Doxorubicin-Immersed Skeletal Muscle Grafts Promote Peripheral Nerve Regeneration Across a 10-mm Defect in the Rat Sciatic Nerve

Abstract

Background The treatment of peripheral nerve defects requires bridging materials. Skeletal muscle grafts have been studied as an alternative to nerve autografts because they contain longitudinally aligned basal laminar tubes that are similar to axons. Several pretreatment methods for muscle grafts have promoted axonal regeneration. Here, a new method of doxorubicin pretreatment was used, and the efficacy of the pretreated muscle graft was evaluated in a rat model of a sciatic nerve defect. Methods A rat model of a 10-mm sciatic nerve defect was analyzed in three settings: muscle grafts with and without doxorubicin pretreatment (M-graft-w-Dox and M-graft-w/o-Dox groups, respectively) and a nerve autograft group (N-graft) (n = 6/group). The M-graft-w-Dox group was immersed in a doxorubicin solution for 10 minutes and rinsed with saline. Analyses of target muscle atrophy, electrophysiology, and histology were performed 8 weeks after grafting. Results Electrophysiological parameters and target muscle atrophy were significantly superior in the M-graft-w-Dox group compared with the M-graft-w/o-Dox group. Histological assessment revealed the presence of a significantly greater number of regenerated axons in the M-graft-w-Dox group versus the M-graft-w/o-Dox group, while there were no significant differences between the M-graft-w-Dox and N-graft groups. The diameter of myelinated axons of the regenerated nerve in the M-graft-w-Dox group was significantly larger than that in the M-graft-w/o-Dox group, while it was not significantly different compared with the N-graft group. Conclusion Pretreatment of muscle grafts with doxorubicin promoted significant peripheral nerve regeneration. This method may represent a new option for the treatment of peripheral nerve defects.

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

“…We weighed the 10-mm graft before and after doxorubicin pretreatment and found that the weight was increased by 0.007 mg on average after the procedure. 24 According to the U.S. Food and Drug Administration drug information, a systemic adverse effect is observed at a total cumulative dose of 300 mg/m 2 . Considering the dosage, adverse effects are thus unlikely to occur.…”

“…Specimens were assessed with light microscopy or examined using transmission electron microscopy (TEM; Model H-7000; Hitachi High-Technologies, Tokyo, Japan), according to the previous studies. 4 24 The total number of myelinated axons (TAN), the shortest myelinated axon diameter (MAD; a), the shortest axon diameter (AD; b), and the myelin thickness (MT; [a − b]/2) were measured using ImageJ software.…”

“…TUNEL staining and immunofluorescent study were assessed in the same manner as the previous reports. 24 Briefly, a TUNEL assay was performed using a kit (In situ Apoptosis Detection Kit; Takara Bio, Shiga, Japan), and the number of double-positive (TUNEL and DAPI) cells was counted with the captured images using Image J (National Institutes of Health, Bethesda, MD). Immunofluorescent studies were performed using primary antibodies (rabbit polyclonal antilaminin antibodies [1:50; Abcam, Cambridge, UK], mouse monoclonal anti-CD68 antibodies [1:50; Abcam, Cambridge, UK], and mouse monoclonal antirat endothelial cell cytoplasm antigen [RECA-1] antibodies [1:50; AbD Serotec, Kidlington, UK]) and secondary antibodies (donkey antimouse IgG [H þ L] CF TM 543 antibody, 1:200; Biotium, Richmond, CA).…”

“…In our previous study, superior outcomes in the 1st and 8th weeks of assessment were confirmed using a 10-mm rat sciatic nerve defect model. 24 Thus, to more completely assess this method, 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 assess the muscle graft with doxorubicin pretreatment (M-graft-w-Dox) in a critical-sized nerve defect model (20 mm, Study III).…”

The efficacy of combining a vascularized biogenic conduit and a decellularized nerve graft in the treatment of peripheral nerve defects: An experimental study using the rat sciatic nerve defect model

“…Fifteen rats were divided into three equal groups: a decellularized nerve graft (DNG) group, a decellularized nerve graft with a vascularized biogenic conduit (DNG-w-VBC) group, and an autologous nerve graft (ANG) group. Right sciatic nerve defect model rats and decellularized nerve grafts were prepared as described in our previous studies (Kaizawa et al, 2016(Kaizawa et al, , 2017Takeuchi et al, 2019). Briefly, the right sciatic nerve was exposed, and a 10-mm nerve segment of the right sciatic nerve was removed.…”