38 results on '"Organotypic hippocampal slices"'
Search Results
2. Cannabidiol inhibits microglia activation and mitigates neuronal damage induced by kainate in an in-vitro seizure model
- Author
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Elisa Landucci, Costanza Mazzantini, Daniele Lana, Maura Calvani, Giada Magni, Maria Grazia Giovannini, and Domenico E. Pellegrini-Giampietro
- Subjects
Cannabinoid ,Neurodegeneration ,Neuroprotection ,Kainate ,Organotypic hippocampal slices ,CA3 region ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Background: Epilepsy is one of the most common brain disorder and, despite the possible use of several therapeutic options, many patients continue to have seizures for their entire lifespan and they need new therapeutic approaches. In the last years the interest on the non-psychoactive compounds present in Cannabis sativa has massively increased, and cannabidiol (CBD) has been shown to be effective in the treatment of different types of neurological disorders and neurodegenerative diseases such as epilepsy, ischemia, multiple sclerosis and Alzheimer's Disease. Methods: We investigated the effects of the selected cannabinoids, Δ9-tetrahydrocannabinol (THC), CBD and cannabigerol (CBG) in rat organotypic hippocampal slices exposed to kainate, an in vitro seizure model. Cell death in the cornu Ammonis 3 (CA3) hippocampal subregion was quantified by propidium iodide fluorescence. Morphological analysis and tissue organization were examined by immunohistochemistry and confocal microscopy and microglia activation and polarization was evaluated using flow cytometry and morphology analysis. Results: When present in the incubation medium, cannabidiol reduced dose-dependent CA3 injury induced by kainate. Conversely, incubation with THC exacerbated hippocampal damage. The neuroprotective effects of cannabidiol were blocked by TRPV1, TRPV2, 5-HT1A, and PPARγ antagonists. Confocal microscopy confirmed that CBD but not THC had a significant protective effect against neuronal damage and tissue disorganization caused by kainate. Cannabidiol incubation significantly block the microglia activation from the M0 to M1 phenotype observed in the kainate in-vitro seizure model, pushing toward a transition from M0 to M2. Conclusions: Our results suggest that CBD mitigated neuronal damage induced by kainate and blocked the transition from the M0 to the M1 phenotype.
- Published
- 2022
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3. The Protective Effect of CBD in a Model of In Vitro Ischemia May Be Mediated by Agonism on TRPV2 Channel and Microglia Activation.
- Author
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Lana, Daniele, Landucci, Elisa, Mazzantini, Costanza, Magni, Giada, Pellegrini-Giampietro, Domenico Edoardo, and Giovannini, Maria Grazia
- Subjects
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CANNABIDIOL , *TRPV cation channels , *CANNABIS (Genus) , *CANNABINOID receptors , *MICROGLIA , *ISCHEMIA , *CANNABINOIDS , *NEUROLOGICAL disorders - Abstract
Cannabinoids, used for centuries for recreational and medical purposes, have potential therapeutic value in stroke treatment. Cannabidiol (CBD), a non-psychoactive compound and partial agonist of TRPV2 channels, is efficacious in many neurological disorders. We investigated the effects of CBD or Δ9-tetrahydrocannabinol (THC) in rat organotypic hippocampal slices exposed to oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Neuronal TRPV2 expression decreased after OGD, but it increased in activated, phagocytic microglia. CBD increased TRPV2 expression, decreased microglia phagocytosis, and increased rod microglia after OGD. THC had effects contrary to those of CBD. Our results show that cannabinoids have different effects in ischemia. CBD showed neuroprotective effects, mediated, at least in part, by TRPV2 channels, since the TRPV2 antagonist tranilast blocked them, while THC worsened the neurodegeneration caused by ischemia. In conclusion, our results suggest that different cannabinoid molecules play different roles in the mechanisms of post-ischemic neuronal death. These different effects of cannabinoid observed in our experiments caution against the indiscriminate use of cannabis or cannabinoid preparations for recreational or therapeutic use. It was observed that the positive effects of CBD may be counteracted by the negative effects caused by high levels of THC. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
4. Differential pattern of neurotoxicity induced by the gliadin peptides p31-43 and p57-68 in in vitro model of epilepsy.
- Author
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Gerace, Elisabetta, Resta, Francesco, Curti, Lorenzo, Di Domizio, Alessandro, Ranieri, Giuseppe, Becatti, Matteo, Renzi, Daniela, Calabrò, Antonino, and Mannaioni, Guido
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GLIADINS , *NEUROTOXICOLOGY , *PEPTIDES , *EPILEPSY , *CENTRAL nervous system , *NEUROLOGICAL disorders - Abstract
[Display omitted] Epilepsy is a central nervous system (CNS) disorder causing repeated seizures due to a transient excessive or synchronous alteration in the electrical activity of the brain. Several neurological disorders have been associated to gluten-related diseases (GRD), including epilepsy. However, the molecular mechanisms that associate GRD and epileptogenesis are still unknown. Our previous data have shown that the gliadin peptide 31–43 (p31-43) enhanced number and duration of seizures induced by kainate in mice and exacerbated CA3-kainate-induced neurotoxicity in organotypic hippocampal slices. Here, we investigated whether another important gliadin peptide p57-68 may exerts effects similar to p31-43 on kainate-induced neurotoxicity. We find that both peptides exacerbate kainate-induced damage in the CA3 region once simultaneously challenged. However, after pre-incubation, p31-43 additionally exacerbates neurotoxicity in the CA1 region, while p57-68 does not. These data suggested differential intracellular mechanisms activated by the peptides. Indeed, analysing intracellular signalling pathways we discover that p31-43 induces significant intracellular changes, including increased phosphorylation of Akt, Erk1/2, and p65, decreased p38 phosphorylation, and deacetylation of nuclear histone-3. Based on these observations, we demonstrate that p31-43 likely activates specific intracellular signaling pathways involved in neuronal excitability, inflammation, and epigenetic regulation, which may contribute to its exacerbation of kainate-induced neurotoxicity. In contrast, p57-68 appears to exert its effects through different mechanisms. Further research is necessary to elucidate the precise mechanisms by which these peptides influence neurotoxicity and understand their implications for neurological disorders. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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- View/download PDF
5. Experimental Models for Testing the Efficacy of Pharmacological Treatments for Neonatal Hypoxic-Ischemic Encephalopathy.
- Author
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Landucci, Elisa, Pellegrini-Giampietro, Domenico E., and Facchinetti, Fabrizio
- Subjects
CEREBRAL anoxia-ischemia ,DRUG therapy ,TREATMENT effectiveness ,THERAPEUTIC hypothermia ,REPERFUSION injury - Abstract
Representing an important cause of long–term disability, term neonatal hypoxic-ischemic encephalopathy (HIE) urgently needs further research aimed at repurposing existing drug as well as developing new therapeutics. Since various experimental in vitro and in vivo models of HIE have been developed with distinct characteristics, it becomes important to select the appropriate preclinical screening cascade for testing the efficacy of novel pharmacological treatments. As therapeutic hypothermia is already a routine therapy for neonatal encephalopathy, it is essential that hypothermia be administered to the experimental model selected to allow translational testing of novel or repurposed drugs on top of the standard of care. Moreover, a translational approach requires that therapeutic interventions must be initiated after the induction of the insult, and the time window for intervention should be evaluated to translate to real world clinical practice. Hippocampal organotypic slice cultures, in particular, are an invaluable intermediate between simpler cell lines and in vivo models, as they largely maintain structural complexity of the original tissue and can be subjected to transient oxygen–glucose deprivation (OGD) and subsequent reoxygenation to simulate ischemic neuronal injury and reperfusion. Progressing to in vivo models, generally, rodent (mouse and rat) models could offer more flexibility and be more cost-effective for testing the efficacy of pharmacological agents with a dose–response approach. Large animal models, including piglets, sheep, and non-human primates, may be utilized as a third step for more focused and accurate translational studies, including also pharmacokinetic and safety pharmacology assessments. Thus, a preclinical proof of concept of efficacy of an emerging pharmacological treatment should be obtained firstly in vitro, including organotypic models, and, subsequently, in at least two different animal models, also in combination with hypothermia, before initiating clinical trials. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
6. NIR Laser Photobiomodulation Induces Neuroprotection in an In Vitro Model of Cerebral Hypoxia/Ischemia.
- Author
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Gerace, Elisabetta, Cialdai, Francesca, Sereni, Elettra, Lana, Daniele, Nosi, Daniele, Giovannini, Maria Grazia, Monici, Monica, and Mannaioni, Guido
- Abstract
Brain photobiomodulation (PBM) is an innovative treatment for a variety of neurological conditions, including cerebral ischemia. However, the capability of PBM for ischemic stroke needs to be further explored and its mechanisms of action remain currently unclear. The aim of the present research was to identify a treatment protocol capable of inducing neuroprotection and to investigate the molecular mechanisms activated by a dual-wavelength near infrared (NIR) laser source in an organotypic hippocampal slice model of hypoxia/ischemia. Hippocampal slices were exposed to oxygen and glucose deprivation (OGD) for 30 min followed by NIR laser light (fluence 3.71, 7.42, or 14.84 J/cm
2 ; wavelengths 808 nm and 905 nm) delivered immediately or 30 min or 60 min after OGD, in order to establish a therapeutic window. Neuronal injury was assessed by propidium iodide fluorescence 24 h later. Our results show that NIR laser irradiation attenuates OGD neurotoxicity once applied immediately or 30 min after OGD. Western blot analysis of proteins involved in neuroinflammation (iNOS, COX-2, NFkB subunit p65, and Bcl-2) and in glutamatergic-mediated synaptic activity (vGluT1, EAAT2, GluN1, and PSD95) showed that the protein modifications induced by OGD were reverted by NIR laser application. Moreover, CA1 confocal microscopy revealed that the profound morphological changes induced by OGD were reverted by NIR laser radiation. In conclusion, NIR laser radiation attenuates OGD neurotoxicity in organotypic hippocampal slices through attenuation of inflammatory mechanisms. These findings shed light on molecular definition of NIR neuroprotective mechanisms, thus underlining the potential benefit of this technique for the treatment of cerebral ischemia. [ABSTRACT FROM AUTHOR]- Published
- 2021
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7. Ethanol neurotoxicity is mediated by changes in expression, surface localization and functional properties of glutamate AMPA receptors.
