The RS504393 Influences the Level of Nociceptive Factors and Enhances Opioid Analgesic Potency in Neuropathic Rats

Abstract

Increasing evidence has indicated that activated glial cells releasing nociceptive factors, such as interleukins and chemokines, are of key importance for neuropathic pain. Significant changes in the production of nociceptive factors are associated with the low effectiveness of opioids in neuropathic pain. Recently, it has been suggested that CCL2/CCR2 signaling is important for nociception. Here, we studied the time course changes in the mRNA/protein level of CD40/Iba-1, CCL2 and CCR2 in the spinal cord/dorsal root ganglia (DRG) in rats following chronic constriction injury (CCI) of the sciatic nerve. Moreover, we examined the influence of intrathecal preemptive and repeated (daily for 7 days) administration of RS504393, CCR2 antagonist, on pain-related behavior and the associated biochemical changes of some nociceptive factors as well as its influence on opioid effectiveness. We observed simultaneous upregulation of Iba-1, CCL2, CCR2 in the spinal cord on 7th day after CCI. Additionally, we demonstrated that repeated administration of RS504393 not only attenuated tactile/thermal hypersensitivity but also enhanced the analgesic properties of morphine and buprenorphine under neuropathy. Our results proof that repeated administration of RS504393 reduced the mRNA and/or protein levels of pronociceptive factors, such as IL-1beta, IL-18, IL-6 and inducible nitric oxide synthase (iNOS), and some of their receptors in the spinal cord and/or DRG. Furthermore, RS504393 elevated the spinal protein level of antinociceptive IL-1alpha and IL-18 binding protein. Our data provide new evidence that CCR2 is a promising target for diminishing neuropathic pain and enhancing the opioid analgesic effects.

Keywords: Buprenorphine; CCR2; Interleukins; Morphine; RS504393.

Scheme 1

All behavioral experiments were preceded by intrathecal implantation of catheters since all drugs were administered via intrathecal (i.t.) injection. One week after implantation, all rats were subjected to sciatic nerve chronic constriction injury (CCI; day 0). a The single injection of RS504393 (20 μg/5 μl) or vehicle was performed 7 days after CCI after which behavioral tests were conducted 30 min (von Frey) and 35 min (cold plate) (data presented on Fig. 2a and c). b The repeated injection of RS504393 (20 μg/5 μl) or vehicle were provided once a day 16 h (day −1) and 1 h (day 0) before CCI and for 7 next days. On the 7th day post-CCI behavioral tests were performed 30 min (von Frey) and 35 min (cold plate) after drug administration and then 4 h (for RT-qPCR analysis) or 6 h (for Western blot analysis) after the last RS/V injection the tissue was collected (data presented on Fig. 2b and d). c Additionally, the coadministration of repeated RS504393 with opioids were performed. The group of chronically RS- or V-treated rats received on the 7th day after CCI (approximately 60 min after RS/V injection) a single injection of morphine or buprenorphine (2.5 μg/5 μl), and then, the von Frey test (30 min) and the cold plate test (35 min) were performed (data presented on Fig. 7a and b). Abbreviations: CCI, chronic constriction injury; DRG, dorsal root ganglia; i.t., intrathecal; RT-qPCR, quantitative reverse transcriptase real-time polymerase chain reaction; RS, RS504393; WB, Western blot; V, vehicle

Fig. 1

The time course changes in CD40, CCL2, CCR2 mRNA (a–c, g–i) and Iba-1, CCL2, CCR2 protein (d–f, j–l) levels in the spinal cord (a–f) and DRG (g–l) tissue on 2nd, 7th, and 14th days after chronic constriction injury (CCI) in rats. The data are presented as the mean ± SEM of 5–8 and 5–6 samples per group in RT-qPCR and Western blot analysis, respectively. The intergroup differences were analyzed using ANOVA with Bonferroni”s multiple comparisons test.^* p < 0.05, ^** p < 0.01 and ^*** p < 0.001 indicate differences between naive rats and CCI-exposed rats. Abbreviations: CCI, chronic constriction injury; N, naive

Fig. 2

The influence of the single (5–8 rats per group; a, c) and repeated (14–20 rats per group; b, d) administration of RS504393 (RS; 20 μg/5 μl; i.t.; 16 h and 1 h before CCI and then once a day for 7 days) on pain-related behavior (a, b von Frey test; c, d, cold plate test) on the 7th day post-CCI, 30 min after RS or V injection. Tactile and thermal hypersensitivity were assessed at 30 min and 35 min, respectively. The data are presented as the mean ± SEM. The horizontal dotted line shows the cut-off value. The intergroup differences were analyzed using ANOVA with Bonferroni”s multiple comparisons test. ^*** p < 0.001 indicates differences between naive rats and V-treated/RS-treated CCI-exposed rats; ^### p < 0.001 indicates differences vs. V-treated CCI-exposed rats. Abbreviations; CCI, chronic constriction injury; N, naive; RS, RS504393; V, vehicle

