Distribution of glial fibrillary acidic protein and fibronectin in primary astroglial cultures from rat brain

Brain Research, 199 (1980) 493-500 © Elsevier/North-Holland BiomedicalPress

493

Distribution of glial fibrillary acidic protein and fibronectin in primary astroglial cultures from rat brain

P. E. STIEG, H. K. KIMELBERG*, J. E. MAZURKIEWICZ and G. A. BANKER Division of Neurosurgery and Departments of Anatomy and Biochemistry, Albany Medical College, Albany, N.Y. 12208 (U.S.A.)

(Accepted June 19th, 1980) Key words: glial fibrillary acidic protein (GFAP) -- astroglial cultures -- fibronectin

Certain types of primary monolayer cultures from dissociated neonatal rat brains have been found to possess a number of the properties of astroglia in vivo2,a,6-11,14,18. The cells in such primary cultures are likely to show more of the characteristics of normal astroglia than do glial cell lines derived from tumors. Because the morphological characteristics of cells in culture are quite variable, immunocytochemical techniques using markers specific for particular cell types have proved a useful way of determining the composition of such primary cultures, as recently shown for cultures from the CNS 14. Using such methods to identify astroglia by the presence of glial fibrillary acidic protein (GFAP) 4,16, we have determined the proportion of astroglia present in primary monolayer cultures under different conditions. Other cell types present in these primary cultures would likely include leptomeningeal cells from residual meninges, endothelial cells or fibroblasts. Since all of these cells have been shown to stain positively for fibronectin 5,14,17, we stained simultaneously for GFAP and fibronectin to positively identify non-GFAP staining cells and also to see whether cultured astroglial cells, which stain for GFAP, do not stain for fibronectin, as reported for these cells in vivo 17. Since cell surface fibronectin cross-reacts with antisera prepared against cold-insoluble globulin (CIg) 15, we used antiserum to CIg to stain for fibronectin. It has been shown that many of the cells in primary cultures from neonatal rat brain undergo a morphological transfomation when exposed to norepinephrine (NE) 13 or dibutyryl 3',5'-cyclic adenosine monophosphate (DBcAMP) 7,1°,11, developing radially oriented processes so that the cells closely resemble astroglia in vivo. We were also interested in determining the correlation between GFAP staining and the ability of the cells to undergo such a morphological transformation in response to treatment with NE. Primary astroglial cultures were prepared from the cerebral hemispheres of 1-3day-old Sprague-Dawley rats after removal of meninges and maintained as previously described 7,s,13. Cells were grown on 18 mm round glass cover slips contained in 60 mm * To whom reprints should be addressed Division of Neurosurgery.

