Vascular endothelial growth factor promotes transdifferentiation of astrocytes into neurons via activation of the MAPK/Erk-Pax6 signal pathway.

Reactive astrocytes can be transformed into new neurons. Vascular endothelial growth factor (VEGF) promotes the transformation of reactive astrocytes into neurons in ischemic brain. Therefore, in this study, the molecular mechanism of VEGF's effect on ischemia/hypoxia-induced astrocyte to neuron transformation was investigated in the models of rat middle cerebral artery occlusion (MCAO) and in astrocyte culture with oxygen and glucose deprivation (OGD). We found that VEGF enhanced ischemia-induced Pax6, a neurogenic fate determinant, expression and Erk phosphorylation in reactive astrocytes and reduced infarct volume of rat brain at 3 days after MCAO, which effects could be blocked by administration of U0126, a MAPK/Erk inhibitor. In cultured astrocytes, VEGF also enhanced OGD-induced Erk phosphorylation and Pax6 expression, which was blocked by U0126, but not wortmannin, a PI3K/Akt inhibitor, or SB203580, a MAPK/p38 inhibitor, suggesting VEGF enhanced Pax6 expression via activation of MAPK/Erk pathway. OGD induced the increase of miR365 and VEGF inhibited the increase of OGD-induced miR365 expression. However, miR365 agonists blocked VEGF-enhanced Pax6 expression in hypoxic astrocytes, but did not block VEGF-enhanced Erk phosphorylation. We further found that VEGF promoted OGD-induced astrocyte-converted to neuron. Interestingly, both U0126 and Pax6 RNAi significantly reduced enhancement of VEGF on astrocytes-to-neurons transformation, as indicated Dcx and MAP2 immunopositive signals in reactive astrocytes. Moreover, those transformed neurons become mature and functional. We concluded that VEGF enhanced astrocytic neurogenesis via the MAPK/Erk-miR-365-Pax6 signal axis. The results also indicated that astrocytes play important roles in the reconstruction of neurovascular units in brain after stroke.

BMECs Ameliorate High Glucose-Induced Morphological Aberrations and Synaptic Dysfunction via VEGF-Mediated Modulation of Glucose Uptake in Cortical Neurons.

It has been demonstrated that diabetes cause neurite degeneration in the brain and cognitive impairment and neurovascular interactions are crucial for maintaining brain function. However, the role of vascular endothelial cells in neurite outgrowth and synaptic formation in diabetic brain is still unclear. Therefore, present study investigated effects of brain microvascular endothelial cells (BMECs) on high glucose (HG)-induced neuritic dystrophy using a coculture model of BMECs with neurons. Multiple immunofluorescence labelling and western blot analysis were used to detect neurite outgrowth and synapsis formation, and living cell imaging was used to detect uptake function of neuronal glucose transporters. We found cocultured with BMECs significantly reduced HG-induced inhibition of neurites outgrowth (including length and branch formation) and delayed presynaptic and postsynaptic development, as well as reduction of neuronal glucose uptake capacity, which was prevented by pre-treatment with SU1498, a vascular endothelial growth factor (VEGF) receptor antagonist. To analyse the possible mechanism, we collected BMECs cultured condition medium (B-CM) to treat the neurons under HG culture condition. The results showed that B-CM showed the same effects as BMEC on HG-treated neurons. Furthermore, we observed VEGF administration could ameliorate HG-induced neuronal morphology aberrations. Putting together, present results suggest that cerebral microvascular endothelial cells protect against hyperglycaemia-induced neuritic dystrophy and restorate neuronal glucose uptake capacity by activation of VEGF receptors and endothelial VEGF release. This result help us to understand important roles of neurovascular coupling in pathogenesis of diabetic brain, providing a new strategy to study therapy or prevention for diabetic dementia. Hyperglycaemia induced inhibition of neuronal glucose uptake and impaired to neuritic outgrowth and synaptogenesis. Cocultured with BMECs/B-CM and VEGF treatment protected HG-induced inhibition of glucose uptake and neuritic outgrowth and synaptogenesis, which was antagonized by blockade of VEGF receptors. Reduction of glucose uptake may further deteriorate impairment of neurites outgrowth and synaptogenesis.

