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Differential uptake of dextran beads by astrocytes, macrophages and oligodendrocytes in mixed glial-cell cultures from brains of neonatal rats
Neuroscience Letters 248 (1998) 159–162
Differential uptake of dextran beads by astrocytes, macrophages and oligodendrocytes in mixed glial-cell cultures from brains of neonatal rats Francine A. Tansey, Wendy Cammer* Department of Neurology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA Received 22 January 1998; received in revised form 2 April 1998; accepted 21 April 1998
Abstract The present study addresses a controversy over the abilities of astrocytes to perform phagocytosis. Primary glial-cell cultures were prepared from the brains of neonatal rats and were incubated with fluorescently-labeled dextran beads (molecular weights ~10 and ~40 kDa). Astrocytes and oligodendrocytes were double-labeled by immunofluorescence staining of cell-specific markers, and microglia by lectin histochemistry. Cells were permitted to take up beads for 1 h, fixed, and incubated with primary antibodies, followed by fluorescent secondary antibodies or fluorescently-labeled lectin. Macrophages and astrocytes internalized beads of both sizes. In astrocyte processes the beads appeared to line up along glial filaments. The results, which provide direct evidence for uptake of beads by astrocytes in vitro and against equally rapid, if any, uptake by oligodendrocytes, bear upon issues of acid/base balance and glial cell development and are relevant to neuropathological observations in human disease. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Glial cells; Phagocytosis; Astrocytes; Oligodendrocytes; Macrophages; Glial-fibrillary-acidic protein
The abilities of microglia to phagocytose particles are well-known (reviewed in Ref. [16]). Diverse observations have left it uncertain, however, whether astrocytes are capable of doing so. One or more oligodendrocytes have been observed inside of hypertrophic astrocytes in vivo in brain tissue affected by multiple sclerosis, stroke, Krabbe’s disease and AIDS encephalitis, but ultrastructural studies have been unable to firmly distinguish whether the astrocytes had enwrapped or phagocytosed the enclosed cells [11,19,20], nor could it be distinguished whether astrocytes protect or degrade oligodendrocytes after engulfment [19]. Internalized axonal fragments, myelin membranes and nerve terminals have also been observed in astrocytes after injury to the brain or optic nerve [3,6,9,10], and some neurons are phagocytosed by astrocytes in amyotrophic lateral sclerosis, although most are found in macrophages [17]. There is some evidence for true phagocytosis by astrocytes in
* Corresponding author. Tel.: +1 718 4302013; fax: +1 718 4308790; e-mail: [email protected]
vitro, e.g. astrocytes can internalize myelin membranes in explant cultures [18]. There is only sparse information about the possibility that oligodendrocytes might be phagocytic. Using cell-specific markers, Ludwin [13] found myelin debris in oligodendrocyte processes but not in astrocytes in optic nerves undergoing Wallerian degeneration. Typically, however, most debris was removed by macrophages. We have now tested the abilities of astrocytes to phagocytose standardized fluorescently-labeled dextran beads. Microglia were also identified in order to serve, if necessary, as positive controls, and oligodendrocytes as a secondary matter of interest. The protocol for animal usage was approved by the Institute for Animals Studies, which is the AAALAC-approved animal-care facility at the Albert Einstein College of Medicine. Sprague–Dawley rats with 2-day-old litters were purchased from Taconic Farms (Germantown, NY), and cultures were prepared from ten 2-day-old pups. Pups were anesthetized by hypothermia and decapitated, and the cerebral hemispheres dissected out and placed in ice-
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00373- 5
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F.A. Tansey, W. Cammer / Neuroscience Letters 248 (1998) 159–162
