Department of Anesthesia, Bradford Teaching Hospitals NHS Foundation Trust, Bradford BD9 6RJ, United Kingdom
Plasticity is inherent within the nervous system. Such plasticity underpins the development of persistent pain following a primary insult. The mechanism by which this occurs is complex and has components within the peripheral and central nervous system. In general, nociceptive neuronal hypersensitivity develops as a result of positive feedback loops, descending facilitation, and loss of inhibitory modulation at the spinal and supraspinal level. This article will focus on various neurobiological elements currently felt to be key to this process. Understanding this process as a pathological phenomenon as opposed to passive transmission has identified numerous targets for therapeutic intervention (pharmacological, psychological, and surgical) and allowed an advance towards rational treatment within the field of pain medicine.
Persistent or chronic pain can be inflammatory or neuropathic and is the result of aberrant functioning of the peripheral or central nervous systems (CNS) that has been pathologically modified. It is not directly related to a noxious stimulus and persists beyond the tissue damage that initiated the pain. The characteristics of the pain, hyperalgesia, and allodynia reflect peripheral and central sensitization of the nociceptive pathways. The fundamental science underpinning persistent pain perception is a continuously evolving field with a bewildering complexity. The diverse range of chemical mediators, cell types, receptors, ion channels, secondary messenger pathways, and novel neural connectivity investigated and implicated, makes a clear sequential explanation that links stimulus to perception, a target far beyond the capacity of a single chapter. Instead, using a simplistic pain pathway as a clothes line, we will peg an overview of current concepts along its length and hope the reader and the line are not weighed down by the meandering and sometime overlapping explanations of this pathological phenomenon.
FROM THE NOCICEPTOR TO THE SPINAL CORD
In the majority of cases, persistent pain begins with a peripheral insult producing an inflammatory response with or without nerve injury. There is then increased excitability of primary nociceptive afferents towards threshold for action potential generation, and in some instance, to the point where action potentials develop spontaneously. The resulting increased action potential frequency in combination with facilitation of excitation-secretion coupling in the dorsal horn increases release of excitatory neurotransmitters and so activation of the central components of the nociceptive pathway. Critical to this peripheral sensitization is an inflammatory process that amplifies and prolongs the impact of the initial insult: the inflammatory soup—the receptors that mediate the influence of this soup become hypersensitive to noxious and non-noxious stimuli and allow drift of the membrane potential toward threshold; transient receptor potential (TRP) ion channels and acid sensing ion channels (ASICs); and the voltage-gated ion channels—the activity of which influences the resting membrane potential, action potential initiation and propagation, and neurotransmitter release—voltage-gated Na+, K+, and Ca2+ channels (Figure 1).
The Inflammatory Soup
Apart from activating the nociceptive neuron and producing an action potential, noxious stimuli can also cause tissue damage and inflammation. The activated and in some cases damaged neurons, along with resident and recruited inflammatory cells (including mast cells, monocytes and macropahges, neutrophils, platelets, and endothelial cells) and the released inflammatory mediators [including arachidonic acid derivatives, cytokines, chemokines, peptides (substance P, bradykinin, calcitonin gene-related peptide), proteases, protons, and purinergic acids] form what is collectively described as an inflammatory soup.
The presence of inflammation is significant in both inflammatory and neuropathic pain, meaning that pathophysiological distinction between the two syndromes is blurring. Immune cells and the released mediators appear to have pivotal roles in the development of peripheral sensitization.1
Figure 1: Factors influencing primary afferent neuron sensitivity and excitability. Injury activates inflammatory cells and neurons causing the release of inflammatory mediators (IL-1β, IL-6, TNF-α, proteases, PAR, NGF, endovanilloids, H+, PGE2, and CLL2) and neuropeptides (ATP and substance P). These factors bind to a number of receptors (NK1, P2X, GPCR G, PAR, trkA, ASIC, and CCR2) in the cell membrane of the afferent neuron. This leads to sensitization of the TRPV1 and TRPA1, and increased neuronal excitation by inhibiting M channels and stimulating Nav.
Apart from the classical immune cells, mast cells, neutrophils, macrophages, and T-cells in nerve injury Schwann cells appear to be fulfilling an immunological role releasing cytokine and phagocytosing debris. Stabilization of mast cells and reduction in infiltration by neutrophils, macrophages, and T cells have all been shown to reduce the development of hyperalgesia and allodynia.2+-5 The role of Schwann cells is slightly more complex. Reduction in tumor necrosis factor-α (TNF-α) expression by Schwann cells reduces neuropathic pain6 and disruption of ErbB expression; a tyrosine kinase important in Schwann cell–neuron interaction caused loss of thermal hypersensitivity in nonmyelianted cells.7 However, disruption of ErbB expression in myelinated cells produced mechanical allodynia.8
With respect to inflammatory mediators, significant candidates include interleukin-1β (IL-1β), TNF-α, IL-6, chemokine ligand 2 (CLL2), prostaglandin E2 (PGE2), adenosine triphosphate (ATP), and nerve growth factor (NGF). IL-1β and TNF-α both induce neuropathic pain on direct injection,9,10 and impairment of their signaling attenuates neuropathic phenomenon.11–13 IL-1β has a number of speculated sites of action: inducing the release of more inflammatory mediators generating positive feedback14 as well as causing direct neuronal excitation15 and stimulating the release of the neuropeptides substance P and calcitonin gene related peptide (CGRP),16,17 which activate voltage-gated sodium (Nav) channels. TNF-α may increase excitation through p38 mitogen kinase mediated phosphorylation of Nav channels (see below). The role of IL-6 is a little less clear since knockout mice exhibit reduced hyperalgesia and allodynia,18 but peripheral application has an antinociceptive effect.19
Two chemokines having a significant role in persistent pain are CLL2 and fractalkine. CLL2 is expressed by macrophages, Schwann cells, and neurons.1 Fractalkine is secreted primarily by neurons and mediates a significant interaction between neurons and microglia in the dorsal horn (see below). CCL2 is upregulated following nerve injury.20 Null mutants without the required receptor [chemokine receptor 2 (CCR2)] have significantly attenuated pain behavior21 and intraneural administration produces transient mechanical allodynia and thermal hyperalgesia.2+ There is evidence that CLL2 is able to excite dorsal root ganglion (DRG) neurons directly23 and reduces inhibitory γ-aminobutyric acid (GABA)-ergic currents.24 PGE2 activates Nav channels and has a role in central sensitization (see below).
