u-0126 and Brain-Injuries

Moderate hypothermia (MH) used as treatment for neurological diseases has a protective effect; however, its mechanism remains unclear. Neuronal autophagy is a fundamental pathological process of early brain injury in subarachnoid hemorrhage (SAH). We found that moderate activation of autophagy can reduce nerve cells damage. In this study, We found that MH can moderately increase the level of autophagy in nerve cells and improve the neurological function in rats. This type of autophagy activation is dependent on extracellular signal-regulated kinase (ERK) signaling pathways. The level of neuronal autophagy was down-regulated significantly by using U0126, an ERK signaling pathway inhibitor. In summary, these results suggest that MH can moderately activate neuronal autophagy through ERK signaling pathway, reduce nerve cell death, and produce neuroprotective effects.

Topics: Animals; Autophagy; Brain Edema; Brain Injuries; Butadienes; CA1 Region, Hippocampal; Disease Models, Animal; Hypothermia, Induced; Male; MAP Kinase Signaling System; Neurons; Neuroprotection; Nitriles; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage

This study was undertaken to examine the function of extracellular signal-regulated kinase (ERK) signaling pathway on the proliferation and activation of microglia/macrophage and astrocytes after brain injury in mice. The result of Western blot showed that p-ERK was immediately activated after injury (<4h), but the duration was short (<4 days). According to immunofluorescence double staining, it was found that at 4 and 8h after injury, p-ERK was expressed in microglia/macrophages, and that more cells were co-expressed by p-ERK and IBA-1 (microglia/macrophage marker) at 8h; at days 1 and 4, p-ERK was expressed in astrocytes, and more cells were co-expressed by p-ERK and GFAP (astrocyte marker) at day 4. After injury, the mice were injected with U0126 (MAPK/ERK signaling pathway inhibitor) via the femoral vein. Compared with those injected with DMSO, the cell number co-expressed by p-ERK and IBA-1 or GFAP significantly decreased (P<0.05). The increase of microglia/macrophage and astrocyte caused by injury was remitted, and the positive cell number significantly decreased (P<0.05). Western blot showed that the expression quantity of IBA-1 and GFAP significantly decreased (P<0.05). Furthermore, the ERK signaling pathway was involved in the proliferation and activation of the two glial cells types and improved long-term neurobehavioral function after brain injury. Therefore, the exploration of the formation mechanism of glial scar after injury and further research on the therapeutic method of neural regeneration are essential.

Topics: Animals; Astrocytes; Blotting, Western; Brain Injuries; Butadienes; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Macrophages; Male; MAP Kinase Signaling System; Mice; Microglia; Neuralgia; Neuroglia; Nitriles; Phosphorylation; Signal Transduction

Radiation-induced normal brain injury is associated with acute and/or chronic inflammatory responses, and has been a major concern in radiotherapy. Recent studies suggest that microglial activation is a potential contributor to chronic inflammatory responses following irradiation; however, the molecular mechanism underlying the response of microglia to radiation is poorly understood. c-Jun, a component of AP-1 transcription factors, potentially regulates neural cell death and neuroinflammation. We observed a rapid increase in phosphorylation of N-terminal c-Jun (on serine 63 and 73) and MAPK kinases ERK1/2, but not JNKs, in irradiated murine microglial BV2 cells. Radiation-induced c-Jun phosphorylation is dependent on the canonical MEK-ERK signaling pathway and required for both ERK1 and ERK2 function. ERK1/2 directly interact with c-Jun in vitro and in cells; meanwhile, the JNK binding domain on c-Jun is not required for its interaction with ERK kinases. Radiation-induced reactive oxygen species (ROS) potentially contribute to c-Jun phosphorylation through activating the ERK pathway. Radiation stimulates c-Jun transcriptional activity and upregulates c-Jun-regulated proinflammatory genes, such as tumor necrosis factor-α, interleukin-1β, and cyclooxygenase-2. Pharmacologic blockade of the ERK signaling pathway interferes with c-Jun activity and inhibits radiation-stimulated expression of c-Jun target genes. Overall, our study reveals that the MEK-ERK1/2 signaling pathway, but not the JNK pathway, contributes to the c-Jun-dependent microglial inflammatory response following irradiation.

