IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 52, NO. 10, OCTOBER 2005 1771 Pulsed Doppler Signal Processing for Use in Mice: Applications Anilkumar K. Reddy*, Member, IEEE, George E. Taffet, Yi-Heng Li, Sang-Wook Lim, Thuy T. Pham, Jennifer S. Pocius, Mark L. Entman, Lloyd H. Michael, and Craig J. Hartley, Senior Member, IEEE Abstract—We have developed a high-frequency, high-resolution Doppler spectrum analyzer (DSPW) and compared its performance against an adapted clinical Medasonics spectrum analyzer (MSA) and a zero-crossing interval histogram (ZCIH) used previously by us to evaluate cardiovascular physiology in mice. The aortic velocity (means SE: 92.7 2.5 versus 82.2 1.8 cm/s) and aortic acceleration (8194 319 versus 5178 191 cm/s2 ) determined by the DSPW were significantly higher compared to those by the MSA. Aortic ejection time was shorter 1.8 ms) and the isovolumic relaxation (48.3 0.9 versus 64.6 was longer (17.6 0.6 versus 13.5 0.6 ms) when determined by the DSPW because it generates shorter temporal widths in the velocity spectra when compared to the MSA. These data indicate that the performance of the DSPW in evaluating cardiovascular physiology was better than that of the MSA. There were no significant differences between the aortic pulse wave velocity determined by using the ZCIH (391 16 cm/s) and the DSPW (394 20 cm/s). Besides monitoring cardiac function, we have used the DSPW for studying peripheral vascular physiology in normal, transgenic, and surgical models of mice. Several applications such as the detection of high stenotic jet velocities ( 4 m/s), vortex shedding frequencies (250 Hz), and subtle changes in wave shapes in peripheral vessels which could not obtained with clinical Doppler systems are now made possible with the DSPW. Index Terms—High-frequency pulsed Doppler ultrasound, mouse cardiac function, mouse cardiovascular physiology, pulse-wave velocity, stenotic jet velocities, vortex shedding frequencies. I. INTRODUCTION T RANSGENIC and gene-targeted (knockout) models of mice are increasingly used to study cardiovascular disease processes and to some extent have replaced the traditional large animal models , . Although it is easy to breed, house, and maintain them, the small size of mice makes the measurement of hemodynamic parameters difficult. Despite Manuscript received August 20, 2004; revised March 26, 2005. This work was supported in part by National Institutes of Health under Grant HL-52364, Grant HL-22512, and Grant AG-13251, and in part by the Texas Advanced Technology Program. The work of Y.-H. Li was supported in part by a grant from National Science Council, Taipei, Taiwan, and the College of Medicine, National Cheng Kung University, Tainan, Taiwan. Dr. Lim was supported by Pochon CHA University, South Korea. Asterisk indicates corresponding author. *A. K. Reddy is with the Baylor College of Medicine, Houston, TX, 77030 USA (e-mail: [email protected]). Y.-H. Li was with the Baylor College of Medicine and is currently with National Cheng Kung University, Tainan, Taiwan. S-W. Lim was with the Baylor College of Medicine and is currently with Pochon CHA University, Bundang-gu, South Korea. G. E. Taffet, T. T. Pham, J. S. Pocius, M. L. Entman, L. H. Michael and C. J. Hartley are with Baylor College of Medicine and DeBakey Heart Center, Houston, TX, 77030 USA. Digital Object Identifier 10.1109/TBME.2005.855709 the fact that mice have high heart rates ( 600 beats/min) many researchers continue to measure blood velocities in mice using clinical systems that have poor spatial resolution because they operate at low frequencies (2–5 MHz), have Doppler probes with large footprints that cannot be oriented properly, and use spectrum analyzers that have poor temporal resolution –. Previously we have used custom made 10- and 20-MHz pulsed Doppler probes and a clinical Medasonics (Vasculab SP25A) spectrum analyzer (MSA) to measure blood velocities from various vessels in mice . Cardiac (aortic and mitral) blood velocity signals of mice were measured using a 10-MHz pulsed Doppler probe and parameters such as the peak and average velocities, and timing of various events in the cardiac cycle were calculated from the screen-captured images of real-time display of velocity spectrograms obtained using the MSA –. The 10- and 20-MHz Doppler probes provided high spatial resolutions and small foot prints. However, the temporal resolution was limited to about 8 ms because of the fast Fourier transform (FFT) window length and the slow sweep speed , . This time resolution is not adequate to measure cardiac timing in mice where the total cardiac period is on the order of 100 ms. The maximum bandwidth (frequency range) of the MSA is 24 kHz which is barely adequate to measure velocities in most cases. However, in mouse models of aortic stenosis the peak blood flow velocities just past the site at the arch can exceed 400 cm/s producing Doppler shifts greater than 100 kHz . Blood flow velocity signals from the aortic arch and abdominal aorta were measured using a 20-MHz pulsed Doppler probe, to calculate pulse wave velocity (PWV). An indicator of arterial compliance, PWV was determined by measuring the difference in time of onset of the blood velocity signals at two sites along a vessel separated by a known distance. Given the fast rise times of the blood flow velocity signal in mice, a zero-crossing interval histogram (ZCIH)  was used to maximize the temporal resolution of the Doppler signals, and the PWVs obtained by this method in mice were reported . The time resolution of the ZCIH improves with the increase in Doppler frequency, but the frequency resolution is nonquantitative even with noise free Doppler signals, allowing only for estimates of spectral information . Thus the need arises for a Doppler data acquisition and analysis system which can sample the data at high rates, have a high bandwidth, provide high temporal resolution, and be capable of acquiring and analyzing multiple cardiovascular signals from a mouse. In the preceding article  we described such a system. Here, we discuss the performance and applications of the above system in evaluating mouse cardiovascular physiology. 0018-9294/$20.00 © 2005 IEEE 1772 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 52, NO. 10, OCTOBER 2005 II. METHODS A. Instrumentation 1) Doppler Signal/Spectrum Analyzers: The Doppler signal processing workstation (DSPW), a real-time signal acquisition and spectrum analyzer system (Indus Instruments, Houston, Texas) was described in the previous paper (Design and Evaluation) . The MSA is a real-time spectrum analyzer and has a frequency response from 500 Hz to 24 kHz, with a temporal resolution of 8 ms, a highest sweep speed of 1-s full scale, and eight sampling rates extending from 3.2 kHz to 38.4 kHz. The sampling rates depend upon the displayed frequency ranges with a sampling rate of 3.2 kHz in the range 563 to 2038 Hz and up to a sampling rate of 38.4 kHz in the range 6.75 to 24.45 kHz. Also, the MSA has a 280 Hz high-pass filter to eliminate low-frequency noise, and two anti-aliasing filters at 8 kHz for ranges up to 8 kHz, and at 24 kHz for ranges 12 kHz and higher. The spectrogram is displayed on a Sanyo RGB color monitor (Model DMC 8500). Since the ECG signal could not be displayed along with the Doppler spectrogram, a 1-ms R-peak trigger pulse generated by the ECG module was added to one of the quadrature audio signals. This R-trigger pulse generated a vertical line in the spectrogram after processing by the FFT spectrum analyzer. This vertical line served as an ECG timing marker. The screen-captured image was printed using a video printer (model UP-870MD, Sony) to obtain hard-copy output which was later scanned