Name: invasive hemodynamic assessment for the right ventricular system and hypoxia-induced pulmonary arterial hypertension in mice
Uploaded: 2019-10-24T13:30:00.000+00:00
Duration: 4 min 54 s
Description: Watch this Scientific Journal Video about Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice at JoVE.com

This research is supported by the Postgraduate Education and Teaching Reform Project of Peking Union Medical College (10023-2016-002-03), the Fuwai Hospital Youth Fund (2018-F09), and the Director Fund of Beijing Key Laboratory of Pre-clinical Research and Evaluation for Cardiovascular Implant Materials (2018-PT2-ZR05).

The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee at Fuwai Hospital, Chinese Academy of Medical Science, Peking Union Medical College (NO.0000287). The experimental animals were housed and fed according to the guidelines of animal welfare in China.
NOTE: Eight- to 12-week-old male C57BL mice were housed in an environment with a 12 h dark/ 12 h light cycle. The PAH mice were housed for 4 weeks under an oxygen concentration of 10%, maintained by an oxygen-controlled hypoxia chamber to induce pulmonary hypertension, and control mice were housed in room air (21% oxygen) under identical conditions. RVP and PAP measurements were performed at the end of the 4 weeks of hypoxia challenge.
1. Preoperative preparation
<ol>
	<li>Soak the pressure transducer catheter (size: 1 Fr) in 0.9% saline at room temperature for at least 30 min before the hemodynamic experiment.</li>
	<li>Filter 2,2,2-Tribromoethanol solution with 0.22 &#956;m filter and store in 4-degree refrigerator.</li>
	<li>Prepare cleaned surgery tools and supplies such as gloves for surgery.</li>
	<li>Prepare 10 mL of 1.0% digestive enzyme solution for catheter cleaning.</li>
	<li>Connect the pressure transducer catheter to a pressure-volume system.</li>
	<li>Calibrate the pressure transducer prior to obtaining pressure measurements for each mouse.
	<ol>
		<li>Turn the calibration knob to 0 mmHg and 25 mmHg to send a verification pressure signal to the data acquisition software and configure the calibration setting in the software.</li>
		<li>Turn the knob to Transducer and adjust the Balance knob to zero baseline.</li>
	</ol>
	</li>
	<li>Set up a standard stereomicroscope and a temperature-controlled small animal surgical table for body temperature maintenance during the surgery.</li>
	<li>Set up a light illumination system for microsurgery to provide enough light over the surgical area.</li>
</ol>
2. Open-Chest surgery and hemodynamic measurement
<ol>
	<li>Anesthetize mice with 250 mg/kg of 2,2,2-Tribromoethanol via intraperitoneal (i.p.) injection. If needed, repeat supplemental doses at 1/3 to 1/2 of the original dose during the procedure.</li>
	<li>Remove chest and neck fur using a shaver and hair removal lotion (Figure 1A, 2A).</li>
	<li>Secure each mouse in the supine position on a temperature-controlled small animal surgical table to help maintain body temperature (37 &#176;C) during surgery.</li>
	<li>Clean the surgical site with 70% ethanal.</li>
	<li>Once anesthesia is in effect, confirm adequate anesthesia induction using a toe pinch.</li>
	<li>Make a midline incision on the neck skin (Figure 1A).</li>
	<li>Dissect the skeletal muscle using curved forceps and expose the trachea (Figure 1B, 1C).</li>
	<li>Perform intubation through the mouth using a modified 22 G intravenous sheath catheter. Confirm that the tubing is in the trachea using forceps (Figure 1D).</li>
	<li>Connect the tubing to a small animal ventilator. Calculate and set respiration rate and tidal volume based on body weight according to the ventilator user manual10. For example, set respiration rate to 133/min and tidal volume to 180 &#956;L for a 30 g mouse based on the described calculation.</li>
	<li>Secure the tubing for ventilation using tape.</li>
	<li>Confirm adequate anesthesia induction using a toe pinch.</li>
	<li>Make a midline incision on the chest skin and carefully dissect the chest muscles using a cautery tool (Figure 2B, 2C).</li>
	<li>Cut the sternum using scissors across the middle and expose the thoracic cavity (Figure 2D).</li>
	<li>Prevent any bleeding using the cautery tool during the open-chest surgery procedure.</li>
	<li>Expose the right ventricle with retractors (Figure 2E).</li>
	<li>Insert the saline-soaked pressure transducer catheter through a small tunnel created with a 25 G needle into the right ventricle to measure RVP (Figure 2F and Figure 3A, 3C).</li>
	<li>Hold the catheter cable and cross the pulmonary valve in a coaxial manner with the pulmonary artery. Observe the pressure waveform and obtain a stable PAP signal (Figure 3B, 3D).</li>
	<li>Record hemodynamic data using the data acquisition system and software.</li>
	<li>After the final measurements, euthanize mice humanely through i.p. injection of an excess dose of 2,2,2-Tribromoethanol solution.</li>
	<li>Carefully remove catheter from the right heart system and place into a 1 mL syringe containing 1% digestive enzyme solution.</li>
	<li>Use distilled water to continuously flush the catheter carefully and store it in the original box.</li>
</ol>
3. Data analysis for hemodynamics
NOTE: The hemodynamic data were recorded and analyzed using analysis software11&#160;(Table of Materials).
<ol>
	<li>For each mouse, select at least 10 continuous and stable heartbeat cycles without noise to obtain the average data of RVP or PAP data for each parameter.</li>
	<li>Use Student&#8217;s t-test to compare the normal air control and hypoxia groups. NOTE: p &#60; 0.05 was considered statistically significant. Data are presented as the mean &#177; SD.</li>
</ol>

