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The Effect of Short Nerve Grafts in Series on Axonal Regeneration Across Isografts or Acellular Nerve Allografts
SCIENTIFIC ARTICLE
The Effect of Short Nerve Grafts in Series on Axonal Regeneration Across Isografts or Acellular Nerve Allografts Ying Yan, MD, PhD,* Matthew D. Wood, PhD,* Daniel A. Hunter,* Xueping Ee, MD,* Susan E. Mackinnon, MD,* Amy M. Moore, MD*
Purpose To evaluate the regenerative effect of the additional suture line when using either isografts (ISOs) or acellular nerve allografts (ANAs) placed end-to-end to span a short gap in a rat model. Methods Rat sciatic nerves were transected and repaired with 2-cm nerve grafts (ISO or ANA). The grafts were 2 cm in length or a 1-cm segment was connected end-to-end to a 1-cm segment to yield a 2-cm length. At 8 weeks, extensor digitorum longus (EDL) muscle force and mass were measured. Nerves were harvested for histomorphometry. In a separate parallel study, the nerves were harvested 2 weeks following graft implantation to assess gene expression changes. Results All grafts demonstrated regeneration across the 2-cm segment(s). The additional suture line did not result in statistical differences in the number of myelinated nerve fibers that reached the distal nerve. However, when the graft types were compared, there was a significant decrease in nerve fibers in the ANA groups. The EDL muscle mass was significantly greater by using nerve ISOs compared with ANAs, regardless of an additional suture line, but there were no statistical differences noted in EDL muscle force. Gene expression analysis did not differ owing to an additional suture line. Conclusions Minimal axonal loss and no functional deficits were identified with an additional suture line in this rodent short nerve gap model. Clinical relevance Placing nerve grafts in series is a viable option for treating short nerve gaps; however, the use of autografts remains preferable over the use of ANAs. (J Hand Surg Am. 2016;-(-):-e-. Copyright Ó 2016 by the American Society for Surgery of the Hand. All rights reserved.) Key words Coaptation site, nerve regeneration, peripheral nerve, processed nerve allograft, suture line.
From the *Division of Plastic and Reconstructive Surgery, Washington University School of Medicine, St. Louis, MO. Received for publication September 28, 2015; accepted in revised form January 8, 2016. No benefits in any form have been received or will be received related directly or indirectly to the subject of this article. Corresponding author: Amy M. Moore, MD, Division of Plastic and Reconstructive Surgery, Washington University School of Medicine, 660 South Euclid, Campus Box 8238, St. Louis, MO. 63110; e-mail: [email protected]. 0363-5023/16/---0001$36.00/0 http://dx.doi.org/10.1016/j.jhsa.2016.01.009
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direct endto-end coaptation is not possible owing to a segmental nerve injury or tension. Nerve grafting inherently predisposes regenerating axons to an additional coaptation site increasing the risk of axonal loss from scarring.1,2 Sutures activate a local inflammatory response, which, if large enough, can generate considerable fibrosis hindering the progression of regenerating axons.3 The primary coaptation site linking the distal and proximal nerve is associated ERVE GRAFTING IS PERFORMED WHEN A
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with slowed or staggered axonal regeneration.4 Thus, it follows that additional coaptation sites may generate similar effects. The impact of an additional suture line on axonal regeneration through nerve grafts is not clearly understood. This information holds clinical value in the event of limited donor nerve supply such as that seen with major injuries involving multiple limbs with large segmental nerve defects. In these devastating cases, the end-to-end linking of multiple nerve grafts in series (autograft and/or acellular nerve allografts [ANAs]) to repair long nerve gaps have been described and clinically referred to as “daisy-chaining.”5 The clinical use of ANAs has increased markedly since the U.S. Food and Drug Administration’s approval of the Avance Nerve Graft (AxoGen, Inc., Alachua, FL) was obtained.6 The decellularized nerve allografts offer an off-the-shelf alternative to autografting and avoid donor site morbidity and prolonged operative time. The potential for using multiple decellularized nerve grafts placed in series is an attractive option, especially in the severely traumatized patient. Our study sought to determine how the additional suture lines affect axonal regeneration across short nerve autografts and acellular nerve allografts. In this study, short nerve grafts were used to avoid additional confounding issues affecting axonal regeneration, such as complete regenerative failure associated with long ANAs.7 The present study evaluated axonal regeneration and functional recovery across isografts (ISOs; animal model equivalent of an autograft8,9) and ANAs repaired using a whole, unmodified graft or a graft of comparable length with an additional suture line. To determine how the additional suture line exerted its influence, gene expression products related to nerve regeneration were measured in the grafts.
with consistent diameter and no additional branch points, eliminating confounders such as axonal loss and graft size. In study A, 32 rats were divided into 4 groups (n ¼ 8 per group) consisting of a whole ISO, an ISO with an additional suture line (ISO in-series), a whole ANA (ANA), or an ANA with an additional suture line (ANA in-series). At 8 weeks after surgery, extensor digitorum longus (EDL) muscle force testing was performed, and EDL muscle mass was measured. The sciatic nerves were also harvested for histomorphometric analysis (Table 1). Eight weeks was chosen based on previous studies that suggest this time point maximizes the sensitivity to measure differences between groups receiving 2-cm nerve grafts.10,11 In a parallel study B, 16 rats were randomized to the same 4 groups (n ¼ 4 per group) and underwent similar procedures as in study A. These rats were used to assess gene expression within the graft area affected by the suture line (or in the midgraft of the whole grafts). At 2 weeks after surgery, gene expression was measured using quantitative reverse-transcriptaseepolymerase chain reaction (qRT-PCR; Table 1). For all groups, Lewis male rats served as the experimental animals and donor animals for ISO groups. In ANA graft groups, Sprague-Dawley rats (250 g; Charles River Laboratories, Wilmington, MA) were used as donor nerve for processing to generate ANAs. The Sprague-Dawley (SD) (RT-1b major histocompatibility complex [MHC]) rat strain is MHCincompatible with Lewis (RT-11 MHC) and were used as allograft donors. Lewis rats are a well-accepted rat strain used to derive ISOs because they are inbred leading to syngeneic donors. Sciatic nerve allografts harvested from donor rats were chemically processed and decellularized using a series of detergents as previously described.7,12,13 Surgical procedures and perioperative care measures were conducted in compliance with the Institutional Animal Studies Committee and National Institutes of Health guidelines. All animals were housed in a central animal care facility and provided with food (PicoLab rodent diet 20; Purina Mills Nutrition International, St. Louis, MO) and water ad libitum.
MATERIALS AND METHODS Animals and experimental design The sciatic nerves of adult male Lewis rats (250 g; Charles River Laboratories, Wilmington, MA) were transected and then immediately repaired with 2-cm nerve grafts. These grafts were either left unmodified (whole) or cut into equal 1-cm pieces and sutured endto-end, thus generating an equivalent 2-cm length. A 2-cm nerve gap was chosen to eliminate the confounding effects of graft length and to focus on the effect of the additional suture line on regeneration. Long nerve grafts (autograft and ANA) have been associated with a significant decline in axonal regeneration in comparison with short nerve grafts.7 Further, a 2-cm graft can be obtained from the sciatic nerve J Hand Surg Am.
