JOURNAL OF APPLIED TOXICOLOGY. VOL. 12(4), 235-238 (1992)

In vivo Evaluation of Airway and Pulmonary Tissue Response to Inhaled Methacholine in the Rat R. S. Sakae', M. A. Martins', P. M. P. Criado', W. A. Zin3 and P. H. N. Saldiva'*2*t 'Laboratbrio de Poluicio Atniosfkrica Experimental and 'Instituto do Coraqio. FMUSP. Sfio Paulo; and 'Instituto de Biofisica. UFRJ, Rio de Janeiro, Brazil ~~

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Key words lung mechanics, airway resistmce, tissue resistance, bronchial reactivity

The current study was designed to assess the methacholine dose-response behaviour of the airways and pulmonary parenchyma with the aid of alveolar capsules. The experiments were performed in eight adult female Wistar rats (155-250 g). The animals were anaesthetized with sodium pentobarbital (30 mg kg-' i.p.) and mechanically ventilated. Measurements of tracheal (pl,) and alveolar (pA)pressures and the pressure change across the airway (paw) were performed prior to and after exposing rats to aerosols generated from sequentially increasing concentrations of methacholine chloride solution (2, 4, 8, 16, 32, 64 and 128 mg m1-I) through the breathing circuit. Baseline pawand pA mean ( ? SD) values (6.44 2.06 and 8.25 3.44 cmH,O, respectively) were not statistically different ( P = 0.220). The increases in plr and p,, were significant during the dose-response study ( P = 0.001), whereas pawwas not increased. The increase in pA was significantly higher than that of paw( P < 0.001). The relationship between the mean ( * SE) values of fir and pA could be well described by a straight line ( r = 0.990, P < 0.001). There were also significant correlations based on regression equations between plr and paw( r = 0.947, P < 0.001) and p,, and paw( r = 0.913, P = 0.004). These findings suggest that the pulmonary tissue of rats is a major component responsible for the increase in lung impedance observed after methacholine challenge. In addition, airway and pulmonary parenchyma pressure changes were correlated, suggesting that both lung regions have a similar sensitivity to the agonist. Our results indicate that the response of peripheral tissues should be considered during bronchial challenge protocols in rats.

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INTRODUCTION

Dose-response curves have been widely used in toxicological studies to assess the magnitude of bronchial reactivity both in humans and animals. 1.2 Airway response to an agonist is usually expressed in terms of increased lung resistance or reduced expiratory volume measured after 1 s of forced expiration, whereas changes in lung compliance have been considered to be a good estimate of mechanical alterations of lung paren~hyma.~ The use of indirect indicators of airway and tissue responses is due to the difficulty in the direct determination of alveolar pressure in vivo. This latter procedure would allow characterization and differentiation of pressure losses across the airways and those due to pulmonary tissue. Recently, alveolar pressure was directly measured in living animals by the use of alveolar capsules.4-s The alveolar pressure measurement has not been reported in rats, probably because of technical difficulties. For example, in a small lung with its intrinsically large pleural curvature, the fixation of the capsule could be impaired and a larger amount of tissue distortion might be expected because of the capsule-transducer's large weight as compared to that t Author to whom corresDondence should be -addressed - - ....- at Departamento d e Patologia, kaculdade de Medicina da USP, Av. Dr Arnaldo 455, CEP 01246, Sio Paulo, Brazil

026W37x/92/040235-O4$07.00 0 1992 by John Wiley & Sons, Ltd.

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of the lung. Recently, we have reported an alveolar capsule technique modified for rodents.h Using the alveolar capsule technique, it has been demonstrated in dogs' that lung tissue viscoelastic properties contribute significantly to the increase in lung resistance observed after histamine inhalation. If this finding is also valid for other species, it has important implications for the assessment of airway responsiveness, since reactions traditionally ascribed to changes in airway calibre may be influenced by tissue mechanical events. In the present study the usefulness of the alveolar capsule technique for partitioning airway and tissue reactivity to agonists in small animals was investigated. This procedure was used to assess airway and tissue pressure changes in rats after inhalation of methacholine. MATERIALS AND METHODS

