Pain is a universal sensation necessary for survival. From a purely sensory perspective, “nociception”, the neural process of encoding and processing noxious stimuli, has been observed in the simplest to the most complex organism.1,2 Although acute pain is an important warning signal for bodily harm, pain that persists long after tissue damage may become a clinical problem.

Chronic pain can be defined as any pain lasting more than three to six months.2 The World Health Organization has declared chronic pain to be one of the most common world health problems, with more than a quarter of the population suffering from chronic pain.3 In Canada, estimates indicate that approximately 29% of the population suffers from chronic pain, with 80% of those individuals suffering from moderate to severe pain.4 Despite the large prevalence of this condition, chronic pain is still poorly understood and notoriously difficult to treat. Acute pain is easily modelled in experimental settings as physiological or behavioural responses to noxious stimuli or tissue damage, while chronic pain can arise from different types of tissue damage. Chronic pain may spontaneously arise without tissue damage or it can paradoxically exist in areas where damaged tissue is no longer present (i.e., phantom limb pain).1,3,5 Chronic pain also violates the boundaries of the thresholds for what normally causes pain, resulting in “allodynia” (pain in response to a stimuli that does not normally provoke pain), “hyperalgesia” (increased sensitivity to stimulation), and “sensitization” (an increased responsiveness of nociceptive neurons to their normal input and/or recruitment of a response to normally subthreshold inputs).1 Overall, chronic pain is a paradoxical phenomenon with no adaptive purpose when compared with acute pain.

Understanding the difficulty in treating chronic pain compared with treating acute pain has been the focus of an intense research effort. The main focus of research in this area has been on trying to understand the transition from injury and acute pain to the development of chronic pain.

Although this transition is still not fully understood, many advances have been made at the cellular and molecular levels using model systems that have advanced our understanding of the basic mechanisms underlying this process. The plastic nature of the central nervous system has been well established, and both functional and structural changes in pain pathways play a role in the development of chronic pain. The plasticity of the nervous system can be seen in processes such as peripheral and central sensitization, which increase sensitivity and lower the threshold to stimuli that cause pain through various mechanisms.6-8 In addition to changes in neurons, non-neuronal components of the nervous system, glia, and immune cells also have a role in the development of chronic pain.9-15 In this review, we consider the current body of research examining the mechanisms that mediate the transition from acute to chronic pain with particular focus on spinal pathways, the plastic changes involved, and the role of glial cells in this process. Although there are many other reviews on the development of chronic pain from acute pain, in this review, we examine the various roles played by neuron, glial, and immune cells and how they work together and interact at the molecular level to cause cellular changes that result in a chronic pain state. In addition, we explore how research in the transition from acute to chronic pain has led to the identification of new therapeutic targets and has revealed new roles for sex differences and bacteria in the development of persistent pain.

Peripheral sensitization: how neuron, immune, and glial cells interact in chronic pain

Neurons in peripheral sensitization

Peripheral sensitization is responsible for the initial sensitization of nociceptors and the development of primary hyperalgesia. It is thought that peripheral sensitization has a role in altered thermal sensitivity.16,17 There are essentially three main steps in the development of peripheral sensitization. First, there is an initial release of inflammatory mediators due to tissue damage. These mediators then act on G-protein-coupled receptors (GPCRs) or tyrosine kinase receptors (TKRs) at nociceptor terminals. This leads to an activation of different intracellular signalling pathways that culminates in the phosphorylation of different receptors and ion-channels. The net result of this cascade is a change in the threshold and kinetics of the nociceptor.18-20 For example, inflammation causes the release of prostaglandin E2, bradykinin, and nerve growth factor (NGF).18-20 Prostaglandin E2 and bradykinin then bind to receptors expressed on nociceptor nerve terminals, which leads to the activation of cyclic adenosine monophosphate (cAMP) dependent protein kinase A (PKA) and the Ca2+/phospholipid-dependent protein kinase C (PKC).18,19 Subsequent phosphorylation of the transient receptor potential vanilloid receptor subtype 1 (TRPV1), an endogenous receptor for “heat” by PKA/PKC, results in a lower activation threshold for the channel (< 40°C).18,19,21,22

