Introduction

The ability to detect noxious (painful) stimuli is a highly evolutionarily conserved function of the nervous system designed to alert us to the presence of environmental dangers and potentially harmful stimuli. Peripheral nociceptive sensory neurons are responsible for the initial detection of noxious stimuli [1]. These cells, termed peripheral nociceptors, represent a heterogeneous neuron population that can be further defined according to their somal diameter, degree of myelination, cell surface markers, gene expression, and electrophysiological properties, each of which contributes to the specialized role each nociceptor class plays in detecting a variety of noxious stimuli [2]. The cell bodies of nociceptors are located in the dorsal root ganglia (DRG) and trigeminal ganglia (TG), which project afferent fibers to all the body and the head, respectively, ending in nerve terminals specialized to detect noxious stimuli. These cells also extend centrally-projecting fibers to the dorsal horn of the spinal cord (for DRG neurons) or spinal trigeminal nucleus of the medulla (for TG neurons), where they transmit information to second-order nociceptive neurons through a variety of neurotransmitters and neuropeptides [3].

The normal physiological role of pain during homeostasis can be perturbed following injuries or insults to the sensory nervous system. This can result in the uncoupling of pain from the degree or presence of noxious stimuli, leading to the presence of pain at rest (spontaneous pain), the over-amplification of the response to painful stimuli (hyperalgesia), or pain elicited by normally innocuous stimuli (allodynia). This dysregulation can result in pain that persists well-after the initial causative injury or lesion has healed, producing chronic pain which can last for months or years [4]. While acute pain is an important physiological system serving protective functions, chronic pain is maladaptive, offering no benefits to organismal survival and wellbeing. Chronic pain has become a global health epidemic: it is the leading cause of disability worldwide, and more than 100 million Americans suffer from at least one chronic pain condition annually [5]. Additionally, the emergence of the opioid crisis has necessitated the removal of opioids from the war chest of healthcare providers, restricting these agents to treat acute and chronic pain in only the most severe circumstances, and often at great cost to patients [6, 7]. Thus, patients and healthcare providers have been left with fewer options for pain therapies than existed even a decade ago. For this reason, the need to identify new potential pain targets and to develop novel neurotherapeutics capable of providing safe, efficacious relief from chronic pain is both critical and urgent.

Our understanding of the mechanisms underlying chronic pain has emerged over the last three decades. It is now understood to result from localized neural plasticity in the peripheral nervous system (PNS, peripheral sensitization) or central nervous systems (CNS, central sensitization) [8, 9]. Initially, our knowledge of the synaptic changes resulting in sensitization were thought to involve only the peripheral and central nociceptive neurons themselves. However, a plethora of evidence now exists which demonstrates that painful injuries cause the activation of various non-neuronal cell types along the pain circuitry, including immune cells and glial cells, producing a localized form of inflammation in the PNS and CNS (neuroinflammation). These activated cells, in turn, form bi-directional interactions with nociceptors and play a highly active role in the initiation and maintenance of chronic pain [10]. In this review, we will discuss the non-neuronal cell types that become activated and contribute to chronic pain pathogenesis, with particular focus on central glial cells. Additionally, we will highlight newly emerging targets in the central glial cell which show promise for the development of novel pain neurotherapeutics.

Central Glia in Homeostasis and Disease

Within the central nervous system, three major classes of glial cells are present under normal conditions, with oligodendrocytes being the most abundant, followed by astrocytes, with microglia being the least abundant [11]. While studies have begun to emerge to suggest that oligodendrocytes may contribute to chronic pain pathogenesis [12, 13], our understanding of their role in this process remains in its infancy, which we briefly discuss. Thus, this review will focus primarily on the well-established role of microglia and astrocytes in chronic pain pathogenesis, with a selective emphasis on prospective microglial and astrocytic targets.

Microglia are the archetypical CNS-resident immune cells, emulating peripheral macrophages in their phagocytic capabilities. Like peripheral macrophages, microglia constantly surveil their environment, hel** to promote clearance of debris, damaged cells, or infectious agents [14]. However, microglia are also appreciated to possess canonical “glia-like” functions to maintain homeostasis and proper neuronal function in the CNS. To this end, microglia play specialized roles in synaptic pruning during development, neural circuit maintenance and synaptic plasticity in adults, and the regulation of adult neurogenesis under normal physiological functions [15]. Interestingly, Liu et al. recently demonstrated that the morphology and function of microglia changes dynamically in response to neuronal activity. In particular, high noradrenergic tone in awake mice reduced microglial process surveillance, indicating that neuronal function can gate some fundamental activities of microglia [16]. Beyond homeostasis, microglia are associated with disease control and/or pathogenesis in a variety of neurodegenerative (e.g., Alzheimer’s disease, Parkinson’s disease, stroke) and neuropsychiatric (e.g., depression, anxiety) diseases [17, 18]. When microglia become activated in disease states or following injury (e.g., peripheral nerve injury), they undergo “microgliosis,” characterized by profound morphological changes (hypertrophy), proliferation, and functional changes, which correlates with changes in gene expression and function [19, 20].

