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Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is a cell adhesion molecule responsible for cell-to-cell interactions between immune cells and endothelial cells. In our previous paper, we have shown that Ninj1 plays an important role in the infiltration of neutrophils in the postischemic brain and that the dodecamer peptide harboring the Ninj1 N-terminal adhesion motif (N-NAM, Pro 26 -Asn 37 ) inhibits infiltration of neutrophils in the postischemic brain and confers robust neuroprotective and anti-inflammatory effects. In the present study, we examinedt the pro-angiogenic effect of N-NAM using human umbilical vein endothelial cells (HUVECs) and rat MCAO (middle cerebral artery occlusion) model of stroke. We found that N-NAM promotes proliferation, migration, and tube formation of HUVECs and demonstrate that the suppression of endogenous Ninj1 is responsible for the N-NAM-mediated pro-angiogenic effects. Importantly, a pull-down assay revealed a direct binding between exogenously delivered N-NAM and endogenous Ninj1 and it is N-terminal adhesion motif dependent. In addition, N-NAM activated the Ang1-Tie2 and AKT signaling pathways in HUVECs, and blocking those signaling pathways with specific inhibitors suppressed N-NAM-induced tube formation, indicating critical roles of those signaling pathways in N-NAM-induced angiogenesis. Moreover, in a rat MCAO model, intranasal administration of N-NAM beginning 4 days post-MCAO (1.5 µg daily for 3 days) augmented angiogenesis in the penumbra of the ipsilateral hemisphere of the brain and significantly enhanced total vessel lengths, vessel densities, and pro-angiogenic marker expression. These results demonstrate that the 12-amino acid Ninj1 peptide, which contains the N-terminal adhesion motif of Ninj1, confers pro-angiogenic effects and suggest that those effects might contribute to its neuroprotective effects in the postischemic brain.

Ethyl pyruvate (EP) is a simple aliphatic ester of pyruvic acid and has been shown to have protective properties, which have been attributed to its anti-inflammatory, anti-oxidative, and anti-apoptotic functions. In an effort to develop better derivatives of EP, we previously synthesized DEOPA (N,N-diethyl-2-oxopropanamide, a novel isoster of EP) which has greater neuroprotective effects than EP, probably due to its anti-inflammatory and anti-excitotoxic effects. In the present study, we synthesized 3 DEOPA derivatives, in which its diethylamino group was substituted with diisopropylamino, dipropylamino, or diisobutylamino groups. Among them, DIPOPA (N,N-diisopropyl-2-oxopropanamide) containing diisopropylamino group had a greater neuroprotective effect than DEOPA or EP when administered intravenously to a rat middle cerebral artery occlusion (MCAO) model at 9 h after MCAO. Furthermore, DIPOPA had a wider therapeutic window than DEOPA and a marked reduction of infarct volume was accompanied by greater neurological and behavioral improvements. In particular, DIPOPA exerted robust anti-inflammatory effects, as evidenced by marked suppressions of microglia activation and neutrophil infiltration in the MCAO model, in microglial cells, and in neutrophil-endothelial cocultures at lower concentration, and did so more effectively than DEOPA. In particular, DIPOPA remarkably suppressed neutrophil infiltration into brain parenchyma, and this effect was attributed to the expressional inhibitions of cell adhesion molecules in neutrophils of brain parenchyma and in circulating neutrophils via NF-κB inhibition. Together, these results indicate the robust neuroprotective effects of DIPOPA are attributable to its anti-inflammatory effects and suggest that DIPOPA offers a potential therapeutic means of ameliorating cerebral ischemic injury and other inflammation-related pathologies.

