Accuracy of Motor Axon Regeneration Across Autograft, Single Lumen, and Multichannel Poly(lactic-co-glycolic Acid) (PLGA) Nerve Tubes

doi:10.1227/01.NEU.0000335081.47352.78

Neurosurgery. Author manuscript; available in PMC 2012 October 3.

Published in final edited form as:

Neurosurgery. 2008 July; 63(1): 144–155.

doi: 10.1227/01.NEU.0000335081.47352.78

PMCID: PMC3463233

NIHMSID: NIHMS80959

Accuracy of Motor Axon Regeneration Across Autograft, Single Lumen, and Multichannel Poly(lactic-co-glycolic Acid) (PLGA) Nerve Tubes

Godard C. de Ruiter, MD, Robert J. Spinner, MD, Martijn J. A. Malessy, MD, PhD, Michael J. Moore, PhD, Eric J. Sorenson, MD, Bradford L. Currier, MD, Michael J. Yaszemski, MD, PhD, and Anthony J. Windebank, MD

From the Cellular Neurobiology Laboratory (G.C.de R., A.J.W.), the Department of Neurologic Surgery (G.C.de R., R.J.S.), the Department of Physiology and Biomedical Engineering (M.J.M., M.J.Y), the Department of Neurology (E.J.S., A.J.W.), and the Department of Orthopedic Surgery (B.L.C., M.J.Y.), Mayo Clinic, Rochester, Minnesota; the Department of Neurosurgery (M.J.A.M.), Leiden University Medical Center, Leiden, the Netherlands

Address reprint requests to Anthony J. Windebank, MD, Department of Neurology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. Email: windebank.anthony/at/mayo.edu

The publisher's final edited version of this article is available at Neurosurgery

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Abstract

Objective

Accuracy of motor axon regeneration becomes an important issue in the development of a nerve tube for motor nerve repair. Dispersion of regeneration across the nerve tube may lead to misdirection and polyinnervation. In this study, we present a series of methods to investigate the accuracy of regeneration, which we used to compare regeneration across autografts and single lumen poly(lactic-co-glycolic acid) (PLGA) nerve tubes. We also present the concept of the multichannel nerve tube that may limit dispersion by separately guiding groups of regenerating axons.

Methods

Simultaneous tracing of the tibial and peroneal nerves with fast blue (FB) and diamidino yellow (DY), 8 weeks after repair of a 1-cm nerve gap in the rat sciatic nerve, was performed to determine the percentage of double-projecting motoneurons. Sequential tracing of the peroneal nerve with DY 1 week before and FB 8 weeks after repair was performed to determine the percentage of correctly directed peroneal motoneurons.

Results

In the cases in which there was successful regeneration across single lumen nerve tubes, more motoneurons had double projections to both the tibial and peroneal nerve branches after single lumen nerve tube repair (21.4%) than after autograft repair (5.9%). After multichannel nerve tube repair, this percentage was slightly reduced (16.9%), although not significantly. The direction of regeneration was nonspecific after all types of repair.

Conclusion

Retrograde tracing techniques provide new insights into the process of regeneration across nerve tubes. The methods and data presented in this study can be used as a basis in the development of a nerve tube for motor nerve repair.

Keywords: misdirection, axon targeting, double labeling, peripheral nerve regeneration, rat sciatic nerve model, retrograde tracing

Introduction

Single lumen or hollow nerve tubes have been developed as an alternative to nerve gap repair with an autologous nerve graft. Advantages of repair with a nerve tube are unlimited, right-off-the-shelf availability, in a range of sizes without additional donor-site morbidity. The first biodegradable nerve tubes are now available for clinical use (29). These nerve tubes are mainly used in the repair of small sensory nerves, such as digital nerve lesions with gaps up to 3 cm (36) and have recently been used in the repair of larger motor nerves (8,23). Single lumen nerve tube repair, however, may lead to inappropriate target reinnervation by the dispersion of regenerating axons across the graft (5). This dispersion may result in 1) misdirection of regenerating axons or 2) polyinnervation of different targets by dispersion of axonal branches originating from the same motoneuron. Dispersion probably occurs less after autograft repair by separate guidance of axonal branches inside the basal lamina tubes, resulting in more grouped muscle fiber reinnervation (35). Multichannel conduits (Fig. 1) that have been developed for both experimental peripheral nerve repair (2,32,39) and spinal cord repair (13,22,30) may also limit this dispersion by separately guiding groups of regenerating axons inside the channels.

