TET1 Participates in Complete Freund's Adjuvant-induced Trigeminal Inflammatory Pain by Regulating Kv7.2 in a Mouse Model

Abstract

Trigeminal inflammatory pain is one of the most severe pain-related disorders in humans; however, the underlying mechanisms remain largely unknown. In this study, we investigated the possible contribution of interaction between ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and the voltage-gated K⁺ channel Kv7.2 (encoded by Kcnq2) to orofacial inflammatory pain in mice. We found that complete Freund's adjuvant (CFA) injection reduced the expression of Kcnq2/Kv7.2 in the trigeminal ganglion (TG) and induced orofacial inflammatory pain. The involvement of Kv7.2 in CFA-induced orofacial pain was further confirmed by Kv7.2 knockdown or overexpression. Moreover, TET1 knockdown in Tet1^flox/flox mice significantly reduced the expression of Kv7.2 and M currents in the TG and led to pain-like behaviors. Conversely, TET1 overexpression by lentivirus rescued the CFA-induced decreases of Kcnq2 and M currents and alleviated mechanical allodynia. Our data suggest that TET1 is implicated in CFA-induced trigeminal inflammatory pain by positively regulating Kv7.2 in TG neurons.

Keywords: Facial pain; KCNQ2 potassium channel; TET1 protein.

Fig. 1

Mechanical allodynia in the whisker pad region in CFA model mice and alterations in M currents and Kcnq2. A The animal before (upper) and after (lower) CFA injection in the whisker pad. B Mechanical allodynia was tested on naïve mice (n = 6) and on model mice on day 3 or day 7 after CFA injection (n = 6). The data are presented as the mean ± SEM. C, D Two sets of sample traces of CFA (purple) and naïve (black) mice depicting the action potential (AP) of TG neurons. E–H Summary data of resting membrane potential (RMP; P = 0.0005, E), rheobase (P = 0.0218, F), AP threshold (P = 0.2720, G), and AP amplitude (P = 0.2514, H) in the TG neurons in naïve (n = 15) and CFA (n = 14) mice. Data are presented as the mean ± SEM. I Two sample traces depicting the M current recorded in the TG neurons in the CFA group (purple) vs the naïve group (black). J Summary data of M currents in the TG neurons in naïve (n = 15) and CFA (n = 15) mice (P = 0.0076). The data are presented as the mean ± SEM. K Relative mRNA expression of Kcnq2 and Kcnq3 in the trigeminal ganglia in naïve and CFA mice at day 3 and day 7. Data are presented as the mean ± SEM. L Representative western blots and summarized results of Kv7.2 protein in the trigeminal ganglia in naïve and CFA mice at day 3 and day 7. The data are presented as the mean ± SEM. M, N TG slices double-stained with Kv7.2 (red) and NF200/CGRP/Ib4 (green) antibodies. Scale bars, 50 µm.

Fig. 2

Behavior, M currents, and APs in si-Kcnq2 and si-scrambled mice. A Mechanical allodynia was tested in si-Kcnq2 (n = 6) and si-scrambled (n = 6) mice. The data are presented as the mean ± SEM. B Two sample traces depicting the M currents recorded in TG neurons in the si-Kcnq2 group (purple) vs the control group (black). C Summary data of M currents in the TG neurons in si-scrambled (n = 15) and si-Kcnq2 (n = 15) mice (P = 0.0110). The data are presented as the mean ± SEM. D Two sets of sample traces of si-Kcnq2 (purple) and control (black) mice depicting the action potential (AP) in TG neurons. E–H Summary data of RMP (P = 0.0004, E), rheobase (P = 0.0003, F), AP threshold (P = 0.0013, G), and AP amplitude (P = 0.0749, H) in the TG neurons in si-scrambled (n = 15) and si-Kcnq2 (n = 14) mice. The data are presented as the mean ± SEM.

Fig. 3

Behavior, M currents, and APs in mice with Kv7.2 agonist ML213 injection after CFA injection. A Mechanical allodynia tested in ML213 (0.2, 1, 2, 10, 20 nmol) + CFA mice and CFA mice (n = 6). The data are presented as the mean ± SEM. B Two sample traces depicting the M currents recorded in TG neurons in the ML213 (2 nmol) + CFA group (purple) vs the CFA group (black). C Summary data of M currents in TG neurons in the CFA (n = 17) and ML213 (2 nmol) + CFA (n = 17) mice (P = 0.0010). The data are presented as the mean ± SEM. D Two sets of sample traces of ML213 (2 nmol) + CFA (purple) and control (black) mice depicting the action potential (AP) in TG neurons. E–H Summary data of RMP (P = 0.0109, E), rheobase (P = 0.0189, F), AP threshold (P = 0.1913, G), and AP amplitude (P = 0.8500, H) in the TG neurons in CFA (n = 15) and ML213 (2 nmol) + CFA (n = 14) mice. The data are presented as the mean ± SEM.

Fig. 4

Change in TET1 expression levels and coexpression of TET1 and Kcnq2. A Relative mRNA expression of TET1–3 in the trigeminal ganglia in naïve and CFA mice. The data are presented as the mean ± SEM. B Representative western blots and summarized results of TET1 protein in the trigeminal ganglia in naïve and CFA mice. The data are presented as the mean ± SEM. C, D TG sections from mice doubly stained with anti-TET1 (red) and anti-NF200/CGRP/Ib4 (green) antibodies. Scale bars, 50 µm. E TG sections were doubly stained with Kv7.2 (red) and TET1 (green) antibodies. Scale bar, 50 µm.

Fig. 5

Behavior, Kv7.2 protein, M currents, and APs in selective TET1 knockdown mice. A Mechanical allodynia in TET1 knockdown and negative-control mice. The data are presented as the mean ± SEM. B, C Representative western blots and summarized results of TET1 and Kv7.2 protein in the trigeminal ganglia in TET1 knockdown and negative control mice. The data are presented as the mean ± SEM. D Two sample traces depicting the M currents recorded in TG neurons in the TET1 knockdown group (purple) vs the control group (black). E Summary data of M currents in TG neurons in TET1 knockdown (n = 16) and control (n = 15) mice (P = 0.0047). The data are presented as the mean ± SEM. F Two sets of sample traces of TET1 knockdown (purple) and control (black) mice depicting the action potential (AP) in the TG neurons. G–J Summary data of RMP (P = 0.0600, G), rheobase (P = 0.0075, H), AP threshold (P = 0.0990, I), and AP amplitude (P = 0.7819, J) in TG neurons in TET1 knockdown (n = 13) and control (n = 13) mice. The data are presented as the mean ± SEM.

Fig. 6

Behavior, Kv7.2 protein, M currents, and APs in TET1-overexpressing and CFA mice. A Mechanical allodynia. The data are presented as the mean ± SEM. B, C Representative western blots and summarized results of TET1 and Kv7.2 protein in the trigeminal ganglia. The data are presented as the mean ± SEM. D Two sample traces depicting the M currents recorded in the TG neurons in the TET1-overexpressing group (purple) vs the control group (black). E Summary data of M currents in the TG neurons in TET1-overexpressing (n = 17) and control (n = 18) mice (P = 0.0252). The data are presented as the mean ± SEM. F Two sets of sample traces of TET1-overexpressing mice (purple) and control mice (black) depicting the action potential (AP) in the TG neurons. G–J Summary data of RMP (P = 0.0408, G), rheobase (P = 0.0382, H), AP threshold (P = 0.4559, I), and AP amplitude (P = 0.8986, J) in the TG neurons in TET1-overexpressing (n = 19) and control (n = 18) mice. The data are presented as the mean ± SEM.