3.1. CTK 01512-2 intrathecal treatment reduced mechanical hypersensitivity and thermal allodynia induced by CPIP
CTK 01512-2 intrathecal treatment has shown its therapeutically potential in several preclinical studies related to pain and inflammatory models [26,22,54,55,12,28,29,23,14,56,25,57,24]. Herein, the CTK 01512-2 treatment (25, 50, and 100 pmol/site; i.t.) reduced mechanical hypersensitivity on day 7 (Fig. 2A; two-way ANOVA showed interaction [F (20, 118) = 6.00; P < 0.0001****], row [F (5, 118) = 70.92; P < 0.001****], and column [F (4, 118) = 71.36; P < 0.0001****] effects), and on day 21 (Fig. 2C; two-way ANOVA showed interaction [F (20, 118) = 7.27; P < 0.0001****], row [F (5, 118) = 130.3; P < 0.0001****], and column [F (4, 118) = 140.0; P < 0.0001****] effects) when the subjects were submitted to CPIP model. On days 7 and 21, Bonferroni's multiple comparisons post hoc test showed that CPIP-untreated group increased the mechanical hypersensitivity in comparison to sham control group at 0, 1, 2, 3, and 4 h. On day 7, post hoc test demonstrated that the doses of 25 and 50 pmol/site reduced the mechanical hypersensitivity in comparison to CPIP-untreated group at 1 and 2 h, and the dose of 50 and 100 pmol/site reduced the mechanical hypersensitivity in comparison to CPIP-untreated group at 3 h. On day 21, post hoc test showed that only the dose of 50 pmol/site reduced the mechanical hypersensitivity in comparison to CPIP-untreated group at 1, 2, and 3 h. Moreover, the CTK 01512-2 treatment also reduced the AUC (Σ1 – 3 h) on day 7 (Fig. 2B; one-way ANOVA showed treatment effect [F (4, 10) = 11.70; P = 0.0009***]), and on day 21 (Fig. 2D; one-way ANOVA showed treatment effect [F (4, 10) = 34.74; P < 0.0001****]). On day 7, post hoc test showed that CPIP-untreated group depicted higher AUC (Σ1 – 3 h) in comparison to sham control group, and that both groups (CTK 01512-2 – 50 and 100 pmol/site, i.t.) demonstrated lower AUC (Σ1 – 3 h) in comparison to CPIP-untreated group. On day 21, post hoc test showed that CPIP-untreated group depicted higher AUC (Σ1 – 3 h) in comparison to sham control group, and only the CTK 01512-2 – 50 pmol/site group demonstrated lower AUC (Σ1 – 3 h) in comparison to CPIP-untreated group.
Concerning the thermal allodynia, the obtained results showed that the CTK 01512-2 treatment (50 pmol/site; i.t.) reduced thermal allodynia on day 7 (Fig. 3A; two-way ANOVA showed interaction [F (15, 72) = 1.27; P=0.2428], row [F (5, 72) = 8.72; P < 0.001****], and column [F (3, 72) = 19.74; P < 0.0001****] effects, and Fig 3B; one-way ANOVA demonstrated treatment [F (3, 8) = 19.41; P=0.0005***] effect), on day 14 (Fig. 3C; two-way ANOVA showed interaction [F (15, 72) = 1.55; P=0.1108], row [F (5, 72) = 6.29; P < 0.001****], and column [F (3, 72) = 13.08; P < 0.0001****] effects, and Fig 3D; one-way ANOVA demonstrated treatment [F (3, 8) = 18.63; P=0.0006***] effect), and on day 21 (Fig. 3E; two-way ANOVA showed interaction [F (15, 72) = 2.20; P=0.0141*], row [F (5, 72) = 15.53; P < 0.0001****], and column [F (3, 72) = 26.54; P < 0.0001****] effects, and Fig 3F; one-way ANOVA demonstrated treatment [F (3, 8) = 83.59; P < 