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.

Macrophage Epithelial Reprogramming Underlies Mycobacterial Granuloma Formation and Promotes Infection

“…CLARITY processing followed by E-cadherin staining demonstrated that individual granulomas were isolated cleanly and were not disrupted during dissection (Chung et al, 2013; Cronan et al, 2015; Yang et al, 2014). Isolated granulomas had central necrotic foci replete with bacteria surrounded by intact layers of E-cadherin-positive cells (Figure 4D), in agreement with findings in tissue sections.…”

“…To better assess the morphological transformations within the granuloma, we used CLARITY to clear dissected zebrafish organs (Figure 5K) (Chung et al, 2013; Cronan et al, 2015; Yang et al, 2014). In control (Tg(mfap4:icre; ubb:BSR); Gt(jup-citrine) ct520a ) animals, the bacteria-laden core of the granuloma was surrounded by adherens junctions (Figure 5L, Movie S3).…”

A beginner’s guide to tissue clearing

“…The founder of this family of methods is CLARITY (Chung et al, 2013; Tomer et al, 2014; Ye et al, 2016), which has been modified by other labs to yield, among others, PACT-PARS (which reduces cost and increases speed of clearing using constant perfusion (Treweek et al, 2015)), ACT (which speeds up clearing with custom electrophoresis equipment (Lee et al, 2016)), and SWITCH (which accelerates clearing using high temperatures (Murray et al, 2015)). These methods have been applied successfully to map brain-wide activity and neural projections (CLARITY (Ye et al, 2016)), visualize Mycobacterium tuberculosis infections deep in whole lungs (PACT (Cronan et al, 2015)), and can even be used to clear bones (PACT-deCAL (Treweek et al, 2015)).…”

High content analysis of granuloma histology and neutrophilic inflammation in adult zebrafish infected with Mycobacterium marinum

“…Advances in optical clearing of whole zebrafish for 3-dimensional in situ analysis of granulomas is a powerful tool for relatively artifact-free microscopy but requires access to expensive multiphoton microscopes to access deep tissues (Cronan et al, 2015). Our method attempts to census the granuloma load of individual zebrafish by creating a catalog of thin section snapshots at 200 μm intervals providing confidence that granulomas are not double counted.…”