Name: assessment of plasma coagulation on liver tissue in a large animal model in vivo
Uploaded: 2018-08-04T13:00:00.000+00:00
Duration: 6 min 23 s
Description: Watch this Scientific Journal Video about Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo at JoVE.com

The authors have no acknowledgements.

Rules governed by German legislation for animal studies as well as Principles of Laboratory Animal Care (National Institutes of Health publication ed. 8, 2011) were followed. Official permission is granted from the governmental animal care office (Landesamt f&#252;r Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Recklinghausen, Germany).
1. Animals
<ol>
	<li>Use female German landrace pigs (weighing 25-30 kg) housed in open cages.</li>
	<li>Use 5 animals per group (argon and helium).</li>
	<li>Allow the animals to acclimatize to the surroundings for at least one week before the experiments. Fast animals for 24 h prior to surgery with free access to water.</li>
</ol>
2. Anesthesia
<ol>
	<li>Premedicate the animals with an intramuscular injection of ketamine (15 mg/kg body weight [BW]), xylazine (10 mg/kg BW), and atropine (0.1 mg/kg BW) 10 min before induction of anesthesia.</li>
	<li>Peripheral venous access is established by placement of a 22-gauge cannula into an ear vein.</li>
	<li>Induce general anesthesia by i.v. injection of propofol 2 mg/kg body weight.</li>
	<li>Place the animal in the supine position and perform a longitudinal skin incision in the jugular groove over a length of 2 cm. Locate vein through the blunt preparation of the subcutaneous tissue. Insert cannula, then Seldinger wire.</li>
	<li>Retract cannula and insert 14 Fr. catheter over the guide wire. Retract guide wire. Connect catheter to the extension and fixate the catheter by a strap or a suture.</li>
	<li>Pull out the tongue and insert straight laryngoscope. Use the tip of the laryngoscope to pull down the epiglottis. Insert the tube through the vocal cords. Place cuff under glottis and inflate.</li>
	<li>Ventilate with 40% oxygen at 20-26 breaths/min and a tidal volume of 10 ml/kg to keep the end-tidal partial carbon dioxide tension between 36 and 42 mm Hg.</li>
	<li>Maintain anesthesia with isoflurane at a concentration of 1-1.5% and fentanyl at a concentration of 3-4 &#181;g/kg/h.</li>
	<li>Supply Ringer&#39;s lactate solution at an initial rate of 4 mL/kg/h, and increase after laparotomy to a constant infusion rate of 8 mL/kg/h.</li>
</ol>
3. Surgery and Plasma Coagulation
<ol>
	<li>Place the animal in a supine position on a standard surgical table.</li>
	<li>Disinfect skin by applying a standard surgical disinfectant (2-Propanol 45 g /100 g, 1-Propanol 10 g / 100g, Biphenyl-2-ol 0.2 g / 100 g) with a surgical swab for 3 times.</li>
	<li>Perform a wide midline laparotomy from the xiphoid process to the pubis with a scalpel and install surgical retractors.</li>
	<li>Switch on the plasma coagulation device, open argon or helium gas bottle, depending on the used carrier gas. Adjust gas flow to 3 L/min. Select coagulation device output power as desired. 
	NOTE: Both noble gases, Argon or Helium, can be used for plasma coagulation. Coagulation effects are comparable. See reference7 for details.</li>
	<li>Perform plasma coagulation on the left liver lobe as previously described7. Use a titanium mold (square aperture 1 x 1 cm2) to standardize the coagulation zone. Coagulate for 5 s with a probe distance of 1 cm. The coagulations with different power settings can be performed side by side with a short distance between the coagulations of 5 mm (Figure 1).</li>
	<li>For harvesting of the liver, divide all ligamentous connections to the liver. Isolate and divide the hepatic pedicle above the superior duodenal flexure leaving long portions of portal vein and common bile duct. Divide the caval vein above and below the liver and retrieve the organ.</li>
	<li>After harvesting the liver, the pigs were euthanized by i.v. administration of 0.16 g/kg BW pentobarbital.</li>
	<li>For the burst pressure experiments, resect half of the left medial liver lobe with sharp scissors. Plasma-coagulate the cut surface (100W output power) or seal the cut surface with fibrin sealant (Figure 2).</li>
</ol>
4. Microcirculation Measurement
NOTE: Laser Doppler spectroscopy can determine blood flow in tissue through measuring the Doppler shift caused by the movement of erythrocytes. The Laser signal correlates with the number of moving erythrocytes. Laser Doppler spectroscopy is in clinical use (e.g. transplant medicine) and has been validated multiple times15.
