Copyright © 2006, The European Society of Cardiology
Doppler myocardial imaging in adult male rats: Reference values and reproducibility of velocity and deformation parameters
aDepartment of Cardiology, University Hospital AZ VUB, Laarbeeklaan 101, 1090 Brussels, Belgium
bDepartment of Nuclear Medicine, University Hospital AZ VUB, Laarbeeklaan 101, 1090 Brussels, Belgium
cDepartment of Cardiology and Cardiac Imaging Research, University Hospital Gasthuisberg, Leuven, Belgium
Received 22 December 2005; received in revised form 7 March 2006; accepted after revision 19 March 2006.
* Corresponding author. Tel.: +32 2 477 6312; fax: +32 2 477 6840. igwsc{at}az.vub.ac.be
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionConclusionReferences |
|---|
Aim Limited data are available about the use of Doppler myocardial imaging (DMI) in small animals. We intend to provide reference values for velocity, strain and strain rate in a large group of healthy rats and studied the reproducibility and repeatability of these parameters.
Methods and results A total of 33 male Wistar rats (503±41g) underwent baseline transthoracic echocardiography with DMI of the anterior and inferior wall in a short-axis view using a 13MHz linear probe. Adequate tissue Doppler measurements could be obtained in 30 rats. On average 10±4 consecutive cycles were studied in post-processing using dedicated software (SPEQLE). Mean radial peak systolic velocity, strain and strain rate were respectively –0.8±0.3cm/s, 38±8% and 9.1±2.0/s in the anterior wall and 3.1±0.6cm/s, 49±10% and 13.7±3.7/s in the inferior wall. The reproducibility and repeatability of the DMI measurements assessed in 10 rats was good.
Conclusion DMI is feasible and reproducible in healthy rats. Establishing reference values opens new perspectives towards the use of strain and strain rate imaging in small rodents in the assessment of myocardial diseases.
Keywords: Echocardiography; Rats; Doppler myocardial imaging; LV function; Reproducibility
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionReferences |
|---|
Transthoracic echocardiography has been established as a safe and reliable non-invasive technique to assess cardiac anatomy and function in a large series of species.
In humans and large animals Doppler myocardial imaging (DMI) can be used to quantify regional myocardial function accurately and more objectively.1–5 Application of this non-invasive tool in small animals is challenging because of the small heart size and fast heart rate. New ultrasound technologies have been developed with increasing resolution capacities resulting in good image quality in small animals.
Very recently the feasibility of measuring myocardial velocity and deformation parameters was demonstrated in small groups of mice and rats.6,7
The purpose of our study was, by studying a large group healthy rat, to provide reference values for myocardial velocity, strain and strain rate. The reproducibility and repeatability of these parameters was tested.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionReferences |
|---|
Animals
A total of 33 adult male Wistar rats (22–24weeks old, weight 503±41g) were studied. The study was conforming to the guidelines of the American Heart association on research animal use. The animal research committee of our institution approved the study protocol.
Anesthesia protocol
Rats were anesthetized with pentobarbital 50mg/kg administered intraperitoneally after short gas anesthesia with isoflurane 2%. To avoid a negative effect of isoflurane on cardiac function, image acquiring was started half an hour after induction with this anesthetic gas.8
Echocardiogram protocol
All rats received baseline echocardiograms under controlled anesthesia and spontaneous respiration, using a Vivid 7 (GE VingMed, Horten, Norway) with a linear 13MHz probe (i13L). The anterior chest hair was removed using a shaver and the rats were positioned in left lateral decubitus on a wooden bench (Fig. 1). Recordings were made under continuous ECG monitoring by fixing the electrodes on the limbs.
|
Grey scale imaging
Grey scale images were recorded in a parasternal short axis view at a depth of 2cm. M-Mode tracings were recorded at the level of the papillary muscles at a speed of 200mm/s. Measurements were done offline using EchoPAC PC (GE Vingmed, version 3.1.3). Left ventricular (LV) dimensions were measured from three consecutive cardiac cycles on the M-Mode tracings. The Teichholz formula was used to calculate LV volumes: Pi*D3/6a (D=diameter of the ventricle in short axis view; a=ellipticity factor. An ellipticity factor of 1/3 was used, assuming that L=3D (L=total length of the ellipse). LVEF was calculated as LV end-diastolic volume – LV end-systolic volume/LV end-diastolic volume and expressed in %.
