Wide therapeutic time window for nimesulide neuroprotection in a model of transient focal cerebral ischemia in the rat

Results from several studies indicate that cyclooxygenase-2 (COX-2) is involved ischemic brain injury. The purpose of this study was to evaluate the neuroprotective effects of the selective COX-2 inhibitor nimesulide on cerebral infarction and neurological deficits in a standardized model of transient focal cerebral ischemia in rats. Three doses of nimesulide (3, 6 and 12 mg/kg; i.p.) or vehicle were administered immediately after stroke and additional doses were given at 6, 12, 24, 36 and 48 h after ischemia. In other set of experiments, the effect of nimesulide was studied in a situation in which its first administration was delayed for 3 to 24 h after ischemia. Total, cortical and subcortical infarct volumes and functional outcome (assessed by neurological deficit score and rotarod performance) were determined 3 days after ischemia. The effect of nimesulide on prostaglandin E2 (PGE2) levels in the injured brain was also investigated. Nimesulide dose-dependently reduced infarct volume and improved functional recovery when compared to vehicle. Of interest is the finding that neuroprotection conferred by nimesulide (reduction of infarct size and neurological deficits and improvement of rotarod performance) was also observed when treatment was delayed until 24 h after ischemia. Further, administration of nimesulide in a delayed treatment paradigm completely abolished PGE2 accumulation in the postischemic brain, suggesting that COX-2 inhibition is a promising therapeutic strategy for cerebral ischemia to target the late-occurring inflammatory events which amplify initial damage

Regional distribution of the prostaglandin E2 receptor EP1 in the rat brain: accumulation in Purkinje cells of the cerebellum

Prostaglandin E2 (PGE2), is a major prostanoid produced by the activity of cyclooxygenases (COX) in response to various physiological and pathological stimuli. PGE2 exerts its effects by activating four specific E-type prostanoid receptors (EP1, EP2, EP3 and EP4). In the present study, we analyzed the expression of the PGE2 receptor EP1 (mRNA and protein) in different regions of the adult rat brain (hippocampus, hypothalamus, striatum, prefrontal cerebral cortex, parietal cortex, brain stem and cerebellum) using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemical methods. On a regional basis, levels of EP1 mRNA were the highest in parietal cortex and cerebellum. At the protein level, we found a very strong expression of EP1 in cerebellum as revealed by Western blotting experiments. Furthermore, the present study provides for the first time evidence that the EP1 receptor is highly expressed in the cerebellum, where the Purkinje cells displayed a very high immunolabeling of their perikaryon and dendrites as observed in the immunohistochemical analysis. Results from the present study indicate that the EP1 prostanoid receptor is expressed in specific neuronal populations, which possibly determine the region specific response to PGE2

Post-ischaemic treatment with the cyclooxygenase-2 inhibitor nimesulide reduces blood-brain barrier disruption and leukocyte infiltration following transient focal cerebral ischaemia in rats

Several studies suggest that cyclooxygenase (COX)-2 plays a pivotal role in the progression of ischemic brain damage. In the present study, we investigated the effects of selective inhibition of COX-2 with nimesulide (12 mg/kg) and selective inhibition of COX-1 with valeryl salicylate (VAS, 12-120 mg/kg) on prostaglandin E2 (PGE2) levels, myeloperoxidase (MPO) activity, Evans Blue (EB) extravasation and infarct volume in a standardized model of transient focal cerebral ischemia in the rat. Postischemic treatment with nimesulide markedly reduced the increase in PGE2 levels in the ischemic cerebral cortex 24 h after stroke and diminished infarct size by 48 % with respect to vehicle-treated animals after 3 days of reperfusion. Furthermore, nimesulide significantly attenuated the blood-brain barrier (BBB) damage and leukocyte infiltration (as measured by EB leakage and MPO activity, respectively) seen at 48 h after the initial ischemic episode. These studies provide the first experimental evidence that COX-2 inhibition with nimesulide is able to limit BBB disruption and leukocyte infiltration following transient focal cerebral ischemia. Neuroprotection afforded by nimesulide is observed even when the treatment is delayed until 6 h after the onset of ischemia, confirming a wide therapeutic window of COX-2 inhibitors in experimental stroke. On the other hand, selective inhibition of COX-1 with VAS had no significant effect on the evaluated parameters. These data suggest that COX-2 activity, but not COX-1 activity, contributes to the progression of focal ischemic brain injury, and that the beneficial effects observed with non-selective COX inhibitors are probably associated to COX-2 rather than to COX-1 inhibition

Ascorbic acid enhances the inhibitory effect of aspirin on neuronal cyclooxygenase-2-mediated prostaglandin E2 production.

