Wild type and EGFP+ Bone Marrow Mononuclear Cells: a useful tool for peripheral axonal regeneration and remyelination in sciatic nerve injury

PIÑERO, GONZALO; USACH, VANINA; SOTO, PAULA; SETTON - AVRUJ, PATRICIA

Chicago

Congreso; 45th Annual Meeting of the Society for Neuroscience; 2015

In order to assess bone marrow cell remyelination ability, we used two models of Wallerian Degeneration (WD): ligation of sciatic nerve as an irreversible model, and sciatic nerve crush as a reversible one (Setton et al., 2007; Usach et al., 2011). In both models, we demonstrated spontaneous migration of endogenous and/or transplanted wild type bone marrow mononuclear cells (wt-BMMCs) ?tracked with CMTMR (5-(and-6)-(((4-Chloromethyl)Benzoyl)Amino)Tetramethylrhodamine)? to the injured nerve; in the reversible model, once the cells arrived at the ipsilateral nerve, some of them colocalized with Schwann cell (S100β and MBP) and nerve fiber markers (PGP 9.5). Also, wt-BMMCs were shown to exert a beneficial effect by accelerating axonal regeneration and, subsequently, remyelination.In this work, and in order to attain long-lasting cell labeling and study longer survival times, we used BMMCs isolated from adult rats of the transgenic strain [Wistar-TgN(CAG-GFP)184ys] (BMMCs-EGFP+). The aim of this work was to reproduce our wild type model and evaluate BMMC-EGFP+ remyelination ability in adult rat undergoing 8 seconds sciatic nerve crush. Initially, when comparing wt-BMMC and BMMC-EGFP+ performance, results showed a lower yield in the isolation of BMMCs-EGFP+. However, flow cytometry analysis confirmed that cells isolated from adult rats of the transgenic strain exhibited similar expression levels of multipotent (CD34 and CD90) and Schwann cell (MBP, S100β and p75NTR) markers as wt-BMMCs. In addition, BMMCs-EGFP+ retained their migration capacity, arrived exclusively at the injured nerve and rendered no significant differences from wt-BMMCs in terms of MBP and PGP 9.5 levels at 7 and 14 days post-injury. Epifluorescence and confocal microscopy and Western blot analyses were later used to evaluate BMMC-EGFP+ effect on sciatic nerve remyelination at longer survival times and their possible beneficial effects on axonal regeneration and remyelination. Results revealed that some of the BMMCs-EGFP+ that arrived at the ipsilateral nerve colocalized with Schwann cell and nerve fiber markers. Optical and electronic microscopy studies are currently ongoing in order to demonstrate axonal recovery and remyelination promoted by cell transplant. On the basis of these findings, BMMCs-EGFP+ appear to be a useful tool to evaluate peripheral nervous system demyelination-remyelination process.