0.0001****] effect). On days 7 and 14, Bonferroni's multiple comparisons post hoc test showed that the CPIP-untreated group increased the thermal allodynia compared to the sham control group at 3 and 4 h. On day 21, a post hoc test showed that the CPIP-untreated group increased the thermal allodynia compared to the sham control group at 2, 3, and 4 h. On day 7, the post hoc test showed that only the positive control (+ω-conotoxin MVIIA; 100 pmol/site, i.t.) significantly reduced the thermal allodynia compared to the CPIP-untreated group at 1, 2, 3, and 4 h. On day 14, a post hoc test revealed that besides the thermal allodynia caused by the positive control group at 4 h, the CTK 01512-2 treatment (50 pmol/site, i.t.) also reduced the thermal allodynia compared to the CPIP-untreated group at 4 h. On day 21, the post hoc test showed a significant treatment effect neither in the positive control group nor in the CTK 01512-2 group compared to the CPIP-untreated group. Regarding the AUC (Σ2 – 4 h) on day 7 (Fig. 3B), day 14 (Fig. 3D), and day 21 (Fig. 3F), the post hoc test showed that in all observed days, the CPIP-untreated group increased the thermal allodynia in comparison to the sham control group. On day 7, the post hoc test showed that only the positive control group reduced the thermal allodynia; however, on days 14 and 21, besides the positive control group, CTK 01512-2 group also reduced the thermal allodynia. Altogether, this set of results showed that the intrathecal treatment with CTK 01512-2 (mainly at 50 pmol/site) reduced both mechanical hypersensitivity and thermal allodynia induced by CPIP.
3.2. CTK 01512-2 systemic treatment reduced mechanical hypersensitivity induced by CPIP
Previous data showed that CTK 01512-2 systemic treatment reduced reserpine-induced mechanical hypersensitivity and thermal hyperalgesia in a mouse model of fibromyalgia [31]. To test whether the CTK 01512-2 systemic treatment was also capable of interfering with mechanical hypersensitivity induced by CPIP, CTK 01512-2 treatment was administered intravenously. It was observed that the CTK 01512-2 treatment (0.2 mg/kg, i.v.) reduced mechanical hypersensitivity on day 7 (Fig. 4A; two-way ANOVA showed interaction [F (10, 84) = 4.55; P < 0.0001****], row [F (5, 84) = 12.55; P < 0.001****], and column [F (2, 84) = 41.99; P < 0.0001****] effects). The Bonferroni's multiple comparisons post hoc test demonstrated that the CPIP-untreated group increased the frequency response (%) in comparison to the sham control group at 1, 2, 3, and 4 h. Moreover, the post hoc test showed that the systemic treatment with CTK 01512-2 reduced the frequency response compared to the CPIP-untreated group at 2, 3, and 4 h. When the AUC (Σ2 – 4 h) was analyzed (Fig. 4B), one-way ANOVA showed treatment effect [F (2, 6) = 140.2; P < 0.0001****]. Post hoc test revealed that the CPIP-untreated group demonstrated a higher AUC (Σ2 – 4 h) compared to the sham control group and that CTK 01512-2 (0.2 mg/kg, i.v.) treatment showed a lower AUC (Σ2 – 4 h) compared to CPIP-untreated group. Together, this set of results revealed that CTK 01512-2 systemic reduced the mechanical hypersensitivity induced by CPIP.
