Respiratory Physiology & Neurobiology 205 (2015) 21–27

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Interactions of mechanically induced coughing and sneezing in cat Michal Simera a,∗ , Ivan Poliacek a , Boris Dobrolubov a , Marcel Veternik a , Jana Plevkova b , Jan Jakus a a b

Institute of Medical Biophysics, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Malá Hora 4, 036 01 Martin, Slovak Republic Institute of Pathophysiology, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Sklabinska 26, 037 53 Martin, Slovak Republic

a r t i c l e

i n f o

Article history: Accepted 19 September 2014 Available online 28 September 2014 Keywords: Tracheobronchial cough Sneeze Mutual interactions Styloglossus muscle Electromyogram

a b s t r a c t Mutual interactions of cough and sneeze were studied in 12 spontaneously breathing pentobarbitone anesthetized cats. Reflexes were induced by mechanical stimulation of the tracheobronchial and nasal airways, respectively. The amplitude of the styloglossus muscle EMG moving average during the sneeze expulsion was 16-fold higher than that during cough (p < 0.01). Larger inspiratory efforts occurred during coughing (p < 0.01) vs. those in sneeze. The number of reflexes during simultaneous mechanical stimulation of the nasal and tracheal airways was not altered significantly compared to controls (p > 0.05) and there was no modulation in temporal characteristics of the behaviors. When both reflexes occurred during simultaneous stimuli the responses were classified as either sneeze or cough (no hybrid responses occurred). During simultaneous stimulation of both airway sites, peak diaphragm EMG and inspiratory esophageal pressures during sneezes were significantly increased. The expiratory maxima of esophageal pressure and amplitudes of abdominal EMGs were increased in coughs and sneezes during simultaneous mechanical stimulation trials compared to control reflexes. © 2014 Published by Elsevier B.V.

1. Introduction Sneezing and coughing are important airway defensive reflexes. Both reflexes consist of an inspiration phase, compression phase, and an expulsion phase (Korpas and Tomori, 1979). The inspiratory phase for both behaviors ensures that there is large volume of air inhaled in the lungs and is produced by vigorous activity of inspiratory pump muscles, mainly the diaphragm. During the transition from inspiration to expiration there is a marked increase in expiratory pump muscle activities (all anterolateral abdominal muscles are involved; Bolser et al., 2000). When abdominal activation, resulting in powerful expiratory efforts, is accompanied by vocal fold adduction (laryngeal adductors e.g. thyroarytenoids are vigorously activated) subglottic pressure abruptly increases and the compressive phase occurs. Continuous abdominal muscle contraction with rapid laryngeal abduction resulting in explosive expiratory airflows characterizes the expiratory phase of both behaviors (Korpas and Tomori, 1979).

∗ Corresponding author. Tel.: +421 432633431. E-mail address: [email protected] (M. Simera). http://dx.doi.org/10.1016/j.resp.2014.09.011 1569-9048/© 2014 Published by Elsevier B.V.

The cough reflex protects the lungs against penetration of inhaled particles and reduces the risk of aspiration. The powerful expiration of coughing ejects potentially harmful irritants and mucus out of the respiratory tract through the mouth. Coughing can be initiated by relevant stimulation (mechanical, chemical or electrical) of laryngeal and tracheal–bronchial sensory receptors or their axons and in contrast to the sneeze reflex it can be initiated voluntarily (Hegland et al., 2012; Jakus et al., 2004). The afferent pathways for cough consist of vagus nerve fibers arising from two main groups of sensory nerve endings: the rapidly adapting mechano- and acid sensing cough receptors, which send information via A␦ nodose fibers, and nociceptive-like free sensory endings of jugular C-fibers (Canning et al., 2006; Widdicombe, 1995). The sneeze reflex can be evoked mechanically or chemically from the nasal mucosa, where free nerve endings of trigeminal origin (the anterior ethmoidal and posterior nasal nerves) are distributed (Cauna et al., 1969; Wallois et al., 1991). After initial inspiratory and compression phases a powerful sneeze expiration enables the flow of air mostly through the nose (“expulsive phase”) in order to eliminate the stimulus provoking it (e.g. mucus, irritants). This sneeze-related expiratory nasal airflow, which does not occur in coughing (Korpas and Tomori, 1979), is caused by activation of additional auxiliary muscles, e.g. the elevator of the back of

