Role of dorsal root ganglion K2p1.1 in peripheral nerve injury-induced neuropathic pain

Abstract

Peripheral nerve injury-caused hyperexcitability and abnormal ectopic discharges in the primary sensory neurons of dorsal root ganglion (DRG) play a key role in neuropathic pain development and maintenance. The two-pore domain background potassium (K2P) channels have been identified as key determinants of the resting membrane potential and neuronal excitability. However, whether K2P channels contribute to neuropathic pain is still elusive. We reported here that K2P1.1, the first identified mammalian K2P channel, was highly expressed in mouse DRG and distributed in small-, medium-, and large-sized DRG neurons. Unilateral lumbar (L) 4 spinal nerve ligation led to a significant and time-dependent reduction of K2P1.1 mRNA and protein in the ipsilateral L4 DRG, but not in the contralateral L4 or ipsilateral L3 DRG. Rescuing this reduction through microinjection of adeno-associated virus-DJ expressing full-length K2P1.1 mRNA into the ipsilateral L4 DRG blocked spinal nerve ligation-induced mechanical, thermal, and cold pain hypersensitivities during the development and maintenance periods. This DRG viral microinjection did not affect acute pain and locomotor function. Our findings suggest that K2P1.1 participates in neuropathic pain development and maintenance and may be a potential target in the management of this disorder.

Figure 1.

K_2P1.1 is expressed exclusively in dorsal root ganglion (DRG) neurons of naive mice. (a). K_2P1.1 is co-expressed with βIII-tubulin in the DRG neurons. (b) K_2P1.1 is undetected in GS-labeled DRG satellite glial cells. Scale bar: 40 µm in (a); 20 µm in (b). GS: glutamine synthetase.

Figure 2.

K_2P1.1 is distributed in the small-, medium-, and large-sized DRG neurons of naive mice. (a) A representative image from immunohistochemical staining showing the distribution of K_2P1.1-positive neurons. Scale bar: 50 µm. (b) Histogram showing the distribution of K_2P1.1-positive somata.

Figure 3.

Co-location of K_2P1.1 with NF200, CGRP, and IB4 in DRG neurons. Double immunohistochemical staining shows that approximately 45.8% of K_2P1.1-labeled neurons are positive for NF200 (a), 31.9% for CGRP (b), and 18.9% for IB4 (c). Scale bar: 40 µm.

Figure 4.

Peripheral nerve injury leads to the decreases in the levels of K_2P1.1 mRNA and protein in the injured DRG. (a) K_2P1.1 protein expression in the ipsilateral L4 DRG after SNL or sham surgery. Representative Western blots (left panels) and a summary of densitometric analysis (right graphs). n = 8 mice/time point. *P < 0.05 vs. the corresponding control group (Sham), one-way ANOVA followed by post hoc Tukey test. (b) K_2P1.1 mRNA expression in the ipsilateral and contralateral L4 DRGs after SNL. n = 8 mice/time point. *P < 0.05 vs. the corresponding control group (0 day), one-way ANOVA followed by post hoc Tukey test. (c) K_2P1.1 protein expression in the ipsilateral L3 DRG after SNL. Representative Western blots (Top panels) and a summary of densitometric analysis (bottom graphs). n = 8 mice/time point. One-way ANOVA followed by post hoc Tukey test. (d) Number of K_2P1.1-labeled neurons in the ipsilateral and contralateral L4 DRGs on day 7 after SNL or sham surgery. Representative immunohistochemical staining (left panels) and a summary of the analysis on the number of K_2P1.1-labeled neurons (right graphs). n = 5 mice/group. *P < 0.05 vs. the corresponding sham group by two-tailed paired t-test. Scale bar: 60 µm. (e) K_2P1.1 protein expression in the ipsilateral L3/4 DRGs on day 7 after axotomy or sham surgery. Representative Western blots (top panels) and a summary of densitometric analysis (bottom graphs). n = 8 mice/group. *P < 0.05 vs. the corresponding control group (Sham) by two-tailed paired t-test. SNL = spinal nerve ligation.

Figure 5.

EGFP-labeled AAV-DJ is limited in L4 DRG and its fibers and terminals on the ipsilateral side after AAV-EGFP injection into the unilateral L4 DRG. (a–f). Time course of EGFP-labeled AAV-DJ expression in the ipsilateral L4 DRG after viral injection. (g). EGFP-labeled AAV-DJ expression in the ipsilateral L4 spinal cord on day 28 after viral injection. (h). High magnification of the outlined region from g. Scale bars: 50 µm in a–f; 100 µm in g; 25 µm in h.

Figure 6.

Rescuing K_2P1.1 expression in the injured DRG blocked SNL-induced pain hypersensitivities during the development period. SNL or sham surgery was carried out four weeks after microinjection of PBS, AAV-K_2P1.1, or AAV-EGFP into the ipsilateral L4 DRG. (a) K_2P1.1 protein expression in the ipsilateral L4 DRG on day 7 after SNL or sham surgery from the treated groups as indicated. Representative Western blots (left panels) and a summary of densitometric analysis (right graphs). n = 8 mice/group. *P < 0.05 vs. naive mice. ^#P < 0.05 vs. the PBS plus SNL group, one-way ANOVA followed by post hoc Tukey test. (b)–(h) Paw withdrawal responses to 0.07 g von Frey filament (b), 0.4 g von Frey filament (c), thermal stimulation (d), and cold stimulation (e) on the ipsilateral side and paw withdrawal responses to 0.07 g von Frey filament (f), 0.4 g von Frey filament (g), and thermal stimulation (h) on the contralateral side from the treated groups as indicated. n = 8 mice/group. *P < 0.05 vs. the PBS plus SNL group at the corresponding time points, by two-way ANOVA followed by post hoc Tukey test.

Figure 7.

Rescuing K_2P1.1 expression in the injured DRG blocked SNL-induced pain hypersensitivities during the maintenance period. SNL or sham surgery was carried out two weeks after microinjection of AAV-K_2P1.1 or AAV-EGFP into the ipsilateral L4 DRG. (a)–(g) Paw withdrawal responses to 0.07 g von Frey filament (a), 0.4 g von Frey filament (b), thermal stimulation (c), and cold stimulation (d) on the ipsilateral side and paw withdrawal responses to 0.07 g von Frey filament (e), 0.4 g von Frey filament (f), and thermal stimulation (g) on the contralateral side from the treated groups as indicated. n = 6 mice/group. *P < 0.05 vs. the AAV-EGFP plus SNL group at the corresponding time points by two-way ANOVA followed by post hoc Tukey test.