Meng-Ling Wang

Increased expression of cysteinyl leukotriene receptor-1 in the brain mediates neuronal damage and astrogliosis after focal cerebral ischemia in rats.

Cysteinyl leukotrienes are potent pro-inflammatory mediators. Cysteinyl leukotriene receptor 1 is one of the two cysteinyl leukotriene receptors cloned. We recently reported that cysteinyl leukotriene receptor 1 antagonists protected against cerebral ischemic injury, and an inducible expression of cysteinyl leukotriene receptor 1 was found in neuron- and glial-appearing cells after traumatic injury in human brain. To determine the role of cysteinyl leukotriene receptor 1 in ischemic brain injury, we investigated the temporal and spatial profile of cysteinyl leukotriene receptor 1 expression in rat brain from 3 h to 14 days after 30 min of middle cerebral artery occlusion, and observed the effect of pranlukast, a cysteinyl leukotriene receptor 1 antagonist, on the ischemic injury. We found that cysteinyl leukotriene receptor 1 mRNA expression was up-regulated in the ischemic core both 3-12 h and 7-14 days, and in the boundary zone 7-14 days after reperfusion. In the ischemic core, cysteinyl leukotriene receptor 1 was primarily localized in neurons 24 h, and in macrophage/microglia 14 days after reperfusion; while in the boundary zone it was localized in proliferated astrocytes 14 days after reperfusion. Pranlukast attenuated neurological deficits, reduced infarct volume and ameliorated neuron loss in the ischemic core 24 h after reperfusion; it reduced infarct volume, ameliorated neuron loss and inhibited astrocyte proliferation in the boundary zone 14 days after reperfusion. Thus, we conclude that cysteinyl leukotriene receptor 1 mediates acute neuronal damage and subacute/chronic astrogliosis after focal cerebral ischemia.

Pranlukast, a cysteinyl leukotriene receptor-1 antagonist, protects against chronic ischemic brain injury and inhibits the glial scar formation in mice.

We have recently reported the neuroprotective effect of pranlukast (ONO-1078), a cysteinyl leukotriene receptor-1 (CysLT1) antagonist, on cerebral ischemia in rats and mice. In this study, we further determined whether the effect of pranlukast is long lasting and related to the formation of a glial scar in cerebral ischemic mice. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). After ischemia, pranlukast (0.1 mg/kg) was injected intraperitoneally for 5 consecutive days. Neurological deficits and sensorimotor function were determined during 70 days after ischemia. Brain lesion and glial scar formation were detected at the end of the experiment. Pranlukast did not reduce mortality, but significantly improved neurological deficits and promoted sensorimotor recovery during 70 days. At the end of the experiment, pranlukast significantly reduced lesion volume, and increased neuron densities in the cortex and hippocampal CA1 region in the ischemic hemispheres. Importantly, pranlukast also remarkably reduced the thickness of a scar wall in the ischemic hemispheres. These findings indicate that pranlukast has a long-lasting protective effect on focal cerebral ischemia in mice, and inhibit the ischemia-induced glial scar formation, providing further evidence of the therapeutic potential of pranlukast in the treatment of ischemic stroke.

Minocycline inhibits 5-lipoxygenase activation and brain inflammation after focal cerebral ischemia in rats

AbstractAim:To determine whether the anti-inflammatory effect of minocycline on postischemic brain injury is mediated by the inhibition of 5-lipoxygenase (5-LOX) expression and enzymatic activation in rats.Methods:Focal cerebral ischemia was induced for 30 min with middle cerebral artery occlusion, followed by reperfusion. The ischemic injuries, endogenous IgG exudation, the accumulation of neutrophils and macrophage/microglia, and 5-LOX mRNA expression were determined 72 h after reperfusion. 5-LOX metabolites (leukotriene B4 and cysteinyl leukotrienes) were measured 3 h after reperfusion.Results:Minocycline (22.5 and 45 mg/kg, ip, for 3 d) attenuated ischemic injuries, IgG exudation, and the accumulation of neutrophils and macrophage/microglia 72 h after reperfusion. It also inhibited 5-LOX expression 72 h after reperfusion and the production of leukotrienes 3 h after reperfusion.Conclusion:Minocycline inhibited postischemic brain inflammation, which might be partly mediated by the inhibition of 5-LOX expression and enzymatic activation.

