are glial fibrillary acidic protein (GFAP)-positive and 80% are vimentin-positive by immunohistochemical staining, suggesting that they are present de novo and ...
GLIA 5:75-80 (1992)
Preparation and Characterization of Astrocytes Cultured From Adult Rat Cortex, Cerebellum, and Striatum JOAN P. SCHWARTZ AND DELORES J. WILSON Clinical Neuroscience Branch, NINDS, NIH Bethesda, Maryland 20892
KEY WORDS
Cyclic AMP, Glial fibrillary acidic protein, Vimentin
ABSTRACT Astrocytes have been prepared from adult rat cortex, cerebellum, and striatum, using a modification of the McCarthy-DeVellis (J Cell Biol 85:890, 1980) method. The cultures consist of 99%type 1 polygonal astrocytes, which divide more slowly than cells from newborn animals. One day after preparing the cultures, 90%of the cells are glial fibrillary acidic protein (GFAP)-positive and 80% are vimentin-positive by immunohistochemical staining, suggesting that they are present de novo and not derived from precursor cells. The astrocytes from adult brain respond to an elevation of intracellular cyclic AMP, following treatment with forskolin, by becoming more stellate in shape and putting out fine ramified processes. They contain the same amount of GFAP per mg protein, measured by immunoblot, as cells from newborn animals. These cultures thus offer the possibility of comparing the biochemical properties of astrocytes derived from adult animals with those from newborn animals, or with cultures of reactive astrocytes isolated from lesioned brain.
INTRODUCTION Cultures of essentially pure type 1 polygonal astrocytes are readily obtained from neonatal rat brain using the McCarthy-DeVellis (1980) procedure or modifications thereof (Dubois-Dalcq, 1987; Noble and Murray, 1984). However, comparable success has not been achieved for preparation of cultures of astrocytes from adult brain. Glial cell lines, established from either normal human brain or human gliomas, consisted of a mixture of cell types that included morphologically identified astrocytes (Ponten and Macintyre, 1968).Astrocytes have been identified, using glial fibrillary acidic protein (GFAP) immunofluorescence, in mixed cultures prepared from adult human brain (Gilden et al., 19761, from adult rat optic nerve (Kennedy and Lisak, 19801, and from adult mouse cerebral hemispheres (Vernadakis et al., 1984):none of these cultures was a pure population of astrocytes. Lindsay et al. (1982) obtained cultures from adult corpus callosum that were 60-90% GFAP-positive, but only after the callosum had been lesioned by a knife cut: such cultures @I992 Wiley-Liss, Inc.
may thus be closer to reactive astrocytes in biochemical characteristics. Norton et al. (1988) described a delayed appearance of GFAP-positive cells in cultures from adult bovine or rat brain: with their preparation methods, the cells appeared over the course of 2-3 weeks in culture, suggesting that they were arising from progenitor cells. In this paper we describe cultures of type 1astrocytes prepared from adult rat cortex, cerebellum, and striatum by a modification of the McCarthy-DeVellis (1980) method. GFAP-positive cells are detected within 1day in culture. Following the shake-off and cytosine arabinoside treatment, the cultures are 99% type 1 astrocytes, indistinguishable in most respects from cultures of 3-day rat astrocytes. Such cultures offer the possibility of comparing astrocytes from normal adult brain with the “reactive” cultures obtained following brain injury (Lindsay et al., 1982). Received October 12,1990; accepted May 3,1991 Address reprint requests to Dr. J.P. Schwartz, CNB, DIR, NINDS, NIH, Building 9, Room 1W115, Bethesda, MD 20892.
