Pulmonary Vascular Congestion Selectively ... - ATS Journals

Pulmonary Vascular Congestion Selectively Potentiates Airway Responsiveness in Piglets TORSTEN UHLIG, JOHANNES H. WILDHABER, NEIL CARROLL, DEBRA J. TURNER, PETER R. GRAY, NIGEL DORE, ALAN L. JAMES, and PETER D. SLY Division of Clinical Sciences, TVW Telethon Institute for Child Health Research; Department of Respiratory Medicine, Princess Margaret Hospital for Children; Department of Pulmonary Physiology, Sir Charles Gairdner Hospital; Departments of Physiology and Paediatrics, University of Western Australia, Perth, Australia

The influence of pulmonary vascular congestion on the response of the airways and lung tissue to low doses of inhaled methacholine (MCh) was studied by inflating a balloon catheter in the left atrium of the heart in six piglets, with an additional five piglets serving as control animals. Congestion alone resulted in small increased in baseline airway (Raw) (14.6 ⫾ 3.7%) and tissue (Rti) resistance (8.1 ⫾ 6.5%). Low-dose inhaled MCh (0.3 mg/ml) increased Raw and Rti in the control group by 10.8 ⫾ 10.3% and 42.2 ⫾ 29.5%, respectively. The increase in Raw with MCh in the presence of vascular engorgement was significantly greater (67.8 ⫾ 18.9%) but the increase in Rti (38.1 ⫾ 13.2%) was similar to that seen in the control group. Morphometric measurements were performed on transverse sections of large and small airways from nine additional piglets (three congested only, three MCh only, and three congestion plus MCh). The thickness of the inner airway wall was similar in all groups. Compared with MCh only piglets, the thickness of the outer airway wall (between the outer border of the smooth muscle and the surrounding lung parenchyma) was increased (p ⬍ 0.05) in engorged only and engorged plus MCh piglets. Compared with MCh only and engorgement only, the amount of airway smooth muscle shortening was greater (p ⬍ 0.05) in all airway size groups in piglets that underwent engorgement plus MCh challenge. The results of this study demonstrate that pulmonary vascular engorgement, induced by increased left atrial pressure, selectively enhances the airway, but not the parenchymal, response to inhaled MCh. These changes are associated with increased thickness of the outer airway wall in response to vascular congestion, suggesting that uncoupling of the mechanical interdependence between the airway smooth muscle and the lung parenchyma may have occurred. Mechanical uncoupling may reduce the load opposing smooth muscle shortening resulting in increased airway narrowing in response to low doses of inhaled methacholine.

latation of pulmonary vessels (1), and edema of the airway wall (5) have all been suggested as possibly contributing to airway hyperresponsiveness (AHR). Thickening of the airway wall inside the muscle layer may potentiate the airway narrowing following bronchoconstricting stimuli (6), i.e., producing an increased response for a given amount of muscle shortening, while causing only minimal changes in baseline resistance (7). Alternatively interstitial edema involving the outer airway wall may uncouple the airways from the lung parenchyma, thus decreasing the elastic load against which bronchial smooth muscle contracts (8) resulting in an increase in muscle shortening for a given constrictor stimulus. The site and mechanism of AHR during pulmonary congestion remain unclear. The responses of airway smooth muscle to cholinergic agonists undergo developmental changes during early postnatal life, as experiments in animals have shown (9); thus, responses in children may differ from those in adults. We have previously demonstrated that, in contrast to adult animals (10), in developing piglets the baseline airway and lung tissue mechanics as well as their responses to pulmonary vascular congestion are not influenced by vagotomy (11). In the present study we aimed to determine the influence of pulmonary vascular congestion on the response to inhaled methacholine (MCh) and sought to determine whether congestion primarily enhanced the response to MCh in airways or in pulmonary tissues. In addition, we measured airway dimensions in subgroups of animals to ascertain whether vascular congestion resulted in changes in airway dimensions that were associated with functional responses to MCh.

