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Relationship of Airway Responsiveness with Airway Morphometry in Normal and Immunized Rabbits
Pulmonary Pharmacology & Therapeutics (2001) 14, 75–83 doi:10.1006/pupt.2000.0265, available online at http://www.idealibrary.com on
PULMONARY PHARMACOLOGY & THERAPEUTICS
Relationship of Airway Responsiveness with Airway Morphometry in Normal and Immunized Rabbits F. E. Woisin∗, C. M. Herd∗1, G. J. Douglas∗2, K. Raynor∗, D. Spina∗, H. W. Mitchell†, C. P. Page∗ ∗Sackler Institute of Pulmonary Pharmacology, Division of Pharmacology and Therapeutics and Department of Respiratory Medicine and Allergy, Kings College, London, UK, †Department of Physiology, University of Western Australia, Nedlands, WA, Australia
SUMMARY: Airway responses to chemical stimuli occur over a wide range of concentrations, with overlap between severe, moderate and mild asthmatic groups and with normal healthy individuals. Mathematical modelling has suggested that relative thickness of the airway wall may account for this range of responsiveness. We have investigated whether in vivo airway responsiveness varies as a function of airway wall thickness in terms of airway smooth muscle area in normal and immunized New Zealand White (NZW) rabbits. Airway responsiveness to inhaled methacholine (MCh) was determined in vivo under neuroleptanalgesia. Subsequently, ex vivo responsiveness to MCh (pD2=−log EC50) of isolated bronchi from the same animal was established. Smooth muscle area per mm basement membrane (SM/mmBM) was also measured morphometrically in the tested bronchi and the findings related to in vivo and ex vivo responsiveness. We found no relationship between airway responsiveness in vivo and pD2 values in either immunized or control rabbits. In both control and immunized rabbits, no correlation was found between SM/mmBM and in vivo airway responsiveness. Only in immunized animals with a PCA titre >0, was there a significant correlation (=−0.5986, P<0.05) between SM/mmBM and pD2. We conclude that airway smooth muscle area per se is not the sole contributor of airway responsiveness in vivo in normal rabbits. 2001 Academic Press
KEY WORDS: Airway smooth muscle, Methacholine, Airway remodelling.
usually determined as the provocative concentration of histamine or methacholine inducing a 20% fall in FEV1 (PC20), and has been suggested to correlate with asthma severity.4 However, the PC20 values of individual asthmatics to such stimuli occur over a wide range of concentrations, with overlap between severe, moderate and mild asthmatic groups and even with normal healthy individuals.4,5 The mechanisms underlying the variability and the wide range of airway responsiveness in vivo are not yet determined, but may be due in part to individual differences in the characteristics of the airway wall secondary to structural changes. Asthma has become recognized as a chronic inflammatory disease, with the airways of even mild asthmatics demonstrating increases in inflammatory cell infiltrate.6 Primed and activated mast cells, eosinophils, macrophages and lymphocytes have been found in bronchoalveolar lavage fluid obtained from
INTRODUCTION Airway hyperresponsiveness in asthmatic individuals is characterized by two components of the response to non-specific stimuli such as histamine, methacholine, cold air or exercise: an increased sensitivity (or leftward shift of the concentration–response curve) and an increased maximal airways responsiveness (heightened or unobtainable plateau of the concentration–response curve).1–3 Clinical assessment of airway responsiveness is ∗Author for correspondence: C.P. Page, Sackler Institute of Pulmonary Pharmacology, Division of Pharmacology and Therapeutics, GKT School of Biomedical Sciences, King’s College, London, 5th Floor, Hodgkin Building, Guy’s Campus, London SE1 9RT, UK. Fax: + 44 (0) 20 7848 6097. E-mail: [email protected] 1Present address: AstraZeneca (UK) Ltd., Charnwood, Leicestershire, UK. 2Present address: Pneumolabs (UK) Ltd., NPIMR, Y Block, Watford Road, Harrow, Middlesex, HA1 3UJ, UK. 1094–5539/01/02075+09 $35.00/0
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Fig. 1 In vivo airway responses of individual rabbits to methacholine challenge, indicating that the parameters measured for each rabbit (R PC50; CdynPC35) occur over a 100-fold range. Data represent measurement of (A) RL PC50; mg/ml and (B) Cdyn PC35; mg/ml from individual immunized rabbits (Ο; n=18), those with PCA>0 (Χ; n = 12) or saline control rabbits (Β; n=13).
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Fig. 2 Concentration–response curve of perfused main bronchi of rabbits to methacholine. Curve represents the change in flow rate in response to methacholine applied to bronchi obtained from control (Β; n=13) and immunized PCA>0 (Χ; n=12) rabbits. Values represent mean±SEM.
asthmatics.7 The products of these cells can act as bronchoconstrictors, chemotactic factors and/or damage the basement membrane and epithelium in the airway. However, clinical observations have led investigators to suggest that persistent airways hyperresponsiveness may occur in the absence of overt airways inflammation, as in subjects undergoing longterm treatment with glucocorticosteroids.8 It is therefore possible that repeated inflammatory insults may lead to more permanent structural changes in the airways. Mediators released by inflammatory cells may perpetuate tissue damage and thus contribute to the airway wall remodelling observed in lung tissue obtained post-mortem9–12 and in lung tissue biopsies from asthmatics.13 In asthmatics, the increased airway reactivity and loss of the dose–response curve plateau to inhaled spasmogens are thought to be a consequence of airway wall thickening.10 Mathematical modelling suggests
that increased wall thickness may account for bronchial hyperresponsiveness in asthmatics.2,14 Mathematical modelling has also suggested that increases in muscle mass may account for the airway hyperresponsiveness observed in asthma.15 However, these data from mathematical modelling are not supported by direct biological evidence relating these morphometric characteristics to in vivo airway function. We have reported previously the range of airway responsiveness to inhaled spasmogens in normal and neonatally immunized rabbits is remarkably similar to the pattern observed in humans.16 Therefore, in the present experiments we have investigated whether in vivo airway responsiveness varies as a function of airway smooth muscle thickness by studying the relationships between in vivo airway responsiveness, ex vivo responsiveness of the isolated bronchi and airway wall morphometry in normal and immunized New Zealand White rabbits.
MATERIALS AND METHODS Study design Rabbits used in this study were randomized at birth to different treatment regimes. Each experimental procedure was performed blind in that no experimenter was aware of the immunological status of the rabbits until all tests (in vivo, in vitro and morphometric measurements) were completed. For each rabbit, in vivo airway responsiveness to methacholine was measured. Subsequently the right main bronchus of the rabbit was isolated and its responsiveness to methacholine in vitro was measured using a perfused bronchial segment preparation. Finally, the bronchus was studied morphometrically to determine the airway wall thickness and smooth muscle area per mm of
Airways in Vivo, in Vitro and Morphometry
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Fig. 3 Relationship between in-vivo airway responsiveness and ex-vivo sensitivity to methacholine of isolated bronchi from the rabbits studied. Figures show (A) MCh Cdyn PC35 and (B) MCh R PC50 vs. MCh pD2 (−log10 EC50) in control (Β, n=13) and immunized PCA>0 (Χ, n=12) rabbits. The correlation coefficients () and associated P values were calculated using Spearman rank order correlations. A. Control, =−0.088, P=n.s., PCA>0, =0.112, P=n.s. B. Control, =0.043, P=n.s., −PCA>0, =−0.112, P=n.s. 7
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Fig. 4 Relationship between the in-vitro responsiveness of perfused rabbit bronchi to methacholine (pD2) and airway smooth muscle thickness of bronchi (SM/mmBM) in control (Β, n=13) and immunized PCA>0 (Χ, n=12) rabbits. The correlation coefficient () and associated P value were calculated using Spearman rank order correlation. Control, =0.098, P= n.s., PCA>0, =−0.599, P<0.05.
