Calcium imaging in intact mouse acinar cells in acute pancreas tissue slices

Abstract

The physiology and pathophysiology of the exocrine pancreas are in close connection to changes in intra-cellular Ca2+ concentration. Most of our knowledge is based on in vitro experiments on acinar cells or acini enzymatically isolated from their surroundings, which can alter their structure, physiology, and limit our understanding. Due to these limitations, the acute pancreas tissue slice technique was introduced almost two decades ago as a complementary approach to assess the morphology and physiology of both the endocrine and exocrine pancreas in a more conserved in situ setting. In this study, we extend previous work to functional multicellular calcium imaging on acinar cells in tissue slices. The viability and morphological characteristics of acinar cells within the tissue slice were assessed using the LIVE/DEAD assay, transmission electron microscopy, and immunofluorescence imaging. The main aim of our study was to characterize the responses of acinar cells to stimulation with acetylcholine and compare them with responses to cerulein in pancreatic tissue slices, with special emphasis on inter-cellular and inter-acinar heterogeneity and coupling. To this end, calcium imaging was performed employing confocal microscopy during stimulation with a wide range of acetylcholine concentrations and selected concentrations of cerulein. We show that various calcium oscillation parameters depend monotonically on the stimulus concentration and that the activity is rather well synchronized within acini, but not between acini. The acute pancreas tissue slice represents a viable and reliable experimental approach for the evaluation of both intra- and inter-cellular signaling characteristics of acinar cell calcium dynamics. It can be utilized to assess many cells simultaneously with a high spatiotemporal resolution, thus providing an efficient and high-yield platform for future studies of normal acinar cell biology, pathophysiology, and screening pharmacological substances.

Ca2+ signals in pancreatic acinar cells in response to physiological stimulation in vivo

“…These characteristics have been thoroughly defined in tissue derived from both rodents and humans (Murphy et al., 2008; Williams et al., 1978). However, exclusively the experiments were performed in dissociated single cells, isolated acini or more recently in organotypic lobule preparations (Gryshchenko et al., 2021; Liang et al., 2017; Marolt et al., 2022; Won et al., 2011) . The impact of these noted caveats associated with ex vivo studies and the spatiotemporal properties of Ca 2+ signals were not known.…”

From Isles of Königsberg to Islets of Langerhans: Examining the Function of the Endocrine Pancreas Through Network Science

“…Multicellular recordings of membrane potential or intracellular calcium concentration are typically used as input signals. Network principles have thus been used to elucidate the connectivity patterns in neurons ( 40 – 44 ), astrocytes ( 45 ), pituitary endocrine cells ( 46 , 47 ), lens epithelium cells ( 48 , 49 ), hepatocytes ( 50 ), mammary epithelium cells ( 51 ), endothelial cells ( 52 ), and pancreatic acinar cells ( 53 ). Network analyses were introduced to the islet community a decade ago and are increasingly seen as a valuable tool to examine and quantify collective activity patterns in the pancreatic islets of Langerhans ( 18 , 54 – 69 ).…”

Western diet-induced ultrastructural changes in mouse pancreatic acinar cells

“…In our present study, we have established a new preclinical methodological pipeline for studying changes in acinar cell structure during the development of diet-induced obesity and T2DM and found that early during the development of obesity and progression from compensated to decompensated diabetes ( Paschen et al, 2019 ), acinar cells already show striking ultrastructural signs of functional adaptation to increased workload and dysfunction affecting intracellular organelles in a rather non-selective manner and before changes in tissue or plasma levels and activity of amylase become apparent. Our approach is methodologically compatible with functional studies of acinar cells ( Marolt et al, 2022 ), with morphological and functional studies of other cell types in the exocrine and endocrine parts of the pancreas ( Marciniak et al, 2014 ; Stožer et al, 2021 ), as well as with more quantitative image-analysis approaches ( Klemen et al, 2022 ). These should be combined in the future to obtain more answers regarding the relationship between structural and functional changes in endocrine, ductal, and acinar cells during disease progression, regarding the relationship between the various cell types, as well as regarding the reversibility of these changes.…”