Role of astrocyte-derived tissue-type plasminogen activator in the regulation of endotoxin-stimulated nitric oxide production by microglial cells

In mixed glial cell cultures from cerebral cortices of newborn rats, endotoxin induces nitric oxide (NO) production in microglial cells. Earlier we demonstrated that endotoxin induced NO production by microglial cells is inhibited in the presence of astroglial cells by transforming growth factor β (TGFβ). Both microglial and astroglial cells produce TGFβ in a biologically inactive form, which can be activated by plasmin generated by plasminogen activators (PA). In the present paper we describe studies on the mechanism by which glial cells may activate inactive TGFβ and its potential inhibitory effect on NO production by microglial cells. Inhibition of plasmin increased NO production in endotoxin-treated mixed glial cell cultures. Subsequently, antibodies against tissue-type plasminogen activator (tPA) increased NO production in endotoxin-treated mixed glial cell cultures while amiloride, an inhibitor for urokinase (uPA), had no effect. We hereby concluded that tPA is the crucial PA involved in plasmin production resulting in inhibition of NO production in mixed glial cell cultures. Zymography and Northern blot analysis of purified astroglial, microglial, and mixed glial cell cultures demonstrated that astroglial cells produce tPA and a plasminogen activator inhibitor (PAI-1) and are thereby responsible for the production of plasmin which may activate the inactive TGFβ in these cultures. In conclusion, astroglial-derived tPA plays a major role in the inhibition of NO production by endotoxin-treated microglial cells through enhanced plasmin production and possible subsequent TGFβ activation. GLIA 22:130–137, 1998. © 1998 Wiley-Liss, Inc.     

Inhibition of endotoxin-induced nitric oxide synthase production in microglial cells by the presence of astroglial cells: A role for transforming growth factor β

In mixed glial cell cultures from cerebral cortices of newborn rats, endotoxin induces inducible nitric oxide (iNOS), nitric oxide (NO), and interleukin-1β (IL-1β) production in microglial cells. Earlier we demonstrated that endotoxin induced iNOS but not IL-1β expression in microglial cells is inhibited by the presence of astroglial cells. In the present paper we describe studies on the mechanism by which astroglial cells exert selective suppressive action on iNOS expression by microglial cells. Expression of iNOS and IL-1β was studied by single or double label immunocytochemical techniques and cell identification was performed with GSA-I-B₄-isolectin and an antibody against GFAP. Production of IL-1β and NO was determined by measurement of IL-1β and nitrite concentrations in cell lysates and the culture medium, respectively. TGFβ, a cytokine known to inhibit NO production by endotoxin challenged macrophages, was measured in culture medium of mixed glial cell cultures using a bioassay. Microglial, astroglial, and mixed glial cell cultures produced similar concentrations of TGFβ. The potential effect of TGFβ was studied by using immunoneutralizing antibodies against TGFβ1 and TGFβ2 on the induction of iNOS in microglial cells in the presence of astroglial cells. Incubation of the mixed glial cell culture with these TGFβ antibodies (3 μg/ml) markedly increased endotoxin-induced NO production and iNOS expression in microglial cells, whereas the production of IL-1β was not affected. The antibodies against TGFβ1 and TGFβ2 marginally increased NO production in pure microglial cell cultures, nonetheless in cultures of purified microglial cells recombinant TGFβ1 and TGFβ2 together with endotoxin inhibited NO production. We conclude that the presence of astroglial cells is essential for the inhibitory effect of TGFβ on NO production by microglial cells (possibly) by activation of TGFβ or by increasing the sensitivity of microglial cells for TGFβ. GLIA 19:190–198, 1997. © 1997 Wiley-Liss, Inc.     

Modulation of glial cell signaling by adenosine and pharmacological reinforcement

In view of the increasing evidence that a pathological glial activation plays a significant role in the development of neurodegenerative diseases, we investigated the underlying molecular signaling as a possible target for a pharmacological therapy. Here, we are particularly focusing on the endogenous modulation of the Ca²⁺ and cyclic nucleotide-dependent signaling by the nucleoside adenosine and its reinforcement by the xanthine derivative propentofylline (PPF). As an experimental model, we used cultured rat microglial cells and astrocytes that are immature, show a high proliferation rate, and resemble in several aspects pathologically activated glial cells. A prolonged increase of the cellular cAMP level favored the differentiation of cultured astrocytes and associated properties required for the physiological nerve cell function. On the other hand, a strengthening of the cyclic nucleotide-dependent signaling inhibited potentially neurotoxic properties of cultured microglial cells. Similar effects were obtained by treatment with propentofylline, which mimicked modulatory adenosine effects and increased the intracellular level of cAMP and cGMP. Such a pharmacological glial cell conditioning, obtained by modifying the strength and the timing of these second messengers, may provide a therapy of neurodegenerative diseases in which a pathological activation of microglial cells and astrocytes is discussed to play a pathogenic role.     

