While considering surgical treatment for lung cancer, attention to the line of resection is essential. This is usually carried out by naked eye examination using flexible bronchoscope. However, it is also mandatory to clarify the depth of tumor invasion into the surrounding bronchial wall. For intraluminal tumor staging of small cancers, it is also necessary to know if the tumor is extending into or beyond the cartilage or just limited inside the submucosal layers. Therefore, it is essential to identify all the layers of the bronchial wall for staging and for decision strategy. By endobronchial ultrasonography (EBUS), using the 20 MHz radial mechanical ultrasonic miniature probe, the multilayer structure of the central airway wall can be clearly analyzed.1–7 It has been shown that exact staging by EBUS can provide a much higher cure rate in bronchoscopic treatment of early lung cancer as compared with conventional imaging.8 On the other hand, a tumor that is in the close proximity to the mediastinum and to the central airways, infiltration of the airway wall reduces the chances of curative resection. Thus, it is essential to thoroughly investigate the outer layers of the airway wall of the central airways for better outcomes. It has been shown that EBUS is the only modality to provide this information.5 However, there is a controversy related to the exact sonographic layer structure of the central airway wall. Some investigators have claimed to have observed only 5 layers,2,7 others found 64 or even 7 layers.1,6 Whereas there is no disagreement about the imaging of mucosa, submucosa, and internal surface of the cartilages, the differences are mainly concerning the interpretation of the external layers. The purpose of this experiment was to clarify the sonographic layer structure of the central airway wall in vitro by comparing ultrasound (US) information with the histology. For simplification, we excluded the membranaceous dorsal wall of the central airways, which is lacking the cartilage layer.
MATERIALS AND METHODS
Five specimens of postmortem tracheas were used for the in vitro examination. The tracheal specimens were cut horizontally into segments of about 3-cm length. Of these, we prepared 3 different kinds of specimens. Type A: a whole structure with adjacent mediastinal tissue, also containing fat, type B: after removal of mediastinal and fat tissue, and type C: after additional removal of the external fibroelastic connective tissue sheath surrounding the central airways, so that the external surface of the cartilages lay bare. For the investigation, we used an Olympus 20 MHz mechanical radial scanning probe (Olympus UM-BS20-26R) with MH-240 driving unit with balloon catheter sheath (Olympus MAJ-643R) and EU-M20 processor. Each specimen was totally immersed in water during ultrasonographic observation. Afterward the EBUS images were compared with the histology (hematoxylin and eosin stain).
Second, we observed a denuded tracheal cartilage without any adherent soft tissue, that had been collected from a tracheotomized patient. This piece of cartilage was also immersed into water and the radial probe was applied for sonographic examination.
In addition, we also investigated the airway from the main bronchus down to segmental bronchi using 4 resected and 1 postmortem specimen.
In each of the 5 tracheal specimens, in all images of specimens type A, 7 layers and an adjacent hazy structure were identified. In the specimens type B, also 7 layers, but without the hazy surrounding structure were observed, and in specimens of type C, 5 layers lacking the external 2 layers were observed. However, in these fixed specimens, clear identification of the structure, especially of the outer layers was difficult, owing to shrinkage of the tissue.
In the native unfixed specimens, we could differentiate 7 layers in the main and lobar bronchi down to the segmental branching. From there the multilayer structure became less distinct toward the periphery as the cartilages were more patchy and finally disappeared altogether leaving a 3-layer sonographic structure. In Figure 1, the US images and corresponding histology of a trachea are demonstrated. In the type A specimen, one can see 7 layers, representing mucosa and submucosa, then cartilage with its inner and outer perichondrium, and finally 2 external layers of the fibroelastic connective tissue. Attached to the latter one can see the adjacent hazy structure describing loose connective tissue and fat. Type B specimen is also described as 7 layers, just the same as type A, but without the adjacent hazy structure. In type C specimens, only 5 layers are left, lacking the external 2 layers.
These observations suggest that sonographic image of the wall of the trachea exhibits as 7-layer structure which corresponds to the anatomic structures of the bronchial wall itself; the sixth and seventh layers correspond to the external fibroelastic connective tissue. Especially, the sixth hypoechoic layer corresponds to the loose part of the external fibroelastic connective tissue.
In the bare cartilage without any attached soft tissue, the spongiform internal hypoechoic structure is accompanied on both sides by a hyperechoic layer, representing endochondrium and perichondrium or internal and external tabula as seen in the bones, and thus is described as 3 layers by endobronchial ultrasound (EBUS) (Fig. 2).
It was apparent that the first and second internal layers correspond to mucosa and submucosa, third, forth, and fifth layer to the cartilage, sixth and seventh layer to the external fibroelastic connective tissue. However, as the sixth layer is very thin, frequently the fifth, sixth, and seventh layers look like 1 continuous layer, especially when lower resolution is applied.
Also in main bronchi of resected specimens, as in the postmortem specimens, usually 7 layers were clearly demonstrated from trachea down to the lobar bronchi. From here the external 2 layers were progressively tapering and thinning away, and the 7-layer structure could be only seen on some parts of the wall. As one advanced more distally, the image revealed a 5-layer structure. Finally, when the cartilages became patchy and began to disappear; only 3 layers could be detected or even the wall condensed with the surrounding structures. Thus, there was no clear demarcation between mucosa, submucosa, and underlying connective tissue structure.
