Jin A. Shin

Nanosized titanium dioxide enhanced inflammatory responses in the septic brain of mouse

Nanosized titanium dioxide (TiO(2)) is used widely in various everyday products and can be applied to the medical field for diagnostic or therapeutic tools. However, its neurobiological responses have not been defined completely in the brain. To evaluate the acute inflammatory response to TiO(2) particles of two different sizes in normal and septic brains, male C57BL/6 mice were given intraperitoneal injections of fine (<1 microm) or ultrafine (21 nm) TiO(2), 30 min after vehicle or lipopolysaccaride (LPS). In the normal brain, neither fine nor ultrafine TiO(2) induced inflammation. However, in the brains of LPS-exposed mice, ultrafine TiO(2) significantly elevated proinflammatory cytokine interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) mRNAs, and IL-1beta protein levels. Also ultrafine TiO(2) increased the levels of reactive oxygen species and activated microglia 24 h after LPS challenge. In BV2 microglial cells stimulated with LPS, ultrafine TiO(2) enhanced TNF-alpha production and augmented nuclear factor-kB binding activity. These findings suggest that nanosized TiO(2) promotes an exaggerated neuroinflammatory responses by enhancing microglial activation in the pre-inflamed brain, in part.

Anti-Inflammatory Mechanism of Compound K in Activated Microglia and Its Neuroprotective Effect on Experimental Stroke in Mice

Microglial activation plays a pivotal role in the pathogenesis of various neurologic disorders, such as cerebral ischemia, Alzheimers disease, and Parkinsons disease. Thus, controlling microglial activation is a promising therapeutic strategy for such brain diseases. In the present study, we found that a ginseng saponin metabolite, compound K [20-O-d-glucopyranosyl-20(S)-protopanaxadiol], inhibited the expressions of inducible nitric-oxide synthase, proinflammatory cytokines, monocyte chemotactic protein-1, matrix metalloproteinase-3, and matrix metalloproteinase-9 in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and primary cultured microglia. Subsequent mechanistic studies revealed that compound K suppressed microglial activation via inhibiting reactive oxygen species, mitogen-activated protein kinases, and nuclear factor-κB/activator protein-1 activities with enhancement of heme oxygenase-1/antioxidant response element signaling. To address the anti-inflammatory effects of compound K in vivo, we used two brain disease models of mice: sepsis (systemic inflammation) and cerebral ischemia. Compound K reduced the number of Iba1-positive activated microglia and inhibited the expressions of tumor necrosis factor-α and interleukin-1β in the LPS-induced sepsis brain. Furthermore, compound K reduced the infarct volume of ischemic brain induced by middle cerebral artery occlusion and suppressed microglial activation in the ischemic cortex. The results collectively suggest that compound K is a promising agent for prevention and/or treatment of cerebral ischemia and other neuroinflammatory disorders.

Therapeutic effects of resveratrol during acute periods following experimental ischemic stroke.

We defined whether resveratrol administration during the acute phase of ischemic stroke reduces brain injury in mice. Infarct volumes were decreased significantly in both sexes with different doses of resveratrol (5mg/kg for males and 1mg/kg for females) administered 3h after ischemic stroke. Administration of resveratrol 6h after insult was also effective to decrease infarct volumes. Resveratrol suppressed expressions of IL-1β and TNF-α, microglial activation, and ROS production in the ischemic cortex. The findings suggest that the suppression of inflammation is partly associated with the neuroprotective effects of resveratrol, and resveratrol can be developed as a therapeutic drug for acute ischemic stroke.

Anti‐inflammatory mechanism of ginsenoside Rh1 in lipopolysaccharide‐stimulated microglia: critical role of the protein kinase A pathway and hemeoxygenase‐1 expression

J. Neurochem. (2010) 115, 1668–1680.

Acute Resveratrol Treatment Modulates Multiple Signaling Pathways in the Ischemic Brain

Resveratrol has several beneficial effects, including reductions of oxidative stress, inflammatory responses and apoptosis. It has been known that resveratrol is a sirtuin 1 (SIRT1) activator and protective effects of resveratrol are mediated by Akt and mitogen-activated protein kinases. However, it is not examined whether these pathways are regulated by resveratrol in the ischemic brain. Previously, we found that acute resveratrol treatment reduces brain injury induced by transient focal ischemic stroke. In the present study, we defined the signaling pathways modulated by resveratrol in ischemia by examining SIRT1 expression and phosphorylation of Akt, ERK1/2 and p38 in the ischemic cortex. Resveratrol increased expression of SIRT1 and phosphorylation of Akt and p38 but inhibited the increase in phosphorylation of ERK1/2. Gene and protein levels of peroxisome proliferator-activated receptor γ coactivator 1α, a downstream molecule of SIRT1, and mRNA levels of its target genes antioxidative superoxide dismutase 2 and uncoupling protein 2 were elevated. Resveratrol also increased phosphorylation of cyclic AMP-response-element-binding protein and transcription of the anti-apoptotic gene Bcl-2. These results suggest that various neuroprotective actions of resveratrol, including anti-oxidative, anti-apoptotic and inflammatory effects, are mediated via modulation of multiple signaling pathways in the ischemic brain.

