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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..

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.

In this study, we investigated the cross-talk between mGlu1 and CB1 receptors in modulating GABA hippocampal output in whole-cell voltage clamp recordings in rat hippocampal acute slices, in organotypic hippocampal slices exposed to oxygen and glucose deprivation (OGD) and in gerbils subjected to global ischemia. CB1 receptor expression was studied using immunohistochemistry and the CA1 contents of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by LC-MS/MS. Our results show that mGlu1 receptor antagonists enhance sIPSCs in CA1 pyramidal cells and the basal and ischemic hippocampal release of GABA in vivo in a manner that is mediated by CB1 receptor activation. In hippocampal slices exposed to OGD and in ischemic gerbils, mGlu1 receptor antagonists protected CA1 pyramidal cells against post-ischemic injury and this effect was reduced by CB1 receptor activation. OGD induced a transient increase in the hippocampal content of AEA and this effect is prevented by mGlu1 receptor antagonist. Finally, OGD induced a late disruption of CB1 receptors in the CA1 region and the effect was prevented when CA1 pyramidal cells were protected by mGlu1 antagonists. Altogether, these results suggest a cooperative interaction between mGlu1 receptors and the endocannabinoid system in the mechanisms that lead to post-ischemic neuronal death.

(1) Background: Over the past 10 years, a number of scientific studies have demonstrated the therapeutic potential of cannabinoid compounds present in the Cannabis Sativa and Indica plants. However, their role in mechanisms leading to neurodegeneration following cerebral ischemia is yet unclear. (2) Methods: We investigated the effects of Cannabis extracts (Bedrocan, FM2) or selected cannabinoids (Δ 9 -tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabigerol) in rat organotypic hippocampal slices exposed to oxygen-glucose deprivation (OGD), an in vitro model of forebrain global ischemia. Cell death in the CA1 subregion of slices was quantified by propidium iodide fluorescence, and morphological analysis and tissue organization were examined by immunohistochemistry and confocal microscopy. (3) Results: Incubation with the Bedrocan extract or THC exacerbated, whereas incubation with the FM2 extract or cannabidiol attenuated CA1 injury induced by OGD. Δ 9 -THC toxicity was prevented by CB1 receptor antagonists, the neuroprotective effect of cannabidiol was blocked by TRPV2, 5-HT1A, and PPARγ antagonists. Confocal microscopy confirmed that CBD, but not THC, had a significant protective effect toward neuronal damage and tissue disorganization caused by OGD in organotypic hippocampal slices. (4) Conclusions: Our results suggest that cannabinoids play different roles in the mechanisms of post-ischemic neuronal death. In particular, appropriate concentrations of CBD or CBD/THC ratios may represent a valid therapeutic intervention in the treatment of post-ischemic neuronal death.

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.

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.)

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.

Background: The physiopathology of bilirubin-induced neurological disorders is not completely understood., Objectives: The aim of our study was to assess the effect on bilirubin neurotoxicity of the maturity or immaturity of exposed cells, the influence of different unconjugated bilirubin (UCB) and human serum albumin (HSA) concentrations, and time of UCB exposure., Methods: Organotypic hippocampal slices were exposed for 48 h to different UCB and HSA concentrations after 14 (mature) or 7 (immature) days of in vitro culture. Immature slices were also exposed to UCB and HSA for 72 h. The different effects of exposure time to UCB on neurons and astrocytes were evaluated., Results: We found that 48 h of UCB exposure was neurotoxic for mature rat organotypic hippocampal slices while 72 h of exposure was neurotoxic for immature slices. Forty-eight-hour UCB exposure was toxic for astrocytes but not for neurons, while 72-h exposure was toxic for both astrocytes and neurons. HSA prevented UCB toxicity when the UCB:HSA molar ratio was ≤1 in both mature and immature slices., Conclusions: We confirmed UCB neurotoxicity in mature and immature rat hippocampal slices, although immature ones were more resistant. HSA was effective in preventing UCB neurotoxicity in both mature and immature rat hippocampal slices., (© 2019 S. Karger AG, Basel.)

