u-0126 and Subarachnoid-Hemorrhage

Vascular pathophysiological changes after haemorrhagic stroke, such as phenotypic modulation of the cerebral arteries and cerebral vasospasms, are associated with delayed cerebral ischemia (DCI) and poor outcome. The only currently approved drug treatment shown to reduce the risk of DCI and improve neurologic outcome after aneurysmal subarachnoid haemorrhage (SAH) is nimodipine, a dihydropyridine L-type voltage-gated Ca2+ channel blocker. MEK1/2 mediated transcriptional upregulation of contractile receptors, including endothelin-1 (ET-1) receptors, has previously been shown to be a factor in the pathology of SAH. The aim of the study was to compare intrathecal and subcutaneous treatment regimens of nimodipine and intrathecal treatment regimens of U0126, a MEK1/2 inhibitor, in a single injection experimental rat SAH model with post 48 h endpoints consisting of wire myography of cerebral arteries, flow cytometry of cerebral arterial tissue and behavioural evaluation. Following ET-1 concentration-response curves, U0126 exposed arteries had a significantly lower ET-1max than vehicle arteries. Arteries from both the intrathecal- and subcutaneous nimodipine treated animals had significantly higher ET-1max contractions than the U0126 arteries. Furthermore, Ca2+ concentration response curves (precontracted with ET-1 and in the presence of nimodipine) showed that nimodipine treatment could result in larger nimodipine insensitive contractions compared to U0126. Flow cytometry showed decreased protein expression of the ETB receptor in U0126 treated cerebral vascular smooth muscle cells compared to vehicle. Only U0126 treatment lowered ET-1max contractions and ETB receptor levels, as well as decreased the contractions involving nimodipine-insensitive Ca2+ channels, when compared to both intrathecal and subcutaneous nimodipine treatment. This indicate that targeting gene expression might be a better strategy than blocking specific receptors or ion channels in future treatments of SAH.

Topics: Animals; Butadienes; Calcium Channel Blockers; Cerebral Arteries; Disease Models, Animal; Male; Mitogen-Activated Protein Kinase Kinases; Muscle Contraction; Muscle, Smooth, Vascular; Nimodipine; Nitriles; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptor, Endothelin B; Subarachnoid Hemorrhage; Up-Regulation; Vasoconstriction

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

Subarachnoid hemorrhage (SAH) is a serious clinical condition where leakage of blood into the subarachnoid space causes an acute rise in intracranial pressure and reduces cerebral blood flow, which may lead to delayed cerebral ischemia and poor outcome. In experimental SAH, we have previously shown that the outcome can be significantly improved by early inhibition of the MAPK/ERK kinase/extracellular signal-regulated kinase (MEK/ERK1/2) pathway. The aim of this study was to apply mass spectrometry to investigate the overall late effects of experimental SAH on cerebrovascular protein expression. SAH was induced in rats that were treated with the MEK1/2 inhibitor U0126 or vehicle. Neurological outcome was assessed using a battery of behavioral tests. Specific protein expression of large cerebral arteries was analyzed quantitatively with high-throughput tandem mass spectrometry. SAH resulted in a marked reduction of neurological scores, which was counteracted by U0126 treatment. Mass spectrometry analysis demonstrated regulation of 184 proteins after SAH, regulations that were in part prevented by U0126 treatment. Network analysis identified several protein networks including a strong structural network centered around 14-3-3. Additionally, protein networks with functions in mRNA metabolism and protein folding were identified. Treatment with U0126 inhibited cerebral vessel wall pERK1/2 expression and significantly improved outcome of the rats. In conclusion, we show that SAH induces a broad array of specific changes in the overall protein networks in cerebral artery smooth muscle cells and suggest that this is essential for understanding the vascular pathophysiology after SAH.

Topics: Animals; Butadienes; Cerebral Arteries; Enzyme Inhibitors; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Proteome; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage

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

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

Subarachnoid hemorrhage (SAH) is associated with high morbidity and mortality. It is suggested that the associated inflammation is mediated through activation of the mitogen-activated protein kinase (MAPK) pathway which plays a crucial role in the pathogenesis of delayed cerebral ischemia after SAH. The aim of this study was first to investigate the timecourse of altered expression of proinflammatory cytokines and matrix metalloproteinase in the cerebral arteries walls following SAH. Secondly, we investigated whether administration of a specific mitogen-activated protein kinase kinase (MEK)1/2 inhibitor, U0126, given at 6 h after SAH prevents activation of the MEK/extracellular signal-regulated kinase 1/2 pathway and the upregulation of cerebrovascular inflammatory mediators and improves neurological function.. SAH was induced in rats by injection of 250 μl of autologous blood into basal cisterns. U0126 was given intracisternally using two treatment regimens: (A) treatments at 6, 12, 24 and 36 h after SAH and experiments terminated at 48 h after SAH, or (B) treatments at 6, 12, and 24 h after SAH and terminated at 72 h after SAH. Cerebral arteries were harvested and interleukin (IL)-6, IL-1β, tumor necrosis factor α (TNF)α, matrix metalloproteinase (MMP)-9 and phosphorylated ERK1/2 (pERK1/2) levels investigated by immunohistochemistry. Early activation of pERK1/2 was measured by western blot. Functional neurological outcome after SAH was also analyzed.. Expression levels of IL-1β, IL-6, MMP-9 and pERK1/2 proteins were elevated over time with an early increase at around 6 h and a late peak at 48 to 72 h post-SAH in cerebral arteries. Enhanced expression of TNFα in cerebral arteries started at 24 h and increased until 96 h. In addition, SAH induced sensorimotor and spontaneous behavior deficits in the animals. Treatment with U0126 starting at 6 h after SAH prevented activation of MEK-ERK1/2 signaling. Further, U0126 significantly decreased the upregulation of inflammation proteins at 48 and 72 h following SAH and improved neurological function. We found no differences between treatment regimens A and B.. These results show that SAH induces early activation of the MEK-ERK1/2 pathway in cerebral artery walls, which is associated with upregulation of proinflammatory cytokines and MMP-9. Inhibition of the MEK-ERK1/2 pathway by U0126 starting at 6 h post-SAH prevented upregulation of cytokines and MMP-9 in cerebral vessels, and improved neurological outcome.

Topics: Animals; Astrocytes; Blood Transfusion, Autologous; Butadienes; Cerebral Arteries; Cytokines; Disease Models, Animal; Enzyme Inhibitors; Exploratory Behavior; Extracellular Signal-Regulated MAP Kinases; Glial Fibrillary Acidic Protein; Male; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinases; Motor Activity; Nervous System Diseases; Nitriles; Rats; Rats, Sprague-Dawley; Signal Transduction; Statistics, Nonparametric; Subarachnoid Hemorrhage; Time Factors; Tumor Necrosis Factor-alpha

Delayed cerebral ischemia after subarachnoid hemorrhage (SAH) remains a major cause of death and disability. It has been hypothesized that cerebrovascular upregulation of vasoconstrictor receptors is a key step in the development of delayed cerebral ischemia. Upregulation of endothelin-B (ET(B)) and 5-hydroxytryptamine 1B (5-HT(1B)) receptors has been demonstrated in cerebral artery smooth muscles in the delayed ischemic phase after experimental SAH, and intracellular signaling via the mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase 1/2 pathway has been shown to be involved in this upregulation. The aim in the present study was to determine whether treatment with the MEK1/2 inhibitor U0126 can prevent cerebrovascular receptor upregulation and improve functional outcome after experimental SAH in rats.. Subarachnoid hemorrhage was induced in male Sprague-Dawley rats by the injection of 250 μl of autologous blood into the basal cisterns. Either U0126 or vehicle was intracisternally administered at 6, 12, 24, and 36 hours after SAH. Smooth muscle ET(B) and 5-HT(1B) receptor upregulation was studied in isolated cerebral artery segments through immunohistochemical and myographic studies of contractile responses to receptor-specific agonists. Gross sensorimotor function in the rats after SAH was assessed using a rotating pole test.. Contractile concentration-response curves for middle cerebral artery (MCA) and basilar artery (BA) segments to endothelin-1 (ET-1) and 5-carboxamidotryptamine (5-CT) were shifted leftward for SAH-induced compared with shamoperated rats due to enhanced contractile responses to individual doses of the agonists (for example, contractile responses of the BA to 3 × 10(-10) M of ET-1 and 3 × 10(-7) M of 5-CT were 9.98 ± 5.01% and 16.75 ± 3.62% of the maximal contractile capacity, respectively, in sham-operated rats and 62.78 ± 9.9% and 45.44 ± 10.62%, respectively, in SAH-induced rats). In vivo treatment with 0.19 μg/kg U0126 normalized responses in the SAH-induced rats to levels in the sham-operated rats. Protein expression of ET(B) and 5-HT(1B) receptors in cerebrovascular smooth muscles from SAH-induced rats was increased to 175 ± 33.17% and 167.7 ± 24.74%, respectively, of the levels in sham-operated rats. Endothelin-B and 5-HT(1B) expression levels in U0126-treated SAH-induced rats were at the levels in sham-operated rats (101.9 ± 13.38% and 91.44 ± 16.75%, respectively). In a rotating pole test used to assess gross sensorimotor function on the 2nd day after surgery, sham-operated rats achieved an average score of 5.37 ± 0.23, SAH-induced rats scored 3.35 ± 0.67, and SAH-induced U0126-treated rats scored 5.00 ± 0.4.. The authors demonstrated that experimental SAH induces upregulation of ET(B) and 5-HT(1B) receptors in cerebrovascular smooth muscles and that treatment with the MEK1/2 inhibitor U0126 abolishes this receptor upregulation. They also demonstrated that experimental SAH results in sensorimotor deficits as assessed by a rotating pole test. These deficits were alleviated by U0126 treatment, suggesting that cerebrovascular receptor upregulation is critical for the functional outcome of delayed cerebral ischemia. The authors suggest that inhibition of MEK1/2 may be a promising new SAH treatment strategy.