Matrix Metalloproteinase-8 Plays a Pivotal Role in Neuroinflammation by Modulating TNF-α Activation

Matrix metalloproteinases (MMPs) play important roles in normal brain development and synaptic plasticity, although aberrant expression of MMPs leads to brain damage, including blood–brain barrier disruption, inflammation, demyelination, and neuronal cell death. In this article, we report that MMP-8 is upregulated in LPS-stimulated BV2 microglial cells and primary cultured microglia, and treatment of MMP-8 inhibitor (M8I) or MMP-8 short hairpin RNA suppresses proinflammatory molecules, particularly TNF-α secretion. Subsequent experiments showed that MMP-8 exhibits TNF-α–converting enzyme (TACE) activity by cleaving the prodomain of TNF-α (A74/Q75, A76/V77 residues) and, furthermore, that M8I inhibits TACE activity more efficiently than TAPI-0, a general TACE inhibitor. Biochemical analysis of the underlying anti-inflammatory mechanisms of M8I revealed that it inhibits MAPK phosphorylation, NF-κB/AP-1 activity, and reactive oxygen species production. Further support for the proinflammatory role of microglial MMP-8 was obtained from an in vivo animal model of neuroinflammatory disorder. MMP-8 is upregulated in septic conditions, particularly in microglia. Administration of M8I or MMP-8 short hairpin RNA significantly inhibits microglial activation and expression/secretion of TNF-α in brain tissue, serum, and cerebrospinal fluid of LPS-induced septic mice. These results demonstrate that MMP-8 critically mediates microglial activation by modulating TNF-α activity, which may explain neuroinflammation in septic mouse brain.

Ischemic preconditioning-induced neuroprotection is associated with differential expression of IL-1β and IL-1 receptor antagonist in the ischemic cortex

Ischemic preconditioning (IP) is a phenomenon that organs develop a tolerance toward subsequent lethal ischemic insults. Among the factors that are involved in IP, IL-1beta and its endogenous receptor antagonist IL-1ra have been identified as important players in the induction of IP. The present study investigated whether IP affects the levels of these two antagonistic proteins during tolerance and reperfusion periods after ischemic stroke. The IP 24 h prior to ischemic stroke resulted in neuroprotection in the cortex. IP-induced protection is accompanied by increased IL-1beta gene and IL-1ra gene and protein levels during the tolerance period. In the post-ischemic cortex, IP resulted in the suppression of IL-1beta mRNA and protein levels at 6 h without affecting IL-1ra expression and the up-regulation of IL-1ra protein at 24 h. These findings demonstrate that IP differentially regulates cortical IL-1beta and IL-1ra expression before and after ischemic stroke and suggest that the shift toward an anti-inflammatory state in the post-ischemic cortex may contribute to IP-induced neuroprotection.

Conserved aquaporin 4 levels associated with reduction of brain edema are mediated by estrogen in the ischemic brain after experimental stroke

Aquaporin 4 (AQP4), the most abundant water channel protein in the brain, is involved in brain edema induced by ischemic insults. To evaluate whether the neuroprotective effects of estrogen are associated with AQP4 expression and edema formation, changes in AQP levels and ischemic edema were examined in the brains of male and female mice subjected to transient middle cerebral artery occlusion. Infarct volume and edema formation were markedly less in females than in males. AQP4 expression in the ischemic cortex of females was relatively well preserved, whereas it was significantly decreased in males. These effects disappeared in ovariectomized females but were reversed by estrogen replacement. Furthermore, AQP4 expression was decreased with increased brain edema in females treated with ICI182,780, an estrogen receptor antagonist. These findings suggest that the estrogen effect on the reduction of ischemic brain edema is associated with the preserved level of AQP4 that is partly mediated by estrogen receptors.

Noggin improves ischemic brain tissue repair and promotes alternative activation of microglia in mice.

We previously reported that bone morphogenetic proteins (BMPs) and their endogenous antagonist noggin are expressed in the brain weeks after an ischemic insult. Here, to define their roles in ischemic brain tissue repair and remodeling, we infused recombinant BMP7 or noggin into the ipsilateral ventricle of mice for 2weeks starting 2weeks after transient middle cerebral artery occlusion (MCAO). Four weeks after MCAO, we measured ischemic brain volume, functional recovery, and molecules related to neurogenesis and angiogenesis such as synaptophysin, GAP-43, and VEGF. Noggin-treated mice but not BMP7-treated mice showed preserved ipsilateral brain volume and reduced neurological deficits compared with artificial cerebrospinal fluids (aCSF)-treated mice. Noggin treatment also decreased glial scar thickness, increased levels of GAP-43 and VEGF protein, and increased the number of Iba1-positive activated microglia in the ipsilateral brain. Furthermore, noggin treatment decreased M1 markers (IL-1β, TNF-α, IL-12, CCL2 and CD86) and increased M2 markers (IL-1ra, IL-10, arginase 1, CD206 and Ym1) of activated microglia, suggesting a shift from M1 to M2 phenotypes. These results suggest that noggin improves functional recovery from ischemic stroke and enhances alternatively activated microglia, thereby promoting tissue repair and remodeling.

Activation of estrogen receptor β reduces blood–brain barrier breakdown following ischemic injury

Estrogen receptors (ERs) play important roles in estrogen-mediated neuroprotection. However, their effects on blood-brain barrier (BBB) disruption with vasogenic edema after ischemic stroke have not been determined. We evaluated a role for ERβ in the brain without effects in the peripheral reproductive organs for the amelioration of vasogenic edema following ischemic stroke. Transient focal ischemic stroke was induced in ovariectomized female C57BL/6 mice (age 10-11weeks) that were treated with the ERβ-selective agonist diarylpropionitrile (DPN). BBB breakdown as determined by the extravasation of endogenous immunoglobulin G (IgG), vasogenic edema, and the infarct volume was significantly reduced by DPN compared to vehicle. Protein expressions of endothelial tight junction proteins (occludin and claudin-5) and the water channel protein aquaporin 4 in the ischemic cortex were not changed by DPN. However, protein levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-1α), a transcription factor that increases VEGF expression, were significantly decreased in the ischemic cortex by DPN. These results suggest that ERβ contributes to the reduction of vasogenic edema caused by BBB breakdown via the inhibition of HIF-1α and VEGF following ischemic stroke.