2.1 Mechanisms of acupuncture analgesia
There are many proposed mechanisms regarding to acupuncture analgesia. Among which, the most acknowledged is the neural mechanism.32 It is proposed since acupuncture analgesia can be blocked if procaine is injected into acupoint in advance of acupuncture. The neural regulation mechanism can be divided as the central or the peripheral (local) mechanism depending on the site of regulation.17
Furthermore, the central regulation can be divided into the spinal mechanism and the supraspinal mechanism. For the spinal mechanism, it is believed that the acupuncture stimulation gives inhibitory regulation to noxious stimulation-induced impulses. This was demonstrated in cat that electroacupuncture at ST36 produce the inhibitory post-synaptic potentials (IPSPs) and long-lasting membrane hyperpolarization in nociceptive spinal dorsal horn neuron.33 For the supraspinal mechanism, it is believed that acupuncture impulse transfer signals from spinal dorsal horn through ventrolateral funiculus to pain regulating areas in the brain and then modulate pain impulse after transferring inhibitory impulse by way of descending inhibitory pathway to pain related lamina in dorsal horn. This hypothesis of acupuncture analgesia was strengthen by experiments demonstrated that the acupuncture effect was attenuated after creating section or lesion to the corresponding ascending and descending tracts.34,35 During the past decades, many nuclei in the brain were identified to participate in acupuncture analgesia,17 these include, RVM (mainly NRM), periaqueductal gray (PAG), locus coeruleus (LC), arcuate nucleus (Arc), preoptic area (Po), centromedian nucleus (CM), nucleus submedius (Sm), anterior pretectal nucleus (APtN), habenular nucleus (Hab), nucleus accumbens (Ac), caudate nucleus (Cd), septal area (Sp), amygdale, anterior
cingulated cortex (ACC), and hypothalamic paraventricular nucleus (PVH).
Among them, the Arc-PAG-NRM-spinal dorsal horn pathway and the opioid receptors seem to play a vital role during acupuncture analgesia.
Recently, a localized mechanism for acupuncture analgesia was raised by Goldman et al.19 They demonstrated with microdialysis and high performance liquid chromatography (HPLC) that during manual acupuncture (MA), ATP was release abundantly at the acupoint after manipulation. This localized ATP release was particularly evident in the acupoint compared to the non-acupoint. They also showed that ATP was later degraded to pain relieving adenosine by maybe prostatic acid phosphatase and resulted in local analgesia. The participant of adenosine is further supported by their use of adenosine A1 receptor knock-out mice. After inducing inflammatory pain or neuropathic pain on the adenosine A1 receptor knock-out mice, MA on the adenosine A1R knock-out mice failed to repeat analgesia as in wild-type mice. This is a strong evidence for involvement of adenosine A1 receptor during acupuncture analgesia. Works from Goldmen et al. showed that ATP was released after manipulation on acupoint. After then, ATP was dephosphated to adenosine and result in acupuncture analgesia by activating inhibitory adenosine A1 receptor. Their study sure is a milestone in acupuncture research, but questions remained to be answered as how ATP is released during MA and why in inflammatory pain model acupuncture-induced inhibitory effect on innervated nerve of ST36 (deep peroneal nerve) is related to desensitize on innervated nerve of paw (tibia nerve)?
2.2 Properties of TRPV1 channel
The transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation permeable channel belong to the transient receptor potential vanilloid family. It can react to various stimulations including capsaicin, heat (>43°C), low pH, voltage, and endogenous lipid.36 Although TRPV1 is permeable to cations as sodium, potassium, and calcium, TRPV1 showed preference for calcium. The TRP family channels are composed of six transmembrane segments (S1-6) with a pore forming loop between S5 and S6. Like most TRPV channels, a six ankyrin repeats is found at its cytosolic N-terminus, which is the site for phosphorylation and to sensitize the channel. 37
Before first recognition, its specific agonist, capsaicin, has long been used as reagent to activate the pain-related C-fibers. Interestingly, after activation of C-fibers, an initial pain sensation is caused. But when capsaicin is given repeatedly or at a high dose, within seconds, an inhibitory effect named conduction analgesia or desensitization is induced.38,39 The conduction analgesia is generated by calcium influx into cell which interact with calmodulin to cause dephosphorylation of TRPV140,41 and by tonic inhibition from PIP2.42,43 However, the conduction analgesia is not the only mechanism for analgesia by capsaicin; another mechanism called noxious stimulus-induced analgesia (NSIA) also participated in capsaicin induced analgesia and will be discussed later.
It is worth mentioning that ATP released to surrounding was reported after activating TRPV1 on urothelial cells.20,21 It is yet not certain of the mechanism for ATP release after TRPV1 activation. Nonetheless, the mechanism for ATP release after mechanical stimulation was reported for another TRPV channel, TRPV4. In the case of TRPV4, Rho kinase was showed to participate after channel activation and trigger ATP release by hemichannel pannxin 1.22
2.3 Properties of TRPV4 channel
The transient receptor potential vanilloid 4 (TRPV4) is another mechanosensitive channel from the TRPV channel family. It is structurally similar to TRPV1 and also has preference to permit calcium.44 Other than heat (>24-27˚C), TRPV4 can be activated by chemicals as 4α-PDD, GSK1016790A, epoxyeicosatrienoic acids, bisandrographolide as well as physical stimulations such as osmolality change and mechanostimulation.