Recurrent attacks of wheezing, dyspnea, and cough characterized as “cardiac asthma” are a common feature in patients with cardiac diseases associated with lung congestion. In adults, there is increasing evidence that impaired left ventricular function is not only responsible for airway obstruction but also for increased responsiveness of the airways to inhaled cholinergic agents (1). In children with congestive heart diseases respiratory resistance and elastance are increased (2), and hyperresponsiveness to histamine has been demonstrated in the majority of these patients (3). Several factors may contribute to changes in airway responsiveness in left heart failure. Decreased airway caliber (4), di-

Animal Preparation

(Received in original form July 22, 1997 and in revised form September 21, 1999) Supported in part by research grants from the Deutsche Forschungsgemeinschaft (DFG), Germany and the NH&MRC, Australia. Correspondence and requests for reprints should be addressed to Peter D. Sly, M.D., TVW Telethon Institute for Child Health Research, P.O. Box 855, West Perth, Western Australia, 6872. E-mail: [email protected] Am J Respir Crit Care Med Vol 161. pp 1306–1313, 2000 Internet address: www.atsjournals.org

METHODS Twenty piglets (weight mean 7.3 ⫾ 0.4 kg, age 3.9 ⫾ 0.4 wk) were studied. Anesthesia was induced with halothane (ICI Pharmaceuticals, East Melbourne, VIC, Australia) and maintained with fentanyl (1 g/kg/h intravenously; Astra Pharmaceuticals, North Ryde, NSW, Australia) and pentobarbitone sodium (10 mg/kg intravenously; Boehringer Ingelheim, Castle Hill, NSW, Australia). The piglets underwent a tracheotomy, were intubated and mechanically ventilated (Model 708; Harvard Apparatus, South Natick, MA) with a tidal volume (VT) of 10 ml/kg body weight. Paralysis was obtained with pancuronium bromide (0.2 mg/kg intravenously; Astra Pharmaceuticals). After insertion of central venous and femoral arterial catheters the chest was widely opened by midline sternotomy. Blood pressure and heart rate were monitored continuously. Once set, VT and ventilator frequency were kept constant throughout the study. The study protocol was approved by the institutional animal ethics committee. Catheters were inserted into the pulmonary artery and the left atrium of the heart, the former to measure pulmonary artery blood pressure continuously and the latter to elevate the left atrial pressure when inflated. The atrial catheter consisted of a self-made latex balloon, capable of being inflated with up to 10 ml of fluid, connected to a modified 20-gauge venous cannula (Insyte, Becton Dickinson, Sandy, UT).

Uhlig, Wildhaber, Carroll, et al.: Congestion Enhances Airway Responsiveness Airway opening pressure (Pao) was measured with a piezoresistive pressure transducer (8510 B-2; Endevco, San Juan Capistrano, · CA). Flow ( V ) was measured with a pneumotachograph (Hans Rudolph Inc., Kansas City, MO) and numerically integrated to provide volume (V).

Alveolar Capsule Technique The alveolar capsule technique (12) was used to measure alveolar pressure (Palv) in the open-chest piglets. Three small plastic capsules were glued to the pleural surface of the right middle and left upper lobe with cyanoacrylate glue (41621; Loctite Corp., Caringbah, NSW, Australia). The visceral pleura was punctured four times to a depth of approximately 0.5 mm with a 19-gauge needle to bring the underlying alveoli into communication with the capsule chamber. A piezoresistive pressure transducer (8507C-2; Endevco, San Juan Capistrano, CA) was lodged into each capsule to measure Palv.

Measurement of Respiratory Mechanics Mechanical ventilation. Respiratory mechanics were calculated from · the Pao, V , and V signals using a multilinear regression implementation of the equation of motion of a single compartment model of the respiratory system, as follows: ˙ + Palv,ee Pao = EL ⋅ V + RL ⋅ V

(1)

where EL is the elastance of the lung, RL is the resistance of the lung, and Palv,ee is the alveolar pressure at end-expiration (13). The tissue contribution to energy dissipation (Rti) during regular mechanical ventilation was calculated using the equation ˙ + Palv,ee Palv = Eti ⋅ V + Rti ⋅ V

(2)

where Eti is tissue elastance. RL, EL, Rti, and Eti were calculated by multiple linear regression fitted to 30-s data epochs containing periods of regular mechanical ventilation. Data were acceptable if the multiple linear regression correlation coefficient was greater than 0.97. Airway resistance (Raw) was then calculated by subtracting tissue resistance (Rti) from RL, i.e., (3)

Raw = R L – Rti

Signal conditioning. All signals were amplified (PR signal conditioner 4423, Endevco), low pass filtered (902 LPF, 8 pole Bessel, Fc ⫽ 10 Hz; Frequency Devices Inc., Haverhill, MA), and recorded by a 12bit analogue-to-digital converter at a rate of 100 Hz. The data were stored on a computer for later analysis using a commercial acquisition and analysis package (Anadat 5.1; RHT-Infodat Inc., Montreal, PQ, Canada).

