Comparisons of effects of intravenous and inhaled methacholine on airway physiology in a murine asthma model

Respiratory Physiology & Neurobiology 165 (2009) 229–236

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Comparisons of effects of intravenous and inhaled methacholine on airway physiology in a murine asthma model Sofia Jonasson ∗ , Göran Hedenstierna, Hans Hedenström, Josephine Hjoberg Department of Medical Sciences, Clinical Physiology, Uppsala University, Uppsala, Sweden

a r t i c l e

i n f o

Article history: Accepted 13 December 2008 Keywords: Lung mechanics Airway reactivity Asthma models Murine strains Ovalbumin Airway inflammation

a b s t r a c t Airway responses to intravenous (i.v.) and inhaled (i.h.) delivery of methacholine (MCh) in BALB/c and C57BL/6 mouse strains have been compared with and without ovalbumin (OVA)-induced airway inflammation. Bronchial reactivity to MCh was assessed in anaesthetised and tracheostomised animals by using an animal ventilator (flexiVent). We partitioned the response of the lungs into airway and parenchymal components in order to compare the contributions of the airways with those of the lung parenchyma to the pulmonary mechanical responses resulting from different routes of MCh administration. Our results indicate disparate physiological responses. Intravenous MCh delivery induced a higher maximum lung resistance than i.h. MCh in OVA-treated BALB/c mice but not in C57BL/6 mice. Inhaled MCh delivery led to a significantly larger fall in lung compliance and a greater impact on peripheral airways than i.v. MCh in both strains. In conclusion, i.v. and i.h. MCh produced disparate effects in different murine strains and variant responses in inflamed airways and healthy controls. The two methods of MCh delivery have important advantages but also certain limitations with regard to measuring airway reactivity in a murine model of allergic asthma. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Airway hyperresponsiveness (AHR) together with eosinophilic inflammation is a hallmark of allergic asthma. The inbred mouse is suitable for certain asthma models and some strains are more susceptible to the development of AHR to allergic sensitisation than others. Many invasive technologies are available for the measurement of airway function in small animals and the measurement of lung mechanics can offer an overall evaluation of lung function in mice. Sensitisation and subsequent airway challenge with an antigen is commonly used to develop AHR in murine models of asthma and this mimics the inflammatory characteristics of human asthma (Kumar and Foster, 2002; Kips et al., 2003; Epstein, 2004). Possible mechanisms for AHR include altered neural pathways, structural changes in the airway wall, airway smooth-muscle shortening, and the presence of inflammatory mediators (Bousquet et al., 2000; Robinson, 2004; Southam et al., 2007). Changes in the parenchyma due to airway inflammation can lead to increased

∗ Corresponding author at: Department of Medical Sciences, Clinical Physiology, University Hospital, Entr. 40 3rd floor, SE-751 85 Uppsala, Sweden. Tel.: +46 18 6115165; fax: +46 18 6114153. E-mail addresses: sofi[email protected] (S. Jonasson), [email protected] (G. Hedenstierna), [email protected] (H. Hedenström), [email protected] (J. Hjoberg). 1569-9048/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2008.12.005

airway reactivity, increased tissue resistance and decreased tissue compliance (Jonasson et al., 2008). External bronchoconstrictor agonists can simulate asthma responses, and the mode of administration and type of spasmogen can have different effects on the lung mechanics (Peták et al., 1997; Fernandez et al., 1999; Collins et al., 2005; Wagers et al., 2007). Our aim was to characterise the response to intravenous (i.v.) and inhaled (i.h.) delivery of bronchoconstrictor stimuli in mice with and without airway inflammation, using two commonly used murine strains, C57BL/6 and BALB/c mice. MCh is a bronchoconstrictive agent that has been widely used in the diagnosis of airway narrowing and AHR. Previous studies focusing on AHR have suggested that C57BL/6 mice are hyporesponsive to MCh compared to other strains (Levitt and Mitzner, 1989; Held and Uhlig, 2000; Takeda et al., 2001; Shinagawa and Kojima, 2003; Whitehead et al., 2003; Fukunaga et al., 2007), but this mouse appears to develop AHR, strong eosinophilic inflammation and also vascular and parenchymal changes after having been sensitised and challenged to ovalbumin. BALB/c mice are good responders to bronchoconstrictor stimulus (Wagers et al., 2004; Busse et al., 2007; Lundblad et al., 2007). Challenging this mouse with allergens results in an eosinophilic inflammation and AHR. We have chosen these two murine strains since they are commonly used for murine asthma models and are widely used in studies of lung physiology. Both strains are also well characterised in their response to ovalbumin and are commonly used for generating genetically manipulated mice.

