Name: Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse (Video) | JoVE Journal
Uploaded: 2022-03-09T12:30:00.000+00:00
Duration: 5 min 30 s
Description: Learn ex vivo hepatic perfusion through the portal vein in mouse for metabolic liver studies. This technique enables precise control of nutrients and hormones to study diabetes, NAFLD, and NASH models.

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5 minutes

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&#39;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.

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
<ol>
	<li>Perfuse livers with perfusate containing Krebs-Henseleit electrolytes6(25 mM NaHCO3, 112 mM NaCl, 4.7 mM KCl, 1.2 mM each of MgSO4, KH2PO4, and 0.5 mM sodium-EDTA, 1.25 mM CaCl2), 6 mM sodium lactate, 0.6 mM sodium pyruvate, 0.2 mM [U-13C] sodium propionate, 10% (v/v) D2O, and 0.63 mM mixed fatty acids (containing palmitic acid (22.1% of total), palmitoleic acid (5.2%), stearic acid (2.7%), oleic acid (27%), linoleic acid (37.7%), &#947;-linolenic acid (2.4%), and decosahexanoic acid (2.8%)) along with 2% (w/v) bovine serum albumin. Set final pH of perfusate to 7.3 using HCl (and NaOH, if necessary).</li>
</ol>
2. Pre-surgery set-up
<ol>
	<li>Assemble two 1 mL syringes with a 23G needle which are 19.05 mm long. Fill one syringe with 0.01 mL of 1000 units/mL heparin and 0.19 mL of saline (0.9% (w/v) NaCl in water; Table 1).</li>
	<li>Fill the second syringe with 0.2 mL of 2% lidocaine and 0.6 mL of 0.9% saline (Table 1). In another 1 mL syringe with a 27 G 38.1 mm needle, fill with the perfusate and maintain at 37 &#176;C.</li>
</ol>
3. Perfusate column set-up
<ol>
	<li>Set the glass bottle containing 500 mL of perfusate into the water bath (Figure 1B). Turn the water bath on and set the temperature to ~42 &#176;C. The higher temperature in the water bath enables maintaining 37 &#176;C in the perfusion column.</li>
	<li>Once the water heats up to 42 &#176;C, turn on the two pumps to circulate the perfusate from the bottle throughout the thin film oxygenator and the water-jacketed 100 cm glass column (Figure 1A-E).</li>
	<li>Turn on the oxygenating gas (95% oxygen and 5% carbon dioxide) to pressurize the oxygenator7 (Figure 1C). Adjust perfusate column height to achieve a flow rate of 8 mL/min with the catheter attached (see step 9 for flow rate measurement)5,8,9. 
	&#8203;NOTE: Flow rate refers to the rate at which perfusate is being expelled by the liver.</li>
</ol>
4. Anesthetization of the mouse
<ol>
	<li>Don PPE as required by IACUC protocol and other appropriate safety guidelines. 
	NOTE: The following steps have been optimized for mice that are 9 - 13 weeks old.</li>
	<li>Place the mouse in the isoflurane chamber. Turn the delivery gas to 100% oxygen, a flow rate of 1 L/min, and the isoflurane to 2%10. Wait till breathing slows down and is steady. 
	NOTE: For the mouse to reach a stable surgical plane, as evidenced by a slow and steady respiratory rate and lack of toe pinch reflex, the oxygen flow rate can be adjusted to ~1.5 L/min to ~3 L/min and the isoflurane concentration from&#160;1 - 3%. The delivery rate of carrier gas and isoflurane concentration depends on the animal&#39;s age and weight and factors such as noise and light.</li>
	<li>Disinfect the abdomen with 70% alcohol. Administer heparin through a deep subcutaneous injection in the abdominal fat layer (Figure 2). Place the mouse back into the anesthesia chamber for 10 min. 
	&#8203;NOTE: Shaving is not required as this procedure is terminal.</li>
</ol>
5. Celiotomy
<ol>
	<li>Transfer the mouse from the anesthesia chamber to the surgery platform and place it in the supine position (Figure 3).</li>
	<li>Place the nose of the mouse into a nose cone and tape the paws down. Take care to not apply any strain on the neck that may lead to suffocation.</li>
	<li>Administer lidocaine through a subcutaneous injection bilaterally in the anterior iliac crest region11 (Figure 3). Perform a toe pinch test to confirm the absence of all pain reflexes.</li>
	<li>Perform celiotomy to expose the internal organs (Figure 4). Make a 3 cm wide incision (the width of the entire abdomen of the mouse). 
	&#8203;NOTE: Width will change with the age and diet of the mouse.</li>
	<li>Expand the incision by using a hemostat that pulls traction by clamping on the xiphoid process (Figure 4).</li>
</ol>
6. Cannulation of portal vein
<ol>
	<li>Use a cotton-tipped applicator to clear the small and large intestines covering the portal vein. Position a silk suture under the arch of the portal vein proximal to the liver (Figure 4A).</li>
	<li>Depending on the anatomical structure, place the second silk suture proximal or distal of the inferior mesenteric vein distal from the liver (Figure 4A)12,13. Use a 2-0 suture for both sutures.</li>
	<li>Once the sutures are in place, cannulate the portal vein with a 22G catheter14 (Figure 4B). When inserting the catheter, keep the bevel pointed up. Enter the portal vein at no more than a 15&#176; angle.</li>
	<li>Tie the first suture past the catheter tapper. After the portal vein is cannulated, anchor the catheter 2-3 mm distal from the branch of the portal vein with the silk suture (Figure 4B). 
	NOTE: The assistant should roll shoulders and wrists to prevent dislodging the catheter or tearing of the portal vein. Each suture requires two knots.</li>
	<li>Next, secure the lower portion of the catheter with the second suture. With the help of the surgical assistant, tie a knot with the suture to secure the catheter to the distal portion of the portal vein and the surrounding tissue.</li>