Fig. 1

Drawing of a multichannel nerve tube with 7 channels (400 μm in diameter) and sleeves on both ends for implantation. (Used with permission of Mayo Foundation for Medical Education and Research.)

In this study, we used simultaneous and sequential tracing techniques to investigate the accuracy of motor axon regeneration across autograft, single lumen, and multichannel poly(lactic-co-glycolic acid) (PLGA) nerve tubes in a 1-cm gap of the rat sciatic nerve model. Simultaneous tracing of tibial and peroneal nerves with fast blue (FB) and diamidino yellow (DY), respectively, was performed to quantify axonal dispersion for the percentage of motoneurons with double projections to both branches (Fig. 2). Sequential tracing of peroneal nerve with DY injection before and FB application 8 weeks after repair was used to quantify the percentage of peroneal motoneurons correctly directed to the peroneal nerve branch (Fig. 3). In addition, quantitative results of regeneration were analyzed with compound muscle action potential (CMAP) recordings and nerve and muscle morphometry.

Fig. 2

The technique of simultaneous tracing and the concepts for the dispersion of regenerating motor axons after autograft, single lumen, and multichannel nerve tube repair.

Fig. 3

The technique of sequential tracing and the concepts for misdirection of regenerating axons after autograft, single lumen, and multichannel nerve tube repair.

Material and Methods

Experimental Groups

Sprague-Dawley rats weighing between 250 and 275 g were randomly assigned to one of the experimental groups for autograft (n=17), single lumen (n=30), or multichannel (n=30) PLGA nerve tube repair. For autograft repair, 7 animals were used for simultaneous tracing and 6 animals were used for CMAP recording and nerve and muscle morphometry. Results for sequential tracing (n=4) were obtained from another study (7). For single lumen and multichannel nerve tube repair, 12 animals were used for simultaneous tracing, 12 animals for sequential tracing, and 6 animals for CMAP recording and nerve and muscle morphometry. Control animals were included for simultaneous tracing (n=4) and for CMAP recording and nerve and muscle morphometry (n=4). All procedures were approved by and performed according to the animal care guidelines of the Mayo Foundation Institutional Animal Care and Use Committee.

Single Lumen and Multichannel Nerve Tube Fabrication

Single lumen and multichannel PLGA nerve tubes were fabricated through an injection-molding solvent evaporation technique (6,22) using the same Teflon mold for the fabrication of single lumen and multichannel nerve tubes, except with different mold assemblies. The mold consisted of cylindrical voids (2.1 mm in diameter) with caps on both ends through which either 1 stainless steel wire (1.6 mm in diameter), for the fabrication of single lumen nerve tubes, or 7 stainless steel wires (400 μm in diameter), for the fabrication of multichannel nerve tubes, were inserted (wires from Small Parts Inc, Miami Lakes, Florida; mold designed and produced at Mayo Clinic). The caps for the fabrication of multichannel nerve tubes provided for an extra 1-mm sleeve on each end for implantation (Fig. 1).

A solution of PLGA (copolymer ratio, 75:25; 92 kD; Fisher Scientific, Hampton, New Hampshire) in methylene chloride (300 mg/450 μL) was injected into the mold. The mold was placed in a vacuum for rapid solvent evaporation, thus creating a highly porous nerve tube structure. Nerve tubes were sterilized in ethanol and prehydrated in sterile phosphate-buffered saline (PBS) before implantation.

Surgical Techniques and Animal Care

Animals were deeply anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (2.5 mg/kg) injected intraperitoneally. The sciatic nerve was exposed through a dorsal gluteal splitting approach with the aid of an operating microscope (Zeiss, Germany). For autograft repair, the nerve was transected at 2 sites, 1 cm apart, and immediately repaired with fascicular alignment using 10-0 sutures (Ethilon, Ethicon Inc, Piscataway, New Jersey) (proximal, 4 sutures, and distal, 3 or 4 sutures for the tibial branch; 2 or 3 sutures for the peroneal branch). For single lumen and multichannel nerve tube repair, the nerve was also transected at 2 sites, 8 mm apart. First, the proximal end was pulled 1 mm into a 12-mm nerve tube (with a single 10-0 suture) while preserving the original alignment; then the same procedure was performed distally for insertion of the tibial and peroneal branches separately, creating a 1-cm gap. Fibrin glue (Tisseel VH fibrin sealant, Baxter, Deerfield, Illinois) was applied to both ends of the tube to seal the lumen and sleeve. Multichannel nerve tubes were always implanted with the same orientation of the channels in relation to the nerve. The wound was closed in layers. Animals received buprenorphine hydrochloride (Reckitt Benckiser Healthcare, Slough, England) to control pain.

