Transforming growth factor-beta 1 enhances discharge activity of cortical neurons

Transforming growth factor-beta 1(TGF-β1)has been extensively studied for its pleiotropic effects on central nervous system diseases.The neuroprotective or neurotoxic effects of TGF-β1 in specific brain areas may depend on the pathological process and cell types involved.Voltage-gated sodium channels(VGSCs)are essential ion channels for the generation of action potentials in neurons,and are involved in various neuroexcitation-related diseases.However,the effects of TGF-β1 on the functional properties of VGSCs and firing properties in cortical neurons remain unclear.In this study,we investigated the effects of TGF-β1 on VGSC function and firing properties in primary cortical neurons from mice.We found that TGF-β1 increased VGSC current density in a dose-and time-dependent manner,which was attributable to the upregulation of Nav1.3 expression.Increased VGSC current density and Nav1.3 expression were significantly abolished by preincubation with inhibitors of mitogen-activated protein kinase kinase(PD98059),p38 mitogen-activated protein kinase(SB203580),and Jun NH2-terminal kinase 1/2 inhibitor(SP600125).Interestingly,TGF-β1 significantly increased the firing threshold of action potentials but did not change their firing rate in cortical neurons.These findings suggest that TGF-β1 can increase Nav1.3 expression through activation of the ERK1/2-JNK-MAPK pathway,which leads to a decrease in the firing threshold of action potentials in cortical neurons under pathological conditions.Thus,this contributes to the occurrence and progression of neuroexcitatory-related diseases of the central nervous system.

Effects of Rhynchophylline on L type Calcium Channels in Isolated Rat Cortical Neurons During Acute Hypoxia

Aim: To study the effects of rhynchophylline (Rhy) on the L type calcium channels in freshly dissociated cortical neurons of Wistar rats during acute hypoxia. Methods: Cell attached configuration of patch clamp technique. L type calcium channel was activated by stepping from 40 mV to 0 mV. Results: The results showed that the L type calcium channels of cortical neurons were activated by acute hypoxia. The mean open time of the channel was increased, the mean close time decreased and the open state probability raised during acute hypoxia. Rhy (15 and 30μmol·L -1 ) in concentration dependent manner blocked activity of the channels. The drug shortened the mean open time of the channels from 8 87 ms to 3 03 ms and 2 23 ms ( P 【0 001), prolonged the mean close time from 9 23 ms to 38 84 ms and 54 43 ms ( P 【0 001), and decreased the open state probability from 0 142 to 0 031 and 0 025 ( P 【0 001) under the hypoxia condition, respectively. The effects of Rhy were similar to but weaker than those of verapamil (15 μmol·L -1 ). Conclusion: The study confirmed that Rhy has the blockade effects on L type calcium channels in cortical neurons of rats during hypoxia, by which it protects the brain from hypoxic injury.

Oxidative Stress and Apoptotic Changes of Rat Cerebral Cortical Neurons Exposed to Cadmium in Vitro

Objective To investigate the cytotoxic mechanism of cadmium（Cd） on cerebral cortical neurons.Methods The primary cultures of rat cerebral cortical neurons were treated with different concentrations of cadmium acetate（0,5,10,and 20 μmol/L）,and then the cell viability,apoptosis,ultrastructure,intracellular [Ca2＋]i and reactive oxygen species（ROS） levels,mitochondrial membrane potential（ΔΨ）,activities of catalase（CAT） and superoxide dismutase（SOD） were measured.Results A progressive loss in cell viability and an increased number of apoptotic cells were observed.In addition,Cd-induced apoptotic morphological changes in cerebral cortical neurons were also demonstrated by Hoechst 33258 staining.Meanwhile,ultrastructural changes were distortion of mitochondrial cristae and an unusual arrangement.Simultaneously,elevation of intracellular [Ca2＋]i and ROS levels,depletion of ΔΨ were revealed in a dose-dependent manner during the exposure.Moreover,CAT and SOD activities in the living cells increased significantly.Conclusion Exposure of cortical neurons to different doses of Cd led to cellular death,mediated by an apoptotic mechanism,and the apoptotic death induced by oxidative stress may be a potential reason.And the disorder of intracellular homeostasis caused by oxidative stress and mitochondrial dysfunction may be a trigger for apoptosis in cortical neurons.

