Journal of Neurocytology

, Volume 23, Issue 7, pp 433–452 | Cite as

Regrowth of axons in lesioned adult rat spinal cord: promotion by implants of cultured Schwann cells

  • C. L. Paíno
  • C. Fernandez-Valle
  • M. L. Bates
  • M. B. Bunge
Article
Received:
Revised:
Accepted:

Summary

Highly purified populations of Schwann cells were grafted into lesioned adult rat spinal cord to determine if they promote axonal regeneration. Dorsal spinal cord lesions were created by a photochemical lesioning technique. Schwann cells derived from E16 rat dorsal root ganglia, either elongated and associated with their extracellular matrix or dissociated and without matrix, were rolled in polymerized collagen to form an implant 4–6 mm long which was grafted at 5 or 28 days after lesioning. No immunosuppression was used. Acellular collagen rolls served as controls. At 14, 28 and 90 days and 4 and 6 months after grafting, animals were analysed histologically with silver and Toluidine Blue stains and EM. The grafts often filled the lesion and the host borders they apposed exhibited only limited astrogliosis. By 14 days, bundles of unmyelinated and occasional thinly myelinated axons populated the periphery of Schwann cell implants. By 28 days and thereafter, numerous unmyelinated and myelinated axons were present in most grafts. Silver staining revealed sprouted axons at the implant border at 28 days and long bundles of axons within the implant at 90 days. Photographs of entire 1 μm plastic cross-sections of nine grafted areas were assembled into montages to count the number of myelinated axons at the graft midpoint; the number of myelinated axons ranged from 517–3214. Electron microscopy of implants showed typical Schwann cell ensheathment and myelination, increased myelin thickness by 90 days, and a preponderance of unmyelinated over myelinated axons. Random EM sampling of five Schwann cell grafts snowed that the ratio of unmyelinated to myelinated axons was highest (20∶1) at 28 days. These ratios implied that axons numbered in the thousands at the graft midpoint. Dissociated Schwann cells without matrix promoted axonal ingrowth and longitudinal orientation as effectively as did elongated Schwann cells accompanied by matrix. There was a suggestion that axonal ingrowth was at least as successful, if not more so, when the delay between lesioning and grafting was 28 rather than 5 days. Acellular collagen grafts did not contain axons at 28 days, the only interval assessed. In sum, grafts of Schwann cells in a rolled collagen layer filled the lesion and were well tolerated by the host. The Schwann cells stimulated rapid and abundant growth of axons into grafts and they ensheathed and myelinated these axons in the normal manner.