Postoperatively, animals were housed in individual cages with a 12-hour light-dark cycle and water and food available ad libitum. The operated limb was sprayed daily with bitter spray (Chew Guard; Butler Corporation, Greensboro, North Carolina) to prevent autotomy. A wire mesh was placed inside the cage to prevent contractures of the ankle and foot (31).

Simultaneous and Sequential Tracing

In the simultaneous tracing experiment, FB and DY tracers (both from EMS-Chemie, Mannedorf, Switzerland) were applied to the tibial and peroneal nerve branches, respectively, 8 weeks after implantation (Fig. 2). First, the tibial nerve was transected and placed in a cup containing 1.5 μL of 5% FB solution for 30 minutes, followed by peroneal nerve transection and capsule application with 1.5 μL of 5% DY solution for 30 minutes. The nerve ends were cleaned with 0.9% saline and sutured into surrounding fat tissue to prevent tracer leakage and cross-contamination. The same procedure was performed on the right side in 4 animals to obtain normal values.

In the sequential tracing experiment, 1 μL of 5% DY solution was injected into the peroneal branch 1 week before implantation using a scaled glass syringe (Hamilton Company, Reno, Nevada) with a 25-gauge needle (Fig. 3). Eight weeks after implantation, the sciatic nerve was reexposed, and the peroneal nerve was transected proximal to the previous injection site and placed in a cup containing 1.5 μL of 5% FB solution for 30 minutes. Again, the nerve end was cleaned with 0.9% saline and sutured into surrounding fat tissue to prevent tracer leakage.

Animals were allowed to survive for 6 days after tracer application and then were perfused with PBS and a solution containing 4% paraformaldehyde and 10% sucrose. Spinal cord segments L1–6 were removed and postfixed overnight. Tissue was embedded in tissue-freezing medium (Triangle Biomedical Services, Inc, Durham, North Carolina) and stored at −80°C until sectioning. Sagittal longitudinal 30-μm-thick sections were cut on a cryostat at −20°C. Slides were immediately evaluated under a fluorescent microscope (Axioplan 2, Carl Zeiss, Inc, Oberkochen, Germany) with a DAPI filter set (360/400-nm bandpass excitation filter, 440-nm long pass emission filter, and 400-nm dichroic beamsplitter) at 20× magnification with a planapochromatically corrected microscope 20×/0.75 objective (Plan Apochromat, Zeiss). Neuronal profiles were counted in every section by 1 observer (G.C.de R.) who was blinded to the experimental groups. Only profiles with a visible nucleus were counted. Profiles with blue cytoplasm and a dark nucleus were counted as FB-labeled motoneurons, profiles with a yellow nucleus and dark cytoplasm as DY-labeled motoneurons, and profiles with a yellow nucleus and blue cytoplasm as FB-DY–double-labeled motoneurons. All sections were counted. No corrections were made for the possibility of counting split motoneurons. Persistence of tracer in the sequential tracing experiment was analyzed from the distribution of double-labeled profiles. If double-labeled profiles were present in an area of the anterior horn that was normally exclusively occupied by tibial motoneurons (determined in control animals in the simultaneous tracing experiment), the case was excluded (7).

In the simultaneous tracing experiment, the percentage of double projections was calculated by dividing the total number of double-labeled (FB-DY) motoneurons by the total number of labeled motoneurons (FB, DY, and FB-DY). In the sequential tracing experiment, the percentage of correctly directed peroneal motoneurons was calculated by dividing the total number of FB-DY–labeled motoneurons by the total number of DY-labeled motoneurons (DY and FB-DY).