basement membrane. These data were collated and examined to determine if any correlation existed between any of these parameters tested. Animals New Zealand White (NZW) rabbits of either sex (2.6–3.8 kg) were obtained from Froxfield Farms UK (Petersfield, Hampshire, UK). The methods described in this study were subject to Home Office approval and were performed under the Animals (Scientific Procedures) Act, 1986. Immunization protocol The immunization procedure has been described in detail previously16 and is briefly outlined here. Within
24 h of birth an equal number of pups from each litter were randomly assigned to either of two groups: control or immunized. Control rabbits were given 0.5 ml saline (sterile, pyrogen free 0.9% sodium chloride solution, Baxter Health Care, Norfolk, UK) intraperitoneally (ip) and their littermates were immunized by injection of 0.5 ml Alternaria tenuis extract (40,000 PNU/ml; Greer Laboratories, North Carolina, USA) mixed with the sterile adjuvant aluminium hydroxide (Al(OH)3) gel (Rehydragel, Dublin, Eire). This regime was repeated weekly for the first month and then biweekly for the following 2 months. At 3 months of age, the rabbits were transferred from the breeding unit to our laboratory. Sensitization assessment through determination of passive cutaneous anaphylaxis An index of the serum IgE levels from all rabbits used in this study was obtained by titration of sera sample dilutions in the passive cutaneous anaphylaxis (PCA) test. Serum was prepared from 10 ml of blood obtained by cardiac puncture when the animals were killed prior to the ex vivo experiments. Recipient naive male NZW rabbits (2.0–3.0 kg) were anaesthetized with Hypnorm (a mixture of fentanyl citrate, 0.315 mg/ ml and fluanisone, 10 mg/ml; Janssen Pharmaceutical Ltd., Oxfordshire, UK) (0.4 ml/kg injected im) and the dorsal skin hair was closely clipped. A pattern of six blocks of six sites for 36 intradermal (id) injections was marked on the dorsal skin. Serial two-fold dilutions (between 1:1–1:64) in PBS were made of six test sera and injected (0.1 ml) id into three recipient rabbits. After a 72-h fixation period the recipient rabbits were anaesthetized with sodium pentobarbitone (30 mg/kg, iv) via an indwelling cannula (23 gauge Butterfly) in the marginal ear vein. Evans blue
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Fig. 5 Relationship between in-vivo responsiveness to methacholine (A) MCh Cdyn PC35 and (B) MCh R PC50 and airway smooth muscle thickness of main bronchi (SM/mmBM) in control (Β, n=13) and immunised with PCA>0 (Χ, n=12) rabbits. The correlation coefficients () and associated P values were calculated using Spearman rank order correlations. A. Control, =0.455, P=n.s., PCA>0, =−0.549, P=n.s. B. Control, =0.461, P=n.s., PCA>0, =−0.430, P=n.s.
dye (10 mg/kg) was administered intravenously followed 10 min later by iv antigen (A. tenuis extract; 7500 PNU/kg). Thirty minutes after antigen challenge the rabbits were killed with an overdose of sodium pentobarbitone (60 mg/kg, iv) and the back skin removed. The titration end-point was taken as the highest dilution at which the test serum caused a positive response when the skin was viewed from the underside. Pulmonary function measurement Measurements of pulmonary function and airway responsiveness were made in spontaneously breathing rabbits under neuroleptanalgesia using an established method.16 Rabbits premedicated with diazepam (2.5 mg/kg, Roche Products Ltd., Hertfordshire, UK) underwent neuroleptanalgesia with 0.4 ml/kg Hypnorm (Janssen Pharmaceutical Ltd., Oxfordshire, UK) and were placed supine on a padded animal board. Neuroleptanalgesia was maintained throughout the course of the experiment by further administration of Hypnorm (0.2–0.3 ml) every 20 min. The rabbits were intubated with a cuffed endotracheal tube which was then attached to a heated Fleisch pneumotachograph connected to a Validyne differential pressure transducer (model MP 45-14-871; Validyne Engineering Corp., California, USA) from which measurements of flow and tidal volume were obtained. A latex oesophageal balloon was inserted into the lower third of the oesophagus and attached to a second Validyne differential pressure transducer (model MP 45-24-871) by way of a cannula for the measurement of transpulmonary pressure. From these measurements total lung resistance (R) and dynamic compliance (Cdyn) were calculated using an on-line
respiratory analyser [Pulmonary Monitoring System (PMS) Version 5.1; Mumed Ltd., London, UK]. Measurement of airways responsiveness in vivo Airway responsiveness to inhaled methacholine hydrochloride (Sigma Chemical Co., Dorset, UK) was measured. Aerosols of methacholine were generated using an ultrasonic nebuliser (de Vilbiss Ultraneb 99) and delivered directly into the lungs via the endotracheal tube. Initially, basal lung function parameters were measured before aerosolised saline was administered for 2 min. After saline inhalation lung function parameter measurements were made again as a baseline with which to compare responses to methacholine. Cumulative concentration–response curves to methacholine (1.25–80 mg/ml), administered for 2-min periods in doubling doses, were established. The provocation concentrations of methacholine that produced a 50% increase in total lung resistance (R PC50) and a 35% fall in dynamic compliance (Cdyn PC35) were determined for each rabbit. These values were used as indices of airway responsiveness in vivo. Measurement of airway smooth muscle responsiveness in vitro In vitro airway responsiveness was determined by measuring changes in flow through a perfused bronchial segment.17 Rabbits were killed by injection of an overdose of sodium pentobarbitone (60 mg/kg, iv; Sigma Chemical Co., Dorset, UK). The right main bronchus was isolated from the lung by careful dissection of the tissue, tying off side branches with surgical silk as the parenchyma was removed. Tissues used for the experiment were approximately
Airways in Vivo, in Vitro and Morphometry
1.8–2.0 cm in length with an internal diameter of 2 mm at the distal end. The tissue was mounted on cannulae that were approximately the same size or slightly larger than the internal diameter of the relaxed segment. The bronchus was then placed in a bath of Krebs– Heinseleit solution containing (in mM concentrations): NaCl (117), NaHCO3 (25.0), -glucose (11.1), KH2PO4 (1.03), MgSO4 ·7H2O (0.57), KCl (5.4) and CaCl2 (2.5). Krebs–Heinseleit solution was maintained at 37°C, gassed with 5% CO2 in O2 and was exchanged for fresh solution at least five times over an equilibration period of 30 min. The tissue was also perfused through the lumen with Krebs– Heinseleit solution, at a transmural pressure of 4 cmH2O and a driving pressure of 2 cmH2O. A transducer measured the flow rate through the segment and changes in flow rate were detected by a custombuilt flow head (Ugo Basile, Italy) set up in series with the tissue to which the transducer was connected.17 The flowhead and transducer were previously checked for linearity and were calibrated over the range of flows encountered in the experiment. Decreases in the flow rate of the perfusate reflected constriction of the rabbit bronchus. Concentration–response curves for methacholine (10−8–10−3) applied serosally to the tissues were obtained. After the flow rate was returned to baseline by repeated washing, tissues were then fixed in 10% formaldehyde for subsequent morphometric analysis to determine the extent of airway wall and airway smooth muscle thickening. Morphometry Fixed bronchial segments of 2.5 mm internal diameter were blocked in paraffin, sectioned every 7 m and then stained with Masson’s trichrome. Five sections taken from the distal third of the segment were studied morphometrically using VideoPro image analysis software, (Leading Edge Pty. Ltd., South Australia). From each section, four sectors of the airway wall were examined under a ×20 objective. This allowed morphometric examination of approximately 75% of the airway wall circumference. In each sector we measured the length of the basement membrane (Pbm), total inner wall area and area of the smooth muscle. Smooth muscle area and total inner wall area measurements were divided by the Pbm to derive a measurement of smooth muscle thickness (SM/mmBM) and airway wall thickness (IWA/mmBM). Mean values were calculated for each section and airway.
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methacholine for in vitro experiments is expressed as a pD2 value (−log10 EC50). Differences between groups were compared using unpaired Student’s t-test. Spearman rank order correlation coefficients () and associated P values were calculated for all correlations. All statistical analyses were determined using the Prism GraphPad computer program, and P values of <0.05 were considered significant. RESULTS PCA titres Passive cutaneous anaphylaxis titres provide an index of serum IgE levels and were expressed as the reciprocal of the highest dilution of serum that gave a positive response. Whilst none of the sera from the control group of rabbits exhibited a PCA titre, 12 of 18 (66%) sera obtained from immunized animals gave a positive PCA response. In vivo airway responsiveness The in vivo airway responsiveness measurements for the rabbits as measured by MCh log10 RL PC50 immunized (n=18) vs. control (n=13) were 0.587±0.096 and 0.502±0.148, respectively and MCh log10 Cdyn PC35 immunized vs. control were 0.544±0.096 and 0.387±0.124, respectively (Fig. 1). Hence, basal airway responsiveness did not differ significantly between immunized and control groups. In rabbits exhibiting a PCA titre >0 (n=12), the in vivo airway responsiveness measurements for MCh log10 Cdyn PC35 and MCh log10 RL PC50 were, respectively, 0.491±0.109 and 0.5392±0.089 and these values did not significantly differ from those of all immunized rabbits or those of the saline-treated controls (Fig. 1). In vitro bronchial responsiveness Methacholine induced a concentration-dependent reduction in flow when applied to the bronchial segments from all rabbits (Fig. 2). The contractile potency (pD2) to methacholine in bronchial segments obtained from control rabbits was not significantly different to that observed in bronchial preparations from immunized rabbits (control, 5.23±0.12, n=13 vs. immunized, 5.30±0.14, n=20, P>0.05). The contractile potency to methacholine in bronchial segments from rabbits with PCA titres >0 (5.22±0.17, n=12) was not significantly different from control (P>0.05).
