Airway wall thickening in patients suffering from an asthmatic attack results from inflammation with edema and inflammatory cell infiltration and from structural changes, such as subepithelial fibrosis, mucous gland and goblet cell hyperplasia, and smooth muscle hypertrophy and hyperplasia.1,2 These structural changes are considered to be features of airway wall remodeling, which result from chronic inflammation.3–5 Airway wall thickening might be as important as smooth muscle shortening in determining the airway responsiveness of asthmatic patients.1
Two methods have been used to assess airway wall remodeling. First, the remodeling of the airway wall is assessed using bronchial biopsy specimens.3,6–12 Invasive diagnostic tools such as bronchial biopsy using flexible bronchoscopy have a risk of hemorrhage from the biopsied bronchial wall. Furthermore, it is often difficult to analyze the laminar structure of the bronchial wall because of insufficient and/or damaged specimens. Second, the whole bronchial wall thickness has been assessed by using high-resolution computed tomography (HRCT),13–18 but assessment using the HRCT technique involves a radiation risk and is not applicable for examining the bronchial mural structure.
Endobronchial ultrasonography (EBUS) is successfully used for the assessment of mediastinal lymph nodes and other mediastinal structures.19,20 Recently, EBUS was shown to be useful for assessing the laminar structure of the bronchial wall.21–27 Experimental needle puncture studies showed a correlation between ultrasonographic images and the bronchial wall structure.21,23 To our knowledge, however, the relationship between the thickness of the bronchial layer measured by ultrasonographic images and bronchial hyperresponsiveness has not been studied in patients with asthma.
The purpose of this study was to evaluate the relationship between the wall structure assessed by using EBUS and bronchial hyperresponsiveness in patients with asthma.
PATIENTS AND METHODS
Twenty-four patients with stable asthma diagnosed according to the American Thoracic Society criteria28 and 11 subjects without asthma and no respiratory symptoms were randomly enrolled. All the patients with asthma received inhaled corticosteroids therapy and the amount of inhaled corticosteroids was 370.8±322.3 μg/d (mean±SD). The duration of asthma was 16.7±12.6 years (mean±SD). On the basis of the severity of asthma, the number of patients (step 2/3/4) in each group was 6, 17, and 1, respectively. The study was approved by the Ethics Committee of the National Hospital Organization Kanazawa Medical Center (Kanazawa, Japan) and written informed consent was obtained from the subjects.
The subjects were administered an intramuscular injection of 25 mg hydroxyzine and an intravenous injection of 0.1 mg/kg midazolam. Topical anesthesia was obtained by inhalation of 4% lidocaine and 2% lidocaine sprayed into the oral passage, directly instilled onto the vocal cords, and used as needed on the bronchial mucosa.
EBUS and the Assessment of Bronchial Wall Thickness
For EBUS (EU-M 20 Endoscopic Ultrasound System; Olympus, Tokyo, Japan), a 2.6-mm-diameter, 20-MHz radial mechanical transducer-type ultrasonic probe (UM-BS20-26R; Olympus) and a flexible balloon sheath equipped with a balloon at the tip (MAJ-643R; Olympus) were used. The probes were introduced through the 2.8-mm-diameter channel of a flexible bronchoscope (model IT-260; Olympus). The balloon sheath was inflated 3 times in the center of the intermediate bronchus, with a minimum amount of saline required to ensure contact with the airway wall to obtain a 360-degree image. The EBUS image showed the layered structure of the bronchial wall, which was recorded on paper (UP-880 Video Graphic Printer; Sony, Tokyo, Japan).21
The cartilaginous portion of the extrapulmonary bronchi was visualized as 5 layers20 and we examined these 5 layers in detail. Three EBUS images showing well-defined laminar structures of the cartilage-containing parts of the bronchial wall were selected by an investigator blind to the patient's diagnosis. The measurements of the whole bronchial wall and the absolute values of the thickness of each layer were taken by 3 observers in a blind manner. The lines of the whole bronchial wall and each layer were drawn on recorded papers by the 3 observers by hand, respectively. First, they measured the outer diameter (DO) and luminal diameter (DL) of 3 selected EBUS images recorded on paper with calipers. The whole bronchial wall thickness was defined as (DO–DL) (Fig. 1). Percentage wall thickness (WT%) was defined as [(DO-DL)/DO]×100.25 WT% was calculated from the mean of the 3 EBUS images. Second, they measured the thickness of each layer. The absolute values of the thickness of each layer were measured with calipers and the mean values of the 3 EBUS images were used. Finally, the mean values of these parameters including WT% and the thickness of each layer measured by the 3 observers were analyzed.
