Microglial Cannabinoid CB₂ Receptors in Pain Modulation

Abstract

Pain, especially chronic pain, can strongly affect patients’ quality of life. Cannabinoids ponhave been reported to produce potent analgesic effects in different preclinical pain models, where they primarily function as agonists of G_i/o protein-coupled cannabinoid CB₁ and CB₂ receptors. The CB₁ receptors are abundantly expressed in both the peripheral and central nervous systems. The central activation of CB₁ receptors is strongly associated with psychotropic adverse effects, thus largely limiting its therapeutic potential. However, the CB₂ receptors are promising targets for pain treatment without psychotropic adverse effects, as they are primarily expressed in immune cells. Additionally, as the resident immune cells in the central nervous system, microglia are increasingly recognized as critical players in chronic pain. Accumulating evidence has demonstrated that the expression of CB₂ receptors is significantly increased in activated microglia in the spinal cord, which exerts protective consequences within the surrounding neural circuitry by regulating the activity and function of microglia. In this review, we focused on recent advances in understanding the role of microglial CB₂ receptors in spinal nociceptive circuitry, highlighting the mechanism of CB₂ receptors in modulating microglia function and its implications for CB₂ receptor- selective agonist-mediated analgesia.

1. Introduction

Chronic pain is a complex web of emotional experiences and subjective senses that brings patients enormous physical and psychological burdens. Due to the current unmet demand for pain relief, attention has been highly focused on effective analgesics without major central adverse effects. The endocannabinoid system serves as an important neuromodulator signaling, pathway that is deeply involved in the complex modulation of endogenous homeostasis and a variety of pathological processes. Currently, in parallel to the great interest in the endocannabinoid system, cannabinoid compounds, including endocannabinoids, plant-derived, and synthetic cannabinoids, are increasingly recognized as potential therapeutic alternatives for pathological pain, because of their vital roles in modulating nociceptive information processing [1,2].

The biological effects of these cannabinoids are mainly mediated by two endocannabinoid receptors, cannabinoid receptors 1 (CB₁R) and 2 (CB₂R). However, currently available cannabis-based medicines are largely targeting CB₁R, which is detected abundantly in both the peripheral and central nervous systems (PNS and CNS, respectively). Their activation can result in a spectrum of adverse effects, such as the development of tolerance [3], addiction, and psychotomimetic effects [4], thus limiting their therapeutic potential. Subsequently, several strategies have been developed for reducing the central side effects of cannabinoid compounds acting as analgesics, including peripherally restricted CB₁R agonists, topically applied cannabinoids, cannabinoid metabolic enzyme inhibitors, bifunctional cannabinoids ligands, as well as selective CB₂R agonists. For example, previously reported VF13, a bifunctional chimeric peptide containing the pharmacophores of the endogenous cannabinoid peptide VD-hemopressin(α) and neuropeptide VF, produced non-tolerance forming antinociception in multiple pain models with reduced cannabinoid-related side effects [5]. In addition, since CB₂R is primarily expressed in immune cells and relative paucity in neurons, the advantageous profile of CB₂R agonists devoid of centrally CB₁R-mediated negative side effects suggests that CB₂R may represent an attractive target for alleviating pain [6,7]. A large number of CB₂R selective agonists such as JWH-133, JWH-015, AM1241, and GW405833 have been demonstrated to exert significant antinociception in various preclinical pain model, including inflammatory pain [8,9], postoperative pain [10], neuropathic pain [8,9,11,12,13,14], and bone cancer pain [15,16], without eliciting tolerance [8,17], hypoactivity [18], hypothermia [18,19], catalepsy [19], psychotropic effects and physical dependence [20,21,22]. Therefore, amounts of research efforts focused on the discovery and optimization of synthetic high-affinity CB₂R agonists to promote selective binding to the CB₂R are ongoing, such as the acyl hydrazone derivative MDA7, a more promising CB₂R agonist for severe pain therapy [23,24,25,26].

Historically, CB₂R was thought to be mainly expressed in peripheral immune cells. However, with the increasing interest in CB₂R-related research, converging evidence over the last couple of decades has indicated that CB₂R is also expressed in microglia and some neurons in the CNS. Meanwhile, it is markedly upregulated in activated microglia in response to injury or chronic pain states [10,27,28,29,30,31,32]. Microglia are the macrophage-like cells in the CNS that providing immunomodulatory functions in response to injury or disease. In recent years, increasing studies have demonstrated that spinal microglial activation and microgliosis induced by some inflammatory mediators released from the central nerve terminals of nociceptor neurons play an active role in pathological pain processing [33,34]. Along with rapid morphological changes in microglia, numerous signaling molecules were also altered in microglia in response to microenvironment changes [33,34]. However, strong evidence now supports the hypothesis that the extent of the spinal CB₂R marked increase in activated microglia may contribute to the shift of microglia to adopt an anti-inflammatory phenotype, with important consequences for the surrounding neural circuitry of nociceptive transmission [35,36,37].

Accumulating studies have shown that targeting spinal CB₂R represents a promising strategy for pain relief without the classical cannabinoid side effects. However, the underlying mechanisms involved in microglial CB₂R activation and its contributions to pain processing remain poorly understood. This review will focus on the current research on the roles of microglial CB₂R in pain processing in vitro and in vivo. We emphasize the involvement of CB₂R signaling cascades in spinal microglia in the pain processes, as well as the molecular mechanisms involved in mediating the analgesic effects of cannabinoids, especially synthetic CB₂R agonists.

