Biomarkers for microglia activation in neuropathic pain

Putu Eka Widyadharma1, Aurelia Vania2, Jimmy FA Barus2, Yudiyanta3, Thomas Eko Purwata1
1Department of Neurology, Faculty of Medicine, Udayana University and Sanglah General Hospital, Bali, (Indonesia)
2Department of Neurology, School of Medicine and Health Sciences, Atma Jaya Catholic University, Jakarta, (Indonesia)
3Department of Neurology, Faculty of Medicine, Public Health, and Nursing, Gadjah Mada University, Yogyakarta, (Indonesia)

Correspondence: Putu Eka Widyadharma, Department of Neurology, Sanglah General Hospital, Diponegoro Street, Denpasar, Bali 80113, (Indonesia);
E-mail: eka.widyadharma@unud.ac.id

ABSTRACT

Neuropathic pain (NP) is a result of direct disturbances of somatosensory pathways. Its pathophysiology includes various mechanisms. Recent studies have reported an important role of microglia in the NP mechanism. There are several chemical molecules which are involved in microglia activation. The activated microglia will, in turn, enhance some receptors expression that can be used as markers of its activation. Though we still need future studies about precise microglia role in NP mechanism, the chemical mediators that initiate microglia activation and the alteration of some receptors in the activated microglia which have been found from previous studies can be the interesting future research materials and the promising target for a new therapy for NP.
Key words: Neuropathic pain; Microglia activation; Biomarker
Citation: Widyadharma PE, Vania A, Barus JFA, Yudiyanta, Purwata TE. Biomarkers for microglia activation in neuropathic pain. Anaesth pain intensive care 2020;24(1):___

DOI: https://doi.org/10.35975/apic.v24i1.

Received – 12 June 2019, Reviewed – 15 September 2019, 29 February 2020, Accepted – 2 March 2020

INTRODUCTION

Neuropathic pain (NP) was defined by IASP as “pain emerging as a direct consequence of a lesion or a disease of the somatosensory system”.1 NP is commonly characterized by electric-shock-like sensation, burning sensation, hot or cold pain, spontaneous ongoing pain, paresthesia or allodynia. If the symptoms persist, NP has a tendency to become a chronic pain. The prevalence of chronic pain caused by NP has been reported to be in the range of 7-10%.2
NP does not occur through the same mechanism with other conditions that lead to chronic pain caused by inflammatory pain. The primary cause of the latter condition is an inflammatory process with chemical mediators alteration at the site of injury.2 Lesions of nervous system which lead to NP most commonly involve nociceptive pathways.1 Disturbances of the somatosensory system can induce abnormal changes in transmission of sensory signals from peripheral system to spinal cord and brain.2 NP includes some complex mechanisms. Some different mechanisms could appear in one patient with NP, and these different mechanisms can cause similar symptoms. At this time, most of analgesic drugs do not completely eliminate symptoms of NP. The management of NP is a real challenge and it drives researchers to learn more about the mechanism and more appropriate therapy for NP. Therapeutic approach with the target based on the mechanism of pain in one patient is the most important thing to get efficient analgesic therapy.
NP is caused by peripheral and central mechanisms. Peripheral mechanism includes ectopic discharges in lesioned fibers and their corresponding ganglia, abnormal activity in axons undamaged by the lesion, alterations in the expression and regulation of intracellular calcium ion and modulatory receptors on primary afferent terminals, neuro-immune interactions resulting in enhanced and/or altered production of inflammatory signaling molecules, sensory-sympathetic coupling and other alterations in receptor signaling. Continuous activities from sensory peripheral system cause the changes in sensory central nervous system that include alteration of presynaptic and postsynaptic molecules, dysfunction of inhibitory interneuron and descendent inhibition system. Finally, all of these alterations result in central sensitization.1,2
Inflammation in the peripheral or central systems has been known to have a contribution to ectopic neuron activity and central sensitization.1 Recent studies have found some evidence of a vital role of microglia as a central nervous system immune cell in NP mechanism.1,3-7 These findings promote more studies for new NP therapeutic approach with microglia activation as the target. This article has been written to learn more about chemical mediators that have roles as the initiation factors or markers of microglia activation and some pathways that contribute to cause NP after microglia activation has occurred. These biochemical markers could be expected to be a new target for NP therapy or as a new tool to monitor the therapy that inhibits microglia activation.

