Search

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12264-019-00371-y) contains supplementary material, which is available to authorized users.

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.

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.

Abnormal expression and dysfunction of methyl-CpG binding protein 2 (MeCP2) cause Rett syndrome (RTT). The diverse phosphorylation modifications modulate MeCP2 function in neural cells. Using western blot and immunohistochemistry, we examined the expression patterns of MeCP2 and three phospho-MeCP2s (pMeCP2s) in the developing rat brain. The expression of MeCP2 and phospho-S80 (pS80) MeCP2 increased while pS421 MeCP2 and pS292 MeCP2 decreased with brain maturation. In contrast to the nuclear localization of MeCP2 and pS80 MeCP2, pS421 MeCP2 and pS292 MeCP2 were mainly expressed in the cytoplasmic compartment. Apart from their distribution in neurons, they were also detected at a low level in astrocytes. Postnatally-initiated MeCP2 deficiency affected cerebellar neural cell development, as determined by the abnormal expression of GFAP, DCX, Tuj1, MAP-2, and calbindin-D28k. Together, these results demonstrate that MeCP2 and diverse pMeCP2s have distinct features of spatio-temporal expression in the rat brain, and that the precise levels of MeCP2 in the postnatal period are vital to cerebellar neural cell development.

Taraxasterol is an effective component of dandelion that has anti-inflammatory effects in vivo and in vitro. The present study was performed to explore whether taraxasterol exhibits a protective effect against rheumatoid arthritis through the modulation of inflammatory responses in mice. Eight-week-old CCR9-deficient mice were injected with a collagen II monoclonal antibody cocktail to create a rheumatoid arthritis model. In the experimental group, arthritic model mice were treated with 10 mg/kg taraxasterol once per day for 5 days. Treatment with taraxasterol significantly increased the pain thresholds and reduced the clinical arthritic scores of the mice in the experimental group compared with those of the model group. Furthermore, treatment with taraxasterol significantly suppressed tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and nuclear factor-κB protein expression levels compared with those in the rheumatoid arthritis model mice. Taraxasterol treatment also significantly reduced nitric oxide, prostaglandin E2 and cyclooxygenase-2 levels compared with those in the rheumatoid arthritis model group. These observations indicate that the protective effect of taraxasterol against rheumatoid arthritis is mediated via the modulation of inflammatory responses in mice.

Glioma stem cells belong to a special subpopulation of glioma cells that are characterized by strong proliferation, invasion and drug resistance capabilities. Magnetic nanoparticles are nanoscale biological materials with magnetic properties. In this study, CD133(+) primary glioma stem cells were isolated from patients and cultured. Then, magnetic nanoparticles were used to mediate the transfection and expression of a microRNA-374a overexpression plasmid in the glioma stem cells. Transmission electron microscopy detected the presence of significant magnetic nanoparticle substances within the CD133(+) glioma stem cells after transfection. The qRT-PCR and Northern blot results showed that the magnetic nanoparticles could be used to achieve the transfection of the microRNA-374a overexpression plasmid into glioma stem cells and the efficient expression of mature microRNA-374a. The MTT and flow cytometry results showed that the proliferation inhibition rate was significantly higher in cells from the microRNA-374a transfection group than in cells from the microRNA-mut transfection group; additionally, the former cells presented significant cell cycle arrest. The Transwell experiments confirmed that the overexpression of microRNA-374a could significantly reduce the invasiveness of CD133(+) glioma stem cells. Moreover, the high expression of microRNA-374a mediated by the magnetic nanoparticles effectively reduced the tumourigenicity of CD133(+) glioma stem cells in nude mice. The luciferase assays revealed that mature microRNA-374a fragments could bind to the 3'UTR of Neuritin (NRN1), thereby interfering with Neuritin mRNA expression. The qRT-PCR and Western blotting results showed that the overexpression of microRNA-374a significantly reduced the expression of genes such as NRN1, CCND1, CDK4 and Ki67 in glioma stem cells. Thus, magnetic nanoparticles can efficiently mediate the transfection and expression of microRNA expression plasmids in mammalian cells. The overexpression of microRNA-374a can effectively silence NRN1 expression, thereby inhibiting the proliferation, invasion and in vivo tumourigenicity of human glioma stem cells.

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 βIII‐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. GLIA 2015;63:1660–1670

Purpose Astrocytes can be reactivated after cerebral ischemia by expressing nestin and other characteristic markers of neural stem cells (NSCs). However, the epigenetic features of reactive astrocytes are not well known. Methyl-CpG-binding protein 2 (MeCP2) is a vital transcriptional modulator in brain development. Although the expression and function of some phosphorylated MeCP2 isoforms have been clarified, phospho-serine 292 (pS292) MeCP2 has not yet drawn much attention. In this study, we used western blot analysis and immunohistochemical and immunofluorescent staining to reveal the expressive features of pS292 MeCP2 and MeCP2 in the adult rat striatum following transient middle cerebral artery occlusion (MCAO). Results We first discovered that the ischemia-induced expression of cytoplasmic pS292 MeCP2 is primarily accumulated in nestin-positive reactive astrocytes in the stroke-injured striatum. Moreover, the enhancement of astrocytic pS292 MeCP2 was correlated with the augmentation of VEGF in astrocytes, as determined by the substantial co-localization of pS292 MeCP2 and VEGF after stroke. Finally, the exogenous overproduction of VEGF further promoted the expression of pS292 MeCP2 in reactive astrocytes, and this effect was accompanied by a marked increase in reactive astrocytes. On the contrary, MeCP2 was predominantly expressed in the neuronal nucleus, and the level of this protein was not significantly altered after ischemic injury and VEGF overproduction. Conclusion Our data provide the first demonstration that overexpression of VEGF enhances the accumulation of pS292 MeCP2 in reactive astrocytes in the ischemic-injured rat striatum, implicating a pS292 MeCP2-related epigenetic role of exogenous VEGF in reactive astrocytes following 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.

