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In This Article

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

Summary

The protocol describes a straightforward method of resectioning an intact mouse liver for metabolic studies through portal vein perfusion.

Abstract

Metabolic diseases such as diabetes, pre-diabetes, non-alcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH) are becoming increasingly common. Ex vivo liver perfusions allow for a comprehensive analysis of liver metabolism using nuclear magnetic resonance (NMR), in nutritional conditions that can be rigorously controlled. As in silico simulations remain a primarily theoretical means of assessing hormone actions and the effects of pharmaceutical intervention, the perfused liver remains one of the most valuable test beds for understanding hepatic metabolism. As these studies guide basic insights into hepatic physiology, results must be accurate and reproducible. The greatest factor in the reproducibility of ex vivo hepatic perfusion is the quality of surgery. Therefore, we have introduced an organized and streamlined method to perform ex vivo mouse liver perfusions in the context of in situ NMR experiments. We also describe a unique application and discuss common issues encountered in these studies. The overall purpose is to provide an uncomplicated guide to a technique we have refined over several years that we deem the golden standard for obtaining reproducible results in hepatic resections and perfusions in the context of in situ NMR experiments. The distance to the center of the field for the magnet as well as the inaccessibility of the tissue to intervention during the NMR experiment makes our methods novel.

Introduction

Ex vivo perfusions are crucial in the study of hepatic metabolism, and perfusion through the portal vein is the standard for these studies. In order to study hepatic metabolism in isolation, the liver must be resected from the body to avoid complications arising from metabolism in other organs (i.e., whole-body metabolism) and to exert control over hormone availability (insulin, glucagon, etc.). This approach can be essential for understanding the effects of diseases such as diabetes, NAFLD, and NASH on hepatic metabolism as well as mechanisms of drug action. This article serves as a guide to hepatic resection and perfusion. We have developed a streamlined pr....

Protocol

Experiments involving mice were handled in compliance with the University of Florida Institutional Animal Care and Use Committee (protocol number #201909320). The mouse strain used was C57BL/6J; all mice were male. This method is generally applicable for studies using other standard mouse strains as well. This surgery is optimally performed by two individuals working together.

1. Initial set-up

  1. Perfuse livers with perfusate containing Krebs-Henseleit electrolytes

Representative Results

Liver function is primarily assessed by oxygen consumption and flow rate. A flow rate of 4-8 mL/min and oxygen consumption of 1 µmol/min.g is typical. These measures will vary depending on specific experimental conditions and biological differences.

The exact amount of isoflurane used will depend on the type of anesthesia system being used as well as the environment and age/weight of the mouse. During the surgery, the isoflurane and delivery gas do not change, although some changes may be.......

Discussion

This surgical procedure is challenging and requires extensive practice to achieve reproducible results. Isoflurane and carrier gas should be adjusted as needed to maintain the viability of the animal through as much of the surgical procedure as possible. Environment, time of day, age, weight, and several other factors will affect anesthesia. Weight, diet, strain of mice and age could affect surgery as fat buildup can interfere with visualizing the portal vein. When taping the paws down, care must be taken to not apply an.......

Acknowledgements

This work was supported by funding from the National Institutes of Health (R01-DK105346, P41-GM122698, 5U2C-DK119889). A portion of this work was performed in the McKnight Brain Institute at the National High Magnetic Field Laboratory's Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) Facility, which is supported by the National Science Foundation Cooperative Agreement No. DMR-1644779 and the State of Florida.

....

Materials

NameCompanyCatalog NumberComments
1 mL Luer-Lock Single Use Sterile Disposable SyringeN/AN/ANon-specific Brand
100 cm Water Jacketed Glass ColumnN/AN/ACustom Made
2-0 Silk SutureBraintree ScientificN/A
22 Gauge Catherter 1 in. Without SafetyTerumoSRFF2225
23 G 0.75 in. Hypodemeric NeedlesExel International26407
27 G 1.5 in. Hypodemeric NeedlesExel International26426
4x4 in. Surgical PlatformN/AN/ACustom Made
70% Alcohol WipeN/AN/ANon-specific Brand
Circulating Water BathMS LaudaN/AModel no longer manufactured
Cotton Tip ApplicatorN/AN/ANon-specific Brand
Delicate Operating Scissors; Straight; Sharp-Sharp; 30mm Blade Length; 4 3/4 "RobozRS-6702
Dumont #5/45 ForcepsFine Scientific Tools11251-35
Dumont #7 - Fine ForcepsFine Scientific Tools11274-20
HemostatsFine Scientific Tools13015-14
Heparin Sodium Injectable 1000 units/mLRX Generics71288-0402-02
IsofluranePatterson Veterinary14043-0704-06
Lidocaine HCl 2%VEDCO Inc.50989-0417-12
Membrane-Thin-Layer OxygenatorRadnotiN/A
Metzenbaum Scissors; Curved; Blunt; 27 mm Blade Length; 5 "RobozRS-6013
Oxygen Meter SystemHanstech Instruments Ltd.N/A
Saline 0.9% SolutionN/AN/ASaline is made in lab
ScaleN/AN/ANon-specific Brand
 Variable Speed Analog Console Pump SystemsCole PalmerN/AModels are custom per application
Weigh boatsN/AN/ANon-specific Brand

References

  1. Ragavan, M., McLeod, M. A., Giacalone, A. G., Merritt, M. E. Hyperpolarized Dihydroxyacetone Is a Sensitive Probe of Hepatic Gluconeogenic State. Metabolites. 11 (7), 441 (2021).
  2. Lumata, L.

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