Alessandra OTTANI - personale UniMoRe

Nuova ricerca

Alessandra OTTANI

Professore Associato
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

Pubblicazioni

2023 - Melanocortin receptor agonist NDP-α-MSH improves cognitive deficits and microgliosis but not amyloidosis in advanced stages of AD progression in 5XFAD and 3xTg mice [Articolo su rivista]
Daini, E.; Vandini, E.; Bodria, M.; Liao, W.; Baraldi, C.; Secco, V.; Ottani, A.; Zoli, M.; Giuliani, D.; Vilella, A.
abstract

Introduction: Alzheimer's disease (AD) is the most frequent cause of dementia and still lacks effective therapy. Clinical signs of AD include low levels of endogenous melanocortins (MCs) and previous studies have shown that treatment with MC analogs induces neuroprotection in the early stages of AD.Methods: We investigated the neuroprotective role of MCs in two transgenic mouse models of severe AD using 5 and 7 month-old (mo) 5XFAD mice and 9 and 12 mo 3xTg mice. These mice were subjected to a chronic stimulation of MC receptors (MCRs) with MC analogue Nle4-D-Phe7-alpha-melanocyte stimulating hormone (NDP-alpha-MSH, 340 mu g/kg, i.p.). Mouse behavior and ex-vivo histological and biochemical analyses were performed after 50 days of treatment.Results:Our analysis demonstrated an improvement in cognitive abilities of AD mice at late stage of AD progression. We also showed that these protective effects are associated with decreased levels of hyperphosphorylated Tau but not with A beta burden, that was unaffected in the hippocampus and in the cortex of AD mice. In addition, an age-dependent NDP effect on glial reactivity was observed only in 3xTg mice whereas a global downregulation of p38 mitogen-activated protein kinase was selectively observed in 7 mo 5XFAD and 14 mo 3xTg mice.Conclusion: Our results suggest that MCR stimulation by NDP-alpha-MSH could represent a promising therapeutic strategy in managing cognitive decline also at late stage of AD, whereas the effects on neuroinflammation may be restricted to specific stages of AD progression.

2020 - Oxidative stress in Alzheimer's disease: In vitro therapeutic effect of amniotic fluid stem cells extracellular vesicles [Articolo su rivista]
Gatti, M.; Zavatti, M.; Beretti, F.; Giuliani, D.; Vandini, E.; Ottani, A.; Bertucci, E.; Maraldi, T.
abstract

Alzheimer's disease (AD) is characterized by abnormal protein aggregation, deposition of extracellular β-amyloid proteins (Aβ), besides an increase of oxidative stress. Amniotic fluid stem cells (AFSCs) should have a therapeutic potential for neurodegenerative disorders, mainly through a paracrine effect mediated by extracellular vesicles (EV). Here, we examined the effect of EV derived from human AFSCs (AFSC-EV) on the disease phenotypes in an AD neuron primary culture. We observed a positive effect of AFSC-EV on neuron morphology, viability, and Aβ and phospho-Tau levels. This could be due to the apoptotic and autophagic pathway modulation derived from the decrease in oxidative stress. Indeed, reactive oxygen species (ROS) were reduced, while GSH levels were enhanced. This modulation could be ascribed to the presence of ROS regulating enzymes, such as SOD1 present into the AFSC-EV themselves. This study describes the ROS-modulating effects of extracellular vesicles alone, apart from their deriving stem cell, in an AD in vitro model, proposing AFSC-EV as a therapeutic tool to stop the progression of AD.

2019 - Mechanisms of Hydrogen Sulfide against the Progression of Severe Alzheimer’s Disease in Transgenic Mice at Different Ages [Articolo su rivista]
Vandini, Eleonora; Ottani, Alessandra; Zaffe, Davide; Calevro, Anita; Canalini, Fabrizio; Cavallini, Gian Maria; Rossi, Rosario; Guarini, Salvatore; Giuliani, Daniela
abstract

Abstract Backgroud: Alzheimer disease is an age-related severe neurodegenerative pathology. The level of the third endogenous gas, hydrogen sulfide (H2S), is decreased in the brain of Alzheimer’s disease (AD) patients compared with the brain of the age-matched normal individuals; also, plasma H2S levels are negatively correlated with the severity of AD. Recently, we have demonstrated that systemic H2S injections are neuroprotective in an early phase of preclinical AD. Objectives: This study focuses on the possible neuroprotection of a chronic treatment with an H2S donor and sulfurous water (rich of H2S) in a severe transgenic 3×Tg-AD mice model. Method: 3×Tg-AD mice at 2 different ages (6 and 12 months) were daily treated intraperitoneally with an H2S donor and sulfurous water (rich of H2S) for 3 months consecutively. We investigated the cognitive ability, brain morphological alterations, amyloid/tau cascade, excitotoxic, inflammatory and apoptotic responses. Results: Three months of treatments with H2S significantly protected against impairment in learning and memory in a severe 3×Tg-AD mice model, at both ages studied, and reduced the size of Amyloid β plaques with preservation of the morphological picture. This neuroprotection appeared mainly in the cortex and hippocampus, associated with reduction in activity of c-jun N-terminal kinases, extracellular signal-regulated kinases and p38, which have an established role not only in the phosphorylation of tau protein but also in the inflammatory and excitotoxic response. Conclusion: Our findings indicate that appropriate treatments with various sources of H2S, might represent an innovative approach to counteract early and severe AD progression in humans.

2018 - Melanocortin receptor-4 and glioblastoma cells: effects of the selective antagonist ML00253764 alone and in combination with temozolomide on cell proliferation and apoptosis. [Articolo su rivista]
Vaglini, F; Pardini, C; Di Desidero, T; Orlandi, P; Pasqualetti, F; Ottani, A; Giuliani, D; Guarini, S; Bocci, G
abstract

Currently, no description of melanocortin receptor-4 (MC4R) expression or activity is available in human cancer cells, including glioblastoma (GBM). The aim of this study is to evaluate the presence of MC4Rs in GBM cells and the selective inhibition of their activity through the MC4R antagonist ML00253764 alone and in association with temozolomide in vitro and in vivo. MC4R genotyping and gene expression were performed on human GBM cells (U-87 and U-118) with real-time PCR. MC4R western blotting, immunohistochemistry, and immunofluorescence were obtained in both cell lines and in human tissues. Proliferation, cell cycle, and apoptotic assays were performed with ML00253764, whereas the synergism of the simultaneous combination with temozolomide was evaluated by the combination index method. ERK1/2 and Akt phosphorylation were quantified by ELISA. In vivo experiments were performed in U-87 xenografted nude mice. Both GBM cell lines and tumor tissues expressed MC4R receptors. The selective antagonist ML00253764 determined an antiproliferative and proapoptotic activity through the inhibition of the phosphorylation of ERK1/2 and Akt. Moreover, the simultaneous combination of temozolomide and ML00253764 determined a highly synergistic effect on GBM cells. The same combination in vivo showed a strong and significant decrease of GBM tumor volumes if compared to the single drug treatments, with an excellent tolerability profile. In conclusion, MC4R is present in GBM cells and its selective inhibition determined antiproliferative and proapoptotic effects, through the inhibition of ERK1/2 and Akt phosphorylation, and the synergistic enhancement of temozolomide effects in vitro and in vivo.

2017 - Effects of COX1-2/5-LOX blockade in Alzheimer transgenic 3xTg-AD mice [Articolo su rivista]
Bitto, Alessandra; Giuliani, Daniela; Pallio, Giovanni; Irrera, Natasha; Vandini, Eleonora; Canalini, Fabrizio; Zaffe, Davide; Ottani, Alessandra; Minutoli, Letteria; Rinaldi, Mariagrazia; Guarini, Salvatore; Squadrito, Francesco; Altavilla, Domenica
abstract

Objective and design: Alzheimer’s disease (AD) is associated with amyloid plaques (Aβ) and hyperphosphorylated tau protein tangles in the brain. We investigated the possible neuroprotective role of flavocoxid, a dual inhibitor of cyclooxygenases-1/2 (COX-1/2) and 5-Lipoxygenase (5-LOX), in triple-transgenic (3xTg-AD) mice. Subjects: Mice were 3 months at the beginning of the study. Treatment: Animals received once daily for 3-month saline solution or flavocoxid (20 mg/kg/ip). Methods: Morris water maze was used to assess learning and memory. Histology was performed to evidence Aβ plaques and neuronal loss, while inflammatory proteins were determined by western blot analysis. Results: Saline-treated 3xTg-AD mice showed an impairment in spatial learning and memory (assessed at 6 months of age), and increased expression of inflammatory and apoptotic molecules. Treatment of 3xTg-AD mice with flavocoxid reduced: (1) learning and memory loss; (2) the increased eicosanoid production and the phosphorylation level of amyloid precursor protein (APP-pThr668), Aβ 1–42, p-tau (pThr181), pERK, and the activation of the NLRP3 inflammasome; (3) Aβ plaques; and (4) neuronal loss, compared to saline-treated animals. Conclusions: Pharmacological blockade of both COX-1/2 and 5-LOX was able to counteract the progression of AD by targeting pathophysiological mechanisms up- and downstream of Aβ and tau.

2017 - Multiple beneficial effects of melanocortin MC4 receptor agonists in experimental neurodegenerative disorders: Therapeutic perspectives [Articolo su rivista]
Giuliani, Daniela; Ottani, Alessandra; Neri, Laura; Zaffe, Davide; Grieco, Paolo; Jochem, Jerzy; Cavallini, Gian Maria; Catania, Anna; Guarini, Salvatore
abstract

Melanocortin peptides induce neuroprotection in acute and chronic experimental neurodegenerative conditions. Melanocortins likewise counteract systemic responses to brain injuries. Furthermore, they promote neurogenesis by activating critical signaling pathways. Melanocortin-induced long-lasting improvement in synaptic activity and neurological performance, including learning and memory, sensory-motor orientation and coordinated limb use, has been consistently observed in experimental models of acute and chronic neurodegeneration. Evidence indicates that the neuroprotective and neurogenic effects of melanocortins, as well as the protection against systemic responses to a brain injury, are mediated by brain melanocortin 4 (MC4) receptors, through an involvement of the vagus nerve. Here we discuss the targets and mechanisms underlying the multiple beneficial effects recently observed in animal models of neurodegeneration. We comment on the potential clinical usefulness of melanocortin MC4 receptor agonists as neuroprotective and neuroregenerative agents in ischemic stroke, subarachnoid hemorrhage, traumatic brain injury, spinal cord injury, and Alzheimer's disease.

2016 - Involvement of the histaminergic system in the resuscitating effect of centrally acting leptin in haemorrhagic shock in rats [Articolo su rivista]
Jochem, J.; Altinbas, B.; Yalcin, M.; Ottani, Alessandra; Giuliani, Daniela; Savci, V.; Kasperska Zajac, A.; Guarini, Salvatore
abstract

Leptin, acting centrally as a neuromodulator, induces the activation of the sympathetic nervous system, which may lead to a pressor action in normotensive animals. In haemorrhagic shock, leptin administered intracerebroventricularly (icv.) evokes the resuscitating effect, with long-lasting rises in mean arterial pressure (MAP) and heart rate (HR), subsequent increase in peripheral blood flows, and a 100% survival at 2 h. Since leptin is able to activate histaminergic neurons, and centrally acting histamine also induces the resuscitating effect with the activation of the sympathetic nervous system, in the present study, we investigated an involvement of the histaminergic system in leptin-evoked cardiovascular effects in haemorrhagic shock. The model of irreversible haemorrhagic shock, with MAP decreased to and stabilised at 20 - 25 mmHg, has been used. Leptin (20 μg) given icv. at 5 min of critical hypotension evoked 181.5% increase in extracellular hypothalamic histamine concentration during the first 10 min after injection. Rises in MAP, HR and renal, mesenteric and hindquarters blood flows induced by leptin were inhibited by icv. pre-treatment with histamine H1 receptor antagonist chlorpheniramine (50 nmol). In contrast, there was no effect of H2, H3 and H4 receptor antagonists ranitidine (25 nmol), VUF 5681 (25 nmol) and JNJ 10191584 (25 nmol), respectively. In conclusion, the histaminergic system is involved in centrally-acting leptin-induced resuscitating effect in haemorrhagic shock in rats.

2015 - INVOLVEMENT OF THE HISTAMINERGIC SYSTEM IN CENTRALLY-ACTING LEPTIN-EVOKED RESUSCITATING EFFECT IN HAEMORRHAGIC SHOCK IN RATS [Abstract in Rivista]
Jochem, J; Altinbas, B; Yalcin, M; Ottani, Alessandra; Giuliani, Daniela; Savci, V; Kasperska Zajac, A; Guarini, Salvatore
abstract

INVOLVEMENT OF THE HISTAMINERGIC SYSTEM IN CENTRALLY-ACTING LEPTIN-EVOKED RESUSCITATING EFFECT IN HAEMORRHAGIC SHOCK IN RATS

2015 - Melanocortins promote neurogenesis and counteract cognitive decline in a transgenic mouse model of moderate Alzheimer’s disease [Abstract in Rivista]
Neri, Laura; Canalini, Fabrizio; Calevro, Anita; Ottani, Alessandra; Vandini, Eleonora; Sena, Paola; Zaffe, Davide; Giuliani, Daniela; Guarini, Salvatore
abstract

Alzheimer's disease (AD), both sporadic and genetic, is a chronic disorder characterized by activation of the amyloid/tau cascade in the hippocampus and isocortex. Besides neuroprotective approaches, also neurorestorative strategies for AD are under intensive investigations. [1] The melanocortin system consists of endogenous neuropeptides of the adrenocorticotropin/melanocyte-stimulating hormone (ACTH/MSH) family, acting via five different metabotropic melanocortin receptor subtypes (MC1-MC5). Melanocortins also induce neuroprotection associated with long-lasting functional recovery and counteraction of cognitive decline, as found in acute experimental neurodegenerative conditions and more recently in a chronic neurodegenerative disease as AD. [2] Further, these endogenous peptides have been by us reported to stimulate neurogenesis in an acute neurodegenerative disorder as ischemic stroke. [3] Here we investigated the possible neuroprotective and neurogenic effect of melanocortins in AD with a medium level of severity by using 24 week-old (at the start of the study) APPSwe transgenic mice (Tg2576). METHODS: Tg2576 mice were treated (once daily on days 1-50) with a nanomolar dose of the melanocortin analog [Nle4,D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH). Animals were prepared for 5-bromo-2’-deoxyuridine (BrdU) labeling of proliferating cells at days 1-11 of the study, and histological and immunohistochemical studies of the brain were performed for the assessment of neurogenesis. Further, the mouse ability to learn and recall was evaluated by means of the Morris water-maze test at the twenty-seventh week (starting 14 days after the first BrdU injection) and thirty-first week of age. Within 90 min the end of the last behavioural test (day 50 of the study; 31 week-old mice) animals were killed and the brains were removed and processed for histological examination. The whole hippocampi were dissected from brains of some animals to perform western blot analysis of the Zif268 protein (Zif268 protein is transiently expressed after synaptic activation). All values were analyzed by means of two-way repeated measures ANOVA (behavioral data) or one-way ANOVA (all other data), both followed by the Student-Newman-Keuls’ test. A value of p < 0.05 was considered significant. RESULTS: Treatment of Tg2576 mice with the melanocortin analog [Nle4,D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH) reduced cerebral cortex/hippocampus level of Aβ deposit (p < 0.001), increased hippocampus Zif268 expression (p <0.001), improved brain histological picture and cognitive functions (p <0.001), relative to saline-treated Tg2576 animals, and no signs of toxicity were recorded. Further, immunohistochemical examination of the hippocampus on day 50 (end of the study) showed, in the dentate gyrus of NDP-α-MSH-treated Tg2576 mice, a very elevated number of BrdU immunoreactive cells colocalized with NeuN (indicator of mature neurons) and Zif268 (indicator of functionally integrated neurons), in comparison with saline-treated Tg2576 animals (p <0.001); no newly formed astrocytes were found. Animal pretreatment (before each administration of NDP-α-MSH) with the selective melanocortin MC4 receptor antagonist HS024 prevented all favourable effects of NDP-α-MSH (p <0.001). CONCLUSIONS: Our data suggest that MC4 receptor-stimulating melanocortins are able to counteract cognitive decline in experimental AD not only by affording neuroprotection, but also by inducing intense neurogenesis. These agents could be candidates for an innovative and safe strategy to counteract AD progression in humans.

