Littérature scientifique sur le sujet « NEUROINFLAMMATION NEUROPATHIC PAIN MICROGLIA »

Neuropathic pain is a serious clinical problem that is difficult to treat. Purinoceptors (P2Rs) transduce pain perception from the peripheral to the central nervous system and play an important role in the transmission of neuropathic pain signals. We previously found that the crude extracts of Hericium erinaceus mycelium (HE-CE) inhibited P2R-mediated signaling in cells and reduced heat-induced pain in mice. The present study explored the effects of HE-CE on neuropathic pain. We used adenosine triphosphate (ATP) as a P2R agonist to generate Ca2+ signaling and neuronal damage in a cell line. We also established a neuropathic mouse model of L5 spinal nerve ligation (L5-SNL) to examine neuropathic pain and neuroinflammation. Neuropathic pain was recorded using the von Frey test. Neuroinflammation was evaluated based on immunohistofluorescence observation of glial fibrillary acidic protein (GFAP) levels in astrocytes, ionized calcium-binding adaptor molecule1 (iba1) levels in microglia, and IL-6 levels in plasma. The results show that HE-CE and erinacine-S, but not erinacine-A, totally counteracted Ca2+ signaling and cytotoxic effects upon P2R stimulation by ATP in human osteosarcoma HOS cells and human neuroblastoma SH-SY5Y cells, respectively. SNL induced a decrease in the withdrawal pressure of the ipsilateral hind paw, indicating neuropathic pain. It also raised the GFAP level in astrocytes, the iba1 level in microglia, and the IL-6 level in plasma, indicating neuroinflammation. HE-CE significantly counteracted the SNL-induced decrease in withdrawal pressure, illustrating that it could relieve neuropathic pain. It also reduced SNL-induced increases in astrocyte GFAP levels, microglial iba1 levels, and plasma IL-6 levels, suggesting that HE-CE reduces neuroinflammation. Erinacine-S relieved neuropathic pain better than HE-CE. The present study demonstrated that HE inhibits P2R and, thus, that it can relieve neuropathic pain and neuroinflammation.

Neuropathic pain is relatively less responsive to opioids than other types of pain, which is possibly due to a disrupted opioid system partially caused by the profound microglial cell activation that underlines neuroinflammation. We demonstrated that intrathecally injected biphalin, a dimeric enkephalin analog, diminished symptoms of neuropathy in a preclinical model of neuropathic pain in rats (CCI, chronic constriction injury of the sciatic nerve) at day 12 postinjury. Using primary microglial cell cultures, we revealed that biphalin did not influence cell viability but diminished NO production and expression of Iba1 in LPS-stimulated cells. Biphalin also diminished MOP receptor level, as well as pronociceptive mediators (iNOS, IL-1β, and IL-18) in an opioid receptor-dependent manner, and it was correlated with diminished p-NF-κB, p-IκB, p-p38MAPK, and TRIF levels. Biphalin reduced IL-6, IL-10, TNFα, p-STAT3, and p-ERK1/2 and upregulated SOCS3, TLR4, and MyD88; however, this effect was not reversed by naloxone pretreatment. Our study provides evidence that biphalin diminishes neuropathy symptoms, which might be partially related to reduced pronociceptive mediators released by activated microglia. Biphalin may be a putative drug for future pain therapy, especially for the treatment of neuropathic pain, when the lower analgesic effects of morphine are correlated with profound microglial cell activation.

Microglia activation and subsequent pro-inflammatory responses play a key role in the development of neuropathic pain. The process of microglia polarization towards pro-inflammatory phenotype often occurs during neuroinflammation. Recent studies have demonstrated an active role for the gut microbiota in promoting microglial full maturation and inflammatory capabilities via the production of Short-Chain Fatty Acids (SCFAs). However, it remains unclear whether SCFAs is involved in pro-inflammatory/anti-inflammatory phenotypes microglia polarization in the neuropathic pain. In the present study, chronic constriction injury (CCI) was used to induce neuropathic pain in mice, the mechanical withdrawal threshold, thermal hyperalgesia were accomplished. The levels of microglia markers including ionized calcium-binding adaptor molecule 1 (Iba1), cluster of differentiation 11b (CD11b), pro-inflammatory phenotype markers including CD68, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and anti-inflammatory phenotype markers including CD206, IL-4 in the hippocampus and spinal cord were determined on day 21 after CCI. The results showed that CCI produced mechanical allodynia and thermal hyperalgesia, and also increased the expressions of microglia markers (Iba1, CD11b) and pro-inflammatory phenotype markers (CD68, IL-1β, and TNF-α), but not anti-inflammatory phenotype marker (CD206, IL-4) in the hippocampus and spinal cord, accompanied by increased SCFAs in the gut. Notably, antibiotic administration reversed these abnormalities, and its effects was also bloked by SCFAs administration. In conclusion, data from our study suggest that CCI can lead to mechanical and thermal hyperalgesia, while SCFAs play a key role in the pathogenesis of neuropathic pain by regulating microglial activation and subsequent pro-inflammatory phenotype polarization. Antibiotic administration may be a new treatment for neuropathic pain by reducing the production of SCFAs and further inhibiting the process of microglia polarization.

Neuropathic pain responds poorly to drug treatments, and partial relief is achieved in only about half of the patients. Puerarin, the main constituent ofPuerariae Lobatae Radix, has been used extensively in China to treat hypertension and tumor. The current study examined the effects of puerarin on neuropathic pain using two most commonly used animal models: chronic constriction injury (CCI) and diabetic neuropathy. We found that consecutive intrathecal administration of puerarin (4–100 nM) for 7 days inhibited the mechanical and thermal nociceptive response induced by CCI and diabetes without interfering with the normal pain response. Meanwhile, in both models puerarin inhibited the activation of microglia and astroglia in the spinal dorsal horn. Puerarin also reduced the upregulated levels of nuclear factor-κB (NF-κB) and other proinflammatory cytokines, such as IL-6, IL-1β, and TNF-α, in the spinal cord. In summary, puerarin alleviated CCI- and diabetes-induced neuropathic pain, and its effectiveness might be due to the inhibition of neuroinflammation in the spinal cord. The anti-inflammation effect of puerarin might be related to the suppression of spinal NF-κB activation and/or cytokines upregulation. We conclude that puerarin has a significant effect on alleviating neuropathic pain and thus may serve as a therapeutic approach for neuropathic pain.

