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The dorsal striatum, apart from controlling voluntary movement, displays a recently demonstrated pain inhibition. It is connected to the descending pain modulatory system and in particular to the rostral ventromedial medulla through the medullary dorsal reticular nucleus. Diseases of the basal ganglia, such as Parkinson's disease, in addition to being characterized by motor disorders, are associated with pain and hyperactivation of the excitatory transmission. A way to counteract glutamatergic hyperactivation is through the activation of group III metabotropic glutamate receptors (mGluRs), which are located on presynaptic terminals inhibiting neurotransmitter release. So far the mGluRs of group III have been the least investigated, owing to a lack of selective tools. More recently, selective ligands for each mGluR of group III, in particular positive and negative allosteric modulators, have been developed and the role of each subtype is starting to emerge. The neuroprotective potential of group III mGluRs in pathological conditions, such as those characterized by elevate glutamate, has been recently shown. In the dorsal striatum, mGluR7 and mGluR8 are located at glutamatergic corticostriatal terminals and their stimulation inhibits pain in pathological conditions such as neuropathic pain. The two receptors in the dorsal striatum have instead a different role in pain control in normal conditions. This review will discuss recent results focusing on the contribution of mGluR7 and mGluR8 in the dorsal striatal control of pain. The role of mGluR4, whose antiparkinsonian activity is widely reported, will also be addressed.

Chronic pain is associated with cognitive deficits. Palmitoylethanolamide (PEA) has been shown to ameliorate pain and pain-related cognitive impairments by restoring glutamatergic synapses functioning in the spared nerve injury (SNI) of the sciatic nerve in mice. SNI reduced mechanical and thermal threshold, spatial memory and LTP at the lateral entorhinal cortex (LEC)-dentate gyrus (DG) pathway. It decreased also postsynaptic density, volume and dendrite arborization of DG and increased the expression of metabotropic glutamate receptor 1 and 7 (mGluR1 and mGluR7), of the GluR1, GluR1s845 and GluR1s831 subunits of AMPA receptor and the levels of glutamate in the DG. The level of the endocannabinoid 2-arachidonoylglycerol (2-AG) was instead increased in the LEC. Chronic treatment with PEA, starting from when neuropathic pain was fully developed, was able to reverse mechanical allodynia and thermal hyperalgesia, memory deficit and LTP in SNI wild type, but not in PPAR alpha null, mice. PEA also restored the level of glutamate and the expression of phosphorylated GluR1 subunits, postsynaptic density and neurogenesis. Altogether, these results suggest that neuropathic pain negatively affects cognitive behavior and related LTP, glutamatergic synapse and synaptogenesis in the DG. In these conditions PEA treatment alleviates pain and cognitive impairment by restoring LIT and synaptic maladaptative changes in the LEC-DG pathway. These outcomes open new perspectives for the use of the N-acylethanolamines, such as PEA, for the treatment of neuropathic pain and its central behavioural sequelae.

Neuropathic pain (NP) remains an untreatable disease due to the complex pathophysiology that involves the whole pain neuraxis including the forebrain. Sensory dysfunctions such as allodynia and hyperalgesia are only part of the symptoms associated with neuropathic pain that extend to memory and affectivity deficits. The development of multi-target molecules might be a promising therapeutic strategy against the symptoms associated with NP. 2-pentadecyl-2-oxazoline (PEA-OXA) is a plant-derived agent, which has shown effectiveness against chronic pain and associated neuropsychiatric disorders. The molecular mechanisms by which PEA-OXA exerts its effects are, however, only partially known. In the current study, we show that PEA-OXA, besides being an alpha2 adrenergic receptor antagonist, also acts as a modulator at histamine H3 receptors, and report data on its effects on sensory, affective and cognitive symptoms associated with the spared nerve injury (SNI) model of neuropathic pain in mice. Treatment for 14 days with PEA-OXA after the onset of the symptoms associated with neuropathic pain resulted in the following effects: (i) allodynia was decreased; (ii) affective/cognitive impairment associated with SNI (depression, spatial, and working memories) was counteracted; (iii) long-term potentiation in vivo in the lateral entorhinal cortex-dentate gyrus (perforant pathway, LPP) was ameliorated, (iv) hippocampal glutamate, GABA, histamine, norepinephrine and dopamine altered levels after peripheral nerve injury were reversed, (v) expression level of the TH positive neurons in the Locus Coeruleus were normalized. Thus, a 16-day treatment with PEA-OXA alleviates the sensory, emotional, cognitive, electrophysiological and neurochemical alterations associated with SNI-induced neuropathic pain.

