OBJECTIVE

Traumatic neuromas represent a prevalent source of neuropathic pain. As of yet, there has been no single treatment method that can guarantee permanent relief of symptoms. Although nerve-capping techniques have shown promise, their exact mechanisms remain elusive. The authors’ aim was to examine the role of the RhoA/ROCK signaling pathway in the prevention of neuroma formation after neurectomy utilizing a nerve-capping technique.

METHODS

An aligned nanofiber tube was fabricated to cap the sciatic nerve in Sprague Dawley rats. The rats (n = 60) were randomly divided into the aligned SF/P (LLA-CL) capping group (capping group, n = 20), the capping andY-27632 (ROCK pathway inhibitor) intervention group (intervention group, n = 20), and the no-capping group (control group, n = 20). The authors undertook a comprehensive assessment of the capping group, examining the animals’ behavior, the extent of neuroma development, histology, gene and protein expression, and ultrastructural changes associated with the RhoA/ROCK signaling pathway. These findings were compared with those in the intervention and control groups.

RESULTS

The inciting injury resulted in the expression of the RhoA/ROCK signaling pathway, as well as its further upregulation in peripheral neurons. Axon outgrowth was significantly increased when RhoA/ROCK signaling pathway was suppressed. The average autotomy score in the capping group was observed to be much lower than that of the intervention and control groups. At 30 days postneurectomy, the capping group displayed no obvious neuroma formation, while a bulbous neuroma was found in the nerve stumps of both the control and intervention groups. Quantitative realtime polymerase chain reaction and the Western blot analysis demonstrated that the expression of myelin-associated glycoprotein was substantially upregulated in the capping group; in contrast, the expression of NF-200 was significantly downregulated. The expression of myosin light chain was notably lower in the intervention group, but there was no significant difference when compared with the control group (p > 0.05).

CONCLUSIONS

The RhoA/ROCK signaling pathway has emerged as a critical player in the process of traumatic neuroma formation after neurectomy. It is possible that the nerve-capping technique could generate a “regenerative brake” based on the regulation of the RhoA/ROCK signaling pathway in this event. These findings may provide concrete evidence that could help develop new strategies for the management of painful neuromas.

KEYWORDS:RhoA/ROCK signaling pathway; painful traumatic neuroma; Y-27632; aligned nanofiber nerve conduit; peripheral nerve

TraumaTic neuromas represent a prevalent source of neuropathic pain and are a challenge that many surgeons must confront in the operating room.5,29 One treatment method for this condition involves altering the region around the nerve stump so as to prevent addition-al neuroma formation after the resection of a neuroma.10 Despite the publication of numerous experimental and clinical treatment modalities, no single treatment method for painful neuromas has emerged to guarantee permanent symptom relief.12,24 Nerve capping, one of the most common surgical treatments for neuroma, is purported to facilitate nerve regeneration of the severed nerve fibers within the conduit, decreasing the potential for additional neuromas.5,10,20,29 In our previous studies, we found that nerve capping with an aligned nanofiber conduit could prevent the formation of traumatic neuromas.29 Nonetheless, the mechanism of this technique is still under investigation.RhoA, a small GTPase, belongs to a subfamily of proteins with highly conserved signaling pathways that are part of the Ras superfamily of G proteins.4,28 RhoA, a regulator of many cellular functions, facilitates cell motility, cytoskeleton rearrangement, intracellular trafficking, transcriptional regulation, cellular clearing through phagocytosis, and cell growth.11,14 The literature contains several animal studies indicating that RhoA/ROCK signaling is involved in a number of CNS diseases.11,19,22 Evidence exists that RhoA GTPase is present and further activated in response to trauma in peripheral neurons, resulting in a higher concentration of axon overgrowth when RhoA/ Rho-associated coiled coil-forming kinase (ROCK) signaling pathway is suppressed. Given this, a more in-depth understanding of the alterations in RhoA’s gene expression may offer novel and useful information needed for the management of traumatic neuroma.

