Under normal conditions, acute pain processing consists of well-characterized neuronal signaling events. When dysfunctional pain signaling occurs, pathological pain ensues. Glial activation and their released factors participate in the mediation of pathological pain. The use of cannabinoid compounds for pain relief is currently an area of great interest for both basic scientists and physicians. These compounds, bind mainly either the cannabinoid receptor subtype 1 (CB₁R) or cannabinoid receptor subtype 2 (CB₂R) and are able to modulate pain. Although cannabinoids were initially only thought to modulate pain via neuronal mechanisms within the central nervous system, strong evidence now supports that CB₂R cannabinoid compounds are capable of modulating glia, (e.g. astrocytes and microglia) for pain relief. However, the mechanisms underlying cannabinoid receptor-mediated pain relief remain largely unknown. An emerging body of evidence supports that CB₂R agonist compounds may prove to be powerful novel therapeutic candidates for the treatment of chronic pain.

2.1. Acute Pain Signaling

Acute pain processing is distinct from the etiology underlying chronic pathological pain. Distinguishing the cellular responses and underlying signaling cascades that are unique to pathological pain may prove critical in understanding why many neuronally targeted treatments do not prove to be effective in relieving chronic pathological pain in the clinical setting. In acute pain, such as that caused by high intensity stimuli from mechanical stimulation (e.g., pinprick), unmyelinated C and lightly myelinated Aδ nociceptive nerve fiber terminals in the body depolarize and transduce this information into action potentials that travel through the peripheral axon to the dorsal root ganglia (DRG). The centrally projecting terminals of these nociceptors predominantly enter the spinal cord dorsal horn to reach the superficial (laminae I–II) and deeper lamina IV–V and synapse onto second order pain projection neurons located in lamina I, IV, and V [9–11]. The classical neurotransmitter primarily responsible for synaptic communication between nociceptors and pain projection neurons is the excitatory amino acid glutamate. Glutamate then binds and activates the ionotropic α-amino-3hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors as well as metabotropic glutamate receptors (mGluR 1, 3, 5, and 7) [12]. Additionally, a number of nociceptive-related neuropeptides acting in the spinal cord dorsal horn have been identified to play key roles in pain neurotransmission. For example, the classic neuropeptide, substance P, is released from primary nociceptive afferents [13]. Substance P then binds and activates its receptor, neurokinin 1 (NK1), which is present in high concentrations on dorsal horn lamina I neurons. Both substance P and its NK1 receptor are widely known to play a significant role in nociceptive processing [14]. These spinal cord nociceptive neurotransmitters, along with their receptors, are critical for activating second-order neurons, which communicate to supraspinal pain-processing centers and elicit reflexive and protective responses to avoid potential or further tissue damage.

2.2. Central Sensitization

However, under some circumstances, incoming nociceptive signaling is prolonged leading to clinical manifestations of pathological neuronal signaling. Examples of such pathological states are hyperalgesia, which is decreased threshold to nociceptive stimuli, and dynamic tactile allodynia, which is increased sensitivity to nonnociceptive light touch. Both pain states often occur in regions beyond the tissue-injured site. The underlying neurobiological events initiated by prolonged nociceptive signaling include increased synaptic function triggered within the central nervous system. Specifically, these events are known to occur within the dorsal horn of the spinal cord and culminate in a process termed spinal sensitization of pain projection neurons [15, 16]. Once triggered, this central sensitization is sustained despite the termination of noxious input. Experimentally, continued activity is substantially extended following the end of the stimulus application [17, 18]. These seminal early studies suggested that pain may be experienced even in the absence of peripheral noxious stimuli.