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the tongue the styloglossus muscle (SG). SG is explosively activated during the sneeze expulsion (Satoh et al., 1998). Thus increased oral airway resistance due to elevation of the back of the tongue causes a strong nasal airflow during sneezing (Satoh et al., 1998). Although the neural mechanisms of coughing have been systematically studied for the last few decades, the differences between coughing and sneezing as well as the neuronal mechanisms of the sneeze reflex in general are still poorly understood (Satoh et al., 1998; Shannon et al., 1996). It is assumed in a recent model of the central neuronal circuits for the cough reflex (Bolser and Davenport, 2002; Shannon et al., 1998, 2000) that the cough-related sensory signal is processed by relay neurons in the nucleus of the solitary tract (NTS) and then this information drives the respiratory/cough central pattern generator (CPG) via the cough gating mechanism. It is not known if a similar behavioral control element (such as cough gating) exists for sneeze (Bolser et al., 2006). It is also unclear how the two behaviors interact when actuated simultaneously. The cough reflex can be modulated by many secondary afferent inputs including the nasal afferents (Plevkova et al., 2004a, 2004b), in particular those which are stimulated by irritants and inflammatory mediators. Such stimulation results in peripheral and central plasticity of neuronal cough components (Buday et al., 2012; McAlexander and Carr, 2009) affecting the expression of actual cough. We hypothesized that simultaneous mechanical stimulation of the upper and lower airways would modulate the cough and sneeze reflexes with no changes in the phase durations of the two behaviors.

2. Material and methods 2.1. Experimental protocol All procedures were performed in accordance with the laws, rules, and regulations of the Slovak Republic and EU. Protocols were approved by the Ethics Committee of Jessenius Faculty of Medicine in Martin. Experiments were performed on 12 spontaneously breathing cats (3.5 ± 0.2 kg) anesthetized with sodium pentobarbital (Biowet, Pulavy, Poland; 40 mg/kg, i.p.). Supplementary doses of the anesthetic agent were administered (1–3 mg/kg, i.v.) as needed if the withdrawal responses to the paw pinch appeared. Atropine (Biotika; 0.15 mg/kg, i.v.) was given at the beginning of the experiment in order to reduce bronchial secretions. The trachea, femoral artery and vein were cannulated. The animals were allowed to breathe spontaneously a gas mixture of 30–60% oxygen. Arterial blood pressure (BP), end-tidal CO2 concentration (ETCO2 ), respiratory rate (RR) and body temperature were monitored continuously. Body temperature was maintained at 38.0 ± 0.5 ◦ C. Samples of arterial blood were periodically (before and after the protocol sequence) removed for blood gas and pH analyses. Bipolar fine wire hook electrodes were placed in the sternal diaphragm (DIA), bilaterally in the transversus abdominis, or the external oblique abdominal muscles (ABD) and in SG for electromyogram (EMG) recordings. A soft balloon was inserted into the esophagus for a measurement of the intrathoracic pressure changes (esophageal pressure – EP). The cough reflex was induced by mechanical stimulation of the tracheobronchial mucosa with a soft nylon fiber or a soft catheter (with external diameter 1.0 mm). The stimulating fiber/catheter was inserted into the trachea and moved back and forth and rotated approximately once per second during 10 s stimulation trial. Sneezing was induced by punctate mechanical stimulation of the nasal septum with an approximately 1.5 cm long soft nylon fiber (external diameter ∅ 0.25 mm). Mechanical probing was always performed by the same person, with the same pattern of the probe movement during the trials. The sneeze