<ol>
	<li>Switch on the laser Doppler flowmeter and spectrophotometer. Use a flat probe.</li>
	<li>Take baseline measurements for flow and velocity. Save or note the values.</li>
	<li>Perform coagulation as described under 3.5.</li>
	<li>Place the flat probe on coagulation sites and measure flow and velocity. Again, save or note values.</li>
	<li>Repeat for all power settings of the coagulator device.</li>
</ol>
5. Temperature Measurement
<ol>
	<li>Switch the system on (thermographic camera, notebook, and infrared thermometer) and let it run for at least 1 h before performing measurements.</li>
	<li>Adjust focus and view frame on the thermographic camera on the coagulation site. Infrared sequences can be detected with the spatial resolution of 1024 x 768 pixels with a temperature resolution bigger than 20 mK. Take into account, that the region of coagulation and the surrounding tissue &#8212; affected by heat transfer &#8212; is located in the middle of the view. 
	NOTE: It should include as many pixels of the frame as possible for an optimal spatial resolution.</li>
	<li>Record coagulation process with the plasma coagulator on the liver surface with the thermographic camera over a 2-min period.</li>
	<li>Analyze image sequences with the thermography analysis software: Define regions of interest. 
	NOTE: Software calculates the course of corresponding mean temperature over time.</li>
</ol>
6. Coagulation Depth Measurement
<ol>
	<li>Harvest the left medial liver lobe with sharp scissors.</li>
	<li>Excise the coagulation sites with 1 cm thickness. Cut into 3 mm thick longitudinal segments for further processing.</li>
	<li>Fix tissue samples at 4 &#176; C overnight with neutral 10% buffered formalin. Heat paraffin 2 &#176;C over the melting point and embed slices. Process overnight.</li>
	<li>Perform Hematoxylin/Eosin staining.
	<ol>
		<li>Deparaffinize and hydrate the tissue by subsequently dipping in 2x Xylene, 100% ethanol (EtOH), 95% EtOH, 70% EtOH, deionized H2O for 2 min each.</li>
		<li>Stain the tissue sample with Meyer&#39;s Hematoxylin solution for 3 min.</li>
		<li>Now rinse in tap water for 5 min.</li>
		<li>Stain the tissue with Eosin solution for 3 min.</li>
		<li>Rinse in 2x EtOH 95% and then Xylene for 3 min each. Mount with the standard mounting medium.</li>
	</ol>
	</li>
	<li>Switch the system on (microscope connected camera, imaging software). View all sections with 40X magnification.</li>
	<li>Take an image of an object micrometer at a magnification of 40X. Press Recalibrate button in the Objectives window. Select manual calibration. Draw a line on the micrometer image of 100 &#181;m. Enter 0,1 mm in the dialog box and press OK.</li>
	<li>Select Length in the View&#62;Analysis Controls&#62;Annotations and Measurements window. Measure from the liver surface to the coagulation margin with the mouse. Export or note the result. Repeat measurement on another location on the same slide. 
	NOTE: Coagulation depth can easily be differentiated from the normal liver tissue by the sharp margin between the normal hepatocyte cords and the zone of necrosis with shrunken cytoplasm, pyknotic nuclei, and hemorrhage zones.</li>
	<li>Calculate mean out of two measurements.</li>
</ol>
7. Burst Pressure Measurement
<ol>
	<li>Switch the system on (automatic pumps, pressure meter). Prepare the liver samples according to the step 3.7. 
	NOTE: Use two parallel pumps connected via a 3-way-stopcock. The maximal pressure of 1,500 mm Hg cannot be obtained with a single pump.</li>
	<li>Isolate portal vein, common hepatic artery and bile duct with scissors in the haptic pedicle. Clamp portal vein with an overholt forceps and ligate with a monofil suture 4-0. Clamp common hepatic artery an overholt forceps and ligate with a monofil suture 4-0.</li>
	<li>Insert Ch-16 catheter into the common bile duct and ligate with a 2-0 silk suture. Connect the catheter to the automatic pumps, install 3-way stopcock with pressure meter (Figure 3).</li>
	<li>Fill the perfusion syringe with saline.</li>
	<li>Start automatic pumps with a delivery rate of 99 mL/h.</li>
	<li>Monitor liver cut surface and pressure meter for leakage and record burst pressure. 
	NOTE: For easier recognition of leakage, patent blue can be added to saline (2 mL patent blue + 18 mL saline). It is easier to observe burst pressure by noticing the time of loss of pressure on the pressure meter.</li>
</ol>

