Simultaneous detection of blood volume, oxygenation, and intracellular calcium changes during cerebral ischemia and reperfusion in vivo using diffuse reflectance and fluorescence

We describe an approach to measure changes in intracellular calcium along with changes in blood volume and oxygenation directly from the exposed rat cortex in vivo during cerebral ischemia and reperfusion. Measurements were made using a catheter-based optical system. The endface of a Y-shaped bifurcated fiber optic bundle was mounted on the cortical surface. It delivered the light at three wavelengths of 548, 555, and 572 nm to the brain through a fast monochromator coupled to a xenon lamp, and collected the calcium-dependent fluorescence emission from Rhod2 at 589 nm (excited at 548 nm) along with the diffuse reflections at the wavelengths of 555 and 572 nm to determine the changes in blood volume and hemoglobin oxygenation. The feasibility of this approach was experimentally examined by inducing transient cerebral ischemia and reperfusion in the rat. The ischemia induced an 8.5% ± 1.7% fluorescence increase compared with the preischemic control values. Blood volume and tissue hemoglobin oxygenation decreased by 57.4% ± 12.6% and 47.3% ± 12.5%, respectively. All signals normalized on reperfusion. The ischemia-induced change in Rhod2-Ca²⁺ fluorescence was blocked using a calcium channel blocker, nimodipine, confirming that intracellular changes in calcium were responsible for the fluorescence changes. Thus, changes in cerebral hemodynamics and intracellular calcium concentration changes were measured simultaneously, facilitating future studies of the interrelationship between neuronal activation and metabolic and vascular processes in normal and diseased brain.