Discrimination between upper versus lower airway components to the rise of total airway resistance measured by Pennock’s method after nasal irritant challenge ()
1. INTRODUCTION
Nasal cavity is the first part of the respiratory system which is exposed to the ambient air [1]. Changes of the temperature, humidity and the presence of air born irritants could be potentially dangerous for the respiratory mucosa. Therefore these stimuli could initiate airway protective and defensive reflexes via activation of trigeminal afferents with the properties of nociceptors [2-4].
It is known that trigeminal excitation by different chemicals initiates the sensation of irritation, pain and lacrimation [5]. Furthermore exposure to chemicals such as irritant vapours, air born pollutants or oxidizing substances frequently induces sensation of chest tightness or dyspnoea, sometimes accompanied by wheezing [6]. It is believed that nasal exposure to irritants induces changes in the lower airway resistance named nasobronchial reflex, which is believed to contribute to the mentioned chest sensations. Nasobronchial reflex, as a phenomenon, remains controversial, because the data about its existence share conflicting evidence.
A nasobronchial reflex has been hypothesized to explain the relationship between events occurring in the upper and the lower airways [7]. Sercer suggested the existence of such a reflex after he observed that blowing air into one nostril induced expansion of the ipsilateral thorax [5]. In another study, bronchoconstriction occurred in individuals who had silica dust blown into their nose, and in one subject who had previously undergone the unilateral trigeminal nerve resection, bronchoconstriction occurred only when the dust was blown through the neurologically intact side [8]. Also bronchoconstriction provoked by nasal cold dry air, histamine and ammonia challenges had been reported [9-11]. Some investigators, however, have been unable to confirm nasobronchial reflex [12-14]. Another debate on nasobronchial interaction is that nasal irritation may not be initiating the bronchial response, but it may potentiate a neural reflex initiated in the lower airways [15].
While in the human studies the bronchial narrowing after nasal irritation is measured by spirometry or plethysmography, the results of animal studies are not easy to interpret because of methodological bias. In our research we use the Pennock’s method, which measures the airway resistance in vivo, in restrained conscious animals. This method is commonly used to study effects of bronchoactive substances [16,17], however, interpretation of the data obtained by Pennock’s method must be considered. There are two main resistors in the respiratory system-upper airway (nasal cavity with the turbinates and the mucosa which can modulate the patency very effectively) and the lower airways with the smooth muscles, which can contract or relax as a response to neural and/or humoral signalling. Pennock’s method measures the total airway resistance as it is explained in the original US patent application [18]. We postulated a hypothesis that pharmacological intervention, selectively blocking upper or lower airways component could contribute to the higher accuracy and better interpretation of obtained data. We decided to test the presence of nasobronchial reflex in conscious animal model and relevance of Pennock’s method in this observation.
2. METHODS
This study was conducted on male Dunking Hartley guinea pigs, obtained from accredited breeding facility (L. Sobota, Městec Králové, Czech Republic). Animals were housed in an approved animal holding facility maintained at controlled room temperature 21˚C - 22˚C, humidity 60% - 70%, ventilation, 12-h light-dark cycle and had free access to water and standard animal food.
Animal care was provided and experiments were conducted in agreement with the Animal Welfare Guidelines of the Comenius University and statutes and rules of Slovak Republic legislation. Current study was approved by the decision No: 2999/07-221.
Animals were adapted to laboratory conditions twice. They stayed in plethysmograpic box in order to get familiar with the environment and people responsible for the experimental manipulations. In the plethysmographic box, they had been exposed to the aerosol of buffered saline for 2 minutes that correspond to future experimental procedure and significantly reduces future stress.
2.1. Nasal Challenges
Based on the literature data, we decided to use TRPA1 agonist allyl-isothiocyanate (AITC), hot component of horseradish, mustard or wasabi (Sigma). Stock solution of AITC in 1% DMSO (dimethylsufloxide) was prepared and then further diluted at the day of its use to 10 mM concentrations (maximal effective concentration in conscious guinea pigs for nasal challenge, Brozmanova, personal communication). As a molecule with opposite effects-anti-irritating, calming and cooling-(-) menthol (10−3 M) TRPM8 agonist has been used. Stock solution prepared in ethanol was further diluted to 10−3 M. As a negative control to both of these agonists isotonic saline (0.015 ml) warmed up to nearly body temperature (32˚C) was used. All of the solutions were administered bilaterally into the each nostril of experimental animals using a thin plastic flexible catheter (external diameter 1 mm) with maximal caution to avoid damage of nasal mucosa (Figure 1).
2.2. Assessment of Nasal Symptoms
The symptoms intensity was evaluated by trained persons used a nasal symptom score system which was developed and validated by Brozmanova et al. (2008) matching symptoms intensity to numeric values (maximum 6 and minimum 0).
Nasal symptoms score was calculated based on validated scale system. Observed processes were count of sneezes and:
1) nasal discharge: no discharge 0, mild/moderate 1, discharge falls in drops from nose 2.
2) eye/conjunctival reaction: no changes 0, hazy eyes1, visible lacrimation 2.
Figure 1. represents the way of administration of nasal stimuli in restrained conscious guinea pigs.
3) nasal phenomenon: no crackles 0, audible crackles 1, crackles audible from a distance 2.
2.3. Measurement of Specific Airway Resistance in Awake Guinea Pigs
Specific airway resistance (Saw) was measured by a simple non-invasive plethysmographic technique, Pennock’s method [18]. Conscious animals were placed in a double chamber body plethysmograph composed of a head chamber and a body chamber (type 855, Hugo Sachs Electronic, Germany). The nasal airflow was measured in the head chamber; the thoracic respiratory airflow was calculated from the pressure changes in the body chamber. Both measurements were made with differential pressure transducers (Simsoft, Martin, Slovakia) and recorded to the PC for Pulmodyn Pennock W software analysis.