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Besides our general aim of characterising the response to i.v. MCh and i.h. MCh, we have also made a more specific analysis of where the effects of MCh occur in murine airways. We wished to examine whether the route of administration of MCh has any effect on the site of action, and for this purpose we partitioned the response of the lungs into airway and parenchymal components. Our results indicate that there are differences in the physiological responses in animals with and without acute airway inflammation depending on the route of administration of the MCh. 2. Materials and methods 2.1. Animals Female mice were obtained from Taconic (M&B), Denmark. The mice were housed in plastic cages with absorbent bedding material and were maintained on a 12 h daylight cycle. Food and water were provided ad libitum. Their care and the experimental protocols were approved by the Regional Ethics Committee on Animal Experiments in Uppsala, Sweden (C86/05). The mice were 9.4 ± 0.4 weeks old and weighed 19.9 ± 0.3 g when their airway physiology was assessed. There were no significant differences in weight or age between strains. 2.2. Preparation of animals On the day of study, animals were weighed and anaesthetised intraperitoneally (i.p.) with pentobarbital sodium (BALB/c, 90 mg/kg and C57BL/6, 68 mg/kg, from local suppliers). The dose of pentobarbital sodium was carefully adjusted to obtain an optimal sedation for each strain. Mice were tracheostomised with an 18gauge cannula and mechanically ventilated in a quasi-sinusoidal fashion with a small animal ventilator (flexiVent, Scireq, Montreal, PQ, Canada) at a frequency of 2.5 Hz and a tidal volume (VT ) of 12 ml/kg body weight. Once ventilation was established, bilateral holes were cut in the chest wall so that pleural pressure would equal body surface pressure and the rib cage would not interfere with lung movement. This made strict lung mechanics measurement possible. Positive end-expiratory pressure (PEEP) of 3 cmH2 O was applied by submerging the expiratory line in water. A warming pad prevented cooling of the animal. Four sigh manoeuvres at three times the tidal volume were performed at the beginning of the experiment to establish stable baseline lung mechanics and to ensure a similar volume history before the experiments. The mice were then allowed a 5-min resting period before the experiment began. 2.3. Analysis of respiratory mechanics Dynamic lung mechanics were measured by applying a sinusoidal standardised breath and the resulting data were analysed using the single compartment model and multiple linear regression, to give lung resistance (RL ) and compliance (CL ) (Irvin and Bates, 2003). RL and CL were measured continuously using a standardised script and the maximum response to a given concentration of MCh is reported. Responsiveness was also expressed as the effective dose (ED) of MCh required to induce a 200% increase in RL (ED200 ). More thorough evaluations of the lung mechanics were made using the forced oscillation technique (FOT), where the ventilator piston delivers superimposed sinusoidal frequencies, ranging from 0.25 to 20.5 Hz, over 16 seconds (Prime 16) or 4 seconds (Prime 4, 13 frequencis), at the mouse’s airway opening. The Prime 4 perturbation was applied to the animal immediately before RL and CL measurements.