Axon-transport plays an important role in neuronal activity and survival. Reduced endogenous VEGF can cause neuronal damage and axon degeneration. It is unknown at this time if VEGF can be transported within the axon or whether it can be released by axonal depolarization. We transfected VEGF-eGFP plasmids in cultured hippocampal neurons and tracked their movement in the axons by live-cell confocal imaging. Then, we co-transfected phVEGF-eGFP and kinesin-1B-DsRed vectors into neurons and combined with immunoprecipitation and two-color imaging to study the mechanism of VEGF axon-trafficking. We found that VEGF vesicles morphologically co-localized and biochemically bounded with kinesin-1B, as well as co-trafficked with it in the axons. Moreover, the capacity for axonal trafficking of VEGF was reduced by administration of nocodazole, an inhibitor of microtubules, or kinesin-1B shRNA. In addition, we found that VEGF could release from the cultured neurons under acute depolarizing stimulation with potassium chloride. Therefore, present findings suggest that neuronal VEGF is stored in the vesicles, actively released, and transported in the axons, which depends on the presence of kinesin-1B and functional microtubules. These results further help us to understand the importance of neuronal VEGF in the maintenance of neuronal activity and survival throughout life.

In this file, the influence of the concentration of additive MPS on the Nuclei Population Density of copper electrocrystallization on a glass carbon electrode (GCE) with different potentials has been studied. The exponential curve has been used to fit test data. From this function, the most nuclei population density of copper electrocrystallization can be estimated under certain experimental condition. From analysis results, we can see that with different potentials, the nuclei population density has different limitations. When the potential is about-0.8 Volt, the largest value of the limitation of the nuclei population density can be achieved.

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.

Influences of the concentration of additive PEG and overpotential on the Nuclei population density of copper electrocrystallization on a glass carbon electrode (GCE) have been studied in this file. We characterized the experimental data extracted from Li’s work [10] with the least square method and exponential curves. The relationship of nucleation number density and overpotential follows the exponential function basically has been obtained by carrying on the data fitting to experimental data. And with a given overpotential, when inject PEG into acidic cupric sulphate electrolyte with a lower concentration, nucleation number density is reduced, but when the injection concentration of PEG is high enough, nucleation number density is increased instead. While with a certain concentration of PEG, as the overpotential more negative, the nuclei population density increased gradually, but if the injection concentration is low, the change of the growth rate of nucleation number density is not significant.

This study examined the effect of neuron-endothelial coupling on the survival of neurons after ischemia and the possible mechanism underlying that effect. Whole-cell patch-clamp experiments were performed on cortical neurons cultured alone or directly cocultured with brain microvascular endothelial cells (BMEC). Propidium iodide (PI) and NeuN staining were performed to examine neuronal death following oxygen and glucose deprivation (OGD). We found that the neuronal transient outward potassium currents (IA) decreased in the coculture system, whereas the outward delayed-rectifier potassium currents (IK) did not. Sodium nitroprusside, a NO donor, enhanced BMEC-induced IA inhibition and nitro-l-arginine methylester, a NOS inhibitor, partially prevented this inhibition. Moreover, the neurons directly cocultured with BMEC showed more resistance to OGD-induced injury compared with the neurons cultured alone, and that neuroprotective effect was abolished by treatment with NS5806, an activator of the IA. These results indicate that vascular endothelial cells assist neurons to prevent hypoxic injury via inhibiting neuronal IA by production of NO in the direct neuron-BMEC coculture system. These results further provide direct evidence of functional coupling between neurons and vascular endothelial cells. This study clearly demonstrates that vascular endothelial cells play beneficial roles in the pathophysiological processes of neurons after hypoxic injury, suggesting that the improvement of neurovascular coupling or functional remodeling may become an important therapeutic target for preventing brain injury.

Objective It has been reported that B-cell lymphoma 2 (Bcl-2) enhances neurogenesis as well as supporting axonal growth after injury. In the present study, we investigated whether Bcl-2 overexpression plays a role in the formation of newborn striatonigral projection neurons in the adult rat brain after transient middle cerebral artery occlusion (MCAO).