Vascular endothelial growth factor increases the function of calcium-impermeable AMPA receptor GluA2 subunit in astrocytes via activation of protein kinase C signaling pathway.

Astrocytic calcium signaling plays pivotal roles in the maintenance of neural functions and neurovascular coupling in the brain. Vascular endothelial growth factor (VEGF), an original biological substance of vessels, regulates the movement of calcium and potassium ions across neuronal membrane. In this study, we investigated whether and how VEGF regulates glutamate-induced calcium influx in astrocytes. We used cultured astrocytes combined with living cell imaging to detect the calcium influx induced by glutamate. We found that VEGF quickly inhibited the glutamate/hypoxia-induced calcium influx, which was blocked by an AMPA receptor antagonist CNQX, but not D-AP5 or UBP310, NMDA and kainate receptor antagonist, respectively. VEGF increased phosphorylation of PKCα and AMPA receptor subunit GluA2 in astrocytes, and these effects were diminished by SU1498 or calphostin C, a PKC inhibitor. With the pHluorin assay, we observed that VEGF significantly increased membrane insertion and expression of GluA2, but not GluA1, in astrocytes. Moreover, siRNA-produced knockdown of GluA2 expression in astrocytes reversed the inhibitory effect of VEGF on glutamate-induced calcium influx. Together, our results suggest that VEGF reduces glutamate-induced calcium influx in astrocytes via enhancing PKCα-mediated GluA2 phosphorylation, which in turn promotes the membrane insertion and expression of GluA2 and causes AMPA receptors to switch from calcium-permeable to calcium-impermeable receptors, thereby inhibiting astrocytic calcium influx. The present study reveals that excitatory neurotransmitter glutamate-mediated astrocytic calcium influx can be regulated by vascular biological factor via activation of AMPA receptor GluA2 subunit and uncovers a novel coupling mechanism between astrocytes and endothelial cells within the neurovascular unit.

Endothelial cells promote excitatory synaptogenesis and improve ischemia-induced motor deficits in neonatal mice.

Brain microvascular endothelial cells (BMEC) are highly complex regulatory cells that communicate with other cells in the neurovascular unit. Cerebral ischemic injury is known to produce detectable synaptic dysfunction. This study aims to investigate whether endothelial cells in the brain regulate postnatal synaptic development and to elucidate their role in functional recovery after ischemia. Here, we found that in vivo engraftment of endothelial cells increased synaptic puncta and excitatory postsynaptic currents in layers 2/3 of the motor cortex. This pro-synaptogenic effect was blocked by the depletion of VEGF in the grafted BMEC. The in vitro results showed that BMEC conditioned medium enhanced spine and synapse formation but conditioned medium without VEGF had no such effects. Moreover, under pathological conditions, transplanted endothelial cells were capable of enhancing angiogenesis and synaptogenesis and improved motor function in the ischemic injury model. Collectively, our findings suggest that endothelial cells promote excitatory synaptogenesis via the paracrine factor VEGF during postnatal development and exert repair functions in hypoxia-ischemic neonatal mice. This study highlights the importance of the endothelium-neuron interaction not only in regulating neuronal development but also in maintaining healthy brain function.

Soluble cpg15 from Astrocytes Ameliorates Neurite Outgrowth Recovery of Hippocampal Neurons after Mouse Cerebral Ischemia.

