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1.
This paper explores the long term development of networks of glia and neurons on patterns of Parylene-C on a SiO(2) substrate. We harvested glia and neurons from the Sprague-Dawley (P1-P7) rat hippocampus and utilized an established cell patterning technique in order to investigate cellular migration, over the course of 3 weeks. This work demonstrates that uncontrolled glial mitosis gradually disrupts cellular patterns that are established early during culture. This effect is not attributed to a loss of protein from the Parylene-C surface, as nitrogen levels on the substrate remain stable over 3 weeks. The inclusion of the anti-mitotic cytarabine (Ara-C) in the culture medium moderates glial division and thus, adequately preserves initial glial and neuronal conformity to underlying patterns. Neuronal apoptosis, often associated with the use of Ara-C, is mitigated by the addition of brain derived neurotrophic factor (BDNF). We believe that with the right combination of glial inhibitors and neuronal promoters, the Parylene-C based cell patterning method can generate structured, active neural networks that can be sustained and investigated over extended periods of time. To our knowledge this is the first report on the concurrent application of Ara-C and BDNF on patterned cell cultures. 相似文献
2.
A. C. Massarelli F. Dainous D. Hoffmann S. Mykita L. Freysz H. Dreyfus R. Massarelli 《Neurochemical research》1986,11(1):29-36
The uptake of radioactive ethanolamine has been studied in exclusively neuronal and glial cell cultures from dissociated cerebral hemispheres of chick embryos. Both cell types show saturable kinetics; neurons have an apparentK
m of 6.7 M,V
max 41.4 pmol mg prot.–1 min–1 and glial cells aK
m of 119.6 M,V
max 3,917 pmol mg prot–1 min–1. The lower affinity of the transport and the 100 fold increase inV
max observed in glial cells correlated with a more important accumulation of free ethanolamine found in glial cells and with a higher degree of phosphorylation of ethanolamine. The uptake appeared to be temperature and Na+ ions dependent but was not affected by CN– or ouabain. Monomethyl-, dimethylethanolamine and choline were effective in inhibiting the uptake. Little or no effect was observed with serine, methionine, carnitine, alanine or glutamate. 相似文献
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Abdu E Bruun DA Yang D Yang J Inceoglu B Hammock BD Alkayed NJ Lein PJ 《Journal of neurochemistry》2011,117(4):632-642
It has recently been reported that soluble epoxide hydrolase (sEH), the major enzyme that metabolizes epoxyeicosatrienoic acids (EETs), is expressed in axons of cortical neurons; however, the functional relevance of axonal sEH localization is unknown. Immunocytochemical analyses demonstrate predominant axonal localization of sEH in primary cultures of not only cortical but also sympathetic and sensory neurons. Morphometric analyses of cultured sensory neurons indicate that exposure to a regioisomeric mixture of EETs (0.01-1.0 μM) causes a concentration-dependent increase in axon outgrowth. This axon promoting activity is not a generalized property of all regioisomers of EETs as axonal growth is enhanced in sensory neurons exposed to 14,15-EET but not 8,9- or 11,12-EET. 14,15-EET also promotes axon outgrowth in cultured cortical neurons. Co-exposure to EETs and either of two structurally diverse pharmacological inhibitors of sEH potentiates the axon-enhancing activity of EETs in sensory and cortical neurons. Mass spectrometry indicates that sEH inhibition significantly increases EETs and significantly decreases dihydroxyeicosatrienoic acid metabolites in neuronal cell cultures. These data indicate that EETs enhance axon outgrowth and suggest that axonal sEH activity regulates EETs-induced axon outgrowth. These findings suggest a novel therapeutic use of sEH inhibitors in promoting nerve regeneration. 相似文献
4.
