The studies and Nicolai J. Wewer Albrechtsen were supported by Novo Nordisk Foundation Excellence Emerging Investigator Grant - Endocrinology and Metabolism (Application No. NNF19OC0055001), European Foundation for the Study of Diabetes Future Leader Award (NNF21SA0072746) and Independent Research Fund Denmark, Sapere Aude (1052-00003B). Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (Grant agreement NNF14CC0001). Figure 1B was created with biorender.com.&#160;We thank Dr. Rune E. Kuhre (Novo Nordisk A/S) for fruitful discussions on the perfused mouse liver.&#160;

All animal experiments were conducted with permission from the Danish Animal Experiments Inspectorate, Ministry of Environment and Food of Denmark (permit 2018-15-0201-01397), and the local ethics committee in accordance with the EU directive 2010/63/EU, the National Institutes of Health (publication No. 85-3) and following the guidelines of Danish legislation governing animal experimentation (1987). This is a terminal procedure, and the cause of death is exsanguination and perforation of the diaphragm under deep anesthesia.
1. Experimental animals
<ol>
	<li>Obtain mice of desired strain, age, and gender. This study used male C57BL/6JRj mice aged 11-16 weeks. House up to 5 male or 8 female mice per cage with ad libitum access to chow and water and maintain a 12 h/12 h light-dark cycle with lights on from 6 AM to 6 PM.</li>
</ol>
2. Preoperative preparations
<ol>
	<li>Make liver perfusion buffer.
	<ol>
		<li>Krebs-Henseleit Buffer. Mix and dissolve 118 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L MgSO4, and 1.2 mmol/L KH2PO4 in dH2O. Dissolve 1.25 mmol/L CaCl2 in a separate beaker and add the buffer.</li>
		<li>Dissolve 25 mmol/L NaHCO3 and add slowly to the buffer while stirring. Store the buffer at 4 deg. The buffer is stable for at least 1 month. 
		NOTE: Precipitation may occur if CaCl2 and NaHCO3 are not dissolved individually before being added.</li>
	</ol>
	</li>
	<li>Filter the perfusion buffer through a 2 &#181;m filter and adjust pH to 7.5 using HCl. 
	NOTE: This step should be performed on the experimental day, as pH increases over time, even when stored in closed flasks.</li>
	<li>Prepare test compounds in a higher concentration than the desired final concentration (e.g., 20x concentration when infused at a rate of 0.175 mL/min via a sidearm pump) in filtered and pH-adjusted Krebs-Henseleit perfusion buffer. If relevant, dilute test compounds in Krebs-Henseleit perfusion buffer with 1% BSA added as a carrier (to avoid adhesion to tubing and glassware, essential for all peptides), filtered through an appropriate size filter.</li>
</ol>
3. Operation and perfusion
NOTE: An illustration of the perfusion setup used in this study is provided in Figure 1.
<ol>
	<li>Gas the perfusion buffer (95% O2, 5% CO2) for at least 30 min to supply sufficient oxygen to the liver and to maintain the correct pH from the start of the operation (the bicarbonate buffer system will reach a pH of 7.4 under continuous gassing, see Supplementary Figure 1).</li>
	<li>Anesthetize a mouse by administering ketamine (90 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection.</li>
	<li>Place the mouse supine on a heated operation table and confirm the lack of reflexes in response to the toe pinch. Spray with 70% ethanol to keep hair from sticking to the scissors. Fixing the mouse to the warm surface helps to increase stability for the following procedures.</li>
	<li>Make an incision with scissors at the base of the abdomen and cut upwards to the ribcage on both sides to expose the abdominal cavity. Move the intestines to the right using a cotton swab, exposing the portal vein.</li>
	<li>Place two ligatures under the portal vein using curved forceps and prepare a loose knot for each ligature.</li>
	<li>Insert a 0.7 mm catheter into the portal vein. When perforated, remove the needle from the catheter and guide the catheter through the vein until the tip of the catheter is close to the liver, as illustrated in Figure 2. Blood flows into the catheter.</li>
	<li>Tighten the ligatures. If the catheter is not filled with blood, fill it up with perfusion buffer to avoid the introduction of an air bubble.</li>
	<li>Attach the perfusion tube and initiate perfusion of the liver with Krebs-Henseleit bicarbonate buffer at 37 &#176;C by starting the roller pump at a perfusion flow rate of 0.8 mL/min. The liver turns pale within seconds.</li>
	<li>Cut the ribcage and diaphragm using scissors. At this point, the animal is euthanized. Place a ligature under the suprahepatic inferior vena cava using fine point forceps. Place a pen, rolled gauze, or another disposable item under the back of the mouse to make the vein more accessible.</li>
	<li>Insert a catheter into the suprahepatic inferior vena cava via the right atrium of the heart. When perforated, remove the needle from the catheter and guide the catheter through the vein until the tip of the catheter is close to the liver. Blood and perfusion buffer runs out immediately.</li>
	<li>Tighten the ligature and attach a tube for the collection of perfusion effluent. Secure all tubes with waterproof tape (sleek tape or similar).</li>
	<li>Use a vessel clamp adaptor to place a vessel clamp across the infrahepatic vena cava immediately above the right renal vein to prevent admixture (Figure 2).</li>
	<li>Increase the perfusion flow rate to 3.5 mL/min and start a timer. Start the pressure recording. Successful perfusion usually results in a pressure of ~10 mm/Hg.</li>
	<li>Cover the liver with a sterile drape moistened with saline and add saline during the experiment to prevent it from drying.</li>
