Fig. 2

Astrocytic ApoE4 in spinal dorsal horn contributed to the increased pain sensitivity. (A) Schematic of experimental design, AAV administration, and transgenic mice information. (B) Western blots showing the expression of Flag and ApoE after the injection of AAV-GFAP-EGFP-Cre virus. n = 3 independent biological replicates per group. (C) Representative images of the spinal dorsal horn labeled with anti-ApoE (red) and anti-GFAP (gray) in LSL-hApoE3 and LSL-hApoE4 mice injected with AAV-GFAP-EGFP-Cre virus. Scale bar, 50 μm (left), 10 μm (right). (D) Mechanical thresholds before and after SNI in LSL-hApoE3 and LSL-hApoE4 mice following virus injection. n = 6 mice per group. Cohen’s d for LSL-hApoE3 vs. LSL-hApoE4 post-SNI: 1.74 (D7), 0.96 (D14), 1.48 (D28). (E, F) Quantification of foot swing (E) and foot cycle (F) during DigiGait™ analysis of LSL-hApoE3 mice and LSL-hApoE4 mice after viral injection, before and 14 days after the SNI. n = 6 mice per group. Cohen’s d = 2.26 for foot swing and 2.28 for foot cycle (LSL-hApoE4 SNI vs. LSL-hApoE4 SNI). (G) Experimental scheme of the fiber photometry analysis. (H) Heatmap of mechanical pain-evoked Ca²⁺ signals from LSL-hApoE3 and LSL-hApoE4 mice following injection of GFAP-Cre and GCaMP6f viruses. n = 10 mice per group. (I) Peri-event plot of GCaMP6f ΔF/F after pain stimulation. (J) Quantification of mean AUC value for LSL-hApoE3 and LSL-hApoE4 mice. n = 10 mice per group. All data are expressed as mean ± SEM. Statistical comparisons were conducted with two-way ANOVA followed by Bonferroni’s post hoc test (D); one-way ANOVA followed by Tukey’s post hoc test (E, F); and unpaired Student’s t-test (J). *P < 0.05, **P < 0.01, and ***P < 0.001