title: CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections.
abstract: 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.
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA mark.cronan@duke.edu david.tobin@duke.edu.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Cronan
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA.
authors.forename: Allison F
authors.initials: AF
authors.lastname: Rosenberg
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA.
authors.forename: Stefan H
authors.initials: SH
authors.lastname: Oehlers
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA.
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Saelens
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA.
authors.forename: Dana M
authors.initials: DM
authors.lastname: Sisk
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA Duke Human Vaccine Institute and Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA.
authors.forename: Kristen L
authors.initials: KL
authors.lastname: Jurcic Smith
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA Duke Human Vaccine Institute and Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA.
authors.forename: Sunhee
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA Department of Immunology, Duke University School of Medicine, Durham, NC 27710, USA mark.cronan@duke.edu david.tobin@duke.edu.
authors.forename: David M
authors.initials: DM
authors.lastname: Tobin
mesh_terms: D000375:Aging / Q000473:pathology*; D000818:Animals; D001808:Blood Vessels / Q000473:pathology; D004195:Disease Models, Animal; D005260:Female; D005453:Fluorescence; D006099:Granuloma / Q000382:microbiology / Q000473:pathology; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008168:Lung / Q000382:microbiology / Q000473:pathology; D008810:Mice, Inbred C57BL; D009169:Mycobacterium tuberculosis / Q000502:physiology; D018919:Neovascularization, Physiologic; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology* / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015027:Zebrafish / Q000382:microbiology*
chemical_list: D014409:Tumor Necrosis Factor-alpha
keywords: CLARITY; Imaging; Infection; Mouse; Mycobacteria; PACT; Tuberculosis; Zebrafish