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Microglia and microglia-derived brain macrophages in culture: generation from axotomized rat facial nuclei, identification and characterization in vitro
Brain Research, 492 (1989) 1-14 Elsevier BRE 14617
1
Research Reports
Microglia and microglia-derived brain macrophages in culture: generation from axotomized rat facial nuclei, identification and characterization in vitro Ellen Rieske, Manuel B. Graeber, Wolfram Tetzlaff, Anna Czlonkowska 1, Wolfgang J. Streit and Georg W. Kreutzberg Department of Neuromorphology, Max-Planck-lnstitutefor Psychiatry, Martinsried (E R. G.) (Accepted 13 December 1988) Key words: Ramified microglia; Brain macrophage; Facial nucleus explant; Cell culture
In order to study microgliai cells and microglia-derived brain macrophages in vitro, a method has been developed which allows the transfer of mitotic microglial cells from adult rat brain into tissue culture. The studies were performed on facial motor nuclei which were explanted after axotomy of the facial nerve. Outgrowing cells were identified and characterized by (i) morphological criteria using light and electron microscopy, (ii) in vivo [3H]thymidine labeling combined with subsequent in vitro autoradiography, (iii) immunocytochemistry for vimentin, GFAP, Fc and complement receptors, MHC antigens, laminin, fibronectin, factor VIII related- and 04 antigen as well as lectin histochemistry, and (iv) functional in vitro tests. In addition, a mieroglial cell line was established from proliferating cells. The results indicate that perineuronal microglia rather than astrocytes, perivascular cells, oligodendrocytes or endothelial cells may become phagocytic after having been activated by axotomy in situ. INTRODUCTION The first attempts to study microglia in culture can be traced back to the early 1930s4'34'36. However, it was not before the present decade that a rapidly growing interest in the intrinsic defense mechanisms of the CNS led to systematic research on microglia in vitro 8'1°'24. Whereas the normal brain parenchyma does not contain active macrophages, phagocytic cells can be observed in neural tissue cultures 8'1°'24 as well as under various pathological conditions in vivo 6. Traumatic CNS injuries are usually accompanied by disruption of the vasculature and a subsequent influx of blood-derived phagocytic cells into the parenchyma 17, but in addition resident microglia represent a major source of endogenous brain macrophages 6'26. Aside from their phagocytic potential microglial cells are thought to be involved in
synaptic changes 2, gliosis al, CNS development 9, and immunological processes 8'9, the latter including antigen presentation in vivo 15, as well as HIV-1 infections 22. However, the great mutability of the microglial cell shape 1'5'13'21 together with the lack of a microglia-specific marker that would allow them to be distinguished from other macrophages have made it difficult in the past to positively identify and study microglia in vitro. Proliferating cells in a central m o t o r nucleus following peripheral nerve injury have been identified as microglia independently by several groups s' 18,35 In view of a recent study employing [3H]thymidine labeling at the ultrastructural level 14, there can now be little doubt that it is indeed endogenous microglia which undergo mitosis in response to axotomy. Following transection of the rat facial nerve, the affected m o t o r neurons not only
] On leave of absence from the Institute of Psychiatry and Neurology, Cerebrovascular Diseases Clinic, Sobieskiego 1/9, 02957 Warsaw, Poland. Correspondence: G.W. Kreutzberg, Max Planck Institute for Psychiatry, Dept. of Neuromorphology, Am Klopferspitz 18a, D-8033 Martinsried, F.R.G. 0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
survive but regenerate. Concomitantly, microglia are ~activated' in a sense that they proliferate but do not transform into macrophages 2'~2"29. Since there is no breakdown of the blood-brain barrier within a regenerating motor nucleus 27, and since an invasion of blood-borne cellular elements does n o t o c c u r 14"25, this model is clearly superior for studying microglia than other approaches employing cryogenic lesions or stab wounds where such a contamination cannot be avoided ~6. The unique advantage and elegance of the axotomy paradigm is that the brain tissue can be challenged without touching it. Based on this model we have now established a combined explant-cell culture system which allows the in vitro identification and characterization of genuine microglia derived from adult rat brain. MATERIALS AND METHODS
Animals and tissue preparation The right facial nerve of male Wistar rats (180200 g, 8-10 weeks old) was transected at the stylomastoid foramen. The contralateral unoperated side served as a control. Following survival times of 4-6 days the animals were perfused through the heart with sterile Hank's solution and the regenerating and contralateral unoperated facial nuclei were removed for explant culture (Fig. 1). Excised facial nuclei were carefully freed from meninges and neighboring tissue under sterile conditions, and each nucleus was cut into about 30 explants measuring approx. 0.125 mm 3. Two to four explants were placed 1 mm apart on coverslips and cultured in 35 mm Falcon tissue culture dishes at 36 °C in a humidified 1% CO2-air atmosphere which adjusted the pH of the medium to 7.4. The medium consisted of Dulbecco's MEM containing 20% human placental serum, 10% chick embryo extract and 5% fetal calf serum. The cultures were fed twice a week with fresh medium and kept as primary cultures for about 3 months. One, two and three,weeks after explantation the number of proliferating cells per explant culture was determined. No antibiotics were applied.
Facial nucleus cell cultures Cell cultures were established from rat facial nucleus explants after one to two weeks in vitro. Following removal of explant debris the layer of
growing cells was disaggregated either by incubating for 2-5 min at 36 °C in 0.25% trypsin solution (GIBCO), or by mechanical aspiration with fine fire-polished pipettes. Culture conditions were the same as for the explants. The following culture substrates were tested: LUX polystyrene and LUX Thermanox (Miles Lab. Inc., Naperville, 1L, U.S.A.) or collagen-coated glass, polystyrene-, polyornithine- or polylysine-coated glass, or polystyrene and a gas-permeable foil (Petriperm; Heraeus, Hanau, F.R.G.). In addition, the growth of primary explant cultures and cell cultures of rat facial nuclei was assessed in serum-free Dulbecco's MEM with the following additives: 2.5S Nerve Growth Factor (10 ng/mi; GIBCO), Epidermal Growth Factor (10 ng/ml; GIBCO), Fibroblast Growth Factor (10 ng/ ml; Collaborative Res., Lexington, MA, and GIBCO), Tuftsin (0.4 nmol; Sigma), TSH (thyroid stimulating hormone, 1 mU/liter; Sigma), and dibutyryl-cyclic AMP (1 raM; Boehringer, Mannheim, F.R.G.).
Cultures of cells prelabeled in vivo by [3H]thymidine These experiments were designed to test whether the cells proliferating in vitro from axotomized rat facial nuclei explants were related to the mitotic microglia of the facial nucleus in situ. Six rats which had undergone a facial nerve transection were injected intravenously with [3H]thymidine (1 mCi/kg b. wt.; Amersham, U.K.) 4 days later, a time at which microglial mitotic activity reaches its peak ~9. Ten hours after [3H]thymidine injection facial nuclei were explanted and cultivated as described above. At 3, 7, 9, 10, and 13 days in vitro (DIV) 2-8 explant cultures of each animal were rinsed with PBS, fixed in 70% alcohol and air-dried. The cultures were then processed for light microscopical autoradiography as described elsewhere 3°, and the total cell number was determined. Cells labeled with [3H]thymidine were classed according to the number of silver grains overlying their nucleus: 3-5, 6-10, 11-20, and 20 or more silver grains per nucleus.
