Distinct roles of ASIC3 and TRPV1 receptors in electroacupuncture-induced segmental and systemic analgesia

Abstract

Previous studies have demonstrated the effects of different afferent fibers on electroacupuncture (EA)-induced analgesia. However, contributions of functional receptors expressed on afferent fibers to the EA analgesia remain unclear. This study investigates the roles of acid-sensing ion channel 3 (ASIC3) and transient receptor potential vanilloid 1 (TRPV1) receptors in EA-induced segmental and systemic analgesia. Effects of EA at acupoint ST36 with different intensities on the C-fiber reflex and mechanical and thermal pain thresholds were measured among the ASIC3^−/−, TRPV1^−/−, and C57BL/6 mice. Compared with C57BL/6 mice, the ipsilateral inhibition of EA with 0.8 C-fiber threshold (0.8Tc) intensity on C-fiber reflex was markedly reduced in ASIC3^−/− mice, whereas the bilateral inhibition of 1.0 and 2.0Tc EA was significantly decreased in TRPV1^−/− mice. The segmental increase in pain thresholds induced by 0.3 mA EA was significantly reduced in ASIC3^−/− mice, whereas the systemic enhancement of 1.0 mA EA was markedly decreased in TRPV1^−/− mice. Thus, segmental analgesia of EA with lower intensity is partially mediated by ASIC3 receptor on Aβ-fiber, whereas systemic analgesia induced by EA with higher intensity is more likely induced by TRPV1 receptor on Ad- and C-fibers.

Keywords

electroacupuncture / analgesia / ASIC3 / TRPV1 / C-fiber reflex

Introduction

Acupuncture analgesia is one of the most intriguing subjects in acupuncture therapy which has been well-documented in the last few decades. The neural mechanism underlying acupuncture analgesia has been demonstrated, including the peripheral afferent pathways, central interaction, and relevant transmitters and modulators [ 1, 2]. The distinct roles of non-nociceptive large afferent fiber (Ab-fiber) and nociceptive small afferent fibers (Ad- and C-fibers) in the peripheral neural pathways mediated electroacupuncture (EA)-induced analgesic effects have been studied with the application of selective blockers [ 3]. However, contributions of the sensory transduction-related ion channels expressed on afferent fibers to the EA analgesia remain unclear. Acid sensing ion channel 3 (ASIC3) known to mediate acid and mechanical responsiveness is located mainly in Ab-fiber innervating the skin and muscle [ 4]. The deletion of ASIC3 channel in mice alters the electrophysiological properties of mechanoreceptors [ 5]. Moreover, the effects of ASIC3 channel on modulating the dynamic mechanical stimuli sensing in physiological condition have been verified [ 6]. Transient receptor potential vanilloid 1 (TRPV1) ion channel belongs to the TRPV subfamily and is highly expressed in sensory Ad- and C-fibers. It can be activated by capsaicin, noxious heat (>42 °C), low pH and voltage, and has been shown to be closely related to noxious physical detection [ 7]. The predominant location of EA-induced TRPV1 expression in unmyelinated C-fiber and thinly myelinated Ad- fiber was confirmed by double staining of TRPV1 with nNOS and peripherin [ 8, 9]. Furthermore, it was shown that TRPV1 is highly expressed in sub-epidermal nerve fibers, and its upregulation following EA stimulation may be critical to the transduction of EA signals to the nervous system [ 10]. Therefore, ASIC3 and TRPV1 receptors are hypothesized to be involved in the peripheral detection of EA stimulation, based on their distribution and functional properties. Both ASIC3 gene knockout (ASIC3^-/-) mice and TRPV1 gene knockout (TRPV1^-/-) mice were used in the present study to investigate the potential roles of the two receptors in segmental and systemic EA analgesia using electromyographic recording of biceps femoris muscle reflex (C-fiber reflex) and mechanical and thermal pain threshold testing.

Materials and methods

Animal preparation

Male ASIC3^-/- mice (n = 9), TRPV1^-/- mice (n = 9), and C57BL/6J mice (n = 9), weighing 20‒25 g, were purchased from Jackson Laboratory (US) and bred at the China Academy of Chinese Medical Science Animal Care Facility. The animals were housed under a 12 h light/dark with ad libitum access to food and water. The tail tips of gene knockout mice were cut off after the experiment, and genome DNA was extracted for further genotype identification by polymerase chain reaction (PCR) test. All animals were treated according to the Guide for Use and Care of Medical Laboratory Animals from Ministry of Public Health of the People’s Republic of China.

