Name: invasive hemodynamic characterization of the portal-hypertensive syndrome in cirrhotic rats
Uploaded: 2018-08-01T13:00:00
Description: Watch this Scientific Journal Video about Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats at JoVE.com

We thank the veterinarians, nurses, and animal keepers at the Center of Biomedical Research for their continuous support during our research projects. The authors acknowledge the important input of all reviewers of this protocol. Some of the research was funded by the &#34;Young Science Award&#34; of the Austrian Society of Gastroenterology and Hepatology (&#214;GGH) to PS and the &#34;Skoda Award&#34; of the Austrian Society of Internal Medicine to TR.

All methods described here have been approved by the ethics committee of the Medical University of Vienna and the Austrian Ministry of Science, Research and Economy (BMWFW). Procedures must be performed in aseptic conditions in an operation room or similar clean working area since the hemodynamic measurements represent surgical interventions. Generally, working in sterile conditions is recommended. When using an inhalation anesthesia, consider adequate ventilation of the surgery room for work safety. A time period of 40&...

<ol>
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M., Van Steenkiste, C., Van Vlierberghe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+Changes+in+Splanchnic+Blood+Vessels+in+Portal+Hypertension.">Hemodynamic Changes in Splanchnic Blood Vessels in Portal Hypertension.</a> Advances in Integrative Anatomy and Evolutionary Biology. 291 (6), 699-713 (2008).</li><li>Laleman, W., Van Landeghem, L., Wilmer, A., Fevery, J., Nevens, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portal+hypertension:+from+pathophysiology+to+clinical+practice.">Portal hypertension: from pathophysiology to clinical practice.</a> Liver International. 25 (6), 1079-1090 (2005).</li><li>Franchis, R. d. . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Updating+Consensus+in+Portal+Hypertension:+Report+of+the+Baveno+III+Consensus+Workshop+on+definitions,+methodology+and+therapeutic+strategies+in+portal+hypertension.">Updating Consensus in Portal Hypertension: Report of the Baveno III Consensus Workshop on definitions, methodology and therapeutic strategies in portal hypertension.</a> Journal of Hepatology. 33 (5), 846-852 (2000).</li><li>Zardi, E. 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PP is the main outcome parameter for evaluation of the portal hypertensive syndrome and reflects the severity of underlying cirrhosis. Both matrix deposition (i.e., fibrosis) and sinusoidal vasoconstriction (due to increased hepatic expression of vasoconstrictors and decreased responsiveness to vasodilators) cause increased IHVR. The importance of PP and its impact on chronic liver disease has been shown in multiple preclinical11,12,<sup class...

PHT is defined as pathologically increased blood pressure in the portal venous system that can cause severe complications in patients with cirrhosis such as variceal bleeding and ascites1. While pre-hepatic (e.g., portal vein thrombosis) and post-hepatic (e.g., Budd-Chiari Syndrome) PHT are rare, intrahepatic PHT due to liver cirrhosis represents the most common cause of PHT2.
In liver cirrhosis, PP is primarily increased as a consequence of elevated IHVR3. In advanced stages, PHT is aggravated by the increased PVBF due to increased cardiac output and decreased systemic and splanchnic vascular resistance &#8212; defining the portal hypertensive syndrome4. Ohm&#39;s law (&#916;P = Q * R) implies that the IHVR and blood flow are proportional to PP5. In patients, direct measurement of PP is risky and not routinely performed; instead, the hepatic venous pressure gradient (HVPG) is used as an indirect measure of PP6,7. The HVPG is calculated by subtracting the free hepatic venous pressure (FHVP) from the wedged hepatic venous pressure (WHVP), which are measured using a balloon catheter placed in a hepatic vein8. The physiological HVPG ranges between 1&#8211;5 mmHg, while an HVPG &#8805;10 mmHg defines clinically significant portal hypertension (CSPH) and indicates increased risk for PHT-related complications, such as variceal bleeding, ascites, and hepatic encephalopathy9. Although PP (i.e., HVPG) is the most relevant parameter for PHT severity, information about other components of PHT, including the severity of hyperdynamic circulation (HR, MAP), splanchnic/mesenteric arterial blood flow, and IHVR, are critical to obtain a comprehensive understanding of the distinct underlying mechanism of PHT.
Thus, in contrast to indirect measurements of PP in humans, the introduced methodology for rats offers the advantage of a direct measurement of PP and allows the recording of additional hemodynamic parameters characterizing the portal hypertensive syndrome. In addition, the direct measurement of PP is an excellent integrative readout of the amount of liver fibrosis (a major determinant of IHVR) and overcomes certain limitations of fibrosis quantification related to liver tissue sampling errors.
The most commonly used rodent models of cirrhotic PHT include surgical bile duct ligation (BDL), toxin-induced liver injury (i.e., by carbon tetrachloride, thioacetamide, or dimethylnitrosamine administration), and diet-induced metabolic liver disease models. Prehepatic (non-cirrhotic) PHT can be induced by partial portal vein ligation (PPVL)10.
Small rodents are well suited for the presented method, including mice, hamsters, rats, or rabbits, and are associated with relatively low maintenance costs. Despite that all the hemodynamic assessments are feasible to perform in mice, better accuracy and reproducibility of results are seen with rats or larger rodents due to the obvious advantage of animal size. In addition, specific micro-instruments and devices are needed to obtain similar hemodynamic parameters in mice. Finally, rats are more robust with lower associated morbidity and mortality and thus, the drop-out rates are likely lower in rats than in mice.
The presented methodology is well-suited for evaluating specific treatments of liver disease (i.e., anti-fibrotic or anti-inflammatory drugs) or novel pharmacological approaches that influence vascular tone and/or endothelial biology; and thus, likely effect hemodynamic parameters in PHT.

