Neuroinflammation and Central Sensitization in Chronic and Widespread Pain

Ru-Rong Ji, Ph.D.; Andrea Nackley, Ph.D.; Yul Huh, B.S., M.S.; Niccolò Terrando, Ph.D.; William Maixner, D.D.S., Ph.D.


Anesthesiology. 2018;129(2):343-366. 

In This Article

Glial Activation as a Primary Feature of Neuroinflammation and a Driver of Chronic Pain

Glial Activation, Gliosis, and Gliopathy After Painful Injuries

Neuroinflammation is characterized by activation of peripheral glia including Schwann cells in the nerve and satellite glial cells in the dorsal root ganglia and trigeminal ganglia, and central glia including microglia, astrocytes, and oligodendrocytes in the spinal cord and brain.[107,108] In this review we focus on central glia, especially microglia and astrocytes and the mechanisms by which glia-produced mediators modulate synaptic plasticity and central sensitization. The previous decade has seen an exponential increase in literature documenting the role of microglia and astrocytes in the pathogenesis of chronic pain; "glial activation" is emerging as a powerful mechanism underlying pathogenesis of chronic pain,[109–115] and chronic pain may also manifest as a "gliopathy."[108]

After painful injuries, there are different activation states of microglia and astrocytes. The morphologic activation (glial reaction or gliosis) is the most studied activation state, although this type of glial activation may not directly cause pain.[108] Glial reaction is characterized by increased expression of microglial markers such as cluster of differentiation molecule 11B, ionized calcium-binder adapter molecule 1, and CX3C chemokine receptor 1 and astroglial markers glial fibrillary acid protein, as well as morphologic changes such as hypertrophy or process retraction/extension of microglia and astrocytes. Microglial reaction in the spinal cord is very rapid and dramatic, whereas astrocyte reaction in the spinal cord is more persistent and occurs in more painful conditions (Figure 3, A–F).[114,116] Subcutaneous formalin injection into a hind paw of rat or mouse is probably the most studied animal model of inflammatory pain, which lasts for less than 1 h. Of interest, in 1999, Fu et al.[117] showed that subcutaneous formalin also caused a robust microglial reaction (labeled with cluster of differentiation molecule 11B) in the spinal cord days after injection. This microglial reaction from formalin-induced nerve injury is associated with the development of mechanical allodynia. This is one of the earliest reports to support a possible role of spinal microglia in pain regulation. Functional imaging also reveals glial activation in patients with chronic pain.[118] In 2003, Zhu et al.[119] and Zhu and Eisenach[120] showed that incision and nerve injury causes upregulation of cyclooxygenase-1 in spinal glial cells, which is important for the development of postoperative pain and neuropathic pain.

Figure 3.

Distinct and time-dependent activation of microglia and astrocytes in the spinal cord after nerve injury. Microglia activation revealed by increased CX3C chemokine receptor 1 (CX3CR1) expression in the spinal cord 10 days (A) and 21 days (B) after nerve injury in Cx3cr1-GFP mice. Scale bar = 100 μm. (C) Phosphorylation of p38 mitogen-activated protein kinase (P-p38) in cluster of differentiation molecule 11B (CD11b)+ microglia in the spinal cord dorsal horn 7 days after nerve injury. Scale bar = 20 μm. Astroctye activation revealed by increased glial fibrillary acidic protein (GFAP) expression in the spinal cord 10 days (D) and 21 days (E) after nerve injury in mice. Scale bar = 100 μm. (F) Expression of connexin 43 (Cx43) in GFAP-positive astrocytes in the spinal cord dorsal horn 21 days after nerve injury. Scale bar = 20 μm. d = days.

