Muscle Grafts with Doxorubicin Pretreatment Produce “Empty Tubes” in the Basal Laminae, Promote Contentious Maturation of the Regenerated Axons, and Bridge 20-mm Sciatic Nerve Defects in Rats

Abstract

Background We newly developed a muscle graft that employs a doxorubicin pretreatment technique. The aims of this study were to reveal the biological and morphological features of the muscle tissue in the second week (Study I), to reveal the regeneration outcomes of functional and kinematic assessments of longer-term follow-up (16 weeks, Study II), and to make assessments of the muscle graft with doxorubicin pretreatment in the critical-sized nerve defect model (20 mm, Study III).

Methods A total of 26 adult rats were used in this study. Doxorubicin treatment was accomplished by immersion in a doxorubicin solution for 10 minutes followed by a rinsing procedure. The rats were divided into three groups: the muscle graft with and without doxorubicin pretreatment (M-graft-w-Dox and M-graft-w/o-Dox) groups and the autologous nerve graft (N-graft) group. Assays of apoptosis, immunofluorescent histochemistry including CD68 (macrophage marker), scanning electron microscopy (SEM), morphometrical studies of the regenerated axons, nerve conduction studies, and kinematic studies were performed.

Results The M-graft-w-Dox group contained significantly larger numbers of apoptotic cells and CD68-positive cells. SEM revealed the existence of the basal lamina, so called “empty tubes,” in the M-graft-w-Dox group. Study II showed contentious maturation of the regenerated axons, especially in the compound muscle action potentials. Study III showed that even at 20 mm, the M-graft-w-Dox group promoted axonal regeneration and functional regeneration.

Conclusion The M-graft-w-Dox group showed superior regeneration results, and this easy and short-term procedure can expand the muscle graft clinical indication for the treatment of peripheral nerve defects.

Keywords

muscle - doxorubicin - peripheral nerve

Author's note

Akio Sakamoto and Ryosuke Ikeguchi contributed equally to this work.

