Ray F. White

BACKGROUND AND PURPOSE: Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine that rapidly upregulates in the brain after injury. The present study was designed to explore the pathophysiological significance of brain TNF-alpha in the ischemic brain by systematically evaluating the effects of lateral cerebroventricular administration of exogenous TNF-alpha and agents that block the effects of TNF-alpha on focal stroke and by examining the potential direct toxic effects of TNF-alpha on cultured neurons to better understand how TNF-alpha might mediate stroke injury. METHODS: TNF-alpha (2.5 or 25 pmol) was administered intracerebroventricularly to spontaneously hypertensive rats 24 hours before permanent or transient (80 minutes and 160 minutes) middle cerebral artery occlusion (MCAO). Animals were examined 24 hours later for neurological deficits and ischemic hemisphere necrosis and swelling. In some of these studies, neutralizing anti-TNF-alpha monoclonal antibody (mAb) (60 pmol) was injected intracerebroventricularly 30 minutes before exogenous TNF-alpha (25 pmol). In addition, the effects of blocking endogenous TNF-alpha on permanent focal ischemic injury were determined 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 neurotoxic 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 hemispheric 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 1.6 +/- 0.2 to 2.3 +/- 0.2 (P < 0.1). TNF-alpha (2.5 pmol) also increased the infarction induced by 80 or 160 minutes 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 infarct size, swelling, and neurological deficit after exogenous TNF-alpha was reversed by preinjection of 60 pmol mAb. Blocking endogenous TNF-alpha also significantly reduced focal ischemic brain injury. Treatment with 60 pmol mAb before and after permanent MCAO significantly reduced 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 26.1%, P < .05) sTNF-RI before and after focal stroke. The intracerebroventricular administration of TNF-alpha or sTNF-RI did not alter brain or 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 release or glutamate or oxygen free radical toxicity. CONCLUSIONS: These studies 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 antagonism 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 oxygen radicals and apparently is mediated through nonneuronal cells. These data suggest that inhibiting TNF-alpha may represent a novel pharmacological strategy to treat ischemic stroke.

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.

Sublethal insults can induce tolerance to subsequent stressors in neurons. As cell death activators such as ROS generation and decreased ATP can initiate tolerance, we tested whether other cellular elements normally associated with neuronal injury could add to this process. In an in vivo model of ischemic tolerance, we were surprised to observe widespread caspase 3 cleavage, without cell death, in preconditioned tissue. To dissect the preconditioning pathways activating caspases, and the mechanisms by which these proteases are held in check, we developed an in vitro model of excitotoxic tolerance. In this model, antioxidants and caspase inhibitors blocked ischemia-induced protection against N-methyl-d-aspartate toxicity. Moreover, agents that blocked preconditioning also attenuated induction of HSP 70; transient overexpression of a constitutive form of this protein prevented HSP 70 up-regulation and blocked tolerance. We outline a neuroprotective pathway where events normally associated with apoptotic cell death are critical for cell survival.

Salicylic acid (SA) is absolutely required for establishment of acquired resistance in non‐infected tissues following localized challenge of other leaves with a necrotizing pathogen. Although not directly responsive to SA, or induced systemically following pathogen challenge, the expression of defence gene promoter fusions AoPR1—GUS and PAL‐3—GUS after wounding or pathogen challenge could be enhanced by pre‐treating tobacco plants hydroponically with SA, a phenomenon designated ‘potentiation’. Potentiation of AoPR1—GUS wound‐responsiveness was also demonstrated locally, but not systemically, in tobacco tissue exhibiting acquired resistance following infection with either viral or bacterial pathogens. Potentiation of wound‐responsive expression by prior wounding could not be demonstrated. In contrast, potentiation of pathogen‐responsive AoPR1—GUS expression was exhibited both locally and systemically in non‐infected tissue. The spatial and temporal exhibition of defence gene potentiation correlated directly with the acquisition of resistance in non‐infected tissue. Pathogen‐responsive potentiation was obtained at about 10‐fold lower levels of salicylic acid than wounding‐responsive potentiation in AoPR1—GUS tobacco plants prefed with salicylate. These results may explain the failure to observe systemic potentiation of the wound‐responsive defence gene expression. The data suggest a dual role for SA in terms of gene induction in acquired immunity: a direct one by induction of genes such as pathogenesis‐related proteins, and an indirect one by potentiation of expression of other local defence genes (such as PAL and AoPR1) which do not respond directly to SA but become induced on pathogen attack or wounding.