Additive antinociceptive effects of a combination of vitamin C and vitamin E after peripheral nerve injury

Abstract

Accumulating evidence indicates that increased generation of reactive oxygen species (ROS) contributes to the development of exaggerated pain hypersensitivity during persistent pain. In the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin C and vitamin E in mouse models of inflammatory and neuropathic pain. We show that systemic administration of a combination of vitamins C and E inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by Complete Freund's Adjuvant. In contrast, vitamin C or vitamin E given alone failed to affect the nociceptive behavior in all tested models. The attenuated neuropathic pain behavior induced by the vitamin C and E combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. Moreover, the vitamin C and E combination ameliorated the allodynia induced by an intrathecally delivered ROS donor. Our results suggest that administration of vitamins C and E in combination may exert synergistic antinociceptive effects, and further indicate that ROS essentially contribute to nociceptive processing in special pain states.

Figure 1. Antinociceptive effects of a Vit C and Vit E combination in the formalin test.

Drugs (15 mg Vit C, 7.5 mg Vit E, the combination of 15 mg Vit C and 7.5 mg Vit E, or saline) were intraperitoneally administered 20 min prior to injection of formalin (15 µl, 5%) into a hindpaw. Sum of paw-licking time in phase 1 (1–10 min) and phase 2 (11–60 min). Note that the licking behavior in phase 1 is considerably reduced in mice treated with the Vit C+E combination. n = 6–8 per group; *significantly different from saline group, p<0.05.

Figure 2. CFA-induced inflammatory pain behavior is not affected by Vit C and Vit E treatment.

Mice were injected with 20 µl CFA into a hindpaw. Drugs (15 mg Vit C, 7.5 mg Vit E, the combination of 15 mg Vit C and 7.5 mg Vit E, or saline) were i.p. administered 24 h (time point ‘0’) and 48 h (time point ‘24’) after CFA injection. Paw withdrawal latency times upon mechanical stimulation are expressed as difference to baseline (i.e. prior to the first drug injection). Statistical analyses revealed no significant differences between groups. n = 7–8 per group.

Figure 3. A combination of Vit C and Vit E inhibits SNI-induced neuropathic pain behavior.

Mice were subjected to SNI surgery and drugs (Vit C, Vit E, the combination of Vit C and Vit E, or saline) were i.p. administered at the indicated doses 14 days (time point ‘0’) and 15 days (time point ‘24’) after SNI surgery. Paw withdrawal latency times upon mechanical stimulation are expressed as difference to baseline (i.e. prior to the first drug injection). Note that the Vit C+E combination dose-dependently inhibited SNI-induced mechanical allodynia, and that a sustained effect occurred after a second injection of Vit C+E. n = 8 per group; *significantly different from saline group, p<0.05.

Figure 4. Intrathecally administered Vit C and E inhibit SNI-induced neuropathic pain behavior.

Same experimental setting as described in Figure 3, but with intrathecal drug administration at the indicated doses. The Vit C+E combination effectively inhibited SNI-induced mechanical allodynia after i.t. administration. n = 6–8 per group; *,^#significantly different from saline group (p<0.05) for Vit C+E and Vit E alone, respectively.

Figure 5. Vit C and Vit E treatment attenuates phosphorylation of p38, but not of p42 or p44, in the spinal cord and DRGs.

Mice were subjected to SNI surgery and received two i.p. injections of saline or the combination of Vit C (30 mg) and Vit E (15 mg) at days 14 and 15 after SNI. The protein expression of phospho-p38 (p-p38) and p-38 (A), and of phospho-p42 (p-p42), p42, phospho-p44 (p-p44) and p44 (B) in the spinal cord and DRGs was analyzed by western blotting of tissues obtained 3 h after the second drug injection. Calnexin was used as loading control. Representative western blots are shown on the left, densitometric analyses are shown on the right. n = 3 animals per group; *p<0.05, **p<0.01.

Figure 6. Vit C and Vit E pretreatment attenuates mechanical allodynia induced by intrathecal TBHP.

Mice were i.p. pretreated with a combination of 30 mg Vit C and 15 mg Vit E or saline. One hour thereafter, the ROS donor TBHP (100 µg) was i.t. injected (time point ‘0’), and paw withdrawal latency times upon mechanical stimulation were measured for 105 min. n = 6 per group; *significantly different from saline group, p<0.05.

Figure 7. Long-term treatment with low dose Vit C and Vit E inhibits SNI-induced neuropathic pain behavior.

Mice were subjected to SNI surgery and drugs were i.p. (A) or orally (B) administered at the indicated doses once daily for 12 days starting immediately after SNI surgery. Paw withdrawal latency times upon mechanical stimulation are shown. Note that both doses of the Vit C+E combination inhibited the neuropathic pain behavior after i.p. and oral administration. n = 8 per group; *,^#significantly different from saline group (p<0.05) for the 3 mg + 1.5 mg combination and the 0.75 mg + 0.375 mg combination, respectively.

Figure 8. Long-term treatment with low dose Vit C and Vit E does not inhibit CFA-induced inflammatory pain behavior.

Mice were injected with 20 µl CFA into a hindpaw and drugs were i.p. administered at the indicated doses once daily for 12 days starting immediately after CFA injection. Paw withdrawal latency times upon mechanical stimulation are shown. Statistical analyses revealed no significant differences between groups. n = 8 per group.