Pathological pain results from inflammation and/or trauma to peripheral nerve(s), tissue(s), or the central nervous system (CNS) and may arise as a complication to numerous medical conditions. Various animal models have been developed to induce conditions similar to those observed clinically. Neuropathic pain is commonly studied in models of peripheral nerve injury/inflammation. Models of diabetic neuropathy, chemotherapy-induced pain, post-surgical pain, and osteoarthritis pain are well-established examples, and reports of these are cited throughout this paper. Although distinct in disease etiology, peripheral neuropathies share in the manifestation of pathological pain. This pathological processing is initially triggered by incoming noxious signals from nociceptors leading to central sensitization. One classically known mechanism for spinal sensitization involves excitation of pain projection neurons in the superficial laminae of the spinal cord dorsal horn as well as wide dynamic range neurons (WDR) located in deeper lamina IV and V that process the rapid and intense nerve depolarizations. Following prolonged and significant depolarization by the actions of glutamate and substance P, spinal pain projection neurons become sensitized, leading to the activation of N-Methyl-D-Aspartic Acid (NMDA) receptors that are normally inactive due to a Mg²⁺ plug within the cation channel. Prolonged depolarization induces Mg²⁺ release followed by enhanced influx of Ca²⁺ [19, 20]. A cascade of intracellular events occurs, which ultimately leads to postsynaptic enhancement of AMPA and mGlu receptor action, thereby increasing synaptic efficacy [21].

2.3. Sensory Changes in Pathological Pain

Increasing synaptic efficacy exerts profound changes in dorsal horn sensory processing [16, 22]. Indeed, enhanced synaptic efficacy, initiated by low intensity mechanosensitive Aβ fibers, occurs at synapses on pain projection neurons in the dorsal horn [23], creating the perceptual equivalent of a noxious stimulus. Activated low-intensity Aβ fibers, that carry nonpainful information such as light touch, are now capable of activating high intensity nociceptive neurons resulting in the clinical phenomenon known as allodynia. That is, nonpainful light touch is coded as painful, leading to a pain sensation that occurs in the absence of noxious input. Despite the fact that the stimulus is initiated in the periphery, its manifestation is a consequence of central changes like sensitization in the spinal cord [15]. Both allodynia and hyperalgesia are a hallmark of pathological pain [15, 16].

3.1. Glial Activation

While it is clear that neuronal processes are critical for spinal sensitization leading to pathological pain signaling, nonneuronal glial mechanisms are also important [24]. Under persistent pathological conditions, the availability of neuropeptides, such as substance P and amino acid neurotransmitters like glutamate is increased and able to bind their receptors not only on neurons, but also on astrocytes as well as parenchymal and perivascular microglia. Glial “activation” ensues and sets in motion a cascade of excitatory signaling events [25].

At the onset and during pathological pain conditions, multiple signaling cascades within glia are triggered including the activation of p38 mitogen-activated protein kinase (p-p38MAPK) and the c-Jun N-terminal kinase (JNK) pathways via phosphorylation events. Consequently, downstream cascades are initiated, including NF-κβ activation, a cytokine nuclear transcription factor, and lead to the subsequent production of proinflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), as well as chemokines (chemoattractant cytokines) [25–30]. It is important to note that multiple signaling pathways can activate NF-κβ leading to altered gene expression. For example, glially released TNF-α, when bound to its receptor, leads to phosphorylation of p38MAPK (p-p38MAPK) and NF-κβ activation. Alternatively, IL-1β, when bound to its receptor, can directly activate NF-κβ [31, 32]. In the spinal cord, IL-1β and TNF-α can further directly excite neurons because neurons express receptors for these cytokines. Indirect neuronal stimulation occurs by cytokine-induced release of additional excitatory mediators such as prostaglandins and nitric oxide (NO). It has been reported that spinal p38MAPK, JNK, and the extracellular signal-regulated kinase, ERK1/2, also referred to as MAPK3/1, are critical mediators of pathological pain in animal models [33–37]. For example, sciatic nerve ligation (SNL), a well-characterized rodent model of peripheral nerve injury, leads to increased p38MAPK in spinal astrocytes and microglia, and upon spinal pharmacological blockade with the p-p38MAPK inhibitor SB203580, p38MAPK activation with associated neuropathic pain is diminished [34]. Although very little is known about oligodendrocyte signaling in chronic pain, emerging evidence suggests that these cells are not merely passive observers to chronic pain, but rather these cells may also upregulate (phosphorylated) p-AKT, a factor that has been found to mediate apoptosis, cell migration, and motility in the dorsal horn of the spinal cord. Phosphorylated-AKT may be critical to the previously discussed spinal cord neuronal sensitization process [38].