Heat hyperalgesia (e.g., after a sunburn) can also be caused by changes in posttranscriptional regulation. This is seen when tissue injury leads to the release of the inflammatory mediator, NGF, causing an induced activation of p38 mitogen-activated protein kinase (MAPK) in primary sensory neurons.20 Increased activity of p38 MAPK can lead to an increased expression and transport of the TRPV1 channel which results in increased heat hyperalgesia.20

Immune responses in the periphery

While the role of neurons in the development of chronic pain is essential, it is also important to acknowledge the role of glial and immune cells in this process. Immune cells in the peripheral nervous system and dorsal root ganglion are critical for mediating the initial responses to injury and the development of pain. This initial response is followed by activation of glial cells in the central nervous system (see below), specifically microglial and astrocytes.

In the early immune response to injury, neutrophil granulocytes (the most common type of white blood cells that make up the innate immune response) are attracted to the site of injury by the release of chemokine ligand 1 and leukotriene-B4.23-27 Though the initial neutrophil response is brief, neutrophils may release chemoattractants and cytokines to potentiate the recruitment of macrophages. The recruitment and activation of resident macrophages at the injury site can be mediated by chemokine (C-C motif) ligand 2 (CCL2, also known as MCP-1) and chemokine (C-C motif) ligand 3 (CCL3, also known as MIP-1α).28,29 Macrophages can release prostaglandins and cytokines, such as interleukin 1β (IL-1β), interleukin 6 (IL-6), tumour necrosis factor (TNF)α, and leukemia inhibitory factor (LIF). Tumour necrosis factor α has autocrine effects that increase cytokine synthesis and release, promoting further macrophage infiltration.30

Other growth factors, such as neuregulin bind to v-erb-b2- erythroblastic leukemia viral oncogene homolog 2 and 3 receptors (ERBB2 and ERBB3) on Schwann cells.31 Early ERBB2 activation results in demyelination, while late activation of ERBB2 causes Schwann cell proliferation. Proliferating Schwann cells release NGF as well as the neurotrophin glial-derived neurotrophic factor, prostaglandins, and cytokines, such as IL-1β and IL-6. All of these factors can sensitize nociceptors and alter gene transcription and expression in sensory neurons.31

Activated macrophages along with Schwann cells also release matrix metalloproteins that attack and break down the blood nerve barrier.12 Vasoactive mediators, such as nitric oxide (NO), are also released, causing hyperemia and swelling, and they have also been implicated in the development of neuropathic pain.32 In addition, breakdown of the blood nerve barrier and the release of these vasoactive mediators also potentiate the infiltration of macrophages into the dorsal root ganglia.

The ability of macrophages to infiltrate and evoke changes in the dorsal root ganglia is essential to the development of central sensitization, a main component of chronic pain states (see following section). The invasion of macrophages into the dorsal root ganglia is mediated by the release of chemokine (C-X3-C motif) ligand 1 (CX3CL1, also known as fractalkine) and CCL2 from dorsal root ganglion neurons.28,33,34 Once they have infiltrated into the ganglia, macrophages can enhance excitatory tetrodotoxin-resistant voltage-gated sodium channel currents in sensory neurons via a TNFα-mediated pathway.12,35 Tumour necrosis factor α binds to the TNFα receptor 1 (TNFR1) and activates the p38 MAPK signalling pathway.36,37 This is important, as multiple studies have shown that increased activation of p38 contributes to the development of pain hypersensitivity.13,38-41 Additional activation of the purinergic receptors, P2X2, P2X3, and P2X7, on immune cells can lead to increased release of IL-1β, IL-6, and LIF.37,42 The release of IL-6 can trigger sprouting of sympathetic nerve fibres into the dorsal root ganglia.43 The sprouting of sympathetic fibres around dorsal root ganglia neurons is thought to enhance communication between the two types of neurons as well as contribute to the long-term maintenance of neuropathic pain, showing how the nervous and immune systems can interact in the periphery to lead to chronic pain development.43

