摘要:

AbstractSeveral morphological and functional properties of microglial cells, the resident immunoeffector cells of the central nervous system (CNS), differ from those of monocytes/macrophages in other tissues. Microglia are assumed to derive from the myelomonocytic lineage, possibly as a distinct subpopulation that diverges from a common cell line early in ontogeny, invades the CNS, proliferates, and differentiates into ameboid and then ramified microglia. We tested the hypothesis that some morphological and functional properties of microglia are induced in myelomonocytic cells by nervous tissue, specifically astrocytes. In the present in vitro studies we compared the differentiation of microglia, blood monocytes, and spleen macrophages on acellular substrates and on monolayers of astrocytes and fibroblasts.On acellular substrates, microglial cells at first acquire an ameboid morphology; later they show a few short, unbranched processes. On monolayers of pure astrocytes, microglial cells at first also differentiate inot ameboid cells, but after 5 to 7 days they start to develop processes with large lamellopodial tips. These lengthen and branch continuously during the next 2 weeks in vitro, demarcating a round to oval territory around the small ellipsoid cell body. By contrast, on monolayers of fibroblasts the microglial cells develop an ameboid morphology, but do not grow the typical long branched processes of the ramified form. Blood monocytes and spleen macrophages behave indistinguishably from microglia both on acellular and cellular substrates, i.e., on astroglia they develop the ramified form, while on fibroblasts they retain the ameboid shape. When microglia, macrophages, or monocytes are cultured on coverslips on top of astrocytic monolayers, i.e., physically separated from the astroglia, but exposed to the medium conditioned by astrocytes, a significant proportion of them also develop the ramified shape. These findings indicate that the ramified shape of microglia is induced by astrocytes. Since this morphology can also be induced in blood monocytes and macrophages, we take this to be further evidence for the proposition that microglial cells are derived from the myelomonocytic lineage, and, moreover, that properties of resident macrophages are largely determined by tissue components of their host organ.

ISSN:0894-1491    

DOI:10.1002/glia.440120402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractMicroglia, the resident macrophages of the central nervous system (CNS), can be distinguished from most other cells of the myelomonocytic lineage by a distinct pattern of memberane currents. In the accompanying paper we have shown that the characteristic morphological feature of microglia, ramification, develops both in microglia and other myelomonocytic cells when they are cocultured with astrocytes. We therefore propose that the electrophysiological properties of microglia also develop under the influence of astrocytes, and moreover, that these properties can also be induced in other cells of the myelomonocytic lineage. Microglia cultured on poly‐d‐lysine or on a monolayer of fibroblasts possess an inwardly rectifying K+‐current only, which is of composite nature. In single‐channel recordings two types of K+‐channels are found: (i) a non‐inactivating channel with a conductance of 43pS, and (ii) an inactivating channel with 32pS. Microglia cultured on a monolayer of astrocytes additionally develop an outward K+‐current and a Na+‐current. The electric parameters of activation and inactivation of the microglial Na+‐current are identical to those of the neuronal Na+‐current. Monocytes from peripheral blood and macrophages from spleen exhibit no inward currents. However, when these cells are cocultured with astrocytes they develop microglia‐like membrane currents, including the inward and outward K+‐rectifyer and the Na+‐current. By contrast, on fibroblasts they retain their macrophage current profile. The expression of the microglia‐like membrane currents in the monounclear phagocytes is induced by a diffusible factor released from the astrocytes into the culture medium, since monocytes and microglia develop the mature microglial current profile, when cultured in astrocyte conditioned medium. These findings show that the current profile of microglia develops only, when they are in association with astrocytes, and that it is induced in myelomonocytic cells from blood and spleen when these also are associated with astrocytes. These findings add to the growing body of evidence that microglia are derived from the myelmonocytic lineage and indicate that the astrocytes are involved in regulating their morphologica