Traveling wave ultrasonic motor (TWUSM) is driven by friction force between stator and rotor. As the friction materials of contact layer in traveling wave ultrasonic motor(TWUSM), tribological properties of Ekonol composites(EK2) have important effects on motor driving characteristics and service life. Then EK2 was stuck on the stator tooth of 40 type disc-shape TWUSM. Under different driven mode and friction combination, tribological properties of EK2 was tested，and the wear morphologies of EK2 surfaces are observed by SEM. Results show that there are ultrasonic antifriction phenomenon in stator/rotor contact interface under ultrasonic drive. Ultrasonic fatigue is the main wear mechanism of contact layer. When Ek2 combine with Cu rotor， contact layer can obtain bigger friction coefficient, higher output torque and better wear properties. Then Ek2 and Cu rotor is a good friction combination which can satisfy the actual running needs of TWUSM.

Traveling wave ultrasonic motor (TWUSM) is driven by friction force between stator and rotor. Then friction material properties are very important on the output characteristics of TWUSM. Under the guidance of an optimization design principle of friction material, a kind of Ekonol composite friction material (EK2) was developed by modification study, orthogonal experimental design and regression analysis. Through the comparison between EK2 and other common friction materials, it was proved that the TWUSM with EK2 can get better running stability, higher output torque and longer lifetime. Therefore, EK2 is a good friction material which can satisfy the practical running needs of TWUSM.

Carbon fiber(CF) and Kevlar fiber(KF) were used as reinforcing agents to fill Ekonol(EK) respectively. Mechanical and tribological properties of composite systems were tested. When CF was used as the EK reinforced filler, the mechanical properties of EK composites couldn’t be enhanced obviously, and the tribological properties were reduced. When KF was filled in EK matrix, it could greatly improve the impact strength of EK composite by reducing crack formation and inhibiting crack propagation. The KF/EK composite has good tribological properties. It keeps the high friction low wear performance advantage of EK, and solves its problem of forming difficulty. In EK composite, KF plays the role of reinforcing agents and friction modifier at the same time, and then it is a kind of excellent EK reinforced filler.

Traveling wave ultrasonic motor (TWUSM) is driven by friction force between stator and rotor, so friction coefficient is an important parameter of contact interface. In fact, there have mutual influence and restriction in various parameters of contact interface. For a comprehensive study on friction coefficient, the interaction between friction coefficient and other parameters should be considered. Base on a new contact model with visco-elastic stator contact layer of TWUSM，coupling effects of two parameters on motor output characteristics were simulated with MATLAB. According to the simulation results, a design principle of friction coefficient was present. A kind of Ekonol composite friction material (EK2) was developed under the guidance of this principle. Comparing with other common friction material of TWUSM, it was proved that motor with EK2 can get better running stability, higher output torque and longer lifetime. Therefore, EK2 is a good friction material which can satisfy the actual running needs of TWUSM.

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-1β (IL-1β). 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-1β 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.

To determine if DNA excision repair enzymes oxoguanine glycosylase 1 (OGG1) and xeroderma pigmentosum group F protein (XPF) are involved in the pathogenesis of Parkinson's disease (PD) in a cell model.PC12 cells were treated with 1-Methyl-4-phenylpyridine ion (MPP(+)) for various periods of time to induce oxidative DNA damage. MTT assay was used to determine cell viability. Immunocytochemistry with antibody against 8-hydroxy-2'-deoxyguanosine (8-oxodG) was used to evaluate oxidative DNA damage. Immunoblotting was used to detect the protein levels of OGG1 and XPF.MPP(+) treatment (1 mmol/L) for 18 h and 24 h reduced cell viability to 78.6% and 70.3% of the control, respectively, in a time-dependent way. MPP(+) increased the immunoreactivity of 8-oxodG in the cytoplasm at 3 h and in the nucleus at 24 h of treatment. With the treatment of MPP(+), the expression of OGG1 was significantly increased at 1 h, reaching a peak at 3 h, and then it was decreased at 24 h, as compared to that with vehicle treatment. The same effect was exerted on XPF level, except that the XPF level reached a peak at 18 h of MPP(+) treatment. Moreover, the maximally-increased protein level of OGG1 by MPP(+) was approximately 2-fold higher than that of XPF.MPP(+) treatment could time-dependently induce increases in OGG1 and XPF expressions in PC12 cells. Also, this study indicates that the base and nucleotide excision repair pathways may be compensatory activated in the early stage of pathogenesis in the cells after MPP(+) treatment.

To study the effects of excision repair cross-complementing 1 (ERCC1) on the pathophysiological process of brain ischemia, we examined the changes in ERCC1 expression, as well as the functional significance of ERCC1 in the rat brain following middle cerebral artery occlusion (MCAO). The results were as follows: (1) ERCC1 immunopositive cells were widely distributed in various brain regions. ERCC1 expression was localized to the nuclei of neurons and astrocytes. (2) ERCC1 expression, as determined by western blot, increased at 3 days, remaining until 14 days, in the ipsilateral cortex and striatum following MCAO. Immunohistochemical analysis demonstrated that ischemia induced increased ERCC1 expression within the periinfarct core, with increasingly less expression toward the core. (3) Knockdown of ERCC1 expression by intraventricular injection of antisense plasmids increased DNA damage and infarct volume in the ischemic brain. (4) ERCC1 overproduction, by injection of expression plasmids, significantly reduced infarct volume and the accumulation of DNA-damaged neurons. Taken together, these results indicate that both endogenous ERCC1 and exogenous ERCC1 have an important neuroprotective function in the brain. In addition, administration of ERCC1 to the brain could prove to be a successful strategy for neuronal protection against ischemic injury.