It is notable that activated microglia respond to and produce inducible nitric oxide synthase (iNOS), and likewise activated astrocytes release NO [39, 40]. The production of NO by both neurons and glia is characteristic of neuroinflammation [41–44]. Thus, upon spinal glial activation from NO (among several other activating factors), intracellular signaling cascades lead to increases in cytokines and diffusible factors that further activate neighboring neurons and glia. That is, IL-1β and TNF-α lead to a feed-forward loop of further JNK and MAPK signaling, NF-κβ activation, and increased NO, cytokine, and chemokine production, which all contribute to ongoing pathological pain.

While microglia and immune-like astrocytes respond to spinal IL-1β and TNF-α resulting in pathological pain, increased peripheral immune cell (neutrophils, lymphocytes, monocytes, macrophages) migration to critical regions of nociceptive processing, such as the DRG and the spinal cord dorsal horn also occurs in response to cytokines [39, 45–47]. The specific underlying mechanisms are poorly understood. What is known, however, is that cellular enrichment at these critically important anatomical sites takes place via increased immune cell actin remodeling and proliferation in response to chemotactic signaling [46, 48, 49].

3.2. Glial Morphology and Activation Markers

Activated glial cells typically undergo changes in morphology, proliferation, and migration, termed gliosis. For example, astrocytes upregulate vimentin and glial fibrillary acidic protein (GFAP) and become highly arborized with thickened processes [50]. These changes in morphology and increased GFAP expression are often considered a sign of spinal cord pathogenesis during the expression of neuropathic pain in animal models and are thought to be indicative of CNS inflammatory processes [26,50, 51]. A report examining cellular enrichment of the spinal cord in a peripheral nerve injury ratmodel of pain identified that microglial cells are more proliferative and undergo more clustering than astrocytes [52]. Microglia, when activated, typically upregulate the cellular makers, ionized calcium binding adaptor molecule-1 (Iba-1), and CD11b/c, also known as OX42 [52–57]. However, the upregulation of these proteins is not always indicative of proinflammatory phenotypic processes of glial cells. For example, activated microglia can additionally express ED2, a classic anti-inflammatory marker, suggesting that activated microglia are not solely engaged in proinflammatory processes [58,59].

Although these cellular changes have been widely documented in animal models of chronic pain, less is known about whether glial activation always reflects inflammation and whether it contributes to chronic pain in humans. What is known is that gliosis occurs within the spinal cord of patients with neuroimmune diseases such as ALS, MS, and spodylotic myopathy [60, 61]. It is noteworthy that these patients often report chronic pain symptoms [4, 6]. Furthermore, postmortem tissue analysis from these patients often reveals gliosis concomitant with the disease, and as such, these glial changes may contribute to chronic pain in these patients. However, the role of these cellular markers in animal models of chronic pain are not fully understood, as reports show a disconnection between glial marker upregulation, proinflammatory signaling markers, and behavior associated with pain. For example, while fluorocitrate attenuated upregulation of GFAP in mice with the chronic constriction injury of the sciatic nerve (CCI), another commonly used model of chronic neuropathic pain, chronic pain symptoms remained unchanged [55]. Additionally, in separate studies utilizing a paw incision model of postsurgical pain, chronic morphine administered subcutaneously delayed the normal resolution of allodynia and hyperalgesia, which was observed with saline-injected controls. Tissues from the corresponding groups in this study were analyzed for GFAP, Iba-1, p-ERK, and p-p38MAPK, with saline-injected animals showing clear behavioral resolution, which was absent in the morphine-treated groups. Strikingly, no differences in GFAP or Iba-1 immunoreactivity were observed between saline-or chronic morphine-treated groups. However, p-ERK and p-38MAPK were increased in the chronic morphine-treated groups, corresponding to their behavioral profile [62]. Conversely, perivascular microglia have been shown to remain in an activated state as assessed by immunohistochemical detection of ED2 during the presence of pain reversal [63]. From these studies, and as noted previously, the presence or absence of glial activation, per se, is too simplistic to fully understand a glial role in chronic pain. It is possible that microglia can remain activated while producing and releasing anti-inflammatory factors that ultimately lead to pain suppression [64, 65].