ISSN:0894-1491    

DOI:10.1002/glia.440120403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe effect of glutamatergic agonists on the intracellular free Ca2+concentration ([Ca2+]i) of neuropile glial cells and Retzius neurones in intact segmental ganglia of the medicinal leechHirudo medicinaliswas investigated by using iontophoretically injected fura‐2. In physiological Ringer solution the [Ca2+]ilevels of both cell types were almost the ssame (glial cells: 58 ± 30 nM, n = 51; Retzius neurones: 61 ± 27 nM, n = 64).In both cell types glutamate, kainate, and quisqualate induced an increase in [Ca2+]iwhich was inhibited by 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX). This increase was caused by a Ca2+influx from the extracellular space because the response was greatly diminished upon removal of extracellular Ca2+.The glutamate receptors of neuropile glial cells and Retzius neurones differed with respect to the relative effectiveness of the agonists used, as well as with regard to the inhibitory strenght of DNQX. In Retzius neurones the agonist‐induced [Ca2+]iincrease was abolished after replacing extracellular Na+by organic cations or by mM amounts of Ni2+, whereas in glial cells the [Ca2+]iincrease was largely preserved under both conditions. It is concluded that in Retzius neurones the Ca2+influx is predominantly mediated by voltage‐dependent Ca2+channels, whereas in neuropile glial cells the major influx occurs via the ion channels that are associated with the glutam

ISSN:0894-1491    

DOI:10.1002/glia.440120404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe expression of the glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. In order to characterize DNA sequences involved in such regulations, we produced transgenic mice bearing 2 kb of the 5′ flanking region of the murine GFAP gene linked to theEscherichia coliβ‐galactosidase (β‐gal) reporter gene. Seven transgenic lines were obtained. We observed that the regulatory elements present in the transgene GFAP‐nls‐LacZ direct an expression in the neural and non‐neural tissue and target in vivo an unexpected subpopulation of astrocyte. In the developing brain, β‐gal activity and GFAP appeared simultaneously and in the same region, on embryonic day 18 (E18), suggesting that the 2 kb of the promoter contains the regulatory sequences responsible for the perinatal vimentin/GFAP switch. In addition, we demonstrated that the 2 kb sequence of the GFAP promoter used in the transgene possess elements which are activated after a surgical injury, thus permitting to study some aspects of reactive gliosis in these transgenic mice. These transgenic lines provide a useful tool by enabling further studies of astroglial and, probably, neur

ISSN:0894-1491    

DOI:10.1002/glia.440120405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe development of the rat fimbria over the first postnatal month is associated with an approximate doubling of the tract diameter, a large increase in the number of glial cells, and the transformation of the prenatal radial glial skeleton into the adult interfascicular glial rows of solitary astrocytes and contiguous myelinating oligodendrocytes.The ventricular zone is reduced from a heterogeneous germinal layer of three or more cells thick at birth to the mature adult unicellular ependyma of homogeneous pale, mitotically inactive cells by the end of the second postnatal week. Mitoses are present throughout the body of the tract at all times, and persist, at reduced levels, in the adult.At birth the interior of the fimbria has only few scattered glial cell nuclei, largely solitary, or at most in longitudinal pairs. Over the first two postnatal weeks, the numbers and density of the interfascicular glia increase continuously. The scattered cells and cell clusters become progressively transformed into longer unicellular rows, which are aligned along the longitudinal axis of the tract, and which finally coalesce to form the continuous regular astrocyte/oligodendrocyte units that make up the interfascicular glial rows of the adult fimbrial glial skeleton.The increased cell packing density of the developing fimbrial glia is associated with a substantial decrease in nuclear and cytoplasmic size. From the end of the second postnatal week, the characteristic, large pale solitary astrocytes, and the smaller, more numerous, densely stained, closely packed oligodendrocytes are recognisable. Immunostaining for glial fibrillary acidic protein shows that immediately after birth the characteristic embryonic pattern of regular parallel radial glial processes starts to be modified by the progressive accumulation of longitudinal astrocytic processes, so the prenatal radial glial framework is rapidly transformed into the adult type of rectilinear array of radial and longitudinal processes.The development of the oligodendrocytes is shown clearly by immunostaining for myelin basic protein in enlarged, cytoplasm‐rich, symmetrically placed cell pairs first seen at around P7. At P8‐P10, there is a characteristic pattern of simultaneous multifocal maturation in which a single oligodendrocyte in each cluster develops a full complement of parallel, rather varicose myelinating processes. By P14 myelination is becoming confluent, oligodendrocytes are smaller, darker, with little cytoplasm, and individual myelinating processes cannot be discerned. Even at the end of the first postnatal month there are still many immature glia of indeterminate morphology. Myelination tends at first to be concentrated in the region adjacent to the hippocampus, and only reaches strengthen the hope that it may in future become possible to devise some form of self‐reconstruction of the damaged adult glial tract structure in traumatic lesions completion by the end of the second month.During the first postnatal week, the entire array of fimbrial axons is traversed by the entire population of dentate neuroblasts. The highly vascular connective tissue on the pial surface of the fimbria is occupied by a prominent but transient population of mast cells which disappear in the