Recent evidence points to the gut microbiota as a regulator of brain and behavior, although it remains to be determined if gut bacteria play a role in chronic pain. The endocannabinoid system is implicated in in- flammation and chronic pain processing at both the gut and central nervous system (CNS) levels. In the present study, we used low Vitamin D dietary intake in mice and evaluated possible changes in gut microbiota, pain processing and endocannabinoid system signaling. Vitamin D deficiency induced a lower microbial diversity characterized by an increase in Firmicutes and a decrease in Verrucomicrobia and Bacteroidetes. Concurrently, vitamin D deficient mice showed tactile allodynia associated with neuronal hyperexcitability and alterations of endocannabinoid system members (endogenous mediators and their receptors) at the spinal cord level. Changes in endocannabinoid (anandamide and 2-ara- chidonoylglycerol) levels were also observed in the duodenum and colon. Remarkably, the anti-inflammatory anandamide congener, palmitoylethanolamide, counteracted both the pain behaviour and spinal biochemical changes in vitamin D deficient mice, whilst increasing the levels of Akkermansia, Eubacterium and Enterobacteriaceae, as compared with vehicle-treated mice. Finally, induction of spared nerve injury in normal or vitamin D deficient mice was not accompanied by changes in gut microbiota composition. Our data suggest the existence of a link between Vitamin D deficiency – with related changes in gut bacterial composition – and altered nociception, possibly via molecular mechanisms involving the endocannabinoid and related mediator signaling systems.

Traumatic brain injury (TBI) represents an important public health problem and is followed by neuroinflammation and neurological dysfunctions. It has been suggested that brain trauma is often associated to deep behavioral alterations and chronic pain-like syndrome. Despite inducing minimal brain damage, mild TBI (mTBI) leads to persistent behavioral changes, including anxiety, depression, social interaction impairment, and aggressiveness. The clinical management of these symptoms is still unsatisfactory and new pharmacological treatments are needed, especially for the aggressiveness and depression. In a mouse model of mTBI, we investigated the effect of 2-Pentadecyl-2-Oxazoline (PEA-OXA), a natural compound, that is a secondary metabolite, found in green and roasted coffee beans, on both the pain perception, and neuropsychiatric dysfunctions. We found that the compound acts as a α2 adrenergic antagonist and this mechanism is here described for the first time. Mild TBI mice, starting from 14-d post-trauma, developed anxious and aggressive behavior, whilst depressive-like behavior and impaired social interactions were observed from the 60th d onward. PEA-OXA normalized all the behavioral changes investigated. We also investigated the memory impairments through Morris Water Maze (MWM) test. Both sham and mTBI mice treated with PEA-OXA showed amelioration in the reversal task of the MWM. Nevertheless, the main symptom of the long-term mTBI is represented by the depressive-like behavior, which was completely reversed by PEA-OXA repeated administration. In humans, mTBI-induced depression precedes the appearance of dementias and is characterized by a massive deficit of GABAergic transmission in the cortices. We found that PEA-OXA normalized the GABA changes in the prefrontal cortex. In order to prove the α2-mediated effect of the PEA-OXA we have performed open field test in naïve animals by microinjecting into the medial prefrontal cortex the dexomedetomidine, a selective α2 agonist with or without PEA-OXA co-injection. We found that PEA-OXA antagonized the α2 agonist effect on the locomotor activity. Moreover, PEA-OXA microinjection into the medial prefrontal cortex induced an enhancement of dopamine release. Collectively, these data suggest that this natural compound, through its multi-target activity is able to: i) ameliorate behavioral alterations (i.e. depression), ii) selectively normalize cortical GABA levels, iii) rescue the impaired neuronal activity in the prefrontal cortex.