In our previous research, we made use of an aligned nanofiber tube to cap the nerve, which altered the outgrowth status of severed nerves and inhibited the occurrence of neuromas.29 We found that the nerve conduit could improve the maturity of Schwann cells, upregulate the expression levels of myosin light chain (MLC) and other cytokines, induce the axial extension of regenerated nerve and collagen fibers, and inhibit their overregeneration, contributing to the prevention of traumatic neuroma formation.29 We hypothesized that the creation of the “regenerative brake” originated from the peripheral neurons—brought on by morphological and structural changes in the nerve stump—played a vital role in the capping technique and thus in the prevention of painful neuromas.29 Nonetheless, in our previous studies, we only found that the nerve-capping procedure could prevent the formation of traumatic neuromas. There remain many unknowns regarding how the “regenerative brake” occurs and its exact mechanism is still uncertain. Therefore, in this investigation, we hypothesized that activation of the RhoA/ROCK signaling pathway might contribute to the initiation of the “brake” in the nerve-capping procedure and might point the way toward a new strategy for treating painful neuromas.

Methods

Experimental Design

This research was authorized by the Ethical Committee of Wenzhou Medical University. Sprague Dawley (SD) rats were treated in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. All rats were purchased from the Wenzhou Medical University Centre for Laboratory Animals. During the experiments, the animals were individually kept at 26。C ± 2。C and had ad libitum access to food and water; we utilized a 12-hour light-dark cycle with the lights turned on at 6:00 am. Adult male SD rats (n = 60, weighing 250– 300 g) were randomly divided into one of the following groups: aligned silk fibroin (SF) mixed with poly(l-lactic acid-co-e-caprolactone) (SF/P [LLA-CL]) capping group (capping group), capping and Y-27632 (ROCK pathway inhibitor) intervention group (intervention group), and the no-capping group (control group).

Surgical Procedure

The surgical procedure was performed based on our previous study.29 Rats were anesthetized using an intraperitoneal injection of sodium pentobarbital (50 mg/kg) and were then placed in the prone position. After both lower limbs were shaved, the animals were disinfected using povidone iodine and then draped. The right sciatic nerve was dissected between the gluteal and the biceps femoris muscles. To quantitatively assess neuroma growth, a marker was made using a 7-0 suture at the point where the posterior gluteal nerve originates under microscopy. We then undertook a sharp transection of the sciatic nerve 1 cm distal to the marked point with the help of a 1-cmlong plastic tube segment. In each case, the transected elements of the distal nerve were resected at least 2 cm long to avoid spontaneous nerve regeneration. As in our previous study, in both the capping and intervention groups, an open-ended aligned SF/P (LLA-CL) nanofiber conduit with a length of 15 mm and an inner diameter of 1.5 mm was placed over the nerve stump of 4 mm in length.9,16, 26,27,29 The nerve stump was then secured to the conduit using an 11-0 monofilament nylon suture (Fig. 1). After hemostasis, the wounds were sutured with 4-0 nylon, and the animals were returned to their cages separately. All operations were carried out using an operative microscope by the same surgeons on one team; ibuprofen was given daily for 7 days after surgery. Each rat was injected with Y-27632 (a volume of 10 mg/[kg x day]) intraperitoneally according to published studies8,9,17 at 1, 4, 7, 10, 13, 16, 19, 22, 25, and 28 days after surgery in the intervention group, whereas the same volume of normal saline was administered in the capping and control groups following the same protocol for 28 days, respectively.9 Of the 60 rats, 1 died 3 days (capping group) and 1 at 3 and 5 days (1 died at 3 days [capping group] and 1 died in 5 days [control group]) after surgery in each group. The rats in all groups were used for behavioral analysis after surgery; the animals were sacrificed 30 days after the operation. The proximal nerve stump (PNS) was cut on both sides (the marked site and the contralateral side) into 1-cm-long segments. Of the 19 specimens harvested in each group at the end of the experiment, 6 were selected at random for histological studies. In the center of the neuroma, a segment measuring approximately 1 x 1 mm was first harvested and then put under a transmission electron microscope. The remaining 13 specimens were divided into one of two groups: for quantitative real-time polymerase chain reaction (RT-PCR) study (n = 5 per group) for the expression of RhoA, MAG, and MLC; and for Western blot analysis (n = 8 per group) of the level of myelin-associated glycoprotein (MAG) and neurofilament 200 (NF-200), respectively.