Light and electron microscopical characterization of proliferating cells Light microscopy. The morphological characteristics of cells were determined by bright field, phase contrast and differential interference contrast light
microscopy as well as by time lapse microcinematography. Immunocytochemistry for bright field and fluorescence microscopy was performed by applying the following antibodies: (1) MRC Ox-42 (Serotec, MCA 275) is a monoclonal mouse IgG2a recognizing the rat CR3 complement receptor; (2) MRC-Ox 6 (Serotec, MCA 46) recognizes a monomorphic determinant of rat MHC class II (Ia) antigen; (3) MRC Ox-17 (Serotec, MCA 50) is a monoclonal mouse IgG1 antibody recognizing a determinant on the e-chain of rat Ia antigen, a homologue of mouse Ia-E product; (4) MRC Ox-18 (Serotec, MCA 51) is a monocional mouse IgG1 antibody recognizing a monomorphic determinant of rat class I MHC antigens; (5) anti-vimentin (monoclonal mouse IgG; Code 814318, Boehringer Mannheim, F.R.G.); (6) anti-GFAP (polyclonal rabbit IgG; Code Z334, Dako, Hamburg, ER.G.); (7) anti-laminin (polyclonal rabbit IgG, Dr. U. Kiihl, Max Planck Institute for Biochemistry, Martinsried, F.R.G.); (8) antifibronectin (polyclonai rabbit IgG, Code A245, DAKO); (9) anti-factor VIII related antigen (Code A082, Dako); (10) 0428, kindly provided by Prof. M. Schachner, Dept. of Neurobiology, University of Heidelberg, F.R.G. Fc receptors were detected by incubation with tetramethylrhodamine isothiocyanate (TRITC) conjugated goat anti-mouse IgG (Sigma, T-5393) diluted 1:10 at 37 °C. In addition, the cells were stained with the lectin from Griffonia simplicifolia (GSA-I-B4)29. Following fixation in 3.7% formaldehyde only or successive fixation in 3.7% formaldehyde, 3.7% formaldehyde containing 0.06% Triton X-100 (10 rain each), and acetone (50, 100, and 50%; 2 min each) immunocytochemical staining of cultures was carded out according to standard procedures using the alkaline phosphatase (Code D314, DAKO) sandwich technique or biotinylated secondary antibodies followed by ABC-alkaline phosphatase (Code AK-5000; Vector, Burlingame, CA). Ultrastructural examination. For transmission electron microscopic examination the explant and cell cultures were grown on glass, polystyrene (Thermanox, Miles Lab. Inc.) or biofoil (Petriperm) and were fixed in culture medium containing 3.5% glutaraldehyde at pH 7.2 for 20 min at room temperature. They were then washed in 0.05 M cacodylate buffer/8.5% sucrose, postfixed in Dal-
ton's osmium and embedded into Araldite or LR White. For scanning electron microscopical examination the cultures were washed in warm serum-free medium and fixed in 3% glutaraldehyde at room temperature. Subsequently, they were washed in phosphate buffer, postfixed in Dalton's osmium for 60 min, and critical point dried with CO 2 followed by gold coating.
Functional characterization of proliferating cells In order to test the phagocytic nature of cells 4 experiments were performed: (i) Presentation of suspended latex beads. Cultures were incubated at 36 °C for 60 min with latex particle solution (1/~m Dow-latex beads, 1.05 g/ml; Dow-Corning, Seneffe, Belgium), diluted 1:100 with medium. (ii) Immunoglobulin-coated ox erythrocytes as indicators of Fc receptor mediated rosetting and particle ingestion. The lowest non-agglutinating dilution of rabbit antiserum against opsonized ox red blood cells (IgG anti-E, Seralab) was tested (1:5 to 1:75) and combined with an equal volume of 5% opsonized ox erythrocytes in physiological salt solution. After incubation at 36 °C for 10-30 min and washing with warm medium the number of rosettes was determined. Ingested erythrocytes were demonstrated by phase contrast microscopy after lysis of extracellular erythrocytes by 3% acetic acid. (iii) Phagocytosis of laser-killed cells. Laser microirradiation was used to selectively kill individual cells among live neighboring cells. Single cells were killed by 8 ns shots of 4 x 1011 W/cm2 at 337 nm during observation through a 40x UV-objective23. Phagocytosis of dead cells by neighboring macrophages was examined by time lapse microcinematography. (iv) The lysosomotropic agent L-leucine-methylester (Sigma, L-9000) was used for selective killing of phagocytic cells 33.
Generation of macrophages from resident parenchyreal cells Three experiments were performed in order to test whether macrophages present in axotomized explant cultures were generated from resident parenchymal cells rather than from blood-derived macrophages invading the facial nucleus prior to explantation: (i) The number of macrophages present in the explants at the beginning of cultivation was determined in semithin sections. (ii) It was tested
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Fig. 2. Cellular outgrowth from axotomized and unoperated control facial nuclei explants in culture. Overall cell numbers in mixed primary cultures were counted. Data were taken from facial nucleus explants of 9 randomly chosen animals undergoing perfusion 4 or 5 days after facial nerve transection. A: n = 149 explants, B: n = 132 explants. C: logarithmic transformation of the geometric mean of data shown in plots A and B.
Fig. 3. A: scanning electron micrograph of an axotomized facial nucleus explant 16 DIV. Numerous spindle-shaped cells and macrophages can be seen within and around the explant. B: in early stages of explantation (10 DIV) spindle-shaped cells (long arrows) predominate over ramified forms. Macrophages (short arrows) are rounded with short cell processes. C: compared to B, older cultures (26 DIV) contain many more differentiated ceils. Spindle-shaped cells have developed ramified cell processes. Macrophages are large and flattened. B and C phase contrast light microscopy. Magnification: x60 (A), x180 (a), x68 (C).
Fig. 1. Culturing of rat facial nucleus explants following axotomy in viva. A: schematic representation of the model. B: control culture of an unoperated rat facial nucleus 12 DIV. Outgrowth of cells is insignificant. C: emigration and proliferation of cells around an axotomized facial nucleus explant 12 DIV. Magnification: x57 (B,C).
Fig. 4. Ultrastructural characteristics of ramified cells ( A - D ) and macrophages (E,F) in cultures of axotomized facial nucleus explants. A: scanning electron micrograph of ramified microglia in a 44-day-old culture. B: transmission electron micrograph of the same type of cell 15 DIV. Note the presence of many rough endoplasmic reticulum cisternae and liposomes. The cell nucleus shows large clusters of heterochromatin in its center and apposed to the nuclear membrane similar to microglia in vivo. C: transmission electron micrograph of a LR White-embedded ramified cell. Numerous filaments of intermediate size (long arrows) can be seen. D: ramified cell. High power micrograph of prominent wide rough endoplasmic reticulum cisternae (short arrows) typical of microglia (37 DIV). E: scanning electron micrograph of a facial nucleus macrophage 14 DIV. Note its undulating cytoplasmic membrane. F: transmission electron micrograph of a young macrophage in a 6-day-old axotomized facial nucleus explant culture. The nucleus is rich in heterochromatin, and cytoplasmic filopodia can be seen. Golgi complexes, lysosomes and vacuoles are also prominent. Magnification: x1500 (A), ×7250 (B), x43,000 (C), ×47,000 (D), ×2400 (E), x5350 (F).
TABLE I
Immunological characteristics of ramified cells and macrophages in cultures of ratfacial nucleus explants Marker Vimentin GFAP
Fc receptor CR3 receptor MHC class I antigen MHC class II antigen GSA-I-B4-isolectin Laminin Fibronectin Factor VIII 04 antigen
Ramified cells Macrophages + -
+ -
+ (+) + + + + + -
+ + + + + -
whether macrophages could be generated in vitro when all mitotic activity in the explants was inhibited by adding 5-fluorodeoxyuridine (FDU, 80 /~M; Sigma, F-0503) together with uridine (200 /~M; Sigma, U-3750). (iii) The degree of [methyl3H]thymidine accumulation in the explants during the first 6 DIV was determined (Warnhoff et al., in preparation) using the method of Murphy et al. 2°. In order to separate free [methyl-aH]thymidine in the medium from the explants, they were washed 3 times with Hank's balanced salt solution (HBSS) followed by lysis with 0.1 N NaOH. [Methyl-3H]thymidine incorporated into the DNA of proliferating cells was separated from the intracellular pools by adding 250 /~g bovine serum albumin (BSA; Sigma, A-7030) as a carrier and precipitating protein with 10% TCA. After centrifugation at 10,000 g for 5 min the protein pellet was dissolved in I N NaOH, liquid scintillation cocktail was added (Ready-Solve HP; Beckman, CA), and the radioactivity was measured in a liquid scintillation counter (efficacy 55%). Blank values, obtained from explants in medium lacking [methyl3H]thymidine during incubation, were determined by adding [methyl-aH]thymidine shortly before processing of the explants as described.
