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A Method for Identifying Mouse Pancreatic Ducts.

ABSTRACT: Proper identification of pancreatic ducts is a major challenge for researchers performing partial duct ligation (PDL), because pancreatic ducts, which are covered with acinar cells, are translucent and thin. Although damage to pancreatic ducts may activate quiescent ductal stem cells, which may allow further investigation into ductal stem cells for therapeutic use, there is a lack of effective techniques to visualize pancreatic ducts. In this study, we report a new method for identifying pancreatic ducts. First, we aimed to visualize pancreatic ducts using black, waterproof fountain pen ink. We injected the ink into pancreatic ducts through the bile duct. The flow of ink was observed in pancreatic ducts, revealing their precise architecture. Next, to visualize pancreatic ducts in live animals, we injected fluorescein-labeled bile acid, cholyl-lysyl-fluorescein into the mouse tail vein. The fluorescent probe clearly marked not only the bile duct but also pancreatic ducts when observed with a fluorescent microscope. To confirm whether the pancreatic duct labeling was successful, we performed PDL on Neurogenin3 (Ngn3)-GFP transgenic mice. As a result, acinar tissue is lost. PDL tail pancreas becomes translucent almost completely devoid of acinar cells. Furthermore, strong activation of Ngn3 expression was observed in the ligated part of the adult mouse pancreas at 7 days after PDL.

SUBMITTER: Nakajima C

PROVIDER: S-EPMC6088256 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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A Method for Identifying Mouse Pancreatic Ducts.

Nakajima Chiemi C Kamimoto Kenji K Miyajima Katsuhiro K Matsumoto Masahito M Okazaki Yasushi Y Kobayashi-Hattori Kazuo K Shimizu Makoto M Yamane Takumi T Oishi Yuichi Y Iwatsuki Ken K

Tissue engineering. Part C, Methods 20180801 8

Proper identification of pancreatic ducts is a major challenge for researchers performing partial duct ligation (PDL), because pancreatic ducts, which are covered with acinar cells, are translucent and thin. Although damage to pancreatic ducts may activate quiescent ductal stem cells, which may allow further investigation into ductal stem cells for therapeutic use, there is a lack of effective techniques to visualize pancreatic ducts. In this study, we report a new method for identifying pancrea ...[more]

PMID: 29993334

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Project description:Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

Project description: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.

Project description:BackgroundLittle is known about what factors predict better outcomes for patients who undergo minimally invasive pancreaticoduodenectomy (MIPD) versus open pancreaticoduodenectomy (OPD). We hypothesized that patients with dilated pancreatic ducts have improved postoperative outcomes with MIPD compared to OPD.MethodsAll patients undergoing pancreaticoduodenectomy were prospectively followed over a time period of 47 months, and perioperative and pathologic covariates and outcomes were compared. Ideal outcome after PD was defined as follows: (1) no complications, (2) postoperative length of stay < 7 days, and (3) negative (R0) margins on pathology. Patients with dilated pancreatic ducts (≥ 3 mm) who underwent MIPD were 1:3 propensity score-matched to patients with dilated ducts who underwent OPD and outcomes compared. Likewise, patients with non-dilated pancreatic ducts (< 3 mm) who underwent MIPD were 1:3 propensity score-matched to patients with non-dilated ducts who underwent OPD and outcomes were compared.Results371 patients underwent PD-74 (19.9%) MIPD and 297 (80.1%) underwent OPD. Overall, patients who underwent MIPD had significantly less intraoperative blood loss. After 1:3 propensity score matching, patients with dilated pancreatic ducts who underwent MIPD (n = 45) had significantly lower overall complication and 90-day readmission rates compared to matched OPD patients (n = 135) with dilated ducts. Patients with dilated duct who underwent MIPD were more likely to have an ideal outcome than patients with OPD (29 vs 15%, p = 0.035). There were no significant differences in postoperative outcomes among propensity score-matched patients with non-dilated pancreatic ducts who underwent MIPD (n = 29) compared to matched patients undergoing OPD (n = 87) with non-dilated ducts.ConclusionsMIPD is safe with comparable perioperative outcomes to OPD. Patients with pancreatic ducts ≥ 3 mm appear to derive the most benefit from MIPD in terms of fewer complications, lower readmission rates, and higher likelihood of ideal outcome.

Dataset Information

A Method for Identifying Mouse Pancreatic Ducts.

Publications

A Method for Identifying Mouse Pancreatic Ducts.

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