Journal: Neural Regeneration Research

Article Title: Mutual regulation of microglia and astrocytes after Gas6 inhibits spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01130

Figure Lengend Snippet: Pro-inflammatory phenotype microglia and cytotoxic phenotype astrocyte cells are increased in the harsh inflammatory micro-environment of SCI. (A) UMAP visualization plot of 66,178 spinal cord cells sequenced from the uninjured spinal cord and injured spinal cord at 1, 3, and 7 dpi, color-coding defined nine major cell types based on signature gene expression. (B) Violin plots showing the smoothed expression distribution of marker genes in nine cell types. (C) The dot plot shows the change of the marker genes for microglia cluster of uninjured group and SCI group (1, 3, and 7 dpi groups). Dot color intensity represents the average expression values, and dot size represents percent of cells with at least one UMI detected per gene. (D) The dot plot is shown the change of the marker genes for astrocyte cluster of the uninjured group and SCI group (1, 3, and 7 dpi groups). (E) Hematoxylin-eosin staining and immunofluorescence analysis of CD68 + (red, Alexa Fluor 594) and GFAP + (green, FITC) cells in the injured spinal cord 3 days post SCI. CD68 + and GFAP + cells increased after SCI. Scale bars: 100 μm (upper and middle), 20 μm (lower). (F) Quantification of CD68 + cells and GFAP + cells in E. (G) Real-time PCR analysis of mRNA expressions of CD68 and GFAP in the injured spinal cord of uninjured group and 3 dpi group. (H) Western Blot analysis of protein expressions of CD68 and GFAP in the injured spinal cord of uninjured group and 3 dpi group. (I) Quantification of protein expressions of CD68 and GFAP in H. The data are presented as the means ± SD ( n = 3). ** P < 0.01 (unpaired t -test). DAPI: 4′,6-Diamidino-2-phenylindole; dpi: day(s) post injury; GFAP: glial fibrillary acidic protein; PCR: polymerase chain reaction; SCI: spinal cord injury; UMAP: uniform manifold approximation and projection.

Article Snippet: After washing three times in TBST, primary antibodies were probed with secondary antibodies: goat anti-rabbit IgG (HRP) (1:4000, Abcam, Cat# ab7090, RRID: AB_955417), Alexa Fluor 647 donkey anti-rabbit IgG (H+L) (1:200, Abcam, Cat# ab150075, RRID: AB_2752244), Alexa Fluor 594 donkey anti-mouse IgG (H+L) (1:400, Invitrogen, Cat# A-21203, RRID: AB_141633), and fluorescein isothiocyanate isomer I (FITC; 1:300, Thermo Fisher Scientific, Waltham, MA, USA, Cat# F-2761, RRID: AB_2536524; Cat# F-2765, RRID: AB_2536525) for 1 hour at room temperature (25°C).

Techniques: Expressing, Marker, Staining, Immunofluorescence, Real-time Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Mutual regulation of microglia and astrocytes after Gas6 inhibits spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01130

Figure Lengend Snippet: Proinflammatory phenotype microglia and cytotoxic phenotype astrocytes reinforce each other. (A) Immunofluorescence analysis of GFAP + (green, FITC) and C3 + (red, Alexa Fluor 594) cells in astrocytes co-cultured with microglia. GFAP + cells and C3 + cells were increased in astrocytes co-cultured with proinflammatory microglia and were decreased in astrocytes co-cultured with anti-inflammatory microglia. (B) Quantification of GFAP + and C3 + cells in A. (C) Real time PCR analysis of mRNA expressions of GFAP and C3 in astrocytes co-cultured with microglia. (D) Western blot analysis of protein expressions of GFAP and C3 in astrocytes co-cultured with microglia. (E) Quantification of protein expressions of GFAP and C3 in D. (F) Immunofluorescence analysis of iNOS + (green, FITC) and Arg1 + (red, Alexa Fluor 594) cells in microglia co-cultured with astrocytes. iNOS + cells were increased in microglia co-cultured with cytotoxic astrocytes, while Arg1 + cells were not alert. Scale bars: 20 μm. (G) Quantification of iNOS + and Arg1 + cells in F. (H) Real-time PCR analysis of mRNA expressions of iNOS and Arg1 in microglia co-cultured with astrocytes. (I) Western blot analysis of protein expressions of iNOS and Arg1 in microglia co-cultured with astrocytes. (J) Quantification of protein expressions of iNOS and Arg1 in I. The data are presented as the means ± SD from one representative experiment of three independent experiments performed in triplicate. ** P < 0.01 (one-way analysis of variance followed by Bonferroni post hoc test [B, C, E] or unpaired t- test [G, H, J]). Arg1: Arginase-1; C3: complement C3; DAPI: 4′,6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate isomer I; GFAP: glial fibrillary acidic protein; iNOS: inducible nitric oxide synthase; PCR: polymerase chain reaction.