cold Dulbecco’s minimal essential medium (DMEM)(from Gibco, Grand Island, NY). The meninges were dissected off and discarded, and the cerebral hemispheres dissociated and placed in culture using minor variations on a conventional method [8]. The cells were plated on poly-d-lysine-coated coverslips in the wells of 24-well tissue-culture plates (COSTAR, Cambridge, MA) at 4 × 104 cells/ml (10 ml per rat brain). The plating medium contained DMEM/F12 medium (from Gibco) (1:1), containing 15 mM HEPES buffer, pH 7.4, 10% fetal bovine serum (Gibco) and 1 × antibiotic-antimycotic, also from Gibco. The plates were
incubated, undisturbed, at 37°C in 5% CO2, 95% air for 3 days. The plating medium was changed at 3 and 6 days in vitro (DIV). By 7 DIV a confluent layer of astrocytes was overlain by small cells. The experiments were performed at 8 DIV. Dextran beads labeled with fluoresceinisothiocyanate (FITC) or rhodamine were purchased from Molecular Probes (Eugene, OR). Beads of molecular weights 10 and 40 kDa, bearing FITC or rhodamine, were used in the respective experiments, which were performed in the original tissue-culture wells. Three experiments were performed
Fig. 1. Phagocytosis of dextran beads by cells in mixed primary glial-cell cultures. The cultures were incubated for 1 h with FITC-labelled 40-kDa dextran beads (a–e,h,i) or rhodamine-labelled 10-kDa dextran beads (f,g), fixed and incubated with antibodies or a lectin directed against cellspecific markers. Confocal images, made at 60 × magnification, represent 1.5-mm slices. (a,c) FITC-labelled 40-kDa beads in astrocyte processes that contain rhodamine-labelled GFAP-positive glial filaments; (b) phase contrast of the field shown in (a). The asterisks indicate the same location in all three fields, and the arrows point to separate locations where the beads appear to line up along intermediate filaments. (d) FITC-labelled 40-kDa beads in cells other than the rhodamine-labelled small O4-positive oligodendrocytes; (e), phase contrast of the same field shown in (d). The arrows in (e–g) point to oligodendrocytes. Note that the microglial cell near the top right side in (e) contains FITC-labelled beads (d). (f,g), Rhodamine-labelled 10-kDa beads are also excluded from FITC-labelled O4-positive oligodendrocytes during the 1-h incubation. (h,i), FITC-labelled 40-kDa dextran beads in rhodamine-BSI-B4 labeled microglia (e.g. arrows).
F.A. Tansey, W. Cammer / Neuroscience Letters 248 (1998) 159–162
on different sets of primary cultures, and in each case FITClabeled antibodies and rhodamine labeled beads were use, and the procedures duplicated with rhodamine-labelled antibodies and FITC-labelled beads. Beads were suspended in phosphate-buffered saline at 0.6 mg/ml, and 30 ml were added to a total volume of 1 ml in each well. The cultures were maintained at 37°C for 1 h, the medium was removed, and the cells were washed and then fixed with 4% (w/v) paraformaldehyde at room temperature for 30 min. The fixed cells were blocked with 20% normal goat serum and incubated with mouse anti-glial fibrillary acidic protein (GFAP)(Boehringer, Indianapolis, IN) (1:100), for astrocytes; monoclonal antibody O4 (1:1), which is a mouse IgM that marks small cells in the oligodendrocyte lineage [2]; or buffer alone, for 1 h at room temperature. The cells were washed and, in Fig. 1, the media replaced with the following, in the respective wells: rhodamine-labelled anti-mouse IgG (Southern Biotechnology, Birmington, AL) in wells that had contained anti-GFAP; FITC-labeled anti-mouse IgM (Kirkegaard and Perry, Gaithersburg, MD) in the wells labeled with O4; or, to label macrophages [15] in the cultures that had been incubated with buffer in place of primary antibody, biotinylated Bandeiria simplicifolia lectin (BSI-B4) (Sigma, St. Louis, MO) that had been labeled further with rhodamine-streptavidin (Vector Laboratories, Burlingame, CA). Astrocytes internalized 40-kDa beads (Fig. 1a) or 10-kDa beads (not shown). Often the beads appeared to line up on or between GFAP-positive intermediate filaments (e.g. arrows in different positions in Fig. 1a,c). During the 1-h incubation period the oligodendrocytes did not internalize 40-kDa (Fig. 1d) or 10-kDa (Fig. 1f) beads. However, the data do not rule out the possibility that they may phagocytose at a slower rate. Microglia took up numerous beads of both sizes (e.g. 40 kDa in Fig. 1h). The heavy (green) labeling of microglia is particularly dramatic in Fig. 1d,e (large