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Resolution of inflammation is an emerging new strategy to reduce damage following ischemic stroke. Lipoxin A4 ( LXA4) is an anti-inflammatory, pro-resolution lipid mediator with high affinity binding to ALX, the lipoxin A4 receptor. Since LXA4 is rapidly inactivated, potent analogs have been created, including the ALX agonist BML-111. We hypothesized that post-ischemic intravenous administration of BML-111 would provide protection to the neurovascular unit and reduce neuroinflammation in a rat stroke model. Animals were subjected to 90 min of middle cerebral artery occlusion ( MCAO) and BML-111 was injected 100 min and 24 h after stroke onset and animals euthanized at 48 h. Post-ischemic treatment with BML-111 significantly reduced infarct size, decreased vasogenic edema, protected against blood-brain barrier disruption, and reduced hemorrhagic transformation. Matrix metalloproteinase-9 and matrix metalloproteinase-3 were significantly reduced following BML-111 treatment. Administration of BML-111 dramatically decreased microglial activation, as seen with CD68, and neutrophil infiltration and recruitment, as assessed by levels of myeloperoxidase and intracellular adhesion molecule-1. The tight junction protein zona occludens-1 was protected from degradation following treatment with BML-111. These results indicate that post-ischemic activation of ALX has pro-resolution effects that limit the inflammatory damage in the cerebral cortex and helps maintain blood-brain barrier integrity after ischemic stroke. [ABSTRACT FROM AUTHOR]

Although hemin-mediated neurotoxicity has been linked to the production of free radicals and glutamate excitotoxicity, the role of the prostaglandin E2 (PGE2)-EP1 receptor remains unclear. Activation of the EP1 receptor in neurons results in increased intracellular calcium levels; therefore, we hypothesize that the blockade of the EP1 receptor reduces hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wild-type (WT) and EP1-/- mice, we investigated the EP1 receptor role in hemin neurotoxicity measured by lactate dehydrogenase (LDH) cell survival assay. Hemin (75μM) induced greater release of LDH in WT (34.7 ± 4.5%) than in EP1-/- (27.6 ± 3.3%) neurons. In the presence of the EP1 receptor antagonist SC-51089, the hemin-induced release of LDH decreased. To further investigate potential mechanisms of action, we measured changes in the intracellular calcium level [Ca2+]i following treatment with 17-phenyl trinor PGE2 (17-pt-PGE2) a selective EP1 agonist. In the WT neurons, 17-pt-PGE2 dose-dependently increased [Ca2+]i. However, in EP1-/- neurons, [Ca2+]i was significantly attenuated. We also revealed that hemin dose-dependently increased [Ca2+]i in WT neurons, with a significant decrease in EP1-/- neurons. Both 17-pt-PGE2 and hemin-induced [Ca2+]i were abolished by N-methyl-D-aspartic (NMDA) acid receptor and ryanodine receptor blockers. These results suggest that blockade of the EP1 receptor may be protective against hemin neurotoxicity in vitro. We speculate that the mechanism of hemin neuronal death involves [Ca2+]i mediated by NMDA acid receptor-mediated extracellular Ca2+ influx and EP1 receptor-mediated intracellular release from ryanodine receptor-operated Ca2+ stores. Therefore, blockade of the EP1 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin. [ABSTRACT FROM AUTHOR]

Although hemin-mediated neurotoxicity has been linked to the production of free radicals and glutamate excitotoxicity, the role of the prostaglandin E2 (PGE2)-EP1 receptor remains unclear. Activation of the EP1 receptor in neurons results in increased intracellular calcium levels; therefore, we hypothesize that the blockade of the EP1 receptor reduces hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wild-type (WT) and EP1-/- mice, we investigated the EP1 receptor role in hemin neurotoxicity measured by lactate dehydrogenase (LDH) cell survival assay. Hemin (75 μM) induced greater release of LDH in WT (34.7 ± 4.5%) than in EP1-/- (27.6 ± 3.3%) neurons. In the presence of the EP1 receptor antagonist SC-51089, the hemin-induced release of LDH decreased. To further investigate potential mechanisms of action, we measured changes in the intracellular calcium level [Ca2+]i following treatment with 17-phenyl trinor PGE2 (17-pt-PGE2) a selective EP1 agonist. In the WT neurons, 17-pt-PGE2 dose-dependently increased [Ca2+]i. However, in EP1-/- neurons, [Ca2+]i was significantly attenuated. We also revealed that hemin dose-dependently increased [Ca2+]i in WT neurons, with a significant decrease in EP1-/- neurons. Both 17-pt-PGE2 and hemin-induced [Ca2+]i were abolished by N-methyl-D-aspartic acid receptor and ryanodine receptor blockers. These results suggest that blockade of the EP1 receptor may be protective against hemin neurotoxicity in vitro. We speculate that the mechanism of hemin neuronal death involves [Ca2+]i mediated by Nmethyl- D-aspartic acid receptor-mediated extracellular Ca2+ influx and EP1 receptor-mediated intracellular release from ryanodine receptor-operated Ca2+ stores. Therefore, blockade of the EP1 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin. [ABSTRACT FROM AUTHOR]

