Microglial inflammation after chronic spinal cord injury is enhanced by reactive astrocytes via the fibronectin/β1 integrin pathway

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Fibronectin; Glial scar; Microglia; Reactive astrocyte; Spinal cord injury.

Fig. 1

The administration of anti-β1 integrin antibody to injured spinal cord in the sub-acute phase suppressed the glial scar formation in the chronic phase, leading to changes in the microglial distribution within the glial scar. a Time schedule of our in vivo experiments. Injured mice were intralesionally administered anti-β1 integrin antibody or control antibody at 9, 11, and 13 days post-injury (dpi). At 42 dpi, the injured spinal cord was analyzed. b Sagittal sections of the chronically injured spinal cords. Magnification of the inset is shown in the right figure. Asterisk indicates the lesion epicenter. GFAP, red; ColIαI, green. Scale bars, 250 μm on the left and 100 μm on the right. c Time course of the Basso Mouse Scale score after SCI. Error bar indicates mean ± SEM. Star indicates statistical significance (p < 0.05). A two-way repeated-measures ANOVA with the Tukey-Kramer post hoc test. n = 14 mice per group. d Peri-lesional glial scar of the chronically injured spinal cord. Microglia are mainly the lesion epicenter. β1Ab administration changed the microglial distribution within glial scars. Asterisk indicates the lesion epicenter. GFAP, red, TMEM119, white. Scale bar, 200 μm. e Gating strategy of fluorescence-activated cell sorting for selective isolation of microglia from injured spinal cord. Red-boxed population, Gr-1^nega-int/CD11b^high/CD45^int, indicates microglial population. f The number of resident microglia in the injured spinal cord counted by FACS. Error bar indicates mean ± SEM. n.s. indicates not significant. Wilcoxon’s rank-sum test. n = 3 per each group, triplicate. F = 0.266

Fig. 2

The administration of anti-β1 integrin antibody to injured spinal cord in the sub-acute phase suppressed microglial inflammation within the glial scar in the chronic phase after SCI. a Sagittal section before and after selective isolation of the glial scars using a laser-captured microdissection system. Purple dots indicate the cutting line of peri-lesional area. The width of the glial scar area was set at approximately 200 μm. Asterisk indicates the lesion epicenter. GFAP, red. Scale bar, 500 μm. b The heatmap indicates the mRNA expression profile of representative pro- and anti-inflammatory markers. The mRNA expression of TNFα and Msr1 was markedly different between the control- and β1Ab-treated groups. n = 4 per each group, duplicate. c Analyses of the mRNA expression of the glial scars by qPCR. Error bar indicates mean ± SEM. Star indicates statistical significance (p < 0.05). Wilcoxon’s rank-sum test. n = 8 per each group. TNFα: F = 0.0002, Msr1: F = 0.0201. d Analyses of the TNFα and Msr1 mRNA expression of microglia by qPCR. Error bar indicates mean ± SEM. Star indicates statistical significance (p < 0.05). Wilcoxon’s rank-sum test. n = 4 per each group. TNFα: F = 0.0365, Msr1: F = 0.0427. e TUNEL-stained sections of chronically injured spinal cords. Asterisk indicates the lesion epicenter. TUNEL-positive, red. Scale bar, 200 μm. f The number of TUNEL-positive apoptotic cells within glial scars in the injured spinal cord counted by the Image J software program. Error bar indicates mean ± SEM. Star indicates statistical significance (p < 0.05). Wilcoxon’s rank-sum test. n = 5 per each group, triplicate. F = 0.0274