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data epochs were then recorded for calculation of baseline RL, Rti, and EL, as described previously. The influence of vascular engorgement on the response to low-dose inhaled MCh was studied in 11 piglets (5 control animals, 6 engorged). Inflation of the left atrial balloon. For engorgement, the left atrial balloon was inflated with isotonic saline until mean pulmonary arterial pressure increased by approximately 15 mm Hg. Systemic blood pressure was continuously monitored to avoid significant depression. After stabilization of pulmonary and systemic blood pressures three data epochs were collected. Measurements of airway responses to MCh. Initially, the piglets were challenged with a 0.9% saline aerosol for 2 min; this was followed by measurements at 2-min intervals until a stable baseline was achieved. Subsequently, the animals received aerosol challenges of increasing concentrations of MCh (0.1 mg/ml and 0.3 mg/ml in 0.9% phosphate-buffered saline [PBS]) for 2 min with measurements taken in 2-min intervals. Fresh MCh solution was used for each animal. Aerosols were generated with a jet nebulizer (LC PLUS; Pari-Werk GmbH, Starnberg, Germany) at an airflow of 10 L/min, and administered to the airways through a separate ventilatory system bypassing the pneumotachograph to prevent deposition of aerosol droplets. During disconnection and reconnection to the pneumotachograph a shutter was used to prevent the lungs from collapsing. The pulmonary response to MCh was defined by the mean values of RL and EL of three subsequent measurements after the challenge. These values were expressed as percentage of the baseline values after saline aerosolization for purposes of comparison between animals.

Airway Morphometry Morphometric measurements of airways were performed in nine additional piglets consisting of three piglets with vascular engorgement only, three after MCh challenge (0.1 mg/ml and 0.3 mg/ml in 0.9% PBS) only, and three after MCh challenge (0.1 mg/ml and 0.3 mg/ml in 0.9% PBS) in the presence of vascular engorgement. Immediately after the appropriate experimental manipulation, the trachea was clamped at FRC and hilar bronchi and major blood vessels of each lung were double clamped. Subsequently the heart and lungs were removed en bloc by cutting between the clamps. Immediately after excision, the lungs were fixed by immersion in 10% buffered formalin for 48 h. All tissue obtained was treated in an identical manner to avoid fixation and/or processing artefact.

Experimental Protocol Baseline measurements. After surgical preparation, a stable ventilation pattern was established with VT of 10 ml/kg. Three discrete 30-s TABLE 1 MEAN BASELINE VALUES*

Body weight, kg Ventilation parameters Tidal volume, ml Frequency, min⫺1 Inspiratory time, s Expiratory time, s Palv,ee, kPa Respiratory mechanics RL, kpa · L⫺1 · s Raw, kPa · L⫺1 · s Rti, kPa · L⫺1 · s El, kPa · L⫺1 Raw/RL, %

Controls

Vascular Engorgement

7.3 ⫾ 0.5

7.0 ⫾ 0.5

90.2 ⫾ 6.4 41.4 ⫾ 0.01 0.65 ⫾ 0.02 0.65 ⫾ 0.01 0.43 ⫾ 0.02

98.3 ⫾ 5.1 41.4 ⫾ 0.01 0.64 ⫾ 0.01 0.66 ⫾ 0.01 0.40 ⫾ 0.03

1.40 ⫾ 0.10 1.01 ⫾ 0.15 0.48 ⫾ 0.09 10.82 ⫾ 2.72 68.3 ⫾ 6.2

1.33 ⫾ 0.16 0.91 ⫾ 0.14 0.42 ⫾ 0.05 8.78 ⫾ 1.04 67.4 ⫾ 4.0

Definition of abbreviations: EL ⫽ lung elastance; Palv,ee ⫽ end-expiratory alveolar pressure; Raw ⫽ airway resistance; RL ⫽ total lung resistance; Rti ⫽ tissue resistance. * Values are expressed as mean ⫾ SEM. None of the differences between groups were statistically significant.

Figure 1. Mean (SEM) pulmonary pressure of animals undergoing vascular engorgement (n ⫽ 6) and control piglets (n ⫽ 5) during the experiment. bs ⫽ baseline; VE ⫽ vascular engorgement. Asterisks indicate significant differences between groups (*p ⬍ 0.005; **p ⬍ 0.001).

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Figure 2. Effect of vascular engorgement of RL, Raw, and Rti. Asterisks indicate significant differences compared with baseline (*p ⬍ 0.02; **p ⬍ 0.001).