It has been reported that the innate immune response plays important roles in brain ischemia and that the infiltration of blood-derived immune cells is a key initiator of this response. Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is a cell adhesion molecule responsible for cell-to-cell interactions between immune cells and endothelial cells. In the present study, we investigated the proinflammatory and neuroprotective effects of Ninj1 and a dodecamer peptide harboring Ninj1 N-terminal adhesion motif (N-NAM, Pro26~Asn37) in a rat middle cerebral artery occlusion (MCAO) model of stroke. Ninj1 was predominantly induced in neutrophils and endothelial cells in the ischemic hemispheres around 12 h to 1 day post-MCAO, which coincided with a massive neutrophil influx. We demonstrated that intranasal administration of Ninj1 small interfering RNA (siRNA) or N-NAM significantly blocked neutrophil infiltration in postischemic brains. In addition, intranasal administration of Ninj1 siRNA or N-NAM reduced the mean infarct volume to 46.5 ± 9.2 or 30.6 ± 11.7% of that of the PBS-treated MCAO controls, respectively, which was accompanied by significant amelioration of neurological and motor deficits. We showed that N-NAM or Ninj1 siRNA effectively blocked the adhesion and transendothelial migration of TNF-α-stimulated human myelocytic leukemia cells to human umbilical vein endothelial cells and similarly suppressed adhesion and migration of monocytes. Activations of phosphoinositide 3-kinase and Ras-related C3 botulinum toxin substrate 1 are involved in these Ninj1-mediated processes and can be inhibited by N-NAM or Ninj1 siRNA. These results indicate that Ninj1 plays an important role in neutrophil infiltration in the postischemic brain and N-NAM confers robust neuroprotective and anti-inflammatory effects by inhibiting Ninj1-mediated infiltration of neutrophils.

4-Hydroxybenzyl alcohol (4-HBA) is an important phenolic constituent of Gastrodia elata (GE) Blume, which is used as a traditional herbal medicine in East Asia. Many activities have been reported to underlie the beneficial effects of 4-HBA in brain, such as, anti-oxidative, anti-inflammatory, anti-excitotoxic, and anti-apoptotic effects in neurons and microglia. Here, the authors demonstrate the robust neuroprotective effects of 4-HBA in rat middle cerebral artery occlusion (MCAO) model of stroke, and showed anti-Zn 2+ toxicity in neurons and astrocytes as a molecular mechanism contributing to these effects. Intraperitoneal administration of 4-HBA (20 mg/kg) in Sprague-Dawley rats 1 h after MCAO reduced infarct volumes to 27.1 ± 9.2% of that of MCAO controls and significantly ameliorated motor impairments and neurological deficits. Significant suppressions of Zn 2+ -induced cell death, ROS generation, and PARP-1 induction by 4-HBA were observed in primary cortical cultures. 4-HBA also protected astrocytes from Zn 2+ -induced toxicity and suppressing ROS generation by employing slightly different molecular mechanisms, i.e., suppressing PARP-1 induction and NAD depletion under acute Zn 2+ -treatment and suppressing p67 NADPH oxidase subunit induction under chronic Zn 2+ -treatment. Results indicate that the protective effects of 4-HBA against Zn 2+ -toxicity in neurons and astrocytes contribute to its robust neuroprotective effects in the postischemic brain. Considering the pleiotropic effects of 4-HBA, which have been reported in previous reports and added in the present study, it has therapeutic potential for the amelioration of ischemic brain damage.

4-Hydroxybenzyl alcohol (4-HBA) is an important phenolic constituent of Gastrodia elata Blume (GEB), a traditional herbal medicine used in East Asia. Many activities have been reported to underlie the beneficial effects of 4-HBA in the brain, and in particular, its anti-inflammatory, anti-oxidative, and anti-zinc-toxic effects have been implicated in the postischemic brain. Here, the authors investigated the anti-oxidative effect of 4-HBA on astrocytes and sought to identify the underlying molecular mechanisms involved. 4-HBA dose-dependently suppressed H2O2-induced astrocyte cell death. More specifically, pre-incubation of C6 cells (an astrocyte cell line) with 100 μM 4-HBA for 6 hrs increased survival when cells were treated with H2O2 (100 μM, 1 hr) from 54.2±0.7% to 85.9±1.5%. In addition, 4-HBA was found to up-regulate and activate Nrf2, and subsequently, to induce the expressions of several anti-oxidative genes, such as, HO-1, NQO1, and GCLM. Notably, HO-1 was induced by 3.4-fold in 4-HBA-treated C6 cells, and siRNA-mediated HO-1 knockdown demonstrated that Nrf2 activation and HO-1 induction were responsible for the observed cytoprotective effect of 4-HBA. ERK and Akt signaling pathways were activated by 4-HBA in C6 cells, suggesting their involvements in protective effect of 4-HBA. In addition, 4-HBA-conditioned astrocyte culture medium was found to have neuroprotective effects on primary neuronal cultures or fresh C6 cells exposed to oxidative stress, and these effects seemed to be mediated by glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), which both accumulated in 4-HBA-treated astrocyte culture media. Thus, the 4-HBA-mediated activation of Nrf2 and induction of HO-1 in astrocytes were found to act via autocrine and paracrine mechanisms to confer protective effects. Furthermore, given the pleiotropic effects of 4-HBA with respect to its targeting of various brain cell types and functions, it would appear that 4-HBA has therapeutic potential for the prevention and amelioration of various brain diseases.

