Measuring Exocytosis in Endocrine Tissue Slices

Klemen, Maša Skelin; Dolenšek, Jurij; Stožer, Andraž; Rupnik, Marjan Slak

doi:10.1007/978-1-62703-676-4_7

Cited by 5 publications

(2 citation statements)

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“…This holds true for both control and disease conditions, e. g., genetic and dietary models of diabetes. Additionally, other cell types and other relevant physiological parameters could be addressed systematically, such as electrophysiological properties, coupling, exocytosis, and functional connectivity ( 51 , 79 , 106 , 107 ). Second, while BMI is often considered as a surrogate measure of body fat, it is more accurately a measure of excess weight, rather than fat.…”

Section: Discussionmentioning

confidence: 99%

Glucose-Stimulated Calcium Dynamics in Beta Cells From Male C57BL/6J, C57BL/6N, and NMRI Mice: A Comparison of Activation, Activity, and Deactivation Properties in Tissue Slices

et al. 2022

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Although mice are a very instrumental model in islet beta cell research, possible phenotypic differences between strains and substrains are largely neglected in the scientific community. In this study, we show important phenotypic differences in beta cell responses to glucose between C57BL/6J, C57BL/6N, and NMRI mice, i.e., the three most commonly used strains. High-resolution multicellular confocal imaging of beta cells in acute pancreas tissue slices was used to measure and quantitatively compare the calcium dynamics in response to a wide range of glucose concentrations. Strain- and substrain-specific features were found in all three phases of beta cell responses to glucose: a shift in the dose-response curve characterizing the delay to activation and deactivation in response to stimulus onset and termination, respectively, and distinct concentration-encoding principles during the plateau phase in terms of frequency, duration, and active time changes with increasing glucose concentrations. Our results underline the significance of carefully choosing and reporting the strain to enable comparison and increase reproducibility, emphasize the importance of analyzing a number of different beta cell physiological parameters characterizing the response to glucose, and provide a valuable standard for future studies on beta cell calcium dynamics in health and disease in tissue slices.

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Section: Discussionmentioning

confidence: 99%

Glucose-Stimulated Calcium Dynamics in Beta Cells From Male C57BL/6J, C57BL/6N, and NMRI Mice: A Comparison of Activation, Activity, and Deactivation Properties in Tissue Slices

et al. 2022

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“…Although there are several in vitro approaches for the isolation of both islets and acini from the pancreas, importantly, the greatest advantages of tissue slice preparation technique is that it does not require enzymatic digestion and the architecture and viability of the cells are retained in an intact, nearly physiological environment. It is also important to emphasize that this technique is suitable for both morphological and functional imaging, as well as for electrophysiological studies and investigating interactions between neighboring cells or between the exocrine and endocrine part of the pancreas (Marciniak et al, 2013; Klemen et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

A Novel in situ Approach to Studying Pancreatic Ducts in Mice

et al. 2019

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Introduction: The tissue slice technique offers several benefits compared to isolated cells and cell clusters that help us understand the (patho)physiology of several organs in situ . The most prominent features are preserved architecture and function, with intact homotypic and heterotypic interactions between cells in slices. In the pancreas, this technique has been utilized successfully to study acinar and endocrine islet cells. However, it has never been used to investigate ductal function. Since pancreatic ductal epithelial cells (PDECs) play an essential role in the physiology of the pancreas, our aim was to use this technique to study PDEC structure and function in situ . Materials and methods: Eight- to sixteen weeks old C57BL/6 mice were used for preparation of pancreas tissue slices. Low melting point agarose was injected into the common bile duct and the whole organ was extracted. For morphological studies, pieces of tissue were embedded in agarose and cryosectioned to obtain 15 μm thick slices. In order to visualize pancreatic ducts, (i) the Giemsa dye was added to the agarose and visualized using light microscopy or (ii) immunostaining for the cystic fibrosis transmembrane conductance regulator (CFTR) was performed. For functional characterization, agarose-embedded tissue was immediately cut to 140 μm thick tissue slices that were loaded with the cell permeant form of the Oregon Green 488 BAPTA-1 dye and used for confocal calcium imaging. Results: Giemsa staining has shown that the injected agarose reaches the head and body of the pancreas to a greater extent than the tail, without disrupting the tissue architecture. Strong CFTR expression was detected at the apical membranes of PDECs and acinar cells, whereas islet cells were completely negative for CFTR. Stimulation with chenodeoxycholic acid (CDCA, 1 mM) resulted in a robust transient increase in intracellular calcium concentration that was readily visible in >40 ductal cells per slice. Conclusion: Our results confirm that the acutely-isolated pancreas tissue slice technique is suitable for structural and functional investigation of PDECs and their relationship with other cell types, such as acini and endocrine cells in situ . In combination with different genetic, pharmacological or dietary approaches it could become a method of choice in the foreseeable future.

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