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The authors have nothing to disclose.

Rodent models for liver surgery are established for a long time16. Nevertheless, large animal models offer certain advantages: no microsurgical equipment is needed as standard operative equipment for humans can be applied, surgical techniques are comparable to clinical use and standard clinical evaluation methods can be transferred to the experiments. For example, standard clinical blood tests can be carried out without the need for special laboratory test methods (Figure 10</...

Argon plasma coagulation (APC) is a widely used instrument in abdominal surgery for more than three decades1,2. It is a standard technique for the achievement of secondary hemostasis after major hepatectomy by sealing the liver cut surface to prevent later hemorrhages3. Plasma coagulation is a specialized form of radiofrequency electrocautery, which delivers the electrical energy through an arc of ionized gas. Providing monopolar electrothermal hemostasis, this noncontact technique has the advantage of preventing the electrode to stick to the tissue4. The ionized gas beam is automatically directed to the area of the lowest electrical resistance and is turned away when resistance rises due to desiccation to other areas not yet desiccated. This produces a uniform limited depth of coagulation5,6. Factors influencing the coagulation effect are the activation time, the power setting of the coagulation device and the distance from the probe to the tissue. Helium is another carrier gas, which can be used for plasma coagulation7. Recent clinical studies concentrated on clinical outcomes rather than histological and functional findings3,8,9, while experimental studies focused on in vitro investigations10 or experiments on isolated perfused organs11.
The underlying protocol allows the study of the effects of plasma coagulation in a large animal model close to the clinical application using standard human equipment on pigs: Microcirculation is assessed noninvasively by a laser Doppler flowmeter and spectrophotometer, which is a standard clinical tool for this indication12,13. Temperature changes during coagulation are monitored with an infrared thermometer and a thermographic camera. The depth of coagulation is measured on histological hematoxylin/eosin stained sections after harvesting of tissue samples. For the comparison with other means for secondary hemostasis, burst pressure experiments are performed. In contrast to previously described techniques14, these are conducted on explanted organs to exclude blood pressure related effects. In addition to the described investigations on the local effects of plasma coagulation, standard blood tests can also be undertaken in the porcine model.

