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ABSTRACT: The role of the purinergic system in the modulation of pain mechanisms suggests that it might be promising target for treating neuropathic pain. In this study we evaluated the effects of two different dialdehydic compounds: a modified stable adenosine (2-[1-(6-amminopurin-9-il)-2-osso-etossi]prop-2-enale, named MED1101), and oxidized ATP (Ox-ATP), in two different neuropathic pain rat models: the sciatic spared nerve injury (SNI) and paclitaxel evoked painful peripheral neuropathy (pPPN). Neuropathic animals were divided in groups as follows: (a) treated with intraperitoneal (i.p.) MED1101 or Ox-ATP for 21 days; (b) receiving vehicle (VEH) and (c) control (CTR) rats. The allodynic and hyperalgesic behavior was investigated by Von Frey filament test and thermal Plantar test, respectively. We evaluated by immunocytochemistry the astrocytic (GFAP) and microglial (Iba1) response on lumbar spinal cord sections. In either experimental models and using either substances, treated animals showed reduced allodynia and thermal hyperalgesia paralleled by a significant reduction of glial reaction in the spinal cord. These data prompt to hypothesize a potential role of dialdehydes as analgesic agent in chronic neuropathic pain and a possible role as anti-gliotic molecules.
Neuroscience Letters 09/2012; · 2.11 Impact Factor
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ABSTRACT: Astrocytic Ca(2+) dynamics have been extensively studied in ex vivo models; however, the recent development of two-photon microscopy and astrocyte-specific labeling has allowed the study of Ca(2+) signaling in living central nervous system. Ca(2+) waves in astrocytes have been described in cultured cells and slice preparations, but evidence for astrocytic activation during sensory activity is lacking. There are currently few methods to image living spinal cord: breathing and heart-beating artifacts have impeded the widespread application of this technique. We here imaged the living spinal cord by two-photon microscopy in C57BL6/J mice. Through pressurized injection, we specifically loaded spinal astrocytes using the red fluorescent dye sulforhodamine 101 (SR101) and imaged astrocytic Ca(2+) levels with Oregon-Green BAPTA-1 (OGB). Then, we studied astrocytic Ca(2+) levels at rest and after right electrical hind paw stimulation. Sensory stimulation significantly increased astrocytic Ca(2+) levels within the superficial dorsal horn of the spinal cord compared to rest. In conclusion, in vivo morphofunctional imaging of living astrocytes in spinal cord revealed that astrocytes actively participate to sensory stimulation.
Neural Plasticity 01/2012; 2012:425818. · 2.00 Impact Factor
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Maria Rosaria Bianco,
Miluscia Berbenni,
Flavio Amara,
Sandra Viggiani,
Martina Fragni,
Valentina Galimberti,
Daniele Colombo, Giovanni Cirillo,
Michele Papa,
Lilia Alberghina,
Anna Maria Colangelo
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ABSTRACT: Neuronal death has been reported to involve mitochondrial dysfunction and cell cycle reentry. In this report, we used Nerve Growth Factor (NGF)-differentiated PC12 cells to investigate mechanisms linking mitochondrial dysfunction and cell cycle activation during neuronal death induced by NGF withdrawal and/or oxidative stress. We found that loss of survival following H(2) O(2) -induced oxidative stress or NGF deprivation was preceded by a decrease in mitochondrial membrane potential (ΔΨm), increase in reactive oxygen species (ROS), and up-regulation of cyclin D1 and phosphorylation (Ser-780) of protein retinoblastoma (P-pRb), without an increase of proliferation rates. Treatment with H(2) O(2) , but not NGF deprivation, also induced the phosporylation (Ser-10) of p27(kip1) and the appearance of a cleaved P-p27(kip1) fragment of about 15 kDa. The extent of cell cycle activation appeared to be inversely correlated to the duration of toxic stimuli (pulse/continuous). H(2) O(2) -induced mitogenic responses appeared to be mediated by induction of P-MAPK and P-Akt and were blocked by p38MAPK and JNK inhibitors as well as by the CDK inhibitor flavopiridol (Flav) and by sodium selenite (Sel), a component of selenoproteins, including glutathione peroxidases. Inhibition of p38MAPK and JNK, instead, did not affect cyclin D1 changes following NGF deprivation. Finally, both Flav hydrochloride and Sel partially prevented mitochondrial dysfunction and cell death following NGF withdrawal or H(2) O(2) toxicity, but not during oxidative stress in the absence of NGF. Taken together, these data suggest that H(2) O(2) -induced oxidative stress can determine distinct patterns of mitogenic responses as a function of mitochondrial dysfunction depending on 1) intensity/duration of stress stimuli and/or 2) presence of NGF.