3.3. Downstream Glial Signaling of Cytokines

Both IL-1β and TNF-α induce chemotactic activity on CNS microglia and astrocytes. Indeed, once activated, microglia and astrocytes are well known to undergo migration and proliferation in the spinal cord under conditions of chronic pain [52]. Recently, it has been shown that an increase in glial cell numbers occurs within the ipsilateral dorsal horn of the spinal cord following unilateral peripheral nerve injury [66].

During pathological pain states, peripheral immune cells additionally migrate to critical CNS pain processing sites. However, the contribution of peripheral versus CNS immune cell actions with subsequent cytokine signaling to neuropathic pain is not fully understood. Rat spinal cord meninges contain peripheral immunocompetent cells such as macrophages, and following in vitro stimulation of isolated meninges with the administration of the HIV-1 envelope glycoprotein gp120, IL-1β, and TNF-α were released [67]. These data suggest that meningeal cells, characterized to include peripheral immune cells like macrophages, contribute to ongoing spinal cord glial activation via proinflammatory cytokine actions. Given these compelling data, we explored the possibility that anatomically intact meninges contain macrophages that express IL-1β. Here, we utilized immunofluorescent histochemical procedures followed by detection with confocal microscopy and demonstrated that IL-1β is indeed present within the meningeal layers surrounding the spinal cord of neuropathic rats (Figures 1(a), 1(b), and 1(c)). Histologically, these data confirm prior reports showing, via in situ hybridization, that IL-1β mRNA was colabeled with Iba-1 [68], indicating infiltrating monocytes/macrophages. Within deeper dorsal horn laminae, IL-1β is colabeled with Iba-1 that identifies microglia (Figures 1(d), 1(e), and 1(f)). While we found some colabeling of IL-1β with GFAP, no co-labeling with NF-H (data not shown) within the dorsal horn of the spinal cord was observed. Given the evidence that immune cell and glial-derived IL-1β (as well as other cytokines discussed, above) has a critical role in animal models of pathological pain, targeting neurons alone is now thought to be an incomplete approach. Immune and glial cells within the CNS may serve as novel targets to modulate enduring pathological pain.

Figure 1

Qualitative confocal images of cellular immunostaining. (a) Immunostaining of Iba-1 (red) for infiltrating macrophages and microglia in the meninges and superficial white matter of the spinal cord in a rat with ongoing neuropathy. (b) Immunostaining with …

