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Cerebral Blood Flow Methods
Introduction
Although the brain represents only 2% of the total body mass in humans, it accounts for a fifth of the body’s basal O 2 consumption and a quarter of its glucose use. A continuous supply of blood and nutrients is essential for its functioning and cerebral blood flow (CBF) must constantly match the demands of brain activity. Because it lacks energy stores, the brain is particularly vulnerable not only to acute ischemic events, but also to chronic impairment in the coupling between CBF and neuronal activity. It is therefore essential to have accurate methods to determine absolute CBF, or at least to monitor changes in CBF following various interventions. Many methods have been developed to monitor CBF clinically as well as in experimental animal models. Because of space constraints, this review will focus on the latter. Methods aimed at monitoring CBF in patients have been reviewed elsewhere .
Owing to their small size, rodents (the most commonly used animals in cerebrovascular research) present unique technical challenges when compared with humans. For instance, a 1-mm spatial resolution that would be considered excellent in humans does not resolve many mouse brain structures, and only a limited volume of blood can be sampled from small animals to measure the arterial input function, which is essential for quantitative CBF analysis using tracers. In addition, many biomedical research laboratories do not have access to technologies used routinely in a clinical setting. The emphasis of this review will therefore be on CBF methods that are both easier and relatively less costly to implement.
Methods Requiring Brain Penetration
These methods require the implantation of probes in one site (or at most a few discrete sites) and are therefore ill-suited for a regional assessment of CBF changes in laboratory animals.
Thermal Clearance or Thermal Diffusion Flowmetry
Since Gibbs’ demonstration in 1933 that changes in CBF could be detected with the use of a heated thermocouple, many investigators have developed thermal systems to measure CBF . Although they initially only provided an assessment of flow in arbitrary units and, hence, of relative changes, they can now achieve reliable CBF measurements in absolute terms. Thermal clearance flowmetry relies on the continuous measurement of the changes in brain thermal conductivity caused by CBF changes. Two thermistors are implanted, one of which is heated to approximately 2°C above the tissue temperature. The proximal thermosensor is located several millimeters away from the distal sensor, outside of its thermal field, thereby allowing continuous monitoring of tissue temperature and compensation of baseline fluctuations. The power dissipated by the heated element to keep itself at a constant elevated temperature provides a direct measure of the brain’s ability to transport heat. This thermal transfer includes not only convective effects induced by blood flow, but also the intrinsic conductive properties of the tissue. These two components must therefore be separated to measure CBF. This was initially achieved by euthanizing the animal to determine the zero flow current. Modern systems enable quantification of tissue perfusion in absolute units by determining the conductive properties of the tissue from the initial rate of propagation of the thermal field and by subtracting this component from the total heat transfer as the determinant of the thermal convection component, making no-flow calibration unnecessary. The geometric arrangement of the two thermistors required for determination of thermal conductivity and convection makes this technique impractical to measure CBF in rodents. But progresses in microfabrication methods have led to the development of thermal flow microsensors small enough to be implanted in rat cortex that can yield continuous, real-time, and quantitative CBF measurement with high long-term accuracy and high temporal resolution .
Hydrogen Clearance
Kety and Schmidt were the first to attempt to measure global CBF in 1945 . They used nitrous oxide to determine the global CBF based on Fick’s principle, which relies on the premise that the total uptake of a tracer by an organ is equal to the product of the blood flow to the organ and the arterial-venous concentration difference of the tracer. The hydrogen clearance is a variation of Kety and Schmidt’s technique that uses H 2 to assess the CBF. H 2 is not normally present in the body, is metabolically inert, dissolves readily into lipids, diffuses rapidly in tissues, such as brain, and is rapidly eliminated by the pulmonary circulation, thereby fulfilling the key criteria for tracer clearance studies developed by Kety and Schmidt. To measure H 2 clearance, one or several electrodes are inserted into the brain (polarized to +400 mV with respect to a subcutaneous reference Ag/AgCl electrode), H 2 is administered either by respiration or intraarterially, it is allowed to be cleared from arterial blood, and the exponential clearance rate of H 2 from the tissue is monitored . This method can be used in both anesthetized and conscious animals to simultaneously measure local CBF at multiple sites, and measurements can be repeated many times over periods ranging from hours to days. It is important to note that absolute CBF values measured with this technique are often significantly lower than those found with other methods. This is likely to reflect actual CBF alterations that occur as a result of electrode insertion, rather than an artifact of the technique. Indeed, a transient flow reduction in the whole cerebral hemisphere has been linked to the induction of spreading depressions after electrode insertion . These spreading depressions, and their effect on measured CBF, can be minimized by using thinner electrodes (50 μm) and/or by implanting the electrodes 1–2 weeks ahead of the experiments.
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