The novel therapeuthic targets in the treatment of chronic pain.

Effective treatment for neuropathic pain is still lacking, because of poor understanding of pathological mechanisms at the molecular level. Chronic pain (inflammatory and neuropathic pain) is believed to be caused by aberrant neuronal responses along the pain transmission pathways. Both peripheral and central origins are likely to be involved in chronic pain, although their contribution may be different depending on the various forms of chronic pain. Glial cells have recently been implicated in neuropathic pain. These cells form close interactions with neurons and thus may modulate nociceptive transmission under pathological conditions. We will first examine the recent progress in the role of glia in neuropathic and inflammatory pain, with particular emphasis on microglia. Finally, we will discuss how the study of the interaction between neuronal and microglial mechanisms can open the door to new therapeutic opportunities, designed to act on the mechanisms underlying the disease ("disease-oriented") using natural endogenous substances.

INTRODUCTION

To develop a better treatment for neuropathic pain, a comprehensive understanding of its pathogenesis is required. Chronic pain (such as inflammatory and neuropathic pain) is believed to be caused by aberrant neuronal responses along the pain transmission pathway from dorsal root ganglion (DRG) to spinal cord, thalamus and cortex. Both peripheral and central origins are likely to be involved in chronic pain, although their contribution may be different depending on the various forms of chronic pain. It has been recently reported that neurons are not the only cell type involved in chronic pain states. Glial cells, including astrocytes and microglia, are emerging as possible additional players in the initiation and maintenance of neuropathic and inflammatory pain. These glial cells have close interactions with neurons and thus modulate pain transmission particularly under pathological conditions [1-2].

THE NOVEL TARGETS

Current therapeutic strategies for neuropathic pain aim to reduce the excitability of neurons by modulating ion channel activity (such as gabapentin and lidocaine) or by enhancing endogenous inhibitory mechanisms (such as tricyclic antidepressants and opioids). As we know, NMDA receptors play a critical role in synaptic plasticity within pain transmission pathways and are thus likely to be important in neuropathic pain. NR2B subunit-containing NMDA receptors are localized in pain-relevant structures, such as in superficial layers of the dorsal spinal horn, thalamus, hippocampus and cortex. The restricted distribution of NR2Bmakes it promising as a candidate target of side effect free analgesic drugs. Indeed, NR2B antagonists, such as ifenprodil and related compounds, are effective in neuropathic pain in animals, and show better separation between efficacy and side effects in human patients than non-selective NMDA receptor blockers.

Our understanding of pathological pain has evolved from solely neuronal mechanisms to neuronglial interactions. In particular, astrocytes and microglia act as possible modulators of neuropathic pain by releasing a number of cytokines and chemokines. Astrocytes and microglia play different roles in relation to neuronal activity; however, they do have some overlapping functions in mediating CNS innate immune response [3]. Both astrocytes and microglia are activated in neuropathic pain, and their activation leads to pro-inflammatory responses with pathological effects, such as neuronal hyperexcitability, neurotoxcity and chronic inflammation. Astrocytes are the most abundant glial cell type in the CNS. In addition to their neuron-supportive functions, astrocytes also directly alter neuronal communication because they completely encapsulate synapses and are in close contact with neuronal somas [3,4]. The close astrocyte–neuron contact allows for astrocyte activation by neurotransmission as astrocytes express various functional neurotransmitter receptors. These include ionotropic non-NMDA and NMDA receptors as well as metabotropic glutamate (mGluR3 and mGluR5), purinergic and substance P receptors. After astrocyte activation, the extracellular signal-regulated kinase (ERK; also known as mitogenactivated protein kinase 1 (MAPK1)) and c-Jun N-terminal kinase (JNK; also known as MAPK8) signalling pathways are activated [5].

These lead to an increase in the synthesis of inflammatory factors (interleukin 1β (IL-1 β), IL-6, tumour-necrosis factor-α (TNF α), prostaglandin E2 (PGE2) and nitric oxide(NO), which finally alter glial glutamate transporter function and gap-junction proteins, which are known to facilitate astrocyte–astrocyte activation through Ca2+ cascades. Although similar pathways are activated in microglia after nerve injury, the temporal patterns of enzyme activation and pro-inflammatory cytokine release are distinct for microglia and astrocytes.. Furthermore, chronic astrocyte activation after nerve injury has been shown to involve ERK activation and subsequent down-regulation of excitatory amino acid transporters (glutamate transporter 1 (GLT1) and glutamate–aspartate transporter (GLAST) leading to a decrease in glutamate uptake and an increase in excitatory synaptic transmission. During chronic neuropathic conditions, astrocytes remain activated in response to the initial microglia-derived inflammatory factors; this is likely to account for their ongoing responses during these conditions.

