Intracisternal increase of superoxide anion production in a canine subarachnoid hemorrhage model

Abstract

Background and Purpose - Reactive oxygen species (ROS) are thought to be primary in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH). However, as direct evidence of ROS has not yet been demonstrated in cerebral vasospasm, we sought to substantiate superoxide anion (·O2-) generation in the subarachnoid space after SAH using a modification of Karnovsky's manganese/diaminobenzidine (Mn2+/DAB) technique. Methods - SAH or sham operation was induced according to a 2-hemorrhage model in a total of 24 beagle dogs. On day 2 or 7 after SAH or sham operation, dogs were intrathecally infused with buffer containing Mn2+ and DAB, and the brain stem was prepared for light and electron microscopy. Possible colocalization of ferrous (Fe2+) or ferric (Fe3+) iron ions with ·O2- was also examined with the use of Turnbull blue or Berlin blue staining, respectively. Results - Light microscopy revealed amorphous, amber deposits within the subarachnoid hematoma, the periarterial space, and the tunica adventitia of the basilar artery on days 2 and 7 after SAH. ·O2- deposits were eliminated by addition of superoxide dismutase or exclusion of either Mn2+ or DAB from the perfusate, confirming the specificity of the reaction. These deposits were colocalized with blue reaction deposits indicating Fe2+ and Fe3+. Within the subarachnoid space, ·O2- indicating electron-dense fine granules were preferentially located around degenerated erythrocytes and, secondarily, infiltrating macrophages and neutrophils. Conclusions - We show direct evidence for enhanced production of ·O2- and Fe2+/Fe3+ iron ions in the subarachnoid space after SAH, lending further support to the pathogenic role of ROS in cerebral vasospasm after SAH.