ATP is released from damaged cells and binds to distinct purinergic receptors found on nociceptive neurons and immune cells. Blocking these receptors blunts neuronal excitability and thermal and mechanical hypersensitivity. It appears that ATP acts by activating Nav channels and stimulating the release of inflammatory cytokines.25
NGF has a significant and multifaceted role. NGF sensitizes nociceptive neurons as emphasized by the mutation of its tropomyosin receptor kinase A (trkA) resulting in insensitivity to pain26 and systemic administration triggering thermal and mechanical hyperalgesia.27 NGF sensitizes TRP vanilloid 1 (TRPV1) receptors via phospholipase C (PLC) activation and stimulation of an increase in TRPV1 expression (see below). It also increases the expression of Nav channels28 and stimulates proliferation of immune cells (mast cell, lymphocytes, and neutrophil).1 An NGF antagonist, tanezumab, a monoclonal antibody is currently being trialed for osteoarthritis and other chronic pain conditions (e.g., low back pain, diabetic peripheral neuropathy, and interstitial cystitis). These trials had been temporarily suspended due accelerated osteoarthritis and osteonecrosis. However, subsequent case review indicated that these phenomenona were due to pretreatment disease rates, increased utilization of the effected limb due to improved analgesia, and the inhibition of bone repair by coadministered nonsteroidal anti-inflammatories.29
The primary afferents have a role as a nociceptor converting the physical experience of a noxious stimulus into an action potential. This is achieved via a variety of transducing receptors broadly divided into those detecting temperature changes, mechanical stimuli, and chemicals. The majority of these receptors are members of the TRP ion channel family. Twenty unique ion channels have been identified.30 They are made of subunits with 6 membrane spanning domains and the amino acids linking the 5th and 6th domains forming an ion channel. This ion channel is relatively nonselective being permeable to monovalent cations and calcium, and allows these to enter along their concentration gradient when the cell is hyperpolarized. This increases neuronal excitability by 2 mechanisms. It shift the membrane potential towards 0 mV and so towards the threshold for action potential initiation, and also via calcium dependent mechanisms, leading to transduction of ion channels to the membrane as well as increasing their activation and expression.
Activation and upregulation of 2 TRPs, TRPV1, and TRP ankyrin 1 (TRPA1) has been implicated in peripheral and central sensitization. Apart from responding to noxious stimuli, they are also activated by inflammatory mediators and protons. Clearly, supraphysiological activation is not a ubiquitous process, since only minorities of patients with nerve lesions, for example, develop neuropathic pain. This may relate to genetic susceptibility, and there is evidence to suggest that polymorphism in TRP genes is significant at least in determining pain characteristics, if not overall susceptibility.31
ASIC are another ligand-gated ion channel that may have a role in persistent pain. ASIC are cationic channels, largely sodium specific that are activated by the binding of protons allowing an influx of sodium ions and membrane depolarization.32 They have been specifically linked to inflammatory pain,33 but as discussed, the inflammation is also integral to neuropathic pain.
Transient Receptor Potential Channels
TRPV1 is found on the peripheral nerve terminal, in all sensory ganglia, in the dorsal horn and various regions of the brain [rostral ventral medulla (RVM), periaqueductal gray (PAG), amygdala, nucleus tractus solitarius, somatosensory cortex, anterior cingulated cortex (ACC), and insula]. It is classified as a thermoreceptor along with TRPV2-4, TRP melastin 8 (TRPM8), and TRP ankyrin 1 (TRPA1), and is activated by a heat (>40°C) causing opening of the cation channel. Numerous studies have indicated that TRPV1 plays a significant role in noxious heat detection and thermal hyperalgesia but knockout animals retain some sensitivity which is attributed to the other TRPV subtypes (TRPV2 around 52°C and TRPV3 and TRPV4, 25–35°C).34 The mechanism of heat mediated opening remains unclear. There is a suggestion that conformational change is solely a thermodynamic phenomenon but transferring the C-terminal domain between channel types transfers heat threshold characteristics.35 TRPV1 is also gated by protons (pH <5.2) and numerous chemical ligands. These ligands include vanillioids (e.g., capsaicin—chilli) and other natural compounds (piperine—black pepper; zingerone—horseradish, and allicin—garlic and onions), and endovanilloid (anandamides) found in the inflammatory soup. The presence of hydrogen ions and endovanilloids in the inflammatory soup lowers the temperature threshold contributing to thermal hyperalgesia in inflammation.36,37 Upregulation of TRPV1 has been demonstrated in inflammatory and neuropathic conditions and models, and there is attenuation of hyperalgesia in knockout animal suggesting a significant role in persistent pain.38–41 The influence of inflammatory mediators is largely via modulation of mechanisms that control TRPV1 sensitivity to ligand binding and expression.
The sensitivity of TRPV1 is dependent on a number of factors, none of which have definitive control. Firstly, phosphorylation mediated by protein kinase C (PKC), protein kinase A (PKA), and Ca2+/calmodulin (CaM)-dependent kinase/calneurin phosphatase balance. Secondly, phosphatidylinositol bisphosphonate (PIP2), CaM, and ATP binding, and thirdly, the protease activated receptors 1 and 4 (PAR 1 and 4) (Figure 2).
Figure 2: Modulation of TRPV1 activity. TRPV1 is activated by heat, H+, exogenous (capsaicin), and endogenous (endovanilloids) factors. Sensitivity is controlled by phosphorylation by PKC, PKA, and CaMKII. PKC is activated by Ca2+, PLC activated by Ca2+ and Gq-coupled receptor binding inflammatory mediators, and PAR. PKA is activated by a Gs-coupled receptor binding PGE2. CaMKII is activated by Ca2+. TRPV1 is inhibited by dephosphorylation mediated by Ca2+ activated calcineurin and by hydrolysis of PIP2.
