Bone marrow mononuclear cells participation in the Wallerian degeneration process.

VANINA USACH; BEL¨¦N GOITIA; PATRICIA SETTON-AVRUJ

Washington, Estados Unidos de Norteam¨¦rica

Congreso; 38th annual meeting of the Society for Neuroscience's; 2008

Society for Neuroscience's

Bone marrow mononuclear cells participation in the Wallerian degeneration process Vanina Usach, Bel¨¦n Goitia and Patricia Setton-Avruj Department of Biological Chemistry, School of Pharmacy and Biochemistry, University of Buenos Aires, IQUFIB, UBA-CONICET, IIMHNO-UBA, Buenos Aires, Argentina. We have previously described the reorganization as clusters of myelin major proteins in the distal stump of the ligated sciatic nerve. Concerning myelin composition, we have observed a decrease in P0 and MBP levels and an increase in a 14 kDa band that was resolved as ¦Á - and ¦Â- globin. In the same model we have demonstrated the migration, either spontaneous or after intraortical injection, of fresh bone marrow mononuclear cells (BMMC) CD34+ to the distal stump of the nerve. According to these, the aim of the present work was to study the temporary window where the 14 kDa band is present in a reversible model of Wallerian degeneration (WD) promoted by the crush of the sciatic nerve; to evaluate the migration of BMMC and mesenchymal stem cells to the distal zone of the crushed sciatic nerve, and to characterise both groups as to identify the population that would migrate to the injured nerve. Adult Wistar rats were submitted to the crush of the right sciatic nerve and were sacrificed at different survival times. The nerve was dissected into proximal and distal areas; the left sciatic nerve, contralateral nerve, was taken as a control. Myelin isolation was done according to Iyer et al. procedure (J Neurosci. Res. 46: 531-539, 1996). BMMC were isolated from the bone marrow extruded from tibia and femur bones. A group was immediately processed and the other was seeded. After 24-48hs the non-adherent cells were separated and the adherent cells were grown to confluence. Both groups were analyzed by Western blot and immunocytochemistry. Fresh isolated or cultured BMMC were dyed with a fluorescent probe and injected intravenously immediately after crushing the sciatic nerve in order to evaluate the migration of these cells to the injured area. Immunohystochemical analyses of the crushed nerve were done to study the changes suffered by the nerve. Our results demonstrate the appearance of the 14 kDa band in isolated myelin of the distal zone and the migration of fresh isolated BMMC to the crushed area of the sciatic nerve during the first week post injury. The hystological analysis of the nerve confirmed the degeneration process from the crush area to the distal zone. The characterisation of cultured BMMCs shows a heterogeneous population of small-rounded, spindle-shaped and large-flattened morphology; they showed fibroblast-like morphology at reaching confluence.  After 24-48hs in culture the adherent and non-adherent cells are CD34+, while adherent cells are CD45-, CD11b-, vimentin+, Thy 1.1+. Our results demonstrate the existence of a progenitor population in BMMC that spontaneously migrates to the injured nerve to participate in the degeneration-regeneration process.