u-0126 and Brain-Ischemia

N-methyl-D-aspartate (NMDA) receptors are key signaling molecules that mediate excitotoxicity during cerebral ischemia. GluN2A-containing NMDA receptors, which are mostly located in the intrasynaptic region, mediate normal physiological processes and promote neuronal survival. GluN2B-containing NMDA receptors, which are mostly located in the extrasynaptic region, mediate excitotoxicity injury and promote neuronal death during ischemia. This study investigated the ability of icaritin (ICT) to protect against cerebral ischemia‒reperfusion injury (CI/RI) by regulating GluN2B-containing NMDA receptors through extracellular signaling regulatory kinases/death associated protein kinase 1 (ERK/DAPK1) signaling. A rat CI/RI model was established by transient middle cerebral artery occlusion (tMCAO). Following treatment with ICT and the ERK-specific inhibitor U0126, cerebral infarction, neurological function, and excitotoxicity-related molecule expression were assessed 24 h after reperfusion. ICT treatment significantly decreased cerebral infarct volume, improved neurological function, and regulated NMDA receptor subtype expression and ERK/DAPK1 signaling activation. The ability of ICT to increase GluN2A and postsynaptic density protein 95 (PSD95) mRNA and protein expression, inhibit GluN2B expression, and regulate DAPK1 activation was reversed after administration of the ERK-specific inhibitor U0126. These data indicated that ICT inhibited excitotoxicity injury and exerted a protective effect against CI/RI that was likely mediated by increased ERK signaling pathway activation and regulation of extrasynaptic and intrasynaptic NMDA receptor function, providing a new therapeutic target for ischemic encephalopathy.

Topics: Animals; Brain Ischemia; Neurons; Rats; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Signal Transduction

BACKGROUND Long-non-coding RNA (lncRNA) SNHG15 has been reported to be an aberrantly expressed lncRNA in patients with ischemic stroke, but its role in neuronal injury following ischemic stroke remains unclear. We hypothesized that this lncRNA is associated with the pathogenesis of ischemic stroke. MATERIAL AND METHODS A mouse model of ischemic stroke was established by middle cerebral artery occlusion (MCAO). A neurogenic mouse cell line Neuro-2a (N2a) was subjected to oxygen-glucose deprivation (OGD) for in vitro experiments. Expression of SNHG15, microRNA-18a (miR-18a), and CXCL13 in mouse brain and in OGD-treated N2a cells was determined. Altered expression of SNHG15 and miR-18a was introduced to detect their roles in N2a cell viability and apoptosis. Targeting relationships between miR-18a and SNHG15 or CXCL13 were validated by luciferase assays. Cells were treated with the ERK/MEK antagonist U0126 to assess the role of the ERK/MEK signaling pathway in N2a cell growth. RESULTS SNHG15 and CXCL13 were overexpressed and miR-18a was underexpressed in MCAO-induced mice and OGD-treated N2a cells. Silencing of SNHG15 or overexpression of miR-18a promoted cell viability, while decreased cell apoptosis induced by OGD; however, subsequent disruption of the ERK/MEK signaling pathway reversed these effects. SNHG15 was found to bind to miR-18a, which could further target CXCL13. CONCLUSIONS Silencing of SNHG15 led to CXCL13 upregulation through sequestering miR-18a and the following ERK/MEK activation, thus enhancing viability while reducing apoptosis of N2a cells. SNHG15 may serve as a novel target for ischemic stroke treatment.

Topics: Animals; Apoptosis; Brain Ischemia; Butadienes; Chemokine CXCL13; Gene Expression; Male; Mice; Mice, Inbred C57BL; MicroRNAs; Mitogen-Activated Protein Kinases; Nitriles; Protein Kinase Inhibitors; RNA, Long Noncoding; RNA, Small Nucleolar; Stroke