Proinflammatory mediator, such as NGF, ATP, bradykinin, serotonin, histamine, proteases, and chemokines cause phosphorylation of TRPV1, increasing the channels sensitivity.42 These mediators activate PLC via a Gq linked receptor. PIP2 is hydrolyzed to diacylglycerol (DAG) and inositol triphosphate (IP3), and DAG activates PKC which then phosphorylates a serine residue on TRPV1.43 In addition, PLC hydrolyses an inhibitory PIP2 constitutively associated with the receptor contributing to sensitization.44
However, PIP2 probably also has an excitatory role. Following high dose capsaicin, which desensitizes TRPV1 receptors, PIP2 synthesis is important for recovery.45 Also, high dose capsaicin appears to produce TRPV1 desensitization by inducing Ca2+ influx via the receptor and activating PLC which depletes PIP2 levels.42
PIP2 binding occurs at the C-terminus of the receptor. Binding at the N-terminus are CaM and ATP in an area called the ankyrin repeat domain (ARD). Their binding in the ARD causes desensitization of the receptor. Cysteine residues in the N-terminus also appear to be the target for the naturally occurring compounds, including capsaicin (although this also binds to residues between the 2nd and 3rd and 4th and 5th domains)46 (Figure 3).
PKA appears to sensitize TRPV1 by phosphorylating combinations of serine and threonine residues.47 PKA is activated by a G protein-linked prostaglandin receptor that increases cyclic adenosine monophosphate (cAMP).47 TRPV1 does not have a specific binding site for PKA, so for this to occur, PKA is brought into proximity with the TRPV1 receptor by protein kinase anchoring protein, which is coexpressed with the receptor and may bind directly to it.48
Figure 3: Structure of transient receptor potential ion channel vanilloid 1. Consists of 6 membrane spanning domains. The ion channel is located between domain 1 and 2.
The activation of PKA and so TRPV1 sensitization can be inhibited by the binding of opioids to μ-receptors.49
Calcium influx and increasing intracellular calcium may regulate further TRPV1 sensitization by calcium-dependent activation of PLC and 2 other opposing enzymes calcineurin and calmodulin dependent protein kinase. Calcineurin, a CaM-dependent phosphatase inactivates TRPV1 by dephosphorylation.50 CaM-dependent protein kinase activates the channel by phosphorylation of sites targeted by PKC and PKA.51 Apart from reducing the sensitivity of TRPV1 receptors, capsaicin also reduces nerve action potential transmission by reducing the density of all voltage-gated calcium channels. It appears that this is mediated by calcineurin-mediated dephosphorylation.52
PARs are G-protein-linked receptors activated by proteases (e.g., thrombin and trypsin) released as a result of inflammatory processes. PAR1 and PAR4 are located on a fraction of primary sensory neurons (15 and 8%, respectively). Activation leads to translocation of PKC to the cell membrane and sensitization of the TRPV1 channels. It also enhances the release of a proinflammatory neuropeptide CGRP. NGF increases the number of PAR1 and PAR4 expressing neurons.53
In addition to being associated with opening of TRPV1 channels in the membrane, sensitization in inflammatory and neuropathic pain is also the result of upregulation of gene translation and movement of assembled TRPV1 channels from intracellular storage to the cell membrane. These processes are stimulated by NGF and mediated by p38 mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase-src kinase.54–56 In neuropathic pain, there appears to be downregulation of TRPV1 in the damaged neurons and upregulation in the undamaged ones.39
TRPV1 channels are also found on glial cells in lamina I and II and may contribute to their proliferation, which is known to be significant in central sensitization (see below). TRPV1 knockout mice show fewer cells in models of inflammatory and neuropathic pain.57
Within the CNS, TRPV1 has an inhibitory role.58 Within the dorsal horn, TRPV1 receptors are found postsynaptically on lamina II cell bodies and excite inhibitory neurons enhancing GABA and glycine release. Also within the brain, TRPV1 channels are found in areas that mediate antinociception (locus coeruleus neurons, ACC, and PAG). Application of agonists and antagonists topically, systemically, and via intracerebroventricular injection, and microinjection into particular brain areas has demonstrated the significance of these channels in this role. Immunoreactivity has indicated that the TRPV1 channels are located on glutamatergic neurons that may receive input from GABAergic neurons. The antinociceptive effects of TRPV1 activation in these areas appear to be glutamate dependent.
TRPV1 receptors are currently a target for treatment in the form of capasacin. The rationale for using an agonist, as alluded to above, is that induced opening of TRPV1 channels leads to depletion of PIP2 precluding further pathological activation and decreases excitability via reduction in voltage-gated calcium channels. However, this induces unpleasant sensations in the first instance and so is not always well tolerated. An alternative is systemic antagonists, which have been shown to be effective in models of neuropathic and inflammatory pain.59,60 A dose-limiting factor for many, however, has been hyperthermia as TRPV1 also appears to be critical for temperature control, although an antagonist without this problem has been identified.61
An alternative approach to desensitization and antagonism is hypersen-sitization. Persistent TRPV1 activation leads to a prolonged increase in intracellular calcium, which has a deleterious effect on the TRPV1 expressing cells. Therefore, targeting neurons, which are overexpressing TRPV1 leading to hyperalgesia and allodynia with a potent agonist would eliminate those neurons and the pain. Resiniferatoxin (RTX), an example of such an agonist has been shown to selectively ablate vanilloid sensitive neurons while leaving other adjacent neurons unaffected.62 As stated previously, it is the undamaged neurons that exhibit TRPV1 upregulation, and so targeting these for destruction appears counterintuitive. However, RTX injected into the dorsal root and trigeminal ganglia has produced analgesia in inflammatory and neuropathic models without effect on touch, proprioception, mechanociception, or motor function.62
Polymorphism in TRPV1 may contribute to variation in persistent pain presentation. Analysis of TRPV1 gene variants in neuropathic patients has identified two key loci, 1911 lying within the ion channel and 1103 within the ARD.31 1911 A>G polymorphism was associated with altered heat pain threshold; AA and AG genotypes showing hyperalgesia. 1103 C>G polymorphism was associated with altered cold threshold, and CG genotype exhibited cold hypesthesia.
