Journal of Acupuncture and Meridian Studies
Volume 2, Issue 3 , Pages 228-235, September 2009

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

Department of Oriental Medicine, Daejeon University, Daejeon, Korea

Received 15 June 2009; accepted 14 July 2009.

Article Outline

Abstract 

The present study evaluates the potential effects of Sanyak (Dioscoreae rhizome) on the regenerative capacity of the peripheral sciatic nerve after crush injury in rats. Focal application of Sanyak extract at the injury site increased GAP-43 and Cdc2 protein levels in the distal portion of the injured nerve. Immunohistochemical analysis showed that the signals of phospho-vimentin as Cdc2 substrate protein were almost colocalized with Cdc2. Retrograde DiI-tracing revealed enhancement in distal elongation of regenerating axons. Furthermore, the number of non-neuronal cells was higher in Sanyak-treated animals than saline controls. Thus, these data suggest that Sanyak extract is effective for promoting regenerative responses in injured peripheral neurons.

Key Words:  axonal regeneration , Dioscoreae rhizome (SY) , rat , sciatic nerve

 

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1. Introduction 

Although injured axons in the peripheral nervous system (PNS) can regenerate and lead to functional recovery by innervating the original target, the extent of regeneration varies depending on the type of nerves and injuries 1, 2. For instance, a complete axotomy of peripheral nerve results in wrong targeting of regenerating axons between cutaneous sensory and motor organs [3]. Numerous studies have identified intrinsic and extrinsic molecular factors involved in regeneration processes of the injured peripheral nerves 3, 4. Some of the prototypical examples of intrinsic factors include axonal growth-associated protein with an apparent molecular weight of 43 kDa (GAP-43) and trophic factors such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), and neurotrophic factor 3 and 4 (NT-3 and -4) 5, 6, 7, 8. GAP-43 is highly induced at the gene expression level in the injured nerve and is thus recognized as a regeneration marker protein rather than a regeneration trigger 9, 10. Besides intrinsic influences, regenerating peripheral axons are greatly affected by non-neuronal environments. Schwann cells are activated around the injury area and are most likely the most important non-neuronal influence on injured nerves. It has been well demonstrated that injured axons at the distal portion undergo Wallerian degeneration in which the debris from degenerating tissues is cleared by phagocytic activity of Schwann cells and macrophages that have infiltrated from blood vessels [1]. Schwann cells are also known to migrate along the injury area and guide regrowing axons 12, 13. Finally, they are involved in remyelination of newly formed axons. Thus, physical and chemical interactions between Schwann cells and injured axons are critical for successful regrowth.

Recent studies have begun to provide evidence that natural herbal drugs such as ginsenoside Rb and Hominis placenta (Jahageo) may facilitate axonal regeneration after peripheral nerve injury 14, 15. Considering that herbal drugs contain diverse chemical ingredients that might be involved in the responsiveness of injured nerves, potential effects of herbal drugs on axonal regeneration could be highly feasible.

Here, we investigated whether Sanyak (SY; Dioscoreae rhizoma) has any growth-promoting activity on sciatic nerve axons after injury in the rat. In oriental medicinal therapy, SY has been prescribed for diverse physiological abnormalities including muscle pain, renal, digestive and hepatic problems. A recent study indicated its inhibitory activity on proinflammatory cytokines, and antioxidant activity, for the protection of DNA 16, 17, 18. Our study demonstrates that SY administration into injured peripheral nerve induces activation of several molecular components that are involved in axonal regeneration, and thus promote nerve regrowth.

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2. Materials and Methods 

2.1. Experimental animals 

Sprague-Dawley rats (8 weeks old) were used in this experiment. They were placed in an animal room with regulated temperature (22°C), 60% of humidity, and a 12 hour light and 12 hour dark cycle. They were allowed to eat commercial rat chow (Samyang Co., Korea) and drink water ad libitum. Rat care and all experimental procedures were in accordance with the Animal Use Statement and Ethics Committee Approval Statement for Animal Experiments at Daejeon University (Daejeon, Korea).