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MATERIALS AND METHODS Cell Culture Cortical, cerebellar, and striatal astrocytes were prepared according to the method of McCarthy and DeVellis (1980) from a litter of 3-day old or from three to four adult (10-15-week-old) Sprague-Dawley rats with minor modifications (Dubois-Dalcq, 1987; Noble and Murray, 1984).After dissection of the tissue and removal of the meninges, the tissue was triturated with a 5 ml pipette, followed by five to ten passes through a 20 gauge needle, a 22 gauge needle, and a 25 gauge needle sequentially. The cells were passed through a 60 pm mesh, pelleted, and transferred in complete medium to 75 ml tissue culture flasks (three flasks per cortex, two per cerebellum, and one per striatum). Viability of cells, determined by trypan blue exclusion, was 88% for cells obtained from 3-day animals and 11%for cells obtained from adult animals. The plating density was 1-2.5 x lo4 live cells/cm2. When the cells became confluent (2-3 weeks), the flasks were shaken a t 225 rpm overnight at 37°C and the medium changed the next morning. Following the third overnight shake, the cells were trypsinized and cultured for 24 h in 10 pM cytosine arabinoside (Sigma Chemical Co.) (Dubois-Dalcq, 1987). The shaking removes -50% of the cells in the flask, which were identified by immunohistochemistry as 0,A progenitor cells, type 2 astrocytes, and microglia. Almost no neurons or oligodendrocytes were present, presumably because the growth conditions are not favorable for survival of these cell types (neuronssurvive less well in 10% CO,, while the presence of serum promotes type 2 astrocyte differentiation over that of oligodendrocytes) (Dubois-Dalcq, 1987; Raff et al., 1983). The cytosine arabinoside treatment kills off fibroblasts, which divide more rapidly than astrocytes. After the trypsinization, the viability of cells from both neonatal and adult was approximately 80%:the plating density a t this step was 2-3 x 104/cm2. When flasks became confluent again (7-14 days), they were subcultured for experiments. Culture conditions used were Dulbecco's Modified Eagle's Medium (GIBCO) containing 10% fetal bovine serum (Whittaker-MA Bioproducts Hybridoma Serum) and 25 kg/ml gentamycin in a humidified atmosphere of 90% air-10% COz. After the second subculture, the cultures are 99% astrocytes (GFAP-positive). Indirect immunofluorescence showed less than 1 4 0 0 cells to be microglia (Mac-1-positive; Springer and Ho, 1982); type 2 astrocytes or neurons (A2B5-positive; Dubois-Dalcq, 1987); oligodendrocytes (galactocerebroside-positive; Raff et al., 1983);or fibroblasts (fibronectin-positive). For analyses of cultures at early time points (within 1-7 days after preparation), cells were seeded directly into chamber slides (Lab-Tek plastic) at 2.5 x lo4 cells/cm2. The cells were fixed with 2% paraformaldehyde directly on the slides prior to staining.
A
Fig. 1. Analysis of adult cortical astrocyte culture by GFAP immunofluorescence and phase contrast. Cultures were prepared, subcultured twice, and stained 1-2 weeks later, as described in Materials and Methods. A: GFAP immunofluorescence stain. B: Phase contrast photograph of same field as in A. Magnification X200.
Immunohistochemistry Antibodies used for immunofluorescence studies were as follows: GFAF', rabbit antibody (Lipsky and Silverman, 1987) at 1500; A2B5, mouse monoclonal supernatant at 1500; galactocerebroside, rabbit antibody (Advanced Immunochemical Services) at 1:200; Mac-1, mouse monoclonal (Boehringer-Mannheim) at 1:25; vimentin, mouse monoclonal (Signet Laboratories) a t 1:25; and fibronectin, rabbit antibody (CalBiochem) at 1:250. Secondary antibodies used were either goat anti-rabbit or goat anti-mouse labelled with fluorescein (FITC) or rhodamine (TRITC) from Cappel (Organon Teknika) or Jackson Immunoresearch Labs. Before staining for GFAF' or vimentin, fixed cells were treated with acid-ethanol (5% glacial acetic acid:95% ethanol) for 10 min at -20°C. Normal rabbit or mouse serum at a comparable dilution was used for the negative controls; no staining was ever detected.
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Fig. 2. Glial fibrillary acidic protein (GFAPjstaining of astrocyte cultures prepared from 3-day and adult rat brain. Cultures were prepared, subcultured twice, and stained 1-2 weeks later, as described in Materials and Methods. A. Astrocytes from 3-day cortex. B: Adult cortex. C: Three-day cerebellum. D: Adult cerebellum. E: Three-day striatum. F: Adult striaturn. Magnification ~ 2 0 0 .
RESULTS Immunohistochemical characterization of astrocyte cultures prepared from cortex, cerebellum, and stria-
tum of both 3-day-old and adult rats, after the second subculture, demonstrated that virtually all cells at this point were GFAF'( +) (Fig. 1).Most of them exhibited a flat polygonal morphology and were also vimentin(+ ).