Keywords

Schwann Cell Myelinated Axon Spinal Cord Lesion Dorsal Spinal Cord Culture Schwann Cell 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
This is a preview of subscription content, log in to check access.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Acheson, A., Barker, P. A., Alderson, R. F., Miller, F. D. &Murphy, R. A. (1991) Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF.Neuron 7, 265–75.Google Scholar
  2. Aebischer, P., Guénard, V., Winn, S. R., Valentini, R. F. &Galletti, P. M. (1988) Semi-permeable guidance channels allow peripheral nerve regeneration in the absence of a distal nerve stump.Brain Research 454, 179–87.Google Scholar
  3. Aebischer, P., Guénard, V. &Brace, S. (1989) Peripheral nerve regeneration through semipermeable guidance channels: effect of the molecular weight cut-off.Journal of Neuroscience,9, 3590–5.Google Scholar
  4. Aebischer, P., Guénard, V. &Valentini, R. F. (1990) The morphology of regenerating peripheral nerves is modulated by the surface microgeometry of polymeric guidance channels.Brain Research 351, 211–18.Google Scholar
  5. Aguayo, A. J. (1985) Axonal regeneration from injured neurons in the adult mammalian central nervous system. In:Synaptic Plasticity (edited byCotman, C. W.), pp. 457–84. New York: Guilford Press.Google Scholar
  6. Bandtlow, C. E., Heumann, R., Schwab, M. E. &Thoenen, H. (1987) Cellular localization of nerve growth factor synthesis byin situ hybridization.EMBO Journal 6, 891–9.Google Scholar
  7. Barde, Y.-A. (1989) Trophic factors and neuronal survival.Neuron 2, 1525–34.Google Scholar
  8. Berry, M., Hall, S., Follows, R., Rees, L., Gregson, N. &Sievers, J. (1988) Response of axons and glia at the site of anastomosis between the optic nerve and cellular or acellular sciatic nerve grafts.Journal of Neurocytology 17, 727–44.Google Scholar
  9. Bixby, J. L. &Harris, W. A. (1991) Molecular mechanisms of axon growth and guidance.Annual Review of Cell Biology 7, 117–59.Google Scholar
  10. Bixby, J. L., Lilien, J. &Reichardt, L. F. (1988) Identification of the major proteins that promote neuronal process outgrowth on Schwann cellsin vitro.Journal of Cell Biology 107, 353–61.Google Scholar
  11. Bunge, M. B. (1993) Schwann cell regulation of extracellular matrix biosynthesis and assembly. In:Peripheral Neuropathy (3rd Edition), (edited byDyck, P. J., Thomas, P. K., Griffin, J., Low, P. A. &Poduslo, J.). pp. 299–316. Philadelphia: W. B. Saunders.Google Scholar
  12. Bunge, M. B., Johnson, M. I., Ard, M. D. &Kleitman, N. (1987) Factors influencing the growth of regenerating nerve fibers in culture.Progress in Brain Research 71, 61–74.Google Scholar
  13. Bunge, M. B., Bunge, R. P., Kleitman, N. &Dean, A. C. (1989) Role of peripheral nerve extracellular matrix in Schwann cell function and in neurite regeneration.Developmental Neuroscience 11, 348–60.Google Scholar
  14. Bunge, M. B., Holets, V. R., Bates, M. L., Clarke, T. S. &Watson, B. D. (1993) Characterization of photochemically induced spinal cord injury in the rat by light and electron microscopy.Experimental Neurology (in press).Google Scholar
  15. Bunge, R. P. (1975) Changing uses of nerve tissue culture 1950–1975. In:The Nervous System. Vol.1: The Basic Neurosciences (edited byTower, D. B.), pp. 31–42. New York: Raven Press.Google Scholar
  16. Bunge, R. P., Bunge, M. B. &Eldridge, C. F. (1986) Linkage between axonal ensheathment and basal lamina production by Schwann cells.Annual Review of Neuroscience 9, 305–28.Google Scholar
  17. Cameron, T., Prado, R., Watson, B. D., Gonzalez-Carvajal, M. &Holets, V. R. (1990) Photochemically induced cystic lesion in the rat spinal cord. I. Behavioral and morphological analysis.Experimental Neurology 109, 214–23.Google Scholar
  18. Campenot, R. B. (1977) Local control of neurite development by nerve growth factor.Proceedings of the National Academy of Sciences (USA) 74, 4516–19.Google Scholar
