CCR2 Regulates Referred Somatic Hyperalgesia by Mediating T-Type Ca2+ Channel Currents of Small-Diameter DRG Neurons in Gastric Ulcer Mice

Abstract

Background: Referred pain frequently co-exists with visceral pain. However, the exact mechanism governing referred somatic hyperalgesia remains elusive. Methods: By injecting 20% acetic acid into the stomach, we established a mouse model of gastric ulcer (GU). Hematoxylin and eosin (H&E) staining was used as the evaluation criterion for the gastric ulcer model. Evan's blue (EB) and von Frey tests detected the somatic sensitized area. The DRG neurons distributed among the spinal segments of the sensitized area were prepared for biochemical and electrophysiological experiments. The CCR2 antagonist was intraperitoneally (i.p.) injected into GU mice to test the effect of blocking CCR2 on somatic neurogenic inflammation. Results: GU not only instigated neurogenic plasma extravasation and referred somatic allodynia in the upper back regions spanning the T9 to T11 segments but also augmented the co-expression of T-type Ca²⁺ channels and CCR2 and led to the gating properties of T-type Ca²⁺ channel alteration in T9-T11 small-diameter DRG neurons. Moreover, the administration of the CCR2 antagonist inhibited the T-type Ca²⁺ channel activation, consequently mitigating neurogenic inflammation and referred somatic hyperalgesia. The application of the CCR2 agonist to normal T9-T11 small-diameter DRG neurons simulates the changes in the gating properties of T-type Ca²⁺ channel that occur in the GU group. Conclusions: Therefore, these findings indicate that CCR2 may function as a critical regulator in the generation of neurogenic inflammation and mechanical allodynia by modulating the gating properties of the T-type Ca²⁺ channels.

Keywords: Gastric ulcer; T-type Ca2+ channels; dorsal root ganglion; referred somatic hyperalgesia; the C-C motif chemokine receptor 2.

Figure 1

Pathological changes in the gastric ulcer (GU) mice induced by acetic acid. (A) Experimental process diagram. (B) Representative images of H&E staining of the stomach in control and GU mice. The right panels provide higher-magnification pictures of the boxed regions in the left panels [left: 10× (magnified), scale bar, 250 μm; right: 20× (magnified), scale bar, 100 μm] (n = 6 mice per group). (C,F) Representative images of EB plasma extravasation points scattered in the regions of operative incision and upper back following GU, as compared to the control. (D,G) Schematic representation of EB points in the skin from control and GU mice. (E,H) Schematics of merged EB points in the skin from the control and GU group. (I) The number of EB points in the dermatomes of the T2-L2 spinal segments. (J) Quantification of total EB points in two groups (n = 6 mice per group). (K) Withdrawal threshold to mechanical stimulation in the T9–T11 upper-back exudation points of each group (n = 6 mice per group). The red arrows indicate the locations of EB points, and the blue dots represent the EB points. Compared with the control group, **** p < 0.0001.