Surgical procedures Surgical procedures were performed under aseptic conditions and with the aid of an operating microscope under magnifications of 10-25X.7 A single surgeon (Y.Y.) performed all operations. Anesthesia was provided by subcutaneous delivery of ketamine (75 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA) and dexmedetomidine (0.5 mg/kg; Pfizer Animal r
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TABLE 1. Groups
Study Design and Groups Animal Numbers
Recipient
Group 1-A Group 2-A Group 3-A
Lewis
ISO in series
Sprague-Dawley
ANA in series
Lewis
ISO in series
Analysis
8 wk
EDL muscle force, muscle mass, and histomorphometry
2 wk
qRT-PCR
ISO
Lewis
4
End Point
ANA in series ANA
8
Group 1-B Group 3-B
Nerve Graft
Sprague-Dawley
Group 4-A Group 2-B
Donor
ANA
Group 4-B
ISO
Health, Exton, PA). The right sciatic nerve was exposed using a muscle-splitting technique. For donor nerve grafts, approximately 3.5 cm of sciatic nerve was harvested, which was later trimmed to appropriate length. Sciatic nerves were transferred to aseptic tubes to undergo acellular processing13 or immediately used as fresh nerve ISOs. In experimental rats, the recipient sciatic nerve was transected 5 mm proximal to the trifurcation of the sciatic nerve. The defect was reconstructed with an ISO or ANA secured to the proximal and distal nerve stumps using 4 interrupted 9e0 nylon epineurial sutures at each suture line. For groups with in-series nerve grafts, the 2 1-cm graft segments were secured first using 4 9-0 nylon epineurial sutures to yield a 2-cm graft before coaptation to the proximal and distal nerve (16 magnification). The muscle and skin were closed in a layered fashion. Animals recovered and were housed in a central animal care facility.
bloc 5 mm proximal and 5 mm distal to the interposed graft. The recipient/donor sciatic nerve complexes were stored in 3% glutaraldehyde and followed by processing and assessment for evidence of nerve regeneration.15 An observer blinded to the experimental groups obtained all measurements. Quantitative reverse-transcriptaseepolymerase chain reaction After 2 weeks in a separate set of animals, nerve grafts (4-mm segments either containing an additional suture line or left whole) were harvested for gene expression analysis. Samples were taken at the center of the graft or directly from the suture line area, which included 2 mm proximal and 2 mm distal (4 mm total) of the suture line area within the graft. Samples for qRT-PCR analysis were stored in RNAlater solution (QIAGEN, Valencia, CA) and stored at 80 C prior to RNA extraction. Total RNA was extracted by using Trizol Kit (QIAGEN, Valencia, CA) according to the manufacturer’s direction. RNA was then reverse transcribed into complementary DNA strands (cDNAs) using the protocol described in the High Capacity RNA-tocDNA kit (Applied Biosystems, Foster City, CA). Gene expression of Col1a1, Cd31, Ang2, Jag1, Dll4, and Gdnf were analyzed using qRT-PCR. Primers shown on Appendix A (available on the Journal’s Web site at www.jhandsurg.org) were purchased from Life Technologies (Grand Island, NY). Amplification of each target cDNA was done with Taqman primers (Appendix A; available on the Journal’s Web site at www.jhandsurg.org) using a Step One Plus thermocycler (Applied Biosystems, Foster City, CA) with normalization to a housekeeping gene (b-actin), and the data analyzed on Step One Software v2.2.2 (Applied Biosystems). A value of 2 or greater was selected as the minimum criteria for a significant difference in expression levels between graft groups and the normal fresh nerve.16
Functional assessment and muscle recovery Functional recovery of muscle force was assessed at 8 weeks. The measurements were performed without blinding because this procedure is not possible without the group identity being revealed. Evoked motor response in reinnervated EDL muscle upon electrical stimulation of the repaired sciatic nerve was measured.7 Maximum specific isometric tetanic force was calculated as the maximum isometric force normalized to physiological muscle cross-sectional area.14 Following force assessment, the EDL muscle was harvested from both the experimental and the contralateral sides and weighed. Histomorphometry After muscle force testing was performed, the animals were killed to analyze axonal regeneration in the grafts and distal nerve. The sciatic nerve was harvested en J Hand Surg Am.
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Graft 1 cm
1 cm
Proximal
Distal Suture line
Additional suture line Graft proximal GP
Suture line
Graft distal GD
Distal section Distal
FIGURE 1: Diagram of surgical model and nerve analysis. The effect of an additional suture line on axonal regeneration across nerve grafts was considered by comparing in-series nerve grafts of comparable length. Histomorphometric analysis was performed at 3 indicated areas: Distal, GD, and GP.
FIGURE 2: AeC Histomorphometric analysis of axonal regeneration 8 weeks following graft reconstructions. Data represented as mean SD; n ¼ 8 per group. *P < .05.
RESULTS Histomorphometric analysis of axonal regeneration To assess the impact of an additional suture line on axonal regeneration across grafts, 3 areas (GP, graft proximal; GD, graft distal; Distal, distal nerve) from each animal were analyzed at 8 weeks after the reconstruction (Fig. 1). As a benchmark for graft reconstruction success, the number of myelinated nerve
Statistical analyses Data were first assessed for normality using a Kolmogorov-Smirnov test. After normality was established, analysis of variance was performed to determine differences between groups with post hoc comparisons determined by a Newman-Keuls test. Significance was established at P less than .05. All results are reported as mean SD. J Hand Surg Am.
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myelinated nerve fibers in the GD section compared to GP (Fig. 2B). Conversely, both ANA groups contained a marked decline in the number of myelinated nerve fibers in GD compared with GP. For whole ANAs, the number of fibers in GD significantly decreased compared with GP (P < .05). In in-series ANAs, the number of fibers in GD decreased compared with GP. Retrospective evaluation of suture line in long ISO model We previously reported limited axonal regeneration across long nerve grafts.7 Similar to the current study, nerve grafts were sutured together end-to-end to generate long 6-cm grafts (3-cm sutured to 3-cm grafts). Based on our current results, we retrospectively assessed how the additional suture line affected axonal regeneration within these long grafts 20 weeks following their implantation.7 Analysis of the ANAs was not performed because ANAs did not support axonal regeneration at this length. Whereas long ANAs failed to regenerate, long ISOs supported a moderate amount of axonal regeneration. In this retrospective analysis, the number of myelinated nerve fibers trended toward a slight decrease (P > .05), reflected by the GD/GP ratio of 73% (Fig. 2C). Functional recovery and EDL muscle mass At 8 weeks, functional motor recovery was assessed by measuring the extent of evoked EDL muscle force as previously described.7 Muscle force production in ISO, ISO in-series, and ANA were comparable (Fig. 3A). The tetanic specific muscle force of the ANA in-series group showed a strong trend of decreased muscle functional recovery in comparison with the other groups (Fig. 3A). However, owing to the large SD, statistical significance was not reached. Measurements of EDL muscle mass were used to assess the amount of muscle atrophy and recovery. The ratio of experimental/contralateral uninjured muscle was used to normalize and quantify this process as percent recovery. There was no difference between ISO and ISO in-series groups or between either ANA group. However, ANA groups yielded significantly decreased muscle mass when compared with ISO groups (Fig. 3B).
