Sridhar Madala

Doppler estimation of reduced coronary flow reserve in mice with pressure overload cardiac hypertrophy

Aortic banding produces pressure overload cardiac hypertrophy in mice, leading to decompensated heart failure in four to eight weeks, but the effects on coronary blood flow velocity and reserve are unknown. To determine whether coronary flow reserve (CFR) was reduced, we used noninvasive 20-MHz Doppler ultrasound to measure left main coronary flow velocity at baseline (B) and at hyperemia (H) induced by low (1%) and high (2.5%) concentrations of isoflurane gas anesthesia. Ten mice were studied before (Pre) and at 1 d, 7 d, 14 d and 21 d after constricting the aortic arch to 0.4 mm diameter distal to the innominate artery. We also measured cardiac inflow and outflow velocities at the mitral and aortic valves and velocity at the jet distal to the aortic constriction. The pressure drop as estimated by 4V2 at the jet was 51 +/- 5.1 (mean +/- SE) mm Hg at 1 d, increasing progressively to 74 +/- 5.2 mm Hg at 21 d. Aortic and mitral blood velocities were not significantly different after banding (p = NS), but CFR, as estimated by H/B, dropped progressively from 3.2 +/- 0.3 before banding to 2.2 +/- 0.4, 1.7 +/- 0.3, 1.4 +/- 0.2 and 1.1 +/- 0.1 at 1 d, 7 d, 14 d and 21 d, respectively (all p < 0.01 vs. Pre). There was also a significant and progressive increase the systolic/diastolic velocity ratio (0.17 Pre to 0.92 at 21 d, all p < 0.01 vs. Pre) suggesting a redistribution of perfusion from subendocardium to subepicardium. We show for the first time that CFR, as estimated by the hyperemic response to isoflurane and measured by Doppler ultrasound, can be measured serially in mice and conclude that CFR is virtually eliminated in banded mice after 21 d of remodeling and hypertrophy. These results demonstrate that CFR is reduced in mice as in humans with cardiac disease but before the onset of decompensated heart failure.

Noninvasive blood pressure measurement in mice using pulsed Doppler ultrasound.

Existing tail-cuff pressure devices for mice use tail flow sensors that measure only systolic and mean pressure. We developed a method to obtain systolic and diastolic pressure in mice using a pulsed Doppler flow velocity sensor and a tail-cuff and validated the method against pressure signals obtained simultaneously from a fluid-filled catheter. The tail-cuff was pressurized to suprasystolic levels to completely occlude the tail artery and then released gradually. The pressure at which the tail flow reappeared was recorded as systolic and the pressure at which the tail flow became continuous was recorded as diastolic. Regression analysis of tail-cuff pressures over catheter pressures obtained from healthy mice (n = 16) showed a high degree of association (r(sys) = 0.95, r(dia) = 0.94, both at p < 0.001). Bland-Altman analysis showed good agreement between the two methods, with a mean difference of -13 ( +/- 12 SD) mmHg and 3 ( +/- 10 SD) mmHg in the systolic (58 to 250 mmHg) and diastolic (48 to 178 mmHg) pressure measurements, respectively. Bland-Altman plots of tail-cuff blood pressures of a second group of mice (n = 20) showed good agreement between repeated measurements obtained on the same day, but had higher variability between measurements made on different days.

Doppler velocity measurements from large and small arteries of mice

With the growth of genetic engineering, mice have become increasingly common as models of human diseases, and this has stimulated the development of techniques to assess the murine cardiovascular system. Our group has developed nonimaging and dedicated Doppler techniques for measuring blood velocity in the large and small peripheral arteries of anesthetized mice. We translated technology originally designed for human vessels for use in smaller mouse vessels at higher heart rates by using higher ultrasonic frequencies, smaller transducers, and higher-speed signal processing. With these methods one can measure cardiac filling and ejection velocities, velocity pulse arrival times for determining pulse wave velocity, peripheral blood velocity and vessel wall motion waveforms, jet velocities for the calculation of the pressure drop across stenoses, and left main coronary velocity for the estimation of coronary flow reserve. These noninvasive methods are convenient and easy to apply, but care must be taken in interpreting measurements due to Doppler sample volume size and angle of incidence. Doppler methods have been used to characterize and evaluate numerous cardiovascular phenotypes in mice and have been particularly useful in evaluating the cardiac and vascular remodeling that occur following transverse aortic constriction. Although duplex ultrasonic echo-Doppler instruments are being applied to mice, dedicated Doppler systems are more suitable for some applications. The magnitudes and waveforms of blood velocities from both cardiac and peripheral sites are similar in mice and humans, such that much of what is learned using Doppler technology in mice may be translated back to humans.