2. The Endocannabinoid System

The endocannabinoid system (ECS) is comprised of multiple receptors, endogenous lipid ligands, and various enzymes that regulate endocannabinoid synthesis and degradation, as summarized in Figure 1. As a neuromodulatory system widely involved in lipid signaling responses, the ECS has shown great potential in the development of therapeutic drugs targeting pain management [38]. The endocannabinoids are generated from the hydrolysis of membrane polyunsaturated fatty acids, which is mediated by different enzymes. Arachidonoyl ethanolamide (AEA) and 2-arachidonoyl-glycerol (2-AG) were the first discovered and extensively studied endocannabinoids. Most AEA appears to be derived from its membrane precursor, N-acyl phosphatidyl ethanolamine (NAPE), which is hydrolyzed by N-acyl-phosphatidyl ethanolamine-phospholipase D (NAPE-PLD) [39]. After its release and uptake, the degradation of AEA is primarily via the enzyme fatty acid amino hydrolase (FAAH) [40] or cyclooxygenase-2 (COX-2) [39]. The 2-AG is mainly produced from the hydrolysis of an arachidonoyl-containing phosphatidyl inositol bis-phosphate (PIP₂) mediated by phosphoinositol-phospholipase C (PLC) and two diacylglycerol lipases α/β (DAGLα/β) [41]. Sequentially, 2-AG is hydrolyzed primarily by monoacylglycerol lipase (MAGL) and to a lesser extent by α/β domain hydrolases (ABDH) 6 and 12. Occasionally, FAAH and COX-2 also degrade 2-AG under some conditions [42,43,44]. In addition to the best-known 2-AG and AEA, some neuropeptides are also considered to be endocannabinoids and have been described to have CB₁R and CB₂R activity, such as hemopressin, a fragment derived from the α-chain of hemoglobin [45,46,47,48].

The CB₁R and CB₂R are the two identified and best-characterized cannabinoid receptor subtypes. Both are seven transmembrane inhibitory G_i/o protein-coupled receptors involved in classical signal transduction pathways, including inhibition of adenylyl cyclase (AC) activity, reduction of cyclic adenosine monophosphate (cAMP) production, as well as signal pathways associated with mitogen-activated protein kinases (MAPK) [49]. The 2-AG acts as a high efficacy full agonist for both CB₁R and CB₂R. However, AEA exhibits low efficacy and can act as a partial agonist preferentially for CB₁R with 4-fold selectivity compared with CB₂R [50,51,52]. In addition, previous studies demonstrated that the putative non-CB₁R/CB₂R orphan receptor GPR55 might represent the third cannabinoid receptor (CB₃R), though GPR55 shares a low amino acid sequence with both CB₁R (13.5%) and CB₂R (14.4%) [53,54]. Subsequently, lysophosphatidylinositol (LPI) is identified as an endogenous agonist of GPR55 [55], while the physiological role and pharmacology of GPR55 remain unclear [56]. Recent studies in animals suggest that GPR55 also plays an important regulatory role in pain signal processing in the CNS [57].

It is acknowledged that CB₁R is abundantly expressed in neurons of the brain cortex, cerebellum, basal ganglia, and hippocampus within CNS areas. These brain areas not only regulate movement and memory functions, but also intricate pain signaling, which accounts for the impact of CB₁R agonists on psychoactivity, cognition, memory, and locomotive activity [57,58,59]. In the spinal cord, CB₁R was found in the glutamatergic interneurons, glycinergic interneurons, GABAergic inhibitory interneurons, and a subset of spinal projection neurons [60,61]. The CB₁R functions primarily by modulating neuropeptide and neurotransmitter release to inhibit synaptic transmission in the brain and spinal cord. Activation of CB₁R results in the activation of inwardly rectifying K⁺ channels, promoting cell hyperpolarization, which leads to the inhibition of the presynaptic neurons. Meanwhile, activation of CB₁R can inhibit Ca²⁺ channels to decrease neurotransmitter release [62] (Figure 1). Additionally, CB₁R has been detected in the peripheral nervous system like the dorsal root ganglion (DRG) and other tissues, such as the spleen [63], leukocytes [64], tonsils [65], skin [66], testes, heart, lungs, small intestine, urinary bladder, prostate, uterus, ovary, and vas deferens [67,68,69]. Recently, CB₁R has also been reported as functionally expressed in different glial cells, specifically at a low level in microglia and mainly in different neuronal subtypes. Whether and how CB₁R regulates microglial function remains unclear. Moreover, CB₁R is also expressed in astrocytes, shaping synaptic transmission and memory [35,70,71,72,73].

The CB₂R shows a distinctly different distribution compared to the CB₁R, which was initially identified predominantly at the periphery, mainly in immunocytes, such as lymphocytes, neutrophils, leukocytes CD8 and CD4, and macrophages [2,4,35,74,75,76,77] in various tissues, such as the liver [78], pancreas [79], bone [80], skin [66], spleen, and vascular elements [81]. Subsequent data indicated that CB₂R can also be detected in nervous systems, but the expression level of CB₂R is 100-fold lower than that of CB₁R under normal physiological conditions, which also suggests that the activation of CB₂R may have a different pharmacological profile and not be involved in the unwanted cannabis-like effects [82]. Within the CNS, the CB₂R appears to be expressed by some neurons specifically within the cortex, striatum, hippocampus, amygdala, olfactory, cerebellum, thalamus, spinal nuclei, and substantia nigra [69,83,84]. Meanwhile, the accumulating reports showed that CB₂R can also be found in brain glial cells, particularly microglia [85,86,87]. When compared with CB₁R, CB₂R exhibit one unique feature that is a dynamic expression manner under certain pathological conditions (e.g., inflammation, anxiety, epilepsy, and nerve injury) in neurons and microglia, suggesting the alteration of CB₂R expression and function are closely related to these neurological diseases [9,32,83,84,88,89,90,91].

Notably, the spinal cord distribution of the CB₂R (mRNA or protein) has been identified primarily in microglia, especially activated microglia, and poorly expressed in neurons by the preponderance of evidence using quantitative real-time PCR [6,9], in situ hybridization [28], western blotting [92,93], immunohistochemical staining [17], or single-cell sequencing [60]. Intriguingly, similar to peripheral CB₂R, which is mainly detected in immune cells, central CB₂R is predominantly expressed in microglia, which indicates that CB₂R is critically involved in the modulation of immune-related responses, including immune-neuron interactions and the occurrence of neuroinflammatory and pathological pain [94].

3. Microglia Express the CB₂R-Related Functional Endocannabinoid System

In acting as the central custodians protected by the blood-brain barrier, microglia are derived from primitive macrophages that emanate from the embryonic yolk sac and invade the CNS via the circulatory system during development [95,96]. Over recent decades, a large amount of strong evidence has supported the hypothesis that microglia-dependent phagocytosis/degradation and neuroimmune response critically contributes to the alterations in synaptic remodeling and signaling pathways for chronic pain development [33,34,97,98]. In order to maintain stability in an ever-changing environment, microglia adopt different phenotype states characterized by distinct morphological types to perform their functions.