Modulation of glial cell signaling by adenosine and pharmacological reinforcement

In view of the increasing evidence that a pathological glial activation plays a significant role in the development of neurodegenerative diseases, we investigated the underlying molecular signaling as a possible target for a pharmacological therapy. Here, we are particularly focusing on the endogenous modulation of the Ca²⁺ and cyclic nucleotide-dependent signaling by the nucleoside adenosine and its reinforcement by the xanthine derivative propentofylline (PPF). As an experimental model, we used cultured rat microglial cells and astrocytes that are immature, show a high proliferation rate, and resemble in several aspects pathologically activated glial cells. A prolonged increase of the cellular cAMP level favored the differentiation of cultured astrocytes and associated properties required for the physiological nerve cell function. On the other hand, a strengthening of the cyclic nucleotide-dependent signaling inhibited potentially neurotoxic properties of cultured microglial cells. Similar effects were obtained by treatment with propentofylline, which mimicked modulatory adenosine effects and increased the intracellular level of cAMP and cGMP. Such a pharmacological glial cell conditioning, obtained by modifying the strength and the timing of these second messengers, may provide a therapy of neurodegenerative diseases in which a pathological activation of microglial cells and astrocytes is discussed to play a pathogenic role.     

Astrocytes enhance lipopolysaccharide-induced nitric oxide production by microglial cells

Several stimuli result in glial activation and induce nitric oxide (NO) production in microglial and astroglial cells. The bacterial endotoxin lipopolysaccharide (LPS) has been widely used to achieve glial activation in vitro, and several studies show that both microglial and, to a lesser extent, astroglial cell cultures produce NO after LPS treatment. However, NO production in endotoxin-treated astrocyte cultures is controversial. We characterized NO production in microglial, astroglial and mixed glial cell cultures treated with lipopolysaccharide, measured as nitrite accumulation in the culture media. We also identified the NO-producing cells by immunocytochemistry, using specific markers for the inducible NO synthase (iNOS) isoform, microglial and astroglial cells. Only microglial cells showed iNOS immunoreactivity. Thus, contaminating microglial cells were responsible for NO production in the secondary astrocyte cultures. We then analysed the effect of astrocytes on NO production by microglial cells using microglial-astroglial cocultures, and we observed that this production was clearly enhanced in the presence of astroglial cells. Soluble factors released by astrocytes did not appear to be directly responsible for such an effect, whereas nonsoluble factors present in the cell membrane of LPS-treated astrocytes could account, at least in part, for this enhancement.     

Glial activation modulates glutamate neurotoxicity in cerebellar granule cell cultures

We studied the influence of glial cells on the neuronal response to glutamate toxicity in cerebellar granule cell cultures. We compared the effect of glutamate on neuronal viability in neuronal vs. neuronal-glial cultures and determined this effect after pretreating the cultures with the lipopolysaccharide (LPS) of Escherichia coli, agent widely used to induce glial activation. Morphological changes in glial cells and nitric oxide (NO) production were evaluated as indicators of glial activation. We observed that glutamate neurotoxicity in neuronal-glial cultures was attenuated in a certain range of glutamate concentration when compared to neuronal cultures, but it was enhanced at higher glutamate concentrations. This enhanced neurotoxicity was associated with morphological changes in astrocytes and microglial cells in the absence of NO production. LPS treatment induced morphological changes in glial cells in neuronal-glial cultures as well as NO production. These effects occurred in the absence of significant neuronal death. However, when LPS-pretreated cultures were treated with glutamate, the sensitivity of neuronal-glial cultures to glutamate neurotoxicity was increased. This was accompanied by additional morphological changes in glial cells in the absence of a further increase in NO production. These results suggest that quiescent glial cells protect neuronal cells from glutamate neurotoxicity, but reactive glial cells increase glutamate neurotoxicity. Therefore, glial cells play a key role in the neuronal response to a negative stimulus, suggesting that this response can be modified through an action on glial cells.     

