Attenuation of Glutamate-Induced Excitotoxicity by Withanolide-A in Neuron-Like Cells: Role for PI3K/Akt/MAPK Signaling Pathway.

Glutamate-induced excitotoxicity is one of the major underlying mechanisms for neurodegenerative diseases. Efforts are being made to treat such conditions with an array of natural compounds that can modulate the release of glutamate or the underlying mechanisms associated with it. Withania somnifera extract has potent pharmacologic activity similar to that of Korean Ginseng tea and is used to treat several neuronal disorders. However, to date, little efforts have been made to evaluate individual constituents of this plant for neurodegenerative disorders. Present study was carried out to investigate withanolide-A, one of the active constituents of Withania somnifera against glutamate-induced excitotoxicity in retinoic acid differentiated Neuro2a neuroblastoma cells. The results indicated that glutamate treatment for 2 h induced death in cells that was significantly attenuated by pre-treatment with MK-801 (specific NMDA receptor antagonist) and different concentrations of withanolide-A. Withanolide-A abated the glutamate-induced influx of intracellular calcium and excessive ROS production significantly. Further on, glutamate treatment resulted in increased levels of pro-apoptotic and decreased levels of anti-apoptotic proteins, and these protein levels were normalized by various doses of withanolide-A. All of these protective effects were partly due to inhibition of MAPK family proteins and activation of PI3K/Akt signaling. Thus, our results suggest that withanolide-A may serve as potential neuroprotective agent.

Effectiveness of Dioscorea hispida Dennst as antibacterial and antibiofilm agent

Aims@#Antimicrobial resistance (AMR) is a growing threat to public health, where treatments using conventional drugs are becoming ineffective. One viable but underexplored alternative is through the use of Dioscorea hispida, a wild plant that exhibits antimicrobial properties. This study aims to explore D. hispida effectiveness as an antibacterial and antibiofilm agent against selected pathogenic and non-pathogenic bacteria. @*Methodology and results@#Different concentrations of D. hispida crude extracts (0 – 2.5 mg/mL) were tested against the growth of planktonic bacterial cells over 24 h incubation, and the half maximal effective concentration (EC50) obtained was used in the antibiofilm test over 24 and 48 h. All bacteria treated with D. hispida showed significant (P<0.05) reduction in planktonic cell and biofilm densities against the negative control starting at 0.3 mg/mL. However, in comparison to the antibiotic, only certain bacteria were significantly affected by D. hispida, implying the plant has a ‘moderate’ biocidal activity in general. Furthermore, Atomic Force Microscopy imaging of S. aureus biofilm with D. hispida revealed increased height and width of cell clusters despite reduction in volume compared to the negative control, suggesting unique biofilm resistance behaviour against the plant. @*Conclusion, significance and impact of study@#This study demonstrated D. hispida capability as a natural antimicrobial and antibiofilm agent. The plant could complement current antimicrobials to maximise killing efficiency and minimise occurrences of resistance. Unique biofilm behaviour against D. hispida also warrants further investigation on the effect of biocides towards biofilm structure. Overall, this research provides new insights into a traditional plant-based antimicrobial activity in combating infectious diseases and AMR.

Withanone, an Active Constituent from Withania somnifera, Affords Protection Against NMDA-Induced Excitotoxicity in Neuron-Like Cells.

Withania somnifera has immense pharmacologic and clinical uses. Owing to its similar pharmacologic activity as that of Korean Ginseng tea, it is popularly called as Indian ginseng. In most cases, extracts of this plant have been evaluated against various diseases or models of disease. However, little efforts have been made to evaluate individual constituents of this plant for neurodegenerative disorders. Present study was carried out to evaluate Withanone, one of the active constituents of Withania somnifera against NMDA-induced excitotoxicity in retinoic acid, differentiated Neuro2a cells. Cells were pre-treated with 5, 10 and 20 µM doses of Withanone and then exposed to 3-mM NMDA for 1 h. MK801, a specific NMDA receptor antagonist, was used as positive control. The results indicated that NMDA induces significant death of cells by accumulation of intracellular Ca2+, generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential, crashing of Bax/Bcl-2 ratio, release of cytochrome c, increased caspase expression, induction of lipid peroxidation as measured by malondialdehyde levels and cleavage of poly(ADP-ribose) polymerase-1 (Parp-1), which is indicative of DNA damage. All these parameters were attenuated with various doses of Withanone pre-treatment. These results suggest that Withanone may serve as potential neuroprotective agent.