</ol>
7. Resection of liver post portal vein cannulation
<ol>
	<li>After the catheter is secured, insert a 1 mL syringe with a 27G needle which is 38.1 mm long into the catheter to flush blood and air bubbles. 
	NOTE: There is usually a backflow of blood out of the catheter from the pressure.</li>
	<li>Use a 1 mm I.D. x 5 mm O.D. silicone tube with a fixed stopcock to couple the perfusion column (Figure 1A) to the catheter allowing the flow of the buffer into the liver marking the start of the perfusion. Start a timer at this point to mark the start of the perfusion.</li>
	<li>Relieve increased vascular pressure by making an incision, using scissors, into the inferior vena cava.</li>
	<li>Confirm flow of perfusate through the liver by observing the homogenous change in liver color from pink/red to a pale yellow. Once the flow is confirmed, excise the stomach, small intestine, large intestine, and the right kidney from surrounding tissue.</li>
	<li>With the help of the surgical assistant, maneuver the liver around the abdominal and thoracic cavity as the surgeon cuts through the parietal peritoneum and thoracic tissue to resect the liver</li>
	<li>Lastly, lift the liver upwards and cut the remaining connective tissues holding the liver in place with scissors. Slowly manipulate the liver for ease of view. Remove any fur that sticks to the liver by rinsing off with perfusate before encapsulating it within the NMR tube. 
	NOTE: In this procedure, only the liver is removed. All other organs are left in the body of the animal. The bile duct can be removed based on the protocol of the experiment. Although for this experiment, it was left in place.</li>
</ol>
8. Hanging the liver from the column
<ol>
	<li>Once the surgeon hands the liver and tubing to the assistant, the assistant disconnects the tubing from the catheter and column.</li>
	<li>Fill the catheter with perfusate until a meniscus is formed on the top of the catheter. Attach the catheter to the column for the liver to hang and perfuse. 
	NOTE: The bead of perfusate on the catheter provides sufficient volume for the liver to function until its connected.The catheter attached to the liver is press fitted to the bottom of the column.</li>
	<li>Screw a 20 mm NMR tube onto the 100 cm glass column to encapsulate the liver (Figure 5). To avoid torsion of the liver and the portal vein, slowly screw on the NMR tube. If torsion does occur and flow is stopped, unscrew and rescrew the NMR tube. This will remedy the occlusion and flow will return.</li>
	<li>Perfuse the liver for 30-60 min based on the details of the study. 
	&#8203;NOTE: Time is based on the perfusion experiment. For this experiment, metabolic turnover was measured within 30 min. The liver perfusion can take up to 10 min to reach a steady state. The time to steady-state starts once the inferior vena cava has been cut and hepatic flow is established.</li>
</ol>
9. Flow measurement
<ol>
	<li>Place a weigh boat on a top loading balance and zero the balance. Place the tubing from the roller pump pulling efferent perfusate from the NMR into the weigh boat and start the timer.</li>
	<li>Weigh the mass of liquid accumulated over 1 min which yields the flow rate of the liver. Place the tube back into the waste/collection container.</li>
</ol>
10. Oxygen Measurement
NOTE: Oxygen meter measurements were set up according to the manufacturer&#39;s instructions15.
<ol>
	<li>Place the electrode containing 20 &#181;L of 50% KCl saturated solution on the platinum dome and place five 10 &#181;L drops around the lower platinum ring of the electrode.</li>
	<li>Remove the adhesive off the cigarette paper. Lay a piece of polytetrafluoroethylene membrane over the cigarette paper.</li>
	<li>Place the two pieces on top of the electrode. Fit a small O-ring around the top of the electrode. Trim the paper to lay flat onto the lower platinum ring on the electrode. 
	NOTE: Some overhang is acceptable. It&#39;s essential to cover the silver of the electrode.</li>
	<li>Place the larger O-ring on the electrode. Couple the electrode to the water chamber and tighten the base to hold the electrode in place. Turn on the water bath and allow to heat up to 37 &#176;C.</li>
	<li>Open oxygen meter software. Click on Calibrate &#62; Air-Saturated Water. Set stirrer speed to 75 and the temperature to 37 &#176;C.</li>
	<li>Fill a 50 mL vial halfway with water and shake vigorously for 2 min. This is air-saturated water, use it as 100% standard. Fill the oxygen meter chamber with ~2 mL of water and place the two-piece stopper on.</li>
	<li>Click Okay on the screen and allow the signal to plateau. Once the signal has reached a plateau, click Okay. Dispose of liquid in the chamber and dry with tissue paper.</li>
	<li>Repeat steps 10.6-10.7 but with 200 mM sodium sulfite (0% standard). Click on Save calibration. 
	NOTE: No vigorous shaking is needed for the 0% standard.</li>
	<li>During perfusion, use two 5 mL syringes. One syringe for circulating perfusate (oxygen in) and the second for efferent perfusate from the NMR tube (oxygen out).</li>
	<li>When drawing up perfusate for both in and out measurements, draw 3-4 mL each time. 
	NOTE: This column has glass tubing to allow for access into the NMR tube to withdraw the perfusate that has flowed through the liver.</li>
	<li>Measure the circulating perfusate, first just as water in step 10.6 and dispose of in step 10.7. Repeat the same steps for the efferent perfusate.</li>
	<li>Perform oxygen measurements every 10 min.</li>
</ol>

