1,721,154 research outputs found
Nucleotide receptors in trigeminal satellite glial cells as new targets for the pharmacological control of migraine pain: in vitro and in vivo studies
No full textBackground and Purpose - The main aim of my PhD research project is to study pain transduction mechanisms in trigeminal ganglia (TG) in migraine, to understand why sensory trigeminal neurons become hyperactive in this pathological status, and how their activity can be pharmacologically modulated. We focused our studies on the role of the purinergic system in the neuron-to-glial cells communication within the TG, and its cross-talk with known pro-algogenic systems (such as bradykinin, BK, calcitonin gene-related peptide, CGRP, and prostaglandins). The final goal is the identification of new cellular and molecular players in the onset and maintenance of trigeminal-associated pain, for the development of new effective therapeutic strategies for migraine.
Methods and Results – For the in vitro studies, we set up primary mixed neuron-glia or purified satellite glial cell (SGCs) cultures from theTG of C57BL6 mice. G protein-coupled P2Y receptor function was evaluated by single cell calcium imaging, and the extracellular concentrations of CGRP and PGE2 were measured by ELISA assays. Western blot experiments on P2Y1 and P2Y2 receptor subtypes were also performed. Concerning the role of metabotropic purinergic receptors, our data show that exposure of mixed-neuron glia cultures to BK induced the neuronal release of CGRP, which in turn significantly potentiated the ADP-responsive P2Y1 and the UTP-sensitive P2Y2 receptor subtypes on surrounding SGCs. The increased activity of P2 receptors was related to increased receptor protein expression. Interestingly, the anti-migraine drug sumatriptan fully inhibited both CGRP release and glial P2Y-receptor potentiation. Moreover, exposure to BK led to increased production of PGE2, an effect completely abolished by the COX-1 inhibitor acetylsalicylic acid (ASA), whichalso blocked neuronal CGRP release. Taken together, these results suggest that a complex cross-talk between neuronal and glial cells takes place in the TG, involving pain mediators and extracellular nucleotides. Modulation of this network by known anti-migraine drugs, such as triptans and COX inhibitors, suggests that it might play an important role in the development of migraine pain.
In vivo studies were aimed at evaluating the pro- or anti-algogenic role of P2Y receptors through their selective inhibition. To this purpose, we set up a sub-chronic model of inflammatory trigeminal hypersensitivity, by injecting complete Freund adjuvant (CFA) into the temporomandibular joint (TMJ) of rats. CFA-injected animals showed ipsilateral mechanical allodynia and TMJ edema. Glial cell activation was evaluated in the spinal-trigeminal system by immunohistochemistry and western blotting analysis of GFAP protein expression, a typical marker of activated SGCs. A significant glial activation within the TG was observed starting from 24 hours up to 11 days after CFA injection, thus confirming that our model leads to TG sensitization. Moreover, upregulation of P2Y1 and P2Y2 protein expression was also observed, thus corroborating our in vitro data. Interestingly, the non-selective P2Y antagonist PPADS showed a strong analgesic effect on CFA-induced TG inflammation, which is comparable to ASA-mediated analgesia.
Conclusions - These results suggest a possible the pro-algogenic role for P2Y receptors in the development of trigeminal sensitization and migraine pain, opening the future perspective of identifying innovative and more selective pharmacological approaches for the sake of those migraineurs who are insensitive to currently available drugs.
References
[1] G. Magni, S. Ceruti. P2Y purinergic receptors: new targets for analgesic and antimigraine drugs. Biochem Pharmacol, 85(4):466-77, 2013.