Central sensitization

Although peripheral sensitization is an important mechanism in the development of short-term pain sensitivity, sensitization of peripheral nociceptors is normally short-lived, reversible, and confined to the site of injury (Table). It is the development of central sensitization after injury that leads to the spread of pain, allodynia, and secondary hyperalgesia. Central sensitization can be defined as a “facilitated excitatory synaptic response and depressed inhibition, causing amplified responses to noxious and innocuous inputs.”44 This heightened synaptic transmission causes a reduced pain threshold, an amplification of pain responses, and the spread of pain to non-injured areas.45 These changes typically occur in somatosensory neurons of the spinal cord dorsal horn after exposure to intense peripheral noxious stimuli, tissue injury, or nerve damage. Normally, excitatory input is subthreshold, but the increased sensitivity of spinal neurons caused by central sensitization results in the recruitment of inputs that can now cause the neurons to generate action potentials in response to these normally ineffective inputs.45,46 This results in pain being produced by low-threshold afferent inputs and hypersensitivity in undamaged areas of tissue around the primary injury site.

TABLE Comparison of the components in peripheral and central sensitization that contribute to the development of chronic pain
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There are several main triggers of central sensitization that are neuronal, immune, and glial related. The principal neuronal changes in central sensitization involved the recruitment of N-methyl-D-aspartate receptors (NMDARs) and their activation in the dorsal horn.47-49 After a repeated train of stimuli that activates peripheral nociceptors, there is a release of glutamate, substance P (SP), calcitonin gene-related peptide (CGRP), and brain-derived neurotrophic factor (BDNF) at central synapses.50-54 Each of these neuromodulators binds to different receptors and all play a part in the development of central sensitization.

Glutamate, the major excitatory neurotransmitter in the central nervous system, binds both to ionotropic receptors (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors [AMPARs] and NMDARs) and to metabotropic glutamate receptors (mGluRs). When glutamate binds to its respective ionotropic receptors, levels of Ca2+, Na+, and calcium/calmodulin-dependent protein kinase II (CaMKII) increase.55,56 This leads to increased depolarization and excitability. The binding of glutamate to its metabotropic receptor, mGluR, and the binding of other neuromodulators to other GPCRs, such as the binding of SP to neurokinin-1 receptors (NK1R) and CGRP to the CGRP receptors 1 (CGRP1), ultimately lead to increases in PKA and PKC as well as increases in intracellular Ca2+.55,56 Protein kinase A and PKC, along with CaMKII, then act to boost synaptic efficiency by phosphorylating different subunits on NMDARs and AMPARs.47,57 Protein kinase A phosphorylation of AMPAR increases its calcium permeability, and this has been shown to increase synaptic strength.57 Phosphorylation of different AMPAR subunits by PKC and CaMKII also contributes to increasing nociceptor excitability.57 Protein kinase A/PKC phosphorylation of NMDAR subunits facilitates hypersensitivity by increasing the response of the receptor to glutamate.47 Protein kinase C also acts to reduce the Mg2+ blockade of NMDA, increasing the channel’s open probability, again prolonging its active state.48 Also, it is important to highlight that the activation of mGluRs and NK1Rs and the subsequent increase in levels of intracellular Ca2+ and PKC can also lead to the activation of the sarcoma (Src) family kinases (a family of non-receptor kinases).58,59 Sarcoma family kinases act to phosphorylate tyrosine restudies on the NR2B subunit of the NMDA receptor to increase NMDA activity and hyperalgesia.56,58

Sarcoma family kinases can also act through receptor tyrosine kinases (TrKs) to increase NMDA activity and produce hyperalgesia.59,60 This occurs when tissue damage detected by primary somatosensory neurons causes a redistribution and accumulation of ephrinB in dorsal horn neurons.60 This redistribution leads to a postsynaptic cascade, which is initiated by the binding of ephrinB to its receptor EphB to cause activation of the Src family kinase signalling and tyrosine phosphorylation of the NMDAR.60 Phosphorylation results in increased NMDA activation and the development of hyperalgesia.59,60 Other signalling pathways involved in the development of pain also act through receptor tyrosine kinases mediated by modulators such as BDNF. Once released, BDNF binds to the tyrosine receptor kinase B (TrkB) and acts to increase levels of phosphorylated extracellular signal-regulated kinase (pERK).61,62 Phosphorylated extracellular signal-regulated kinase acts to decrease potassium currents by phosphorylating Kv 4.2 channels to increase membrane excitability.61,62 In addition, pERK is involved in the sustainment of long-term pain, a process discussed below. Overall, multiple signalling pathways are involved in the development of central sensitization and pain hypersensitivity.

