Chemokine Receptor CXCR3 in the Spinal Cord Contributes to Chronic Itch in Mice

Abstract

Recent studies have shown that the chemokine receptor CXCR3 and its ligand CXCL10 in the dorsal root ganglion mediate itch in experimental allergic contact dermatitis (ACD). CXCR3 in the spinal cord also contributes to the maintenance of neuropathic pain. However, whether spinal CXCR3 is involved in acute or chronic itch remains unclear. Here, we report that Cxcr3 ^-/- mice showed normal scratching in acute itch models but reduced scratching in chronic itch models of dry skin and ACD. In contrast, both formalin-induced acute pain and complete Freund's adjuvant-induced chronic inflammatory pain were reduced in Cxcr3 ^-/- mice. In addition, the expression of CXCR3 and CXCL10 was increased in the spinal cord in the dry skin model induced by acetone and diethyl ether followed by water (AEW). Intrathecal injection of a CXCR3 antagonist alleviated AEW-induced itch. Furthermore, touch-elicited itch (alloknesis) after compound 48/80 or AEW treatment was suppressed in Cxcr3 ^-/- mice. Finally, AEW-induced astrocyte activation was inhibited in Cxcr3 ^-/- mice. Taken together, these data suggest that spinal CXCR3 mediates chronic itch and alloknesis, and targeting CXCR3 may provide effective treatment for chronic pruritus.

Keywords: Alloknesis; CXCL10; CXCR3; Chronic itch; Dry skin; Spinal cord.

Fig. 1

CXCR3 is not required in models of acute itch in the neck and cheek. A Acute itch induced by intradermal injection of compound 48/80 (48/80, 100 μg) or chloroquine (CQ, 200 μg) into the neck skin was comparable in WT and Cxcr3 ^−/− mice. B, C Acute itch and pain, which were induced by intradermal injection of 48/80 (B) or CQ (C) into the cheek, induced similar wipes and scratches in WT and Cxcr3 ^−/− mice.

Fig. 2

CXCR3 is required for chronic itch in the dry skin and allergic contact dermatitis models. A AEW treatment induced chronic itch in WT mice, which was reduced in Cxcr3 ^−/− mice (*P < 0.05, two-way repeated measures ANOVA). B Area under the curve of (A) (*P < 0.05, Student’s t test). C Chronic itch induced by DCP was substantially reduced in Cxcr3 ^−/− mice (*P < 0.05 two-way repeated measures ANOVA). D Area under the curve of (C) (*P < 0.05, Student’s t test). E Chronic itch induced by DNFB was markedly reduced in Cxcr3 ^−/− mice (*P < 0.05, two-way repeated measures ANOVA). F Area under the curve of (E) (*P < 0.05, Student’s t test).

Fig. 3

CXCR3 is required for both formalin-induced and CFA-induced inflammatory pain. A Intraplantar injection of formalin-induced spontaneous pain was reduced in Cxcr3 ^−/− mice (*P < 0.05, two-way repeated measures ANOVA). B Spontaneous pain in the first phase (0–10 min) was comparable between WT and Cxcr3 ^−/− mice, but that in the second phase (10–45 min) was reduced in Cxcr3 ^−/− mice (*P < 0.05, Student’s t test). C Mechanical allodynia induced by intraplantar injection of CFA declined from day 3 to day 10 in Cxcr3 ^−/− mice (*P < 0.05, two-way repeated measures ANOVA followed by Bonferroni test). D CFA-induced heat hyperalgesia was greatly attenuated from day 1 to day 10 in Cxcr3 ^−/− mice (*P < 0.05, two-way repeated measures ANOVA followed by Bonferroni test).

Fig. 4

The expression of CXCR3 and CXCL10 is upregulated in the spinal cord after AEW treatment. A qPCR showed increased Cxcr3 and Cxcl10 mRNA 7 days after AEW (*P < 0.05, Student’s t test, n = 6–8 mice/group). B, C The intensity of CXCR3-IR (B) and CXCL10-IR (C) was also increased 7 days after AEW (*P < 0.05, student’s t test, n = 3–4 mice/group). D, E Representative images of CXCR3 immunostaining from control (D) and AEW-treated (E) mice. F, G Representative images of CXCL10 immunostaining from control (F) and AEW-treated (G) mice.

Fig. 5

Spinal CXCR3 is required for AEW-induced chronic itch. A Intrathecal injection of the CXCR3 antagonist NBI-74330 (20 μg) on day 7 of AEW treatment reduced scratching behavior (*P < 0.05, two-way repeated measures ANOVA followed by Bonferroni test). B The same dose of NBI-74330 did not affect motor function as assessed by the rota-rod test.

Fig. 6

CXCR3 is required for touch-evoked scratching (alloknesis) after compound 48/80 or AEW treatment. A Alloknesis, induced 30 min after compound 48/80 treatment, was reduced in Cxcr3 ^−/− mice (*P < 0.05, two-way repeated measures ANOVA). B The total alloknesis score was reduced in Cxcr3 ^−/− mice (*P < 0.05, Student’s t test). C Alloknesis, induced after AEW-induced dry skin, was reduced in Cxcr3 ^−/− mice (*P < 0.05, two-way repeated measures ANOVA). D The total alloknesis score was reduced in Cxcr3 ^−/− mice (*P < 0.05, Student’s t test).

Fig. 7

AEW-induced astrocyte activation is reduced in Cxcr3-deficient mice. A Immunostaining of GFAP shows activated astrocytes 7 days after AEW treatment in WT mice, but not in Cxcr3 ^−/− mice. B Quantitative analysis of GFAP-IR in WT and Cxcr3 ^−/− mice (*P < 0.05, Student’s t test, n = 3–4 mice/group).