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.

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+-GAD67+), 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.Conclusion—Ischemia-induced newly formed striatal GABAergic and cholinergic neurons could become functionally integrated into neural networks in the brain of adult rats after stroke.

Beta-catenin, a protein that functions in both cell adhesion and Wnt signaling, plays vital roles in mammalian neural development. To investigate the roles of beta-catenin in stroke-induced neurogenesis, we injected beta-catenin siRNA into ipsilateral ischemic lateral ventricle. We found that inactivation of beta-catenin by siRNA caused the decline of beta-catenin in the ischemic striatum, enlarged stroke-induced infarct volume, reduced SVZ expansion, and inhibited striatal neurogenesis in adult rat brain following a transient middle cerebral artery occlusion (tMCAO). These results show that beta-catenin-mediated transcriptional activation functions in the decision of subventricular zone precursors to proliferate or differentiate during stroke-induced striatal neurogenesis, and suggest that the signaling activity of beta-catenin may control the production of newborn neurons and thus regulate the autonomous repair in the striatum after ischemia.

In this research, we investigated striatal neurogenesis in 3-, 6-, 12-, and 18-month-old rats after cerebral ischemic injury. All rats were subjected to a 20-min middle cerebral artery occlusion (MCAO), given 5′-bromodeoxyuridine (BrdU, 30 mg/kg, i.p.) once daily during days 4–7 and sacrificed 2 weeks after MCAO. Neurogenesis was assessed with double immunohistochemical/immunofluorescence labeling of BrdU and doublecortin (DCX), microtubule-associated protein 2 (MAP-2), or 67-kDa glutamic acid decarboxylase (GAD 67 ). In 6-, 12-, and 18-month-old rats, the numbers of nestin + , BrdU + –DCX + (a marker of newborn neuronal progenitors/immature neuron), BrdU + –MAP-2 + (a marker of newborn mature neuron), and BrdU + –GAD 67 + (a marker of newborn GABAergic neuron) cells decreased dramatically in the ipsilateral striatum to MCAO compared with that in 3-month-old rats. The results indicated that stroke-induced striatal neurogenesis still existed in aging rats. However, the capacity of neurogenesis in older rats was considerably lower than that in young adults. Meanwhile, the apoptosis of neural precursors and immature neurons, indicated by double labeling of active caspase-3 and nestin/DCX/Tuj-1(β-tubulin III)/CRMP-4 (collapsin response-mediated protein-4), increased noticeably in the ipsilateral striatum of older rats. Taken together, the results suggested that aging-related attenuation of ischemia-induced striatal neurogenesis might be related to decrease of neural precursors and increase of apoptosis of newborn neurons.

Accumulating evidence indicates that the aberrant re-entry of post-mitotic neurons into the G2/M phase of cell cycle and the resulting mitotic catastrophe may contribute to the pathogenesis of Alzheimer's disease. However, the cellular event that drives the differentiated neurons to abnormally enter G2/M phase remains elusive. Similarly, whether mitotic catastrophe is indeed one of the death pathways for differentiated neurons is not clear. Previous studies revealed that okadaic acid (OA), a phosphatase inhibitor that induces AD like pathological changes, evokes mitotic changes in neuroblastoma cells. In this study, we examined the in vivo effects of OA on cyclin B1 expression, the induction of mitosis, and subsequent mitotic catastrophe. We found that cyclin B1 expression in adult neurons was significantly increased after injecting OA into rat frontal cortex, which also increased tau protein phosphorylation. Interestingly, cyclin B1 and phosphorylated tau were well co-localized around the OA injection site, but were only partially co-localized in other brain regions. Staining with toluidine blue, Giemsa dye or propidium iodide revealed typical mitotic and mitotic catastrophe-like morphological changes with irregular arrangement of condensed chromatin and chromosome fibers in a few cells. Furthermore, the strong cyclin B1 staining in these cells suggests that cyclin B1 promoted G2 to M phase transition is required for the mitotic catastrophe. The detection of neuron-specific enolase in a portion of these cells demonstrated that at least part them are neuron. All together, our results suggest that the disturbance of the protein kinase-phosphatase system caused by OA is sufficient to induce neuronal cyclin B1 expression, force neurons into the mitotic phase of cell cycle, and cause mitotic catastrophe.

Inhibition of Qo site of mitochondrial complex III under hypoxia has received attention, but its downstream pathways remain unclear. We used Qo site inhibitor myxothiazol to mimic the inhibition of the Qo site of complex III and studied the effects of the inhibition of this site on persistent and transient sodium currents and neuron excitability in rat hippocampal CA1 cells. The results showed myxothiazol apparently increased persistent sodium currents but with a weak effect on transient sodium currents; the effect of myxothiazol on persistent sodium currents was blocked by protein kinase C inhibitor and superoxide scavengers, but not by hydrogen peroxide scavenger and hydroxyl radical formation inhibitor; myxothiazol could increase the activity of protein kinase C and neuron excitability. These results suggest that the inhibition of Qo site of mitochondrial complex III increases persistent sodium currents via superoxide production and protein kinase C activation.