The experiments were performed in eight adult female Wistar rats (155-250 8). The animals were anaesthetized with sodium pentobarbital (30 mg kg-' i.p.). A tracheal cannula (1.7 mm I.D.) was introduced into the trachea. The animals were ventilated using a mechanical ventilator with a tidal volume of 2.5 ml and a respiratory frequency of 100 cycles min-'. A wide bilateral thoracotomy was performed to expose both lungs, and warm (37°C) isotonic saline was poured Received 5 November 1990 Accepted (revised) 8 October 1991

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R. S. SAKAE ET

on the exposed regions to prevent dehydration. After thoracic opening, a positive end-expiratory pressure of 5 cmH20 was applied to the breathing circuit to avoid lung collapse. Tracheal pressure (PI,) was measured with a Validyne MP 45 differential pressure transducer connected to the end of the tracheal cannula. Alveolar pressure (PA) was measured by the alveolar capsule technique as previously described for rats.h The capsules were made out of the tips of 3-ml plastic syringes, and attached to the pleural surface of the lung with cyanoacrylate glue. The capsule body was connected to a Validyne MP 45 differential pressure transducer through a 15cm long polyethylene catheter (1.6 mm I.D.) to avoid parenchymal distortion due to the weight of the capsule-transducer assembly. This set-up has been shown to provide reliable information up to frequencies of 20 H z . ~One capsule was used in each animal. Capsules were placed in the left lung (1 animal), in the cranial lobe (2 animals), in the middle lobe (4 animals) and in the caudal lobe (1 animal). ptr and p A signals were registered in a Gould-Brush 2400 ink recorder and computed manually as the total swing in pressure between end-expiration and end-inspiration. The pressure drop across the airways (paw) was computed manually as the difference between ptr and

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imethacholinel (mg/ml) Figure 1. Changes in tracheal, alveolar and airway pressures along the dose-response curves. Each point represents the mean ( 2SE) of eight rats. ptr and pn presented statistically significant increases ( P < 0.001 1.

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After a 5-min period for the baseline measurements, sequentially increasing concentrations of methacholine chloride (Sigma Chemical Co, USA) diluted in saline to 2 , 4 , 8 , 16,32, 64 and 128 mg ml-' were aerosolized using an ultrasonic nebulizer (ICEL US-1000, Sio Paulo, Brazil). Methacholine aerosols were administered into the breathing circuit through the inlet port of the ventilator. Each dose was delivered for 60 s and pawand p a measurements were performed for 1 min, starting immediately after completion of methacholine administration. The progressively increasing concentrations of methacholine were administered immediately after the end of the 1-min data collection period. without allowing pressure profiles to return to baseline levels. Dose-response curves were prepared in terms of absolute changes in ptr,paw,andp,. Statistical analysis was done by means of analysis of variance and linear regression analysis, with a significance level of 5 % .

RESULTS

Figure 1 shows the mean (k SE) values of p,,, paw and pA measured during the dose-response studies for the eight animals. Baseline pawand pA mean (? SD) values (6.44 -C 2.06 and 8.25 5 3.44 cmH20, respectively) were not statistically different ( P = 0.220). The increases in ptr and pA were significant during the dose-response study ( P = 0.001), whereas Paw did not increase significantly. The increase in pA was significantly higher than that of paw ( P < 0.001). Figure 2 shows the relationship between the mean (f SE) values of ptr and pA for the eight rats at different doses of methacholine. This relationship could be described by a straight line (r = 0.990, P < 0.001). Figure 3 shows the correlation between the mean

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alveolar pressure (cmH10)

Figure2. Relationship between the mean (2SE) values of prr and pn

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Figure 3. Relationship between the mean ( 2SE)values of pt, and P a w

INHALED METHACHOLINE IN THE RAT (k SE) ptr and paw values ( r = 0.947, P < 0.001), which could also be described in terms of a linear function. There was also a good correlation betweenp, and p;lw,as depicted in Fig. 4 ( r = 0.913, P = 0.004). 0

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airway pressure (cmH20)

Figure4. Relationship between the mean ( 2 SE) values of pn and paw.