Involvement of PI3K/Akt Pathway in the Neuroprotective Effect of Sonic Hedgehog on Cortical Neurons under Oxidative Stress

The Sonic hedgehog (SHH) signaling pathway plays a pivotal role in neurogenesis and brain damage repair. Our previous work demonstrated that the SHH signaling pathway was involved in the neuroprotection of cortical neurons against oxidative stress. The present study was aimed to further examine the underlying mechanism. The cortical neurons were obtained from one-day old Sprague-Dawley neonate rats. Hydrogen peroxide (H2O2, 100 μmol/L) was used to treat neurons for 24h to induce oxidative stress. Exogenous SHH (3μg/mL) was employed to activate the SHH pathway, and cyclopamine (20 μmol/L), a specific SHH signal inhibitor, to block SHH pathway. LY294002 (20 μmol/L) were used to pretreat the neurons 30 min before H2O2 treatment and selectively inhibit the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. The cell viability was measured by MTT and apoptosis rate by flow cytometry analysis. The expression of p38, p-p38, ERK, p-ERK, Akt, p-Akt, Bcl-2, and Bax in neurons was detected by immunoblotting. The results showed that as compared with H2O2 treatment, exogenous SHH could increase the expression of p-Akt by 20% and decrease the expression of p-ERK by 33%. SHH exerted no significant effect on p38 mitogen-activated protein kinase (p38 MAPK) pathway. Blockade of PI3K/Akt pathway by LY294002 decreased the cell viability by 17% and increased the cell apoptosis rate by 2-fold. LY294002 treatment could up-regulate the expression of the proapoptotic gene Bax by 12% and down-regulate the expression of the antiapoptotic gene Bcl-2 by 54%. In conclusion, SHH pathway may activate PI3K/Akt pathway and inhibit the activation of the ERK pathway in neurons under oxidative stress. The PI3K/Akt pathway plays a key role in the neuroprotection of SHH. SHH/PI3K/Bcl-2 pathway may be implicated in the protection of neurons against H2O2-induced apoptosis.

Neuroprotective effect of interleukin-6 regulation of voltage-gated Na^+ channels of cortical neurons is time-and dose-dependent

Interleukin-6 has been shown to be involved in nerve injury and nerve regeneration, but the effects of long-term administration of high concentrations of interleukin-6 on neurons in the central nervous system is poorly understood. This study investigated the effects of 24 hour expo-sure of interleukin-6 on cortical neurons at various concentrations （0.1, 1, 5 and 10 ng/mL） and the effects of 10 ng/mL interleukin-6 exposure to cortical neurons for various durations （2, 4, 8, 24 and 48 hours） by studying voltage-gated Na＋ channels using a patch-clamp technique. Volt-age-clamp recording results demonstrated that interleukin-6 suppressed Na＋ currents through its receptor in a time- and dose-dependent manner, but did not alter voltage-dependent activation and inactivation. Current-clamp recording results were consistent with voltage-clamp recording results. Interleukin-6 reduced the action potential amplitude of cortical neurons, but did not change the action potential threshold. The regulation of voltage-gated Na＋channels in rat corti-cal neurons by interleukin-6 is time- and dose-dependent.

Neuroprotective effects of combined lead and cadmium,as well as N-acetylcysteine,on cerebral cortical neurons following lipid peroxidation injury