2015 - NDP-α-MSH attenuates heart and liver responses to myocardial reperfusion via the vagus nerve and JAK/ERK/STAT signaling [Articolo su rivista]
Ottani, Alessandra; Giuliani, Daniela; Neri, Laura; Calevro, Anita; Canalini, Fabrizio; Vandini, Eleonora; Cainazzo, Maria Michela; Ruberto, Ippazio Antonio; Barbieri, Alberto; Rossi, Rosario; Guarini, Salvatore
abstract

Melanocortin peptides afford cardioprotection during myocardial ischemia/reperfusion via janus kinases (JAK), extracellular signal-regulated kinases (ERK) and signal transducers/activators of transcription (STAT) pathways. Here we investigated whether melanocortin-induced modulation of the JAK/ERK/STAT signaling occurs via the cholinergic anti-inflammatory pathway, focusing our study on cardiac and hepatic responses to prolonged myocardial ischemia/reperfusion. Ischemia was produced in rats by ligature of the left anterior descending coronary artery for 30min; effects of ischemia/reperfusion were evaluated using Western blot of heart and liver proteins. Intravenous treatment, during coronary artery occlusion, with the melanocortin analog (Nle(4), D-Phe(7))α-melanocyte-stimulating hormone (NDP-α-MSH) induced a left ventricle up-regulation of the cardioprotective transcription factors pJAK2, pERK1/2 and pTyr-STAT3 (JAK-dependent), and a reduction in the levels of the inflammatory mediators tumor necrosis factor-α (TNF-α) and pJNK (a transcription factor also involved in apoptosis), as assessed at the end of the 2-h reperfusion period. Further, these beneficial effects of NDP-α-MSH were associated with heart over-expression of the pro-survival proteins heme oxygenase-1 (HO-1) and Bcl-XL, and decrease of ventricular arrhythmias and infarct size. In the liver NDP-α-MSH induced a decrease in the pJAK2 and pTyr-STAT3 levels, and strongly reduced pERK1/2 expression. In the liver of ischemic rats NDP-α-MSH also blunted pJNK activity and TNF-α expression, and up-regulated Bcl-XL. Bilateral cervical vagotomy prevented all effects of NDP-α-MSH, both in the heart and liver. These results indicate that melanocortins inhibit heart and liver damage triggered by prolonged myocardial ischemia/reperfusion likely, as main mechanism, via the vagus nerve-mediated modulation of the JAK/STAT/ERK signaling pathways.

2015 - NDP-α-MSH induces intense neurogenesis and cognitive recovery in Alzheimer transgenic mice through activation of melanocortin MC4 receptors [Articolo su rivista]
Giuliani, Daniela; Neri, Laura; Canalini, Fabrizio; Calevro, Anita; Ottani, Alessandra; Vandini, Eleonora; Sena, Paola; Zaffe, Davide; Guarini, Salvatore
abstract

Melanocortins exert neuroprotection in a variety of experimental neurodegenerative disorders, including Alzheimer's disease (AD). Further, in previous research we showed that these endogenous peptides stimulate neurogenesis in an acute neurodegenerative disorder such as ischemic stroke. In the present research, we investigated the potential neurogenic effect of melanocortins in AD using APPSwe transgenic mice (Tg2576). To this purpose, 24week-old animals were prepared for 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells on days 1-11 of the study. Treatment of Tg2576 mice with nanomolar doses of the melanocortin analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH), administered once daily from day 1 to 50, improved brain histology and cognitive functions relative to saline-treated Tg2576 animals. No signs of toxicity were observed. Immunohistochemical examination of the hippocampus at the end of the study (day 50) showed that NDP-α-MSH-treated Tg2576 mice had a greater number of BrdU immunoreactive cells colocalized with NeuN (an indicator of mature neurons) and Zif268 (an indicator of functionally integrated neurons) in the dentate gyrus, relative to saline-treated Tg2576 animals; no newly formed astrocytes were found. Animal pretreatment with selective melanocortin MC4 receptor antagonist HS024 before each NDP-α-MSH administration prevented all the beneficial effects of the peptide. The present data indicate that MC4 receptor stimulation by a melanocortin prevents cognitive decline in experimental AD, this effect being associated not only with neuroprotection but also with an intense neurogenesis. MC4 receptor agonists could be innovative and safe candidates to counteract AD progression in humans.

2014 - Melanocortins protect against brain damage and counteract cognitive decline in a transgenic mouse model of moderate Alzheimer׳s disease. [Articolo su rivista]
Giuliani, Daniela; Galantucci, M; Neri, L; Canalini, F; Calevro, Anita; Bitto, A; Ottani, Alessandra; Vandini, E; Sena, Paola; Sandrini, Maurizio; Squadrito, F; Zaffe, Davide; Guarini, Salvatore
abstract

We previously reported that melanocortins induce neuroprotection in experimental acute and chronic neurodegenerative conditions, including Alzheimer׳s disease (AD) of mild severity. Here we investigated whether melanocortins afford neuroprotection and counteract cognitive decline in AD with a medium level of severity by using 24 week-old (at the start of the study) APPSwe transgenic mice (Tg2576). Saline-treated (days 1-50) control Tg2576 mice showed an impairment in spatial learning and memory, associated (at day 50, end of the study) with hippocampus at low levels of the synaptic activity-dependent gene Zif268, relevant brain changes such as cerebral cortex/hippocampus increased level of β-amyloid (Aβ) deposit, and neuronal loss, in comparison with wild-type animals. Treatment of Tg2576 mice (once daily at days 1-50) with a nanomolar dose of the melanocortin analog [Nle4,D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH) reduced cerebral cortex/hippocampus level of Aβ deposit, decreased neuronal loss, increased hippocampus Zif268 expression and improved cognitive functions, relative to saline-treated Tg2576 mice. Pharmacological blockade of melanocortin MC4 receptors with the MC4 receptor antagonist HS024 prevented all favorable effects of NDP-α-MSH. Our data indicate that MC4 receptor-stimulating melanocortins are able to counteract cognitive decline in experimental AD of medium severity through induction of neuroprotection and improvement of synaptic transmission. After further studies, these agents could gain a role as disease modifying therapeutics for AD.

2014 - Melanocortins protect against progression of Alzheimer's disease in triple-transgenic mice by targeting multiple pathophysiological pathways [Articolo su rivista]
Giuliani, Daniela; A., Bitto; M., Galantucci; Zaffe, Davide; Ottani, Alessandra; N., Irrera; L., Neri; Cavallini, Gian Maria; D., Altavilla; A. R., Botticelli; F., Squadrito; Guarini, Salvatore
abstract

Besides specific triggering causes, Alzheimer's disease (AD) involves pathophysiological pathways that are common to acute and chronic neurodegenerative disorders. Melanocortins induce neuroprotection in experimental acute neurodegenerative conditions, and low melanocortin levels have been found in occasional studies performed in AD-type dementia patients. Here we investigated the possible neuroprotective role of melanocortins in a chronic neurodegenerative disorder, AD, by using 12-week-old (at the start of the study) triple-transgenic (3xTg-AD) mice harboring human transgenes APP(Swe), PS1(M146V), and tau(P301L). Treatment of 3xTg-AD mice, once daily until the end of the study (30 weeks of age), with the melanocortin analog [Nle(4),D-Phe(7)]-alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) reduced cerebral cortex/hippocampus phosphorylation/level of all AD-related biomarkers investigated (mediators of amyloid/tau cascade, oxidative/nitrosative stress, inflammation, apoptosis), decreased neuronal loss, induced over-expression of the synaptic activity-dependent gene Zif268, and improved cognitive functions, relative to saline-treated 3xTg-AD mice. Pharmacological blockade of melanocortin MC4 receptors prevented all neuroprotective effects of NDP-alpha-MSH. Our study identifies, for the first time, a class of drugs, MC4 receptor-stimulating melanocortins, that are able to counteract the progression of experimental AD by targeting pathophysiological mechanisms up- and down-stream of beta-amyloid and tau. These data could have important clinical implications. (C) 2014 Elsevier Inc. All rights reserved.

2014 - Protective effects of the melanocortin analog NDP-α-MSH in rats undergoing cardiac arrest [Articolo su rivista]
Ottani, Alessandra; Neri, Laura; Canalini, Fabrizio; Calevro, Anita; Rossi, Rosario; Cappelli, Gianni; Ballestri, M; Giuliani, Daniela; Guarini, Salvatore
abstract

We previously reported that melanocortins afford cardioprotection in conditions of experimental myocardial ischemia/reperfusion, with involvement of the janus kinases (JAK), extracellular signal-regulated kinases (ERK) and signal transducers and activators of transcription (STAT) signalings. We investigated the influence of the melanocortin analog [Nle(4), D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH) on short-term detrimental responses to cardiac arrest (CA) induced in rats by intravenous (i.v.) administration of potassium chloride, followed by cardiopulmonary resuscitation (CPR) plus epinephrine treatment. In CA/CPR rats i.v. treated with epinephrine (0.1mg/kg) and returned to spontaneous circulation (48%) we recorded low values of mean arterial pressure (MAP) and heart rate (HR), alteration of hemogasanalysis parameters, left ventricle low expression of the cardioprotective transcription factors pJAK2 and pTyr-STAT3 (JAK-dependent), increased oxidative stress, up-regulation of the inflammatory mediators tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and down-regulation of the anti-inflammatory cytokine IL-10, as assessed at 1h and 3h after CPR. On the other hand, i.v. treatment during CPR with epinephrine plus NDP-α-MSH (340μg/kg) almost completely restored the basal conditions of MAP and HR, reversed metabolic acidosis, induced left ventricle up-regulation of pJAK2, pTyr-STAT3 and IL-10, attenuated oxidative stress, down-regulated TNF-α and IL-6 levels, and improved survival rate by 81%. CA/CPR plus epinephrine alone or in combination with NDP-α-MSH did not affect left ventricle pSer-STAT3 (ERK1/2-dependent) and pERK1/2 levels. These results indicate that melanocortins improve return to spontaneous circulation, reverse metabolic acidosis, and inhibit heart oxidative stress and inflammatory cascade triggered by CA/CPR, likely via activation of the JAK/STAT signaling pathway.

2013 - Hydrogen sulfide slows down progression of experimental Alzheimer's disease by targeting multiple pathophysiological mechanisms. [Articolo su rivista]
Giuliani, Daniela; Ottani, Alessandra; Zaffe, Davide; Galantucci, M; Strinati, F; Lodi, Renzo; Guarini, Salvatore
abstract

It has been previously reported that brain hydrogen sulfide (H2S) synthesis is severely decreased in Alzheimer's disease (AD) patients, and plasma H2S levels are negatively correlated with the severity of AD. Here we extensively investigated whether treatment with a H2S donor and spa-waters rich in H2S induces neuroprotection and slows down progression of AD. Studies with sodium hydrosulfide (a H2S donor) and Tabiano's spa-water were carried out in three experimental models of AD. Short-term and long-term treatments with sodium hydrosulfide and/or Tabiano's spa-water significantly protected against impairment in learning and memory in rat models of AD induced by brain injection of β-amyloid1-40 (Aβ) or streptozotocin, and in an AD mouse model harboring human transgenes APPSwe, PS1M146V and tauP301L (3xTg-AD mice). The improvement in behavioral performance was associated with hippocampus was size of Aβ plaques and preservation of the morphological picture, as found in AD rats. Further, lowered concentration/phosphorylation levels of proteins thought to be the central events in AD pathophysiology, namely amyloid precursor protein, presenilin-1, Aβ1-42 and tau phosphorylated at Thr181, Ser396 and Ser202, were detected in 3xTg-AD mice treated with spa-water. The excitotoxicity-triggered oxidative and nitrosative stress was counteracted in 3xTg-AD mice, as indicated by the decreased levels of malondialdehyde and nitrites in the cerebral cortex. Hippocampus reduced activity of c-jun N-terminal kinases, extracellular signal-regulated kinases and p38, which have an established role not only in phosphorylation of tau protein but also in inflammation and apoptosis, was also found. Consistently, decrease in tumor necrosis factor-α level, up-regulation of Bcl-2, and down-regulation of BAX and the downstream executioner caspase-3, also occurred in the hippocampus of 3xTg-AD mice after treatment with Tabiano's spa-water, thus suggesting that it is also able to modulate inflammation and apoptosis. Our findings indicate that appropriate treatments with H2S donors and Tabiano's spa-waters, and may be other spa-waters rich in H2S content, might represent an innovative approach to slow down AD progression in humans by targeting multiple pathophysiological mechanisms.

2013 - Modulation of the JAK/ERK/STAT signaling in melanocortin-induced inhibition of local and systemic responses to myocardial ischemia/reperfusion [Articolo su rivista]
Ottani, Alessandra; Maria, Galantucci; Ettore, Ardimento; Laura, Neri; Canalini, Fabrizio; Calevro, Anita; Zaffe, Davide; Ettore, Novellino; Paolo, Grieco; Giuliani, Daniela; Guarini, Salvatore
abstract

The janus kinases (JAK), extracellular signal-regulated kinases (ERK) and signal transducers and activators of transcription (STAT) pathways have been shown to play a cardioprotective role. We previously gave evidence that melanocortins afford cardioprotection in conditions of myocardial ischemia/reperfusion. Here we aimed to investigate the influence of melanocortins on the JAK/ERK/STAT signaling in cardiac and systemic responses to prolonged myocardial ischemia/reperfusion. Ischemia was produced in rats by ligature of the left anterior descending coronary artery for 30 min. At the end of the 2-h reperfusion, western blot analysis of the cardioprotective transcription factors pJAK2, pERK1/2, pTyr-STAT3 and pSer-STAT3, the inflammatory mediator tumor necrosis factor-α (TNF-α), the pro-apoptotic factors BAX and c-jun N-terminal kinases (pJNK), the anti-apoptotic protein Bcl-XL, as well as of the cardioprotective enzyme heme oxygenase-1 (HO-1), was performed in the left ventricle and spleen. Intravenous treatment, during coronary artery occlusion, with the melanocortin analogs [Nle4, D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH) and adrenocorticotropic hormone 1-24 [ACTH-(1-24)], induced a left ventricle up-regulation of pJAK2, pERK1/2 and pTyr-STAT3 (JAK-dependent), and a reduction in pJNK and TNF-α levels; these effects of NDP-α-MSH and ACTH-(1-24) were associated with over-expression of the pro-survival proteins HO-1 and Bcl-XL, and marked decrease of the myocardial infarct size. Melanocortin treatment did not affect left ventricle pSer-STAT3 (ERK1/2-dependent) and BAX levels. In the spleen, NDP-α-MSH and ACTH-(1-24) induced similar effects on the expression of the above transcription factors/proteins, except for pERK1/2 (down-regulated) and HO-1 (unaffected). Blockade of JAK and ERK pathways with AG490 and U0126, respectively, abrogated the myocardial infarct size reduction by NDP-α-MSH. These results indicate that melanocortins inhibit local and systemic inflammatory and apoptotic cascades triggered by prolonged myocardial ischemia/reperfusion, with consequent reduction in myocardium infarct size, seemingly via activation of the JAK/STAT signaling and with modulation of an ERK (STAT unrelated) signaling pathway.