Journal of Neuroscience Research 08/2011; 89(8):1302-15. · 2.74 Impact Factor
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ABSTRACT: Neuroglial cells are fundamental for control of brain homeostasis and synaptic plasticity. Decades of pathological and physiological studies have focused on neurons in neurodegenerative disorders, but it is becoming increasingly evident that glial cells play an irreplaceable part in brain homeostasis and synaptic plasticity. Animal models of brain injury and neurodegenerative diseases have largely contributed to current understanding of astrocyte-specific mechanisms participating in brain function and neurodegeneration. Specifically, gliotransmission (presence of glial neurotransmitters, and their receptors and active transporters), trophic support (release, maturation and degradation of neurotrophins) and metabolism (production of lactate and GSH components) are relevant aspects of astrocyte function in neuronal metabolism, synaptic plasticity and neuroprotection. Morpho-functional changes of astrocytes and microglial cells after traumatic or toxic insults to the central nervous system (namely, reactive gliosis) disrupt the complex neuro-glial networks underlying homeostasis and connectivity within brain circuits. Thus, neurodegenerative diseases might be primarily regarded as gliodegenerative processes, in which profound alterations of glial activation have a clear impact on progression and outcomes of neuropathological processes. This review provides an overview of current knowledge of astrocyte functions in the brain and how targeting glial-specific pathways might ultimately impact the development of therapies for clinical management of neurodegenerative disorders.
Biotechnology advances 07/2011; 30(1):261-71. · 8.25 Impact Factor
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ABSTRACT: Peptidomimetics hold a great promise as therapeutic agents for neurodegenerative disorders. We previously described a Nerve Growth Factor (NGF)-like peptide, now named BB14, which was found to act as a strong TrkA agonist and to be effective in the sciatic nerve injury model of neuropathic pain. In this report we present the effects of BB14 in reducing reactive astrocytosis and reverting neuroplastic changes of the glutamate/GABAergic circuitry in the lumbar spinal cord following spared nerve injury (SNI) of the sciatic nerve. Immunohistochemical analysis of spinal cord sections revealed that SNI was associated with increased microglial (Iba1) and astrocytic (GFAP) responses, indicative of reactive gliosis. These changes were paralleled by (i) decreased glial aminoacid transporters (GLT1 and GlyT1) and increased levels of (ii) neuronal glutamate transporter EAAC1, (iii) neuronal vesicular GABA transporter (vGAT) and (iv) the GABAergic neuron marker GAD65/67. A remarkable increase of the Glutamate/GABA ratio and the reduction of glutathione (GSH) levels were also indicative of modifications of glial function in neuroprotection. All these molecular changes were found to be linked to an alteration of endogenous NGF metabolism, as demonstrated by decreased levels of mature NGF, increase of proNGF and increased activity of NGF-degrading methallo-proteinases (MMPs). Biochemical alterations and SNI-related neuropathic behavior, characterized by allodynia and hyperalgesia, were reversed by 7-days i.t. administration of the NGF-like peptide BB14, as well as by increasing endogenous NGF levels by i.t. infusion of GM6001, a MMPs inhibitor. All together, while confirming the correlation between reactive astrogliosis and perturbation of synaptic circuitry in the SNI model of peripheral nerve injury, these data strongly support the beneficial effect of BB14 in reducing reactive astrogliosis and restoring synaptic homeostasis under pathological conditions linked to alteration of NGF availability and signaling, thereby suggesting a potential role of BB14 as a therapeutic agent.
