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Age-related differences in proliferative responses of Schwann cells during Wallerian degeneration
Brain Research, 573 (1992) 267-275 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
267
BRES 17474
Age-related differences in proliferative responses of Schwann cells during Wallerian degeneration Atsushi Komiyama and Kinuko Suzuki Department of Pathology, and Brain and Development Research Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525 (U.S.A.) (Accepted 15 October 1991) Key words: Schwann cell; Fibroblast; Wallerian degeneration; Proliferation; Development; Cell culture; Autoradiography
Age-related differences in proliferative responses of Schwann cells during Wallerian degeneration were investigated in the mouse sciatic nerves after nerve-transection at 3, 10 and 60 days of age, corresponding to the periods of early myelination, active myelination and postmyelination. As assessed by thymidine incorporation for the first 24 h in culture, Schwann cells from adult nerve proliferated rapidly within day 1 post-transection and reached a peak at day 3. In the nerves from neonatal or suckling mice, however, division rate of Schwann cells declined after transection, and was even less in the transected nerves than in the contralateral uninjured nerves. The reduction in thymidine uptake by Schwann cells was more pronounced in nerves sectioned at postnatal day 3 than those sectioned at day 10. By contrast, fibroblasts divided rapidly following transection regardless of age. These data suggest that mitogens from myelin components are important for proliferation of Schwann cells and that in the degenerating nerves of young mice, mitotic capacity of Schwann cells declined due to not only a loss of axonal mitogens but also the paucity of mitogens from myelin components. Proliferation of fibroblasts is likely to be stimulated by more general growth-promoting polypeptides common to any other tissues during wound repair. INTRODUCTION During Wallerian d e g e n e r a t i o n of p e r i p h e r a l nerves, Schwann cells are stimulated to proliferate vigorously 1' 5,18. Most of these Schwann cell responses after injury have b e e n investigated in adult animals 1'5'1s. Conversely, studies on gliogenesis of the p e r i p h e r a l nervous system have b e e n carried out on n o r m a l pups 2'7'11. Therefore, little is known about the influence of insult on mitotic capacity of Schwann cells in nerves of young animals. W i t h respect to mitogens for Schwann cells, myelin components 'processed' by m a c r o p h a g e s were suggested to p r o m o t e Schwann cell proliferation during Wallerian degeneration 4'16. A Schwann cell mitogen during developm e n t or r e g e n e r a t i o n has b e e n shown to be an axonal surface proteoglycan-growth factor complex 2°. These studies led us to test a hypothesis that Schwann cells in the degenerating nerves of young mice m a y lose their proliferative capacity because of a loss of axonal mitogens as well as the paucity of mitogen from myelin components. To test this hypothesis, we investigated the differences in proliferative responses to Wallerian degeneration of Schwann cells from mice of different ages. O u r assay was b a s e d on the previous observation 8'16 that the mitotic rate of Schwann cells from dissociated nerves,
as assessed in vitro by thymidine a u t o r a d i o g r a p h y for the first 24 h, reflects the state of in vivo proliferation at the time of dissociation. A progressive loss of proliferative capacity of Schwann cells was d e m o n s t r a t e d in the degenerating nerves of young mice. A b y p r o d u c t of this study was the d e m o n s t r a t i o n that regardless of age, fibroblasts proliferated vigorously after nerve-transection. These differential proliferative responses of non-neuronal cells in the degenerating nerves of young mice suggest that the mitogen responsible for the proliferation of fibroblasts is not necessarily the same as that involved in the proliferation of Schwann cells. Alternatively, Schwann cells m a y need additional specific mitogen from myelin c o m p o n e n t s to proliferate in response to insults. MATERIALS AND METHODS Animal surgery The sciatic nerve of C3HeB/FeJ mice (The Jackson Laboratory, Bar Harbor, Maine, U.S.A.) was permanently transected at postnatal day (P) 3, 10 and 60 as described previously14'16. Except for 3-day-old neonates, male mice were used to rule out a possible effect of sex on Schwann cell proliferation. In 3- and 10-day old mice, the right sciatic nerve was transected and the left nerve served as an uninjured control. In adult mice bilateral sciatic nerves were transected in test mice and uninjured nerves from other mice were used as control.
Correspondence: K. Suzuki, Department of Pathology, and Brain and Development Research Center, School of Medicine, CB~7525, 409 Brinkhous-Bullitt Bldg., University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7527, U.S.A. Fax: (1) (919) 966-6718.
268
Cell culture
in cultures prepared from adult mice (P60) (Fig. 1E).
Uninjured nerves or distal segments of sciatic nerves, 1-7 days after nerve-transection were used for studies. Schwann cells were cultured on polylysine-coatedcoverslips at a density of 6 x 104 cells in 24-well dishes as described previouslyx4-16. They were maintained in Dulbecco's modified Eagle's medium supplemented with 15% (v/v) fetal bovine serum (JRH Biosciences, Lexena, KS, Lot #OH2115), 25 mM HEPES, 100 U/ml penicillin and 100/~g/ml streptomycin at 37°C in a 5% CO2-gassed incubator. The same batch of fetal bovine serum was used throughout the study to eliminate a possible difference in mitogenic capability for Schwann cells and fibroblasts.
