Neuroma Management: Capping Nerve Injuries With an Acellular Nerve Allograft Can Limit Axon Regeneration

Abstract

Background: Management of painful neuromas continues to challenge clinicians. Controlling axon growth to prevent neuroma has gained considerable traction. A logical extension of this idea is to therefore develop an approach to control and arrest axon growth. Given the limits in axonal regeneration across acellular nerve allografts (ANAs), these constructs could provide a means to reliably terminate axon regeneration from an injured nerve. The purpose of this study was to determine if attaching an ANA to an injured nerve could provide a means to control and limit axon regeneration in a predictable manner. Methods: Twenty (20) adult rats received a sciatic nerve transection, where only the proximal nerve was repaired using an ANA of variable length (0.5, 2.5, and 5.0 cm) or left unrepaired (control). The nerves were harvested 5 weeks post-operatively for gross and histomorphometric analysis. The extent of myelinated axons in regenerated tissue was quantified. Results: At 5 weeks, limited axon regeneration within the ANAs was observed. All lengths of ANAs lead to reduced myelinated axon numbers in the most terminal tissue region compared to untreated injured nerve (P = .002). Additionally, ANA length 2.5 cm or greater did not contain any axons at the most terminal tissue region. Conclusions: This study demonstrates a proof of concept that ANAs attached to the proximal end of an injured nerve can limit axon growth in a controlled manner. Furthermore, the extent of axon growth from the injured nerve into the ANA is dependent on the ANA length.

Introduction

Neuromas are non-neoplastic, but aberrant growths of nerve tissue that form on injured peripheral nerves.^1-3 Although they are not always symptomatic, they have the ability to cause significant pain and disability impacting patient’s quality of life.^4-9 The management of neuroma still poses a considerable challenge for surgeons.^7,10,11 While numerous surgical techniques have been proposed and tested, no single method has been able to completely eliminate neuroma-associated pain.¹⁰

The mechanisms by which neuromas lead to pain have been assessed in studies ranging from changes in nerve electrophysiological, structure, and molecular biology.^12-16 In brief summary, neuromas have been found to be a mass consisting mainly of axons, fibroblasts, and Schwann cells regenerating in a chaotic fashion in locations where epineurium and perineurium have been compromised. The spontaneous and provoked firings due to traction or pressure of the nerve endings along with abnormal concentrations of nociceptors is implicated in pain associated with neuromas.^13,16 Given the disorganized directionality of the axons within the neuroma, it is reasonable to assume that these axons may be hypersensitive to strain, which would correspond to the symptoms associated with neuroma-like pain clinically.^14,17

Among the many attempts to alleviate neuroma pain, several notable methods include the transposition of transected nerve into muscle or bone, centro-central anastomosis, and nerve caps made of various materials.^10,18-22 Common to all these methods is their effort to isolate the site of potential neuroma formation, where axons would then be relatively protected from trauma, or to prevent nerve regeneration, and thus avoid neuroma formation altogether. Expanding on the latter, it has been found that axons will not regenerate through epineurium, and thus attempts have been made to cap the nerve using epineurial flaps or to ligate the epineurium allowing no place for axonal regeneration.^21-23 In practice, however, the peripheral nerve’s incredible propensity to regenerate its axons can result in unwanted axon growth outside this closure accompanied with pain.^21,22

Meanwhile, research into nerve repair using nerve grafts has revealed findings applicable to developing a new approach to control axon outgrowth from injured nerve. Repair of nerve gaps using acellular nerve allografts (ANAs) has shown that regenerating axons were unable to cross long ANAs. Specifically, ANAs with lengths in excess of 3.0 cm result in limited axon regeneration within the ANA and axon growth arrest before crossing the length of the ANA.^24,25 While not ideal for nerve repair to reconnect the proximal and distal nerve stumps, alternatively this knowledge could be applied to avert neuroma formation. In theory, ANAs could provide a basal lamina scaffold for axon growth yet still provide termination of axon growth in a controlled fashion within this ANA scaffold.

While previous studies established that long ANAs can terminate axon growth, the aforementioned studies were conducted with the intent to repair nerve injuries, and thus the distal end of the ANA was anastomosed to the distal nerve stump of the injured nerve. For the treatment of neuroma, the ANA would not attach to a distal nerve stump. Therefore, in our studies, we attached an ANA to the injured proximal nerve to determine (1) whether an ANA could “cap” the nerve, thereby acting as a guide to contain axonal regeneration within the ANA even without the distal nerve, and (2) what length of ANA is effective to arrest regenerating axons with minimal indications that axons would regenerate across the ANA.