<table><tbody><tr><td>Xylazine 20 mg/mL</td><td>Vetoquinol GmbH</td><td>Xylapan</td><td></td></tr><tr><td>Ketamine 100 mg/mL</td><td>Ceva GmbH</td><td>Ceva Ketamine Injection</td><td></td></tr><tr><td>Atropine 100 mg / 10 mL</td><td>Dr. Franz K&amp;ouml;hler Chemie GmbH</td><td>Atropinsulfat K&amp;ouml;hler 100mg Amp.</td><td></td></tr><tr><td>Propofol</td><td>Fresenius Kabi GmbH</td><td>Propofol 1% MCT Fresenius</td><td></td></tr><tr><td>Fentanyl</td><td>KG Rotexmedica GmbH</td><td>Fentanyl 0,5mg Rotexmedica</td><td></td></tr><tr><td>Isoflurane</td><td>Abbot GmbH</td><td>Forene 100% (V/V) 250 mL</td><td></td></tr><tr><td>Ringer&#039;s lactate solution</td><td>Baxter Deutschland GmbH</td><td></td><td>sodium 131mmol/l, potassium 5 mmol/l, calcium 2 mmol/l, cloride 111 mmol/l, lactate 29 mmol/l</td></tr><tr><td>Surgical disinfactant</td><td>Sch&amp;uuml;lke &amp;amp; Mayr GmbH</td><td>Kodan Tinktur forte gef&amp;auml;rbt 1l 104804</td><td></td></tr><tr><td>Motorized microscope</td><td>Nikon Instruments Europe</td><td>Eclipse TE2000-E</td><td></td></tr><tr><td>Microscope camera</td><td>Nikon Instruments Europe</td><td>Digitalsight DS-Qi1Mc</td><td></td></tr><tr><td>Imaging software</td><td>Nikon Instruments Europe</td><td>NIS elements Vers. 4.40</td><td></td></tr><tr><td>Plasma coagulator</td><td>S&amp;ouml;ring GmbH</td><td>CPC-1000</td><td></td></tr><tr><td>Argon gas</td><td>Linde AG</td><td>Argon 4.8&amp;nbsp;</td><td></td></tr><tr><td>Helium gas</td><td>Linde AG</td><td>Helium 4.8</td><td></td></tr><tr><td>O2C</td><td>LEA Medizintechnik GmbH</td><td>O2C Version 1212</td><td>with LF-2 or LF-3 probe</td></tr><tr><td>Infrared thermometer</td><td>Voltcraft</td><td>VOLTCRAFT IR 260-8S</td><td></td></tr><tr><td>Thermographic camera</td><td>InfraTec GmbH</td><td>VarioCAM HD head 820</td><td></td></tr><tr><td>Thermographic analysis software</td><td>InfraTec GmbH</td><td>IRBIS 3</td><td></td></tr><tr><td>Mayer&#039;s Hematoxylin solution</td><td></td><td>Merck 1.09249</td><td></td></tr><tr><td>Eosin solution</td><td>VWR International GmbH</td><td>Merck 1.09844</td><td></td></tr><tr><td>Rollerpump Masterflex L/S easy Load</td><td>Cole-Parmer Instrument Company</td><td>model 7518-10</td><td></td></tr><tr><td>Perfusorpump</td><td>B. Braun Melsungen AG</td><td>Perfusor secura FT</td><td></td></tr><tr><td>Digital pressure meter</td><td>Greisinger electronic</td><td>GMH 3161</td><td></td></tr><tr><td>Perfusorsyringe, 50 mL</td><td>B. Braun Melsungen AG</td><td>REF 8728810 F</td><td></td></tr><tr><td>Perfusor line, Type IV Standard, PVC Luer lock</td><td>B. Braun Melsungen AG</td><td>REF 8722960</td><td></td></tr><tr><td>3-Way stopcock, Dicofix C35C</td><td>B. Braun Melsungen AG</td><td>REF 16494 C</td><td></td></tr><tr><td>Silk 2-0. 3 metric</td><td>Resorba</td><td>REF H5F</td><td></td></tr><tr><td>Vicryl 4-0 Sutupak</td><td>Ethicon</td><td>V1224H</td><td></td></tr><tr><td>NaCl 0.9 %</td><td>B. Braun Melsungen AG</td><td></td><td></td></tr></tbody></table>

assessment of plasma coagulation on liver tissue in a large animal model in vivo

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent hemorrhages at a later stage. The exact effects of plasma coagulation on liver tissue are only poorly examined. In our porcine model, the coagulation effects can be examined close to the clinical application. A combined laser Doppler flowmeter and spectrophotometer documents microcirculation changes during coagulation at 8 mm tissue depth noninvasively, providing quantifiable information about hemostasis beyond the subjective clinical impression. The temperature at coagulation site is assessed with an infrared thermometer prior and post coagulation and with a thermographic camera during coagulation, a measurement of the gas beam temperature is not possible due to the upper threshold of the devices. The depth of coagulation is measured microscopically on hematoxylin/eosin stained sections after calibration with an object micrometer and gives an exact information about the power setting-coagulation depth-relation. The sealing effect is examined on the bile ducts as it is not possible for a plasma coagulator to seal larger blood vessels. Burst pressure experiments are carried out on explanted organs to rule out blood pressure related effects.