To investigate the relationship between the Erk1/2 signal pathway and neuronal apoptosis in ischemic stroke rats. Male SD(Sprague Dawley)  rats (n = 24) were randomly divided into three groups, each containing 8 rats: sham-operated group, MCAO(Midle cerebral artery oclusion)  group, and MCAO + U0126 intervention group (U0126 group). In in vitro trial, primary cortical nerve cells were divided into three groups: control group, OGD(Oxygen and glucose deprivation)  group, and U0126 intervention group (U0126 group). In vivo protein expression levels of Erk1/2, p-Erk1/2 and Bcl-2 were determined using western blot. The expressions of Bcl-2, Bcl-xl and Bax were assayed using immunohistochemical staining. Nerve cell mortality in cerebral tissue was detected using TUNEL staining. In in vitro trials, cell apoptosis was assayed with flow cytometry and LDH release. The activity of caspase-3 was determined. Nerve cell apoptosis was determined using Hoechst33258 staining method. In in vivo trial, it was found that the protein expression level of p-ERK1/2 in cerebral tissue in the MCAO group was significantly increased, when compared with that of the sham-operated group, while the protein expression level of p-Erk1/2 in the U0126 group was significantly lower than that in the MCAO group. The expression levels of Bcl-2 and Bcl-xl in the MCAO group were significantly lower than the corresponding expression levels in the sham-operated group, while the expressions of Bcl-2 and Bcl-xl in the U0126 group were significantly lower than those in MCAO group. In MCAO group, the expression of Bax was significantly higher than that in the sham-operated group, while Bax expression was higher in U0126 than in MCAO group. There were significantly higher number of dead nerve cells in MCAO group than in the sham-operated group, while nerve cell mortality in U0126 group was significantly lower than in MCAO group. In in vitro trials, flow cytometry revealed significantly higher apoptosis of OGD-treated nerve cells, relative to the control group. Nerve cells exposed to U0126 and treated with ODR (Oxygen-dependent repressor)  were significantly decreased in population, when compared with single OGD treatment group. The LDH release level of nerve cells treated OGD was significantly increased, when compared with that of the control group. However, LDH release level of nerve cells treated with OGD after U0126 intervention was significantly decreased, relative to the single OGD treatment group.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Brain Ischemia; Butadienes; Caspase 3; Cerebral Cortex; In Situ Nick-End Labeling; Male; MAP Kinase Signaling System; Nitriles; Proto-Oncogene Proteins c-bcl-2; Random Allocation; Rats; Rats, Sprague-Dawley

Cerebral ischemia can trigger the ERK1/2 signaling cascade that enables the brain to adapt to ischemic injury. However, the mechanism of ERK1/2 in ischemic brain injury remains unclear. The aim of this study was to examine the roles of the ERK1/2 signaling pathway and NMDA receptors in the apoptosis of CA1 pyramidal neurons after ischemia/reperfusion (I/R).. Male Wistar rats were subjected to a sham or transient forebrain ischemia procedure. Animals received the intracerebroventricular injection of U0126 (5 μl, 0.2 μg/μl) or vehicle 30 min before ischemia. Homogenates of the hippocampal CA1 field were obtained from sham-operated and ischemic rats 6, 12 or 48 h after ischemia/reperfusion (n = 6 per group) and then subjected to Western blotting analysis and TUNEL staining. Caspase-3 activity was assayed with a colorimetric assay kit.. We found that the phosphorylation level of ERK1/2 is increased in the CA1 region following transient I/R. Blocking the ERK1/2 signaling pathway by administration U0126 attenuated apoptotic neuronal cell death via inhibition of NMDA receptors.. These findings suggest a novel mechanism by which the ERK1/2 signaling pathway affects the post-I/R apoptosis of CA1 pyramidal neurons, which will provide a therapeutic target for the treatment of stroke.

Topics: Animals; Apoptosis; Brain Ischemia; Butadienes; CA1 Region, Hippocampal; Male; MAP Kinase Signaling System; Neurons; Neuroprotective Agents; Nitriles; Phosphorylation; Prosencephalon; Rats, Wistar; Receptors, N-Methyl-D-Aspartate

Multi-protein complexes, termed "inflammasomes," are known to contribute to neuronal cell death and brain injury following ischemic stroke. Ischemic stroke increases the expression and activation of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) Pyrin domain containing 1 and 3 (NLRP1 and NLRP3) inflammasome proteins and both interleukin (IL)-1β and IL-18 in neurons. In this study, we provide evidence that activation of either the NF-κB and MAPK signaling pathways was partly responsible for inducing the expression and activation of NLRP1 and NLRP3 inflammasome proteins and that these effects can be attenuated using pharmacological inhibitors of these two pathways in neurons and brain tissue under in vitro and in vivo ischemic conditions, respectively. Moreover, these findings provided supporting evidence that treatment with intravenous immunoglobulin (IVIg) preparation can reduce activation of the NF-κB and MAPK signaling pathways resulting in decreased expression and activation of NLRP1 and NLRP3 inflammasomes, as well as increasing expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary cortical neurons and/or cerebral tissue under in vitro and in vivo ischemic conditions. In summary, these results provide compelling evidence that both the NF-κB and MAPK signaling pathways play a pivotal role in regulating the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons and brain tissue under ischemic conditions. In addition, treatment with IVIg preparation decreased the activation of the NF-κB and MAPK signaling pathways, and thus attenuated the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons under ischemic conditions. Hence, these findings suggest that therapeutic interventions that target inflammasome activation in neurons may provide new opportunities in the future treatment of ischemic stroke.

