ARSP and ATP are supported by the fellowships SFRH/BD/121684/2016 and SFRH/BPD/123181/2016, respectively, from Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia.

All animal procedures were performed according to Directive 2010/63/EU. The procedures were approved by the institutional Animal Welfare Body, licensed by DGAV, the Portuguese competent authority for animal protection (license number 0421/000/000/2018). 
NOTE: Female Wistar Han IGS (Crl:WI(Han) from Charles River Laboratories (12-16 weeks-old) were used. This protocol is specific for rats independently of their strain, age or gender.
1. Preparing Rats for Echocardiography: Anesthesia and Reversion Protocol
<ol>
	<li>Weigh rats.</li>
	<li>Prepare a three-component anesthetic composed by midazolam (4.76 mg/kg), medetomidine (0.356 mg/kg) and fentanyl (0.012 mg/kg), according to rat weight.</li>
	<li>Inject anesthesia intraperitoneally. Check for the absence of pedal withdrawal reflexes to evaluate the depth of anesthesia.</li>
	<li>Shave the hair from the torso area.</li>
	<li>Apply durable gel to both eyes to prevent drying of the sclera.</li>
	<li>Place the anesthetized rat in a supine position atop a heating pad in order to maintain body temperature (37.0 &#176;C &#177; 0.5 &#176;C).</li>
	<li>Apply a layer of preheated (close to body temperature) ultrasound gel to the chest, mainly in the area overlying the heart. Avoid air bubbles in the gel that can interfere with ultrasonic imaging.</li>
	<li>Reverse anesthesia through subcutaneous injection using atipamezole (0.94 mg/kg) and flumazenil (0.2532 mg/kg), immediately after the end of the echocardiography. 
	NOTE: This anesthetic combination provides up to 45 min for echocardiography imaging. The echocardiographic protocol described below is compatible with any other anesthesia protocol.</li>
</ol>
2. Echocardiography
NOTE: The echocardiograms are performed with a conventional clinical echocardiographic equipment, with a 12 MHz cardiac probe, and include acquired still images and loops in parasternal (long axis and short axis views), apical (4, 5, 2 and 3 chambers) and suprasternal views. An electrocardiogram is recorded to identify end-systole and end-diastole, for measurement procedures and loop acquisition (ECG triggered)11,12. A preset is used to keep image definition stable between rats: frequency 5-10 MHz, depth 2.5 cm, frame rate 125 fps, Doppler sample 1.0 mm and color Doppler aliasing velocity 40 cm/s. Loops were recorded with at least 3 heart beats.
<ol>
	<li>Left parasternal long axis view 
	NOTE: Position the probe on the left side of the sternum and the index mark turned to the right shoulder.
	<ol>
		<li>Record M-mode images at the aortic valve, mitral valve leaflets and left ventricular mid-cavity (cursor at mitral valve tips or cordal level)1,2,3,4. The M-Mode cursor should be perpendicular to the structure of interest1,3,10.</li>
		<li>Record a 2D loop of all views.</li>
		<li>Record a 2D loop with zoom at the left ventricular outflow tract.</li>
		<li>Record a 2D loop with color Doppler imaging simultaneously at the aortic and mitral valves.</li>
	</ol>
	</li>
	<li>Left parasternal short-axis view 
	NOTE: Place the probe on the left side of the sternum with the index mark rotated to the left shoulder.
	<ol>
		<li>Obtain an image at the aortic valve level by tilting the probe slightly cranially.</li>
		<li>Record a 2D loop of all views.</li>
		<li>Record a 2D loop with color Doppler imaging simultaneously at the aortic and pulmonary valves.</li>
		<li>Acquire a spectral pulsed Doppler image at the pulmonary artery. The cursor should be parallel to flow1,3.</li>
