Maria Domenica Sanna

Pleiotropic effect of histamine H4 receptor modulation in the central nervous system

The histamine H4 receptor (H4R) is expressed primarily on cells involved in inflammation and immune responses. Recently, it has been reported the functional expression of H4R within neurons of the central nervous system, but their role has been poorly understood. The present study aimed to elucidate the physiopathological role of cerebral H4R in animal models by the intracerebroventricular administration of the H4R agonist VUF 8430 (20-40 μg per mouse). Selectivity of results was confirmed by the prevention of the effects produced by the H4R antagonist JNJ 10191584 (3-9 mg/kg p.o.). Neuronal H4R activation induced acute thermal antinociception, indicating that neuronal histamine H4R might be involved in the production of antinociception in the absence of an inflammatory process. An anxiolytic-like effect of intensity comparable to that exerted by diazepam, used as reference drug, was produced in the light-dark box test. VUF 8430 reversed the scopolamine-induced amnesia in the passive avoidance test and showed anorexant activity in food deprived mice. Conversely, the H4R activation did not modify the immobility time in the tail suspension test. Rotarod performance test was employed to demonstrate that the effects observed following the administration of VUF 8430 and JNJ 10191584 were not due to impaired motor function of animals. Furthermore, both compounds did not alter spontaneous mobility and exploratory activity in the hole board test. These results show the antinociceptive, antiamnesic, anxiolytic and anorexant effects induced by neuronal H4R agonism, suggesting that H4 modulators may have broader utility further the control of inflammatory and immune processes.

Behavioural phenotype of histamine H4 receptor knockout mice: Focus on central neuronal functions

ABSTRACT The functional expression of H4 receptors (H4R) within neurons of the central nervous system has been recently reported, but their role is poorly understood. The present study aims to elucidate the role of neuronal H4R by providing the first description of the behavioural phenotype of H4R‐deficient (H4R knockout, H4R‐KO) mice. Mice lacking H4R underwent behavioural studies to evaluate locomotor activity, pain perception, anxiety, depression, memory and feeding behaviour. H4R‐KO mice showed a significant increase in ambulation in an open field as well as in exploratory activity in the absence of any modification of motor coordination. The sensitivity of mutant mice to a thermal or a mechanical stimulus was identical to that of the wild type mice, but H4R‐KO showed sensory hypersensitivity toward a condition of neuropathic pain. The lack of H4R is associated with the promotion of anxiety in the light‐dark box test. H4R‐KO mice showed an increased immobility time in the tail suspension test, experimental procedure used to evaluate the response of H4R deficient mice to a behavioural despair paradigm. Cognitive function parameters of H4R deficient mice, examined using the passive avoidance and the novel object recognition tests, were unaltered showing the lack of influence of H4R on working and recognition memory. Finally, H4R‐deficient mice showed an orectic phenotype. These results illustrate that H4R modulates various neurophysiological functions such as locomotor activity, anxiety, nociception and feeding behaviour, confirming the importance of the integrity and functionality of neuronal H4R in the histaminergic regulation of neuronal functions. Graphical abstract Figure. No Caption available. HighlightsThe behavioural phenotype of H4R‐KO has been defined.H4R modulates locomotor activity, anxiety, pain and feeding behaviour.Neuronal H4R has a prominent role in the histaminergic regulation of neuronal functions.

Inhibition of spinal ERK1/2-c-JUN signaling pathway counteracts the development of low doses morphine-induced hyperalgesia

Morphine-induced hyperalgesia is a pharmacological phenomenon often hindering its prolonged applications in the clinic. It has been shown that systemic administration of morphine induced a hyperalgesic response at an extremely low dose. Extracellular signal-regulated kinase (ERK) pathway contributes to pain sensitization, and its phosphorylation under pain conditions results in the induction and maintenance of pain hypersensitivity. The present study was designed to determine whether low dose morphine treatment in mice could influence the spinal activity of ERK. The data showed that morphine (1 µg/kg) induced a marked increase in ERK phosphorylation. Intrathecal pre-treatment with a selective mitogen-activated and extracellular signal-regulated kinase (MEK) inhibitor PD98059, attenuated morphine-associated thermal hyperalgesia. Morphine exposure increased phosphorylation of c-JUN, that was prevented by the inhibition of ERK pathway. In addition, double immunofluorescence studies revealed that, p-ERK and p-c-JUN are localized on neurons of the spinal dorsal horn expressing µ receptors. These data suggest that ERK contributes to the morphine-induced hyperalgesia by regulating the activation of c-JUN.

Histamine H4 receptor agonist-induced relief from painful peripheral neuropathy is mediated by inhibition of spinal neuroinflammation and oxidative stress

Neuropathic pain is under‐treated, with a detrimental effect on quality of life, partly because of low treatment efficacy, but also because pathophysiological mechanisms are not fully elucidated. To clarify the pathobiology of neuropathic pain, we studied the contribution of neuroinflammation and oxidative stress in a model of peripheral neuropathy. We also assessed an innovative treatment for neuropathic pain by investigating the effects of histamine H4 receptor ligands in this model.