Biotechnology advances 05/2011; 30(1):223-32. · 8.25 Impact Factor
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ABSTRACT: Repeated exposure to psychostimulant drugs induces complex molecular and structural modifications in discrete brain regions of the meso-cortico-limbic system. This structural remodeling is thought to underlie neurobehavioral adaptive responses. Administration to adolescent rats of methylphenidate (MPH), commonly used in attention deficit and hyperactivity disorder (ADHD), triggers alterations of reward-based behavior paralleled by persistent and plastic synaptic changes of neuronal and glial markers within key areas of the reward circuits. By immunohistochemistry, we observe a marked increase of glial fibrillary acidic protein (GFAP) and neuronal nitric oxide synthase (nNOS) expression and a down-regulation of glial glutamate transporter GLAST in dorso-lateral and ventro-medial striatum. Using electron microscopy, we find in the prefrontal cortex a significant reduction of the synaptic active zone length, paralleled by an increase of dendritic spines. We demonstrate that in limbic areas the MPH-induced reactive astrocytosis affects the glial glutamatergic uptake system that in turn could determine glutamate receptor sensitization. These processes could be sustained by NO production and synaptic rearrangement and contribute to MPH neuroglial induced rewiring.
European neuropsychopharmacology: the journal of the European College of Neuropsychopharmacology 05/2011; 22(1):53-63. · 3.68 Impact Factor
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ABSTRACT: Reactive gliosis has been implicated in both inflammatory and neurodegenerative diseases. However, mechanisms by which astrocytic activation affects synaptic efficacy have been poorly elucidated. We have used the spared nerve injury (SNI) of the sciatic nerve to induce reactive astrocytosis in the lumbar spinal cord and investigate its potential role in disrupting the neuro-glial circuitry. Analysis of spinal cord sections revealed that SNI was associated with an increase of microglial (Iba1) and astrocytic (GFAP) markers. These changes, indicative of reactive gliosis, were paralleled by (i) a decrease of glial amino acid transporters (GLT1 and GlyT1) and increased levels of (ii) neuronal glutamate transporter EAAC1, (iii) neuronal vesicular GABA transporter (vGAT) and (iv) the GABAergic neuron marker GAD65/67. Besides the increase of Glutamate/GABA ratio, indicative of the perturbation of synaptic circuitry homeostasis, the boost of glutamate also compromised glial function in neuroprotection by up-regulating the xCT subunit of the glutamate-cystine antiport system and reducing glutathione (GSH) production. Finally, this study also shows that all these structural changes were linked to an alteration of endogenous NGF metabolism, as demonstrated by the decrease of endogenous NGF expression levels and increased activity of the NGF-degrading metalloproteinases. All the changes displayed by SNI-animals were reversed by a 7-days i.t. administration of NGF or GM6001, a generic metalloproteinase inhibitor, as compared to vehicle (ACSF)-treated animals. All together, these data strongly support the correlation between reactive astrogliosis and mechanisms underlying the perturbation of the synaptic circuitry in the SNI model of peripheral nerve injury, and the essential role of NGF in restoring both synaptic homeostasis and the neuroprotective function of glia.