CMAP Recording

In the experiment on CMAP recording and nerve and muscle morphometry, CMAPs were recorded at 4, 6, 8, 10, and 12 weeks after implantation. CMAPs were recorded using electromyography (Nicolet Viking IV, Viasys Healthcare Inc, Conshohocken, Pennsylvania) in the tibial and peroneal nerve–innervated foot muscles of the operated limb. Needle recording electrodes were placed in the plantar or dorsal foot muscles referenced to needle electrodes placed distally in the foot digits. Needle stimulating electrodes were placed directly posterior to the tibia with approximately 5 mm between the distal cathode and proximal anode. The stimulating electrodes were adjusted locally to produce the maximal CMAP amplitude. The stimulus was increased incrementally to produce a supramaximal response. CMAPs were recorded and analyzed for the amplitude, the area under the curve, and latency of the action potential.

Nerve Morphometry

The graft was reexposed 12 weeks after autograft, single lumen, and multichannel nerve tube repair and fixed in situ with a 2.5% glutaraldehyde solution in PBS for 30 minutes (9). The graft was resected and placed in the same fixative overnight. Specimens (1 mm) were selected 2 mm proximal, at the midpoint, and 2 mm distal to the graft and embedded in spur resin for postfixation in 1% osmium tetroxide. Sections (1 μm) were cut with a glass knife on an ultramicrotome and stained with 1% phenylenediamine. The number of myelinated axons and mean size were analyzed at all 3 levels using the imaging system for nerve morphometry (Peripheral Nerve Laboratory of Dr Peter J. Dyck, Mayo Clinic Rochester). Between 500 and 600 myelinated axons were randomly selected in the slide and analyzed at 63× magnification (9).

Muscle Morphometry

After resection of the graft, the soleus muscle was resected, placed in a plastic cup containing tissue-freezing medium, frozen with isopentane and liquid nitrogen, and stored at −80°C until sectioning. Transverse 10-μm sections were cut on the cryostat at −20°C. Sections taken from the mid-belly of the muscle were stained for myofibrillar ATPase at pH 9.4 according to the method described by Brooke and Kaiser (4), staining slow (type I) fibers light and fast (type II) fibers dark. The total muscle fiber surface area was determined using an image analysis system (KS400, version 3.0, Zeiss, Germany) (35). The numbers of type I and type II fibers were counted. The mean muscle fiber size was calculated by dividing the total muscle fiber surface area by the total number of muscle fibers (type I and II).

Statistical Analysis

Statistical analysis was performed using 1-way analysis of variance with post-hoc Bonferroni tests. P values less than .05 were considered significant.

Results

Number of Analyzed Animals per Experimental Group

Successful regeneration across the nerve graft (determined from the presence of FB-, DY-, or FB-DY–labeled profiles in the anterior horn after simultaneous tracing, the presence of FB-labeled profiles after sequential tracing, or the presence of CMAPs and myelinated axons distal to the graft) was found in 17 of 17 animals (100%) after autograft repair, in only 16 of 30 animals (53.3%) after single lumen nerve tube repair, and in 13 of 30 (43.3%) after multichannel nerve tube repair. Animals that had no signs of regeneration across the nerve tube were excluded from further analysis because of the confounding effect on the results for the cases with successful regeneration (for example, including the cases with no signs of regeneration would lead to low percentages of double-projecting motoneurons because of 0% double-labeling in these cases). In addition, in the simultaneous tracing experiment, 2 cases of single lumen PLGA nerve tube repair and 2 cases of multichannel PLGA nerve tube repair were excluded from further analysis because of exclusive regeneration to the tibial branch (presence of only FB-labeled profiles). In the sequential tracing experiment, 1 case of single lumen nerve tube repair was excluded from further analysis because of persistence of DY tracer, and 1 case of multichannel nerve tube repair was excluded because of failure to label the original peroneal motoneuron pool.

Simultaneous Tracing

Spinal organization of single-labeled FB and DY profiles in control animals demonstrated that the tibial and peroneal motoneuron pool are normally present in 2 separate, nonintermingling spinal nuclei in the anterior horn at the levels L2-L6. After autograft, single lumen, and multichannel nerve tube repair, this spinal organization was disturbed, and FB-, DY-, and FB-DY–labeled motoneurons were found intermingled.