In this system, the specific airway resistance is proportional to phase difference between nasal airflow and thoracic respiratory flow [18]. Specific airway resistance was calculated from the phase difference between the thoracic respiratory flow and nasal airflow based on Lissajous loop presentation of the thoracic respiratory flow and nasal airflow on the Xand Y-axis. Specific airway resistance was measured before the challenges assigned as baseline, then after nasal challenges of AITC, menthol and saline respectively. Finally, resistance was measured also after inhalation of histamine and methacholine aerosols.
In the second round of experiments, resistance was measured before nasal challenges assigned as baseline, then nasal administration of oxymetazoline 1% and inhalation of salbutamol (0.1 mg/ml) were used to observe separately upper and lower airway components of the airway resistance after nasal administration of selected agonists AITC (10 mM) and menthol (10 mM).
2.4. Statistical Analysis of the Data
Data representing the airway resistance are expressed as mean and standard error of mean. Analysis of variance (ANOVA for repeated measurements) was used to analyze the data. As with any ANOVA, repeated measures ANOVA tests the equality of means. However, repeated measures ANOVA is used when all members of a random sample are measured under a number of different conditions. As the sample is exposed to each condition in turn, the measurement of the dependent variable is repeated. Using a standard ANOVA in this case is not appropriate because it fails to model the correlation between the repeated measures: the data violate the ANOVA assumption of independence. p < 0.05 was considered as being statistically significant.
3. RESULTS
3.1. Nasal Symptoms Score Induced by Selected Nasal Challenges
Nasal administration of saline warmed up to body temperature (0.015 ml, bilateral challenge using thin plastic catheter) induced only a sneezing reflex, 2 - 3 sneezes per nostril in average, very likely because of mechanical irritation of the nerves on the turbinate mucosa. No other symptoms were observed. Administration of 0.015 ml of l-menthol isomer (10−3 M, bilateral challenge) induced similar sneezing response, and additionally, 4/15 guinea pigs rubbed their noses by the front limb, this reaction was transient, restricted to the interval immediately after the administration. Nasal administration of an airway irritant allyl-isothiocyanate (AITC) induced massive sneezing response, significantly higher than that observed after nasal saline or menthol challenges (3 ± 1 vs 10 ± 4 sneezes per observed period of 15 minutes, p < 0.05). Except robust activation of sneezing reflex, animals developed rhinorrhoea with nasal discharge, they rubbed their noses by the front limb, and they also developed nasal acoustic phenomena (crackles) which are signs of nasal obstruction with secretion in the nasal passages.
These reactions were taken as an evidence for robust activation of nasal trigeminal afferents, particularly nociceptors. Menthol in this concentration was expected to stimulate distinct population of neurons, (TRPM8+/ TRPV1-population of trigeminal afferents), therefore no nocifensive reactions were observed.
3.2. Specific Airway Resistance Measured by Pennock’s Method after Nasal Challenges
Specific resistance was measured before nasal challenges (assigned as basal), then after nasal challenges of saline (0.015 ml), l-menthol and AITC (bilateral challenges) (assigned as after nasal challenge) and finally, animals in plethysmographic box were exposed to histamine aerosol, and methacholine aerosol to observe airway reactivity to most frequently used bronchoactive substances—more in methods section). Figure 2 represents the change of specific airway resistance after nasal AITC, saline and l-menthol challenges.
The specific resistance measured by Pennock’s method was not significantly influenced after nasal challenges with saline and menthol. The rise of resistance was spotted after nasal AITC challenge, suggesting for nasobronchial reflex. Resistance rose even higher after inhalation of histamine and methacholine respectively, what was expected (5.3 vs 10.18 vs 11.26 vs 17.32 cmH2O. s−1, p < 0.05). Animals with nasal saline and l-menthol challenges responded to histamine and methacholine less significantly than animals with nasal AITC
Figure 2. Represents the rise of the airway resistance after nasal administration of an irritant AITC 10 mM. Inhalation of histamine and methacholine provoked significant increase when comparing to baseline, however not significantly different with the value obtained after nasal AITC challenge. Additional rise of the resistance after histamine and methacholine could be attributed to the bronchomotor response, as the upper airway component is already involved. Nasal administration of saline and menthol did not provoke the rise of the airway resistance just by themselves. Further inhalation of histamine and methacholine provoked the rise of airway resistance probably by the combined upper and lower airway influencing mechanisms.*p < 0.05.
(data in saline and menthol lines were not significantly changed). This is a circumstantial evidence confirming increased responsiveness of the lower airway once the nasal afferents are robustly activated.
3.3. Specific Airway Resistance Measured by Pennock’s Method after Nasal AITC—Effects of Nasal Decongestant versus (2 Mimetics Pre-Treatment
In further studies we focused on the effects provoked by nasal irritant AITC after selective blocks of nasal reactivity by nasal pre-treatment with 1% oxymetazoline (a1 agonist and potent decongestant), which blocks nasal vascular reactivity and salbutamol—b2 agonist, which has bronchodilatatory effects.
Figure 3 documents the decrease of airway resistance measured by Pennock’s method from baseline (5.3 ± 0.25 to 3.7 ± 0.3 cmH2O·s−1, p < 0.05) after the nasal cavity was pre-treated with 1% oxymetazoline. Nasal administration of AITC 10 mM to the nose pre-treated with oxymetazoline 1% did not induce significant rise of the airway resistance. Further inhalation of salbutamoll did not modulate the airway resistance in this model, confirming the role of the upper rather than a lower airway component to the total airway resistance after nasal challenge of airway irritants.