Statistical analysis In vivo methacholine provocation concentration values were log10 transformed and expressed as the geometric mean ±SEM. Contractile potency to
Morphometry The mean area of airway smooth muscle per mm of basement membrane did not differ between the
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immunized and control rabbits, 0.0238±0.0018 mm (n=18) and 0.0243±0.0015 mm (n=13), respectively. Mean inner wall thickness per mm basement membrane for the immunized and control rabbits were, respectively, 0.075±0.005 mm (n=18) and 0.083±0.007 mm (n=13), which were not significantly different. In animals with a positive PCA, the mean airway smooth muscle area per mm of basement membrane was 0.022±0.0023 mm (n = 12) and the mean inner wall thickness was 0.073±0.007 mm (n= 12), which were not significantly different from the values obtained for the control group. Correlation of in vivo and ex vivo airways responsiveness Since no differences were observed between the immunized rabbits and the rabbits exhibiting a PCA titre >0 in any of the parameters observed, the correlations presented are measurements made using data obtained from the control and the immunised rabbits with a PCA titre >0, as this group of rabbits represent successfully immunised rabbits. There was no correlation between the in vivo airways responsiveness and ex vivo bronchial responsiveness to methacholine in either the saline treated control group (Cdyn PC35 vs. pD2, P>0.05; RL PC50 vs. pD2, P>0.05; Fig. 3) or in immunized rabbits with a PCA>0 (Cdyn PC35 vs. pD2, P>0.05; RL PC50 vs. pD2, P>0.05; Fig. 3). Correlation of ex vivo airway responsiveness with airway morphometry No significant correlation was observed between ex vivo bronchial responsiveness to methacholine and airway smooth muscle per mm basement membrane in the control group (Fig. 4). However, in animals with a PCA titre >0, there was a significant correlation (=−0.5986, P<0.05) between SM/mmBM and pD2. Correlation of in vivo airway responsiveness with airway morphometry There were no correlations between in vivo airway responsiveness to methacholine and morphometry in either the control rabbits (Cdyn PC35 vs. SM/mmBM, P>0.05; RL PC50 vs. SM/mmBM, P>0.05; Fig. 5) or rabbits with a PCA titre>0 (Cdyn PC35 vs. SM/mmBM, P>0.05; RL PC50 vs. SM/mmBM, P>0.05; Fig. 5).
DISCUSSION The distribution of airway responsiveness in a random population to direct acting bronchoconstrictors such as histamine or methacholine is continuous.18, In a
study of the bronchial reactivity to histamine in nonasthmatics and four subgroups of asthmatic patients, a range of responsiveness with considerable overlap between groups was observed.4, Mathematical modelling of asthmatic airways suggests that the thickening of the airway wall observed in histological studies of asthmatic tissue 6,7,10,11,19, can account for the exaggerated airway-narrowing characteristic of asthma14 and that airway smooth muscle mass alone can account for this excessive airway narrowing.15 However, whilst mathematical modelling predicts a relationship between airway wall thickening/ smooth muscle mass and responsiveness in asthmatic airways, few studies have been undertaken in vivo to confirm this hypothesis. Rabbits immunized neonatally produce IgE antibodies preferentially16 and the success of the sensitization protocol can be measured through the use of the PCA reaction. We have previously reported that the distribution of airway responsiveness in a random population to direct acting bronchoconstrictors is continuous (approximately 100-fold range) in both normal and sensitised rabbits with considerable overlap between the groups16 and this is confirmed in the present study. The mathematical model studies of airway physiology imply that variations in airway smooth muscle area may explain this range of responsiveness. In order to investigate the application of this theory to in vivo biology, immunized and non-immunized rabbits were studied. In vivo airway responsiveness and ex vivo bronchial responsiveness to methacholine was measured and then airway smooth muscle area of each bronchus was determined in an attempt to relate the responsiveness to smooth muscle area. The PCA reaction was used to assess the percentage of rabbits successfully immunized and to be able to study the successfully immunized rabbits as a separate population. In the present study, 66% percent of the rabbits that underwent the immunization procedure exhibited a PCA titre >0, yet in terms of in vivo and ex vivo parameters, no differences in responsiveness were observed between the group of all immunised rabbits, non-immunized rabbits and those exhibiting a PCA titre >0. The present data would not support the hypothesis that a relationship exists between airway smooth muscle thickness and airway responsiveness. No differences were observed in airway smooth muscle area between control rabbits, immunized rabbits and rabbits with positive PCA titres in the present study, and hence it is unlikely that changes in airway smooth muscle thickness per se account for the wide variation in airway responsiveness observed in the different populations of rabbits studied. Observation of relationships between in vivo airway
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responsiveness and smooth muscle area may be dependent upon the size of the airway examined morphometrically. A study using sensitized Brown Norway rats suggested that the relationship between airway smooth muscle area and in vivo responsiveness is dependent on the size of the airway studied.20 Although this study showed no correlation between airway smooth muscle area and in vivo responsiveness in this species overall, when large airways (2–2.99 mm id) were studied in isolation, a statistically significant relationship was found between EC200 R (concentration of methacholine required to double pulmonary resistance) and smooth muscle area. Since in that study all animals were sensitized, it is unknown whether such a relationship exists in non-sensitised rats. However, in a study of normal guinea pigs a relationship was observed between the response to methacholine administered in vivo and the quantity of airway smooth muscle in larger, cartilaginous airways only.21 Others have shown that in rats subjected to prolonged hyperoxia, the subsequent airway hyperresponsiveness to acetylcholine was associated with airway wall thickening.22,23 It should be noted, however, that a later study showed that such hyperresponsiveness can occur at a time point prior to the development of the thickened airway wall,24 again suggesting that factors other than airway wall thickness per se may contribute to airways hyperresponsiveness. Furthermore, in a study of three strains of mice, it was determined that velocity of airway smooth muscle shortening and not airway morphometry was related to the inter-strain differences in airway responsiveness in this species.25 Interestingly, it has recently been suggested that differences in mechanical responses of asthmatic airways cannot be explained solely by the amount of smooth muscle in the airway wall.26 Using a novel method to study the amount of airway smooth muscle in asthmatic and non-asthmatic tissues, it was concluded that there was no increase in the quantity of airway smooth muscle in the asthmatic airways over that of the non-asthmatics.26 These observations are in agreement with the present findings, as we have observed no difference between immunized and nonimmunised rabbits in terms of smooth muscle area. Furthermore, a recent study of airway hyperresponsiveness in various inbred and cross-bred strains of mice indicate that airway hyperresponsiveness (measured as the effective dose required to increase R to 200% of control values) was inherited and that the data are consistent with that of polygenic control of airway responsiveness in some of the cross strains of mice.27 Indeed, these researchers were able to identify three trait loci that influence airway responsiveness, one of which maps very closely to the acetylcholine receptor.27