FIG. 1. Demonstration of surgical procedures. The white arrows on the left show the site where the labeled suture was placed for quantitative analysis and the origin of the posterior gluteal nerve; the arrow on the right indicates the length maker (the silicone tube in the middle) for accurate nerve cut. The numbers 1 and 1.5 in green and the inset pictures (left, center, and right) show the details of preparation of PNS. The PNS was inserted 4 mm into the 1.5-cm-long SF/P (LLA-CL) nanofiber conduit (internal diameter, 1.5 mm) (lower right). Figure is available in color online only.

Behavioral Analysis

All rats were monitored for Family medical history their autotomy scores (n = 19) over a period of 30 days. Scores were recorded 3 times/week by dual, blinded observers in order to assess neuropathic pain levels. The modified Wall Scale was adopted to document points based on the severity of autotomy30 (Fig. 2).

Quantitative Morphological Analysis

At end of the experiment, the PNS was transected from the previously labeled point on the operated side, and a corresponding nerve segment of 1 cm in length from the contralateral side was harvested for quantitative measurement of neuroma growth. To represent neuroma growth, we used weight ratios (WRs), calculating them with the following equation:[NW (the weight of neuroma) NNW (the weight of the excised normal nerve segment)]/NNW × 100%.

Histological Analysis

The PNSs were first weighed after excision and then transected at about 5 mm distal to the proximal end. The nerve specimens were placed in a solution of 10% formalin overnight and then washed several times in a phosphate-buffered saline. Following this, they were dehydrated, embedded in paraffin, and cut at 8 lm perpendicular to the main nerve axis. From the distal end of the specimens, we randomly chose the sections at lengths ranging from 400 to 600 μm. After excluding excessively small or improperly cut sections, we obtained a minimum of 30 sections from each sample (thus, 20 sections remained at least, from which we randomly narcissistic pathology selected 10). These sections were used for Masson’s trichrome staining, and the rest were labeled with anti-NF-200 antibodies.

Real-Time PCR

Isolation of total RNA from the neuromas was performed using the Absolutely RNA Microprep Kit (Stratagene). The amount of RNA was determined using a NanoDrop UV spectrophotometer (NanoDrop Technologies). The primers necessary for quantitative RT-PCR were obtained from Sigma-Genosys. Transcription and RT-PCR were performed using the SYBR Green Premix Ex Taq (Takara Korea Biomedicala) on a StepOne Real-Time PCR System (Applied Biosystems). The formation of PCR products was confirmed via dissociation curve analysis at the end of each PCR. To assess the relative amount of product, we used the ΔΔCt method, normalizing for the expression of the housekeeping gene—actin —and related to the treatment of control animals.

FIG. 2. Modified Wall’s score in rats. No autotomy (score 0) (A); loss of half of the second toe (score 1) (B); loss of the first and the third toes (score 1) or the second toe was totally missing (score 2), score 1 + 2 = 3 in total (C); loss of the first and the second toes (score 2 + 2 = 4) (D); loss of the first and the second toes, and the third toe was half missing, score 2 + 2 + 1 = 5 in total (E); loss of the first, second, and the third toes, score 2 × 3 = 6 in total (F). Figure is available in color online only.