- Author
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Gerace, Elisabetta, Ilari, Alice, Caffino, Lucia, Buonvicino, Daniela, Lana, Daniele, Ugolini, Filippo, Resta, Francesco, Nosi, Daniele, Grazia Giovannini, Maria, Ciccocioppo, Roberto, Fumagalli, Fabio, Pellegrini‐Giampietro, Domenico E., Masi, Alessio, and Mannaioni, Guido
- Subjects
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AMPA receptors , *GLUTAMATE receptors , *NEUROTOXICOLOGY , *PYRAMIDAL neurons , *SODIUM dichromate , *ETHANOL - Abstract
Modifications in the subunit composition of AMPA receptors (AMPARs) have been linked to the transition from physiological to pathological conditions in a number of contexts, including EtOH‐induced neurotoxicity. Previous work from our laboratory showed that EtOH withdrawal causes CA1 pyramidal cell death in organotypic hippocampal slices and changes in the expression of AMPARs. Here, we investigated whether changes in expression and function of AMPARs may be causal for EtOH‐induced neurotoxicity. To this aim, we examined the subunit composition, localization and function of AMPARs in hippocampal slices exposed to EtOH by using western blotting, surface expression assay, confocal microscopy and electrophysiology. We found that EtOH withdrawal specifically increases GluA1 protein signal in total homogenates, but not in the post‐synaptic density‐enriched fraction. This is suggestive of overall increase and redistribution of AMPARs to the extrasynaptic compartment. At functional level, AMPA‐induced calcium influx was unexpectedly reduced, whereas AMPA‐induced current was enhanced in CA1 pyramidal neurons following EtOH withdrawal, suggesting that increased AMPAR expression may lead to cell death because of elevated excitability, and not for a direct contribution on calcium influx. Finally, the neurotoxicity caused by EtOH withdrawal was attenuated by the non‐selective AMPAR antagonist 2,3‐dioxo‐6‐nitro‐1,2,3,4‐tetrahydrobenzo[f]quinoxaline‐7‐sulfonamide disodium salt as well as by the selective antagonist of GluA2‐lacking AMPARs 1‐naphthyl acetyl spermine. We conclude that EtOH neurotoxicity involves changes in expression, surface localization and functional properties of AMPARs, and propose GluA2‐lacking AMPARs as amenable specific targets for the development of neuroprotective drugs in EtOH‐withdrawal syndrome. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
8. Experimental Models for Testing the Efficacy of Pharmacological Treatments for Neonatal Hypoxic-Ischemic Encephalopathy
- Author
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Elisa Landucci, Domenico E. Pellegrini-Giampietro, and Fabrizio Facchinetti
- Subjects
cerebral ischemia ,hypoxia ,oxygen and glucose deprivation ,cell cultures ,organotypic hippocampal slices ,neonate animal models ,Biology (General) ,QH301-705.5 - Abstract
Representing an important cause of long–term disability, term neonatal hypoxic-ischemic encephalopathy (HIE) urgently needs further research aimed at repurposing existing drug as well as developing new therapeutics. Since various experimental in vitro and in vivo models of HIE have been developed with distinct characteristics, it becomes important to select the appropriate preclinical screening cascade for testing the efficacy of novel pharmacological treatments. As therapeutic hypothermia is already a routine therapy for neonatal encephalopathy, it is essential that hypothermia be administered to the experimental model selected to allow translational testing of novel or repurposed drugs on top of the standard of care. Moreover, a translational approach requires that therapeutic interventions must be initiated after the induction of the insult, and the time window for intervention should be evaluated to translate to real world clinical practice. Hippocampal organotypic slice cultures, in particular, are an invaluable intermediate between simpler cell lines and in vivo models, as they largely maintain structural complexity of the original tissue and can be subjected to transient oxygen–glucose deprivation (OGD) and subsequent reoxygenation to simulate ischemic neuronal injury and reperfusion. Progressing to in vivo models, generally, rodent (mouse and rat) models could offer more flexibility and be more cost-effective for testing the efficacy of pharmacological agents with a dose–response approach. Large animal models, including piglets, sheep, and non-human primates, may be utilized as a third step for more focused and accurate translational studies, including also pharmacokinetic and safety pharmacology assessments. Thus, a preclinical proof of concept of efficacy of an emerging pharmacological treatment should be obtained firstly in vitro, including organotypic models, and, subsequently, in at least two different animal models, also in combination with hypothermia, before initiating clinical trials.
- Published
- 2022
- Full Text
- View/download PDF
9. Differential mechanisms of tolerance induced by NMDA and 3,5‐dihydroxyphenylglycine (DHPG) preconditioning.
- Author
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Gerace, Elisabetta, Zianni, Elisa, Landucci, Elisa, Scartabelli, Tania, Berlinguer Palmini, Rolando, Iezzi, Daniela, Moroni, Flavio, Di Luca, Monica, Mannaioni, Guido, Gardoni, Fabrizio, and Pellegrini‐Giampietro, Domenico E.
- Subjects
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AMPA receptors , *WESTERN immunoblotting , *METHYL aspartate receptors , *PYRAMIDAL neurons , *LIPASE inhibitors , *GLUTAMATE receptors - Abstract
We investigated the molecular events triggered by NMDA and 3,5‐dihydroxyphenylglycine (DHPG) preconditioning, that lead to neuroprotection against excitotoxic insults (AMPA or oxygen and glucose deprivation) in rat organotypic hippocampal slices, with particular attention on glutamate receptors and on cannabinoid system. We firstly evaluated the protein expression of NMDA and AMPA receptor subunits after preconditioning using western blot analysis performed in post‐synaptic densities. We observed that following NMDA, but not DHPG preconditioning, the expression of GluA1 was significantly reduced and this reduction appeared to be associated with the internalization of AMPA receptors. Whole‐cell voltage clamp recordings on CA1 pyramidal neurons of organotypic slices show that 24 hr after exposure to NMDA and DHPG preconditioning, AMPA‐induced currents were significantly reduced. To clarify the mechanisms induced by DHPG preconditioning, we then investigated the involvement of the endocannabinoid system. Exposure of slices to the CB1 antagonist AM251 prevented the development of tolerance to AMPA toxicity induced by DHPG but not NMDA. Accordingly, the MAG‐lipase inhibitor URB602, that increases arachidonoylglycerol (2‐AG) content, but not the FAAH inhibitor URB597, that limits the degradation of anandamide, was also able to induce tolerance versus AMPA and OGD toxicity, suggesting that 2‐AG is responsible for the DHPG‐induced tolerance. In conclusion, preconditioning with NMDA or DHPG promotes differential neuroprotective mechanisms: NMDA by internalization of GluA1‐AMPA receptors, DHPG by producing the endocannabinoid 2‐AG. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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- View/download PDF
10. Tolerance Induced by (S)-3,5-Dihydroxyphenylglycine Postconditioning is Mediated by the PI3K/Akt/GSK3β Signalling Pathway in an In Vitro Model of Cerebral Ischemia.
- Author
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Gerace, Elisabetta, Scartabelli, Tania, Pellegrini-Giampietro, Domenico E., and Landucci, Elisa
- Subjects
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CEREBRAL ischemia , *GLYCOGEN synthase kinase , *GLUTAMATE receptors , *REACTIVE oxygen species - Abstract
• DHPG postconditioning (PostC) induces tolerance by reducing ROS formation. • DHPG PostC induces neuroprotection through PI3K/Akt/GSK3β activation. • The GSK3β inhibitors are neuroprotective in OGD. • The GSK3β inhibitors can be used as PostC agents. Ischemic postconditioning (PostC) is an endogenous neuroprotective strategy for cerebral ischemia induced by low activation of glutamate receptors. We have previously shown that the application of the mGluR1/5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) 5 min after 30 min of oxygen and glucose deprivation (OGD) reduces CA1 damage in organotypic hippocampal slices by activating the PI3K–Akt signalling pathway. In order to extend these data, we analysed the production of reactive oxygen species (ROS) and the glycogen synthase kinase 3β (GSK3β) signalling pathway. Our results show that DHPG PostC was associated with a reduction in the formation of ROS that is massively increased 24 h after OGD exposure. This reduction was prevented by the PI3K inhibitor LY294002, indicating that there is a link between the PI3K/Akt pathway and the formation of ROS in the protective mechanisms of PostC. DHPG PostC also induces a transient increased in GSK3β phosphorylation and inactivation that is followed by nuclear accumulation of β-catenin, that probably lead to the up-regulation of neuroprotective genes. Our results propose GSK3β as new target for neuroprotection, therefore, we verified that the two GSK3β inhibitors N-(3-Chloro-4-methylphenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazol-2-amine (TC-G 24) and LiCl are neuroprotective agents in OGD and also can be used as PostC agents. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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11. Tight coupling of astrocyte energy metabolism to synaptic activity revealed by genetically encoded FRET nanosensors in hippocampal tissue.
- Author
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Ruminot, Iván, Schmälzle, Jana, Leyton, Belén, Barros, L. Felipe, and Deitmer, Joachim W.
