Our laboratory has developed a technique of serially monitoring systolic and diastolic function of the left ventricle utilizing pulsed Doppler studies that have allowed us to distinguish changes involved with cardiac injury [Michael et al, 1999], inotropic treatment [Hartley et al, 1997], and aging [Gould et al, 2002]. In addition to the Doppler studies, echocardiographic assessment of mouse cardiovascular function utilizing echocardiography can be serially followed [Dewald et al, 2003; Hartley et al, 2002]. Finally, we have developed a method of calibrated aortic constriction [Hartley et al, 2002] to impose a consistent and defined stress on the circulation (see above); the ability of animals to withstand this stress is a additional function which can be followed serially. The power of serial measurement as a function of intervention has proven a powerful method of assessing treatment regimens.
Noninvasive Serial/Longitudinal Measurement of Cardiac Function
We have developed the instrumentation and techniques for measuring and quantifying ECG’s in mice. For standardization, an ECG amplifier was designed with extended frequency response and lead switching to generate any standard lead configuration. For measurements, mice are anesthetized and taped with electrode paste to a PC board which contains ECG lead pads under each limb. The board also contains a resistive heating element (surface-mount resistors) under the mouse which is connected to a custom-designed temperature controller to regulate and display the board and/or mouse temperatures. This general setup and board are used during surgery, Doppler monitoring, and echocardiographic imaging. VisualSonics uses our ECG board in their Vevo 770 mouse ultrasound system.
Doppler Flow Studies
Cardiac Flow Velocity Measurement - Cardiac Function
We have developed technology to allow noninvasive ultrasonic monitoring of blood flow velocity in the heart and peripheral vessels of anesthetized mice [Hartley et al, 1995; Hartley et al, 1997; Kurrelmeyer et al, 2000]. The system currently used was developed in collaboration with Indus Instruments, Houston, TX and consists of a modular Baylor ultrasonic mainframe with high-PRF 10 and 20 MHz pulsed Doppler modules, a mouse ECG amplifier with extended frequency response, a temperature monitoring and control module, a PC board with ECG electrodes and heater, several miniature pulsed Doppler probes, and an Indus Work Station for collection, storing, and analyzing ECG and Doppler signals from mice.
The system was designed with high spatial (0.1mm) and temporal (0.1 ms) resolution for monitoring blood flow velocity in small animals with high heart rates. The Indus analyzer was developed by Dr. Hartley for mice and performs complex FFT’s from the quadrature Doppler signals both in real-time for operator feedback during data acquisition and on stored signals for more detailed and higher resolution analysis. The system can measure velocities up to 5 m/s using a 10MHz probe with a 125 KHz sampling rate with temporal resolutions to 0.1 ms with full operator control of the FFT window and number of points (64-1024). The system is configured to detect the peak Doppler shift and to semi-automatically extract features such as peak and average velocities, slopes and accelerations, and areas under portions of the waveform.
Mice are anesthetized in a chamber with isoflurane gas and maintained by delivery through a nasal cone and taped to a temperature-controlled laminated plastic board with copper electrodes placed such that the 3 bipolar limb leads allow electrocardiographic monitoring. Body fur at the left lower sternal border is clipped and the skin wetted with warm electrode gel to improve sound transmission. Cardiac Doppler signals are normally acquired by placing a 10 MHz probe over the cardiac apex below the sternum and pointing the sound beam toward the LV inflow track to record mitral velocity signals or toward the LV outflow track to record aortic velocity signals. The pulsed Doppler range gate depth is set at 4 to 7 mm to obtain optimal signals from the LV inflow and outflow tracks substernally. Repeated measures are made from each animal to allow for observation at different heart rates and to ascertain the reproducibility of the measurements. For each study, 4-6 beats are analyzed. The pulsed Doppler instrument and probes are custom made in our laboratories [Hartley et al, 1995].
From these signals we simultaneously determine peak and mean aortic velocities and acceleration as indices of cardiac output by Doppler studies and LV systolic function, and mitral E and A velocities and their ratio E/A as indices of LV diastolic function [Taffet et al, 1996]. We found these indices to be altered in systematic ways in many of the disease models studied. For instance, in hyperthyroid mice, both systolic and diastolic indices were increased; in senescent mice, systolic indices were normal and diastolic indices were depressed. In myocardial coronary occlusions, permanently occluded mice had more depressed indices than those with reperfusion after occlusion [Michael et al, 1999].
Peripheral Vascular Measurements – Flow Velocity and Vessel Stiffness
Some of the mouse models we study have alterations in peripheral vascular function, arterial compliance, vascular tone, vascular impedance, and regional blood flow. In order to characterize these models we have developed several noninvasive ultrasonic methods to assess blood flow velocity in many peripheral vessels including carotid and coronary [Hartley et al, 2002; Hartley et al, 2007] and the mechanical properties of the aorta and carotid arteries. These include methods to measure pulse wave velocity as an index of vascular stiffness [Hartley et al, 1997], the direct measurement of the diameter pulsations of vessel walls [Hartley et al, 2004], and the measurement of vascular impedance spectra [Reddy et al, 2003], and the measurement of coronary blood flow velocity and coronary flow reserve [Hartley et al, 2007; Hartley et al, 2008]. We have used these methods to characterize atherosclerotic mice [Hartley et al, 2000] and the peripheral vascular adaptations to aortic banding [Li et al, 2003] and aging [Reddy et al, 2003].
Coronary Flow Reserve
Through several recent studies, we have determined that defective angiogenesis is a potential source of intolerance of increased systolic cardiac load. We have developed a method of assessing this by measuring coronary flow reserve in a noninvasive way. Coronary flow is measured with a 20-MHz Doppler ultrasound probe. This probe has been designed so that we can easily assess coronary flow velocity in the main coronary artery of the mice. To induce maximum cardiac dilatation, the mouse is briefly exposed to high concentrations of isofluorane gas anesthesia (2.5 percent). The technique has already been used in several ongoing studies and is now a routine option for mice undergoing models of cardiac overload [Hartley et al, 2008].
Assessment of Mouse Cardiovascular Function
We use a VisualSonics Vevo 770 with a high frequency transducer to perform 2-D-directed M-mode echocardiography in mice. This instrument was purchased under a Shared Instrumentation Grant specifically for the mouse laboratory and is available full-time for research applications. The laboratory routinely uses this system to assess cardiac structure and function [Dewald et al, 2003; Kurrelmeyer et al, 2000; Medrano et al, 2016; Cieslik et al, 2011; Haudek et al, 2010].
Invasive Measurement of Cardiac Function
Mouse Cardiac Catheterization
Mice will be anesthetized with isoflourane. LV function will be assessed using a 1.0-Fr micro-tipped Millar catheter, as described in Reddy et al, 2007. The right carotid artery will be dissected using a dissecting microscope (SZ40, Olympus Inc., Tokyo, Japan), and cannulated with a 1.0-Fr micro-tipped Millar (Millar Instruments Inc., Houston, Texas). The 1.0-F high-fidelity micro-manometer catheter will be calibrated with a mercury manometer at the beginning of each experiment. Baseline zero reference will be obtained by placing the sensor in normal saline prior to insertion. LV pressure (LVP), HR, and the positive and negative first derivative of LV pressure with respect to time (+dP/dt max, -dP/dt max) will be determined. Given that heart rate modifies isovolumic indices of cardiac contractile performance, such as LV dP/dt, the animals will be given dobutamine or isoproterenol intraperitoneally to increase heart rate and contractility at which dP/dt is maximal, as defined by the force frequency curves for each animal.