As heart disease in western society and around the world becomes more conspicuous as a source of illness and death, so do genetically engineered mice designed to facilitate the study of the cardiovascular system and the effects of specific genes on its various failings. Methods to assess the cardiovascular system are varied in their complexity and versatility. Investigators should carefully select those testing techniques best suited to the specific genes and questions being addressed. In all cases, evaluating cardiovascular parameters at rest and under stress is an important aspect of phenotypic characterization, because many abnormalities will not be discovered until the cardiovascular system is pushed beyond normal metabolic constraints.
One of the easiest systems for tracking and evaluating the cardiovascular system is telemetric monitoring. Implantable telemetric transmitters are placed inside the mouse while under general anesthesia. Depending on the type, they can be placed subcutaneously, intraperitoneally, or in specific blood vessels such as the abdominal aorta or carotid artery. Telemetry can be used to monitor body temperature, blood pressure, heart rate, and heart electrical activity through constant EKG readings. The major advantage with these systems is in the fact that 5 days postimplantation, cardiovascular parameters can be measured in freely moving awake mice for an extended duration. Mice with telemetry transmitters can function in all ways like normal mice, even reproductively. Female mice with telemetry devices implanted in the thoracic aorta are able to conceive, gestate, deliver, and provide postnatal care for pups without interference by the transmitter and with constant cardiovascular monitoring throughout.23
Echocardiography in mice has been more widely used in recent years to accurately assess cardiac structure and function as well as measure cardiac volume and output. At this point, two-dimensionally directed M-mode echocardiography is believed to be the leading imaging method for small animal cardiovascular work. To adequately visualize the heart and surrounding structures, the required linear array, broadband transducer should operate at no less than 12 to 15 MHz. Newer scanners operating at higher frame rates per second allow high resolution real-time imaging in multiple planes, allowing for calculations of two-dimensional left ventricular volume and mass. Mice are most frequently anesthetized for echocardiography as imaging conscious mice is difficult and prone to error. The investigator should be aware of the changes to the various cardiovascular parameters induced by anesthesia and factor these differences into any results obtained through the use of this procedure. Murine echocardiography is still a new technology, and there are limitations in its capacity to phenotype genetically engineered mice, such as decreased ability to view the right heart chambers and required operator expertise.24
MRI, or magnetic resonance imaging, is another form of imaging that can be used to assess cardiac structure and function. As with echocardiography, mice undergoing an MRI exam should be anesthetized. MRI provides noninvasive, highly accurate three-dimensional imaging. Unlike echo, the right chambers can be readily visualized, and operator expertise is inconsequential. In addition, serial assessment of cardiac structure and function in all age groups is reliable and accurate and provides reproducible serial measurements of cardiac output. However, MRI is costly, resource-intensive, and has limited availability to many investigators.24
A noninvasive exercise stress test can be used to assess mitochondrial physiology and cardiac function. Mice are exercised on a modular variable speed and angle treadmill enclosed in an airtight chamber, so that a known composition and constant flow atmosphere can be maintained. Paramagnetic sensors are used to monitor the VO2 and VCO2 of the outflow. It can readily be determined when the mouse begins making the transition from aerobic to anaerobic exercise by the rapid increase in exhaled CO 2. Anaerobic exercise produces excess lactate, which is buffered by HCO3- as a result of the increased exhalation of CO2. It is therefore suggested that the exercise stress test in the mouse provides a quantitative indication of the mitochondrial bioenergetic capacity.25
In addition to the aforementioned more generalized cardiovascular assessment techniques, there are numerous more specific procedures that can be used to characterize genetically engineered mice. Conductance volumetry catheterization permits in vivo determination of instantaneous pressure-volume relationships and calculation of load-independent indices of left ventricular function. Sonomi-crometry measures immediate estimates of left ventricular cavity dimension with excellent resolution. X-ray contrast microangiography can be used to quantitate right ventricular dilatation and dysfunction as well as tricuspid regurgitation. Whole-animal elecrophysiologic studies with octapolar catheters can determine the molecular basis of conduction and arrhythmic disorders.24 Quantitative fluorescence microscopy using individual artery perfusion of fluorescently labeled molecules measures microvessel and macrovessel permeability and reactivity. The interaction between macromolecules and the vascular wall can be determined using optical coherence tomography and 2-photon fluorescence. Other assays in vascular tissue include measurement of hydraulic conductivity and macromolecular flux across capillary endothelium, macromolecular localization in the vascular wall, adhesion molecule properties and signal transduction in leukocytes and platelets, atheroma quantification, and assessment of lipo-protein metabolism.
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