Histology

The heart and lungs were removed en bloc under isoflurane anesthesia. Lungs were fixed with paraformaldehyde in the inflated state. Four-micron thick slices of paraffin-embedded, fixed whole-mount coronal sections of left and right lung were stained for hematoxylin and eosin. Extent of bronchiolitis obliterans organizing pneumonia (BOOP) was quantified by assessing percentage of the cross sectional area of the lung involved at low power, morphology confirmed with high resolution JPEG images, as we previously reported (6). Histology consistent with BOOP (Masson bodies, inflammation, and young fibroblasts filling the distal airways and alveoli) was assessed with high power images. Using a scale developed for quantification of BOOP (6, 21), we also performed grading of epithelial injury, fibrosis, and inflammatory changes (EFI) in the most severely affected areas of cross sectional lung images, each on a zero to three scale. The presence or absence of pulmonary vascular changes (thickening of intima or media or vascular drop out) and necrosis were noted in each image (Table 1). Two blinded reviewers graded all images. Average values were used for analysis.

To quantify BOOP, we developed a grading scale for our studies (6) based on methods to quantify BOOP in human transplant patients (21). This scale includes grading epithelial injury, fibrosis, and inflammatory changes, each on a scale from zero to three, and estimation of the percentage area of the lung exhibiting BOOP. Images were graded by two investigators blinded to the treatment group.