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markers. In the 3-day rat cultures, 40-60% of the fluorescent-labelled cells were GFAP(+ ) whereas 90-100% were vimentin(+)-thus, about half expressed both markers. In contrast, in the adult cultures, most cells were positive for both markers. Representative results from 3-day (Fig. 3A) and adult (Fig. 3B) cerebellar cultures are illustrated in Figure 3. Almost none of the cells in the adult cultures was A2B5(+) over the period from 1-7 days in culture, nor were there significant numbers of fibroblasts (fibronectin-positive)in any of the cultures (S2%). Other differences between astrocytes prepared from 3-day versus adult rat brain include the initial viability, the plating efficiency and/or survival rate, and the doubling time. When identical numbers of total live cells were seeded (2.5 x lo4 cells/cm2),the number of astrocytes in the 3-day cultures exceeded that in the adult cultures by five- to ten-fold over the first 4 days in culture. However, after 2-3 weeks in culture, both had become confluent. Following the shake-off and cytosine arabinoside treatment, when essentially all cells in the cultures are type 1astrocytes, the cells from adult brain took twice as many days to reach confluence when identical numbers of live cells were plated. In two other respects, the cells from neonatal and adult brain behave identically. GFAP content per mg protein, measured by an immunoblot method (Lipsky and Silverman, 1987), is virtually the same (3-day cortical astrocytes: 0.10 mg GFAP/mg protein; adult cortex: 0.12; 3-day cerebellum: 0.11; adult cerebellum: 0.13). The morphological response of the adult astrocytes to treatment with forskolin, which elevates intracellular cyclic AMP, is illustrated in Figure 4.Elevated cyclic AMP causes the appearance of stellate-shaped cells with fine ramified processes (Fig. 4B), as has been described for younger cultures (Kimelberg et al., 1978; Korinkova and Loden, 1977; Moonen et al., 1975; Schousboe, 1980; Sensenbrenner et al., 1980; Tardy et al., 1981; Trimmer et al., 1982).
Fig. 3. Expression of GFAP and vimentin by 3-day and adult astrocytes in the first 7 days of culture. Cultures in chamber slides were fixed in paraformaldehyde within 1-7 days after the initial preparation of the cells and stained for both GFAP and vimentin, as described in Materials and Methods. Five to ten fields per chamber were evaluated for numbers of total cells, GFAP(+) cells, and vimentin(+) cells, using phase, fluorescein, and rhodamine filters and objectives, respectively. Data are expressed as percent cells positive for GFAP [GFAP(+) celldtotal cells, hatched bar] or for vimentin [vimentin(+) celldtotal cells, open bar]. Six to seven slides, from three cultures prepared at different times, were evaluated for each time period. The total number of cells counted for each time period ranged from 200 to 450. A Astrocytes from 3-day rat cerebellum. B: Astrocytes from adult cerebellum.
DISCUSSION We describe in this paper the use of a modified The astrocytes from adult brain (Fig. 2B,D,F) were essentially indistinguishable from those obtained from McCarthy-DeVellisprocedure for routine preparation of 3-day animals (Fig. 2A,C,E). Additional staining with type 1 astrocyte cultures from adult rat brain. Astroantibodies to potentially contaminating cell types dem- cytes have been cultured from cortex, cerebellum, and onstrated the absence of oligodendrocytes,type 2 astro- striatum, and all show identical characteristics. No cytes, neurons, fibroblasts, or microglia (less than 1/100 priming lesion, as described by Lindsay et al. (19821, was needed, suggesting that the cells may resemble cells: results not shown). In order to characterize the cultures further, the time normal astrocytes more than reactive astrocytes. In course for the appearance of GFAP and vimentin in cells addition, there is no question as to the anatomical origin during the first week after preparation was compared of the cells, since no knife cuts (i.e., priming lesion) were for 3-day versus adult brain. Cells were stained for both required. Several differences were noted relative to astrocytes GFAP and vimentin, using double label immunohistochemistry, 1-7 days after being put into culture. Both prepared from 3-day rat brain. The initial plating effiGFAP(+) and vimentin(+) cells could already be de- ciency and/or survival of the cells from adult brain was tected at 1 day in culture-the total number of both lower, only 10-20% that of the 3-day cells over the first 4 increased over the 7 days of culture and 80-100% of all days in culture. However, cells that had attached to the cells in a given field were positive for one or both chamber within the first day were predominantly astro-
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Fig. 4. Effect of forskolin on astrocyte morphology. Adult cortical astrocytes were prepared as described and used 2-3 weeks after the second subculture. The cells were incubated for 3 h without (A)or with (B) 5 x M forskolin. Photographs were taken under phase-magnification ~ 4 0 .
