Fig 2: Representative images of the clinical MC group and control group with (A) HE staining (scale bar = 1.25 mm), (B) immunofluorescence of GFAP and Iba-1 (scale bar = 200 µm), and (C) immunofluorescence of Tuj-1 and NeuN (scale bar = 200 µm); images in the lower right corner are magnified from the boxed area of the corresponding images, with target protein indicated by coloured arrows. (d)–(f) show quantitative measurement of GFAP/Iba-1/NeuN-positive cells (means ± SD of a single slice, * indicates P < 0.0.5). MCs: Modic changes.

Fig 3: Representative images of cocultured AFCs and hippocampal neuron in bright-field, Tuj-1/NeuN/DAPI/merged immunofluorescence, and neurite length measurement (20x, A–D, scale bar = 100 µm, the white arrows indicate where the neurite formed between neurons and AFCs). (e) shows the quantitative measurement of neurite length (means ± SD, *P < 0.0.5). (f) Nerve-related gene expression in hippocampal neuron-AFC coculture environment (means ± SD, * indicates P < 0.0.5). AFCs: annulus fibrosus cells; C. a: Cutibacterium acnes.

Fig 4: Representative images of the SD rat MC group and control group with immunofluorescence of GFAP/iba-1 (A and C, scale bar = 100 µm) and Tuj-1/NeuN (B and D, scale bar = 100 µm); images in the lower right corner are magnified from the boxed area of the corresponding images, and white arrows indicate the GFAP/Iba-1/NeuN-positive neurons. (E) and (F) show the location of GFAP/Iba-1/NeuN-positive neurons in the IVD (cells with positive immunofluorescence were marked as bright yellow dots (scale bar = 1 mm)); (g)–(i) show quantitative measurement of GFAP/Iba-1/NeuN-positive cells (means ± SD of a single motion segment, * indicates P < 0.0.5). MCs: Modic changes.

Fig 5: Behavioral effects of Gq pathway activation in astrocytes of hippocampal CA1 region. (A) Bilateral hippocampal CA1 regions were injected with rAAV-GfaABC1D-hM3D(Gq)-mCherry (n = 20) or rAAV-GfaABC1D-mCherry (n = 10) 21 days before surgery. (B) hM3D(Gq) was specifically expressed in astrocytes. GFAP was used to label astrocytes, NEUN to label neurons, Iba-1 to label microglia, mCherry to label hM3D(Gq), and DAPI to label nuclei, respectively. Red arrows indicate double-staining positivity, while white arrows indicate mCherry single-staining positivity. Scale bar = 25 µm. (C) The ratio of GFAP and mCherry double-positive cells to GFAP-positive cells is the epistasis rate. (D) The ratio of GFAP and mCherry double-positive cells to mCherry-positive cells is the specificity rate. (E) Locomotor activity was measured on day 7 after tibial fracture by open-field assay after 30 min of intraperitoneal injection of CNO or saline. (F–H) Quantification of total distance travelled, speed of movement, and time spent moving in specific areas in 3 groups of mice. (I) Novel object recognition tests were performed on the 7th and 8th day after tibial fracture. (J,K) Quantitative analysis of discrimination index and percentage of new object recognition in the 3 groups of mice. (L,M) Three cycles of training were performed on postoperative day 9, and the percentage of freezing time was assessed on day 10 in the Context test and the Cue test. Data were expressed as mean ± SD and analyzed by ANOVA test with a Tukey post hoc test. The p-values are shown in the picture.