Topics: Animals; Base Sequence; Brain Injuries; Butadienes; Cell Line; DNA Primers; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Inflammation; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Microglia; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Radiation Injuries, Experimental; Reactive Oxygen Species; Recombinant Fusion Proteins; Transcription, Genetic

N-methyl-D-aspartate (NMDA)-induced pial artery dilation (PAD) is reversed to vasoconstriction after fluid percussion brain injury (FPI). Tissue type plasminogen activator (tPA) is up-regulated and the tPA antagonist, EEIIMD, prevents impaired NMDA PAD after FPI. Mitogen-activated protein kinase (MAPK), a family of at least three kinases, ERK, p38, and JNK, is also up-regulated after traumatic brain injury (TBI). We hypothesize that tPA impairs NMDA-induced cerebrovasodilation after FPI in a MAPK isoform-dependent mechanism.. Lateral FPI was induced in newborn pigs. The closed cranial window technique was used to measure pial artery diameter and to collect cerebrospinal fluid (CSF). ERK, p38, and JNK MAPK concentrations in CSF were quantified by ELISA.. CSF JNK MAPK was increased by FPI, increased further by tPA, but blocked by JNK antagonists SP600125 and D-JNKI1. FPI modestly increased p38 and ERK isoforms of MAPK. NMDA-induced PAD was reversed to vasoconstriction after FPI, whereas dilator responses to papaverine were unchanged. tPA, in post-FPI CSF concentration, potentiated NMDA-induced vasoconstriction while papaverine dilation was unchanged. SP 600125 and D-JNKI1, blocked NMDA-induced vasoconstriction and fully restored PAD. The ERK antagonist U 0126 partially restored NMDA-induced PAD, while the p38 inhibitor SB203580 aggravated NMDA-induced vasoconstriction observed in the presence of tPA after FPI.. These data indicate that tPA contributes to impairment of NMDA-mediated cerebrovasodilation after FPI through JNK, while p38 may be protective. These data suggest that inhibition of the endogenous plasminogen activator system and JNK may improve cerebral hemodynamic outcome post-TBI.

Topics: Animals; Animals, Newborn; Anthracenes; Brain Injuries; Butadienes; Drug Interactions; Female; Imidazoles; JNK Mitogen-Activated Protein Kinases; Male; Mitogen-Activated Protein Kinases; N-Methylaspartate; Nitriles; Oligopeptides; Papaverine; Peptides; Pia Mater; Pyridines; Swine; Tissue Plasminogen Activator; Up-Regulation; Vasoconstriction; Vasodilation

Aquaporin-4 (AQP4) is the principle water channel and the primary route for water transport across astrocytic membranes. AQP4 co-localizes with Kir4.1 channels at astrocytic endfeet, and it has been suggested that these channels cooperate in K(+) and water homeostasis. In response to injury, two additional aquaporins, AQP1 and AQP9, can be detected in astrocytes, yet neither is found in cultured astrocytes, and therefore their contribution to astrocyte water uptake and biology is poorly investigated. In this study, we used a cortical stab wound assay to demonstrate an upregulation of AQP1 following injury in reactive glia. We were able to mimic such injury in astrocytic cultures and show that AQP1 expression is induced within 16 h following injury in vitro. This induction could be blocked by inhibition of MEK1/2 using U0126, and suggests that AQP1 is specifically induced in reactive astrocytes via the mitogen-activated protein kinases signaling pathway.

Topics: Animals; Aquaporin 1; Aquaporin 4; Astrocytes; Brain Injuries; Butadienes; Cells, Cultured; Enzyme Inhibitors; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Nitriles; Rats; Rats, Sprague-Dawley; RNA, Messenger; Time Factors; Wounds, Stab

