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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Published: May 6th, 2021

DOI:

10.3791/62538

1Department of Emergency Medicine, Seoul National University Bundang Hospital, 2Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, 3KI for Health Science and Technology, Korea Advanced Institute of Science and Technology

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

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.

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 ....

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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.......

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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.......

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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.......

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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).

....

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Name Company Catalog Number Comments
Alexa Fluor 647 Succinimidyl Esters (NHS esters) Invitrogen A20006 Fluorescent probe for conjugate with antibody
BALB/C Nude OrientBio BALB/C Nude BALB/C Nude
BD Intramedic polyethylene tubing BD Biosciences 427401 PE10 catheter for connection with needle
C57BL/6N OrientBio C57BL/6N C57BL/6N
Cover glasses circular Marienfeld 0111520 Cover glass for pancreatic imaging window
FITC Dextran 2MDa Merck (Former Sigma Aldrich) FD200S For vessel identification
IMARIS 8.1 Bitplane IMARIS Image processing
Intravital Microscopy IVIM tech IVM-C Intravital Microscopy
IRIS Scissor JEUNGDO BIO & PLANT CO, LTD S-1107-10 This product can be replaced with the product from other company
Loctite 401 Henkel 401 N-butyl cyanoacrylate glue
Micro Needle holder JEUNGDO BIO & PLANT CO, LTD H-1126-10 This product can be replaced with the product from other company
Micro rectractor JEUNGDO BIO & PLANT CO, LTD 17004-03 This product can be replaced with the product from other company
Microforceps JEUNGDO BIO & PLANT CO, LTD F-1034 This product can be replaced with the product from other company
MIP-GFP The Jackson Laboratory 006864 B6.Cg-Tg(Ins1-EGFP)1Hara/J
Nylon 4-0 AILEE NB434 Non-Absorbable Suture
Omnican N 100 30G B BRAUN FT9172220S For Vascular Catheter, Use only Needle part
PANC-1 NucLightRed Custom-made Custom-made Made in laboratory
Pancreatic imaging window Geumto Engineering Custom order Pancreatic imaging window - custom order
Physiosuite Kent Scientific PS-02 Homeothermic temperature controller
Purified NA/LE Rat Anti-Mouse CD31 BD Biosciences 553708 Antibody for in vivo vessel labeling
Ring Forceps JEUNGDO BIO & PLANT CO, LTD F-1090-3 This product can be replaced with the product from other company
Rompun Bayer Rompun Anesthetic agent
TMR Dextran 65-85kDa Merck (Former Sigma Aldrich) T1162 For vessel identification
Window holder Geumto Engineering Custom order Window holder - custom order
Zoletil Virbac Zoletil 100 Anesthetic agent

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