Name: hemodynamic characterization of rodent models of pulmonary arterial hypertension
Uploaded: 2016-04-11T14:00:00.000+00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension at JoVE.com

SR is supported by NIH K08HL114643, Gilead Research Scholars in Pulmonary Arterial Hypertension and a Burroughs Wellcome Fund Career Award for Medical Scientists.

All procedures described follow the animal care guidelines of Duke University School of Medicine.
1. Prior to Starting the Procedure
Note: Prior to any animal procedures, ensure that appropriate institutional permission has been obtained. As with all procedures, use appropriate pain medication to ensure that there is no animal suffering.
<ol>
	<li>Flush catheters with heparinized sterile saline (100 U/ml) to ensure the patency. Mark a point from the tip of the catheter equivalent to the length from the heart to the mid of the neck (approximately 4 cm for rats and 2 cm for mice).</li>
	<li>Anesthetize the mouse or rat. Choices of anesthetic include isoflurane (induction 3-4%, maintenance 1.5% mixed with 100% oxygen), ketamine/xylazine (80-120/10 mg/kg) and pentobarbital (40-80 mg/kg)6.
	<ol>
		<li>For example, with ketamine:xylazine (80-120 mg/kg: 10-16 mg/kg IP for mice and 80-100 mg/kg: 5-10 mg/kg IP for rats), a single dose lasts for 20-50 min of anesthesia. For echocardiography, anesthetize mouse or rat with isoflurane (3-4% for induction and 1.5% for maintenance). Assess anesthetic depth by pinching the rodent in the surgical area to confirm that withdrawal reflexes are absent. Use veterinary ointment on eyes to prevent dryness while under anesthesia. 
		Note: Different anesthetic agents can be used to obtain reliable results with proper use and optimization (reviewed elsewhere6). Our preference for catheterization is to use ketamine:xylazine. Overdose with ketamine/xylazine may profoundly decrease heart rate and cardiac function, so it is critical to maintain proper temperature and respiratory control. To maintain heart rate (&#62; 400/min) in mice, we routinely perform bilateral vagotomy.&#160;The amount of ketamine/xylazine here will typically last 20-30 min, which is sufficient to perform either open- or closed-chest heart catheterization followed by euthanizing the animal.</li>
	</ol>
	</li>
	<li>Prepare the rat/mouse for the surgical procedure (Figure 1).
	<ol>
		<li>Shave the fur from the chest (to allow echocardiography) and from the surgical region, in the right neck.</li>
		<li>Scrub the shaved surgical regions with a circular sweep from the center outwards using betadine, followed by cleansing with a 70% alcohol swab.</li>
		<li>Place the animal on a surgical platform with a warming pad underneath. Set the heating level to maintain a body temperature of 37-37.5 &#176;C. Monitor body temperature with a rectal probe. Hypothermia can result in significant bradycardia and hyperthermia results in significant tachycardia.</li>
	</ol>
	</li>
</ol>
2. Echocardiography
Note: A full description of rodent echocardiography is described elsewhere7. For the mouse, prior to anesthesia, images can be obtained on the awake, manually restrained animal. For the rat, anesthesia prior to echocardiography is preferred as rats are too large to be manually restrained while awake).
<ol>
	<li>Parasternal Long Axis (PLAX) View.
	<ol>
		<li>Place the animal in a supine position on the platform or restrain it manually.</li>
		<li>Select B Mode to project a 2D live image.</li>
		<li>Align the ultrasound transducer with a frequency of 40 MHz for mouse or 25 MHz for Rats to the left parasternal line, and then rotate the transducer counterclockwise 30&#176; with the probe indicator pointing in the caudal direction (5 to 11 o&#39;clock line position). Angle the transducer slightly (rocking along the short axis of the transducer in the same tomographic plane) to obtain a full LV chamber view in the center of the screen.</li>
