| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Dept of Physiology, University of Western Australia and 2 Dept of Pathology, Murdoch University, Perth, Western Australia, Australia
CORRESPONDENCE: D.J. Turner, Dept of Physiology, University of Western Australia, Nedlands, WA 6907, Australia. Fax: 61 893801025
Keywords: acetylcholine, airway hyperresponsiveness, allergen, bronchi, smooth muscle
Received: March 20, 2001
Accepted July 30, 2001
This work was supported by the National Health and Medical Research Council of Australia.
 |
Abstract |
|---|
 TOP Abstract Materials and methodsResultsDiscussionReferences |
|---|
Responsiveness to acetylcholine (ACh) was measured in eight ovalbumin (OA)-sensitized/challenged pigs (tests) and eight saline-challenged controls. In vivo responsiveness to ACh was determined from pulmonary resistance (RL). In vitro responsiveness to ACh was determined from airway pressure in isovolumic bronchial segments, after exposure via the adventitial or the luminal surface.
Test pigs had lung (255±26% increase in RL, p<0.0001) and skin responses to OA, and AHR to ACh (p<0.0001). In vitro, test bronchi were less sensitive than controls to ACh applied to the airway adventitia (negative log of the ACh concentration producing half the maximum response (pD2)=4.18 and 4.58 respectively, p<0.01), but not the lumen. Test bronchi had an increased amount of smooth muscle normalized for airway size versus controls (p<0.05). Maximum responses to lumenal ACh in vitro showed a weak positive correlation with maximum changes to ACh in vivo (r=0.599, p=0.05).
This study concludes that the effect of antigen challenge on bronchial responsiveness varies with the route of exposure to acetylcholine. In vitro responses to lumenal acetylcholine are increased despite a possible reduction in responsiveness of airway smooth muscle. Responsiveness of the bronchial wall only partially explains responsiveness of the lungs in vivo.
Despite the central role of airway narrowing in the response of the lung to provocative stimuli, its importance in airway hyperresponsiveness (AHR) has not been established. Since smooth muscle contraction is a major determinant of airway diameter, one proposal has been that AHR is due to altered contractile behaviour of airway smooth muscle (ASM). Studies in allergic animal models indicate that ASM contraction might be increased 1–3, while the responses of smooth muscle isolated from asthmatics are variable 4, 5. In vivo, ASM is subject to additional factors arising from outside the airway, such as load from parenchymal tethering 6. Some studies suggest that asthmatic bronchi have a reduced susceptibility to forces from parenchymal tethering, which could favour greater airway narrowing when the airway is stimulated 6, 7.
Many studies have focused on the responses of ASM after its isolation from the, airway, usually the trachea 8. In contrast, physiological responses described in intact bronchial airways isolated from asthmatics or animals models are scarce and the contributions that the bronchial wall may make to AHR in vivo remains controversial. A porcine model of AHR is presented in this study, which because of its size and ready availability allows the functional properties of different size bronchi to be investigated 9, 10. Early allergen-induced responses have previously been reported in the pig 7, 11. The preliminary aim of this current study was, therefore, to determine whether sensitization and repeated allergen exposure in pigs subsequently led to AHR. A further aim was to establish for the first time whether or not physiological responses in intact bronchi isolated from hyperresponsive pigs were increased. It was hypothesized that if AHR in vivo is due to physiological or morphological changes to the bronchial wall, then there should be an increase in responsiveness of bronchi isolated from the lung. In vitro responses were assessed in a bronchial segment, which preserves the structural components of the wall in their normal geometric relationship. This allows the smooth muscle to be exposed to contractile stimuli directly, via the adventitial surface, or via the airway lumen to simulate in vivo aerosol challenge.
|
Materials and methods |
|---|
|
TOPAbstractMaterials and methodsResultsDiscussionReferences |
|---|
Cutaneous responses
Success of sensitization was determined on day 14 and 20 using 50 µL intradermal injections of OA in 10-fold dilutions (0.001–1 mg·mL–1). The lowest concentration of OA at which a positive response (red wheal >5 mm diameter) occurred was recorded and used as an index of reactivity to the allergen 12.
