Tesis sobre el tema "NEUROINFLAMMATION NEUROPATHIC PAIN MICROGLIA"

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.

La douleur chronique est une pathologie invalidante de longue durée notamment caractériséepar trois symptômes : l’allodynie (un stimulus non douloureux est perçu comme douloureux),l’hyperalgésie (un stimulus douloureux est perçu comme encore plus douloureux) et desdouleurs ambulatoires. Quand cette douleur est due à une lésion ou une dysfonction du systèmenerveux on parle de douleur neuropathique. Chez les patients et les modèles animaux dedouleurs neuropathiques, les études ont montré que les neurones thalamiques étaienthyperexcitables. Les cellules gliales, astrocytes et microglies, sont des partenaires synaptiquesimpliqués dans la transmission et la plasticité synaptique et pourraient être impliqués dans cephénomène. En effet, ces cellules peuvent modifier leur phénotype lorsque le système nerveuxest affecté, elles sont réactives : leur morphologie est hypertrophiée, l’expression d’ARNm et deprotéines comme iba-1 (ionized binding-adaptor molecule 1) et CD11b/c (cluster ofdifferentiation 11b/c) pour les cellules microgliales et GFAP (glial fibrillary acidic protein) etS100β (S100 calcium binding protein β) pour les cellules astrocytaires est augmentée. Ellespeuvent également libérer des molécules pro-inflammatoires. Tout ceci pourrait générer ouamplifier l’hyperexcitabilité des neurones présents dans le thalamus.Mon travail de thèse a consisté en l’étude des astrocytes et de la microglie thalamique dans lemodèle de douleurs neuropathiques de ligature des nerfs spinaux L5-L6 du nerf sciatique (spinalnerve ligation, SNL). Les symptômes d’allodynie et d’hyperalgésie mécaniques ont étécaractérisés par le test des filaments de von Frey et les douleurs ambulatoires par le test dedistribution pondéral dynamique. L’expression des ARNm de marqueurs gliaux a été étudiée parune approche de qRT-PCR sur des prélèvements thalamiques et sur des noyaux thalamiquesobtenus par microdissection au laser. L’expression neurochimique des marqueurs iba-1,CD11b/c, Cathepsine S, GFAP et S100β a été étudié par immunohistofluorescence en quantifiantle nombre de cellules immunopositives et la surface occupée par les marqueurs. Toutes cesexpériences ont été réalisées à J14 et J28 après la chirurgie.A J14, les animaux SNL développent des symptômes d’allodynie et d’hyperalgésie mécaniqueainsi que des douleurs ambulatoires. Chez ces animaux, les cellules microgliales thalamiquesprésentent des signes de réactivité avec l’augmentation de l’expression des ARNm desmarqueurs CTSS et CX3CR1, le récepteur de la fractalkine, marqueurs connus pour leursimplications dans l’hyperexcitabilité neuronale spinale en conditions de douleursneuropathiques. De plus, l’expression neurochimique des marqueurs gliaux étudiés est diminuéece qui se traduit notamment par une diminution du nombre de cellules immunopositives pources marqueurs chez les animaux SNL. A J28, les symptômes douloureux sont maintenus. De plus,la réactivité microgliale décelée à J14 par qRT-PCR est toujours présente avec l’augmentation del’expression de l’ARNm codant pour la fractalkine (CX3CL1), partenaire de la voieCTSS/CX3CR1/CX3CL1. La diminution de l’expression neurochimique thalamique desmarqueurs gliaux chez les animaux SNL était transitoire et n’est plus présente à J28. Enrevanche, des signes de réactivité astrocytaire thalamique ont été mis en évidence chez lesanimaux SNL.Ainsi, ce travail dévoile une ambivalence au niveau des altérations de la glie thalamique dans cemodèle SNL: une diminution précoce de l’expression des marqueurs gliaux thalamiques suivied’une réactivité astrocytaire plus tardive concomitante à des signes de réactivité microgliale. Denombreuses expériences sont encore nécessaires pour appréhender l’impact de cetteambivalence gliale thalamique inédite dans un contexte de douleur neuropathique