<table>
	<tbody>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart 7 Pro software</td>
			<td>ADInstruments, Colorado Springs, CO, USA</td>
			<td>&#160;-</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>ML870 PowerLab 8/30</td>
			<td>ADInstruments, Colorado Springs, CO, USA</td>
			<td>&#160;-</td>
			<td>Electronic multichannel recorder</td>
		</tr>
		<tr>
			<td>MLT0380/D</td>
			<td>ADInstruments, Colorado Springs, CO, USA</td>
			<td>&#160;-</td>
			<td>Pressure transducer (x2: for Portal Pressure and Arterial Pressure)</td>
		</tr>
		<tr>
			<td>ML112 Quad Bridge Amplifier</td>
			<td>ADInstruments, Colorado Springs, CO, USA</td>
			<td>&#160;-</td>
			<td>Bridge amplifier</td>
		</tr>
		<tr>
			<td>TS420</td>
			<td>Transonic Systems Inc., Ithaca, NY, USA</td>
			<td>&#160;-</td>
			<td>Flowmeter module</td>
		</tr>
		<tr>
			<td>Biological Research Apparatus 7025</td>
			<td>UGO BASILE S.R.L., Comerio, Italy</td>
			<td>&#160;-</td>
			<td>Ventilator</td>
		</tr>
		<tr>
			<td>Vapor 2000</td>
			<td>Dr&#228;ger Medical AG &#38; Co. KG, L&#252;beck, Germany</td>
			<td>&#160;-</td>
			<td>Isofluran Vaporizer</td>
		</tr>
		<tr>
			<td>Perivascular probes (rat) for Transonic systems (Superior Mesenteric Artery)</td>
			<td>Transonic Systems Inc., Ithaca, NY, USA</td>
			<td>#MA1PRB</td>
			<td>Ultrasonic flow probe (1mm)</td>
		</tr>
		<tr>
			<td>Perivascular probes (rat) for Transonic systems (Portal Vein)</td>
			<td>Transonic Systems Inc., Ithaca, NY, USA</td>
			<td>#MA2PSB</td>
			<td>Ultrasonic flow probe (2mm)</td>
		</tr>
		<tr>
			<td>1st for intubation &#38; 2nd for clean skin incisions</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>Mayo scissor [x2]</td>
		</tr>
		<tr>
			<td>Metzenbaum scissor</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
		</tr>
		<tr>
			<td>Cuticle scissor</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
		</tr>
		<tr>
			<td>e.g. Adson Brown tissue forceps</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>Tissue Forceps</td>
		</tr>
		<tr>
			<td>High precision 45&#176; angle broad point forceps [x2]</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
		</tr>
		<tr>
			<td>Hemostat [x4]</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
		</tr>
		<tr>
			<td>e.g. Mikulicz peritoneal clamp</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>Curved clamp</td>
		</tr>
		<tr>
			<td>e.g. Dieffenbach clamp</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>Micro clamp</td>
		</tr>
		<tr>
			<td>e.g. micro spatula with flat ends, width 4 mm,</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>Micro metal spatula</td>
		</tr>
		<tr>
			<td>for transbuccal suture at intubation</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>Needle holder</td>
		</tr>
		<tr>
			<td>Scalpel grip</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
		</tr>
		<tr>
			<td>selfmade</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>Intubation desk</td>
		</tr>
		<tr>
			<td>blut, flexible and with a suitable diameter for arterial cannula and venflow</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>Blunt steel wire</td>
		</tr>
		<tr>
			<td>modified arterial line 20G with Flowstich</td>
			<td>Becton Dickinson, Farady Road, Swindon, UK</td>
			<td>#682245</td>
			<td>Arterial line</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
		</tr>
		<tr>
			<td>Rectal temerature probe</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
		</tr>
		<tr>
			<td>Saline heater</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
		</tr>
		<tr>
			<td>Laryngoscope (specific for animal size, e.g. rat)</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
		</tr>
		<tr>
			<td>Inductionbox for inhalation anesthesia</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
		</tr>
		<tr>
			<td>Scale (able to measure mg)</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
		</tr>
		<tr>
			<td>Hair clipper</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
			<td>&#160;-</td>
		</tr>
		<tr>
			<td>Name </td>