Release of growth stimulating factors from axotomized facial nuclei In order to test whether axotomized facial nuclei themselves release growth stimulating factors, 3 experiments were performed: (i) The time interval between nerve transection and explantation of facial
nuclei was varied from 5 h to 42 days. (ii) Control explants of 6 rats were fed with serum-free Dulbecco's MEM supplemented with 10-20% saline extract from axotomized facial nuclei (200 mg ww/ml Hank's solution); saline extract was obtained by homogenizing freshly excised rat facial nuclei 5 days after axotomy at 4 °C. (iii) Control explants of two rats were co-cultured with explants of axotomized facial nuclei by setting 4-6 axotomized explants around two control explants on coverslips. RESULTS
Enhanced cellular outgrowth from axotomized facial motor nuclei explants At all time points investigated the number of proliferating cells per axotomized explant culture by far exceeded those in controls, ranging from 10-fold at the end of the first week to more than 200-fold during the third week (Fig. 2). Whereas cultures of axotomized facial nucleus explants exhibited exponential growth characteristics, growth in control cultures was insignificant (Fig. 2). After 2-3 weeks in vitro, celt cultures from the operated facial nucleus demonstrated two large populations of cells clearly distinguishable and outnumbering all other cell types in these explant cultures: ramified cells and macrophages (Figs. 3 and 4). Ramified cells grew out from axotomized explants beginning late in the first week in vitro. In control cultures they were found only occasionally and not before the second week.
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Fig. 5. Proliferation kinetics of in vivo [3H]thymidine-labeled cells of axotomized facial nuclei in vitro. The rapid clearance of heavily labeled cells (closed triangles) suggests strong proliferative activity. Labeled cells were of the ramified type but absent from controls.
Fig. 7. Immunophenotypic characteristics of ramified microglia (A-C,E) and macrophages (D,F). A: vimentin. Fluorescence microscopy (fluorescein). B: Fc receptors. Fluorescence microscopy (rhodamine). C,D: Ia-antigen (Ox-6 mab) and alkaline phosphatase immunocytochemistry. E: GSA-I-B4-isolectin positive ramified microglia on top of a monolayer of lectin negative cells, 29 DIV. F: lectin-positive macrophages in the peripheral zone of outgrowth within the same culture as in E. Phase contrast microscopy and peroxidase/diaminobenzidine. Magnification: x900 (A), ×460 (B), ×290 (C,D,F), x 180 (E).
Macrophages also grew out of virtually all axotomized facial nucleus explants. They not only migrated, but also proliferated and covered large areas around the explants (Figs. 1 and 3). After 3 weeks,
1-5 x 103 macrophages per explant culture were present on average whereas in control cultures none or less than 15 macrophages per explant could be found (Fig. 1). There was no outgrowth at all from
<..Fig. 6. Functional tests on phagocytic activity of proliferating cells from axotomized facial nucleus explants. A: latex beads (1 am) are selectively taken up by macrophages and are concentrated around the cell nucleus and along cell processes. 14 DIV, and 1 h after incubation with latex beads. B: ingested opsonized ox erythrocytes within the cytoplasm of macrophages. Extracellularly bound erythrocytes were removed by washing with acetic acid (8 DIV). C: rosetting of opsonized ox erythrocytes around macrophages (thick arrow); ramified cells are free of ox erythrocytes (long arrow); 8 DIV. D: differentiated macrophages with flattened perikarya, 26 DIV. Phase contrast microscopy. Magnification: x243.
10
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11 control and axotomized explants when the nutrient medium contained less than 20% of biological ingredients, e.g. serum or chick embryo extract. It further appeared that rich medium formulas as well as high pH (>7.4) favored the proliferation of ramified cells rather than that of macrophages. However, under standard conditions the number of macrophages was approximately the same as that of ramified cells. There was no outgrowth of neurites or long term survival of neurons.
Morphology, functional characteristics and immunophenotypic characterisation of proliferating cells Macrophages were easy to recognize with phase contrast optics by both their irregular shape and high content of bright perinuclear droplets (Fig. 3B). Time lapse microcinematography revealed typical locomotion behavior. Their undulating membranes (Fig. 6D) and finger-like projections (Fig. 4F) were easily visible. Macrophages within and near the explants (Figs. 3A and 8B) were loaded with cellular debris after about 5 weeks. In contrast, macrophages migrating into the zone of outgrowth became increasingly flattened (Fig. 6D) and eventually acquired a stellate shape. They were able to survive for more than 8 weeks. Ultrastructurally, the cytoplasm of macrophages contained well-developed Golgi complexes, many dense bodies, lysosomes, mitochondria and membrane-bound vacuoles of different size (Fig. 4F). Their nuclei were folded with dense appositions of heterochromatin to the nuclear membrane (Fig. 4F). All phagocytosis assays were positive, e.g. latex beads as well as IgG-coated ox erythrocytes were readily taken up (Fig. 6A-C). Presentation of cells killed in vitro by laser microirradiation, resulted in rapid phagocytosis (within hours) by neighboring macrophages. Selective elimination of macrophages could be achieved with L-leucine-methylester which caused lysosomal disruption, cytoplasmic swelling and resulting death of macrophages within less than 2 days. Immunostain-
ing was positive with all macrophage markers but antibodies against other glial or endothelial cell markers, e.g. GFAP, 04, and factor VIII, failed to stain the cells (Table I). Ramified cells were 4 times more common in cultures of axotomized facial nucleus explants than in controls although in the latter they were by far the most frequent cell type. Ramified cells were positive for Fc receptors although they showed no signs of phagocytic activity neither in functional testing nor ultrastructurally (Fig. 4B). Their immunophenotype (Table I) appeared to be related to the monocyte family rather than to astrocytes, endothelia or oligodendrocytes (cf. Fig. 7). Ramified cells were also rich in microtubuli, microfilaments and filaments of intermediate size (Fig. 4C). They were easily passaged and could be established as a permanent line. Although difficult to observe directly, there seemed to be occasional transitions from ramified cells into macrophages especially at later stages of cultivation. Most strikingly, in all of the in vivo labeling experiments (Fig. 5) ramified cells appeared to be exclusively labeled.
Origin of brain macrophages in vitro Axotomy did not increase the number of macrophages in facial motor nuclei at the onset of culturing (Fig. 8A). It took at least 24 h for noticeable differences to become apparent between cultures of axotomized and control facial nuclei, i.e. macrophages were growing out only from axotomized explants. In some cases the explants were removed at this time, and numerous flat macrophages were visible covering most of the area of former explant attachment. Similarly, there was increased generation of macrophages within the axotomized explants themselves. No macrophages could be detected in control explants and cultures at this stage. At the beginning of the second week, semithin sections demonstrated densely packed macrophages in axotomized explants (Fig. 8B). Even after FDU
Fig. 8. In vitro generation of macrophages from resident parenchymal cells of axotomized facial nucleus explants. Semithin sections of explants embedded into Araldite (A) or LR White (B). A: chromatolytic facial motoneurons are closely surrounded by reactive microglia (arrows). Note the absence of macrophages. Axotomized facial nucleus explant at the very beginning of culturing. B: axotomized explant overcrowded by macrophages after 8 DIV. Magnification: ×270. C: mitotic activity in facial nucleus explants was determined by measuring [methyl-3H]thymidine uptake after 6 days of incubation. Accumulation of [3H]thymidine is significantly higher in axotomized facial nucleus explants. The mean _+ S.D. of 19 measurements is given.
12 treatment more than 150 macrophages on the average grew out from each mitotically inhibited axotomized explant after 3 weeks in culture. In inhibited cultures of normal facial nuclei none or maximally 10 macrophages per explant culture were found. In contrast to controls, axotomized facial nuclei explants continued to take up [methyl-3H]thymidine (Fig. 8C).