reflex has a long latency period in cat; thus, during combined stimulation of cough and sneeze, the nasal stimulation was applied first and then stimulation of the tracheal bronchial mucosa was applied during ongoing nasal stimulation. All control trials were performed before and after stimulation protocol. The cough and sneeze reflexes were defined by a large augmenting burst of DIA EMG activity immediately followed (and partially overlapped) by a burst of expiratory ABD EMG activity (Jakus et al., 1987, 2004; Tomori and Stransky, 1973; Tomori, 1979), with corresponding inspiratory–expiratory (I–E) oscillations of EP. The sneeze is distinguished from the cough reflex by a large SG EMG activity during the expulsive phase, which matches the occurrence of ballistic-like ABD discharge (Satoh et al., 1998; see also results or Fig. 1). 2.2. Data processing and analysis All EMGs were amplified, filtered (300–3000 Hz; GRASS), digitized (12-bit multi-function plug-in ISA card, Dataq Instruments, sampling frequency of 20 kHz), and recorded along with the waveforms of BP and EP. EMGs were then rectified and integrated (moving average) with a time constant of 200 ms (using Spike 2 software, CED, Cambridge, England). The number of cough efforts induced during the mechanical probing of airways (number of coughs – CN per 10 s stimulation) and the number of sneeze efforts in response to stimulation of the nasal septum (number of sneezes – SN per 10 s stimulation of nasal mucosa) were analyzed in each sequence of trials. The amplitudes of DIA, ABD, and SG integrated EMGs as well as the inspiratory and expiratory EPs during coughs and sneezes were normalized to the first sequence of cough trials. In the temporal analysis, the durations of cough and sneeze related DIA and ABD activations, augmenting part of DIA (TI = inspiratory cough/sneeze phase) and ABD activity, the time from the maximum of DIA activity to the end of cough/sneeze related ABD activity (TE1 = active expiratory cough/sneeze phase), the time from the maximum of DIA activity to the end of the cough/sneeze cycle (TE = expiratory phase), the overlapping of DIA and ABD burst in the I–E transition, the time between maxima of DIA and ABD activity, the quiescent period of the cough/sneeze cycle (cough/sneeze TE2 phase), the duration of all cough/sneeze related EMG activity (Tactive), and the whole cough/sneeze cycle duration were analyzed in each stimulation period (Ttot). The results (EMG activities, corresponding pressure changes of EP as well as temporal parameters) are expressed as means ± SE. For statistical analysis a paired t-test and repeated measures ANOVA were applied as appropriate. The differences of variables were considered significant at p < 0.05. In order to quantify the differences that we found between cough and sneeze elicited separately and during simultaneous stimuli, we performed linear and non-linear correlation analysis of selected parameters. Such analysis on the cough reflex performed by Wang et al. (2009) revealed limited correlations among the majority of cough characteristics. 3. Results 3.1. Spatio-temporal analysis data Mechanical stimulation of the tracheal–bronchial mucosa resulted in repetitive coughing while nasal stimulation exclusively produced sneezing (Fig. 1). Reflexes were classified (coughs vs. sneezes) based on the region of the airway that was stimulated (tracheal–bronchial or nasal). The amplitude of SG integrated EMG during the sneeze expulsion was 1630 ± 320% of that during cough (p < 0.001; 10 cats). This activity became main differentiating

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Fig. 1. The cough reflex (left) and the sneeze reflex (right). EP – esophageal pressure; DIA EMG, ABD EMG, SG EMG – EMG activities of the diaphragm, abdominal, and styloglossus muscles, respectively. RF – integrated (moving average) form of related EMG activities. Timescale is in seconds.