AbstractNociceptive and neuropathic pain signals are known to result from noxious stimuli, which are converted into electrical impulses within tissue nociceptors. There is a complex equilibrium of pain-signalling and pain-relieving pathways connecting PNS and CNS. Drugs against long-term pain are today directed against increased neuronal excitability, mostly with less success.An injury often starts with acute physiological pain, which becomes inflammatory, nociceptive, or neuropathic, and may be transferred into long-term pain. Recently a low-grade inflammation was identified in the spinal cord and along the pain pathways to thalamus and the parietal cortex. This neuroinflammation is due to activation of glial cells, especially microglia, with production of cytokines and other inflammatory mediators within the CNS. Additionally, substances released to the blood from the injured region influence the blood–brain barrier, and give rise to an increased permeability of the tight junctions of the capillary endothelial cells, leading to passage of blood cells into the CNS. These cells are transformed into reactive microglia. If the inflammation turns into a pathological state the astrocytes will be activated. They are coupled into networks and respond to substances released by the capillary endothelial cells, to cytokines released from microglia, and to neurotransmitters and peptides released from neurons. As the astrocytes occupy a strategic position between the vasculature and synapses, they monitor the neuronal activity and transmitter release. Increased release of glutamate and ATP leads to disturbances in Ca2+ signalling, increased production of cytokines and free radicals, attenuation of the astrocyte glutamate transport capacity, and conformational changes in the astrocytic cytoskeleton, the actin filaments, which can lead to formation and rebuilding of new synapses. New neuronal contacts are established for maintaining and spreading pain sensation with the astrocytic networks as bridges. Thereby the glial cells can maintain the pain sensation even after the original injury has healed, and convert the pain into long-term by altering neuronal excitability. It can even be experienced from other parts of the body. As astrocytes are intimate co-players with neurons in the CNS, more knowledge on astrocyte responses to inflammatory activators may give new insight in our understanding of mechanisms of low-grade inflammation underlying long-term pain states and pain spreading. Novel treatment strategies would be to restore glial cell function and thereby attenuate the neuroinflammation.

Microglia, the resident macrophages, act as the first and main form of active immune defense in the central nervous system. Arginase 2 (Arg2) is an enzyme involved in L-arginine metabolism and is expressed in macrophages and nervous tissue. In this study, we determined whether the absence of Arg2 plays a beneficial or detrimental role in the neuroinflammatory process. We then investigated whether the loss of Arg2 potentiated microglia activation and pain behaviors following nerve injury-induced neuropathic pain. A spinal nerve transection (SNT) experimental model was used to induce neuropathic pain in mice. As a result of the peripheral nerve injury, SNT induced microgliosis and astrogliosis in the spinal cord, and upregulated inflammatory signals in both wild-type (WT) and Arg2 knockout (KO) mice. Notably, inflammation increased significantly in the Arg2 KO group compared to the WT group. We also observed a more robust microgliosis and a lower mechanical threshold in the Arg2 KO group than those in the WT group. Furthermore, our data revealed a stronger upregulation of M1 pro-inflammatory cytokines, such as interleukin (IL)-1β, and a stronger downregulation of M2 anti-inflammatory cytokines, including IL4 and IL-10, in Arg2 KO mice. Additionally, stronger formation of enzyme-inducible nitric oxide synthase, oxidative stress, and decreased expression of CD206 were detected in the Arg2 KO group compared to the WT group. These results suggest that Arg2 deficiency contributes to inflammatory response. The reduction or the loss of Arg2 results in the stronger neuroinflammation in the spinal dorsal horn, followed by more severe pain behaviors arising from nerve injury-induced neuropathic pain.

AbstractBackgroundAcute pain in response to injury is an important mechanism that serves to protect living beings from harm. However, persistent pain remaining long after the injury has healed serves no useful purpose and is a disabling condition. Persistent postsurgical pain, which is pain that lasts more than 3 months after surgery, affects 10–50% of patients undergoing elective surgery. Many of these patients are affected by neuropathic pain which is characterised as a pain caused by lesion or disease in the somatosen-sory nervous system. When established, this type of pain is difficult to treat and new approaches for prevention and treatment are needed.A possible contributing mechanism for the transition from acute physiological pain to persistent pain involves low-grade inflammation in the central nervous system (CNS), glial dysfunction and subsequently an imbalance in the neuron–glial interaction that causes enhanced and prolonged pain transmission.AimThis topical review aims to highlight the contribution that inflammatory activated glial cell dysfunction may have for the development of persistent pain.MethodRelevant literature was searched for in PubMed.Results Immediately after an injury to a nerve ending in the periphery such as in surgery, the inflammatory cascade is activated and immunocompetent cells migrate to the site of injury. Macrophages infiltrate the injured nerve and cause an inflammatory reaction in the nerve cell. This reaction leads to microglia activation in the central nervous system and the release of pro-inflammatory cytokines that activate and alter astrocyte function. Once the astrocytes and microglia have become activated, they participate in the development, spread, and potentiation of low-grade neuroinflammation. The inflammatory activated glial cells exhibit cellular changes, and their communication to each other and to neurons is altered. This renders neurons more excitable and pain transmission is enhanced and prolonged. Astrocyte dysfunction can be experimentally restored using the combined actions of a μ–opioid receptor agonist, a μ–opioid receptor antagonist, and an anti-epileptic agent. To find these agents we searched the literature for substances with possible anti-inflammatory properties that are usually used for other purposes in medicine. Inflammatory induced glial cell dysfunction is restorable in vitro by a combination of endomorphine-1, ultralow doses of naloxone and levetiracetam. Restoring inflammatory-activated glial cells, thereby restoring astrocyte-neuron interaction has the potential to affect pain transmission in neurons. ConclusionSurgery causes inflammation at the site of injury. Peripheral nerve injury can cause low-grade inflammation in the CNS known as neuroinflammation. Low-grade neuroinflammation can cause an imbalance in the glial-neuron interaction and communication. This renders neurons more excitable and pain transmission is enhanced and prolonged. Astrocytic dysfunction can be restored in vitro by a combination of endomorphin-1, ultralow doses of naloxone and levetiracetam. This restoration is essential for the interaction between astrocytes and neurons and hence also for modulation of synaptic pain transmission.ImplicationsLarger studies in clinical settings are needed before these findings can be applied in a clinical context. Potentially, by targeting inflammatory activated glial cells and not only neurons, a new arena for development of pharmacological agents for persistent pain is opened.