The present study focuses on the function of cpg15, a neurotrophic factor, in ischemic neuronal recovery using transient global cerebral ischemic (TGI) mouse model and oxygen-glucose deprivation (OGD)-treated primary cultured cells. The results showed that expression of cpg15 proteins in astrocytes, predominantly the soluble form, was significantly increased in mouse hippocampus after TGI and in the cultured astrocytes after OGD. Addition of the medium from the cpg15-overexpressed astrocytic culture into the OGD-treated hippocampal neuronal cultures reduces the neuronal injury, whereas the recovery of neurite outgrowths of OGD-injured neurons was prevented when cpg15 in the OGD-treated astrocytes was knocked down, or the OGD-treated-astrocytic medium was immunoadsorbed by cpg15 antibody. Furthermore, lentivirus-delivered knockdown of cpg15 expression in mouse hippocampal astrocytes diminishes the dendritic branches and exacerbates injury of neurons in CA1 region after TGI. In addition, treatment with inhibitors of MEK1/2, PI3K, and TrkA decreases, whereas overexpression of p-CREB, but not dp-CREB, increases the expression of cpg15 in U118 or primary cultured astrocytes. Also, it is observed that the Flag-tagged soluble cpg15 from the astrocytes transfected with Flag-tagged cpg15-expressing plasmids adheres to the surface of neuronal bodies and the neurites. In conclusion, our results suggest that the soluble cpg15 from astrocytes induced by ischemia could ameliorate the recovery of the ischemic-injured hippocampal neurons via adhering to the surface of neurons. The upregulated expression of cpg15 in astrocytes may be activated via MAPK and PI3K signal pathways, and regulation of CREB phosphorylation.SIGNIFICANCE STATEMENT Neuronal plasticity plays a crucial role in the amelioration of neurological recovery of ischemic injured brain, which remains a challenge for clinic treatment of cerebral ischemia. cpg15 as a synaptic plasticity-related factor may participate in promoting the recovery process; however, the underlying mechanisms are still largely unknown. The objective of this study is to reveal the function and mechanism of neuronal-specific cpg15 expressed in astrocytes after ischemia induction, in promoting the recovery of injured neurons. Our findings provided new mechanistic insight into the neurological recovery, which might help develop novel therapeutic options for cerebral ischemia via astrocytic-targeting interference of gene expression.

MicroRNA-365 modulates astrocyte conversion into neuron in adult rat brain after stroke by targeting Pax6.

Reactive astrocytes induced by ischemia can transdifferentiate into mature neurons. This neurogenic potential of astrocytes may have therapeutic value for brain injury. Epigenetic modifications are widely known to involve in developmental and adult neurogenesis. PAX6, a neurogenic fate determinant, contributes to the astrocyte-to-neuron conversion. However, it is unclear whether microRNAs (miRs) modulate PAX6-mediated astrocyte-to-neuron conversion. In the present study we used bioinformatic approaches to predict miRs potentially targeting Pax6, and transient middle cerebral artery occlusion (MCAO) to model cerebral ischemic injury in adult rats. These rats were given striatal injection of glial fibrillary acidic protein targeted enhanced green fluorescence protein lentiviral vectors (Lv-GFAP-EGFP) to permit cell fate mapping for tracing astrocytes-derived neurons. We verified that miR-365 directly targets to the 3'-UTR of Pax6 by luciferase assay. We found that miR-365 expression was significantly increased in the ischemic brain. Intraventricular injection of miR-365 antagomir effectively increased astrocytic PAX6 expression and the number of new mature neurons derived from astrocytes in the ischemic striatum, and reduced neurological deficits as well as cerebral infarct volume. Conversely, miR-365 agomir reduced PAX6 expression and neurogenesis, and worsened brain injury. Moreover, exogenous overexpression of PAX6 enhanced the astrocyte-to-neuron conversion and abolished the effects of miR-365. Our results demonstrate that increase of miR-365 in the ischemic brain inhibits astrocyte-to-neuron conversion by targeting Pax6, whereas knockdown of miR-365 enhances PAX6-mediated neurogenesis from astrocytes and attenuates neuronal injury in the brain after ischemic stroke. Our findings provide a foundation for developing novel therapeutic strategies for brain injury.

[VEGF enhances reconstruction of neurovascular units in the brain after injury].

Vascular endothelial growth factor (VEGF) was originally recognized as a substance predominantly with vascular permeability and angiogenesis. Recently, more and more evidence indicated that VEGF is expressed in the neurons of the developing and adult brains. Functional investigation demonstrated that VEGF shows several important effects on the neuronal development and physiological function. For example, VEGF accelerates the development of neurons and neural dendritic and axon growth. Besides, VEGF directly and acutely regulates the functions of multiple ion channels of the neuron membrane and changes neural excitability. In traumatic or ischemic injured brains, VEGF produces neuroprotection, enhances capacity of adult neurogenesis and transformation of astroglial cells into new neurons, which are fundamental basis for re-establishment of neural network. Based on the knowledge obtained from the literatures, we propose that VEGF may play very important roles in neural plasticity in the normal brain, and the reconstruction of neurovascular units and neural repair in the traumatic injured brain. This review mainly focuses on neural activity and repair roles of VEGF in adult mammalian brains. Further study on the mechanism of VEGF's neurobiological effects in the brain will be helpful for understanding the regulation of brain functions and developing new therapeutic strategy for prevention of neurodegeneration of the brain.

