Geum Sil Cho

Methyleugenol reduces cerebral ischemic injury by suppression of oxidative injury and inflammation

Abstract The present study tested the cytoprotective effect of methyleugenol in an in vivo ischemia model (i.e. middle cerebral artery occlusion (MCAO) for 1.5 h and subsequent reperfusion for 24 h) and further investigated its mechanism of action in in vitro cerebral ischemic models. When applied shortly after reperfusion, methyleugenol largely reduced cerebral ischemic injury. Methyleugenol decreased the caspase-3 activation and death of cultured cerebral cortical neurons caused by oxygen-glucose deprivation (OGD) for 1 h and subsequent re-oxygenation for 24 h. Methyleugenol markedly reduced superoxide generation in the ischemic brain and decreased the intracellular oxidative stress caused by OGD/re-oxygenation. It was found that methyleugenol elevated the activities of superoxide dismutase and catalase. Further, methyleugenol inhibited the production of nitric oxide and decreased the protein expression of inducible nitric oxide synthase. Methyleugenol down-regulated the production of pro-inflammatory cytokines in the ischemic brain as well as in immunostimulated mixed glial cells. The results indicate that methyleugenol could be useful for the treatment of ischemia/inflammation-related diseases.

Activated Microglia Are Less Vulnerable to Hemin Toxicity due to Nitric Oxide-Dependent Inhibition of JNK and p38 MAPK Activation

In intracerebral hemorrhage, microglia become rapidly activated and remove the deposited blood and cellular debris. To survive in a harmful hemorrhagic or posthemorrhagic condition, activated microglia must be equipped with appropriate self-defensive mechanism(s) to resist the toxicity of hemin, a component released from damaged RBCs. In the current study, we found that activation of microglia by pretreatment with LPS markedly reduced their vulnerability to hemin toxicity in vitro. Similarly, intracorpus callosum microinjection of LPS prior to hemin treatment reduced the brain tissue damage caused by hemin and increased microglial density in the penumbra in rats. LPS induced the expressions of inducible NO synthase (iNOS) and heme oxygenase (HO)-1, the rate-limiting enzyme in heme degradation in microglia. The preventive effect by LPS was significantly diminished by an iNOS inhibitor, L-N6-(1-iminoethyl)lysine, whereas it was mimicked by a NO donor, diethylamine-NONOate, both suggesting the crucial role of NO in the modulation of hemin-induced toxicity in activated microglia. We further found that NO reduced hemin toxicity via inhibition of hemin-induced activation of JNK and p38 MAPK pathways in microglia. Whereas HO-1 expression in LPS-stimulated microglia was markedly blocked by L-N6-(1-iminoethyl)lysine, the HO-1 inhibitor, tin protoporphyrin, increased iNOS expression and decreased the susceptibility of LPS-activated microglia to hemin toxicity. The data indicate that the mutual interaction between NO and HO-1 plays a critical role in modulating the adaptive response of activated microglia to hemin toxicity. Better understanding of the survival mechanism of activated microglia may provide a therapeutic strategy to attenuate the devastating intracerebral hemorrhagic injury.

Neuroprotective effect of fucoidin on lipopolysaccharide accelerated cerebral ischemic injury through inhibition of cytokine expression and neutrophil infiltration

In our previous study, we reported that lipopolysaccharide (LPS) activated microglia and accelerated cerebral ischemic injury in the rat brain through the overexpression of cytokines in microglia. In the present study, we investigated the effect of the intraperitoneal administration of fucoidin, a potent inhibitor of leukocyte rolling and anti-inflammatory agent, against accelerated cerebral ischemic injury by LPS pretreatment using rats. We found that fucoidin treatment inhibited the expressions of some brain cytokine or chemokine mRNA such as IL-8, TNF-α and iNOS in the brain of the rats treated only with LPS. We also observed that fucoidin treatment dramatically decreased the infarct size in accelerated cerebral ischemic injury induced by LPS treatment at an early time after ischemic injury. In addition, the immunoreactivity of myleoperoxidase (MPO), a marker for quantifying neutrophil accumulation, was distinctively decreased in the ischemic brain of the fucoidin-treated rat. In brief, our results indicate that fucoidin showed a neuroprotective effect on LPS accelerated cerebral ischemic injury through inhibiting the expression of some cytokine/chemokine and neutrophil recruitments.