Histologic preparation. After fixation, specimens of all identifiable lobar, segmental, and subsegmental airways that could be cut in transverse section and free from branching were obtained from each lung. The lungs were then cut in half and six parenchymal sections were taken at random from the midsagittal slice of the lung. Tissue blocks were processed through a series of graded alcohols and embedded in paraffin wax, sectioned (5 ␮m), and stained with hematoxylin–eosin for morphometric analysis. Airway dimensions. On all airways cut in transverse section (defined as an even thickness of epithelium, and even thickness from the basement membrane to the smooth muscle layer) and free from branching, the following measurements were made using a microscope, sidearm, and digitizer. The basement membrane area (Abm) and perimeter (Pbm), defined by the basement membrane; the outer muscle area (Amo) and perimeter (Pmo), defined by the outer border of the airway smooth muscle; and the outer airway area (Ao) and perimeter (Po), defined by the border of the lung parenchyma surrounding the airways (14) (Figure 4A). Calculations. Compartmental areas of the airway wall were calculated. The inner wall area (WAi) between the basement membrane and the outer border of smooth muscle ⫽ Amo ⫺ Abm. The outer wall area (WAo) between the outer border of the smooth muscle and the surrounding border of the lung parenchyma ⫽ Ao ⫺ Amo. To calculate the percent muscle shortening (PMS) present in each airway, measured airway smooth muscle length given by Pmo was subtracted from the calculated “relaxed” muscle length (Pmor). “Relaxed” airway dimensions were derived using Pbm and WAi because these dimensions are independent of smooth muscle tone and lung volume. The relaxed lumen area (Abmr) is a circle with a circumference equal to the measured Pbm; thus Abmr ⫽ (Pbm)2/4␲. By adding the measured area of wall between the basement membrane and outer muscle border (Amo ⫺ Abm) to Abmr the relaxed outer muscle area (Amor) was obtained, from which Pmor is derived. Therefore, PMS ⫽ (Pmor ⫺ Pmo)/Pmor ⫻ 100.

Statistical Analysis The data are presented as means ⫾ SEM, and statistical comparisons between groups for measurements of airway and tissue mechanics were performed using single-factor or two-factor analysis of variance (ANOVA) as appropriate. For morphometric analysis, the airways

Figure 3. Mean (SEM) values of resistance after aerosolization of saline, MCh 0.1 mg/ml, and MCh 0.3 mg/ml, respectively. Asterisks indicate significant differences (p ⬍ 0.05) in comparison to control piglets.

were divided into three airway size groups: Pbm ⬎ 3 mm, Pbm 1 to 3 mm, and Pbm ⬍ 1 mm. The mean data for measurements of airway dimensions made within each size group for each piglet were used because multiple measurements from the same lung are not independent measurements. Because airway dimensions were not normally distributed, comparisons of airway dimensions and percent smooth muscle shortening were performed using a Kruskal-Wallis one-way ANOVA on ranks. A p value ⬍ 0.05 was considered statistically significant.

RESULTS Baseline Airway and Tissue Mechanics

Initial total lung resistance (RL) was 1.40 ⫾ 0.10 kPa ⭈ L⫺1 ⭈ s with Raw accounting for 68.3 ⫾ 6.2% of RL. There were no significant differences in pulmonary baseline mechanics or ventilation parameters between the control and engorged animals (Table 1). Effects of Vascular Engorgement

Inflation of the left atrial balloon resulted in an increase of mean pulmonary artery pressure from 18.5 ⫾ 0.6 mm Hg to 34.3 ⫾ 1.8 mm Hg (p ⬍ 0.005). Mean pulmonary artery pressure remained significantly higher in the group of animals undergoing vascular engorgement than the control group throughout the experiment (Figure 1). Vascular engorgement resulted in an increase of RL by 10.4 ⫾ 1.7% (p ⬍ 0.0005) with increases of

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Uhlig, Wildhaber, Carroll, et al.: Congestion Enhances Airway Responsiveness TABLE 2 PERCENTAGE CHANGES FROM BASELINE VALUES IN PULMONARY MECHANICS AFTER AEROSOLIZATION OF MCh* Control

Vascular Engorgement

MCh 0.1 mg/ml

MCh 0.3 mg/ml

MCh 0.1 mg/ml

MCh 0.3 mg/ml

7.5 ⫾ 4.6 ⫺3.6 ⫾ 9.7 31.7 ⫾ 14.2 15.9 ⫾ 6.3

16.0 ⫾ 3.8 10.8 ⫾ 10.3 42.2 ⫾ 29.5 18.5 ⫾ 8.6

25.1 ⫾ 5.2† 35.0 ⫾ 12.4† 6.7 ⫾ 5.3 12.8 ⫾ 8.0

59.7 ⫾ 14.2† 67.8 ⫾ 18.9† 38.1 ⫾ 13.2 24.8 ⫾ 12.2

RL, % Raw, % Rti, % EL %

* Values are expressed as mean ⫾ SEM. Baseline was defined as the response to aerosolization of isotonic saline. † p ⬍ 0.05 when compared with control animals.

