GLIA 4:529-533 (1991)

In Vitro Myelination of Regenerating Adult Rat Retinal Ganglion Cell Axons by Schwann Cells M.B m R , ’ J.M. HOPKINS? AND R.P. BUNGE2 ‘Neurologische Uniuersitatsklinik and Max-Planck-Institut f u r Entwicklungsbiologie Tubingen, 7400 Tubingen, Germany and “The Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami. Florida 33136

ABSTRACT Schwann cell cultures provide a highly favorable substrate for retinal ganglion cell (RGC) survival and axon growth in vitro (Bahr and Bunge, Exp Neurol 106:27, 1989; Hopkins and Bunge, Glia 4:46, 1991). In this report we have extended former studies to obtain axon regeneration, long-term survival, and myelination of adult rat RGC axons in co-cultures of retinal explants with purified Schwann cells. By using modified co-culture conditions, we observed myelination of regenerating adult RGC axons by Schwann cells after 3-4 weeks in vitro. Myelination was associated with a one-to-one Schwann cell-axon relationship, characteristic of the formation of peripheral myelin. Under culture conditions that supported myelination, long-term survival (more than 12 weeks) of a small population of RGCs was observed. These findings highlight the remarkable ability of Schwann cells to support long-term survival of adult rat RGCs in the absence of either central nervous system (CNS)target tissue or other peripheral nervous system (PNS) components. This tissue culture system may serve as a model for the systematic study of the molecular mechanisms which are involved in axon regeneration and myelination of adult CNS neurons.

Unlike oligodendrocytes [the myelinating glia of the atic nerves as described by Brockes et al. (1979).Briefly, mammalian central nervous system (CNSII, Schwann purified Schwann cell populations were obtained by cells (the myelinating glia found in peripheral nerves) treatment of the mixed cell population obtained from provide a favorable substrate for survival and axonal neonatal rat sciatic nerves with two cycles of cytosinegrowth of adult CNS neurons in vivo and in vitro (Bahr arabinoside (Sigma, St. Louis, MO, lop5 M). Subseand Bunge, 1989; Berry et al., 1988; Bray et al., 1987; quently the cultures were treated with anti-Thy 1.1 David and Aguayo, 1981). It has been previously dem- antibody (collected as a supernatant from hybridoma onstrated that retinal ganglion cells (RGCs) in retinal cells, American Type Culture Collection, Rockville, MD) explants, obtained from adult rats 1 week after optic and rabbit complement (Organon Technica, West nerve crush, regenerate axons and survive for a limited Chester, PA) to remove contaminating fibroblasts that time when cultured on acellular substrates (Bahr et al., remained after the antimitotic treatment (Bahr and 1988; Ford-Holevinsky et al., 1986). When retinal ex- Bunge, 1989). Purified Schwann cells were seeded on plants were co-cultured with Schwann cells, both neu- ammoniated collagen-covered Petriperm dishes (Herrite growth and RGC survival were enhanced compared aeus) and maintained in Dulbecco’s modified Eagle’s to explants cultured on acellular substrates (Bahr and medium (DMEM, Gibco, Grand Island, NY). Purity of Bunge, 1989). In the present study, we asked whether Schwann cells would support sustained survival of adult rat RGCs in order to allow ensheathment and Received February 13,1991; accepted April 16,1991 myelination of the regenerated RGC axons. Address reprint requests to Dr. M. Bahr, Max-Planck-Institut Tubingen, Schwann cells were prepared from neonatal rat sci- Spemannstr. 35/I, 7400 Tubingen, FRG. 01991 Wiley-Liss, Inc.