The study investigated the role of the metabotropic glutamate receptor subtype 7 (mGluR7) in pain signalling in the dorsal striatum of sham and neuropathic rats. Supraspinal circuitries involved in the dorsal striatum control of pain were also explored. In the sham rats, microinjection of N,N'-bis(diphenylmethyl)-1,2-ethanediamine (AMN082), a selective mGluR7 positive allosteric modulator, into the dorsal striatum, facilitated pain, increased the activity of the ON cells and inhibited the activity of the OFF cells in the rostral ventromedial medulla, and decreased glutamate levels in the dorsal striatum. Conversely, AMN082 inhibited pain and the activity of the ON cells while increased the activity of the OFF cells in rats with spared nerve injury (SNI) of the sciatic nerve. AMN082 also decreased glutamate levels in the dorsal striatum of SNI rats. The effect of AMN082 on mechanical allodynia and glutamate release was blocked by 6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydro-4(5H)-benzoxazolone (ADX71743), a selective mGluR7 negative allosteric modulator. Moreover, in the sham rats, AMN082 increased the activity of total nociceptive convergent neurons in the dorsal reticular nucleus while in the SNI rats, such activity was decreased. The administration of lidocaine into the subthalamic nucleus abolished the effect of AMN082 on the total nociceptive convergent neurons in the sham rats but not in the SNI rats. Thus, the dual effect of mGluR7 in facilitating or inhibiting pain responses may be due to the recruitment of different pathways of the basal ganglia, the indirect or direct pathway, in physiological or pathological conditions, respectively.

// Flavio Fazioli 1, * , Gianluca Colella 2, * , Roberta Miceli 3 , Mariano Giuseppe Di Salvatore 1 , Michele Gallo 1 , Serena Boccella 4 , Annarosaria De Chiara 5 , Carlo Ruosi 2, * and Filomena de Nigris 6, * 1 Division of Musculoskeletal Oncology Surgery, National Cancer Institute G. Pascale, Naples, Italy 2 Department of Human Health, Federico II University of Naples, Naples, Italy 3 S.C. Cellular Biology and Biotherapy, National Cancer Institute G. Pascale, Naples, Italy 4 Department of Experimental Medicine, University of Campania “Luigi Vanvitelli”, Naples Italy 5 Division of Pathology, National Cancer Institute G. Pascale Foundation, Naples, Italy 6 Department of Biochemistry, Biophysics and General Pathology, University of Campania “Luigi Vanvitelli”, Naples Italy * These authors have contributed equally to this work Correspondence to: Filomena de Nigris, email: Filomena.denigris@unina2.it Keywords: recurrences, sarcoma, angiogenesis, stem cells, AKT Received: March 31, 2017 Accepted: May 29, 2017 Published: June 28, 2017 ABSTRACT Giant cell tumors of bone (GCTB) are rare sarcomas with a high rate of unpredictable local relapse. Studies suggest that surgical methods affect recurrence, supporting the idea that local disease develops from re-growth of residual cancer cells. To identify early prognostic markers of individual risk of recurrence, we evaluated the effect of post-surgery fluids from a cohort of GCTB patients on growth of primary and established sarcoma cell lines, and mice xenograph. Post-surgery fluids increased cell growth and enhanced expression of CD44 ++ , the principal receptor for the extracellular matrix component hyaluronan and the mesenchymal stem marker CD117 + . Cancer cells became highly invasive and tumorigenic, acquiring stemness properties, and activated AKT/mTOR pathway. Prolonged stimulation with post-surgery fluids down-regulated the mesenchymal gene TWIST1 and Vimentin protein, and transdifferentiated cells into tubule-like structures positive to the endothelial markers VE-Cadherin and CD31 + . In mice, post-surgery fluids gave rise to larger and more vascularized tumors than control, while in patients AKT/mTOR pathway activation was associated with recurrence by logistic regression (Kaplan-Meier; P

Inflammation plays a key role in the pathogenesis of several chronic diseases. The urokinase plasminogen activator receptor (uPAR) exerts a plethora of functions in both physiological and pathological processes, including inflammation. In this study, we evaluated the anti-inflammatory effect of a novel peptide ligand of uPAR, UPARANT, in different animal models of inflammation. Rats and mice were divided in different groups (n = 5) for single or repeated administration of vehicle (9% DMSO in 0.9% NaCl), UPARANT (6, 12 and 24 mg/kg) or dexamethasone (2 mg/kg). Animals were subjected to carrageenan-induced paw oedema or zymosan-induced peritonitis. UPARANT effects were tested on: (1) the carrageenan-induced paw oedema volume, (2) the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the nitrite/nitrate (NOx) levels in the paw exudates, (3) cells recruitment into the peritoneal cavity after zymosan injection and (4) NOx levels in the peritoneal lavage. UPARANT (12 and 24 mg/kg) reduced inflammation in both experimental paradigms. Analysis of pro-inflammatory enzymes revealed that administration of UPARANT reduced iNOS, COX2 and NO over-production. Our study provides a solid evidence that UPARANT reduces the severity of inflammation in diverse animal models, thus representing a novel anti-inflammatory drug with potential advantages with respect to the typical steroidal agents.

