Glial Glutamate Transporter Modulation Prevents Development of Complete Freund's Adjuvant-Induced Hyperalgesia and Allodynia in Mice

Abstract

Glial glutamate transporter (GLT-1) modulation in the hippocampus and anterior cingulate cortex (ACC) is critically involved in nociceptive pain. The objective of the study was to investigate the effects of 3-[[(2-methylphenyl) methyl] thio]-6-(2-pyridinyl)-pyridazine (LDN-212320), a GLT-1 activator, against microglial activation induced by complete Freund's adjuvant (CFA) in a mouse model of inflammatory pain. Furthermore, the effects of LDN-212320 on the protein expression of glial markers, such as ionized calcium-binding adaptor molecule 1 (Iba1), cluster of differentiation molecule 11b (CD11b), mitogen-activated protein kinases (p38), astroglial GLT-1, and connexin 43 (CX43), were measured in the hippocampus and ACC following CFA injection using the Western blot analysis and immunofluorescence assay. The effects of LDN-212320 on the pro-inflammatory cytokine interleukin-1β (IL-1β) in the hippocampus and ACC were also assessed using an enzyme-linked immunosorbent assay. Pretreatment with LDN-212320 (20 mg/kg) significantly reduced the CFA-induced tactile allodynia and thermal hyperalgesia. The anti-hyperalgesic and anti-allodynic effects of LDN-212320 were reversed by the GLT-1 antagonist DHK (10 mg/kg). Pretreatment with LDN-212320 significantly reduced CFA-induced microglial Iba1, CD11b, and p38 expression in the hippocampus and ACC. LDN-212320 markedly modulated astroglial GLT-1, CX43, and, IL-1β expression in the hippocampus and ACC. Overall, these results suggest that LDN-212320 prevents CFA-induced allodynia and hyperalgesia by upregulating astroglial GLT-1 and CX43 expression and decreasing microglial activation in the hippocampus and ACC. Therefore, LDN-212320 could be developed as a novel therapeutic drug candidate for chronic inflammatory pain.

Keywords: LDN-212320; astroglia; chronic pain; glutamate transporter; mice; microglia.

Figure 1

The diagram depicts the timeline of the experiments. CFA, complete Freund’s adjuvant; LDN, 3-[[(2-methylphenyl) methyl] thio]-6-(2-pyridinyl)-pyridazine; DHK, dihydrokainic acid.

Figure 2

Effects of LDN-212320 or gabapentin on the 50% paw withdrawal threshold (50% PWT) during CFA-induced chronic inflammatory pain: (A) the 50% PWT was assessed using von Frey filaments 3 h, 1 day, 3 days, and 7 days after CFA (1 mg/mL, i.pl) administration; (C) effects of LDN-212320 or gabapentin on latency time on hotplate test (51 ± 0.5 °C) during CFA-induced chronic inflammatory pain, where the latency time on the hotplate was measured 3 h, 1 day, 3 days, and 7 days after CFA (1 mg/mL, i.pl) administration (B,D). The area under the curve (AUC) of the time course of the different behavioral tests was calculated by the trapezoidal rule. Control animals received an equal volume of vehicle (i.pl). Data are expressed as means ± S.E.M. (n = 4–6 mice/group). Note: *** p < 0.001, **** p < 0.0001, control (20 µL of vehicle, i.pl) vs. CFA/vehicle (1 mg/mL), ^$ p < 0.05, ^$$$ p < 0.001, or ^$$$$ p < 0.0001, CFA/vehicle (1 mg/mL) vs. CFA/LDN-212320 (20 mg/kg) and ^### p < 0.001 or ^#### p < 0.0001, CFA/vehicle (1 mg/mL) vs. CFA/gabapentin (100 mg/kg). AUC *** p < 0.001, **** p < 0.0001.

Figure 3

Effects of the astroglial GLT-1 antagonist, dihydrokainic acid (DHK), on LDN-212320-induced anti-allodynic and anti-hyperalgesic effects during CFA-induced chronic inflammatory pain: (A) the 50% paw withdrawal threshold (50% PWT) was assessed using von Frey filaments 3 h, 1 day, 3 days, and 7 days after CFA (1 mg/mL, i.pl) administration; (C) latency times on a hotplate test (51 ± 0.5 °C) were measured 3 h, 1 day, 3 days, and 7 days after CFA (1 mg/mL, i.pl) administration, while DHK (10 mg/kg) was injected 30 min before all behavioral experiments (B,D). The area under the curve (AUC) of the time course of the different behavioral tests was calculated using the trapezoidal rule. Control animals received an equal volume of vehicle (i.pl). Data are expressed as means ± S.E.M. (n = 4–6 mice/group). Note: **** p < 0.0001, control (20 µL of vehicle, i.pl) vs. CFA/vehicle (1 mg/mL), ^$$$ p < 0.001 or ^$$$$ p < 0.0001, CFA/vehicle (1 mg/mL) vs. CFA/LDN-212320 (20 mg/kg) and ^### p < 0.001 or ^#### p < 0.0001, CFA/LDN-212320 (20 mg/kg) vs. CFA/LDN-212320 (20 mg/kg) plus DHK (10 mg/kg). AUC ** p < 0.01, *** p < 0.001, or **** p < 0.0001.