Aerosol challenge
Test animals received OA aerosol (50 mg·mL–1 in saline) on days 14, 17 and 20 while anaesthetized and intubated, as described previously 7. Control animals received aerosols of saline alone. Pulmonary resistance (RL) and dynamic compliance (CL) were measured using an established protocol 7. Measurements were made at baseline and for 30 min following challenge to detect early allergen responses.
Assessment of airways hyperresponsiveness in vivo
Airway responsiveness was determined from concentration-response curves (CRCs) to doubling concentrations of acetylcholine (ACh; Sigma Castlehill, NSW, Australia) dissolved in saline. Solutions were delivered as aerosols into the airways for 60 s, as described for allergen delivery 7. RL and CL were measured before and after each concentration. Increasing concentrations of ACh were administered until RL reached a plateau. Baseline responsiveness was measured on day 14, prior to the first challenge, and again on day 20, 1 hr after the final challenge. Reactivity to ACh was determined from the maximal RL recorded during the CRC. Sensitivity to ACh was determined from the concentration of ACh required to increase RL 50% between baseline and the maximum RL value obtained (PC50).
Assessment of airways hyperresponsiveness in vitro
The stem bronchus was dissected from the left and right lower lung lobes of euthanized pigs on day 20 following completion of in vivo measurements. The parenchyma was removed, side branches were ligated and a 25 mm-long segment was mounted in an organ bath, as previously described 7, 13. These segments were classified as small/medium sized based on internal diameter (2–3 mm), location (spanning generations 9–14), and the presence of cartilage 14. The segment adventitia was bathed in Kreb's solution and the lumen filled with Kreb's from a separate reservoir. Stopcocks at either end of the segment allowed the airway lumen to be sealed so that intralumenal pressure could be monitored using a calibrated pressure transducer 10. Segments were checked for pressure leaks throughout each experiment.
Bronchi were electrically field stimulated (EFS, 60V, 20 Hz and 3 ms pulses) using platinum ring electrodes and a Grass S44 stimulator (Grass Instruments, Quincy, MA, USA). EFS responses were used to assess the optimum passive pressure and stretch of segments (5 cmH2O and 110% relaxed length, respectively, fig. 1
) and to confirm that bronchial segments had recovered from the previous ACh concentration.
|
Morphology
Bronchi were fixed at an intralumenal pressure of 5 cmH2O with 10% buffered formalin solution. Fixed tissues were paraffin embedded, 5 µm sections were cut and stained with haematoxylin and eosin. ASM area, inner and outer wall area 15 and the lumen perimeter (Pi) were determined using image analysis software (Leading Edge Pty Ltd, Adelaide, South Australia). Measurements were averaged from five randomly-selected sections of each bronchus. Area was normalized 16 for differences In airway size by 
area/Pi. The intra-observer coefficient of variation was <1.3%.
Histology
Representative sections of lower lung lobes were fixed in 10% buffered formalin. Fixed tissues were paraffin embedded, sectioned (5 µm) and stained with haematoxylin and eosin. Tissue samples were assigned a code number and examined by the pathologist. Visual assessment of the overall architecture of the alveolar and bronchiolar tissue, degree of vascular disturbance (oedema, hyperaemia and haemorrhage) and the presence of inflammatory cells (neutrophils, eosinophils, macrophages and lymphocytes) were noted.
Statistical analyses
Cutaneous and lung responses to OA or saline, and the reactivity and sensitivity to ACh were compared within groups using paired t-tests and between groups using unpaired t-tests. In vivo and in vitro CRCs to ACh were compared between groups using nonlinear regression (F-test) Unpaired t-tests were used to compare morphological parameters.
Correlations were determined from data obtained in controls, test and nonresponder animals, using the method of least squares. A p-value <0.05 was regarded as significant. Data are shown throughout as mean±sem, unless otherwise stated
|
Results |
|---|
|
TOPAbstractMaterials and methodsResultsDiscussionReferences |
|---|
In vivo responses
Naïve and sham-sensitized control animals showed no differences in lung function or responsiveness to ACh (table 1). The data was combined into one group (n=8) for comparison with test animals.
|
). Control animals showed no changes to saline aerosol (RL increased 11±4%, CL decreased 1±6%). Peak responses occurred in all animals within 5 min of completing the aerosol.
