Modulation of oxidative stress/NMDA/nitric oxide pathway by topiramate attenuates morphine dependence in mice

Abstract

Morphine belongs to the class of opioids and is known for its potential to cause dependence and addiction, particularly with prolonged use. Due to the associated risks, caution must be taken when prescribing and limiting its clinical use. Overexpression of N-methyl-D-aspartate (NMDA) receptors, nitric oxide and cGMP pathway has been implicated in exacerbate the development of morphine dependence and withdrawal. Topiramate, an antiepileptic drug, interacts with various receptors, ion channels and certain enzymes. In this study, we investigated the effects of topiramate on morphine dependence in mice, specifically targeting NMDA/Nitric oxide/cGMP pathway. Mice were administered different doses of topiramate (intraperitoneally) during the development phase, 45 min prior to morphine administration. Topiramate (20 mg/kg) significantly reduced naloxone-induced withdrawal symptoms in morphine-dependent mice. Additionally, subeffective doses of topiramate, when co-administered with NMDA receptor antagonist MK-801 (0.05 mg/kg) or nitric oxide synthase inhibitors such as L-NAME (10 mg/kg, a non-specific NOS inhibitor) and 7-NI (20 mg/kg, a selective nNOS inhibitor), showed a marked reduction in withdrawal signs. However, the effect of topiramate (20 mg/kg) was abolished when co-administered with NMDA (75 mg/kg, an NMDA receptor agonist) or L-arginine (60 mg/kg, a NOS substrate). Ex-vivo analysis revealed that topiramate significantly reduced oxidative stress and downregulated the gene expression of nNOS, NR1, and NR2B in morphine-treated mice. Furthermore, the expression of NR1 and NR2B proteins in the hippocampus and cortex was significantly reduced in topiramate-pretreated mice. Hence, this finding suggest that topiramate mitigates morphine dependence and withdrawal by inhibiting oxidative stress and modulating the NMDA/NO pathway.

Keywords: Dependence; MK-801; Morphine; NMDA; Nitric oxide; Oxidative stress.

Graphical abstract

Fig. 1

Experimental design and various treatment groups.

Fig. 2

(A–D). Effect of different doses of topiramate (Topi) on morphine (MOR) dependence and withdrawal, administered (i.p.) 45 min before each dose of MOR administration for 5 days. Data are expressed as mean ± S.E.M. for withdrawal signs such as jumping, Number of grooming, Diarrhea and Weight loss after naloxone injection for 8–9 mice. ^###p < 0.001 for Top 20 mg/kg compared to morphine treated group. ∗∗p < 0.01, ∗∗∗p < 0.001 for morphine compared to control/vehicle treated group.

Fig. 3

(A–D). Effect of Topiramate on naloxone-induced withdrawal signs in morphine-dependent mice. Topiramate (Topi) (15 mg/kg) was administered 45 min while L-NAME (10 mg/kg) was administered 30 min before each dose of MOR injection for 5 days. Data are presented as mean ± S.E.M for withdrawal signs of 8–9 mice. A: Number of jumping after naloxone injection. B: Number of grooming. C: Diarrhea. D: Weight loss after naloxone injection. ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 for Topiramate (10 mg/kg) compared to the morphine treated group.

Fig. 4

Effect of combined administration of topiramate (15 mg/kg) and 7-NI (20 mg/kg) on withdrawal symptoms in morphine-dependent mice. The results are expressed as mean ± S.E.M for withdrawal signs in 8–9 mice per group. No significant differences were observed. A: Number of jumps after naloxone administration ∗∗∗p > 0.05, B: Number of grooming episodes after naloxone ∗∗p > 0.05, C: Diarrhea score after naloxone ∗p > 0.05, and D: Weight loss after naloxone ∗∗∗p > 0.05 for the group treated with topiramate (10 mg/kg) + 7-NI compared to the morphine-only group.

Fig. 5

(A–D)Effect of co-administration of topiramate (Top) with Aminoguanidine (50 mg/kg) on morphine (MOR) withdrawal signs. Data are presented as mean ± S.E.M for withdrawal syndrome of 8–9 mice. A: Number of jumping after. B: Number of grooming. C: Diarrhea and D: Weight loss (g) after naloxone injection. ^#p < 0.05, ^##p < 0.01 for Topiramate 10 mg/kg + Aminoguanidine compared to morphine-treated group.