Minocycline protects PC12 cells against NMDA-induced injury via inhibiting 5-lipoxygenase activation

Recently, we have reported that minocycline, a semi-synthetic tetracycline with neuroprotective effects, inhibits the in vitro ischemic-like injury and 5-lipoxygenase (5-LOX) activation in PC12 cells. In the present study, we further determined whether minocycline protects PC12 cells from excitotoxicity via inhibiting 5-LOX activation. We used N-methyl-d-aspartate (NMDA, 200 microM) to induce early (exposure for 6 h) and delayed (exposure for 6 h followed by 24 h recovery) injuries. We found that NMDA receptor antagonist ketamine, 5-LOX inhibitor caffeic acid and minocycline concentration dependently attenuated NMDA-induced early and delayed cell injuries (viability reduction and cell death). However, only ketamine (1 microM) inhibited NMDA-evoked elevation of intracellular calcium. In addition, immunohistochemical analysis showed that NMDA induced 5-LOX translocation to the nuclear membrane after 1- to 6-h exposure which was confirmed by Western blotting, indicating that 5-LOX was activated. Ketamine, caffeic acid and minocycline (each at 1 microM) inhibited 5-LOX translocation after early injury. After delayed injury, PC12 cells were shrunk, and 5-LOX was translocated to the nuclei and nuclear membrane; ketamine, caffeic acid and minocycline inhibited both cell shrinking and 5-LOX translocation. As a control, 12-LOX inhibitor baicalein showed a weak effect on cell viability and death, but no effect on 5-LOX translocation. Therefore, we conclude that the protective effect of minocycline on NMDA-induced injury is partly mediated by inhibiting 5-LOX activation.

Caffeic acid ameliorates early and delayed brain injuries after focal cerebral ischemia in rats.

AbstractAim:To investigate the effects of caffeic acid on early and delayed injuries after focal cerebral ischemia in rats, and the possible relation to 5-lipoxygenase inhibition.Methods:Transient focal cerebral ischemia was induced by middle cerebral artery occlusion in Sprague-Dawley rats. Caffeic acid (10 and 50 mg/kg) was ip injected for 5 d after ischemia. The brain injuries were observed, and the levels of cysteinyl leukotrienes and leukotriene B4 in the brain tissue were measured.Results:Caffeic acid (50 mg/kg) ameliorated neurological dysfunction and neuron loss, and decreased infarct volume 24 h after ischemia; it attenuated brain atrophy, infarct volume, and particularly astrocyte proliferation 14 d after ischemia. In addition, it reduced the production of leukotrienes (5-lipoxygenase metabolites) in the ischemic hemispheres 3 h and 7 d after ischemia.Conclusion:Caffeic acid has protective effect on both early and delayed injuries after focal cerebral ischemia in rats; and this effect may partly relate to 5-lipoxygenase inhibition.

Expression patterns of 5‐lipoxygenase in human brain with traumatic injury and astrocytoma

5‐Lipoxygenase (5‐LOX) is a key enzyme in the metabolism of arachidonic acid to leukotrienes. The levels of leukotrienes increase after brain injury and when tumors are present. It has been reported that 5‐LOX is widely expressed in the brain and that 5‐LOX inhibition provides neuroprotection. However, there is still no information available for the expression patterns of 5‐LOX in human brain following trauma or with astrocytomas. We investigated its expression patterns by immunohistochemistry. We found that 5‐LOX is normally expressed in neurons and glial cells. In neurons, it was expressed in two patterns: in the cytosol and nucleus or only in the cytosol. In traumatic brain injury, 5‐LOX expression increased in glial cells and neutrophils. Double‐labeling immunohistochemistry showed that part of the 5‐LOX‐positive glial cells were GFAP positive. No 5‐LOX expression was found in brain microvessel endothelia, except in the regenerated endothelia of a patient 8 days following brain trauma. Furthermore, 5‐LOX expression increased and showed a granular pattern in high‐grade (grade III/IV) astrocytoma. These results indicate that 5‐LOX has multiple expression patterns, and can be induced by brain injury, which implies that 5‐LOX might have pathophysiological roles in the human brain.

Spatio-temporal expression of cysteinyl leukotriene receptor-2 mRNA in rat brain after focal cerebral ischemia.

Cysteinyl leukotrienes (CysLTs) induce inflammatory responses mediated by activating CysLT(1) and CysLT(2) receptors. We have recently reported that CysLT(1) receptor expression is increased in rat brain after focal cerebral ischemia and the increased expression is spatio-temporally related to acute neuronal injury and late astrocyte proliferation. Here we report spatio-temporal expression of CysLT(2) receptor mRNA in rat brain after focal cerebral ischemia induced by 30min of middle cerebral artery occlusion. We found that the neuron density was gradually decreased or disappeared in the ischemic core and boundary zone during 14 days after reperfusion, and the astrocyte population in the boundary zone was increased 3-14 days after reperfusion. In the ischemic core, the expression of CysLT(2) receptor mRNA was increased at 6, 12 and 24h and then recovered at 3, 7 and 14 days after reperfusion. In the boundary zone, the expression was significantly increased 3, 7 and 14 days after reperfusion. The results suggest that CysLT(2) receptor may be related to the acute neuronal injury and late astrocyte proliferation in the ischemic brain.

