CXCL12-mediated monocyte transmigration into brain perivascular space leads to neuroinflammation and memory deficit in neuropathic pain

Abstract

Emerging clinical and experimental evidence demonstrates that neuroinflammation plays an important role in cognitive impairment associated with neuropathic pain. However, how peripheral nerve challenge induces remote inflammation in the brain remains largely unknown. Methods: The circulating leukocytes and plasma C-X-C motif chemokine 12 (CXCL12) and brain perivascular macrophages (PVMs) were analyzed by flow cytometry, Western blotting, ELISA, and immunostaining in spared nerve injury (SNI) mice. The memory function was evaluated with a novel object recognition test (NORT) in mice and with Montreal Cognitive Assessment (MoCA) in chronic pain patients. Results: The classical monocytes and CXCL12 in the blood, PVMs in the perivascular space, and gliosis in the brain, particularly in the hippocampus, were persistently increased following SNI in mice. Using the transgenic CCR2^RFP/+ and CX3CR1^GFP/+ mice, we discovered that at least some of the PVMs were recruited from circulating monocytes. The SNI-induced increase in hippocampal PVMs, gliosis, and memory decline were substantially prevented by either depleting circulating monocytes via intravenous injection of clodronate liposomes or blockade of CXCL12-CXCR4 signaling. On the contrary, intravenous injection of CXCL12 at a pathological concentration in naïve mice mimicked SNI effects. Significantly, we found that circulating monocytes and plasma CXCL12 were elevated in chronic pain patients, and both of them were closely correlated with memory decline. Conclusion: CXCL12-mediated monocyte recruitment into the perivascular space is critical for neuroinflammation and the resultant cognitive impairment in neuropathic pain.

Keywords: CXCL12; chronic pain; hippocampus perivascular macrophage; memory deficit; monocyte transmigration; neuroinflammation.

Figure 1

SNI causes a persistent increase in circulating classical monocytes. (A) Representative flow cytometry profiles of different subtypes of leukocytes gated by CD45⁺ in sham (Sh) and SNI mice. The inserted number shows the percentages of the classical monocytes (CMo: CD45⁺CD11b⁺L6C^high, red), CD45⁺CD11b⁺L6C^low cells (M, blue), granulocytes (G: CD45⁺CD11b⁺L6C^med, purple) and lymphocytes (L: CD45⁺CD11b^-, golden) in all CD45⁺ leukocytes. Sh: sham group, S1d, S3d, S9d, S14d, S21d show the groups harvested at different days after SNI. (B, C) Percentages of blood classical monocytes (CMo), CD45⁺CD11b⁺L6C^low cells (M), granulocytes (G) and lymphocytes (L) in all leukocytes (CD45⁺) from different groups. n = 10 for naïve and S9d groups. In sham mice, the experiments were performed at three time points (1 d, 3 d, 9 d after sham operation, 3 mice at each time point). As no difference was detected, the data from sham mice were used together; n = 4 in other groups. (D-F) Changes in nonclassical monocytes (NCMo: CD45⁺CD11b⁺Ly6C^lowCD49b^-, C), NK cells (CD45⁺CD3^-CD49b⁺, D), and neutrophils (CD45⁺SSC^medLy6G⁺, F) in sham and SNI groups (n = 3-5 mice/group). *P < 0.05, **P < 0.01, ***P < 0.001, and n.s.: not significant, vs. sham group, two-way ANOVA with Bonferroni's post hoc test used for B and C, one-way ANOVA with Bonferroni's post hoc test used for D-F.

Figure 2

SNI increases perivascular macrophages (PVMs) in the bilateral hippocampi. (A) Representative micrographs of CD3 (T lymphocytes marker), Ly6G (neutrophils marker) or CD49b (NK cells marker) staining of hippocampal sections from sham (Sh) or SNI 9 d groups and spleen sections (positive controls). Scale bar: 100 µm. (B) Expression of CD68 and P2Y12 in the hippocampal sulcus on day 9 after sham or SNI surgery. P2Y12⁺ cells with weaker CD68 staining are microglia (yellow) and with stronger CD68 staining (CD68^high) are PVMs located predominantly in perivascular spaces (green). Scale bar: 50 µm. (C, D) Numbers of PVMs (CD68^high, green) and the relative integrated density (IntDen) of CD68^high or PECAM-1 (magenta) in the hippocampus with time after SNI compared to the sham group. Arrows indicate monocytes in the process of migrating into the perivascular space. Scale bars, 100 µm and 25 µm (bottom). n = 3 mice/group, 3-4 slices/mice. (E) Triple-staining confocal merge image (left) and multiple plane images (right) of the CD68^high monocyte and endothelium (marked by PECAM-1) at 1 µm intervals showing monocyte migration across the endothelium into the perivascular space. Scale bar = 5 µm. (F, G) Changes of CD68^high cells and PECAM-1 in the bilateral hippocampi in sham and SNI 3 d groups (n = 3 mice/group, 3-4 images/mice). I: ipsilateral hippocampus, C: contralateral hippocampus. Scale bar: 250 µm. * P < 0.05, **P < 0.01, ***P < 0.001 vs. sham group, one-way ANOVA with Bonferroni's post hoc test.