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Sparrow and Mitchell have developed a method to study perfused isolated airway segments that allows airway narrowing in response to spasmogens to be studied via changes in our flow through the lumen.17, 28 This technique has been shown to account for differences in tissue responses due to size of segment, presence or absence of epithelium and wall-to-lumen ratio.29 Measuring responses of isolated airway preparations using isometric and isotonic techniques may not be physiologically relevant to airway narrowing in the intact lung because in vivo narrowing is neither isometric nor isotonic. Instead, as the airway narrows, both the load placed on the lung and the muscle length change. In the perfused bronchial segment the capacity of the airway smooth muscle strip to develop force, measured isometrically in response to spasmogens, was found to be only partly related to its capacity to narrow the isolated segment, dependent on the size of the airway used.28 Measuring the flow through a perfused segment of airway appears to mimic more closely airway narrowing in vivo, rather than isometric and/or isotonic in-vitro techniques. We have used the perfused airway segment preparation to determine whether airway responsiveness in vivo was related to airways responsiveness ex vivo. However, we have found no correlations between in vivo and ex vivo responsiveness in the control rabbits, and other investigators have reported similar findings. Studies of asthmatic and non-asthmatic subjects found a lack of correlation between in vivo responsiveness with ex vivo tissue responsiveness.30–32 Furthermore, airway responsiveness to methacholine in vivo in immunized and control guinea pigs did not correlate with in-vitro sensitivity.33 The lack of correlation reported here and in previous studies may be due to alterations in the structural integrity of the airways that occurs when they are used in vitro. Removal of the parenchyma during dissection results in the loss of the series elastic component of the load that is present on the airways in vivo. Removal of tissue would also disrupt some of the reflex and/or central inhibitory neuronal mechanisms suggested to be present in situ, for example, vagally mediated effects,34 which may contribute to the lack of correlation between in vivo and ex vivo responsiveness. Indeed, in rabbit lungs challenged in situ with methacholine, significantly greater airway narrowing was induced in the lungs of immature rabbits than in those of mature rabbits.35 The authors suggested that the greater tendency for airway closure in the immature rabbits may reflect maturational changes in the properties of the lung parenchyma or in the coupling between airways and lung parenchymal tissue, since parenchyma undergoes significant post-natal development whilst airways are already fully formed at birth.35 Interestingly, the relationship between ex vivo responsiveness of bronchi isolated from rabbits ex-
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hibiting a PCA titre >0 and airway smooth muscle area of the bronchi was significant and negative. In other words, tissues that were less sensitive to methacholine ex vivo contained more airway smooth muscle area than tissue that was more sensitive. This is difficult to rationalize since, according to the mathematical modelling theory, one might expect that smooth muscle area would positively correlate with increased sensitivity.15 However, as this relationship was observed only in animals exhibiting a PCA titre >0, it is most likely a result of some aspect of the sensitization process. In summary, we have demonstrated that airway responsiveness to methacholine was not correlated with airway smooth muscle thickness in immunized and non-immunized rabbits, suggesting that airway smooth muscle area per se is not the sole contributor to the wide range of airway responsiveness observed to inhaled spasmogens in vivo. REFERENCES 1. Woolcock A J, Salome C M, Yan K. The shape of the doseresponse curve to histamine in asthmatic and normal subjects. Am Rev Respir Dis 1984; 130: 71–75. 2. Moreno R H, Hogg J C, Pare P D. Mechanics of airway narrowing. Am Rev Respir Dis 1986; 133: 1171–1180. 3. Bel E H, Timmers M C, Zwinderman A H, Dijkman J H, Sterk P J. The effect of inhaled corticosteroids on the maximal degree of airway narrowing to methacholine in asthmatic subjects. Am Rev Respir Dis 1991; 143: 109–113. 4. Cockroft D W, Killian D N, Mellon J J A, Hargreave F E. Bronchial reactivity to inhaled histamine: a method and clinical survey. Clin Allergy 1977; 7: 235–243. 5. Holgate S T, Pattermore P K. Asthma in Childhood. In: Page C P, Gardiner P J, eds. Airway Hyperresponsiveness: is it really important for asthma? Oxford: Blackwell Scientific Publications 1993; 55–78. 6. Jeffery P K, Godfrey R W, Adelroth E, Nelson F, Rogers A, Johansson S A. Effects of treatment on airway inflammation and thickening of basement membrane reticular collagen in asthma. A quantitative light and electron microscopic study. Am Rev Respir Dis 1992; 145: 890–899. 7. Jeffery P K, Wardlaw A J, Nelson F C, Collins J V, Kay A B. Bronchial biopsies in asthma. An ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis 1989; 140: 1745–1753. 8. Lundgren R, Soderberg M, Horstedt P, Stenling R. Morphological studies of bronchial mucosal biopsies from asthmatics before and after ten years of treatment with inhaled steroids. Eur Respir J 1988; 1: 883–889. 9. Dunnill M S. The pathology of asthma, with special reference to changes in the bronchial mucosa. J Clin Pathol 1960; 13: 27–33. 10. James A L, Pare P D, Hogg J C. The mechanics of airway narrowing in asthma. Am Rev Respir Dis 1989; 139: 242–246. 11. Ebina M, Yaegashi H, Chiba R, Takahashi T, Motomiya M, Tanemura M. Hyperreactive site in the airway tree of asthmatic patients revealed by thickening of bronchial muscles. A morphometric study. Am Rev Respir Dis 1990; 141: 1327–1332. 12. Ebina M, Takahashi T, Chiba T, Motomiya M. Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma. A 3-D morphometric study. Am Rev Respir Dis 1993; 148: 720–726.
13. Jeffery P K. Structural Changes in Asthma. In: Page C. P., Black J., eds. Airways and Vascular Remodelling in Asthma and Cardiovascular Disease: Implications for Therapeutic Intervention. London: Academic Press 1994; 3–19. 14. Wiggs B R, Bosken C, Pare P D, James A, Hogg J C. A model of airway narrowing in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 1992; 145: 1251–1258. 15. Lambert R K, Wiggs B R, Kuwano K, Hogg J C, Pare P D. Functional significance of increased airway smooth muscle in asthma and COPD. J Appl Physiol 1993; 74: 2771–2781. 16. Minshall E M, Riccio M M, Herd C M et al. A novel animal model for investigating persistent airway hyperresponsiveness. J Pharmacol Toxicol Methods 1993; 30: 177–188. 17. Sparrow M P, Mitchell H W. Modulation by the epithelium of the extent of bronchial narrowing produced by substances perfused through the lumen. Br J Pharmacol 1991; 103: 1160–1164. 18. Cockroft D W. Airway Responsiveness. In: Barnes P J, Grunstein M M, Leff A R, Woolcock A J, eds. Asthma. Philadelphia: Lippincott-Raven 1997; 1253–1266. 19. Carroll N, Elliot J, Morton A, James A. The structure of large and small airways in nonfatal and fatal asthma. Am Rev Respir Dis 1993; 147: 405–410. 20. Sapienza S, Du T, Eidelman D H, Wang N S, Martin J G. Structural changes in the airways of sensitized Brown Norway rats after antigen challenge. Am Rev Respir Dis 1991; 144: 423–427. 21. Martin J G, Opazo-Saez A, Du T, Tepper R, Eidelman D H. In vivo airway reactivity: predictive value of morphological estimates of airway smooth muscle. Can J Physiol Pharmacol 1991; 70: 597–601. 22. Hershenson M B, Garland A, Kelleher M D, Zimmermann A, Hernandez C, Solway J. Hyperoxia-induced airway remodeling in immature rats. Correlation with airway responsiveness [published erratum appears in Am Rev Respir Dis 1993; 148]. Am Rev Respir Dis 1992; 146: 1294–1300. 23. Hershenson M B, Aghili S, Punjabi N et al. Hyperoxiainduced airway hyperresponsiveness and remodeling in immature rats [published erratum appears in Am J Physiol 1993 Aug; 265]. Am J Physiol 1992; 262: L263–9. 24. Szarek J L, Ramsay H L, Andringa A, Miller M L. Time course of airway hyperresponsiveness and remodeling induced by hyperoxia in rats. Am J Physiol 1995; 269: L227–33. 25. Duguet A, Biyah K, Minshall E M et al. Bronchial responsiveness among inbred mouse strains. Am J Respir Crit Care Med 2000; 161: 839–848. 26. Thomson R J, Bramley A M, Schellenberg R R. Airway muscle stereology: implications for increased shortening in asthma. Am J Respir Crit Care Med 1996; 154: 749–757. 27. De Sanctis G T, Merchant M, Beier D R et al. Quantitative locus analysis of airway hyperresponsiveness in A/J and C57BL/6J mice. Nature Genetics 1995; 11: 150–154. 28. Mitchell H W, Sparrow M P The relevance of pharmacological dose-response curves to airway narrowing. Trends Pharmacol Sci 1989; 10: 488–491. 29. Mitchell H W, Sparrow M P. Increased responsiveness to cholinergic stimulation of small compared to large diameter cartilaginous bronchi. Eur Respir J 1994; 7: 298–305. 30. Cerrina J, Le Roy Ladurie M, Labat C, Raffestin B, Bayol A, Brink C. Comparison of human bronchial muscle responses to histamine in vivo with histamine and isoproterenol agonists in vitro. Am Rev Respir Dis 1986; 134: 57–61. 31. Taylor S M, Pare P D, Armour C L, Hogg J C, Schellenberg R R. Airway reactivity in chronic obstructive pulmonary disease. Failure of in vivo methacholine responsiveness to correlate with cholinergic, adrenergic, or nonadrenergic responses in vitro. Am Rev Respir Dis 1985; 132: 30–35. 32. Armour C L, Lazar N M, Schellenberg R R et al. A comparison of in vivo and in vitro human airway reactivity to histamine. Am Rev Respir Dis 1984; 129: 907–910. 33. Featherstone R L, Hutson P A, Holgate S T, Church M K.
Airways in Vivo, in Vitro and Morphometry Active sensitization of guinea-pig airways in vivo enhances in vivo and in vitro responsiveness. Eur Respir J 1988; 1: 839–845. 34. Costello R W, Evans C M, Yost B L et al. Antigen-induced hyperreactivity to histamine: role of the vagus nerves and eosinophils. Am J Physiol (Lung Cell Mol Physiol) 1999; 276: L709–L714. 35. Tepper R S, Gunst S J, Doerschuk C M et al. Effect of
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transpulmonary pressure on airway closure in immature and mature rabbits. J Appl Physiol 1995; 78: 505–512.
