Visualization of the membrane surface and cytoskeleton of oligodendrocyte progenitor cell growth cones using a combination of scanning ion conductance and four times expansion microscopy.

Growth cones of oligodendrocyte progenitor cells (OPCs) are challenging to investigate with conventional light microscopy due to their small size. Especially substructures such as filopodia, lamellipodia and their underlying cytoskeleton are difficult to resolve with diffraction limited microscopy. Light microscopy techniques, which surpass the diffraction limit such as stimulated emission depletion microscopy, often require expensive setups and specially trained personnel rendering them inaccessible to smaller research groups. Lately, the invention of expansion microscopy (ExM) has enabled super-resolution imaging with any light microscope without the need for additional equipment. Apart from the necessary resolution, investigating OPC growth cones comes with another challenge: Imaging the topography of membranes, especially label- and contact-free, is only possible with very few microscopy techniques one of them being scanning ion conductance microscopy (SICM). We here present a new imaging workflow combining SICM and ExM, which enables the visualization of OPC growth cone nanostructures. We correlated SICM recordings and ExM images of OPC growth cones captured with a conventional widefield microscope. This enabled the visualization of the growth cones' membrane topography as well as their underlying actin and tubulin cytoskeleton.

Deficiency of Thyroid Hormone Reduces Voltage-Gated Na+ Currents as Well as Expression of Na+/K+-ATPase in the Mouse Hippocampus.

Mice lacking functional thyroid follicular cells, Pax8-/- mice, die early postnatally, making them suitable models for extreme hypothyroidism. We have previously obtained evidence in postnatal rat neurons, that a down-regulation of Na+-current density could explain the reduced excitability of the nervous system in hypothyroidism. If such a mechanism underlies the development of coma and death in severe hypothyroidism, Pax8-/- mice should show deficits in the expression of Na+ currents and potentially also in the expression of Na+/K+-ATPases, which are necessary to maintain low intracellular Na+ levels. We thus compared Na+ current densities in postnatal mice using the patch-clamp technique in the whole-cell configuration as well as the expression of three alpha and two beta-subunits of the Na+/K+-ATPase in wild type versus Pax8-/- mice. Whereas the Na+ current density in hippocampal neurons from wild type mice was upregulated within the first postnatal week, the Na+ current density remained at a very low level in hippocampal neurons from Pax8-/- mice. Pax8-/- mice also showed significantly decreased protein expression levels of the catalytic α1 and α3 subunits of the Na+/K+-ATPase as well as decreased levels of the ß2 isoform, with no changes in the α2 and ß1 subunits.

Adjusting the neuron to astrocyte ratio with cytostatics in hippocampal cell cultures from postnatal rats: A comparison of cytarabino furanoside (AraC) and 5-fluoro-2'-deoxyuridine (FUdR).

Cell culture studies offer the unique possibility to investigate the influence of pharmacological treatments with quantified dosages applied for defined time durations on survival, morphological maturation, protein expression and function as well as the mutual interaction of various cell types. Cultures obtained from postnatal rat brain contain a substantial number of glial cells that further proliferate with time in culture leading to an overgrowth of neurons with glia, especially astrocytes and microglia. A well-established method to decrease glial proliferation in vitro is to apply low concentrations of cytosine arabinoside (AraC). While AraC primarily effects dividing cells, it has been reported repeatedly that it is also neurotoxic, which is the reason why most protocols limit its application to concentrations of up to 5 µM for a duration of 24 h. Here, we investigated 5-fluoro-2'-deoxyuridine (FUdR) as a possible substitute for AraC. We applied concentrations of both cytostatics ranging from 4 µM to 75 µM and compared cell composition and cell viability in cultures prepared from 0-2- and 3-4-day old rat pups. Using FUdR as proliferation inhibitor, higher ratios of neurons to glia cells were obtained with a maximal neuron to astrocyte ratio of up to 10:1, which could not be obtained using AraC in postnatal cultures. Patch-clamp recordings revealed no difference in the amplitudes of voltage-gated Na+ currents in neurons treated with FUdR compared with untreated control cells suggesting replacement of AraC by FUdR as glia proliferation inhibitor if highly neuron-enriched postnatal cultures are desired.

Novel Tools towards Magnetic Guidance of Neurite Growth: (I) Guidance of Magnetic Nanoparticles into Neurite Extensions of Induced Human Neurons and In Vitro Functionalization with RAS Regulating Proteins.

