Objectives: Osteopontin (OPN) is an inflammatory cytokine inducer involved in cell proliferation and migration in inflammatory diseases or tumors. To investigate the function of OPN in astrocyte activation during brain injury, we compared OPN-deficient (OPN/KO) with wild-type (WT) mouse brains after stab wound injury and primary culture of astrocytes.
Methods: Primary cultures of astrocytes were prepared from either WT or OPN/KO postnatal mouse brains. Activation efficiency of astrocytes in primary culture was accessed using Western blotting by examining the protein levels of glial fibrillary acidic protein (GFAP) and tenascin-C (TN-C), which are markers for reactive astrocytes, following lipopolysaccharide (LPS) stimulation. Furthermore, the stab wound injury on the cerebral cortex as a brain traumatic injury model was used, and activation of astrocytes and microglial cells was investigated using immunofluorescent analysis on fixed brain sections.
Results: Primary cultures of astrocytes prepared from WT or OPN/KO postnatal mouse brains showed that only 25% of normal shaped astrocytes in a flask were produced in OPN/KO mice. The expression levels of both GFAP and TN-C were downregulated in the primary culture of astrocytes from OPN/KO mice compared with that from WT mice. By the immunofluorescent analysis on the injured brain sections, glial activation was attenuated in OPN/KO mice compared with WT mice.
Discussion: Our data suggest that OPN is essential for proper astrocytic generation in vitro culture prepared from mouse cerebral cortex. OPN is indispensable for astrocyte activation in the mouse brain injury model and in LPS stimulated primary culture.
Abbreviations: AQP4: aquaporin 4; BBB: blood brain barrier; BrdU: bromo-deoxy uridine; CNS: central nervous system; GFAP: glial fibllirary acidic protein; IgG: immunoglobulin G; LPS: lipopolysaccharide; OPN: osteopontin; OPN/KO: osteopontin-deficient; TN-C: tenascin-C.
Keywords: Blood-brain barrier; IgG; bromo-deoxy uridine; glial fibllirary acidic protein; lipopolysaccharide; primary culture of astrocytes; primary culture of microglial cells; tenascin-C.