			<td>Company </td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Consumables </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>e.g. modified BD Venflon Pro Safety 14GA</td>
			<td>Becton Dickinson Infusion Therapy, AB, SE251 06 Helsingborg, Sweden</td>
			<td>#393230</td>
			<td>Peripheral venous catheter (14G)</td>
		</tr>
		<tr>
			<td>Fine-Bore Polyethylene Tubing, ID 0.58mm, OD 0.96mm, Portex,</td>
			<td>Smiths Medical International Ltd., Kent, UK</td>
			<td>#800/100/200</td>
			<td>Catheter tube (PE-50)</td>
		</tr>
		<tr>
			<td>e.g. Omnifix-F Solo</td>
			<td>B. Braun Melsungen AG, Melsungen, Germany</td>
			<td>#9161406V</td>
			<td>Syringe 1mL</td>
		</tr>
		<tr>
			<td>e.g. Injekt Solo</td>
			<td>B. Braun Melsungen AG, Melsungen, Germany</td>
			<td>#4606051V</td>
			<td>Syringe 5mL</td>
		</tr>
		<tr>
			<td>e.g. Injekt Solo</td>
			<td>B. Braun Melsungen AG, Melsungen, Germany</td>
			<td>#4606205V</td>
			<td>Syringe 20mL</td>
		</tr>
		<tr>
			<td>e.g. BD Microlance 3, 18G - 1 1/2&#34;</td>
			<td>Becton Dickinson S.A., Fraga, Spain</td>
			<td>#304622</td>
			<td>Cannula (18G)</td>
		</tr>
		<tr>
			<td>e.g. BD Microlance 3, 23G - 1&#34;</td>
			<td>Becton Dickinson S.A., Fraga, Spain</td>
			<td>#300800</td>
			<td>Cannula (23G)</td>
		</tr>
		<tr>
			<td>e.g. BD Microlance 3, 30G - 1/2&#34;</td>
			<td>Becton Dickinson S.A., Fraga, Spain</td>
			<td>#304000</td>
			<td>Cannula (30G) </td>
		</tr>
		<tr>
			<td>e.g. Leukoplast S</td>
			<td>BSN medical GmbH, Hamburg,&#160; Germany</td>
			<td>#47619-00</td>
			<td>Adhesive tape</td>
		</tr>
		<tr>
			<td>e.g. Gazin RK Mullkompressen (18x8cm)</td>
			<td>Lohmann &#38; Rauscher, Vienna, Austria</td>
			<td>#10972</td>
			<td>Gauze compress (small)</td>
		</tr>
		<tr>
			<td>e.g. Gazin RK Mullkompressen (5x5cm)</td>
			<td>Lohmann &#38; Rauscher, Vienna, Austria</td>
			<td>#10961</td>
			<td>Gauze compress (big)</td>
		</tr>
		<tr>
			<td>Silk Braided black, USP 4/0, EP 1.5</td>
			<td>SMI AG, St. Vith, Belgium</td>
			<td>#2021-04</td>
			<td>Suture (Silk 4/0, EP 1.5)</td>
		</tr>
		<tr>
			<td>e.g. Mersilk, 2-0 (3 Ph. Eur.), PS-1 Prime</td>
			<td>Johnson &#38; Johnson Medical GmbH - Ethicon Deutschland, Germany</td>
			<td>#EH7552</td>
			<td>Transbuccal suture</td>
		</tr>
		<tr>
			<td>e.g. Cottonbuds (2.2mm, 15cm)</td>
			<td>Paul Hartmann AG, Heidenheim, Germany</td>
			<td>#967936</td>
			<td>Cotton buds</td>
		</tr>
		<tr>
			<td>e.g. Vue Ultrasoundgel</td>
			<td>Optimum Medical Limited, UK</td>
			<td>#1157</td>
			<td>Ultrasound gel</td>
		</tr>
		<tr>
			<td>e.g. Glubran 2</td>
			<td>Gem srl, Viareggio, Italy</td>
			<td>#G-NB2-50</td>
			<td>Tissue glue</td>
		</tr>
		<tr>
			<td>e.g. Surgical scalpell knife Nr. 10 - carbon steel</td>
			<td>Swann-Morton, England, B.S.</td>
			<td>#202</td>
			<td>Scalpel Knife</td>
		</tr>
		<tr>
			<td>Heparin, 5000 i.E./mL (Natriumheparin)</td>
			<td>Medicamentum Pharma GmbH, Allerheiligen im M&#252;rztal, Austria</td>
			<td>&#160;-</td>
			<td>Heparin</td>
		</tr>
		<tr>
			<td>Florane</td>
			<td>Aesica Queenborough Ltd., Queenborough, UK</td>
			<td>&#160;-</td>
			<td>Isoflurane</td>
		</tr>
		<tr>
			<td>OeloVital (5g)</td>
			<td>Fresenius Kabi Austira Gmbh, Graz, Austria</td>
			<td>&#160;-</td>
			<td>Eye gel</td>
		</tr>
		<tr>
			<td>Ketasol</td>
			<td>aniMedica GmbH, Senden-B&#246;sensell, Germany</td>
			<td>&#160;-</td>
			<td>Ketamine</td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Bayer Austria Ges.m.b.H., Vienna, Austria</td>
			<td>&#160;-</td>
			<td>Xylazine</td>
		</tr>
		<tr>
			<td>Xylocain 10% Pumpspray</td>
			<td>AstraZeneca &#214;sterreich GmbH, Vienna, Austria</td>
			<td>&#160;-</td>
			<td>Lidocaine pump spray</td>
		</tr>
		<tr>
			<td>Dipidolor</td>
			<td>Jansen-Cilag Pharma GmbH, Vienna, Austria</td>
			<td>&#160;-</td>
			<td>Piritramide</td>
		</tr>
		<tr>
			<td>NaCl 0.9% Fresenius, 1L</td>
			<td>Fresenius Kabi Austira GmbH, Graz, Austria</td>
			<td>#13LIP132</td>
			<td>Physiological saline solution</td>
		</tr>
	</tbody>
</table>