FIGURE 3: A, B Evoked muscle force measurement of EDL muscle at 8 weeks after reconstruction. Data represented as mean SD; n ¼ 8 per group. ***P < .001. SpF, specific muscle force.
fibers that crossed the grafts reaching the distal nerve was evaluated. All nerve grafts demonstrated regeneration. The number of myelinated nerve fibers that crossed either the ISO or the ANA groups to the distal nerve was comparable; but when comparing graft type, the ANA groups had significantly decreased numbers of fibers (Fig. 2A). To assess for subtle differences that an additional suture line might have on nerve grafts, the areas just before (GP) and after (GD) the additional suture line within the nerve grafts were also evaluated (Fig. 1). These numbers were directly compared with each another to see if the number of myelinated nerve fibers declined after encountering the additional suture line. For both ISO groups, there was no decrease in J Hand Surg Am.
Gene expression profile analysis with qRT-PCR In order to understand how the additional suture line in grafts affected the regenerative environment, gene expression was analyzed in the area affected by the additional suture line. This analysis was performed at 2 weeks after graft implantation, which allowed us to identify potential barriers during the active portion of regeneration. Groups that received grafts with an r
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FIGURE 4: Quantification of gene expression related to peripheral nerve regeneration in the middle segment of nerve graft. Data represented as mean SD; n ¼ 4 per group. *P < .05.
additional suture line were compared with whole grafts at the midline (similar point on chained grafts) to determine differences in gene expression due to the additional suture line. Genes related to scar formation (Col1a1), angiogenesis and vascular remodeling (Cd31, Ang2, Jag1, and Dll4), and neurotrophic factors (Gdnf) were analyzed. All groups were differentially compared with the ISO group and represented as fold changes shown in Figure 4. The expression of Gdnf, a potent neurotrophic factor for axonal regeneration, was expressed at levels significantly greater in the ISO groups compared with either ANA group. An additional suture line in nerve grafts did not result in an obvious change in the regenerative properties to the graft.
of an additional suture line on axonal regeneration, gene expression, and functional recovery across either short ISOs or ANAs. Our model employed an additional suture line for in-series nerve grafts to generate a longer graft. Overall, we found that regeneration occurred across all grafts and the number of myelinated nerve fibers that crossed the graft and regenerated within the graft were not affected by an additional suture line. Muscle force and mass recovery were also not directly affected by an additional suture line. Mirroring these results, we found that mRNA expression levels from selected genes did not differ due to an additional suture line with respect to genes associated with scar formation, angiogenesis and vascular remodeling, and axonal regeneration. Conversely, we found regenerative outcome and gene expression differed based upon the choice of nerve graft material: ISOs compared with ANAs. In nerve reconstruction following injury, sutures are employed to coapt the nerve ends. As with any foreign material, sutures can elicit a physiological
DISCUSSION Limited donor nerve supply or long segmental nerve defects create situations in which the linking of multiple nerve grafts could be considered to repair the defect. We designed our study to evaluate the effect J Hand Surg Am.
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less ideal outcomes in ANAs.11 Our study identified changes in gene expression in the neurotrophic factor glial cell lineederived neurotrophic factor due to graft choice. Glial cell lineederived neurotrophic factor is a major component of the regenerative process after peripheral nerve injury.34,35 Beyond previous studies’ observations, the greater Gdnf expression in ISOs compared with ANAs provides a possible explanation as to why ISOs outperformed ANAs. Animal models, such as rodent peripheral nerve injury and repair models, may not be directly extrapolated to the clinical realm. The rodent has a superlative rate and capacity for axonal regeneration compared with that of humans. A large animal study is the necessary next step for better clinical extrapolation and comparison. Thus, care should be placed regarding directly applying these results to the clinical setting.
inflammatory reaction, including proliferation and migration of macrophages and fibroblasts, as well as production, deposit, and remodeling of collagen near the suture site.17,18 Damaged nerve ends are particularly susceptible to scarring,19 where scar formation (collagen accumulation) has a major negative impact on axonal regeneration.19,20 Therefore, scar formation generated owing to suture lines is considered a possible factor affecting axonal regeneration after repair. In addition, owing to suture lines, blood flow is temporarily disrupted, requiring vascular remodeling to store blood perfusion. This disruption of blood flow due to an additional suture line is also a potential concern. However, direct causal evidence of the impact that additional suture lines have on axonal regeneration is limited. Epineurial sutures lead to increased collagen deposition in the epineurium but not the endoneurium.21 In addition, Atkins et al3 demonstrated a correlation of intraneural collagen content (scar) with decreased axonal regeneration across the scarred area, but their data also suggested that a noteworthy amount of endoneurial scar was needed to impact axonal regeneration across the scar zone. In our current studies, an additional suture in nerve grafts placed in series did not directly affect axonal regeneration. We also assessed gene expression associated with fibrosis (Col1a1) and angiogenesis and vascular remodeling (Cd31, Ang2, Jag1, Dll4).22e25 The expression of collagen type I or vascular remodeling genes was unchanged across additional suture lines in both the ISO and the ANA groups. Therefore, we infer that, because an additional suture line within grafts did not increase collagen expression or vascular remodeling, axonal regenerative outcome was not affected owing to this mechanism. Although we did not observe differences in regenerative outcome or gene expression due directly to an additional suture line in nerve grafts, we did notice strong regenerative differences when we compared the efficacy of regeneration across graft type: ISOs versus ANAs. The in-series ANA graft reconstruction led to significantly decreased regeneration when compared with whole or in-series ISOs. Previous studies have observed similar findings of inferiority.7,10,26,27 To prepare ANAs, donor nerves are processed with detergents that remove critical glial cells including Schwann cells, endoneurial fibroblasts, and endothelial cells.12,13 Schwann cells are critical for efficient regeneration of peripheral nerve following injury. They support regeneration in vivo by producing myelin, basal lamina, growth factors, and cell adhesion molecules.28e33 There is strong evidence that the removal of these key components are responsible for J Hand Surg Am.
