Effects of nefopam on streptozotocin-induced diabetic neuropathic pain in rats

Abstract

Background: Nefopam is a centrally acting non-opioid analgesic agent. Its analgesic properties may be related to the inhibitions of monoamine reuptake and the N-methyl-D-aspartate (NMDA) receptor. The antinociceptive effect of nefopam has been shown in animal models of acute and chronic pain and in humans. However, the effect of nefopam on diabetic neuropathic pain is unclear. Therefore, we investigated the preventive effect of nefopam on diabetic neuropathic pain induced by streptozotocin (STZ) in rats.

Methods: Pretreatment with nefopam (30 mg/kg) was performed intraperitoneally 30 min prior to an intraperitoneal injection of STZ (60 mg/kg). Mechanical and cold allodynia were tested before, and 1 to 4 weeks after drug administration. Thermal hyperalgesia was also investigated. In addition, the transient receptor potential ankyrin 1 (TRPA1) and TRP melastatin 8 (TRPM8) expression levels in the dorsal root ganglion (DRG) were evaluated.

Results: Pretreatment with nefopam significantly inhibited STZ-induced mechanical and cold allodynia, but not thermal hyperalgesia. The STZ injection increased TRPM8, but not TRPA1, expression levels in DRG neurons. Pretreatment with nefopam decreased STZ-induced TRPM8 expression levels in the DRG.

Conclusions: These results demonstrate that a nefopam pretreatment has strong antiallodynic effects on STZ-induced diabetic rats, which may be associated with TRPM8 located in the DRG.

Keywords: TRPA1; TRPM8; allodynia; nefopam; painful diabetic neuropathy; streptozotocin.

Fig. 1

Changes of blood glucose levels and body weights after streptozotocin (STZ) treatment in rats. After overnight fasting, an intraperitoneal injection of a vehicle (Control) or STZ (60 mg/kg) was performed. Nefopam was injected intraperitoneally at a dose of 30 mg/kg 30 min before the administration of STZ (Nefo-STZ). The induction of diabetes was assessed by a serial measurement of the tail vein blood glucose level using a blood glucose meter (A). Body weight was also measured (B). The data are presented as the means ± standard error. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 compared to the control group.

Fig. 2

Effect of a nefopam pretreatment on the withdrawal response to mechanical and thermal stimuli in streptozotocin (STZ)-induced diabetic rats. Pretreatment with nefopam (30 mg/kg) was given intraperitoneally 30 min prior to an intraperitoneal injection of STZ (60 mg/kg). Behavioral tests were performed before, and 1 to 4 weeks after the final administration of drugs. Systemic pretreatment with 30 mg/kg of nefopam significantly inhibited STZ-induced mechanical allodynia at 4 weeks (A). Pretreatment with nefopam significantly inhibited STZ-induced cold allodynia from 2 weeks after the nefopam treatment (B). However, a nefopam pretreatment did not attenuate STZ-induced thermal hyperalgesia (C). The data are presented as the means ± standard error. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 compared to the control group. ^†P < 0.05 and ^††P < 0.01 compared to the STZ group.

Fig. 3

Effect of a nefopam pretreatment on the transient receptor potential ankyrin 1 (TRPA1) and melastatin 8 (TRPM8) protein expression levels in the dorsal root ganglion (DRG) of streptozotocin (STZ)-induced diabetic rats. Pretreatment with nefopam (30 mg/kg) was performed intraperitoneally 30 min prior to an intraperitoneal injection of STZ (60 mg/kg). To evaluate the expressions of TRPA1 (A) and TRPM8 (B) proteins located in the L4-L6 DRG, a Western blot analysis was performed 4 weeks after the final drug administration. The specific signals for TRPA1 and TRPM8 were quantified and plotted (lower panel). β-actin was used as an internal loading control. STZ injections did not significantly affect the basal level of the TRPA1 protein in the DRG, which was not altered by the nefopam pretreatment. The TRPM8 protein level in the DRG significantly increased 4 weeks after the STZ injection, which was reduced by the nefopam pretreatment. ^***P < 0.001 compared to the control group. ^†††P < 0.001 compared to the STZ group.

Fig. 4

Typical double immunofluorescence staining of transient receptor potential melastatin 8 (TRPM8) and neurofilament 200 (NF200) in the rat dorsal root ganglion (DRG) of streptozotocin (STZ)-induced diabetic rats. Pretreatment with nefopam (30 mg/kg) was performed intraperitoneally 30 min prior to an intraperitoneal injection of STZ (60 mg/kg). Four weeks after the final drug treatment, L5 DRG samples were immunostained with TRPM8 and neurofilament 200 (NF200), a marker of myelinated neurons. The colocalization of TRPM8 was visualized in a merged image.