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.

<ol><li>Ripoll, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatic+venous+pressure+gradient+predicts+clinical+decompensation+in+patients+with+compensated+cirrhosis%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Hepatic venous pressure gradient predicts clinical decompensation in patients with compensated cirrhosis.</a> Gastroenterology. 133 (2), 481-488 (2007).</li>
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<li>Abraldes, J. G., Pasarín, M., García-Pagán, J. C. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/17075968">Animal models of portal hypertension.</a> World Journal of Gastroenterology : WJG. 12 (41), 6577-6584 (2006).</li>
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<li>Schwabl, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pioglitazone+decreases+portosystemic+shunting+by+modulating+inflammation+and+angiogenesis+in+cirrhotic+and+non-cirrhotic+portal+hypertensive+rats%5BTitle%5D)+AND+%22Journal+of+Hepatology%22%5BJournal%5D)">Pioglitazone decreases portosystemic shunting by modulating inflammation and angiogenesis in cirrhotic and non-cirrhotic portal hypertensive rats.</a> Journal of Hepatology. 60 (6), 1135-1142 (2014).</li>
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<li>Schwabl, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+FXR+agonist+PX20606+ameliorates+portal+hypertension+by+targeting+vascular+remodelling+and+sinusoidal+dysfunction%5BTitle%5D)+AND+%22Journal+of+Hepatology%22%5BJournal%5D)">The FXR agonist PX20606 ameliorates portal hypertension by targeting vascular remodelling and sinusoidal dysfunction.</a> Journal of Hepatology. 66 (4), 724-733 (2017).</li>
<li>Mandorfer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sustained+virologic+response+to+interferon-free+therapies+ameliorates+HCV-induced+portal+hypertension%5BTitle%5D)+AND+%22J+Hepatol%22%5BJournal%5D)">Sustained virologic response to interferon-free therapies ameliorates HCV-induced portal hypertension.</a> J Hepatol. 65 (4), 692-699 (2016).</li>
<li>Schwabl, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interferon-free+regimens+improve+portal+hypertension+and+histological+necroinflammation+in+HIV%2FHCV+patients+with+advanced+liver+disease%5BTitle%5D)+AND+%22Aliment+Pharmacol+Ther%22%5BJournal%5D)">Interferon-free regimens improve portal hypertension and histological necroinflammation in HIV/HCV patients with advanced liver disease.</a> Aliment Pharmacol Ther. 45 (1), 139-149 (2017).</li>
<li>Reiberger, T., Mandorfer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beta+adrenergic+blockade+and+decompensated+cirrhosis%5BTitle%5D)+AND+%22Journal+of+Hepatology%22%5BJournal%5D)">Beta adrenergic blockade and decompensated cirrhosis.</a> Journal of Hepatology. 66 (4), 849-859 (2017).</li>
<li>Reiberger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Carvedilol+for+primary+prophylaxis+of+variceal+bleeding+in+cirrhotic+patients+with+haemodynamic+non-response+to+propranolol%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">Carvedilol for primary prophylaxis of variceal bleeding in cirrhotic patients with haemodynamic non-response to propranolol.</a> Gut. 62 (11), 1634-1641 (2013).</li>
<li>Reiberger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Austrian+consensus+guidelines+on+the+management+and+treatment+of+portal+hypertension+%28Billroth+III%29%5BTitle%5D)+AND+%22Wiener+klinische+Wochenschrift%22%5BJournal%5D)">Austrian consensus guidelines on the management and treatment of portal hypertension (Billroth III).</a> Wiener klinische Wochenschrift. 129 (3), 135-158 (2017).</li>
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</ol>

The authors have nothing to disclose.