Chronic pain is an incapacitating and long lasting pathology mainly characterized by threesymptoms: allodynia (a non painful stimulus is perceived as painful), hyperalgesia (a painfulstimulus is perceived as more painful) and ambulatory pains. When chronic pain is due to alesion or dysfunction of nervous system it is called neuropathic pain. In both patients and animalmodels of neuropathic pain, researchers found that thalamic neurons are hyperexcitable. Glialcells, astrocytes and microglia, are strong synaptic partners involved in synaptic transmissionand plasticity and therefore could be involved in this phenomenon. Indeed, these cells canmodify their phenotype when nervous system is damaged. They become reactive: theirmorphology is hypertrophied, mRNA and protein expression of iba-1 (ionized binding-adaptormolecule 1) and CD11b/c (cluster of differentiation 11b/c) for microglia and GFAP (glialfibrillary acidic protein) and S100β (S100 calcium binding protein β) for astrocytes is increased.They could also release pro-inflammatory molecules. All of these could contribute to generate oramplify the thalamic neuronal hyperexcitability.In my PhD work I studied thalamic astrocytes and microglia in a rat neuropathic pain model ofL5-L6 spinal nerves ligation (SNL). Mechanical allodynia and hyperalgesia were characterizedwith von Frey filament test and ambulatory pain with dynamic weight bearing apparatus. mRNAexpression of glial markers were studied with qRT-PCR technique on thalamic punches andlaser-microdissected nuclei. Neurochemical expressions of iba-1, CD11b/c, cathepsin S, GFAPand S100β markers were quantified using an immunohistofluorescence approach to count thenumber of immunopositive cells and surface stained by these markers. All these experimentswere done at D14 and D28 after surgery.At D14, SNL animals develop mechanical allodynia and hyperalgesia as well as ambulatory pain..For these animals, thalamic microglial cells showed signs of reactivity with the increase mRNAexpression of CTSS and CX3CR1, fractalkine receptor, well known markers involved in spinalneuronal hyperexcitability under neuropathic pain conditions. In addition, the number ofimmunopositive cells for the glial markers is decreased in SNL animals. At D28, the neuropathicpain symptoms are still present. Furthermore, thalamic microglial reactivity found at D14 withqRT-PCRm method is still present with the increased mRNA expression of fractalkine (CX3CL1),partner of CTSS/CX3CR1/CX3CL1 pathway. The decreased neurochemical expression of glialmarkers found at D14 was transient as I didn’t find this result at D28. However, thalamicastrocytic reactivity was found at D28 in SNL animals.So, this work reveal a new glial process at thalamic level in this SNL model of neuropathic pain :an early decreased expression of glial markers and then a later thalamic astrocytic reactivityconcomitant with signs of thalamic microglial reactivity. Numerous studies are required toexplore the role of such novel ambivalent glial alterations in the context of neuropathic pain

Under pathological conditions microglia (resident central nervous system (CNS) immune cells) become activated, and produce reactive oxygen and nitrogen species and pro-inflammatory cytokines: molecules that can contribute to disorders including stroke, traumatic brain injury, progressive neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, and several retinal diseases. Given that ATP is frequently released from CNS neurons during tissue damage and inflammation, its actions on microglia-mediated toxicity are especially pertinent. For example, the ATP-gated P2X7 purinergic receptor (P2X7R) cation channel is up-regulated around amyloid beta-peptide plaques in transgenic mouse models of Alzheimer's disease and co-localizes to microglia and astrocytes. Upregulation of P2X7R on microglia occurs also following spinal cord injury and after brain ischemia. ATP, via activation of P2X7R, is one of the most powerful stimuli for secretion of the key pro-inflammatory cytokine interleukin-1β (IL-1β) in its mature form. This project investigates the pharmacological and biochemical behaviors of P2X7R on microglia and astrocytes cultured from rat cerebral cortex, spinal cord and cerebellum, and the relationship between these two glial cell types. ATP is an efficient stimulus for IL-1β secretion only after the cells have undergone a short 'priming' with endotoxin (lipopolysaccharide (LPS)). Indeed LPS, but not ATP caused release of IL-1β from cortical microglia. However, it is known that the greater part of the IL-1β thus released is the precursor (biologically inactive) form. Purified (>99%) cortical microglia and enriched (>95%) astrocytes were primed for 2 hours with LPS, followed by addition of ATP for 1 hour. Culture medium was then collected and the content of IL-1β quantified by ELISA. The effects of LPS and ATP were concentration-dependent; although LPS alone (but not ATP) modestly stimulated IL-1β release, levels of cytokine release were much higher from primed cells incubated with ATP. The ATP-dependent component was fully blocked by selective P2X7R antagonists, and followed their known rank order of target potency. The P2X7R priming response was also seen with spinal cord and cerebellar microglia, a