Evaluation of a Mouse Model of Necrotic Granuloma Formation Using C3HeB/FeJ Mice for Testing of Drugs against Mycobacterium tuberculosis

Other manifestations of experimental Erdman infection of C3HeB/FeJ mice include neovascularization, individual foamy macrophage necrosis, cholesterol cleft formation, and lesion cavitation. (A) Higher magnification of the fibrous capsule from lung lesions with liquefactive necrosis shows an increase in blood-filled capillaries and infiltrates of mixed inflammatory cells.(B) A higher magnification of the region depicted by the square in panel A showing numerous blood-filled capillaries (arrowheads). (C) Besides liquefactive necrosis, lesions have individual foamy macrophage (blue arrow) necrosis with intracellular and extracellular cholesterol clefts (black arrows). (D) Encapsulated lesions with liquefactive necrosis occasionally have central cavitation (Ca). (E) Higher-magnification image of the inner surface of the cavitary lesions depicted by the square in panel D showing a dense accumulation of neutrophils (arrowhead) admixed with foamy macrophages and lymphocytes. Stain: hematoxylin and eosin.

Lung lesions with liquefactive necrosis from C3HeB/FeJ mice experimentally infected with the Erdman strain of M. tuberculosis are delineated by fibrous connective tissue. (A) Newer, immature collagen fibrils (F) are admixed with more mature collagen (yellow, birefringence). (B) Higher-magnification image of the fibrous capsule depicted by the rectangle in panel A showing both immature collagen fibrils (yellow, birefringence) and mature collagen fibers (red staining). Stain: picrosirius red.

Different staining techniques reveal phenotypic differences in bacilli in the late stage of experimental infection of C3HeB/FeJ mice with the Erdman strain of M. tuberculosis. Images A and D are serial sections of lesions from mice 6 weeks after infection. Images B and E are serial sections of lesions from mice 10 weeks after infection, and images C and F are serial sections of lesions from untreated control mice at 13 weeks after infection. Kinyoun-stained sections A, B and C detect numerous intra- and extracellular bacilli. (A) At 6 weeks of infection, bacilli are primarily intracellular, whereas at 10 weeks (B), the majority of bacteria are located within necrotic granulomas. (C) Thirteen weeks postinfection showing the center of liquefactive necrotic lung lesion positive for AFB. Auramine-rhodamine-stained sections (D, E, and F) lose the ability to detect bacilli over infection. (D) At 6 weeks, intracellular bacilli are clearly seen. Included is a magnification inset of intracellular staining. (E) At 10 weeks, the central region of necrotic granuloma is devoid of fluorescent bacilli. In inset 1, the center of the necrotic lesion has reduction in AR-positive bacilli. In inset 2, the edge of the necrotic lesion retains the majority of detectable staining. (F) At 13 weeks, the entirety of the necrotic lung lesion is without stain, except for the very edge of the rim, depicted in the inset.

Lung lesions with liquefactive necrosis from C3HeB/FeJ mice experimentally infected with the Erdman strain of M. tuberculosis show evidence of hypoxia. (A) Immunohistochemical staining for the hypoxia marker pimonidazole within a lung lesion with liquefactive necrosis (N) showing multiple positive stained cells. (B) Higher magnification of the region depicted by the square in panel A showing multiple cells staining positive for pimonidazole adducts. (C) Viable remnants of interalveolar septal walls within a region with necrotizing alveolitis have residual cells that stain positive for pimonidazole. Counterstain: hematoxylin.

Isoniazid (INH) treatment at 25 mg/kg is less effective in C3HeB/FeJ mice against the Erdman strain of M. tuberculosis than in the BALB/c mouse strain. (A) In BALB/c mice, the reduction in CFU in the lung (filled symbols) and spleen (open symbols) was biphasic, whereas, in the C3HeB/FeJ mouse strain (B), the killing kinetics in the lung were attenuated, as was the efficacy in the spleen. *, untreated control mice were euthanized at day 26 due to morbidities. ND, no bacterial colonies detected (detection limit, 50 CFU).

Rifampin (RIF) treatment at 10 mg/kg is less effective in C3HeB/FeJ mice against the Erdman strain of M. tuberculosis than in the BALB/c mouse strain. (A) In BALB/c mice, the reduction in CFU in the lung (filled symbols) and spleen (open symbols) was biphasic, whereas, in the C3HeB/FeJ mouse strain (B), the killing kinetics in the lung were attenuated, as was the efficacy in the spleen. *, untreated control mice were euthanized at day 26 due to morbidities. ND, no bacterial colonies detected (detection limit, 50 CFU).

Linezolid (LZD) treatment at 100 mg/kg is less effective in C3HeB/FeJ mice against the Erdman strain of M. tuberculosis than in the BALB/c mouse strain. (A) In BALB/c mice, the reduction in CFU in the lung (filled symbols) and spleen (open symbols) was biphasic, whereas, in the C3HeB/FeJ mouse strain (B), the killing kinetics in the lung were attenuated, as was the efficacy in the spleen. *, untreated control mice were euthanized at day 26 due to morbidities.

Pyrazinoic acid (PZA) treatment at 150 mg/kg is bacteriostatic in C3HeB/FeJ mice against the Erdman strain of M. tuberculosis compared the activity in the BALB/c mouse strain. (A) In BALB/c mice, the reductions in CFU in the lung (filled symbols) and spleen (open symbols) were more pronounced in the early stages of infection, but PZA was less effective late. (B) PZA treatment of C3HeB/FeJ mice prevented the progressive bacterial growth seen in control animals (diamonds) and therefore was bacteriostatic in the lung and spleen. *, untreated control mice were euthanized at day 26 due to morbidities.