Doppler myocardial imaging
Color Doppler myocardial images were recorded in a parasternal short axis view at the mid-ventricular level at a depth of 2cm. The tissue Doppler frequency was 6.4MHz. The nyquist limit was set as low as possible avoiding aliasing. High frame rate was obtained by reducing beam width.
All measurements were performed offline using dedicated software (SPEQLE). Strain rate was calculated from the spatial derivative of the myocardial velocities over the computation area. Natural strain profiles were obtained by time integrating the strain rate profile. Lateral averaging of 3 to 5 beams/pixels was performed and the values for peak systolic velocity, strain, and strain rate were averaged over a mean of 10±4 consecutive cycles. A strain estimation length of 0.8–1.2mm was used depending on the thickness of the wall. The software allows M-mode tracking of the wall to ensure the sample volume stays in the middle of the myocardium. Timings of the beginning and ending of the ejection phase were obtained using the ECG and the velocity trace as previously demonstrated in humans.3
Repeatability and reproducibility
To assess the repeatability of the tissue Doppler parameters, echocardiography was repeated in 10 rats with an interval of 1week and analyzed by the same observer (inter study variability).
Intra- and inter observer variability was assessed by analyzing the color-coded images from 10 consecutive exams by two independent observers and by the same observer with an interval of 2months. The first observer was experienced in clinical and small animal echocardiography and in tissue Doppler analysis. The second observer was a medical doctor in training.
Statistical analysis
Data were averaged and presented as means±SD.
Reproducibility was assessed as the mean difference between two measurements and presented as mean percent error±SD (absolute difference/average of both observations). Differences between the repeated measurements were evaluated by the paired Student's t-test. P<0.05 was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionReferences |
|---|
All rats survived the protocol and no heart rate abnormalities could be detected. The mean heart rate was 308±19 beats per minute.
M-Mode parameters
Fig. 2 shows an example of an M-Mode image obtained in a parasternal short axis view. Image quality was excellent in all rats. Means and SD of the different anatomical parameters are given in Table 1. As can be derived from Fig. 2 and from the M-Mode measurements wall thickening in the anterior wall was lower than in the inferior wall.
|
|
Doppler myocardial imaging
Adequate regional systolic velocity, strain and strain rate curves could be obtained in 30 rats. In 3 rats the tracings were not good enough to measure peak values of strain and strain rate accurately. The nyquist limit ranged between 8 and 12cm/s. The frame rate averaged 404±82 frames per second. An example of a tracing of the anterior and inferior is given in Fig. 3. Normal values for the anterior and inferior wall are given in Table 2.
|
|
As all animals were about the same age and weight no significant correlation between these characteristics and the deformation parameters could be found (R2<0.2).
Reproducibility and repeatability for radial peak systolic velocity, strain and strain rate is given in Table 3. We found a good reproducibility for all TDI measurements in the anterior and inferior wall (between 8 and 19%). In the anterior wall the repeatability was low for the velocity measurements (35%), but good for the deformation parameters. The mean values±SD of the repeated measurements are given in Table 4. There was no significant difference between the mean values of the repeated measurements.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionReferences |
|---|
This study illustrates the feasibility and reproducibility of Doppler myocardial imaging in the evaluation of LV function using high-resolution echocardiography in a large group of healthy rats. Reference values are given for radial systolic velocities, strain and strain rate of the anterior and inferior wall.
Standard measurements of LV wall thickness and systolic and diastolic LV diameters by M-mode have extensively been described in normal and diseased rat models.9–12 The ratio data such as fractional shortening and ejection fraction have been demonstrated to be similar in rat and human echocardiography.9 Comparable results were obtained in the present study by using high-resolution technology.