ROLE OF MICROGLIA CELL IN NP

Microglia are macrophage-like cells in the central nervous system that regulate homeostasis in the brain and spinal cord. These immune cells are emerging as the key to the pathogenesis of some neurodegenerative diseases. These cells contribute to the pathogenesis of the disease via neuroinflammatory reactions.6 Lesion in both peripheral and central system will trigger the inflammatory reaction. Although neuroinflammatory reaction has neuroprotective and neurotrophic effects, it is also associated with the nerve damage that causes NP. Microglia are the first immune cells that are activated in dorsalis root ganglion after nerve injury.1
Microglia activation can be induced by several chemical mediators that are released from neurons after nerve injury, such as colony stimulating factor-1, fractalkine, chemokine (C-C motif) ligand 2 (CCL2), neuregulin-1, and matrix metalloproteinase (MMP).6 The activated microglia will proliferate and change its morphology from “resting” state to “amoeboid” state. This activation will also increase microglia markers expression (for example CD11b, Iba1, P2X4, CX3CR1, TLR4).7,9 Microglia will also migrate to the site of injury and stimulate the production of proinflammatory cytokines such as IL-6, IL-1β, TNFα, colony stimulating factor-1 (CSF1), nitric oxide, brain-derived neurotrophic factor (BDNF), excitatory amino acids, ATP, and prostaglandins.1,3,6 These inflammatory mediators are the key to the occurrence of NP.6 Activated microglia will increase nerve excitation and decrease the inhibition by interneurons.
Activation of microglia occurs during the early phase of NP and before astrogliosis. It supports the hypothesis that microglia could be important for the initiation of NP, whereas astrocytes are essential for the alteration of acute pain to chronic pain.9 One of the studies in animal has found that microglia activation will cause spinal hyperexcitability and is associated with pain and allodynia conditions. That inhibiting microglia activation diminished allodynia and hypersensitivity after nerve injury has been reported in several studies.3
BIOCHEMICAL MARKERS IN THE PROCESS OF MICROGLIA ACTIVATION
  1. Neuregulin-1 dan erbB2
Neuregulin-1 is a family member of growth factors released after nerve injury and involved in neuronal development. It is one of the important mediators for microglia activation. Calvo et al. found the release of neuregulin-1 from primary afferent within dorsal horn following spinal nerve ligation in mice and it activated erbB2 receptor. Receptors of neuregulin-1 including erbB 2, 3, and 4 are found on the surface of microglia cells, suggesting that there is a neuron-glial interaction. Calvo et al. also reported that activation of erbB2 by neuregulin-1 will stimulate MEK/ERK pathway in microglia. MEK/ERK activation will drive proliferation of microglia cells and the release of inflammatory mediators, and contribute to the development of NP. Some studies have shown that inhibition of neuregulin-1, erbB2, MEK/ERK pathway will reduce microglial proliferation as well as mechanical and cold pain related hypersensitivity. These studies proposed that neuregulin-1 and erbB2 could be the potential target of NP therapy.3,10,11
  1. Matrix Metalloproteinase
The MMPs are a family of zinc-containing endoproteinases that share structural domains but differ in substrate specificity, cellular sources, and inducibility. The major function of MMPs is the degradation and remodeling of extracellular matrix component. MMPs are synthesized in their inactive proform, then activated extracellularly by proteolytic cleavage that is regulated by several inflammatory mediators, such as cytokines, chemokines, free radicals, and steroid. As proteolytic enzymes, MMPs take part in the development and physiological activities of central nervous system, such as myelin formation, axonal growth, angiogenesis, and regeneration. In general, if there is an abnormal expression or overproduction of MMPs, they will cause tissue destruction.12
One of the variants of MMPs, MMP-9, play a role in microglia activation. Some studies have reported there is an increased of MMP-9 in dorsalis ganglion following nerve injury. A study from Kawasaki et al. proved this hypothesis. They found a transient and rapid upregulation of MMP-9 with its peak at day 1 and started to decline after three days in dorsal root ganglion (DRG) primary sensory neuron after spinal nerve ligation in mice.13 Liou et al. also found rapid increased MMP-9 expression in peripheral nerve and spinal cord after partial sciatic nerve ligation in mice.14 These studies concluded that MMP-9 is involved in starting the NP rather than maintenance of NP.3,5
MMP-2, the other variant of MMPs, has also been found to be associated with NP mechanism. Unlike MMP-9, MMP-2 has more important role in maintenance of NP than initiation of NP. Kawasaki et al. showed upregulation of MMP-2 occurred at day seven and was still detected on day 21. They concluded that MMP-9 and MMP-2 take part in NP through IL-1β cleavage and microglia activation.13 In one study, MMP-2 intrathecal injection lead to NP condition in mice and peritoneal administration of MMP inhibitor, GM6001, diminished thermal hyperalgesia and allodynia triggered by nerve injury. Therefore, either MMP-2 or MMP-9 were considered as important chemicals for the development and maintenance of NP, as they play a role in the initiation and persistence of NP.3