2013 - Up-regulation of the canonical Wnt-3A and Sonic hedgehog signaling underlies melanocortin-induced neurogenesis after cerebral ischemia [Articolo su rivista]
Spaccapelo, Luca; Galantucci, Maria; Neri, Laura; Contri, Miranda; R., Pizzala; D'Amico, Roberto; Ottani, Alessandra; Sandrini, Maurizio; Zaffe, Davide; Giuliani, Daniela; Guarini, Salvatore
abstract

In experimental cerebral ischemia, melanocortin MC4 receptor agonists induce neuroprotection and neurogenesis with subsequent long-lasting functional recovery. Here we investigated the molecular mechanisms underlying melanocortin-induced neurogenesis. Gerbils were subjected to transient global cerebral ischemia, then they were treated every 12 h, and until sacrifice, with 5-bromo-2'-deoxyuridine (BrdU; to label proliferating cells), and the melanocortin analog [Nle(4),D-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) or saline. NDP-alpha-MSH increased hippocampus dentate gyrus (DG) expression of Wnt-3A, beta-catenin, Sonic hedgehog (Shh), Zif268, interleukin-10 (IL-10) and doublecortin (DCX), as detected at days 3, 6 and 10 after the ischemic insult. Further, an elevated number of BrdU immunoreactive cells was found at days 3 and 10, and an improved histological picture with reduced neuronal loss at day 10, associated with learning and memory recovery. Pharmacological blockade of the Wnt-3A/beta-catenin and Shh pathways, as well as of melanocortin MC4 receptors, prevented all effects of NDP-alpha-MSH. These data indicate that, in experimental brain ischemia, treatment with melanocortins acting at MC4 receptors induces neural stem/progenitor cell proliferation in the DG by promptly and effectively triggering the canonical Wnt-3A/beta-catenin and Shh signaling pathways. Activation of these pathways is associated with up-regulation of the repair factor Zif268 and the neurogenesis facilitating factor IL-10, and it seems to address mainly toward a neuronal fate, as indicated by the increase in DCX positive cells.

2012 - Centrally acting leptin induces a resuscitating effect in haemorrhagic shock in rats. [Articolo su rivista]
Jochem, J; Kalarus, Z; Spaccapelo, Luca; Canalini, F; Ottani, Alessandra; Giuliani, Daniela; Guarini, Salvatore
abstract

Centrally acting leptin induces the activation of the sympathetic nervous system with a pressor effect in normotensive rats. The purpose of the study was to examine central leptin-evoked action in critical haemorrhagic hypotension. In anaesthetized male Wistar rats subjected for irreversible haemorrhagic shock with mean arterial pressure (MAP) 20-25 mmHg haemodynamic parameters and plasma concentrations of adrenaline and noradrenaline were measured. Leptin given intracerebroventricularly (20 μg) evoked long-lasting rises in MAP and heart rate (HR), with a subsequent increase in renal, mesenteric and hindquarters blood flows and a 100% survival at 2 h. MAP and peripheral blood flow changes were inhibited by a pre-treatment with α(1)- and α(2)-adrenoceptor antagonists prazosin (0.5 mg/kg) and yohimbine (1 mg/kg), while β-adrenoceptor antagonist propranolol (1 mg/kg) blocked leptin-induced HR changes, without influence on MAP, peripheral blood flows and survival. Twenty min after leptin treatment, there were higher plasma concentrations of noradrenaline, but not adrenaline, in comparison with the saline-treated control group. In conclusion, centrally acting leptin induces a long-lasting pressor effect with an improvement in the survival rate in haemorrhage-shocked rats. The effect may be associated with the activation of the sympathetic nervous system.

2012 - Melanocortins as potential therapeutic agents in severe hypoxic conditions. [Articolo su rivista]
Giuliani, Daniela; Minutoli, L; Ottani, Alessandra; Spaccapelo, L; Bitto, A; Galantucci, M; Altavilla, D; Squadrito, F; Guarini, Salvatore
abstract

Melanocortin peptides with the adrenocorticotropin/melanocyte-stimulating hormone (ACTH/MSH) sequences and synthetic analogs have protective and life-saving effects in experimental conditions of circulatory shock, myocardial ischemia, ischemic stroke, traumatic brain injury, respiratory arrest, renal ischemia, intestinal ischemia and testicular ischemia, as well as in experimental heart transplantation. Moreover, melanocortins improve functional recovery and stimulate neurogenesis in experimental models of cerebral ischemia. These beneficial effects of ACTH/MSH-like peptides are mostly mediated by brain melanocortin MC(3)/MC(4) receptors, whose activation triggers protective pathways that counteract the main ischemia/reperfusion-related mechanisms of damage. Induction of signaling pathways and other molecular regulators of neural stem/progenitor cell proliferation, differentiation and integration seems to be the key mechanism of neurogenesis stimulation. Synthesis of stable and highly selective agonists at MC(3) and MC(4) receptors could provide the potential for development of a new class of drugs for a novel approach to management of severe ischemic diseases.

2012 - Molecular Changes Induced in Rat Liver by Hemorrhage and Effects of Melanocortin Treatment. [Articolo su rivista]
Lonati, C; Sordi, A; Giuliani, Daniela; Spaccapelo, Luca; Leonardi, P; Carlin, A; Ottani, Alessandra; Galantucci, Maria; Grieco, P; Catania, A; Guarini, Salvatore
abstract

BACKGROUND: Melanocortin peptides improve hemodynamic parameters and prevent death during severe hemorrhagic shock. In the present research we determined influences of a synthetic melanocortin 1/4 receptor agonist on the molecular changes that occur in rat liver during hemorrhage.METHODS: Controlled-volume hemorrhage was performed in adult rats under general anesthesia by a stepwise blood withdrawal until mean arterial pressure fell to 40 mmHg. Then rats received either saline or the synthetic melanocortin 1/4 receptor agonist Butir-His-D-Phe-Arg-Trp-Sar-NH2 (Ro27-3225; n = 6-8 per group). Hemogasanalysis was performed throughout a 60-min period. Gene expression in liver samples was determined at 1 or 3 h using quantitative real-time polymerase chain reaction.RESULTS: At 1 h, in saline-treated shocked rats, there were significant increases in activating transcription factor 3 (Atf3), early growth response 1 (Egr1), heme oxygenase (decycling) 1 (Hmox1), FBJ murine osteosarcoma viral oncogene homolog (Fos), and jun oncogene (Jun). These changes were prevented by Ro27-3225 (mean ± SEM: Atf3 152.83 ± 58.62 vs. 579.00 ± 124.13, P = 0.002; Egr1 13.21 ± 1.28 vs. 26.63 ± 1.02, P = 0.001; Hmox1 3.28 ± 0.31 vs. 166.54 ± 35.03, P = 0.002; Fos 4.36 ± 1.03 vs. 14.90 ± 3.44, P < 0.001; Jun 6.62 ± 1.93 vs. 15.07 ± 2.09, P = 0.005; respectively). Increases in alpha-2-macroglobulin (A2m), heat shock 70kD protein 1A (Hspa1a), erythropoietin (Epo), and interleukin-6 (Il6) occurred at 3 h in shocked rats and were prevented by Ro27-3225 treatment (A2m 6.90 ± 0.82 vs. 36.73 ± 4.00, P < 0.001; Hspa1a 10.34 ± 3.28 vs. 25.72 ± 3.64, P = 0.001; Epo 0.49 ± 0.13 vs. 2.37 ± 0.73, P = 0.002; Il6 1.05 ± 0.15 vs. 1.88 ± 0.23, P < 0.001; respectively). Further, at 3 h in shocked rats treated with Ro27-3225 there were significant increases in tight junction protein 1 (Tjp1; 27.30 ± 2.43 vs. 5.03 ± 1.68, P < 0.001) and nuclear receptor subfamily 4, group A, member 1 (Nr4a1; 91.03 ± 16.20 vs. 30.43 ± 11.0, P = 0.01) relative to sham animals. Treatment with Ro27-3225 rapidly restored blood pressure, hemogasanalysis parameters, and lactate blood levels.CONCLUSIONS: Melanocortin treatment significantly prevents most of the systemic and hepatic detrimental changes induced by hemorrhage.

2012 - Protective effects of melanocortins on short-term changes in a rat model of traumatic brain injury [Articolo su rivista]
Alessandra, Bitto; Francesca, Polito; Natasha, Irrera; Margherita, Calo`; Luca, Spaccapelo; Herbert R., Marini; Giuliani, Daniela; Ottani, Alessandra; Mariagrazia, Rinaldi; Letteria, Minutoli; Guarini, Salvatore; Francesco, Squadrito; Domenica, Altavilla
abstract

Objective: Treatment for traumatic brain injury remains elusivedespite compelling evidence from animal models for a variety oftherapeutic targets. Melanocortins have established neuroprotective effects against experimental ischemic stroke. We investigated whether melanocortin treatment of traumatic brain injury induces neuroprotection and promotes functional recovery.Design: Randomized experiment.Setting: Research laboratory at a university hospital.Subjects: Male Sprague-Dawley rats (n=215).Interventions: Experimental rat model of diffuse traumaticbrain injury, the impact-acceleration model.Measurement and Main Results: Brain tissue nitrites, phosphorylation level of extracellular signal-regulated kinases, and c-jun N-terminal kinases; and expression of active caspase-3,tumor necrosis factor-alpha, BAX, and Bcl-2 as well as serum levels of interleukin-6, high mobility group box-1, interleukin-10, and brain histologic damage were evaluated 24 or 48 hrs after theinsult. Sensorimotor orientation and limb use were evaluated atday 7 and learning and memory at days 23–30 after injury.Posttraumatic treatment every 12 hrs with the melanocortin analog [Nle4, D-Phe7]-alpha-melanocyte-stimulating hormone (starting 3 or 6 hrs after injury) inhibited traumatic brain injury-induced upregulation of nitric oxide synthesis, phosphorylation level of extracellular signal-regulated kinases, phosphorylation level of c-jun N-terminal kinases, and active caspase-3; reduced expressions/levels of tumor necrosis factor-alpha, BAX, interleukin-6, and high mobility group box-1; and increased those of Bcl-2 and interleukin-10. These molecular changes were associated with a reduction in brain tissue damage, as highlighted by histopathological findings and improved functional recovery. Pretreatment with the melanocortin MC4 receptor antagonist HS024 abated the positive effects of [Nle4, D-Phe7]-alpha-melanocyte-stimulating hormone.Conclusions: Our data indicate that melanocortins protectagainst traumatic brain injury, in a broad time window andthrough activation of MC4 receptors, by counteracting the maintraumatic brain injury-related mechanisms of damage. Thesefindings could have major clinical implications.

2011 - Melanocortin MC(4) receptor agonists counteract late inflammatory and apoptotic responses and improve neuronal functionality after cerebral ischemia. [Articolo su rivista]
Spaccapelo, L; Bitto, A; Galantucci, M; Ottani, Alessandra; Irrera, N; Minutoli, L; Altavilla, D; Novellino, E; Grieco, P; Zaffe, Davide; Squadrito, F; Giuliani, Daniela; Guarini, Salvatore
abstract

Indirect evidence indicates that, in cerebral ischemia, melanocortins have neuroprotective effects likely mediated by MC(4) receptors. To gain direct insight into the role of melanocortin MC(4) receptors in ischemic stroke, we investigated the effects of a highly selective MC(4) receptor agonist. Gerbils were subjected to transient global cerebral ischemia by occluding both common carotid arteries for 10min. In saline-treated stroke animals, an impairment in learning and memory occurred that, at day 11 after stroke, was associated with hippocampus up-regulation of tumor necrosis factor-α (TNF-α), BAX, activated extracellular signal-regulated kinases (ERK1/2), c-jun N-terminal kinases (JNK1/2) and caspase-3, down-regulation of Bcl-2, and neuronal loss. Treatment for 11days with the selective melanocortin MC(4) receptor agonist RO27-3225, as well as with the well known non-selective [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH) as a reference non-selective melanocortin, counteracted the inflammatory and apoptotic responses, as indicated by the changes in TNF-α, BAX, ERK1/2, JNK1/2, caspase-3 and Bcl-2 protein expression. Furthermore, melanocortin treatment reduced neuronal loss and dose-dependently improved learning and memory. These positive effects were associated with overexpression of Zif268, an immediate early gene involved in injury repair, synaptic plasticity and memory formation. Pharmacological blockade of MC(4) receptors with the selective MC(4) receptor antagonist HS024 prevented all effects of RO27-3225 and NDP-α-MSH. These data give direct evidence that stimulation of MC(4) receptors affords neuroprotection and promotes functional recovery from stroke, by counteracting prolonged and/or recurrent inflammatory and apoptotic responses, and likely by triggering brain repair pathways.

2011 - Melanocortin 4 receptor activation protects against testicular ischemia-reperfusion injury by triggering the cholinergic antiinflammatory pathway. [Articolo su rivista]
Minutoli, L.; Bitto, Alessandra; Squadrito, F.; Irrera, N.; Rinaldi, M.; Nicotina, P. A.; Arena, S.; Magno, C.; Marini, H.; Spaccapelo, Luca; Ottani, Alessandra; Giuliani, Daniela; Romeo, C.; Guarini, Salvatore; Antonuccio, P.; Altavilla, D.
abstract

Melanocortins (MC) trigger a vagus nerve-mediated cholinergic-antiinflammatory pathway projecting to the testis. We tested whether pharmacological activation of brain MC receptors might protect the testis from the damage induced by ischemia-reperfusion. Adult male rats were subjected to 1-h testicular ischemia, followed by 24-h reperfusion [testicular ischemia-reperfusion (TI/R)]. Before TI/R, groups of animals were subjected to bilateral cervical vagotomy, or pretreated with the nicotinic acetylcholine receptor antagonist chlorisondamine or the selective MC(4) receptor antagonist HS024. Immediately after reperfusion, rats were ip treated with saline or the MC analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH) (340 μg/kg). We evaluated testicular IL-6 and TNF-α by Western blot analysis and organ damage by light microscopy. Some experimental groups were prepared for neural efferent activity recording along the vagus nerve starting 30 min after treatment with NDP-α-MSH or saline, and for a 30-min period. Additional groups of TI/R rats were treated for 30 d with saline, NDP-α-MSH, chlorisondamine plus NDP-α-MSH, or HS024 plus NDP-α-MSH to evaluate spermatogenesis, organ damage, and the apoptosis machinery. After a 24-h reperfusion, in TI/R saline-treated rats, there was an increase in IL-6 and TNF-α expression and a marked damage in both testes. NDP-α-MSH inhibited IL-6 and TNF-α expression, decreased histological damage, and increased neural efferent activity. Furthermore, NDP-α-MSH administration for 30 d greatly improved spermatogenesis, reduced organ damage, and inhibited apoptosis. All positive NDP-α-MSH effects were abrogated by vagotomy, chlorisondamine, or HS024. Our data suggest that selective MC(4) receptor agonists might be therapeutic candidates for the management of testicular torsion.