Brain angiotensin enhances dopaminergic cell death via microglial activation and NADPH-derived ROS

Angiotensin II (AII) plays a major role in the progression of inflammation and NADPH-derived oxidative stress (OS) in several tissues. The brain possesses a local angiotensin system, and OS and inflammation are key factors in the progression of Parkinson's disease. In rat mesencephalic cultures, AII increased 6-OHDA-induced dopaminergic (DA) cell death, generation of superoxide in DA neurons and microglial cells, the expression of NADPH-oxidase mRNA, and the number of reactive microglial cells. These effects were blocked by AII type-1 (AT1) antagonists, NADPH inhibitors, or elimination of glial cells. DA degeneration increased angiotensin converting enzyme activity and AII levels. In rats, 6-OHDA-induced dopaminergic cell loss and microglial activation were reduced by treatment with AT1 antagonists. The present data suggest that AII, via AT1 receptors, increases the dopaminergic degeneration process by amplifying the inflammatory response and intraneuronal levels of OS, and that glial cells play a major role in this process.     

Microglial cell upregulation of HIV-1 expression in the chronically infected promonocytic cell line U1: the role of tumor necrosis factor-α

Culture supernatants from lipopolysaccharide (LPS)-treated murine microglial cells were found to markedly induce the expression of human immunodeficiency virus (HIV)-1 in the chronically infected human promonocytic cell line U1 as detected by measurements of HIV-1 p24 antigen release into U1 culture supernatants. Antibody to tumor necrosis factor (TNF)-α had an inhibitory effect on the induction of virus by microglial cell supernatants. Also, treatment of microglia with pentoxifylline, an inhibitor of TNF-α production, resulted in suppressed amounts of TNF in the supernatants of LPS-treated microglia and in a reduced stimulatory capacity of these supernatants on HIV-1 expression in U1 cells. These findings support the concept that TNF-α production by glial cells plays a pathogenetic role in HIV-1-associated brain disease by promoting the expression of the virus in infected cells.     

Secretory products of central nervous system glial cells induce Schwann cell proliferation and protect from cytokine-mediated death

There continues to be interest in Schwann cells (SC) as a possible source of myelinating cells for transplantation into the central nervous system (CNS) of patients with multiple sclerosis (MS) and spinal cord injury. It has been suggested that CNS glial cells interfere with SC migration, survival, maturation, and clinically significant remyelination in the CNS. To investigate the effects of CNS glial cells on SC, we examined the effects of serum-free supernatants obtained from rat mixed CNS glial cultures on rat neonatal SC cultures. Supernatants from 1-, 3-, and 5-day CNS glial cultures induced proliferation of SC assayed at 5 days in vitro but did not induce SC differentiation as measured by induction of surface expression of galactolipids (GalL). High concentrations of cAMP simulate many of the effects of axolemma on SC; CNS glial cell supernatants did not inhibit cAMP induction of SC differentiation. CNS glial cell supernatants had no apparent effect on SC viability at 48 hr as measured by trypan blue exclusion. We have previously demonstrated that incubation of SC with transforming growth factor-beta1 (TGF-beta1) + tumor necrosis factor-alpha (TNF-alpha) induces SC death via apoptosis. We now show that CNS glial supernatants inhibits TGF-beta1/TNF-alpha-induced SC death. Our data show that soluble products of CNS glial cells do not induce or inhibit SC differentiation or increase cell death but have the potential to increase proliferation of SC and their resistance to cytokine-mediated death, and thus may affect the outcome of SC transplantation into the CNS.     

Generation and characterization of mouse microglial cell lines

A murine cell line (MMGT1) has been established after transfection of primary microglial cell cultures with a v-myc-containing plasmid. This cell line was comparable with primary microglial cells with respect to morphology, presence of acetylated low density lipoprotein receptor, non-specific estrase, CD63, major histocompatibility complex antigens and CD11, and binding for Ricinus communis agglutinin. Primary microglia as well as MMGT1 cells were negative for glial fibrillary acidic protein. Different MMGT1 strains were obtained after subcloning, two of which resembled histocytes (F4/80 and BM-8). These cell strains, MMGT12 and 16, were able to opsonize latex beads, and could be induced by endotoxins (LPS) to secrete TNF-α, IL-1, IL-6, TGF-β, and EGF. The other subclones had intermediate (MCA519, ER-MP20) or mixed macrophage characteristics and did not react to endotoxin by an increase in TNF-α, IL-1, and TGF-β. Our newly established murine microglia lines may prove to be useful models to study inflammation and repair in the brain.     

