Pergamon Brain Research Bulletin, Vol. 34, No. 2, pp. 137-141, 1994 Copyright Q 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0361-9230/94 $6.00 + .OO 0361.923O(!M)EOO26-V Leukocyte Involvement in Cerebral Infarct Generation After Ischemia and Reperfusion LYNN A. HEINEL, SHARON RUBIN, ROBERT H. ROSENWASSER, USHA S. VASTHARE AND RONALD F. TUMA’ Cerebrovascular Laboratory, Departments of Physiology and Neurosurgery, Temple University School of Medicine and Hospital, Philadelphia, PA 19140 Received 30 August 1993; Accepted 21 January 1994 HEINEL, L. A., S. RUBIN, R. H. ROSENWASSER, U. S. VASTHARE AND R. F. TUMA. Leukocyte involvemenr in cerebrul infarct generation after ischemia and reperfusion. BRAIN RES BULL 34(2) 137-141, 1994.-White blood cell involvement in the generation of cerebral infarcts was evaluated following ischemia and repertitsion injury in the rat. Control and leukopenic rats (induced by vinblastine, WBC counts < 1500/mm3) were compared in a global forebrain ischemic model after 1 h of ischemia and 1 h 15 min of reperfusion. Cerebral infarcts were defined on coronal brain sections using Triphenyl tetrazolium chloride (TX) staining. Electroencephalographic activity (EEG) and somatosensory evoked potentials (SSEP) were also compared. Results in- dicate that the area infarcted in leukopenic rats was signi&antly less than infarcts generated in corresponding controls (21 + 16% vs. 70 ? 16%). In addition, EEG was preserved in all leukopenic animals when compared to controls, both during ischemia and after reperfusion. The cortical peak component of the SSEP was also better preserved in the leukopenic animals both during &hernia and at 60 min of repertinsion. These results indicate white blood cell participation in the generation of cerebral damage in a model of global forebrain ischemia and reperfusion as indicated by TIC staining of cerebral infarcts. Cerebral infarcts EEG Reperfusion injury SSEP TTC staining White blood cells THE involvement of white blood cells in &hernia and reperfu- sion injury has been suggested and documented in various tissues including heart, brain, skeletal muscle, intestine, and kidney (4,5,10,13,22-24). The white blood cells have been suggested to be a major participant in the inflammatory cascade that is initiated by ischemia and may be even further deleterious to ischemic tissue upon reperfusion. The role of the white blood cell, in par- ticular the neutrophil, has been reported to be of significance during early reperfttsion; however, the effect may be transient and may or may not result in permanent injury (7). In the brain, white blood cells have been found to accumulate under low flow conditions (4,8,25) and to affect cortical functional activity fol- lowing cerebral &hernia and reperfusion (24). However, whether these cells are important in influencing irreversible ce- rebral injury and the conditions under which this may occur is unknown. Previous evidence using antineutrophil serum to di- minish white cells in a rabbit model of permanent thromboem- bolic stroke combined with systemic hypotension has shown a reduction in infarct size when compared to corresponding con- trols (3). It is the purpose of this study to determine if white blood cells are involved in irreversible cerebral injury as determined by infarct size in a model of global forebrain &hernia in the rat where blood flow is interrupted and restored by reperfusion. Triphenyl tetrazolium chloride staining, a widely accepted technique for determining viable tissue in the heart and brain (2,3,6,X-18), was used in control and leukopenic rats to deter- 1 Requests for reprints should be addressed to Ronald F. Tuma. mine if white blood cells contribute to the generation of cerebral infarcts following 60 min of cerebral ischemia and 75 min of reperfusion. METHOD Barrier reared Sprague-Dawley rats (250-275 g) were main- tained on food and water ad lib. The rats were randomly selected to serve as a control (normopenic) or to be rendered leukopenic by a single bolus intravenous injection of vinblastine sulfate (8 mg/kg) (21,24). This dose produces a gradual reduction in the circulating levels of white blood cells until the fourth day when the nadir of the white blood cell reduction was reached. At this time, the animals were subjected to the experimental protocol for cerebral ischemia and reperfusion. Circulating levels of white blood cells and platelets were determined prior to experimenta- tion using a Spencer hemacytometer. A protocol for inducing cerebral &hernia and reperfusion injury, previously documented in various laboratories including our own (7,20,24), was used to randomly evaluate control and leukopenic rats. An inhalation mixture of 1.5% enflurane (Ethrane), with a 70% nitrous oxide and oxygen was initially used to anesthesize each animal. The animals were subsequently tracheostomized and placed on a small animal ventilator. Ventilation was adjusted to maintain arterial blood gases within physiological range. At- ropine sulfate (50 &kg, IM) was administered to reduce bron- 137