  19. Daniloff, J. K., Levi, G., Grumet, M., Rieger, F. &Edelman, G. M. (1986) Altered expression of neuronal cell adhesion molecules induced by nerve injury and repair.Journal of Cell Biology 103, 929–45.Google Scholar
  20. Davis, G. E., Varon, S., Engvall, E. &Manthorpe, M. (1985) Substratum binding neurite-promoting factors: relationship to laminin.Trends in Neurosciences 8, 528–32.Google Scholar
  21. Eldridge, C. F., Bunge, M. B., Bunge, R. P. &Wood, P. M. (1987) Differentiation of axon-related Schwann cellsin vitro. I. Ascorbic acid regulates basal lamina assembly and myelin formation.Journal of Cell Biology 105, 1023–34.Google Scholar
  22. Franklin, R. J. M., Crang, A. J. &Blakemore, W. F. (1992) The behaviour of meningeal cells following glial cell transplantation into chemically-induced areas of demyelination in the CNS.Neuropathology and Applied Neurobiology 18, 189–200.Google Scholar
  23. Garcia-Rill, E., Houle, J. D., Reese, N. B. &Skinner, R. D. (1992) Modulation of locomotion by a nerve graft across a spinal transection in rat.Restorative Neurology and Neuroscience 4, 419–24.Google Scholar
  24. Guénard, V., Morrissey, T. K., Kleitman, N., Bunge, R. P. &Aebischer, P. (1991) Cultured syngeneic adult Schwann cells seeded in synthetic guidance channels enhance sciatic and optic nerve regeneration.Society for Neuroscience Abstracts 17, 565.Google Scholar
  25. Guénard, V., Kleitman, N., Morrissey, T. K., Bunge, R. P. &Aebischer, P. (1992) Syngeneic Schwann cells derived from adult nerves seeded in semipermeable guidance channels enhance peripheral nerve regeneration.Journal of Neuroscience 12, 3310–20.Google Scholar
  26. Guénard, V., Xu, X. M. &Bunge, M. B. (1993) The use of Schwann cell transplantation to foster central nervous system repair.Seminars in the Neurosciences (in press).Google Scholar
  27. Gundersen, R. W. &Barrett, J. N. (1980) Characterization of the turning response of dorsal root neurites toward nerve growth factor.Journal of Cell Biology 87, 546–54.Google Scholar
  28. Hall, S. &Berry, M. (1989) Electron microscopic study of the interaction of axons and glia at the site of anastomosis between the optic nerve and cellular and acellular sciatic nerve grafts.Journal of Neurocytology 18, 171–84.Google Scholar
  29. Heumann, R., Korsching, S., Bandtlow, C. &Thoenen, H. (1987) Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection.Journal of Cell Biology 104, 1623–31.Google Scholar
  30. Hopkins, J. M. &Bunge, R. P. (1991a) Regeneration of axons from adult rat retinal ganglion cells on cultured Schwann cells is not dependent on basal lamina.Glia 4, 46–55.Google Scholar
  31. Hopkins, J. M. &Bunge, R. P. (1991b) Regeneration of axons from adult human retinain vitro.Experimental Neurology 112, 243–51.Google Scholar
  32. Hopkins, J. M., Schachner, M. &Bunge, R. P. (1989) Antibodies against the cell adhesion molecule L1 do not block growth of adult rat retinal ganglion cell axons on Schwann cells.Society for Neuroscience Abstracts 15, 331.Google Scholar
  33. Houle, J. D. (1991) Demonstration of the potential for chronically injured neurons to regenerate axons into intraspinal peripheral nerve grafts.Experimental Neurology 113, 1–9.Google Scholar
  34. Kaufman, L. M. &Barrett, J. N. (1983) Serum factor supporting long-term survival of rat central neurons in culture.Science 220, 1394–6.Google Scholar
  35. Kleitman, N., Wood, P., Johnson, M. I. &Bunge, R. P. (1988a) Schwann cell surfaces but not extracellular matrix organized by Schwann cells support neurite outgrowth from embryonic rat retina.Journal of Neuroscience 8, 653–63.Google Scholar
  36. Kleitman, N., Simon, D. K., Schachner, M. (1988b) Growth of embryonic retinal neurites elicited by contact with Schwann cell surfaces is blocked by antibodies to L1.Experimental Neurology 102, 298–306.Google Scholar