Endophytes and Neurodegenerative Diseases: A Hope in Desperation.

Neurodegeneration is progressive loss of functional and structural integrity of the central nervous system. Neurodegenerative disorders are yet without any reliable therapy because the neurons of the central nervous system have limited ability to regenerate. Current therapeutic approaches rely mainly on abrogation of symptoms and leave the dying neurons to their fate. Protective and/or rescuing treatments need to be explored fully to suppress neuronal death that will automatically alleviate the symptoms. Adequate precedent exists in literature regarding the neuroprotective activity of endophytes. Endophytes are a class of microorganisms which colonize healthy plant tissues without causing any apparent harm to the host. Chemical moieties from known endophytes have been used against many disease models including neurodegenerative diseases. There is great hope that novel bioactive molecules from newer endophytes can impede pathogenic mechanisms and progression of many diseases. In this review, we will discuss promising pharmacological or clinical relevance of endophytes against various neurodegenerative diseases.

Dynamics of GFP-Fusion p110α and p110ß Isoforms of PI3K Signaling Pathway in Normal and Cancer Cells.

Cancer therapeutics is a hot subject and PI3K class 1A isoforms (p110α and p110ß) are pursued as major targets. Genetic analysis, biochemical approaches, and structural studies have demonstrated crucial roles for these isoforms in several physiological processes. p110α is critical for insulin signaling, whereas p110ß is essential for the growth and differs from p110α in many ways. Here, we have generated GFP-fusion clones of wildtype and mutant version of p110α and p110ß and expressed them in HEK293 and cancer cells to examine their subcellular localization and their impact on downstream signaling. In HEK293 cells, p110ß GFP-fusion protein is translocated into the nucleus, whereas p110α-GFP stays exclusively in the cytoplasm. This study demonstrates that p110α and p110ß oncogenecity, kinase activity, and interaction with p85 regulatory subunit does not have any impact on their subcellular localization. PI3K pathway specific inhibitor, LY294002, abrogated PI3K signaling by reducing pAkt levels, however, the subcellular localization of p110α and p110ß remained unchanged. Furthermore, we analyzed the expression of recombinant p110α and p110ß in a panel of human cancer cells and observed remarkable differences in their expression levels. The differential expression of recombinant p110α and p110ß was observed to be mainly regulated by the endogenous levels of pAkt. Unlike in HEK293, p110α showed nuclear localization in cancer cells in a similar fashion to p110ß. Moreover, we observed the PI3K signaling activities in low pAkt expressing cells are mediated by PDK1 and S6K proteins. Finally, p110α and p110ß were seen to play an essential role in promoting the cell cycle progression in MCF-7 and HCT-116 cells. J. Cell. Biochem. 117: 2864-2874, 2016. © 2016 Wiley Periodicals, Inc.

Promise of Retinoic Acid-Triazolyl Derivatives in Promoting Differentiation of Neuroblastoma Cells.

Retinoic acid induces differentiation in various types of cells including skeletal myoblasts and neuroblasts and maintains differentiation of epithelial cells. The present study demonstrates synthesis and screening of a library of retinoic acid-triazolyl derivatives for their differentiation potential on neuroblastoma cells. Click chemistry approach using copper(I)-catalyzed azide-alkyne cycloaddition was adopted for the preparation of these derivatives. The neurite outgrowth promoting potential of retinoic acid-triazolyl derivatives was studied on neuroblastoma cells. Morphological examination revealed that compounds 8a, 8e, 8f, and 8k, among the various derivatives screened, exhibited promising neurite-outgrowth inducing activity at a concentration of 10 µM compared to undifferentiated and retinoic acid treated cells. Further on, to confirm this differentiation potential of these compounds, neuroblastoma cells were probed for expression of neuronal markers such as NF-H and NeuN. The results revealed a marked increase in the NF-H and NeuN protein expression when treated with 8a, 8e, 8f, and 8k compared to undifferentiated and retinoic acid treated cells. Thus, these compounds could act as potential leads in inducing neuronal differentiation for future studies.