Electromyographic recording

Electromyographic recording was carried out as previously described [ 11, 12]. The ipsilateral biceps femoris muscle reflex was evoked by electrical stimulation of the C-fiber within the receptive field of the sural nerve. Under 4% chloral hydrate (0.1 ml/g dose, i.p.) anesthesia, a pair of non-insulated platinum–iridium stimulating electrodes was inserted into the medial part of the fourth and lateral part of the fifth toes. Electromyographic (EMG) responses were recorded via another pair of needle electrodes inserted through the skin into the biceps femoris muscle. Single-square shocks of electrical stimuli with 1 ms duration were delivered once every 5 s by a constant-current stimulator. EMG responses were fed to an oscilloscope for continuous monitoring, captured online, and analyzed offline by a data acquisition system (Power-Laboratory/4s, AD Instruments) and Chart 5.2 software. The minimal electrical stimulations that can evoke the C-fiber-mediated reflex of EMG response were considered as the thresholds of C-fiber (Tc) and taken as the values of the EA intensities in the following pain threshold test section. Electrical stimulation with intensity of 1.2 times of Tc was administered to elicit stable C-fiber-mediated reflex of EMG responses. Pain threshold tests were applied to the animals following a 48 h recovery.

Mechanical pain threshold test

Mechanical pain threshold test was performed in animals placed in individual test compartments in an elevated mesh-bottomed platform with a grid [ 13]. The animals were acclimatized to the testing environment for 30 min. Mechanical pain thresholds were measured using a dynamic plantar aesthesiometer (UGO Basile, Italy) by applying increasing pressure (36.5 g 36 s Ramp) to the plantar surface of bilateral hindpaws until the withdrawal response was elicited. The test was repeated three times in each hindpaw, and the mean value of paw withdrawal latency was calculated.

Thermal pain threshold test

Thermal pain thresholds of bilateral hindpaws were assessed by exposing the plantar surface of hindpaw to a beam of radiant heat using a plantar analgesia apparatus (UGO Basile, Italy) [ 14]. The stimulus intensity (IR30) was adjusted to give 5‒7 s withdrawal latency in the C57BL/6 mice (baseline). A cut-off of 10 s was set to avoid potential tissue damage. Paw withdrawal latency was determined as the mean value of three consecutive trials with 180 s intervals.

EA protocol in electromyographic test

EA used in this section was quantified as multiples of the threshold for the activation of C-fiber (Tc) to produce the anti-nociceptive responses, including 0.8, 1.0 and 2.0 times of the threshold (0.8, 1.0 and 2 Tc, respectively). The EMG responses elicited by 1.2 Tc electrical stimulation were kept stable before EA stimulation (as control). EA (50 Hz) at ipsilateral or contralateral acupoint ST36 with different intensities was administered for 20 min in mice anesthetized by 4% chloral hydrate, and the EMG responses were recorded at 0, 1, 2, 3, 4, and 5 min after EA stimulation. The frequency of EMG responses following EA stimulation was normalized and expressed as the percentage of the control value.

EA protocol in behavioral tests

EA stimuli (50 Hz) were delivered via a pair of non-insulated needle electrodes inserted into the right ST36 with 0.3 cm distance. Distinct EA stimuli (0.3 and 1.0 mA, determined by the value of Tc from the EMG recording) were applied at ST36 for 20 min in conscious mice stabilized by self-made sack. Mechanical and thermal pain thresholds of the bilateral hindpaws were detected before and immediately after EA application. Changes in the mechanical and thermal pain thresholds were calculated according to (post-EA − pre-EA)/pre-EA × 100%.

Statistical analysis

Data were expressed as mean±SE. Differences in pain thresholds between groups were analyzed for statistical significance by paired t-test. The results of EMG recording were compared using two-way ANOVA followed by Tukey post-hoc test. The two factors considered were EA treatments and time period. A value of P<0.05 was considered significant.

Results

General characteristics of noxious C-fiber-mediated reflex responses of EMG

Electrical stimulation with 1.2 times of the threshold for the activation of C-fiber-mediated reflex (1.2 Tc) on the sural nerve territory evoked a stable reflex EMG response in the ipsilateral biceps femoris muscle. As shown in Figs. 1–3 (A, B), the EMG responses recorded in three groups of mice were generally with a certain latency (18.5±0.7 ms), duration (10.3±0.9 ms), and threshold (0.54±0.04 mA, i.e., Tc). If one considers velocity of C-fiber (1- 2 m/s) [ 15] and the distance of the afferent pathway from the stimulating needles in the territory of the sural nerve to its projecting termination in the lumbar spinal cord, the following can be stated: neglecting synaptic delays occurring within the spinal cord, then responses elicited by the activation of unmyelinated fibers should occur at latencies from 15 ms to 30 ms. Given the mean latency recorded in this study, it could be reasoned that the reflex firing was mediated by activation of unmyelinated C-fiber afferents.