  37. Kleitman, N., Wood, P. M. &Bunge, R. P. (1991) Tissue culture methods for the study of myelination. In:Neuronal Cell Culture (edited byBanker, G. A. &Goslin, K.) pp. 337–77. Boston: MIT Press.Google Scholar
  38. Kromer, L. F. &Cornbrooks, C. J. (1985) Transplants of Schwann cell cultures promote axonal regeneration in the adult mammalian brain.Proceedings of the National Academy of Sciences (USA) 82, 6330–4.Google Scholar
  39. Kromer, L. F. &Cornbrooks, C. J. (1987) Identification of trophic factors and transplanted cellular environments that promote CNS axonal regeneration.Annals of the New York Academy of Sciences 495, 207–23.Google Scholar
  40. Kuffler, D. P. (1986) Isolated satellite cells of a peripheral nerve direct the growth of regenerating frog axons.Journal of Comparative Neurology 249, 57–64.Google Scholar
  41. Kuhlengel, K. R., Bunge, M. B. &Bunge, R. P. (1990) Implantation of cultured sensory neurons and Schwann cells into lesioned neonatal rat spinal cord. I. Methods for preparing implants from dissociated cells.Journal of Comparative Neurology 293, 63–73.Google Scholar
  42. Leoz-Ortín, G. &Arcaute, L. R. (1913) Procesos regenerativos del nervio óptico y retina con ocasión de ingertos nerviosos.Trabajos del Laboratorio de Investigaciones Biológicas de la Universidad de Madrid 11, 239–54.Google Scholar
  43. Lu, X. &Richardson, P. M. (1991) Inflammation near the nerve cell body enhances axonal regeneration.Journal of Neuroscience 11, 972–8.Google Scholar
  44. Martin, D., Schoenen, J., Delrée, P., Leprince, P., Rogister, B. &Moonen, G. (1991) Grafts of syngeneic cultured, adult dorsal root ganglion-derived Schwann cells to the injured spinal cord of adult rats: preliminary morphological studies.Neuroscience Letters 124, 44–8.Google Scholar
  45. Mirsky, R. &Jessen, K. R. (1990) Schwann cell development and the regulation of myelination.Seminars in the Neurosciences 2, 423–35.Google Scholar
  46. Montgomery, C. T. &Robson, J. A. (1990) New method of transplanting purified glial cells into the brain.Journal of Neuroscience Methods 32, 135–41.Google Scholar
  47. Montgomery, C. T. &Robson, J. A. (1993) Implants of cultured Schwann cells support axonal growth in the central nervous system of adult rats.Experimental Neurology 122, 107–24.Google Scholar
  48. Moya, F., Bunge, M. B. &Bunge, R. P. (1980) Schwann cells proliferate but fail to differentiate in defined medium.Proceedings of the National Academy of Sciences (USA) 77, 6902–6.Google Scholar
  49. Nieke, J. &Schachner, M. (1985) Expression of the neural cell adhesion molecules L1 and N-CAM and their common carbohydrate epitope L2/HNK-1 during development and after transection of the mouse sciatic nerve.Differentiation 30, 141–51.Google Scholar
  50. Nornes, H., Björklund, A. &Stenevi, U. (1984) Transplantation strategies in spinal cord regeneration. In:Neural Transplants (edited bySladek, J. R. Jr &Gash, D. M.) pp. 407–21. New York: Plenum Press.Google Scholar
  51. Paíno, C. L. &Bunge, M. B. (1990) Axon growth into implants of Schwann cells placed in lesioned spinal cord.Society for Neuroscience Abstracts 16, 1282.Google Scholar
  52. Paíno, C. L. &Bunge, M. B. (1991) Induction of axon growth into Schwann cell implants grafted into lesioned adult rat spinal cord.Experimental Neurology 114, 254–7.Google Scholar
  53. Paíno, C. L., Fernandez-Valle, C. &Bunge, M. B. (1991) Axon growth into Schwann cell grafts placed in lesioned adult rat spinal cord.Society for Neuroscience Abstracts 17, 236.Google Scholar
  54. Porter, S., Clark, M. B., Glaser, L. &Bunge, R. P. (1986) Schwann cells stimulated to proliferate in the absence of neurons retain full functional capability.Journal of Neuroscience 6, 3070–8.Google Scholar
  55. Ramön Y Cajal, S. (1928)Degeneration and Regeneration of the Nervous System. May, R. M. (Translator), London: Hafner Publishing Co.Google Scholar