		<li>Locate and view these anatomic structures (Figure 2A): the lumen of the left ventricle (LV); Interventricular septum (IVS); the lumen of the right ventricle (RV); Ascending aorta (AO); and Left atrium (LA).</li>
		<li>Switch to M Mode, once these above structures are clearly visualized. Place the indicator line through the widest part of LV lumen using AO as the reference point and also make the focus depth lie in the center of LV Chamber (Figure 2B). Make similar measurements of the RV by changing angulation of the transducer and obtaining M mode measurements.</li>
		<li>Use cine store to create a video loop to record the data for offline measurement (LV chamber dimension, FS and LV wall thickness).</li>
		<li>Obtain a doppler tracing of the aortic outflow in PW Doppler Mode by placing the PW cursor in the aorta and recording (Figure 2C).</li>
	</ol>
	</li>
	<li>&#160;Parasternal Short-axis View (PSAX) at the Aortic Level.
	<ol>
		<li>Switch to B Mode.</li>
		<li>Rotate the transducer 90&#176; clockwise from the parasternal long axis view to obtain the parasternal short-axis view (Figure 3). Move and angle the transducer toward the cranium to identify the aortic valve cross section view.</li>
		<li>Identify the right ventricular outflow tract (RVOT) as a crescent-shaped structure localized to the upper right to the aorta, continued with pulmonary valve leaflets and pulmonary artery.</li>
		<li>Hold steady at the same position manually. Switch to PW Doppler Mode. 
		Note: A station platform for holding the rodent and probe can be used to minimize movement and variation in transducer position.</li>
		<li>Place the sample volume proximal to the level of pulmonary valve in the center of the right ventricular outflow tract and then position the cursor parallel to the direction of blood flow through the vessel (Figure 3B). 
		Note: It is important to adjust the sampling angle to the direction of blood flow or use the ultrasound software to correct for a change in angle. Without correction, the maximum angle to the vessel is 30&#176;, which corresponds to ~ 15% underestimate of the velocity.</li>
		<li>Adjust the scale (the velocity of blood flow) as needed to obtain a &#34;good&#34; Doppler envelope, which has white borders and a dark hollow inside indicating laminar blood flow (Figure 3C). Record the Doppler tracing. 
		Note: A &#34;bad&#34; Doppler envelope does not accommodate sufficient white borders and a dark hollow.</li>
		<li>If catheterization is not performed at this juncture, allow the rodent to recover if anesthesia was used. Do not leave the rodent unattended until it has regained sufficient consciousness to maintain sternal recumbency and do not return it to the company of other animals until it is fully recovered. If catheterization is performed, proceed to Section 3.</li>
	</ol>
	</li>
</ol>
3. Right Heart Catheterization
<ol>
	<li>&#160;Closed-chest approach for RV pressure measurement
	<ol>
		<li>Setup:
		<ol>
			<li>Connect the pressure transducer to input channel 1. In the software, set channel 1 for pressure and channel 2 for heart rate.</li>
			<li>To convert units to mmHg, record the baseline trace, perform a pressure calibration manually using a pressure gauge (if using a blood pressure transducer and PE tubing). Then perform units conversion under channel 1.</li>
			<li>To set heart rate, turn off the input of channel 2. Select cyclic measurements under channel 2 and choose channel 1 for source and rate for measurement.</li>
		</ol>
		</li>
		<li>Place the mouse/rat underneath a dissection microscope with the focus in depth and a magnification of 5x.</li>
		<li>Incise the skin from the mandible to the sternum (Figure 1). Place a pair of retractors to each side of the incision to fully expose the cervical area.</li>
