Method for morphometric analysis of axons in experimental peripheral nervereconstruction

A new method for morphometric analysis of axons in experimental peripheral nerve reconstruction is presented. Twelve adult female rabbits were used. I n nine animals the saphenous nerve was transected and stitched epineurially . Three animals functioned as control, After 3, 6, and 12 months, the nerve s were harvested, fixed in Kryofix and embedded in Histowax. Transverse sec tions of 6 mu m were cut, immunohistochemically stained for NF 90, and coun terstained by Sirius Red. Quantification of nerve fibers in cross sections was performed by using a confocal laser scanning microscope (CLSM), and the images were stored digitally. Data analyzing was performed by the Optimas program (5.2). Calculations were done with Microsoft Excel. The total numbe r of axons, the mean axon diameter and the percentage axon area/fascicle ar ea were evaluated statistically. This method for morphologic analysis provi des automatically complete registration of axons and so different methods o f experimental nerve reconstruction can be compared in a fast and reliable way. (C) 2000 Wiley-Liss, Inc.