The cardiovascular complications reported to be associated with cyclooxygenase inhibitor use have shifted our focus toward prostaglandins and their respective receptors. Prostaglandin D2 and its DP1 receptor have been implicated in various normal and pathologic conditions, but their role in stroke is still poorly defined. Here, we tested whether DP1 deletion aggravates N-methyl- d-aspartic acid (NMDA)-induced acute toxicity and whether DP1 pharmacologic activation protects mice from acute excitotoxicity and transient cerebral ischemia. Moreover, since the elderly are more vulnerable to stroke-related damage than are younger patients, we tested the susceptibility of aged DP1 knockout (DP1−/−) mice to brain damage. We found that intrastriatal injection of 15 nmol NMDA caused significantly larger lesion volumes (27.2 ± 6.4%) in young adult DP1−/− mice than in their wild-type counterparts. Additionally, intracerebroventricular pretreatment of wild-type mice with 10, 25, and 50 nmol of the DP1-selective agonist BW245C significantly attenuated the NMDA-induced lesion size by 19.5 ± 5.0%, 39.6 ± 7.7%, and 28.9 ± 7.0%, respectively. The lowest tested dose of BW245C also was able to reduce middle cerebral artery occlusion-induced brain infarction size significantly (21.0 ± 5.7%). Interestingly, the aggravated NMDA-induced brain damage was persistent in older DP1−/− mice as well. We conclude that the DP1 receptor plays an important role in attenuating brain damage and that selective targeting of this receptor could be considered as an adjunct therapeutic tool to minimize stroke damage. [ABSTRACT FROM AUTHOR]

Death-associated protein kinase (DAPK) is a key player in multiple cell death signaling pathways. We report that DAPK is regulated by DANGER, a partial MAB-21 domain-containing protein.DANGERbinds directly toDAPKand inhibitsDAPKcatalytic activity. DANGERdeficient mouse embryonic fibroblasts and neurons exhibit greater DAPK activity and increased sensitivity to cell death stimuli than do wild-type control cells. In addition, DANGER-deficient mice manifest more severe brain damage after acute excitotoxicity and transient cerebral ischemia than do control mice. Accordingly, DANGER may physiologically regulate the viability of neurons and represent a potential therapeutic target for stroke and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Background: The prostaglandin E2 EP2 receptor has been shown to be important in dictating outcomes in various neuroinflammatory disorders. Here, we investigated the importance of the EP2 receptor in short- and long-term ischemic outcomes by subjecting wildtype (WT) and EP2 knockout (EP2-/-) mice to two distinct and complementary stroke models [transient and permanent middle cerebral artery occlusion (tMCAO and pMCAO)] and by using the EP2 receptor agonist ONO-AE1-259-01. Methods: First, WT and EP2-/- mice were subjected to 90-min tMCAO with a monofilament followed by 4-day reperfusion. Second, WT mice were infused intracerebroventricularly with vehicle or ONO-AE1-259-01 45-50 min before being subjected to tMCAO. Finally, WT and EP2-/- mice were subjected to pMCAO and allowed to survive for an extended period of 7 days. Results: Infarct volumes in EP2-/- mice were 55.0 ± 9.1% larger after tMCAO and 33.3 ± 8.6% larger after pMCAO than those in WT mice. Neurobehavioral deficits also were significantly greater in the EP2-/- mice. These results suggest that EP2 is beneficial and that activation is sustained for days after the stroke. We also found that pharmacologic activation of EP2 with 1.0- and 2.0-nmol doses of ONO-AE1-259-01 was sufficient to significantly reduce the infarct volume in WT mice compared with that in vehicle-treated controls (20.1 ± 3.9% vs. 37.1 ± 4.6%). This reduction correlated with improved neurologic scores. No significant effect on physiologic parameters was observed. Conclusion: Together, our results reveal that pharmacologic stimulation of the EP2 receptor has an important beneficial role in cerebral ischemia and might be considered as an adjunct therapy for ischemic stroke. [ABSTRACT FROM AUTHOR]

Background: The enzyme cytosolic phospholipase A2 alpha (cPLA2α) has been implicated in the progression of cerebral injury following ischemia and reperfusion. Previous studies in rodents suggest that cPLA2α enhances delayed injury extension and disruption of the blood brain barrier many hours after reperfusion. In this study we investigated the role of cPLA2α in early ischemic cerebral injury. Methods: Middle cerebral artery occlusion (MCAO) was performed on cPLA2α+/+ and cPLA2α-/- mice for 2 hours followed by 0, 2, or 6 hours of reperfusion. The levels of cPLA2α, cyclooxygenase-2, neuronal morphology and reactive oxygen species in the ischemic and contralateral hemispheres were evaluated by light and fluorescent microscopy. PGE2 content was compared between genotypes and hemispheres after MCAO and MCAO and 6 hours reperfusion. Regional cerebral blood flow was measured during MCAO and phosphorylation of relevant MAPKs in brain protein homogenates was measured by Western analysis after 6 hours of reperfusion. Results: Neuronal cPLA2α protein increased by 2-fold immediately after MCAO and returned to pre-MCAO levels after 2 hours reperfusion. Neuronal cyclooxygenase-2 induction and PGE2 concentration were greater in cPLA2α+/+ compared to cPLA2α-/- ischemic cortex. Neuronal swelling in ischemic regions was significantly greater in the cPLA2α+/+ than in cPLA2α-/- brains (+/+: 2.2 ± 0.3 fold vs. -/-: 1.7 ± 0.4 fold increase; P < 0.01). The increase in reactive oxygen species following 2 hours of ischemia was also significantly greater in the cPLA2α+/+ ischemic core than in cPLA2α-/- (+/+: 7.12 ± 1.2 fold vs. -/-: 3.1 ± 1.4 fold; P < 0.01). After 6 hours of reperfusion ischemic cortex of cPLA2α+/+, but not cPLA2α-/-, had disruption of neuron morphology and decreased PGE2 content. Phosphorylation of the MAPKs-p38, ERK ½, and MEK ½-was significantly greater in cPLA2α+/+ than in cPLA2α-/- ischemic cortex 6 hours after reperfusion. Conclusions: These results indicate that cPLA2α modulates the earliest molecular and injury responses after cerebral ischemia and have implications for the potential clinical use of cPLA2α inhibitors. [ABSTRACT FROM AUTHOR]