Ethyl pyruvate (EP) is a simple aliphatic ester of pyruvic acid and has been shown to have robust neuroprotective effects via its anti-inflammatory, anti-oxidative, and anti-apoptotic functions. In an effort to develop novel EP derivatives with greater protective potencies than EP, we generated four EP isosteres, among them the neuroprotective potency of N,N-diethyl-2-oxopropanamide (DEOPA), in which the ethoxy group of EP was replaced with diethylamine, was far greater than that of EP. When DEOPA was administered intravenously (5 mg/kg) to rat middle cerebral artery occlusion (MCAO) model at 6 hrs post-surgery, it suppressed infarct formation, ameliorated neurological and sensory/motor deficits, and inhibited microglial activation and neutrophil infiltrations in the postischemic brain more effectively than EP. In particular, DEOPA markedly suppressed LPS-induced nitrite production and cytokine/chemokine inductions in microglia, neutrophils, and endothelial cells and these effects are attributable to inhibition of the activity of NF-κB by suppressing IκB-α degradation and p65 to DNA binding. In addition, DEOPA suppressed NMDA-induced neuronal cell death in primary cortical neuron cultures by NAD replenishment and suppression of NF-κB activity. Together, these results indicate DEOPA has multi-modal protective effects against ischemic brain damage targeting numerous cell types in the brain and also against other inflammation-related diseases.

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system and is mediated by complement-dependent cytotoxicity (CDC) of NMO-specific immunoglobulin G (IgG) antibodies (NMO-IgG). Glycyrrhizic acid (GA) has numerous pharmacological effects including inhibition of the complement pathway. We aimed to study the influence of GA on NMO-IgG-induced CDC. NMO-IgG samples from 7 patients with NMO, together with human complement, induced CDC in an aquaporin 4 M23-overexpressing glial cell line, an in vitro NMO model. GA attenuated NMO-IgG-induced CDC in a dose-dependent manner. The mechanism of the GA-related CDC inhibition was sequentially dissected and found to involve inhibition of C1q binding to NMO-IgG. Consequently, GA attenuates NMO-IgG-induced CDC and may be a promising novel therapeutic agent against NMO., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Postischemic brain damage in stroke is proceded with complicated pathological events, and so multimodal drug treatments may offer better therapeutic means for improving clinical outcomes. Here, we report robust neuroprotective effects of a novel compound, 2-((2-oxopropanoyl)oxy)-4-(trifluoromethyl)benzoic acid (OPTBA), a 2-hydroxy-4-trifluoromethyl benzoic acid (HTB, a metabolite of triflusal)-pyruvate ester. Intravenous administration of OPTBA (5 mg/kg) 3 or 6 h after middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats reduced infarct volumes to 38.5 ± 11.4% and 46.5 ± 15.3%, respectively, of that of MCAO controls, and ameliorated motor impairment and neurological deficits. Importantly, neuroprotective effects of OPTBA were far greater than those afforded by combined treatment of HTB and pyruvate. Furthermore, OPTBA suppressed microglial activation and proinflammatory cytokine inductions more effectively than HTB/pyruvate co-treatment in the postischemic brain and LPS-treated cortical slice cultures and also attenuated NMDA-induced neuronal death in hippocampal slice cultures. LC-MS analysis demonstrated that OPTBA was hydrolyzed to HTB and pyruvate with a t1/2 of 38.6 min in blood and 7.2 and 2.4 h in cortex and striatum, respectively, and HTB was maintained for more than 24 h both in blood and brain tissue. Together these results indicate OPTBA acts directly and via its hydrolysis products, thus acting as a multimodal neuroprotectant in the postischemic brain.

