Involvement of Macrophage Inflammatory Protein-1 Family Members in the Development of Diabetic Neuropathy and Their Contribution to Effectiveness of Morphine

Abstract

Current investigations underline the important roles of C-C motif ligands in the development of neuropathic pain; however, their participation in diabetic neuropathy is still undefined. Therefore, the goal of our study was to evaluate the participation of macrophage inflammatory protein-1 (MIP-1) family members (CCL3, CCL4, CCL9) in a streptozotocin (STZ)-induced mouse model of diabetic neuropathic pain. Single intrathecal administration of each MIP-1 member (10, 100, or 500 ng/5 μl) in naïve mice evoked hypersensitivity to mechanical (von Frey test) and thermal (cold plate test) stimuli. Concomitantly, protein analysis has shown that, 7 days following STZ injection, the levels of CCL3 and CCL9 (but not CCL4) are increased in the lumbar spinal cord. Performed additionally, immunofluorescence staining undoubtedly revealed that CCL3, CCL9, and their receptors (CCR1 and CCR5) are expressed predominantly by neurons. In vitro studies provided evidence that the observed expression of CCL3 and CCL9 may be partially of glial origin; however, this observation was only partially possible to confirm by immunohistochemical study. Single intrathecal administration of CCL3 or CCL9 neutralizing antibody (2 and 4 μg/5 μl) delayed neuropathic pain symptoms as measured at day 7 following STZ administration. Single intrathecal injection of a CCR1 antagonist (J113863; 15 and 20 μg/5 μl) also attenuated pain-related behavior as evaluated at day 7 after STZ. Both neutralizing antibodies, as well as the CCR1 antagonist, enhanced the effectiveness of morphine in STZ-induced diabetic neuropathy. These findings highlight the important roles of CCL3 and CCL9 in the pathology of diabetic neuropathic pain and suggest that they play pivotal roles in opioid analgesia.

Keywords: CCL3; CCL4; CCL9; CCR1 antagonist (J113863); neutralizing antibody.

Figure 1

The effects of single intrathecal (i.t.) administration of macrophage inflammatory protein-1 family members on nociceptive transmission in naïve mice. The effects of single i.t. CCL3 (A,B), CCL4 (C,D), or CCL9 (E,F) administration (10, 100, or 500 ng/5 μl) on mechanical nociceptive threshold [von Frey test; (A,C,E)] and thermal nociceptive threshold [cold plate test; (B,D,F)] were measured at 60, 240, and 1,440 min following administration. The data are presented as the means ± the SEM (4–7 mice per group). The results were evaluated using two-way analysis of variance followed by Bonferroni’s test for multiple comparisons; *(P < 0.05), **(P < 0.01), and ***(P < 0.001) indicate significant differences compared with the naïve animals.

Figure 2

The effects of single intraperitoneal (i.p.) streptozotocin (STZ; 200 mg/kg) administration on the protein levels of macrophage inflammatory protein-1 members measured at day 7 following STZ administration in STZ-induced diabetic neuropathic mice. The protein analysis was performed with a protein microarray for CCL3 (A) and CCL9 (E) and by western blotting for CCL3 (B), CCL4 (D), and CCL9 (F), CCL4 (C) not presented on the membrane. Biochemical analysis was performed on lumbar spinal cords dissected from naïve and STZ-injected diabetic neuropathic mice on day 7 following STZ injection. The data are presented as fold changes relative to the control (naïve) ± the SEM (8–12 samples per group). The results were evaluated using Student’s t-test; *(P < 0.05) and ***(P < 0.001) indicate significant differences compared with the control group (naïve animals).

Figure 3

Immunohistochemical staining of macrophage inflammatory protein-1 members in the streptozotocin (STZ)-induced model of diabetic neuropathic pain. Immunofluorescent staining performed on paraffin-embedded microtome slices of spinal cords from STZ-treated mice. (A–C)—co-localization of immunoreactivity for CCL3 (green) and NeuN (red); (D–F)—co-localization of immunoreactivity for CCL9 (green) and neuronal marker, NeuN (red); (G–O)—co-localization of immunoreactivity for CCL3/CCL9 (green) and microglia marker, IBA1 (red); (P–U)—co-localization of immunoreactivity for CCL3/CCL9 (green) and astroglia marker, GFAP (red). Scale bars: 25 µm.

