Brief Electrical Stimulation Accelerates Axon Regeneration and Promotes Recovery Following Nerve Transection and Repair in Mice.

BACKGROUND: Clinical outcomes following nerve injury repair can be inadequate. Pulsed-current electrical stimulation (ES) is a therapeutic method that facilitates functional recovery by accelerating axon regeneration. However, current clinical ES protocols involve the application of ES for 60 minutes during surgery, which can increase operative complexity and time. Shorter ES protocols could be a strategy to facilitate broader clinical adoption. The purpose of the present study was to determine if a 10-minute ES protocol could improve outcomes. METHODS: C57BL/6J mice were randomized to 3 groups: no ES, 10 minutes of ES, and 60 minutes of ES. In all groups, the sciatic nerve was transected and repaired, and, in the latter 2 groups, ES was applied after repair. Postoperatively, changes to gene expression from dorsal root ganglia were measured after 24 hours. The number of motoneurons regenerating axons was determined by retrograde labeling at 7 days. Histomorphological analyses of the nerve were performed at 14 days. Function was evaluated serially with use of behavioral tests up to 56 days postoperatively, and relative muscle weight was evaluated. RESULTS: Compared with the no-ES group, both ES groups demonstrated increased regeneration-associated gene expression within dorsal root ganglia. The 10-minute and 60-minute ES groups demonstrated accelerated axon regeneration compared with the no-ES group based on increased numbers of labeled motoneurons regenerating axons (mean difference, 202.0 [95% confidence interval (CI), 17.5 to 386.5] and 219.4 [95% CI, 34.9 to 403.9], respectively) and myelinated axon counts (mean difference, 559.3 [95% CI, 241.1 to 877.5] and 339.4 [95% CI, 21.2 to 657.6], respectively). The 10-minute and 60-minute ES groups had improved behavioral recovery, including on grid-walking analysis, compared with the no-ES group (mean difference, 11.9% [95% CI, 3.8% to 20.0%] and 10.9% [95% CI, 2.9% to 19.0%], respectively). There was no difference between the ES groups in measured outcomes. CONCLUSIONS: A 10-minute ES protocol accelerated axon regeneration and facilitated functional recovery. CLINICAL RELEVANCE: The brief (10-minute) ES protocol provided similar benefits to the 60-minute protocol in an acute sciatic nerve transection/repair mice model and merits further studies.

Liposomes embedded within fibrin gels facilitate localized macrophage manipulations within nerve.

BACKGROUND: Understanding the role of macrophages at discrete spatial locations during nerve regeneration after injury is important. But, methodologies that systemically manipulate macrophages can obscure their roles within discrete spatial locations within nerve. NEW METHOD: Liposomes were embedded within fibrin gels to construct a delivery system that facilitated macrophage-specific manipulations at a sole spatial region, as macrophages accumulated within the fibrin. Clodronate liposomes were characterized for their toxicity to specific cells composing nerve in vitro, then tested for macrophage-specific depletion in vivo. This delivery system using clodronate liposomes was used to repair a mouse sciatic nerve gap to evaluate its efficacy and effects. RESULT: Clodronate liposomes showed specific toxicity to macrophages without affecting dorsal root ganglia (DRG)-derived neurons, endothelial cells, or Schwann cells in culture. The delivery system demonstrated sustained release of liposomes for more than 7 days while still retaining liposomes within the fibrin. In vivo, the delivery system demonstrated macrophages were targeted by liposomes, and the use of clodronate liposomes minimized macrophage accumulation within fibrin, while not affecting macrophage accumulation within DRG. Nerve regeneration across the nerve gap repaired using this delivery system was associated with decreased angiogenesis, Schwann cell accumulation, axon growth, and reinnervation of affected muscle. COMPARISON WITH EXISTING METHODS: This delivery system allowed specific perturbation of macrophages locally in nerve. This method could be applicable across species without the need for genetic manipulations or systemic pharmaceuticals. CONCLUSION: Liposomes embedded within fibrin gels locally target macrophages at the site of nerve injury, which enables greater precision in conclusions regarding their roles in nerve.

The CCL2/CCR2 axis is critical to recruiting macrophages into acellular nerve allograft bridging a nerve gap to promote angiogenesis and regeneration.

Acellular nerve allografts (ANAs) are increasingly used to repair nerve gaps following injuries. However, these nerve scaffolds have yet to surpass the regenerative capabilities of cellular nerve autografts; improved understanding of their regenerative mechanisms could improve design. Due to their acellular nature, both angiogenesis and diverse cell recruitment is necessary to repopulate these scaffolds to promote functional regeneration. We determined the contribution of angiogenesis to initial cellular repopulation of ANAs used to repair nerve gaps, as well as the signaling that drives a significant portion of this angiogenesis. Wild-type (WT) mice with nerve gaps repaired using ANAs that were treated with an inhibitor of VEGF receptor signaling severely impaired angiogenesis within ANAs, as well as hampered cell repopulation and axon extension into ANAs. Similarly, systemic depletion of hematogenous-derived macrophages, but not neutrophils, in these mice models severely impeded angiogenesis and subsequent nerve regeneration across ANAs suggesting hematogenous-derived macrophages were major contributors to angiogenesis within ANAs. This finding was reinforced using CCR2 knockout (KO) models. As macrophages represented the majority of CCR2 expressing cells, a CCR2 deficiency impaired angiogenesis and subsequent nerve regeneration across ANAs. Furthermore, an essential role for CCL2 during nerve regeneration across ANAs was identified, as nerves repaired using ANAs had reduced angiogenesis and subsequent nerve regeneration in CCL2 KO vs WT mice. Our data demonstrate the CCL2/CCR2 axis is important for macrophage recruitment, which promotes angiogenesis, cell repopulation, and subsequent nerve regeneration and recovery across ANAs used to repair nerve gaps.

Combination of Electrospun Nanofiber Sheet Incorporating Methylcobalamin and PGA-Collagen Tube for Treatment of a Sciatic Nerve Defect in a Rat Model.

BACKGROUND: For peripheral nerve defects, autografting is considered the therapeutic gold-standard treatment. However, this procedure leads to donor-site morbidity. While various artificial conduits have been recently developed, treatment outcome has been demonstrated to be poorer than that with autograft. In our previous study using a rat sciatic nerve crush injury model, we demonstrated that the delivery of electrospun nanofiber sheets incorporating methylcobalamin (MeCbl sheet) to the local site of a peripheral nerve injury promoted peripheral nerve regeneration. In this study, we examined the effects of combination therapy using an MeCbl sheet and a polyglycolic acid tube filled with collagen sponge (PGA-c) in a rat model of a 10-mm sciatic nerve defect. METHODS: The rats were divided into 4 groups: (1) sham group (n = 10); (2) PGA-c group (n = 9), in which the gap was bridged using a PGA-c; (3) PGA-c/Sheet group (n = 8), in which the gap was bridged using a PGA-c wrapped in an MeCbl sheet; and (4) autograft group (n = 10), in which the gap was bridged using a reversed autograft. Motor and sensory function were evaluated, electrophysiological analysis was performed, and histomorphological findings were analyzed at 12 weeks postoperatively. RESULTS: Compared with the PGA-c group, the PGA-c/Sheet group demonstrated significant improvements in the paw-withdrawal threshold expressed as a ratio relative to the contralateral side (mean difference [MD], -1.51; 95% confidence interval [CI], -2.64 to -0.38), terminal latency (MD, -0.86 ms; 95% CI, -1.56 to -0.16 ms), myelinated axon area (MD, 4.97%; 95% CI, 0.14% to 9.80%), proportion of myelinated axons (MD, 8.453%; 95% CI, 0.001% to 16.905%), and g-ratio (MD, -0.018; 95% CI, -0.035 to -0.001). No significant improvements were observed regarding motor function, electrophysiological findings with the exception of terminal latency, and axon numbers. CONCLUSIONS: An MeCbl sheet in combination with a PGA-c significantly accelerated recovery with respect to sensory function, electrophysiology, and histomorphometry. CLINICAL RELEVANCE: An MeCbl sheet may represent an effective therapeutic strategy for promoting regeneration across a nerve gap bridged with an artificial conduit.

A Nanofiber Sheet Incorporating Vitamin B12 Promotes Nerve Regeneration in a Rat Neurorrhaphy Model.

Outcomes of peripheral nerve repair after injury are often suboptimal. Therefore, developing biological approaches to augment nerve regeneration is important. In this in vivo study, we tested the hypothesis that augmentation with an electrospun nanofiber sheet incorporating methylcobalamin (MeCbl) would be effective for regeneration after peripheral nerve transection and repair. METHODS: Rats were divided into 3 groups that either underwent sciatic nerve repair with or without the MeCbl sheet, or a sham operation. At 4 and/or 8 weeks after the operation, sensory and motor functional recovery, along with histological findings, were compared among the groups using the toe-spreading test, mechanical and thermal algesimetry tests, tibialis anterior muscle weight measurements, electrophysiological analyses, which included nerve conduction velocity (NCV), compound muscle action potential (CMAP), and terminal latency (TL), and histological analyses involving the myelinated axon ratio, axon diameter, and total axon number. RESULTS: Compared with the repair group without the MeCbl sheet, the repair group with the MeCbl sheet showed significant recovery in terms of tibialis anterior muscle weight, NCV and CMAP, and also tended to improve in the toe-spreading test, mechanical and thermal algesimetry tests, and TL. Histological analyses also demonstrated that the myelinated axon ratios and axon diameters were significantly higher. Among these findings, the repair group with the MeCbl sheet demonstrated the same recovery in NCV as the sham group. CONCLUSION: This study demonstrated that electrospun nanofiber MeCbl sheets promoted nerve regeneration and functional recovery, indicating that this treatment strategy may be viable for human peripheral nerve injuries.

Neurotropin® Accelerates the Differentiation of Schwann Cells and Remyelination in a Rat Lysophosphatidylcholine-Induced Demyelination Model.

Neurotropin® (NTP), a non-protein extract of inflamed rabbit skin inoculated with vaccinia virus, is clinically used for the treatment of neuropathic pain in Japan and China, although its effect on peripheral nerve regeneration remains to be elucidated. The purpose of this study was to investigate the effects of NTP on Schwann cells (SCs) in vitro and in vivo, which play an important role in peripheral nerve regeneration. In SCs, NTP upregulated protein kinase B (AKT) activity and Krox20 and downregulated extracellular signal-regulated kinase1/2 activity under both growth and differentiation conditions, enhanced the expression of myelin basic protein and protein zero under the differentiation condition. In a co-culture of dorsal root ganglion neurons and SCs, NTP accelerated myelination of SCs. To further investigate the influence of NTP on SCs in vivo, lysophosphatidylcholine was injected into the rat sciatic nerve, leading to the focal demyelination. After demyelination, NTP was administered systemically with an osmotic pump for one week. NTP improved the ratio of myelinated axons and motor, sensory, and electrophysiological function. These findings reveal novel effects of NTP on SCs differentiation in vitro and in vivo, and indicate NTP as a promising treatment option for peripheral nerve injuries and demyelinating diseases.