A clearing-free protocol for imaging intact whole adipose tissue innervation in mice.
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ABSTRACT:
SUBMITTER: Willows JW
PROVIDER: S-EPMC8784403 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:ObjectiveAdipose tissue, via sympathetic and possibly sensory neurons, communicates with the central nervous system (CNS) to mediate energy homeostasis. In contrast to the sympathetic nervous system, the morphology, role and regulation of the sensory nervous system in adipose tissue are poorly characterized.Methods and resultsTaking advantage of recent progress in whole-mount three-dimensional imaging, we identified a network of calcitonin gene-related protein (CGRP)-positive sensory neurons in murine white adipose tissue (WAT). We found that adipose mammalian target of rapamycin complex 2 (mTORC2), a major component of the insulin signaling pathway, is required for arborization of sensory neurons, but not of sympathetic neurons. Time course experiments revealed that adipose mTORC2 is required for maintenance of sensory neurons. Furthermore, loss of sensory innervation in WAT coincided with systemic insulin resistance. Finally, we established that neuronal protein growth-associated protein 43 (GAP43) is a marker for sensory neurons in adipose tissue.ConclusionOur findings indicate that adipose mTORC2 is necessary for sensory innervation in WAT. In addition, our results suggest that WAT may affect whole-body energy homeostasis via sensory neurons.
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Project description:Fast Free-of-Acrylamide Clearing Tissue (FACT) is a new sodium dodecyl sulfate (SDS)-based clearing protocol for the chemical clearing and imaging of brain tissue containing transgenic or immunolabeled fluorescent proteins. In the present study, we have developed this new method and optimized multiple dimensions of the workflow, including reduced clearing time, improved efficiency of fluorescent signals without the need for electrophoretic or complex instrumentations, preservation of cytoarchitectural details, optimized confocal microscopy, and accelerated data collection. We systematically compared seven clearing protocols with the FACT protocol, using transgenic mouse brains with fluorochrome expression in microglia. Only six days were required for detecting transgene-labeled markers in a 1-mm thick brain slice from adult mice, and 14 days were required for detecting antibody-labeled markers in the same-sized tissue. Preservation of fluorescent signal was achieved by decreasing clearing time, adjusting the pH of the SDS solution, and using the appropriate temperature for tissue clearing, all of which contributed to the superiority of our method. We conclude that the FACT protocol can be successfully applied to the fluorescent imaging of mouse brain tissue, and will facilitate structural analyses and connectomics of large assemblies of cells and their networks in the context of three-dimensional organ systems.
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Project description:The spinal cord contains a diverse array of sensory and motor circuits that are essential for normal function. Spinal cord injury (SCI) permanently disrupts neural circuits through initial mechanical damage, as well as a cascade of secondary injury events that further expand the spinal cord lesion, resulting in permanent paralysis. Tissue clearing and 3D imaging have recently emerged as promising techniques to improve our understanding of the complex neural circuitry of the spinal cord and the changes that result from damage due to SCI. However, the application of this technology for studying the intact and injured spinal cord remains limited. Here, we optimized the passive CLARITY technique (PACT) to obtain gentle and efficient clearing of the murine spinal cord without the need for specialized equipment. We demonstrate that PACT clearing enables 3D imaging of multiple fluorescent labels in the spinal cord to assess molecularly defined neuronal populations, acute inflammation, long-term tissue damage, and cell transplantation. Collectively, these procedures provide a framework for expanding the utility of tissue clearing to enhance the study of spinal cord neural circuits, as well as cellular- and tissue-level changes that occur following SCI.
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Project description:The mouse eye is used to model central nervous system development, pathology, angiogenesis, tumorigenesis, and regenerative therapies. To facilitate the analysis of these processes, we developed an optimized tissue clearing and depigmentation protocol, termed InVision, that permits whole-eye fluorescent marker tissue imaging. We validated this method for the analysis of normal and degenerative retinal architecture, transgenic fluorescent reporter expression, immunostaining and three-dimensional volumetric (3DV) analysis of retinoblastoma and angiogenesis. We also used this method to characterize material transfer (MT), a recently described phenomenon of horizontal protein exchange that occurs between transplanted and recipient photoreceptors. 3D spatial distribution analysis of MT in transplanted retinas suggests that MT of cytoplasmic GFP between photoreceptors is mediated by short-range, proximity-dependent cellular interactions. The InVision protocol will allow investigators working across multiple cell biological disciplines to generate novel insights into the local cellular networks involved in cell biological processes in the eye.
