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Rewiring of regenerated axons by combining treadmill training with semaphorin3A inhibition.

Zhang L, Kaneko S, Kikuchi K, Sano A, Maeda M, Kishino A, Shibata S, Mukaino M, Toyama Y, Liu M, Kimura T, Okano H, Nakamura M - Mol Brain (2014)

Bottom Line: However, these effects were limited because most regenerated axons likely do not connect to the right targets.Although extensive treadmill training combined with SM-345431 administration did not further improve axon regeneration, hindlimb motor performance was restored, as evidenced by the significant improvement in the execution of plantar steps on a treadmill.This study highlights the importance of combining treatments that promote axon regeneration with specific and appropriate rehabilitations that promote rewiring for the treatment of spinal cord injury.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. hidokano@a2.keio.jp.

ABSTRACT

Background: Rats exhibit extremely limited motor function recovery after total transection of the spinal cord (SCT). We previously reported that SM-216289, a semaphorin3A inhibitor, enhanced axon regeneration and motor function recovery in SCT adult rats. However, these effects were limited because most regenerated axons likely do not connect to the right targets. Thus, rebuilding the appropriate connections for regenerated axons may enhance recovery. In this study, we combined semaphorin3A inhibitor treatment with extensive treadmill training to determine whether combined treatment would further enhance the "rewiring" of regenerated axons. In this study, which aimed for clinical applicability, we administered a newly developed, potent semaphorin3A inhibitor, SM-345431 (Vinaxanthone), using a novel drug delivery system that enables continuous drug delivery over the period of the experiment.

Results: Treatment with SM-345431 using this delivery system enhanced axon regeneration and produced significant, but limited, hindlimb motor function recovery. Although extensive treadmill training combined with SM-345431 administration did not further improve axon regeneration, hindlimb motor performance was restored, as evidenced by the significant improvement in the execution of plantar steps on a treadmill. In contrast, control SCT rats could not execute plantar steps at any point during the experimental period. Further analyses suggested that this strategy reinforced the wiring of central pattern generators in lumbar spinal circuits, which, in turn, led to enhanced motor function recovery (especially in extensor muscles).

Conclusions: This study highlights the importance of combining treatments that promote axon regeneration with specific and appropriate rehabilitations that promote rewiring for the treatment of spinal cord injury.

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Related in: MedlinePlus

SM-345431 enhanced axonal regeneration in vivo, but combined treatment had only a limited effect on further axon regeneration. (A-I) Sagittal sections from SCT rats immunostained for GAP-43. Low-magnification images of the control (A), SM-345431 (B) and combined treatment (C) groups. Scale bars = 500 μm. (D-F) Magnified images of the boxed areas shown in A-C. Scale bars = 10 μm. (G-I) Additional magnified images of the boxed areas shown in A-C. Scale bars = 10 μm. (J) Quantitative analysis of GAP-43-positive areas at 1 mm rostral to the lesion, 1 mm caudal to the lesion and at the epicenter of the lesion. Immunohistochemistry was performed using DAB with nickel enhancement. *P < 0.05, **P < 0.01. Statistical analyses were performed using one-way ANOVA and Bonferroni post hoc tests. Data are represented as the mean ± S.E.M. (K-P) Sagittal sections of SCT rats double-stained for 5-HT and GFAP. Scar tissue is outlined by GFAP staining, which also allowed for confirmation of total transection of the spinal cord in each animal. (K-M) Low-magnification images of the control (K), SM-345431 (L) and combined treatment (M) groups. Scale bars = 500 μm. (N-P) Magnified images of the boxed areas shown in K-M. Scale bars = 10 μm. Arrowheads represent 5-HT-positive (serotonergic) axons. (Q-S) Quantitative analyses of 5-HT-positive axons that penetrated into the scar tissue. (Q) Quantitative analysis of the number of 5-HT-positive axons that penetrated into the lesion site. (R,S) Quantitative analysis of the 5-HT-positive area within the scar tissue area. Immunohistochemistry was performed by double-staining using DAB with or without nickel enhancement. The left side is rostral in all images. **P < 0.01. Statistical analyses were performed using a Kruskal-Wallis H test. Data represent the mean ± S.E.M.
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Figure 2: SM-345431 enhanced axonal regeneration in vivo, but combined treatment had only a limited effect on further axon regeneration. (A-I) Sagittal sections from SCT rats immunostained for GAP-43. Low-magnification images of the control (A), SM-345431 (B) and combined treatment (C) groups. Scale bars = 500 μm. (D-F) Magnified images of the boxed areas shown in A-C. Scale bars = 10 μm. (G-I) Additional magnified images of the boxed areas shown in A-C. Scale bars = 10 μm. (J) Quantitative analysis of GAP-43-positive areas at 1 mm rostral to the lesion, 1 mm caudal to the lesion and at the epicenter of the lesion. Immunohistochemistry was performed using DAB with nickel enhancement. *P < 0.05, **P < 0.01. Statistical analyses were performed using one-way ANOVA and Bonferroni post hoc tests. Data are represented as the mean ± S.E.M. (K-P) Sagittal sections of SCT rats double-stained for 5-HT and GFAP. Scar tissue is outlined by GFAP staining, which also allowed for confirmation of total transection of the spinal cord in each animal. (K-M) Low-magnification images of the control (K), SM-345431 (L) and combined treatment (M) groups. Scale bars = 500 μm. (N-P) Magnified images of the boxed areas shown in K-M. Scale bars = 10 μm. Arrowheads represent 5-HT-positive (serotonergic) axons. (Q-S) Quantitative analyses of 5-HT-positive axons that penetrated into the scar tissue. (Q) Quantitative analysis of the number of 5-HT-positive axons that penetrated into the lesion site. (R,S) Quantitative analysis of the 5-HT-positive area within the scar tissue area. Immunohistochemistry was performed by double-staining using DAB with or without nickel enhancement. The left side is rostral in all images. **P < 0.01. Statistical analyses were performed using a Kruskal-Wallis H test. Data represent the mean ± S.E.M.