Neurobiology of Disease 03/2011; 41(3):630-9. · 5.40 Impact Factor
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ABSTRACT: Reactive astrocytosis seems to be strongly implicated in the development and maintenance of inflammatory and neurodegenerative disorders. We design a new toxic model treatment with 3-nitropropionic acid (3-NP), a mitochondrial complex II irreversible inhibitor, to induce in rats Huntington's disease (HD) like syndrome, characterized by hindlimb dystonia, involuntary choreiform movements and reduced global activity. In an attempt to find out whether molecular and morphological changes in the neuro-glial network could be involved in the pathogenesis of this disease, we developed a protocol of subchronic intra-peritoneal 3-NP intoxication. Moreover we set up specific, highly discriminative, behavioral tests to detect very early mild motor disabilities in 3-NP treated rats. This treatment did not cause severe cell death. However, in the Caudate-Putamen (CPu) of all 3-NP treated animals we found a massive astrogliosis, revealed by increased GFAP levels, paralleled by changes of the glial glutamate transporter GLAST distribution. To these glial changes we detected a transcriptional upregulation of c-fos and Sub-P in the striatal medium spiny neurons (MSN). We propose that this model of 3-NP intoxication along with the designed set of behavioral analyses allow to unmask in a very early phase the motor deficits and the underlying morpho-molecular changes associated to the onset of motor disabilities in the HD-like syndrome. Therefore this model unveil the key role played by the different components of the tripartite synapse in the pathogenesis of the HD, a putative non-cell-autonomous disease.
Neurochemistry International 09/2009; 56(1):152-60. · 2.86 Impact Factor
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ABSTRACT: Nerve growth factor (NGF), an essential peptide for sensory neurons, seems to have opposite effects when administered peripherally or directly to the central nervous system. We investigated the effects of 7-days intrathecal (i.t.) infusion of NGF on neuronal and glial spinal markers relevant to neuropathic behavior induced by chronic constriction injury (CCI) of the sciatic nerve. Allodynic and hyperalgesic behaviors were investigated by Von Frey and thermal Plantar tests, respectively. NGF-treated animals showed reduced allodynia and thermal hyperalgesia, compared to control animals. We evaluated on lumbar spinal cord the expression of microglial (ED-1), astrocytic (GFAP and S-100beta), and C- and Adelta-fibers (SubP, IB-4 and Cb) markers. I.t. NGF treatment reduced reactive astrocytosis and the density of SubP, IB4 and Cb positive fibers in the dorsal horn of injured animals. Morphometric parameters of proximal sciatic nerve stump fibers and cells in DRG were also analyzed in CCI rats: myelin thickness was reduced and DRG neurons and satellite cells appeared hypertrophic. I.t. NGF treatment showed a beneficial effect in reversing these molecular and morphological alterations. Finally, we analyzed by immunohistochemistry the expression pattern of neurotrophin receptors TrkA, pTrkA, TrkB and p75(NTR). Substantial alterations in neurotrophin receptors expression were observed in the spinal cord of CCI and NGF-treated animals. Our results indicate that i.t. NGF administration reverses the neuro-glial morphomolecular changes occurring in neuropathic animals paralleled by alterations in neurotrophin receptors ratio, and suggest that NGF is effective in restoring homeostatic conditions in the spinal cord and maintaining analgesia in neuropathic pain.
Cellular and Molecular Neurobiology 08/2009; 30(1):51-62. · 1.97 Impact Factor
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ABSTRACT: Methylphenidate (MPH) administration to adolescent rodents produces persistent region-specific changes in brain reward circuits and alterations of reward-based behavior. We show that these modifications include a marked increment of serotonin (5-hydroxy-tryptamine) receptor type 7 (Htr7) expression and synaptic contacts, mainly in the nucleus accumbens, and a reduction of basal behavioral impulsivity. We show that neural and behavioral consequences are functionally related: administration of a selective Htr7 antagonist fully counteracts the MPH-reduced impulsive behavior and enhances impulsivity when administered alone in naive rats. Agonist-induced activation of endogenous Htr7 significantly increases neurite length in striatal neuron primary cultures, thus suggesting plastic remodeling of neuronal morphology. The mixed Htr (1a/7) agonist, 8-OH-DPAT, reduces impulsive behavior in adolescent rats and in naive adults, whose impulsivity is enhanced by the Htr7 antagonist. In summary, behavioral pharmacology experiments show that Htr7 mediates self-control behavior, and brain primary cultures experiments indicate that this receptor may be involved in the underlying neural plasticity, through changes in neuronal cytoarchitecture.