494 diameter plastic petri dishes (Corning), and reached a saturation density of around 5 × 104 cells/sq, cm by the third week. For meningeal cultures, the leptomeninges were removed from 10 neonatal Sprague-Dawley rats, separated into a single-cell suspension with 0.25 ~ Trypsin (GIBCO) at 37 °C, centrifuged and then re-suspended in modified Eagle's medium containing 20 ~ fetal calf serum (FCS). They were plated onto 18 m m round glass cover slips contained in 60 m m petri dishes and maintained exactly as were the primary astroglial cultures. These cultures grew rapidly and reached a saturation density of around 1 × 105 cells/sq, cm by the second to third week. Antigen localization was performed on cells grown on cover slips following the unlabeled antibody method of Sternberger 19 using rabbit antiserum directed against bovine G F A P 4 and rabbit antiserum directed against rat plasma-CIg 1, respectively. All photographs for the present publication were made from single-labeled cultures, and positive staining for both G F A P and fibronectin is seen as a black precipitate. Simultaneous localization of fibronectin and G F A P was performed by a modification of the methods of Nakane 12 and Tramu et al. 21 and were used for the quantitative data shown in Table II which has been presented elsewhere 20. For the quantitative studies, fixed cells were counted on a Zeiss Photo-toreroscope equipped for phase microscopy. Cells stained for G F A P alone with or without N E treatment were counted individually in random areas of the cover slip. A total of 400 cells on each cover slip was counted. Each cell counted was classified as G F A P positive (GFAP(~-)) or negative ( G F A P (--))based on control slides and its morphology was classified as process- or non-process-bearing. Cells were classified as processbearing if they had2 or more processes which were at least twice as long as the diameter of the cell body la. In double-label experiments, random areas of the cover slip rather than individual cells were counted, using an eyepiece grid containing 36 squares, 55 /~m on a side. A total of 400-900 squares was counted on each cover slip. A square was scored as positive for fibronectin, G F A P or unstained cells if one-half or more of its area showed positive staining for either antigen or contained unstained cells. Because the cells are stratified, these criteria resulted in some squares being counted as positive for more than one category. To determine the effects of 0.1 m M NE on cell morphology and G F A P localization, complete growth medium was removed from the petri dish and the cover slips were washed 3 times in growth medium without FCS. The cells were then treated with N E in growth medium without FCS for 2 h at 37°C in a 95 ~ air/5 ~ CO2 humidified incubator. Cells not treated with N E were incubated under identical conditions in growth medium without FCS for 2 h as controls. The cells were then stained for GFAP. Two major types of cells were observed in cultures which had been maintained for 4-28 days. The morphology of the predominant cell type appeared to be density- or age-dependent. At low densities, these cells were typically broad, flat and polygonal in shape. In confluent cultures the cells were smaller, more regular in shape and arranged in a pavemented pattern. Fig. la is a bright-field micrograph of a low density area of a 14day culture stained for G F A P showing this predominant cell type. It can be seen that

Fig. 1. a: bright-field photomicrograph of 14-day primary astroglial cultures stained for G F A P alone as described in Materials and Methods. b: bright-field micrograph of 14-day cultures treated with 0.1 m M N E for 2 h as described in Materials and Methods and stained for GFAP. c: phase micrograph of confluent, 21-day astroglial culture stained for fibronectin, d: bright-field micrograph from the same field as c but with different orientation. The single fibronectin-positive cell is marked by arrows in c and d.

496 these cells were G F A P ( + ) , showing the characteristic filamentous cytoplasmic staining and no nuclear staining 14,a6. We have previously reported that up to 60°//o of the cells in these cultures undergo a morphologic transformation when exposed to NE with the cells developing radially oriented processes and closely resembling astroglia in vivo 13. It can be seen in Fig. I b that these process-bearing cells showed intense staining for GFAP, whereas the G F A P ( ÷ ) flat cells stained less intensely. Figs. lc and l d are micrographs from a typical area of a confluent 21-day culture stained for fibronectin. Fig. l c is a phase micrograph showing that this area contains the predominant cell type with the pavement appearance mentioned above. As seen in Fig. ld, only one cell from this field showed the extracellular surface staining pattern for fibronectin. Also, Fig. l d shows the control background level of staining generally obtained. Table I summarizes quantitative data on the relationship between G F A P staining and process formation in response to NE treatment. It can be seen that an overall mean of 94 -~ 4 ° / ( ± S . D . ) of all the cells in cultures ranging in age from 7 to 21 days which responded to NE by forming processes, were GFAP(~-). The percentage of the cells in the control group which formed processes and were G F A P ( + ) was 85 ± 11 ~,i. This value was not significantly different from the 94 -~ 4 o~ found after addition of N E, as calculated by the Student's t-test. The percentage of the non-process-bearing ceils which were GFAP(~-) was less than for process-bearing cells both in the presence and absence of NE. However, these cells still constituted the majority with an overall mean of around 70 % for all cultures. Taking the values for process- and non-process-bearing cells, the overall mean for GFA P ( + ) cells was 74 70/ for cultures not treated with NE and 79 ~ for cultures treated with NE. Thus, treatment with NE did not seem to significantly affect the proportion of cells in the cultures which were G F A P ( + ) , but that the cells which transform morphologically are largely, and perhaps exclusively, astroglia. The total percentages of the process-bearing plus non-process-bearing cells which were GFAP(q-) in 7-, 14- and 21-day cultures (data not shown) were 64, 66 and 92 for control and 73, 79 and 84 for NE-treated cultures, TABLE I Relationship between process formation in response to norepinephrine and GFAP staining