In the healthy adult CNS, homeostatic microglia with a small soma and highly ramified processes classically display a “resting” phenotype to continuously palpate the environment [95]. In nerve injury or chronic pain conditions, spinal microglia may rapidly proliferate and become activated to a pro-inflammatory (M1, classically activated state) phenotype with shorter processes and hypertrophic soma to respond to some inflammatory mediators, including neurotransmitters (e.g., calcitonin gene-related peptide, CGRP), growth factors (e.g., colony-stimulating factor-1, CSF-1), adenosine triphosphate (ATP), chemokines (e.g., CX3CL1), and enzymes (e.g., Caspase-6), released from central nerve terminals of nociceptor neurons [99,100,101]. Meanwhile, microglia produce and secrete various pro-inflammatory mediators, including interleukin 1β (IL-1β), IL-6, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), and prostaglandin E₂ (PGE₂), which can promote immunological actions and can also act on neurons to lead to central sensitization of surrounding neuronal circuits, contributing to the manifestation of chronic pain [102].

In addition, after this M1 response, aimed at eliminating noxious stimuli and restraining the initial inflammation from the area, the resolution of the inflammatory process is essential to bypass neurotoxicity and chronic inflammation [103]. For this resolving phase to occur, a shift in microglia phenotype along a spectrum of activation states from pro-inflammatory to phagocytic anti-inflammatory (M2, alternative activation state) is typically observed, which could increase the production of anti-inflammatory mediators, including transforming growth factor (TGF-β), interleukin 10 (IL-10), IL-13, and IL-4 [104,105]. This process can dampen the pro-inflammatory cytokine levels and promote the resolution of inflammation and the recovery of tissue homeostasis [105,106].

Previous studies in CB₂R^−/− mice have demonstrated that an exacerbated microgliosis occurs in the spinal cord after peripheral nerve injury [29,107], which indicates the relevant role of CB₂R in controlling microglial proliferation and reactivity. Indeed, accumulating reports have shown that microglia express functional endocannabinoid signaling, which could modulate the activity of microglia to restore CNS homeostasis during pathological neuroinflammatory conditions [108,109]. Firstly, classic studies have shown that cultured microglia from humans [103,110], rats [77,111], or mouse tissues [9,85] and the BV-2 microglia cell line [85], express large amounts of CB₂R. As previously mentioned in Section 2, CB₂R is predominantly expressed in activated microglia within the CNS, which is consistent with CB₂R upregulation in activated macrophages in the peripheral [77,112]. By contrast, the expression of CB₁R in microglia is ambiguous. Preliminary evidence from early studies suggested that CB₁R is expressed in culture microglia prepared from mollusks and rodents [111,113], but not in humans [110]. However, it has recently been shown that, unlike Cnr2 (CB₂R) transcripts, Cnr1 (CB₁R) expression was not detected in naïve microglia by microfluidic RT-qPCR [114]. These results require further functional and anatomical evidence to investigate whether CB₁R can regulate microglial function. To date, Agnès Nadjar et al. have provided the only evidence of CB₁R functional expression on microglia [115].Their results showed that neither male nor female animals exhibited any peculiar behavioral phenotype when CB₁R was selectively deleted in CX3CR1-positive cells, including motor activity, anxiety levels, learning, and memory. These results suggested that there is a possibility that low expression of CB₁R in microglia has no clear role on microglia function. Futhermore, Maciej Pietr et al. found that GPR55 is significantly expressed in both the BV-2 cells and primary mouse microglia. Their expression pattern response to the state of microglia is very similar to that of CB₂R, suggesting it might also be involved in the neuroinflammatory process [116].

Microglia not only express CB₂R responding to cannabinoid ligands but also produce and inactivate the endocannabinoids AEA and 2-AG by the complex cellular machinery [111,117,118]. The endocannabinoids are highly lipophilic molecules, that are naturally synthesized from glia and neuron membrane phospholipids during physiological events in the CNS. As a result, endocannabinoids are released immediately after their production to stimulate CB₁R and CB₂R, and in turn, they are inactivated by re-uptake and the ensuing hydrolysis [119,120]. Notably, under neuroinflammatory conditions in vitro, microglia are capable of producing 20 times more endocannabinoids than other glial cells and neurons. Therefore, microglia may be the primary cellular source of endocannabinoids in vivo [35,85]. Note that the expression of AEA and 2-AG is significantly upregulated when microglia are activated and switch to a protective phenotype (M2) under pathological conditions [68,103,121]. Further studies have demonstrated that the production of 2-AG and AEA is dependent on sustained rises in intracellular Ca²⁺ concentration, which may be mediated by the activation of ionotropic P₂X purinergic receptors P₂X₄ and P₂X₇ [118,122]. The molecular mechanism underlying this process involves the fact that an increase in intracellular Ca²⁺ can directly increase NAPE-PLD and DAGL activity while inhibiting MAGL activity in microglia [85,123,124]. The inverse sensitivity of DAGL and MAGL to Ca²⁺ constitutes a primitive and efficient modality for microglia to continuously accumulate 2-AG [118,125]. Additionally, microglia also express the metabolic enzyme FAAH, which is responsible for the degradation of 2-AG and AEA [123]. Several reports have detailed that under neuropathological conditions, activated spinal microglia may produce significantly more 2-AG and AEA by upregulating DAGL and NAPE-PLD, while downregulating FAAH [103,126,127]. Meanwhile, it has been suggested that the exposure of human or rat microglia to 2-AG and AEA at low concentrations increases the expression of the M2 microglial marker arginase 1 (Arg-1), together with other markers such as suppressor of cytokine signaling 3 (SOCS₃) [103]. These results support that the production and actions of endocannabinoids are closely related to the phenotype of microglia, and the endocannabinoid system also plays an important role in microglial immunomodulation by inducing an M2 phenotype. It also explains that some of the neuroprotective effects of the endocannabinoid system in diverse pathological states of inflammatory models may be mediated by their immunomodulatory actions, such as disrupting pro-inflammatory processes [128,129,130].

