Treatment with 5-fluoro-2-oxindole Increases the Antinociceptive Effects of Morphine and Inhibits Neuropathic Pain

Abstract

The efficacy of µ-opioid receptors (MOR) in neuropathic pain is low and with numerous side effects that limited their use. Chronic neuropathic pain is also linked with emotional disorders that aggravate the sensation of pain and which treatment has not been resolved. This study investigates whether the administration of an oxindole, 5-fluoro-2-oxindole, could inhibit the nociceptive and emotional behaviors and increase the effectiveness of morphine via modulating the microglia and activating the nuclear factor erythroid-2 related factor 2 (Nrf2) signaling pathway and MOR expression. In C57BL/6 mice with neuropathic pain provoked by the total constriction of sciatic nerve we studied the effects of 10 mg/kg 5-fluoro-2-oxindole in: (i) the allodynia and hyperalgesia caused by the injury; (ii) the anxiety- and depressive-like behaviors; (iii) the local antinociceptive actions of morphine; (iv) the expression of CD11b/c (a microglial marker), the antioxidant and detoxificant enzymes Nrf2, heme oxygenase 1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1), and of MOR in the spinal cord and hippocampus. Results showed that the inhibition of the main nociceptive symptoms and the anxiety- and depressive-like behaviors induced by 5-fluoro-2-oxindole were accompanied with the suppression of microglial activation and the activation of Nrf2/HO-1/NQO1 signaling pathway in the spinal cord and/or hippocampus. This treatment also potentiated the pain-relieving activities of morphine by normalizing the reduced MOR expression. This work demonstrates the antinociceptive, anxiolytic and antidepressant effects of 5-fluoro-2-oxindole, suggests a new strategy to enhance the antinociceptive actions of morphine and proposes a new mechanism of action of oxindoles during chronic neuropathic pain.

Keywords: Anxiety; Depression; Neuropathic pain; Opioid receptors; Oxidative stress; Oxindole.

Fig. 1

Effects of the acute administration of 5-fluoro-2-oxindole in the mechanical allodynia, thermal hyperalgesia and cold allodynia induced by sciatic nerve injury. Mechanical antiallodynic (a), thermal antihyperalgesic (b) and cold antiallodynic (c) effects produced by the intraperitoneal administration of different doses (logarithmic axis) of 5-fluoro-2-oxindole (FLUO) or vehicle in sciatic nerve-injured mice. Data are expressed as mean values of maximal possible effect (%) for mechanical allodynia and thermal hyperalgesia, or inhibition (%) for cold allodynia ± SEM (6 animals for dose). For each test, *denotes significant differences as compared with vehicle treated mice, + denotes significant differences as compared with the effects produced by 10 mg/kg FLUO and # denotes significant differences as compared with the effects produced by 20 mg/kg FLUO (p < 0.05; one-way ANOVA followed by the Student–Newman–Keuls test)

Fig. 2

Repetitive administration of 5-fluoro-2-oxindole inhibits the mechanical allodynia, thermal hyperalgesia and cold allodynia induced by sciatic nerve injury. The effects of the repeated administration of 10 mg/kg 5-fluoro-2-oxindole (FLUO) or vehicle in the mechanical allodynia (a), thermal hyperalgesia (b) and cold allodynia (c) in the ipsilateral paw of sciatic nerve-injured or sham-operated mice at 17, 18, 20, 22, 25 and 28 days after surgery are shown. For each test and time assessed: *indicates significant differences as compared with sham-operated mice treated with vehicle, + indicates significant differences as compared with sham-operated mice treated with FLUO, and # indicates significant differences as compared with sciatic nerve-injured mice treated with FLUO (p < 0.05; one-way ANOVA followed by the Student–Newman–Keuls test). Results are presented as the mean values ± SEM; n = 6 animals per experimental group