Topics: Adaptor Proteins, Signal Transducing; Animals; Anthracenes; Apoptosis Regulatory Proteins; Brain; Brain Ischemia; Butadienes; Extracellular Signal-Regulated MAP Kinases; Imidazoles; Inflammasomes; Mice; Neurons; NF-kappa B; Nitriles; NLR Family, Pyrin Domain-Containing 3 Protein; Pyridines; Signal Transduction; Stroke; Sulfones

Cerebral vasospasm and late cerebral ischemia (LCI) remain leading causes of mortality in patients experiencing a subarachnoid hemorrhage (SAH). This occurs typically 3 to 4 days after the initial bleeding and peaks at 5 to 7 days. The underlying pathophysiology is still poorly understood. Because SAH is associated with elevated levels of endothelin-1 (ET-1), focus has been on counteracting endothelin receptor activation with receptor antagonists like clazosentan, however, with poor outcome in clinical trials. We hypothesize that inhibition of intracellular transcription signaling will be an effective approach to prevent LCI. Here, we compare the effects of clazosentan versus the MEK1/2 blocker U0126 in a rat model of SAH. Although clazosentan directly inhibits the contractile responses in vivo to ET-1, it did not prevent SAH-induced upregulation of ET receptors in cerebral arteries and did not show a beneficial effect on neurologic outcome. U0126 had no vasomotor effect by itself but counteracts SAH-induced receptor upregulation in cerebral arteries and improved outcome after SAH. We suggest that because SAH induces elevated expression of several contractile receptor subtypes, it is not sufficient to block only one of these (ET receptors) but inhibition of transcriptional MEK1/2-mediated upregulation of several contractile receptors may be a viable way towards alleviating LCI.

Topics: Animals; Brain Ischemia; Butadienes; Cerebral Arteries; Dioxanes; Disease Models, Animal; Endothelin-1; Enzyme Inhibitors; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Nitriles; Pyridines; Pyrimidines; Rats; Rats, Sprague-Dawley; Receptors, Endothelin; Subarachnoid Hemorrhage; Sulfonamides; Tetrazoles; Up-Regulation

Global cerebral ischemia following cardiac arrest is associated with increased cerebral vasoconstriction and decreased cerebral blood flow, contributing to delayed neuronal cell death and neurological detriments in affected patients. We hypothesize that upregulation of contractile ETB and 5-HT1B receptors, previously demonstrated in cerebral arteries after experimental global ischemia, are a key mechanism behind insufficient perfusion of the post-ischemic brain, proposing blockade of this receptor upregulation as a novel target for prevention of cerebral hypoperfusion and delayed neuronal cell death after global cerebral ischemia. The aim was to characterize the time-course of receptor upregulation and associated neuronal damage after global ischemia and investigate whether treatment with the MEK1/2 inhibitor U0126 can prevent cerebrovascular receptor upregulation and thereby improve functional outcome after global cerebral ischemia. Incomplete global cerebral ischemia was induced in Wistar rats and the time-course of enhanced contractile responses and the effect of U0126 in cerebral arteries were studied by wire myography and the neuronal cell death by TUNEL. The expression of ETB and 5-HT1B receptors was determined by immunofluorescence.. Enhanced vasoconstriction peaked in fore- and midbrain arteries 3 days after ischemia. Neuronal cell death appeared initially in the hippocampus 3 days after ischemia and gradually increased until 7 days post-ischemia. Treatment with U0126 normalised cerebrovascular ETB and 5-HT1B receptor expression and contractile function, reduced hippocampal cell death and improved survival rate compared to vehicle treated animals.. Excessive cerebrovascular expression of contractile ETB and 5-HT1B receptors is a delayed response to global cerebral ischemia peaking 3 days after the insult, which likely contributes to the development of delayed neuronal damage. The enhanced cerebrovascular contractility can be prevented by treatment with the MEK1/2 inhibitor U0126, diminishes neuronal damage and improves survival rate, suggesting MEK1/2 inhibition as a novel strategy for early treatment of neurological consequences following global cerebral ischemia.

Topics: Animals; Brain Ischemia; Butadienes; Cerebrovascular Circulation; Drug Evaluation, Preclinical; Hypoxia, Brain; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Nitriles; Rats; Rats, Wistar; Receptor, Endothelin B; Receptor, Serotonin, 5-HT1B; Treatment Outcome; Up-Regulation; Vasoconstriction

Upregulation of vasoconstrictor receptors in cerebral arteries, including endothelin B (ETB) and 5-hydroxytryptamine 1B (5-HT(1B)) receptors, has been suggested to contribute to delayed cerebral ischemia, a feared complication after subarachnoid hemorrhage (SAH). This receptor upregulation has been shown to be mediated by intracellular signalling via the mitogen activated protein kinase kinase (MEK1/2)--extracellular regulated kinase 1/2 (ERK1/2) pathway. However, it is not known what event(s) that trigger MEK-ERK1/2 activation and vasoconstrictor receptor upregulation after SAH.We hypothesise that the drop in cerebral blood flow (CBF) and wall tension experienced by cerebral arteries in acute SAH is a key triggering event. We here investigate the importance of the duration of this acute CBF drop in a rat SAH model in which a fixed amount of blood is injected into the prechiasmatic cistern either at a high rate resulting in a short acute CBF drop or at a slower rate resulting in a prolonged acute CBF drop.. We demonstrate that the duration of the acute CBF drop is determining for a) degree of early ERK1/2 activation in cerebral arteries, b) delayed upregulation of vasoconstrictor receptors in cerebral arteries and c) delayed CBF reduction, neurological deficits and mortality. Moreover, treatment with an inhibitor of MEK-ERK1/2 signalling during an early time window from 6 to 24 h after SAH was sufficient to completely prevent delayed vasoconstrictor receptor upregulation and improve neurological outcome several days after the SAH.. Our findings suggest a series of events where 1) the acute CBF drop triggers early MEK-ERK1/2 activation, which 2) triggers the transcriptional upregulation of vasoconstrictor receptors in cerebral arteries during the following days, where 3) the resulting enhanced cerebrovascular contractility contribute to delayed cerebral ischemia.