Background: 3-Iodothyroacetic acid (TA1) is among the thyroid hormone (T3) metabolites that can acutely modify behavior in mice. This study aimed to investigate whether TA1 is also able to reduce neuron hyper-excitability and protect from excitotoxic damage., Methods: CD1 male mice were treated intraperitoneally with saline solution or TA1 (4, 7, 11, or 33 μg/kg) before receiving 90 mg/kg pentylenetrazole subcutaneously. The following parameters were measured: latency to first seizure onset, number of mice experiencing seizures, hippocampal levels of c-fos, and PI3K/AKT activation levels. Organotypic hippocampal slices were exposed to vehicle or to 5 μM kainic acid (KA) in the absence or presence of 0.01-10 μM TA1. In another set of experiments, slices were exposed to vehicle or 5 μM KA in the absence or presence of 10 μM T3, 3,5,3'-triiodothyroacetic acid (TRIAC), T1AM, thyronamine (T0AM), or thyroacetic acid (TA0). Neuronal cell death was measured fluorimetically. The ability of TA1 and T3, TRIAC, T1AM, T0A, and TA0 to activate the PI3K/AKT cascade was evaluated by Western blot. The effect of TA1 on KA-induced currents in CA3 neurons was evaluated by patch clamp recordings on acute hippocampal slices., Results: TA1 (7 and 11 μg/kg) significantly reduced the number of mice showing convulsions and increased their latency of onset, restored pentylenetrazole-induced reduction of hippocampal c-fos levels, activated the PI3K/AKT, and reduced GSK-3β activity. In rat organotypic hippocampal slices, TA1 reduced KA-induced cell death by activating the PI3K/AKT cascade and increasing GSK-3β phosphorylation levels. Protection against KA toxicity was also exerted by T3 and other T3 metabolites studied. TA1 did not interact at KA receptors. Both the anticonvulsant and neuroprotective effects of TA1 were abolished by pretreating mice or organotypic hippocampal slices with pyrilamine, an histamine type 1 receptor antagonist (10 mg/kg or 1 μM, respectively)., Conclusions: TA1 exerts anticonvulsant activity and is neuroprotective in vivo and in vitro. These findings extend the current knowledge on the pharmacological profile of TA1 and indicate possible novel clinical use for this T3 metabolite.

We previously demonstrated that the non-steroidal anti-inflammatory agent meloxicam has neuroprotective effects in an oxygen and glucose deprivation model (OGD) of rat organotypic hippocampal slice cultures. We wondered if GABAergic transmission changed the neuroprotective effects of meloxicam and if meloxicam was able to modulate endoplasmic reticulum stress (ER stress) in this model. Mortality was measured using propidium iodide. Western blot assays were performed to measure levels of cleaved and non-cleaved caspase-3 to quantify apoptosis, while levels of GRP78, GRP94 and phosphorylated eIF2α were used to detect unfolded protein response (UPR). Transcript levels of GRP78, GRP94 and GABAergic receptor α, β, and γ subunits were measured by real-time quantitative polymerase chain reaction (qPCR). In the present study, we show that the presence of meloxicam in a 30 min OGD assay, followed by 24 h of normoxic conditions, presented an antiapoptotic effect. The simultaneous presence of the GABA A receptor antagonist, bicuculline, in combination with meloxicam blocked the neuroprotective effect provided by the latter. However, in light of its effects on caspase 3 and PARP, bicuculline did not seem to promote the apoptotic pathway. Our results also showed that meloxicam modified the unfolded protein response (UPR), as well as the transcriptional response of different genes, including the GABA A receptor, alpha1, beta3 and gamma2 subunits. We concluded that meloxicam has a neuroprotective anti-apoptotic action, is able to enhance the UPR independently of the systemic anti-inflammatory response and its neuroprotective effect can be inhibited by blocking GABA A receptors., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Purpose: To investigate the feasibility of a study based on treatment with topiramate (TPM) added to moderate hypothermia in newborns with hypoxic ischemic encephalopathy (HIE)., Materials and Methods: Multicenter randomized controlled trial. Term newborns with precocious metabolic, clinical and electroencephalographic (EEG) signs of HIE were selected according to their amplified integrated EEG pattern and randomized to receive either TPM (10 mg/kg once a day for the first three days of life) plus moderate hypothermia or hypothermia alone. Safety was assessed by monitoring cardiorespiratory parameters and blood samples collected to check renal, liver, metabolic balance and TPM pharmacokinetics. Efficacy was evaluated by the combined frequency of mortality and severe neurological disability as primary outcome. Incidence of magnetic resonance injury, epilepsy, blindness, hearing loss, neurodevelopment at 18-24 months of life was assessed as secondary outcomes., Results: Forty-four asphyxiated newborns were enrolled in the study. Twenty one newborns (10 with moderate and 11 with severe HIE) were allocated to hypothermia plus TPM and 23 (12 moderate and 11 severe HIE) to hypothermia. No statistically or clinically significant differences were observed for safety, primary or secondary outcomes. However, a reduction in the prevalence of epilepsy was observed in newborns co-treated with TPM., Conclusions: Results of this pilot trial suggest that administration of TPM in newborns with HIE is safe but does not reduce the combined frequency of mortality and severe neurological disability. The role of TPM co-treatment in preventing subsequent epilepsy deserves further studies.

Hypoxic-ischemic encephalopathy (HIE) is a major cause of perinatal mortality and subsequent severe neurological sequelae. Mild hypothermia is a standard therapy for HIE, but is used only in selected Reference Centers and in neonates >1800 g. Since neuronal death following HIE occurs by a cascade of events triggered by activation of glutamate receptors, we used in vitro and in vivo models of HIE to examine whether the AMPA/kainate receptor antagonist topiramate and the NMDA receptor antagonist memantine could exert neuroprotective effects, alone or in combination with hypothermia. For the in vitro experiments, rat organotypic hippocampal slices were exposed to a 30 min duration of oxygen-glucose deprivation (OGD): treatment with topiramate (1 μM) and memantine (10-30 μM) or hypothermia (35 °C or 32 °C) significantly attenuated CA1 damage after 24 h. The combination of hypothermia with topiramate and memantine enhanced their protective effect. For the in vivo experiments, we used 7 day-old rat pups subjected to permanent left common carotid artery occlusion followed by 120 min of hypoxia. Administration of topiramate or memantine (i.p., 20 mg/kg) immediately and 2 h after hypoxia or exposure to hypothermia (32 °C for 4 h beginning 1 h after hypoxia) significantly reduced the extent of the resulting infarct. The combination of topiramate or memantine with hypothermia elicited a reduction of the infarct that was greater than that produced by drugs or hypothermia alone. Notably, memantine displayed a higher degree of neuroprotection as compared to topiramate both in vitro and in vivo and, when used alone at 20 mg/kg in vivo, produced a greater reduction in brain damage than observed using topiramate in combination with hypothermia. These results suggest that memantine may be more advantageous than topiramate as a therapeutic agent in neonates with HIE treated with hypothermia., (Copyright © 2018 Elsevier B.V. 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.)

