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Multipotent mesenchymal stromal cells (MMSCs) demonstrated a measurable therapeutic effect following transplantation into animal models of periventricular leukomalacia (PVL), brain white-matter degeneration resulting from hypoxic-ischemic incidents and/or inflammation. However, the mechanisms by which transplanted MMSCs promote cell survival and/or functional recovery remain indeterminate. In this work we used organotypic brain slices for PVL model in vitro (PVLmiv) subjecting cultures to oxygen-glucose deprivation (OGD) and endotoxin lipopolysaccharide (LPS). This approach allowed us to simulate important pathogenic factors both responsible for PVL, hypoxic-ischemic component and inflammation. Based on the cell viability and the glial reaction, we evaluated distant effects of MMSCs on brain slices with PVLmiv in the non-contact co-culture. Cell viability was assessed by the measurement of cytoplasmic enzyme lactate dehydrogenase (LDH) released into the culture medium. Glial reaction in the periventricular regions of slices was analyzed immunochistochemically using specific antibodies to glial markers of oligodendrocytes, astrocytes and microglia (Rip, GFAP and Iba-1, respectively). We showed that the PVLmiv resulted in a significant release of the cytosolic enzyme LDH into medium demonstrating substantial cell damage. A decrease of Rip-immunoreactivity indicated deterioration within oligodenrocytic population of cells, while an increase in GFAP and Iba-1 immunoreactivity reflected pronounced astroand microgliosis. The presence of MMSCs in the co-culture diminished PVLmiv effects improving cell viability, preventing degradation of oligodendocytes and extensive astroand microgliosis in brain slices. Our data suggest that protective capacity of MMSCs can be executed distantly most likely via released biomodulatory compounds.

The effects of multipotent mesenchymal stromal cells on mouse brain slices at their co-culture in an in vitro model of periventricular leukomalacia