TRPV4 was detected in various diameters of neurons in the DRG, therefore the expression is not restricted to small diameter fibers as C-fibers.45
Similar to TRPV1, TRPV4 is related to local ATP release from cell after mechanical stimulations.20,22,23 As reported by Seminario-Vidal et al. in airway epithelial cells, Rho kinase activation occurred after TRPV4 activation and trigger ATP release by hemichannel pannxin 1.22
2.4 Properties of ASIC3 channel
The acid-sensing ion channels 3 (ASIC3) is one of seven member of the ASIC channel family (ASIC1a, ASIC1b, ASICb2, ASIC2a, ASIC2b, ASIC3, and ASIC4), which can be activated by extracellular proton and make them pH sensors for cells. The distributions of the ASIC family are different as ASIC1a and ASIC2 are presented both in CNS and PNS; however ASIC1b and ASIC3 are restricted to PNS.46 Beside from proton sensing, ASIC3 channel is also suggested to be a mechanosensitive channel.47,48 Similar to TRPV1 and TRPV4, ASIC3 is related to local ATP release after stimulation.20
ASIC3 was reported to play a vital role in inducing acid-induced muscle pain created by repeated pH 4 saline injection at gastrocnemius muscle.
Jerzy Karczewski et al.49 reported APETx2, an ASIC3 specific antagonist, reduced mechanical hypersensitivity in rodent acid-induced muscle pain
model created by repeated pH 4 saline injection. They concluded that ASIC3 is the major sensing component when injecting pH 4 saline to muscle. This implies pH4 saline injection is capable of activating ASIC3 in vivo.
2.5 Relationship between acupoint and the mechanosensitive channels : TRPV1, TRPV4, and ASIC3
Recently there are papers implying the involvement of the mechanosensitive TRPV1, TRPV4, and ASIC3 channels during acupuncture.
First, as demonstrated by Yamamoto et al.25 that when applying manual acupuncture (MA) or electroacupuncture (EA) to ST36 of rats, both heart rate and blood pressure would transiently attenuate during acupuncture stimulation. However, if the stretch-activated channel blocker gadolinium was given systemically during acupuncture, the attenuation effects by acupuncture were depressed. This strongly suggests that at least a stretch-activated channel is participated during MA. Notably, TRPV1, TRPV4, and ASIC3 are channels that could be non-selectively blocked by gadolinium. Furthermore, Abraham et al.50 revealed that TRPV1 is more abundantly expressed in subepidermal nerve fibers of BL40 and BL57 acuppoints compared to non-acupoint. Also, TRPV1 in subepidermal nerve fibers of acupoint increased after EA.
2.6 Noxious stimulus-induced analgesia (NSIA)
The noxious stimulus-induced analgesia (NSIA) is a heterosegmental antinociception in which pain sensation is reduced by another pain impulse.
This pain-induced analgesia can be generated by hot water (50°C) or subdermal capsaicin injection and can be equivalent in magnitude to that of high dose morphine in jaw-opening reflex.51 Depending on the site of noxious regulation, mechanism for NSIA could be divided into the spinal pathway and the supraspinal pathway.
For the spinal pathway, when capsaicin is injected into subcutis, TRPV1 activates primary afferent fiber and leads to release of substance P at the laminae I, III and IV of spinal cord. This in turn activates Neurokinin 1 (NK1) receptor of substance P on inhibitory interneuron.52 Other than substance P, NSIA can also lead to release of glutamate at spinal lamina which also causes activation of inhibitory interneuron by ionotropic glutamate receptors.53 The activated inhibitory interneuron responses by increases γ-aminobutyric acid (GABA) or glycine release at nociceptive related lamina II. As a result, noxious impulse to the lamina II is negatively regulated.
For the supraspinal pathway, modulation between the spinal lamina and the nucleus accumbens plays a major role. In normal physiological status, tonic activity by spino-supraspinal projection inhibits activity from the nucleus accumbens, which after activation turns on pain modulating Habenula-PAG-NRM-spinal dorsal horn pathway.54,55 However, when NSIA was introduced by capsaicin, primary afferent stimulated by capsaicin release glutamate at spinal lamina and activate inhibitory interneuron by NMDA receptor, AMPA/kainite receptor and mGluR5 receptor. The activated interneuron then suppresses the inhibitory spino-supraspinal projection by releasing opioid and GABA. As a result, the nucleus accumbens is freed from inhibition and turns on the opioid related pain
modulating Habenula-PAG-NRM-spinal dorsal horn pathway.56
It’s noteworthy that aside from capsaicin, NSIA can also be activated by agonist of P2X receptor, αβ-methylene-ATP. This was demonstrated by increased frequency of the spontaneous inhibitory postsynaptic currents (sIPSC) from the spinal lamina II after applying αβ-methylene-ATP as when capsaicin was applied.53
2.7 Calcium wave propagation (CWP)
It’s worth mentioning that the local ATP released is related to intercellular purinergic signaling called calcium wave propagation (CWP).26,57 Once activated by extracellular ATP via purinergic P2Y receptors (P2Y1 or P2Y2 receptors), the stimulated cells are then processed through intracellular calcium signaling, resulting in ATP release by hemi-channels (e.g., pannexin 1 or connexin 43). ATP released from the cells then stimulates purinergic receptors in the nearby cells in a paracrine manner and causes again both intracellular calcium signaling and ATP release. This chain-like process can continue for a certain distance before termination. The phenomenon is universal and is reported among glia,26 salivary glands,27 nephrons,28 fibroblasts,29 etc. Recently, CWP during acupuncture was reported at acupoint in both neural and non-neural cell.31
It was proposed in keratinocytes30 and subepithelial fibroblasts29 of villi that cells respond to mechanical stimulation by local ATP release. Then, by way of CWP, the stimulation signal was conduct to nearby neuron indirectly.
This demonstrates an alternative sensing pathway other than direct neural stimulation.