criterion for cough and sneeze induced during simultaneous stimulation of both airway regions (“combined” responses; Fig. 3). Other differences in parameters between control coughing and sneezing were: weaker sneeze inspiratory efforts vs. cough (amplitudes of DIA EMGs during sneeze were 72 ± 8% of that during cough; p < 0.01; inspiratory amplitude of EP −0.98 ± 0.15 kPa and −1.44 ± 0.15 kPa, respectively; p < 0.01) and longer TI (1.35 ± 0.17 s vs. 1.01 ± 0.12 s, p < 0.05) and the duration of DIA activation (1.60 ± 0.17 s vs. 1.27 ± 0.15 s, p < 0.05) in sneeze. Mechanical stimulation trials of the nasal airway that induced no sneeze reflex (and started at least 10 s before the cough stimulus) had no effect on the parameters of coughing elicited by tracheal–bronchial stimulation (seven cats). When both reflexes were executed (cough and sneeze; eight cats) during simultaneous stimuli the responses were classified as either sneeze or cough (no hybrid responses occurred) and they were potentiated compared to control reflexes (Fig. 3). DIA EMG moving average maxima (Table 1) and inspiratory amplitude of EP (Fig. 2) were increased in sneezes induced during combined trials compared to control sneezing (p < 0.05). Expiratory maxima of the esophageal pressure (Fig. 2) and amplitudes of ABD EMG moving averages (Table 1) were increased in coughs and sneezes induced during combined trials compared to control coughs and sneezes (both p < 0.05). In coughs, the SG activity during the expulsion represents a tonic component of the SG muscle activity (supposedly a baseline muscle tonus, see Fig. 1). Consistent with higher cough E efforts during dual

stimulation, the levels of SG EMG activity in the cough TE1 phase reached 150% of that in control coughs (p < 0.05). The number of reflexes during combined trials (either coughs or sneezes) was not altered significantly compared to controls (p > 0.05). Temporal analysis of cough and sneeze phases revealed no other differences in timing features of the reflexes than longer TI and DIA activation in sneezing including those during combined stimuli (Table 2). 3.2. Correlation analysis data We found a weak to moderate linear correlation (r2 ≤ 0.45, / 0) for all of following pairs (for cough, p > 0.065 for the slope = sneeze, combined cough, and combined sneeze): TI vs. the amplitude of DIA or ABD, TE, TE2; TE1 vs. the amplitude of DIA or ABD, TE2; the amplitude of DIA vs. the amplitude of ABD, TE, TE2, Ttot; the amplitude of ABD vs. TE, TE2, Ttot (for sneeze, combined cough, and combined sneeze). However, a strong linear correlation (r2 > 0.5, slopes = 0.43 ± 0.15 and 0.81 ± 0.32, respectively; p < 0.05 the slopes = / 0) was found between TI and TE1 for sneeze (either control or combined), unlike for cough (0.3 < r2 < 0.45, p > 0.075 for the slope = / 0). We also found a strong linear correlation for TE2 vs. Ttot (r2 > 0.55, the slope = 1.01 ± 0.26 and 1.49 ± 0.51, respectively; p < 0.05 the slope = / 0) for cough (control as well as those induced during simultaneous stimulation of both airway regions), not for sneeze (0.3 < r2 < 0.4, p > 0.05 for the slope = / 0). Simultaneous mechanical stimulation of both airway regions was associated with very strong linear correlations of TI vs. Ttot for both reflexes (r2 ≥ 0.7, the slope = 2.05 ± 0.47 and 3.43 ± 0.92, Table 1 Peak diaphragm (DIA) and abdominal (ABD) EMG moving average activities during combined coughing and sneezing normalized to control cough responses (eight cats).

Fig. 2. Peak inspiratory and expiratory esophageal pressures during coughing and sneezing (control and combined stimuli) black – tracheal–bronchial cough; white – sneeze; light gray – cough during combined stimuli; dark gray – sneezing during combined stimuli; I – peak inspiration; E – peak expiration.

Control cough Control sneeze Combined cough Combined sneeze * $ #

% DIA

% ABD

100 74 ± 9* 103 ± 9 115 ± 15$

100 207 ± 55 184 ± 24* 335 ± 76*,$,#

p < 0.05 in comparison with control cough. p < 0.05 in comparison with control sneeze. p < 0.05 in comparison with combined cough.