Anti-ischemic and anti-inflammatory activity of (S)-cis-verbenol

Abstract (S)-cis-verbenol, a natural metabolite from (-)-alpha-pinene of host pine tree, has been suggested to have anti-ischemic activity. However, the exact mechanism for the anti-ischemic activity of (S)-cis-verbenol remains unclear yet. In the present study, (S)-cis-verbenol reduced cerebral ischemic injury caused by 1.5-h middle cerebral artery occlusion followed by 24-h reperfusion. Furthermore, (S)-cis-verbenol significantly prevented neuronal cell death caused by oxygen-glucose deprivation (OGD, 1 h) and subsequent re-oxygenation (5 h). While (S)-cis-verbenol did not inhibit the NMDA-stimulated calcium influx, it reduced the intracellular level of reactive oxygen species (ROS) elevated by OGD/re-oxygenation. ORAC assay indicated that (S)-cis-verbenol potently eliminated peroxyl radicals. In DPPH and DHR123 fluorescence assays, however, (S)-cis-verbenol did not show a direct ROS scavenging effect. Furthermore, (S)-cis-verbenol reduced the expression levels of pro-inflammatory cytokines in ischemic brain and immunostimulated glial cells. The present results indicate that (S)-cis-verbenol may be a useful therapeutic agent due to its anti-oxidative and anti-inflammatory activities.

Neuroprotection of Ischemic Preconditioning is Mediated by Thioredoxin 2 in the Hippocampal CA1 Region Following a Subsequent Transient Cerebral Ischemia

Preconditioning by brief ischemic episode induces tolerance to a subsequent lethal ischemic insult, and it has been suggested that reactive oxygen species are involved in this phenomenon. Thioredoxin 2 (Trx2), a small protein with redox‐regulating function, shows cytoprotective roles against oxidative stress. Here, we had focused on the role of Trx2 in ischemic preconditioning (IPC)‐mediated neuroprotection against oxidative stress followed by a subsequent lethal transient cerebral ischemia. Animals used in this study were randomly assigned to six groups; sham‐operated group, ischemia‐operated group, IPC plus (+) sham‐operated group, IPC + ischemia‐operated group, IPC + auranofin (a TrxR2 inhibitor) + sham‐operated group and IPC + auranofin + ischemia‐operated group. IPC was subjected to a 2 minutes of sublethal transient ischemia 1 day prior to a 5 minutes of lethal transient ischemia. A significant loss of neurons was found in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) in the ischemia‐operated‐group 5 days after ischemia‐reperfusion; in the IPC + ischemia‐operated‐group, pyramidal neurons in the SP were well protected. In the IPC + ischemia‐operated‐group, Trx2 and TrxR2 immunoreactivities in the SP and its protein level in the CA1 were not significantly changed compared with those in the sham‐operated‐group after ischemia‐reperfusion. In addition, superoxide dismutase 2 (SOD2) expression, superoxide anion radical ( O2− ) production, denatured cytochrome c expression and TUNEL‐positive cells in the IPC + ischemia‐operated‐group were similar to those in the sham‐operated‐group. Conversely, the treatment of auranofin to the IPC + ischemia‐operated‐group significantly increased cell damage/death and abolished the IPC‐induced effect on Trx2 and TrxR2 expressions. Furthermore, the inhibition of Trx2R nearly cancelled the beneficial effects of IPC on SOD2 expression, O2− production, denatured cytochrome c expression and TUNEL‐positive cells. In brief, this study shows that IPC conferred neuroprotection against ischemic injury by maintaining Trx2 and suggests that the maintenance or enhancement of Trx2 expression by IPC may be a legitimate strategy for therapeutic intervention of cerebral ischemia.