Differential sensitivity of GABAergic and glycinergic inputs to orexin-A in preganglionic cardiac vagal neurons of newborn rats

The effects of melatonin on the mitochondrial DNA (mtDNA) damage induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine ion (MPP(+)) were investigated both in vivo and in vitro. MPTP (24 mg/kg, s.c.) induced a rapid increase in the immunoreactivity of 8-hydroxyguanine (8-oxoG), a common biomarker of DNA oxidative damage, in the cytoplasm of neurons in the Substantia Nigra Compact of mouse brain. Melatonin preinjection (7.5, 15 or 30 mg/kg, i.p.) dose-dependently prevented MPTP-induced DNA oxidative damage. In SH-SY5Y cells, MPP(+) (1 mm) increased the immunoreactivity of 8-oxoG in the mitochondria at 1 hr and in the nucleus at 3 hr after treatment. Melatonin (200 microm) preincubation significantly attenuated MPP(+)-induced mtDNA oxidative damage. Furthermore, MPP(+) time-dependently increased the accumulation of mitochondrial oxygen free radicals (mtOFR) from 1 to 24 hr and gradually decreased the mitochondrial membrane potential (Psim) from 18 to 36 hr after incubation. At 72 hr after incubation, MPP(+) caused cell death in 49% of the control. However, melatonin prevented MPP(+)-induced mtOFR generation and Psim collapse, and later cell death. The present results suggest that cytoprotection of melatonin against MPTP/MPP(+)-induced cell death may be associated with the attenuation of mtDNA oxidative damage via inhibition of mtOFR generation and the prevention of Psim collapse.

Hypoxia-induced inhibition of Qi site of mitochondrial complex III under hypoxia has received attention, but its downstream pathways remain unclear. In this paper, we used Qi site inhibitor antimycin A to mimic the inhibition of the Qi site of mitochondrial complex III and studied the effects of the inhibition of this site on persistent sodium currents, transient sodium currents, and neuronal excitability in rat hippocampal CA1 cells with whole cell patch-clamp methods. The results showed that antimycin A decreased the amplitude of both persistent and transient sodium currents; antioxidant 2-mercaptopropionylglycine or 1,10 phenanthroline abolished the effect of antimycin A; the complex III Qo site inhibitor stigmatellin, the protein kinase C inhibitor chelerythrine, but not the protein kinase A inhibitor H89, canceled the effect of antimycin A; antimycin A decreased the amplitude of both persistent and transient sodium currents only at more depolarized membrane potentials and the decrease percentage of both persistent and transient sodium currents after antimycin A at potentials above -50 mV increased with the change in potentials toward more depolarized direction; exogenous application of H2O2 inhibited the amplitude of both persistent and transient sodium currents; the amount of current required to trigger spikes was increased and the number of spikes produced by varying levels of currents was decreased by antimycin A. These results suggest that the inhibition of Qi site of mitochondrial complex III decreases both persistent and transient sodium currents via reactive oxygen species and protein kinase C in rat hippocampal CA1 cells.

The present paper studied the effect and mechanism of neurosteroid pregnenolone sulfate (PREGS) on spontaneous glutamate release using electrophysiological and biochemical methods combined with a pharmacological approach. The results suggested that PREGS had a selective enhancing effect on spontaneous glutamate release in the prelimbic cortex and the hippocampus but not in the striatum. The effect of PREGS in the prelimbic cortex appeared to be via modulation of alpha1-adrenergic and sigma1 receptors, but in the hippocampus it might be dependent on sigma1 receptors only. The activation of alpha1-adrenergic receptors synergized sigma1 receptor activation in the prelimbic cortex. Intracellular calcium released from the endoplasmic reticulum, protein kinase C, adenylyl cyclase and protein kinase A played a key role in the effect of PREGS. Intracellular calcium, protein kinase C and adenylyl cyclase might be upstream events in the activation of protein kinase A after PREGS.

Cytochromec-initiated activation of apoptotic protease activating factor-1 (Apaf-1) is a key step in the mitochondrial-signaling pathway for the activation of death-executing caspases in apoptosis. This signaling pathway has been implicated in the pathophysiology of various neurological disorders, including ischemic brain injury. In this study, we have cloned a novel rat gene product, designated as Apaf-1-interacting protein (AIP), which functions as a dominant-negative inhibitor of the Apaf-1-caspase-9 pathway. AIP is constitutively expressed in the brain, but at substantially lower levels than Apaf-1 and caspase-9. AIP can directly bind to Apaf-1in vitrothrough its N-terminal caspase-recruiting domain, and this protein interaction was increased in cells undergoing apoptosis. Cytosolic extracts from cells overexpressing AIP were highly resistant to cytochromec- dATP-induced activation of caspase-9 and caspase-3. Gene transfection of AIP into cell lines, including the neuronal-differentiated PC12 cells, potently suppressed apoptosis induced by various pro-apoptotic stimuli. To further investigate the functional role of AIP in primary neurons and in the brain, an adeno-associated virus (AAV) vector carrying the AIP cDNA was constructed. AAV-mediated overexpression of AIP in primary cortical- hippocampal neurons markedly reduced cell death and caspase-3 activation triggered by protein kinase C inhibition, DNA damage, or oxygen- glucose deprivation. Moreover, intracerebral infusion of the AAV vector resulted in robust AIP expression in the hippocampus and significantly promoted CA1 neuronal survival after transient global cerebral ischemia. These results suggest that molecular targeting of the Apaf-1-caspase-9 signaling pathway may be a feasible neuroprotective strategy to enhance the endogenous threshold for caspase activation and prevent neuronal loss in stroke and related disorders.

