A modified KOH-collagenase method applied to scanning electron microscopic observations of peripheral nerves

Certain parts of the peripheral nervous system were observed by a modification of the KOH-collagenase method by Miller et al. (1982). The sciatic nerve of the mouse, and dorsal root ganglion, lingual muscle and jejunum of the rat were fixed over one day at room temperature in a fixative containing paraformaldehyde and glutaraldehyde. After fixation, specimens were treated with 5N KOH solution for 5-10 min at 60 degrees C, immersed in a collagenase solution for 3-5 h at 37 degrees C and processed for scanning electron microscopy (SEM). In adequately treated specimens, connective tissue matrices and basal laminae were completely removed without causing any severe tissue damage. The three-dimensional visualization of cellular elements of peripheral nerves was enabled with the following results: 1) Individual nerve fibers of the sciatic nerve were clearly exposed. Myelinated fibers were non-branching cords with local annular constrictions at the node of Ranvier, while unmyelinated ones branched and anastomosed with one another to form loose networks. The sites of accumulation of the Schwann cell cytoplasm swelled on the surface of the myelinated fibers. Mesaxons were visualized as longitudinal furrows. 2) Ganglion cells covered by satellite cells were observed in the dorsal root ganglion. The ganglion cells were covered by their own convoluted dendro-axonal processes, thus forming the initial glomeruli of Cajal. Satellite cells at the glomeruli extended many finger-like projections surrounding the dendro-axonal processes. 3) Motor endplates were observed in lingual muscles. Terminal Schwann cells (teloglia) at the endplate were clearly visualized: the round perikaryon extended cytoplasmic processes along the axonal branches within the endplate. The processes issued fine finger-like projections from their margins. 4) Vascular autonomic plexuses were clearly demonstrated in lingual muscles. Unmyelinated nerves branched and anastomosed to form elaborate nerve networks around the vessels. Neuronal processes and associated Schwann cells were identifiable at high magnification. 5) Submucous nerve plexuses in the jejunum consisted of numbers of ganglia and interconnecting strands of fibers which formed very complicated networks. These observations indicate that this modified KOH-digestion method is useful for the SEM study of the three-dimensional cellular organization of nervous elements in various tissues.