Genes Brain and Behavior 03/2009; 8(3):356-68. · 3.48 Impact Factor
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ABSTRACT: The interactions between the mother/parents and their offspring provides socioemotional input, which is essential for the establishment and maintenance of synaptic networks in prefrontal and limbic brain regions. Since glial cells are known to play an important role in developmental and experience-driven synaptic plasticity, the effect of an early adverse emotional experience induced by maternal separation for 1 or 6 h on the expression of the glia specific proteins S100beta and glial fibrillary acidic protein (GFAP) was quantitatively analyzed in anterior cingulate cortex, hippocampus, and precentral medial cortex. Three animal groups were analyzed at postnatal day 14: (i) separated for 1 h; (ii) separated for 6 h; (iii) undisturbed (control). Twenty-four hours after stress exposure, the stressed brains showed significantly reduced numbers of S100beta-immunoreactive (ir) cells in the anterior cingulate cortex (6-h stress) and in the precentral medial cortex (1- and 6-h stress). Significantly reduced numbers of GFAP-ir cells were observed only in the medial precentral cortex (1- and 6-h stress); no significant changes were observed in the anterior cingulate cortex. No significant changes of the two glial markers were observed in the hippocampus. Double-labeling experiments with GFAP and pCREB revealed pCREB labeling only in the hippocampus, where the stressed brains (1 and 6 h) displayed significantly reduced numbers of GFAP/pCREB-ir glial cells. The observed downregulation of glia-specific marker proteins is in line with our hypothesis that emotional experience can alter glia cell activation in the juvenile limbic system.
Developmental Neurobiology 02/2009; 69(4):203-11. · 3.55 Impact Factor
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Anna Maria Colangelo,
Maria Rosaria Bianco,
Luigi Vitagliano,
Carlo Cavaliere, Giovanni Cirillo,
Luca De Gioia,
Donatella Diana,
Daniele Colombo,
Cristina Redaelli,
Laura Zaccaro,
Giancarlo Morelli,
Michele Papa,
Paolo Sarmientos,
Lilia Alberghina,
Enzo Martegani
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ABSTRACT: Analysis of the structure of nerve growth factor (NGF)-tyrosine kinase receptor A (TrkA) complex, site-directed mutagenesis studies and results from chemical modification of amino acid residues have identified loop 1, loop 4, and the N-terminal region of the NGF molecule as the most relevant for its biological activity. We synthesized several peptides mimicking the two loops (1 and 4) linked together with an appropriate spacer, with or without the N-terminal region. Two peptides named NL1L4 and L1L4 demonstrated good NGF agonist activity at a concentration as low as 3 mum. They induced differentiation of chick dorsal root ganglia and stimulated tyrosine phosphorylation of TrkA, but not TrkB, receptor. In addition L1L4 was able to induce differentiation of PC12 cells. More interestingly, the peptide with the highest "in vitro" activity (L1L4) was shown to reduce neuropathic behavior and restore neuronal function in a rat model of peripheral neuropathic pain, thereby suggesting a potential therapeutic role for this NGF-mimetic peptide.
Journal of Neuroscience 04/2008; 28(11):2698-709. · 7.11 Impact Factor
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ABSTRACT: Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeats within the coding sequence of the ataxin-1 protein. In the present study, we used a conditional transgenic mouse model of SCA1 to investigate very early molecular and morphological changes related to the behavioral phenotype. In mice with neural deficits detected by rotarod performance, and simultaneous spatial impairments in exploratory activity and uncoordinated gait, we observed both significant altered expression and patchy distribution of excitatory amino acids transporter 1. The molecular changes observed in astroglial compartments correlate with changes in synapse morphology; synapses have a dramatic reduction of the synaptic area external to the postsynaptic density. By contrast, Purkinje cells demonstrate preserved structure. In addition, severe reactive astrocytosis matches changes in the glial glutamate transporter and synapse morphology. We propose these morpho-molecular changes are the cause of altered synaptic transmission, which, in turn, determines the onset of the neurological symptoms by altering the synaptic transmission in the cerebellar cortex of transgenic animals. This model might be suitable for testing drugs that target activated glial cells in order to reduce CNS inflammation.
