There is increasing evidence that reactive oxygen species (ROS) are involved in hypoxic/ischemic tissue damage in a number of organs, including the brain. Due to methodological difficulties in the in vivo detection of the extremely shortlived ROS many issues regarding the role of ROS in ischemia remain unclear. In particular, the temporal pattern and the cellular sources of ROS production in ischemia/reperfusion of the brain is unknown. Methods currently available for monitoring ROS production in the brain in vivo lack temporal resolution, and are therefore not well suited to study the temporal dynamics of ROS production. In the process of chemiluminescence (CL), specific chemical compounds (i.e. luminol or lucigenin) react with ROS to produce light. CL is a well established principle for monitoring ROS production in vitro (Kricka, 1993) with high sensitivity. We use CL for the first time to study brain cortex surface ROS production in vivo from brain slices and from the intact brain.
Anesthetized and ventilated rats are equipped with a closed cranial window over the right parietal cortex (dura removed, superfused with artificial cerebrospinal fluid (aCSF). The animals are then positioned in a dark box for chemiluminescence recording (see fig. 1). Physiological variables are monitored and kept within normal limits. Regional cortical blood flow (rCBF) is measured continuously in the brain cortex with Laser-Doppler flowmetry (LDF).
Brain slices are prepared in standard technique from rats and transferred to a warmed chamber (37 oC). In the chamber, the brain slices are superfused with artificial cerebrospinal fluid (aCSF). The O2-tension and temperature inside the chamber are monitored with a Licox pO2-monitor.
The animal with the cranial window or the brain slice chamber is transferred to a dark box and positioned under a reflector, reflecting the photons from the exposed brain onto the photon-sensitive area of the photocathode (fig.1) of a cooled photomultiplier. To record ROS production optically, the CL enhancer Lucigenin, which is paritcularly sensitive to superoxide, is topically superfused on the brain tissue in the slice chamber or the cranial window.
Results of ongoing studies are shown in the figures. Figure 3 shows that after global cerebral ischemia in the rat there is a CL burst with reperfusion. CL returns to baseline in the 10 minute ischemia group, whereas it remains elevated in the 20 minutes group.Figure 4 shows that the burst occurs also in brain slices with reoxygenation after hypoxia, and that this burst is dependent on the developmental age of the rats.
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. The production of ROS is preceded by reperfusion, burstlike, and dependent on the duration of the ischemic interval.
Setup for in vivo chemiluminescence. Shown is the setup for CL from brain of experimental animals. For brain slice CL, a slice chamber is positioned in the dark box.
A: Effect of inhalation of 100 % oxygen on the lucigenin enhanced chemiluminescence (CL) recorded from the brain cortex in vivo. B: Effect of inhalation of 100 % nitrogen on the lucigenin enhanced CL recorded from the brain cortex. C: Effect of the i.v. injection of 1 ml saturated KCl solution on the lucigenin enhanced CL recorded from the brain cortex. D. Typical recording of hypoxia (15 min) and reoxygenation in the brain slice preparation.
Lucigenin enhanced chemiluminescence recordings from group I (10 minutes of global cerebral ischemia) and group II (20 minutes of global cerebral ischemia) animals. n=8 in each group.
Age dependency of OFR production after hypoxia in the brain slice. A: 12 day old rats (n=5), B: 24 day old rats (n=5) C: adult rats (n=5). Note that the CL burst develops with the age of the animal.
Supported by the DFG and Wilhelm Sander Stiftung