Growth-promoting Activity of Sanyak (Dioscoreae rhizoma) Extract on Injured Sciatic Nerve in Rats

Analysis of GAP-43 protein levels in the injured sciatic nerve. (A) Western blot analysis of GAP-43 protein. The sciatic nerve lysate at the injury area (1 cm length) was prepared 5 days after injury, and the lysate from the sciatic nerve from intact animal was used as a negative control. 15 μg of protein was used for individual samples, and Western analysis with anti-actin antibody was used as an internal loading control. (B) Comparison of GAP-43 protein levels between saline- and SY-treated animals in the sciatic nerves by immunofluorescence staining. GAP-43 protein signal (in red) was analyzed in longitudinal nerve sections 5 days after injury. GAP-43 protein signal was compared at the same distances from the injury site between saline and SY-treated groups.

Identification of regenerating axons in the sciatic nerve 5 days after injury. Immunofluorescence images of NF-200-stained axons in the longitudinal sciatic nerve sections were examined at the injury site, and the areas 3 mm proximal, and 3 mm and 5 mm distal to the injury site.

Retrograde analysis of DiI-labeled neurons after sciatic nerve injury. (A) Retrograde tracing of DRG sensory neurons 5 days after sciatic nerve injury. DRG at lumbar 4 and 5 was prepared 5 days after surgical procedure and 20 μm sections were prepared for further analysis. DiI-impregnated sensory neurons were visualized under the fluorescence microscope (red). The images on the right side are the enlarged view of the corresponding images to the left side. (B) Retrograde tracing of motor neuron cell bodies at the ventral horn of the spinal cord 5 days after sciatic nerve injury. The longitudinal spinal cord sections were prepared 5 days after surgical procedure and analyzed under the fluorescence microscope (red). The images on the right side are the enlarged view of the images corresponding to the left side. (C) Quantitation of DiI-labeled DRG sensory neurons and motor neurons in the spinal cord. The number of labeled cells was compared between saline- and SY-treated cells. Mean ± SEM (n = 4, *p = 0.042, **p = 0.0087).

Cdc2 and phospho-vimentin protein analysis in the sciatic nerve. (A) Western blot analysis of Cdc2 in the injured sciatic nerve. Proximal and distal sciatic nerve stumps (0.5 cm length) were prepared from the intact animal, saline (Sal), and SY-treated animals. 15 μg of protein lysates was analyzed for Western blot analysis with anti-Cdc2 and anti-actin antibodies. Western analysis for actin protein was performed as an internal loading control. (B) The longitudinal sciatic nerve sections were prepared 5 days after injury from saline (Sal) and SY-treated animals, and used for double immunofluorescence staining with anti-Cdc2 (red) and anti-phospho-vimentin antibodies (green). The stained nerve sections among different groups were compared at the injury site, and 3 mm and 5 mm distal to the injury site. (C) Representative immunofluorescence images, prepared from SY-treated animals, for phospho-vimentin and Cdc2 in nerve sections 5 mm distal to the injury site.

Nuclear staining to count the number of non-neuronal cells in the sciatic nerve. The sciatic nerve sections were prepared 5 days after injury from saline- or SY-treated animals. (A) In saline- and SY-treated groups, representative images from 3 mm proximal, 0 mm (i.e. injury site), and 3 mm and 5 mm distal to the injury site are shown. (B) Quantitative comparison of Hoechst-stained cell counts between saline and SY-treated groups. Mean ± SEM (n = 4), *p = 0.043, **p = 0.0063 (Student's t-test).