3.4. Glia in DRG

Glial satellite cells in the DRG are also important in mediating pathological pain in addition to spinal cord glial cytokine actions. Satellite glia completely surround DRG neurons and together form a functional unit [69]. Glial satellite cells become activated and contribute to pathological pain in response to peripheral injury by several possible mechanisms [69–73]. For example, glial satellite cells generate cytokines, including IL-1β and TNF-α, which have been characterized to activate peripheral immune cells [71, 73–75]. DRG invasion by peripheral immune cells [76–79] occurs as a consequence of peripheral nerve injury [29, 80, 81]. Neuroimmune activity is a potential mechanism because DRG neurons have receptors for these cytokines and, when stimulated, lead to the production of the chemokine monocyte chemoattractant protein-1 (MCP-1), which induces peripheral immune cell migration to the DRG [49, 81]. In addition, neuroactive IL-1β and other immune signals released from satellite glia act in a paracrine fashion to stimulate neighboring sensory ganglia and their axons, creating allodynia [69, 73, 82–84]. Indeed, stimulating sensory neurons in the DRG with IL-1β leads to further axonal release of substance P [85] within the dorsal horn of the spinal cord. IL-1β acts in a p-p38MAPK-dependent manner in the DRG [82], and increased p-p38MAPK expression is well characterized in the DRG following peripheral nerve injury that produces pathological pain [82, 86, 87]. Here we show an example of DRG IL-1β in close proximity with sensory neurons. IL-1β is colabeled with GFAP-positive satellite cells within a DRG from an animal with ongoing CCI-induced neuropathy which is shown (Figures 1(g), 1(h), and 1(i)). The actions of glially released cytokines such as IL-1β on nearby neuronal processing in both spinal cord and DRG indicate that not only neuronal, but also glial systems are altered during conditions that lead to and promote chronic pain. These data strongly suggests that in order to efficiently control chronic or pathological pain associated with numerous disease states, including diabetic neuropathy and cancer, promising therapeutics will need to address this underlying glial contribution.

3.5. Modulating Glial Activation for Pain Relief

Several compounds specifically targeting glial activation have been developed with the potential for the treatment of pain. While a full discussion of such compounds is beyond the scope of this paper (for review, see [88]), one example drug is discussed here to underscore the supposition that altering glial activation states is a highly promising approach to control pathological pain. An example of a compound that targets microglial activation is minocycline, a well-characterized microglial inhibitor [89]. In numerous animal models, minocycline robustly produces antiallodynia and hyperalgesia [55,89–91]. However, globally disrupting the function of microglia as well as peripheral immune cells may produce unintended side effects, such as increased susceptibility to CNS infection [92]. An alternative approach using cannabinoid-related compounds appears to be very promising for clinical pain relief. Cannabinoids may act in an anti-inflammatory manner, and these anti-inflammatory actions may have a glial role [7, 93]. Intriguingly, the cannabinoid receptor subtype 2, CB₂R, has been identified primarily on microglia [94]. Published reports strongly suggest that activation of this receptor subtype leads to pain control [95, 96]. In the remainder of this paper we will provide a brief overview of cannabinoids, specifically discussing published data in support of cannabinoid-related compounds for pain control with a glial-centric view.

4.1. Components of the Endocannabinoid System

The endogenous cannabinoid (endocannabinoid) system is comprised of multiple components, including receptors, ligands, and degradative enzymes. Each will be discussed in turn, below. Within the past 6 years, an explosion of reports has occurred on the endocannabinoid system and its potential role in modulating numerous disease processes, including those associated with pathological pain conditions. This is due, in part, following the identification of cells that express cannabinoid receptors and subsequent signaling mechanisms. In general, endocannabinoid signaling was thought to involve only neurons [97–99]. Glia in the CNS had no role. However, immune cells, including microglia are now known to be involved in endocannabinoid signaling cascades (discussed further, below). While the underlying mechanisms involved in mediating the therapeutic effects of the endocannabinoid system are still a mystery, new breakthroughs have elucidated the bioavailability of endocannabinoids and cannabinoid receptor action with regard to the mediation of pain processing.

The two widely acknowledged cannabinoid receptors are the CB₁R and the CB₂R. Both have shown great potential for the development of therapeutics targeted at pain control. The putative cannabinoid receptor subtype of the “orphan” receptor, GPR55 [100], remains controversial as several reports indicate opposite pharmacological profiles [101–104]. Research targeting this receptor with cannabinoid ligands has just begun to gain momentum [105–107]. However, there are reports that at least five distinct cannabinoid receptors have been identified [8]. The most well-characterized cannabinoid receptor, the CB₁R, is primarily found on neurons within the heart, small intestine, urinary bladder and vas deferens in the periphery and, within the CNS, has the highest concentrations in the cerebellum, hippocampus, basal ganglia and cerebral cortex [108–110]. However, the CB₂R has a distinctly different distribution and is primarily found on immune cells [111–113]. Current evidence demonstrates that the endocannabinoid system may have potential as a target for pain control, and thus the remainder of this paper will focus on the endocannabinoid system relative to pain therapeutics.