The increased production of endocannabinoids may also upregulate the expression of microglial CB₂R, which in turn may activate more CB₂R to dampen nociceptive signaling cascades, amplifying the anti-inflammatory responses [103]. Notably, the activation of CB₂R has been reported to transform microglia from the M1 to the M2 phenotype [131,132], suggesting that CB₂R signaling is very important for microglia to polarize towards the M2 phenotype with phagocytic capacity by morphology alterations [103]. These findings are consistent with previous studies that showed that upregulation of CB₂R in activated microglia has been associated with improvement of the disease consequences in specific neuroinflammatory conditions [126,133,134].

In summary, the overall consequence of CB₂R activation on microglia by endocannabinoids AEA and 2-AG or by exogenous cannabinoids appears to be to exert beneficial properties of microglia, such as the release of anti-inflammatory mediators, by promoting the generation of neuroprotective microglia phenotype (M2). The M2 phenotype could reduce neuronal hyperexcitation causally involved in central sensitization, with the capacity of phagocytosis and reduction of releasing detrimental factors like pro-inflammatory cytokines and free radicals [15,124,127,135,136,137,138,139,140,141]. The expression profiles of CB₂R and endocannabinoids in homeostatic and activated microglia are summarized in Figure 2.

4. The Role of Microglial CB₂R in Pathological Pain

4.1. Antinociceptive Effects of Well-Characterized CB₂R Selective Agonists

Currently, intense interest has been focused on the use of cannabinoid compounds typically acting upon the CB₁R and CB₂R for the treatment of pathological pain. These widely researched compounds include endocannabinoids like AEA and 2-AG, phytocannabinoids like Δ-9-tetrahydrocannabinol and cannabidiol, as well as a large number of synthetic cannabinoids [20,142,143,144,145,146]. However, in parallel with a deeper understanding of the endocannabinoid system’s underlying expression profiles and the physiological and pharmacological properties of cannabinoid receptors, CB₂R selective agonist compounds are increasingly recognized as safer novel therapeutic candidates, with their properties to bypass certain centrally mediated unwanted effects associated with the activation of CB₁R.

The following section summarizes the antinociceptive effects of various well-characterized CB₂R selective agonists, including HU308, JWH-015, JWH-133, GW405833, AM1241, and MDA7, in different pain states to provide direct support for the hypothesis that CB₂R can serve as a promising therapeutic target for pain relief (Table 1).

The HU308 is the first CB₂R selective synthetic compound (K_i = 22.7 ± 3.9 nM) that exhibits low affinity for CB₁R (K_i > 10 μM), which exerted anti-inflammatory and peripheral antinociceptive activities in an arachidonic acid-induced mouse inflammatory pain model and the late phase of the mouse formalin pain model. These activities were significantly inhibited by the use of selective CB₂R antagonist SR144528 [147]. In the rat postoperative pain model, surgical incision-induced tactile allodynia was significantly suppressed by HU308 [148]. The topical HU308 also has been found to reduce corneal hyperalgesia and inflammation in wild-type mice, but not in CB₂R^−/− mice, further validating that CB₂R is a drug target of this compound [149].

The JWH-015, one of the earliest discovered compounds, was found to have improved selectivity for the CB₂R (K_i = 13.8 nM at CB₂R and K_i = 383 nM at CB₁R) from the aminoalkylindole classification of CB₂R agonists, which is effective in alleviating pain-related behaviors and reducing inflammatory responses without inducing psychotropic effects even when intrathecally applied [21,52,150,151,152]. The intrathecal administration of JWH015 reduced the paw incision and caused postoperative hypersensitivity and microglial activation in the spinal cord without inducing behavioral side effects. This effect was prevented by intrathecal injection of the CB₂R selective antagonist AM630 [10,153], indicating a CB₂R-dependent mechanism of action. In spinal nerve ligation (SNL) or lumbar 5 nerve transection (L5NT) neuropathic pain models, intrathecal JWH015 treatment significantly reduced nerve injury-induced hypersensitivity, which can also be blocked by intrathecal AM630 [13,17]. Additionally, it has also been reported that intrathecal or intraperitoneal injection of JWH-015 displayed an analgesic effect to attenuate bone cancer-induced spontaneous pain and mechanical allodynia [15,92,154].

The JWH-133 is also a well-characterized CB₂R agonist, one of the most highly selective ligands for the CB₂R (K_i = 677 ± 132 nM at CB₁R and K_i = 3.4 ± 1.0 nM at CB₂R) [155,156], which inhibits both inflammatory and neuropathic hyperalgesia through a CB₂R-selective mechanism. For example, spinal (i.t.) or local (i.pl.) administration of JWH-133 reduced noxious mechanical stimulation evoked responses in wide dynamic range neurons recorded in SNL neuropathic pain, carrageenan-induced inflammatory pain and osteoarthritis pain, in a manner that was prevented by SR144528 [157,158,159]. The JWH-133, administered systemically (s.c.), can increase weight bearing and decrease peripheral edema or allodynia in the carrageenan-inflamed paw and osteoarthritis pain [160]. However, the JWH-133 has been shown to functionally interact with opioids to modulate antinociception in the formalin test without inducing tolerance, and can also attenuate cross-tolerance with morphine [161]. Furthermore, recent studies using CB₂R constitutive knockout and tissue-specific genetic deletion mice suggested that self-administration of JWH-133 not only attenuated spontaneous pain and anxiety-associated behavior in the partial sciatic nerve ligation (PSNL) induced neuropathic pain model but also void of reinforcing effects in animals without pain, indicating the absence of abuse liability [14].