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ACKNOWLEDGMENTS This work was supported in part by National Institutes of Neurological Disorders and Stroke of the National Institutes of Health under award number R56 NS33406. Salary support was provided in part by the Barnes-Jewish Hospital Foundation for M.D.W. The content is solely the responsibility of the authors and does not necessarily represent the official views of Washington University. REFERENCES 1. Bratton BR, Kline DG, Coleman W, Hudson AR. Experimental interfascicular nerve grafting. J Neurosurg. 1979;51(3):323e332. 2. Hudson AR, Hunter D, Kline DG, Bratton BR. Histological studies of experimental interfascicular graft repairs. J Neurosurg. 1979;51(3): 333e340. 3. Atkins S, Smith KG, Loescher AR, et al. Scarring impedes regeneration at sites of peripheral nerve repair. Neuroreport. 2006;17(12): 1245e1249. 4. Al-Majed AA, Neumann CM, Brushart TM, Gordon T. Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J Neurosci. 2000;20(7):2602e2608. 5. Mackinnon SE, Hudson AR. Clinical application of peripheral nerve transplantation. Plast Reconstr Surg. 1992;90(4):695e699. 6. Brooks DN, Weber RV, Chao JD, et al. Processed nerve allografts for peripheral nerve reconstruction: a multicenter study of utilization and outcomes in sensory, mixed, and motor nerve reconstructions. Microsurgery. 2012;32(1):1e14. 7. Saheb-Al-Zamani M, Yan Y, Farber SJ, et al. Limited regeneration in long acellular nerve allografts is associated with increased Schwann cell senescence. Exp Neurol. 2013;247:165e177. 8. Mackinnon SE, Hudson AR, Falk RE, Kline D, Hunter D. Peripheral nerve allograft: an immunological assessment of pretreatment methods. Neurosurgery. 1984;14(2):167e171. 9. Mackinnon SE, Hudson AR, Falk RE, Hunter DA. The nerve allograft response—an experimental model in the rat. Ann Plast Surg. 1985;14(4):334e339. 10. Whitlock EL, Tuffaha SH, Luciano JP, et al. Processed allografts and type I collagen conduits for repair of peripheral nerve gaps. Muscle Nerve. 2009;39(6):787e799.
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24. Kim I, Kim JH, Ryu YS, Jung SH, Nah JJ, Koh GY. Characterization and expression of a novel alternatively spliced human angiopoietin-2. J Biol Chem. 2000;275(24):18550e18556. 25. Suchting S, Freitas C, le Noble F, et al. The Notch ligand deltalike 4 negatively regulates endothelial tip cell formation and vessel branching. Proc Natl Acad Sci U S A. 2007;104(9): 3225e3230. 26. Moore AM, MacEwan M, Santosa KB, et al. Acellular nerve allografts in peripheral nerve regeneration: a comparative study. Muscle Nerve. 2011;44(2):221e234. 27. Wood MD, Kemp SW, Liu EH, Szynkaruk M, Gordon T, Borschel GH. Rat-derived processed nerve allografts support more axon regeneration in rat than human-derived processed nerve xenografts. J Biomed Mater Res A. 2014;102(4):1085e 1091. 28. Araki T, Milbrandt J. Ninjurin, a novel adhesion molecule, is induced by nerve injury and promotes axonal growth. Neuron. 1996;17(2): 353e361. 29. Bunge RP. The role of the Schwann cell in trophic support and regeneration. J Neurol. 1994;242(1 Suppl 1):S19eS21. 30. Bunge RP, Bunge MB, Eldridge CF. Linkage between axonal ensheathment and basal lamina production by Schwann cells. Annu Rev Neurosci. 1986;9:305e328. 31. Friedman B, Scherer SS, Rudge JS, et al. Regulation of ciliary neurotrophic factor expression in myelin-related Schwann cells. in vivo. Neuron. 1992;9(2):295e305. 32. Levi AD, Bunge RP. Studies of myelin formation after transplantation of human Schwann cells into the severe combined immunodeficient mouse. Exp Neurol. 1994;130(1):41e52. 33. Levi AD, Guenard V, Aebischer P, Bunge RP. The functional characteristics of Schwann cells cultured from human peripheral nerve after transplantation into a gap within the rat sciatic nerve. J Neurosci. 1994;14(3 Pt 1):1309e1319. 34. Henderson CE, Phillips HS, Pollock RA, et al. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science. 1994;266(5187):1062e1064. 35. Hammarberg H, Piehl F, Cullheim S, Fjell J, Hokfelt T, Fried K. GDNF mRNA in Schwann cells and DRG satellite cells after chronic sciatic nerve injury. Neuroreport. 1996;7(4):857e860.
11. Hoben G, Yan Y, Iyer N, et al. Comparison of acellular nerve allograft modification with Schwann cells or VEGF. Hand (N Y). 2015;10(3):396e402. 12. Hudson TW, Liu SY, Schmidt CE. Engineering an improved acellular nerve graft via optimized chemical processing. Tissue Eng. 2004;10(9e10):1346e1358. 13. Hudson TW, Zawko S, Deister C, et al. Optimized acellular nerve graft is immunologically tolerated and supports regeneration. Tissue Eng. 2004;10(11e12):1641e1651. 14. Urbanchek MS, Chung KC, Asato H, Washington LN, Kuzon WM Jr. Rat walking tracks do not reflect maximal muscle force capacity. J Reconstr Microsurg. 1999;15(2):143e149. 15. Hunter DA, Moradzadeh A, Whitlock EL, et al. Binary imaging analysis for comprehensive quantitative histomorphometry of peripheral nerve. J Neurosci Methods. 2007;166(1):116e124. 16. Hoke A, Redett R, Hameed H, et al. Schwann cells express motor and sensory phenotypes that regulate axon regeneration. J Neurosci. 2006;26(38):9646e9655. 17. Sunderland SS. Nerve Injuries and Their Repair. 2nd ed. New York: Churchill Livingstone Inc; 1991. 18. DeLee JC, Smith MT, Green DP. The reaction of nerve tissue to various suture materials: a study in rabbits. J Hand Surg Am. 1977;2(1):38e43. 19. Lane JM, Bora FW Jr, Pleasure D. Neuroma scar formation in rats following peripheral nerve transection. J Bone Joint Surg Am. 1978;60(2):197e203. 20. Pleasure D, Bora FW Jr, Lane J, Prockop D. Regeneration after nerve transection: effect of inhibition of collagen synthesis. Exp Neurol. 1974;45(1):72e78. 21. Martins RS, Teodoro WR, Simplicio H, et al. Influence of suture on peripheral nerve regeneration and collagen production at the site of neurorrhaphy: an experimental study. Neurosurgery. 2011;68(3): 765e772. discussion 772. 22. Patan S. Vasculogenesis and angiogenesis as mechanisms of vascular network formation, growth and remodeling. J Neurooncol. 2000;50(1e2):1e15. 23. Hellstrom M, Phng LK, Hofmann JJ, et al. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature. 2007;445(7129):776e780.