Microglia are the resident macrophages and main immune-response cells in the CNS [6]. They comprise 5–10% of the glial cell population and are quite evenly distributed in the brain. Under pathological conditions, these cells are activated and exhibit chemotactic, phagocytoxic and secretory responses to various stimuli.

Resting ramified microglia rapidly transform into an activated state in most pathological conditions, including host defense against infectious organisms, autoimmune inflammation, ischemia, trauma, neurodegeneration and neuropathic pain [6,7]. Activation of microglia is accompanied by: changes in morphology, characterized by hypertrophy with retracted processes and associated with proliferation or microgliosis; upregulation of immune surface antigens included CD11b, P2X4 receptors, toll-like receptor 4, CD44, and MHC II. Finally, the activation of spinal microglia in neuropathic pain is characterized by phosphorylation of MAP kinases, including the p38 and Src-family kinases. Interestingly, spinal microglia activation occurs during the early phase of neuropathic pain and precedes astrogliosis, supporting the current hypothesis that microglia may be important for initiation, while astrocytes are important for the maintenance of neuropathic pain [8,10]. It is important to point out that none of the spinal glial cells project to the brain. Thus, the influence of glial changes may act through ascending neuronal transmission.

Using transgenic mice in which microglia are selectively labeled with GFP, recent studies[ 9] performed systematic mapping of microglia in major pain-related brain areas following nerve injury. They have confirmed the microglial activation occurs only in spinal cord but not in supraspinal structures; Zhang et all showed the possibility that microglia are altered at the biochemical and molecular levels in neuropathic pain. Indeed, upregulation of microglial and astrocytic markers such as OX-42 and GFAP was observed in rat brain after peripheral administration of complete Freunds adjuvant (CFA) to produce inflammation [11]. Interestingly, astrogliosis was observed in the ACC after sciatic nerve ligation.

In the setting of neuropathic pain, peripheral neurons transmit signals to spinal dorsal horn neurons, releasing neurotransmitters such as calcitonin gene-related protein (CGRP), substance P, glutamate, and ATP. Locally in the dorsal horn, there are also other neurotransmitters involved, such as GABA, glycine, serotonin.

Spinal microglia activation happens via several signaling pathways, which include ATP and its receptors (P2X and P2Y receptor), fractalkine and CX3CR1, monocyte chemotactic protein (MCP-1) and CCR2. Similar to microglia in the brain, spinal microglia show fast chemotaxis in response to local application of ATP [12]. Microglia are known to express both ionotropic receptors, such as P2X4 and P2X7, and metabotropic receptors, such as P2Y6 and P2Y12 [13,14]. Activation of P2X4 in microglia facilitates BDNF release [15], while activation of P2X7 in microglia induces IL1β release and CXCL2 production [16,17]. Interestingly, The P2Y12 receptor in microglia is reported to mediate ATP-induced microglial chemotaxis [18,19] while P2Y6 may mediate microglial phagocytosis. Moreover, ATP induced both inward and outward current in resting microglia, which may be mediated by P2X and P2Y receptors, respectively [14]. In models of neuropathic pain, both P2X4 and P2Y12 receptors are upregulated in microglia, but not in neurons or astrocytes in the dorsal horn.

Fractalkine and its receptor CX3CR1 are also involved in microglial activation associated with neuropathic pain. Fractalkine is a neuronal transmembrane glycoprotein that can be released after being cleaved by proteolysis; fractalkine activates p38 in spinal microglia and produces mechanical allodynia and thermal hyperalgesia [20]. The cleavage of fractalkine may involve cathepsin S, a cysteine protease that is expressed in spinal microglia. It has been shown that noxious stimulation of primary afferent fibers induces release of cathepsin S from microglia; the process may require P2X7 activation [21]. Microglial cells constitutively express CX3CR1, and its expression is markedly upregulated in models of neuropathic pain.