cell near top without arrow) and in Fig. 1h, where the red labeling with BSIB4 and the green beads result in the appearance of yellow cells. The data show that during a relatively short incubation period astrocytes can internalize dextran beads that are 40 kDa in molecular size, whereas oligodendrocytes from the same animals may internalize beads, even as small as 10 kDa, more slowly or not at all. The observation that astrocytes can take up 40-kDa beads in vitro bears upon several issues in the neuroscience literature. (1) It does not distinguish whether astrocytes surround or phagocytose whole cells, such as oligodendrocytes or neurons [17,19,20], but does support the possibility that astrocytes phagocytose smaller particles, such as myelin debris or fragments of axons [3,6,18]. (2) Astrocytic carbonic anhydrase II (CAII) is believed to help regulate the acid/ base balance around neurons in the gray matter (reviewed in Refs. [4,7]), but it is controversial whether the effective CAII is in astrocyte processes or whether the astrocytes release it into the extracellular space. The efficacy of
161
CAII inhibitors bound to dextran beads in blocking fluctuations in pH in vitro has been used to argue in favor of extracellular CAII [5]. The present data suggest, however, that the astrocytes could have phagocytosed the beads, allowing the inhibitor access to intracellular CAII. (3) In the area of glial cell development most studies indicate separate postnatal development of GFAP- positive astrocytes and O4-positive oligodendrocytes [14]. However, a few observations of GFAP + /O4 + cells have called that conclusion into question [1,12]. The present observations suggest that phagocytosis or engulfment of O4-positive oligodendrocyte precursors by GFAP-positive astrocytes may account for the latter, seemingly contradictory, finding. We thank the staff of the Analytical Imaging Facility at Albert Einstein College of Medicine for assisted use of the confocal microscope. This work was supported by grant no. RO1 NS12890 from the National Institutes of Health and by grant no. 2971 from the National Multiple Sclerosis Society. [1] Armstrong, R., Friedrich, V.L., Holmes, K.V. and Dubois-Dalq, M., In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination, J. Cell Biol., 111 (1990) 1183–1195. [2] Bansal, R., Warrington, A.E., Gard, A.L., Ranscht, B. and Pfeiffer, S.E., Multiple and novel specificities of monoclonal antibodies O1, O4 and RMAb used in the analysis of oligodendrocyte development, J. Neurosci. Res., 24 (1989) 548–557. [3] Bechmann, I. and Nitsch, R., Astrocytes and microglial cells incorporate degenerating fibers following entorhinal lesion: a light, confocal, and electron microscopical study using a phagocytosis-dependent labeling technique, Glia, 20 (1997) 145– 154. [4] Cammer, W. and Brion, L.P., Carbonic anhydrase in the nervous system. In W.R. Chegwidden, N.D. Carter, and Y.H. Edwards (Eds.), The Carbonic Anhydrases: New Horizons. Birkhauser, Basel, in press. [5] Chen, J.C.T. and Chesler, M., pH transients evoked by excitatory synaptic transmission are increased by inhibition of extracellular carbonic anhydrase, Proc. Natl. Acad. Sci. USA, 89 (1992) 7786–7790. [6] Cheng, H.W., Jiang, T., Brown, S.A., Pasinetti, G.M., Finch, C.E. and Mc Neill, T.H., Response of striatal astrocytes to neuronal deafferentation: an immunocytochemical and ultrastructural study, Neuroscience, 62 (1994) 425–439. [7] Chesler, M., The regulation and modulation of pH in the nervous system, Prog. Neurobiol., 34 (1990) 401–427. [8] Chiu, F.-C. and Goldman, J.E., Synthesis and turnover of cytoskeletal proteins in cultured astrocytes, J. Neurochem., 42 (1984) 166–174. [9] Cook, R.D. and Wisniewski, H.M., The role of oligodendroglia and astroglia in Wallerian degeneration of the optic nerve, Brain Res., 61 (1973) 191–206. [10] Frank, M. and Wolburg, H., Cellular reactions at the lesion site after crushing of the rat optic nerve, Glia, 16 (1996) 227–240. [11] Ghatak, N.R., Occurrence of oligodendrocytes within astrocytes in demyelinating lesions, J. Neuropathol. Exp. Neurol., 51 (1992) 40–46. [12] Godfraind, C., Friedrich, V.L., Holmes, K.V. and Dubois-Dalq, M., In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice, J. Cell Biol., 109 (1989) 2405–2416.