Pharmacologic overview of Withania somnifera, the Indian Ginseng.

Withania somnifera, also called 'Indian ginseng', is an important medicinal plant of the Indian subcontinent. It is widely used, singly or in combination, with other herbs against many ailments in Indian Systems of Medicine since time immemorial. Withania somnifera contains a spectrum of diverse phytochemicals enabling it to have a broad range of biological implications. In preclinical studies, it has shown anti-microbial, anti-inflammatory, anti-tumor, anti-stress, neuroprotective, cardioprotective, and anti-diabetic properties. Additionally, it has demonstrated the ability to reduce reactive oxygen species, modulate mitochondrial function, regulate apoptosis, and reduce inflammation and enhance endothelial function. In view of these pharmacologic properties, W. somnifera is a potential drug candidate to treat various clinical conditions, particularly related to the nervous system. In this review, we summarize the pharmacologic characteristics and discuss the mechanisms of action and potential therapeutic applications of the plant and its active constituents.

Bacopa monniera ameliorates cognitive impairment and neurodegeneration induced by intracerebroventricular-streptozotocin in rat: behavioral, biochemical, immunohistochemical and histopathological evidences.

The standardized extract of Bacopa monniera (BM) is a complex mixture of ingredients with a uniquely wide spectrum of neuropharmacological influences upon the central nervous system including enhanced learning and memory with known antioxidant potential and protection of the brain from oxidative damage. The present study demonstrates the therapeutic efficacy of BM on cognitive impairment and oxidative damage, induced by intracerebroventricular injection of streptozotocin (ICV-STZ) in rat models. Male Wistar rats were pre-treated with BM at a selected dose (30 mg/Kg) given orally for 2 weeks and then were injected bilaterally with ICV-STZ (3 mg/Kg), while sham operated rats were received the same volume of vehicle. Behavioral parameters were subsequently monitored 2 weeks after the surgery using the Morris water maze (MWM) navigation task then were sacrificed for biochemical, immunohistochemical (Cu/Zn-SOD) and histopathological assays. ICV-STZ-infused rats showed significant loss in learning and memory ability, which were significantly improved by BM supplementation. A significant increase in thiobarbituric acid reactive species and a significant decrease in reduced glutathione, antioxidant enzymes in the hippocampus were observed in ICV-STZ rats. Moreover, decrease in Cu/Zn-SOD expression positive cells were observed in the hippocampus of ICV-STZ rats. BM supplementation significantly ameliorated all alterations induced by ICV-STZ in rats. The data suggest that ICV-STZ might cause its neurotoxic effects via the production of free radicals. Our study demonstrates that BM is a powerful antioxidant which prevents cognitive impairment, oxidative damage, and morphological changes in the ICV-STZ-infused rats. Thus, BM may have therapeutic value for the treatment of cognitive impairment.

Inflammation in ischemic stroke: mechanisms, consequences and possible drug targets.

Ischemic stroke is caused when blood flow to the brain is hampered, leading to instant deficiency of nutrients and oxygen required for normal brain functioning. Reperfusion can alleviate damage from stroke if performed immediately after the onset of ischemia however the efficacy of reperfusion is tempered by secondary injury mechanisms. This multifarious sequence of events leads to the commencement of deleterious cycles of inflammation, oxidant stress and apoptosis that finally culminate in delayed death of neuronal cells even when the brain is effectively reperfused. Wealth of data from clinical as well as experimental studies points to a prominent role of inflammation in secondary injury. In this review we will discuss, in detail, the cellular and molecular mediators of inflammation and their possible therapeutic targets in both experimental and clinical forms of stroke.

