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. 2023 Mar 29;13(4):579.
doi: 10.3390/brainsci13040579.

Pharmacological Modulation of the MIP-1 Family and Their Receptors Reduces Neuropathic Pain Symptoms and Influences Morphine Analgesia: Evidence from a Mouse Model

Agata Ciechanowska  1 , Katarzyna Pawlik  1 , Katarzyna Ciapała  1 , Joanna Mika  1
Affiliations

Pharmacological Modulation of the MIP-1 Family and Their Receptors Reduces Neuropathic Pain Symptoms and Influences Morphine Analgesia: Evidence from a Mouse Model

Agata Ciechanowska et al. Brain Sci. .

Abstract

Neuropathic pain pathophysiology is not fully understood, but it was recently shown that MIP-1 family members (CCL3, CCL4, and CCL9) have strong pronociceptive properties. Our goal was to examine how pharmacological modulation of these chemokines and their receptors (CCR1 and CCR5) influence hypersensitivity after nerve injury in Albino Swiss male mice. The spinal changes in the mRNA/protein levels of the abovementioned chemokines and their receptors were measured using RT-qPCR and ELISA/Western blot techniques in a mouse model of chronic constriction injury of the sciatic nerve. Behavioral studies were performed using the von Frey and cold plate tests after pharmacological treatment with neutralizing antibodies (nAbs) against chemokines or antagonists (CCR1-J113863, CCR5-TAK-220/AZD-5672) alone and in coadministration with morphine on Day 7, when the hypersensitivity was fully developed. Our results showed enhanced protein levels of CCL3 and CCL9 1 and 7 days after nerve injury. The single intrathecal administration of CCL3 or CCL9 nAb, J113863, TAK-220, or AZD-5672 diminished neuropathic pain symptoms and enhanced morphine analgesia. These findings highlight the important roles of CCL3 and CCL9 in neuropathic pain and additionally indicate that these chemokines play essential roles in opioid analgesia. The obtained results suggest CCR1 and CCR5 as new, interesting targets in neuropathy treatment.

Keywords: AZD-5672); CCL3; CCL4; CCL9; CCR1 antagonist (J113863); CCR5 antagonist (TAK-220.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest.