4.2. Classical Cannabinoid Receptor Signaling

Both the CB₁R and CB₂R belong to the G-protein coupled receptor (GPCR) superfamily and couple to the inhibitory G_i/o and G_i, respectively. Activation of either receptor leads to p42/44 MAPK signaling and inhibits adenylate cyclase, limiting the ATP production of cyclic AMP (cAMP) and leading to lessened activity of protein kinase A (PKA) [108, 114, 115]. CB₁R activation, but not CB₂R activation, can modulate ionic Ca²⁺ and K⁺ channels, which is blocked with pertussis toxin, indicating that the CB₁RG_i/o proteins are directly responsible for modulation of these ion channels [108, 116–118]. Evidence exists that CB₁R activation can activate p38MAPK in vitro [119, 120]. However, this is a paradoxical finding, because activation of p38MAPK can lead to increased pain signaling, which opposes the therapeutic efficacy of CB₁R agonists for pain control. A mechanism for these findings has not been elucidated, but may include or be wholly dependent on noncannabinoid receptor signaling cascades. No similar in vivo report exists detailing p38MAPK activation from CB₁R activation.

Although a few of the above mentioned signaling properties of the CB₁R have proven to be sufficient in leading to pain control, the practical implications of CB₁R agonists in a clinical setting are limited. The CB₁R was first discovered as the receptor for the major psychoactive ingredient in Cannabis sativa, Δ⁹-tetrahydrocannabinol (THC) which was first isolated in 1965 [121–123]. The attractiveness for clinical application of compounds selectively acting on the CB₁R is limited by the development of tolerance [124] and its psychotropic effects [7, 125], which include cognitive impairment [126], catalepsy [127–129], hypothermia [127–129], and negative impacts on learning and memory [130, 131]. This is in contrast to the effects of cannabidiol, another active compound of marijuana [132]. Cannabidiol does not produce unwanted CNS side effects by itself, but it is not widely thought to act robustly at either the CB₁R or the CB₂R due to low binding affinities observed in vitro [132]. Despite low CB₁R and CB₂R binding properties that cannabidiol possesses, it remains as a promising therapeutic for chronic pain treatment based on its anti-inflammatory actions.

In vivo, cannabidiol within the CNS may still produce CB₂R activation resulting in anti-inflammatory properties. It was recently demonstrated in a mouse model of diabetic neuropathy that intranasal administration of cannabidiol produces anti-inflammatory actions via downregulation of p-p38MAPK in spinal glia [133]. In this animal model, spinal glia are characterized to become activated and contribute to neuropathic conditions resulting in mechanical sensitivity and thermal hyperalgesia through the activation of proinflammatory signaling cascades like p38MAPK [134–136]. Cannabidiol is sufficient to produce neuropathic pain relief that is dependent on CB₂R activation [133]. These data demonstrate that there is a critical link between cannabidiol’s therapeutic action, which includes a CB₂R role, to spinal glial activation and pain control.