Harmonic distortion in the system was avoided by using mutually prime frequencies (Hantos et al., 1992). Knowing the dynamic calibration signal characteristics, the Fourier transformations of the recordings of pressure and volume displacement within the ventilator cylinder can be used (Pcyl and Vcyl ) to calculate the respiratory system input impedance (Zrs) (Gomes et al., 2000). Fitting the Zrs to an advanced model of respiratory mechanics, the constant phase model (Hantos et al., 1992) allows the lung mechanics to be divided into central and peripheral components. The primary parameters obtained were tissue damping (G), which is closely related to tissue resistance and reflects energy dissipation in the lung tissues, and tissue elastance (H), which is characteristic of tissue stiffness and reflects energy storage in the tissues (Tomioka et al., 2002; Bates and Irvin, 2003; Irvin and Bates, 2003; Bates and Lutchen, 2005). 2.4. Experimental protocols The 17-day-long airway inflammation protocol started with the intraperitoneal injection of 10 ␮g ovalbumin (OVA, Sigma–Aldrich) emulsified in Al(OH)3 (Sigma–Aldrich) on days 0 and 7. Mice were then challenged with 1% OVA diluted in PBS. The animals were subjected to aerosolised OVA for 30 min on days 14–16. Aerosol exposure was performed in a chamber coupled to a nebuliser (DeVilbiss UltraNeb® , Sunrise Medical Ltd., U.K.), the chamber being divided into pie-shaped compartments with individual boxes for each animal, providing equal and simultaneous exposure to allergen. The experiment ended on day 17 with an assessment of lung mechanics and eosinophil counts in bronchoalveolar lavage. Control mice (PBS) were sensitised with OVA i.p. and challenged with aerosolised PBS using the same protocol as the OVA-treated animals described above. Control mice showed no airway inflammation. Animals were randomised and allocated to the following groups: (1) Intravenous delivery of MCh: To assess airway responsiveness, BALB/c mice (OVA n = 8, PBS n = 8) and C57BL/6 mice (OVA n = 7, PBS n = 6), were given incremental doses of MCh i.v. in the lateral tail vein (0 = PBS, 0.03, 0.1, 0.3, 1, and 3 mg/kg) at 4 min intervals. MCh (MCh, acetyl-␤-methylcholine chloride, Sigma–Aldrich) was diluted in phosphate-buffered saline (PBS, Sigma–Aldrich) with 10 U ml−1 of heparin, and a volume of 2000 ␮l/kg was given at each injection. Heparin was added to prevent clotting in the i.v. catheters. The lung resistance was allowed to return to the baseline level between each dose of MCh. (2) Inhaled delivery of MCh: The concentrations of i.h. MCh were chosen so that the maximum dose would produce an increase in RL similar to that produced by the maximum dose of i.v. MCh in healthy BALB/c mice. These doses were then used for all groups and strains receiving i.h. MCh. To investigate the effect of incremental doses of i.h. MCh delivery into the trachea on BALB/c mice (OVA n = 8, PBS n = 8) and C57BL/6 mice (OVA n = 6, PBS n = 6), doses of MCh (0 = PBS, 1.67, 5, 25, 50, 100, and 200 mg/ml) were given at 4 min intervals. MCh was diluted in PBS and a volume of 10 ␮l was given over 10 s with an aerosol with a volume diameter between 4 and 6 ␮m (AeronebTM , Scireq). Each solution was aerosolised without any interference with the ventilation pattern. Between each dose of PBS and MCh, the lung resistance was allowed to return to the baseline level before the next aerosol dose was administered. 2.5. Bronchoalveolar lavage After completion of the lung mechanics experiment (24 h after the final aerosol challenge), the mice were exsanguinated and subjected to bronchoalveolar lavage (BAL). The lungs were lavaged