Methacholine Challenge Test
Methacholine was dissolved in physiological saline solution to produce doubling concentrations of 0.04, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 mg/mL. Saline and each methacholine concentration were inhaled from a DeVilbiss 646 nebulizer (DeVilbiss Co, Somerset, PA) operated by compressed air at 5 L/min. The nebulizer output was 0.14 mL/min. Saline was inhaled first for 2 min and forced expiratory volume in 1 second (FEV1) was measured. If the change in FEV1 from the baseline value was less than 10%, inhalation of methacholine was started, and if the saline solution caused a 10% or greater change in FEV1, the test was stopped or postponed. Methacholine was inhaled for 2 minutes by tidal mouth breathing, followed immediately by 3 measurements of flow-volume curves at 1-minute intervals; the largest flow volume curve was retained for analysis. Increasing concentrations of methacholine were inhaled until there was a decrease of 20% or more in FEV1. Bronchial hyperresponsiveness was expressed as the provocative concentration of methacholine causing a decrease of 20% or more in FEV1 (PC20 in mg/mL).
Data were expressed as means (SD) and were analyzed with the StatView 5.0 program (SAS Institute, Cary, NC). An unpaired t test or the Mann-Whitney U test was used to compare the groups. The Spearman rank correlation test or the Pearson correlation test was used to analyze the relationship between the variables. P values of less than 0.05 were considered significant.
The characteristics of the patients with asthma and subjects without asthma are summarized in Table 1. In the 11 subjects without asthma, bronchofiberscopy was performed to diagnose hemoptysis and persistent cough or to exclude malignancy. The intermediate bronchus of each individual without asthma was not involved in inflammation and/or malignancy as these conditions could affect the EBUS-layered structure. The mean (SD) FEV1, percentage predicted FEV1, FEV1/forced vital capacity ratio, and the geometric mean (geometric standard error of the mean) value of PC20 were significantly lower than those in the subjects without asthma (P<0.01).
WT% Measured by EBUS
The mean (SD) WT% measured by EBUS images was 27.2% (3.9%) in the patients with asthma and 18.9% (2.6%) in subjects without asthma (Fig. 2). WT% measured by EBUS images was significantly greater in patients with asthma than in subjects without asthma (P<0.01).
Details of the 5 Airway Layers and Bronchial Responsiveness to Methacholine
Thickness of the 5 layers (1-hyperechoic marginal echo, 2-hypoechoic submucosal tissue, 3-hyperechoic inner marginal echo of the cartilage, 4-hypoechoic cartilage, and 5-hyperechoic outer marginal echo of the cartilage) was measured by EBUS in the patients with asthma and in the subjects without asthma (Fig. 3). The mean (SD) thickness was 0.35 (0.09 mm), 0.37 (0.07 mm), 0.31 (0.09 mm), 0.59 (0.15 mm), and 0.38 mm (0.13 mm) for layers 1, 2, 3, 4, and 5 in patients with asthma and 0.29 (0.08 mm), 0.28 (0.06 mm), 0.27 (0.08 mm), 0.55 (0.14 mm), and 0.37 mm (0.11 mm) in subjects without asthma, respectively. The thickness of the second layer in patients with asthma was significantly greater than that in subjects without asthma (P<0.05; Fig. 4); in contrast, the other layers were not significant.
In patients with asthma, PC20 was significantly and negatively correlated with the thickness of the second layer (r=0.52, P<0.01; Fig. 5). In contrast, there was no correlation between PC20 and the thickness of any other layer (1, 3, 4, and 5 layer and whole bronchial wall).
To our knowledge, this is the first study to investigate the relationship between submucosal thickness assessed by EBUS and bronchial responsiveness in patients with asthma. Airway wall thickness (WT% in the large airway) measured by EBUS images was significantly greater in patients with asthma than in subjects without asthma. Furthermore, on EBUS images, the thickness of the second layer in the large airway, such as the intermediate bronchus, was greater in patients with asthma than in subjects without asthma. The thickness of the second layer assessed by EBUS examination was positively correlated with nonspecific bronchial responsiveness in patients with asthma.
