This work was supported by NHLBI K01 HL155241 and AHA CDA849387 awarded to the author P.C.R.

All experiments in this protocol were performed following the animal care guidelines of the University of Illinois at Chicago, Chicago Institutional Animal Care and Use Committee. Male Sprague Dawley (SD) rats weighed between 0.200-0.240 kg at the time of MCT injection; however, the protocol described in this article can be used with a wider body weight range. The animals were obtained from a commercial source (see Table of Materials).
1. Study design
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<ol>
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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five-year+outcomes+of+patients+enrolled+in+the+REVEAL+registry.">Five-year outcomes of patients enrolled in the REVEAL registry.</a> Chest. 148 (4), 1043-1054 (2015).</li><li>Zolty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+experimental+therapies+for+treatment+of+pulmonary+arterial+hypertension.">Novel experimental therapies for treatment of pulmonary arterial hypertension.</a> Journal of Experimental Pharmacology. 13, 817-857 (2021).</li><li>Jasmin, J. F., Lucas, M., Cernacek, P., Dupuis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+a+nonselective+ET(A/B)+and+a+selective+ET(A)+antagonist+in+rats+with+monocrotaline-induced+pulmonary+hypertension.">Effectiveness of a nonselective ET(A/B) and a selective ET(A) antagonist in rats with monocrotaline-induced pulmonary hypertension.</a> Circulation. 103 (2), 314-318 (2001).</li><li>Stenmark, K. R., Meyrick, B., Galie, N., Mooi, W. J., McMurtry, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+pulmonary+arterial+hypertension:+the+hope+for+etiological+discovery+and+pharmacological+cure.">Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure.</a> American Journal of Physiology Lung Cellular and Molecular Physiology. 297 (6), 1013-1032 (2009).</li><li>Muresian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+anatomy+of+the+right+ventricle.">The clinical anatomy of the right ventricle.</a> Clinical Anatomy. 29 (3), 380-398 (2016).</li><li>Rudski, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+echocardiographic+assessment+of+the+right+heart+in+adults:+a+report+from+the+American+Society+of+Echocardiography+endorsed+by+the+European+Association+of+Echocardiography,+a+registered+branch+of+the+European+Society+of+Cardiology,+and+the+Canadian+Society+of+Echocardiography.">Guidelines for the echocardiographic assessment of the right heart in adults: a report from the American Society of Echocardiography endorsed by the European Association of Echocardiography, a registered branch of the European Society of Cardiology, and the Canadian Society of Echocardiography.</a> Journal of the American Society of Echocardiography. 23 (7), 685-713 (2010).</li><li>Jones, N., Burns, A. T., Prior, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+assessment+of+the+right+ventricle-state+of+the+art.">Echocardiographic assessment of the right ventricle-state of the art.</a> Heart Lung and Circulation. 28 (9), 1339-1350 (2019).</li><li>Spyropoulos, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+markers+of+pulmonary+hemodynamics+and+right+ventricular+hypertrophy+in+rat+models+of+pulmonary+hypertension.">Echocardiographic markers of pulmonary hemodynamics and right ventricular hypertrophy in rat models of pulmonary hypertension.</a> Pulmonary Circulation. 10 (2), 2045894020910976 (2020).</li><li>Armstrong, W. F., Ryan, T., Feigenbaum, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Feigenbaum's echocardiography. 7th edn. , (2010).</li><li>Kimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+right+ventricle+by+speckle+tracking+and+conventional+echocardiography+in+rats+with+right+ventricular+heart+failure.">Evaluation of right ventricle by speckle tracking and conventional echocardiography in rats with right ventricular heart failure.</a> International Heart Journal. 56 (3), 349-353 (2015).</li><li>Cheng, H. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+right+ventricular+structure+and+function+in+mouse+model+of+pulmonary+artery+constriction+by+transthoracic+echocardiography.">Assessment of right ventricular structure and function in mouse model of pulmonary artery constriction by transthoracic echocardiography.</a> Journal of Visualized Experiments. 84, e51041 (2014).</li><li>Mazurek, J. A., Vaidya, A., Mathai, S. C., Roberts, J. D., Forfia, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follow-up+tricuspid+annular+plane+systolic+excursion+predicts+survival+in+pulmonary+arterial+hypertension.">Follow-up tricuspid annular plane systolic excursion predicts survival in pulmonary arterial hypertension.</a> Pulmonary Circulation. 7 (2), 361-371 (2017).</li><li>Grapsa, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+and+hemodynamic+predictors+of+survival+in+precapillary+pulmonary+hypertension:+seven-year+follow-up.">Echocardiographic and hemodynamic predictors of survival in precapillary pulmonary hypertension: seven-year follow-up.</a> Circulation: Cardiovascular Imaging. 8 (6), 002107 (2015).</li><li>Bernardo, I., Wong, J., Wlodek, M. 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Echocardiographic evaluation of the RV is a valuable discovery tool for the screening of the effectiveness of novel treatments in animal models of PAH. In-depth characterization of the RV structure and function is necessary as novel targets in treating PAH address RV remodeling4,14. This study describes a detailed protocol that allows for the successful characterization of RV structure and function.
The complex structural geometry and ...