Date received: 18 April 2000 Date accepted: 6 November 2000 Published electronically: 10 January 2001.
PULMONARY PHARMACOLOGY & THERAPEUTICS
Relationship of Airway Responsiveness with Airway Morphometry in Normal and Immunized Rabbits F. E. Woisin∗, C. M. Herd∗1, G. J. Douglas∗2, K. Raynor∗, D. Spina∗, H. W. Mitchell†, C. P. Page∗ ∗Sackler Institute of Pulmonary Pharmacology, Division of Pharmacology and Therapeutics and Department of Respiratory Medicine and Allergy, Kings College, London, UK, †Department of Physiology, University of Western Australia, Nedlands, WA, Australia
SUMMARY: Airway responses to chemical stimuli occur over a wide range of concentrations, with overlap between severe, moderate and mild asthmatic groups and with normal healthy individuals. Mathematical modelling has suggested that relative thickness of the airway wall may account for this range of responsiveness. We have investigated whether in vivo airway responsiveness varies as a function of airway wall thickness in terms of airway smooth muscle area in normal and immunized New Zealand White (NZW) rabbits. Airway responsiveness to inhaled methacholine (MCh) was determined in vivo under neuroleptanalgesia. Subsequently, ex vivo responsiveness to MCh (pD2=−log EC50) of isolated bronchi from the same animal was established. Smooth muscle area per mm basement membrane (SM/mmBM) was also measured morphometrically in the tested bronchi and the findings related to in vivo and ex vivo responsiveness. We found no relationship between airway responsiveness in vivo and pD2 values in either immunized or control rabbits. In both control and immunized rabbits, no correlation was found between SM/mmBM and in vivo airway responsiveness. Only in immunized animals with a PCA titre >0, was there a significant correlation (=−0.5986, P<0.05) between SM/mmBM and pD2. We conclude that airway smooth muscle area per se is not the sole contributor of airway responsiveness in vivo in normal rabbits. 2001 Academic Press
KEY WORDS: Airway smooth muscle, Methacholine, Airway remodelling.
usually determined as the provocative concentration of histamine or methacholine inducing a 20% fall in FEV1 (PC20), and has been suggested to correlate with asthma severity.4 However, the PC20 values of individual asthmatics to such stimuli occur over a wide range of concentrations, with overlap between severe, moderate and mild asthmatic groups and even with normal healthy individuals.4,5 The mechanisms underlying the variability and the wide range of airway responsiveness in vivo are not yet determined, but may be due in part to individual differences in the characteristics of the airway wall secondary to structural changes. Asthma has become recognized as a chronic inflammatory disease, with the airways of even mild asthmatics demonstrating increases in inflammatory cell infiltrate.6 Primed and activated mast cells, eosinophils, macrophages and lymphocytes have been found in bronchoalveolar lavage fluid obtained from
INTRODUCTION Airway hyperresponsiveness in asthmatic individuals is characterized by two components of the response to non-specific stimuli such as histamine, methacholine, cold air or exercise: an increased sensitivity (or leftward shift of the concentration–response curve) and an increased maximal airways responsiveness (heightened or unobtainable plateau of the concentration–response curve).1–3 Clinical assessment of airway responsiveness is ∗Author for correspondence: C.P. Page, Sackler Institute of Pulmonary Pharmacology, Division of Pharmacology and Therapeutics, GKT School of Biomedical Sciences, King’s College, London, 5th Floor, Hodgkin Building, Guy’s Campus, London SE1 9RT, UK. Fax: + 44 (0) 20 7848 6097. E-mail: [email protected] 1Present address: AstraZeneca (UK) Ltd., Charnwood, Leicestershire, UK. 2Present address: Pneumolabs (UK) Ltd., NPIMR, Y Block, Watford Road, Harrow, Middlesex, HA1 3UJ, UK. 1094–5539/01/02075+09 $35.00/0
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Fig. 1 In vivo airway responses of individual rabbits to methacholine challenge, indicating that the parameters measured for each rabbit (R PC50; CdynPC35) occur over a 100-fold range. Data represent measurement of (A) RL PC50; mg/ml and (B) Cdyn PC35; mg/ml from individual immunized rabbits (Ο; n=18), those with PCA>0 (Χ; n = 12) or saline control rabbits (Β; n=13).
Flow rate (ml/min)
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Fig. 2 Concentration–response curve of perfused main bronchi of rabbits to methacholine. Curve represents the change in flow rate in response to methacholine applied to bronchi obtained from control (Β; n=13) and immunized PCA>0 (Χ; n=12) rabbits. Values represent mean±SEM.
asthmatics.7 The products of these cells can act as bronchoconstrictors, chemotactic factors and/or damage the basement membrane and epithelium in the airway. However, clinical observations have led investigators to suggest that persistent airways hyperresponsiveness may occur in the absence of overt airways inflammation, as in subjects undergoing longterm treatment with glucocorticosteroids.8 It is therefore possible that repeated inflammatory insults may lead to more permanent structural changes in the airways. Mediators released by inflammatory cells may perpetuate tissue damage and thus contribute to the airway wall remodelling observed in lung tissue obtained post-mortem9–12 and in lung tissue biopsies from asthmatics.13 In asthmatics, the increased airway reactivity and loss of the dose–response curve plateau to inhaled spasmogens are thought to be a consequence of airway wall thickening.10 Mathematical modelling suggests
that increased wall thickness may account for bronchial hyperresponsiveness in asthmatics.2,14 Mathematical modelling has also suggested that increases in muscle mass may account for the airway hyperresponsiveness observed in asthma.15 However, these data from mathematical modelling are not supported by direct biological evidence relating these morphometric characteristics to in vivo airway function. We have reported previously the range of airway responsiveness to inhaled spasmogens in normal and neonatally immunized rabbits is remarkably similar to the pattern observed in humans.16 Therefore, in the present experiments we have investigated whether in vivo airway responsiveness varies as a function of airway smooth muscle thickness by studying the relationships between in vivo airway responsiveness, ex vivo responsiveness of the isolated bronchi and airway wall morphometry in normal and immunized New Zealand White rabbits.
MATERIALS AND METHODS Study design Rabbits used in this study were randomized at birth to different treatment regimes. Each experimental procedure was performed blind in that no experimenter was aware of the immunological status of the rabbits until all tests (in vivo, in vitro and morphometric measurements) were completed. For each rabbit, in vivo airway responsiveness to methacholine was measured. Subsequently the right main bronchus of the rabbit was isolated and its responsiveness to methacholine in vitro was measured using a perfused bronchial segment preparation. Finally, the bronchus was studied morphometrically to determine the airway wall thickness and smooth muscle area per mm of
Airways in Vivo, in Vitro and Morphometry
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Fig. 3 Relationship between in-vivo airway responsiveness and ex-vivo sensitivity to methacholine of isolated bronchi from the rabbits studied. Figures show (A) MCh Cdyn PC35 and (B) MCh R PC50 vs. MCh pD2 (−log10 EC50) in control (Β, n=13) and immunized PCA>0 (Χ, n=12) rabbits. The correlation coefficients () and associated P values were calculated using Spearman rank order correlations. A. Control, =−0.088, P=n.s., PCA>0, =0.112, P=n.s. B. Control, =0.043, P=n.s., −PCA>0, =−0.112, P=n.s. 7
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Fig. 4 Relationship between the in-vitro responsiveness of perfused rabbit bronchi to methacholine (pD2) and airway smooth muscle thickness of bronchi (SM/mmBM) in control (Β, n=13) and immunized PCA>0 (Χ, n=12) rabbits. The correlation coefficient () and associated P value were calculated using Spearman rank order correlation. Control, =0.098, P= n.s., PCA>0, =−0.599, P<0.05.
