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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., (Copyright © 2020 IBRO. Published by Elsevier Ltd. All rights reserved.)

Previous studies have demonstrated that antagonists of mGluR1, but not mGluR5, are neuroprotective in models of cerebral ischemia. To investigate the individual roles of mGlu1 and mGlu5 receptors in in vitro model of cerebral ischemia we used low doses of the non-selective group I agonist DHPG and mGlu1 and mGlu5 selective positive allosteric modulators (PAMs). In hippocampal slices subjected to 30 min oxygen-glucose deprivation (OGD), DHPG (1 μM) and the mGluR5 PAM (VU0092273) significantly reduced OGD-induced CA1 injury monitored by propidium iodide staining of the slices and quantitative analysis of CA1 neurons. In contrast, the mGluR1 PAM (VU0483605) showed no neuroprotection. These protective effects of DHPG and VU0092273 were prevented by inhibition of PI3K/Akt pathway by LY294002. The mGluR5 PAM (VU0092273) also prevented GluA2 down-regulation triggered by ischemic injury, via PI3K/Akt pathway, revealing a further contribution to its neuroprotective effects by reducing the excitotoxic effects of increased Ca 2+ influx through GluA2-lacking AMPA receptors. Furthermore, immunohistochemical assays confirmed the neuroprotective effect of VU0092273 and revealed activation of glia, indicating the involvement reactive astrogliosis in the mechanisms of neuroprotection. Our data suggest that selective activation/potentiation of mGluR5 signalling represents a promising strategy for the development of new interventions to reduce or prevent ischemia-induced neuronal death., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bv8/prokineticin 2 (PK2) is a member of a bioactive family of peptides that regulate multiple functions in the CNS including hyperalgesia, neurogenesis, neuronal survival and inflammation. Recent studies have associated PK2 and prokineticin receptors (PKR) with human diseases, but because their role in neuropathology is still debated we examined whether prokineticins exert a protective or deleterious role in models of cerebral ischemia and ischemic tolerance in vitro. In order to mimic cerebral ischemia, we exposed primary murine cortical cell cultures or rat organotypic hippocampal slices to appropriate periods of oxygen-glucose deprivation (OGD), which leads to neuronal damage 24 h later. Ischemic tolerance was induced by exposing hippocampal slices to a preconditioning subtoxic pharmacological stimulus (3 μM NMDA for 1 h) 24 h before the exposure to OGD. Bv8 (10-100 nM) attenuated OGD injury in cortical cultures and hippocampal slices, and the effect was prevented by the PKR antagonist PC7. The development of OGD tolerance was associated with an increase in the expression of PK2, PKR1 and PKR2 mRNA and proteins and was prevented by addition of the antagonist PC7 into the medium during preconditioning. Both Bv8 at protective concentrations and the NMDA preconditioning stimulus promoted the phosphorylation of ERK1/2 and Akt. These findings indicate that the prokineticin system can be up-regulated by a defensive preconditioning subtoxic NMDA stimulus and that PK2 may act as an endogenous neuroprotective factor through the activation of the ERK1/2 and Akt transduction pathways., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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 © 2010 Elsevier Ltd. All rights reserved.)

An emerging body of evidence supports a key role for the endocannabinoid system in numerous physiological and pathological mechanisms of the central nervous system. In the recent past, many experimental studies have examined the putative protective or toxic effects of drugs interacting with cannabinoid receptors or have measured the brain levels of endocannabinoids in in vitro and in vivo models of cerebral ischemia. The results of these studies have been rather conflicting in supporting either a beneficial or a detrimental role for the endocannabinoid system in post-ischemic neuronal death, in that cannabinoid receptor agonists and antagonists have both been demonstrated to produce either protective or toxic responses in ischemia, depending on a number of factors. Among these, the dose of the administered drug and the specific endocannabinoid that accumulates in each particular model appear to be of particular importance. Other mechanisms that have been put forward to explain these discrepant results are the effects of cannabinoid receptor activation on the modulation of excitatory and inhibitory transmission, the vasodilatory and hypothermic effects of cannabinoids, and their activation of cytoprotective signaling pathways. Alternative mechanisms that appear to be independent from cannabinoid receptor activation have also been suggested. Endocannabinoids probably participate in the mechanisms that are triggered by the initial ischemic stimulus and lead to delayed neuronal death. However, further information is needed before pharmacological modulation of the endocannabinoid system may prove useful for therapeutic intervention in stroke and related ischemic syndromes.