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The authors have nothing to disclose.

Tracheal intubation is the first important step for open-chest surgeries. The classic method of tracheal intubation for small animals, such as rats or mice, involves making a T-shaped incision on the trachea and directly inserting Y-type tracheal tubing into the trachea. In practice, we find that this method is not easy during operation. The Y-type tracheal tubing is too large for small animals and forms an angle with the trachea. Thus, it is difficult to fix the tubing in place. Additionally, once the intubation tubing accidentally comes out from the airway during open-chest surgery, it usually results in animal death because of loss of mechanical ventilation support. Therefore, we modified the method of endotracheal intubation14&#160;by making an incision on the skin, separating the muscle layer to expose the trachea (Figure 1C), and directly inserting the tracheal tubing into the airway through the animal&#39;s mouth. The placement of tubing in the trachea can be conveniently confirmed by clamping the trachea using forceps (Figure 1D). After removing the guide needle and only using the sheath catheter, a 22 G intravenous catheter is used as the intubation tubing. The tubing can be easily secured after intubation. This is a safe way to manage intubation during surgery and can significantly improve the success rate of small animal open-chest surgery. However, this method requires training and practice.
The closed-chest approach for right heart hemodynamic measurement has been described in detail15,16. One limitation of the closed-chest method is that it can be used to only evaluate RVP, because the catheter cannot access the pulmonary artery in mice. We use a midline chest incision where the right ventricular free wall is located, just below the sternum (Figure 2D). After right ventricular catheterization to obtain RVP, it is easy to insert the catheter in a coaxial manner with the pulmonary artery to get PAP (Figure 2E). When the sternum is cut during the open-chest surgery, an electrocoagulation tool is used to avoid sternal cutting section bleeding to prevent artificial blood pressure decrease caused by blood loss (Figure 2C). It is optional for this open-chest surgery to use a P-V loop catheter to get both RVP and volume information17. However, it is best to not use it to obtain PAP because of its bigger size. Although this method is best performed by a well-trained surgeon, it is preferable to the closed-chest approach because it allows for the maintenance of tracheal intubation and prevention of bleeding during open-chest surgery to avoid animal death.
Additionally, the right ventricular free wall is punctured with a 25 G or smaller needle to reduce resistance during the insertion of the catheter into the ventricle. During catheterization, &#160;the pressure sensor surface must not deviate from the bevel of the needle to prevent accidental damage to the catheter sensor by the sharp metal surface. It is preferable to not use a large needle to puncture the ventricular free wall, as it usually causes further bleeding, and the insufficient blood volume in circulation also causes artificial pressure data.
Because of the small volume of the ventricle and the irregular size of the right ventricular chamber in mice, the pressure sensor of the catheter easily touches the right ventricular free wall during the high heartbeat rate. This generates noise on the ventricular pressure curve (Figure 4), directly affecting ventricular pressure analysis. In this case, the angle and depth of the catheter should be adjusted until the noise disappears to obtain a smooth ventricular pressure waveform again.
The small size of the 1 Fr pressure transducer catheter7 makes it a very precise, accurate pressure transducer. Zero drift is generally not experienced during a standard catheter test in saline solution in vitro unless the catheter is faulty or damaged. However, in the presence of body blood, blood components adhering to the pressure sensor surface may cause the catheter to undergo zero drift during an in vivo experiment (Figure 5). To address this issue, we do the following: temporarily remove the catheter out from the ventricular chamber and place the sensor tip of the catheter into warm 1.0% digestive enzyme solution; incubate it to digest the blood components attached to the sensor surface; and after gently wiping the catheter with saline-soaked gauze, insert the catheter back to the ventricular chamber to obtain a stable, non-zero drift ventricular pressure waveform.
The preparation of a pressure transducer catheter is also essential to obtain stable data. The pressure sensor tip of the catheter must be soaked for at least 30 min in 0.9% saline at room temperature before the in vivo procedure to maintain the stability of the catheter. In this way, the electrical characteristics of the pressure transducer catheter can be optimally stabilized.
Finally, the hypoxia period is viable from 3 to 4 weeks for the hypoxia-induced hypertension model in mice6,14,17,18. Our data showed that 4 weeks of hypoxia can induce a stable pulmonary hypertension model in C57BL mice, and the PAP and RVP levels are comparable with the literature. Further study is needed to address how long the PAH model can be maintained if the mice are put back in normoxic conditions for different hypoxia protocols.