		<li>Bluntly dissect to separate the salivary glands to expose the right external jugular vein using fine blunt tip forceps (Figure 4A, B).</li>
		<li>Carefully isolate the right external jugular vein from the surrounding connective tissue.</li>
		<li>Place two pieces of silk suture (4-0 for rats; 6-0 for mice) underneath the right external jugular vein, ligate the vein distally (as close to the mandible as possible), and then tie a loose knot proximally (Figure 4C).</li>
		<li>Use iris scissors to make a small &#34;nick&#34; (cut) proximal to the distal tied knot.</li>
		<li>Hold the catheter with a forceps and insert the catheter into the cut of the vein, and then tighten the proximal knot. 
		Note: We usually use polyethylene (PE)-10 tubing (~2 Fr size) for mice and PE-50 (~3 Fr size) for rats, which is connected to the regular pressure transducer through a 31 G or 21 G needle and calibrated. Mark the catheter with a marker at a length roughly corresponding to placement of the tip in the right ventricle. As an alternative to PE tubing, a micromanometer catheter can be used. Gently pulling the distal knot can help introduce the catheter.</li>
		<li>Gently push the catheter into the right heart and monitor the depth of advancement according to the mark. Monitor the pressure trace in the software to verify the catheter location and identify the RV pressure (Figure 5).</li>
		<li>Keep the catheter immobile and collect the data (toggle data recording next to the Start button) for 2 min.</li>
		<li>Proceed to sample collection (Section 4).</li>
	</ol>
	</li>
	<li>Open-chest Approach for RV P-V Loop Analysis. 
	Note: P-V loop analysis of the right ventricle can&#39;t be performed with a closed-chest approach due to the stiffness of the conductance catheter, which does not pass from the SVC to the RA. Commercially available conductance catheters are designed for LV P-V loop analysis.
	<ol>
		<li>In the software set Channel 1 for conductance; Channel 2 for pressure; and Channel 3 for heart rate.</li>
		<li>Intubate the rats with a 16 G Teflon tube and connect the tube to a mechanical ventilator. Calculate and set the ventilation parameters for mice or rats using the following formulas6: tidal volume (Vt, ml) = 6.2 x M1.01(M = animal mass, kg); respiration rate (RR, min-1) = 53.5 x M-0.26(Figure 6A).</li>
		<li>Spread 70% alcohol onto the fur to reduce the spread of fur onto the surgical field.</li>
		<li>Make an incision below the xyphoid process and bilaterally dissect the skin with scissors towards the flank.</li>
		<li>Cut through the abdominal wall and open the abdominal cavity by bilateral dissection along the diaphragm.</li>
		<li>Open the diaphragm to expose the apex of the heart and bilaterally cut the rib cage (Figure 6A). Prevent evaporation and tissue drying by spraying saline into the thoracic and peritoneal cavities using a syringe. 
		Note: We usually use a dissection scissor to open the abdominal cavity and rib cage. Bleeding is usually not significant, but if there is bleeding, electrocautery can be used.</li>
		<li>Carefully isolate the inferior vena cava (IVC) from the surrounding connective tissue.</li>
		<li>Place a piece of silk suture (4-0 for rats; 6-0 for mice) around the IVC, and then tie a loose knot (or thread the suture through a 16 G Teflon tube) (Figure 6B).</li>
		<li>Puncture the apical RV free wall with a 27-30 gauge needle parallel to the RV free wall and remove the needle. Be careful not to push the needle in more than 4 mm. 
		Note: Alternatively, a small piece of PE-60 tubing can be used to guide puncture of the conductance catheter into the RV apex.</li>
		<li>Insert the conductance catheter tip through the stab wound on the apical RV free wall until all electrodes are inside the ventricle (Figure 6C).</li>