Parkinson's disease (PD) is a neurodegenerative disease associated with loss or dysfunction of dopaminergic neurons located in the substantia nigra (SN), and there is no cure available. An emerging new approach for treatment is to transplant human induced dopaminergic neurons directly into the denervated striatal brain target region. Unfortunately, neurons grafted into the substantia nigra are unable to grow axons into the striatum and thus do not allow recovery of the original connectivity. Towards overcoming this general limitation in guided neuronal regeneration, we develop here magnetic nanoparticles functionalized with proteins involved in the regulation of axonal growth. We show covalent binding of constitutive active human rat sarcoma (RAS) proteins or RAS guanine nucleotide exchange factor catalytic domain of son of sevenless (SOS) by fluorescence correlation spectroscopy and multiangle light scattering as well as the characterization of exchange factor activity. Human dopaminergic neurons were differentiated from neural precursor cells and characterized by electrophysiological and immune histochemical methods. Furthermore, we demonstrate magnetic translocation of cytoplasmic γ-Fe2O3@SiO2 core-shell nanoparticles into the neurite extensions of induced human neurons. Altogether, we developed tools towards remote control of directed neurite growth in human dopaminergic neurons. These results may have relevance for future therapeutic approaches of cell replacement therapy in Parkinson's disease.

Differential expression patterns of sodium potassium ATPase alpha and beta subunit isoforms in mouse brain during postnatal development.

The sodium potassium ATPase (Na+/K+ ATPase) is essential for the maintenance of a low intracellular Na+ and a high intracellular K+ concentration. Loss of function of the Na+/K+ ATPase due to mutations in Na+/K+ ATPase genes, anoxic conditions, depletion of ATP or inhibition of the Na+/K+ ATPase function using cardiac glycosides such as digitalis, causes a depolarization of the resting membrane potential. While in non-excitable cells, the uptake of glucose and amino acids is decreased if the function of the Na+/K+ ATPase is compromised, in excitable cells the symptoms range from local hyper-excitability to inactivating depolarization. Although several studies have demonstrated the differential expression of the various Na+/K+ ATPase alpha and beta isoforms in the brain tissue of rodents, their expression profile during development has yet to be thoroughly investigated. An immunohistochemical analysis of postnatal day 19 mouse brain showed ubiquitous expression of Na+/K+ ATPase isoforms α1, ß1 and ß2 in both neurons and glial cells, whereas α2 was expressed mostly in glial cells and the α3 and ß3 isoforms were expressed in neurons. Furthermore, we examined potential changes in the relative expression of the different Na+/K+ ATPase isoforms in different brain areas of postnatal day 6 and in adult 9 months old animals using immunoblot analysis. Our results show a significant up-regulation of the α1 isoform in cortex, hippocampus and cerebellum, whereas, the α2 isoform was significantly up-regulated in midbrain. The ß3 isoform showed a significant up-regulation in all brain areas investigated. The up-regulation of the α3 isoform matched that of the ß2 isoform which were both significantly up-regulated in cortex, hippocampus and midbrain, suggesting that the increased maturation of the neuronal network is accompanied by an increase in expression of α3/ß2 complexes in these brain structures.

Regulation of neuronal excitability by release of proteins from glial cells.

Effects of glial cells on electrical isolation and shaping of synaptic transmission between neurons have been extensively studied. Here we present evidence that the release of proteins from astrocytes as well as microglia may regulate voltage-activated Na(+) currents in neurons, thereby increasing excitability and speed of transmission in neurons kept at distance from each other by specialized glial cells. As a first example, we show that basic fibroblast growth factor and neurotrophin-3, which are released from astrocytes by exposure to thyroid hormone, influence each other to enhance Na(+) current density in cultured hippocampal neurons. As a second example, we show that the presence of microglia in hippocampal cultures can upregulate Na(+) current density. The effect can be boosted by lipopolysaccharides, bacterial membrane-derived stimulators of microglial activation. Comparable effects are induced by the exposure of neuron-enriched hippocampal cultures to tumour necrosis factor-α, which is released from stimulated microglia. Taken together, our findings suggest that release of proteins from various types of glial cells can alter neuronal excitability over a time course of several days. This explains changes in neuronal excitability occurring in states of thyroid hormone imbalance and possibly also in seizures triggered by infectious diseases.

Long-term, long-distance recording of a living migrating neuron by scanning ion conductance microscopy.

