Photobiomodulation Promotes Repair Following Spinal Cord Injury by Regulating the Transformation of A1/A2 Reactive Astrocytes

Abstract

After spinal cord injury (SCI), reactive astrocytes can be classified into two distinctive phenotypes according to their different functions: neurotoxic (A1) astrocytes and neuroprotective (A2) astrocytes. Our previous studies proved that photobiomodulation (PBM) can promote motor function recovery and improve tissue repair after SCI, but little is known about the underlying mechanism. Therefore, we aimed to investigate whether PBM contributes to repair after SCI by regulating the activation of astrocytes. Male rats subjected to clip-compression SCI were treated with PBM for two consecutive weeks, and the results showed that recovery of motor function was improved, the lesion cavity size was reduced, and the number of neurons retained was increased. We determined the time course of A1/A2 astrocyte activation after SCI by RNA sequencing (RNA-Seq) and verified that PBM inhibited A1 astrocyte activation and promoted A2 astrocyte activation at 7 days postinjury (dpi) and 14 dpi. Subsequently, potential signaling pathways related to A1/A2 astrocyte activation were identified by GO function analysis and KEGG pathway analysis and then studied in animal experiments and preliminarily analyzed in cultured astrocytes. Next, we observed that the expression of basic fibroblast growth factor (bFGF) and transforming growth factor-β (TGF-β) was upregulated by PBM and that both factors contributed to the transformation of A1/A2 astrocytes in a dose-dependent manner. Finally, we found that PBM reduced the neurotoxicity of A1 astrocytes to dorsal root ganglion (DRG) neurons. In conclusion, PBM can promote better recovery after SCI, which may be related to the transformation of A1/A2 reactive astrocytes.

Keywords: A1/A2 astrocytes; basic fibroblast growth factor; photobiomodulation; spinal cord injury; transforming growth factor-β.

FIGURE 1

Photobiomodulation promoted motor recovery, reduced the lesion cavity size and increased the number of surviving neurons after SCI. (A) Timeline of the experimental design. (B,C) BBB scores and LSS scores were used to evaluate the motor function of each group (n = 6 individuals per group). (D) Representative footprints from each group at 14 and 28 dpi obtained by gait analysis. The average step width was calculated (n = 6 individuals per group). (E) Representative images of immunofluorescence staining of GFAP in spinal cord tissue from the SCI + vehicle and SCI + PBM groups at 7, 14, and 28 dpi. Quantification of the cavity area of spinal cord lesions at each time point (n = 5 individuals per group). Scale bar: 400 μm. (F) Representative images of immunofluorescence staining for NeuN in the ventral horn of the spinal cord within ± 150 μm from the lesion epicenter. Quantification of the number of NeuN⁺ cells in the SCI + vehicle group and SCI + PBM group at each time point (n = 6 individuals per group). Scale bar: 200 μm. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Changes in astrocyte-specific transcript levels after SCI. (A) The number of differentially expressed genes in the control, SCI-1d, SCI-3d, and SCI-7d groups. (B) A heatmap of selected significantly changed PAN-reactive, A1-specific, and A2-specific genes. (C–E) Fold changes in the levels of the representative PAN-reactive transcripts (Vim, Timp1, and GFAP) at each time point after SCI. (F–H) Fold changes in the levels of the representative A1-specific reactive transcripts (Serping1, Fkbp5, and Gbp2) at each time point after SCI. (I–K) Fold changes in the levels of the representative A2-specific transcripts (Emp1, S100a10, and Ptgs2) at each time point after SCI. N = 3 individuals per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. the sham control group.

FIGURE 3

Photobiomodulation modulated the transformation of A1/A2 astrocytes. (A) Representative images of immunofluorescence staining for GFAP (green) and C3 (red) or S100a10 (red) in the lesion area 150 μm from the epicenter in the SCI + vehicle and SCI + PBM groups at 7 and 14 dpi. Scale bar: 200 μm. (B) Western blot analysis and quantification of the expression levels of C3 and S100a10 in each group at 7 dpi (n = 3 individuals per group). (C) Representative immunofluorescence images of GFAP (green) and C3 (red) or S100a10 (red) in astrocytes in the control, control + PBM, A1, and A1 + PBM groups. Con, control; A1, A1 astrocytes. Scale bar: 200 mm. (D) Representative blots and quantification of the expression levels of C3 and S100a10 in each group. The experiments were independently repeated three times. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