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the Schwann cell cultures was demonstrated by immunostaining with an antibody against the surface protein 217C, a selective Schwann cell marker (Fields and Dammermann, 1985; see Fig. 1).Axotomized retinae were obtained from five adult rats which had received a “conditioning”lesion (unilateral optic nerve crush, carried out under general chloral hydrate anesthesia; 420 mg/kg body weight) 1 week before removal of the retinae. Retinal explants were prepared as described recently (Bahr et al., 1988). The retinal explants (n = 40) were placed on purified Schwann cell cultures which had been prepared before. Previous investigations of myelination of sensory neurites by Schwann cells in tissue culture (Eldridge et al., 1987, 1989) pointed out the importance of supplementing culture media with serum and ascorbate to achieve optimal myelination by Schwann cells in vitro (Eldridge et al., 1987). We therefore used a modification of the myelinating medium described by Eldridge et al. (1987) in our co-cultures of adult rat retinae and Schwann cells [E 15 medium supplemented with 15% human placental serum (or fetal bovine) serum, nerve growth factor (NGF) (50 U/ml), d-glucose (to a final concentration of approx. 5 mglml), and ascorbate (50 p.g/ml)l. We used NGF as a culture supplement because we did not observe myelination in standard DMEM medium (n = 16). This suggests that NGF either supports RGC survival or influences the Schwann cell-axon interaction during myelination (Urschel and Hulsebrosch, 1990). Medium was added every 3 days to the cultures, which were maintained in an oxygen-enriched atmosphere (5% COz, approximately 70% 02,balance nitrogen). Under these conditions dense outgrowth of RGC axons was observed after several days in vitro (Fig. 1). RGC neurites were visualized by incubation with mouse antineurofilament mAB (SMI-31,Sternberger & Meyer Immunocytochemicals,Jarretsville),followedbyfluoresceinconjugated secondary antibodies and viewed under epifluorescence illumination. Axotomized RGC axons in our cultures usually regenerate from the tips of the retinal explants, which correspond to the former optic disc region (Bahr et al., 1988). There, Schwann cells were often found to be arranged in linear arrays, and dense neuritic outgrowth could be observed (Fig. 2A). After 3 to 4 weeks in vitro, individual Schwann cells could be observed in close association with RGC axons (Fig. 2B), which is characteristic of ensheathment and myelination. To confirm that the ensheathed RGC axons were myelinated, the cultures (n = 24) were fixed Fig. 1. A: Phase contrast micrograph of a purified Schwann cell population, seeded on a collagen substrate. The Schwann cells were prepared from neonatal rat sciatic nerves and purified as described by Brockes et al. (1979). B: Only Schwann cells are present in this preparation, as demonstrated by immunostaining against the 217 C antigen, a cell surface marker specific for Schwann cells. C: Phase contrast micrograph of a Schwann cell monolayer, co-cultured with retinal explants from adult rats for 2 weeks. D: Immunostaining with anti-neurofilament antibodies reveals dense RGC axon growth extending from retinal explants. The axons always grow in close association with Schwann cell surfaces (arrow), avoiding the acellular collagen substrate. Scale bars = 20 p m for A and B and 50 pm for C and D, respectively.

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Fig. 2. A: After 3 weeks in vitro thick bundles of regenerating RGC neurites on Schwann cell surfaces can be observed. RGC axons are visualized by anti-neurofilament antibodies. B. Phase contrast micrograph showing Schwann cell ensheathment of regenerating RGC axons. The arrowhead indicates a Schwann cell nucleus; note the characteristic profile of the internodal region (flanking the nucleus) associated with the presence of myelin. Scale bars = 100 pm in A and 20 pm in B.

(2%glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, 80 mM sucrose), postfixed in 2% osmium tetroxide, dehydrated, and embedded in Polybed (Polysciences, Warrington, PA). Areas near the explant tips, where dense axon growth could be observed, were chosen for the presence of Schwann cell profiles (as seen in Fig. 2B), sectioned, stained with lead citrate and uranyl acetate, and viewed in a Phillips transmission electron microscope. RGC axons were found in different stages of

association with Schwann cells. Small axons were apposed to Schwann cell surfaces but were not ensheathed. Large axons were either ensheathed or myelinated by Schwann cells. Both ensheathed and myelinated axon-Schwann cell units were surrounded by a typical basal lamina. Myelinated axons were wrapped by varying numbers of lamellae with the characteristic features of peripheral myelin (n = 16,see Fig. 3). These results show that Schwann cells do not only

Fig. 3. Electron micrographs of adult retinaschwann cell co-cultures after 3 and 5 weeks in vitro. A. Three stages in the myelination of RGC axons by Schwann cells are illustrated: ensheathment (indicated by f?), formation of the first compact myelin lamellae (A-f?), and relatively mature compact myelin with many lamellae (bzl*).Note that axon-Schwann cell units are surrounded by basal lamina (arrow-

heads). Magnification XZ1,500. B: Two myelinated RGC axons are seen, both of which are surrounded by Schwann cell processes and a basal lamina after 5 weeks in vitro (arrowhead),In the upper right part of the electron micrograph single RGC axons are visible in close contact with Schwann cell surfaces. Magnification x30,OOO.