Bias-free, three-dimensional imaging of entire living cellular specimen is required for investigating shape and volume changes that occur during cellular growth or migration. Here we present fifty consecutive recordings of a living cultured neuron from a mouse dorsal root ganglion obtained by Scanning ion conductance microscopy (SICM). We observed a saltatory migration of the neuron with a mean velocity of approximately 20 µm/h. These results demonstrate the non-invasiveness of SICM, which makes it unique among the scanning probe microscopes. In contrast to SICM, most scanning probe techniques require a usually denaturating preparation of the cells, or they exert a non-negligible force on the cellular membrane, impeding passive observation. Moreover, the present series of recordings demonstrates the potential use of SICM for the detailed investigation of cellular migration and membrane surface dynamics even of such delicate samples as living neurons.

Effect of sample slope on image formation in scanning ion conductance microscopy.

Scanning ion conductance microscopy (SICM) is a scanning probe technique that allows investigating surfaces of complex, convoluted samples such as living cells with minimal impairment. This technique monitors the ionic current through the small opening of an electrolyte-filled micro- or nanopipet that is approached toward a sample, submerged in an electrolyte. The conductance drops in a strongly distance-dependent manner. For SICM imaging, the assumption is made that positions of equal conductance changes correspond to equal tip-sample distances and thus can be utilized to reconstruct the sample surface. Here, we examined this assumption by investigating experimental approach curves toward silicone droplets, as well as finite element modeling of the imaging process. We found that the assumption is strictly true only for perpendicular approaches toward a horizontal sample and otherwise overestimates the sample height by up to several pipet opening radii. We developed a method to correct this overestimation and applied it to correct images of fixed cellular structures and living entire cells.

Migrating oligodendrocyte progenitor cells swell prior to soma dislocation.

The migration of oligodendrocyte progenitor cells (OPCs) to the white matter is an indispensable requirement for an intact brain function. The mechanism of cell migration in general is not yet completely understood. Nevertheless, evidence is accumulating that besides the coordinated rearrangement of the cytoskeleton, a finetuned interplay of ion and water fluxes across the cell membrane is essential for cell migration. One part of a general hypothesis is that a local volume increase towards the direction of movement triggers a mechano-activated calcium influx that regulates various procedures at the rear end of a migrating cell. Here, we investigated cell volume changes of migrating OPCs using scanning ion conductance microscopy. We found that during accelerated migration OPCs undergo an increase in the frontal cell body volume. These findings are supplemented with time lapse calcium imaging data that hint an increase in calcium content the frontal part of the cell soma.

Strategies for repair of white matter: influence of osmolarity and microglia on proliferation and apoptosis of oligodendrocyte precursor cells in different basal culture media.

The aim of the present study has been to obtain high yields of oligodendrocyte precursor cells (OPCs) in culture. This is a first step in facilitation of myelin repair. We show that, in addition to factors, known to promote proliferation, such as basic fibroblast growth factor (FGF-2) and platelet derived growth factor (PDGF) the choice of the basal medium exerts a significant influence on the yield of OPCs in cultures from newborn rats. During a culture period of up to 9 days we observed larger numbers of surviving cells in Dulbecco's Modified Eagle Medium (DMEM), and Roswell Park Memorial Institute Medium (RPMI) compared with Neurobasal Medium (NB). A larger number of A2B5-positive OPCs was found after 6 days in RPMI based media compared with NB. The percentage of bromodeoxyuridine (BrdU)-positive cells was largest in cultures maintained in DMEM and RPMI. The percentage of caspase-3 positive cells was largest in NB, suggesting that this medium inhibits OPC proliferation and favors apoptosis. A difference between NB and DMEM as well as RPMI is the reduced Na(+)-content. The addition of equiosmolar supplements of mannitol or NaCl to NB medium rescued the BrdU-incorporation rate. This suggested that the osmolarity influences the proliferation of OPCs. Plating density as well as residual microglia influence OPC survival, BrdU incorporation, and caspase-3 expression. We found, that high density cultures secrete factors that inhibit BrdU incorporation whereas the presence of additional microglia induces an increase in caspase-3 positive cells, indicative of enhanced apoptosis. An enhanced number of microglia could thus also explain the stronger inhibition of OPC differentiation observed in high density cultures in response to treatment with the cytokines TNF-α and IFN-γ. We conclude that a maximal yield of OPCs is obtained in a medium of an osmolarity higher than 280 mOsm plated at a relatively low density in the presence of as little microglia as technically achievable.