DISCUSSION

The current study was designed to assess the methacholine concentration dose-response behaviour of the airways and pulmonary parenchyma. Before discussing the results, some comments are needed on the methods used in this study. Several authors have shown that mechanical properties of the lung exhibit regional differences.x." It was also demonstrated that mechanical unevenness became more evident after bronchoconstrictive stimuli. l o Mechanical inhomogeneities are more pronounced at high respiratory frequencies and low amplitude oscillations."' Low tidal volumes may cause a less hornogeneous distribution of inhaled aerosol" and increase the regional mechanical differences during challenge with agonists. In these circumstances, it is important to note that the measurements of p A that we performed were indicative of the entire lung. It was previously demonstrated in normal open-chest rats that there are n o significant regional lung mechanical differences." In addition, to minimize mechanical inhomogeneities eventually induced by methacholine inhalation, our rats were ventilated with a relatively high tidal volume (2.5 ml) and a breathing frequency slightly lower than the usual (80 cycles min-I). The alveolar capsules placed in different regions of pulmonary parenchyma provided the same basic information, i.e. a larger increase in p , in comparison to p.,,,,. This fact suggests that there were no areas of exclusion of ventilation due to severe airway narrowing along the bronchial challenge protocol. In this context. Ludwig et al.' demonstrated that dog lungs exhibit no marked regional mechanical differences after histamine-induced bronchoconstriction, since the coefficient of variation between capsules located in three different lung regions was ca. 1%. The changes in p A after methacholine challenge is due to modifications of lung tissue mechanics. Lung

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tissue exhibits viscoelastic behaviour, i.e. its mechanical profile is dependent on the magnitude and frequency of stretching.".I3 Lung dynamic elasticity increases with volume, and tissue resistance decreases with increasing breathing frequency.12-'3Considering this behaviour, it is important to stress that our measurements were performed in paralysed animals, with fixed ventilatory settings. The tidal volume employed was within the limits of the linear segment of the lung pressure-volume curve. The present results indicate that the pressure changes attributable to lung tissue following methacholine inhalation are significantly higher than that of the airways (Fig. 1). The increase in lung tissue impedance cannot be explained by air trapping and displacement of end-expiratory lung volume along the pressure-volume curve, since p A swings did not shift from baseline at end-expiration in all animals. The observed increase in lung tissue impedance after methacholine inhalation is in agreement with previous findings in dogs,7 also using alveolar capsules, where lung tissue viscoelastic pressure losses increase significantly after histamine inhalation. Measurements of lung stress relaxation in guinea pigs have also shown that parenchymal forces are the main factor responsible for the increase in lung resistance after allergen-induced asthma. l 4 The observed increase in overall lung impedance was due to both lung tissue and airway resistance. There was a significant correlation, based on regression equations, between ptrand pA and ptr and paw(Figs 2 and 3 , respectively). The p A and pClw relationship also could be well described by a straight line (Fig. 4). which means that airway and tissue sensitivity (i.e. the dose of the agonist needed to cause half-maximal response)I5 to methacholine is similar, despite the fact that their maximal responses are significantly different. The increase in pdm could be ascribed to the contraction of airway smooth muscle. However, the precise structural elements that are responsible for the rise of tissue impedance are still not identified. Contractile elements are present in alveolar walls"' and in alveolar ducts.17 Theoretically, contraction of peripheral vasculature could also be invoked to explain the rise in pA. Our finding that lung tissue responds to cholinergic stimuli suggests that some aspects of the assessment of airway reactivity in rats should be revised. Traditionally, the increase in lung impedance has been ascribed to changes in airway calibre. Moreover, airway calibre and the extent of smooth-muscle shortening are affected by the tethering action of the surrounding parenchyma'K and the load represented by the radial traction of lung tissue on the airways.I9 Clearly, further studies are needed to clarify the role of lung tissue on overall lung impedance increase during bronchial challenge protocols. In conclusion. we have shown that pulmonary tissue of rats is a major factor responsible for the increase in lung impedance observed after methacholine challenge. In addition, we demonstrated that the reactivity of airway and pulmonary parenchyma is correlated, suggesting that both lung regions have a similar sensitivity to the agonist. Our results indicate that peripheral events should be considered during bronchial challenge protocols in rats.

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R. S. SAKAE ET A L . This work was supported hy the following Brazilian Institutions: FAPESP, CNPq and COOPERSUCAR.

Acknowledgements The authors are grateful to Professor Gyorgy Miklos Bohm for his opinions on the manuscript.

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