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Fig. 3. Cough and sneeze reflexes during combined stimulation. EP – esophageal pressure; DIA EMG, ABD EMG, SG EMG – EMG activities of the diaphragm, abdominal, and styloglossus muscles, respectively. RF – integrated (moving average) form of related EMG activities. S – sneeze response with accompanying styloglossus muscles activity; C – cough response. *The beginning and the end of stimulation of nasal mucosa, # stimulation of tracheal–bronchial mucosa.

respectively; p < 0.01 the slopes = / 0), and no significant correlation for control coughs and sneezes (r2 ≤ 0.25). The correlation between the amplitude of ABD vs. TE, TE2, and Ttot was nonlinear in our control cough dataset (Spearman non-parametric correlation 0.43 < r < 0.71; only the slope of the relationship between amplitude of ABD vs. TE2 was different from 0, p < 0.05). We found a very strong linear association of TE1 among the responses: cough vs. combined cough, sneeze vs. combined sneeze, and cough vs. sneeze (r2 > 0.75, the slope = 0.64 ± 0.15, 1.78 ± 0.33, / 0), but not for and 0.87 ± 0.18, respectively; p < 0.01 the slopes = combined cough vs. combined sneeze (r2 < 0.35, p > 0.14 for the / 0). Linear correlations were also found for TI for control slope = vs. combined cough and control cough vs. control sneeze (r2 ≥ 0.45, the slope = 0.74 ± 0.18 and 0.99 ± 0.39, respectively; p < 0.05 the slopes = / 0). For control vs. combined sneeze as well as for combined cough vs. combined sneeze the correlations were weaker (0.25 < r2 < 0.4). The amplitudes of DIA were moderately correlated (r2 > 0.43) / 0) and for control vs. combined cough (p > 0.07 for the slope = cough vs. sneeze (the slope = 0.83 ± 0.33, p < 0.05 the slope = / 0) and those of ABD for control vs. combined cough (r2 > 0.55, the / 0). No correlation was slope = 0.83 ± 0.30, p < 0.05 the slope = found for the amplitudes of ABD for cough vs. sneeze and combined responses (r2 ≤ 0.01).

4. Discussion The main finding of our study is that in anesthetized cats the temporal patterns of individual sneezes and coughs are unchanged during the dual upper/lower airway mechanical stimulation paradigm, but rather amplitudes of muscle motor drive were significantly increased. Our dual stimulation trials showed no effect on the number of coughs and sneezes with no modulation in their temporal characteristics. 4.1. Generation of cough and sneeze motor pattern The patterns of individual cough and sneeze reflexes induced during dual stimulation including their timing, and shaping of EMGs was preserved. Consequently, we identified these behaviors as coughs or sneezes (no hybrid responses) according to the presence of an SG burst of EMG activity concurrent with vigorous ABD activation. We presumed that hybrid responses, resulting from the temporal overlap of two different behaviors, would be characterized by mixing of typical cough and sneeze features. We did not observe hybrid responses in our experiments. For example, longlasting sequential activations of DIA (typical for sneeze; Korpas and Tomori, 1979) would be followed by an expulsion without SG burst of electrical activity (as seen in coughs) or shorter ramp-like DIA EMG could be followed by a strong SG activity during the expulsion.

Table 2 Temporal analysis of cough and sneeze. TDIA, duration of diaphragm activity; TI, duration of cough inspiratory phase = augmenting part of DIA activity; TABD, duration of abdominal muscles activity; Overlap, overlapping of DIA and ABD discharge; Dif, duration from DIA to ABD activity maximum; TE1, TE2, and TE, duration of active expiratory, quiescent “inter-cough” period, and the whole E phase of cough, respectively; Tactive, duration of active cough phase = TI + TE1; Ttot, the whole cough cycle duration. Time [s] TDIA TI = Iaug TABD Overlap Dif TE1 TE TE2 Tactive Ttot * #

Control cough 1.27 1.01 0.88 0.37 0.33 0.77 1.85 1.08 1.77 2.85

± ± ± ± ± ± ± ± ± ±

0.15 0.12 0.15 0.11 0.04 0.11 0.29 0.26 0.20 0.33

p < 0.05 compared to the control cough. p < 0.05 compared to the control sneeze (unpaired test).