Raw by 14.6 ⫾ 3.7% (p ⬍ 0.05) and Rti by 8.1 ⫾ 6.5% (p ⬍ 0.005) compared with the baseline values (Figure 2). MCh Challenge

In the control animals, there were small but significant increases in RL and Rti after inhalation of MCh 0.1 mg/ml and MCh 0.3 mg/ml and in Raw after MCh 0.3 mg/ml compared with values after the inhalation of saline (p ⬍ 0.05). In the animals undergoing vascular engorgement, the increases in RL and Raw after inhalation of MCh 0.1 mg/ml and MCh 0.3 mg/ml were significantly greater than those seen in the control piglets (Figure 3). There were no differences between the animals undergoing vascular engorgement and the control group with respect to Rti (Figure 3). The corresponding percentage changes of resistance and elastance are given in Table 2. The relative contribution of Raw to RL at baseline values (68.3 ⫾ 6.2%) did not change significantly after vascular engorgement (versus 67.4 ⫾ 4.0%, p ⫽ 0.24) or, in the nonengorged animals after inhalation of 0.1 mg/ml MCh (67.1 ⫾ 4.1%, p ⫽ 0.35) or 0.3 mg/ml MCh (68.2 ⫾ 7.2%, p ⫽ 0.94). However, when engorgement and MCh were combined there was a significant increase in the contribution of Raw to RL compared with baseline (engorgement ⫹ MCh 0.1 mg/ml: 74.9 ⫾ 7.5%, p ⬍ 0.01; engorgement ⫹ MCh 0.3 mg/ml: 75.8 ⫾ 8.7%, p ⬍ 0.001). Airway Morphometry

The results of morphometric measurements for the three treatment groups for three airway size categories are shown in Ta-

ble 3. Mean airway sizes (Pbm) in each category were similar in the treatment groups, as were WAi. Compared with the MCh only group (Figures 4A and 4B, WAo was increased (p ⬍ 0.05) in all size groups in piglets undergoing engorgement only (Figures 5A and 5B), or with the addition of inhaled MCh (Figures 6A and 6B). In midsized airways (Pbm ⫽ 1 to 3 mm), administration of MCh in addition to engorgement was associated with greater WAo (p ⬍ 0.05) than engorgement alone. PMS was significantly greater (p ⬍ 0.05) in piglets with vascular engorgement and MCh than those with either alone.

DISCUSSION The results of the present study confirm earlier observations that congestion of the pulmonary vessels and/or airway edema caused by partial outflow obstruction at the left atrial level of the heart leads to increases in both airway and lung tissue resistance. In addition, in the immature piglets studied, pulmonary vascular engorgement selectively enhanced the responsiveness of the airways to low doses of MCh, whereas the lung tissue did not substantially contribute to the hyperresponsiveness observed. This increase in airway responsiveness cannot be accounted for by the small (14.6 ⫾ 3.7%) increase in baseline Raw seen after vascular engorgement. The increase in outer airway wall thickness observed in this study, secondary to pulmonary vascular congestion, may have reduced the load opposing airway smooth muscle shortening provided by the elastic recoil pressure of the lung parenchyma. AHR has been reported in adult patients with impaired left