BACKGROUND： Studies have reported the antioxidant effects of lead and cadmium in the central nervous system, but very few have addressed the combined toxicity of lead and cadmium. The mechanisms by which these combined heavy metals are toxic, as well as how to protect cells from these agents, remains poorly understood. OBJECTIVE： Primary cultured rat cortical neurons were used to determine the effects of combined lead and cadmium on levels of glutathione peroxidase （GSH-Px）, superoxide dismutase （SOD）, catalase （CAT）, and acetylcholinesterase （ACHE）, as well as malondialdehyde （MDA）, and to evaluate the neuroprotective effects of N-acetylcysteine （NAC）. DESIGN, TIME AND SETTING An in vitro toxicological observation was performed at the Comparative Medicine Center of Yangzhou University from August 2007 to April 2008. MATERIALS： Lead acetate, cadmium acetate, and NAC were purchased from Sigma-Aldrich （St. Louis, USA）. Commercial kits of GSH-Px, SOD, CAT, ACHE, and MDA were purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. METHODS： The cerebral cortical neurons were isolated from newborn Sprague dawley rats at 24 hours after birth and primary cultured for 6 days. Thereafter, the cells were treated with a range of cadmium doses （0, 5.0, and 10.0μmol/L）, lead doses （0, 1.0, and 2.0 μmol/L）, or a combination of the two for 12 hours at 37℃in a 5% CO2 incubator, respectively. In addition, the cells were incubated with different doses of cadmium and/or lead and （0 and 50 μmol/L） NAC for 12 hours to assess the protective effects on cell survival. MAIN OUTCOME MEASURES： The activity of SOD, GSH-Px, CAT, and ACHE, as well as MDA content, in the cell lysates was detected using commercial kits. RESULTS： At 12 hours after treatment, compared to the control group, activity of GSH-Px, SOD, and AChE in the lead, cadmium, or combined treated cells was significantly decreased with increasing doses of cadmium/or lead （P 〈 0.05）, but CAT activity and MDA levels were significantly increased （P 〈 0.05）. The combination of cadmium and lead led to higher levels of toxicity than individual exposure. CONCLUSION： The degree of oxidative damage increased when the two heavy metals were combined. NAC protected neonatal cortical neurons by increasing activity of anti-oxidative enzymes and reducing lipid peroxidation, but the reduction was not statistically significant.

Neuroprotective effects of human telomerase reverse transcriptase on beta-amyloid fragment 25-35-treated human embryonic cortical neurons

BACKGROUND： Numerous current studies have suggested that human telomerase reverse transcriptase （hTERT） gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the mechanism of action remains unknown. OBJECTIVE： To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 （AI325-35）. DESIGN, TIME AND SETTING： The randomized, controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research, Zhongshan School of Medicine, Sun Yat-sen University, China, from September 2005 to June 2008. MATERIALS： AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University, China. Human cortical neurons were derived from 12-20 week old aborted fetuses, obtained from the Guangzhou Maternal and Child Health Hospital, China. Mouse anti-Odk5 and mouse anti-p16 monoclonal antibodies （Lab Vision, USA）, and mouse anti-hTERT monoclonal antibody （Epitomics, USA）, were used in this study. METHODS： （1） Recombinant adenovirus vectors, encoding hTERT （Ad-hTERT） and green fluorescent protein （Ad-GFP）, were constructed using the AdEasy-1 Expression System. Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise, human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days. Human embryonic cortical neurons in the control group were cultured as normal. （2） Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 pmol/L Aβ25-35 for 24 hours. Normal human embryonic cortical neurons treated with 10 pmol/Lβ25.35 for 24 hours served as a model group. Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35. MAIN OUTCOME MEASURES： Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol （TRAP） ELISA kit. Neural activity in human embryonic cortical neurons was examined by MTT assay; apoptosis was measured using TUNEL assay; and Cdk5 and p16 protein expressions were measured by Western blot. RESULTS： Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group. No hTERT expression was detected in the Ad-GFP and control groups. Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups （P 〈 0.01）. Compared with the control group, cell activity was significantly decreased （P 〈 0.05）, and cell apoptotic rate, Cdk5 and p16 expression were significantly increased （P 〈 0.01） in the model group. Compared with the model group, cell activity was increased in the Ad-hTERT group, and peaked at day 3 post-transfection （P 〈 0.05）. Neuroprotective effects also peaked at day 3 post-transfection; and the apoptotic rate, Cdk5 and p16 expression significantly decreased （P 〈 0.01）. CONCLUSION： Expression of hTERT in human embryonic cortical neurons can relieve Aβ25-35-induced neuronal apoptosis. The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.

Injury of cortical neurons is caused by the advanced glycation end products-mediated pathway

Advanced glycation end products lead to cell apoptosis, and cause cell death by increasing endoplasmic reticulum stress. Advanced glycation end products alone may also directly cause damage to tissues and cells, but the precise mechanism remains unknown. This study used primary cultures of rat cerebral cortex neurons, and treated cells with different concentrations of glycation end products （50, 100, 200, 400 mg/L）, and with an antibody for the receptor of advanced glycation end products before and after treatment with advanced glycation end products. The results showed that with increasing concentrations of glycation end products, free radical content increased in neurons, and the number of apoptotic cells increased in a dose-dependent manner. Before and after treatment of advanced glycation end products, the addition of the antibody against advanced glycation end-products markedly reduced hydroxyl free radicals, malondialdehyde levels, and inhibited cell apoptosis. This result indicated that the antibody for receptor of advanced glycation end-products in neurons from the rat cerebral cortex can reduce glycation end product-induced oxidative stress damage by suppressing glycation end product receptors. Overall, our study confirms that the advanced glycation end products-advanced glycation end products receptor pathway may be the main signaling pathway leading to neuronal damage.