Late-onset central sensitization

The long-term maintenance of chronic pain involves late-onset central sensitization, so named because the changes involved in its development can take hours to occur. These processes require the activation of transcription. One signal that may trigger changes in transcription is the release of BNDF.38 Brain-derived neurotrophic factor release plays a key role in the long-term maintenance of central sensitization as this pathway can modulate transcriptional processes in the cell. Binding of BNDF to TrkB leads to phosphorylation of ERK through the mitogen kinase activated pathway (MAPK pathway: Ras-Raf-MEK-MAPK/ERK). Phosphorylated extracellular signal-regulated kinase can enter the nucleus of the cell, resulting in transcriptional changes by increasing levels of RSK2 (an AGC group kinase of the ribosomal s6 kinase [RSK] family), which allows for the phosphorylation of cAMP response element-binding protein at the serine 133 residue.63,64 Transcriptional changes then occur in the dorsal root ganglion of the spinal cord that contribute to pain.63,65 Although there are hundreds of gene modifications that contribute to pain, their specific roles and pathways are still poorly understood.66 It has been shown that increased transcription of the genes for c-Fos, cyclooxygenase-2 (known as early gene encoding), neurokinin (NK), TrkB, and prodynorphin (known as late-response gene encoding) contribute to long-term central sensitization and pain.10,38,67-69

It has also been shown that a phenotypic switch in dorsal root ganglia neurons in the periphery can occur, possibly due to changes in transcription.70 It has been observed that large-diameter Aβ neurons may begin to express SP under pathological conditions associated with pain hypersensitivity.70 Since these Aβ fibres now express high levels of SP, they now function like C-fibres and can increase central excitability.70 In addition, as Aβ fibres terminate in the lamina III of the dorsal horn, an area where there are NK1R-expressing cells, this phenotypic switch in the afferent cell body may be involved in postsynaptic alterations in excitability.70 It has been suggested that phenotypic switches of this nature serve as a key mechanism whereby previously non-nociceptive afferents now have the ability to induce central sensitization and tactile pain hypersensitivity.11,71

Glial recruitment and central sensitization

Phenotypic switches and the maintenance of chronic pain can also be influenced by the actions of glial cells in the spinal cord dorsal horn. Although injury initially triggers changes in the periphery, it can also trigger major changes to the reactivity and functioning of spinal microglia and astrocytes in the dorsal horn. Microglia and astrocytes are normally activated by pathogens through their recognition of pathogenic protein patterns using different pattern-recognition receptors, such as toll-like receptors (TLRs). Toll-like receptors 2 expressed on astrocytes and microglia and TLR4 expressed on microglia mediate neuropathic pain processing.14,72 Toll-like receptors 2 and TLR4 activation leads to an immune-like response that results in the release of pro-inflammatory cytokines (such as IL-1β, TNFα, and IL-6) and an increase in phagocytosis.14,15,72 Release of these cytokines can result in an excitatory positive feedback loop that modulates glial-neuronal communications where TLRs are also directly activated by IL-1β, TNFα, IL-6, and NO.14,15,72 Tumour necrosis factor α and Il-1β released from astrocytes also act to increase neuronal excitability by increasing synaptic strength as a result of increased AMPAR/NMDAR number and connectivity.73,74 Interleukin-1β does this by increasing the inward Ca2+ conductance of NMDARs.73 The increased Ca2+ conductance induces phosphorylation of NMDARs for a further increase in Ca2+ influx.75 Increases in NMDARs can create a feedback loop where increased production of NO increases neuronal excitability.76,77

