Doppler Ultrasound in Mice

C© 2007, the AuthorsJournal compilation C© 2007, Blackwell Publishing, Inc.DOI: 10.1111/j.1540-8175.2006.00358.x

Doppler Ultrasound in MiceJorg Stypmann∗†‡∗Department of Cardiology and Angiology, Hospital of the University of Munster, Germany,†Interdisciplinary Center for Clinical Research (IZKF), Munster, Central Project Group 4a(ZPG4a), Germany, ‡Collaborative Research Center (SFB) 656, Project C3, Munster, Germany

Color, power, spectral, and tissue Doppler have been applied to mice. Due to the noninvasive na-ture of the technique, serial intraindividual Doppler measurements of cardiovascular function arefeasible in wild-type and genetically altered mice before and after microsurgical procedures or tofollow age-related changes. Fifty-megahertz ultrasound biomicroscopy allows to record the first beatsof the embryonic mouse heart at somite stage 5, and the first Doppler-flow signals can be recordedafter the onset of intrauterine cardiovascular function at somite stage 7. Using 10- to 20-MHz ultra-sound transducers in the mouse embryo, cardiac, and circulatory function can be studied as earlyas 7.5 days after postcoital mucous plug. Postnatal Doppler ultrasound examinations in mice arepossible from birth to senescent age. Several strain-, age-, and gender-related differences of Dopplerultrasound findings have been reported in mice. Results of Doppler examinations are influenced bythe experimental settings as stress testing or different forms of anesthesia. This review summarizesthe present status of Doppler ultrasound examinations in mice and animal handling in the frame-work of a comprehensive phenotype characterization of cardiac contractile and circulatory function.(ECHOCARDIOGRAPHY, Volume 24, January 2007)

review, tutorial, Doppler, echocardiography ultrasound, mice

Different Doppler techniques such as colorDoppler, power Doppler, spectral Doppler, andtissue Doppler have been used alternatively orin combination to assess global and regionalsystolic and diastolic cardiac function, globalcirculatory performance, and vessel propertiesin mice. The study of small mammals requiresspecially adapted ultrasound equipment forrecording and data acquisition, which must ful-fill the high demands of spatial and temporalresolution. Therefore, knowledge of the spatialand temporal resolution of the used equipmentis fundamental for interpretation of results. Theend-diastolic length of an adult mouse heart isaround 6–7 mm, left ventricular end-diastolicdiameter is close to 4 mm, and end-diastolic wallthickness of the posterior wall is around 1 mm.During normal heart rates up to 12 heart beatsper second can be measured.

For transthoracical Doppler echocardiogra-phy, special attention must to be put on the tech-

Address for correspondence and reprint requests: Dr. JorgStypmann, Medizinische Klinik und Poliklinik C, Kar-diologie und Angiologie, Universitatsklinikum Munster,Albert-Schweitzer-Str. 33, D-48149 Munster, Germany.Fax: 49-(0)251-8347684; E-mail: [email protected]

nical presettings of the echo platform to achievebest quality of the Doppler signals. For ade-quate spatial resolution, the transducer shouldwork within a range of 10–20 MHz and zoombox should be set at 2 to 3 cm depth. For ade-quate temporal resolution, the frame rate of the2D mode should not be lower than 200 framesper second and should not drop below 80–100frames per second during Doppler mode. Sweepspeed of the monitor should be set high at 100–200 mm/sec. For pulsed-wave Doppler exami-nations, the gate length of the sample volumeshould be minimized 0.6 mm or less and ve-locity scale settings should be adapted around120 cm/sec. During color flow Doppler exam-inations, aliasing due to the Nyquist relationshould be set at twofold maximum flow velocityof the examined valve in the region of interestfor best demonstration of valvular or septal de-fects.

General Considerations on Animal Careand Management with Influence on

Doppler Examinations in Mice

The knowledge of strain-dependent normalvalues of parameters of cardiac and vascular

Vol. 24, No. 1, 2007 ECHOCARDIOGRAPHY: A Jrnl. of CV Ultrasound & Allied Tech. 97

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function is crucial for the interpretation of re-sults.1,2

Circadian rhythms and resting or feedingconditions may modify the results of Dopplerexaminations. Mice are active during the night.Therefore, physiological parameters such asheart rate, blood pressure, stroke volume, orbody temperature show important diurnal vari-ations.3,4

All these problems can be minimized if themice are conventionally housed in a controlledenvironment with a temperature of 21 ± 1◦Cand a 12-hour day/night cycle. Mice should bekept in macrolon cages and have access to stan-dard food pellets and tap water ad libitum.All subsequent experiments including Dopplerechocardiographic recordings should be per-formed in accordance with approved protocolsof the local animal welfare committees. The in-vestigations should conform to the guide for theCare and Use of Laboratory Animals publishedby the US National Institutes of Health.

In addition, changes in the experimentalsetting from the choice of anesthetic agent(Table I,5–8), room temperature, or atmosphericcontent to the surrounding noise can influencethe parameters to be measured. To avoid sys-tematical error, Doppler ultrasound measure-ments in transgenic mice—like all functionalmeasurements—are therefore best performedat the same time of the day in age- and sex-matched pairs of transgenic mice and their lit-termate wild-type siblings.

TABLE I

Range of Normal Values for Functional Cardiac Parameters at Rest and during Acute ß-Adrenergic Stimulation andInfluence of the Type of Sedation, Analgesia, or Anesthesia Used

Base Base Base Isoprenaline Isoprenaline Isoprenaline

Anesthesia Ket/xyl Isoflurane Diazepam Ket/ Xyl Isoflurane DiazepamHeart rate (bpm) 320 ± 6 457 ± 17 520 ± 26 560 ± 18∗ 556 ± 15∗ 645 ± 13∗Cardiac output (ml/min) 12 ± 1 23 ± 3 17 ± 1 24 ± 1∗ 27 ± 4 24 ± 2∗Stroke volume µl [VTI] 39 ± 1 50 ± 6 32 ± 2 58 ± 6∗ 49 ± 7 37 ± 0∗Stroke volume (µl) [Teichholz] 54 ± 4 52 ± 4 34 ± 2 38 ± 3∗ 52 ± 3 27 ± 3∗FS (%) 36 ± 1 39 ± 1 50 ± 2 70 ± 3∗ 87 ± 4∗ 58 ± 2∗Vcf (circ/s) 6 ± 0 7 ± 0 9 ± 1 12 ± 1∗ 11 ± 2∗ 13 ± 1∗EF (%) 66 ± 2 72 ± 3 82 ± 2 95 ± 2∗ 88 ± 2∗ 89 ± 2∗MNSER 0.8 ± 0.1 1.1 ± 0.0 1.4 ± 0.1 1.6 ± 0.1∗ 1.6 ± 0.1∗ 2.1 ± 0.1∗E velocity (cm/s) 65 ± 2 73 ± 4 84 ± 6 103 ± 2∗ 85 ± 5∗ 106 ± 6∗A velocity (cm/s) 26 ± 2 41 ± 6 63 ± 7∗ 66 ± 0∗ 47 ± 5 FusedE/A 2.7 ± 0.3 1.2 ± 0.1 2.5 ± 0.2 1.6 ± 0.0∗ 1.8 ± 0.2∗ Fused

Studies in adult CD1 mice show different absolute values in cardiac Doppler measurement depending on the anestheticagent chosen. An increase in contractility or cardiac output is, however, evident with all anesthetic agent used. Asterisksindicate significant differences between baseline and isoprenaline stimulation, p< 0.05, n = 5–10 per group. Ketaminexylazine i.p., isoflurane inhalation 2%, diazepam 17.5 mg/kg i.p.

