Blockade of the swelling-induced chloride current attenuates the mouse neonatal hypoxic-ischemic brain injury in vivo.

Activation of swelling-induced Cl- current (ICl,swell) during neonatal hypoxia-ischemia (HI) may induce brain damage. Hypoxic-ischemic brain injury causes chronic neurological morbidity in neonates as well as acute mortality. In this study, we investigated the role of ICl,swell in hypoxic-ischemic brain injury using a selective blocker, 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl) oxybutyric acid (DCPIB). In primary cultured cortical neurons perfusion of a 30% hypotonic solution activated ICl,swell, which was completely blocked by the application of DCPIB (10 µmol/L). The role of ICl,swell in neonatal hypoxic-ischemic brain injury in vivo was evaluated in a modified neonatal hypoxic-ischemic brain injury model. Before receiving the ischemic insult, the mouse pups were injected with DCPIB (10 mg/kg, ip). We found that pretreatment with DCPIB significantly reduced the brain damage assessed using TTC staining, Nissl staining and whole brain imaging, and improved the sensorimotor and vestibular recovery outcomes evaluated in neurobehavioural tests (i.e. geotaxis reflex, and cliff avoidance reflex). These results show that DCPIB has neuroprotective effects on neonatal hypoxic-ischemic brain injury, and that the ICl,swell may serve as a therapeutic target for treatment of hypoxic-ischemic encephalopathy.

Transient receptor potential melastatin 2 channels (TRPM2) mediate neonatal hypoxic-ischemic brain injury in mice.

Transient receptor potential melastatin 2 (TRPM2), a calcium-permeable non-selective cation channel, is reported to mediate brain damage following ischemic insults in adult mice. However, the role of TRPM2 channels in neonatal hypoxic-ischemic brain injury remains unknown. We hypothesize that TRPM2+/- and TRPM2-/- neonatal mice have reduced hypoxic-ischemic brain injury. To study the effect of TRPM2 on neonatal brain damage, we used 2,3,5-triphenyltetrazolium chloride (TTC) staining to assess the infarct volume and whole brain imaging to assess morphological changes in the brain. In addition, we also evaluated neurobehavioral outcomes for sensorimotor function 7days following hypoxic-ischemic brain injury. We report that the infarct volumes were significantly smaller and behavioral outcomes were improved in both TRPM2+/- and TRPM2-/- mice compared to that of wildtype mice. Next, we found that TRPM2-null mice showed reduced dephosphorylation of GSK-3ß following hypoxic ischemic injury unlike sham mice. TRPM2+/- and TRPM2-/- mice also had reduced activation of astrocytes and microglia in ipsilateral hemispheres, compared to wildtype mice. These findings suggest that TRPM2 channels play an essential role in mediating hypoxic-ischemic brain injury in neonatal mice. Genetically eliminating TRPM2 channels can provide neuroprotection against hypoxic-ischemic brain injury and this effect is elicited in part through regulation of GSK-3ß.

GSK-3ß inhibitor TDZD-8 reduces neonatal hypoxic-ischemic brain injury in mice.

AIMS: Glycogen synthase kinase 3ß (GSK-3ß) is activated following hypoxic-ischemic (HI) brain injury. TDZD-8 is a specific GSK-3ß inhibitor. Currently, the impact of inhibiting GSK-3ß in neonatal HI injury is unknown. We aimed to investigate the effect of TDZD-8 following neonatal HI brain injury. METHODS: Unilateral common carotid artery ligation followed by hypoxia was used to induce HI injury in postnatal day 7 mouse pups pretreated with TDZD-8 or vehicle. The infarct volume, whole-brain imaging, Nissl staining, and behavioral tests were used to evaluate the protective effect of TDZD-8 on the neonatal brain and assess functional recovery after injury. Western blot was used to evaluate protein levels of phosphorylated protein kinase B (Akt), GSK-3ß, and cleaved caspase-3. Protein levels of cleaved caspase-3, neuronal marker, and glial fibrillary acidic protein were detected through immunohistochemistry. RESULTS: Pretreatment with TDZD-8 significantly reduced brain damage and improved neurobehavioral outcomes following HI injury. TDZD-8 reversed the reduction of phosphorylated Akt and GSK-3ß, and the activation of caspase-3 induced by hypoxia-ischemia. In addition, TDZD-8 suppressed apoptotic cell death and reduced reactive astrogliosis. CONCLUSION: TDZD-8 has the therapeutic potential for hypoxic-ischemic brain injury in neonates. The neuroprotective effect of TDZD-8 appears to be mediated through its antiapoptotic activity and by reducing astrogliosis.

