logy 115 (2007) 164–170www.elsevier.com/locate/ijcard

International Journal of Cardio

Doppler echocardiography and Tissue Doppler Imaging in thehealthy rabbit: Differences of cardiac function during awake and

anaesthetised examination

Jörg Stypmann a,b,⁎,1, Markus A. Engelen a,c,1, Anne-Kristin Breithardt d,2,3, Peter Milberg a,Markus Rothenburger e, Ole A. Breithardt f, Günter Breithardt a,b, Lars Eckardt a,b,

Poulsen Nautrup Cordula c

a Department of Cardiology and Angiology, University Hospital Münster, Germanyb Interdisciplinary Center for Clinical Research, Central Project Group (ZPG 4a), Westfälische Wilhelms Universität, Münster, Germany

c University Medical Center Utrecht, Department of Medical Physiology, The Netherlandsd Veterinary Anatomy I, Ludwig-Maximilian-University, München, Germany

e Department of Thoracic and Cardiovascular Surgery, University Hospital Münster, Germanyf Department of Cardiology, University Hospital Mannheim, Germany

Received 19 October 2005; received in revised form 7 January 2006; accepted 11 March 2006Available online 27 June 2006

Abstract

Objective: In the past years, Doppler echocardiography has evolved into a commonly used technique. More recent sophisticated advances inimaging quality have substantially improved spatial and temporal resolution allowing the adaptation of this technique to small animal models,particularly in rabbits but even in mice. Recently, parameters obtained by Tissue Doppler Imaging (TDI) have been shown to be moreindependent of pre- and afterload than classic hemodynamic Doppler measurements. Exploration of animal models may require anaesthesiabut there is only very little information on the effect of anaesthesia on echocardiographic parameters in rabbits.Methods: We therefore performed Doppler-echocardiographic examinations of 20 wild-type New Zealand White rabbits in awake state andunder light ketamine–xylazine anaesthesia. Special focus was put on the evaluation of global and regional left ventricular systolic anddiastolic function using TDI and the myocardial performance index (Tei-index).Results: Doppler-echocardiographic measurements including TDI in rabbits were feasible to assess cardiac morphology and function within ashort examination time. There were some distinct changes of functional parameters during anaesthesia. Exemplary for systolic function,fractional shortening, cardiac output and systolic TDI velocity of the lateral wall decreased distinctly. Global left ventricular functionmeasured by the Tei-index deteriorated.Conclusions: Doppler echocardiography and TDI can be performed easily, quickly and safely in the rabbit. Anaesthesia with thecardiodepressive ketamine–xylazine shows some distinct Doppler-echocardiographically measurable negative effects on cardiac function.Thus, echocardiography with less cardiodepressive anaesthetic regimes or even without anaesthesia after training of the animals should beconsidered as alternatives whenever possible.© 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Echocardiography; Tissue Doppler Imaging; Rabbit; Anaesthesia; Awake; Narcosis

⁎ Corresponding author. UniversitätsklinikumMünster Medizinische Klinik und Poliklink C Kardiologie und Angiologie Albert-Schweitzer-Str. 33 D - 48149Münster, Germany. Tel.: +49 251 8347617; fax: +49 251 8347684.

E-mail address: Stypmann@mednet.uni-muenster.de (J. Stypmann).1 These authors contributed equally.2 These data are part of the doctoral thesis of A.-K. Breithardt.3 Current address: Von-Esmarch-Str. 117, 48149 Münster, Germany.

0167-5273/$ - see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.ijcard.2006.03.006

165J. Stypmann et al. / International Journal of Cardiology 115 (2007) 164–170

1. Introduction

Recent developments in animal models of cardiovasculardiseases have made them an increasingly important tool incardiovascular research. Rabbit models have vastly beenused, especially in the study of arrhythmogenesis asarrhythmias in relatively large rabbit hearts are morecomparable to human arrhythmias, particularly due to more“critical mass” for reentry as compared to smaller animalmodels like mice. Doppler echocardiography has evolvedinto a commonly used technique in various animal modelsdue to recent advances in the imaging processes that haveimproved spatial and temporal resolution. This as well asminiaturization has led to the adaptation of this technique tothe study of the physiology of animal models that nowreliably allow the non-invasive assessment of cardiovascularmorphology and function.