finding not described in the literature until now. To rule out a contribution by the minor population of microglia in our astrocyte cultures, the latter were treated with the lysosomotropic agent L-leucyl-L-leucine methyl ester (L-LME) which selectively eliminates cells with cytotoxic potential (e.g. macrophages, microglia). Immunocytochemical and molecular biological evaluation showed L-LME-treated cortical and spinal cord astrocytes to be fully depleted of microglia. These purified astrocytes failed to respond to LPS, and did not show the ATP priming behavior. Responsiveness was recovered upon addition of microglia to the L-LME-treated astrocytes and, moreover, a far more robust release of IL-1β occurred than that achieved with the same numbers of microglia alone. This astrocyte-microglia interaction was also observed for LPS-stimulated release of nitric oxide and IL-6, and was not mediated by astrocyte-derived soluble factors. Lastly, the LPS/ATP priming behavior was studied by examining the ability of other agents, linked to neuropathology, to replace either LPS or ATP. Neither ethanol (ethanol intoxication; in place of LPS) nor amyloid beta-peptides (Alzheimer disease; in place of ATP) were able to provoke IL-1β release from microglia. However, both zymosan and poly(I:C), agonists of Toll-like receptors -2 and -3, respectively, were capable of substituting LPS (a Toll-like receptor 4 agonist) in the P2X7R priming response. Release of IL-1β in all these cases was antagonized by inhibitors of p38 mitogen-activated protein kinase (a stress response kinase). TLRs contribute to CNS immunocompetent cell activation and the resulting pro-inflammatory cascade producing pathological pain. TLR4 recognizes not only LPS, but also ligands called damage associated molecular patterns, released by the injured tissue The involvement of extracellular TLR4 and TLR2, as well as TLR3 in preclinical pain models has been demonstrated. The findings described here further support the notion of astrocyte/microglia interaction, which may improve our understanding in how these cells respond to CNS injury or inflammation, in particular where TLRs are involved
αLe ricerche svolte hanno riguardato lo studio dei comportamenti della microglia e degli astrociti derivati da corteccia, midollo spinale e cervelletto di ratto in presenza di uno stimolo infiammatorio, in particolare quello associato alla famiglia dei recettori toll-like (TLR) e il recettore purinergico P2X7 (P2X7R). E’ ormai riconosciuto il coinvolgimento della glia nello sviluppo delle malattie neurodegenerative e nel dolore neuropatico, sia nel midollo spinale che nelle aree corticali. In particolare, il rilascio di ATP da cellule danneggiate o morenti (cellule gliali, neuroni e cellule endoteliali) può agire come un segnale di 'pericolo' attraverso un sottotipo di recettori purinergici come il P2X7. I recettori P2X7 possono influenzare la morte cellulare attraverso la capacità di regolare il processo e il rilascio di interluchina-1β (IL-1β), un mediatore chiave nella neurodegenerazione, infiammazione e dolore cronico. L’IL-1β e' rilasciata dai macrofagi e dalla microglia esposta a endotossina batterica (lipopolisaccaride, LPS). Scopo della ricerca svolta è stato quindi la caratterizzazione di queste popolazioni di cellule gliali con metodi immunologici, molecolari, biochimici e farmacologici in presenza o assenza di stimolo infiammatorio (LPS) e l'identificazione del meccanismo del 'priming' (le cellule di microglia trattate per breve tempo con LPS diventano più sensibili all’azione dell’ATP) in termine di rilascio di IL-1β. Fino ad ora la maggior parte degli studi presenti in letteratura hanno esaminato il ruolo della microglia ma non degli astrociti nella neuroinfiammazione. Il lavoro svolto nel primo anno è consistito principalmente nella caratterizzazione del comportamento della microglia e degli astrociti ottenuti da corteccia, midollo spinale e cervelletto, in termini di risposta ad ATP, in presenza o assenza di uno stimolo infiammatorio come LPS. Per questo scopo è stato utilizzato un modello in vitro di microglia preparata da corteccia cerebrale di ratto di 1-2 giorni d’eta' o da midollo spinale. Dopo 1-2 settimane la popolazione di cellule di microglia vengono separate dagli astrociti e raccolte. Queste colture secondarie di microglia purificate (>99%) e astrociti arricchiti (>95%) (come determinato mediante colorazione immunocitochimica e l'espressione genica (RT-PCR), sono state trattate e il terreno di coltura è stato utilizzato per l’analisi (ELISA) delle citochine (IL-1β, TNF-α, IL-6), rilasciate. Inoltre, nel terreno di coltura si è provveduto a valutare la quantità di ossido d’azoto (NO). La microglia e gli astrociti di controllo e trattati con ATP (fino a 5 mM) non producono una quantità misurabile di IL-1β. Il LPS, alla dose di 100 ng/ml, produce invece un considerevole aumento di NO e IL-1β in funzione del tempo in tutte e due i tipi cellulari. Successivamente abbiamo analizzato la risposta della microglia e degli astrociti esposti per tempi brevi prima a LPS e poi ad ATP per stimolare il rilascio di una quantità maggiore di IL-1β matura ('priming'). Sia la microglia che gli astrociti, stimolati con LPS e ATP, rilasciano IL-1β . La parte della risposta data dall’ATP e non dall’LPS viene bloccata dagli antagonisti del P2X7R. L’ SB-202190, un inibitore selettivo della miogeno-activated-protein (MAP) chinasi p38, riduce il rilascio di IL-1β indotto da LPS nel 'priming', ma lo aumenta (da 6 a 10 volte) se incubato con LPS in presenza di siero per 24 ore. L’ SB-202190 inoltre, inibisce il rilascio di NO in cellule trattate con LPS per 24 ore. L’SP600125, un inibitore del c-Jun N-terminale chinasi (JNK), aumenta il rilascio di IL-1β indotto da LPS (24 ore) in microglia. In contrasto, nel priming l’SP600125 riduce il rilascio di IL-1β. I due meccanismi di rilascio di IL-1β sono quindi presumibilmente diversi, ma legati alla cascata del segnale della MAP chinasi. La risposta 'priming' è stata anche dimostrata in microglia ottenuta da cervelletto di ratto. Questi resultati rappresentano la prima descrizione della risposta 'priming' nelle cellule gliali del midollo spinale e del cervelletto collegato al P2X7R. Il lavoro svolto nel secondo anno è consistito principalmente nel caratterizzare dal punto di vista molecolare e farmacologico il rapporto tra microglia e astrociti in queste culture. Siccome le colture arricchite in astrociti contengono una piccola percentuale di microglia (<5%), abbiamo voluto verificare se il rilascio di citochine in seguito a uno stimolo infiammatorio dipende da una azione diretta sugli astrociti o una azione indiretta tramite la presenza della microglia. Per eliminare la microglia residua le colture arricchite in astrociti (corticali o spinali), sono state trattate per un ora con l’agente lisosomotropico L-leucine methyl ester (L-LME) (50 mM), che è tossico per i macrofagi. Dopo un ora di incubazione la soluzione di L-LME è stata sostituita da terreno di coltura per 24 ore, al termine delle quali sono stati iniziati i trattamenti con lo stimolo infiammatorio. Da ora in poi definiamo tali colture come ‘astrociti purificati’. L’RNA messaggero (mRNA) per l’ Iba1, proteina espressa solo nella microglia, non è stato rilevato tramite la tecnica RT-PCR negli astrociti purificati, mentre il segnale è presente nelle cellule prima del trattamento con L-LME. Il trattamento con L-LME elimina anche la risposta LPS/ATP (priming) negli astrociti corticali e spinali purificati. L’aggiunta di una quantità nota di microglia agli astrociti purificati, ripristina la risposta degli astrociti al priming. Tuttavia, lo stesso numero di microglia, da sola, senza la presenza degli astrociti, è insufficiente a produrre il segnale. Lo studio dell’interazione tra microglia e astrociti purificati, è stato esteso anche alla produzione dell’NO e mRNA per iNOS (NO sintasi inducibile). E’stato dimostrato che l’aumento della produzione di NO in seguito a stimolazione con LPS, è stata abolita negli astrociti purificati. L’aggiunta di una quantità nota di microglia alle cellule di astrociti purificati, ripristina la risposta degli astrociti al LPS e di conseguenza un aumento di iNOS e NO. Inoltre, il terreno di coltura dagli astrociti trattati con LPS non è grado a stimolare il rilascio di NO da microglia. Un comportamento simile è stato osservato per IL-6 mRNA e proteine. Questi dati mostrano chiaramente che la microglia, ma non gli astrociti rispondono al LPS, e che il comportamento degli astrociti dipende dalla presenza di microglia. L'obiettivo ultimo era quello di esaminare, oltre al LPS o ATP, la capacità di altri agenti potenzialmente patologici, ad esempio il peptide beta-amiloide (Aβ), l’etanolo, e agonisti del recettore TLR2 e TLR3 nella risposta 'priming' della microglia. Aβ, il principale costituente delle placche che si trovano nel cervello dei pazienti affetti dalla malattia Alzheimer (AD), può attivare la microglia. E’ stato pubblicato che la proteina Aβ (25-35) aumenta la secrezione di IL-1β nella microglia esposta a LPS. Siccome nel cervello dei pazienti affetti da AD si accumula la forma (1-42) della Aβ e non la (25-35), tutte e due le forme sono state testate per vedere se sono in grado di sostituire l’ATP nella microglia pretrattata con LPS. Nessuno dei due peptidi però si sono dimostrati efficaci. L’ intossicazione da etanolo cronica e acuta promuove i processi infiammatori nel cervello e nelle cellule gliali agendo sul TLR4. L’etanolo, dunque, potrebbe attivare la microglia e sostituire LPS nella risposta 'priming' per l’ATP. Per dimostrare questa teoria, la microglia corticale e da midollo spinale è stata incubata con 100 mM etanolo o 1 microg/ml LPS per due ore, seguita da incubazione con 5 mM ATP per un' ora. Successivamente è stato misurato il rilascio di IL-β nel mezzo di coltura che ha dimostrato l’effetto dell’ATP, ma non dell’etanolo, sulle cellule pretrattate con LPS. La microglia oltre al TLR4 esprime anche TLR2 e TLR3 funzionali che, quando attivati, partecipano al dolore neuropatico causato da lesioni nervose. Finora non sono stati fatti studi che dimostrano il coinvolgimento degli agonisti TLR2 e TLR3 sulla microglia ‘primed’ e il rilascio di citochina IL-1β. L’agonista TLR2 (Zymosan) e TLR3 (Poly(I:C)), sono stati dunque testati nella microglia al posto del LPS e si sono dimostrati capaci di attivare la microglia, corticale, spinale e cerebellare. In presenza di ATP sono stati in grado, come il LPS, di essere efficaci come ‘priming’ in termini di rilascio di IL-1β. Un inibitore selettivo della MAP chinasi p38 (SB-202190) riduce il rilascio di IL-1β indotto da Zymosan o Poly(I:C) nel 'priming', ma lo aumenta (circa 4 volte) se incubato per 24 ore, in presenza di siero. Questo studio costituisce un contributo originale alla ricerca nel campo della neuroinfiammazione a livello cellulare e in particolare il rapporto tra astrociti e microglia. Dato che gli astrociti sono molto più numerosi della microglia nel sistema nervoso centrale, questi dati suggeriscono che una simile interazione tra astrociti e microglia in vivo può essere un elemento importante per l'evoluzione di una patologia infiammatoria. Inoltre, questo lavoro dimostra per la prima volta che l'attivazione del P2X7R si verifica in microglia da midollo spinale e da cervelletto, che può verificarsi con agonisti TL2 e TLR3 oltre che con TLR4. Questo amplia notevolmente le possibilità di partecipazione del recettore P2X7 nella neuroinfiammazione

Chronic pain affects 20 % of the global population, causes suffering, is difficult to treat, and constitutes a large economic burden for society. So far, the characterization of molecular mechanisms of chronic pain-like behaviors in animal models has not translated into effective treatments. In this thesis, consisting of five studies, pain patient biofluids were analyzed with modern proteomic methods to identify biomarker candidates that can be used to improve our understanding of the pathophysiology chronic pain and lead to more effective treatments. Paper I is a proof of concept study, where a multiplex solid phase-proximity ligation assay (SP-PLA) was applied to cerebrospinal fluid (CSF) for the first time. CSF reference protein levels and four biomarker candidates for ALS were presented. The investigated proteins were not altered by spinal cord stimulation (SCS) treatment for neuropathic pain. In Paper II, patient CSF was explored by dimethyl and label-free mass spectrometric (MS) proteomic methods. Twelve proteins, known for their roles in neuroprotection, nociceptive signaling, immune regulation, and synaptic plasticity, were identified to be associated with SCS treatment of neuropathic pain. In Paper III, proximity extension assay (PEA) was used to analyze levels of 92 proteins in serum from patients one year after painful disc herniation. Patients with residual pain had significantly higher serum levels of 41 inflammatory proteins. In Paper IV, levels of 55 proteins were analyzed by a 100-plex antibody suspension bead array (ASBA) in CSF samples from two neuropathic pain patient cohorts, one cohort of fibromyalgia patients and two control cohorts. CSF protein profiles consisting of levels of apolipoprotein C1, ectonucleotide pyrophosphatase/phosphodiesterase family member 2, angiotensinogen, prostaglandin-H2 D-isomerase, neurexin-1, superoxide dismutases 1 and 3 were found to be associated with neuropathic pain and fibromyalgia. In Paper V, higher CSF levels of five chemokines and LAPTGF-beta-1were detected in two patient cohorts with neuropathic pain compared with healthy controls. In conclusion, we demonstrate that combining MS proteomic and multiplex antibody-based methods for analysis of patient biofluid samples is a viable approach for discovery of biomarker candidates for the pathophysiology and treatment of chronic pain. Several biomarker candidates possibly reflecting systemic inflammation, lipid metabolism, and neuroinflammation in different pain conditions were identified for further investigation.