2.2. Drugs 

SY was obtained from Daejeon University Oriental Medicine Hospital (Daejeon, Korea). SY (58 g dry weight) was resuspended in 2 liters of water, heat-extracted with 2 liters of water for 3 hours, and filtered three times. The filtered fluid was distilled using the rotary vacuum evaporator (Büchi 461, Eyela, USA). Concentrated solutions were frozen at −70°C for 4 hours, and freeze-dried for 24 hours. The yield for SY was 3.2 g from 58 g of the initial raw materials. The product was kept at 4°C, and dissolved in water. The stock solution (10 mg/mL in phosphate buffered saline) was stored at −20°C and used for experiments by diluting with physiological saline solution (0.9% NaCl in water) before use. SY (5 μL of 10 mg/mL) or an equivalent volume of saline (0.9% NaCl) was directly applied into the crush sites.

2.3. Surgery and tissue preparation 

Rats were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (5 mg/kg). Sciatic nerve was exposed and crushed with a pair of forceps held tightly for 30 seconds twice at 1 minute intervals. Animals were allowed to recover from anesthesia and were sacrificed 5 days later. Animals were deeply anesthetized again with a mixture of ketamine and xylazine, and the proximal and distal portions of the injury site of the sciatic nerves were dissected separately, immediately frozen, and kept at −70°C until their use for immunohistochemistry or Western blot analysis.

2.4. Immunohistochemistry 

For immunohistochemistry experiments, dissected nerve tissue was immediately frozen at −75°C and embedded into OCT medium. Sciatic nerve sections (20 μm) were cut using a cryostat and mounted on positively charged slides. Sections on a slide were fixed with 4% paraformaldehyde, 4% sucrose in phosphate-buffered saline (PBS) at room temperature for 40 minutes, permeabilized with 0.5% nonidet P-40 in PBS, and blocked with 2.5% horse serum and 2.5% bovine serum albumin for 4 hours at room temperature. Sections were incubated with primary antibody, and then incubated with fluorescein-goat anti-mouse (Molecular Probes, Eugene, OR, USA) or rhodamine-goat anti-rabbit secondary antibodies (Molecular Probes, USA) in 2.5% horse serum and 2.5% bovine serum albumin for 1 hour at room temperature. Slides were subsequently cover-slipped with gelatin mount medium. The primary antibody reaction was performed with single or double antibodies, depending on experimental purposes, and were followed with corresponding specific secondary antibody reactions. The primary antibodies used were anti-NF-200 antibody (Sigma, St. Louis, MO, USA), anti-Cdc2 antibody (Santa Cruz Biotech, Santa Cruz, CA, USA), anti-phospho-vimentin antibody (MBL, USA,) and anti-GAP-43 antibody (Santa Cruz Biotech, Santa Cruz, CA, USA). In some studies, Hoechst staining for nuclear visualization was also performed following the first washing step after the secondary antibody reaction. Tissue sections were treated with 25 μg/mL of Hoechst 33258 dye (Sigma, St. Louis, MO, USA) in 0.1% Triton X-100 in PBS for 10 minutes. Sections were observed with a Nikon fluorescence microscope (E-600, Nikon, Japan) and the images were captured using a Nikon camera (DXM 1200F, Nikon, Japan). The merged images were produced by using layer blending mode options on Adobe Photoshop (version 5.5).

2.5. Western blot analysis 

Nerve tissue was washed with ice-cold PBS, and sonicated in 50-200 μL of Triton lysis buffer (20 mM Tris, pH 7.4, 137 mM NaCl, 25 mM β-glycerophosphate, pH 7.14, 2 mM sodium pyrophosphate, 2 mM EDTA, 1 mM Na3VO4, 1% Triton X-100, 10% glycerol, 5 μg/mL leupeptin, 5 μg/mL aprotinin, 3 μM benzamidine, 0.5 mM DTT, 1 mM PMSF). Protein (15 μg) was resolved in 12% SDS polyacrylamide gel and transferred to Immobilon polyvinylidenedifluoride membranes (Millipore, Billerica, MA, USA). Blots were blocked with 5% nonfat dry milk in PBST (17 mM KH2PO4, 50 mM Na2HPO4, 1.5 mM NaCl, pH 7.4, and 0.05% Tween-20) for 1 hour at room temperature and incubated overnight at 4°C in 0.1% Triton X-100 in PBS plus 5% nonfat dry milk containing antibodies. Protein bands were detected using the Amersham ECL kit (Amersham Pharmacia Biotech, USA), with horseradish peroxidase-conjugated secondary goat anti-rabbit or goat anti-mouse antibodies (Transduction Laboratories, Lexington, KY, USA). The primary antibodies used in the present study were an anti-GAP-43 antibody (Santa Cruz Biotech, Santa Cruz, CA, USA), an anti-Cdc2 antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) and an anti-actin antibody (Santa Cruz Biotech, Santa Cruz, CA, USA).