Doppler Signals of the AorticValve in Mice

Normal Valvular Function, Systolic Function,and Assessment of CO

Normal aortic valve function in mice can beassessed from apical four-chamber views andparasternal long-axis views. The combined useof continuous wave aortic Doppler, pulsed-waveaortic Doppler, and color flow Doppler is opti-mal to noninvasively describe valvular hemo-dynamic function. To calculate maximum pres-sure gradients via the continuation equationover the aortic valve, peak aortic velocities withcontinuous wave Doppler are recorded.9 Dur-ing examination, the ultrasound beam shouldbe positioned at an angle with minimum devia-tion from parallel to blood flow as signal veloc-ities are otherwise underestimated subject tocosine function of the deviation angle. This hasto be considered when transgenic animals withmalformations of the aortic arch are studied aschanges in aortic anatomy can cause systematicmeasurement errors.

The aortic Doppler waveform typically ex-hibits a rapid acceleration within the first thirdof the signal followed by immediate decelera-tion. The peak aortic Doppler flow velocitiesof adult mice at rest range from 60 cm/sec to120 cm/sec under physiological settings. Ejec-tion time (as the sum of acceleration time plusdeceleration time) and peak aortic flow veloc-ity have been shown to reflect systolic work

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DOPPLER ULTRASOUND IN MICE

and parallel changes of contractile performancewhen assessed serially in the same animal.These indices are influenced by preload, after-load, and heart rate. Doppler echocardiographicstroke volume of the left ventricle is character-ized by the velocity time integral of the aor-tic Doppler echocardiographic signal. To calcu-late stroke volume, the cross-sectional area ofthe left ventricular outflow tract is multipliedwith the velocity time integral of the pulsed-wave Doppler signal in the outflow tract. COthen is the product of stroke volume and heartrate. Bland–Altman analyses showed excellentlimits of agreement in noninvasive assessmentof CO in mice by magnetic resonance imag-ing (MRI) and Doppler echocardiography incomparison to invasive measurement by ther-modilution or electromagnetic flowmeter. COin MRI and flowmeter showed good correla-tion (r = 0.80–0.84, P < 0.05).10,11 But despiteits advantages, MRI is cost intensive, resourceintensive, and not everywhere available.12–14

CO measured by Doppler echocardiographyhas difficulties in estimating the exact cross-sectional area and good signal quality of thevelocity time integral. Comparison of Dopplerechocardiographic-determined CO with CO de-rived from electromagnetic and transit timeflow probes showed no significant differences(r2 = 0.51, P < 0.05).15

Doppler Ultrasound during Prenatal andPostnatal Development

From birth to senescent age, the use of manydifferent commercially available conventionalultrasound systems for cardiovascular pheno-typing of genetically targeted mice has been ex-tensively reported.1,12,13,16 The use of 15-MHztransducers allows spatial resolutions between100 µm axial direction and 250 µm lateral di-rection. This resolution is sufficient to detectchanges in left ventricular wall thickness or car-diac chamber size of approximately 10 to 20%,and of left atrial size.17–21 For the recording oflocal flows, the sample volume of the pulsed-wave Doppler has to be minimized to less than0.6 mm.

In ultrasound biomicroscopy systems thatemploy up to 55 MHz, axial resolutions as lowas 28 µm and lateral resolutions as low as 62µm can be achieved, but this high spatial res-olution comes at the price of a poor and ofteninsufficient temporal resolution of 10 imagesper second (i.e., 1 to 1.5 images per cardiac cy-cle). These systems have been used for semi-

invasive and noninvasive evaluation of the de-velopment of cardiovascular function in the em-bryonic mouse. Ultrasound biomicroscopy ex-aminations at an embryonic age of 3 somitesshow no heartbeat and no Doppler flow sig-nals. At an embryonic age of 5 somites, car-diac contractions can be seen at a frequencyof 100 to 130 beats per minute (bpm) but stillno Doppler flow signals can be found. The firstDoppler flow signals are recorded with ultra-sound biomicroscopy systems at an age of early7 somites, equaling an embryonic age of 8 daysafter postcoital mucous plug with a heart rateof 137 ± 19 bpm and a Doppler flow velocity of25 cm/sec of the embryonic heart prior to sep-tation (Fig. 1). Doppler waveforms closely re-semble those of larger animals in these firststages. At an embryonic age of 10 days, heartrate rises to 173 ± 16 bpm and the velocitiesof the Doppler flow signals increase to 50 to60 cm/sec.22 Sonomorphometric characteriza-tion of mouse embryos has been reported with14-Mz transducers starting at an embryonic ageof 7.5 days with follow-up to embryonic day 15.5.First noninvasive intrauterine Doppler flow sig-nals with 15 MHz-transducers in mice can re-producibly be registered at an embryonic age of11–12 days with a size of the sample volumesmaller than 1 mm.23,24

Impact of Anesthesia, Heart Rate andRhythm on Doppler Signals in Mice

In addition to technical issues related tothe optimal positioning of the Doppler ultra-sound transducer, the type of sedation, anal-gesia, and/or anesthesia affects the normalrange of Doppler and echocardiographic mea-surements.8

The effect of different types of sedation oranesthesia on heart rate and parameters of car-diac function is illustrated in Table I.

Heart rate has a modest effect on indexes ofcontractility in mice, especially at physiologi-cally low heart rates. Heart rate during exam-ination should be carefully assessed and bestbe kept well above 350 bpm. Comparable heartrates should be selected for analysis of wild-typeand transgenic mice. Nemoto et al. described anoptimal CO at heart rates of 505 ± 14 bpm.25

As E- and A-waves fuse at heart rates above600 bpm (Fig. 6), diastolic function can be mea-sured in a range between 300 and 600 bpm.Doppler flow measurements are dependent oncardiac rhythm. Figure 2 shows examples ofreduced transaortic Doppler flow due to atrial


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Figure 1. Arrows show the embryonic heart. Correlation of ultrasound biomicroscopy system-Doppler cardiac functionalparameters with in situ hybridization. Representative data at 3-somites, 5-somites, and 8-somites are shown. At 3-somites,neither a heartbeat nor Doppler flow was detectable. At 5-somites, only a heartbeat was present by ultrasound biomicroscopysystems; the panel shows maternal respirations, distinct from embryonic Doppler blood flow. At 3-somites, when there is noheartbeat, no erythroblasts are seen within the embryo; at 5-somites, with a heartbeat, few erythroblasts; and at 8-somites, withDoppler flow, many erythroblasts. In the bottom panel, ultrasound biomicroscopy systems ultrasound biomicroscopy systems-Doppler functional category is plotted against erythroblasts density ranking (bars show mean rank). No embryos lacking aheartbeat showed any cells, whereas maturation of cardiac function as characterized by ultrasound biomicroscopy systems-Doppler correlated with erythroblast density ranking. (Reprinted from: Ji et al.22 with permission from the American HeartAssociation.)

fibrillation and bigeminus, emphasizing thatreal-time measurements of aortic flow are cru-cial to obtain valid results in transgenic modelswith cardiac arrhythmias. Simultaneous elec-trocardiogram (ECG) recordings help to eluci-date the underlying rhythm.