TRPM8 is a cold transducer. Like TRPV1, it is activated by temperature change (<25°C) but also by natural ligands, such as menthol. It is located on separate neurons to TRPV1.63 Knockout mice demonstrate a significant but not complete deficit in cold response at the cellular, nerve fiber, and behavioral level. The residual sensitivity is accounted for by another TRP channel TRPA1 (<15°C).64
PKC appears to inactivate TRPM8.65 This occurs directly by hydrolysis of PIP2 which maintains TRPM8 in an open state,66 and indirectly by the activation of a calcineurin, which dephosphorylates TRPM8.65
Cold allodynia is a feature of neuropathic pain and it is logical that sensitization and increased expression of DRG, TRPM8, and TRPA1 would play a part in this. However, in neuropathic models that demonstrate allodynia in response to cold and menthol, TRPM8 and TRPA1 expression decreases, and there is no increase in channel activity as measured by calcium influx.67 This phenomenon, therefore, may develop at the spinal67 or supraspinal level as suggested by recruitment of the dorsolateral prefrontal cortex (DLPFC) and midbrain noted on functional magnetic resonance imaging (fMRI).68 TRPM8 actually appears to have an analgesic role, since cooling and low dose menthol, which reduce the thermal hyperalgesia and mechanical allodynia in neuropathic models, are ineffective in TRPM8 knockouts.69
Noxious cold sensation may also be influenced by potassium channels. A specific potassium channel blocker has been shown to increase the threshold for cold sensitive neurons and reduce the behavioral response to cold without influencing effect of heat and mechanical stimuli.70
TRPA1 is a TRP channel implicated in the transduction of cold (<15°C), mechanical, and chemical stimuli. It is coexpressed on peripheral nociceptive afferents with TRPV1.63 A structurally diverse range of compounds are able to activate the channel, binding covalently to cysteine residues in the extended ARD at the N-terminus.71 These include an allicin (garlic and onions), cinnamaldehyde (cinnamon oil), allyl isothiocyanate (mustard oil), and acrolein (wood and tobacco smoke, tear gas, and car exhaust).
Unlike TRPV1, which with exception of protons and endovanilloids, is only sensitized by inflammatory mediators, TRPA1 can be directly activated and indirectly sensitized by multiple endogenous inflammatory mediators.72 Like TRPV1, sensitization is mediated by PLC and PIP2 hydrolysis73 and upregulation in TRPA1 membrane expression occurs via a p38 MAPK pathway triggered by NGF.74
TRPA1 appears to have a significant role in persistent pain. Mutation in the gene coding TRPA1 has been recently identified in an autosomal dominant condition with episodes of debilitating upper body pain triggered by fasting and physical stress (familial episodic pain syndrome).75 Knockdown and antagonist studies in models of inflammatory and neuropathic pain demonstrate that TRPA1 mediates mechanical hypersensitivity and cold hyperalgesia.64,74,76,77 Peripheral and intrathecal infiltration of antagonists suggest that hyperalgesia is a result of peripheral expression and allodynia a central expression.78 As with TRPV1, polymorphism may be relevant. Analysis of TRPA1 gene variants in neuropathic pain demonstrated that 710G>A was associated with paradoxical heat sensation.31
Activation of TRPA1 and TRPV1 by anesthesia could contribute to postoperative pain. Airway irritation associated with isoflurane and desflurane and pain on intravenous injection of propofol and etomidate have been attributed to TRPA1 activation.79 Local anesthetic infiltration (lignocaine and bupivacaine) activates TRPV1 and to a lesser extent TRPA1.80 While these effects are short lived, such activation may lead to peripheral and central sensitization.79
Acid-sensing Ion Channels
ASIC3 is a subtype located on nociceptive neurons in cardiac and skeletal muscle.81 It is particularly sensitive to lactic acid81 and mediates cardiac ischemic pain82 and chronic hyperalgesia in injured skeletal muscle.83 In cardiac muscle, 80% of ASIC3 muscle afferent neurons co-express CGRP, which is a vasodilatory peptide, suggesting capability for modulating blood flow in response to ischemia.84
There is, however, some conflicting evidence with respect to persistent pain. ASIC1, 2, and 3 knockout studies have demonstrated no or increased primary hyperalgesia rather than a decrease as might be expected.85,86 There is evidence, however, that ASIC3 may have a role in central and peripheral sensitization. Inflammation initiated in the muscle of one limb increases ASIC3 expression in the DRG bilaterally and this expression is required for secondary hyperalgesia in the ipsilateral and contralateral limb.33 ASIC3 micro-RNA (miRNA) (inhibits native mRNA translation) introduced into a muscle by a viral vector prior to inducing inflammation prevents primary and secondary hyperalgesia and reduced ASIC3 mRNA expression in the DRG.87 ASIC1 also mediates primary hyperalgesia in the inflamed muscle.33
The role of ASIC2 may be modulatory. Like ASIC3, its expression is increased bilaterally in the DRG during inflammation. However, inhibition leads to hyperalgesia. It may, therefore, have a central inhibitory role.87
The Voltage-gated Ion Channels
Nav channels are opened,once a threshold has been reached in the primary afferent. The resulting influx of extracellular sodium ions down their electrochemical gradient brings about membrane depolarization and propagation of an action potential from the periphery to the dorsal horn and beyond. Clearly, a decrease in the threshold of these channels by activation and an increase in their number will increase the rate at which action potentials are formed and transmitted, increasing pain sensitivity and intensity.
Nav channels exist in a number of different isoforms. Data collected from human and transgenic mice studies have suggested that Nav1.3, Nav1.7, Nav1.8, and Nav1.9 have differential roles in peripheral and central sensitization and inflammatory and neuropathic pain. Nav1.8 and Nav1.9 are expressed exclusively in nociceptive neurons.