Western Blot Analysis

We extracted the MAG and NF-200 from the neuromas. To achieve total protein extraction from the cells, the tissues were placed in an ice-cold homogenization buffer containing 50 mmol/L Tris-HCl, protease inhibitors, pH 6.8, 100 mmol/L dithiothreitol, and 10% glycerol and 2% SDS. Protein levels were quantified by Western blotting. A bicinchoninic acid (BCA) assay was used to assess the overall concentration of protein. We resolve the lysates with equal amounts of protein by using the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) technique, and the total protein was maintained at -80。C for further analysis. SDS-PAGE separated the equal amounts of protein and then transferred them onto a PVDF (polyvinylidene difluoride) membrane. Milk powder 5% was diluted as a blocking agent in Tris-buffered saline for 3 hours on each blot. Blots were incubated overnight with rabbit anti-NF-200 (1:000, Abcam) and monoclonal antibodies against rabbit anti-MAG (1:1000, Abcam) at 4。C. The blots were initially incubated in the primary antibody, and then for 2 hours they were incubated with a secondary antibody (horseradish peroxidase–conjugated goat anti–rabbit IgG [1:8000]) at room temperature. Protein amounts loaded into each lane were verified by reprobing the blots with beta-actin (dilution 1:1000, Santa Cruz Biotechnology). All signals were scanned with the ChemiDoc XRS + Bio-Rad Imaging System. The Zegocractin densities of each protein were normalized by their corresponding actin signal.

Statistical Analysis

The Mann-Whitney U-test, a nonparametric method, was employed for the statistical analysis between groups using SPSS version 19.0 (IBM Corp.). All values are expressed as the mean ± SEM. Statistical significance was set at the level of p > 0.05.

Results

All animals in the three groups were in good health during the experimental period.

Autotomy Observation

The animals displayed autotomous behavior on the right foot (operated side). Statistically significant differences in the average autotomy scores were noted between the capping group and the control group at each of the time points (p < 0.05). Furthermore, the average autotomy score in the capping group was found to be significantly lower than that in the control and intervention groups (Fig. 3 and see Fig. 5A). In the control group, 16 (84.2%) of the 19 remaining rats developed differing degrees of autotomy (scores ranging from 3 to 6, mean 5.55 ± 0.32); in contrast, 13 rats in the intervention group (68.4%; autotomy score 2–5, mean 4.19 ± 0.49) and only 5 of 19 rats remaining in the capping group (26.3%; autotomy score 1–3, mean 1.97 ± 0.24) showed evidence of autotomy. Autotomy onset occurred between 5 and 30 days after surgery in all animals. No significant difference, however, was seen between the control and intervention groups throughout the experiment.

FIG. 3. Photographs of neuromas in all the groups. At 30 days after neurectomy, a typical bulbous neuroma was observed in both the control group (C) and intervention group (A), while no such findings were noted in the capping group (B). Figure is available in color online only.

Gross Evaluation

Neuromas in the capping group presented a slightly lower weight ratio (0.745 ± 0.058) compared to those in the intervention (1.637 ± 0.218) and control groups (2.111 ± 0.391). The differences were significant between the two groups in terms of weight ratios (capping group vs control group, p = 0.021; intervention group vs capping group, p = 0.013). However, there was no significant difference between the intervention and control groups in weight ratio (p = 0.160).

Histological Findings and General Observations

At 30 days postneurectomy, a typical bulbous neuroma was noted at the end of the PNS in both the control and intervention groups. In 9 rats, a mildly spindle-shaped stump was also seen in the other 2 cases, whereas no such clinical manifestation was seen in the capping group. We only found a bullet-shaped neuroma in the conduit in all animals of this group.The light microscope revealed the unorganized growth of cells in the neuroma, the regeneration of axons, infiltration of muscle, and ensheathing cells that formed a small patch and funicular structure in both the intervention and control groups. In the capping group, however, there was less proliferation of nerve fibers with a regular arrangement (Fig. 4A).Masson’s trichrome staining revealed that highly proliferated collagen (stained in blue) was mixed with disorganized nerve fascicles in the intervention and control groups, while in the capping group, a somewhat orderly tissue configuration was found with organized nerve fibers and slightly blue-stained collagen (Fig. 4B).