The percentages of double projections were significantly different after autograft repair and for the cases with successful regeneration after single lumen and multichannel PLGA nerve tube repair (F[2,14]=16.5; P=.0002) (Figure 4). After single lumen nerve tube repair, more motoneurons had double projections to both the tibial and peroneal nerve branches (21.4%±4.9%) than after autograft repair (5.9%±2.9%) (P<.001 for posttest). The percentage after multichannel nerve tube repair was slightly lower (16.9%±6.0%) compared with that after single lumen nerve tube repair, but this difference was not significant. The total numbers of motoneurons from which axons had regenerated into the tibial and/or peroneal nerve branch were significantly different from the normal number of motoneurons (1,246±43) (F[3,17]=48.3; P<.0001), except after autograft repair (1,140±179). The total numbers of single lumen (448±108) and multichannel (406±156) nerve tube repair were not significantly different.

Fig. 4

Results of simultaneous tracing for the mean number of fast blue (FB)–, diamidine yellow (DY)–, and FB-DY–labeled profiles and the percentages of double-projecting motoneurons in normal animals (n=4) and after autograft (n=7), (more ...)

Sequential Tracing

Differently labeled profiles (FB, DY, or FB-DY) were also found to be intermingled in the anterior horn after sequential tracing and autograft, single lumen, and multichannel tube repair. The size of the nucleus of profiles was variable, especially for DY profiles (ranging from 10 to 20 μm in diameter). Therefore, no corrections were made for the profile counts on the basis of the size of the nucleus (1).

The percentages of correct direction were significantly different after autograft repair and for the cases with successful regeneration after single lumen and multichannel PLGA nerve tube repair (F[2,8]=9.4; P=.008) (Figure 5). The percentage after autograft repair (25.1%±6.6%) was significantly higher than with single lumen (11.5%±3.8%) and multichannel (11.6%±3.7%) nerve tubes (P<.05 for both posttests); however, these percentages were probably underestimated because of the decreased number of regenerated motoneurons after single lumen and multichannel nerve tube repair, resulting in a relatively high number of single DY-labeled profiles. After correction for the total number of regenerated profiles found with simultaneous tracing, the percentages of correctly directed peroneal motoneurons are similar (27.4% for autograft, 32.0% for single lumen, and 35.6% for multichannel nerve tube repair). Considering the sizes of the peroneal and tibial motoneuron pool found with simultaneous tracing (487 [61%] and 760 [39%] motoneurons, respectively), these percentages indicate that regeneration was nonspecific after all types of repair.

Fig. 5

Results of sequential tracing for the mean number of fast blue (FB)–, diamidine yellow (DY)–, and FB-DY–labeled profiles, and the percentages of correctly directed peroneal motoneurons after autograft (n=4), single lumen (n=5), (more ...)

Compound Muscle Action Potentials

The first CMAPs were detected at 8 weeks after autograft repair, compared with 10 weeks in the cases with successful regeneration after single lumen and multichannel PLGA nerve tubes (Fig. 6 A, B). The CMAP amplitude and area recorded at 12 weeks were only significantly different in the dorsal foot muscles (F[2,9]=9.7 (P=.0057) and F[2,9]=6.2 (P=.0199), respectively); they were not significantly different in the plantar foot muscles (F[2,9]=4.1 (P=.05) and F[2,9]=3.3 (P=.08), respectively) (Fig. 6 A-D), with a significantly larger CMAP amplitude and area after autograft repair than with single lumen and multichannel nerve tube repair (P<.05 for both posttests, except for the comparison of the CMAP area after autograft and single lumen nerve tube repair). The CMAP latency decreased with time after all repair techniques (Fig. 6 E, F).

Fig. 6

Results of compound muscle action potential (CMAP) recordings for the amplitude (A, B), area (C, D), and latency (E, F) recorded in the plantar (A, C, E) and dorsal (B, D, F) foot muscles 8, 10, and 12 weeks after autograft (gray), single lumen (white), (more ...)