2011 - Melanocortin 4 receptor stimulation decreases pancreatitis severity in rats by activation of the cholinergic anti-inflammatory pathway [Articolo su rivista]
Minutoli, L.; Squadrito, F.; Nicotina, P. A.; Giuliani, Daniela; Ottani, Alessandra; Polito, F.; Bitto, Alessandra; Irrera, N.; Guzzo, G.; Spaccapelo, Luca; Fazzari, C.; Macrì, A.; Marini, H.; Guarini, Salvatore; Altavilla, D.
abstract

OBJECTIVE: Acute pancreatitis is an inflammatory condition that may lead to multisystemic organ failure. Melanocortin peptides have been successfully used in experimental models of organ failure and shock, and their protective effect occurs through the activation of a vagus nerve-mediated cholinergic anti-inflammatory pathway by acting at brain melanocortin 4 receptors. In the light of these observations, we studied the effects of the selective melanocortin 4 receptor agonist RO27-3225 in an experimental model of cerulein-induced pancreatitis.DESIGN: Randomized experiment.SETTING: Research laboratory at a university hospital.SUBJECT: Experimental pancreatitis in rats.INTERVENTIONS: Acute pancreatitis was induced in male Sprague-Dawley rats by intraperitoneal injections of cerulein (80 μg/kg, four injections at hourly intervals). Before pancreatitis induction, groups of animals were subjected to bilateral cervical vagotomy, pretreated with the nicotinic acetylcholine receptor antagonist chlorisondamine or the selective melanocortin 4 receptor antagonist HS024, or not pretreated. Thirty minutes after the first cerulein injection, rats were intraperitoneally treated with a nanomolar dose of RO27-3225 or vehicle. Some experimental groups were prepared for neural efferent activity recording along the vagus nerve starting 30 mins after treatment with RO27-3225 or vehicle, and for a 30-min period.MEASUREMENTS AND MAIN RESULTS: Serum lipase and amylase activity, tumor necrosis factor-α and interleukin-6 expression, pancreatic myeloperoxidase activity, and histologic damage were evaluated; neural efferent activity of vagal fibers was also assessed. RO27-3225 reduced cerulein-induced serum lipase and amylase activity, blunted the expression of tumor necrosis factor-α and interleukin-6, abated the increase in pancreatic myeloperoxidase activity, and protected against histologic damage. Furthermore, RO27-3225 markedly increased neural efferent activity along the vagus nerve. Vagotomy, chlorisondamine, and HS024 abated these protective effects of RO27-3225.CONCLUSIONS: Our data show that melanocortin 4 receptor agonists reduce pancreatitis severity through the activation of the cholinergic anti-inflammatory pathway. These findings could be of particular interest in the clinical setting.

2011 - Melanocortins protect against multiple organ dysfunction syndrome in mice [Articolo su rivista]
BITTO, ALESSANDRA; Polito, F; Altavilla, D; Irrera, N; GIULIANI, Daniela; OTTANI, Alessandra; Minutoli, L; SPACCAPELO, Luca; GALANTUCCI, Maria; LODI, Renzo; Guzzo, G; GUARINI, Salvatore; Squadrito, F.
abstract

Background and purpose: Melanocortins reverse circulatory shock and improve survival by counteracting the systemic inflammatory response, and through the activation of the vagus nerve-mediated cholinergic anti-inflammatory pathway. To gain insight into the potential therapeutic value of melanocortins against multiple organ damage following systemic inflammatory response, here we investigated the effects of the melanocortin analogue [Nle(4) , D-Phe(7) ]α-MSH (NDP-α-MSH) in a widely used murine model of multiple organ dysfunction syndrome (MODS). Experimental approach: MODS was induced in mice by a single intraperitoneal injection of lipopolysaccharide followed, 6 days later (= day 0), by zymosan. After MODS or sham MODS induction, animals were randomized to receive intraperitoneally NDP-α-MSH (340 µg kg(-1) day) or saline for up to 16 days. Additional groups of MODS mice were concomitantly treated with the melanocortin MC(4) receptor antagonist HS024, or the nicotinic acetylcholine receptor antagonist chlorisondamine, and NDP-a-MSH. Key results: At day 7, in the liver and lung NDP-α-MSH significantly reduced mRNA expression of tumour necrosis factor-α (TNF-α), increased mRNA expression of interleukin-10 and improved the histological picture, as well as reduced TNF-α plasma levels; furthermore, NDP-α-MSH dose-dependently increased survival rate, as assessed throughout the 16-day observation period. HS024 and chlorisondamine prevented all the beneficial effects of NDP-a-MSH in MODS mice. Conclusions and Implications: These data indicate that NDP-a-MSH protects against experimental MODS by counteracting the systemic inflammatory response, probably through brain MC(4) receptor-triggered activation of the cholinergic anti-inflammatory pathway. These findings reveal previously undescribed effects of melanocortins and could have clinical relevance in the MODS setting.

2011 - Treatment of cerebral ischemia with melanocortins acting at MC(4) receptors induces marked neurogenesis and long-lasting functional recovery. [Articolo su rivista]
Giuliani, Daniela; Zaffe, Davide; Ottani, Alessandra; Spaccapelo, L; Galantucci, M; Minutoli, L; Bitto, A; Irrera, N; Contri, Miranda; Altavilla, D; Botticelli, Ar; Squadrito, F; Guarini, Salvatore
abstract

Melanocortins produce neuroprotection against ischemic stroke with subsequent long-lasting functional recovery, through melanocortin MC(4) receptor activation. Here we investigated whether the long-lasting beneficial effect of melanocortins in stroke conditions is associated with a stimulation of neurogenesis. Gerbils were subjected to transient global cerebral ischemia by occluding both common carotid arteries for 10 min; then, they were prepared for 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells. Delayed treatment (up to 9 h after the ischemic injury) for 11 days with the melanocortin analog [Nle(4),D: -Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH) improved learning and memory throughout the 50-day observation period. Immunohistochemical examination of the hippocampus on day 50 showed, in the dentate gyrus, an elevated number of BrdU immunoreactive cells colocalized with NeuN (used as indicator of mature neurons) and Zif268 (used as indicator of functionally integrated neurons). Retrospective analysis during the early stage of neural stem/progenitor cell development (days 3 and 4 after stroke) showed, in NDP-α-MSH-treated gerbils, a high degree of daily cell proliferation and revealed that NDP-α-MSH favorably affects Wnt-3A signaling pathways and doublecortin expression. Pharmacologic blockade of MC(4) receptors prevented all effects of NDP-α-MSH. These data indicate that treatment of cerebral ischemia with MC(4) receptor agonists induces, with a broad window of therapeutic opportunity, long-lasting functional recovery associated with a large number of mature and likely functional newborn neurons in brain injured areas. Our findings reveal previously undescribed effects of melanocortins which might have major clinical implications

2010 - High mobility group box-1 expression correlates with poor outcome in lung injury patients [Articolo su rivista]
Bitto, Alessandra; Barone, M; David, A; Polito, F; Familiari, D; Monaco, F; Giardina, M; David, T; Messina, R; Noto, A; Di Stefano, V; Altavilla, D; Bonaiuto, A; Minutoli, L; Guarini, Salvatore; Ottani, Alessandra; Squadrito, F; Venuti, F. S.
abstract

Chest trauma is frequently followed by pulmonary contusion and sepsis. High mobility group box-1 (HMGB-1) is a late mediator of severe sepsis that has been associated with mortality under experimental conditions. We studied HMGB-1 mRNA expression in patients with lung injury and its relationship with the severity of trauma and survival.A total of 24 consecutive patients with chest trauma referring to the Intensive Care Unit of Messina University Hospital, were enrolled. Lung trauma was established on the basis of chest X-ray and computed tomography. Injury Severity Score (ISS), Revised Trauma Score (RTS) and Glasgow Coma Scale (GCS) were also assessed. Accordingly to these results 6 patients were considered as controls because of no penetrating trauma and low ISS.Blood and broncho-alveolar lavage fluid (BALF) from chest trauma patients were withdrawn at admission and 24 h after the beginning of the standard therapeutic protocol.HMGB-1 mRNA increased significantly in blood (r = 0.84) and BALF (r = 0.87) from patients with trauma and pulmonary contusion and positively correlated with the severity of trauma (based on ISS and RTS) and the final outcome. HMGB-1 protein levels were also elevated in BALF macrophages from severe trauma patients compared to control subjects, furthermore TNF-α and its receptor TNFR-1 mRNA levels were also markedly increased in patients with a poor outcome respect to other subjects.Our study suggests that HMGB-1 may be an early indicator of poor clinical outcome in patients with chest trauma.

2010 - Involvement of the brain histaminergic system in the melanocortin MC4 receptor agonist RO27-3225-induced resuscitating effect in haemorrhage-shocked rats – haemodynamic studies. [Abstract in Rivista]
Jochem, J.; Giuliani, Daniela; Ottani, Alessandra; Galantucci, Maria; Krawitowski, M.; Spaccapelo, Luca; Guarini, Salvatore
abstract

Involvement of the brain histaminergic system in the melanocortin MC4 receptor agonist RO27-3225-induced resuscitating effect in haemorrhage-shocked rats – haemodynamic studies.

2010 - Melanocortins and the cholinergic anti-inflammatory pathway. [Capitolo/Saggio]
Giuliani, Daniela; Ottani, Alessandra; Altavilla, D; Bazzani, Carla; Squadrito, F; Guarini, Salvatore
abstract

Experimental evidence indicates that small concentrations of inflammatory molecules produced by damaged tissues activate afferent signals through ascending vagus nerve fibers, that act as the sensory arm of an "inflammatory reflex". The subsequent activation of vagal efferent fibers, which represent the motor arm of the inflammatory reflex, rapidly leads to acetylcholine release in organs of the reticuloendothelial system. Acetylcholine interacts with α7 subunit-containing nicotinic receptors in tissue macrophages and other immune cells and rapidly inhibits the synthesis/release of tumor necrosis factor-α and other inflammatory cytokines. This neural anti-inflammatory response called "cholinergic anti-inflammatory pathway" is fast and integrated through the central nervous system. Preclinical studies are in progress, with the aim to develop therapeutic agents able to activate the cholinergic anti-inflammatory pathway. Melanocortin peptides bearing the adrenocorticotropin/α-melanocyte-stimulating hormone sequences exert a protective and life-saving effect in animals and humans in conditions of circulatory shock. These neuropeptides are likewise protective in other severe hypoxic conditions, such as prolonged respiratory arrest, myocardial ischemia, renal ischemia and ischemic stroke, as well as in experimental heart transplantation. Moreover, experimental evidence indicates that melanocortins reverse circulatory shock, prevent myocardial ischemia/reperfusion damage and exert neuroprotection against ischemic stroke through activation of the cholinergic anti-inflammatory pathway. This action occurs via stimulation of brain melanocortin MC3/MC4 receptors. Investigations that determine the molecular mechanisms of the cholinergic anti-inflammatory pathway activation could help design of superselective activators of this pathway.

2010 - Melanocortins counteract inflammatory and apoptotic responses to prolonged myocardial ischemia/reperfusion through a vagus nerve-mediated mechanism [Articolo su rivista]
Ottani, Alessandra; Giuliani, Daniela; Galantucci, Maria; Spaccapelo, Luca; Novellino, E; Grieco, P; Jochem, J; Guarini, Salvatore
abstract

Recently we reported that an efferent vagal fibre-mediated cholinergic protective pathway, activated by melanocortins acting at brain melanocortin MC3 receptors, is operative in a condition of short-term myocardial ischemia/reperfusion associated with a high incidence of severe arrhythmias and death. Here we investigated melanocortin effects, and the role of the vagus nerve-mediated cholinergic protective pathway, in a rat model of prolonged myocardial ischemia/reperfusion associated with marked inflammatory and apoptotic reactions, and a large infarct size. Ischemia was produced in rats by ligature of the left anterior descending coronary artery for 30 min. At the end of the 2-h reperfusion, western blot analysis of the inflammatory and apoptotic markers tumor necrosis factor-α (TNF-α), c-jun N-terminal kinases (JNK) and caspase-3, as well as of the anti-apoptotic extracellular signal-regulated kinases (ERK 1/2), was performed in the left ventricle. In saline-treated ischemic rats there was an increase in TNF-α levels and in the activity of JNK and caspase-3 accompanied, despite an appreciable ERK 1/2 activation, by a large infarct size. Intravenous treatment, during coronary artery occlusion, with the melanocortin analog [Nle4, D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH) produced a reduction in TNF-α levels and in the activity of JNK and caspase-3, associated with marked activation of the pro-survival kinases ERK 1/2, and consequent attenuation of infarct size. Bilateral cervical vagotomy blunted the protective effects of NDP-α-MSH. These results indicate that melanocortins modulate the inflammatory and apoptotic cascades triggered by prolonged myocardial ischemia/reperfusion, and reduce infarct size, seemingly by activation of the vagus nerve-mediated cholinergic protective pathway.

2009 - Functional recovery after delayed treatment of ischemic stroke with melanocortins is associated with overexpression of the activity-dependent gene Zif268 [Articolo su rivista]
Giuliani, Daniela; Ottani, Alessandra; Letteria, Minutoli; Vincenzo Di, Stefano; Maria, Galantucci; Alessandra, Bitto; Zaffe, Davide; Domenica, Altavilla; Annibale R., Botticelli; Francesco, Squadrito; Guarini, Salvatore
abstract

Melanocortin peptides afford strong neuroprotection and improve functional recovery in experimental ischemic stroke; they also have established neurotrophic actions. The expression of the immediate early gene Zif268 is dependent on synaptic activity and is involved in injury repair and memory formation. Here, we investigated the role of Zif268 in learning and memory recovery after delayed treatment of ischemic stroke with the melanocortin analog [Nle4, d-Phe7]α-MSH (NDP-α-MSH). A 10-min period of global cerebral ischemia was induced by occluding both common carotid arteries in gerbils. Treatment with a nanomolar dose of NDP-α-MSH (every 12 h for 11 days) was performed starting 3 h or 9 h after stroke induction; where indicated, gerbils were pretreated with the melanocortin MC4 receptor antagonist HS024. Animals were subjected to the Morris water-maze test (four sessions from 4 to 50 days after the ischemic episode). Fifty days after stroke, histological damage and Zif268 expression were investigated in the hippocampus. Treatment with NDP-α-MSH significantly reduced hippocampal damage, including neuronal death, and improved learning and memory recovery. This protective effect was long-lasting (50 days, at least) and associated with Zif268 overexpression, with both schedules of NDP-α-MSH treatment. Pharmacological blockade of MC4 receptors prevented these effects. Our data indicate that MC4 receptor-mediated actions of melanocortins could trigger repair mechanisms able to improve neuronal functionality and synaptic plasticity, and to promote long-lasting functional recovery from ischemic stroke with Zif268 gene involvement.

2009 - Vagus nerve mediates the protective effects of melanocortins against cerebral and systemic damage after ischemic stroke. [Articolo su rivista]
Ottani, Alessandra; Giuliani, Daniela; Mioni, C; Galantucci, M; Minutoli, L; Bitto, A; Altavilla, D; Zaffe, Davide; Botticelli, Ar; Squadrito, F; Guarini, Salvatore
abstract

A vagus nerve-mediated, efferent cholinergic protective pathway activated by melanocortins is operative in circulatory shock and myocardial ischemia. Moreover, melanocortins have neuroprotective effects against brain damage after ischemic stroke. Here we investigated cerebral and systemic pathophysiologic reactions to focal cerebral ischemia in rats induced by intrastriatal microinjection of endothelin-1, and the possible protective role of the melanocortin-activated vagal cholinergic pathway. In the striatum and liver of saline-treated control rats, the activation of extracellular signal-regulated kinases, c-jun N-terminal kinases, and caspase-3, the increase in tumor necrosis factor-α (TNF-α) concentration and DNA fragmentation, as well as the increase in TNF-α plasma levels, occurred 10 and 20 h after the ischemic insult suggesting an activation of inflammatory and apoptotic responses. Treatment with [Nle4, D-Phe7]α-melanocyte-stimulating hormone (NDP-α-MSH; 3 or 9 h after stroke) suppressed the inflammatory and apoptotic cascades at central and peripheral level. Bilateral vagotomy and pharmacologic blockade of peripheral nicotinic acetylcholine receptors blunted the protective effect of NDP-α-MSH. The present results show that focal brain ischemia in rats causes significant effects not only in the brain, but also in the liver. Moreover, our data support the hypothesis that a protective, melanocortin-activated, vagal cholinergic pathway is likely operative in conditions of ischemic stroke.

2008 - Regulation of hypothalamic endocannabinoid levels by neuropeptides and hormons involved in food intake and metabolism: insulin and melanocortins [Articolo su rivista]
Matias, I; Vergoni, Anna Valeria; Petrosino, S; Ottani, Alessandra; Pocai, A; Bertolini, Alfio; DI MARZO, V.
abstract

Endocannabinoids are paracrine/autocrine lipid mediators with several biological functions. One of these, i.e. the capability to stimulate food intake via cannabinoid CB1 receptors, has been particularly studied, thus leading to the development of the first CB1 receptor blocker, rimonabant, as a therapeutic tool against obesity and related metabolic disorders. Hypothalamic endocannabinoids stimulate appetite by regulating the expression and release of anorexic and orexigenic neuropeptides via CB1 receptors. In turn, the tone of the latter receptors is regulated by hormones, including leptin, glucocorticoids and possibly ghrelin and neuropeptide Y, by modulating the biosynthesis of the endocannabinoids in various areas of the hypothalamus. CB1 receptor stimulation is also known to increase blood glucose during an oral glucose tolerance test in rats. Here we investigated in the rat if insulin, which is known to exert fundamental actions at the level of the mediobasal hypothalamus (MBH), and the melanocortin system, namely α-melanocyte stimulating hormone (α-MSH) and melanocortin receptor-4 (MCR-4), also regulate hypothalamic endocannabinoid levels, measured by isotope-dilution liquid chromatography coupled to mass spectrometry. No effect on anandamide and 2-arachidonoylglycerol levels was observed after 2 h infusion of insulin in the MBH, i.e. under conditions in which the hormone reduces blood glucose, nor with intra-cerebroventricular injection of α-MSH, under conditions in which the neuropeptide reduces food intake. Conversely, blockade of MCR-4 receptors with HS014 produced a late (6 h after systemic administration) stimulatory effect on endocannabinoid levels as opposed to a rapid and prolonged stimulation of food-intake (observable 2 and 6 h after administration). These data suggest that inhibition of endocannabinoid levels does not mediate the effect of insulin on hepatic glucose production nor the food intake-inhibitory effect of α-MSH, although stimulation of endocannabinoid levels might underlie part of the late stimulatory effects of MCR-4 blockade on food intake

2007 - Azione neuroprotettiva dei peptici melanocortinici nell’ictus ischemico sperimentale [Articolo su rivista]
Giuliani, Daniela; Mioni, C.; Bazzani, Carla; Ottani, Alessandra; Galantucci, M.; Zaffe, Davide; Botticelli, A. R.; Lodi, Renzo; Guarini, Salvatore
abstract

L’ictus ischemico è una delle cause principali di disabilità e di morte nei paesi occidentali. Negli ultimi anni abbiamo dimostrato che dosi nanomolari di peptidi melanocortinici, somministrati per via sistemica nel gerbillo e nel ratto, promuovono (verosimilmente in modo definitivo) il recupero funzionale dopo un attacco ischemico cerebrale globale o focale. Infatti, il trattamento con [Nle4, D-Phe7]--MSH (NDP--MSH, agonista sintetico dei recettori melanocortinici MCI, MC3, MC4 e MC5), causa una riduzione della risposta infiammatoria, come indicato dalla diminuzione dell’attività dei fattori regolatori della trascrizione JNKs, p38 ed ERKs, e dei livelli delle citochine proinfiammatorie TNF- (tumour necrosis factor-) e interleukina-6 (IL-6); NDP- -MSH riduce anche l’attività della caspasi-3 (proteina proapoptotica effettrice) e la frammentazione del DNA nelle aree cerebrali danneggiate. Inoltre, NDP--MSH dimostra un’ampia finestra terapeutica, infatti il trattamento è efficace anche quando inizia 12 ore dopo l’insulto ischemico e probabilmente 18 ore sono il tempo limite per la somministrazione del neuropeptide. I meccanismi di neuroprotezione sembrano coinvolgere direttamente I’attivazione dei recettori melanocortinici MC4, Infatti, il blocco farmacologico di questi recettori non solo previene l’effetto neuroprotettivo dell' NDP-a-MSH, ma addirittura peggiora il recupero funzionale. I nostri dati suggeriscono che agonisti melanocortinici, ahmente selettivi per i recettori MC4 e capaci di superare la barriera ematoencefalica, potrebbero rappresentare il mezzo per un approccio più mirato, innovativo e sicuro nell'ictus umano.

2007 - Dual acting anti-inflammatory drugs [Articolo su rivista]
Leone, Sheila; Ottani, Alessandra; Bertolini, Alfio
abstract

Drugs able to inhibit both cyclooxygenases (COX-1 and COX-2) and 5-lipoxygenase (5-LOX) (dual acting anti-inflammatory drugs) have been designed in order to obtain compounds that retain the activity of classical nonsteroidal anti-inflammatory drugs (NSAIDs) while avoiding their main drawbacks. The classical NSAIDs display their anti-inflammatory action mainly through inhibition of COX and one of their main drawbacks is the curtailed production of gastroprotective prostaglandins (PGs) being associated with the concurrent increased production of the gastro-damaging and bronchoconstrictive leukotrienes (LTs). Leukotrienes and cysteinyl-leukotrienes are moreover pro-inflammatory and increase microvascular permeability. One of the leukotrienes (LTB4) is the most potent chemotactic agent and it induces chemotaxis of eosinophils, neutrophils and monocytes in the inflamed tissue, increases superoxide generation and proinflammatory cytokines production. It is further advantageous for a drug to have both COX and 5-LOX inhibiting activities because prostaglandins enhance leukotriene-mediated inflammation. Various structural families of dual inhibitors have been designed and several compounds are currently undergoing clinical development. In the post-COX-2 selective inhibitors era, these dual acting inhibitors may turn out to be promising new drugs to treat inflammatory diseases and possibly other diseases. Indeed, both COX-2 and 5-LOX are also involved in the development and progression of several types of cancer; in these conditions, selective inhibition of COX-2 alone may lead to a shunt of arachidonic acid metabolism towards the leukotriene pathway, and therefore the blockade of both COX-2 and 5-LOX may produce a better anticancer response. In addition, the dual inhibition of both COX and 5-LOX is neuroprotective by suppressing toxic actions of reactive microglia and macrophages, that are increased in aging brain and in age-related degenerative conditions, particularly Alzheimer's and Parkinson's diseases. Finally, the blockade of 5-LOX does not impair the synthesis of lipoxins (LXs), which are mainly produced by further lipoxygenation of 15-HPETE, and which have potent anti-inflammatory properties and can be considered as stop-signal mediators. Leukocyte 15-LOX and platelet 12-LOX by intercellular mechanism via leukocyte/platelet cell-cell interaction convert 15-HPETE into lipoxins.

2007 - Neuroprotection in focal cerebral ischemia owing to delayed treatment with melanocortins. [Articolo su rivista]
Giuliani, Daniela; Ottani, Alessandra; C., Mioni; Bazzani, Carla; M., Galantucci; L., Minutoli; A., Bitto; Zaffe, Davide; A. R., Botticelli; F., Squadrito; Guarini, Salvatore
abstract

In gerbils subjected to transient global cerebral ischemia, melanocortin peptides produce long-lasting protection with a broad time window, and through the activation of central nervous system melanocortin MC(4) receptors. Here we aimed to investigate whether melanocortins are neuroprotective also in a rat model of focal cerebral ischemia induced by intrastriatal microinjection of endothelin-1. The vasoconstrictor agent endothelin-1 caused a significant impairment in spatial learning and memory, as well as in sensory-motor orientation and limb use, associated with severe striatal morphological damage including intense neuronal death and an almost complete myelin degradation. Treatment of ischemic rats with a nanomolar dose (340 mug/kg/day i.p. for 11 days, beginning 3 h or 9 h after endothelin-1 microinjection) of the melanocortin analog [Nle(4), d-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) significantly reduced striatal damage, and improved subsequent functional recovery, with all scheduled NDP-alpha-MSH treatments. Pharmacological blockade of melanocortin MC(4) receptors prevented the protective effect of NDP-alpha-MSH. Our findings give evidence that melanocortins are neuroprotective, with a broad time window, also in a severe model of focal cerebral ischemia, and suggest that melanocortin MC(4) receptor agonists could produce neuroprotection in different experimental models of ischemic stroke.

2007 - Preference for palatable food is reduced by the gamma-hydroxybutyrate analogue GET73, in rats [Articolo su rivista]
Ottani, Alessandra; Leone, Sheila; Vergara Garcia, Fb; Tacchi, Raffaella; Loche, A; Bertolini, Alfio
abstract

Palatability and variety of foods are major reasons for “hedonic” eating, and hence for overeating and obesity. Palatable food and drugs of abuse share a common reward mechanism, and compounds that block the reinforcing effect of drugs of abuse preferentially suppress the intake of palatable foods. This research was aimed at studying the inﬂuence of the gamma-hydroxybutyrate analogue N-(4-triﬂuoromethylbenzyl)-4¬methoxybutanamide (GET73) – that inhibits alcohol consumption – on consumption and reinforcing effect of palatable food. Adult male rats were used. For place preference conditioning, sweetened corn ﬂakes were used as the reinforcer, and GET73 (50, 100 and 200 mg kg−1) or vehicle were orally (p.o.) administered either 30 min before each training session and the test session, or only before the test session. To study the inﬂuence on con¬sumption, GET73 was given p.o. at the same doses once daily for 12 days to rats given free access to both palatable and varied food (cafeteria diet) or to standard chow. Both acquisition and expression of palatable food-induced conditioned place preference were inhibited by GET73, either adminis¬tered throughout the conditioning period or only before the test session. GET73 reduced also the consumption of cafeteria food, while that of standard chow was increased. At these doses, GET73 had no detrimental effect on open-ﬁeld behaviour. GET73 seems to speciﬁcally attenuate the gratiﬁcation produced by varied and palatable food, without affecting the consumption of not particularly palatable chow. Since, overweight and obesity are mostly due to the overeating of palatable and varied foods, drugs like GET73 could represent a somewhat ideal and rational approach to obesity treatment.

2007 - Similarities and differences between chronic migraine and episodic migraine [Articolo su rivista]
Ferrari, Anna; S., Leone; Vergoni, Anna Valeria; Bertolini, Alfio; G., Sances; C. P. R., Coccia; Ottani, Alessandra; Pinetti, Diego; Sternieri, Emilio
abstract

Objective.-To quantify and characterize the similarities and the differences between chronic migraine (CM) patients with medication overuse and episodic migraine (EM) patients with only occasional analgesic use.Background.-Population-level epidemiology, characteristics, mechanisms of chronic daily headache, and medication-overuse headache have been widely studied but patient characteristics have received less attention.Methods.-We compared sociodemographic data, family history, physiological and medical history, health services utilized, drugs taken/prescribed, and outcome of 2 groups of subjects: 150 patients, suffering from CM, complicated by probable medication-overuse headache (CM group), consecutively admitted during 2005 to the inpatients' ward of the Headache Centre of the University Hospital of Modena and Reggio Emilia, Italy, to undergo withdrawal from their overused medications; 100 patients suffering from EM, uncomplicated by medication overuse (EM group), consecutively referred to the outpatients' ward of the Headache Centre during November and December 2005.Results.-All sociodemographic characteristics were significantly different between the 2 groups. As a whole, the CM group began to suffer from migraine earlier than the EM group. Drug and/or alcohol abuse was significantly higher among first-degree relatives of CM (19%) than of EM (6%) patients. The most frequent comorbid disorders were psychiatric (67%) and gastrointestinal diseases (43%) in the CM group, and allergies in the EM group (31%). Seventy percent of CM patients and 42% of EM patients were taking daily at least another drug, besides those for headache treatment. Most overused medications in the CM group were triptans (43%); the EM group used above all single NSAIDs (56%). At 3-month follow-up, prophylactic treatments reduced, by at least 50%, the frequency of headache in about three-fourths of patients of both the groups; however, headache remained significantly more frequent in the CM than in EM group: only a minority (15%) of CM patients reverted to a headache frequency comparable to that of the EM group.Conclusions.-CM patients present more multiple comorbid disorders, polypharmacy, and social impediments than EM patients. These associated conditions complicate CM clinical management. Even after withdrawal from medication overuse, CM could not be completely reverted by current prophylactic treatments.

2006 - Paracetamol: new vistas of an old drug [Articolo su rivista]
Bertolini, Alfio; Ferrari, Anna; Ottani, Alessandra; Guerzoni, Simona; Tacchi, Raffaella; S., Leone
abstract

Paracetamol (acetaminophen) is one of the most popular and widely used drugs for the treatment of pain and fever. It occupies a unique position among analgesic drugs. Unlike NSAIDs it is almost unanimously considered to have no antinflammatory activity and does not produce gastrointestinal damage or untoward cardiorenal effects. Unlike opiates it is almost ineffective in intense pain and has no depressant effect on respiration. Although paracetamol has been used clinically for more than a century, its mode of action has been a mystery until about one year ago, when two independent groups (Zygmunt and colleagues and Bertolini and colleagues) produced experimental data unequivocally demonstrating that the analgesic effect of paracetamol is due to the indirect activation of cannabinoid CBI receptors. In brain and spinal cord, paracetamol, following deacetylation to its primary amine (p-aminophenol), is conjugated with arachidonic acid to form N-arachidonoylphenolamine, a compound already known (AM404) as an endogenous cannabinoid. The involved enzyme is fatty acid amide hydrolase. N-arachidonoylphenolamine is an agonist at TRPV1 receptors and an inhibitor of cellular anandamide uptake, which leads to increased levels of endogenous cannabinoids; moreover, it inhibits cyclooxygenases in the brain, albeit at concentrations that are probably not attainable with analgesic doses of paracetamol. CB1 receptor antagonist, at a dose level that completely prevents the analgesic activity of a selective CB1 receptor agonist, completely prevents the analgesic activity of paracetamol. Thus, paracetamol acts as a pro-drug, the active one being a cannabinoid. These findings finally explain the mechanism of action of paracetamol and the peculiarity of its effects, including the behavioral ones. Curiously, just when the first CB I agonists are being introduced for pain treatment, it comes out that an indirect cannabinomimetic had been extensively used (and sometimes overused) for more than a century

2006 - The analgesic activity of paracetamol is prevented by the blockade of cannabinoid CB1 receptors [Articolo su rivista]
Ottani, Alessandra; S., Leone; Sandrini, Maurizio; Ferrari, Anna; Bertolini, Alfio
abstract

The mechanism of the analgesic activity of paracetamol (acetaminophen), one of the most widely used drugs for the treatment of pain, is still not clear. Here we show that in rats, using the hot plate test, the analgesic effect of paracetamol is prevented by two antagonists at cannabinoid CB I receptors (AM281 and SR141716A) at doses that prevent the analgesic activity of the cannabinoid wCB(1) agonist HU210. Our present results suggest that paracetamol-induced antinociception involves the cannabinoid system.

2005 - Adverse reactions related to drugs for headache treatment: clinical impact [Articolo su rivista]
Ferrari, Anna; Ottani, Alessandra; Bertolini, Alfio; A. F. G., Cicero; C. P. R., Coccia; S., Leone; Sternieri, Emilio
abstract

Objective: To assess the clinical impact of adverse reactions related to drugs for primary headache treatment.Methods: We examined the adverse reactions to 360 medications prescribed by the specialists of the Headache Centre of the University of Modena and Reggio Emilia to 256 consecutive outpatients (214 female, 42 male; mean age: 38.88+/-14.06 years; range 10-72 years). Adverse reactions were reported by patients during scheduled follow-up visits, classified by specialists and reassessed by a clinical pharmacologist.Results: Adverse reactions with a causal relationship classified as definite/probable/possible were 202 (56%): 62% (80/129) were due to acute treatments and 53% (122/231) to prophylactic treatments (chi(2) test, P=0.115 ns). More than 90% of the adverse reactions were of limited intensity [mild (58%) or moderate (36%)]. Only 5% were severe, and two reactions (1%) were serious. The most affected apparatus was the nervous system (41%). Of these adverse reactions, 43% caused the discontinuance of the treatment, especially of prophylaxis (54%). Patients evaluated 70% of the medications as effective, but, at the same time, they considered most of the adverse reactions (69%) unacceptable.Conclusion: Adverse reactions related to headache medications have a strong impact on patients' management, even if their real intensity and severity are usually very limited. Drugs for headache treatment are still far from being ideal drugs. To prevent the discontinuance of effective medications, the physician, prior to prescribing, should assess, together with the patient, the acceptability of the more common adverse drug reactions.

2004 - Different effect of sumatriptan in models of thermal, chemical or cephalalgic pain, in rats [Abstract in Rivista]
Ottani, Alessandra; Ferraris, E.; Bertolini, Alfio; Ferrari, Anna
abstract

Triptans have represented a real progress in the acute therapy of migraine. The beneficial effect of this new class of drugs depends on their agonist activity on 5-HT1B/1D/1F receptors. However, some sparse experimental data have been produced showing that triptans may have a non specific, systemic analgesic activity in virtue of other mechanisms of action. Aim of our present study was to evaluate the effect of sumatriptan (chosen as the lead of this class) in two standard algesimetric tests and in an animal model of cephalalgia. Adult male Wistar rats were used. The response to a thermal painful stimulus was evaluated by means of the hot-plate test (temperature of the plate: 54±0.4°C; cut-off time: 30 s); the response to a chemical painful stimulus was evaluated by means of the abdominal constriction test (writhing test) (intraperitoneal injection of 2 ml 0.5% acetic acid); cephalalgia was produced by injecting bradykinin (8 µg in a volume of 8 µl) into a common carotid artery. Sumatriptan was subcutaneously (s.c.) injected at the doses of 4, 8, 24 and 42 mg/kg. 10 min before testing. Morphine (5 or 10 mg/kg s.c.) and indomethacin (10 mg/kg per os), used as standard analgesic drugs, were administered 20 or 30 min, respectively, before testing. Groups of 8-12 rats per dose were used; each rat was used for only one treatment. Control rats received by the same routes identical volumes of saline. Results were analyzed by one-way ANOVA followed by Student-Newman-Keuls’ test, or by Friedman test followed by Mann-Withney U test, or by Chi-square test, where appropriate. Sumatriptan had no analgesic activity at any of the doses used either in the hot-plate test [F(7,80)=11.82, P=0.000] or in the writhing test [F(7,63)=17.37, P=0.000], while morphine and indomethacin were significantly effective. On the other hand, sumatriptan, at the doses of 24 and 42 mg/kg, significantly reduced the consequences of the intracarotid injection of bradikinin (vocalization, tachypnea, ECG alterations) [x 2=8.55, P=0.014]. These data demonstrate that sumatriptan is significantly effective in a reliable animal model of cephalalgia, while being devoid – at the same doses and in the same animal species – of any systemic analgesic activity.

2004 - Effect of late treatment with gamma-hydroxybutyrate on the histological and behavioral consequences of transient brain ischemia in the rat. [Articolo su rivista]
Ottani, Alessandra; Vergoni, Anna Valeria; Saltini, Sabrina; Mioni, Chiara; Giuliani, Daniela; M., Bartiromo; A. R., Botticelli; Bertolini, Alfio; Genedani, Susanna
abstract

It has been previously described that gamma-hydroxybutyrate (GHB) provides significant protection against transient global cerebral ischemia in the rat (four vessel occlusion model), when given 30 min before or 10 min after artery occlusion. Here, we show that in the same rat model, significant protection can also be obtained when treatment is started 2 h after the ischemic episode. In saline-treated animals, 30 min of global ischemia followed by reperfusion caused a massive loss of neurons in the hippocampal CA1 subfield (examined 63 days after the ischemic episode), and an impairment of sensory-motor performance (tested on the 51st and 63rd days after ischemia) and of spatial learning and memory (evaluated starting 46 days after the ischemic episode). Treatment with GHB--300 mg/kg intraperitoneally (i.p.) 2 h after the ischemia-reperfusion episode, followed by 100 mg/kg i.p. twice daily for the following 10 days--afforded a highly significant protection, against both histological damage and sensory-motor and learning-memory impairments. These data further suggest the possible therapeutic effectiveness of GHB in brain ischemia, and indicate that the underlying mechanism of action involves non-immediate steps of the ischemia-induced cascade of events.

2004 - Effect of sumatriptan in different models of pain in rats [Articolo su rivista]
Ottani, Alessandra; Ferraris, E; Giuliani, Daniela; Mioni, C; Bertolini, Alfio; Sternieri, Emilio; Ferrari, Anna
abstract

The effect of sumatriptan in two standard algesimetric tests and in a model of cephalalgia was evaluated in rats. The pain threshold was measured by the hot-plate and the writhing tests; cephalalgia was produced by injecting bradykinin (10 μg in a volume of 10 μl) into a common carotid artery. Sumatriptan was subcutaneously (s.c.) injected at the doses of 4, 8, 24 or 42 mg/kg; morphine (5 or 10 mg/kg s.c.) and indomethacin (5 or 10 mg/kg s.c) were used as standard analgesic drugs. Sumatriptan had no analgesic activity either in the hot-plate test or in the writhing test. On the other hand, at 24 and 42 mg/kg it dose-dependently reduced the response to the intracarotid injection of bradykinin (vocalization and tachypnea), this effect being prevented by the 5-HT1B receptor antagonist, isamoltane. The 5-HT1D receptor antagonist BRL15572 prevented the effect of sumatriptan on bradykinin-induced tachypnea, but not the effect of sumatriptan on bradykinin-induced vocalization. These data demonstrate that sumatriptan is significantly effective in a reliable animal model of cephalalgia, while having no systemic analgesic activity

2004 - Headache treatment before and after the consultation of a specialized centre: a pharmacoepidemiology study [Articolo su rivista]
Ferrari, Anna; G., Pasciullo; G., Savino; A. F. G., Cicero; Ottani, Alessandra; Bertolini, Alfio; Sternieri, Emilio
abstract

Our aim was to study and compare pharmacoepidemiology of headache treatment in two different settings: inside and outside a specialized Centre. We analysed the differences in headache treatment between 612 subjects admitted for the first visit ('naive') (F/M: 2.41; mean age = 37.31 +/- 14.09 years) and 620 subjects admitted for a control visit (F/M: 3.18; mean age = 44.30 +/- 15.37 years) to the Headache Centre of the University of Modena and Reggio Emilia. Most patients suffered from migraine. As acute treatment, on the first visit, 49.4% of them were taking drugs prescribed by a doctor; 41.5% were taking over-the-counter analgesics (OTCAs); 9.1% were not taking any drug. On the control visit, 81.3% of patients were taking prescription drugs; 15.8% OTCAs; 2.9% were not taking drugs (overall chi-square = 139.229, P < 0.001). Non-selective analgesics were the most-used drugs. Triptans were used by 9.1% of 'naive' patients and by 31.8% of patients attending for the control visit (Fisher's Z = 7.655, P < 0.001). Nimesulide was the most-used drug. A prophylactic treatment was made by 16.8% of 'naive' patients, and by 58.2% of patients admitted to the control visit (Fisher's Z = 12.135, P < 0.001). Antidepressants were the class of drugs most used for prophylaxis. Amitriptyline was the drug for prophylaxis most frequently used by patients attending the control visit, while flunarizine was the most frequently used by 'naive' patients. Before being examined in a specialized centre, few patients take prescription drugs, triptans, or prophylactic drugs; specialized care increases the proportion of patients taking prophylactic drugs, and changes the type of acute treatment used into disease-specific medication for headache.

2004 - Selective COX-2 inhibitors and dual acting anti-inflammatory drugs: critical remarks [Capitolo/Saggio]
Bertolini, Alfio; Ottani, Alessandra; Sandrini, Maurizio
abstract

Non steroidal anti-inflammatory drugs (NSAIDs) are still the most commonly used remedies for rheumatic diseases. But NSAIDs produce serious adverse effects, the most important being gastric injury up to gastric ulceration and renal damage. Several strategies have been adopted in order to avoid these shortcomings, especially gastrointestinal toxicity. So, non steroidal anti-inflammatory drugs have been associated with gastroprotective agents that counteract the damaging effects of prostaglandin synthesis suppression: however, a combination therapy introduces problems of pharmacokinetics, toxicity, and patient s compliance. Also incorporation of a nitric oxide (NO)-generating moiety into the molecule of several NSAIDs was shown to greatly attenuate their ulcerogenic activity: however, several findings suggest a possible involvement of NO in the pathogenesis of arthritis and subsequent tissue destruction. A most promising approach seemed to be the preparation of novel NSAIDs, specific for the inducible isoform of cyclooxygenase (COX-2): they appear to be devoid of gastrointestinal toxicity, in that they spare mucosal prostaglandin synthesis. However, a number of recent studies raised serious questions about the two central tenets that support this approach, namely that the prostaglandins that mediate inflammation and pain are produced solely via COX-2 and that the prostaglandins that are important in gastrointestinal and renal function are produced solely via COX-1. So, increasing evidence shows that COX-2 (not only COX-1) also plays a physiological role in several body functions and that, conversely, COX-1 (not only COX-2) may also be induced at sites of inflammation. Moreover, COX-2 selective NSAIDs have lost the cardiovascular protective effects of non-selective NSAIDs, effects which are mediated through COX-1 inhibition (in addition, COX-2 has a role in sustaining vascular prostacyclin production). The products generated by the 5-lipoxygenase pathway (leukotrienes) are particularly important in inflammation: indeed, leukotrienes increase microvascular permeability and are potent chemotactic agents; moreover, inhibition of 5-lipoxygenase indirectly reduces the expression of TNF-alpha (a cytokine that plays a key role in inflammation). This explains the efforts to obtain drugs able to inhibit both 5-lipoxygenase and cyclooxygenases: the so-called dual acting anti-inflammatory drugs. Such compounds retain the activity of classical NSAIDs, while avoiding their main drawbacks, in that curtailed production of gastroprotective prostaglandins is associated with a concurrent curtailed production of the gastro-damaging and bronchoconstrictive leukotrienes. Moreover, thanks to their mechanism of action, dual acting anti-inflammatory drugs could not merely alleviate symptoms of rheumatic diseases, but might also satisfy, at least in part, the criteria of curative drugs. Indeed, leukotrienes are pro-inflammatory, increase microvascular permeability, are potent chemotactic agents and attract eosinophils, neutrophils and monocytes into the synovium. Finally, recent data strongly suggest that dual inhibitors may have specific protective activity also in neurodegeneration.

2003 - Effect of gamma-hydroxybutyrate in two rat models of focal cerebral damage [Articolo su rivista]
Ottani, Alessandra; Saltini, S.; Bartiromo, M.; Zaffe, Davide; Botticelli, A. R.; Ferrari, Anna; Bertolini, Alfio; Genedani, Susanna
abstract

Gamma-Hydroxybutyrate (GHB) and its lactone, gamma-butyrolactone (GBL) have been previously shown to produce a protective effect in animal models of cerebral ischaemia/hypoxia, as well as in human conditions of head injury-induced coma. The aim of the present research was to study the effect of GHB in experimental conditions of focal cerebral damage, either induced by ischaemia or excitotoxicity. Under general anaesthesia, rats were injected into the right striatum with either endothelin-1 (ET-1, 0.43 nmol) or kainic acid (7.5 nmol) in a volume of 1 mul. Sham-lesioned rats received 1 mul of the solvent. Both ET-1- and kainic acid-lesioned rats were randomly assigned to one of the following intraperitoneal (i.p.) treatments: (i) and (ii) GHB, 100 or 300 mg kg(-1) 2 h after the lesion, followed by 50 or 100 mg kg(-1), respectively, every 12 h; (iii) saline, 2 ml kg(-1), same schedule. Sham animals were treated with saline, 2 ml kg(-1), same schedule. Treatments lasted for 10 days. The higher dose of GHB produced a significant protection against the ET-1-induced impairments in sensory-motor orientation and coordinated limb use (evaluated 24 and 42 days after the lesion) and in place learning and memory (Morris test, performed 19 and 39 days after the lesion). The same dose regimen reduced the circling behaviour induced by apomorphine in kainate-lesioned rats (10 days after the lesion), and limited or prevented at all the histological damage produced either by ET-1 or by kainic acid (evaluated 43 or 10 days after the lesion, respectively). These results show that GHB limits both histological and functional consequences of a focal ischaemic or excitotoxic insult of the brain, in rats, even if the treatment is started 2 h after the lesion.

2002 - Influence of sildenafil on central dopamine-mediated behaviour in male rats [Articolo su rivista]
F., Ferrari; Ottani, Alessandra; Giuliani, Daniela
abstract

Two experiments were performed to evaluate the effects of sildenafil on spontaneous or dopamine agonist-induced behaviour in male rats. Data obtained in experiment 1 show that oral administration of the drug, at 1 mg/kg, significantly increased the occurrence of penile erections, anogenital self-grooming and homosexual mounting in grouped sexually-experienced, but not inexperienced, animals. In experiment 2, pre-treatment with sildenafil (0.5, 1 or 10 mg/kg) dose-dependently modified several behavioural signs, centrally evoked by the D-2/D-3 dopamine agonists, 7-OH-DPAT or B-HT 920 (both at 0.1 mg/ka), in experimentally naive male rats. While sildenafil at 1 mg/kg significantly increased the number of penile erection and stretching-yawning episodes induced by 7-OH-DPAT or B-HT 920, at 10 mg/kg it elicited low stereotyped behaviour, antagonizing stretching-yawning and sedation in 7-OH-DPAT treated rats. Discussion centres on the modulatory activity of sildenafil on central dopaminergic pathways and, possibly, on nitric oxide production. (C) 2002 Elsevier Science Inc. All rights reserved.

2002 - Influence of sildenafil on copulatory behaviour in sluggish or normal ejaculator male rats: a central dopamine mediated effect? [Articolo su rivista]
Giuliani, Daniela; Ottani, Alessandra; Ferrari, Francesca
abstract

The present study investigates the effects induced by sildenafil (1 mg/kg, p.o.) and the dopamine agonist, SND 919 (0.1 mg/kg, i.p.) on copulatory behaviour of male rats, categorized, on the basis of seven consecutive mating pre-tests, as sluggish and normal ejaculators (SE and NE, respectively). The data obtained show that sildenafil modifies both sexual arousal and ejaculatory mechanisms of copulation. It appears that, although it induced a facilitatory effect on ejaculation of all rats, similarly to SND 919, the lowering of ejaculatory threshold was achieved by means of a reduction of mount frequency and intromission frequency in SE and NE groups, respectively. Differently from SND 919, sildenafil increased sexual arousal, diminishing post ejaculatory interval in SE animals and inter-intromission interval in both SE and NE rats. As the dopamine antagonist, (-)eticlopride (0.02 mg/kg, s.c.), significantly inhibited sildenafil-induced enhancement of sexual arousal in SE rats, it is suggested that the drug acts both peripherally and centrally.

2002 - Modulatory activity of sildenafil on copulatory behaviour of both intact and castrated male rats [Articolo su rivista]
Ottani, Alessandra; Giuliani, Daniela; Ferrari, Francesca
abstract

The first experiment of the present study investigates the effects induced by sildenafil (1 or 10 mg/kg po) on the copulatory behaviour of intact male rats, categorized, on the basis of seven consecutive mating pretests, as sluggish or normal ejaculators (SE or NE, respectively). The data obtained show that sildenafil modifies both sexual arousal and the ejaculatory mechanisms of copulation, diminishing ejaculation latency in both categories and increasing copulatory efficacy in SE rats; in addition, it reduced the inter-intromission interval in both SE and NE animals and the post-ejaculatory interval only in SE animals. The second experiment, conducted on rats 3 weeks after their castration, shows that sildenafil alone (1 or 10 mg/kg) did not modify copulatory failure. However, 3 months after castration, and 24 h after the last injection of testosterone (25 microg/kg sc) given twice weekly for 4 weeks, sildenafil (1 or 10 mg/kg) ameliorated rat copulatory performance.

2002 - Neuroleptic-like profile of the cannabinoid agonist, HU 210, on rodent behavioural models [Articolo su rivista]
Ottani, Alessandra; Ferrari, Francesca; Giuliani, Daniela
abstract

The present study was performed to assess the effects exerted by the cannabinoid (CB) agonist, ()11-hydroxy-D8-tetrahydrocannabinol-dymethylheptyl (HU 210; 12.5–50 mg/kg ip), on rodent behavioural tests involving dopamine (DA) transmission;in comparison, the DA D2 antagonist, S()-3-chloro-5-ethyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-hydroxy-2-methoxy-benzamide hydrochloride(()eticlopride; 50 mg/kg sc), was used. (2) In rats, HU 210, at all doses, potently antagonized penile erection (PE) and stretching–yawning (SY) typically elicited by the DA D2/D3 agonists, 6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d]-azepine (B-HT 920) and±7-hydroxy-N,N-di-n-propylaminotetralin hydrobromide (7-OH-DPAT) both at 100 mg/kg ip. (3) In nonreserpinized mice, HU 210 impairedmotor ability assessed by means of a motor test battery, and B-HT 920 (1 mg/kg ip) worsened the phenomenon. (4) In reserpinized mice, HU210 at 50 mg/kg counteracted the amelioration exerted by B-HT 920 (1 mg/kg ip) on reserpine-induced akinesia. (5) As all these effects weresimilarly displayed by ()eticlopride (50 mg/kg sc), our data suggest a neuroleptic-like profile of acute HU 210 in animal behavioural tests.

2002 - Selective COX-2 inhibitors and dual acting anti-inflammatory drugs: Critical remarks [Articolo su rivista]
Bertolini, Alfio; Ottani, Alessandra; Sandrini, Maurizio
abstract

Non steroidal anti-inflammatory drugs (NSAIDs) are still the most commonly used remedies for rheumatic diseases. But NSAlDs produce serious adverse effects, the most important being gastric injury up to gastric ulceration and renal damage. Several strategies have been adopted in order to avoid these shortcomings, expecially gastrointestinal toxicity. So, non steroidal anti-inflammatory drugs have been associated with gastroprotective agents that counteract the damaging effects of prostaglandin synthesis suppression: however, a combination therapy introduces problems of pharmacokinetics, toxicity, and patient's compliance. Also incorporation of a nitric oxide (NO)generating moiety into the molecule of several NSAIDs was shown to greatly attenuate their ulcerogenic activity: however, several findings suggest a possible involvement of NO in the pathogenesis of arthritis and subsequent tissue destruction. A most promising approach seemed to be the preparation of novel NSAIDs, specific for the inducible isoform of cyclooxygenase (COX-2): they appear to be devoid of gastrointestinal toxicity, in that they spare mucosal prostaglandin synthesis. However, a number of recent studies raised serious questions about the two central tenets that support this approach, namely that the prostaglandins that mediate inflammation and pain are produced solely via COX-2 and that the prostaglandins that are important in gastrointestinal and renal function are produced solely via COX-1. So, increasing evidence shows that COX-2 (not only COX-1) also plays a physiological role in several body functions and that, conversely, COX-1 (not only COX-2) may also be induced at sites of inflammation. Moreover, COX-2 selective NSAIDs have lost the cardiovascular protective effects of non-selective NSAIDs, effects which are mediated through COX-1 inhibition (in addition, COX-2 has a role in sustaining vascular prostacyclin production). The products generated by the 5-lipoxygenase pathway (leukotrienes) are particularly important in inflammation: indeed, leukotrienes increase microvascular permeability and are potent chemotactic agents; moreover, inhibition of 5-lipoxygenase indirectly reduces the expression of TNF-alpha (a cytokine that plays a key role in inflammation). This explains the efforts to obtain drugs able to inhibit both 5-lipoxygenase and cyclooxygenases: the so-called dual acting anti - inflammatory drugs. Such compounds retain the activity of classical NSAIDs, while avoiding their main drawbacks, in that curtailed production of gastroprotective prostaglandins is associated with a concurrent curtailed production of the gastro-damaging and bronchoconstrictive leukotrienes. Moreover, thanks to their mechanism of action, dual acting anti-inflammatory drugs could not merely alleviate symptoms of rheumatic diseases, but might also satisfy, at least in part, the criteria of curative drugs. Indeed, leukotrienes are pro-inflammatory, increase microvascular permeability, are potent chemotactic agents and attract eosinophils, neutrophils and monocytes into the synovium. Finally, recent data strongly suggest that dual inhibitors may have specific protective activity also in neurodegeneration.

2001 - Dual acting anti-inflammatory drugs: a reappraisal [Articolo su rivista]
Bertolini, Alfio; Ottani, Alessandra; Sandrini, Maurizio
abstract

Rheumatic diseases are the most prevalent causes of disability in western countries, and nonsteroidal anti-inflammatory drugs (NSAIDs) are still the most commonly used remedies. However, NSAIDs cause several serious adverse effects, the most important being from gastric injury to gastric ulceration and renal damage. Attempts to develop non-steroidal anti-inflammatory remedies devoid of these shortcomings-especially gastrointestinal toxicity-have followed several strategies. Non-steroidal antiinflammatory drugs have, therefore, been associated with gastroprotective agents that counteract the damaging effects of prostaglandin synthesis suppression; however, a combination therapy introduces other problems of pharmacokinetics, toxicity, and patient´s compliance. More recently, incorporation of a nitric oxide (NO)-generating moiety into the molecule of several NSAIDs was shown to greatly attenuate their ulcerogenic activity; however, several findings suggest a possible involvement of NO in the pathogenesis of arthritis and subsequent tissue destruction. A most promising approach seemed to be the preparation of novel NSAIDs, targeted at the inducible isoform of prostaglandin synthase (COX-2); they appear to be devoid of gastrointestinal toxicity, in that they spare mucosal prostaglandin synthesis. However, a number of recent studies have raised serious questions about the two central tenets that support this approach, namely that the prostaglandins that mediate inflammation and pain are produced solely via COX-2 and that the prostaglandins that are important in gastrointestinal and renal function are produced solely via COX-1. So, a growing body of evidence shows that COX-2 (riot only COX-1) also plays a physiological role in several body functions and that, conversely, COX-1 (not only COX-2) may also be induced at sites of inflammation. More recent and puzzling data shows that COX-2 is induced during the resolution of an inflammatory response, and at this point it produces anti-inflammatory (PGD(2) and PGF(2 alpha)), but not proinflammatory (PGE(2)) prostaglandins; inhibition of COX-2 at this point thus results in persistence of the inflammation. Moreover, COX-2 selective NSAIDs have lost the cardiovascular protective effects of nonselective NSAIDs, effects which are mediated through COX-1 inhibition (in addition, COX-2 has a role in sustaining vascular prostacyclin production). The generation of other very important products of the arachidonic acid cascade (besides cyclooxygenase-produced metabolites) is inhibited neither by non-selective nor by COX-2 selective NSAIDs. The products generated by the 5-lipoxygenase pathway (leukotrienes) are particularly important in inflammation; indeed, leukotrienes increase microvascular permeability and are potent chemotactic agents. Moreover, inhibition of 5-lipoxygenase indirectly reduces the expression of TNF-alpha (a cytokine that plays a key role in inflammation). These data and considerations explain the efforts to obtain drugs able to inhibit both 5-lipoxygenase and cyclooxygenases, the so-called dual acting anti-inflammatory drugs. Such compounds retain the activity of classical NSAIDs, while avoiding their main drawbacks, in that curtailed production of gastroprotective prostaglandins is associated with a concurrent curtailed production of the gastro-damaging and bronchoconstrictive leukotrienes. Moreover. thanks to their mechanism of action, dual acting anti-inflammatory drugs could not merely alleviate symptoms of rheumatic diseases, but might also satisfy, at least in part, the criteria of a more definitive treatment. Indeed, leukotrienes are pro-inflammatory, increase microvascular permeability, are potent chemotactic agents and attract eosinophils, neutrophils and monocytes into the synovium.

2001 - HU 210: A potent tool for investigations of the cannabinoid system [Articolo su rivista]
Ottani, Alessandra; Giuliani, Daniela
abstract

The synthetic compound HU 210 displays a multiplicity of biochemical, pharmacological, and behavioral effects, most of which have been demonstrated to be dependent on a selective agonistic activity at CB1 and CB2 cannabinoid receptors and to involve the main neurotransmitter systems. Results obtained in various studies suggest a potential clinical application of this highly potent drug (e.g., as antipyretic, antiinflammatory, analgesic, antiemetic, and antipsychotic agent) as well as its usefulness in research aimed to develop a better understanding of the involvement of the endogenous cannabinoid system in a number of physiopathological functions.

2000 - Effect of the cannabinoid receptor agonist, HU 210, on body weight and feeding behaviour of rats [Relazione in Atti di Convegno]
Giuliani, Daniela; Ottani, Alessandra; Ferrari, Francesca
abstract

The effect of the synthetic cannabinoid receptor agonist, y.11-hydroxy-D8-tetrahydrocannabinol-dymethylheptylHU 210., on ratbody weight and eating and drinking behaviour was examined. In Experiment 1, the drug25, 50 or 100 mgrkg., sub-chronicallyadministered for 4 days, produced a dose- and time-dependent loss of body weight that, at the highest dose, was not regained by 7 daysafter the drug was stopped, and remained markedly below that of vehicle-treated animals. In Experiment 2, food and water intakes, whichwere evaluated in fasted rats, tested as in Experiment 1, were significantly inhibited only by the dose of 100 mgrkg, and this effect wasstill present 7 days after the last injection of the drug. The possibility that the effects observed are not directly dependent on the control ofappetite and might be ascribable to stress-related phenomena is discussed.

2000 - EFFECTS OF THE CANNABINOID RECEPTOR AGONIST, HU 210, ON INGESTIVE BEHAVIOUR AND BODY WEIGHT OF RATS [Articolo su rivista]
Giuliani, Daniela; Ottani, Alessandra; Ferrari, Francesca
abstract

The effect of the synthetic cannabinoid receptor agonist, (-)11-hydroxy-Delta(8)-tetrahydrocannabinol-dymethylheptyl (HU 210), on rat body weight and eating and drinking behaviour was examined. In Experiment 1, the drug (25, 50 or 100 microg/kg), sub-chronically administered for 4 days, produced a dose- and time-dependent loss of body weight that, at the highest dose, was not regained by 7 days after the drug was stopped, and remained markedly below that of vehicle-treated animals. In Experiment 2, food and water intakes, which were evaluated in fasted rats, tested as in Experiment 1, were significantly inhibited only by the dose of 100 microgram/kg, and this effect was still present 7 days after the last injection of the drug. The possibility that the effects observed are not directly dependent on the control of appetite and might be ascribable to stress-related phenomena is discussed.

2000 - Inhibitory effects of the cannabinoid agonist HU 210 on rat sexual behaviour [Articolo su rivista]
F., Ferrari; Ottani, Alessandra; Giuliani, Daniela
abstract

The present study investigates the effects induced by the cannabinoid agonist HU 210 (25-100 mu g/kg, administered intraperitoneally [i.p.]) on the following parameters: (a) sexual behaviour of male rats, categorised on the basis of seven consecutive mating pretests as sexually active (SA) and sexually inactive (SI) and (b) sexual receptivity of ovariectomised female rats displaying hormonally induced heat. The data obtained show that HU 210, administered in acute or subchronic mode (once daily for 7 and 14 days), impaired the copulatory pattern of Sh rats in a dose- and mode-dependent manner, decreasing their sexual drive, mainly as represented by an increase in mount and intromission latencies, and affecting ejaculation mechanisms (represented as a decrease in intromission frequency and increase in ejaculation latency). After subchronic treatment with the highest dose had been suspended for 2 weeks, SA males´ performance was still impaired. In SI rats, acute injections of the drug (25 and 50 mu g/kg, i.p.) at the higher dose increased contact latency and decreased genital exploration time towards the female. Acute HU 210 (25-100 mu g/kg, ip) also inhibited female sexual behaviour, potently reducing lordosis quotient and lordosis intensity.