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APPENDIX A. Technologies Genes
Primers for qRT-PCR From Life Assay Identification
Col1a1
Rn01463848_m1
Cd31
Rn01467262_m1
Ang-2
Rn02349499_g1
Jag1
Rn00569647_m1
Dll4
Rn01512886_m1
Gdnf
Rn00569510_m1
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The Effect of Short Nerve Grafts in Series on Axonal Regeneration Across Isografts or Acellular Nerve Allografts Ying Yan, MD, PhD,* Matthew D. Wood, PhD,* Daniel A. Hunter,* Xueping Ee, MD,* Susan E. Mackinnon, MD,* Amy M. Moore, MD*
Purpose To evaluate the regenerative effect of the additional suture line when using either isografts (ISOs) or acellular nerve allografts (ANAs) placed end-to-end to span a short gap in a rat model. Methods Rat sciatic nerves were transected and repaired with 2-cm nerve grafts (ISO or ANA). The grafts were 2 cm in length or a 1-cm segment was connected end-to-end to a 1-cm segment to yield a 2-cm length. At 8 weeks, extensor digitorum longus (EDL) muscle force and mass were measured. Nerves were harvested for histomorphometry. In a separate parallel study, the nerves were harvested 2 weeks following graft implantation to assess gene expression changes. Results All grafts demonstrated regeneration across the 2-cm segment(s). The additional suture line did not result in statistical differences in the number of myelinated nerve fibers that reached the distal nerve. However, when the graft types were compared, there was a significant decrease in nerve fibers in the ANA groups. The EDL muscle mass was significantly greater by using nerve ISOs compared with ANAs, regardless of an additional suture line, but there were no statistical differences noted in EDL muscle force. Gene expression analysis did not differ owing to an additional suture line. Conclusions Minimal axonal loss and no functional deficits were identified with an additional suture line in this rodent short nerve gap model. Clinical relevance Placing nerve grafts in series is a viable option for treating short nerve gaps; however, the use of autografts remains preferable over the use of ANAs. (J Hand Surg Am. 2016;-(-):-e-. Copyright Ó 2016 by the American Society for Surgery of the Hand. All rights reserved.) Key words Coaptation site, nerve regeneration, peripheral nerve, processed nerve allograft, suture line.
From the *Division of Plastic and Reconstructive Surgery, Washington University School of Medicine, St. Louis, MO. Received for publication September 28, 2015; accepted in revised form January 8, 2016. No benefits in any form have been received or will be received related directly or indirectly to the subject of this article. Corresponding author: Amy M. Moore, MD, Division of Plastic and Reconstructive Surgery, Washington University School of Medicine, 660 South Euclid, Campus Box 8238, St. Louis, MO. 63110; e-mail: [email protected]. 0363-5023/16/---0001$36.00/0 http://dx.doi.org/10.1016/j.jhsa.2016.01.009
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direct endto-end coaptation is not possible owing to a segmental nerve injury or tension. Nerve grafting inherently predisposes regenerating axons to an additional coaptation site increasing the risk of axonal loss from scarring.1,2 Sutures activate a local inflammatory response, which, if large enough, can generate considerable fibrosis hindering the progression of regenerating axons.3 The primary coaptation site linking the distal and proximal nerve is associated ERVE GRAFTING IS PERFORMED WHEN A
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with slowed or staggered axonal regeneration.4 Thus, it follows that additional coaptation sites may generate similar effects. The impact of an additional suture line on axonal regeneration through nerve grafts is not clearly understood. This information holds clinical value in the event of limited donor nerve supply such as that seen with major injuries involving multiple limbs with large segmental nerve defects. In these devastating cases, the end-to-end linking of multiple nerve grafts in series (autograft and/or acellular nerve allografts [ANAs]) to repair long nerve gaps have been described and clinically referred to as “daisy-chaining.”5 The clinical use of ANAs has increased markedly since the U.S. Food and Drug Administration’s approval of the Avance Nerve Graft (AxoGen, Inc., Alachua, FL) was obtained.6 The decellularized nerve allografts offer an off-the-shelf alternative to autografting and avoid donor site morbidity and prolonged operative time. The potential for using multiple decellularized nerve grafts placed in series is an attractive option, especially in the severely traumatized patient. Our study sought to determine how the additional suture lines affect axonal regeneration across short nerve autografts and acellular nerve allografts. In this study, short nerve grafts were used to avoid additional confounding issues affecting axonal regeneration, such as complete regenerative failure associated with long ANAs.7 The present study evaluated axonal regeneration and functional recovery across isografts (ISOs; animal model equivalent of an autograft8,9) and ANAs repaired using a whole, unmodified graft or a graft of comparable length with an additional suture line. To determine how the additional suture line exerted its influence, gene expression products related to nerve regeneration were measured in the grafts.
with consistent diameter and no additional branch points, eliminating confounders such as axonal loss and graft size. In study A, 32 rats were divided into 4 groups (n ¼ 8 per group) consisting of a whole ISO, an ISO with an additional suture line (ISO in-series), a whole ANA (ANA), or an ANA with an additional suture line (ANA in-series). At 8 weeks after surgery, extensor digitorum longus (EDL) muscle force testing was performed, and EDL muscle mass was measured. The sciatic nerves were also harvested for histomorphometric analysis (Table 1). Eight weeks was chosen based on previous studies that suggest this time point maximizes the sensitivity to measure differences between groups receiving 2-cm nerve grafts.10,11 In a parallel study B, 16 rats were randomized to the same 4 groups (n ¼ 4 per group) and underwent similar procedures as in study A. These rats were used to assess gene expression within the graft area affected by the suture line (or in the midgraft of the whole grafts). At 2 weeks after surgery, gene expression was measured using quantitative reverse-transcriptaseepolymerase chain reaction (qRT-PCR; Table 1). For all groups, Lewis male rats served as the experimental animals and donor animals for ISO groups. In ANA graft groups, Sprague-Dawley rats (250 g; Charles River Laboratories, Wilmington, MA) were used as donor nerve for processing to generate ANAs. The Sprague-Dawley (SD) (RT-1b major histocompatibility complex [MHC]) rat strain is MHCincompatible with Lewis (RT-11 MHC) and were used as allograft donors. Lewis rats are a well-accepted rat strain used to derive ISOs because they are inbred leading to syngeneic donors. Sciatic nerve allografts harvested from donor rats were chemically processed and decellularized using a series of detergents as previously described.7,12,13 Surgical procedures and perioperative care measures were conducted in compliance with the Institutional Animal Studies Committee and National Institutes of Health guidelines. All animals were housed in a central animal care facility and provided with food (PicoLab rodent diet 20; Purina Mills Nutrition International, St. Louis, MO) and water ad libitum.
MATERIALS AND METHODS Animals and experimental design The sciatic nerves of adult male Lewis rats (250 g; Charles River Laboratories, Wilmington, MA) were transected and then immediately repaired with 2-cm nerve grafts. These grafts were either left unmodified (whole) or cut into equal 1-cm pieces and sutured endto-end, thus generating an equivalent 2-cm length. A 2-cm nerve gap was chosen to eliminate the confounding effects of graft length and to focus on the effect of the additional suture line on regeneration. Long nerve grafts (autograft and ANA) have been associated with a significant decline in axonal regeneration in comparison with short nerve grafts.7 Further, a 2-cm graft can be obtained from the sciatic nerve J Hand Surg Am.