In addition, MCP-1 and its receptor CCR2 are involved in microglial activation and neuropathic pain. Intrathecal injection of MCP-1 produced tactile allodynia, Microglial cells in the dorsal horn express CCR2, which may mediate MCP-1's effect: microglial morphology transitions and p38 activation in microglia. How might activated microglia contribute to neuropathic pain? One intriguing pathway involving ATP and the P2X4 receptor has been proposed. Peripheral nerve injury leads to an upregulation of P2X4 receptors in activated microglia. ATP acts on the P2X4 receptor in microglia and induces intracellular Ca2+ elevation and phosphorylation of p38, which subsequently increase BDNF synthesis and release. BDNF released from microglia may produce a shift in neuronal anion gradient with potassium-chloride exporter KCC2 reduced in dorsal horn neurons. This prompts a disinhibition and thus facilitates mechanical allodynia after nerve injury.

It has been shown that p38 activation turns on the transcription factor NF-κB, which leads to the expression of IL-1β, IL6, and COX-2 [22]. These and other cytokines and chemokines released by activated microglia, such as tumor necrosis factor-α(TNFα), PGE2, and nitric oxide, will amplify microglial activation in an autocrine manner and may act directly on dorsal horn neurons to cause behavioral sensitization.

Among the receptors we know to be upregulated on immune response cells under neuropathic pain conditions, there are also the cannabinoids receptors (CB1 and CB2).

Cannabinoids suppress behavioural responses to noxious stimulation and suppress nociceptive transmission through activation of CB1 and CB2 receptor subtypes. CB1 receptors are mainly expressed at high levels in the central nervous system (CNS), whereas CB2 receptors are found predominantly, but not exclusively, on immune cells outside the CNS.

Activation of CB1 and CB2 receptors inhibits adenylyl cyclase [23,24] and activates mitogen-activated protein kinase [25] through binding of the α-subunit of the Gi/o protein.

Only CB1 receptors suppress calcium currents and activate inward-rectifying potassium channels: these effects are associated with depression of neuronal excitability and transmitter release. Thus, differences in receptor distribution and signal transduction mechanisms are likely to account for the relative absence of the CNS side effects induced by CB2 agonists.

Microglia or macrophages exspress CB2 receptors in a different way in vivo or in vitro: in vitro its levels depend on the local environment and the combination of inflammatory molecules [26]; in vivo CB2 is not expressed equally in all microglial populations, but rather it is predominantly present in perivascular or activated microglia [27]. Treatment of activated microglia cultures with anandamide, the main endogenous CB1-ligand, decreases the expression of the inducible NOS-2 and the production of nitrites in a CB1- and CB2-dependent manner [28]. In addition, through CB2 receptors, cannabinoids inhibit the expression of TNF-a, IL-1b and the p40 subunit of IL-12 and IL-23 by microglia/macrophages [29]. Astrocytes express CB1 and CB2 receptors and they respond to cytokines secreted by the immune cells by regulating the production of molecules involved both in the bystander injury and in the protection of CNS tissue. Among these molecules, astrocytes express NOS-2 and they produce NO in response to several inflammatory signals. Cannabinoids inhibit the release of nitrites by astrocytes through a mechanism involving the CB2 receptor. In these cells, cannabinoids also inhibit the inflammation-induced expression of TNF-a, IL-1b and IL-6 through CB1 and CB2 receptors [28].

THE NOVEL THERAPEUTHIC STRATEGIES

All these considerations together suggest that novel pharmacotherapies targeting CB2 receptors may have considerable therapeutic potential for suppressing inflammatory and neuropathic pain states. In animal models of tissue and nerve injury-induced nociception, CB2-selective agonists suppress hyperalgesia and allodynia and normalize nociceptive thresholds without inducing analgesia [30]. CB2 selective agonists may also be more efficacious in suppressing hypersensitivity to mechanical as opposed to thermal stimulation for reasons that remain incompletely understood. As mentioned above, a particularly beneficial aspect of the pharmacological profile of CB2 agonists is the failure of these compounds to induce adverse CNS side effects associated with activation of CB1 receptors.