4.3. Cannabinoid 2 Receptors

The CB₂R is predominantly found on immune cells, such as lymphocytes, neutrophils, and macrophages, with the highest peripheral concentrations in the spleen, lymph nodes, and testes [94,111–113].Within the CNS, the CB₂R is found primarily on microglia, and some neurons specifically within the hippocampus, cortex, and substantia nigra [110, 137, 138]. However, the spinal cord and DRG distribution of the CB₂R is an area of much debate, as the current literature frequently reports conflicting data. One report suggests that the CB₂R is expressed only on neurons [139], while a separate report reveals CB₂R expression on microglia, and to a lesser extent on neurons [96], and yet another report demonstrates CB₂R on microglia with no neuronal expression [138]. The discrepancy between studies identifying the CB₂R expression on specific cell types may be partially due to the recent discovery of two separate isoforms of the CB₂R. The most prevalent isoform of the CB₂R within the periphery, termed “CB2B”, is found extensively within the spleen, and to a lesser degree, in the liver, intestines, and leukocytes. The shorter isoform, “CB2A”, is predominately found within the brain, testes, and to a lesser degree in the spleen, kidney, muscle, and leukocytes [140]. As most commercially available antibodies for the CB₂R utilize CB₂R isolated from spleen, it is possible that the CB2B isoform, found in much greater abundance within the spleen than the CB2A isoform, is the isoform recognized by most commercially available antibodies for immunohistochemistry. For example, Wotherspoon and colleagues, utilizing immunohistochemistry and CB₂R null mice, showed that CB₂R expression was induced by nerve ligation and was localized to the spinal cord superficial lamina ipsilateral to the nerve damage, while null CB₂R mice revealed no upregulation [139]. The authors suggest that CB₂R was expressed on sensory afferent terminals because colocalization with growth-associated protein-43 and the neuropeptide galanin, was observed. However, Romero-Sandoval and colleagues demonstrated, also through immunohistochemistry, that the CB₂R was primarily found on parenchymal and perivascular microglial cells. The authors additionally noted very limited and sparse staining in neurons [63, 96]. Lastly, in a study using in situ hybridization (ISH), CB₂R mRNA was present on immunohistochemically identified microglia [138].

Based on the above-noted discrepancies of the cellular localization of the CB₂R in the spinal cord, one possible consideration should be the animal model that is utilized. It has been demonstrated that the CB₂R may not be upregulated in the dorsal horn of the spinal cord in inflammatory pain models, but rather in chronic neuropathic pain models [138]. This suggests that the degree of CB₂R upregulation in chronic pain is heavily dependent on the model. The type of the injury induced in a specific model may dictate the overall receptor upregulation and the cellular colocalization of the CB₂R. The CB₂R isoform distribution within the spinal cord and DRG under basal and chronic inflammatory pain conditions has not been systematically examined. Given these potential confounds, identifying the cellular distribution of the CB₂R within the spinal cord remains elusive.

4.4. Bioavailability of Endocannabinoids

The endogenous cannabinoid system is also comprised of a number of endogenous ligands for the CB₁R and CB₂R, which includes anandamide (AEA), 2-arachadonyl glycerol (2-AG), as well as degradative enzymes [141, 142]. The endocannabinoids AEA and 2-AG are produced and released from neurons and microglia [94], which are controlled by enzymatic hydrolysis of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively [141, 143]. The enzyme MAGL has been identified on presynaptic axon terminals in brain, suggesting that it can terminate 2-AG activity in presynaptic nerve terminals [142, 144] of centrally projecting afferent nociceptors in the spinal cord dorsal horn. Interestingly, it has been found that microglia release 2-AG, and functional MAGL has been described in primary microglial cell cultures [145]. Recently, a novel isoform of MAGL has been described in BV-2 microglial cell cultures, although it is uncertain if this isoform occurs within microglial cells in vivo [146]. To date, MAGL cellular coexpression utilizing immunohistological techniques has not been performed on pain-relevant spinal cord dorsal horn regions or spinal cord tissue. We show here, utilizing confocal microscopy, that within the meningeal layer surrounding the spinal cord taken from behaviorally verified neuropathic rats, MAGL is colabeled with Iba-1-positive infiltrating monocytes. In superficial laminae, MAGL is colabeled with either resident microglia or infiltrating monocytes/macrophages (Figures 1(j), 1(k), and 1(l)).We additionally observed in the deeper dorsal horn laminae, that MAGL is colabeled with Iba-1-positive microglia and morphologically identifiable neuronal cell bodies (data not shown).