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[13] Ludwin, S.K., Phagocytosis in the rat optic nerve following Wallerian degeneration, Acta Neuropathol., 80 (1990) 266–273. [14] Skoff, R.P. and Knapp, P.E., The origins and lineages of macroglial cells. In H. Kettenmann and B.R. Ransom (Eds.) Neuroglia, Oxford University Press, New York, 1995, pp. 135–148. [15] Streit, W.J., An improved staining method for rat microglial cells using the lectin Griffonia simplicifolia (GSA I-B4), J. Histochem. Cytochem., 38 (1990) 1683–1686. [16] Thomas, W.E., Brain macrophages: evolution of microglia and their functions, Brain Res. Rev., 17 (1992) 61–74. [17] Troost, D., Claessen, N., van den Oord, J.J., Swaab, D.F. and de Jong, J.M., Neuronophagia in the motor cortex in amyotrophic lateral sclerosis, Neuropathol. Appl. Neurobiol., 19 (1993) 390–397.
[18] Vinores, S.A. and Herman, M.M., Phagocytosis of myelin by astrocytes in explants of adult rabbit cerebral white matter maintained on Gelfoam matrix, J. Neuroimmunol., 43 (1993) 169–176. [19] Wu, E. and Raine, C.S., Multiple sclerosis: interactions between oligodendocytes and hypertrophic astrocytes and their occurrence in other, non-demyelinating conditions, Lab. Invest., 67 (1992) 88–99. [20] Wu, E., Brosnan, C.F. and Raine, C.S., SP-40, 40 immunoreactivity in inflammatory CNS lesions displaying astrocyte/oligodendrocyte interactions, J. Neuropathol. Exp. Neurol., 52 (1993) 129–134.
Differential uptake of dextran beads by astrocytes, macrophages and oligodendrocytes in mixed glial-cell cultures from brains of neonatal rats Francine A. Tansey, Wendy Cammer* Department of Neurology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA Received 22 January 1998; received in revised form 2 April 1998; accepted 21 April 1998
Abstract The present study addresses a controversy over the abilities of astrocytes to perform phagocytosis. Primary glial-cell cultures were prepared from the brains of neonatal rats and were incubated with fluorescently-labeled dextran beads (molecular weights ~10 and ~40 kDa). Astrocytes and oligodendrocytes were double-labeled by immunofluorescence staining of cell-specific markers, and microglia by lectin histochemistry. Cells were permitted to take up beads for 1 h, fixed, and incubated with primary antibodies, followed by fluorescent secondary antibodies or fluorescently-labeled lectin. Macrophages and astrocytes internalized beads of both sizes. In astrocyte processes the beads appeared to line up along glial filaments. The results, which provide direct evidence for uptake of beads by astrocytes in vitro and against equally rapid, if any, uptake by oligodendrocytes, bear upon issues of acid/base balance and glial cell development and are relevant to neuropathological observations in human disease. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Glial cells; Phagocytosis; Astrocytes; Oligodendrocytes; Macrophages; Glial-fibrillary-acidic protein
The abilities of microglia to phagocytose particles are well-known (reviewed in Ref. [16]). Diverse observations have left it uncertain, however, whether astrocytes are capable of doing so. One or more oligodendrocytes have been observed inside of hypertrophic astrocytes in vivo in brain tissue affected by multiple sclerosis, stroke, Krabbe’s disease and AIDS encephalitis, but ultrastructural studies have been unable to firmly distinguish whether the astrocytes had enwrapped or phagocytosed the enclosed cells [11,19,20], nor could it be distinguished whether astrocytes protect or degrade oligodendrocytes after engulfment [19]. Internalized axonal fragments, myelin membranes and nerve terminals have also been observed in astrocytes after injury to the brain or optic nerve [3,6,9,10], and some neurons are phagocytosed by astrocytes in amyotrophic lateral sclerosis, although most are found in macrophages [17]. There is some evidence for true phagocytosis by astrocytes in