The GW405833, another highly selective CB₂R ligand, is also classified as an aminoalkylindole [162]. In addition, the GW405833 was determined to be a selective human CB₂R agonist in a recombinant binding assay (K_i = 2043 ± 183 nM at CB₁R and K_i = 14 ± 6 nM at CB₂R), while its selectivity appeared to be lower for the rat CB₂R (K_i = 273 ± 42.6 nM at CB₁R and K_i = 3.6 ± 1.1 nM at CB₂R) [163,164]. In both rats and mice, pharmacological characterization of GW405833 has been previously shown to elicit efficacious antihyperalgesic and anti-inflammatory effects in several pain models, including PSNL, hind paw incision, and complete Freund’s adjuvant (CFA)-induced inflammatory pain, without eliciting the centrally CB₁R-mediated side effects [163,165]. Similarly, systemic administration of GW405833 reduced the late phase of formalin pain and allodynia elicited by SNL in a dose-dependent manner [9]. Additionally, hind paw incision, chronic constriction injury (CCI)-induced tactile allodynia, and carrageenan-evoked peripheral edema or weight bearing were relieved by GW405833 [148,166,167]. These above-mentioned effects were demonstrated to be dependent upon CB₂R activation rather than the activation of CB₁R or opioid receptors by the experiments performed in CB₂R^−/− mice or utilizing CB₂R and opioid receptor selective antagonists. Interestingly, analgesic effects of high-dose GW405833 (i.p. 100 mg/kg) were also evident in the tail flick and hot plate tests in CB₂R^−/− mice, which might be attributed to both moderate affinities for CB₁R and significant CNS penetration [163,165]. Moreover, in stark contrast to treatment with WIN55,212-22, a mixed CB₁R/CB₂R agonist, chronic repeated injection of GW405833 was able to provide sustained reversal of allodynia following SNL without tolerance development [168].

The AM1241, another agonist possessing a high affinity for the CB₂R (K_i = 3.4 ± 0.5 nM at the CB₂R and K_i = 280 ± 41 nM at CB₁R), belong to the aminoalkylindole class [169]. There is growing evidence supporting the hypothesis that AM1241 produced antinociceptive effects in preclinical inflammatory and neuropathic pain models lacking CNS side effects in a tetrad of behavioral tests that is used to assess cardinal signs of central CB₁R [169]. The systemic (i.p.) and local (i.pl.) administration of AM1241 exhibited a thermal antinociceptive effect in the acute pain model of rats, which was significantly blocked by the CB₂R selective antagonist AM630 [19]. These actions of CB₂R have been confirmed by further studies, in which acute nociception of AM1241 was lost in the tail flick and hot plate tests of CB₂R^−/− mice [22]. In the rat postoperative pain model, AM1241 also obviously suppressed tactile allodynia [148]. Moreover, systemic (i.p.) or local (i.pl.) administration of AM1241 suppressed allodynia, hyperalgesia, and peripheral edema in the carrageenan-evoked rat inflammatory pain model in a CB₂R-dependent manner because SR144528 or AM630 specifically blocked these effects [170,171,172,173]. Similarly, intravenously administered AM1241 reduced the late phase of formalin pain, which also depends upon CB₂R activation [9]. In the SNL or CCI of sciatic nerve-induced neuropathic pain models and the CFA-induced chronic inflammatory pain model, AM1241 (i.p., i.DRG., or i.t.) produced a significant reversal of established mechanical and thermal hypersensitivity in rats or CB₁R^−/− mice [6,9,169,174]. The AM1241 could also reduce pain symptoms in a CB₂R dependent manner in the vincristine-induced neuropathic pain model and bone the cancer-induced pain model [93,175,176].

The MDA7, one of the acylhydrazone derivatives, is a more promising CB₂R selective agonist (hCB₁R K_i > 10,000 nM; hCB₂R K_i = 422 nM; rCB₁R K_i = 2565 nM; rCB₂R K_i = 238 nM) for the treatment of pain [24,177]. It has been shown that systemic administration of MDA7 exhibits an attenuated SNL-induced tactile allodynia in rats in a dose-dependent way. The target specificity of MDA7 was confirmed by pretreatment with selective antagonists, while attenuation of the antiallodynic effects was mediated by AM630 but not either the CB₁R selective antagonist AM251 or the opioid antagonist naloxone [24]. This molecule has also been shown to effectively suppress mechanical allodynia rats and mice in paclitaxel (PTX)-induced neuropathic pain models. In addition, MDA7 can produce a modest thermal antinociceptive effect in naive rats without affecting locomotor activity. These effects were blocked after pretreatment with AM630 in wild type mice or were absent in CB₂R^−/− mice, which indicates that the action of MDA7 directly involves the activation of CB₂R [24,25,26].

As a result of the great potential of targeting CB₂R, new potential drugs are constantly being developed. Some of them are being tested in clinical trials. For example, Olorinab, an oral and highly selective full agonist of CB₂R, reached phase II trials for abdominal pain in Crohn’s disease and for irritable bowel syndrome [178]. However, there is still no CB₂R selective agonist on the market as a new analgesic drug. This situation resulted from many reasons. Firstly, most CB₂R ligands were highly lipophilic and, as such, not optimal for clinical application due to unfavorable physicochemical properties, which potentially contributed to modest or lack of clinical efficacy. Secondly, these compounds will be required to have high affinity and selectivity for CB₂R to avoid the adverse effects of activating CB₁R, while the target engagement of current CB₂R ligands is poor. Aside from the development and optimization of CB₂R ligands, the fact that human and rodent CB₂R sequences have relatively low homology should be considered, which may give rise to differences in ligand engagement and efficacy [179]. Furthermore, preclinical pain models in animals might not fully and accurately reflect human pathological mechanisms, which may also affect the clinical translation of CB₂R agonists. Nonetheless, substantial efforts to better optimize CB₂R ligands for clinical application are ongoing, and many existing ligands have reached the most advanced phases, such as JBT-101 [180,181]. There is no doubt that specifically activating CB₂R is considered a good strategy for developing new analgesic agents with fewer side effects.

Table 1. Efficacy of some well-characterized CB₂R selective ligands in different pain models.