Signaling pathways involved in A1/A2 astrocyte activation were regulated by PBM. (A) GO function analysis between the SCI-7d group and sham control group. The inflammatory response and immune system process were dramatically activated. (B) The histogram shows the –log10 (p-value) of the NF-κB pathway, PI3K–Akt pathway, JAK–STAT pathway, and Notch pathway (comparison between multiple groups vs. the sham control group), as determined by KEGG enrichment analysis. (C) Western blot analysis of the expression of proteins related to the NF-κB signaling pathway, JAK2–STAT3 signaling pathway, and PI3K–Akt signaling pathway in the sham control, SCI + vehicle, and SCI + PBM groups at 7 dpi. Quantification of the relative protein levels of pNF-κB, NF-κB, pJAK2, JAK2, pSTAT3, STAT3, pPI3K, PI3K, pAkt, and Akt. n = 3 individuals per group. **p < 0.01, ***p < 0.001, the SCI + vehicle group vs. the sham control group; ^##p < 0.01, the SCI + PBM group vs. the SCI + vehicle group. (D) Representative blots and quantification of the relative levels of pNF-κB, NF-κB, Notch1, pJAK2, JAK2, pSTAT3, STAT3, pPI3K, PI3K, pAkt, and Akt in the control, control + PBM, A1, and A1 + PBM groups. Con, control; A1, A1 astrocytes. The experiments were independently repeated three times. **p < 0.01, ***p < 0.001, ****p < 0.0001, the A1 group vs. the control group; ^##p < 0.01, ^###p < 0.001, the A1 + PBM group vs. the A1 group. (E) Representative blots and quantification of the relative levels of C3 and S100a10 in astrocytes treated with different inhibitors. The experiments were independently repeated three times. **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. the A1 group.

FIGURE 5

Basic fibroblast growth factor and TGF-β expression was upregulated by PBM and inhibited by A1 astrocyte activation. (A) The mRNA levels of proinflammatory factors and neurotrophic factors in each group at 3 dpi. n = 5 individuals per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, the SCI + vehicle group vs. the sham control group; ^#p < 0.05, ns, not significant, the SCI + PBM group vs. the SCI + vehicle group. (B,C) The levels of bFGF and TGF-β in the indicated medium were measured by ELISA. The experiments were independently repeated four times. *p < 0.05, **p < 0.01, ***p < 0.001. (D,F) The effect of different concentrations of bFGF and TGF-β on reactive astrocyte-related transcript levels in resting astrocytes. (E,G) The effects of different concentrations of bFGF and TGF-β on reactive astrocyte-related transcript levels in A1 astrocytes 24 h after induction. The data are shown as the fold change compared to the control group, as determined by RT-PCR. The experiments were independently repeated three times.

FIGURE 6

Photobiomodulation alleviated the neurotoxic effect of A1 astrocytes on DRG neurons. (A) DRG neurons treated with ACM collected 24 h after different treatments. Con, medium from control resting astrocytes; Con + PBM: medium from resting astrocytes treated with PBM; A1CM, medium from A1 astrocytes; PBM + A1CM, medium from A1 astrocytes treated with PBM. Representative immunofluorescence images of NeuN (red) and β III tubulin (green) in DRG neurons. Scale bar: 100 mm. (B,C) The length of axons in a single neuron and the number of neurons per field were analyzed. Five visual fields from each sample were randomly selected and analyzed. The experiments were independently repeated three times. *p < 0.01; ns, not significant.

FIGURE 7

Schematic diagram showing that PBM regulates the transformation of A1/A2 reactive astrocytes. An implantable fiber was embedded in the rat vertebral plate after SCI, and PBM improved motor recovery and promoted tissue repair. At 7 dpi, A1 astrocyte activation was suppressed, and A2 astrocyte activation was promoted after PBM treatment. The mechanism may be related to the ability of PBM to reduce the toxicity of A1 astrocytes to neurons by altering the activation of specific signaling pathways and factors associated with A1/A2 astrocyte activation.