Toll-like receptors 4 is also specifically involved in pain processing via activation by its endogenous ligand, fibronectin. Fibronectin is produced in response to tissue injury and is also involved in the up-regulation of the purinergic receptor, P2X4, in microglia.78 Purinergic receptors on microglia also promote neuropathic pain signalling when activated by adenosine triphosphate (ATP) released in the dorsal horn. It has been shown that direct intrathecal injections of ATP or the intrathecal administration of ATP-stimulated microglia in rats can cause mechanical hypersensitivity.79,80

Adenosine triphosphate action on spinal microglial P2X4 receptors has also been shown to cause a biphasic BDNF release.81 The release of BDNF is thought to occur both through an extracellular Ca2+ dependent SNARE- (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediated pathway and through activation of the p38-MAPK pathway.81 Brain-derived neurotrophic factor released from microglia has been shown to play a major role in chronic pain development.82-84 The release of BDNF from spinal microglia leads to a depolarizing shift in dorsal horn lamina I neurons (Figure A & B).81,82 This is caused by an inversion of normally inhibitory gamma-aminobutyric acid (GABA) signals to excitatory ones, which facilitates pain.81,83 This inversion in GABA inhibitory signalling is thought to be due to a decrease in the levels of the potassium-chloride exchanger KCC2, which results in an increase in intracellular chloride and a depolarizing shift.83 The decrease in KCC2 is mediated by the binding of BDNF to TrKB.83 Overall, the decrease in KCC2 and reversal of inhibitory signalling results in hypersensitivity.83

Figure
figure 1

Role of KCC2 in chronic pain. A) Binding of ATP to its purinergic receptor, P2X4, on activated microglia in the spinal cord causes the release of BDNF. B) The released BDNF binds to its TrkB receptor, leading to a decrease in KCC2. A lack of KCC2 at the plasma membrane causes an increase in [Cl(-)]i. This leads to a depolarizing shift whereby the binding of GABA released from inhibitory neurons is now depolarizing. This depolarizing shift causes hyperexcitability, which contributes to chronic pain. C) Administration of the new compound, CLP257, a chloride extrusion enhancer, has been shown to help re-establish KCC2 function. CLP257 is thought to do this by increases in plasma membrane levels of KCC2, which would decrease the high levels of [Cl(-)]i seen in chronic pain, meaning there would no longer be a depolarizing shift. GABAergic input would now be hyperpolarizing as normal, and there would no longer be any hyperexcitability. KCC2 = potassium-chloride exchanger 2; ATP = adenosine triphosphate; BDNF = brain-derived neurotrophic factor; TrkB = tyrosine kinase B; GABA = gamma-aminobutyric acid

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Discovery of this microglia-mediated pathway has recently led to the development of a new class of therapeutic targets, i.e., chloride extrusion enhancers.84 High-throughput screening led to the discovery of KCC2 activators that help reduce the high levels of intracellular chloride seen with chronic pain.84 A KCC2-specific activator (CLP257) has been shown to restore chloride transport, lower intracellular chloride, restore plasma membrane KCC2 levels, and normalize sensitized spinal nociceptive pathways (Figure C).84 Most importantly, the target compound successfully reversed hypersensitivity in a rat model of neuropathic pain, demonstrating that chloride extrusion enhancers may prove to be a successful therapeutic target for chronic pain.84