2.6. Retrograde tracing 

The sciatic nerve of rats anesthetized with ketamine and xylazine was exposed and fluorescent lipophilic carbocyanine dye l,l′dioctodecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate (DiI, Molecular Probe; 5 μL of 3% in DMSO) was applied 0.5 cm distal to the injury site with a microsyringe. The incision was sutured, and the animals were returned to their cages after they recovered from narcosis. DiI-labeled sensory neurons in the dorsal root ganglion (DRG) were visualized and measured under fluorescence microscope by an observer unaware of the experimental treatment.

2.7. Statistical analysis 

Comparisons among experimental groups were made by two sample Student's t-test. A criterion of p values less than 0.05 was applied to assess statistical significance.

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3. Results 

3.1. Changes in levels of GAP-43 production 

In order to study possible effects of SY on facilitated axonal regeneration, protein lysates prepared from proximal and distal portions of the injured sciatic nerves were analyzed by Western and immunofluorescence staining. GAP-43 was not detected in the intact sciatic nerve, but largely induced after injury (Figure 1A). SY treatment further elevated GAP-43 levels in the injured nerve. The intensity of the GAP-43 protein band was higher in the distal sciatic nerve than proximal nerve in both saline and SY-treated groups. To investigate the distribution of induced GAP-43 protein in the injured nerve tissue, immunofluorescence staining was performed (Figure 1B). In the saline-treated group, moderate levels of GAP-43 protein signal was detected throughout the nerve with a pattern of gradual decrease toward the distal end. In the SY-treated group, levels of GAP-43 signal were consistent throughout the distal portion of the nerve, thus revealing higher signals in the 5 mm distal area compared with saline control. This demonstrates elevated GAP-43 protein levels in the SY group compared with the saline control, as was also demonstrated by Western blot analysis.

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  • Figure 1. 

    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.

3.2. Histological analysis of regenerative responses 

To determine whether SY treatment affects axonal regeneration, growing axons in the distal portion of the nerve were analyzed by immunofluorescence staining. Five days after nerve injury in the saline-treated group, axonal staining with NF-200 protein was clearly observed at the injury site, but was greatly reduced at 3 mm and 5 mm portions distal to the injury site (Figure 2). In contrast, SY treatment showed consistent axonal staining at the 3 mm and 5 mm distal areas.

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  • Figure 2. 

    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.

To further investigate changes in axonal regeneration following SY treatment, neuronal cell bodies corresponding to sciatic nerve axons were analyzed from the DRG at lumbar 4 and 5 and the spinal cord between the lower thoracic and upper lumbar levels by retrograde tracing. As shown in Figure 3A, DiI-labeled neuronal cell bodies were clearly observed in DRG sections. In the saline-treated group, mostly round shape labeled cells together with a few weakly stained small cells, were seen in the tissue. The number of DiI-labeled cells was significantly higher in the SY-treated group than saline control (Figure 3C). Moreover, the processes around the cell bodies were observed more frequently in the SY-treated group. Regeneration responses of the motor neuron axons in the injured sciatic nerve were similarly investigated by measuring DiI-labeled motor neurons in the spinal cord ventral horn. As shown in Figure 3B, DiI-labeled motor neurons were clearly seen in the spinal cord sections. The number of labeled cells was significantly higher in the SY-treated group than saline control (Figure 3C).

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  • Figure 3. 

    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).

3.3. Changes in Cdc2 activity and cell proliferation in regenerating nerves 

The cell cycle protein, Cdc2, was investigated in the injured sciatic nerves. Cdc2 protein was not detected in the intact nerve but was highly induced in the distal sciatic nerve after injury (Figure 4A). SY treatment further increased Cdc2 in the distal nerve. To examine Cdc2 phosphorylation of its substrate protein, vimentin, immunofluorescence staining was performed in distal sciatic nerve sections. Low Cdc2 and phospho-vimentin protein signals were detected at the injury site and the signals were slightly elevated in the 3 mm and 5 mm distal portions of the nerve (Figure 4B). SY treatment increased both Cdc2 and phospho-vimentin signals at the 3 mm and 5 mm distal portion of the nerve. Merged images of Cdc2 and phospho-vimentin showed high colocalization of protein signals in the nerve tissue (Figure 4C).

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  • Figure 4. 

    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.

