Post-fixation lung treatment and assessment of post-pneumonectomy acinar morphometry with multi-resolution micro computed tomography

Mammalian respiration occurs within sac-like structures called alveoli, which occur within pulmonary acinar structures present at the ends of airways within the lungs. Alveoli form the terminal gas exchange unit called an acinus, which comprise the functional portions of lung tissue, meaning that it is where oxygen is moved into, and carbon dioxide is removed from. Efforts have been made to quantify the architecture of lung tissue, as its structure is closely tied to its role in gas exchange. This includes the chronic ramifications to the loss of functional lung tissue due to conditions such as COPD and lung trauma, and the replacement lung growth (compensatory lung growth - CLG) response that may arise to compensate for lost lung tissue volume. Surgical removal of one lung (pneumonectomy) has been used to induce CLG as a means of investigating the parameters leading to growth. However, to date, anatomic assessments have been limited to 2D microscopy. Techniques have previously been developed for the stabilization of lung tissue using fixative chemicals while maintaining normal lung volumes for the ex-vivo analysis of lung architecture. The development of nondestructive μCT imaging allows for the measurement of the 3D microscopic structures within the acinus at micrometer resolutions.

We present methodologies for processing lung tissue for μCT imaging and the analysis of the 3D architecture of the acinus for the extraction of structural features to quantify changes in these regions of gas exchange following CLG. This method results in the creation of stable lung samples that are suitable for μCT imaging of pulmonary acini for the comparison of architectural differences in canine control acini and in acini following CLG in response to pneumonectomy. Using these techniques, acinar airway diameters were observed to be both larger in diameter and length following CLG than in control airways, while the number of airways present in either group was found to be similar and peaked in airway counts within generations 8-10 in both groups. Airway counts peaked one generation later in acini following CLG compared to control acini for most pairs of acini. Due to the limited number of samples measured, future investigations are needed to validate these initial assessments of the control and post-PNX acini. No significant differences were observed in acinar volumes following CLG. Through the techniques described in this work, with their effectiveness demonstrated by these findings, we seek to extend current knowledge of mammalian lung architecture and provide a method for the analysis of the effects of CLG on respiration.