Striatal astrocytes transdifferentiate into functional mature neurons following ischemic brain injury.

To determine whether reactive astrocytes stimulated by brain injury can transdifferentiate into functional new neurons, we labeled these cells by injecting a glial fibrillary acidic protein (GFAP) targeted enhanced green fluorescence protein plasmid (pGfa2-eGFP plasmid) into the striatum of adult rats immediately following a transient middle cerebral artery occlusion (MCAO) and performed immunolabeling with specific neuronal markers to trace the neural fates of eGFP-expressing (GFP(+)) reactive astrocytes. The results showed that a portion of striatal GFP(+) astrocytes could transdifferentiate into immature neurons at 1 week after MCAO and mature neurons at 2 weeks as determined by double staining GFP-expressing cells with ßIII-tubulin (GFP(+)-Tuj-1(+)) and microtubule associated protein-2 (GFP(+)-MAP-2(+)), respectively. GFP(+) neurons further expressed choline acetyltransferase, glutamic acid decarboxylase, dopamine receptor D2-like family proteins, and the N-methyl-D-aspartate receptor subunit R2, indicating that astrocyte-derived neurons could develop into cholinergic or GABAergic neurons and express dopamine and glutamate receptors on their membranes. Electron microscopy analysis indicated that GFP(+) neurons could form synapses with other neurons at 13 weeks after MCAO. Electrophysiological recordings revealed that action potentials and active postsynaptic currents could be recorded in the neuron-like GFP(+) cells but not in the astrocyte-like GFP(+) cells, demonstrating that new GFP(+) neurons possessed the capacity to fire action potentials and receive synaptic inputs. These results demonstrated that striatal astrocyte-derived new neurons participate in the rebuilding of functional neural networks, a fundamental basis for brain repair after injury. These results may lead to new therapeutic strategies for enhancing brain repair after ischemic stroke.

Splicing factor NSSR1 reduces neuronal injury after mouse transient global cerebral ischemia.

This study focuses on the function of NSSR1, a splicing factor, in neuronal injury in the ischemic mouse brain using the transient global cerebral ischemic mouse model and the cultured cells treated with oxygen-glucose deprivation (OGD). The results showed that the cerebral ischemia triggers the expression of NSSR1 in hippocampal astrocytes, predominantly the dephosphorylated NSSR1 proteins, and the Exon3 inclusive NCAM-L1 variant and the Exon4 inclusive CREB variant. While in the hippocampus of astrocyte-specific NSSR1 conditional knockdown (cKD) mice, where cerebral ischemia no longer triggers NSSR1 expression in astrocytes, the expression of Exon3 inclusive NCAM-L1 variant and Exon4 inclusive CREB variant were no longer triggered as well. In addition, the injury of hippocampal neurons was more severe in astrocyte-specific NSSR1 cKD mice compared with in wild-type mice after brain ischemia. Of note, the culture media harvested from the astrocytes with overexpression of NSSR1 or the Exon3 inclusive NCAM-L1 variant, or Exon4 inclusive CREB variant were all able to reduce the neuronal injury induced by OGD. The results provide the evidence demonstrating that: (1) Splicing factor NSSR1 is a new factor involved in reducing ischemic injury. (2) Ischemia induces NSSR1 expression in astrocytes, not in neurons. (3) NSSR1-mediated pathway in astrocytes is required for reducing ischemic neuronal injury. (4) NCAM-L1 and CREB are probably mediators in NSSR1-mediated pathway. In conclusion, our results suggest for the first time that NSSR1 may provide a novel mechanism for reducing neuronal injury after ischemia, probably through regulation on alternative splicing of NCAM-L1 and CREB in astrocytes.

Regulation of RAGE splicing by hnRNP A1 and Tra2ß-1 and its potential role in AD pathogenesis.