Materials and Methods

All materials were purchased from Sigma Aldrich, St. Louis, MO, unless otherwise specified.

Animals

Adult male Lewis rats weighing 226 to 250 g (Charles River Laboratories, Wilmington, MA) were included in the study. Sprague-Dawley rats were used as donor animals to generate ANAs. The Sprague–Dawley (SD) (RT-1^b MHC) rat strain is MHC incompatible with Lewis (RT-1¹ MHC) yielding an appropriate allograft donor. The animals were housed in a central housing facility and received food (PicoLab rodent diet 20, Purina Animal Nutrition LLC, St. Louis, MO) and water ad libitum. Animals were monitored postoperatively for signs of weight loss and infection. All procedures were carried out in strict accordance with the guidelines set forth by the National Institutes of Health and were approved by the Division of Comparative Medicine at the institution. Specifically, surgical procedures and peri-operative care measures were conducted in compliance with the AAALAC accredited Washington University Institutional Animal Care and Use Committee (IACUC) under animal protocol #20150120.

Experimental Design

Twenty adult male Lewis rats were randomized into four groups (Groups I-IV), where each group contained five animals each (Figure 1). Group I served as a control in which the sciatic nerve was transected but not repaired, as uncontrolled axon growth would result.²⁶ Groups II, II, and IV were experimental groups, wherein the transected sciatic nerve was repaired using various length ANAs of 0.5 cm, 2.5 cm, and 5.0 cm, respectively. ANAs were harvested from 10 Sprague-Dawley donor rats.

Figure 1.

Schematic of experimental design. Experimental groups (n = 5 per group) had their sciatic nerve transected and left unrepaired (Group I; control) or transected and repaired with an ANA (Groups II-IV).

Note. At a 5-week endpoint, the entire nerve and ANA (when applicable) was harvested and assessed for the extent of axon regeneration. Areas where axon regeneration was evaluated are indicated by dotted lines for each group. Dotted trace for Group I represents post-operative tissue regenerated to provide perspective as to how this group was evaluated. ANA = acellular nerve allograft.

Surgical Procedure

Surgical procedures were performed using aseptic conditions with the aid of an operating microscope (JEDMED/KAPS, St. Louis, MO). Rats were anesthetized with a subcutaneous injection of ketamine (75 mg/kg, Ketaset®; Fort Dodge Animal Health, Fort Dodge, IA) and medetomidine (0.5 mg/kg, Dormitor®; Orion Corporation, Espoo, Finland). The right sciatic nerve was exposed through a gluteal muscle-splitting incision and transected 5 mm proximal to the trifurcation with surgical scissors. The distal nerve stump was ligated with 4-0 silk suture (Ethicon, Somerville, NJ) and then moved away from the proximal stump and secured to the muscle bed using 9-0 nylon (Surgical Specialties Corporation, Wyomissing, PA). In Groups II to IV, ANA was coapted via the epineurium to the proximal stump with four to five 9-0 nylon sutures. For longer ANAs (2.5 cm and 5.0 cm), the distal end of the ANA was secured by the epineurium with 9-0 nylon away from both the proximal stump and ligated distal stump to nearby muscle tissue. Upon completion of the procedures, the incision was irrigated with bacteriostatic 0.9% sodium chloride (Hospira, Inc., Lake Forest, IL) and the muscle fascia and skin were closed in layers using 4-0 vicryl and nylon suture (Ethicon). The animals were recovered with atipamezole hydrochloride (1 mg/kg, Antisedan®; Orion Corporation, Espoo, Finland) on warmed heating station and closely monitored before returning to a central housing facility. Postoperative pain was managed using Buprenorphine SR^TM (0.05 mg/kg; ZooPharm, Windsor, CO) q 8 to 12 hours prn.