Agonist	In vitro binding profile		Pain Model	Route of Administration	Species	Efficacy	Reference
	CB1R	CB2R
HU308	Ki > 10 μM Rat brain	Ki = 22.7 ± 3.9 nM Transfected cells	Formalin test	i.p., 50 mg/kg	Mouse	Antinociception	[148]
			Arachidonic acid-induced ear Inflammation	i.p., 50 mg/kg	Mouse	Reduce ear swelling	[148]
			Post-operative pain	i.p., 0.3–30 mg/kg	Rat	Antiallodynic effect	[149]
JWH-015	Ki = 383 nM	Ki = 13.8 nM	Tail flick test	i.p., 1–100 mg/kg	Mouse	Antinociception	[21]
			Tail immersion test/Paw pressure test	i.p., 5–20 mg/kg	Rat	Antinociception	[153]
			Formalin test	i.p., 0.1–100 mg/kg	Mouse	Antinociception	[153]
			Post-operative pain	i.t., 2–10 μg	Rat	Antiallodynic effect	[10,21,154]
				i.p., 1–10 mg/kg	Rat	Antinociception
			Inflammatory pain (CFA)	i.p., 5–10 mg/kg	Rat	Antinociception	[153]
			Neuropathic pain (SNI, SNL, L5NT, or bone cancer induced)	i.p., 1–10 mg/kg	Rat	Antinociception	[13,15,17,21,92,155]
				i.t., 0.4–50 μg	Rat	Antiallodynic effect
JWH-133	Ki = 677 ± 132 nM Rat brain	Ki = 3.4 ± 1.0 nM Human embryonic kidney 293 cells	Formalin test	i.p., 0.1–10 mg/kg	Mouse	Antinociception	[162]
			Inflammatory pain (Carrageenan or osteoarthritis induced)	s.c. 10 mg/kg	Rat	Increase weight bearing	[158,161]
				i.pl., 5–15 μg	Rat	Inhibits mechanically evoked neuron responses
			Neuropathic pain (SNL or PSNL induced)	i.pl., 5–15 μg	Rat	Inhibits mechanically evoked neuron responses	[14,158,159,160]
				i.t., 8–486 ng	Rat	Inhibits mechanically evoked neuron responses
				s.c. 1 mg/kg	Rat	Antiallodynic effect
				i.v., 0.15–0.3 mg/kg	Mouse	Antinociception
GW405833	Ki = 2043 ± 183 nM Cos-7 cells Ki = 273 ± 42.6 nM Rat brain	Ki = 14 ± 6 nM Cos-M6 cells Ki = 3.6 ± 1.1 nM Rat spleen	Hot plate test/Tail flick test	i.p., 100 mg/kg	Mouse	Antinociception	[166]
			Formalin test	i.v., 3–10 mg/kg	Mouse	Antinociception	[9]
			Post-operative pain	i.p., 0.3–30 mg/kg	Rat	Antiallodynic effect	[149,164]
			Inflammatory pain (Carrageenan or CFA)	i.p., 3–30 mg/kg	Mouse	Antiallodynic effect	[164,166,167]
				i.p., 0.1–100 mg/kg	Rat	Antinociception
			Neuropathic pain model (PSNL, L5NT, or CCI)	i.p., 3–30 mg/kg	Mouse	Antiallodynic effect	[9,164,166,168,169]
				i.p., 0.01–30 mg/kg	Rat	Antiallodynic effect
AM1241	Ki = 280 ± 41 nM Rat brain	Ki = 3.4 ± 0.5 nM Mouse spleen	Hargreaves acute thermal stimulation	i.p., 0.3–3 mg/kg	Mouse	Antinociception	[22]
				i.p., 0.033–0.33 mg/kg	Rat	Antinociception	[19]
			Hot plate test/Tail flick test	i.p., 0.3–10 mg/kg	Mouse	Antinociception	[22]
			Formalin test	i.p., 3–10 mg/kg	Mouse	Antinociception	[9]
				i.v., 0.3–3 mg/kg	Mouse	Antinociception
			Post-operative pain	i.p., 3–30 mg/kg	Rat	Antiallodynic effect	[149]
			Inflammatory pain (Carrageenan, capsaicin, or CFA induced)	i.p., 0.033–1 mg/kg	Rat	Antinociception	[6,171,172,173]
				i.pl., 0.033–4mg/kg	Rat	Antinociception/Reduce paw edema
				i.DRG, 100 nmol	Rat	Antinociception
				i.t., 100 nmol	Rat	Antinociception
			Neuropathic pain (SNL, bone cancer, vincristine-induced )	i.t., 0.03–0.3 μg	Mouse	Antinociception	[93,176,177] [6,9,170,175]
				i.p., 0.3–10 mg/kg	Mouse	Antiallodynic effect
				i.p., 0.1–3 mg/kg	Rat	Antinociception
				i.DRG, 100 nmol	Rat	Antinociception
				i.t., 0.01–10 μg	Rat	Antinociception
				i.v., 3–6 mg/kg	Rat	Antiallodynic effect
MDA7	hKi > 10,000 nM CHO-K1 cells rKi = 2565 nM CHO-K1 cells	hKi = 422 nM CHO-K1 cells rKi = 238 nM CHO-K1 cells	Hargreaves acute thermal stimulation	i.p., 1–10 mg/kg	Rat	Antinociception	[24]
			Neuropathic pain (SNL or PTX)	i.p., 5–15 mg/kg	Rat	Antiallodynic effect	[24,25,26]

Abbreviations: CFA: complete Freund’s adjuvant; CCI: chronic constriction injury; i.t.: intratecal; i.p.: intraperitoneal; i.pl.: intraplantar; i.v.: intravenous; i.DRG.: intra Dorsal Root Ganglion, s.c.: subcutaneous; PSNL: partial sciatic nerve ligation; SNL: spinal nerve ligation; L5NT: L5 nerve transection.

Table 1. Efficacy of some well-characterized CB₂R selective ligands in different pain models.

