T Liu

BACKGROUND AND PURPOSE: Tumor necrosis factor-alpha (TNF-alpha) 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-alpha messenger RNA (mRNA) and peptide are expressed in the brain after experimental focal stroke and before leukocyte accumulation. METHODS: TNF-alpha mRNA and protein expression were monitored in the ischemic and nonischemic cerebral cortex of rats after focal ischemia produced by permanent middle cerebral artery occlusion. The effect of TNF-alpha administered by microinjection into the brain cortex on leukocyte adherence to brain capillaries was also studied. RESULTS: Induction of TNF-alpha mRNA, normalized to a standard reference rat macrophage TNF-alpha mRNA, was detected as early as 1 hour after middle cerebral artery occlusion. TNF-alpha 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-alpha mRNA levels in ischemic cortex were still significantly elevated (38 +/- 5%; P < .05). Also, TNF-alpha mRNA expression was greater in the ischemic cortex of spontaneously hypertensive rats than in normotensive rats (P < .05). Double-labeling, immunohistochemical studies revealed the presence of TNF-alpha protein localized within nerve fibers in the evolving infarct at 6 and 12 hours after ischemia and further expression in the tissues immediately adjacent to the infarct 24 hours after ischemia. After 5 days, the neuronally localized peptide had diminished greatly, but macrophages located within the infarcted tissues were immunoreactive. Cortical microinjections of TNF-alpha (10 ng in 1 microL) produced a significant neutrophil adherence/accumulation in capillaries and small blood vessels 24 hours later. CONCLUSIONS: These results represent the first demonstration that focal cerebral ischemia in rats results in elevated TNF-alpha mRNA and protein in ischemic neurons. The neuronal expression of peptide appears to facilitate the infiltration of inflammatory cells that can further exacerbate tissue damage in cerebral ischemia and might contribute to increased sensitivity and risk in focal stroke.

BACKGROUND AND PURPOSE: Interleukin-1 beta is a proinflammatory cytokine produced by blood-borne and resident brain inflammatory cells. The present study was conducted to determine if interleukin-1 beta mRNA was produced in the brain of rats subjected to permanent focal ischemia. METHODS: Rat interleukin-1 beta cDNA, synthesized from stimulated rat peritoneal macrophage RNA by reverse transcription and polymerase chain reaction and cloned in plasmid Bluescript KS+, was used to evaluate the expression of interleukin-1 beta mRNA in cerebral cortex from spontaneously hypertensive rats and normotensive rats subjected to permanent middle cerebral artery occlusion. Interleukin-1 beta mRNA was quantified by Northern blot analysis and compared with rat macrophage RNA standard. To correct for gel loading, blots were also analyzed with cyclophilin cDNA, which encodes an abundant, conserved protein that was unchanged by the experimental conditions. RESULTS: Interleukin-1 beta mRNA produced in the ischemic zone was significantly increased from 6 hours to 120 hours, with a maximum of 211 +/- 24% of interleukin-1 beta reference standard, ie, 0.2 ng stimulated rat macrophage RNA, mRNA compared with the level in nonischemic cortices (4 +/- 2%) at 12 hours after ischemia (P < .01; n = 6). Interleukin-1 beta mRNA at 12 hours after ischemia was markedly elevated in hypertensive rats over levels found in two normotensive rat strains. Neurological deficits were also apparent only in the hypertensive rats. CONCLUSIONS: Brain interleukin-1 beta mRNA is elevated acutely after permanent focal ischemia and especially in hypertensive rats. These data suggest that this potent proinflammatory and procoagulant cytokine might have a role in brain damage following ischemia.