Blockade of the MEK/ERK Pathway with a Raf Inhibitor Prevents Activation of Pro-Inflammatory Mediators in Cerebral Arteries and Reduction in Cerebral Blood Flow after Subarachnoid Hemorrhage in a Rat Model

Abstract

Cerebral ischemia that develops after subarachnoid hemorrhage (SAH) carries high morbidity and mortality. Inflammatory mediators are involved in the development of cerebral ischemia through activation of the mitogen-activated protein kinase pathway. We hypothesized that blockade of the MAPkinase/ERK (MEK)/extracellular signal-regulated kinase (ERK) pathway upstream with a specific raf inhibitor would prevent SAH-induced activation of the cerebrovascular inflammatory response. The raf inhibitor SB-386023-b was injected intracisternally in our rat model at 0, 6, or 12 hours after the SAH. After 48 hours, cerebral arteries were harvested, and iNOS, interleukin (IL)-6, IL-1b, matrix metalloproteinase (MMP)-9, tissue inhibitors of metalloproteinase (TIMP)-1, and phosphorylated ERK1/2 were investigated by immunofluorescence, real-time polymerase chain reaction (PCR), and Western blot analysis. Cerebral blood flow (CBF) was measured using autoradiography. Protein levels of MMP-9, TIMP-1, iNOS, IL-6, and IL-1b were increased after SAH, as were mRNA levels of IL-6, MMP-9, and TIMP-1. After SAH, pERK1/2 was increased, but CBF was reduced. Treatment with SB-386023-b at 0 or 6 hours after SAH normalized CBF and prevented SAH-induced upregulation of MMPs, pro-inflammatory cytokines, and pERK1/2 proteins. These results suggested that inhibition of MEK/ERK signal transduction by a specific raf inhibitor administered up to 6 hours after SAH normalized the expression of pro-inflammatory mediators and extracellular matrix-related genes.

Vascular plasticity in cerebrovascular disorders

“…However, the authors did not find increased cytokine mRNA levels in the cerebral artery walls after SAH (Maddahi et al, 2011). This suggests that the cytokines in the smooth muscle layer are either synthesized outside the vessels or by a small subpopulation of infiltrating cells in the smooth muscle cell layer, such as leukocytes.…”

“…In experimental SAH, the delayed global cerebral ischemia 2 days after the SAH was also shown to be associated with enhanced protein levels of IL-1b, IL-6, and nitric oxide synthase in the smooth muscle layer of cerebral arteries (Maddahi et al, 2011). Blockage of the MEK/ERK pathway prevented this response in both models, and this effect could also be seen when treatment with the blocker was delayed for up to 6 hours after the insult (Maddahi and Edvinsson, 2010;Maddahi et al, 2011).…”

“…Thus, 2 hours of MCAO followed by reperfusion for 46 hours resulted in enhanced levels of tumor necrosis factor-a, IL-1b, IL-6, and nitric oxide synthase in the smooth muscle layer of the ischemic MCA and in associated intracerebral microvessels (Maddahi and Edvinsson, 2010). In experimental SAH, the delayed global cerebral ischemia 2 days after the SAH was also shown to be associated with enhanced protein levels of IL-1b, IL-6, and nitric oxide synthase in the smooth muscle layer of cerebral arteries (Maddahi et al, 2011). Blockage of the MEK/ERK pathway prevented this response in both models, and this effect could also be seen when treatment with the blocker was delayed for up to 6 hours after the insult (Maddahi and Edvinsson, 2010;Maddahi et al, 2011).…”

“…In experimental MCAO and SAH, enhanced protein and mRNA expression of MMP-9 and TIMP-1 has been demonstrated in cerebral vessel walls. Interestingly, MMP-9 and TIMP-1 was especially associated with smooth muscle actin but there was no overlap with the astrocyte/ glial cell marker GFAP in the vessel walls, indicating that the transcriptional upregulation takes place in the vascular smooth muscle cells themselves (Maddahi et al, 2009(Maddahi et al, , 2011. Treatment with inhibitors of the MEK-ERK1/2 pathway normalized the expression of MMP-9 and TIMP-1 (Maddahi et al, 2009(Maddahi et al, , 2011, indicating that both upregulation of pro-inflammatory mediators and extracellular-matrix-related proteins is governed by at least some of the same signaling pathways as cerebrovascular receptor upregulation.…”

Genetic Elimination of eNOS Reduces Secondary Complications of Experimental Subarachnoid Hemorrhage

“…Subarachnoid hemorrhage increased iNOS in both wild-type and eNOS KO mice, which is consistent with other studies in wild-type animals. 26,27 Increased iNOS activity could produce more ROS and aggravate brain injury and large artery vasospasm. [28][29][30] Yatsushige et al, 31 however, found that inhibiting the increase in iNOS after SAH in rats did not reduce blood-brain barrier breakdown, brain edema, neuron death, or mortality.…”