Browsing by Author "Amantea, Diana"

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    Caratterizzazione pre-clinica di una nuova strategia terapeutica per l'ischemia cerebrale identificata mediante drug repurposing di un antibiotico macrolide
    (2013-11-30) Certo, Michelangelo; Amantea, Diana; Sisci, Diego
    Cerebral ischemia is one of the most common causes of disability and mortality worldwide and the only pharmacological treatment currently available is thrombolysis. The understanding of the mechanisms underlying ischemic injury has led to the identification of several neuroprotective compounds aimed at the recovery of the damaged brain tissue. However, most of these drugs have produced disappointing results in clinical trials because of the high toxicity or lack of efficacy in patients. Therefore, there is a real need to develop novel therapeutic strategies that do not consider neurons as the only target. In fact, the neuronal damage is strongly influenced by the inflammatory and immune processes that develop both locally and systemically after ischemia. The inflammatory response evolves slowly, and this allows to significantly expand the time window for pharmacological intervention, highlighting the therapeutic potential of anti-inflammatory and immunomodulatory drugs. Therefore, the first objective of this work was to characterize central and peripheral inflammatory responses that occur following an ischemic insult in rodents. In particular, in order to identify potential targets, we have evaluated the temporal profile of activation of specific inflammatory cells. By immunofluorescence analysis, we observed an early activation of microglia and astrocytes in the ischemic hemisphere, as a result of transient middle cerebral artery occlusion (MCAo) in rodents. We have also detected a massive brain recruitment of neutrophils and macrophages, with a peak of infiltration 48 hours after the insult, whereas T lymphocytes have been identified only at later times. Together with evidence from microarray studies demonstrating that the majority of genes modulated acutely in the blood of stroke patients resides in neutrophils and monocytes, our findings suggest that these cells may be useful therapeutic targets. Using the repurposing approach we have selected a drug, azithromycin, that is able to modulate the functions of macrophages and neutrophils in pathological conditions other than ischemia. Pre-treatment with azithromycin (150 mg/kg, orally) produces a significant reduction of the cerebral infarct volume induced by transient or permanent MCAo in rats. This suggests a potential prophylactic use of the drug during surgical procedures associated to a high risk of ischemic tissue damage. We have also observed the neuroprotective activity of azithromycin when the drug is administered systemically after a transient ischemic insult. The reduction of the infarct volume induced by transient MCAo is dose-dependent (ED50 = 0.59 mg/kg in mice, ED50 = 1.19 mg/kg in rats) and is approximately 60% (compared to vehicle) with the most effective dose of azithromycin (150 mg/kg, i.p.). The neuroprotective doses in rodents are therefore much lower than the antibiotic ones. We have also documented that the reduction of the infarct volume and the improvement of the neurological deficit due to azithromycin post-treatment (150 mg/kg, ip) are maintained up to 7 days after the insult. Furthermore, the time window of efficacy is rather wide, since neuroprotection is observed with the drug administered up to 6 hours after the insult both in rats and in mice subjected to transient MCAo. The characterization of the neuroprotective effects of azithromycin, demonstrated by the present study in models of focal ischemia in rodents, provides the basis for the validation of the drug efficacy in patients suffering from ischemic stroke.
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    Charatterization of store-operated calcium entry in the neuroimmune response evoked by ischemic preconditioning in mice subjected to middle cerebral artery occlusion
    (Università della Calabria, 2021-06-10) Frisina, Marialaura; Andò, Sebastiano; Amantea, Diana
    Cerebral ischemia is one of the leading causes of death and long-term disability worldwide. Currently approved therapies for ischemic stroke are limited to reperfusion through mechanical recanalization and/or pharmacological thrombolysis; however, only a small percentage of eligible patients may benefit from this treatment due to its contraindications and, furthermore, it does not provide neuroprotective effects. In this context, several studies have highlighted the potential of inducing ischemic tolerance by stimulating endogenous neuroprotection. To this aim, brain ischemic preconditioning (PC), namely a sublethal ischemic event able to increase the resistance of the brain against a subsequent, more intense ischemic insult, has been considered as a useful experimental paradigm to investigate the mechanisms implicated in brain tolerance. A deep comprehension of endogenous neuroprotection elicited by ischemic PC represents a promising approach to identify novel targets that can be translated into stroke therapy. One of the main factors involved in the progression of neuronal damage during cerebral ischemia is the alteration of cellular Ca2+ homeostasis. Indeed, cytosolic Ca2+ overload due to increased membrane permeability or to its leak from intracellular organelles could result in neuronal demise. Detrimental effects involve the activation of a series of Ca2+-dependent enzymes that degrade cellular components or activate death pathways, and the formation of cytotoxic products that cause irreversible mitochondrial damage and cellular demise. The main objective of the present research work was to investigate the involvement of store-operated calcium entry (SOCE) in brain ischemia and ischemic preconditioning in mice subjected to focal cerebral ischemia. Following an ischemic insult and depletion of Ca2+ stores, the endoplasmic reticulum Ca2+ sensor stromal interaction molecule (STIM)1 interacts with the Ca2+ selective plasmamembrane channel Orai1, to promote SOCE, that may protect neurons by re-establishing Ca2+ homeostasis or could also be the source of excessive Ca2+ influx, thus causing nonexcitotoxic neuronal death. This Ca2+ influx is regulated by SOCE-associated regulatory factor (SARAF) that associates with STIM1 and promotes a slow Ca2+- dependent inactivation of SOCE, or directly interacts with Orai1 to promote SOCE activation in the absence of STIM1. Furthermore, SOCE represents the main source of Ca2+ in immune cells, regulating several of their critical functions. Besides the pivotal role played by immune mediators in the evolution of cerebral ischemic damage, it has been demonstrated that the innate immune system is also an essential component of the delayed ischemic tolerance elicited in the brain by ischemic PC. Therefore, we investigated whether central and peripheral innate immune responses contribute to PC-induced ischemic tolerance and if modulation of different SOCE components occurs in ischemic damage (1h middle cerebral artery occlusion, MCAo, followed by 24h of reperfusion) and/or in neuroprotection conferred by ischemic PC (15 min MCAo, 72h before) in C57BL/6J adult male mice. Ischemic PC significantly reduced histological damage and neurological deficits produced in mice by a more severe ischemia of 1h. Western blot analysis revealed that Orai1 expression is not affected by the ischemic insult preceded or not by the PC stimulus in the frontoparietal ischemic cortex. However, Orai1 expression was detected in neurons, but also in Ly6B.2+ myeloid cells infiltrating the ischemic hemisphere. By contrast, STIM1 and SARAF expression, mainly found in NeuN+ neurons, was significantly reduced in the ischemic cortex. Interestingly, ischemic PC prevented SARAF downregulation in the ischemic cortex, thus suggesting that this regulatory factor may play a crucial role in SOCE-mediated tolerance. To assess the immunomodulatory effects of ischemic PC, we performed ELISA assay to demonstrate that cerebral damage was associated with increased protein levels of the proinflammatory cytokine IL-1β in the ischemic cortex, while this effect was prevented by the PC stimulus. Regarding alternatively-activated phenotypes, western blot analysis revealed a significant elevation of the expression of Ym1, marker of M2-polarized microglia/macrophages, in the ischemic cortex as compared to contralateral tissue. Interestingly, ischemic PC further increased Ym1 expression in the ipsilateral cortex as compared to MCAo group. Immunohistochemical analysis revealed that the majority of Ym1+ cells are mainly amoeboid CD11b+ myeloid cells, very likely monocytes/macrophages infiltrating from blood vessels. Thus, elevated brain infiltration of these phenotypes is very likely involved in the protective effects of ischemic PC. The involvement of the peripheral immune response was confirmed by the evidence that the 70% increase in spleen weight observed after 1h MCAo was abolished in mice pre-exposed to PC. Accordingly, flow cytometry analysis revealed that PC significantly attenuates elevation of neutrophil counts (Ly-6G+ events) induced by 1h MCAo in blood. Since the Ca2+-selective plasmamembrane channel Orai1 is crucial in the recruitment of immune cells during inflammation, we have analysed its expression in the whole population of circulating leukocytes and in neutrophils, demonstrating that the number of Orai1+ cells, mainly corresponding to Ly-6G+ neutrophils, was significantly enhanced in the blood after the ischemic insult, as compared to sham, regardless of whether mice received or not ischemic PC. In conclusion, this research project reaffirms that cerebral ischemic tolerance induced by PC involves both central and peripheral modulation of the innate immune system, further underscoring the relevance of exploiting immunomodulatory approaches for the development of effective stroke therapies and originally demonstrates that preventing SARAF downregulation could represent an important neuroprotective mechanism aimed at preserving SOCE functions, making SARAF a valuable target to protect neurons from the ischemic damage.
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    Cratterizzazione degli effetti neuroprotettivi della leptina in un modello sperimentale di ischemia cerebrale focale nel ratto
    (2011) Petrelli, Francesco; Sisci, Diego; Amantea, Diana
    La leptina, oltre ad avere effetti sull’ipotalamo per il controllo del peso corporeo, è coinvolta nella regolazione della funzionalità, dello sviluppo e della sopravvivenza neuronale. Studi recenti hanno evidenziato i suoi effetti neuroprotettivi nel danno ischemico cerebrale, ma fino ad oggi il ruolo del fattore di trasduzione ed attivatore trascrizionale (STAT)-3, il principale mediatore della via di trasduzione del segnale di ObR nel cervello, non è stato chiarito. I nostri dati dimostrano che la somministrazione sistemica acuta di leptina è neuroprotettiva in ratti sottoposti ad occlusione permanente dell’arteria cerebrale media (MCAo), come documentato dalla riduzione significativa del volume di infarto cerebrale e del deficit neurologico fino a 7 giorni dopo l’induzione di ischemia. Mediante analisi di immunofluorescenza e tecniche di frazionamento subcellulare abbiamo osservato che la neuroprotezione da leptina è associata con la modulazione dei livelli di fosforilazione di STAT-3 in differenti tipi cellulari nella corteccia cerebrale ischemica. Infatti, poche ore dopo l’insulto la leptina aumenta i livelli di p-STAT3 nel nucleo degli astrociti della penombra ischemica contribuendo così agli effetti benefici di queste cellule sull’evoluzione del danno. L’aumentata espressione di homer-1a che osserviamo negli astrociti fino a 7 giorni dopo l’induzione di ischemia, sottolinea ulteriormente il loro ruolo benefico. Mediante ricostruzione 3D di immagini di microscopia elettronica, combinata con analisi morfometrica, abbiamo osservato che gli astrociti reattivi mostrano un ridotto coverage bilaterale, mentre la percentuale di contatto con le sinapsi glutammatergiche rimane invariata. Inoltre, l’aumento di p-STAT3 indotto dalla leptina nei neuroni dopo 24h di MCAo è associato con un aumento dell’espressione dell’inibitore tissutale delle metalloproteasi della matrice (TIMP)-1 nella corteccia, suggerendo un suo coinvolgimento nella neuroprotezione indotta dall’adipochina