MYELINATION OF REGENERATING RETINAL GANGLION CELL AXONS

provide growth-promoting factors on their surfaces (Bahr and Bunge, 1989) but are also capable of ensheathing and myelinating these regenerating CNS axons in vitro. The understanding of the molecular mechanisms involved in myelination of regenerating (or demyelinated) CNS axons and the trophic support of CNS neurons by Schwann cells is important because Schwann cells might be used as cellular grafts in the damaged adult CNS (Bunge, 1975). This report describes a model system that allows examination of Schwann cell myelination and trophic support of adult CNS neurons in the relatively controlled environment of tissue culture. The regulation of neuronal survival after axotomy, especially in the CNS, remains an interesting and unresolved question. Following transection of the optic nerve in adult rats, approximately 90%of the RGC population present in the normal retina will die within 4 weeks (Bray et al., 1987). The presence of a segment of sciatic nerve, grafted to the transected optic nerve stump, supports survival of 2040% of the original RGC population (Berry et al., 1989; Bray et al., 1987). Interestingly, Maffei et al. (1990) have shown that intraocular injection of viable Schwann cells after optic nerve transection in adult rats can result in a four-fold increase in surviving RGCs, as judged by retrograde transport of horseradish peroxidase (HRP). Our studies suggest that the ability of Schwann cells to rescue injured RGC neurons in vitro may be mediated by physical contact rather than by soluble factors, since Schwann-cell-conditionedmedium did not support longterm survival of adult rat RGCs cultured on acellular substrates (Bahr and Bunge, 1989). Even in the presence of viable Schwann cells or astrocytes (Bahr and Bunge, 1990)it was not possible to achieve considerable RGC survival for more than 2 weeks in vitro, unless the chemically defined medium was substituted by E l 5 medium. This medium allows ensheathment and myelination of RGC axons by Schwann cells. Therefore, extended RGC survival in our system appears to be mediated by contact between Schwann cells and RGC axons. This is consistent with earlier observations on the maintenance of dorsal root ganglion neurons by Schwann cells in vitro. There it could be demonstrated that even in the presence of NGF, direct contact between sensory neurons grown in culture and Schwann cells was necessary for trophic support of the neuronal soma (Bunge et al., 1988).Although the nature of the trophic support for adult CNS neurons by Schwann cells is unknown, factors other than NGF seem to be involved (Bahr et al., 1989; Thanos et al., 1989). On the other hand NGF seems to be an important co-factor for ensheathment and myelination in our culture system. Interestingly, it has recently been reported that NGF not only acts as a neurotrophic molecule but might also be important for the initiation and progress of normal myelination by Schwann cells (Urschel and Hulsebrosch, 1990). Therefore, NGF might be required as a co-factor for Schwann cell myelination of regenerating adult RGC axons. Since we have now shown that axotomized, regenerating adult rat RGCs can be maintained in co-cultures