Psarra A.-M. G. Pfeiffer B. Giannakopoulou M. Sotiroudis T. G. Stylianopoulou F. Hamprecht B. 《Brain Cell Biology》1998,27(11):779-790
Summary. The physiological function of brain glycogen and the role of phosphorylase kinase as a regulatory enzyme in the cascade of reactions associated with glycogenolysis in the brain have not been fully elucidated. As a first step toward elucidating such a function, we studied the localization of phosphorylase kinase in glial and neuronal primary cell cultures, and in adult rat brain slices, using a rabbit polyclonal antibody against skeletal muscle glycogen phosphorylase kinase. Immunocytochemical examination of rat astroglia-rich primary cultures revealed that a large number of cells were positive for glycogen phosphorylase kinase immunoreactivity. These cells were also positive for vimentin, a marker for immature glia, while they were negative for glial fibrillary acidic protein, a marker for mature astroglia, and for galactocerebroside, an oligodendroglial marker. Neurons in rat neuron-rich primary cultures did not show any kinase-positive staining. In paraformaldehyde-fixed adult rat brain sections, phosphorylase kinase immunoreactivity was detected in glial-like cells throughout the brain, with relatively high staining found in the cerebral cortex, the cerebellum, and the medulla oblongata. Phosphorylase kinase immunoreactivity could not be detected in neurons, with the exception of a group of large neurons in the brain stem, most likely belonging to the mesencephalic trigeminal nucleus. Phosphorylase kinase was also localized in the choroid plexus and to a lesser degree in the ependymal cells lining the ventricles. Phosphorylase kinase thus appears to have the same cellular distribution in nervous tissue as its substrates, i.e. glycogen phosphorylase and glycogen, which suggests that the physiological role of brain phosphorylase kinase is the mobilization of glycogen stores to fuel the increased metabolic demands of neurons and astrocytes. 相似文献
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Immunocytochemical localization of protein kinase C in developing brain tissue and in primary neuronal cultures 总被引:2,自引:0,他引:2
Antisera to protein kinase C (PKC) have been used to examine the presence and distribution of the enzyme in developing cerebellar cortex of postnatal rat and in cultures of rat sympathetic ganglia. In the cerebellar cortex of 2-,4-, and 6-day old rats, immunostaining was observed in areas of early-forming presynaptic terminals and growth cones. No staining was evident in the cortical proliferative zone. Beginning at 10 days postnatal, nuclear staining, not apparent at earlier stages, was prominent in Purkinje cells. In neuronal cultures of dissociated rat sympathetic ganglia, PKC was immunolocalized in cell bodies and bundles of neuronal processes. Immunoreactivity was particularly striking in growth cones of extending neurites and in axonal varicosities. These results suggest a role for PKC in neuronal growth following cell proliferation and in synaptic function. The appearance of nuclear staining in later developmental stages suggests that the enzyme may be involved in the promotion and maintenance of the differentiated state of neurons. 相似文献
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In a basic approach to investigations of neuronal–glial interactions during both normal brain development and its pathogenesis, embryonic brain cell populations were fractionated into purified neuronal and glial components. Using separation procedures based on differential adhesion and cytotoxicity, the isolated neuronal and glial phenotypes could be identified by distinct morphological and biochemical characteristics, including the visualization of glial fibrillary acid protein (GFA) within glial cells in immunohistochemical assays with monospecific anti-GFA serum. When unfractionated cerebrum cells dissociated from 10-day chick or 14-day mouse embryos were plated as monolayers and cultured for 1-14 days, monospecific antiserum against fibronectin (LETS glycoprotein) was found to react with many, but not all, of the cells as revealed by indirect immunofluorescence microscopy. The isolated neuronal and glial components of these populations were used to determine whether the appearance of membrane-associated fibronectin was characteristic of one cell type or the other, or both, and if neuronal–glial cell interaction was required for its expression. It was found that the surfaces of glial cells, completely isolated from neurons, showed an intense fluorescent reaction to the anti-fibronectin serum. In contrast, the purified neuronal cultures showed no fluorescence with either the anti-GFA or anti-fibronectin sera. These results demonstrate fibronectin as a cell surface protein associated primarily with glial cells and independent of neuronal–glial cell interaction for its expression. Furthermore, the results indicate that the fibronectin observed on glial cell surfaces in these cultures is produced endogenously and is not due to the preferential binding of fibronectin present in the culture medium. The role of fibronectin as an adhesive molecule in neuronal–glial interactions is discussed. 相似文献
8.