		<li>Monitor the pressure volume loop in the software and then adjust the position of the catheter to obtain consistently shaped loops that do not demonstrate significant respiratory variation (Figure 7B, C).</li>
		<li>Record baseline P-V loops (toggle data recording next to the Start button) for at least 10 sec to obtain a number of P-V loops.</li>
		<li>Pull the suture placed around the IVC to alter the preloads and record the P-V loops. Analyze the data off-line and derive various parameters of RV systolic function (Figure 7D). This analysis has been described previously8. 
		Note: IVC can alternatively be occluded by forceps. Monitor the RV pressure trace to confirm the reduction of preload.</li>
		<li>Perform saline and cuvette calibrations as previously described to allow a conversion from conductance units to volume units6.</li>
		<li>After recording the data, gently pull the catheter out and place the tip of the catheter immediately into a water bath with saline. Upon finishing, clean the catheter per manufacturer&#39;s instructions.</li>
	</ol>
	</li>
</ol>
4. Collection of Heart and Lung Samples
Note: As the procedures here are described as terminal, the animal must be euthanized after either closed- or open-chest right heart catheterization.
<ol>
	<li>Euthanize the mice by opening the thorax (bilateral thoracotomy) if a closed-chest approach was used, exanguination, or by turning off the ventilator after anesthetic overdose. 
	Note: Cervical dislocation is not recommended.</li>
	<li>To perform inflation perfusion of the lung, connect the inflation tubing onto a ringstand set to inflate the lung with a pressure of 20 cmH2O (but do not open the valve yet to inflate the lungs).</li>
	<li>Bluntly dissect the trachea from surrounding muscle and connective tissue.</li>
	<li>Place a piece of silk suture (4-0 for rats; 6-0 for mice) around the trachea, and then tie a loose knot.</li>
	<li>Gently stretch the trachea by pressing the head and make a cut (70% of circumference) close to the mandible.</li>
	<li>Keep the gentle stretch and insert the tracheal cannula (20 G for mice or 16 G for rats). Secure the cannula using the suture. Connect the cannula onto the inflation tubing and tie the suture around the cannula to prevent the backflow of fixatives.</li>
	<li>Flush the lungs with PBS by using a 10 ml syringe to stab the RV free wall and inject toward the pulmonary artery. Nick the left atrium once the lungs start to blanch.</li>
	<li>Harvest the heart by cutting at the root of the aorta.</li>
	<li>Clamp the right lower lobe of lung using a mosquito hemostat and cut the right lower lobe. Place the pieces into microcentrifuge tubes and snap freeze in liquid nitrogen.</li>
	<li>Inflate the lung with 10% buffered-neutral formalin for 5 min and remove the trachea cannula followed by ligating the trachea.</li>
	<li>Dissect the lung out of the thorax and fix with 10% buffered-neutral formalin. 
	Note: Alternatively, inflate the lung with optimal cutting media (OCT, diluted 1:1 with PBS) and freeze in undiluted OCT for later preparation of frozen sections.</li>
	<li>Carefully separate the atria from the ventricles and isolate the right ventricle free wall by dissecting alongside the interventricular septum.</li>
	<li>Weigh the RV and LV + septum (LV+S) to calculate a Fulton index (RV/LV+S)9, which quantifies the degree of RV hypertrophy. 
	Note: TheFulton Index varies in different models of PH. Rat10: control, 0.28 &#177;0.01; hypoxia-induced, 0.57 &#177;0.02; MCT-treated, 0.51 &#177;0.03. C57BL6/J mouse11: control, 0.26 &#177;0.01; SuHx (14 days), 0.40 &#177;0.02; SuHx (21 days), 0.43 &#177;0.01; SuHx (28 days), 0.44 &#177;0.03.</li>
	<li>Snap freeze the RV and LV+S in liquid nitrogen or fix in 10% buffered-neutral formalin.</li>
</ol>