[2] S. Ceruti, G. Villa, M. Fumagalli, L. Colombo, G. Magni, M. Zanardelli, E. Fabbretti, C. Verderio, A. M. J. M. van den Maagdenberg, A. Nistri, M. P. Abbracchio. CGRP−mediated enhancement of purinergic neuron/glia communication by the algogenic factor bradykinin in mouse trigeminal ganglia from wild type and R192Q Cav2.1 knock-in mice: implications for basic mechanisms of migraine pain. J Neuroscience, 31(10):3638 –3649, 2011
[3] G. Villa, S. Ceruti, M. Zanardelli, G. Magni, L. Jasmin, P. T. Ohara, M. P. Abbracchio.. Temporomandibular join inflammation activates glial and immune cells in both the trigeminal ganglia and the spinal trigeminal nucleus. Mol Pain, 6:89, 2010
BBB and the purinergic system
No full textThe blood-brain barrier (BBB) is composed of endothelial cells, pericytes and astrocytes [1], and serves as interface between the blood flow and the central nervous system (CNS). Since BBB deregulation plays important roles in the pathogenesis of several CNS diseases spanning from brain tumors to stroke, understanding the molecular mechanisms controlling BBB functions might help to unveil new therapeutic targets to brain pathologies. Extracellular nucleotides are important signaling molecules both in physiological and pathological conditions. Their actions are mediated by 7 ionotropic P2X and 8 metabotropic P2Y purinergic receptors, and terminated by metabolizing enzymes, named ectonucleotidases (NTPDases) and 5’-nucleotidase [2]. To date, the role of purinergic transmission in controlling BBB functions is not fully understood yet. Therefore, we used a new in vitro cell culture model of BBB [3] to investigate the expression and distribution of NTPDases and P2Y receptors either in control conditions or following exposure to ischemia. Semi quantitative RT-PCR analysis showed that astrocytes and pericytes expressed all the cloned P2Y receptors, however endothelial cells showed the presence only the P2Y1,2,4 subtypes. NTPDase1 and 2 enzymes were expressed at different level but by all the three types of cell. Application of oxygen-glucose deprivation (OGD), which mimics cytotoxicity induced by ischemia in vivo, showed extremely high susceptibility to cell death of endothelial cells, whereas astrocytes and pericytes were more resistant. Semi-quantitative densitometry assay highlighted increased ecto-ATPase activity following exposure to OGD in the three types of cell population, either they were grown separately or in triple co-culture. Our data show the usefulness of this new in vitro model to demonstrate a role for extracellular nucleotides in modulating BBB responses to ischemic events, and to determine if the purinergic system could represent a new target for the development of effective therapies to brain pathologies.
Figure 1: Table of contents. (A) ATP hydrolyzing enzymes and some of the pharmacological targets of ATP metabolites. (B) schematic representation of the blood brain barrier in vivo and of its reproduction in vitro. (C) experimental plan.
Figure 2: Schematic drawing of the BBB model and its electron microscopic picture. (A) Endothelial cells (green), pericytes (brown) and astrocytes (blue) as they are located in the well of Transwell cell culture plate. (B) Electron micrograph: single layer of endothelial cells on the top and multilayer of pericytes on the bottom of the 400 nm thick membrane.
References
[1] N. Abbott, L. Rönnbäck, E. Hansson, Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci, 7, 41-53, 2006.
[2] G. Yegutkin, Nucleotide- and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade. Biochim Biophys Acta, 1783, 673-694, 2008.
[3] S. Nakagawa, M. Deli, H. Kawaguchi, T. Shimizudani, T. Shimono, A. Kittel, K. Tanaka, M. Niwa, A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem Int, 54, 253-263, 2009
P2Y purinergic receptors: new targets for analgesic and antimigraine drugs
Full text linkMillions of individuals worldwide suffer from acute and, more severely, chronic pain conditions (e.g., neuropathic pain, and migraine). The latter bear tremendous personal, familial, and social costs, since sufferers and their relatives undergo a complete turnaround of their lives with the search of relief from pain becoming their pivotal thought. Sadly, to date no effective pharmacological approaches are available which can alleviate chronic pain significantly or in the long run in all patients. The current central strategy for the development of new and effective painkillers lies in the hypothesis that cellular and/or molecular players in nociception must exists that are not targeted by "classical" analgesics, and therefore researchers have put tremendous efforts into the in-depth analysis of the pathways leading to pain development and maintenance over time. In this complex scenario, two outsiders are now taking the center stage: glial cells in sensory ganglia and in the central nervous system, thanks to their ability to communicate with neurons and to modulate their firing, and the purinergic system. Extracellular purine and pyrimidine nucleotides are involved in the physiology of virtually every body district, and their extracellular concentrations massively increase under pathological situations, suggesting that they might represent potential targets for the modulation of disease-associated symptoms, like pain. Here, we provide an overview of the present knowledge of the role of nucleotides in nociception, with a particular emphasis on G protein-coupled P2Y receptors and their involvement in the communication between first- and second-order neurons in sensory nerve pathways and surrounding glial cells
A one-step procedure for the purification of uridine phosphorylase from Escherichia coli.