Nav1.3 expression increases in the DRG following primary afferent injury88 and in the second order neurons following spinal injury.89 However, knockdowns only inhibited hypersensitivity following spinal injury, suggesting a more significant role in central sensitization in neuropathic pain.89
Nav1.7 is preferentially expressed in DRG and sympathetic ganglion and is activated close to the resting potential; therefore, can move the membrane potential toward threshold on activation. In humans, a mutation of the gene encoding Nav1.7 causing gain of function produces severe burning pain (eyrthromelagia), and one causing loss of function leads to insensitivity to pain altogether.90 Nav1.7 knockout mice demonstrate hyposensitivity to innocuous mechanical and thermal stimuli in inflammatory91 but not neuropathic pain.92 Nav1.7 appears to be activated by PKC and upregulated by a p38 mitogen kinase mediated pathway processes initiated by binding of proinflamatory cytokines.93 Binding of opioids to δ-receptors decreases PKC and p38 activity with a resultant reduction in Nav1.7 expression.93
Nav1.8 contributes significantly to the DRG action potential,94 hence, increased sensitivity increases neural excitability. Nav1.8 knockout mice demonstrate decreased sensitivity to noxious mechanical and cold stimuli in inflammatory pain,95 and Nav1.8 knockdown reversed neuropathic pain.96 Nav1.8 is activated by PKA and PKC mediated phosphorylation. This is initiated by the neuropetides, CGRP (PKA and PKC)97 and substance P (PKC),98 and by PGE2 (PKC facilitating PKA mediated activation),99 all released in response to nerve injury and inflammation.
Nav1.9 is active at the resting potential. Therefore, increasing opening leads to a rise in the potential, increasing neuronal excitability. Nav1.9 knockout mice demonstrated decreased thermal but not mechanical hypersensitivity in inflammatory models.100 They also have decreased thermal hypersensitivity in response to injection of inflammatory mediators (PGE2, bradykinin, and IL-1β but not NGF) suggesting that Nav1.9 has a role in facilitating the effects of these mediators.100 Activation of Nav1.9, like 1.8, results from phosphorylation mediated by PKC and PKA.100 Nav1.9 knockouts do not have reduced excitatability in neuropathic pain models,100 suggesting that role of Nav1.9 is limited to inflammatory persistent pain.
Currently, Nav channels are targeted nonspecifically, utilizing local anesthetic compounds and the anticonvulsants, such as carbamazepine or lamotrigine, but the global effects on membrane excitability narrows their therapeutic range. This is circumvented in the case of local anesthetics with topical application, but the area covered is limited by practicality and potential systemic toxicity. However, isoform specific antagonists (Nav1.7-NeP1 and Nav1.8-A-887826) are being studied in rodent models of neuropathic pain.101,102 In addition, tricyclic antidepressants (TCAs) and serotonin uptake inhibitors also antagonize Nav1.7, TCAs at therapeutic doses, which may contribute to their antihyperalgesic efficacy.103
Voltage-gated potassium (Kv) channels also have a significant role in membrane excitability and nociceptive transmission.104 Resting membrane potential is largely determined by the concentrations of intra- and extracellular potassium with ions reaching equilibrium along an electrochemical gradient. There are Kv channels, “M channels” (Kv7/KCNQ) that increase the resting potential and so reduce excitability. The M channels are influenced by Gq protein-linked receptors that bind neurotransmitters (acetylcholine and glutamate), serotonin, purines (ATP and adenosine diphosphate), and peptides (substance P, bradykinin, and angiotensin). Gq activates PLC, converting PIP2 to DAG and IP3. However, it is not these second messengers that influences the M channels, rather the decrease in PIP2 levels which causes closure and so increased membrane excitability.
In inflammatory pain, there is M channel inhibition mediated by PAR2/PLC activation and PIP2 hydrolysis.105 In neuropathic pain, M channel transcription is downregulated.106 Selective enhancement of M channel activity produces analgesia in animal models of inflammatory and neuropathic pain.107
Calcium-ion channels are the final group of voltage-gated channels. They have a role in action potential initiation and propagation like the sodium and potassium channels, but also influence neurotransmitter release within the dorsal horn. The role of two channels are relatively well defined within nociception; T-type channels, expressed in the cell bodies and nerve endings of afferent fibers and N-type channels found presynaptically in lamina 1 and 2 of the dorsal horn.108 T-type contributes to the regulation of membrane excitability and so the initiation of the action potential. Opening of the T-type channels lowers the membrane threshold, increasing excitability by movement of calcium inward. The activity of this channel, therefore, has an impact on nociception and persistent pain; as reflected in the upregulation of expression in models of neuropathic pain109 and hypoalgesia in gene knockout models and following intrathecal injection of antagonists.110
N-type channels are opened by the arrival of the action potential at the nerve terminal. The subsequent influx of calcium then triggers exocytosis of vesicles and activation of the spinothalamic neurons via release of glutamate, calcitonin gene- related protein, and substance P. These channels are a target for the inhibitory effects of opioids111 and descending adrenergic pathway112 via a G-protein dependent mechanism. N-type channels are significant in inflammatory and neuropathic pain since knockouts demonstrated decreased pain responses.113 There is also downregulation of these channels following nerve ligation,114 which may act as an intrinsic protection mechanism. ω-conotoxin produced by cone snails is a nonspecific blocker of N-type receptors. The synthetic form, ziconotide, is used intrathecally in cancer pain and refractory neuropathic pain. Various channel subtypes are generated by variant splicing of mRNA and analysis of these by intrathecal injection of inhibitory RNA that blocks translation demonstrated their differential role in persistent pain, thermal and mechanical hyperalgesia (exon 37a), and mechanical allodynia (exon 37a and 37b).114
There are other voltage-gated calcium channels, L-, P/Q -, and R-type voltage, which have been less-extensively investigated but also may have notable roles in physiological and pathological nociception. The role of L-type channels has been investigated using administration of channel specific antagonists and knockout mice. There is potentiation of μ-opioid induced analgesia when coadministered with antagonists,115 suggesting a nociceptive role. L-type gene knockout reversing allodynia and dorsal horn neuron excitability in a model neuropathic pain116 would suggest this was pathological. Unlike T- and N-type channels, excitation is most probably the result of calcium induced gene activation with long term effects on excitation and plasticity116 as oppose to change in membrane polarity or increased exocytosis. Also, unlike N-type channels that are downregulated, L-type channels are upregulated in neuropathic pain, and it is suggested that this plays a prominent role in the maintenance of long-term neuropathic sensitization.116
P/Q channels along with N and R channels are thought to work in a coordinated way to release neurotransmitter at most synapses in the CNS. Unlike the others, mutations in P/Q-type channels are the only ones to be linked to specific disease states, including epilepsy, ataxia, and migraine (familial hemiplegic migraine type 1).117 There is evidence to suggest these channels have a physiological and pathological role in nociception, but the observations are contradictory and difficult to rationalise.117 Blocking the channel at a spinal level has been shown to decrease118 as well as increase excitability,119 and gene knockout not only reduced acute nociception threshold but also reduced behavioral response to noxious stimulus.120
Within the spinal cord R-type channels are located in the DRG and lamina 1, 2, and 3 of the dorsal horn. R-type knockout had no effect on the response to acute mechanical or noxious thermal stimuli but significantly reduced the response to somatic and visceral inflammation.121 There was also an exaggerated response after presensitization, suggesting a role in inhibiting the effects of central sensitization.121 This inhibitory pathway, which is thought to be serotonergic, rather than opioid mediated, is derived in the RVM. R-type channels are located in the PAG, which is thought to control the excitability of the RVM.121
The Dorsal Horn
Release of presynaptic neurotransmitters, glutamate, substance P, and CGRP activate postsynaptic ion channels and receptors that lead to influx of sodium and calcium ions. Summation of peripheral action potentials is required for sufficient influx of cations and the threshold reached for postsynaptic Nav channels to open and initiate an action potential. Central sensitization is produced by mechanisms, which increase the rate at which this threshold is achieved either by augmenting excitatory processes or restricting inhibitory processes.