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The authors have nothing to disclose.

The protocols outlined here describe a comprehensive characterization of hemodynamics and right ventricular function in rodent models of pulmonary hypertension. While right heart catheterization as described here is a terminal procedure, the mortality associated with echocardiography is minimal, which allows for screening and follow-up of disease progression. However, similar to patients with PH having markedly increased mortality with anesthesia17, in our experience, rats with severe PH do not tolerate anesth...

Pulmonary arterial hypertension (PAH) is a disease of the pulmonary vasculature associated with inflammatory cell infiltration, smooth muscle proliferation and endothelial cell apoptosis. These changes result in obliteration of pulmonary arterioles, subsequently leading to right ventricular (RV) dysfunction and heart failure. In order to understand the pathophysiology underlying PAH and RV failure in PAH, a number of different models, including genetic and pharmacologic models, for studying this disease have been developed (reviewed elsewhere1,2).
Of these models, the most popular are hypoxia-induced (Hx) PAH in the mouse and the monocrotaline (MCT) and SU5416-hypoxia (SuHx) models in the rat. In the mouse Hx model, mice are exposed to 4 weeks of hypoxia (either normobaric or hypobaric, corresponding to an altitude of 18,000 feet with a FiO2 of 0.10), with the resultant development of medial proliferation, increased RV systolic pressures and the development of RV hypertrophy3. MCT at a single dose of 60 mg/kg results in injury to pulmonary endothelial cells through an unclear mechanism that then results in the development of PAH4. SU5416 is a an inhibitor of the vascular endothelial growth factor receptors (VEGFR) 1 and 2 blocker, and treatment with a single subcutaneous injection of 60 mg/kg followed by exposure to chronic hypoxia for 3 weeks results in permanent pulmonary hypertension with pathologic changes similar to that seen in the human disease, with the formation of obliterative vascular lesions5. In the past years, several transgenic mouse models for pulmonary hypertension have been developed. These include knockout and mutations of the bone morphogenetic protein receptor 2 (BMPR2), as BMPR2 gene mutations are found in both familial and idiopathic forms of PAH, heme oxygenase-1 knockout and IL-6 overexpression (reviewed elsewhere1,2).
These different rodent models of PH have different levels of pulmonary hypertension, RV hypertrophy and RV failure. While the hypoxia and various transgenic mouse models result in much milder PAH than the either rat model1, it does allow testing of different genetic mutations and their associated molecular signaling pathways. The MCT model does result in severe PAH, although MCT appears to be toxic to endothelial cells in multiple tissues4. The SuHx model is characterized by vascular changes more similar to that seen in idiopathic PAH in humans, although requires both pharmacologic manipulation and hypoxia exposure. Moreover, in all of these models, there may be a disconnection between the histopathologic changes, pulmonary pressures and RV function associated with the development of PAH. This is in contrast to the human disease, where there is usually a proportionate relationship between histopathologic changes, the severity of pulmonary hypertension and the degree of RV failure. Thus, a comprehensive characterization of these rodent models of PH is required, and involves assessments of RV function (typically by echocardiography), hemodynamics (by cardiac catheterization) and histopathology of the heart and lungs (from tissue harvesting).
In this protocol, we describe the basic techniques used for hemodynamic characterization of PAH models in the rat and the mouse. These general techniques can be applied to any study of the right ventricle and pulmonary vasculature and is not limited to models of PAH. Visualizing the RV by echocardiography is relatively straightforward in rats, but is more challenging in mice due to their size and the complex geometry of the RV. Moreover, some surrogates used for quantifying RV function, such as TAPSE, pulmonary artery (PA) acceleration time and PA Doppler waveform notching, are not well validated in humans and correlate only weakly with assessment of pulmonary hypertension and RV function by invasive hemodynamics. Determination of the RV hemodynamics is best done with a closed-chest, to maintain the effects of a negative intrathoracic pressure with inspiration, although open chest catheterization with an impedance catheter allows determination of pressure-volume (PV) loops and a more detailed hemodynamic characterization. As with any procedure, developing experience with the procedures is critical to experimental success.

