Background and Purpose-Cyclooxygenase-2 (COX-2) is implicated in ischemic neuronal death. In focal ischemia, its mRNA induction is mediated through N-methyl-D-aspartic acid (NMDA) receptors and phospholipase A 2 . Because mechanisms of neuronal death involving COX-2 in global ischemia are unclear, we studied the time course and regulation of COX-2 expression in rat brain global ischemia. Methods-Global ischemia was induced by a 4-vessel occlusion method. COX-2 mRNA levels were demonstrated with in situ hybridization and COX-2 protein with immunocytochemistry. Several animals were pretreated with MK-801, an NMDA receptor antagonist; 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX), an ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist; and dexamethasone. Results-In the cortex, the CA3 hippocampal region and dentate gyrus expression of COX-2 mRNA peaked at 4 to 8 hours, while in the CA1 region COX-2 mRNA levels were high at 4 to 24 hours. COX-2 protein was induced in the corresponding regions at 12 to 24 hours, but in the CA1 neurons the protein was still seen at 3 days. COX-2 mRNA induction in the cortex was inhibited by NBQX and dexamethasone and in CA1 neurons was inhibited by NBQX. MK-801 did not suppress COX-2 induction. Conclusions-COX-2 is differentially induced in the cortex and hippocampal structures after global ischemia. The prolonged COX-2 expression in the vulnerable CA1 neurons is regulated by AMPA receptors, suggesting that COX-2 expression is likely to be associated with AMPA receptor-mediated neuronal death in global ischemia. Glucocorticoids may not be efficiently used to inhibit ischemia-induced COX-2 expression in the hippocampus. Key Words: free radicals Ⅲ gene expression Ⅲ hippocampus Ⅲ prostaglandins Ⅲ rats C yclooxygenase-2 (COX-2) is the inducible form of the 2 prostaglandin-synthesizing enzymes and is exclusively present in excitatory neurons, such as glutamatergic cells in the normal brain. [1][2][3][4] Previous studies indicate that prostaglandins have several functions that are potentially protective in brain ischemia. For example, prostanoids produce dilatation of cerebral arteries, 5-7 which could increase cerebral blood flow in ischemic tissue. In cultured microglia, prostaglandin E 2 has been reported to inhibit expression of inducible nitric oxide synthase (iNOS) 8 and production of interleukin-1␤, 9 2 key mediators of inflammation. In addition, prostaglandins protect cultured cortical neurons against glutamate toxicity. 10 However, COX-2 is believed to play a negative role in brain injury, including ischemia, because inflammatory cytokines cause a rapid induction of COX-2, 11,12 COX enzymes generate superoxide and cause inflammation, 1,2 and, most importantly, COX inhibitors reduce experimental brain edema 13,14 and reduce brain damage after global [15][16][17] 18,19 Recently, COX-2 gene expression has been shown to be induced in spreading depression and focal brain ischemia by activation of N-methyl-D-aspartic acid (NMDA) receptors and ph...