In order to obtain more heart rate- and load-independent parameters of cardiac contractilityderived from the aortic Doppler signals, indexesin B-mode rate-corrected velocity of LV fibershortening (Vcfc), normalized systolic ejectionrate (MNSER), and Doppler myocardial



Figure 2. Doppler signals of the aorta during atrial ar-rhythmia show irregular Doppler wave forms with chang-ing size and cycle length. Time interval between dots 200 ms.Scale on right is in m/s (own data).

performance index (MPI), according to Tei,can be used in mice.26 MPI can be calculatedfrom transmitral and transaortic Doppler flowmeasurements according to Tei 27 following thesimple formula (ICT + IVRT)/ET. ICT stands forthe isovolumetric contraction time from closureof the mitral valve to opening of the aortic valveand IVRT for the isovolumetric relaxation timefrom closure of the aortic valve until openingof the mitral valve. ET is the ejection time fromopening to closure of the aortic valve. This indexis relatively independent of changes in heartrate and preload.26 Such parameters, althoughindirect and therefore prone to multiple system-atic confounding influences, may at times beuseful to compare Doppler measurements ob-tained in different mouse strains and/or underdifferent experimental conditions.25,28–33

Aortic Insufficiency in Mice

Aortic insufficiency in mice has been de-scribed in various genetically altered mice andafter artificial induction. Clear detection of aor-tic regurgitation in vivo in mice is possiblewith pulsed-wave and color flow Doppler. The

Doppler waveforms of aortic insufficiency showa diastolic regurgitation into the left ventriclewith maximum velocities of 3 to 4 m/sec. Thesignals show a deceleration in cw-Doppler sig-nals and aliasing in pw- and cf-Doppler sig-nals. We18 could recently show that left ventric-ular dilation due to dilatative cardiomyopathyin mice deficient of lysosomal cysteine peptidasecathepsin L may be a causative factor for thedevelopment of aortic insufficiency. A total of50% of the mice with LV dilatation showed se-vere aortic insufficiency that was most likelysecondary to the general cardiac dilatation.In a mouse model with cardiac pathologies ofmucopolysaccharidosis type VI,34 aortic insuffi-ciency in a 1-year-old mouse has been reported.It was caused by thickening of the aortic cuspthrough the underlying disease with storage ofglycosaminoglycan. Bibin et al.35 showed com-bined aortic stenosis and insufficiency in micemutant for Egfr and Shp2. These mice have de-fective semilunar valvulogenesis.

However, these findings have to be comparedto the natural age-related prevalence of aorticinsufficiency. Patten et al.36 studied 20 senes-cent C57Bl/6J mice (>1 year of age) and de-tected three cases of aortic insufficiency, equal-ing a 15% incidence. These aortic insufficiencieswere mild and not as severe as the experimen-tally induced insufficiencies in the control groupof young mice. In aortic insufficiency, significantmarked changes with reduced fractional short-ening and enlarged intraventricular diastolicand systolic diameters can be found. In view ofthe aforementioned abnormalities in “normal”mice, the significance of these findings needs tobe confirmed in larger series.

Aortic Stenosis in Mice

Doppler signals of valvular aortic stenosis inmice so far have been published in differentmouse strains but again no age-related preva-lence has been reported. The acceleration timeof the Doppler signal shortens with increasingdegrees of stenosis, and the blood flow veloc-ity as assessed by Doppler shows maximumvalues well above 3 m/sec (Fig. 3). Mice fromthe 129/Sv strain heterozygous for mutationsof the home box gene Nkx2.5 show aortic steno-sis with a bicpusid aortic valve. In 10-month-old mice, aortic Doppler showed a maximumvelocity of 3.24 m/sec, and a mean velocity of2.17 m/sec.35 As already stated above, mice mu-tant for Egfr and Shp2 have defective semilunarvalvugenesis,37 thus leading to aortic stenosis


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Figure 3. Functional assessment of heart valves by pulse-waved Doppler-echocardiography. (A) Wild-type mouseshowing normal antegrade flow through the aortic valve. (B)Aortic regurgitation and high velocity of aortic antegradeflow as hallmarks of aortic valve insufficiency in a ctsl−/−mouse (own data).

and regurgitation similar to the clinical entitiyof “combined aortic valvular defect.”

Doppler Signals of the MitralValve in Mice

Normal Mitral Flow and Diastolic Function inMice

Doppler assessment of mitral flow is used toexamine the mitral valve but even more so to es-timate diastolic function. Doppler signals of themitral valve can be obtained from apical and leftparasternal transducer positions. The mitralinflow in mice is directed to the septal wall. Theapical acoustic window can be achieved from anepigastric acoustic transducer position angeledcranially giving the equivalent of an apical four-chamber view and the left parasternal acousticwindow can be achieved in a supine mouse po-sition angeled 45◦ to the left giving a paraster-nal long-axis view of the left ventricle. In mice,left ventricular diastole can echocardiograph-ically be divided into four phases using time-and velocity-based measures of the transmitralinflow Doppler signals. Phase one starts afterclosure of the aortic valve when left ventricu-lar pressure declines without changes in vol-

ume until left atrial pressure exceeds LV pres-sure and opens the mitral valve (isovolumetricrelaxation measured as IVRT). The second so-called early rapid filling phase (represented asthe E-wave or early wave) begins and is drivenby the atrioventricular pressure gradient acrossthe mitral valve and further relaxation of theleft ventricle. Phase 3, the so-called diastasisof diastole, reflects the equilibration with atrialpressure. Finally, in phase 4, the atrial contrac-tion measured by the A-wave or atrial wave isseen in transmitral Doppler signals. The ampli-tude, duration, and shape of the signals dependon ventricular diastolic pressure and stiffnessas well as on atrial contractility.

Impact of Heart Rate on Doppler Signalsof the Mitral Valve in Mice

Even more than systolic function, diastolicfunction is frequency dependent.

The typical Doppler waveform within physi-ological heart rates at rest shows the E-waveduring relaxation of left ventricle and theA-wave during atrial contraction. Waveformsare dependent on preload conditions. If heartrates exceed 600 bpm (e.g., during stress), dis-crimination of E- and A-wave in mice can bedifficult as both waves move together and fuse(Fig. 6). Incremental atrial pacing can be usedto confirm that this fusion phenomenon is re-ally a frequency-dependent effect rather thansecondary to the effects of catecholamine stim-ulation (Fig. 7). Peak E-wave velocity, peakA-wave velocity, E/A ratio, IVRT, and IVRT cor-rected for differences in heart rate, decelera-tion time of the E-wave, and deceleration ofE-wave have all been used to describe dias-tolic properties of the left ventricle. These time-and velocity-based measurements are, again,dependent on preload conditions. As heart ratecorrection causes additional sources of system-atic error, comparisons at equal heart rates areoptimal. Diastolic color M-mode flow propaga-tion velocity into the left ventricle determinedby the slope of the first aliasing isovelocity lineduring E-wave adds spatial, allocative informa-tion about Doppler signals in the left ventri-cle. It seems to be relatively inert to changes inheart rate and left atrial pressure.38 If wild-typeand transgenic mouse differ in heart rate, pac-ing, preferably of the right atrium, may be help-ful for proper evaluation. Pacing can be usedto control heart rate during the measurements(Fig. 6).



Figure 4. Typical Doppler flow spectra obtained from the mitral orifice (A–E) and from the tricuspid orifice (a–e) in mouseneonates on the first, third, fifth, and seventh day after birth and in a mouse at weaning age (4-wk old). Arrows in (e) marktricuspid waves acquired near the end of inspiration. Note the changes in waveform shape and amplitude. Time and velocityscales for all Doppler flow spectra are the same. MV, mitral valvular orifice; TV, tricuspid valvular orifice. (Reprinted from:Zhou et al.62 used with permission from The American Physiological Society.)

