A Novel in situ Approach to Studying Pancreatic Ducts in Mice

Abstract

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.

A Novel in situ Approach to Studying Detrusor Smooth Muscle Cells in Mice

“…3). Interestingly, the slight differences in loading or basal calcium levels in different cells actually helped identify individual cells from one another, especially in regions where they were more closely packed together, similarly to tissue preparations from other organs [15][16][17][18][19] .…”

Experimental Acute Pancreatitis Models: History, Current Status, and Role in Translational Research

“…Similarly, the protocol of pancreatic duct isolation was initially developed in the 80's and it is also based on enzymatic digestion of the tissue (Argent et al, 1986). However, this enzymatic processing could affect the physiology of ductal epithelia and thus may require additional steps facilitating ductal regeneration, which extends and complicates the entire procedure (Gal et al, 2019). In order to address this limitation, Gal et al recently proposed a novel in situ approach that aims to preserve the physiological environment and the ductal structure Lampel and Kern, 1977;Niederau et al, 1985;Saluja et al, 2007 L-arginine Tashiro et al, 2001;Hegyi et al, 2004;Dawra et al, 2007 *Lieber-DeCarli diet plus other stimuli (rats/mice) Pandol et al, 2003;Perides et al, 2005;Lugea et al, 2010 Ethonal Schmidt et al, 1992a,b;Laukkarinen et al, 2007;Perides et al, 2010 PD ligation (rats/mice) or CPBD ligation (opossum) Lerch et al, 1993;Mooren et al, 2003;Meyerholz and Samuel, 2007;Samuel et al, 2010 Post-ERCP pancreatitis (rats/mice) He et al, 2003;Hackert et al, 2004;Xiong et al, 2007;Noble et al, 2008;Jin et al, 2015;Radadiya et al, 2019 *Lieber-DeCarli diet is to mix alcohol into a liquid diet (supplemented with calories); FFA: fatty acid acid; PD, pancreatic duct; CPBD, common pancreatic biliary duct; ERCP, endoscopic retrograde cholangiopancreatography.…”

“…Frontiers in Physiology | www.frontiersin.org of the mouse pancreatic tissue (Gal et al, 2019). By injecting (post-mortem) low melting point agarose into the pancreatic duct and a subsequent cooling of the whole organ, agarose settles in the pancreatic ductal system and helps to maintain its three dimensional architecture.…”

Formation and Function of Mammalian Epithelia: Roles for Mechanosensitive PIEZO1 Ion Channels

“…The mature exocrine pancreas consists of two main epithelial cell types: pancreatic ductal epithelial cells and acinar cells, with acinar cells representing the predominant cell type (>90%) (Romac et al, 2018; Gál et al, 2019). Unique to the pancreas, is its exquisite sensitivity to touch (Romac et al, 2018).…”

“…Furthermore, damage caused by high intraductal pressure is reduced in the presence of GsMTx4 as well as by targeted deletion of Piezo1 in acinar cells (Romac et al, 2018). While levels of Piezo1 mRNA specifically in pancreatic ductal epithelial cells have not been reported, perfusion of mouse pancreas tissue slices with the bile acid, chenodeoxycholic acid, is associated with transient calcium increases in these cells (Gál et al, 2019). Given its role in the transport of pancreatic secretions and this tissue’s sensitivity to physical obstruction and compression (Hegyi and Petersen, 2013; Romac et al, 2018), it would be valuable to know whether PIEZO1 is also active in epithelial cells lining the pancreatic duct.…”