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The protocol describes a straightforward method of resectioning an intact mouse liver for metabolic studies through portal vein perfusion.
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.
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....
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
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.......
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.......
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.
....Name | Company | Catalog Number | Comments |
1 mL Luer-Lock Single Use Sterile Disposable Syringe | N/A | N/A | Non-specific Brand |
100 cm Water Jacketed Glass Column | N/A | N/A | Custom Made |
2-0 Silk Suture | Braintree Scientific | N/A | |
22 Gauge Catherter 1 in. Without Safety | Terumo | SRFF2225 | |
23 G 0.75 in. Hypodemeric Needles | Exel International | 26407 | |
27 G 1.5 in. Hypodemeric Needles | Exel International | 26426 | |
4x4 in. Surgical Platform | N/A | N/A | Custom Made |
70% Alcohol Wipe | N/A | N/A | Non-specific Brand |
Circulating Water Bath | MS Lauda | N/A | Model no longer manufactured |
Cotton Tip Applicator | N/A | N/A | Non-specific Brand |
Delicate Operating Scissors; Straight; Sharp-Sharp; 30mm Blade Length; 4 3/4 " | Roboz | RS-6702 | |
Dumont #5/45 Forceps | Fine Scientific Tools | 11251-35 | |
Dumont #7 - Fine Forceps | Fine Scientific Tools | 11274-20 | |
Hemostats | Fine Scientific Tools | 13015-14 | |
Heparin Sodium Injectable 1000 units/mL | RX Generics | 71288-0402-02 | |
Isoflurane | Patterson Veterinary | 14043-0704-06 | |
Lidocaine HCl 2% | VEDCO Inc. | 50989-0417-12 | |
Membrane-Thin-Layer Oxygenator | Radnoti | N/A | |
Metzenbaum Scissors; Curved; Blunt; 27 mm Blade Length; 5 " | Roboz | RS-6013 | |
Oxygen Meter System | Hanstech Instruments Ltd. | N/A | |
Saline 0.9% Solution | N/A | N/A | Saline is made in lab |
Scale | N/A | N/A | Non-specific Brand |
Variable Speed Analog Console Pump Systems | Cole Palmer | N/A | Models are custom per application |
Weigh boats | N/A | N/A | Non-specific Brand |
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