  1. CCL2 and Chemotactic Cytokine Receptor-2
CCL2, also known as monocyte chemoattractant protein 1 (MCP1), will be upregulated in dorsalis ganglion after nerve injury. CCL2 can induce microglia activation.15 CCL2 has low expression in normal condition, but it will increase its level after nerve injury. CCL2 binds to the chemotactic cytokine receptor-2 (CCR2) which is a G-protein coupled receptor in microglia cells. Zhang et al. using partial sciatic nerve ligation in mice model showed that CCR2 knocked out mice had reduced microglia activation.16 Zhu et al. study using a rat model with lumbar disk herniation reported that there was an increased CCR2 expression in DRG and spinal cord. They also found that intratechal injection of CCR2 inhibitor could diminish pain hypersensitivity induced by NP.17
Thacker et al. found an increased CCL2 concentration at day 1 in DRG after chronic constriction injury and spinal nerve ligation in rats.18 White et al. in their study using chronic compression of DRG in mice reported that CCL2 will cause neuron depolarization, trigger excitatory condition in neuron of dorsalis ganglion that will result in NP symptoms such as allodynia.19 The results of several studies have found that intrathecal injection of CCL2 would result in allodynia condition in 24 hours, while injection of CCL2 neutralizing antibody or CCR2 antagonist would prevent allodynia. These studies concluded CCL2 and NP interaction occurs by neuron excitation and microglia activation.3 CCL2, however, also has direct effects on neurons in dorsal root ganglion demonstrating that the role of this chemokine might be various and not only limited to the microglia activation.10
  1. P2X4 receptor
P2X4 receptor (P2X4r) is belonged to purinergic system that has intrinsic pores that open on binding of extracellular ATP.5 P2X4r is exclusively expressed in activated microglia after peripheral nerve injury.1,4,5 P2X4r expression upregulation in microglia is known to have a positive interaction with pain hypersensitivity. Some studies have shown that the blockage of P2X4 receptor would attenuate allodynia after nerve lesion, while injection of microglia with P2X4r expression would trigger allodynia in naïve animal.1,9 Role of P2X4r was first known in 2003. In that year, Tsuda et al. published a report that described an increased expression of P2X4r in microglia cells that have been activated after spinal nerve ligation in rats. They also reported that intrathecal administration of P2X4r inhibitor reduced tactile allodynia and intrathecal administration of P2X4r-stimulated microglia was sufficient enough to develop tactile allodynia in normal rats.20 Their study result was supported by Ulmann et al. who also found upregulation of P2X4r in microglia after partial sciatic nerve ligation in mice and P2X4r knock out mice were more resistant to tactile allodynia induced by peripheral nerve injury.21
Activated P2X4r will increase the release of BDNF via calcium ion influx and activation of P38 mitogen-activated kinase, and it also increases synthesis and SNARE-mediated exocytosis of BDNF. BDNF causes disinhibition in the dorsalis horn through increasing level of intracellular chloride by downregulating KCC2 function as neuronal chloride transporter.3,5,22-23 This process triggered by BDNF would change the GABA and glycine effects to be neurons depolarization rather than hyperpolarization.4,5
At last, activated P2X4r will result in increasing of neuron discharge, response to non-nociceptive stimuli, and spontaneous neuron activity.23 The activation of P2X4r itself is mediated by upregulated IRF8-IRF5 cascade and chemical mediators released by the damaged neurons, such as chemokine, fractalkine, CCL2, and CCL21.22,24 The result from several studies reported P2X4r has a crucial role for microglia to be an important cause of NP mechanism.
  1. Toll-Like Receptor-4
Toll-Like Receptor-4 (TLR4) involved in innate immune system is mainly expressed in microglia.8 Activated microglia will increase TLR4 expression on its surface. TLR4 is a transmembrane receptor protein receptor with extracellular leucine-rich repeat domains and cytoplasmic repeat domains.15 Lehnardt et al. reported that TLR4 is expressed exclusively in microglia.25 Some microglia markers, including TLR4, can be used to detect when the microglia activation is initiated and the duration of microglia activation during the episode of NP. Tanga et al. used real-time PCR identification to detect CD14 and TLR4 in their research of microglia activation, and found the highest peak at day four and a return to normal level by day 28 after spinal nerve transection in rats.26 While Jurga et al. found enhanced expression of TLR4 in spinal medulla and dorsal ganglion at day seven and day 14 after chronic constriction injury in rats in their study.27 Tanga et al. in their later study using spinal nerve transection model in mice reported TLR4 antisense ODN could reduce pain hypersensitivity, microglial activation, and production of proinflammatory cytokines.28
