Objective To identify the protective effect of lipopolysaccharide （LPS） preconditioning against LPS-induced inflammatory damage in dopaminergic neurons of midbrain slice culture and the possible mechanisms. Methods ...Objective To identify the protective effect of lipopolysaccharide （LPS） preconditioning against LPS-induced inflammatory damage in dopaminergic neurons of midbrain slice culture and the possible mechanisms. Methods After cultured in vitro for 14 d, the rat organotypic midbrain slices were pretreated with different concentrations （0, 1, 3, 6 or 10 ng/mL） of LPS for 24 h followed by treatment with 100 ng/mL LPS for 72 h. The whole slice viability was detelmined by measurement of the activity of lactic acid dehydrogenase （LDH）. Tyrosine hydroxylase-immunoreactive （TH-IR） neurons and CD 1 1 b/c equivalent-immunoreactive （OX-42-IR） microglia in the slices were observed by immunohistochemical method, and tumor necrosis factor-α （TNF-α levels in the culture media were detected by enzymelinked immunosorbent assays （ELISA）. Results In the slices treated with 100 ng/mL LPS for 72 h, the number of TH-IR neurons reduced from 191± 12 in the control slices to 46±4, and the LDH activity elevated obviously （P 〈 0.01）, along with remarkably increased number of OX-42-IR cells and production of TNF-α （P 〈 0.01）. Preconditioning with 3 or 6 ng/mL LPS attenuated neuron loss （the number of TH-IR neurons increased to 126± 12 and 180± 13, respectively） and markedly reduced LDH levels （P 〈 0.05）, accompanied by significant decreases of OX-42-IR microglia activation and TNF-α production （P 〈 0.05）. Conclusion Low-dose LPS preconditioning could protect dopaminergic neurons against inflammatory damage in rat midbrain slice culture, and inhibition of microglial activation and reduction of the proinflammatory factor TNF-α production may contribute to this protective effect. Further understanding the underlying mechanism of LPS preconditioning may open a new window for treatment of Parkinson＇s disease.展开更多
Objective： To establish a simple, effective and high-purity primary culture method for fetal rat hippocampalneurons. Methods： Wistar rats of gestational age 18 days were taken and the brain tissue was separated unde...Objective： To establish a simple, effective and high-purity primary culture method for fetal rat hippocampalneurons. Methods： Wistar rats of gestational age 18 days were taken and the brain tissue was separated under themicroscope. Single neuronal cells were obtained by digestion with Brain Dissociation Kit, and then were seeded incell plates to observe the basic morphologic structure after 24h, 3d, and 5d. Immunofluorescence of microtubuleassociated protein 2 was applied to assess cell purity of the culture. Results： The hippocampal neurons obtained inthis culture method are in good condition and grow vigorously. On the 7th day after culture, the purity of neuronswas up to 99.62%. Conclusion： The method is simple and effective for obtaining the high-purity and stableneurons.展开更多
Objective Muncl8-1 has an important role in neurotransmitter release, and controls every step in the exocy- totic pathway in the central nervous system. In the present study, whether epileptic seizure causes a change ...Objective Muncl8-1 has an important role in neurotransmitter release, and controls every step in the exocy- totic pathway in the central nervous system. In the present study, whether epileptic seizure causes a change of Muncl8 localization in neuronal nuclei was analyzed. Methods Epilepsy models were established by injection of kainic acid （KA） solution into hippocampus of Sprague-Dawley （SD） rats or intraperitoneal injection of KA in Kunming mice. The hippocampal neurons were prepared from embryonic day 18 SD rats, and cultured in neurobasal medium, followed by treatment with glutamate for 3 h. Neuronal and glial nuclei of hippocampus were separated by sucrose density gradient centrifugation. The nucleus-enriched fractions were stained with 0.1% Cresyl Violet for morphological assay. Immuno- chemistry and immunoelectron microscopy with anti-Muncl 8-1 antibody were used to determine the nuclear locatization of Munc 18-1. Immunoblotting was used to detect the protein level of Munc 18-1. Results The localization of Munc 18-1 in nucleus of rat hippocampal neuron was confirmed by immunochemistry, immunoelectron