Neuron Glia Biology 11/2007; 3(4):335-51. · 1.34 Impact Factor
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ABSTRACT: Gliosis is strongly implicated in the development and maintenance of persistent pain states following chronic constriction injury of the sciatic nerve. Here we demonstrate that in the dorsal horn of the spinal cord, gliosis is accompanied by changes in glial amino acid transporters examined by immunoblot, immunohistochemistry and RT-PCR. Cytokines, proinflammatory mediators and microglia increase up to postoperative day (pd) 3 before decreasing on pd 7. Then, spinal glial fibrillary acidic protein increases on pd 7, lasting until pd 14 and later. Simultaneously, the expression of glial amino acid transporters for glycine and glutamate (GlyT1 and GLT1) is reduced on pd 7 and pd 14. Consistent with a reduced expression of GlyT1 and GLT1, high performance liquid chromatography reveals a net increase in the concentration of glutamate and glycine on pd 7 and pd 14 in tissue from the lumbar spinal cord of neuropathic mice. In this study we have confirmed that microglial activation precedes astrogliosis. Such a glial cytoskeletal rearrangement correlates with a marked decrease in glycine and glutamate transporters, which might, in turn, be responsible for the increased concentration of these neurotransmitters in the spinal cord. We speculate that these phenomena might contribute, via over-stimulation of NMDA receptors, to the changes in synaptic functioning that are responsible for the maintenance of persistent pain.
Neuron Glia Biology 05/2007; 3(2):141-53. · 1.34 Impact Factor
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ABSTRACT: Cyclosporine A (CsA), a neutral, highly hydrophobic cyclic peptide with 11 amino acids, is currently the most widely used immunosuppressive drug for preventing graft rejection and autoimmune diseases. Despite its efficacy, the use of CsA is limited by severe side effects, mainly nephrotoxicity and arterial hypertension. Single cell microfluorimetry was used to evaluate the role of CsA on Ca(2+) signaling pathway in intact cells of the porcine proximal tubule-like cell line LLC-PK1; the assay of the in vitro activity of the plasma membrane Ca(2+) pump (PMCA) was carried out through the preparation and isolation of membranes. The addition of CsA to incubation medium at doses ranging from 0.1 to 2 microM did not change the basal level of intracellular calcium ([Ca(2+)](i)), whereas it affected the [Ca(2+)](i) response to thapsigargin (TG), a powerful inhibitor of microsomal Ca(2+) pump. In control studies, 5 microM TG produced a biphasic response: [Ca(2+)](i) peaked with a 60-s lag, and it then declined to a plateau of elevated [Ca(2+)](i), which remains above basal. However, it became evident that CsA strengthened the Ca(2+) response to TG because the addition of 5 microM TG to cells exposed to 400 nM CsA did not affect the peak response to TG, but it markedly affected the subsequent sustained phase ([Ca(2+)](i) = 156 +/- 4.84 versus 130 +/- 3.28 nmol, mean +/- SEM, n = 6, P < 0.001). In membrane preparations, 200 nM CsA brought about, in the presence of 10 microM calmodulin (CaM), a significant decrease of plasma membrane Ca(2+) pump (PMCA) activity (46.96 +/- 0.26 versus 53.48 +/- 1.96 nmol x mg of protein(-1) x min(-1), n = 6, P < 0.02), a value similar to that obtained in the presence of equimolar amounts of cyclosporine H (CsH), a non-immunosuppressive analogue of CsA. These findings suggest that in this cell line CsA affects the Ca(2+) export pathway through the reduction of the PMCA activity with consequent amplification and strengthening of [Ca(2+)](i) response after exposure to agents that trigger intracellular Ca(2+) release. The increased cell sensitivity during Ca(2+) signaling events ensuing from the impairment of this "defense system" may be regarded as one of the basic mechanisms involved in the development of the side effects induced by CsA.
Journal of the American Society of Nephrology 07/2003; 14(6):1435-42. · 9.66 Impact Factor