Tideglusib, a chemical inhibitor of GSK3ß, attenuates hypoxic-ischemic brain injury in neonatal mice.

BACKGROUND: Hypoxia-ischemia is an important cause of brain injury and neurological morbidity in the newborn infants. The activity of glycogen synthase kinase-3ß (GSK-3ß) is up-regulated following neonatal stroke. Tideglusib is a GSK-3ß inhibitor which has neuroprotective effects against neurodegenerative diseases in clinical trials. However, the effect of tideglusib on hypoxic-ischemic (HI) brain injury in neonates is still unknown. METHODS: Postnatal day 7 (P7) mouse pups subjected to unilateral common carotid artery ligation followed by 1h of hypoxia or sham surgery was performed. HI animals were administered tideglusib (5mg/kg) or vehicle intraperitoneally 20min prior to the onset of ischemia. The brain infarct volume and whole brain images, were used in conjunction with Nissl staining to evaluate the protective effects of tideglusib. Protein levels of glial fibrillary acidic protein (GFAP), Notch1, cleaved caspase-3/9, phosphorylated signal transducer and activator of transcription 3 (STAT3), GSK-3ß and protein kinase B (Akt) were detected to identify potentially involved molecules. RESULTS: Tideglusib significantly reduced cerebral infarct volume at both 24h and 7days after HI injury. Tideglusib also increased phosphorylated GSK-3ß(Ser9) and Akt(Ser473), and reduced the expression of GFAP and p-STAT3(Tyr705). In addition, pretreatment with tideglusib also enhanced the protein level of Notch1. Moreover, tideglusib reduced the cleavage of pro-apoptotic signal caspase proteins, including caspase 3 and caspase 9 following HI. CONCLUSION: These results indicate that tideglusib shows neuroprotection against hypoxic-ischemic brain injury in neonatal mice. GENERAL SIGNIFICANCE: Tideglusib is a potential compound for the prevention or treatment of hypoxic-ischemic brain injury in neonates.

Xyloketal B suppresses glioblastoma cell proliferation and migration in vitro through inhibiting TRPM7-regulated PI3K/Akt and MEK/ERK signaling pathways.

Glioblastoma, the most common and aggressive type of brain tumors, has devastatingly proliferative and invasive characteristics. The need for finding a novel and specific drug target is urgent as the current approaches have limited therapeutic effects in treating glioblastoma. Xyloketal B is a marine compound obtained from mangrove fungus Xylaria sp. (No. 2508) from the South China Sea, and has displayed antioxidant activity and protective effects on endothelial and neuronal oxidative injuries. In this study, we used a glioblastoma U251 cell line to (1) explore the effects of xyloketal B on cell viability, proliferation, and migration; and (2) investigate the underlying molecular mechanisms and signaling pathways. MTT assay, colony formation, wound healing, western blot, and patch clamp techniques were employed. We found that xyloketal B reduced cell viability, proliferation, and migration of U251 cells. In addition, xyloketal B decreased p-Akt and p-ERK1/2 protein expressions. Furthermore, xyloketal B blocked TRPM7 currents in HEK-293 cells overexpressing TRPM7. These effects were confirmed by using a TRPM7 inhibitor, carvacrol, in a parallel experiment. Our findings indicate that TRPM7-regulated PI3K/Akt and MEK/ERK signaling is involved in anti-proliferation and migration effects of xyloketal B on U251 cells, providing in vitro evidence for the marine compound xyloketal B to be a potential drug for treating glioblastoma.