Microcirculation: Utilizing the diagnostic device for hemostasis following plasma coagulation can be demonstrated by microcirculation changes. Capillary blood flow (displayed as arbitrary units (AU)) decreases from a baseline value of 142.7 &#177; 76.08 AU to 57.78 &#177; 49.57 AU at 25 W device output power, to 48.5 &#177; 7.26 AU at 50 W and to 5.04 &#177; 1.31 AU at 100 W (Figure 4).
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Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

Plasma coagulation as a form of electrocautery is used in liver surgery for decades to seal the large liver cut surface after major hepatectomy to prevent ...

assessment-plasma-coagulation-on-liver-tissue-large-animal-model

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8.8K Views.  University of Bonn. Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

Video: Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Bioengineering

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via a liver laceration that results in consistent blood loss, hemodynamic alterations, and survival.
Mice undergo a standardized resection of the left-middle lobe of the liver. They are allowed to bleed without mechanical intervention. Hemostatic agents can be administered as pre-treatment or rescue therapy depending on the interest of the investigator. During the time of hemorrhage, real-time hemodynamic monitoring via a left femoral arterial line is performed. Mice are then sacrificed, blood loss is quantified, blood is collected for further analysis, and organs are harvested for analysis of injury. Experimental design is described to allow for simultaneous testing of multiple animals.
Liver hemorrhage as a model of uncontrolled hemorrhage exists in the literature, primarily in rat and porcine models. Some of these models utilize hemodynamic monitoring or quantify blood loss but lack consistency. The present model incorporates quantification of blood loss, real-time hemodynamic monitoring in a murine model that offers the advantage of using transgenic lines and a high-throughput mechanism to further investigate the pathophysiologic mechanisms in uncontrolled hemorrhage.

Uncontrolled hemorrhage, an important cause of mortality among trauma patients, can be modeled using a standard liver laceration in a murine model. This model results in consistent blood loss, survival, and allows for testing hemostatic agents. This article provides the step-by-step process to perform this valuable model.

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. Portal hypertension (PHT) due to cirrhosis is responsible for the most severe complications in patients with liver disease. The full picture of the portal hypertensive syndrome is characterized by increased portal pressure (PP) due to the increased intrahepatic vascular resistance (IHVR), hyperdynamic circulation, and increased splanchnic blood flow. Progressive splanchnic arterial vasodilation and increased cardiac output with elevated heart rate (HR) but low arterial pressure characterizes the portal hypertensive syndrome.
Novel therapies are currently being developed that aim to decrease PP by either targeting IHVR or increased splanchnic blood flow &#8212; but side effects on systemic hemodynamics may occur. Thus, a detailed characterization of portal venous, splanchnic, and systemic hemodynamic parameters, including measurement of PP, portal venous blood flow (PVBF), mesenteric arterial blood flow, mean arterial pressure (MAP), and HR is needed for preclinical evaluation of the efficacy of novel treatments for PHT. Our video article provides the reader with a structured protocol for performing invasive hemodynamic measurements in cirrhotic rats. In particular, we describe the catheterization of the femoral artery and the portal vein via an ileocolic vein and the measurement of portal venous and splanchnic blood flow via perivascular Doppler-ultrasound flow probes. Representative results of different rat models of PHT are shown.

Here we describe a detailed protocol for invasive measurements of hemodynamic parameters including portal pressure, splanchnic blood flow, and systemic hemodynamics in order to characterize the portal hypertensive syndrome in rats.

This is a detailed protocol describing invasive hemodynamic measurements in cirrhotic rats for the characterization of portal hypertensive syndrome. ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic 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.