microscopy, and immunob- lotting detection of neuronal nucleus fraction. In animals receiving intrahippocampal or intraperitoneal injection of KA, immunostaining revealed that the expression of Muncl 8-1 decreased in pyramidal cell layer of CA regions, as well as in hilus and granular cell layer of dentate gyrus in hippocampus. Moreover, immunoblotting analysis showed that the expres- sion level of Muncl 8-1 in nucleus fraction of hippocampus significantly decreased in KA-treated animals. The relation- ship between the change of Muncl8-1 expression in neuronal nuclei and neuronal over-activation was also tested in pri- mary cultured neurons. After treatment with 50 ~tmol/L glutamate acid for 3 h, Muncl8-1 level was decreased in nucleus fraction and increased in cytoplasmic fraction of primary cultured neurons. Conclusion These results suggest that excit- atory stimulation can induce the distribution change of Munc 18-1 in neuron, which may subsequently modulate neuronal functions in brain.展开更多
Objective:To investigate the culture method of skin-derived precursors (SKPs) and to explore a new cell source for cell transp lantation of central nervous system. Methods:Cells from skins of juvenile and adult mice w...Objective:To investigate the culture method of skin-derived precursors (SKPs) and to explore a new cell source for cell transp lantation of central nervous system. Methods:Cells from skins of juvenile and adult mice were iso lated and cultured in serum-free medium. A mechanical method was chosen to pass age these cells and they were identified by the immunocytochemistry assay. Results:SKPs could be isolated from adult and neonatal skins . They could be maintained in vitro for long periods with stable proliferation, and expanded as undifferentiated cells in culture for more than 12 passages. Abo ut 50% of SKPs expressed nestin and majority of these cells expressed fibronecti n when they were plated on polyornithine and laminin coated plates. About 5% cel ls showed neuronal differentiation and expressed neurofilament-M (NF-M) and NS E when SKPs were plated in serum-containing medium, and these cells could also differentiate into adipocytes and fibroblast-like cells. Conclusions:The data support the hypothesis that adult skin contains stem cells capable of differentiating into neurons, adipocytes, and fib roblast-like cells. They may represent an alternative autologous stem cell sour ce for CNS cell transplantation.展开更多
基金the Foundation of Beijing Municipal Commission of Education,China (No.200410025011)
文摘Objective To identify the protective effect of lipopolysaccharide （LPS） preconditioning against LPS-induced inflammatory damage in dopaminergic neurons of midbrain slice culture and the possible mechanisms. Methods After cultured in vitro for 14 d, the rat organotypic midbrain slices were pretreated with different concentrations （0, 1, 3, 6 or 10 ng/mL） of LPS for 24 h followed by treatment with 100 ng/mL LPS for 72 h. The whole slice viability was detelmined by measurement of the activity of lactic acid dehydrogenase （LDH）. Tyrosine hydroxylase-immunoreactive （TH-IR） neurons and CD 1 1 b/c equivalent-immunoreactive （OX-42-IR） microglia in the slices were observed by immunohistochemical method, and tumor necrosis factor-α （TNF-α levels in the culture media were detected by enzymelinked immunosorbent assays （ELISA）. Results In the slices treated with 100 ng/mL LPS for 72 h, the number of TH-IR neurons reduced from 191± 12 in the control slices to 46±4, and the LDH activity elevated obviously （P 〈 0.01）, along with remarkably increased number of OX-42-IR cells and production of TNF-α （P 〈 0.01）. Preconditioning with 3 or 6 ng/mL LPS attenuated neuron loss （the number of TH-IR neurons increased to 126± 12 and 180± 13, respectively） and markedly reduced LDH levels （P 〈 0.05）, accompanied by significant decreases of OX-42-IR microglia activation and TNF-α production （P 〈 0.05）. Conclusion Low-dose LPS preconditioning could protect dopaminergic neurons against inflammatory damage in rat midbrain slice culture, and inhibition of microglial activation and reduction of the proinflammatory factor TNF-α production may contribute to this protective effect. Further understanding the underlying mechanism of LPS preconditioning may open a new window for treatment of Parkinson＇s disease.
