Ependymal cells did not transform to astrocytes in our transgenic mice.

(A), construction strategies of transgenic mice. (B), ependymal cells both in fourth ventricle (4V) and lateral ventricle (LV) had been labeled by reporter gene EGFP (green). n = 6 biological repeats. (C), ependymal cells did not produce astrocytes in the lesion area. n = 3 biological repeats. (D-E), ependymal cells were observed to produce offspring of neurons (Tuj1+, red), Oligo2 (white) negative and S100A10 (white in D or red in E) positive cells in epicenter. n = 3 biological repeats. 4V=fourth ventricle, LV=lateral ventricle, CB= Cerebellum. Scale bars had been indicated in pictures.

Resident astrocytes in lumbar enlargement were selectively targeted and eliminated.

(A), the experiment flowchart. (B), the histogram and statistical results of GFAP fluorescence intensity. The location of three largest ascending tracts of lumbar enlargements, including (1) CU, GR; (2) LST, DLST, VST; (3) DSC, VSC, along with SDH and gray matter. CU=cuneate fasciculus, GR=gracile fasciculus, LST=lateral spinothalamic tract, DLST=dorsolateral spinothalamic tract, VST=ventral spinothalamic tract, DSC=dorsal spinocerebellar tract, VSC=ventral spinocerebellar tract, SDH=superficial dorsal horn. n = 3 biological repeats. Scale bar, 200μm. Values are the mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Student Newman–Keuls post hoc test. **P < 0.01, ***P < 0.001, Sham group vs. SCI Pain positive (+) group. (C), AAV2/5-GfaABC1D-Cre targeted astrocytes were mainly in dorsal side and reflected by tdTomato and GFAP double positive cells in SCI+AAV group, no tdTomato and GFAP double positive cells were found in SCI+AAV+DT group. n = 3 biological repeats. Scale bars had been indicated in pictures. (D), the efficiency of astrocytes labeling and elimination in dorsal side of spinal cord. Immunofluorescence analysis results of GFAP after selective astrocyte elimination. Values are the mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Student Newman–Keuls post hoc test. n = 3 biological repeats. TdTomato positive cells indicated AAV. 2/5-GfaABC1D-Cre targeted astrocytes. ***P < 0.001.

Selective astrocyte elimination in lumbar enlargement attenuated neuropathic pain.

Change of weight (A), BMS scores (B), mechanical allodynia (C-D), cold hyperalgesia (E) and thermal hyperalgesia (F) in mice after astrocyte elimination. Sham group, n=30; sham+AAV group, n=30; SCI+AAV group, n=36; SCI+AAV+DT group, n=36. Arrow, 500 ng DT injection was performed on 31, 32 and 34 dpi (days post-injury). Left, left hindlimbs; right, right hindlimbs. i.p., intraperitoneal injection. Values are the mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Student Newman–Keuls post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, SCI+AAV+DT group vs. SCI+AAV group.

Microglia in lumbar enlargement were activated after selective astrocyte elimination.

(A-B), images of immunofluorescent staining using GFAP (green), Tuj1 (red) and IBA1 (white) as characteristic markers of astrocyte, neuron and microglia, respectively. n = 3 biological repeats. Scale bar, 200μm. (C), the histogram and statistical results of GFAP, IBA1 and Tuj1 fluorescence intensity in lesion area. (D), the heatmap showed that the transcripts of pro-inflammatory and anti-inflammatory microglial marker genes. n = 3 biological repeats. (E-H), the histogram and statistical results of IBA1 fluorescence intensity in lumbar enlargement. CU=cuneate fasciculus, GR=gracile fasciculus, LST=lateral spinothalamic tract, DLST=dorsolateral spinothalamic tract, VST=ventral spinothalamic tract, DSC=dorsal spinocerebellar tract, VSC=ventral spinocerebellar tract, SDH=superficial dorsal horn. Values are the mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Student Newman–Keuls post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, SCI+AAV+DT group vs. SCI+AAV group.

