CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections.
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Visualization of infection and the associated host response has been challenging in adult vertebrates. Owing to their transparency, zebrafish larvae have been used to directly observe infection in vivo; however, such larvae have not yet developed a functional adaptive immune system. Cells involved in adaptive immunity mature later and have therefore been difficult to access optically in intact animals. Thus, the study of many aspects of vertebrate infection requires dissection of adult organs or ex vivo isolation of immune cells. Recently, CLARITY and PACT (passive clarity technique) methodologies have enabled clearing and direct visualization of dissected organs. Here, we show that these techniques can be applied to image host-pathogen interactions directly in whole animals. CLARITY and PACT-based clearing of whole adult zebrafish and Mycobacterium tuberculosis-infected mouse lungs enables imaging of mycobacterial granulomas deep within tissue to a depth of more than 1 mm. Using established transgenic lines, we were able to image normal and pathogenic structures and their surrounding host context at high resolution. We identified the three-dimensional organization of granuloma-associated angiogenesis, an important feature of mycobacterial infection, and characterized the induction of the cytokine tumor necrosis factor (TNF) within the granuloma using an established fluorescent reporter line. We observed heterogeneity in TNF induction within granuloma macrophages, consistent with an evolving view of the tuberculous granuloma as a non-uniform, heterogeneous structure. Broad application of this technique will enable new understanding of host-pathogen interactions in situ.
Disease Models, Animal
Mice, Inbred C57BL
Tumor Necrosis Factor-alpha
Published Version (Please cite this version)10.1242/dmm.021394
Publication InfoCronan, Mark R; Rosenberg, Allison F; Oehlers, Stefan H; Saelens, Joseph W; Sisk, Dana M; Jurcic Smith, Kristen L; ... Tobin, David M (2015). CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections. Dis Model Mech, 8(12). pp. 1643-1650. 10.1242/dmm.021394. Retrieved from https://hdl.handle.net/10161/12636.
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Adjunct Associate Professor in the Department of Molecular Genetics and Microbiology
Tuberculosis remains a major global health problem, despite the widespread use of the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine and drug therapies. Coinfection with Mycobacterium tuberculosis and HIV, as well as multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, makes TB control difficult, complex and challenging. M. tuberculosis is an extraordinarily successful pathogen that has managed to latently infect nearly one third of humanity and is responsible
Associate Professor of Molecular Genetics and Microbiology
Tuberculosis: Mycobacterial Pathogenesis and Host Susceptibility Tuberculosis kills 1.5 million people annually. Our laboratory aims to understand the intricate interplay between mycobacteria and their hosts using a combination of model organism genetics, human genetics, pharmacology and high-resolution microscopy. By identifying key pathways utilized by the infecting bacteria and the host innate immune system, we hope to discover new therapeutic targets and interventi
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