to save the image on a computer. The zero crossing interval histogram (ZCIH) analyzer is a time-domain Doppler signal analyzer, which continuously displays the frequency of consecutive zero crossings of the Doppler signal. The output of the ZCIH was displayed on an oscilloscope and simultaneously acquired by a data acquisition system at a sampling rate of 37 kHz. 2) Analog Doppler Signal Processors: The high-frequency pulsed Doppler analog mainframe (Baylor College of Medicine, Houston, Texas) can generate pulse repetition frequencies (PRF) of 31.25, 62.5, or 125 kHz and can operate at ultrasonic frequencies of 5, 10, or 20 MHz. For velocity measurements in mice 10 or 20 MHz are typically used. The mainframe accommodates several modules which include 10and 20-MHz pulsed Doppler modules, blood pressure module, ECG module, and temperature control module. The Doppler modules generate 10- or 20-MHz signals pulsed at either 62.5 kHz or 125 kHz allowing for the measurement of blood velocity signals with Doppler shifts of up to 31.25 kHz or 62.5 kHz respectively, to be resolved without aliasing . measured in frequency (kHz) were conThe Doppler shifts verted to velocities (cm/s) using the Doppler equation , where is the speed of sound (1540 m/s) is the ultrasonic frequency (10 or 20 MHz). A 10-MHz and pulsed Doppler probe is used to obtain cardiac (aortic outflow and mitral inflow) blood velocity signals from mice while a 20 MHz probe is used to obtain signals from peripheral vessels that are within 2–4 mm of the skin surface in mice. We used custom built 10- and 20-MHz Doppler probes with a 1.0 mm (20 MHz) or a 1.5 mm (10 MHz) square of piezoelectric material (PZT-5A) mounted at the end of a 2.25-mm-diameter 10-cm-long stainless-steel tubing. An epoxy lens is molded to the front face of the crystal to focus the sound beam at 4 mm (20 MHz) or 6 mm (10 MHz). The sample volume is about 0.3–0.5 mm in length and 0.3–0.5 mm in diameter at the focal point of the lens giving an estimated volume of 0.02–0.08 l. The sample volume dimensions are on the order of or slightly smaller than the diameter of the mouse aorta . 3) Pressure Amplifier Module: The blood pressure module consists of a custom built bridge amplifier which has a frequency response of 0–2 kHz with balance and gain adjustments and the option to obtain mean pressure. Pressure sensors used in mice include the RADI Pressure Wire (RADI Medical Systems) for aortic  and ventricular pressure measurements, Millar Mikro-tip catheter transducer (SPR-671, Millar Instruments, Houston, Texas) for tonometric pressure measurements, and an external pressure transducer (Meritrans #K09-03 243P, Merit Medical Systems, Inc.) connected to a fluid filled catheter for the measurement of carotid  and iliac artery pressures. The RADI catheter was designed as an angioplasty guide wire and has a 3-cm-long floppy wire tip distal to the pressure sensor. The distal segment was removed and epoxy was used to smooth the tip before the catheter was used in the mouse. A custom signal conditioner was built to interface the RADI catheter to our pressure amplifier module . The catheters and the pressure amplifier were calibrated for each experiment from 0 to 250 mmHg using a mercury manometer. 4) ECG Amplifier Module: The ECG module consisting of a custom built differential amplifier module with frequency response extending from 0.1 to 2 kHz was used to record ECG’s (Lead I, II, III, AVR, AVF, AVL) from limb electrodes. A mouse ECG board (Baylor College of Medicine, Houston, Texas) was designed and built for use in mouse studies. The ECG board has a large ground plane and contains four electrodes, a DC powered heating pad consisting of an array of surface mount resistors, and a temperature sensor. A custom-built temperature control module was used to regulate the temperature of the heating pad using the mouse body temperature or the board temperature as feedback . B. Animal Protocols All studies (invasive and noninvasive) were performed on anesthetized mice. The mice were anesthetized with either gas anesthesia or two different intraperitoneally (IP) administered anesthetics depending upon the type of study. The gas anesthesia was administered at a continuous flow rate (20 ml/min) of a mixture containing 1% isoflurane combined with 99% oxygen. One of the IP administered anesthetics used was a rodent cocktail (ketamine 42.8 mg/ml, xylazine 8.6 mg/ml, and acepromazine 1.4 mg/ml) given at 0.5 l/g of body weight. The other IP administered anesthetic was pentobarbital cocktail (1.6 ml of 50 mg/ml pentobarbital sodium, 4.0 ml of 200 proof ethyl alcohol, and 16.0 ml of 0.9% saline) given at 10 l/g of body weight. The mouse was anesthetized and placed on the ECG/heater board with the board temperature adjusted to maintain mouse body temperature at 37 1 C. The limbs of the mouse were taped to the four electrodes as shown in Fig. 1, the quality of ECG assessed, and the electrode contact optimized if needed. All animal protocols were approved by the Institutional Animal Care and Use Committee of Baylor REDDY et al.: PULSED DOPPLER SIGNAL PROCESSING FOR USE IN MICE: APPLICATIONS 1773 Fig. 1. Experimental setup for the measurement of Doppler flow velocity signals along with ECG and blood pressure signals in mice. Several possible configurations are shown in this setup. For example, three Doppler probes are shown but only one Doppler probe can be used at any time to measure blood velocity signals. Fig. 2. (a). Aortic and mitral flow velocity signals from an anesthetized mouse. Four cardiac cycles of aortic velocity signal were simultaneously processed by the DSPW and the MSA (above), and displayed at same sweep speed of 1-s full scale. The vertical dashed lines before each aortic wave processed by the MSA is the ECG marker which overlaps the mitral valve click which can be clearly seen in the signal processed by the DSPW. The mitral signals were similarly processed and displayed (below). (b). Flow velocity signals measured at the aortic arch and at the abdominal aorta in an anesthetized mouse. Four cardiac cycles of each velocity signal were simultaneously processed by the DSPW and the ZCIH, and displayed at the same sweep speed of 1-s full scale. A vertical dashed line is drawn to indicate the alignment of the ECG R-peak which was used as a reference to measure the pulse transit time. College of Medicine in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (DHHS Publication no. 85–23, Revised 1985, Office of Science and Health Reports, Bethesda, MD). C. Noninvasive Measurements Cardiac Doppler signals were obtained from the aortic root and the mitral inflow track using a 10-MHz probe (Fig. 1). 