Compared to microglia, astrocytes are approximately 2–4 times more abundant, accounting for 19–40% of all glial cells in the CNS [11], and are endowed with several unique and non-overlap** homeostatic functions. First, astrocytes are physically coupled to one another through gap junctions, allowing for the permissive exchange of ions and small molecules between adjacent cells [21]. Astrocytes also form unique and extensive contacts with synapses, allowing them to provide structural and metabolic support for neurons to aid in neurotransmission [22]. Under normal physiological conditions, astrocytes play active roles in regulating the extracellular environment, maintaining the proper balance of glutamate, potassium, and water homeostasis [23]. Of particular importance, glutamatergic synaptic transmission is tightly controlled by astrocytic expression of excitatory amino acid transporters EAAT1 and EAAT2 which remove extracellular glutamate, thereby controlling the extent and duration of glutamatergic synaptic transmission [24]. Similar to microglia, astrocytes become activated in a variety of pathological conditions, leading to reactive states (classically termed “astrogliosis”) characterized by morphological changes, profound upregulation of the astrocyte marker glial fibrillary acidic protein (GFAP), and increased proliferation [25, 26]. These changes are also thought to be coupled with a loss of the aforementioned homeostatic functions of astrocytes. However, it is important to note that the activation of astrocytes under pathological conditions is increasingly considered to result in a variety of reactive astrocyte states which vary depending on the initiating disease process [27], although further support for this hypothesis is necessary.

Distinct Roles of Microglia and Astrocytes in Chronic Pain

Similar to their involvement in other pathological disease processes, microglia [20, 28] and astrocytes [23, 29, 30] each play an active role in the pathogenesis of chronic pain. Following nerve injury, hallmarks of microgliosis in the ipsilateral dorsal horn of the spinal cord can be observed rapidly [31], although the precise time course may be dependent on the definition of microgliosis (Fig. 1). Interestingly, spinal microglia activation following nerve injury requires the activity of peripheral sensory afferents [72,73,74,75,76]. Mechanistically, microglia and astrocyte-derived IL-1β can produce pain by both enhancing excitatory AMPA and NMDA-mediated synaptic transmission and concurrent inhibition of GABAergic and glycinergic neurotransmission in the superficial layers of the dorsal spinal cord [72]. Another inflammation-modulating cytokine and IL-1 family member, interleukin-18 (IL-18), is also produced by activated microglia and astrocytes in the spinal dorsal horn following nerve injury and bone cancer, likely driving central sensitization and pain through similar mechanisms to IL-1β [77,78,79].

Both IL-1β and IL-18 are initially produced as cytosolic pro-protein forms which require proteolytic cleavage at specific sites to activate their biological function and enable their extracellular release. The caspase family of cysteine proteases governs the cleavage and subsequent secretion of these factors, which function within a specialized inflammation-driving multimeric protein complex called the inflammasome [80]. Pro-caspase-1 is a component of the canonical inflammasome, linked to one of many specialized inflammasome sensor proteins through an adapter protein, ASC. The inflammasome sensor proteins are variable, conveying functional specificity leading to unique inflammasome subtypes [81]. These inflammasome sensor proteins recognize signals associated with infection, toxic chemicals, cell damage, and stress, driving sensor protein oligomerization, ASC recruitment, and polymerization. This process subsequently enables docking of pro-caspase-1 at the inflammasome, enabling dimerization and subsequent autocleavage to generate the fully active cleaved form, caspase-1. Mature caspase-1 is subsequently active and present, enabling cleavage of pro-IL-1β and pro-IL-18 to their mature forms, which are rapidly released into the extracellular environment to drive inflammation and pain [80, 81].