N-Methyl-d-aspartate receptor antagonists memantine and MK-801 attenuate the cerebral infarct accelerated by intracorpus callosum injection of lipopolysaccharides

Inflammatory responses have been shown to modulate the pattern and degree of ischemic injury. Previously, we demonstrated that intracorpus callosum microinjection of lipopolysaccharide (LPS, a well-known endotoxin) markedly induced inflammatory responses confined to ipsilateral hemisphere and aggravated cerebral ischemic injury. Here we report that LPS injection increases the degree of N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, one of major causes of cerebral ischemic injury. Intracorpus callosum microinjection of LPS 1 day prior to ischemic insults augmented intraneuronal Ca(2+) rise in rat brains subjected to transient occlusion of middle cerebral artery. Intraperitoneal administration of memantine, a NMDA receptor antagonist, reduced the LPS-enhanced calcium response as well as ischemic tissue damage. Western blot and immunohistochemistry data showed that the level of IL-1β was enhanced in LPS-injected rat brains, particularly in isolectin-B4 immunoreactive cells. Intraventricular microinjection of recombinant rat IL-1β aggravated cerebral ischemic injury, which was significantly reduced by memantine. Intraventricular injection of anti-IL-1β antibody significantly reduced the cerebral infarction aggravated by LPS preinjection. The results indicate that IL-1β released from isolectin-B4 immunoreactive cells enhanced excitotoxicity, consequently aggravating ischemic brain injury.

Changes in the expression of DNA-binding/differentiation protein inhibitors in neurons and glial cells of the gerbil hippocampus following transient global cerebral ischemia

Inhibitors of DNA-binding/differentiation (ID) proteins bind to basic helix-loop-helix (bHLH) transcription factors, including those that regulate differentiation and cell-cycle progression during development, and regulate gene transcription. However, little is known about the role of ID proteins in the brain under transient cerebral ischemic conditions. In the present study, we examined the effects of ischemia-reperfusion (I-R) injury on the immunoreactivity and protein levels of IDs 1–4 in the gerbil hippocampus proper Cornu Ammonis regions CA1–3 following 5 min of transient cerebral ischemia. Strong ID1 immunoreactivity was detected in the nuclei of pyramidal neurons in the hippocampal CA1–3 regions; immunoreactivity was significantly changed following I-R in the CA1 region, but not in the CA2/3 region. Five days following I-R, ID1 immunoreactivity was not detected in the CA1 pyramidal neurons. ID1 immunoreactivity was detected only in GABAergic interneurons in the ischemic CA1 region. Weak ID4 immunoreactivity was detected in non-pyramidal cells, and immunoreactivity was again only changed in the ischemic CA1 region. Five days following I-R, strong ID4 immunoreactivity was detected in non-pyramidal cells, which were identified as microglia, and not astrocytes, in the ischemic CA1 region. Furthermore, changes in the protein levels of ID1 and ID4 in the ischemic CA1 region studied by western blot were consistent with patterns of immunoreactivity. In summary, these results indicate that immunoreactivity and protein levels of ID1 and ID4 are distinctively altered following transient cerebral ischemia only in the CA1 region, and that the changes in ID1 and ID4 expression may relate to the ischemia-induced delayed neuronal death.

Simvastatin Reduces Lipopolysaccharides-Accelerated Cerebral Ischemic Injury via Inhibition of Nuclear Factor-kappa B Activity

Preceding infection or inflammation such as bacterial meningitis has been associated with poor outcomes after stroke. Previously, we reported that intracorpus callosum microinjection of lipopolysaccharides (LPS) strongly accelerated the ischemia/reperfusion-evoked brain tissue damage via recruiting inflammatory cells into the ischemic lesion. Simvastatin, 3-hydroxy-3-methylgultaryl (HMG)-CoA reductase inhibitor, has been shown to reduce inflammatory responses in vascular diseases. Thus, we investigated whether simvastatin could reduce the LPS-accelerated ischemic injury. Simvastatin (20 mg/kg) was orally administered to rats prior to cerebral ischemic insults (4 times at 72, 48, 25, and 1-h pre-ischemia). LPS was microinjected into rat corpus callosum 1 day before the ischemic injury. Treatment of simvastatin reduced the LPS-accelerated infarct size by 73%, and decreased the ischemia/reperfusion-induced expressions of pro-inflammatory mediators such as iNOS, COX-2 and IL-1β in LPS-injected rat brains. However, simvastatin did not reduce the infiltration of microglial/macrophageal cells into the LPS-pretreated brain lesion. In vitro migration assay also showed that simvastatin did not inhibit the monocyte chemoattractant protein-1-evoked migration of microglial/macrophageal cells. Instead, simvastatin inhibited the nuclear translocation of NF-κB, a key signaling event in expressions of various proinflammatory mediators, by decreasing the degradation of IκB. The present results indicate that simvastatin may be beneficial particularly to the accelerated cerebral ischemic injury under inflammatory or infectious conditions.

