Expression of tumor necrosis factor alpha after focal cerebral ischaemia in the rat

Induction of tumor necrosis factor α was studied in the brain of rats after focal cerebral ischaemia by occlusion of the left middle cerebral artery. Using a specific antisense riboprobe for situ hybridization histochemistry, cells positive for tumor necrosis factor α messenger RNA were detected within 30 min in the brain regions known to be necrotic within one to two days after onset of ischaemia. Their number increased over a time period of 1–8 h and then declined. Only a few tumor necrosis factor α 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 α messenger RNA level in the ischaemic brain hemisphere occurred 3 h after occlusion of the middle cerebral artery. Immunocytochemical experiments using an anti-tumor necrosis factor α antibody showed the presence of tumor necrosis factor α immunopositive cells as early as 30 min after occlusion of the middle cerebral artery in the same brain regions where tumor necrosis factor α messenger RNA positive cells were detected. Tumor necrosis factor α 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 ischaemia. Specificity of the anti-tumor necrosis factor α antibody and of the induction of tumor necrosis factor α protein was confirmed by western blot analysis. Tumor necrosis factor α messenger RNA- and protein-positive cells were also detected in the watershed zone and in some structures of the contralateral brain hemisphere. According to their morphology, tumor necrosis factor α-positive cells could be identified as microglial cells and macrophages at different states of activation. This assumption was further confirmed by double-labeling studies using the isolectin B 4 from Griffonia simplicifolia, a specific microglial/macrophage cell marker. These results demonstrate that expression of tumor necrosis factor α is part of an intrinsic inflammatory reaction of the brain after ischaemia.