LPS induces stefin A3 expression in mouse primary cultured glial cells

We searched for a gene that is up-regulated in response to LPS at a later time point in primary cultured glial cells. Using a Gene Chip Probe Array, we identified stefin A3, which is known as a cysteine protease inhibitor. As assessed by RT-PCR, we observed a time-dependent (2 to 48 h) up-regulation of stefin A3. The results indicate that stefin A3 is involved in infection and inflammation at a later time point.     

Early damage of sympathetic neurons after co-culture with macrophages: a model of neuronal injury in vitro

Since activated immune cells may damage peripheral nerves during inflammation, we developed a co-culture model that permits the direct study of macrophage-induced neuronal damage. Sympathetic neurons were enzymatically isolated from neonatal mice and co-cultured with increasing numbers of peritoneal macrophages for 24 h. This caused rapid neuronal cell death, reducing neuronal number by 24.1 +/- 4% with the addition of 11.5 x 10(3) macrophages, representing a ratio of 8 macrophages per neuron. Nuclear analysis showed that cell death occurred by both apoptosis and necrosis. These effects were not mimicked by addition of macrophage-conditioned medium, and were prevented by 10 microM dexamethasone. Although no appreciable neuronal death occurred beyond 24 h, the density of neurites was decreased between 1 and 2 days of co-culture (p < 0.05). There is, therefore, a rapid induction of cytotoxicity by macrophages after their addition to the neuronal cultures, followed by axonal damage without neuronal cell death.     

Traumatic injury of spinal cord cells in vitro reduced by GM1 ganglioside

GM1 ganglioside (monosialoganglioside) is a significant endogenous component of central nervous system (CNS) cellular membranes, thereby contributing to the membranes' integrity and function. Exogenous gangliosides have been shown to be incorporated into plasma membranes and can exert neuroprotective effects on damaged neuronal tissue(s). An in vitro method of physical injury (trauma) previously described which used cultures derived from fetal mouse spinal cord [38] was adapted for these studies in order for us to assess GMl's neuroprotective efficacy. Injury was induced by uniformly crosshatching the spinal cell cultures with a 1 mm plastic pipette tip. The extent of injury and the effects of GM1 ganglioside posttreatment (80 μM) was assessed after 48 h by measuring lactate dehydrogenase (LDH) released and by observing changes in the plasma membrane surface distribution of endogenous GM1 using cholera toxin/antitoxin/fluorescent antibody immunohistochemistry. A gradient of injury, from the zone of maximum injury to partially traumatized or non-injured areas, was seen using immunohistochemistry. The primary injury zone in this gradient was characterized by areas of swollen or dead cells and abnormal or degenerating cell processes. At further distances, cells were observed to be nearly normal, with intact fibers. This gradient of injury may reflect proximate (at the locus of trauma) and distant effects of the release of neurotoxic levels of endogenous glutamate (Glu) and other excitatory amino acids. Ganglioside GM1 treatment resulted in a significantly reduced (>75%) release of LDH as well as enhanced cell and process integrity indicative of reduced tissue injury. These initial results indicate that GMl's previously documented neuroprotective effects using neuronal culture systems can be generalized to injured spinal cells in vitro wherein there is evidence for preservation (rescue) of cellular plasma membranes after injury as reflected by reduced cell loss, swelling, and process degeneration, as well as decreased LDH release.     

成人骨髓基质细胞体外诱导成神经细胞实验研究

目的探讨骨髓基质细胞的体外培养、扩增及诱导其向神经元样细胞定向分化的方法。方法采用改进的全骨髓培养法,培养来源于正常成人献髓者的骨髓基质细胞。传至第5代,用全反式维甲酸(RA)等细胞因子诱导分化,于诱导后第7d和第14d行神经元细胞特异性抗原神经元特异性烯醇化酶(NSE)、微管相关蛋白-2(MAP-2)、神经丝蛋白(β-tublin)和胶质纤维酸性蛋白(GFAP)鉴定,计数诱导前后的细胞。结果成人骨髓基质细胞经RA 碱性成纤维细胞生长因子(bFGF) 脑源性神经营养因子(BDNF)诱导后可见神经元样细胞出现,经NSE、MAP-2及β-tublin免疫组化和荧光鉴定阳性,NSE表达率达到48.5%±0.2%。结论骨髓基质细胞在体外扩增迅速,纯化需时短,并可在全反式维甲酸等细胞因子诱导下向神经元样细胞分化。     

Calcium rise in cultured neurons from medial septum elicits calcium waves in surrounding glial cells

One prerequisite for understanding the physiological relevance of intercellular calcium waves in glia is the examination of mechanisms that trigger these waves. Here, we show that stimulation of cultured septal neurons to produce a large and sustained calcium rise in the soma can initiate calcium waves in surrounding glial cells. The initiation of calcium waves is dependent on calcium influx through voltage-gated calcium channels on the neuron. The waves are not due to direct stimulation of the glial cells or to loss of neuronal membrane integrity. Mechanism of wave initiation is distinct from that involved in wave propagation and does not involve glutamate or acetylcholine release. Communication via gap junctions, or nitric oxide production, is not involved in the initial signaling between a stimulated neuron and the surrounding astrocytes. Suramin, a blocker of P2 receptors blocked the waves but failed to abolish the responses in glial cells immediately surrounding the stimulated neuron. Our results suggest that patterns of calcium rises on neurons, like those seen in glutamate cytotoxicity, can cause calcium waves in surrounding glial cells.     