The receptor for advanced glycation end products (RAGE) gene expresses two major alternative splicing isoforms, full-length membrane-bound RAGE (mRAGE) and secretory RAGE (esRAGE). Both isoforms play important roles in Alzheimer's disease (AD) pathogenesis, either via interaction of mRAGE with ß-amyloid peptide (Aß) or inhibition of the mRAGE-activated signaling pathway. In the present study, we showed that heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and Transformer2ß-1 (Tra2ß-1) were involved in the alternative splicing of mRAGE and esRAGE. Functionally, two factors had an antagonistic effect on the regulation. Glucose deprivation induced an increased ratio of mRAGE/esRAGE via up-regulation of hnRNP A1 and down-regulation of Tra2ß-1. Moreover, the ratios of mRAGE/esRAGE and hnRNP A1/Tra2ß-1 were increased in peripheral blood mononuclear cells from AD patients. The results provide a molecular basis for altered splicing of mRAGE and esRAGE in AD pathogenesis. The receptor for advanced glycation end products (RAGE) gene expresses two major alternative splicing isoforms, membrane-bound RAGE (mRAGE) and secretory RAGE (esRAGE). Both isoforms play important roles in Alzheimer's disease (AD) pathogenesis. Mechanism for imbalanced expression of these two isoforms in AD brain remains elusive. We proposed here a hypothetic model to illustrate that impaired glucose metabolism in AD brain may increase the expression of splicing protein hnRNP A1 and reduce Tra2ß-1, which cause the imbalanced expression of mRAGE and esRAGE.

Diabetes-induced central neuritic dystrophy and cognitive deficits are associated with the formation of oligomeric reticulon-3 via oxidative stress.

Diabetes is a high risk factor to dementia. To investigate the molecular mechanism of diabetic dementia, we induced type 2 diabetes in rats and examined potential changes in their cognitive functions and the neural morphology of the brains. We found that the diabetic rats with an impairment of spatial learning and memory showed the occurrence of RTN3-immunoreactive dystrophic neurites in the cortex. Biochemical examinations revealed the increase of a high molecular weight form of RTN3 (HW-RTN3) in diabetic brains. The corresponding decrease of monomeric RTN3 was correlated with the reduction of its inhibitory effects on the activity of ß-secretase (BACE1), a key enzyme for generation of ß-amyloid peptides. The results from immunoprecipitation combined with protein carbonyl detection showed that carbonylated RTN3 was significantly higher in cortical tissues of diabetic rats compared with control rats, indicating that diabetes-induced oxidative stress led to RTN3 oxidative damage. In neuroblastoma SH-SY5Y cells, high glucose and/or H2O2 treatment significantly increased the amounts of carbonylated proteins and HW-RTN3, whereas monomeric RTN3 was reduced. Hence, we conclude that diabetes-induced cognitive deficits and central neuritic dystrophy are correlated with the formation of aggregated RTN3 via oxidative stress. We provided the first evidence that oxidative damage caused the formation of toxic RTN3 aggregates, which participated in the pathogenesis of central neuritic dystrophy in diabetic brain. Present findings may offer a new therapeutic strategy to prevent or reduce diabetic dementia.

Alleviating sleep disturbances and modulating neuronal activity after ischemia: Evidence for the benefits of zolpidem in stroke recovery.

AIMS: Sleep disorders are prevalent among stroke survivors and impede stroke recovery, yet they are still insufficiently considered in the management of stroke patients, and the mechanisms by which they occur remain unclear. There is evidence that boosting phasic GABA signaling with zolpidem during the repair phase improves stroke recovery by enhancing neural plasticity; however, as a non-benzodiazepine hypnotic, the effects of zolpidem on post-stroke sleep disorders remain unclear. METHOD: Transient ischemic stroke in male rats was induced with a 30-minute middle cerebral artery occlusion. Zolpidem or vehicle was intraperitoneally delivered once daily from 2 to 7 days after the stroke, and the electroencephalogram and electromyogram were recorded simultaneously. At 24 h after ischemia, c-Fos immunostaining was used to assess the effect of transient ischemic stroke and acute zolpidem treatment on neuronal activity. RESULTS: In addition to the effects on reducing brain damage and mitigating behavioral deficits, repeated zolpidem treatment during the subacute phase of stroke quickly ameliorated circadian rhythm disruption, alleviated sleep fragmentation, and increased sleep depth in ischemic rats. Immunohistochemical staining showed that in contrast to robust activation in para-infarct and some remote areas by 24 h after the onset of focal ischemia, the activity of the ipsilateral suprachiasmatic nucleus, the biological rhythm center, was strongly suppressed. A single dose of zolpidem significantly upregulated c-Fos expression in the ipsilateral suprachiasmatic nucleus to levels comparable to the contralateral side. CONCLUSION: Stroke leads to suprachiasmatic nucleus dysfunction. Zolpidem restores suprachiasmatic nucleus activity and effectively alleviates post-stroke sleep disturbances, indicating its potential to promote stroke recovery.