The most common neurological complication in patients receiving successful combination antiretroviral therapy (cART) is peripheral neuropathic pain. Data show that distal symmetric polyneuropathy (DSP) also develops along with murine acquired immunodeficiency syndrome (MAIDS) after infection with the LP-BM5 murine retrovirus mixture. Links between cannabinoid receptor 2 (CB2R) and peripheral neuropathy have been established in animal models using nerve transection, chemotherapy-induced pain, and various other stimuli. Diverse types of neuropathic pain respond differently to standard drug intervention, and little is currently known regarding the effects of modulation through CB2Rs. In this study, we evaluated whether treatment with the exogenous synthetic CB2R agonists JWH015, JWH133, Gp1a, and HU308 controls neuropathic pain and neuroinflammation in animals with chronic retroviral infection. Hind-paw mechanical hypersensitivity in CB2R agonist-treated versus untreated animals was assessed using the MouseMet electronic von Frey system. Multicolor flow cytometry was used to determine the effects of CB2R agonists on macrophage activation and T-lymphocyte infiltration into dorsal root ganglia (DRG) and lumbar spinal cord (LSC). Results demonstrated that, following weekly intraperitoneal injections starting at 5 wk p.i., JWH015, JWH133, and Gp1a, but not HU308 (5 mg/kg), significantly ameliorated allodynia when assessed 2 h after ligand injection. However, these same agonists (2x/wk) did not display antiallodynic effects when mechanical sensitivity was assessed 24 h after ligand injection. Infection-induced macrophage activation and T-cell infiltration into the DRG and LSC were observed at 12 wk p.i., but this neuroinflammation was not affected by treatment with any CB2R agonist. Activation of JAK/STAT3 has been shown to contribute to development of neuropathic pain in the LSC and pretreatment of primary murine microglia (2 h) with JWH015-, JWH133-, or Gp1a-blocked IFN-gamma-induced phosphorylation of STAT1 and STAT3. Taken together, these data show that CB2R agonists demonstrate acute, but not long-term, antiallodynic effects on retrovirus infection-induced neuropathic pain.

This work is focused in the study of the pathophysiology of neuropathic pain, particularly in the role of the endogenous opioid and cannabinoid systems. Neuropathic pain is a chronic illness with a high prevalence in the population and is characterized by the presence of spontaneous pain and abnormal stimulus-evoked pain responses, among other symptoms. It is a clinical pain manifestation that has shown to be poorly treated with the available pharmacological treatment. Even with the existence of many therapeutic approaches, there is not an adequate effective treatment for palliating all symptoms of neuropathic pain. This situation leads us to study the specific involvement of the endogenous opioid and cannabinoid systems in the pathophysiology of the development and maintenance of neuropathic pain. In the present study, we have evaluated the role of delta opioid receptor (DOR) in the central nervous system (CNS) and peripheral nociceptive neurons, as well as the participation of cannabinoid receptor type 2 (CB2) in the activated microglia at the spinal cord. The results show that DOR and CB2 may be pharmacological targets for the development of new drugs with analgesic activity, but devoid of the psychotropic side effects of traditional opioids and cannabinoid agonists.
Aquest treball es centra en l’estudi de la fisiopatologia del dolor neuropàtic, en particular en el paper dels sistemes endògens opioide i cannabinoide. El dolor neuropàtic és una malaltia crònica amb una alta prevalença en la població i es caracteritza per la presència de dolor espontani i percepció anormal del dolor, entre d’altres símptomes. És una manifestació clínica del dolor que ha demostrat ser mal tractada amb el tractament farmacològic disponible. Malgrat l’existència de molts enfocs terapèutics, no hi ha un tractament eficaç adequat per pal•liar els símptomes del dolor neuropàtic. Aquesta situació ens porta a estudiar la participació específica dels sistemes endògens opioide i cannabinoide en la fisiopatologia del desenvolupament i manteniment del dolor neuropàtic. En el present estudi, hem avaluat el paper del receptor opioide delta (DOR) en el sistema nerviós central (SNC) i perifèric en neurones nociceptives, així com la participació dels receptors cannabinoides tipus 2 (CB2) a la micròglia activada a la medul•la espinal. Els resultats mostren que DOR i CB2 poden ser dianes farmacològiques per al desenvolupament de nous fàrmacs amb activitat analgèsica, però amb menys efectes psicotròpics secundaris dels opioides tradicionals i els agonistes cannabinoides.

Nerve damage leads to the development of disabling neuropathic pain in susceptible individuals, where patients present with pain as well as co-morbid behavioural changes, such as anhedonia, decreased motivation and depression. The pathophysiology of neuropathic pain remains unknown, however accumulating evidence suggests that neuroimmune interactions play a key role in its pathogenesis and development of co-morbid behavioural disturbances. Complex regional pain syndrome (CRPS) is a debilitating neuropathic disorder where trauma to a limb results in chronic pain. Mass cytometry (CyTOF) was used to systematically analyse circulating immune cells with a panel of 38 phenotypic and activation markers in the blood of CRPS patients and healthy controls. CyTOF revealed an expansion and increased activation of signalling pathways in several distinct populations of central memory CD8+ and CD4+ T lymphocytes. Regarding emotional state, CD8+ T lymphocytes were correlated with clinical scores for stress and CD4+ Th1 lymphocytes correlated with clinical scores for anxiety. There was also a reduction in circulating Dendritic cells (DC), indicative of DC tissue trafficking and potential involvement in lymphocyte activation. These data highlight a pathogenic role for T lymphocyte mediated chronic inflammation in CRPS and co-morbid behavioural disabilities. To further explore to role of neuroimmune interactions in the development of neuropathic pain and co-morbid behavioural changes, a rodent nerve injury model was utilized to evaluate whether individual differences in radial maze behaviour and neuroimmune interactions in the hippocampus (HP) and medial prefrontal cortex (mPFC) occurred in rats after sciatic nerve chronic constriction injury (CCI). CCI reduced mechanical withdrawal thresholds in all rats, whilst pellet-seeking behaviours were altered in some but not all rats. One group, termed ‘No effect’, had no behavioural changes compared to sham rats. Another group, termed ‘Acute effect’, had a temporary alteration to their exploration pattern, displaying more risk-assessment behaviour in the early phase post-injury. In a third group, termed ‘Lasting effect’, exploratory behaviours were remarkably different for the entire post-injury period, showing a withdrawal from pellet-seeking. Immunohistochemical analysis throughout the dorso-ventral axis of the HP revealed that the withdrawal from pellet-seeking observed in Lasting effect rats was concomitant with distinct glial-cytokine-neuronal adaptations within the contralateral ventral HP, including; increased expression of IL-1b and MCP-1; astrocyte atrophy and decreased area in the dentate gyrus (DG); reactive microglia and increased FosB/DFosB expression in the cornu ammonis (CA) subfield. These data highlight that glial-cytokine-neuronal adaptations in the ventral HP may mediate individual differences in radial maze behaviour following CCI. A follow up experiment explored whether pre-injury learning on the maze altered the effects of nerve injury on exploratory behaviour and spatial memory function. Whilst CCI again produced three distinct patterns of behaviour on the radial maze, Acute effect rats had improved working spatial memory outcomes after CCI. This indicates that the increased risk-assessment behaviours employed by Acute effect rats after injury may be considered advantageous when pellet-seeking, as it reduces unnecessary exploration during reward-seeking. The behavioural disruptions observed in Lasting effect rats were accompanied by neuroimmune activation within the contralateral ventral HP and mPFC. Multiplex immunoassay analysis revealed an increase in IL-1b, IL-6 and MCP-1 within the contralateral mPFC and ventral HP. Detailed immunohistochemical analysis of the mPFC and HP revealed an increased expression of IL-6, increased phospho-p38 MAPK expression in neurons and microglia, and a shift to a reactive microglial morphology in the caudal prelimbic and infralimbic cortex, ventral CA1 and DG. There was also a reduction in astrocyte cell size and BDNF expression in the contralateral ventral DG. These data provide further evidence that neuroinflammation in the mPFC and ventral HP may influence individual differences in radial maze behaviour following CCI. Collectively, these data provide evidence that individual differences in circulating immune cell activation and neuroimmune signature in the interconnected ventral HP-mPFC circuitry may play a significant role in the divergent behavioural trajectories in the neuropathic pain state, contributing to co-morbid behavioural changes in susceptible individuals.