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.

Depending on the animal model and the severity of liver disease, the degree of PHT and severity of the portal hypertensive syndrome is different (Figure 7).
The BDL model causes biliary cirrhosis due to cholestasis. Accordingly, PP increases over time and a hyperdynamic circulation develops, as seen by an increase of HR and decrease of MAP. In cirrhotic animals, SMABF, PVBF and IHVR also increase co...

Watch this Scientific Journal Video about Invasive Hemodynamic Characterization of the Portal-hypertensive Syndrome in Cirrhotic Rats at JoVE.com

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

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. ...

This method can help answer key questions in the field of advanced liver fibrosis and portal hypertension. It offers a unique opportunity to evaluate pharmacological approaches that target vascular tone, fibrogenesis, or endothelial dysfunction in rats with portal-hypertensive syndrome. The main advantages of this technique include the direct measurement of portal pressure and the opportunity to obtain additional hemodynamic parameters in contrast to the indirect measurement of portal pressure by hepatic venous pressure gradient, HVPG, in patients.Begin by sedating the rat, using short-time isoflurane anesthesia and optionally additional anesthetics. Quickly proceed to intubation, using a suitable self-made endotracheal tube. Use two fingers to grab the tongue, and gently pull it.Then, carefully moisten the laryngeal area with a cotton swab soaked in lidocaine spray. Next, gently insert the endotracheal tube between the vocal folds and into the trachea. Fix the endotracheal loop by a transbuccal suture through the cheek and the adhesive tape loop of the tube.After administering analgesia and injectable anesthesia, apply eye ointment. Then, clip the body hair at the abdominal region and both inner thighs, and disinfect the skin. Assess the anesthesia depth by lid-closure reflex and toe-pinch test prior to any intervention or surgery.Use tissue forceps to lift the skin on one of the shaved thighs above the presumed location of the femoral artery. Use Mayo scissors to remove a two-centimeter area of skin. Then, use a hemostat to blunt dissect the artery-vein-nerve complex from the connective tissue.For better visibility of the artery-vein-nerve complex, the cuticle scissor can be used to remove parts of fat tissue. Next, use two high-precision, 45-degree angle, broad-point forceps to separate the femoral artery from the femoral vein and nerve. The femoral vein is the most medial structure, and the femoral artery is located more lateral.Place a ligature on the femoral artery as distal as possible, and use a curved clamp on the suture to apply gentle longitudinal traction to the femoral artery. Then, place a second, loosely pre-knotted suture on the femoral artery as proximal as possible. Compress the femoral artery at the proximal end of the exposed section, using a micro clamp to temporarily stop the arterial blood flow.Then, place a supporting micro metal spatula underneath the femoral artery, and use a bent 23-gauge cannula to perforate the anterior arterial wall distal to the occluded section. Carefully catheterize the femoral artery through the perforation with the slanted tip of the catheter facing upward. Advance the catheter until it reaches the micro clamp.Fix the catheter in its intraluminal location by closing the prepared proximal ligature around the femoral artery and its introduced catheter. Then, open the micro clamp, and check for pulsatile arterial blood flow into the catheter. Fix the catheter position along the vessel, using the ends of the distal ligature to prevent dislocation and to ensure a longitudinal position of the catheter.Use the digital interface to start recording the heart rate and mean atrial pressure. Begin by performing a median laparotomy. First, lift the skin layer with tissue forceps five to six centimeters below the xiphoid, and use a Mayo scissor to remove a thin strip of skin, using above the linea alba until the xiphoid is reached.Then, use tissue forceps to lift the muscular layer at the center of the linea alba to create distance between the abdominal