In the present study, we investigated the effect of melatonin on the outward delayed rectifier potassium currents (IK) in CA1 pyramidal neurons of rat hippocampal slices using patch-clamp technique in whole-cell configuration. In a concentration-dependent manner, melatonin caused a reduction of IK with a half-maximal inhibitory concentration (IC50) of 3.75 mm. The inhibitory effect had rapid onset and was readily reversible. Melatonin shifted steady-state inactivation of IK in hyperpolarizing direction but did not alter its steady-state activation. Neither luzindole, an MT1/MT2 receptor antagonist, nor prazosin, an MT3 receptor antagonist, blocked melatonin-induced current reduction. The results indicate that melatonin-induced IK inhibition was not via activation of its own membrane receptors. 5-Hydroxytryptamine (5-HT), a melatonin precursor and an agonist of serotonin receptors, when it was given in pipette internal solution but not bath solution, produced a similar inhibitory effect to that of melatonin. Moreover, indole, a major component of melatonin, reversibly and dose dependently inhibited IK with an IC50 of 3.44 mm. Present results suggest that melatonin inhibits IK in hippocampal CA1 pyramidal neurons probably through its interaction with the intracellular indole-related domains of potassium channels.

We examined the effects of progesterone on frequency of miniature excitatory postsynaptic currents (mEPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs), and dopamine-induced increase in the frequency of sEPSCs in pyramidal cells of layers V-VI of the rat prelimbic cortex using whole-cell patch-clamp techniques in slices. The results showed that progesterone 100 microM had no effects on the frequency of mEPSCs and sEPSCs, but significantly inhibited dopamine-induced increase in frequency of sEPSCs. This was in contrast to the effect of progesterone on the effect of 5-HT, which showed no changes after progesterone. When studying the mechanism of the progesterone effect, we observed that GABA(A) receptor antagonist and progesterone receptor antagonist did not influence the effect of progesterone; progesterone had no effects on D1 receptor agonist, protein kinase A and protein kinase C activator-induced increase in the frequency of sEPSCs. Interestingly, sigma(1) receptor antagonist could inhibit the effect of dopamine and sigma(1) receptor agonist had a synergistic effect on the effect of D1 receptor agonist. These results suggest that progesterone may inhibit dopamine-induced increase in frequency of sEPSCs in rat prelimbic cortical neurons via inhibition of sigma(1)/D1 receptor synergism because progesterone has been known to be an antagonist of sigma(1) receptor.

Our studies observed that, consistent with the literature, ischemic/hypoxic insults increased the expression of voltage-gated potassium channel (Kv) 1.2 potassium channel as well as elevating the endogenous level of vascular endothelial growth factor (VEGF) in neurons of adult rat brain following middle cerebral artery occlusion and in SH-SY5Y cells after hypoxia and glucose deprivation. Concomitantly, we also observed that ischemic injury increased the tyrosine phosphorylation of Kv 1.2 in in vivo and in vitro; the introduction of exogenous VEGF could attenuate cell death in in vitro models. Furthermore, we found that the protective effect of VEGF is mediated through its up-regulative actions on the tyrosine phosphorylation of Kv 1.2, which in turn has a direct influence on cell viability after ischemic insult. In substantiation of this result, we used anti-sense methodology to suppress the expression of endogenous VEGF, which significantly inhibited the tyrosine phosphorylation of Kv 1.2 and increased cell death elicited by ischemic/hypoxic injury. Finally, the enhancement of the tyrosine phosphorylation of the channel by VEGF in neuronal cells was significantly attenuated in the presence of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), or genestin, an inhibitor of tyrosine kinase, thus suggesting that the phosphorylation of Kv 1.2 induced by VEGF is mechanistically linked to the PI3-K pathway.

Double-fluorescence staining was combined with confocal laser scanning microscopy to localize fetal liver kinase-1 (Flk-1) and fms-like tyrosine kinase-1 (Flt-1) in the neonatal rat brain. The results showed that Flk-1 and Flt-1 immunostaining was observed in the cells with neuron-specific enolase, a neuronal marker, and with factor VIII (F VIII), an endothelium marker, but not in cells with glial fibrillary acidic protein (GFAP), a glial marker, of brain sections from rats on postnatal day 7 (P7). This indicates that both vascular endothelial growth factor (VEGF) receptors were distributed in the neurons and the vascular endothelium. A regional analysis showed that Flt-1 was distributed most densely in the hippocampus, followed by the retrosplenial agranular cortex and the striatum, and Flk-1 was evenly distributed throughout the brain. In a comparison of the density of immunopositive staining neurons, Flt-1 was much higher than Flk-1 in most of the brain regions. A time-course analysis showed that both Flt-1 and Flk-1 were highly expressed in the cerebral vessel of rats on P1, P7, and P14, and then declined in adults, consistent with the development of angiogenesis in neonates. In the neurons, Flt-1 was highest in the cerebral cortex and hippocampus of P1-P14 rats, and then gradually decreased, whereas Flk-1 abruptly increased and reached its highest level in adults. The results suggest that Flt-1 and Flk-1 are expressed in the neurons with their individual time-dependent manners and regional distribution in the brain. However, the significance of the neuronal distribution of Flt-1 and Flk-1 remains to be determined.