Neuropathic pain is a chronic and debilitating disease that occurs secondarily to injury of the peripheral and/or central nervous system. This pathology affects million people in the world and can be classified as an incurable disease for the lack of valid treatments. Neuronal injuries often arise from a nerve trauma or metabolic disease, such as diabetes, and neuropathic patients, whatever the cause, typically exhibit a mixture of sensory loss with ongoing spontaneous pain and enhanced sensitivity either to innocuous or painful stimuli. Although the underlying mechanisms are far to being elucidated, it is well established that neuronal injury not only results in profound modifications in the activity of sensory neurons and their central projection pathways, but is also coupled to a sustained immune response at different anatomical locations associated to chronic pain processing with an important contribution of cytokines and chemokines (Calvo et al., 2012; Sacerdote et al., 2013). Since intensive researches over the past years have identified the prokineticins (PKs) as possible candidates for mediating these pathological neuro-immune interactions in pain, in these years of PhD school my research was focused on the characterization of the PKs system in the development of experimental neuropathic pain. PKs family comprehends small chemokines-like proteins highly conserved across the species including the mammalian prokineticin 1 (PK1) and prokineticin 2 (PK2). These proteins modulate a large spectrum of biological activities in the organism. In particular it is well documented the pro-nociceptive/proinflammatory activity of the ligand PK2 (Negri et al., 2007). Two G protein-coupled receptors (PKR1 and PKR2) mediate PK2 actions. PK2, binding to PKR1 and PKR2 widely distributed in the central nervous system, DRG, sensory neurons and in cells participating to immune and inflammatory responses, exerts in fact a critical role in pain perception inducing nociceptor sensitization and increasing the release of neuromediators implicated in pain processing such as CGRP and SP (Negri et al., 2007; DeFelice et al., 2012; Vellani et al., 2006). Moreover the ligand influences macrophages and T lymphocytes activity inducing a pro-inflammatory phenotype in the macrophage and skewing the Th1/Th2 balance towards a Th1 response (Martucci et al., 2006; Franchi et al., 2008). In order to understand if PK2, PKR1 and PKR2 activities were necessary for the onset, maintenance and resolution of neuropathic pain, in this study, in vivo and ex-vivo experiments were performed using a non-peptidic PKR antagonist, named PC1, proved capable of antagonizing all pro-nociceptive effects induced by PK2 (Balboni et al., 2008; Giannini et al., 2009; Negri and Lattanzi, 2012). The efficacy of PC1 treatment was evaluated in two different mouse models of painful neuropathy: a mononeuropathy induced by the chronic constriction injury (CCI) of sciatic nerve and a diabetic polyneuropathy induced by the injection of a pancreatic β cell toxin, streptozotocin (STZ). CCI procedure was performed through three loose ligatures around the right common sciatic nerve while the diabetic painful neuropathy was induced in animals by the administration of either a single high dose (200 mg/kg) or repeated multi-lower doses (80 mg/kg) of STZ. Changes in pain behavior were evaluated measuring the paw withdrawal thresholds after noxious (hyperalgesia) and/or innocuous (allodynia) stimulation with the Plantar Test Apparatus and the Dynamic Plantar Aesthesiometer, respectively. To check the efficacy of PC1 to counteract painful manifestations, 3 days after CCI surgery and 21 days after STZ administrations, time points corresponding to full neuropathic pain development, CCI-operated and STZ-injected mice were subjected to a therapeutic treatment with the antagonist PC1 (150 µg/kg). The first major finding of this study was that, independently from neuropathic pain etiology, PC1 treatment was effective in alleviating established painful symptoms in mice without producing tolerance. Repeated systemic injections of PC1 from day 3 to 9 after surgery or from day 21 to 34 after diabetes induction in fact abolished thermal hyperalgesia and mechanical allodynia in nerve injured mice, and mechanical allodynia in diabetic animals. The fact that painful symptoms were completely reversed by the chronic administration of the PKR antagonist unequivocally indicated the involvement of the PKs system in neuropathic pain. Moreover, interestingly, in STZ-injected mice the anti-allodynic effect induced by the antagonist was still evident two weeks after the treatment discontinuation leading us to suppose that blocking PK2 signaling could induce permanent changes in neuronal circuits involved in the maintenance of neuropathic pain. At the end of treatments, i.e. on day 10 after CCI surgery and at different time points from diabetes induction (7, 14, 35 and 56 days after STZ injection) when the anti-hyperalgesic and anti-allodynic effects of PC1 were evident, biochemical evaluations were performed in neuropathic animals (CCI-operated and STZ-injected mice) treated with either PC1 or saline and in the respective controls to determine the expression of PK2 and its receptors, PKR1 and PKR2, at the peripheral and central sites of pain transmission. Real Time PCR analysis performed on sciatic nerve and spinal cord from neuropathic animals revealed a general up-regulation of PK2 and PKRs in these tissues furthermore demonstrating the close correlation between the PKs system and the development of neuropathic pain. In particular, in STZ model, an over expression of PK2 in spinal cord was present since the appearance of painful symptoms and was observed for all the persistence of allodynia. In addition, we also exactly discriminated in the spinal cord and in periphery, the cells mainly involved in the CCI-induced PKs system activation. In the spinal cord of injured nerve mice the expression of PK2 and PKRs was observed in the superficial layers of the spinal cord, at the levels of the presynaptic terminals. PK2 as well as PKR2 were also mostly expressed in proliferating and activated astrocytes. In periphery, at the level of the injured nerve, the expression of PK2 was evident in Schwann cells, neutrophils and macrophages, while PKR1 and PKR2 were highly expressed on activated inflammatory cells and on Schwann cells, respectively. In CCI animals the therapeutic treatment with the antagonist PC1 succeeded in decreasing the neuropathy-induced PK2 up-regulation both in the spinal cord and in the injured nerve, without significantly affecting PKR1 and PKR2 mRNA levels. In particular, a significant reduction of PK2 immunoreactivity was observed at the presynaptic terminals of the dorsal horns, in the reactive spinal astrocytes and in infiltrating neutrophils, mirroring the lower PK2 mRNA levels. In STZ mice, the therapeutic treatment with the antagonist was also able to counteract the PK2 augmentation in the spinal cord and to significantly reduce the neuropathy-induced PKR1 up-regulation in the sciatic nerve. Since PKR1 is the receptor mostly implicated in the immune response and it was previously demonstrated to mediate macrophage migration (Martucci et al., 2006), it can be assumed that blocking PKRs with PC1 could affect macrophage chemotaxis, reducing or preventing the recruitment of inflammatory cells expressing PKR1 in the nerve with a consequence reduction of neuroinflammation. Considering the pro-inflammatory activity of PK2 and the presence of the PKRs in Schwann and immune cells in the nerve and the PKR2 in the spinal astrocytes, it was examined the efficacy of PC1 to counteract also the neuroinflammation associated to neuropathic pain development, evaluating by Real Time PCR and ELISA, the levels of the pro-inflammatory cytokine IL-1β and anti-inflammatory cytokine IL-10 in the sciatic nerve and the spinal cord from neuropathic mice. The release of inflammatory mediators, such as cytokines and chemokines, from glia and immune cells plays in fact an important role in the genesis of neuropathic pain and it was demonstrated that an altered balance of some pro- and anti-inflammatory cytokines in nervous tissues linked to pain transmission, such as the nerve, the DRG and the spinal cord is well correlated with the presence of neuropathic pain either in CCI or STZ mice (Sacerdote et al., 2013; Valsecchi et al., 2011). In agreement with what already published, in presence of high levels of PK2 and consistently with its immunomodulatory activity, an augmentation of the pro-nociceptive cytokine IL-1β was observed both in the central and peripheral nervous system of CCI and STZ neuropathic mice, while the levels of the anti-inflammatory cytokine IL-10 appeared lower respect to the basal levels of controls. Repeated PC1 administration induced a clear reduction of the neuropathy-induced IL-1β increase observed in the sciatic nerve and in the spinal cord from neuropathic mice. In addition, PC1 enhanced the levels of IL-10, which is likely to participate in the therapeutic effects observed. These data clearly demonstrated the implication of the PKs system in neuropathic pain suggesting its possible implication not only in the maintenance but also in the onset of the pathology. In order to confirm this hypothesis, we performed a precocious blocking of the PKRs in STZ mice not yet neuropathic. Early PC1 administrations from day 0, time point corresponding to first STZ injection, to 13 days after diabetes induction, prevented in fact the development of mechanical allodynia in STZ mice and the spinal cord up-regulation of PK2. Glutamate is one of the main mediator in pain processing and it is known to participate in the alteration of the synaptic transmission during neuropathic pain (Iwata et al., 2007; Daulhac et., 2011). In order to further support the anti-allodynic effect of PC1, we analyzed the expression of glutamate NMDA and AMPA receptor subunits in spinal cord of STZ mice treated with preventive PC1 administrations. Western blot analysis revealed that in presence of a fully developed allodynia, a decrease of the spinal NMDA subunit N2A was present, while the expression of the subunit N2B significantly increased. Early PC1 administration was effective in preventing N2B up-regulation in spinal cord of diabetic mice, without affecting the levels of the subunit N2A. Finally, considering the precocious involvement of the PKs system in the onset of the diabetic neuropathy it was interesting to investigate whether a preventive blocking of the PKRs positively influenced also the course of the diabetic pathology itself, modulating the hyperglycaemic state of the animals or reducing the peripheral inflammatory component which is known to be associated to diabetic status (Agrawal and Kant, 2014). Early PC1 administrations from day 0 to 13 after diabetes induction were not effective either in reducing high glucose levels in STZ mice or in re-establishing the plasmatic insulin levels. However, blocking the PKs system was effective in ameliorating the general pro-inflammatory status that was present in diabetic mice. The antagonist was in fact able to prevent the dysregulation of the IL-1β and IL-10 levels in the pancreas, which appeared drastically diminished in the STZ mice. Moreover, in the diabetic animals we observed a significant alteration of both innate and acquired immunity, characterized by elevated levels of IL-1β produced by macrophages, and a Th1 pro-inflammatory profile. The PC1 treatment reduced the peripheral inflammatory status, decreasing macrophagic IL-1β and switching Th1/Th2 balance towards Th2. In conclusion, considering the efficacy of PC1 to contrast painful symptoms and the neuroinflammation associated to the development of neuropathic pain, blocking PKRs signalling could represent a new possible therapeutic strategy to treat neuropathic pain. In addition, beyond reducing the neuropathy-induced pain hypersensitivity, the anti-inflammatory properties of the antagonist PC1 could be useful to ameliorate other pathologies, characterized by a sustained inflammatory component.