Recent evidence indicated essential role of microglia with TLR2 and TLR4 expression in NP mechanism. Neuronal damaged can lead to the release of proinflammatory factors that activate spinal microglia, one of the pathways is via TLR4/NF-κB signaling pathway. Studies reported the decreasing microglia activation, reduction of cytokines production, increasing pain resistance, and decreasing pain hypersensitivity in TLR4 deficient animals.15,27,29 Diminished mechanical allodynia and thermal hyperalgesia found in TLR4 knockout mice were showed in Bettoni et al. study using chronic constrictive injury model.29
Jurga et al. found the administration of LPS-RS (TLR2 and TLR4 antagonists) and LPS-RS ultrapure (TLR4 antagonist) could diminish pain after chronic constriction injury, in which they used mice with nerve injury-induced NP model. These TLR antagonists enhanced buprenorphine activity as antiallodynia and antihyperalgesia, but they did not show the same effect in morphine.27 Previous studies reported important contribution of TLR4 in initiation and maintenance of microglia activation and NP.
  1. Fractalkine and CX3CR1 Receptor
Fractalkine (FKN) is expressed primarily in neuron of the central nervous system. The expression of FKN can also be observed in the cell body of peripheral sensory neurons in dorsalis ganglion. FKN is a neuron transmembrane glycoprotein and can be cleaved from neuron as a result of increased neuronal activity.3,30 FKN can be cleaved by the protease Cathepsin S which is released by activated microglia.3,15 The cleaved FKN binds to CX3CR1 receptor in microglia, creating a positive feedback loop for the activation of microglia cells.3 CX3CR1 is seven-transmembrane domained G-protein coupled receptor and is expressed abundantly in both blood leucocyte cells and microglia. Upregulation of CX3CR1 occurs in spinal microglia following injury to the peripheral nerve.30 Level of FKN and Capthesin S activity in cerebrospinal fluid increased significantly after peripheral nerve injury, associated with microglia activation.15
Several studies reported that there is increased FKN concentration and role of CX3CR1 expression in NP occurrence after nerve injury.3,9,30 Zhuang et al. found there was an upregulation of CX3CR1 in microglia that started at day 1 and reduction of mechanical allodynia after injection of neutralizing antibody of CX3CR1 in mice after spinal nerve ligation.31 Thermal hyperalgesia and mechanical allodynia were attenuated in CX3CR1 knockout mice, compared to naïve mice after partial sciatic nerve ligation. This finding was showed in a study by Staniland et al.32 While Hu et al. reported that there were delayed appearance of mechanical allodynia, depressed microglia activation, and inhibition of p38MAPK expression after intrathecal administration of CX3CR1 neutralizing antibody in rats with bone cancer pain model.33
FKN and CX3CR1 activation result in phosphorylation of p38MAPK, which subsequently stimulate the release of inflammatory mediators such as IL-1β, IL-6 and NO in microglia that are involved in increasing pain sensation.3,30 The pro-nociceptive effects of the CatS/FKN/CX3CR1 pathway are crucial for the maintenance phase of NP. Inhibition of CX3CR1, p38MAPK, dan Cathepsin S could diminish hypersensitivity condition in chronic pain model.3,30
  1. CD11b and Iba1
CD11b and Iba1 have been used most frequently for the study of microglia activation. CD11b is recognized by using OX-42 antibody. Upregulation of OX-42 or Iba1 are the marker of microglia activation.
CD11 is a family member of integrin and is expressed highly in monocytes or macrophages, common dendritic cells, and microglia cells. CD11b in common dendritic cells and macrophages could inhibit T-cells activation and inflammatory process. Whereas CD11 in microglia and its role in NP mechanism has not been known.34 In the pain model that did not involve nerve injury, OX-42 expression did not increase significantly. Nerve injury, for example, lesion in sciatic nerve, caused enhancing of OX-42 expression.7 Mika et al. used C1q and OX-42 antibody for binding CD11b to study about microglia activation and found that the microglia activation occurred on the second day after nerve injury, and reached its peak on day 7-9. They found the reduction of NP symptoms and microglia activation on day 17-21.3,35 Clark et al. also found increased CD11b expression following microglia activation after partial sciatic nerve ligation in mice.36 Although CD11b is a marker of microglia activation, the pain condition itself may not be related to CD11b changes in some cases. The increased CD11b expression could be more related to nerve injury rather than the pain symptoms.7
Ionized calcium-binding adapter molecule-1 (Iba1) is a protein bounded to calcium ion and is expressed exclusively in microglia in the brain. Iba1 is one of the important keys for the migration and phagocyte activity of microglia. Anti Iba1 antibody is used to detect microglia in dorsalis ganglion. Romero-Sandoval et al. has used Iba1 as microglia marker and found that microglia is important for maintenance of neuropathic process.37 Leinders et al. found evidence for an increased Iba1 expression in microglia that could be detected for at least 14 weeks after spinal nerve transection in rats.38 But, the other study has reported that this marker was not enough to trigger hypersensitivity after microglia activation. These have been shown from a study using the mutant mice without P2X4r, whereas increasing of Iba1 and other morphology changes were not different from other normal mice, mechanical hypersensitivity did not occurred in mice without P2X4r.23
FUTURE THERAPEUTIC IMPLICATIONS