RMP was reported to regulate transcription via competing with HBx to bind the general transcription factor IIB (TFIIB) and interacting with RPB5 subunit of RNA polymerase II as a corepressor of transcription regulator. However, our present research uncovered that RMP also regulates the transcription through interaction with the general transcription factors IIF (TFIIF), which assemble in the preinitiation complex and function in both transcription initiation and elongation. With in vitro pull-down assay and Far-Western analysis, we demonstrated that RMP could bind with bacterially expressed recombinant RAP30 and RAP74 of TFIIF subunits. In the immunoprecipitation assay in COS1 cells cotransfected with FLAG-tagged RMP or its mutants, GST-fused RAP30 and RAP74 were co-immunoprecipitated with RMP in approximately equal molar ratio, which suggests that RAP30 and RAP74 interact with RMP as a TFIIF complex. Interestingly both RAP30 and RAP74 interact with the same domain (D5) of the C-terminal RMP of 118-amino-acid residuals which overlaps with its TFIIB-binding domain. Internal deletion of D5 region of RMP abolished its binding ability with both subunits of TFIIF, while D5 domain alone was sufficient to interact with TFIIF subunits. The result of luciferase assay showed that overexpression of RMP, but not the mutant RMP lacking D5 region, suppressed the transcription activated by Gal-VP16, suggesting that interaction with TFIIF is required for RMP to suppress the activated transcription. The interaction between RMP and TFIIF may be an additional passway for RMP to regulate the transcription, or alternatively TFIIF may cooperate with RPB5 and TFIIB for the corepressor function of RMP.

We recently indicated that the vascular endothelial growth factor (VEGF) protects neurons against hypoxic death via enhancement of tyrosine phosphorylation of Kv1.2, an isoform of the delayed-rectifier potassium channels through activation of the phosphatidylinositol 3-kinase (PI3-K) signaling pathway. The present study investigated whether VEGF could attenuate ischemia-induced increase of the potassium currents in the hippocampal pyramidal neurons of rats after ischemic injury. Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) to induce brain ischemia. The whole-cell patch-clamp technique was used to record the potassium currents of hippocampal neurons in brain slices from the ischemically injured brains of the rats 24h after MCAO. We detected that transient MCAO caused a significant increase of voltage-gated potassium currents (Kv) and outward delayed-rectifier potassium currents (IK), but not outward transient potassium currents (IA), in the ipsilateral hippocampus compared with the sham. Moreover, we found that VEGF could acutely, reversibly and voltage-dependently inhibit the ischemia-induced IK increase. This inhibitory effect of VEGF could be completely abolished by wortmannin, an inhibitor of PI3-K. Our data indicate that VEGF attenuates the ischemia-induced increase of IK via activation of the PI3-K signaling pathway.

The antisense knockdown technique and confocal laser scanning microscopic analysis were used to elucidate vascular endothelial growth factor (VEGF) induction and its effect on DNA damage and repair in rat brain following a transient middle cerebral artery occlusion. Immunohistochemical study and in situ hybridization showed that the expression of VEGF and its mRNA was enhanced in the ischemic core and penumbra of ischemic brain. Western blot analysis further illustrated that VEGF induction was time-dependently changed in these areas. Double-staining analysis indicated that VEGF-positive staining existed in the neuron, but not in the glia, and it colocalized with excision repair cross-complementing group 6 (ERCC6) mRNA, a DNA repair factor. VEGF antisense oligodeoxynucleotide infusion reduced VEGF induction and resulted in an enlargement of infarct volume of the brain caused by ischemia. Moreover, it also increased the number of DNA damaged cells and lessened the induction of ERCC6 mRNA in ischemic brains. These results suggest that the induction of endogenous VEGF in ischemic neurons plays a neuroprotective role probably associated with the expression of ERCC6 mRNA.

In the present study, double fluorescence staining combined with confocal laser scanning microscopy analysis were used to examine the effects of melatonin on ischemia-induced neuronal DNA strand breaks and its possible mechanisms in a transient middle cerebral artery (MCA) occlusion model. Results showed that melatonin dose-dependently reduced infarct areas and decreased both DNA double and single strand breaks (DSB and SSB) and enhanced cell viability in the peri-ischemic brain regions. Furthermore, Bcl-2 induction in the ischemic brain was further enhanced by melatonin treatment. Double staining analysis indicated that the cells costained for Bcl-2 and TdT-mediated-deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL), a DSB marker, displayed a relative regular morphology compared with the cells only stained with TUNEL. Transient ischemia induced an expression of excision repair cross-complementing factor 6 (ERCC6) mRNA, a gene essential for the preferential repair of nuclear excision repair, in the injured neurons. Double labeling showed that ERCC6 only co-localized with proliferating cell nuclear antigen (PCNA), a member of the nuclear excision repair complex, but not with TUNEL. Melatonin further and statistical significantly up-regulated ERCC6 mRNA expression in the peri-ischemic region of rat brains. The results suggest that neuroprotection by melatonin against ischemic injury may be related to modulation of apoptosis and DNA repair capacity.