* Corresponding author. Tel.: +1 718 4302013; fax: +1 718 4308790; e-mail: [email protected]
vitro, e.g. astrocytes can internalize myelin membranes in explant cultures [18]. There is only sparse information about the possibility that oligodendrocytes might be phagocytic. Using cell-specific markers, Ludwin [13] found myelin debris in oligodendrocyte processes but not in astrocytes in optic nerves undergoing Wallerian degeneration. Typically, however, most debris was removed by macrophages. We have now tested the abilities of astrocytes to phagocytose standardized fluorescently-labeled dextran beads. Microglia were also identified in order to serve, if necessary, as positive controls, and oligodendrocytes as a secondary matter of interest. The protocol for animal usage was approved by the Institute for Animals Studies, which is the AAALAC-approved animal-care facility at the Albert Einstein College of Medicine. Sprague–Dawley rats with 2-day-old litters were purchased from Taconic Farms (Germantown, NY), and cultures were prepared from ten 2-day-old pups. Pups were anesthetized by hypothermia and decapitated, and the cerebral hemispheres dissected out and placed in ice-
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00373- 5
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F.A. Tansey, W. Cammer / Neuroscience Letters 248 (1998) 159–162
cold Dulbecco’s minimal essential medium (DMEM)(from Gibco, Grand Island, NY). The meninges were dissected off and discarded, and the cerebral hemispheres dissociated and placed in culture using minor variations on a conventional method [8]. The cells were plated on poly-d-lysine-coated coverslips in the wells of 24-well tissue-culture plates (COSTAR, Cambridge, MA) at 4 × 104 cells/ml (10 ml per rat brain). The plating medium contained DMEM/F12 medium (from Gibco) (1:1), containing 15 mM HEPES buffer, pH 7.4, 10% fetal bovine serum (Gibco) and 1 × antibiotic-antimycotic, also from Gibco. The plates were
incubated, undisturbed, at 37°C in 5% CO2, 95% air for 3 days. The plating medium was changed at 3 and 6 days in vitro (DIV). By 7 DIV a confluent layer of astrocytes was overlain by small cells. The experiments were performed at 8 DIV. Dextran beads labeled with fluoresceinisothiocyanate (FITC) or rhodamine were purchased from Molecular Probes (Eugene, OR). Beads of molecular weights 10 and 40 kDa, bearing FITC or rhodamine, were used in the respective experiments, which were performed in the original tissue-culture wells. Three experiments were performed
Fig. 1. Phagocytosis of dextran beads by cells in mixed primary glial-cell cultures. The cultures were incubated for 1 h with FITC-labelled 40-kDa dextran beads (a–e,h,i) or rhodamine-labelled 10-kDa dextran beads (f,g), fixed and incubated with antibodies or a lectin directed against cellspecific markers. Confocal images, made at 60 × magnification, represent 1.5-mm slices. (a,c) FITC-labelled 40-kDa beads in astrocyte processes that contain rhodamine-labelled GFAP-positive glial filaments; (b) phase contrast of the field shown in (a). The asterisks indicate the same location in all three fields, and the arrows point to separate locations where the beads appear to line up along intermediate filaments. (d) FITC-labelled 40-kDa beads in cells other than the rhodamine-labelled small O4-positive oligodendrocytes; (e), phase contrast of the same field shown in (d). The arrows in (e–g) point to oligodendrocytes. Note that the microglial cell near the top right side in (e) contains FITC-labelled beads (d). (f,g), Rhodamine-labelled 10-kDa beads are also excluded from FITC-labelled O4-positive oligodendrocytes during the 1-h incubation. (h,i), FITC-labelled 40-kDa dextran beads in rhodamine-BSI-B4 labeled microglia (e.g. arrows).