The use of M-Mode parameters for the evaluation of global LV function is limited because of their load dependency. Visual assessment of global and regional LV function is very subjective and gives rise to high inter and intra observer variability. In humans and large animals Doppler myocardial imaging can be used to analyze quantitatively regional LV function with good inter and intra observer variability.1–5 In small animals the use of this technique is challenging because of the small heart size and fast heart rate and demands high spatial and high temporal resolution capacities. Therefore, in our experiment, a high frequency probe (up to 14MHz) was used and high frame rate images (80 frames/heartbeat; temporal resolution 2.5msec) were obtained. This frame rate is high enough to get reliable velocity and deformation traces.1,2,13
Till now Doppler myocardial imaging in small animals has especially been used to assess mitral annulus and regional myocardial velocities.14–16 Radial myocardial velocities of the inferior (inferolateral) wall are likely to be similar in rodents when compared to adult echocardiography and range between 2.2 and 5.2cm/s depending on the size of the animal and the location of the sample volume.6,11,15,17,18 Radial anterior (anteroseptal) wall velocities are usually more difficult to measure and result in more artifacts in the velocity traces, which may partially be due to near field clutter. In the study by Sebag et al. radial anterior myocardial velocities were not published because of the large inter observer variability making the analysis unreliable.6 As anterior wall velocities in small animals are close to zero it can be expected that the use of "mean percent errors" to assess variability will easily result in higher variability or repeatability values although the absolute errors are very small. In our study the repeatability for the anterior wall velocity was low but the inter and intra observer reproducibility of the velocity measurements was good and comparable to the results of the inferior wall. We are the first authors to demonstrate that measuring anterior wall velocities is feasible in small animals and this is a relevant issue as the spatial derivative of local velocities is used to measure the deformation parameters.
The use of strain and strain rate imaging has become more promising in the analysis of regional contractility as it eliminates motion artifacts of the wall. Only few data have been published about deformation imaging in rodents.6,7,17,18
Derumeaux et al. first published the use of velocity gradients in the diagnosis of pathological LV hypertrophy in a small group of rats.17 Sebag et al. demonstrated recently that radial strain rate can be measured in mice with good inter and intra observer variability and validated the technique with sonomicrometry.6 As invasive techniques were used in both studies, only a small group of normal animals (n
10) were assessed and the analysis was restricted to the radial function of the inferolateral wall.
Hirano et al. recently used deformation imaging in rats for serial assessment of radial deformation parameters in response to dobutamine.7 In this first part of the study baseline values for peak systolic strain and strain rate of the inferolateral wall were 27±12% and 7.6±2.2/s, respectively. The inter and intra observer variability of the repeated measurements was low and ranged between 3.2 and 6.4%. As notified by the authors, the strain length used for the analysis was 2mm, which is probably too large for the rat's myocardium. Using a strain length that is larger than the myocardium can result in an underestimation of the radial deformation parameters. This has nicely been demonstrated recently in an open-chest pig model: the higher the strain length the lower the absolute values of the deformation parameters.5 This can be explained by the spatial gradient in strains through the thickness of the wall. Reducing the strain length on the other hand can lead to larger variations in measured strain because higher values are obtained when the sample volume is placed closer to the endocardium and lower values are obtained when measuring closer to the epicardium. Matre et al. stated in their conclusions that ideally the strain length should be half of the thickness of the wall.5 In our study a strain length 0.8 to 1.2mm was used for a diastolic wall thickness ranging between 1.6 and 2.3mm. Using this strain length resulted in higher but probably more correct mean values for the deformation parameters (radial peak systolic strain and strain rate 49±10% and 13.7±3.7/s, respectively). As mentioned earlier this also explains the higher but more realistic variability of the repeated measurements (between 14 and 19%) when compared to those described by Hirano et al.
The second part of the study performed by Hirano et al. evaluates the spatial distribution of the deformation parameters in a partially ischemic anterior wall.7 Because peak systolic strain values varied with different Doppler angles the corrected values were used as ratio of peak strain during ischemia to peak strain pre-ischemia. Reference values for the anterior wall measurements could therefore not be given and the reproducibility of the anterior wall parameters was not tested. We are the first authors to give reference values, with good reproducibility, for radial deformation parameters in the anterior wall in small animals.
