Nuova ricerca

Daniela GIULIANI

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

Home | Curriculum(pdf) | Didattica |


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.

2022 - Bioresorbable Nanostructured Chemical Sensor for Monitoring of pH Level In Vivo [Articolo su rivista]
Corsi, Martina; Paghi, Alessandro; Mariani, Stefano; Golinelli, Giulia; Debrassi, Aline; Egri, Gabriella; Leo, Giuseppina; Vandini, Eleonora; Vilella, Antonietta; Dähne, Lars; Giuliani, Daniela; Barillaro, Giuseppe
abstract

Here, the authors report on the manufacturing and in vivo assessment of a bioresorbable nanostructured pH sensor. The sensor consists of a micrometer-thick porous silica membrane conformably coated layer-by-layer with a nanometer-thick multilayer stack of two polyelectrolytes labeled with a pH-insensitive fluorophore. The sensor fluorescence changes linearly with the pH value in the range 4 to 7.5 upon swelling/shrinking of the polymer multilayer and enables performing real-time measurements of the pH level with high stability, reproducibility, and accuracy, over 100 h of continuous operation. In vivo studies carried out implanting the sensor in the subcutis on the back of mice confirm real-time monitoring of the local pH level through skin. Full degradation of the pH sensor occurs in one week from implant in the animal model, and its biocompatibility after 2 months is confirmed by histological and fluorescence analyses. The proposed approach can be extended to the detection of other (bio)markers in vivo by engineering the functionality of one (at least) of the polyelectrolytes with suitable receptors, thus paving the way to implantable bioresorbable chemical sensors.

2022 - Implantable and Bioresorbable Nanostructured Fluorescence Sensor for In vivo pH Monitoring [Relazione in Atti di Convegno]
Corsi, M.; Paghi, A.; Mariani, S.; Golinelli, G.; Debrassi, A.; Egri, G.; Leo, G.; Vandini, E.; Vilella, A.; Dahne, L.; Giuliani, D.; Barillaro, G.
abstract

Here we report on a bioresorbable fluorescence sensor for in vivo pH monitoring. The sensor leverages a nanometer-thick multilayer stack of polyelectrolytes labelled with a pH-insensitive fluorophore conformably deposited within a porous silica membrane-thickness of a few micrometers-to increase fluorescence intensity up to 600 times and enable reliable measurements through skin.

2022 - PCSK9 Affects Astrocyte Cholesterol Metabolism and Reduces Neuron Cholesterol Supplying In Vitro: Potential Implications in Alzheimer’s Disease [Articolo su rivista]
Papotti, B.; Adorni, M. P.; Marchi, C.; Zimetti, F.; Ronda, N.; Panighel, G.; Lupo, M. G.; Vilella, A.; Giuliani, D.; Ferri, N.; Bernini, F.
abstract

The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) involvement in Alzheimer's disease (AD) is poorly investigated. We evaluated the in vitro PCSK9 modulation of astrocyte cholesterol metabolism and neuronal cholesterol supplying, which is fundamental for neuronal functions. Moreover, we investigated PCSK9 neurotoxic effects. In human astrocytoma cells, PCSK9 reduced cholesterol content (-20%; p < 0.05), with a greater effect in presence of beta amyloid peptide (A beta) (-37%; p < 0.01). PCSK9 increased cholesterol synthesis and reduced the uptake of apoE-HDL-derived cholesterol (-36%; p < 0.0001), as well as the LDL receptor (LDLR) and the apoE receptor 2 (ApoER2) expression (-66% and -31%, respectively; p < 0.01). PCSK9 did not modulate ABCA1- and ABCG1-cholesterol efflux, ABCA1 levels, or membrane cholesterol. Conversely, ABCA1 expression and activity, as well as membrane cholesterol, were reduced by A beta (p < 0.05). In human neuronal cells, PCSK9 reduced apoE-HDL-derived cholesterol uptake (-41%; p < 0.001) and LDLR/apoER2 expression (p < 0.05). Reduced cholesterol internalization occurred also in PCSK9-overexpressing neurons exposed to an astrocyte-conditioned medium (-39%; p < 0.001). PCSK9 reduced neuronal cholesterol content overall (-29%; p < 0.05) and increased the A beta-induced neurotoxicity (p < 0.0001). Our data revealed an interfering effect of PCSK9, in cooperation with A beta, on brain cholesterol metabolism leading to neuronal cholesterol reduction, a potentially deleterious effect. PCSK9 also exerted a neurotoxic effect, and thus represents a potential pharmacological target in AD.

2021 - Identification of a Thyroid Hormone Derivative as a Pleiotropic Agent for the Treatment of Alzheimer’s Disease [Articolo su rivista]
Runfola, M.; Perni, M.; Yang, X.; Marchese, M.; Bacci, A.; Mero, S.; Santorelli, F. M.; Polini, B.; Chiellini, G.; Giuliani, D.; Vilella, A.; Bodria, M.; Daini, E.; Vandini, E.; Rudge, S.; Gul, S.; Wakelam, M. O. J.; Vendruscolo, M.; Rapposelli, S.
abstract

The identification of effective pharmacological tools for Alzheimer’s disease (AD) represents one of the main challenges for therapeutic discovery. Due to the variety of pathological processes associated with AD, a promising route for pharmacological intervention involves the development of new chemical entities that can restore cellular homeostasis. To investigate this strategy, we designed and synthetized SG2, a compound related to the thyroid hormone thyroxine, that shares a pleiotropic activity with its endogenous parent compound, including autophagic flux promotion, neuroprotection, and metabolic reprogramming. We demonstrate herein that SG2 acts in a pleiotropic manner to induce recovery in a C. elegans model of AD based on the overexpression of Aβ42 and improves learning abilities in the 5XFAD mouse model of AD. Further, in vitro ADME-Tox profiling and toxicological studies in zebrafish confirmed the low toxicity of this compound, which represents a chemical starting point for AD drug development.

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.

2019 - Regenerative potential of human dental pulp stem cells in the treatment of stress urinary incontinence: In vitro and in vivo study [Articolo su rivista]
Zordani, Alessio; Pisciotta, Alessandra; Bertoni, Laura; Bertani, Giulia; Vallarola, Antonio; Giuliani, Daniela; Puliatti, Stefano; Mecugni, Daniela; Bianchi, Giampaolo; De Pol, Anto; Carnevale, Gianluca
abstract

OBJECTIVES: To evaluate the regenerative potential of human dental pulp stem cells (hDPSCs) in an animal model of stress urinary incontinence (SUI). SUI, an involuntary leakage of urine, is due to physical stress involving an increase in bladder pressure and a damage of external urethral sphincter affecting muscles and nerves. Conventional therapies can only relieve the symptoms. Human DPSCs are characterized by peculiar stemness and immunomodulatory properties and might provide an alternative tool for SUI therapy. MATERIALS AND METHODS: In vitro phase: hDPSCs were induced towards the myogenic commitment following a 24 hours pre-conditioning with 5-aza-2'-deoxycytidine (5-Aza), then differentiation was evaluated. In vivo phase: pudendal nerve was transected in female rats to induce stress urinary incontinence; then, pre-differentiated hDPSCs were injected in the striated urethral sphincter. Four weeks later, urethral sphincter regeneration was assayed through histological, functional and immunohistochemical analyses. RESULTS: Human DPSCs were able to commit towards myogenic lineage in vitro and, four weeks after cell injection, hDPSCs engrafted in the external urethral sphincter whose thickness was almost recovered, committed towards myogenic lineage in vivo, promoted vascularization and an appreciable recovery of the continence. Moreover, hDPSCs were detected within the nerve, suggesting their participation in repair of transected nerve. CONCLUSIONS: These promising data and further investigations on immunomodulatory abilities of hDPSCs would allow to make them a potential tool for alternative therapies of SUI.

2018 - Melanocortin Receptor-4 Gene Polymorphisms in Glioblastoma Patients Treated with Concomitant Radio-Chemotherapy. [Articolo su rivista]
Pasqualetti, F; Orlandi, P; Simeon, V; Cantarella, M; Giuliani, Daniela; Di Desidero, T; Gonnelli, A; Delishaj, D; Lombardi, G; Sechi, A; Sanson, M; Zagonel, V; Paiar, F; Danesi, R; Guarini, Salvatore; Bocci, G.
abstract

Melanocortins are peptides with well-recognized antiinflammatory and neuroprotective activity. No data are currently available on melanocortin receptor-4 (MC4R) gene polymorphisms and tumors, including glioblastomas (GBMs), or their relationship with radiotherapy or chemotherapy. The aim of this study was to evaluate the possible predictive/prognostic role of the MC4R SNPs on GBM patients. Fifty-five patients with a proven diagnosis of GBM, treated with radiotherapy and temozolomide, were consecutively enrolled. MC4R gene SNPs (rs17782313, rs489693, rs8087522, rs17700633) were analyzed by a validated TaqMan® SNP genotyping assays. Univariate and multivariate analyses were performed. A P < 0.0125 (Bonferroni's correction) was considered significant ( Clinicaltrial.gov identifier NCT02458508). The median progression-free survival (PFS) and median overall survival (OS) of these patients were 9.54 (95% CI 5.4-14.3) months and 24.9 (95% CI 17.8-34.6) months, respectively. The MC4R rs489693 AA genotype was significantly associated with a shorter PFS and OS. Indeed, with regard to PFS, patients harboring the rs489693 AA genotype had a median PFS of 2.99 months whereas patients with AC/CC genotypes had a median PFS of 10.82 months (P = 0.009). Interestingly, the rs489693 AA patients also had a lower median OS as compared with the median OS of the AC/CC genotypes (10.75 vs. 29.5 months, respectively, P = 0.0001). This study suggests that the MC4R rs489693 AA genotype is significantly associated with a shorter PFS and OS in patients treated with radiotherapy and temozolomide. These findings represent a relevant effort to identify novel clinical markers for RT-CT therapy in GBM to be validated in future pharmacogenetic clinical trials.

