Verification of the two-dimensional disector, a method for the unbiased estimation of density and number of myelinated nerve fibers in peripheral nerves

Quantification of the number of myelinated fibers in peripheral nerves is a common requirement in quantitative morphology. This parameter provides important information on the consequences of various physiological, pathological and experimental conditions on the nerve structure and is one of the main indicators of success of peripheral nerve repair. In this paper, the theoretical rationale for the application of stereological principles to obtain unbiased estimates of the density and total number of myelinated fibers in peripheral nerves is discussed and a simple stereological method is described. The method is applied together with a systematic random sampling scheme, that was optimized for the purposes of the present study, and with sampling scheme analysis by calculating the coefficient of error (CE). The stereological method, which consists of a two-dimensional variation of the classical disector procedure (two-dimensional disector), and the sampling scheme are verified by comparing estimates with the true density and total number of myelinated fibers in peripheral nerve trunks where true values have been accurately determined by extensive counting. The verification of the 2-D disector method, both of normal and regenerated nerves, showed that estimates of density and total number of myelinated nerve fibers are unbiased. The method also proved to be efficient (timesaving): Estimation of density and total number of myelinated fibers in a single nerve takes about 2-3 hours.