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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H., Inui, H., Kamiyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+laparoscopic+haemostasis+using+an+argon+beam+coagulator+for+blunt+traumatic+splenic+injury.">Successful laparoscopic haemostasis using an argon beam coagulator for blunt traumatic splenic injury.</a> EurJSurg. 167, 316-318 (2001).</li><li>Frilling, A., 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=Effectiveness+of+a+new+carrier-bound+fibrin+sealant+versus+argon+beamer+as+haemostatic+agent+during+liver+resection:+a+randomised+prospective+trial.">Effectiveness of a new carrier-bound fibrin sealant versus argon beamer as haemostatic agent during liver resection: a randomised prospective trial.</a> Langenbecks ArchSurg. 390, 114-120 (2005).</li><li>Raiser, J., Zenker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Argon+plasma+coagulation+for+open+surgical+and+endoscopic+applications:+state+of+the+art.">Argon plasma coagulation for open surgical and endoscopic applications: state of the art.</a> J Phys Appl Phys. 39 (16), 3520-3523 (2006).</li><li>Farin, G., Grund, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technology+of+argon+plasma+coagulation+with+particular+regard+to+endoscopic+applications.">Technology of argon plasma coagulation with particular regard to endoscopic applications.</a> EndoscSurgAllied Technol. 2, 71-77 (1994).</li><li>Grund, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Argon+plasma+coagulation+(APC):+ballyhoo+or+breakthrough?.">Argon plasma coagulation (APC): ballyhoo or breakthrough?.</a> Endoscopy. 29, 196-198 (1997).</li><li>Glowka, T. R., Standop, J., Paschenda, P., Czaplik, M., Kalff, J. C., Tolba, R. 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U., Blum, A., Schlegel, A., Farin, G., Garbe, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+argon+plasma+coagulation+in+skin+surgery.">Application of argon plasma coagulation in skin surgery.</a> Dermatology. 197, 152-157 (1998).</li><li>Carus, T., Rackebrandt, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collateral+tissue+damage+by+several+types+of+coagulation+(monopolar,+bipolar,+cold+plasma+and+ultrasonic)+in+a+minimally+invasive,+perfused+liver+model.">Collateral tissue damage by several types of coagulation (monopolar, bipolar, cold plasma and ultrasonic) in a minimally invasive, perfused liver model.</a> ISRNSurg. , 518924 (2011).</li><li>Bludau, M., Vallbohmer, D., Gutschow, C., Holscher, A. 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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 border="0" cellpadding="0" cellspacing="0" width="824">
	<tbody>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Xylazine 20 mg/mL</td>
			<td style="width:184px;">Vetoquinol GmbH</td>
			<td style="width:192px;">Xylapan</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Ketamine 100 mg/mL</td>
			<td style="width:184px;">Ceva GmbH</td>
			<td style="width:192px;">Ceva Ketamine Injection</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Atropine 100 mg / 10 mL</td>
			<td style="width:184px;">Dr. Franz K&#246;hler Chemie GmbH</td>
			<td style="width:192px;">Atropinsulfat K&#246;hler 100mg Amp.</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;">Propofol</td>
			<td style="width:184px;">Fresenius Kabi GmbH</td>
			<td style="width:192px;">Propofol 1% MCT Fresenius</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;">Fentanyl</td>
			<td style="width:184px;">KG Rotexmedica GmbH</td>
			<td style="width:192px;">Fentanyl 0,5mg Rotexmedica</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;">Isoflurane</td>
			<td style="width:184px;">Abbot GmbH</td>
			<td style="width:192px;">Forene 100% (V/V) 250 mL</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;">Ringer&#39;s lactate solution</td>
			<td style="width:184px;">Baxter Deutschland GmbH</td>
			<td style="width:192px;"></td>
			<td style="width:232px;">sodium 131mmol/l, potassium 5 mmol/l, calcium 2 mmol/l, cloride 111 mmol/l, lactate 29 mmol/l</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Surgical disinfactant</td>
			<td style="width:184px;">Sch&#252;lke &#38; Mayr GmbH</td>
			<td style="width:192px;">Kodan Tinktur forte gef&#228;rbt 1l 104804</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Motorized microscope</td>
			<td style="width:184px;">Nikon Instruments Europe</td>
			<td style="width:192px;">Eclipse TE2000-E</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Microscope camera</td>
			<td style="width:184px;">Nikon Instruments Europe</td>
			<td style="width:192px;">Digitalsight DS-Qi1Mc</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Imaging software</td>
			<td style="width:184px;">Nikon Instruments Europe</td>
			<td style="width:192px;">NIS elements Vers. 4.40</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Plasma coagulator</td>
			<td style="width:184px;">S&#246;ring GmbH</td>
			<td style="width:192px;">CPC-1000</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Argon gas</td>
			<td style="width:184px;">Linde AG</td>
			<td style="width:192px;">Argon 4.8&#160;</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Helium gas</td>
			<td style="width:184px;">Linde AG</td>
			<td style="width:192px;">Helium 4.8</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">O2C</td>
			<td style="width:184px;">LEA Medizintechnik GmbH</td>
			<td style="width:192px;">O2C Version 1212</td>
			<td style="width:232px;">with LF-2 or LF-3 probe</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;">Infrared thermometer</td>
			<td style="width:184px;">Voltcraft</td>
			<td>VOLTCRAFT IR 260-8S</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Thermographic camera</td>
			<td>InfraTec GmbH</td>
			<td>VarioCAM HD head 820</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Thermographic analysis sofrtware</td>
			<td>InfraTec GmbH</td>
			<td style="width:192px;">IRBIS 3</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Mayer&#39;s Hematoxylin solution</td>
			<td></td>
			<td style="width:192px;">Merck 1.09249</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Eosin solution</td>
			<td style="width:184px;">VWR International GmbH</td>
			<td style="width:192px;">Merck 1.09844</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Rollerpump Masterflex L/S easy Load</td>
			<td style="width:184px;">Cole-Parmer Instrument Company</td>
			<td style="width:192px;">model 7518-10</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Perfusorpump</td>
			<td style="width:184px;">B. Braun Melsungen AG</td>
			<td style="width:192px;">Perfusor secura FT</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Digital pressure meter</td>
			<td style="width:184px;">Greisinger electronic</td>
			<td style="width:192px;">GMH 3161</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Perfusorsyringe, 50 mL</td>
			<td style="width:184px;">B. Braun Melsungen AG</td>
			<td style="width:192px;">REF 8728810 F</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Perfusor line, Type IV Standard, PVC Luer lock</td>
			<td style="width:184px;">B. Braun Melsungen AG</td>
			<td style="width:192px;">REF 8722960</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">3-Way stopcock, Dicofix C35C</td>
			<td style="width:184px;">B. Braun Melsungen AG</td>
			<td style="width:192px;">REF 16494 C</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Silk 2-0. 3 metric</td>
			<td style="width:184px;">Resorba</td>
			<td style="width:192px;">REF H5F</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">Vicryl 4-0 Sutupak</td>
			<td style="width:184px;">Ethicon</td>
			<td>V1224H</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">NaCl 0.9 %</td>
			<td style="width:184px;">B. Braun Melsungen AG</td>
			<td style="width:192px;"></td>
			<td style="width:232px;"></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