Figure 2

Both the expression and gating properties of the T-type channel in small-diameter DRG neurons are increased following acetic acid-induced GU. (A) Representative IHC images of DAPI (blue) and T-type Ca²⁺ channels (Ca_v3.2) (green) in DRGs of control and GU groups. The right panels provide higher-magnification pictures of the boxed regions in the left panels [left: 10× (magnified), scale bar, 250 μm; right: 40× (magnified), scale bar, 75 μm]. The numbers of Ca_v3.2-positive DRG neurons are shown in two groups (n = 12 DRGs from eight mice per group). Blue represents the cell nucleus, and green represents the T-type (Ca_v3.2)-positive neurons. (B) Representative 40× (magnified) images of recording from a small-diameter (<20 μm) and medium-diameter (20–30  μm)  DRG neurons in a whole-mount DRG preparation are recorded [scale bar, 10 μm]. The dashed red circles represent the morphology of the clamped cells. (C) Top: Representative traces of T-type channel activation curves from different diameter DRG neurons in two groups. Bottom: The voltage protocol used to activate the T-type channels. (D,E) An overview of the normalized (pA/pF) I_T-type density versus voltage relationship from DRG small-diameter (D) and medium-dimeter (E) neurons [two-way RM ANOVA with multiple comparisons tests: small-diameter DRG neurons, F _{(1, 11)} = 11.53, p = 0.0060; medium-diameter DRG neurons, F _{(1, 16)} = 0.1683, p = 0.6870]. (F–I) Boltzmann fits for normalized conductance, G/G_max, voltage relations for voltage-dependent activation (F,H), and inactivation (G,I) of small- and medium-diameter DRG neurons in two groups [two-way RM ANOVA with multiple comparisons tests: small-diameter DRG neurons, activation curves: F _{(1, 11)} = 6.172, p = 0.0303; inactivation curves: F _{(1, 22)} = 0.2834, p = 0.5998; medium-diameter DRG neurons, activation curves: F _{(1, 16)} = 0.01542, p = 0.9027; inactivation curves: F _{(1, 21)} = 1.001, p = 0.3284]. (Small-diameter DRG neurons: control, n = 5–12 cells from five mice, GU, n = 8–12 from five mice; medium-diameter DRG neurons: control, n = 10–12 cells from five mice, GU, n = 8–12 from four mice.). Compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Effects of inhibition of the chemokine CCR2 on I_T-type and referred somatic hyperalgesia in gastric ulcer mice. (A) Representative IHC images of DAPI (blue), T-type Ca²⁺ channels (green), and CCR2 (red) in DRGs of control and GU model. The right panels provide higher-magnification pictures of the boxed regions in the left panels [left: 10× (magnified), scale bar, 250 μm; right: 40× (magnified), scale bar, 75 μm]. The numbers of T-type (Ca_v3.2)⁺ and CCR2⁺ DRG neurons are shown in two groups (n = 12 DRGs from eight mice per group). Blue represents the cell nucleus, green represents the T-type (Ca_v3.2)-positive neurons and red represents the CCR2-positive neurons. (B) The schematic shows drugs and CCR2 antagonists added to the bath. (C) Representative 40× (magnified) image of recording from a small-diameter (<20  μm) DRG neurons in a whole-mount DRG preparation is recorded [scale bar, 10 μm]. The dashed red circles represent the morphology of the clamped cell. (D) Top: Representative traces of T-type channel activation curves from small-diameter DRG neurons treated with vehicle or RS102895 in GU mice. Bottom: The voltage protocol is used to activate the T-type channels. (E) An overview of the normalized (pA/pF) I_T-type density versus voltage relationship from DRG small-diameter neurons [two-way RM ANOVA with multiple comparisons tests: F _{(1, 13)} = 8.014, p = 0.0142]. (F) Boltzmann fits for normalized conductance (G/G_max) and voltage-dependent activation. (G) Boltzmann fits for voltage-dependent inactivation [two-way RM ANOVA withmultiple comparisons tests: activation curves, F _{(1, 13)} = 3.154, p = 0.1011; inactivation curves, F _{(1, 22)} = 0.006003, p = 0.9391] (vehicle, n = 8–12 cells from five mice; RS102895, n = 6–9 cells from four mice). (H) Quantification of total EB points in each group. (I) Withdrawal threshold to mechanical stimulation in the T9–T11 upper-back exudation points of each group (n = six mice per group). (J,K) Representative images on the left showing EB plasma extravasation points after i.p. of vehicle (J) or i.p. of RS102895; (K) 100 μL. Schematic representations of EB sites on the body surface of i.p. of vehicle (J) or i.p. of RS102895 (K) are on the right (n = six mice per group). The red arrows indicate the locations of EB points, and the blue dots represent the EB points. Compared with the vehicle group, * p < 0.05, *** p < 0.001, **** p < 0.0001.