The mitotic stimulus in axotomized facial nucleus explants. Growth factors Neither the application of extracts obtained from operated facial nuclei nor cocultivation of control with axotomized explants resulted in an increase in the number of macrophages emerging from control explants. Furthermore, of all growth factors tested only E G F appeared to exert a slightly positive effect. When the time interval between nerve transection and explantation of facial motor nuclei for cultivation was varied from 5 h to 42 days, a significant increase in macrophage generation could only be found when the interval was at least 9 h. There was almost no reaction at an interval of 37 and 42 days. DISCUSSION The objective of the present study was to investigate the microglia of adult rat brain in vitro. The reason for choosing exptanted axotomized facial nuclei as a source of microglia rather than other tissue preparations 7'8'~° lies in the unique properties of the facial nerve experimental model. As was to be expected from our in vivo data 14'1s'19 the mitotic activity of cell cultures derived from axotomized facial nuclei exceeded that of controls by several orders of magnitude. Not only was the number of proliferating cells higher in cultures of axotomized facial nuclei explants, but also the onset of proliferation was earlier and macrophages appeared very rapidly. Since there was no evidence indicating an infiltration of blood-borne cellular elements in vivo, we suggest that all cells outgrowing from control and axotomized facial nucleus explants were of endogenous origin. This view is strengthened by the fact that all animals were perfused before explantation in order to remove blood cells present in the vasculature. More importantly, however, the in vivo labeling of facial nucleus microglia with [3H]thymidine
resulted in high numbers of labeled cells proliferating in vitro and was confined to cultures obtained from the operated nucleus. Based on morphological criteria, the majority, if not all, of these labeled cells were non-phagocytic and of the ramified type. Cellular markers specific for astrocytes, oligodendrocytes or endothelial cells did not stain ramified cells, and their immunophenotype and ultrastructural appearance were similar to that of microglia in vivo 1"12-14"21"29"31"32 (cf. Table I). However, permanent lines of ramified cells expressed common microglia markers, e.g. CR3 or GSA-1-B 4, only at low levels. In this context, the high number of macrophages exclusively present in cultures of axotomized facial nucleus explants needs to be explained. While activated microglial cells in vivo are non-phagocytic following facial nerve axotomy 2"~2~9, it has recently been shown that resident microglia have the capacity to develop into macrophages during degeneration of facial motoneurons lethally injured by injection of toxic ricin into the facial nerve 3°. Since the transfer of facial nuclei into culture leads to neuronal death in the explants, the development of microglia into macrophages in this situation appears to be analogous to that of ricininduced degeneration in vivo. Interestingly, macrophage generation from controls was not only delayed but also very much reduced. This may reflect not only the low number of microglia present in the normal facial nucleus, but also the different state of activation of these cells as compared to their proliferating counterparts 12A3"31. Furthermore, by explanting previously axotomized motoneurons they are injured for a second time which is likely to accelerate neuronal death. Activated microglia may thus be stimulated to become phagocytic. The hypothesis that brain macrophages in axotomized facial nucleus explants may be derived from activated microglia is further supported by the fact that high doses of anti-mitotic FDU were 15 times less effective in suppressing the generation of macrophages in cultures of axotomized facial nucleus explants than in controls. Moreover, macrophages were clearly absent from axotomized facial nuclei at the very beginning of culturing but [3H]thymidine uptake by axotomized facial nucleus explants was strongly elevated. Finally, in vivo microglia appear to be the only population of cells occurring in the rat
13 facial nucleus which have the capacity to proliferate and to develop into phagocytes 14"3°. Therefore, we conclude that resident parenchymal microglia represent precursors of brain macrophages also in vitro. As suggested by their immunophenotypic characteristics we further consider the proliferating ramified type of cell to be a transitional form in the development of true brain macrophages. However, culture conditions yielding pure populations of ramified microglia have not yet been established. In culture as well as under the influence of pathological stimuli in vivo, microglia appear to have a strong tendency to develop into phagocytes. With regard to the mitotic stimulus elaborated in axotomized facial nuclei, it may be important to note that the interval between facial nerve transection and explantation of its nucleus had to be at least 9 h, but not longer than 37 days. Interestingly, mitotic activity in vivo increases later (days 2-3) but is diminished earlier (day 14) than these data would suggest ~s'19. Thus, the putative mitogen may be present for longer periods than originally expected from in vivo experiments. Frei et al. proposed astrocyte-derived interleukin 3 to be a growth factor for microglia 7. However, our failure to obtain significant growth stimulating effects by both facial nucleus extracts and cocultured facial nuclei might indicate the possible existence of a direct cellular mechanism of glial activation. Furthermore, since growth of macrophages in control cultures was insignificant it seems reasonable to suppose that the REFERENCES 1 Blakemore, W.F., The ultrastructure of normal and reactive microglia, Acta Neuropathol., Suppl. 6 (1975) 273278. 2 Blinzinger, K. and Kreutzberg, G.W., Displacement of synaptic terminals from regenerating motoneurons by microglial cells, Z. Zellforsch., 85 (1968) 145-157. 3 Cammermeyer, J., Juxtavascular karyokinesis and microglia cell proliferation during retrograde reaction in the mouse facial nucleus, Ergeb. Ant. Entwicklgs. Gesch., 38 (1965) 1-22. 4 Costero, I., Estudios sobre la explantacion de tejido nervioso. Cuitivo 'in vitro' de microglia, Bol. Roy. Soc. Espan. Hist. Nat., 30 (1930) 165-171. 5 Del Rio-Hortega, P., Microglia. In W. Penfield (Ed.), Cytology and Cellular Pathology of the Central Nervous System, Vol. 2, P.B. Hoeber, New York, 1932, pp. 481-534. 6 Duchen, L.W., General pathology of neurons and neuroglia. In J.H. Adams, J.A.N. Corsellis and L.W. Duchen
presence of neuronal debris by itself is not a sufficient stimulus for microglia to develop into macrophages. A certain amount of time and the appropriate challenge, as in the case of preceding axotomy, may be needed by both neurons and microglia in order to pass through a threshold of activation before being committed to interact. In conclusion, we suggest that microglia activated by peripheral nerve injury can be transferred into culture, and that microglia represent precursors of brain macrophages in vitro as well as in vivo. In culture, however, the cells may be found in at least two phenotypic forms, i.e. ramified cells and macrophages, a finding which is in agreement with studies on microglia and microglia-derived brain macrophages from neonatal brain 8,1°. Further work is needed to establish cellular markers specific for microglia. The culture model introduced here which provides a highly enriched source of microglia may prove to be a useful tool.
ACKNOWLEDGEMENTS We wish to thank Dr. M. Warnhoff for providing some of his data and Dr. G. Raivich for statistical advice. We greatly appreciate the excellent technical assistance of Angelika Ertlmaier, Karin St61tzing, Liselotte Konrad, Dietmute Bfiringer, Irmtraud Milojevi6 and Waltraud Komp. We thank Dr. M. Reddington for reading the manuscript. (Eds.), Greenfield's Neuropathology, Edward Arnold, London, 1984, pp. 1-52. 7 Frei, K., Bodmer, S., Schwerdel, C. and Fontana, A., Astrocyte-derived interleukin 3 as a growth factor for microglia cells and peritoneal macrophages, J. Immunol., 137 (1986) 3521-3527. 8 Frei, K., Siepl, C., Groscurth, P., Bodmer, S., Schwerdel, C. and Fontana, A., Antigen presentation and tumor cytotoxicityby interferon-y-treated microglial cells, Eur. J. lmmunol., 17 (1987) 1271-1278. 9 Giulian, D., Ameboid microglia as effectors of inflammation in the central nervous system, J. Neurosci. Res., 18 (1987) 155-171. 10 Giulian, D. and Baker, T.J., Characterization of ameboid microglia isolated from developing mammalian brain, J. Neurosci., 6 (1986) 2163-2178. 11 Giulian, D. and Lachman, L.B., Interleukin-1 stimulation of astroglial proliferation after brain injury, Science, 228 (1985) 497-499. 12 Graeber, M.B., Streit, W.J. and Kreutzberg, G.W., Axotomy of the rat facial nerve leads to increased CR3
14
13
14
15
16
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18
19
20
21
22
23
24
complement receptor expression by activated microglial cells, J. Neurosci. Res., 21 (1988) 18-24. Graeber, M.B., Streit, W.J. and Kreutzberg, G.W., The microglial cytoskeleton: vimentin is localized within activated cells in situ, J. Neurocytol., 17 (1988) 573-580. Graeber, M.B., Tetzlaff, W., Streit, W.J. and Kreutzberg, G.W., Microglial cells but not astrocytes undergo mitosis following rat facial nerve axotomy, Neurosci. Lett., 85 (1988) 317-321. Hickey, W.F. and Kimura, H., Perivascular microglial cells of the CNS are bone marrow-derived and present antigen in vivo, Science, 239 (1988) 290-292. Kaur, C., Ling, E.A. and Wong, W.C., Origin and fate of neural macrophages in a stab wound of the brain of the young rat, J. Anat. 154 (1987) 215-227. Konigsmark, B.W. and Sidman, R.L., Origin of brain macrophages in the mouse, J. Neuropathol. Exp. Neurol., 22 (1963) 643-676. Kreutzberg, G.W., Autoradiographische Untersuchung fiber die Beteiligung von Gliazellen an der axonalen Reaktion im Facialiskern der Ratte, Acta Neuropathol., 7 (1966) 149-161. Kreutzberg, G.W., Ober perineuronale Mikrogliazellen (Autoradiographische Untersuchungen), Acta Neuropathol., Suppl. 4 (1968) 141-145. Murphy, S., MeCabe, N., Morrow, C. and Pearce, B., Phorbol ester stimulates proliferation of astroeytes in primary culture, Dev. Brain Research, 31 (1987) 133-135. Peters, A., Palay, S.L. and Webster, H. deE, The Fine Structure of the Nervous System: The Neurons and Supporting Cells, W.B. Saunders, Philadelphia, 1976, 406 pp. Price, R.W., Brew, B., Sidtis, J., Rosenblum, M., Scheck, A.C. and Cleary, P., The brain in AIDS: central nervous system HIV-1 infection and AIDS dementia complex, Science, 239 (1988) 586-592. Rieske, E. and Kreutzberg, G.W., Neurite regeneration after cell surgery with laser microbeam irradiation, Brain Research, 148 (1978) 478-483. Rieske-Shows, E., Tetzlaff, W., Cztonkowska, A., Graeber, M. and Kreutzberg, G.W., Microglia in culture. In H.H. Althaus and W. Seifert (Eds.), Glial-Neuronal Communication in Development and Regeneration, NA TO AS1 Series, H, Vol. 2, Springer, Berlin, 1987, pp. 41-51.
