A clearing-free protocol for imaging intact whole adipose tissue innervation in mice

Willows, Jake W.; Blaszkiewicz, Magdalena; Townsend, Kristy L.

doi:10.1016/j.xpro.2021.101109

Cited by 9 publications

(7 citation statements)

References 4 publications

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“…Finally, tissues were mounted on glass slides and imaged. For additional details see [2] and the accompanying peer-reviewed protocol [19].…”

Section: Methodsmentioning

confidence: 99%

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The Presence of Myelinated Nerves and Schwann Cells in White Adipose Tissue: Proximity to Synaptic Vesicle Containing Nerve Terminals and Potential Role in BTBR ob/ob Demyelinating Diabetic Neuropathy

Willows

Gunsch

Paradi

et al. 2022

Preprint

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Peripheral neuropathy is a pathophysiological state of nerve degeneration and loss of tissue innervation. The most prominent cause of small fiber neuropathy is diabetes which can be demyelinating in nature, but this has not yet been explored in adipose tissue. Both demyelinating neuropathies and axonopathies implicate Schwann cells (SCs), the peripheral glial required for nerve myelination and regeneration after injury. Here, we perform a comprehensive assessment of SCs and myelination patterns of subcutaneous white adipose tissue (scWAT) nerves, including changes that occur with obesity and other imbalanced energy states in mice and humans. We found that mouse scWAT is densely innervated by both myelinated and unmyelinated sensory and sympathetic nerves. Accordingly, scWAT is home to both myelinating and non-myelinating SCs; the greater proportion of which are myelinating. Furthermore, SCs were found closely associated with synaptic vesicle-containing nerve terminals in scWAT. Obese BTBR ob/ob mice exhibit diabetic peripheral neuropathy in scWAT, and display concordant demyelination specific to small fibers, which was also associated with a decrease in the pan-SC marker Sox10 and compensatory increase in Krox20 gene expression. Together this suggests that adipose SCs may be involved in regulating the plasticity or the neuropathy of adipose tissue nerves.

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“…Finally, tissues were mounted on glass slides and imaged. For additional details see [2] and the accompanying peer-reviewed protocol [19].…”

Section: Methodsmentioning

confidence: 99%

“…Blots were visualized with enhanced chemiluminescence (ECL; Pierce) on a Syngene G:BOX. TH and SOX10 expression were normalized to Cyclophilin B, and MPZ was normalized to β-Tubulin and quantified by densitometry in Fiji [19].…”

Section: Western Blot (Wb)mentioning

confidence: 99%

The Presence of Myelinated Nerves and Schwann Cells in White Adipose Tissue: Proximity to Synaptic Vesicle Containing Nerve Terminals and Potential Role in BTBR ob/ob Demyelinating Diabetic Neuropathy

Willows

Gunsch

Paradi

et al. 2022

Preprint

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“…The iWAT was also collected for whole‐mount processing as described previously 16 . The samples were incubated with primary antibodies against S100 (1:200 dilution, Signalway Antibody), CGRP (1:1000 dilution, Abcam), PGP9.5 (1:1000 dilution, Abcam), and TRPV1 (1:1000 dilution, Abcam).…”

Section: Methodsmentioning

confidence: 99%

“…1:1000 dilution, Abcam), recombinant uncoupling protein 1 (UCP1) The iWAT was also collected for whole-mount processing as described previously. 16 The samples were incubated with primary antibodies against S100 (1:200 dilution, Signalway Antibody), CGRP…”

Section: Western Blottingmentioning

confidence: 99%

Remodelling the inguinal adipose sensory system to switch on the furnace: Electroacupuncture stimulation induces brown adipose thermogenesis