Chronic pain is a major health problem worldwide. We have recently demonstrated the analgesic effect of the nitroxyl donor, Angeli's salt (AS) in models of inflammatory pain. In the present study, the acute and chronic analgesic effects of AS was investigated in chronic constriction injury of the sciatic nerve (CCI)-induced neuropathic pain in mice. Acute (7th day after CCI) AS treatment (1 and 3 mg/kg; s.c.) reduced CCI-induced mechanical, but not thermal hyperalgesia. The acute analgesic effect of AS was prevented by treatment with 1H-[1,2, 4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor), KT5823 (an inhibitor of protein kinase G [PKG]) or glibenclamide (GLB, an ATP-sensitive potassium channel blocker). Chronic (7-14 days after CCI) treatment with AS (3 mg/kg, s.c.) promoted a sustained reduction of CCI-induced mechanical and thermal hyperalgesia. Acute AS treatment reduced CCI-induced spinal cord allograft inflammatory factor 1 (known as Iba-1), interleukin-1β (IL-1β), and ST2 receptor mRNA expression. Chronic AS treatment reduced CCI-induced spinal cord glial fibrillary acidic protein (GFAP), Iba-1, IL-1β, tumor necrosis factor-α (TNF-α), interleukin-33 (IL-33) and ST2 mRNA expression. Chronic treatment with AS (3 mg/kg, s.c.) did not alter aspartate aminotransferase, alanine aminotransferase, urea or creatinine plasma levels. Together, these results suggest that the acute analgesic effect of AS depends on activating the cGMP/PKG/ATP-sensitive potassium channel signaling pathway. Moreover, chronic AS diminishes CCI-induced mechanical and thermal hyperalgesia by reducing the activation of spinal cord microglia and astrocytes, decreasing TNF-α, IL-1β and IL-33 cytokines expression. This spinal cord immune modulation was more prominent in the chronic treatment with AS. Thus, nitroxyl limits CCI-induced neuropathic pain by reducing spinal cord glial cells activation.