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 preclinical11,12,13,14 and clinical studies15,16,17,18. Hence, in cirrhotic patients, PP is a hard outcome parameter, and its reduction is recommended by treatment guidelines19,20 and a main research goal of current hepatology. Comprehensive animal models are needed to characterize and translate16,21 novel treatment options of PHT22. This protocol presents the methodology needed for a detailed hemodynamic characterization, including assessment of portal pressure, the hyperdynamic circulation, splanchnic vasodilation and intrahepatic resistance. To achieve a representative and full hemodynamic data set from rodent models, experience and training of the performing operator is of utmost importance.
Prevention and control of severe bleedings are especially key skills. Blunt and precise preparations of the vascular sections of interest is critical in order to avoid cannulation failures and severe bleedings. Significant blood loss has an impact on hemodynamics and precludes accurate measurements of PP or may even result in death of the laboratory animal. Document bleedings that have occurred during the measurements in the protocols and characterize the severity and location of bleeding.
Of note, using perivascular ultrasound flow probes to assess blood flow generates only an approximation and might be subjected to reading errors, due to different vessel sizes and incorrect probe alignment. Another technique to measure blood flow, and especially blood-flow distribution (including calculation of portosystemic shunting) is the colored microsphere technique23. However, whole organs must be harvested, dissolved, and analyzed, and this omits the possibility to perform histological or expression analysis. Hence, the ultrasound technique supports the principles of the &#39;three Rs&#39; in animal research (reduce, refine, and replace) by Russell and Burch24. In addition, flow probes are suitable to monitor splanchnic blood flow in real-time and parallel to other hemodynamic parameters, while the colored microsphere technique requires integrating organ (mesenteric blood) flow over time. Moreover, colored microspheres, which usually have a diameter of 15 &#181;m, require a normal distribution of micro-vessels with a diameter &#60; 15 &#181;m in the respective organs to avoid becoming trapped and immobile, which might be not the case in cirrhotic livers.
The main limitation of this method is the need for a state of unconsciousness and anesthesia during the hemodynamic characterization of the PHT syndrome in animals. The most common and widely used injection anesthesia ketamine/xylazine often requires redosing after 30&#8211;45 min to obtain a necessary depth of anesthesia25,26; this adds time pressure especially if troubleshooting is needed. Using inhalation anesthesia involves many advantages, but special equipment is required, and safety regulations related to volatile anesthetics must be followed. The depth of anesthesia can be rapidly adapted without interfering with the surgery procedures by adjusting the anesthesia concentration. The endotracheal tube secures airways especially after activation of salvation by ketamine and the ventilation ensures sufficient oxygenation and ventilation of the animal to lower the risk of anesthesia-induced death27. While ketamine/xylazine is still widely used, low-dose isoflurane anesthesia causes no significant changes of hemodynamic or cardiovascular parameters in rats28,29.
Local experience and regulations provide stateoftheart recommendations and best practices of anesthesia, and the researchers must continuously reconsider the type of anesthesia used to perform these hemodynamic assessments30. Future experiments might use telemetry with implanted wireless pressure transducers that will overcome the current limitations related to general anesthesia and allow hemodynamic characterization of conscious animals.