Experimental evidence indicates that metabotropic glutamate (mGlu) receptors of the mGlu1 and mGlu5 subtypes play a differential role in models of cerebral ischemia and that only mGlu1 receptors are implicated in the pathways leading to postischemic neuronal injury. The localization of mGlu1 receptors in GABA-containing interneurons rather than in hippocampal CA1 pyramidal cells that are vulnerable to ischemia has prompted experimental studies that have demonstrated mGlu1 receptor antagonist agents attenuate postischemic injury by enhancing GABA-mediated neurotransmission, thus providing a new viewpoint on the neuroprotective mechanism of these pharmacological agents. In view of the recent discovery of a functional interaction between group I mGlu receptors and the cannabinoid system in the modulation of synaptic transmission, we propose a novel mechanism that predicts that the neuroprotective effects of mGlu1 receptor antagonists on CA1 pyramidal cells are mediated by a mechanism that overcomes the "synaptic circuit break" operated by endocannabinoids on GABAergic transmission.

Ischemic postconditioning is defined as a repetitive series of brief interruptions of reperfusion applied immediately after ischemia. In this study, postconditioning was investigated by first exposing rat organotypic hippocampal slices to 30min oxygen-glucose deprivation (OGD), which promotes selective CA1 pyramidal cell death, and 5min later to either a brief period (3min) of OGD or to a low dose (10microM) of 3,5-dihydroxyphenylglycine (DHPG) for 30min. Both protocols attenuated CA1 neuronal injury, as revealed 24h later by measuring the intensity of propidium iodide fluorescence in this region. The beneficial effects were observed when DHPG postconditioning was applied up to 15min after OGD, but not at later time points, and was not additive with the neuroprotective effects of a preconditioning DHPG treatment. The attenuation of the OGD-induced CA1 injury evoked by postconditioning was prevented when mGlu1 and mGlu5 receptor antagonists and inhibitors of phosphatidylinositol 3-kinase and Akt activity were present in the incubation medium during the 5min recovery period after OGD and the 30min exposure to DHPG. The PI3K inhibitor was also able to prevent the reduction of NMDA toxicity induced by the DHPG treatment. Finally, DHPG increased the phosphorylation of Akt in a transient and mGlu1/mGlu5-dependent manner. Our results show that activation of the mGlu1/mGlu5-PI3K-Akt signaling pathway plays a crucial role in the mechanisms of postconditioning evoked by DHPG and point to this strategy as a possible novel therapeutic tool for stroke and cerebral ischemia.

Background: Propofol (2,6-diisopropylphenol) has been shown to attenuate neuronal injury in a number of experimental conditions, but studies in models of cerebral ischemia have yielded conflicting results. Moreover, the mechanisms involved in its neuroprotective effects are yet unclear., Methods: The authors evaluated the neuroprotective effects of propofol in rat organotypic hippocampal slices exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. To investigate its possible mechanism of action, the authors then examined whether propofol could reduce Ca2+-induced rat brain mitochondrial swelling, an index of mitochondrial membrane permeability, as well as the mitochondrial swelling evoked by oxygen-glucose deprivation in CA1 pyramidal cells by transmission electron microscopy. Finally, they evaluated whether propofol could attenuate the infarct size and improve the neurobehavioral outcome in rats subjected to permanent middle cerebral artery occlusion in vivo., Results: When present in the incubation medium during oxygen-glucose deprivation and the subsequent 24 h recovery period, propofol (10-100 microM) attenuated CA1 injury in hippocampal slices in vitro. Ca2+-induced brain mitochondrial swelling was prevented by 30-100 microM propofol, and so were the ultrastructural mitochondrial changes in CA1 pyramidal cells exposed to oxygen-glucose deprivation. Twenty-four hours after permanent middle cerebral artery occlusion, propofol (100 mg/kg, intraperitoneal) reduced the infarct size by approximately 30% when administered immediately after and up to 30 min after the occlusion. Finally, propofol administered within 30 min after middle cerebral artery occlusion was unable to affect the global neurobehavioral score but significantly preserved spontaneous activity in ischemic rats., Conclusions: These results show that propofol, at clinically relevant concentrations, is neuroprotective in models of cerebral ischemia in vitro and in vivo and that it may act by preventing the increase in neuronal mitochondrial swelling.