New striatal neurons form projections to substantia nigra in adult rat brain after stroke.

Previous studies have demonstrated that newborn striatal neurons can functionally integrate with local neural networks in adult rat brain after injury. In the present study, we determined whether these newly generated striatal neurons can develop projections to the substantia nigra, a target of striatal projection neurons. We used 5'-bromodeoxyuridine (BrdU) and a retroviral vector expressing green fluorescent protein (GFP) combined with multiple immunostaining labels of newborn striatal neurons, and nigral microinjection of fluorogold (FG) to trace the striatonigral projection in adult rat brain at different weeks following a transient middle cerebral artery occlusion (MCAO). We found that FG positive (FG(+)) cells could be detected in newly generated neurons (BrdU(+)-NeuN(+) and GFP(+)-NeuN(+)) in ipsilateral striatum clearly at 12, but not 2 weeks after MCAO. The data suggest that ischemia-induced newborn striatal projection neurons could form long axons that targeted the substantia nigra (striatonigral projection pathway) and that have intact axonal transport from the nerve terminal to cell body. These new striatal neurons express glutamate NR2 and dopamine D2L receptors, which form the molecular basis for responding to the inputs from cortical glutamatergic and nigral dopaminergic projection neurons. Our data provide the first morphological evidence that newborn neurons in the striatum, a non-neurogenic region, can establish new striatonigral neural circuits, important pathways for the maintenance of motor function. These results help us to understand endogenous cellular mechanisms of brain repair, and suggest that increasing adult neurogenesis could be a practical strategy for enhancing the efficacy of rehabilitative therapy in stroke patients.

Neuroprotective Effect of Angiopoietin2 Is Associated with Angiogenesis in Mouse Brain Following Ischemic Stroke.

Angiogenic factors play an important role in protecting, repairing, and reconstructing vessels after ischemic stroke. In the brains of transient focal cerebral ischemic mice, we observed a reduction in infarct volume after the administration of Angiopoietin 2 (Angpt2), but whether this process is promoted by Angpt2-induced angiogenesis has not been fully elaborated. Therefore, this study explored the angiogenic activities, in reference to CD34 which is a marker of activated ECs and blood vessels, of cultured ECs in vitro and in ischemic damaged cerebral area in mice following Angpt2 administration. Our results demonstrate that Angpt2 administration (100 ng/mL) is neuroprotective by significantly increasing the CD34 expression in in vitro-cultured ECs, reducing the infarct volume and mitigating neuronal loss, as well as enhancing CD34+ vascular length and area. In conclusion, these results indicate that Angpt2 promotes repair and attenuates ischemic injury, and that the mechanism of this is closely associated with angiogenesis in the brain after stroke.

Melatonin supplementation in the subacute phase after ischemia alleviates postischemic sleep disturbances in rats.

BACKGROUND: Sleep disorders are highly prevalent among stroke survivors and impede stroke recovery. It is well established that melatonin has neuroprotective effects in animal models of ischemic stroke. However, as a modulator of endogenous physiological circadian rhythms, the effects of melatonin on poststroke sleep disorders remain unclear. In the present study, we investigated how melatonin delivered intraperitoneally once daily in the subacute phase after stroke onset, influencing neuronal survival, motor recovery, and sleep-wake profiles in rats. METHODS: Transient ischemic stroke in male Sprague-Dawley rats was induced with 30 min occlusion of the middle cerebral artery. Melatonin or vehicle was delivered intraperitoneally once daily in the subacute phase, from 2 to 7 days after stroke. Electroencephalogram and electromyogram recordings were obtained simultaneously. RESULTS: Compared to the effects observed in the vehicle-treated ischemic group, after 6 daily consecutive treatment of melatonin at 10 mg/kg starting at ischemic/reperfusion day 2, the infarct volume was significantly decreased (from 39.6 to 26.2%), and the degeneration of axons in the ipsilateral striatum and the contralateral corpus callosum were significantly alleviated. Sensorimotor performances were obviously improved as evidenced by significant increases in the latency to falling off the wire and in the use of the impaired forelimb. In addition to those predictable results of reducing brain tissue damage and mitigating behavioral deficits, repeated melatonin treatment during the subacute phase of stroke also alleviated sleep fragmentation through reducing sleep-wake stage transitions and stage bouts, together with increasing stage durations. Furthermore, daily administration of melatonin at 9 a.m. significantly increased the nonrapid eye movement sleep delta power during both the light and dark periods and decreased the degree of reduction of the circadian index. CONCLUSIONS: Melatonin promptly reversed ischemia-induced sleep disturbances. The neuroprotective effects of melatonin on ischemic injury may be partially associated with its role in sleep modulation.