There are various pathways occurring before and after microglia activation in NP mechanism. Which pathways is the core mechanism of neuropathic pain remains to be determined. Nevertheless, these findings of the crucial role of microglia have been interesting many researchers to find new therapeutic drugs for NP. Some researches have been done to know the effectiveness of some drugs associated with microglia activity.
Recently, minocycline, an antibiotic that has been used for a long time, is being studied as a future potential NP therapy. Rojewska et al. reported that minocycline could reduce MMP-2 and MMP-9 level in spinal cord and DRG. They administered minocycline repeatedly and via intraperitoneal pathway to mice before injury occurred. In their study, minocycline caused the change in the ratio of pronociceptive and antinociceptive factors.39 Minocycline has also been studied in human in some studies, but the results of these studies have not shown a consistent effect of minocycline. Sumitani et al. in their study concluded that minocycline was not succeeded to decrease pain intensity, but it improved the affective disorders associated with NP.40 Based on previous research, minocycline is known to have the ability to prevent the development of NP, but it is not effective to reduce NP after it occurs.6 Therefore, we still need to do more research about minocycline as the NP preventive and therapeutic drug.
There are several chemicals that have been studied to target pathway involved in microglia activation. Japanese researches have been used NP-1815-PX compound as potential P2X4r antagonist. Intrathecal administration of NP-1815-PX could reduce mechanical allodynia induced by herpetic pain in a mouse model.41 Yamashita et al. investigated duloxetine effect and found that duloxetine, one of the SNRI inhibitor agents, could inhibit recombinant P2X4r that they got from cultured cells. Intrathecal administration of duloxetine could also reverse mechanical allodynia by inhibiting microglia P2X4r in mice and rat.42 Recently, there is another potent P2X4r antagonist that is being studied in Japan.24 Hewitt et al. studied MIV-247, selective cathepsin S inhibitor, and reported that MIV-247 could attenuate allodynia and enhance antiallodynic properties of pregabalin and gabapentin without increasing the side effects of these drugs in mice model of NP.43 Naltrexone and naloxone were shown as potential inhibitor of microglia activation and TLR-4 signaling pathway. They reduced production of ROS and phagocytosis induced by microglia activation.44
The results of microglia modulators in human still shows inconsistent effects that may be due to the different underlying cellular mechanisms in each NP cases or due to the study design. There were some other approaches that have been studied such as neuromodulation or stem cell therapy.6 There are various chemical molecules involved in microglia activation and NP mechanisms. Effective analgesic drugs should target all these various mediators.

CONCLUSION

Microglia have been emerging as an essential role in the pathophysiology of NP. Various receptor and inflammatory mediators are involved in microglia activation in the pathophysiology of NP. Both the receptor and their chemical mediators can be the useful biochemical markers for the future study about the pathophysiology of microglia activation and its relationship with the mechanism and progressivity of NP. Although we need to learn more, the studies which have been done before about microglia activation and biochemical markers involved in it can be the basis of the development of new therapy for NP.

Declarations of interest: We declare no competing interests.
Conflict of interest: None declared by the author Authors’ contribution:
IPEW: Corresponding Author, Concept, Manuscript Writing
AV, JFAB: Manuscript Writing
Y, THP: Manuscript Editing
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