Signaling Mechanisms in Glial Regulation of Allodynia and Hyperalgesia

Several neuromodulators such as adenosine triphosphate (ATP), chemokines (CL1, chemokine (CC motif) ligand 2, and chemokine (CXC motif) ligand 13), and neuropeptides (substance P and calcitonin gene–related peptide), and colony-stimulating factor 1 are involved in glial activation after painful insults[108,121,122] (Table 2). The upregulation of glia-specific receptors and channels are functionally correlated with pain hypersensitivity. ATP modulates glial activation via stimulation of ionotropic P2X receptors and metabotropic P2Y receptors.[123,124] Peripheral nerve injury increases the expression of ATP P2Y receptors (P2X4, P2X7, and P2Y12) in spinal microglia, and each upregulation was implicated in neuropathic pain sensitization (mechanical allodynia).[125,126] Accumulating evidence suggests that after tissue and nerve injury, ATP is generated from different cell types including astrocytes, neurons, and microglia. Astrocyte-expressing hemichannels connexin 43 are permeable to ATP.[127] Glucocorticoids induce ATP release from spinal astrocytes, leading to microglial activation and diurnal exacerbation of allodynia.[128] Vesicular nucleotide transporter regulates ATP release from spinal cord neurons after nerve injury.[129] Microglial pannexin 1 channel was also shown to facilitate ATP release in neuropathic pain.[130] Nerve injury results in cleavage and activation of chemokine CX3C ligand 1/fractalkine by protease cathepsin S, leading to microglia activation through stimulation of CX3C chemokine receptor 1 receptor.[113] CX3C chemokine receptor 1, one of the best-known markers of microglia, is strongly upregulated after nerve injury, as revealed in Cx3cr1-GFP mice (Figure 3, A and B). This microglial receptor is also critical for the development of neuropathic pain symptoms, because mechanical allodynia after nerve injury is reduced after spinal administration of the CX3C chemokine receptor 1 neutralizing antibody and abrogated in Cx3cr1 knockout mice.[113,131,132] After chronic constriction injury of the sciatic nerve, CX3C chemokine receptor 1 upregulation peaks within 10 days. In the late phase of mechanical allodynia (greater than 3 weeks), microglial CX3C chemokine receptor 1 expression markedly declines (Figure 3, A and B). Microglia may play a role in maintaining persistent hyperalgesia and allodynia after bone cancer.[133] Orthopedic surgery and bone fracture also may result in nerve injury and microglial activation in the spinal cord. It was proposed that spinal microglial activation also contributes to postoperative cognitive dysfunction such as delirium.[134] Substance P signaling from C-fiber afferent terminals in the spinal cord results in microglia activation and central sensitization after bone fracture.[135]

After painful insults, gliopathy is also characterized by dysfunction of astrocytes, such as downregulation of glutamate transporters (glutamate transporter 1 and glutamate aspartate transporter) in spinal cord astrocytes, resulting in glutamate accumulation in synaptic clefts causing neuronal hyperactivity.[108,136] Connexin 43 is a critical astrocytic signaling molecule that controls the release of astroglial mediators including glutamate and ATP.[137] Of note, connexin 43 upregulation in spinal cord astrocytes is sustained after spinal cord injury and nerve injury (Figure 3F) and contributes to the development and maintenance of mechanical allodynia.[138,139] Additionally, chronic pain-associated gliopathy could manifest as a functional switch of connexin 43 from gap junction communication to hemichannel regulation, so that astrocytes become "leaky" during this switch, resulting in increased secretion of cytokines (interleukin-1β) and chemokines ([CC motif] ligand 2, [CXC motif] ligand 1).[139,140] Chemokines regulate bidirectional interactions of neurons and glial cells.[141] Astrocytes not only produce chemokines that can "talk to" neurons by modulating neuronal activity but also "listen to" neurons by responding to chemokines (e.g., [CXC motif] ligand 13) derived from neurons.[122] Astrocytes also release thrombospondin 4 to modulate synapse formation, synaptic plasticity, and behavioral hypersensitivity (Table 2).[142]

A critical step of glial activation in persistent pain is the activation of intracellular signaling pathways, especially the mitogen-activated protein kinase pathways. There are three major members in the mitogen-activated protein kinase family: extracellular signal-regulated kinase 1 and 2, p38, and c-Jun N-terminal kinase.[143] Phosphorylation of p38 in spinal microglia occurs in different pain conditions after surgery, nerve injury (Figure 3C), and opioid tolerance, resulting in increased synthesis and release of microglial mediators (tumor necrosis factor, interleukin-1β, and brain-derived growth factor) and pain hypersensitivity.[132,133,144–147] Inflammation or nerve injury also activates c-Jun N-terminal kinase in astrocytes,[148,149] leading to increased secretion of chemokine (CC motif) ligand 2 and chemokine (CXC motif) ligand 1 and enhanced pain states.[139,150] Nerve injury also causes sequential activation of extracellular signal-regulated kinase in microglia (early phase) and astrocytes (late phase),[143] indicating distinct involvement of these two glial cell types in chronic pain induction and maintenance.