ISSN:0894-1491    

DOI:10.1002/glia.440120406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractProliferation of microglia/macrophages is a common finding in many central nervous system diseases. To identify mitogenic signals for human microglia, we examined primary cultures of human fetal and adult microglia after stimulation with cytokines, colony stimulating factors (CSFs), or LPS, using proliferating cell nuclear antigen (PCNA) expression as an index of cell proliferation. The results showed that both M‐CSF and GM‐CSF induced microglial proliferation in fetal and adult human cultures, but that GM‐CSF provided a much stronger stimulus. At 96 h post‐stimulation, the mean PCNA labeling index was 2.4 for M‐CSF and 13.3 for GM‐CSF in fetal microglia; in adult microglia, the PCNA labeling index was 4.7 for M‐CSF and 9.0 for GM‐CSF. The effect of GM‐CSF on fetal microglia was dose dependent and synergistic with M‐CSF. LPS abolished the basal level of PCNA labeling in adult microglia, but in fetal microglia, caused a slight increase in PCNA labeling (1.9) at 96 h and consistently enhanced microglial cell survival and differentiation into highly branched cells. The production of GM‐CSF in purified human fetal astrocyte and microglial cultures was examined after stimulation with LPS, TNF‐α, or IL‐1β. Unlike M‐CSF, neither cell type produced GM‐CSF in unstimulated cultures; however, when stimulated with IL‐1β, astrocytes expressed GM‐CSF mRNA and protein, which accumulated in the culture through 72 h. In microglia, LPS was the only effective inducing agent. An immunocytochemical study performed to identify in vivo sources of GM‐CSF revealed selective labeling of reactive astrocytes in active lesions of multiple sclerosis and senile plaques of Alzheimer's disease. Our data demonstrate that both fetal and adult human microglia are capable of proliferation in response to CSFs, GM

ISSN:0894-1491    

DOI:10.1002/glia.440120407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractThe glial population of the lumbosacral spinal cord of the rat can be markedly depleted by exposure to ionizing radiation during the first postnatal week. Identification of specific cell populations which survive the exposure to radiation is difficult in situ; therefore, the present investigation used in vitro approaches to address issues related to specific phenotypes and maturational states of glia in cultures derived from non‐irradiated (control) and irradiated (experimental) lumbosacral spinal cords of 3‐day‐old rats. Cultures were established from the spinal cords 2 to 4 hours following irradiation and were compared to cultures from non‐irradiated, littermate controls. By 4 days in vitro (DIV) the numbers of cells in experimental cultures were profoundly reduced when compared to controls, and this reduction persisted through the termination of the study (8 DIV). In addition to reduction in numbers, astrocyte phenotypes were altered in experimental cultures, with greater proportions of the astrocyte population being constituted by the flat angular, large angular, and pancake types and a lesser proportion by stellate cells. The non‐astrocytic cell types were dramatically reduced as evidenced by the paucity of oligodendrocytes immunoreactive for galactocerebroside and of small, non‐process bearing cells binding the lectin, Griffonia (Bandeiraea) simplicifolia, a marker for microglia. Experimental cultures contained an increased incidence of binucleate astrocytes, an increase not restricted to a particular astrocyte phenotype. This study established the feasibility of utilizing this combined in vivo/in vitro approach in assessment of glial populations in immature spinal cords, and further investigations are in progress using