The mean radial strain values obtained in our study are comparable to the mean values obtained in human echocardiography (anterior wall 41±17%19; inferior wall: 48±12%2). Due to the higher heart rate the mean radial values for strain rate are much higher in small animals compared to humans as the same amount of deformation has to be acquired in a much shorter period of time (anterior wall: 9.1±2.0 versus 3.8±0.6%19; inferior wall: 13.7±3.7 versus 3.1±0.7/s2).
In our study the anterior wall velocity, strain and strain rate values are smaller when compared to the inferior wall. The same regional differences in deformation have already been demonstrated in large animals.3,4 This is in concurrence with the differences in wall thickening as measured by M-Mode and is probably a consequence of interdependency of the anteroseptal wall with the right ventricle.
Limitations
Our results are restricted to the analysis of radial LV function. Cardiac imaging of the left ventricle in rodents is limited to a few echocardiographic views: parasternal short- and long- axis view and apical 4-chamber view. For tissue Doppler analysis the anteroseptal and inferolateral mid segments, in short axis view, are the most easily to analyze. Although it is possible to obtain an apical 4-chamber view, good alignment with the beam is difficult and the lateral wall is rarely visualized. The quality of the longitudinal velocity and deformation traces that we experienced is poor and therefore we could not give data about the longitudinal function assessed by DMI.
The type of anesthesia used for animal protocols can influence heart rate and intrinsic myocardial contractility and result in lower regional deformation parameters.8 Therefore reference values should always take into account the type of anesthesia used during the experiment. To avoid cardio-toxic effects of the anesthetic gas that we used, measurements were taken half an hour after the induction of the anesthesia.
|
Conclusion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionReferences |
|---|
This study determines the feasibility of measuring Doppler myocardial imaging parameters in the assessment of systolic LV function in adult male rats. We were able to establish reference values for radial velocity, strain and strain rate with good reproducibility and repeatability. Knowledge of these values permits more accurate quantification of echocardiographic data obtained in rat models with different cardiac disease such as heart failure models.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionConclusionReferences |
|---|
- Urheim S., Edvardsen T., Torp H., Angelsen B., Smiseth O.A. Myocardial strain by Doppler echocardiography validation of a new method to quantify regional myocardial function. Circulation (2000) 102:1158–1164.
[Abstract/Free Full Text] - Kowalski M., Kukulski T., Jamal F., D'hooge J., Weidemann F., Rademaekers F., et al. Can natural strain and strain rate quantify regional myocardial deformation? A study in healthy subjects. Ultrasound Med Biol (2001) 27(8):1087–1097.[CrossRef][ISI][Medline]
- Jamal F., Strotmann J., Weidemann F., Kukulski T., D'hooge J., Bijnens B., et al. Non-invasive quantification of the contractile reserve of stunned myocardium by ultrasonic strain rate and strain. Circulation (2001) 104:1059–1065.
[Abstract/Free Full Text] - Turschner O., D'hooge J., Dommke C., Claus P., Verbeken E., De scheerder I., et al. The sequential changes in myocardial thickness and thickening, which occur during acute transmural infarction, infarct reperfusion and the resultant expression of reperfusion injury. Eur Heart J (2004) 25:714–715.
[Free Full Text] - Matre K., Fannelop T., Dahle G.O., Heimdal A., Grong K. Radial strain gradient across normal myocardial wall in open chest pigs measured with Doppler strain rate imaging. J Am Soc Echocardiogr (2005) 18:1066–1073.[CrossRef][ISI][Medline]
- Sebag I., Handschumacher M., Ichinose F., Morgan J., Hataishi R., Rodrigues A., et al. Quantitative assessment of regional myocardial function in mice by tissue Doppler imaging. Comparison with hemodynamics and sonomicrometry. Circulation (2005) 111:2611–2616.
