Wen-Zhen Shi

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.

The new P2Y-like receptor G protein-coupled receptor 17 mediates acute neuronal injury and late microgliosis after focal cerebral ischemia in rats.

G protein-coupled receptor 17 (GPR17), the new P2Y-like receptor, is phylogenetically related to the P2Y and cysteinyl leukotriene receptors, and responds to both uracil nucleotides and cysteinyl leukotrienes. GPR17 has been proposed to be a damage sensor in ischemic stroke; however, its role in brain inflammation needs further detailed investigation. Here, we extended previous studies on the spatiotemporal profiles of GPR17 expression and localization, and their implications for brain injury after focal cerebral ischemia. We found that in the ischemic core, GPR17 mRNA and protein levels were upregulated at both 12-24 h and 7-14 days, but in the boundary zone the levels increased 7-14 days after reperfusion. The spatiotemporal pattern of GPR17 expression well matched the acute and late (subacute/chronic) responses in the ischemic brain. According to previous findings, in the acute phase, after ischemia (24 h), upregulated GPR17 was localized in injured neurons in the ischemic core and in a few microglia in the ischemic core and boundary zone. In the late phase (14 days), it was localized in microglia, especially in activated (ED1-positive) microglia in the ischemic core, but weakly in most microglia in the boundary zone. No GPR17 was detectable in astrocytes. GPR17 knockdown by a small interfering RNA attenuated the neurological dysfunction, infarction, and neuron loss at 24 h, and brain atrophy, neuron loss, and microglial activation at 14 days after reperfusion. Thus, GPR17 might mediate acute neuronal injury and late microgliosis after focal cerebral ischemia.

CysLT2 receptor-mediated AQP4 up-regulation is involved in ischemic-like injury through activation of ERK and p38 MAPK in rat astrocytes

AIMS We previously reported that cysteinyl leukotriene receptor 2 (CysLT(2)) mediates ischemic astrocyte injury, and leukotriene D(4)-activated CysLT(2) receptor up-regulates the water channel aquaporin 4 (AQP4). Here we investigated the mechanism underlying CysLT(2) receptor-mediated ischemic astrocyte injury induced by 4-h oxygen-glucose deprivation and 24-h recovery (OGD/R). MAIN METHODS Primary cultures of rat astrocytes were treated by OGD/R to construct the cell injury model. AQP4 expression was inhibited by small interfering RNA (siRNA). The expressions of AQP4 and CysLTs receptors, and the MAPK signaling pathway were determined. KEY FINDINGS OGD/R induced astrocyte injury, and increased expression of the CysLT(2) (but not CysLT(1)) receptor and AQP4. OGD/R-induced cell injury and AQP4 up-regulation were inhibited by a CysLT(2) receptor antagonist (Bay cysLT2) and a non-selective CysLT receptor antagonist (Bay u9773), but not by a CysLT(1) receptor antagonist (montelukast). Knockdown of AQP4 by siRNA attenuated OGD/R injury. Furthermore, OGD/R increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the cell injury and AQP4 up-regulation. SIGNIFICANCE The CysLT(2) receptor mediates AQP4 up-regulation in astrocytes, and up-regulated AQP4 leads to OGD/R-induced injury, which results from activation of the ERK1/2 and p38 MAPK pathways.

Montelukast, a cysteinyl leukotriene receptor-1 antagonist, attenuates chronic brain injury after focal cerebral ischaemia in mice and rats.

Objectives  Previously we demonstrated the neuroprotective effect of montelukast, a cysteinyl leukotriene receptor‐1 (CysLT1) antagonist, on acute brain injury after focal cerebral ischaemia in mice. In this study, we have determined its effect on chronic brain injury after focal cerebral ischaemia in mice and rats.

Cysteinyl leukotriene receptor 2 is spatiotemporally involved in neuron injury, astrocytosis and microgliosis after focal cerebral ischemia in rats.