wall and the splanchnic organs. Open the peritoneal cavity by incising the abdominal wall with a scalpel at the linea alba. Extend the incision along the linea alba, over the same distance as the skin layer.Next, carefully excavate the intestine using wet cotton buds. Start with the caecum, and lay it on a large gauze compress, soaked in sterile physiological saline solution placed next to the incision. Wrap the intestine with the gauze compress, and make sure it is moistened with sterile physiological saline solution.The cirrhotic liver should now be visible. Then, dissect the superior mesenteric artery with two self-made blunt Schwabl hooks, lift the artery with the first hook, and place the second one through the tissue tunnel. Expose the superior mesenteric artery along a five-millimeter distance to ensure the flow probe can be placed around it.Next, apply ultrasound gel to the ultrasonic flow probe sensor. Attach the sensor to the splanchnic mesenteric artery so that it is aligned to the natural route of the superior mesenteric artery. Measure the superior mesenteric artery blood flow, and assess the accordance of the pulsatile flow signal to the systolic peaks of the femoral artery recording.To measure portal vein blood flow, first locate and expose the portal vein at the dorsal face of the mesentery that is close to the liver hilum. Then, use high-precision, 45-degree angle, broad-point forceps to gently dissect the portal vein from the surrounding tissue. Enlarge the tissue tunnel, and expose the portal vein along a distance of five to six millimeters to allow the placement of the perivascular flow probe.Apply ultrasound gel to the ultrasonic flow probe sensor, and attach it to the portal vein aligned with its natural route. Then, close the flow probe, and start recording the portal vein blood flow. To measure portal pressure, wet gloves with physiological saline, and spread the intestine across the fingers.Optimize the view of the mesenteric vascular bed close to the small intestine. Insert the catheter into the mesenteric tissue by perforating the visceral peritoneum of the mesentery close to the vascular junction chosen for catheterization. Then, carefully advance the slanted tip of the catheter closer to a junction of the ileocolic vein until a slight impression of the vessel junction is seen.Finally, catheterize the venous system in line with the joining vessel route by perforating the vascular wall at the crossing angle of the vessels. Record all hemodynamic parameters simultaneously under stable conditions for several minutes before euthanizing the animal. The bile duct ligation model causes biliary cirrhosis due to cholestasis.Accordingly, portal pressure increases over time, and a hyperdynamic circulation develops, as evident by an increased heart rate and decrease of mean arterial pressure. In cirrhotic animals, intrahepatic vascular resistance and superior mesenteric arterial blood flow increase, while subsequently portal venous blow flow rises, depending on the amount of portosystemic collateralization. In contrast, partial portal vein ligation causes prehepatic, non-cirrhotic portal hypertension, which is characterized by an immediate increase in portal pressure.However, during the time course, portosystemic collaterals develop that may lower portal pressure. In addition, we see corresponding changes in mean arterial pressure and heart rate. For healthy, sham-operated animals, their hemodynamic values remain at physiological levels and do not significantly change over time.The portal pressure in healthy, SO animals is at maximum five to six millimeters of mercury. Once mastered, these techniques can be performed within 40 to 50 minutes, depending on the animal model, need for troubleshooting, and optimal pre-surgical preparations. While performing the surgical procedure, it's very important to monitor the temperature of the animal, as well as the heart rate and the arterial pressure in order to assess the level of anesthesia.After watching this video, you should have a good understanding of how to cannulate the femoral artery and the portal vein and how to attach ultrasound flow probes to splanchnic blood vessels in a safe way that will guarantee accurate and reproducible results.