Soluble epoxide hydrolase inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid is neuroprotective in rat model of ischemic stroke.

Soluble epoxide hydrolase (sEH) diminishes vasodilatory and neuroprotective effects of epoxyeicosatrienoic acids by hydrolyzing them to inactive dihydroxy metabolites. The primary goals of this study were to investigate the effects of acute sEH inhibition by trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) on infarct volume, functional outcome, and changes in cerebral blood flow (CBF) in a rat model of ischemic stroke. Focal cerebral ischemia was induced in rats for 90 min followed by reperfusion. At the end of 24 h after reperfusion rats were euthanized for infarct volume assessment by triphenyltetrazolium chloride staining. Brain cortical sEH activity was assessed by ultra performance liquid chromatography-tandem mass spectrometry. Functional outcome at 24 and 48 h after reperfusion was evaluated by arm flexion and sticky-tape tests. Changes in CBF were assessed by arterial spin-labeled-MRI at baseline, during ischemia, and at 180 min after reperfusion. Neuroprotective effects of t-AUCB were evaluated in primary rat neuronal cultures by Cytotox-Flour kit and propidium iodide staining. t-AUCB significantly reduced cortical infarct volume by 35% (14.5 ± 2.7% vs. 41.5 ± 4.5%), elevated cumulative epoxyeicosatrienoic acids-to-dihydroxyeicosatrienoic acids ratio in brain cortex by twofold (4.40 ± 1.89 vs. 1.97 ± 0.85), and improved functional outcome in arm-flexion test (day 1: 3.28 ± 0.5 s vs. 7.50 ± 0.9 s; day 2: 1.71 ± 0.4 s vs. 5.28 ± 0.5 s) when compared with that of the vehicle-treated group. t-AUCB significantly reduced neuronal cell death in a dose-dependent manner (vehicle: 70.9 ± 7.1% vs. t-AUCB0.1µM: 58 ± 5.11% vs. t-AUCB0.5µM: 39.9 ± 5.8%). These findings suggest that t-AUCB may exert its neuroprotective effects by affecting multiple components of neurovascular unit including neurons, astrocytes, and microvascular flow.

Prostaglandin D2 toxicity in primary neurons is mediated through its bioactive cyclopentenone metabolites.

Prostaglandin D2 (PGD2) is the most abundant prostaglandin in brain but its effect on neuronal cell death is complex and not completely understood. PGD2 may modulate neuronal cell death via activation of DP receptors or its metabolism to the cyclopentenone prostaglandins (CyPGs) PGJ2, Δ(12)-PGJ2 and 15-deoxy-Δ(12,14)-PGJ2, inducing cell death independently of prostaglandin receptors. This study aims to elucidate the effect of PGD2 on neuronal cell death and its underlying mechanisms. PGD2 dose-dependently induced cell death in rat primary neuron-enriched cultures in concentrations of ≥10µM, and this effect was not reversed by treatment with either DP1 or DP2 receptor antagonists. Antioxidants N-acetylcysteine (NAC) and glutathione which contain sulfhydryl groups that can bind to CyPGs, but not ascorbate or tocopherol, attenuated PGD2-induced cell death. Conversion of PGD2 to CyPGs was detected in neuronal culture medium; treatment with these CyPG metabolites alone exhibited effects similar to those of PGD2, including apoptotic neuronal cell death and accumulation of ubiquitinated proteins. Disruption of lipocalin-type prostaglandin D synthase (L-PGDS) protected neurons against hypoxia. These results support the hypothesis that PGD2 elicits its cytotoxic effects through its bioactive CyPG metabolites rather than DP receptor activation in primary neuronal culture.

Increased generation of cyclopentenone prostaglandins after brain ischemia and their role in aggregation of ubiquitinated proteins in neurons.