Age-Dependent Mitral Flow Alterationsin Mice

Embryonic Doppler measurements with 20MHz in an open abdominal approach39 and 40MHz in C57Bl/6J mice40 have been performed(Fig. 4). At embryonic day 15 and 19, theyshowed E/A ratios of approximately 0.4 and atembryonic day 19, E/A ratios of 0.43. Inflow ve-locities ranged from 62.33 ± 4.06 mm/sec at em-bryonic day 10.5 to 106.23 ± 11.6 mm/sec at em-bryonic day 14.5.24,39 Age-related alterations inlate diastolic function in mice were shown tobe improved by caloric restriction.41 A model offunctional cardiac aging was described in youngadult mice with mild overexpression of serumresponse factor.42

Pathologic Mitral Flow Alterations inMice

Diastolic function can be altered by changesin calcium homeostasis,43,44 but also by is-chemia, hibernation, or fibrotic ventricular re-modeling. In diabetic mice, reduced circumfer-ential fiber shortening and reduced E/A ra-tio in 12-week old mice were interpreted as asign of systolic and diastolic impairment.45 In amouse model of hypertrophic cardiomyopathy,M-mode and Doppler echocardiography showednormal left ventricular dimensions and normalsystolic function but a diastolic impairment asevidenced by a 50% reduction in the E/A ra-tio of mitral inflow velocity.46,47 Diastolic dys-function as reflected by changes in mitral inflowpattern was also shown in another model of fa-milial hypertrophic cardiomyopathy.48 Gender-

specific ventricular diastolic dysfunction withreduction of the E/A ratio was reported in malerelaxin deficient mice.49 Significant changes inE/A ratio were documented in hyperthyroidmice with a 47% increase and senescent micewith a 59% decrease.41 Diastolic color M-modeflow propagation velocity of the mitral inflow(Fig. 5) was shown to be a useful tool for eval-uation of left ventricular diastolic function ingenetically altered mice.31

Atrial cardiomyopathy which is accompa-nied by atrial bradyarrhythmias is reflectedby altered mitral valve Doppler waveforms.17,38

The A-wave may already be diminished inearly stages of atrial dysfunction (Fig. 7).Mice with heart-directed overexpression of theA3-adenosine-receptor already showed signifi-cantly reduced A-waves in young age. Signif-icant reduction persists lifelong.17 The subse-quent development of atrial enlargement andfibrosis is illustrated in Figure 8, whereas thefunctional relevance of this atrial cardiomyo-pathy is documented in Table II. Mitral waveDoppler can be used to detect atrial fibrillationdue to the loss of the A-wave in the fibrillat-ing atria. Arrhythmias change CO on a beat-to-beat basis (see Fig. 2) and thereby influencefunctional measurements.

Mitral Regurgitation and Stenosis inMice

Mitral regurgitation with or without en-largement of the left atrium has been de-tected by transthoracic Doppler echocardiogra-phy. Systematic data on gender- and age-related


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Figure 5. Diastolic color M-mode flow propagation velocity vp into left ventricle, which is determined by slope of first aliasingisovelocity line during early filling. A: Representative tracings of PLB/KO (left), WT (middle), and PLB/N27A mice (right).In WT animal, early filling progresses rapidly into LV toward apex. This is further enhanced in PLB/KO mice. In contrast,in PLB/N27A, slope of early filling is low and does not reach apex, relatively high velocity is observed during A-wave. B:Mean values for color M-mode flow propagation (±SEM). ∗P < .05 versus WT; #P < .05 versus PLB/KO. LV, Left ventricular;PLB/KO, phosholamban knockout; WT, wild type; PLB/N27A, mutant form of PLB. (Reprinted from: Schmidt et al.31withpermission from American Society of Echocardiography.)

prevalence in different mouse strains are notavailable. Senescent over 1-year-old C57Bl/6Jmice showed mitral insufficiency in two outof 20 animals, detected in both cases with cf-Doppler and once confirmed with pw-Doppler.36

This could be a hint that among old C57Bl/6Jmice there is a low prevalence of mitral regurgi-tation. In addition to strain-related influences,the conditions of long-term animal housing mayinfluence the rate of mitral insufficiency inlaboratory animals (e.g., stress-related alter-ations in blood pressure and rate of infections).Thus, as stated above for assessment of aor-tic valve disease, comparisons between age-andsex-matched littermates are crucial to detecta genotype-associated phenotype. Determining

the degree and functional relevance of valvu-lar abnormalities will allow to detect genotype-associated differences. In mice with deficiencyof arylsulfatase B, mitral insufficiency (Fig. 9)was more often and more severely present intransgenic mice.34

To the best of our knowledge, mitral stenosisin mice identified by Doppler has so far not beenreported.

The Pulmonary Vein in Mice

Marked differences exist between the mouseheart and the human heart in the atrial struc-ture. The human left atrium usually has fourpulmonary vein orifices whereas in mice there



Figure 6. Example of cardiac Doppler measurements in amouse during atrial pacing via a transjugular octapolarelectrophysiologic catheter. During incremental atrial pac-ing at the beginning of the Doppler registration with cyclelength 100 ms, E-wave and A-wave still can be well recog-nized. At the end of the registration with cycle length 90 ms,E-wave and A-wave fuse. Time scale between dots is 200 ms.Scale on right is in m/s (own data).

is only a single pulmonary vein that connectswith the left atrium. The inflow of the pul-monary vein into the left atrium was reportedto be best recorded in a left parasternal longitu-dinal section with ultrasound biomicroscopy.50

The recorded Doppler flow spectrum showeda small retrograde wave, followed by two an-tegrade waves.50 The waveforms were modu-lated by ventilation of the lungs.50 ConventionalDoppler techniques are not sufficient to detectsuch low-flow Doppler phenomena.

Tissue Doppler Imaging (TDI) in Mice

In humans, TDI is considered to allow mea-surement of load-independent diastolic left ven-tricular function. Whether in small animals likemice TDI really is load independent has not yetbeen formally tested. Its feasibility and valuein mice is of interest. In C57Bl/6 mice, TDI per-formed at the LV posterior wall at the level ofthe papillary muscles could detect diastolic dys-function caused by chronic pressure overload 4weeks after aortic banding. Peak early diastolicvelocity and the ratio of peak-to-late filling ve-locities were significantly reduced after band-ing.20,30,51–54 Doppler flow measurements of mi-tral inflow also provided evidence for diastolicdysfunction.29,30,52,53,55,56

Murine Coronary Arteries

Murine coronary circulation still is largelyunknown. By use of an Acuson Sequoia 512with a Microson 15L8 transducer, left coronaryartery (LCA) anatomy in 10-week old C57Bl/6mice could be imaged. Reproducible Doppler

Figure 7. The upper panel shows normal mitral inflow pat-terns in a control mouse, the lower panel shows decreasedA-wave amplitude reflecting descreased atrial function ina model of atrial bradycardiomyopathy [(Reprinted from:Fabritz et al.17with permission from The European Societyof Cardiology), see also Figure 8]. Anesthesia with ketaminand xylazine, littermates aged 8 weeks. Dots mark 200 mstime intervals. Scale on right reflects m/s (see table for val-ues).

flow signals of the proximal part of the LCAand its anterior and lateral branches could berecorded. Peak flow velocity, mean flow velocity,and velocity time integrals were significantlyhigher in the proximal LCA compared to distalbranches. Similar to systolic and diastolic car-diac function, there were striking similaritiesbetween murine and human flow velocity pro-files.57–59 These findings suggest that despitehigh heart rates both systemic blood pressureand the relation between systole and diastoleare strikingly similar between mice and men.Corresponding to these data, another study us-ing ultrasound biomicroscopy showed that theDoppler flow spectrum of the proximal LCA im-aged from a left parasternal transverse sectionhad a continuous and pulsatile flow patternthroughout the cardiac cycle.50

Right Heart Measurements in Mice

Doppler echocardiographic access to the rightheart is possible from the apical four- chamberview and the parasternal short axis. Further-more, transesophageal echo has been used to


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Figure 8. Atrial dilatation and fibrosis with concomitant decrease in A-wave amplitude. (A) Representative echocardiographicparasternal long axis views of a WT mouse heart and A3-Adenosine receptor overexpressing hearts (A3high) at 8, 12, and 21weeks of age. White bars indicate left atrial diameter. (B) Mean left atrial diameter in WT (open bars) and A3high (filled bars).Asterisks (∗) indicate significant differences between A3high and WT. For Doppler values see table. (C) Sirius red staining ofleft atria from littermates aged 14 weeks indicating fibrosis in A3high atria. (Reprinted from: Fabritz et al.17with permissionfrom The European Society of Cardiology.)