25 Schelper, R.L. and Adrian, E.K., Non-specific esterase activity in reactive cells in injured nervous tissue labeled with 3H-thymidine or ~25iododeoxyuridine injected before injury, J. Comp. Neurol., 194 (1980) 829-844. 26 Schelper, R . L and Adrian, E.K., Monocytes become macrophages; they do not become microglia: a light and electron microscopic study using 125-iododeoxyuridine. J. Neuropathol. Exp. Neurol., 45 (1986) 1-19. 27 Sj6strand, J., Studies on glial cells in the hypoglossal nucleus of the rabbit during nerve regeneration, Acta Physiol. Scand., 67 (Suppl. 270) (1966) 1-17. 28 Sommer, I. and Sehachner, M., Monoclonal antibodies (01 to 04) to oligodendrocyte cell surfaces: an immunoeytological study in the central nervous system, Dev. Biol., 83 (1981) 311-327. 29 Streit, W.J. and Kreutzberg, G.W., Lectin binding by resting and reactive microglia, J. Neurocytol., 16 (1987) 249-260. 30 Streit, W.J. and Kreutzberg, G.W., Response of endogenous glial cells to motor neuron degeneration induced by toxic ricin, J. Comp. Neurol., 268 (1988) 248-263. 31 Streit, W.J., Graeber, M.B. and Kreutzberg, G.W., Peripheral nerve lesion produces increased levels of MHC antigens in the CNS, J. Neuroimmunol., 21 (1989) 117123. 32 Streit, W.J., Graeber, M.B. and Kreutzberg, G.W., Functional plasticity of microglia: a review, Glia, 1 (1988) 301-307. 33 Thiele, D.L., Kurosaka, M. and Lipsky, EE., Phenotype of the accessory cell necessary for mitogen-stimulated T and B cell response in human peripheral blood: delineation by its sensitivity to the lysosomotropic agent, L-leucin methyl ester, J. lmmunol., 131 (1983) 2282-2290. 34 Von Michalik, P., Uber, die Nervengewebekulturen, mit besonderer Beriicksichtigung der Neuronenlehre und der Mikrogliafrage, Arch. Exp. Zellforsch., 17 (1935) 119-176. 35 Watson, W.E., An autoradiographic study of the incorporation of nucleic-acid precursors by neurones and glia during nerve regeneration, J. Physiol. (Lond.), 180 (1965) 741-753. 36 Wells, A.Q. and Carmichael, E.A., Microglia: an experimental study by means of tissue culture and vital staining, Brain, 53 (1930) 1-10.
1
Research Reports
Microglia and microglia-derived brain macrophages in culture: generation from axotomized rat facial nuclei, identification and characterization in vitro Ellen Rieske, Manuel B. Graeber, Wolfram Tetzlaff, Anna Czlonkowska 1, Wolfgang J. Streit and Georg W. Kreutzberg Department of Neuromorphology, Max-Planck-lnstitutefor Psychiatry, Martinsried (E R. G.) (Accepted 13 December 1988) Key words: Ramified microglia; Brain macrophage; Facial nucleus explant; Cell culture
In order to study microgliai cells and microglia-derived brain macrophages in vitro, a method has been developed which allows the transfer of mitotic microglial cells from adult rat brain into tissue culture. The studies were performed on facial motor nuclei which were explanted after axotomy of the facial nerve. Outgrowing cells were identified and characterized by (i) morphological criteria using light and electron microscopy, (ii) in vivo [3H]thymidine labeling combined with subsequent in vitro autoradiography, (iii) immunocytochemistry for vimentin, GFAP, Fc and complement receptors, MHC antigens, laminin, fibronectin, factor VIII related- and 04 antigen as well as lectin histochemistry, and (iv) functional in vitro tests. In addition, a mieroglial cell line was established from proliferating cells. The results indicate that perineuronal microglia rather than astrocytes, perivascular cells, oligodendrocytes or endothelial cells may become phagocytic after having been activated by axotomy in situ. INTRODUCTION The first attempts to study microglia in culture can be traced back to the early 1930s4'34'36. However, it was not before the present decade that a rapidly growing interest in the intrinsic defense mechanisms of the CNS led to systematic research on microglia in vitro 8'1°'24. Whereas the normal brain parenchyma does not contain active macrophages, phagocytic cells can be observed in neural tissue cultures 8'1°'24 as well as under various pathological conditions in vivo 6. Traumatic CNS injuries are usually accompanied by disruption of the vasculature and a subsequent influx of blood-derived phagocytic cells into the parenchyma 17, but in addition resident microglia represent a major source of endogenous brain macrophages 6'26. Aside from their phagocytic potential microglial cells are thought to be involved in
synaptic changes 2, gliosis al, CNS development 9, and immunological processes 8'9, the latter including antigen presentation in vivo 15, as well as HIV-1 infections 22. However, the great mutability of the microglial cell shape 1'5'13'21 together with the lack of a microglia-specific marker that would allow them to be distinguished from other macrophages have made it difficult in the past to positively identify and study microglia in vitro. Proliferating cells in a central m o t o r nucleus following peripheral nerve injury have been identified as microglia independently by several groups s' 18,35 In view of a recent study employing [3H]thymidine labeling at the ultrastructural level 14, there can now be little doubt that it is indeed endogenous microglia which undergo mitosis in response to axotomy. Following transection of the rat facial nerve, the affected m o t o r neurons not only
] On leave of absence from the Institute of Psychiatry and Neurology, Cerebrovascular Diseases Clinic, Sobieskiego 1/9, 02957 Warsaw, Poland. Correspondence: G.W. Kreutzberg, Max Planck Institute for Psychiatry, Dept. of Neuromorphology, Am Klopferspitz 18a, D-8033 Martinsried, F.R.G. 0006-8993/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)
survive but regenerate. Concomitantly, microglia are ~activated' in a sense that they proliferate but do not transform into macrophages 2'~2"29. Since there is no breakdown of the blood-brain barrier within a regenerating motor nucleus 27, and since an invasion of blood-borne cellular elements does n o t o c c u r 14"25, this model is clearly superior for studying microglia than other approaches employing cryogenic lesions or stab wounds where such a contamination cannot be avoided ~6. The unique advantage and elegance of the axotomy paradigm is that the brain tissue can be challenged without touching it. Based on this model we have now established a combined explant-cell culture system which allows the in vitro identification and characterization of genuine microglia derived from adult rat brain. MATERIALS AND METHODS
Animals and tissue preparation The right facial nerve of male Wistar rats (180200 g, 8-10 weeks old) was transected at the stylomastoid foramen. The contralateral unoperated side served as a control. Following survival times of 4-6 days the animals were perfused through the heart with sterile Hank's solution and the regenerating and contralateral unoperated facial nuclei were removed for explant culture (Fig. 1). Excised facial nuclei were carefully freed from meninges and neighboring tissue under sterile conditions, and each nucleus was cut into about 30 explants measuring approx. 0.125 mm 3. Two to four explants were placed 1 mm apart on coverslips and cultured in 35 mm Falcon tissue culture dishes at 36 °C in a humidified 1% CO2-air atmosphere which adjusted the pH of the medium to 7.4. The medium consisted of Dulbecco's MEM containing 20% human placental serum, 10% chick embryo extract and 5% fetal calf serum. The cultures were fed twice a week with fresh medium and kept as primary cultures for about 3 months. One, two and three,weeks after explantation the number of proliferating cells per explant culture was determined. No antibiotics were applied.