Topics: Analysis of Variance; Animals; Antipyrine; Area Under Curve; Blood Pressure; Brain Ischemia; Butadienes; Carbon Isotopes; Cerebral Arteries; Cerebrovascular Circulation; Disease Models, Animal; Enzyme Inhibitors; Laser-Doppler Flowmetry; Male; MAP Kinase Signaling System; Motor Activity; Nervous System Diseases; Nitriles; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Receptor, Serotonin, 5-HT1B; Signal Transduction; Subarachnoid Hemorrhage; Up-Regulation

Our previous data indicated that hypoxic preconditioning (HPC) ameliorates transient global cerebral ischemia (tGCI)-induced neuronal death in hippocampal CA1 subregion of adult rats. However, the possible molecular mechanisms for neuroprotection of this kind are largely unknown. This study was performed to investigate the role of the mitogen-activated protein kinase/extra-cellular signal-regulated kinase kinase (MEK)/extra-cellular signal-regulated kinase (ERK) pathway in HPC-induced neuroprotection. tGCI was induced by applying the four-vessel occlusion method. Pretreatment with 30 min of hypoxia applied 1 day before 10 min tGCI significantly decreased the level of MEK1/2 and ERK1/2 phosphorylation in ischemic hippocampal CA1 subregion. Also, HPC decreased the expression of phosphorylated ERK1/2 in degenerating neurons and astrocytes. However, the administration of U0126, a MEK kinase inhibitor, partly blocked MEK1/2 and ERK1/2 phosphorylation induced by tGCI. Meanwhile, neuronal survival was improved, and glial cell activation was significantly reduced. Collectively, these data indicated that the MEK/ERK signaling pathway might be involved in HPC-induced neuroprotection following tGCI. Also, HPC resulted in a reduction of glial activation.

Topics: Aging; Animals; Brain Ischemia; Butadienes; CA1 Region, Hippocampal; Cell Death; Cytoprotection; Hypoxia; Male; MAP Kinase Signaling System; Neurons; Neuroprotective Agents; Nitriles; Phosphorylation; Rats; Rats, Wistar

Our previous data indicate that the inhibition of L-type calcium channels (LTCCs) might be the cause of post-ischemic neuronal injury and that the activation of LTCCs can give rise to neuroprotection. In the present study, we aimed to profile the intervention window of Bay K8644, an LTCC agonist, and determine the involved mechanisms. The four vessel occlusion and oxygen-glucose deprivation models were employed to mimic ischemia/reperfusion damage in vivo and in vitro. Neuronal injury was analyzed using Nissl and Fluoro-Jade B staining in vivo and Hoechst 33342 and propidium iodide staining in vitro. The behavioral effects were tested using the Morris water maze. The phosphorylation of P38, Jun N-terminal kinase, and extracellular-regulated kinase (ERK) was detected by Western blotting. Our results show that Bay K8644 administered as late as 24 h after reperfusion prevented CA1 neuronal death and ameliorated the deficiencies in spatial learning performance induced by global ischemia. In oxygen-glucose deprivation (OGD), Bay K8644 delivered from 1 to 12 h after re-oxygenation reduced neuronal death. The decrease in p-ERK1/2 that was observed at 1 h after OGD was reversed by Bay K8644, and the effect of Bay K8644 was blocked by treatment with U0126 and MEK kinase dead transfection. Moreover, similar to Bay K8644, FPL 64176, another potent LTCC agonist, extends the window of intervention against neuronal injury in an in vitro model of ischemia. In conclusion, our data suggest that opening LTCCs may be a practicable approach for stroke therapy.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Brain Ischemia; Butadienes; Calcium Channels, L-Type; Extracellular Signal-Regulated MAP Kinases; Maze Learning; Memory; Neurons; Neuroprotective Agents; Nitriles; Phosphorylation; Pyrroles; Rats; Rats, Sprague-Dawley; Rats, Wistar