Several core inflammasome components have been demonstrated to play a role in inflammatory and neuropathic pain. Mice lacking caspase-1 demonstrated attenuated mechanical allodynia in an acute inflammatory pain model induced by intraplantar carrageenin, with a corresponding reduction in hindpaw IL-1β [82], supporting a role of peripheral inflammasome-derived IL-1β the induction of acute pain. In a nerve injury model of neuropathic pain, Li et al. demonstrated the activation of a specialized NALP1+ inflammasomes in spinal astrocytes and neurons in the superficial dorsal horn, which corresponded with heightened IL-1β production. Additionally, administration of a caspase-1 inhibitor could attenuate nerve injury–induced IL-1β production and chronic pain [83]. NALP1+ inflammasomes have also been observed in activated microglia and astrocytes following spinal cord injury (SCI) and traumatic brain injury [84, 85], but their consequence on chronic pain in these models has not been determined. In addition, the NOD-like receptor protein 3 (NLRP3)-containing inflammasome, the prototypical inflammasome driving IL-1β-mediated inflammation in response to sterile injury, has emerged as a novel contributor to pain pathogenesis [86]. Formation of the NLRP3 inflammasome in peripheral sensory neurons has been demonstrated to contribute to acute inflammatory pain, postoperative pain, and neuropathic pain in the chemotherapy-induced peripheral neuropathy (CIPN) model [87,88,136]. However, there is some controversy surrounding the localization and function of BDNF in pain regulation, as BDNF has been challenging to detect in spinal microglia but can readily be detected in DRG neurons and primary afferents in the spinal dorsal horn [137]. Moreover, Sikander et al. demonstrated that sensory neuron-derived BDNF contributes to the acute-to-chronic pain transition in both male and female mice [138], raising the possibility that the sexual dimorphic contributions of BDNF may also be dependent on the pain model analyzed.

Interestingly, while sex appears to dictate the contribution of microglia to pain pathogenesis, inhibition of astrocyte activation attenuates pain in preclinical models equally in males and females, indicating astrocytes may contribute equally [20, 23, 139]. These studies demonstrate that in addition to neuropathic pain model, stage, and genetic background, our understanding of the glial/immune cell contribution to pain pathogenesis is also influenced by sex, underscoring the importance of sex as a biological variable in pain research. It is important to note, however, that clinical studies in humans have yet to conclude that microglia contribute to pain in a sex-specific manner, and both microglia and astrocytes have been found to be activated in both males and females [136]. In future studies, clarification of whether microglia or other immune cells contribute to pain in a sexually dimorphic manner in humans is an important question. Moreover, it will be interesting to identify additional cell types and mechanisms that differentially contribute to pain in males and females through future preclinical and clinical studies.

Emerging Role of Oligodendrocytes in Pain

Oligodendrocytes are the most abundant glial cell type in the CNS, accounting for 45–75% of all glia. However, relatively few studies have focused on the role of oligodendrocytes in the pathogenesis of chronic pain. In patients with HIV-associated peripheral neuropathy, a condition characterized by chronic pain, markers of oligodendrocyte precursor cells (NG2 and Olig2) and mature oligodendrocytes (PDGFRa and MBP) were upregulated in the spinal dorsal horn of human postmortem tissues [13]. Additionally, a subset of patients suffering from neuromyelitis optica, a painful demyelinating disorder affecting the optic nerve and spinal cord, have autoantibodies targeting myelin oligodendrocyte glycoprotein (MOG) [140]. Notably, chronic pain occurs in many patients with multiple sclerosis [141], another demyelinating disorder characterized by autoimmune-mediated loss of oligodendrocytes, suggesting a possible interaction between oligodendrocyte destruction and pain in humans. Experimental ablation of oligodendrocytes in adult mice using diphtheria toxin was found to trigger neuropathic pain for several weeks, which was found to be independent of adaptive immune cells or reactive microglia and astrocytes [142], indicating oligodendrocytes may contribute to the maintenance of pain during homeostasis. Additionally, Zarpelon et al. found that sciatic nerve injury in mice resulted in the upregulation of IL-33 primarily by oligodendrocytes in the dorsal spinal cord, and mice lacking the IL-33 receptor ST2 exhibit reduced pain. Additionally, intrathecal administration of IL-33 evoked hypersensitivity in naïve mice and potentiated mechanical allodynia following nerve injury, which was dependent on TNF-α and IL-1β [12]. Thus, the role of oligodendrocyte in pain modulation is likely interwoven with that of primary afferents, microglia, and astrocytes.

Conclusions and Future Directions

In summary, we have reviewed several targets which have dual functions in the pathogenesis of chronic pain: 1) a direct role in activating sensory neurons in the nociceptive circuitry, thereby acutely promoting pain; and 2) in activating or sustaining the activation of central glia, such as microglia and astrocytes, which themselves produce inflammatory mediators that sensitize nociceptive sensory neurons, leading to pain chronification. Thus, a therapeutic strategy aimed at the pro-inflammatory mediators contributing to both acute and chronic aspects of persistent pain is, in theory, a tenable approach to achieving both acute pain relief and promoting pain resolution. To this end, this review focused on a small cohort of particularly promising, and potentially “druggable,” targets to treat chronic pain. IL-1β is a critical regulator of sensitization and neuroinflammation in the PNS and CNS and thus may be one promising target, especially given the relatively recent emergence of IL-1β-targeting therapies. However, given the protective role of IL-1β in host immunity, targeting IL-1β may yield undesirable consequences such as increased infection or immunosuppression [80, 86, 91]. To avoid such effects, central blockade (e.g., intrathecal administration) of spinal cord microglia and astrocyte-derived IL-1β could be one strategy, also enabling the administration of lower doses of drugs. In addition, targeting other central glia pathways involved in IL-1β synthesis, maturation, or release, such as MMP-2 or MMP-9, Cx43, Panx1, or selective components of the inflammasome machinery may also be potential strategies (summarized in Fig. 4). Several of these potential targets have already been the target of considerable research and development efforts, resulting in inhibitors and antagonists in various stages of clinical trials for human disease, which may facilitate their application to testing their efficacy in chronic pain conditions in humans. The list of targets covered in this review is in no way all-inclusive, and many other therapeutic strategies have been discussed elsewhere, including neuromodulation [143], intrathecal cell therapy [144], glial modulators (e.g., ibudilast) [145], TLR4 antagonists, IL-10 gene therapy [146, 147], and β-blockers, each of which represents an exciting prospective strategy to treat pain through modulation of central glia and neuronal function.

