Global cerebral ischemia in the rat: online monitoring of oxygen free radical production using chemiluminescence in vivo.

Using online in vivo chemiluminescence (CL), we studied for the first time continuously the production of reactive oxygen species (ROS) after global cerebral ischemia and the relationship of ROS production to CBF. In anesthetized rats equipped with a closed cranial window, the CL enhancer, lucigenin (1 mM), was superfused onto the brain topically. CL was measured through the cranial window with a cooled photomultiplier, and CBF was measured simultaneously with laser-Doppler flowmetry. Reperfusion after 10 min (n = 8) of global cerebral ischemia led to a CL peak to 188 +/- 77% (baseline = 100%) within 10 +/- 4 min. After 2 h of reperfusion, CL had returned to 102 +/- 28%. Reperfusion after 20 min (n = 8) of ischemia increased CL to 225 +/- 48% within 12 +/- 3 min. After 2 h, CL was still increased (150 +/- 44%, p < 0.05 compared with 10 min of ischemia). CL after 10 min of ischemia was neither affected by brain topical free CuZn-superoxide dismutase (SOD) (100 U/ml, n = 3) nor by i.v. administration of free CuZn-SOD (104 U/kg, followed by 104 U/kg/h, n = 3). The CBF hyperfusion peak on reperfusion preceded the CL peak in all experiments by several minutes. In additional in vitro experiments we investigated the source of CL: Intracellular loading of lucigenin was demonstrated in cultured CNS cells, and a very similar pattern of CL as in the in vivo preparation after ischemia developed in rat brain slices after 15 min of hypoxia, which was unaffected by free CuZn-SOD (100 U/ml) but strongly attenuated by liposome-entrapped CuZn-SOD. We conclude that lucigenin-enhanced CL is a promising tool to study ROS production continuously from the in vivo brain of experimental animals and brain slices, and that the CL signal most likely derives from the intracellular production of superoxide. The production of ROS is preceded by reperfusion, is burst-like, and is dependent on the duration of the ischemic interval.

Capillary perfusion of the rat brain cortex. An in vivo confocal microscopy study.

Confocal laser-scanning microscopy was used to visualize subsurface cerebral microvessels labeled with intravascular fluorescein in a closed cranial window model of the anesthetized rat. In noninvasive optical sections up to 250 microns beneath the brain surface, plasma perfusion and blood cell perfusion of individual capillaries were studied. Under resting conditions, in all cerebral capillaries the presence of plasma flow as demonstrated by the appearance of an intravenously injected fluorescent tracer within 20 seconds after injection. Plasma flow was verified even in capillaries that contained stationary erythrocytes or leukocytes; 91.1% of the capillaries contained flowing blood cells, 5.2% contained stationary blood cells, and no blood cells were seen in 3.6%. Mean blood cell velocity was 498.3 +/- 443.9 microns/s, and the mean blood cell supply rate was 35.75 +/- 28.01 cells per second. When capillaries were continuously observed for 1 minute, "on" and "off" periods of blood cell flow were noted. During hypercapnia (increase of PCO2 from 33.25 to 50.26 mm Hg), mean blood cell flux increased from 38.6 +/- 17.2 to 55.5 +/- 12.2 per second (P < .005, paired t test of mean values in six animals), and blood cell velocity increased from 519.5 +/- 254.8 to 828.5 +/- 460.8 microns/s (P = .074, paired t test of mean values in six animals). Homogeneity of blood cell flux increased as indicated by the coefficient of variation decreasing from 44.6% to 22.0%, and the portion of poorly perfused capillaries (blood cell flux, < 40 per second) decreased from 59.2% to 22.4%.(ABSTRACT TRUNCATED AT 250 WORDS)

Global forebrain ischaemia in the rat: controlled reduction of cerebral blood flow by hypobaric hypotension and two-vessel occlusion.

We developed and characterized a model of global forebrain ischaemia in rats, permitting control of CBF at any desired ischaemic level with minimum surgery and without anticoagulation. Both common carotid arteries are occluded temporarily and systemic arterial pressure is lowered by pooling venous blood by lower body negative pressure with a cheap suction device. By measuring rCBF continuously (laser-Doppler-flowmetry) and regulating systemic arterial pressure, the model was used to automatically control cortical rCBF at predetermined ischaemic levels at 50, 30, 15, and 5% of normal rCBF (n = 5). When both common carotid arteries were occluded and systemic arterial pressure was lowered to 55 mmHg with hypobaric hypotension (n = 5), cortical CBF always fell to less than 5% of normal rCBF (n = 5). Prompt recirculation was achieved after reopening of the carotid arteries and return to normobaric body pressure. Hypobaric hypotension with bilateral common carotid occlusion requires only carotid surgery and measurement of systemic arterial pressure; it produces global forebrain ischaemia without anticoagulation as a true step function type insult. If rCBF is measured continuously, the model can be used to control ischaemic CBF to predetermined values.

Imaging of leukocytes within the rat brain cortex in vivo.

Confocal laser scanning microscopy was used in a rat closed cranial window preparation in order to study rhodamin 6G-labeled leukocytes within the brain cortex in vivo. Leukocytes were visualized up to 150 microns beneath the rat brain surface in noninvasive optical sections. In pial venules, leukocytes were seen flowing with the blood stream, rolling along or sticking to the endothelium, and migrating through the vessel wall. Within cerebral capillaries, leukocyte flux, velocities, and leukocyte plugging were measured. After additional intravenous administration of fluorescein, the plasma, leukocytes, and erythrocytes were visualized simultaneously. Based on stacks of optical sections of fluorescein-labeled capillaries, the individual capillaries were localized within the three-dimensional microvascular network. The usefulness of this technique was illustrated in a feasibility study in which leukocyte sticking to the vascular walls of venules, leukocyte extravasation, and intracapillary leukocyte plugging were monitored in a model of global cerebral ischemia.