Fig. 3

The administration of anti-β1 integrin antibody significantly suppressed pro-inflammatory polarization of BV-2 microglial cells mediated by fibronectin as well as conditioned medium of reactive astrocytes in vitro. a Schematic illustration of our in vitro experiments. At 2 h after stimulation of primary astrocyte culture, the conditioned medium (RACM) was collected, concentrated, and incubated with BV-2 microglia. BV-2 cells were subjected to analyses at 48 h after the stimulation. b Phase contrast images of BV-2 cells before and after RACM stimulation. c Analyses of the mRNA expression of BV-2 cells after pre-treatment with control antibody or anti-β1 integrin antibody and RACM stimulation by qPCR. The concentration of 1× RACM was 10 times denser than 10×-diluted RACM. Error bar indicates mean ± SEM. Star indicates statistical significance (p < 0.05). Wilcoxon’s rank-sum test. n = 3 per each group, triplicate. TNFα: F = 0.306, 0.0256, and 0.0014, respectively. Msr1: F = 0.872, 0.047, and 0.032, respectively

Fig. 4

Reactive astrocytes expressed fibronectin both in vitro and in vivo. a Sagittal sections of chronically injured spinal cord. Asterisk indicates the lesion epicenter. Fibronectin, Laminin, white. Scale bar, 500 μm. b Sagittal section of naïve spinal cord. Magnification of the inset is shown in d. GFAP, red. c Sagittal section of injured spinal cord at 7 days post-injury. Magnification of the inset is shown in e. GFAP, red. Asterisk indicates the lesion epicenter. Scale bar, 500 μm. d, e GFAP-positive astrocytes (marked by white arrow-heads) were isolated marginally (surrounded area by white dots) by laser-captured microdissection (LMD). GFAP, red; Hoechst, blue. Scale bar, 20 μm. f Both NAs and RAs were isolated from spinal cord by LMD in vivo. Purple dots indicate the cutting line of the peri-lesional area. The mRNA expression of fibronectin and laminin, ligands of β1 integrin receptor, was analyzed by qPCR. Error bar indicates mean ± SEM. Star indicates statistical significance (p < 0.05). Wilcoxon’s rank-sum test. n = 3 per each group, triplicate. Fibronectin: F = 1.3 × 10⁻⁶. Laminin: F = 0.0385. g Both NAs and RAs were collected from primary cultures in vitro. The mRNA expression of fibronectin and laminin was analyzed by qPCR. Error bar indicates mean ± SEM. Star indicates statistical significance (p < 0.05). Wilcoxon’s rank-sum test. n = 3 per each group, triplicate. Fibronectin: F = 0.0432. Laminin: F = 0.0002

Fig. 5

Fibronectin expressed by reactive astrocytes is associated with intercellular interaction between astrocytes and microglia in vivo. a Peri-lesional glial scar of the chronically injured spinal cord. Asterisk indicates the lesion epicenter. GFAP, red; Fibronectin, green; TMEM119, white. Scale bar, 200 μm. b Magnification of inset b in a. GFAP, red; Fibronectin, green. Scale bar, 100 μm. c Magnification of inset c in a. CD11b, white; Fibronectin, green. Scale bar, 100 μm. d The administration of anti-β1 integrin antibody had no effect on the mRNA expression of Fn1 within glial scars. The error bar indicates mean ± SEM. Star indicates statistical significance (p < 0.05). n.s., not significant. Wilcoxon’s rank-sum test. n = 4 per each group, duplicate. F = 0.192. e The TNFα mRNA expression of BV-2 cells after fibronectin stimulation with or without β1Ab pre-treatment. Error bar indicates mean ± SEM. Star indicates statistical significance (p < 0.05). Wilcoxon’s rank-sum test. n = 3 per each group, triplicate. f Our hypothesis of the novel glial scar pathology and therapeutic effects of anti-β1 integrin antibody. Fibronectin is suggested to be expressed by reactive astrocytes and recognized by the β1 integrin receptor in microglia. Microglia attain a pro-inflammatory phenotype by fibronectin. As previously reported, the antibody blocked the interaction between reactive astrocytes and collagen, leading to the suppression of glial scar formation. The present findings suggested that the antibody also blocked interaction between reactive astrocytes and fibronectin, leading to microglia polarization within the glial scar to an anti-inflammatory condition. These integrated effects of anti-β1 integrin antibody administration can modulate the glial scar pathology and improve the chronic microenvironment after SCI