		<li>Obtain an image of the left ventricle at the papillary muscle level by tilting the probe slightly downward.</li>
		<li>Record a 2D loop of all views.</li>
	</ol>
	</li>
	<li>Apical 4-chamber view 
	NOTE: Position the probe at the apical area in the anterior axillary line and with the index mark turned to left shoulder.
	<ol>
		<li>Record a 2D loop of all views.</li>
		<li>Record a loop of 2D and tissue Doppler imaging including all 4 chambers.</li>
		<li>Focus on the left cardiac chambers.
		<ol>
			<li>Record a 2D loop with zoom at the left atrium.</li>
			<li>Record a 2D loop with color Doppler imaging at the mitral valve and left atrium.</li>
			<li>Record simultaneous M-mode and color Doppler images for left ventricular propagation flow.</li>
			<li>Get a spectral pulsed wave (PW) Doppler at the mitral valve for left ventricular inflow. Place the sample at the mitral leaflet tips, in their fully open diastolic position1,2,3,11,12.</li>
			<li>Add a continuous wave (CW) Doppler image at the mitral valve, if there is mitral valve regurgitation.</li>
			<li>Obtain a spectral pulsed tissue Doppler image at the mitral annulus (left ventricular lateral and septal walls). Align the PW Doppler cursor with the long axis of the heart in order to produce the maximum Doppler signal1,2,3,13.</li>
			<li>Record M-mode of mitral annulus for mitral annular plane systolic excursion measurement (cursor at lateral left ventricle wall).</li>
		</ol>
		</li>
		<li>Focus on right cardiac chambers.
		<ol>
			<li>Record a 2D loop with zoom at the right atrium.</li>
			<li>Record a 2D loop with color Doppler imaging at the tricuspid valve and the right atrium.</li>
			<li>Obtain a spectral pulsed tissue Doppler image at the tricuspid annulus (right ventricular wall).</li>
			<li>Record M-mode for tricuspid annular plane systolic excursion (TAPSE) by placing the 2D cursor at the tricuspid lateral annulus.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Apical 5-chamber view 
	NOTE: From the 4-chamber view, tilt the probe slightly anterior to the chest.
	<ol>
		<li>Record a 2D loop of all views.</li>
		<li>Record a 2D loop with color Doppler imaging at the aortic valve and left ventricular outflow tract.</li>
		<li>Get a spectral pulsed wave Doppler image at the left ventricular outflow tract. Place the cursor parallel to the flow and place the sample at the left ventricular outflow tract4,14.</li>
		<li>Get a spectral pulsed wave Doppler image at the left ventricle mid-cavity for simultaneous left ventricular inflow and outflow waves.</li>
		<li>Obtain a spectral continuous wave Doppler image at aortic valve. Transvalvular flow is recorded below baseline and regurgitation, if present, above baseline.</li>
	</ol>
	</li>
	<li>Apical 2-chamber view 
	NOTE: Return to a 4-chamber view and rotate the probe 90&#176; counterclockwise.
	<ol>
		<li>Record a 2D loop of all views.</li>
		<li>Record a 2D loop with color Doppler imaging at the mitral valve.</li>
	</ol>
	</li>
	<li>Apical 3-chamber view 
	NOTE: Tilt the probe slightly cranially.
	<ol>
		<li>Record a 2D loop of all views.</li>
		<li>Record a 2D loop with color Doppler simultaneously at the aortic and mitral valves.</li>
	</ol>
	</li>
	<li>Suprasternal window 
	NOTE On the left side of supraclavicular space with probe directed downward
	<ol>
		<li>Record a 2D loop of the aortic arch.</li>
		<li>Get spectral pulsed wave Doppler image at the ascending aorta.</li>
		<li>Get spectral pulsed wave Doppler image at the descending aorta.</li>
	</ol>
	</li>
</ol>
3. Measurements
<ol>
	<li>Proceed to measurements, including global longitudinal strain. Perform these measurements offline in order to reduce the anesthesia time.</li>
</ol>