Figures

Figure 1
Figure 1
Development of thermal hypersensitivity in CCI-exposed mice (from 1 to 35 days) as measured via the cold plate test (B). Timeline of tissue collection at the indicated time points for behavioral and biochemical analyses (D). Time-dependent changes in the mRNA levels of Olig2 (A), TMEM119 (C), IBA1 (E), and GFAP (G) measured via RT-qPCR, and in the protein levels of IBA1 (F) and GFAP (H) measured via Western blot in the spinal cord of naive and CCI-exposed mice (from 1 to 35 days). The data (n = 5–10) are presented as the mean fold changes relative to the control ± SEM for the biochemical assays and the mean ± SEM for the behavioral tests. The results were evaluated using one-way ANOVA followed by Bonferroni’s post hoc test for comparisons of selected pairs. * p  <  0.05; ** p  <  0.01; and *** p  <  0.001 indicate significant differences vs. the naive group. Abbreviations: -: naive; CCI: chronic constriction injury of the sciatic nerve.
Figure 2
Figure 2
Time-dependent changes in the mRNA levels of CCL3 (A), CCL4 (C), and CCL9 (E) measured via RT-qPCR and changes in the protein levels of CCL3 (B), CCL4 (D), and CCL9 (F) measured using ELISA in the spinal cord of naive and CCI-exposed mice (from 1 to 35 days). The data are presented as the mean fold changes relative to the control ± SEM (n = 4–10). The results were evaluated using one-way ANOVA followed by Bonferroni’s post hoc test for comparisons of selected pairs. * p  <  0.05, ** p  <  0.01, and *** p  <  0.001 indicate significant differences vs. the naive group. Abbreviations: -: naive; CCI: chronic constriction injury of the sciatic nerve.
Figure 3
Figure 3
The effects of CCL3 neutralizing antibody administered intrathecally (i.t.) according to timeline (A), at doses of 0.5, 2, and 4 μg/5 μL on mechanical (B) and thermal (C) hypersensitivity, and the influence of a CCL3 neutralizing antibody at a dose of 2 μg/5 μL plus morphine 2.5 μg/5 μL on mechanical (E) and thermal (F) hypersensitivity, administered according to timeline (D), 7 days after CCI in mice. The data are presented as the mean ± SEM (naive n = 5; CCI n = 7–8). The results were evaluated using one-way ANOVA followed by Bonferroni’s post hoc test for comparisons of selected pairs. * p  <  0.05, ** p  <  0.01, and *** p  <  0.001 indicate significant differences vs. V-treated group at each of the investigated time points: 1, 4, and 24 h for (B,C) graphs; * p  <  0.05 and *** p  <  0.001 indicate significant differences vs. V + W-treated group for (E,F) graphs; # p  <  0.05 indicates significant differences between the V + M- and nAb + M-treated groups for (E,F) graphs; and & p  <  0.05 and && p  <  0.01 indicate significant differences between the nAb + W- and nAb + M-treated groups. Abbreviations: V: vehicle (PBS); W: vehicle (aqua pro injectione); nAb: neutralizing antibody; M: morphine; CCI: chronic constriction injury of the sciatic nerve.
Figure 4
Figure 4
The effects of CCL9 neutralizing antibody administered intrathecally (i.t.) according to timeline (A), at doses of 0.5, 2, and 4 μg/5 μL on mechanical (B) and thermal (C) hypersensitivity, and the influence of a CCL9 neutralizing antibody at a dose of 2 μg/5 μL plus morphine 2.5 μg/5 μL on mechanical (E) and thermal (F) hypersensitivity, administered according to timeline (D), 7 days CCI in mice. The data are presented as the mean ± SEM (naive n = 5; CCI n = 5–8). The results were evaluated using one-way ANOVA followed by Bonferroni’s post hoc test for comparisons of selected pairs. * p  <  0.05, ** p  <  0.01, and *** p  <  0.001 indicate significant differences vs. V-treated group at each of the investigated time points: 1, 4, and 24 h for (B,C) graphs; * p  <  0.05, ** p  <  0.01, and *** p  <  0.001 indicate significant differences vs. V + W-treated group for (E,F) graphs; # p  <  0.05 indicates significant differences between the V + M- and nAb + M-treated groups for (E,F) graphs; and & p  <  0.05 indicates significant differences between the nAb + W- and nAb + M-treated groups. Abbreviations: V: vehicle (PBS); W: vehicle (aqua pro injectione); nAb: neutralizing antibody; M: morphine; CCI: chronic constriction injury of the sciatic nerve.
Figure 5
Figure 5
Time-dependent changes in the mRNA levels of CCR1 (A) and CCR5 (C) measured via RT-qPCR and changes in the protein levels of CCR1 (B) and CCR5 (D) measured using Western blot in the spinal cord of naive and chronic CCI-exposed mice (from 1 to 35 days). The data are presented as the mean fold changes relative to the control ± SEM (n = 4–9). The results were evaluated using one-way ANOVA followed by Bonferroni’s post hoc test for comparisons of selected pairs. * p  <  0.05, ** p  <  0.01, and *** p  <  0.001 indicate significant differences vs. the naive group. Abbreviations: -: naive; CCI: chronic constriction injury of the sciatic nerve.
Figure 6
Figure 6