Glucose is the primary energy substrate for neurons. Glucose transporter 3 (Glut3) localizes at the neuronal cellular membrane, which transports glucose from the extracelluar space into neurons. Ischemia results in an increased energy demand that is associated with profound changes in brain energy metabolism. Magnesium sulfate (MgSO(4)) ameliorates ischemia-induced neuronal death in the rat and gerbil model. We investigated the effects of MgSO(4) administration on the expression of Glut3 in cortex and hippocampus of gerbils during ischemia. The focal cerebral ischemia was produced by unilateral occlusion of the right common carotid artery and right middle cerebral artery. Following ischemia, Glut3 expression increased significantly versus non-ischemic (contra-lateral) cortex and hippocampus. MgSO(4) treatment significantly increased the level of Glut3 expression in the non-ischemic and ischemic cortex and hippocampus. We found that the MgSO(4)-induced increase in Glut3 expression was not reversed by administration of U0126, a MEK kinase inhibitor. These results suggest that other factors may function to modulate the MgSO(4)-induced Glut3 response. In all, our data showed that MgSO(4) increases the expression of Glut3 in the cortex and hippocampus of gerbil brains both in non-ischemia and ischemia status. However, the MEK signaling pathway might not be involved in MgSO(4)-induced Glut3 expression following focal ischemia.

Topics: Animals; Brain Ischemia; Butadienes; Cerebral Cortex; Enzyme Inhibitors; Gerbillinae; Glucose Transporter Type 3; Hippocampus; Magnesium Sulfate; Male; Nitriles; Rats

Our previous study demonstrated that pretreatment with electroacupuncture (EA) elicited protective effects against transient cerebral ischemia through cannabinoid receptor type 1 receptor (CB1R). In the present study, we investigated whether or not the extracellular signal regulated-kinase 1/2 (ERK1/2) pathway was involved in the ischemic tolerance induced by EA pretreatment through CB1R. At 24h after the end of the last EA pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion for 120min in rats. The neurological scores and infarct volumes were evaluated at 24h after reperfusion. The expression of p-ERK1/2 in the brains was also investigated in the presence or absence of CB1R antagonist AM251. EA pretreatment reduced infarct volumes and improved neurological outcome at 24h after reperfusion, and the beneficial effects were abolished by U0126. The blockade of CB1R by AM251 reversed the up-regulation of p-ERK1/2 expression induced by EA pretreatment. Our findings suggest that the ERK1/2 pathway might be involved in EA pretreatment-induced cerebral ischemic tolerance via cannabinoid CB1 receptor in rats.

Topics: Animals; Behavior, Animal; Blotting, Western; Brain Ischemia; Butadienes; Electroacupuncture; Enzyme Activation; Enzyme Inhibitors; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neuroprotective Agents; Nitriles; Piperidines; Pyrazoles; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB1; Reperfusion Injury; Signal Transduction; Up-Regulation

Two pathways that have been shown to mediate cerebral ischemic damage are the MEK/ERK cascade and the pro-apoptotic deltaPKC pathway. We investigated the relationship between these pathways in a rat model of focal ischemia by observing and modifying the activation state of each pathway. The ERK1/2 inhibitor, U0126, injected at ischemia onset, attenuated the increase in phosphorylated ERK1/2 (P-ERK1/2) after reperfusion. The deltaPKC inhibitor, deltaV1-1, delivered at reperfusion, did not significantly change P-ERK1/2 levels. In contrast, the deltaPKC activator, psi deltaRACK, injected at reperfusion, reduced ERK1/2 phosphorylation measured 4 h after reperfusion. Additionally, U0126 pretreatment at ischemia onset reduced infarct size compared with vehicle, but U0126 injected at the onset of reperfusion had no protection. Finally, combination of U0126 injection at ischemia onset plus deltaV1-1 injection at reperfusion further reduced infarct size, while combination of U0126 delivered at ischemia onset with psi deltaRACK injected at reperfusion increased infarct size compared with U0126 alone. In conclusion, we find that inhibiting both the MEK/ERK and the deltaPKC pathways offers greater protection than either alone, indicating they likely act independently.

Topics: Animals; Brain Ischemia; Butadienes; Disease Models, Animal; Drug Administration Schedule; Drug Therapy, Combination; Enzyme Inhibitors; Male; Mitogen-Activated Protein Kinase 3; Neuroprotective Agents; Nitriles; Phosphorylation; Protein Kinase C-delta; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Stroke; Up-Regulation

Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. However, Molecular mechanisms that regulate neuronal cell proliferation in the dentate gyrus (DG) following ischemic stroke insult are poorly understood. This study was designed to investigate the potential regulatory capacity of non-receptor tyrosine kinase Src on ischemia-stimulated cell proliferation in the adult DG and its underlying mechanism.. Src kinase activated continuously in the DG 24 h and 72 h after transient global ischemia, while SU6656, the Src kinase inhibitor significantly decreased the number of bromodeoxyuridine (BrdU) labeling-positive cells of rats 7 days after cerebral ischemia in the DG, as well as down-regulated Raf phosphorylation at Tyr(340/341) site, and its down-stream signaling molecules ERK and CREB expression followed by 24 h and 72 h of reperfusion, suggesting a role of Src kinase as an enhancer on neuronal cell proliferation in the DG via modifying the Raf/ERK/CREB cascade. This hypothesis is supported by further findings that U0126, the ERK inhibitor, induced a reduction of adult hippocampal progenitor cells in DG after cerebral ischemia and down-regulated phospho-ERK and phospho-CREB expression, but no effect was detected on the activities of Src and Raf.. Src kinase increase numbers of newborn neuronal cells in the DG via the activation of Raf/ERK/CREB signaling cascade after cerebral ischemia.