P2X7 receptors belong to a family of membrane bound ion channels which are activated by extracellular ATP, resulting in the opening of a non-selective cation channel. After prolonged or repeated exposure to agonist, functional and cellular changes can occur, including the formation of a large pore, cell lysis and the release of mature, biologically active interleukin-1β. It is this diversity of functions that underlies the significance of this receptor in pain processing. P2X7 receptors are expressed on microglia, which when activated, release a host of mediators which contribute to central sensitisation, a phenomenon associated with neuropathic pain. The role of P2X7 receptors in the activation of microglia is less well established and is the main subject of this thesis. Before considering the interaction between P2X7 receptors and microglia, the first aim was to establish whether P2X7 receptors played a role in a pathological process known to be associated with microglial activation. An additional aim was to establish whether the site of action was in the central nervous system (CNS), where microglia are located. These aims were accomplished using a surgery-based rat model of neuropathic pain, the chronic constriction injury (CCI) model, and by comparing the effects of different P2X7 receptor antagonists when dosed peripherally or directly into the spinal cord. The results indicated that P2X7 receptor antagonists produced efficacy in the CCI model via a mechanism located in the CNS. To further investigate the association between P2X7 receptors and microglia, a different experimental paradigm was explored. Chronically dosed morphine is known to activate microglia, the consequence of which is thought to underlie morphine tolerance and reduced morphine analgesia. By administering a P2X7 receptor antagonist to CCI-operated rats treated with chronic morphine, the interaction between the P2X7 receptor and morphine tolerance and analgesia was explored. The results showed that P2X7 receptor antagonism delayed morphine tolerance and increased the efficacy of low doses of morphine, suggesting an association between P2X7 receptors and microglia. It was intended to confirm the interaction between a P2X7 receptor antagonist and morphine in another neuropathic pain model, namely varicella zoster virus-induced neuropathy. However due to a lack of reproducibility, this model was not used for pharmacological studies. Having demonstrated an association between P2X7 receptor antagonist and morphine in a chronic pain setting, studies were initiated to explore whether this interaction occurred in other morphine-related behaviours. The effect on body weight, motor coordination and single dosed morphine-induced analgesia was assessed in rats co-administered with P2X7 receptor antagonist and morphine. Results demonstrated that the blockade of P2X7 receptors enhanced morphine acute dose-induced analgesia, but had no influence on motor-impairment and body weight. The final part of the thesis used immunohistochemical and molecular techniques to confirm that microglia played a role in established allodynia induced by CCI-surgery and that P2X7 receptors directly influenced microglia-activation. In conclusion, the data in this thesis has illustrated an association between centrally activated P2X7 receptors and microglia, as well as an association between the P2X7 receptor and morphine-induced tolerance and analgesia. It is possible that co-administration of a P2X7 receptor antagonist with morphine could reduce the effective dose of morphine clinically, thereby reducing the side effects of this commonly used analgesic.

Neuregulin 1 (NRG1) is a growth factor required for peripheral nerve development and functional recovery following nerve injury. However, its importance in regulating neuropathic pain via microglial signaling remains unclear. Previous pharmacological studies suggest NRG1 regulates microglial proliferation, mechanical allodynia and cold hypersensitivity through binding to extracellular tyrosine kinase receptors (e.g. erbB2, erbB3 and erbB4) found on microglia. The aim of this thesis was to further dissect the role of NRG1 in regulating pain behaviour during neuropathic pain by using transgenic systems that conditionally ablate NRG1 expression in adulthood or erbB receptor expression specifically within microglia. In our hands it was determined that the CX3CR1 Cre is more efficient than the Cd11b Cre system in effectively targeting tissue specific gene ablation. Using animal models of nerve injury, gene expression analysis showed that NRG1 and erbB gene expression levels are dysregulated in the peripheral nervous system and the spinal cord, in neuropathic pain models. A novel cold pain behaviour assay was optimised to measure cold pain behaviour. With conditional NRG1 ablation, and the use of the spared nerve injury model, it was determined that NRG1 regulates cold hypersensitivity in the delayed stages of nerve injury but does not regulate mechanical hypersensitivity or attenuate microglial proliferation. Similarly, conditional ablation of erbB3 and erbB4 receptors in microglia suggests that the NRG1-erbB signaling pathway does not regulate mechanical hypersensitivity or microglial proliferation. However, NRG1-erbB signaling does regulate cold hypersensitivity in the delayed stages of nerve injury through microglia. The work presented in this thesis has further refined the role of the NRG1-erbB signaling pathway in the context of peripheral nerve injury neuropathic pain.