Background
Organotypic brain slice culturing techniques are extensively used in a wide range of experimental procedures and are particularly useful in providing mechanistic insights into neurological disorders or injury. The cellular and morphological alterations associated with hippocampal brain slice cultures has been well established, however, the neuronal response of mouse cortical neurons to culture is not well documented.Methods
In the current study, we compared the cell viability, as well as phenotypic and protein expression changes in cortical neurons, in whole brain slice cultures from mouse neonates (P4–6), adolescent animals (P25–28) and mature adults (P50+). Cultures were prepared using the membrane interface method.Results
Propidium iodide labeling of nuclei (due to compromised cell membrane) and AlamarBlue™ (cell respiration) analysis demonstrated that neonatal tissue was significantly less vulnerable to long-term culture in comparison to the more mature brain tissues. Cultures from P6 animals showed a significant increase in the expression of synaptic markers and a decrease in growth-associated proteins over the entire culture period. However, morphological analysis of organotypic brain slices cultured from neonatal tissue demonstrated that there were substantial changes to neuronal and glial organization within the neocortex, with a distinct loss of cytoarchitectural stratification and increased GFAP expression (p<0.05). Additionally, cultures from neonatal tissue had no glial limitans and, after 14 DIV, displayed substantial cellular protrusions from slice edges, including cells that expressed both glial and neuronal markers.Conclusion
In summary, we present a substantial evaluation of the viability and morphological changes that occur in the neocortex of whole brain tissue cultures, from different ages, over an extended period of culture. 相似文献9.
Terry Ritchie Sheila A. Scully Jean de Vellis Ernest P. Noble 《Neurochemical research》1986,11(3):383-392
The enzymatic activities in post-mortem rat brain kept at 4°C and at 25°C were determined for a number of enzymes localized in specific cell types in the central nervous system. Choline acetyltransferase (CAT), glycerol-3-phosphate dehydrogenase (GPDH), glutamine synthetase (GS), lactate dehydrogenase (LDH) and 2,3-cyclic nucleotide phosphohydrolase (CNPase) were found to be very stable at both 4°C and 25°C with only slight, if any, losses of activity being seen even at periods as long as 72 hr. Glutamic acid decarboxylase (GAD) activity was less stable than that of the other enzymes. In brains kept at 4°C GAD activity was stable out to 24 hr after which it began to decline rapidly to 65% of control at 72 hr. In brains kept at 25°C, GAD activity was stable for 6–8 hr and then began to steadily decline to 58% of control at 24 hr and 29% of control at 72 hr. Assuming that these enzymes have similar stabilities in post-mortem human brain, the effect of post-mortem delay in processing tissues may be of lesser significance than other factors with regard to the measured enzyme activities in human brain samples. 相似文献
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The inhibition of the growth of Yersinia enterocolitica by other gram-negative bacteria in mixed cultures at 32 degrees C was not the consequence of a depletion in essential nutrients, an unfavorable change in pH or oxygen tension or the production of toxic metabolic products. The inability of Y. enterocolitica to attain its potential maximum population in mixed cultures appeared instead to result from "metabolic crowding," which occurred when the faster-growing antagonistic organism reached stationary-phase density. Lowering the incubation temperature, a technique commonly used in "cold" enrichment for isolation of Y. enterocolitica, tended to equalize growth rates and thereby allowed Y. enterocolitica to achieve a higher population. 相似文献
15.
Identification of prothymosin-alpha1, the necrosis-apoptosis switch molecule in cortical neuronal cultures 下载免费PDF全文
We initially identified a nuclear protein, prothymosin-alpha1 (ProTalpha), as a key protein inhibiting necrosis by subjecting conditioned media from serum-free cultures of cortical neurons to a few chromatography steps. ProTalpha inhibited necrosis of cultured neurons by preventing rapid loss of cellular adenosine triphosphate levels by reversing the decreased membrane localization of glucose transporters but caused apoptosis through up-regulation of proapoptotic Bcl(2)-family proteins. The apoptosis caused by ProTalpha was further inhibited by growth factors, including brain-derived neurotrophic factor. The ProTalpha-induced cell death mode switch from necrosis to apoptosis was also reproduced in experimental ischemia-reperfusion culture experiments, although the apoptosis level was markedly reduced, possibly because of the presence of growth factors in the reperfused serum. Knock down of PKCbeta(II) expression prevented this cell death mode switch. Collectively, these results suggest that ProTalpha is an extracellular signal protein that acts as a cell death mode switch and could be a promising candidate for preventing brain strokes with the help of known apoptosis inhibitors. 相似文献
16.