Fig. 4
figure 4

Sensory neuron interactions with central glial cells in neuropathic pain. (a) Following nerve injury, primary afferent nerve fibers release pro-inflammatory mediators such as Csf1, caspase-6 and MMP-9, activating microglia (microgliosis). In turn, activated microglia produce and release mature IL-1β and IL-18 and TNF-α, which act on primary afferent fibers and spinal dorsal horn nociceptive neurons and contribute to sensitization. Microglial-derived TNF-α also acts on nearby astrocytes, contributing to their activation. Activation of astrocytes causes release of mature IL-1β and IL-18, as well as Cx43-mediated release of ATP, glutamate, and chemokines such as CXCL1 and CCL2, all of which induce sensitization of primary afferents and excitatory spinal dorsal horn nociceptors. IL-1β can also suppress GABAergic and glycinergic synaptic transmission in inhibitor spinal dorsal horn neurons (not shown), thus producing central sensitization through both enhanced excitation and central disinhibition. Activation of microglial purinergic receptors also leads to their production and release of BDNF, which drives neuropathic pain via central disinhibition. (b) Astrocyte-derived ATP can also amplify microglial activation by binding to microglial purinergic receptors, contributing to inflammasome activation and subsequent IL-1β and IL-18 maturation (cleavage) and release by the pore-forming inflammasome as well as by microglial Panx1 channels. In addition to caspase-1/inflammasome-mediated activation of IL-1β and IL-18, activated microglia and astrocytes can activate these cytokines through alternative mechanisms involving MMP-9 (microglia) or MMP-2 (astrocytes)

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For each therapeutic agent, a consideration must be made on where the therapeutic effects are most advantageous (e.g., peripheral or central route of administration), where likely side effects would be least desirable, and the pharmacokinetic properties of each agent, including the CNS penetrance. Small nonpolar molecules, for example, are typically much more adept at gaining CNS entry than large biologics such as neutralizing antibodies. It is important to note, however, that blood–brain barrier (BBB) permeability can be altered in many disease states, including chronic pain states [148], and penetrance of large biologics into the CNS can be increased [149]. However, even if BBB permeability is increased, whether systemic administration of large biologics could reach sufficient concentrations to achieve their therapeutic effects is questionable, emphasizing the need to develop new small molecule antagonists which can penetrate the BBB and/or consider alternative routes of administration (e.g., intrathecal).

This review also highlights the crucial role that central glial cells play in the pathogenesis of chronic pain. While the role of oligodendrocytes is still emerging, the contribution of microglia and astrocytes to pain pathogenesis is substantial and irrefutable. Importantly, neither microgliosis nor astrogliosis exists as an “all-or-none” phenomenon. Similarly, the absence of immunohistochemical signatures of microgliosis or astrogliosis does not necessarily imply the absence of a functional change in microglia and astrocytes. In addition, neither reactive microglia nor reactive astrocytes are likely to exist as a homogeneous cell state, but rather, as a dynamic and heterogeneous functional state that is dependent on the evoking stimulus and the local cellular environment [20, 23]. Indeed, even under resting conditions, profound region-specific heterogeneity has been observed when analyzing microglia [150] and astrocytes [151] using single-cell sequencing techniques. Thus, future studies aimed at understanding the molecular and phenotypic changes in microglia and astrocytes at baseline and in various chronic pain states—beyond simple parameters of glial cell activation—will greatly enhance our understanding of the pathophysiological mechanisms involved. Towards this goal, Renthal et al. recently conducted a comprehensive analysis of the transcriptional changes occurring within DRGs by performing single nucleus RNA sequencing on over 100,000 single cells across several different injury models [152]. Similar studies analyzing transcriptomic changes in the central nervous system are needed to facilitate the identification of new glial cell targets for pain treatment.