1774 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 52, NO. 10, OCTOBER 2005 Fig. 3. A cardiac cycle showing both aortic and mitral flow velocity spectrograms from an anesthetized mouse. The segments were captured offline when upper and lower panels were displayed at a sweep speed of 500-ms full scale. The aortic and mitral signals were obtained separately using a 10-MHz probe. The probe was oriented to place sample volume in the aortic outlet region and then reoriented to place sample volume in the mitral inlet region. The Doppler signals were sampled at 125 kilo samples/s. A 512-sample cFFT window was used to generate the velocity spectrogram. Several cardiac parameters are identified and defined. Doppler signals from each mouse were acquired and processed by both the DSPW and MSA (at about the same time if not simultaneously). Cardiac parameters were extracted during analysis offline from data obtained by both systems and compared. Doppler signals from many peripheral vessels (carotid, femoral, tail, renal, aortic arch, abdominal aorta) were obtained using a 20-MHz probe, the most common being the measurements from two aortic sites to determine aortic PWV as described elsewhere . Doppler signals from the aortic arch and abdominal aorta were acquired and processed by both the DSPW and the ZCIH systems. Aortic PWV determined from signals obtained by both systems was compared. The noninvasive measurement of systolic and diastolic blood pressure in mice using a tail-cuff and a 20-MHz Doppler probe is important. The procedure to measure blood pressure by tail-cuff has been described in detail elsewhere . Briefly, a tail-cuff (10 mm long and 7 mm diameter) was placed close to the base of the tail of an anesthetized mouse. A 3–4 mm epoxy cuff with a 20-MHz piezo-electric crystal (1 mm ) embedded at an angle of 45 was placed distally near the tail-cuff and adjusted to obtain the best velocity signal. The tail-cuff pressure was increased until the velocity signal sensed by the Doppler probe in the tail disappeared (above systolic). Then the pressure in the cuff was decreased slowly ( 3–5 mmHg/s). The pressure at which the signal first appeared was defined as systolic pressure and the pressure at which the signal became continuous was defined as diastolic pressure. The tail blood flow velocity signal sensed by the Doppler probe and the tail-cuff pressure signal sensed by the Meritrans pressure sensor were processed and stored for analysis offline. D. Invasive Measurements The most common invasive measurement made in mice is blood pressure. Blunt dissection techniques were used on an anesthetized mouse to expose and isolate the carotid artery which was then cannulated with either a fluid-filled catheter connected to a Meritrans pressure sensor to measure pressure in the carotid artery or with RADI pressure catheter to measure pressures in the ascending aorta and left ventricle. Similar surgical procedure was performed in the lower abdominal section to access the iliac artery for cannulation with a fluid-filled catheter. For the measurement of tonometric pressure using a Millar catheter the artery of interest was exposed but unopened. The pressure sensor was placed under the exposed artery such that the arterial wall was positioned flat on the sensor. E. Data Analysis All data are presented in the form of mean SE. Comparisons of the cardiac parameters analyzed by the DSPW and the MSA, and PWV determined from peripheral velocity signals measured by the DSPW and the ZCIH were made with paired Student’s t-test using a significance level of 0.05. REDDY et al.: PULSED DOPPLER SIGNAL PROCESSING FOR USE IN MICE: APPLICATIONS 1775 TABLE I CARDIAC PARAMETERS EXTRACTED FROM AORTIC AND MITRAL FLOW VELOCITY SIGNALS PROCESSED USING THE MSA AND THE DSPW SYSTEMS III. RESULTS Typical mouse cardiac and peripheral Doppler signals are shown in Fig. 2. The cardiac signals (aortic and mitral) were obtained from the same mouse, with the same cardiac cycles processed by the DSPW and the MSA, and displayed at the same sweep speed of 1-s full scale [Fig. 2(a)]. Similarly, each of the peripheral signals (aortic arch and abdominal aorta) was obtained from the same mouse and the data from the same cardiac cycles were processed by the DSPW and the ZCIH [Fig. 2(b)]. Shown in Fig. 3 is an expanded cycle of composite cardiac Doppler signal (both aortic and mitral components) of a mouse. The Doppler audio signals were sampled at 125 ksps, the spectrogram was calculated using a 256-point complex FFT (cFFT) window, and displayed at a sweep speed of 200 ms full scale. Several parameters that can be measured from both aortic and mitral signals are also shown. Normally the aortic and mitral signals are measured separately [as shown in Fig. 2(a)] to maximize their peak velocities. Some of the important cardiac parameters obtained from normal mice using the DSPW and the MSA are summarized in Table I. Also shown in the table are % changes in parameter values when measured by DSPW from the values measured by MSA. The “p-value” is the statistical probability value obtained from the paired Student t-test comparisons. The peak E-velocity and peak aortic velocity were significantly higher when measured by the DSPW. Although not significant, peak A-velocity was also higher when measured by the DSPW (The large variability in peak A-velocity resulted in the lack of statistical significance). On the other hand, timing parameters such as time to aortic peak and ejection time (ET) were significantly greater when measured by the MSA. A similar trend, albeit not significant, was observed in the measurement of time to E-peak, E-deceleration time, and pre- Fig. 4. A cardiac cycle showing both aortic arch and abdominal aorta flow velocity waves from an anesthetized mouse The Doppler signals corresponding to these waves were obtained using a 20-MHz probe and processed simultaneously by the DSPW (left) and the ZCIH (right). ejection time when measured by the MSA. Isovolumic relaxation time (IVRT) was significantly higher when measured by the DSPW. We were unable to measure isovolumic contraction time (IVCT) with the MSA but could consistently measure it with the DSPW which allowed us to calculate the TEI index. A reliable robust measure of cardiac function, the TEI index is calculated as (IVRT IVCT)/ET . Mean velocity and mean acceleration of aortic flow were significantly higher when measured by the MSA. Fig. 4 shows the calculation of PWV using a single cycle of aortic arch and abdominal velocity signals processed by the DSPW and the ZCIH to illustrate the calculation of PWV. No significant differences were observed in the aortic PWV determined in 24 mice using the two systems (ZCIH: 391 16, DSPW: 394 20). 1776 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 52, NO. 10, OCTOBER 2005 Fig. 5. Cardiac and peripheral blood flow velocity signals along with ECG signal in a mouse. The cardiac (aortic and mitral) signals were obtained using a 10-MHz probe and the peripheral signals were obtained using a 20-MHz probe. All the Doppler signals were sampled at 125 ksps and processed using a 512-sample cFFT to generate the velocity spectrogram. Part of the figure was adapted from Hartley et al.,  and reprinted with permission from ILAR (Institute for Laboratory Animal Research) journal. IV. DISCUSSION A high-speed, high-bandwidth, real-time Doppler signal processing workstation (DSPW) which provides high temporal resolution and is capable of acquiring and analyzing multiple cardiovascular signals from a mouse was developed and evaluated. In the initial part of this section we discuss the performance of the DSPW versus the MSA in the measurement of cardiac signals, and versus the ZCIH in the measurement of pulse wave velocity. In the later part we discuss several applications of the DSPW that are used for cardiovascular phenotyping in the mouse. A. Performance of the DSPW Versus the MSA and the ZCIH The velocity and timing parameters shown in Table I are the important ones among the parameters listed in Fig. 3 and are routinely used to evaluate cardiac function in a mouse. The velocity parameters extracted from cardiac (aortic and mitral) flow velocity signals are comparable to those measured in humans , rats , and dogs . Also, the aortic PWV in mice is similar to that of humans  and other animals , . 1) Measurement of Peak Velocities: We found that higher peak velocities were obtained when the Doppler signals were processed by the DSPW versus those processed by the MSA. While the DSPW uses up to 95% energy level to detect the peak velocities the level used by the MSA could be lower than 95%. Typically 75–95% is energy level range used for detecting peak velocity . Another possibility is that in the MSA the baseline noise could have been subtracted before displaying the spectrogram thereby reducing the maximum frequencies. In addition, the fastest sweep speed is limited to 1-s full scale in the MSA thus limiting pixel resolution at the spectrogram peaks. 