<ol>
	<li>Armstrong, W. F., Ryan, T. <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. , (2010).</li><li>Douglas, P. S., 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=ACCF/ASE/ACEP/ASNC/SCAI/SCCT/SCMR+2007+appropriateness+criteria+for+transthoracic+and+transesophageal+echocardiography:+a+report+of+the+American+College+of+Cardiology+Foundation+Quality+Strategic+Directions+Committee+Appropriateness+Criteria+Working+Group,+American+Society+of+Echocardiography,+American+College+of+Emergency+Physicians,+American+Society+of+Nuclear+Cardiology,+Society+for+Cardiovascular+Angiography+and+Interventions,+Society+of+Cardiovascular+Computed+Tomography,+and+the+Society+for+Cardiovascular+Magnetic+Resonance+endorsed+by+the+American+College+of+Chest+Physicians+and+the+Society+of+Critical+Care+Medicine.">ACCF/ASE/ACEP/ASNC/SCAI/SCCT/SCMR 2007 appropriateness criteria for transthoracic and transesophageal echocardiography: a report of the American College of Cardiology Foundation Quality Strategic Directions Committee Appropriateness Criteria Working Group, American Society of Echocardiography, American College of Emergency Physicians, American Society of Nuclear Cardiology, Society for Cardiovascular Angiography and Interventions, Society of Cardiovascular Computed Tomography, and the Society for Cardiovascular Magnetic Resonance endorsed by the American College of Chest Physicians and the Society of Critical Care Medicine.</a> Journal of the American College of Cardiology. 50 (2), 187-204 (2007).</li><li>Otto, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Textbook of Clinical Echocardiography. , (2018).</li><li>Liu, J., Rigel, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+examination+in+rats+and+mice.">Echocardiographic examination in rats and mice.</a> Cardiovascular Genomics. Methods in Molecular Biology (Methods and Protocols). 573, 139-155 (2009).</li><li>Ram, R., Mickelsen, D. M., Theodoropoulos, C., Blaxall, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+in+small+animal+echocardiography:+imaging+the+sounds+of+silence.">New approaches in small animal echocardiography: imaging the sounds of silence.</a> American Jounal of Physiology- Heart and Circulatory Physiology. 301 (5), H1765-H1780 (2011).</li><li>Aronsen, J. M., 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=Noninvasive+stratification+of+postinfarction+rats+based+on+the+degree+of+cardiac+dysfunction+using+magnetic+resonance+imaging+and+echocardiography.">Noninvasive stratification of postinfarction rats based on the degree of cardiac dysfunction using magnetic resonance imaging and echocardiography.</a> American Jounal of Physiology - Heart and Circulatory Physiology. 312 (5), H932-H942 (2017).</li><li>Forman, D. E., Cittadini, A., Azhar, G., Douglas, P. S., Wei, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+morphology+and+function+in+senescent+rats:+gender-related+differences.">Cardiac morphology and function in senescent rats: gender-related differences.</a> Journal of the American College of Cardiology. 30 (7), 1872-1877 (1997).</li><li>Walker, E. M., 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=Age-associated+changes+in+hearts+of+male+Fischer+344/Brown+Norway+F1+rats.">Age-associated changes in hearts of male Fischer 344/Brown Norway F1 rats.</a> Annals Of Clinical And Laboratory Science. 36 (4), 427-438 (2006).</li><li>Watson, L. E., Sheth, M., Denyer, R. F., Dostal, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Baseline+echocardiographic+values+for+adult+male+rats.">Baseline echocardiographic values for adult male rats.</a> Journal of the American Society of Echocardiography. 17 (2), 161-167 (2004).</li><li>Lang, R. M., 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=Recommendations+for+cardiac+chamber+quantification+by+echocardiography+in+adults:+an+update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging.">Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging.</a> Journal of the American Society of Echocardiography. 28 (1), 1-39 (2015).</li><li>Galderisi, M., 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=Standardization+of+adult+transthoracic+echocardiography+reporting+in+agreement+with+recent+chamber+quantification,+diastolic+function,+and+heart+valve+disease+recommendations:+an+expert+consensus+document+of+the+European+Association+of+Cardiovascular+Imaging.">Standardization of adult transthoracic echocardiography reporting in agreement with recent chamber quantification, diastolic function, and heart valve disease recommendations: an expert consensus document of the European Association of Cardiovascular Imaging.</a> European Heart Journal - Cardiovascular Imaging. 18 (12), 1301-1310 (2017).</li><li>Nagueh, S. 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=Recommendations+for+the+Evaluation+of+Left+Ventricular+Diastolic+Function+by+Echocardiography:+An+Update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging.">Recommendations for the Evaluation of Left Ventricular Diastolic Function by Echocardiography: An Update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging.</a> European Heart Journal - Cardiovascular Imaging. 29 (4), 277-314 (2016).</li><li>Weytjens, C., 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=Doppler+myocardial+imaging+in+adult+male+rats:+reference+values+and+reproducibility+of+velocity+and+deformation+parameters.">Doppler myocardial imaging in adult male rats: reference values and reproducibility of velocity and deformation parameters.</a> European Journal of Echocardiography. 7 (6), 411-417 (2006).</li><li>Scheer, P., 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=Basic+values+of+M-mode+echocardiographic+parameters+of+the+left+ventricle+in+outbreed+Wistar+rats.">Basic values of M-mode echocardiographic parameters of the left ventricle in outbreed Wistar rats.</a> Veterinarni Medicina. 57 (1), 42-52 (2012).</li><li>Lancellotti, P., 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=Recommendations+for+the+echocardiographic+assessment+of+native+valvular+regurgitation:+an+executive+summary+from+the+European+Association+of+Cardiovascular+Imaging.">Recommendations for the echocardiographic assessment of native valvular regurgitation: an executive summary from the European Association of Cardiovascular Imaging.</a> European Heart Journal - Cardiovascular Imaging. 14 (7), 611-644 (2013).</li><li>Albrecht, M., Henke, J., Tacke, S., Markert, M., Guth, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+repeated+anaesthesia+on+physiological+parameters+in+male+Wistar+rats:+a+telemetric+study+about+isoflurane,+ketamine-xylazine+and+a+combination+of+medetomidine,+midazolam+and+fentanyl.">Influence of repeated anaesthesia on physiological parameters in male Wistar rats: a telemetric study about isoflurane, ketamine-xylazine and a combination of medetomidine, midazolam and fentanyl.</a> BMC Veterinary Research. 10, 310 (2014).</li><li>Dragoi Galrinho, R., 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=New+Echocardiographic+Protocol+for+the+Assessment+of+Experimental+Myocardial+Infarction+in+Rats.">New Echocardiographic Protocol for the Assessment of Experimental Myocardial Infarction in Rats.</a> Maedica (Bucharest). 10 (2), 85-90 (2015).</li></ol>