Lu,

Yu,

Yang

et al. 2024

Diabetes Obesity Metabolism

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Brown and white adipose tissue mediate thermogenesis through the thermogenetic centre of the brain, but safe methods for activating thermogensis and knowledge of the associated molecular mechanisms are lacking. We investigated body surface electroacupuncture stimulation (ES) at ST25 (targeted at the abdomen) induction of brown adipose thermogenesis and the neural mechanism of this process. Inguinal white adipose tissue (iWAT) and interscapular brown adipose tissue (iBAT) were collected and the thermogenic protein expression levels were measured to evaluate iBAT thermogenesis capacity. The thermogenic centre activating region and sympathetic outflow were evaluated based on neural electrical activity and c‐fos expression levels. iWAT sensory axon plasticity was analysed with whole‐mount adipose tissue imaging. ES activated the sympathetic nerves in iBAT and the c‐fos‐positive cells induced sympathetic outflow activation to the iBAT from the medial preoptic area (MPA), the dorsomedial hypothalamus (DM) and the raphe pallidus nucleus (RPA). iWAT denervation mice exhibited decreased c‐fos‐positive cells in the DM and RPA, and lower recombinant uncoupling orotein 1 peroxisome proliferator‐activated receptor, β3‐adrenergic receptor, and tyrosine hydroxylase expression. Remodelling the iWAT sensory axons recovered the signal from the MPA to the RPA and induced iBAT thermogenesis. The sympathetic denervation attenuated sensory nerve density. ES induced sympathetic outflow from the thermogenetic centres to iBAT, which mediated thermogenesis. iWAT sensory axon remodelling induced the MPA–DM–RPA–iBAT thermogenesis pathway.

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“…Intact scWAT depots were excised from mice, fixed overnight in 2% PFA at 4°C, and processed following the Z‐depth reduction method as described previously (Willows et al, 2021 ) with accompanying protocol (Willows, Blaszkiewicz, & Townsend, 2022 ). Tissues were stained with the sympathetic nerve marker tyrosine hydroxylase (TH, 1:200, EMD Millipore, AB152), secondary antibody (goat anti‐rabbit IgG Alexa Fluor Plus 594, 1:1000, ThermoFisher Cat#A32740), and the vascular marker Isolectin IB 4 conjugated to Alexa Fluor 488 (2.5 μg/ml, Thermofisher, Cat# I21411).…”

Section: Methodsmentioning

confidence: 99%

Age‐related changes to adipose tissue and peripheral neuropathy in genetically diverse HET3 mice differ by sex and are not mitigated by rapamycin longevity treatment

et al. 2023

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Neural communication between the brain and adipose tissues regulates energy expenditure and metabolism through modulation of adipose tissue functions. We have recently demonstrated that under pathophysiological conditions (obesity, diabetes, and aging), total subcutaneous white adipose tissue (scWAT) innervation is decreased (‘adipose neuropathy’). With advanced age in the C57BL/6J mouse, small fiber peripheral nerve endings in adipose tissue die back, resulting in reduced contact with adipose‐resident blood vessels and other cells. This vascular neuropathy and parenchymal neuropathy together likely pose a physiological challenge for tissue function. In the current work, we used the genetically diverse HET3 mouse model to investigate the incidence of peripheral neuropathy and adipose tissue dysregulation across several ages in both male and female mice. We also investigated the anti‐aging treatment rapamycin, an mTOR inhibitor, as a means to prevent or reduce adipose neuropathy. We found that HET3 mice displayed a reduced neuropathy phenotype compared to inbred C56BL/6 J mice, indicating genetic contributions to this aging phenotype. Compared to female HET3 mice, male HET3 mice had worse neuropathic phenotypes by 62 weeks of age. Female HET3 mice appeared to have increased protection from neuropathy until advanced age (126 weeks), after reproductive senescence. We found that rapamycin overall had little impact on neuropathy measures, and actually worsened adipose tissue inflammation and fibrosis. Despite its success as a longevity treatment in mice, higher doses and longer delivery paradigms for rapamycin may lead to a disconnect between life span and beneficial health outcomes.

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A clearing-free protocol for imaging intact whole adipose tissue innervation in mice

Cited by 9 publications

References 4 publications

The Presence of Myelinated Nerves and Schwann Cells in White Adipose Tissue: Proximity to Synaptic Vesicle Containing Nerve Terminals and Potential Role in BTBR ob/ob Demyelinating Diabetic Neuropathy

The Presence of Myelinated Nerves and Schwann Cells in White Adipose Tissue: Proximity to Synaptic Vesicle Containing Nerve Terminals and Potential Role in BTBR ob/ob Demyelinating Diabetic Neuropathy

Remodelling the inguinal adipose sensory system to switch on the furnace: Electroacupuncture stimulation induces brown adipose thermogenesis

Age‐related changes to adipose tissue and peripheral neuropathy in genetically diverse HET3 mice differ by sex and are not mitigated by rapamycin longevity treatment

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