F.A. Tansey, W. Cammer / Neuroscience Letters 248 (1998) 159–162
on different sets of primary cultures, and in each case FITClabeled antibodies and rhodamine labeled beads were use, and the procedures duplicated with rhodamine-labelled antibodies and FITC-labelled beads. Beads were suspended in phosphate-buffered saline at 0.6 mg/ml, and 30 ml were added to a total volume of 1 ml in each well. The cultures were maintained at 37°C for 1 h, the medium was removed, and the cells were washed and then fixed with 4% (w/v) paraformaldehyde at room temperature for 30 min. The fixed cells were blocked with 20% normal goat serum and incubated with mouse anti-glial fibrillary acidic protein (GFAP)(Boehringer, Indianapolis, IN) (1:100), for astrocytes; monoclonal antibody O4 (1:1), which is a mouse IgM that marks small cells in the oligodendrocyte lineage [2]; or buffer alone, for 1 h at room temperature. The cells were washed and, in Fig. 1, the media replaced with the following, in the respective wells: rhodamine-labelled anti-mouse IgG (Southern Biotechnology, Birmington, AL) in wells that had contained anti-GFAP; FITC-labeled anti-mouse IgM (Kirkegaard and Perry, Gaithersburg, MD) in the wells labeled with O4; or, to label macrophages [15] in the cultures that had been incubated with buffer in place of primary antibody, biotinylated Bandeiria simplicifolia lectin (BSI-B4) (Sigma, St. Louis, MO) that had been labeled further with rhodamine-streptavidin (Vector Laboratories, Burlingame, CA). Astrocytes internalized 40-kDa beads (Fig. 1a) or 10-kDa beads (not shown). Often the beads appeared to line up on or between GFAP-positive intermediate filaments (e.g. arrows in different positions in Fig. 1a,c). During the 1-h incubation period the oligodendrocytes did not internalize 40-kDa (Fig. 1d) or 10-kDa (Fig. 1f) beads. However, the data do not rule out the possibility that they may phagocytose at a slower rate. Microglia took up numerous beads of both sizes (e.g. 40 kDa in Fig. 1h). The heavy (green) labeling of microglia is particularly dramatic in Fig. 1d,e (large cell near top without arrow) and in Fig. 1h, where the red labeling with BSIB4 and the green beads result in the appearance of yellow cells. The data show that during a relatively short incubation period astrocytes can internalize dextran beads that are 40 kDa in molecular size, whereas oligodendrocytes from the same animals may internalize beads, even as small as 10 kDa, more slowly or not at all. The observation that astrocytes can take up 40-kDa beads in vitro bears upon several issues in the neuroscience literature. (1) It does not distinguish whether astrocytes surround or phagocytose whole cells, such as oligodendrocytes or neurons [17,19,20], but does support the possibility that astrocytes phagocytose smaller particles, such as myelin debris or fragments of axons [3,6,18]. (2) Astrocytic carbonic anhydrase II (CAII) is believed to help regulate the acid/ base balance around neurons in the gray matter (reviewed in Refs. [4,7]), but it is controversial whether the effective CAII is in astrocyte processes or whether the astrocytes release it into the extracellular space. The efficacy of
161
CAII inhibitors bound to dextran beads in blocking fluctuations in pH in vitro has been used to argue in favor of extracellular CAII [5]. The present data suggest, however, that the astrocytes could have phagocytosed the beads, allowing the inhibitor access to intracellular CAII. (3) In the area of glial cell development most studies indicate separate postnatal development of GFAP- positive astrocytes and O4-positive oligodendrocytes [14]. However, a few observations of GFAP + /O4 + cells have called that conclusion into question [1,12]. The present observations suggest that phagocytosis or engulfment of O4-positive oligodendrocyte precursors by GFAP-positive astrocytes may account for the latter, seemingly