The authors have nothing to disclose.

This protocol allows a complete echocardiographic study using conventional medical ultrasound equipment and a high-frequency probe in adult rats. This is an important aspect of the protocol, since ultrasound equipment dedicated for small animals is expensive and the investment is not always justifiable.
As longitudinal imaging studies require repeated anesthesia, a combination of medetomidine-midazolam-fentanyl was proposed in this protocol since it is more suitable for serial use when compared to isoflurane or a mixture of ketamine-xylazine, in Wistar rats. However, the proposed echocardiographic protocol is compatible with any other anesthesia protocol16. As described, our echocardiography protocol includes the evaluation of several parameters that enables the identification of anatomic and functional cardiac changes.
Focusing on the anatomical characterization, it is possible to evaluate the dimensions of all heart chambers and their dilatations, left ventricle hypertrophy, valvular fibrosis or calcifications. Concerning the cardiac function, the left ventricular systolic and diastolic function and right ventricular systolic function can be analyzed1,3,4. Also, the cardiac valve&#39;s anatomy and function are studied, using 2D-echo for anatomic characterization (identifying fibrosis, calcification or abnormal opening) and using Doppler imaging for functional characterization and detection of stenosis or regurgitations. Color Doppler imaging enables detection of flow direction and turbulences and spectral Doppler waves allow measurements of velocities and gradients1,3.
Adequate image quality was obtained in almost all rats (smallest weight of 200 g), although due to inter-individual differences in anatomy, echocardiographic views may not be obtained with the exact same definition between rats, which may have an impact in cavity dimension measurements. There is 5% intra-observer reported variability on left ventricle M-mode measurements17. Particularly, when using M-Mode for left ventricular measurements, the following limitations may exist: difficulties in getting a perpendicular angle; including only basal segments (resulting in inaccurate measurements in the presence of asymmetric hypertrophy or regional systolic dysfunction); and geometric assumptions (considering that the left ventricle is a prolate ellipsoid with a 2:1 long/short axis ratio and symmetric distribution of hypertrophy). Also, inclusion of cubed measurements may impact accuracy, since even a small error in dimensions may lead to overestimated mass1,3,10. Even when using volumes and the ejection fraction calculated by the Simpson&#39;s method, there are disadvantages: the apex is frequently foreshortened; the endocardial dropout can bias the measurement and is blind to shape distortions not visualized in the apical 4 and 2-chamber views1,3,10.
Importantly, this protocol highlights the use of advanced measurements and evaluations, such as the left ventricle strain and the strain rate, assessed by speckle tracking, to achieve more complete information about myocardial fibers behavior1,3. For a more accurate strain and strain rate evaluation, the optimization of image quality, maximizations of frame rate, and minimization of apex foreshortening are required. Midwall global longitudinal strain is used as it agrees with more published available data and has been shown in several clinical studies to be robust and reproducible10. The electrocardiographic monitorization integrated in the equipment is very prone to artifacts, which is a constraint. Also, it is very important to state that the functional or hemodynamic cardiac status of the rat can depend on variables such as temperature, blood pressure and heart rate4,5,6,7,8,9,13,14,17.
Since resolution is related to the probe-frequency, future developments are expected to develop higher frequency probes and consequently higher resolution and image definition in non-invasive cardiovascular imaging in small animals, with this type of equipment. Standardization of methods and measurements is considered critical in this field of research, reaching more precise echocardiographic diagnosis of experimental rat models and resulting in a better understanding of molecular biology of human cardiovascular diseases.