2) Measurement of Timing of Cardiac Events: The timing of cardiac events determined from the spectrograms obtained by both the DSPW and the MSA were longer than the actual time. This is because the measurement of time events from the spectrogram is influenced by the cFFT-window sample size in addition to the sampling rate and sweep speed used to calculate the spectrogram. While the cFFT window of the MSA is limited to 8-ms , , the cFFT window of the DSPW can be made as small as 0.5 ms at a sampling rate of 125 kHz. Therefore, with a 256-sample cFFT the timing events measured by the MSA are much longer when compared to those measured by the DSPW with the exception of isovolumic relaxation time. Since each spectral window in the MSA is longer, the trailing edge of the velocity spectrum becomes much wider than that of the REDDY et al.: PULSED DOPPLER SIGNAL PROCESSING FOR USE IN MICE: APPLICATIONS 1777 Fig. 6. Spectral envelope signals from the aortic arch of a wild-type mouse and an apolipoprotein E knockout (ApoE-/-) mouse showing systemic differences in acceleration of the velocity waveforms. Acceleration can be divided into two phases, A1 and A2, with A2 being smaller than A1 in wild-type mice and larger than A1 in ApoE-/- mice. We hypothesized that the differences were due to altered peripheral wave reflections. Reproduced from Hartley et al., Am. J. Physiol. Heart Circ. Physiol., 279:H2326–H2334, 2000, with permission from American Physiological Society. DSPW thus making the period of isovolumic relaxation time to appear shorter when determined using the MSA. We could not determine isovolumic contraction time using the MSA because the exact locations of the trailing edge of the inflow and leading edge of the outflow velocities were difficult to discern (see Fig. 3 for the definitions of the above parameters). 3) Measurement of Pulse Wave Velocity: Aortic PWV has been used in humans and large animals to evaluate stiffness of the vessel wall segment between two measurement sites , . We determined PWV using the time intervals from R-peak to the onset of aortic arch and abdominal velocity signals (Fig. 4) and the separation distance between these sites. No significant differences were observed in the PWV determined by both systems. The values of PWV are similar to those previously reported in mice , . Although the time resolution of the ZCIH system is good , it only provides an average of center frequency  and, therefore, cannot be used to measure peak velocities. B. Applications of DSPW Cardiac inflow and outflow velocity signals obtained with a 10-MHz pulsed Doppler probe and various peripheral arterial velocity signals obtained with a 20-MHz pulsed Doppler probe in mice and processed using the DSPW are shown in Fig. 5. The velocity signals from various locations were obtained based on the knowledge of the anatomy of the mouse and the estimated angles of the Doppler beam with the axial direction of flow. The origination of most of these signals has been previously confirmed with invasive procedures. These signals obtained using the DSPW are of exceptional quality and detail and could not be obtained using the MSA or the ZCIH. 1) Peripheral Vascular Signals: Blood velocity signals from peripheral arterial sites can be analyzed by the DSPW for alterations in peak and mean velocities, and specifically for changes in the shapes of the waveform caused by specific phenotypes which would be not possible with the MSA. For Fig. 7. (a). Analysis of a mouse peripheral velocity signal. The above signal was obtained from a mouse carotid artery using a 20-MHz pulsed Doppler probe, sampled at 125 ksps, and the velocity spectrogram was calculated using a 256-sample cFFT window. Also shown in the figure are the calculations for pulsatility index (PI) and resistance index (RI) that characterize the local compliance and downstream resistance of the artery. (b). Doppler velocity signals from the right and left carotid arteries before aortic banding and from the same locations after banding. While the pulsatility index of the left and right carotid velocity was similar before aortic banding, it increased 5-fold after banding. (b) reproduced from Li et al., J. Gerontol., 58A:895–899, 2003, Copyright © The Gerontological Society of America. Reproduced by permission of the publisher. example, we could not have detected the biphasic upstroke of the aortic arch velocity signal in apolipoprotein-E knockout mice using the MSA or other clinical Doppler systems because their best temporal resolution is longer than the duration of the initial phase of the velocity upstroke (see Fig. 6) . We hypothesized that this change in the wave shape could have been caused by stronger wave reflections from the stenosed carotid bifurcation in ApoE-KO mice . Peripheral vascular signals are analyzed to evaluate the function of the proximal and distal arterial systems which are responsible for maintaining adequate peripheral perfusion. Typically peripheral vessel function is characterized by pulsatility index and resistance index which are used to estimate local compliance and distal arterial resistance. Shown in Fig. 7(a) is the 1778 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 52, NO. 10, OCTOBER 2005 Fig. 8. Doppler velocity signals across aortic band and pressure tracings from both carotid arteries (left panel). Peak value (V) of jet velocity and instantaneous carotid pressure gradient ( P) were measured. Pressure gradient estimated by simplified Bernoulli equation ( P 4V ) was plotted against the carotid catheter pressure gradient (right lower panel). The right upper panel shows the position of the probe and sample volume (SV) for measuring the jet velocity across aortic band. LCPr, left carotid pressure; RCPr, right carotid pressure. The right lower panel shows correlation between the pressure gradient estimated by simplified Bernoulli equation from Doppler studies (4V ) and measured from carotid catheterization studies ( P). The five different symbols represent data from five different mice. Reproduced from Li et al., Ultrasound Med. Biol., 29(9):128l–1289, 2003, Elsevier. 1 1= 1 analysis process of a peripheral (carotid artery in this case) arterial velocity signal. Using such analysis we have shown that 16 month old mice take longer time to establish adequate and equal mean flow velocity in the carotid arteries in response to transverse aortic stenosis [Fig. 7(b)] when compared to mice aged 4 months . 