Ethyl pyruvate (EP) is a simple aliphatic ester of pyruvic acid and has been shown to have robust protective effect in various pathological conditions. A variety of mechanisms have been reported to underlie the protective effects of EP, which include anti-inflammatory, anti-oxidative, and anti-apoptotic functions. Recently, we reported that EP suppressed high mobility group box 1 (HMGB1) release from primary microglial cells via direct Ca(2+) chelation. In the present study, we investigated whether and how EP chelates Zn(2+) in neurons when it is present at toxic levels. In cortical neurons treated with 40 μM of Zn(2+) for 24 h, both EP and pyruvate significantly suppressed neuronal cell death, although the potency of pyruvate was greater than that of EP, and that NAD replenishment contributed to the neuroprotective effects of both pyruvate and EP. However, when cortical neurons were exposed to acute treatment of Zn(2+) (400 μM, 15 min), EP, but not pyruvate, significantly suppressed neuronal death, despite the fact that NAD replenishment by EP was weaker than that by pyruvate. Spectrophotometric studies revealed that EP directly chelates Zn(2+), and this was confirmed in physiological contexts, such as, NMDA-treated primary cortical cultures and OGD-subjected hippocampal slice cultures, in which EP suppressed intracellular Zn(2+) elevation and neuronal cell death. In addition, EP markedly reduced the expressions of PARP-1 and of the NADPH oxidase subunit in Zn(2+)-treated primary cortical neurons, well known Zn(2+)-induced downstream processes. Together, these results show EP suppresses Zn(2+) induced neurotoxicity via dual functions, chelating Zn(2+) and promoting NAD replenishment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cerebral ischemia leads to brain injury via a complex series of pathophysiological events, and therefore, multi-drug treatments or multi-targeting drug treatments provide attractive options with respect to limiting brain damage. Previously, we reported that a novel multi-functional compound oxopropanoyloxy benzoic acid (OBA-09, a simple ester of pyruvate and salicylic acid) affords robust neuroprotective effects in the postischemic rat brain. OBA-09 exhibited anti-oxidative effects that appeared to be executed by OBA-09 and by the salicylic acid afforded by hydrolysis. Here, we report the anti-inflammatory effects of OBA-09. Microglial activation observed at 2 days post-middle cerebral artery occlusion (MCAO, 90 min) and at 1 day after a LPS injection (0.5 mg/kg, intravenously) in the brains of Sprague-Dawley rats were markedly suppressed by the administration of OBA-09 (10 mg/kg). Inductions of proinflammatory markers (TNF-α, IL-1β, iNOS, and COX-2) were also suppressed by OBA-09 in both the LPS and MCAO models. Moreover, the anti-inflammatory effect of OBA-09 was accompanied by the suppression of infarct formation in the postischemic brain, but appeared to be independent of neuroprotection in LPS-treated rats. The inductions of proinflammatory markers were also inhibited by OBA-09 in LPS-treated BV2 cells (a microglia cell line) and in LPS-treated-primary neutrophils, possibly due to the suppression of NF-κB activity. Interestingly, the anti-inflammatory effect of OBA-09 was greater than that of equivalent co-treatment with pyruvate and salicylic acid. Together these results indicate that OBA-09 is a potent multi-modal neuroprotectant in the postischemic brain, and that its anti-inflammatory effect contributes to its neuroprotective function., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Noninvasive intranasal drug administration has been noted to allow direct delivery of drugs to the brain. In the present study, the therapeutic efficacy of intranasal small interfering RNA (siRNA) delivery was investigated in the postischemic rat brain. Fluorescein isothiocyanate (FITC)-labeled control siRNA was delivered intranasally in normal adult rats using e-PAM-R, a biodegradable PAMAM dendrimer, as gene carrier. Florescence-tagged siRNA was found in the cytoplasm and processes of neurons and of glial cells in many brain regions, including the hypothalamus, amygdala, cerebral cortex, and striatum, in 1 hour after infusion, and the FITC-fluorescence was continuously detected for at least 12 hours. When siRNA for high mobility group box 1 (HMGB1), which functions as an endogenous danger molecule and aggravates inflammation, was delivered intranasally, the target gene was significantly depleted in many brain regions, including the prefrontal cortex and striatum. More importantly, intranasal delivery of HMGB1 siRNA markedly suppressed infarct volume in the postischemic rat brain (maximal reduction to 42.8 ± 5.6% at 48 hours after 60 minutes middle cerebral artery occlusion (MCAO)) and this protective effect was manifested by recoveries from neurological and behavioral deficits. These results indicate that the intranasal delivery of HMGB1 siRNA offers an efficient means of gene knockdown-mediated therapy in the ischemic brain.

