Role of TRPM8 in dorsal root ganglion in nerve injury-induced chronic pain

Abstract

Background: Chronic neuropathic pain is an intractable pain with few effective treatments. Moderate cold stimulation can relieve pain, and this may be a novel train of thought for exploring new methods of analgesia. Transient receptor potential melastatin 8 (TRPM8) ion channel has been proposed to be an important molecular sensor for cold. Here we investigate the role of TRPM8 in the mechanism of chronic neuropathic pain using a rat model of chronic constriction injury (CCI) to the sciatic nerve.

Results: Mechanical allodynia, cold and thermal hyperalgesia of CCI rats began on the 4th day following surgery and maintained at the peak during the period from the 10th to 14th day after operation. The level of TRPM8 protein in L5 dorsal root ganglion (DRG) ipsilateral to nerve injury was significantly increased on the 4th day after CCI, and reached the peak on the 10th day, and remained elevated on the 14th day following CCI. This time course of the alteration of TRPM8 expression was consistent with that of CCI-induced hyperalgesic response of the operated hind paw. Besides, activation of cold receptor TRPM8 of CCI rats by intrathecal application of menthol resulted in the inhibition of mechanical allodynia and thermal hyperalgesia and the enhancement of cold hyperalgesia. In contrast, downregulation of TRPM8 protein in ipsilateral L5 DRG of CCI rats by intrathecal TRPM8 antisense oligonucleotide attenuated cold hyperalgesia, but it had no effect on CCI-induced mechanical allodynia and thermal hyperalgesia.

Conclusions: TRPM8 may play different roles in mechanical allodynia, cold and thermal hyperalgesia that develop after nerve injury, and it is a very promising research direction for the development of new therapies for chronic neuroapthic pain.

Figure 1

Assessment of behavioral reflex sensitization in CCI model of neuropathic pain. A, Cold sensitivity assessed by cold plate test. B, Thermal sensitivity assessed by hot plate test. C, Mechanical sensitivity assessed by Von Frey filament assay. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. sham-operated rats (t test).

Figure 2

TRPM8 expression in ipsilateral L5 DRGs of CCI and sham-operated rats. A, TRPM8 protein staining in L5 DRG sections. Arrows indicate TRPM8 positive neurons. The graph shows the percentage of TRPM8 positive neurons. B, TRPM8- immunoblotting of L5 DRGs. β-actin expression is shown as a control. The graph shows the ratio of density of TRPM8 band to that of β-actin band. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. sham-operated rats (t test). Magnification of immunohistochemical images is 400 ×, and scale bars represent 25 μm.

Figure 3

Effect of menthol on behavioral reflex sensitization of CCI rats. A, Mechanical sensitivity assessed by Von Frey filament assay. B, Thermal sensitivity assessed by hot plate test. C, Cold sensitivity assessed by cold plate test. Data are expressed as mean ± SEM. **P < 0.01 vs. pre-drug (paired t test).

Figure 4

Effect of menthol on expression of TRPM8 protein in ipsilateral L5 DRGs of CCI rats. Immunohistochemical staining (A) and western blot analysis (B) of TRPM8 protein in L5 DRGs from sham-operated rats treated with normal saline or menthol and CCI rats treated with normal saline or menthol. Data are expressed as mean ± SEM. **P < 0.01 vs. Sham-NS rats; ^§§P < 0.01 vs. Sham-Menthol rats (one-way ANOVA followed by SNK test). Magnification of immunohistochemical images is 400 ×, and scale bars represent 25 μm. Arrows indicate TRPM8 positive neurons.

Figure 5

Effect of TRPM8 ASODN on behavioral reflex sensitization of CCI rats. A, Cold sensitivity assessed by cold plate test. B, Mechanical sensitivity assessed by Von Frey filament assay. C, Thermal sensitivity assessed by hot plate test. Data are expressed as mean ± SEM. **P < 0.01 vs. CCI-MMODN rats (one-way ANOVA followed by SNK test).

Figure 6

Effect of TRPM8 ASODN on expression of TRPM8 protein in ipsilateral L5 DRGs of CCI rats. Immunohistochemical staining (A) and western blot analysis (B) of TRPM8 protein in L5 DRGs from sham-operated rats treated with MMODN or ASODN and CCI rats treated with MMODN or ASODN. TRPV1 immunoreactivity (B) is used to examine the specificity of TRPM8 antisense experiment. Data are expressed as mean ± SEM. **P < 0.01 vs. CCI-MMODN rats; ^§§P < 0.01 vs. Sham-MMODN rats (one-way ANOVA followed by SNK test). Magnification of immunohistochemical images is 400 ×, and scale bars represent 25 μm. Arrows indicate TRPM8 positive neurons.