2) Validation of the Simplified Bernoulli Equation in Mice: One of the important applications of the DSPW was the measurement of stenotic jet velocity in mice with transverse aortic banding to estimate the pressure gradient across the aortic stenosis using the simplified Bernoulli equation ( P 4V ). Previously, we were unable to measure high-velocity signals across the stenotic jet using the MSA because its frequency range was limited to 24 kHz (92.4 cm/s with 20-MHz pulsed Doppler with speed of sound in soft tissue/blood 1540 cm/s). Using the DSPW we recorded stenotic jet velocities which were higher than 110 kHz ( 400 cm/s with 20-MHz pulsed Doppler) , . In contrast, Fard et al. measured stenotic jet velocities of about 3.1 m/s in mice using a 12-MHz clinical Doppler system (Hewlett-Packard 5500, Andover, MA) . Another group reported stenotic jet velocities of about 4.5 m/s in mice (with angle correction) using a 10-MHz clinical Doppler system (HDI3000/5000, ATL, Inc., Bothell, WA) . However, they acknowledged that higher correction angles may result in the overestimation of velocities. While a 10 change in angle within 0–20 range results in maximum error of 4.5%, the same in 60–75 range results in a maximum error of 16.4%. Clinical Doppler systems typically use angle corrections in the 60–70 range making velocity measurement less precise. Continuous wave Doppler has been used in some cases to measure blood velocities in mice  to overcome the frequency range limitation of some clinical pulsed Doppler systems. The accuracy of the simplified Bernoulli equation has been demonstrated in the estimation of the pressure gradient across stenotic valves in patients –. However, some of the un- Fig. 9. The detection of vortex shedding frequencies in the peak aortic velocity distal to the location of an aortic stenosis caused by banding. The signal was obtained using a 20-MHz pulsed Doppler probe with the crystal mounted to the side of a 22-gauge needle and passed down the esophagus. The transient oscillations in peak velocity occur at a frequency of about 250 Hz. derlying assumptions used in deriving P 4V may not hold in mice due to higher wall shear stress resulting in a short inlet length and ultimately a nearly parabolic velocity profile in the aortic arch. Despite these questions our data (Fig. 8)  indicate that the accuracy of the simplified Bernoulli equation in estimating the pressure drop in banded mice is similar to that of clinical systems used in patients. 3) Detection of Vortex Shedding Frequencies: In the mouse model of aortic banding we were able to detect high-frequency velocity fluctuations in peak aortic velocity distal to a stenosis (Fig. 9). We used a 20-MHz esophageal pulse Doppler probe to measure this signal. Given that the aortic diameter in a mouse is less than 1 mm, the vortices have to be smaller than 1 mm. The ability to detect these 250 Hz fluctuations in velocity demonstrates that the sample volume of the 20-MHz Doppler probe is quite small and that the DSPW can detect these vortex shedding frequencies. Gosling and King  briefly discussed the changes in peak blood velocity due to vortices in flow distal to stenosis. However, the signals were of poor resolution and the vortex shedding frequencies were not discernible. To our knowledge no other group has reported such findings in mice thus reflecting the lack of studies on such significant hemodynamic perturbations in this mouse model . 4) Arterial Pulse Wave Velocity: One of the important and commonly used applications of peripheral blood velocity measurements is the determination of arterial PWV as an index of arterial stiffness . Pressure and velocity waves travel faster in stiffer vessels than in compliant vessels. Determination of PWV (Fig. 4) requires the measurement of velocity signals from two arterial sites separated by a known distance (SD). The transit time (PTT) of the velocity pulse from the first site to the second site is measured and PWV is calculated as SD/PTT. Previously we reported elevated PWV in Apolipoprotein E knockout mice compared to wild-type mice  but such differences were not apparent at rest in other models such as the -smooth muscle actin knockout mouse . However the differences can be brought out with pharmacological interventions (Fig. 10). Other mouse models such as Matrix GLA protein KO mice have greatly elevated PWV at rest. REDDY et al.: PULSED DOPPLER SIGNAL PROCESSING FOR USE IN MICE: APPLICATIONS Fig. 10. Pulse wave velocity in normal and transgenic mice (-SMA-/-, Matrix GLA-/-). Reprinted with permission from ILAR journal, 43(3):147–158, 2002, Institute for Laboratory Animal Research, National Academies, 500 Fifth Street NW, Washington DC 20001 (www.national-academies.org/ilar). Fig. 11. Peak aortic flow velocity followed for 5–6 months in mice subjected , 2-hour occlusion followed by reperfusion , and to sham operation permanent occlusion . Data are % of preoperative values and are expressed as means SE. Preoperative values: sham (n 15), 104 20 cm/s; permanent occlusion (n 24), 111 17 cm/s; reperfusion (n 13), 102 10 cm/s. -p<0.05, permanent occlusion versus sham; #-p<0.05, reperfusion versus sham. Reproduced from Michael et al., Am. J. Physiol. 277:H660–H668, 1999, with permission from American Physiological Society. 6 z = () (1) 6 = () 6 = 6 5) Myocardial Infarction and Remodeling in Mice: In the mouse studies of myocardial infarction and ventricular remodeling following reperfusion we quantified myocardial tissue repair through the measurement of peak aortic flow velocity (systolic function) and peak early filling velocity (diastolic function) in mice subjected to sham operation, permanent coronary occlusion, and 2-hour occlusion followed by reperfusion. While the mice with permanent occlusion showed significant and sustained reduction in peak aortic flow velocity, the reperfusion group recovered to preocclusion levels of peak velocity in 2 weeks (Fig. 11). Also, peak early velocity was significantly reduced in mice with permanent occlusion whereas the reperfused animals recovered to about 90% of preocclusion levels in 2 weeks. These data demonstrate that mice with permanent coronary occlusion are incapable of compensating unrelieved ischemia . 6) Aortic Input Impedance in Mice: Aortic input impedance is defined as pressure/flow, but it is often calculated using velocity instead of flow because velocity is a measured parameter 1779 and conversion to flow is prone to errors . Normally the luthe value of which depends on minal average flow velocity, the velocity profile in lumen of the vessel is used in calculation. (spatial peak velocity) For a blunt velocity profile and for a parabolic velocity profile . Shown in Fig. 12 is the procedure to calculate input impedance using pressure and . Using aortic input impedance we characterized the effects of aging on aortic stiffness in mice. We found that the impedance curves resemble those from humans; and, as in man, the peripheral resistance (modulus of impedance at zero frequency), characteristic impedance (average of impedance modulus from second to tenth harmonics) and location of first minimum, all increase with age . 7) Noninvasive Blood Pressure Measurement in Mice: We recently developed, validated, and reported a noninvasive method to obtain systolic and diastolic pressures in mice using a Doppler flow sensor and a tail-cuff. The principle of operation is described briefly earlier in Methods and in detail elsewhere . Data were obtained from normal mice (n 17) at baseline and after methoxamine administration. Regression analysis of the data showed high correlation between tail-cuff and catheter pressures (R 0.91 systolic, R 0.89 diastolic). We used the method to differentiate phenotypes of seven unmarked mice. Based on pressures obtained at baseline and in response to changes in blood volume and methoxamine administration, three mice were correctly identified as -smooth muscle actin knockouts and four as controls (Fig. 13). The genotypes were confirmed with DNA tests. Thus, this method can reliably measure systolic and diastolic pressures in mice to differentiate phenotypes. 