LIPUS Enhance Elongation of Neurites in Rat Cortical Neurons through Inhibition of GSK-3β

Objective Low-intensity pulsed ultrasound （LIPUS） has been reported to enhance proliferation and to alter protein production in various kinds of cells. In the present study, we measured the neurites length after LIPUS treatment to define the effectiveness of LIPUS stimulation on neurons, and then we examined the acticity of GSK-3β to study the intracellular mechanism of neurite＇s outgrowth. Methods LIPUS was applied to cultured primary rat cortical neurons for 5 minutes every day with spatial- and temporal average intensities （SATA） of 10 mW/cm^2, a pulse width of 200 microseconds, a repetition rate of 1.5 KHz, and an operation frequency of 1 MHz. Neurons were photographed on the third day after LIPUS treatment and harvested at third, seventh, and tenth days for immnoblot and semi-quantitative RT-PCR analysis. Results Morphology change showed that neurite extension was enhanced by LIPUS. There was also a remarkable decrease of proteins, including p-Akt, p-GSK-3β, and p-CRMP-2, observed on the seventh and tenth days, and of GSK-3β mRNA expression, observed on the seventh day, in neurons treated with LIPUS. Conclusion LIPUS can enhance elongation of neurites and it is possible through the decreased expression of GSK-3β.

Protective effects of berberine against amyloid beta-induced toxicity in cultured rat cortical neurons

Berberine, a major constituent of Coptidis rhizoma, exhibits neural protective effects. The present study analyzed the potential protective effect of berberine against amyloid β-induced cytotoxicity in rat cerebral cortical neurons. Alzheimer＇s disease cell models were treated with 0.5 and 2 μmol/L berberine for 36 hours to inhibit amyloid β-induced toxicity. Methyl thiazolyl tetrazolium assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining results showed that berberine significantly increased cell viability and reduced cell apoptosis in primary cultured rat cortical neurons. In addition, western blot analysis revealed a protective effect of berberine against amyloid β-induced toxicity in cultured cortical neurons, which coincided with significantly decreased abnormal up-regulation of activated caspase-3. These results showed that berberine exhibited a protective effect against amyloid β-induced cytotoxicity in cultured rat cortical neurons.

Electrical stimulation of cortical neurons promotes oligodendrocyte development and remyelination in the injured spinal cord

Background and early studies： Endogenous tri-potential neural stem cells （NSCs） exist in the adult mammalian central nervous system （CNS）. In the spinal cord, NSCs distribute throughout the entire cord, but exist predominately in white matter tracts. The phenotypic fate of these cells in white matter is glial, largely oligodendrocyte, but not neuronal.

Effect of polygonatum polysaccharide on the hypoxia-induced apoptosis and necrosis in in vitro cultured cerebral cortical neurons from neonatal rats

BACKGROUND： Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE ： To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN： A comparative experiment.SETTING： Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS： The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats （male or female） of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College （certification number was 021-97-03）. METHODS：① Preparation of cerebral cortical neurons of rats： The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons： After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages （1-20 g/L） was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③Grouping： After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours.④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES： ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons;② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining;③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④DNA agarose gel electrophoresis ladder-like strap appeared or not;⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS：① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons （P 〉 0.05）. ②The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [（13.00±4.52）%,（12.72±2.15）%, （11.80±1.18）%,（38.03±1.05）%, P 〈 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture （36.77±1.45）%, （36.60±1.61）%, （36.37±2.02）%, （38.03±1.05）%, P 〉 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L （P 〈 0.01）. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation （P 〈 0.01）. ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia;⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased （P 〈 0.01）, and those of Bax protein and Caspase-3 protein were reduced （P 〈 0.01）, and the ratio of Bcl-2/Bax was increased （P 〈 0.01）. CONCLUSION： Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.

