Microglial inflammation after chronic spinal cord injury is enhanced by reactive astrocytes via the fibronectin/[beta]1 integrin pathway

Background After spinal cord injury (SCI), glial scarring is mainly formed around the lesion and inhibits axon regeneration. Recently, we reported that anti-[beta]1 integrin antibody ([beta]1Ab) had a therapeutic effect on astrocytes by preventing the induction of glial scar formation. However, the cellular components within the glial scar are not only astrocytes but also microglia, and whether or not [beta]1Ab treatment has any influence on microglia within the glial scar remains unclear. Methods To evaluate the effects of [beta]1Ab treatment on microglia within the glial scar after SCI, we applied thoracic contusion SCI to C57BL/6N mice, administered [beta]1Ab in the sub-acute phase, and analyzed the injured spinal cords with immunohistochemistry in the chronic phase. To examine the gene expression in microglia and glial scars, we selectively collected microglia with fluorescence-activated cell sorting and isolated the glial scars using laser-captured microdissection (LMD). To examine the interaction between microglia and astrocytes within the glial scar, we stimulated BV-2 microglia with conditioned medium of reactive astrocytes (RACM) in vitro, and the gene expression of TNF[alpha] (pro-inflammatory M1 marker) was analyzed via quantitative polymerase chain reaction. We also isolated both naïve astrocytes (NAs) and reactive astrocytes (RAs) with LMD and examined their expression of the ligands for [beta]1 integrin receptors. Statistical analyses were performed using Wilcoxon's rank-sum test. Results After performing [beta]1Ab treatment, the microglia were scattered within the glial scar and the expression of TNF[alpha] in both the microglia and the glial scar were significantly suppressed after SCI. This in vivo alteration was attributed to fibronectin, a ligand of [beta]1 integrin receptors. Furthermore, the microglial expression of TNF[alpha] was shown to be regulated by RACM as well as fibronectin in vitro. We also confirmed that fibronectin was secreted by RAs both in vitro and in vivo. These results highlighted the interaction mediated by fibronectin between RAs and microglia within the glial scar. Conclusion Microglial inflammation was enhanced by RAs via the fibronectin/[beta]1 integrin pathway within the glial scar after SCI. Our results suggested that [beta]1Ab administration had therapeutic potential for ameliorating both glial scar formation and persistent neuroinflammation in the chronic phase after SCI. Keywords: Spinal cord injury, Glial s