Control sneeze 1.60 1.35 1.10 0.53 0.37 0.76 1.87 1.11 2.11 3.22

± ± ± ± ± ± ± ± ± ±

*

0.17 0.17* 0.30 0.16 0.02 0.10 0.26 0.28 0.25 0.30

Combined cough 1.11 0.89 0.92 0.42 0.28 0.72 1.77 1.05 1.61 2.66

± ± ± ± ± ± ± ± ± ±

#

0.14 0.12 0.15 0.10 0.03 0.10 0.19 0.15 0.20 0.28

Combined sneeze 1.45 1.12 1.25 0.63 0.43 0.95 2.16 1.21 2.07 3.28

± ± ± ± ± ± ± ± ± ±

0.28 0.21 0.22 0.12 0.08 0.24 0.37 0.32 0.42 0.51

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Our correlation data strongly support “selection” of coughs and sneezes in the dual stimuli trials. The TI as well as the amplitudes of ABD were strongly correlated between control coughs and coughs observed during the simultaneous mechanical stimulation trials. The strong correlation of cough TE2 and Ttot, reported before by Wang et al. (2009), was preserved for coughing in the combined cough and sneeze protocol. There were strong linear correlations between TI and TE1 for control and combined sneeze, unlike that for coughing. TE1 was strongly correlated among the responses, besides that between combined coughs and sneezes (see Section 3), suggesting the difference between combined coughs and sneezes. In addition, no association of the ABD amplitudes between cough and sneeze (either control or combined responses) occurred and some correlation of ABD and SG amplitudes for sneeze (almost none for cough) was seen. Differences in the regulation of breathing and other motor responses suggest the existence of one or more novel central regulatory elements essential for the production, control, and coordination of airway defensive motor acts e.g. cough (Bolser and Davenport, 2002). These presumptive behavioral control neuronal assemblies, which are thought to represent functionally distinct populations of neurons that participate in the control of different airway defensive behaviors (Bolser et al., 2006; Pitts et al., 2013), allow just one behavior to occur at any one time. We propose that either there are two different CPGs for cough and for sneeze and just one of them at a time generates the related motor pattern, or alternatively there is a common CPG that reconfigures for generation of either cough or sneeze. The occurrence of well discriminated coughs and sneezes with stable temporal features and maintenance of well-known differences in TI for sneeze and cough during dual stimuli are consistent with two functionally and probably anatomically distinct CPGs producing the motor pattern of the reflexes. This concept is supported by other significant anatomical and functional differences between the cough and sneeze reflexes (Korpas and Tomori, 1979). Anatomical neuronal substrates explored by c-fos immunoreactivity in cats showed high neuronal activation during coughing within several areas of the brainstem (Gestreau et al., 1997; Jakus et al., 2008). Within contrast, in response to sneeze stimulation, c-fos immunoreactivity was observed mainly in the trigeminal sensory complex at the levels where nasal afferents project, in the solitary complex, and in the parabrachial area (Masmoudi et al., 1997; Wallois et al., 1995). It has been found, that expiratory neurons in rostral ventrolateral medulla in Bötzinger complex area were inactive during sneezing in awake cats (Orem et al., 1986) although these expiratory neurons have an essential role in shaping of cough expulsion in anesthetized animals (Bongianni et al., 1998). In rabbits kainic acid lesions in the raphe nuclei reduced the number of coughs although it had no effect on the incidence of sneeze (Simera et al., 2013). Application of antitussives such as codeine had low or no suppressive effects on sneeze while cough was greatly reduced (Korpas and Tomori, 1979; Simera et al., 2010). Our analysis of the DIA EMG activities during cough and sneeze in rabbits showed marked differences in their frequency composition (Knocikova et al., 2009). Moreover, cough can be produced voluntarily (i.e. patients are coughing on request), however, voluntary sneezes have never been reported (Korpas and Tomori, 1979). An alternative functional design would comprise a common CPG for cough and sneeze that is reconfigured for the generation of individual behaviors. This would be accomplished by different sensory and/or feedback afferent inputs from the periphery and central behavioral control elements. For example, it has been proposed that the respiratory CPG is reconfigured for the production of cough by lower airway stimulation (Bolser and Davenport, 2002; Bolser et al., 2006). However, our simultaneous stimulation protocol produced

