Wei-Ping Zhang

Intranasal recombinant human erythropoietin protects rats against focal cerebral ischemia.

Erythropoietin (EPO) is a hematopoietic growth factor with tissue-protective properties, and can protect animals from cerebral ischemic injury. However, the central nervous effects of EPO as a glycoprotein is limited by the potential complication resulted from its erythropoietic activity and the problem of the penetration through blood-brain barrier (BBB). To avoid these limitations, in this study we administered recombinant human EPO (rhEPO) intranasally (i.n.) to evaluate its neuroprotective effect in the rats with focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO). We found that rhEPO i.n. at doses of 4.8, 12 and 24 U (administered 10 min after MCAO and 1h after reperfusion) reduced infarct volume, brain swelling and cell damage in the ischemic hemispheres, and improved behavioral dysfunction 24 h after cerebral ischemia. Intraperitoneal rhEPO (5000 U/kg) also showed the protective effect, but the heat-inactivated rhEPO did not show any effect. Thus, intranasal administration of relatively small doses of rhEPO protects rats from acute injury after focal cerebral ischemia, suggesting that intranasal rhEPO may be a more effective and safer administration route for treatments of ischemic or other brain diseases.

Montelukast, a cysteinyl leukotriene receptor-1 antagonist, dose- and time-dependently protects against focal cerebral ischemia in mice

Our previous studies showed that cysteinyl leukotriene receptor-1 (CysLT1) antagonist pranlukast has a neuroprotective effect on cerebral ischemia in rats and mice. However, whether the neuroprotective effect of pranlukast is its special action or a common action of CysLT1 receptor antagonists remains to be clarified. This study was performed to determine whether montelukast, another CysLT1 receptor antagonist, has the neuroprotective effect on focal cerebral ischemia in mice, and to observe its dose- and time-dependent properties. Permanent focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Montelukast was injected intraperitoneally either as multiple doses (once a day for 3 days and 30 min before MCAO) or as a single dose (at 30 min before, 30 min after, or 1 h after MCAO), respectively, and pranlukast and edaravone were used as controls. The neurological deficits, infarct volumes, brain edema, neuron density, and Evans blue extravasation in the brain were determined 24 h after MCAO. Pretreatments with multiple doses or a single dose of montelukast (0.1 and 1.0 mg/kg) before MCAO significantly attenuated all the ischemic insults. Post-treatment with a single dose of montelukast (0.1 and 1.0 mg/kg) at 30 min after MCAO also significantly decreased brain edema and infarct volume, but not neurological deficits. However, post-treatment with a single dose of montelukast at 1 h after MCAO had no significant effect. Pranlukast showed the same effects as montelukast, but edaravone attenuated the ischemic insults only with multiple doses before MCAO. Thus, montelukast has a dose- and time-dependent neuroprotective effect on permanent focal cerebral ischemia in mice, with an effective dose range of 0.1–1.0 mg/kg and a therapeutic window of 30 min. These findings further support the therapeutic potential of CysLT1 receptor antagonists in the treatment of cerebral ischemia at earlier phases.

Expression of cysteinyl leukotriene receptor 1 in human traumatic brain injury and brain tumors

Cysteinyl leukotrienes (CysLTs) are potent proinflammatory mediators. CysLT receptor 1 (CysLT(1)) is one of the two CysLT receptors that has been cloned. Although the expression of CysLT(1) in the brain has been demonstrated by Northern blot and RT-PCR analyses, the location of CysLT(1) in the brain remains unknown. The objective of this study was to examine the distribution of CysLT(1) by immunohistochemical analysis in human brains with traumatic injury or tumors. CysLT(1) was expressed intensely in the microvascular endothelial cells in both normal and abnormal conditions. At 8 days after traumatic injury, microvascular regeneration was found and all of the endothelial cells highly expressed CysLT(1). In gray and white matters of the normal regions of the brain, CysLT(1) was expressed weekly or not at all. However, the CysLT(1) expression increased in the neuron- and glial-appearing cells in gray and white matters after traumatic brain injury. CysLT(1) was also detected in astrocytoma, ganglioglioma and metastatic adenocarcinoma, and the expression in the neuron- and glial-appearing cells around brain tumors increased robustly.

Minocycline protects PC12 cells from ischemic-like injury and inhibits 5-lipoxygenase activation

Minocycline protects animals against cerebral ischemia by inhibiting inflammation. To determine whether minocycline protects PC12 cells from in vitro ischemic-like injury and affects pro-inflammatory 5-lipoxygenase activation, the cell viability and 5-lipoxygenase translocation to nuclear membrane were observed after oxygen-glucose deprivation (OGD). We found that OGD reduced cell viability, which was attenuated by minocycline and 5-lipoxygenase inhibitor caffeic acid. 5-Lipoxygenase protein was detected in PC12 cells by immunohistochemical and Western blot analyses. OGD induced 5-lipoxygenase translocation to nuclear membranes, which was abolished by minocycline and caffeic acid. Thus, minocycline can protect PC12 cells from in vitro ischemic-like injury, and this effect may be partly related to the inhibition of 5-lipoxygenase activation.