Publikationsverlauf

Eingereicht: 19. Juni 2021

Angenommen: 23. April 2022

Artikel online veröffentlicht:
18. Juli 2022

• References

• 1 Huckhagel T, Nüchtern J, Regelsberger J, Lefering R. TraumaRegister DGU. Nerve injury in severe trauma with upper extremity involvement: evaluation of 49,382 patients from the TraumaRegister DGU® between 2002 and 2015. Scand J Trauma Resusc Emerg Med 2018; 26 (01) 76-78
• 2 Bekelis K, Missios S, Spinner RJ. Falls and peripheral nerve injuries: an age-dependent relationship. J Neurosurg 2015; 123 (05) 1223-1229
• 3 Tos P, Battiston B, Ciclamini D, Geuna S, Artiaco S. Primary repair of crush nerve injuries by means of biological tubulization with muscle-vein-combined grafts. Microsurgery 2012; 32 (05) 358-363
• 4 Kaizawa Y, Kakinoki R, Ikeguchi R. et al. Bridging a 30 mm defect in the canine ulnar nerve using vessel-containing conduits with implantation of bone marrow stromal cells. Microsurgery 2016; 36 (04) 316-324
• 5 Takeuchi H, Ikeguchi R, Watanabe M, Yasuda T, Matsuda S. Postoperative Evaluation of patient satisfaction and tingling sensation after replantation surgery without nerve repair for complete digital amputation. J Reconstr Microsurg Open 2017; 2 (01) e19-e22
• 6 Jesuraj NJ, Santosa KB, Macewan MR. et al. Schwann cells seeded in acellular nerve grafts improve functional recovery. Muscle Nerve 2014; 49 (02) 267-276
• 7 Moore AM, MacEwan M, Santosa KB. et al. Acellular nerve allografts in peripheral nerve regeneration: a comparative study. Muscle Nerve 2011; 44 (02) 221-234
• 8 Deumens R, Bozkurt A, Meek MF. et al. Repairing injured peripheral nerves: bridging the gap. Prog Neurobiol 2010; 92 (03) 245-276
• 9 Dalamagkas K, Tsintou M, Seifalian A. Advances in peripheral nervous system regenerative therapeutic strategies: a biomaterials approach. Mater Sci Eng C 2016; 65: 425-432
• 10 Marcoccio I, Vigasio A. Muscle-in-vein nerve guide for secondary reconstruction in digital nerve lesions. J Hand Surg Am 2010; 35 (09) 1418-1426
• 11 Meek MF, Varejão ASP, Geuna S. Use of skeletal muscle tissue in peripheral nerve repair: review of the literature. Tissue Eng 2004; 10 (7–8): 1027-1036
• 12 Whitworth IH, Doré C, Hall S, Green CJ, Terenghi G. Different muscle graft denaturing methods and their use for nerve repair. Br J Plast Surg 1995; 48 (07) 492-499
• 13 Ide C. Nerve regeneration through the basal laminae scaffold of the skeletal muscle. Neurosci Res 1984; 1: 379-391
• 14 Chu C, Liu L, Rung S. et al. Modulation of foreign body reaction and macrophage phenotypes concerning microenvironment. J Biomed Mater Res A 2020; 108 (01) 127-135
• 15 Mligiliche N, Kitada M, Ide C. Grafting of detergent-denatured skeletal muscles provides effective conduits for extension of regenerating axons in the rat sciatic nerve. Arch Histol Cytol 2001; 64 (01) 29-36
• 16 Mligiliche N, Tabata Y, Endoh K, Ide C. Peripheral nerve regeneration through a long detergent-denatured muscle autografts in rabbits. Neuroreport 2001; 12 (08) 1719-1722
• 17 McGowan JV, Chung R, Maulik A, Piotrowska I, Walker JM, Yellon DM. Anthracycline chemotherapy and cardiotoxicity. Cardiovasc Drugs Ther 2017; 31 (01) 63-75
• 18 Zhao J, Du J, Pan Y. et al. Activation of cardiac TrkB receptor by its small molecular agonist 7,8-dihydroxyflavone inhibits doxorubicin-induced cardiotoxicity via enhancing mitochondrial oxidative phosphorylation. Free Radic Biol Med 2019; 130: 557-567
• 19 Mohajeri M, Sahebkar A. Protective effects of curcumin against doxorubicin-induced toxicity and resistance: a review. Crit Rev Oncol Hematol 2018; 122: 30-51
• 20 Segredo MP, Salvadori DM, Rocha NS. et al. Oxidative stress on cardiotoxicity after treatment with single and multiple doses of doxorubicin. Hum Exp Toxicol 2014; 33 (07) 748-760
• 21 Min K, Kwon O-S, Smuder AJ. et al. Increased mitochondrial emission of reactive oxygen species and calpain activation are required for doxorubicin-induced cardiac and skeletal muscle myopathy. J Physiol 2015; 593 (08) 2017-2036
• 22 Campbell TL, Quadrilatero J. Data on skeletal muscle apoptosis, autophagy, and morphology in mice treated with doxorubicin. Data Brief 2016; 7: 786-793
• 23 Damiani RM, Moura DJ, Viau CM, Caceres RA, Henriques JAP, Saffi J. Pathways of cardiac toxicity: comparison between chemotherapeutic drugs doxorubicin and mitoxantrone. Arch Toxicol 2016; 90 (09) 2063-2076
• 24 Takeuchi H, Sakamoto A, Ikeguchi R. et al. Doxorubicin-immersed skeletal muscle grafts promote peripheral nerve regeneration across a 10-mm defect in the rat sciatic nerve. J Reconstr Microsurg 2020; 36 (01) 41-52
• 25 Kakinoki R, Nishijima N, Ueba Y, Oka M, Yamamuro T. Relationship between axonal regeneration and vascularity in tubulation–an experimental study in rats. Neurosci Res 1995; 23 (01) 35-45
• 26 Tajino J, Ito A, Tanima M. et al. Three-dimensional motion analysis for comprehensive understanding of gait characteristics after sciatic nerve lesion in rodents. Sci Rep 2018; 8 (01) 13585-13589
• 27 Yurie H, Ikeguchi R, Aoyama T. et al. The efficacy of a scaffold-free Bio 3D conduit developed from human fibroblasts on peripheral nerve regeneration in a rat sciatic nerve model. PLoS One 2017; 12 (02) e0171448
• 28 Pereira JH, Bowden REM, Gattuso JM, Norris RW. Comparison of results of repair of digital nerves by denatured muscle grafts and end-to-end sutures. J Hand Surg [Br] 1991; 16 (05) 519-523
• 29 Manoli T, Schulz L, Stahl S, Jaminet P, Schaller HE. Evaluation of sensory recovery after reconstruction of digital nerves of the hand using muscle-in-vein conduits in comparison to nerve suture or nerve autografting. Microsurgery 2014; 34 (08) 608-615
• 30 Ueno M, Kakinuma Y, Yuhki K. et al. Doxorubicin induces apoptosis by activation of caspase-3 in cultured cardiomyocytes in vitro and rat cardiac ventricles in vivo. J Pharmacol Sci 2006; 101 (02) 151-158
• 31 Weigert A, Tzieply N, von Knethen A. et al. Tumor cell apoptosis polarizes macrophages role of sphingosine-1-phosphate. Mol Biol Cell 2007; 18 (10) 3810-3819
• 32 Cattin A-L, Burden JJ, Van Emmenis L. et al. Macrophage-induced blood vessels guide Schwann cell-mediated regeneration of peripheral nerves. Cell 2015; 162 (05) 1127-1139
• 33 Dodla MC, Bellamkonda RV. Differences between the effect of anisotropic and isotropic laminin and nerve growth factor presenting scaffolds on nerve regeneration across long peripheral nerve gaps. Biomaterials 2008; 29 (01) 33-46
• 34 Bozkurt A, Boecker A, Tank J. et al. Efficient bridging of 20 mm rat sciatic nerve lesions with a longitudinally micro-structured collagen scaffold. Biomaterials 2016; 75: 112-122
• 35 Boecker AH, van Neerven SG, Scheffel J. et al. Pre-differentiation of mesenchymal stromal cells in combination with a microstructured nerve guide supports peripheral nerve regeneration in the rat sciatic nerve model. Eur J Neurosci 2016; 43 (03) 404-416