After nerve-transection, small round Schwann cells were increased in n u m b e r in cultures prepared from neonatal
In vitro autoradiograpghy Three hours after plating, [methyl-3H]thymidine (final concentration, 10 pCi/ml; 50-90 Ci/ml, New England Nuclear, Boston, MA) was added to cultures. Twenty-four hours later the cultures were fixed and processed for autoradiography 14'1s. Small round cells (0 10 pm) as well as bipolar/tripolar cells were identified as Schwann cells with the positive immunofluorescence for S-100 and galactocerebroside (GalC), and negative staining for a macrophage marker, Mac-114"15. Flat cells were considered as fibroblasts by a characteristic staining with anti-fibronectin (Gibco, Grand Island, NY: 1:120). A minimum of 500 cells (or total Schwann cells or fibroblasts when the numbers of these cells were less than 500) were evaluated and the labeling index was calculated (labeled cells/total cells x 100) on quadruplicate coverslips.
lmmunofluorescence The amount of myelin components phagocytosed by Schwann cells or macrophages was estimated using triple labeling immunofluorescence with GalC, myelin basic protein (MBP) and Mac-116. In brief, unfixed cells were first incubated with anti-Mac-1 (Boehringer, Indianapolis, IN; 1:120), followed by AMCA (7-amino-4methylcourmarin-3-acetate)-labeled goat anti-rat IgG (Jackson Immunoresearch, West Grove, PA; 1:40). After fixation with 2.0% paraformaldehyde and subsequent extraction with 0.05% Triton X-100, cells were incubated with rabbit polyclonal anti-GalC (a gift from Dr. Joyce A. Benjamins; 1:20) and mouse monoclonal antiMBP (Boehringer; 1:20), followed by fluorescein-labeled anti-rabbit IgG (Chemicon, Temecula, CA; 1:40) and rhodamine-labeled anti-mouse IgG (Chemicon; 1:40). The mounting medium contained 1,4-diazobicyclo-(2,2,2)octane to inhibit photobleaching ~2. The slides were examined on a Microphoto-FXA (Nikon) equipped with phase-contrast, selective filters for fluorescein, rhodamine and AMCA, and epi-illumination.
Light microscopy in vivo One to two weeks after nerve-transection, mice were killed and the transected or uninjured sciatic nerves were fixed in situ with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. The nerves were dissected, postfixed in 0.2% osmium tetroxide, and embedded in poly/Bed 812 (Polyscience Inc., Warrington, PA). One-/~m thick cross sections were stained with Toluidine blue, and nuclei were counted at a magnification of × 1000 with oil immersion under bright-field light microscopy.
RESULTS
Morphological observation in vitro Schwann cells isolated from nerves undergoing Wallerian degeneration showed some morphological differences in culture depending on the age of the mice. Most of the Schwann cells from uninjured nerves of neonatal (P3) or suckling (P10) mice assumed a typical spindleshaped configuration at 1 day in culture (Fig. 1A,C), whereas the n u m b e r of small r o u n d cells were n u m e r o u s
or suckling nerves (Fig. 1B,D). In cultures from adult nerves, by contrast, the n u m b e r of Schwann cells with typical spindle morphology was increased after transection during the period we examined (Fig. 1F). A n increase of small r o u n d Schwann cells suggested a reduced mitotic rate of Schwann cells in these cultures, since our previous study TM showed that these small round Schwann cells did not incorporate thymidine. Irrespective of age, cultures prepared from transected nerves contained numerous macrophages and fibroblasts 14'15 (Fig. 1B,D).
Schwann cell division after nerve-transection Counts of labeled Schwann cells from adult nerves showed that division rate rose within 1 day post-transection and reached a peak at day 3, followed by a gradual decrease (Figs. 2, 5E,F). As reported previouslyTM, Schwann cells from uninjured adult nerves were barely labeled for the first 24 h in culture (Figs. 2, 5E). Schwann cells from neonatal or suckling nerves responded to nerve-transection in a different m a n n e r from adult nerves. The labeling indices of Schwann cells from uninjured nerves decreased rapidly from P10 to P17 (Fig. 3), reflecting active myelin formation and consequent differentiation of Schwann cells during this period 15. Schwann cells prepared from transected nerves tended to incorporate less thymidine than those from uninjured contralateral nerves (Figs. 3, 5C,D). Decrease in the n u m b e r of labeled Schwann cells was gradual in uninjured nerves of mice from P3 to P107'15, whereas labeling indices of Schwann cells from the transected nerves declined rapidly (Figs. 4, 5A,B).
Fibroblast division after nerve-transection Fibroblasts are known to proliferate in the degenerating nerves of adult animals z. To examine whether Schwann cells and fibroblasts in nerves of young mice responded similarly to Wallerian degeneration, labeling indices of these cells from transected nerves were compared with those from uninjured control nerves. In neonatal or suckling nerves, although the division rate of Schwann cells from the transected nerves was lower than those from the control nerves, fibroblasts divided more vigorously in the transected nerves (Table I, Fig. 5 A - D ) . In adult mice, however, fibroblast proliferation was stimulated in a m a n n e r similar to that of Schwann cell proliferation in the transected nerves (Fig. 5E,F).
Phagocytosed myelin components in Schwann cells or macrophages Immunoreactivity to anti-GalC and -MBP was clearly
269
Fig. 1. Phase-contrast photomicrographs showing the difference in morphology between cells prepared from uninjured nerves (A,C,E) and nerves 5 days after nerve-transection (B, D, F). Nerves were transected at P3 (B), P10 (D) or P60 (F). Cells were cultured for 24 h. In cultures from transected neonatal or suckling nerves (B,D), the majority of Schwann cells assume a round configuration (arrowheads) and the number of fibroblasts (F) and macrophages (M) are increased. In cultures prepared from transected adult nerves (F), by contrast, the number of Schwann cells with spreading processes are pronounced. Bar = 50 ~m.
270
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Three hours after plating, cells were pulsed with 10 /~Ci/ml of [3H]thymidine for 24 h. Values are the mean + S.E.M. of values of quadruplicate coverslips from each of two separate experiments.
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Comparison of thymidine uptake by Schwann cells and fibroblasts between transected and uninjured control nerves
I
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2 3 4 5 7 DAYS AFTER NERVE-TRANSECTION
Fibroblasts(%)
Transected Control Transected
Without transection Neonatal (P3) 37.0+4.0 Suckling (P10) 24.8+4.3 Adult (P60) 0.4+0.2
-
3 days after transection* Neonatal (P6)* 27.5+3.1 Suckling (P13)* 18.7+3.1 Adult (P63) -
13.3+3.0 13.6+3.2 28.4+ 1.2
56.3+5.1 37.9+4.8 9.5+4.2
I
Fig. 2. Changes in thymidine uptake by Schwann cells from nerves transected at P60. Three hours after plating, cells were pulsed with 10/tCi/ml of [3H]thymidine for 24 h. Each data point is the mean + S.E.M. of values of quadruplicate coverslips from each of two separate experiments.