basement membrane. These data were collated and examined to determine if any correlation existed between any of these parameters tested. Animals New Zealand White (NZW) rabbits of either sex (2.6–3.8 kg) were obtained from Froxfield Farms UK (Petersfield, Hampshire, UK). The methods described in this study were subject to Home Office approval and were performed under the Animals (Scientific Procedures) Act, 1986. Immunization protocol The immunization procedure has been described in detail previously16 and is briefly outlined here. Within
24 h of birth an equal number of pups from each litter were randomly assigned to either of two groups: control or immunized. Control rabbits were given 0.5 ml saline (sterile, pyrogen free 0.9% sodium chloride solution, Baxter Health Care, Norfolk, UK) intraperitoneally (ip) and their littermates were immunized by injection of 0.5 ml Alternaria tenuis extract (40,000 PNU/ml; Greer Laboratories, North Carolina, USA) mixed with the sterile adjuvant aluminium hydroxide (Al(OH)3) gel (Rehydragel, Dublin, Eire). This regime was repeated weekly for the first month and then biweekly for the following 2 months. At 3 months of age, the rabbits were transferred from the breeding unit to our laboratory. Sensitization assessment through determination of passive cutaneous anaphylaxis An index of the serum IgE levels from all rabbits used in this study was obtained by titration of sera sample dilutions in the passive cutaneous anaphylaxis (PCA) test. Serum was prepared from 10 ml of blood obtained by cardiac puncture when the animals were killed prior to the ex vivo experiments. Recipient naive male NZW rabbits (2.0–3.0 kg) were anaesthetized with Hypnorm (a mixture of fentanyl citrate, 0.315 mg/ ml and fluanisone, 10 mg/ml; Janssen Pharmaceutical Ltd., Oxfordshire, UK) (0.4 ml/kg injected im) and the dorsal skin hair was closely clipped. A pattern of six blocks of six sites for 36 intradermal (id) injections was marked on the dorsal skin. Serial two-fold dilutions (between 1:1–1:64) in PBS were made of six test sera and injected (0.1 ml) id into three recipient rabbits. After a 72-h fixation period the recipient rabbits were anaesthetized with sodium pentobarbitone (30 mg/kg, iv) via an indwelling cannula (23 gauge Butterfly) in the marginal ear vein. Evans blue
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Fig. 5 Relationship between in-vivo responsiveness to methacholine (A) MCh Cdyn PC35 and (B) MCh R PC50 and airway smooth muscle thickness of main bronchi (SM/mmBM) in control (Β, n=13) and immunised with PCA>0 (Χ, n=12) rabbits. The correlation coefficients () and associated P values were calculated using Spearman rank order correlations. A. Control, =0.455, P=n.s., PCA>0, =−0.549, P=n.s. B. Control, =0.461, P=n.s., PCA>0, =−0.430, P=n.s.
dye (10 mg/kg) was administered intravenously followed 10 min later by iv antigen (A. tenuis extract; 7500 PNU/kg). Thirty minutes after antigen challenge the rabbits were killed with an overdose of sodium pentobarbitone (60 mg/kg, iv) and the back skin removed. The titration end-point was taken as the highest dilution at which the test serum caused a positive response when the skin was viewed from the underside. Pulmonary function measurement Measurements of pulmonary function and airway responsiveness were made in spontaneously breathing rabbits under neuroleptanalgesia using an established method.16 Rabbits premedicated with diazepam (2.5 mg/kg, Roche Products Ltd., Hertfordshire, UK) underwent neuroleptanalgesia with 0.4 ml/kg Hypnorm (Janssen Pharmaceutical Ltd., Oxfordshire, UK) and were placed supine on a padded animal board. Neuroleptanalgesia was maintained throughout the course of the experiment by further administration of Hypnorm (0.2–0.3 ml) every 20 min. The rabbits were intubated with a cuffed endotracheal tube which was then attached to a heated Fleisch pneumotachograph connected to a Validyne differential pressure transducer (model MP 45-14-871; Validyne Engineering Corp., California, USA) from which measurements of flow and tidal volume were obtained. A latex oesophageal balloon was inserted into the lower third of the oesophagus and attached to a second Validyne differential pressure transducer (model MP 45-24-871) by way of a cannula for the measurement of transpulmonary pressure. From these measurements total lung resistance (R) and dynamic compliance (Cdyn) were calculated using an on-line
respiratory analyser [Pulmonary Monitoring System (PMS) Version 5.1; Mumed Ltd., London, UK]. Measurement of airways responsiveness in vivo Airway responsiveness to inhaled methacholine hydrochloride (Sigma Chemical Co., Dorset, UK) was measured. Aerosols of methacholine were generated using an ultrasonic nebuliser (de Vilbiss Ultraneb 99) and delivered directly into the lungs via the endotracheal tube. Initially, basal lung function parameters were measured before aerosolised saline was administered for 2 min. After saline inhalation lung function parameter measurements were made again as a baseline with which to compare responses to methacholine. Cumulative concentration–response curves to methacholine (1.25–80 mg/ml), administered for 2-min periods in doubling doses, were established. The provocation concentrations of methacholine that produced a 50% increase in total lung resistance (R PC50) and a 35% fall in dynamic compliance (Cdyn PC35) were determined for each rabbit. These values were used as indices of airway responsiveness in vivo. Measurement of airway smooth muscle responsiveness in vitro In vitro airway responsiveness was determined by measuring changes in flow through a perfused bronchial segment.17 Rabbits were killed by injection of an overdose of sodium pentobarbitone (60 mg/kg, iv; Sigma Chemical Co., Dorset, UK). The right main bronchus was isolated from the lung by careful dissection of the tissue, tying off side branches with surgical silk as the parenchyma was removed. Tissues used for the experiment were approximately
Airways in Vivo, in Vitro and Morphometry
1.8–2.0 cm in length with an internal diameter of 2 mm at the distal end. The tissue was mounted on cannulae that were approximately the same size or slightly larger than the internal diameter of the relaxed segment. The bronchus was then placed in a bath of Krebs– Heinseleit solution containing (in mM concentrations): NaCl (117), NaHCO3 (25.0), -glucose (11.1), KH2PO4 (1.03), MgSO4 ·7H2O (0.57), KCl (5.4) and CaCl2 (2.5). Krebs–Heinseleit solution was maintained at 37°C, gassed with 5% CO2 in O2 and was exchanged for fresh solution at least five times over an equilibration period of 30 min. The tissue was also perfused through the lumen with Krebs– Heinseleit solution, at a transmural pressure of 4 cmH2O and a driving pressure of 2 cmH2O. A transducer measured the flow rate through the segment and changes in flow rate were detected by a custombuilt flow head (Ugo Basile, Italy) set up in series with the tissue to which the transducer was connected.17 The flowhead and transducer were previously checked for linearity and were calibrated over the range of flows encountered in the experiment. Decreases in the flow rate of the perfusate reflected constriction of the rabbit bronchus. Concentration–response curves for methacholine (10−8–10−3) applied serosally to the tissues were obtained. After the flow rate was returned to baseline by repeated washing, tissues were then fixed in 10% formaldehyde for subsequent morphometric analysis to determine the extent of airway wall and airway smooth muscle thickening. Morphometry Fixed bronchial segments of 2.5 mm internal diameter were blocked in paraffin, sectioned every 7 m and then stained with Masson’s trichrome. Five sections taken from the distal third of the segment were studied morphometrically using VideoPro image analysis software, (Leading Edge Pty. Ltd., South Australia). From each section, four sectors of the airway wall were examined under a ×20 objective. This allowed morphometric examination of approximately 75% of the airway wall circumference. In each sector we measured the length of the basement membrane (Pbm), total inner wall area and area of the smooth muscle. Smooth muscle area and total inner wall area measurements were divided by the Pbm to derive a measurement of smooth muscle thickness (SM/mmBM) and airway wall thickness (IWA/mmBM). Mean values were calculated for each section and airway.
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methacholine for in vitro experiments is expressed as a pD2 value (−log10 EC50). Differences between groups were compared using unpaired Student’s t-test. Spearman rank order correlation coefficients () and associated P values were calculated for all correlations. All statistical analyses were determined using the Prism GraphPad computer program, and P values of <0.05 were considered significant. RESULTS PCA titres Passive cutaneous anaphylaxis titres provide an index of serum IgE levels and were expressed as the reciprocal of the highest dilution of serum that gave a positive response. Whilst none of the sera from the control group of rabbits exhibited a PCA titre, 12 of 18 (66%) sera obtained from immunized animals gave a positive PCA response. In vivo airway responsiveness The in vivo airway responsiveness measurements for the rabbits as measured by MCh log10 RL PC50 immunized (n=18) vs. control (n=13) were 0.587±0.096 and 0.502±0.148, respectively and MCh log10 Cdyn PC35 immunized vs. control were 0.544±0.096 and 0.387±0.124, respectively (Fig. 1). Hence, basal airway responsiveness did not differ significantly between immunized and control groups. In rabbits exhibiting a PCA titre >0 (n=12), the in vivo airway responsiveness measurements for MCh log10 Cdyn PC35 and MCh log10 RL PC50 were, respectively, 0.491±0.109 and 0.5392±0.089 and these values did not significantly differ from those of all immunized rabbits or those of the saline-treated controls (Fig. 1). In vitro bronchial responsiveness Methacholine induced a concentration-dependent reduction in flow when applied to the bronchial segments from all rabbits (Fig. 2). The contractile potency (pD2) to methacholine in bronchial segments obtained from control rabbits was not significantly different to that observed in bronchial preparations from immunized rabbits (control, 5.23±0.12, n=13 vs. immunized, 5.30±0.14, n=20, P>0.05). The contractile potency to methacholine in bronchial segments from rabbits with PCA titres >0 (5.22±0.17, n=12) was not significantly different from control (P>0.05).