Fig. 3

Treatment with 5-fluoro-2-oxindole inhibits the anxiety- and depressive-like behaviors associated with neuropathic pain. The effects of the administration of 10 mg/kg 5-fluoro-2-oxindole (FLUO) or vehicle during 11 consecutive days on the anxiety- and depressive-like behaviors associated with chronic neuropathic pain at 28 days after surgery are shown. The effects of FLUO or vehicle in sham-operated mice are also shown. In the EPM test, the number of entries into the open arms (a), the percentage of time spent in the open arms (b), and the number of entries into the closed arms (c) are shown. For TST (d) and FST (e), the immobility time (s) are shown. For each test, *indicates significant differences as compared with sham-operated mice treated with vehicle, + indicates significant differences as compared with sham-operated mice treated with FLUO, and # indicates significant differences as compared with sciatic nerve-injured mice treated with FLUO (p < 0.05; one-way ANOVA followed by the Student–Newman–Keuls test). Results are presented as the mean values ± SEM; n = 6–8 animals per experimental group

Fig. 4

Administration of 5-fluoro-2-oxindole increases the local antinociceptive effects of morphine during neuropathic pain. The mechanical antiallodynic (a), thermal antihyperalgesic (b) and cold antiallodynic effects (c) of the acute administration of 10 mg/kg 5-fluoro-2-oxindole (FLUO) or vehicle (VEH) combined with 50 µg morphine (MS) or saline (SS) in the ipsilateral paw of sciatic nerve-injured mice at 28 days after surgery are shown. For each test, *indicates significant differences as compared with VEH + SS group, + indicates significant differences as compared with VEH + MS group, and #indicates significant differences as compared with FLUO + SS group (p < 0.05; one-way ANOVA followed by the Student–Newman–Keuls test). Results are presented as the mean values ± SEM; n = 6–8 animals per experimental group. Data are expressed as the mean values of the maximal possible effect (%) ± SEM for mechanical allodynia and thermal hyperalgesia and as the mean values of inhibition (%) ± SEM for cold allodynia

Fig. 5

Effects of 5-fluoro-2-oxindole in the expression of Nrf2, HO-1, NQO1, CD11b/c and MOR in the spinal cord of sciatic nerve-injured mice. The relative protein levels of Nrf2 (a), HO-1 (b), NQO1 (c), CD11b/c (d) and MOR (e) in the spinal cord of the sciatic nerve-injured (CCI) mice treated with 5-fluoro-2-oxindole (FLUO) or vehicle are represented. Sham-operated (SHAM) mice treated with vehicle were used as controls. All proteins are expressed relative to the GAPDH levels. Representative blots for Nrf2, HO-1, NQO1, CD11b/c, MOR and GAPDH are shown (f). In all panels, *represents significant differences as compared with sham-operated mice treated with vehicle, + represents significant differences as compared with sciatic nerve-injured mice treated with vehicle and # represents significant differences as compared with sciatic nerve-injured mice treated with FLUO (p < 0.05; one-way ANOVA followed by the Student–Newman–Keuls test). Mean values ± SEM; n = 4 samples per group

Fig. 6

Effects of 5-fluoro-2-oxindole in the expression of Nrf2, HO-1, NQO1, CD11b/c and MOR in the hippocampus of sciatic nerve-injured mice. The relative protein levels of Nrf2 (a), HO-1 (b), NQO1 (c), CD11b/c (d) and MOR (e) in the hippocampus of the sciatic nerve-injured (CCI) mice treated with 5-fluoro-2-oxindole (FLUO) or vehicle are represented. Sham-operated (SHAM) mice treated with vehicle were used as controls. All proteins are expressed relative to the GAPDH levels. Representative blots for Nrf2, HO-1, NQO1, CD11b/c, MOR and GAPDH are shown (f). In all panels, *represents significant differences as compared with sham-operated mice treated with vehicle, + represents significant differences as compared with sciatic nerve-injured mice treated with vehicle and # represents significant differences as compared with sciatic nerve-injured mice treated with FLUO (p < 0.05; one-way ANOVA followed by the Student–Newman–Keuls test). Mean values ± SEM; n = 4 samples per group