Diabetes mellitus is one of the most common and serious chronic disease in the world. Although the number of available agents to manage diabetes continues to rapidly expand, treatment of diabetes complications, such as neuropathy that is one of the most frequent complication of diabetes mellitus, remains a substantial challenge [Aring et al., 2005]. Pathophysiology of diabetic neuropathy is complex and not fully elucidated; it has multipathogenic mechanisms that cause a diversity of physical symptoms: allodynia, hyperalgesia, numbness and cutaneous ulceration [Vinik et al., 1995]. Persistent Neuropathic Pain (NP) interferes significantly with quality of life, impairing sleep, and emotional well-being, and is a significant causative factor for anxiety, loss of sleep, and non-compliance with treatment. Recent advances in the mechanisms involved in NP have demonstrated that pro- and anti-inflammatory cytokines produced by immune cells as well as by glia and microglia in nerve, dorsal root ganglia (DRG) and spinal cord are common denominators in neuropathic pain [Sacerdote et al., 2013; Old et al., 2015]. These start a cascade of neuroinflammation-related events that may maintain and worsen the original injury, participating in pain generation and chronicization [Valsecchi et al., 2011; Sommer and Kress, 2004; Austin and Moaelem-Taylor, 2010]. Activation of inflammatory cascade, pro- inflammatory cytokines upregulation, and neuroimmune communication pathways play a vital role in structural and functional damage of the peripheral nerves leading to the diabetic peripheral neuropathy. Unfortunately, most of the available analgesic drugs appear to be relatively ineffective in controlling diabetic neuropathic pain, both for insufficient efficacy and side effects [Galer et al., 2000; Kapur, 2003]. Thus, there is a clear need for new disease-modifying therapeutic approaches. Mesenchymal stem/stromal cells (MSCs) may offer a novel therapeutic option to treat diabetic neuropathy. MSCs modulate the nervous system injured environment and promote repair as they secrete anti-inflammatory, anti-apoptotic molecules, and trophic factors to support axonal growth, immunomodulation, angiogenesis, remyelination, and protection from apoptotic cell death [Ma et al., 2014]. Transplanted MSCs not only directly differentiate into endogenous cells on administration, but also secrete a broad range of biologically active factors, generally referred to as the MSCs secretome; in fact even if initially MSCs were proposed for cell therapy based on their differentiation potential, the lack of correlation between functional improvement and cell engraftment or differentiation at the site of injury has led to the proposal that MSCs exert their effects not through their differentiation potential but through their secreted products [Makridakis, 2016; Blaber et al., 2012]. For these reasons in the present study we analyze in a Streptozotocin mouse model of type 1 diabetes the therapeutic effect of hASC (human adipose stem/stromal cells) and their conditioned media (CM-hASC/ secretome) on allodynia and hyperalgesia, on pro- and anti- inflammatory cytokines expression in the main tissue stations involved in nociception transmission as well as in peripheral immune responses. Type 1 diabetes was induced in mice by intraperitoneal (i.p.) injection of moderate low doses of Streptozotocin (STZ, 80 mg/kg, daily for three consecutive days) while control mice were injected with vehicle (citrate buffer). In all groups, mechanical allodynia was evaluated by Von Frey test before diabetes induction and every week after STZ until the end of protocol (14 weeks after STZ). When allodynia was established (2 weeks after STZ) animals were treated with 106 hASC that have been mechanically dissociated to a single cell suspension in PBS solution with 2.5% heparin; CM-hASC from 2x106 cells was also re-suspended in PBS solution with 2.5% heparin and both hASC and CM-hASC were intravenously injected in the tail vein to mice. Animals injected with vehicle only were considered as controls. Our data demonstrated that hASC and CM-hASC treatments were able to reduce allodynia, although the effect of hASC was significantly higher than that elicited by CM-hASC. The effect of both hASC and their secretome was very fast, since a significant reduction of mechanical allodynia was evident already 3 hours after the injection. The antiallodynic effect was maximal beetwen 1 and 2 weeks after treatments and it was extremely long lasting: a significant reduction of allodynia was still present 12 weeks after a single hASC and CM-hASC treatment. Moreover, 4 weeks after the first hASC/CM-hASC treatment (6 weeks after STZ) we decided to treat again a group of diabetic animals with hASC or CM-hASC; repeated hASC treatment did not further ameliorate allodynia. On the other hand, already few hours after the second CM-hASC injection, the antiallodynic effect was significantly potentiated and it completely mimicked the effect evoked by hASC. In order to discover whether hASC and CM-hASC treatments were effective also in a more advanced stage of the disease, when a severe loss of nerve function is reported, we treated animals 6 weeks after diabetes induction. Also in this situation both treatments were efficacious in providing a fast and irreversible antiallodynic effect. Futhermore, in order to verify whether stemness is a fundamental prerequisite for obtaining pain relief a group of STZ-mice was treated with CM obtained from 2x106 human fibroblasts (CM-hF). CM-hF did not exert any effect on mechanical allodynia, demonstrating that only secretome from stem cell cultures is biologically active. It is very important also to consider preparation method of secretome, because lyophilized CM-hASC was unable to provide pain relief, suggesting that during the lyophilization process some essential bioactive factors may be lost. Moreover, since in patients sensory alterations associated to diabetic neuropathy are often diverse in order to ascertain whether the effects of hASC and CM-hASC were limited only to mechanical allodynia, we evaluated thermal hyperalgesia (hot stimuli) and thermal allodynia (cold stimuli) by plantar test and acetone test, respectively. In STZ-mice cold allodynia was present and both treatments were able to significantly reduce it. As regards to heat hyperalgesia, it was present in diabetic mice until 3 weeks from STZ administration, but subsequently we observed hypoalgesia appearance and both treatments were able to avoid hypoalgesia development; these results demostrate the ability of stem cells and their secretome to relieve and prevent the typical diabetic hypersensitivity in response to different types of stimuli. In order to evaluate the impact of treatments on pro- and anti- inflammatory cytokines, animals were sacrificed at different time points: 2 weeks after STZ, i.e. 3 hours after hASC/CM-hASC treatment; 3 weeks after STZ, i.e. 1 week from treatments and 14 weeks after STZ, i.e. 12 or 8 weeks from treatments. From each animal, sciatic nerves, dorsal root ganglia, spinal cord and spleens were collected. IL-1β, TNF-α, IL-6 and IL-10, were evaluated as protein in nervous tissues by ELISA assay. Three weeks after neuropathy induction pro-inflammatory cytokines IL-1β, TNFα and IL-6 resulted overexpressed in peripheral (sciatic nerve and DRG) and central (spinal cord) nervous system of diabetic mice, both hASC and CM-hASC were similarly able to restore pro-inflammatory cytokine levels that 1 week from treatments were back to basal levels; while in all nervous tissues IL-10 levels appeared instead significantly reduced in diabetic animals and both hASC and CM-hASC significantly increased IL-10 concentrations, reaching physiological levels in DRG and spinal cord, while it exceeded basal levels in the sciatic nerve, indicating a switch towards an anti-inflammatory environment in all these tissues. Fourteen weeks after STZ, spinal cord IL-1β, TNF-α and IL-6 levels were still significantly elevated and IL-10 levels reduced in comparison to non diabetic mice, indicating the persistence of neuroinflammation. As observed for the antiallodynic effect, also cytokine modulation induced by hASC and CM-hASC was long lasting. Twelve weeks after treatments performed 2 weeks from STZ, IL-1β, TNF-α and IL-6 levels were still significantly reduced by hASC and CM-hASC treatments, while hASC-treated mice showed a significant normalization of IL-10 levels. Similar effects were observed also in double treatments (2 and 6 weeks after STZ) and both treatments were effective in modulating cytokine levels also when they were administered in an advanced pathological state (6 weeks after STZ). Moreover, to investigate the timing of cytokines modulation exerted by both treatments IL-1 and IL-10 levels in scatic nerves, DRG and spinal cord were measured. Two weeks after diabetic induction, STZ mice were characterized by pro- inflammatory profile and only 3 hours after hASC and CM-hASC administration , both treatments were able to modulate cytokines levels. To further demonstrate the modulation of treatments on pain-related mediators we demonstrated the ability of hASC and CM-hASC to normalize calcitonin gene related peptide level (CGRP), that was elevated in DRG from diabetic animals. Moreover, we evaluated loss of nerve fibers and skin thickness 1 and 12 weeks after a single hASC/CM-hASC administration at 2 weeks after STZ. Both treatments were able to contrast loss of nerve fibers and skin thickness, although hASC treatment was more effective. Since STZ multiple low-doses protocol that we utilized is able to develop an autoimmune response against pancreatic tissue sustained by a T-helper 1 pattern of activation, we studied whether a T-helper polarization was present in splenocytes from diabetic mice and whether hASC or their secretome did exert any immunomodulatory activity. Two weeks after STZ, Con-A stimulated splenocytes released higher levels of IFN-γ, while IL-10 release was significant reduced; both hASC and CM-hASC treatments 3 hours after administration were already able to augment IL-10 levels. Th1/Th2 cytokines unbalance was more evident 3 weeks after STZ and both tretaments appeared able to restablish a correct IFNγ/IL-10 balance. When cytokine levels were measured at longer time from diabetes induction, i.e. 14 weeks after STZ, a clear shift toward a Th1 pattern, characterized by higher IFN-γ and IL-2 secretion and lower levels of IL-4 and IL-10, was present and both hASC/CM-hASC treatments were able to normalize cytokine levels. In the whole, the data indicate that both hASC and CM-hASC treatments are able to block Th1 polarization that develops in this experimental model of diabetes. Moreover, throughout the experiment, blood glucose levels and weight were monitored. In respect to non-diabetic control animals, a significant body weight loss was observed in diabetic mice, that started to be significant 3 weeks after STZ. In STZ-mice the administration of hASC or CM-hASC, 2 weeks after diabetes induction significantly prevented the loss of body weight. Neither treatments did modify blood glucose levels that were elevated in STZ-mice nor glucose tolerance test response. Moreover both hASC and CM-hASC did ameliorate nephropathy that was present in diabetic animals, indicating that the treatments may be useful for treating also other diabetes complications. Our results demonstrated that hASC can control diabetic complications such as neuropathic pain, acting on several peripheral and central mechanisms involved in development and maintenance of this condition, such as neural and immune elements. Moreover the significant new positive results observed also with hASC conditioned medium strongly suggest that their effect is likely to be mediated by their secreted products.