It is a common opinion that metabotropic glutamate receptor subtype 6 (mGluR6) is expressed exclusively in the retina, and in particular in the dendrites of ON-bipolar cells. Glutamate released in darkness from photoreceptors activates mGluR6, which is negatively associated with a membrane non-selective cation channel, the transient receptor potential melanoma-related 1, TRPM1, resulting in cell hyperpolarization. The evidence that mGluR6 is expressed not only in the retina but also in other tissues and cell populations has accumulated over time. The expression of mGluR6 has been identified in microglia, bone marrow stromal and prostate cancer cells, B lymphocytes, melanocytes and keratinocytes and non-neural tissues such as testis, kidney, cornea, conjunctiva, and eyelid. The receptor also appears to be expressed in brain areas, such as the hypothalamus, cortex, hippocampus, nucleus of tractus solitarius, superior colliculus, axons of the corpus callosum and accessory olfactory bulb. The pharmacological activation of mGluR6 in the hippocampus produced an anxiolytic-like effect and in the periaqueductal gray analgesic potential. This review aims to collect all the evidence on the expression and functioning of mGluR6 outside the retina that has been accumulated over the years for a broader view of the potential of the receptor whose retinal confinement appears understimated.

Neurological dysfunctions are the most impactful and persistent consequences of traumatic brain injury (TBI). Indeed, previous reports suggest that an association between TBI and chronic pain syndromes, as well anxio-depressive behaviors, tends to be more common in patients with mild forms of TBI. At present, no effective treatment options are available for these symptoms. In the present study, we used a weight drop mild TBI mouse model to investigate the effect of a commercially available 10% Cannabidiol (CBD) oil on both the sensorial and neuropsychiatric dysfunctions associated with mild TBI through behavioral and biomolecular approaches. TBI mice developed chronic pain associated with anxious and aggressive behavior, followed by a late depressive-like behavior and impaired social interaction. Such behaviors were related with specific changes in neurotransmitters release at cortical levels. CBD oral treatment restored the behavioral alterations and partially normalized the cortical biochemical changes. In conclusion, our data show some of the brain modifications probably responsible for the behavioral phenotype associated with TBI and suggest the CBD as a pharmacological tool to improve neurological dysfunctions caused by the trauma.

Non-small-cell lung cancer (NSCLC) accounts for 85%–90% of all cases of lung cancer that is the most deadly type of cancer. Despite advances in chemotherapy and radiotherapy, severe side effects and frequent drug resistance limit the success of the treatments, and the identification of new therapeutic options still represents a crucial challenge. Here, we provide the evidence for the therapeutic potential of an aptamer-microRNA (miR) complex (AmiC) composed by an aptamer (GL21.T), able to bind and antagonize the oncogenic receptor Axl, and the miR-137, downregulated in lung cancer and involved in cell survival and proliferation. We found that, when applied to Axl-expressing NSCLC cancer cells, the complex is effectively internalized, increasing miR cellular levels and downregulating miR targets. Most importantly, the complex combines the inhibitory function of the GL21.T aptamer and miR-137, leading to a negative impact on NSCLC migration and growth. The described AmiC thus represents a promising tool for the development of new therapeutic approaches for NSCLC. Keywords: aptamers, miRNAs, NSCLC, targeted delivery

ObjectiveThe role of the hydroxycarboxylic acid receptor 2 (HCA2) in the retinal damage induced by diabetes has never been explored. In this context, the present study highlights an upregulation of retinal HCA2 receptors in diabetic C57BL6J mice. Moreover, we illustrate that HCA2 receptors exert an anti-inflammatory effect on the retinal damage induced by diabetes when activated by the endogenous ligand β-hydroxybutyrate.MethodologySeven-to-10-week-old C57BL6J mice were rendered diabetic by a single intraperitoneal injection of streptozotocin (75 mg/kg of body weight) and monitored intermittently over a 10-week period extending from the initial diabetes assessment. Mice with a fasting blood glucose level higher than 250 mg/dl for 2 consecutive weeks after streptozotocin injection were treated twice a week with intraperitoneal injections of 25-50-100 mg/kg β-hydroxybutyrate.ResultsInterestingly, while the retinal endoplasmic reticulum stress markers (pPERK, pIRE1, ATF-6α) were elevated in diabetic C57BL6J mice, their levels were significantly reduced by the systemic intraperitoneal treatment with 50 mg/kg and 100 mg/kg β-hydroxybutyrate. These mice also exhibited high NLRP3 inflammasome activity and proinflammatory cytokine levels. In fact, the elevated levels of retinal NLRP3 inflammasome activation markers (NLRP3, ASC, caspase-1) and of the relative proinflammatory cytokines (IL-1β, IL-18) were significantly reduced by 50 mg/kg and 100 mg/kg β-hydroxybutyrate treatment. These doses also reduced the high apoptotic cell number exhibited by the diabetic mice in the retinal outer nuclear layer (ONL) and increased the ONL low connexin 43 expression, leading to an improvement in retinal permeability and homeostasis.ConclusionsThese data suggest that the systemic treatment of diabetic C57BL6J mice with BHB activates retinal HCA2 and inhibits local damage.