N-Methyl-D-aspartic Acid Receptors
N-methyl-D-aspartic acid (NMDA) receptors are found throughout the periphery and CNS, in the dorsal horn, and also in the brainstem and forebrain (see below). Their peripheral activation may contribute further to peripheral sensitization, but the principal focus is on their role with the dorsal horn in central sensitization.
They are heteromeric receptor and consist of an NR1 subunit forming a nonspecific ion channel bound to a NR2 (A, B, C, and D), plus or minus an NR3 (A and B) subunit responsible for ligand binding.122 Variation in composition results in receptors with diverse properties. In general, the receptors have 3 distinct characteristics. Firstly, they are permeable to monovalent cations and calcium ions. Secondly, they bind both glutamate and glycine agonists. Thirdly, at the resting membrane potential, the receptor is blocked by magnesium and opens when there is simultaneous ligand binding and membrane depolarization.123 Changes in the NR2 subunit produces change in sensitivity to glutamate activation and magnesium blockade as well as variation in magnitude and duration of ion conductance.124,125 The presence of NR3 subunits forms a receptor that is insensitive to glutamate altogether, i.e., a glycine receptor that is impermeable to calcium ions and resistant to magnesium blockade.126
NMDA receptors are one of 3 ionotropic receptors binding glutamate, and within the dorsal horn, it is a-amino-3-hydroxy-5-methyl-4-isoxazole-propanoic acid (AMPA) and kainate that are the principal receptors responsible for postsynaptic membrane excitation during a painful stimulus by facilitation of sodium ion influx. Following injury, a greater frequency of primary afferent impulses produces postsynaptic depolarization to a threshold capable of displacing the magnesium ion and activating the NMDA. This produces increased cation influx and potentiates postsynaptic excitation in response to the noxious stimulus (hyperalgesia)34 (Figure 4). NMDA receptors are also located on myelinated and unmyelinated primary afferents at both the peripheral and central terminals. Centrally, presynaptic NMDA receptor activation by glutamate binding increases calcium influx and so substance P and glutamate release, contributing to central sensitization via the subsequent increase in postsynaptic membrane excitation (Figure 4).
Peripheral NMDA receptors are integral to peripheral sensitization in inflammation with expression increased127 and reversal of hyperalgesia and spontaneous pain behavior with peripheral infiltration of NMDA antagonists.128 Peripheral NMDA antagonists have no effect on neuropathic hyperalgesia.129 It is debatable how the physiological process of NMDA mediated postsynaptic excitation becomes pathological central sensitization. Differential NR2 expression, activation of a protein kinase cascade, and reduced postsynaptic inhibition are 3 possibilities.
Figure 4: Activation of NMDAR after tissue injury. Normal synaptic transmission. NMDARs are blocked by magnesium and glutamate released presynaptically opens AMPARs. After tissue injury, increased postsynaptic depolarization leads to decreased magnesium inhibition and NMDAR activation by glutamate. Presynaptic, postsynaptic, and extrasynaptic NMDAR activation leads to Ca2+ influx. Presynaptically, this increases glutamate release (positive feedback). Postsynaptically, this increases membrane potential and neuronal excitability. Extrasynaptically, this activates post-translational and transcriptional changes.
In inflammatory pain, NR2A receptor expression increases and NR2C receptor expression decreases.130 In neuropathic pain, NR2A receptor expression decreases.131 However, the relevance of this is unclear, as there is no change in overall receptor expression130 or dose response curve,131 and NR2A expression has no effect on neuropathic or inflammatory pain presentation.132,133 Also the NR2B receptor, the expression of which is unchanged, has a significant role in neuropathic133 and inflammatory134 pain.
Electrophysiological studies imply that NR2A receptors predominate postsynaptically, and so it is felt that NR2B receptor are located extrasynaptically.135 NR2B receptors produce central sensitization, not via direct facilitation of membrane depolarization, but by activation of protein kinase cascades that positively feedback to sensitize NR2B receptors, and also have post-translation and transcriptional influences that contribute to immediate and long-term excitation (Figure 5).
NMDAR, N-methyl-D-aspartic acid receptor; CaMKII, Ca2+/calmodulin-dependent protein kinase II; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole-propanoic acid; Glum, metabotropic glutamate receptor; PKA, protein kinase A; PKC, protein kinase C; ERK, extracellular signal-regulating kinase; CREB, cAMP response element binding; NOS, nitric oxide synthase; COX2, cyclooxygenase-2; NK1, neurokinin 1.
Figure 5: Extrasynaptic effect of NMDAR activation. Influx of Ca2+ mediated by NMDAR activates CaMKII. CaMKII binds to NMDAR and then activates PKC, PKA, and Src kinase. These kinases then activate ERK. ERK downregulates Kv4.2 and upregulates AMPAR by post-translational effects increasing membrane excitability, and CREB (cAMP response element-binding), a transcription factor.
