EXPRESSION OF TUMOR-NECROSIS-FACTOR-ALPHA AFTER FOCAL CEREBRAL-ISCHEMIA IN THE RAT

Induction of tumor necrosis factor alpha was studied in the brain of r ats after focal cerebral ischaemia by occlusion of the left middle cer ebral artery. Using a specific antisense riboprobe for in situ hybridi zation histochemistry, cells positive for tumor necrosis factor alpha messenger RNA were detected within 30 min in the brain regions known t o be necrotic within one to two days after onset of ischaemia. Their n umber increased over a time period of 1-8 h and then declined. Only a few tumor necrosis factor alpha messenger RNA positive cells could be detected four days after the onset of ischaemia. Reverse-transcription polymerase chain reaction experiments showed that maximal increase of tumor necrosis factor alpha messenger RNA level in the ischaemic brai n hemisphere occurred 3 h after occlusion of the middle cerebral arter y. Immunocytochemical experiments using an anti-tumor necrosis factor alpha antibody showed the presence of tumor necrosis factor alpha immu nopositive cells as early as 30 min after occlusion of the middle cere bral artery in the same brain regions where tumor necrosis factor alph a messenger RNA positive cells were detected. Tumor necrosis factor al pha positive cells were highly abundant in the infarcted brain 8-24 h, but only few of them were detectable four days after the onset of isc haemia. Specificity of the anti-tumor necrosis factor alpha antibody a nd of the induction of tumor necrosis factor alpha protein was confirm ed by western blot analysis. Tumor necrosis factor alpha messenger RNA - and protein-positive cells were also detected in the watershed zone and in some structures of the contralateral brain hemisphere. Accordin g to their morphology, tumor necrosis factor alpha-positive cells coul d be identified as microglial cells and macrophages at different state s of activation. This assumption was further confirmed by double-label ing studies using the isolectin B-4 from Griffonia simplicifolia, a sp ecific microglial/macrophage cell marker. These results demonstrate th at expression of tumor necrosis factor alpha is part of an intrinsic i nflammatory reaction of the brain after ischaemia.