<table><tbody><tr><td>Vevo 2100 Imaging System (120V)&amp;nbsp;</td><td>VisualSonics, inc.&amp;nbsp;</td><td>VS-11945</td><td></td></tr><tr><td>Vevo 2100 Imaging Station&amp;nbsp;</td><td>VisualSonics, inc.&amp;nbsp;</td><td></td><td></td></tr><tr><td>High-frequency Mechanical Transducers</td><td>VisualSonics, inc.&amp;nbsp;</td><td>MS250, MS550D, MS400</td><td></td></tr><tr><td>Ultrasound Gel Parker&amp;nbsp;</td><td>Laboratories Inc.&amp;nbsp;</td><td>01-08</td><td></td></tr><tr><td>PowerLab 4/35</td><td>ADInstruments</td><td>ML765</td><td></td></tr><tr><td>Labchart 8</td><td>ADInstruments</td><td></td><td></td></tr><tr><td>BP transducer with stopcock and cable</td><td>ADInstruments</td><td>MLT1199</td><td></td></tr><tr><td>BP transducer calibration kit</td><td>ADInstruments</td><td>MLA1052</td><td></td></tr><tr><td>Mikro-Tip Pressure Catheter for mouse</td><td>Millar</td><td>SPR-1000</td><td>Alternative catheter available from Scisense FT111B (mouse) and FT211B (rat)</td></tr><tr><td>Mikro-Tip Pressure Catheter for rat</td><td>Millar</td><td>SPR-513</td><td>Alternative catheter available from Scisense FT111B (mouse) and FT211B (rat)</td></tr><tr><td>Millar Mikro-Tip&amp;nbsp;ultra-miniature PV loop catheter for mice</td><td>Millar</td><td>PVR-1035</td><td>Alternative catheter available from Scisense FT112 (mouse)</td></tr><tr><td>Millar Mikro-Tip ultra miniature PV loop catheter for rats</td><td>Millar</td><td>SPR-869</td><td>Alternative catheter available from Scisense FT112 (mouse)</td></tr><tr><td>Millar PV system MPVS-300&amp;nbsp;</td><td>Millar</td><td>MPVS-300</td><td></td></tr><tr><td>4-0 Silk Black Braid 100 Yard Spool</td><td>Roboz Surgical</td><td>SUT-15-2</td><td></td></tr><tr><td>6-0 Silk Black Braid 100 Yard Spool</td><td>Roboz Surgical</td><td>SUT-14-1</td><td></td></tr><tr><td>Iris Scissors, Delicate, Integra Miltex</td><td>VWR</td><td>21909-248</td><td></td></tr><tr><td>VWR Dissecting Scissors, Sharp/Blunt Tip</td><td>VWR</td><td>82027-588</td><td></td></tr><tr><td>VWR Delicate Scissors, 4 1/2&quot;</td><td>VWR</td><td>82027-582</td><td></td></tr><tr><td>Two star Hemostats, Excelta</td><td>VWR</td><td>63042-090</td><td></td></tr><tr><td>Neutral-buffered formalin</td><td>VWR</td><td>89370-094</td><td></td></tr><tr><td>Crotaline</td><td>Sigma</td><td>C2401</td><td></td></tr><tr><td>SU5416</td><td>Tocris Biosciences</td><td>3037</td><td></td></tr><tr><td>3.5X-45X Boom Stand Trinocular Zoom Stereo Microscope&amp;nbsp;</td><td>AmScope</td><td>SM-3BX</td><td></td></tr><tr><td>PE (Polyethylene Tubing)-10</td><td>Braintree Scientific Inc</td><td>PE10 36 FT</td><td></td></tr><tr><td>PE (Polyethylene Tubing)-50</td><td>Braintree Scientific Inc</td><td>PE50 36 FT</td><td></td></tr><tr><td>PE (Polyethylene Tubing)-60</td><td>Braintree Scientific Inc</td><td>PE60 36 FT</td><td></td></tr><tr><td>Tabletop Isoflurane Anesthesia Unit</td><td>Kent Scientific</td><td>ACV-1205S</td><td></td></tr><tr><td>Surgisuite multi-functional surgical platform</td><td>Kent Scientific</td><td>Surgisuite</td><td></td></tr><tr><td>Retractor set</td><td>Kent Scientific</td><td>SURGI-5002</td><td></td></tr><tr><td>Anesthesia induction chamber</td><td>VetEquip</td><td>941443</td><td></td></tr><tr><td>Anesthesia Gas filter canister</td><td>Kent Scientific</td><td>ACV-2001</td><td></td></tr><tr><td>Rodent nose cone</td><td>VetEquip</td><td>921431</td><td></td></tr></tbody></table>

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.