基金Natural Science Foundation of China （NO.81373703 NO. 81674042）Basic research project of natural science in shaanxi province - major basic research project （NO. 2017zdjc-15）
文摘Objective： To establish a simple, effective and high-purity primary culture method for fetal rat hippocampalneurons. Methods： Wistar rats of gestational age 18 days were taken and the brain tissue was separated under themicroscope. Single neuronal cells were obtained by digestion with Brain Dissociation Kit, and then were seeded incell plates to observe the basic morphologic structure after 24h, 3d, and 5d. Immunofluorescence of microtubuleassociated protein 2 was applied to assess cell purity of the culture. Results： The hippocampal neurons obtained inthis culture method are in good condition and grow vigorously. On the 7th day after culture, the purity of neuronswas up to 99.62%. Conclusion： The method is simple and effective for obtaining the high-purity and stableneurons.
基金supported by grants from the National Natural Science Foundation of China (No. 81071017, 30470536, 90919004)
文摘Objective Muncl8-1 has an important role in neurotransmitter release, and controls every step in the exocy- totic pathway in the central nervous system. In the present study, whether epileptic seizure causes a change of Muncl8 localization in neuronal nuclei was analyzed. Methods Epilepsy models were established by injection of kainic acid （KA） solution into hippocampus of Sprague-Dawley （SD） rats or intraperitoneal injection of KA in Kunming mice. The hippocampal neurons were prepared from embryonic day 18 SD rats, and cultured in neurobasal medium, followed by treatment with glutamate for 3 h. Neuronal and glial nuclei of hippocampus were separated by sucrose density gradient centrifugation. The nucleus-enriched fractions were stained with 0.1% Cresyl Violet for morphological assay. Immuno- chemistry and immunoelectron microscopy with anti-Muncl 8-1 antibody were used to determine the nuclear locatization of Munc 18-1. Immunoblotting was used to detect the protein level of Munc 18-1. Results The localization of Munc 18-1 in nucleus of rat hippocampal neuron was confirmed by immunochemistry, immunoelectron microscopy, and immunob- lotting detection of neuronal nucleus fraction. In animals receiving intrahippocampal or intraperitoneal injection of KA, immunostaining revealed that the expression of Muncl 8-1 decreased in pyramidal cell layer of CA regions, as well as in hilus and granular cell layer of dentate gyrus in hippocampus. Moreover, immunoblotting analysis showed that the expres- sion level of Muncl 8-1 in nucleus fraction of hippocampus significantly decreased in KA-treated animals. The relation- ship between the change of Muncl8-1 expression in neuronal nuclei and neuronal over-activation was also tested in pri- mary cultured neurons. After treatment with 50 ~tmol/L glutamate acid for 3 h, Muncl8-1 level was decreased in nucleus fraction and increased in cytoplasmic fraction of primary cultured neurons. Conclusion These results suggest that excit- atory stimulation can induce the distribution change of Munc 18-1 in neuron, which may subsequently modulate neuronal functions in brain.
基金ThisworkwassupportedbyNaturalScienceFoundationofGuangdongProvince (No .01245200001)andtheNationalNaturalScienceFoundationofChina(KeyProjectNo .3 9993 4 3 0 )
文摘Objective:To investigate the culture method of skin-derived precursors (SKPs) and to explore a new cell source for cell transp lantation of central nervous system. Methods:Cells from skins of juvenile and adult mice were iso lated and cultured in serum-free medium. A mechanical method was chosen to pass age these cells and they were identified by the immunocytochemistry assay. Results:SKPs could be isolated from adult and neonatal skins . They could be maintained in vitro for long periods with stable proliferation, and expanded as undifferentiated cells in culture for more than 12 passages. Abo ut 50% of SKPs expressed nestin and majority of these cells expressed fibronecti n when they were plated on polyornithine and laminin coated plates. About 5% cel ls showed neuronal differentiation and expressed neurofilament-M (NF-M) and NS E when SKPs were plated in serum-containing medium, and these cells could also differentiate into adipocytes and fibroblast-like cells. Conclusions:The data support the hypothesis that adult skin contains stem cells capable of differentiating into neurons, adipocytes, and fib roblast-like cells. They may represent an alternative autologous stem cell sour ce for CNS cell transplantation.