[The physiological and pathological significance of epigenetic regulation in neural development].

Epigenetics refers to changes in gene expression that do not entail a change in DNA sequence, namely phenotype changed, other than the genotype. These changes may remain through cell division for the remainder of the cell's life and may also last for multiple generations. It contains DNA methylation, histone modification, chromatin remodeling, gene imprinting and so on. Methylation is one of the chief epigenetic modify patterns in genomic DNA. DNA methylation via the interaction of methylation-CpG-binding proteins will affect the gene expression. Furthermore, the dysfuction of epigenetic mechanisms may lead to multiple genetic neurodevelopmental disorders and degenerative diseases. Here we mainly rewiewed the physiological and pathological significance of epigenetics regulations in neural development and neurodegeneration.

Vascular endothelial growth factor acutely reduces calcium influx via inhibition of the Ca2+ channels in rat hippocampal neurons.

Vascular endothelial growth factor (VEGF) protects neurons against ischemic injury. An overload of intracellular calcium ions (Ca(2+)) caused by the excessive release of glutamate is widely considered to be one of the molecular mechanisms of ischemic neuronal death. In the present study, we investigated whether VEGF could modulate the activity of Ca(2+) channels on the neuronal membrane. We used the Fluo-3 image method assisted by confocal laser scan microscopy to detect any Ca(2+) influx in primary cultured hippocampal neurons. Whole-cell patch-clamp techniques were used to record the activity of the high-voltage-activated (HVA) Ca(2+) currents in the CA1 pyramidal neurons of hippocampal slices that were freshly prepared from neonatal brains of rats. The results obtained from the Fluo-3 image experiments showed that VEGF pretreatment of cultured neurons at a final concentration of 50, 100, or 200 ng/ml acutely and dose dependently attenuated the Ca(2+) influx induced by application of KCl (60 mM) or glutamate (50 microM). This effect was blocked by SU1498, an antagonist of Flk-1 VEGF receptor. The influx of Ca(2+) returned to basal levels after removal of VEGF. Furthermore, electrophysiological recording data showed that VEGF could acutely reduce the amplitudes of the HVA Ca(2+) currents in a dose- and voltage-dependent manner. The HVA Ca(2+) currents also returned to the levels of the control after removal of VEGF from the system. Taken together, the results obtained from the present study demonstrated that VEGF specifically reduced the influx of Ca(2+) via the inhibitory activity of the HVA Ca(2+) channels in hippocampal neurons.

Necrostatin-1 Prevents Necroptosis in Brains after Ischemic Stroke via Inhibition of RIPK1-Mediated RIPK3/MLKL Signaling.