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>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LabChart 7 Pro software</td><td>ADInstruments, Colorado Springs, CO, USA</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Software&lt;/em&gt;</td></tr><tr><td>ML870 PowerLab 8/30</td><td>ADInstruments, Colorado Springs, CO, USA</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Electronic multichannel recorder&lt;/em&gt;</td></tr><tr><td>MLT0380/D</td><td>ADInstruments, Colorado Springs, CO, USA</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Pressure transducer (x2: for Portal Pressure and Arterial Pressure)&lt;/em&gt;</td></tr><tr><td>ML112 Quad Bridge Amplifier</td><td>ADInstruments, Colorado Springs, CO, USA</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Bridge amplifier&lt;/em&gt;</td></tr><tr><td>TS420</td><td>Transonic Systems Inc., Ithaca, NY, USA</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Flowmeter module&lt;/em&gt;</td></tr><tr><td>Biological Research Apparatus 7025</td><td>UGO BASILE S.R.L., Comerio, Italy</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Ventilator&lt;/em&gt;</td></tr><tr><td>Vapor 2000</td><td>Dr&amp;auml;ger Medical AG &amp;amp; Co. KG, L&amp;uuml;beck, Germany</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Isofluran Vaporizer&lt;/em&gt;</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>&lt;em&gt;Ultrasonic flow probe (1mm)&lt;/em&gt;</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>&lt;em&gt;Ultrasonic flow probe (2mm)&lt;/em&gt;</td></tr><tr><td>1st for intubation &amp;amp; 2nd for clean skin incisions</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Mayo scissor [x2]&lt;/em&gt;</td></tr><tr><td>&lt;em&gt;Metzenbaum scissor&lt;/em&gt;</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td></tr><tr><td>&lt;em&gt;Cuticle scissor&lt;/em&gt;</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td></tr><tr><td>e.g. Adson Brown tissue forceps</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Tissue Forceps&lt;/em&gt;</td></tr><tr><td>&lt;em&gt;High precision 45&amp;deg; angle broad point forceps [x2]&lt;/em&gt;</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td></tr><tr><td>&lt;em&gt;Hemostat [x4]&lt;/em&gt;</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td></tr><tr><td>e.g. Mikulicz peritoneal clamp</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Curved clamp&lt;/em&gt;</td></tr><tr><td>e.g. Dieffenbach clamp</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Micro clamp&lt;/em&gt;</td></tr><tr><td>e.g. micro spatula with flat ends, width 4 mm,</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Micro metal spatula&lt;/em&gt;</td></tr><tr><td>for transbuccal suture at intubation</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Needle holder&lt;/em&gt;</td></tr><tr><td>&lt;em&gt;Scalpel grip&lt;/em&gt;</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td></tr><tr><td>selfmade</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Intubation desk&lt;/em&gt;</td></tr><tr><td>blut, flexible and with a suitable diameter for arterial cannula and venflow</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Blunt steel wire&lt;/em&gt;</td></tr><tr><td>modified arterial line 20G with Flowstich</td><td>Becton Dickinson, Farady Road, Swindon, UK</td><td>#682245</td><td>&lt;em&gt;Arterial line&lt;/em&gt;</td></tr><tr><td>&lt;em&gt;Heating pad&lt;/em&gt;</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td></tr><tr><td>&lt;em&gt;Rectal temerature probe&lt;/em&gt;</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td></tr><tr><td>&lt;em&gt;Saline heater&lt;/em&gt;</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td></tr><tr><td>&lt;em&gt;Laryngoscope (specific for animal size, e.g. rat)&lt;/em&gt;</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td></tr><tr><td>&lt;em&gt;Inductionbox for inhalation anesthesia&lt;/em&gt;</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td></tr><tr><td>&lt;em&gt;Scale (able to measure mg)&lt;/em&gt;</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td></tr><tr><td>&lt;em&gt;Hair clipper&lt;/em&gt;</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td></tr><tr><td>&lt;strong&gt;Name &lt;/strong&gt;</td><td>&lt;strong&gt;Company &lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumables &lt;/strong&gt;</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>&lt;em&gt;Peripheral venous catheter (14G)&lt;/em&gt;</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>&lt;em&gt;Catheter tube (PE-50)&lt;/em&gt;</td></tr><tr><td>e.g. Omnifix-F Solo</td><td>B. Braun Melsungen AG, Melsungen, Germany</td><td>#9161406V</td><td>&lt;em&gt;Syringe 1mL&lt;/em&gt;</td></tr><tr><td>e.g. Injekt Solo</td><td>B. Braun Melsungen AG, Melsungen, Germany</td><td>#4606051V</td><td>&lt;em&gt;Syringe 5mL&lt;/em&gt;</td></tr><tr><td>e.g. Injekt Solo</td><td>B. Braun Melsungen AG, Melsungen, Germany</td><td>#4606205V</td><td>&lt;em&gt;Syringe 20mL&lt;/em&gt;</td></tr><tr><td>e.g. BD Microlance 3, 18G - 1 1/2&quot;</td><td>Becton Dickinson S.A., Fraga, Spain</td><td>#304622</td><td>&lt;em&gt;Cannula (18G)&lt;/em&gt;</td></tr><tr><td>e.g. BD Microlance 3, 23G - 1&quot;</td><td>Becton Dickinson S.A., Fraga, Spain</td><td>#300800</td><td>&lt;em&gt;Cannula (23G)&lt;/em&gt;</td></tr><tr><td>e.g. BD Microlance 3, 30G - 1/2&quot;</td><td>Becton Dickinson S.A., Fraga, Spain</td><td>#304000</td><td>&lt;em&gt;Cannula (30G) &lt;/em&gt;</td></tr><tr><td>e.g. Leukoplast S</td><td>BSN medical GmbH, Hamburg,&amp;nbsp; Germany</td><td>#47619-00</td><td>&lt;em&gt;Adhesive tape&lt;/em&gt;</td></tr><tr><td>e.g. Gazin RK Mullkompressen (18x8cm)</td><td>Lohmann &amp;amp; Rauscher, Vienna, Austria</td><td>#10972</td><td>&lt;em&gt;Gauze compress (small)&lt;/em&gt;</td></tr><tr><td>e.g. Gazin RK Mullkompressen (5x5cm)</td><td>Lohmann &amp;amp; Rauscher, Vienna, Austria</td><td>#10961</td><td>&lt;em&gt;Gauze compress (big)&lt;/em&gt;</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>&lt;em&gt;Suture (Silk 4/0, EP 1.5)&lt;/em&gt;</td></tr><tr><td>e.g. Mersilk, 2-0 (3 Ph. Eur.), PS-1 Prime</td><td>Johnson &amp;amp; Johnson Medical