C3 peptide promotes axonal regeneration and functional motor recovery after peripheral nerve injury
Stefanie C Huelsenbeck et al. Neurotherapeutics. 2012 Jan.
Abstract
Peripheral nerve injuries are frequently seen in trauma patients and due to delayed nerve repair, lifelong disabilities often follow this type of injury. Innovative therapies are needed to facilitate and expedite peripheral nerve regeneration. The purpose of this study was to determine the effects of a 1-time topical application of a 26-amino-acid fragment (C3(156-181)), derived from the Clostridium botulinum C3-exoenzyme, on peripheral nerve regeneration in 2 models of nerve injury and repair in adult rats. After sciatic nerve crush, different dosages of C3(156-181) dissolved in buffer or reference solutions (nerve growth factor or C3(bot)-wild-type protein) or vehicle-only were injected through an epineurial opening into the lesion sites. After 10-mm nerve autotransplantation, either 8.0 nmol/kg C3(156-181) or vehicle were injected into the proximal and distal suture sites. For a period of 3 to 10 postoperative weeks, C3(156-181)-treated animals showed a faster motor recovery than control animals. After crush injury, axonal outgrowth and elongation were activated and consequently resulted in faster motor recovery. The nerve autotransplantation model further elucidated that C3(156-181) treatment accounts for better axonal elongation into motor targets and reduced axonal sprouting, which are followed by enhanced axonal maturation and better axonal functionality. The effects of C3(156-181) are likely caused by a nonenzymatic down-regulation of active RhoA. Our results indicate the potential of C3(156-181) as a therapeutic agent for the topical treatment of peripheral nerve repair sites.
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Figures Fig. 1.
Schematic drawing of the sciatic nerve injury and repair models used in the presented study. (a) After sciatic nerve crush, 1 of 6 different solutions was injected into the crush lesion through an epineurial window, which was closed afterward by a single epineurial suture. (b) After bridging a 10-mm sciatic nerve gap with an autotransplant, either vehicle alone or 8 nmol/kg C3156-181 was injected into each suture site. PBS = phosphate buffered saline Fig. 2.
Results after injection of toluidine vital dye into the crush lesion. Photographs were taken directly (a) and (c) and 24 h (b) and (d) after injection of 40 μL of toluidine blue in 2 different nerves. The thin suture marks the closed epineurial window. (a-d) Scale bar = 5 mm. (e, f) 10-μm longitudinal cryosections through a third sample revealed that the vital dye was distributed also inside the nerve approximately 10-mm proximal to the injection site Fig. 3.
Analysis of motor recovery after nerve crush injury, as evaluated by the Static Sciatic Index (SSI) and calculation of the gastrocnemius muscle weight ratio. (a) Mean ± SEM values of the SSI determined 2 weeks and 3 weeks after surgery. A significant difference to the phosphate buffered saline (PBS) control group is seen at first in the 40 nmol/kg C3156-181 group 2 weeks after surgery. Best motor recovery is indicated in the C3156-181-treated groups at 3 weeks after surgery. (b) The mean ± SEM value of the gastrocnemius muscle weight ratio is correlated to the motor recovery level determined by the SSI. Treatment with 8.0 nmol/kg and 40 nmol/kg C3156-181 significantly improved the obtained regeneration values. NGF = nerve growth factor Fig. 4.
Nerve fiber densities as determined by nerve morphometry 4-mm distal to the crush lesion at 3 weeks after surgery. (a) Bar graph depicting the number of regenerated myelinated axons/mm2. Treatment with C3156-181 significantly increased axonal regeneration. (b) and (c) Details of semi-thin cross section through regenerated nerves after phosphate buffered saline (PBS) (b), or 1.6 nmol/kg C3156-181 (c) treatment. Arrowheads point to onion bulb formations of degenerated axons that are still present. Scale bar = 20 μm. NGF = nerve growth factor Fig. 5.
Analysis of regeneration parameters 4 weeks after 10-mm nerve autotransplantation. (a) Bar graph depicting a significant right shift of the evocable movement score after treatment with C3156-181. (b) Four weeks after surgery, significantly more animals of the C3156-181 group, as in the phosphate buffered saline (PBS) group displayed a gastrocnemius muscle weight ratio greater than the control group median. (c) The density of regenerated myelinated axons at 1-mm distal to the autotransplants at 4 weeks after surgery was significantly reduced after C3156-181 treatment Fig. 6.
Analysis of regeneration parameters at 10 weeks after 10-mm nerve autotransplantation. (a) 10 weeks after surgery significantly more animals of the C3156-181 group, as in the phosphate buffered saline (PBS) group displayed a motor nerve conduction velocity ratio greater than the control group median. (b) and (c) Nerve morphometry at 1-mm distal to the autotransplants 10 weeks after surgery. Treatment with 8.0 nmol/kg C3156-181 significantly increased axonal diameters (b) and myelin thickness (c) of the regenerated myelinated axons Fig. 7.
Prevention of RhoA activation in vivo by C3156-181 peptide or C3bot-wild-type protein. (a) GTP-RhoA immunoprecipitation and Western blot analysis demonstrating reduced levels of active GTP-RhoA in contralateral and ipsilateral sciatic nerves after treatment with C3156-181 peptide and C3bot-wild-type protein in comparison to phosphate buffered saline (PBS) treatment. (c) Densitometry and normalization against β-actin allowed the calculation of the ratio between total RhoA input (middle level) in (b) and GTP-RhoA levels (upper level) in (b). The bar graph underscores the prevented activation of RhoA by treatment with C3156-181 peptide or C3bot-wild-type protein in contrast to application of PBS
References
Noble J, Munro CA, Prasad VS, Midha R. Analysis of upper and lower extremity peripheral nerve injuries in a population of patients with multiple injuries. J Trauma. 1998;45:116–122. doi: 10.1097/00005373-199807000-00025. - DOI - PubMed
Asplund M, Nilsson M, Jacobsson A, von Holst H. Incidence of traumatic peripheral nerve injuries and amputations in Sweden between 1998 and 2006. Neuroepidemiology. 2009;32:217–228. doi: 10.1159/000197900. - DOI - PubMed
Ciardelli G, Chiono V. Materials for peripheral nerve regeneration. Macromol Biosci. 2006;6:13–26. doi: 10.1002/mabi.200500151. - DOI - PubMed
Deumens R, Bozkurt A, Meek MF, et al. Repairing injured peripheral nerves: Bridging the gap. Prog Neurobiol. 2010;92:245–276. doi: 10.1016/j.pneurobio.2010.10.002. - DOI - PubMed
Lundborg G, Rosen B. Nerve injury and repair — a challenge to the plastic brain. J Peripher Nerv Syst. 2003;8:209–226. doi: 10.1111/j.1085-9489.2003.03027.x. - DOI - PubMed