Anaesthesia may have a profound effect on cardiacfunction, but there is only scarce information on its effect oncardiac function in the rabbit. Although Doppler echocardi-ography is widely used, only scarce echo data on rabbitmodels are available. Tissue Doppler Imaging has theadvantage to be rather independent of preload [1]. We,therefore, systematically performed Doppler echocardiogra-phy including M-Mode, 2D and TDI in 20 wild-type NewZealand White rabbits of either sex in the awake state as wellas during anaesthesia to obtain normal values and to evaluatethe effect of anaesthesia on cardiac function.

2. Methods

Animal experimental procedures were performed inaccordance with institutional guidelines and approved bylocal authorities. The investigation conforms to theGuide forthe Care and Use of Laboratory Animals published by theUS National Institutes of Health (NIH Publication No. 85-23, revised 1996). We studied twenty 16-week-old wild-typeNew Zealand White rabbits (10 male, 10 female, Peter Rolli,Oelde, Germany). They were housed in the Central AnimalFacility of the Medical Faculty of the University of Münsterat 20 °C at 60% humidity with a 12:12-h light–dark cycleand fed with a standard diet and water ad libidum.

2.1. Echocardiography

Doppler echocardiography was performed in awakeanimals and under light ketamine–xylazine anaesthesia ineach animal. The thoracic fur was completely removed and a1-lead ECG was obtained during all measurements tomonitor heart rate continuously.

For the awake examination, animals were held on the lapof the examiner (A.K.B.) and first allowed to adapt to thissituation for 5 min. This situation was trained with theanimals several times before the examination to train themand to avoid too much stress. Echocardiography wasperformed in a sitting position.

For the examination in light anaesthesia, animals wereanesthetized by intramuscular injection of a mixture ofketamine (50 mg/kg) und xylazine (4 mg/kg) and allowed tobreathe spontaneously. The anesthetized animals wereplaced in a left lateral position on a custom-built echo-table on a feedback-controlled heating pad to maintain bodytemperature at 37 °C.

Transthoracic Doppler echocardiography was performedusing a commercially available digital cardiac ultrasoundplatform equipped with a 12-MHz short focal-length phasedarray transducer (SONOS 5500, B2 software package,Philips Medical Systems, The Netherlands). A smooth layerof ultrasound gel previously centrifuged at 3000 rpm for10 min to avoid air bubbles that would disturb acousticalcoupling was placed on the chest of the rabbits. The probewas gently dipped into this layer of gel avoiding any pressureon the thorax.

Short axis and apical four- and five-chamber views in thelong axis were obtained to measure the length of the leftventricle in end-diastole and diameters of the LV outflowtract, aortic root and left atrium. B-mode guided M-modeechocardiography was performed in the parasternal long-axisview at the mid level of the papillary muscles. End-diastolicand end-systolic dimensions of the LV-cavity as well asanterior and posterior wall thickness were assessed accord-ing to the leading-edge method of the American Society ofEchocardiography [2].

The percentage of fractional shortening was calculated as

FS %ð Þ ¼ LVEDD� LVESDLVEDD

� �� �d 100

For determination of systolic outflow of the left ventricle,pulsed wave Doppler signals were obtained by placing thesample volume parallel to the flow in the long-axis 5-chamber view into the left ventricular outflow tract. Mitralinflow was assessed in the apical 4-chamber view with thesample volume placed at the level of the mitral leaflet tips.

Monoplane left ventricular ejection fraction (EF) wascalculated from the apical 4-chamber view using the methodof discs (modified Simpson rule) [3,4]. In addition, EF wasmeasured using the automatic-border-detection (ABD)function of the ultrasound platform [5].

Left ventricular stroke volume (SV) was calculated as

SV ¼ p� ðLVOT=2Þ2 � VTI

π=3.14, VTI = velocity time integral of the aortic flow.Cardiac output (CO) was calculated as

CO ¼ SV� HR

SV = stroke volume, HR = heart rate

Isovolumetric relaxation time (IVRT) was measured asthe time interval between end of aortic outflow and onset ofthe mitral inflow by PW-Doppler. Isovolumetric contraction

166 J. Stypmann et al. / International Journal of Cardiology 115 (2007) 164–170

time (IVCT) was measured as the time interval between theend of mitral inflow and onset of aortic outflow by PW-Doppler (Fig. 1).

The myocardial performance index according to Tei [6–9] (Tei Index) was calculated as

MPIðTeiÞ ¼ IVCTþ IVRTLVET

with IVCT=isovolumetric contraction time, IVRT=isovo-lumetric relaxation time, LVET=left ventricular ejectiontime.