GmbH - Ethicon Deutschland, Germany</td><td>#EH7552</td><td>&lt;em&gt;Transbuccal suture&lt;/em&gt;</td></tr><tr><td>e.g. Cottonbuds (2.2mm, 15cm)</td><td>Paul Hartmann AG, Heidenheim, Germany</td><td>#967936</td><td>&lt;em&gt;Cotton buds&lt;/em&gt;</td></tr><tr><td>e.g. Vue Ultrasoundgel</td><td>Optimum Medical Limited, UK</td><td>#1157</td><td>&lt;em&gt;Ultrasound gel&lt;/em&gt;</td></tr><tr><td>e.g. Glubran 2</td><td>Gem srl, Viareggio, Italy</td><td>#G-NB2-50</td><td>&lt;em&gt;Tissue glue&lt;/em&gt;</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>&lt;em&gt;Scalpel Knife&lt;/em&gt;</td></tr><tr><td>Heparin, 5000 i.E./mL (Natriumheparin)</td><td>Medicamentum Pharma GmbH, Allerheiligen im M&amp;uuml;rztal, Austria</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Heparin&lt;/em&gt;</td></tr><tr><td>Florane</td><td>Aesica Queenborough Ltd., Queenborough, UK</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Isoflurane&lt;/em&gt;</td></tr><tr><td>OeloVital (5g)</td><td>Fresenius Kabi Austira Gmbh, Graz, Austria</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Eye gel&lt;/em&gt;</td></tr><tr><td>Ketasol</td><td>aniMedica GmbH, Senden-B&amp;ouml;sensell, Germany</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Ketamine&lt;/em&gt;</td></tr><tr><td>Rompun</td><td>Bayer Austria Ges.m.b.H., Vienna, Austria</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Xylazine&lt;/em&gt;</td></tr><tr><td>Xylocain 10% Pumpspray</td><td>AstraZeneca &amp;Ouml;sterreich GmbH, Vienna, Austria</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Lidocaine pump spray&lt;/em&gt;</td></tr><tr><td>Dipidolor</td><td>Jansen-Cilag Pharma GmbH, Vienna, Austria</td><td>&amp;nbsp;-</td><td>&lt;em&gt;Piritramide&lt;/em&gt;</td></tr><tr><td>NaCl 0.9% Fresenius, 1L</td><td>Fresenius Kabi Austira GmbH, Graz, Austria</td><td>#13LIP132</td><td>&lt;em&gt;Physiological saline solution&lt;/em&gt;</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 concordantly to the hepatic and hemodynamic alterations (Figure 7A-F).
In contrast, PPVL causes prehepatic, non-cirrhotic PHT, which is characterized by an immediate increase in PP and corresponding changes in systemic hemodynamics (Figure 7G-I). However, during the time-course portosystemic collaterals develop which may lower PP.
The hemodynamic values of sham-operated animals remain at physiological levels and do not significantly change over time. The portal pressure in healthy SO animals is at maximum 5 to 6 mmHg (Figure 7J-L).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57261/57261fig1.jpg" /> 
Figure 1: Self-made intubation devices: (A) Endotracheal tube. (B) Guide wire device (C) Intubation desk. (D) Tube attached to guide wire device. <a href="/files/ftp_upload/57261/57261fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57261/57261fig2.jpg" /> 
Figure 2: Pre-surgical Preparations: (A) Intubation of animal. (B) Intramuscular and subcutaneous injection for anesthesia. (C) Fixation of animal on heating mat. (D) Placing and fixing rectal temperature probe. <a href="/files/ftp_upload/57261/57261fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57261/57261fig3.jpg" /> 
Figure 3: Heart Rate (HR) and Mean Arterial Pressure (MAP): (A) Skin incision. (B) Preparation of the femoral vascular and nerve structures. (C-F) Dissection of the femoral artery. (G) Distal suture and fixation - proximal pre-knot proximal suture. (H) Preparation of the femoral catheter. (I) Placement of the vascular micro clamp. (J) Perforation of the femoral artery with a bend needle. (K) Catheterization of the femoral artery. (L) Opening of the micro clamp for assessment of pulse. (M) Proximal fixation of catheter. (N) Distal fixation of catheter. (O) Measurement of MAP and HR. (P) Covering the surgical field with soaked small gauze compress. <a href="/files/ftp_upload/57261/57261fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57261/57261fig4.jpg" /> 
Figure 4: Superior Mesenteric Arterial Blood Flow (SMABF): (A-C) Median laparotomy. (D) Excavation of coecum. (E-F) Excavation of intestine. (G) Wrapping of the intestines in soaked gauze compress. (H-K) Preparation of the splanchnic mesenteric artery with blunt cannula hooks. (L, M) Attachment of the flow probe. (N) Application of ultrasound gel on the flow probe sensor. (O) Correct &#39;non-constrictive&#39; placement of the of the flow probe. (P) Measurement of SMABF. <a href="/files/ftp_upload/57261/57261fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57261/57261fig5.jpg" /> 
Figure 5: Portal Venous Blood Flow (PVBF): (A) Optimized dorsal view on portal vein (B) Dissection of the portal vein from mesenteric fat tissue. (C) Creation of a tissue tunnel for the portal vein flow probe. (D, E) Attachment of the flow probe to portal vein. (F) Application of ultrasound gel on the flow probe sensor. (G) Correct &#39;non-constrictive&#39; placement of the flow probe. (H) Measurement of PVBF. <a href="/files/ftp_upload/57261/57261fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57261/57261fig6.jpg" /> 
Figure 6: Portal Pressure (PP): (A) Preparation of catheter. (B) Preparation of the intestines. (C) Optimized view on the main mesenteric venous vasculature. (D) Perforation of the visceral peritoneum and advancement of the catheter closer to suitable vascular branch. (E) Catheterization of the ileocolic vein at junction angle between the main branch and a side-branch. (F) Advancement of the catheter tip into the portal vein closer to liver hilum. (G, H) Measurement of PP. <a href="/files/ftp_upload/57261/57261fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57261/57261fig7v2.jpg" /> 
Figure 7: Representative Results: Time course of (A) PP, (B) MAP, and (C) HR in BDL rats. Accordingly, changes in (D) SMABF, (E) PVBF, and (F) IHVR are observed. In the PPVL, hemodynamic changes of (G) PP, (H) MAP, and (I) HR are most pronounced in the early days after surgery. In healthy sham-operated (SO) animals, (J) PP, (K) MAP and (L) HR remain within physiological values and do not change over time. <a href="/files/ftp_upload/57261/57261fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