TABLE II

Mice with Atrial Cardiomyopathy Show Decreased A-wave Amplitude and Increased E/A Ratios. Studies Were Performed inSedation with Ketamine and Xylazine

8 weeks 21 weeksAge WT A3

high WT A3high

Genotype n = 14 N=14 n = 10 n = 10

Heart rate (beats/min) 330 ± 12 303 ± 9 312 ± 16 272 ± 16MV E-wave peak (cm/s) 72.7 ± 2.4 73.7 ± 3.0 65.9 ± 3.4 67.1 ± 3.1MV A-wave peak (cm/s) 35.7 ± 2.5 28.3 ± 1.9∗ 29.1 ± 1.8 23.4 ± 1.6∗MV E/A 2.14 ± 0.14 2.71 ± 0.17∗ 2.33 ± 0.19 2.97 ± 0.20∗

assess right heart function in normoxic and hy-poxic mice.60,61 Developmental changes of rightventricular diastolic filling patterns via the tri-cuspid valve have been described with varioustechniques from embryonic day 9.5 over post-natal day one to senescent age.62 In general,right heart echocardiographic visualization fol-lows the same principles as left heart visual-ization, with the anatomic limitation that thenormal right ventricle has a much smaller wallthickness. Assessment of systolic Doppler flowshas been used to compare CO in the right heartvia pulmonary artery flow and in the left heart

Figure 9. Functional assessment of mouse mitral valves by Doppler echocardiography. (A) Pulse-wave Doppler echocardio-graphy in a wild-type mouse at 12 mo of age. (B) Pulse-wave Doppler echocardiography in a MPS VI mouse at 12 mo of age.E-wave (∗) in wild-type mice is twice as fast compared with MPS VI, while there is no difference in the A-waves (†). (C) Colorflow Doppler echocardiography of the same mouse as in (B). Insufficiency backflow (white arrows) was only detected in theheart of this MPS VI mouse. RV = right ventricle; IVS = interventricular septum; AML = anterior mitral leaflet; LA = leftatrium; AoV = aortic valve. (Reprinted from: Strauch et al.34with permission from Lippencott, Williams & Wilkens, Invoice #B43384362.)

via aortic flow which, if divergent, represent in-direct evidence of intracardiac shunts.35,63

The Pulmonary Valve in Mice

A left parasternal longitudinal section and aleft parasternal transverse section at the levelof the cardiac base have been reported to givebest Doppler signal recordings of the pulmonaryartery.50 Doppler recordings on the level of thepulmonary valve in 8- to 12-month-old 129/svJmice have yielded a maximum velocity of 65± 12 cm/sec and a mean velocity of 40 ± 7


STYPMANN

cm/sec. The velocity time integral of the pul-monary artery is 70% of the aortic velocity timeintegral.35

The Tricuspid Valve in Mice

Doppler signals of the tricuspid valve canbe obtained from a right parasternal trans-verse section, the apical four-chamber view, andin longitudinal sections of the left parasternalwindow. The tricuspid Doppler flow spectrumshows a lower early diastolic E-wave and ahigher A-wave. As expected, the waves increaseconsiderably with inspiration.62,64–66 Develop-mental changes of diastolic inflow through thetricuspid valve into the right ventricle of micehave been recorded from embryonic day 14.5 upto postnatal week 12. Doppler signals of the tri-cuspid valve at embryonic day 14.5 show a peakE-wave velocity of 8.9 ± 0.6 cm/sec that grad-ually increases to 28.2 ± 1.1 cm/sec. The am-plitude of this signal is similar after birth un-til week 12. The peak A-wave velocities at thesame time points are around 35 cm/sec. The am-plitude of the E-wave increases during the first4 postnatal weeks.50,62,64–66

Inflow into the Right Atrium in Mice

The right atrial inflow in mice is threefold.In addition to the inferior vena cava and theright superior vena cava, a persisting left su-perior vena cava is present and can be visual-ized from a left parasternal longitudinal win-dow. The right superior vena cava can be as-sessed from a right parasternal longitudinalwindow. The entrance of the vena cava inferiorinto the right atrium is often not visualized dueto acoustical shadowing by the inferior lungs.The best Doppler flow spectrum is derived fromthe right upper vena cava and shows a smallretrograde A-wave, a medium D-wave preced-ing the A-wave, and a relatively larger S-wavefollowing the A-wave.50

Atrial and Ventricular Septal Defects inMice

Color-coded 2D Doppler measurements canbe used to detect atrial and ventricular sep-tal defects in mice. One example are eNOS–/–mice that show atrial or ventricular septal de-fects. Atrial septal defects in these mice havethe typical signs and location of a septum se-cundum defect. Ventricular septal defects weremembranous or muscular defects. In addition,

in eNOS–/– mice there was ventricular hy-pertrophy and atrial enlargement compared toeNOS+/+. The histological data also show thatatrial septal defects in normal C57Bl/6 mice oc-cur in nearly 5% and lead to enlargement of theatria.67

Vascular Doppler in Mice

Ultrasound-guided localization of murinevessels allows repeated serial Doppler ultra-sound measurement at different peripheral ar-terial sites. Examination time from putting themouse into anesthesia to returning the mouseto its cage takes 30 minutes, and offline anal-yses of the derived Doppler curves may takeanother 20–60 minutes depending on the num-ber of Doppler measurements. Thus, althoughstill time consuming, it becomes feasible toscreen and phenotype large numbers of mice us-ing these technically sophisticated, noninvasiveDoppler techniques to assess regional changesin the systemic circulation. Aorta ascendens,aortic arch, and aorta descendens can be visual-ized with color flow Doppler from a suprasternaland right clavicular window with a 12–15 MHztransducer. Using a 20-MHz Doppler probe, theaorta ascendens, the aortic arch, and the aortadescendens, the left and right carotid arteries,the abdominal aorta, the celiac truncus, andthe left and right renal arteries can be visual-ized and blood flow measurements can be per-formed.68–70

Peripheral Doppler measurements of bloodflow velocity can be used to determine arterialpulse wave velocity as an index of arterial stiff-ness. Stiffer vessels propagate pressure and ve-locity waves faster.68–71 Doppler signals havebeen used to quantify flow in transgenic micewith aortic aneurysms and cardiovascular mal-function.72

Aortic Banding in Mice

Nakamura et al.73,74 were able to show thetime course of aortic flow velocity and pressuregradient and peak pressure gradient as well aswall thickness and parameters of contraction atdays 1, 3, 10, and 20 after aortic banding. Basedon wall thickness and chamber width and flowparameters wall stress (g/cm2) could be calcu-lated, showing a sudden increase in wall stressafter aortic constriction with a subsequent de-cline in wall stress with evolving cardiac hy-pertrophy. Li et al.75 used Doppler to describe



age-related effects on peripheral vascular re-sponse to transverse aortic banding.