50.6+3.8 74.1+3.4 33.0+5.4 62.7+5.1 48.2+3.4
* Right sciatic nerve was transected and left served as control.
evidence of scanty myelin formation in neonatal sciatic demonstrated in cytoplasm of Schwann cells or macrophages from adult nerves 4 days after nerve-transection (Fig. 6D-F), implying the presence of phagocytozed myelin components in these cells. Although GalC or MBP was also recognizable in cells from neonatal nerves 4 days post-transection (Fig. 6A-C), immunoreactivity to these myelin components in cells from neonatal nerves appeared weaker than that in cells from adult nerves. These findings were in agreement with morphological
n e r v e s TM"
Morphology and nuclear density of transected nerves in vivo
To correlate the mitotic rate of Schwann cells and fibroblasts in vivo (as assessed by the cells 24 h in culture) with changes in morphology and cell density in situ, we examined 1-#m thick cross sections of sciatic nerves. In sciatic nerves of adult mice, nuclear density increased
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2 3 4 5 7 DAYS AFTER NERVE-TRANSECTION Fig. 3. Changes in thymidine uptake by Schwann ceils from nerves transected at P10. Three hours after plating, cells were pulsed with 10 ~Ci/ml of [3H]thymidine for 24 h. Each data point is the mean + S.E.M. of values of quadruplicate coverslips from each of two separate experiments. *P < 0.05 vs uninjured control, Student's t-test. Contralateral nerves of the same mice were used as uninjured controls.
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I 2 5 4 5 7 DAYS AFTER NERVE-TRANSECTION Fig. 4. Changes in thymidine uptake by Schwann cells from nerves transected at P3. Three hours after plating, cells were pulsed with 10/~Ci/ml of [3H]thymidine for 24 h. Each data point is the mean _+ S.E.M. of values of quadruplicate coverslips from each of two separate experiments. *P < 0.05 vs uninjured control, Student's t-test. Contralateral nerves of the same mice were used as uninjured controls.
271
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A
Fig. 5. Bright-field autoradiographs counter-stained with Mayer's hematoxylin showing the difference in thymidine labeling of Schwann cells (S) and fibroblasts (F). Cells were prepared from nerves 3 days after transection (B,D,F) or uninjured nerves (A,C,E). Nerves were transected at P3 (B), P10 (D), and P60 (F). Three hours after plating, cells were pulsed with 10 #Ci/ml of [3H]thymidine for 24 h. Bar = 50/~m.
272
Fig. 6. Triple labeling immunofluorescence showing staining of Schwann cells and macrophages with antibodies against galactocerebroside (GalC) or myelin basic protein (MBP). Cultures were prepared from neonatal (P3) (A-C) or adult (P60) nerves (D-F) 3 days after transection. Cells were cultured for 24 h. A,D: GalC with fluorescein conjugate; B, E: MBP with rhodamine conjugate; C,F: Mac-1 with AMCA conjugate. Bar = 50 #m.
3-fold at 1 week after nerve-transection and 5-fold at 2 weeks (Fig. 7). Cell density in the uninjured nerves of neonatal o r suckling mice were higher than that in the adult. H o w e v e r , an increase in cell density after nervetransection was less than 2-fold of the control uninjured nerves. F u r t h e r m o r e , in neonatal nerves cell density at 2 weeks t e n d e d to be less than that at 1 week (Fig. 7). Precise identification of the cell in the transected adult nerves was difficult, but the majority of cells showed a round or oval configuration suggesting Schwann cells (Fig. 8C, D). On the o t h e r hand, in the transected nerves of young mice there were numerous cells with attenuated cytoplasmic processes suggestive of firoblasts 26 (Fig. 8A,B).
DISCUSSION In accordance with the previous in v i v o 6'18 o r in vitro 8'16 studies, Schwann cell mitosis reached a maxim u m at day 3 post-transection in sciatic nerves of adult mice. In neonatal or suckling nerves, however, the labeling indices of Schwann cells decreased after transection, and r e m a i n e d less in the transected nerves than in the contralateral uninjured nerves. (This is also in contrast to enhanced proliferation of oligodendrocytes in the degenerating optic nerve of young rats19.) Thymidine uptake was far less in nerves sectioned at P3 than those sectioned at P10. These findings are in agreement with the in vitro study of Salzer and Bunge 23 demonstrating
273
25
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~60d lOd 3d
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0 WEEKS
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AFTER NERVE-TRANSECTION
Fig. 7. Comparison of changes in nuclear density between uninjured nerves and the nerves after transection. Cell nuclei were counted with oil immersion at x 1000 magnification under brightfield microscopy. Each data is the mean _+ S.E.M. for each of 4 nerves.
that Schwann cells undergoing proliferation after nerve transection were the myelin-related Schwann cells and that Schwann cell proliferation rapidly terminated if the young neurites without fully ensheated myelin were sectioned. Therefore, the capacity of Schwan cells to proliferate during Wallerian degeneration may be dependent on the stage of myelination. A t P10, when a certain amount of myelin has already been formed (Fig. 8A), approximately 15% of Schwann cells in uninjured nerves incorporated thymidine in situ as assessed by a single pulse method (unpublished observation). The question of why a majority of quiescent Schwann cells in suckling nerves did not enter S-phase during Wallerian degeneration remains unclear. In a previous in vitro study, we observed that removal of axorg myelin debris from cultures, 3 h after plating, reduced thymidine uptake by Schwann cells from the mice at P30, but far less from P1015. This experimental system, 'in vitro Wallerian degeneration', indicates that physiological amounts of axon/myelin debris derived from nerves stimulated Schwann cells of older mice but to a
Fig. 8. Difference in cellular responses to Wallerian degeneration in situ between suckling (P10) (A,B) and adult (P60) (C,D) nerves. Cross sections (1/~m) of uninjured nerves (A,C) or nerves 7 days after transection (B,D) are shown. Note that fibroblast-like cells with attenuated cytoplasmic processes (arrowheads) are increased in suckling nerves after nerve-transection. Toluidine blue stain. Bar = 20 gin.
