Resumo

Stroke is the third leading cause of death worldwide and the largest cause of disability in adults. Our group demonstrated beneficial effects of treatment with bone marrow mononuclear cells (BMMCs) in a rat model of cortical ischemia induced by thermocoagulation. Significant recovery of sensorimotor function was observed when animals were treated 1 or 7 days after ischemia but not after 14 days. However, the mechanism(s) of action of this treatment are still unknown. In order to investigate these mechanisms, we evaluated the possible anti-inflammatory effect of BMMCs transplantation. In order, to monitor the transplanted cells we pre-labeled them with SPIONS (Superparamagnetic iron oxide nanoparticles). BMMCs were incubated for 3h with 25µg/mL nanoparticles coated with Dextran and 5µg/mL Protamina in serum free media at 37ºC under agitation. The assay for viability was performed using the Live/Dead Kit and approximately 90% were viable at the end of the protocol. The labeling of the cells with SPIONS was identified by immunofluorescence (antibody anti-Dextran) and transmission electronic microscopy (TEM). With this protocol 60% of the BMMCs were labeled with SPIONS (p<0.01; n=3) and TEM analysis showed the nanoparticles located in cytoplasmic compartments. The animals received 3x107 BMMCs or saline into the jugular vein 1 day after ischemia and were allowed to survive for 2 or 6 days. Cells expressing CD68 (ED1) antigen (a marker of activated microglia and macrophages), or positive for SPIONs or both were identified and quantified in 3 sections at the level of the lateral ventricle (2.20 mm, 1.00 mm and 0.40 mm from Bregma). In each section 6 fields were counted using a 20x objective. A large number of activated microglia with a phagocytic morphology was observed 3 days after ischemia in both treated and untreated animals. Seven days after ischemia both phagocitic and ramified activated microglia were found at the lesioned hemisphere in treated and untreated animals and the number of activated microglia was significantly reduced in the animals that received BMMCs when compared with the saline treated ones (n=3 BMMCs, n=4 saline; p<0.05). Only a few SPION labeled cells were founded at the lesion site suggesting that the modulatory effect can be due to the release of factors by the transplanted cells and that the presence of BMMCs at the site is not necessary for the beneficial effect to occur.