FIG 5.
Distribution of CFZ, RIF, and PZA in centrally necrotizing granulomas of BALB/c IL-13tg mice. BALB/c IL-13tg mice were infected with 263 CFU of M. tuberculosis H37Rv. After 9 weeks, animals were treated with CFZ/PZA/RIF for 10 days, and 1 h after the last administration, lung tissue was collected and serial cryosections were prepared for histological characterization of lesions and MALDI-MS imaging analysis. Correlation of lesion pathology (A to C; G to J) and drug distribution (D to F) in neighboring lung cryosections of a BALB/c IL-13tg mouse are shown. (A) HE staining revealed cellular, inflammatory lesions (c) and highly organized centrally necrotizing granulomas (n), which are surrounded by a rim of macrophages (C) detected by CD68/ZN staining and encapsulated by a fibrous cuff (B) detected by trichrome staining. (D) Distribution of PZA [M + 2H]+· (m/z 125.05836), pixel size of 35 μm, DHB matrix. (E) Distribution of RIF [M-H]− (m/z 821.39784), pixel size of 35 μm, DHAP matrix. (F) Distribution of CFZ [M+H]+ (m/z 473.12942), pixel size of 35 μm. (G to J) Higher magnifications of selected regions from panel C (rectangles) for detection of AFB within the rim of macrophages (G) and the center of necrotic granulomas (H to J). The MALDI-MS imaging measurements of PZA and CFZ (shifted by 15 μm in x and y direction) and also immunohistochemical CD68/ZN staining were conducted on the same cryosection. Red arrows indicate cord formation. Scale bars, 1 mm (A to F); 10 μm (G to J).