Stress Testing in Mice

Phenotyping of transgenic models understress is considered to be crucial to determinethe clinical relevance of a phenotype.76 Exerciserepresents an established physical stress to theintact cardiovascular system which is a majordeterminant of the utilization of metabolic sub-strates. Exercise adaptation is the result of a co-ordinated response of multiple organ systems,including cardiovascular, pulmonary, endocrinemetabolic, immunologic, and skeletal muscleadaptions. If changes in mice like cardiac andskeletal muscle hypertrophy mimic normal hu-man reactions to training, genetically alteredmice may provide useful models for exploringcardiovascular regulation under stress.77 Manystressors have been used and described in mice.During the first trimester of pregnancy, hy-potension and blunted pressor response to an-giotensin II with a decrease in hematocrit de-velop in mice. At late pregnancy there is amarked increase in CO due to an increase instroke volume by one third and an increase inheart rate by approximately 20%.78 In addi-tion to this physiological long-term stress forfemale mice, standardized experimental stressprotocols have been developed in mice: Sub-maximal stress testing by swimming17,79 ormental stressors such as repetitive blasts ofair48,80 or intensity-controlled treadmill exer-cise77 have been used for functional cardio-vascular phenotyping of mice. Doppler mea-surements (transaortic, transmitral, and TDI,among others) can be recorded during acute cat-echolamine stress. Table I illustrates the effectsof ß-adrenergic stimulation on parameters ofDoppler echocardiography using different anes-thetic agents on adult CD1 wild-type mice. ß-adrenergic stimulation increases CO. The in-crease in CO is mainly mediated by an increasein heart rate.

Summary and Perspectives

Doppler ultrasound provides noninvasivemeasurements of flow and movement velocities.The magnitudes and the waveforms of pressureand velocities in small mammal and vertebraterecordings are almost indistinguishable fromsignals from similar sites in humans when ex-trapolated to a similar time scale, and add valu-able functional data for assessment of ventricu-

lar systolic and diastolic function as well as foratrial, valvular, and vascular pathologies.

For experiments needing a transgenic orknockout background, mice provide the bestsmall animal model. Ultrasound equipment hasbeen adapted to mouse physiology and down-sized to meet the demands of murine spatialand temporal resolution from newborn miceto senescent age. For adult mice, commer-cially available ultrasound platforms give sat-isfactory Doppler ultrasound examinations, butwith further interest in cardiovascular devel-opmental changes a new perspective for ul-trasound examinations is ultrasound biomicro-scope with higher spatial resolution. Tempo-ral resolution on these systems still is poor forDoppler examinations but ongoing technical ad-vancement may, in future, provide better framerates.

Doppler ultrasound in specialized mouseclinics is one of the first-line techniques forcardiovascular phenotyping in small animals.These measurements can be performed at restand during defined stress tests. Such facilitiesshould be capable of assessing hemodynamic,morphologic, functional, and electrophysiologi-cal measurements, as these parameters influ-ence each other. In addition, quality of Dopplerultrasound imaging is dependent on experienceof the examiner. Therefore, centralization ofsuch measurements has taken place in manylarger research facilities. Supplemental tech-niques are pressure–volume loops assessed bycentral impedance catheters, MRI,50 and radi-ologic computer tomography or high-resolutionpositron emission tomography which have beenadapted to mice.81 Their complementary useis estimated to push imaging of cardiovascu-lar function in mice and other small animalsforward to new frontiers. Still, the real-timecapabilities of ultrasound measurements andtheir noninvasive nature make them unique forthe study of transgenic small mammals.

Acknowledgments: This paper was funded by the Inter-disciplinary Center for Clinical Research (IZKF) Munster,project number ZPG 4a, and partly supported by grantsfrom the Deutsche Forschungsgemeinschaft (DFG), Sonder-forschungsbereich 656 MoBil Munster, Germany (projectC3).

References

1. Hoit BD, Kiatchoosakun S, Restivo J, et al: Naturallyoccurring variation in cardiovascular traits among in-bred mouse strains. Genomics 2002;79(5):679–685.


STYPMANN

2. Hoit BD: Murine physiology: Measuring the pheno-type. J Mol Cell Cardiol 2004;37(2):377–387.

3. Desai KH, Schauble E, Luo W, Kranias E, BernsteinD: Phospholamban deficiency does not compromise ex-ercise capacity. Am J Physiol 1999;276(4 Pt 2):H1172–H1177.

4. Tiemann K, Weyer D, Djoufack PC, et al: Increasingmyocardial contraction and blood pressure in C57BL/6mice during early postnatal development. Am J Phys-iol Heart Circ Physiol 2003;284(2):H464–H474.

5. Chaves AA, Weinstein DM, Bauer JA: Non-invasiveechocardiographic studies in mice: influence of anes-thetic regimen. Life Sci 2001;69(2):213–222.

6. Hart CY, Burnett JC Jr., Redfield MM: Effects ofavertin versus xylazine-ketamine anesthesia on car-diac function in normal mice. Am J Physiol Heart CircPhysiol 2001;281(5):H1938–H1945.

7. Rottman JN, Ni G, Khoo M, et al: Temporal changes inventricular function assessed echocardiographicallyin conscious and anesthetized mice. J Am Soc Echocar-diogr 2003;16(11):1150–1157.

8. Yang XP, Liu YH, Rhaleb NE, Kurihara N, Kim HE,Carretero OA: Echocardiographic assessment of car-diac function in conscious and anesthetized mice. AmJ Physiol 1999;277(5 Pt 2):H1967–H1974.

9. Luo W, Grupp IL, Harrer J, et al: Targeted ablation ofthe phospholamban gene is associated with markedlyenhanced myocardial contractility and loss of beta-agonist stimulation. Circ Res 1994;75(3):401–409.

10. Franco F, Thomas GD, Giroir B, et al: Magnetic res-onance imaging and invasive evaluation of develop-ment of heart failure in transgenic mice with myocar-dial expression of tumor necrosis factor-alpha. Circu-lation 1999;99(3):448–454.

11. Nahrendorf M, Hiller KH, Hu K, Ertl G, Haase A,Bauer WR: Cardiac magnetic resonance imaging insmall animal models of human heart failure. Med Im-age Anal 2003;7(3):369–375.

12. Hoit BD: Murine physiology: Measuring the pheno-type. J Mol Cell Cardiol 2004;37(2):377–387.

13. Hoit BD: Echocardiographic characterization of thecardiovascular phenotype in rodent models. ToxicolPathol 2006;34(1):105–110.

14. Hoit BD, Nadeau JH: Phenotype-driven genetic ap-proaches in mice: High-throughput phenotyping fordiscovering new models of cardiovascular disease.Trends Cardiovasc Med 2001;11(2):82–89.

15. Janssen B, Debets J, Leenders P, Smits J: Chronicmeasurement of cardiac output in consciousmice. Am J Physiol Regul Integr Comp Physiol2002;282(3):R928–R935.

16. Hoit BD: Spectral and color M-mode Doppler in genet-ically altered mice. Assessment of diastolic function.Minerva Cardioangiol 2003;51(6):609–618.

17. Fabritz L, Kirchhof P, Fortmuller L, et al: Gene dose-dependent atrial arrhythmias, heart block, and brady-cardiomyopathy in mice overexpressing A(3) adeno-sine receptors. Cardiovasc Res 2004;62(3):500–508.

18. Stypmann J, Glaser K, Roth W, et al: Dilated car-diomyopathy in mice deficient for the lysosomal cys-teine peptidase cathepsin L. Proc Natl Acad SciU S A 2002 30;99(9):6234–6239.