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft failure, ongoing organ donor shortages, and the increased use of marginal grafts call for the improvement of current concepts, such as the implementation of organ machine perfusion. In order to evaluate new methods of graft reconditioning and modulation, translational models are required. With respect to anatomical and physiological similarities to humans and recent progress in the field of xenotransplantation, pigs have become the main large animal species used in transplantation models. After the initial introduction of a porcine orthotopic liver transplant model by Garnier et al. in 1965, several modifications have been published over the past 60 years.
Due to specifies-specific anatomical traits, a veno-venous bypass during the anhepatic phase is regarded as a necessity to reduce intestinal congestion and ischemia resulting in hemodynamic instability and perioperative mortality. However, the implementation of a bypass increases the technical and logistical complexity of the procedure. Furthermore, associated complications such as air embolism, hemorrhage, and the need for a simultaneous splenectomy have been reported previously.
In this protocol, we describe a model of porcine orthotopic liver transplantation without the use of a veno-venous bypass. The engraftment of donor livers after static cold storage of 20 h - simulating extended criteria donor conditions - demonstrates that this simplified approach can be performed without significant hemodynamic alterations or intraoperative mortality and with regular uptake of liver function (as defined by bile production and liver-specific CYP1A2 metabolism). The success of this approach is ensured by an optimized surgical technique and a sophisticated anesthesiologic volume and vasopressor management.
This model should be of special interest for workgroups focusing on the immediate postoperative course, ischemia-reperfusion injury, associated immunological mechanisms, and the reconditioning of extended criteria donor organs.

In this protocol, a model of porcine orthotopic liver transplantation after static cold storage of donor organs for 20 h without the use of a veno-venous bypass during engraftment is described. The approach uses a simplified surgical technique with minimization of the anhepatic phase and sophisticated volume and vasopressor management.

Liver transplantation is regarded as the gold standard for the treatment of a variety of fatal hepatic diseases. However, unsolved issues of chronic graft ...

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) survival model has been preferred in many settings due to sensitivity to clinically relevant doses of FVIII, whereas other established models, such as the tail clip model, require higher levels of procoagulant compounds. To avoid using survival as an endpoint, we developed a TVT model establishing blood loss and bleeding time as endpoints and full anesthesia during the entire experiment. Briefly, anesthetized mice are positioned with the tail submerged in temperate saline (37&#176;C) and dosed with the test compound in the right lateral tail vein. After 5 min, the left lateral tail vein is transected using a template guide, the tail is returned to the saline, and all bleeding episodes are monitored and recorded for 40 min while collecting the blood. If no bleeding occurs at 10 min, 20 min, or 30 min post-injury, the clot is challenged gently by wiping the cut twice with a wet gauze swab. After 40 min, blood loss is quantified by the amount of hemoglobin bled into the saline. This fast and relatively simple procedure results in consistent and reproducible bleeds. Compared to the TVT survival model, it uses a more humane procedure without compromising sensitivity to pharmacological intervention. Furthermore, it is possible to use both genders, reducing the total number of animals that need to be bred, in adherence with the principles of 3R&#39;s. A potential limitation in bleeding models is the stochastic nature of hemostasis, which can reduce the reproducibility of the model. To counter this, manual clot disruption ensures that the clot is challenged during monitoring, preventing primary (platelet) hemostasis from stopping bleeding. This addition to the catalog of bleeding injury models provides an option to characterize procoagulant effects in a standardized and humane manner.

The refined tail vein transection (TVT) bleeding model in anesthetized mice is a sensitive in vivo method for the assessment of hemophilic bleeding. This optimized TVT bleeding model uses blood loss and bleeding time as endpoints, refining other models and avoiding death as an endpoint.

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) ...

Biology

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the end pathway for both inflammation and thrombosis, it is not capable of showing the temporal changes that occur throughout the time course of these processes. Intravital microscopy (IVM) is the use of live-animal imaging to gain temporal insight into physiologic processes in vivo. This method is particularly powerful when assessing cellular and protein interactions within the circulation due to the rapid and sequential events that are often necessary for these interactions to occur. While IVM is an extremely powerful imaging methodology capable of viewing complex processes in vivo, there are a number of methodological factors that are important to consider when planning an IVM study. This paper outlines the process of conducting intravital imaging of the liver, identifying important considerations and potential pitfalls that may arise. Thus, this paper describes the use of IVM to study platelet-leukocyte-endothelial interactions in liver sinusoids to study the relative contributions of each in different models of acute liver injury.

Intravital microscopy is a powerful tool that provides insight into both the temporal and spatial relationships of rapid and/or sequential processes. Herein, we describe a protocol to assess both protein-protein interactions and platelet-neutrophil-endothelial interactions in liver sinusoids in a murine model of experimental sepsis (endotoxemia).

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the ...

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo

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Chapters in this video

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Porcine Liver Transplantation Without Veno-Venous Bypass As an Extended Criteria Donor Model

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver