Intraluminal increase of superoxide anion following transient focal cerebral ischemia in rats

Using a modification of Karnovsky's Mn²⁺/diaminobenzizine (DAB) technique, we examined the production of superoxide anion (O⁻₂) in the vascular lumen following transient occlusion and reperfusion of the left middle cerebral artery (MCA) in Sprague–Dawley rats. The MCA was occluded for 2 h using an intraluminal suture method. Zero, 15, 30, and 60 min after reperfusion, animals were perfused transcardially with buffer containing Mn²⁺ and DAB, and brain samples were prepared for light and electron microscopic examination. The amber reaction deposits of O⁻₂ were observable to the naked eye along the major cerebral vessels of the ischemic hemisphere after each reperfusion period. Upon microscopic examination the deposits were revealed to be within arterial, capillary, and venular lumen. The amount of reaction deposits in the ischemic hemisphere corresponded to the duration of reperfusion. The formation of O⁻₂ was suppressed when the perfusate contained superoxide dismutase and when either Mn²⁺ or DAB was omitted, confirming that the reaction products produced are due to the enhanced production of O⁻₂. These results show that there is a progressive increase in intraluminal O⁻₂ during reperfusion following an ischemic insult which may participate in the aggravation of cerebral damage.