The available literature supports the efficacy of CB2 agonists in suppressing persistent pain states following acute administration. However, the impact of long-term treatment with CB2 agonists on antihyperalgesic efficacy and immune system function remains largely unknown [31]. More work is needed to identify the limitations associated with therapeutic strategies targeting CB2 receptors and to explore the therapeutic potential of multimodal analgesic strategies that combine CB2-mediated pharmacotherapies for pain with other agents directed at different analgesic targets. Such strategies offer the potential to produce synergistic antihyperalgesic effects with a more beneficial therapeutic ratio compared to conventional analgesics (for example, by combining a CB2-selective agonist with lower doses of opiates, CB1 agonists or nonsteroidal anti-inflammatory drugs that are below the threshold for inducing undesirable side effects).

Taking into account that the endocannabinoids are synthesized only “on demand” and that tissue accumulation of N-acylated glycerophospholipids, and free N-acylamides, such as anandamide (AEA) and palmitoylethanolamide(PEA), occurs in some pathological conditions known to be associated with inflammatory and pain reactions, several studies have been carried out to identify the beneficial effects associated with therapeutic use of PEA [32].

PEA is an endogenous fatty acid and with properties comparable to anandaminde, as well as other endogenous cannabinoids. PEA may behave as local autacoids capable of negatively modulating mast cell activation (ALIA mechanism) [33]. In keeping with this hypothesis, the direct anti-inflammatory effect of PEA is parly linked to its modulation of mast cells degranulation, thus to inhibition of the release of several pro-inflammatory enzymes such as iNOS, chynase and metalloproteinase MMP-9, as well as mediators such as nitric oxide and TNF-α. PEA pharmacological effects could be mediated by its interactions with CB2 receptors, or CB2 like-receptors, highly exspressed on mast cells and neuronal cells [32].

Lipids like N-palmitoylethanolamine can act as signaling molecules, activating intracellular and membrane-associated receptors to regulate physiological functions. The signaling lipid PEA is known to activate and membrane-associated, intracellular and nuclear receptors (PPAR-alpha) and regulate many physiological functions related to the inflammatory cascade, and thus is of high interest in the treatment of neuropathic, or gliopathic pain.

Meanwhile it is an established fact that anandamide (AEA) and PEA regulate directly or indirectly many of the same pathophysiological processes, including pain perception, inflammation, convulsions and neuroprotection. There are a number of biological effects of endocannabinoids which can be enhanced by related endogenous fatty acid derivatives which are devoid of some of these primary effects. The last mechanism of action is called the entourage effect [34].

The fatty acids as PEA seem to be co-synthesized and co-released with the endocannabinoids such as anandaminde. Anandamide and PEA both are present in the spinal cord, but the concentration of PEA is most probably 10 times higher. The entourage effect of PEA most probably is due to its function of amplifier activity anti-inflammatory and antinociceptive of other endogenous compounds such as endocannabinoid anandamide. It has been suggested that PEA enhances the effects of AEA by acting as a competitive inhibitor of the enzymatic degradation of endocannabinoids, and increaseing the affinity of the AEA for the CB1 and vanilloid receptors (TRPV1), highly exspressed on neuronal and mast cells [34].

Transient Receptor Potential Vanilloid 1 (TRPV1) is a Ca2+ permeant non-selective cation channel and they are exspressed highly on sensorial fiber nerve endings (FNE) and they can act as a transducers of noxious temperature and chemical stimuli [35].

TRPV1 expression is increased in inflammation and neuropathic pain because of the retrograde transport of nerve growth factor (NGF) released at the site of peripheral tissue injury to the DRG soma.

The possibility of using TRPV1 receptor agonists, such as capsaicin, to alleviate pain is an interesting concept. Capsaicin acts first exciting FNE, than leading to TRPV1 down-regulation. As a result of this FNE show a loss of pain sensibility known as analgesia. Moreover capsaicin inhibits the retrograde transport of NGF. Activation of TRPV1 by capsaicin can induce persistent depolarization of the nerve terminals causing a decrease in their ability to generate and propagate action potentials [36]. On the other hand, TRPV1 activation causes a massive influx of Ca2+ that in the long-term can cause nerve terminal degeneration. The use of transdermal capsaicin has been shown to reduce FNE density in epidermis in a reversible manner and to cause a long-lasting loss of thermal sensitivity without affecting mechanical sensitivity. This effect is dose dependent and it suggests the use of a novel generation of analgesics suche as the patch to high concentrations of capasaicin in the treatment of neuropathic or gliopathic pain.