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rather consistent afferent drive from both airway sites that does not seem to be consistent with alternating sequences of coughs and sneezes induced (see Fig. 3). The CPGs, including the respiratory/cough rhythm/pattern generator, have to be sufficiently plastic to permit appropriate modifications during changes in behavior and the environment (Dutschmann and Paton, 2002). The respiratory/cough CPG reconfigures in a fast and reversible manner in response to reflex sensory activation or descending commands from higher brain centers. Consequently, the cough reflex is not a stereotypic phenomenon, but rather a plastic behavior, which undergoes modulation, which recently has been recognized as “cough plasticity” (Buday et al., 2012; McAlexander and Carr, 2009). Coughing is modulated (Hanacek et al., 2006) by several behaviors executed simultaneously with it, e.g. the aspiration reflex (Poliacek et al., 2009a, 2009b) and swallowing (Pitts et al., 2013). Our previous results (Poliacek et al., 2009a, 2009b) indicate reduced excitability and rhythmicity of mechanically induced tracheobronchial cough or alterations in the cough motor pattern by aspiration reflexes induced by nasopharyngeal stimulation in anesthetized cats. Pitts et al. (2013) showed that in anesthetized cats the coughs, which occurred after the introduction of a stimulus that promotes swallowing, had significantly greater motor drive. Our present data showed execution of either discrete coughs or sneezes, supporting the notion that the neural substrates for these behaviors may involve reciprocal inhibition. Thus, the modulation of behaviors (similar to interaction of coughs and swallows; Pitts et al., 2013) is likely executed at the level of proposed behavioral control elements. Execution of swallows and sneezes among coughs within a sequential series of coughs resulted in more pronounced expiratory efforts of both reflexes. This fact supports mutual modulation of the responses and their potentiation consistent with previous findings (Pitts et al., 2013). 4.2. Modulation of cough and sneeze Simultaneous execution of coughing and sneezing resulted in no modulation of the temporal characteristics of either reflex. Similarly, aspiration reflexes (Poliacek et al., 2009a, 2009b) and swallows (Pitts et al., 2013) had no significant effects on TI and TE1 (the duration of active phases) of coughing. As we suggested earlier the cough temporal pattern is pre-programmed, i.e. the CPG produces a cough pattern that is resistant to alteration by the actuation of non-cough behaviors (Poliacek et al., 2009a, 2009b). The correlation data differentiating cough and sneeze (including those executed during dual stimulation) are consistent with this hypothesis. The variability of Ttot of the reflex response is caused mainly by the variability in TE2 (Wang et al., 2009). The most striking difference between control and dual stimuli responses in our correlation data is the increase in the linear association of Ttot vs. TI. We interpret this finding as an increased cycling characteristic (lower variability in Ttot) under dual stimuli resulting in more stable TE2, consistent with potentiation of both reflexes. Inspiratory efforts during control sneezes were weaker than those during control coughs. Expulsions also were stronger in sneeze (our results; Tomori, 1965). Several findings relate deeper cough inspirations to stronger and more effective cough expulsions (Korpas and Tomori, 1975) suggesting the modulatory effect of the Hering–Breuer inflation reflex and possibly myotatic reflexes on the E effort (Bucher, 1958; Korpas and Tomori, 1958; Widdicombe, 1964). However, the volume timing relationship typical for breathing does not apply for coughing (Bolser and Davenport, 2000). Our data (weaker I associated with stronger E in sneeze vs. cough) are not consistent with a dominant role of the magnitude of I for the development of expulsion in the studied reflexes. There is very weak to no correlation between the amplitudes of DIA and ABD (see