19. Stypmann J, Janssen PM, Prestle J, et al: LAMP-2 de-ficient mice show depressed cardiac contractile func-tion without significant changes in calcium handling.Basic Res Cardiol 2006;101(4):281–291.

20. Stypmann J, Engelen MA, Epping C, et al: Ageand gender related reference values for transthoracic

Doppler-echocardiography in the anesthetized CD1mouse. Int J Cardiovasc Imaging 2006;22(3–4):353–362.

21. Stypmann J, Engelen M, Orwat S, et al: Cardiovascu-lar characterization of Pkd2+/LacZ mice, an animalmodel for the Autosomal Dominant Polycystic KidneyDisease type 2 (ADPKD2). Int J Cardiol 2006;in press.

22. Ji RP, Phoon CK, Aristizabal O, McGrath KE, Palis J,Turnbull DH: Onset of cardiac function during earlymouse embryogenesis coincides with entry of primi-tive erythroblasts into the embryo proper. Circ Res2003;92(2):133–135.

23. Chang CP, Chen L, Crabtree GR: Sonographic stag-ing of the developmental status of mouse embryos inutero. Genesis 2003;36(1):7–11.

24. Ishiwata T, Nakazawa M, Pu WT, Tevosian SG,Izumo S: Developmental changes in ventricular di-astolic function correlate with changes in ventricularmyoarchitecture in normal mouse embryos. Circ Res2003;93(9):857–865.

25. Nemoto S, Defreitas G, Mann DL, Carabello BA: Ef-fects of changes in left ventricular contractility on in-dexes of contractility in mice. Am J Physiol Heart CircPhysiol 2002;283(6):H2504–H2510.

26. Broberg CS, Pantely GA, Barber BJ, et al: Validationof the myocardial performance index by echocardiog-raphy in mice: a noninvasive measure of left ventricu-lar function. J Am Soc Echocardiogr 2003;16(8):814–823.

27. Tei C, Ling LH, Hodge DO, et al: New index of com-bined systolic and diastolic myocardial performance: asimple and reproducible measure of cardiac function—a study in normals and dilated cardiomyopathy. J Car-diol 1995;26(6):357–366.

28. Gergs U, Boknik P, Buchwalow I, et al: Overex-pression of the catalytic subunit of protein phos-phatase 2A impairs cardiac function. J Biol Chem2004;279(39):40827–40834.

29. Schaefer A, Klein G, Brand B, Lippolt P, DrexlerH, Meyer GP: Evaluation of left ventricular diastolicfunction by pulsed Doppler tissue imaging in mice. JAm Soc Echocardiogr 2003;16(11):1144–1149.

30. Schaefer A, Meyer GP, Hilfiker-Kleiner D, Brand B,Drexler H, Klein G: Evaluation of Tissue Doppler Teiindex for global left ventricular function in mice af-ter myocardial infarction: Comparison with PulsedDoppler Tei index. Eur J Echocardiogr 2005;6(5):367–375.

31. Schmidt AG, Gerst M, Zhai J, et al: Evaluation of leftventricular diastolic function from spectral and colorM-mode Doppler in genetically altered mice. J Am SocEchocardiogr 2002;15(10 Pt 1):1065–1073.

32. Jin D, Takai S, Yamada M, et al: Impact of chymase in-hibitor on cardiac function and survival after myocar-dial infarction. Cardiovasc Res 2003;60(2):413–420.

33. Pennock GD, Yun DD, Agarwal PG, Spooner PH,Goldman S: Echocardiographic changes after myocar-dial infarction in a model of left ventricular diastolicdysfunction. Am J Physiol 1997;273(4 Pt 2):H2018–H2029.

34. Strauch OF, Stypmann J, Reinheckel T, Martinez E,Haverkamp W, Peters C: Cardiac and ocular patholo-gies in a mouse model of mucopolysaccharidosis typeVI. Pediatr Res 2003;54(5):701–708.

35. Biben C, Weber R, Kesteven S, et al: Cardiac septaland valvular dysmorphogenesis in mice heterozygousfor mutations in the homeobox gene Nkx2-5. Circ Res2000;87(10):888–895.



36. Patten RD, Aronovitz MJ, Bridgman P, Pandian NG:Use of pulse wave and color flow Doppler echocardio-graphy in mouse models of human disease. J Am SocEchocardiogr 2002;15(7):708–714.

37. Chen B, Bronson RT, Klaman LD, et al: Mice mutantfor Egfr and Shp2 have defective cardiac semilunarvalvulogenesis. Nat Genet 2000;24(3):296–299.

38. Skryabin BV, Holtwick R, Fabritz L, et al: Hy-pervolemic hypertension in mice with systemic in-activation of the (floxed) guanylyl cyclase-A geneby alphaMHC-Cre-mediated recombination. Genesis2004;39(4):288–298.

39. Keller BB, MacLennan MJ, Tinney JP, Yoshigi M: Invivo assessment of embryonic cardiovascular dimen-sions and function in day-10.5 to -14.5 mouse embryos.Circ Res 1996;79(2):247–255.

40. Srinivasan S, Baldwin HS, Aristizabal O, et al: Non-invasive, in utero imaging of mouse embryonic heartdevelopment with 40-MHz echocardiography. Circula-tion 1998;98(9):912–918.

41. Taffet GE, Pham TT, Hartley CJ: The age-associatedalterations in late diastolic function in mice are im-proved by caloric restriction. J Gerontol A Biol Sci MedSci 1997;52(6):B285–B290.

42. Zhang X, Azhar G, Furr MC, Zhong Y, Wei JY: Model offunctional cardiac aging: Young adult mice with mildoverexpression of serum response factor. Am J PhysiolRegul Integr Comp Physiol 2003;285(3):R552–R560.

43. Kirchhefer U, Neumann J, Bers DM, et al: Impairedrelaxation in transgenic mice overexpressing junctin.Cardiovasc Res 2003;59(2):369–379.

44. Kirchhefer U, Jones LR, Begrow F, et al: Transgenictriadin 1 overexpression alters SR Ca2+ handlingand leads to a blunted contractile response to beta-adrenergic agonists. Cardiovasc Res 2004;62(1):122–134.

45. Semeniuk LM, Kryski AJ, Severson DL: Echocardio-graphic assessment of cardiac function in diabeticdb/db and transgenic db/db-hGLUT4 mice. Am J Phys-iol Heart Circ Physiol 2002;283(3):H976–H982.

46. Oberst L, Zhao G, Park JT, et al: Dominant-negativeeffect of a mutant cardiac troponin T on cardiac struc-ture and function in transgenic mice. J Clin Invest1998;102(8):1498–1505.

47. Freeman K, Colon-Rivera C, Olsson MC, et al: Pro-gression from hypertrophic to dilated cardiomyopa-thy in mice that express a mutant myosin transgene.Am J Physiol Heart Circ Physiol 2001;280(1):H151–H159.

48. Knollmann BC, Blatt SA, Horton K, et al: Inotropicstimulation induces cardiac dysfunction in transgenicmice expressing a troponin T (I79N) mutation linkedto familial hypertrophic cardiomyopathy. J Biol Chem2001;276(13):10039–10048.

49. Du XJ, Samuel CS, Gao XM, Zhao L, Parry LJ, TregearGW: Increased myocardial collagen and ventricular di-astolic dysfunction in relaxin deficient mice: a gender-specific phenotype. Cardiovasc Res 2003;57(2):395–404.

50. Zhou YQ, Foster FS, Nieman BJ, Davidson L, Chen XJ,Henkelman RM: Comprehensive transthoracic car-diac imaging in mice using ultrasound biomicroscopywith anatomical confirmation by magnetic resonanceimaging. Physiol Genomics 2004;18(2):232–244.