Cardiovascular disease is the leading cause of death in Europe, responsible for over 4 million yearly deaths, despite advances in therapy, diagnosis, and monitoring that have improved patient outcomes in recent years. A rapid technological evolution has contributed to progress in cardiovascular patient care. Within these diagnostic tools, particular attention has been paid to biomedical imaging, which allows an anatomic and functional evaluation in a non-invasive way1,2,3. Similarly, medicine benefits from the results of biomedical research. Experimental animal models are very useful for testing hypotheses derived from the clinical setting and to develop innovative therapies4,5.
There is increasing interest in the use of echocardiography as a research tool in experimental animal models, allowing the acquisition of multiple measurements from a single animal in longitudinal studies. It is important to note that there are some advantages in using murine or rodent models. The short gestation period, low cost of breeding and housing, the knowledge of their genome and possibility to develop transgenic animals are the main advantages of these species, making them attractive to study the mechanisms involved in cardiovascular disease4,5,6,7,8,9. Although rat and mouse models show similar advantages, rats are the classical choice in cardiovascular studies due to their larger physical dimension and lower heart rate that provides better images in echocardiography studies4,5,6,7,8,9,10.
We describe an echocardiography protocol using conventional medical ultrasound equipment to evaluate cardiac chambers and heart valves (anatomy and function) using Wistar rats. This is a concise and complete protocol for short time acquisition images and loops that allow offline measurements, which can be later revised to integrate new variables or measurements over time.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12S-RS Probe</td>
			<td>GE Medical Systems</td>
			<td>H44901AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Antisedan (5 mg/ml)</td>
			<td>Esteve</td>
			<td>P01B9003</td>
			<td></td>
		</tr>
		<tr>
			<td>EKG monitoring unit</td>
			<td>GE Medical Systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrodes</td>
			<td>FIAB</td>
			<td>F9089/100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fentanilo (0.05 mg/ml)</td>
			<td>B.Braun</td>
			<td>BB3644960</td>
			<td></td>
		</tr>
		<tr>
			<td>Flumazenilo (0.1 mg/ml)</td>
			<td>Generis</td>
			<td>MUEH5933080</td>
			<td></td>
		</tr>
		<tr>
			<td>Insuline Syringe 1ml</td>
			<td>SOL M</td>
			<td>1612912</td>
			<td></td>
		</tr>
		<tr>
			<td>Lubrithal gel (10mg)</td>
			<td>Dechra</td>
			<td>NC519</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetor (1 mg/ml)</td>
			<td>Vibarc</td>
			<td>P01B0308</td>
			<td></td>
		</tr>
		<tr>
			<td>Midazolan (5 mg/ml)</td>
			<td>Labesfal</td>
			<td>MUEH5506191</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver Razor AESCULAP Isis GT608</td>
			<td>Braun</td>
			<td>90200714</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Animal Heated Pad 120volts</td>
			<td>K&#38;H Manufacturing inc.</td>
			<td>655199010608</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound Gel</td>
			<td>Parker Laboratories</td>
			<td>REF 01-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound machine</td>
			<td>GE Medical Systems</td>
			<td>VIVID T8</td>
			<td></td>
		</tr>
		<tr>
			<td>Underpads</td>
			<td>Henry Schein</td>
			<td>900-8132</td>
			<td></td>
		</tr>
	</tbody>
</table>