|
). In contrast, control animals showed no change between day 14 and 20 (fig. 3).
|
, the concentration of ACh causing a 50% increase between baseline and maximal RL (PC50) decreased in test pigs from a baseline of 9.9±1.5 mg·mL–1 on day 14 to 5.1±0.9 on day 20, p=0.01, while controls showed no change (8.3±0.6 mg·mL–1 and 8.4±1.1). PC50 was significantly different between the groups on day 20 (p<0.05), but not on day 14. Mean maximal RL increased from day 14 to 20 (289±36% and 387±68% respectively, p<0.05) in test animals but did not change in controls (295±49% on day 14 to 332±60% on day 20).
Nonresponder
One of the eight sensitized pigs showed minimal responses to aerosol OA on day 20 (fig. 4), no change in responsiveness following OA exposure (fig. 4), and had a very weak response to skin-prick testing (1 mg·mL–1). The animal was classed as a nonresponder and excluded from the test group for analyses.
|
).
|
); however, neither pD2 nor Emax differed between the groups (pD2: controls 2.84±0.13 and tests 3.05±0.17; Emax: controls 60.1±4.1 cmH2O and tests 61.1±4.9 cmH2O). In contrast to lumenal data, test bronchi showed a significant 2.5-fold loss in contractile potency to adventitial ACh compared to controls (p<0.0001, fig. 5). pD2 was significantly reduced (control 4.58±0.10, test 4.18±0.07, p<0.01), although Emax was not different between the two groups (control 82.1±3.4 cmH2O, test 75.6±7.5 cmH2O, fig. 5).
|
). There were no differences in inner, outer or total wall area between the two groups (table 3). The epithelium was >85% intact in both control and test bronchi, and of normal appearance.
|
Histology
Lymphocytes, neutrophils and eosinophils were evident in the parenchyma of test pigs. There was frequent, mild granulocytic cuffing around small bronchioles and an increase in cellularity within interstitial areas. Control animals showed occasional areas of increased cellularity, primarily due to atelectasis of the parenchyma. Larger airways were normal in appearance in both test and control pigs. No differences were observed in overall architecture of the tissue, or the degree of vascular disturbance between the two groups.
|
Discussion |
|---|
|
TOPAbstractMaterials and methodsResultsDiscussionReferences |
|---|
Bronchi from sensitized pigs had increased ASM, which is consistent with airway remodelling as seen in asthmatics 16. However, when assessed in vitro bronchi from sensitized pigs were less responsive to adventitial ACh than controls. Responsiveness could be influenced by inflammatory stimulation and also by the presence of smooth muscle cells with different contractile or secretory phenotypes 17. In some animal models, tracheal smooth muscle contraction is increased 1–3. There are fewer studies in bronchi, but one human study showed decreased smooth muscle force in bronchi obtained post mortem 5. The reduced bronchial response to adventitial ACh in the present study suggests that ASM force and sensitivity is decreased despite the presence of more ASM. At present, the authors have no information on contractions in more peripheral airways, which might show different levels of responsiveness. In contrast to the adventitial route, there was a modest rightward shift in the lumenal CRC of sensitized bronchi, the route considered to be more physiological. The small increase in lumenal sensitivity suggests the presence of some mechanism(s) within the airway wall which act to increase activation of ASM. Of particular importance is the lining epithelium, which if damaged leads to major increases in lumenal responsiveness due to increased drug penetration to the underlying smooth muscle 13. Although no gross alterations to epithelial morphology were observed in the test bronchi of this study, more subtle changes in epithelial permeability may have been present.
The prevalence of early airway responses to inhaled allergen using this porcine model was 87%, which compares favourably with other allergic animal models 18. One sensitized pig failed to respond to OA aerosol and showed a poor cutaneous response compared to the other seven test animals. This biological variation is typical of species that are not highly inbred, hence some research groups use screening tests, such as cutaneous responses, to preselect the animals they study 11, 18–20. The antigen-sensitized animals developed a typical wheal and flare response, suggesting a production of antigen-specific immunoglobulin-E (IgE) antibodies 12; whereas, neither naïve nor sham-sensitized control pigs responded to cutaneous antigen. A more sensitive index of sensitization would be advantageous. To date, it is not possible to measure porcine IgE concentrations directly, as there are no commercially-available antiporcine IgE monoclonal antibodies. However, significant advances have recently been made with the isolation of the pig IgE epsilon chain 21.