Figure 3

Circulating inflammatory monocytes contribute to increased PVMs in the hippocampus in neuropathy. (A, B) CCR2^RFP/+ monocytes (red) in sham (Sh) and SNI 1d, 3d, 9d groups (n = 3-4 slices/mice, 3 mice/group). The insets in A are magnified from the white dotted boxes. (C) Triple-staining confocal merged image showing the relationship of a CCR2^RFP cell (red) with the endothelium (marked by PECAM-1, white), microglia (marked by CX3CR1^GFP, green and P2Y12, white), astrocytes (GFAP, white) and nucleus (DAPI, blue). Scale bars: 200 µm (A) and 20 µm (C). (D) Diagram showing the experimental procedure. Vehicle (Vehi) or clodronate liposomes (CLL, 15 mL/kg, i.v.) were injected on 1 d before and 3 d after sham or SNI surgery. The efficacy of CLL on monocytes was verified at 18 h after first injection of CLL by blood flow cytometry (FC) and brain tissue immunofluorescence (IF). The short-term memory (STM) index analyzed with the novel object recognition test (NORT) and IF was performed 9 d after surgery. (E-I) Percentages of different circulating leukocyte subpopulations at 1 d after injection of vehicle or clodronate (n = 3 for each group). (J, K) PVM (CD68^high) numbers around the hippocampal sulcus from different groups (n = 3-4 slices/mice, 3 mice/group). The insets were magnified from the white dotted boxes. Scale bars: 100 µm in F, 20 µm in insets. **P < 0.01, ***P < 0.001 vs. vehicle or sham group, ^###P < 0.001 vs. vehicle SNI group, one-way ANOVA with Bonferroni's post hoc test (B, K), two-way ANOVA with Bonferroni's post hoc test (F, G), and two-tailed Student's t-test (H, I).

Figure 4

SNI causes progressive gliosis in many brain regions, especially in bilateral hippocampi. (A, B) Temporal change of the morphology, normalized integrated density (IntDen) of CD11b and GFAP, and numbers in microglia and astrocytes in bilateral hippocampi at 1, 3, and 9 d after SNI. (C-E) Fluorescent IntDen of CD11b and GFAP in different brain regions as indicated in sham and SNI groups at 9 d after surgery. CD11b- and GFAP- immunostaining micrographs in both groups are from the same section. The white line boxed areas in C are magnified in A to show the morphological changes of glial cells. The images in D are magnified from dotted boxes in C. RawIntDen analysis of CD11b and GFAP staining was used for statistics in E. Hip: hippocampus, S1: primary somatosensory cortex, RSGc: retrosplenial granular cortex, Thal: Thalamus, Hypo: hypothalamus, Amy: amygdala. Scale bars: 100 µm (A), 500 µm (C) and 25 µm (D). n = 3 mice/group, 3-4 images/mice, *P < 0.05, **P < 0.001, ***P < 0.001 vs. sham group, two-way ANOVA with Bonferroni's post hoc test.

Figure 5

Depletion of circulating monocytes abolishes memory deficit and gliosis induced by SNI (A) Recognition index accessed by NORT in mice in the sham group (Sh) and in groups 9 d after SNI with clodronate or vehicle (Vehi) injection are shown (n = 7 in vehicle and CLL sham groups, n = 8 in other groups). (B, C) Effect of clodronate on PECAM-1 expression and activation of microglia (CD11b) and astrocytes (GFAP) at 9 d after SNI. Scale bar = 100 µm. n = 3 mice/group, 3-4 images/mice, **P < 0.01, ***P < 0.001 vs. vehicle sham group, ^##P < 0.01, ^###P < 0.001 vs. vehicle SNI group, one-way ANOVA with Bonferroni's post hoc test (A) and two-way ANOVA with Bonferroni's post hoc test (C).