Cerebral ischemia leads to brain injury via a complex series of pathophysiological events. Therefore, multidrug treatments or multitargeting drug treatments are attractive options in efficiently limiting brain damage. Here, we report a novel multifunctional compound oxopropanoyloxy benzoic acid (OBA-09), a simple ester of pyruvate and salicylic acid. This protective effect was manifested by recoveries from neurological and behavioral deficits. OBA-09 exhibited antioxidative effects in the postischemic brain, which was evidenced by remarkable reduction of lipid peroxidation and 4-hydroxy-2-nonenal staining in OBA-09-administered animals. Reactive oxygen species generation was markedly suppressed in primary cortical cultures under oxygen-glucose deprivation. More interestingly, OBA-09 was capable of scavenging hydroxyl radical in cell-free assays. High-performance liquid chromatography results demonstrated that OBA-09 was hydrolyzed to salicylic acid and pyruvate with t(1/2) = 43 min in serum and 4.2 h in brain parenchyma, indicating that antioxidative function of OBA-09 is executed by itself and also by salicylic acid after the hydrolysis. In addition to antioxidative function, OBA-09 exerts anti-excitotoxic and anti-Zn(2+)-toxic functions, which might be attributed to attenuation of ATP and nicotinamide adenine dinucleotide depletion and to the suppression of nuclear factor-κB activity induction. Together these results indicate that OBA-09 has a potent therapeutic potential as a multimodal neuroprotectant in the postischemic brain and these effects were conferred by OBA-09 itself and subsequently its hydrolyzed products.

Although RNA interference (RNAi)-mediated gene silencing provides a powerful strategy for modulating specific gene functions, difficulties associated with siRNA delivery have impeded the development of efficient therapeutic applications. In particular, the efficacy of siRNA delivery into neurons has been limited by extremely low transfection efficiencies. e-PAM-R is a biodegradable arginine ester of PAMAM dendrimer, which is readily degradable under physiological conditions (pH 7.4, 37 degrees C). In the present study, we investigated the efficiency of siRNA delivery by e-PAM-R in primary cortical cultures and in rat brain. e-PAM-R/siRNA complexes showed high transfection efficiencies and low cytotoxicities in primary cortical cultures. Localization of fluorescence-tagged siRNA revealed that siRNA was delivered not only into the nucleus and cytoplasm, but also along the processes of the neuron. e-PAM-R/siRNA complex-mediated target gene reduction was observed in over 40% of cells and it was persistent for over 48 h. The potential use of e-PAM-R was demonstrated by gene knockdown after transfecting High mobility group box-1 (HMGB1, a novel cytokine-like molecule) siRNA into H(2)O(2)- or NMDA-treated primary cortical cultures. In these cells, HMGB1 siRNA delivery successfully reduced both basal and H(2)O(2)- or NMDA-induced HMGB1 levels, and as a result of that, neuronal cell death was significantly suppressed in both cases. Furthermore, we showed that e-PAM-R successfully delivered HMGB1 siRNA into the rat brain, wherein HMGB1 expression was depleted in over 40% of neurons and astrocytes of the normal brain. Moreover, e-PAM-R-mediated HMGB1 siRNA delivery notably reduced infarct volume in the postischemic rat brain, which is generated by occluding the middle cerebral artery for 60 min. These results indicate that e-PAM-R, a novel biodegradable nonviral gene carrier, offers an efficient means of transfecting siRNA into primary neuronal cells and in the brain and of performing siRNA-mediated gene knockdown., ((c) 2009 Elsevier B.V. All rights reserved.)