We previously demonstrated that N-methyl-D-aspartate (NMDA) treatment (50 microM, 3 h) induced astrocytic production of monocyte chemoattractant protein-1 (MCP-1, CCL2), a CC chemokine implicated in ischemic and excitotoxic brain injury, in rat corticostriatal slice cultures. In this study, we investigated the signaling mechanisms for NMDA-induced MCP-1 production in slice cultures. The results showed a close correlation between NMDA-induced neuronal injury and MCP-1 production, and an abrogation of NMDA-induced MCP-1 production in NMDA-pretreated slices where neuronal cells had been eliminated. These results collectively indicate that NMDA-induced neuronal injury led to astrocytic MCP-1 production. NMDA-induced MCP-1 production was significantly inhibited by U0126, an inhibitor of extracellular signal-regulated kinase (ERK). Immunostaining for phosphorylated ERK revealed that transient neuronal ERK activation was initially induced and subsided within 30 min, followed by sustained ERK activation in astrocytes. Treatment with U0126 during only the early phase (U0126 was washed out at 15 or 30 min after NMDA administration) suppressed early activation of ERK in neuronal cells, but not later activation of ERK in astrocytes. In this case, MCP-1 production was not suppressed, suggesting that activation of neuronal ERK is not necessary for MCP-1 production. In contrast, delayed application of U0126 at 3 h after the beginning of NMDA treatment inhibited MCP-1 production to the same degree as that observed when U0126 was applied from 3 h before NMDA administration. These findings suggest that sustained activation of the ERK signaling pathway in astrocytes plays a key role in neuronal injury-induced MCP-1 production.

Topics: Animals; Astrocytes; Brain Injuries; Butadienes; Cerebral Cortex; Chemokine CCL2; Corpus Striatum; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; In Vitro Techniques; MAP Kinase Signaling System; N-Methylaspartate; Neurons; Nitriles; Phosphorylation; Rats; Rats, Wistar; Time Factors

The extracellular signal-regulated kinase (ERK) pathway, which modulates the activity of many transcriptional factors leading to the proliferation of various cells, is activated in lesions in regions of selective vulnerability after traumatic brain injury (TBI). In the present study, using the ERK inhibitor U0126, we investigated the role of the ERK pathway in histopathological and behavioral outcomes after TBI. Adult male Sprague-Dawley rats, weighing 300-400 g were subjected to lateral fluid percussion brain injury. The ERK inhibitor U0126 was injected intravenously before injury at 100, 200 and 400 microg/kg. The severity of CA3 neuronal damage was evaluated by the number of surviving CA3 neurons 7 days after injury. The contusional lesion volume 72 h after injury was analysed using a computer-assisted analysis system. Three different motor skill tasks were measured on days 1-5, 7, 14 and 21 after injury. Pretreatment with U0126 significantly reduced both CA3 neuronal damage and contusional lesion volume after injury. In addition, administration of U0126 ameliorated motor function recovery on days 3, 4 and 5 after injury. Therefore, inhibition of ERK phosphorylation could be a potentially effective therapeutic target after TBI.

Topics: Animals; Behavior, Animal; Blotting, Western; Body Weight; Brain; Brain Injuries; Butadienes; Cell Count; Cell Survival; Cerebral Cortex; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Hippocampus; Immunohistochemistry; Male; Neurons; Nitriles; Postural Balance; Rats; Rats, Sprague-Dawley

We have studied the involvement of the thrombin receptor [protease-activated receptor-1 (PAR-1)] in astrogliosis, because extravasation of PAR-1 activators, such as thrombin, into brain parenchyma can occur after blood-brain barrier breakdown in a number of CNS disorders. PAR1-/- animals show a reduced astrocytic response to cortical stab wound, suggesting that PAR-1 activation plays a key role in astrogliosis associated with glial scar formation after brain injury. This interpretation is supported by the finding that the selective activation of PAR-1 in vivo induces astrogliosis. The mechanisms by which PAR-1 stimulates glial proliferation appear to be related to the ability of PAR-1 receptor signaling to induce sustained extracellular receptor kinase (ERK) activation. In contrast to the transient activation of ERK by cytokines and growth factors, PAR-1 stimulation induces a sustained ERK activation through its coupling to multiple G-protein-linked signaling pathways, including Rho kinase. This sustained ERK activation appears to regulate astrocytic cyclin D1 levels and astrocyte proliferation in vitro and in vivo. We propose that this PAR-1-mediated mechanism underlying astrocyte proliferation will operate whenever there is sufficient injury-induced blood-brain barrier breakdown to allow extravasation of PAR-1 activators.