To clarify the relationship between melatonin's hydroxyl radical (*OH) scavenging ability and its protective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuronal injury, in the present study, the salicylate trapping method combined with high-performance liquid chromatography (HPLC)-electrochemical detection were used to measure the contents of dihydroxybenzoic acid (DHBA) and dopamine (DA) in brain tissues of C57BL/6 mice. Immunocytohistochemistry was used to detect tyrosine hydroxylase (TH)-like positive staining neurons. Results show that MPTP treatment induced an increase in the content of DHBA and decrease in the level of DA as well as the number of TH positive stained neurons in the mouse brain. However, melatonin dose-dependently inhibited the increase of DHBA levels in ventral midbrain tissues, the decrease of DA content and the loss of dopaminergic neurons. Moreover, the relationship between the changes of DHBA and DA levels in the brain of mice following MPTP and melatonin treatment showed a statistically significant negative correlation. Present results suggest that melatonin can ameliorate MPTP-induced dopaminergic neuronal lesions probably, at least partially, because of its inhibition of *OH generation.

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.

Background The neurotransmitter glutamate is involved in fast excitatory synaptic transmission in the mammalian brain. Glutamate released from presynaptic terminals must be removed rapidly from the synaptic cleft by high affinity, sodium-dependent glutamate transporters to keep the extracellular glutamate concentration low to protect neuron from glutamate excitotoxicity, which is the major pathological mechanism of brain ischemia. GLAST is one of the identified four subtypes of the glutamate transporter system and has been suggested to play an important role in some pathological conditions. But until recently, very little information existed the concerning relationship between GLAST expression and cerebral ischemia. Methods Nonradioactive in situ hybridization was employed to evaluate the changes of glutamate transporter GLAST mRNA expression in rat cerebral cortex and hippocampus following photochemically induced focal cortical ischemia. Results GLAST mRNA expression in cerebral pyramid cells below the infarcted area did not change at 3 h, significantly decreased at 12 h, recovered to the control level at 24 h, and significantly increased at 72 h following the ischemic lesion. No changes in GLAST mRNA expression were observed in all subfields of the hippocampal complex. Conclusions The present findings suggest that the time-course changes of GLAST mRNA expression after ischemia may be correlated with the pathogenesis of photosensitive ischemic brain damage. Anat. Rec. 251:9–14, 1998. © 1998 Wiley-Liss, Inc.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

N-methyl- d -aspartate (NMDA) receptor-mediated events have been implicated in the pathophysiology of posttraumatic spinal cord injury. In the present study, [ 3 H]MK801 was used to analyse the changes in NMDA receptor-binding sites in rat spinal cord after impact trauma at T9. In contrast to brain, which showed only a single binding site, spinal cord showed both high-affinity ( K d1 = 0.47 ± 0.24nM ) and low-affinity ( K d2 = 7.75 ± 1.82nM ) binding sites with relatively low binding density ( B max1 = 0.11 ± 0.04pmol/mg protein and B max2 = 0.84 ± 0.11pmol/mg protein ). Time-course studies demonstrated significant decreases in the binding of [ 3 H]MK801 at the thoracic and lumbar segments at 4 h after spinal cord injury with recovery by 24 h. Scatchard analyses indicate that these changes likely involve both high- and low-affinity binding sites. The transitory reduction in [ 3 H]MK801-binding after trauma may reflect downregulationn of NMDA receptors as a consequence of posttraumatic glutamate release and may serve to limit excitotoxin-induced injury.

Our previous experiments suggest that treatment with Bcl-2 increases proliferation and differentiation of neuronal progenitors induced by ischemic injury and ameliorates neurological functional deficits after stroke. However, in addition to its traditional anti-apoptotic effect, little is known about the concrete molecular modulation mechanism. In this study, Bcl-2-expressing plasmids were injected into the lateral ventricle of rat brains immediately following a 30-min occlusion of the middle cerebral artery to determine the role of Bcl-2 in adult neurogenesis. Bcl-2 overexpression reduced ischemic infarct and astrogenesis, and enhanced ischemia-induced striatal neurogenesis. We further found that Bcl-2 increased β-catenin, a key mediator of canonical Wnt/β-catenin signaling pathway, and reduced bone morphogenetic proteins-4 (BMP-4) expression in the ipsilateral striatum following ischemia. Treatment of stroke with β-catenin siRNA (i.c.v.) showed that β-catenin siRNA antagonized Bcl-2 neuroprotection against ischemic brain injury. More interestingly, β-catenin siRNA simultaneously abolished Bcl-2-mediated reduction of BMP-4 expression and enhancement of neurogenesis in the ipsilateral striatum. This effect is independent of Noggin, the known BMP antagonist. These findings highlight a new regulatory mechanism that Bcl-2 elevates ischemia-induced striatal neurogenesis by down-regulating expression of BMP-4 via activation of the Wnt/β-catenin signaling pathway in adult rat brains.