Accuracy of sampling methods in morphometric studies of the sural nerve in man

Abstract

A variety of sampling methods are used in quantitative studies of myelinated sural nerve fibres, however there is no consensus as to which method is most accurate. This study compares whole fascicular sampling and systematic sampling of myelinated fibres with evaluation of the total myelinated nerve fibre population. Two control and eighteen pathological sural nerves showing varying degrees of demyelination/remyelination and axonal degeneration were examined. The fascicular area, number of myelinated fibres, myelinated fibre density, fibre diameter and axonal diameter were measured in each fascicle of all the nerves using I micron plastic cross sections stained with osmium tetroxide. Each fascicle was divided into measuring frames, and the number and size of myelinated fibres in each frame (field) counted using the Quantimet 500MC computer-assisted image analysis system (Leica-Cambridge, UK). Frequency distributions of myelinated fibre density and size were calculated. The mean values and frequency distributions of fibre density, fibre diameter and axonal diameter of each sample were compared to the whole population by the Wilcoxon Rank-Sum test and Kolmogorov- Smirnov goodness-of-fit test. Fascicular sampling of the two control sural nerves (14 fascicles) showed that 8 fascicles had different myelinated fibre density (P<0.05), and 8 fascicles had different fibre diameter and/or axonal diameter (P<0.05) when compared to the whole population. In the 18 pathological nerves there were 168 fascicles. When compared to the whole population, 61 fascicles had different myelinated fibre density (P<0,05), and 90 fascicles had different fibre diameter and/or axonal diameter (P<0.05). There was no relationship between the inated fibre density of each fascicle and the fascicle diameter or area in either control and pathological sural nerves. It is concluded that morphometric study of myelinated fibres of one or part of a fascicle cannot accurately represent the whole myelinated fibre population in the sural nerve. Systematic sampling of one third to half of the total transverse fascicular area in control and pathological sural nerves did not accurately depict the fibre diameter or axonal diameter of the whole myelinated fibre population. The myelinated fibre density derived from systematic sampling was more accurate than that derived from fascicular sampling. The spatial distribution of the number and size of myelinated fibres within and between fascicles is heterogeneous in the sural nerve. It is necessary to quantitate more than half the area of every fascicle to acquire accurate data about myelinated fibres that is representative of the whole myelinated fibre population.