Inhibition of neuronal cyclooxygenase-2 (COX-2) and hence prostaglandin E2 (PGE2) synthesis by non-steroidal anti-inflammatory drugs has been suggested to protect neuronal cells in a variety of pathophysiological situations including Alzheimer's disease and ischemic stroke. Ascorbic acid (vitamin C) has also been shown to protect cerebral tissue in a variety of experimental conditions, which has been attributed to its antioxidant capacity. In the present study, we show that ascorbic acid dose-dependently inhibited interleukin-1beta (IL-1beta)-mediated PGE2 synthesis in the human neuronal cell line, SK-N-SH. Furthermore, in combination with aspirin, ascorbic acid augmented the inhibitory effect of aspirin on PGE2 synthesis. However, ascorbic acid had no synergistic effect along with other COX inhibitors (SC-58125 and indomethacin). The inhibition of IL-1beta-mediated PGE2 synthesis by ascorbic acid was not due to the inhibition of the expression of COX-2 or microsomal prostaglandin E synthase (mPGES-1). Rather, ascorbic acid dose-dependently (0.1-100 microM) produced a significant reduction in IL-1beta-mediated production of 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), a reliable indicator of free radical formation, suggesting that the effects of ascorbic acid on COX-2-mediated PGE2 biosynthesis may be the result of the maintenance of the neuronal redox status since COX activity is known to be enhanced by oxidative stress. Our results provide in vitro evidence that the neuroprotective effects of ascorbic acid may depend, at least in part, on its ability to reduce neuronal COX-2 activity and PGE2 synthesis, owing to its antioxidant properties. Further, these experiments suggest that a combination of aspirin with ascorbic acid constitutes a novel approach to render COX-2 more sensitive to inhibition by aspirin, allowing an anti-inflammatory therapy with lower doses of aspirin, thereby avoiding the side effects of the usually high dose aspirin treatment

Rice bran derivatives alleviate microglia activation: possible involvement of MAPK pathway

(A-C). Effects of RBE on the phosphorylation of p38MAPK, ERK, and JNK in non-activated microglia. Cells were treated with RBE (50–300 μg/ml) for 24 h followed by cell lyses and protein estimation. During stimulation, one of the wells in 6-well plate was incubated with LPS (10 ng/ml) for 30 min to be used as positive control to validate the functionality of antibodies against activated state of kinases. Whole cell lysates were subjected to western blots analyses. Representative blots (upper panel) and densitometry analyses (lower panel) are shown: A) p38 MAPK, B) pERK, and C) pJNK. Statistical analyses were carried out by using one-way ANOVA with post hoc Student-Newman-Keuls test (multiple comparisons). Results are expressed as means ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared control cells. (TIF 963 kb

Inhibition of neuroinflammation by thymoquinone requires activation of Nrf2/ARE signalling

Thymoquinone is an antioxidant phytochemical that has been shown to inhibit neuroinflammation. However, little is known about the potential roles of intracellular antioxidant signalling pathways in its anti-inflammatory activity. The objective of this study was to elucidate the roles played by activation of the Nrf2/ARE antioxidant mechanisms in the anti-inflammatory activity of this compound. Thymoquinone inhibited lipopolysaccharide (LPS)-induced neuroinflammation through interference with NF-B signalling in BV2 microglia. Thymoquinone also activated Nrf2/ARE signalling by increasing nuclear localisation, DNA binding and transcriptional activity of Nrf2, as well as increasing protein levels of HO-1 and NQO1. Suppression of Nrf2 activity through siRNA or with the use of trigonelline resulted in the loss of anti-inflammatory activity by thymoquinone. Taken together, our studies show that thymoquinone inhibits NF-kappaB-dependent neuroinflammation in BV2 microglia, by targeting antioxidant pathway involving activation of both Nrf2/ARE. We propose that activation of Nrf2/ARE signalling pathway by thymoquinone probably results in inhibition of NF-kappaB-mediated neuroinflammation

Cyclooxygenase Inhibition Limits Blood-Brain Barrier Disruption following Intracerebral Injection of Tumor Necrosis Factor-alpha in the Rat