Transmission Electron Microscopy

Transmission electron microscopy revealed numerous axons in the neuromas, more collagen fiber, more fusoid fibroblasts, and degenerated myelin sheath and hyperplasia in the intervention and control groups. In the capping group, there were fewer myelin sheaths in the proximal end of injured nerves, fewer Schwann cells, and sparse collagen fibers and fibroblasts (Fig. 4C).

Gene Expression of MAG, RhoA/ROCK, and MLC

We observed that the expression of MAG was markedly unregulated in the neuromas 30 days after surgery in the intervention and capping groups compared with the control group, while the expression of RhoA/ROCK and MLC was significantly downregulated in the intervention group. In all groups, the gene expression of RhoA in neuromas was vastly upregulated after surgery when compared with the uninjured side at 30 days after surgery (Fig. 5B).

MAG and NF-200 Measurement by Western Blot Assay

Western blotting revealed MAG and NF-200 expression in the PNS (Fig. 5C andD). At 30 days, the expression of NF-200 was significantly lower in the capping group as compared to the
control and intervention groups. In contrast, the protein content of MAG in the capping and intervention groups was significantly higher than that in the control group.

Discussion

A peripheral nerve lesion is an injury frequently encountered by surgeons. When a peripheral nerve is transected, wallerian degeneration occurs in the area distal to the lesion site, whereas proximally regenerating axons sprout into the surrounding tissue and may produce a tangled mass of nerves known as a neuroma.5,7 In some cases, surgeons have attempted to cover the nerve stump in order to isolate it from the inflammatory cascade and from the production of neurotrophic factors that is triggered by nerve trauma in the surrounding tissues.Surgeons have the option of covering a nerve either by burying the stump into a neighboring anatomical structure or by capping it with either a biological or synthetic nerve cannula. We hypothesized that the orientation of the nanofiber nerve cannula may be mediated by a signal pathway in order to inhibit the proliferation of collagen scar tissue.

FIG. 4. A: H & E staining of neuromas at 30 days after surgery. Using light microscopy, H & E staining of the traumatic neuroma in the capping group (A1) showed that the number of fibroblasts was minimal, and there were a large number of red fibers in the intervention group and control group (A2 and A3, red arrows). The neuroma is composed of a haphazard proliferation of nerve fascicles, including axons and fibroblasts (A2). Original magnification ×200. B: Trichrome Masson’s staining of neuromas at 30 days after surgery. B1: Slightly blue-stained collagens with relatively orderly arranged nerve fibers (red arrow) in the capping group. B2: Dense blue-stained collagens with haphazardly arranged nerve fascicles (red arrow) in the control group with a lot of axons in neuromas with more fusoidfibroblasts and collagen fibers. B3: Dense blue-stained collagens with regularly arranged nerve fascicles (red arrow) in the intervention group with fewer fusoidfibroblasts and collagen fibers than the control group. Original magnification ×300. C: General observation by transmission electron microscopy at 30 days after surgery. C1: Capping group: plenty of thick myelinated fibers with few fibroblasts were observed. The red arrow shows the scattered unmyelinated fibers; the blue arrow shows the myelinated fiber. Only a few collagen fibers can be seen randomly. C2: Control group: many unmyelinated fibers with abundant fibroblasts are seen. Abundant transverse and oblique collagen fibers are distributed randomly. The markers show the dense transverse collagen fibers (red arrow) and the myelin sheath (blue arrow). C3: Intervention group: at the bottom, there were distributed collagen fibers; the blue arrow shows the Schwann cell with mitochondria and endoplasmic reticulum inside and the red arrow depicts an unmyelinated nerve fiber. Magnification ×3000. Figure is available in color online only.