8) Flow Velocity Measurements in Prenatal, Neonatal, and Juvenile Mice: The usefulness of the DSPW in quantifying the velocities and time events in the cardiac cycle of mice to study the developmental changes in left and right ventricular diastolic function from prenatal to juvenile mice has been demonstrated by Zhou et al. . Flow velocity signals from mitral and tricuspid orifices were measured in embryonic, neonatal, preweaning, and postweaning stages (Fig. 14) using an image-guided 20-MHz Doppler (ultrasound biomicroscope VS40, VisualSonics, Toronto, Canada) and the signals were acquired and analyzed by the DSPW. Parameters such as peak velocities of E- and A-waves, peak E/A ratio, peak E/E-A area, and isovolumic relaxation time were extracted from the mitral and tricuspid inflow signals. The progression of these parameters showed that age-related changes were mainly associated with increases in peak-E velocity up to 3 weeks postnatal at which time maturation of diastolic function was observed . C. Limitations and Recommendations The limitation of our system is that we measure velocity signals by placing the sample volume in the blood stream at a desired location without image guidance. We overcome this limitation with knowledge of mouse anatomy and the shape and timing of the signals with respect to ECG signal. However, in-utero measurements (in fetal mice) such as those described by Zhou et al.  cannot be made with our system without the use of image guidance. 1780 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 52, NO. 10, OCTOBER 2005 Fig. 12. Determination of input impedance. Discrete Fourier transform (DFT) was performed separately on the digitized pressure and average velocity waveforms followed by the calculation of pressure and velocity moduli. Impedance modulus was computed by dividing the pressure modulus by velocity modulus at each harmonic. The magnitudes of P(f) and V(f) at higher harmonics (although distinct) are not quite discernible in the figure due the large amplitudes at the first and second harmonics. Reproduced from Reddy et al., Proceedings of the 25th IEEE/EMBS Conference, 276–278, 2003, with permission from IEEE. j j j j 6 Fig. 13. Mean ( SE) systolic and diastolic tail-cuff pressures from -smooth muscle actin knockout (n = 3) and littermate control (n = 4) mice at chronological events; baseline (Bsl), 600 l blood withdrawal (W6), 600 l blood re-infusion (16), stabilized (Stb), 3 min post-methoxamine (Mx3), 6 min post-methoxamine (Mx6), and final withdrawal of 200 l of blood (W2). V. CONCLUSION We have developed a high-frequency high-speed Doppler spectrum analyzer specifically targeted for use in mice. This system provides a significant improvement over the previously used adapted clinical spectrum analyzer and zero crossing interval histogram with respect to temporal resolution and frequency range. The DSPW system has a time resolution of 0.1 ms and 100 ms sweep speed, both about 10 times better than the best clinical Doppler systems used in mice. The small foot prints of the 10- and 20-MHz Doppler probes allow us to position the ultrasound beam almost parallel to the flow. This kind of positioning in not possible with clinical probes and small errors in angle corrections (in the range of 30 –90 ) may cause significant errors in the estimations of peak velocities. In disturbed flows it is important to have the ultrasound beam point to the direction of main flow in order to measure the Fig. 14. Developmental changes in the flow velocity signals at the mitral and tricuspid orifices starting from embryonic (14.5 day of gestation) to adult (12 weeks). The waveform in each cardiac cycle has two peaks. The first peak is the early (E) flow and the second peak is the atrial (A) flow. Except for the embryonic stage the direction of flow was toward the tip of the probe. The velocity signals were obtained using a 20-MHz pulsed Doppler probe and processed by DSPW. Reproduced from Zhou et al., Am. J. Physiol. 285:H1563–H1575, 2003, with permission from American Physiological Society. maximum velocities. With our 20-MHz probe and the DSPW system (0–125 kHz frequency response), we were able to obtain high-resolution stenotic jet velocity signals with peaks greater than 4 m/s in mice. In clinical systems where the frequency range of pulsed Doppler is limited continuous wave Doppler is used to measure high-velocity signals. However, we can use pulsed Doppler at very high frequencies in mice which cannot be done even in humans with clinical Doppler systems. Detection of vortex shedding frequencies in the flow distal to the aortic stenosis in mice by our 20-MHz esophageal probe and the ability of the DSPW to process and display such signals demonstrate that the performance of our system is much better than that of the clinical systems used in mice. REDDY et al.: PULSED DOPPLER SIGNAL PROCESSING FOR USE IN MICE: APPLICATIONS ACKNOWLEDGMENT The authors wish to acknowledge the assistance of L. N. Ochao, R. Kwun, M. Theresa, R. Hartley, A. Tumang on this project. They wish to thank Dr. Y.-Q. Zhou and Dr. L. Adamson of the University of Toronto, Toronto, ON, Canada, for providing them figures from their work. Finally, they would like to thank J. Brooks for editorial review. REFERENCES  J. F. James, T. E. Hewett, and J. Robbins, “Cardiac physiology in transgenic mice,” Circ. Res., vol. 82, pp. 407–415, 1998.  P. A. Doevendans, M. J. Daemen, E. D. de Muinck, and J. F. Smits, “Cardiovascular phenotyping in mice,” Cardiovasc. Res., vol. 39, pp. 34–49, 1998.  A. Nakamura, D. G. Rokosh, M. Paccanaro, R. R. Yee, P. C. Simpson, W. Grossman, and E. Foster, “LV systolic performance improves with development of hypertrophy after transverse aortic constriction in mice,” Am. J. Physiol. Heart Circ. Physiol., vol. 281, pp. H1104–H1112, 2001.  C. S. Broberg, G. A. Pantely, B. J. Barber, G. K. Mack, K. Lee, T. Thigpen, L. E. Davis, D. Sahn, and A. R. Hohimer, “Validation of the myocardial performance index by echocardiography in mice: A noninvasive measure of left ventricular function,” J. Am. Soc. Echocardiogr., vol. 16, pp. 814–823, 2003.  A. Schaefer, G. Klein, B. Brand, P. Lippolt, H. Drexler, and G. P. Meyer, “Evaluation of left ventricular diastolic function by pulsed Doppler tissue imaging in mice,” J. Am. Soc. Echocardiogr., vol. 16, pp. 1144–1149, 2003.  C. J. Hartley, L. H. Michael, and M. L. Entman, “Noninvasive measurement of ascending aortic blood velocity in mice,” Am. J. Physiol. Heart Circ. Physiol., vol. 268, pp. H499–H505, 1995.  G. E. Taffet, C. J. Hartley, X. Wen, T. T. Pham, L. H. Michael, and M. L. Entman, “Noninvasive indexes of cardiac systolic and diastolic function in hyperthyroid and senescent mouse,” Am. J. Physiol. Heart Circ. Physiol., vol. 270, pp. H2204–H2209, 1996.  G. E. Taffet, T. T. Pham, and C. J. Hartley, “The age-associated alterations in late diastolic function in mice are improved by caloric restriction,” J. Gerontol. A. Biol. Sci. Med. Sci., vol. 52, pp. B285–B290, 1997.  C. J. Hartley, G. E. Taffet, A. K. Reddy, M. L. Entman, and L. H. Michael, “Noninvasive cardiovascular phenotyping in mice,” Inst. Lab. Anim. Res., vol. 43, pp. 147–158, 2002.  C. J. Hartley, G. E. Taffet, L. H. Michael, T. T. Pham, and M. L. Entman, “Noninvasive determination of pulse-wave velocity in mice,” Am. J. Physiol., vol. 273, pp. H494–H500, 1997.  A. K. Reddy, A. D. Jones, C. Martono, W. Caro, S. Madala, and C. J. Hartley, “Pulsed Doppler signal processing for use in mice: Design and evaluation,” IEEE Trans. Biomed. Eng., vol. 52, no. 10, pp. 1764–1770, Oct. 2005.  C. J. Hartley, “Resolution of frequency aliases in the ultrasonic pulsed Doppler velocimeters,” IEEE Trans. Biomed. Eng., vol. BME-28, pp. 69–75, 1981.  Y.-H. Li, A. K. Reddy, G. E. Taffet, L. H. Michael, M. L. Entman, and C. J. Hartley, “Doppler evaluation of peripheral vascular adaptations to transverse aortic banding in mice,” Ultrasound Med. Biol., vol. 29, pp. 1281–1289, 2003.  A. K. Reddy, Y.-H. Li, T. T. Pham, L. N. Ochoa, M. T. Treviño, C. J. Hartley, L. H. Michael, M. L. Entman, and G. E. Taffet, “Measurement of aortic input impedance in mice: Effect of age on aortic stiffness,” Am. J. Physiol. Heart Circ. Physiol., vol. 285, pp. H1464–H1470, 2003.  A. K. Reddy, G. E. Taffet, S. Madala, L. H. Michael, M. L. Entman, and C. J. Hartley, “Noninvasive blood pressure measurement in mice using pulsed Doppler ultrasound,” Ultrasound Med. Biol., vol. 29, pp. 379–385, 2003.  