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.

Effects of carnosine on amygdaloid-kindled seizures in Sprague–Dawley rats

The effects of carnosine (beta-alanyl-L-histidine) on amygdaloid-kindled seizures were investigated in rats. I.p. injection of carnosine (500, 1000, 1500 mg/kg, i.p.) significantly decreased seizure stage, afterdischarge duration and generalized seizure duration, and significantly prolonged generalized seizure latency of amygdaloid-kindled seizures, in a dose-dependent, and time-related manner. The protective effect of carnosine (1500 mg/kg) was completely antagonized by histamine H1-antagonists pyrilamine (2, 5 mg/kg, i.p.) and diphenhydramine (5, 10 mg/kg, i.p.), but not by histamine H2-antagonist zolantidine even at a high dose of 10 mg/kg. Carnosine (1500 mg/kg, i.p.) caused a significant increase of carnosine and histidine levels in the hypothalamus, thalamus, hippocampus, amygdala and cortex, as well as histamine levels in the hippocampus and amygdala. I.c.v. injection of alpha-fluoromethylhistidine (50 microg, i.c.v.), a selective and irreversible histidine decarboxylase inhibitor, only partially reversed the inhibition of amygdaloid-kindled seizures induced by carnosine. In addition, carnosine significantly decreased glutamate contents in the amygdala and hippocampus. These results indicate that carnosine could protect against amygdaloid-kindled seizures in rats, and its action may be due to the activation of histamine postsynaptic H1-receptors via two different mechanisms, one being carnosines direct action, and the other being indirectly mediated by histaminergic pathway. The study suggests that carnosine may be an endogenous anticonvulsant factor in the brain and could be used as a new antiepileptic drug in the future.

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.

Activation of CysLT receptors induces astrocyte proliferation and death after oxygen-glucose deprivation.

We recently found that 5‐lipoxygenase (5‐LOX) is activated to produce cysteinyl leukotrienes (CysLTs), and CysLTs may cause neuronal injury and astrocytosis through activation of CysLT1 and CysLT2 receptors in the brain after focal cerebral ischemia. However, the property of astrocyte responses to in vitro ischemic injury is not clear; whether 5‐LOX, CysLTs, and their receptors are also involved in the responses of ischemic astrocytes remains unknown. In the present study, we performed oxygen‐glucose deprivation (OGD) followed by recovery to induce ischemic‐like injury in the cultured rat astrocytes. We found that 1‐h OGD did not injure astrocytes (sub‐lethal OGD) but induced astrocyte proliferation 48 and 72 h after recovery; whereas 4‐h OGD moderately injured the cells (moderate OGD) and led to death 24–72 h after recovery. Inhibition of phospholipase A2 and 5‐LOX attenuated both the proliferation and death. Sub‐lethal and moderate OGD enhanced the production of CysLTs that was inhibited by 5‐LOX inhibitors. Sub‐lethal OGD increased the expressions of CysLT1 receptor mRNA and protein, while moderate OGD induced the expression of CysLT2 receptor mRNA. Exogenously applied leukotriene D4 (LTD4) induced astrocyte proliferation at 1–10 nM and astrocyte death at 100–1,000 nM. The CysLT1 receptor antagonist montelukast attenuated astrocyte proliferation, the CysLT2 receptor antagonist BAY cysLT2 reversed astrocyte death, and the dual CysLT receptor antagonist BAY u9773 exhibited both effects. In addition, LTD4 (100 nM) increased the expression of CysLT2 receptor mRNA. Thus, in vitro ischemia activates astrocyte 5‐LOX to produce CysLTs, and CysLTs result in CysLT1 receptor‐mediated proliferation and CysLT2 receptor‐mediated death.

Dihydroartemisinin exerts cytotoxic effects and inhibits hypoxia inducible factor-1α activation in C6 glioma cells

Artemisinin and its analogue dihydroartemisinin exert cytotoxic effects in some kinds of cancer cell lines. Here we determined whether dihydroartemisinin inhibits the growth and induces apoptosis of rat C6 glioma cells. We found dihydroartemisinin (5–25 μM) inhibited the growth and induced apoptosis of C6 cells in a concentration‐ and time‐dependent manner; however, it was much less toxic to rat primary astrocytes. Dihydroartemisinin (5–25 μM) also increased the generation of reactive oxygen species in C6 cells. These effects of dihydroartemisinin were enhanced by ferrous ions (12.5–100 μM) and reduced by the iron chelator deferoxamine (25–200 μM). Immunoblotting analysis revealed that dihydroartemisinin (5–25 μM) significantly reduced hypoxia‐ and deferoxamine‐induced expression of hypoxia inducible factor‐1α and its target gene protein, vascular endothelial growth factor, in C6 cells. The results showed that dihydroartemisinin exerts a selective cytotoxic effect on C6 cells by increasing the reactive oxygen species and inhibiting hypoxia inducible factor‐1α activation.

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.