Tissue Doppler imaging was performed from the apical 4-chamber view as previously described [10–12]. In brief, thesample volume was placed at the septal and lateral insertionsite of the mitral annulus. Gain and filter settings wereadjusted to eliminate background noise and allow forrecording of clear tissue signals. Measurements includedearly and late diastolic as well as systolic maximal velocity.

2.2. Statistical analysis

For each Doppler-echocardiographic parameter, the meanof at least three beats was calculated. All results are given asmeans±standard deviation. Statistical analysis was per-formed using SPSS software (Version 13, SPSS, Chicago).Whenever appropriate, the data of the awake and theanesthetized animals were compared with Student's t-testfor matched pairs. Two-sided P-values<0.05 were consid-ered as significant.

Fig. 1. Measurement of IVCT, IVRT and calculation of m

3. Results

Technically adequate measurements could be obtainedin all 20 animals. No rabbit died during or afterexamination. Mean body weight was 2.92 kg (range2.45–3.35 kg). Weight did not change significantlybetween the examination under narcosis and in the awakestate. As expected, heart rate was significantly lower in theanesthetized (198±37 bpm) as compared to the awakeanimals (234±26 bpm, p<0.0001). Heart rate was stableduring the whole examination as well in the awake state asunder anaesthesia. All echocardiographic studies werecompleted within 20 min. Representative examples areshown in Fig. 2.

Results of the B- and M-mode measurements are shownin Table 1. Fractional shortening was 17.4% lower duringanaesthesia as compared to the awake state ( p=0.001),mainly due to an increase of the LV inner diameter in systole(+6.4%, p=0.032).

Doppler measurements including TDI and calculatedindices are shown in Table 2. Several significant differencesare found between the awake and anaesthetised state.Exemplary for systolic function, cardiac output (−27%,p=0.006) and systolic TDI velocity of the lateral walldecreased distinctly (−23%, p=0.038). Global left ventric-ular function measured by Tei index deteriorated (0.57±0.20versus 0.71±0.16, p=0.011). In some animals (3 of 20), E/Aratio was reversed under anaesthesia. There was a significantunderestimation of ejection fraction using automatic borderdetection compared to the EF determined according to

yocardial performance index according to Tei [8].

Fig. 2. Representative echocardiographic examples. White bars represents 200 ms. PW–Doppler of the aortic outflow (awake A, narcosis B), mitral LV inflow(awake C, narcosis D with reversal of the E/A ratio), M–Mode of the LV (E) and aorta+LA (F), TDI (awake G, narcosis H with reversal of the E/A ratio).

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Simpson's rule (for examination of awake animals–11%,p=0.0042 and for animals during anaesthesia–17%,p=0.006).

Table 1B- and M-mode measurements

Measurement Awake Narcosis p

LVOT (cm) 0.723±0.065 0.714±0.064 nsLVAWd (cm) 0.217±0.056 0.199±0.033 nsLVPWd (cm) 0.274±0.041 0.267±0.047 nsLVIDd (cm) 1.540±0.112 1.480±0.081 nsLVIDs (cm) 1.009±0.091 1.074±0.089 0.032FS (%) 34.5±4.9 28.5±3.8 0.001

LVOT=LV outflow tract; LVAWd=LV anterior wall in diastole;LVPWd=LV posterior wall in diastole; LVIDd/LVIDs=LV inner diameterin diastole/systole; FS=fractional shortening; ns=not significant.

4. Discussion

Doppler echocardiography of animal models more andmore evolved to be an important non-invasive tool infundamental and clinical cardiovascular research. Acomplete Doppler-echocardiographic examination includ-ing TDI can be obtained in the awake as well as in theanaesthetised rabbit. Examination in the awake state leadsto more physiological results as compared to echocardi-ography in ketamine–xylazine anaesthesia whereas exam-ination in the awake status is more difficult, more time-consuming, and needs special training of the animals sothat they are accustomed to the examination situation andof the examiner to perform the examination carefully andwithout abruptness.