Fig. 3

The influence of the repeated administration of RS504393 (RS; 20 μg/5 μl; i.t.; 16 h and 1 h before CCI and then once a day for 7 days) on protein (a–f) levels of Iba-1 (a, d), CD4 (b, e), CD8 (c, f) in the spinal cord and DRG on the 7th day after CCI. The data are presented as the mean fold changes of control ± SEM (5–9 samples per group). The intergroup differences were analyzed using ANOVA with Bonferroni’s multiple comparisons test. ^*** p < 0.001 indicate differences between naive rats and V-treated/RS-treated CCI-exposed rats. ^# p < 0.05 and ^### p < 0.001 indicate differences vs. V-treated CCI-exposed rats. Abbreviations: CCI, chronic constriction injury; N, naive; V, vehicle; RS, RS504393

Fig. 4

The influence of the repeated administration of RS504393 (RS; 20 μg/5 μl; i.t.; 16 h and 1 h before CCI and then once a day for 7 days) on mRNA (a–d, i–l) and protein (e–h, m–p) levels of IL-1alpha (a, e, i, m), IL-1beta (b, f, j, n), IL-1RA (c, g, k, o) and IL-1R1 (d, h, l, p) in the spinal cord and DRG on the 7th day after CCI. The data are presented as the mean fold changes of control ± SEM of 4–7 and 5–9 samples per group in RT-qPCR and Western blot analysis, respectively. The intergroup differences were analyzed using ANOVA with Bonferroni’s multiple comparisons test. ^* p < 0.05, ^** p < 0.01 and ^*** p < 0.001 indicate differences between naive rats and V-treated/RS-treated CCI-exposed rats. ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 indicate differences vs. V-treated CCI-exposed rats. Abbreviations: CCI, chronic constriction injury; N, naive; V, vehicle; RS, RS504393

Fig. 5

The influence of the repeated administration of RS504393 (RS; 20 μg/5 μl; i.t.; 16 h and 1 h before CCI and then once a day for 7 days) on mRNA (a–c, g–i) and protein (d–f, j–l) levels of IL-18 (a, d, g, j), IL-18BP (b, e, h, k) and IL-18R (c, f, i, l) in the spinal cord and dorsal root ganglia on the 7th day after CCI. The data are presented as the mean fold changes of control ± SEM of 4–7 and 5–9 samples per group in RT-qPCR and Western blot analysis, respectively. The intergroup differences were analyzed using ANOVA with Bonferroni’s multiple comparisons test. ^* p < 0.05, ^** p < 0.01 and ^*** p < 0.001 indicate differences between naive rats and V-treated/RS-treated CCI-exposed rats. ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 indicate differences vs. V-treated CCI-exposed rats. Abbreviations: CCI, chronic constriction injury; N, naive; V, vehicle; RS, RS504393

Fig. 6

The influence of the repeated administration of RS504393 (RS; 20 μg/5 μl; i.t.; 16 h and 1 h before CCI and then once a day for 7 days) on mRNA (a–c, g–i) and protein (d–f, j–l) levels of IL-6 (a, d, g, j), IL-10 (b, e, h, k) and iNOS (c, f, i, l) in the spinal cord and DRG on the 7th day after CCI. The data are presented as the mean fold changes of control ± SEM of 4–7 and 5–9 samples per group in RT-qPCR and Western blot analysis, respectively. The intergroup differences were analyzed using ANOVA with Bonferroni’s multiple comparisons test. ^* p < 0.05, ^** p < 0.01 and ^*** p < 0.001 indicate differences between naive rats and V-treated/RS-treated CCI-exposed rats. ^# p < 0.05 and ^## p < 0.01 indicate differences vs. V-treated CCI-exposed rats. Abbreviations: CCI, chronic constriction injury; N, naive; V, vehicle; RS, RS504393

Fig. 7

The influence of the repeated administration of RS504393 (RS; 20 μg/5 μl; i.t.; 16 h and 1 h before CCI and then once a day for 7 days) on pain-related behavior (A, von Frey test; B, cold plate test) and the analgesic effects of morphine (M; 2.5 μg/5 μl; single dose i.t.; on the 7th day post-CCI, 60 min after RS or V injection) and buprenorphine (B; 2.5 μg/5 μl; single dose i.t.; on the 7th day post-CCI, 60 min after RS or V injection) in CCI-exposed rats. Tactile and thermal hypersensitivity were assessed at 30 min and 35 min, respectively, after the last RS504393 injection and once again after single morphine or buprenorphine injections. The data are presented as the mean ± SEM of 5–14 rats per group. The horizontal dotted line shows the cut-off value. The intergroup differences were analyzed using ANOVA with Bonferroni”s multiple comparisons test. ^### p < 0.001 indicates differences vs. V + V-treated CCI-exposed rats; ^&&& p < 0.001 indicates differences vs. RS + V-treated CCI-exposed rats; ^^^p < 0.001 indicates differences between V + M- or V + B-treated vs. RS + M- or RS + B-treated, CCI-exposed rats. Abbreviations: B, buprenorphine; CCI, chronic constriction injury; M, morphine; N, naive; RS, RS504393; V, vehicle