Cysteinyl leukotrienes (CysLTs), potent inflammatory mediators, are released from ischemic brain, and may regulate ischemic injury through activating CysLT1 and CysLT2 receptors. The CysLT1 receptor is closely associated with ischemic injury and post-ischemic repair; however, the CysLT2 receptor-mediated responses remain unknown. Here, we investigated the spatiotemporal profiles and implications of CysLT2 receptor expression and localization in rat brain after focal cerebral ischemia. CysLT2 receptors were normally localized in astrocytes in the cortex and around the ventricles. After focal cerebral ischemia, CysLT2 receptor expression was up-regulated in concert with neuronal and glial responses. In the acute phase (6-24 h), up-regulated CysLT2 receptors were restricted to injured neurons in the ischemic core; while in the late phase (3-28 days), the up-regulation was restricted to hypertrophic microglia (ischemic core) and mainly localized in hypertrophic astrocytes (boundary zone). Thus, the spatiotemporal profiles of CysLT2 receptor expression suggest that it plays regulatory roles in acute neuron injury, and astrocytosis and microgliosis in the late phase.

Aggravated chronic brain injury after focal cerebral ischemia in aquaporin-4-deficient mice

The water channel aquaporin-4 (AQP4) is important in brain water homeostasis, and is also involved in astrocyte growth and glial scar formation. It has been reported that AQP4 deficiency attenuates acute ischemic brain injury as a result of reducing cytotoxic edema. Here, we determined whether AQP4 deficiency influences chronic brain injury after focal cerebral ischemia induced by 30 min of middle cerebral artery occlusion (MCAO). AQP4(-/-) mice exhibited a lower survival rate and less body weight gain than wild-type mice, but their neurological deficits were similar to wild-type mice during 35 days after MCAO. At 35 days after MCAO, AQP4(-/-) mice showed more severe brain atrophy and cavity formation in the ischemic hemisphere as well as more neuronal loss in the hippocampus. Furthermore, astrocyte proliferation and glial scar formation were impaired in AQP4(-/-) mice. Therefore, AQP4 deficiency complicated by astrocyte dysfunction aggravates chronic brain injury after focal cerebral ischemia, suggesting that AQP4 may be important in the chronic phase of the post-ischemic recovery process.

Aquaporin-4 deficiency attenuates acute lesions but aggravates delayed lesions and microgliosis after cryoinjury to mouse brain

ObjectiveTo determine whether aquaporin-4 (AQP4) regulates acute lesions, delayed lesions, and the associated microglial activation after cryoinjury to the brain.MethodsBrain cryoinjury was applied to AQP4 knockout (KO) and wild-type mice. At 24 h and on days 7 and 14 after cryoinjury, lesion volume, neuronal loss, and densities of microglia and astrocytes were determined, and their changes were compared between AQP4 KO and wild-type mice.ResultsLesion volume and neuronal loss in AQP4 KO mice were milder at 24 h following cryoinjury, but worsened on days 7 and 14, compared to those in wild-type mice. Besides, microglial density increased more, and astrocyte proliferation and glial scar formation were attenuated on days 7 and 14 in AQP4 KO mice.ConclusionAQP4 deficiency ameliorates acute lesions, but worsens delayed lesions, perhaps due to the microgliosis in the late phase.

Aggravated inflammation and increased expression of cysteinyl leukotriene receptors in the brain after focal cerebral ischemia in AQP4-deficient mice

ObjectiveAquaporin-4 (AQP4), the main water channel protein in the brain, plays a critical role in water homeostasis and brain edema. Here, we investigated its role in the inflammatory responses after focal cerebral ischemia.MethodsIn AQP4-knockout (KO) and wild-type mice, focal cerebral ischemia was induced by 30 min of middle cerebral arterial occlusion (MCAO). Ischemic neuronal injury and cellular inflammatory responses, as well as the expression and localization of cysteinyl leukotriene CysLT2 and CysLT1 receptors, were determined at 24 and 72 h after MCAO.ResultsAQP4-KO mice showed more neuronal loss, more severe microglial activation and neutrophil infiltration, but less astrocyte proliferation in the brain after MCAO than wild-type mice. In addition, the protein levels of both CysLT1 and CysLT2 receptors were up-regulated in the ischemic brain, and the up-regulation was more pronounced in AQP4-KO mice. The CysLT1 and CysLT2 receptors were primarily localized in neurons, microglia and neutrophils; those localized in microglia and neutrophils were enhanced in AQP4-KO mice.ConclusionAQP4 may play an inhibitory role in postischemic inflammation.