Uppsala Berzelii Technology Centre for Neurodiagnostics

The immune system in mammals contains a wide array of cells which serves to maintain normal physiologic conditions and promote the repairing processes of damaged tissues. Many of these activities are mediated by soluble factors, such as chemokines and cytokines, which are released from immune cells and bind to specific receptors or interact with other cellular targets, resulting in the activation of selected genes. This cross talk between different cell types maintains the homoeostatic balance of the immune system. Microglia cells, located in the brain and spinal cord, represent the immune cells of the central nervous system (CNS) and, along with astrocytes, constitute the first line of defense in the CNS. Several studies developed over the years show that a lesion of peripheral nerves can lead to the variation of plasticity in central synapses at structural and functional level. Specifically, these alterations lead to an imbalance between excitatory and inhibitory synapses in the dorsal horns of the spinal cord where we find the pain pathways, which could be the cause of the phenomenon of hypersensitivity at the base of the pathophysiological process. The origin of this imbalance is still not fully clarified, but with increasing certainty it has emerged how the activation of microglia cells present at the spinal level could play a fundamental role. Neuropathic pain (NP) is the most difficult to treat type of pain; the patient hardly achieves a complete analgesic effect, but only obtains a reduction in symptoms. Therefore, therapy is often unsatisfactory due to both the poor efficacy and side effects of the drugs used. The purpose of this thesis is precisely to find innovative therapies that target factors involved in microglial activation and its shift into pro-inflammatory microglia. In NP, microglia represent a key modulator of the various processes that characterize this chronic and disabling disease, which can be summarized as: neuroinflammation, neurodegeneration, and demyelination. The multiple mechanisms of the innovative treatments studied in this work for reducing microglial activation produced two main effects: reduction of neuroinflammation by promoting the phenotypic shift of microglia, and counteraction of demyelination. Our findings highlight the key role of microglia in the neuronal maintenance and open a broader vision of NP therapy through an approach that can resolve the causes rather than being a simple symptomatic intervention. The targets on which we have mainly focused our attention are modulators of genetic transcription: HDAC1 and HuR, and their silencing with different therapeutical approaches, induced a slow-down of pathology. Innovation is also brought by the type of administration used, the intranasal delivery, which have an effect at the level of the CNS in a non-invasive way compared to more complex and disabling routes such as intrathecal. In fact, the use of non-invasive routes favors an increase in compliance with the patient, which also ensures a higher effectiveness of therapies and increases quality of life. Neuropathies are chronic diseases whose main symptoms are associated with pain, but which involve, given their difficulty in resolving, the onset of long-term comorbidities such as anxiety, depression, insomnia, and loss of cognitive ability. Using a marker of cellular senescence, we evaluated the possible development of microglial aging at the level of the spinal cord (central site of pain) and of the hippocampus (important site of the development of anxiety and depression). Indeed, preventing microglial senescence means reducing the possibility of having an alteration in the homeostasis of the CNS, that leads to aging and deterioration of the cognitive abilities of the person. Another aspect that has emerged rather recently is the possible connection between microglia and all kind of addictive conditions, including those produced by alcohol. Indeed, about 50% of individuals who exhibit alcohol dependence develops the so called “alcoholic neuropathy”. In the spinal cords of alcohol-dependent animals that develop neuropathy, we observed an increase in the expression of microglial markers, thus opening a new avenue in the possible involvement of microglia also in these forms of neuropathy. In conclusion, a stressful correlation between neuroinflammation, demyelination, and degeneration in central and peripheral neuropathies was observed in this thesis. Given that microglia activation is an important crossroads in the onset and regulation of these processes, this work suggest that its role should be more deeply investigated and considered as an important drug target. Indeed, restoring normal physiological conditions in the nervous environment through the modulation of specific microglial targets is an innovative approach to move towards more effective, safe, and personalized therapies.