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three times via the tracheal tube with a total volume of 1 ml PBS containing 0.6 mM EDTA (EDTA, ethylenediaminetetraacetic acid, Sigma–Aldrich). The BAL fluid was then immediately centrifuged (10 min, 4 ◦ C, 1200 rpm). After removing the supernatant, the cell pellet was resuspended for 2 min at room temperature in 100 ␮l of erythrocyte lysis buffer containing 0.15 M NH4 Cl, 1.0 mM KHCO3 , and 0.1 mM EDTA. The suspension was then diluted with 1 ml PBS and recentrifuged (10 min, 4 ◦ C, 1200 rpm). Leukocytes were counted manually in a hemacytometer so that 50,000 cells could be loaded and centrifuged using a Cytospin® centrifuge (Shandon© cytospin 3 cyto-centrifuge, cell preparation system). Cytocentrifuged preparations were stained with May-Grünwald-Giemsa reagent and differential cell counts of pulmonary inflammatory cells (macrophages, neutrophils, lymphocytes, and eosinophils) were made using standard morphological criteria and counting 300 cells per cytospin preparation. 2.6. Histology Following BAL, the lungs were inflated with 4% paraformaldehyde solution to a pressure of 20 cmH2 O without removing the lungs from the chest. The trachea was tied off, and the lungs were then removed and stored overnight in 4% paraformaldehyde, and then stored in 70% ethanol at room temperature until the time for embedding. After embedding in paraffin, the tissue was cut into 4 ␮m thick sections and mounted on positively charged slides. To assess inflammatory cell infiltration, the sections were deparaffinised, dehydrated, and stained with hematoxylin and eosin (H&E). H&E-stained sections were examined by bright field microscopy (Axioskop 40 microscope, Carl Zeiss, Göttingen, Germany) and images were captured with a high-resolution digital camera system (AxioCam, Axiovison 2007, Carl Zeiss Imaging Systems, Göttingen, Germany). Repeated blind histopathological analyses were performed and representative images have been selected. 2.7. Statistical analysis Results are presented as the mean ± standard error of mean (S.E.M.). Statistical significance was assessed by parametric methods using two-way analysis of variance (ANOVA) to determine differences between groups, followed by a Bonferroni post hoc test. When appropriate, one-way ANOVA or Student’s unpaired t-test was used. A statistical result with p < 0.05 was considered significant. The statistical analysis was carried out and graphs were prepared with GraphPad Prism (version 4.0 GraphPad software Inc., San Diego, CA, USA). 3. Results

Fig. 1. Total cell count and differential cell counts in bronchoalveolar lavage from animals subjected to an ovalbumin (OVA) challenge protocol or a control protocol with phosphate-buffered saline (PBS). Values are expressed as mean ± S.E.M. OVA and PBS groups within each strain have been compared, and significant differences have been found in the total cell count and in the number of eosinophils, ***p < 0.001.

perivascular infiltration of inflammatory cells, mainly eosinophils, than BALB/c mice. 3.3. Lung resistance, RL For both strains, the RL value increased in a similar manner up to an i.v. MCh dose of 1 mg/kg. There were no differences in RL between PBS-treated BALB/c and C57BL/6 mice after either i.v. or i.h. MCh delivery. The maximum RL values for each combination of treatment and strain are shown in Table 1. 3.3.1. BALB/c mice OVA-treated mice had a significantly higher maximum RL with i.v. MCh delivery than with i.h. MCh delivery (p < 0.0001, Fig. 3 and Table 1). OVA significantly shifted both the i.v. and i.h. dose response curve to the left (i.v. MCh; PBS:ED200 0.28 ± 0.04 vs. OVA:ED200 0.10 ± 0.02, p < 0.001) (i.h. MCh; PBS:ED200 15.9 ± 2.3 vs. OVA:ED200 8.2 ± 2.3, p < 0.03). There was no such shift in the i.h. group. 3.3.2. C57BL/6 mice In OVA-treated mice, there was no difference between i.v. and i.h. MCh delivery with regard to the maximum RL . OVA significantly shifted the i.v. dose response curve to the left (PBS:ED200 0.25 ± 0.04 vs. OVA:ED200 0.12 ± 0.01, p < 0.004), this shift was absent in the i.h. group.

3.1. Cellular data 3.4. Lung compliance, CL Animals undergoing the OVA-challenge protocol showed clear signs of airway inflammation and a markedly larger total BAL cell count than PBS-challenged mice (Fig. 1). In OVA-treated mice, cell differentials from BAL fluid confirmed the inflammatory profile with an increased number of eosinophils. There were no significant differences between OVA-treated BALB/c and C57BL/6 mice with regard to the number of eosinophils, regardless of whether they had received i.h. or i.v. delivered MCh. 3.2. Histological evaluation Light microscopic examination of hematoxylin and eosin sections from OVA-treated BALB/c and C57BL/6 animals, revealed an eosinophilic inflammation with eosinophils surrounding the airways and within the alveolar spaces (Fig. 2). C57BL/6 animals sensitised and challenged with OVA had comparatively more