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Research

JoVE Journal

Medicine

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle proliferation and obliteration of pulmonary arterioles. This in turn results in right ventricular (RV) failure, with significant morbidity and mortality. Rodent models of PAH, in the mouse and the rat, are important for understanding the pathophysiology underlying this rare disease. Notably, different models of PAH may be associated with different degrees of pulmonary hypertension, RV hypertrophy and RV failure. Therefore, a complete hemodynamic characterization of mice and rats with PAH is critical in determining the effects of drugs or genetic modifications on the disease. 
Here we demonstrate standard procedures for assessment of right ventricular function and hemodynamics in both rat and mouse PAH models. Echocardiography is useful in determining RV function in rats, although obtaining standard views of the right ventricle is challenging in the awake mouse. Access for right heart catheterization is obtained by the internal jugular vein in closed-chest mice and rats. Pressures can be measured using polyethylene tubing with a fluid pressure transducer or a miniature micromanometer pressure catheter. Pressure-volume loop analysis can be performed in the open chest. After obtaining hemodynamics, the rodent is euthanized. The heart can be dissected to separate the RV free wall from the left ventricle (LV) and septum, allowing an assessment of RV hypertrophy using the Fulton index (RV/(LV+S)). Then samples can be harvested from the heart, lungs and other tissues as needed.

Pulmonary arterial hypertension (PAH) is a disease of pulmonary arterioles that leads to their obliteration and the development of right ventricular failure. Rodent models of PAH are critical in understanding the pathophysiology of PAH. Here we demonstrate hemodynamic characterization, with right heart catheterization and echocardiography, in the mouse and rat.

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature characterized by endothelial cell apoptosis, smooth muscle ...

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 ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, there is no cure for PAH. It is important to discover new compounds that can be used to treat PAH. The mouse hypoxia-induced PAH model is a widely used model for PAH research. This model recapitulates human clinical manifestations of PAH Group 3 disease and is an important research tool to evaluate the effectiveness of new experimental therapies for PAH. Research using this model often requires the administration of compounds in mice. For a compound that needs to be given directly into the bloodstream, optimizing intravenous (IV) administration is a key part of the experimental procedures. Ideally, the IV injection system should permit multiple injections over a set time course. Although the mouse hypoxia-induced PAH model is very popular in many laboratories, it is technically challenging to perform multiple IV bolus dosing and invasive hemodynamic assessment in this model. In this protocol, we present step-by-step instructions on how to carry out multiple IV bolus dosing via mouse jugular vein and perform arterial and right ventricle catheterization for hemodynamic assessment in mouse hypoxia-induced PAH model.

This protocol provides a step-by-step procedure for executing multiple intravenous bolus dose administration and invasive hemodynamic monitoring in mice. Investigators can use this protocol for future therapeutic compound screening for pulmonary artery hypertension.

Pulmonary arterial hypertension (PAH) is a progressive life-threatening disease, primarily affecting small pulmonary arterioles of the lung. Currently, ...