echocardiographic assessment of cardiac anatomy and function in adult rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

Figure 1 shows the probe position on the chest to display the parasternal window long axis view (Figure 2). This view allows accurate measurements of left ventricle cavity and wall thickness, systolic function (Figure 3), left ventricle outflow diameter (to apply in other formulas such as in cardiac output), ascending aorta diameter and left atrium diameter. All chamber dimensions were indexed to body weight. The parasternal long axis view allows anatomical (with 2D-Echo) and functional (with color Doppler imaging) evaluation of the aortic and mitral valves. This view also allows the identification and measurement of the pericardial effusion, if present. M-Mode can be used for left ventricle measurements (Figure 3): septum and posterior walls dimensions, left ventricle dimensions, left ventricle systolic function and left ventricle mass1,3,4,10,14.
Left ventricle systolic function is evaluated by fractional shortening and also by visualizing the excursion and thickening of walls during cardiac cycle (assessed by the ECG). Left ventricle mass is obtained by the formula: 
LV mass = 0.8 x 1.04 x [(IVS + LVID + PWT)3 - LVID3] 
(IVS: interventricular septum thickness; LVID: left ventricle internal diameter; PWT: posterior wall thickness, with measurements made at end-diastole)1,3,4,10,14.
Figure 4 shows the probe position on the chest to display the parasternal window short axis view. This view allows the visualization of the right ventricular outflow, the aortic valve, the pulmonary valve, the pulmonary artery (Figure 5), and the left ventricular mid-cavity size (Figure 6) and function (with 2D visualization of segmental contractility)1,3,4,10,11.
Figure 7 shows the probe position on the chest to display the apical views. In the apical 4-chamber view (Figure 8), all 4-chamber dimensions (areas of all 4-chambers and volume of left ventricle) and function can be assessed. The anatomic and functional characterization of mitral and tricuspid valves can be also evaluated. The left ventricular outflow, aortic valve flow and ascending aorta were obtained with the apical 5-chamber view. The apical 2-chamber view (Figure 9) focuses on the left atrium and ventricular size and function. Apical 3-chamber and 5-chamber views allow aortic valve and left ventricular outflow evaluation. All views combined to allow the assessment of the different left ventricular walls and segments and the study of different systolic and diastolic function parameters1,3,4,10,11.
Left ventricular diastolic function can be assessed by pulsed Doppler imaging at the mitral valve (Figure 10), isovolumetric relaxation time of the left ventricle, and tissue Doppler imaging at the mitral annulus1,3,12. Normal mitral inflow consists of biphasic flow from the left atrium to the left ventricle. In normal conditions, the early flow coincident with E-wave is higher than the later flow that occurs with atrial contraction (A-wave).
Left ventricular diastolic function can also be studied with tissue Doppler imaging, which analyzes myocardial velocities (Figure 11). Spectral tissue Doppler imaging studies systolic and diastolic function over a cardiac cycle and has 3 peaks: one positive systolic peak (s&#39;-wave) representing myocardial contraction and two negative diastolic peaks (e&#39;-wave of early diastolic myocardial relaxation and a&#39;-wave of active atrial contraction in late diastole) assessed at the mitral annular level, from septal or lateral annulus1,3,4,10,14.
Characterization of left ventricular diastolic function by pulsed Doppler imaging at the mitral valve and tissue Doppler imaging at the mitral annulus should include the following parameters: E-wave velocity, A-wave velocity, E/A ratio, e&#39; velocity, a&#39; velocity, E/e&#39; ratio and deceleration time of E-wave1,3,4,10,14.
Left ventricular systolic function can be studied by mitral annular plane systolic excursion measurement, fractional shortening (Figure 3), ejection fraction, stroke volume, cardiac output, systolic tissue s&#39;-wave velocity (Figure 11) and global longitudinal strain by myocardial deformation with strain and strain rate analysis (Figure 12)1,3,4,10.
Ejection fraction is calculated with volumes by a modified Simpson method based on visual tracings of the blood and tissue interface using the apical 4 and 2-chamber views. At the basal or mitral valve level, the contour is closed by connecting the two opposite sections of the mitral ring with a straight line1,3,4,10. The volume of blood that forms the ejection fraction represents the stroke volume. If the mitral valve is competent, then this can be multiplied by heart rate to calculate the cardiac output1,3,4. Stroke volume is based on the measurements of blood flow through the left ventricle outlet tract during cardiac cycle, using this formula: 
SV = &#960; x (LVOT diameter /2)2 x VTI (LVOT) 
(LVOT: left ventricle outflow tract; LVOT diameter is measured in the parasternal long axis view. VTI(LVOT): velocity time integral traced from pulsed wave Doppler at LVOT in apical 5-chamber view)1,3.