	<li>Collect the perfusion effluent for 1 min and measure the volume. The volume should be approximately 3.5 mL/min. Admixture of blood is not expected at this stage, and the heart does not pump any longer.</li>
	<li>Wait for an equilibration period of 30 min before starting the experiment.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65596/65596fig01.jpg" /> 
Figure 1: An illustration of the perfusion setup. (A)The operating table is elevated on a tripod stand and heated to 37 &#176;C. The perfusion buffer is gassed (95% O2, 5% CO2), pumped via a peristaltic roller pump, and heated in the heat exchanger with a built-in bubble trap. The system furthermore consists of a pressure gauge and spindle pump for adjustment of the perfusion pressure. The perfusion pressure is continuously recorded and visualized via a transducer on a PC, a pressure recording program. (B) The red box captures the connections of three-way stopcocks. The first three-way stopcock is open for the infusion of a test compound via a syringe pump, and the second is closed. The third is open for continuous pressure measurements. The fourth stopcock may be used to collect input samples, for e.g., gas analysis across the perfused liver. The connectors can be modified as needed for specific experiments requiring more or fewer infusion lines. <a href="https://www.jove.com/files/ftp_upload/65596/65596fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65596/65596fig02.jpg" /> 
Figure 2: Photos of the mouse abdominal cavity before and during liver perfusion. (A)The green dot indicates the location of the tip of the portal vein catheter. It is important that the tip of the catheter is positioned just below the branching point of the portal vein into the left and right hepatic portal veins but above the pancreato-duodenal branch to avoid leakage. The yellow dot indicates the correct location of the vessel clamp on the infrahepatic inferior vena cava between the right renal vein and the liver to avoid the backflow of blood into the perfused liver. (B, C) A perfused mouse liver with the two catheters inserted in the portal vein (B) and suprahepatic inferior vena cava (C) and the vessel clamp on the infrahepatic inferior vena cava (B). <a href="https://www.jove.com/files/ftp_upload/65596/65596fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Experiment
<ol>
	<li>Start the experiment by collecting the first baseline samples using a fraction collector at the end of the equilibration period. Collect samples at the desired time interval and place them on ice immediately.&#160;</li>
	<li>Check the bubble trap regularly and refill with perfusion buffer when close to empty.</li>
	<li>Collect a sample of the buffer through a three-way stopcock immediately before it enters the organ and from the collecting catheter inserted in the inferior vena cava (after it has been perfused through the liver).</li>
	<li>Measure the samples with a blood-gas analyzer to confirm that the organ is metabolically active (indicated by an increase in the partial pressure of CO2 and a decrease in pH).</li>
	<li>Repeat steps 4.3-4.4 at the end of the experiment to assess viability throughout the experiment (Supplementary Figure 2). 
	NOTE: The decrease in oxygen partial pressure does not provide a reliable measure of respiration because of losses of oxygen from tubing, the organ etc.</li>
	<li>After 15 min of baseline perfusion, start the first stimulation by infusing a test substance through a three-way stopcock at desired flow rate using a syringe pump (e.g., 20x concentration of the test substance when infused at a rate of 0.175 mL/min via a sidearm pump). Alternatively, switch the baseline buffer to a new (oxygenated and heated) buffer containing the test compound in the final concentration.</li>
	<li>Stop the stimulation and collect baseline samples for 20-30 min before starting the second stimulation.</li>
	<li>At the end of the experiment, infuse an appropriate positive control for 5-10 min.</li>
	<li>After the experiment, excise the perfused liver and weigh it to normalize the output to liver weight. Snap-freeze the liver in liquid nitrogen for potential measurement of protein content to normalize the output to protein content.</li>
</ol>
5. Biochemical measurements
<ol>
	<li>Quantify the concentration of the molecule of interest using assays appropriate for measurements in perfusion buffer (in-house or commercially available colorimetric or ELISA assays). The buffer is compatible with most omics-based techniques such as metabolomics and proteomics. 
	NOTE: In this study, urea was measured based on a colorimetric assay previously described14. Glucose and non-esterified fatty acids were quantified using commercially available kits.</li>
</ol>
6. Data analysis
<ol>
	<li>Present the data in XY graphs showing the secretory output over time. 
	NOTE: It is one of the virtues of the in vitro perfusion system that it is possible to express data as the actual output (concentration &#215; flow rate, e.g., &#181;mol/min) instead of the measured concentration in the output (mmol/L) as in in vivo studies. Consider normalizing the output to the liver weight when comparing different mouse models or to the total protein content (measured by BCA) when comparing control mice to mouse models with obesity or liver diseases where an increase in liver weight may be due to increased fat content.</li>
	<li>Present summary data as individual dots per animal representing the mean or total output during baseline and over a stimulation period (typically 15-30 min) or incremental output using the preceding baseline for each stimulation period dependent on the study design.</li>
	<li>Analyze the data using paired t-test (two groups) or one-way ANOVA with repeated stimulations (more than two groups) with an appropriate post-hoc test for multiple testing.</li>
</ol>