Scanning ion conductance microscopy for studying biological samples.

Scanning ion conductance microscopy (SICM) is a scanning probe technique that utilizes the increase in access resistance that occurs if an electrolyte filled glass micro-pipette is approached towards a poorly conducting surface. Since an increase in resistance can be monitored before the physical contact between scanning probe tip and sample, this technique is particularly useful to investigate the topography of delicate samples such as living cells. SICM has shown its potential in various applications such as high resolution and long-time imaging of living cells or the determination of local changes in cellular volume. Furthermore, SICM has been combined with various techniques such as fluorescence microscopy or patch clamping to reveal localized information about proteins or protein functions. This review details the various advantages and pitfalls of SICM and provides an overview of the recent developments and applications of SICM in biological imaging. Furthermore, we show that in principle, a combination of SICM and ion selective micro-electrodes enables one to monitor the local ion activity surrounding a living cell.

Normal sulfation levels regulate spinal cord neural precursor cell proliferation and differentiation.

BACKGROUND: Sulfated glycosaminoglycan chains are known for their regulatory functions during neural development and regeneration. However, it is still unknown whether the sulfate residues alone influence, for example, neural precursor cell behavior or whether they act in concert with the sugar backbone. Here, we provide evidence that the unique 473HD-epitope, a representative chondroitin sulfate, is expressed by spinal cord neural precursor cells in vivo and in vitro, suggesting a potential function of sulfated glycosaminoglycans for spinal cord development. RESULTS: Thus, we applied the widely used sulfation inhibitor sodium chlorate to analyze the importance of normal sulfation levels for spinal cord neural precursor cell biology in vitro. Addition of sodium chlorate to spinal cord neural precursor cell cultures affected cell cycle progression accompanied by changed extracellular signal-regulated kinase 1 or 2 activation levels. This resulted in a higher percentage of neurons already under proliferative conditions. In contrast, the relative number of glial cells was largely unaffected. Strikingly, both morphological and electrophysiological characterization of neural precursor cell-derived neurons demonstrated an attenuated neuronal maturation in the presence of sodium chlorate, including a disturbed neuronal polarization. CONCLUSIONS: In summary, our data suggest that sulfation is an important regulator of both neural precursor cell proliferation and maturation of the neural precursor cell progeny in the developing mouse spinal cord.

A boundary delimitation algorithm to approximate cell soma volumes of bipolar cells from topographical data obtained by scanning probe microscopy.

BACKGROUND: Cell volume determination plays a pivotal role in the investigation of the biophysical mechanisms underlying various cellular processes. Whereas light microscopy in principle enables one to obtain three dimensional data, the reconstruction of cell volume from z-stacks is a time consuming procedure. Thus, three dimensional topographic representations of cells are easier to obtain by scanning probe microscopical measurements. RESULTS: We present a method of separating the cell soma volume of bipolar cells in adherent cell cultures from the contributions of the cell processes from data obtained by scanning ion conductance microscopy. Soma volume changes between successive scans obtained from the same cell can then be computed even if the cell is changing its position within the observed area. We demonstrate that the estimation of the cell volume on the basis of the width and the length of a cell may lead to erroneous determination of cell volume changes. CONCLUSIONS: We provide a new algorithm to repeatedly determine single cell soma volume and thus to quantify cell volume changes during cell movements occuring over a time range of hours.

Backstep scanning ion conductance microscopy as a tool for long term investigation of single living cells.

Scanning ion conductance microscopy (SICM) is a suitable tool for imaging surfaces of living cells in a contact-free manner. We have shown previously that SICM in backstep mode allows one to trace the outlines of entire cell somata and to detect changes in cellular shape and volume. Here we report that SICM can be employed to quantitatively observe cellular structures such as cell processes of living cells as well as cell somata of motile cells in the range of hours.

The use of size-defined DNA-functionalized calcium phosphate nanoparticles to minimise intracellular calcium disturbance during transfection.