[Abstract/Free Full Text] - Hirano T., Asanuma T., Azakami R., Okuda K., Ishikura F., Beppu S. Non-invasive quantification of regional ventricular function in rats: assessment of serial change and spatial distribution using ultrasound strain analysis. J Am Soc Echocardiogr (2005) 18:907–912.[CrossRef][ISI][Medline]
- Roth D.M., Swaney J.S., Dalton N.D., Gilpin E.A., Ross J. Jr. Impact of anesthesia on cardiac function during echocardiography in mice. Am J Physiol Heart Circ Physiol (2002) 282:H2134–H2140.
[Abstract/Free Full Text] - Watson L., Sheth M., Denyer R., Dostal D. Baseline echocardiographic values for adult male rats. J Am Soc Echocardiogr (2004) 17(2):161–167.[CrossRef][ISI][Medline]
- Slama M., Ahn, Varagic J., Susic D., Frohlich E. Long-term left ventricular echocardiography follow-up of SHR and WKY rats: effects of hypertension and age. Am J Physiol Heart Circul Physiol (2004) 286:181–185.[CrossRef]
- Pires M., Salemi V., Cestari I.A., Picard M., Leirne A., Mady C., et al. Non-invasive assessment of Hemodynamic Parameters in experimental stenosis of the ascending aorta. Artif Organs (2003) 27:695–700.[CrossRef][ISI][Medline]
- Litwin S.E., Katz S.E., Weinberg E.O., Lorell B.H., Aurigemma G.P., Douglas P.S. Serial echocardiographic assessment of left ventricular function in rats with pressure-overload hypertrophy. Circulation (1995) 91:2642–2654.
[Abstract/Free Full Text] - Palka P., Lange A., Flemming A.D., Sutherland G.R., Fenn L.N., Mc Dicken W.N. Doppler tissue imaging: normal wall motion velocities in normal subjects. J Am Soc Echocardiog (1995) 8(5):659–668.[CrossRef][Medline]
- Nagueh S.F., Kopelen H.A., Lim D., Zoghbi W.A., Quinones M.A., Roberts R., et al. Tissue Doppler Imaging consistently detects myocardial contraction and relaxation abnormalities irrespective of cardiac hypertrophy in a transgenic rabbit model of human hypertrophic cardiomyopathy. Circulation (2000) 102(12):1346–1350.
[Abstract/Free Full Text] - Shimizu M., Konstantinov I., Suess A., Cheung M., McCrindle B., Vogel M., et al. Non-invasive analysis of myocardial function using high-resolution Doppler tissue echocardiography in rats. J Am Soc Echocardiogr (2005) 18:461–467.[CrossRef][ISI][Medline]
- Schaefer J.A., Klein G., Brand B., Lippolt P., Drexler H., Meyer G.M. Evaluation of left ventricular diastolic function by pulsed Doppler tissue in mice. J Am Soc Echocardiogr (2003) 16(11):1144–1149.[CrossRef][ISI][Medline]
- Derumeaux G., Mulder P., Richard V., Chargraoui A., Nafeh C., Bauer F., et al. Tissue Doppler imaging differentiates physiological from pathological pressure-overload left ventricular hypertrophy in rats. Circulation (2002) 105:1602–1608.
[Abstract/Free Full Text] - D'hooge J., Thijs D., Sipido K., Claus P., Bijnens B., Van de Werf F., et al. Ultrasound strain and strain rate imaging, a new tool to quantify regional myocardial function in mice. Eur Heart J (2004) 25:475. Abstract.
- Boettler P., Claus P., Herbots L., Mc Laughlin M., D'hooge J., Bijnens B., et al. New aspects of the ventricular septum and its function: an echocardiographic study. Heart (2005) 91:1343–1348.
[Abstract/Free Full Text]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Weytjens, P. R. Franken, J. D'hooge, S. Droogmans, B. Cosyns, T. Lahoutte, and G. Van Camp Doppler myocardial imaging in the diagnosis of early systolic left ventricular dysfunction in diabetic rats Eur J Echocardiogr, May 1, 2008; 9(3): 326 - 333. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||