ERK1/2 phosphorylation is involved in the antidepressant-like action of 2,5-diphenyl-3-(4-fluorophenylseleno)-selenophene in mice

We investigated the antidepressant-like action of 5 compounds belonging to the selenophene class. The involvement of ERK and CREB activation in this action was also demonstrated. In the experiment 1, time-course and dose-response effect of H-DPS, CH3-DPS, Cl-DPS, F-DPS and CF3-DPS were accompanied in the mouse forced swimming test (FST). Firstly, animals received compounds at a dose of 50mg/kg, by intragastric (i.g.) route, at different times (15-240 min) before test. Results showed that the peak of maximum anti-despair behavior induced by Cl-DPS, F-DPS and CF3-DPS was at 30 min; maximum effect of H-DPS and CH3-DPS was found at 60 min, which was maintained until 120 min. Regarding dose-response effect, all compounds reduced immobility time and increased latency for the first episode of immobility at a dose of 50mg/kg. In addition, F-DPS also showed antidepressant-like action at a dose of 25mg/kg, whilst H-DPS, CH3-DPS, Cl-DPS and CF3-DPS were not effective at lower doses. Thus, F-DPS was chosen for further investigation of its mechanism of action. Experiment 2 showed that treatment of animals with F-DPS (50 mg/kg, i.g.) significantly increased phosphorylated ERK1/2 levels in the prefrontal cortex and hippocampus; however, pCREB levels were not affected. Additionally, in the experiment 3 anti-immobility effect of F-DPS was completely blocked by pretreatment of animals with PD 98,059, an inhibitor of ERK phosphorylation, suggesting that ERK signalling activation is involved in its antidepressant-like action in mice. Together our data appoint F-DPS as a promising molecule for the development of a new antidepressant therapy.

HuD-mediated distinct BDNF regulatory pathways promote regeneration after nerve injury

Up-regulation of brain-derived neurotrophic factor (BDNF) synthesis is an important mechanism of peripheral nerve regeneration after injury. However, the cellular and molecular mechanisms underlying this process are not fully understood. This study examines the role of BDNF in the spared nerve injury (SNI) mice model. Protein expression and cellular localization were investigated in the dorsal root ganglia (DRG) and spinal cord by western blotting and immunofluorescence experiments respectively. BDNF protein was markedly increased 3 and 7days post-injury in the spinal cord and DRG. Following nerve injury sensory neurons produce molecules to promote regeneration, such as growth-associated protein 43 (GAP-43) and cytoskeletal proteins. Our results show that the expression of GAP-43 was increased in the DRG and spinal cord while, an increased of p-NFH content was detected in the spinal cord, with no modification in the DRG. Both events were counteracted by the administration of an anti-BDNF antibody. In DRG of SNI mice we also detected an increase of HuD expression, a RNA-binding protein known to stabilize BDNF and GAP-43 mRNA. Silencing of HuD prevented the nerve injury-induced BDNF and GAP-43 enhanced expression in the DRG. HuD-mediated BDNF synthesis in the primary sensory neurons, is followed by an anterograde transport of the neurotrophin to the central terminals of the primary afferents in the spinal dorsal horn, to modulate GAP-43 and NFH activation. Our data suggest that BDNF, GAP-43 and p-NFH proteins increase are linked events required for the enhanced regeneration after nerve injury.

Silencing of the RNA-binding protein HuR attenuates hyperalgesia and motor disability in experimental autoimmune encephalomyelitis

&NA; Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system associated with progressive neuronal loss and axonal degeneration. Neuronal lesions and dysfunction lead often to neuropathic pain, the most prevalent and difficult to treat pain syndrome observed in MS patients. Despite its widespread occurrence, the underlying neural mechanisms for MS pain are not fully understood. For a better clarification of the pathophysiology of MS‐associated pain, we investigated the role of HuR, an RNA‐binding protein that positively regulates the stability of many target mRNAs, including several cytokines. The influence of HuR in the generation of the hypernociceptive response in a mouse model of relapsing‐remitting experimental autoimmune encephalomyelitis (RR‐EAE), an experimental model of MS, was investigated. HuR silencing, obtained through the repeated intrathecal administration of an antisense oligonucleotide (aODN) anti‐HuR, completely attenuated hindpaw mechanical allodynia and thermal hyperalgesia developed by RR‐EAE mice. Anti‐HuR aODN also reduced severity of motor deficits as reflected by a reduction of clinical EAE score and improvement of rotarod performance. RR‐EAE mice showed demyelination in spinal cord sections that was significantly reduced by HuR silencing. Double‐staining immunofluorescence studies showed a neuronal localization of HuR within dorsal horn spinal cord, consistent with a neuronal mechanism of action. Our findings suggest the involvement of HuR in the hypernociceptive behaviour of RR‐EAE mice providing the first pharmacological assessment of an antiallodynic and antihyperalgesic effect of HuR silencing. These data may provide support for HuR modulation as a therapeutic perspective for the management of MS‐related neuropathic pain. HighlightsSpinal HuR silencing relieves mechanical and thermal allodynia in relapsing‐remitting EAE mice.Spinal HuR silencing attenuates EAE clinical score and motor disability of EAE mice.Spinal HuR silencing reduces spinal cord demyelination and increased histological scores.Targeting HuR would represent an innovative therapeutic approach for intervention in EAE and MS.