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&nbsp;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 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 - 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 - 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 - 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 - Effects of aglycone genistein in a rat experimental model of postmenopausal metabolic syndrome. [Articolo su rivista]
Bitto, Alessandra; Altavilla, D; Bonaiuto, A; Polito, F; Minutoli, L; Di Stefano, V; Giuliani, Daniela; Guarini, Salvatore; Arcoraci, V; Squadrito, F.
abstract

Genistein aglycone, a soy derived isoflavone, has been demonstrated to be effective in reducing cardiovascular risk in postmenopausal women. We therefore investigated its effects in an experimental model of postmenopausal metabolic syndrome. Female spontaneously hypertensive obese rats (SHROB, n=40), a genetic model of syndrome X, and age-matched Wistar Kyoto (WKY, n=40) rats were used. A group of SHROB (n=20) and WKY (n=20) animals were ovariectomized (OVX). Four weeks after surgery all animals were randomized to receive either genistein (54 mg/human equivalent dose/day for 4 weeks), or vehicle. Body weight, food intake, systolic blood pressure (SBP), heart rate, plasma glucose, insulin resistance (HOMA-IR), total plasma cholesterol and triglycerides, and uterine weights were studied. Furthermore, we investigated acetylcholine- and sodium nitroprusside-induced relaxation of aortic rings as well as NG-L-arginine (L-NMA: 10–100 mM) induced vasoconstriction in phenylephrine-precontracted aortic segments. Liver expression of the peroxisome proliferator-activated receptor alpha (PPARA and gamma (PPARG was also assessed. OVX animals had a slight increase in SBP, body weight, insulin resistance, and plasma cholesterol. OVX-SHROB rats showed also impaired endothelial responses, blunted L-NMA induced contraction (L-NMA 100 mM, WKY=2.2±0.3 g/mg tissue; OVX-SHROB=1.1±0.4 g/mg tissue). Genistein treatment decreased SBP and plasma lipids, ameliorated endothelial dysfunction and insulin resistance, increased HDL cholesterol, and enhanced liver expression of PPARA and PPARG. Our data suggest that genistein is effective in ameliorating cardiovascular profiles in an experimental model of postmenopausal metabolic syndrome, attenuating the features of this disease. The effects of genistein are likely mediated by PPARA and PPARG receptors. This evidence would support the rationale for some pilot clinical trials using genistein in postmenopausal women affected by metabolic syndrome.

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.

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 - 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 - Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage [Articolo su rivista]
Giuliani, Daniela; C., Mioni; Bazzani, Carla; Zaffe, Davide; A. R., Botticelli; S., Capolongo; A., Sabba; M., Galantucci; Iannone, Anna; P., Grieco; E., Novellino; G., Colombo; Tomasi, Aldo; A., Catania; Guarini, Salvatore
abstract

Background and purpose: In circulatory shock, melanocortins have life-saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists. Experimental approach: Severe haemorrhagic shock was produced in rats under general anaesthesia. Rats were then treated with either the non-selective agonist [Nle(4), D-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) or with the selective MC4 agonists RO27-3225 and PG-931. Cardiovascular and respiratory functions were continuously monitored for 2 h; survival rate was recorded up to 24 h. Free radicals in blood were measured using electron spin resonance spectrometry; tissue damage was evaluated histologically 25 min or 24 h after treatment. Key results: All shocked rats treated with saline died within 30-35 min. Treatment with NDP-alpha-MSH, RO27-3225 and PG-931 produced a dose-dependent (13-108 nmol kg(-1) i.v.) restoration of cardiovascular and respiratory functions, and improved survival. The three melanocortin agonists also markedly reduced circulating free radicals relative to saline-treated shocked rats. All these effects were prevented by i.p. pretreatment with the selective MC4 receptor antagonist HS024. Moreover, treatment with RO27-3225 prevented morphological and immunocytochemical changes in heart, lung, liver, and kidney, at both early (25 min) and late (24 h) intervals. Conclusions and Implications: Stimulation of MC4 receptors reversed haemorrhagic shock, reduced multiple organ damage and improved survival. Our findings suggest that selective MC4 receptor agonists could have a protective role against multiple organ failure following circulatory shock.

2007 - The disaccharide trehalose inhibits proinflammatory phenotype activation in macrophages and prevents mortality in experimental septic shock [Articolo su rivista]
L., Minutoli; D., Altavilla; A., Bitto; F., Polito; E., Bellocco; G., Lagana; Giuliani, Daniela; T., Fiumara; S., Magazu; P., Ruggeri; Guarini, Salvatore; F., Squadrito
abstract

Proinflammatory phenotype activation in macrophages (M phi s) after sepsis orchestrates an inflammatory response leading to multiple organ dysfunction. Trehalose preserves cell viability during exposure to a range of environmental stresses. We investigated whether trehalose may inhibit endotoxin-induced activation of the inflammatory phenotype in M phi s. Rat peritoneal M phi s were stimulated with 50 mu g/mL of Salmonella enteritidis lipopolysaccharide (LPS). Stimulated M phi s were coincubated with trehalose (25, 50, and 100 mmol), sucrose (100 mmol), or RPMI alone. Macrophages cultures were used for Western blot analysis of extracellular-regulated kinase, c-jun-N terminal kinase, and inducible nitric oxide synthase; interleukin (IL) 1 beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) gene expression by real-time reverse transcriptase-polymerase chain reaction, and supernatants for measuring the release of inflammatory cytokines and nitrite content. In vitro trehalose significantly blunted LPS-induced extracellular-regulated kinase (LPS 21 +/- 6 integrated intensity; LPS + trehalose 100 mmol 2 +/- 0.3 integrated intensity), c-jun-N terminal kinase (LPS 15 +/- 5 integrated intensity; LPS + trehalose 100 mmol 3.5 +/- 0.9 integrated intensity), and inducible nitric oxide synthase activation (LPS = 12 +/- 3 integrated intensity; LPS + trehalose 100 mmol = 1 +/- 0.09 integrated intensity), blunted IL-1 beta (LPS = 5 +/- 1.9 n-folds/beta-actin; LPS + trehalose 100 mmol = 1.5 +/- 0.8 n-folds/beta-actin), IL-6 (LPS = 4 +/- 1.5 n-folds/beta-actin; LPS + trehalose 100 mmol = 1.4 +/- 0.5 n-folds/beta-actin), and TNF-alpha (LPS = 4.2 +/- 1.6 n-folds/beta-actin; LPS + trehalose 100 mmol = 1.1 +/- 0.7 n-folds/beta-actin) gene expression, and markedly reduced the release of inflammatory cytokines and nitrite content. Furthermore, in vivo trehalose prevented mortality in rats challenged with a lethal dose (20 mg/kg; LD90) of LPS (80% survival rate and 70% survival rate 24 and 72 h after LPS injection, respectively) and reduced serum TNF-alpha. Sucrose did not modified inflammatory phenotype in vitro nor in vivo protected against enclotoxin-induced mortality. Our study suggests that trehalose inhibits proinflammatory phenotype activation in M phi s and prevents enclotoxin-induced mortality.

2006 - Activation of the cholinergic anti-inflammatory pathway reduces NF-kappa B activation, blunts TNF-alpha production, and protects against splanchnic artery occlusion shock [Articolo su rivista]
D., Altavilla; Guarini, Salvatore; A., Bitto; Mioni, Chiara; Giuliani, Daniela; Bigiani, Albertino; G., Squadrito; L., Minutoli; Fs, Venuti; F., Messineo; V., De Meo; Bazzani, Carla; F., Squadrito
abstract

The cholinergic anti-inflammatory pathway has not yet been studied in splanchnic artery occlusion (SAO) shock. We investigated whether electrical stimulation (STIM) of efferent vagus nerves suppresses the inflammatory cascade in SAO shock. Animals were subjected to clamping of the splanchnic arteries for 45 min, followed by reperfusion. This surgical procedure resulted in an irreversible state of shock (SAO shock). Sham-operated animals were used as controls. Two minutes before the start of reperfusion, rats were subjected to bilateral cervical vagotomy (VGX) or sham surgical procedures. Application of constant voltage pulses to the caudal vagus ends (STIM: 5 V, 2 ms, 6 Hz for 15 min, 5 min after the beginning of reperfusion) increased survival rate (VGX + SAO + Sham STIM = 0% at 4 h of reperfusion; VGX + SAO + STIM = 90% at 4 h of reperfusion), reverted the marked hypotension, inhibited I kappa B alpha liver loss, blunted the augmented nuclear factor-kappa B activity, decreased hepatic tumor necrosis factor (TNF)-alpha mRNA (VGX + SAO + Sham STIM = 1.0 +/- 1.9 TNF-alpha/ glyceraldehyde-3-phosphate dehydrogenase ratio; VGX + SAO + STIM = 0.3 +/- 0.2 TNF-alpha/glyceraldehyde-3-phosphate dehydrogenase ratio), reduced plasma TNF-alpha (VGX + SAO + Sham STIM = 118 +/- 19 pg/mL; VGX + SAO + STIM = 39 +/- 8 pg/mL), ameliorated leukopenia, and decreased leukocyte accumulation, as revealed by means of myeloperoxidase activity in the ileum (VGX + SAO + Sham STIM = 7.9 +/- 1 U/g tissue; VGX + SAO + STIM = 3.1 +/- 0.7 U/g tissue) and in the lung (VGX + SAO + Sham STIM = 8.0 +/- 1.0 U/g tissue; VGX + SAO + STIM = 3.2 +/- 0.6 U/g tissue). Chlorisondamine, a nicotinic receptor antagonist, abated the effects of vagal stimulation. Our results show a parasympathetic inhibition of nuclear factor-kappa B and TNF-alpha in SAO shock.