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with vimentin( + ) cells always appearing earlier and faster than GFAP(+) cells. They concluded that the GFAP(+) astrocytes derived from vimentin(+)/ GFAP(- ) cells, which in turn arose from glial precursor cells (Norton et al., 1988).Furthermore, they referred to earlier studies demonstrating that “astrocytes swell and lyse during the trypsinization procedure” (Snyder et al., 19801,suggesting the loss of astrocytes during cell isolation. Comparison of our data with those of Norton et al. (1988) raises an important point. We could detect cells positive for either GFAP, vimentin, or both, in cultures prepared from adult brain within 1 day after preparation: their number then increased continuously with time in culture. Furthermore, the fact that 90-100% of the cells were GFAP(+) after 1 day in culture suggests that they are astrocytes that were resident in the brain at the time of tissue dissection, rather than deriving from vimentin( + )/GFAP(-1 precursor cells during culture. In contrast, only 40430% of the cells in cultures from neonatal brain were GFAP(+)himentin(+ ) astrocytes 1day after cell preparation. Most of the remainder of the cells in the neonatal cultures, which were vimentin( + )/GFAP- 1, morphologically resemble 0,A progenitor cells as well as microglia, both of which have been shown to express vimentin (Graeber et al., 1988; Raff et al., 1984). We believe that these differences reflect the preparation protocols: thus, the use of trypsin by Norton et al. (1988) may have killed the astrocytes present in adult brain, as they suggested, and so GFAP(+) cells appeared only after precursor cells had divided and differentiated. We conclude that one can readily prepare pure cultures of type l astrocytes from various regions of adult rat brain. Such cultures offer the possibility to compare biochemical properties, such as neurotrophic factor production, of astrocytes from adult brain with those of astrocytes from neonatal animals, in order to ascertain whether “adult”astrocytes exhibit different biochemical properties. Since the cells from adult brain respond morphologically to elevated cyclic AMP in the same way as cells from neonatal brain, it will be of great interest to determine whether biochemical responses to cyclic AMP, such as nerve growth factor synthesis (Schwartz and Mishler, 19901, enzyme induction (Narumi et al., 19781, and GFAP and vimentin phosphorylation (McCarthy et al., 1985) also occur to the same extent. Finally, comparison of these cultures of astrocytes prepared from normal adult brain to those derived from injured brain (“reactive”astrocytes) may provide clues as to the changes induced in glia following neuronal injury.
cytes as demonstrated by expression of both GFAP (90-100% of cells positive) and vimentin (70-90%). The low incidence of other cell types suggests that adult neurons do not survive under these conditions and that microglia, fibroblasts, and other cells either are present in normal adult brain in low numbers, do not readily survive the isolation procedure, or are incompletely dissociated without enzymatic digestion. After the cells had achieved the initial state of confluency, and had been shaken and treated with cytosine arabinoside, they were essentially all type 1 astrocytes [GFAP(+) and A2B5(-)]. At this point, the astrocytes from adult brain required twice the amount of time to achieve confluency as the 3-day cells. Norton et al. (1988) described the preparation of astrocyte cultures from 30-day rat and adult bovine ACKNOWLEDGMENTS brain, using a method that included trypsinization and sucrose density gradient purification. They reported We thank Ms. Joan Darcey for help in manuscript that they could never detect GFAP(+) or vimentin( + 1 cells after 1 day in culture and that both markers preparation and Dr. Ann Marini for assistance in carryincreased in number slowly after 2-3 weeks in culture, ing out the GFAP immunoblot.