The effects of a CCR1 antagonist (J113863) administered intrathecally (i.t.) according to timeline (A), at doses of 1, 15, 30, and 60 µg/5 µL on mechanical (B) and thermal (C) hypersensitivity and the influence of J113863 at a dose of 15 μg/5 μL plus morphine 2.5 μg/5 μL on mechanical (E) and thermal (F) hypersensitivity, administered according to timeline (D), 7 days after CCI in mice. The data are presented as the mean ± SEM (naive n = 5; CCI n = 5–8). The results were evaluated using one-way ANOVA followed by Bonferroni’s post hoc test for comparisons of selected pairs. * p  <  0.05, ** p  <  0.01, and *** p  <  0.001 indicate significant differences vs. V-treated group at each of the investigated time points: 1, 4, and 24 h for (B,C) graphs; * p  <  0.05 and *** p  <  0.001 indicate significant differences vs. V + W-treated group for (E,F) graphs; ## p  <  0.01 indicates significant differences between the V + M- and J11 + M-treated groups for (E,F) graphs; and & p  <  0.05 indicates significant differences between the J11 + W- and J11 + M-treated groups. Abbreviations: V: vehicle (DMSO); W: vehicle (aqua pro injectione); M: morphine, J11: J113863; CCI: chronic constriction injury of the sciatic nerve.
Figure 7
Figure 7
The effects of CCR5 antagonists (TAK-220) administered intrathecally (i.t.) according to timeline (A), at doses of 0.5, 2, 4, and 15 µg/5 µL on mechanical (B) and thermal (C) hypersensitivity and the influence of TAK-220 at a dose of 4 μg/5 μL plus morphine 2.5 μg/5 μL on mechanical (E) and thermal (F) hypersensitivity, administered according to timeline (D), 7 days after CCI in mice. The data are presented as the mean ± SEM (naive n = 5; CCI n = 8–10). The results were evaluated using one-way ANOVA followed by Bonferroni’s post hoc test for comparisons of selected pairs. * p  <  0.05, ** p  <  0.01, and *** p  <  0.001 indicate significant differences vs. V-treated group at each of the investigated time points: 1, 4, and 24 h for (B,C) graphs; * p  <  0.05, ** p  <  0.01, and *** p  <  0.001 indicate significant differences vs. V + W-treated group for (E,F) graphs; ## p  <  0.01 indicates significant differences between the V + M- and TAK + M-treated groups for (E,F) graphs; and & p  <  0.05 and && p  <  0.01 indicate significant differences between the TAK + W- and TAK-220 + M-treated groups. Abbreviations: V: vehicle (DMSO); W: vehicle (aqua pro injectione); M: morphine, TAK: TAK-220; CCI: chronic constriction injury of the sciatic nerve.
Figure 8
Figure 8
The effects of CCR5 antagonists (AZD-5672) administered intrathecally (i.t.) according to timeline (A), at doses of 0.5, 2, 4, and 15 µg/5 µL on mechanical (B) and thermal (C) hypersensitivity and the influence of AZD-5672 at a dose of 4 μg/5 μL plus morphine 2.5 μg/5 μL on mechanical (E) and thermal (F) hypersensitivity, administered according to timeline (D), 7 days after CCI in mice. The data are presented as the mean ± SEM (naive n = 5; CCI n = 8–10). The results were evaluated using one-way ANOVA followed by Bonferroni’s post hoc test for comparisons of selected pairs. * p  <  0.05, ** p  <  0.01, and *** p  <  0.001 indicate significant differences vs. V-treated group at each of the investigated time points: 1, 4, and 24 h for (B,C) graphs; * p  <  0.05, ** p  <  0.01, and *** p  <  0.001 indicate significant differences vs. V + W-treated group for (E,F) graphs; ### p  <  0.001 and && p  <  0.01 indicates significant differences between the V + M- and AZD + M-treated groups for (E,F) graphs; and p  <  0.01 indicates significant differences between the AZD + W- and AZD + M-treated groups. Abbreviations: V: vehicle (DMSO); W: vehicle (aqua pro injectione); M: morphine; AZD: AZD -5672; CCI: chronic constriction injury of the sciatic nerve.
Figure 9
Figure 9
Comparison of the effects of intrathecal (i.t.) administration of substances targeting CCR1 (J113863), CCR5 (TAK-220 or AZD-5672), and their combination (J11 + TAK-220 or J11 + AZD-5672) at a dose of 15 µg/5 µL (timeline (A)) on mechanical and thermal hypersensitivity measured after 1 h (B,C) and after 4 h (D,E), 7 days after chronic CCI in mice. The data are presented as the mean ± SEM (naive n = 5; CCI n = 7–8). The results were evaluated using one-way ANOVA followed by Bonferroni’s post hoc test for comparisons of selected pairs. ● p  <  0.05, ●● p  <  0.01, and ●●● p  <  0.001 indicate significant differences vs. J11-treated group; ♦ p  <  0.05 and ♦♦♦ p  <  0.001 indicate significant differences vs. TAK-treated group; ■ p  <  0.05 indicates significant differences vs. AZD-treated group; and ▲ p  <  0.05 indicates significant differences vs. J11 + TAK-treated group. Abbreviations: N: naive; V: vehicle (DMSO); J11: J113863; TAK: TAK-220; AZD: AZD -5672; CCI: chronic constriction injury of the sciatic nerve.
Scheme 1
Scheme 1
Pharmacological modulation of chemokines from MIP-1 family (CCL3 and CCL9) via neutralizing antibodies and their receptors (CCR1 by J113863, CCR5 by TAK-220 or AZD -5672) reduces neuropathic pain symptoms and influences morphine analgesia—evidence from mice model evoked by chronic constriction injury of the sciatic nerve.
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