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with Schwann cells for periods up to 12 weeks, it should be possible to use this in vitro system to characterize the factors produced by Schwann cells that regulate the survival of adult CNS neurons after axotomy. ACKNOWLEDGMENTS This study was supported by the Max-Planck society (M.B.), NIH postdoctoral fellowship EY 06073-02 (J.M.H.), and NS 09923 (R.P.B.). The authors thank Ulrike Kirsch, Mrs. Zimmermann, and Margaret Bates for technical help. M. Bahr thanks Drs. J. Dichgans and F. Bonhoeffer for supporting his work. REFERENCES Bahr, M. and Bunge, R.P. (1989) Functional status influences the ability of Schwann cells to support adult rat retinal ganglion cell survival and axonal regrowth. Ezp. Neurol., 106:27--10. Bahr, M. and Bunge, R.P. (1990) Growth of adult rat retinal ganglion cell neurites on astrocytes. Gliu, 3:293-300. Bahr, M., Vanselow, J., and Thanos, S. (1988) In vitro regeneration of adult rat ganglion cell axons from retinal explants. Exp. Brain Res., 73:393-401. Bahr, M., Vanselow, J., and Thanos, S. (1989) Ability of adult rat ganglion cells to regrow axons in vitro can be influenced by fibroblast growth factor and gangliosides. Neurosci. Lett., 96:197-201. Berry, M., Rees, L., Hall, S., Yiu, P., and Sievers, J . (1988)Optic axons regenerate into sciatic nerve isografts only in the presence of Schwann cells. Brain Res. Bull., 20:223-231. Bray, G.M., Villegaz-Perez, M.P.,Vidal-Sanz, M., and Aguayo, A.J. (1987) The use of peripheral nerve grafts to enhance neuronal survival, promote growth and permit terminal reconnection in the central nervous system of adult rats. J.Ezp. Biol., 1325-19. Brockes, J.P., Fields, K.L., and Raff, M.C. (1979) Studies on cultured rat Schwann cells. I. Establishment of purified populations from cultures of peripheral nerves. Brain Res., 165:105-118. Bunge, R.P. (1975) Changing uses of nerve tissue culture 1950-1975. In: The Nervous System, D.B. Tower, ed. Vol. I: The Basic Neurosciences. Raven Press, New York. Bunge, R.P., Eldridge, C.F., Ard, M.D., and Kleitman, N. (1988) Schwann cell contact as a factor in neuronal trophic support and the promotion of neurite growth. In: Neurobiology of Amino Acids, Peptides and Trophic Factors. J.A. Ferendelli, R.C. Collins, and E.M. Johnson, eds. Fluwer Academic Publishers, pp. 115-126. David, S. and Aguayo, A.J. (1981) Axonal elongation into peripheral nervous system bridges after central nervous injury - - in adult rats. Science, 2413931-933. Eldridrre. C.F.. Bunae. M.B.. Bunrre. R.P.. and Wood, P.M. (1987) Diff&entiation of %on-related Sihwann'cells in vitro. I. Ascorbic acid regulates basal lamina assembly and myelin formation. J. Cell Biol., 1051023-1034. Eldridge, C.F., Bunge, M.B., and Bunge, R.P. (1989) Differentiation of axon-related Schwann cells in vitro 11: Control of myelin formation by basal lamina. J.Neurosci., 9:625-638. Fields, KL. and Dammermann, M. (1985) A monoclonal antibody equivalent to anti-rat antigen 1 as a marker for Schwann cells. Neuroscience, 15877-885. Ford-Holevinski,T.S., Hopkins, J.M., McCoy, J.P., and Agranoff, B.W. (1986) Laminin supports neurite outgrowth from explants of adult retinal neurons. Deu. Brain Res., 28:121-126. Hopkins, J.M. and Bunge, R.P. (1991)Regeneration of s o n s from adult rat retinal ganglion cells on cultured Schwann cells is not dependent on basal lamina. Glia, 4:46-55. Maffei, L., Carmignoto, G., Perry, V.H., Candeo, P., and Ferrari, G. (1990) Schwann cells promote the survival of rat retinal ganglion cells after optic nerve section. Proc. Nutl. Acad. Sci. USA, 87~1855-1859. Thanos, S., Bahr, M., Barde, Y.-A., and Vanselow, J. (1989) Survival and axonal elongation of adult rat retinal ganglion cells: In vitro effects of lesioned sciatic nerve brain derived neurotrophic factor. Eur. J.Neurosci., 1:19-26. Urschel, B.A. and Hulsebrosch, C.E. (1990) Schwann cell-neuronal interactions in the rat involve nerve growth factor. J.Comp. Neurol., 296:114-122.

In vitro myelination of regenerating adult rat retinal ganglion cell axons by Schwann cells.

Schwann cell cultures provide a highly favorable substrate for retinal ganglion cell (RGC) survival and axon growth in vitro (Bähr and Bunge, Exp Neur...
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