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Glioblastoma is a terminal disease with no effective treatment currently available. Among the new therapy candidates are oncolytic viruses capable of selectively replicating in cancer cells, causing tumor lysis and inducing adaptive immune responses against the tumor. However, tumor antiviral responses, primarily mediated by type I interferon (IFN-I), remain a key problem that severely restricts viral replication and oncolysis. We show here that the Semliki Forest virus (SFV) strain SFV4, which causes lethal encephalitis in mice, is able to infect and replicate independent of the IFN-I defense in mouse glioblastoma cells and cell lines originating from primary human glioblastoma patient samples. The ability to tolerate IFN-I was retained in SFV4-miRT124 cells, a derivative cell line of strain SFV4 with a restricted capacity to replicate in neurons due to insertion of target sites for neuronal microRNA 124. The IFN-I tolerance was associated with the viral nsp3-nsp4 gene region and distinct from the genetic loci responsible for SFV neurovirulence. In contrast to the naturally attenuated strain SFV A7(74) and its derivatives, SFV4-miRT124 displayed increased oncolytic potency in CT-2A murine astrocytoma cells and in the human glioblastoma cell lines pretreated with IFN-I. Following a single intraperitoneal injection of SFV4-miRT124 into C57BL/6 mice bearing CT-2A orthotopic gliomas, the virus homed to the brain and was amplified in the tumor, resulting in significant tumor growth inhibition and improved survival. IMPORTANCEAlthough progress has been made in development of replicative oncolytic viruses, information regarding their overall therapeutic potency in a clinical setting is still lacking. This could be at least partially dependent on the IFN-I sensitivity of the viruses used. Here, we show that the conditionally replicating SFV4-miRT124 virus shares the IFN-I tolerance of the pathogenic wildtype SFV, thereby allowing efficient targeting of a glioma that is refractory to naturally attenuated therapy vector strains sensitive to IFN-I. This is the first evidence of orthotopic syngeneic mouse glioma eradication following peripheral alphavirus administration. Our findings indicate a clear benefit in harnessing the wild-type virus replicative potency in development of nextgeneration oncolytic alphaviruses. G lioblastoma (GBM) is the most common primary brain tumor and a devastating disease with a median survival of only 15 months despite best available therapy (1). Oncolytic virotherapy provides a novel option to treat malignant central nervous system (CNS) tumors, as many of the potential oncolytic viruses are tumor homing, self-amplifying, and may elicit antitumor Tcell responses (2). Oncolytic viruses harnessed recently in virotherapy of human glioblastoma include herpes simplex virus (3), reovirus (Reolysin) (4), Newcastle disease virus (NDV-HUJ) (5), and poliovirus (PVS-RIPO) (6). Apart from anecdotal reports of successful cases and despite a relatively good tolerability of the vectors by the patien...

Excitotoxicity through stimulation of N-methyl-D-aspartate (NMDA) receptors contributes to neuronal death in brain injuries, including stroke. Several lines of evidence suggest a role for protein kinase C (PKC) isoforms in NMDA excitotoxicity. We have used specific peptide inhibitors of classical PKCs (a, b, and c), novel PKCs d and e, and an atypical PKCf in order to delineate which subspecies are involved in NMDAinduced cell death. Neuronal cell cultures were prepared from 15-day-old mouse embryos and plated onto the astrocytic monolayer. After 2 weeks in vitro the neurons were exposed to 100 lM NMDA for 5 min, and 24 h later the cell viability was examined by measuring the lactate dehydrogenase release and bis-benzimide staining. While inhibitors directed to classical (a, b, and c) or novel PKCs (d or e) had no effect, the PKCf inhibitor completely prevented the NMDA-induced necrotic neuronal death. Confocal microscopy confirmed that NMDA induced PKCf translocation, which was blocked by the PKCf inhibitor. The NMDA-induced changes in intracellular free Ca 2+ were not affected by the peptides. In situ hybridization experiments demonstrated that PKCf mRNA is induced in the cortex after focal brain ischemia. Altogether, the results indicate that PKCf activation is a downstream signal in NMDA-induced death of cortical neurons.

The delayed death of CA1 neurons after global brain ischemia is associated with induction of apoptosis genes and is inhibited by protein synthesis inhibitors, suggesting that the degeneration of CA1 pyramidal neurons is an active process that requires new gene expression. The transient global ischemia model has been extensively used to identify enzymes and other proteins underlying delayed neuronal cell death. The expression of protein kinase C (PKC) subspecies after 20 minutes of global brain ischemia produced by a four-vessel occlusion model in the rat was studied. From the multiple PKC subspecies studied, only PKCδ mRNA was significantly up-regulated in CA1 pyramidal neurons at 24 hours and in activated microglia at 3 to at least 7 days after ischemia. The induction of PKCδ mRNA was also found in the cortex at 8 hours and 3 days after ischemia. This cortical but not hippocampal induction was regulated by an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/kainate receptor antagonist, 6-nitro-7-sulfamobenzo[ f]quinoxaline-2,3-dione, and glucocorticoids. An N-methyl-d-aspartate receptor antagonist, MK-801, was without effect on the induction of PKCδ subspecies. The selective and prolonged induction of the PKCδ mRNA and protein first in CA1 pyramidal neurons and at a later stage in activated microglia suggests that the PKCδ isozyme may take part in regulation of the delayed death of CA1 neurons after transient global brain ischemia.