TABLE 3 AIRWAY MORPHOMETRY* Parameter

Study Group

Airway Size (Pbm) ⬎ 3 mm

Airway Size (Pbm) 1–3 mm

Airway Size (Pbm) ⬍ 1 mm

Pbm, mm

MCh only Engorgement only MCh ⫹ engorgement

4.80 ⫾ 0.98 5.03 ⫾ 1.0 5.23 ⫾ 1.1

1.62 ⫾ 0.48 1.57 ⫾ 0.44 1.70 ⫾ 0.70

0.66 ⫾ 0.18 0.67 ⫾ 0.14 0.66 ⫾ 0.14

Wai, mm2


0.28 ⫾ 0.03 0.30 ⫾ 0.038 0.30 ⫾ 0.043

0.036 ⫾ 0.005 0.037 ⫾ 0.002 0.042 ⫾ 0.007

0.006 ⫾ 0.001 0.007 ⫾ 0.001 0.006 ⫾ 0.001

Wao, mm2


1.22 ⫾ 0.27 1.53 ⫾ 0.26† 1.56 ⫾ 0.29†

0.071 ⫾ 0.021 0.094 ⫾ 0.008† 0.148 ⫾ 0.04†‡

0.012 ⫾ 0.001 0.021 ⫾ 0.002† 0.019 ⫾ 0.001†

PMS, %


11 ⫾ 2 12 ⫾ 1 20 ⫾ 4§

13 ⫾ 3 12 ⫾ 2 29 ⫾ 4§

6⫾2 5⫾1 24 ⫾ 4§

Definition of abbreviations: MCh ⫽ methacholine; Pbm ⫽ basement membrane perimeter; PMS ⫽ percent of airway smooth muscle shortening; WAi ⫽ inner wall area; WAo ⫽ outer wall area. * Values are expressed as mean ⫾ SEM. † p ⬍ 0.05 when compared with MCh only. ‡ p ⬍ 0.05 MCh ⫹ engorgement versus engorgement only. § p ⬍ 0.05 MCh ⫹ engorgement versus MCh only and engorgement only.

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Figure 4. (A) Transverse section of a midsized cartilaginous airway (Pbm ⫽ 4.2 mm) from a lung after MCh challenge only. The dotted lines represent the boundaries defining the outer muscle border (Pmo) and the parenchymal border (Po) measured in this study. There is little folding of the mucosal surface and little proteinaceous fluid in the airspaces. Original magnification: ⫻25. (B) Transverse section of a small membranous airway (Pbm ⫽ 0.9 mm) from a lung after MCh challenge only. The dotted lines represent the boundaries defining the Pmo and the Po measured in this study. There is little folding of the mucosal surface and no proteinaceous fluid in the airspaces. Original magnification: ⫻100.

ventricular function, due to mitral valve disease (15), coronary artery disease, or dilated cardiomyopathy (1), in the presence of moderate pulmonary congestion (16). The clinical symptoms of many of those patients resemble asthma. There seems to be no direct correlation between hyperresponsiveness and the degree of airway obstruction reported in such patients, but a relationship between airway responsiveness and intrapulmonary vascular pressures has been reported (15). In children with congenital heart disease, abnormalities in pulmonary function are well documented (2, 17) and a substantial proportion of these patients have a history of recurrent episodes of cough and wheezing. A recent study demonstrated increased airway reactivity to histamine in the majority of children with congestive heart diseases (3). Before discussing the implications of the results of the present study, a number of technical factors need to be considered. In the present study we deliberately chose low doses of

MCh to avoid deterioration of the already compromised vascular system and to minimize heterogeneities in peripheral airway narrowing which may occur with higher doses of cholinergic agonists (18). Under control conditions these doses caused only minimal increases in baseline resistance (Table 2), which were largely the result of an increase in the tissue component (42.2% increase in Rti after 0.3 mg/ml MCh). These data are consistent with reports in other species where the lung tissues have been found to be more sensitive to inhaled MCh than the airways (19). The lung tissues account for a variable part of RL in different animal species and humans and have been shown to respond as an active element after exogenous constriction (19, 20). We found that Rti accounted for almost 30% of RL under baseline conditions. This value is higher than those reported by Dreshaj and coworkers (21) in 2- to 3-wk-old and 10-wk-old piglets. As the ratio between Raw and Rti is sensitive to ventilatory parameters (22), differ-

Figure 5. (A) Morphologic effects of vascular engorgement alone on a midsized cartilaginous airway (Pbm ⫽ 4.9 mm). Proteinaceous fluid is seen between the lung parenchymal border and the cartilage plates in the airway and in the alveolar spaces (arrowheads). The dotted lines represent the boundaries defining the Pmo and the Po measured in this study. There is limited mucosal folding with some epithelial detachment. Original magnification: ⫻25. (B) Morphologic effects of vascular engorgement alone on a small membranous airway (Pbm ⫽ 0.9 mm). The dotted lines represent the boundaries defining the Pmo and the Po measured in this study. Some proteinaceous fluid is observed in the alveolar spaces (arrowheads) and lung interstitium. There is minimal folding of the airway mucosa and engorgement of vessels in the alveolar walls. Original magnification: ⫻100.