C. Tei, “New noninvasive index for combined systolic and diastolic ventricular function,” J. Cardiol., vol. 26, no. 2, pp. 135–136, 1995.  L. L. Huntsman, D. K. Stewart, S. R. Barnes, S. B. Franklin, J. S. Colocousis, and E. A. Hessel, “Noninvasive Doppler determination of cardiac output in man,” Circulation, vol. 67, pp. 593–604, 1983.  S. M. Gardiner, A. M. Compton, T. Bennet, and C. J. Hartley, “Can the pulsed Doppler technique measure changes in aortic blood flow in conscious rats?,” Am. J. Physiol., vol. 259, pp. H448–H456, 1990. 1781  M. I. M. Noble, D. Trenchard, and A. Guz, “Left ventricular ejection in conscious dogs: Measurement and significance of the maximum acceleration of blood from the left ventricle,” Circ. Res., vol. 19, pp. 139–147, 1966.  R. D. Latham, N. Westerhof, P. Sipkema, B. J. Rubal, P. Reuderink, and J. P. Murgo, “Regional wave travel and reflections along the human aorta: A study with six simultaneous micromanometric pressures,” Circulation, vol. 72, pp. 1257–1269, 1985.  J. Atkinson, P. Poitevin, J. Chillon, I. Lartaud, and B. Levy, “Vascular ca overload produced by vitamin D3 plus nicotine diminishes arterial distensibility in rats,” Am. J. Physiol. Heart Circ. Physiol., vol. 266, pp. H540–H547, 1994.  B. P. Cholley, S. G. Shroff, C. Korcarz, and R. M. Lang, “Aortic eleastic properties with transesophageal echocardiography with automated border detection: Validation according to regional differences between proximal and distal thoracic aorta,” J. Am. Soc. Echocardiogr., vol. 9, no. 4, pp. 539–548, 1996.  D. H. Evans and W. N. McDicken, “Blood flow,” in Doppler Ultrasound: Physics, Instrumentation, and Signal Processing, 2nd ed, D. H. Evans and W. N. McDicken, Eds. New York: Wiley, 2000, vol. 180, pp. 6–7.  C. J. Hartley, A. K. Reddy, S. Madala, B. Martin-McNulty, R. Vergona, M. E. Sullivan, M. Halks-Miller, G. E. Taffet, L. H. Michael, M. L. Entman, and Y.-X. Wang, “Hemodynamic changes in apolipoprotein E-knockout mice,” Am. J. Physiol. Heart Circ. Physiol., vol. 279, pp. H2326–H2334, 2000.  Y.-H. Li, A. K. Reddy, L. N. Ochoa, T. T. Pham, C. J. Hartley, L. H. Michael, M. L. Entman, and G. E. Taffet, “Effect of age on peripheral vascular response to transverse aortic banding in mice,” J. Gerontol. A., vol. 58, pp. B895–B899, 2003.  A. Fard, C. Y. Wang, S. Takuma, H. A. Skopicki, D. J. Pinsky, M. R. Di Tullio, and S. Homma, “Noninvasive assessment and necropsy validation of changes in left ventricular mass in ascending aortic banded mice,” J. Am. Soc. Echocardiogr., vol. 13, pp. 582–587, 2000.  R. D. Patten, M. J. Aronovitz, P. Bridgman, and N. G. Pandian, “Use of pulse wave and color flow Doppler echocardiography in mouse models of human disease,” J. Am. Soc. Echocardiogr., vol. 15, pp. 708–714, 2002.  R. C. Fentzke, C. E. Korcarz, S. G. Shroff, H. Lin, J. Sandelski, J. M. Leiden, and R. M. Lang, “Evaluation of ventricular and arterial hemodynamics in anesthetized closed-chest mice,” J. Am. Soc. Echocardiogr., vol. 10, pp. 915–925, 1997.  M. Berger, R. L. Berdorf, P. E. Galterstein, and E. Goldberg, “Evaluation of aortic stenosis by continuous wave Doppler ultrasound,” J. Am. Coll. Cardiol., vol. 3, pp. 150–156, 1984.  L. Hatle, B. A. Angelsen, and A. Tromsdal, “Non-invasive assessment of aortic stenosis by Doppler ultrasound,” Br. Heart J., vol. 43, pp. 284–292, 1980.  M. D. Smith, P. L. Dawson, J. L. Elion, T. Wisenbaugh, O. L. Kwan, S. Handshoe, and A. N. DeMaria, “Systemic correlation of continuous wave Doppler and hemodynamic measurements in patients with aortic stenosis,” Am. Heart J., vol. 111, pp. 245–252, 1986.  R. G. Gosling and D. H. King, “Processing arterial Doppler signals for clinical data,” in Handbook of Clinical Ultrasound, M. deVlieger, J. H. Holmes, A. Kratochwill, E. Kazner, R. Kraus, G. Kossoff, J. Poujol, and D. E. Strandness, Eds. New York: Wiley, 1978, pp. 613–646.  L. A. Schildmeyer, R. Braun, G. E. Taffet, M. De Biasi, A. E. Burns, A. Bradley, and R. J. Schwartz, “Impaired vascular contractility and blood pressure homeostasis in the smooth muscle -actin null mouse,” FASEB J., vol. 14, pp. 2213–2220, 2000.  L. H. Michael, C. M. Ballantyne, J. P. Zachariah, K. E. Gould, J. S. Pocius, G. E. Taffet, C. J. Hartley, T. T. Pham, S. L. Daniel, E. Funk, and M. L. Entman, “Myocardial infarction and remodeling in mice: Effect of reperfusion,” Am. J. Physiol. Heart Circ. Physiol., vol. 277, pp. H660–H668, 1999.  W. W. Nichols and M. F. O’Rourke, “Vascular impedance,” in McDonald’s Blood Flow in Arteries: Theoretical, Experimental, and Clinical Principles, W. W. Nichols and M. F. O’Rourke, Eds. London, UK: Edward Arnold, 1998, pp. 281–282.  W. W. Nichols, M. F. O’Rourke, A. P. Avolio, T. Yaginuma, J. P. Murgo, C. J. Pepine, and C. R. Conti, “Effects of age on ventricular-vascular coupling,” Am. J. Cardiol., vol. 55, pp. 1179–1184, 1985.  Y.-Q. Zhou, F. S. Foster, R. Parkes, and S. L. Adamson, “Developmental changes in left and right ventricular diastolic filling patterns,” Am. J. Physiol. Heart Circ. Physiol., vol. 285, pp. H1563–H1575, 2003. 1782 IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 52, NO. 10, OCTOBER 2005 Anilkumar K. Reddy (S’93–M’97) was born in Kalwakole, Andhra Pradesh, India, in 1963. He received the B.E. degree in electronics and communication engineering from Osmania University (Chaitanya Bharathi Institute of Technology), Hyderabad, India, in 1985, the M.S. degree in biomedical engineering from The University of Akron, Akron, OH, in 1991, and the Ph.D. degree in bioengineering from Texas A&M University, College Station, in 1996. From 1997 to 2001, he was a Postdoctoral Fellow in the Section of Cardiovascular Sciences in the Department of Medicine, Baylor College of Medicine, Houston, TX, as a Postdoctoral Associate, and worked on the development of ultrasound instrumentation for small animal research. Since 2001, has been a member of the faculty and is currently Assistant Professor of Medicine. His past experience includes working as a Teaching and a Research Assistant, an Instructor, and a Research/Design Engineer. His interests include development of instrumentation for use in mice and the study the cardiovascular system of normal, disease, and transgenic models of mice. His past work involved analysis of physiological signals. Dr. Reddy is a member of American Physiological Society, Houston Society for Engineering in Medicine and Biology, and since 2002 has served on a National Institutes of Health (NIH) study section that reviews Small Business Grants. He is an ad hoc reviewer for IEEE TBME, ABME, and AJP journals. He was an invited speaker at the 3rd International Congress on Cardiovascular Disease in Taiwan in 2004. He is a recipient of a Research Career Development Award (2005–10) from NIH. Yi-Heng Li was born in Taipei, Taiwan, in 1963. He received the M.D. degree from the Department of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, in 1988, and the Ph.D. degree from the Institute of Basic Medical Science, National Cheng Kung University (NCKU), Tainan, Taiwan, in 2000. He is currently an Associate Professor in the Cardiology Division, Department of Internal Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan. His past work included Military Service (Taiwan), Resident in Internal Medicine, Fellow and Chief Resident, Attending Physician, Lecturer, and Visiting Postdoctoral Fellow. His research interests are atherosclerosis and related vascular diseases. Dr. Li is a member of Formosan Medical Association and Societies of Internal Medicine, Cardiology, Ultrasound in Medicine, and Critical Care & Emergency Medicine in Taiwan. He has received Young Investigator Award (Taiwan Soc. of Cardiology), Best Paper of the Year