Mentions: To examine the regeneration of axons after SM-345431 treatment and SM-345431 treatment combined with extensive treadmill training, we evaluated axons in the injured spinal cord with immunostaining using antibodies against GAP43 and serotonin (5-HT) (Figure 2), GAP43 is widely used as a marker for regenerated axons. In both treatment groups, a marked increase in the number of GAP43-positive axons was observed at the epicenter of the injury (Figure 2D-F) and in the surrounding area (Figure 2G-I). Compared with the control group, the number of GAP43 axons was significantly increased in both the SM-345431 treatment group and the combined treatment group, especially at 1 mm caudal to the injury epicenter (Figure 2J). No significant difference was observed between the 2 treatment groups. Thus, administration of the semaphorin3A inhibitor SM-345431 using this DDS enhanced axonal regeneration. However, no additional axonal regeneration was observed when SM-345431 treatment was combined with treadmill training.


Rewiring of regenerated axons by combining treadmill training with semaphorin3A inhibition.

Zhang L, Kaneko S, Kikuchi K, Sano A, Maeda M, Kishino A, Shibata S, Mukaino M, Toyama Y, Liu M, Kimura T, Okano H, Nakamura M - Mol Brain (2014)

SM-345431 enhanced axonal regeneration in vivo, but combined treatment had only a limited effect on further axon regeneration. (A-I) Sagittal sections from SCT rats immunostained for GAP-43. Low-magnification images of the control (A), SM-345431 (B) and combined treatment (C) groups. Scale bars = 500 μm. (D-F) Magnified images of the boxed areas shown in A-C. Scale bars = 10 μm. (G-I) Additional magnified images of the boxed areas shown in A-C. Scale bars = 10 μm. (J) Quantitative analysis of GAP-43-positive areas at 1 mm rostral to the lesion, 1 mm caudal to the lesion and at the epicenter of the lesion. Immunohistochemistry was performed using DAB with nickel enhancement. *P < 0.05, **P < 0.01. Statistical analyses were performed using one-way ANOVA and Bonferroni post hoc tests. Data are represented as the mean ± S.E.M. (K-P) Sagittal sections of SCT rats double-stained for 5-HT and GFAP. Scar tissue is outlined by GFAP staining, which also allowed for confirmation of total transection of the spinal cord in each animal. (K-M) Low-magnification images of the control (K), SM-345431 (L) and combined treatment (M) groups. Scale bars = 500 μm. (N-P) Magnified images of the boxed areas shown in K-M. Scale bars = 10 μm. Arrowheads represent 5-HT-positive (serotonergic) axons. (Q-S) Quantitative analyses of 5-HT-positive axons that penetrated into the scar tissue. (Q) Quantitative analysis of the number of 5-HT-positive axons that penetrated into the lesion site. (R,S) Quantitative analysis of the 5-HT-positive area within the scar tissue area. Immunohistochemistry was performed by double-staining using DAB with or without nickel enhancement. The left side is rostral in all images. **P < 0.01. Statistical analyses were performed using a Kruskal-Wallis H test. Data represent the mean ± S.E.M.
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Related In: Results  -  Collection