Metabotropic glutamate receptors of the mGlu(1) and mGlu(5) subtypes exhibit a high degree of sequence homology and are both coupled to phospholipase C and intracellular Ca(2+) mobilization. However, functional differences have been detected for these receptor subtypes when they are coexpressed in the same neuronal populations. Experimental evidence indicates that mGlu(1) and mGlu(5) receptors play a differential role in models of cerebral ischemia and that only mGlu(1) receptors are implicated in the pathways leading to post-ischemic neuronal injury. The localization of mGlu(1) receptors in GABA-containing interneurons rather than in hippocampal CA1 pyramidal cells that are vulnerable to ischemia has prompted studies that have provided a new viewpoint on the neuroprotective mechanism of mGlu(1) receptor antagonists. The hypothesis predicts that these pharmacological agents attenuate post-ischemic injury by enhancing GABA-mediated neurotransmission.

Poly(ADP-ribose) polymerases (PARPs) are a group of protein-modifying and nucleotide-polymerizing enzymes able to catalyze the transfer of multiple ADP-ribose units from NAD to substrate proteins. In the human genome, 16 different genes encoding for members of this emerging family of enzymes have been identified. Known family members are PARP-1, PARP-2, PARP-3, vPARP, tankyrase 1 and tankyrase 2, each of them with a possible specific role in cell biology. The most studied member of the family is PARP-1, which is abundantly present in the nucleus and is involved in the maintenance of genomic stability. In pathological conditions, highly reactive radical species may cause DNA damage and PARP-1 hyperactivation. This may lead to necrotic cell death through massive NAD consumption. We show that following middle cerebral artery occlusion, rats treated with PARP inhibitors displayed reduced brain infarct volumes. Similarly, PARP inhibitors reduced neuronal death induced by oxygen-glucose deprivation (OGD) or excitotoxins in primary cultures of murine cortical cells. On the contrary, PARP inhibitors did not attenuate the OGD-induced selective loss of CA1 pyramidal cells in rat organotypic hippocampal slices. In addition, they were not neuroprotective against transient bilateral carotid occlusion in gerbils. We observed that post-ischemic brain damage was predominally necrotic in cultured cortical cells, whereas a caspase-dependent apoptotic process was responsible for the CA1 pyramidal cell loss in hippocampal slices. Hence, it appears reasonable to propose PARP inhibitors as useful therapeutic agents in pathological brain conditions were necrosis predominates.

Kynurenine 3-mono-oxygenase (KMO) inhibitors facilitate kynurenic acid (KYNA) neosynthesis and reduce the formation of 3OH-kynurenine (3-HK) and quinolinic acid (QUIN). They also attenuate post-ischemic brain damage and decrease glutamate (Glu) content in brain extracellular spaces. To investigate KMO mechanism(s) of neuroprotection, we performed experiments in gerbils subjected to bilateral carotid occlusion and in organotypic rat hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD). In gerbils, direct application of KYNA (100 nM, through reverse microdialysis in the hippocampus) completely prevented the increase in Glu output induced by transient (5 min) occlusion of the carotids. In rat hippocampal slices exposed for 30 min to OGD, KMO inhibitors (m-nitrobenzoyl)-alanine (mNBA, 30-100 microM) or 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (Ro 61-8048, 1-10 microM) reduced post-ischemic neuronal death and increased KYNA concentrations in the incubation medium. KYNA may antagonize glycineb or alpha7 nicotinic acetylcholine receptors but the concentrations in the incubation medium never reached values that could efficiently antagonize receptor function. On the contrary, 3-HK (1-10 microM) added to slices exposed to OGD in the presence of KMO inhibitors completely prevented the neuroprotective effects of the inhibitors. Our findings suggest that KMO inhibitors reduce OGD-induced pyramidal cell death by decreasing 3-HK (and possibly QUIN) synthesis.

Kynurenine 3-mono-oxygenase (KMO) inhibitors reduce 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN) neosynthesis and facilitate kynurenine metabolism towards kynurenic acid (KYNA) formation. They also reduce tissue damage in models of focal or transient global cerebral ischemia in vivo. We used organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD) to investigate KMO mechanism(s) of neuroprotective activity. Exposure of the slices to 30 min of OGD caused CA1 pyramidal cell death and significantly decreased the amount of KYNA released in the incubation medium. The KMO inhibitors (m-nitrobenzoyl)-alanine (30-100 micro m) or 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (1-10 micro m) reduced post-ischemic neuronal death and increased KYNA concentrations in slice incubation media. The maximal concentration of KYNA detected in the incubation media of slices treated with KMO inhibitors was approximately 50 nm and was too low to efficiently interact with alpha7 nicotinic acetylcholine receptors or with the glycineb site of N-methyl-d-aspartate (NMDA) receptors. On the other hand, the addition of either 3-HK or QUIN (1-10 micro m) to OGD-exposed hippocampal slices prevented the neuroprotective activity of KMO inhibitors. Our results suggest that KMO inhibitors reduce the neuronal death found in the CA1 region of organotypic hippocampal slices exposed to 30 min of OGD by decreasing the local synthesis of 3-HK and QUIN.

In order to investigate the involvement of mGlu1 and mGlu5 metabotropic glutamate receptors in the development of postischemic neuronal death, we examined the effects of selective agonists and antagonists in models of cerebral ischemia in vitro and in vivo. In murine cortical cell cultures and rat organotypic hippocampal slices exposed to oxygen and glucose deprivation (OGD), the mGlu1 antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA; 300 microM), (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG; 300 microM), 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt; 10-30 microM) and (+)-2-methyl-4-carboxyphenylglycine (LY367385; 30-100 microM) reduced neuronal loss when added to the medium during OGD and the subsequent 24-h recovery period. On the contrary, the potent and selective mGlu5 antagonist methyl-6-(phenylethynyl)-pyridine (MPEP; 0.1-1 microM) did not exhibit neuroprotection in any of these in vitro models. Incubation with the nonselective mGlu1 and mGlu5 agonist 3,5-dihydroxyphenylglycine (3,5-DHPG; 300 microM) but not with the mGlu5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG; 1 mM) enhanced the severity of OGD-induced neuronal damage. In gerbils subjected to global ischemia, intracerebroventricular administration of AIDA (100 nmol two times) or CBPG (300 nmol, two times) afforded consistent protection against CA1 pyramidal cell death, whereas MPEP (10 pmol i.c.v two times and 10 mg/kg i.p two times) failed to reduce postischemic hippocampal damage. Our results suggest that activation of mGlu1 but not mGlu5 receptor contributes to postischemic neuronal injury.

Selective antagonists of mGlu1 metabotropic glutamate receptors attenuate neuronal death in models of cerebral ischemia. Because GABAergic mechanisms have recently been proposed to contribute to these neuroprotective effects, we examined the effects of selective mGlu1 antagonists characterized in our laboratory on GABAergic transmission in three different models of neuropathology. In rat organotypic hippocampal slices exposed to oxygen-glucose deprivation, the mGlu1 antagonists AIDA, CBPG and 3-MATIDA reduced CA1 pyramidal cell loss when added to the medium during the insult and the subsequent recovery period. This effect was mimicked by the GABA(A) and GABA(B) agonists muscimol and baclofen and partially prevented by the antagonists bicuculline and CGP 55845. In gerbils subjected to global ischemia, protection of CA1 pyramidal cells by transdialytic perfusion of AIDA and CBPG was associated with a significant increase in the basal and ischemic output of GABA and minor changes in the output of glutamate. In a mouse cortical wedge model, both muscimol and 3-MATIDA reduced the frequency of spontaneous bursts induced by 4-aminopyridine and this reduction was prevented by co-perfusion with bicuculline. Taken together, our results suggest that the release of GABA, and the subsequent activation of GABA receptors, may contribute to the attenuation of post-ischemic neuronal damage and epileptiform activity induced by mGlu1 receptor antagonists.

We examined the pharmacological properties of 3-methyl-aminothiophene dicarboxylic acid (3-MATIDA) by measuring second messenger responses in baby hamster kidney cells stably transfected with mGlu1a, mGlu2, mGlu4a or mGlu5a receptors and ionotropic glutamate receptor agonist-induced depolarizations in mouse cortical wedges. 3-MATIDA was a potent (IC(50)=6.3 microM, 95% confidence limits 3-15) and relatively selective mGlu1 receptor antagonist. When tested on mGlu2, mGlu4 or mGlu5 receptors its IC(50) was >300 microM. When tested in cortical wedges, however, 3-MATIDA was also able to antagonize AMPA or NMDA responses with an IC(50) of 250 microM. When present in the incubation medium of cultured murine cortical cells, 3-MATIDA (1-100 microM) significantly reduced the death of neurons induced by 60 min of oxygen and glucose deprivation (OGD), even when added up to 60 min after OGD. A similar neuroprotective activity was observed when 3-MATIDA was present at 10-100 microM in the medium of rat organotypic hippocampal slice cultures exposed to 30 min OGD. Systemic administration of 3-MATIDA (3-10 mg/kg, immediately and 1 h after the onset of ischemia) reduced the volume of brain infarcts following permanent middle cerebral artery occlusion in rats. Our results show that 3-MATIDA is a potent and possibly selective mGlu 1 receptor antagonist that may be considered as a novel prototype neuroprotective agent.