Nerve Morphometry

The number of myelinated axons at the midpoint and distal to the graft was significantly different compared with normal after autograft and single lumen and multichannel nerve tube repair (F[3,11]=88.6 (P<.0001) and F[3,8]=23.9 (P=.0002); 4 samples were lost because of longitudinal imbedding) (Table 1). After autograft repair, these numbers were significantly increased (P<.001 and P<.05, respectively). In the cases with successful regeneration after single lumen and multichannel PLGA nerve tubes, these numbers were significantly decreased (P<.001 and P<.05, and P<.01 and P<.05, respectively). There was no significant difference for single lumen and multichannel PLGA nerve tube repair, despite the more than 2-fold smaller cross-sectional lumen area available for regeneration in the multichannel nerve tube compared with the single lumen nerve tube (0.8 mm² for 7 channels 400 μm in diameter compared with 2.0 mm² for a single lumen nerve tube with a lumen diameter of 1.6 mm), and the fact that only 3 of 7 channels were filled with myelinated axons (Fig. 7). The number of myelinated axons proximal to the graft was increased after all types of repair, although not significantly. The mean size of myelinated axons was significantly decreased compared with normal after all types of repair (proximal to the graft, F[2,9]=31.4 (P<.0001); mid, F[3,11]=190.2 (P<.0001); and distal, F[3,8]=257.0 (P<.0001), with no significant difference for autograft, single lumen, and multichannel nerve tube repair (P>.05 for all posttests, except for the comparison of single lumen and multichannel nerve tubes distal to the graft [P<.05]).

Table 1

Number and Mean Size of Myelinated Axons in Normal Rats, Proximal to, at the Middle of, and Distal to the Graft 12 Weeks After Autograft, Single Lumen, and Multichannel Nerve Tube Repair

Fig. 7

Microscopic sections from the middle of a single lumen (A and C) and multichannel (B and D) nerve tube 3 months after implantation. Stained with toluidine blue for myelinated axons and taken at 40× (A, B) and 160× (C, D) magnification. (more ...)

Muscle Morphometry

The number of muscle fibers was not significantly different in normal animals, after autograft, and in the cases with successful regeneration after single lumen and multichannel nerve tube repair (Table 2). The mean size of the muscle fibers was significantly different from normal after all types of repair (F[3,10]=13.2; P=.0008), with no significant difference for the autograft, single lumen, and multichannel PLGA nerve tube repair. The distribution of type I and type II muscle fibers had changed from more type I than type II in normal soleus muscle (10:1) to more type II than type I after autograft and single lumen nerve tube repair (F[3,10]=30.8 (P<.0001), with P<.001 for both posttests) (Fig. 8). For the successful cases of multichannel nerve tube repair, this distribution (ratio 3:1) was not significantly different from normal.

Table 2

Mean Muscle Fiber Size and Distributions of Type I and Type II Fibers in Normal Rats and 12 Weeks After Autograft, Single Lumen, and Multichannel Nerve Tube Repair

Fig. 8

Microscopic sections taken from the mid–muscle belly of normal soleus muscle (A) and after autograft repair (B), single lumen nerve tube repair (C), and multichannel nerve tube repair (D). Distribution of type I (light) and type II (dark) fibers (more ...)

Discussion

Accuracy of regeneration, ie, the event that axons find their original or related end organ, is essential to obtain functional recovery when a nerve gap has to be bridged. Autografts are routinely used. In recent years, different single lumen nerve tubes have been introduced to serve as guidance for axonal outgrowth (29). Little is known about the potential effect of single lumen nerve tube structure on the accuracy of regeneration as compared to the autograft (35), and even less is known about the effect of more complex structures as the multichannel nerve tube. In this study, we used simultaneous and sequential retrograde tracing techniques to compare the accuracy of motor axon regeneration across autograft, single lumen, and multichannel PLGA nerve tube repair in a 1-cm gap of the rat sciatic nerve model.

Single Lumen Nerve Tube Repair Versus Autograft Repair

Simultaneous tracing in our study showed increased dispersion of regenerating axons in the cases in which there was successful regeneration across the single lumen nerve tube, with more double-projecting motoneurons (21.4%) than after autograft repair (5.9%). Sequential tracing showed no difference in the direction of regenerating axons with similar percentages of correctly directed peroneal motoneurons (after correction for the decreased number of regenerated profiles). Although various methods have been used to investigate the accuracy of reinnervation after single lumen nerve tube repair (3,12,28,34,41), this is, to our knowledge, the first study that compares the accuracy of motor axon regeneration across single lumen nerve tubes and autografts using simultaneous and sequential tracing.

Technical Note

Numerous factors have to be considered in the interpretation of our results from tracing (including the size of the nerve gap, time point of evaluation, technique of tracing, and physical properties of the nerve tube). Dispersion has been found to increase with gap length (5). Also, the number of double-projecting motoneurons has been found to decrease again with time (18). Axonal branching with polyinnervation, followed by pruning, may therefore actually be a mechanism to correct for misdirection. This can explain the lower percentages of multiple-projecting motoneurons that were found by Valero-Cabre et al (34) 90 days after repair of 8-mm nerve gaps with single lumen poly-L-lactid-ε-caprolactone (6.0%) and silicone nerve tubes (10%) (compared with 5.6% after autograft repair). More research is needed to further investigate the influence of nerve gap size and the effect of the time point of evaluation in the analysis of the accuracy of regeneration across nerve tubes.