For Sprague-Dawley donor animals, similar procedures were performed to allow for bilateral nerve exposure and harvest. Immediately following these procedures, animals were euthanized by sodium pentobarbital (>200 mg/kg, Vortech Pharmaceutical Ltd, Dearborn, MI). Sciatic nerve harvested from donor rats were chemically processed and decellularized using a series of detergents as previously described to generate ANAs.²⁵

Five weeks post-operatively, all experimental animals were euthanized, and the proximal sciatic nerve along with the grafts and any regenerated tissue were harvested en bloc for visual assessment and histomorphometric analysis. Five weeks was chosen as the endpoint as it was determined in previous work by our group that nerve regeneration in ANAs arrested between 2 and 4 weeks (Poppler 2016).²⁷ The harvested nerves were marked with a proximal suture to indicate orientation and fixed in 3% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA) in 0.1 M pH 7.2 phosphate buffer (Fisher Scientific, Fair Lawn, NJ) at 4°C until processing was performed.

Histomorphometry

The explanted nerves were analyzed as described previously (Hunter 2007).²⁸ Briefly, the nerves were preserved in 3% glutaraldehyde, postfixed in 1% osmium tetroxide (Polysciences, Inc., Warrington, PA), and serially dehydrated in 90% ethanol (Thermo Fischer Scientific, Winmill Hill, UK). The nerves were then embedded in grade 502 epoxy (Polysciences, Inc., Warrington, PA) and sectioned to 1 µm with an ultramicrotome. The slides were stained with 1% toluidine blue and analyzed under light microscopy at 1000x (Leitz Laborlux S, Leica, Buffalo Grove, IL) using the IA32 Image Analysis System (Leco, St. Joseph, MI) in order to quantify nerve fiber counts, fiber width, fiber density, and percent neural tissue. For all groups, the most terminal portion of the harvested tissue was assessed for the extent of axon regeneration and quantified. Additionally, for Groups III and IV, sections were obtained 5 mm distal to the repair site to determine the extent of axon regeneration within the proximal portion of the ANAs (Figure 1). All analysis was conducted by a blinded observer to the experimental groups.

Statistical Analysis

All data were compiled in Microsoft Excel (Microsoft, Redmond, WA), and statistical analysis was performed using SPSS (IBM, Armonk, NY). Data are reported as mean ± standard deviation and tested for normality using the Kolmogorov-Smirnov test. One-way analysis of variance (ANOVA) was used to examine means from histomorphometry. If analysis demonstrated significant differences, Tukey’s post-hoc test was performed to isolate these differences while correcting for multiple comparisons. Significance was set at α = .05 (P < .05).

Results

To assess whether an ANA could control axon regeneration when used to “cap” a transected nerve, the extent of regeneration within the ANA was assessed 5 weeks following proximal nerve repair with attachment of the ANA. In Group I, which was injured nerve left unrepaired, all rats had evidence of nerve regeneration distal to the site of injury, which presented as a conical termination of tissue past the original site of transection (Figure 2). Conversely, nerve regeneration differed in rats receiving an ANA attached to the injured proximal nerve. A portion of rats in Group II showed macroscopic evidence of regeneration distal to the ANA. In Groups III and IV, the ANA had no macroscopic evidence of regeneration distal to the ANA. Overall, qualitative examination of injured nerve with an attached ANA demonstrated evidence of limited and controlled nerve regeneration.

Figure 2.

Intraoperative photos from experimental groups immediately post-operation and during tissue harvest at 5 weeks post-operation. Yellow arrows indicate proximal nerve stump and injury site. Orange asterisks indicate regenerated tissue regions.

Based on qualitative examination demonstrating efficacy of an ANA to control the regenerative response, we assessed the extent of axon regeneration using histology. We first examined nerve regeneration at regions just distal (~3-5 mm) to the injury (Group I) or injury/repair (Groups II-IV) sites. All groups had robust axon regeneration at these regions (Figure 3a-3d). For groups receiving ANAs (Groups II-IV), these results demonstrated that axons were able to regenerate into ANAs despite the lack of a distal nerve attachment to the ANA. Additionally, for Groups I and II, this region represented the terminal portion of the harvested tissue.

Figure 3.

Representative histological nerve sections distal to the injury site. Toluidine blue counterstained, nerve cross-sections show myelinated axons in all sections taken just distal (~3-5 mm) to the injury site (a-d). (a) Group I, (b) Group II, (c) Group III, and (d) Group IV. Additionally, Groups III and IV contained additional terminal regenerated tissue. Sections taken at this terminal region demonstrated no axon regeneration (e-f). (e) Group III and (f) Group IV.

