ABSTRACT Thesis (Ph.D. - University of Birmingham, UK) ABSTRACT The hypothesis that chronic admin... more ABSTRACT Thesis (Ph.D. - University of Birmingham, UK) ABSTRACT The hypothesis that chronic administration of nicotine may result in pharmacological modifications of GABAB receptors in the rat mesocorticolimbic system was investigated. In vitro brain autoradiography revealed that GABAB receptor expression and affinity in the ventral tegmental area, the nucleus accumbens and the prefrontal cortex were not significantly altered by chronic (14 days) or subchronic (4 days) treatment with daily subcutaneous injections of nicotine, or in rats that self-administered the drug by the intravenous route. However, reduced G-protein coupling to the GABAB receptor was detected in the prefrontal cortex and the nucleus accumbens, core and shell, of rats receiving chronic subcutaneous injections of nicotine, when compared to saline-injected controls. In order to characterise the functional status of the GABAB receptor, the effect of baclofen on the release of preloaded [3H]dopamine from mesocorticolimbic regions was investigated. Baclofen did not modify the nicotine-induced release of [3H]dopamine from accumbal and cortical slices obtained either from nicotine-treated rats or from saline-controls. By contrast, the GABAB agonist dose-dependently inhibited the [3H]dopamine outflow, evoked by electrical stimulation, from ventral tegmental area slices of naïve animals. The inhibitory effect of baclofen was significantly reduced in midbrain slices obtained from rats pretreated for 14 days with subcutaneous injections of nicotine. This finding was consistent with preliminary in vivo observations revealing that the inhibitory effect of baclofen on the basal and nicotine-evoked somatodendritic release of dopamine in the ventral tegmental area was abolished in rats pre-exposed to chronic nicotine treatment. Thus, chronic administration of nicotine may be associated with a subsensitivity of GABAB receptors in rat brain regions belonging to the mesocorticolimbic system. This would suggest that altered GABAB receptor function might underlie some of the addictive properties of nicotine.
Ischemic stroke is a devastating condition primarily caused by reduced blood supply to the brain.... more Ischemic stroke is a devastating condition primarily caused by reduced blood supply to the brain. Interleukin (IL)-1β is a pro-inflammatory cytokine that plays a pivotal role in the detrimental inflammatory processes that participate to cerebral ischemic damage. After injury, it is produced by distinct cells of the neurovascular unit as an inactive precursor, pro-IL-1β. Although previous studies have suggested that caspase-1 is the main enzyme implicated in the cleavage of pro-IL-1β into the biologically active cytokine, recent work has demonstrated that, under ischemia-reperfusion conditions, other mechanisms may be involved in cytokine maturation. Indeed, we have shown that in rats subjected to transient middle cerebral artery occlusion (MCAo), elevation of IL-1β levels is paralleled by an elevation of gelatinolytic, but not caspase-1 activity in the injured hemisphere and pharmacological inhibition of gelatinases, i.e. matrix metalloproteases (MMP)-2 and MMP-9...
Innate immune cells play a dualistic role in the evolution of ischemic brain damage, with classic... more Innate immune cells play a dualistic role in the evolution of ischemic brain damage, with classic phenotypes promoting injury, and alternatively activated M2 microglia/macrophages or N2 neutrophils providing tissue remodelling and repair. Recently, a number of drugs commonly used for other indications (i.e., azithromycin, minocycline, bexarotene, rosiglitazone, metformin) was reported to provide neuroprotection in preclinical stroke models by promoting immune polarization towards non-inflammatory, protective phenotypes. Repurposing drugs with a well-established safety profile should allow a reduction in the risk of clinical trial failure that has dominated the unsuccessful development of neuroprotective drugs in stroke during the last decades. The clinical validation of the proof of concept, followed by the assessment of safety and efficacy of immune-polarizing repurposed drugs will definitively offer new opportunities for the acute treatment of ischemic stroke.
The identification of novel drug targets for the treatment of ischemic stroke is currently an urg... more The identification of novel drug targets for the treatment of ischemic stroke is currently an urgent challenge. Recent experimental findings have highlighted the neuroprotective potential of immunomodulatory strategies, based on polarization of myeloid cells toward non-inflammatory, beneficial phenotypes. Given the role of retinoid X receptors (RXR) in myeloid cells differentiation and polarization, here we have explored the neuroprotective potential of the RXR agonist bexarotene in mice subjected to focal cerebral ischemia. Acute administration of bexarotene significantly reduced blood brain barrier leakage, brain infarct damage and neurological deficit produced by transient middle cerebral artery occlusion in mice, without affecting cerebral blood flow. The rexinoid exerted neuroprotection with a wide time-window, being effective when administered up to 4.5h after the insult. The amelioration of histological outcome, as well as the ability of bexarotene to revert middle cerebral artery occlusion (MCAo)-induced spleen atrophy, was antagonised by BR1211, a pan-RXR antagonist, or by the selective peroxisome proliferator-activated receptor (PPAR)γ antagonist bisphenol A diglycidyl ether (BADGE), highlighting the involvement of the RXR/PPARγ heterodimer in the beneficial effects exerted by the drug. Immunofluorescence analysis revealed that bexarotene elevates Ym1-immunopositive N2 neutrophils both in the ipsilateral hemisphere and in the spleen of mice subjected to transient middle cerebral artery occlusion, pointing to a major role for peripheral neutrophil polarization in neuroprotection. Thus, our findings suggest that the RXR agonist bexarotene exerts peripheral immunomodulatory effects under ischemic conditions to be effectively repurposed for the acute therapy of ischemic stroke.