Figure 4

Effects of LDN-212320 on microglial Iba1 expression in the hippocampus (A) and anterior cingulate cortex (ACC) (B) on CFA-induced chronic inflammatory pain. Representative Western blot images for Iba1 expression from the hippocampus (A) and ACC (B) (top panel). CFA (i.pl) injection increased the Iba1 expression in the hippocampus (A) and ACC (B) compared to the control. Treatment of LDN-212320 (10 or 20 mg/kg) reversed the CFA-induced increased Iba1 expression in the hippocampus (A) or LDN-212320 (20 mg/kg) in the ACC (B). Control animals received an equal volume of vehicle (i.pl). Data are expressed as means ± S.E.M. (n = 4–6 mice/group). Note: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 5

Effects of LDN-212320 on microglial CD11b expression in the hippocampus (A) and anterior cingulate cortex (ACC) (B) during CFA-induced chronic inflammatory pain. Representative Western blot images for CD11b expression from the hippocampus (A) and ACC (B) (top panel). CFA (i.pl) injection increased CD11b expression in the hippocampus (A) and ACC (B) compared to the control. Treatment of LDN-212320 (10 or 20 mg/kg) reversed the CFA-induced increased CD11b expression in the hippocampus (A) or LDN-212320 (20 mg/kg) in the ACC (B). Control animals received an equal volume of vehicle (i.pl). Data are expressed as means ± S.E.M. (n = 4–6 mice/group). Note: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 6

Effects of LDN-212320 on microglial p38 immunoreactivity in the CA1 and dentate gyrus (DG) of the hippocampus and anterior cingulate cortex (ACC) in CFA (1 mg/mL. i.pl)-induced chronic inflammatory pain. CFA (i.pl) injection increased the p38 immunoreactivity in the CA1 (A), DG (B), and ACC (C) compared to the control. LDN-212320 (20 mg/kg) treatment decreased the CFA-induced increased p38 immunoreactivity in the CA1 (A), DG (B), and ACC (C). Magnification 20×, scale bars, 50 µm. Representative images of immunofluorescence in (A–C) for the CA1 and DG of the hippocampus and ACC, respectively. Control animals received an equal volume of vehicle (i.pl). Data are expressed as means ± S.E.M. (n = 4–6 mice/group). Note: *** p < 0.001.

Figure 6

Effects of LDN-212320 on microglial p38 immunoreactivity in the CA1 and dentate gyrus (DG) of the hippocampus and anterior cingulate cortex (ACC) in CFA (1 mg/mL. i.pl)-induced chronic inflammatory pain. CFA (i.pl) injection increased the p38 immunoreactivity in the CA1 (A), DG (B), and ACC (C) compared to the control. LDN-212320 (20 mg/kg) treatment decreased the CFA-induced increased p38 immunoreactivity in the CA1 (A), DG (B), and ACC (C). Magnification 20×, scale bars, 50 µm. Representative images of immunofluorescence in (A–C) for the CA1 and DG of the hippocampus and ACC, respectively. Control animals received an equal volume of vehicle (i.pl). Data are expressed as means ± S.E.M. (n = 4–6 mice/group). Note: *** p < 0.001.

Figure 7

Effects of LDN-212320 on astroglial GLT-1 expression in the hippocampus (A) and anterior cingulate cortex (ACC) (B) during CFA-induced chronic inflammatory pain. Representative Western blot images for GLT-1 expression from the hippocampus (A) and ACC (B) (top panel). CFA (i.pl) injection decreased the GLT-1 expression in the hippocampus (A) and ACC (B) compared to the control. Treatment with LDN-212320 (20 mg/kg) reversed the CFA-induced decreased GLT-1 expression in the hippocampus (A) and ACC (B). Control animals received an equal volume of vehicle (i.pl). Data are expressed as means ± S.E.M. (n = 4–6 mice/group). Note: **** p < 0.0001.

Figure 8

Effects of LDN-212320 on astroglial CX43 immunoreactivity in the CA1 and dentate gyrus (DG) of the hippocampus and anterior cingulate cortex (ACC) in CFA (1 mg/mL. i.pl)-induced chronic inflammatory pain. CFA (i.pl) injection decreased the CX43 immunoreactivity in the CA1 (A), DG (B), and ACC (C) compared to the control. LDN-212320 (20 mg/kg) treatment increased the CFA-induced decreased CX43 immunoreactivity in the CA1 (A), DG (B), and ACC (C). Magnification 20×, scale bars, 50 μm. Representative images of immunofluorescence in (A–C) for the CA1 and DG of the hippocampus and ACC, respectively. Control animals received an equal volume of vehicle (i.pl). Data are expressed as means ± S.E.M. (n = 4–6 mice/group). Note: *** p < 0.001.

Figure 8

Effects of LDN-212320 on astroglial CX43 immunoreactivity in the CA1 and dentate gyrus (DG) of the hippocampus and anterior cingulate cortex (ACC) in CFA (1 mg/mL. i.pl)-induced chronic inflammatory pain. CFA (i.pl) injection decreased the CX43 immunoreactivity in the CA1 (A), DG (B), and ACC (C) compared to the control. LDN-212320 (20 mg/kg) treatment increased the CFA-induced decreased CX43 immunoreactivity in the CA1 (A), DG (B), and ACC (C). Magnification 20×, scale bars, 50 μm. Representative images of immunofluorescence in (A–C) for the CA1 and DG of the hippocampus and ACC, respectively. Control animals received an equal volume of vehicle (i.pl). Data are expressed as means ± S.E.M. (n = 4–6 mice/group). Note: *** p < 0.001.

Figure 9

Effects of LDN-212320 on IL-1β levels in the hippocampus and anterior cingulate cortex (ACC). CFA (i.pl) injection increased the IL-1β levels in the hippocampus (A) and ACC (B) compared to the control. Treatment with LDN-212320 (20 mg/kg) decreased the IL-1β level in the hippocampus (A) or (10 or 20 mg/kg) ACC (B). Control animals received an equal volume of vehicle (i.pl). Data are expressed as means ± S.E.M. (n = 4–6 mice/group). Note: ** p < 0.01, *** p < 0.001.