Factors concerning the techniques of tracing must also be considered. In this study, we used FB and DY tracers for both simultaneous and sequential tracing. This combination of tracers has been investigated in detail by Puigdellivol-Sanchez et al (25–27). The practical advantage is that both tracers can be visualized with the same filter (27). The sequential tracing technique with DY injection followed by FB application 8 weeks after repair has a high labeling efficiency (86.9%) (25) that is better than the technique of tracer muscle injection (17), which has been used previously to investigate the accuracy of muscle reinnervation after nerve tube repair (3,28). There is also no significant fading of the first tracer (DY) or blockage of uptake of the second one (FB) (27). Problems of the nerve injection technique though are 1) persistence of DY tracer that may lead to overestimation of the percentage of correctly directed motoneurons (26) and 2) damage to the nerve before nerve injury and repair that may affect regeneration to the peroneal nerve branch. In our study, only 1 case of significant persistence of tracer was found. The number of motoneurons from which axons had regenerated into the peroneal branch did not appear to be different after simultaneous and sequential tracing (compare the number of DY- plus FB-DY–labeled motoneurons in Fig. 4 with the number of FB-and FB-DY–labeled motoneurons in Fig. 5). In addition, the size of the nerve used for sequential tracing must be considered. Recently, Puigdellivol-Sanchez et al (24) found a much higher percentage of correctly directed tibial motoneurons after direct coaptation repair (87%) using the same technique and time point of evaluation. The difference with our results might be explained by the difference in repair techniques (direct coaptation vs graft repair (7) and by the larger size of the tibial motoneuron pool.

Different physical properties of the nerve tube may also affect results. In our study, nerve tubes were made of PLGA using an injection-molding solvent evaporation technique (6,22). This biomaterial has been approved by the US Food and Drug Administration; is biodegradable, biocompatible, and sterilizable; can be used for sustained-release drug delivery; and has been used previously to fabricate both single lumen and multichannel nerve tubes (11,32,37). We recently found in vitro, however, that PLGA nerve tubes swell extensively, especially for lower lactic acid–to–glycolic acid ratios (6). Swelling may block the lumen or channels for regeneration or compress regenerated axons. In addition, acidic products formed during the degradation process may interfere with regeneration, and PLGA nerve tubes may elongate and collapse (10,11). These factors can explain the disappointing results found after PLGA nerve tube repair in our study with successful regeneration across the nerve tube in only ~50% of the cases and, in these cases, with significantly decreased numbers of motoneurons from which axons had regenerated across the nerve tube compared with normal and autograft repair. Currently, we are investigating the use of novel biomaterials, for example, poly(caprolactone fumarate), which self-cross-links and therefore can also be used for the injection-molding technique of fabrication (20). Another physical property that may affect the accuracy of regeneration is the permeability or porosity of the nerve tube, which controls the exchange of external and internal neurotrophic factors. Both single lumen and multichannel PLGA nerve tubes used in this study were highly permeable because of the solvent evaporation technique of fabrication (6).

Finally, it must be noted that in this study we used an optimized technique for autografting with an immediate direct repair of matched size nerve stumps. Results might differ from clinical nerve repair with interposition of multiple sural nerve grafts after a time delay. Moreover, in clinical nerve repair, it is not always possible to accurately determine and reconstruct the fascicular architecture of the nerve.

Although different factors must thus be considered in the interpretation of these results, our study demonstrates that simultaneous and sequential tracing techniques provide new insights into the accuracy of regeneration across nerve tubes. Of course, conventional evaluation methods (including CMAP recording and nerve and muscle morphometry) and functional analysis will still have to be performed, especially in the development of a nerve tube for possible clinical application.

Single Lumen Versus Multichannel Nerve Tube Repair

To reduce dispersion, we designed a multichannel nerve tube and compared it to a single lumen nerve tube made of the same material using the same fabrication technique. In this study, no significant differences between single lumen and multichannel PLGA nerve tubes were found. In our opinion, however, the concept of a multichannel nerve tube that might limit dispersion remains appealing for the following reasons.