- Abstract
The potassium ion, K+, a neuronal signal that is released during excitatory synaptic activity, produces acute activation of glucose consumption in cultured astrocytes, a phenomenon mediated by the sodium bicarbonate cotransporter NBCe1 (SLC4A4). We have explored here the relevance of this mechanism in brain tissue by imaging the effect of neuronal activity on pH, glucose, pyruvate and lactate dynamics in hippocampal astrocytes using BCECF and FRET nanosensors. Electrical stimulation of Schaffer collaterals produced fast activation of glucose consumption in astrocytes with a parallel increase in intracellular pyruvate and biphasic changes in lactate. These responses were blocked by TTX and were absent in tissue slices prepared from NBCe1-KO mice. Direct depolarization of astrocytes with elevated extracellular K+ or Ba2+ mimicked the metabolic effects of electrical stimulation. We conclude that the glycolytic pathway of astrocytes in situ is acutely sensitive to neuronal activity, and that extracellular K+ and the NBCe1 cotransporter are involved in metabolic crosstalk between neurons and astrocytes. Glycolytic activation of astrocytes in response to neuronal K+ helps to provide an adequate supply of lactate, a metabolite that is released by astrocytes and which acts as neuronal fuel and an intercellular signal. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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12. Neuroprotective Effects of Thymoquinone by the Modulation of ER Stress and Apoptotic Pathway in In Vitro Model of Excitotoxicity
- Author
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Elisa Landucci, Costanza Mazzantini, Daniela Buonvicino, Domenico E. Pellegrini-Giampietro, and Maria Camilla Bergonzi
- Subjects
thymoquinone ,excitotoxicity ,neuroprotection ,organotypic hippocampal slices ,ER stress ,apoptosis ,Organic chemistry ,QD241-441 - Abstract
Experimental evidence indicates that the activation of ionotropic glutamate receptors plays an important role in neurological disorders’ models such as epilepsy, cerebral ischemia and trauma. The glutamate receptor agonist kainic acid (KA) induces seizures and excitotoxic cell death in the CA3 region of the hippocampus. Thymoquinone (TQ) is the most important component of the essential oil obtained from black cumin (Nigella sativa L.) seeds. It has many pharmacological actions including antioxidant, anti-inflammatory, and anti-apoptotic effects. TQ was used in an in vitro experimental model of primary cultures where excitotoxicity was induced. Briefly, rat organotypic hippocampal slices were exposed to 5 µM KA for 24 h. Cell death in the CA3 subregions of slices was quantified by measuring propidium iodide fluorescence. The cross-talk between TQ, ER stress and apoptotic pathways was investigated by Western blot. In untreated slices TQ (10 µM) induced a significant increase on the PSD95 levels and it decreased the excitotoxic injury induced by KA. Additionally, TQ was able to ameliorate the KA-induced increase in unfolded proteins GRP78 and GRP94 expression. Finally, TQ was able to partially rescue the reduction of the KA-induced apoptotic pathway activation. Our results suggest that TQ modulates the processes leading to post-kainate neuronal death in the CA3 hippocampal area.
- Published
- 2021
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13. Glutamate Receptor-Mediated Neurotoxicity in a Model of Ethanol Dependence and Withdrawal in Rat Organotypic Hippocampal Slice Cultures
- Author
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Elisabetta Gerace, Elisa Landucci, Daniele Bani, Flavio Moroni, Guido Mannaioni, and Domenico E. Pellegrini-Giampietro
- Subjects
ethanol withdrawal ,glutamate receptors ,organotypic hippocampal slices ,CA1 injury ,neuroprotection ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Long-term alcohol use can lead to alterations in brain structure and functions and, in some cases, to neurodegeneration. Several mechanisms have been proposed to explain ethanol (EtOH)-related brain injury. One of the most relevant mechanisms of alcohol-induced neurodegeneration involves glutamatergic transmission, but their exact role is not yet fully understood. We investigated the neurochemical mechanisms underlying the toxicity induced by EtOH dependence and/or withdrawal by exposing rat organotypic hippocampal slices to EtOH (100–300 mM) for 7 days and then incubating the slices in EtOH-free medium for the subsequent 24 h. EtOH withdrawal led to a dose-dependent CA1 pyramidal cell injury, as detected with propidium iodide fluorescence. Electron microscopy of hippocampal slices revealed that not only EtOH withdrawal but also 7 days chronic EtOH exposure elicited signs of apoptotic cell death in CA1 pyramidal cells. These data were supported by electrophysiological recordings of spontaneus Excitatory Post Synaptic Currents (sEPSCs) from CA1 pyramidal cells. The average amplitude of sEPSCs in slices treated with EtOH for 7 days was significantly increased, and even more so during the first 30 min of EtOH withdrawal, suggesting that the initial phase of the neurodegenerative process could be due to an excitotoxic mechanism. We then analyzed the expression levels of presynaptic (vGlut1, vGlut2, CB1 receptor, synaptophysin) and postsynaptic (PSD95, GluN1, GluN2A, GluN2B, GluA1, GluA2, mGluR1 and mGluR5) proteins after 7 days EtOH incubation or after EtOH withdrawal. We found that only GluA1 and mGluR5 expression levels were significantly increased after EtOH withdrawal and, in neuroprotection experiments, we observed that AMPA and mGluR5 antagonists attenuated EtOH withdrawal-induced toxicity. These data suggest that chronic EtOH treatment promotes abnormal synaptic transmission that may lead to CA1 pyramidal cell death after EtOH withdrawal through glutamate receptors and increased excitotoxicity.
- Published
- 2019
- Full Text
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14. Glutamate Receptor-Mediated Neurotoxicity in a Model of Ethanol Dependence and Withdrawal in Rat Organotypic Hippocampal Slice Cultures.
- Author
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Gerace, Elisabetta, Landucci, Elisa, Bani, Daniele, Moroni, Flavio, Mannaioni, Guido, and Pellegrini-Giampietro, Domenico E.
- Subjects
GLUTAMATE receptors ,NEUROTOXICOLOGY ,NEURODEGENERATION ,ELECTRON microscopy ,SYNAPTOPHYSIN - Abstract
Long-term alcohol use can lead to alterations in brain structure and functions and, in some cases, to neurodegeneration. Several mechanisms have been proposed to explain ethanol (EtOH)-related brain injury. One of the most relevant mechanisms of alcohol-induced neurodegeneration involves glutamatergic transmission, but their exact role is not yet fully understood. We investigated the neurochemical mechanisms underlying the toxicity induced by EtOH dependence and/or withdrawal by exposing rat organotypic hippocampal slices to EtOH (100–300 mM) for 7 days and then incubating the slices in EtOH-free medium for the subsequent 24 h. EtOH withdrawal led to a dose-dependent CA1 pyramidal cell injury, as detected with propidium iodide fluorescence. Electron microscopy of hippocampal slices revealed that not only EtOH withdrawal but also 7 days chronic EtOH exposure elicited signs of apoptotic cell death in CA1 pyramidal cells. These data were supported by electrophysiological recordings of spontaneus Excitatory Post Synaptic Currents (sEPSCs) from CA1 pyramidal cells. The average amplitude of sEPSCs in slices treated with EtOH for 7 days was significantly increased, and even more so during the first 30 min of EtOH withdrawal, suggesting that the initial phase of the neurodegenerative process could be due to an excitotoxic mechanism. We then analyzed the expression levels of presynaptic (vGlut1, vGlut2, CB1 receptor, synaptophysin) and postsynaptic (PSD95, GluN1, GluN2A, GluN2B, GluA1, GluA2, mGluR1 and mGluR5) proteins after 7 days EtOH incubation or after EtOH withdrawal. We found that only GluA1 and mGluR5 expression levels were significantly increased after EtOH withdrawal and, in neuroprotection experiments, we observed that AMPA and mGluR5 antagonists attenuated EtOH withdrawal-induced toxicity. These data suggest that chronic EtOH treatment promotes abnormal synaptic transmission that may lead to CA1 pyramidal cell death after EtOH withdrawal through glutamate receptors and increased excitotoxicity. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
15. PARP-1 activation causes neuronal death in the hippocampal CA1 region by increasing the expression of Ca2+-permeable AMPA receptors
- Author
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E. Gerace, A. Masi, F. Resta, R. Felici, E. Landucci, T. Mello, D.E. Pellegrini-Giampietro, G. Mannaioni, and F. Moroni
- Subjects
MNNG ,PARP ,Organotypic hippocampal slices ,Hippocampus ,Cell death ,GluA1 ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
An excessive activation of poly(ADP-ribose) polymerases (PARPs) may trigger a form of neuronal death similar to that occurring in neurodegenerative disorders. To investigate this process, we exposed organotypic hippocampal slices to N-methyl-N′-nitro-N′-nitrosoguanidine (MNNG, 100 μM for 5 min), an alkylating agent widely used to activate PARP-1. MNNG induced a pattern of degeneration of the CA1 pyramidal cells morphologically similar to that observed after a brief period of oxygen and glucose deprivation (OGD). MNNG exposure was also associated with a dramatic increase in PARP-activity and a robust decrease in NAD+ and ATP content. These effects were prevented by PARP-1 but not PARP-2 inhibitors. In our experimental conditions, cell death was not mediated by AIF translocation (parthanatos) or caspase-dependent apoptotic processes. Furthermore, we found that PARP activation was followed by a significant deterioration of neuronal membrane properties. Using electrophysiological recordings we firstly investigated the suggested ability of ADP-ribose to open TRPM2 channels in MNNG-induced cells death, but the results we obtained showed that TRPM2 channels are not involved. We then studied the involvement of glutamate receptor-ion channel complex and we found that NBQX, a selective AMPA receptor antagonist, was able to effectively prevent CA1 neuronal loss while MK801, a NMDA antagonist, was not active. Moreover, we observed that MNNG treatment increased the ratio of GluA1/GluA2 AMPAR subunit expression, which was associated with an inward rectification of the IV relationship of AMPA sEPSCs in the CA1 but not in the CA3 subfield. Accordingly, 1-naphthyl acetyl spermine (NASPM), a selective blocker of Ca2+-permeable GluA2-lacking AMPA receptors, reduced MNNG-induced CA1 pyramidal cell death. In conclusion, our results show that activation of the nuclear enzyme PARP-1 may change the expression of membrane proteins and Ca2+ permeability of AMPA channels, thus affecting the function and survival of CA1 pyramidal cells.