Selective astrocyte elimination activated Type I IFNs signal.

(A), the heatmap showed the DEGs between SCI+AAV+DT group and SCI+AAV group. (B), PPI networks analysis showed the interaction of the DEGs between SCI+AAV+DT group and SCI+AAV group. (C), GO enrichment analysis revealed the dominant biological process of DEGs between SCI+AAV+DT group and SCI+AAV group. (D), KEGG pathway showed dysregulated signaling pathways between SCI+AAV+DT group and SCI+AAV group. (E-F), GSEA analysis showed the dysregulated signaling pathways between SCI+AAV+DT group and SCI+AAV group. (G), the heatmap showed that the critical genes expression involved in type I IFNs signal. n = 3 biological repeats.

The type I IFNs signal was activated in microglia after selective resident astrocyte elimination.

(A-B), RT-PCR identified the effects of selective astrocyte elimination and DT injection alone on mRNA expression of type I IFNs signal genes. n = 3 biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001. (C-D), images of immunofluorescent staining and statistical results using IRF7, ISG15 and STAT1 as critical proteins of type I IFNs signal and microglia marker IBA1. Scale bar, 200μm. n = 3 biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001, SCI+AAV+DT group vs. SCI+AAV group. (E), images of immunofluorescent staining using IRF7, ISG15 and IBA1. Scale bar, 200μm. n = 3 biological repeats. (F-G), the mRNA expression of pro- and anti-inflammatory microglial markers and type I IFNs signal critical genes after 0.2 μg/mL LPS stimulation. n = 3 biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001, Control group vs. LPS 0.2μg group. All values are the mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Student Newman–Keuls post hoc test.

Interferon agonist (Ploy:IC) an STING agonist (ADU-S100 and DMXAA) significantly activated the type I IFNs signal in microglia.

A-F, the mRNA expression levels of IRF7, IRF7, IFNb, STAT2, ISG15, STAT1, and IRF9 after stimulating with different concentrations of Ploy:IC, ADU-S100 and DMXAA. n = 3 biological repeats. *P < 0.05, **P < 0.01, ***P < 0.001, treatment group vs. control group. All values are the mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Student Newman–Keuls post hoc test.

The potential schematic diagram of selective resident astrocytes elimination attenuated neuropathic pain after SCI.

(A), astrocytes in lumbar enlargement were targeted and selectively eliminated through transgenic mice injected with an adeno-associated virus vector (AAV2/5-GfaABC1D-Cre) and diphtheria toxin. Selective astrocyte elimination in lumbar enlargement could attenuate neuropathic pain after SCI, which were associated with type I IFNs signal and microglia activation. (B), the production of type I IFNs. Type I IFNs production is mainly caused by the contact of innate immune cells (mainly macrophages, microglia and astrocyte in CNS) surface or internal receptors (RIG-I receptor IFIH1/MDA5, cGAS, etc.) with virus specific antigenic substances (DNA, RNA), then through intracellular signal molecule transmission (STING, TBK, IKK, etc.), and finally activate the transcription factor IRF3/7 to promote the expression of type I IFNs, including IFN-α and IFN-β. (C), the signal transduction of type I IFNs. Type I IFNs bind to the same two membrane spanning polypeptide chains type I IFNs receptor 1/2 (IFNAR1/2) (Borden et al., 2007; Owens et al., 2014) and lead to cross phosphorylation and activation of Tyk2/JaK1. Activation of Tyk2 and JAK1 phosphorylate STAT1/2 to form a heterodimer, which then translocate to the nucleus and associate with IRF9 to further form the heterotrimeric transcription factor complex IFN-stimulated gene factor-3 (ISGF3). Finally, ISGF3 translocate to the nucleus and bind to specific IFN-response elements (ISREs) to control the expression of IFNs-stimulated genes (ISGs) (Rothhammer et al., 2016), including ISG15, MX1, OAS1, OAS2, IFIT1, ZBP1.