Osmic acid staining of myelin sheath in normal and regenerated peripheral nerves

Objective ： To introduce a practical, economical, and time-saving method to stain （with osmic acid） the myelin sheath in normal and regenerated peripheral nerves. Methods： A total of 12 Sprague Dawley rats, weighing 250-320 g （ mecan = 276 g ± 38 g ）, were divided into two groups： a normal nerve group （n = 6 ） and a regenerated nerve group （ n = 6 ）. In the normal nerve group, the ventral and dorsal roots of L4 to L6 and their sciatic nerves were harvested for histological analysis. While in the regenerated nerve group, the right sciatic nerves were severed and then repaired with an epineurial microsuture method. The repaired nerves were harvested 12 weeks postoperatively. All the specimens were fixed in 4 % paraformaldehyde and transferred to 2 % osmic acid for 3-5 days. Then the specimens were kept in 75% alcohol before being embedded in paraffin. The tissues were cut into sections of 3 ten in thickness with a conventional microtome. Results： Under a light microscope, myelin sheaths were clearly visible at all magnifications in both groups. They were stained in clear dark colour with a light yellow or colorless background, which provided high contrast images to allow reliable morphometric measurements. Morphological assessment was made in both normal and regenerated sciatic nerves. The ratios of the myelin area to the fibre area were 60. 28 % ± 7.66 % in the normal nerve group and 51. 67% ± 6. 85% in the regenerated nerve group, respectively （P〈0.01）. Conclusions： Osmic acid staining is easy to perform and a very clear image for morphometrical assessment is easy to obtain. Therefore, it is a reliable technique for quantitative evaluation of nerve morphology.