Additional contributors to the development of chronic pain

Bacteria and hypersensitivity

In chronic wounds, there is often a prolonged inflammatory response that can result in persistent pain.85 Bacterial infections can further contribute to this enhanced pain state in the patient.86 It had been previously thought that bacterial infections exacerbate the inflammatory response, causing an increase in pro-inflammatory mediators that irritate nerve endings. New research suggests, however, that bacteria can directly activate nociceptors.87 This work showed that pain is correlated with bacterial load in mice infected with Staphylococcus aureus, bacteria responsible for many in-hospital wound infections.87 The bacteria were also found in close contact with dermal nociceptors and mirrored the time course of hyperalgesia, suggesting a direct bacteria-neuron interaction.87 This idea of a direct bacteria-nerve interaction was furthered when it was determined that host defences, such as adaptive immunity through T and B cells, were not required for pain.87 Instead, the study found that bacterial molecules, such as N-formulated peptides and pore-forming toxin, α-hemolysin, act to induce calcium flux and action potentials in nociceptors.87 This suggests that bacteria can act directly on nociceptors to cause pain. Interestingly, it was shown that increased hypersensitivity caused by bacteria may actually act to decrease immune responses.87 This is contrary to the idea that hypersensitivity is usually associated with increased immune responses and pro-inflammatory cytokine release. Through the ablation of nociceptors, it was found that there was increased immune activity that may be mediated through different neuropeptides, such as CGRP, galanin, and somatostatin, which are known to have immunosuppressive qualities.87 Research into the mechanisms by which bacterial infections can induce hypersensitivity provide new insight into an additional factor that can contribute to some forms of chronic pain. In addition, a new nervous-immune system interaction has also been revealed whereby pain acts to decrease the immune response. Further understanding into how different types of bacteria interact with nociceptors could lead to the development of better therapeutics to help those with increased hypersensitivity and pain after bacterial wound infections.

Sex differences in chronic pain

Sex differences between males and females also have an important role in chronic pain. Research into these differences has revealed interesting and clinically relevant differences that have increased our understanding of the underlying mechanisms of chronic pain. Many studies have shown that there is a slightly higher prevalence of chronic pain conditions in females.88,89 Despite knowing this, the role of sex in the development of chronic pain is still poorly understood. Some research into this area has, however, revealed different roles of sex hormones in inflammatory pain through the use of the formalin test in both male and female mice and rats. Research in this area has shown that female rats and mice seem to be more sensitive to formalin pain compared with their male counterparts and that this occurs in the later more chronic phase of the formalin test.90-93 Interestingly, it appears as though it is not the day-to-day changes in the estrous cycles of female animals that cause these differences, but rather, it is the underlying differences in testosterone and estrogen/progesterone in males and females.90,94 It has been suggested that testosterone has a more protective role in this model of tonic inflammatory pain, while female sex hormones may act to increase inhibitory mechanisms in pain.91,94,95 These findings are interesting as they suggest possible different underlying mechanisms for tonic inflammatory pain in males and females.

Other research in this area has also revealed a potential sex-related mechanism in glial cell activation through TLR4. As previously discussed, TLRs, such as TLR4, are essential in the activation of glial cells needed for the development of central sensitization. Mice with dysfunctional TLR4s show reduced allodynia and hyperalgesia in a nerve injury model.72,96 Nevertheless, when the TLR4 gene itself is mutated, only male mice exhibit a reduction in pain sensitivity.96,97 Furthermore, when TLR4 activity is manipulated using an antagonist in females, no change in pain sensitivity is observed.97 This suggests that there may be different underlying mechanisms in glial activation mediating central sensitization in males and females. Understanding the underlying mechanistic differences between the sexes is important for the development of targeted therapeutics, as it is possible that only one of the sexes may benefit from a new drug if not tested first in both sexes. Further research in this area is needed for a better understanding of how sex differences can contribute to the development of chronic pain states.

Summary

Overall, a number of mechanisms are in place that can mediate the transition from acute to chronic pain. This transition can occur in the peripheral or central nervous system, and it has become increasingly clear that the underlying mechanisms include more than just neuronal signalling pathways. Although chronic pain is a complex condition, research in this area has come a long way in understanding its underlying mechanisms. Improvements in understanding how neurons and immune cells interact have already begun to lead to the development of new therapeutic strategies like chloride extrusion enhancers.

Key points

  • The transition from acute to chronic pain is complicated and involves more than just neurons.

  • Changes in neuronal plasticity result in the development of different types of sensitization, such as peripheral and central sensitization, which lead to chronic pain states.

  • Infiltration of immune cells into the dorsal horn of the spinal cord contributes to the development of chronic pain and is mediated by different chemoattractants.

  • Glial cells play an important role in the development of pain, and their response is mediated by the release of different chemokines and cytokines.

  • Other factors, such as sex differences and bacterial infection, can contribute to chronic pain in unique ways.