Tumor necrosis factor reduces cAMP production in rat microglia

cAMP has been reported to exert a neuroprotective role in several in vivo and in vitro models of brain pathologies, mainly by regulating microglial activation and orienting these cells toward a neuroprotective phenotype. In order to elucidate the intracellular pathways regulated by tumor necrosis factor (TNF) in glial cells, I have studied the modulation of cAMP accumulation by TNF in microglia and astrocyte cultures obtained from the neonatal rat brain. Pre-treatment of microglia with TNF reduced in a dose- and time-dependent manner cAMP accumulation induced by forskolin (FSK), in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). The TNF inhibitory action was 90% reverted by a neutralizing polyclonal anti-TNF antibody and was not prevented by a 16 h pre-treatment of microglial cultures with the Gi protein inhibitor pertussis toxin (PTx). These results suggest that TNF acts at a step of the cAMP transduction pathway other than receptors, G proteins, and phosphodiesterases. The target of TNF appeared to be adenylyl cyclase, whose ability to synthesize cAMP was markedly reduced (up to 50%) in membranes prepared from TNF-treated microglial cells, both in basal conditions and after stimulation with FSK. TNF induced a time-dependent degradation of IkappaB-alpha in microglial cells that was reverted by two inhibitors of nuclear factor kappaB activation, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-CBZ-Leu-Leu-Leu-al (MG132). The same inhibitors also markedly prevented the reduction of FSK-evoked cAMP accumulation by TNF, suggesting the involvement of NFkappaB in the regulation of adenylyl cyclase by TNF in microglia. Conversely, cAMP accumulation in astrocytes was not affected by TNF. Based on these findings, it is proposed that the ability of TNF to inhibit cAMP synthesis in microglia may exacerbate its response and contribute to cell damage in neuroinflammation and neurodegeneration, possibly through enhanced release of proinflammatory and/or cytotoxic factors.     

Induction of metallothionein in astrocytes and microglia in the spinal cord from the myelin-deficient jimpy mouse

Jimpy is a shortened life-span murine mutant whose genetic disorder results in severe pathological alterations in the CNS, including hypomyelination, oligodendrocyte death and strong astroglial and microglial reaction. The knowledge of metallothionein (MT) regulation in the CNS and especially of MT presence in specific glial cell types under pathological conditions is scarce. In the present study, immunocytochemical detection of MT-I+II has been performed in spinal cord sections from 10–12- and 20–22-day-old jimpy and normal animals. The identification of MT-positive glial cells was achieved through double labeling combining MT immunocytochemistry and selective markers for oligodendrocytes, astrocytes and microglia. MT was found in glial cells and was present in the spinal cord of jimpy and normal mice at both ages, but there were remarkable differences in MT expression and in the nature of MT-positive glial cells depending on the type of mouse. The number of MT-positive cells was higher in jimpy than in normal spinal cords. This was apparent in all spinal cord areas, although it was more pronounced in white than in the gray matter and at 20–22 days than at 10–12 days. The mean number of MT-positive glia in the jimpy white matter was 1.9-fold (10–12 days) and 2.4-fold (20–22 days) higher than in the normal one. Astrocytes were the only parenchymal glial cells that were positively identified as MT-producing cells in normal animals. Interestingly, MT in the jimpy spinal cord was localized not only in astrocytes but also in microglial cells. The occurrence of MT induction in relation to reactive astrocytes and microglia, and its role in neuropathological conditions is discussed.     

Microglial development is altered in immature spinal cord by exposure to radiation

Previous studies in this laboratory have documented that the microglial environment of the immature spinal cord is altered by exposure to ionizing radiation. As a result, the lumbosacral spinal cord is markedly depleted of both oligodendrocytes and astrocytes, while leaving axons and the overall cytoarchitecture intact. The status of the microglia in the irradiated region is unknown and is of interest given the interactions between microglia and astrocytes recently elucidated by others. This study uses both in vivo and in vitro approaches to examine the microglial population in normal and irradiated immature spinal cord. The lectin, Griffonia (Bandeiraea) simplicifolia, was selected since it marks microglia both in paraffin embedded sections and in cell cultures. Light microscopic examination of spinal cord sections revealed a reduced microglial population in the irradiated region when compared to littermate controls, and a change in morphology of the remaining microglia to that described by others as ‘‘activated’’. Cultures prepared from lumbosacral spinal cords harvested from 3-day-old rats within 2–4 hr following irradiation were compared with cultures derived from their non-irradiated littermates after 8 days in vitro. Cultures from the irradiated spinal cords revealed trends similar to those observed in vivo, i.e. a reduced microglial population and altered morphology. Although all glial cell types were reduced in cultures from irradiated spinal cords, the few microglia present were usually positioned atop astrocytes. The consistency of reduction in all glial populations in this model shows the microglia to be a novel microenvironment for further studies of roles of microglia within the spinal cord.     

