The Use of Fixed and Stained Sections in Quantitative Studies of Peripheral Nerve

Abstract

ABSTRACT A technique which has been widely used in quantitative studies of peripheral nerve involves fixation in Flemming’s fluid, embedding in paraffin wax, and staining with haematoxylin. An appraisal of the size changes induced by a standardized form of this technique has been made. Observations have been confined to changes in the thickness and external diameter of the internodal compact myelin sheath of the non-fasciculated nervus gastrocnemius medialis of the adult rabbit. For a comparison of external diameters, sections from adjacent parts of the chosen nerve were prepared (a) by the standardized Flemming-Wolter technique; (b) by the rapid freezing technique, previously shown to reproduce accurately the dimensions present in fresh teased unfixed nerve-fibres. There was no significant difference in distribution of external diameters between sections prepared in the two ways. For a comparison of sheath thicknesses, a regression line of 2 × myelin sheath thickness on external diameter was derived from each type of preparation. As external diameter was not appreciably affected by processing, a direct comparison of these regression lines could be made. It was found that regardless of external diameter or original sheath thickness, processing caused a reduction of sheath thickness of some 20% as a result of increase in internal diameter. This latter increase ranged from about 50% to under 20% with increasing fibre size. Single frozen sections were passed through the series of reagents used in the Flemming-Wolter technique and photographed after each of seven stages. The principal changes in sheath thickness occurred in the first and last stages of processing. External diameter was not appreciably affected whereas the internal margin of the sheath was extremely labile. Observations suggest that slight inconsistencies in technique may be accompanied by relatively gross errors when measurements are undertaken. All stages need careful standardization, the most important in this respect being fixation, differentiation, final dehydration, and photographic exposure.