Regular growth medium was removed from cell cultures and replaced with growth medium without FCS, with or without 0.1 mM NE as indicated in the table and described in Materials and Methods. Each value represents the overall mean & S.D. for cultures ranging in age from 7 to 21 days. n represents the total number of coverslips counted within each category. 400 individual cells on each cover slip were counted in random areas. The average ~ cells forming processes was 36~ and 18 ~ in the presence and absence of NE, respectively. % Cells GFAP-positive Process bearing

Non-process-bearing

Control (--)NE

85 ± 11 (n -- 6)

70 ± 17 (n ~ 6)

(÷)NE

94 ± 4 (n -- 6)

68 ± 14 (n 6) -

-

Fig. 2. A 21-day primary astroglial culture from cerebral hemispheres kept in growth medium without FCS for phase microscopy, or stained for GFAP. a: bright-field micrograph showing the GFAPpositive cells stained black lying underneath the unstained diagonally arranged bundle of cells growing on top. It can be seen that some of the astroglia have formed processes due to removal of FCS. b: phase micrograph of the same field, c: 21-day primary astroglial cultures from cerebral hemispheres stained for fibronectin, d: phase micrograph of same field showing fibronectin positive cells on top of the unstained astroglial cells.

498 respectively. The values for the per cent G F A P ( ÷ ) cells in confluent, 21-day cultures are in reasonable agreement with the 95 ~ value Manthorpe et al. TM repoIt for late, dense cultures. However, our double-label data (see below) suggest that these figures may overestimate the purity of these older cultures. The second type of cell seen in these cultures was typically spindle shaped and grew in a more fibroblastic pattern on top of the G F A P ( + ) ceils. These cells did not stain for G F A P as can be seen in Figs. 2a and 2b. When cultures were stained for fibronectin, these cells showed the typical surface staining pattern described for cultured fibroblasts 5, as shown in Figs. 2c and 2d. Double-label experiments clearly showed that the fibronectin positive cells typically grew on top of the flat G F A P ( + ) cells. There are several possible origins for the GFAP(--), fibronectin(+) cells. One possible source could well have been from residual leptomeningeal fragments that we cannot be sure we totally removed during culture preparation1< In support of this it has been reported 14, and we also found, that cultures prepared from the leptomeninges alone were G F A P (--) but fibronectin ( + ) . Table II summarizes data on the relative amounts of GFAP(-~ ) and fibronectin(-?) cells, estimated by counting areas which were G F A P ( + ) or fibronectin(+) after simultaneous localization of both antigens. It can be seen that the proportion of fibronectin(+) cells increased in older confluent cultures. The proportion of G F A P ( + ) areas showed a more variable behavior. At 7 days, 53 }~i of the areas were GFAP(-?), but this was associated with 35 ~; of the areas which did not stain for either antigen. At 14 and 21 days there was an increase in the G F A P ( + ) areas to 74% and 68 %~, respectively, associated with a marked decrease in unstained areas. No cells appeared to stain for both antigens. It should be noted that the percentages of GFAP(-?) cells at 14 days estimated by the single- and double-label techniques are in reasonable agreement (66~,/, and 74 jo/0, respectively). However, in confluent, 21-day cultures the single-label method appears to overestimate significantly the percentage of G F A P ( + ) cells (92 ~o for single-label versus 68 % for double-label). This suggests that in the dense, confluent cultures the single-label technique is particularly prone to underestimate the fibronectin(-?) cells which grow mainly on top of G F A P ( - - ) cells, because of difficulties in distinguishing the G F A P ( - - ) cells by phase microscopy when TABLE 11

Simultaneous localization o f fibronectin and GFAP Random areas on each coverslip were counted on the days indicated as described in Materials and Methods. Each value is the overall per cent area (mean :t: S.D.) covered by the two cell types on the total number of cover slips shown, n represents the number of coverslips counted from different dishes of the same age culture.