Roles of ASIC3 and TRPV1 receptors in the inhibitory effects on C-fiber-mediated reflex response of EMG induced by EA with different intensities.

The effects of the EA at ipsilateral or contralateral ST36 on the C-fiber reflex were investigated in this section. Fig. 1 shows that 0.8 Tc EA at ipsilateral ST36 produced a slight but significant reduction of the C-fiber reflex, lasting for only 1‒2 min among the three groups of mice (P<0.01, P<0.001). However, the reduction of the C-fiber reflex by 0.8 Tc EA at ipsilateral ST36 in ASIC3^-/- mice was significantly lower than that in C57BL/6 mice (P<0.01, P<0.001). Notably, 0.8Tc EA at contralateral ST36 had no marked effect on the C-fiber reflex among the three groups of mice. These results suggested that EA with lower intensity (<Tc) produced regional inhibition of C-fiber reflex, in which the ASIC3 receptor was highly involved.

The 1.0 and 2.0 Tc EA at either ipsilateral or contralateral ST36 produced significant inhibition on the C-fiber reflex among three groups of mice (P<0.05, P<0.01, and P<0.001, Fig. 2C and 2D, Fig. 3C and 3D). However, the reduction in TRPV1^-/- group was significantly lower than those in the two other groups (P<0.01, P<0.001). In addition, the inhibitory effects induced by 1.0 and 2.0 Tc EA stimuli lasted much longer (4‒5 min) than that of 0.8 Tc after EA application. These results implied that stronger systemic inhibitory effects on C-fiber reflex induced by EA stimuli with intensities higher than or equal to Tc were partially mediated by TRPV1 receptor.

Roles of ASIC3 and TRPV1 receptors in modulating pain thresholds produced by EA in conscious mice

The 0.3 and 1 mA EA stimuli with a duration of 1 ms and frequency of 50 Hz were administered at right ST36 on mice. Changes in mechanical and thermal pain thresholds induced by distinct EA stimuli were investigated among C57BL/6, ASIC3^-/-, and TRPV1^-/- mice. Bilateral mechanical and thermal pain thresholds were significantly increased by 1.0 mA EA among the three groups of mice (P<0.05, P<0.01, and P<0.001; Fig. 4). The analgesic effect of 0.3 mA EA was found only on the ipsilateral side in C57BL/6 and TRPV1^-/- mice (P<0.05, P<0.01; Fig. 4A and 4B, 4E and 4F). However, 0.3 mA EA had no impact on pain thresholds in ASIC3^-/- mice (Fig. 4C and 4D).

The enhancement of mechanical and thermal pain thresholds after EA was normalized with the pre-EA values. Compared with C57BL/6 mice, the enhancement on ipsilateral mechanical and thermal pain thresholds induced by 0.3 mA EA at ST36 was significantly reduced in ASIC3^-/- mice (increase in mechanical pain threshold: C57BL/6: 9.67%±2.49% vs. ASIC3^-/- : 0.44%±2.20%, P<0.05; increase in thermal pain threshold: C57BL/6: 12.39%±3.39% vs. ASIC3^{- / -} : 0.42%±1.53%, P<0.05, Fig. 5A and 5C). Moreover, the analgesic effects of 1.0 mA EA were markedly decreased bilaterally in TRPV1^-/- mice (increase in ipsilateral mechanical pain threshold by 1 mA: C57BL/6: 15.79%±2.13% vs. TRPV1^-/- : 8.03%±2.15%, P<0.05; contralateral: C57BL/6: 6.67%±1.24% vs. TRPV1^-/- : 2.98%±1.10%, P<0.05; increase in ipsilateral thermal pain threshold by 1 mA: C57BL/6: 16.74%±3.17% vs. TRPV1^-/- : 7.38%±2.40%, P<0.05; contralateral: C57BL/6: 11.87%±3.74% vs. TRPV1^-/- : 2.83%±1.10%, P<0.05; Fig. 5B and 5D). These results further indicated that the analgesic effects of 0.3 mA EA were segmental and closely related with ASIC3 receptor on Ab-fiber, whereas TRPV1 receptor on Ad-/C-fibers was more likely to be involved in systemic analgesia induced by 1.0 mA EA.