Neuropathic pain is a debilitating persistent (chronic) pain condition which affects 2% of the total population, characterised by spontaneous pain (stimulus independent), allodynia (pain generated from non-noxious stimuli) and hyperalgesia (heightened sense of pain to noxious stimuli). Unlike other types of pain such as nociceptive or inflammatory, neuropathic pain is maladaptive and therefore neither protects or supports healing or repair. It is defined as “pain caused by a lesion or disease of the somatosensory nervous system” and can develop following an array of aetiologies such as peripheral or central nerve lesions, diabetes, herpes zoster, HIV and cancer, to name a few. However, resolution of the underlying disease and/or healing of the injury often does not alleviate the associated neuropathic pain symptoms suggesting that central maladaptive plasticity may occur in people with neuropathic pain. Compounding this situation, this maladaptive plasticity often renders traditional analgesics used for nociceptive and inflammatory pain ineffective, thus reducing the treatment options available for neuropathic pain sufferers. The spinal mechanisms which lead to persistent pain development have yet to be fully elucidated. It is well understood that adaptations in the reactivity of spinal glial cells (microglia and astrocytes) may also contribute to central neuronal plasticity, by releasing inflammatory mediators such as nitric oxide and other reactive nitrogen species, that enhance excitatory and/or reduce inhibitory neuronal signalling (also referred to as neuro-immune signalling). Previous limitations in methodology have limited our understanding of longitudinal changes in spinal glial during critical developmental stages in persistent pain pathology. Whether there is a correlation between glial reactivity and neuropathic pain severity during the development of the disease model, has yet to be established. Therefore, the initial aim of this thesis was to determine if reactivity characteristics of spinal microglia may correlate with peripheral injury severity and subsequent neuropathic pain symptoms, in mouse models of persistent pain (Chapter 5). Studies suggest that following peripheral injury, there may be alternative reactive nitrogen species, other than nitric oxide, released by highly reactive glial cells which may facilitate neural plasticity within the spinal cord. The recent development of novel fluorescent tools for measuring reactive nitrogen species, such as nitroxyl, have yet to be used to identify the endogenous presence of this reactive nitrogen species in neuropathic pain development. Therefore, the second aim of this thesis was to validate the use of a novel fluorescent probe for the detection of endogenous nitroxyl in mouse models of persistent pain (Chapter 3). The role of nitroxyl in persistent pain development, has been complicated by recent reports whereby exogenous application of high concentration of this reactive nitrogen species, can act as therapeutic agent for persistent pain. The mechanism of action has yet to be fully elucidated, however nitroxyl is highly reactive towards thiols and metalloproteases which have been implicated in various persistent pain pathways. This led to the subsequent aim of this thesis, which was to determine whether the exogenous nitroxyl donor (Angelis’s salt) may reduce allodynia via its ability to cleave active cysteine residues on lysosomal proteasomes and thus reduce their enzyme function (such as Cathepsin B) in persistent pain mouse models (Chapter 4). The studies offered herein demonstrate that: both the onset time post-injury, and level of microglial reactivity is closely correlated with the severity of peripheral injury and subsequent allodynia; endogenous nitroxyl is produced in models of persistent pain (and other diseases) and can be detected in multiple imaging platforms using novel fluorescent probes; and exogenous