This study investigated whether metabotropic glutamate receptor (mGluR) 5 and 8 are involved in the effect of ultramicronizedpalmitoylethanolamide (um-PEA) on the cognitive behavior and long term potentiation (LTP) at entorhinal cortex (LEC)-dentate gyrus (DG) pathway in mice rendered neuropathic by the spare nerve injury (SNI). SNI reduced discriminative memory and LTP. Um-PEA treatment started after the development of neuropathic pain had no effects in sham mice, whereas it restored cognitive behavior and LTP in SNI mice. 2-Methyl-6-(phenylethynyl) pyridine (MPEP), a selective mGluR5 antagonist, improved cognition in SNI mice and produced a chemical long term depression of the field excitatory postsynaptic potentials (fEPSPs) in sham and SNI mice. After theta burst stimulation (TBS) MPEP restored LTP in SNI mice. In combination with PEA, MPEP antagonized the PEA effect on discriminative memory and decreased LTP in SNI mice. The (RS)-4-(1-amino-1-carboxyethyl)phthalic acid (MDCPG), a selective mGluR8 antagonist, did not affect discriminative memory, but it induced a chemical LTP and prevented the enhancement of fEPSPs after TBS in SNI mice which were treated or not treated with PEA. The effect of PEA on LTP and cognitive behavior was modulated by mGluR5 and mGluR8. In particular in the SNI conditions, the mGluR5 blockade facilitated memory and LTP, but prevented the beneficial effects of PEA on discriminative memory while the mGluR8 blockade, which was ineffective in itself, prevented the favorable action of the PEA on LTP. Thus, although their opposite roles (excitatory/inhibitory of the two receptor subtypes on the glutamatergic system), they appeared to be required for the neuroprotective effect of PEA in conditions of neuropathic pain.

The mechanisms underlying neuropathic pain are poorly understood. Here we show the unexplored role of the hydroxyl carboxylic acid receptor type 2 (HCAR2) in 2 models of neuropathic pain. We used an oral treatment with dimethyl fumarate and the HCAR2 endogenous ligand β-hydroxybutyrate (BHB) in wild-type (WT) and HCAR2-null mice. We found an up-regulation of the HCAR2 in the sciatic nerve and the dorsal root ganglia in neuropathic mice. Accordingly, acute and chronic treatment with dimethylfumarate (DMF) and BHB reduced the tactile allodynia. This effect was completely lost in the HCAR2-null mice after a 2-d starvation protocol, in which the BHB reached the concentration able to activate the HCAR2-reduced tactile allodynia in female WT mice, but not in the HCAR2-null mice. Finally, we showed that chronic treatment with DMF reduced the firing of the ON cells (cells responding with an excitation after noxious stimulation) of the rostral ventromedial medulla. Our results pave the way for investigating the mechanisms by which HCAR2 regulates neuropathic pain plasticity.-Boccella, S., Guida, F., De Logu, F., De Gregorio, D., Mazzitelli, M., Belardo, C., Iannotta, M., Serra, N., Nassini, R., de Novellis, V., Geppetti, P., Maione, S., Luongo, L. Ketones and pain: unexplored role of hydroxyl carboxylic acid receptor type 2 in the pathophysiology of neuropathic pain.