Spinal astrocytic c-Jun N-terminal kinase (JNK) activation as counteracting mechanism to the amitriptyline analgesic efficacy in painful peripheral neuropathies.

ABSTRACT Several drugs and agents are currently used for the treatment of neuropathic pain. Among them amitriptyline, a tricyclic antidepressant drug, represent a first line treatment. Despite its well‐documented clinical efficacy, amitriptyline is ineffective in some animal models of neuropathic pain. The aim of this study was to investigate into amitriptyline poor efficacy in neuropathic pain and to determine the role of c‐Jun N‐terminal kinase (JNK) activation as counteracting mechanism to the analgesic effects of this drug. Experiments were performed in mice with painful peripheral neuropathies due to the antiretroviral agent 2,3‐dideoxycytidine (ddC), and with the partial sciatic nerve injury produced in the spared nerve injury model (SNI). In mice subjected to SNI and antiretroviral treatment, amitriptyline did not attenuate mechanical allodynia and thermal hyperalgesia. Conversely, intrathecal injection of the JNK inhibitor SP600125 prevented SNI and ddC‐induced nociceptive behavior and, its inactive dose co‐administrated with amitriptyline induced an antinociceptive effect. Western blotting analysis showed an upregulation of p‐JNK in the lumbar spinal cord of SNI and ddC‐exposed mice, that was further enhanced after amitriptyline administration. Additionally, amitriptyline further promoted astrocyte activation in neuropathic mice, as illustrated by the increased expression of glial fibrillary acidic protein (GFAP), that was attenuated by intrathecal injection of the JNK inhibitor. These data indicate astrocyte JNK activation as counteracting pathway to amitriptyline analgesic response. Targeting the JNK pathway in spinal astroglia may present an efficient way to improve the analgesic efficacy of amitriptyline in the neuropathic pain treatment.

Effect of amitriptyline treatment on neurofilament-H protein in an experimental model of depression

It has been proposed that depression is associated with dysfunction of hippocampal plasticity. Novel hypotheses suggest that antidepressants induce neuronal structural plasticity, although the underlying mechanisms still remain unclear. Therefore, the aim of this study was to investigate the effects of amitriptyline on levels of phosphorylated heavy neurofilament subunit (NF-H) in the hippocampus of mice exposed to acute and chronic behavioral despair paradigms. Immunoblotting experiments showed that animals exposed to the tail suspension test (TST) displayed diminished levels of pNF-H 24h after testing. Repeated administration of amitriptyline (10mg/kg i.p.) prevented this decreased hippocampal phosphorylation of NF-H. Conversely, administration of citalopram (10mg/kg i.p.) left unchanged pNF-H levels. The expression of pNF-H was also analyzed by immunofluorescence in mice exposed to the unpredictable chronic mild stress paradigm (UCMS), an experimental model of depression. Mice that developed a depressive-like behavior showed a decreased pNF-H immunostaining selectively in the hippocampal CA3 region. Chronic administration of amitriptyline reversed the despaired behavior induced by exposure to UCMS paradigm and, fully recovered pNF-H labeling to control values. Our results indicate that the cytoskeletal remodeling induced by amitriptyline in the hippocampal CA3 region might underpin its behavioral efficacy. Hippocampal alterations of the NF appeared associated with the mechanism of this antidepressant drug and may contribute to its psychotherapeutic actions.

St. John’s Wort Potentiates anti-Nociceptive Effects of Morphine in Mice Models of Neuropathic Pain

Objective In this study, we compared the efficacy of a combination of PKC-blocker St. Johns Wort (SJW) and morphine in mice with painful antiretroviral (2,3-dideoxycitidine [ddC]) and chemotherapic (oxaliplatin) neuropathy. Methods Morphine (1 and 5 mg/Kg i.p.), SJW (1 and 5 mg/Kg o.s.), or their combination was administered by systemic injection, and antinociception was determined by using the hot and cold plate tests. Results Here we demonstrate the ability of SJW to relieve neuropathic pain in mice neuropathic models and a potentiation of morphine antinociception in thermal pain. The potentiating effect shown by SJW was not secondary to its antinociceptive activity as the increase of the morphine antinociceptive effect was produced at a dose (1mg/kg o.s.) devoid of any capability to modulate the pain threshold in neuropathic pain mice. Further examinations of the SJW main components revealed that hypericin was responsible for the potentiating properties whereas flavonoids were ineffective. Conclusions These results show that SJW has notable antinociceptive activity for both neuropathic pain models and could be used in neuropathic pain relief alone or in combination with morphine. These data support the utility of combination SJW/opioid therapy in pain management for antinociceptive efficacy by enhancing opioid analgesia.