Hemoproteins undergo degradation during hypoxic/ischemic conditions, but the pro-oxidant free heme that is released cannot be recycled and must be degraded. The extracellular heme associates with its high-affinity binding protein, hemopexin (HPX). Hemopexin is shown here to be expressed by cortical neurons and it is present in mouse cerebellum, cortex, hippocampus, and striatum. Using the transient ischemia model (90-min middle cerebral artery occlusion followed by 96-h survival), we provide evidence that HPX is protective in the brain, as neurologic deficits and infarct volumes were significantly greater in HPX−/− than in wild-type mice. Addressing the potential protective HPX cellular pathway, we observed that exogenous free heme decreased cell survival in primary mouse cortical neuron cultures, whereas the heme bound to HPX was not toxic. Heme–HPX complexes induce HO1 and, consequently, protect primary neurons against the toxicity of both heme and pro-oxidant tert-butyl hydroperoxide; such protection was decreased in HO1−/− neuronal cultures. Taken together, these data show that HPX protects against heme-induced toxicity and oxidative stress and that HO1 is required. We propose that the heme–HPX system protects against stroke-related damage by maintaining a tight balance between free and bound heme. Thus, regulating extracellular free heme levels, such as with HPX, could be neuroprotective.Journal of Cerebral Blood Flow & Metabolism (2009) 29, 953–964; doi:10.1038/jcbfm.2009.19; published online 11 March 2009 [ABSTRACT FROM AUTHOR]

Carbon monoxide (CO) is associated with central nervous system toxicity. However, evidence also indicates that CO can be protective, depending on its concentration. To determine if CO can be neuroprotective after ischemic brain injury, we subjected mice to transient middle cerebral artery occlusion and exposed them to different concentrations of CO. We found that in mice, low CO levels protected the brain from injury following 90-min transient focal ischemia and 48 h of reperfusion. When inhalation of 125 or 250 ppm CO began immediately at the onset of reperfusion, total hemispheric infarct volume was reduced by 32.1 ± 8.9% and 62.2 ± 14.4%, respectively; with an extended therapeutic window of 1–3 h after ischemia, CO inhalation also attenuated infarct volume significantly. Furthermore, early CO exposure limited brain edema formation by 3.2 ± 0.8% (125 ppm) and 2.6 ± 0.3% (250 ppm). Finally, CO inhalation significantly improved neurological deficit scores at 48 h of survival time after ischemia. Transient elevation of carboxyhemoglobin levels returned rapidly to baseline when CO exposure was stopped. These findings suggest a potential application of CO to treat brain ischemic stroke. [ABSTRACT FROM AUTHOR]

Anti-inflammatory drugs reduce the risk of Alzheimer's disease but fail to slow its progression. Studying the expression of prostaglandin E2 synthases downstream of cyclooxygenase-2 is important. Here, the expression patterns of cytosolic prostaglandin E2 synthases, an immediate prostaglandin E2 source was investigated. Sections taken from the middle frontal gyrus of brains of 10 patients with Alzheimer's and 5 age-matched controls were examined by immunostaining for the presence of the synthases. Immunofluorescence analysis of control brains showed that cytosolic prostaglandin E2 synthases co-localize with microglia, neurons, and endothelium markers, but not with astrocytes or smooth muscle cells. Immunohistochemical staining for the synthases was positive in the pyramidal neurons of controls but barely detectable in the brain of Alzheimer's patients. These findings revealed that cytosolic prostaglandin E2 synthases is found in microglia, neurons, and endothelium of control human middle frontal gyrus and that its levels decrease in pyramidal cells of Alzheimer's disease brains. [ABSTRACT FROM AUTHOR]

Although some of the COX-2 metabolites and prostaglandins have been implicated in stroke and excitotoxicity, the role of prostaglandin F2a (PGF2a) and its FP receptor have not been elucidated in the pathogenesis of ischemic-reperfus ion (I/R) brain injury. Here we investigated the FP receptor's contribution in a unilateral middle cerebral artery (MCA) occlusion model of focal cerebral ischemia in mice. The MCA in wildtype (WT) and FP knockout (FP-/-) C57BL/6 male mice was transiently occluded with a monofilament for 90 min. After 96 h of reperfusion, the FP-/- mice had 25.3% less neurological deficit (p<0.05) and 34.4% smaller infarct volumes (p<0.05) than those of the W T mice. In a separate cohort, physiological parameters were monitored before, during, and after ischemia, and the results revealed no differences between the groups. Because excitotoxicity is an acute mediator of stroke outcome, the effect of acute NMDA- induced neurotoxicity was also tested. Forty-eight hours after unilateral intrastriatal NMDA injection, excitotoxic brain damage was 20.8% less extensive in the FP-/- mice (p<0.05) than in the WT counterparts, further supporting the toxic cont ribution of the FP receptor in I/R injury. Additionally, we investigated the effect of post-treatment with the FP agonist latanoprost in mice subjected to MCA occlusion; such treatment resulted in an increase in neurological deficit and infarct size in W T mice (p<0.05), though no effects were observed in the latanoprost-treated FP-/- mice. Together, the results suggest that the PGF2a FP receptor significantly enhances cerebral ischemic and excitotoxic brain injury and that these re sults are of importance when planning for potential development of therapeutic drugs to treat stroke and its acute and/or long term consequences. [ABSTRACT FROM AUTHOR]