Influx of calcium ions mediated by AMPA and NMDA receptors and PLC activated by a metabotropic glutamate receptor activate PKC and CaM-dependent protein kinase II (CaMKII).136 NR2B receptors are activated by phosphorylation that reduces Mg2+ blockade.137 This is mediated by PKC directly138 but also by Src kinase activated by PKC.139 Activated CaMKII binds to the phosphorylated NR2B subunit,140 which then maintains CaMKII in a persistently active state.141 The NR2B-CaMKII complex allows CaMKII autophosphorylation critical to its role in neuropathic pain.142 PD95/SAP90 is a scaffolding protein beneath the membrane, which is essential for this interaction and neuropathic pain,143,144 and also facilitates the interaction between Src kinase and NRB2.134
PKC, Src kinase, and PKA activated by CaMKII, phosphorylate and activate extracellular signaling regulating kinase (ERK). ERK has post-translational effects upregulating AMPA receptors and downregulating Kv4.2 channels, which increases membrane excitability. It also, along with a small contribution from CaMKII, phosphorylates and activates cAMP response element binding (CREB)protein. CREB is a transcription regulator, which is felt to influence long-term excitability.136
Postsynaptic excitation is also amplified in the presence of injury by decreased inhibitory activity. There is a reduction in a GABA activity that results from a decreased GABA release, secondary to enzyme downregulation (GAD65), and to a lesser extent inhibitory neuron loss, which are mediated by NMDA receptor activation.145,146
Glial cells are subdivided into astrocytes, microglia, and oligodendrocytes. They are non-neuronal cells that have a role in homeostasis, support and protection, acting as immunological cells performing phagocytosis and releasing immunological mediators. In addition to this, they appear to have a pivotal role in neuropathic pain. There is a reciprocal mediator and neurotransmitter release by the glial cells and neuronal cells which maintains the glial cell in active state and potentiates neuronal excitability (Figure 6).25,147
Astrocytes encase the synapse and have a number of receptors for synaptic neurotransmitters (glutamate, substance P, and ATP). Binding to these receptors stimulates intracellular kinases (MAPK1 and c-Jun N-terminal kinase), leading to increased secretion of inflammatory factors [(IL1β, IL6, TNF-α, PGE2, and nitric oxide (NO)] implicated in increasing pain, neuronal excitability, and central sensitization.147
TNF-α and IL-1β increase neuronal excitability and synaptic strength by increasing NMDA and AMPA receptor conductivity and number.148 IL-1β, IL-6, and TNF-α have been implicated in DRG sensitization and pain hypersensitivity (see above). NO contributes to central sensitization by CREB phosphorylation149,150 and PGE2 contributes by activation of glutamate receptors151 and inhibition of glycine receptors.152 The astrocytes also release glutamate and ATP leading to further neuronal and glial activation.147
Microglia usually has a role in antigen phagocytosis, antigen presentation, and release of pro- and anti-inflammatory cytokine. Like astrocytes, they also have receptors for neurotransmitters and can be activated by excitatory transmitters and potentiate central sensitization via the same mechanisms. In addition, ATP binds to a purinergic ionotropic receptor (P2X4) on the microglia stimulating the release of ATP and brain-derived neurotropic factor (BDNF). BDNF downregulates KCC2, a potassium chloride cotrans‑ porter responsible for maintaining a low chloride ion level within the cells causing inversion of the chloride gradient. Therefore, when GABA binds, this inhibitory transmitter has an excitatory effect allowing chloride efflux rather than influx.153
Figure 6: Influence of astrocytes and microglia on neuronal excitability. For both astrocytes and microglia, neuropeptides activate MAPKI and c-Jun that lead to release of TNF-α, IL-1β, IL-6,nitric oxide. These mediators increase presynaptic neuronal excitability (peripheral sensitization) and increase NMDAR and AMPAR number and activity postsynaptically (central sensitization). Microglia alone are activated by ATP leading to the release of BDNF, which inhibits a potassium/chloride cotransporter. GABA then has an excitatory rather than inhibitory effect. Microglia are also activated by CX3L1 (Fractalkine) binding to a specific receptor and RNA and HSP binding to TLR 2 and 4.
If glial cells are capable of this interaction with neurons, why does persistent excitability only occur following injury? It is perhaps because of a need for an initial activation process and a signal to support ongoing activation. Glial cells have toll-like receptors (TLRs) that have a role in pathogen recognition and initiate the release of proinflammatory mediators. mRNA and heat shock proteins released from damaged cells are likely endogenous ligand.34 TLR2 and 4 appear to be integral to the development of pathological pain,154,155 and TLR4 antagonism has been shown to reverse neuropathic pain.156
TLR4 also binds opioids.156 Recurrent activation of glial cells by opioids stimulates the release of neuroexcitatory proinflammatory mediators, which counteract their effects. Suppressing glial activation enhances acute opioid analgesia and suppresses tolerance, dependency, and withdrawal.147
Chemokines may act to prolong activation of the glia and neurons. Following nerve injury, there is an upregulation of chemokine production and CCRs on the glial cells and neurons. Fractalkine (CX3CL1) released by spinal cord neuron, binds to a receptor (CX3CR1) principally found on microglia.157 It is likely that CX3CL1 has a significant role since blockage of the receptors prevents development and persistence of neuropathic pain158 and CX3CR1 knockout mice show a deficit in inflammatory and neuropathic nociceptive responses.159
DESCENDING PATHWAYS AND SUPRASPINAL INFLUENCE
On the face of it, the peripheral mechanisms appear to consist of endless positive feedbacks between the immune cells and neurons that could maintain neuronal hyperexcitability indefinitely. However, in general, there is healing and so the inflammation, be it related to nerve injury or not, comes to an end. It is likely, therefore, that peripheral sensitization and the resulting afferent hyperactivity plays a transient role as an initiator of central sensitisation. With this in mind, it may be illogical in the chronic pain setting to target components of peripheral sensitization. Diseases, such as osteoarthritis, where there is an ongoing destructive process, may be an exception to this.
Central sensitization has similar feedback characteristics. It could, therefore, be self-perpetuating. However, there is evidence that descending facilitation is required to maintain excitability. It would also appear that such supraspinal modulation not only influences perception of a stimulus after sensitization but may also provide a mechanism by which persistent pain can occur in the absence of peripheral sensitization (e.g., fibromyalgia). Placebo can produce analgesia, psychological factors can influence persistent pain, and pain can influence cognition.