Zaprinast, an inhibitor of cGMP-selective phosphodiesterases, enhances the secretion of TNF-alpha and IL-1beta and the expression of iNOS and MHC class II molecules in rat microglial cells

Proinflammatory cytokines produced by activated glial cells may in turn augment the immune/inflammatory reactions of glial cells through autocrine and paracrine routes. The NO/cGMP signaling represents one of the reactions of activated glial cells. We investigated whether the production of proinflammatory cytokines by glial cells is affected by NO-dependent downstream cGMP signaling. In primary cultures of mixed astrocytes and microglial cells, zaprinast (0.1 mM), an inhibitor of cGMP-selective phosphodiesterases, enhanced the basal and LPS (1.0 microg/ml)-induced secretion of TNF-alpha and IL-1beta. Zaprinast also enhanced NO production induced by LPS or IFN-gamma (100 U/ml), and in microglial cell cultures, but not in astrocyte cultures, zaprinast enhanced the basal and the IFN-gamma-induced production of the cytokines, TNF-alpha and IL-1beta, and of NO. This upregulation by zaprinast was partially inhibited by KT5823 (1.0 microM), an inhibitor of protein kinase G. The LPS-induced production of TNF-alpha, IL-1beta, and NO was inhibited by ODQ (50 microM), an inhibitor of soluble guanylyl cyclase, and by KT5823. Immunohistochemical analysis of mixed glial cell cultures showed that LPS/IFN-gamma-induced iNOS expression and the enhanced expression of iNOS by zaprinast were restricted to microglial cells. Zaprinast enhanced the IFN-gamma (200 U/ml)-induced expression of MHC Class II molecules in astrocytes and microglial cells in mixed cultures, but did not enhance this IFN-gamma-induced expression in pure astrocytes, which lacked paracrine TNF-alpha from microglial cells. Summarizing, zaprinast, which is associated with cGMP/protein kinase G signaling, may augment central immune/inflammatory reactions, possibly via the increased production of TNF-alpha and IL-1beta by activated microglial cells.     

Oligodendrocyte differentiation and progenitor cell proliferation are independently regulated by cyclic AMP

Oligodendrocytes, the glial cells specialized to synthesize myelin in the central nervous system, differentiate in primary rat brain cell cultures on a schedule similar to that observed in vivo. The schedule of oligodendrocyte differentiation and the rate of oligodendroglial progenitor cell proliferation in vitro are both modulated by 3′,5′-cyclic AMP (cAMP). A 24-hour exposure to 1 mM N⁶,2′O-dibutyryladenosine 3′,5′-cyclic monophosphate (dbcAMP) induced a wave of oligodendrocyte differentiation but inhibited proliferation of oligodendroglial progenitors, and reduced by 30-fold the proliferation of progenitors in response to platelet-derived growth factor (PDGF). When cells were grown in the presence of maximally stimulating concentrations of PDGF, the inhibitory effect of cAMP on progenitor cell proliferation was abolished while the stimulatory effect of cAMP on oligodendrocyte differentiation remained, demonstrating that these two cAMP-regulated events are independent. © 1993 Wiley-Liss, Inc.     

An efficient method to limit microglia-dependent effects in astroglial cultures

Microglia, the CNS resident macrophages, and astrocytes, the most abundant glial cell population, are both implicated in brain pathologies and can exhibit a pro-inflammatory phenotype. Microglial cells are known to rapidly and strongly react to brain insults. They will promote astrocyte activation and may lead to a vicious, self-perpetuating cycle of chronic inflammation. To obtain a better understanding of the individual role of both cell types, primary cells are frequently used in in vitro studies, but the purity of specific cell cultures remains rarely investigated. The aim of this study is to determine the effect of specific removal of microglial cells on the inflammatory properties of different glial cultures. Here, the removal of microglial contamination from mixed glial cultures to obtain astrocyte-enriched cultures was achieved using a magnetic cell sorting approach. Compared to mixed cultures, we clearly showed that these enriched cultures are only weakly activated by pro-inflammatory agents (lipopolysaccharide, interferon-γ or beta-amyloid peptide). This finding was confirmed using twice-sorted astrocyte-enriched cultures and microglia-free cultures composed of neurosphere-derived astrocytes. Thus, we present evidence that the magnitude of the pro-inflammatory response is linked to the percentage of microglia in cultures. Due to their high reactivity to various insults or pro-inflammatory stimuli, microglia-derived effects could be credited to astrocytes in mixed glial cultures. Therefore, we highlight the importance of monitoring the presence of microglia in glial cultures since they can affect the interpretation of the results, especially when inflammatory processes are studied.     