<ol><li>Ragavan, M., McLeod, M. A., Giacalone, A. G., Merritt, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hyperpolarized+Dihydroxyacetone+Is+a+Sensitive+Probe+of+Hepatic+Gluconeogenic+State%5BTitle%5D)+AND+%22Metabolites%22%5BJournal%5D)">Hyperpolarized Dihydroxyacetone Is a Sensitive Probe of Hepatic Gluconeogenic State.</a> Metabolites. 11 (7), 441(2021).</li>
<li>Lumata, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hyperpolarized+%2813%29C+Magnetic+Resonance+and+Its+Use+in+Metabolic+Assessment+of+Cultured+Cells+and+Perfused+Organs%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Hyperpolarized (13)C Magnetic Resonance and Its Use in Metabolic Assessment of Cultured Cells and Perfused Organs.</a> Methods in Enzymology. 561, 561-573 (2015).</li>
<li>Moreno, K. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+detection+of+hepatic+gluconeogenic+and+glycogenolytic+states+using+hyperpolarized+%5B2-13C%5D+dihydroxyacetone%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Real-time detection of hepatic gluconeogenic and glycogenolytic states using hyperpolarized [2-13C] dihydroxyacetone.</a> The Journal of Biological Chemistry. 289 (52), 35859-35867 (2014).</li>
<li>Moreno, K. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hyperpolarized+%CE%B4-%5B1-13C%5D+gluconolactone+as+a+probe+of+the+pentose+phosphate+pathway%5BTitle%5D)+AND+%22NMR+in+Biomedicine%22%5BJournal%5D)">Hyperpolarized δ-[1-13C] gluconolactone as a probe of the pentose phosphate pathway.</a> NMR in Biomedicine. 30 (6), (2017).</li>
<li>Merritt, M. E., Harrison, C., Sherry, A. D., Malloy, C. R., Burgess, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flux+through+hepatic+pyruvate+carboxylase+and+phosphoenolpyruvate+carboxykinase+detected+by+hyperpolarized+13C+magnetic+resonance%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Flux through hepatic pyruvate carboxylase and phosphoenolpyruvate carboxykinase detected by hyperpolarized 13C magnetic resonance.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (47), 19084-19089 (2011).</li>
<li>Bailey, L. E., Ong, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Krebs-Henseleit+solution+as+a+physiological+buffer+in+perfused+and+superfused+preparations%5BTitle%5D)+AND+%22Journal+of+Pharmacological+Methods%22%5BJournal%5D)">Krebs-Henseleit solution as a physiological buffer in perfused and superfused preparations.</a> Journal of Pharmacological Methods. 1 (2), 171-175 (1978).</li>
<li>Kolwicz, S. C. Jr, Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+Cardiac+Function+and+Energetics+in+Isolated+Mouse+Hearts+Using+31P+NMR+Spectroscopy%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy.</a> Journal of Visualized Experiments: JoVE. (42), e2069(2010).</li>
<li>Hwang, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protective+effect+of+butylated+hydroxylanisole+against+hydrogen+peroxide-induced+apoptosis+in+primary+cultured+mouse+hepatocytes%5BTitle%5D)+AND+%22Journal+of+Veterinary+Science%22%5BJournal%5D)">Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes.</a> Journal of Veterinary Science. 16 (1), 17-23 (2015).</li>
<li>Bessems, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+isolated+perfused+rat+liver%3A+standardization+of+a+time-honoured+model%5BTitle%5D)+AND+%22Laboratory+Animals%22%5BJournal%5D)">The isolated perfused rat liver: standardization of a time-honoured model.</a> Laboratory Animals. 40 (3), 236-246 (2006).</li>
<li>Beal, E. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Small+Animal+Model+of+Ex+Vivo+Normothermic+Liver+Perfusion%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">A Small Animal Model of Ex Vivo Normothermic Liver Perfusion.</a> Journal of Visualized Experiments: JoVE. (136), e57541(2018).</li>
<li>Collins, J. B., Song, J., Mahabir, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Onset+and+duration+of+intradermal+mixtures+of+bupivacaine+and+lidocaine+with+epinephrine%5BTitle%5D)+AND+%22The+Canadian+Journal+of+Plastic+Surgery%22%5BJournal%5D)">Onset and duration of intradermal mixtures of bupivacaine and lidocaine with epinephrine.</a> The Canadian Journal of Plastic Surgery. 21 (1), 51-53 (2013).</li>
<li>Medical Dictionary. , Merriam-Webster. Available from: 
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<li>Thorpe, D. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aThorpe%2C+DR+%22A+Dissection+in+Color%3A+The+Rat+%28and+the+Sheep%27s+Brain%29%22">A Dissection in Color: The Rat (and the Sheep's Brain)</a>. , National Press Books. Palo Alto, CA. (1968).</li>
<li>Cabral, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+of+Hepatocytes+and+Sinusoidal+Endothelial+Cells+from+Mouse+Liver+Perfusion%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion.</a> Journal of Visualized Experiments: JoVE. (132), e56993(2018).</li>
<li>Operations Manual Setup, Installation and Maintenance. , Hansatech Instruments. Available from: 
 <a target="_blank" href="https://www.chem.ucla.edu/dept/Faculty/merchant/pdf/electrode_prep_maintenance.pdf">https://www.chem.ucla.edu/dept/Faculty/merchant/pdf/electrode_prep_maintenance.pdf</a> (2006).</li>
<li>Heparin. , DrugBank Online. Available from: 
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The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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...