The lung compliance data are shown in Fig. 4, calculated as the percentage difference from the initial PBS value, and the maximum values are given in Table 1. 3.4.1. BALB/c mice The baseline CL was not affected by OVA-treatment. After both i.h. and i.v. MCh delivery, OVA-treated mice exhibited a larger decline in CL than the PBS-treated mice (p < 0.01, Fig. 4 and Table 1). The maximal drop in CL was significantly greater after i.h. MCh than after i.v. MCh delivery in both OVA- and PBS-treated mice (p < 0.001, Fig. 4 and Table 1). 3.4.2. C57BL/6 mice In C57BL/6 mice, OVA-treatment led to a decrease in the baseline CL by 22% (p < 0.0001). OVA-treated mice exhibited a larger decline

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Fig. 2. Representative histological sections (hematoxylin and eosin-stained) from animals with OVA-induced airway inflammation and from PBS-challenged controls. Examination of sections from animals with airway inflammation revealed a significant inflammation surrounding the airways and within the alveolar spaces. PBS-challenged mice showed no signs of inflammation. Photomicrographs (40×) of: (A) BALB/c, OVA; (B) BALB/c, PBS; (C) C57BL/6, OVA; (D) C57BL/6, PBS. Table 1 Maximum values of lung resistance, RL , lung compliance, CL , tissue damping, G, and tissue elastance, H, after i.v. MCh or i.h. MCh delivery for PBS-treated mice and animals with airway inflammation (OVA) for both BALB/c and C57BL/6 strains of mice. Strain

BALB/c

Treatment

OVA −1

MCh-induced RL i.v. MCh (cmH2 O s ml ) MCh-induced RL i.h. MCh (cmH2 O s ml−1 ) MCh-induced CL i.v. MCh % decrease from PBS dose MCh-induced CL i.h. MCh % decrease from PBS dose G i.v. MCh (cmH2 O s ml−1 ) (Prime 4) G i.h. MCh (cmH2 O s ml−1 ) (Prime 4) H i.v. MCh (cmH2 O s ml−1 ) (Prime 4) H i.h. MCh (cmH2 O s ml−1 ) (Prime 4) Baseline G (cmH2 O s ml−1 ) (Prime 16) Baseline H (cmH2 O s ml−1 ) (Prime 16)

7.0 4.2 55 84.6 5.7 15.9 40.8 108.0 5.2 30.9

C57BL/6 PBS

± ± ± ± ± ± ± ± ± ±

*,#,␥

0.6 0.6* 4.4* , ␥ 3.9 0.36# , ␥ 2.6* 3.9# , ␥ 13.8* , # 0.14# 1.3#

3.7 3.1 34.5 73.7 5.8 10.2 31.5 64.9 5.9 29.8

OVA ± ± ± ± ± ± ± ± ± ±

␦

0.3 0.1 2.3␥ 3.5 0.4␥ 0.9 1.9␥ ,␦ 5.5 0.2 1.2

5.5 5.2 60.6 83.0 10.2 17.7 73.2 155.3 9.2 48.1

PBS ± ± ± ± ± ± ± ± ± ±

*

0.6 1.2 6.5* , ␥ 3.2 1.3* , ␥ 2.2* 6.1* , ␥ 24.7* 1.2* 2.3*

2.9 4.1 35.4 72.9 6.2 9.9 46.2 79.6 5.8 34.5

± ± ± ± ± ± ± ± ± ±

0.3␥ 0.8 1.6␥ 1.5 0.3␥ 2.0 3.5␥ 15.9 0.1 1.1

Results are expressed as mean ± S.E.M. * p < 0.05, OVA vs. PBS within strains. # p < 0.05, OVA vs. OVA between strains. ␦ p < 0.05, PBS vs. PBS between strains. ␥ p < 0.05, i.v. vs. i.h. within strains and within the same treatment.

in CL than the PBS-treated mice after i.v. MCh (p < 0.01), but not after i.h. MCh delivery. OVA- and PBS-treated mice subjected to i.h. MCh delivery exhibited a markedly larger drop in CL than those subjected to i.v. MCh (p < 0.0001, Fig. 4 and Table 1). 3.5. Peripheral lung components 3.5.1. Baseline airway physiology (FOT, Prime 16) As shown in Table 1 and Fig. 5, there were significant increases in tissue damping (G) and tissue elastance (H) before RL and CL

assessment in C57BL/6 mice, but this was not seen in BALB/c mice. 3.5.2. Peripheral lung components (FOT, Prime 4) 3.5.2.1. BALB/c mice. OVA- and PBS-treated mice exhibited significantly higher maximum G values after i.h. MCh than after i.v. MCh delivery (OVA, p < 0.001; PBS, p < 0.0002, Fig. 5 and Table 1). OVA and PBS-treated mice also exhibited higher maximum H values following i.h. MCh than following i.v. MCh delivery (OVA, p < 0.001; PBS, p < 0.001, Fig. 5 and Table 1).