Surgical procedures Surgical procedures were performed under aseptic conditions and with the aid of an operating microscope under magnifications of 10-25X.7 A single surgeon (Y.Y.) performed all operations. Anesthesia was provided by subcutaneous delivery of ketamine (75 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA) and dexmedetomidine (0.5 mg/kg; Pfizer Animal r
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TABLE 1. Groups
Study Design and Groups Animal Numbers
Recipient
Group 1-A Group 2-A Group 3-A
Lewis
ISO in series
Sprague-Dawley
ANA in series
Lewis
ISO in series
Analysis
8 wk
EDL muscle force, muscle mass, and histomorphometry
2 wk
qRT-PCR
ISO
Lewis
4
End Point
ANA in series ANA
8
Group 1-B Group 3-B
Nerve Graft
Sprague-Dawley
Group 4-A Group 2-B
Donor
ANA
Group 4-B
ISO
Health, Exton, PA). The right sciatic nerve was exposed using a muscle-splitting technique. For donor nerve grafts, approximately 3.5 cm of sciatic nerve was harvested, which was later trimmed to appropriate length. Sciatic nerves were transferred to aseptic tubes to undergo acellular processing13 or immediately used as fresh nerve ISOs. In experimental rats, the recipient sciatic nerve was transected 5 mm proximal to the trifurcation of the sciatic nerve. The defect was reconstructed with an ISO or ANA secured to the proximal and distal nerve stumps using 4 interrupted 9e0 nylon epineurial sutures at each suture line. For groups with in-series nerve grafts, the 2 1-cm graft segments were secured first using 4 9-0 nylon epineurial sutures to yield a 2-cm graft before coaptation to the proximal and distal nerve (16 magnification). The muscle and skin were closed in a layered fashion. Animals recovered and were housed in a central animal care facility.
bloc 5 mm proximal and 5 mm distal to the interposed graft. The recipient/donor sciatic nerve complexes were stored in 3% glutaraldehyde and followed by processing and assessment for evidence of nerve regeneration.15 An observer blinded to the experimental groups obtained all measurements. Quantitative reverse-transcriptaseepolymerase chain reaction After 2 weeks in a separate set of animals, nerve grafts (4-mm segments either containing an additional suture line or left whole) were harvested for gene expression analysis. Samples were taken at the center of the graft or directly from the suture line area, which included 2 mm proximal and 2 mm distal (4 mm total) of the suture line area within the graft. Samples for qRT-PCR analysis were stored in RNAlater solution (QIAGEN, Valencia, CA) and stored at 80 C prior to RNA extraction. Total RNA was extracted by using Trizol Kit (QIAGEN, Valencia, CA) according to the manufacturer’s direction. RNA was then reverse transcribed into complementary DNA strands (cDNAs) using the protocol described in the High Capacity RNA-tocDNA kit (Applied Biosystems, Foster City, CA). Gene expression of Col1a1, Cd31, Ang2, Jag1, Dll4, and Gdnf were analyzed using qRT-PCR. Primers shown on Appendix A (available on the Journal’s Web site at www.jhandsurg.org) were purchased from Life Technologies (Grand Island, NY). Amplification of each target cDNA was done with Taqman primers (Appendix A; available on the Journal’s Web site at www.jhandsurg.org) using a Step One Plus thermocycler (Applied Biosystems, Foster City, CA) with normalization to a housekeeping gene (b-actin), and the data analyzed on Step One Software v2.2.2 (Applied Biosystems). A value of 2 or greater was selected as the minimum criteria for a significant difference in expression levels between graft groups and the normal fresh nerve.16
Functional assessment and muscle recovery Functional recovery of muscle force was assessed at 8 weeks. The measurements were performed without blinding because this procedure is not possible without the group identity being revealed. Evoked motor response in reinnervated EDL muscle upon electrical stimulation of the repaired sciatic nerve was measured.7 Maximum specific isometric tetanic force was calculated as the maximum isometric force normalized to physiological muscle cross-sectional area.14 Following force assessment, the EDL muscle was harvested from both the experimental and the contralateral sides and weighed. Histomorphometry After muscle force testing was performed, the animals were killed to analyze axonal regeneration in the grafts and distal nerve. The sciatic nerve was harvested en J Hand Surg Am.
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Graft 1 cm
1 cm
Proximal
Distal Suture line
Additional suture line Graft proximal GP
Suture line
Graft distal GD
Distal section Distal
FIGURE 1: Diagram of surgical model and nerve analysis. The effect of an additional suture line on axonal regeneration across nerve grafts was considered by comparing in-series nerve grafts of comparable length. Histomorphometric analysis was performed at 3 indicated areas: Distal, GD, and GP.
FIGURE 2: AeC Histomorphometric analysis of axonal regeneration 8 weeks following graft reconstructions. Data represented as mean SD; n ¼ 8 per group. *P < .05.
RESULTS Histomorphometric analysis of axonal regeneration To assess the impact of an additional suture line on axonal regeneration across grafts, 3 areas (GP, graft proximal; GD, graft distal; Distal, distal nerve) from each animal were analyzed at 8 weeks after the reconstruction (Fig. 1). As a benchmark for graft reconstruction success, the number of myelinated nerve
Statistical analyses Data were first assessed for normality using a Kolmogorov-Smirnov test. After normality was established, analysis of variance was performed to determine differences between groups with post hoc comparisons determined by a Newman-Keuls test. Significance was established at P less than .05. All results are reported as mean SD. J Hand Surg Am.
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myelinated nerve fibers in the GD section compared to GP (Fig. 2B). Conversely, both ANA groups contained a marked decline in the number of myelinated nerve fibers in GD compared with GP. For whole ANAs, the number of fibers in GD significantly decreased compared with GP (P < .05). In in-series ANAs, the number of fibers in GD decreased compared with GP. Retrospective evaluation of suture line in long ISO model We previously reported limited axonal regeneration across long nerve grafts.7 Similar to the current study, nerve grafts were sutured together end-to-end to generate long 6-cm grafts (3-cm sutured to 3-cm grafts). Based on our current results, we retrospectively assessed how the additional suture line affected axonal regeneration within these long grafts 20 weeks following their implantation.7 Analysis of the ANAs was not performed because ANAs did not support axonal regeneration at this length. Whereas long ANAs failed to regenerate, long ISOs supported a moderate amount of axonal regeneration. In this retrospective analysis, the number of myelinated nerve fibers trended toward a slight decrease (P > .05), reflected by the GD/GP ratio of 73% (Fig. 2C). Functional recovery and EDL muscle mass At 8 weeks, functional motor recovery was assessed by measuring the extent of evoked EDL muscle force as previously described.7 Muscle force production in ISO, ISO in-series, and ANA were comparable (Fig. 3A). The tetanic specific muscle force of the ANA in-series group showed a strong trend of decreased muscle functional recovery in comparison with the other groups (Fig. 3A). However, owing to the large SD, statistical significance was not reached. Measurements of EDL muscle mass were used to assess the amount of muscle atrophy and recovery. The ratio of experimental/contralateral uninjured muscle was used to normalize and quantify this process as percent recovery. There was no difference between ISO and ISO in-series groups or between either ANA group. However, ANA groups yielded significantly decreased muscle mass when compared with ISO groups (Fig. 3B).
