Summary
Current methods of morphological analysis do not permit detailed imaging of individual myelinated fibres over substantial lengths without disruption of neighbouring, potentially significant, cellular and extracellular relationships. We report a new method which overcomes this limitation by combining aldehyde-induced fluorescence with confocal microscopy. Myelin fluorescence was intense relative to that from other tissue components, enabling individual myelinated nerve fibres to be traced for distances of many millimeters in whole PNS nerve trunks. Images obtained with a Bio-Rad MRC-600 confocal laser scanning microscope clearly displayed features of PNS and CNS myelinated fibres including nodes of Ranvier; fibre diameter; sheath thickness and contour; branch points at nodes; as well as (in the PNS) Schmidt-Lanterman incisures and the position of Schwann cell nuclei. Direct comparisons using the same specimens (whole nerve trunks; also teased fibres) showed confocal imaging to be markedly superior to conventional fluorescence microscopy in terms of contrast, apparent resolution and resistance to photobleaching. Development of the fluorophore was examined systematically in sciatic nerves of young adult rats. In separate experiments, animals were perfused systemically using (1) 5% glutaraldehyde; (2) Karnovsky's solution; (3) 4% paraformaldehyde; buffered with either 0.1 M sodium phosphate or sodium cacodylate (pH 7.4). The concentration of glutaraldehyde in the fixative solution was the principal determinant of fluorescence intensity. Confocal imaging was achieved immediately following perfusion with 5% glutaraldehyde or Karnovsky's. Fluorescence intensity increased markedly during overnight storage in these fixatives and continued to increase during subsequent storage in buffer alone. The fluorophore was stable and resistant to fading during storage (15 months at least), enabling data collection over extended periods. To demonstrate application of the method in neuropathology, individual fibres in transected sciatic nerve trunks were traced through multiple successive internodes: Classical features of Wallerian degeneration (axonal swelling and debris; ovoid formation and incisure changes; variation among fibres in the extent of degeneration) were displayed. The method is compatible with subsequent ultrastructural examination and will complement existing methods of investigation of myelinated fibre anatomy and pathology, particularly where preservation of 3-dimensional relationships or elucidation of spatial gradients are required.
Similar content being viewed by others
References
Bacallao, R., Bomsel, M., Stelzer, E. H. K. &De Mey, J. (1989) Guiding principles of specimen preservation for confocal fluorescence microscopy. In:The Handbook of Biological Confocal Microscopy (edited byPawley, J.) pp. 181–7. Madison: IMR Press.
Bacallao, R. &Stelzer, E. H. K. (1989) Preservation of biological specimens for observation in a confocal fluorescence microscope and operational principles of confocal fluoresence microscopy.Methods in Cell Biology 31, 437–52.
Björklund, A. (1983) Fluorescence histochemistry of biogenic monoamines. In:Handbook of Chemical Neuroanatomy Vol. 1.Methods in Chemical Neuroanatomy (edited byBjörklund, A. &Hokfelt, T.) pp. 50–121. Oxford: Elsevier.
Brakenhoff, G. J., Vandervoort, H. T. M., Van Spronsen, E. A. &Nanninga, N. (1986) Three-dimensional imaging by confocal scanning fluorescence microscopy.Annals of the New York Academy of Sciences 483, 405–15.
Bürge, P. (1987) Plastic and reconstructive surgery: peripheral nerve injuries.Hospital Update 13, 525–36.
Chaudhry, V. &Cornblath, D. R. (1992) Wallerian degeneration in human nerves: serial electrophysiological studies.Muscle and Nerve 15, 687–93.
Dyck, P. J., Giannini, C. &Lais, A. (1993) Pathologic alterations of nerves. In:Peripheral Neuropathy Vol. 1.3rd Edition (edited byDyck, P. J., Thomas, P. K., Griffin, J. W., Low, P. A. &Poduslo, J. F.) pp. 514–95. Philadelphia: W. B. Saunders.
Elberger, A. J. &Honig, M. G. (1990) Double-labelling of tissue containing the carbocyanine dye Dil for immunocytochemistry.The Journal of Histochemistry and Cytochemistry 38, 735–9.
Fraher, J. P. (1978a) Quantitative studies on the maturation of central and peripheral parts of individual ventral motoneuron axons.Journal of Anatomy 126, 509–33.
Fraher, J. P. (1978b) Quantitative studies on the maturation of central and peripheral parts of individual ventral motoneuron axons II. Internodal length.Journal of Anatomy 127, 1–15.
Friede, R. L., Meier, T. &Diem, M. (1981) How is the exact length of an internode determined?Journal of the Neurological Sciences 50, 217–28.
Furness, J. B., Heath, J. W. &Costa, M. (1978) Aqucous aldehyde (Faglu) methods for the fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue.Histochemistry 57, 285–95.
Gabreëls-Festen, A. A. W. M., Joosten, E. M. G., Gabreëls, F. J. M., Jennekens, F. G. I. &Janssen-Van Kempen, T. W. (1991) Early morphological features in dominantly inherited demyelinating motor and sensory neuropathy (HMSN type I).Journal of the Neurological Sciences 107, 145–54.
Gabreëls-Festen, A. A. W. M., Joosten, E. M. G., Gabreëls, F. J. M., Stegeman, D. F., Vos, A. J. M. &Busch, H. F. M. (1990) Congenital demyelinating motor and Sensory neuropathy with focally folded myelin sheaths.Brain 113, 1629–43.
Ghabriel, M. N. &Allt, G. (1979) The role of Schmidt-Lanterman incisures in Wallerian degeneration I. A quantitative teased fibre study.Acta Neuropathologica (Berlin) 48, 83–93.