Topics: Amides; Analysis of Variance; Animals; Animals, Newborn; Astrocytes; Blotting, Northern; Blotting, Western; Brain Injuries; Bromodeoxyuridine; Butadienes; Cell Count; Cell Movement; Cell Proliferation; Cells, Cultured; Coculture Techniques; Colforsin; Cyclin D1; Disease Models, Animal; Drug Interactions; Enzyme Inhibitors; Functional Laterality; Glial Fibrillary Acidic Protein; Gliosis; Immunohistochemistry; Male; MAP Kinase Kinase Kinases; Mice; Mice, Knockout; Microglia; Nitriles; Oligopeptides; Pyridines; Receptor, PAR-1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thrombin; Time Factors

Emerging data suggest that matrix metalloproteinase-9 (MMP-9) plays a critical role in the pathophysiology of brain injury. However, the regulatory mechanisms involved in vivo remain unclear. In this study, we focus on a mitogen activated protein kinase (MAPK) pathway that may trigger MMP-9 after traumatic brain injury. We aim to show that inhibition of the extracellular signal regulated kinase (ERK) would attenuate MMP-9 levels, reduce blood-brain barrier damage, and attenuate edema after trauma induced by controlled cortical impact in mouse brain. Western blots showed that phospho-ERK was rapidly upregulated after trauma. Treatment with U0126, which inhibits MEK, the kinase upstream of ERK, effectively prevented the activation of ERK. After trauma, gelatin zymography showed an increase in MMP-9. U0126 significantly reduced trauma-induced MMP-9 levels. Correspondingly, U0126 ameliorated the degradation of the tight junction protein ZO-1, which is an MMP-9 substrate, and significantly attenuated tissue edema. At 7 days after trauma, traumatic lesion volumes were significantly reduced by U0126 compared with saline-treated controls. These data indicate that the ERK MAPK pathway triggers the upregulation in MMP-9 after trauma, and further suggest that targeting the upstream signaling mechanisms that regulate deleterious MMP-9 activity may reveal new therapeutic opportunities for traumatic brain injury.

Topics: Animals; Brain Injuries; Butadienes; Down-Regulation; Edema; Enzyme Inhibitors; Male; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Nitriles

Matrix metalloproteinases (MMP) are involved in the pathophysiology of brain injury. We recently showed that knockout mice deficient in MMP-9 expression were protected against traumatic brain injury. However, the cellular sources of MMP activity after trauma remain to be fully defined. In this study, we investigated the hypothesis that resident brain cells secrete MMP after mechanical trauma injury in vitro, and mitogen-activated protein (MAP) kinase signal transduction pathways are involved in this response. Rat primary cortical neurons, astrocytes, and co-cultures were subjected to needle scratch mechanical injury, and levels of MMP-2 and MMP-9 in conditioned media were assayed by zymography. MMP-2 and MMP-9 were increased in cortical astrocytes and co-cultures, whereas only MMP-2 was increased in neurons. Western blots showed that phosphorylated extracellular signal regulated kinase (ERK1/2) and p38 were rapidly upregulated in co-cultures after mechanical injury. No change in phosphorylated c-jun N-terminal kinase (JNK) was observed. In-gel kinase assays confirmed this lack of response in the JNK pathway. Treatment with either 10 microM of U0126 (a MAP kinase/ERK1/2 kinase inhibitor) or 10 microM of SB203580 (a p38 inhibitor) had no detectable effect on MMP-2 and MMP-9 levels after mechanical injury. However, combination treatment with both inhibitors significantly reduced secretion of MMP-9. Herein, we demonstrate that (1) resident brain cells secrete MMP after mechanical injury, (2) astrocytes are the main source of MMP-9 activity, and (3) ERK and p38 MAP kinases are upregulated after mechanical injury, and mediate the secretion of MMP-9.

Topics: Animals; Brain Injuries; Butadienes; Cells, Cultured; Cerebral Cortex; Enzyme Inhibitors; Imidazoles; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neurons; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyridines; Rats; Rats, Sprague-Dawley; Up-Regulation