Focal cerebral ischemia in the TNF-transgenic rat: Regional expression of TNF protein, TNF converting enzyme, and TNF receptor 1

Background and Aims: Tumor necrosis factor-alpha (TNFa) is an inflammatory mediator that becomes elevated in ischemic brain. TNF converting enzyme (TACE) cleaves active TNFa from a transmembrane precursor. Active TNFa will bind to its receptor, TNFR1, and may promote apoptosis through activation of caspase 8. We tested the hypothesis that overexpression of the murine TNFa gene in the brain of a transgenic (TNFa-Tg) rat will affect TNFa protein levels and alter regional expression of TACE and TNFR1 after focal ischemia. Methods: TNFa-Tg and wild type (WT) rats underwent middle cerebral artery occlusion (MCAO) for 1 hr. Samples of ischemic core (IC) and penumbral (PN) tissue obtained after 30 min or 3/24 hr of reperfusion were assayed for TNFa (Linco multiplex) and expression of TACE and TNFR1 (western blot). Results: The TNFa levels in IC and PN of TNFa-Tg rat brain were unchanged at 30 min and 3 hr but fell below non-ischemic control levels at 24 hr (p<0.02; n=3 ? 5 per group; Fisher's test; Table 1). The TNFa levels in IC and PN of WT rats could be measured only at 24 hrs (p=NS compared to non-ischemic WT controls). TNFa levels were significantly higher in IC (p<0.003) and PN (p<0.01) of the TNFa-Tg rat than in WT animals at all 3 sampling times. Non-ischemic TNFa-Tg and WT rats were equivalent in expression of active TACE isotype and TNFR1. After MCAO, TACE expression increased in both IC and PN for TNFa-Tg and WT rats at 30 min (p<0.003) and at 24 hrs (p<0.03) but only in TNFa-Tg animals at 3 hrs (p<0.03). In ischemic TNFa-Tg rats, TNFR1 expression increased in both IC and PN at 30 min (p<0.004) and remained upregulated at 3 (p<0.05) and 24 (p<0.0001) hrs in IC. For ischemic WT animals, TNFR1 expression remained unchanged at 30 min but was increased in both IC and PN at 3 (p<0.01) and 24 hrs (p<0.006). Conclusions: TNFa increased in both IC and PN after MCAO in TNFa-Tg rats and remained several fold higher than in WT rats 24 hrs afterward. TACE was not constitutively upregulated but became increased in response to ischemic stress more robustly than in WT animals. TNFR1 expression became elevated in the TNFa-Tg animal during early reperfusion and showed regional heterogeneity not observed in WT rats. We conclude that cerebral ischemic injury in the TNFa-Tg rat may amplify basal TNFa synthesis through upregulation of TACE and could augment cellular death through enhanced TNFR1 activation.