274 lesser degree the Schwann cells from suckling mice in culture, a finding consistent with the present observation. Yoshino et al. 27 also have shown that Schwann cells prepared from sciatic nerves of 6-day-old rat pups proliferated more vigorously than those from 2-day-old pups in response to myelin enriched-fractions. Several mitogenic signals have been implicated to be relevant to Schwann cell proliferation during Wallerian degeneration: chemical stimulus diffusible from autolysing nerves 1, axonal fragments 24, loss of axonal components TM and myelin 4. Our results and others 23 indicate that loss of axonal components has an inhibitory but not stimulatory effect for Schwann cells in actively myelinating nerves. In adult nerves in which myelin is already well formed, Schwann cells undergo vigorous mitotic responses after nerve-transection. Therefore, as has been suggested, it is likely that myelin components 'processed' by macrophages play a primary role in Schwann cell mitosis in situ during Wallerian degeneration 4'~6. This hypothesis is well supported by the report on the absence of Schwann cell division during degeneration of unmyelinated nerves ~3'22, and rapid proliferation of non-myelin-forming Schwann cells in the sciatic nerves after transection 8. In contrast to reduced mitotic responses of Schwann cells in transected nerves of young mice, fibroblasts divided vigorously during Wallerian degeneration of nerves at any ages. The high mitotic capacity expressed by fibroblasts may contradict the contention that high cellular densities in nerves of young mice inhibited cellular proliferation. Furthermore, this finding may suggest that
mitogens of fibroblasts during Wallerian degeneration are different from those of Schwann cells, possibly related to mitogens c o m m o n to any other tissues during the wound healing process 6'17. This observation may be of importance, when we discuss mitogens primarily responsible for Schwann cells in situ during Wallerian degeneration. A number of mitogens for Schwann cells in vitro have been reported: platelet-derived growth factor (PDGF) 9, fibroblast growth factor (FGF) 9, and transforming growth factor-fl (TGF-fl) 1°'21. The main source of these mitogenic polypeptides in situ comes from macrophages activated during wound healing. Interestingly, these mitogens are also stimulatory for fibroblast growth, at least in some condition, and implicated as playing a regulatory role in fibrosis during wound healing 6'17. It is, therefore, conceivable that in degenerating nerves of young mice these polypeptides function as growth-control factors for fibroblasts but not for Schwann cells. Another possibility is that although these growth factors are released into injured sites and promote Schwann cell/fibroblast proliferation synergistically with macrophage-mediated myelin-related factors 4 in adult nerves, a loss of axonal mitogens and paucity of myelin-related mitogenic factors may lead to reduced Schwann cell division in degenerating nerves of young mice.
REFERENCES
9 Davis, J.B. and Stroobant, E, Platelet-derived growth factors and fibroblast growth factors are mitogens for rat Schwann cells, J. Cell Biol., 110 (1990) 1353-1360. 10 Eccleston, P.A., Jessen, K.R. and Mirsky, R., Transforming growth factor-fl and r-interferon have dual effects on growth of peripheral glia, J. Neurosci. Res., 24 (1989) 524-530. 11 Friede, R.L. and Samorajski, T., Myelin formation in the sciatic nerve of the rat. A quantitative electron microscopic, histochemical and radioautographic study, J. Neuropathol. Exp. Neurol., 27 (1968) 546-570. 12 Johnson, G.D., Davidson, R.S., McNamee, K.C., Russell, G., Goodwin, D. and Holborow, E.J., Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy, J. Immunol. Methods, 55 (1982) 231-242. 13 Jospeh, J., Absence of cell multiplication during Wallerian degeneration of non-myelinated nerves, J. Anat., 81 (1947) 135139. 14 Komiyama, A., Novicki, D.L. and Suzuki, K., Adhesion and proliferation are enhanced in vitro in Schwann cells from nerve undergoing Wallerian degeneration, J. Neurosci. Res., 29 (1991) 308-318. 15 Komiyama, A. and Suzuki, K., Age-related changes in attachment and proliferation of mouse Schwann cells in vitro, Dev. Brain Res., 82 (1991) 7-16, 16 Komiyama, A. and Suzuki, K., Normal rate of Schwann cell proliferation in the MBP-deficient shiverer mouse during Wallerian degeneration, Brain Res., 563 (1991) 345-348.
1 Abercrombie, M. and Johnson, M.L., Quantitative histology of Wallerian degeneration. I. Nuclear population in rabbit sciatic nerves, J. Anat., 80 (1946) 37-50. 2 Asbury, A.K., Schwann cell proliferation in developing mouse sciatic nerve. A radioautographic study, J. Cell Biol., 34 (1967) 735-743. 3 Asbury, A.K. and Arnason, B.G., Experimental allergic neuritis: a radioautographic study, J. Neuropathol. Exp. Neurol., 27 (1968) 581-590. 4 Baichwal, R.R., Bigbee, J.W. and DeVries, G.H., Macrophagemediated myelin-related mitogenic factor for cultured Schwann cells, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 1701-1705. 5 Bradley, W.G. and Asbury, A.K., Duration of synthesis phase in neurilemma cells in mouse sciatic nerves during degeneration, Exp. Neurol., 26 (1970) 275-282. 6 Brandes, M.E. and Finkelstein, J.N., The production of alveolar macrophage-derived growth-regulating proteins in response to lung injury, Toxicol. Lett., 54 (1990) 3-22. 7 Brown, M.J. and Asbury, A.K., Schwann cell proliferation in the postnatal mouse: timing and topography, Exp. Neurol., 74 (1981) 170-186. 8 Clemence, A., Mirskey, R. and Jessen, K.R., Non-myelinforming Schwann cells proliferate rapidly during Wallerian degeneration in the rat sciatic nerve, J. Neurocytol., 18 (1989) 185-192.
