Understanding whether lung development occurs by increase in cell size or cell number can lead to relevant clinical implications in phenomena such catch up growth, chronic neonatal lung disease and cancer. Although alveolar type 1 (AT1) and type 2 (AT2) cells have been quantified in the past, these methods are not up to par with current software and 3D molecular imaging. Here, novel stereological methods are proposed for alveolar cell quantification in the murine lung. Lungs were harvested from postnatal FVB Wild Type Mice at two time points: days 2 (P2) and 37 (P37). Volumes were measured using two different methods: fluid displacement and Cavalieri Principle, the latter with the use of the Stepanizer® software. Immunofluorescence staining with ECAD and HOPX using whole mount staining led to visualization of 3D structures using confocal microscopy. These images were used to quantify and calculate the numerical density of AT1 and AT2 in the Imaris® software. With the application of the Cavalieri Principle, the total absolute number of AT1 and AT2 cells was analyzed at both time points of postnatal lung development. When comparing P2 and P37, we found that the left lung was 19 uL and 98 uL, respectively. The numerical density of alveolar cells for left lung in a P2 was 188.6 AT1 cells/3.0 x 106 um3 and 166.6 AT2 cells/3.0 x 106 um3, and in a P37 was 280.75 AT1 cells/6.0 x 106 um3 and 724.1 AT2 cells/6.0 x 106 um3. A total number alveolar cells in a P2 were 1.11 x 106 AT1 cells and 9.83 x 105 AT2 cells, and in a P37 were 4.5 x 106 AT1 cells and 1.18 x 107 AT2 cells. These results suggest that lung development occurs by an increase in cell size over time.

This  presentation is supported by the National Cancer Institute through the U54 CA096297/CA096300: UPR/MDACC Partnership for Excellence in Cancer Research Training Program.

Researchers should cite this work as follows:

• Odemaris Narvaez del Pilar (2016), "Novel Stereological Methods for Alveolar Cell Quantification in the Mouse Lung," https://ncihub.cancer.gov/resources/1704.

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