u-0126 and Ischemic-Attack--Transient

Hyperbaric oxygen (HBO) is emerging as a therapy for brain ischemia, although its benefits are still debated. The present study aimed to investigate the effect of HBO on brain damage in a rat model of transient focal cerebral ischemia and its underlying mechanism of action. Male Wistar rats, which had suffered 1.5h of transient middle cerebral artery occlusion (tMCAO) and had a Longa's neuron score of 3, were given pure oxygen at 3.0 atm absolute, for 60 min after the third hour of reperfusion. After 24h of reperfusion, rat brains were removed and studied. 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin and eosin staining revealed that the infarct ratio in the HBO group increased remarkably when compared with the MCAO group. Up-regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation was detected in the HBO group because of reactive oxygen species (ROS) generation. Autophagy appeared to be obstructed in the HBO group. Administration of the ERK1/2 inhibitor U0126 decreased the infarct ratio and improved protein clearance by autophagy in the HBO group. Collectively, these results suggest that HBO enlarges the area of brain damage via reactive oxygen species-induced activation of ERK1/2, which interrupts autophagy flux.

Topics: Animals; Autophagy; Brain; Butadienes; Disease Models, Animal; Enzyme Inhibitors; Hyperbaric Oxygenation; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Rats; Rats, Wistar; Reactive Oxygen Species; Up-Regulation

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

It is well documented that cannabinoid receptor agonist WIN 55,212-2 had protective effect against cerebral ischemic injury. Our previous study indicated that WIN 55,212-2 pretreatment induced ischemic tolerance to focal cerebral ischemia in a dose-dependent manner. The aim of the present study was to investigate the time-effect relationship of the WIN 55,212-2 pretreatment and explore the role of phosphorylated extracellular signal-regulated kinase 1/2. Rats were pretreated with 1mg/kg WIN 55,212-2 once a day for 1, 3 and 5 days. Twenty four hours after the end of pretreatment, focal cerebral ischemia was induced by the middle cerebral artery occlusion. Brain ischemic injury was evaluated by neurological function scores and infarction volumes. The effect of U0126, a potent and specific inhibitor of mitogen-activated protein kinase kinase, on WIN 55,212-2 pretreatment was also studied. Moreover, the expression of phosphorylated extracellular signal-regulated kinase 1/2 in the penumbra of ischemic side 4h after reperfusion was investigated by immunohistochemistry and Western blotting. The results showed that WIN 55,212-2 pretreatment can protect the rat brain against transient focal cerebral ischemia injury, and its protective effect was enhanced gradually with increasing numbers of pretreatment, and was partially reversed by U0126. We further found that WIN 55,212-2 pretreatment up-regulated the levels of phosphorylated extracellular signal-regulated kinase 1/2. These findings suggest that the neuroprotective effect of WIN 55,212-2 pretreatment against focal cerebral ischemia is through the activation of extracellular signal-regulated kinases in rats.

Topics: Animals; Benzoxazines; Brain; Butadienes; Cannabinoid Receptor Agonists; Dose-Response Relationship, Drug; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Male; Morpholines; Naphthalenes; Neuroprotective Agents; Nitriles; Phosphorylation; Rats; Rats, Sprague-Dawley; Time Factors; Up-Regulation

Recent studies have demonstrated that lithium has a neuroprotective effect against brain ischemia. Whether this effect is mediated by hippocampal neurogenesis remains unknown. The ERK (extracellular signal-regulated kinase) pathway plays an essential role in regulating neurogenesis. The present study was undertaken to investigate whether lithium regulates hippocampal neurogenesis by the ERK pathway and improves spatial learning and memory deficits in rats after ischemia. Rats were daily injected with lithium (1 mmol/kg) and 2 weeks later subjected to 15-min ischemia induced by four-vessel occlusion method. 5-bromo-2'-deoxyuridine (Brdu; 50mg/kg) was administrated twice daily at postischemic day 6, or for 3 days from postischemic day 6 to 8. We found that lithium increased the ERK1/2 activation after ischemia by western blotting analysis. There was a significant increase in Brdu-positive cells in the hippocampal dentate gyrus after lithium treatment, compared with ischemia group at postischemic days 7 and 21; furthermore, the survival rate of Brdu-positive cells was elevated by lithium. Inhibition of the ERK1/2 activation by U0126 diminished these effects of lithium. The percentages of Brdu-positive cells that expressed a neuronal marker or an astrocytic marker were not significantly influenced by lithium. Moreover, lithium improved the impaired spatial learning and memory ability in Morris water maze, and U0126 attenuated the behavioral improvement by lithium. These results suggest that lithium up-regulates the generation and survival of new-born cells in the hippocampus by the ERK pathway and improves the behavioral disorder in rats after transient global cerebral ischemia.

