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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarized+Dihydroxyacetone+Is+a+Sensitive+Probe+of+Hepatic+Gluconeogenic+State.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarized+(13)C+Magnetic+Resonance+and+Its+Use+in+Metabolic+Assessment+of+Cultured+Cells+and+Perfused+Organs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+detection+of+hepatic+gluconeogenic+and+glycogenolytic+states+using+hyperpolarized+[2-13C]+dihydroxyacetone.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarized+&#948;-[1-13C]+gluconolactone+as+a+probe+of+the+pentose+phosphate+pathway.">Hyperpolarized &#948;-[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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flux+through+hepatic+pyruvate+carboxylase+and+phosphoenolpyruvate+carboxykinase+detected+by+hyperpolarized+13C+magnetic+resonance.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Krebs-Henseleit+solution+as+a+physiological+buffer+in+perfused+and+superfused+preparations.">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., Tian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Cardiac+Function+and+Energetics+in+Isolated+Mouse+Hearts+Using+31P+NMR+Spectroscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+butylated+hydroxylanisole+against+hydrogen+peroxide-induced+apoptosis+in+primary+cultured+mouse+hepatocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolated+perfused+rat+liver:+standardization+of+a+time-honoured+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Small+Animal+Model+of+Ex+Vivo+Normothermic+Liver+Perfusion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Onset+and+duration+of+intradermal+mixtures+of+bupivacaine+and+lidocaine+with+epinephrine.">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 Available from: <a target="_blank" href="https://www.merriam-webster.com/medical">https://www.merriam-webster.com/medical</a> (2022)</li><li>Thorpe, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Dissection in Color: The Rat (and the Sheep's Brain). , (1968).</li><li>Cabral, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Hepatocytes+and+Sinusoidal+Endothelial+Cells+from+Mouse+Liver+Perfusion.">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 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>. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#34;</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 &#34;</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>&#160;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

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 &#181;mol/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...

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Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

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

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 ...

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Research

JoVE Journal

Biochemistry

8.3K Views.  University of Florida. The protocol describes a straightforward method of resectioning an intact mouse liver for metabolic studies through portal vein perfusion.

Video: Ex Vivo Hepatic Perfusion Through the Portal Vein in Mouse

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

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.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis&#8212;in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before&#8212;and provide some representative results.

Here we present a protocol to perform polysome profiling on the isolated perfused mouse heart. We describe methods for heart perfusion, polysome profiling, and analysis of the polysome fractions with respect to mRNAs, miRNAs, and the polysome proteome.

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning display increased atherosclerotic lesions in the aorta compared with female mice with intact ovarian function. However, laboratory models involving estrogen-deficient mice with atherosclerosis-prone status are lacking. This deficit is crucial because clinical estrogen deficiency in menopausal women may aggravate the incidence of pre-existing or ongoing lipid disruption and atherosclerosis. In this study, we establish an in vivo estrogen-deficient mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apolipoprotein E (apoE)-/- mice. We then compare the effects of 17&#946;-estradiol and pseudoprotodioscin (PPD) (a phytoestrogen) perorally administered via hazelnut spread. We find that although PPD exerts some effect on reducing final body weight and plasma TG in OVX apoE-/- mice, it has anti-atherosclerotic and cardiac-protective capacities comparable with its 17&#946;-estradiol counterpart. PPD is a phytoestrogen that has been reported to exert anti-tumor properties. Thus, the proposed method is applicable for screening phytoestrogens via peroral administration to substitute for traditional hormone replacement therapy in postmenopausal women, which has been reported to have potentially deleterious tumorigenetic capacity. Peroral administration via hazelnut spread is noninvasive, rendering it widely applicable to many patients. This article contains step-by-step demonstrations of bilateral ovariectomy via the double dorsal-lateral incision in apoE-/- mice and peroral 17&#946;-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

Clinically, estrogen deficiency in menopausal women may aggravate the incidence of lipid disruption and atherosclerosis. We established an in vivo estrogen deficiency model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice. The mouse model is applicable for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. Biomolecules such as proteins, carbohydrates, vitamins, fatty acids, nucleic acids, and enzymes are fundamental building blocks of living beings; they assemble into complex networks in biosystems to synchronize a myriad of life events. Proteins typically utilize complex interactome networks to carry out their functions; hence it is mandatory to evaluate such interactions to unravel their importance in cells at both cellular and organism levels. Toward this goal, we introduce a rapidly emerging technology, field-effect biosensing (FEB), to determine specific biomolecular interactions. FEB is a benchtop, label-free, and reliable biomolecular detection technique to determine specific interactions and uses high-quality electronic-based biosensors. The FEB technology can monitor interactions in the nanomolar range due to the biocompatible nanomaterials used on its biosensor surface. As a proof of concept, the protein-protein interaction (PPI) between heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) was elucidated. Hsp90 is an ATP-dependent molecular chaperone that plays an essential role in the folding, stability, maturation, and quality control of many proteins, thereby regulating multiple vital cellular functions. Cdc37 is regarded as a protein kinase-specific molecular chaperone, as it specifically recognizes and recruits protein kinases to Hsp90 to regulate their downstream signal transduction pathways. As such, Cdc37 is considered a co-chaperone of Hsp90. The chaperone-kinase pathway (Hsp90/Cdc37 complex) is hyper-activated in multiple malignancies promoting cellular growth; therefore, it is a potential target for cancer therapy. The present study demonstrates the efficiency of FEB technology using the Hsp90/Cdc37 model system. FEB detected a strong PPI between the two proteins (KD values of 0.014 &#181;M, 0.053 &#181;M, and 0.072 &#181;M in three independent experiments). In summary, FEB is a label-free and cost-effective PPI detection platform, which offers fast and accurate measurements.

Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. ...