Cyclooxygenase-2 (COX-2) enzyme increases abnormally during excitotoxicity and cerebral ischemia and promotes neurotoxicity. Although COX-2 inhibitors could be beneficial, they have significant side effects. We and others have shown that the EP1 receptor is important in mediating PGE2 toxicity. Here, we tested the hypothesis that pretreatment with a highly selective EP1 receptor antagonist, ONO-8713, would improve stroke outcome and that post-treatment would attenuate NMDA-induced acute excitotoxicity and protect organotypic brain slices from oxygen-glucose deprivation (OGD)-induced toxicity. Male C57BL/6 mice were injected intracerebroventricularly with ONO-8713 before being subjected to 90-min middle cerebral artery occlusion (MCAO) and 96-h reperfusion. Significant reduction in infarct size was observed in groups given 0.1 (25.9 ± 4.7%) and 1.0 nmol (27.7 ± 2.8%) ONO-8713 as compared with the vehicle-treated control group. To determine the effects of ONO-8713 post-treatment on NMDA-induced excitotoxicity, mice were given a unilateral intrastriatal NMDA injection followed by one intraperitoneal injection of 10 µg/kg ONO- 8713, 1 and 6 h later. Significant attenuation of brain damage (26.6 ± 4.9%) was observed at 48 h in the ONO-8713-treated group. Finally, brain slice cultures were protected (25.5 ± 2.9%) by the addition of ONO-8713 to the medium after OGD. These findings support the notion that the EP1 receptor propagates neurotoxicity and that selective blockade could be considered as a potential preventive and/or therapeutic tool against ischemic/ hypoxic neurological conditions. [ABSTRACT FROM AUTHOR]

Carbon monoxide derived from heme oxygenase (HO) may participate in cerebrovascular regulation under specific circumstances. Previous work has shown that HO contributes to feline pial arteriolar dilation to acetyicholine after transfusion of a cell-free polymeric hemoglobin oxygen carrier. The role of constitutive HO2 in the pial arteriolar dilatory response to acetylcholine was determined by using 1) HO2-null mice (HO2-/-), 2) the HO inhibitor tin protoporphyrin IX (SnPPIX), and 3) 4,5,6,7- tetrabromobenzotriazole (TBB), an inhibitor of casein kinase-2 (CK2)-dependent phosphorylation of HO2. In anesthetized mice, superfusion of a cranial window with SnPPIX decreased arteriolar dilation produced by 10 µM acetylcholine by 51%. After partial polymeric hemoglobin exchange transfusion, the acetylcholine response was normal but was reduced 72% by SnPPIX and 95% by TBB. In HO2-/- mice, the acetylcholine response was modestly reduced by 14% compared with control mice and was unaffected by SnPPIX. After hemoglobin transfusion in HO2-/- mice, acetylcholine responses were also unaffected by SnPPIX and TBB. In contrast, nitric oxide synthase inhibition completely blocked the acetylcholine responses in hemoglobin-transfused HO2-/- mice. We conclude 1) that HO2 activity partially contributes to acetylcholine-induced pial arteriolar dilation in mice, 2) that this contribution is augmented in the presence of a plasma-based hemoglobin polymer and appears to depend on a CK2 kinase mechanism, 3) that nitric oxide synthase activity rather than HO1 activity contributes to the acetylcholine reactivity in HO2-/- mice, and 4) that plasma-based polymeric hemoglobin does not scavenge all of the nitric oxide generated by cerebrovascular acetylcholine stimulation. [ABSTRACT FROM AUTHOR]

To investigate whether HT008-1, a prescription used in traditional Korean medicine to treat mental and physical weakness, has a neuroprotective effect on a rat model of global brain ischemia and an enhancing effect against memory deficit following ischemia. Global brain ischemia was induced for 10 min by using 4-vessel occlusion (4-VO). HT008-1 was orally administered at doses of 30, 100, and 300 mg/kg respectively twice at 0 and 90 min after ischemia. The effect on memory deficit was investigated by using a Y-maze neurobehavioral test 4 days after brain ischemia, and the effect on neuronal damage was measured 7 days after ischemia. The mechanism of action was studied immunohistochemically using an anti-CD11b (OX-42) antibody. The oral administration of HT008-1 at 100 and 300 mg/kg significantly reduced hippocampal neuronal cell death by 49% and 53%, respectively, compared with a vehicle-treated group, and also improved spatial memory function in the Y-maze test. Immunohistochemically, HT008-1 inhibited OX-42 expression in the hippocampus. The effects of HT008-1 were more pronounced than those of its individual herb components. The herbal mixture HT008-1 protects the most vulnerable CA1 pyramidal cells of the hippocampus and enhances spatial memory function against global brain ischemia; an anti-inflammatory effect may be one of the mechanisms of action. [ABSTRACT FROM AUTHOR]