As right heart catheterization in rodents is typically a terminal procedure that is not applicable to longitudinal follow-up, echocardiography is an excellent noninvasive alternative for screening and follow-up12. While pulmonary artery systolic pressure in human PAH on echocardiography is usually derived from tricuspid regurgitation that is usually straightforward to be obtained in the apical view, such a view is not reliably obtained in rodents, preventing the estimation of pulmonary artery systolic pressure...

Watch this Scientific Journal Video about Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension at JoVE.com

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

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 ...

hemodynamic-characterization-rodent-models-pulmonary-arterial

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

20.8K Views.  Duke University Medical Center. 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.

Video: Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Immunology and Infection

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

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 ...

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

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

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

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

The Left Pneumonectomy Combined with Monocrotaline or Sugen as a Model of Pulmonary Hypertension in Rats

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats with the additional administration of monocrotaline (MCT) or SU5416 (Sugen). We also compare these two models to other PAH models commonly used in research. In the last few years, the focus of animal PAH models has moved towards studying the mechanism of angioproliferation of plexiform lesions, in which the role of increased pulmonary blood flow is considered as an important trigger in the development of severe pulmonary vascular remodeling. One of the most promising rodent models of increased pulmonary flow is the unilateral left pneumonectomy combined with a "second hit" of MCT or Sugen. The removal of the left lung leads to increased and turbulent pulmonary blood flow and vascular remodeling. Currently, there is no detailed procedure of the pneumonectomy surgery in rats. This article details a step-by-step protocol of the pneumonectomy surgical procedure and post-operative care in male Sprague-Dawley rats. Briefly, the animal is anesthetized and the chest is opened. Once the left pulmonary artery, pulmonary vein, and bronchus are visualized, they are ligated and the left lung is removed. The chest then closed and the animal recovered. Blood is forced to circulate only on the right lung. This increased vascular pressure leads to a progressive remodeling and occlusion of small pulmonary arteries. The second hit of MCT or Sugen is used one week post-surgery to induce endothelial dysfunction. The combination of increased blood flow in the lung and endothelial dysfunction produces severe PAH. The primary limitation of this procedure is that it requires general surgical skills.

The rodent left pneumonectomy is a valuable technique in pulmonary hypertension research. Here, we present a protocol to describe the rat pneumonectomy procedure and postoperative care to ensure minimal morbidity and mortality.

In this protocol, we detail the correct procedural steps and necessary precautions to successfully perform a left pneumonectomy and induce PAH in rats ...

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heart&#160;catheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response to&#160;treatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the right&#160;ventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

This protocol describes the induction of pulmonary hypertension (PH) in mice based on the exposure to hypoxia and the injection of a VEGF receptor antagonist. The animals develop PH and right ventricular (RV) hypertrophy 3 weeks after the initiation of the protocol. The functional and morphometrical characterization of the model is also presented.

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by ...

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. ...

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, ...