To compare the cell number between different treatments, Hoechst nuclear staining of the sciatic nerve was performed. After nerve injury, the number of nuclei increased to a large extent, particularly at the injury site and the adjacent distal area (Figure 5A). When the number of nuclei were compared between saline and SY-treated groups at the same distances from the injury site, nucleus counts were similar at the proximal nerve area between the two groups, but were significantly increased at the injury site and at the 3 mm distal nerve area in SY-treated groups compared with the saline control (Figure 5B).

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  • Figure 5. 

    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).

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4. Discussion 

According to the principles of oriental medicine, SY is believed to strengthen Qi in the spleen, lung and kidneys, and is thus used for treatment of diverse symptoms in related organs 16, 17. Here, we found that SY treatment improved axonal regeneration responses toward the distal end of the sciatic nerve and upregulated production of several key proteins associated with axonal regeneration.

Biochemical and histological analyses strongly indicate that SY may facilitate axonal regeneration. First, SY treatment increased GAP-43 protein levels in the distal portion of the sciatic nerve. GAP-43 is strongly induced at the gene expression level in neurons after axonal injury, and transported into the growth cone which is involved in the process of regrowth 9, 10. Thus, our data on higher levels of GAP-43 at the distal nerve stump compared with the proximal area support the observation that SY treatment may be positively associated with the growth process connected with GAP-43 activity in the distal nerve. Second, our data showed that SY treatment increased staining for NF-200 in axons. Furthermore, quantitative analysis of axonal regeneration by counting DiI-labeled neurons whose axons underwent axonal regeneration revealed that SY increased axonal regeneration. Careful observation of DiI-labeled sensory neurons in the DRG or motor neurons in the spinal cord revealed more intense DiI-labeling intensity in the SY-treated group than saline control. This might suggest that individual regrowing axons at the distal area were repaired more efficiently by SY treatment, rendering enhanced labeling of individual neurons with DiI.

The present data showed that SY treatment up-regulated production of Cdc2 protein levels in the injured sciatic nerve. Cdc2 is the prototypical cell cycle protein and plays a critical role in the transition between growth phase 2 to the mitotic phase [19]. Previous studies have shown that Cdc2 protein and its kinase activity were not found in the intact nerve tissues, but strongly induced in Schwann cells after nerve injury [20]. The current study showed the same pattern of Cdc2 in the injured nerve. Biochemical studies have identified numerous proteins as Cdc2 kinase substrates, and most of them are primarily involved in the progression of mitosis [21]. The vimentin protein is one of the targets for Cdc2 kinase and was, therefore, investigated in this study. Vimentin is an intermediate protein which is involved in maintaining the mechanical stability of cells 22, 23. Vimentin can be phosphorylated by several kinases [24], but its phosphorylation by Cdc2 at Serine 55 has been suggested to play an important role in subsequent phosphorylation of vimentin at another site leading to mitotic separation of dividing cells 23, 25, 26, 27. Furthermore, the phosphorylated form of vimentin, created by Cdc2, was found in glial cells undergoing migration activity [28]. Thus, it is conceivable that Cdc2 phosphorylation of vimentin may function for mitosis and cell migratory activity, as has been demonstrated by Cdc2 phosphorylation of caldesmon in Schwann cells and kidney cell lines 20, 29. The present data showed that SY treatment elevated levels of phospho-vimentin in the distal area of injured sciatic nerve. Since the activation of the Cdc2-phospho-vimentin pathway may be functionally involved in Schwann cell proliferation and migration leading to facilitated axonal re-growth, growth-promoting activity by SY treatment could be mediated through Schwann cell activation. Indeed, our measurement of individual nonneuronal cells in the sciatic nerve by Hoechst nuclear staining showed increased cell counts following SY treatments in the distal nerve area, implicating possible involvement of the Cdc2-vimentin pathway in Schwann cells. In conclusion, SY appears to activate several molecules known to be important for axonal regeneration in the injured peripheral nerve, and thus play an important role in facilitated axonal regeneration. Further studies to identify key molecular components in SY may be potentially critical for the development of therapeutic targets.

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Acknowledgments 

This work was supported by grants from Korea Pharmacopuncture Institute 2008 (to K. Hong) and Korea Research Foundation (E00116) to U. Namgung.

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PII: S2005-2901(09)60059-5

doi:10.1016/S2005-2901(09)60059-5

Journal of Acupuncture and Meridian Studies
Volume 2, Issue 3 , Pages 228-235, September 2009

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