Impaired neuropathic pain and preserved acute pain in rats overexpressing voltage-gated potassium channel subunit Kv1.2 in primary afferent neurons

Abstract

Voltage-gated potassium (Kv) channels are critical in controlling neuronal excitability and are involved in the induction of neuropathic pain. Therefore, Kv channels might be potential targets for prevention and/or treatment of this disorder. We reported here that a majority of dorsal root ganglion (DRG) neurons were positive for Kv channel alpha subunit Kv1.2. Most of them were large and medium, although there was a variety of sizes. Peripheral nerve injury caused by lumbar (L)5 spinal nerve ligation (SNL) produced a time-dependent reduction in the number of Kv1.2-positive neurons in the ipsilateral L5 DRG, but not in the contralateral L5 DRG. Such reduction was also observed in the ipsilateral L5 DRG on day 7 after sciatic nerve axotomy. Rescuing nerve injury-induced reduction of Kv1.2 in the injured L5 DRG attenuated the development and maintenance of SNL-induced pain hypersensitivity without affecting acute pain and locomotor function. This effect might be attributed to the prevention of SNL-induced upregulation of endogenous Kv1.2 antisense RNA, in addition to the increase in Kv1.2 protein expression, in the injured DRG. Our findings suggest that Kv1.2 may be a novel potential target for preventing and/or treating neuropathic pain.

Figure 1

Kv1.2 expression predominantly in the large and medium DRG neurons of normal rats. (A) A representative image from immunohistochemical staining showing the distribution of Kv1.2-positive neurons. Scale bar: 200 μm. (B) Histogram showing the distribution of Kv1.2-positive somata.

Figure 2

Co-localization of Kv1.2 with NF200, IB4, P2X3, SP, and CGRP in DRG neurons. Double immunohistochemical staining shows that approximately 80.3% of Kv1.2-labeled neurons are positive for NF200 (A–C), 2.45% for IB4 (D–F), 11.11% for P2X3 (G–I), 3.97% for SP (J–L), and 10.7% for CGRP (M–O). Arrows: double-labeled neurons. Scale bar: 70 μm.

Figure 3

Time-dependent decreases in the number of Kv1.2-positive neurons in the injured DRG after peripheral nerve injury. Representative Kv1.2 immunohistochemical staining (Top) and statistical summary (Bottom) of the percentage of Kv1.2-positive neurons in the contralateral (Contra) and ipsilateral (Ipsi) L₅ DRGs on days 3 (A), 7 (B), and 14 (C) after L₅ spinal nerve ligation (SNL) or sham surgery and on day 7 after axotomy or sham surgery (D). Scale bar: 100 μm. * P < 0.05, ** P < 0.01 compared to the number of Kv1.2-positive neurons on the contralateral side of sham-operated rats.

Figure 4

In vitro expression and current of Kv1.2 in HEK-293 cells transfected with Kv1.2 vector. (A) Representative Western blot showing Kv1.2 expression at the expected size in HEK-293 cells transfected with full-length Kv1.2 vector. β-actin was used as a loading control. (B) Representative traces of Kv1.2 current recorded by whole-cell voltage patch clamp in HEK-293 cells transfected with full-length Kv1.2 vector before or after bath perfusion of 100 nM maurotoxin (MTX). (C) Current–voltage curves of HEK-293 cells transfected with full-length Kv1.2 before or after treatment with 100 nM MTX. The current density was plotted against each step testing voltage. n = 5 cells.

Figure 5

Overexpressing Kv1.2 in the injured DRG mitigates neuropathic pain. Kv1.2 was delivered into the injured DRG through microinjection of AAV5-Kv1.2. AAV5-EGFP was used as a control. (A-C) Behavioral tests show the effect of overexpressing Kv1.2 on the development of spinal nerve ligation (SNL)-induced pain hypersensitivities. Ipsilateral and contralateral paw withdrawal responses to mechanical (A), cold (B), and thermal (C) stimuli at the times shown before and after SNL. n = 5/group. *P < 0.05, **P < 0.01 vs. the ipsilateral side of the AAV5-EGFP-treated group at the corresponding time point. (D-F) Behavioral tests show the effect of overexpressing Kv1.2 on the maintenance of SNL-induced pain hypersensitivities. Ipsilateral and contralateral paw withdrawal responses to mechanical (D), cold (E), and thermal (F) stimuli at the times shown before and after SNL. n = 5/group. *P < 0.05, **P < 0.01 vs. the ipsilateral side of the AAV5-EGFP-treated group at the corresponding time point.

Figure 6

Overexpressing Kv1.2 in the DRG does not alter capsaicin-induced acute nociceptive pain. (A) The duration of capsaicin-induced paw licking/lifting was recorded for 5 minutes after capsaicin injection in the AAV5-EGFP-injected and AAV5-Kv1.2-injected groups. n = 5/group. (B-D) Paw withdrawal responses of AAV5-EGFP-injected and AAV5-Kv1.2-injected rats to mechanical (B), heat (C), and cold (D) stimuli on the ipsilateral side prior to viral injection (baseline), before capsaicin injection (Pre-Cap; 5 weeks after viral injection), and 30 minutes after capsaicin injection (Post-Cap). n = 5/group.

Figure 7

Overexpressing Kv1.2 rescues SNL-induced Kv1.2 downregulation and blocks SNL-induced Kv1.2 AS RNA upregulation in the injured DRG. (A) Quantitative RT-PCR shows Kv1.2 AS RNA, Kv1.2 mRNA, and Kv1.4 mRNA expression in the ipsilateral and contralateral L₅ DRGs on day 14 after spinal nerve ligation (SNL) or sham surgery in the AAV5-EGFP-injected and AAV5-Kv1.2-injected groups. n = 12 rats/group. **P < 0.01 vs. the corresponding AAV5-EGFP-injected group after sham surgery; ##P < 0.01 vs. the corresponding AAV5-EGFP-injected group after SNL. (B) Western blot analysis shows Kv1.2 and Kv1.4 protein expression in the ipsilateral (Ipsi) and contralateral (Con) L₅ DRGs on day 14 after SNL in the AAV5-EGFP-injected and AAV5-Kv1.2-injected groups. n = 8 rats/group. **P < 0.01 vs. the contralateral side of the AAV5-EGFP-injected group; #P < 0.05 vs. the ipsilateral side of the AAV5-EGFP-injected group. (C) Western blot analysis shows Kv1.2 and Kv1.4 protein expression in the ipsilateral and contralateral L₅ DRGs on day 14 after sham surgery in the AAV5-EGFP-injected and AAV5-Kv1.2-injected groups. n = 8 rats/group. **P < 0.01 vs. the contralateral side of the AAV5-EGFP-injected group.