreflex and after recovery of control breathing in one anesthetized rabbit. The pulmonary chemoreflex was evoked by a right atrial injection of 240 g PBG (arrow). Traces are: Phr IN, phrenic integrated neurogram; Phr N, phrenic neurogram; Abd IEMG, abdominal integrated electromyographic activity; Abd EMG, abdominal electromyographic activity; BP, arterial blood pressure; TP, tracheal pressure. Tussigenic stimulation marked by filled bars.
PBG or capsaicin intravenously in a bolus, probably targeting pulmonary C-fibres, resulting in profound depression of respiration, i.e. apnea, followed by rapid and shallow breathing. Given that the neural network implicated in the generation of the eupneic pattern of breathing is also involved in the production of the cough motor pattern [1,3,4,15–18], it is not surprising that coughing is abolished under conditions where central respiratory drive is profoundly depressed. However, in agreement with Widdicombe and Singh [25], it seems unlikely that the suppression of cough observed in cats and dogs is related to the apnea per se. In fact, Tatar et al. [22] have reported that the strength of cough was significantly reduced during the phase of rapid and shallow breathing and that cough was decreased, but not abolished during apnea induced by intravenous injections of veratrine in the cat [20]. In keeping with this interpretation, we have observed that the cough reflex evoked by mechanical stimulation of the tracheobronchial tree is still present, although greatly attenuated during apnea induced by severe hyperventilatory hypocapnia (Bongianni F, Mutolo D, Fontana GA, Pantaleo T, unpublished observations). We do not know the reasons of the discrepancies between present and previous results. However, we believe that differences in the animal species employed may have played the major role. Present results do not allow us to establish whether cough-related changes in timing are directly dependent upon the activation of pulmonary C-fibres or are secondary to the increases in respiratory frequency. Nevertheless, the observed changes in the time components of the cough motor pattern support for the first time the hypothesis that the neural mechanisms generating cough are dependent upon the characteristics of the ongoing respiratory activity. In particular, it appears that the timing of the cough motor pattern is, to some extent, related to the timing of baseline respiratory activity, thus implying that the reconfigured respiratory pattern generator during coughing (see e.g. Refs. [1,4,16,18]) retains memory of the preceding respiratory pattern. Furthermore, it seems reasonable to hypothesize that decreases in duration of cough-related TI and TE beyond a certain limit may lead to reductions in the peak amplitude of inspiratory and expiratory cough efforts due to the lack of sufficient time for their development. As an extreme consequence of this phenomenon at very high respiratory frequency or even during tonic activity [11,14], reductions in the cough number or cough suppression could be produced without any involvement of an inhibitory action of pulmonary C-fibres. Interestingly, the results also suggest that further studies may increase our knowledge on the distinctive features of the pulmonary chemoreflex in different animal species. In addition, comparative studies performed by using not only mechanical but also other tussigenic stimuli, such as inhalation of citric acid or
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