Abstract: Background: The proinflammatory prostaglandin E2 (PGE2) fluctuates over time in the cerebrospinal fluid of patients with Alzheimer’s disease (AD), but the cerebral distribution and expression patterns of microsomal prostaglandin-E synthase (mPGES)–1 have not been compared with those of normal human brains. Methods: Middle frontal gyrus tissue from AD and age-matched control brains was analyzed by Western blot, immunofluorescence, and immunohistochemistry with mPGES-1–specific antibodies. Results: Western blotting revealed that mPGES-1 expression was significantly elevated in AD tissue. Furthermore, immunofluorescence of mPGES-1 was observed in neurons, microglia, and endothelial cells of control and AD tissue. Although mPGES-1 was consistently present in astrocytes of control tissue, it was present in only some astrocytes of AD tissue. Immunohistochemical staining suggested that mPGES-1 was elevated in pyramidal neurons of AD tissue when compared with controls. Conclusions: The results suggest that mPGES-1 is normally expressed constitutively in human neurons, microglia, astrocytes, and endothelial cells but is up-regulated in AD. [Copyright &y& Elsevier]

Prostaglandin D2 is the most abundant prostaglandin in the brain. It has long been described as a modulator of the neuroinflammatory process, but little is known regarding the role of its Gαs-coupled receptor, DP1. Therefore, in this study, the effect of the DP1 receptor on the outcome of cerebral ischemia in wildtype (WT) and DP1 knockout (DP1–/–) C57Bl/6 mice was investigated. Ischemia-reperfusion injury was produced by a 90-min occlusion of the right middle cerebral artery followed by a 4-day reperfusion. Infarct size was 49.0 ± 11.0% larger in DP1–/– mice ( n = 11; P < 0.01) than in WT mice ( n = 9 per group). However, no differences were detected in the relative cerebral blood flow (CBF) or any of the physiological parameters measured ( n = 5 per group) or in the large blood vessel anatomy ( n = 3 per group). To further address whether the DP1 protective role in the brain could be extended to neurons, mouse primary corticostriatal neuronal cultures were exposed to the DP1-selective agonist, BW245C, which provided dose-dependent protection against excitotoxicity induced by glutamate. Protection was significant at a dose as low as 0.05 µm. The results indicate that the DP1 receptor is neuroprotective in both in vivo and in vitro paradigms. Development of drugs to stimulate the DP1 receptor in brain could provide a new therapeutic strategy against cerebral ischemia and potentially other neurological conditions. [ABSTRACT FROM AUTHOR]

Intracerebral hemorrhage (ICH) is a devastating clinical event without effective therapies. Increasing evidence suggests that inflammatory mechanisms are involved in the progression of ICH-induced brain injury. Inflammation is mediated by cellular components, such as leukocytes and microglia, and molecular components, including prostaglandins, chemokines, cytokines, extracellular proteases, and reactive oxygen species. Better understanding of the role of the ICH-induced inflammatory response and its potential for modulation might have profound implications for patient treatment. In this review, a summary of the available literature on the inflammatory responses after ICH is presented along with discussion of some of the emerging opportunities for potential therapeutic strategies. In the near future, additional strategies that target inflammation could offer exciting new promise in the therapeutic approach to ICH.Journal of Cerebral Blood Flow & Metabolism (2007) 27, 894–908. doi:10.1038/sj.jcbfm.9600403; published online 11 October 2006 [ABSTRACT FROM AUTHOR]

Intraluminal occlusion of the middle cerebral artery in rodents is widely used for investigating cerebral ischemia and reperfusion injury. Two types of filaments used for occlusion were tested in terms of surgical success, incidence of subarachnoid hemorrhage, and mortality: a standard 6-0 monofilament coated with methyl methacrylate glue (rigid probe) and an 8-0 monofilament coated with silicone (flexible probe). In 98 wild-type (WT) mice, the flexible probe produced significantly (P < .05) more successful strokes (73.5%) than the rigid probe (46.6%). The incidences of subarachnoid hemorrhage (3.7%) and mortality (5.6%) with the flexible probe were significantly lower than those with the rigid probe (26.6% and 11.1%, respectively). Rigid and flexible probes were also compared in heme oxygenase 1 knockout (n = 17) and WT littermates (n = 17), because knockout mice have been suggested to have more fragile blood vessels. All mice receiving the flexible probe had successful strokes, with no cases of subarachnoid hemorrhage or mortality; however, with the rigid probe, the success rate was only 80% in the WT mice and 60% in the knockout mice. The rates of subarachnoid hemorrhage and mortality were also significantly higher with the rigid probe in both genotypes, but the infarct volumes produced by each type of probe did not differ significantly between the 2 groups. We conclude that the flexible silicone-coated 8-0 probe is superior to the more rigid glue-coated probe, because it produces infarct volumes of equal size with a higher success rate and lower risk of subarachnoid hemorrhage and mortality. [Copyright &y& Elsevier]

