Department of Orthopedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
Department of Neuroscience & Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
J Neuroinflammation. 2021 Jan 6;18(1):12. doi: 10.1186/s12974-020-02059-x.
After 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.
To 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.
After 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.
Microglial 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.
脊髓损伤（SCI）后，胶质瘢痕主要围绕损伤部位形成，并抑制轴突再生。最近，我们报道抗β1 整合素抗体（β1Ab）通过阻止胶质瘢痕形成的诱导，对星形胶质细胞具有治疗作用。然而，胶质瘢痕中的细胞成分不仅是星形胶质细胞，还有小胶质细胞，β1Ab 治疗是否对胶质瘢痕中的小胶质细胞有任何影响尚不清楚。
为了评估β1Ab 治疗对 SCI 后胶质瘢痕中小胶质细胞的影响，我们对 C57BL/6N 小鼠进行了胸段挫伤 SCI，在亚急性期给予β1Ab，并在慢性期通过免疫组织化学分析损伤的脊髓。为了研究小胶质细胞和胶质瘢痕中的基因表达，我们使用荧光激活细胞分选选择性收集小胶质细胞，并使用激光捕获显微切割（LMD）分离胶质瘢痕。为了研究胶质瘢痕中小胶质细胞和星形胶质细胞之间的相互作用，我们在体外用反应性星形胶质细胞（RACM）的条件培养基刺激 BV-2 小胶质细胞，并通过定量聚合酶链反应分析 TNFα（促炎 M1 标志物）的基因表达。我们还使用 LMD 分离了原代星形胶质细胞（NAs）和反应性星形胶质细胞（RAs），并研究了它们对β1 整合素受体配体的表达。使用 Wilcoxon 秩和检验进行统计分析。
在进行β1Ab 治疗后，小胶质细胞在胶质瘢痕中分散，SCI 后小胶质细胞和胶质瘢痕中的 TNFα 表达均显著受到抑制。这种体内变化归因于纤维连接蛋白，它是β1 整合素受体的配体。此外，体外研究表明，RACM 以及纤维连接蛋白可调节小胶质细胞中 TNFα 的表达。我们还证实纤维连接蛋白由反应性星形胶质细胞在体外和体内分泌。这些结果突出了胶质瘢痕中小胶质细胞和星形胶质细胞之间由纤维连接蛋白介导的相互作用。
SCI 后，胶质瘢痕中的反应性星形胶质细胞通过纤维连接蛋白/β1 整合素途径增强小胶质细胞炎症。我们的结果表明，β1Ab 给药具有改善 SCI 后慢性期胶质瘢痕形成和持续神经炎症的治疗潜力。
