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 Measurements for Longitudinal Noninvasive 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 and Echocardiography
During the last several years, we have been active in developing 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].
Echocardiographic Assessment of Mouse Cardiovascular Function
We have been using an VisualSonics Vevo 700 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 has recently upgraded our capabilities with the purchase of a VisualSonics Vevo-770 that is in place since 2007. Dr. Hartley has worked with the manufacturers to allow us to interface the instrument with our future technologies.
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.
We have had great success analyzing infarct size, cardiac repair and remodeling post myocardial infarction utilizing a serial section technique that allows quantitation of ventricular expansion and hypertrophy. This method has allowed us to describe important differences in response to cardiac injury relating to cardiac age [Bujak and Frangogiannis, 2007; Gould et al, 2002] and to the presence or absence of reperfusion [Michael et al, 1999]. In addition, we use techniques in which we measure cardiac function in isolated perfused hearts taken at specific times. This technique allows measurement of global cardiac function. It is feasible to utilize ischemic and reperfused hearts in this protocol. Specific methods follow below.
Terminal Analysis of Cardiac Function and Structure Anatomical Methods
The following sections outline the methods used to assess anatomic changes in the mouse heart associated with tissue repair and adaptation to injury as described in our recent publications.
Infarct Size Measurements
After the desired reperfusion time (5h to several days), hearts are removed and area at risk and infarct size measured. Briefly, hearts are removed at euthanasia by intravenous injection of a cardioplegic solution ((in g/L) 4.0 NaCl, 5.5 KCl, 1.0 NaHCO3, 2.0 glucose, 3.0 2,3-butanedione monoxime, 3.8 ethylene glycol-bis(b-aminoethyl) - N,N,N’,N’-tetraacetic acid (EGTA) and 0.0002 nifedipine). The aorta is cannulated with a 22 gauge Luer stub and 50ul of one percent Evans blue perfused into the aorta and coronary arteries with distribution throughout the ventricular wall proximal to the site of coronary artery ligation. The transverse section cutter used in these studies has blades spaced 1000 μm apart, therefore each heart section between the blades has section thickness of 1000 μm whereas the apical tip and base may vary outside these thicknesses since the first cutter blade is positioned at the site of coronary artery occlusion. In a typical 12 week old mouse this cutting gives 3 equal 1000 μm thick pieces, an approximately 2000 μm thick basal section and an apical section generally less than 1000 μm thick.
After staining with 1.5 percent triphenyl tetrazolium chloride and image analysis, the infarction is determined by the following equations: weight of infarction = (A1 x WT1) + (A2 x WT2) + (A3 x WT3) + (A4 x WT4) + (A5 x WT5), where A is percent area of infarction by planimetry from subscripted numbers 1-5 representing sections, and WT is weight of the same numbered sections. Percentage of infarcted LV is (WT of infarction/WT of LV) x 100. Area at risk is calculated as percentage of LV (WT of LV - WT of LV stained blue/WT of LV). The weight of LV stained blue is calculated in a similar fashion by sum of products of the percent area of each slice x the weight of the respective slice.
Perfusion Fixation and Histology
For assessment of changes in cardiac structure cardioplegic solution is perfused through the jugular vein to promote quiescence and relaxation. After excising and rinsing in cold cardioplegic solution, the aorta is cannulated with a 22 gauge blunt needle and the left atrial appendage with a polyethylene 50 catheter pushed across the mitral valve into the left ventricle and secured in place. Hearts are fixed for 10 minutes by aortic perfusion of 10 percent zinc buffered formalin with the left ventricular drain held vertically 16 cm above the base of the heart to maintain constant left ventricular pressure at approximately 16 cm H2O. After removing the cannulae, fixation by immersion is continued for two hours after which the hearts are dehydrated, cleared and embedded in paraffin. The entire heart is cross-sectioned into 5 mm sections from base to apex [Gould et al, 2002; Hartley et al, 2002].