Glia Activation in the Brain After Painful Injuries

Accumulating evidence also suggests a role of glial activation, revealed in different brain regions, in regulating neuroinflammation and pain sensitivity. Sciatic nerve ligation induces astrocyte activation in the S1 sensory cortex, which is associated with upregulation of metabotropic glutamate receptor 5. Activation of this glutamate receptor subtype in astroglia induces spontaneous somatic Ca2+ transients and secretion of thrombospondin 1 from astrocytes, leading to new synapse formation and mechanical allodynia.[151] Toll-like receptor 4 plays a critical role in glial activation and neuroinflammation in the spinal cord, as well as hyperalgesia and allodynia.[152,153] Toll-like receptor 4 also contributes to neuroinflammation in the prefrontal cortex and visceral pain after chronic stress. Increased expression of Toll-like receptor 4 is associated enhanced glia activation in the prefrontal cortex and increased levels of proinflammatory cytokines. Administration of the Toll-like receptor 4–specific antagonist TAK-242 in the prefrontal cortex is sufficient to attenuate visceral hypersensitivity.[154] Peripheral nerve injury causes microglia activation within the mesolimbic reward circuitry, leading to a disruption of dopaminergic signaling and reward behavior.[155] Furthermore, nerve injury causes activation of microglia and astrocytes in the anterior cingulate cortex, and administration of microglial inhibitor minocycline in this brain region inhibited mechanical allodynia.[151] However, earlier studies showed that nerve injury did not cause microgliosis in the anterior cingulate cortex and that long-term synaptic plasticity (long-term potentiation) was not altered by minocycline.[156,157] Future studies are warranted to investigate how microglia and astrocytes regulate different forms of synaptic plasticity in different brain regions after painful insults in the peripheral tissues and the central nervous system.

Sex Dimorphism in Glial Regulation of Allodynia and Hyperalgesia

Chronic pain such as chronic orofacial pain associated with temporomandibular disorder occurs more frequently in women.[158,159] In 1993, Maixner and Humphrey[160] reported sex differences in pain and cardiovascular responses to forearm ischemia in humans. Paradoxically, the majority, if not all, pain-related studies were conducted in male animals.[161] However, it was fortunate that males were tested because spinal microglia play little or no role in regulating inflammatory and neuropathic pain primarily in female rodents[162–165] (Figure 4). Sorge[162] demonstrated that spinal Toll-like receptor 4, an important receptor for microglia activation, regulates hyperalgesia and allodynia resulting from inflammation or nerve injury exclusively in male mice. Of note, morphologic activation and proliferation of spinal microglia are identical in males and females after nerve injury. Nerve injury–evoked mechanical allodynia is also equivalent in both sexes during the tested times.[163,164] However, mechanical allodynia after nerve injury was exclusively attenuated in male mice after intrathecal injection of microglial inhibitor (minocycline), microglial toxin, or P2X4 blocker, or after special deletion of Bdnf in microglia.[163] Furthermore, nerve injury activates p38 in spinal microglia of male but not female mice; in agreement, spinal administration of p38 inhibitor reduces neuropathic pain only in male mice.[164] Caspase-6 is a microglial activator and released from axonal terminals in the spinal cord after tissue and nerve injury, which can act on microglia to release tumor necrosis factor.[166] Sex dimorphism was also revealed in caspase-6–mediated microglial signaling, and caspase-6 regulates neuropathic pain exclusively in males.[165] It appears some male-specific microglial responses require testosterone, because minocycline reduces allodynia in testosterone-treated females but not in castrated males. In female rodents, the role of microglia in neuropathic pain appears to be replaced by T cells.[163]

Figure 4.

Schematic illustration of local and remote central sensitization induced by glial activation and neuroinflammation in the spinal cord. Activation of spinal microglia and astrocytes by painful insults results in secretion of glial mediators such as tumor necrosis factor (TNF), interleukin (IL)-1β, chemokine (CC motif) ligand 2 (CCL2), chemokine (CXC motif) ligand 1 (CXCL1), brain-derived neurotrophic factor (BDNF), and D-serine, which can act as neuromodulators to induce local central sensitization in surrounding excitatory synapses (facilitation) and inhibitory synapses (disinhibition). During neuroinflammation, these glial mediators also affect synapses in different spinal segments to cause remote central sensitization and extraterritorial and widespread pain beyond the initial injury site. It is also possible that central sensitization may further promote peripheral sensitization via neuroinflammation. AMPAR = α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; CSF = cerebrospinal fluid; GABA = γ-aminobutyric acid; GABAR = γ-aminobutyric acid receptor; Glu = glutamate; GlyR = glycine receptor; NMDAR = N-methyl-D-aspartate receptor.