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to act as an anti-inflammatory molecule in various pathological conditions, which include sepsis or hemorrhagic shock. Recently, we showed that ethyl pyruvate has a neuroprotective effect in the postischemic brain and also in KA-induced pathogenesis in the brain. In this study, we examined whether aspirin augments neuroprotective effect of ethyl pyruvate in transient focal ischemia model by complementing the neuroprotective effects of ethyl pyruvate. Although, most of neuroprotective effect of aspirin has been attributed to the anti-platelet action, aspirin also has direct neuroprotective effects, including NF-kappaB inhibition. Ethyl pyruvate dose-dependently suppressed infarct formation in the postischemic brain, wherein intravenous administration of 5 mg/kg ethyl pyruvate 30 min after the occlusion reduced infarct volume to 34.5 +/- 15.5% (n = 6, P < 0.01) of that of the untreated control. In combination with aspirin (5 mg/kg, i.v.), the neuroprotective effect was enhanced, resulting in 16.0 +/- 5.9% (n = 6, P < 0.01) infarct volume. The time window for synergistic neuroprotection by ethyl pyruvate and aspirin extended to 9 h post-MCAO. The synergistic reduction in infarct volume was accompanied by suppression of the clinical manifestations associated with cerebral ischemia including motor impairment and neurological deficits. Inflammatory processes including microglial activation and proinflammatory cytokine expression were notably suppressed by the combination treatment in the postischemic brain and in primary microglia cultures, wherein ethyl pyruvate and aspirin modulate NF-kappaB signaling differentially. Aspirin interferes with IkappaB phosphorylation and degradation in the cytoplasm, possibly by specifically inhibiting IkappaB kinase-beta, whereas, the effect of ethyl pyruvate seems to occur in the nucleus, where it may interfere with the binding of NF-kappaB to responsive promoter elements in the target genes. Similar enhancement in neuroprotective effect was also observed in primary cortical cultures after NMDA or Zn(2+) treatment or oxygen-glucose deprivation. Together, these results indicate that combination treatment of ethyl pyruvate and aspirin affords synergistic neuroprotection in the postischemic brain with a wide therapeutic window, in part via differential modulation of the NF-kappaB signaling pathway.

Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) and one of the commonly prescribed antidepressants. Numerous clinical observations and animal studies indicate that fluoxetine enhances the anticonvulsant potencies of several antiepileptic drugs. In the previous report, we showed that fluoxetine strongly protects against delayed cerebral ischemic injury. In the present study, the authors investigated whether fluoxetine has a beneficial effect on KA-induced neuronal cell death. An intracerebroventricular (i.c.v.) injection of 0.94 nmol (0.2 microg) of KA produced typical neuronal cell death both in CA1 and CA3 regions of the hippocampus. Although, there was no significant difference in the time course or severity of epileptic behavior, the systemic administration of fluoxetine 30 min before KA administration significantly attenuated this neuronal cell death. Fluoxetine was found to suppress neuronal cell loss when injected at 10 mg/kg and the effect was enhanced at 50 mg/kg. Furthermore, this fluoxetine-induced neuroprotection was accompanied by marked improvements in memory impairment, as determined by passive avoidance tests. KA-induced gliosis and proinflammatory marker (COX-2, IL-1beta, and TNF-alpha) inductions were also suppressed by fluoxetine administration. It is interesting to note here that fluoxetine treatment suppressed NF-kappaB activity dose-dependently in KA-treated mouse brains, suggesting that this explains in part its anti-inflammatory effect. Together, these results suggest that fluoxetine has therapeutic potential in terms of suppressing KA-induced pathogenesis in the brain, and that these neuroprotective effects are associated with its anti-inflammatory effects.

Fluoxetine is a selective serotonin reuptake inhibitor that is widely used in the treatment of major depression including after stroke. In this study, we tested whether fluoxetine protects neuronal death in a rat cerebral ischemia model of middle cerebral artery occlusion (MCAO). The administration of fluoxetine intravenously (10 mg/kg) at 30 min, 3 hr, or 6 hr after MCAO reduced infarct volumes to 21.2+/-6.7%, 14.5+/-3.0%, and 22.8+/-2.9%, respectively, of that of the untreated control. Moreover, the neuroprotective effect of fluoxetine was evident when it was administered as late as 9 hr after MCAO/reperfusion. These neuroprotective effects were accompanied by improvement of motor impairment and neurological deficits. The fluoxetine-treated brain was found to show marked repressions of microglia activation, neutrophil infiltration, and proinflammatory marker expressions. Moreover, fluoxetine suppressed NF-kappaB activity dose-dependently in the postischemic brain and also in lipopolysaccharide-treated primary microglia and neutrophil cultures, suggesting that NF-kappaB activity inhibition explains in part its anti-inflammatory effect. These results demonstrate that curative treatment of fluoxetine affords strong protection against delayed cerebral ischemic injury, and that these neuroprotective effects might be associated with its anti-inflammatory effects.

