Comparative analysis of numerical estimation methods of epithelial nerve fibers using tissue sections

The proper assessment of neuron numbers in the nervous system during physiological and pathological conditions, as well as following various treatments, has always been an important part of neuroscience. The present paper evaluates three methods for numerical estimates of nerves in epithelium: I) unbiased nerve fiber profile and nerve fiber fragment estimation methods, II) the traditional method of counting whole nerve fibers, and III) the nerve fiber estimation method. In addition, an unbiased nerve length estimation method was evaluated. Of these four methods, the nerve length per volume method was theoretically optimal, but more time-consuming than the others. The numbers obtained with the methods of nerve fiber profile, nerve fragment and nerve fiber estimation are dependent on the thickness of the epithelium and the sections as well as certain shape factors of the counted fiber. However for those, the actual counting can readily be performed in the microscope and is consequently quick and relatively inexpensive. The statistical analysis showed a very good correlation (R > 0.96) between the three numerical methods, meaning that basically any method could be used. However, dependent on theoretical and practical considerations and the correlation statistics, it may be concluded that the nerve fiber profile or fragment estimation methods should be employed if differences in epithelial and section thickness and the nerve fibers shape factors can be controlled. Such drawbacks are not inherent in the nerve length estimation method and, thus, it can generally be applied.