Facial nucleus cell cultures Cell cultures were established from rat facial nucleus explants after one to two weeks in vitro. Following removal of explant debris the layer of
growing cells was disaggregated either by incubating for 2-5 min at 36 °C in 0.25% trypsin solution (GIBCO), or by mechanical aspiration with fine fire-polished pipettes. Culture conditions were the same as for the explants. The following culture substrates were tested: LUX polystyrene and LUX Thermanox (Miles Lab. Inc., Naperville, 1L, U.S.A.) or collagen-coated glass, polystyrene-, polyornithine- or polylysine-coated glass, or polystyrene and a gas-permeable foil (Petriperm; Heraeus, Hanau, F.R.G.). In addition, the growth of primary explant cultures and cell cultures of rat facial nuclei was assessed in serum-free Dulbecco's MEM with the following additives: 2.5S Nerve Growth Factor (10 ng/mi; GIBCO), Epidermal Growth Factor (10 ng/ml; GIBCO), Fibroblast Growth Factor (10 ng/ ml; Collaborative Res., Lexington, MA, and GIBCO), Tuftsin (0.4 nmol; Sigma), TSH (thyroid stimulating hormone, 1 mU/liter; Sigma), and dibutyryl-cyclic AMP (1 raM; Boehringer, Mannheim, F.R.G.).
Cultures of cells prelabeled in vivo by [3H]thymidine These experiments were designed to test whether the cells proliferating in vitro from axotomized rat facial nuclei explants were related to the mitotic microglia of the facial nucleus in situ. Six rats which had undergone a facial nerve transection were injected intravenously with [3H]thymidine (1 mCi/kg b. wt.; Amersham, U.K.) 4 days later, a time at which microglial mitotic activity reaches its peak ~9. Ten hours after [3H]thymidine injection facial nuclei were explanted and cultivated as described above. At 3, 7, 9, 10, and 13 days in vitro (DIV) 2-8 explant cultures of each animal were rinsed with PBS, fixed in 70% alcohol and air-dried. The cultures were then processed for light microscopical autoradiography as described elsewhere 3°, and the total cell number was determined. Cells labeled with [3H]thymidine were classed according to the number of silver grains overlying their nucleus: 3-5, 6-10, 11-20, and 20 or more silver grains per nucleus.
Light and electron microscopical characterization of proliferating cells Light microscopy. The morphological characteristics of cells were determined by bright field, phase contrast and differential interference contrast light
microscopy as well as by time lapse microcinematography. Immunocytochemistry for bright field and fluorescence microscopy was performed by applying the following antibodies: (1) MRC Ox-42 (Serotec, MCA 275) is a monoclonal mouse IgG2a recognizing the rat CR3 complement receptor; (2) MRC-Ox 6 (Serotec, MCA 46) recognizes a monomorphic determinant of rat MHC class II (Ia) antigen; (3) MRC Ox-17 (Serotec, MCA 50) is a monoclonal mouse IgG1 antibody recognizing a determinant on the e-chain of rat Ia antigen, a homologue of mouse Ia-E product; (4) MRC Ox-18 (Serotec, MCA 51) is a monocional mouse IgG1 antibody recognizing a monomorphic determinant of rat class I MHC antigens; (5) anti-vimentin (monoclonal mouse IgG; Code 814318, Boehringer Mannheim, F.R.G.); (6) anti-GFAP (polyclonal rabbit IgG; Code Z334, Dako, Hamburg, ER.G.); (7) anti-laminin (polyclonal rabbit IgG, Dr. U. Kiihl, Max Planck Institute for Biochemistry, Martinsried, F.R.G.); (8) antifibronectin (polyclonai rabbit IgG, Code A245, DAKO); (9) anti-factor VIII related antigen (Code A082, Dako); (10) 0428, kindly provided by Prof. M. Schachner, Dept. of Neurobiology, University of Heidelberg, F.R.G. Fc receptors were detected by incubation with tetramethylrhodamine isothiocyanate (TRITC) conjugated goat anti-mouse IgG (Sigma, T-5393) diluted 1:10 at 37 °C. In addition, the cells were stained with the lectin from Griffonia simplicifolia (GSA-I-B4)29. Following fixation in 3.7% formaldehyde only or successive fixation in 3.7% formaldehyde, 3.7% formaldehyde containing 0.06% Triton X-100 (10 rain each), and acetone (50, 100, and 50%; 2 min each) immunocytochemical staining of cultures was carded out according to standard procedures using the alkaline phosphatase (Code D314, DAKO) sandwich technique or biotinylated secondary antibodies followed by ABC-alkaline phosphatase (Code AK-5000; Vector, Burlingame, CA). Ultrastructural examination. For transmission electron microscopic examination the explant and cell cultures were grown on glass, polystyrene (Thermanox, Miles Lab. Inc.) or biofoil (Petriperm) and were fixed in culture medium containing 3.5% glutaraldehyde at pH 7.2 for 20 min at room temperature. They were then washed in 0.05 M cacodylate buffer/8.5% sucrose, postfixed in Dal-
ton's osmium and embedded into Araldite or LR White. For scanning electron microscopical examination the cultures were washed in warm serum-free medium and fixed in 3% glutaraldehyde at room temperature. Subsequently, they were washed in phosphate buffer, postfixed in Dalton's osmium for 60 min, and critical point dried with CO 2 followed by gold coating.
Functional characterization of proliferating cells In order to test the phagocytic nature of cells 4 experiments were performed: (i) Presentation of suspended latex beads. Cultures were incubated at 36 °C for 60 min with latex particle solution (1/~m Dow-latex beads, 1.05 g/ml; Dow-Corning, Seneffe, Belgium), diluted 1:100 with medium. (ii) Immunoglobulin-coated ox erythrocytes as indicators of Fc receptor mediated rosetting and particle ingestion. The lowest non-agglutinating dilution of rabbit antiserum against opsonized ox red blood cells (IgG anti-E, Seralab) was tested (1:5 to 1:75) and combined with an equal volume of 5% opsonized ox erythrocytes in physiological salt solution. After incubation at 36 °C for 10-30 min and washing with warm medium the number of rosettes was determined. Ingested erythrocytes were demonstrated by phase contrast microscopy after lysis of extracellular erythrocytes by 3% acetic acid. (iii) Phagocytosis of laser-killed cells. Laser microirradiation was used to selectively kill individual cells among live neighboring cells. Single cells were killed by 8 ns shots of 4 x 1011 W/cm2 at 337 nm during observation through a 40x UV-objective23. Phagocytosis of dead cells by neighboring macrophages was examined by time lapse microcinematography. (iv) The lysosomotropic agent L-leucine-methylester (Sigma, L-9000) was used for selective killing of phagocytic cells 33.
Generation of macrophages from resident parenchyreal cells Three experiments were performed in order to test whether macrophages present in axotomized explant cultures were generated from resident parenchymal cells rather than from blood-derived macrophages invading the facial nucleus prior to explantation: (i) The number of macrophages present in the explants at the beginning of cultivation was determined in semithin sections. (ii) It was tested
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Fig. 2. Cellular outgrowth from axotomized and unoperated control facial nuclei explants in culture. Overall cell numbers in mixed primary cultures were counted. Data were taken from facial nucleus explants of 9 randomly chosen animals undergoing perfusion 4 or 5 days after facial nerve transection. A: n = 149 explants, B: n = 132 explants. C: logarithmic transformation of the geometric mean of data shown in plots A and B.
Fig. 3. A: scanning electron micrograph of an axotomized facial nucleus explant 16 DIV. Numerous spindle-shaped cells and macrophages can be seen within and around the explant. B: in early stages of explantation (10 DIV) spindle-shaped cells (long arrows) predominate over ramified forms. Macrophages (short arrows) are rounded with short cell processes. C: compared to B, older cultures (26 DIV) contain many more differentiated ceils. Spindle-shaped cells have developed ramified cell processes. Macrophages are large and flattened. B and C phase contrast light microscopy. Magnification: x60 (A), x180 (a), x68 (C).