- Published
- 2014
- Full Text
- View/download PDF
16. Maturation of neural stem cells and integration into hippocampal circuits: functional study in post-ischemia in situ.
- Author
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Kopach, Olga, Rybachuk, Oksana, Krotov, Volodymyr, Kyryk, Vitalii, Voitenko, Nana, and Pivneva, Tatyana
- Subjects
- *
HIPPOCAMPUS (Brain) , *NEURAL stem cells , *CELL death , *PREVENTION - Abstract
The hippocampus is the most susceptible region of the brain to ischemic lesion, with highly vulnerable pyramidal interneurons to ischemic cell death. A restricted brain neurogenesis limits a withdrawal of massive cell death after stroke that endorses cell-based therapies for neuronal replacement strategies following cerebral ischemia. Neurons differentiated from neural stem/progenitor cells (NSPCs) matured and integrated into host circuitry, improving recovery after stroke. However, how host environment regulates the NSPC behaviour in post-ischemic tissue remains unknown. Here we studied functional maturation of NSPCs in control and post-ischemic hippocampal tissue after modelling cerebral ischemia in situ. We traced maturation of electrophysiological properties and integration of the NSPC-derived neurons into the host circuits, developing appropriate activity that takes 3 weeks or less after engraftment. In the ischemic-injured tissue, the NSPC-derived neurons exhibited functional deficits and differentiation of embryonic NSPCs was boosted to glial type - oligodendrocytes and astrocytes. Our findings of the delayed neuronal maturation whilst the promoted NSPC differentiation towards glial cell type in post-ischemic conditions provide new insights into stem-cell-therapy for replacement strategies in cerebral ischemia. [ABSTRACT FROM AUTHOR]
- Published
- 2018
17. Virtual Screening-Accelerated Discovery of a Phosphodiesterase 9 Inhibitor with Neuroprotective Effects in the Kainate Toxicity In Vitro Model.
- Author
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Landucci E, Ribaudo G, Anyanwu M, Oselladore E, Giannangeli M, Mazzantini C, Lana D, Giovannini MG, Memo M, Pellegrini-Giampietro DE, and Gianoncelli A
- Subjects
- 3',5'-Cyclic-AMP Phosphodiesterases, Kainic Acid, Ligands, Phosphoric Diester Hydrolases metabolism, Hippocampus metabolism, Phosphodiesterase Inhibitors pharmacology, Neuroprotective Agents pharmacology
- Abstract
In the central nervous system, some specific phosphodiesterase (PDE) isoforms modulate pathways involved in neuronal plasticity. Accumulating evidence suggests that PDE9 may be a promising therapeutic target for neurodegenerative diseases. In the current study, computational techniques were used to identify a nature-inspired PDE9 inhibitor bearing the scaffold of an isoflavone, starting from a database of synthetic small molecules using a ligand-based approach. Furthermore, docking studies supported by molecular dynamics investigations allowed us to evaluate the features of the ligand-target complex. In vitro assays confirmed the computational results, showing that the selected compound inhibits the enzyme in the nanomolar range. Additionally, we evaluated the expression of gene and protein levels of PDE9 in organotypic hippocampal slices, observing an increase following exposure to kainate (KA). Importantly, the PDE9 inhibitor reduced CA3 damage induced by KA in a dose-dependent manner in organotypic hippocampal slices. Taken together, these observations strongly support the potential of the identified nature-inspired PDE9 inhibitor and suggest that such a molecule could represent a promising lead compound to develop novel therapeutic tools against neurological diseases..
- Published
- 2023
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18. Ethanol Toxicity During Brain Development: Alterations of Excitatory Synaptic Transmission in Immature Organotypic Hippocampal Slice Cultures.
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Gerace, Elisabetta, Landucci, Elisa, Totti, Arianna, Bani, Daniele, Guasti, Daniele, Baronti, Roberto, Moroni, Flavio, Mannaioni, Guido, and Pellegrini‐Giampietro, Domenico E.
- Subjects
- *
PROTEIN analysis , *FETAL alcohol syndrome , *ANIMAL experimentation , *BRAIN , *ELECTROPHYSIOLOGY , *ETHANOL , *GAS chromatography , *HIPPOCAMPUS (Brain) , *MASS spectrometry , *RESEARCH methodology , *MICROSCOPY , *NEURAL transmission , *NEUROTRANSMITTER receptors , *PROBABILITY theory , *RATS , *RESEARCH funding , *STATISTICS , *TISSUE culture , *WESTERN immunoblotting , *DATA analysis , *DATA analysis software , *DESCRIPTIVE statistics , *ONE-way analysis of variance - Abstract
Background The developing brain is particularly vulnerable to alcohol: Drinking during pregnancy can lead to a number of physical, learning, and behavioral disorders in the newborn. It has been demonstrated that immature and mature brain tissues display a differential sensitivity to ethanol (EtOH) toxicity and that cerebral structure and function are diversely impaired according to the stage of synaptic maturation. Methods Rat organotypic hippocampal slice cultures were exposed for 7 days to EtOH (100 to 300 mM) after 2 days (immature) or 10 days (mature) of culture in vitro; EtOH was then removed from the medium, and 24 hours later, slices were analyzed by fluorescence microscopy, Western blotting, electrophysiology, and electron microscopy to explore the molecular mechanisms of EtOH toxicity in the developing hippocampus. Results EtOH withdrawal elicited a selective CA1 pyramidal cell injury in mature slices, but not in immature slices. A significant increase in the expression of pre- and postsynaptic proteins in mature slices revealed that slice maturation is presumably associated with the development of new synapses. Incubation with chronic EtOH for 7 days and its removal from the medium induced a significant decrease in GluA1 and GluA2 expression levels; a significant reduction in the expression of synaptophysin and GluN2A was observed only after EtOH withdrawal. Whole-cell patch-clamp recordings showed that incubation with EtOH for 7 days induced a significant decrease in spontaneous excitatory postsynaptic current (sEPSC) frequency in CA1 pyramidal cells of immature slices and a trend toward a decrease in sEPSC amplitude. Electron microscopy revealed a disorganization of neurotubuli in immature slices after chronic exposure to EtOH. Conclusions These results indicate that prolonged incubation with EtOH and its subsequent withdrawal from the medium induce an impairment of excitatory synaptic transmission and possibly an incorrect formation of neuronal circuits in developing hippocampus in vitro, which is suggestive of mechanisms that may lead to mental retardation in fetal alcohol spectrum disorders. [ABSTRACT FROM AUTHOR]
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- 2016
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19. Desflurane impairs outcome of organotypic hippocampal slices in an in vitro model of traumatic brain injury.
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Krings, Matthias, Jingjin Liu, Grüsser, Linda, Rossaint, Rolf, Coburn, Mark, and Höllig, Anke
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NEUROPROTECTIVE agents , *BRAIN injuries , *TISSUE slices - Abstract
Decreased mortality and disability after traumatic brain injury is a significant medical challenge. Desflurane, a widely used volatile anesthetic has proven to be neuroprotective in a variety of in vitro and in vivo models of ischemic brain injury. The aim of this study was to investigate whether desflurane exhibits neuroprotective properties in an in vitro model of traumatic brain injury. Organotypic hippocampal slice cultures were prepared from brains of 5-7-day-old C57/BL6 mouse pups. After 14 days of culture, the slices were subjected to a focal mechanical trauma and thereafter incubated with three different concentrations of desflurane (2, 4 and 6%) for 2, 24 and 72 hours. Cell injury was assessed with propodium iodide uptake. Our results showed that after 2 hours of desflurane exposure, no significant change in trauma intensity was observed. However, 2% and 4% desflurane could reduce the trauma intensity significantly in the no trauma group than in the no desflurane and trauma group. Incubation with 4% desflurane for 24 hours doubled the trauma intensity in comparison to the trauma control group and the trauma intensity further increased after 72 hours of incubation. Furthermore, a dose-dependent increase of trauma intensity after 24 hours exposure was observed. Our results suggest that a general neuroprotective attribute of desflurane in an in vitro model of traumatic brain injury was not observed. [ABSTRACT FROM AUTHOR]
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- 2016
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20. Neurogenic and Neuroprotective Potential of Stem/Stromal Cells Derived from Adipose Tissue
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Klaudia Radoszkiewicz, Anna Figiel-Dabrowska, Dorota Sulejczak, Anna Sarnowska, Paulina Rybkowska, and Natalia Ewa Krzesniak
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Nervous system ,Stromal cell ,neurotrophic factors ,neural differentiation ,QH301-705.5 ,oxygen–glucose deprivation injury ,Neurogenesis ,Cell ,DFAT cells ,Adipose tissue ,Biology ,ASC ,Neuroprotection ,Hippocampus ,Article ,organotypic hippocampal slices ,Neurotrophic factors ,In vivo ,medicine ,Adipocytes ,Humans ,Cell Lineage ,Biology (General) ,neuroprotective potential ,Cells, Cultured ,indirect co-culture ,low oxygen concentration ,Mesenchymal stem cell ,Cell Differentiation ,Mesenchymal Stem Cells ,General Medicine ,medicine.anatomical_structure ,Neuroprotective Agents ,Adipose Tissue ,Cancer research - Abstract
Currently, the number of stem-cell based experimental therapies in neurological injuries and neurodegenerative disorders has been massively increasing. Despite the fact that we still have not obtained strong evidence of mesenchymal stem/stromal cells’ neurogenic effectiveness in vivo, research may need to focus on more appropriate sources that result in more therapeutically promising cell populations. In this study, we used dedifferentiated fat cells (DFAT) that are proven to demonstrate more pluripotent abilities in comparison with standard adipose stromal cells (ASCs). We used the ceiling culture method to establish DFAT cells and to optimize culture conditions with the use of a physioxic environment (5% O2). We also performed neural differentiation tests and assessed the neurogenic and neuroprotective capability of both DFAT cells and ASCs. Our results show that DFAT cells may have a better ability to differentiate into oligodendrocytes, astrocytes, and neuron-like cells, both in culture supplemented with N21 and in co-culture with oxygen–glucose-deprived (OGD) hippocampal organotypic slice culture (OHC) in comparison with ASCs. Results also show that DFAT cells have a different secretory profile than ASCs after contact with injured tissue. In conclusion, DFAT cells constitute a distinct subpopulation and may be an alternative source in cell therapy for the treatment of nervous system disorders.