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also Wang et al., 2009). Moreover, during dual stimuli both I and E efforts of sneeze were enhanced, while in cough only expiratory efforts increased. Our experiments were conducted on pentobarbital anesthetized cats. Besides the reduction of reflex behaviors by general anesthesia (including respiratory responses; Korpas and Tomori, 1979) this animal model expresses stable and vigorous coughing and sneezing under this anesthetic (Korpas and Tomori, 1979; Jakus et al., 1998). Mechanical stimulation (for more details see Section 2.1) was the most suitable (stable and potent) because chemical stimuli are largely ineffective in inducing coughing under general anesthesia (Canning, 2007). Tracheostomy in our animals eliminated laryngeal airflow during the reflexes as well as the contribution of the laryngeal valve to the compression phase of coughing and sneezing. However, cough DIA and ABD (and laryngeal muscles) discharge were not significantly affected (including timing) by tracheostomy (airflow through the larynx vs. through the tracheostomy) during coughing (Poliacek, 2000; Sant’Ambrogio et al., 1997). Laryngeal activities and analysis of compression phase was not the goal of this paper. Bilateral vagotomy increased the duration of sneeze I (unilateral vagotomy increased the duration of cough Ttot; Hanacek et al., 1984), suggesting an important role of vagal afferent input in shaping of the sneeze reflex (Batsel, 1981; Wallois et al., 1992; Wallois and Macron, 1994). Complex convergent inputs from both nasal and vagal receptors can modulate the sneeze reflex Wallois et al. (1991). Vice versa, the stimulation of nasal afferents in animals enhances cough responsiveness to subsequent lower airway challenges (Canning, 2008; Plevkova et al., 2004a, 2004b). Afferent drive from the nasal cavity can also modulate coughing in relation to the pathogenesis of upper airway cough syndrome (Plevkova et al., 2004a, 2004b; Plevkova and Song, 2013). It was documented that administration of TRPA1 relevant agonists to the nasal mucosa induce sneezing and promote urge to cough in humans (Buday et al., 2012). However, our data showed a very limited effect of nasal mechanosensors on cough induced in lower airways (unless the sneeze response had been already initiated). These findings are consistent with well discriminated mechano- vs. chemosensors in the nasal mucosa and their markedly different modulatory effect on coughing.

4.3. Concluding remarks Cough and sneeze are important airway defensive reflexes in human. Their mechanisms are similar and they consist of the same phases (inspiration, compression, and expulsion). Therefore, they can presumably serve the same role in evacuating mucus, tissue debris and other irritants out of the respiratory tract. Under situations of diminished coughing (e.g. diabetic patients, stroke or neurodegenerative diseases patients, muscle dystrophy patients; Fontana et al., 1998; Ebihara and Ebihara, 2011) a reduced effectiveness of airway protection leads to respiratory complications such as infections and aspiration pneumonia. Induction of sneeze by sensory stimulation of nasal mucosa can be potentially used as a preventive strategy against respiratory complications in patients with impaired cough. Although the modulation of coughing by secondary sensory input and by other reflex responses is broadly discussed, the use of this approach to reduce possible respiratory complications in patients has not been systematically studied in clinical settings. We conclude that mechanosensitive nasal afferents do not modulate the tracheobronchial cough response. However, coexpression of coughing and sneezing results in enhanced muscle recruitment in both cough and sneeze representing improved airway defense. The cough and sneeze reflex are likely generated by

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