Pulsed Doppler signal processing for use in mice: design and evaluation

We have developed and evaluated a high-frequency, real-time pulsed Doppler and physiological signal acquisition and analysis system specifically for use in mice. The system was designed to provide sampling rates up to 125 kilosamples/s (ksps) with software controlled data acquisition and analysis in real-time. Complex fast Fourier transforms are performed every 0.1 ms (or longer up to 10 ms) to provide 0.1-ms time resolution and using 64-1024 sample segments of the Doppler audio signals resulting in frequency resolution ranging from 122-1953 Hz. The system was evaluated by its response to frequency swept signals with slopes (accelerations) and magnitudes (velocities) comparable to actual blood velocity signals in mice. Signals up to a maximum frequency of 125 kHz and a maximum acceleration of 20 MHz/s were processed and displayed. This corresponds to a maximum velocity of 480 (960) cm/s and a maximum acceleration of 750 (1500) m/s/sup 2/ when Doppler shifts are measured with a 20- (10-) MHz probe, thereby allowing us to measure high stenotic jet velocities. The directional transitions of the spectrogram across zero frequency and across Nyquist frequency (sampling rate/2) were smooth with no discernible artifacts. Signals with period as low as 2 ms were processed and displayed at sweep speed that is ten times that in clinical Doppler systems, so that measurements of small temporal events can be made with precision. Thus, the new system can measure higher blood velocities with higher spatial and temporal resolution than is possible using clinical Doppler systems adapted for use in mice.

Multichannel pulsed Doppler signal processing for vascular measurements in mice.

The small size, high heart rate and small tissue displacement of a mouse require small sensors that are capable of high spatial and temporal tissue displacement resolutions and multichannel data acquisition systems with high sampling rates for simultaneous measurement of high fidelity signals. We developed and evaluated an ultrasound-based mouse vascular research system (MVRS) that can be used to characterize vascular physiology in normal, transgenic, surgically altered and disease models of mice. The system consists of multiple 10/20MHz ultrasound transducers, analog electronics for Doppler displacement and velocity measurement, signal acquisition and processing electronics and personal computer based software for real-time and off-line analysis. In vitro testing of the system showed that it is capable of measuring tissue displacement as low as 0.1mum and tissue velocity (mum/s) starting from 0. The system can measure blood velocities up to 9m/s (with 10MHz Doppler at a PRF of 125kHz) and has a temporal resolution of 0.1 milliseconds. Ex vivo tracking of an excised mouse carotid artery wall using our Doppler technique and a video pixel tracking technique showed high correlation (R(2)=0.99). The system can be used to measure diameter changes, augmentation index, impedance spectra, pulse wave velocity, characteristic impedance, forward and backward waves, reflection coefficients, coronary flow reserve and cardiac motion in murine models. The system will facilitate the study of mouse vascular mechanics and arterial abnormalities resulting in significant impact on the evaluation and screening of vascular disease in mice.

Noninvasive ultrasonic measurement of arterial wall motion in mice

To facilitate assessment of arterial function, we developed a noninvasive Doppler method for measuring vessel motion in genetically altered mice. A 20 MHz probe was held by an alligator clip and positioned over the carotid arteries of 16 mice including six 3 to 5-month old wild-type (WT), four 30-month old senescent (Old), two apolipoprotein-E null (ApoE), and four alpha smooth muscle actin null (αSMA) mice. Doppler signals were obtained simultaneously from both vessel walls and from blood flow using one or two probes. The displacement signals from the near and far walls were subtracted to generate a diameter signal from which the excursion and an augmentation index were calculated. The excursion ranged between 13 μm (in ApoE) and 95 μm (in αSMA). The augmentation index was lowest in the WT mice (0.06) and highest in the Old mice (0.29). This noninvasive method is able to identify and confirm characteristic changes in arterial properties associated with age, atherosclerosis, and the absence of vascular tone.