In Situ Mouse Liver Perfusion Model to Study Metabolic Responses to Substrates

<ol>
	<li>Bale, S. S., Geerts, S., Jindal, R., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+co-culture+of+rat+parenchymal+and+non-parenchymal+liver+cells+to+evaluate+cellular+interactions+and+response.">Isolation and co-culture of rat parenchymal and non-parenchymal liver cells to evaluate cellular interactions and response.</a> Scientific Reports. 6, 25329 (2016).</li><li>Lauschke, V. 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=Massive+rearrangements+of+cellular+MicroRNA+signatures+are+key+drivers+of+hepatocyte+dedifferentiation.">Massive rearrangements of cellular MicroRNA signatures are key drivers of hepatocyte dedifferentiation.</a> Hepatology. 64 (5), 1743-1756 (2016).</li><li>Seirup, 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=Rapid+changes+in+chromatin+structure+during+dedifferentiation+of+primary+hepatocytes+in+vitro.">Rapid changes in chromatin structure during dedifferentiation of primary hepatocytes in vitro.</a> Genomics. 114 (3), 110330 (2022).</li><li>Gupta, R., 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=Comparing+in+vitro+human+liver+models+to+in+vivo+human+liver+using+RNA-Seq.">Comparing in vitro human liver models to in vivo human liver using RNA-Seq.</a> Archive of Toxicology. 95 (2), 573-589 (2021).</li><li>Bell, C. C., 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=Characterization+of+primary+human+hepatocyte+spheroids+as+a+model+system+for+drug-induced+liver+injury,+liver+function,+and+disease.">Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function, and disease.</a> Scientific Reports. 6, 25187 (2016).</li><li>Dewyse, L., Reynaert, H., van Grunsven, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Best+practices+and+progress+in+precision-cut+liver+slice+cultures.">Best practices and progress in precision-cut liver slice cultures.</a> International Journal of Molecular Sciences. 22 (13), 7137 (2021).</li><li>Li, X., George, S. M., Vernetti, L., Gough, A. H., Taylor, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+glass-based,+continuously+zonated+and+vascularized+human+liver+acinus+microphysiological+system+(vLAMPS)+designed+for+experimental+modeling+of+diseases+and+ADME/TOX.">A glass-based, continuously zonated and vascularized human liver acinus microphysiological system (vLAMPS) designed for experimental modeling of diseases and ADME/TOX.</a> Lab on a Chip. 18 (17), 2614-2631 (2018).</li><li>Broutier, L., 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=Culture+and+establishment+of+self-renewing+human+and+mouse+adult+liver+and+pancreas+3D+organoids+and+their+genetic+manipulation.">Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.</a> Nature Protocols. 11 (9), 1724-1743 (2016).</li><li>Barto&#353;ek, I., Guaitani, A., Miller, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Isolated Liver Perfusion and its Applications. , (1973).</li><li>Gores, G. J., Kost, L. J., LaRusso, N. F. <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:+conceptual+and+practical+considerations.">The isolated perfused rat liver: conceptual and practical considerations.</a> Hepatology. 6 (3), 511-517 (1986).</li><li>Mischinger, H. J., 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=An+improved+technique+for+isolated+perfusion+of+rat+livers+and+an+evaluation+of+perfusates.">An improved technique for isolated perfusion of rat livers and an evaluation of perfusates.</a> Journal of Surgical Research. 53 (2), 158-165 (1992).</li><li>Vairetti, 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=Correlation+between+the+liver+temperature+employed+during+machine+perfusion+and+reperfusion+damage:+role+of+Ca2.">Correlation between the liver temperature employed during machine perfusion and reperfusion damage: role of Ca2.</a> Liver Transplantation. 14 (4), 494-503 (2008).</li><li>Ferrigno, A., Richelmi, P., Vairetti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troubleshooting+and+improving+the+mouse+and+rat+isolated+perfused+liver+preparation.">Troubleshooting and improving the mouse and rat isolated perfused liver preparation.</a> Journal of Pharmacological and Toxicological Methods. 67 (2), 107-114 (2013).</li><li>Zawada, R. J. X., Kwan, P., Olszewski, K. L., Llinas, M., Huang, S. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+of+urea+concentrations+in+cell+culture+medium.">Quantitative determination of urea concentrations in cell culture medium.</a> Biochemistry and Cell Biology. 87 (3), 541-544 (2009).</li></ol>