Agonist	In vitro binding profile		Pain Model	Route of Administration	Species	Efficacy	Reference
	CB1R	CB2R
HU308	Ki > 10 μM Rat brain	Ki = 22.7 ± 3.9 nM Transfected cells	Formalin test	i.p., 50 mg/kg	Mouse	Antinociception	[148]
			Arachidonic acid-induced ear Inflammation	i.p., 50 mg/kg	Mouse	Reduce ear swelling	[148]
			Post-operative pain	i.p., 0.3–30 mg/kg	Rat	Antiallodynic effect	[149]
JWH-015	Ki = 383 nM	Ki = 13.8 nM	Tail flick test	i.p., 1–100 mg/kg	Mouse	Antinociception	[21]
			Tail immersion test/Paw pressure test	i.p., 5–20 mg/kg	Rat	Antinociception	[153]
			Formalin test	i.p., 0.1–100 mg/kg	Mouse	Antinociception	[153]
			Post-operative pain	i.t., 2–10 μg	Rat	Antiallodynic effect	[10,21,154]
				i.p., 1–10 mg/kg	Rat	Antinociception
			Inflammatory pain (CFA)	i.p., 5–10 mg/kg	Rat	Antinociception	[153]
			Neuropathic pain (SNI, SNL, L5NT, or bone cancer induced)	i.p., 1–10 mg/kg	Rat	Antinociception	[13,15,17,21,92,155]
				i.t., 0.4–50 μg	Rat	Antiallodynic effect
JWH-133	Ki = 677 ± 132 nM Rat brain	Ki = 3.4 ± 1.0 nM Human embryonic kidney 293 cells	Formalin test	i.p., 0.1–10 mg/kg	Mouse	Antinociception	[162]
			Inflammatory pain (Carrageenan or osteoarthritis induced)	s.c. 10 mg/kg	Rat	Increase weight bearing	[158,161]
				i.pl., 5–15 μg	Rat	Inhibits mechanically evoked neuron responses
			Neuropathic pain (SNL or PSNL induced)	i.pl., 5–15 μg	Rat	Inhibits mechanically evoked neuron responses	[14,158,159,160]
				i.t., 8–486 ng	Rat	Inhibits mechanically evoked neuron responses
				s.c. 1 mg/kg	Rat	Antiallodynic effect
				i.v., 0.15–0.3 mg/kg	Mouse	Antinociception
GW405833	Ki = 2043 ± 183 nM Cos-7 cells Ki = 273 ± 42.6 nM Rat brain	Ki = 14 ± 6 nM Cos-M6 cells Ki = 3.6 ± 1.1 nM Rat spleen	Hot plate test/Tail flick test	i.p., 100 mg/kg	Mouse	Antinociception	[166]
			Formalin test	i.v., 3–10 mg/kg	Mouse	Antinociception	[9]
			Post-operative pain	i.p., 0.3–30 mg/kg	Rat	Antiallodynic effect	[149,164]
			Inflammatory pain (Carrageenan or CFA)	i.p., 3–30 mg/kg	Mouse	Antiallodynic effect	[164,166,167]
				i.p., 0.1–100 mg/kg	Rat	Antinociception
			Neuropathic pain model (PSNL, L5NT, or CCI)	i.p., 3–30 mg/kg	Mouse	Antiallodynic effect	[9,164,166,168,169]
				i.p., 0.01–30 mg/kg	Rat	Antiallodynic effect
AM1241	Ki = 280 ± 41 nM Rat brain	Ki = 3.4 ± 0.5 nM Mouse spleen	Hargreaves acute thermal stimulation	i.p., 0.3–3 mg/kg	Mouse	Antinociception	[22]
				i.p., 0.033–0.33 mg/kg	Rat	Antinociception	[19]
			Hot plate test/Tail flick test	i.p., 0.3–10 mg/kg	Mouse	Antinociception	[22]
			Formalin test	i.p., 3–10 mg/kg	Mouse	Antinociception	[9]
				i.v., 0.3–3 mg/kg	Mouse	Antinociception
			Post-operative pain	i.p., 3–30 mg/kg	Rat	Antiallodynic effect	[149]
			Inflammatory pain (Carrageenan, capsaicin, or CFA induced)	i.p., 0.033–1 mg/kg	Rat	Antinociception	[6,171,172,173]
				i.pl., 0.033–4mg/kg	Rat	Antinociception/Reduce paw edema
				i.DRG, 100 nmol	Rat	Antinociception
				i.t., 100 nmol	Rat	Antinociception
			Neuropathic pain (SNL, bone cancer, vincristine-induced )	i.t., 0.03–0.3 μg	Mouse	Antinociception	[93,176,177] [6,9,170,175]
				i.p., 0.3–10 mg/kg	Mouse	Antiallodynic effect
				i.p., 0.1–3 mg/kg	Rat	Antinociception
				i.DRG, 100 nmol	Rat	Antinociception
				i.t., 0.01–10 μg	Rat	Antinociception
				i.v., 3–6 mg/kg	Rat	Antiallodynic effect
MDA7	hKi > 10,000 nM CHO-K1 cells rKi = 2565 nM CHO-K1 cells	hKi = 422 nM CHO-K1 cells rKi = 238 nM CHO-K1 cells	Hargreaves acute thermal stimulation	i.p., 1–10 mg/kg	Rat	Antinociception	[24]
			Neuropathic pain (SNL or PTX)	i.p., 5–15 mg/kg	Rat	Antiallodynic effect	[24,25,26]

Abbreviations: CFA: complete Freund’s adjuvant; CCI: chronic constriction injury; i.t.: intratecal; i.p.: intraperitoneal; i.pl.: intraplantar; i.v.: intravenous; i.DRG.: intra Dorsal Root Ganglion, s.c.: subcutaneous; PSNL: partial sciatic nerve ligation; SNL: spinal nerve ligation; L5NT: L5 nerve transection.

4.2. Molecular Mechanisms Involved in the Action of Microglial CB₂R in Pain Processing

The dorsal horn of the spinal cord is the vital site for controlling pain intensity because it is where efficient transmission of nociceptive information occurs between the central terminals of primary afferents and second-order interneurons. Additionally, the crosstalk between spinal neurons, astrocytes, oligodendrocytes, and microglia is indispensable to mediating central pain sensitization of neuronal circuits. A considerable amount of evidence has implicated the crucial role of selective agonists of CB₂R in treating pathologic pain symptoms through the modulation of microglia in a CB₂R dependent manner [17,25,26,29,177,182,183,184,185]. Therefore, microglial CB₂R can serve as a promising therapeutic target for pain relief because of the important role of spinal microglia in regulating central sensitization. Later in the remainder of this review, we will focus on the molecular mechanism of targeting spinal cord CB₂R to inhibit neuroinflammatory signaling pathways for pain relief with a microglial-centric view.