Statistical analysis In vivo methacholine provocation concentration values were log10 transformed and expressed as the geometric mean ±SEM. Contractile potency to
Morphometry The mean area of airway smooth muscle per mm of basement membrane did not differ between the
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immunized and control rabbits, 0.0238±0.0018 mm (n=18) and 0.0243±0.0015 mm (n=13), respectively. Mean inner wall thickness per mm basement membrane for the immunized and control rabbits were, respectively, 0.075±0.005 mm (n=18) and 0.083±0.007 mm (n=13), which were not significantly different. In animals with a positive PCA, the mean airway smooth muscle area per mm of basement membrane was 0.022±0.0023 mm (n = 12) and the mean inner wall thickness was 0.073±0.007 mm (n= 12), which were not significantly different from the values obtained for the control group. Correlation of in vivo and ex vivo airways responsiveness Since no differences were observed between the immunized rabbits and the rabbits exhibiting a PCA titre >0 in any of the parameters observed, the correlations presented are measurements made using data obtained from the control and the immunised rabbits with a PCA titre >0, as this group of rabbits represent successfully immunised rabbits. There was no correlation between the in vivo airways responsiveness and ex vivo bronchial responsiveness to methacholine in either the saline treated control group (Cdyn PC35 vs. pD2, P>0.05; RL PC50 vs. pD2, P>0.05; Fig. 3) or in immunized rabbits with a PCA>0 (Cdyn PC35 vs. pD2, P>0.05; RL PC50 vs. pD2, P>0.05; Fig. 3). Correlation of ex vivo airway responsiveness with airway morphometry No significant correlation was observed between ex vivo bronchial responsiveness to methacholine and airway smooth muscle per mm basement membrane in the control group (Fig. 4). However, in animals with a PCA titre >0, there was a significant correlation (=−0.5986, P<0.05) between SM/mmBM and pD2. Correlation of in vivo airway responsiveness with airway morphometry There were no correlations between in vivo airway responsiveness to methacholine and morphometry in either the control rabbits (Cdyn PC35 vs. SM/mmBM, P>0.05; RL PC50 vs. SM/mmBM, P>0.05; Fig. 5) or rabbits with a PCA titre>0 (Cdyn PC35 vs. SM/mmBM, P>0.05; RL PC50 vs. SM/mmBM, P>0.05; Fig. 5).
DISCUSSION The distribution of airway responsiveness in a random population to direct acting bronchoconstrictors such as histamine or methacholine is continuous.18, In a
study of the bronchial reactivity to histamine in nonasthmatics and four subgroups of asthmatic patients, a range of responsiveness with considerable overlap between groups was observed.4, Mathematical modelling of asthmatic airways suggests that the thickening of the airway wall observed in histological studies of asthmatic tissue 6,7,10,11,19, can account for the exaggerated airway-narrowing characteristic of asthma14 and that airway smooth muscle mass alone can account for this excessive airway narrowing.15 However, whilst mathematical modelling predicts a relationship between airway wall thickening/ smooth muscle mass and responsiveness in asthmatic airways, few studies have been undertaken in vivo to confirm this hypothesis. Rabbits immunized neonatally produce IgE antibodies preferentially16 and the success of the sensitization protocol can be measured through the use of the PCA reaction. We have previously reported that the distribution of airway responsiveness in a random population to direct acting bronchoconstrictors is continuous (approximately 100-fold range) in both normal and sensitised rabbits with considerable overlap between the groups16 and this is confirmed in the present study. The mathematical model studies of airway physiology imply that variations in airway smooth muscle area may explain this range of responsiveness. In order to investigate the application of this theory to in vivo biology, immunized and non-immunized rabbits were studied. In vivo airway responsiveness and ex vivo bronchial responsiveness to methacholine was measured and then airway smooth muscle area of each bronchus was determined in an attempt to relate the responsiveness to smooth muscle area. The PCA reaction was used to assess the percentage of rabbits successfully immunized and to be able to study the successfully immunized rabbits as a separate population. In the present study, 66% percent of the rabbits that underwent the immunization procedure exhibited a PCA titre >0, yet in terms of in vivo and ex vivo parameters, no differences in responsiveness were observed between the group of all immunised rabbits, non-immunized rabbits and those exhibiting a PCA titre >0. The present data would not support the hypothesis that a relationship exists between airway smooth muscle thickness and airway responsiveness. No differences were observed in airway smooth muscle area between control rabbits, immunized rabbits and rabbits with positive PCA titres in the present study, and hence it is unlikely that changes in airway smooth muscle thickness per se account for the wide variation in airway responsiveness observed in the different populations of rabbits studied. Observation of relationships between in vivo airway
Airways in Vivo, in Vitro and Morphometry
responsiveness and smooth muscle area may be dependent upon the size of the airway examined morphometrically. A study using sensitized Brown Norway rats suggested that the relationship between airway smooth muscle area and in vivo responsiveness is dependent on the size of the airway studied.20 Although this study showed no correlation between airway smooth muscle area and in vivo responsiveness in this species overall, when large airways (2–2.99 mm id) were studied in isolation, a statistically significant relationship was found between EC200 R (concentration of methacholine required to double pulmonary resistance) and smooth muscle area. Since in that study all animals were sensitized, it is unknown whether such a relationship exists in non-sensitised rats. However, in a study of normal guinea pigs a relationship was observed between the response to methacholine administered in vivo and the quantity of airway smooth muscle in larger, cartilaginous airways only.21 Others have shown that in rats subjected to prolonged hyperoxia, the subsequent airway hyperresponsiveness to acetylcholine was associated with airway wall thickening.22,23 It should be noted, however, that a later study showed that such hyperresponsiveness can occur at a time point prior to the development of the thickened airway wall,24 again suggesting that factors other than airway wall thickness per se may contribute to airways hyperresponsiveness. Furthermore, in a study of three strains of mice, it was determined that velocity of airway smooth muscle shortening and not airway morphometry was related to the inter-strain differences in airway responsiveness in this species.25 Interestingly, it has recently been suggested that differences in mechanical responses of asthmatic airways cannot be explained solely by the amount of smooth muscle in the airway wall.26 Using a novel method to study the amount of airway smooth muscle in asthmatic and non-asthmatic tissues, it was concluded that there was no increase in the quantity of airway smooth muscle in the asthmatic airways over that of the non-asthmatics.26 These observations are in agreement with the present findings, as we have observed no difference between immunized and nonimmunised rabbits in terms of smooth muscle area. Furthermore, a recent study of airway hyperresponsiveness in various inbred and cross-bred strains of mice indicate that airway hyperresponsiveness (measured as the effective dose required to increase R to 200% of control values) was inherited and that the data are consistent with that of polygenic control of airway responsiveness in some of the cross strains of mice.27 Indeed, these researchers were able to identify three trait loci that influence airway responsiveness, one of which maps very closely to the acetylcholine receptor.27