2000 - Neuroprotective effect of gamma-hydroxybutyrate in transient global cerebral ischemia in the rat [Articolo su rivista]
Vergoni, Anna Valeria; Ottani, Alessandra; Botticelli, A. R.; Zaffe, Davide; Guano, L.; Loche, A.; Genedani, Susanna; Gessa, G. L.; Bertolini, Alfio
abstract

The effect of gamma-hydroxybutyrate on the histological and behavioral consequences of transient brain ischemia was studied in the four vessel occlusion rat model. In saline-treated animals, 30 min ischemia caused a massive loss of neurons in the hippocampal CA1 subfield (normal neurons: 14%, 5%, 23% and 30% on the 3rd, 10th 15th and 65th day after ischemia, respectively). gamma-Hydroxybutyrate - 300 mg/kg intraperitoneally (i.p.) 30 min before or 10 min after arteries occlusion, followed by 100 mg/kg i.p. twice daily for the following 10 days - afforded a highly significant protection (normal neurons on the 3rd, 10th, 15th and 65th day after ischemia: 88% and 91%, 80% and 80%, 91% and 90%, 72% and 71% in rats receiving the first dose before or after arteries occlusion, respectively). The ischemia-induced sensory-motor impairment was significantly attenuated in rats receiving the first dose of gamma-hydroxybutyrate before arteries occlusion. Finally, the ischemia-induced impairment in spatial learning and memory, evaluated starting 27 days after the ischemic episode, was significantly attenuated by gamma-hydroxybutyrate, either injected first at 30 min before or 10 min after arteries occlusion. Lower doses of gamma-hydroxybutyrate had no significant effect. In conclusion, these results indicate that gamma-hydroxybutyrate provides significant protection against both histological and behavioral consequences of transient global cerebral ischemia in rats. (C) 2000 Elsevier Science B.V. All rights reserved.

2000 - Rat cognitive function during and after treatment with the cannabinoid agonist, HU 210 [Relazione in Atti di Convegno]
Ottani, Alessandra; Giuliani, Daniela; Ferrari, Francesca
abstract

To ascertain whether the cannabinoidagonist HU 210 (25, 50, or 100mg/kg, IP) influences rat spatial learning, water-maze performance was examined in theplace (hidden platform)—and cue (visible platform)—versions of the Morris water maze. In addition, other unlearned behaviorswere examined, namely, vocalization and wall hugging during the place task, and motor abilities during a motor test battery.The results obtained show that HU 210 at 50 or 100mg/kg (once daily for 4 days, 60 min before a daily session) impairedlearning in the place version but not in the cue one; wall hugging and enhanced vocalization were also displayed by the animalsin the fourth session. Motor activity was compromised by the same treatment schedule. When the drug was discontinued,the effects produced by HU 210 at 50mg/kg reversed in 3 days, while disruption of acquisition and vocalization caused byHU 210 at 100mg/kg remained after 7 days’ abstinence. Discussion centers on the possible specific cognitive mechanisms affectedby the drug and on aspecific factors (i.e., anxiety-like state), which may contribute to the impairment of spatial learning.

2000 - THE CANNABINOID AGONIST HU 210 MODIFIES RAT BEHAVIOURAL RESPONSES TO NOVELTY AND STRESS [Articolo su rivista]
Giuliani, Daniela; Ferrari, Francesca; Ottani, Alessandra
abstract

Experiments were performed on groups of rats after acute and sub-chronic treatment (once daily for 9 days) with the cannabinoid agonist HU 210 (25-100 µg kg(-1), i.p.) as well as 24 h and 7 days after the last drug injection. The animals underwent three behavioural tests in novel environments. In the observation cages (Test 1), rat locomotor activity was found to be dose-dependently reduced after acute and sub-chronic treatment at all doses and virtually unchanged during abstinence; grooming was potently inhibited by acute treatment but potentiated by the sub-chronic one at doses of 50 and 100 µg kg(-1), the effect of the higher dose persisting after 24 h and 7 days abstinence. Vocalization in animals in response to a tactile stimulus was highest after HU 210 at 100 µg kg(-1)in all experimental modes except after 7 days abstinence. In the X-maze (Test 2), sub-chronic HU 210 dose- dependently enhanced rat natural aversion for open arms, and this behaviour persisted during abstinence after the highest dose. Grooming in the X-maze was completely absent in rats acutely injected with HU 210 but potentiated in those sub-chronically treated or abstinent. In the swimming test (Test 3) rats sub-chronically treated at 50 and 100 pg kg(-1)displayed relevant wall-hugging and the same occurred 24 h after last injection. On the whole, our results are indicative of an anxiogenic-like effect of sub-chronic HU 210 at high doses and reflect the persistence of enhanced emotional response to novel environments when the treatment is discontinued.

1999 - Cannabimimetic activity in rats and pigeons of HU 210, a potent antiemetic drug [Articolo su rivista]
Ferrari, Francesca; Ottani, Alessandra; Giuliani, Daniela
abstract

Preliminary behavioral experiments in rats with thecannabinoid agonist HU 210 (12.5–100mg/kg IP) showed that it has a potent cannabimimetic profile similar to that ofD9THC;the drug dose dependently depressed locomotor activity, rearing, and grooming and elicited vocalization and circling at thehighest doses. In subsequent studies on pigeons, HU 210 (12.5–50mg/kg SC) confirmed its sedative effects; it also affordedprotection against vomiting induced by cisplatin (7.5 mg/kg IV) and emetine (20 mg/kg SC) and emetine-induced headshake.

1999 - Gamma-hydroxybutyrate increases gastric emptying in rats [Articolo su rivista]
Poggioli, Rosanna; Vitale, Giovanni; G., Colombo; Ottani, Alessandra; Bertolini, Alfio
abstract

The influence of gamma-hydroxybutyrate (GHB; 10, 50 or 100 mg/kg orally) and of its receptor antagonist, NCS-382 (25, 100 or 200 mg/kg orally, and 100 or 200 mg/kg intraperitoneally), on gastric emptying was studied in rats by measuring the serum level of acetaminophen (20 mg/rat orally, 30 min after GHB or NCS-382) 15, 30, 45 and 60 min after acetaminophen administration, or the amount of acetaminophen still present in the stomach 30 min after its administration. The highest dose of GHB produced a significant increase in 15 and 30 min serum levels of acetaminophen, indicating an acceleration of gastric emptying. A similar result was obtained with the prokinetic drug cisapride, at the oral dose of 2 mg/kg. On the other hand, NCS-382 significantly and dose-dependently reduced the serum levels of acetaminophen at every time of blood sampling, indicating a delay of gastric emptying, an effect confirmed by the amount of acetaminophen still present in the stomach 30 min after administration. Moreover, NCS-382 antagonized the prokinetic effect of GHB. These results may suggest for GHB (and/or possibly for its metabolites) a role in rat stomach motility.

1999 - Influence of the cannabinoid agonist HU 210 on cocaine- and CQP 201-403-induced behavioural effects in rat [Articolo su rivista]
Ferrari, Fabrizio; Ottani, Alessandra; Giuliani, Daniela
abstract

Acute injection of the cannabinoid agonist HU 210 (6.25-100 mu g/kg, i.p.) dose-dependently inhibited rat locomotor activity and rearing, while subchronic treatment with the drug (once daily for 7 days) at the same doses only diminished locomotion. Acute but not subchronic administration of HU 210 (12.5-50 mu g/kg, i.p.) potently counteracted acute and subchronic cocaine (15 mg/kg, i.p.)- induced hyperlocomotion and enhanced rearing. The acute cannabinoid (6.25-100 mu g/kg, i.p.) also inhibited locomotor activity, stereotyped behaviour and shaking elicited by the D-1/D-2 agonist CQP 201-403 (500 mu g/kg, i.p.). On the contrary, subchronic treatment's with HU 210 enhanced CQP 201-403-induced locomotor activity and potently stimulated escape attempts. Discussion centers on the influence of cannabinoids on experimental models of psychosis.

1999 - Learning impairment produced in rats by the cannabinoid agonist HU 210 in a water-maze task [Articolo su rivista]
Ferrari, F; Ottani, Alessandra; Vivoli, R; Giuliani, Daniela
abstract

To ascertain whether the cannabinoid agonist HU 210 (25, 50, or 100 mu g/kg, IP) influences rat spatial learning, water-maze performance was examined in the place (hidden platform)-and cue (visible platform)-versions of the Morris water maze. In addition, other unlearned behaviors were examined, namely, vocalization and wall hugging during the place task, and motor abilities during a motor test battery. The results obtained show that HU 210 at 50 or 100 mu g/kg (once daily for 4 days, 60 min before a daily session) impaired learning in the place version but not in the cue one: wall hugging and enhanced vocalization were also displayed by the animals in the fourth session. Motor activity was compromised by the same treatment schedule. When the drug was discontinued, the effects produced by HU 210 at 50 mu g/kg reversed in 3 days, while disruption of acquisition and vocalization caused by HU 210 at 100 mu g/kg remained after 7 days´ abstinence. Discussion centers on the possible specific cognitive mechanisms affected by the drug and on aspecific factors (i.e., anxiety-like state), which may contribute to the impairment of spatial learning.

1999 - The potentiation of analgesic activity of paracetamol plus morphine involves the serotonergic system in rat brain [Articolo su rivista]
Sandrini, Maurizio; Vitale, Giovanni; Ottani, Alessandra; Pini, Luigi Alberto
abstract

Objective and Design: We investigated the antinociceptive effect of subactive doses of paracetamol and morphine, given in combination. Material and Treatment: Male Wistar rats were injected with paracetamol (50 or 100 mg/kg i.p.) and morphine (2, 3 or 5 mg/kg s.c.) 10 min later and subjected to algesimetric tests 20 min thereafter. Methods: Pain threshold was evaluated in the hot-plate and formalin tests. 5-HT2 receptor binding capacity and 5-HT and 5-HIAA levels were measured in cortical and pontine areas of the brain by means of radioligand binding technique and by HPLC, respectively. Statistical analysis was done using Student-Neuman-Keul's test and 2 x 2 factorial analysis. Results: Only when given in combination, paracetamol(100 mg/kg) and morphine (2 and 3 mg/kg) were able to evoke an antinociceptive effect in both tests associated with an increase in 5-HT levels and a decrease in 5-HT2 receptors In the cortex. These effects were prevented by i,p. pretreatment with naloxone (I mg/kg i.p,). Conclusions: Subactive doses of paracetamol and morphine exert an analgesic effect when given in combination in the rat and indicate an involvement of both serotonergic and opiatergic systems.

1998 - Acetylsalicyclic acid potentiates the antinociceptive effect of morphine in the rat:involvement of the central serotonergic system. [Articolo su rivista]
Sandrini, Maurizio; Ottani, Alessandra; Vitale, Giovanni; Pini, Luigi Alberto
abstract

Acetylsalicylic acid and morphine are the most widely distributed and most frequently used drugs in the relief of pain, but their analgesic activity has adverse side-effects. Mixtures containing these two drugs are frequently used to relieve mild to moderate pain despite the paucity of relevant experimental evidence so far published. We set out to study the possible antinociceptive effect of a combination of subactive doses of the two drugs in rats. A combination of low doses of acetylsalicylic acid (50 mg/kg i.p.) and morphine (3 mg/kg s.c.) was administered and the pain threshold was evaluated in the hot-plate and formalin tests, and 5-HT2 receptor binding capacity, 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels were measured in the cortex and pontine areas of the brain. The combination of acetylsalicylic acid and morphine had an analgesic effect in both tests that was associated with an increase in 5-HT levels and a decrease in 5-HT2 receptors in the cortex. These effects were either completely abolished or partially prevented by i.p. pretreatment with naloxone (1 mg/kg i.p.). Our results demonstrate that subactive doses of acetylsalicylic acid and morphine can exert analgesic and biochemical effects when given in combination in the rat and suggest an involvement of serotonergic and opiatergic systems.

1998 - Effect of acetylsalicylic acid on formalin test and on serotonin system in the rat brain. [Articolo su rivista]
Pini, Luigi Alberto; Sandrini, Maurizio; Vitale, Giovanni; Ottani, Alessandra
abstract

Acetylsalicylic acid (ASA; 400 mg/kg, i.p.) increased serotonin (5-HT) content in rat brain but did not modify the number or the affinity of 5-HT1A receptors in the pons and the cerebral cortex, whereas the number of cortical 5-HT2 receptors decreased significantly. 2. Pretreatment with parachlorophenylaline (100 mg/kg/day for 4 days) depleted 5-HT brain content but modified neither the serum levels of salicylates nor the 5-HT2 cortical receptor characteristics, and it abolished the antinociceptive effect of ASA, 400 mg/kg, in the first phase of the formalin test. 3. These data support the involvement of the central serotonergic system in the antinociceptive activity of ASA

1997 - Naloxone-reverisble antinociception by paracetamol in the rat. [Articolo su rivista]
Pini, Luigi Alberto; Vitale, Giovanni; Ottani, A; Sandrini, M.
abstract

Paracetamol at the dose of 400 mg/kg i.p. displayed antinociceptive activity in the hot-plate test and the formalin test. Moreover, it induced a significant increase in brain serotonin (5-HT) concentration and a reduction in the number of 5-HT2 receptors in cortical membranes. Pretreatment with naloxone abolished this antinociceptive activity both in the hot-plate test and in the first phase of the formalin test without affecting the serum concentration of paracetamol. At the same time, naloxone prevented the increase in 5-HT concentration in the central nervous system and the reduction in 5-HT2 receptors in cortical membranes. Competition experiments demonstrated that paracetamol possesses affinity for [3H]naloxone binding sites. The action of morphine on nociception and on the serotonergic system was similar to that of paracetamol; all morphine-induced effects were blocked by naloxone. These data provide further evidence for a central antinociceptive effect of paracetamol and support the hypothesis that paracetamol exerts its antinociceptive activity through the serotonergic system. Moreover, our results point to the relationship between serotonergic and opiatergic systems in the antinociceptive activity of paracetamol.

1997 - Streptozotocin-induced diabetes provokes changes in serotonin concentration and on 5-HT1A and 5-HT2 receptors in the rat brain [Articolo su rivista]
Sandrini, Maurizio; Vitale, Giovanni; Vergoni, Anna Valeria; Ottani, Alessandra; Bertolini, Alfio
abstract

Since reduced levels of brain serotonin are known to cause behavioural abnormalities, to which diabetics are also prone, we investigated the effect, in rats, of chronic diabetes on brain serotonin concentration and on the numbers of 5-HT1A and 5-HT2 receptors in cerebral cortex and brainstem. Our data show that streptozotocin induces a longlasting hyperglicemia that is associated with a decrease in cerebral concentration of serotonin and with an accompanying increase in the maximum number of 5-HT1A and 5-HT2 receptors in the brain areas studied. Our results may suggest that changes in serotonergic transmission in the CNS play a role in diabetes-related behavioural abnormalities.

1996 - Effect of acute and chronic treatment with triiodothyronine on serotonin levels and serotonergic receptor subtypes in the rat brain [Articolo su rivista]
Sandrini, Maurizio; Vitale, Giovanni; Vergoni, Anna Valeria; Ottani, Alessandra; Bertolini, Alfio
abstract

Hyperthyroidism is often associated with behavioral disorders, and thyroid hormones modify receptor sensitivity as well as the synthesis and/or turnover rate of many neurotransmitters. We evaluated the influence in adult rats of triiodothyronine (T-3), administred s.c. (100 mu g/kg) acutely (once only) or chronically (once a day for 3 or 7 consecutive days), on brain serotonin concentration and on the density and affinity of two brain serotonin (5-HT) receptor subtypes mainly involved in behavioral effects. After both acute and chronic T-3 treatment, serotonin levels increased in the cerebral cortex but not in the hippocampus. The density and affinity of 5-HT1A receptors (using [H-3]-8-OH-DPAT as ligand) were not affected, while there was a significant decrease in the number of 5-HT2 receptors in the cerebral cortex (using [H-3]ketanserin as ligand). This observation might indicate that thyroid hormones enhance 5-HT concentration in certain brain areas, thus causing a down-regulation of 5-HT2 receptors. The serotonergic system could be involved in the complex brain-neurotransmitter imbalance underlying hyperthyroidism-linked behavioral changes.

Università degli studi di Modena e Reggio Emilia

Pubblicazioni