The cyclopentenone prostaglandin (CyPG) J2 series, including prostaglandin J2 (PGJ2), Δ¹²-PGJ2, and 15-deoxy-∆¹²,¹4-prostaglandin J2 (15d-PGJ2), are active metabolites of PGD2, exerting multiple effects on neuronal function. However, the physiologic relevance of these effects remains uncertain as brain concentrations of CyPGs have not been precisely determined. In this study, we found that free PGD2 and the J2 series CyPGs (PGJ2, Δ¹²-PGJ2, and 15d-PGJ2) were increased in post-ischemic rat brain as detected by UPLC-MS/MS with 15d-PGJ2 being the most abundant CyPG. These increases were attenuated by pre-treating with the cyclooxygenase (COX) inhibitor piroxicam. Next, effects of chronic exposure to 15d-PGJ2 were examined by treating primary neurons with 15d-PGJ2, CAY10410 (a 15d-PGJ2 analog lacking the cyclopentenone ring structure), or vehicle for 24 to 96 h. Because we found that the concentration of free 15d-PGJ2 decreased rapidly in cell culture medium, freshly prepared medium containing 15d-PGJ2, CAY10410, or vehicle was changed twice daily to maintain steady extracellular concentrations. Incubation with 2.5 µM 15d-PGJ2, but not CAY10410, increased the neuronal cell death without the induction of caspase-3 or PARP cleavage, consistent with a primarily necrotic mechanism for 15d-PGJ2-induced cell death which was further supported by TUNEL assay results. Ubiquitinated protein accumulation and aggregation was observed after 96 h 15d-PGJ2 incubation, accompanied by compromised 20S proteasome activity. Unlike another proteasome inhibitor, MG132, 15d-PGJ2 treatment did not activate autophagy or induce aggresome formation. Therefore, the cumulative cytotoxic effects of increased generation of CyPGs after stroke may contribute to delayed post-ischemic neuronal injury.

Modification of ubiquitin-C-terminal hydrolase-L1 by cyclopentenone prostaglandins exacerbates hypoxic injury.

Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ(12,14) -prostaglandin J(2) (15d-PGJ(2)), are active prostaglandin metabolites exerting a variety of biological effects that may be important in the pathogenesis of neurological diseases. Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain specific deubiquitinating enzyme whose aberrant function has been linked to neurodegenerative disorders. We report that [15d-PGJ(2)] detected by quadrapole mass spectrometry (MS) increases in rat brain after temporary focal ischemia, and that treatment with 15d-PGJ(2) induces accumulation of ubiquitinated proteins and exacerbates cell death in normoxic and hypoxic primary neurons. 15d-PGJ(2) covalently modifies UCH-L1 and inhibits its hydrolase activity. Pharmacologic inhibition of UCH-L1 exacerbates hypoxic neuronal death while transduction with a TAT-UCH-L1 fusion protein protects neurons from hypoxia. These studies indicate that UCH-L1 function is important in hypoxic neuronal death and that excessive production of CyPGs after stroke may exacerbate ischemic injury by modification and inhibition of UCH-L1.

Neuroprotection Offered by Majun Khadar, a Traditional Unani Medicine, during Cerebral Ischemic Damage in Rats.

Stroke results in damages to many biochemical, molecular and behavioral deficits. Present study provides evidence of the protective efficacy of a Unani herbal medicine, Majun Khadar (MK), against cerebral ischemia-induced behavioral dysfunctions and neurochemical alterations in the hippocampus (HIP). Transient focal cerebral ischemia was induced for 2 h followed by reperfusion for 22 h in a rat model. Rats were divided into four groups: sham, middle cerebral artery occluded (MCAO), drug sham (MK; 0.816 g kg(-1) orally for 15 days) and MK pre-treated ischemic group (MK + MCAO). Levels of enzymatic and non-enzymatic antioxidants were estimated in HIP along with behavioral testing. MK pre-treatment significantly (P < .05-.001) restored the activities of glutathione peroxidase (GP×), glutathione reductase (GR), glutathione S-transferase (GST) and decreased the level of lipid peroxidation (LPO) and H2O2 content in HIP in the MK + MCAO group which were severely altered in the MCAO group. The content of glutathione (GSH), total thiols (TT) and ascorbic acid (AsA) was significantly depleted in the MCAO group; pretreatment with MK was able to restore its levels. Also in the MK + MCAO group, significant (P < .5-.001) recovery in behavioral testing by rota rod and open-field activities was seen as compared with the MCAO group. MK alone did not show any change neither in the status of various antioxidants nor behavioral functions over sham values. Although detailed studies are required for the evaluation of exact neuroprotective mechanism of MK against cerebral ischemia these preliminary experimental findings conclude that MK exhibits neuroprotective effect in cerebral ischemia by potentiating the antioxidant defense system of the brain.