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 procedure to perform these metabolic liver studies with sufficient rigor and reproducibility. If the surgery is not performed correctly, there is pronounced variability in the metabolic data obtained. We describe an organized method to perform portal vein catheterization and liver resection in the context of metabolic studies in situ in a nuclear magnetic resonance (NMR) spectrometer, as described in the literature1,2,3,4,5.
Currently, there is no literature describing an ex vivo hepatic perfusion using a glass column within an NMR. Nor is there a video or text publication providing a clear example of how to perform the procedure with the mouse liver, specifically, demonstrating how to catheterize the portal vein, resect a liver, transfer, and hang the liver onto a glass column. As the genetically modified mouse is ubiquitously used for studying liver metabolism, this is an essential procedure that deserves a complete description. Liver perfusion surgeries are not new, but this article is a gold standard method accompanied by a video demonstrating the technical excellence described in this paper to aid everyone interested in this procedure. The method presented here would be best applied to real-time metabolism to detect the function and turnover of metabolites in disease models.
This method uses a 100 cm water-jacketed glass column, which allows the liver to hang at the bottom of the cannula encapsulated by perfusate inside an NMR tube. Heated water in the glass jacket is used to control perfusate temperature. A thin layer oxygenator is pressurized with 95%/5% O2/CO2 for pH control. By using three separate pumps, the perfusate column height is set, which provides constant pressure to the liver. Flow rates are not controlled beyond the application of constant pressure (Figure 1). To confirm the liver is appropriately functioning, oxygen measurements are taken along with flow rates. In our hands, this set of preconditions leads to highly repeatable NMR experiments for the assessment of liver metabolic function.