Recent studies have shown that ethyl pyruvate (EP) acts as an anti-inflammatory molecule in several cell lines including RAW264.7 macrophages. However, the potential therapeutic value of EP for the treatment of the pathologic brain has not been investigated fully. In the present study, we examined whether EP has a beneficial effect on KA-induced neuronal cell death. Intracerebroventricular (i.c.v.) injection of 0.94 nmol (0.2 mug) of KA produced typical neuronal cell death in the CA1 and CA3 pyramidal layers of the hippocampus, and the systemic administration of EP significantly attenuated KA-induced neuronal cell death in these regions. Ethyl pyruvate was found to exert a protective effect when it was injected as late as 12 hr after KA-injection. Moreover, this EP-induced neuroprotection was accompanied by reduced levels of reactive gliosis and COX-2, IL-1beta, and TNF-alpha in the hippocampus. In addition, in passive avoidance tests, KA-induced memory impairment was improved markedly by EP. These results suggest that EP has a therapeutic potential for suppressing KA-induced pathogenesis in the brain.

Ethyl pyruvate (EP) is a pyruvate derivative, and has recently been reported to prevent lethality in mice with established lethal sepsis and systemic inflammation. In a previous study, we reported that EP has a neuroprotective effect in a rat cerebral ischemia model of middle cerebral artery occlusion (MCAO), in which it was found to be effective when injected as late as 12 h after MCAO/reperfusion. In the present study, we show that therapeutic window of pyruvate in this MCAO animal model is limited to 1 h (30 min before and 30 min after MCAO). Moreover, both pyruvate and EP have a neuroprotective effect during oxygen-glucose deprivation (OGD) or H2O2 challenge in primary cortical culture. In contrast, EP suppressed the LPS-induced activation of primary microglia in culture, but pyruvate did not. The suppression of microglia activation was evidenced by a reduction in nitric oxide release and by a proinflammatory factor induction in primary microglia culture, which were accompanied by the repression of nuclear factor-kappaB activation. These results suggest that EP has a strong protective effect and a wide therapeutic window, and that this protective effect of EP is related to its anti-inflammatory action.

Gastrodin (GAS) is a predominant bioactive constituent of the Chinese herbal medicine Tianma (Gastrodia elata Blume). Many authors have reported GAS has the beneficial effect on diverse diseases of the CNS, including epilepsy, Alzheimer's disease, Parkinson's disease, and cerebral ischemia. Here, we report GAS exhibited a robust neuroprotective effect in an Sprague-Dawley rat model of stroke (transient middle cerebral artery occlusion, tMCAO), and show that the underlying molecular mechanism involves its protective effect against Zn2+-toxicity and its anti-oxidative effects in astrocytes. Intraperitoneal administration of GAS (40 mg/kg) after MCAO reduced mean infarct volume to 30.1 ± 5.9% of that of MCAO controls and this neuroprotective effect was accompanied by neurological function recoveries which was measured using modified neurological severity score (mNSS). Interestingly, GAS induced up-regulation and nuclear translocation of Nrf2, and subsequently increased the expressions of anti-oxidative genes, such as, HO-1 and GCLM, in astrocytes. Furthermore, GAS co- or pre-treatment markedly suppressed Zn2+-induced cell death caused by excessive ROS production and PARP-1 induction. We found that GAS suppressed p67 expression and PAR formation in astrocytes, which might underlie the anti- Zn2+-toxicity and anti-oxidative effects of GAS in astrocytes. These findings indicate GAS protects astrocytes from Zn2+-induced toxicity and oxidative stress and these effects contribute to its neuroprotective effects in the postischemic brain. [ABSTRACT FROM AUTHOR]