Topics: Animals; Behavior, Animal; Butadienes; Cerebral Arteries; Endothelin B Receptor Antagonists; Immunohistochemistry; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Muscle Contraction; Muscle, Smooth, Vascular; Nervous System Diseases; Nitriles; Postural Balance; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptor, Serotonin, 5-HT1B; Serotonin; Serotonin Antagonists; Serotonin Receptor Agonists; Subarachnoid Hemorrhage; Up-Regulation; Vasoconstriction

Mitogen-activated protein kinase (MAPK) may be involved in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage. This study was conducted to investigate the ability of the MAPK inhibitors PD-98059 and U-0126 to reverse vasospasm in a double-hemorrhage model in dogs.. Twenty-two adult mongrel dogs of either sex, each weighing 18 to 24 kg, were divided randomly into four groups: control SAH (four dogs), vehicle- (dimethyl sulfoxide, six dogs), PD-98059- (six dogs), and U-0126-treated groups (six dogs). The double-hemorrhage model was created by an autologous blood injection into the cisterna magna on Days 0 and 2. An intracisternal injection of MAPK inhibitors was administered once per day on Days 3 through 6. Cerebral angiography was performed on Days 0 and 7 before the animals were killed. Western blot analysis was used to study the effects of hemorrhage and drug treatment on the MAPK immunoprecipitation. Severe vasospasm developed in the dogs in the control and vehicle-treated groups (basilar artery [BA] diameter reduction 46.6 +/- 5.5% and 49.3 +/- 4.6%, respectively). In the PD-98059-treated group, most of the dogs developed mild vasospasm (18.9 +/- 6.2%). In the U-0126-treated group, severe vasospasm was observed despite treatment (39.6 +/- 6.4%). The PD-98059 but not the U-0126 abolished MAPK immunoprecipitation in the spastic BAs. However, treatment with either PD-98059 or U-0126 improved the clinical scores of the dogs.. The present study is the first in which the effects of MAPK inhibitors on vasospasm have been investigated in vivo. The authors demonstrate that MAPK may play a role in vasospasm and that PD-98059 is a potential candidate for the treatment of cerebral vasospasm.

Topics: Animals; Butadienes; Cerebral Angiography; Cerebral Arteries; Disease Models, Animal; Dogs; Flavonoids; Mitogen-Activated Protein Kinases; Nitriles; Subarachnoid Hemorrhage; Vasodilator Agents; Vasospasm, Intracranial

Vasospasm in the penetrating arteries contributes to ischemic neurological deficit. It may be as important as angiographic vasospasm because it would explain the discrepancies between angiographic vasospasm and clinical symptoms in some patients. It may also underlie the different effects of vasodilators. The present study examined this hypothesis by looking at the effect of the inhibitors of mitogen-activated protein kinase (MAPK) on vasospasm of the penetrating arteries.. Twenty-two adult mongrel dogs of either sex were used for the dog double-hemorrhage model. The dogs were randomly divided into four groups: control-hemorrhage, vehicle-treated, PD98059-treated, and U0126-treated groups. The drug injections were started on Day 3 after the first subarachnoid hemorrhage (SAH). The clinical status of the dogs was studied, based on their activity, appetite, and focal neurological symptoms. On Day 7, all the dogs were sacrificed, and the penetrating arteries from the brain stem were prepared for transmission electron microscopy.. (1) Severe vasospasm developed in the basilar arteries in the SAH-without-treatment group (control), in the DMSO-treated group (DMSO), and in the U0126 treatment group with mean reduction of the basilar artery diameter of 46.57%, 49.3%, and 39.6%, respectively. In the PD98059-treatment group only a mild vasospasm was observed and the mean reduction of the basilar artery diameter was 18.9%. (2) All the dogs in the control SAH and vehicle-treated groups developed severe angiographic and clinical vasospasm. The penetrating arteries were contracted, and the endothelial and smooth muscle cells were dystrophic. (3) The dogs in the U0126-treated group developed severe angiographic, but not clinical, vasospasm. The penetrating arteries were not contracted, and the endothelial and smooth muscle cells were not dystrophic. (4) The dogs in the PD98059 group developed mild angiographic vasospasm. No dog developed clinical symptoms that could be attributed to vasospasm. In morphological studies, the penetrating arteries were slightly contracted, but the cells were not dystrophic.. Vasospasm of the penetrating arteries, but not angiographic vasospasm, is consistent with the clinical symptoms and signs of vasospasm. MAPK may be important in maintaining vasospasm of both major and penetrating cerebral arteries. The correlation of the improvement in the clinical score with the reduction of vasospasm in the penetrating arteries demonstrated an important role of penetrating arteries in the morbidity and mortality caused by SAH.

Topics: Animals; Basilar Artery; Butadienes; Cerebral Arteries; Dogs; Enzyme Inhibitors; Female; Flavonoids; Male; Mitogen-Activated Protein Kinases; Models, Animal; Nitriles; Random Allocation; Subarachnoid Hemorrhage; Vasospasm, Intracranial