- Published
- 2021
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21. Neuroprotective effects of thymoquinone by the modulation of ER stress and apoptotic pathway in in vitro model of excitotoxicity
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Maria Camilla Bergonzi, Daniela Buonvicino, Elisa Landucci, Costanza Mazzantini, and Domenico E. Pellegrini-Giampietro
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Male ,Excitotoxicity ,thymoquinone ,Pharmaceutical Science ,Pharmacology ,medicine.disease_cause ,Analytical Chemistry ,chemistry.chemical_compound ,0302 clinical medicine ,organotypic hippocampal slices ,Drug Discovery ,Benzoquinones ,Excitatory Amino Acid Agonists ,Thymoquinone ,0303 health sciences ,Kainic Acid ,Neuronal Plasticity ,Glutamate receptor ,apoptosis ,Endoplasmic Reticulum Stress ,CA3 Region, Hippocampal ,Neuroprotective Agents ,Chemistry (miscellaneous) ,Molecular Medicine ,Female ,neuroprotection ,ER stress ,Disks Large Homolog 4 Protein ,excitotoxicity ,Ionotropic effect ,Kainic acid ,Programmed cell death ,In Vitro Techniques ,Neuroprotection ,Article ,lcsh:QD241-441 ,03 medical and health sciences ,lcsh:Organic chemistry ,medicine ,Animals ,Propidium iodide ,Rats, Wistar ,Physical and Theoretical Chemistry ,030304 developmental biology ,Epilepsy ,Organic Chemistry ,Rats ,Disease Models, Animal ,chemistry ,030217 neurology & neurosurgery - Abstract
Experimental evidence indicates that the activation of ionotropic glutamate receptors plays an important role in neurological disorders’ models such as epilepsy, cerebral ischemia and trauma. The glutamate receptor agonist kainic acid (KA) induces seizures and excitotoxic cell death in the CA3 region of the hippocampus. Thymoquinone (TQ) is the most important component of the essential oil obtained from black cumin (Nigella sativa L.) seeds. It has many pharmacological actions including antioxidant, anti-inflammatory, and anti-apoptotic effects. TQ was used in an in vitro experimental model of primary cultures where excitotoxicity was induced. Briefly, rat organotypic hippocampal slices were exposed to 5 µM KA for 24 h. Cell death in the CA3 subregions of slices was quantified by measuring propidium iodide fluorescence. The cross-talk between TQ, ER stress and apoptotic pathways was investigated by Western blot. In untreated slices TQ (10 µM) induced a significant increase on the PSD95 levels and it decreased the excitotoxic injury induced by KA. Additionally, TQ was able to ameliorate the KA-induced increase in unfolded proteins GRP78 and GRP94 expression. Finally, TQ was able to partially rescue the reduction of the KA-induced apoptotic pathway activation. Our results suggest that TQ modulates the processes leading to post-kainate neuronal death in the CA3 hippocampal area.
- Published
- 2021
22. Ethanol neurotoxicity is mediated by changes in expression, surface localization and functional properties of glutamate AMPA receptors
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Maria Grazia Giovannini, Domenico E. Pellegrini-Giampietro, Lucia Caffino, Elisabetta Gerace, Alice Ilari, Daniele Nosi, Daniele Lana, Roberto Ciccocioppo, Fabio Fumagalli, Alessio Masi, Guido Mannaioni, Francesco Resta, Filippo Ugolini, and Daniela Buonvicino
- Subjects
0301 basic medicine ,Male ,Programmed cell death ,Protein subunit ,Glutamic Acid ,AMPA receptor ,Hippocampal formation ,Biochemistry ,Hippocampus ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Organ Culture Techniques ,mental disorders ,medicine ,Animals ,Receptors, AMPA ,Rats, Wistar ,Ethanol ,Chemistry ,musculoskeletal, neural, and ocular physiology ,Antagonist ,Glutamate receptor ,Neurotoxicity ,medicine.disease ,Flow Cytometry ,Cell biology ,Rats ,Electrophysiology ,030104 developmental biology ,Gene Expression Regulation ,nervous system ,AMPA receptors ,CA1 injury ,ethanol withdrawal ,organotypic hippocampal slices ,scaffold proteins ,Female ,Excitatory Amino Acid Antagonists ,030217 neurology & neurosurgery - Abstract
Modifications in the subunit composition of AMPA receptors (AMPARs) have been linked to the transition from physiological to pathological conditions in a number of contexts, including EtOH-induced neurotoxicity. Previous work from our laboratory showed that EtOH withdrawal causes CA1 pyramidal cell death in organotypic hippocampal slices and changes in the expression of AMPARs. Here, we investigated whether changes in expression and function of AMPARs may be causal for EtOH-induced neurotoxicity. To this aim, we examined the subunit composition, localization and function of AMPARs in hippocampal slices exposed to EtOH by using western blotting, surface expression assay, confocal microscopy and electrophysiology. We found that EtOH withdrawal specifically increases GluA1 protein signal in total homogenates, but not in the post-synaptic density-enriched fraction. This is suggestive of overall increase and redistribution of AMPARs to the extrasynaptic compartment. At functional level, AMPA-induced calcium influx was unexpectedly reduced, whereas AMPA-induced current was enhanced in CA1 pyramidal neurons following EtOH withdrawal, suggesting that increased AMPAR expression may lead to cell death because of elevated excitability, and not for a direct contribution on calcium influx. Finally, the neurotoxicity caused by EtOH withdrawal was attenuated by the non-selective AMPAR antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt as well as by the selective antagonist of GluA2-lacking AMPARs 1-naphthyl acetyl spermine. We conclude that EtOH neurotoxicity involves changes in expression, surface localization and functional properties of AMPARs, and propose GluA2-lacking AMPARs as amenable specific targets for the development of neuroprotective drugs in EtOH-withdrawal syndrome.
- Published
- 2021
23. Prolonged ampakine exposure prunes dendritic spines and increases presynaptic release probability for enhanced long-term potentiation in the hippocampus.
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Chang, Philip K.‐Y., Prenosil, George A., Verbich, David, Gill, Raminder, and McKinney, R. Anne
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AMPAKINES , *PRESYNAPTIC receptors , *HIPPOCAMPUS (Brain) , *DENDRITIC cells , *ALLOSTERIC regulation , *IMMUNOMODULATORS , *PROPIONIC acid , *GLUTAMATE receptors - Abstract
CX 546, an allosteric positive modulator of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type ionotropic glutamate receptors (AMPARs), belongs to a drug class called ampakines. These compounds have been shown to enhance long-term potentiation ( LTP), a cellular model of learning and memory, and improve animal learning task performance, and have augmented cognition in neurodegenerative patients. However, the chronic effect of CX546 on synaptic structures has not been examined. The structure and integrity of dendritic spines are thought to play a role in learning and memory, and their abnormalities have been implicated in cognitive disorders. In addition, their structural plasticity has been shown to be important for cognitive function, such that dendritic spine remodeling has been proposed as the morphological correlate for LTP. Here, we tested the effect of CX546 on dendritic spine remodeling following long-term treatment. We found that, with prolonged CX546 treatment, organotypic hippocampal slice cultures showed a significant reduction in CA3- CA1 excitatory synapse and spine density. Electrophysiological approaches revealed that the CA3- CA1 circuitry compensates for this synapse loss by increasing synaptic efficacy through enhancement of presynaptic release probability. CX546-treated slices showed prolonged and enhanced potentiation upon LTP induction. Furthermore, structural plasticity, namely spine head enlargement, was also more pronounced after CX546 treatment. Our results suggest a concordance of functional and structural changes that is enhanced with prolonged CX546 exposure. Thus, the improved cognitive ability of patients receiving ampakine treatment may result from the priming of synapses through increases in the structural plasticity and functional reliability of hippocampal synapses. [ABSTRACT FROM AUTHOR]
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- 2014
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24. Blocking brain-derived neurotrophic factor inhibits injury-induced hyperexcitability of hippocampal CA3 neurons.
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Gill, Raminder, Chang, Philip K.‐Y., Prenosil, George A., Deane, Emily C., and McKinney, Rebecca A.