4.5. The Implications of the Endocannabinoid System in Pain Modulation

Following peripheral nerve or tissue injury, increased expression of endocannabinoids, CB₂R, FAAH, and MAGL occurs in DRG and spinal cord [94, 109, 147]. For example, AEA and 2-AG are upregulated in DRG following L5 spinal nerve ligation (SNL), a well-described animal model that leads to neuropathic pain [109]. Additionally, in a paw incision model of pain, 2-AG, widely characterized to produce analgesia, was found to be upregulated in the ipsilateral lumbar spinal cord on days 3 and 9 and in the contralateral lumbar spinal cord on days 1 and 9 after surgery [63]. These data suggest that endocannabinoid compounds may act to counterbalance the cytokine actions known to mediate neuropathic pain.

Recent studies show that exogenous application of the endocannabinoids, AEA and 2-AG, leads to pain control. Exogenous AEA administered spinally reverses carageenan-induced nociception [148], and exogenous 2-AG injected into the hindpaw blocks nociceptive responses due to formalin injection [149,150]. Paradoxically, administration of exogenous AEA to the hindpaw or high intrathecal doses produce nociceptive behavior and, in both cases, is mediated by the ionotropic transient receptor potential cation channel, superfamily V subtype 1 (TRPV-1) [148, 151]. Several reports detail that the actions of AEA may be mediated by TRPV-1, and as such, caution must be taken when assigning endocannabinoid actions to only the CB₁R or the CB₂R.

Manipulating the enzymes responsible for the bioavailability of AEA or 2-AG is additionally effective for pain control. Altering endocannabinoid levels by inhibiting the actions of MAGL and/or FAAH increases available endogenous AEA and 2-AG and results in therapeutic actions. Following localized administration of MAGL inhibitors (JZL184, URB602), into rat hindpaws increases local levels of 2-AG, with simultaneous attenuation of formalin-induced pain in rats [149, 150]. Additionally, systemic administration of FAAH inhibitors (PF-3845, URB597), MAGL inhibitors (JZL184, URB602) or the dual FAAH/MAGL inhibitor, JZL195, increases CNS levels of AEA and 2-AG, with attenuation of CCI-induced pain in mice [152, 153]. Specifically, the pharmacological FAAH inhibitor, PF-3845, decreased allodynia and hyperalgesia in CCI-induced neuropathic mice without the development of tolerance [129]. Numerous studies have demonstrated that MAGL inhibitors increase 2-AG accumulation [129,152, 154].However, recent studies indicate that following challenge with CB₁R agonists, increased 2-AG availability leads to CB₁R downregulation, desensitization, and lessened CB₁R effects [127, 129]. These data suggest that significantly increasing the levels of 2-AG may not be a clinically viable approach for treating chronic pain conditions.

Although pain behavior is suppressed following exogenous administration of 2-AG and AEA or by increased levels of endocannabinoids via enzyme inhibitors, the exact mechanisms of these cannabinoids underlying the modulation of inflammation and pain are not well understood. Studies are currently underway by several groups to elucidate the mechanisms whereby the endocannabinoid system is able to lead to pain control [63, 129, 152, 153]. Alkaitis and colleagues, utilizing dual CB₁R and CB₂R antagonists, AM281 and AM630, respectively, recently found that the endocannabinoid system plays critical roles in the resolution of allodynia from surgical hindpaw incision, an animal model of postsurgical pain [63]. In addition, blocking the activation of both the CB₁R and the CB₂R resulted in increased p-p38MAPK levels in this model of postsurgical pain, suggesting that constitutive endocannabinoid actions play a role in modulating p-p38MAPK. These findings support that the endocannabinoid system alters factors which are critical mediators of inflammatory processes underlying pain responses in a wide range of medical conditions where chronic pain is a component. Although speculative, CB₂R actions during chronic pain may be primed for enhanced activity to ultimately produce pain modulation following CB₂R stimulation, as downregulation and desensitization previously noted to occur with the CB₁R have not been observed with the CB₂R [7, 95, 155]. Numerous synthetic CB₂R agonists are currently being explored as potential therapeutic interventions for the treatment of chronic pain.

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