PAH is a progressive disease defined as a mean pulmonary arterial pressure at rest of greater than 20 mmHg1. Pathological changes in PAH include pulmonary artery (PA) remodeling, vasoconstriction, inflammation, and fibroblast activation and proliferation. These pathological changes lead to increased pulmonary vascular resistance and, consequently, right ventricular remodeling, hypertrophy, and failure2. PAH is a complex disease that involves crosstalk between several signaling pathways. The currently approved drugs for treating PAH mostly target vasodilator pathways, including the nitric oxide-cyclic guanosine monophosphate pathway, prostacyclin pathway, and endothelin pathway. Therapeutics targeting these pathways have been used as both monotherapies and in combination therapies3,4. Despite the advances in treatment for PAH in the last decade, findings from the US-based REVEAL registry show a poor 5 year survival rate for newly diagnosed patients5. More recently, emerging therapeutic modalities have focused on disease-modifying agents that can impact the multifactorial pathophysiology of the vascular remodeling occurring in PAH in the hope of disrupting the disease6.
Animal models of PAH are invaluable tools in assessing the efficacy of new drug treatments. The MCT-induced PAH rat model is a widely used animal model characterized by remodeling of the pulmonary arterial vessels, which in turn leads to increased pulmonary vascular resistance and right ventricular hypertrophy and dysfunction7,8. To assess the efficacy of new treatments, researchers normally focus on the terminal assessment of RV pressure without considering the longitudinal evaluation of PA pressure, RV morphology, and RV function. The use of noninvasive and non-terminal imaging techniques is crucial for a comprehensive examination of disease progression in animal models. Transthoracic echocardiography remains the standard approach to evaluating heart morphology and function in animal models due to its low cost and ease of use compared to other imaging modalities, such as magnetic resonance imaging. However, echocardiographic evaluation of the RV can be challenging due to the RV positioning beneath the sternum shadow, its well-developed trabeculation, and its anatomical shape, all of which make it difficult to delineate the endocardial border9,10,11.
This article aims to describe a comprehensive protocol to evaluate RV dimensions, areas and volumes, and systolic and diastolic function in naive and MCT-induced PAH in Sprague Dawley (SD) rats. Additionally, this protocol details a method to assess echocardiographic dimensions in the normal and dilated right atrium.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% sodium cloride injection USP</td>
			<td>Baxter</td>
			<td>2B1324</td>
			<td></td>
		</tr>
		<tr>
			<td>Braided cotton rolls</td>
			<td>4MD Medical Solutions</td>
			<td>RIHD201205</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilating agent</td>
			<td>Wallgreens</td>
			<td>Nair Hair Remover&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode gel</td>
			<td>Parker Laboratories&#160;</td>
			<td>15-60</td>
			<td></td>
		</tr>
		<tr>
			<td>High frequency ultrasound image system and imaging station</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td>Vevo 2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>MedVet</td>
			<td>RXISO-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Male sprague Dawley rats</td>
			<td>Charles River Laboratories</td>
			<td>CD 001</td>
			<td>CD IGS Rats (Crl:CD(SD))</td>
		</tr>
		<tr>
			<td>Monocrotaline (MCT)</td>
			<td>Sigma-Aldrich</td>
			<td>C2401</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectal temperature probe &#160;</td>
			<td>Physitemp&#160;</td>
			<td>RET-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sealed induction chambers</td>
			<td>Scivena Scientific</td>
			<td>RES644&#160;</td>
			<td>3 L size</td>
		</tr>
		<tr>
			<td>Solid-state array ultrasound transducer</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td>Vevo MicroScan transducer MS250S</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel digital calipers</td>
			<td>VWR Digital Calipers</td>
			<td>62379-531</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound gel&#160;</td>
			<td>Parker Laboratories&#160;</td>
			<td>11-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo Lab software</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td></td>
			<td>Verison 5.5.1</td>
		</tr>
	</tbody>
</table>

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.

In this study, MCT-treated rats were used as a model of PAH. Echocardiographic analysis was performed on Study Day 23 post-MCT administration, and all measurements and calculations represented averages from three consecutive cycles. Echocardiographic parameters obtained from control (vehicle: deionized water) and MCT-treated (60 mg/kg) rats are shown in Table 1.
Representative images of the PLAX view in control and MCT-treated rats are shown in Figure 1A</...

Watch this Scientific Journal Video about Comprehensive Echocardiographic Assessment of Right Ventricle Function in a Rat Model of Pulmonary Arterial Hypertension at JoVE.com

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

The present protocol describes the echocardiographic characterization of right ventricular morphology and 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 ...