Topics: Animals; Butadienes; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Hippocampus; Ischemic Attack, Transient; Learning; Lithium Chloride; Male; Memory; Neurons; Nitriles; Rats; Rats, Sprague-Dawley; Space Perception

Brain subjected to acute ischemic attack caused by an arterial blockage needs immediate arterial recanalization. However, restoration of cerebral blood flow can cause tissue injury, which is termed reperfusion injury. It is important to inhibit reperfusion injury to achieve greater brain protection. Because oxidative stress has been shown to activate mitogen-activated protein kinases (MAPKs), and because oxidative stress contributes to reperfusion injury, MAPK may be a potential target to inhibit reperfusion injury after brain ischemia. Here, we demonstrate that reperfusion after forebrain ischemia dramatically increases phosphorylation level of extracellular signal-regulated kinase 2 (ERK2) in the gerbil hippocampus. In addition, i.v. administration of U0126 (100-200 mg/kg), a specific inhibitor of MEK (MAPK/ERK kinase), protects the hippocampus against forebrain ischemia. Moreover, treatment with U0126 at 3 h after ischemia significantly reduces infarct volume after transient (3 h) focal cerebral ischemia in mice. This protection is accompanied by reduced phosphorylation level of ERK2, substrates for MEK, in the damaged brain areas. Furthermore, U0126 protects mouse primary cultured cortical neurons against oxygen deprivation for 9 h as well as nitric oxide toxicity. These results provide further evidence for the role of MEK/ERK activation in brain injury resulting from ischemia/reperfusion, and indicate that MEK inhibition may increase the resistance of tissue to ischemic injury.

Topics: Animals; Butadienes; Cells, Cultured; Enzyme Inhibitors; Gerbillinae; In Vitro Techniques; Ischemic Attack, Transient; Male; MAP Kinase Kinase Kinase 1; Mice; Mice, Inbred ICR; Nitriles; Protein Serine-Threonine Kinases; Reperfusion; Time Factors

In transient forebrain ischemia, sodium orthovanadate as well as insulinlike growth factor-1 (IGF-1) rescued cells from delayed neuronal death in the hippocampal CA1 region. Adult Mongolian gerbils were subjected to 5-minute forebrain ischemia. Immunoblotting analysis with anti-phospho-Akt/PKB (Akt) antibody showed that phosphorylation of Akt at serine-473 (Akt-Ser-473) in the CA1 region decreased immediately after reperfusion, and in turn transiently increased 6 hours after reperfusion. The decreased phosphorylation of Akt-Ser-473 was not observed in the CA3 region. The authors then tested effects of intraventricular injection of orthovanadate and IGF-1, which are known to activate Akt. Treatment with orthovanadate or IGF-1 30 minutes before ischemia blocked delayed neuronal death in the CA1 region. The neuroprotective effects of orthovanadate and IGF-1 were associated with preventing decreased Akt-Ser-473 phosphorylation in the CA1 region observed immediately after reperfusion. Immunohistochemical studies with the anti-phospho-Akt-Ser-473 antibody also demonstrated that Akt was predominantly in the nucleus and was moderately activated in the cell bodies and dendrites of pyramidal neurons after orthovanadate treatment. The orthovanadate treatment also prevented the decrease in phosphorylation of mitogen-activated protein kinase (MAPK). Pretreatment with combined blockade of phosphatidylinositol 3-kinase and MAPK pathways totally abolished the orthovanadate-induced neuroprotective effect. These results suggest that the activation of both Akt and MAPK activities underlie the neuroprotective effects of orthovanadate on the delayed neuronal death in the CA1 region after transient forebrain ischemia.

Topics: Androstadienes; Animals; Butadienes; Cell Death; Enzyme Inhibitors; Gerbillinae; Hippocampus; Immunohistochemistry; Injections, Intraventricular; Insulin-Like Growth Factor I; Ischemic Attack, Transient; Male; Mitogen-Activated Protein Kinases; Neurons; Neuroprotective Agents; Nitriles; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Vanadates; Wortmannin