Topics: Analysis of Variance; Animals; Blotting, Western; Brain Ischemia; Butadienes; Cell Count; Cell Proliferation; Cyclic AMP Response Element-Binding Protein; Dentate Gyrus; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Immunohistochemistry; Indoles; Male; Neurons; Nitriles; Phosphorylation; raf Kinases; Rats; Rats, Sprague-Dawley; Reperfusion; Signal Transduction; src-Family Kinases; Sulfonamides

MEK1/2 is a serine/threonine protein that phosphorylates extracellular signal-regulated kinase (ERK1/2). Cerebral ischemia results in enhanced expression of cerebrovascular contractile receptors in the middle cerebral artery (MCA) leading to the ischemic region. Here we explored the role of the MEK/ERK pathway in receptor expression following ischemic brain injury using the specific MEK1 inhibitor U0126.. Rats were subjected to a 2-h middle cerebral artery occlusion (MCAO) followed by reperfusion for 48-h and the ischemic area was calculated. The expression of phosphorylated ERK1/2 and Elk-1, and of endothelin ETA and ETB, angiotensin AT1, and 5-hydroxytryptamine 5-HT1B receptors were analyzed with immunohistochemistry using confocal microscopy in cerebral arteries, microvessels and in brain tissue. The expression of endothelin ETB receptor was analyzed by quantitative Western blot. We demonstrate that there is an increase in the number of contractile smooth muscle receptors in the MCA and in micro- vessels within the ischemic region. The enhanced expression occurs in the smooth muscle cells as verified by co-localization studies. This receptor upregulation is furthermore associated with enhanced expression of pERK1/2 and of transcription factor pElk-1 in the vascular smooth muscle cells. Blockade of transcription with the MEK1 inhibitor U0126, given at the onset of reperfusion or as late as 6 hours after the insult, reduced transcription (pERK1/2 and pElk-1), the enhanced vascular receptor expression, and attenuated the cerebral infarct and improved neurology score.. Our results show that MCAO results in upregulation of cerebrovascular ETB, AT1 and 5-HT1B receptors. Blockade of this event with a MEK1 inhibitor as late as 6 h after the insult reduced the enhanced vascular receptor expression and the associated cerebral infarction.

Topics: Animals; Blotting, Western; Brain; Brain Ischemia; Butadienes; Enzyme Inhibitors; ets-Domain Protein Elk-1; Immunohistochemistry; Infarction, Middle Cerebral Artery; Injections, Intraperitoneal; Male; MAP Kinase Kinase 2; Microscopy, Confocal; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Nitriles; Phosphorylation; Rats; Rats, Wistar; Receptor, Angiotensin, Type 1; Receptor, Endothelin A; Receptor, Endothelin B; Receptor, Serotonin, 5-HT1B; Receptors, Cell Surface; Reperfusion Injury; Signal Transduction

Hyperglycaemia aggravates ischaemic brain injury, possibly due to activation of signalling pathways involving mitogen-activated protein kinases (MAPK). In this study, the activation of MAPK/ERK was inhibited using the upstream inhibitor of MAPK-ERK-kinase (MEK) U0126, and the effects on focal brain ischaemia were evaluated during normo- and hyperglycaemia.. Temporary (90 min) middle cerebral artery occlusion (MCAO) was induced in five groups of rats. U0126 (400 microg kg(-1)) or vehicle was given as 60-min intravenous infusions starting either 30 min prior to MCAO or 30 min prior to reperfusion. The infarct size was determined by perfusion with tetrazolium red after 24 h of survival, and the neurology was tested with the 4-level scale of Bederson and performance on an inclined plane. The inhibitory effect on the targeted MEK enzyme was investigated by analysing the phosphorylation of the downstream target ERK with western immunoblotting. Two subgroups were investigated with magnetic resonance imaging (MRI), including diffusion-weighted (DWI) and perfusion-weighted imaging (PWI).. U0126 effectively reduced the infarct size and improved neurology in hyperglycaemic rats both when given before and after ischemic onset. This effect was not accompanied by any detectable changes in cerebral blood flow on MRI. Normoglycaemic rats had generally milder injuries compared with the hyperglycaemic and there was a nonsignificant trend for U0126 to reduce damage also in the nonhyperglycaemic groups.. In conclusion, U0126 appears to be neuroprotective in this model of hyperglycaemic ischaemic brain injury. The findings support the pathogenic importance of the MEK-ERK pathway in hyperglycaemic-ischaemic brain injury.

