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. 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 — 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.
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 — defining the portal hypertensive syndrome4. Ohm's law (ΔP = 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 1–5 mmHg, while an HVPG ≥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.
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 40–50 min/animal has to be considered in the case all hemodynamic readouts presented in this protocol.
1. Pre-surgical Preparations
2. Measurement of HR and MAP
3. Superior Mesenteric Artery Blood Flow (SMABF)
4. PVBF
5. PP
6. IHVR
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).
Figure 1: Self-made intubation devices: (A) Endotracheal tube. (B) Guide wire device (C) Intubation desk. (D) Tube attached to guide wire device. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 'non-constrictive' placement of the of the flow probe. (P) Measurement of SMABF. Please click here to view a larger version of this figure.
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 'non-constrictive' placement of the flow probe. (H) Measurement of PVBF. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 'three Rs' 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 µm, require a normal distribution of micro-vessels with a diameter < 15 µm 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 30–45 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.
The authors have nothing to disclose.
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 "Young Science Award" of the Austrian Society of Gastroenterology and Hepatology (ÖGGH) to PS and the "Skoda Award" of the Austrian Society of Internal Medicine to TR.
Instruments | |||
LabChart 7 Pro software | ADInstruments, Colorado Springs, CO, USA | - | Software |
ML870 PowerLab 8/30 | ADInstruments, Colorado Springs, CO, USA | - | Electronic multichannel recorder |
MLT0380/D | ADInstruments, Colorado Springs, CO, USA | - | Pressure transducer (x2: for Portal Pressure and Arterial Pressure) |
ML112 Quad Bridge Amplifier | ADInstruments, Colorado Springs, CO, USA | - | Bridge amplifier |
TS420 | Transonic Systems Inc., Ithaca, NY, USA | - | Flowmeter module |
Biological Research Apparatus 7025 | UGO BASILE S.R.L., Comerio, Italy | - | Ventilator |
Vapor 2000 | Dräger Medical AG & Co. KG, Lübeck, Germany | - | Isofluran Vaporizer |
Perivascular probes (rat) for Transonic systems (Superior Mesenteric Artery) | Transonic Systems Inc., Ithaca, NY, USA | #MA1PRB | Ultrasonic flow probe (1mm) |
Perivascular probes (rat) for Transonic systems (Portal Vein) | Transonic Systems Inc., Ithaca, NY, USA | #MA2PSB | Ultrasonic flow probe (2mm) |
1st for intubation & 2nd for clean skin incisions | - | - | Mayo scissor [x2] |
Metzenbaum scissor | - | - | - |
Cuticle scissor | - | - | - |
e.g. Adson Brown tissue forceps | - | - | Tissue Forceps |
High precision 45° angle broad point forceps [x2] | - | - | - |
Hemostat [x4] | - | - | - |
e.g. Mikulicz peritoneal clamp | - | - | Curved clamp |
e.g. Dieffenbach clamp | - | - | Micro clamp |
e.g. micro spatula with flat ends, width 4 mm, | - | - | Micro metal spatula |
for transbuccal suture at intubation | - | - | Needle holder |
Scalpel grip | - | - | - |
selfmade | - | - | Intubation desk |
blut, flexible and with a suitable diameter for arterial cannula and venflow | - | - | Blunt steel wire |
modified arterial line 20G with Flowstich | Becton Dickinson, Farady Road, Swindon, UK | #682245 | Arterial line |
Heating pad | - | - | - |
Rectal temerature probe | - | - | - |
Saline heater | - | - | - |
Laryngoscope (specific for animal size, e.g. rat) | - | - | - |
Inductionbox for inhalation anesthesia | - | - | - |
Scale (able to measure mg) | - | - | - |
Hair clipper | - | - | - |
Name | Company | Catalog Number | Comments |
Consumables | |||
e.g. modified BD Venflon Pro Safety 14GA | Becton Dickinson Infusion Therapy, AB, SE251 06 Helsingborg, Sweden | #393230 | Peripheral venous catheter (14G) |
Fine-Bore Polyethylene Tubing, ID 0.58mm, OD 0.96mm, Portex, | Smiths Medical International Ltd., Kent, UK | #800/100/200 | Catheter tube (PE-50) |
e.g. Omnifix-F Solo | B. Braun Melsungen AG, Melsungen, Germany | #9161406V | Syringe 1mL |
e.g. Injekt Solo | B. Braun Melsungen AG, Melsungen, Germany | #4606051V | Syringe 5mL |
e.g. Injekt Solo | B. Braun Melsungen AG, Melsungen, Germany | #4606205V | Syringe 20mL |
e.g. BD Microlance 3, 18G – 1 1/2" | Becton Dickinson S.A., Fraga, Spain | #304622 | Cannula (18G) |
e.g. BD Microlance 3, 23G – 1" | Becton Dickinson S.A., Fraga, Spain | #300800 | Cannula (23G) |
e.g. BD Microlance 3, 30G – 1/2" | Becton Dickinson S.A., Fraga, Spain | #304000 | Cannula (30G) |
e.g. Leukoplast S | BSN medical GmbH, Hamburg, Germany | #47619-00 | Adhesive tape |
e.g. Gazin RK Mullkompressen (18x8cm) | Lohmann & Rauscher, Vienna, Austria | #10972 | Gauze compress (small) |
e.g. Gazin RK Mullkompressen (5x5cm) | Lohmann & Rauscher, Vienna, Austria | #10961 | Gauze compress (big) |
Silk Braided black, USP 4/0, EP 1.5 | SMI AG, St. Vith, Belgium | #2021-04 | Suture (Silk 4/0, EP 1.5) |
e.g. Mersilk, 2-0 (3 Ph. Eur.), PS-1 Prime | Johnson & Johnson Medical GmbH – Ethicon Deutschland, Germany | #EH7552 | Transbuccal suture |
e.g. Cottonbuds (2.2mm, 15cm) | Paul Hartmann AG, Heidenheim, Germany | #967936 | Cotton buds |
e.g. Vue Ultrasoundgel | Optimum Medical Limited, UK | #1157 | Ultrasound gel |
e.g. Glubran 2 | Gem srl, Viareggio, Italy | #G-NB2-50 | Tissue glue |
e.g. Surgical scalpell knife Nr. 10 – carbon steel | Swann-Morton, England, B.S. | #202 | Scalpel Knife |
Heparin, 5000 i.E./mL (Natriumheparin) | Medicamentum Pharma GmbH, Allerheiligen im Mürztal, Austria | - | Heparin |
Florane | Aesica Queenborough Ltd., Queenborough, UK | - | Isoflurane |
OeloVital (5g) | Fresenius Kabi Austira Gmbh, Graz, Austria | - | Eye gel |
Ketasol | aniMedica GmbH, Senden-Bösensell, Germany | - | Ketamine |
Rompun | Bayer Austria Ges.m.b.H., Vienna, Austria | - | Xylazine |
Xylocain 10% Pumpspray | AstraZeneca Österreich GmbH, Vienna, Austria | - | Lidocaine pump spray |
Dipidolor | Jansen-Cilag Pharma GmbH, Vienna, Austria | - | Piritramide |
NaCl 0.9% Fresenius, 1L | Fresenius Kabi Austira GmbH, Graz, Austria | #13LIP132 | Physiological saline solution |