In breast cancer, Lysine-specific demethylase-1 (LSD1) and other lysine demethylases (KDMs), such as Lysine-specific demethylase 6A also known as Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), are co-expressed and co-localize with estrogen receptors (ERs), suggesting the potential use of hybrid (epi)molecules to target histone methylation and therefore regulate/redirect hormone receptor signaling. Here, we report on the biological activity of a dual-KDM inhibitor (MC3324), obtained by coupling the chemical properties of tranylcypromine, a known LSD1 inhibitor, with the 2OG competitive moiety developed for JmjC inhibition. MC3324 displays unique features not exhibited by the single moieties and well-characterized mono-pharmacological inhibitors. Inhibiting LSD1 and UTX, MC3324 induces significant growth arrest and apoptosis in hormone-responsive breast cancer model accompanied by a robust increase in H3K4me2 and H3K27me3. MC3324 down-regulates ER&alpha, in breast cancer at both transcriptional and non-transcriptional levels, mimicking the action of a selective endocrine receptor disruptor. MC3324 alters the histone methylation of ER&alpha, regulated promoters, thereby affecting the transcription of genes involved in cell surveillance, hormone response, and death. MC3324 reduces cell proliferation in ex vivo breast cancers, as well as in breast models with acquired resistance to endocrine therapies. Similarly, MC3324 displays tumor-selective potential in vivo, in both xenograft mice and chicken embryo models, with no toxicity and good oral efficacy. This epigenetic multi-target approach is effective and may overcome potential mechanism(s) of resistance in breast cancer.

This study sought to determine the possible detrimental effects of several low- or non-caloric sweeteners on endothelial progenitor cells (EPCs), inflammation and behavioural changes in mice. C57BL/6 male mice received low and high dose of natural and artificial sweeteners for 4 weeks. EPCs, physical and biochemical variables, inflammation and behavioural changes were evaluated. A significant reduction of about 25% of EPCs was found when mice received a moderate amount of all sweeteners (p p p p

The cellular microenvironment plays a critical role in the maintenance of bone marrow-derived mesenchymal stem cells (BM-MSCs) and their subsequent cell lineage differentiation. Recent studies suggested that individuals with adipocyte-related metabolic disorders have altered function and adipogenic potential of adipose stem cell subpopulations, primarily BM-MSCs, increasing the risk of heart attack, stroke or diabetes. In this study, we explored the potential therapeutic effect of some of the most abundant non-euphoric compounds derived from the Cannabis sativa plant (or phytocannabinoids) including tetrahydrocannabivarin (THCV), cannabidiol (CBD), cannabigerol (CBG), cannabidiolic acid (CBDA) and cannabigerolic acid (CBGA), by analysing their pharmacological activity on viability of endogenous BM-MSCs as well as their ability to alter BM-MSC proliferation and differentiation into mature adipocytes. We provide evidence that CBD, CBDA, CBGA and THCV (5 µM) increase the number of viable BM-MSCs; whereas only CBG (5 µM) and CBD (5 µM) alone or in combination promote BM-MSCs maturation into adipocytes via distinct molecular mechanisms. These effects were revealed both in vitro and in vivo. In addition, phytocannabinoids prevented the insulin signalling impairment induced by palmitate in adipocytes differentiated from BM-MSCs. Our study highlights phytocannabinoids as a potential novel pharmacological tool to regain control of functional adipose tissue in unregulated energy homeostasis often occurring in metabolic disorders including type 2 diabetes mellitus (T2DM), aging and lipodystrophy.

The chronic constriction injury (CCI) of the sciatic nerve is a nerve injury-based model of neuropathic pain (NP). Comorbidities of NP such as depression, anxiety, and cognitive deficits are associated with a functional reorganization of the medial prefrontal cortex (mPFC). Here, we have employed an adapted model of CCI by placing one single loose ligature around the sciatic nerve in mice for investigating the alterations in sensory, motor, affective, and cognitive behavior and in electrophysiological and biochemical properties in the prelimbic division (PrL) of the mPFC. Our adapted model of CCI induced mechanical allodynia, motor, and cognitive impairments and anxiety- and depression-like behavior. In the PrL division of mPFC was observed an increase in GABA and a decrease in d-aspartate levels. Moreover an increase in the activity of neurons responding to mechanical stimulation with an excitation, mPFC (+), and a decrease in those responding with an inhibition, mPFC (-), was found. Altogether these findings demonstrate that a single ligature around the sciatic nerve was able to induce sensory, affective, cognitive, biochemical, and functional alterations already observed in other neuropathic pain models and it may be an appropriate and easily reproducible model for studying neuropathic pain mechanisms and treatments.