Acknowledgements. This work was supported in part by U.S.P.H.S. Grants NS-24453, HD-03110 and ES-01104. We thank Dr. Joyce A. Benjamins for providing us with the anti-GalC antibodies, Mrs. Clarita Langaman for 1-/~mthick sections from sciatic nerves and Mrs. Deborah H. Sears for manuscript preparation.
275 17 Nathan, C.F., Secretory products of macrophages, J. Clin. Invest., 79 (1987) 319-326. 18 Pellegrino, R.G., Politis, M.J., Ritchie, J.M. and Spencer, ES., Events in degenerating cat peripheral nerve: induction of Schwann cell S phase and its relation to nerve fibre degeneration, J. Neurocytol., 15 (1986) 17-28. 19 Privat, A., Valat, J. and Fulcrand, J., Proliferation of neuroglial cell lines in the degenerating optic nerve of young rats. A radioautographic study, J. Neuropathol. Exp. Neurol., 40 (1981) 46-60. 20 Ratner, N., Hong, D., Lieberman, M.A., Bunge, R.P. and Glaser, L., The neuronal cell-surface molecule mitogenic for Schwann cells is a heparin-binding protein, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 6992-6996. 21 Ridley, A.J., Davis, J.B., Stroobant, P. and Land, H., Transforming growth factors-ill and -f12 are mitogens for rat Schwann cells, J. Cell Biol., 109 (1989) 3419-3424. 22 Romine, J.S., Bray, G.M. and Aguayo, A.J., Schwann cell
23
24
25 26 27
multiplication after crush injury of unmyelinated fibers, Arch. Neurol., 33 (1976) 49-54. Salzer, J.L. and Bunge, R.E, Studies of Schwann cell proliferation. I. An analysis in tissue culture of proliferation during development, Wallerian degeneration, and direct injury, J. Cell Biol., 84 (1980) 739-752. Sobue, G., Kreider, B,, Asbury, A. and Pleasure, D., Specific and potent mitogenic effects of axolemmal fraction on Schwann cells from rat sciatic nerves in serum-containing and defined media, Brain Research, 280 (1983) 263-275. Thomas, G.A., Quantitative histology of Wallerian degeneration. II. Nuclear population in two nerves of different fibre spectrum, J. Anat., 82 (1948) 135-145. Weinberg, H.J. and Spencer, ES., The fate of Schwann cells isolated from axonal contact, J. Neurocytol., 7 (1978) 555-569. Yoshino, J.E., Mason, P.W. and DeVries, G.H., Developmental changes in myelin-induced proliferation of cultured Schwann cells, J. Cell Biol., 104 (1987) 655-660.
267
BRES 17474
Age-related differences in proliferative responses of Schwann cells during Wallerian degeneration Atsushi Komiyama and Kinuko Suzuki Department of Pathology, and Brain and Development Research Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525 (U.S.A.) (Accepted 15 October 1991) Key words: Schwann cell; Fibroblast; Wallerian degeneration; Proliferation; Development; Cell culture; Autoradiography
Age-related differences in proliferative responses of Schwann cells during Wallerian degeneration were investigated in the mouse sciatic nerves after nerve-transection at 3, 10 and 60 days of age, corresponding to the periods of early myelination, active myelination and postmyelination. As assessed by thymidine incorporation for the first 24 h in culture, Schwann cells from adult nerve proliferated rapidly within day 1 post-transection and reached a peak at day 3. In the nerves from neonatal or suckling mice, however, division rate of Schwann cells declined after transection, and was even less in the transected nerves than in the contralateral uninjured nerves. The reduction in thymidine uptake by Schwann cells was more pronounced in nerves sectioned at postnatal day 3 than those sectioned at day 10. By contrast, fibroblasts divided rapidly following transection regardless of age. These data suggest that mitogens from myelin components are important for proliferation of Schwann cells and that in the degenerating nerves of young mice, mitotic capacity of Schwann cells declined due to not only a loss of axonal mitogens but also the paucity of mitogens from myelin components. Proliferation of fibroblasts is likely to be stimulated by more general growth-promoting polypeptides common to any other tissues during wound repair. INTRODUCTION During Wallerian d e g e n e r a t i o n of p e r i p h e r a l nerves, Schwann cells are stimulated to proliferate vigorously 1' 5,18. Most of these Schwann cell responses after injury have b e e n investigated in adult animals 1'5'1s. Conversely, studies on gliogenesis of the p e r i p h e r a l nervous system have b e e n carried out on n o r m a l pups 2'7'11. Therefore, little is known about the influence of insult on mitotic capacity of Schwann cells in nerves of young animals. W i t h respect to mitogens for Schwann cells, myelin components 'processed' by m a c r o p h a g e s were suggested to p r o m o t e Schwann cell proliferation during Wallerian degeneration 4'16. A Schwann cell mitogen during developm e n t or r e g e n e r a t i o n has b e e n shown to be an axonal surface proteoglycan-growth factor complex 2°. These studies led us to test a hypothesis that Schwann cells in the degenerating nerves of young mice m a y lose their proliferative capacity because of a loss of axonal mitogens as well as the paucity of mitogen from myelin components. To test this hypothesis, we investigated the differences in proliferative responses to Wallerian degeneration of Schwann cells from mice of different ages. O u r assay was b a s e d on the previous observation 8'16 that the mitotic rate of Schwann cells from dissociated nerves,
as assessed in vitro by thymidine a u t o r a d i o g r a p h y for the first 24 h, reflects the state of in vivo proliferation at the time of dissociation. A progressive loss of proliferative capacity of Schwann cells was d e m o n s t r a t e d in the degenerating nerves of young mice. A b y p r o d u c t of this study was the d e m o n s t r a t i o n that regardless of age, fibroblasts proliferated vigorously after nerve-transection. These differential proliferative responses of non-neuronal cells in the degenerating nerves of young mice suggest that the mitogen responsible for the proliferation of fibroblasts is not necessarily the same as that involved in the proliferation of Schwann cells. Alternatively, Schwann cells m a y need additional specific mitogen from myelin c o m p o n e n t s to proliferate in response to insults. MATERIALS AND METHODS Animal surgery The sciatic nerve of C3HeB/FeJ mice (The Jackson Laboratory, Bar Harbor, Maine, U.S.A.) was permanently transected at postnatal day (P) 3, 10 and 60 as described previously14'16. Except for 3-day-old neonates, male mice were used to rule out a possible effect of sex on Schwann cell proliferation. In 3- and 10-day old mice, the right sciatic nerve was transected and the left nerve served as an uninjured control. In adult mice bilateral sciatic nerves were transected in test mice and uninjured nerves from other mice were used as control.