Discussion

In the present study, the distinct roles of ASIC3 and TRPV1 receptors in EA analgesia were investigated using electromyographic recording of biceps femoris muscle reflex (C-fiber reflex) and mechanical and thermal pain threshold testing in mutant mice. The thresholds for activation of C-fiber were measured (Tc, 0.54±0.04 mA) by recording of C-fiber reflex responses of EMG. The intensities of EA stimuli were determined based on Tc values to activate different afferent fibers. Combined EMG recording with the detection of mechanical and thermal pain thresholds, our data showed that the segmental analgesia induced by 0.8 Tc or 0.3 mA EA at ST36 was significantly decreased in ASIC3^-/- mice, however, the systemic analgesia caused by 1.0 Tc, 2.0 Tc, as well as 1.0 mA EA, was markedly reduced in TRPV1^-/- mice. Consequently, the results suggested that ASIC3 receptor played a critical role in the segmental analgesia induced by EA with low intensity (<Tc), whereas TRPV1 receptor was more likely to be involved in the systemic analgesia elicited by EA with higher intensity (>Tc).

Increasing evidence has shown that EA can activate various primary afferent fibers including groups II – III [ 16], groups III – IV [ 17], and groups II – IV [ 18]. As suggested by gate control theory, it is generally believed that regional analgesia induced by local EA with low intensity is effective by activating Ab-fiber, whereas systemic analgesia elicited by remote EA stimulation with higher intensity is achieved by exciting Ad-/C-fibers via the diffuse noxious inhibitory control (DNIC) system [ 19]. Cutaneous afferent sensory (Ab-, Ad-, or C-) fibers can be classified according to sensory modality; light touch is mediated mainly by Ab-fiber with low mechanical thresholds, whereas the perception of painful touch is initiated by high-threshold Ad- and C-fibers. Moreover, recent work has revealed a number of ion channels that are candidate transducers of somatosensation in these fibers [ 20]. However, ion channels responsible for the transduction of EA signal still remain elusive.

Degenerin/epithelial Na⁺ channels (DEG/ENaC) and transient receptor potential (TRP) families of ion channels form molecular components of the mechanotransduction complex which detects mechanical stimuli [ 21]. These channels locate within nerve endings in a multitude of species, and disruption of these channels results in alteration of the mechanical detection [ 22– 24]. ASIC3 receptor was expressed in dorsal root ganglia neurons and innocuous Ab-fiber which correspond to low threshold mechano-detection [ 5] and the sensing of dynamic mechanical stimuli in physiological condition [ 6]. On the contrary, the sub-epidermal nerve fibers showed a colocalization of TRPV1 with peripherine, a marker for the noxious Ad- and C-fibers. Furthermore, the expression of TRPV1 in sub-epidermal nerve fibers is significantly increased by EA, implying that TRPV1 in local nerve terminals may play a vital role in response to EA stimulation [ 10]. Although there is no evidence that TPRV1 receptor directly mediates the cutaneous mechanotransduction process [ 25], the release of neuroactive mediators induced by EA stimulation can activate or sensitize TRPV1 in afferent terminals [ 26]. Therefore, in the present study, the detection of EA with lower intensities (0.3 mA and 0.8Tc) in ASIC3^-/- mice was impaired so that the segmental analgesia induced by activation of Ab-fibers was reduced. However, the perception of EA with higher intensities (1.0 and 2.0 Tc, as well as 1.0 mA) was weakened in TRPV1^-/- mice, leading to the decreased systemic analgesia elicited by activation of Ad-/C-fibers.

The EMG responses of biceps femoris muscle elicited by electrical stimulation on the sural nerve receptive field (C-fiber reflex) were recorded as a pain index in the present study. Animal studies have shown that the C-fiber reflex is strongly inhibited by distant noxious stimulation applied to paw and tail [ 12] or by nerve stimulation [ 27]. In humans, the painful sensation and nociceptive reflex (R_III) evoked by electrical stimulation on the sural nerve are inhibited not only by EA below R_III threshold applied at regional acupoints but also by heterotopic noxious stimuli [ 19, 28]. The results in the present study also indicate that the C-fiber reflex could be profoundly inhibited by both EA at ipsilateral ST36 with the intensity below Tc and EA at the contralateral ST36 with intensities equal to or higher than Tc. Moreover, it could not be decreased by innocuous EA at the contralateral ST36, as consistent with previous studies’ findings. Collectively, the inhibition of C-fiber reflex and R_III reflex are closely related to both the intensity and location of EA stimulation.

In summary, the results indicated that the segmental analgesic effects of EA with low intensity are mediated by afferent Ab-fiber, and the systemic analgesic effects of EA with high intensity are attributed to the activation of Ad/C-fibers. Furthermore, ASIC3 receptor plays a critical role in the segmental analgesia induced by EA with low intensity (<Tc), whereas TRPV1 receptor is involved in the systemic analgesia elicited by EA with higher intensity (>Tc).

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