Vascular endothelial growth factor (VEGF)-C and its receptor, vascular endothelial growth factor receptor (VEGFR)-3, are responsible for lymphangiogenesis in both embryos and adults. In epilepsy, the expression of VEGF-C and VEGFR-3 was significantly upregulated in the human brains affected with temporal lobe epilepsy. Moreover, pharmacologic inhibition of VEGF receptors after acute seizures could suppress the generation of spontaneous recurrent seizures, suggesting a critical role of VEGF-related signaling in epilepsy. Therefore, in the present study, the spatiotemporal expression of VEGF-C and VEGFR-3 against pilocarpine-induced status epilepticus (SE) was investigated in C57BL/6N mice using immunohistochemistry. At 1 day after SE, hippocampal astrocytes and microglia were activated. Pyramidal neuronal death was observed at 4 days after SE. In the subpyramidal zone, VEGF-C expression gradually increased and peaked at 7 days after SE, while VEGFR-3 was significantly upregulated at 4 days after SE and began to decrease at 7 days after SE. Most VEGF-C/VEGFR-3-expressing cells were pyramidal neurons, but VEGF-C was also observed in some astrocytes in sham-manipulated animals. However, at 4 days and 7 days after SE, both VEGFR-3 and VEGF-C immunoreactivities were observed mainly in astrocytes and in some microglia of the stratum radiatum and lacunosum-moleculare of the hippocampus, respectively. These data indicate that VEGF-C and VEGFR-3 can be upregulated in hippocampal astrocytes and microglia after pilocarpine-induced SE, providing basic information about VEGF-C and VEGFR-3 expression patterns following acute seizures.

The present study investigated the neuroprotective effects of anthocyanins extracted from black soybean (cv. Cheongja 3, Glycine max (L.) MERR.) seed coat against oxygen-glucose deprivation (OGD) and glutamate-induced cell death in rat primary cortical neurons. Lactate dehydrogenase (LDH) release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assays were employed to assess cell membrane damage and viability of primary neurons, respectively. OGD-induced cell death in 7 d in vitro primary cortical neurons was found to be OGD duration-dependent, and approximately 3.5 h of OGD resulted in ≈60% cell death. Treatment with black soybean anthocyanins dose-dependently prevented membrane damage and increased the viability of primary neurons that were exposed to OGD. Glutamateinduced neuronal cell death was dependent on the glutamate concentration at relatively low concentrations and the number of days the cells remained in culture. Interestingly, black soybean anthocyanins did not protect against glutamate-induced neuronal cell death. They did, however, inhibit the excessive generation of reactive oxygen species (ROS) and preserve mitochondrial membrane potential (MMP) in primary neurons exposed to OGD. In agreement with the neuroprotective effect of crude black soybean anthocyanins, purified cyanidin-3-glucoside (C3G), the major component of anthocyanins, also offered dose-dependent neuroprotection against OGD-induced neuronal cell death. Moreover, black soybean C3G markedly prevented excessive generation of ROS and preserved MMP in primary neurons that were exposed to OGD. Collectively, these results suggest that the neuroprotection of primary rat cortical neurons by anthocyanins that were extracted from black soybean seed coat might be mediated through oxidative stress inhibition and MMP preservation but not through glutamate-induced excitotoxicity attenuation.

Aspirin (acetylsalicylic acid) is one of the most widely used therapeutic agents based on its pharmacological actions, including anti-inflammatory, analgesic, anti-pyretic, and anti-thrombotic effects. In this study, we investigated the effects of aspirin on seizure susceptibility and hippocampal neuropathology following pilocarpine-induced status epilepticus (SE). SE was induced by pilocarpine hydrochloride (280 mg/kg, i.p.) administration in C57BL/6 mice (aged 8 weeks). Aspirin was administered daily (15 mg/kg or 150 mg/kg, i.p.) for 10 days starting 3 days before SE, continuing until 6 days after SE. After pilocarpine injection, SE onset time and mortality were recorded. Neuronal cell death was examined using cresyl violet and Fluoro-Jade staining, and glial responses were observed 7 days post SE using immunohistochemistry. In the aspirin-treated group, the onset time of SE was significantly shortened and mortality was markedly increased compared to the control group. However, in this study, aspirin treatment did not affect SE-induced neuronal cell death or astroglial and microglial responses in the hippocampus. In conclusion, these results suggest that the safety of aspirin should be reevaluated in some patients, especially with neurological disorders such as temporal lobe epilepsy.

The Bcl-2-interacting death suppressor (Bis) protein is involved in antiapoptosis and antistress pathways. However, its roles after neonatal hypoxia-ischemia remain obscure. Therefore, we investigated the effects of Bis deletion on hippocampal cell death following neonatal hypoxia-ischemia. We transected the right common carotid artery of bis(+/+) and bis(-/-) mice at postnatal Day 7 and subjected them to hypoxia for 35 min. Cresyl violet staining showed that hypoxia-ischemia induced progressive cell death in the hippocampi of bis(+/+) mice. Moreover, Bis was expressed in astrocytes, not microglia, in sham-manipulated hippocampi of bis(+/+) mice, and was markedly enhanced after hypoxia-ischemia. Immunoblotting showed that Bis expression significantly increased 3 and 7 days following hypoxia-ischemia. Unexpectedly, 7 days after hypoxia-ischemia, the number of hippocampal NeuN-positive cells was higher in the bis(-/-) mice than in the bis(+/+) mice. We subsequently performed transcriptomic analysis and quantitative real time polymerase chain reaction to search for the underlying genes responsible for resistance to hypoxia-ischemia in the bis(-/-) hippocampus. These studies showed that 6 h after hypoxia-ischemia, galectin 3 and filamin C levels increased to a lesser extent in the bis(-/-) hippocampi compared with the bis(+/+) hippocampi. Finally, our in vitro hypoxia-ischemia model, using A172 glioma cells and primary astrocytes, showed that downregulation of Bis blocked the enhanced expression of galectin 3 after oxygen-glucose deprivation. This study demonstrated that Bis was upregulated in the astrocytes after hypoxia-ischemia. In addition, we showed that hippocampal neurons are less vulnerable to hypoxia-ischemia in mice lacking Bis, possibly because of the modulation of galectin 3 induction.

Many neurodevelopmental disorders feature learning and memory difficulties. Regulation of neurite outgrowth during development is critical for neural plasticity and memory function. Here, we show a novel regulator of neurite outgrowth during cortical neurogenesis, Lin28, which is an RNA-binding protein. Persistent Lin28 upregulation by in utero electroporation at E14.5 resulted in neurite underdevelopment during cortical neurogenesis. We also showed that Lin28-overexpressing cells had an attenuated response to excitatory inputs and altered membrane properties including higher input resistance, slower action potential repolarization, and smaller hyperpolarization-activated cation currents, supporting impaired neuronal functionality in Lin28-electroporated mice. When we ameliorated perturbed Lin28 expression by siRNA, Lin28-induced neurite underdevelopment was rescued with reduction of Lin28-downstream molecules, high mobility group AT-Hook 2, and insulin-like growth factor 1 receptor. Finally, Lin28-electroporated mice showed significant memory deficits as assessed by the Morris water maze test. Taken together, these findings demonstrate a new role and the essential requirement of Lin28 in developmental control of neurite outgrowth, which has an impact on synaptic plasticity and spatial memory. These findings suggest that targeting Lin28 may attenuate intellectual disabilities by correction of impaired dendritic complexity, providing a novel therapeutic candidate for treating neurodevelopmental disorders.