Post-ischemic treatment of WIB801C, standardized Cordyceps extract, reduces cerebral ischemic injury via inhibition of inflammatory cell migration.

ETHNOPHARMACOLOGICAL RELEVANCE Anti-inflammatory therapy has been intensively investigated as a potential strategy for treatment of cerebral stroke. However, despite many positive outcomes reported in animal studies, anti-inflammatory treatments have not proven successful in humans as yet. Although immunomodulatory activity and safety of Cordyceps species (Chinese caterpillar fungi) have been proven in clinical trials and traditional Asian prescriptions for inflammatory diseases, its anti-ischemic effect remains elusive. AIM OF THE STUDY In the present study, therefore, we investigated the potential therapeutic efficacy of WIB801C, the standardized extract of Cordyceps militaris, for treatment of cerebral ischemic stroke. MATERIALS AND METHODS The anti-chemotactic activity of WIB801C was assayed in cultured rat microglia/macrophages. Sprague-Dawley rats were subjected to ischemic stroke via either transient (1.5-h tMCAO and subsequent 24-h reperfusion) or permanent middle cerebral artery occlusion (pMCAO for 24-h without reperfusion). WIB801C was orally administered twice at 3- and 8-h (50mg/kg each) after the onset of MCAO. Infarct volume, edema, blood brain barrier and white matter damages, neurological deficits, and long-term survival rates were investigated. The infiltration of inflammatory cells into ischemic lesions was assayed by immunostaining. RESULTS WIB801C significantly decreased migration of cultured microglia/macrophages. This anti-chemotactic activity of WIB-801C was not mediated via adenosine A3 receptors, although cordycepin, the major ingredient of WIB801C, is known as an adenosine receptor agonist. Post-ischemic treatment with WIB801C significantly reduced the infiltration of ED-1-and MPO-positive inflammatory cells into ischemic lesions in tMCAO rats. WIB801C-treated rats exhibited significantly decreased infarct volume and cerebral edema, less white matter and blood-brain barrier damages, and improved neurological deficits. WIB801C also improved survival rates over 34 days after ischemia onset. A significant reduction in infarct volume and neurobehavioral deficits by WIB801C was also observed in rats subjected to pMCAO. CONCLUSIONS In summary, post-ischemic treatment of WIB801C reduced infiltration of inflammatory cells into ischemic lesions via inhibition of chemotaxis, which confers long-lasting histological and neurological protection in ischemic brain. WIB801C may be a promising anti-ischemic drug candidate with clinically relevant therapeutic time window and safety.

Effects of chronic scopolamine treatment on cognitive impairment and neurofilament expression in the mouse hippocampus

Neurofilaments (NFs) including neurofilament-200 kDa (NF-H), neurofilament-165 kDa (NF-M) and neurofilament-68 kDa (NF-L) are major protein constituents of the brain, and serve important roles in the regulation of axonal transport. NF alteration is a key feature in the pathogenesis of neurological disorders involving cognitive dysfunction. In the present study, cognitive impairments were investigated, via assessments using the Morris water maze and passive avoidance tests, in mice following chronic systemic treatment with 1 mg/kg scopolamine (SCO) for 4 weeks. SCO-induced cognitive impairments were significantly observed 1 week following the SCO treatment, and these cognitive deficits were maintained for 4 weeks. However, the NF immunoreactivities and levels were altered differently according to the hippocampal subregion following SCO treatment. NF-H immunoreactivity and levels were markedly altered in all hippocampal subregions, and were significantly increased 1 week following the SCO treatment; thereafter, the immunoreactivity and levels significantly decreased with time. NF-M immunoreactivity and levels gradually decreased in the hippocampus and were significantly decreased 4 weeks following SCO treatment. NF-L immunoreactivity and levels gradually decreased in the hippocampus, and were significantly decreased 2 and 4 weeks following SCO treatment. In conclusion, the results of the present study demonstrated that chronic systemic treatment with SCO induced cognitive impairment from 1 week following SCO treatment, and NF expression was diversely altered according to the hippocampal subregion from 1 week following SCO treatment. These results suggest that SCO-induced changes in NF expression may be associated with cognitive impairment.