Article Snippet: After washing three times in TBST, primary antibodies were probed with secondary antibodies: goat anti-rabbit IgG (HRP) (1:4000, Abcam, Cat# ab7090, RRID: AB_955417), Alexa Fluor 647 donkey anti-rabbit IgG (H+L) (1:200, Abcam, Cat# ab150075, RRID: AB_2752244), Alexa Fluor 594 donkey anti-mouse IgG (H+L) (1:400, Invitrogen, Cat# A-21203, RRID: AB_141633), and fluorescein isothiocyanate isomer I (FITC; 1:300, Thermo Fisher Scientific, Waltham, MA, USA, Cat# F-2761, RRID: AB_2536524; Cat# F-2765, RRID: AB_2536525) for 1 hour at room temperature (25°C).

Techniques: Immunofluorescence, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: Mutual regulation of microglia and astrocytes after Gas6 inhibits spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01130

Figure Lengend Snippet: Gas6/Axl signal pathway suppresses the cross-regulation between pro-inflammatory phenotype microglia and cytotoxic phenotype astrocytes. (A) Bar plots of the ranking of signal pathway axes by over all information flow differences in the interaction networks between normal and injured sample. The signal pathways with red colored are more enriched in the normal sample. The signal pathways with green colored are more enriched in the injured sample. (B) Immunofluorescence analysis of Gas6 (red, Alexa Fluor 594) and Axl (green, FITC) immunopositivities at the injured spinal cord after SCI. Gas6 + and Axl + cells were decreased after SCI. Scale bars: 100 μm (upper), 20 μm (lower). (C, D) Quantification of Gas6 and Axl immunopositivities in E. (E) Enzyme linked immunosorbent assay of Gas6 concentration of astrocytes and microglia. (F) Immunofluorescence analysis of GFAP + (green, FITC) cells in astrocytes co-cultured with microglia and additional Gas6 treatment. GFAP + cells were increased in astrocytes co-cultured with proinflammatory microglia and were decreased in astrocytes co-cultured with proinflammatory microglia and additional Gas6. Scale bar: 20 μm. (G) Quantification of GFAP + cells in F. (H) Immunofluorescence analysis of iNOS + (red, Alexa Fluor 594) cells in microglia co-cultured with astrocytes and additional Gas6 treatment. iNOS + cells were increased in microglia co-cultured with cytotoxic astrocytes and decreased in microglia co-cultured with cytotoxic astrocytes and additional Gas6. Scale bar: 20 μm. (I) Quantification of iNOS + cells in H. The data are presented as the means ± SD from one representative experiment of three independent experiments performed in triplicate. ** P < 0.01 (unpaired t -test [B, C, E] or one-way analysis of variance followed by Bonferroni post hoc test [G, I]). A.U.: Arbitrary unit; Axl: AXL receptor tyrosine kinase; DAPI: 4′,6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate isomer I; Gas6: growth arrest specific protein 6; GFAP: glial fibrillary acidic protein; iNOS: inducible nitric oxide synthase; SCI: spinal cord injury.

Article Snippet: After washing three times in TBST, primary antibodies were probed with secondary antibodies: goat anti-rabbit IgG (HRP) (1:4000, Abcam, Cat# ab7090, RRID: AB_955417), Alexa Fluor 647 donkey anti-rabbit IgG (H+L) (1:200, Abcam, Cat# ab150075, RRID: AB_2752244), Alexa Fluor 594 donkey anti-mouse IgG (H+L) (1:400, Invitrogen, Cat# A-21203, RRID: AB_141633), and fluorescein isothiocyanate isomer I (FITC; 1:300, Thermo Fisher Scientific, Waltham, MA, USA, Cat# F-2761, RRID: AB_2536524; Cat# F-2765, RRID: AB_2536525) for 1 hour at room temperature (25°C).

Techniques: Immunofluorescence, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture

Journal: Neural Regeneration Research

Article Title: Mutual regulation of microglia and astrocytes after Gas6 inhibits spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01130

