Imaging myelinated nerve fibres by confocal fluorescence microscopy: individual fibres in whole nerve trunks traced through multiple consecutive internodes

Abstract

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.