To develop novel and effective treatments for ischemic stroke, we investigated the neuroprotectiv... more To develop novel and effective treatments for ischemic stroke, we investigated the neuroprotective effects of the macrolide antibiotic azithromycin in a mouse model system of transient middle cerebral artery occlusion. Intraperitoneal administration of azithromycin significantly reduced blood-brain barrier damage and cerebral infiltration of myeloid cells, including neutrophils and inflammatory macrophages. These effects resulted in a dose-dependent reduction of cerebral ischemic damage, and in a remarkable amelioration of neurological deficits up to 7days after the insult. Neuroprotection was associated with increased arginase activity in peritoneal exudate cells, which was followed by the detection of Ym1- and arginase I-immunopositive M2 macrophages in the ischemic area at 24-48h of reperfusion. Pharmacological inhibition of peritoneal arginase activity counteracted azithromycin-induced neuroprotection, pointing to a major role for drug-induced polarization of migratory macrophages towards a protective, non-inflammatory M2 phenotype.
Cerebral ischemia-reperfusion (I/R) injury is associated with the production of reactive oxygen s... more Cerebral ischemia-reperfusion (I/R) injury is associated with the production of reactive oxygen species (ROS) (Halliwell & Gutteridge, 1989). Although ROS are well known for their role in neurotoxicity, there is emerging evidence that under physiological conditions they also play an important role in cell function (Klann & Thiels, 1999). Moreover, we have recently shown a protective role of hydrogen peroxide (H 2 O 2) in oxygen-deprived dopaminergic neurones of the rat substantia nigra (Geracitano et al., 2005). Neuroprotection by H 2 O 2 (3mM) was also observed in rat hippocampal slices exposed to an ischemic insult. Particularly, we observed that the rescuing action of H 2 O 2 against oxygen/glucose deprivation (OGD) insult in hippocampal slices was mediated by catalase (CAT), as pre-treatment with the CAT irreversible inhibitor 3-amino-1,2,4-triazole (3-AT) blocked this effect. Additionally, we showed that an increase of the endogenous levels of H 2 O 2 , due to a combination of ...
The innate immune system plays a dualistic role in the evolution of ischemic brain damage and has... more The innate immune system plays a dualistic role in the evolution of ischemic brain damage and has also been implicated in ischemic tolerance produced by different conditioning stimuli. Early after ischemia, perivascular astrocytes release cytokines and activate metalloproteases (MMPs) that contribute to blood-brain barrier (BBB) disruption and vasogenic oedema; whereas at later stages, they provide extracellular glutamate uptake, BBB regeneration and neurotrophic factors release. Similarly, early activation of microglia contributes to ischemic brain injury via the production of inflammatory cytokines, including tumor necrosis factor (TNF) and interleukin (IL)-1, reactive oxygen and nitrogen species and proteases. Nevertheless, microglia also contributes to the resolution of inflammation, by releasing IL-10 and tumor growth factor (TGF)-β, and to the late reparative processes by phagocytic activity and growth factors production. Indeed, after ischemia, microglia/macrophages differentiate toward several phenotypes: the M1 pro-inflammatory phenotype is classically activated via toll-like receptors or interferon-γ, whereas M2 phenotypes are alternatively activated by regulatory mediators, such as ILs 4, 10, 13, or TGF-β. Thus, immune cells exert a dualistic role on the evolution of ischemic brain damage, since the classic phenotypes promote injury, whereas alternatively activated M2 macrophages or N2 neutrophils prompt tissue remodeling and repair. Moreover, a subdued activation of the immune system has been involved in ischemic tolerance, since different preconditioning stimuli act via modulation of inflammatory mediators, including toll-like receptors and cytokine signaling pathways. This further underscores that the immuno-modulatory approach for the treatment of ischemic stroke should be aimed at blocking the detrimental effects, while promoting the beneficial responses of the immune reaction.
