Free radicals are known as an important factor which may act on reperfusion injury after transient or permanent brain ischemia. Numerous studies about cytotoxic function of free radical have been done. Most of these studies demonstrate the function of free radical in reperfusion injury by using radical scavenger or antioxidant as inhibitor of radicals. We used a modification of Karnovsky's Mn2 /diaminobenzidine (DAB) technique to demonstrate intravascular free radicals following transient occlusion and reperfusion of one middle cerebral artery in Sprague-Dawley rats. The MCA was occluded for 2 hours using an intraluminal suture method. The reperfusion time after transient ischemia was 1 hour, 6 hours, and 24 hours, respectively. Animals were perfused transcardially with solution containing Mn2 and DAB. After DAB perfusion, the brains were removed promptly, sectioned in frozen, and stained with methylene blue for light microscopic examination. Upon light microscopic examination, free radicals were confined within intravascular lumen and the amount of deposits increased according to the duration of reperfusion. Upon electron microscopic examination, free radicals were located in nuclear membrane and membrane of mitochondria and RER, and demonstrated as electron dense deposits. In addition, cell processes of the neuron revealed an electron dense deposits beneath the inner side of the membrane. In conclusion, free radicals demonstrated in the reperfusion injury area indicate that free radical acts as an important cytotoxic factor. Intracellular localization of free radicals may explain the relationship between free radical and delayed neuronal injury.
Animals ; Brain Ischemia* ; Brain* ; Free Radicals* ; Ischemia ; Membranes ; Methylene Blue ; Middle Cerebral Artery ; Mitochondria ; Neurons ; Nuclear Envelope ; Perfusion ; Rats, Sprague-Dawley ; Reperfusion Injury* ; Reperfusion* ; Sutures