The only slight reduction in dispersion after multichannel nerve tube repair in this study might be explained by the findings that 1) some axons had probably already branched before entering the channel (as can be concluded from the increased number of axons proximal to the nerve graft); 2) results for the number of FB- and DY-labeled profiles were variable (in some cases even exclusive regeneration to the tibial branch was found); and 3) only 3 of 7 channels were filled with myelinated axons 3 months after multichannel PLGA nerve tube repair (compared with the large number of basal lamina tubes in an autograft, ~1,000 in the sciatic nerve in mice [14]). Different modifications to the multichannel nerve tube, including more channels and different channel fillings, may further reduce dispersion. A total of 7 channels (400 μm in diameter) was the maximum number that fit into a tube with a 1.6-mm inner diameter, because of the minimum distance of 100 μm needed to drill holes in the end caps through which the wires were inserted. Currently, we are investigating the use of 3-dimensional printing. With this fabrication technique, nerve tubes with any shape can be built in a layer-by-layer fashion. In the future, it might even be possible with this technique to reconstruct the fascicular architecture of the nerve that often does not consist of longitudinally aligned fascicles, but instead forms an intraneural plexus (33).

Different channel fillings, including surface coatings, growth factors, and Schwann cells, may increase both the number of axons that regenerate and the number of channels across which axons regenerate. An additional advantage of the multichannel nerve tube thereby is that it provides more luminal surface area than single lumen nerve tubes for cell attachment and local release of incorporated growth factors. The finding that quantitative results of regeneration in this study were similar to repair with empty single lumen and multichannel nerve tubes (despite the more than 2-fold smaller cross-sectional lumen area for multichannel nerve tubes) encourages us to investigate further the use of the different channel fillings, including neurotrophic factors that might also be of interest to guide regenerating axons, which often wander at the suture site (38), straight into the channels (21).

Finally, of interest in this study was the distribution of type I and type II fibers that was closer to normal (10:1) after multichannel nerve tube repair (3:1) than after both single lumen and autograft repair (both 2:3) (Fig. 8). Although this may be explained by various factors, it may indicate more accurate muscle reinnervation after multichannel nerve tube repair. Both the small slow soleus muscle and large fast gastrocnemic muscle (40) are innervated by the lateral gastrocnemic nerve (15), and after nerve injury and repair, the fiber type distribution in soleus muscle may change as a result of misdirection (3,16,19). Despite similar percentages of correct direction, multichannel nerve tube repair may therefore lead to improved muscle reinnervation, for example, by a more organized distribution of regenerated motoneurons in the anterior horn. In the present study, differences in spinal organization could not be analyzed because of the small number of regenerated profiles. More research is therefore needed to investigate this finding further.

Conclusions

Retrograde tracing in this study demonstrated the importance of investigating the accuracy of motor axon regeneration in the development of a nerve tube for motor nerve repair. We also presented the concept of the multichannel nerve tube that might limit dispersion, although in this study no statistical difference in accuracy of regeneration across single lumen and multichannel nerve tubes was found.

Acknowledgments

Dr de Ruiter was supported by the Sundt Fellowship from the Mayo Clinic Department of Neurosurgery and grants from the Netherlands (VSB Fonds, Leids Universitair Fonds, Janneke Fruin-Helb beurs, Stichting Fundatie van de Vrijvrouwe van Renswoude te’s Gravenhage, Stichting Mitialto, Stichting Dr. Hendrik Muller’s Vaderlandsch Fonds, Lustra and Jo Keur Fonds). Dr Windebank was supported by National Institutes of Health grant (EB02390) and Mayo Clinic College of Medicine.

We thank LouAnn Gross for advice on embedding and staining techniques, Dr Peter J. Dyck and JaNean Engelstad for advice on nerve morphometry, and Tony Koch for excellent animal care.

Baxter Healthcare Corporation, Westlake Village, California, provided the fibrin glue.

Editing, proofreading, and reference verification were provided by the Section of Scientific Publications, Mayo Clinic.

Abbreviations


CMAP	compound muscle action potential

DY	diamidino yellow

FB	fast blue

PBS	phosphate-buffered saline

PLGA	poly(lactic-co-glycolic acid)

Footnotes

The authors declare no financial interest in the multichannel nerve tube.

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