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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Abstract

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Introduction

The pancreas is an abdominal organ with an exocrine function in the digestive tract and an endocrine function of secreting hormones into the bloodstream. High-resolution cellular imaging of the pancreas could reveal the pathophysiology of various diseases involving the pancreas, including pancreatitis, pancreatic cancer, and diabetes mellitus1. Conventional diagnostic imaging tools such as computed tomography, magnetic resolution imaging, and ultrasonography are widely available in the clinical field1,2. However, these imaging modalities are restricted to visualizing only structural or ....

Protocol

All procedures described in this paper were conducted in accordance with the 8th edition of the Guide for the Care and Use of Laboratory Animals (2011)26 and approved by the Institutional Animal Care and Use Committee at the Korea Advanced Institute of Science and Technology (KAIST) and Seoul National University Bundang Hospital (SNUBH).

1. Preparation of the window and other materials

  1. Custom design the pancreatic intravital imaging window to secl.......

Representative Results

Intravital microscopy combined with the supporting base integrated pancreatic intravital imaging window enables longitudinal cellular level imaging of the pancreas in a mouse. This protocol with the pancreatic intravital imaging window provides long-term tissue stability that enables the acquisition of high-resolution imaging to track individual islets for up to 3 weeks. As a result, mosaic imaging for an extended field of view, three-dimensional (3D) reconstruction of z-stack imaging, and longitudinal tracking of the sa.......

Discussion

The protocol described here consists of intravital imaging of the pancreas using a novel supporting base integrated pancreatic intravital imaging window modified from an abdominal imaging window. Among the protocols described above, the first critical step is the implantation of the intravital pancreatic imaging window in the mouse. For the application of the glue in the window, it is important to apply the glue between the margin of the window and the cover glass, but not on the pancreatic tissue, as it may significantl.......

Disclosures

The authors have nothing to disclose.

Acknowledgements

This study was supported by grant No. 14-2020-002 from the SNUBH Research Fund and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2020R1F1A1058381, NRF-2020R1A2C3005694).

....

Materials

NameCompanyCatalog NumberComments
Alexa Fluor 647 Succinimidyl Esters (NHS esters)InvitrogenA20006Fluorescent probe for conjugate with antibody
BALB/C NudeOrientBioBALB/C NudeBALB/C Nude
BD Intramedic polyethylene tubingBD Biosciences427401PE10 catheter for connection with needle
C57BL/6NOrientBioC57BL/6NC57BL/6N
Cover glasses circularMarienfeld0111520Cover glass for pancreatic imaging window
FITC Dextran 2MDaMerck (Former Sigma Aldrich)FD200SFor vessel identification
IMARIS 8.1BitplaneIMARISImage processing
Intravital MicroscopyIVIM techIVM-CIntravital Microscopy
IRIS ScissorJEUNGDO BIO & PLANT CO, LTDS-1107-10This product can be replaced with the product from other company
Loctite 401Henkel401N-butyl cyanoacrylate glue
Micro Needle holderJEUNGDO BIO & PLANT CO, LTDH-1126-10This product can be replaced with the product from other company
Micro rectractorJEUNGDO BIO & PLANT CO, LTD17004-03This product can be replaced with the product from other company
MicroforcepsJEUNGDO BIO & PLANT CO, LTDF-1034This product can be replaced with the product from other company
MIP-GFPThe Jackson Laboratory006864B6.Cg-Tg(Ins1-EGFP)1Hara/J
Nylon 4-0AILEENB434Non-Absorbable Suture
Omnican N 100 30GB BRAUNFT9172220SFor Vascular Catheter, Use only Needle part
PANC-1 NucLightRedCustom-madeCustom-madeMade in laboratory
Pancreatic imaging windowGeumto EngineeringCustom orderPancreatic imaging window - custom order
PhysiosuiteKent ScientificPS-02Homeothermic temperature controller
Purified NA/LE Rat Anti-Mouse CD31BD Biosciences553708Antibody for in vivo vessel labeling
Ring ForcepsJEUNGDO BIO & PLANT CO, LTDF-1090-3This product can be replaced with the product from other company
RompunBayerRompunAnesthetic agent
TMR Dextran 65-85kDaMerck (Former Sigma Aldrich)T1162For vessel identification
Window holderGeumto EngineeringCustom orderWindow holder - custom order
ZoletilVirbacZoletil 100Anesthetic agent

References

  1. Dimastromatteo, J., Brentnall, T., Kelly, K. A. Imaging in pancreatic disease. Nature Reviews. Gastroenterology & Hepatology. 14 (2), 97-109 (2017).
  2. Cote, G. A., Smith, J., Sherman, S., Kelly, K. Technologies for imaging the normal and diseased pancreas.

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