32-Channel System to Measure the Electrophysiological Properties of Bioengineered Cardiac Muscle

The purpose of this study was to develop, assess, and validate a custom 32-channel system to analyze the electrical properties of 3-D artificial heart muscle (3D-AHM). In this study, neonatal rat cardiac cells were cultured in a fibrin gel to drive the formation of 3D-AHM. Once the tissues were fully formed, the customized electrocardiogram (EKG) sensing system was used to obtain the different electrophysiological characteristics of the muscle constructs. Additionally, this system was used to evaluate the electrical properties of native rat hearts, for comparison to the fabricated tissues and native values found in the literature. Histological evaluation showed extensive cellularization and cardiac tissue formation. EKG data analysis yielded time delays between the signals ranging from 0 to 7 ms. Optical maps exhibited slight trends in impulse propagation throughout the fabricated tissue. Conduction velocities were calculated longitudinally at 277.81 cm/s, transversely at 300.79 cm/s, and diagonally at 285.68 cm/s for 3D-AHM. The QRS complex exhibited an R-wave amplitude of 438.42 ± 36.96 μV and an average duration of 317.5 ± 16.5 ms for the tissue constructs. The data collected in this study provide a clearer picture about the intrinsic properties of the 3D-AHM while proving our systems efficacy for EKG data procurement. To achieve a viable and permanent solution, the bioengineered heart muscle must physiologically resemble native heart tissue as well as mimic its electrical properties for proper contractile function. This study allows us to monitor such properties and assess the necessary changes that will improve construct development and function.

Characterization of Arterial Wave Propagation and Reflection in Mice

Wave propagation through the arterial system changes with age and disease state, and mutant mice are often used to study these conditions. We have developed several noninvasive ultrasonic techniques to measure blood velocity and vessel wall motion from which we can calculate aortic pulse wave velocity (PWV), local compliance, impedance spectra, characteristic impedance (Zc), augmentation index (AI), and forward and backward waves in intact anesthetized mice. We found altered vascular mechanics in many mutant strains of mice. In old mice PWV, AI, and Zc are increased. In atherosclerotic mice PWV, Zc, and AI are increased; peripheral resistance and arterial compliance are decreased; and wave reflections are enhanced. We find that the initial deceleration of carotid velocity is caused by peripheral reflections, and that increased acceleration of velocity in the aortic arch in atherosclerotic mice is caused by enhanced carotid reflections returning to the heart and traveling forward in the aorta. We conclude that when scaled for heart period, the mouse arterial system and its responses to age and disease are similar to those in man. The ability to evaluate arterial mechanics in mice will expand their use as models to study human arterial diseases and conditions

Feasibility of dual Doppler velocity measurements to estimate volume pulsations of an arterial segment.

If volume flow was measured at each end of an arterial segment with no branches, any instantaneous differences would indicate that volume was increasing or decreasing transiently within the segment. This concept could provide an alternative method to assess the mechanical properties or distensibility of an artery noninvasively using ultrasound. The goal of this study was to determine the feasibility of using Doppler measurements of pulsatile velocity (opposed to flow) at two sites to estimate the volume pulsations of the intervening arterial segment. To test the concept over a wide range of dimensions, we made simultaneous measurements of velocity in a short 5 mm segment of a mouse common carotid artery and in a longer 20 cm segment of a human brachial-radial artery using a two-channel 20 MHz pulsed Doppler and calculated the waveforms and magnitudes of the volume pulsations during the cardiac cycle. We also estimated pulse wave velocity from the velocity upstroke arrival times and measured artery wall motion using tissue Doppler methods for comparison of magnitudes and waveforms. Volume pulsations estimated from Doppler velocity measurements were 16% for the mouse carotid artery and 4% for the human brachial artery. These values are consistent with the measured pulse wave velocities of 4.2 m/s and 10 m/s, respectively, and with the mouse carotid diameter pulsation. In addition, the segmental volume waveforms resemble diameter and pressure waveforms as expected. We conclude that with proper application and further validation, dual Doppler velocity measurements can be used to estimate the magnitude and waveform of volume pulsations of an arterial segment and to provide an alternative noninvasive index of arterial mechanical properties.

Hemodynamics of atherosclerotic mice

Apolipoprotein-E knockout mice are often used as models of human atherosclerosis, but little is known of the hemodynamic consequences. Using noninvasive Doppler methods, it was found that heart rate and blood pressure were normal but that aortic pulse wave velocity, mean aortic and mitral blood velocities, and peripheral wave reflections were increased. It was also demonstrated that heart weight/body weight ratio was increased, and that hematocrit was decreased. The changes are consistent with decreased peripheral vascular resistance and compliance and with volume overload cardiac hypertrophy.