Note. Scale bar = 20 µm.

The extent of axon regeneration at the terminal portion of the harvested tissue was quantified. Myelinated axons regenerated beyond the most distal region of ANA for Group II, yet no axons were observed in the distal tissue region of the original implanted ANA for Groups III or IV (Figure 3e and 3f). Using histomorphometric analysis, total myelinated axon numbers were quantified and revealed that all groups with ANAs contained significantly less axons in the terminal tissue region compared to Group I (F = 7.640, df = 3, 16, P < .005; Figure 4). While no statistical differences were observed between Groups II and IV, Groups III and IV did not contain any myelinated axons in the terminal tissue. Overall, this analysis revealed that sufficiently long ANAs (ANA length > 0.5 cm) were effective to limit axon regeneration from reaching the most terminal portion of regenerated tissue.

Figure 4.

Histomorphometric evaluation of axon regeneration at the terminal regenerated tissue region 5 weeks following nerve injury. Groups receiving ANAs (Groups II-IV) contained significantly less axons in their terminal regenerated tissue region compared to Group I.

Note. Data represented as mean ± standard deviation. ANA = acellular nerve allograft.

*Indicates statistical significance compared to Group I (P < .05).

Discussion

The overarching principle guiding our research is a surgical premise that isolating and preventing unwanted axon growth, when nerve regeneration is not an option, can minimize neuroma formation and the onset of pain. Unfortunately, this experimental premise has not yet been established, nor have approaches to robustly test this premise. Toward testing this premise, our group has been developing approaches to control and arrest axon regeneration in order to minimize neuroma formation. In our current work, we hypothesized that guiding regenerating axons using sufficiently long ANAs would limit axon regeneration. The current study investigated the use of ANA to “cap” a nerve injury. Using a rat sciatic nerve transection model repaired with ANAs of variable length, we determined two major findings. First, ANAs can successfully be used to “cap” transected nerves, whereby the ANA guided regenerating axons into the ANA. Second, a sufficiently long ANA limits regenerating axons from reaching the terminal portion of the ANA, suggesting that axon regeneration is permanently arrested within the ANA.

The ANA lengths examined were based upon previous work that established ANAs > 3.0 cm do not support axon regeneration across the ANA.^24,25 Furthermore, previous work has also demonstrated that attaching a nerve autograft without connection to a distal nerve stump reduced the ability for axons to regenerate within the autograft, suggesting that an ANA length shorter than 3.0 cm could achieve axon growth arrest.²⁹ Therefore, ANA lengths of 0.5, 2.5, and 5.0 cm were tested based upon the clinical availability of “off-the-shelf” products. We found that regenerating myelinated axons did not reach the distal end of either 2.5 or 5.0 cm ANAs indicating that a sufficiently long ANA can limit and control axon regeneration. Furthermore, these results suggest that a sufficiently long ANA could arrest axon regeneration within the length of the ANA for an extended period. This surgical approach can now be more rigorously tested to evaluate its potential to prevent axon growth over the long term, as well as the onset of pain, which would in turn be invaluable in managing neuromas in the clinic.

A current limitation of this work is the available data to determine the complete extent of axon regeneration. A 5-week endpoint was carefully chosen based on two previous studies using ANAs in an injury/repair model. In one study, axon regeneration within ANAs was noted to only reach ~10 to 20 mm in long (>3.0 cm) ANAs by 10 weeks.²⁴ In another study, axon growth arrest was noted to occur by 4 weeks in rats repaired using long (>3.0 cm) ANAs, where again axons only advanced ~10 mm into the ANA.²⁵ The results of these studies suggest that the cessation of axon regeneration within an ANA occurs within 4 weeks, and thus our endpoint of 5 weeks was assumed to be of sufficient duration to capture an important first outcome, given the variety of ANA lengths that we evaluated for their effectiveness. Studies of longer duration are ongoing to conclude that ANAs without a distal nerve connection can stably arrest myelinated, as well as unmyelinated, axon regeneration.

Conclusion

Surgical treatments to minimize neuroma formation and control nerve regeneration are desired. This study serves as a proof of concept that an ANA can reliably serve as a cap. Although future studies are warranted, the use of ANAs as a tool to control neuroma formation may be viable clinical option.