Microglia play a major role in immune response in the brain. Recent progress in studies for microglia suggests that stress causes morphological alterations in microglia and affects microglial humoral release and phagocytosis. In this review, we present a molecular mechanism by which stress impacts microglia. Then, we describe current findings for the involvement of microglia in stress-related mental disorders including posttraumatic stress disorder (PTSD), depression, and pain enhancement. We focus on preclinical and clinical studies. Preclinical PTSD studies using animal models with fear memory dysregulation show neuroinflammation by microglia and altered microglial phagocytosis, two imaging studies and a postmortem study assessing neuroinflammation in PTSD patients show contradictory results. Imaging studies suggest neuroinflammation in depressed patients, postmortem studies show no microglial inflammatory changes in non-suicidal depressed patients. Although it has been established that microglia in the spinal cord play a pivotal role in chronic neuropathic pain, several preclinical studies suggest microglia also participate in stress-induced pain. A clinical study with induced microglia-like (iMG) cells and an imaging study indicate neuroinflammation by microglia in fibromyalgia patients. We believe that progress in interactive research between humans and animals elucidates the role of microglia in the pathophysiology of stress-related mental disorders.

Microglia maintain cellular, synaptic, and myelin homeostasis during development and normal function and response to injury. Surveilling icroglia actively explore their environment by dynamically extending thin processes that respond to local signals. Activated (“reactive,” or “effector”) microglia constitute a heterogeneous population that dynamically change in phenotype depending on their environmental context and may mediate either injury or neuroprotection, repair, and circuit refinement. Any type of injury in the CNS elicits activation of microglia, astrocytes, and oligodendrocyte precursors, which together with infiltrating cells from the blood in the case of blood-brain barrier disruption interact via several signals to elicit elimination of pathogens, limit the spatial extent of the lesion, and eventually promote tissue remodeling, repair, and remyelination. Neuroinflammation is a feature of essentially all types of neurologic disorders, including traumatic, vascular, and inflammatory/demyelinating lesions; autoimmune encephalitis; and neurodegenerative disorders and has a major role in mechanisms of epilepsy and pain.

Neuropathic pain arises as a direct consequence of a lesion or a disease affecting the somatosensory system. The mechanisms of neuropathic pain are often complex and difficult to study given the diversity of causes, pathology, and clinical presentation of various neuropathic pain conditions. Common mechanisms include peripheral and central sensitizations. Peripheral sensitization refers to increased responsiveness and reduced threshold of nociceptive neurons in the periphery to the stimulation of their receptive fields. Central sensitization refers to the augmented response of central signaling neurons. The mechanisms of peripheral and central sensitization are understood at the cellular and molecular levels. The processes of neuroplasticity involve activation of inflammatory cells, such as macrophages (and microglia in the central nervous system) and other immune cells, and release of inflammatory mediators, such as cytokines, chemokines, and a host of other mediators. Interactions of these mediators with specific receptors in the nociceptors or the spinal cord neurons may lead to phosphorylation or changes in expression of ion channels, receptors, transporters, and other effectors through specific signaling pathways. These events ultimately lead to changes in excitability, conductivity, and transmissibility of neurons in the pain processing pathways. Other factors may include disinhibition of interneurons, changes in descending inhibitory and excitatory pathways, and reorganization of the cortical areas and their interconnections.