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Figure 6. (A) Large cartilaginous airway (Pbm ⫽ 5.2 mm) with prominent mucosal folding and proteinaceous fluid and red blood cells in the alveolar space (arrowheads) from a piglet challenged with a low dose of MCh after vascular engorgement. The dotted lines represent the boundaries defining the Pmo and the Po measured in this study. Original magnification: ⫻25. (B) Large membranous airway (Pbm ⫽ 1.9 mm) with prominent mucosal folding and proteinaceous fluid and red blood cells in the alveolar space or airway adventitia and airway lumen (arrowheads) from a piglet challenged with a low dose of methacholine after vascular engorgement. The dotted lines represent the boundaries defining the Pmo and the Po measured in this study. Original magnification: ⫻100.

ences in ventilation patterns (the animals of the present study have been ventilated with a higher frequency and slightly higher end-expiratory pressure) are very likely to be responsible for this. Because the bronchoconstrictor response to MCh is highly dependent on lung volume (23), we attempted to keep lung volume constant throughout the experiment. We believe that we were reasonably successful in this endeavor as the level of end-expiratory pressure did not change and the changes induced in lung mechanics were modest. In a previous study we have shown that increases in Raw and Rti caused by a transient inflation of a balloon in the left atrium of the heart are completely and almost immediately reversible (11) suggesting dilatation of the pulmonary vessels rather than edema as the responsible mechanism. In the present study, however, the balloon was inflated throughout the experiment for a much longer period of time (approximately 1 h). This raises the possibility that we may have induced a degree of interstitial edema in the present study. An increase in the outer airway wall area and the presence of proteinaceous fluid and red blood cells in the alveolar spaces support this (Figures 5 and 6). Thus, the results of our previous study (11) may not be completely applicable to the present study. The most striking finding in the present study was the difference in the pattern of mechanical response to inhaled MCh with and without vascular engorgement. In the absence of vascular engorgement a modest increase in RL in response to lowdose MCh, which was predominantly due to an increase in Rti (Table 2), was seen. However, in the presence of vascular engorgement, the increase in RL was much greater (59.7% versus 16.0% at 0.3 mg/ml MCh), with a marked increase in Raw (67.8% versus 10.8%). The increases in Rti were similar (38.1% versus 42.2%). There are two mechanisms that could explain this marked difference in airway response to low doses of MCh. Thickening of the airway wall inside the muscle layer would be expected to increase Raw for a given amount of muscle shortening (24). However, there were no significant differences in WAi between the engorgement and the engorgement plus MCh groups for any of the three airway size groups (Table 3). Alternatively, uncoupling of the muscle from the impedance to shortening provided by parenchymal attachments could result in a greater degree of muscle short-

ening in response to a given stimulus (8, 24). Macklem (8) has proposed that airway smooth muscle is inhibited from shortening in vivo to the degree it is capable of shortening to in vitro by the mechanical load it has to contract against, provided by parenchymal attachments and cartilage in the airway wall. This concept has been supported by the demonstration of an increased response to inhaled MCh in human subjects if lung volume is decreased below FRC and a decreased response if lung volume is increased above FRC (23). In the present study, WAo, defined as the area between the outer border of the airway smooth muscle and the surrounding lung parenchyma (Figures 4–6), was increased in piglets undergoing vascular engorgement compared with those that were not engorged. The response to MCh during congestion was associated with an increased degree of airway smooth muscle shortening compared with piglets receiving engorgement alone or MCh alone (Table 3). The increase in WAo in response to pulmonary vascular engorgement might occur via different mechanisms. First, pulmonary edema might result in accumulation of fluid within the airway wall between the outer border of the airway smooth muscle and the lung parenchyma, thereby resulting in an overall increase in the total cross-sectional area of the airway (i.e., Ao) with no change to the inner airway wall dimensions. Compared with MCh only animals, the total cross-sectional area of the airway (Ao) in animals with engorgement only was 3.2 mm2 versus 2.8 mm2 in airways ⬎ 3 mm Pbm, 0.27 mm2 versus 0.24 mm2 in airways 1 to 3 mm2, and 0.064 mm2 versus 0.051 mm2 in airways ⬍ 1 mm Pbm. Therefore, engorgement resulted in an increase in WAo by expansion of the total cross-sectional area with little encroachment on the airway lumen and therefore no significant change in Raw. This occurred by accumulation of fluid within the airway wall and between the adventitia of the airway and the surrounding lung parenchyma (Figures 5 and 6). Wagner (25) induced similar changes in the small airways of sheep. Airway wall edema induced by bradykinin resulted in an increase in the total cross-sectional wall area from 32% to 42% with no change in the smooth muscle perimeter and less than 5% change in the luminal area and no change in baseline Raw. She found no difference in airway responses to MCh challenge but found a limited relaxation response in animals with induced airway wall edema. That study differs from the current