2-Hydroxy-4-trifluoromethylbenzoic acid (HTB) is a metabolite of triflusal (TF), and has been reported to exert anti-inflammatory effect. In this study, the authors investigated whether HTB has a neuroprotective effect against ischemic brain injuries. We showed that intravenous administration of HTB (5 mg/kg) 30 min before or 1, 3, or 6 h after middle cerebral artery occlusion (MCAO) reduced brain infarct to 10.4 ± 3.3%, 16.9 ± 2.3%, 22.2 ± 1.5% and 40.7 ± 7.5%, respectively, of that of treatment-naive MCAO controls, and the therapeutic time window extended to 9 h after MCAO (40.7 ± 7.5%). Furthermore, HTB suppressed infarct formation, protected motor activities, and ameliorated neurological deficits more effectively than by TF or salicylic acid (SA). HTB markedly suppressed microglial activation and proinflammatory cytokines expressions in the postischemic brain and in BV2 cells and suppressed LPS-induced nitrite production by inhibiting IkB degradation. In addition, HTB suppressed NMDA-induced neuronal cell death more effectively than TF or SA in primary cortical neuron cultures. Together, these results indicate that HTB has multi-modal protective effects against ischemic brain damage that encompass anti-inflammatory, anti-excitotoxicity, and anti-Zn 2+ -toxicity effects. [ABSTRACT FROM AUTHOR]

Abstract: High mobility group box 1 (HMGB1) is an endogenous danger signal molecule. In a previous report, we showed that HMGB1 is massively released during NMDA-induced acute damaging process in the postischemic brain and triggers inflammatory processes, like microglial activation. siRNA-mediated HMGB1 knockdown markedly reduced infarct volumes, confirming the crucial role played by HMGB1 in the postischemic brain. In the present study, we showed neuroprotective effects of glycyrrhizin (GL) in the postischemic rat brain after middle cerebral artery occlusion (MCAO). GL, a triterpene present in the roots and rhizomes of licorice, Glycyrrhiza glabra, has been shown to have anti-inflammatory and anti-viral effects. It has been reported that GL binds directly to HMGB1, and inhibits its chemoattractant and mitogenic activities. The administration of GL (10mg/kg) intravenously at 3 or 6h after MCAO reduced infarct volumes to 12.9±4.2% and 46.2±9.9%, respectively, of untreated control. This neuroprotective effect was accompanied by improvements in motor impairment and neurological deficits and suppressions of microglia activation and proinflammatory cytokine induction. Interestingly, GL almost completely blocked HMGB1 secretion in the postischemic brain and in lipopolysaccharide (LPS)-treated microglia cells. Furthermore, HMGB1 phosphorylation, which is the initial step for HMGB1 secretion, and the interaction between HMGB1 and protein kinase C (PKC) or calcium/calmodulin-dependent protein kinase IV (CaMKIV) were suppressed dose-dependently by GL. Here, we hypothesized that the blockage for the putative phosphorylation sites in HMGB1 by GL might be attributed to this suppression. In addition to the anti-inflammatory effects, we found that GL has anti-excitotoxic and anti-oxidative effects in neurons. Together these results indicate that GL has neuroprotective efficacy in the postischemic brain via its anti-inflammatory, anti-excitotoxic, and anti-oxidative effects and in particular, it exerts anti-inflammatory effect, at least in part, by inhibiting HMGB1 secretion. [Copyright &y& Elsevier]

Abstract: High mobility group box 1 (HMGB1) is a family of endogenous molecules that is released by necrotic cells and causes neuronal damages by triggering inflammatory processes. In the cerebral ischemic brain, sustained and regulated suppression of HMGB1 has been emerged as a therapeutic means to grant neuroprotection. HMGB1 consists of two HMG boxes (A and B) and an acidic C-terminal tail, and the A box peptide antagonistically competes with HMGB1 for its receptors. In the middle cerebral artery occlusion (MCAO) in rats, a murine model of transient cerebral ischemia, administration of HMGB1 A box intraparenchymally, after encapsulated in biodegradable gelatin microspheres (GMS), which enhances the stability of peptide inside and allows its sustained delivery, at 1 h, 3 h, or 6 h after MCAO, reduced mean infarct volumes by, respectively, 81.3%, 42.6% and 30.7% of the untreated MCAO-brain, along with remarkable improvement of neurological deficits. Furthermore, the administration of HMGB1 A box/GMS suppressed proinflammatory cytokine inductions more strongly than the injection of non-encapsulated HMGB1 A box. Given that insulted brains-like ischemia have enhanced gelatinase activity than the normal brain, our results suggest that GMS-mediated delivery of therapeutic peptides is a promising means to provide efficient neuroprotection in the postischemic brain. [ABSTRACT FROM AUTHOR]