Pharmacological studies have indirectly shown that necroptosis participates in ischemic neuronal death. However, its mechanism has yet to be elucidated in the ischemic brain. TNFα-triggered RIPK1 kinase activation could initiate RIPK3/MLKL-mediated necroptosis under inhibition of caspase-8. In the present study, we performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia in rats and used immunoblotting and immunostaining combined with pharmacological analysis to study the mechanism of necroptosis in ischemic brains. In the ipsilateral hemisphere, we found that ischemia induced the increase of (i) RIPK1 phosphorylation at the Ser166 residue (p-RIPK1), representing active RIPK1 kinase and (ii) the number of cells that were double stained with P-RIPK1 (Ser166) (p-RIPK1+) and TUNEL, a label of DNA double-strand breaks, indicating cell death. Furthermore, ischemia induced activation of downstream signaling factors of RIPK1, RIPK3 and MLKL, as well as the formation of mature interleukin-1ß (IL-1ß). Treatment with necrostatin-1 (Nec-1), an inhibitor of necroptosis, significantly decreased ischemia-induced increase of p-RIPK1 expression and p-RIPK1+ neurons, which showed protection from brain damage. Meanwhile, Nec-1 reduced RIPK3, MLKL and p-MLKL expression levels and mature IL-1ß formation in Nec-1 treated ischemic brains. Our results clearly demonstrated that phosphorylation of RIPK1 at the Ser166 residue was involved in the pathogenesis of necroptosis in the brains after ischemic injury. Nec-1 treatment protected brains against ischemic necroptosis by reducing the activation of RIPK1 and inhibiting its downstream signaling pathways. These results provide direct in vivo evidence that phosphorylated RIPK1 (Ser 166) plays an important role in the initiation of RIPK3/MLKL-dependent necroptosis in the pathogenesis of ischemic stroke in the rodent brain.

MicroRNA-365 Knockdown Prevents Ischemic Neuronal Injury by Activating Oxidation Resistance 1-Mediated Antioxidant Signals.

MicroRNA-365 (miR-365) is upregulated in the ischemic brain and is involved in oxidative damage in the diabetic rat. However, it is unclear whether miR-365 regulates oxidative stress (OS)-mediated neuronal damage after ischemia. Here, we used a transient middle cerebral artery occlusion model in rats and the hydrogen peroxide-induced OS model in primary cultured neurons to assess the roles of miR-365 in neuronal damage. We found that miR-365 exacerbated ischemic brain injury and OS-induced neuronal damage and was associated with a reduced expression of OXR1 (Oxidation Resistance 1). In contrast, miR-365 antagomir alleviated both the brain injury and OXR1 reduction. Luciferase assays indicated that miR-365 inhibited OXR1 expression by directly targeting the 3'-untranslated region of Oxr1. Furthermore, knockdown of OXR1 abolished the neuroprotective and antioxidant effects of the miR-365 antagomir. Our results suggest that miR-365 upregulation increases oxidative injury by inhibiting OXR1 expression, while its downregulation protects neurons from oxidative death by enhancing OXR1-mediated antioxidant signals.

Functional integration of newly generated neurons into striatum after cerebral ischemia in the adult rat brain.

BACKGROUND AND PURPOSE: Ischemic injury can induce neurogenesis in the striatum. Those newborn neurons can express glutamic acid decarboxylase and choline acetyltransferase, markers of GABAergic and cholinergic neurons, respectively. The present study investigated whether these GABAergic and cholinergic new neurons could differentiate into functional cells. METHODS: Retrovirus containing the EGFP gene was used to label dividing cells in striatal slices prepared from adult rat brains after middle cerebral artery occlusion. EGFP-targeted immunostaining and immunoelectron microscopy were performed to detect whether newborn neurons could anatomically form neuronal polarity and synapses with pre-existent neurons. Patch clamp recording on acute striatal slices of brains at 6 to 8 weeks after middle cerebral artery occlusion was used to determine whether the newborn neurons could display functional electrophysiological properties. RESULTS: EGFP-expressing (EGFP(+)) signals could be detected mainly in the cell body in the first 2 weeks. From the fourth to thirteenth weeks after their birth, EGFP(+) neurons gradually formed neuronal polarity and showed a time-dependent increase in dendrite length and branch formation. EGFP(+) cells were copositive for NeuN and glutamic acid decarboxylase (EGFP(+)-NeuN(+)-GAD(67)(+)), MAP-2, and choline acetyltransferase (EGFP(+)-MAP-2(+)-ChAT(+)). They also expressed phosphorylated synapsin I (EGFP(+)-p-SYN(+)) and showed typical synaptic structures comprising dendrites and spines. Both GABAergic and cholinergic newborn neurons could fire action potentials and received excitatory and inhibitory synaptic inputs because they displayed spontaneous postsynaptic currents in picrotoxin- and CNQX-inhibited manners. CONCLUSIONS: Ischemia-induced newly formed striatal GABAergic and cholinergic neurons could become functionally integrated into neural networks in the brain of adult rats after stroke.