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Figure 2: SM-345431 enhanced axonal regeneration in vivo, but combined treatment had only a limited effect on further axon regeneration. (A-I) Sagittal sections from SCT rats immunostained for GAP-43. Low-magnification images of the control (A), SM-345431 (B) and combined treatment (C) groups. Scale bars = 500 μm. (D-F) Magnified images of the boxed areas shown in A-C. Scale bars = 10 μm. (G-I) Additional magnified images of the boxed areas shown in A-C. Scale bars = 10 μm. (J) Quantitative analysis of GAP-43-positive areas at 1 mm rostral to the lesion, 1 mm caudal to the lesion and at the epicenter of the lesion. Immunohistochemistry was performed using DAB with nickel enhancement. *P < 0.05, **P < 0.01. Statistical analyses were performed using one-way ANOVA and Bonferroni post hoc tests. Data are represented as the mean ± S.E.M. (K-P) Sagittal sections of SCT rats double-stained for 5-HT and GFAP. Scar tissue is outlined by GFAP staining, which also allowed for confirmation of total transection of the spinal cord in each animal. (K-M) Low-magnification images of the control (K), SM-345431 (L) and combined treatment (M) groups. Scale bars = 500 μm. (N-P) Magnified images of the boxed areas shown in K-M. Scale bars = 10 μm. Arrowheads represent 5-HT-positive (serotonergic) axons. (Q-S) Quantitative analyses of 5-HT-positive axons that penetrated into the scar tissue. (Q) Quantitative analysis of the number of 5-HT-positive axons that penetrated into the lesion site. (R,S) Quantitative analysis of the 5-HT-positive area within the scar tissue area. Immunohistochemistry was performed by double-staining using DAB with or without nickel enhancement. The left side is rostral in all images. **P < 0.01. Statistical analyses were performed using a Kruskal-Wallis H test. Data represent the mean ± S.E.M.
Mentions: To examine the regeneration of axons after SM-345431 treatment and SM-345431 treatment combined with extensive treadmill training, we evaluated axons in the injured spinal cord with immunostaining using antibodies against GAP43 and serotonin (5-HT) (Figure 2), GAP43 is widely used as a marker for regenerated axons. In both treatment groups, a marked increase in the number of GAP43-positive axons was observed at the epicenter of the injury (Figure 2D-F) and in the surrounding area (Figure 2G-I). Compared with the control group, the number of GAP43 axons was significantly increased in both the SM-345431 treatment group and the combined treatment group, especially at 1 mm caudal to the injury epicenter (Figure 2J). No significant difference was observed between the 2 treatment groups. Thus, administration of the semaphorin3A inhibitor SM-345431 using this DDS enhanced axonal regeneration. However, no additional axonal regeneration was observed when SM-345431 treatment was combined with treadmill training.

Bottom Line: However, these effects were limited because most regenerated axons likely do not connect to the right targets.Although extensive treadmill training combined with SM-345431 administration did not further improve axon regeneration, hindlimb motor performance was restored, as evidenced by the significant improvement in the execution of plantar steps on a treadmill.This study highlights the importance of combining treatments that promote axon regeneration with specific and appropriate rehabilitations that promote rewiring for the treatment of spinal cord injury.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. hidokano@a2.keio.jp.

ABSTRACT

Background: Rats exhibit extremely limited motor function recovery after total transection of the spinal cord (SCT). We previously reported that SM-216289, a semaphorin3A inhibitor, enhanced axon regeneration and motor function recovery in SCT adult rats. However, these effects were limited because most regenerated axons likely do not connect to the right targets. Thus, rebuilding the appropriate connections for regenerated axons may enhance recovery. In this study, we combined semaphorin3A inhibitor treatment with extensive treadmill training to determine whether combined treatment would further enhance the "rewiring" of regenerated axons. In this study, which aimed for clinical applicability, we administered a newly developed, potent semaphorin3A inhibitor, SM-345431 (Vinaxanthone), using a novel drug delivery system that enables continuous drug delivery over the period of the experiment.

Results: Treatment with SM-345431 using this delivery system enhanced axon regeneration and produced significant, but limited, hindlimb motor function recovery. Although extensive treadmill training combined with SM-345431 administration did not further improve axon regeneration, hindlimb motor performance was restored, as evidenced by the significant improvement in the execution of plantar steps on a treadmill. In contrast, control SCT rats could not execute plantar steps at any point during the experimental period. Further analyses suggested that this strategy reinforced the wiring of central pattern generators in lumbar spinal circuits, which, in turn, led to enhanced motor function recovery (especially in extensor muscles).

Conclusions: This study highlights the importance of combining treatments that promote axon regeneration with specific and appropriate rehabilitations that promote rewiring for the treatment of spinal cord injury.

Show MeSH
Related in: MedlinePlus