The clinical side effects associated with the inhibition of cyclooxygenase enzymes under pathologic conditions have recently raised concerns. A better understanding of neuroinflammatory mechanisms and neuronal survival requires knowledge of cyclooxygenase downstream pathways, especially PGE2 and its G-protein-coupled receptors. In this study, we postulate that EP1 receptor is one of the mechanisms that propagate neurotoxicity and could be a therapeutic target in brain injury. This hypothesis was tested by pretreating C57BL/6 wildtype mice with the EP1 receptor selective agonist ONO-DI-004 and the selective antagonist ONO-8713, followed by striatal unilateral NMDA injection. Results revealed that ONO-DI-004 increased NMDA-induced lesion volume up to 128.7 ± 12.0%, while ONO-8713 significantly decreased lesion volume to 71.3 ± 10.9%, as compared to the NMDA-control group. Neurotoxic EP1 receptor properties were also studied using C57BL/6 EP1 receptor knockout (EP1−/−) mice, which revealed a significant decrease to 74.5 ± 8.2%, as compared to wildtype controls. The protective effect of the antagonist ONO-8713 was also tested in the EP1−/− mice, revealing no additional protection in these mice. Together, these results support the selectivity of ONO-8713 toward EP1 receptor and suggest the neurotoxic role of EP1 receptor. Furthermore, the EP1 receptor role in ischemic brain damage was investigated using a model of middle cerebral artery occlusion (MCAO) and reperfusion. The infarct volume was significantly reduced to 56.9 ± 11.5% in EP1−/− mice, as compared to wildtype controls. This is the first study that demonstrates that EP1 receptor aggravates neurotoxicity and that modulation of this receptor can determine the outcomes in both excitotoxic and focal ischemic neuronal damage. [ABSTRACT FROM AUTHOR]

Differential neurological outcomes due to prostaglandin E2 activating G-protein-coupled prostaglandin E (EP) receptors have been observed. Here, we investigated the action of the EP4/EP3 agonist 1-hydroxyPGE1 (1-OHPGE1) in modulating transient ischemic brain damage. C57BL/6 mice were pretreated 50 min before transient occlusion of the middle cerebral artery with an intraventricular injection of 1-OHPGE1 (0.1, 0.2, 2.0 nmol/0.2 µL). Brain damage 4 days after reperfusion, as estimated by infarct volume, was significantly reduced by more than 19% with 1-OHPGE1 in the two higher-dose groups ( P < 0.05). To further address whether protection also was extended to neurons, primary mouse cultured neuronal cells were exposed to N-methyl-d-aspartate. Co-treatment with 1-OHPGE1 resulted in significant neuroprotection ( P < 0.05). To better understand potential mechanisms of action and to test whether changes in cyclic adenosine monophosphate (cAMP) levels and downstream signaling would be neuroprotective, we measured cAMP levels in primary neuronal cells. Brief exposure to 1-OHPGE1 increased cAMP levels more than twofold and increased the phosphorylation of extracellular-regulated kinases at positions Thr-202/Tyr-204. In a separate cohort of animals, 1-OHPGE1 at all doses tested produced no significant effect on the physiological parameters of core body temperature, mean arterial pressure and relative cerebral blood flow observed following drug treatment. Together, these results suggest that modulation of PGE2 receptors that increase cAMP levels and activate extracellular-regulated kinases 1/2 caused by treatment with 1-OHPGE1 can be protective against neuronal injury induced by focal ischemia. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is associated with gliosis, neuroinflammation and higher levels of prostaglandins. Conflicting roles for cyclooxygenases and prostaglandins in the etiopathology of AD have been reported. We hypothesized that PGE2 signaling through EP2 and EP4 G-protein-coupled receptors could protect against amyloid β-peptide (Aβ) neurotoxicity by increasing the cAMP signaling cascade. Using primary neuronal cultures, we investigated the presence of EP receptors (EP1–4) and the action of PGE2 and EP receptor agonists on neuronal susceptibility to Aβ1−42 toxicity. Low concentrations (1 µ m) of PGE2, butaprost (EP2 agonist), and 1-hydroxy-PGE1 (EP4/EP3 agonist) were neuroprotective against Aβ1−42 toxicity, while sulprostone (EP3/EP1 agonist) at similar doses had no detectable effects. EP2 and EP4 receptor-mediated neuroprotection would involve changes in cAMP levels, as both EP2 and EP4 agonists increased intracellular cAMP concentration by approximately doubling basal levels, and both exhibited neuroprotective actions against Aβ-induced toxicity. The protein kinase A (PKA) inhibitor RpcAMPS significantly attenuated the neuroprotection by butaprost, but not that by 1-hydroxy-PGE1, implying differences between EP2 and EP4 receptor protective mechanisms. Additionally, the increase in reactive oxygen species generated following exposure to Aβ was reduced by stimulation of both EP2 and EP4 receptors. Together, these results indicate that PGE2 can protect neurons against Aβ toxicity by acting on given receptors and stimulating a cascade of intracellular events, including the cAMP–PKA pathway. We propose that development and testing of specific PGE2 receptor agonists downstream of cyclooxygenase could lead to therapeutic applications. [ABSTRACT FROM AUTHOR]

