High-resolution 3D micro-CT reveals an additional muscle layer in esophageal atresia

Background Esophageal atresia (EA) is a congenital malformation intrinsically associated with esophageal dysmotility. Its etiology is poorly understood and it is unclear why motility is better preserved in some patients than in others. Esophageal motility is a three-dimensional (3D) process orchestrated by microscale structures, but conventional histology offers two-dimensional insight in the organization of the esophageal wall, hindering the identification of subtle, but potentially relevant, microarchitectural abnormalities that may contribute to understanding of the etiology of EA and variability in motility patterns. We aimed to evaluate the 3D microscale architecture of the esophagus in EA patients and identify potential anomalies using microfocus computed tomography (micro-CT). Methods Full thickness esophageal samples from the proximal and distal pouch of eleven neonates undergoing EA repair and three fetal control esophagus samples (gestational age: 24 weeks) were collected, contrast-enhanced, and imaged with high-resolution micro-CT (2–5 μm voxel size). Muscle layers were segmented using the machine learning software RootPainter and EA samples were compared with the fetal controls. Findings were validated by histology. Results Micro-CT enabled clear visualization of epithelial, submucosal, vascular, and muscular structures in all samples. 3D reconstructions consistently revealed a previously unreported oblique muscle layer situated medial to the circular muscle layer in all EA specimens. This layer varied in prominence and was not recognizable on 2D histology alone. Fetal control samples displayed only the typical circular and longitudinal layers, with oblique muscle fibres confined to the stomach. Conclusion Three-dimensional assessment of esophageal microarchitecture using micro-CT allows differentiation between epithelium, musculature and vascularization. Moreover, it facilitated the identification of an oblique muscle layer in proximal pouch and distal trachea-esophageal fistula specimens from EA patients. It remains to be studied whether these findings can explain the observed variation in motility patterns in EA patients.