Supraspinal modulation involves areas within the cortex and brainstem with the RVM as the final relay. Neurons originating in the RVM have a significant role in the development of persistent neuropathic pain. In a rodent model of spinal nerve ligation, injection of lignocaine into the RVM and lesion of the dorsolateral funiculus arising from it did not prevent initial tactile and thermal hypersensitivity, but did block these effects after 3 days, causing restoration of normal thresholds.160 The descending facilitation appears to be serotinergic since intrathecal 5-dihydroxytryptamine (5-HT) depletion161 and inhibition of RVM 5-HT expression162 attenuated this delayed hypersensitivity. However, this may not be the only mechanism. Spinal nerve ligation increases RVM CCK levels, and microinjection of CCK into the RVM induces tactile hypersensitivity, which appears to be mediated by increased spinal PGE2 and 5-HT.163
RVM also has an inhibitory role. In rodents that do not develop allodynia in response to nerve ligation, injection of lignocaine into the RVM induces allodynia. This appears to be mediated by noradrenergic neurons, since allodynia was also induced by spinal α2 antagonism.164
Neuroimaging, including fMRI with blood-oxygen-level-dependent response and positron emission tomography have allowed the identification of areas of the brain that become active during placebo and pain perception, and indicated changes in connectivity between areas which may affect persistent pain perception. Also, having identified key areas representing pain perception these may act as biomarkers to give an objective indication of drug efficacy.165
Understanding the biological mechanism in placebo is of relevance to persistent pain since it is a demonstration of the descending inhibitory pathway and has a powerful influence on analgesic efficacy. In response to magnitude of placebo, there is increased activity in cortical structures (the rostral ACC and DLPFC)and brainstem structures (the amygdala, hypothalamus, PAG, and RVM). There is also increased connectivity between the ACC and PAG, the strength of which predicted stronger RVM activity and magnitude of placebo. All of this appears to be mediated by endogenous opioids, since naloxone reduced the placebo effect and the activity and connectivity of the pathway.166
The strength of an individual's response to a placebo correlates with their response to exogenous opioids.167 This may be a reflection of augmentation of this pathway in addition to classical opioid targets.
The analgesic power of this pathway is significant. Positive treatment expectancy has been shown to double the efficacy of a remifentanil infusion during a noxious thermal stimulus in association with activation of this pathway.168 This pathway also appears to have a number of psychological influences. In terms of positive influence, anticipation of pain increases activity within the DLPFC and PAG and has analgesic effect correlating with increased placebo induced pain relief;169 and distraction induces analgesia by increasing PAG activity.170 In terms of negative influence, anxiety and pain vigilance prestimulus are associated with decreased connectivity between the ACC and PAG, suggesting a mechanism for pain susceptibility in anxious individuals.171
In terms of persistent pain perception, the medial prefrontal cortex (mPFC), dorsal ACC, insula, and amygdala appear to have activity which implicate them in pain intensity and duration, pain memory, as well as significant roles in cognitive change and emotional-affective components.172 In back pain, mPFC ctivity strongly correlated with pain intensity. However, this does not apply to all chronic pain conditions. In osteoarthritis knee, for example, pain rating is reportedly linked to insula activity. It is likely that the areas activated will reflect the type of pain (inflammatory or neuropathic), the nature of the stimulus, and other characteristics like the duration of the pain.172
ACC is responsible for aversive memory, and is persistently active in neuropathic173 and inflammatory pain.174 This activity is glutamate mediated,173,174 and direct application of NR2B antagonist in an inflammatory model reverses hypersensitivity.174 It is hypothesized that persistent pain is a state of continuous learning, in which aversive emotional associations are continually made with incidental events due to the persistent presence of the pain. An inability to extinguish these aversive associations may perpetuate the condition,172 perhaps by maintaining ACC activation.
The PFC areas and the amygdala appear to be of critical importance in cognitive function, such as decision making.175 Chronic pain is associated with impaired decision making in tasks that require adaptation of behavior with the expectation of achieving greater long-term benefit.176 This is perhaps due to gray matter loss in these areas in persistent pain.177 The altered expectation may be an early contributor to depression in persistent pain prior to the impact of loss of function and role.178
Loss of gray matter in persistent pain implies that it may be a central neurodegenerative condition.172 In studies of chronic back pain, the 2 areas most effected were the bilateral DLPFC and right thalamus. Focusing on the DLPFC, it was found that pain intensity, duration, and characteristics correlated with extent of loss. There was also a greater degree of DLPFC degeneration in the presence of neuropathic pain. The implications of this are as follows: Firstly, persistent pain may be related to loss of elements of the descending inhibitory pathway.179 Secondly, regional atrophy may explain the transition between acute and chronic pain.177 Thirdly, a lower volume of gray matter at time zero when compared to healthy subjects may indicate a genetic predisposition.172
Our current understanding of the neurobiology of persistent pain is by no means the complete picture. Overall, a cascade of peripheral sensitization, central sensitization, followed by descending facilitation appears common to inflammatory and neuropathic persistent pain. Evidence suggests that TRP and ASIC channels, voltage-gated ion channels, NMDA receptors, and glial cells have a significant role in the peripheral and spinal components of this process. It also appears that supraspinal facilitation and loss of supraspinal inhibition is also significant and provides a route via which placebo and psychological factors may not only effect interpretation of painful stimuli but also have a direct influence on neuronal sensitization.
Genetic studies and brain imaging indicates potential individual susceptibility to persistent pain as a result of protein polymorphism, variation in supraspinal neuronal connectivity, and gray matter mass. Imaging also suggests the possibility that persistent pain may be a neurodegenerative condition.
A greater understanding of persistent pain neurobiology provides insight into the mechanisms behind the efficacy of current therapy and presents new targets for the future.
The ability of functional brain imaging to visualize the perception of pain may have potential to objectively quantify drug efficacy, making evidence more robust. Also, if stereotypical neuronal activity can be linked to particular pain presentations and the technique becomes technically and economically accessible, functional imaging may also have a diagnostic role and facilitate more targeted treatment.
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