In Vivo Measurement of Superoxide in the Cerebral Cortex Utilizing Cypridina luciferin analog (MCLA) Chemiluminescence

Summary

Temproal profile of superoxide (O₂ ⁻) generation following cerebral hypoxia and ischemia has been obscure, although it has been implicated in the progression of reperfusion injury. We have examined the time course of O ⁻₂ generation in the cat cortex following reversible hypoxia and forebrain incomplete ischomia. We used 20 cats anesthetized with halothane inhalation. A closed cranial window was placed over the exposed temporoparietal cortex, and Cypridina luciferin analog (MCLA), a chemiluminescence probe for the measurement of O ⁻₂ , was dissolved in the artificial cerebrospinal fluid (ACSF) and superfused continuously throughout the experiments. We simultaneously monitored a reflectance (398 nm) from the cortex by means of an in vivo fluoromicroscope with two photomultiplier tubes. We subtracted a hemodynamic artifact from the MCLA chemiluminescence. Hypoxia was induced by pure N₂ inhalation for 1 min, and forebrain incomplete ischemia was induced by 30-min ligation of both common carotid arteries combined with hypotension (= 50 mmHg). As a result, the MCLA chemiluminescence increased following reoxygenation and reperfusion, indicating an enhanced O ⁻₂ generation. It was significantly reduced during hypoxia and severe ischemia, but fluctuated during mild ischemia. We speculate a breakdown of arachidonic acid and a biochemical interaction between endothelial cells and polymorphonuclear leukocytes as a source of O ⁻₂ generation.