The microbiota-gut-brain axis (MGBA) regulates the reciprocal interaction between chronic inflammatory bowel and psychiatric disorders. This interaction involves multiple pathways that are highly debated. We examined the behavioural, biochemical and electrophysiological alterations, as well as gut microbiota composition in a model of antibiotic-induced experimental dysbiosis. Inflammation of the small intestine was also assessed. Mice were exposed to a mixture of antimicrobials for 2 weeks. Afterwards, they received Lactobacillus casei DG (LCDG) or a vehicle for up to 7 days via oral gavage. Perturbation of microbiota was accompanied by a general inflammatory state and alteration of some endocannabinoidome members in the gut. Behavioural changes, including increased immobility in the tail suspension test and reduced social recognition were observed, and were associated with altered BDNF/TrkB signalling, TRPV1 phosphorylation and neuronal firing in the hippocampus. Moreover, morphological rearrangements of non-neuronal cells in brain areas controlling emotional behaviour were detected. Subsequent probiotic administration, compared with vehicle, counteracted most of these gut inflammatory, behavioural, biochemical and functional alterations. Interestingly, levels of Lachnospiraceae were found to significantly correlate with the behavioural changes observed in dysbiotic mice. Our findings clarify some of the biomolecular and functional modifications leading to the development of affective disorders associated with gut microbiota alterations.

Our previous work discovered that combining the appropriate 5′- and N6-substitution in adenosine derivatives leads to the highly selective human A1 adenosine receptor (hA1AR) agonists or highly potent dual hA1AR agonists and hA3AR antagonists. In order to explore novel dual adenosine receptor ligands, a series of N6-substituted-5′-pyrazolyl-adenosine and 2-chloro-adenosine derivatives were synthesized and assayed in vitro at all ARs. The N6-(±)-endo-norbornyl derivative 12 was the most potent and selective at A1AR and effective as an analgesic in formalin test in mice, but none of the 5′-pyrazolyl series compounds showed a dual behavior at hA1 and hA3AR. Molecular modeling studies rationalized the structure–activity relationships and the selectivity profiles of the new series of A1AR agonists. Interestingly, an unexpected inverted binding mode of the N6-tetrahydrofuranyl derivative 14 was hypothesized to explain its low affinity at A1AR.

This chapter describes surgical procedures for the induction of neuropathic pain using an animal model (rat or mouse) of spared nerve injury. In addition to technical details of the surgical technique, details of anesthesia and perioperative care are also included.

The endogenous fatty acid amide palmitoylethanolamide (PEA) has been shown to exert anti-inflammatory actions mainly through inhibition of the release of pro-inflammatory molecules from mast cells, monocytes and macrophages. Indirect activation of the endocannabinoid (eCB) system is among the several mechanisms of action that have been proposed to underlie the different effects of PEA in vivo. In this study, we used cultured rat microglia and human macrophages to evaluate whether PEA affects eCB signaling. PEA was found to increase CB2 mRNA and protein expression through peroxisome proliferator-activated receptor-α (PPAR-α) activation. This novel gene regulation mechanism was demonstrated through: (i) pharmacological PPAR-α manipulation, (ii) PPAR-α mRNA silencing, (iii) chromatin immunoprecipitation. Moreover, exposure to PEA induced morphological changes associated with a reactive microglial phenotype, including increased phagocytosis and migratory activity. Our findings suggest indirect regulation of microglial CB2R expression as a new possible mechanism underlying the effects of PEA. PEA can be explored as a useful tool for preventing/treating the symptoms associated with neuroinflammation in CNS disorders.

Depressive symptoms and other neuropsychiatric dysfunctions are common in neurodegenerative disorders, including chronic pain and dementia. A correlation between the β-amyloid protein accumulation and the development of depression has been suggested, however the underlying mechanisms are unknown. d-Aspartate (d-Asp) is a free d-amino acid found in the mammalian brain and involved in neurological and psychiatric processes, such as cognition and affective disorders. In this study we have investigated the effects of a repeated treatment with d-Asp in a long-lasting (12 months) model of neuropathic pain, the spared nerve injury (SNI), in mice. Specifically, we evaluated i) the pain sensitivity and related emotional/cognitive dysfunctions induced by SNI, ii) possible changes in the β-amyloid protein accumulation in specific brain regions involved in pain mechanisms ii) possible changes in steroids level in neuropathic animals with or without d-Asp in the same brain areas. SNI mice showed an increase of the insoluble form of Aβ1-42 at hippocampal level and displayed cognitive impairments, stereotypical and depressive-like behaviours. d-Asp treatment reduced abnormal behaviours and normalized the β-amyloid protein expression. Moreover, d-Asp dramatically increased steroids level measured in the prefrontal cortex and in the hippocampus. Our findings provide new insights into pain mechanisms and suggest a possible role of β-amyloid protein in neuropsychiatric dysfunctions associated with chronic pain.