Previous research has found that RhoA is involved in a number of cellular activities, such as axon regeneration and guidance, neuronal migration, and macrophage infiltration.4,28 Evidence from animal studies has determined that RhoA/ROCK signaling is involved in numerous diseases of the peripheral nervous system.11,22,27 Given that RhoA/ROCK plays a critical role in the pathophysiology of peripheral nervous system diseases, it is anticipated that the development of therapeutic agents targeting this pathway would make a substantial contribution to the treatment of peripheral nervous system diseases.19 The RhoA/ ROCK signaling pathway mediates the effects of myelinassociated axon growth inhibitors—Nogo, MAG, oligo-dendrocyte-myelin glycoprotein, and repulsive guidance molecule.15 By blocking RhoA/ROCK signaling it is possible to reverse the inhibitory effects of these molecules on axon outgrowth and to promote axonal sprouting and functional recovery in animal models4,6 (Fig. 6).

Remodeling of the NF-200 in axon tips/growth cones is considered to be a vital part of successful axon growth and elongation. In the RhoA/ROCK signaling pathway activated form, the upregulation of MAG activates RhoA/ ROCK, which results in the phosphorylation of a number of target proteins—including MLC—which downregulates its downstream effector NF-200. The suppression of NF200 expression in axons can result in the prevention of neuroma formation.Y-27632 has been well established as a ROCK inhibitor in a variety of systems.9,13,28 A pyridine derivative, Y-27632 was the first specific inhibitor of the Rho-kinase family of enzymes to be synthesized and reported.1,2 Y-27632 works by inhibiting ROCK functioning, competitively binding with adenosine triphosphate to the catalytic domain. Prior research has found that Y-27632 has a much higher specificity for ROCK than for other protein kinases, such as myosin light chain kinase and protein kinases A and C.

FIG. 5. A: Results of daily average autotomy scores. Statistically significant differences were noted between the capping group and the control group at all of the time points (p < 0.05) except at the end of 10 days (p = 0.283). B: Gene expression of MAG,RhoA, ROCK, and MLC. When comparing the capping and control groups, RT-PCR demonstrated that the expression of RhoA
and ROCK in the neuroma was dramatically upregulated after surgery in the capping group in comparison with the control group, but more obviously in MAG and MLC (*capping group vs control group, p < 0.001). After injecting Y-27632, we found that the gene expressions of RhoA, ROCK, and MLC were significantly downregulated; however, the MAG was more or less the same (▽ capping group vs intervention group, p < 0.001). The gene expression of RhoA (▽) and ROCK in the capping group is significantly higher than that in the control group and intervention group (both p < 0.05). In addition, the expression of MLC (Δ) in the intervention group is significantly lower than that in the other two groups (both p < 0.05). C: Relative expression of MAG in the neuromas. The protein content of MAG expression in the capping group and intervention group was significantly higher than that in the control group (*capping group and ⊗intervention group vs control group, p < 0.001), but not obviously between the capping and intervention groups (*capping group vs intervention group, p = 0.328). D: Relative expression of NF-200 in the neuromas. The protein content of NF-200 expression in the control group was significantly higher than that in the capping group and intervention group (*both groups vs control group, p < 0.001).

