EXPRESS: Methylcobalamin ameliorates neuropathic pain induced by vincristine in rats: Effect on loss of peripheral nerve fibers and imbalance of cytokines in the spinal dorsal horn

Abstract

Background: Vincristine, a widely used chemotherapeutic agent, often induces painful peripheral neuropathy and there are currently no effective drugs to prevent or treat this side effect. Previous studies have shown that methylcobalamin has potential analgesic effect in diabetic and chronic compression of dorsal root ganglion model; however, whether methylcobalamin has effect on vincristine-induced painful peripheral neuropathy is still unknown.

Results: We found that vincristine-induced mechanical allodynia and thermal hyperalgesia, accompanied by a significant loss of intraepidermal nerve fibers in the plantar hind paw skin and an increase in the incidence of atypical mitochondria in the sciatic nerve. Moreover, in the spinal dorsal horn, the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the protein expression of p-p65 as well as tumor necrosis factor a was increased, whereas the protein expression of IL-10 was decreased following vincristine treatment. Furthermore, intraperitoneal injection of methylcobalamin could dose dependently attenuate vincristine-induced mechanical allodynia and thermal hyperalgesia, which was associated with intraepidermal nerve fibers rescue, and atypical mitochondria prevalence decrease in the sciatic nerve. Moreover, methylcobalamin inhibited the activation of NADPH oxidase and the downstream NF-kB pathway. Production of tumor necrosis factor a was also decreased and production of IL-10 was increased in the spinal dorsal horn following methylcobalamin treatment. Intrathecal injection of Phorbol-12-Myristate-13-Acetate, a NADPH oxidase activator, could completely block the analgesic effect of methylcobalamin.

Conclusions: Methylcobalamin attenuated vincrinstine-induced neuropathic pain, which was accompanied by inhibition of intraepidermal nerve fibers loss and mitochondria impairment. Inhibiting the activation of NADPH oxidase and the downstream NF-kB pathway, resulting in the rebalancing of proinflammatory and anti-inflammatory cytokines in the spinal dorsal horn might also be involved. These findings might provide potential target for preventing vincristine-induced neuropathic pain.

Figure 1.

Effects of MeCbl on mechanical allodynia produced by vincristine. (a) and (b) Intraperitoneal injection of vincristine (100 µg/kg for 10 consecutive days, n = 7) but not saline (n = 5) to rats induced persistent decrease in 50% PWT in bilateral hind paw, which lasted at least 19 days. MeCbl alone had no effect on the 50% PWT in saline-treated rats. (c) and (d) 1 mg/kg MeCbl injected intraperitoneally, started 15 days before, and then every other day thereafter until the 19th day after onset of vincristine significantly increased the bilateral 50% PWT (n = 11). MeCbl at the doses of 0.5 and 0.12 mg/kg had no effect on the decrease of 50% PWTs in the bilateral hind paw (p > 0.05) except that 0.5 mg/kg MeCbl significantly increased the 50% PWTs on the left hind paw at day 19 after onset of vincristine (p < 0.01). ^***p < 0.001 compared with baseline; ^##p < 0.01, ^###p < 0.001, and ^$$p < 0.01 compared with vincristine-treated group. n = 5–11 for each group.

Figure 2.

Effects of MeCbl on thermal hyperalgesia produced by vincristine. (a) and (b) Intraperitoneal injection of vincristine (100 µg/kg for 10 consecutive days, n = 7) but not saline (n = 5) to rats induced persistent decrease in PWL in bilateral hind paw, which lasted at least 19 days. MeCbl alone had no effect on the paw withdrawal latency in saline-treated rats. (c) and (d) All the three doses of MeCbl that tested, including 1, 0.5, and 0.12 mg/kg significantly increased the bilateral paw withdrawal latency (n = 5 or 11). ^***p < 0.001 compared with baseline; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, and ^$$p < 0.01 compared with vincristine-treated group. n = 5–11 for each group.

Figure 3.

Effect of intraperitoneal injection of MeCbl on loss of IENF induced by vincristine. (a) PGP9.5-labeled IENF emerged from cutaneous nerves and traveled vertically into the epidermis where they branched into terminals. (b) MeCbl alone did not affect the density of IENF in saline-treated rats. (c) A significant loss of IENF was found on day 19 after vincristine treatment. (d) 0.5 mg/kg MeCbl significantly inhibited the loss of IENF induced by vincristine on day 19. ^***p < 0.001 compared with saline-treated group. ^###p < 0.001 compared to the vincristine-treated group. n = 5 for each group. Scale bar: 100 µm.

Figure 4.

Effects of MeCbl on the prevalence of atypical mitochondria in A- and C- fiber. (a) to (h) Representative ultrastructural imaging showing the prevalence of atypical mitochondria in A- fiber (a–d) and C- fiber (e–h) from the four different groups as indicated. The histogram shows the summary data (n = 5/group, means ± SEM) of abnormal mitochondria on day 19 after onset of vincristine treatment. ^**p < 0.01, ^***p < 0.001 compared with saline-treated group. ^##p < 0.01, ^###p < 0.001 compared with vincristine-treated group. Scale bar: 200 nm. SN: saline-treated group; SN + M: saline and MeCbl-treated group; VCR: vincristine-treated group; VCR + M: vincristine and MeCbl-treated group.

Figure 5.

MeCbl inhibited the activation NADPH oxidase in spinal dorsal horn induced by vincristine, and intrathecal injection of NADPH oxidase activator blocked the effects of MeCbl on mechanical allodynia and thermal hyperalgesia. (a) The histogram shows NADPH oxidase activity in the lumbar spinal dorsal horn from the four different groups, as indicated (n = 5/group, means ± SEM). (b) to (e) PMA (0.2 µg/5 µl) administrated intrathecally 10 min before MeCbl could block the inhibitory effect of MeCbl on the mechanical allodynia (b) to (c) and on thermal hyperalgesia (d) to (e) in bilateral hind paw. (a) ^*p < 0.05 compared with saline-treated group; ^##p < 0.01 compared with vincristine- treated group; (b) to (e): ^#p < 0.05; ^##p < 0.01; ^###p < 0.001 compared with vincristine-treated group that had received vehicle and MeCbl administration.

Figure 6.

Effects of MeCbl on the activation of NF-κB pathway in spinal dorsal horn induced. The bands show the expression of p-p65, p65, and β-actin from protein samples of bilateral lumbar spinal dorsal horn in saline (SN)-treated group, saline and MeCbl (SN + MeCbl)-treated group, vincristine (VCR)-treated group, and vincristine-treated group that had received MeCbl administration. The histogram shows the quantification of p-p65 normalized by β-actin (n = 6/group). ^*p < 0.05 compared with saline-treated group; ^##p < 0.01 compared with vincristine-treated group.

Figure 7.

Effects of MeCbl on TNF-α overexpression and IL-10 downregulation in spinal dorsal horn induced. (a) The bands show the expression of TNF-α and β-actin from protein samples of bilateral lumbar spinal dorsal horn in the four groups (saline group, saline + MeCbl group, VCR group and MeCbl + VCR group). The histogram shows the quantification of TNF-α normalized by β-actin (n = 6/group). (b) The bands show the expression of IL-10 and β-actin from protein samples of bilateral lumbar spinal dorsal horn in the above four groups. The histogram shows the quantification of IL-10 normalized by β-actin (n = 6/group). ^*p < 0.05 compared with saline-treated group; ^#p < 0.05 compared with vincristine-treated group.