Figure 4

The effects of single intraperitoneal (i.p.) streptozotocin (STZ; 200 mg/kg) administration on the protein levels and neuronal localization of CCR1 and CCR5 measured at day 7 following STZ administration in STZ-induced diabetic neuropathic mice. (A,B) Protein analysis performed by western blotting for CCR1 (A) and CCR5 (B). (C) The co-localization of CCR1 and CCR5 with NeuN (A–F), IBA1 (G–L), and GFAP (M–R) assessed by immunohistochemical staining and visualized in the superimposed pictures. The data are presented as fold changes relative to the control (naïve) ± the SEM (8–10 samples per group). The results were evaluated using Student’s t-test. Scale bars: 25 µm.

Figure 5

The effects of lipopolysaccharide (LPS; 100 ng/ml) stimulation on the mRNA and protein levels of macrophage inflammatory protein-1 members in primary microglial and astroglial cultures. The mRNA expression of Ccl3 (A,C), Ccl4 (E,G), and Ccl9 (I,K) was measured by quantitative reverse transcriptase real-time PCR (qRT-PCR) in vehicle(−)- and LPS(+)-treated microglia (A,E,I) and astroglia (C,G,K) at 24 h following LPS stimulation. The protein levels of CCL3 (B,D), CCL4 (F,H), and CCL9 (J,L) were evaluated by western blotting in vehicle(−)- and LPS(+)-treated microglia (B,F,J) and astroglia (D,H,L) at 24 h following LPS stimulation. The data are presented as the means ± the SEM (4–12 samples per group). The results were evaluated using Student’s t-test; **(P < 0.01) and ***(P < 0.001) indicate significant differences compared with the vehicle(−)-treated cells.

Figure 6

The effects of lipopolysaccharide (LPS; 100 ng/ml) stimulation on the mRNA and protein levels of CCR1 and CCR5 in primary microglial and astroglial cultures. The mRNA expression of Ccr1 (A,C) and Ccr5 (E,G) was measured by quantitative reverse transcriptase real-time PCR (qRT-PCR) in the vehicle(−)- and LPS(+)-treated microglia (A,E) and astroglia (C,G) at 24 h following LPS stimulation. The protein levels of CCR1 (B,D) and CCR5 (F,H) were evaluated by western blotting in the vehicle(−)- and LPS(+)-treated microglia (B,F) and astroglia (D,H) at 24 h following LPS stimulation. The data are presented as the means ± the SEM (6–12 samples per group). The results were evaluated using Student’s t-test; *(P < 0.05), **(P < 0.01), and ***(P < 0.001) indicate significant differences compared with the vehicle(−)-treated cells.

Figure 7

The effects of single intrathecal (i.t.) CCL3 or CCL9 neutralizing antibody (nAb) administration on the mechanical and thermal nociceptive thresholds in streptozotocin (STZ; 200 mg/kg; i.p.)-induced diabetic neuropathic mice at day 7 following STZ injection. The effects of single i.t. vehicle, nAb CCL3 (A,B) or nAb CCL9 (C,D) administration (0.5, 2, and 4 μg/5 μl) on the mechanical nociceptive threshold [von Frey test; (A,C)] and thermal nociceptive threshold [cold plate test; (B,D)] were measured at 60, 240, 1,440, and 2,880 min following administration at day 7 following STZ injection. The data are presented as the means ± the SEM (5–7 mice per group). The results were evaluated using two-way analysis of variance followed by Bonferroni’s test for multiple comparisons; ^#(P < 0.05), ^##(P < 0.01), and ^###(P < 0.001) indicate significant differences compared with the vehicle-treated STZ-injected diabetic neuropathic mice.

