Background and Purpose Tumor necrosis factor-a (TNF-a) is a cytokine with diverse proinflammatory actions, including endothelial leukocyte adhesion molecule expression. Since leukocytes infiltrate into ischemic brain lesions, the present study was conducted to examine whether TNF-a messenger RNA (mRNA) and peptide are expressed in the brain after experimental focal stroke and before leukocyte accumulation.Methods TNF-a mRNA and protein expression were monitored in the ischeraic and nonischemic cerebral cortex of rats after focal ischemia produced by permanent middle cerebral artery occlusion. The effect of TNF-a administered by microinjection into the brain cortex on leukocyte adherence to brain capillaries was also studied.Results Induction of TNF-a mRNA, normalized to a standard reference rat macrophage TNF-a mRNA, was detected as early as 1 hour after middle cerebral artery occlusion. TNF-a mRNA was elevated by 3 hours (29±6% versus 2±1% in sham-operated rats) only in the ischemic cortex, with peak expression at 12 hours (104±8%; P<.01). Five days after middle cerebral artery occlusion, TNF-a mRNA levels in ischemic cortex were still significantly elevated (38±5%; P<.05). Also, TNF-a mRNA expression was greater in the

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Two different techniques were utilized to identify the infiltration of polymorphonuclear leukocytes (PMN) into cerebral tissue following focal ischemia: histologic analysis and a modified myeloperoxidase (MPO) activity assay. Twenty-four hours after producing permanent cortical ischemia by occluding and severing the middle cerebral artery of male spontaneously hypertensive rats, contralateral hemiparalysis and sensory-motor deficits were observed due to cerebral infarction of the frontal and parietal cortex. In hematoxylin-and-eosin-stained histologic sections, PMN, predominantly neutrophils, were identified at various stages of diapedesis from deep cerebral and meningeal vessels at the periphery of the infarct, into brain parenchyma. When MPO activity in normal brain tissue was studied initially, it could not be demonstrated in normal tissues extracted from non-washed homogenates. However, if tissue was homogenized in phosphate buffer (i.e., washed), MPO activity was expressed upon extraction. Utilizing this modified assay, MPO activity was significantly increased only in the infarcted cortex compared to other normal areas of the brain. This was observed in non-perfused animals and after perfusion with isotonic saline to remove blood constituents from the vasculature prior to brain removal. The increased PMN infiltration and MPO activity were not observed in forebrain tissue of sham-operated control rats. Also, MPO activity was not increased in the ischemic cortex of MCAO rats perfused immediately after middle cerebral artery occlusion, indicating that blood was not trapped in the ischemic area. By using a leukocyte histochemical staining assay, activity of peroxidases was identified within vascular-adhering/infiltrating PMN in the infarcted cortex 24 hr after focal ischemia. An evaluation of several blood components indicated that increased MPO activity was selective for PMN. The observed increase of approximately 0.3 U MPO/g wet weight ischemic tissue vs. nonischemic cerebral tissues probably reflects the increased vascular adherance/infiltration of approximately 600,000 PMN/g wet weight infarcted cortex 24 hr after focal ischemia. This combined biochemical and histological study strongly suggests that PMN adhere within blood vessels and infiltrate into brain tissue injured by focal ischemia and that the associated inflammatory response might contribute to delayed progressive tissue damage in focal stroke. This modified MPO assay is a useful, quantitative index of PMN that can be utilized to elucidate the potential deleterious consequences of neutrophils infiltrating into the central nervous system after cerebral ischemia, trauma, or other pro-inflammatory stimuli.

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