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BRAIN injury treatment , *BRAIN-derived neurotrophic factor , *TROPOMYOSINS , *HIPPOCAMPUS injuries , *AXONS , *TRAUMATIC epilepsy , *COMPLICATIONS of brain injuries - Abstract
Brain trauma can disrupt synaptic connections, and this in turn can prompt axons to sprout and form new connections. If these new axonal connections are aberrant, hyperexcitability can result. It has been shown that ablating tropomyosin-related kinase B ( Trk B), a receptor for brain-derived neurotrophic factor ( BDNF), can reduce axonal sprouting after hippocampal injury. However, it is unknown whether inhibiting BDNF-mediated axonal sprouting will reduce hyperexcitability. Given this, our purpose here was to determine whether pharmacologically blocking BDNF inhibits hyperexcitability after injury-induced axonal sprouting in the hippocampus. To induce injury, we made Schaffer collateral lesions in organotypic hippocampal slice cultures. As reported by others, we observed a 50% reduction in axonal sprouting in cultures treated with a BDNF blocker ( Trk B- Fc) 14 days after injury. Furthermore, lesioned cultures treated with Trk B- Fc were less hyperexcitable than lesioned untreated cultures. Using electrophysiology, we observed a two-fold decrease in the number of CA3 neurons that showed bursting responses after lesion with Trk B- Fc treatment, whereas we found no change in intrinsic neuronal firing properties. Finally, evoked field excitatory postsynaptic potential recordings indicated an increase in network activity within area CA3 after lesion, which was prevented with chronic Trk B- Fc treatment. Taken together, our results demonstrate that blocking BDNF attenuates injury-induced hyperexcitability of hippocampal CA3 neurons. Axonal sprouting has been found in patients with post-traumatic epilepsy. Therefore, our data suggest that blocking the BDNF-TrkB signaling cascade shortly after injury may be a potential therapeutic target for the treatment of post-traumatic epilepsy. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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25. REVERSAL OF FIBRONECTIN-INDUCED HIPPOCAMPAL DEGENERATION WITH ENCAPSULATED MESENCHYMAL STROMAL CELLS.
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DOLLÉ, JEAN-PIERRE, BARMINKO, JEFFREY, VERUVA, SAI, MOURE, CASEY, SCHLOSS, RENE, and YARMUSH, MARTIN L.
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FIBRONECTINS , *STROMAL cells , *NANOPORES , *BLOOD plasma , *CELL death - Abstract
Mesenchymal stromal cells (MSC) can promote tissue protection following injury, in part by modulating inflammatory cell responses. The aim of this study was to investigate the potential tissue protective properties of encapsulated MSCs (eMSC) in an organotypic injury model induced by fibronectin culture. MSC were encapsulated in alginate beads containing a network of nanopores, which segregate the cells from the extracapsular milieu, while still permitting diffusion into and out of the capsule. An increase in blood brain barrier permeability during pathological conditions permits the influx of blood plasma constituents that can be quite harmful to surrounding tissues. In particular, increased concentrations of fibronectin have been shown in a number of diseases and CNS traumas, co-localizing in areas of activated microglia. We observed over a 14-day period, a consistent increase in OHC degradation in the presence of fibronectin measured by a significant decrease in slice area, the breakdown in OHC pyramidal layers, and consistent cell death over the culture period. Microglial ionized calcium-binding adapter molecule 1 (IBA-1) expression remained elevated throughout the culture period with the majority found within the pyramidal layers. When eMSC were added to the cultures, a significant decrease in OHC degradation was observed as reflected by a reduction in OHC area shrinkage and in the amount of cell death. In the presence of eMSC, pyramidal layer structure was maintained and axonal extension from the periphery of the OHCs was observed. Therefore, MSC, delivered in a nanoporous alginate matrix, can modulate responses to injury by reversing fibronectin-induced OHC degradation. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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26. Acute and sustained isoflurane neuroprotection: The effect of culture age and duration of oxygen and glucose deprivation.
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Wise-Faberowski, Lisa and Osorio-Lujan, Suzanne
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ANALYSIS of variance , *ANIMAL experimentation , *BIOLOGICAL models , *CELL death , *HIPPOCAMPUS (Brain) , *IMMUNOHISTOCHEMISTRY , *ISOFLURANE , *RESEARCH methodology , *MICROSCOPY , *PROBABILITY theory , *RATS , *RESEARCH funding , *STAINS & staining (Microscopy) , *STATISTICS , *TISSUE culture , *DATA analysis , *NEUROPROTECTIVE agents , *CASE-control method , *DESCRIPTIVE statistics , *IN vitro studies , *PHARMACODYNAMICS - Abstract
Objectives: Organotypic hippocampal slice (OHS) cultures provide the opportunity to dissect factors influencing volatile anaesthetic neuroprotective efficacy. It was hypothesized that three conditions-OHS culture age, oxygen glucose deprivation (OGD) duration and day of evaluation for cell death after OGD-influence isoflurane's ability to provide acute and sustained protection against OGD-induced cell death. Methods: OHS were prepared from PND 9-11 rat pups and maintained in vitro for 0.5, 1, 2 or 3 weeks. The slices were exposed to OGD for 0, 10, 30, 60 or 90 minutes with or without 1.5% isoflurane. Sytox staining was used to determine the amount of cell death on post-OGD days 1, 3 and 7 and was compared to the amount of cell death in culture-age matched controls (no OGD). Results: The duration of OGD necessary to produce cell death was inversely related to culture age. All culture ages showed evidence of both acute and sustained neuroprotection, but the magnitude of protection depended on OHS culture age, duration of OGD and post-OGD day of evaluation. In 1 and 2-week old slices early isoflurane neuroprotection was best observed with 90 minutes OGD and late isoflurane protection was best observed with 10 minutes OGD. Conclusions: In OHS, acute and sustained isoflurane neuroprotection in OGD-induced cell death is dependent on the conditions being studied. [ABSTRACT FROM AUTHOR]
- Published
- 2013
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27. CB1 receptors and post-ischemic brain damage: Studies on the toxic and neuroprotective effects of cannabinoids in rat organotypic hippocampal slices
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Landucci, Elisa, Scartabelli, Tania, Gerace, Elisabetta, Moroni, Flavio, and Pellegrini-Giampietro, Domenico E.
- Subjects
- *
BRAIN damage , *NEUROPROTECTIVE agents , *CANNABINOIDS , *HIPPOCAMPUS (Brain) , *CENTRAL nervous system , *CEREBRAL ischemia , *NEUROTOXIC agents , *LABORATORY rats - Abstract
Abstract: Cannabinoids (CBs) are implicated in a number of physiological and pathological mechanisms in the central nervous system, but their exact role in post-ischemic brain injury is unclear. The toxic and neuroprotective effects of synthetic and endogenous CBs were evaluated in rat organotypic hippocampal slices exposed to 20 min oxygen–glucose deprivation (OGD) and in gerbils subjected to bilateral carotid occlusion for 5 min. When present in the incubation medium, the synthetic CB agonists WIN 55212-2 and CP 55940 (1–30 μM) and the CB1 agonist ACEA exacerbated CA1 injury induced by OGD, whereas the CB1 receptor antagonists AM 251 and LY 320135 were neuroprotective with maximal activity at 1 μM. AM 251 (at 3 mg/kg, i.p.) also attenuated CA1 pyramidal cell death in gerbils in vivo. The endocannabinoid 2-arachidonoylglycerol (2-AG) reduced OGD injury in hippocampal slices at 0.1–1 μM, whereas anandamide (AEA) was neurotoxic at the same concentrations. The effects of WIN 55212-2, AEA and 2-AG in slices were all dependent on the activation of CB1 but not CB2 receptors, except for the toxic effects of AEA that were also dependent on vanilloid TRPV1 receptors. Our results suggest that exogenous administration of CB1 agonists and the production of endocannabinoids “on demand” may produce different, if not opposite, effects on the fate of neurons following cerebral ischemia. [Copyright &y& Elsevier]
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- 2011
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28. Differential expression of TRPM2 and TRPV4 channels and their potential role in oxidative stress-induced cell death in organotypic hippocampal culture
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Bai, Ji-Zhong and Lipski, Janusz
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OXIDATIVE stress , *CELL death , *HIPPOCAMPUS (Brain) , *GENE expression , *TRP channels , *BRAIN physiology , *PHYSIOLOGICAL effects of hydrogen-ion concentration , *POLYMERASE chain reaction , *IMMUNOCYTOCHEMISTRY , *SULFOXIMINES - Abstract
Abstract: TRPM2 and TPPV4 channels, two members of TRP channel family, are known to be widely expressed in the brain but their exact expression pattern and function are not well understood. Due to their high Ca2+ permeability and gating by reactive oxygen species (TRPM2), or cell swelling, low pH and high temperature (TRPV4), they are likely to be involved in cell damage associated with various brain pathologies. The aim of this study was to investigate the expression of these channels and their potential role in oxidative stress-induced cell damage in organotypic hippocampal slice cultures, a model that retains the complex interaction between neurons and astrocytes. Channel expression was confirmed with RT-PCR and western blotting, while immunocytochemistry demonstrated TRPM2 in CA1–CA3 pyramidal neurons and TRPV4 in astrocytes. Oxidative stress induced by exogenous application of H2O2 (600μM) caused preferential damage of pyramidal neurons, while oxidative stress evoked with mercaptosuccinate (MCS; 400μM) or buthionine sulfoximine (BSO; 4μM) mainly damaged astrocytes, as identified by propidium iodide fluorescence. Antioxidants (Trolox 500μM; MitoE 2μM) reduced both neuronal and astrocytic cell death. Blockers of TRPV4 channels (Gd3+ 500μM; Ruthenium red 1μM) increased the viability of astrocytes following MCS or BSO treatments, consistent with the expression pattern of these channels. Blockers of TRPM2 channels clotrimazole (20μM), N-(p-amylcinnomoyl)anthranilic acid (ACA, 25μM) or flufenamic acid (FFA, 200μM) failed to protect pyramidal neurons from damage caused by exogenous H2O2, and increased damage of these neurons caused by MCS and BSO. The differential expression of stress-sensitive TRPM2 and TRPV4 channels in hippocampal neurons and astrocytes that show distinct differences in vulnerability to different forms of oxidative stress suggests the specific involvement of these channels in oxidative stress-induced cell damage. However, the exact relationship between TRPM2 channel activation and cell death still remains to be determined due to the lack of protective effects of TRPM2 channel blockers. [Copyright &y& Elsevier]
- Published
- 2010
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29. Carbamylated erythropoietin is neuroprotective in an experimental model of traumatic brain injury.
