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Visualization and analysis of whole depot adipose tissue neural innervation.

ABSTRACT: Little is known about the diversity and function of adipose tissue nerves, due in part to the inability to effectively visualize the tissue's diverse nerve subtypes and the patterns of innervation across an intact depot. The tools to image and quantify adipose tissue innervation are currently limited. Here, we present a method of tissue processing that decreases tissue thickness in the z-axis while leaving cells intact for subsequent immunostaining. This was combined with autofluorescence quenching techniques to permit intact whole tissues to be mounted on slides and imaged by confocal microscopy, with a complementary means to perform whole tissue neurite density quantification after capture of tiled z-stack images. Additionally, we demonstrate how to visualize nerve terminals (the neuro-adipose nexus) in intact blocks of adipose tissue without z-depth reduction. We have included examples of data demonstrating nerve subtypes, neurovascular interactions, label-free imaging of collagen, and nerve bundle digital cross-sections.

SUBMITTER: Willows JW

PROVIDER: S-EPMC8479257 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Visualization and analysis of whole depot adipose tissue neural innervation.

Willows Jake W JW Blaszkiewicz Magdalena M Lamore Amy A Borer Samuel S Dubois Amanda L AL Garner Emma E Breeding William P WP Tilbury Karissa B KB Khalil Andre A Townsend Kristy L KL

iScience 20210911 10

Little is known about the diversity and function of adipose tissue nerves, due in part to the inability to effectively visualize the tissue's diverse nerve subtypes and the patterns of innervation across an intact depot. The tools to image and quantify adipose tissue innervation are currently limited. Here, we present a method of tissue processing that decreases tissue thickness in the z-axis while leaving cells intact for subsequent immunostaining. This was combined with autofluorescence quench ...[more]

PMID: 34622172

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Project description:Peripheral nerves allow a bidirectional communication between brain and adipose tissues, and many studies have clearly demonstrated that a loss of the adipose nerve supply results in tissue dysfunction and metabolic dysregulation. Neuroimmune cells closely associate with nerves in many tissues, including subcutaneous white adipose tissue (scWAT). However, in scWAT, their functions beyond degrading norepinephrine in an obese state remain largely unexplored. We previously reported that a myeloid-lineage knockout (KO) of brain-derived neurotrophic factor (BDNF) resulted in decreased innervation of scWAT, accompanied by an inability to brown scWAT after cold stimulation, and increased adiposity after a high-fat diet. These data underscored that adipose tissue neuroimmune cells support the peripheral nerve supply to adipose and impact the tissue's metabolic functions. We also reported that a subset of myeloid-lineage monocyte/macrophages (Ly6c+CCR2+Cx3cr1+) is recruited to scWAT in response to cold, a process known to increase neurite density in adipose and promote metabolically healthy processes. These cold-induced neuroimmune cells (CINCs) also expressed BDNF. Here we performed RNAseq on CINCs from cold-exposed and room temperature-housed mice, which revealed a striking and coordinated differential expression of numerous genes involved in neuronal function, including neurotrophin signaling and axonal guidance, further supporting that CINCs fulfill a nerve-supporting role in adipose. The increased expression of leukocyte transendothelial migration genes in cold-stimulated CINCs also confirms prior evidence that they are recruited to scWAT and are not tissue resident. We now provide whole-depot imaging of scWAT from LysM-BDNF KO mice, revealing a striking reduction of innervation across the depot fitting with their reduced energy expenditure phenotype. By contrast, Cx3cr1-BDNF KO mice (a macrophage subset of LysM+ cells) exhibited increased thermogenesis and energy expenditure, with compensatory increased food intake and no change in adiposity or body weight. While these KO mice also exhibit a significantly reduced innervation of scWAT, especially around the subiliac lymph node, they displayed an increase in small fiber sympathetic neurite branching, which may underlie their increased thermogenesis. We propose a homeostatic role of scWAT myeloid-lineage neuroimmune cells together in nerve maintenance and neuro-adipose regulation of energy expenditure.