Creating a Murine Model of RV Hypertrophy and Failure by Pulmonary Trunk Banding

A Murine Model of Pressure Overload-Induced Right Ventricular Hypertrophy and Failure by Pulmonary Trunk Banding

Right ventricular (RV) failure caused by pressure overload is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary diseases. The pathogenesis of RV failure is complex and remains inadequately understood. To identify new therapeutic strategies for the treatment of RV failure, robust and reproducible animal models are essential. Models of pulmonary trunk banding (PTB) have gained popularity, as RV function can be assessed independently of changes in the pulmonary vasculature.
In this paper, we present a murine model of RV pressure overload induced by PTB in 5-week-old mice. The model can be used to induce different degrees of RV pathology, ranging from mild RV hypertrophy to decompensated RV failure. Detailed protocols for intubation, PTB surgery, and phenotyping by echocardiography are included in the paper. Furthermore, instructions for customizing instruments for intubation and PTB surgery are given, enabling fast and inexpensive reproduction of the PTB model.
Titanium ligating clips were used to constrict the pulmonary trunk, ensuring a highly reproducible and operator-independent degree of pulmonary trunk constriction. The severity of PTB was graded by using different inner ligating clip diameters (mild: 450 &#181;m and severe: 250 &#181;m). This resulted in RV pathology ranging from hypertrophy with preserved RV function to decompensated RV failure with reduced cardiac output and extracardiac manifestations. RV function was assessed by echocardiography at 1 week and 3 weeks after surgery. Examples of echocardiographic images and results are presented here. Furthermore, results from right heart catheterization and histological analyses of cardiac tissue are shown.

Author Spotlight: Investigating the Underlying Mechanisms of Right Ventricular Failure in Pulmonary Hypertension

We describe a murine model of right ventricular pressure overload-induced by pulmonary trunk banding. Detailed protocols for intubation, surgery, and phenotyping by echocardiography are included in the paper. Custom-made instruments are used for intubation and surgery, allowing for fast and inexpensive reproduction of the model.

Right ventricular (RV) failure caused by pressure overload is strongly associated with morbidity and mortality in a number of cardiovascular and pulmonary ...

Optimizing Pulmonary Artery Banding for Consistent Right Heart Failure Induction

Rat Model of Right-Sided Cardiac Remodeling and Arrhythmia Using Pulmonary Artery Banding

Clinical conditions, including chronic obstructive pulmonary disease or pulmonary arterial hypertension (PAH), can lead to chronic right ventricle pressure overload and progressive right heart failure (RHF). RHF can be identified by right-sided cardiac hypertrophy and dilation associated with abnormal myocardial function affecting the RV and the right atrium (RA). We recently demonstrated that severe RHF is accompanied by an increased risk of atrial inflammation, atrial fibrosis, and atrial fibrillation (AF), the most common type of cardiac arrhythmia (CA). Recent studies have shown that RV and RA inflammation plays an important role in the arrhythmogenesis of CA, including AF. However, the impact of inflammation in the development of CA and AF in RHF is poorly described.
Experimental models of RHF are required to better understand the association between right-sided myocardial inflammation and CA. The rat model of monocrotaline (MCT)-induced pulmonary hypertension (PH) is well-established to provoke RHF. However, MCT triggers severe pneumo-toxicity and pulmonary inflammation. Hence, MCT-induced RHF does not help to distinguish whether the subsequent myocardial inflammation originates from the RHF per se or circulating inflammatory signals secreted by the injured lung.
In this article, a mechanical method involving pulmonary artery trunk banding (PAB) was used to provoke right-sided cardiac arrhythmogenesis. The PAB consists of performing a permanent suture of the pulmonary artery trunk for 3 weeks. Such an approach generates increased right-sided pressure overload. At D21 post-PAB, the suture results in hypertrophied, dilated, and inflamed RV and RA. The PAB-induced RHF is also accompanied by vulnerability to ventricular and atrial arrhythmias, including AF.

Right heart failure (RHF) is characterized by right-sided cardiac dilation and hypertrophy, leading to ventricular and atrial malfunction. Cardiopulmonary conditions associated with RHF are accompanied by an increased risk for cardiac arrhythmias. This article describes a standardized model of pulmonary artery banding-induced RHF associated with enhanced ventricular and atrial arrhythmogenesis.