The most commonly used strain-based measure of LV global systolic function is global longitudinal strain obtained by myocardial deformation with strain and strain rate analysis1,3,4,10. It is usually assessed by speckle-tracking echocardiography, where the peak of global longitudinal strain describes the relative length change of the LV myocardium between end-diastole and end-systole: 
GLS(%) = (MLs &#8722; MLd)/MLd 
(MLs: myocardial length at end-systole; MLd: myocardial length at end-diastole).
Measurements should begin with the apical 3-chamber view to visualize aortic valve closure, using opening and closing clicks of the aortic valve in spectral Doppler imaging or aortic valve opening and closing on M-mode imaging1,3,4,10. Apical 4 and 2-chamber views are also evaluated, and all three views&#39; measurements are averaged. Right ventricular systolic function is evaluated by tricuspid annular plane systolic excursion (TAPSE) and tissue Doppler imaging at tricuspid annulus. All valves are studied by color Doppler imaging, allowing direct visualization of stenosis or regurgitation (Figure 13). If aortic valve regurgitation is present, it can be studied and quantified by vena contracta and half-pressure time with continuous Doppler imaging (Figure 14)15. Figure 15 shows the ascending aorta, the aortic arch and the proximal descending aorta visualized in suprasternal window.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60404/60404fig1a.jpg" /> 
Figure 1: Probe positioning for parasternal long-axis view. <a href="https://www.jove.com/files/ftp_upload/60404/60404fig1large.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/60404/60404fig2a.jpg" /> 
Figure 2: 2D parasternal long-axis view of left atrium (LA), left ventricle (LV), aortic valve, ascending aorta (Ao) and mitral valve (MV). <a href="https://www.jove.com/files/ftp_upload/60404/60404fig2large.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/60404/60404fig3a.jpg" /> 
Figure 3: M-Mode of left ventricle with measurements, including interventricular septum thickness in diastole (IVSd), left ventricle internal diameter in diastole (LVIDd) and systole (LVIDs), posterior wall thickness (LVIPWd), fractional shortening (%FS), ejection fraction calculated with Teichholz method [EF(Teich)], left ventricle mass (LVdMass), parietal thickness (EPR) and left ventricle mass with calculation adapted to rodent (LVM Mouse). <a href="https://www.jove.com/files/ftp_upload/60404/60404fig3large.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/60404/60404fig4a.jpg" /> 
Figure 4: Probe positioning for parasternal short-axis view. <a href="https://www.jove.com/files/ftp_upload/60404/60404fig4large.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/60404/60404fig5a.jpg" /> 
Figure 5: 2D parasternal short-axis view at aortic valve (Ao), left atrium (LA), right atrium (RA), right ventricle (RV) and pulmonary artery (PA). <a href="https://www.jove.com/files/ftp_upload/60404/60404fig5large.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/60404/60404fig6a.jpg" /> 
Figure 6: Parasternal short-axis view at left ventricle papillary muscles level. <a href="https://www.jove.com/files/ftp_upload/60404/60404fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60404/60404fig7a.jpg" /> 
Figure 7: Probe positioning for apical 4-chamber view. <a href="https://www.jove.com/files/ftp_upload/60404/60404fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60404/60404fig8a.jpg" /> 
Figure 8: 2D of 4-chamber view including left atrium (LA), left ventricle (LV), right atrium (RA) and right ventricle (RV). <a href="https://www.jove.com/files/ftp_upload/60404/60404fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/60404/60404fig9a.jpg" /> 
Figure 9: 2D Echo of apical 2-chamber view including left atrium (LA), ventricle (LV) and mitral valve (MV). <a href="https://www.jove.com/files/ftp_upload/60404/60404fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/60404/60404fig10a.jpg" /> 
Figure 10: Pulsed wave Doppler at mitral valve, showing E-wave velocity = 0.49 m/s, A-wave velocity = 0.33 m/s, E-wave deceleration time = 35 ms and E/A ratio = 1.48. <a href="https://www.jove.com/files/ftp_upload/60404/60404fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/60404/60404fig11a.jpg" /> 
Figure 11: Spectral Doppler tissue at septal mitral annulus, showing myocardial tissue waves of diastole (e&#39; and a&#39;) and of systole (s&#39;). <a href="https://www.jove.com/files/ftp_upload/60404/60404fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/60404/60404fig12a.jpg" /> 
Figure 12: Myocardial deformation analysis with longitudinal strain evaluated at 4-chamber view. <a href="https://www.jove.com/files/ftp_upload/60404/60404fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/60404/60404fig13a.jpg" /> 
Figure 13: Visualization of aortic regurgitation with Color Doppler. <a href="https://www.jove.com/files/ftp_upload/60404/60404fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/60404/60404fig14a.jpg" /> 
Figure 14: Continuous Doppler of aortic valve at apical 5-chamber view, showing regurgitation above baseline with half-pressure time measured of 95 ms. <a href="https://www.jove.com/files/ftp_upload/60404/60404fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/60404/60404fig15a.jpg" /> 
Figure 15: Suprasternal view of ascending aorta (Asc), aortic arch (Arch) and descending aorta (Desc). <a href="https://www.jove.com/files/ftp_upload/60404/60404fig15large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats at JoVE.com

