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A6212 - Flow Cytometry Underestimates and Planimetry Overestimates AEC2 Expansion After Lung Injury in Mice
Author Block: R. Zemans1, N. Jansing2, J. McClendon2, E. Redente3, R. Tuder4, D. Hyde5, J. Nyengaard6; 1Medicine, University of Michigan, Ann Arbor, MI, United States, 2Medicine, National Jewish Health, Denver, CO, United States, 3Pediatrics, National Jewish Health, Denver, CO, United States, 4Pulmonary Dept, Univ of Colorado Denver Sch of Med, Aurora, CO, United States, 5Univ of California At Davis, Davis, CA, United States, 6Clinical Medicine, Core Center for Molecular Morphology, Section for Stereology and Microscopy, Aarhus University, Aarhus, Denmark.
Introduction. An active area of investigation is lung regeneration after injury, notably expansion of the alveolar epithelial cell (AEC)2 population. AEC2 expansion is typically assessed by flow cytometry of cells recoverable from a lung digest or by counting cell profiles in arbitrary fields of immunostained lung sections (planimetry). However, these methods have not been validated and have theoretical limitations. Here, we employed stereology, an unbiased approach recommended by the American Thoracic Society, as the gold standard for quantitation of AEC2 expansion during repair after lung injury. The results were compared with flow cytometric and planimetric analyses. Methods. SPCCreERT2+/-;mTmG+/- mice induced with tamoxifen were treated with LPS and BrdU. Lungs were inflation-fixed and immunostained for GFP and BrdU. Stereology: The optical fractionator was used to estimate the absolute number of AEC2s, the key parameter for alveolar regeneration, and the number of BrdU+ AEC2s. Planimetry: Total AEC2s or BrdU+ AEC2s per HPF were counted. Flow Cytometry: Lungs were digested with dispase, elastase, and/or collagenase, and the number of AEC2s or BrdU+ AEC2s was determined. Results. Stereologic analysis revealed 14.3±1.7x106 AEC2 in the naive mouse lung, similar to previous estimates. AEC2 number increased by 50% during repair after injury. By flow cytometry, only 2.6±1.4x106 AEC2s were identifiable in the naive lung, and this number declined by 65% during repair. Planimetric analysis revealed a 90% increase in the number of AEC2s per HPF during repair. The percent of BrdU+ AEC2s peaked at 9.9%, 12.2%, and 3.2% by stereologic, flow cytometric, and planimetric analysis, respectively. Discussion. In comparison to stereology, flow cytometry underestimated the number of AEC2s in the naive lung, with a further decline in AEC2 number during regeneration. Moreover, the percent of cycling AEC2s was higher by flow cytometry than by stereology, suggesting that the AEC2s detectable in the digest may not be representative of all AEC2s: cycling cells may be preferentially recoverable. Planimetric analysis overestimated AEC2 expansion during repair by 40%, perhaps because AEC2s hypertrophy and/or because the reduced compliance of injured lungs limits inflation, thereby decreasing lung volume and increasing cell density. These key findings raise concern that apparent reduced AEC2 expansion in diseased lungs or knockout mice may be confounded by impaired recoverability from the digest of injured lungs or biases inherent to planimetry due to differential tissue inflation, overrepresentation of larger cells in two-dimensional sections, and nonrandom sampling. We conclude that studies of lung regeneration should include stereologic approaches.