Optimized murine lung preparation for detailed structural evaluation via micro-computed tomography

Abstract

Utilizing micro-X-ray CT (μCT) imaging, we sought to generate an atlas of in vivo and intact/ex vivo lungs from normal murine strains. In vivo imaging allows visualization of parenchymal density and small airways (15–28 μm/voxel). Ex vivo imaging of the intact lung via μCT allows for improved understanding of the three-dimensional lung architecture at the alveolar level with voxel dimensions of 1–2 μm. μCT requires that air spaces remain air-filled to detect alveolar architecture while in vivo structural geometry of the lungs is maintained. To achieve these requirements, a fixation and imaging methodology that permits nondestructive whole lung ex vivo μCT imaging has been implemented and tested. After in vivo imaging, lungs from supine anesthetized C57Bl/6 mice, at 15, 20, and 25 cmH2O airway pressure, were fixed in situ via vascular perfusion using a two-stage flushing system while held at 20 cmH2O airway pressure. Extracted fixed lungs were air-dried. Whole lung volume was acquired at 1, 7, 21, and >70 days after the lungs were dried and served as validation for fixation stability. No significant shrinkage was observed: +8.95% change from in vivo to fixed lung ( P = 0.12), −1.47% change from day 1 to day 7 ( P = 0.07), −2.51% change from day 1 to day 21 ( P = 0.05), and −4.90% change from day 1 to day 70 and thereafter ( P = 0.04). μCT evaluation showed well-fixed alveoli and capillary beds correlating with histological analysis. A fixation and imaging method has been established for μCT imaging of the murine lung that allows for ex vivo morphometric analysis, representative of the in vivo lung.