Ghabriel, M. N. &Allt, G. (1981) Incisures of Schmidt-Lanterman.Progress in Neurobiology 17, 25–58.
Griffin, J. W. &Huffman, P. N. (1993) Degeneration and regeneration in the peripheral nervous system. In:Peripheral Neuropathy Vol. 1.3rd Edition (edited byDyck, P. J., Thomas, P. K., Griffin, J. W., Low, P. A. &Poduslo, J. F.) pp. 361–76. Philadelphia: W B Saunders.
Hayat, M. A. (1989)Principles and Techniques of Electron Microscopy Biological Applications. 3rd Edition. London: Macmillan Press.
Hirano, A. &Dembitzer, H. M. (1967) A structural analysis of the myelin sheath in the central nervous system.Journal of Cell Biology 34, 555–67.
Kidd, G. J. &Heath, J. W. (1988a) Double myelination of axons in the sympathetic nervous system of the mouse. I. Ultrastructural features and distribution.Journal ofNeurocytology 17, 245–61.
Kidd, G. J. &Heath, J. W. (1988b) Double myelination of axons in the sympathetic nervous system of the mouse. II. Mechanisms of formation.Journal of Neurocytology 17, 263–76.
Lubinska, L. (1977) Early course of Wallerian degeneration in myelinated fibres of the rat phrenic nerve.Brain Research 130, 47–63.
Lunn, E. R., Brown, M. C. &Perry, V. H. (1990) The pattern of axonal degeneration in the peripheral nervous system varies with different types of lesion.Neuroscience 35, 157–65.
Malbouisson, A. M. B., Ghabriel, M. N. &Allt, G. (1984) The non-directional pattern of axonal changes in Wallerian degeneration: a computer-aided morphometric analysis.Journal of Anatomy 139, 159–74.
Mårtensson, R. &Björklund, A. (1984) Low-power photography in the fluorescence microscope using an automatic dark-field condenser-scanner. In:Handbook of Chemical Neuroanatomy Vol. 2: Classical Transmitters in the CNS, Part 1 (edited byBjörklund, A. &Hokfelt, T.) pp. 380–6. Oxford: Elsevier.
Murray, J. M. (1992) Neuropathology in depth: the role of confocal microscopy.Journal of Neuropathology and Experimental Neurology 51, 475–87.
Ohi, T., Kyle, R. A. &Dyck, P. J. (1985) Axonal attenuation and secondary segmental demyelination in myeloma neuropathies.Annals of Neurology 17, 255–61.
Ormerod, M. G. &Imrie, S. F. (1989) Immunohistochemistry. In:Light Microscopy in Biology: A Practical Approach (edited byLacey, A. J.) pp. 103–36. Oxford: IRL Press.
Ploem, J. S. (1989) Fluorescence Microscopy. In:Light Microscopy In Biology: A Practical Approach (edited byLacey, A. J.) pp. 163–85. Oxford: IRL Press.
Reynolds, R. J., Heath, J. W. &Little, G. J. (1993) Visualising individual myelinated nerve fibres by confocal fluorescence microscopy.Abstract, The 1993 International Conference On Confocal Microscopy & 3D Image Processing, Sydney, p. 134.
Rost, F. W. D. (1992)Fluorescence Microscopy Vol. 1. Sydney: Cambridge University Press.
Seckel, B. R. (1990) Enhancement of peripheral nerve regeneration.Muscle and Nerve 13, 785–800.
Sobue, G., Doyu, M., Watanabe, M., Hayashi, F. &Mitsuma, T. (1992) Extensive demyelinating changes in the peripheral nerves of Crow-Fukase syndrome: a pathological study of one autopsied case.Acta Neuropathologica 84, 171–7.
Spencer, P. S. &Thomas, P. K. (1970) The examination of isolated nerve fibres by light and electron microscopy with observations on demyelination proximal to neuromas.Acta Neuropathologica 16, 177–86.
Stämpfli, R. (1981) Ultrastructural observations on nodes of Ranvier from isolated single frog peripheral nerve fibres.Brain Research 215, 93–101.
Sugimura, K., Takahashi, A., Watanabe, M., Mano, K. &Watanabe, H. (1990) Demyelinating changes in sural nerve biopsy of patients with HTLV-I-associated myelopathy.Neurology 40, 1263–6.
Tuisku, F. &Hildebrand, C. (1992) Nodes of Ranvier and myelin sheath dimensions along exceptionally thin myelinated vertebrate PNS axons.Journal of Neurocytology 21, 796–806.
Waxman, S. G., Pappas, G. D. &Bennett, M. V. L. (1972) Morphological correlates of functional differentiation of nodes of Ranvier along single fibers in the neurogenic electric organ of the knife fish‘sternarchus’.Journal of Cell Biology 53, 210–24.
White, J. G., Amos, W. B. &Fordham, M. (1987) An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy.Journal of Cell Biology 105, 41–8.
Wright, S. J. &Schatten, G. (1991) Confocal fluorescence microscopy and three-dimensional reconstruction.Journal of Electron Microscope Techniques 18, 2–10.
Wright, S. J., Walker, J. S., Schatten, H., Simerly, C., Mccarthy, J. J. &Schatten, G. (1989) Confocal fluorescence microscopy with the tandem scanning light microscope.Journal of Cell Science 94, 617–24.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Reynolds, R.J., Little, G.J., Lin, M. et al. Imaging myelinated nerve fibres by confocal fluorescence microscopy: individual fibres in whole nerve trunks traced through multiple consecutive internodes. J Neurocytol 23, 555–564 (1994). https://doi.org/10.1007/BF01262056
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01262056