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

echocardiographic-assessment-cardiac-anatomy-function-adult

Research

JoVE Journal

Medicine

20.4K Views.  Universidade de Lisboa. A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

Video: Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

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, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Normothermic Ex Situ Heart Perfusion in Working Mode: Assessment of Cardiac Function and Metabolism

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation time and increases the risk of adverse post-transplantation outcomes. Moreover, the static nature of CS does not allow for organ evaluation or intervention during the preservation interval. Normothermic ex situ heart perfusion (ESHP) is a novel method for preservation of the donated heart that minimizes cold ischemia by providing oxygenated, nutrient-rich perfusate to the heart. ESHP has been shown to be non-inferior to CS in the preservation of standard-criteria donor hearts and has also facilitated the clinical transplantation of the hearts donated after the circulatory determination of death. Currently, the only available clinical ESHP device perfuses the heart in an unloaded, non-working state, limiting assessments of myocardial performance. Conversely, ESHP in working mode provides the opportunity for comprehensive evaluation of cardiac performance by assessment of functional and metabolic parameters under physiologic conditions. Moreover, earlier experimental studies have suggested that ESHP in working mode may result in improved functional preservation. Here, we describe the protocol for ex situ perfusion of the heart in a large mammal (porcine) model, which is reproducible for different animal models and heart sizes. The software program in this ESHP apparatus allows for real-time and automated control of the pump speed to maintain desired aortic and left atrial pressure and evaluates a variety of functional and electrophysiological parameters with minimal need for supervision/manipulation.

Normothermic ex situ heart perfusion (ESHP), preserves the heart in a beating, semi-physiologic state. When performed in a working mode, ESHP provides the opportunity to perform sophisticated assessments of donor heart function and organ viability. Here, we describe our method for myocardial performance evaluation during ESHP.

The current standard method for organ preservation (cold storage, CS), exposes the heart to a period of cold ischemia that limits the safe preservation ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...