ISSN:0894-1491    

DOI:10.1002/glia.440120408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractSpecies difference in the susceptibility to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) was investigated in cultured rat and mouse astrocytes, where 1‐methyl‐4‐phenylpyridinium (MPP+), the toxic mediator of MPTP, is formed. Type A monoamine oxidase (MAO) predominated in both rat and mouse astrocytes, while its activity was slightly higher in mouse cells compared to rat cells; MAO‐B activity, on the other hand, was significantly lower in mouse astrocytes than in rat astrocytes. Because both types of MAO have been reported to make similar contributions to MPP+production in astrocytes, their total activity was examined and results indicated that there was no significant difference between these two species. In additon, MPP±caused a dose dependent loss of cell viability as judged by the amount of lactate dehydrogenase released into the incubation medium. The toxicity of MPP±on astrocytes started to be seen after a 2 day incubation period. Mouse astrocytes were more vulnerable to MPP±than rat astrocytes. The threshhold values for MPP±toxicity in mouse and rat cultures were 10 ±M and 70 ±M, respectively. After addition of [3H] MPP±to the medium, intracellular [3H] MPP±was found to increase in both cultures. Mouse astrocytes accumulated more MPP±than rat astrocytes (150 pmol/mg protein vs. 65 pmol/mg protein). When astrocytes were allowed to accumulate [3H] MPP±and then incubated in fresh medium medium not containing [3H] MPP±, intracellular levels of [3H] MPP±in both cells rapidly declined (110 pmol/protein in mouse vs. 40 pmol/mg protein in rat of MPP±been released). These results indicated that (1) MPP±could cross the plasma membrane of astrocytes despite of its charged chemical structure, (2) mouse astrocytes had a higher capacity for MPP±accumulation (approximately 2‐fold), as well as release (approximately 2.7‐fold), than rat astrocytes, and (3) mouse astrocytes were more v

ISSN:0894-1491    

DOI:10.1002/glia.440120409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

摘要:

AbstractNa+‐Ca2+exchange activity in its reverse mode was demonstrated in cultured rat astrocytes. Combination of ouabain (1 mM) and monensin (20 ±M) caused a marked increase in45Ca2+uptake in astrocytes.45Ca2+uptake was also stimulated by lowering the external Na+concentration. Ouabain plus monensin‐stimulated45Ca2+uptake was blocked by 3,4‐dichlorobenzamil (IC50, 16 ±M), an inhibitor of Na+‐Ca2+exchanger, but not by nifedipine (0.1 ±M). The stimulated‐45Ca2+uptake was observed even in K+‐free medium, and external K+at 5‐10 mM caused a 2.2‐fold increase in the uptake. Microspectrofluorimetry using the Ca2+‐sensitive dye fura‐2 showed that ouabain plus monensin increased intracellular Ca2+concentration in single astrocytes. The Ca2+signal was dependent on external Ca2+(EC50, 1.4 mM), and blocked by 20 ±M 3,4‐dichlorobenzamil, but not by Ca2+channel blockers (Cd2+, 20 ±M; Ni2+, 100 ±M). Antiserum of cardiac Na+‐Ca2+exchanger recognized 160 and 120‐135 kDa proteins on SDS‐polyacrylamide gel electrophoresis of astrocyte homogenate. Northern blot analysis revealed the presence of mRNA for the exchanger protein in astrocytes. These findings indicate that Na+‐Ca2+exchanger which is modulated by K+is

ISSN:0894-1491    

DOI:10.1002/glia.440120410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY

ISSN:0894-1491    

DOI:10.1002/glia.440120401

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1994

数据来源: WILEY