Correspondence: K. Suzuki, Department of Pathology, and Brain and Development Research Center, School of Medicine, CB~7525, 409 Brinkhous-Bullitt Bldg., University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7527, U.S.A. Fax: (1) (919) 966-6718.
268
Cell culture
in cultures prepared from adult mice (P60) (Fig. 1E).
Uninjured nerves or distal segments of sciatic nerves, 1-7 days after nerve-transection were used for studies. Schwann cells were cultured on polylysine-coatedcoverslips at a density of 6 x 104 cells in 24-well dishes as described previouslyx4-16. They were maintained in Dulbecco's modified Eagle's medium supplemented with 15% (v/v) fetal bovine serum (JRH Biosciences, Lexena, KS, Lot #OH2115), 25 mM HEPES, 100 U/ml penicillin and 100/~g/ml streptomycin at 37°C in a 5% CO2-gassed incubator. The same batch of fetal bovine serum was used throughout the study to eliminate a possible difference in mitogenic capability for Schwann cells and fibroblasts.
After nerve-transection, small round Schwann cells were increased in n u m b e r in cultures prepared from neonatal
In vitro autoradiograpghy Three hours after plating, [methyl-3H]thymidine (final concentration, 10 pCi/ml; 50-90 Ci/ml, New England Nuclear, Boston, MA) was added to cultures. Twenty-four hours later the cultures were fixed and processed for autoradiography 14'1s. Small round cells (0 10 pm) as well as bipolar/tripolar cells were identified as Schwann cells with the positive immunofluorescence for S-100 and galactocerebroside (GalC), and negative staining for a macrophage marker, Mac-114"15. Flat cells were considered as fibroblasts by a characteristic staining with anti-fibronectin (Gibco, Grand Island, NY: 1:120). A minimum of 500 cells (or total Schwann cells or fibroblasts when the numbers of these cells were less than 500) were evaluated and the labeling index was calculated (labeled cells/total cells x 100) on quadruplicate coverslips.
lmmunofluorescence The amount of myelin components phagocytosed by Schwann cells or macrophages was estimated using triple labeling immunofluorescence with GalC, myelin basic protein (MBP) and Mac-116. In brief, unfixed cells were first incubated with anti-Mac-1 (Boehringer, Indianapolis, IN; 1:120), followed by AMCA (7-amino-4methylcourmarin-3-acetate)-labeled goat anti-rat IgG (Jackson Immunoresearch, West Grove, PA; 1:40). After fixation with 2.0% paraformaldehyde and subsequent extraction with 0.05% Triton X-100, cells were incubated with rabbit polyclonal anti-GalC (a gift from Dr. Joyce A. Benjamins; 1:20) and mouse monoclonal antiMBP (Boehringer; 1:20), followed by fluorescein-labeled anti-rabbit IgG (Chemicon, Temecula, CA; 1:40) and rhodamine-labeled anti-mouse IgG (Chemicon; 1:40). The mounting medium contained 1,4-diazobicyclo-(2,2,2)octane to inhibit photobleaching ~2. The slides were examined on a Microphoto-FXA (Nikon) equipped with phase-contrast, selective filters for fluorescein, rhodamine and AMCA, and epi-illumination.
Light microscopy in vivo One to two weeks after nerve-transection, mice were killed and the transected or uninjured sciatic nerves were fixed in situ with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2. The nerves were dissected, postfixed in 0.2% osmium tetroxide, and embedded in poly/Bed 812 (Polyscience Inc., Warrington, PA). One-/~m thick cross sections were stained with Toluidine blue, and nuclei were counted at a magnification of × 1000 with oil immersion under bright-field light microscopy.
RESULTS
Morphological observation in vitro Schwann cells isolated from nerves undergoing Wallerian degeneration showed some morphological differences in culture depending on the age of the mice. Most of the Schwann cells from uninjured nerves of neonatal (P3) or suckling (P10) mice assumed a typical spindleshaped configuration at 1 day in culture (Fig. 1A,C), whereas the n u m b e r of small r o u n d cells were n u m e r o u s
or suckling nerves (Fig. 1B,D). In cultures from adult nerves, by contrast, the n u m b e r of Schwann cells with typical spindle morphology was increased after transection during the period we examined (Fig. 1F). A n increase of small r o u n d Schwann cells suggested a reduced mitotic rate of Schwann cells in these cultures, since our previous study TM showed that these small round Schwann cells did not incorporate thymidine. Irrespective of age, cultures prepared from transected nerves contained numerous macrophages and fibroblasts 14'15 (Fig. 1B,D).
Schwann cell division after nerve-transection Counts of labeled Schwann cells from adult nerves showed that division rate rose within 1 day post-transection and reached a peak at day 3, followed by a gradual decrease (Figs. 2, 5E,F). As reported previouslyTM, Schwann cells from uninjured adult nerves were barely labeled for the first 24 h in culture (Figs. 2, 5E). Schwann cells from neonatal or suckling nerves responded to nerve-transection in a different m a n n e r from adult nerves. The labeling indices of Schwann cells from uninjured nerves decreased rapidly from P10 to P17 (Fig. 3), reflecting active myelin formation and consequent differentiation of Schwann cells during this period 15. Schwann cells prepared from transected nerves tended to incorporate less thymidine than those from uninjured contralateral nerves (Figs. 3, 5C,D). Decrease in the n u m b e r of labeled Schwann cells was gradual in uninjured nerves of mice from P3 to P107'15, whereas labeling indices of Schwann cells from the transected nerves declined rapidly (Figs. 4, 5A,B).