Increased permeability of the blood-brain barrier (BBB) is important in neurological disorders. Neuroinflammation is associated with increased BBB breakdown and brain injury. Tumor necrosis factor-alpha (TNF-a) is involved in BBB injury and edema formation through a mechanism involving matrix metalloproteinase (MMP) upregulation. There is emerging evidence indicating that cyclooxygenase (COX) inhibition limits BBB disruption following ischemic stroke and bacterial meningitis, but the mechanisms involved are not known. We used intracerebral injection of TNF-a to study the effect of COX inhibition on TNF-a-induced BBB breakdown, MMP expression/activity and oxidative stress. BBB disruption was evaluated by the uptake of 14C-sucrose into the brain and by magnetic resonance imaging (MRI) utilizing Gd-DTPA as a paramagnetic contrast agent. Using selective inhibitors of each COX isoform, we found that COX-1 activity is more important than COX-2 in BBB opening. TNF-a induced a significant upregulation of gelatinase B (MMP-9), stromelysin-1 (MMP-3) and COX-2. In addition, TNF-a significantly depleted glutathione as compared to saline. Indomethacin (10 mg/kg; i.p.), an inhibitor of COX-1 and COX-2, reduced BBB damage at 24 h. Indomethacin significantly attenuated MMP-9 and MMP-3 expression and activation, and prevented the loss of endogenous radical scavenging capacity following intracerebral injection of TNF-a. Our results show for the first time that BBB disruption during neuroinflammation can be significantly reduced by administration of COX inhibitors. Modulation of COX in brain injury by COX inhibitors or agents modulating prostaglandin E2 formation/signaling may be useful in clinical settings associated with BBB disruption

Cyclooxygenase inhibition in ischemic brain injury

Neuroinflammation is one of the key pathological events involved in the progression of brain damage caused by cerebral ischemia. Metabolism of arachidonic acid through cyclooxygenase (COX) enzymes is known to be actively involved in the neuroinflammatory events leading to neuronal death after ischemia. Two isoforms of COX, termed COX-1 and COX-2, have been identified. Unlike COX-1, COX-2 expression is dramatically induced by ischemia and appears to be an effector of tissue damage. This review article will focus specifically on the involvement of COX isozymes in brain ischemia. We will discuss issues related to the biochemistry and selective pharmacological inhibition of COX enzymes, and further refer to their expression in the brain under normal conditions and following excitotoxicity and ischemic cerebral injury. We will review present knowledge of the relative contribution of each COX isoform to the brain ischemic pathology, based on data from investigations utilizing selective COX-1/COX-2 inhibitors and genetic knockout mouse models. The mechanisms of neurotoxicity associated with increased COX activity after ischemia will also be examined. Finally, we will provide a critical evaluation of the therapeutic potential of COX inhibitors in cerebral ischemia and discuss new targets downstream of COX with potential neuroprotective ability

Targeting oxidative stress: Novel coumarin-based inverse agonists of GPR55

Oxidative stress is associated with different neurological and psychiatric diseases. Therefore, development of new pharmaceuticals targeting oxidative dysregulation might be a promising approach to treat these diseases. The G-protein coupled receptor 55 (GPR55) is broadly expressed in central nervous tissues and cells and is involved in the regulation of inflammatory and oxidative cell homeostasis. We have recently shown that coumarin-based compounds enfold inverse agonistic activities at GPR55 resulting in the inhibition of prostaglandin E2. However, the antioxidative effects mediated by GPR55 were not evaluated yet. Therefore, we investigated the antioxidative effects of two novel synthesized coumarin-based compounds, KIT C and KIT H, in primary mouse microglial and human neuronal SK-N-SK cells. KIT C and KIT H show antioxidative properties in SK-N-SH cells as well as in primary microglia. In GPR55-knockout SK-N-SH cells, the antioxidative effects are abolished, suggesting a GPR55-dependent antioxidative mechanism. Since inverse agonistic GPR55 activation in the brain seems to be associated with decreased oxidative stress, KIT C and KIT H possibly act as inverse agonists of GPR55 eliciting promising therapeutic options for oxidative stress related diseases

Modulation of neuroinflammation and oxidative stress by targeting GPR55 – new approaches in the treatment of psychiatric disorders

Pharmacological treatment of psychiatric disorders remains challenging in clinical, pharmacological, and scientific practice. Even if many different substances are established for treating different psychiatric conditions, subgroups of patients show only small or no response to the treatment. The neuroinflammatory hypothesis of the genesis of psychiatric disorders might explain underlying mechanisms in these non-responders. For that reason, recent research focus on neuroinflammatory processes and oxidative stress as possible causes of psychiatric disorders. G-protein coupled receptors (GPCRs) form the biggest superfamily of membrane-bound receptors and are already well known as pharmacological targets in various diseases. The G-protein coupled receptor 55 (GPR55), a receptor considered part of the endocannabinoid system, reveals promising modulation of neuroinflammatory and oxidative processes. Different agonists and antagonists reduce pro-inflammatory cytokine release, enhance the synthesis of anti- inflammatory mediators, and protect cells from oxidative damage. For this reason, GPR55 ligands might be promising compounds in treating subgroups of patients suffering from psychiatric disorders related to neuroinflammation or oxidative stress. New approaches in drug design might lead to new compounds targeting different pathomechanisms of those disorders in just one molecule