Eggenreich K Zeipper U Schwendenwein E Hadju S Kaltenecker G Laslo I Lang S Roschger P Vecsei V Wintersteiger R 《Journal of biochemical and biophysical methods》2002,53(1-3):51-59
A highly specific and sensitive isocratic reversed-phase high performance liquid chromatography (HPLC) method for the determination of the major component of teicoplanin in tissue is reported. Comparing fluorescamine and o-phthalaldehyde (OPA) as derivatizing agents, the derivative formed with the latter exhibits superior fluorescence intensity allowing detection of femtomole quantities. Pretreatment for tissue samples is by solid-phase extraction which uses Bakerbond PolarP C(18) cartridges and gives effective clean up from endogenous by-products. Linearity was given from 0.6 to 100 ng per injection. The coefficient of variation did not exceed 5.8% for both interday and intraday assays. It was found that when bone defects are repaired with a hydroxyapatite-teicoplanin mixture, the antibiotic does not degrade, even when it is in the cement for several months. The stability of teicoplanin in bone cement was determined fluorodensitometrically. 相似文献
17.
Di Loreto S Maccarone R Corvetti L Sebastiani P Piancatelli D Adorno D 《European cytokine network》2003,14(2):97-102
We analysed the specific effects of IL-1beta immunoneutralization on the expression of IL-6 in different pure cultures of neurones and glia after both experimental subliminal hypoxia and recovery. Whereas the IL-1beta-deprivation signal induced a decrease in IL-6 expression and release of normoxic neurones, it provoked an increase in IL-6 protein in hypoxic neurones. Moreover, the direct correlation between IL-1beta and IL-6, observed in normal and recovering neuronal cultures, was reversed in hypoxic conditions. These reversals were not observed in glial cells, in which IL-1beta immunosuppression led to a decrease in IL-6 under all conditions considered. In conclusion, the IL-1beta modulates IL-6 in different ways according to the ambient physiological or pathological conditions, and also acts via different mechanisms, depending on the cellular phenotype. 相似文献
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Knutsen AK Chang YV Grimm CM Phan L Taber LA Bayly PV 《Journal of biomechanical engineering》2010,132(10):101004
Folding of the cerebral cortex is a critical phase of brain development in higher mammals but the biomechanics of folding remain incompletely understood. During folding, the growth of the cortical surface is heterogeneous and anisotropic. We developed and applied a new technique to measure spatial and directional variations in surface growth from longitudinal magnetic resonance imaging (MRI) studies of a single animal or human subject. MRI provides high resolution 3D image volumes of the brain at different stages of development. Surface representations of the cerebral cortex are obtained by segmentation of these volumes. Estimation of local surface growth between two times requires establishment of a point-to-point correspondence ("registration") between surfaces measured at those times. Here we present a novel approach for the registration of two surfaces in which an energy function is minimized by solving a partial differential equation on a spherical surface. The energy function includes a strain-energy term due to distortion and an "error energy" term due to mismatch between surface features. This algorithm, implemented with the finite element method, brings surface features into approximate alignment while minimizing deformation in regions without explicit matching criteria. The method was validated by application to three simulated test cases and applied to characterize growth of the ferret cortex during folding. Cortical surfaces were created from MRI data acquired in vivo at 14 days, 21 days, and 28 days of life. Deformation gradient and Lagrangian strain tensors describe the kinematics of growth over this interval. These quantitative results illuminate the spatial, temporal, and directional patterns of growth during cortical folding. 相似文献
20.
Antagonism by gram-negative bacteria to growth of Yersinia enterocolitica in mixed cultures. 总被引:3,自引:1,他引:2 下载免费PDF全文
The inhibition of the growth of Yersinia enterocolitica by other gram-negative bacteria in mixed cultures at 32 degrees C was not the consequence of a depletion in essential nutrients, an unfavorable change in pH or oxygen tension or the production of toxic metabolic products. The inability of Y. enterocolitica to attain its potential maximum population in mixed cultures appeared instead to result from "metabolic crowding," which occurred when the faster-growing antagonistic organism reached stationary-phase density. Lowering the incubation temperature, a technique commonly used in "cold" enrichment for isolation of Y. enterocolitica, tended to equalize growth rates and thereby allowed Y. enterocolitica to achieve a higher population. 相似文献