Fig. 1. Culturing of rat facial nucleus explants following axotomy in viva. A: schematic representation of the model. B: control culture of an unoperated rat facial nucleus 12 DIV. Outgrowth of cells is insignificant. C: emigration and proliferation of cells around an axotomized facial nucleus explant 12 DIV. Magnification: x57 (B,C).
Fig. 4. Ultrastructural characteristics of ramified cells ( A - D ) and macrophages (E,F) in cultures of axotomized facial nucleus explants. A: scanning electron micrograph of ramified microglia in a 44-day-old culture. B: transmission electron micrograph of the same type of cell 15 DIV. Note the presence of many rough endoplasmic reticulum cisternae and liposomes. The cell nucleus shows large clusters of heterochromatin in its center and apposed to the nuclear membrane similar to microglia in vivo. C: transmission electron micrograph of a LR White-embedded ramified cell. Numerous filaments of intermediate size (long arrows) can be seen. D: ramified cell. High power micrograph of prominent wide rough endoplasmic reticulum cisternae (short arrows) typical of microglia (37 DIV). E: scanning electron micrograph of a facial nucleus macrophage 14 DIV. Note its undulating cytoplasmic membrane. F: transmission electron micrograph of a young macrophage in a 6-day-old axotomized facial nucleus explant culture. The nucleus is rich in heterochromatin, and cytoplasmic filopodia can be seen. Golgi complexes, lysosomes and vacuoles are also prominent. Magnification: x1500 (A), ×7250 (B), x43,000 (C), ×47,000 (D), ×2400 (E), x5350 (F).
TABLE I
Immunological characteristics of ramified cells and macrophages in cultures of ratfacial nucleus explants Marker Vimentin GFAP
Fc receptor CR3 receptor MHC class I antigen MHC class II antigen GSA-I-B4-isolectin Laminin Fibronectin Factor VIII 04 antigen
Ramified cells Macrophages + -
+ -
+ (+) + + + + + -
+ + + + + -
whether macrophages could be generated in vitro when all mitotic activity in the explants was inhibited by adding 5-fluorodeoxyuridine (FDU, 80 /~M; Sigma, F-0503) together with uridine (200 /~M; Sigma, U-3750). (iii) The degree of [methyl3H]thymidine accumulation in the explants during the first 6 DIV was determined (Warnhoff et al., in preparation) using the method of Murphy et al. 2°. In order to separate free [methyl-aH]thymidine in the medium from the explants, they were washed 3 times with Hank's balanced salt solution (HBSS) followed by lysis with 0.1 N NaOH. [Methyl-3H]thymidine incorporated into the DNA of proliferating cells was separated from the intracellular pools by adding 250 /~g bovine serum albumin (BSA; Sigma, A-7030) as a carrier and precipitating protein with 10% TCA. After centrifugation at 10,000 g for 5 min the protein pellet was dissolved in I N NaOH, liquid scintillation cocktail was added (Ready-Solve HP; Beckman, CA), and the radioactivity was measured in a liquid scintillation counter (efficacy 55%). Blank values, obtained from explants in medium lacking [methyl3H]thymidine during incubation, were determined by adding [methyl-aH]thymidine shortly before processing of the explants as described.
Release of growth stimulating factors from axotomized facial nuclei In order to test whether axotomized facial nuclei themselves release growth stimulating factors, 3 experiments were performed: (i) The time interval between nerve transection and explantation of facial
nuclei was varied from 5 h to 42 days. (ii) Control explants of 6 rats were fed with serum-free Dulbecco's MEM supplemented with 10-20% saline extract from axotomized facial nuclei (200 mg ww/ml Hank's solution); saline extract was obtained by homogenizing freshly excised rat facial nuclei 5 days after axotomy at 4 °C. (iii) Control explants of two rats were co-cultured with explants of axotomized facial nuclei by setting 4-6 axotomized explants around two control explants on coverslips. RESULTS
Enhanced cellular outgrowth from axotomized facial motor nuclei explants At all time points investigated the number of proliferating cells per axotomized explant culture by far exceeded those in controls, ranging from 10-fold at the end of the first week to more than 200-fold during the third week (Fig. 2). Whereas cultures of axotomized facial nucleus explants exhibited exponential growth characteristics, growth in control cultures was insignificant (Fig. 2). After 2-3 weeks in vitro, celt cultures from the operated facial nucleus demonstrated two large populations of cells clearly distinguishable and outnumbering all other cell types in these explant cultures: ramified cells and macrophages (Figs. 3 and 4). Ramified cells grew out from axotomized explants beginning late in the first week in vitro. In control cultures they were found only occasionally and not before the second week.
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Fig. 5. Proliferation kinetics of in vivo [3H]thymidine-labeled cells of axotomized facial nuclei in vitro. The rapid clearance of heavily labeled cells (closed triangles) suggests strong proliferative activity. Labeled cells were of the ramified type but absent from controls.
Fig. 7. Immunophenotypic characteristics of ramified microglia (A-C,E) and macrophages (D,F). A: vimentin. Fluorescence microscopy (fluorescein). B: Fc receptors. Fluorescence microscopy (rhodamine). C,D: Ia-antigen (Ox-6 mab) and alkaline phosphatase immunocytochemistry. E: GSA-I-B4-isolectin positive ramified microglia on top of a monolayer of lectin negative cells, 29 DIV. F: lectin-positive macrophages in the peripheral zone of outgrowth within the same culture as in E. Phase contrast microscopy and peroxidase/diaminobenzidine. Magnification: x900 (A), ×460 (B), ×290 (C,D,F), x 180 (E).
Macrophages also grew out of virtually all axotomized facial nucleus explants. They not only migrated, but also proliferated and covered large areas around the explants (Figs. 1 and 3). After 3 weeks,
1-5 x 103 macrophages per explant culture were present on average whereas in control cultures none or less than 15 macrophages per explant could be found (Fig. 1). There was no outgrowth at all from
<..Fig. 6. Functional tests on phagocytic activity of proliferating cells from axotomized facial nucleus explants. A: latex beads (1 am) are selectively taken up by macrophages and are concentrated around the cell nucleus and along cell processes. 14 DIV, and 1 h after incubation with latex beads. B: ingested opsonized ox erythrocytes within the cytoplasm of macrophages. Extracellularly bound erythrocytes were removed by washing with acetic acid (8 DIV). C: rosetting of opsonized ox erythrocytes around macrophages (thick arrow); ramified cells are free of ox erythrocytes (long arrow); 8 DIV. D: differentiated macrophages with flattened perikarya, 26 DIV. Phase contrast microscopy. Magnification: x243.
10
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6 Days incubation time
11 control and axotomized explants when the nutrient medium contained less than 20% of biological ingredients, e.g. serum or chick embryo extract. It further appeared that rich medium formulas as well as high pH (>7.4) favored the proliferation of ramified cells rather than that of macrophages. However, under standard conditions the number of macrophages was approximately the same as that of ramified cells. There was no outgrowth of neurites or long term survival of neurons.
Morphology, functional characteristics and immunophenotypic characterisation of proliferating cells Macrophages were easy to recognize with phase contrast optics by both their irregular shape and high content of bright perinuclear droplets (Fig. 3B). Time lapse microcinematography revealed typical locomotion behavior. Their undulating membranes (Fig. 6D) and finger-like projections (Fig. 4F) were easily visible. Macrophages within and near the explants (Figs. 3A and 8B) were loaded with cellular debris after about 5 weeks. In contrast, macrophages migrating into the zone of outgrowth became increasingly flattened (Fig. 6D) and eventually acquired a stellate shape. They were able to survive for more than 8 weeks. Ultrastructurally, the cytoplasm of macrophages contained well-developed Golgi complexes, many dense bodies, lysosomes, mitochondria and membrane-bound vacuoles of different size (Fig. 4F). Their nuclei were folded with dense appositions of heterochromatin to the nuclear membrane (Fig. 4F). All phagocytosis assays were positive, e.g. latex beads as well as IgG-coated ox erythrocytes were readily taken up (Fig. 6A-C). Presentation of cells killed in vitro by laser microirradiation, resulted in rapid phagocytosis (within hours) by neighboring macrophages. Selective elimination of macrophages could be achieved with L-leucine-methylester which caused lysosomal disruption, cytoplasmic swelling and resulting death of macrophages within less than 2 days. Immunostain-
ing was positive with all macrophage markers but antibodies against other glial or endothelial cell markers, e.g. GFAP, 04, and factor VIII, failed to stain the cells (Table I). Ramified cells were 4 times more common in cultures of axotomized facial nucleus explants than in controls although in the latter they were by far the most frequent cell type. Ramified cells were positive for Fc receptors although they showed no signs of phagocytic activity neither in functional testing nor ultrastructurally (Fig. 4B). Their immunophenotype (Table I) appeared to be related to the monocyte family rather than to astrocytes, endothelia or oligodendrocytes (cf. Fig. 7). Ramified cells were also rich in microtubuli, microfilaments and filaments of intermediate size (Fig. 4C). They were easily passaged and could be established as a permanent line. Although difficult to observe directly, there seemed to be occasional transitions from ramified cells into macrophages especially at later stages of cultivation. Most strikingly, in all of the in vivo labeling experiments (Fig. 5) ramified cells appeared to be exclusively labeled.