Days ineulture

n

Squares > 1/2 fibronectin ( + )

Squares > 1/2 GFAP ( + )

Squares >_ 1/2 unstained

7 14 21

5 6 4

8~ ± 5 24~ ~6 2 8 ~ ~: 10

5 8 ~ i 23 74%± 7 68% ~: 7

3 5 ~ ± 18 1~:t: 1 4~ ± 5

499 they are unstained. These fibronectin(+) cells are not the same as the small phase dark process-bearing cells described by Manthorpe et al. 1° as comprising up to 1 6 ~ of G F A P ( - - ) cells in their early cultures. Under our conditions such phase dark cells are only seen in young cultures but their proportion does not exceed 5 ~ and is usually less. Such cells may correspond to the oligodendroglia described by Raft et al. la as being present in CNS primary cultures. In conclusion, our results support the use of immunohistochemical characterization for quantification of the composition of primary CNS cultures, and confirms that such cultures prepared as described in this paper and elsewhere from neonatal rat cerebral hemispheres, are predominantly astroglia and are therefore good models for studying the properties of normal astroglial cells. Between 14 and 21 days, the time period in which we generally use the cultures, astroglia comprise 70-80 ~ of the cells and the remaining cells may be derived from residual meninges and other sources. However, the proportion of astroglia could not be consistently increased by being as meticulous as possible in removing all visible meninges under a dissecting microscope during preparation. Thus, to reproducibly obtain pure astroglial primary cultures from these preparations, additional steps for their purification are necessary. In addition, under the present growth conditions and using antiserum against G F A P alone, we have shown that around 90 ~ of the process-bearing cells in the presence or absence of N E are G F A P ( + ) and therefore astroglia. Also, the results from the double-label method confirm 14 that astroglial cells in culture do not appear to stain positively for fibronectin. This work was supported by Grant 13042 from N I N C D S (P.E.S. and H.K.K.), a grant from the Sinsheimer Fund (G.A.B.) and Grant S07 RR-05394 from N1H (J.E.M.). We thank Dr, L. F. Eng for generously providing the anti-GFAP serum and Dr. F. Blumenstock for the anti-CIg serum. 1 Blumenstock, F. A., Saba, T. M. and Weber, P., An affinity method for the rapid purification of opsonic a2SB glycoprotein from serum, Advanc. Shock Res., 2 (1979) 55-71. 2 Bock, E., Moiler, M., Nissen, C. and Sensenbrenner, M., Glial fibrillary acidic protein in primary astroglial cell cultures derived from newborn rat brain, FEBS Lett., 83 (1977) 207-211. 3 Booher, J. and Sensenbrenner, M., Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures, Neurobiology, 2 (1972) 97-105. 4 Eng, L. F. and Rubinstein, L. J., Contribution of immunohistochemistry to diagnostic problems of human cerebral tumors, J. Histochem. Cytoehem., 26 (1978) 513-522. 5 Hedman, K., Vaheri, A. and Wartiovaara, J., External fibronectin of cultured human fibroblasts is predominantly a matrix protein, J. Cell Biol., 76 (1978) 748-760. 6 Hertz, L., Bock, E. and Schousboe, A., GFA content, glutamate uptake and activity of glutamate metabolizing enzymes in differentiating mouse astrocytes in primary cultures, Develop. Neurosci., 1 (1978) 226-238. 7 Kimelberg, H. K., Narumi, S. and Bourke, R. S., Enzymatic and morphological properties of primary rat brain astrocyte cultures and enzyme development in vivo, Brain Research, 153 (1978) 55-77. 8 Kimelberg, H. K., Bowman, C., Biddlecome, S. and Bourke, R. S., Cation transport and membrane potential properties of primary astroglial cultures from neonatal rat brains, Brain Research, 177 (1979) 533-550. 9 Lira, R., Troy, S. S. and Turriff, D. E., Fine structure of cultured glioblasts before and after stimulation by a glia maturation factor, Exp. Cell Res., 106 (1977) 357-372.

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