Transport of L-arginine in cultured glial cells

Uptake of radiolabelled L-arginine was studied in four different kinds of glial cultures, in astroglia-rich primary cultures derived from neonatal rat and mouse brains, in pure murine astrocyte cultures, and in rat glioma cells C6-BU-1. A saturable component of uptake was found in all cases with K_M values between 15 and 35 μM and V_max values between 0.8 and 2.5 nmol · min^?1 · (mg protein)^?1. In addition, in all cell types a non-saturable component dominated total uptake at high concentrations of extracellular arginine. Rates of uptake of arginine were not affected when Na⁺ or Cl^? were absent from the incubation buffer. Carrier-mediated uptake of arginine was reduced by depolarizing concentrations of K⁺ and strongly inhibited by an excess of lysine or ornithine. Histidine, asparagine, glutamine, citrulline, creatine, N^G-nitro-L-arginine, N^G-monomethyl-L-arginine, or L-canavanine inhibited L-arginine transport to various degrees. Uptake of arginine was not reduced in the presence of serine or alanine, cysteic acid, N-methyl-α-aminoisobutyric acid, or 2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid. Rates of uptake of arginine were increased when cells had been preloaded with lysine. Preincubation of primary cultures, but not glioma cells, with bacterial lipopolysaccharide stimulated transport of arginine by increasing the V_max value of uptake. This stimulation was dependent on protein synthesis. The results suggest that, at physiological concentrations, arginine is taken up into the glial cells with the help of the transport system “y⁺” for basic amino acids. In glial primary cultures, uptake of arginine appears to be regulated by compounds which also exert influence on nitric oxide synthesis. © 1994 Wiley-Liss, Inc.     

In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system

This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip–mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.     

Adult human spinal cord harbors neural precursor cells that generate neurons and glial cells in vitro

Adult human and rodent brains contain neural stem and progenitor cells, and the presence of neural stem cells in the adult rodent spinal cord has also been described. Here, using electron microscopy, expression of neural precursor cell markers, and cell culture, we investigated whether neural precursor cells are also present in adult human spinal cord. In well-preserved nonpathological post-mortem human adult spinal cord, nestin, Sox2, GFAP, CD15, Nkx6.1, and PSA-NCAM were found to be expressed heterogeneously by cells located around the central canal. Ultrastructural analysis revealed the existence of immature cells close to the ependymal cells, which display characteristics of type B and C cells found in the adult rodent brain subventricular region, which are considered to be stem and progenitor cells, respectively. Completely dissociated spinal cord cells reproducibly formed Sox2(+) nestin(+) neurospheres containing proliferative precursor cells. On differentiation, these generate glial cells and gamma-aminobutyric acid (GABA)-ergic neurons. These results provide the first evidence for the existence in the adult human spinal cord of neural precursors with the potential to differentiate into neurons and glia. They represent a major interest for endogenous regeneration of spinal cord after trauma and in degenerative diseases.     

Protein synthesis in neurons and glial cells by extrahepatic cholestasis in rats

Cats with lesions of the dorsal columns at the cervical level were compared to sham-operated controls in their ability to jump over barriers while being carried on a moving conveyor belt. The results showed that cats with the lesions fail to initiate jumping movements at the appropriate time even when it is visually obvious that they have to be made. The cats are consistently carried by the conveyor belt directly up to the barrier, so that they make contact with it before jumping over. The paws of two cats were jammed between the conveyor belt and the rear edge of the apparatus because of this failure to begin the response at the appropriate time. In contrast, the control cats usually initiate their jump at a considerable distance in front of the barrier and thereby avoid making contact with it. The data suggest that the dorsal column projection system is necessary for the anticipatory components of sequential behavior patterns.     

TWEAK is expressed by glial cells,induces astrocyte proliferation and increases EAE severity

TWEAK is a new TNF family member with proinflammatory and proliferative effects on different cell types, mediated by the recently identified Fn14 receptor. TWEAK expression was analyzed on mouse microglial cells and astrocytes. Both cell types express TWEAK mRNA. Astrocytes expressed Fn14 and proliferated in the presence of rTWEAK. TWEAK mRNA is expressed in normal CNS and its steady state level increases in spinal cord during EAE. Finally, EAE severity is enhanced in soluble TWEAK-overexpressing transgenic mice. These results support the contention that TWEAK is involved in CNS inflammation.     

Effect of HEPES on the taurine uptake by cultured glial cells

HEPES inhibited the taurine uptake in glial cells. A different kind of inhibition was observed when HEPES was present in the culture medium or in the incubation medium used for the taurine-uptake measurement. As an example of a possible interference of HEPES in pharmacological experiments, we have studied the effect of this buffer on the modulation of taurine uptake by beta agonists or ionic concentration.     

体外培养原代神经元损伤模型的建立及损伤后脑红蛋白的表达

目的建立原代神经元机械性损伤的体外细胞模型并探讨神经元损伤后脑红蛋白（NGB）的表达变化。方法取出生12h内Wistar鼠的大脑皮质，培养10-12d后，采用所设计的标准模板，直视下进行损伤，采用免疫组化方法和图像分析技术观测损伤前、后不同时间点神经元的NGB免疫组化反应及其阳性信号的灰度值。结果皮质神经元损伤2h后，NGB阳性信号强度即开始增加，16h后达高峰，随后逐渐下降，128h后恢复至损伤前水平。结论皮质神经元体外损伤模型可用于在体外观察神经元受到机械性损伤后细胞内蛋白表达变化的一些分子事件，简单实用。机械性损伤可以诱导神经元NGB在损伤后一定时间段内表达上调。