Topics: Animals; Blotting, Western; Brain; Brain Ischemia; Butadienes; Enzyme Inhibitors; Hyperglycemia; Magnetic Resonance Imaging; Mitogen-Activated Protein Kinase Kinases; Nitriles; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Treatment Outcome

Hyperglycemia aggravates ischemic brain injury, possibly due to the activation of signaling pathways involving reactive oxygen species, Src and mitogen-activated protein kinases. The aim of this study was to investigate the effects of the spin trap agent alpha-phenyl-N-tert-butyl nitrone (PBN), the Src family kinase inhibitor PP2 and the MEK1-inhibitor U0126 on focal hyperglycemic ischemic brain injury. Temporary middle cerebral artery occlusion (90 min) was induced in four groups of rats (PBN, PP2, and U0126 vs. control). Neurological testing and tetrazolium red staining were performed after 1 day. PBN decreased the infarct volume by 70% compared with the control (P<0.05) and a tendency towards reduced infarcts was seen in the PP2 or U0126 groups. Furthermore, neurological testing was consistent with the volumetric analysis. In conclusion, PBN appears to be a potential neuroprotective agent in hyperglycemic, focal ischemic brain injury, while the efficacy of PP2 and U0126 could not be confirmed by the present data.

Topics: Animals; Blood Glucose; Body Temperature; Brain Ischemia; Butadienes; Carbon Dioxide; Cyclic N-Oxides; Enzyme Inhibitors; Hyperglycemia; Male; Neurologic Examination; Neuroprotective Agents; Nitriles; Nitrogen Oxides; Oxygen; Pyrimidines; Rats; Rats, Sprague-Dawley; Tetrazolium Salts; Time Factors

It has been proposed that mitogen-activated protein kinase (MAPK) pathways may play a role in the regulation of pro-inflammatory cytokines, such as interlukine-1, during cerebral ischemia. Our previous study showed that extracellular-signal-regulated kinases 1 and 2 (ERK 1/2) were activated during focal cerebral ischemia in mice [J. Cereb. Blood Flow Metab. 20 (2000) 1320]. However, the effect of ERK 1/2 activation in focal cerebral ischemia is still unclear. In this study we reported that in vivo phospho-ERK 1/2 expression increased following 30 min of middle cerebral artery occlusion (MCAO) in the mouse brain in both the ischemic core and perifocal regions. Western blot analysis and immunohistochemistry demonstrated that pro-treatment with 1,4-diamino-2,3-dicyano-1,4-bis butadiene (U0126) [J. Biol. Chem. 273 (1998) 18623] could significantly inhibit mouse brain phospho-MEK 1/2 and phospho-ERK 1/2 expression after 1-2 h of MCAO (p<0.05). Compared to the control group of mice, brain infarct volume was significantly decreased after 24 h of MCAO in the U0126-treated mice (27+/-6 vs. 46+/-9 mm(2), p<0.05). Inhibition of the MEK/ERK 1/2 pathway also prevented downstream kinase Elk-1 phosphorylation, and further reduced cytokine IL-1beta mRNA, but not TNFalpha, IL-1alpha, or chemokine MIP-1alpha mRNA expression. Our data demonstrates that in vivo the close linking of MEK 1/2, ERK 1/2, Elk-1, and IL-1 mRNA expression in the cerebral ischemia animals suggests that ERK 1/2 pathway activation is important in pro-inflammatory cytokine IL-1beta signaling, which induces an inflammatory response and exacerbates ischemic brain injury. Inhibiting the ERK 1/2 pathway may therefore provide a novel approach for the reduction of ischemia-induced IL-1beta overexpression.

Topics: Animals; Blotting, Western; Brain; Brain Ischemia; Butadienes; Chemokine CCL3; Chemokine CCL4; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Immunohistochemistry; Infarction, Middle Cerebral Artery; Interleukin-1; Macrophage Inflammatory Proteins; Male; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase Kinases; Nitriles; Phosphorylation; Time Factors; Tumor Necrosis Factor-alpha