Inflammation after stroke: mechanisms and therapeutic approaches.

Reperfusion of ischemic brain can reduce injury and improve outcome, but secondary injury due to inflammatory mechanisms limits the efficacy and time window of such treatments for stroke. This review summarizes the cellular and molecular basis of inflammation in ischemic injury as well as possible therapeutic strategies.

Prostaglandin D2 DP1 receptor is beneficial in ischemic stroke and in acute exicitotoxicity in young and old mice.

The cardiovascular complications reported to be associated with cyclooxygenase inhibitor use have shifted our focus toward prostaglandins and their respective receptors. Prostaglandin D(2) 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.

The PGE2 EP2 receptor and its selective activation are beneficial against ischemic stroke.

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.

The cyclooxygenase site, but not the peroxidase site of cyclooxygenase-2 is required for neurotoxicity in hypoxic and ischemic injury.

Cyclooxygenase-2 (COX-2) activity has been implicated in the pathogenesis of ischemic injury, but the exact mechanisms responsible for its toxicity remain unclear. Infection of primary neurons with an adenovirus expressing wild type (WT) COX-2 increased the susceptibility of neurons to hypoxia. Infection with an adenoviral vector expressing COX-2 with a mutation at the cyclooxygenase site did not increase susceptibility to hypoxia, whereas over-expression of COX-2 with a mutation in the peroxidase site produced similar susceptibility to hypoxia as WT COX-2. Primary neuronal cultures obtained from transgenic mice bearing a mutation in the COX-2 cylooxygenase site were protected from hypoxia. Mice with a mutation in the cyclooxygenase site had smaller infarctions 24 h after 70 min of middle cerebral artery occlusion than WT control mice. COX-2 activity had no effect on the formation of protein carbonyls. Ascorbate radicals were detected by electron paramagnetic resonance as a product of recombinant COX-2 activity and were blocked by COX-2 inhibitors. Similarly, formation of ascorbate radicals was inhibited in the presence of COX-2 inhibitors and in homogenates obtained from COX-2 null mice. Taken together, these results indicate that the cyclooxygenase activity of COX-2 is necessary to exacerbate neuronal hypoxia/ischemia injury rather than the peroxidase activity of the enzyme.

Prolonged opportunity for neuroprotection in experimental stroke with selective blockade of cyclooxygenase-2 activity.

The post-treatment effects of the selective cyclooxygenase (COX)-2 inhibitor, valdecoxib, were investigated in a rat model of temporary focal ischemia. Valdecoxib reduced basal brain prostaglandin E(2) concentrations at dosages that did not affect serum thromboxane B(2), consistent with a selective COX-2 effect. Temporary focal cerebral ischemia was produced in rats by middle cerebral artery occlusion for 90 min. There was increased expression of COX-2 protein detected by Western blot and immunocytochemistry within neurons in the ischemic cortex at 4 and 24 h after ischemia. Rats were treated with vehicle or valdecoxib 15 min before or 1.5, 3 and 6 h after cerebral ischemia. Rats were sacrificed and brain infarction volume determined 24 h after ischemia. Valdecoxib treatment was associated with a decrease in infarction volume when administered 15 min before, and 1.5 or 3 h but not 6 h after cerebral ischemia. There were no differences in physiological parameters during the procedure. Valdecoxib administered at 1.5 h after ischemia significantly reduced the concentrations of prostaglandin E(2) in ischemic penumbral cortex as compared to the vehicle-treated group and contralateral non-ischemic cortex. These results suggest that COX-2 inhibition with valdecoxib is effective when initiated both before and after middle cerebral artery occlusion.