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

Vasilescu DM, Knudsen L, Ochs M, Weibel ER, Hoffman EA. Optimized murine lung preparation for detailed structural evaluation via micro-computed tomography. J Appl Physiol 112: 159-166, 2012. First published August 4, 2011; doi:10.1152/japplphysiol.00550.2011.Utilizing micro-X-ray CT (mu CT) 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 mu m/voxel). Ex vivo imaging of the intact lung via mu CT allows for improved understanding of the three-dimensional lung architecture at the alveolar level with voxel dimensions of 1-2 mu m. mu CT 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 mu CT imaging has been implemented and tested. After in vivo imaging, lungs from supine anesthetized C57Bl/6 mice, at 15, 20, and 25 cmH(2)O 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). mu CT evaluation showed well-fixed alveoli and capillary beds correlating with histological analysis. A fixation and imaging method has been established for mu CT imaging of the murine lung that allows for ex vivo morphometric analysis, representative of the in vivo lung.