2006 - Both early and delayed treatment with melanocortin 4 receptor-stimulating melanocortins produces neuroprotection in cerebral ischemia [Articolo su rivista]
Giuliani, Daniela; Mioni, C.; Altavilla, D.; Leone, S.; Bazzani, Carla; Minutoli, L.; Bitto, A.; Cainazzo, M. M.; Marini, H.; Zaffe, Davide; Botticelli, A. R.; Pizzala, R.; Savio, M.; Necchi, D.; Schioth, H. B.; Bertolini, Alfio; Squadrito, F.; Guarini, Salvatore
abstract

Ischemic stroke is one of the main causes of death and disability. We investigated whether melanocortin peptides, which have protective effects in severe hypoxic conditions, also produce neuroprotection in a gerbil model of ischemic stroke. A 10-min period of global cerebral ischemia, induced by occluding both common carotid arteries, caused impairment in spatial learning and memory that was associated with activation of inflammatory and apoptotic pathways, including severe DNA damage and delayed neuronal death, in the hippocampus. Treatment with nanomolar doses of the melanocortin analog [Nle(4), D-Phe(7)] alpha-MSH [which activates the melanocortin receptor subtypes ( MC) mainly expressed in central nervous system, namely MC3 and MC4] modulated the inflammatory and apoptotic cascades and reduced hippocampus injuries even when delayed up to 9 h after ischemia, with consequent dose-dependent improvement in subsequent functional recovery. The selective MC3 receptor agonist gamma(2)-MSH had no protective effects. Pharmacological blockade of MC4 receptors prevented the neuroprotective effects of [Nle(4), D-Phe(7)] alpha-MSH and worsened some ischemia outcomes. Together, our findings suggest that MC4 receptor-stimulating melanocortins might provide potential to develop a class of drugs with a broad treatment window for a novel approach to neuroprotection in ischemic stroke.

2006 - Broad therapeutic treatment window of [Nle(4), D-Phe(7)]alpha-melanocyte-stimulating hormone for long-lasting protection against ischemic stroke, in Mongolian gerbils [Articolo su rivista]
Giuliani, Daniela; Leone, S.; Mioni, C.; Bazzani, Carla; Zaffe, Davide; Botticelli, A. R.; Altavilla, D.; Galantucci, M.; Minutoli, L.; Bitto, A.; Squadrito, F.; Guarini, Salvatore
abstract

Melanocortin peptides have been shown to produce neuroprotection in experimental ischemic stroke. The aim of the present investigation was to identify the therapeutic treatment window of melanocortins, and to determine whether these neuropeptides chronically protect against damage consequent to brain ischemia. A 10-min period of global cerebral ischemia in gerbils, induced by occluding both common carotid arteries, caused impairment in spatial learning and memory (Morris test: four sessions from 4 to 67 days after the ischemic episode), associated with neuronal death in the hippocampus. Treatment with a nanomolar dose (340 mu g/kg i.p., every 12 h for 11 days) of the melanocortin analog [Nle(4), D-Phe(7)]alpha-metanocyte-stimulating hormone (NDP-alpha-MSH), starting 3-18 h after the ischemic episode, reduced hippocampal damage with improvement in subsequent functional recovery. The protective effect was long-lasting (67 days, at least) with all schedules of NDP-alpha-MSH treatment; however, in the latest treated (18 h) gerbils, some spatial memory deficits were detected. Pharmacological blockade of melanocortin MC4 receptors prevented the protective effects of NDP-alpha-MSH. Our findings indicate that, in conditions of brain ischemia, melanocortins can provide strong and longlasting protection with a broad therapeutic treatment window, and with involvement of melanocortin MC4 receptors, 18 h being the approximately time-limit for stroke late treatment to be effective.

2006 - I neuropeptidi melanocortinici nell’approccio cardioprotettivo contro il danno da ischemia e da riperfusione [Articolo su rivista]
Mioni, C.; Giuliani, Daniela; Bazzani, Carla; Zaffe, Davide; Botticelli, A. R.; Bigiani, Albertino; Lodi, Renzo; Guarini, Salvatore
abstract

In questi ultimi anni abbiamo documentato che dosi nanomolari di melanocortine (neuropeptidi appartenenti al gruppo ACTH/MSH) hanno la capacità di attenuare in modo significativo, nel ratto, il danno conseguente ad ischemia/riperfusione miocardica e ad occlusione coronarica permanente, Dai nostri dati emerge che le melanocortine, previa stimolazione di recettorimelanocortinici MC3 localizzati nel sistema nervoso centrale, durante l’episodio ischemico innescherebbero l'attivazione di una via vagale efferente cardioprotettiva. La tappa finale di tale via cardioprotettiva sembra essere rappresentata dall’attivazione di recettori muscarinici periferici. I nostri dati suggeriscono che le melanocortine potrebbero fornire il potenziale per lo sviluppo di una nuova classe di farmaci per un approccio innovativo alla patologia ischemica cardiaca.

2006 - Melanocortins protect against myocardial ischemia/reperfusion injury through the activation of an efferent cholinergic pathway [Abstract in Atti di Convegno]
Giuliani, Daniela; Bazzani, Carla; Mioni, Chiara; Zaffe, Davide; Squadrito, F; Guarini, Salvatore
abstract

A vagus nerve-mediated brain cholinergic protective mechanism, is operative in circulatory schock. We investigated, therefore, role and functional mechanism of such vagal efferent pathway (s) in a model of ischemic heart disease. Anesthetized rats were subjected to transient coronary artery occlusion (5 min) followed by reperfusion: occurrence of ventricular tachycardia (VT), ventricular fibrillation (VF), and lethality, were recorded up to the 5th min after reperfusion. Electrical stimulation of efferent vagal fibres (5 V, 2 ms, 1-9 Hz, for the whole period of ischemia/reperfusion) reduced the high incidence of VT , VF and lethality, the increase in free radical blood levels and left ventricle histological alteration. Treatment with same melanocortin peptides (162 nmol/kg i.v. or 16.2 nmol/kgi.c.v.) produced the same protective effects of electrical stimulation, and with the same muscarinic receptor-dependent mechanism, seemingly through brain activation (mediated by melanocortin MC3 receptors) of such efferent vagal pathway. These findings could provide the potential for a novel approach to management of ischemic heart disease.

2006 - Recombinant human erythropoietin improves angiogenesis and wound healing in experimental burn wounds [Articolo su rivista]
M., Galeano; D., Altavilla; A., Bitto; L., Minutoli; M., Calo; P., Lo Cascio; F., Polito; G., Giugliano; G., Squadrito; Mioni, Chiara; Giuliani, Daniela; F. S., Venuti; F., Squadrito
abstract

Objective., Erythropoietin interacts with vascular endothelial growth factor (VEGF) and stimulates endothelial cell mitosis and motility; thus it may be of importance in the complex phenomenon of wound healing. The purpose of this study was to investigate the effect of recombinant human erythropoietin (rHuEPO) on experimental bum wounds. Design: Randomized experiment. Setting. Research laboratory. Subjects. C57BL/6 male mice weighing 25-30 g. Interventions. Mice were immersed in 80 degrees C water for 10 secs to achieve a deep-dermal second degree burn. Animals were randomized to receive either rHuEPO (400 units/kg/day for 14 days in 100 mu L subcutaneously) or its vehicle alone (100 mu l/day distilled water for 14 days subcutaneously). On day 14 the animals were killed. Burn areas were used for histologic examination, evaluation of neoangiogenesis by immunohistochemistry, and expression (Western blot) of the specific endothelial marker CD31 as well as quantification of microvessel density, measurement of VEGF wound content (enzyme-linked immunosorbent assay), expression (Western blot) of endothelial and inducible nitric oxide synthases, and determination of wound nitric oxide (NO) products. Measurements and Main Results. rHuEPO increased burn wound reepithelialization and reduced the time to final wound closure. These effects were completely abated by a passive immunization with specific antibodies against erythropoietin. rHuEPO improved healing of burn wound through increased epithelial proliferation, maturation of the extracellular matrix, and angiogenesis. The hematopoietic factor augmented neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of the specific endothelial marker CD31. Furthermore, rHuEPO enhanced the wound content of VEGF caused a marked expression of endothelial and inducible nitric oxide synthases and increased wound content of nitric oxide products. Conclusions: Our study suggests that rHuEPO may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

2005 - Activation of an efferent cholinergic pathway produces strong protection against myocardial ischemia/reperfusion injury in rats [Articolo su rivista]
Mioni, C.; Bazzani, Carla; Giuliani, Daniela; Altavilla, D.; Leone, S.; Ferrari, Anna; Minutoli, L.; Bitto, A.; Marini, H.; Zaffe, Davide; Botticelli, Ar; Iannone, Anna; Tomasi, Aldo; Bigiani, Albertino; Bertolini, Alfio; Squadrito, F.; Guarini, Salvatore
abstract

Objective: A vagus nerve-mediated, brain cholinergic protective mechanism activated by melanocortin peptides is operative in conditions of circulatory shock; moreover, there is anatomical evidence of dual vagal-cardiac efferent pathways in rats, which could play different roles in controlling heart function. Therefore, we investigated the role and functional mechanism of such vagal efferent pathway(s) in an experimental model of ischemic heart disease. Design: Randomized experimental study. Setting: Research laboratory. Subjects: Adult Wistar rats of either sex. Interventions: After bilateral cervical vagotomy (with or without pretreatment with atropine), efferent vagal fibers were electrically stimulated in rats subjected to coronary artery occlusion (5 mins) followed by reperfusion (5 mins). Other rats (intact, vagotomized, or pretreated with atropine) were treated with nanomolar doses of melanocortin peptides. Measurements and Main Results: Electrical stimulation of efferent vagal fibers (5 V, 2 msecs,1-9 Hz, for the whole period of ischemia/reperfusion) strongly reduced the high incidence of severe arrhythmias and lethality, reduced the increase in free radical blood levels and left-ventricle histologic alterations, and augmented the extracellular signal-regulated kinase activation. Treatment with the melanocortin peptides adrenocorticotropin and gamma(2)-melanocyte-stimulating hormone (162 nmol/kg intravenously or 16.2 nmol/kg intracerebroventricularly, during coronary occlusion) produced the same protective effects of electrical stimulation and with the same muscarinic acetylcholine receptor-dependent mechanism, seemingly through brain activation (mediated by melanocortin MC3 receptors, as previously described) of such efferent vagal pathway. Conclusions: The present results give evidence for the identification of a protective, melanocortin-activated, efferent vagal cholinergic pathway, operative in conditions of myocardial ischemia/reperfusion. These data suggest that melanocortins and pertinent compounds able to activate such a pathway could provide the potential for development of a new class of drugs for a novel approach to management of ischemic heart disease.

2005 - Neuroprotection against ischemic stroke by early or delayed treatment with MC4 receptor-stimulating melanocortins [Abstract in Atti di Convegno]
Giuliani, Daniela; Bazzani, C; Mioni, C; Altavilla, D; Leone, S; Minutoli, L; Bitto, A; Zaffe, D; Botticelli, Ar; Pizzala, R; Ferrari, Anna; Squadrito, F; Bertolini, A; Guarini, Salvatore
abstract

Here we investigated whether melanocortin peptides, which have protective effects in severe hypoxic conditions, are able to produce neuroprotection in ischemic stroke. Our findings suggest that melanocortins that are agonist at the MC4 receptors may represent a class of drugs with a broad therapeutic window for a new approach to neuroprotection in ischemic stroke

2004 - Adrenocorticotropin reverses hemorrhagic shock in anesthetized rats through the rapid activation of a vagal anti-inflammatory pathway [Articolo su rivista]
Guarini, Salvatore; Cainazzo, Maria Michela; Giuliani, Daniela; Mioni, Chiara; D., Altavilla; H., Marini; Bigiani, Albertino; Ghiaroni, Valeria; M., Passaniti; Leone, Sheila; Bazzani, Carla; Ap, Caputi; F., Squadrito; Bertolini, Alfio
abstract

Objective: Several melanocortin peptides have a prompt and sustained resuscitating effect in conditions of hemorrhagic shock. The transcription nuclear factor kB (NF-kB) triggers a potentially lethal systemic inflammatory response, with marked production of tumor necrosis factor-alpha (TNF-alpha), in hemorrhagic shock. Here we investigated whether the hemorrhagic shock reversal produced by the melanocortin ACTH-(1-24) (adrenocorticotropin) depends on the activation of the recently recognized, vagus nerve-mediated, brain cholinergic anti-inflammatory pathway. Methods and results: Anesthetized rats were stepwise bled until mean arterial pressure (MAP) atabilized at 20-25 turn Hg. The severe hypovolemia was incompatible with survival, and all saline-treated animals died within 30 min. In rats intravenously (i.v.) treated with ACTH-(1-24), neural efferent activity along vagus nerve (monitored by means of a standard system for extracellular recordings) was markedly increased, and the restoration of cardiovascular and respiratory functions was associated with blunted NF-kB activity and with decreased TNF-alpha mRNA liver content and TNF-alpha plasma levels. Bilateral cervical vagotomy, pretreatment with the melanocortin MC4 receptor antagonist HS014, atropine sulfate or chlorisondamine, but not with atropine methylbromide, prevented the life-saving effect of ACTH-(1-24) and the associated effects on NF-kB activity and TNF-alpha levels. HS014 and atropine sulfate prevented, too, the ACTH-(1-24)-induced increase in neural efferent vagal activity, and accelerated the evolution of shock in saline-treated rats. Conclusions: The present data show, for the first time, that the melanocortin ACTH-(I -24) suppresses the NF-kB-dependent systemic inflammatory response triggered by hemorrhage, and reverses shock condition, by brain activation (in real-time) of the cholinergic anti-inflammatory pathway, this pathway seeming to be melanocortin-dependent. (C) 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

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 - Effects of ketamine anesthesia on central nociceptive processing in the rat: a 2-deoxyglucose study. [Articolo su rivista]
Porro, Carlo Adolfo; Cavazzuti, Milena; Giuliani, Daniela; Vellani, Vittorio; Lui, Fausta; Baraldi, Patrizia
abstract

Ketamine is a dissociative anesthetic with complex actions on the CNS. We investigated here the effects of ketamine anesthesia on somatosensory processing in the rat spinal cord, thalamus, and cerebral cortex, using the quantitative 2-deoxyglucose mapping technique. Unanesthetized or ketamine-anesthetized male Sprague-Dawley rats received a s.c. injection of a dilute formaldehyde solution (5%, 0.08 ml) into a forepaw, inducing prolonged noxious afferent input, or an equal volume of isotonic saline as a control stimulus. The 2-deoxyglucose experiments started 30 min after the injection. In the cervical enlargement of the spinal cord, ketamine had no significant effect on glucose metabolic rates in saline-injected animals, whereas it prevented the metabolic increases elicited by prolonged noxious stimulation in unanesthetized animals. At the thalamic level, ketamine increased glucose uptake in both saline- and formalin-injected rats in the lateral posterior, lateral dorsal, medial dorsal, gelatinosus, antero-ventral and antero-medial thalamic nuclei, whereas it decreased metabolic activity in the ventro-basal complex. At the cortical level, the drug increased metabolic activity in both control and formalin groups in the lacunosus-molecularis layer of the dorsal hippocampus, posterior parietal, retrosplenial, cingulate and frontal cortex; significant metabolic decreases were found in the CA1 region of the dorsal hippocampus and in the parietal 1 and 2 cortical areas. In the investigated brain regions, ketamine did not abolish noxious-evoked increases in glucose uptake, which were in fact enhanced in the forelimb cortex and in the lacunosus-molecularis layer of the hippocampus. The dissociation between the spinal and supraspinal effects of ketamine suggests a specific antinociceptive action on spinal circuits, in parallel with complex changes of the activity of brain circuits involved in somatosensory processing. More generally, this study shows that functional imaging techniques are able to quantitatively assess the effects of anesthetic drugs on nociceptive processing at different levels of the neuraxis. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

2004 - Erratum: Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors (European Journal of Pharmacology (2003) 477 (227-234)) [Articolo su rivista]
Mioni, C.; Giuliani, D.; Cainazzo, M. M.; Leone, S.; Iannone, A.; Bazzani, C.; Grieco, P.; Novellino, E.; Tomasi, A.; Bertolini, A.; Guarini, S.
abstract

2004 - Sull'identificazione di una via colinergica anti-shock fisiologica e dipendente dalle melanocortine. [Articolo su rivista]
Bazzani, Carla; Cainazzo, Maria Michela; Giuliani, Daniela; Mioni, Chiara; Leone, Sheila; Pagliai, Francesca; Ghiaroni, Valeria; Bigiani, Albertino; Altavilla, D.; Squadrito, F.; Bertolini, Alfio; Guarini, Salvatore
abstract

Da tempo abbiamo documentato che dosi nanomolari di melanocortine (peptidi appartenenti al gruppo ACTH/a-MSH) hanno un pronto effetto salvavita, indipendente dal surrene, sia nell'animale che nell'uomo in condizioni di shock circolatorio. Recentemente abbiamo dimostrato che, in condizioni di shock emorragico, il sistema nervoso centrale (SNC) modula rapidamente la risposta infiammatoria sistemica attraverso l attivazione di una via colinergica anti-infiammatoria mediata dal nervo vago e che l'azione salvavita delle melanocortine è dovuta all'attivazione di tale via, con inibizione dell’attivazione del fattore di trascrizione nucleare NF-kB a livello epatico e diminuzione dei livelli di "tumour necrosis fictor-" (TNF-) mRNA epatico e TNF- circolante. Le nostre ricerche hanno anche dimostrato che la via colinergica anti-infiammatoria coinvolge (come tappa iniziale) l'attivazione di recettori melanocortinici MC4, localizzati nel SNC e (come principale tappa finale) recettori nicotinici periferici molto probabilmente localizzati sui macmfagi epatici. I nostri dati suggeriscono che questa rapida via salvavita potrebbe essere fisiologica e melanocortined-dipendente, con ovvie ed importanti implicazioni cliniche.

2003 - Efferent vagal fibre stimulation blunts nuclear factor-kappa B activation and protects against hypovolemic hemorrhagic shock [Articolo su rivista]
Guarini, Salvatore; D., Altavilla; Mm, Cainazzo; Giuliani, Daniela; Bigiani, Albertino; H., Marini; G., Squadrito; L., Minutoli; Bertolini, Alfio; R., Marini; Eb, Adamo; Fs, Venuti; F., Squadrito
abstract

Background-We investigated whether electrical stimulation (STIM) of efferent vagus nerves may suppress nuclear factor (NF)-kappaB activation and the inflammatory cascade in hemorrhagic (Hem) shock. Methods and Results-Rats were subjected to bilateral cervical vagotomy (VGX) or sham surgical procedures. Hem shock was induced by intermittent withdrawing of blood until mean arterial pressure stabilized within the range of 35 to 40 mm Hg. Application of constant voltage pulses to the caudal vagus ends (STIM; 5 V, 2 ms, 1 Hz for 12 minutes, 5 minutes after mean arterial pressure stabilization) increased survival time (VGX+Hem+Sham STIM=38+/-3 minutes; VGX+Hem+STIM&gt;180 minutes), reverted the marked hypotension (VGX+Hem+Sham STIM=33+/-3 mm Hg; VGX+Hem+STIM=66+/-5 mm Hg), inhibited IkappaBalpha liver loss, and blunted the augmented NF-kappaB activity, decreased hepatic tumor necrosis factor (TNF)-alpha mRNA (VGX+Hem+Sham STIM=1.42+/-0.5 amount of TNF-alpha m-RNA; VGX+Hem+STIM=0.51+/-0.2 amount of TNF-alpha mRNA), and reduced plasma TNF-alpha (VGX+Hem+Sham STIM=190+/-24 pg/mL; VGX+Hem+STIM=87+/-15 pg/mL). Chlorisondamine, a nicotinic receptor antagonist, abated the effects of vagal stimulation. Conclusions-Our results show a parasympathetic inhibition of NF-kappaB by which the brain opposes NF-kappaB activation in the liver and modulates the inflammatory response during acute hypovolemic hemorrhagic shock.

2003 - Further evidence that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors [Articolo su rivista]
Mioni, Chiara; Giuliani, Daniela; Mm, Cainazzo; Leone, Sheila; Iannone, Anna; Bazzani, Carla; P., Grieco; E., Novellino; Tomasi, Aldo; Bertolini, Alfio; Guarini, Salvatore
abstract

In rats subjected to myocardial ischemia/reperfusion, melanocortin peptides, including gamma(1)-melanocyte-stimulating hormone (gamma(1)-MSH), are able to exert a protective effect by stimulating brain melanocortin MC3 receptors. A non-melanocortin receptor belonging to a group of receptors for Phe-Met-Arg-Phe-NH2 (FMRFamide)-like peptides may be involved in some of the cardiovascular effects of the gamma-MSHs. FMRFamide-like peptides and gamma(1)-/gamma(2)-MSH share, among other things, the C-terminal Arg-Phe sequence, which seems to be essential for cardiovascular effects in normal animals. So we aimed to further investigate which receptor and which structure are involved in the protective effects of melanocortins in anesthetized rats subjected to myocardial ischemia by ligature of the left anterior descending coronary artery (5 min), followed by reperfusion. In saline-treated rats, reperfusion induced, within a few seconds, a high incidence of ventricular tachycardia and ventricular fibrillation, and a high percentage of death within the 5 min of observation period. Reperfusion was associated with a massive increase in free radical blood levels and with an abrupt and marked fall in systemic arterial pressure. The i.v. treatment (162 nmol/kg) during the ischemic period with the adrenocorticotropin fragment 1-24 [ACTH-(1-24): the reference protective melanocortin which binds all melanocortin receptors], as well as with both the melanocortin MC3 receptor agonists gamma(2)-MSH and [D-Trp(8)]gamma(2)-MSH, reduced the incidence of ventricular tachycardia, ventricular fibrillation and death, the increase in free radical blood levels and the fall in arterial pressure. On the contrary, gamma(2)-MSH-(6-12) (a fragment unable to bind melanocortin receptors) was ineffective. Such protective effect was prevented by the melanocortin MC3/MC4 receptor antagonist SHU 9119. In normal (i.e., not subjected to myocardial ischemia/reperfusion) rats, the same i.v. dose (162 nmol/kg) of gamma(2)-MSH, [D-Trp(8)]gamma(2)-MSH and gamma(2)-MSH-(6-12) provoked a prompt and transient increase in arterial pressure; on the other hand, ACTH-(I -24), which lacks the C-terminal Arg-Phe sequence, decreased arterial pressure, but only at higher doses. Heart rate of normal rats was not affected by any of the assayed peptides. The present data confirm and extend our previous findings that melanocortins prevent myocardial reperfusion injury by activating melanocortin MC3 receptors. Moreover, they further support the notion that, in normal rats, cardiovascular effects of gamma-MSHs are mediated by receptors for FMRFamide-like peptides, for whose activation, but not for that of melanocortin MC3 receptors, the C-terminal Arg-Phe structure being relevant.

2003 - Independent time courses of supraspinal nociceptive activity and spinally mediated behavior during tonic pain [Articolo su rivista]
Porro, Carlo Adolfo; Cavazzuti, Milena; Lui, Fausta; Giuliani, Daniela; Pellegrini, Maddalena; Baraldi, Patrizia
abstract

The behavioral response to acute tissue injury is usually characterized by different phases, but the brain mechanisms underlying changes in pain-related behavior over time are still poorly understood. We aimed to analyze time-dependent changes in metabolic activity levels of 49 forebrain structures in the formalin pain model, using the autoradiographic 2-deoxyglucose method in unanesthetized, freely moving rats. We examined rats during the first phase of pain-related reactions ('early' groups), or during the third recovery phase, 60 min later, when the supraspinally mediated behavioral responses were reduced ('late' group). In the early groups, metabolic rates were bilaterally increased over control values in the periaqueductal gray, zona incerta and in several thalamic nuclei (anteroventral, centrolateral, lateral dorsal, parafascicular, posteromedial, submedius, ventromedial, and ventrobasal complex), as well as in the habenulae and in the parietal, cingulate, antero-dorsal insular, and anterior piriform cortex. A contralateral, somatotopically specific activation was found in the putative hindlimb representation area of the somatosensory cortex. In the late group, noxious-induced activation declined in most structures. However, metabolic rates were higher than controls in the periaqueductal gray and zona incerta and in two other structures not previously active: the prerubral area/field of Forel and the arcuate hypothalamic nucleus. These findings provide a time-dependent functional map of nociceptive and anti-nociceptive forebrain circuits during tonic pain. The parallel decrease in licking behavior and forebrain activity, at times when spinally mediated limb flexion responses were still present, suggests that endogenous antinociceptive systems may differently modulate spinal and supraspinal nociceptive networks following acute tissue injury. (C) 2003 International Association for the Study of Pain Published by Elsevier Science B.V. All fights reserved.

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 - MC3 receptors are involved in the protective effect of melanocortins in myocardial ischemia/reperfusion-induced arrhythmias [Articolo su rivista]
Guarini, Salvatore; Hb, Schioth; Mioni, Chiara; Mm, Cainazzo; G., Ferrazza; Giuliani, Daniela; Bertolini, Alfio; Bazzani, Carla; Es, Wikberg
abstract

Myocardial ischemia/reperfusion induces ventricular tachycardia (VT), ventricular fibrillation (VF) and a high degree of lethality. Since ACTH-(1-24) (adrenocorticotropin) protects against such injuries in rats, we investigated which melanocortin MC receptor is involved. Ischemia was produced in anesthetized rats by ligature of the left anterior descending coronary artery (5 min), and reperfusion-induced VT, VF, lethality and time-course of arterial blood pressure within the 5 min following reperfusion were evaluated. I.v. administration of the selective MC3 receptor agonist gamma(1)-melanocyte-stimulating hormone (gamma(1)-MSH), as well as of an equimolar dose (162 nmol/kg) of both the non-selective agonist ACTH-(1-24) and alpha-MSH, significantly prevented VT and VF, and increased survival. Coronary reperfusion was followed by an abrupt and massive fall in mean arterial pressure and pulse pressure, in saline-treated rats. Treatment either with ACTH(1-24) or gamma(1)-MSH completely prevented such fall. The protective effect of ACTH-(1-24) against the occurrence of VT, VF and lethality was neither affected by adrenalectomy, nor by i.v. pretreatment with the selective MC4 receptor antagonist HS014 and the MC4-MC5 antagonist HS059. On the other hand, the MC3-MC4 receptor antagonist SHU 9119 prevented such protective effect. Moreover, the selective MC1 receptor agonist MS05 (162 nmol/kg i.v.) failed to reduce the incidence of arrhythmias and lethality. These data demonstrate that MC3 receptors mediate the protective effect of melanocortins in myocardial ischemia/reperfusion-induced arrhythmias, in rats.

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.

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 - 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 - 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 - 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 - 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 - CNS pattern of metabolic activity during tonic pain: evidence for modulation by beta-endorphin [Articolo su rivista]
Porro, Carlo Adolfo; Cavazzuti, Milena; Baraldi, Patrizia; Giuliani, Daniela; Ae, Panerai; Corazza, Ruggero
abstract

CNS correlates of acute prolonged pain, and the effects of partial blockade of the central beta-endorphin system, were investigated by the quantitative 2-deoxyglucose technique in unanaesthetized, freely moving rats. Experiments were performed during the second, tonic phase of the behavioural response to a prolonged chemical noxious stimulus (s.c. injection of dilute formalin into a forepaw), or after minor tissue injury (s.c. saline injection). During formalin-induced pain, local glucose utilization rates in the CNS were bilaterally increased in the grey matter of the cervical spinal cord, in spinal white matter tracts and in several supraspinal structures, including portions of the medullary reticular formation, locus coeruleus, lateral parabrachial region, anterior pretectal nucleus, the medial, lateral and posterior thalamic regions, basal ganglia, and the parietal, cingulate, frontal, insular and orbital cortical areas. Pretreatment with anti-beta-endorphin antibodies, injected i.c.v., led to increased metabolism in the tegmental nuclei, locus coeruleus, hypothalamic and thalamic structures, putamen, nucleus accumbens, diagonal band nuclei and dentate gyrus, and in portions of the parietal, cingulate, insular, frontal and orbital cortex. In formalin-injected rats, pretreated with anti-beta-endorphin, behavioural changes indicative of hyperalgesia (increased licking response) were found, which were paralleled by a significant enhancement of functional activity in the anterior pretectal nucleus and in thalamo-cortical systems. A positive correlation was found between the duration of the licking response and metabolic activity of several forebrain regions. These results provide a map of the CNS pattern of metabolic activity during tonic somatic pain, and demonstrate a modulatory role for beta-endorphin in central networks that process somatosensory inputs.

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 - 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.

1997 - Effects of )eticlopride and 7-OH-DPAT on the tail-suspension test in mice [Articolo su rivista]
Ferrari, Francesca; Giuliani, Daniela
abstract

The dopamine (DA) D-2/D-3 antagonist (-)eticlopride (0.02, 0.05 and 0.1 mg/kg), dose-dependently increased immobility in the mouse tail-suspension test. Chronic treatment with the same compound (0.05, 0.1 mg/kg, x 14 days) produced a different effect, decreasing immobility when animals were tested 24 h after the last injection. The DA D-3 agonist, 7-OH-DPAT, acutely administered before the same test, behaved biphasically, increasing and decreasing mice immobility at low (0.05 and 0.1mg/kg) and high (1 and 2 mg/kg) doses, respectively. Chronically administered 7-OH-DPAT reduced the immobility time at 2 mg/kg but not at 0.1mg/kg. These effects, coupled with measurements of locomotor activity and evaluation of mice behaviour in different conditions, are discussed in the light of putative DA involvement in depressive states and are considered as predicting antidepressant potential.

1997 - INVOLVEMENT OF DOPAMINE D2 RECEPTORS IN THE EFFECT OF COCAINE ON SEXUAL BEHAVIOUR AND STRETCHING-YAWNING OF MALE RATS. [Articolo su rivista]
Ferrari, Francesca; Giuliani, Daniela; .,
abstract

The effect of cocaine (7.5, 15 and 30 mg/kg) administered in acute or subchronic mode, on the mating behaviour of sexually active male rats varied in a dose- and mode-dependent manner. Regardless of mode of treatment, 30 mg/kg markedly impaired the rats´ copulatory ability and impairment continued for a week after suspension of subchronic treatment. An acute dose of 15 mg/kg reduced intromission frequency, while in subchronic mode it also reduced ejaculation latency. Mount frequency was increased by 7.5 and 15 mg/kg, but only on first injection. In the case of sexually-naive male rats, acute administration of cocaine (3-30 mg/kg) stimulated penile erections at 7.5 mg/kg and motor hyperactivity at all doses. (-) Eticlopride (0.025 and 0.05 mg/kg), a DA D-2 antagonist, counteracted cocaine-induced motor hyperactivity but not penile erection, which it enhanced. (-)Eticlopride at the same doses also antagonized cocaine potentiation of lisuride (0.2 mg/kg)-induced behavioural effects. When male rats treated with subchronic cocaine (15 mg/kg) were injected with the DA D-2 agonist SND 919 (0.1 mg/kg), they displayed a more marked stretching-yawning behaviour than control animals receiving SND 919 at the same dose. The involvement of DA D-2 receptors in cocaine-induced effects is suggested.

1997 - Involvement of dopamine receptors in the antipsychotic profile of )eticlopride [Articolo su rivista]
Giuliani, Daniela; Ferrari, Francesca
abstract

The present study was performed to assess the effects exerted by the dopamine (DA) D-2/D-3 antagonist (-) eticlopride on rodent behavioral models considered to be predictive of antipsychotic activity, namely, antagonism toward DA agonist-induced stereotyped behavior (SB), and ketamine- and cocaine-induced hypermotility. (-) Eticlopride (10-50 mu g/kg) dose-dependently inhibited SB elicited by SND 919 (10 mg/kg), CQP 201-403 (0.5 mg/kg), and 7-OH-DPAT (5 mg/kg); moreover, it significantly counteracted the hypermotility induced in rats and mice by ketamine(5 and 10 mg/kg). When (-) eticlopride was injected before cocaine(15 mg/kg) either acutely or subchronically administered in male rats, it also potently antagonized the hypermotility typically induced by the drug. These results are discussed in the light of putative D-2/D-3 receptor involvement, and are considered as predictive of antipsychotic potential.

1996 - Behavioral effects induced by the dopamine D-3 agonist 7-OH-DPAT in sexually-active and -inactive male rats [Articolo su rivista]
Ferrari, Francesca; Giuliani, Daniela
abstract

The present study investigates the effects induced by the putative DA D-3 agonist 7-OH-DPAT (0.1 and 1 mg/kg, s.c.) on: (1) the sexual behavior of male rats, categorized on the basis of seven consecutive mating pre-tests as sexually-active (SA) and sexually-inactive (SI); and (2) stretching-yawning, penile erection, sedation and stereotyped behavior of the same animals. The data obtained show that 7-OH-DPAT at both doses modifies the copulatory pattern of SA rats, facilitating ejaculation mechanisms, but fails to increase the sexual drive of the animals as is evident from the ineffectiveness in SI rats. The second major finding is that the two groups of rats, which are markedly different as regards sexual typology, exhibit different behavioral responses to 7-OH-DPAT. Copyright

1996 - Differential behavioral response to dopamine D-2 agonists by sexually naive, sexually active, and sexually inactive male rats [Articolo su rivista]
GIULIANI, Daniela; FERRARI, Francesca
abstract

This study was performed with male rats categorized as sexually naive (SN), sexually active (SA), or sexually inactive (SI). In a first experiment the effects of the dopamine (DA) D-2 agonist SND 919 (0.05, 1, and 10 mg/kg) on the copulatory behavior of SN, SA, and SI rats were assessed. In a second experiment the DA D-2 agonist B-HT 920 (0.2 mg/kg) was used, and examination was limited to SN and SA rats. The effects exerted on stretching-yawning, penile erection, and sedation by the same compounds at the same doses in these three rat categories were also investigated. The main findings were that SND 919 and B-HT 920 facilitated ejaculation in SA rats, and that the rats that were different as regards level of sexual activity Exhibited different behavioral responses to the two DA agonists.

1996 - Influence of eticlopride on cocaine- and DA D-2 agonist-induced behavioral effects in rats [Articolo su rivista]
F., Ferrari; Giuliani, Daniela
abstract

The influence of the DA D-2 antagonist (-) eticlopride on cocaine- and DA D-2 agonist-induced behavioral effects was investigated by means of two series of experiments, in rats. In the first 10-day series, coadministration of (-) eticlopride (10 and 50 mu g/kg, SC) always potently inhibited cocaine (15 mg/kg, IP)-induced hypermotility but did not modify the penile erection (PE)-enhancement produced by the drug at the first injection; it actually counteracted the inhibitory effect of subchronic cocaine on PE. In the second series, (-) eticlopride, at the same doses, antagonized PE elicited by various DA D-2 agonists at nonstereotyping doses; when, along with PE, stereotyped behavior was induced, only the latter was inhibited by (-) eticlopride, which even increased PE.

1996 - Synthesis, resolution, and preliminary evaluation of trans-2-amino-6(5)-hydroxy-1-phenyl-2,3-dihydro-1H-indenes and related derivatives as dopamine receptors ligands [Articolo su rivista]
F., Claudi; Gm, Cingolani; A., Distefano; G., Giorgioni; F., Amenta; P., Barili; Ferrari, Francesca; Giuliani, Daniela
abstract

The present work reports the synthesis of enantiomeric pairs of the trans-2-amino-6-hydroxyl-phenyl-2,3-dihydro-1H-indene [(+)-14a, (-)-14a] and trans-2-amino-5-hydroxy-1-phenyl-2,3-dihydro-1H-indene [(+)-14b, (-)-14b] and their N,N-di-n-propyl [(+)- and (-)-15a,b], N-methyl-N-allyl [(+)- and (-)-16a,b], and N-methyl-N-n-propyl [(+) and (-)-17a,b] derivatives obtained by a combination of stereospecific reactions and optical resolution. The new compounds were evaluated for their affinity at the dopamine D-1 and D-2 receptors. The amines (+)- and (-)-14a, incorporating the D-1 pharmacophore 2-phenyl-2-(3-hydroxyphenyl)ethylamine in a trans extended conformation, and their derivatives displayed D-1 and D-2 affinity in the nanomolar range. On the other hand, the enantiomers (+)- and (-)-14b, (+)- and (-)-15b displayed high affinity and selectvity for the D-1 receptor. In a preliminary behavioral study on rats (+)-14b, and to a greater extent (+)-15b, promoted episodes of intense grooming, thus indicating that they act as central D-1 agonists. The trans-2-amino-5-hydroxy-1-phenyl-2,3-dihydro-1H-indenes (+)-14b and (+)-15b represent selective D-1 agonists lacking a catechol group, which should meet the prerequisites for a central nervous system penetration.

1995 - BEHAVIOURAL ASSESSMENT IN RATS OF THE ANTIPSYCHOTIC POTENTIAL OF THE POTENT DOPAMINE D2 RECEPTOR ANTAGONIST, (-) ETICLOPRIDE [Articolo su rivista]
F., Ferrari; Giuliani, Daniela
abstract

The effects of the selective D-2 DA receptor antagonist, (-)eticlopride, a drug belonging to the benzamide class, were investigated on the D-2 DA agonist SND 919- and CQP 201-403-induced stereotyped behaviour and on CQP 201-403-induced shaking, in rats, and on isolation-induced aggression, in mice. (-)Eticlopride was also tested over a wide dose range (5-1200 mu g kg(-1), s.c.) for sedative and cataleptic activity, in rats, For comparison, some experiments were performed with (-)sulpiride (10 and 40 mg kg(-1), s.c.) The data obtained show that (-)eticlopride differs from (-)sulpiride and potentially modifies animal behaviour, whether spontaneous or induced; moreover, they suggest a potential clinical use for this neuroleptic in the management of psychotic states.

1995 - BEHAVIOURAL EFFECTS OF THE DOPAMINE D3 RECEPTOR AGONIST, 7-OH-DPAT IN RATS. [Articolo su rivista]
Ferrari, Francesca; Giuliani, Daniela
abstract

The putative selective dopamine (DA) D3 receptor agonist, 7-OH-DPAT (25-4000 micrograms kg-1), enhanced stretching-yawning and penile erection in male rats, besides respectively increasing and decreasing sedation at low (25-200 micrograms kg-1) and high (1600 and 4000 micrograms kg-1) doses and inducing stereotypy from 800 micrograms kg-1 upwards. The DA D2 antagonist, (-) eticlopride (10 and 20 micrograms kg-1), antagonized stretching-yawning and penile erection induced by a low dose of 7-OH-DPAT (50 micrograms kg-1) but not those produced by high doses (1600 and 4000 micrograms kg-1), when stereotyped behaviour, on the other hand, was potently inhibited. Comparative experiments performed with the DA agonist SND 919 gave similar results.

1995 - Sexual attraction and copulation in male rats: effects of the dopamine agonist SND 919 [Articolo su rivista]
Ferrari, Francesca; Giuliani, Daniela; .,
abstract

Behavioral differences towards receptive females, involving latency to the first contact, amicable behavior, genital exploration, and copulatory pattern, were seen in sexually active (A), sluggish (S), and inactive (I) male rats classified on the basis of 11 consecutive mating tests. The D-2 dopamine agonist SND 919 (1 mg/kg) was intraperitoneally administered to the three groups 25 min before a 12th test; the drug stimulated the copulatory behavior of A and S but not of I rats in which it diminished genital exploration and amicable behavior. In a 13th test, conducted 1 week later, 31% of I initiated mating, 16% of them reaching ejaculation. The stimulant effect of SND 919 on copulation in A rats was confirmed in further experiments where it was injected at 0.1 mg/kg, a dose selective for the D-2 autoreceptors.

1994 - EFFECT ON RAT-FEEDING BEHAVIOR OF 2 SELECTIVE D-2-DOPAMINE AGONISTS [Articolo su rivista]
Ferrari, Francesca; Giuliani, Daniela
abstract

B-HT 958 and SND 919, two selective agonists at D-2 dopamine receptors, were examined for their influence on the feeding behavior of fasted rats. When food intake was determined in the rat's individual home cage, it was found to be reduced by both drugs at low sedative doses during the first hour after treatment and by SND 919 at the highest dose (which also elicits stereotypy) only 24 h later. However, SND 919 and B-HT 958 had no significant effect on feeding evaluated according to the X-maze and tube feeding tests. Analysis of the results, seen in the context of other behavioral signs produced by the drugs, suggests that data on feeding may vary depending on the experimental model used and can be modified by extraneous factors that interfere with a specific effect on food intake.

1994 - INABILITY OF )DEPRENYL TO MODIFY COPULATORY PERFORMANCE IN THE MALE-RAT, WHETHER OR NOT STIMULATED BY THE SELECTIVE D-2 DOPAMINE AGONIST SND-919 [Articolo su rivista]
F., Ferrari; Giuliani, Daniela
abstract

INABILITY OF )DEPRENYL TO MODIFY COPULATORY PERFORMANCE IN THE MALE-RAT, WHETHER OR NOT STIMULATED BY THE SELECTIVE D-2 DOPAMINE AGONIST SND-919

1994 - THE SELECTIVE D-2-DOPAMINE-RECEPTOR ANTAGONIST ETICLOPRIDE COUNTERACTS THE EJACULATIO-PRAECOX INDUCED BY THE SELECTIVE D-2-DOPAMINE AGONIST SND-919 IN THE RAT [Articolo su rivista]
Ferrari, Francesca; Giuliani, Daniela
abstract

The selective D-2 antagonist eticlopride, at a dose (0.01 mg/kg, s.c.) that fails to modify the normal behavior of rats, significantly reversed all the behavioral effects exerted by the selective D-2 agonist SND 919 (0.1 mg/kg, i.p.), namely, the stimulation of stretching-yawning, penile erection and sedation and the inhibition of grooming. In the copulatory test, eticlopride at the same dose did not affect animal sexual behavior but potently counteracted the reduction in mount and intromission frequency and latency to ejaculation induced by SND 919 at 0.1 mg/kg, a behavioral pattern which might possibly be proposed as an animal model for human ejaculatio praecox.

1993 - A COMPARATIVE BEHAVIOURAL ANALYSIS IN RATS OF THE D2 AGONIST RU 24213 AND TWO NEW STRUCTURALLY SIMILAR COMPOUNDS [Articolo su rivista]
Ferrari, Francesca; F., Claudi; G., Giorgioni; Giuliani, Daniela
abstract

The present study reports a comparative analysis of the behavioural effects induced in rats by three structurally similar compounds. One of them is the D2 dopamine agonist RU 24213, the others are newly-synthesized drugs whose selective affinity for D2 receptors has been established in priliminary binding studies. Discussion centers on the possible relationship between their chemical structure and behavioural activity.

1993 - BEHAVIORAL EVIDENCE THAT DIFFERENT NEUROCHEMICAL MECHANISMS UNDERLY STRETCHING-YAWNING AND PENILE ERECTION INDUCED IN MALE-RATS BY SND-919, A NEW SELECTIVE D-2 DOPAMINE-RECEPTOR AGONIST [Articolo su rivista]
F., Ferrari; F., Pelloni; Giuliani, Daniela
abstract

The behavioural effects induced in male Wistar rats by SND 919, a new drug reputed to have selective agonistic activity at D-2 dopamine (DA) receptors, were studied. The following aspects of behaviour were considered: motor activity, stretching-yawning (SY), penile erection (PE) and stereotyped behaviour (SB). Intraperitoneal injection (IP) of the drug (0.01-20 mg/kg) induced an SY syndrome in the form of a bell-shaped dose-response curve, the effect being maximal at the dose of 0.1 mg/kg and disappearing completely at 10 mg/kg. SND 919 also potently elicited PE; this latter effect, however, was not coincident with SY induction, being maximal at 1 mg/kg and persisting at 10 and 20 mg/kg. SND 919-induced SY was potently antagonized by pretreatment not only with the D-2 antagonist, L-sulpiride (20 mg/kg), but also with the alpha(2) antagonist, yohimbine (1, 3 mg/kg), and the more selective alpha(2) antagonist, idazoxan (1, 2 and 5 mg/kg). While sulpiride also decreased SND 919-induced PE, idazoxan at all doses and yohimbine at 1 mg/kg did not affect this behaviour. Inhibition of motor activity was induced by the D-2 agonist at low doses (0.05, 0.1 mg/kg), while at high doses (1, 10 and 20 mg/kg), it was actually replaced by a form of SB characterized by downward sniffing and licking. When, for comparison, the D-2 agonist, RU 24213 (0.1-20 mg/kg IF), was tested for PE, SY, motor activity and SB, it displayed a behavioural pattern very similar to that obtained with SND 919. Idazoxan (2 mg/kg), administered before RU 24213 (10 mg/kg), significantly antagonized the drug-induced SY, but not PE. The discussion centres on the specific neurochemical mechanisms presumably underlying the various forms of SND 919-induced behaviour and, in particular, PE and SY, which seem to differ, at least with respect to alpha(2) involvement.

1993 - BEHAVIOURAL EFFECTS INDUCED IN RATS AND CHICKS BY D2 DOPAMINE AGONISTS [Articolo su rivista]
F., Ferrari; Giuliani, Daniela
abstract

In a first series of experiments, different selective dopamine D2 receptor agonists (B-HT 920, B-HT 958, SND 919, CQ 32-084, CQP 201-403, and lisuride) and the D1/D2 agonist apomorphine were IP injected into adult male rats. At low doses, they elicited repeated episodes of penile erection and stretching-yawning; at all doses tested, B-HT 920, B-HT 958, and CQ 32-084 also induced hypomotility, a sign that, in the case of high doses of SND 919, CQP 201-403, lisuride, and apomorphine, was replaced by stereotyped behaviour. In a second series of experiments, the same D2 agonists and the mixed D1/D2 agonist apomorphine were IP injected at the same doses into chicks. The following behavioural signs were observed: hypomotility, sleep-like state, and stereotyped pecking. The results show that: 1. there are similarities between the behavioural effects induced by the DA agonists in rats and chicks; and 2. in both species some behavioural signs elicited by the DA ergic compounds are useful pointers to their specific neurochemical activity.

1993 - BEHAVIOURAL PROFILE IN CHICKEN OF CQ 32-084 AND CQP 201-403, TWO DOPAMINE AGONISTS [Articolo su rivista]
Ferrari, Francesca; F., Pelloni; Giuliani, Daniela
abstract

CQ 32-084 and CQP 201-403, two ergot derivatives that previous behavioural studies in rats had suggested to be differently active on dopamine (DA) receptors, were IP injected into male chickens. Both compounds strongly modified the animals' behaviour. CQ 32-084 led to sedation, increased yawning, and decreased preening, while CQP 201-403 exerted a biphasic activity: At a low dose, it elicited sedation and yawning; at high doses, however, it induced a state of excitation manifested by diminished sedation and yawning, enhanced preening, and pecking. The sedation, increased yawning, and decreased preening induced by the two DA agonists were reversed by the D2-selective antagonist, sulpiride. The present studies indicate that, from a behavioural point of view, chickens respond similarly to rats to the DA agonists CQ 32-084 and CQP 201-403, which differ in their selectivity of action on the various DA receptor subtypes.

1993 - INFLUENCE OF IDAZOXAN ON THE DOPAMINE D(2) RECEPTOR AGONIST-INDUCED BEHAVIORAL-EFFECTS IN RATS [Articolo su rivista]
F., Ferrari; Giuliani, Daniela
abstract

The behavioural effects in rats of the dopamine D2 receptor agonists. lisuride, B-HT 920 and SND 919. were variously influenced by pre-treatment with the selective alpha2-adrenoceptor antagonist, idazoxan (2 mg/kg), depending on the nature of the effect in question and the doses of agonist employed. The influence of idazoxan on drug-induced stretching-yawning, penile erection, sedation, stereotyped behaviour, aggressiveness and mounting is described and tentatively interpreted in neurochemical terms, account being taken of the activity of respective alpha2-adrenoceptor antagonist and dopamine receptor agonists used, at alpha2-adrenoceptors and at different dopamine D2 receptor subtypes, pre- and postsynaptically located.

1992 - A COMPARATIVE-STUDY OF B-HT 920 AND DIAZEPAM IN THE X-MAZE FEEDING TEST [Articolo su rivista]
Giuliani, Daniela; F., Pelloni; Tartoni, Pier Luigi; Ferrari, Francesca
abstract

Food-deprived rats injected with B-HT 920, an azepine agonist of dopamine D2 receptors, were observed for thier feeding behaviour using a novel a novel experimental model, X-maze test. Our sydy shows that the new procedure, though rapid, provides an indication of the emotional state and feeding of animals, and that B-HT 920 increses feeding behaviour in rats, it is very similar to elicited by diazepam.

1992 - B-HT 920 STIMULATES FEEDING AND ANTAGONIZES ANOREXIA INDUCED BY ACTH AND IMMOBILIZATION [Articolo su rivista]
F., Ferrari; F., Pelloni; Giuliani, Daniela
abstract

The influence of immobilisation and i.c.v. injection of ACTH on feeding in rats was examined using a new experimental model. An X-maze with alternate open and covered arms, each baited with standard laboratory chow was used, where individual rats were placed and observed for 5 min. Two essential aspects of the behaviour towards food were considered, namely, lasting and feeding. A number of parameters were applied to demonstrate the anorectic activity of ACTH and immobilisation, in accordance with data obtained using classical procedures for feeding analysis. ACTH at the dose used did not modify rat exploratory activity and grooming in the X-maze without food pellets. In the same X-maze feeding test, B-HT 920, a selective agonist of dopamine D2 receptors and alpha-2-adrenoceptors, shown earlier to have anxiolytic- and antidepressive-like properties in rats, enhanced appetite and exerted anxiolytic activity when injected ia.p. Pretreatment with B-HT 920 counteracted the restraint- and ACTH-induced effects. The results are discussed in the light of the relation between control of feeding and affective disorders. B-HT 920 activity seems to be of particular interest in view of its antagonism towards the anorexia elicited by two different agents reputed to have in common a key role in the stress-related disturbances of food intake.

1992 - B-HT 920-INDUCED EFFECTS ON RAT FEEDING BEHAVIOUR [Articolo su rivista]
Ferrari, Francesca; F., Pelloni; Giuliani, Daniela
abstract

Wistar rats, deprived of food for 15 h, were injected with B-HT 920 and 20 min later presented with their normal diet in their individual home cages. The parameters considered were latency to feeding and food intake which was determined 0.5, 1, 2, 3, 6 and 24 h later. B-HT 920 significantly reduced latency to feeding at 0.1, 0.5 and 1 mg/kg; food intake was increased by doses of 0.05 and 0.1 mg/kg 3 and 6 h after treatment and decreased by a dose of 1 mg/kg at 3 h. The amount of food eaten over a 24 h period by the various groups did not differ. At this time rats received a second injection of the drug at the same dosages, and preweighed food was presented again 20 min later. We confirmed that latency to feeding is lowered by B-HT 920 at 0.1, 0.5 and 1 mg/kg, doses which also induced feeding in sated rats within the first half-hour and even after 1 h in the case of the highest dose. Since penile erection and stretching and yawning, signs typically induced by all DA D2 agonists, were observed after B-HT 920 at 0.05, 0.1 and 0.5 mg/kg, discussion centres on the possible mechanisms involved in the B-HT 920-induced effects.