Table 2Doppler-echocardiographic measurements including TDI and calculatedindices

Measurement Awake Narcosis p

Ao Vmax (cm/s) 74.9±19.5 72.3±17.0 nsLVET (ms) 126±14 146±17 0.001SV (ml) 1.8±0.46 1.6±0.6 nsEF (%) (Simpson) 54±9 53±9 nsEF (%) (ABD) 48±8 44±9 0.055CO (ml/min) 421±100 307±99 0.0006E-wave max (cm/s) 71.5±13.8 55.6±10.3 0.043A-wave max (cm/s) 51.4±14.5 37.9±9.2 0.031E/A ratio 1.44±0.28 1.51±0.31 nsIVRT (ms) 38±5 43±10 nsIVCT (ms) 24±11 36±10 0.0348IVCT+IVRT (ms) 107±18 73±30 0.0258MPI (Tei) 0.57±0.20 0.71±0.16 0.011TDI syst LW (cm/s) 5.5±1.3 4.9±1.0 0.041TDI E LW (cm/s) 6.7±1.9 5.7±2.2 nsTDI A LW (cm/s) 3.9±0.7 4.6±1.1 nsTDI syst septal (cm/s) 5.3±1.0 4.08±0.5 0.038TDI E septal (cm/s) 5.0±1.7 4.1±1 nsTDI A septal (cm/s) 3.2±0.9 3.9±1.2 ns

Ao Vmax=maximum flow velocity aorta; LET=LV ejection time;SV=stroke volume; EF=ejection fraction; CO=cardiac output; E-wavemax=maximum early flow velocity mitralis; A-wave max=maximum late(atrial) flow velocity mitralis; IVRT/IVCT= isovolumetric relaxation/contraction time; MPI=myocardial performance index; TDI=TissueDoppler Imaging; ns=not significant.

Table 3Comparison of our values with literature

Own(anaesthesia)

Minimum Maximum

Heart rate (bpm) 198±37 145±14 [12] 297±8 [27]LVOT (cm) 0.714±0.064 0.65±0.05 [11] 0.75±0.04 [11]LVAWd (cm) 0.199±0.033 0.2±0.03 [10] 0.33±0.04 [11]LVPWD (cm) 0.267±0.047 0.21±0.01 [28] 0.3±0.06 [11]LVIDd (cm) 1.480±0.081 1.41±0.02 [10] 1.75±0.03 [29]LVIDS (cm) 1.074±0.089 0.88±0.02 [30] 1.08±0.13 [31]FS (%) 28.5±3.8 29±4 [30] 37.6±7.6 [10]EF (%) 53±9 42±6 / 62±6 [30] 73.8±1.9 [32]E-wave max

(cm/s)57.5±11.2 47±6 [12] 54±8 [11]

A-wave max(cm/s)

37.9±9.4 26.5±6 [10] 41±8 [30]

IVRT (ms) 46±19 40±6 [10] 45.3±6 [12]

min/max=minimal and maximal value found in the literature, furtherabbreviations see Tables 1 and 2.

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Heart rate under anaesthesia was significantly lower ascompared to heart rate in the awake state. The higher heartrate in the awake animal is mainly due to the increase ofsympathetic tone which is counteracted by a negativeinotropic effect of the anaesthetics and a well-knownnegative chronotropic effect particularly of xylazine [13–15]. Marano et al. [16] reported the physiologic heart rate ofthe New Zealand White rabbit measured by telemetrywithout any restrainment or drug-effects to be 218±4 bpm,suggesting that the higher rate in our animals in the awakestate was indeed due to an increase in sympathetic tone andthe lower rate during anaesthesia was mainly due to a directnegative chronotropic effect of xylazine. Different sympa-thetic tones should be taken into account when assessingcardiac function of rabbits under awake or anaesthetizedconditions.

To the best of our knowledge, there has been nointernational publication showing echocardiographic datain the awake rabbit. We did not find any difference instructural measurements between animals in the awake stateand under anaesthesia. However, most functional parametersin M-mode and pulsed-wave Doppler measurements werechanged under anaesthesia as compared to the awake state.Particularly, fractional shortening and cardiac output asmeasurement for systolic function decreased significantlyunder narcosis. This can be explained by a direct negativeinotropic and chronotropic effect of the anaesthetic regimeused in this study and by reduction in sympathetic drive[14,15,17,18]. There was no significant change in E/A-ratio

since the maximum velocity of E- and A-wave decreased to asimilar degree. But there was a reversal of the E/A-ratio insome animals under anaesthesia, showing anaesthesia-induced impairment of LV diastolic function [19]. Thehighly significant decrease of cardiac output duringanaesthesia can largely be explained by the significantreduction of heart rate as stroke volume only decreasedslightly without reaching statistical significance. LV ejectionfraction according to Simpson's rule did not show anydifference comparing the examinations of awake rabbits withthe examinations under narcosis whereas there was asignificant underestimation of EF determined by automaticborder detection comparing the different methods ofdetermination. This is probably due to problems inherentin the software algorhythmus of this method, mainlyinaccurate detection of the endocardial border in the smalland fast-beating rabbit heart. Use of automatic borderdetection is also discussed quite controversially in humanpatients [20]. In the future, these problems might beovercome by the use of contrast echocardiography for betterdelineation of the left ventricular cavum and by three-dimensional echocardiography to resolve the necessity ofpresuming a symmetric left cavity for the calculation of leftventricular volumes.