  56. Rende, M., Muir, D., Ruoslahti, E., Hagg, T., Varon, S. &Manthorpe, M. (1992) Immunolocalization of ciliary neuronotrophic factor in adult rat sciatic nerve.Glia 5, 25–32.Google Scholar
  57. Richardson, P. M., Mcguinness, U. M. &Aguayo, A. J. (1980) Axons from CNS neurones regenerate into PNS grafts.Nature 284, 264–5.Google Scholar
  58. Richardson, P. M., Mcguinness, U. M. &Aguayo, A. J. (1982) Peripheral nerve autografts to the rat spinal cord: studies with axonal tracing methods.Brain Research 237, 147–62.Google Scholar
  59. Richardson, P. M., Issa, V. M. K. &Aguayo, A. J. (1984) Regeneration of long spinal axons in the rat.Journal of Neurocytology 13, 165–82.Google Scholar
  60. Schachner, M. (1990) Functional implications of glial cell recognition molecules.Seminars in the Neurosciences 2, 497–507.Google Scholar
  61. Schnell, L. &Schwab, M. E. (1990) Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors.Nature 343, 269–72.Google Scholar
  62. Sevier, A. E. &Munger, B. L. (1965) A silver method for paraffin sections of neural tissue.Journal of Neuropathology and Experimental Neurology 24, 130–5.Google Scholar
  63. Smith, G. V. &Stevenson, J. A. (1988) Peripheral nerve grafts lacking viable Schwann cells fail to support central nervous system axonal regeneration.Experimental Brain Research 69, 299–306.Google Scholar
  64. Taniuchi, M., Clark, H. B. &Johnson, E. M. Jr. (1986) Induction of nerve growth factor receptor in Schwann cells after axotomy.Proceedings of the National Academy of Sciences (USA) 83, 4094–8.Google Scholar
  65. Tello, F. (1911) La influencia del neurotropismo en la regeneración de los centros nerviosos.Trabajos del Laboratorio de Investigaciones Biológicas de la Universidad de Madrid 9, 123–59.Google Scholar
  66. Uzman, B. G. &Villegas, G. M. (1983) Mouse sciatic nerve regeneration through semipermeable tubes: a quantitative model.Journal of Neuroscience Research 9, 325–38.Google Scholar
  67. Venstrom, K. A. &Reichardt, L. F. (1993) Extracellular matrix 2: Role of extracellular matrix molecules and their receptors in the nervous system.FASEB Journal 7, 996–1003.Google Scholar
  68. Watson, B. D., Holets, V. R., Prado, R. &Bunge, M. B. (1993) Laser-driven photochemical induction of spinal cord injury in the rat: methodology, histopathology, and applications.Neuroprotocols 3, 3–15.Google Scholar
  69. Wood, P. M. (1976) Separation of functional Schwann cells and neurons from normal peripheral tissue.Brain Research 115, 361–75.Google Scholar
  70. Wrathall, J. R., Rigamonti, D. D., Braford, M. R. &Kao, C. C. (1982) Reconstruction of the contused cat spinal cord by the delayed nerve cell graft technique and cultured peripheral non-neuronal cells.Acta Neuropathologica 57, 59–69.Google Scholar
  71. Xu, X. M., Guénard, V., Kleitman, N. &Bunge, M. B. (1992) Axonal growth into Schwann cell-seeded guidance channels grafted into transected adult rat spinal cord.Society for Neuroscience Abstracts 18, 1479.Google Scholar
  72. Xu, X. M., Guénard, V., Chen, A., Kleitman, N. &Bunge, M. B. (1993a) Rostral and caudal axonal regeneration into Schwann cell-seeded guidance channels grafted into a gap in adult rat spinal cord.Society for Neuroscience Abstracts 19, 681.Google Scholar
  73. Xu, X. M., Guénard, V., Kleitman, N. &Bunge, M. B. (1993b) Axonal regeneration into Schwann cell-seeded guidance channels grafted into transected adult rat spinal cord. (Submitted)Google Scholar

Copyright information

© Chapman and Hall 1994

Authors and Affiliations

  • C. L. Paíno
    • 1
  • C. Fernandez-Valle
    • 1
    • 2
  • M. L. Bates
    • 1
  • M. B. Bunge
    • 1
    • 3
  1. 1.The Chambers Family Electron Microscopy Laboratory, The Miami Project to Cure ParalysisUniversity of Miami School of MedicineMiamiUSA
  2. 2.The Department of Neurological SurgeryUniversity of Miami School of MedicineMiamiUSA
  3. 3.Department of Cell Biology and AnatomyUniversity of Miami School of MedicineMiamiUSA

Personalised recommendations