One study by Wei et al.28 indicated that the increase of RhoA and collagen fibers in neurons suggests that they are inherently important for the growth, guidance, and branching of axons postinjury. Previous research has demonstrated that changes in RhoA/ROCK and in the expression of MLC can regulate NF-200 expression. Activation of the RhoA pathway was shown to lead to sequential Rho-associated kinase/LIM kinase/cofilin-mediated actin filament depolymerization and collapse of growth cone; these expressions are negative responses to the growth of painful traumatic neuromas. Axonal or neurite elongation after RhoA and Rho-kinase inhibition has been well documented; RhoA inactivation can increase neurite growth and axonal regeneration—both in vivo. The Rho-kinase inhibitor Y-27632 has been shown to promote neurite extension on neurons.In the present study, we found the expression of RhoA/ ROCK in neuromas to be vastly upregulated after surgery compared with the contralateral side at 30 days in all groups; this was more obvious in the capping group (both p < 0.001), which was consistent with our previous findings.29 In addition, RT-PCR detected that the expression of MAG was greatly upregulated in the PNS 30 days after surgery in the capping group compared to the control group (p < 0.001) but not the intervention group (p = 0.328). In the intervention group, weak RhoA and MLC expression was only observed in a few neurons, but in the capping group, RhoA and MLC expression intensity was significantly increased. At the same time, our investigation revealed that the number of protein expressions of NF-200 increased drastically in both the control and intervention groups.Furthermore, a transmission electron microscopic study showed that there were numerous axons in neuromas, with more fusoid fibroblasts, collagen fibers, and degenerated myelin sheaths in the intervention and control groups. In the capping group, however, there were fewer myelin sheaths, fewer Schwann cells, and fibroblasts with sparse collagen fibers. Pain has been reported to be primarily mediated by unmyelinated fibers.3,7 Our results showed that in both the intervention and control groups, there were a multitude of unmyelinated fibers with numerous fibroblasts; in the capping group, we found many thick myelinated fibers with a reduced number of fibroblasts.In our previous study, we observed a positive staining of NF-200 in the traumatic neuromas harvested from the rats with high autotomy scores. In this experiment, the immunofluorescence expression intensity of NF-200 was positively correlated with the injection of Y-27632 with high autotomy scores in both the intervention and control groups. In contrast, only somewhat positive immunofluorescence expression was detected in the capping group (Fig. 7). Gene expressions of RhoA/ROCK and MLC in the capping group were significantly higher than those in the control group. At the same time, gene expressions of RhoA/ROCK in the intervention group were significantly lower than those in the other two groups after injecting Y-27632.

FIG. 6. A schematic review of the RhoA/ROCK signaling pathway. Figure is available in color online only.

One clear limitation of our study is that we used an indirect approach toward the mechanistic goal of evaluating ROCK inhibition. We concluded that the capping (conduit) group works via a so-called regenerative brake. However, inhibiting Rho-kinase affects the axon regeneration of peripheral motor and sensory nerves in different ways. The intervention group with the ROCK inhibitor only provided indirect evidence. Further studies with more direct evidence such as those employing a conditional knockout rodent model are warranted in the future. In addition, although some researchers have adopted the autotomy behavior as a parameter for pain evaluation in rats,18,20 this behavior in the present study was by far an indirect sign for pain assessment, and more specific painrelated measurements are required in the future for further study.In summary, our study determined that the RhoA/ ROCK pathway plays an important role in axon growth in neuromas. Of the 3 groups, the gene expressions of RhoA/ ROCK and MLC were lowest in the intervention group with a higher level of protein content of NF-200 when the RhoA/ROCK pathway was blocked by Y-27632. This supports the rationale that using the nerve-capping technique with the aligned nanofiber conduit in the prevention of neuroma formation is closely associated with the RhoA/ ROCK signal pathway.

FIG. 7. Immunofluorescence staining of NF-200. A: The regenerated axons (marked with NF-200 and stained with tetramethyl-rhodamineisothiocyanate [TRITC]) were densely distributed in a chaotic way in the control group. B: The larger and regenerated axons in the intervention group were arranged more regularly than those in the control group. C: Only a few axons (marked with NF-200 and stained with TRITC) arranged regularly in a linear fashion in the capping group. Original magnification ×3000. Figure is available in color online only.

Conclusions

The RhoA/ROCK signal pathway has emerged as a critical player in the process of traumatic neuroma formation after neurectomy. The nerve-capping technique may generate a “regenerative brake” based on the regulation of the RhoA/ROCK signaling pathway in this event. These findings may provide certain evidence that could further the development of new strategies for the management of painful neuromas.