invasive-hemodynamic-characterization-portal-hypertensive-syndrome

Nanyang Technological University

Research

JoVE Journal

Medicine

15.0K Views.  Medical University of Vienna, Vienna, Austria. Hier beschrijven we een gedetailleerd protocol voor invasieve meting van hemodynamische parameters, met inbegrip van portal druk, splanchnic doorbloeding en systemische hemodynamica teneinde het karakteriseren van de portal hypertensieve syndroom bij ratten.

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

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

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. ...

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 ...

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

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

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

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

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

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

Measurement of the Hepatic Venous Pressure Gradient and Transjugular Liver Biopsy

Here we provide a detailed protocol describing the clinical procedure of hepatic venous pressure gradient (HVPG) measurement in patients with advanced chronic liver disease followed by an instruction for transjugular biopsy. Under local anesthesia and ultrasound guidance, a catheter introducer sheath is placed in the right internal jugular vein. Using&#160;fluoroscopic guidance, a balloon catheter is advanced into the inferior vena cava (IVC) and inserted into a large hepatic vein. Correct and sufficient wedge position of the catheter is ensured by injecting contrast media while the balloon is blocking the outflow of the cannulated hepatic vein. After calibrating the external pressure transducer, continuous pressure recordings are obtained with triplicate recordings of the wedged hepatic venous pressure (WHVP) and free hepatic venous pressure (FHVP). The difference between FHVP and WHVP is referred to as HVPG, with values &#8805;10 mm Hg indicating clinically significant portal hypertension (CSPH). Before removing the catheter, pressure readings obtained in the IVC at the same level, as well as the right atrial pressure are recorded.
Finally, a transjugular liver biopsy can be obtained via the same vascular route. Different systems are available; however, core biopsy needles are preferred over aspiration needles, especially for cirrhotic livers. Again, under fluoroscopic guidance a biopsy needle introducer sheath is advanced into an hepatic vein. Next, the transjugular biopsy needle is gently advanced through the introducer sheath: (i) in case of aspiration biopsy, the needle is advanced into the liver parenchyma under aspiration and then removed quickly, or (ii) in case of a core biopsy, the cutting-mechanism is triggered inside the parenchyma. Several separate passages can be safely performed to obtain sufficient liver specimens via transjugular biopsy. In experienced hands, the combination of these procedures takes about 30-45 min.

Here, we present a protocol for measurement of hepatic venous pressure gradient (HVPG),the gold standard to diagnose clinically significant portal hypertension. Moreover, we describe how to perform a transjugular liver biopsy within the same session.

Here we provide a detailed protocol describing the clinical procedure of hepatic venous pressure gradient (HVPG) measurement in patients with advanced ...

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. ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...

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, ...

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

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