No full textThe Purinergic System and Glial Cells : Emerging Costars in Nociception
Full text linkIt is now well established that glial cells not only provide mechanical and trophic support to neurons but can directly contribute to neurotransmission, for example, by release and uptake of neurotransmitters and by secreting pro- and anti-inflammatory mediators. This has greatly changed our attitude towards acute and chronic disorders, paving the way for new therapeutic approaches targeting activated glial cells to indirectly modulate and/or restore neuronal functions. A deeper understanding of the molecular mechanisms and signaling pathways involved in neuron-to-glia and glia-to-glia communication that can be pharmacologically targeted is therefore a mandatory step toward the success of this new healing strategy. This holds true also in the field of pain transmission, where the key involvement of astrocytes and microglia in the central nervous system and satellite glial cells in peripheral ganglia has been clearly demonstrated, and literally hundreds of signaling molecules have been identified. Here, we shall focus on one emerging signaling system involved in the cross talk between neurons and glial cells, the purinergic system, consisting of extracellular nucleotides and nucleosides and their membrane receptors. Specifically, we shall summarize existing evidence of novel "druggable" glial purinergic targets, which could help in the development of innovative analgesic approaches to chronic pain states
The development of an SPH tree-based algorithm to investigate the evolution of self-gravitating Protoplanetary Disks
Full text linkI Dischi Circumstellari costituiscono un campo di studio molto moderno, per diverse ragioni. Prima di tutto, lo studio dell'evoluzione di tali sistemi mira a spiegare la configurazione delle migliaia di sistemi planetari che sono stati osservati fin ora, e può fornire risposte importanti anche sulla formazione del Sistema Solare.
In secondo luogo, gli sviluppi delle tecnologie interferometriche nella banda Infrarossa e Millimetrica hanno fornito un ampia gamma di possibilità di osservare i dischi con una risoluzione angolare molto piccola. I dischi hanno dunque rappresentato un ampio target osservativo che ha stimolato non in maniera indifferente l'applicazione e il miglioramento delle più avanzate tecniche osservative.
Inoltre, l'evoluzione dei dischi è guidata da diversi processi fisici, sia dinamici sia idrodinamici, e dunque essi figurano tragli oggetti di studio più importanti a cui applciare sofisticate tecniche di modellistica numerica.
In particolare, i dischi Protoplanetari sono diventati, dagli ultimi due decenni, un obbiettivo chiave nel campo dei sistemi stellari multipli. La loro evoluzione in ambienti eterogenei ricchi di stelle e gas rappresenta una nuova frontiera del calcolo numerico applicato all’astronomia, perché implica numerosi problemi ancora irrisolti. Le recenti scoperte di dischi in ammassi aperti ha aperto a nuove prospettive nello studio teorico di sistemi planetari extrasolari nel loro stato primordiale gassoso.
Per queste ragioni, l’attività del mio Dottorato ha come scopo l’implementazione di uno strumento di modellistica numerico, capace di affrontare questi e molti altri problemi legati all’interazione tra gas e stelle. In questo documento, il Sottoscritto presenterà un nuovo algoritmo sviluppato per integrare l’evoluzione temporale di sistemi gassosi (con o senza gravità) e la loro interazione con le stelle. Il codice si basa sul ben noto approccio Smoothed Particles Hydrodynamics (SPH), e contiene un metodo numerico per accoppiare il cas con un limitato numero di punti di massa calcolando, con elevata precisione, la loro mutua interazione gravitazionale. Il calcolo dell’autogravità è fatto approssimando la forza mediante il noto Tree-Scheme.
Dopo aver descritto alcuni test basilari sulla stabilità e sulle prestazioni, il Sottoscritto descriverà il problema astrofisico principale studiato durante il lavoro di dottorato: l’evoluzione di dischi protoplanetari perturbati da stelle che effettuano incontri ravvicinati, che cistituisce uno schema approssimato del più complesso caso di interazione di un disco con l’ambiente stellare in cui esso è immerso.Circumstellar Disks represent a very modern object of investigation, for many reasons. First, the investigation on their evolution aims at explaining the configuration of several thousand of planetary systems detected so far and may give important answers even on the formation of Solar System. Secondly, the early development of the interferometric technique to observe in the infrared and mm wave-length, opened a wide range of possibilities to observe disks with very short angular resolution. Thus, Disks constitute a wide target, stimulating the application and the improvement of the most advanced observational techniques. Moreover, disk evolution is driven by several physical processes, both dynamical and hydrodynamical, they are indeed one of the most important objects of application of several sophisticated numerical techniques.