Project description:BackgroundLow-frequency electroacupuncture (EA) has been shown to ameliorate obesity and reproductive dysfunctions in patients with polycystic ovary syndrome (PCOS), and further explorations in PCOS-like rats showed that EA could affect white adipose tissue. However, the function and neuromodulation of brown adipose tissue (BAT) in PCOS and after EA treatment have remained unknown. The present study focused on the role of BAT in PCOS-like rats and its relationship with EA and characterized the three-dimensional (3D) innervation of BAT associated with activation molecules.MethodsFemale rats (21 days old) were implanted with dihydrotestosterone or fed with a high fat diet to establish PCOS-like and obesity models, respectively, and then EA treatment at "Guilai" (ST 29) and "Sanyinjiao" (SP 6) was carried out for 4 weeks. In the present study, morphological analysis, 3D imaging, molecular biology, and other experimental techniques were used to study the sympathetic nerves and activity of BAT.ResultsPCOS-like rats showed both obvious weight gain and reproductive dysfunction, similar to what was seen in obese rats except for the absence of reproductive dysfunction. The body weight gain was mainly caused by an increase in white adipose tissue, and there was an abnormal decrease in BAT. Because both the lipid metabolism and reproductive disorders could be improved with bilateral EA at "Guilai" (ST 29) and "Sanyinjiao" (SP 6), especially the restoration of BAT, we further investigated the neuromodulation and inflammation in BAT and identified the sympathetic marker tyrosine hydroxylase as one of the key factors of sympathetic nerves. Modified adipo-clearing technology and 3D high-resolution imaging showed that crooked or dispersed sympathetic nerves, but not the twisted vasculature, were reconstructed and associated with the activation of BAT and are likely to be the functional target for EA treatment.ConclusionOur study highlights the significant role of BAT and its sympathetic innervations in PCOS and in EA therapy.

Project description:Adipose tissue (AT) is an endocrine organ with a central role on whole-body energy metabolism and development of metabolic diseases. Single-cell and single-nuclei RNA sequencing (scRNA-seq and snRNA-seq, respectively) analyses in mice and human AT have revealed vast cell heterogeneity and functionally distinct subtypes that are potential therapeutic targets to metabolic disease. In periparturient dairy cows, AT goes through intensive remodeling and its dysfunction is associated with metabolic disease pathogenesis and decreased productive performance. The contributions of depot-specific cells and subtypes to the development of diseases in dairy cows remain to be studied. Our objective was to elucidate differences in cellular diversity of visceral (VAT) and subcutaneous (SAT) AT in dairy cows at the single-nuclei level. We collected matched SAT and VAT samples from three dairy cows and performed snRNA-seq analysis. We identified distinct cell types including four major mature adipocytes (AD) and three stem and progenitor cells (ASPC) subtypes, along with endothelial cells (EC), mesothelial cells (ME), immune cells, and pericytes and smooth muscle cells. All major cell types were present in both SAT and VAT, although a strong VAT-specificity was observed for ME, which were basically absent in SAT. One ASPC subtype was defined as adipogenic (PPARG+) while the other two had a fibro-adipogenic profile (PDGFRA+). We identified vascular and lymphatic EC subtypes, and different immune cell types and subtypes in both SAT and VAT, i.e., macrophages, monocytes, T cells, and natural killer cells. Not only did VAT show a greater proportion of immune cells, but these visceral immune cells had greater activation of pathways related to immune and inflammatory response, and complement cascade in comparison with SAT. There was a substantial contrast between depots for gene expression of complement cascade, which were greatly expressed by VAT cell subtypes compared to SAT, indicating a pro-inflammatory profile in VAT. Unprecedently, our study demonstrated cell-type and depot-specific heterogeneity in VAT and SAT of dairy cows. A better understanding of depot-specific molecular and cellular features of SAT and VAT will aid in the development of AT-targeted strategies to prevent and treat metabolic disease in dairy cows, especially during the periparturient period.

Dataset Information

Visualization and analysis of whole depot adipose tissue neural innervation.

Publications

Visualization and analysis of whole depot adipose tissue neural innervation.

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