51. Schaefer A, Meyer GP, Brand B, Hilfiker-Kleiner D,Drexler H, Klein G: Effects of anesthesia on dias-tolic function in mice assessed by echocardiography.Echocardiography 2005;22(8):665–670.

52. Sebag IA, Handschumacher MD, Ichinose F, et al:Quantitative assessment of regional myocardial func-tion in mice by tissue Doppler imaging: comparisonwith hemodynamics and sonomicrometry. Circulation2005;111(20):2611–2616.

53. Silvestre JS, Mallat Z, Tamarat R, Duriez M,Tedgui A, Levy BI: Regulation of matrix metallo-proteinase activity in ischemic tissue by interleukin-10: role in ischemia-induced angiogenesis. Circ Res2001;89(3):259–264.

54. Tsujita Y, Kato T, Sussman MA: Evaluation of leftventricular function in cardiomyopathic mice by tissueDoppler and color M-mode Doppler echocardiography.Echocardiography 2005;22(3):245–253.

55. Matoba S, Hwang PM, Nguyen T, Shizukuda Y: Eval-uation of pulsed Doppler tissue velocity imaging forassessing systolic function of murine global heart fail-ure. J Am Soc Echocardiogr 2005;18(2):148–154.

56. Tsujita Y, Kato T, Sussman MA: Evaluation of leftventricular function in cardiomyopathic mice by tissueDoppler and color M-mode Doppler echocardiography.Echocardiography 2005;22(3):245–253.

57. Gan LM, Wikstrom J, Bergstrom G, Wandt B: Non-invasive imaging of coronary arteries in living miceusing high-resolution echocardiography. Scand Car-diovasc J 2004;38(2):121–126.

58. Johansson ME, Hagg U, Wikstrom J, Wickman A,Bergstrom G, Gan LM: Haemodynamically significantplaque formation and regional endothelial dysfunc-tion in cholesterol-fed ApoE-/- mice. Clin Sci (Lond)2005;108(6):531–538.

59. Wikstrom J, Gronros J, Bergstrom G, Gan LM:Functional and morphologic imaging of coronaryatherosclerosis in living mice using high-resolutioncolor Doppler echocardiography and ultrasoundbiomicroscopy. J Am Coll Cardiol 2005;46(4):720–727.

60. Scherrer-Crosbie M, Steudel W, Hunziker PR, etal: Determination of right ventricular structure andfunction in normoxic and hypoxic mice: a tran-soesophageal echocardiographic study. Circulation1998;98(10):1015–1021.

61. Steudel W, Scherrer-Crosbie M, Bloch KD, et al: Sus-tained pulmonary hypertension and right ventricularhypertrophy after chronic hypoxia in mice with con-genital deficiency of nitric oxide synthase 3. J ClinInvest 1998;101(11):2468–2477.

62. Zhou YQ, Foster FS, Parkes R, Adamson SL: Develop-mental changes in left and right ventricular diastolicfilling patterns in mice. Am J Physiol Heart Circ Phys-iol 2003;285(4):H1563–H1575.

63. Stennard FA, Costa MW, Lai D, et al: Murine T-box transcription factor Tbx20 acts as a repressorduring heart development, and is essential for adultheart integrity, function and adaptation. Development2005;132(10):2451–2462.

64. Zhou YQ, Zhu Y, Bishop J, et al: Abnormal cardiacinflow patterns during postnatal development in amouse model of Holt-Oram syndrome. Am J PhysiolHeart Circ Physiol 2005;289(3):H992–H1001.

65. Zhou YQ, Davidson L, Henkelman RM, et al:Ultrasound-guided left-ventricular catheterization: Anovel method of whole mouse perfusion for microimag-ing. Lab Invest 2004;84(3):385–389.

66. Zhou YQ, Foster FS, Qu DW, Zhang M, HarasiewiczKA, Adamson SL: Applications for multifrequency ul-trasound biomicroscopy in mice from implantation toadulthood. Physiol Genomics 2002;10(2):113–126.


STYPMANN

67. Feng Q, Song W, Lu X, et al: Development ofheart failure and congenital septal defects in micelacking endothelial nitric oxide synthase. Circulation2002;106(7):873–879.

68. Hartley CJ, Reddy AK, Madala S, Entman ML,Michael LH, Taffet GE: Noninvasive ultrasonic mea-surement of arterial wall motion in mice. AmJ Physiol Heart Circ Physiol 2004;287(3):H1426–H1432.

69. Hartley CJ, Taffet GE, Reddy AK, Entman ML,Michael LH: Noninvasive cardiovascular phenotypingin mice. ILAR J 2002;43(3):147–158.

70. Hartley CJ, Reddy AK, Madala S, et al: Hemody-namic changes in apolipoprotein E-knockout mice.Am J Physiol Heart Circ Physiol 2000;279(5):H2326–H2334.

71. Phoon CK, Aristizabal O, Turnbull DH: Spatial ve-locity profile in mouse embryonic aorta and Doppler-derived volumetric flow: a preliminary model. AmJ Physiol Heart Circ Physiol 2002;283(3):H908–H916.

72. Maki JM, Rasanen J, Tikkanen H, et al: Inactivationof the lysyl oxidase gene Lox leads to aortic aneurysms,cardiovascular dysfunction, and perinatal death inmice. Circulation 2002;106(19):2503–2509.

73. Nakamura A, Rokosh DG, Paccanaro M, et al: LV sys-tolic performance improves with development of hy-pertrophy after transverse aortic constriction in mice.Am J Physiol Heart Circ Physiol 2001;281(3):H1104–H1112.

74. Nakamura Y, Yoshiyama M, Omura T, et al: Transmi-tral inflow pattern assessed by Doppler echocardiogra-

phy in angiotensin II type 1A receptor knockout micewith myocardial infarction. Circ J 2002;66(2):192–196.

75. Li YH, Reddy AK, Ochoa LN, et al: Effect of ageon peripheral vascular response to transverse aor-tic banding in mice. J Gerontol A Biol Sci Med Sci2003;58(10):B895–B899.

76. Bernstein D: Exercise assessment of transgenic mod-els of human cardiovascular disease. Physiol Ge-nomics 2003;13(3):217–226.

77. Kemi OJ, Loennechen JP, Wisloff U, Ellingsen O:Intensity-controlled treadmill running in mice: car-diac and skeletal muscle hypertrophy. J Appl Physiol2002;93(4):1301–1309.

78. Wong AY, Kulandavelu S, Whiteley KJ, Qu D, LangilleBL, Adamson SL: Maternal cardiovascular changesduring pregnancy and postpartum in mice. Am J Phys-iol Heart Circ Physiol 2002;282(3):H918–H925.

79. Kirchhof P, Fabritz L, Fortmuller L, et al: Alteredsinus nodal and atrioventricular nodal function infreely moving mice overexpressing the A1 adeno-sine receptor. Am J Physiol Heart Circ Physiol2003;285(1):H145–H153.

80. Knollmann BC, Kirchhof P, Sirenko SG, et al: Famil-ial hypertrophic cardiomyopathy-linked mutant tro-ponin T causes stress-induced ventricular tachycar-dia and Ca2+-dependent action potential remodeling.Circ Res 2003;92(4):428–346.

81. Schafers KP, Larmann J, Stypmann J, Theilmeier G,Schober O, Schafers M: High resolution positron emis-sion tomography to image myocardial infaction in amouse. Nuclear Medicine 2004;43(1):N6–N7.


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Doppler Ultrasound in Mice