Abnormal expression of matrix metalloproteinases (MMPs) has been implicated in the pathophysiolog... more Abnormal expression of matrix metalloproteinases (MMPs) has been implicated in the pathophysiology of neuroinflammatory processes that accompany most central nervous system disease. In particular, early upregulation of the gelatinases MMP-2 and MMP-9 has been shown to contribute to disruption of the blood-brain barrier and to death of neurons in ischemic stroke. In situ zymography reveals a significant increase in gelatinolytic MMPs activity in the ischemic brain hemisphere after 2-h middle cerebral artery occlusion (MCAo) followed by 2-h reperfusion in rat. Accordingly, gel zymography demonstrates that expression and activity of MMP-2 and MMP-9 are enhanced in cortex and striatum ipsilateral to the ischemic insult. The latter effect appears to be instrumental for development of delayed brain damage since administration of a broad spectrum, highly specific MMPs inhibitor, GM6001, but not by its negative control, results in a significant (50%) reduction in ischemic brain volume. Incr...
The activation of GABAB receptors in the ventral tegmental area (VTA) has been suggested to atten... more The activation of GABAB receptors in the ventral tegmental area (VTA) has been suggested to attenuate the rewarding properties of psychostimulants, including nicotine. However, the neurochemical mechanism that underlie this effect remains unknown. Since GABAB receptors modulate the release of several neurotransmitters in the mammalian brain, we have characterised the effect of the GABAB receptor agonist baclofen on the release of [3H]-dopamine ([3H]-DA) from VTA slices of naive rats and of rats pre-treated with nicotine. In naive rats, baclofen concentration-dependently inhibited the electrically evoked release of [3H]-DA from the isolated VTA (EC50 = 0.103 microM, 95% CI = 0.043-0.249), without affecting the basal [3H]-monoamine overflow. This effect was mediated by activation of GABAB receptors as it was blocked by the selective receptor antagonist CGP55845A. Chronic administration of nicotine (0.4 mg kg(-1), s.c., for 14 days) affected neither the basal nor the electrically evoke...
Cerebral ischemia involves an important immune response, engaging both the innate and the adaptiv... more Cerebral ischemia involves an important immune response, engaging both the innate and the adaptive immunity systems. The activation of innate immunity involves an inflammatory response, which has been associated with increased brain damage and a worse prognosis in patients who have suffered an ischemic infarction. However, it has been described that inflammation is essential to start the process of repair; therefore, a controlled inflammatory response may be necessary and also beneficial. The activation of adaptive immunity has been associated not only to deleterious autoimmune processes but also to processes of repair. Recent advances in the immunomodulation field, together with a better understanding of ischemic tolerance (IT) and the role of immunity in this pathology, open the possibility to apply immunomodulatory therapies in the treatment of cerebral ischemia.
The receptor for advanced glycation end products (RAGE) has a potential role as a damage-sensing ... more The receptor for advanced glycation end products (RAGE) has a potential role as a damage-sensing molecule; however, to date, its involvement in the pathophysiology of stroke and its modulation following neuroprotective treatment are not completely understood. We have previously demonstrated that expression of distinct RAGE isoforms, recognized by different antibodies, is differentially modulated in the brain of rats subjected to focal cerebral ischemia. Here, we focus on the full-length membrane-bound RAGE isoform, showing that its expression is significantly elevated in the striatum, whereas it is reduced in the cortex of rats subjected to transient middle cerebral artery occlusion (MCAo). Notably, the reduction of cortical levels of full-length RAGE detected 24 h after reperfusion is abolished by systemic administration of a neuroprotective dose of the poly(ADP-ribose) polymerase (PARP) inhibitor, N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34). More interestingly, a significant reduction of plasma soluble RAGE (sRAGE) occurs 24 h after reperfusion and this effect is reverted by a neuroprotective dose of PJ34. Soluble forms of RAGE, generated either by alternative splicing or by proteolysis of the full-length form, effectively bind advanced glycation end products, thereby competing with the cell surface full-length RAGE, thus providing a 'decoy' function that may counteract the adverse effects of receptor signaling in neurons and may possibly exert cytoprotective effects. Thus, our data confirm the important role of RAGE in ischemic cerebral damage and, more interestingly, suggest the potential use of sRAGE as a blood biomarker of stroke severity and of neuroprotective treatment efficacy.