There are only scarce data on echocardiography in rabbitsin the literature, especially on TDI. Anaesthesia leads to asignificant increase in LV ejection time and isovolumetriccontraction time whereas isovolumetric relaxation timetended to increase but this trend did not reach statisticalsignificance. This resulted in a significant increase of themyocardial performance index (Tei index), indicating asignificant global impairment of systolic and diastolicfunction under anaesthesia [6–9]. To the best of ourknowledge, no previous study has used the myocardialperformance index as a measure for global systolic anddiastolic LV-function in the rabbit.

We found distinctly lower wall motion velocities ascompared to the scarce data in the literature [10–12]. This is

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probably due to the use of different ultrasound-platforms anddifferent settings. We could show a significant decrease insystolic wall motion velocity under anaesthesia representingimpairment of regional systolic LV-function. Furthermore,there was a distinct increase of atrial contribution to LVfilling as compared to the awake state. This underlines theproblems of anaesthesia in rabbits.

Our measurements are comparable to those reportedpreviously (Table 3). However, we did not find anyinformation on LV ejection time, cardiac output, andisovolumetric contraction in the surveyed literature.

5. Limitations

One limitation of this study is the use of ketamine–xylazine anaesthesia. But on the other hand, this anaesthesiawas chosen deliberately because of the well-knowncardiodepressive effects as one of the major issues of thisstudy was to prove if TDI and MPI could distinguish theglobal and regional left ventricular performance under thedifferent examination settings. Most available anaestheticshave a distinct impact on cardiac function, partly by theirinfluence on autonomic tone, partly due to a directcardiodepressive effect. Newer volatile anaesthetics likeisoflurane or sevoflurane have less impact on cardiacfunction and heart rate but this kind of anaesthesia was notyet readily available when we started our experiments. Theimpact of these anaesthetics on ultrasound examinationsincluding TDI, strain and strain rate is the focus ofsubsequent recent studies in our animal laboratory. Intrave-nous/intramuscular injection narcosis, particularly keta-mine–xylazine, is still very common in research as well asin veterinarian practice. Comparison of echocardiography inthe awake state versus under anaesthesia was part of thescope of this paper.

We did not validate our measurements directly to invasivemeasurements but this was not in the primary scope of thisstudy. Comparisons between echocardiographic parametersincluding TDI and invasive measurements have already beendone extensively in other studies, mainly in human patients(e.g., [21,22]). Finally, invasive measurements in the awakeanimal cannot be easily performed. Data on reproducibilityof echocardiographic examinations in rabbits were notperformed as the main scope of the presented study was toprove the discriminative power of TDI and MPI to detectchanges in left ventricular function. Extensive experience ofour group in qualified repetitive echocardiography's in smallanimals has been published before [23–26].

6. Conclusions and outlook

We could show that a complete Doppler-echocardio-graphic examination of the rabbit is feasible within a shortexamination time during light anaesthesia as well as in theawake state. Since various anaesthetic agents may have adistinct negative impact on cardiovascular function, the

choice whether animals are examined in the awake state orunder light anaesthesia should be reflected carefully.Echocardiographic measurements in the absence of cardi-odepressive effects of anaesthetic drugs allow obtainingmore physiological parameters. On the other hand,examination without any anaesthesia is more cumbersomelyand a cardiovascular effect of a slight increase insympathetic tone cannot be ruled out. These problemsmight in future be overcome by introduction of neweranaesthetic regimes using volatile narcotics as isoflurane,sevoflurane or Xenon.

Tissue Doppler Imaging and calculation of the Tei-indexshould be implemented as part of standard rabbit echocar-diography protocol to judge global and regional systolic anddiastolic LV-function in rabbit models. Thereby, rabbitmodels of cardiovascular diseases can be examined moreindependent of pre- and afterload. Thus, the reliability ofthese measurements is improved and the results are bettertransferable to clinical patients. In the future, TDI measure-ments can be supplemented by the introduction of strain- andstrain-rate measurements.

Acknowledgements

This work was partly supported by grants from theDeutsche Forschungsgemeinschaft (DFG), Sonderforschungs-bereich 656 MoBil Münster, Germany (project C3).

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