HYBRiD: hydrogel-reinforced DISCO for clearing mammalian bodies

Adipose tissues communicate with the central nervous system to maintain whole-body energy homeostasis. The mainstream view is that circulating hormones secreted by the fat convey the metabolic state to the brain, which integrates peripheral information and regulates adipocyte function through noradrenergic sympathetic output1. Moreover, somatosensory neurons of the dorsal root ganglia innervate adipose tissue2. However, the lack of genetic tools to selectively target these neurons has limited understanding of their physiological importance. Here we developed viral, genetic and imaging strategies to manipulate sensory nerves in an organ-specific manner in mice. This enabled us to visualize the entire axonal projection of dorsal root ganglia from the soma to subcutaneous adipocytes, establishing the anatomical underpinnings of adipose sensory innervation. Functionally, selective sensory ablation in adipose tissue enhanced the lipogenic and thermogenetic transcriptional programs, resulting in an enlarged fat pad, enrichment of beige adipocytes and elevated body temperature under thermoneutral conditions. The sensory-ablation-induced phenotypes required intact sympathetic function. We postulate that beige-fat-innervating sensory neurons modulate adipocyte function by acting as a brake on the sympathetic system. These results reveal an important role of the innervation by dorsal root ganglia of adipose tissues, and could enable future studies to examine the role of sensory innervation of disparate interoceptive systems.

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“…To visualize sensory nerves in mouse torso and iWAT samples, tissue samples were cleared using the HYBRiD method as described previously 17 .…”

Section: En Bloc Hybrid Tissue Clearing and Immunolabelling Of Iwatmentioning

confidence: 99%

“…HYBRiD, specifically designed for en bloc fluorescence visualization of large tissues 17 , paving the way to directly characterize the intact sensory innervation from DRGs to fat.…”

mentioning

confidence: 99%

The role of somatosensory innervation of adipose tissues

Wang

Leung

Zhang

et al. 2022

Nature

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“…In the hydrogel-based clearing of bones, EDTA, which is a chelating agent of calcium ions, is applied prior to hydrogel synthesis. 42, 43 Thus, we first tested the pre-gelation EDTA treatment. We treated fixed mouse embryos with 0.5 M EDTA for longer than three weeks and then proceeded to gelation, proteinase K treatment, collagenase mixture treatment, proteinase K treatment again, and then expansion in DI water.…”

Section: Resultsmentioning

confidence: 99%

Nanoscale resolution imaging of the whole mouse embryos and larval zebrafish using expansion microscopy

Sim

et al. 2021

Preprint

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Nanoscale imaging of all anatomical structures over whole vertebrates is needed for a systematic understanding of human diseases, but this has not yet been achieved. Here, we demonstrate whole-ExM, which enables nanoscale imaging of all anatomical structures of whole zebrafish larvae by labeling the proteins of the larvae with fluorophores and expanding them four-fold. We first optimize the fluorophore selection and labeling procedure to visualize a broader range of anatomical structures. We then develop an expansion protocol for zebrafish larvae having calcified body parts. Through this process, we visualize the nanoscale details of diverse larvae organs, which have corresponding organ counterparts in humans, over the intact larvae. We show that whole-ExM retains the fluorescence signals of fluorescent proteins, and its resolution is high enough to visualize various structures that can be imaged only with electron microscopy. Whole-ExM would enable the nanoscale study of the molecular mechanisms of human diseases.

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“…Advanced imaging techniques such as Light-sheet fluorescence microscopy (LSFM) of optically clear tissue samples offer a way to address these questions by visualizing large tissue volumes at cellular resolution. To date LSFM has been applied in three studies visualizing SARS-CoV-2 infection in different animal models [28][29][30]. Zaeck et al [28] used the ferret model for valuable insights into infection initiation in the epithelium of the upper respiratory tract.…”

Section: Introductionmentioning

confidence: 99%

Spatiotemporal analysis of SARS-CoV-2 infection reveals an expansive wave of monocyte-derived macrophages associated with vascular damage and virus clearance in hamster lungs

Bagato

Balkema‐Buschmann

Todt

et al. 2023

Preprint

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Factors of the innate immune response to SARS-CoV-2 in the lungs are pivotal for the ability of the host to deal with the infection. In humans, excessive macrophage infiltration is associated with disease severity. Using 3D spatiotemporal analysis of optically cleared hamster lung slices in combination with virological, immunohistochemical and RNA sequence analyses, we visualized the spread of SARS-CoV-2 through the lungs and the rapid anti-viral response in infected lung epithelial cells, followed by a wave of monocyte-derived macrophage (MDM) infiltration and virus elimination from the tissue. These SARS-CoV-2 induced innate immune processes are closely related to the onset of necrotizing inflammatory and consecutive remodelling responses in the lungs, which manifests as extensive cell death, vascular damage, thrombosis, and cell proliferation. Here we show that MDM are directly linked to virus clearance, and appear in connection with tissue injury and blood vessel damage. Rapid initiation of prothrombotic factor upregulation, tissue repair and alveolar cell proliferation results in tissue remodelling, which is followed by fibrosis development despite a decrease in inflammatory and anti-viral activities. Thus, although the hamsters are able to resolve the infection following the MDM influx and repair lung tissue integrity, longer-term alterations of the lung tissues arise as a result of concurrent tissue damage and regeneration processes.

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HYBRiD: hydrogel-reinforced DISCO for clearing mammalian bodies

Cited by 30 publications

References 31 publications

The role of somatosensory innervation of adipose tissues

The role of somatosensory innervation of adipose tissues

Nanoscale resolution imaging of the whole mouse embryos and larval zebrafish using expansion microscopy

Spatiotemporal analysis of SARS-CoV-2 infection reveals an expansive wave of monocyte-derived macrophages associated with vascular damage and virus clearance in hamster lungs

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