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Project description:Organic solvent dibenzyl ether (DBE)-based protocols have been widely used in adipose tissue clearing. However, benzyl alcohol/benzyl benzoate (BABB)-based clearing has been shown to offer better transparency in other tissues. The addition of diphenyl ether (DPE) to BABB (BABB-D4) is often included to preserve fluorescent signals, but its effects on adipose tissue transparency and shrinkage have not been explored. Distinct adipocyte subpopulations contribute to its cellular composition and biological activity. Here, we compared clearing solvents to create an optimized clearing methodology for the study of adipocyte subpopulations. Adipose tissues were cleared with BABB, BABB-D4, and DBE, and post-clearing transparency and tissue shrinkage were measured. An optimized protocol, including BABB-D4 clearing, delipidation, and extensive immunofluorescence blocking steps, was created to examine the spatial distribution of Wt-1 positive progenitor-derived (Type-1) adipocytes in intact mesenteric fat. Both BABB and BABB-D4 lead to significantly increased tissue transparency with reduced tissue shrinkage compared to DBE-cleared adipose tissue. Type-1 adipocytes are found in a clustered distribution with predominant residence in fat associated with the ileum and colon. This paper details an optimized clearing methodology for adipose tissue with increased tissue transparency and reduced shrinkage, and therefore will be a useful tool for investigating adipose tissue biology.
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Project description:Optical clearing has emerged as a powerful tool for volume imaging. Although volume imaging with immunostaining have been successful in many protocols, yet obtaining homogeneously stained thick samples remains challenging. Here, we propose a method for label-free imaging of brain slices by enhancing the regional heterogeneity of the optical properties using the tissue clearing principle. We used FxClear, a method for delipidation of brain tissue, to retain a larger proportion of lipids at the white matter (WM). Furthermore, the embedding media affected the contrasts for the lipid-rich or extracellular matrix-rich areas, depending on their chemical properties. Thus, we tailored clearing conditions for the enhancement of the refractive indices (RIs) differences between gray and WM, or several pathological features. RI differences can be imaged using conventional light microscopy or optical coherence tomography. We propose that our protocol is simple, reliable, and flexible for label-free imaging, easily implementable to routine histology laboratory.
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Project description:Rationale: Structural remodeling or damage as a result of disease or injury is often not evenly distributed throughout a tissue but strongly depends on localization and extent of damaging stimuli. Skeletal muscle as a mechanically active organ can express signs of local or even systemic myopathic damage, necrosis, or repair. Conventionally, muscle biopsies (patients) or whole muscles (animal models) are mechanically sliced and stained to assess structural alterations histologically. Three-dimensional tissue information can be obtained by applying deep imaging modalities, e.g. multiphoton or light-sheet microscopy. Chemical clearing approaches reduce scattering, e.g. through matching refractive tissue indices, to overcome optical penetration depth limits in thick tissues. Methods: Here, we optimized a range of different clearing protocols. We find aqueous solution-based protocols employing (20-80%) 2,2'-thiodiethanol (TDE) to be advantageous over organic solvents (dibenzyl ether, cinnamate) regarding the preservation of muscle morphology, ease-of-use, hazard level, and costs. Results: Applying TDE clearing to a mouse model of local cardiotoxin (CTX)-induced muscle necrosis, a complete loss of myosin-II signals was observed in necrotic areas with little change in fibrous collagen or autofluorescence (AF) signals. The 3D aspect of myofiber integrity could be assessed, and muscle necrosis in whole muscle was quantified locally via the ratios of detected AF, forward- and backward-scattered Second Harmonic Generation (fSHG, bSHG) signals. Conclusion: TDE optical clearing is a versatile tool to study muscle architecture in conjunction with label-free multiphoton imaging in 3D in injury/myopathy models and might also be useful in studying larger biofabricated constructs in regenerative medicine.
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