The authors declare no conflicts of interest relevant to this article.

The isolated perfused mouse liver is a strong research tool for studies of the dynamics and molecular mechanisms of hepatic metabolism. The possibility of minute-to-minute sample collection provides a detailed evaluation of the direct effect of a test compound on the liver. Compared to in vivo studies, the perfused liver allows us to study liver metabolism in an isolated manner avoiding extra-hepatic factors carried by the blood and with complete control over the experimental conditions. The advantages of liver ...

The liver is an essential organ in metabolism. It plays a key role in the control of whole-body energy balance by regulating glucose, lipid, and amino acid metabolism. The increase in liver diseases worldwide is emerging as a major global health burden, and more knowledge is needed about the pathophysiology and its consequences for liver functions.
Various in vitro models have been developed for research on the liver to complement in vivo studies. Isolated and cultured primary hepatocytes from rodents and humans are widely used. Non-parenchymal cells can be separated from hepatocytes using differential and gradient centrifugation, and the co-culture of different cell types is useful for studying intercellular crosstalk1. Although primary human hepatocytes are considered the golden standard for testing drug toxicity, several studies have shown that the hepatocytes rapidly dedifferentiate in tissue culture resulting in loss of hepatic functions2,3,4. Hepatocyte culture in a 3D spheroid system ameliorates the dedifferentiation, is more stable, and appears to mimic the liver in vivo to a higher degree than the traditional 2D culture systems5. Precision-cut liver slices are another well-established in vitro model that keeps the tissue architecture intact and contains the non-parenchymal cells present in the liver6. More advanced in vitro models include liver-on-a-chip7 and liver organoids8. However, with all these approaches, there is a loss of structural integrity and flow dynamics, including vectorial portal-hepatic vein flow, which likely impacts the generalizability.
The isolated perfused rat liver was first described by Claude Bernard in 18559, and is still used in various scientific fields for studies of liver biology, toxicology, and pathophysiology. Advantages of the perfused liver compared to the above-mentioned in vitro models include the maintenance of the hepatic architecture, the vascular flow, the hepatocyte polarity and zonation, and the interactions between hepatocytes and non-parenchymal cells. Compared to in vivo studies, the perfused liver allows the study of liver metabolism in an isolated manner avoiding extra-hepatic factors carried by the blood and with complete control over the experimental conditions. Several modifications have been made to improve the rat liver perfusion model over the years10,11,12,13. Although mice have been used for isolated perfused liver studies, less literature is available. Here, we present a method for in situ perfusion of the mouse liver by cannulation of the portal vein and the suprahepatic vena cava inferior to study the acute and direct metabolic responses to metabolic substrates and hormones as measured in the hepatic venous effluent from the mouse liver in real-time.

