Transplante alogênico de células-tronco mesenquimais induz alteração do perfil de citocinas pró e antiflamatórias e de fatores neurotróficos no tratamento da lesão espinal

Abstract

This thesis aimed to compare two treatment protocols with cryopreserved allogenic adipose-derived mesenchymal stem cells (AD-MSC) after spinal cord injury. Experiment 1 evaluated pre-freeze and pos-freeze populations of AD-MSCs response to freezing at 30, 60 and 90 days following cryopreservation. Plasticity, cell surface phenotype and cell viability were evaluated at different times after thawing technique. Results showed that MSC do not lose their multipotency, do not alter phenotypic profile and have a high cell viability for up to 90 days of cryopreservation. Experiment 2 evaluated the immunomodulatory and neuroprotective effects of cryopreserved allogenic adipose-derived MSCs after spinal cord injury (SCI). Treatment effects were comparatively evaluated by quantitation of gene expression of interleukin-10 (IL-10), interleukin-1 beta (IL-1), tumor necrosis factor (TNF-), transforming growth factor (TGF -), brain-derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF). Immunohistochemical analyzes were used to quantify inflammatory infiltrate (anti-CD68), neural integrity (anti-NeuN), and astrocytes activation (anti-GFAP). Frozen adipose-derived MSCs were transplanted after three hours (GI) and three hours and seven days after SCI (GII). The results were compared with animals without SCI (GS) and SCI without treatment (GC). MSCs treated groups had higher scores in functional assessments from 72h after injury, being the average of the highest scores achieved by the GII; lower expression of CD68 and GFAP along with reducing of degeneration of the white matter and increased expression of NeuN. The treatment with the cells also resulted in increased IL-10 expression, GDNF, VEGF and reduced of TNF- and TGF- expression. Additionally the GII decreased IL-1 expression and increased BDNF expression. The results suggest that early intravenous injection of AD-MSCs after acute SCI promotes better recovery of function, prevent futher damage through enhancement of anti-inflammatory mechanisms, reduces the inflammatory infiltrate, active anti-inflammatory phenotype of microglia, increase neuron viability, reduces the degeneration of the white matter and astrocyte activation in SCI. In addition, treatment with double applications of AD-MSCs was superior to the single administration of these cells, resulting in improved motor response and secretion of BDNF.