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study in that pulmonary (alveolar) edema was not apparent and MCh was administered as an infusion (1 ␮g/ml) into the bronchial artery. The presence of pulmonary edema in our study may have reduced lung elastic recoil opposing muscle shortening; however, the mechanical properties of the lungs changed little with engorgement and MCh (Table 2). In animals with engorgement and MCh challenge, total cross-sectional area (Ao) was either similar or slightly less (data not shown) than in engorgement only animals. However the cross-sectional area and perimeter defined by the outer border of the smooth muscle layer (Amo and Pmo) were markedly reduced in animals after engorgement and MCh challenge as shown by the significant increase in muscle shortening (Table 3). Mitchell and Gray (26) have demonstrated similar results in organ bath experiments. They showed that the amount that the outer border of the airway (Ao) decreases during smooth muscle contraction is significantly less than the reduction in lumenal size (Abm) in the same airway, suggesting an uncoupling of the inner and outer borders of the airway wall during smooth muscle contraction. Therefore, fluid accumulates passively in the airway adventitia as a result of pulmonary congestion/edema and also during smooth muscle shortening to enlarge the space between the smooth muscle layer and the surrounding lung parenchyma as seen in the midsized airways in the present study (Table 3). However, Table 3 shows that the increase in WAo occurs in the absence of MCh and that both MCh and engorgement were necessary to achieve increased smooth muscle shortening. Sasaki and coworkers (27) found that bronchoconstriction alone did not actually alter airway dimensions and that prolonged bronchoconstriction actually reduced WAo. The effects of pulmonary edema and uneven, localized parenchymal distortion (28) on airway function might also have contributed to our results because pulmonary edema, airway lumenal fluid, and variable alveolar collapse were evident around some airways (Figures 5 and 6). However, it seems likely that these changes were not systematically different between the groups because the mechanical properties of the parenchyma at baseline (Table 1), and the change in Rti in response to MCh in control and engorgement groups (Table 2) were similar. In rat lungs at low volumes and filled with fluid, both increased passive and active smooth muscle shortening occur, compared with air-filled lungs (29). This was not associated with a significant increase in airway wall area as observed in the present study. Therefore, although the accumulation of fluid in the alveoli and airway lumen will alter airspace surface forces, this did not appear to be the mechanism for increased responsiveness to MCh observed in the present study. Similar to our findings, Snapper and coworkers (30) showed that acute (0.5 h) elevation of left atrial pressure did not alter tissue responses to an inhaled bronchoconstrictor, histamine. Finally, it is possible that engorgement resulted in increased stimulation of the airway smooth muscle by reducing the removal or accumulation of MCh, resulting in greater force development. In isolated dog lung lobes, circulatory obstruction has been shown to prolong responses to bronchoconstrictive agents without increasing maximal airway responses (31); however, we cannot exclude increased force development as contributing to our findings. The data from the present study may help clarify the cause of symptoms and increased bronchial responsiveness seen in patients with pulmonary congestion secondary to cardiac disease. Airway wall thickening secondary to bronchial vasodilatation and/or edema has been suggested as a possible cause of wheezing in patients with congestive heart disease (1). In adult humans (32) and animals (5) intravenous infusion of normal

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saline resulted in increased responses to aerosol challenges with MCh. While one must be cautious when extrapolating the results of animal studies to human diseases, the results of the present study suggest that if the increased smooth muscle shortening in response to constrictor stimuli also occurs in patients, bronchodilator agents rather than anti-inflammatory drugs may be the treatment of choice. The present study was conducted in piglets just after the age of weaning. In piglets, contractile responses of the bronchial airways and their topographic distribution have been shown to change during maturation (33). There is a greater contribution of airways to the increases in RL with advancing age in response to histamine (21). Furthermore, development of a mechanical association of extra-alveolar arteries to the lung parenchyma occurs gradually within the first 3 mo of life (34), allowing intrapulmonary arteries of newborn piglets to increase their diameters when kept at a constant intravascular pressure. Muscarinic receptors are present throughout the lung in pigs (35), and Sparrow and coworkers (36) demonstrated that already in the fetal pig lung the airways including the distal and terminal airways narrow in response to cholinergic agonists. However, basal bronchomotor tone is lacking in newborn piglets (37) and the changes in respiratory mechanics in response to vascular engorgement are not mediated by vagal reflex bronchoconstriction in 3- to 5-wk-old piglets (11). Thus, the responses seen in immature animals and the mechanisms producing those responses may be different in immature animals than in adult animals. In summary, our results demonstrate that in developing piglets, pulmonary vascular congestion selectively increases the responsiveness of the airways but not the lung tissues to MCh. This is likely to occur via uncoupling the airways from the mechanical impedance imposed by the parenchymal compartment of the lung. These findings may provide an explanation for the presence of wheezing in many children with impaired left ventricular function.

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