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Adembri, Chiara, Massagrande, Alessandra, Tani, Alessia, Miranda, Marco, Margheri, Martina, De Gaudio, Raffaele, and Pellegrini-Giampietro, Domenico E.
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- *
RECOMBINANT erythropoietin , *BRAIN injuries , *ERYTHROPOIETIN , *NEUROPROTECTIVE agents , *CELL death , *RECOMBINANT blood proteins - Abstract
The article investigates the neuroprotective capability of carbamylated-recombinant human erythropoietin (rhEPO) (CEPO) in an in vitro model of cerebral trauma in which rhEPO was previously shown to reduce posttraumatic cell death. Results suggest that 10 IU/ml CEPO exerts neuroprotective effects comparable with those of rhEPO in an in vitro model of mechanical cerebral trauma. CEPO may represent an pharmacologic tool in treating patients with mechanical injury to the brain.
- Published
- 2008
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30. Differential role of mGlu1 and mGlu5 receptors in rat hippocampal slice models of ischemic tolerance.
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Werner, Claudia G., Scartabelli, Tania, Pancani, Tristano, Landucci, Elisa, Moroni, Flavio, and Pellegrini‐Giampietro, Domenico E.
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- *
HIPPOCAMPUS (Brain) , *ISCHEMIA , *BLOOD circulation disorders , *CEREBRAL cortex , *CELL death , *GLUCOSE , *LIMBIC system , *NEUROTOXICOLOGY - Abstract
Activation of glutamate receptors has been proposed as a key factor in the induction of ischemic tolerance. We used organotypic rat hippocampal slices exposed to 30 min oxygen–glucose deprivation (OGD) to evaluate postischemic pyramidal cell death in the CA1 subregion. In this model, 10 min exposure to OGD 24 h before the exposure to toxic OGD was not lethal and reduced the subsequent OGD neurotoxicity by ∼ 53% (ischemic preconditioning). Similarly, a 30 min exposure to the group I mGlu receptor agonist DHPG (10 µM) significantly reduced OGD neurotoxicity 24 h later (pharmacological preconditioning). Ischemic tolerance did not develop when either the selective mGlu1 antagonists LY367385 and 3-MATIDA or the AMPA/KA antagonist CNQX were present in the incubation medium during exposure to sublethal OGD. Neither the NMDA antagonist MK801 nor the mGlu5 antagonist MPEP affected the preconditioning process. On the other hand, pharmacological preconditioning was prevented not only by LY367385 or CNQX, but also by MPEP. In preconditioned slices, the toxic responses to AMPA or NMDA were reduced. The neurotoxicty of 100 µM DHPG in slices simultaneously exposed to a mild (20 min) OGD was differentially altered in the two preconditioning paradigms. After ischemic preconditioning, DHPG neurotoxicity was reduced in a manner that was sensitive to LY367385 but not to MPEP, whereas after pharmacological preconditioning it was enhanced in a manner that was sensitive to MPEP but not to LY367385. Our results show that mGlu1 and mGlu5 receptors are differentially involved in the induction and expression of ischemic tolerance following two diverse preconditioning stimuli. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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31. Tolerance Induced by (S)-3,5-Dihydroxyphenylglycine Postconditioning is Mediated by the PI3K/Akt/GSK3β Signalling Pathway in an In Vitro Model of Cerebral Ischemia
- Author
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Gerace, E., Scartabelli, T., Pellegrini-Giampietro, D. E., and Landucci, E.
- Subjects
DHPG postconditioning ,Lithium chloride ,organotypic hippocampal slices ,oxygen and glucose deprivation ,PI3K/Akt/GSK3β signaling pathway - Published
- 2020
32. Neurogenic and Neuroprotective Potential of Stem/Stromal Cells Derived from Adipose Tissue.
- Author
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Figiel-Dabrowska, Anna, Radoszkiewicz, Klaudia, Rybkowska, Paulina, Krzesniak, Natalia Ewa, Sulejczak, Dorota, and Sarnowska, Anna
- Subjects
STROMAL cells ,ADIPOSE tissues ,NEUROPROTECTIVE agents ,CELL populations ,NERVOUS system ,FAT cells ,OLIGODENDROGLIA - Abstract
Currently, the number of stem-cell based experimental therapies in neurological injuries and neurodegenerative disorders has been massively increasing. Despite the fact that we still have not obtained strong evidence of mesenchymal stem/stromal cells' neurogenic effectiveness in vivo, research may need to focus on more appropriate sources that result in more therapeutically promising cell populations. In this study, we used dedifferentiated fat cells (DFAT) that are proven to demonstrate more pluripotent abilities in comparison with standard adipose stromal cells (ASCs). We used the ceiling culture method to establish DFAT cells and to optimize culture conditions with the use of a physioxic environment (5% O
2 ). We also performed neural differentiation tests and assessed the neurogenic and neuroprotective capability of both DFAT cells and ASCs. Our results show that DFAT cells may have a better ability to differentiate into oligodendrocytes, astrocytes, and neuron-like cells, both in culture supplemented with N21 and in co-culture with oxygen–glucose-deprived (OGD) hippocampal organotypic slice culture (OHC) in comparison with ASCs. Results also show that DFAT cells have a different secretory profile than ASCs after contact with injured tissue. In conclusion, DFAT cells constitute a distinct subpopulation and may be an alternative source in cell therapy for the treatment of nervous system disorders. [ABSTRACT FROM AUTHOR]- Published
- 2021
- Full Text
- View/download PDF
33. Neuroprotective Effects of Thymoquinone by the Modulation of ER Stress and Apoptotic Pathway in In Vitro Model of Excitotoxicity.
- Author
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Landucci, Elisa, Mazzantini, Costanza, Buonvicino, Daniela, Pellegrini-Giampietro, Domenico E., Bergonzi, Maria Camilla, Patruno, Antonia, and Pesce, Mirko
- Subjects
BLACK cumin ,NEUROLOGICAL disorders ,NEUROPROTECTIVE agents ,GLUTAMATE receptors ,CELL death ,LONG-term synaptic depression ,BACOPA monnieri - Abstract
Experimental evidence indicates that the activation of ionotropic glutamate receptors plays an important role in neurological disorders' models such as epilepsy, cerebral ischemia and trauma. The glutamate receptor agonist kainic acid (KA) induces seizures and excitotoxic cell death in the CA3 region of the hippocampus. Thymoquinone (TQ) is the most important component of the essential oil obtained from black cumin (Nigella sativa L.) seeds. It has many pharmacological actions including antioxidant, anti-inflammatory, and anti-apoptotic effects. TQ was used in an in vitro experimental model of primary cultures where excitotoxicity was induced. Briefly, rat organotypic hippocampal slices were exposed to 5 µM KA for 24 h. Cell death in the CA3 subregions of slices was quantified by measuring propidium iodide fluorescence. The cross-talk between TQ, ER stress and apoptotic pathways was investigated by Western blot. In untreated slices TQ (10 µM) induced a significant increase on the PSD95 levels and it decreased the excitotoxic injury induced by KA. Additionally, TQ was able to ameliorate the KA-induced increase in unfolded proteins GRP78 and GRP94 expression. Finally, TQ was able to partially rescue the reduction of the KA-induced apoptotic pathway activation. Our results suggest that TQ modulates the processes leading to post-kainate neuronal death in the CA3 hippocampal area. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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34. PARP-1 activation causes neuronal death in the hippocampal CA1 region by increasing the expression of Ca2+-permeable AMPA receptors
- Author
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Elisa Landucci, F. Moroni, Domenico E. Pellegrini-Giampietro, Francesco Resta, Elisabetta Gerace, Tommaso Mello, Guido Mannaioni, Alessio Masi, and Roberta Felici
- Subjects
Cell death ,Programmed cell death ,Poly ADP ribose polymerase ,Poly (ADP-Ribose) Polymerase-1 ,TRPM Cation Channels ,AMPA receptor ,Biology ,Poly(ADP-ribose) Polymerase Inhibitors ,Receptors, N-Methyl-D-Aspartate ,Hippocampus ,PARP ,lcsh:RC321-571 ,Tissue Culture Techniques ,chemistry.chemical_compound ,MNNG ,Animals ,TRPM2 ,Receptors, AMPA ,Rats, Wistar ,Hypoxia ,CA1 Region, Hippocampal ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,PI3K/AKT/mTOR pathway ,Pyramidal Cells ,Glutamate receptor ,Organotypic hippocampal slices ,CA3 Region, Hippocampal ,Cell biology ,Glucose ,Neurology ,Biochemistry ,chemistry ,nervous system ,Caspases ,Nerve Degeneration ,NMDA receptor ,NBQX ,Calcium ,Poly(ADP-ribose) Polymerases ,GluA1 - Abstract
An excessive activation of poly(ADP-ribose) polymerases (PARPs) may trigger a form of neuronal death similar to that occurring in neurodegenerative disorders. To investigate this process, we exposed organotypic hippocampal slices to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG, 100μM for 5min), an alkylating agent widely used to activate PARP-1. MNNG induced a pattern of degeneration of the CA1 pyramidal cells morphologically similar to that observed after a brief period of oxygen and glucose deprivation (OGD). MNNG exposure was also associated with a dramatic increase in PARP-activity and a robust decrease in NAD(+) and ATP content. These effects were prevented by PARP-1 but not PARP-2 inhibitors. In our experimental conditions, cell death was not mediated by AIF translocation (parthanatos) or caspase-dependent apoptotic processes. Furthermore, we found that PARP activation was followed by a significant deterioration of neuronal membrane properties. Using electrophysiological recordings we firstly investigated the suggested ability of ADP-ribose to open TRPM2 channels in MNNG-induced cells death, but the results we obtained showed that TRPM2 channels are not involved. We then studied the involvement of glutamate receptor-ion channel complex and we found that NBQX, a selective AMPA receptor antagonist, was able to effectively prevent CA1 neuronal loss while MK801, a NMDA antagonist, was not active. Moreover, we observed that MNNG treatment increased the ratio of GluA1/GluA2 AMPAR subunit expression, which was associated with an inward rectification of the IV relationship of AMPA sEPSCs in the CA1 but not in the CA3 subfield. Accordingly, 1-naphthyl acetyl spermine (NASPM), a selective blocker of Ca(2+)-permeable GluA2-lacking AMPA receptors, reduced MNNG-induced CA1 pyramidal cell death. In conclusion, our results show that activation of the nuclear enzyme PARP-1 may change the expression of membrane proteins and Ca(2+) permeability of AMPA channels, thus affecting the function and survival of CA1 pyramidal cells.
- Published
- 2014
35. Enhanced Neuroprotective Effects of Panax Ginseng G115® and Ginkgo Biloba GK501® Combinations In Vitro Models of Excitotoxicity
- Author
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Domenico E. Pellegrini-Giampietro, Anna Rita Bilia, Elisa Landucci, and Maria Camilla Bergonzi
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Male ,Ginkgo biloba GK501® ,Kainic acid ,cortical cells ,Excitotoxicity ,Panax ,Hippocampal formation ,Pharmacology ,medicine.disease_cause ,Neuroprotection ,Article ,Catalysis ,Inorganic Chemistry ,chemistry.chemical_compound ,Ginseng ,organotypic hippocampal slices ,immune system diseases ,medicine ,Animals ,Propidium iodide ,Rats, Wistar ,Physical and Theoretical Chemistry ,Molecular Biology ,Spectroscopy ,Brain Diseases ,biology ,Plant Extracts ,Chemistry ,Ginkgo biloba ,Organic Chemistry ,Panax ginseng G115®, organotypic hippocampal slices ,General Medicine ,biology.organism_classification ,Panax ginseng G115® ,Rats ,Computer Science Applications ,Disease Models, Animal ,Neuroprotective Agents ,nervous system ,NMDA receptor ,neuroprotection ,Female ,excitotoxicity - Abstract
Neurological-related disorders are seen as an increasingly important aspect of welfare. While conventional medicine is still the mainstay for the treatment of these diseases, it is becoming apparent that patients are also seeking more natural and preventative interventions. Panax ginseng G115®, and Ginkgo biloba GK501®, extracts alone or in combination were used in two in vitro experimental models of primary cultures exposed to excitotoxicity: rat organotypic hippocampal slices exposed to either 5 µ, M kainic acid or 10 µ, M N-Methyl-d-aspartate for 24 hours, and mixed cortical cells exposed to 300 µ, M NMDA for 10 min. Cell death in the Cornu Ammonis areas CA3 or CA1 subregions of slices was quantified by measuring propidium iodide fluorescence, whereas in cortical cells, it was assessed by measuring the amount of lactate dehydrogenase. In slices, treatment with extracts alone or in combination significantly attenuated CA3 and CA1 damage induced by exposure to kainic acid or NMDA, respectively. A similar neuroprotective effect was observed in cortical cells exposed to NMDA. Analysis of cell signaling pathways found that the two extracts induced an increase of the phosphorylation and they reversed the decrease of phosphorylation of ERK1/2 and Akt induced by kainic acid and NMDA in organotypic hippocampal slices. These results suggest that P. ginseng G115®, and G. biloba GK501®, extracts may mediate their effects by activating phosphorylation of ERK1/2 and Akt signaling pathways, protecting against excitotoxicity-induced damage in in vitro models.
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- 2019
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36. Enhanced Neuroprotective Effects of Panax ginseng G115® and Ginkgo biloba GK501® Combinations In Vitro Models of Excitotoxicity.
- Author
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Landucci, Elisa, Pellegrini-Giampietro, Domenico E., Bilia, Anna Rita, and Bergonzi, Maria Camilla
- Subjects
GINKGO ,GINSENG ,HIPPOCAMPUS (Brain) ,KAINIC acid ,CELL analysis ,LACTATE dehydrogenase - Abstract
Neurological-related disorders are seen as an increasingly important aspect of welfare. While conventional medicine is still the mainstay for the treatment of these diseases, it is becoming apparent that patients are also seeking more natural and preventative interventions. Panax ginseng G115
® and Ginkgo biloba GK501® extracts alone or in combination were used in two in vitro experimental models of primary cultures exposed to excitotoxicity: rat organotypic hippocampal slices exposed to either 5 µM kainic acid or 10 µM N-Methyl-d-aspartate for 24 hours, and mixed cortical cells exposed to 300 µM NMDA for 10 min. Cell death in the Cornu Ammonis areas CA3 or CA1 subregions of slices was quantified by measuring propidium iodide fluorescence, whereas in cortical cells, it was assessed by measuring the amount of lactate dehydrogenase. In slices, treatment with extracts alone or in combination significantly attenuated CA3 and CA1 damage induced by exposure to kainic acid or NMDA, respectively. A similar neuroprotective effect was observed in cortical cells exposed to NMDA. Analysis of cell signaling pathways found that the two extracts induced an increase of the phosphorylation and they reversed the decrease of phosphorylation of ERK1/2 and Akt induced by kainic acid and NMDA in organotypic hippocampal slices. These results suggest that P. ginseng G115® and G. biloba GK501® extracts may mediate their effects by activating phosphorylation of ERK1/2 and Akt signaling pathways, protecting against excitotoxicity-induced damage in in vitro models. [ABSTRACT FROM AUTHOR]- Published
- 2019
- Full Text
- View/download PDF
37. N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide (EPPTB) prevents 3-iodothyronamine (T1AM)-induced neuroprotection against kainic acid toxicity.
- Author
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Landucci, Elisa, Gencarelli, Manuela, Mazzantini, Costanza, Laurino, Annunziatina, Pellegrini-Giampietro, Domenico Edoardo, and Raimondi, Laura
- Subjects
- *
KAINIC acid , *WESTERN immunoblotting , *CYCLIC-AMP-dependent protein kinase , *PROTEIN kinase B , *PROPIDIUM iodide , *FORMYLATION - Abstract
Thyroid hormone and thyroid hormone metabolites, including 3-iodothyronamine (T1AM) and 3-iodothyroacetic acid (TA1), activate AKT signaling in hippocampal neurons affording protection from excitotoxic damage. We aim to explore whether the mechanism of T1AM neuroprotection against kainic acid (KA)-induced excitotoxicity included the activation of the trace amine associated receptor isoform 1 (TAAR1), one of T1AM targets. Rat organotypic hippocampal slices were exposed to vehicle (Veh) or to 5 μM kA for 24 h in the absence or presence of 0.1, 1 and 10 μM T1AM or to 0.1, 1 and 10 μM T1AM and 1 μM N-(3-Ethoxy-phenyl)-4-pyrrolidin-1-yl-3-trifluoromethyl-benzamide (EPPTB), the only available TAAR1 antagonist, or to 1 μM T1AM in the absence or in the presence of 10 μM LY294002, an inhibitor of phosphoinositide 3-kinases (PI3Ks). Cell death was evaluated by measuring propidium iodide (PI) levels of fluorescence 24 h after treatment. In parallel, the expression levels of p-AKT and p-PKA were evaluated by Western blot analysis of slice lysates. The activity of mitochondrial monoamine oxidases (MAO) was assayed fluorimetrically. 24 h exposure of slices to T1AM resulted in the activation of AKT and PKA. KA exposure induced cell death in the CA3 region and significantly reduced p-AKT and p-PKA levels. The presence of 1 and 10 μM T1AM significantly protected neurons from death and conserved both kinase levels with the essential role of AKT in neuroprotection. Furthermore, EPPTB prevented T1AM-induced neuroprotection, activation of PKA and AKT. Of note, in the presence of EPPTB T1AM degradation by MAO was reduced. Our results indicate that the neuroprotection offered by T1AM depends, as for TA1, on AKT activation but do not allow to conclusively indicate TAAR1 as the target implicated. Image 1 [ABSTRACT FROM AUTHOR]
- Published
- 2019
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- View/download PDF
38. Oxygen glucose deprivation causes mitochondrial dysfunction in cultivated rat hippocampal slices: protective effects of CsA, its immunosuppressive congener [D-Ser](8)CsA, the novel non-immunosuppressive cyclosporin derivative Cs9, and the NMDA receptor antagonist MK 801.
- Author
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Trumbeckaite S, Gizatullina Z, Arandarcikaite O, Röhnert P, Vielhaber S, Malesevic M, Fischer G, Seppet E, Striggow F, and Gellerich FN
- Subjects
- Animals, Hippocampus drug effects, In Vitro Techniques, Male, Mitochondria drug effects, Mitochondria metabolism, Rats, Rats, Wistar, Cell Respiration, Cyclosporins metabolism, Dizocilpine Maleate metabolism, Glucose metabolism, Hippocampus physiology, Neuroprotective Agents metabolism, Oxygen metabolism
- Abstract
We have introduced a sensitive method for studying oxygen/glucose deprivation (OGD)-induced mitochondrial alterations in homogenates of organotypic hippocampal slice cultures (slices) by high-resolution respirometry. Using this approach, we tested the neuroprotective potential of the novel non-immunosuppressive cyclosporin (CsA) derivative Cs9 in comparison with CsA, the immunosuppressive CsA analog [D-Ser](8)CsA, and MK 801, a N-methyl-d-aspartate (NMDA) receptor antagonist. OGD/reperfusion reduced the glutamate/malate dependent (and protein-related) state 3 respiration to 30% of its value under control conditions. All of the above drugs reversed this effect, with an increase to >88% of the value for control slices not exposed to OGD. We conclude that Cs9, [D-Ser](8)CsA, and MK 801, despite their different modes of action, protect mitochondria from OGD-induced damage., (Copyright © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Published by Elsevier B.V. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
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