FIGURE 3: A, B Evoked muscle force measurement of EDL muscle at 8 weeks after reconstruction. Data represented as mean SD; n ¼ 8 per group. ***P < .001. SpF, specific muscle force.
fibers that crossed the grafts reaching the distal nerve was evaluated. All nerve grafts demonstrated regeneration. The number of myelinated nerve fibers that crossed either the ISO or the ANA groups to the distal nerve was comparable; but when comparing graft type, the ANA groups had significantly decreased numbers of fibers (Fig. 2A). To assess for subtle differences that an additional suture line might have on nerve grafts, the areas just before (GP) and after (GD) the additional suture line within the nerve grafts were also evaluated (Fig. 1). These numbers were directly compared with each another to see if the number of myelinated nerve fibers declined after encountering the additional suture line. For both ISO groups, there was no decrease in J Hand Surg Am.
Gene expression profile analysis with qRT-PCR In order to understand how the additional suture line in grafts affected the regenerative environment, gene expression was analyzed in the area affected by the additional suture line. This analysis was performed at 2 weeks after graft implantation, which allowed us to identify potential barriers during the active portion of regeneration. Groups that received grafts with an r
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FIGURE 4: Quantification of gene expression related to peripheral nerve regeneration in the middle segment of nerve graft. Data represented as mean SD; n ¼ 4 per group. *P < .05.
additional suture line were compared with whole grafts at the midline (similar point on chained grafts) to determine differences in gene expression due to the additional suture line. Genes related to scar formation (Col1a1), angiogenesis and vascular remodeling (Cd31, Ang2, Jag1, and Dll4), and neurotrophic factors (Gdnf) were analyzed. All groups were differentially compared with the ISO group and represented as fold changes shown in Figure 4. The expression of Gdnf, a potent neurotrophic factor for axonal regeneration, was expressed at levels significantly greater in the ISO groups compared with either ANA group. An additional suture line in nerve grafts did not result in an obvious change in the regenerative properties to the graft.
of an additional suture line on axonal regeneration, gene expression, and functional recovery across either short ISOs or ANAs. Our model employed an additional suture line for in-series nerve grafts to generate a longer graft. Overall, we found that regeneration occurred across all grafts and the number of myelinated nerve fibers that crossed the graft and regenerated within the graft were not affected by an additional suture line. Muscle force and mass recovery were also not directly affected by an additional suture line. Mirroring these results, we found that mRNA expression levels from selected genes did not differ due to an additional suture line with respect to genes associated with scar formation, angiogenesis and vascular remodeling, and axonal regeneration. Conversely, we found regenerative outcome and gene expression differed based upon the choice of nerve graft material: ISOs compared with ANAs. In nerve reconstruction following injury, sutures are employed to coapt the nerve ends. As with any foreign material, sutures can elicit a physiological
DISCUSSION Limited donor nerve supply or long segmental nerve defects create situations in which the linking of multiple nerve grafts could be considered to repair the defect. We designed our study to evaluate the effect J Hand Surg Am.
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less ideal outcomes in ANAs.11 Our study identified changes in gene expression in the neurotrophic factor glial cell lineederived neurotrophic factor due to graft choice. Glial cell lineederived neurotrophic factor is a major component of the regenerative process after peripheral nerve injury.34,35 Beyond previous studies’ observations, the greater Gdnf expression in ISOs compared with ANAs provides a possible explanation as to why ISOs outperformed ANAs. Animal models, such as rodent peripheral nerve injury and repair models, may not be directly extrapolated to the clinical realm. The rodent has a superlative rate and capacity for axonal regeneration compared with that of humans. A large animal study is the necessary next step for better clinical extrapolation and comparison. Thus, care should be placed regarding directly applying these results to the clinical setting.
inflammatory reaction, including proliferation and migration of macrophages and fibroblasts, as well as production, deposit, and remodeling of collagen near the suture site.17,18 Damaged nerve ends are particularly susceptible to scarring,19 where scar formation (collagen accumulation) has a major negative impact on axonal regeneration.19,20 Therefore, scar formation generated owing to suture lines is considered a possible factor affecting axonal regeneration after repair. In addition, owing to suture lines, blood flow is temporarily disrupted, requiring vascular remodeling to store blood perfusion. This disruption of blood flow due to an additional suture line is also a potential concern. However, direct causal evidence of the impact that additional suture lines have on axonal regeneration is limited. Epineurial sutures lead to increased collagen deposition in the epineurium but not the endoneurium.21 In addition, Atkins et al3 demonstrated a correlation of intraneural collagen content (scar) with decreased axonal regeneration across the scarred area, but their data also suggested that a noteworthy amount of endoneurial scar was needed to impact axonal regeneration across the scar zone. In our current studies, an additional suture in nerve grafts placed in series did not directly affect axonal regeneration. We also assessed gene expression associated with fibrosis (Col1a1) and angiogenesis and vascular remodeling (Cd31, Ang2, Jag1, Dll4).22e25 The expression of collagen type I or vascular remodeling genes was unchanged across additional suture lines in both the ISO and the ANA groups. Therefore, we infer that, because an additional suture line within grafts did not increase collagen expression or vascular remodeling, axonal regenerative outcome was not affected owing to this mechanism. Although we did not observe differences in regenerative outcome or gene expression due directly to an additional suture line in nerve grafts, we did notice strong regenerative differences when we compared the efficacy of regeneration across graft type: ISOs versus ANAs. The in-series ANA graft reconstruction led to significantly decreased regeneration when compared with whole or in-series ISOs. Previous studies have observed similar findings of inferiority.7,10,26,27 To prepare ANAs, donor nerves are processed with detergents that remove critical glial cells including Schwann cells, endoneurial fibroblasts, and endothelial cells.12,13 Schwann cells are critical for efficient regeneration of peripheral nerve following injury. They support regeneration in vivo by producing myelin, basal lamina, growth factors, and cell adhesion molecules.28e33 There is strong evidence that the removal of these key components are responsible for J Hand Surg Am.
