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Abstract

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Protocol

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Medicine

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Published: May 10th, 2021

DOI:

10.3791/62530

1Department of Surgery, Medical University of South Carolina, 2Georgetown University, 3Ralph H. Johnson Veterans Affairs Medical Center
* These authors contributed equally

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Intraportal islet transplantation (IIT) via the portal vein is the most commonly used method for human islet transplantation in clinical settings. The mouse IIT model offers a great opportunity to study islet transplantation and test promising interventional approaches that can enhance the efficacy of islet transplantation1. IIT was first described in the 1970s and used by several groups1,2,3,4,5. It regained popularity after the breakthrough in human islet transplantation in the yea....

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All procedures were conducted with the approval of the Institutional Animal Care and Use Committees at the Medical University of South Carolina and the Ralph H Johnson Medical Center in Charleston.

1. Diabetes induction using streptozotocin (STZ)

  1. Recipient mice preparation:
    1. Weigh all mice individually.
    2. Check blood glucose levels from a tail vein blood sample using a glucometer.
  2. STZ dose determination for three different scenarios:

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We performed syngeneic and xenogeneic islet transplantations via the portal vein. Islet graft function was observed in a dose-dependent manner in both islet transplantation models. In the syngeneic islet transplantation model using C57BL/6 mice, transplantation of 250 islets led to transitory normoglycemia before mice returned to hyperglycemia. Mice receiving 500 islets reached and maintained normoglycemia beyond 30 days after transplantation (Figure 2A). Mice in both groups showed increased.......

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In this study, two improved procedures that can prevent bleeding and may reduce mouse mortality during mouse IIT have been demonstrated. This study enables researchers to visualize the islet transplantation model that is unique in studying the instant blood mediated inflammatory response after transplantation. The IIT model is a distinctive model for studying islet cell survival and hepatic ischemic injuries in response to islet transplantation19. Here, we refined the procedure based on previous s.......

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This study was supported by the Department of Veterans Affairs (VA-ORD BLR&D Merit I01BX004536), and the National Institute of Health grants # 1R01DK105183, DK120394, DK118529, to HW. We would like to thank you Mr. Michael Lee and Ms. Lindsay Swaby for language editing

....

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Name Company Catalog Number Comments
10% Neutral buffered formalin v/v Fisher Scientific 23426796
1 mL Syringe with needle AHS AH01T
20 mL Syringe BD 301031
25G x 5/8" hypodermic needles BD 305122
Alcohol prep pads, sterile Fisher Scientific 22-363-750
Animal Anesthesia system VetEquip, Inc. 901806
Buprenorphine hydrochloride, injection Par Sterile Products, LLC NDC 42023-179-05
Centrifuge tubes, 15 mL Fisher Scientific 0553859A
CMRL-1066 Corning 15110CV
DMEM Corning 10013CV
Ethanol, absolute (200 proof), molecular biology grade Fisher Scientific BP2818500
Extra fine Micro Dissecting scissors 4” straight sharp Roboz Surgical Instrument Co. RS-5882
Fetal bovine serum (FBS) Corning 35011CV
FreeStyle  Glucose meter Abbott Lite
FreeStyle Blood Glucose test strips Abbott Lite
Gelfoam (absorbable gelatin sponge, USP) Pharmacia & Upjohn Company 34201
Graefe forceps 4” extra delicate tip Roboz Surgical Instrument Co. RS-5136
Heated pad Amazon B07HMKMBKM
Hegar-Baumgartner Needle Holder 5.25” Roboz Surgical Instrument Co. RS-7850
Insulin syringe with 27-gauge needle BD 879588
Iodine prep pads Fisher Scientific 19-027048
Isoflurane Piramal Critical Care NDC 66794-017-25
Penicillin/streptomycin (P/S) HyClone SV30010
Polypropylene Suture 4-0 Med-Vet International MV-8683
Polypropylene Suture 5-0 Med-Vet International MV-8661
Sodium chloride, 0.9% intravenous solution VWR 2B1322Q
Streptozocin (STZ) Sigma S0130
Surgical drape, sterile Med-Vet International DR1826
Tissue Cassette Fisher Scientific 22-272416

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