Increases in bronchial wall thickness result from inflammatory changes, such as edema and inflammatory cell infiltration, and from structual changes, such as mucous gland hyperplasia, reticular basement membrane thickening, vascular proliferation, and airway smooth muscle hypertrophy and hyperplasia. These structural changes are features of airway remodeling associated with chronic inflammation.6 Airway wall thickening might thus lead to airway hyperresponsiveness, an essential feature of asthma.29,30 HRCT has been used to measure airway wall dimensions in patients with asthma.14–18 Patients with asthma have thicker airways on HRCT scans than do healthy controls,15–18 and the degree of thickening is related to the severity of the disease,15,16,18 airflow obstruction,16,17 and airway hyperresponsiveness.31
EBUS is a noninvasive and safe method for assessing the mediastinal lymph nodes, other mediastinal structures, and the laminar structure of the bronchial wall.19–25 The subjects did not complain of discomfort, and dyspnea and coughing were not associated with the procedure in this study. Recently, Irani et al26 reported that EBUS was useful for identifying and quantitatively assessing the bronchial wall structure in lung transplant recipients. Soja et al27 reported that EBUS was useful for assessing bronchial wall thickness of the tenth segment of the right lung in patients with asthma. Although it would have been more appropriate for us to carry out the EBUS study at the tenth segment of the right lung, the tenth segment of bronchus could not be accessed using a 20-MHz ultrasonic probe because it was impossible to clearly distinguish the 5 layers of the bronchial wall.27 Therefore, we performed EBUS at the large airway, intermediate bronchus, but not at the tenth segment of the right lung.27 Ultrasound images obtained in this large airway were clear enough to assess not only each layer of the bronchial wall but also the whole bronchial wall.
We were able to discriminate the multilayer structure of the airway wall at this location as described earlier.19,21–24 Kurimoto et al21 reported that the first layer (hyperechoic layer) is a marginal echo extended from the inner margin of the mucosal epithelium to the inner part of the mucosal tissue; the second layer (hypoechoic) is the outer part of mucosal tissue; the third layer (hyperechoic) is a marginal echo on the inside of the cartilage; the fourth layer (hypoechoic) is cartilage; and the fifth layer (hyperechoic) is a marginal echo on the outside of the cartilage, as determined with a needle puncture experiment. In this study, WT%, especially, of the second layer was greater in patients with asthma than in subjects without asthma. As the second layer is equal to the large part of the submucosal tissue containing airway smooth muscle,21 we found that by using the EBUS technique, the thickness of the submucosal tissue containing smooth muscle in patients with asthma was greater than that in nonasthmatic subjects. These findings could explain the postmortem findings by James and colleagues1 that the increased wall area was because of increased areas in the epithelium, muscle, and submucosa. The thickness of the second layer (submucosal tissue and airway smooth muscle) was positively correlated with bronchial responsiveness in patients with asthma. As the EBUS techinique involved the inflation of a saline-filled balloon around the ultrasound probe, we were concerned that the saline-filled balloon might compress the airway and alter the whole wall thickness measurements. Shaw et al25 reported that ultrasound images recorded from sheep airways in an in-vitro study with the balloon inflated showed a slightly greater airway wall thickness (approximately 0.5 mm) than when it was deflated, although this difference did not reach statistical significance. When the latex balloon sheath is in contact with the airway, its thickness is included in the measured thickness of the first layer. As the thickness of the first layer of this ultrasound image contains at least 3 components (latex baloon, epithelium, and the inner part of the submucosal tissue), we could not precisely evaluate the thickness of the epithelium in patients with asthma. The resolution of the 20-MHz ultrasound probe is limited, which explains why the borders of the particular layers seemed to be blurred. A 30-MHz probe providing a higher image resolution has been developed.24 Future studies using this new equipment are required.
In conclusion, the findings of this study indicate that the thickness of the whole airway, especially, the thickness of the submucosal layer containing smooth muscle measured by EBUS images, is significantly greater in patients with asthma than in subjects without asthma. Furthermore, bronchial responsiveness was negatively correlated with the thickness of the second layer measured by EBUS. These findings indicate that the thickness of the submucosal layer containing smooth muscle might be related to the degree of bronchial responsiveness in patients with asthma. Thus, the EBUS technique is useful for evaluating a distinct laminar airway structure, such as bronchial wall remodeling in patients with asthma.
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Keywords:© 2010 Lippincott Williams & Wilkins, Inc.
airway hyperresponsiveness; airway remodeling; asthma; endobronchial ultrasound; mural structure