IMAGING MYELINATED NERVE-FIBERS BY CONFOCAL FLUORESCENCE MICROSCOPY -INDIVIDUAL FIBERS IN WHOLE NERVE TRUNKS TRACED THROUGH MULTIPLE CONSECUTIVE INTERNODES

Current methods of morphological analysis do not permit detailed imagi ng of individual myelinated fibres over substantial lengths without di sruption of neighbouring, potentially significant, cellular and extrac ellular relationships. We report a new method which overcomes this lim itation by combining aldehyde-induced fluorescence with confocal micro scopy. Myelin fluorescence was intense relative tb that from other tis sue components, enabling individual myelinated nerve fibres to be trac ed for distances of many millimeters in whole PNS nerve trunks. Images obtained with a Bio-Rad MRC-600 confocal laser scanning microscope cl early displayed features of PNS and CNS myelinated fibres including no des of Ranvier; fibre diameter; sheath thickness and contour; branch p oints at nodes; as well as (in the PNS) Schmidt-Lanterman incisures an d the position of Schwann cell nuclei. Direct comparisons using the sa me specimens (whole nerve trunks; also teased fibres) showed confocal imaging to be markedly superior to conventional fluorescence microscop y in terms of contrast, apparent resolution and resistance to photoble aching. Development of the fluorophore was examined systematically in sciatic nerves of young adult rats. In separate experiments, animals w ere 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 glutaral dehyde in the fixative solution was the principal determinant of fluor escence intensity. Confocal imaging was achieved immediately following perfusion with 5% glutaraldehyde or Karnovsky's. Fluorescence intensi ty increased markedly during overnight storage in these fixatives and continued to increase during subsequent storage in buffer alone. The f luorophore was stable and resistant to fading during storage (15 month s at least), enabling data collection over extended periods. To demons trate application of the method in neuropathology, individual fibres i n transected sciatic nerve trunks were traced through multiple success ive internodes: Classical features of Wallerian degeneration (axonal s welling and debris; ovoid formation and incisure changes; variation am ong fibres in the extent of degeneration) were displayed. The method i s compatible with subsequent ultrastructural examination and will comp lement existing methods of investigation of myelinated fibre anatomy a nd pathology, particularly where preservation of S-dimensional relatio nships or elucidation of spa tial gradients are required.