Figure Lengend Snippet: Gas6/Axl signal pathway is inactive in reactive and scar-forming astrocyte after SCI. (A) The violin plot analyses were shown the change of the Gas6 and Axl expression in naive, reactive and scar-forming astrocytes clusters after SCI. (B) Immunofluorescence analysis of Ascc1 + (green, FITC) cells and Gas6 (red, Alexa Fluor 594) immunopositivity in the injured spinal cord. Ascc1 + cells and the immunopositivity of Gas6 were significantly decreased after SCI. (C) Immunofluorescence analysis of Nestin + (green, FITC) cells and Gas6 (red, Alexa Fluor 594) immunopositivity in the injured spinal cord. Nestin + cells were increased and the immunopositivity of Gas6 was significantly decreased after SCI. (D) Immunofluorescence analysis of Bcan + (green, FITC) cells and Gas6 (red, Alexa Fluor 594) immunopositivity in the injured spinal cord. Bcan + cells were increased and the immunopositivity of Gas6 was significantly decreased after SCI. (E) Immunofluorescence analysis of Ascc1 + (red, Alexa Fluor 594) cells and Axl (green, FITC) immunopositivity in the injured spinal cord. Ascc1 + cells and immunopositivity of Axl were significantly decreased after SCI. (F) Immunofluorescence analysis of Nestin + (red, Alexa Fluor 594) cells and Axl (green, FITC) immunopositivity in the injured spinal cord. Nestin + cells were increased and the immunopositivity of Axl was significantly decreased after SCI. (G) Immunofluorescence analysis of Bcan + (red, Alexa Fluor 594) cells and Axl (green, FITC) immunopositivity in the injured spinal cord. Bcan + cells were increased and the immunopositivity of Axl was significantly decreased after SCI. Scale bars: 100 μm (upper), 20 μm (lower). The data are presented as the means ± SD ( n = 3). ** P < 0.01, vs . sham group (unpaired t -test). A.U.: Arbitrary unit; Axl: AXL receptor tyrosine kinase; Bcan: brevican; DAPI: 4′,6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate isomer I; Gas6: growth arrest specific protein 6; SCI: spinal cord injury.

Article Snippet: After washing three times in TBST, primary antibodies were probed with secondary antibodies: goat anti-rabbit IgG (HRP) (1:4000, Abcam, Cat# ab7090, RRID: AB_955417), Alexa Fluor 647 donkey anti-rabbit IgG (H+L) (1:200, Abcam, Cat# ab150075, RRID: AB_2752244), Alexa Fluor 594 donkey anti-mouse IgG (H+L) (1:400, Invitrogen, Cat# A-21203, RRID: AB_141633), and fluorescein isothiocyanate isomer I (FITC; 1:300, Thermo Fisher Scientific, Waltham, MA, USA, Cat# F-2761, RRID: AB_2536524; Cat# F-2765, RRID: AB_2536525) for 1 hour at room temperature (25°C).

Techniques: Expressing, Immunofluorescence

Journal: Neural Regeneration Research

Article Title: Mutual regulation of microglia and astrocytes after Gas6 inhibits spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01130

Figure Lengend Snippet: Gas6 improves the inflammatory micro-environment to promote tissue repair and locomotor recovery. (A) Immunofluorescence analysis of CD68 + (red, Alexa Fluor 594) cells and GFAP (green, FITC) immunopositivity in the injured spinal cord after SCI ( n = 3). CD68 + cells and the immunopositivity of GFAP were increased after SCI and were significantly decreased with additional Gas6. After addition of Gas6 and R428, CD68 + cells and the immunopositivity of GFAP were increased compared with the Gas6 group. Scale bars: 100 μm (upper), 20 μm (lower). (B) Quantification of CD68 + cells and GFAP immunopositivity in A ( n = 3). (C) Hematoxylin-eosin and Nissl staining of the spinal cord in different groups. The cavity area was decreased in the Gas6 group. The number of Nissl bodies was increased in the Gas6 group. (D) Quantification of hematoxylin-eosin and Nissl staining in different groups ( n = 5). (E) Behavioral character images showing hindlimb movement in different groups. Rats in the Gas6 group grabbed and stood up more easily than those in the other groups. (F) Footprint analysis of different groups. Rats in the Gas6 group stepped more easily than those in the other group. Blue indicates the forelimb and red indicates the hindlimb. (G) BBB scores in different groups ( n = 5). The data are presented as the means ± SD. * P < 0.05, ** P < 0.01, vs . SCI group (one-way analysis of variance followed by Bonferroni post hoc test). A.U.: Arbitrary unit; Axl: AXL receptor tyrosine kinase; BBB: Basso, Beattie, and Bresnahan locomotion scale; DAPI: 4′,6-diamidino-2-phenylindole; FITC: fluorescein isothiocyanate isomer I; Gas6: growth arrest specific protein 6; GFAP: glial fibrillary acidic protein; R428: AXL inhibitor; SCI: spinal cord injury.

Article Snippet: After washing three times in TBST, primary antibodies were probed with secondary antibodies: goat anti-rabbit IgG (HRP) (1:4000, Abcam, Cat# ab7090, RRID: AB_955417), Alexa Fluor 647 donkey anti-rabbit IgG (H+L) (1:200, Abcam, Cat# ab150075, RRID: AB_2752244), Alexa Fluor 594 donkey anti-mouse IgG (H+L) (1:400, Invitrogen, Cat# A-21203, RRID: AB_141633), and fluorescein isothiocyanate isomer I (FITC; 1:300, Thermo Fisher Scientific, Waltham, MA, USA, Cat# F-2761, RRID: AB_2536524; Cat# F-2765, RRID: AB_2536525) for 1 hour at room temperature (25°C).

Techniques: Immunofluorescence, Staining