Fibroblast division after nerve-transection Fibroblasts are known to proliferate in the degenerating nerves of adult animals z. To examine whether Schwann cells and fibroblasts in nerves of young mice responded similarly to Wallerian degeneration, labeling indices of these cells from transected nerves were compared with those from uninjured control nerves. In neonatal or suckling nerves, although the division rate of Schwann cells from the transected nerves was lower than those from the control nerves, fibroblasts divided more vigorously in the transected nerves (Table I, Fig. 5 A - D ) . In adult mice, however, fibroblast proliferation was stimulated in a m a n n e r similar to that of Schwann cell proliferation in the transected nerves (Fig. 5E,F).
Phagocytosed myelin components in Schwann cells or macrophages Immunoreactivity to anti-GalC and -MBP was clearly
269
Fig. 1. Phase-contrast photomicrographs showing the difference in morphology between cells prepared from uninjured nerves (A,C,E) and nerves 5 days after nerve-transection (B, D, F). Nerves were transected at P3 (B), P10 (D) or P60 (F). Cells were cultured for 24 h. In cultures from transected neonatal or suckling nerves (B,D), the majority of Schwann cells assume a round configuration (arrowheads) and the number of fibroblasts (F) and macrophages (M) are increased. In cultures prepared from transected adult nerves (F), by contrast, the number of Schwann cells with spreading processes are pronounced. Bar = 50 ~m.
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-
3 days after transection* Neonatal (P6)* 27.5+3.1 Suckling (P13)* 18.7+3.1 Adult (P63) -
13.3+3.0 13.6+3.2 28.4+ 1.2
56.3+5.1 37.9+4.8 9.5+4.2
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Fig. 2. Changes in thymidine uptake by Schwann cells from nerves transected at P60. Three hours after plating, cells were pulsed with 10/tCi/ml of [3H]thymidine for 24 h. Each data point is the mean + S.E.M. of values of quadruplicate coverslips from each of two separate experiments.
50.6+3.8 74.1+3.4 33.0+5.4 62.7+5.1 48.2+3.4
* Right sciatic nerve was transected and left served as control.
evidence of scanty myelin formation in neonatal sciatic demonstrated in cytoplasm of Schwann cells or macrophages from adult nerves 4 days after nerve-transection (Fig. 6D-F), implying the presence of phagocytozed myelin components in these cells. Although GalC or MBP was also recognizable in cells from neonatal nerves 4 days post-transection (Fig. 6A-C), immunoreactivity to these myelin components in cells from neonatal nerves appeared weaker than that in cells from adult nerves. These findings were in agreement with morphological
n e r v e s TM"
Morphology and nuclear density of transected nerves in vivo
To correlate the mitotic rate of Schwann cells and fibroblasts in vivo (as assessed by the cells 24 h in culture) with changes in morphology and cell density in situ, we examined 1-#m thick cross sections of sciatic nerves. In sciatic nerves of adult mice, nuclear density increased
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2 3 4 5 7 DAYS AFTER NERVE-TRANSECTION Fig. 3. Changes in thymidine uptake by Schwann ceils from nerves transected at P10. Three hours after plating, cells were pulsed with 10 ~Ci/ml of [3H]thymidine for 24 h. Each data point is the mean + S.E.M. of values of quadruplicate coverslips from each of two separate experiments. *P < 0.05 vs uninjured control, Student's t-test. Contralateral nerves of the same mice were used as uninjured controls.
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I 2 5 4 5 7 DAYS AFTER NERVE-TRANSECTION Fig. 4. Changes in thymidine uptake by Schwann cells from nerves transected at P3. Three hours after plating, cells were pulsed with 10/~Ci/ml of [3H]thymidine for 24 h. Each data point is the mean _+ S.E.M. of values of quadruplicate coverslips from each of two separate experiments. *P < 0.05 vs uninjured control, Student's t-test. Contralateral nerves of the same mice were used as uninjured controls.
271
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Fig. 5. Bright-field autoradiographs counter-stained with Mayer's hematoxylin showing the difference in thymidine labeling of Schwann cells (S) and fibroblasts (F). Cells were prepared from nerves 3 days after transection (B,D,F) or uninjured nerves (A,C,E). Nerves were transected at P3 (B), P10 (D), and P60 (F). Three hours after plating, cells were pulsed with 10 #Ci/ml of [3H]thymidine for 24 h. Bar = 50/~m.
272
Fig. 6. Triple labeling immunofluorescence showing staining of Schwann cells and macrophages with antibodies against galactocerebroside (GalC) or myelin basic protein (MBP). Cultures were prepared from neonatal (P3) (A-C) or adult (P60) nerves (D-F) 3 days after transection. Cells were cultured for 24 h. A,D: GalC with fluorescein conjugate; B, E: MBP with rhodamine conjugate; C,F: Mac-1 with AMCA conjugate. Bar = 50 #m.
3-fold at 1 week after nerve-transection and 5-fold at 2 weeks (Fig. 7). Cell density in the uninjured nerves of neonatal o r suckling mice were higher than that in the adult. H o w e v e r , an increase in cell density after nervetransection was less than 2-fold of the control uninjured nerves. F u r t h e r m o r e , in neonatal nerves cell density at 2 weeks t e n d e d to be less than that at 1 week (Fig. 7). Precise identification of the cell in the transected adult nerves was difficult, but the majority of cells showed a round or oval configuration suggesting Schwann cells (Fig. 8C, D). On the o t h e r hand, in the transected nerves of young mice there were numerous cells with attenuated cytoplasmic processes suggestive of firoblasts 26 (Fig. 8A,B).