hepatic glucose production, ureagenesis, and lipolysis quantified using the perfused mouse liver model

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused liver allows the study of liver biology and metabolism in the whole liver with an intact hepatic architecture, separated from the influence of extra-hepatic factors. Liver perfusions were originally developed for rats, but the method has been adapted to mice as well. Here we describe a protocol for in situ perfusion of the mouse liver. The liver is perfused antegradely through the portal vein with oxygenated Krebs-Henseleit bicarbonate buffer, and the output is collected from the suprahepatic inferior vena cava with clamping of the infrahepatic inferior vena cava to close the circuit. Using this method, the direct hepatic effects of a test compound can be evaluated with a detailed time resolution. Liver function and viability are stable for at least 3 h, allowing the inclusion of internal controls in the same experiment. The experimental possibilities using this model are numerous and may infer insight into liver physiology and liver diseases.

A steady baseline is required to determine whether a stimulus or substrate leads to the release of the molecule of interest. Figure 3A shows an example of a successful experiment. Production of urea in the perfused liver is measured in 2 min intervals and shown as mean &#177; SEM. The baseline periods preceding each of the two stimulation periods are steady. The mean urea production during the two stimulation periods and the respective preceding baselines are...

Watch this Scientific Journal Video about Investigating Hepatic Adaptations and Prediabetic Progression in Liver Diseases at JoVE.com

Author Spotlight: Investigating Hepatic Adaptations and Prediabetic Progression in Liver Diseases 

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

The liver has numerous functions, including nutrient metabolism. In contrast to other in vitro and in vivo models of liver research, the isolated perfused ...

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1.6K Views.  University of Copenhagen. 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.

Video: Author Spotlight: Investigating Hepatic Adaptations and Prediabetic Progression in Liver Diseases 

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal vascular occlusion, retinal detachment, and diabetic retinopathy. It was the object of this study to introduce a standardized stress model for future preclinical therapeutic testing.
Bovine retinas were prepared and perfused with an oxygen saturated standard solution, and the ERG was recorded. After recording stable b-waves, hypoxia (pure N2) or glutamate stress (250 &#181;m glutamate) was exerted for 45 min. To investigate the effects on photoreceptor function alone, 1 mM aspartate was added to obtain a-waves. ERG-recovery was monitored for 75 min.
For hypoxia, a decrease in a-wave amplitude of 87.0% was noted (p &#60;0.01) after an exposition time of 45 min (decrease of 36.5% after the end of the washout p = 0.03). Additionally, an initial decrease in b-wave amplitudes of 87.23% was recorded, that reached statistical significance (p &#60;0.01, decrease of 25.5% at the end of the washout, p = 0.03).
For 250 &#181;m glutamate, an initial 7.8% reduction of a-wave amplitudes (p &#62;0.05) followed by a reduction of 1.9% (p &#62;0.05). A reduction of 83.7% of b-wave amplitudes (p &#60;0.01) was noted; after a washout of 75 min the reduction was 2.3% (p = 0.62). In this study, a standardized stress model is presented that may be useful to identify possible neuroprotective effects in the future.

With this study, we introduce a standardized stress model for the isolated superfused bovine retina for future preclinical therapeutic testing. The effect of either hypoxia (pure N2) or glutamate stress (250 &#181;M glutamate) on retinal function represented by a- and b-wave amplitudes was evaluated.

Neuroprotection has been a strong field of investigation in ophthalmological research in the past decades and affects diseases such as glaucoma, retinal ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

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

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Chapters in this video

Murine Bioluminescent Hepatic Tumour Model

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

A Preclinical Murine Model of Hepatic Metastases

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Glutamate and Hypoxia as a Stress Model for the Isolated Perfused Vertebrate Retina

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography