Where acute pain protects an individual from further damage, chronic pain serves no adaptive purpose. Approximately 1 in 5 people suffer from chronic pain, which exacts a substantial toll on the both the individual and on national economies [1–3]. Chronic pain can arise due to unresolved inflammation (e.g., rheumatoid arthritis), damage to the nervous system (neuropathic pain), and due to unknown precipitating factors (e.g., fibromyalgia). Specific injuries and diseases that can cause neuropathic pain include traumatic injury (e.g. spinal cord injury), stroke, herpes zoster infection (post-herpetic neuralgia), and multiple sclerosis.
An extensive literature has been devoted to understanding neuropathic pain. Basic science studies have revealed that there are key nervous systems sites for pain modulation. These include the site of injury, the cell bodies of primary afferents (dorsal root ganglia; DRG), the dorsal horn of the spinal cord, and a distributed group of brain regions known as the ‘pain matrix’ [4–7]. Animal studies have predominantly focused on the spinal cord; this site has particular significance for pain, as primary afferent neurons synapse here with pain projection neurons, and are modulated by descending inhibitory neurons and interneurons [5,6]. Therefore, this site will be the focus of this review. Studies in spinal cord have revealed that neuropathic pain is not purely the product of dysfunctional neuronal communication, but that non-neuronal cells, such as microglia, are also critical participants [8,9].
What is the evidence for involvement of microglia in chronic pain?
Microglia are the resident macrophages of the central nervous system (CNS) and play a key role in maintaining homeostasis. Activation of spinal microglia in response to injury of peripheral nerves was first demonstrated in the late 1990s; immunohistochemical and gene expression studies revealed that microglial ‘activation’ markers were rapidly elevated [10,11]. A causal role for microglia in the pain behaviors caused by peripheral nerve injury was then inferred when minocycline treatment was found to prevent pain in rats [12,13]. A key finding of these and subsequent studies (e.g. ) was that expression of microglia activation markers peaked within 2 weeks of injury, while inhibition of microglial signaling could prevent, but not reverse, neuropathic pain. This led to the conclusion that microglia participate in the development of neuropathic pain, but not its maintenance. A recent study using Mac1-saporin depletion has claimed that microglia do contribute to the maintenance of neuropathic pain, as their depletion reverses pain behaviors 3 months after nerve injury . However, the consensus view is that microglia are principally early responders to nerve injury.
A limitation to this early research is that pharmacological agents like minocycline also inhibit neurons and astrocytes; the lack of selectivity for microglia confounds interpretation. However, several studies have reinforced evidence for microglial participation in pain. Demonstrating sufficiency for pain, microglia were activated in vitro and then adoptively transferred to naïve male rats, inducing pain behaviors . We have recently expressed Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in spinal microglia, using a cell-selectively promoter [17,18]. Activation of excitatory DREADDs in naïve male rats induced allodynia (sensitivity to innocuous stimuli), supportive of the sufficiency for microglia activation for pain. We also showed that inhibiting microglia with DREADDs could reverse neuropathic pain in males, which demonstrated that microglia are necessary for neuropathic pain.
There is emerging evidence that microglia may contribute to neuropathic pain in a sex-dependent manner. Acute depletion of microglia or inhibition with minocycline reversed neuropathic pain in male rats only . However, these findings are not without controversy [20,21], and additional studies with longer-term treatments are required; when neuropathic pain is fully developed, repeated treatments are often required for reversal. Nonetheless, the accumulating evidence for males-specific engagement of particular signaling pathways will be discussed.
How do microglia become activated after neuronal injury?
Given the evidence that microglia are important cellular mediators of neuropathic pain, investigators next turned to the question of how microglia can respond to nerve injury.
Microglial activation is understood as a change in cell number, morphology, phenotype and motility, the expression of membrane-bound and intracellular signaling proteins, and the release of immunoregulatory products, such as cytokines and chemokines. Microglia express a range of receptors that detect ligands released as a consequence of neuronal injury, and that lead to their activation (illustrated in Fig. 1).
ATP. Injured neurons release ATP in the dorsal horn of the spinal cord , which is detected by purinergic receptors expressed by microglia; these include ionotropic P2X4 and P2X7 receptors, and metabotropic P2Y12 and P2Y13 receptors [23,24]. Microglia upregulate surface expression of these receptors after peripheral nerve injury. Their involvement in neuropathic pain has been confirmed in genetic knockout and knockdown studies, as pain behaviors are reduced under these conditions [16,25]. However, the P2X4R pathway is specifically engaged in male rather than female rodents [19,26]. This may be due to differential transcriptional regulation of P2rx4 by the transcription factor IRF5 .
Chemokines. Several chemokines are produced and released by injured neurons. At present the strongest evidence supports a role for CSF-1. The cognate receptor CSF-1R is expressed exclusively by microglia in the CNS, and is essential for survival . Peripheral nerve injury induces de novo expression of CSF-1 in the DRG . Conditional knockout of CSF-1 in sensory neurons prevented development of pain behaviors, and reduced microglial activation and proliferation in the spinal dorsal horn . There is evidence that engagement of this pathway may precede activation of microglia via ATP, as the CSF-1R adapter protein DAP12, is upstream of the P2X4R gene.
Other groups have previously suggested that the chemokines CCL2 and CX3CL1 are neuron-glia signals in neuropathic pain [29,30]. However, subsequent studies revealed that microglia express very low levels of CCR2 , suggesting a limited role for its ligand CCL2. While microglia uniquely express the receptor for CX3CL1 in the CNS , the release of this ligand from neurons is now known to be secondary to microglial activation; CX3CL1 is cleaved by cathepsin S derived from already activated microglia . Furthermore, DAP12 is also upstream of CX3CR1 and cathepsin S genes . Thus, CSF-1 appears to be the principal chemokine to mediate microglial activation after peripheral nerve injury.
Damage associated molecular patterns (DAMPs). According to the danger model of immunogenicity , the immune system can respond to cellular damage and the consequent release of DAMPs. Several DAMPs have been implicated in neuropathic pain, including High Mobility Group Box 1 (HMGB1), Heat shock proteins (HSP)-60 and -90, biglycan and fibrinogen . These molecules are typically sequestered within intracellular compartments or the extracellular matrix, and become liberated into the extracellular milieu during stress or damage. It should be noted that the cellular source of many of these DAMPs has not been unequivocally demonstrated. Nonetheless, microglia respond to exogenous administration of DAMPs. Pattern recognition receptors are those capable of detecting such DAMPs, and among this receptor class, Toll-like receptors (TLRs) have been most thoroughly investigated .
Of the 13 TLRs expressed in rodents, most research has focused on TLR4. TLR4 is upregulated after peripheral nerve injury. However, evidence for causal involvement in neuropathic pain comes from pharmacological and genetic manipulations: pain behaviors after nerve injury are attenuated when TLR4 or accessory proteins (required for signaling) are inhibited, knocked out, or knocked down [34–36]. There is an emerging role for other TLRs in neuropathic pain, including TLR2, 3, 5, and 9 .
Finally, TLR4-dependent pain may be sexually dimorphic. Intrathecal injection of the TLR4 agonist LPS induces allodynia in male (but not female) rats . This is in notable contrast to intrathecal disulfide HMGB1, which induces allodynia in both male and female mice . Reversal of intrathecal LPS-evoked tactile allodynia by the small molecule TLR4 antagonist TAK-242 occurs in male mice, while having no effect on females  Similarly, male mice deficient in TLR4 do not develop robust neuropathic pain after peripheral nerve injury, whereas female mice do . This leads to the intriguing possibility of sex differences regarding the involvement of innate immune signaling in the development of neuropathic tactile hypersensitivity. Given the predominance of chronic pain conditions disproportionately afflicting women, it has been argued that interactions between endocrine and immune mechanisms may help explain some of the sex differences observed in the epidemiology of pain disorders [41,42].
Intracellular signaling pathways.Many of the receptors described above converge on common intracellular signaling pathways, including activation of the transcription factor NFB, and p38 and ERK mitogen activated protein kinases (MAPKs). Such activation occurs in spinal microglia after peripheral nerve injury, and leads increased transcription of proinflammatory and pronociceptive cytokines and growth factors [14,43,44]. Other signaling pathways include activation of NADPH oxidase isoform 2 that generate reactive oxygen species . These enzymes and transcription factors are causal to neuropathic pain, as their inhibition or genetic deletion reverses pain behaviors evoked by peripheral nerve injury in male rodents. However, one group has assessed female rodents, and shown that pain behaviors are not reversed when p38 is inhibited [19,46]. This again points to a male-specific role for microglia in neuropathic pain.
How do activated microglia cause pain?
As noted above, a primary consequence of microglial activation is the release of proinflammatory cytokines, growth factors, and reactive oxygen species. These mediators act at central synapses in the spinal cord dorsal horn to disrupt the balance of excitatory and inhibitory neurotransmission (illustrated in Figure 1).
Enhanced excitatory synaptic transmission. Cytokines such as TNF and IL-1 increase the excitatory tone of pain projection neurons by enhancing glutamate release and availability . For example, activation of IL-1 receptors that are functionally coupled to presynaptic NMDA receptors promotes glutamate exocytosis . Both IL-1 and TNF can downregulate transporters expressed by astrocytes that are responsible for uptake of glutamate [48,49]; the consequence is excessive glutamate levels in the synaptic cleft. Cytokines and chemokines can also sensitize post-synaptic terminals. The mechanisms include increased trafficking and surface expression of AMPA receptors, and by phosphorylating NMDA receptor subunits [50–53]. Collectively, these studies show that inflammatory mediators released by microglia have the capacity to facilitate hyperexcitability of pain projection neurons.
Reduced inhibitory synaptic transmission. Mediators derived from microglia can also cause disinhibition. Cytokines, chemokines and reactive oxygen species can diminish release of GABA and glycine from interneurons and descending inhibitory projections in the spinal cord [54–56]. Activated microglia also sculpt synaptic elements in the spinal dorsal horn and excessively prune GABAergic terminals . The growth factor BDNF activates TrkB receptors on postsynaptic terminals, reducing expression of the KCC2 potassium-chloride cotransporter . This leads to increased intracellular Cl- concentrations that weaken GABA receptor mediated hyperpolarization. Only males exhibit BDNF-dependent neuropathic pain ; this result is consistent with BDNF release requiring P2X4 receptor and p38 MAPK activation, which themselves are activated in a sexually dimorphic manner [26,46]. The consequence of these disinhibitory mechanisms is to increase the excitability of pain projection neurons.
Future directions and conclusions
A major challenge for the future is to translate the findings from animal models into humans. Imaging studies are yielding very promising results using radioligand binding to TSPO, a putative marker of glial activation. Binding was increased in thalamic nuclei, somatosensory cortices of patients with low back pain , and the neuroforamina and spinal cords of patients with lumbar radiculopathy . Further work is required to understand the function of TSPO in neuropathic pain and to develop new radioligands. Firstly, it is unclear whether TSPO expression correlates with activation . Secondly, there is some evidence that TSPO has anti-inflammatory function , and thus only a subset of glia may be labelled. Which subset that may be, and its relationship to neuropathic pain, need to be defined. Finally, the selectivity of TSPO to microglia is a matter of debate that needs clarification . Nonetheless, these studies provide compelling evidence for the association of glial activation in human chronic pain states. PET imaging may also be a useful tool to validate target engagement for development of novel therapeutics that target microglia.
The vast majority of studies have focused on microglia in the spinal cord. However, the sensory and affective components of chronic pain are ultimately encoded in the brain, as noted above. Several groups, including my own, are now beginning to investigate how microglia in the brain contribute to the multi-dimensional experience of chronic pain [64,65].
The evidence summarized here highlights a critical role for microglia in the initiation of neuropathic pain, especially in male rodents. Given that women predominantly suffer with chronic pain, studies should now be directed towards identifying whether microglia are truly a relevant clinical target for treatment of chronic pain; dysfunctional synaptic plasticity induced by activated microglia is a promising therapeutic avenue for a disease that is poorly managed and causes untold suffering.
Peter M. Grace, PhD – Department of Symptom Research, University of Texas MD Anderson Cancer Center, Houston, TX 77030; e: firstname.lastname@example.org
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