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder with elevation in pulmonary artery pressure (PAP) and right ventricular pressure (RVP) that is caused by cellular proliferation and fibrosis of small pulmonary arteries1. Pulmonary artery catheters, also called Swan-Ganz catheters2, are commonly used in the clinical monitoring of RVP and PAP. Furthermore, a wireless PAP monitoring system has been used clinically3,4,5. To mimic the disease for study in mice, a hypoxic environment is used to simulate human clinical manifestations of PAH6. In the evaluation of PAP in animals, large animals are relatively easy to monitor through pulmonary artery catheters using the same technique as for human subjects, but small animals such as rats and mice are difficult to assess because of their small body size. Hemodynamic measurement of the right ventricular system in mice is possible with an ultrasmall size 1 Fr catheter7. A method for measuring RVP and PAP in mice has been reported in the literature8,9, but the methodology lacks a detailed description. RVP and PAP are more challenging to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems.
To get both PAP and RVP parameters in the same mouse, we describe an open-chest surgery-based approach for right heart hemodynamic measurements, its validation with healthy and PAH mice, and how to avoid generating artificial data during the complicated open-chest surgery. Although this technique is best performed by a well-trained surgeon, it has the advantage of being able to assess PAP and RVP in the same mouse.

<table><tbody><tr><td>2,2,2-Tribromoethanol</td><td>Sigma-Aldrich</td><td>T48402-5G</td><td>For anesthesia</td></tr><tr><td>Animal temperature controller</td><td>Physitemp Instruments, Inc.</td><td>TCAT-2LV</td><td>For temperature control</td></tr><tr><td>Dissection forceps</td><td>Fine Science Tools, Inc.</td><td>11274-20</td><td>For surgery</td></tr><tr><td>Gemini Cautery System</td><td>Gemini</td><td>GEM 5917</td><td>For surgery</td></tr><tr><td>Intravenous catheter (22G)</td><td>BD angiocath</td><td>381123</td><td>For intubation</td></tr><tr><td>LabChart 7.3</td><td>ADInstruments</td><td></td><td>For data analysis</td></tr><tr><td>Light illumination system</td><td>Olympus</td><td></td><td>For surgery</td></tr><tr><td>Mikro-Tip catheter</td><td>Millar Instruments, Houston, TX</td><td>SPR-1000</td><td>For pressure measurement</td></tr><tr><td>Millar Pressure-Volume Systems</td><td>Millar Instruments, Houston, TX</td><td>MVPS-300</td><td>For pressure measurement</td></tr><tr><td>O2 Controller and Hypoxia chamber</td><td>Biospherix</td><td>ProOx 110</td><td>For chronic hypoxia</td></tr><tr><td>PowerLab Data Acquisition System</td><td>ADInstruments</td><td>PowerLab 16/30</td><td>For data recording</td></tr><tr><td>Scissors</td><td>Fine Science Tools, Inc.</td><td>14084-08</td><td>For surgery</td></tr><tr><td>Small animal ventilator</td><td>Harvard Apparatus</td><td>Mini-Vent 845</td><td>For surgery</td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZ61</td><td>For surgery</td></tr><tr><td>Surgery tape</td><td>3M</td><td></td><td>For surgery</td></tr><tr><td>Terg-a-zyme enzyme</td><td>Sigma-Aldrich</td><td>Z273287-1EA</td><td>For catheter cleaning</td></tr></tbody></table>

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.

The pressure transducer catheter was inserted into the right ventricle (Figure 3A) through a tunnel expanded by a 25 G needle, and a typical RVP waveform (Figure 3C) was obtained. The catheter was continually adjusted and slowly advanced and kept in the same axis as the pulmonary artery while passing through the pulmonary valve (Figure 3B). When the pressure sensor was successfully inserted into the pulmonary artery, a typical PAP waveform with a characteristic dicrotic notch appeared (Figure 3D). To avoid the generation of artificial data, we observed whether the waveform had noise (Figure 4) or whether the zero level of the catheter had drifted (Figure 5). If this occurred, corrections were made, and these segments with noise were excluded from data analysis.