Microglial cell reaction in the gray and white matter in spinal cords from jimpy mice. An enzyme histochemical study at the light and electron microscope level

Jimpy is a genetic disorder which results in a severe hypomyelination in the central nervous system associated with a variety of astroglial and oligodendroglial abnormalities. In this study, we examined the morphology and distribution of microglial cells in spinal cord sections from jimpy and normal mice at 10–12 and 20–22 days postnatal using a specific microglial marker, the nucleoside diphosphatase staining. Compared to those of normal littermates, the spinal cords of jimpy mice showed an intense microglial cell reaction in white and gray matter, as revealed by quantitative analysis and light and electron microscope study. Microglial reactivity was apparent in all spinal cord areas, although it was more pronounced in white than in gray matter. The mean microglial densities in the jumpy white matter were about threefold (10–12 days) and fivefold (20–22 days) higher than in the normal, whereas in the gray matter, microglial density in jimpy was about 60% higher than in normal at both ages. Morphologically, microglial cells in the normal spinal cord showed a ramified appearance, similar in size and ramification pattern to those reported in other normal CNS areas. In contrast, microglial cells in the jimpy spinal cord showed a reactive morphology, characterized by a shortening and coarsening of their cell processes, swelling of their cell body and accumulation of lipid inclusions. Reactive microglial cells were found in close association with axons and oligodendroglial cells. The possible role of microglial cells in hypomyelination is discussed.     

A simple magnetic separation method for high-yield isolation of pure primary microglia

Microglial cells play a dynamic role in the brain beyond their established function of immune surveillance. Activated microglia play key roles in neural development, neuroinflammation, neural repair and neurotoxicity. They are particularly important in several neurodegenerative diseases in which sustained microglial activation contributes to the progression of neurodegenerative processes. Consequently, understanding microglial function in CNS health and disease has become an area of active research in recent years. However, a significant obstacle to progress in this field has been the inherent difficulties in obtaining large amounts of primary microglial cells to routinely perform mechanistic studies and characterize signaling pathways regulating the dynamics of microglial activation. Herein, we describe a novel column-free magnetic separation protocol for high-yield isolation of primary microglia from mouse postnatal mixed glial cultures. The procedure is based on optimized culture conditions that enable high microglial cell densities in confluent mixed glial cultures followed by highly efficient recovery of pure microglia by magnetic separation. The novel column-free magnetic separation system utilizes tetrameric antibody complexes (TAC) with dual specificity for CD11b-PE labeled microglia and dextran magnetic nanoparticles. An FcR blocker (anti-CD16/32) is added to enhance the purity of the microglial separation by preventing non-specific labeling of other cell types. This procedure yields on average >3×10? microglial cells per mouse pup, with a remarkable purity of 97% and recovery of around 87% of microglia from the mixed glial population. Importantly, the microglia obtained by this method are fully functional and respond like cells obtained by conventional isolation techniques.     

The effects of cytochalasin B and colchicine on cell motility and ultrastructure in primary cultures of malignant gliomas

Summary Primary tissue cultures of human gliomas were treated with cytochalasin B (0.5–60 g/ml for 90 min). Cell motility was inhibited irreversibly in glial tumour cells, but the effect was reversible on the mesenchymal cells growing in culture in the lower dose range. Cell adhesion was considerably reduced as the dose was increased, as was the capacity for cells to spread on a surface from suspension. Low concentrations of cytochalasin B caused negligible cell death and little disruption of cell ultrastructure. However, increases in dose were accompanied by a greater predominance of rough endoplasmic reticulum and inclusions and aggregation of microfilament bundles. As seen by scanning electron microscopy, cytochalasin B caused the withdrawal of peripheral cell borders, disappearance of ruffles and the breakdown of cytoplasmic lamellae. Charateristic surface blebs and folds appeared in their place.By comparison, colchicine (1–10 g/ml) caused a less marked and non-specific reversible reduction in cell motility on both glial and mesenchymal cells. No significant change in cell adhesion or spreading took place even at high doses, although at all concentrations gross disruption of the cell surface took place with changes in ultrastructure characterised by loss of cytoplasmic microtubules and aggregation of 10 nm filaments.