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REFERENCES Dubois-Dalcq, M. (1987) Characterization of a slowly proliferative cell along the oligodendrocyte differentiation pathway. EMBO J., 6:2587-2595. Gilden, D.H., Wroblewska, Z., Eng, L.F., and Rorke, L.B. (1976) Human brain in tissue culture. Part 5. Identification of glial cells by immunofluorescence.J . Neurol. Sci., 29:177-184. Graeber, M.B., Streit, W.J., and Kreutzberg, G.W. (1988) The microglial cytoskeleton: Vimentin is localized within activated cells in situ. J.Neurocytol., 17573-580. Kennedy, P.G.E. and Lisak, R.P. (1980) Astrocytes and oligodendrocytes in dissociated culture of adult rat optic nerve. Neurosci. Lett., 16~229-233. Kimelberg, H.K., Narumi, S., and Bourke, R.A. (1978) Enzymatic and morphological properties of primary rat brain astrocyte cultures and enzymatic development in vivo. Brain Res., 153:55-77. Korinkova, P. and Lodin, Z. (1977)A transitional differentiation ofglial cells of cultured corpus collosum caused by dibutyryl cyclic adenosine monophosphate. Neuroscience, 2:1103-1114. Lindsay, R.M., Barber, P.C., Sherwood, M.R., Zimmer, J., and Raisman, G. (1982)Astrocyte cultures from adult rat brain. Derivation, characterization and neurotrophic properties of pure astroglial cells from corpus callosum. Bruin Res., 243:329-343. Lipsky, R.H. and Silverman, S.J.(1987)Effects ofmycophenolic acid on detection of glial filaments in human and rat astrocytoma cultures. Cancer Res., 47:4900-4904. McCarthy, K.D. and DeVellis, J. (1980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J . Cell B i d , 85:890-902. McCarthy, K.D., Prime, J.,Harmon, T., and Pollenz, R. (1985) Receptor-mediated phosphorylation of astroglial intermediate filament proteins in cultured astroglia. J . Neurochem., 44:723-730. Moonen, G., Cam, Y., Sensenbrenner, M., and Mandel, P. (1975) Variability of the effects of serum-free medium, dibutyryl-cyclicAMP or theophylline on the morphology of cultured newborn rat astroblasts. Cell Tissue Res., 163:365-372. Narumi, S., Kimelberg, H.K., and Bourke, R.S. (1978) Effects of norepinephrine on the morphology and some enzyme activities of primary monolayer cultures from rat brain. J . Neurochem., 31:1479-1490.
Noble, M. and Murray, K. (1984) Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell. EMBO J., 3~2243-2247. Norton, W.T., Farooq, M., Chin, F-C., and Bottenstein, J.E. (1988)Pure astrocyte cultures derived from cells isolated from mature brain. Glia, 1:403-414. Ponten, J. andMacintyre, E.H. (1968) Long term culture of normal and neoplastic human glia. Acta Pathol Microbiol. Scand., 74:465-486. Raff, M.C., Miller, R.H., and Noble, M. (1983) A glial progenitor cell that develops in vitro into a n astrocyte or a n oligodendrocyte depending on culture medium. Nature, 303:390-396. Raff, M.C., Williams, B.P., and Miller, R.H. (1984) The in vitro differentiation of a bipolar glial progenitor cell. EMBO J., 3:1857-1864. Schousboe, A. (1980) Primary cultures of astrocytes from mammalian brain as a tool in neurochemical research. Cell. Mol. Biol., 26:505-513. Schwartz, J.P. and Mishler, K. (1990) P-Adrenergic receptor regulation, through cyclic AMP, of nerve growth factor expression in rat cortical and cerebellar astrocytes. Cell. Mol. Neurobiol., 10:447457. Sensenbrenner, M., DeVilliers, G., Bock, E., and Porte, A. (1980) Biochemical and ultrastructural studies of cultured rat astroglial cells: Effect of brain extract and dibutyryl cyclic AMP on glial fibrillary acidic protein and glial filaments. Differentiation, 17:51-61. Snyder, D.S., Raine, C.S., Farooq, M., and Norton, W.T. (1980) The bulk isolation of oligodendroglia from whole rat forebrain: A new procedure using physiologic media. J . Neurochem., 34:1614-1621. Springer, T.A. and Ho, M.K. (1982) In: Hybridomas in Cancer Diagnosis and Treatment. M.S. Mitchell and H.F. Oettgen, eds. Raven Press, New York, pp. 35-46. Tardy, M., Fages, C., Rolland, B., Bardakdjian, J., and Gonnard, P. (1981) Effects of prostaglandins and dibutyryl cyclic AMP on the morphology of cells in primary astroglial cultures and on metabolic enzymes of GABA and glutamate metabolism. Ezperientia, 37~19-21. Trimmer, P.A., Reier, P.J., Oh, T.H., and Eng, L.F. (1982) An ultrastructural and immunocytochemical study of astrocyte differentiation in vitro. J . Neuroimmunol., 2:235-260. Vernadakis, A,, Mangoura, D., Sakellaridis, N., and Linderholm, S. (19841Glial cells dissociated from newborn and aged mouse brain. J . Neurosci. Res., 11:253-262.