All control animals were exposed to an aerosol of saline, rather than OA in saline diluent, as repeated OA aerosol exposure may be sufficient to sensitize the animal in the absence of an initial antigen injection 22. Sham-sensitized pigs showed the same responses as naïve pigs (table 1). A third control group of OA-sensitized and saline-challenged pigs had been previously assessed in the present authors' laboratory with similar results (unpublished data).
Multiple allergen exposure has been used in several animal models 23–25 to induce some of the functional and struclural changes seen in asthma. In this study, three aerosol exposures to OA resulted in a mild inflammation in test animals particularly in the small airways. There was also an increase in ASM area, but no other changes in overall architecture of the tissue or degree of vascular disturbance distinguished test animals from controls.
An aim of the present study was to determine whether differences in the responsiveness of lungs in vivo could be accounted for by properties of the isolated airway wall. Data from control, test and nonresponder pigs were combined providing a broad range of responses. The present study demonstrates a relationship between maximal responses to lumenal challenge in vivo and in vitro. Previous studies using rings or strips of smooth muscle 8 have failed to show such an association, possibly because the dissection procedure may compromise the epithelium and other structural features that contribute to responsiveness in vivo. More recently, Masaki et al. 26 showed a positive correlation between the effect of lumenally-applied ACh on tracheal narrowing in vitro and insufflation pressure in vivo in an OA-sensitized cohort of guinea pigs. In contrast, a study by Woisin et al. 27 found no correlation between in vivo and in vitro responsiveness in control and sensitized rabbits; however, the sensitized cohort failed to show hyperresponsiveness when compared to the controls. The present study extends these findings as it includes a cohort of normal pigs together with sensitized animals exhibiting clear AHR.
The present results suggest that reactivity in vivo may be related to reactivity in the individual bronchus, as determined in vitro. There was no such relationship between sensitivities in vivo and in vitro. These findings are consistent with sensitivity arising as a result of more than one mechanism. Extrinsic airway mechanisms, as well as local airway alterations, could contribute to responsiveness as suggested by others 6. The present authors have previously shown that bronchi from test pigs are less compliant than controls 7, which is consistent with structural remodelling. In the present study, test animals showed airway wall remodelling in the form of increased airway smooth muscle area. This could lead to stiffer airways, which could be less prone to forces of interdependence in the lungs. Any reduction in the after-load on airway smooth muscle contraction, arising from forces of interdependence, might favour airway narrowing in vivo, but would not be apparent in the individual airway in vitro. Subsequent studies using this model may uncover, at the cellular level, mechanisms producing changes to airway function in the bronchial wall.
|
References |
|---|
|
TOPAbstractMaterials and methodsResultsDiscussionReferences |
|---|
chain. Immunogenetics 1997;46:461–468.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) pigs. Responses were calculated as percentages of maximum EFS response in each bronchus and are expressed as a group mean±sem. a) Responses varied with length in test bronchi, but not control (analysis of variance (ANOVA), p<0.02). b) There were no significant differences at different transmural pressures or between control or test bronchi at individual pressures (ANOVA and two-tailed unpaired t-test).
; control: 
) in individual pigs, pre and post in vivo aerosol challenge on day 20. Test animals received an ovalbumin (OA) aerosol (• and 
) compared to baseline measurements on day 14 (
; F-test, p<0.0001). The concentration of ACh causing a 50% increase between baseline and maximal pulmonary resistance (RL) was significantly decreased (paired t-test, p=0.01) and maximal RL significantly increased (paired t-test, p<0.05) on day 20 versus day 14. b) Control animals (n=8) showed no change between day 14 (•) and 20 (
; n=7) and control (adventitia: 
; n=8) pigs. Mean±sem control and test concentration-response curves were significantly different for both adventitial (p<0.0001, F-test) and lumenal (p<0.03, F-test) administration of ACh. The –log concentration of ACh producing half the maximum pressure developed in response to ACh (Emax) was significantly reduced in test animals (p<0.01) compared to controls in response to adventitial ACh only.