Figure 8

The effects of single intrathecal (i.t.) CCL3 or CCL9 neutralizing antibody (nAb) administration on the mechanical nociceptive threshold, the thermal nociceptive threshold and the effectiveness of morphine (M) in streptozotocin (STZ; 200 mg/kg; i.p.)-induced diabetic neuropathic mice at day 7 following STZ injection. Part A presents a scheme of the experiment (A). The effects of single i.t. vehicle, nAb CCL3 [2 μg/5 μl; (B,C)] or nAb CCL9 [4 μg/5 μl; (D,E)] administration on the mechanical nociceptive threshold [von Frey test; (B,D)] and thermal nociceptive threshold [cold plate test; (C,E)] were measured 120 min following administration on day 7 following STZ injection. Morphine (1 μg/5 μl) was administered as a single i.t. injection 135 min after the antibody administration and the completion of the behavioral tests; the tests were repeated 30 min following the morphine administration (B–E). The data are presented as the means ± the SEM (4–10 mice per group). The results were evaluated using one-way analysis of variance followed by Bonferroni’s test for multiple comparisons; ^#(P < 0.05), ^##(P < 0.01), and ^###(P < 0.001) indicate significant differences compared with the vehicle-treated STZ-injected diabetic neuropathic mice; ^$(P < 0.05), ^$$(P < 0.01), and ^$$$(P < 0.001) indicate significant differences compared with the vehicle (water for injection) + vehicle-treated STZ-induced diabetic neuropathic mice; ^○○(P < 0.01) and ^○○○(P < 0.001) indicate significant differences compared with the neutralizing antibody + vehicle-treated STZ-induced diabetic neuropathic mice. ^&&&(P < 0.001) indicates significant differences compared with the vehicle + morphine-treated STZ-induced diabetic neuropathic mice.

Figure 9

The effects of single intrathecal (i.t.) administration of a CCR1 antagonist (J113863) on the mechanical nociceptive threshold, the thermal nociceptive threshold and the effectiveness of morphine (M) in streptozotocin (STZ; 200 mg/kg; i.p.)-induced diabetic neuropathic mice at day 7 following STZ injection. The effects of single i.t. vehicle (5% DMSO) and J113863 [10, 15, and 20 μg/5 μl; (A,B)] administration on the mechanical threshold [von Frey test; (A)] and thermal threshold [cold plate test; (B)] were measured 30 min following administration on day 7 following STZ injection. The time-dependent changes of a 15 µg dose of J113863 was determined by the von Frey test (C) and the cold plate test (D) performed at 30, 90, 240, and 1,440 min following administration on day 7 after STZ injection. Scheme of combined administration of J113863 and morphine (E). Morphine (1 μg/5 μl) was administered as a single i.t. injection 35 min after the J113863 (15 μg/5 μl) administration and the completion of the behavioral tests, and then the tests were repeated 30 min following the morphine administration (F,G). The data are presented as the means ± SEM (4–14 mice per group). The results were evaluated using one-way analysis of variance followed by Bonferroni’s test for multiple comparisons; *(P < 0.05), **(P < 0.01), and ***(P < 0.001) indicate significant differences compared with the naïve animals; ^#(P < 0.05), ^##(P < 0.01), and ^###(P < 0.001) indicate significant differences compared with the vehicle (5% DMSO or water for injections)-treated STZ-injected diabetic neuropathic mice; ^(P < 0.05) and ^^^(P < 0.001) indicate significant differences compared with the J113863-injected STZ-induced diabetic neuropathic mice; ^&&&(P < 0.001) indicates significant differences compared with the vehicle + morphine-treated STZ-induced diabetic neuropathic mice.

Figure 10

The effects of single intrathecal (i.t.) CCR5 antagonist (DAPTA) administration on the mechanical nociceptive threshold and thermal nociceptive threshold and the effectiveness of morphine (M) in streptozotocin (STZ; 200 mg/kg; i.p.)-induced diabetic neuropathic mice at day 7 following STZ injection. The effects of single i.t. vehicle (water for injection) and DAPTA [10 and 30 μg/5 μl; (A,B)] administration on the mechanical nociceptive threshold [von Frey test; (A)] and thermal nociceptive threshold [cold plate test; (B)] were measured 30 min following administration at day 7 following STZ injection. The time-dependent changes of a 30-µg dose of DAPTA was determined by the von Frey test (C) and the cold plate test (D) at 30, 90, 240, and 1,440 min following administration on day 7 following STZ injection. Scheme of combined administration of DAPTA and morphine (E). Morphine (1 μg/5 μl) was administered as a single i.t. injection 35 min after the DAPTA (30 μg/5 μl) administration and the completion of the behavioral tests, and then the tests were repeated 30 min following the morphine administration (F,G). The data are presented as the means ± SEM (5–9 mice per group). The results were evaluated using one-way analysis of variance followed by Bonferroni’s test for multiple comparisons; ***(P < 0.001) indicates significant differences compared with the naïve animals; ^^^(P < 0.001) indicate significant differences compared with the J113863-injected STZ-induced diabetic neuropathic mice.