Possible Role Of Raf-1 Kinase In The Development Of Cerebral Vasospasm And Early Brain Injury After Experimental Subarachnoid Hemorrhage In Rats

Part one: Expression of phosphorylated Raf-1 in the basilar artery after experimental subarachnoid hemorrhage in ratsObjective This study aimed to investigate the expression of phosphorylated Raf-1 in the basilar artery in a rat SAH model and to clarify the potential role of Raf-1 kinase in cerebral vasospasm.Methods A total of 72 rats were randomly divided into four groups: control group; day 3, day 5, and day 7 groups. Day 3, day 5, and day 7 groups were all SAH groups. The animals in SAH groups were subjected to injection of autologous blood into cisterna magna twice on day 0 and day 2 and were sacrificed on days 3, 5, and 7, respectively. Cross-sectional area of basilar artery was measured and the morphological changes of basilar artery were detected with light microscopy. The phosphorylated Raf-1 expression was assessed by immunohistochemistry and Western blot.Results The rat“double-hemorrhage”model of vasospasm was successed to be induced. The cross-sectional area of basilar artery was found to be 57,944±5,581μm2 in control group, 26,100±2,639μm2 in day 3, 19,723±2,412μm2 in day 5, and 28,800±2,980μm2 in day 7 group, respectively. The basilar artery exhibited vasospasm after SAH and became more severe on day 5 and was characterized by arterial norrowing, thickness of arterial wall, degeneration of endothelial cells and smooth muscle cells. The elevated protein of phosphorylated Raf-1 were detected after SAH and peaked on day 5.Conclusion Phosphorylated Raf-1 is increasingly expressed in a parallel time course to the development of cerebral vasospasm in a rat experimental model of SAH. These findings indicate the possible role of Raf-1 in the pathogenesis of cerebral vasospasm after SAH.Part two: Role of Raf-1 kinase in cerebral vasospasm after experimental subarachnoid hemorrhageObjective This study was conducted to examine the effect of BAY 43-9006, a highly specific inhibitor of Raf-1 kinase, on cerebral vasospasm and to clarify the possible role of Raf-1 kinase in cerebral vasospasm.Methods A total of 144 rats were randomly divided into four groups: control, SAH, SAH+Vehicle and SAH+BAY 43-9006. An established rat double-hemorrhage model of SAH was used by injecting autologous arterial blood into the cisterna magna on days 0 and 2 and were sacrificed on day5. Cross-sectional area of basilar artery was measured and the morphological changes of basilar artery were detected with light microscopy. The phosphorylated Raf-1 was assessed by immunohistochemistry and Western blot. The phosphorylated ERK1/2 and the activation of NF-κB along with the IL-6 and IL-1βmRNA in the basilar arteries were also examined.Results Severe vasospasm was observed in the basilar arteries of SAH and Vehicle rats with cross-sectional area of which were 17,225±4,436μm2 and 19,780±7,870μm2, respectively. The enhanced protein of phosphorylated Raf-1 and phosphorylated ERK1/2 were detected in the basilar arteries after SAH along with the activation of NF-κB and the concomitant upregulation of IL-6 and IL-1βmRNA production. Oral administration of BAY 43-9006 decreased phosphorylated Raf-1 and phosphorylated ERK1/2 in the basilar arteries and reduced the activation of NF-κB and the production of IL-6 and IL-1βmRNA with a concomitant reduction of morphological vasospasm.Conclusions These results suggest that Raf-1/ERK1/2 and Raf-1/NF-κB signaling pathway are activated in the basilar arteries after SAH, leading to the development of vasospasm. Suppressing the activity of Raf-1 kinase may have potential in the management of cerebral vasospasm. Part three: Role of Raf-1 kinase in early brain injury after experimental subarachnoid hemorrhageObjective This study was designed to explore the possible role of Raf-1 kinase in early brain injury after SAH.Methods A total of 96 rats were randomly divided into four groups: control, SAH, SAH+DMSO and SAH+BAY 43-9006. The rat SAH model was induced by injecting autologous arterial, non-heparinized blood into the prechiasmatic cistern and was sacrificed at 24 hours. EBI was assessed in terms of brain water content, blood-brain barrier permeability and apoptotic index in the cortex at 24 hours after SAH. The phosphorylated Raf-1 was examined in the cortex by immunohistochemistry and Western blot. The phosphorylated ERK1/2 and the activation of NF-κB along with the MMP-9, VEGF, COX-2, and IκB-αin the cortex were also examined.Results Experimental SAH led to the brain edema, blood-brain barrier impairment, and cortical apoptosis. SAH markedly increased expressions of Raf-1 pathway related agents, such as phosphorylated Raf-1, phosphorylated ERK1/2, NF-κB, MMP-9, VEGF, COX-2, and decreased expression of IκB-αin the cortex. Administration of BAY 43-9006 following SAH significantly decreased the phosphorylated Raf-1, phosphorylated ERK1/2, NF-κB, MMP-9, VEGF, COX-2, and increased expression of IκB-αwith a concomitant amelioration of EBI, such as cortical apoptosis, brain edema, and blood-brain barrier impairment.Conclusions These results demonstrate that Raf-1 kinase contributes to early brain injury after SAH by enhancing the activation of the Raf-1/ERK1/2 and Raf-1/NF-κB signaling pathways, and that the inhibition of these pathways might offer new treatment strategies for early brain injury after SAH.