The application of US in the central airways has been investigated by our group since the early 1990s.1,9 In fact one of the first indications for which we approached Olympus Co in 1989 to develop EBUS was for preoperative assessment of airway wall in patients with centrally localized lung tumor. From our previous surgical experience, we were aware that CT of the chest was inadequate in providing this detail and often was falsely positive. Thus, a better diagnostic modality, which could delineate the airway wall anatomy was very much needed. Using EBUS, we were able to offer potentially curative surgery to a significant number of patients, that according to CT scan would have been treated by palliative measures. In a prospective study, we could prove that EBUS is a highly accurate diagnostic tool and superior to chest CT in evaluating the question of airway involvement by lung cancers, which are localized in the vicinity of the central airways5 (Fig. 3). In this study, confirmation of the integrity of the outer layers of the tracheobronchial wall was as important of a feature for establishing a proper staging as analyzing the internal layers for diagnosing early stage lung cancer. After application of the balloon sheath by Becker,1 which enables the observation of the airway wall and its surrounding structures in 360 degrees, the clinical application of EBUS has gained increasing popularity.1–8,10–13 Miyazu et al8 reported that EBUS can clearly identify whether a tumor is growing beyond the cartilage or is limited to within its layers. This is especially important while selecting patients for photodynamic therapy or other bronchoscopic treatment modalities. As EBUS with the radial scanning mechanical 20 MHz probe has the ability of analysis in the submillimeter order, this is currently the only established method to describe the delicate layers of the airway wall and the immediate surrounding structures.2–8,10
However, concerning the interpretation, there are different observations. Kurimoto et al2 first reported that the wall of the central airways is composed of 5 layers based on his needle-puncture experiment, where he is correlating mucosa and submucosa to the innermost 2 sonographic layers, attributing the third and fourth layer to the cartilage (inner surface and combined internal structure and external surface) and the fifth layer to the adventitia. Later he reported that in several cases he also observed 7 layers in the trachea and main bronchi.6 Baba et al4 found 6 layers. Becker1,3 observed 7 layers in his in vivo and in vitro experiments. Moreover, analyzing isolated cartilages, we could demonstrate that the cartilage itself is composed of 3 ultrasonic layers. Nakamura et al7 reported seeing a total of 5 layers. In most reports there is no dispute in the interpretation of the inner 4 layers; the differences are mainly related to the outer ones. The precise interpretation of the outer layers is important especially to make a decision on the therapeutic strategy for extramural tumors (Fig. 4). EBUS with the radial scanning probe has the ability to clarify this question, whereas the resolution of the 7.5 MHz linear scanner is not sufficient. To communicate among specialists, it is essential that we come to an agreement on the structural anatomy of the airways as seen on the sonographic images.
Detailed information on structure of the airway wall has become of great interest in this era of detection and treatment of early lung cancer.14 In our study, the postmortem trachea specimens were described as having 7 layers. After removal of the external fibroelastic connective tissue, the sixth and seventh layers of the EBUS image disappeared. This fact demonstrates that the sixth and seventh layers correspond to the external fibroelastic tissue. Kurimoto reported that in the cases in whom he observed 7 layers in the trachea, the sixth hypoechoic layer corresponded to a thick loose connective tissue layer covering the cartilage, and the seventh hyperechoic layer corresponded to collagen fibers outside the loose connective tissue. He also described that the membranaceous part of the tracheal wall is composed of 3 ultrasonic layers whereas the cartilaginous portion of the trachea and main bronchi may reveal up to 7 layers.6 This observation is similar to our experiment.
We agree that loose part of external fibroelastic connective tissue just adjacent to the perichondrium is ultrasonographically described as sixth hypoechoic layer, and the dense part, adjacent to the loose part, corresponds to the seventh hyperechoic layer. So, the whole wall is made up of 7 layers. However, the sixth layer is very thin and sometimes difficult to identify, so that the fifth, sixth, and seventh layer occasionally are described as 1 single layer, and some of the disagreement arises from this observation. In Baba's publication, the adventitia was described as a single layer.4 In Nakamura's figure, we see typical 7 layers when we pay attention to the delicate sixth layer. Furthermore, he analyzed the captured images using freeware image analysis software (NIH image, version 1.62; National Institute of Health; Bethesda, MD) and reported that the 5 layers are described as W-shape curve.7 However, at a closer look after the first W-shape, one can observe another smaller W-shape, that indicates the sixth and seventh layer.
Compared with trachea and main bronchi, the external fibroelastic connective tissue of the smaller airway is thinner, lacking the loose part under low resolution. Therefore, the outer part of the wall was described as 1 hyperechoic layer, composing 5 layers in total. Former disagreement might have been caused as the investigators have mainly concentrated on more distal airways.
We have demonstrated that EBUS of the trachea, main bronchi, and proximal lobar bronchi exhibit 7 layers, including an external fibroelastic connective tissue layer, layer of loose tissue, distal lobar bronchi along with a demonstrated 5-layer structure.
The layer structure of airway wall was investigated in vitro by EBUS. The ultrasonic image of the tracheal wall was confirmed as a 7-layer structure. After removal of the external fibroelastic connective tissue, 5 layers remained. The ultrasonographic layers of trachea correspond to the histology. The lobar bronchi are usually described to have 7 layers but distally the external 2 layers taper out and thin, resulting in a 5-layer structure. Further examination of distal airways reveal only 3 layers and finally no more distinct layers can be identified.
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