As described above, reactive microglia express CB₂R [17,26,184]. The protein and mRNA levels of spinal cord CB₂R were both significantly upregulated in chronic pain conditions, including CFA-induced inflammatory pain [106], SNI, SNL, CCI, and chemotherapy-induced neuropathic pain [29,30]. The above studies indicate that CB₂R over-expression in activated microglia at the dorsal horn of the spinal cord under pathological pain conditions may occur as a result of specific neuroinflammatory responses. After that, modulation targeting CB₂R may result in a neuroprotective effect [186]. Accordingly, the preponderance of evidence has implicated that activation of the CB₂R system via spinal administration of CB₂R agonists produces significant control over inflammatory and neuropathic pain in multiple models. For example, intrathecal administration of AM1241 attenuates allodynia or thermal hyperalgesia induced by CFA, SNL [6], CCI, or bone cancer models [93]. The therapeutic utility of other CB₂R selective agonists that exhibit analgesic effects to treat different pains has been thoroughly described in Section 4.1.

Below, we will summarize our current understanding of the cellular and molecular mechanisms involved in the action of microglial CB₂R in pain processing. However, several studies have demonstrated that spinal CB₂R activation limits microglia activity to the ipsilateral dorsal horn, because constitutive knockout of CB₂R results in a spread of microgliosis to the contralateral dorsal horn in an arthritis model or after sciatic nerve injury [187,188,189]. The possible mechanisms involved in this process are discussed in detail. Firstly, the activation of CB₂R can inhibit the activities of adenylyl cyclase [190] and MAPK [174,191]. The CB₂R selective agonists exerted anti-allodynic effects in rats by reducing MAPK (p38 and ERK_1/2) phosphorylation and inducing MAPK phosphatases (MKP-1 and MKP-3, the major regulators of MAPKs) expression in the spinal dorsal horn [13]. The downregulation of the p38 MAPK pathway can lead to a reduction of the cytokines IL-1β, TNF-α, and brain-derived neurotrophic factor (BDNF) [174,192,193], and the suppression of ERK_1/2 can decrease microglia proliferation [26,194,195]. Furthermore, the AMPK pathway is upregulated after CB₂R activation, which can downregulate the synthesis of nitric oxide (NO) [196]. Actually, the activation of CB₂R in microglial cells has been found to lead to spinal decreased iNOS, IL-6, BDNF, CCR2, and TNFα receptor expression during neuropathic pain [25,197], and increased release of anti-inflammatory cytokines, such as IL-10 [174,198,199]. Specifically, the activation of spinal CB₂R by exercise-induced AEA release also reduces the production of IL-1β and TNFα in mice within a carrageenan-induced pain model [136]. This may also be closely related to the reduction of endocannabinoid degradative enzymes at the spinal cord level. It has been shown that intrathecal AM1241 not only modulates critical glial factors but also reduces the expression levels of MAGL, while not altering FAAH [174]. In addition, CB₂R agonist treatment can reduce microglial purinergic receptor P₂X₄ upregulation [25], which may be another mechanism by which CB₂R activation reduces microglial contributions to pain. The P₂X₄ was identified as a microglia-specific molecule that was activated and upregulated after peripheral nerve injury and also plays critical roles in processing nociceptive information and contributing to microglial-dependent central pain sensitization [200].

In addition to modulating microglial immune function by reducing the production of pro-inflammatory cytokines and increasing the release of anti-inflammatory cytokines involved in neuroinflammatory signaling pathways, activation of CB₂R also can switch microglia into a more anti-inflammatory state by limiting migration and promoting phagocytic function. For example, it has been found that 2-AG can induce the recruitment of microglia partly by stimulating CB₂R in BV-2 cells [85]. However, another study showed that CB₂R activation in microglia stimulates MKP phosphatases, which can inhibit the ERK pathway, thus decreasing microglial chemotaxis/migration mediated by ADP [201]. Moreover, it has been found that the activation of CB₂R is capable of inducing the removal of native beta-amyloid both in situ and in vitro by promoting the phagocytic function of macrophages [138]. On the contrary, some results also show that the activation of CB₂R can inhibit the phagocytosis of microglia by activating ERK_1/2/AKT-Nurr1 signal pathways [202]. These different results of microglial migration or phagocytosis mediated by CB₂R activation may be due to the different stages of inflammation development. However, as described in Section 3 of this review, the CB₂R activation can promote the shift of M1 to M2 microglia. All of this may enhance the beneficial properties of microglia and be associate with the restoration of microglial activity.

Overall, accumulating evidence reveals that activation of CB₂R vitally regulates microglial immune function by blocking the normal inflammatory response with increased production of anti-inflammatory mediators and decreased production of proinflammatory mediators causally involved in central sensitization [29,31,127,135,203,204].

5. Conclusions

In this review article, we summarize the analgesic effects mediated by CB₂R and the mechanisms involved in pain regulation. Firstly, it is well known that the endocannabinoid system exerts an important role in neuronal regulation. Within the CNS, CB₂R mainly expresses in homeostatic microglia, while there is a unique feature that their expression is rapidly upregulated in activated microglia under certain pathological conditions. The CB₂R might serve as an intriguing target for the development of drugs for the management of pain because of its ability to mediate analgesia with few psychoactive effects. Indeed, accumulating data have demonstrated that the CB₂R agonists exert analgesic effects in various preclinical pain models, such as inflammatory and neuropathic pain. Additionally, spinal microglia can modulate the activity of spinal cord neurons and have a critical role in the development and maintenance of chronic pain. The activation of CB₂R can reduce pain signaling by regulating the activity of spinal microglia and inhibiting neuroinflammation. Specifically, the CB₂R activation has been reported to transform microglia from the pro-inflammatory M1 to the neuroprotective M2 phenotype by promoting the beneficial properties of microglia, such as the releasing of anti-inflammatory mediators, or the induction of phagocytosis, and reducing their ability to release pro-inflammatory cytokines involved in central sensitization. Overall, we provided an improved understanding of the underlying mechanisms involved in the action of microglial CB₂R in pain processing. However, further studies are needed to dissect the specific role of CB₂R expressed in different phenotype microglia to provide a better alternative to controlling pain by regulating CB₂R.