1992 - EFFECT OF THE D2-AUTORECEPTOR AGONIST B-HT-958 ON BOTH SPONTANEOUS AND ACTH-INDUCED STRETCHING, YAWNING AND GROOMING IN THE RAT [Articolo su rivista]
Ferrari, Francesca; F., Pelloni; M., Filaferro; Giuliani, Daniela
abstract

The D2 autoreceptor agonist B-HT 958, intraperitoneally injected into Wistar male rats in a novel environment, significantly increased stretching and yawning (SY) while inhibiting grooming. Pretreatment with the D2 antagonist sulpiride reversed these effects, antagonizing SY and restoring grooming. Similarly, when B-HT 958 was administered to rats in their home cages, it elicited SY and abolished grooming; moreover, when administered before the i.c.v. injection of adrenocorticotropin hormone, dose-dependently enhanced SY and strongly antagonized the typical syndrome of intensified grooming induced by the peptide. The possible relationship between SY and grooming and the involvement of D2 autoreceptors are discussed.

1992 - EFFECTS OF THE DOPAMINE D(2)-AGONISTS LISURIDE AND CQ 32-084 ON RAT-FEEDING BEHAVIOR [Articolo su rivista]
Ferrari, Fabrizio; F., Pelloni; Giuliani, Daniela
abstract

The influence on rat-feeding behaviour of lisuride and CQ 32-084, agonists at dopamine D2 receptors, was examined using two procedures. In a first series of experiments, the apparatus was an X-maze baited with food pellets where individual fasted rats were observed for 5 min. A number of parameters were recorded: latency to tasting and feeding, interval between tasting and feeding, total feeding time, and total grooming time. Lisuride (0.05 and 0.1 mg/kg) and CQ 32-084 (0.05 and 0.5 mg/kg) behaved as stimulants of eating; lisuride (0.4 mg/kg) inhibited the phenomenon. Both drugs always antagonized grooming. Subsequently, when food intake was determined in the home cages of fasted animals lisuride reduced feeding at all doses during the first hour after treatment, while CQ 32-084 had no effect. The data show that the two compounds display different activity on ingestive behaviour according to the dose and experimental model used. Discussion centres on the possible dependence of feeding enhancement in the X-maze on the anxiolytic activity exerted by low D2 autoreceptorial doses.

1992 - GROOMING AND STRETCHING-YAWNING - 2 BEHAVIORAL POINTERS OF D2 DOPAMINE RECEPTOR AGONISTIC EFFECT [Articolo su rivista]
Giuliani, Daniela; F., Pelloni; Ferrari, Francesca
abstract

A typical stretching-yaning (SY) syndromehas been described to be produce by D2 agonists and D1/D2 agonists when administered at doses incapable of eliciting a marked degree of stereotyped behaviour. Grooming has been reported to be antagonized by some D2 antagonists but stimulated by D1 agonist. Our results show that grooming and SY syndrome, contemporaneous detected, are sensitive behavioural pointers of the D2 selective affinity of the compounds.

1992 - Hyperphagic effects of the α2 adrenoceptor antagonists idazoxan and yohimbine in the x-maze feeding test [Articolo su rivista]
Ferrari, Francesca; Giuliani, Daniela
abstract

1992 - SUPPRESSIVE EFFECT OF THE DOPAMINE D2-RECEPTOR AGONIST B-HT 920 ON RAT GROOMING [Articolo su rivista]
F., Ferrari; F., Pelloni; Giuliani, Daniela
abstract

The effect of the D2 agonist B-HT 920 was examined on three behavioural models of induced grooming in the rat. B-HT 920 potently inhibited the grooming elicited by a novel environment, whereas it stimulated the stretching-yawning syndrome. Pretreatment with the selective dopamine D2 receptor antagonist, sulpiride, reversed the phenomenon. When B-HT 920 was administered to rats before water immersion, it similarly antagonized total grooming; wet-dog shakes, detected in these same animals, were potently inhibited. Finally, B-HT 920 displayed inhibitory activity towards adrenocorticotropin hormone-induced excessive rooming. On the basis of these effects, the role of D2 receptor subtypes in the modulation of grooming is discussed.

1992 - THE D2 AGONIST B-HT 920 POTENTLY ANTAGONIZES RAT GROOMING. [Articolo su rivista]
Ferrari, Francesca; F., Pelloni; Giuliani, Daniela
abstract

Grooming is anormal trait of rat's behaviour which serves different physiological and ethological functions. It has been hypothesized that grooming reflects a state of stress of the animal and antagonized by dopamine D1 agonist. We examined the effects of the selective D2 agonist B-HTt 920 on grooming behaviour. Our results show that B-HT 920 exerts an inhibitory effect of induced-grooming in rats.

1991 - BRAIN ACETYLCHOLINE IS INVOLVED IN THE ACTH-INDUCED BEHAVIORAL SYNDROME IN RATS [Articolo su rivista]
Poggioli, Rosanna; Vergoni, Anna Valeria; Giuliani, Daniela; D., Marrama; E., Rasori; Bertolini, Alfio
abstract

Stretching, yawning, penile erections and excessive grooming induced in adult rats by the intracerebroventricular injection of ACTH1-24 (4-mu-g/rat) were completely abolished by pretreatment with the cholinergic neuron blocking agent, hemicholinium-3 (20-mu-g/rat i.c.v.), and significantly reduced by the non-selective muscarinic antagonist, atropine sulphate (4 mg/kg i.p.) (except for stretching). The M1-muscarinic antagonist, pirenzepine, at the dose of 80-mu-g/rat i.c.v. almost completely prevented all the above behavioral effects of ACTH, while the M3-muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (25-mu-g/rat i.c.v.) had no influence, and the nicotinic antagonist mecamylamine (25 and 50-mu-g/rat i.c.v.) only reduced excessive grooming and the number of penile erections. The M2-antagonist, 11-2[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one, (AF-DX 116), inhibited stretching, yawning and grooming (but not penile erections) at low doses (1-mu-g/rat i.c.v.), while at higher doses (4 or 6-mu-g/rat i.c.v.) it inhibited penile erections but not the other ACTH-induced behaviors. It is concluded that the most typical behavioral effects of ACTH in rats involve brain cholinergic neurons and the activation mainly of central M1- and M2-muscarinic receptors.

1991 - HYPERPHAGIC EFFECT OF B-HT-920 IN A MODIFIED X-MAZE TEST [Articolo su rivista]
Ferrari, Francesca; Pelloni, F; Giuliani, Daniela
abstract

The influence on rat-feeding behavior of B-HT 920 (a selective agonist of D2DA receptors at low doses, but also a potent stimulant of alpha-2-adrenoceptors at high doses) was examined using a new experimental model. The apparatus adopted was an X-maze with alternate open and covered arms, each baited with two food-pellets. Individual rats were placed in the apparatus and observed for 5 min. Two essential aspects of rat behavior in the presence of food were considered: tasting and feeding. A number of parameters were recorded: latency to tasting and feeding; interval between tasting and feeding; total feeding time. We also took into account the type of arm in which the rats indulged in their first bout of tasting and feeding. In the first series of experiments, B-HT 920 was injected intraperitoneally (0.1, 1, 2 and 3 mg/kg) into fed and fasted rats; in the second series, the drug was injected intracerebroventricularly (10, 20 and 80-mu-g/rat) into fasted rats. In both experimental conditions, the drug significantly modified the animals' feeding behavior and affected their natural preference for the closed arms as places of choice in which to feed. Comparison of the results with those obtained using norepinephrine (20-mu-g/rat, intracerebroventricularly), a well-known hyperphagic agent, shows that B-HT 920 strongly stimulates feeding. Also, the results of the intracerebroventricular experiments confirm that the effect on feeding appears at doses that are active both on alpha-2-adrenoceptors and on D2DA receptors, as can be deduced from penile erection and stretching and yawning elicited in rats by the same D2DA stimulant doses. The involvement of the latter receptors in feeding has been investigated by means of a new series of experiments with rats treated with the D2DA antagonist sulpiride, before B-HT 920 at 100-mu-g/kg. The possible mechanisms underlying B-HT 920-hyperphagic effects and the value of the X-maze feeding test as a simple procedure indicative of changes in rat feeding and emotionality are discussed in the light of these latest findings.

1990 - Behavioral and binding studies on the interactions between melanopeptides and brain acetylcholine [Articolo su rivista]
Poggioli, R.; Sandrini, M.; Vergoni, A. V.; Giuliani, D.; Rasori, E.; Marrama, D.; Bertolini, A.
abstract

1990 - Influence of atriopeptin II on behavior and pain sensitivity in rats [Articolo su rivista]
Poggioli, R.; Vergoni, A. V.; Marrama, D.; Giuliani, D.; Rasori, E.; Bertolini, A.
abstract

1990 - NPY-induced inhibition of male copulatory acivity is a direct behavioural effect [Articolo su rivista]
Poggioli, Rosanna; Vergoni, Anna Valeria; D., Marrama; Giuliani, Daniela; Bertolini, Alfio
abstract

In adult, sexually-experienced male rats, the intracerebroventricular injection of NPY caused a dose-related inhibition of copulatory behaviour, all parameters (mount, intromission and ejaculation latencies, mount and intromission frequencies, mean inter-intromission interval, post-ejaculatory interval) being significantly worsened at the dose of 8 micrograms/rat. Since rats were deprived of food during the behavioural test, it is concluded that inhibition of sexual behaviour is a 'true', direct behavioural effect of NPY, not due to a shift towards increased feeding.

1989 - INFLUENCE OF ACTH-(1-24) ON INGESTIVE BEHAVIOURS [Articolo su rivista]
Vergoni, Anna Valeria; Poggioli, Rosanna; D., Marrama; Giuliani, Daniela
abstract

ACTH-(1-24) has a complex, direct and differentiated effect on eating behaviour which seems to be very similar to that already described for CRF. These similarities indicate that both these neuropeptides coud be putative mediators in stress-induced anorexia.