Protoplanetary disks have become, since the last 20 years, a key target in the field of star clusters. Their evolution in heterogeneous environments rich in stars and gas represents a new frontier for the Numerical Astronomy because it carries several issues still not overcome. The recent discoveries of circumstellar disks in open clusters opened to new perspectives to the theoretical investigations of non-isolated exoplanetary systems in their primordial gaseous state.
For such purposes, my PhD activity aimed at building a suitable numerical investigating instrument, able to face this and many other problems related to the interaction between gaseous systems and stars. In this document, I will present a new Algorithm developed to treat the evolution of gaseous systems (both selfgravitating and non-selfgravitating) and their interaction with Stars. The Code is based on the well-known Lagrangian Smoothed Particle Hydrodynamics approach, and contains a numerical technique to couple a gas distribution with a few amounts of point-mass particles, evaluating,
with high precision, their motion. The calculus of self-gravity is accomplished with a tree-scheme approach.
After describing some basic stability and performance tests, I will focus on the main problem investigated during my work: the evolution of protoplanetary disks perturbed by passing-by stars, which constitute an approximated scheme of the more complex disk-stellar environment interaction
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
MODULATION OF GLIAL P2Y RECEPTORS BY ALGOGENIC MEDIATORS IN THE TRIGEMINAL GANGLIA: A ROLE FOR THE PURINERGIC SYSTEM IN THE MECHANISM OF ACTION OF KNOWN ANTI-MIGRAINE DRUGS?
No full textAim of the investigation: to study the role of the purinergic system in the neuron-to-glial cell communication within the trigeminal ganglion, and its cross-talk with known pro-algogenic systems (such as bradikynin, BK, calcitonin gene-related peptide, CGRP, and prostaglandins). The final goal is the identification of new cellular and molecular players in the onset and maintenance of trigeminal-associated pain, for the development of new effective therapeutic strategies for migraine.
Methods: primary mixed neuron-glia or purified glial cultures from trigeminal ganglion (TG) were prepared from C57BL6 mice. G protein-coupled P2Y receptor function was evaluated by single cell calcium imaging,
and the extracellular concentrations of CGRP were measured by an ELISA assay. Western blot experiments on P2Y1 and P2Y2 receptors subtypes were also performed.
Results: exposure of mixed-neuron glia cultures to BK induced neuronal release of CGRP, which, in turn, significantly potentiated P2Y receptor-mediated calcium responses to their cognate ligands ADP and UTP on satellite glial cells. A 30-min pre-treatment with the anti-migraine drug sumatriptan completely inhibited both BK-mediated CGRP release from TG neurons, and the potentiation of P2Y receptors. Acetylsalicylic acid (1 mM) and ibuprofen (50 μM) were also able to inhibit CGRP release from neurons, suggesting that prostaglandins are also involved in the complex network of events leading to the potentiation of glial P2Y receptors. To clearly identify the P2Y receptor subtypes involved, we utilized purified glial cultures where CGRP increased ADP- or UTP-induced intracellular calcium concentrations. The selective P2Y1 receptor antagonist MRS2179 completely inhibited ADP-mediated responses, while UTP-mediated responses were almost completely inhibited by a selective P2Y2 receptor antagonist, both under control condition and after exposure to CGRP. After application of the selective P2Y2 antagonist, about 14% of cells showed a residual response to UTP in control cultures, suggesting a contribution of the P2Y4 receptor subtype to UTP-mediated responses. On the other hand, in CGRP-treated cells, no residual UTP-mediated increases of intracellular calcium concentrations were detected, suggesting that only the P2Y2 subtype is potentiated by CGRP exposure. Western blot analysis using commercial polyclonal antibodies against P2Y1 and P2Y2 receptors showed specific protein bands for both receptor subtypes with a significant increase in P2Y1 receptor protein and a trend to increase in P2Y2 receptor protein expression after CGRP treatment.
Conclusions: our data suggest that a complex cross-talk between neuronal and glial cells takes place in the TG, involving pain mediators and extracellular nucleotides. Modulation of this network by known anti-migraine drugs, such as triptans and COX inhibitors, suggests that it might play an important role in the development of migraine pain. Further studies are in progress to understand the pro- or anti-algogenic role for the different P2Y receptor subtypes, and to test whether innovative and more selective pharmacological approaches can be identified for the sake of those migraineurs who are insensitive to currently available drugs
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