1These authors contributed equally to this study.Abbreviations used: CAT, catalase; CM, conditioned medium; EAE, experimental autoimmune encephalomyelitis; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFAP, glial fibrillary acid protein; GST, glutathione-S-transferase; MOG, myelin oligodendrocyte glycoprotein peptide; MS, multiple sclerosis; MSC, mesenchimal stem cell; MT, metallothionein; NeuN, neuronal nuclei; PARP-1, poly(ADP-ribose) polymerase-1; PBS, phosphate-buffered saline; RPMI, Royal Park Memorial Institute; SOD, superoxide dismutase. AbstractExperimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis, is characterized by demyelination, inflammation and neurodegeneration of CNS in which free radicals play a role. Recently, the efficacy of murine mesenchimal stem cells (MSCs) as treatment of EAE induced in mice by the encephalitogenic peptide MOG(35-55) was demonstrated. The present study analyzed some markers of oxidative stress, inflammation/degeneration and apoptosis such as metallothioneins (MTs), antioxidant enzymes (superoxide dismutase, catalase and glutathione-S-transferase), poly(ADP-ribose) polymerase-1 and p53 during EAE progression and following MSC treatment. Expression of the three brain MT isoforms increased significantly in EAE mice compared with healthy controls, but while expression of MT-1 and MT-3 increased along EAE course, MT-2 was up-regulated at the onset, but returned to levels similar to those of controls in chronic phase. The changes in the transcription and activity of the antioxidant enzymes and in expression of poly(ADP-ribose) polymerase-1 and p53 showed the same kinetics observed for MT-1 and MT-3 during EAE. Interestingly, i.v. administration of MSCs reduced the EAE-induced increases in levels/activities of all these proteins. These results support an antioxidant and neuroprotective activity for MSCs that was also confirmed in vitro on neuroblastoma cells exposed to an oxidative insult.

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Oxidative stress is considered to be one of the main causes of neural damage after injury. However, little is known about the changes in mRNA expression of anti-oxidant molecules that occur after injury and regenera-tion of the peripheral nerve. In the present study, the rat median nerve was transected, and transcriptional changes were studied at day 6 and day 12 after injury in both the proximal and the distal stumps, in the ab-sence or presence of microsurgical repair. The expres-sion profiles of the following genes were investigated: three metallothionein isoforms (MT-1, MT-2, and MT-3), the main antioxidant enzymes (catalase, superoxide dismutase, and glutathione-S-transferase), and the marker of cellular damage poly(ADP-ribose) polymer-ase-1 (PARP-1). The results showed that, in the proxi-mal nerve stump, MT-3 mRNA expression was signifi-cantly and markedly up-regulated in the absence of surgical repair, whereas MT-1 and MT-2 showed signifi-cant down-regulation. In the distal nerve portion, mRNA expression of all MT isoforms decreased significantly in the absence of microsurgical reconstruction, whereas, after repair, MT-3 mRNA expression alone was up-regu-lated. Expression of all the antioxidant enzymes decreased significantly after repair in the proximal nerve portion, but a significant general increase in their mRNA expression was revealed in the distal nerve stump. PARP-1 expression was significantly up-regu-lated in the proximal nerve portion without repair but dramatically reduced after reconstruction. In contrast, PARP-1 expression increased markedly in the distal stump after surgical repair. Taken together, these find-ings indicate that antioxidant molecules are differen-tially modulated and might, therefore, play an important role in peripheral nerve injury and regeneration.

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