Abstract
BackgroundAfter spinal cord injury (SCI), glial scarring is mainly formed around the lesion and inhibits axon regeneration. Recently, we reported that anti-β1 integrin antibody (β1Ab) 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 β1Ab treatment has any influence on microglia within the glial scar remains unclear.MethodsTo evaluate the effects of β1Ab treatment on microglia within the glial scar after SCI, we applied thoracic contusion SCI to C57BL/6N mice, administered β1Ab 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 TNFα (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 β1 integrin receptors. Statistical analyses were performed using Wilcoxon’s rank-sum test.ResultsAfter performing β1Ab treatment, the microglia were scattered within the glial scar and the expression of TNFα in both the microglia and the glial scar were significantly suppressed after SCI. This in vivo alteration was attributed to fibronectin, a ligand of β1 integrin receptors. Furthermore, the microglial expression of TNFα 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.ConclusionMicroglial inflammation was enhanced by RAs via the fibronectin/β1 integrin pathway within the glial scar after SCI. Our results suggested that β1Ab administration had therapeutic potential for ameliorating both glial scar formation and persistent neuroinflammation in the chronic phase after SCI.
Highlights
After spinal cord injury (SCI), glial scarring is mainly formed around the lesion and inhibits axon regeneration
Microglial inflammation was enhanced by Reactive astrocyte (RA) via the fibronectin/β1 integrin pathway within the glial scar after SCI
We demonstrated that the number of microglia as well as the mRNA expression of the pro-inflammatory cytokine TNFα remained increased until the chronic phase after SCI [11]
Summary
After spinal cord injury (SCI), glial scarring is mainly formed around the lesion and inhibits axon regeneration. The main reasons why chronic SCI treatments are ineffective are suggested to be (1) the glial scar, which hinders axonal regeneration through physical and chemical barriers [3], and (2) persistent neuroinflammation, which cause SCI lesions to become refractory to treatment [4]. To modulate these inhibitory factors in the injured spinal cord, including glial scars, many studies have focused on the role of inflammatory cells and astrocytes, which are the major component of lesional scars [3, 5, 6]. As such, clarifying the role of microglial cells as well as their interaction with astrocytes within the glial scars would contribute to a better understanding of the pathophysiology of chronic SCI [14]
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