Ischemic tolerance is a phenomenon in which exposure to a mild preconditioning stress results in resistance to a subsequent lethal ischemic insult. Here we investigated the role of poly(ADP-ribose) polymerase (PARP) in the development of ischemic tolerance by using organotypic rat hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD), which leads to selective injury of the CA1 subregion 24 h later. We developed models of pharmacological preconditioning by exposing slices to subtoxic concentrations of either N-methyl-D-aspartate (NMDA) or (S)-3,5-dihydroxyphenylglycine (DHPG) and then, 24 h later, to 30 min OGD. Under these conditions, we observed a significant reduction in OGD-induced CA1 damage. Exposure of slices to the PARP-1 and -2 inhibitors TIQ-A, PJ-34 and UPF 1069 during preconditioning prevented the development of OGD tolerance in a concentration-dependent manner. NMDA and DHPG preconditioning increased the activity of PARP, as detected by immunoblots using antibodies against the poly(ADP-ribose) polymer product, but was not associated with consumption of cellular NAD(+) or ATP. Neuroprotection induced by preconditioning was also prevented by the caspase inhibitor Z-VAD-FMK. The modest but significant increase in caspase-3/7 induced by preconditioning, however, was not associated with PARP-1 cleavage, as occurred with staurosporine. Finally, TIQ-A prevented the activation of ERK1/2 and Akt induced by NMDA preconditioning, suggesting that the protective mechanism evoked by PARP requires activation of these prosurvival mediators. Our results suggest that preconditioning with appropriate pharmacological stimuli may promote neuroprotective mechanisms triggered by the sublethal activation of two otherwise deleterious executioners such as PARP and caspase-3/7., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Organotypic slices cultured for weeks in vitro represent an extremely valuable strategy for the investigation of the long-term properties of neuronal circuits under physiological and pathological conditions. Here, we describe how to prepare rat organotypic hippocampal slice cultures and how to expose them for appropriate periods of time to excitotoxic agents or to oxygen and glucose deprivation conditions, in order to mimic the pattern of pyramidal cell damage which is observed in vivo and in other in vitro models. This preparation is very useful not only to study synaptic plasticity or the pathways and mechanisms of neurodegeneration but also to evaluate the effects of neuroprotective agents.

The design and synthesis of new N1-substituted 3-carboxy- and 3-phosphonopyrazoline and pyrazole amino acids that target the glutamate binding site of NMDA receptors are described. An analysis of the stereochemical requirements for high-affinity interaction with these receptors was performed. We identified two highly potent and selective competitive NMDA receptor antagonists, (5S,alphaR)-1 and (5S,alphaR)-4, which exhibit good in vitro neuroprotective activity and in vivo anticonvulsant activity by i.p. administration, suggesting that these molecules may have potential use as therapeutic agents.

(+/-)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo [3,4 -d]-isoxazole-4-carboxylic acid (HIP-A) and (+/-)-3-hydroxy-4,5,6, 6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B) are selective inhibitors of excitatory amino acid transporters (EAATs), as potent as DL-threo-beta-benzyloxyaspartic acid (TBOA). We report here that the active isomers are (-)-HIP-A and (+)-HIP-B, being approximately 150- and 10-fold more potent than the corresponding enantiomers as inhibitors of [3H]aspartate uptake in rat brain synaptosomes and hEAAT1-3-expressing cells. Comparable IC(50) values were found on the three hEAAT subtypes. (-)-HIP-A maintained the remarkable property, previously reported with the racemates, of inhibiting synaptosomal glutamate-induced [3H]D-aspartate release (reverse transport) at concentrations significantly lower than those inhibiting [3H]L-glutamate uptake. New data suggest that the noncompetitive-like interaction described previously is probably the consequence of an insurmountable, long-lasting impairment of EAAT's function. Some minutes of preincubation are required to induce this impairment, the duration of preincubation having more effect on inhibition of glutamate-induced release than of glutamate uptake. In organotypic rat hippocampal slices and mixed mouse brain cortical cultures, TBOA, but not (-)-HIP-A, had toxic effects. Under ischemic conditions, a neuroprotective effect was found with 10 to 30 microM (-)-HIP-A, but not with 10 to 30 microM TBOA or 100 microM (-)-HIP-A. The effect of (-)-HIP-A suggests that, under ischemia, EAATs mediate both release (reverse transport) and uptake of glutamate. The neuroprotection with the lower (-)-HIP-A concentrations may indicate a selective inhibition of the reverse transport confirming the data obtained in synaptosomes. The selective interference with glutamate-induced glutamate release might offer a new strategy for neuroprotective action.