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

ex vivo hepatic perfusion through the portal vein in mouse

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

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.

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 nece...

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 ...

ex-vivo-hepatic-perfusion-through-the-portal-vein-in-mouse

Research

JoVE Journal

Biochemistry

This video describes a procedure for mouse liver perfusion through the portal vein using collagenase solution prior to isolating liver cells.

In Vivo Murine Liver Perfusion: A Method to Infuse the Rodent Liver with Solution via the Portal Vein

Liver macrophages, named Kupffer cells, are responsible for the capture of circulating nanoparticles. We describe here a method, of high cell purity and yield, for Kupffer cell isolation. The modified LDH assay is used here to measure the toxicity induced by carbon nanotubes in Kupffer cells.

Kupffer Cell Isolation for Nanoparticle Toxicity Testing

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

A protocol is described for in situ perfusion of the mouse lower body, including the bladder, the prostate, sex organs, bone, muscle and foot skin.

Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

Fatty acid β-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid β-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

The present protocol optimizes the liver in situ perfusion/decellularization and two-photon microscopy methods to establish a reliable platform to visualize the dynamics of extracellular matrix (ECM) remodeling during non-alcoholic steatohepatitis (NASH).

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

A normothermic ex vivo liver perfusion (NEVLP) system was created for mouse livers. This system requires experience in microsurgery but allows for reproducible perfusion results. The ability to utilize mouse livers facilitates the investigation of molecular pathways to identify novel perfusate additives and enables the execution of experiments focused on organ repair.

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

Here, we present a robust method for in situ perfusion of the mouse liver to study the acute and direct regulation of liver metabolism without disturbing the hepatic architecture but in the absence of extra-hepatic factors.

Hepatic Glucose Production, Ureagenesis, and Lipolysis Quantified using the Perfused Mouse Liver Model

Here, we describe a platform that allows noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. The workflow spans from generating spheroids from primary liver cells to transplantation into the mouse eye and in vivo imaging at cellular resolution by confocal microscopy.

In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye

Learn ex vivo hepatic perfusion through the portal vein in mouse for metabolic liver studies. This technique enables precise control of nutrients and hormones to study diabetes, NAFLD, and NASH models.

Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

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