Abstract: Heme oxygenase (HO) catalyzes the rate-limiting step in heme degradation, producing iron, carbon monoxide, and bilirubin/biliverdin. HO consists of two isozymes: HO-1, which is an oxidative stress-response protein, and HO-2, which is constitutively expressed. HO-2 accounts for most HO activity within the nervous system. Its posttranslational modifications and/or interactions with other proteins make HO-2 a unique regulator of cellular homeostasis. Our previous results revealed that brain infarct volume was enlarged in HO-2 knockout mice. A similar neuroprotective role of HO-2 was shown using primary cortical neurons. To better understand the neuroprotective mechanism of HO-2, we used a catalytically inactive mutant, HO-2H45A, and investigated its cellular effects in response to hemin and hydrogen peroxide-induced cytotoxicity. We observed that HO-2WT overexpression in the HEK293 cell lines became less sensitive to hemin, whereas the inactive mutant HO-2H45A was more sensitive to hemin as compared to control. Interestingly, HO-2WT- and HO-2H45A-overexpressing cells were both protected against H2O2-induced oxidative stress and had less oxidatively modified proteins as compared to control cells. These data indicate that when HO-2 cannot metabolize the prooxidant heme, more cytotoxicity is found, whereas, interestingly, the catalytically inactive HO-2H45A was also able to protect cells against oxidative stress injury. These results suggest the multiplicity of action of the HO-2 protein itself. [Copyright &y& Elsevier]

Eicosanoids and the enzymes associated with their metabolism play an active role in the neuroinflammatory process that is often a hallmark of neurodegenerative disorders. Cerebral cortical neurons constitute a highly affected cell population in neurologic disorders. To obtain a cellular model to analyze prostaglandin action and metabolism in cortical neurons, we developed postnatal neuronal cultures from mouse cortex in a serum-free medium. Cultured cortical cells were highly enriched in neurons containing only 5±2% glial cells. The cultures were assayed for expression of several protein markers of neuronal maturity: synaptic markers (synapsin I and synaptophysin) and glutamate receptor subunits (NMDA receptor 1 and glutamate receptor 1). The protein expression of eicosanoid-metabolizing enzymes, including cyclooxygenase-2 and microsomal and cytosolic PGE2 synthases, was investigated as well. Postnatal neurons successfully survived for a long term (up to 40 days) in vitro in serum-free media, as characterized by the expression of synapsin 1, synaptophysin, and microtubule-associated protein 2. Glutamate receptor subunit expression increased over time in cultures, with the highest levels at 15 days. Enzymes involved in the eicosanoid metabolism followed a distinct pattern of expression, suggesting potential regulation of PGE2 synthesis with time in cultures under basal conditions. Use of postnatal brain cultures offers several advantages, especially regarding degree of neuronal maturation, use of postnatal pups instead of pregnant mice, and potentially increased clinical relevance in models of neuroinflammation processes and prostanoid cellular neurobiology. [ABSTRACT FROM AUTHOR]

Heme oxygenase (HO) is the rate-limiting enzyme for the degradation of heme, a prooxidant, coming from a multitude of heme-containing proteins/enzymes. With the action of cytochrome P450 reductase, HO cleaves the heme ring into biliverdin which is converted into bilirubin, both have been shown to have intrinsic radical scavenger activities. Iron is also released from the heme core and in its free form can act as a catalyst for oxidative stress damage or can be sequested by several iron-binding proteins. Under physiological conditions, the newly generated iron can be neutralized within the cell. The third product of the opening of the porphyrin ring is carbon monoxide, which role has been puzzling. It has been reported as a potential neuromodulator, it modulates guanylate cyclase activity and has vasodilation, anti-inflammatory and antiapoptotic effects. In the brain, HO2 accounts for the vast majority of HO activity. By decreasing HO2 activity, one would expect more neuronal damage after oxidative stress injury with possible direct implications to acute and chronic neurodegenerative disorders. Pharmacological ways to increase neuronal HO activity is likely to have therapeutic applications. [Copyright &y& Elsevier]

Hemolysis is a physiological condition in which red blood cells (RBCs) lyse, releasing their contents into the extracellular environment. Hemolysis can be a manifestation of several diseases and conditions, such as sickle cell disease, hemorrhagic stroke, and trauma. Heme and hemoglobin are among the unique contents of RBCs that are released into the environment. Although these contents can cause oxidative stress, especially when oxidized in the extracellular environment, they can also initiate a proinflammatory response because they bind to receptors such as the Toll-like receptor (TLR) family. This review seeks to clarify the mechanism by which TLRs initiate a proinflammatory response to heme, hemoglobin, and their oxidized derivatives, as well as the possibility of using soluble TLRs (sTLRs) as therapeutic agents. Furthermore, this review explores the possibility of using sTLRs in hemorrhagic disorders in which mitigating inflammation is essential for clinical outcomes, including hemorrhagic stroke and its subtypes, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH). [ABSTRACT FROM AUTHOR]