Calcium phosphate-based transfection methods are frequently used to transfer DNA into living cells. However, it has so far not been studied in detail to what extend the different transfection methods lead to a net calcium uptake. Upon subsequent resolution of the calcium phosphate, intracellular free ionic calcium-surges could result, inducing as side effect various physiological responses that may finally result in cell death. Here we investigated the overall calcium uptake by the human bladder carcinoma cell line T24 during the standard calcium phosphate transfection method and also during transfection with custom-made calcium phosphate/DNA nanoparticles by isotope labelling with (45)calcium. (45)Calcium uptake was strongly increased after 7h of standard calcium phosphate transfection but not if the transfection was performed with calcium phosphate nanoparticles. Time lapse imaging microscopy using the calcium-sensitive dye Fura-2 revealed large transient increases of the intracellular free calcium level during the standard calcium phosphate transfection but not if calcium phosphate nanoparticles were used. Consistently, the viability of cells transfected by calcium phosphate/DNA nanoparticles was not changed, in remarkable contrast to the standard method where considerable cell death occurred.

Thyroid hormone (T3)-induced up-regulation of voltage-activated sodium current in cultured postnatal hippocampal neurons requires secretion of soluble factors from glial cells.

We have previously shown that treatment with the thyroid hormone T(3) increases the voltage-gated Na(+)current density (Nav-D) in hippocampal neurons from postnatal rats, leading to accelerated action potential upstrokes and increased firing frequencies. Here we show that the Na(+) current regulation depends on the presence of glial cells, which secrete a heat-instable soluble factor upon stimulation with T(3). The effect of conditioned medium from T(3)-treated glial cells was mimicked by basic fibroblast growth factor (bFGF), known to be released from cerebellar glial cells after T(3) treatment. Neutralization assays of astrocyte-conditioned media with anti-bFGF antibody inhibited the regulation of the Nav-D by T(3). This suggests that the up-regulation of the neuronal sodium current density by T(3) is not a direct effect but involves bFGF release and satellite cells. Thus glial cells can modulate neuronal excitability via secretion of paracrinely acting factors.

Corticosteroids reverse cytokine-induced block of survival and differentiation of oligodendrocyte progenitor cells from rats.

BACKGROUND: Periventricular leukomalacia (PVL) is a frequent complication of preterm delivery. Proinflammatory cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) released from astrocytes and microglia activated by infection or ischemia have previously been shown to impair survival and maturation of oligodendrocyte progenitors and could thus be considered as potential factors contributing to the generation of this disease. The first goal of the present study was to investigate whether exposure of oligodendrocyte precursors to these cytokines arrests the maturation of ion currents in parallel to its effects on myelin proteins and morphological maturation. Secondly, in the search for agents, that can protect differentiating oligodendrocyte precursor cells from cytokine-induced damage we investigated effects of coapplications of corticosteroids with proinflammatory cytokines on the subsequent survival and differentiation of oligodendrocyte progenitor cells. METHODS: To exclude influences from factors released from other cell types purified cultures of oligodendrocyte precursors were exposed to cytokines and/or steroids and allowed to differentiate for further 6 days in culture. Changes in membrane surface were investigated with capacitance recordings and Scanning Ion Conductance Microscopy. Na+- and K+- currents were investigated using whole cell patch clamp recordings. The expression of myelin specific proteins was investigated using western blots and the precursor cells were identified using immunostaining with A2B5 antibodies. RESULTS: Surviving IFN-gamma and TNF-alpha treated cells continued to maintain voltage-activated Na+- and K+ currents characteristic for the immature cells after 6 days in differentiation medium. Corticosterone, dihydrocorticosterone and, most prominently dexamethasone, counteracted the deleterious effects of IFN-gamma and TNF-alpha on cell survival, A2B5-immunostaining and expression of myelin basic protein. The most potent corticosteroid tested, dexamethasone, was shown to counteract cytokine effects on membrane surface extension and capacitance. Furthermore, coapplication of dexamethasone blocked the cytokine-induced downregulation of the inwardly rectifying potassium current in 80% of the precursor cells and restored the cytokine-blocked down-regulation of the voltage activated Na+- and K+ currents during subsequent differentiation. CONCLUSION: Our results show that treatment of oligodendrocyte precursors with the inflammatory cytokines TNF-alpha and IFN-gamma block the differentiation of oligodendrocyte precursors at the level of the differentiation of the voltage-gated ion currents. Co-treatment with corticosteroids at the time of cytokine application restores to a considerable extent survival and differentiation of oligodendrocytes at the level of morphological, myelin protein as well as ion current maturation suggesting the option for a functional restoration of cytokine-damaged immature oligodendrocytes.

Enhancement of dopaminergic properties and protection mediated by neuronal activation of Ras in mouse ventral mesencephalic neurones.