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ACKNOWLEDGMENTS This work was supported in part by National Institutes of Neurological Disorders and Stroke of the National Institutes of Health under award number R56 NS33406. Salary support was provided in part by the Barnes-Jewish Hospital Foundation for M.D.W. The content is solely the responsibility of the authors and does not necessarily represent the official views of Washington University. REFERENCES 1. Bratton BR, Kline DG, Coleman W, Hudson AR. Experimental interfascicular nerve grafting. J Neurosurg. 1979;51(3):323e332. 2. Hudson AR, Hunter D, Kline DG, Bratton BR. Histological studies of experimental interfascicular graft repairs. J Neurosurg. 1979;51(3): 333e340. 3. Atkins S, Smith KG, Loescher AR, et al. Scarring impedes regeneration at sites of peripheral nerve repair. Neuroreport. 2006;17(12): 1245e1249. 4. Al-Majed AA, Neumann CM, Brushart TM, Gordon T. Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J Neurosci. 2000;20(7):2602e2608. 5. Mackinnon SE, Hudson AR. Clinical application of peripheral nerve transplantation. Plast Reconstr Surg. 1992;90(4):695e699. 6. Brooks DN, Weber RV, Chao JD, et al. Processed nerve allografts for peripheral nerve reconstruction: a multicenter study of utilization and outcomes in sensory, mixed, and motor nerve reconstructions. Microsurgery. 2012;32(1):1e14. 7. Saheb-Al-Zamani M, Yan Y, Farber SJ, et al. Limited regeneration in long acellular nerve allografts is associated with increased Schwann cell senescence. Exp Neurol. 2013;247:165e177. 8. Mackinnon SE, Hudson AR, Falk RE, Kline D, Hunter D. Peripheral nerve allograft: an immunological assessment of pretreatment methods. Neurosurgery. 1984;14(2):167e171. 9. Mackinnon SE, Hudson AR, Falk RE, Hunter DA. The nerve allograft response—an experimental model in the rat. Ann Plast Surg. 1985;14(4):334e339. 10. Whitlock EL, Tuffaha SH, Luciano JP, et al. Processed allografts and type I collagen conduits for repair of peripheral nerve gaps. Muscle Nerve. 2009;39(6):787e799.
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24. Kim I, Kim JH, Ryu YS, Jung SH, Nah JJ, Koh GY. Characterization and expression of a novel alternatively spliced human angiopoietin-2. J Biol Chem. 2000;275(24):18550e18556. 25. Suchting S, Freitas C, le Noble F, et al. The Notch ligand deltalike 4 negatively regulates endothelial tip cell formation and vessel branching. Proc Natl Acad Sci U S A. 2007;104(9): 3225e3230. 26. Moore AM, MacEwan M, Santosa KB, et al. Acellular nerve allografts in peripheral nerve regeneration: a comparative study. Muscle Nerve. 2011;44(2):221e234. 27. Wood MD, Kemp SW, Liu EH, Szynkaruk M, Gordon T, Borschel GH. Rat-derived processed nerve allografts support more axon regeneration in rat than human-derived processed nerve xenografts. J Biomed Mater Res A. 2014;102(4):1085e 1091. 28. Araki T, Milbrandt J. Ninjurin, a novel adhesion molecule, is induced by nerve injury and promotes axonal growth. Neuron. 1996;17(2): 353e361. 29. Bunge RP. The role of the Schwann cell in trophic support and regeneration. J Neurol. 1994;242(1 Suppl 1):S19eS21. 30. Bunge RP, Bunge MB, Eldridge CF. Linkage between axonal ensheathment and basal lamina production by Schwann cells. Annu Rev Neurosci. 1986;9:305e328. 31. Friedman B, Scherer SS, Rudge JS, et al. Regulation of ciliary neurotrophic factor expression in myelin-related Schwann cells. in vivo. Neuron. 1992;9(2):295e305. 32. Levi AD, Bunge RP. Studies of myelin formation after transplantation of human Schwann cells into the severe combined immunodeficient mouse. Exp Neurol. 1994;130(1):41e52. 33. Levi AD, Guenard V, Aebischer P, Bunge RP. The functional characteristics of Schwann cells cultured from human peripheral nerve after transplantation into a gap within the rat sciatic nerve. J Neurosci. 1994;14(3 Pt 1):1309e1319. 34. Henderson CE, Phillips HS, Pollock RA, et al. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science. 1994;266(5187):1062e1064. 35. Hammarberg H, Piehl F, Cullheim S, Fjell J, Hokfelt T, Fried K. GDNF mRNA in Schwann cells and DRG satellite cells after chronic sciatic nerve injury. Neuroreport. 1996;7(4):857e860.
11. Hoben G, Yan Y, Iyer N, et al. Comparison of acellular nerve allograft modification with Schwann cells or VEGF. Hand (N Y). 2015;10(3):396e402. 12. Hudson TW, Liu SY, Schmidt CE. Engineering an improved acellular nerve graft via optimized chemical processing. Tissue Eng. 2004;10(9e10):1346e1358. 13. Hudson TW, Zawko S, Deister C, et al. Optimized acellular nerve graft is immunologically tolerated and supports regeneration. Tissue Eng. 2004;10(11e12):1641e1651. 14. Urbanchek MS, Chung KC, Asato H, Washington LN, Kuzon WM Jr. Rat walking tracks do not reflect maximal muscle force capacity. J Reconstr Microsurg. 1999;15(2):143e149. 15. Hunter DA, Moradzadeh A, Whitlock EL, et al. Binary imaging analysis for comprehensive quantitative histomorphometry of peripheral nerve. J Neurosci Methods. 2007;166(1):116e124. 16. Hoke A, Redett R, Hameed H, et al. Schwann cells express motor and sensory phenotypes that regulate axon regeneration. J Neurosci. 2006;26(38):9646e9655. 17. Sunderland SS. Nerve Injuries and Their Repair. 2nd ed. New York: Churchill Livingstone Inc; 1991. 18. DeLee JC, Smith MT, Green DP. The reaction of nerve tissue to various suture materials: a study in rabbits. J Hand Surg Am. 1977;2(1):38e43. 19. Lane JM, Bora FW Jr, Pleasure D. Neuroma scar formation in rats following peripheral nerve transection. J Bone Joint Surg Am. 1978;60(2):197e203. 20. Pleasure D, Bora FW Jr, Lane J, Prockop D. Regeneration after nerve transection: effect of inhibition of collagen synthesis. Exp Neurol. 1974;45(1):72e78. 21. Martins RS, Teodoro WR, Simplicio H, et al. Influence of suture on peripheral nerve regeneration and collagen production at the site of neurorrhaphy: an experimental study. Neurosurgery. 2011;68(3): 765e772. discussion 772. 22. Patan S. Vasculogenesis and angiogenesis as mechanisms of vascular network formation, growth and remodeling. J Neurooncol. 2000;50(1e2):1e15. 23. Hellstrom M, Phng LK, Hofmann JJ, et al. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature. 2007;445(7129):776e780.
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APPENDIX A. Technologies Genes
Primers for qRT-PCR From Life Assay Identification
Col1a1
Rn01463848_m1
Cd31
Rn01467262_m1
Ang-2
Rn02349499_g1
Jag1
Rn00569647_m1
Dll4
Rn01512886_m1
Gdnf
Rn00569510_m1
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