Pranlukast reduces neutrophil but not macrophage/microglial accumulation in brain after focal cerebral ischemia in mice

AbstractAim:To determine whether pranlukast, a cysteinyl leukotriene receptor-1 antagonist, exerts an anti-inflammatory effect on focal cerebral ischemia in mice.Methods:Focal cerebral ischemia in mice was induced by permanent middle cerebral artery occlusion (MCAO). In addition to neurological deficits, infarct volume, degenerated neurons and endogenous IgG exudation, we detected accumulation of neutrophils and macrophage/microglia in the ischemic brain tissue 72 h after MCAO. Pranlukast was ip injected 30 min before and after MCAO.Results:Pranlukast significantly attenuated neurological deficits, infarct volume, neuron degeneration and IgG exudation. Importantly, pranlukast (0.01 and 0.1 mg/kg) inhibited myeloperoxidase-positive neutrophil, but not CD 11b-positive macrophage/microglial accumulation in the ischemic cortical tissue.Conclusion:Pranlukast exerts an anti-inflammatory effect on focal cerebral ischemia in the subacute phase that is limited to neutrophil recruitment through the disrupted blood-brain barrier.

Minocycline inhibits 5-lipoxygenase expression and accelerates functional recovery in chronic phase of focal cerebral ischemia in rats.

AIMS We previously reported that minocycline attenuates acute brain injury and inflammation after focal cerebral ischemia, and this is partly mediated by inhibition of 5-lipoxygenase (5-LOX) expression. Here, we determined the protective effect of minocycline on chronic ischemic brain injury and its relation with the inhibition of 5-LOX expression after focal cerebral ischemia. MAIN METHODS Focal cerebral ischemia was induced by 90 min of middle cerebral artery occlusion followed by reperfusion for 36 days. Minocycline (45 mg/kg) was administered intraperitoneally 2h and 12h after ischemia and then every 12h for 5 days. Sensorimotor function was evaluated 1-28 days after ischemia and cognitive function was determined 30-35 days after ischemia. Thereafter, infarct volume, neuron density, astrogliosis, and 5-LOX expression in the brain were determined. KEY FINDINGS Minocycline accelerated the recovery of sensorimotor and cognitive functions, attenuated the loss of neuron density, and inhibited astrogliosis in the boundary zone around the ischemic core, but did not affect infarct volume. Minocycline significantly inhibited the increased 5-LOX expression in the proliferated astrocytes in the boundary zone, and in the macrophages/microglia in the ischemic core. SIGNIFICANCE Minocycline accelerates functional recovery in the chronic phase of focal cerebral ischemia, which may be partly associated with the reduction of 5-LOX expression.

Protective effects of baicalin on oxygen/glucose deprivation- and NMDA-induced injuries in rat hippocampal slices.

Baicalin is a flavonoid derivative from Scutellaria baicalensis Georgi with various pharmacological effects. Recently, the neuroprotective effect of baicalin was reported. To confirm this effect and explore the possible mechanism, we have investigated the protective effect of baicalin on ischaemiclike or excitotoxic injury and the activation of protein kinase Cα (PKCα) in rat hippocampal slices. In‐vitro ischaemic‐like injury was induced by oxygen/glucose deprivation (OGD) and the excitotoxic injury by N‐methyl‐d‐aspartate (NMDA). The viability and swelling of the slices were detected by triphenyltetrazolium chloride (TTC) staining and image analysis of light transmittance (LT), respectively. The translocation of PKCα was measured by immunoblotting. Baicalin was added during both injuries. Baicalin (0.1, 1, and 10 μmolL−1) concentration‐dependently inhibited OGD‐induced viability reduction and acute neuron swelling, and inhibited the increased portion of PKCα present in the membrane fraction over the total PKCα. Baicalin ameliorated NMDA‐induced viability reduction (not LT elevation) and inhibited the NMDA‐increased membrane portion of PKCα at 1 μmolL−1. We concluded that baicalin had a protective effect on ischaemic‐like or excitotoxic injury in rat hippocampal slices, which might have been partly related to inhibition of PKCα translocation.