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Sparrow and Mitchell have developed a method to study perfused isolated airway segments that allows airway narrowing in response to spasmogens to be studied via changes in our flow through the lumen.17, 28 This technique has been shown to account for differences in tissue responses due to size of segment, presence or absence of epithelium and wall-to-lumen ratio.29 Measuring responses of isolated airway preparations using isometric and isotonic techniques may not be physiologically relevant to airway narrowing in the intact lung because in vivo narrowing is neither isometric nor isotonic. Instead, as the airway narrows, both the load placed on the lung and the muscle length change. In the perfused bronchial segment the capacity of the airway smooth muscle strip to develop force, measured isometrically in response to spasmogens, was found to be only partly related to its capacity to narrow the isolated segment, dependent on the size of the airway used.28 Measuring the flow through a perfused segment of airway appears to mimic more closely airway narrowing in vivo, rather than isometric and/or isotonic in-vitro techniques. We have used the perfused airway segment preparation to determine whether airway responsiveness in vivo was related to airways responsiveness ex vivo. However, we have found no correlations between in vivo and ex vivo responsiveness in the control rabbits, and other investigators have reported similar findings. Studies of asthmatic and non-asthmatic subjects found a lack of correlation between in vivo responsiveness with ex vivo tissue responsiveness.30–32 Furthermore, airway responsiveness to methacholine in vivo in immunized and control guinea pigs did not correlate with in-vitro sensitivity.33 The lack of correlation reported here and in previous studies may be due to alterations in the structural integrity of the airways that occurs when they are used in vitro. Removal of the parenchyma during dissection results in the loss of the series elastic component of the load that is present on the airways in vivo. Removal of tissue would also disrupt some of the reflex and/or central inhibitory neuronal mechanisms suggested to be present in situ, for example, vagally mediated effects,34 which may contribute to the lack of correlation between in vivo and ex vivo responsiveness. Indeed, in rabbit lungs challenged in situ with methacholine, significantly greater airway narrowing was induced in the lungs of immature rabbits than in those of mature rabbits.35 The authors suggested that the greater tendency for airway closure in the immature rabbits may reflect maturational changes in the properties of the lung parenchyma or in the coupling between airways and lung parenchymal tissue, since parenchyma undergoes significant post-natal development whilst airways are already fully formed at birth.35 Interestingly, the relationship between ex vivo responsiveness of bronchi isolated from rabbits ex-
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hibiting a PCA titre >0 and airway smooth muscle area of the bronchi was significant and negative. In other words, tissues that were less sensitive to methacholine ex vivo contained more airway smooth muscle area than tissue that was more sensitive. This is difficult to rationalize since, according to the mathematical modelling theory, one might expect that smooth muscle area would positively correlate with increased sensitivity.15 However, as this relationship was observed only in animals exhibiting a PCA titre >0, it is most likely a result of some aspect of the sensitization process. In summary, we have demonstrated that airway responsiveness to methacholine was not correlated with airway smooth muscle thickness in immunized and non-immunized rabbits, suggesting that airway smooth muscle area per se is not the sole contributor to the wide range of airway responsiveness observed to inhaled spasmogens in vivo. REFERENCES 1. Woolcock A J, Salome C M, Yan K. The shape of the doseresponse curve to histamine in asthmatic and normal subjects. Am Rev Respir Dis 1984; 130: 71–75. 2. Moreno R H, Hogg J C, Pare P D. Mechanics of airway narrowing. Am Rev Respir Dis 1986; 133: 1171–1180. 3. Bel E H, Timmers M C, Zwinderman A H, Dijkman J H, Sterk P J. The effect of inhaled corticosteroids on the maximal degree of airway narrowing to methacholine in asthmatic subjects. Am Rev Respir Dis 1991; 143: 109–113. 4. Cockroft D W, Killian D N, Mellon J J A, Hargreave F E. Bronchial reactivity to inhaled histamine: a method and clinical survey. Clin Allergy 1977; 7: 235–243. 5. Holgate S T, Pattermore P K. Asthma in Childhood. In: Page C P, Gardiner P J, eds. Airway Hyperresponsiveness: is it really important for asthma? Oxford: Blackwell Scientific Publications 1993; 55–78. 6. Jeffery P K, Godfrey R W, Adelroth E, Nelson F, Rogers A, Johansson S A. Effects of treatment on airway inflammation and thickening of basement membrane reticular collagen in asthma. A quantitative light and electron microscopic study. Am Rev Respir Dis 1992; 145: 890–899. 7. Jeffery P K, Wardlaw A J, Nelson F C, Collins J V, Kay A B. Bronchial biopsies in asthma. An ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis 1989; 140: 1745–1753. 8. Lundgren R, Soderberg M, Horstedt P, Stenling R. Morphological studies of bronchial mucosal biopsies from asthmatics before and after ten years of treatment with inhaled steroids. Eur Respir J 1988; 1: 883–889. 9. Dunnill M S. The pathology of asthma, with special reference to changes in the bronchial mucosa. J Clin Pathol 1960; 13: 27–33. 10. James A L, Pare P D, Hogg J C. The mechanics of airway narrowing in asthma. Am Rev Respir Dis 1989; 139: 242–246. 11. Ebina M, Yaegashi H, Chiba R, Takahashi T, Motomiya M, Tanemura M. Hyperreactive site in the airway tree of asthmatic patients revealed by thickening of bronchial muscles. A morphometric study. Am Rev Respir Dis 1990; 141: 1327–1332. 12. Ebina M, Takahashi T, Chiba T, Motomiya M. Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma. A 3-D morphometric study. Am Rev Respir Dis 1993; 148: 720–726.
13. Jeffery P K. Structural Changes in Asthma. In: Page C. P., Black J., eds. Airways and Vascular Remodelling in Asthma and Cardiovascular Disease: Implications for Therapeutic Intervention. London: Academic Press 1994; 3–19. 14. Wiggs B R, Bosken C, Pare P D, James A, Hogg J C. A model of airway narrowing in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 1992; 145: 1251–1258. 15. Lambert R K, Wiggs B R, Kuwano K, Hogg J C, Pare P D. Functional significance of increased airway smooth muscle in asthma and COPD. J Appl Physiol 1993; 74: 2771–2781. 16. Minshall E M, Riccio M M, Herd C M et al. A novel animal model for investigating persistent airway hyperresponsiveness. J Pharmacol Toxicol Methods 1993; 30: 177–188. 17. Sparrow M P, Mitchell H W. Modulation by the epithelium of the extent of bronchial narrowing produced by substances perfused through the lumen. Br J Pharmacol 1991; 103: 1160–1164. 18. Cockroft D W. Airway Responsiveness. In: Barnes P J, Grunstein M M, Leff A R, Woolcock A J, eds. Asthma. Philadelphia: Lippincott-Raven 1997; 1253–1266. 19. Carroll N, Elliot J, Morton A, James A. The structure of large and small airways in nonfatal and fatal asthma. Am Rev Respir Dis 1993; 147: 405–410. 20. Sapienza S, Du T, Eidelman D H, Wang N S, Martin J G. Structural changes in the airways of sensitized Brown Norway rats after antigen challenge. Am Rev Respir Dis 1991; 144: 423–427. 21. Martin J G, Opazo-Saez A, Du T, Tepper R, Eidelman D H. In vivo airway reactivity: predictive value of morphological estimates of airway smooth muscle. Can J Physiol Pharmacol 1991; 70: 597–601. 22. Hershenson M B, Garland A, Kelleher M D, Zimmermann A, Hernandez C, Solway J. Hyperoxia-induced airway remodeling in immature rats. Correlation with airway responsiveness [published erratum appears in Am Rev Respir Dis 1993; 148]. Am Rev Respir Dis 1992; 146: 1294–1300. 23. Hershenson M B, Aghili S, Punjabi N et al. Hyperoxiainduced airway hyperresponsiveness and remodeling in immature rats [published erratum appears in Am J Physiol 1993 Aug; 265]. Am J Physiol 1992; 262: L263–9. 24. Szarek J L, Ramsay H L, Andringa A, Miller M L. Time course of airway hyperresponsiveness and remodeling induced by hyperoxia in rats. Am J Physiol 1995; 269: L227–33. 25. Duguet A, Biyah K, Minshall E M et al. Bronchial responsiveness among inbred mouse strains. Am J Respir Crit Care Med 2000; 161: 839–848. 26. Thomson R J, Bramley A M, Schellenberg R R. Airway muscle stereology: implications for increased shortening in asthma. Am J Respir Crit Care Med 1996; 154: 749–757. 27. De Sanctis G T, Merchant M, Beier D R et al. Quantitative locus analysis of airway hyperresponsiveness in A/J and C57BL/6J mice. Nature Genetics 1995; 11: 150–154. 28. Mitchell H W, Sparrow M P The relevance of pharmacological dose-response curves to airway narrowing. Trends Pharmacol Sci 1989; 10: 488–491. 29. Mitchell H W, Sparrow M P. Increased responsiveness to cholinergic stimulation of small compared to large diameter cartilaginous bronchi. Eur Respir J 1994; 7: 298–305. 30. Cerrina J, Le Roy Ladurie M, Labat C, Raffestin B, Bayol A, Brink C. Comparison of human bronchial muscle responses to histamine in vivo with histamine and isoproterenol agonists in vitro. Am Rev Respir Dis 1986; 134: 57–61. 31. Taylor S M, Pare P D, Armour C L, Hogg J C, Schellenberg R R. Airway reactivity in chronic obstructive pulmonary disease. Failure of in vivo methacholine responsiveness to correlate with cholinergic, adrenergic, or nonadrenergic responses in vitro. Am Rev Respir Dis 1985; 132: 30–35. 32. Armour C L, Lazar N M, Schellenberg R R et al. A comparison of in vivo and in vitro human airway reactivity to histamine. Am Rev Respir Dis 1984; 129: 907–910. 33. Featherstone R L, Hutson P A, Holgate S T, Church M K.
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Date received: 18 April 2000 Date accepted: 6 November 2000 Published electronically: 10 January 2001.