Award (NCKU), First Prize of the Special Grant Excellent Research (The 64th Annual Scientific Meeting of the Japanese Circ. Soc.), and Medical Research Award (Taiwan Soc. of Vascular Disease & Atherosclerosis). Sang-Wook Lim was born in Kwangju Ci, Jeon Nam Do, South Korea, in 1963. He received the M. D. degree from the College of Medicine, Yonsei University, Seoul, South Korea, in 1988 and the Ph.D. degree from the department of Pharmacology, the College of Medicine, Korea University, Seoul, in 1997. He is currently an Associate Professor in the Department of Cardiology, College of Medicine, Pochon CHA University, Bundang-Gu, South Korea, where he is in charge of patient care and cardiac catheterization laboratory. His research interests include therapeutic and diagnostic strategies for advanced heart failure. He authored and co-authored many articles regarding ischemic heart disease and heart failure that were published in Korean journals. Dr. Lim is a member of Korean Circulation Society and a member of Korea Society of Internal Medicine. George E. Taffet received the B.A. degree in mathematics and M.D. degrees from Brown University, Providence, RI, in 1979 and 1982, respectively. From 1982–86 he did his Internship and Residency in Internal Medicine at Baylor College of Medicine, Houston, TX. He was a Clinical Fellow in Geriatric Medicine and then a Research Fellow in Cardiovascular Sciences. Since 1988, he has been a faculty and is currently Associate Professor of Medicine in the Sections of Cardiovascular Sciences and Geriatrics in the Department of Medicine at Baylor College of Medicine. He is an Attending Physician at Houston VA Medical Center. He is Chief of Geriatrics at Baylor College of Medicine and a faculty associate of the Huffington Center on Aging. He is principle investigator on several research grants focused on aging and the cardiovascular system. Dr. Taffet is a member of American Geriatric Society, International Society of Heart Research; Gerontological Society of America; Texas Geriatrics Society (Board Member); American Academy of the Advancement of Science, American Heart Association—Basic Science Council, and Association of Directors of Geriatric Academic Programs. He has been an Expert Panel Member of Heart Failure with Diastolic Dysfunction and Aging in Disparate Species at National Institute on Aging and of Adaptation to Space/Physiological Factors Associated with Normal Aging at NASA. He is a member of the National Institutes of Health (NIH) GRM study section, Ad Hoc Reviewer for National Institute for Nursing Research, USDA, VA Research Service, and AHPCR Guidelines Cardiac Rehabilitation, and Geriatric and Cardiology Spokesman for John A. Hartford Foundation. Thuy T. Pham had received the B.S. degree in biology from the University of Houston, Houston, TX, in 1990. She has been with Baylor College of Medicine, Houston, since 1990 and is currently a Research Technician in the Section of Cardiovascular Sciences in the Department of Medicine. Her expertise includes Murine Ultrasound Sonography and data analysis. Jennifer Pocius received the B.S. degree in Biology from University of Houston, Houston, TX, in 1974. From 1974 to 1975, she worked as a Laboratory Technician at University of Texas and has been at Baylor College of Medicine, Houston, since 1975. and is currently Research Associate in the Section of Cardiovascular Sciences in the Department of Medicine. Her expertise is in mouse surgery and routinely performs coronary LAD occlusion surgeries in mice. In addition, she is also in charge of cardiovascular murine laboratory. Ms. Pocius received an Achievement Award from VA Medical Center in 1994. REDDY et al.: PULSED DOPPLER SIGNAL PROCESSING FOR USE IN MICE: APPLICATIONS Mark L. Entman pursued his Pre-med education at University of Florida, Gainesville, and received the M.D. degree in Medicine from Duke University Medical School, Durham, NC, in 1963. He was an Intern at Johns Hopkins University (Osler Service), Baltimore, MD, from 1963–1964. From 1964–1968, he was a Postdoctoral Fellow and Resident in Cardiology and Internal Medicine. He has been with Baylor College of Medicine, Houston, TX, since 1970 and is currently Professor and Head, Section of Cardiovascular Science, Department of Medicine and Scientific Director of DeBakey Heart Center and the William J. Osher Professor of Cardiovascular Research in 2003. His research interests include role of inflammation in cardiac injury and repair and identification of potential therapeutic targets. The work is done primarily in altered mice. Dr. Entman is a fellow of American College of Cardiology, and a member of American Society of Clinical Investigation and Association of American Physicians. He is a past recipient of American College of Cardiology Young Investigator Award, the Award for Outstanding Research-International Society of Heart Research, The Roussell Award for Cardiology Research, and an NHLBI research MERIT award. He is also a past recipient of the Duke University Medical Center Distinguished Alumnus award. Lloyd H. Michael received the A.A. degree from Keystone College, La Plume, PA, in 1962, the B.S. degree in biology from Moravian College, Bethlehem, PA, in 1964, the M.S. degree in physiology from Kent State University, Kent, OH, in 1966, and the Ph.D. degree in physiology from the University of Ottawa School of Medicine, Ottawa, ON, Canada, in 1973. He completed Postdoctoral Fellowships in myocardial biology at Baylor College of Medicine, Houston, TX, and in clinical immunology at Columbia College of Physicians and Surgeons, New York, NY. He has been with Baylor College of Medicine since 1976 and is Professor of Medicine in the Department of Medicine, Section of Cardiovascular Sciences, Director of the DeBakey Heart Center Core Animal laboratories and Senior Associate Dean of Medical College Admissions. His current research areas include myocardial ischemia and animal models to investigate the role of inflammation, leukocytes, and complement in myocardial damage; the mechanics of cardiac muscle contraction. He is an editorial board member for the American Journal of Physiology, a council member for AAALAC Int’l, and a National Institutes of Health (NIH) study section peer reviewer. 1783 Craig J. Hartley (S’64–M’66–SM’88) received the B.S.E.E. and Ph.D. degrees from the University of Washington, Seattle, in 1966 and 1970, respectively. From 1970 to 1973, he was a Postdoctoral Fellow in Bioengineering at Rice University, Houston, TX. Since 1973, he has been with Baylor College of Medicine, Houston, where he is currently a Professor of Medicine in the section of Cardiovascular Sciences. He is also an adjunct Professor of Bioengineering at Rice University and Professor of Electrical Engineering at the University of Houston. Since 1968, he has been active in the development of ultrasonic methods to measure blood flow and cardiovascular function in man and in animal models of human diseases. He is principle investigator on several research grants and has received a Research Career Development Award and a MERIT award from the National Institutes of Health (NIH). Dr. Hartley is a member of the American Institute of Ultrasound in Medicine, the American Physiological Society, the Cardiovascular Systems Dynamic Society, and since 1993 has served on an NIH study section that reviews Small Business Grants. In 1993, he received the Laufman Prize for career achievement from the Association for the Advancement of Medical Instrumentation, and in 1998 became an AIMBE fellow. He is the regional representative to the EMBS administrative committee, was treasurer of the EMBS-BMES 2002 Joint Conference in Houston, and is currently chair of the EMBS Awards Committee.