DISCUSSION In accordance with the previous in v i v o 6'18 o r in vitro 8'16 studies, Schwann cell mitosis reached a maxim u m at day 3 post-transection in sciatic nerves of adult mice. In neonatal or suckling nerves, however, the labeling indices of Schwann cells decreased after transection, and r e m a i n e d less in the transected nerves than in the contralateral uninjured nerves. (This is also in contrast to enhanced proliferation of oligodendrocytes in the degenerating optic nerve of young rats19.) Thymidine uptake was far less in nerves sectioned at P3 than those sectioned at P10. These findings are in agreement with the in vitro study of Salzer and Bunge 23 demonstrating
273
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Fig. 7. Comparison of changes in nuclear density between uninjured nerves and the nerves after transection. Cell nuclei were counted with oil immersion at x 1000 magnification under brightfield microscopy. Each data is the mean _+ S.E.M. for each of 4 nerves.
that Schwann cells undergoing proliferation after nerve transection were the myelin-related Schwann cells and that Schwann cell proliferation rapidly terminated if the young neurites without fully ensheated myelin were sectioned. Therefore, the capacity of Schwan cells to proliferate during Wallerian degeneration may be dependent on the stage of myelination. A t P10, when a certain amount of myelin has already been formed (Fig. 8A), approximately 15% of Schwann cells in uninjured nerves incorporated thymidine in situ as assessed by a single pulse method (unpublished observation). The question of why a majority of quiescent Schwann cells in suckling nerves did not enter S-phase during Wallerian degeneration remains unclear. In a previous in vitro study, we observed that removal of axorg myelin debris from cultures, 3 h after plating, reduced thymidine uptake by Schwann cells from the mice at P30, but far less from P1015. This experimental system, 'in vitro Wallerian degeneration', indicates that physiological amounts of axon/myelin debris derived from nerves stimulated Schwann cells of older mice but to a
Fig. 8. Difference in cellular responses to Wallerian degeneration in situ between suckling (P10) (A,B) and adult (P60) (C,D) nerves. Cross sections (1/~m) of uninjured nerves (A,C) or nerves 7 days after transection (B,D) are shown. Note that fibroblast-like cells with attenuated cytoplasmic processes (arrowheads) are increased in suckling nerves after nerve-transection. Toluidine blue stain. Bar = 20 gin.
274 lesser degree the Schwann cells from suckling mice in culture, a finding consistent with the present observation. Yoshino et al. 27 also have shown that Schwann cells prepared from sciatic nerves of 6-day-old rat pups proliferated more vigorously than those from 2-day-old pups in response to myelin enriched-fractions. Several mitogenic signals have been implicated to be relevant to Schwann cell proliferation during Wallerian degeneration: chemical stimulus diffusible from autolysing nerves 1, axonal fragments 24, loss of axonal components TM and myelin 4. Our results and others 23 indicate that loss of axonal components has an inhibitory but not stimulatory effect for Schwann cells in actively myelinating nerves. In adult nerves in which myelin is already well formed, Schwann cells undergo vigorous mitotic responses after nerve-transection. Therefore, as has been suggested, it is likely that myelin components 'processed' by macrophages play a primary role in Schwann cell mitosis in situ during Wallerian degeneration 4'~6. This hypothesis is well supported by the report on the absence of Schwann cell division during degeneration of unmyelinated nerves ~3'22, and rapid proliferation of non-myelin-forming Schwann cells in the sciatic nerves after transection 8. In contrast to reduced mitotic responses of Schwann cells in transected nerves of young mice, fibroblasts divided vigorously during Wallerian degeneration of nerves at any ages. The high mitotic capacity expressed by fibroblasts may contradict the contention that high cellular densities in nerves of young mice inhibited cellular proliferation. Furthermore, this finding may suggest that
mitogens of fibroblasts during Wallerian degeneration are different from those of Schwann cells, possibly related to mitogens c o m m o n to any other tissues during the wound healing process 6'17. This observation may be of importance, when we discuss mitogens primarily responsible for Schwann cells in situ during Wallerian degeneration. A number of mitogens for Schwann cells in vitro have been reported: platelet-derived growth factor (PDGF) 9, fibroblast growth factor (FGF) 9, and transforming growth factor-fl (TGF-fl) 1°'21. The main source of these mitogenic polypeptides in situ comes from macrophages activated during wound healing. Interestingly, these mitogens are also stimulatory for fibroblast growth, at least in some condition, and implicated as playing a regulatory role in fibrosis during wound healing 6'17. It is, therefore, conceivable that in degenerating nerves of young mice these polypeptides function as growth-control factors for fibroblasts but not for Schwann cells. Another possibility is that although these growth factors are released into injured sites and promote Schwann cell/fibroblast proliferation synergistically with macrophage-mediated myelin-related factors 4 in adult nerves, a loss of axonal mitogens and paucity of myelin-related mitogenic factors may lead to reduced Schwann cell division in degenerating nerves of young mice.
REFERENCES
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Acknowledgements. This work was supported in part by U.S.P.H.S. Grants NS-24453, HD-03110 and ES-01104. We thank Dr. Joyce A. Benjamins for providing us with the anti-GalC antibodies, Mrs. Clarita Langaman for 1-/~mthick sections from sciatic nerves and Mrs. Deborah H. Sears for manuscript preparation.
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multiplication after crush injury of unmyelinated fibers, Arch. Neurol., 33 (1976) 49-54. Salzer, J.L. and Bunge, R.E, Studies of Schwann cell proliferation. I. An analysis in tissue culture of proliferation during development, Wallerian degeneration, and direct injury, J. Cell Biol., 84 (1980) 739-752. Sobue, G., Kreider, B,, Asbury, A. and Pleasure, D., Specific and potent mitogenic effects of axolemmal fraction on Schwann cells from rat sciatic nerves in serum-containing and defined media, Brain Research, 280 (1983) 263-275. Thomas, G.A., Quantitative histology of Wallerian degeneration. II. Nuclear population in two nerves of different fibre spectrum, J. Anat., 82 (1948) 135-145. Weinberg, H.J. and Spencer, ES., The fate of Schwann cells isolated from axonal contact, J. Neurocytol., 7 (1978) 555-569. Yoshino, J.E., Mason, P.W. and DeVries, G.H., Developmental changes in myelin-induced proliferation of cultured Schwann cells, J. Cell Biol., 104 (1987) 655-660.