Microwave Fixation for the Determination of Cerebral Blood Volume in Rats

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Abstract
The cerebral blood volume (CBV) is sensitive to changing hydrostatic pressures. Thus, measurement methods that rely on removing tissue from unfixed brain may lead to underestimates of the CBV due to the loss of blood from the tissue. In situ fixation of tissue before removal may offer improved accuracy. We employed a triple-label method to measure simultaneously whole brain CBF and CBV in halothane-anesthetized Sprague–Dawley rats, which were then killed either by focused microwave irradiation (≈8 kW of incident power × 770 ms) or by decapitation. CBF was measured with [3H]nicotine while the CBV was determined as the sum of the cerebral red cell volume (CRCV—measured with 99mTc-labeled red cells) and the cerebral plasma volume (CPV—measured with [14C]dextran). Animals were studied during hypocarbic (Paco2 ≈ 25 mm Hg), normocarbic, or hypercarbic (Paco2 ≈ 70 mm Hg) conditions. Added studies were performed to verify that the microwave irradiation scheme used was capable of fixing previously administered tracers in place, and also halting the entry of tracer given after irradiation. Results indicate that the method of killing had no effects on CBF measurements, as assessed either by absolute values during normocarbia or responsiveness to changing Paco2. However, all three volume measurements made using nondiffusible tracers (CRCV, CPV, and CBV) were significantly lower in animals killed by decapitation. Furthermore, CO2 responsiveness for all three variables (as assessed by the slope of the Paco2/volume) was not evident in decapitated animals. We conclude that in situ fixation offers significant advantages when examining the cerebral distribution space of nondiffusible tracers.