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Fig. 3. The effect of the delivery route of methacholine (i.v. MCh or i.h. MCh) on lung resistance (RL ) in PBS-challenged mice and in mice with airway inflammation (OVA). Values are shown as mean ± S.E.M. For each dose, OVA and PBS groups are compared, **p < 0.01; ***p < 0.001.

Fig. 4. The effect of delivery route of methacholine (MCh) on lung compliance (CL ), presented as the MCh-induced CL as the percentage decrease from the initial PBS level (i.h. or i.v. PBS) for both i.v. MCh and i.h. MCh delivery. Values are shown as mean ± S.E.M. For each dose, OVA and PBS groups are compared, *p < 0.05; **p < 0.01; ***p < 0.001.

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Fig. 5. Tissue elastance (H) and tissue damping (G) were measured with a forced oscillation technique (Prime 4 perturbation, Zrs measurements) before each dose of PBS or MCh (i.h. or i.v. delivery). Values are shown as mean ± S.E.M. For each dose, the following groups are compared: *C57BL/6; OVA vs. PBS, # BALB/c; OVA vs. PBS, ␦ BALB/c; OVA vs. C57BL/6 OVA (*p < 0.05; **p < 0.01; ***p < 0.001).

3.5.2.2. C57BL/6 mice. In OVA-treated mice, G and H increased more after i.h. MCh than after i.v. MCh delivery (G, p = 0.013; H, p < 0.001, Fig. 5 and Table 1). There were, however, differences in G and H after i.h. MCh and i.v. MCh delivery in PBS-treated mice at the maximum dose (Table 1). 4. Discussion The aim of this study was to evaluate the roles of different routes of delivery of the bronchoconstrictor agonist methacholine in a murine model of asthma, and to examine whether the site of action of MCh differs depending on the route of administration. MCh was either inhaled or administered intravenously and the response of the lungs was divided into airway and parenchymal components. Both the low-frequency oscillation technique (FOT) and the single compartment model were implemented to characterise the effects of MCh delivery. FOT has the capacity to partition the respiratory properties into central and peripheral airways and also into airway and tissue properties (Hantos et al., 1992; Tomioka et al., 2002). The most important findings in this study are that: (1) in OVA-treated C57BL/6 mice, i.v. and i.h. MCh induced similar maximum lung resistance, while in OVA-treated BALB/c mice i.v. MCh induced a significantly higher maximum lung resistance than i.h. MCh; (2) in both strains and irrespective of OVA-treatment, i.h. MCh led to a significantly greater fall in lung compliance and had a greater impact on peripheral airways than i.v. MCh; (3) i.v. MCh led to a more homogeneous airway constriction with little tissue distortion than i.h. MCh which led to a more heterogeneous airway behaviour and a more pronounced tissue distortion.

