TUMOR-NECROSIS-FACTOR-ALPHA - A MEDIATOR OF FOCAL ISCHEMIC BRAIN INJURY

Background and Purpose Tumor necrosis factor-alpha (TNF-alpha) is a pl eiotropic cytokine that rapidly upregulates in the brain after injury. The present study was designed to explore the pathophysiological sign ificance of brain TNF-alpha in the ischemic brain by systematically ev aluating the effects of lateral cerebroventricular administration of e xogenous TNF-alpha and agents that block the effects of TNF-alpha on f ocal stroke and by examining the potential direct toxic effects of TNF -alpha on cultured neurons to better understand how TNF-alpha might me diate stroke injury. Methods TNF-alpha (2.5 or 25 pmol) was administer ed intracerebroventricularly to spontaneously hypertensive rats 24 hou rs before permanent or transient (80 minutes and 160 minutes) middle c erebral artery occlusion (MCAO). Animals were examined 24 hours later for neurological deficits and ischemic hemisphere necrosis and swellin g. In some of these studies, neutralizing anti-TNF-alpha monoclonal an tibody (mAb) (60 pmol) was injected intracerebroventricularly 30 minut es before exogenous TNF-alpha (25 pmol). In addition, the effects of b locking endogenous TNF-alpha on permanent focal ischemic injury were d etermined with the use of either mAb (60 pmol) or soluble TNF receptor I (sTNF-RI) (0.3 or 0.7 nmol) administered intracerebroventricularly 30 minutes before and 3 and 6 hours after MCAO. Finally, the direct ne urotoxic effects of TNF-alpha were studied in cultured rat cerebellar granule cells exposed to TNF-alpha (10 to 2000 U/mL for 6 to 24 hours) , and neurotransmitter release, glutamate toxicity, and oxygen radical toxicity were studied. Results TNF-alpha increased the percent hemisp heric infarct induced by permanent MCAO in a dose-related manner from 13.1+/-1.3% (vehicle) to 18.9+/-1.7% at 2.5 pmol (P<.05) and 27.1+/-1. 3% at 25 pmol (P<.0001). The high dose of TNF-alpha increased ischemia -induced forelimb deficits from 1.61-0.2 to 2.3+/-0.2 (P<.01). TNF-alp ha (2.5 pmol) also increased the infarction induced by 80 or 160 minut es of transient MCAO from 1.9+/-0.9% to 4.3+/-0.4% (P<.01) and from 14 .2+/-1.3% to 21.6+/-2.2% (P<.05), respectively. The exacerbation of in farct size, swelling, and neurological deficit after exogenous TNF-alp ha was reversed by preinjection of 60 pmol mAb. Blocking endogenous TN F-alpha also significantly reduced focal ischemic brain injury. Treatm ent with 60 pmol mAb before and after permanent MCAO significantly red uced infarct size compared with control (nonimmune) antibody treatment by 20.2% (P<.05). Reduced brain infarction also was produced by brain administration of 0.3 nmol (decreased 18.2%) or 0.7 nmol (decreased 2 6.1%; P<.05) sTNF-RI before and after focal stroke. The intracerebrove ntricular administration of TNF-alpha or sTNF-RI did not alter brain o r body temperature, blood gases or pH, blood pressure, blood glucose, or general blood chemistry. In cultured cerebellar granule cells, the application of TNF-alpha did not directly affect neurotransmitter rele ase or glutamate or oxygen free radical toxicity. Conclusions These st udies demonstrate that exogenous TNF-alpha exacerbates focal ischemic injury and that blocking endogenous TNF-alpha is neuroprotective. The specificity of the action(s) of TNF-alpha was demonstrated by antagoni sm of its effects with specific anti-TNF-alpha tools (ie, mAb and sTNF -RI). TNF-alpha toxicity does not appear to be due to a direct effect on neurons or modulation of neuronal sensitivity to glutamate or oxyge n radicals and apparently is mediated through nonneuronal cells. These data suggest that inhibiting TNF-alpha may represent a novel pharmaco logical strategy to treat ischemic stroke.