Visualization of M. tuberculosis in Animal Models Higher magnification of the inflammatory lesion seen in figure 2J. The lesion shows that the majority of remaining AR+ stained bacilli are intracellular within macrophages (AR, 2006 magnification). (L) High magnification of the inflammatory lesion seen in figure 2K (square). This fluorescent image shows AR+ bacilli were predominantly found within macrophage cells comprising the few remaining inflammatory lesions (AR, 10006magnification). doi:10.1371/journal.pone.0017550.g002 with the hypoxia marker 2-nitroimidazole 1.5 h prior to euthanasia, as we previously described for the guinea pig model [23]. Mice were injected 15, 17, 20, 22, 25, and 29 days after aerosol. Hypoxia was not detected by pimonidazole staining in the lungs of M. tuberculosis infected GKO mice until 25 days post aerosol infection (data not shown). By 29 days, pimonidazole was observed to markedly stain around areas of necrosis in pulmonary lesions of the GKO mice (Fig. 3G, H). Hypoxia was not detected within areas of necrosis due to the absence of viable cells for the pimonidazole to form adducts with. These results indicate that the Figure 3. The progression of cellular necrosis that occurs within pulmonary inflammatory lesions during M. tuberculosis infection in GKO mice and the development of hypoxia as revealed by pimonidazole. (AR) = auramine-rhodamine, hematoxylin QS and DAPI; (H&E) = hematoxylin and eosin. (A) An inflammatory lesion from the lungs of M. tuberculosis infected GKO mice 22 days after aerosol infection. The lesion shows a number of alveolar spaces beginning to accumulate granulocytes prior to the presence of cellular necrosis (H&E staining, 2006 magnification). (B) High magnification of the area demarcated in figure 3A (circle) showing an alveolus completely occluded with inflammatory infiltrate (H&E staining, 10006magnification). (C) Fluorescent image of an inflammatory lesion from the lungs of M. tuberculosis infected GKO mice 18 days post-aerosol infection showing a highly organized tubulin network with intact nuclei (Immunofluorescence for Tubulin (green) and MTB (red) and DAPI (blue). 10006 magnification). (D) Immunofluorescent image of an inflammatory lesion from the lungs of M. tuberculosis infected GKO mice 28 days post-aerosol TB (red) infection showing loss of tubulin (green) architecture and degenerated nuclei (blue) indicating loss of cell viability (10006 magnification). (E) Confocal microscopy of an inflammatory lesion from the lungs of M. tuberculosis infected GKO mice 18 days post-aerosol infection. The nuclei (blue) appear healthy with few numbers of AR+ bacilli (red). (AR staining combined with DAPI; 6306 magnification with additional 2.56 digital zoom). (F) Confocal microscopy of an inflammatory lesion from the lungs of M. tuberculosis infected GKO mice 28 days post- aerosol infection. Degenerated fragments of inflammatory cell nuclei are within alveoli with a high number of extracellular AR+ bacilli (AR staining combined with DAPI; 6306magnification with additional 2.56digital zoom). (G) Immunohistochemistry detecting hypoxia (brown) in a necrotic lung lesion from an M. tuberculosis infected GKO mouse 29 days after aerosol infection. The area of central necrosis (N) is surrounded by epithelioid macrophages near a major airway. (Hematoxylin counterstain, 406 magnification). (H) High magnification of the area demarcated within the inflammatory lesion shown in figure 3G (square). The center of alveoli, which is likely hypoxic, is filled with cellular debris and fails to stain due to the lack of viable cells (Hematoxylin counterstain, 4006 magnification). doi:10.1371/journal.pone.0017550.g003 PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e17550