PAH is characterized by a sustained elevation in PAP and RVP, caused by increased resistance in small pulmonary arteries. PAH is defined by a mean PAP of &#8805;25 mmHg at rest, measured during right heart catheterization in the clinic12. We measured the RVP and PAP in the mice with the induced chronic hypoxia (kept at 10% oxygen for 4 weeks) or a control group (kept in normal air). The results are shown in Figure 6. Compared to those in the normal air control group, systolic PAP (Figure 6A), diastolic PAP (Figure 6B), mean PAP (Figure 6C), and right ventricular systolic pressure (Figure 6D) were all significantly increased in the chronic hypoxia group. Investigators have also reported that compared with hypoxia alone, a combination of a VEGFR inhibitor with chronic hypoxia for 3 weeks to induce severe PAH in mice can result in significantly increased RVP13,18.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60090/60090fig1.jpg" /> 
Figure 1: Intubation for mechanical ventilation support in mice. (A) The neck fur is removed using hair removal lotion to obtain a clean area for surgery. A midline incision is made on the skin of the neck. (B) The skeletal muscle covering the trachea is exposed. (C) The skeletal muscles are bluntly dissected to expose the trachea. The yellow arrow indicates the trachea. (D) The tubing (modified using a 22 G intravenous catheter) is inserted into the airway, with placement confirmed using forceps. The yellow arrow indicates the tubing inside the trachea. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60090/60090fig1large.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/60090/60090fig2.jpg" /> 
Figure 2: Open-chest surgery for hemodynamic measurement at the right ventricular system. (A) The chest fur is removed using hair removal lotion to obtain a clean area for surgery. (B) A midline incision is made to expose chest skeletal muscles and the sternum. (C) A cautery tool is used to minimize bleeding during chest opening (the arrow indicates the cautery tip). (D) The sternum is cut along the midline (the yellow dash line). (E) Two retractors are used to expose the heart (the upper arrow indicates the right atrial wall, and the lower arrow indicates the right ventricular free wall). (F) A pressure transducer catheter (the lower arrow) is inserted into the right ventricular chamber using a puncture tool (25 G size needle, the upper arrow) to produce a small tunnel on the right ventricular free wall. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60090/60090fig2large.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/60090/60090fig3.jpg" /> 
Figure 3: Representative RVP and PAP curves. The pressure transducer catheter is inserted into the right ventricular chamber (A) to obtain the RVP waveform (C). The pressure transducer catheter goes through the pulmonary valve and then stays in the pulmonary artery (B) to generate the PAP waveform. The arrows indicate the characteristic dicrotic notch on the PAP waveform (D), which is a sign of a pulmonary valve closure. RA = right atrium, RV = right ventricle, PA = pulmonary artery, LV = left ventricle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60090/60090fig3large.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/60090/60090fig4.jpg" /> 
Figure 4: RVP waveform noise caused by touching of the pressure sensor surface to the ventricular wall. The arrow point shows a sharp increase in pressure on the RVP curve (the upper channel), which simultaneously produces an artificial change in dP/dt (the lower channel). dP/dt is calculated from RVP. The dashed lines indicate dP/dt noise. If the noise is constantly present, adjustment of the catheter sensor position in the ventricle can prevent noise. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60090/60090fig4large.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/60090/60090fig5.jpg" /> 
Figure 5: Zero drift of pressure transducer during RVP measurement. The left window shows artificially slightly elevated end-diastolic RVP. The right expanded window shows increased end-diastolic RVP (arrows indicate end-diastolic RVP). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60090/60090fig5large.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/60090/60090fig6.jpg" /> 
Figure 6: Hypoxia-induced pulmonary artery hypertension in C57BL mice. (A) Systolic PAP (sPAP). (B) Diastolic PAP (dPAP). (C) Mean PAP (mPAP). (D) Right ventricular systolic pressure (RVSP). (E) and (F), Representative PAP waveforms for control and PAH mice respectively *p &#60; 0.05; Student&#39;s t-test; control group n = 10; hypoxia group n = 3. Data are presented as the mean &#177; SD. PAP = pulmonary artery pressure, RVP = right ventricular pressure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60090/60090fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice at JoVE.com

