Myelinated Axon Analysis of Sciatic Nerve

A 5mm portion of the sciatic nerve immediately distal to the distal suture line of the nerve graft was harvested at the time of sacrifice. The nerve tissues were fixed in formalin for 24 h and then postfixed in 2% osmium tetroxide solution (Electron Microscopy Sciences, Hatfield, PA, USA) for 60 min, dehydrated, and embedded into Epon 812 resin (Electron Microscopy Sciences, Hatfield, PA, USA). 0.5 μm sections were stained with toluidine blue and imaged via light microscopy with a ZEISS AxioScan.Z1 (Oberkochen, Germany). Analysis was performed using ImageJ to determine nerve fascicle area, axon density, and total number of myelinated axons. Stereological techniques were used to obtain unbiased representations of the total number of myelinated axons and axon diameter per cross section [36 (link), 42 (link)].