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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 ?m 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 ?m 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.

SUBMITTER: Gal E

PROVIDER: S-EPMC6668154 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A Novel in situ Approach to Studying Pancreatic Ducts in Mice.

Gál Eleonóra E Dolenšek Jurij J Stožer Andraž A Pohorec Viljem V Ébert Attila A Venglovecz Viktória V

Frontiers in physiology 20190724

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 ducta ...[more]

PMID: 31396104

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Project description:Background & aimsPancreatitis after endoscopic retrograde cholangiopancreatography (PEP) is thought to be provoked by pancreatic ductal hypertension, via unknown mechanisms. We investigated the effects of hydrostatic pressures on the development of pancreatitis in mice.MethodsWe performed studies with Swiss Webster mice, B6129 mice (controls), and B6129 mice with disruption of the protein phosphatase 3, catalytic subunit, βisoform gene (Cnab-/- mice). Acute pancreatitis was induced in mice by retrograde biliopancreatic ductal or intraductal infusion of saline with a constant hydrostatic pressure while the proximal common bile duct was clamped -these mice were used as a model of PEP. Some mice were given pancreatic infusions of adeno-associated virus 6-nuclear factor of activated T-cells-luciferase to monitor calcineurin activity or the calcineurin inhibitor FK506. Blood samples and pancreas were collected at 6 and 24 hours and analyzed by enzyme-linked immunosorbent assay, histology, immunohistochemistry, or fluorescence microscopy. Ca2+ signaling and mitochondrial permeability were measured in pancreatic acinar cells isolated 15 minutes after PEP induction. Ca2+-activated phosphatase calcineurin within the pancreas was tracked in vivo over 24 hours.ResultsIntraductal pressures of up to 130 mm Hg were observed in the previously reported model of PEP; we found that application of hydrostatic pressures of 100 and 150 mm Hg for 10 minutes consistently induced pancreatitis. Pancreatic tissues had markers of inflammation (increased levels of interleukin [IL] 6, IL1B, and tumor necrosis factor), activation of signal transducer and activator of transcription 3, increased serum amylase and IL6, and loss of tight junction integrity. Transiently high pressures dysregulated Ca2+ processing (reduced Ca2+ oscillations and an increased peak plateau Ca2+ signal) and reduced the mitochondrial membrane potential. We observed activation of pancreatic calcineurin in the pancreas in mice. Cnab-/- mice, which lack the catalytic subunit of calcineurin, and mice given FK506 did not develop pressure-induced pancreatic inflammation, edema, or loss of tight junction integrity.ConclusionsTransient high ductal pressure produces pancreatic inflammation and loss of tight junction integrity in a mouse model of PEP. These processes require calcineurin signaling. Calcineurin inhibitors might be used to prevent acute pancreatitis that results from obstruction.

Project description:Cost-effective production of lignocellulosic biofuel requires efficient breakdown of cell walls present in plant biomass to retrieve the wall polysaccharides for fermentation. In-depth knowledge of plant cell wall composition is therefore essential for improving the fuel production process. The precise spatial three-dimensional (3D) organization of cellulose, hemicellulose, pectin and lignin within plant cell walls remains unclear to date since the microscopy techniques used so far have been limited to two-dimensional, topographic or low-resolution imaging, or required isolation or chemical extraction of the cell walls. In this paper we demonstrate that by cryo-immobilizing fresh tissue, then either cryo-sectioning or freeze-substituting and resin embedding, followed by cryo- or room temperature (RT) electron tomography, respectively, we can visualize previously unseen details of plant cell wall architecture in 3D, at macromolecular resolution (∼ 2 nm), and in near-native state. Qualitative and quantitative analyses showed that wall organization of cryo-immobilized samples were preserved remarkably better than conventionally prepared samples that suffer substantial extraction. Lignin-less primary cell walls were well preserved in both self-pressurized rapidly frozen (SPRF), cryo-sectioned samples as well as high-pressure frozen, freeze-substituted and resin embedded (HPF-FS-resin) samples. Lignin-rich secondary cell walls appeared featureless in HPF-FS-resin sections presumably due to poor stain penetration, but their macromolecular features could be visualized in unprecedented details in our cryo-sections. While cryo-tomography of vitreous tissue sections is currently proving to be instrumental in developing 3D models of lignin-rich secondary cell walls, here we confirm that the technically easier method of RT-tomography of HPF-FS-resin sections could be used immediately for routine study of low-lignin cell walls. As a proof of principle, we characterized the primary cell walls of a mutant (cob-6) and wild type Arabidopsis hypocotyl parenchyma cells by RT-tomography of HPF-FS-resin sections, and detected a small but significant difference in spatial organization of cellulose microfibrils in the mutant walls.

Project description:Our earlier studies demonstrated that cysteine414- (zinc-binding site of mCRY1-) alanine mutant mCRY1 transgenic mice (Tg mice) exhibit diabetes characterized by the reduction of β-cell proliferation and by β-cell dysfunction, presumably caused by senescence-associated secretory phenotype- (SASP-) like characters of islets. Earlier studies also showed that atypical duct-like structures in the pancreas developed age-dependently in Tg mice. Numerous reports have described that karyopherin alpha 2 (KPNA2) is highly expressed in cancers of different kinds. However, details of the expression of KPNA2 in pancreatic ductal atypia and in normal pancreatic tissues remain unclear. To assess the feature of the expression of KPNA2 in the development of the ductal atypia and islet architectures, we scrutinized the pancreas of Tg mice histopathologically. Results showed that considerable expression of KPNA2 was observed in pancreatic β-cells, suggesting its importance in maintaining the functions of β-cells. In mature stages, the level of KPNA2 expression was lower in islets of Tg mice than in wild-type controls. At 4 weeks, the expression levels of KPNA2 in islets of Tg mice were the same as those in wild-type controls. These results suggest that the reduction of KPNA2 might contribute to β-cell dysfunction in mature Tg mice. Additionally, the formation of mucin-producing intra-islet ducts, islet fibrosis, and massive T cell recruitment to the islet occurred in aged Tg mice. In exocrine areas, primary pancreatic intraepithelial neoplasias (PanINs) with mucinous pancreatic duct glands (PDGs) emerged in aged Tg mice. High expression of KPNA2 was observed in the ductal atypia. By contrast, KPNA2 expression in normal ducts was quite low. Thus, upregulation of KPNA2 seemed to be correlated with progression of the degree of atypia in pancreatic ductal cells. The SASP-like microenvironment inside islets might play stimulatory roles in the formation of ductal metaplasia inside islets and in islet fibrosis in Tg mice.

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A Novel in situ Approach to Studying Pancreatic Ducts in Mice.

Publications

A Novel <i>in situ</i> Approach to Studying Pancreatic Ducts in Mice.

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