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

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. ...

invasive-hemodynamic-assessment-for-right-ventricular-system-hypoxia

Nanyang Technological University

Research

JoVE Journal

Medicine

9.0K Views.  Chinese Academy of Medical Sciences and Peking Union Medical College. Dit protocol is een open borst chirurgie gebaseerde hemodynamische beoordeling en het is nuttig voor de juiste ventriculaire en longslagader druk evaluatie bij kleine dieren zoals muizen. Het voordeel van deze procedure is dat het zowel VP- als PAP-parameters tegelijkertijd kan genereren. Het heeft dus de voorkeur voor evaluaties van PAK-modellen.Begin met het opzetten van de apparatuur voor chirurgie. Week de druktransducerkatheter in 0,9% zout bij kamertemperatuur gedurende ten minst...

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

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 ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension

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

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

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

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

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

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

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

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 ...

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, ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...

Biology

Comprehensive Echocardiographic Assessment of Right Ventricle Function in a Rat Model of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease caused by vasoconstriction and remodeling of the small arteries in the lungs. This remodeling leads to increased pulmonary vascular resistance, worsened right ventricular function, and premature death. Currently approved therapies for PAH largely target pulmonary vasodilator pathways; however, recent emerging therapeutic modalities are focused on other novel pathways involved in the pathogenesis of the disease, including right ventricle (RV) remodeling. Imaging techniques that allow longitudinal assessment of novel therapeutics are very useful for determining the efficacy of new drugs in preclinical studies. Noninvasive trans-thoracic echocardiography remains the standard approach to evaluating heart function and is widely used in rodent models. However, echocardiographic evaluation of the RV can be challenging due to its anatomical position and structure. In addition, standardized guidelines are lacking for echocardiography&#160;in preclinical rodent models, making it difficult to carry out a uniform assessment of RV&#160;function across studies in different laboratories. In preclinical studies, the monocrotaline (MCT) injury model in rats is widely used to evaluate drug efficacy for treating PAH. This protocol describes the echocardiographic evaluation of the RV in na&#239;ve and MCT-induced PAH rats.

The present protocol describes the echocardiographic characterization of right ventricular morphology and function in a rat model of pulmonary arterial hypertension.

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

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