Traumatic brain injury (TBI) represents a major public health problem, which is associated with neurological dysfunction. In severe or moderate cases of TBI, in addition to its high mortality rate, subjects may encounter diverse behavioural dysfunctions. Previous reports suggest that an association between TBI and chronic pain syndromes tends to be more common in patients with mild forms of brain injury. Despite causing minimal brain damage, mild TBI (mTBI) often leads to persistent psychologically debilitating symptoms, which can include anxiety, various forms of memory and learning deficits, and depression. At present, no effective treatment options are available for these symptoms, and little is known about the complex cellular activity affecting neuronal activity that occurs in response to TBI during its late phase. Here, we used a mouse model to investigate the effect of Palmitoylethanolamide (PEA) on both the sensorial and neuropsychiatric dysfunctions associated with mTBI through behavioural, electrophysiological, and biomolecular approaches. Fourteen-day mTBI mice developed anxious, aggressive, and reckless behaviour, whilst depressive-like behaviour and impaired social interactions were observed from the 60th day onwards. Altered behaviour was associated with changes in interleukin 1 beta (IL-1β) expression levels and neuronal firing activity in the medial prefrontal cortex (m-PFC). Compared with vehicle, PEA restored the behavioural phenotype and partially normalised the biochemical and functional changes occurring at the supraspinal level. In conclusion, our findings reveal some of the supraspinal modifications responsible for the behavioural alterations associated with mTBI and suggest PEA as a pharmacological tool to ameliorate neurological dysfunction induced by the trauma.

Structural determinants of affinity of N(6)-substituted-5'-C-(ethyltetrazol-2-yl)adenosine and 2-chloroadenosine derivatives at adenosine receptor (AR) subtypes were studied with binding and molecular modeling. Small N(6)-cycloalkyl and 3-halobenzyl groups furnished potent dual acting A1AR agonists and A3AR antagonists. 4 was the most potent dual acting human (h) A1AR agonist (Ki = 0.45 nM) and A3AR antagonist (Ki = 0.31 nM) and highly selective versus A2A; 11 and 26 were most potent at both h and rat (r) A3AR. All N(6)-substituted-5'-C-(ethyltetrazol-2-yl)adenosine derivatives proved to be antagonists at hA3AR but agonists at the rA3AR. Analgesia of 11, 22, and 26 was evaluated in the mouse formalin test (A3AR antagonist blocked and A3AR agonist strongly potentiated). N(6)-Methyl-5'-C-(ethyltetrazol-2-yl)adenosine (22) was most potent, inhibiting both phases, as observed combining A1AR and A3AR agonists. This study demonstrated for the first time the advantages of a single molecule activating two AR pathways both leading to benefit in this acute pain model.

Pain perception can become altered in individuals with eating disorders and obesity for reasons that have not been fully elucidated. We show that leptin deficiency in ob/ob mice, or leptin insensitivity in the arcuate nucleus of the hypothalamus in mice with high-fat diet (HFD)-induced obesity, are accompanied by elevated orexin-A (OX-A) levels and orexin receptor-1 (OX1-R)-dependent elevation of the levels of the endocannabinoid, 2-arachidonoylglycerol (2-AG), in the ventrolateral periaqueductal gray (vlPAG). In ob/ob mice, these alterations result in the following: (i) increased excitability of OX1-R-expressing vlPAG output neurons and subsequent increased OFF and decreased ON cell activity in the rostral ventromedial medulla, as assessed by patch clamp and in vivo electrophysiology; and (ii) analgesia, in both healthy and neuropathic mice. In HFD mice, instead, analgesia is only unmasked following leptin receptor antagonism. We propose that OX-A/endocannabinoid cross talk in the descending antinociceptive pathway might partly underlie increased pain thresholds in conditions associated with impaired leptin signaling.Neuropsychopharmacology advance online publication, 15 July 2015; doi:10.1038/npp.2015.173.

The role of neuroinflammatory cells in the establishment of neuropathic pain has been investigated in depth in the last few years. In particular, microglia have been shown to be key players in the induction of tactile allodynia, as they release proinflammatory molecules that, in turn, sensitize nociceptive neurons within the spinal cord. However, the role of peripheral immune cells such as macrophages, infiltrating monocytes, mast cells, and T-cells has been highlighted in the last few studies, even though the data are still conflicting and need to be clarified. Intriguingly, the central (microglia) and peripheral (T-cell)-adaptive immune cells that orchestrate maladaptive process-driven neuropathic pain seem to be involved in a gender-dependent manner. In this review, we highlight the role of the microglia and peripheral immune cells in chronic degenerative disease associated with neuro-immune-inflammatory processes.