contradictory, finding. We thank the staff of the Analytical Imaging Facility at Albert Einstein College of Medicine for assisted use of the confocal microscope. This work was supported by grant no. RO1 NS12890 from the National Institutes of Health and by grant no. 2971 from the National Multiple Sclerosis Society. [1] Armstrong, R., Friedrich, V.L., Holmes, K.V. and Dubois-Dalq, M., In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination, J. Cell Biol., 111 (1990) 1183–1195. [2] Bansal, R., Warrington, A.E., Gard, A.L., Ranscht, B. and Pfeiffer, S.E., Multiple and novel specificities of monoclonal antibodies O1, O4 and RMAb used in the analysis of oligodendrocyte development, J. Neurosci. Res., 24 (1989) 548–557. [3] Bechmann, I. and Nitsch, R., Astrocytes and microglial cells incorporate degenerating fibers following entorhinal lesion: a light, confocal, and electron microscopical study using a phagocytosis-dependent labeling technique, Glia, 20 (1997) 145– 154. [4] Cammer, W. and Brion, L.P., Carbonic anhydrase in the nervous system. In W.R. Chegwidden, N.D. Carter, and Y.H. Edwards (Eds.), The Carbonic Anhydrases: New Horizons. Birkhauser, Basel, in press. [5] Chen, J.C.T. and Chesler, M., pH transients evoked by excitatory synaptic transmission are increased by inhibition of extracellular carbonic anhydrase, Proc. Natl. Acad. Sci. USA, 89 (1992) 7786–7790. [6] Cheng, H.W., Jiang, T., Brown, S.A., Pasinetti, G.M., Finch, C.E. and Mc Neill, T.H., Response of striatal astrocytes to neuronal deafferentation: an immunocytochemical and ultrastructural study, Neuroscience, 62 (1994) 425–439. [7] Chesler, M., The regulation and modulation of pH in the nervous system, Prog. Neurobiol., 34 (1990) 401–427. [8] Chiu, F.-C. and Goldman, J.E., Synthesis and turnover of cytoskeletal proteins in cultured astrocytes, J. Neurochem., 42 (1984) 166–174. [9] Cook, R.D. and Wisniewski, H.M., The role of oligodendroglia and astroglia in Wallerian degeneration of the optic nerve, Brain Res., 61 (1973) 191–206. [10] Frank, M. and Wolburg, H., Cellular reactions at the lesion site after crushing of the rat optic nerve, Glia, 16 (1996) 227–240. [11] Ghatak, N.R., Occurrence of oligodendrocytes within astrocytes in demyelinating lesions, J. Neuropathol. Exp. Neurol., 51 (1992) 40–46. [12] Godfraind, C., Friedrich, V.L., Holmes, K.V. and Dubois-Dalq, M., In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice, J. Cell Biol., 109 (1989) 2405–2416.
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[13] Ludwin, S.K., Phagocytosis in the rat optic nerve following Wallerian degeneration, Acta Neuropathol., 80 (1990) 266–273. [14] Skoff, R.P. and Knapp, P.E., The origins and lineages of macroglial cells. In H. Kettenmann and B.R. Ransom (Eds.) Neuroglia, Oxford University Press, New York, 1995, pp. 135–148. [15] Streit, W.J., An improved staining method for rat microglial cells using the lectin Griffonia simplicifolia (GSA I-B4), J. Histochem. Cytochem., 38 (1990) 1683–1686. [16] Thomas, W.E., Brain macrophages: evolution of microglia and their functions, Brain Res. Rev., 17 (1992) 61–74. [17] Troost, D., Claessen, N., van den Oord, J.J., Swaab, D.F. and de Jong, J.M., Neuronophagia in the motor cortex in amyotrophic lateral sclerosis, Neuropathol. Appl. Neurobiol., 19 (1993) 390–397.
[18] Vinores, S.A. and Herman, M.M., Phagocytosis of myelin by astrocytes in explants of adult rabbit cerebral white matter maintained on Gelfoam matrix, J. Neuroimmunol., 43 (1993) 169–176. [19] Wu, E. and Raine, C.S., Multiple sclerosis: interactions between oligodendocytes and hypertrophic astrocytes and their occurrence in other, non-demyelinating conditions, Lab. Invest., 67 (1992) 88–99. [20] Wu, E., Brosnan, C.F. and Raine, C.S., SP-40, 40 immunoreactivity in inflammatory CNS lesions displaying astrocyte/oligodendrocyte interactions, J. Neuropathol. Exp. Neurol., 52 (1993) 129–134.