Pain Modulation in WAG/Rij Epileptic Rats (A Genetic Model of Absence Epilepsy): Effects of Biological and Pharmacological Histone Deacetylase Inhibitors

Abstract

Epigenetic mechanisms are involved in epilepsy and chronic pain development. About that, we studied the effects of the natural histone deacetylase (HDAC) inhibitor sodium butyrate (BUT) in comparison with valproic acid (VPA) in a validated genetic model of generalized absence epilepsy and epileptogenesis. WAG/Rij rats were treated with BUT (30 mg/kg), VPA (300 mg/kg), and their combination (BUT + VPA) daily per os for 6 months. Rats were subjected at Randall-Selitto, von Frey, hot plate, and tail flick tests after 1, 3, and 6 months of treatment to evaluate hypersensitivity to noxious and non-noxiuous stimuli. Moreover, PPAR-γ (G3335 1 mg/kg), GABA-B (CGP35348 80 mg/kg), and opioid (naloxone 1 mg/kg) receptor antagonists were administrated to investigate the possible mechanisms involved in analgesic activity. The expression of NFkB, glutathione reductase, and protein oxidation (carbonylation) was also evaluated by Western blot analysis. WAG/Rij rats showed an altered pain threshold throughout the study (p < 0.001). BUT and BUT + VPA treatment reduced hypersensitivity (p < 0.01). VPA was significantly effective only after 1 month (p < 0.01). All the three receptors are involved in BUT + VPA effects (p < 0.001). BUT and BUT + VPA decreased the expression of NFkB and enhanced glutathione reductase (p < 0.01); protein oxidation (carbonylation) was reduced (p < 0.01). No effect was reported with VPA. In conclusion BUT, alone or in coadministration with VPA, is a valuable candidate for managing the epilepsy-related persistent pain.

Keywords: WAG/Rij rats; allodynia; epilepsy; histone deacetylase-inhibitors; hyperalgesia; pain; sodium butyrate; valproic acid.

FIGURE 1

Pain threshold measurements. WAG/Rij animals were treated daily p.o. from day 1 with BUT (30 mg/kg), VPA (300 mg/kg), (BUT + VPA, 30 + 300 mg/kg) or vehicle. Wistar rats received vehicles. The response to a non-noxious mechanical stimulus was measured by the von Frey test (A), the response to a noxious mechanical stimulus was measured by the Randall-Selitto test (B), the response to a noxious thermal stimuli was measured by the hot plate test (C) and by the tail flick test (D). The tests were performed after 1, 3, and 6 months. Data are expressed as mean ± S.E.M. of at least six rats per group. Statistical analysis is two-way ANOVA followed by Bonferroni’s post hoc comparison. ⁺⁺⁺ p < 0.001 vs Wistar + Vehicle; *p < 0.05 ** < 0.01 and ***p < 0.001 vs WAG + vehicle.

FIGURE 2

Pain threshold after 1 month of wash out following 6 months of treatments. WAG/Rij animals were treated daily p.o. from day 1 with BUT (30 mg/kg), VPA (300 mg/kg), (BUT + VPA, 30 + 300 mg/kg) or vehicle for 6 months. Thereafter all treatments were suspended for 1 month, pain threshold was measured by von Frey, Randall–Selitto, hot plate, and tail flick tests. Data are expressed as mean ± S.E.M. of at least six rats per group. Statistical analysis is one-way ANOVA followed by Bonferroni’s post hoc comparison.

FIGURE 3

Effect of specific antagonists. Pain threshold, response to a non-noxious mechanical stimulus was measured. In von Frey test, WAG/Rij rats were treated daily p.o. for 1 week with BUT (30 mg/kg), VPA (300 mg/kg), (BUT + VPA, 30 + 300 mg/kg). On day 8, the von Frey test was performed. Separately, the PPAR-γ antagonist G3335 (1 mg/kg), the GABA-B antagonist CGP35348 (80 mg/kg) and the opioid antagonist naloxone (NALO 1 mg/kg) were administered intraperitoneally 30 min before the tests to the BUT + VPA group. Data are expressed as mean ± S.E.M. of at least six rats per group. Statistical analysis is one-way ANOVA followed by Bonferroni’s post hoc comparison. *p < 0.05 vs WAG + vehicle; °p < 0.05 vs WAG + BUT + VPA.

FIGURE 4

Effect of specific antagonists. Pain threshold, response to a noxious mechanical stimulus was measured. WAG/Rij rats were treated daily p.o. for 1 week with BUT (30 mg/kg), VPA (300 mg/kg), (BUT + VPA, 30 + 300 mg/kg). On day 8, the Randall–Selitto test was performed. Separately, the PPAR-γ antagonist G3335 (1 mg/kg), the GABA-B antagonist CGP35348 (80 mg/kg) and the opioid antagonist naloxone (NALO 1 mg/kg) were administered intraperitoneally 30 min before the tests to the BUT + VPA group. Data are expressed as mean ± S.E.M. of at least six rats per group. Statistical analysis is one-way ANOVA followed by Bonferroni’s post hoc comparison.*p < 0.05 vs WAG + vehicle; ^ºº p < 0.001 vs WAG + BUT + VPA.

FIGURE 5

Effect of specific antagonists. Pain threshold, response to a noxious thermal stimulus was measured. WAG/Rij rats were treated daily p.o. for 1 week with BUT (30 mg/kg), VPA (300 mg/kg), (BUT + VPA, 30 + 300 mg/kg). On day 8, the hot plate test was performed. Separately, the PPAR-γ antagonist G3335 (1 mg/kg), the GABA-B antagonist CGP35348 (80 mg/kg) and the opioid antagonist naloxone (NALO 1 mg/kg) were administered intraperitoneally 30 min before the tests to the BUT + VPA group. Data are expressed as mean ± S.E.M. of at least six rats per group. Statistical analysis is one-way ANOVA followed by Bonferroni’s post hoc comparison. *p < 0.05 vs WAG + vehicle; ^ººº p < 0.01 vs WAG + BUT + VPA.

FIGURE 6

Spinal cord, molecular analysis. NFkB, SOD1 and glutathione reductase were measured in the spinal cord of WAG/Rij rats treated daily p.o. from day 1 with BUT (30 mg/kg), VPA (300 mg/kg), (BUT + VPA, 30 + 300 mg/kg) or vehicle for 6 months. Densitometric analysis and representative Western blot are shown. β-Actin, α-tubulin, GAPDH normalization were performed for NFkB, SOD1 and glutathione reductase, respectively. Data are expressed as mean ± S.E.M. of at least six rats per group. Statistical analysis is one-way ANOVA followed by Bonferroni’s post hoc comparison. **p < 0.01 vs WAG + vehicle.

FIGURE 7

Spinal cord, oxidation. Carbonylated proteins were measured in the spinal cord of WAG/Rij rats treated daily p.o. from day 1 with BUT (30 mg/kg), VPA (300 mg/kg), (BUT + VPA, 30 + 300 mg/kg) or vehicle for 6 months. Densitometric analysis (top panel) and representative Western blot (lower panel) are shown. β-Actin normalization was performed for each sample. Data are expressed as mean ± S.E.M. of at least six rats per group. Statistical analysis is one-way ANOVA followed by Bonferroni’s post hoc comparison. **p < 0.01 vs WAG + vehicle.