In depth echocardiographic evaluation of the right ventricular structure and function is a valuable tool to screen the effectiveness of novel therapeutic targets in animal models of pulmonary hypertension. The main advantage of this protocol is that it provides a detailed description of all the echocardiographic views and measurements needed to assess the right atria and right ventricular morphology, as well as to characterize the right ventricle systolic and diastolic functions. This technique describes the echocardiographic characterization of a rat model of pulmonary hypertension induced by Monocrotaline.It is important that the echocardiographer spends enough time practicing and becoming proficient in the right ventricle imaging even if they had previous experience with the left ventricle. Begin by removing the rats from the induction chamber after they lose consciousness and transfer them to the imaging station animal platform in a dorsal decubitus position. Administer isoflurane using a nose cone connected to a vaporizer.Apply electrode gel to each paw and secure the paws in the electrocardiogram lead plates of the animal platform. Remove the fur by shaving the chest and using a depilating agent. Secure a rectal temperature probe in place.Place cotton rolls on the right and left side of the animal and secure them with tape to maintain the position when the platform is tilted. To acquire images, tilt the platform to the right by 10 to 15 degrees and bring it caudally down approximately five degrees. Move the transducer to point to the right parasternal line of the rat, and lower the transducer.Rotate the transducer counterclockwise to ensure the aorta and mitral valve are visible. Place the M-mode sample volume line at the region where the right ventricle is widest and adjust the gate to encompass the right and left ventricle. Lift the transducer and reposition it such that it is slightly inclined toward the right parasternal line of the rat.Move the platform to a position that is slightly tilted to the right. Lower the transducer until it is in contact with the gel. Move the platform caudally into the right or left until the right ventricular outflow track is in view and the pulmonary valve is in focus and clearly visible.Then press Sign Store to record the B-Mode image of the pulmonary artery valve. Maintaining the same B-Mode image location, press Color to aid the identification of flow through the pulmonary valve. Adjust the velocity so that the highest velocity point is visible.Press PW or Pulsed Wave to quantify the blood flow spectrum. Align the PW angle parallel to the direction of the flow through the pulmonary valve. Press update to view the pulmonary velocities.Tilt the platform to the left corner and down cranially as far as possible. Rotate the transducer counterclockwise by 30 to 45 degrees and move the transducer to point at the right shoulder or ear of the animal. Lower the transducer until it is in contact with the gel.Ensure a typical four chamber view where the left ventricle and left atria are visible, but the sternum shadow is over the right ventricle free wall. Move the transducer for adjusting the applicable four chamber view acquiring a focused view of the right ventricle. Move the platform slightly caudally if required until the maximal plane is obtained.Ensure that the right ventricle is not foreshortened and that the left ventricle outflow tract is not opened. Place the M-mode cursor through the tricuspid annulus at the right ventricular free wall to visualize TAPSE and press Update and Sign Store to record the TAPSE data. Press B-mode and then press Color to aid the identification of flow through the TV.Press PW to quantify the blood flow spectrum.Return to B-mode, press tissue, and place the Tissue Doppler sample volume gate at the tricuspid annulus at the RV free wall. Press Update to view the Tissue Doppler image. Using PLAX views, initial assessment of the position of the heart and left ventricular morphology was performed.Assessment of the right ventricle using a modified PLAX view showed that the monocrotaline or MCT-treated rats had an enlarged right ventricle and displaced left ventricle compared to the control rats. A significant increase in RVIDd, RVIDs, and RVFWT was observed in the MCT-treated rats indicating dilation of the right ventricle and thickening of the right ventricular free wall. The modified PLAX view was also used to visualize the pulmonary artery and measure the pulmonary valve diameter.Measurement of pulmonary artery flow velocities using doppler imaging showed that in control rats, pulmonary flow exhibited a symmetrical V shape with a peak velocity in mid-systole. Whereas an MCT-treated rats, peak velocity was slower and occurred earlier in the systole and displayed a notch in late systole. The right ventricle-focused apical four chamber view was used to measure right ventricular and systolic area and right atrial area.MCT-treated rats exhibited right atrial dilation because of increased pulmonary artery pressure. Moreover, the right ventricular pressure was increased resulting in loss of normal curvature of the left ventricle compared to controls. The intraventricular septum also appeared flattened.The bright ventricle-focused apical four chamber view was also used to measure TAPSE from the M-mode interrogation of the tricuspid annulus. TAPSE was significantly reduced in MCT-treated rats suggesting compromised function. The most challenging part of this protocol is obtaining an optimal right ventricle-focused apical four chamber view.We suggest that the echocardiographer ensures that the entire right ventricle is in the view during systole and diastole. Images are obtained with a right ventricle-focused apical four chamber view can be later used for a strain analysis, a powerful tool that may be used to evaluate right ventricle systolic dysfunction at the initial stages.

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3.0K Views.  University of Illinois at Chicago. In depth echocardiographic evaluation of the right ventricular structure and function is a valuable tool to screen the effectiveness of novel therapeutic targets in animal models of pulmonary hypertension. The main advantage of this protocol is that it provides a detailed description of all the echocardiographic views and measurements needed to assess the right atria and right ventricular morphology, as well as to characterize the right ventricle systolic and diastolic functions. This techniq...