Research

JoVE Journal

Medicine

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

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Assessment of Child Anthropometry in a Large Epidemiologic Study

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to understand health effects of child growth and adiposity. Gold standard methods of measuring adiposity, such as dual X-ray absorptiometry (DXA), may not be feasible in large field studies. Research staff can, however, complete anthropometric measurements, such as body circumferences and skinfold measurements, using inexpensive portable equipment. In this protocol we detail how to obtain manual anthropometric measurements from children, including standing and sitting height, weight, waist circumference, hip circumference, mid-upper arm circumference, triceps skinfold thickness, and subscapular skinfold thickness, and procedures to assess the quality of these measurements. To demonstrate accuracy of these measurements, among 1,110 school-aged children in the pre-birth cohort Project Viva we calculated Spearman correlation coefficients comparing manual anthropometric measurements with a gold standard measure of body fat, DXA fat mass1. To address reliability, we evaluate intra-rater technical error of measurement at a quality control session conducted on adult female volunteers.

In epidemiologic studies of children, well-trained research staff can accurately and precisely assess weight, height, sitting height, skinfold thicknesses, and body circumferences.

A high proportion of children have overweight and obesity in the United States and other countries. Accurate assessment of anthropometry is essential to ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Invasive Hemodynamic Monitoring of Aortic and Pulmonary Artery Hemodynamics in a Large Animal Model of ARDS

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to pulmonary hypertension. For a better understanding and treatment of this disease, precise hemodynamic monitoring of left and right ventricular parameters is important. For this reason, it is essential to establish experimental pig models of cardiac hemodynamics and measurements for research purpose. 
This article shows the induction of ARDS by using oleic acid (OA) and consequent right ventricular dysfunction, as well as the instrumentation of the pigs and the data acquisition process that is needed to assess hemodynamic parameters. To achieve right ventricular dysfunction, we used oleic acid (OA) to cause ARDS and accompanied this with pulmonary artery hypertension (PAH). With this model of PAH and consecutive right ventricular dysfunction, many hemodynamic parameters can be measured, and right ventricular volume load can be detected. 
All vital parameters, including respiratory rate (RR), heart rate (HR) and body temperature were recorded throughout the whole experiment. Hemodynamic parameters including femoral artery pressure (FAP), aortic pressure (AP), right ventricular pressure (peak systolic, end systolic and end diastolic right ventricular pressure), central venous pressure (CVP), pulmonary artery pressure (PAP) and left arterial pressure (LAP) were measured as well as perfusion parameters including ascending aortic flow (AAF) and pulmonary artery flow (PAF). Hemodynamic measurements were performed using transcardiopulmonary thermodilution to provide cardiac output (CO). Furthermore, the PiCCO2 system (Pulse Contour Cardiac Output System 2) was used to receive parameters such as stroke volume variance (SVV), pulse pressure variance (PPV), as well as extravascular lung water (EVLW) and global end-diastolic volume (GEDV). Our monitoring procedure is suitable for detecting right ventricular dysfunction and monitoring hemodynamic findings before and after volume administration.

We present a protocol of creating right ventricular dysfunction in a pig model by inducing ARDS. We demonstrate invasive monitoring of left and right ventricular cardiac output using flow probes around the aorta and the pulmonary artery, as well as blood pressure measurements in the aorta and pulmonary artery.

One of the leading causes of morbidity and mortality in patients with heart failure is right ventricular (RV) dysfunction, especially if it is due to ...

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, ...

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 ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...