Software package

Then, 12 weeks after surgery, the gastrocnemius muscles of operated hind limb were harvested and immersed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer at 4°C for 2 weeks. Then five transverse sections (thickness of 50.0 mm) per specimen were prepared and subjected to HE staining. Thereafter, these sections were examined under a light microscope (AH3; Olympus). Five middle-powered fields (200) in each section were randomly chosen for quantitative analysis with a Leica software package. The extent of the atrophy/reinnervation of target muscles was assessed by the percentage of muscle fiber area (P_m), which was calculated according to the equation:
$P_{\text{m}}=A_{\text{m}}/A_{\text{t}}\times 100\mathrm{\%}$
where A_m is the area of muscle fibers in each field (magnification, 100) and A_t is the total area including muscle fibers and other tissues of the field.

The gastrocnemius muscles from the operated limbs were harvested at the 8-week time point. Muscle specimens were fixed with formalin, embedded in paraffin, cut into 6 mm-thick sections and stained with hematoxylin and eosin. Random pictures from 3 fields of view were obtained from each sample and were analyzed with a Leica software package to measure the transverse section area of the muscle fibers. The percentage of muscle fiber area was calculated as muscle fiber area/total area.