Genome-wide profiling of long noncoding RNA expression patterns and CeRNA analysis in mouse cortical neurons infected with different strains of borna disease virus

Borna disease virus 1(BoDV-1)is neurotropic prototype of Bornaviruses causing neurological diseases and maintaining persistent infection in brain cells of mammalian species.Long non-coding RNA(lncRNA)is transcript of more than 200 nucleotides without proteincoding function regulating various biological processes as proliferation,apoptosis,cell migration and viral infection.However,regulatory of lncRNAs in BoDV-1 infection remains unknown.To identify differential expression profiles and predict functions of lncRNA in BoDV-1 infection,microarray data showed that 3528 lncRNAs and 2661 lncRNAs were differentially expressed in Strain V and Hu-H1 BoDV-infected groups compared with control groups,respectively.Gene Ontology(GO)and pathway analysis suggested that differential lncRNAs may be involved in regulation of metabolic,biological regulation,cellular process,endocytosis,viral infections and cell adhesion processes,cancer in both BoDV-infected strains.ENSMUST00000128469 was found down-regulated in both BoDV-infected groups compared with control groups consistent with microarray(p<0.05).ceRNA analysis indicated possible interaction networks as ENSMUST00000128469/miR-22-5p,miR-206-3p,miR-302b-5p,miR-302c-3p,miR-1a-3p/Igf1.Igf1 was found up-regulated in both BoDV-infected groups compared with control groups(p<0.05).Possible functions of predicted target mRNAs and miRNAs of ENSMUST00000128469 were involved in cell proliferation,transcriptional misregulation and proteoglycan pathways enriched in cancer.lncRNA may be involved in regulation of Hu-H1 inhibited cell proliferation and promoted apoptosis through NF-kB,JNK/MAPK signaling,BCL2 and CDK6/E2F1 pathways different from Strain V.Possible interaction networks as ENSMUST00000128469/miR-22-5p,miR-206-3p,miR-302b-5p,miR-302c-3p,miR-1a-3p/Igf1 may involve in regulation of cell proliferation,apoptosis,and cancer.

Neurotrophic effects of 7,8-dihydroxycoumarin in primary cultured rat cortical neurons

Objective Neuronal loss in the central nervous system is central to the occurrence of neurodegenerative diseases. Pharmaceutical companies have devoted much effort to developing new drugs against such diseases, since there are currently no effective drugs for neurodegenerative disease treatment. Promoting the capacity for nerve regeneration is an ideal treatment target. The present study aimed to investigate the neurotrophic effects of 7,8-dihydroxycoumarin （DHC） or daphnetin in primary cultured rat cortical neurons. Methods Cortical neurons were identified by microtubule-associated protein 2 （MAP2） immunostaining. Morphological observation was used to measure the average length of neurite outgrowth. MTT and lactate dehydrogenase assays were used to assess neuronal survival. The mRNA expression of MAP2 and brain-derived neurotrophic factor （BDNF） was measured by RT-PCR. Results MAP2 immunostaining showed that most of the cultured cells were neurons. Compared with the vehicle control group, DHC promoted neurite outgrowth and prolonged neuronal survival time at concentrations ranging from 2 to 8 μmol/L. Expression of both BDNF mRNA and MAP2 mRNAwas increased in the groups treated with 2, 4 and 8 μmol/L DHC. Conclusion DHC significantly increases neurite outgrowth and promotes neuronal survival in primary cultured rat cortical neurons. The neurotrophic effects of DHC are probably associated with increased BDNF expression.

Activation of sigma-1 receptor enhances synaptosomal Ca^(2+) via L-type C^(2+) channel in cortical neuron

Sigma-1 receptors are unique receptors that are postulated to act as intracellular amplifiers for signal transduction within cells of the nervous system. The present paper studied the

SIRT1 Protects Cortical Neurons from Shear Stress Induced Mechanical Injury

Emerging evidence implicates that SIRT1,one of NAD-dependent protein deacetylase,is involved in the regulation of neuronal survival.However,the role of SIRT1 in mechanical injury is still unclear.Here,we first established a mechanical injury model of rat cortical neurons through exposing to 10 dyn/cm 2 laminar shear stress for 4 h.At the same time,we found that SIRT1 expression increased during the process of shear stress induced neuronal injury.SIRT1 overexpression in cortical neurons significantly decreased the percentage of died cells induced by shear stress.Our results demonstrate that SIRT1 may play potential neuroprotective effects in shear stress induced mechanical injury.

