Optical Clearing and 3D Analysis Optimized for Mouse and Human Pancreata

Alvarsson, Alexandra; Jiménez-González, Maria; Li, Rosemary; Rosselot, Carolina; Tzavaras, Nikolaos; Wu, Zhuhao; Stanley, Sarah A.

doi:10.21769/bioprotoc.4103

Cited by 5 publications

(6 citation statements)

References 21 publications

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“…In the mouse, innervation was highly enriched in the endocrine pancreas with regional differences. Moreover, an increase in islet nerve density in nonobese diabetic mice, mice treated with streptozotocin, and pancreata of human donors with T2D was documented 137 . Unexpectedly, nerve contacts with beta cells were preserved in diabetic mice and humans.…”

Section: Tools For Integrated Pancreatic Analysismentioning

confidence: 95%

“…Moreover, an increase in islet nerve density in nonobese diabetic mice, mice treated with streptozotocin, and pancreata of human donors with T2D was documented. 137 Unexpectedly, nerve contacts with beta cells were preserved in diabetic mice and humans. In summary, the development of this technique has revealed dynamic changes in pancreatic innervation in the setting of diabetes.…”

Section: D Imaging Technologymentioning

confidence: 99%

“…In particular, a tissue clearing protocol based on iDisco+ has been developed to facilitate imaging endocrine cells and innervation in intact pancreata from mouse models of T1D as well as human donor pancreatic tissue from individuals with T2D. 137 The use of tissue clearing combined with light sheet microscopy and 3D analysis provided detailed quantification of the abundance of alpha cells, beta cells, and pancreatic nerve fibers, as well as their distribution and heterogeneity within both control and diabetic pancreatic tissue. In the mouse, innervation was highly enriched in the endocrine pancreas with regional differences.…”

Section: D Imaging Technologymentioning

confidence: 99%

“…Understanding the detailed anatomy of the endocrine pancreas, its innervation, and the remodeling that occurs in diabetes can provide new insights into metabolic diseases. In particular, a tissue clearing protocol based on iDisco+ has been developed to facilitate imaging endocrine cells and innervation in intact pancreata from mouse models of T1D as well as human donor pancreatic tissue from individuals with T2D 137 . The use of tissue clearing combined with light sheet microscopy and 3D analysis provided detailed quantification of the abundance of alpha cells, beta cells, and pancreatic nerve fibers, as well as their distribution and heterogeneity within both control and diabetic pancreatic tissue.…”

Section: Tools For Integrated Pancreatic Analysismentioning

confidence: 99%

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Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases

Mastracci¹,

Apte

Ámundadóttir

et al. 2022

Pancreas

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The “Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases” Workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report summarizes the workshop proceedings. The goal of the workshop was to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into 6 major themes, including (a) Pancreas Anatomy and Physiology; (b) Diabetes in the Setting of Exocrine Disease; (c) Metabolic Influences on the Exocrine Pancreas; (d) Genetic Drivers of Pancreatic Diseases; (e) Tools for Integrated Pancreatic Analysis; and (f) Implications of Exocrine-Endocrine Crosstalk. For each theme, there were multiple presentations followed by panel discussions on specific topics relevant to each area of research; these are summarized herein. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of the normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

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Section: Tools For Integrated Pancreatic Analysismentioning

confidence: 95%

Section: D Imaging Technologymentioning

confidence: 99%

Section: D Imaging Technologymentioning

confidence: 99%

Section: Tools For Integrated Pancreatic Analysismentioning

confidence: 99%

See 2 more Smart Citations

Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases

Mastracci¹,

Apte

Ámundadóttir

et al. 2022

Pancreas

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“…Fat pads were then rehydrated with a methanol/B1n buffer gradient (80%, 60%, 40%, 20%) and then washed twice in 100% B1n buffer (1h, o/n). Primary antibodies (EPCAM: 1:250; TH: 1:500) were diluted in modified PTxwH (PBS with 0.5% Trixon X-100, 0.1% Tween-20, 2 µg/ml heparin, as previously described 66 and applied for 6 days at 37 ᵒ C. Following 5 washes with modified PTxwH over 1 day with the last wash performed overnight, secondary antibodies were diluted in modified PTxwH (1:500) and samples incubated for 6 days at 37 ᵒ C. Samples were washed 5 times over 1 day in modified PTxwH at 37 ᵒ C, 5 times over 1 day in PBS at RT, embedded in 1% agarose, dehydrated with a methanol gradient in H2O (12%, 50%, 75%, 100%), washed 3 times for 1 hr in 100% methanol followed by 3 times for 1 hr in DCM, before being transferred to dibenzylether (DBE) (Sigma-Aldrich) to clear.…”

Section: Optical Clearingmentioning

confidence: 99%

Mammary ductal epithelium controls cold-induced adipocyte thermogenesis

Arneson

K³

et al. 2020

Preprint

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Almost four decades of research suggest a dynamic role of ductal epithelial cells in adipocyte adaptation in mammary gland white adipose tissue (mgWAT), but factors that mediate such communication are not known. Here, we identify a complex intercellular crosstalk in mgWAT revealed by single-cell RNA-seq (scRNA-seq) and comprehensive data analysis suggest that epithelial luminal cells during cold exposure undergo major transcriptomic changes that lead to the expression of an array of genes that encode for secreted factors involved in adipose metabolism such as Adropin (Enho), neuregulin 4 (Nrg4), angiopoietin-like 4 (Angptl4), lipocalin 2 (Lcn2), milk fat globule-EGF factor 8 (Mfge8), Insulin-like growth factor-binding protein 1 (Igfbp1), and haptoglobin (Hp). To define the mammary epithelial secretome, we coin the phrase “mammokines”. We validated our cluster annotations and cluster-specific transcriptomics using eight different adipose scRNA-seq datasets including Tabula Muris and Tabula Muris Senis. In situ mRNA hybridization and ex vivo isolated mgWAT luminal cells show highly localized expression of mammokines in mammary ducts. Trajectory inference demonstrates that cold-exposed luminal cells have similar transcriptional profiles to lactation post-involution (PI), a phase defined by reappearance and maintenance of adipocytes in the mammary gland. Concomitantly, we found that under cold exposure female mgWAT maintains more adipogenic and less thermogenic potential than male scWAT. Conditioned media from isolated mammary epithelial cells treated with isoproterenol suppressed thermogenesis in differentiated beige/brown adipocytes and treatment of beige/brown differentiated adipocyte with LCN2 suppresses thermogenesis and increases adipogenesis. Finally, we find that mice lacking LCN2 show markedly higher colddependent thermogenesis in mgWAT than controls, and reconstitution of LCN2 in the mgWAT of LCN2 knockout mice promotes inhibition of thermogenesis. These results show a previously unknown role of mammary epithelium in adipocyte metabolism and suggest a potentially redundant evolutionary role of mammokines in maintaining mgWAT adiposity during cold exposure. Our data highlight mammary gland epithelium as a highly active metabolic cell type and mammokines could have broader implications in mammary gland physiology, glucose and lipid metabolism, and tumorigenesis.

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