Fig. 6

(A–D). Effect of co-administration of topiramate (Topi) with L-arginine (60 mg/kg) on topiramate dependence and withdrawal symptoms. Topiramate and L-arginine were administered 45 min and 30 min before each dose of topiramate injection for 5 consecutive days. The data are presented as mean ± S.E.M. for withdrawal signs in 8–9 mice. A: Number of jumping. B: Number of grooming. C: Diarrhea. D: Weight loss following naloxone injection. Statistical analysis revealed significant differences: ^#p < 0.05, ^##p < 0.01, for the Topi (15 mg/kg) + L-arginine group compared to the topiramate-treated morphine group.

Fig. 7

(A–D). Shows the effect of co-administration of topiramate (Topi, 15 mg/kg) with MK-801 (0.05 mg/kg) on naloxone induced withdrawal symptoms. Topiramate and MK-801 were administered 45 min and 30 min before each dose of morphine (MOR) injection for 5 consecutive days. The data are presented as mean ± S.E.M. for withdrawal signs in 8–9 mice. A: Number of jumping. B: Number of grooming. C: Diarrhea. D: Weight loss following naloxone injection. Statistical analysis revealed significant differences: ^#p < 0.5, ^##p < 0.01, for the Topi (15 mg/kg) + MK-801 group compared to the topiramate-treated group.

Fig. 8

Effect of co-treatment of topiramate (Topi, 20 mg/kg) with NMDA (0.05 mg/kg) on naloxone induced withdrawal symptoms. Topiramate and MK-801 were administered 45 min and 30 min before each dose of morphine (MOR) injection for 5 consecutive days. The data are presented as mean ± S.E.M. for withdrawal signs in 8–9 mice. A: Number of jumping. B: Number of grooming. C: Diarrhea. D: Weight loss following naloxone injection. Statistical analysis revealed significant differences: ^#p < 0.5, ^##p < 0.01, for the Topi (20 mg/kg) + NMDA group compared to the morphine-treated group.

Fig. 9

(A–D). Graphical representation of the effect of Topiramate against (A) glutathione-S-transferase (GST), (B) reduced glutathione (GSH), (C) catalase and (D) lipid peroxidation (LPO) in morphine treated mice cortex and hippocampal tissues. Values are expressed as mean ± SEM (n = 7/group). One-way ANOVA with post-hoc Tukey’s test. ^###p < 0.001 denotes a significant difference from the saline group, only effective topiramate treatment group has no significant difference vs. Saline group. ∗∗∗p < 0.001 denotes a significant difference from the disease group (Morphine).

Fig. 10

(A–C). Inhibitory effect of Topiramate against mRNA of (A) nNOS, (B) NR1 and (C) NR2B expression in morphine treated mice cortex and hippocampal tissues using real-time polymerase chain reaction (RT-PCR) technique. Values expressed as mean ± SEM (n = 8/group). One-way ANOVA with post-hoc Tukey’s test. ^###p < 0.001 denotes a significant difference from the saline group, only effective topiramate treatment group has no significant difference vs. Saline group. ∗∗p < 0.01 and ∗∗∗p < 0.001 denotes a significant difference from the disease group (Morphine).

Fig. 11

Expression of iNOS gene in Hippocampus and Cortex in morphine and topiramate treated group. No significance was found p > 0.05.

Fig. 12

Effect of Topiramate treatment on morphine-induced NMDA-receptor expression using immunohistochemical analysis, showing the effects of NR1 in the mice cortex and hippocampal region. Scale bar 50 μm, magnification 40 × (n = 8/group).

Fig. 13

Inhibitory effect of Topiramate against NR1 expression in Morphine-treated mice cortex and hippocampal region, using the immunohistochemical technique. Values expressed as mean ± SEM (n = 8/group). One-way ANOVA with post-hoc Tukey’s test. ^###p < 0.001 denotes a significant difference from the saline group, ∗∗p < 0.01, ∗∗∗p < 0.001 demonstrate a significant difference from the disease group (Morphine).

Fig. 14

Effect of Topiramate treatment on morphine-induced NMDA-receptor expression using immunohistochemical analysis, showing the effects of NR2B in the mice cortex and hippocampal region. Scale bar 50 μm, magnification 40 × (n = 8/group).

Fig. 15

Inhibitory effect of Topiramate against NR2B expression in Morphine-treated mice cortex and hippocampal region, using the immunohistochemical technique. Values expressed as mean ± SEM (n = 8/group). One-way ANOVA with post-hoc Tukey’s test. ^###p < 0.001 shows a significant difference when morphine treated group was compared to saline group. ∗∗∗p < 0.001 demonstrates a significant difference when drug treated morphine group was compared to disease group (only Morphine treated group).