The extracellular signal-regulated kinase pathway of the mitogen-activated protein kinase signal transduction cascade has been implicated in the neuronal and endothelial dysfunction witnessed following cerebral ischemia-reperfusion injury. Extracellular signal-regulated kinase is activated by mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2. We evaluated the ability of a mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2-specific inhibitor (U0126) to block extracellular signal-regulated kinase activation and mitigate ischemic neuronal damage in a model of deep hypothermic circulatory arrest.. Piglets underwent normal flow cardiopulmonary bypass (control, n = 4), deep hypothermic circulatory arrest (n = 6), and deep hypothermic circulatory arrest with U0126 (n = 5) at 20 degrees C for 60 minutes. The deep hypothermic circulatory arrest with U0126 group was given 200 microg/kg of U0126 45 minutes prior to initiation of bypass followed by 100 microg/kg at reperfusion. Following 24 hours of post-cardiopulmonary bypass recovery, brains were harvested. Eleven distinct cortical regions were evaluated for neuronal damage using hematoxylin and eosin staining. A section of ischemic cortex was further evaluated by immunohistochemistry with rabbit polyclonal antibody against phosphorylated extracellular signal-regulated kinase 1/2.. The deep hypothermic circulatory arrest and deep hypothermic circulatory arrest with U0126 groups displayed diffuse ischemic changes. However, the deep hypothermic circulatory arrest with U0126 group possessed significantly lower neuronal damage scores in the right frontal watershed zone of cerebral cortex, basal ganglia, and thalamus (P < or =.05) and an overall trend toward neuroprotection versus the deep hypothermic circulatory arrest group. This neuroprotection was accompanied by nearly complete blockade of phosphorylated extracellular signal-regulated kinase in the cerebral vascular endothelium.. In this experimental model of deep hypothermic circulatory arrest, U0126 blocked extracellular signal-regulated kinase activation and provided a significant neuroprotective effect. These results support targeting of the extracellular signal-regulated kinase pathway for inhibition as a novel therapeutic approach to mitigate neuronal damage following deep hypothermic circulatory arrest.

Topics: Animals; Animals, Newborn; Brain Ischemia; Butadienes; Cardiopulmonary Bypass; Cerebrovascular Circulation; Disease Models, Animal; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Heart Arrest, Induced; Hypothermia, Induced; Immunohistochemistry; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Models, Cardiovascular; Neurons; Nitriles; Postoperative Complications; Signal Transduction; Swine; Treatment Outcome

The 14-3-3 protein family comprises critical regulatory molecules involved in signaling during cell division, proliferation, and apoptosis. Despite extensive study, the functions of the 14-3-3 proteins in brain remain unclear. 14-3-3gamma, a subtype of the 14-3-3 family of proteins, was thought to be brain- and neuron-specific. Using RNA arbitrarily primed PCR, we identified an upregulated cDNA fragment of the 14-3-3gamma gene in primary cultures of astrocytes. Using Northern blot analysis, we confirmed this fragment was brain-specific. In cultures of astrocytes, 14-3-3gamma genes and proteins were differentially expressed at different ages and the proteins were distributed only in the cytoplasm. These results indicated that 14-3-3gamma was not neuron-specific but also expressed in astrocytes. The function of this protein in brain is unclear. Northern and Western blot analyses demonstrated that 14-3-3gamma mRNA and protein were upregulated in cultured astrocytes in an anaerobic chamber-induced ischemia model. The induction of 14-3-3gamma proteins was neither suppressed by an MAP kinase inhibitor (U0126) nor a PI-3 kinase inhibitor (LY294002). These data indicated that induction of 14-3-3gamma might not involve PI-3 and MAP kinase-dependent pathways. Using coimmunoprecipitation, we demonstrated that endogenous 14-3-3gamma bound to c-Raf-1 and p-Raf 259. As Raf is one of the critical serine/threonine kinases controlling cell growth, differentiation, and death, the binding of 14-3-3gamma to Raf indicates the critical role of this protein in ischemia-induced apoptosis and the changes in signal transduction in astrocytes in culture.

Topics: 14-3-3 Proteins; Animals; Animals, Newborn; Astrocytes; Base Sequence; Brain Ischemia; Butadienes; Cell Death; Cells, Cultured; Cerebral Cortex; Chromones; Cloning, Molecular; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; In Vitro Techniques; Mice; Mice, Inbred ICR; Molecular Sequence Data; Morpholines; Nitriles; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-raf; Tyrosine 3-Monooxygenase; Up-Regulation

To determine the role of extracellular signal-regulated kinase (ERK)1/2 during focal cerebral ischemia.. Left middle cerebral artery occlusion (MCAO) was undergone after the introduction of a nylon suture to the left internal carotid artery in 70 male adult CD-1 mice. ERK 1/2 phosphorylation was detected using Western blot analysis, and the morphological feature was determined by immunohistochemistry. An ERK pathway inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis[2-amino-phenylthio] butadiene (U0126), was administered intravenously 20 minutes before MCAO, and the neurological deficit levels and the infarct volumes were measured 24 hours after MCAO.. Phosphorylated ERK 1/2 (pERK 1/2) activity increased after 30 minutes of MCAO and peaked at 2 hours. The immunohistochemical study displayed a large number of pERK 1/2 positive cells in the ischemic basal ganglion and surrounding cortex. Double-labeled fluorescent staining identified the pERK1/2 positive cells as neurons or astrocytes. In U0126 treated mice which had undergone 24 hours of MCAO, the neurological deficit levels and the infarct volumes were 44.6% and 45.8% respectively, less than those of the control mice.. ERK plays an important role in focal cerebral ischemia and inhibition of the ERK pathway can help protect against ischemic brain injury, which may provide a therapeutic approach for cerebral ischemia.

Topics: Animals; Basal Ganglia; Brain Ischemia; Butadienes; Cerebral Cortex; Immunohistochemistry; Male; Mice; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylation