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Corresponding author. Department of Orthopaedic Surgery and Sports Medical Science, Osaka University, Graduate School of Medicine, Suita, Osaka, Japan. Fax: +81-668793559.
Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, JapanDepartment of Sports Medical Science, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan
Peripheral nerve injuries are common and serious conditions. The effect of Neurotropin® (NTP), a nonprotein extract derived from the inflamed skin of rabbits inoculated with vaccinia virus, on peripheral nerve regeneration has not been fully elucidated. However, it has analgesic properties via the activation of descending pain inhibitory systems. Therefore, the current study aimed to determine the effects of NTP on peripheral nerve regeneration.
Methods
We examined axonal outgrowth of dorsal root ganglion (DRG) neurons using immunocytochemistry in vitro. In addition, nerve regeneration was evaluated functionally, electrophysiologically, and histologically in a rat sciatic nerve crush injury model in vivo. Furthermore, gene expression of neurotrophic factors in the injured sciatic nerves and DRGs was evaluated.
Results
In the dorsal root ganglion neurons in vitro, NTP promoted axonal outgrowth at a concentration of 10 mNU/mL. Moreover, the systemic administration of NTP contributed to the recovery of motor and sensory function at 2 weeks, and of sensory function, nerve conduction velocity, terminal latency, and axon-remyelination 4 weeks after sciatic nerve injury. In the gene expression assessment, insulin-like growth factor 1 and vascular endothelial growth factor expressions were increased in the injured sciatic nerve 2 days postoperatively.
Conclusions
Therefore, NTP might be effective in not only treating chronic pain but also promoting peripheral nerve regeneration after injury.
]. Although there are developments in medical science over the past century, the treatment outcomes of peripheral nerve injuries are often unsatisfactory. In cases of nerve injury with continuity, such as crush injury and entrapment neuropathy, microsurgery is not significantly effective. Therefore, pharmacological therapy is used to primarily enhance nerve regeneration in these cases. Recently, several growth factors have been extensively evaluated [
]. Nevertheless, their optimal administration methods and hazardous properties are still unknown, and they have not yet been assessed in clinical trials. Drug repositioning may be an ideal technique because approved drugs and those routinely used in clinical settings are utilized. This study focused on the effect of Neurotropin® (NTP), a nonprotein extract derived from the inflamed skin of rabbits inoculated with vaccinia virus, which has been widely used in Japan for over 25 years. NTP has been found to be effective against chronic pain and other various neurologic symptoms [
]. NTP is correlated with the neuronal expression of some neurotrophic factors. Fukuda et al. showed that NTP promoted nerve growth factor signaling and stimulated neurite outgrowth via the activation of Trk and downstream signaling molecules in PC12 cells [
]. However, the therapeutic effect of NTP against peripheral nerve injury remains to be elucidated. Therefore, the current study aimed to evaluate whether the administration of NTP can promote nerve regeneration and functional recovery in a rat sciatic nerve crush injury model.
2. Materials and methods
2.1 Isolation of dorsal root ganglion neurons and cell culture
Dorsal root ganglion (DRG) neurons were harvested and cultured, as described in a previous study with minor modifications [
]. In brief, DRG neurons were collected from Wistar rats at postnatal day 10 and were digested with 0.1% collagenase type I, 0.25% trypsin, and DNase I 210 U/mL for 30 min at an incubator with a temperature of 37 °C. Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum was added to inhibit enzymatic reaction. Then, after trituration and centrifugation at 800 rpm for 5 min, DRGs were resuspended in Sato medium [
] and were placed on poly-l-lysine (10 μg/mL)-coated four-well chambers.
2.2 Immunocytochemistry
DRG neurons were washed once with phosphate buffered saline (PBS) and were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. Next, DRG neurons were blocked with PBS containing 0.2% TritonX and 5% bovine serum albumin (BSA; Sigma–Aldrich, St. Louis, MO, the USA) for 30 min at room temperature and were incubated with primary antibodies overnight at 4 °C. Fluorescence-conjugated secondary antibodies were reacted for 1 h at room temperature. The nuclei were stained with PermaFluor Aqueous Mounting Medium containing 4′,6-diamidino-2-phenylindole (DAPI) (D532; DOJINDO, Kumamoto, Japan). The primary and secondary antibodies were neuronal class III β-tubulin (Tuj1) mouse monoclonal antibody (1:1000; Covance, Dedham, MA, the USA) and Alexa Fluor 488 goat anti-mouse IgG (1:1000; Life Technologies, Carlsbad, CA, the USA), respectively.
2.3 Measurement of the axonal length
DRG neurons were placed on four-well chambers with a density of 5 × 104 cells per each well and were cultured in Sato medium. Then, 24 h after incubation, they were treated with NTP (1 μ–100 mNU/mL; Nippon Zoki Pharmaceutical Co., Ltd., Osaka, Japan) for 48 h and were immunostained using the abovementioned methods. The axonal length was assessed using a fluorescent microscope (Eclipse 90i, Nikon, Tokyo, Japan) and a digital camera (DS-Ri1 and DS-U3, Nikon), as described in a previous study [
]. At least 30 neurons were identified for each treatment group. This experiment was replicated three times using different rats.
2.4 Surgical procedures
All animal experiments were approved by the Ethics Review Committee for Animal Experimentation of the authors’ affiliated institutions. In total, 47 male Wistar rats weighing 180–220 g (Charles River Laboratories Japan, Inc., Yokohama, Japan) were used in this study. In all experiments, the animals were deeply anesthetized via the subcutaneous injection of midazolam (2 mg/kg), butorphanol (2.5 mg/kg), and medetomidine (0.15 mg/kg). Under sterile conditions, the left sciatic nerve was exposed from the sciatic notch to its bifurcation into the tibial and peroneal nerves. The sciatic nerve was crushed 5 mm distal from the sciatic notch three times (10 s each), with an interval of 10 s using forceps (Dumont No. 5), as described in a previous study [
Electrospun nanofiber sheets incorporating methylcobalamin promote nerve regeneration and functional recovery in a rat sciatic nerve crush injury model.
]. Then, the fascia and the skin were closed using a nylon suture. In the untreated group (n = 5 for 3 days, n = 6 for 2 weeks, and n = 12 for 4 weeks postoperative experiments), saline was administered systemically with an osmotic minipump immediately after the induction of crush injury (Model 2ML2; Alzet Cupertino, CA, the USA). In the treated group (n = 5 for 3 days, n = 6 for 2 weeks, and n = 13 for 4 weeks postoperative experiments), NTP at a dose of 12 NU/kg/day was administered systemically with an osmotic minipump, which was placed subcutaneously in the back immediately after the induction of nerve injury. The dosage of NTP was determined based on a previous report with a slight modification [
]. Two and four weeks after surgery, the hindpaws were painted with black ink, and the rats were allowed to walk on a narrow track. Using the footprints, the following parameters were measured: print length (PL), which is the distance from the heel to the toe; toe spread (TS), which is the distance from the first to the fifth toes; and intermediary toe spread (ITS), which is the distance from the second to the fourth toes. Then, SFI was calculated as follows: SFI [−38.3 × (EPL − NPL)/NPL] + [109.5 × (ETS − NTS)/NTS] + [13.3 × (EITS − NITS)/NITS] − 8.8 (E = experimental, N = normal).
Mechanical sensitivity, a sensory function, was assessed using calibrated von Frey filaments (0.008–26 g; Touch Test, North Coast Medical Inc., Gilroy, CA, the USA), as described in a previous study [
]. In brief, 2 and 4 weeks after surgery, the rats were placed on an elevated metal mesh, and von Frey filaments were applied to the plantar surface of the hindpaw innervated by the sciatic nerve until they were bent, and paw-withdrawal thresholds were recorded. Then, the ratio of paw-withdrawal thresholds (ipsilateral/contralateral) of each rat was calculated.
However, rats with toe autotomy (two in the untreated group and five in the treated group) were not included in these analyses at 4 weeks postoperatively. No autotomies were observed among the rats sacrificed at 2 weeks postoperatively.
2.6 Electrophysiology
An electrophysiological analysis was performed 4 weeks after surgery, as described in a previous study [
Methylcobalamin promotes the differentiation of Schwann cells and remyelination in lysophosphatidylcholine-induced demyelination of the rat sciatic nerve.
]. Rats were anesthetized and placed in prone position; then, the left sciatic nerve and the left tibialis anterior muscle were exposed. For stimulation, a pair of electrodes was applied noninvasively on the proximal side of the injury site, and the compound muscle action potentials (CMAP) and the terminal latency (TL) were recorded by placing a recording electrode in the tibialis anterior muscle. The nerve conduction velocity (NCV) was calculated by comparing records of two different points across the injury site. These parameters were evaluated using the PowerLab device and software (AD Instruments, Bella Vista, NSW, Australia). However, two rats in the treated group died just before this experiment due to the anesthesia, so these rats were not included in this analysis.
2.7 Histology
In the evaluation of axonal myelination, animals were sacrificed 4 weeks after surgery, as described in a previous study [
Methylcobalamin promotes the differentiation of Schwann cells and remyelination in lysophosphatidylcholine-induced demyelination of the rat sciatic nerve.
]. Briefly, the injured sciatic nerve was harvested, embedded in 4% PFA for 5 days at room temperature, and then stored in 20% sucrose in 0.01 M PBS for 24 h. Each nerve was immersed in the frozen section compound (Leica Biosystems, Wetzlar, Germany) and was frozen. Then, 5-μm axial sections of the damaged site were established and were mounted on glass slides. The slides were permeabilized with 100% methanol, blocked with PBS +0.2% TritonX +5% BSA, and reacted with primary antibodies overnight at 4 °C, followed by incubation with fluorescence-conjugated secondary antibodies for 1 h at room temperature. The nuclei were stained with PermaFluor Aqueous Mounting Medium containing DAPI (D532; DOJINDO). The number of total axons and myelinated axons were calculated from three random microscopic fields of each sample at 200 × magnification using the NIS Elements BR software (Nikon, Tokyo, Japan). The primary antibodies were MBP (1:1000; NE1018; Calbiochem, Tokyo, Japan) and NF200 (1:1000; N4142; Sigma–Aldrich). The secondary antibodies were Alexa Fluor 488 goat anti-rabbit IgG antibody (1:1000; Life Technologies) and Alexa Fluor 568 goat anti-mouse IgG antibody (1:1000; Life Technologies). However, three rats in the untreated group and four rats in the treated group died before the harvest of sciatic nerves due to anesthesia, so these rats were not included in this analysis.
To evaluate the extent of mononuclear cell infiltration, the sciatic nerve was harvested at 3 days after surgery, fixed in 4% PFA, transferred to 20% sucrose, embedded in the frozen section compound, frozen using liquid nitrogen, then the damaged site was sliced into 5-μm axial sections. Sections were stained with hematoxylin-eosin (H&E). Infiltrating inflammatory cells were counted using the Aperio Image Scope software (Leica Biosystems, Wetzlar, Germany). Then cell numbers per square millimeter were calculated from three random microscopic fields at 200 × magnification.
2.8 Gene expression analysis
Sciatic nerves were harvested 2 weeks after surgery, and homogenized with TissueLyser and QIAzol Reagent (QIAGEN, Hilden, Germany). Total RNA was isolated from the homogenate using an RNeasy Micro Kit (QIAGEN) according to the manufacturer's instructions. Total RNA was reverse transcribed using the SuperScript VILO Master Mix (QIAGEN). Real-time PCR was performed using SYBR Green Master Mix (Thermo Scientific) according to the manufacturer's instructions, and data were generated on a StepOnePlus Real-Time PCR System (Thermo Scientific). The amount of RNA in the samples was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers (forward and reverse, respectively) used here were as follows: GAPDH (5′-GAACGGGAAGCTCACTGGC-3′ and 5′-GCATGTCAGATCCACAACGG-3′), nerve growth factor (NGF) (5′-TCCAGGTGCATAGCGTAATG-3′ and 5′-CTCCGGTGAGTCCTGTTGAA-3′), brain-derived neurotrophic factor (BDNF) (5′-GGTATCAAAAGGCCAACTGA-3′ and 5′-GCAGCCTTCCTTGGTGTAAC-3′), glial cell-derived neurotrophic factor (GDNF) (5′-GAGAGAGGAACCGGCAAGCT-3′ and 5′-GCGACCTTTCCCTCTGGAAT-3′), insulin-like growth factor 1 (IGF1) (5′-AGACGGGCATTGTGGATGA-3′ and 5′-ACATCTCCAGCCTCCTCAGATC-3′), platelet-derived growth factor (PDGF) (5′-GTTCGGACGGTGCGAATC-3′ and 5′-GTGTGCTTAAACTTTCGGTGCTT-3′), vascular endothelial growth factor (VEGF) (5′-ACGACAGAAGGGGAGCAG-3′ and 5′-AGATGTCCACCAGGGTCTCA-3′).
2.9 Statistics
Data were expressed as mean ± standard error of measurement. Statistical analyses were performed using one-way analysis of variance followed by the post hoc Dunnett's test for a multiple comparison and the Student's t-test for 2-group comparisons.
3. Results
3.1 NTP promoted axonal outgrowth in vitro
Whether the administration of NTP can promote nerve regeneration and the most effective concentration remain unclear. To determine whether NTP is effective in enhancing axonal outgrowth, DRG neurons were cultured with NTP at stepwise concentrations (range: 1 μNU/mL–100 mNU/mL). NTP was found to promote the axonal outgrowth of DRG neurons with a significant difference at a concentration of 10 mNU/mL compared with the control (Fig. 1A and B).
Fig. 1(A) Representative immunocytochemical images of the axons (green, Tuj1). Scale bars, 50 μm (B) Quantification of the axonal length (Mean ± standard error, n = 3 for each group).
3.2 NTP promoted the recovery of motor and sensory functions after sciatic nerve crush injury
NTP promoted the axonal outgrowth of DRG neurons in vitro, as mentioned above. To determine whether NTP was also effective in promoting peripheral nerve regeneration in vivo, we first assessed motor function in a rat sciatic nerve crush injury model. Motor recovery after sciatic nerve injury was evaluated using the SFI, which is a reliable method for assessing the recovery of foot muscle innervation. In addition, there was no correlation between walking track parameters and maximal muscle forces because maximal effort is not necessary for walking [
]. Therefore, SFI using walking track analysis is effective in assessing the middle stage of recovering muscle forces. The SFI value of the treated group was significantly higher than that of the untreated group 2 weeks after surgery (−50.5 ± 3.6 and −70.9 ± 3.3, respectively), but those at 4 weeks postoperatively did not significantly differ (−15.9 ± 2.3 and −24.7 ± 3.5, respectively, p = 0.06) (Fig. 2A).
Fig. 2(A) Motor and (B) sensory function recovery assessment (Mean ± standard error, n = 6 for the untreated group and n = 6 for the treated group for 2 weeks, n = 10 and 8 for 4 weeks postoperative, respectively).
Next, sensory function was evaluated based on pain reaction elicited after a touch stimulus using von Frey filaments. Rats with crush nerve injury had higher mechanical thresholds in the sciatic territory of the ipsilateral hindpaw. Moreover, mechanical thresholds and sensory function recovery returned to baseline levels [
]. In this study, the ratio of mechanical thresholds (ipsilateral/contralateral) in the treated group was significantly lower than that in the untreated group both 2 weeks (1.33 ± 0.15 and 3.22 ± 0.61, respectively) and 4 weeks postoperatively (1.13 ± 0.19 and 2.14 ± 0.20, respectively) (Fig. 2B). Hence, NTP promoted the recovery of motor and sensory functions after sciatic nerve injury.
3.3 NTP promoted the recovery of TL and NCV after sciatic nerve crush injury
Four weeks after surgery, we performed an electrophysiological analysis to determine whether NTP also promoted muscle reinnervation of the injured nerve. We did not detect a significant difference in CMAP (Fig. 3A) between the untreated group (7.38 ± 0.66 mV) and the treated group (7.83 ± 0.57 mV). On the other hand, the treated group had significantly shorter TL values (3.10 ± 0.10 vs 3.45 ± 0.12 ms) (Fig. 3B) and faster NCV values (29.9 ± 1.5 vs 19.7 ± 1.25 m/s) (Fig. 3C) than the untreated group. These results suggested that NTP promoted nerve regeneration even though the recovery was premature.
Fig. 3Electrophysiological evaluations by (A) compound muscle action potentials, (B) terminal latency, and (C) nerve conduction velocity (Mean ± standard error, n = 12 for the untreated group and n = 11 for the treated group).
3.4 NTP did not influence the number of infiltrating inflammatory cells
Three days after surgery, we performed a histological analysis using H&E staining to evaluate the effect of NTP on the inflammatory cell infiltration in the acute phase. The number of infiltrating mononuclear cells in the NTP-treated group showed no significant difference compared with the untreated group (Fig. 4A and B). Therefore, NTP did not influenced the number of infiltrating inflammatory cells.
Fig. 4(A) Light micrographs (H&E staining). (B) Infiltrating mononuclear cells number (n = 5 for both groups). (C) Fluorescence micrographs labeled for MBP (red) and NF200 (green). (D) Myelinated axon ratio (n = 9 for both groups) (Mean ± standard error).
]. To investigate the process of regeneration, we performed a histological analysis 4 weeks after the injury, and remyelination was assessed. The ratio of myelinated axon in the treated group, compared with the untreated group, significantly increased (79.9% ± 1.1% vs 67.1% ± 1.5%) (Fig. 4C and D). Therefore, NTP promoted nerve regeneration, particularly myelination.
3.6 NTP increased the gene expression of trophic factors within the injured nerve
To evaluate the effect of NTP on the release of trophic factors during the nerve regeneration process, mRNA was extracted from the injured sciatic nerve and DRG 2 weeks postoperatively and gene expression levels were measured. The expression of IGF1 and VEGF were significantly upregulated in the sciatic nerve in the treated group compared with the untreated group (Fig. 5). On the other hand, for NGF, BDNF, GDNF, and PDGF, there was a tendency of higher expression mainly in the sciatic nerve in the treated group, but there were no significant differences.
Fig. 5Relative expression levels of mRNA extracted from the injured sciatic nerves and DRGs (Mean ± standard error, n = 6 for both groups).
To date, the effect of NTP on neurons, particularly axons in vitro, has only been partly identified. Taneda et al. showed that the administration of NTP at doses of 10, 30, 100, and 300 mNU/mL had no effect on neurite outgrowth in DRG neurons in the absence of nerve growth factors [
Neurotropin inhibits both capsaicin-induced substance P release and nerve growth factor-induced neurite outgrowth in cultured rat dorsal root ganglion neurones.
]. Furthermore, NTP was reported to inhibit axonal transport property in cultured mouse DRG neurons at a concentration ranging from 100 μNU/mL to 1 NU/mL [
]. These results have been described as directing the pain suppression and antipruritic effects of NTP. However, whether the administration of NTP influenced axonal outgrowth remains unclear. In the current study, NTP promoted the axonal outgrowth of DRG neurons at a concentration of 10 mNU/mL (Fig. 1A and B). This is not consistent with the above report showing no effect of NTP on neurite outgrowth but may be due to the contents of culture medium used (e.g., serum-free or not). The detailed mechanism of the compatibility of the regeneration-promoting and pain-suppressing effects of NTP on neuronal cells remains to be further investigated.
After peripheral nerve injury, the distal axons undergo Wallerian degeneration. Then, Schwann cells at the injury site dedifferentiate, proliferate, and establish a line to guide axons toward the target organs [
]. No previous study has focused on the efficacy of NTP in promoting peripheral nerve regeneration in a rat sciatic nerve crush injury model. The current study showed that NTP improved motor and sensory functions (Fig. 2), TL, NCV (Fig. 3B and C), and myelinated ratio based on a histological analysis (Fig. 4D), but not CMAP in vivo. Navarro X et al. classified the full process of nerve regeneration into three general phases: regeneration of axons, reinnervation of targets, and recovery of functions. They also recommended that different in vivo tests must be performed to classify the phase of nerve regeneration [
]. Electrophysiological analysis could be a key method for evaluating motor axon regeneration and muscle reinnervation. TL includes the conduction time from stimulating the site to the distal end and the transmission time across the neuromuscular junction. NCV is a local indicator of nerve regeneration near the injury site. The amplitude of the CMAP is correlated with the number of muscle fibers innervated, and full recovery in the sciatic nerve crush injury model requires more than 90 days. In addition, a histological analysis around the injury site is an important method for assessing local nerve regeneration. In the site of crush injury, nerve degeneration was observed, followed by axonal sprouting, enlarging, and remyelination [
]. Our results showed that the administration of NTP might promote not only local regeneration around the injury site, as shown in the histological evaluation, but also accelerated functional recovery, as SFI improved significantly at 2 weeks postoperatively and paw withdrawal threshold was restored to the same level as the contralateral side through 4 weeks after surgery. In SFI test, no significant difference was observed between the two groups at 4 weeks, but SFI is an effective analysis to assess the middle stage of recovering muscle forces as mentioned in the result section. Sufficient recovery of motor function is considered to need more than 4 weeks after crush injury, then a longer follow-up duration after the nerve injury might help identify a significantly better outcome in CMAP as an indicator of mature motor function. A previous study showed that NTP accelerates the differentiation of Schwann cells in vitro and promotes remyelination and functional recovery in a rat lysophosphatidylcholine-induced demyelination model [
]. Hence, NTP may have a therapeutic effect in the current model via not only neurons but also Schwann cells.
A recent report by Fukuda et al. suggested that unidentified lipid-rafts-like fractions formed on PC12 cells by NTP administration act as a site of crosstalk between neurotrophic factor receptors and innate immune receptors [
]. In addition, Nishimoto et al. reported that NTP reduced the local inflammation of injured nerve in a mouse sciatic nerve chronic constriction injury model [
]. These results suggest that NTP may elicit a neuroprotective effect by reducing neuroinflammation. Although NTP did not affect the number of recruited inflammatory cells in the acute phase of the injury (Fig. 4B), it is possible that NTP may have reduced inflammation and promoted axonal growth by exerting neuroprotective effects through the crosstalk described above under inflammatory conditions after inflammatory cell infiltration. Further elucidation is awaited.
Previous studies have reported different pathways for axonal outgrowth mediated by neurotrophins-neurotrophic receptors, such as nerve growth factor-tropomyosin-related kinase A (TrkA), brain-derived neurotrophic factor-TrkB, and neurotrophin-3-TrkC, and their downstream molecules [
]. However, which signaling pathway was closely correlated with NTP-induced axonal outgrowth has not been investigated yet. In this study, we found a significant increase in IGF1 and VEGF gene expression in the damaged sciatic nerve 2 weeks after surgery. Regarding IGF1, its effects of promoting elongation and guidance of corticospinal motor neurons have been reported [
]. In addition, VEGF has been demonstrated to have an axon guidance effect on commissural neurons and neurotrophic properties through retrograde transport in abducens motoneurons [
]. Furthermore, although not significantly, NGF and PDGF also showed a higher tendency compared with the untreated group. This suggests that NTP may also exert its regeneration-promoting activity by increasing these neurotrophic factors mainly in the sciatic nerve.
The current study contained a few limitations. First, the main active ingredient of NTP is yet to be elucidated. One of the reasons is that NTP comprises several components, including nucleic acids, amino acids, and sugars (unpublished observations). Finally, the local concentration of NTP around the affected neurons is not fully elucidated. Although NTP at a dose of 10 mNU/mL significantly promoted axonal outgrowth in vitro, the measurement of local NTP concentration is challenging because the active ingredient has not been identified, as mentioned in the previous text. Therefore, future studies must be conducted to address these limitations.
5. Conclusion
This study aimed to investigate whether NTP is effective in stimulating nerve regeneration and functional recovery in vivo in a rat sciatic nerve crush injury model. Results showed that NTP promoted axonal outgrowth at a concentration of 10 mNU/mL in vitro. Moreover, the systemic administration of NTP contributed to the recovery of motor and sensory functions, NCV, and TL, and to the promotion of myelination after sciatic nerve injury. Therefore, NTP may be effective in not only treating chronic pain but also promoting peripheral nerve regeneration.
Funding
This work was supported by Nippon Zoki Pharmaceutical Co., Ltd., in Japan [J180701127].
Ethics
All animal experiments were approved by the Ethics Review Committee for Animal Experimentation of the Graduate School of Medicine, Osaka University (approval number: 24-026-010).
Declaration of competing interest
The funding sponsor, Nippon Zoki Pharmaceutical Co., Ltd., had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. The authors have declared that no further conflict of interest exists.
Acknowledgments
We thank all the members of Okada's laboratory for the helpful discussion and comments. The authors would like to thank Enago (www.enago.jp) for the English language review.
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Electrospun nanofiber sheets incorporating methylcobalamin promote nerve regeneration and functional recovery in a rat sciatic nerve crush injury model.
Methylcobalamin promotes the differentiation of Schwann cells and remyelination in lysophosphatidylcholine-induced demyelination of the rat sciatic nerve.
Neurotropin inhibits both capsaicin-induced substance P release and nerve growth factor-induced neurite outgrowth in cultured rat dorsal root ganglion neurones.
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