nitroxyl donor can reduce both Cathepsin B enzyme activity and allodynia, however Cathepsin B inactivation does not directly account for the reduced allodynia and may not be the pathway involved in this phenomenon. Collectively, these results highlight that there is a correlation between microglial reactivity and the severity of injury and subsequent allodynia which may suggest that physicians should consider the severity of the injury when prescribing treatment and at which timepoint post-injury to best intervene. In addition, novel tools developed at the ARC Centre of Excellence for Nanoscale Biophotonics, University of Adelaide, have provided a way to demonstrate that stimuli used in persistent pain models can generate endogenous nitroxyl which can be semi-quantitatively measured. Furthermore, exogenous nitroxyl donors may reduce allodynia via the in-activation of key thiols and metalloproteases which are critical to persistent pain development. With future research, these novel fluorescent probes may be used in vivo to measure the endogenous nitroxyl output in central glial cells in relation to peripheral injury severity. Furthermore, future work exploring the mechanisms by which exogenous nitroxyl is able to reduce allodynia, could provide a safe therapeutic tool for treating symptoms in neuropathic pain patients
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2019

Background: Animal models of sciatic nerve injury are commonly used to study neuropathic pain as well as axon regeneration. Inflammation/immune response at the site of nerve lesion is known to be an essential trigger of the pathological changes that have a critical impact on nerve repair and regeneration, moreover the damage to peripheral nerve can cause a loss of sensory function and produces a persistent neuropathic pain. N-acylethanolamines (NAEs) involve a family of lipid molecules existent in animal and plant, of which N-Palmitoylethanolamide (PEA) that arouses great attention owing to its anti-inflammatory, analgesic and neuroprotective activities. The modulation of specific amidases for NAEs (and in particular NAE-hydrolysing acid amidase NAAA, which is more selective for PEA) could be a condition to preserve its levels. Here we investigated, in a mice model of sciatic nerve crush, the effect of 2-Pentadecyl-2-oxazoline (PEA-OXA) the oxazoline of PEA, that reportedly modulates activity of NAAA. Methods: In this experimental model the mice, following the sciatic nerve crush, were treated daily with PEA-OXA at a dose of 10 mg Kg for 14 days. Therefore, we evaluated the effects of PEA- OXA on the degree of injury, on the inhibition of neuropathic pain, and on the inflammatory process, as in the improvement of reparative processes and therefore in the restoration of locomotor function. Results: Ours results showed that PEA-OXA (10mg/kg) treatment, daily, for 14 days after sciatic nerve crush, have an anti-inflammatory and neuroprotective effect, and moreover have an analgesic protective effect on hypersensitivity, and improve the functional recovery after nerve crush. Conclusions: Therefore, treatment with PEA-OXA as a whole has shown a protective effect, which makes it a powerful candidate for the treatment of peripheral nerve injury and neuropathic pain.

Neuropathic pain is one of the most debilitating disorders. Currently available treatments for neuropathic pain are still unsatisfactory as they have only limited treatment effect and patients may suffer from unwanted side effects. Mechanism-based approaches to neuropathic pain treatment are considered to be more effective. Therefore multiple studies are dedicated to study the pathophysiological mechanisms of neuropathic pain. One of the possible underlying mechanism that causes neuropathic pain is neuroinflammation. Recent studies suggested that angiotensin II ( main effector molecule of the renin-angiotensin system) via its receptors in the central nervous system may be involved in the neuroinflammatory processes. The aim of this study was to investigate the role of angiotensin receptor type 1 in the developement and maintenance of neuropathic pain induced in animal model. Spinal nerve ligation (L5) was used as a model of peripheral neuropathy. Our results showed that treatment with AT1R blocker losartan markedly reduced thermal hyperalgesia and reduced increased sensitivity to mechanical stimuli in the SNL-operated rats.This indicates a possibly significant role of AT1 receptors in the development of neuropathic pain, probably due to reduction of neuroinflammation in the nervous system. These findings...