The poor differentiation and survival of dopaminergic neurones are practical constraints in their therapeutic applications. Here we explored the role of neuronally activated Ras in ventral mesencephalon-derived neurospheres generated from synRas mouse embryos. The expression of Val12 Ha-Ras transgene and enhanced Ras activity was evident after differentiation of the neurospheres with a corresponding activating phosphorylation of mitogen-activated protein kinase. Phosphorylation of Akt/PKB, the target kinase of phosphoinositide 3-kinase, along with phosphorylation of Bad and CREB were enhanced in synRas-derived differentiated neurosphere cultures. Furthermore, increased Nurr1 expression was associated with elevated numbers of dopaminergic neurones in synRas-derived cultures compared with the wild-type. Correspondingly, tyrosine hydroxylase promoter assays revealed enhanced transcriptional activation of the promoter in synRas-derived cultures. synRas-derived dopaminergic neurones were greatly resistant to degeneration induced by various noxious stimuli. Consistently, the transgenic expression of activated Ras attenuated the adverse 6-hydroxydopamine effects on dopaminergic neurones. Dopaminergic neurones derived from both wild-type and synRas cultures expressed voltage-gated potassium and sodium currents, fired action potentials and exhibited electrical network activity. Thus, expression of the transgene promotes survival and enhances differentiation towards a dopaminergic cell fate without altering their basic electrical properties. Our results suggest that intracellular cell therapy mimicking trophic signalling may offer potential benefit in models of human disease associated with dopamine neurone dysfunction.

Constant-distance mode scanning potentiometry. 1. Visualization of calcium carbonate dissolution in aqueous solution.

Constant-distance mode scanning potentiometry was established by integrating potentiometric microsensors as ion-selective scanning probes into a SECM setup that was equipped with a piezoelectric shear force-based tip-to-sample distance control. The combination of specially designed micrometer-sized potentiometric tips with an advanced system for tip positioning allowed simultaneous acquisition of both topographic and potentiometric information at solid/liquid interfaces with high spatial resolution. The performance of the approach was evaluated by applying Ca(2+)-selective constant-distance mode potentiometry to monitor the dissolution of calcium carbonate occurring either at the (104) surface of calcite crystals or in proximity to the more complex surface of cross sections of a calcium carbonate shell of Mya arenaria exposed to slightly acidic aqueous solutions. Micrometer-scale heterogeneities in the apparent calcium activity profiles have successfully been resolved for both samples.

Effects of interferon-gamma and tumor necrosis factor-alpha on survival and differentiation of oligodendrocyte progenitors.

OBJECTIVE: There is strong evidence from recent clinical studies that ascending intrauterine infection is associated with an increased incidence of periventricular leukomalacia in very premature fetuses. Periventricular leukomalacia is characterized by disrupted myelination from a loss of oligodendrocyte progenitors. We investigated the effects of proinflammatory cytokines on the survival and differentiation of this cell type. METHODS: Cultures of more than 90% A2B5-positive progenitors were prepared from neonatal rats and kept for 3 days in medium supplemented with factors that stimulate cell proliferation. After 1 day in proliferation medium, cells were treated with interferon-gamma (100 U/mL) and tumor necrosis factor-alpha (100 ng/mL) for 48 hours triggering an increase in apoptotic A2B5 progenitor cells from 3.2 +/- 2.3% to 11.0 +/- 2.6%. After cytokine treatment cultures were transferred to medium containing factors to promote differentiation of progenitors into the myelinating phenotype. RESULTS: In cytokine pretreated cultures, only 2.6 +/- 1.1% of total cells survived after a total of 9 days in vitro, whereas in untreated cultures most cells differentiated as shown by expression of myelin basic protein, myelin-associated glycoprotein, 2,3-cyclic nucleotide 3-phosphodiesterase, and myelin oligodendrocyte-specific protein. Using ten-fold reduced concentrations of combined interferon-gamma (10 U/mL) and tumor necrosis factor-alpha (10 ng/mL) pretreatment resulted in a survival to 11.2 +/- 4.9% of total cells with 36.3 +/- 11.6% A2B5-positive cells at day 9. This indicates a major enrichment of undifferentiated cells compared with untreated controls which harbored only 1.0 +/- 0.3% A2B5-positive cells. CONCLUSION: Inflammatory cytokines not only induced apoptotic cell death but also prevented the differentiation of immature A2B5 oligodendrocyte progenitors into the myelinating phenotype.