As in earlier studies (Singh et al., 2005), C57BL/6 mice exhibited more vascular inflammation after OVA-treatment than BALB/c mice. Singh et al. (2005) have previously shown time- and strainspecific differences in the perivascular recruitment of inflammatory cells in acute OVA-induced airway inflammation. In this study, the total cell count in BAL fluid was much higher in C57BL/6 mice than in BALB/c mice, and the number of eosinophils in C57BL/6 mice exceeded the number in similarly sensitised and challenged BALB/c mice; in agreement with the results of other studies (Takeda et al., 2001; Whitehead et al., 2003). As expected, OVA-treated mice of both strains exhibited acute hyperresponsiveness to i.v. MCh delivery. After i.h. MCh delivery, however, the data demonstrated disparate results; OVA-treated BALB/c mice showed a hyperresponsiveness similar to that with i.v. MCh delivery, whereas C57BL/6 mice showed no hyperresponsiveness to i.h. MCh delivery. Intravenous MCh delivery induced airway constriction in PBS-challenged mice, but i.h. MCh delivery induced a significantly greater response in the parenchymal tissue properties and less airway constriction, as assessed by FOT. Inhaled MCh may be unevenly distributed around the bronchial tree and thus induce a heterogeneous peripheral airway response (Fredberg et al., 1985; Ludwig et al., 1988; Brown et al., 1993; Salerno and Ludwig, 1996). This may lead to more sensitive regions, or regions which receive more MCh and become extremely constricted or even atelectatic, while adjacent regions can be hyperinflated (Nagase et al., 1994). MCh leads to muscle contraction by stimulating the muscarinic cholinerergic receptors that can be found in both airways and lung parenchyma (Barnes, 1993, 1995; Sly et al., 1995; Fisher et al., 2004). Receptors located on the airway

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smooth muscle may be more easily reached by i.v. MCh delivery, since inhaled MCh has to diffuse across the respiratory epithelium before reaching the muscle. It has been suggested that muscarinic receptors located on the alveolar wall are involved in the parenchymal response (Sly et al., 1995) and these are probably reached more easily by inhaled MCh (Peták et al., 1997). Moreover, the distribution of MCh down the airway tree may be limited during i.h. administration but not during of i.v administration. According to previous studies (Nagase et al., 1994; Salerno et al., 1995; Peták et al., 1997; Wagers et al., 2007), the pattern of effect depends on whether the MCh is inhaled or i.v. delivered. According to Salerno and Ludwig (1996), the different changes in peripheral airways induced by i.v. MCh and i.h. MCh delivery could also be due to the size of the murine airways. Salerno et al. claim that i.h. MCh delivery will lead to a higher concentration in the smaller airways than in the larger airways and that more marked effects may therefore be seen in the peripheral parts. Another possible explanation for the heterogeneity induced by i.h. MCh delivery could be due to droplet formation in smaller airways. It should be borne in mind that it is difficult to compare directly the i.v. MCh doses and the actual doses of MCh delivered by inhalation. The reason is, of course, that most of the inhaled nebulised MCh follows the expired air out from the animal so that the exact deposited dose is difficult to measure. In the present study, this problem was solved by determining the dose of i.h. MCh that led to an increase in RL similar to the maximum response to i.v. MCh (3 mg/kg). Different routes of MCh delivery had different effects on Zrs, causing G (tissue damping) and H (tissue elastance) to change to different degrees as the i.v. MCh or i.h. MCh reached the airway smooth muscle and caused it to contract. G reflects the parenchymal distortion that occurs when the airways constrict and, as with H, G increases with an elevated degree of regional airflow heterogeneity throughout the lung. H increases due to airway closure and to increased intrinsic tissue stiffness, which can result from the distortion of the parenchyma as the airways narrow. Another way that tissue stiffness can increase is through the development of regional heterogeneities in the lung tissue (Wagers et al., 2007). Previous studies (Wagers et al., 2004, 2007; Lundblad et al., 2007) have shown that increased airway reactivity in OVA-inflamed mice is entirely due to exaggerated closure of peripheral and small airways. In the present study, the OVA-treated mice were found to be more likely to develop higher increases in G and H after i.h. MCh delivery than after i.v. MCh delivery. OVA-treated C57BL/6 mice showed a more pronounced parenchymal response than OVA-treated BALB/c mice. In conclusion, it is clear that the effects of MCh on a murine airway and the physiological response depend on the administration route. i.v. MCh delivery led to a more homogeneous and marked airway constriction and less tissue distortion than i.h. MCh delivery, which led to more heterogeneous airway behaviour and tissue distortion. It must be emphasised that care should be taken when designing similar MCh provocation protocols. Both methods for the delivery of MCh have important advantages but also certain limitations when studying changes in airway mechanics in murine models of allergic asthma. When deciding on a murine asthma model it is important to consider the choice of strain and the mode of delivery of the bronchoconstrictor.

Acknowledgements This work was supported by the Swedish Heart-Lung Association and the Swedish Medical Research Council. We thank Maria Lundqvist for invaluable help with the animal experiments.

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