3.3. CTK 01512-2 intracerebroventricular treatment mitigated paclitaxel-induced mechanical hypersensitivity and thermal allodynia
Our data demonstrate CTK 01512-2 intracerebroventricular treatment effects in a neuropathic pain model. Our results showed that CTK 01512-2 treatment (30, 50, and 100 pmol/site; i.c.v.) mitigated paclitaxel (2mg/kg, i.p. + vehicle, i.c.v.)-induced mechanical hypersensitivity on day 7 (Fig. 5A; two-way ANOVA showed interaction [F (25, 132) = 1.60; P=0.0475*], row [F (5, 132) = 2.53; P=0.0318*], and column [F (5, 132) = 2.48; P=0.0348*] effects), on day 12 (Fig. 5C; two-way ANOVA showed interaction [F (25, 131) = 2.11; P=0.0037**], row [F (5, 131) = 3.84; P=0.0028**], and column [F (5, 131) = 6.10; P < 0.0001****] effects), on day 14 (Fig. 5E; two-way ANOVA showed interaction [F (25, 132) = 1.52; P=0.0692], row [F (5, 132) = 3.64; P=0.0040**], and column [F (5, 132) = 6.33; P < 0.0001****] effects), and on day 21 (Fig. 5G; two-way ANOVA showed interaction [F (25, 132) = 1.98; P=0.0072**], row [F (5, 132) = 4.72; P=0.0005***], and column [F (5, 132) = 3.06; P=0.0121*] effects). On day 7, Bonferroni's multiple comparisons post hoc test showed that only the positive control (+ω-conotoxin MVIIA; 50 pmol/site, i.c.v.) significantly increased the paw withdrawal threshold compared to PTX-untreated group at 3 h. On day 12, post hoc test demonstrated that besides the increased paw withdrawal threshold of the positive control group compared to PTX-untreated group at 4 and 5 h, the CTK 01512-2 (30 pmol/site; i.c.v.) also increased the paw withdrawal threshold compared to PTX-untreated group at 4 h. On day 14, post hoc test showed that both positive control group and CTK 01512-2 (50 pmol/site; i.c.v.) increased the paw withdrawal threshold compared to PTX-untreated group at 5 h, although on day 21, no statistical difference was observed. Furthermore, when the paw withdrawal threshold was analyzed at the 3rd hour of the day 7 (Fig. 5B), at the 4th hour of day 12 (Fig. 5D), and at the 5th hour of days 14 (Fig. 5F) and 21 (Fig. 5H), one-way ANOVA showed treatment effect [day 7: F (5, 22) = 6.24, P=0.0010*; day 12: F (5, 22) = 5.90, P=0.0013**; day 14: F (5, 22) = 6.88, P=0.0005***; and day 21: F (5, 22) = 2.10, P=0.1041]. Post hoc test demonstrated that at the 3rd hour of the day 7, both positive control and CTK 01512-2 (50 pmol/site; i.c.v.) groups showed a higher paw withdrawal threshold compared to the PTX-untreated group. At the 4th hour of day 12 and at the 5th hour of day 14, positive control and CTK 01512-2 (30 and 50 pmol/site; i.c.v.) groups showed a higher paw withdrawal threshold compared to the PTX-untreated group, although at the 5th hour of day 21, no statistical difference was observed.
Additionally, CTK 01512-2 treatment (30, 50, and 100 pmol/site; i.c.v.) also inhibited paclitaxel (2 mg/kg, i.p. + vehicle, i.c.v.)-induced thermal allodynia on day 7 (Table 1; two-way ANOVA showed time [F (4, 88) = 7.10; P < 0.0001****], and subject [F (22, 88) = 1.67; P=0.0497*] effects), on day 8 (Fig. 6A; two-way ANOVA showed interaction [F (20, 110) = 1.94; P=0.0157*], row [F (4, 110) = 5.44; P=0.0005***], and column [F (5, 110) = 19.95; P<0.0001****] effects, and Fig 6B; one-way ANOVA demonstrated treatment [F (5, 12) = 8.634; P=0.0011**] effect), on day 12 (Table 1; two-way ANOVA showed time [F (4, 88) = 7.58; P < 0.0001****], and subject [F (22, 88) = 4.07; P < 0.0001****] effects), on day 14 (Table 1; two-way ANOVA showed time [F (4, 88) = 7.75; P < 0.0001****], and subject [F (22, 88) = 6.67; P < 0.0001****] effects), on day 15 (Fig. 6C; two-way ANOVA showed interaction [F (20, 110) = 2.93; P=0.0002***], row [F (4, 110) = 8.37; P<0.0001****], and column [F (5, 110) = 17.68; P<0.0001****] effects, and Fig 6D; one-way ANOVA demonstrated treatment [F (5, 6) = 3.92; P=0.0633] effect), on day 21 (Table 1; two-way ANOVA showed subject [F (22, 88) = 3.50; P < 0.0001****] effect), and on day 22 (Fig. 6E; two-way ANOVA showed interaction [F (20, 110) = 1.38; P=0.1476], row [F (4, 110) = 4.18; P=0.0035**], and column [F (5, 110) = 16.20; P<0.0001****] effects, and Fig 6F; one-way ANOVA demonstrated treatment [F (5, 12) = 12.14; P=0.0002***] effect). On day 7, Bonferroni's multiple comparisons post hoc test showed that only the CTK 01512-2 (50 pmol/site; i.c.v.) group significantly increased the thermal allodynia compared to PTX-untreated group at 2 h. On day 8, post hoc test showed that at 1, 2, and 3 h the PTX-untreated group increased the thermal allodynia (time; s) compared to sham control group. Moreover, at 1 h, CTK 01512-2 treatment (100 pmol/site; i.c.v.) group and the positive control (+ω-conotoxin MVIIA; 50 pmol/site, i.c.v.) decreased thermal allodynia compared to PTX-untreated group. At 2 h, CTK 01512-2 treatment (50 and 100 pmol/site; i.c.v.) group and positive control group decreased thermal allodynia compared to PTX-untreated group. At the 3 h, CTK 01512-2 treatment (30, 50 and 100 pmol/site; i.c.v.) group and positive control group decreased thermal allodynia compared to PTX-untreated group. On day 14, post hoc test showed only the positive control (+ω-conotoxin MVIIA; 50 pmol/site, i.c.v.) group significantly increased the thermal allodynia compared to PTX-untreated group at 2 h. On day 15, post hoc test revealed that at 3 h the PTX-untreated group increased the thermal allodynia compared to sham control group. In addition, at 2 h, only the positive control group decreased thermal allodynia compared to PTX-untreated group. At 3 h, CTK 01512-2 treatment (30, 50 and 100 pmol/site; i.c.v.) group and the positive control group decreased thermal allodynia compared to PTX-untreated group. At the 3 h, CTK 01512-2 treatment (30, 50 and 100 pmol/site; i.c.v.) group and positive control group decreased thermal allodynia compared to PTX-untreated group. On day 22, post hoc test showed that at 2 h the PTX-untreated group increased the thermal allodynia compared to sham control group. Moreover, at 1 h, the CTK 01512-2 treatment (100 pmol/site; i.c.v.) group and the positive control group decreased thermal allodynia compared to PTX-untreated group. At 2 h, CTK 01512-2 treatment (50 and 100 pmol/site; i.c.v.) group and the positive control group decreased thermal allodynia in relation to PTX-untreated group. At 3 and 4 h, only the positive control group decreased thermal allodynia compared to PTX-untreated group. In regard to the AUC on day 8 (Fig. 6B), on day 15 (Fig. 6D), and on day 22 (Fig. 6F), post hoc test showed that PTX-untreated group increased the thermal allodynia compared to sham control group on days 8 and 22. On day 8 and 22, post hoc test showed that CTK 01512-2 treatment (50 and 100 pmol/site; i.c.v.) group and the positive control group reduced the thermal allodynia. On day 15, only the positive control group reduced the thermal allodynia. Altogether, this set of results showed that the intracerebroventricular treatment with CTK 01512-2 significantly reduced paclitaxel-induced mechanical hypersensitivity and thermal allodynia.
3.4. CTK 01512-2 altered the astroglial cell viability
It is well established that astroglial cells become activated under pathological conditions to maintain homeostasis of the brain microenvironment [58]. Moreover, previous findings demonstrated that the inhibition of astrocytic activation could be a useful therapeutic strategy for treating neuropathic pain [59]. Herein, we have used the MTT reduction assay to test the effect of toxins on cellular viability of C6 astroglial cells after 24 h considering that the number of viable cells with metabolically active mitochondria were determined based on the mitochondrial reduction of a tetrazolium bromide salt (MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay). One-way ANOVA showed treatment (Fig. 7; F (7, 28) = 5.46; P=0.0005***] effect. Post hoc test showed that LPS did not change the viability of C6 astroglial cells at the concentrations tested (1, 10, and 50 ng/ml; P > 0.05), while CTK 01512-2 (1 and 3 pmol/µl) increased MTT reduction. Although conotoxin also apparently increased MTT reduction, the effect was not statistically significant.