SALVATORE GUARINI - personale UniMoRe

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SALVATORE GUARINI

CULTORE DELLA MATERIA
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

Pubblicazioni

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

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

2018 - Maternal responses and communication development in extremely preterm infants. [Relazione in Atti di Convegno]
Sansavini, A.; Benassi, E.; Guarini, A.; Savini, S.; Caselli, M. C.; Iverson, J. M.
abstract

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 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 - Farmacologia Medica ed Elementi di Terapia [Curatela]
D'Alessandro, N.; De Ponti, F.; Failli, P.; Giraldi, T.; Guarini, Salvatore; Locatelli, V.
abstract

Il presente volume possiede caratteristiche congiunte di completezza e sintesi che lo rendono interessante e adeguato per gli studenti delle facoltà universitarie con corsi di Laurea in medicina e chirurgia e delle professioni sanitarie che si stiano preparando a superare esami di profitto nelle materie farmacologiche.

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

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

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

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

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

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

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

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

2009 - 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.

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 - Lipid peroxidation triggers both c-Jun N-terminal kinase (JNK) and extracellular-regulated kinase (ERK) activation and neointimal hyperplasia induced by cessation of blood flow in the mouse carotid artery [Articolo su rivista]
F., Squadrito; L., Minutoli; M., Esposito; A., Bitto; H., Marini; P., Seminara; A., Crisafulli; M., Passaniti; E. B., Adamo; R., Marini; Guarini, Salvatore; D., Altavilla
abstract

We investigated whether lipid peroxidation might influence activation of the mitogen activated protein kinase (MAPK) extracellular regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) in neointimal hyperplasia induced by flow interruption of carotid artery in mice. C57/BL6 mice were subjected to a complete ligation of the left common carotid artery or to a sham ligation. Animals were randomized to receive either IRFI-042, a Vitamin E-like inhibitor of lipid peroxidation (20 mg/kg/i.p., immediately after artery occlusion) or its vehicle (1 ml/kg of a NaCl-DMSO solution). The extent of lipid peroxidation (investigated by the means of conjugated dienes levels) and JNK and ERK activation were evaluated by Western blot analysis after blood flow interruption. ICAM-1 expression in injured arteries was investigated 4 days after artery ligation by the means of reverse transcriplase polymerase chain reaction (RT-PCR) and quantification of the ICAM-1 protein levels. Morphometric analysis of the structural alteration caused by the disruption of the arterial blood flow was performed 4 weeks after surgery.

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

2005 - Oxidative stress and experimental shock [Abstract in Rivista]
Tomasi, Aldo; Bergamini, Stefania; Bellei, Elisa; Bazzani, Carla; Bertolini, Alfio; Guarini, Salvatore; Iannone, Anna
abstract

In this introductory presentation, some of the recent results of our and other groups on the oxidative stress theory in the development of experimental shock, will be presented and discussed.

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 - 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>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 - Identification of a melanocortin-dependent anti-shock pathway: brain-to-immune system cholinergic communication [Abstract in Rivista]
Squadrito, F.; Altavilla, D.; Bazzani, Carla; Minutoli, L.; Marini, H.; Mioni, C.; Bertolini, Alfio; Venuti, F. S.; Guarini, Salvatore
abstract

Identification of a melanocortin-dependent anti-shock pathway: brain-to-immune system cholinergic communication

2003 - Lipid peroxidation inhibition reduces NF-kappa B activation and attenuates cerulein-induced pancreatitis [Articolo su rivista]
D., Altavilla; C., Famulari; M., Passaniti; Gm, Campo; A., Macri; P., Seminara; H., Marini; M., Calo; Lb, Santamaria; D., Bono; Fs, Venuti; Mioni, Chiara; Leone, Sheila; Guarini, Salvatore; F., Squadrito
abstract

Increased lipid peroxidation, enhanced nuclear factor kappa-B (NF-kappaB) activation and augmented tumor necrosis factor-alpha (TNF-alpha) production have been implicated in cerulein-induced pancreatitis. We investigated whether lipid peroxidation inhibition might reduce NF-kappaB activation and the inflammatory response in cerulein-induced pancreatitis. Male Sprague-Dawley rats of 230-250 g body weight received administration of cerulein (80 mug/kg s.c. for each of four injections at hourly intervals). A control group received four s.c. injections of 0.9% saline at hourly intervals. Animals were randomized to receive either raxofelast, an inhibitor of lipid peroxidation (20 mg/kg i.p. administered with the first cerulein injection) or its vehicle (1 ml/kg of a 10% DMSO/NaCl solution). All these rats were sacrificed 2 h after the last injection of either cerulein or its vehicle. Raxofelast administration (20 mg/kg i.p. with the first cerulein) significantly reduced malondialdehyde (MDA) levels, an index of lipid peroxidation (CER+DMSO=3.075+/-0.54 mumol/g; CER+raxofelast= 0.693+/-0.18 mumol/g; p < 0.001), decreased myeloperoxidase (MPO) activity (CER+DMSO=22.2+/-3.54 mU/g; CER+raxofelast=9.07+/-2.05 mU/g; p < 0.01 ), increased glutathione levels (GSH) (CER+DMSO= 5.21+/-1.79 mumol/g; CER+raxofelast=15.71+/-2.14 mumol/g; p < 0.001 ), and reduced acinar cell damage evaluated by means of histology and serum levels of both amylase (CER+DMSO=4063+/-707.9 U/l; CER+raxofelast=1198+/-214.4 U/l; p < 0.001 ), and lipase (CER+DMSO=1654+/-330 U/l; CER+raxofelast= 386+/-118.2 U/l; p < 0.001 ), Furthermore, raxofelast reduced pancreatic NF-κB activation and the TNF-α mRNA levels and tissue content of mature protein in the pancreas. Indeed, lipid peroxidation inhibition might be considered a potential therapeutic approach to prevent the severe damage in acute pancreatitis.

2003 - New gene therapy for the treatment of burn wounds [Articolo su rivista]
Guarini, Salvatore
abstract

Skin gene therapy is a relatively new approach with great potential because of the accessibility and the possibility to monitor the modified area. Gene therapy may facilitate the transfer of several growth factors. Among them, vascular endothelial growth factor (VEGF) plays a pivotal role in the skin repair process: For wound healing, the induction of controlled neo-angiogenesis is, in fact, a fundamental process. Dr. Galeano and colleagues designed a study, described in this issue of Critical Care Medicine, aimed at evaluating the efficacy of a gene therapy with VEGF for the treatment of burn wounds. For successful gene delivery, the selection of an appropriate vector has been shown to be paramount. Viruses, in particular adenoviruses, with their transfection capabilities, have been used as gene vectors. However, adenoviruses display infection-associated toxicity, immunologic compromise, and possible mutagenic effects that make this approach potentially dangerous . In their experiments of VEGF gene therapy, Dr. Galeano and colleagues used vectors based on the adeno-associated virus (AAV). These vectors, derived from a nonpathogenic Parvovirus, do not have any viral genes and therefore cause no inflammatory or immune reaction in the site of injection.

2002 - ACTH analogue in treatment of acute aortic dissection-Authors' reply. [Articolo su rivista]
Noera, G; Lamarra, M; Guarini, Salvatore; Bertolini, Alfio
abstract

Sir—The fact that the mortality rate inour patients who were assigned to thestandard treatment, without ACTH-(1-24) addition, was higher than thatreported in previous studies might beexplained by their severe haemodynamiccompromise. We selectedsubjects with type A aortic dissectioncomplicated by aortic rupture andcardiac tamponade, and with clinicaland laboratory signs of haemorrhagicshock.We share Olsson’s opinion thatACTH-(1-24) essentially modifies thecomplex pathophysiology of theperioperative period. Not only byimproving the cardiovascular function;indeed, several experimental datasuggest that melanocortins have also apeculiar, adrenal-independent antiinflammatoryactivity. They reduce theproduction of proinflammatorycytokines, such as interleukins 1, 1,and 6, and tumour necrosis factor(TNF) , and inhibit the activation ofthe transcription factor NF-kB, whileincreasing the production of the antiinflammatorycytokines interleukins 8and 10;1 in particular, in conditions ofhaemorrhagic shock, melanocortinsinhibit the overproduction of TNF,nitric oxide, and free radicals.2,3Moreover, melanocortins protectagainst the outcomes either of a shorttermmyocardial ischaemia followed byreperfusion or of the permanentocclusion of a coronary artery in rats.4Our patients underwent fluidreplacement en route and ventilatorysupport but no rescue surgery. Ourcardiac surgery unit is situated roughlyin the middle of a flat territory innorthern Italy, with a high populationdensity (about 1 million people), and areasonable road system. The time lapsefrom emergency call to arrival into thecasualty ward, by ambulance or byhelicopter, is 20–40 min.Our animal data suggest that thetime span of ACTH-(1-24) efficacy isbest if given within 5–15 min of shockinduction. We have fewer human data,but they suggest that treatment must bemade within 1 h of the first signs ofshock. In case of haemorrhagic shock(road or industrial accidents, &c), webelieve ACTH should be given asintravenous bolus injection at thescene, or en route in the ambulance.The effect of ACTH lasts a few hours.

2002 - Cannabinoid CB1 receptor blockade enhances the protective effect of melanocortins in hemorrhagic shock in the rat [Articolo su rivista]
Mm, Cainazzo; G., Ferrazza; Mioni, Chiara; Bazzani, Carla; Bertolini, Alfio; Guarini, Salvatore
abstract

Activation of peripheral cannabinoid CB1 receptors contributes to hemorrhagic hypotension, and endocannabinoids produced by macrophages and platelets may be mediators of this effect. A number of studies have provided evidence that functional links exist in the mechanisms of action of cannabinoids and opioid peptides; and opioids too play an important role in the pathophysiology of hemorrhagic hypotension and shock. On the other hand, melanocortin peptides, which are the main endogenous functional antagonists of opioid peptides, have an antishock effect in animals and humans. Thus, we investigated whether an interaction exists between endocannabinoids and the endogenous opioid/antiopioid system also in a condition of hemorrhagic shock and, particularly, whether the blockade of cannabinoid CB1 receptors potentiates the antishock effect of melanocortins, Urethane-anesthetized rats were stepwise bled until mean arterial pressure decreased to, and stabilized at, 21-23 mm Hg. In this model of hemorrhagic shock, which caused the death of all control rats within 30 min after vehicle (tween 80, 5% in saline) injection, the intravenous (i.v.) bolus injection of the cannabinoid CB1 receptor antagonist N-pip -eridino-5-[4-chlorophenyl]-1-[2,4 dichlorophenyl]-4-methyl-3-pyrazolecarboxaniide (SR141716A) increased mean arterial pressure, pulse pressure, respiratory rate and survival rate in a dose-related manner (0.1-3 mg/kg), an almost complete recovery of mean arterial pressure, pulse pressure and respiratory rate, and 100% survival at the end of the observation period (2 h), occurring with the dose of 3 mg/kg. The melanocortin ACTH-(1-24) (adrenocorticotropin) also produced in a dose-related manner (0.02-0.16 mg/kg i.v.) a restoration of cardiovascular and respiratory functions, and increased survival rate, an almost complete recovery and 100% survival at the end of the observation period (2 h) occurring with the dose of 0.16 mg/kg. When a subactive dose of SR141716A (0.2 mg/kg; 30% survival) was associated with a subactive dose of ACTH-(1-24) (0.02 mg/kg; 12% survival), a complete reversal of the shock condition was obtained with 100% survival at the end of the 2-h observation period. The present results show that the concurrent inhibition of both endogenous opioid and cannabinoid systems produces a reversal of hemorrhagic shock more effective than that produced by the inhibition of either of them. These data suggest that functional interactions between endocannabinoids and opioid/antiopioid are at work also in the pathophysiology of hemorrhagic shock. (C) 2002 Elsevier Science B.V. All rights reserved.

2002 - Evidence for a role of nuclear factor-kappa B in acute hypovolemic hemorrhagic shock [Articolo su rivista]
D., Altavilla; A., Saitta; G., Squadrito; M., Galeano; Sf, Venuti; Guarini, Salvatore; Bazzani, Carla; Bertolini, Alfio; Ap, Caputi; F., Squadrito
abstract

Background. In acute hypovolemic shock, a rapid systemic release of the inflammatory cytokine tumor necrosis factor (TNF-alpha) contributes to vascular failure. Nuclear factor kappaB (NF-kappaB) is an ubiquitous rapid-response transcription factor involved in inflammatory reactions and exerts its effect by expressing cytokines, chemokines, and cell adhesion molecules. The purpose of this study was to evaluate the role of NF-kappaB in acute hypovolemic hemorrhagic shock. Methods. Hemorrhagic shock was induced in anesthetized male rats by intermittently withdrawing blood from an iliac catheter for 20 minutes (bleeding period) until mean arterial blood pressure (MAP) decreased and stabilized within the range of 20 to 30 mm Hg. Two minutes after bleeding was discontinued the rats received tacrolimus (100 mug/kg), an inhibitor of NF-kappaB activation, or its vehicle. We then evaluated survival rate and survival time, liver NF-kappaB activation by means of electrophoretic mobility shaft assay, liver IkappaBalpha protein in the cytoplasm, hepatic TNF-alpha messenger RNA expression, plasma TNF-alpha, arterial blood pressure, and the contractile response of aortic rings to phenylephrine. Results. Rats that underwent hemorrhagic shock died 28+/-2 minutes after bleeding was discontinued, experienced marked hypotension (MAP, 20-30 mm Hg), and had enhanced plasma levels of TNF-alpha (218+/-28 pg/mL 20 minutes after bleeding was discontinued). Aortas taken 20 minutes after bleeding was discontinued in rats that underwent hemorrhagic shock showed marked hyporeactivity to phenylephrine (1 nmol/L-10 mumol/L) compared with aortas harvested from sham shocked rats. Rats that underwent hemorrhagic shock also had increased levels of TNF-alpha messenger RNA in the liver. Furthermore, electrophoretic mobility shaft assay showed that liver NF-kappaB binding activity increased in the nucleus, and Western blot analysis suggested that the levels of inhibitory IkappaBalpha protein in the cytoplasm decreased. Tacrolimus (100 mug/kg, administered 2 minutes after bleeding was discontinued) inhibited the loss of IkappaBalpha protein from the cytoplasm and prevented NF-kappaB binding activity in the nucleus. Moreover; tacrolimus increased survival time (118+/-7 minutes; P<.01) and survival rate (vehicle = 0 and tacrolimus = 90% 240 minutes after bleeding was discontinued), reverted the marked hypotension, decreased liver messenger RNA for TNF-α reduced plasma TNF-α (35&PLUSMN;6 pg/mL), and restored the hyporeactivity to phenylephrine to control values. Conclusions. Our results suggest that acute blood loss (50% of the estimated total blood volume during a 20-minute period) causes activation of NF-κB and that tacrolimus, by inhibiting this transcription factor, protects against acute hypovolemic shock.

2002 - Involvement of the central nervous system in the protective effect of melanocortins in myocardial ischaemia/reperfusion injury [Articolo su rivista]
Bazzani, Carla; Mioni, Chiara; G., Ferrazza; Cainazzo, Maria Michela; Bertolini, Alfio; Guarini, Salvatore
abstract

AbstractMelanocortin peptides exert, in rats, a protective effect in myocardial ischaemia followed by reperfusion, or permanent occlusion of a coronary artery. Moreover, melanocortins have an anti-shock effect. Since the mechanism of the life-saving effect of these peptides in haemorrhagic shock includes an essential brain loop, we aimed to determine whether the central nervous system (CNS) is also involved in the protective effect against the outcome of short-term myocardial ischaemia followed by reperfusion. Ischaemia was produced in anaesthetized rats by ligature of the left anterior descending coronary artery for 5 min. Reperfusion-induced ventricular tachycardia (VT), ventricular fibrillation (VF) and lethality, and the time-course of arterial blood pressure over 5 min following reperfusion were evaluated. Groups of 8-14 rats were used. Intravenous (i.v.) injection of ACTH-(1-24) (0.16-0.48 mg/kg) during the ischaemic period dose dependently reduced the incidence of VT, VF and of lethality. In saline-treated rats, coronary reperfusion caused VT in 100% animals, VF in 86%, and death in 86%, The highest dose of ACTH-(1-24) (0.48 mg/kg) completely prevented the occurrence of VT, VF and death in all rats (P < 0.005). Moreover, the melanocortin peptide prevented the fall in mean arterial pressure (MAP) occurring during reperfusion. Treatment with ACTH-(1-24) by the intracerebroventricular (i.c.v.) route also reduced the incidence of VT, VF and lethality, and prevented the fall in MAP in a dose dependent manner. Complete (100%) protection occurred with an i.c.v. dose (0.048 mg/kg) 10 times less than that needed by the i.v. route. The present data show that in the protective effect of melanocortin peptides against the injury after myocardial ischaemia/reperfusion, the i.c.v. route of administration is more effective than the i.v. route. They suggest that a CNS mechanism, not yet identified, may be involved.

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 - Vagus nerve stimulation blunts NF-kB activation and protects against hypovolemic hemorrhagic shock [Abstract in Rivista]
Altavilla, D.; Cainazzo, Maria Michela; Bazzani, Carla; Bigiani, Albertino; Squadrito, F.; Minutoli, L.; Bertolini, Alfio; Guarini, Salvatore
abstract

Vagus nerve stimulation blunts NF-kB activation and protects against hypovolemic hemorrhagic shock

2001 - Central nervous system is involved in the effect of melanocortins in myocardial ischemia. [Abstract in Rivista]
Bazzani, Carla; Mioni, Chiara; Ferrazza, G.; Cainazzo, Maria Michela; Bertolini, Alfio; Guarini, Salvatore
abstract

Central nervous system is involved in the effect of melanocortins in myocardial ischemia.

2001 - Melanocortin MC3 receptor agonists prevent the myocardial ischemia/reperfusion-induced injury. [Abstract in Rivista]
Guarini, Salvatore; Schiöth, H.; Mioni, Chiara; Cainazzo, Maria Michela; Ferrazza, G.; Wikberg, J. E. S.; Bertolini, Alfio; Bazzani, Carla
abstract

Melanocortin MC3 receptor agonists prevent the myocardial ischemia/reperfusion-induced injury

2001 - Melanocortin peptides prevent the ischemia and reperfusion-induced myocardial damage. [Abstract in Rivista]
Cainazzo, Maria Michela; Bazzani, Carla; Botticelli, A.; Zaffe, Davide; Tomasi, Aldo; Bini, Anna; Ferrazza, G.; Mioni, Chiara; Bertolini, Alfio; Guarini, Salvatore
abstract

Melanocortin peptides prevent the ischemia and reperfusion-induced myocardial damage.

2001 - Nuclear factor-kappa B as a target of cyclosporin in acute hypovolemic hemorrhagic shock [Articolo su rivista]
D., Altavilla; A., Saitta; Guarini, Salvatore; M., Galeano; G., Squadrito; Lb, Santamaria; Fs, Venuti; Bazzani, Carla; Bertolini, Alfio; F., Squadrito
abstract

Background: Cyclosporin is an immunosuppressive drug that blocks Nuclear Factor KB (NF-KB) activation. We investigated the role of NF-KB in acute hypovolemic hemorrhagic (Hem) shock and the effects of cyclosporin in this model of experimental shock. Methods: Hem shock was induced in male anesthetized rats by intermittently withdrawing blood from an iliac catheter over a period of 20 min (bleeding period) until mean arterial blood pressure (MAP) fell and stabilized within the range of 20-30 mmHg. Two minutes after bleeding cessation, animals received intravenously cyclosporin (1 mg kg(-1)) or its vehicle. Survival rate and survival time were evaluated for 120 min after bleeding was discontinued. Plasma TNF-alpha levels were investigated at different time points after bleeding cessation. Moreover we investigated levels of TNF-alpha mRNA in the liver, vascular reactivity, liver NF-KB binding activity and levels of the inhibitory protein I kappaB alpha in the cytoplasm. Results: Hemorrhagic shocked rats died in 27 +/-6 min following the cessation of bleeding, experienced a marked hypotension (mean arterial blood pressure=20-30 mmHg) and had enhanced plasma levels of Tumor Necrosis Factor-alpha (208 +/- 22 pg ml(-1), 20 min after the end of bleeding). Furthermore, aortas taken 20 min after bleeding from hemorrhagic shocked rats showed a marked hypo-reactivity to phenylephrine (PE: 1 nM-10 muM) compared with aortas harvested from sham shocked rats. Hem shocked rats also had increased levels of TNF-ot mRNA in the liver (15-20 min after the end of bleeding). Electrophoretic mobility shift assay showed that liver NF-KB binding activity increased in the nucleus 10 min after the end of hemorrhage and remained elevated until the death of animals. Western blot analysis suggested that the levels of inhibitory protein IKB alpha in the cytoplasm decreased at 5 min after the end of bleeding. Cyclosporin inhibited the loss Of IKB alpha protein from the cytoplasm and prevented NF-kappaB binding activity in the nucleus. Furthermore, cyclosporin increased survival time (118 +/-7 min; P <0.01) and survival rate (vehicle=0% and cyclospofin=80%, at 120 min after the end of bleeding), reverted the marked hypotension, decreased liver mRNA for TNF-alpha, reduced plasma TNF-alpha (28 +/-7 pg ml(-1)), and restored to control values the hypo-reactivity to PE. Conclusions: Our results suggest that acute blood loss (50% of the estimated total blood volume over a period of 20 min) causes early activation of NF-KB which triggers an inflammatory cascade leading to a fatal outcome. Cyclosporin blocks NF-KB activation and protects against hypovolemic hemorrhagic shock. (C) 2001 Elsevier Science B.V. All rights reserved.

2001 - Oxidative stress causes nuclear Factor-kappa B activation in acute hypovolemic hemorrhagic shock [Articolo su rivista]
D., Altavilla; A., Saitta; Guarini, Salvatore; M., Galeano; G., Squadrito; D., Cucinotta; Lb, Santamaria; At, Mazzeo; Gm, Campo; M., Ferlito; L., Minutoli; Bazzani, Carla; Bertolini, Alfio; Ap, Caputi; F., Squadrito
abstract

Nuclear Factor kappaB (NF kappaB) is an ubiquitous rapid response transcription factor involved in inflammatory reactions and exerts its action by expressing cytokines, chemokines, and cell adhesion molecules. We investigated the role of NF-kappaB in acute hypovolemic hemorrhagic (Hem) shock. Hem shock was induced in male anesthetized rats by intermittently withdrawing blood from an iliac catheter over a period of 20 min (bleeding period) until mean arterial blood pressure (MAP) fell and stabilized within the range of 20-30 mmHg, Hemorrhagic shocked rats died in 26.3 +/- 2.1 min following the discontinuance of bleeding, experienced a marked hypotension (mean arterial blood pressure = 20-30 mmHg) and had enhanced plasma levels of Tumor Necrosis Factor-alpha (200 +/- 15 pg/ml, 20 min after the end of bleeding). Furthermore, aortas taken 20 min after bleeding from hemorrhagic shocked rats showed a marked hypo-reactivity to phenylephrine (PE; 1nM to 10 muM) compared with aortas harvested from sham shocked rats. Hem shocked rats also had increased levels of TNF-alpha mRNA in the liver (15-20 min after the end of bleeding) and enhanced plasma levels of 2,5-dihydroxybenzoic acid (2,5-DHBA, 6 +/- 2.2 mum), 2,3-dihydroxybenzoic acid (2,3-DHBA; 13 +/- 2.1 mum), both studied to evaluate: OH. production. Electrophoretic mobility shift assay showed that liver NF-kappaB binding activity increased in the nucleus 10 min after the end of hemorrhage and remained elevated until the death of animals. Western blot analysis suggested that the levels of inhibitory I kappaB alpha protein in the cytoplasm became decreased at 5 min after the end of bleeding. IRFI-042, a vitamin E analogue (20 mg/kg intraperitoneally 2 min after the end of bleeding), inhibited the loss of I kappaB alpha protein from the cytoplasm and blunted the increase in NF-kappaB binding activity. Furthermore IRFI-042 increased survival time (117.8 +/- 6.51 min; p <.01) and survival rate (vehicle = 0% and IRFI-042 = 80%, at 120 min after the end of bleeding), reverted the marked hypotension, decreased liver mRNA for TNF-<alpha>. reduced plasma TNF-alpha (21 +/- 4.3 pg/ml), and restored to control values the hypo-reactivity to PE. Our results suggest that acute blood loss (50% of the estimated total blood volume over a period of 20 min) causes early activation of NF-kappaB, likely through an increased production of reactive oxygen species. This experiment indicates that NF-kappaB-triggered inflammatory cascade becomes early activated during acute hemorrhage even in the absence of resuscitation procedures.

2001 - Protective effect of melanocortin peptides in rat myocardial ischemia [Articolo su rivista]
Bazzani, Carla; Guarini, Salvatore; Botticelli, A. R.; Zaffe, Davide; Tomasi, Aldo; Bini, Anna; Cainazzo, M. M.; Ferrazza, G.; Mioni, C.; Bertolini, Alfio
abstract

The influence of the melanocortin peptide ACTH-(1-24) (adrenocorticotropin) on the consequences of short-term coronary ischemia (5 min) followed by reperfusion, and the effect of the long-acting melanocortin [Nle(4),D-Phe(7)]alpha -melanocyte-stimulating hormone (NDP-MSH) on the damage induced by a permanent coronary occlusion, were investigated in anesthetized rats. Ischemia was produced by ligature of the left anterior descending coronary artery. Reperfusion-induced arrhythmias [ventricular tachycardia (VT), ventricular fibrillation (VF)] and survival rate within the 5 min following reperfusion, blood levels of free radicals detected 2 min after reperfusion by electron spin resonance spectrometry, and amount of healthy myocardial tissue, measured 72 h after permanent coronary occlusion on immunohistologically stained serial sections, were evaluated. Postischemic reperfusion induced VT in all saline-treated rats, and VF and death in a high percentage of animals (87%). In rats treated i.v. (2.5 min after coronary occlusion) with ACTH-(1-24) (0.16-0.48 mg/kg) there was a significantly dose-dependent reduction in the incidence of arrhythmias and lethality. Ischemia/reperfusion caused a large increase in free radical blood levels; treatment with ACTH-(1-24) (0.48 mg/kg i.v.) almost completely prevented this increase. In rats subjected to permanent coronary occlusion, the amount of healthy myocardial tissue was much reduced in saline-treated rats, while in rats treated s.c. with NDP-MSH (0.27 mg/kg every 12 h) it was significantly higher. The present data demonstrate, for the first time, an unforeseen property of melanocortin peptides, i.e., their ability to significantly reduce both heart ischemia/reperfusion injury and size of the ischemic area induced by permanent coronary occlusion.

2001 - Survival rate after early treatment for acute type-A aortic dissection with ACTH-(1-24) [Articolo su rivista]
G., Noera; M., Lamarra; Guarini, Salvatore; Bertolini, Alfio
abstract

Haemorrhagic shock, usually as a consequence of major trauma, is the most frequent cause of death among people younger than 40 years. Reports Indicate that melanocortin peptides are effective in reversing haemorrhagic shock. We found that in patients with aortic-dissection-induced haemorrhagic shock, the addition of an early intravenous bolus Injection of the melanocortin andrenocorticotrophic hormone (ACTH)-(1.24) to standard treatment significantly improved cardiovascular function and increased survival rate. Because administration of ACTH-(1.24) is simple, and because melanocortin peptides have no acute toxicity, their use in the early critical care of patients in shock should be more extensively assessed.

2000 - Adrenocorticotropin inhibits nitric oxide synthase II mRNA expression in rat macrophages [Articolo su rivista]
D., Altavilla; Bazzani, Carla; F., Squadrito; Mm, Cainazzo; Mioni, Chiara; Bertolini, Alfio; Guarini, Salvatore
abstract

During hemorrhagic shock there is a massive overproduction of nitric oxide (NO). In such conditions, the intravenous (i.v.) injection of melanocortin peptides in nanomolar amounts produces a long-lasting restoration of cardiovascular and respiratory functions associated with the normalization of NO blood levels. To clarify the mechanism of such melanocortin-induced inhibition of NO overproduction, the influence of the adrenocorticotropin fragment 1-24 [ACTH(1-24)] on the NO synthesizing activity of rat macrophages was studied in vitro. Nitrite production, an indicator of NO synthesis, was measured in the supernatant of rat macrophages whose inducible NO synthase (NOS II, iNOS) had been stimulated by the addition of S. enteritidis lipopolysaccharide (LPS, 50 mu g/ml). ACTH-(1-24) (25, 50 and 100 nM) inhibited nitrite production when incubated together with LPS, but had no effect when applied 6h after LPS. Further, the effect of ACTH-(1-24) on the expression of iNOS mRNA in rat macrophages activated with LPS was studied by means of a reverse transcriptase-polymerase chain reaction assay. ACTH-(1-24) (25, 50 and 100 nM), applied together with LPS, dose-dependently suppressed iNOS gene activation. The present data suggest that the melanocortin-induced normalization of NO blood levels during hemorrhagic shock is due, at least in part, to a direct inhibition of iNOS induction, at the level of mRNA transcription.

2000 - Dallo studio del meccanismo d'azione delle melanocortine nello shock all'individuazione di trattamenti farmacologici causali, inoovativi ed efficaci per questa condizione patologica [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; Mioni, Chiara; Cainazzo, Maria Michela; Ferrazza, G; Bertolini, Alfio
abstract

I neuropeptidi melanocortinic (ACTH-MSH) hanno un effetto salvavita in condizioni di shock emorragico nell'animale e nell'uomo. Tale effetto è indipendente dal surrene, è mediato da recettori melanocortinici MC4 localizzati nel sistema nervoso centrale ed è accompagnato dalla normalizzazione dei livelli ematici di vari mediatori dello shock.

2000 - Influence of early treatment with ACTH-(1-24) on the outcome of aortic dissection [Abstract in Rivista]
Noera, G.; Bertolini, Alfio; Lamarra, M.; Cainazzo, Maria Michela; Bazzani, Carla; Guarini, Salvatore
abstract

Influence of early treatment with ACTH-(1-24) on the outcome of aortic dissection

1999 - ACTH inhibits iNOS induction in LPS-stimulated macrophages [Abstract in Rivista]
Cainazzo, Maria Michela; Altavilla, D.; Bazzani, Carla; Squadrito, F.; Bertolini, Alfio; Guarini, Salvatore
abstract

ACTH inhibits iNOS induction in LPS-stimulated macrophages

1999 - Adrenocorticotropin reverses vascular dysfunction and protects against splanchnic artery occlusion shock [Articolo su rivista]
F., Squadrito; Guarini, Salvatore; D., Altavilla; G., Squadrito; Gm, Campo; M., Arlotta; C., Quartarone; A., Saitta; D., Cucinotta; Bazzani, Carla; Mm, Cainazzo; Mioni, Chiara; Bertolini, Alfio; Ap, Caputi
abstract

1 Tumour necrosis factor (TNF-alpha) is involved in the pathogenesis of splanchnic artery occlusion (SAO) shock. On the other hand, inhibition of TNF-alpha is an important component of the mechanism of action of melanocortins in reversing haemorrhagic shock. We therefore investigated the effects of the melanocortin peptide ACTH-(1-24) (adrenacorticotropin fragment 1-24) on the vascular failure induced by SAO shack. 2 SAO-shocked rats had a decreased survival rate (0% at 4 h of reperfusion, while sham-shocked rats survived for more than 4 h), enhanced serum TNF-alpha concentrations (755 +/- 81 U ml(-1), decreased mean arterial blood pressure, leukopenia, and increased ileal leukocyte accumulation, as revealed by means of myeloperoxidase activity (MPO = 9.4 +/- 1 U g(-1) tissue). Moreover, aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE, 1 nM-10 mu M) (E-max and ED50 in shocked rats = 7.16 mN mg(-1) tissue and 120 nM, respectively; E-max and ED50 in sham-shocked rats = 16.31 mN mg(-1) tissue and 100 nM, respectively), reduced responsiveness to acetylcholine (ACh, 10 nM-10 mu M) (E-max and ED50 in shocked rats = 30% relaxation and 520 nM, respectively; E-max and ED50 in sham-shocked rats = 82% relaxation and 510 nM, respectively) and increased staining for intercellular adhesion molecule-1 (ICAM-1). 3 ACTH-(1-24) [160 mu g kg(-1) intravenously (i.v.), 5 min after SAO] increased survival rate [SAO + ACTH-(1-24) = 80% at 4 h of reperfusion], reversed hypotension, reduced serum TNF-alpha (55 +/- 13 U ml(-1)), ameliorated leukopenia, reduced ileal MPO (1.2 +/- 0.2 U g(-1) tissue), restored the reactivity to PE, improved the responsiveness to ACh and blunted the enhanced immunostaining for ICAM-1 in the aorta. 4 Adrenalectomy only in part - but not significantly -reduced the ACTH-induced shock reversal, the survival rate of SAO + ACTH-(1-24) adrenalectomized rats being 60% at 4 h of reperfusion; and methylprednisolone (80 mg(-1) i.v., 5 min after SAO) had a non-significant effect (10% survival) at 4 h of reperfusion. 5 The present data show that melanocortins are effective also in SAO shock, their effect being, at least in part, mediated by reduced production of TNF-alpha. Furthermore, they demonstrate, for the first time, that this inhibition is responsible for the adrenocorticotropin-induced reversal of vascular failure and leukocyte accumulation.

1999 - Brain MC4 receptors are involved in the melanocortin-induced hemorrhagic shock reversal, in rats [Abstract in Rivista]
Guarini, Salvatore; Bazzani, Carla; Cainazzo, Maria Michela; Mioni, Chiara; Vergoni, Anna Valeria; Schiöth, H. B.; Bertolini, Alfio
abstract

Brain MC4 receptors are involved in the melanocortin-induced hemorrhagic shock reversal, in rats

1999 - Evidence that melanocortin 4 receptor mediates hemorrhagic shock reversal caused by melanocortin peptides [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; Mm, Cainazzo; Mioni, Chiara; G., Ferrazza; Vergoni, Anna Valeria; Hb, Schioth; Jes, Wikberg; Bertolini, Alfio
abstract

Melanocortin peptides are known to be extremely potent in causing the sustained reversal of different shock conditions, both in experimental animals and humans; the mechanism of action includes an essential brain loop. Three melanocortin receptor subtypes are expressed in brain tissue: MC3, MC4, and MC5 receptors. In a volume-controlled model of hemorrhagic shock in anesthetized rats, invariably causing the death of control animals within 30 min after saline injection, the i.v. bolus administration of the adrenocorticotropin fragment 1-24 (agonist at MC4 and MC5 receptors) at a dose of 160 mu g/kg i.v. (54 nmol/kg) produced an almost complete and sustained restoration of cardiovascular and respiratory functions. An equimolar dose of gamma(1)-melanocyte stimulating hormone (selective agonist at MC3 receptors) was completely ineffective. The selective antagonist at MC4 receptors, HS014, although having no influence on cardiovascular and respiratory functions per se, dose-dependently prevented the antishock activity of adrenocorticotropin fragment 1-24, with the effect being complete either at the i.v. dose of 200 mg/kg or at the i.c.v. dose of 5 mg/rat (17-20 mg/kg). We concluded that the effect of melanocortin peptides in hemorrhagic shock is mediated by the MC4 receptors in the brain.

1999 - High blood levels of nitric oxide in rats subjected to prolonged respiratory arrest and their modulation during adrenocorticotropin-induced resuscitation [Articolo su rivista]
Bazzani, Carla; Bini, Anna; Mm, Cainazzo; Meletti, Eros; Tomasi, Aldo; Bertolini, Alfio; Guarini, Salvatore
abstract

Anaesthetized rats, endotracheally intubated and mechanically ventilated with room air, were subjected to a 5-min period of asphyxia by turning off the ventilator. The ventilator was then turned back on and, simultaneously, the animals were treated with either the adrenocorticotropin fragment 1-24 [ACTH-(1-24), 160 mu g/kg in a volume of 1 ml/kg i.v.] or an equivalent volume of saline. Nitric oxide (NO)-haemoglobin formation was detected ex vivo in arterial blood by electron spin resonance spectrometry; arterial blood pressure, electrocardiogram (ECG) and electroencephalogram (EEG) were monitored for a 60-min observation period, or until prior death. During asphyxia, there was massive formation of NO (red cell concentrations 40-80 mu M), associated with a dramatic fall in mean arterial pressure and pulse pressure, marked bradycardia and ECG signs of ischaemic damage, as well as an isoelectric EEG. Treatment with ACTH-(1-24) produced a prompt (within 15 min) and long-lasting drop in NO blood levels, associated with an almost immediate (within 1 min) restoration of cardiovascular function and with a more gradual recovery of EEG, which became normal after 30-40 min; all parameters remained stable throughout the 60-min observation period. In saline-treated rats, on the other hand, there was a further increase in NO blood levels, as detected 3 min after treatment, and all died within 5-8 min. Moreover, pretreatment and treatment with S-methylisothiourea sulphate (SMT, 3 mg/kg i.v.), a relatively specific inhibitor of inducible NO synthase, inhibited NO formation, but did not affect the mortality rate (100% within 5-8 min). The present results provide the first evidence that prolonged asphyxia is associated with high blood concentrations of NO, and that the life-saving effect of melanocortin peptides in severe hypoxic conditions is associated with a complete normalization of NO blood levels. However, the lack of SMT protection in this experimental model seems to rule out the possibility that the ACTH-(1-24)-induced resuscitation is due to an effect on NO overproduction.

1998 - Adrenocorticotropin counteracts the increase in free radical blood levels, detected by electron spin resonance spectrometry, in rats subjected to prolonged asphyxia [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; Bini, Anna; Cainazzo, Maria Michela; Tomasi, Aldo; Bertolini, Alfio
abstract

We investigated the influence of the adrenocorticotropic fragment 1-24 [ACTH(1-24)] on the blood levels of highly-reactive free radicals in a rat model of prolonged asphyxia. Anesthetized animals were endotracheally intubated and mechanically ventilated with room air; after a 10 min stabilization period, the ventilator was turned off to induce asphyxia for 5 min; then, the ventilator was turned back on, and, simultaneously, the rats were intravenously treated with either ACTH-(1-24) (160 mu g/kg in a volume of 1 ml/kg) or equivolume saline. Free radicals were detected in arterial blood by electron spin resonance spectrometry using an ex vivo method that avoids injection of the spin-trapping agent employed (alpha-phenyl-N-tert-butylnitrone). Arterial pressure, electrocardiogram (ECG) and electroencephalogram (EEG) were monitored for the 60 min observation period, or until prior death. At the end of the 5 min period of respiratory arrest, blood levels of free radicals were about four times higher than those of the basal, pre-asphyxia condition, arterial pressure had dramatically decreased, ECG showed marked bradycardia and signs of ischemic damage and the EEG had become isoelectric. Treatment with ACTH-(1-24) produced an immediate normalization of the blood levels of free radicals, associated with a restoration of cardiovascular function and full recovery of EEG within 30-45 min; all the saline-treated rats, on the other hand, died within 6.89 +/- 0.96 min. These results provide direct evidence that in st severe condition of prolonged asphyxia there is a rapid and massive production of highly-reactive free radicals and suggest that the resuscitating effect of adrenocorticotropin fragments in severe hypoxic conditions may be largely due to the inhibition of free radical overproduction during tissue reoxygenation.

1998 - L'inibizione del tumor necrosis factor-alfa contribuisce all'effetto anti-shock delle melanocortine. [Abstract in Rivista]
Cainazzo, Maria Michela; Bazzani, Carla; Altavilla, D.; Squadrito, F.; Guarini, Salvatore
abstract

L'inibizione del tumor necrosis factor-alfa contribuisce all'effetto anti-shock delle melanocortine.

1998 - Le melanocortine come salvavita in gravi condizioni ipossiche caratterizzate da iperproduzione di radicali liberi. [Abstract in Rivista]
Guarini, Salvatore; Bazzani, Carla; Bini, Anna; Cainazzo, Maria Michela; Mattera Ricigliano, G.; Tomasi, Aldo
abstract

Le melanocortine come salvavita in gravi condizioni ipossiche caratterizzate da iperproduzione di radicali liberi.

1998 - Tumour necrosis factor-alpha as a target of melanocortins in haemorrhagic shock, in the anaesthetized rat [Articolo su rivista]
D., Altavilla; Cainazzo, Maria Michela; F., Squadrito; Guarini, Salvatore; Bertolini, Alfio; Bazzani, Carla
abstract

The cytokine tumour necrosis factor- (TNF-) is involved (mostly through the activation of inducible nitric oxide synthase) in the pathogenesis of circulatory shock. On the other hand, melanocortin peptides are potent and effective in reversing haemorrhagic shock, both in animals (rat, dog) and in humans. This prompted us to study the influence of the melanocortin peptide ACTH-(1–24) on the blood levels of TNF- in haemorrhage-shocked rats and on the in vitro production of TNF- by lipopolysaccharide (LPS)-activated macrophages. Plasma levels of TNF- were undetectable before starting bleeding and greatly increased 20 min after bleeding termination in saline-treated rats. In rats treated with ACTH-(1–24) the almost complete restoration of cardiovascular function was associated with markedly reduced levels of TNF- 20 min after bleeding termination. On the other hand, ACTH-(1–24) did not influence TNF- plasma levels in sham-operated, unbled rats. In vitro, ACTH-(1–24) (25–100 nM) dose-dependently reduced the LPS-stimulated production of TNF- by peritoneal macrophages harvested from untreated, unbled rats. These results indicate that inhibition of TNF- overproduction may be an important component of the mechanism of action of melanocortins in reversing haemorrhagic shock.

1997 - Adrenocorticotropin normalizes the blood levels of nitric oxide in hemorrhage-shocked rats [Articolo su rivista]
Guarini, Salvatore; Bini, Anna; Bazzani, Carla; Gm, Ricigliano; Cainazzo, Maria Michela; Tomasi, Aldo; Bertolini, Alfio
abstract

Anesthetized rats were subjected to volume-controlled hemorrhagic shock by stepwise bleeding. Besides cardiovascular and respiratory functions, nitric oxide (NO)-hemoglobin formation in arterial blood was directly evaluated by means of electron spin resonance spectroscopy. During hemorrhagic shock there was a massive increase in NO-hemoglobin, associated with a fall in mean arterial pressure, pulse pressure, respiratory rate and heart rate, and there was a further increase in NO-hemoglobin 15 min after intravenous (i.v.) treatment with saline. All rats died within 30 min. The reversal of the shock condition induced by the i.v. injection of the adrenocorticotropin (ACTH) fragment 1-24 (160 mu g/kg, 5 min after bleeding termination) was associated with a prompt disappearance of NO-hemoglobin. Also S-methylisothiourea (3 mg/kg i.v.), a selective inhibitor of inducible NO synthase, provoked a disappearance of NO-hemoglobin and reversal of the shock condition. The present results provide a direct demonstration that volume-controlled hemorrhagic shock is associated with highly increased blood levels of NO, as indicated by increased NO-hemoglobin, and indicate that ACTH-induced reversal of the shock condition is associated with the normalization of NO blood levels, and a parallel improvement of cardiovascular and respiratory functions. This occurs probably through the inhibition of inducible NO synthase, as suggested by the fact that S-methylisothiourea, a selective inhibitor of this NO synthase isoform, produced the same results.

1997 - Inhibition of nitric oxide overproduction, directly detected in the blood by ESR spectrometry, is involved in the mechanism of action of ACTH-(1-24) in hemorrhagic shock reversal. [Abstract in Rivista]
Guarini, Salvatore; Bazzani, Carla; Bini, Anna; Tomasi, Aldo; Bertolini, Alfio
abstract

Inhibition of nitric oxide overproduction, directly detected in the blood by ESR spectrometry, is involved in the mechanism of action of ACTH-(1-24) in hemorrhagic shock reversal.

1997 - Inhibition of nitric oxide synthases enhances the effect of ACTH in hemorrhagic shock [Articolo su rivista]
Bazzani, Carla; Bertolini, Alfio; Guarini, Salvatore
abstract

In a model of volume-controlled hemorrhagic shock in rats, invariably leading to death within 30 min of bleeding termination, the intravenous (i.v.) bolus injection of ACTH-(1-24) at the dose of 0.16 mg/kg restored cardiovascular and respiratory functions and greatly prolonged survival. I.v. or intracerebroventricular (i.c.v.) treatment with N-G-nitro-L-arginine methylester (L-NAME), a non-isoform-selective inhibitor of nitric oxide synthases (NOSs), at the doses of 2.5-10 mg/kg i.v. or 0.015-0.135 mg/kg i.c.v., as well as i.v. treatment with S-methylisothiourea (SMT), a selective inhibitor of the inducible isoform of NOS, at the doses of 0.001-3 mg/kg, dose-dependently improved cardiovascular and respiratory functions and potentiated the effect of a subthreshold dose (0.02 mg/kg) of ACTH-(1-24). On the other hand, either intraperitoneal or i.c.v. pretreatment with L-arginine, the substrate of NOSs, prevented the effect of ACTH-(1-24). These data suggest that inhibition of NO overproduction is involved in the mechanism of action of ACTH-(1-24) in shock reversal.

1997 - Life-saving effect of physostigmine in a rat model of prolonged respiratory arrest [Abstract in Rivista]
Bazzani, Carla; Cainazzo, Maria Michela; Bertolini, Alfio; Guarini, Salvatore
abstract

Life-saving effect of physostigmine in a rat model of prolonged respiratory arrest

1997 - Mechanisms of the melanocortin-induced resuscitation in a rat model of prolonged asphyxia [Abstract in Rivista]
Guarini, Salvatore; Bazzani, Carla; Cainazzo, Maria Michela; Bertolini, Alfio
abstract

Mechanisms of the melanocortin-induced resuscitation in a rat model of prolonged asphyxia

1997 - Physostigmine has a life-saving effect in rats subjected to prolonged respiratory arrest [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; Bertolini, Alfio
abstract

We have previously reported that centrally-acting cholinomimetic drugs have a prompt and sustained resuscitating effect in pre-terminal conditions of hemorrhagic shock in rats. Here we have studied the effect of physostigmine in another experimental condition of hypoxia in anesthetized rats, which were endotracheally intubated and subjected to prolonged (5 min) interruption of ventilation. This led to a dramatic fall in mean arterial pressure (MAP), pulse pressure (PP), heart rate (HR), pH, PO2, SO2 and base excess, while PCO2 increased; the electroencephalogram (EEG) became isoelectric, and the electrocardiogram (EGG) showed marked bradycardia, P-wave inversion, partial atrio-ventricular block and S-T segment elevation; all saline-treated rats died of cardiac arrest within 7.01 +/- 0.85 min of ventilation being resumed. When ventilation resumption was associated with the simultaneous intravenous (i.v.) injection of physostigmine (70 mu g/kg) there was an almost immediate and impressive increase in MAP, PP and HR, with normalization of ECG within 4 min and full recovery of EEG after 30-50 min. This was associated with a normalization of blood gases and pH. Fifteen days later 40% of treated animals were still alive and apparently in normal health, the mean survival time of the remaining 60% animals being 22.67 +/- 10.19 h. Pretreatment with atropine sulfate or hemicholinium-3 did not modify the response to physostigmine, which, however, was strongly antagonized by the intracerebroventricular injection of mecamylamine. These results suggest that centrally-acting cholinomimetic agents may have a resuscitating effect in pre-terminal conditions produced by prolonged asphyxia, probably through the direct activation of nicotinic receptors in the central nervous system.

1997 - Resuscitating effect of melanocortin peptides after prolonged respiratory arrest [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; Bertolini, Alfio
abstract

1 The resuscitating activity of melanocortin peptides (MSH-ACTH peptides) was tested in an experimental model of prolonged respiratory arrest. 2 Anaesthetized, endotracheally intubated rats subjected to a 5 min period of ventilation interruption, invariably died from cardiac arrest within 6-9 min of resumption of ventilation. 3 When resumption of ventilation was associated with the simultaneous intravenous (i.v.) injection of a melanocortin peptide (alpha-MSH or ACTH-(1-24)) (160 mu g kg(-1)) there was an almost immediate (within 1 min), impressive increase in cardiac output, heart rate, mean arterial pressure (+560% of the before treatment value) and pulse pressure (+356% of the before-treatment value), with full recovery of electroencephalogram after 30-45 min. Blood gases and pH were normalized within 15-60 min after treatment, and all treated animals eventually recovered completely and survived indefinitely (= more than 15 days). 4 The same response was observed in adrenalectomized animals, as well as in animals pretreated with a beta 1-adrenoceptor blocking agent (atenolol, 3 mg kg(-1), i.v.), or with an al;adrenoceptor blocking agent (prazosin, 0.1 mg kg(-1), i.v.), or with an adrenergic neurone blocking agent (guanethidine, 10 mg kg(-1), intraperitoneally). 5 An effect quite similar to that produced by melanocortins was obtained with ouabain (0.1 mg kg(-1) i.v.); the antioxidant drug, glutathione (75 mg kg(-1), i.v.) also produced 100% resuscitation, but the effect was slower in onset. On the other hand, adrenaline (0.005 mg kg(-1), i.v.) was able to resuscitate only 1 out of 8 rats and dobutamine (0.02 mg kg(-1), i.v.) resuscitated 4 out of 8 rats; moreover, the effect of both catecholamines was much slower in onset than that of melanocortins and the initial, impressive stimulation of cardiovascular function was absent. 6 These results show that melanocortin peptides have a resuscitating effect in a pre-terminal condition produced in rats by prolonged asphyxia. This effect seems primarily due to the restoration of cardiac function, not mediated by catecholamines. These data also suggest that these peptides may have potential therapeutic value in conditions of transient cardiac hypoxia and re-oxygenation such as occur in coronary artery disease.

1996 - A highly reproducible model of arterial thrombosis in rats [Articolo su rivista]
Guarini, Salvatore
abstract

The objective of this investigation was to develop a reproducible and reliable method of arterial thrombosis in a small laboratory animal. Rats were anesthetized with urethane, and a common carotid artery was exposed. A completely occlusive thrombus was produced by applying an electric current to the arterial wall (2 mA for 5 min) while simultaneously constricting the artery with a hemostatic clamp placed immediately downstream from the electrodes. A complete and persistent cessation of blood flow was obtained in all the control rats starting 10-15 min after the thrombogenic lesion. Histological examination revealed a picture of mixed white and red thrombus, stratified and rich in platelets aggregates and fibrin, with piles of red cells trapped in the fibrin network. On the other hand, stasis alone (clamping) was ineffective at all, whereas electric current application alone caused non-occlusive thrombosis only in 30% of animals. An antithrombotic dose of heparin (3 mg/kg i.v., 30 min before thrombus induction) prevented the formation of a persistent thrombus, blood now being progressively restored (up to 59% of basal value within 45 min). Similarly, a thrombolytic dose of urokinase-type plasminogen activator (2 mg/kg, for 120 min, starting 15 min after thrombus induction) caused a rapid and progressive resumption of blood now (up to 80% of basal value). This method gives highly consistent and reproducible results and may be suitable for the study or the screening of antithrombotic as well as thrombolytic agents.

1996 - Antithrombotic activity of a 2-kDa heparin fragment in an experimental model of carotid artery thrombosis in rats [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; Botticelli, Annibale Renzo; A., Balugani; Bertolini, Alfio
abstract

The antithrombotic activity of a 2-kDa heparin fragment was studied in a rat model of common carotid artery thrombosis that causes a completely occlusive thrombus with cessation of the blood flow within 10-15 min. The compound reduced thrombus formation in a dose-dependent manner, starting from an intravenous dose of 5 mg kg(-1). A dose of 20 mg kg(-1) completely prevented thrombus formation and apparently induced the almost complete lysis of the already formed occlusive thrombus. At none of the doses used did the compound cause increased bleeding or the formation of haematomas. The present results indicate that low molecular weight heparins, which have an established, highly beneficial effect in venous thromboembolism, are also highly effective in an animal model of arterial thrombosis.

1996 - Influence of ACTH-(1-24) on free radical levels in the blood of haemorrhage-shocked rats: Direct ex vivo detection by electron spin resonance spectrometry [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; Gm, Ricigliano; Bini, Anna; Tomasi, Aldo; Bertolini, Alfio
abstract

1 The influence of ACTH-(1-24) on the blood levels of highly reactive free radicals in haemorrhagic shock was studied in rats. 2 Volume-controlled haemorrhagic shock was produced in adult rats under general anaesthesia (urethane, 1.25 g kg(-1) intraperitoneally) by stepwise bleeding until mean arterial pressure stabilized at 20-23 mmHg. Rats were intravenously (i.v.) treated with either ACTH-(1-24) (160 mu g kg(-1) in a volume of 1 ml kg(-1)) or equivolume saline. Free radicals were measured in arterial blood by electron spin resonance spectrometry using an ex vivo method that avoids injection of the spin-trapping agent (alpha-phenyl-N-tert-butylnitrone). 3 Blood levels of free radicals were 6490+/-273 [arbitrary units (a.u.) ml(-1) whole blood, before starting bleeding, and 30762+/-2650 after bleeding termination (means+/-s.e.mean of the values obtained in all experimental groups). All rats treated with saline died within 30 min, their blood levels of free radicals being 35450+/-5450 a.u. ml(-1) blood, 15 min after treatment. Treatment with ACTH-(1-24) produced a rapid and sustained restoration of arterial pressure, pulse pressure, heart rate and respiratory function, with 100% survival at the end of the observation period (2 h); this was associated with an impressive reduction in the blood levels of free radicals, that were 12807+/-2995, 10462+/-2850, 12294+/-4120, and 10360+/-2080 a.u. ml(-1) blood, 15, 30, 60 and 120 min after ACTH-(1-24) administration, respectively. 4 These results provide a direct demonstration that (i) in haemorrhagic shock there is a rapid and massive production of highly reactive free radicals, and that (ii) the sustained restoration of cardiovascular and respiratory functions induced by the i.v. injection of ACTH-(1-24) is associated with a substantial reduction of free radical blood levels. It is suggested that ACTH-(1-24) prevents the burst of free radical generation during blood mobilisation and subsequent tissue reperfusion, and this may be an important component of its mechanism of action in effectively preventing death for haemorrhagic shock.

1996 - Serotonin is involved in the ACTH-induced reversal of hemorrhagic shock in anesthetized rats [Articolo su rivista]
Bazzani, Carla; L., Fiore; F., Ferrante; Bertolini, Alfio; Guarini, Salvatore
abstract

In a rat model of volume-controlled hemorrhagic shock (mean arterial pressure = 20-24 mm Hg) causing the death of all saline-treated animals within 30 min, the i.v. bolus injection of ACTH-(1-24) (160 mu g/kg) produced an almost complete and sustained reversal of the shock condition, with recovery of arterial blood pressure, pulse pressure and respiratory rate, and with 100% survival at the end of the experiment (2 h). The serotonin-depleting agent p-chlorophenylalanine (316 mg/kg i.p., administered 66-70 h before hemorrhage) almost completely prevented the effect of ACTH. The 5-HT1/5-HT2 receptor antagonist, methysergide, prevented the effect of ACTH completely when injected i.v. (5 mg/kg), but only in part when injected into a brain ventricle (i.c.v.) (15 mu g/rat); the 5-HT2 antagonist, ketanserin, prevented the effect of ACTH completely when injected i.c.v. (1.5 mu g/rat), but only in part when injected i.v. (0.5 mg/kg); the 5-HT3 antagonist, MDL 72222, largely prevented the effect of ACTH when injected i.c.v. (10 mu g/rat), but had no influence at all when injected i.v. (3 mg/kg); finally, the 5-HT4 antagonist, GR 125487, had no effect when injected i.v. (5 mu g/kg) or when injected i.c.v. (30 ng/rat). Overall, these data indicate that both CNS and peripheral serotonin play an important role in the complex mechanism of the ACTH-induced hemorrhagic shock reversal.

1996 - The ACTH-induced reversal of haemorrhagic shock is associated with an impressive reduction of free radical and nitric oxide levels in blood [Abstract in Rivista]
Bazzani, Carla; Guarini, Salvatore; Bini, Anna; Mattera Ricigliano, G.; Meletti, Eros; Tomasi, Aldo; Bertolini, Alfio
abstract

The ACTH-induced reversal of haemorrhagic shock is associated with an impressive reduction of free radical and nitric oxide levels in blood

1996 - The resuscitating effect of ACTH-(1-24) after prolonged respiratory arrest is associated with normalization of nitric oxide and free radical blood levels. [Abstract in Rivista]
Guarini, Salvatore; Bini, Anna; Bazzani, Carla; Cainazzo, M. M.; Mattera Ricigliano, G.; Tomasi, Aldo; Bertolini, Alfio
abstract

The resuscitating effect of ACTH-(1-24) after prolonged respiratory arrest is associated with normalization of nitric oxide and free radical blood levels.

1996 - The reversal of experimental hemorrhagic shock induced by nicotine and dimethylphenylpiperazinium is adrenal-dependent [Articolo su rivista]
Bazzani, Carla; Bertolini, Alfio; Gm, Ricigliano; Cainazzo, Maria Michela; A., Balugani; Guarini, Salvatore
abstract

In a rat model of volume-controlled hemorrhagic shock causing the death of all control animals within 30 min, the intravenous injection of either nicotine (50 mu g/kg) or dimethylphenylpiperazinium (DMPP) (0.5 mu g/kg) produced a rapid and sustained reversal of the shock condition, with 100% survival 2 h after treatment. Bilateral adrenalectomy completely prevented the anti-shock effect of the two drugs, even though administered at higher doses (150 mu g/kg in the case of nicotine; 10 mu g/kg in the case of DMPP). It is concluded that stimulation of adrenaline release plays a fundamental role in the mechanism of action of nicotine- and DMPP-induced shock reversal.

1995 - Adrenocorticotropin release is not involved in the nicotine-induced reversal of hemorrhagic shock in anesthetized rats [Articolo su rivista]
Bazzani, Carla; Bertolini, Alfio; L., Casalgrandi; E., Bertolini; A., Balugani; Guarini, Salvatore; L., Fiore
abstract

In a model of volume-controlled hemorrhagic shock causing the death of all control animals within 30 min, the intravenous injection of nicotine produced a rapid, sustained and dose-dependent restoration of cardiovascular and respiratory functions, with 60 and 100% survival 2 h after the administration of 3 and 12 micrograms/kg, respectively. An effect similar to that of the highest dose of nicotine were obtained with the intravenous bolus injection of ACTH(1-24) at the dose of 160 micrograms/kg. However, the ACTH plasma levels of hemorrhage-shocked rats treated with nicotine was not different from that of hemorrhage-shocked rats treated with saline, thus excluding the possibility that nicotine-induced shock reversal may be due to the massive release of ACTH. Since in rats pretreated with cycloheximide at a dose (20 mg/kg intraperitoneally) causing an 82% inhibition of protein synthesis, and then bled to hemorrhagic shock, the effect of nicotine was greatly reduced (only the dose of 50 micrograms/kg producing 100% survival 2 h after treatment), protein synthesis, however, seems to be important for the effect of nicotine in hemorrhagic shock, at least at the lowest doses.

1995 - Nitric oxide interferes with the ACTH-induced reversal of hemorrhagic shock in rats. [Abstract in Rivista]
Guarini, Salvatore; Bazzani, Carla; Mattera Ricigliano, G.; Bertolini, Alfio
abstract

Nitric oxide interferes with the ACTH-induced reversal of hemorrhagic shock in rats.

1995 - Reperfusion-induced arrhythmias and lethality are reduced by a 2KDa heparin fragment [Articolo su rivista]
Guarini, Salvatore; Mc, Martini; Bertolini, Alfio
abstract

The influence of a low molecular weight heparin (Oligo-H, m.w. 2KDa) on ventricular arrhythmias and lethality induced by heart reperfusion following a 5 min coronary occlusion was studied in anesthetized rats. Both intravenous (i.v.) and subcutaneous (s.c.) injection of the compound dose- and time-dependently prevented the reperfusion syndrome: in all saline-pretreated animals post-ischemic reperfusion induced ventricular tachycardia (VT), which degenerated into ventricular fibrillation (VF) in 25 out of 30 rats, with a mortality rate of 73%; on the other hand, in rats i.v. or s.c. pretreated with Oligo-H (20 mg/kg, 30 and 90 min, respectively, before coronary occlusion), VT occurred in 4 out of 10-11 animals and degenerated into VF in 2-3 out of 10-11 animals, with a mortality rate of 18-20%. Even more effective was a low molecular weight dermatan sulfate (Oligo-DS, m.w. 2.1 KDa). In rats treated with lidocaine, used as reference compound, at the dose of 5 mg/kg i.v. 10 min before coronary occlusion, VT occurred in 2 out of 10 animals and degenerated into VF in 1 out of 10 animals, with a mortality rate of 10%. It is concluded that low molecular weight glycosaminoglycans significantly reduce the consequences of heart reperfusion.

1995 - Resuscitating effect of melanocortins after prolonged respiratory arrest, in rats. [Abstract in Rivista]
Bertolini, Alfio; Bazzani, Carla; Cainazzo, Maria Michela; Guarini, Salvatore
abstract

Resuscitating effect of melanocortins after prolonged respiratory arrest, in rats.

1995 - Role of brain dopamine systems in the ACTH-induced reversal of hemorrhagic shock [Abstract in Rivista]
Guarini, Salvatore; Bazzani, Carla; Bertolini, Alfio
abstract

Role of brain dopamine systems in the ACTH-induced reversal of hemorrhagic shock

1995 - The anti-shock effect of nicotine and dimethylphenylpiperazinium is adrenal-dependent [Abstract in Rivista]
Bazzani, Carla; Guarini, Salvatore; Martini, M. C.; Bertolini, Alfio
abstract

The anti-shock effect of nicotine and dimethylphenylpiperazinium is adrenal-dependent

1994 - Dopamine D1 receptors are involved in the ACTH-induced reversal of hemorrhagic shock [Articolo su rivista]
Bazzani, Carla; Nardi, Maria Grazia; F., Ferrante; Bertolini, Alfio; Guarini, Salvatore
abstract

AbstractIn an experimental model of volume-controlled hemorrhagic shock causing the death of all rats within 30 min, the intravenous (i.v.) bolus injection of the adrenocorticotropic hormone fragment 1-24 (ACTH-(1-24)) (160 μg/kg) induced a prompt and sustained improvement of cardiovascular and respiratory function, with 100% survival 2 h after treatment. Pretreatment with either haloperidol, 300 μg/kg i.v. (antagonist at dopamine D[1] and D[2] receptors), or (R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hemimaleate (SCH 23390), 50 μg/kg intraperitoneally (selective antagonist at dopamine D[1] receptors), significantly inhibited the effect of ACTH-(1-24)

1994 - Sistemi anti-analgesici endogeni [Articolo su rivista]
Bertolini, Alfio; Poggioli, Rosanna; Bernardi, Mara; Genedani, Susanna; Guarini, Salvatore; Bazzani, Carla; Arletti, Rossana; Benelli, Augusta; Bertolini, E; Balugani, A; Vergoni, Anna Valeria
abstract

The two main anti-analgesic systems - i.e., the melanotropinergic and the cholecystokininergic - are here reviewed for their role, not only in nociception, but in a wide variety of vital functions.

1993 - Comparison of the effects of ACTH-(1-24), methylprednisolone, aprotinin, and norepinephrine in a model of hemorrhagic shock in rats [Articolo su rivista]
Bazzani, Carla; A., Balugani; Bertolini, Alfio; Guarini, Salvatore
abstract

Rats bled to a severe condition of volume-controlled hemorrhagic shock were randomly assigned to one of the following treatments: (1) saline, 1 ml/kg i.v.; (2) saline, 0.2 ml/kg per min i.v. for 10 min; (3) ACTH-(1-24),160 mug/kg i.v.; 4) methylprednisolone, 40 mg/kg i.v.; (5) methylprednisolone, 80 mg/kg i.v.; (6) aprotinin, 10 000 KIU/kg i.v.; (7) norepinephrine, 5 mug/kg per min i.v. for 10 min; (8) norepinephrine, 10 mug/kg per min i.v. for 10 min. All rats treated with saline or with either of the two doses of methylprednisolone, and half of the rats treated with aprotinin, died within the subsequent 2 h. On the other hand, rats treated with norepinephrine, at either dose, or with ACTH-(1-24) were all still alive 2 h later, a similar improvement in cardiovascular and respiratory parameters being obtained with the two treatments. The effect of ACTH on mean arterial pressure was however more sustained throughout the observation period. These results further support the potential usefulness of ACTH-(1-24) as first-aid treatment in cases of severe blood losses.

1993 - Role of neuronal and vascular Ca2+- channels in the ACTH-induced reversal of haemorrhagic shock. [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; Bertolini, Alfio
abstract

1 In a rat model of volume-controlled haemorrhagic shock causing the death of all control (saline-treated) animals within 30 min, the intravenous (i.v.) bolus injection of ACTH-(1-24) at a dose. of 160 mug kg-1 produced an impressive and sustained restoration of arterial pressure, pulse pressure and respiratory function, with 100% survival at the end of the observation period (2 h). 2 Both intracerebroventricular (i.c.v., 0.015-0.06 mug kg-1) and i.v. (5 mug kg-1) pretreatment with the N-calcium channel blocker, omega-onotoxin GVIA, and i.v. (but not i.c.v.) pretreatment with the L-calcium channel blocker, nicardipine (125-500 mug kg-1) dose-dependently prevented the ACTH-induced shock reversal. 3 These results further indicate that the effect of ACTH in haemorrhagic shock may involve a neuronal link and the eventual restoration of vascular tone mediated by N- and L-type calcium channels, respectively.

1992 - Anatomia chimica e funzionale dei sistemi peptidergici. [Capitolo/Saggio]
Bertolini, Alfio; Poggioli, Rosanna; Arletti, Rossana; Benelli, Augusta; Marrama, D.; Bazzani, Carla; Tagliavini, S.; Bernardi, Mara; Rasori, E.; Sandrini, Maurizio; Guarini, Salvatore; Genedani, Susanna; Vergoni, Anna Valeria
abstract

Verranno qui trattati i sistemi melanocortinergico, colecistochininergico, tiroliberinergico, ossitocinergico; verrà oltre fatto cenno ad altri neuropeptidi di possibile potenziale interesse sotto questo particolare punto di vista (galanina, NPY, ecc..).

1992 - Capsaicin prevents the adrenocorticotropin-induced improvement of cardiovascular function and survival in hemorrhage-shocked rats [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; S., Tagliavini; Bertolini, Alfio; Ferrari, William
abstract

A volume-controlled hemorrhagic shock was produced in anesthetized rats by intermittent bleeding from an iliac vein over a period of 20-30 min, until the carotid mean arterial pressure (MAP) stabilized around 20-24 mmHg. In this condition, which caused the death of all saline-treated animals within 25-30 min, the intravenous (i.v.) bolus injection of the adrenocorticotropin fragment 1-24 (ACTH(1-24)) at a dose of 160-mu-g/kg promptly restored MAP, as well as pulse pressure, heart rate and respiratory function, and greatly prolonged the survival time. Capsaicin (125 mg/kg cumulatively, s.c., 1 week before) completely prevented the anti-shock effect of ACTH(1-24), which, on the other hand, was shared by i.v. [Nle11]-substance P (SP) (200-300-mu-g/kg). Finally the SP-antagonist [D-Arg1,D-Pro2,D-Trp7.9,Leu11]-SP prevented the effect of ACTH(1-24). These results suggest that SP-containing nerve fibers are required for the effect of ACTH in hemorrhagic shock.

1992 - Dimethylphenylpiperazinium reverses hemorrhagic shock in rats [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; Bertolini, Alfio; Ferrari, William
abstract

Dimethylphenylpiperazinium reverses hemorrhagic shock in rats

1992 - Influence of ACTH-(1-24) on metabolic acidosis and hypoxemia induced by massive hemorrhage in rats [Articolo su rivista]
Bazzani, Carla; S., Tagliavini; E., Bertolini; Bertolini, Alfio; Guarini, Salvatore
abstract

In anesthetized rats, step-wise bleeding to a severe condition of hemorrhagic shock causes a decrease in arterial and venous pH and in venous PO2 and SO2 and an increase in arterial PO2 and in venous PCO2 and lactic acid. The intravenous bolus injection of ACTH-(1-24) (160 micrograms/kg)--which causes a rapid and sustained reversal of the shock condition--produces a gradual and almost complete recovery (within 60 min) of venous PO2, PCO2 and SO2; on the other hand, the normalization of blood pH and lactate is preceded by a further worsening during the first minutes after treatment. On the whole, these data are compatible with the ACTH-(1-24)-induced mobilization of the residual blood--which is pooled in poorly oxygenated tissues--and with the improved circulatory and respiratory functions.

1992 - Investigations on the mechanisms and site(s) of action of the nicotine-induced reversal of hemorrhagic shock. [Articolo su rivista]
Bertolini, Alfio; Bazzani, Carla; Ferrari, William; Guarini, Salvatore
abstract

Investigations on the mechanisms and site(s) of action of the nicotine-induced reversal of hemorrhagic shock.

1992 - Modello di shock emorragico a volume controllato per lo studio di farmaci attivi in condizioni di ipovelmia acuta [Articolo su rivista]
Bertolini, Alfio; Bazzani, Carla; Nardi, Mg; Parmeggiani, P; Bertolini, Elena; Balugani, A; Guarini, Salvatore
abstract

An experimental model of severe volume-controlled hemorrhage shock in rats and dogs (either conscious or anesthetized is described).

1992 - Omega-Conotoxin prevents the effect of adrenocorticotropin in hypovolemic shock [Abstract in Rivista]
Guarini, Salvatore; Bazzani, Carla; Bertolini, Alfio
abstract

Omega-Conotoxin prevents the effect of adrenocorticotropin in hypovolemic shock

1992 - Reversal of experimental hemorrhagic shock by dimethylphenylpiperazinium (DMPP) [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; S., Tagliavini; Bertolini, Alfio; Ferrari, William
abstract

In a rat model of hemorrhagic shock which caused the death of all control rats within 30 min, i.v. injection of the ganglion-stimulating drug dimethylphenylpiperazinium (DMPP) caused a dose-dependent reversal of the shock condition - without the need for reinfusion of the shed blood - starting from the dose of 4 ng/kg i.v. Shock reversal was associated with the mobilization of residual blood and improvement in blood flow, particularly at the carotid level. These results could influence our thinking on pathophysiology and first-aid management of shock.

1992 - Treatment of experimental hemorrhagic shock: comparison of the effects of ACTH-(1-24), methylprednisolone, aprotinin and norepinephrine. [Articolo su rivista]
Bazzani, Carla; Guarini, Salvatore; Bertolini, Alfio
abstract

Treatment of experimental hemorrhagic shock: comparison of the effects of ACTH-(1-24), methylprednisolone, aprotinin and norepinephrine.

1991 - Afferent vagal fibres and central cholinergic mechanisms are involved in the TRH-induced reversal of haemorrhagic shock [Articolo su rivista]
Vergoni, Anna Valeria; D., Marrama; Guarini, Salvatore; S., Tagliavini; Bazzani, Carla; A., Maugeri; Bertolini, Alfio
abstract

In a model of haemorrhagic shock causing the death of all saline-treated rats within 25.8 +/- 2.7 min after treatment, the intravenous injection of thyrotropin-releasing hormone tartrate (TRH-T) at the dose of 4 mg/kg induces a prompt and sustained increase of arterial pressure and pulse amplitude, with survival of all rats. Bilateral vagotomy, atropine sulphate (2 mg/kg intraperitoneally) and hemicholinium-3 (20 micrograms/rat intracerebroventricularly) partially prevent the TRH-T-induced shock reversal, whereas atropine methylbromide has no effect. These data indicate that afferent vagal fibres, brain cholinergic neurons and central muscarinic receptors play a role in the mechanism of the anti-shock effect of TRH-T.

1991 - Influence of TRH on regional blood flow and metabolic acidosis in a model of volume-controlled hemorrhagic shock in rats. [Articolo su rivista]
S., Tagliavini; E., Bertolini; Bazzani, Carla; Bertolini, Alfio; Guarini, Salvatore
abstract

In anesthetized rats, massive bleeding to a severe condition of hemorrhagic shock (invariably leading to death within 30 min) was obviously associated with a dramatic decrease in tissue blood flow and with profound modifications of several blood parameters leading to metabolic acidosis: decrease in arterial and venous pH, bicarbonate and BE, decrease in arterial pCO2 and in venous pO2 and SO2, increase in arterial pO2, venous pCO2 and venous lactate. The i.v. bolus injection of protirelin tartrate (TRH-T, 4 mg/kg), which produces a prompt and sustained reversal of the shock condition, caused a rapid increase in venous pO2, pCO2 and SO2; on the other hand, arterial and venous pH, bicarbonate and BE continued to decrease--and venous lactate to increase during the first few minutes after treatment. However venous pCO2 and lactate, as well as arterial and venous pH, returned to the pre-bleeding values within 60 min after treatment. The data are in keeping with the TRH-T-induced improvement of circulatory and respiratory functions, with mobilization of the residual blood from its capillary pooling and consequent immission of acid metabolites into the blood stream.

1991 - Nicotine reverses hemorrhagic shock in rats [Articolo su rivista]
Guarini, Salvatore; S., Tagliavini; Bazzani, Carla; Bertolini, Alfio; Ferrari, William
abstract

Cholinergic mechanisms are currently thought to play an essential role in blood pressure homeostasis. Here we show that, in urethane-anaesthetized rats bled to severe hemorrhagic shock, the i.v. administration of nicotine 0.2 - 50-mu-g/kg causes a prompt, sustained and dose-dependent improvement in cardiovascular and respiratory functions, the animals' survival rate being significantly higher than that of animals treated with saline. These effects are prevented by bilateral cervical vagotomy and by concurrent local anaesthesia of the carotid bodies, which suggests that stimulation of visceral afferents is the main mechanism of action of nicotine in hemorrhagic shock.

1991 - TRH reverses the ECG and EEG ischemic changes induced by massive hemorrhage in rats [Articolo su rivista]
S., Tagliavini; Bazzani, Carla; Bertolini, Elena; Guarini, Salvatore; Bertolini, Alfio
abstract

In a rat model of volume-controlled hemorrhagic shock causing the death of all saline-treated animals within 30 min of treatment, the intravenous bolus injection of thyrotropin- releasing hormone tartrate (TRH-T) at the dose of 4 mg/kg induced the prompt and sustained disappearance of the ECG and EEG signs of heart and brain ischemia, along with the reversal of hypotension and respiratory depression and with 100% survival rate at the end of the 2 h observation period. These data confirm that, in a pre-terminal condition induced by massive hemorrhage, timely treatment with TRH-T will restore heart and brain perfusion to levels compatible with survival and with functional recovery from ischemia and maintain it at those levels for some hours.

1990 - Brain M3 muscarinic receptors are involved in the ACTH-induced reversal of hemorrhagic shock [Articolo su rivista]
Guarini, Salvatore; S., Tagliavini; Bazzani, Carla; M., Pasini; Bertolini, Alfio
abstract

In an experimental model of bleeding-induced hemorrhagic shock causing the death of all saline-treated rats within 30 min, the intravenous injection of ACTH-(1-24) at the dose of 160 micrograms/kg induced a sustained reversal of the shock condition, with almost complete recovery of blood pressure, pulse amplitude, respiratory rate, heart rate, and 100% survival, at least for the 2 h of observation. This effect of ACTH-(1-24) was prevented by the intracerebroventricular injection of 4-DAMP (a highly selective antagonist for M1 and M3 muscarinic receptors), but unaffected by the intracerebroventricular injection of pirenzepine (a highly selective antagonist for M1 muscarinic receptors). These data indicate that an essential step in the complex mechanism of the ACTH-induced shock reversal may be the activation of brain M3 muscarinic receptors.

1990 - Circulatory and respiratory consequences of massive hemorrhage are reversed by protoveratrines. [Articolo su rivista]
Bertolini, Alfio; Ferrari, William; Guarini, Salvatore; Tagliavini, S.
abstract

In a rat model of severe hypotension and respiratory depression induced by step-wise bleeding, protoveratrines cause a prompt and sustained improvement of cardiovascular and respiratory functions, both in anesthetized and in conscious animals, seemingly through a magnification of the reflex response originated by the chemoreceptors of aortic and carotid bodies. The restoration of cardiovascular function is attributable to an increase both in total peripheral resistance and cardiac output. The finding could provide the basis for a new approach to the first-aid management of massive blood losses.

1990 - Early treatment with ACTH-(1-24) in a rat model of hemorrhagic shock prolongs survival and extends the time-limit for blood reinfusion to be effective [Articolo su rivista]
Guarini, Salvatore; S., Tagliavini; Bazzani, Carla; Ferrari, William; Bertolini, Alfio
abstract

The ability of ACTH-(1-24) to prolong survival and to extend the deadline for effective blood reinfusion has been studied in a model of lethal hypovolemic shock in the rat. Anesthetized rats were bled to a mean arterial pressure of 18 to 25 mm Hg and then subjected to one of the following iv treatments: a) saline; b) ACTH-(1-24), 160 micrograms/kg; c) blood reinfusion; d) ACTH-(1-24), 160 micrograms/kg; c) blood reinfusion; d) ACTH-(1-24), with saline 5 min after bleeding died within 0.05 h. On the other hand, the treatment with ACTH-(1-24) induced an almost complete and sustained recovery of cardiovascular and respiratory functions associated with a survival time of 44 +/- 18 h, while four of six rats reinfused with the withdrawn blood were still alive 15 days later. The time-lapse between bleeding and treatment was of crucial importance, and neither ACTH-(1-24) injection nor blood reinfusion had any effect if performed 25 min after bleeding. However, treatment with ACTH-(1-24) shortly after bleeding (5 min) greatly improved the effect of a later blood reinfusion. These data indicate that ACTH-(1-24) can prolong survival and permit the time-lapse between blood loss and blood reinfusion to be extended.

1990 - Influence of morphine on the reversal of haemorrhagic shock induced by cholinergic drugs [Articolo su rivista]
S., Tagliavini; Bazzani, Carla; Guarini, Salvatore; Bertolini, Alfio
abstract

In haemorrhage-shocked rats, the recovery of mean arterial pressure (MAP), pulse pressure (PP) and respiratory rate (RR), as well as the improvement of survival rate, induced by the i.v. administration of centrally acting cholinergic drugs (physostigmine, oxotremorine) are not affected by morphine at the dose of 2.5 mg/kg i.v., and only partially (MAP, PP, survival rate) or not at all (RR) prevented by a dose of morphine of 5 mg/kg i.v. These results indicate that the anti-shock effect of cholinergic drugs is largely independent of the opioid tone, this possibly being of practical relevance.

1990 - Intracerebroventricular injection of hemicolinium-3 prevents the ACTH-induced, but not the physostigmine-induced, reversal of hemorrhagic shock in rats [Articolo su rivista]
Guarini, Salvatore; S., Tagliavini; Bazzani, Carla; F., Ferrante; Bertolini, Alfio
abstract

In rats bled to hypovolemic shock, the intracerebroventricular injection of hemicholinium-3 (20 micrograms/rat) completely prevented the shock reversal induced by the intravenous injection of ACTH (1-24) (160 micrograms/kg), but had no influence on the shock reversal induced by the intravenous injection of physostigmine (70 micrograms/kg). These data indicate that brain cholinergic neurons are involved in the anti-shock effect of ACTH-peptides, but not in that of centrally acting cholinergic drugs.

1990 - Role of brain acetylcholine in the ACTH-induced shock reversal [Abstract in Rivista]
Guarini, Salvatore; Tagliavini, S.; Bazzani, Carla; Ferrari, William; Bertolini, Alfio
abstract

Role of brain acetylcholine in the ACTH-induced shock reversal

1989 - A pharmacological study of the cardiovascular effects of TRH-T in haemorrhagic shock in rats. [Articolo su rivista]
Guarini, Salvatore; Gherardi, S; Calabrò, G; Bertolini, Alfio
abstract

An extremely severe hypovolemic shock was produced in urethane-anesthetized rats by massive bleeding. In such preterminal conditions (mean arterial pressure = 19-25 mmHg; pulse pressure = 10-18 mmHg; death of all saline-treated animals in 26.16 +/- 3.18 min) the i.v. injection of protirelin tartrate (TRH-T) within 5 min after shock induction promptly and dose-dependently improved arterial pressure, pulse pressure and respiration, the dose of 4 mg/kg ensuring survival for 402.01 +/- 39.45 min without the need for the restoration of blood volume. The injection of TRH-T 20 min after shock induction, on the other hand, was ineffective. The concurrent administration of morphine (2.5 mg/kg i.v.) partially antagonized the effect of TRH-T; however, if the dose of the peptide was increased, its effect was restored. The effect of TRH-T in haemorrhagic shock was associated with a significant increase in the volume of residual circulating blood. The present data (i) confirm that TRH-T improves cardiovascular function and greatly prolongs survival in an otherwise irreversible model of haemorrhagic shock; (ii) show that this effect is associated with mobilization of residual blood and that respiratory function is greatly improved as well; (iii) indicate that the anti-shock effect of TRH-T is partially antagonized by morphine in a surmountable way. Finally, on the basis of our results, it appears that TRH-T has to be given rapidly after shock induction to be effective.

1989 - Adrenocorticotropic hormone (ACTH) and centrally-acting cholinomimetic drugs improve survival of rats with severe hemorrhagic shock through distinct central cholinergic mechanisms. [Articolo su rivista]
Bertolini, Alfio; Ferrari, William; Guarini, Salvatore
abstract

Pharmacological doses (40-160 micrograms/kg) of adrenocorticotropic hormone (ACTH) intravenously injected to urethane-anesthetized rats subjected to otherwise lethal hemorrhagic shock (mean arterial pressure stabilized at 20-25 mmHg) promptly restore blood pressure to about the pre-bleeding values, and prevent death (anti-shock effect). Hemicholinium-3 (i.c.v. injected) and atropine sulphate, but not atropine methylbromide, antagonize these ACTH effects. Moreover, since pirenzepine, injected i.v. or i.c.v., does not affect the anti-shock activity of ACTH, the central cholinergic mechanism participating in this ACTH action must involve M2, but not M1 brain muscarinic receptors. Intravenous physostigmine, too (but not neostigmine) and oxotremorine have an ACTH-like anti-shock effect, which however is neither affected by hemicholinium-3, nor by atropine methylbromide, nor by atropine sulphate, but only by high i.c.v. doses of gallamine or pancuronium. On the other hand, reserpine, guanethidine, and alpha-adrenoceptor blocking drugs inhibit the anti-shock effect of ACTH as well as that of oxotremorine and physostigmine. It is suggested that, in rats, both ACTH and cholinergic drugs must activate a central cholinergic mechanism(s) in order to exert a sympathetic nerve-mediated anti-shock effect. However, receptors involved are of the muscarinic M2 subtype in the case of ACTH, and probably nicotinic in the case of cholinergic drugs. That ACTH and cholinergic drugs activate different central cholinergic mechanisms is also suggested by the fact that cholinergic drugs have a centrally-mediated hypertensive action in normal animals, which is not shared by ACTH.

1989 - Bombesin reverses bleeding-induced hypovolemic shock, in rats [Articolo su rivista]
Guarini, Salvatore; S., Tagliavini; Bazzani, Carla; Bertolini, Alfio
abstract

In an experimental model of bleeding-induced hypovolemic shock causing the death of all saline-treated rats within 26 +/- 4 min, the intravenous injection of bombesin (2.5, 5 or 10 micrograms/kg) dose-dependently restored blood pressure, pulse amplitude, heart rate and respiratory function, and improved survival rate as assessed at the end of the experiment (2 h). The effect on cardiovascular and respiratory functions was prompt (within 1-2 min) and sustained. The release of cholecystokinin seems to be the main mechanism of action, because the anti-shock effect of bombesin is largely prevented by the CCK-antagonist, L-364,718.

1989 - Central cholinergic mechanisms involved in the shock-reversal activity of ACTH-(1-24) and of cholinomimetic drugs. [Articolo su rivista]
Tagliavini, S.; Guarini, Salvatore; Bazzani, Carla; Bertolini, Alfio; Ferrari, William
abstract

Central cholinergic mechanisms involved in the shock-reversal activity of ACTH-(1-24) and of cholinomimetic drugs.

1989 - Characteristics of brain, heart ventricle and spleen capsule adrenoceptors in rats bled to hypovolemic shock and treated with ACTH-(1-24) [Articolo su rivista]
Sandrini, Maurizio; Guarini, Salvatore; Bertolini, Alfio
abstract

Our data show that massive bleeding causes a significant reduction in the number of heart ventricle and spleen caspusle adrenoceptors, that is reversed by ACTH (1-24).

1989 - Cholecystokinin peptides and bombesin reverse hemorrhagic shock in rats [Articolo su rivista]
Guarini, Salvatore; S., Tagliavini; Bazzani, Carla; Vergoni, Anna Valeria; Bertolini, Alfio
abstract

Our results demonstrated that CCk peptides induce reversal of experimental hemorrhagic ahock, and that such effect is mediated by CCk-A receptors.

1989 - Effect of ACTH-(1-24) on the volume of circulating blood and on regional blood flow in rats bled to hypovolemic shock. [Articolo su rivista]
Guarini, Salvatore; S., Tagliavini; Bazzani, Carla; Benelli, Augusta; Bertolini, Alfio; Ferrari, William
abstract

These data show that the anti-shcok effect of ACTH-(1-24) in bleed rats is associated a massive increse in the volume of circulating blood and with a restoration of the venous blood flow in periferal vascular beds.

1989 - Endogenous antagonists of opioid peptides [Capitolo/Saggio]
Bertolini, Alfio; Poggioli, Rosanna; Guarini, Salvatore; Genedani, Susanna; Vergoni, Anna Valeria
abstract

SEVERAL ENDOGENOUS SUBSTANCES HAVE BEEN CLAIMED TO PLAY THE ROLE OF ENDOGENOUS ANTAGONIST OF OPIOIDS. THE MOST LIKELY ARE THE PEPTIDES OF THE ACTH-MSH FAMILY.

1989 - Protoveratrines restore cardiovascular and respiratory functions, and improve survival, in an experimental model of hemorrhagic shock. [Abstract in Rivista]
Tagliavini, S.; Guarini, Salvatore; Ferrari, William; Bertolini, Alfio
abstract

Protoveratrines restore cardiovascular and respiratory functions, and improve survival, in an experimental model of hemorrhagic shock.

1989 - Reversal of haemorrhagic shock in rats by cholinomimetic drugs. [Articolo su rivista]
Guarini, Salvatore; Tagliavini, S.; Ferrari, William; Bertolini, Alfio
abstract

1. In an experimental model of haemorrhagic shock resulting in the death of all rats within 20-30 min, the intravenous (i.v.) injection of the tertiary amine cholinesterase inhibitor physostigmine (17-70 micrograms kg-1) induced a prompt, sustained and dose-dependent improvement of cardiovascular and respiratory function, with marked increase in the volume of circulating blood and survival of all treated animals, at least for the 2 h of observation. 2. Similar results were obtained with the i.v. injection of the cholinoceptor agonist oxotremorine (5-25 micrograms kg-1), while neostigmine (54 or 70 micrograms kg-1), a quaternary cholinesterase inhibitor which cannot cross the blood-brain barrier, had negligible effects. 3. The anti-shock activities of oxotremorine and physostigmine were blocked by the intracerebroventricular injection of either of the combined nicotinic and M2-muscarinic receptor antagonists gallamine and pancuronium, or of the nicotinic antagonist mecamylamine. They were also blocked by intraperitoneal injection of the adrenergic neurone blocking agent guanethidine, but they were not antagonized by either the combined M1- and M2-muscarinic receptor antagonist atropine, the M1-muscarinic receptor antagonist pirenzepine, or the M2-muscarinic receptor 4-diphenylacetoxy-N-methylpiperidine methobromide. 4. It is concluded that cholinomimetic drugs can reverse hypovolaemic shock through central activation (seemingly mediated by nicotinic receptors) of sympathetic tone, with mobilization and redistribution of the residual blood.

1989 - The adrenocorticotropic hormone (ACTH)-induced reversal of hemorrhagic shock. [Articolo su rivista]
Bertolini, Alfio; Ferrari, William; Guarini, Salvatore
abstract

Adrenocorticotropic hormone (ACTH), while having negligible effects on cardiovascular function in the intact animal, induces a potent and sustained reversal of an otherwise invariably, rapidly fatal condition of hemorrhage-induced hypovolemic shock, in rats and dogs. The main site(s) of action are at the peripheral level; however, subsidiary site(s) of action in the CNS cannot be excluded. The studies on the mechanism of action indicate that the ACTH-induced reversal of hemorrhagic shock (a) is an extra-hormonal, adrenal-independent effect, because it is not affected by adrenalectomy and is shared by many ACTH-fragments practically devoid of corticotropic activity; (b) is antagonized by morphine in a surmontable way; (c) needs the functional integrity of the sympathetic nervous system (it is prevented by guanethidine, reserpine, and clonidine) and the availability of peripheral alpha-adrenoceptors (it is antagonized by dibenamine, prazosin and yohimbine, but not by practolol); (d) requires the integrity of afferent vagal fibers (it is almost completely abolished by vagotomy); (e) involves central cholinergic networks (it is antagonized by atropine sulphate, but not by atropine methyl bromide; and it is prevented by the intracerebroventricular injection of hemicholinium-3); (f) is associated with a massive increase in the volume of circulating blood, likely due to a mobilization from peripheral pooling sites (it is largely prevented by splenectomy or by suprahepatic veins ligature, and is associated with a restoration of the venous blood flow in peripheral vascular beds and with a normalization of venous PO2); (g) is associated with a restoration of heart and spleen adrenoceptors, whose number is significantly decreased during hemorrhagic shock. The survival time of hemorrhage-shocked animals, which is 26 +/- 3 min in controls, is greatly prolonged (44 +/- 18 h) by ACTH, provided that the treatment is made within 5-10 min after bleeding. Finally, in animals treated with ACTH within 5-10 min after bleeding, blood reinfusion retains its effectiveness and reverse shock even if performed 2-5 h later.

1989 - The endogenous antagonists of opioid peptides in the management of hemorrhagic shock. [Articolo su rivista]
Guarini, Salvatore; S., Tagliavini; Bazzani, Carla; Ferrari, William; Bertolini, Alfio
abstract

The endogenous antagonists of opioid peptides in the management of hemorrhagic shock.

1988 - Anti-shock effect of ACTH-(1-24): influence of subtotal hepatectomy [Articolo su rivista]
Guarini, Salvatore; Ferrari, William; Bertolini, Alfio
abstract

Subtotally hepatectomized or sham-operated rats were bled to hypovolemic shock (mean arterial pressure = 18-25 mmHg) and then treated with an intravenous bolus injection of ACTH-(1-24), 160 ug/kg. The treatment caused a prompt and sustained reversal of hypotension, with survival of all sham-operated animals, at least for the first 2 h, while in hepatectomized rats the arterial pressure increase was negligible and there was a 50% mortality within 2 h after treatment. Moreover, the blood volume which could be drained from an arterial catheter prior to death, measured 15-20 min after ACTH injection, was 1.51 +/- 0.12 and 0.64 +/- 0.11 ml/100 g b.w. in sham-operated and hepatectomized rats, respectively. These results further support the idea that the effect of ACTH in haemorrhagic shock is due to the mobilization of blood pooled in peripheral reserve organs.

1988 - Anti-shock effect of CCK-8 mechanis of action [Abstract in Rivista]
Guarini, Salvatore; Bazzani, Carla; Vergoni, Anna Valeria; Tagliavini, S; Bertolini, Alfio
abstract

Anti-shock effect of CCK-8 mechanis of action

1988 - Bombesin reverses experimental hemorrhagic shock [Articolo su rivista]
Bertolini, Alfio; Guarini, Salvatore
abstract

Bombesin reverses experimental hemorrhagic shock

1988 - Centrally acting cholinomimetic drugs reverse experimental hemorrhagic shock [Abstract in Rivista]
Guarini, Salvatore; Tagliavini, S.; Ferrari, William; Bertolini, Alfio
abstract

Centrally acting cholinomimetic drugs reverse experimental hemorrhagic shock

1988 - Effect of TRH-T in an experimental model of haemorrhagic shock. [Capitolo/Saggio]
Bertolini, Alfio; Guarini, Salvatore; Tagliavini, S.
abstract

In experimental models of endotoxic, haemorrhagic, anaphylactic and spinal shock, TRH improves blood pressure and short-term survival.

1988 - Haematological changes induced by the intravenous injection of CCK-8 in rats subjected to haemorrhagic shock [Articolo su rivista]
Guarini, Salvatore; Bazzani, Carla; L., Leo; Bertolini, Alfio
abstract

In rats bled to invariably fatal haemorrhagic shock (mean arterial pressure = 18-24 mmHg), the prompt and sustained improvement of cardiovascular function, obtained with the i.v. injection of cholecystokinin octapeptide (CCK-8, 20 g/kg) is associated with a massive increase in the volume of residual circulating blood (0.69 +/- 0.12 ml/100 g b.w. in saline-treated rats; 1.61 +/- 0.09 ml/100 g b.w. in CCK-8-treated rats). The number of red cells/mm3 and the % Hb content is the same in CCK-8-treated and in control rats. So, in a condition of severe haemorrhage, otherwise incompatible with survival, the i.v. injection of CCK-8 not only induces an impressive increase in arterial pressure and in circulating blood volume, but also greatly improves tissue oxygenation.

1988 - Influence of ACTH-(1-24) and blood reinfusion, or both, on the survival time of hemorrhage-shocked rats [Articolo su rivista]
Guarini, Salvatore; Tagliavini, S.; Bazzani, Carla; Ferrari, William; Bertolini, Alfio
abstract

Influence of ACTH-(1-24) and blood reinfusion, or both, on the survival time of hemorrhage-shocked rats

1988 - Influence of the intravenous administration of ACTH-(1-24) on the characteristics of brain, heart and spleen adrenoceptors of haemorrhage-shocked rats [Articolo su rivista]
Sandrini, Maurizio; Guarini, Salvatore; Bertolini, Alfio
abstract

Urethane-anesthetized rats were bled to otherwise irreversible haemorrhagic shock (mean arterial pressure = 18-25 mmHg) and then i.v. treated with ACTH-(1-24) (160 micrograms/kg) or saline. In comparison with sham-operated, non-shocked controls, bled rats showed a significant reduction in Bmax for [3H]Dihydroalprenolol and [3H]Dihydroergocryptine in heart ventricles, and for [3H]Yohimbine in spleen capsule. Neither the Kd of heart and spleen adrenoceptors nor the Bmax or Kd of brain adrenoceptors were affected. Treatment with ACTH-(1-24) restored to normal the Bmax for [3H]Dihydroalprenolol in heart ventricles, and for [3H]Yohimbine in the spleen capsule. These data indicate that the anti-shock effect of ACTH-(1-24) in bled rats is associated with a restoration of heart and spleen responsiveness to adrenergic stimuli.

1988 - Involvement of the sympathetic nervous system in the cardiovascular effects of ACTH-(1-24) during hemorrhagic shock in rats. [Articolo su rivista]
Guarini, Salvatore; Ferrari, William; Bertolini, Alfio
abstract

In urethane-anesthetized rats, removal of about 50% of the total blood volume over a period of 25-30 min caused hypovolemic shock, with extreme hypotension (MAP = 18-25 mm Hg and death of all animals within 22 +/- 5 min. The i.v. injection of ACTH-(1-24) in the dose range of 40-160 micrograms/kg induced a sustained, dose-dependent, and, at the highest dose used, an almost complete recovery of blood pressure, and 100% survival, at least for 2 h after treatment. The effect of ACTH-(1-24) was completely prevented by reserpine (5 mg/kg) and clonidine (0.1 mg/kg), significantly reduced by prazosin (0.1 mg/kg), dibenamine (15 mg/kg) and i.v. yohimbine (1 mg/kg) and unaffected by i.c.v. yohimbine (0.2 mg/kg) and i.v. practolol (15 mg/kg). These data suggest that the effect of ACTH-(1-24) in hypovolemic shock depends on the functional integrity of the sympathetic nervous system and is mediated through an activation of peripheral alpha-adrenoceptors.

1988 - Mechanism of action of the anti-shock effect of CCK-8: influence of CCK antagonists and of sympatholytic drugs [Articolo su rivista]
Guarini, Salvatore; Vergoni, Anna Valeria; Bertolini, Alfio
abstract

In an experimental model of haemorrhagic shock that causes 100% mortality in rats within 30 min, the intravenous bolus injection (20 micrograms/kg) of sulfated cholecystokinin octapeptide (CCK-8) induces a prompt and sustained rise in blood pressure and pulse amplitude, all treated animals still surviving at the end of the experiment (2 h). This effect of CCK-8 is completely blocked by reserpine (5 mg/kg i.p.), significantly antagonized by prazosin (0.1 mg/kg i.v.) and yohimbine (1 mg/kg i.v.), and unaffected by practolol (15 mg/kg i.v.) and proglumide (0.2 mg/kg i.v.); it is completely antagonized by the intravenous (0.01-0.05 mg/kg), but not by the intracerebroventricular (0.002 mg/kg) injection of the 'peripheral' CCK antagonist, L-364,718. The present data indicate that the cardiovascular effects of CCK-8 in haemorrhagic shock involve peripheral CCK receptors, and require the functional integrity of the sympathetic nervous system.

1988 - Veratrum alkaloids reverse hemorrhagic shock, in rats [Articolo su rivista]
Tagliavini, S.; Guarini, Salvatore; Ferrari, William; Bertolini, Alfio
abstract

Veratrum alkaloids reverse hemorrhagic shock, in rats

1987 - ACTH-(1-24) restores blood pressure in acute hypovolaemia and haemorrhagic shock in humans. [Articolo su rivista]
Bertolini, Alfio; Guarini, Salvatore; Ferrari, William; Noera, G; MASSINI C., DI TIZIO S.
abstract

ACTH-(1-24) restores blood pressure in acute hypovolaemia and haemorrhagic shock in humans.

1987 - Anti-shock effect of ACTH-(1-24): comparison between intracerebroventricular and intravenous route of administration [Articolo su rivista]
Guarini, Salvatore; Vergoni, Anna Valeria; Bertolini, Alfio
abstract

In an experimental model of hypovolemic shock in rats, produced by withdrawing about 50% of the estimated total blood volume, and causing 100% deaths within 30 min, ACTH-(1-24) dose-dependently improved arterial and pulse pressure and increased survival rate. The intracerebroventricular (i.c.v.) route of administration was more effective than the intravenous (i.v.) route: at the dose of 24 micrograms/kg, 45% and 91% of rats were still surviving 2 hr after i.v. and i.c.v. treatment, respectively. At higher doses of ACTH-(1-24), the survival rate rose to 100% regardless of the route of administration, but arterial pressure increased more after i.c.v. than after i.v. injection. These data suggest that the CNS plays an important role in the anti-shock effect of ACTH.

1987 - Anti-shock effect of ACTH: haematological changes and influence of splenectomy. [Articolo su rivista]
Guarini, Salvatore; Ferrari, William; Mottillo, G; Bertolini, Alfio
abstract

ACTH-(1-24), injected i.v. into rats subjected to otherwise invariably fatal bleeding, at the dose of 160 micrograms/kg, causes a prompt and sustained increase in mean arterial and pulse pressure, with survival of all treated animals, at least for the first 2 hr. This is associated with a 100% increase in the volume of circulating blood, which is of normal composition, so that also the number of circulating red cells is doubled as compared to controls. Splenectomy greatly impairs the beneficial effect of ACTH on blood pressure, blood volume and survival. It is concluded that, in cases of acute hypovolemia, ACTH-(1-24) induces a recall of blood from storage sites and its redistribution, though the precise mechanism is as yet unknown.

1987 - Different cholinergic pathways are involved in the improvement induced by CCK-8 and by ACTH-(1-24) in massive acute hemorrhage, in rats. [Articolo su rivista]
Guarini, Salvatore; Bertolini, Alfio; Lancellotti, N; Rompianesi, E.; Ferrari, William
abstract

Cholecystokinin octapeptide (CCK-8) (20 micrograms/kg i.v.) and tetracosactide [ACTH-(1-24)] (160 micrograms/kg i.v.) restore blood pressure and allow rats subjected to otherwise invariably fatal acute hemorrhage to survive. Atropine sulphate (2-8 mg/kg i.p.), which crosses the blood-brain barrier, dose-dependently prevents this effect both in the case of ACTH-(1-24) and in that of CCK-8. On the other hand, atropine methyl bromide (2-8 mg/kg i.p.), which does not cross the blood-brain barrier, prevents the effect in the case of CCK-8, but not in that of ACTH-(1-24). These data suggest that a cholinergic mechanism is involved in the anti-shock effect of both ACTH-(1-24) and CCK-8, though the sites of action appear to be in the CNS, in the case of ACTH-(1-24), and outside the CNS, in that of CCK-8.

1987 - Effetti periferici degli oppioidi [Capitolo/Saggio]
Bertolini, Alfio; Guarini, Salvatore; Balzano, S; Poggioli, Rosanna; Vergoni, Anna Valeria; Genedani, Susanna
abstract

In questo capitolo verranno presi in esame la presenza ed il ruolo degli oppioidi nell'apparato digerente e nelle ghiandole annesse, nell'apparato cardiovascolare, nell'apparato riproduttivo, nella placenta e nel feto, nel sistema immunocompetente.

1986 - Adrenal-independent, anti-shock effect of ACTH-(1-24) in rats [Articolo su rivista]
Bertolini, Alfio; Guarini, Salvatore; Ferrari, William
abstract

Our results demonstrated that the intravenus injection of ACTH-(1-24) improves cardiovascular functional and reverses otherwise fatal hypovolemic shock in rats, effects which are not mediated by the adrenals.

1986 - Adrenocorticotropin reversal of experimental hemorrhagic shock is antagonized by morphine [Articolo su rivista]
Bertolini, Alfio; Guarini, Salvatore; Ferrari, William; Rompianesi, E.
abstract

ACTH-(1-24) dose-dependently improved cardiovascular function in rats and dogs subjected to experimental hemorrhagic shock, and intravenous dose of 160 and 100/microgram/kg, respectively, completely restoring arterial blood pressure and pulse amplitude. All saline-treated animals died within 30 min of bleeding, while all ACTH-treated animals were still alive at the end of the observation period (2 hr). The injection of ACTH-(1-24) also dramatically improved the respiratory function. Morphine, i.v. injected into rats at the dose of 2.5 mg/kg, antagonised the effect of ACTH-(1-24) to a greater or lesser degree, depending on the dose of peptide employed: at 160/microgram/kg, antagonism was complete, at 320/microgram/kg antagonism was only partial, while at 480/microgram/kg antagonism was almost completely overcome. These data further support the idea that melanocortins are physiological antagonists of opioids, and suggest that melanocortin peptides may prove to be rational and effective drugs in the treatment of hypovolemic shock.

1986 - Alpha-MSH and other ACTH fragments improve cardiovascular function and survival in experimental hemorrhagic shock [Articolo su rivista]
Bertolini, Alfio; Guarini, Salvatore; E., Rompianesi; Ferrari, William
abstract

Hypovolemic shock was produced in rats by withdrawing about 50% of the estimated total blood volume. Following mean arterial pressure stabilization in the range of 15-25 mm Hg, with a pulse pressure of 7-12 mm Hg, the rats were given intravenous bolus injections either of ACTH fragments or of saline. The following ACTH fragments or analogs were used: ACTH-(4-10), alpha-MSH, ACTH-(1-16), ACTH-(1-17), ACTH-(1-18), [Nle4,D-Phe7]alpha-MSH, [beta-Ala1,Lys17]ACTH-(1-17)-4-amino-n-butilamide (alsactide). ACTH-(1-24) and human synthetic ACTH-(1-39) were used for comparison. All animals treated with saline died in 22.51 +/- 3.62 min. Treatment with ACTH fragments (160 micrograms/kg i.v.) increased blood pressure and pulse amplitude, the effect starting within a few minutes, gradually increasing, and reaching a maximum in 15-30 min. The blood and pulse pressure increases were sustained, remaining almost stable until the end of the 2 h recording. Two out of nine rats treated with alsactide, which was the least active, died within 2 h after treatment, while all rats treated with the other ACTH fragments or analogs were still surviving at that time. Both on a weight and on a molar basis, the most active was ACTH-(1-24), followed by ACTH-(1-16), by the alpha-MSH analog [Nle4,D-Phe7]ACTH-(1-13), by ACTH-(1-18) and by ACTH-(1-17). The present results show that melanocortins reverse otherwise fatal hypovolemic shock, and suggest a new therapeutic approach for shock treatment.

1986 - Caerulein and cholecystokinin reverse experimental hemorrhagic shock. [Articolo su rivista]
Bertolini, Alfio; Guarini, Salvatore; Ferrari, William; Rompianesi, E.
abstract

Intravenously injected cholecystokinin octapeptide (CCK-8) (5-20/micrograms/kg) and caerulein (1.25-10/micrograms/kg) caused a prompt, dose-dependent and sustained improvement in blood pressure, pulse amplitude and survival in rats subjected to otherwise invariably fatal hemorrhagic shock.

1986 - Evidence that melanocortins are physiological antagonists of opioids [Capitolo/Saggio]
Bertolini, Alfio; Poggioli, Rosanna; Vergoni, Anna Valeria; Castelli, Mario; Guarini, Salvatore
abstract

There is abundant experimental evidence that melanocortins (ACTH-MSH peptides) and opioids modulate in a usually opposive way many functions not only in the central nervous system (neuronal firing, adenylate cyclate activity, Ca++ uptake into synaptosomes, pain sensitivity, body temperature, sexual behavior, memory), but also at the peripheral level.

1986 - Influence of vagotomy and of atropine on the anti-shock effect of adrenocorticotropin [Articolo su rivista]
Guarini, Salvatore; Rompianesi, E; Ferrari, William; Bertolini, Alfio
abstract

ACTH-(1-24), intravenously injected at the dose of 160 micrograms/kg to rats bled to the point of otherwise irreversible hypovolemic shock, causes a prompt and sustained increase in blood pressure and pulse amplitude, all treated rats surviving at the end of the experiment (2 hr). Bilateral vagotomy, as well as atropine sulphate (2 mg/kg i.p. immediately before bleeding), almost completely abolishes the anti-shock activity of ACTH. These data indicate that a central cholinergic pathway and vagal afferent (but not efferent) fibers play an important role in the anti-shock effect of ACTH.

1986 - Studies on epinephrine-induced lung edema in the rat. I. Selective alpha 1-adrenoceptor involvement. [Articolo su rivista]
Ferrari, William; Baggio, Giosuè Gabriele; Guarini, Salvatore
abstract

Phentolamine (Phe) prevents the induction by epinephrine (E: 1800 nmol/kg, i.v.) of lung edema (LE) in urethane-anesthetized and bivagotomized rats in a dose-related manner (from 246 to 3933 nmol/kg, i.v.). Since Phe blocks and E activates both alpha 1- and alpha 2-adrenoceptors, the evidence does not allow us to link LE to selective (alpha 1 or alpha 2) or non-selective (alpha 1 + alpha 2) alpha-adrenoceptor activation. Accordingly, we tried to find out whether: phenylephrine (PE), a selective alpha 1-adrenoceptor agonist, and B-HT 920, a selective alpha 2-adrenoceptor agonist, would cause LE; prazosin (Praz), a selective alpha 1-adrenoceptor antagonist, and yohimbine (Yoh), a selective alpha 2-adrenoceptor antagonist, would protect rats against LE caused by E or PE. We found that: 1) PE (from 736 to 5892 nmol/kg, i.v.), but not B-HT 920 (from 190 to 12200 nmol/kg, i.v.), caused LE, while both drugs increased arterial blood pressure; 2) Praz prevented induction of LE, whether by E (1800 nmol/kg, i.v.) or by PE (5892 nmol/kg, i.v.), in a dose-related manner (from 15 to 119 nmol/kg, i.v.). In contrast, Yoh was ineffective at doses up to 7675 nmol/kg, i.v. We conclude, therefore, that E-induced LE in urethane-anesthetized and bivagotomized rats strictly depends on alpha 1-adrenoceptor activation. The outcome of E-induced LE is usually rat death, the incidence of which depends on the dose. Since alpha 1-adrenoceptor agonists rank in the same order of potency for the induction of death as for that of LE, and since Phe and Praz protect from death at LE-preventing doses, there seems to be some link between LE and death, even though protection against death is obtained with doses of antagonists lower than those abolishing induction of LE. Finally, alpha 1-agonists cause maximum arterial hypertension at all doses used, irrespective of induction of LE and of protective pretreatment against LE.

1986 - Studies on epinephrine-induced lung edema in the rat. II. Hemodynamic changes. [Articolo su rivista]
Guarini, Salvatore; Baggio, Giosuè Gabriele; Ferrari, William
abstract

In order to elucidate the pathogenesis of epinephrine (E)-induced lung edema (LE) as well as the mechanism of protection afforded by alpha-adrenoceptor blockade in urethane-anesthetized and bivagotomized rats, we investigated the influence of phentolamine (Phe) and prazosin (Praz) on arterial hypertension and LE provoked by continuous intravenous infusion of E and on blood pressure changes in left (LHV) and right (RHV) heart ventricles caused by a bolus injection of E at a LE-producing dose (1800 nmol/kg). Our results show that neither LE nor death are related to E-induced hypertension and also that LE-induction is accompanied by significant increases in LHV (telediastolic and systolic) as well as in RHV (systolic) pressures, of which the LHV telediastolic and the RHV systolic pressure increases are prevented by Phe and Praz at a dose capable of counteracting E-induced LE. The significance of this finding is briefly discussed.

1986 - The effect of sodium deoxycholate given by gavage with heparin on the histology of the intestinal mucosa of the rat [Articolo su rivista]
Guarini, Salvatore; Fano, Rita Adriana; Rompianesi, E; Martinelli, Anna Maria; Ferrari, William
abstract

To gain direct insight into the mechanism of sodium deoxycholate (DOC)-induced enhancement of gastroenteral heparin absorption in rats, we performed light and electron microscopic examination of the mucosa of the small intestine of animals treated orally with DOC, heparin or DOC plus heparin. The sole morphological change observed after DOC and DOC plus heparin administration was a marked reduction in the length and distribution of glycocalyx filaments on the microvilli of epithelial cells. The morphological picture had reverted to normal after 24 h, when the promotion of enteral heparin absorption by DOC is greatly reduced. Thus, we suggest that DOC may promote the enteral absorption of heparin in rats by affecting some as yet unidentified barrier mechanism requiring glycocalyx integrity.

1985 - Absorption of heparin injected into various parts of the rat intestinal tract: a bile-dependent mechanism? [Articolo su rivista]
Guarini, Salvatore
abstract

Heparin sodium, dissolved in water, causes plasma clearing activity (PC) and appreciable heparinemia (HE) when directly delivered into the duodenum, the jejunum, the ileum or the large intestine of rats, as well as when administered through an esophageal tube to distal duodenum-ligated animals. However, choledochus occlusion significantly decreases both PC and HE produced by intraesophageal heparin application. It is suggested that, in rats, bile flow participates in intestinal heparin absorption.

1985 - Olive oil-provoked bile-dependent absorption of heparin from gastro-intestinal tract in rats [Articolo su rivista]
Guarini, Salvatore; Ferrari, William
abstract

Aqueous heparin sodium solution dispersed in olive oil administered esophageally to fed rats induced blood plasma clearing activity, inhibition of blood coagulation and increased plasma heparin level. Plasma clearing activity was dependent on heparin dose (125 to 1000 mg/Kg). No plasma clearing activity being observed in pylorus- or coledoch-ligated rats, while heparin absorption reappeared after ox or rat bile administration, we conclude that olive oil favours enteral absorption of heparin by increasing bile function.

1985 - Sodium deoxycholate promotes the absorption of heparin administered orally, probably by acting on gastrointestinal mucosa, in rats. [Articolo su rivista]
Guarini, Salvatore; Ferrari, William
abstract

Sodium deoxycholate (DOC), selected as a promoter of gastrointestinal absorption of heparin, was administered orally to rats, followed, at increasing intervals, by heparin. Maximal plasma clearing activity (PC) was obtained with a 60-min interval, though PC was still elicited after 24 h, suggesting that DOC acts on the gastrointestinal mucosa. Inhibition of blood coagulation was also observed after oral heparin. The suggestion that DOC increases heparin absorption is supported by increased plasma levels of heparin. No signs of several gastrointestinal damage were seen.

1984 - Influence of clonidine on the ACTH-induced behavioral syndrome [Articolo su rivista]
Poggioli, Rosanna; Vergoni, Anna Valeria; Guarini, Salvatore; Bertolini, Alfio
abstract

In male rats, clonidine in a dose range of 1-3000 micrograms/kg i.p. antagonized the stretching-yawning syndrome induced by the intraventricular injection of ACTH-(1-24) (3 micrograms/rat) dose-dependently. On the other hand, the effect of clonidine on ACTH-induced penile erections was potentiation at low doses (5 and 10 micrograms/kg) and inhibition at the highest doses (1000 and 3000 micrograms/kg), the intermediate doses (50 and 100 micrograms/kg) being without effect. There was no relationship between these behavioral effects and the effect on arterial blood pressure.

1984 - Structural restriction in bile acids and non-ionic detergents for promotion of heparin absorption from rat gastro-intestinal tract. [Articolo su rivista]
Guarini, Salvatore; Ferrari, William
abstract

Selected non-ionic surfactants of the polyoxyethylene ether series as well as selected bile acids elicit plasma clearing activity (PC) after oral heparin administration by action on the gastro-intestinal mucosa. Evidence obtained indicates that certain structural characteristics are needed for promoting heparin absorption, since this effect is observed with some but not all non-ionic detergents (cetyl and stearyl ethers but not lauryl) and bile components (deoxycholic, chenodeoxycholic and cholic acids, but not ursodeoxycholic or dehydrocholic acids or lecithin). It is suggested that a specific mechanism of action is involved.

1981 - Diuretic effect of dopaminomimetic agents. [Capitolo/Saggio]
Baggio, Giosuè Gabriele; Ferrari, Francesca; Guarini, Salvatore; Sandrini, Maurizio; Ferrari, William
abstract

The intraperitoneal injection of dopamine (DA), (3,4-dihydroxy-phenylamino)-2-imidazoline (DPI) or noradrenaline (NA) to normally hydrated conscious rats evokes a prompt, marked and short-lasting increase in urine and electrolite elimination. Apomorphine (APO) and (+/-)-N-n-propyl-norapomorphine (NPA) are ineffective.

1980 - Antagonistic effects of Na-3.4.5-trimethoxybenzoyl-epsilon-aminocaproate (Capobenate, TMBAC) on angiotensin II-induced arterial blood hypertension in the anaesthetized rat [Articolo su rivista]
Baggio, Giosuè Gabriele; Guarini, Salvatore; Ferrari, Francesca; Bazzani, Carla; Arletti, Rossana
abstract

Arterial blood hypertension caused by the intravenus administration of angiotensin II to urethane-anaesthetized rats can be antagonized byt he injection of Na-3.4.5-trimethoxybenzoyl-epsilon-aminocaproate (Capobenate, TMBAC) in a dose -dependent manner.

Università degli studi di Modena e Reggio Emilia

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