Figure 6

SNI produces persistent elevation of CXCL12 in plasma and hippocampal perivascular spaces. (A) Cytokine array results showing changes in plasma cytokines and chemokines in sham (Sh) and in 3 d, 9 d after SNI groups. There was no difference in the positive control protein levels (white boxes) among the three groups. (B, C) Plasma CXCL12 expression in different groups of female (F) and male (M) mice. n = 3 mice/group. (D) ELISA results showing changes in plasma CXCL12 concentrations with time after SNI, compared to sham mice (n = 3-6 mice/group). (E) SNI induced upregulation of CXCL12 in bilateral hippocampi (n = 4-6 mice/group). I: ipsilateral hippocampus, C: contralateral hippocampus. (F) CXCL12 mRNA in bilateral hippocampi at different time points after SNI (n = 7-12 mice/group). (G, H) Expression of CXCL12 around the hippocampal sulcus but not in the CA1 area was upregulated from 1 d to 9 d after SNI compared to sham mice (n = 3-4 slices, 3 mice/group). The white dotted boxes in top images are magnified below. Scale bars: 100 µm (top) and 25 µm (below). n.s.: no significant difference, *P < 0.05, **P < 0.01, ***P < 0.001 vs. sham group, two-way ANOVA with Bonferroni's post-hoc test used for C and one-way ANOVA with Bonferroni's post-hoc test used for D-G. (I) Multiple sets of images show the colocalization of CXCL12 (red) with the PVM marker CD68 (green), endothelial cell marker PECAM-1 (green), astrocyte marker GFAP (green) or pericyte maker CD13 (green) in sham and SNI 3d groups. Red arrows showing the images in I are magnified from the red boxes in H. Scale bar = 25 µm.

Figure 7

Blocking the CXCL12-CXCR4 pathway reverses SNI-induced cognitive impairment, PVMs increase, and gliosis in the hippocampus. (A) Experimental protocol showing that anti-CXCL12 neutralizing antibody (20 ng/200 µL, i.v.) or CXCR4 antagonist AMD3100 (200 µg/mL, 1 mg/kg, i.p.) or vehicle (Vehi) was applied 30 min before and daily after sham (Sh) or SNI for 9 successive days. On day 9 after the injection, memory function was analyzed with NORT, and mice were perfused for IF and FC. (B) Anti-CXCL12 neutralizing antibody or AMD3100 injection prevented SNI-induced decline in the recognition index but had no effect in sham mice (n =5-12 mice/group). (C-F) Number of PVMs (CD68^high) and the IntDen of CD68^high, PECAM-1, CD11b, and GFAP in the hippocampus in indicated groups. Scale bar = 100 µm. n = 3 mice/group, 3-4 slices/mice. *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle sham group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. vehicle SNI group, one-way ANOVA with Bonferroni's posthoc test.

Figure 8

Intravenous injection of CXCL12 induces memory deficit and elevates circulating monocytes and plasma CXCL12. (A) CXCL12 (1.0 and 2.5 ng/mL, 200 µL) or same volume of vehicle (Vehi) was injected for successive 9 days via the tail vein of naïve mice, and memory function was analyzed by NORT. The blood and brain tissue were harvested for ELISA, FC, and IF after the behavioral test. (B) Effects of injection of vehicle or different dosages of CXCL12 on STM index are shown (6-9 mice/group). (C) ELISA results revealed that CXCL12 injection at 2.5 ng/mL but not 1.0 ng/mL for 9 days elevated plasma CXCL12 (3-4 mice/group). The plasma CXCL12 was measured 24 h after the last injection of CXCL12. (D-J) Effects of iv injection of CXCL12 at 1.0 and 2.5 ng/mL on circulating leukocyte subpopulations. n = 5-6 mice/group. **P < 0.01, ***P < 0.001 vs. vehicle group, one-way ANOVA with Bonferroni's post hoc test (B, C, H, J) and two-way ANOVA with Bonferroni's post hoc test (E, F).

Figure 9

CXCL12 intravenous injection increases perivascular macrophages and PECAM-1 expression and gliosis in the hippocampus. (A, B) Number of PVMs (CD68^high) and immune fluorescence intensities of CD68^high in the hippocampal perivascular space in vehicle and CXCL12 groups (n = 3 mice, 3-4 slices /mice). (C, D) Effect of vehicle or CXCL12 injection on the CD11b, GFAP, PECAM-1, and CXCL12 expression. n = 3-4 slices/mice, 3 mice/group. Scale bar = 100 µm. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. vehicle group, one-way ANOVA with Bonferroni's post-hoc test.

Figure 10

Circulating monocytes and plasma CXCL12 are increased in patients with chronic pain and are correlated with cognitive decline. (A) Memory function accessed by MoCA in patients with chronic neuropathic pain (Chronic pain, n = 30) was lower than healthy controls (Contr, n = 40). (B-G) Percentages of monocytes (B), granulocytes (C), neutrophils (D), lymphocytes (E), eosinophils (F), and basophils (G) in various groups were determined by routine blood analysis. (H) Concentration of plasma CXCL12 in healthy control subjects and chronic pain patients are shown. ^**P < 0.01, ^***P < 0.001 vs. healthy control group, two-tailed Student's t-test. (I) Scatterplots showing that the percentage of circulating monocytes (Spearman rank correlation, R² = 0.1022, P = 0.007) and plasma CXCL12 concentration (R² = 0.2323, P < 0.0001) was negatively correlated with MoCA scores in healthy controls and patients with chronic pain.