Analysis of Ventricular Mass and Scar Formation
The mass of the left ventricle and septum is obtained by integration of a curve created by plotting the LV area measured in each cross-section against the distance of the cross-section from the base of the heart. The basal cross-section used as the starting point in each heart is identified as the last cross-section containing any part of the aortic valve and every 50th cross-section (intervals of 5mm x 50 = 250mm) is analyzed.
The sum of the areas in each of the partitions is obtained by integration of this curve carried out by dividing the interval of integration (x-axis) into 250mm partitions representing each of the cross-sections, beginning at the first basal cross-section and ending at the last apical cross-section. The sum is then used as an estimate of total ventricular space occupied by the myocardium in each heart. Left ventricular myocardial mass is derived from this space estimate by multiplying by commonly assumed muscle tissue density of 1.065 mg/ml. The mass of the interventricular septum is separately analyzed utilizing identical methods. In the latter case, the LV septum is described in the first basal section and continues through the last section to show evidence of the right ventricular chamber. The edges of the septum are defined by the approximate sites of the anterior and posterior interventricular (longitudinal) sulci on each cross section. The actual line of demarcation is arbitrarily drawn on each section with consideration of the sites of these sulci and with the understanding of a slight rightward (toward R. ventricle) convexity of the interventricular septum. The remaining sections are available for serial histologic or histochemical analysis in the project protocols.
Computation of Ventricular Expansion Ratio and Histopathologic Sampling
Computation of Ventricular Expansion Ratio [Michael et al, 1999]
In each cardiac cross-section, a thin segment is identified in the LV free wall. The thicknesses of these segments is characteristically less than 40 percent of the remaining ventricular free wall in the cross-section. The thicknesses of thin segments varied less than 10 percent when compared in all of the cross-sections from a single heart. The length of the endocardial surface underlying each thin segment is measured as well as the length of the total endocardial surface in the cross-section. Thin segment length and total endocardial surface length are plotted against the distance of the cross-section from the base of the heart. Each plot is integrated by dividing the interval of integration (x-axis) into 250m partitions representing the distance between the cross-sections taken for analysis, beginning at the first basal cross-section and ending at the last apical cross-section, and obtaining the sum of the areas in each of the partitions. The sums are used as estimates for the thin segment area and for the total endocardial surface area in the LV of each heart. The ventricular expansion ratio is calculated by dividing thin segment endocardial area by total endocardial area and multiplying by 100. Intervening sections are stained for pertinent histopathologic assessment.
Mouse Cardiac Catheterization
Mice will be anesthetized intraperitoneally with a mixture of ketamine (100 mg/kg), xylazine (2.5 mg/kg), and heparin (5,000 U/kg); additional doses will be given as needed. LV function will be assessed using a 1.4-Fr micro-tipped Millar catheter, as described [Nemoto et al, 2002a; Nemoto et al, 2002b]. The right carotid artery will be dissected using a dissecting microscope (SZ40, Olympus Inc., Tokyo, Japan), and cannulated with a 1.4-Fr micro-tipped Millar (Millar Instruments Inc., Houston, Texas). The catheter will be advanced into the LV under echocardiographic guidance. The 1.4-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 hearts will be paced from the atrium to a heart rate (HRmax) at which dP/dt is maximal, as defined by the force frequency curves for each animal (Figure 5). Atrial pacing will be achieved by advancing a 1-Fr bipolar mouse pacing catheter (EP118-2, NuMED, Inc., Hopkinton, NY) into the right atrium. Atrial pacing will be established using a stimulator (SD9E, Grass Medical Instrument, Quincy, MA).
We recently purchased catheters to measure left ventricular pressure and volume in mice from both Millar Instruments and from Scisense. We are currently evaluating the characteristics of these two methods along with fluid-filled catheters and RADI pressure wires for use in mice. We now have the capability of measuring left ventricular pressure-volume loops as an alternative method to assess ventricular mechanics and cardiac function in mice as shown in Figure 6. In addition, the VisualSonics Vevo-770 ultrasonic imaging system allows us to record LV pressure along with cardiac M-mode and ECG. We can then use the M-mode-derived chamber dimension signal to estimate an LV volume signal and generate pressure-volume loops. Thus, we will have several options for acquiring pressure-volume signals from mice.