Intracerebral hemorrhage (ICH) is the second most common subtype of stroke, and it is often associated with a high mortality rate and significant morbidity among survivors. Recent studies have shown that bilirubin, a product of heme metabolism, can exhibit cytoprotective, antioxidant and, anti-inflammatory properties. However, little is known about the role of bilirubin in combating several pathophysiological pathways caused by intracerebral bleeding in patients with ICH. In this study, data were collected retrospectively on 276 patients with ICH who were admitted to a university hospital between 5 January 2014 and 31 December 2017. We assessed the relationship between levels of total, direct, and indirect serum bilirubin and assessments of initial stroke severity and clinical outcomes by using Spearman's rank correlation and Kruskal-Wallis H tests. A secondary examination of the carrier protein albumin was also undertaken. Our study found that higher levels of direct bilirubin were correlated with worse admission Glasgow Coma Scales (GCS) (rs = −0.17, p = 0.011), worse admission ICH Scores (rs = 0.19, p = 0.008), and worse discharge modified Rankin Scales (mRS) (rs = 0.15, p = 0.045). Direct bilirubin was still significantly correlated with discharge mRS after adjusting for temperature at admission (rs = 0.16, p = 0.047), oxygen saturation at admission (rs = 0.15, p = 0.048), white blood cell count (rs = 0.18, p = 0.023), or Troponin T (rs = 0.25, p = 0.001) using partial Spearman's correlation. No statistical significance was found between levels of total or indirect bilirubin and assessments of stroke severity and outcomes. In contrast, higher levels of albumin were correlated with better admission GCS (rs = 0.13, p = 0.027), discharge GCS (rs = 0.15, p = 0.013), and discharge mRS (rs = −0.16, p = 0.023). We found that levels of total bilirubin, direct bilirubin, and albumin were all significantly related to discharge outcomes classified by discharge destinations (p = 0.036, p = 0.014, p = 0.016, respectively; Kruskal-Wallis H tests). In conclusion, higher direct bilirubin levels were associated with greater stroke severity at presentation and worse outcomes at discharge among patients with ICH. Higher levels of albumin were associated with lower stroke severity and better clinical outcomes. Future prospective studies on the free bioactive bilirubin are needed to better understand the intricate relationships between bilirubin and ICH. [ABSTRACT FROM AUTHOR]

Circulating hemopexin is the primary protein responsible for the clearance of heme; therefore, it is a systemic combatant against deleterious inflammation and oxidative stress induced by the presence of free heme. This role of hemopexin is critical in hemolytic pathophysiology. In this review, we outline the current research regarding how the dynamic activity of hemopexin is implicated in sickle cell disease, which is characterized by a pathological aggregation of red blood cells and excessive hemolysis. This pathophysiology leads to symptoms such as acute kidney injury, vaso-occlusion, ischemic stroke, pain crises, and pulmonary hypertension exacerbated by the presence of free heme and hemoglobin. This review includes in vivo studies in mouse, rat, and guinea pig models of sickle cell disease, as well as studies in human samples. In summary, the current research indicates that hemopexin is likely protective against these symptoms and that rectifying depleted hemopexin in patients with sickle cell disease could improve or prevent the symptoms. The data compiled in this review suggest that further preclinical and clinical research should be conducted to uncover pathways of hemopexin in pathological states to evaluate its potential clinical function as both a biomarker and therapy for sickle cell disease and related hemoglobinopathies. [ABSTRACT FROM AUTHOR]

Soluble receptors are widely understood to be freestanding moieties formed via cleavage from their membrane-bound counterparts. They have unique structures, are found among various receptor families, and have intriguing mechanisms of generation and release. Soluble receptors' ability to exhibit pleiotropic action by receptor modulation or by exhibiting a dual role in cytoprotection and neuroinflammation is concentration dependent and has continually mystified researchers. Here, we have compiled findings from preclinical and clinical studies to provide insights into the role of soluble/decoy receptors, focusing on the soluble cluster of differentiation 36, the soluble cluster of differentiation 163, and soluble lipoprotein-related protein 1 (sCD36, sCD163, and sLRP1, respectively) and the functions they could likely serve in the management of stroke, as they would notably regulate the bioavailability of the hemoglobin and heme after red blood cell lysis. The key roles that these soluble receptors play in inflammation, oxidative stress, and the related pharmacotherapeutic potential in improving stroke outcomes are described. The precise pleiotropic physiological functions of soluble receptors remain unclear, and further scientific investigation/validation is required to establish their respective role in diagnosis and therapy. [ABSTRACT FROM AUTHOR]

A recent article supports the concept that prostaglandin (PG)E2 EP1-receptor antagonists reduce stroke severity and cell damage; could these agents become a substitute for cyclooxygenase (COX)-2 inhibitors? The total activity of COXs – rate-limiting enzymes of PGE2 synthesis – increases following acute neurological insult. Drugs that offer the beneficial anti-inflammatory and neuroprotective effects of PGs but that limit the negative effects of COX-2 inhibition could provide the next generation of treatment for acute neuronal damage. [Copyright &y& Elsevier]

Cerebral ischemia is a devastating disease with a high incidence of death and disability; however, effective therapeutics remain limited. The transcriptional factor Nrf2 has been shown to play a pivotal role in the endogenous defense against brain oxidative stress and inflammation and therefore represents a promising target for stroke intervention. However, the long-term effects of Nrf2 and the standardized Korean red ginseng (ginseng), a potent Nrf2 natural inducer, on permanent cerebral ischemic damage have not yet been reported. Wildtype (WT) and Nrf2−/− adult mice were pretreated with either vehicle or ginseng and were subjected to permanent distal middle cerebral artery occlusion (pdMCAO). The infarct volume, the reactive astrocytes and microglia, and the water regulatory protein aquaporin 4 (AQP4) were examined at 28 days after stroke. When compared with the WT matched controls, the Nrf2 disruption significantly enlarged the infarct volume (40.4 ± 10.1%) and exacerbated the progression of reactive gliosis and AQP4 protein levels after pdMCAO. In contrast, ginseng significantly reduced the infarct volume and attenuated the reactive gliosis and AQP4 in the ischemic WT mice (47.3 ± 6.9%), but not in the Nrf2−/− mice (25.5 ± 5.6%). In conclusion, Nrf2 plays an important role in the long-term recovery of permanent cerebral ischemic damage and the neuroprotection of ginseng. [ABSTRACT FROM AUTHOR]