The effect of repeated administration of nicotine (0.4 mg/kg, daily, s.c., for 14 days) on GABA B... more The effect of repeated administration of nicotine (0.4 mg/kg, daily, s.c., for 14 days) on GABA B receptor density, affinity and G-protein coupling was investigated in the mesocorticolimbic system of the rat brain. Baclofen-stimulated [ 35 S]GTPgS binding autoradiography revealed that the level of G-protein coupling to GABA B receptors was significantly reduced in the medial prefrontal cortex and the nucleus accumbens of nicotine-treated rats as compared to vehicle-injected controls. By contrast, GABA B receptor density and affinity, as revealed by [ 3 H]GABA saturation binding autoradiography, were not altered by the nicotine exposure in any of the regions examined. Reduced Gprotein coupling to the GABA B receptor may result in disinhibition of mesocorticolimbic dopaminergic neurones, which would contribute to the development of sensitised dopaminergic responses to repeated administration of nicotine. q
Activation of RAGE (receptor for advanced glycation endproducts) and of its subtypes may play a r... more Activation of RAGE (receptor for advanced glycation endproducts) and of its subtypes may play a role in neuronal damage and neuroinflammation associated with brain ischemia, though the underlying mechanisms remain unclear. In this study, we have examined by Western blotting the expression of RAGE isoforms in the cerebral cortex and striatum of Wistar rats subjected to transient (1 or 2 h) middle cerebral artery occlusion (tMCAo). The findings show that the full-length RAGE (~50 kDa) and its isoforms in the 26-43 kDa range are significantly decreased in the ischemic cortex, but not in the striatum, after 1 and 2 h tMCAo when compared to the sham group. By contrast, in the striatum, ischemia-reperfusion injury caused a significant increase of full-length RAGE and its isoforms in the 72-100 kDa range. We also investigated the soluble form of RAGE, which was significantly decreased in the plasma of rats subjected to transient or permanent MCAo. In conclusion, the present data demonstrate that regional brain expression of RAGE is differentially affected by tMCAo in rat. These modifications are accompanied by a decrease in the plasma levels of soluble RAGE, thereby suggesting a potential role for soluble RAGE as a peripheral biomarker of focal ischemia.
OBJECTIVES: To investigate the relationship between use of proton pump inhibitors (PPIs) and inci... more OBJECTIVES: To investigate the relationship between use of proton pump inhibitors (PPIs) and incident dependency in older adults discharged from acute care hospitals. DESIGN: Prospective observational study. SETTING: Eleven geriatric and internal medicine acute care wards located throughout Italy. PARTICIPANTS: Individuals (mean age 79.2 AE 5.5) who were not completely dependent at the time of discharge from participating wards (N = 401). MEASUREMENTS: The outcome of interest was the loss of at least one basic activity of daily living (ADL) from discharge to the end of follow-up (12 months). The relationship between PPI use and functional decline was investigated using logistic regression analysis before and after propensity score matching. RESULTS: Use of PPIs was significantly associated with functional decline before (odds ratio (OR) = 1.75, 95% confidence interval (CI) = 1.17-2.60) and after propensity score matching (OR = 2.44; 95% CI = 1.36-4.41). Other predictors of functional decline were hypoalbuminemia (OR = 3.10, 95% CI = 1.36-7.10 before matching, OR = 2.81, 95% CI = 1.09-7.77 after matching) and cognitive impairment (OR = 4.08, 95% CI = 1.63-10.2 before matching, OR = 6.35, 95% CI = 1.70-24.0 after matching).
Reactive oxygen species (ROS) accumulation has been described in the brain following an ischemic ... more Reactive oxygen species (ROS) accumulation has been described in the brain following an ischemic insult. Superoxide anion is converted by superoxide dismutase into hydrogen peroxide (H2O2), and the latter is then transformed into the toxic hydroxyl radical, through the Haber-Weiss reaction, converted to water by glutathione peroxidase (GPx) or dismuted to water and oxygen through catalase. Accumulation of H2O2 has been suggested to exert neurotoxic effects, although recent in vitro studies have demonstrated either physiological or protective roles of this molecule in the brain. In particular, oxidative stress is critically involved in brain damage induced by transient cerebral ischemia. Here, we demonstrate that inhibition of GPx by systemic (i.p.) administration of mercaptosuccinate (MS, 1.5-150 mg/kg) dose-dependently reduces brain infarct damage produced by transient (2 h) middle cerebral artery occlusion (MCAo) in rat. Neuroprotection was observed when the drug was administered 15 min before the ischemic insult, whereas no effect was detected when the drug was injected 1h before MCAo or upon reperfusion. Furthermore, application of MS (1 mM) to corticostriatal slices limited the irreversible functional derangement of field potentials caused by a prolonged (12 min) oxygen-glucose deprivation. This effect was reverted by concomitant bath application of the catalase inhibitor 3-aminotriazole (20mM), suggesting the involvement of catalase in mediating the neuroprotective effects of MS. Thus, our findings demonstrate that MS is neuroprotective in both in vivo and in vitro ischemic conditions, through a mechanism which may involve increased endogenous levels of H2O2 and its consequent conversion to molecular oxygen by catalase.
ABSTRACT Thesis (Ph.D. - University of Birmingham, UK) ABSTRACT The hypothesis that chronic admin... more ABSTRACT Thesis (Ph.D. - University of Birmingham, UK) ABSTRACT The hypothesis that chronic administration of nicotine may result in pharmacological modifications of GABAB receptors in the rat mesocorticolimbic system was investigated. In vitro brain autoradiography revealed that GABAB receptor expression and affinity in the ventral tegmental area, the nucleus accumbens and the prefrontal cortex were not significantly altered by chronic (14 days) or subchronic (4 days) treatment with daily subcutaneous injections of nicotine, or in rats that self-administered the drug by the intravenous route. However, reduced G-protein coupling to the GABAB receptor was detected in the prefrontal cortex and the nucleus accumbens, core and shell, of rats receiving chronic subcutaneous injections of nicotine, when compared to saline-injected controls. In order to characterise the functional status of the GABAB receptor, the effect of baclofen on the release of preloaded [3H]dopamine from mesocorticolimbic regions was investigated. Baclofen did not modify the nicotine-induced release of [3H]dopamine from accumbal and cortical slices obtained either from nicotine-treated rats or from saline-controls. By contrast, the GABAB agonist dose-dependently inhibited the [3H]dopamine outflow, evoked by electrical stimulation, from ventral tegmental area slices of naïve animals. The inhibitory effect of baclofen was significantly reduced in midbrain slices obtained from rats pretreated for 14 days with subcutaneous injections of nicotine. This finding was consistent with preliminary in vivo observations revealing that the inhibitory effect of baclofen on the basal and nicotine-evoked somatodendritic release of dopamine in the ventral tegmental area was abolished in rats pre-exposed to chronic nicotine treatment. Thus, chronic administration of nicotine may be associated with a subsensitivity of GABAB receptors in rat brain regions belonging to the mesocorticolimbic system. This would suggest that altered GABAB receptor function might underlie some of the addictive properties of nicotine.
Ischemic stroke is a devastating condition primarily caused by reduced blood supply to the brain.... more Ischemic stroke is a devastating condition primarily caused by reduced blood supply to the brain. Interleukin (IL)-1β is a pro-inflammatory cytokine that plays a pivotal role in the detrimental inflammatory processes that participate to cerebral ischemic damage. After injury, it is produced by distinct cells of the neurovascular unit as an inactive precursor, pro-IL-1β. Although previous studies have suggested that caspase-1 is the main enzyme implicated in the cleavage of pro-IL-1β into the biologically active cytokine, recent work has demonstrated that, under ischemia-reperfusion conditions, other mechanisms may be involved in cytokine maturation. Indeed, we have shown that in rats subjected to transient middle cerebral artery occlusion (MCAo), elevation of IL-1β levels is paralleled by an elevation of gelatinolytic, but not caspase-1 activity in the injured hemisphere and pharmacological inhibition of gelatinases, i.e. matrix metalloproteases (MMP)-2 and MMP-9...
Innate immune cells play a dualistic role in the evolution of ischemic brain damage, with classic... more Innate immune cells play a dualistic role in the evolution of ischemic brain damage, with classic phenotypes promoting injury, and alternatively activated M2 microglia/macrophages or N2 neutrophils providing tissue remodelling and repair. Recently, a number of drugs commonly used for other indications (i.e., azithromycin, minocycline, bexarotene, rosiglitazone, metformin) was reported to provide neuroprotection in preclinical stroke models by promoting immune polarization towards non-inflammatory, protective phenotypes. Repurposing drugs with a well-established safety profile should allow a reduction in the risk of clinical trial failure that has dominated the unsuccessful development of neuroprotective drugs in stroke during the last decades. The clinical validation of the proof of concept, followed by the assessment of safety and efficacy of immune-polarizing repurposed drugs will definitively offer new opportunities for the acute treatment of ischemic stroke.
The identification of novel drug targets for the treatment of ischemic stroke is currently an urg... more The identification of novel drug targets for the treatment of ischemic stroke is currently an urgent challenge. Recent experimental findings have highlighted the neuroprotective potential of immunomodulatory strategies, based on polarization of myeloid cells toward non-inflammatory, beneficial phenotypes. Given the role of retinoid X receptors (RXR) in myeloid cells differentiation and polarization, here we have explored the neuroprotective potential of the RXR agonist bexarotene in mice subjected to focal cerebral ischemia. Acute administration of bexarotene significantly reduced blood brain barrier leakage, brain infarct damage and neurological deficit produced by transient middle cerebral artery occlusion in mice, without affecting cerebral blood flow. The rexinoid exerted neuroprotection with a wide time-window, being effective when administered up to 4.5h after the insult. The amelioration of histological outcome, as well as the ability of bexarotene to revert middle cerebral artery occlusion (MCAo)-induced spleen atrophy, was antagonised by BR1211, a pan-RXR antagonist, or by the selective peroxisome proliferator-activated receptor (PPAR)γ antagonist bisphenol A diglycidyl ether (BADGE), highlighting the involvement of the RXR/PPARγ heterodimer in the beneficial effects exerted by the drug. Immunofluorescence analysis revealed that bexarotene elevates Ym1-immunopositive N2 neutrophils both in the ipsilateral hemisphere and in the spleen of mice subjected to transient middle cerebral artery occlusion, pointing to a major role for peripheral neutrophil polarization in neuroprotection. Thus, our findings suggest that the RXR agonist bexarotene exerts peripheral immunomodulatory effects under ischemic conditions to be effectively repurposed for the acute therapy of ischemic stroke.
To develop novel and effective treatments for ischemic stroke, we investigated the neuroprotectiv... more To develop novel and effective treatments for ischemic stroke, we investigated the neuroprotective effects of the macrolide antibiotic azithromycin in a mouse model system of transient middle cerebral artery occlusion. Intraperitoneal administration of azithromycin significantly reduced blood-brain barrier damage and cerebral infiltration of myeloid cells, including neutrophils and inflammatory macrophages. These effects resulted in a dose-dependent reduction of cerebral ischemic damage, and in a remarkable amelioration of neurological deficits up to 7days after the insult. Neuroprotection was associated with increased arginase activity in peritoneal exudate cells, which was followed by the detection of Ym1- and arginase I-immunopositive M2 macrophages in the ischemic area at 24-48h of reperfusion. Pharmacological inhibition of peritoneal arginase activity counteracted azithromycin-induced neuroprotection, pointing to a major role for drug-induced polarization of migratory macrophages towards a protective, non-inflammatory M2 phenotype.
Cerebral ischemia-reperfusion (I/R) injury is associated with the production of reactive oxygen s... more Cerebral ischemia-reperfusion (I/R) injury is associated with the production of reactive oxygen species (ROS) (Halliwell & Gutteridge, 1989). Although ROS are well known for their role in neurotoxicity, there is emerging evidence that under physiological conditions they also play an important role in cell function (Klann & Thiels, 1999). Moreover, we have recently shown a protective role of hydrogen peroxide (H 2 O 2) in oxygen-deprived dopaminergic neurones of the rat substantia nigra (Geracitano et al., 2005). Neuroprotection by H 2 O 2 (3mM) was also observed in rat hippocampal slices exposed to an ischemic insult. Particularly, we observed that the rescuing action of H 2 O 2 against oxygen/glucose deprivation (OGD) insult in hippocampal slices was mediated by catalase (CAT), as pre-treatment with the CAT irreversible inhibitor 3-amino-1,2,4-triazole (3-AT) blocked this effect. Additionally, we showed that an increase of the endogenous levels of H 2 O 2 , due to a combination of ...
The innate immune system plays a dualistic role in the evolution of ischemic brain damage and has... more The innate immune system plays a dualistic role in the evolution of ischemic brain damage and has also been implicated in ischemic tolerance produced by different conditioning stimuli. Early after ischemia, perivascular astrocytes release cytokines and activate metalloproteases (MMPs) that contribute to blood-brain barrier (BBB) disruption and vasogenic oedema; whereas at later stages, they provide extracellular glutamate uptake, BBB regeneration and neurotrophic factors release. Similarly, early activation of microglia contributes to ischemic brain injury via the production of inflammatory cytokines, including tumor necrosis factor (TNF) and interleukin (IL)-1, reactive oxygen and nitrogen species and proteases. Nevertheless, microglia also contributes to the resolution of inflammation, by releasing IL-10 and tumor growth factor (TGF)-β, and to the late reparative processes by phagocytic activity and growth factors production. Indeed, after ischemia, microglia/macrophages differentiate toward several phenotypes: the M1 pro-inflammatory phenotype is classically activated via toll-like receptors or interferon-γ, whereas M2 phenotypes are alternatively activated by regulatory mediators, such as ILs 4, 10, 13, or TGF-β. Thus, immune cells exert a dualistic role on the evolution of ischemic brain damage, since the classic phenotypes promote injury, whereas alternatively activated M2 macrophages or N2 neutrophils prompt tissue remodeling and repair. Moreover, a subdued activation of the immune system has been involved in ischemic tolerance, since different preconditioning stimuli act via modulation of inflammatory mediators, including toll-like receptors and cytokine signaling pathways. This further underscores that the immuno-modulatory approach for the treatment of ischemic stroke should be aimed at blocking the detrimental effects, while promoting the beneficial responses of the immune reaction.
Abnormal expression of matrix metalloproteinases (MMPs) has been implicated in the pathophysiolog... more Abnormal expression of matrix metalloproteinases (MMPs) has been implicated in the pathophysiology of neuroinflammatory processes that accompany most central nervous system disease. In particular, early upregulation of the gelatinases MMP-2 and MMP-9 has been shown to contribute to disruption of the blood-brain barrier and to death of neurons in ischemic stroke. In situ zymography reveals a significant increase in gelatinolytic MMPs activity in the ischemic brain hemisphere after 2-h middle cerebral artery occlusion (MCAo) followed by 2-h reperfusion in rat. Accordingly, gel zymography demonstrates that expression and activity of MMP-2 and MMP-9 are enhanced in cortex and striatum ipsilateral to the ischemic insult. The latter effect appears to be instrumental for development of delayed brain damage since administration of a broad spectrum, highly specific MMPs inhibitor, GM6001, but not by its negative control, results in a significant (50%) reduction in ischemic brain volume. Incr...
The activation of GABAB receptors in the ventral tegmental area (VTA) has been suggested to atten... more The activation of GABAB receptors in the ventral tegmental area (VTA) has been suggested to attenuate the rewarding properties of psychostimulants, including nicotine. However, the neurochemical mechanism that underlie this effect remains unknown. Since GABAB receptors modulate the release of several neurotransmitters in the mammalian brain, we have characterised the effect of the GABAB receptor agonist baclofen on the release of [3H]-dopamine ([3H]-DA) from VTA slices of naive rats and of rats pre-treated with nicotine. In naive rats, baclofen concentration-dependently inhibited the electrically evoked release of [3H]-DA from the isolated VTA (EC50 = 0.103 microM, 95% CI = 0.043-0.249), without affecting the basal [3H]-monoamine overflow. This effect was mediated by activation of GABAB receptors as it was blocked by the selective receptor antagonist CGP55845A. Chronic administration of nicotine (0.4 mg kg(-1), s.c., for 14 days) affected neither the basal nor the electrically evoke...
Cerebral ischemia involves an important immune response, engaging both the innate and the adaptiv... more Cerebral ischemia involves an important immune response, engaging both the innate and the adaptive immunity systems. The activation of innate immunity involves an inflammatory response, which has been associated with increased brain damage and a worse prognosis in patients who have suffered an ischemic infarction. However, it has been described that inflammation is essential to start the process of repair; therefore, a controlled inflammatory response may be necessary and also beneficial. The activation of adaptive immunity has been associated not only to deleterious autoimmune processes but also to processes of repair. Recent advances in the immunomodulation field, together with a better understanding of ischemic tolerance (IT) and the role of immunity in this pathology, open the possibility to apply immunomodulatory therapies in the treatment of cerebral ischemia.
The receptor for advanced glycation end products (RAGE) has a potential role as a damage-sensing ... more The receptor for advanced glycation end products (RAGE) has a potential role as a damage-sensing molecule; however, to date, its involvement in the pathophysiology of stroke and its modulation following neuroprotective treatment are not completely understood. We have previously demonstrated that expression of distinct RAGE isoforms, recognized by different antibodies, is differentially modulated in the brain of rats subjected to focal cerebral ischemia. Here, we focus on the full-length membrane-bound RAGE isoform, showing that its expression is significantly elevated in the striatum, whereas it is reduced in the cortex of rats subjected to transient middle cerebral artery occlusion (MCAo). Notably, the reduction of cortical levels of full-length RAGE detected 24 h after reperfusion is abolished by systemic administration of a neuroprotective dose of the poly(ADP-ribose) polymerase (PARP) inhibitor, N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide (PJ34). More interestingly, a significant reduction of plasma soluble RAGE (sRAGE) occurs 24 h after reperfusion and this effect is reverted by a neuroprotective dose of PJ34. Soluble forms of RAGE, generated either by alternative splicing or by proteolysis of the full-length form, effectively bind advanced glycation end products, thereby competing with the cell surface full-length RAGE, thus providing a 'decoy' function that may counteract the adverse effects of receptor signaling in neurons and may possibly exert cytoprotective effects. Thus, our data confirm the important role of RAGE in ischemic cerebral damage and, more interestingly, suggest the potential use of sRAGE as a blood biomarker of stroke severity and of neuroprotective treatment efficacy.
The effect of repeated administration of nicotine (0.4 mg/kg, daily, s.c., for 14 days) on GABA B... more The effect of repeated administration of nicotine (0.4 mg/kg, daily, s.c., for 14 days) on GABA B receptor density, affinity and G-protein coupling was investigated in the mesocorticolimbic system of the rat brain. Baclofen-stimulated [ 35 S]GTPgS binding autoradiography revealed that the level of G-protein coupling to GABA B receptors was significantly reduced in the medial prefrontal cortex and the nucleus accumbens of nicotine-treated rats as compared to vehicle-injected controls. By contrast, GABA B receptor density and affinity, as revealed by [ 3 H]GABA saturation binding autoradiography, were not altered by the nicotine exposure in any of the regions examined. Reduced Gprotein coupling to the GABA B receptor may result in disinhibition of mesocorticolimbic dopaminergic neurones, which would contribute to the development of sensitised dopaminergic responses to repeated administration of nicotine. q
Activation of RAGE (receptor for advanced glycation endproducts) and of its subtypes may play a r... more Activation of RAGE (receptor for advanced glycation endproducts) and of its subtypes may play a role in neuronal damage and neuroinflammation associated with brain ischemia, though the underlying mechanisms remain unclear. In this study, we have examined by Western blotting the expression of RAGE isoforms in the cerebral cortex and striatum of Wistar rats subjected to transient (1 or 2 h) middle cerebral artery occlusion (tMCAo). The findings show that the full-length RAGE (~50 kDa) and its isoforms in the 26-43 kDa range are significantly decreased in the ischemic cortex, but not in the striatum, after 1 and 2 h tMCAo when compared to the sham group. By contrast, in the striatum, ischemia-reperfusion injury caused a significant increase of full-length RAGE and its isoforms in the 72-100 kDa range. We also investigated the soluble form of RAGE, which was significantly decreased in the plasma of rats subjected to transient or permanent MCAo. In conclusion, the present data demonstrate that regional brain expression of RAGE is differentially affected by tMCAo in rat. These modifications are accompanied by a decrease in the plasma levels of soluble RAGE, thereby suggesting a potential role for soluble RAGE as a peripheral biomarker of focal ischemia.
OBJECTIVES: To investigate the relationship between use of proton pump inhibitors (PPIs) and inci... more OBJECTIVES: To investigate the relationship between use of proton pump inhibitors (PPIs) and incident dependency in older adults discharged from acute care hospitals. DESIGN: Prospective observational study. SETTING: Eleven geriatric and internal medicine acute care wards located throughout Italy. PARTICIPANTS: Individuals (mean age 79.2 AE 5.5) who were not completely dependent at the time of discharge from participating wards (N = 401). MEASUREMENTS: The outcome of interest was the loss of at least one basic activity of daily living (ADL) from discharge to the end of follow-up (12 months). The relationship between PPI use and functional decline was investigated using logistic regression analysis before and after propensity score matching. RESULTS: Use of PPIs was significantly associated with functional decline before (odds ratio (OR) = 1.75, 95% confidence interval (CI) = 1.17-2.60) and after propensity score matching (OR = 2.44; 95% CI = 1.36-4.41). Other predictors of functional decline were hypoalbuminemia (OR = 3.10, 95% CI = 1.36-7.10 before matching, OR = 2.81, 95% CI = 1.09-7.77 after matching) and cognitive impairment (OR = 4.08, 95% CI = 1.63-10.2 before matching, OR = 6.35, 95% CI = 1.70-24.0 after matching).
Reactive oxygen species (ROS) accumulation has been described in the brain following an ischemic ... more Reactive oxygen species (ROS) accumulation has been described in the brain following an ischemic insult. Superoxide anion is converted by superoxide dismutase into hydrogen peroxide (H2O2), and the latter is then transformed into the toxic hydroxyl radical, through the Haber-Weiss reaction, converted to water by glutathione peroxidase (GPx) or dismuted to water and oxygen through catalase. Accumulation of H2O2 has been suggested to exert neurotoxic effects, although recent in vitro studies have demonstrated either physiological or protective roles of this molecule in the brain. In particular, oxidative stress is critically involved in brain damage induced by transient cerebral ischemia. Here, we demonstrate that inhibition of GPx by systemic (i.p.) administration of mercaptosuccinate (MS, 1.5-150 mg/kg) dose-dependently reduces brain infarct damage produced by transient (2 h) middle cerebral artery occlusion (MCAo) in rat. Neuroprotection was observed when the drug was administered 15 min before the ischemic insult, whereas no effect was detected when the drug was injected 1h before MCAo or upon reperfusion. Furthermore, application of MS (1 mM) to corticostriatal slices limited the irreversible functional derangement of field potentials caused by a prolonged (12 min) oxygen-glucose deprivation. This effect was reverted by concomitant bath application of the catalase inhibitor 3-aminotriazole (20mM), suggesting the involvement of catalase in mediating the neuroprotective effects of MS. Thus, our findings demonstrate that MS is neuroprotective in both in vivo and in vitro ischemic conditions, through a mechanism which may involve increased endogenous levels of H2O2 and its consequent conversion to molecular oxygen by catalase.
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Papers by Diana Amantea