Protective Effect of Tetrandrine against Excitatory Amino Acids induced Neuronal Injury in Cortical Culture

The ability of tetrandrine (Tet), an alkaloid isolated from Radix Stephaniae Tetrandrae, to reduce cortical neuronal injury in cortical cultures derived from fetal rats was quantitatively assessed by examination of morphological changes and measurement of lactate dehydrogenase (LDH) released to the extracellular bathing media Cell cultures exposed to the excitatory amino acids (EAA) 50 μmol L 1 glutamate (Glu), 20 μmol L 1 N methyl D aspartate (NMDA), 300 μmol·L 1 β N oxalylamino L alanine (BMAA, NMDA receptor agonist) or 20 μmol·L 1 β N oxaly lamino L alanine (BOAA, non NMDA receptor agonist) for 24 h at 37℃ showed widespread neuronal injury Tet had little effect on the injury induced by 20 μmol·L 1 NMDA but 10 7 and 10 6 μmol·L 1 Tet did partially attenuate the neuronal degeneration, neuronal loss and LDH efflux resulting from prolonged exposures to 100 μmol·L 1 Glu, 300 μmol·L 1 BMAA and 20 μmol·L 1 BOAA respectively The ability of Tet to reduce the neuronal injury induced by prolonged exposure to EAA may contribute, at least in part, to the reduction of Ca 2+ influx through inhibiting the opening of voltagegated Ca 2+ channels Another mechanism that Tet might have a little inhibitory effect on NMDA receptor on neuronal membrane cannot be excluded, as BMAA has been considered to act as a weak NMDA receptor agonist

Curcumin pretreatment and post-treatment both improve the antioxidative ability of neurons with oxygen-glucose deprivation

Recent studies have shown that induced expression of endogenous antioxidative enzymes thr- ough activation of the antioxidant response element/nuclear factor erythroid 2-related factor 2 （Nrf2） pathway may be a neuroprotective strategy. In this study, rat cerebral cortical neurons cultured in vitro were pretreated with 10 ktM curcumin or post-treated with 5 pM curcumin, respectively before or after being subjected to oxygen-glucose deprivation and reoxygenation for 24 hours. Both pretreatment and post-treatment resulted in a significant decrease of cell injury as indicated by propidium iodide/Hoechst 33258 staining, a prominent increase of Nrf2 protein expression as indicated by western blot analysis, and a remarkable increase of protein expression and enzyme activity in whole cell lysates of thioredoxin before ischemia, after ischemia, and after reoxygenation. In addition, post-treatment with curcumin inhibited early DNA/RNA oxidation as indicated by immunocytochemistry and increased nuclear Nrf2 protein by inducing nuclear accumulation of Nrf2. These findings suggest that curcumin activates the expression of thi- oredoxin, an antioxidant protein in the Nrf2 pathway, and protects neurons from death caused by oxygen-glucose deprivation in an in vitro model of ischemia/reperfusion. We speculate that pharmacologic stimulation of antioxidant gene expression may be a promising approach to neu- roprotection after cerebral ischemia.

Cadmium Activates Reactive Oxygen Species-dependent AKT/mT OR and Mitochondrial Apoptotic Pathways in Neuronal Cells

Objective To examine the role of Cd-induced reactive oxygen species（ROS） generation in the apoptosis of neuronal cells. Methods Neuronal cells（primary rat cerebral cortical neurons and PC12 cells） were incubated with or without Cd post-pretreatment with rapamycin（Rap） or N-acetyl-L-cysteine（NAC）. Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3’-kinase/protein kinase B（Akt）/mammalian target of rapamycin（m TOR） and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays. Results Cd-induced activation of Akt/m TOR signaling, including Akt, m TOR, p70 S6 kinase（p70 S6K）, and eukaryotic initiation factor 4E binding protein 1（4E-BP1）. Rap, an m TOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/m TOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein（Bcl-2/Bax） ratio, release of cytochrome c, cleavage of caspase-3 and poly（ADP-ribose） polymerase（PARP）, and nuclear translocation of apoptosis-inducing factor（AIF） and endonuclease G（Endo G）. Conclusion Cd-induced ROS generation activates Akt/m TOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that m TOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.