Abstract
Although peripheral nerves can regenerate, clinical outcomes after peripheral nerve injuries are not always satisfactory, especially in cases of severe or proximal injuries. Further, autologous nerve grafting remains the gold standard for the reconstruction of peripheral nerves, although this method is still accompanied by issues of donor-site morbidity and limited supply. Cell therapy is a potential approach to overcome these issues. However, the optimal cell type for promoting axon regeneration remains unknown. Here, we report a novel experimental model dedicated to elucidation of the axon-promoting effects of candidate cell types using simple and standardized techniques. This model uses rat sciatic nerves and consists of a 25 mm-long acellular region and a crush site at each end. The acellular region was made by repeated freeze/thaw procedures with liquid nitrogen. Importantly, the new model does not require microsurgical procedures, which are technically demanding and greatly affect axon regeneration. To test the actual utility of this model, red fluorescent protein-expressing syngeneic Schwann cells (SCs), marrow stromal cells, or fibroblasts were grafted into the acellular area, followed by perfusion of the rat 2 weeks later. All types of grafted cells survived well. Quantification of regenerating axons demonstrated that SCs, but not the other cell types, promoted axon regeneration with minimum variability. Thus, this model is useful for differentiating the effects of various grafted cell types in axon regeneration. Interestingly, regardless of the grafted cell type, host SCs migrated into the acellular area, and the extent of axon regeneration was strongly correlated with the number of SCs. Moreover, all regenerating axons were closely associated with SCs. These findings suggest a critical role for SCs in peripheral nerve axon regeneration. Collectively, this novel experimental model is useful for elucidating the axon-promoting effects of grafted cells and for analyzing the biology of peripheral nerve axon regeneration.
Highlights
Peripheral nerves can regenerate, clinical outcomes after peripheral nerve injuries are not always satisfactory, especially in severe cases and proximal injury cases (Seddon, 1947; Seddon, 1963; Evans et al, 1994; Lundborg, 2000)
We specified six requirements for an experimental model that can elucidate the effect of test cells on peripheral nerve axon regeneration
To label dead cells following the freeze and thaw (FT) procedure, sciatic nerves were incubated with propidium iodide (PI) for 1 min immediately after the FT procedures and fixed in PFA
Summary
Peripheral nerves can regenerate, clinical outcomes after peripheral nerve injuries are not always satisfactory, especially in severe cases and proximal injury cases (Seddon, 1947; Seddon, 1963; Evans et al, 1994; Lundborg, 2000). For reconstruction following peripheral nerve injury, autologous nerve grafting (ANG) has remained the gold standard for more than 7 decades (Seddon, 1947; Millesi, 1973; Evans et al, 1994; Lundborg, 2000; Isaacs, 2010; Daly et al, 2012). Cell therapy is a potential option, according to recent advancements in regenerative medicine (Behfar et al, 2014; Hundepool et al, 2014; Kotton and Morrisey, 2014; Bussolati and Camussi, 2015; Grayson et al, 2015; Sakai and Andersson, 2015; Bitar and Zakhem, 2016; Kadoya et al, 2016; Assinck et al, 2017; Ellis et al, 2017; Chichagova et al, 2018). The most effective cell type for induction of peripheral nerve regeneration when grafting into an injured nerve or when combined with a scaffold remains to be determined
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