Origin of brain macrophages in vitro Axotomy did not increase the number of macrophages in facial motor nuclei at the onset of culturing (Fig. 8A). It took at least 24 h for noticeable differences to become apparent between cultures of axotomized and control facial nuclei, i.e. macrophages were growing out only from axotomized explants. In some cases the explants were removed at this time, and numerous flat macrophages were visible covering most of the area of former explant attachment. Similarly, there was increased generation of macrophages within the axotomized explants themselves. No macrophages could be detected in control explants and cultures at this stage. At the beginning of the second week, semithin sections demonstrated densely packed macrophages in axotomized explants (Fig. 8B). Even after FDU
Fig. 8. In vitro generation of macrophages from resident parenchymal cells of axotomized facial nucleus explants. Semithin sections of explants embedded into Araldite (A) or LR White (B). A: chromatolytic facial motoneurons are closely surrounded by reactive microglia (arrows). Note the absence of macrophages. Axotomized facial nucleus explant at the very beginning of culturing. B: axotomized explant overcrowded by macrophages after 8 DIV. Magnification: ×270. C: mitotic activity in facial nucleus explants was determined by measuring [methyl-3H]thymidine uptake after 6 days of incubation. Accumulation of [3H]thymidine is significantly higher in axotomized facial nucleus explants. The mean _+ S.D. of 19 measurements is given.
12 treatment more than 150 macrophages on the average grew out from each mitotically inhibited axotomized explant after 3 weeks in culture. In inhibited cultures of normal facial nuclei none or maximally 10 macrophages per explant culture were found. In contrast to controls, axotomized facial nuclei explants continued to take up [methyl-3H]thymidine (Fig. 8C).
The mitotic stimulus in axotomized facial nucleus explants. Growth factors Neither the application of extracts obtained from operated facial nuclei nor cocultivation of control with axotomized explants resulted in an increase in the number of macrophages emerging from control explants. Furthermore, of all growth factors tested only E G F appeared to exert a slightly positive effect. When the time interval between nerve transection and explantation of facial motor nuclei for cultivation was varied from 5 h to 42 days, a significant increase in macrophage generation could only be found when the interval was at least 9 h. There was almost no reaction at an interval of 37 and 42 days. DISCUSSION The objective of the present study was to investigate the microglia of adult rat brain in vitro. The reason for choosing exptanted axotomized facial nuclei as a source of microglia rather than other tissue preparations 7'8'~° lies in the unique properties of the facial nerve experimental model. As was to be expected from our in vivo data 14'1s'19 the mitotic activity of cell cultures derived from axotomized facial nuclei exceeded that of controls by several orders of magnitude. Not only was the number of proliferating cells higher in cultures of axotomized facial nuclei explants, but also the onset of proliferation was earlier and macrophages appeared very rapidly. Since there was no evidence indicating an infiltration of blood-borne cellular elements in vivo, we suggest that all cells outgrowing from control and axotomized facial nucleus explants were of endogenous origin. This view is strengthened by the fact that all animals were perfused before explantation in order to remove blood cells present in the vasculature. More importantly, however, the in vivo labeling of facial nucleus microglia with [3H]thymidine
resulted in high numbers of labeled cells proliferating in vitro and was confined to cultures obtained from the operated nucleus. Based on morphological criteria, the majority, if not all, of these labeled cells were non-phagocytic and of the ramified type. Cellular markers specific for astrocytes, oligodendrocytes or endothelial cells did not stain ramified cells, and their immunophenotype and ultrastructural appearance were similar to that of microglia in vivo 1"12-14"21"29"31"32 (cf. Table I). However, permanent lines of ramified cells expressed common microglia markers, e.g. CR3 or GSA-1-B 4, only at low levels. In this context, the high number of macrophages exclusively present in cultures of axotomized facial nucleus explants needs to be explained. While activated microglial cells in vivo are non-phagocytic following facial nerve axotomy 2"~2~9, it has recently been shown that resident microglia have the capacity to develop into macrophages during degeneration of facial motoneurons lethally injured by injection of toxic ricin into the facial nerve 3°. Since the transfer of facial nuclei into culture leads to neuronal death in the explants, the development of microglia into macrophages in this situation appears to be analogous to that of ricininduced degeneration in vivo. Interestingly, macrophage generation from controls was not only delayed but also very much reduced. This may reflect not only the low number of microglia present in the normal facial nucleus, but also the different state of activation of these cells as compared to their proliferating counterparts 12A3"31. Furthermore, by explanting previously axotomized motoneurons they are injured for a second time which is likely to accelerate neuronal death. Activated microglia may thus be stimulated to become phagocytic. The hypothesis that brain macrophages in axotomized facial nucleus explants may be derived from activated microglia is further supported by the fact that high doses of anti-mitotic FDU were 15 times less effective in suppressing the generation of macrophages in cultures of axotomized facial nucleus explants than in controls. Moreover, macrophages were clearly absent from axotomized facial nuclei at the very beginning of culturing but [3H]thymidine uptake by axotomized facial nucleus explants was strongly elevated. Finally, in vivo microglia appear to be the only population of cells occurring in the rat
13 facial nucleus which have the capacity to proliferate and to develop into phagocytes 14"3°. Therefore, we conclude that resident parenchymal microglia represent precursors of brain macrophages also in vitro. As suggested by their immunophenotypic characteristics we further consider the proliferating ramified type of cell to be a transitional form in the development of true brain macrophages. However, culture conditions yielding pure populations of ramified microglia have not yet been established. In culture as well as under the influence of pathological stimuli in vivo, microglia appear to have a strong tendency to develop into phagocytes. With regard to the mitotic stimulus elaborated in axotomized facial nuclei, it may be important to note that the interval between facial nerve transection and explantation of its nucleus had to be at least 9 h, but not longer than 37 days. Interestingly, mitotic activity in vivo increases later (days 2-3) but is diminished earlier (day 14) than these data would suggest ~s'19. Thus, the putative mitogen may be present for longer periods than originally expected from in vivo experiments. Frei et al. proposed astrocyte-derived interleukin 3 to be a growth factor for microglia 7. However, our failure to obtain significant growth stimulating effects by both facial nucleus extracts and cocultured facial nuclei might indicate the possible existence of a direct cellular mechanism of glial activation. Furthermore, since growth of macrophages in control cultures was insignificant it seems reasonable to suppose that the REFERENCES 1 Blakemore, W.F., The ultrastructure of normal and reactive microglia, Acta Neuropathol., Suppl. 6 (1975) 273278. 2 Blinzinger, K. and Kreutzberg, G.W., Displacement of synaptic terminals from regenerating motoneurons by microglial cells, Z. Zellforsch., 85 (1968) 145-157. 3 Cammermeyer, J., Juxtavascular karyokinesis and microglia cell proliferation during retrograde reaction in the mouse facial nucleus, Ergeb. Ant. Entwicklgs. Gesch., 38 (1965) 1-22. 4 Costero, I., Estudios sobre la explantacion de tejido nervioso. Cuitivo 'in vitro' de microglia, Bol. Roy. Soc. Espan. Hist. Nat., 30 (1930) 165-171. 5 Del Rio-Hortega, P., Microglia. In W. Penfield (Ed.), Cytology and Cellular Pathology of the Central Nervous System, Vol. 2, P.B. Hoeber, New York, 1932, pp. 481-534. 6 Duchen, L.W., General pathology of neurons and neuroglia. In J.H. Adams, J.A.N. Corsellis and L.W. Duchen
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