Fig 2: Infusion of CXCL7 improved PVTGlu neuronal activity and induced pain hypersensitivity in control mice. A Schematic of the experimental procedure.B Image showing the site of the cannula implanted in the lateral ventricle of control mice. Scale bar, 500 μm.C and D Effects of infusion of CXCL7 in the lateral ventricle on the pain threshold in control mice treated with ACSF or CXCL7, using von Frey test (C) and Hargreaves tests (D). n = 5 mice per group, P= 0.0079 (C), P= 0.0007 (D).E Representative heatmaps of travel trajectory of control mice treated with ACSF or CXCL7 in the CPA test. F and G Summarized data of aversion index (F) and preference index (G) for (E), n = 7 mice per group. P< 0.0001 (F and G). (H) Image showing the site of the cannula implanted in the PVT of control mice. Scale bar, 500 μm.I and J Representative images (I) and summarized data (J) of CXCL7 on PVTGlu neurons in control mice treated with ACSF or CXCL7. n = 6 mice, with one slice from each mouse, P< 0.0001. Scale bar, 10 μm.K and L Representative images (K) and summarized data (L) of c-Fos in PVT from control mice treated with ACSF or CXCL7. n = 5 mice, with one slice from each mouse. P= 0.0061. Scale bar, 200 μm.M and N Sample traces (M) and summarized number of spikes (N) in PVTGlu neurons recorded from control mice treated with ACSF or CXCL7. n = 14 cells from 4 mice per group. P(time×column factor)= 0.0019, P(time)< 0.0001, P(column factor)= 0.001, F(1, 26)= 13.79.O and P Effects of the infusion of CXCL7 in PVTGlu neurons on the pain threshold using the von Frey test (O) and Hargreaves tests (P) of control mice treated with ACSF or CXCL7. n = 7 mice per group, P= 0.0002 (O), P= 0.0015 (P).Q Representative heatmaps of travel trajectory of control mice treated with ACSF or CXCL7 within PVT in the CPA test. (R and S) Summarized data of aversion index (R) and preference index (S) for (Q), n = 7 mice per group. P= 0.0003 (R and S). Statistical analysis was performed using Student’s t test in (C), (D), (F), (G), (J), (L), (O), (P), (R) and (S), and two-way ANOVA followed by Bonferroni’s post hoc correction in (N). **P< 0.01, ***P< 0.001, ****P< 0.0001

Fig 3: Increased inflammatory and oxidative stress levels in the PVT of DIO mice. A Quantification of CXCL7 in plasma from control or DIO mice. n = 3 mice per group, P= 0.0003.B and C Representative images (B) and summarized data (C) of the expression of CXCL7 in PVTGlu neurons from control and DIO mice. n = 5 mice per group, with one slice from each mouse. P< 0.0001. Scale bars, 10 μm.D-H Representative images (D) and quantification of IBA1+ cell numbers (E), intensity (F) and Imaris-based semiautomatic quantification of cell morphometry, including total branch length (G) and points (H) of IBA1+-microglia from control and DIO mice. n = 5 mice per group for quantification of cell number and intensity, with one slice from each mouse. P(number)= 0.0081, P(intensity)= 0.0122. n= 6 mice for the control group and n = 7 mice for the DIO group in the quantification of branch length and points, with one slice from each mouse. P(length)= 0.014, P(intensity)= 0.0001. Scale bars, 10 μm (overview) and 3 μm (inset and rendering).I and J Representative images (I) and summarized data (J) of GPX4 in PVTGlu neurons from control and DIO mice. n = 6 mice per group, with one slice from each mouse. P= 0.0012. Scale bars, 10 μm. Statistical analysis was performed using Student’s t test. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001

Fig 4: Mechanism of CXCL7-CXCR2 interaction in PVTGlu→BLAGlu neuronal circuit promotes the onset of obesity-related pain. In obese mice, elevated expression of the chemokine, CXCL7, results in the binding of CXCR2 receptors of PVTGlu neurons, thereby leading to increased activity in PVTGlu→BLAGlu neuronal circuit, which mediate obesity-related pain