TrkB‐mediated activation of the phosphatidylinositol‐3‐kinase/Akt cascade reduces the damage inflicted by oxygen–glucose deprivation in area CA3 of the rat hippocampus

The selective vulnerability of hippocampal area CA1 to ischemia‐induced injury is a well‐known phenomenon. However, the cellular mechanisms that confer resistance to area CA3 against ischemic damage remain elusive. Here, we show that oxygen–glucose deprivation–reperfusion (OGD‐RP), an in vitro model that mimic the pathological conditions of the ischemic stroke, increases the phosphorylation level of tropomyosin receptor kinase B (TrkB) in area CA3. Slices preincubated with brain‐derived neurotrophic factor (BDNF) or 7,8‐dihydroxyflavone (7,8‐DHF) exhibited reduced depression of the electrical activity triggered by OGD‐RP. Consistently, blockade of TrkB suppressed the resistance of area CA3 to OGD‐RP. The protective effect of TrkB activation was limited to area CA3, as OGD‐RP caused permanent suppression of CA1 responses. At the cellular level, TrkB activation leads to phosphorylation of the accessory proteins SHC and Gab as well as the serine/threonine kinase Akt, members of the phosphoinositide 3‐kinase/Akt (PI‐3‐K/Akt) pathway, a cascade involved in cell survival. Hence, acute slices pretreated with the Akt antagonist MK2206 in combination with BDNF lost the capability to resist the damage inflicted with OGD‐RP. Consistently, with these results, CA3 pyramidal cells exhibited reduced propidium iodide uptake and caspase‐3 activity in slices pretreated with BDNF and exposed to OGD‐RP. We propose that PI‐3‐K/Akt downstream activation mediated by TrkB represents an endogenous mechanism responsible for the resistance of area CA3 to ischemic damage.     

Prostaglandin E2 type 1 receptors contribute to neuronal apoptosis after transient forebrain ischemia

Cyclooxygenase-2-derived prostaglandin E₂ (PGE₂) contributes to excitotoxic and ischemic neuronal cell death by engaging neuronal PGE₂ type 1 receptors (EP1R). Our previous studies have shown that EP1R signaling resulted in disturbances of intracellular Ca²⁺ homeostasis and suppression of the pro-survival protein kinase AKT. The aim of this study was to investigate whether these pathophysiological mechanism have a role in the neuronal cell death after transient forebrain ischemia. Mice were subjected to ischemia/reperfusion by bilateral common carotid artery occlusion. Hippocampal cornu ammonis area 1 (CA1) neuronal cell death was determined 5 days after reperfusion. Animals treated with the EP1R antagonist SC51089 or EP1R-deficient mice (EP1^−/−) showed significantly less neuronal injury as compared to vehicle-treated wild-type controls. Benefits of EP1R blockage were still evident 14 days after injury. Better neuronal survival was correlated with reduced neuronal caspase-3 activity and decreased nuclear translocation of the apoptosis-inducing factor . Neuroprotection could be reverted by intracerebroventricular administration of the phosphoinositide 3-kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and was not further increased by the calcineurin inhibitor FK506. These data implicate EP1R in postischemic neuronal apoptosis possibly by facilitating AKT inhibition.     

Neuroprotective effects of the anti-inflammatory compound triflusal on ischemia-like neurodegeneration in mouse hippocampal slice cultures occur independent of microglia

Microglial cells, known to play key roles in neuroinflammation, can be immunotoxically eliminated from hippocampal slice cultures by treatment with saporin coupled to the microglial receptor Mac1. Considering microglial cells as a target for anti-inflammatory treatment we studied the effects of microglial depletion on anti-inflammatory treatment of mouse hippocampal slice cultures subjected to ischemia-like neurodegeneration, induced by oxygen-glucose deprivation (OGD).Hippocampal slice cultures, derived from 7-day-old mice and grown for 2 weeks, were divided into 8 groups: (1) control cultures; (2) sham-OGD cultures; (3) OGD cultures; (4) OGD cultures treated with triflusal during OGD; (5) microglia-depleted control cultures; (6) microglia-depleted sham-OGD cultures; (7) microglia-depleted OGD cultures; and (8) microglia-depleted OGD cultures treated with triflusal during OGD. The resulting neurodegeneration was quantified by densitometric measurements of cellular uptake of propidium iodide (PI), with focus on the hippocampal CA1 subfield.Subjection of regular cultures to OGD for 30 min induced a significant increase in PI uptake in the CA1 pyramidal cell layer, compared to control cultures. The presence of 100 μM triflusal during OGD protected against OGD-induced neurodegeneration, and reduced the number of OGD-induced NFkB positive-cells correspondingly. Cultures treated with the Mac1-saporin complex for 7 days displayed an almost total loss of microglial cells. When subjected to OGD after microglial depletion, these cultures displayed a significant increase in OGD-induced PI uptake compared to non-depleted cultures. The presence of triflusal during OGD of these cultures reduced neurodegeneration of the irrespective absence of microglia. In accordance with that, the presence of triflusal during OGD significantly inhibited the increase in the number of reactive microglia and proliferative cells in the CA1 pyramidal and dentate granule cell layers.We conclude that immunotoxic microglia depletion significantly increases the susceptibility of CA1 pyramidal cells to neurodegeneration and that the anti-inflammatory drug triflusal still can exert its neuroprotective role following depletion of microglia.     

Toxic role of prostaglandin E2 receptor EP1 after intracerebral hemorrhage in mice

Inflammatory mechanisms mediated by prostaglandins may contribute to the progression of intracerebral hemorrhage (ICH)-induced brain injury, but they are not fully understood. In this study, we examined the effect of prostaglandin E₂ receptor EP1 (EP1R) activation and inhibition on brain injury in mouse models of ICH and investigated the underlying mechanism of action. ICH was induced by injecting collagenase, autologous blood, or thrombin into the striatum of middle-aged male and female mice and aged male mice. Effects of selective EP1R agonist ONO-DI-004, antagonist SC51089, and nonspecific Src family kinase inhibitor PP2 were evaluated by a combination of histologic, magnetic resonance imaging (MRI), immunofluorescence, molecular, cellular, and behavioral assessments. EP1R was expressed primarily in neurons and axons but not in astrocytes or microglia after ICH induced by collagenase. In middle-aged male mice subjected to collagenase-induced ICH, EP1R inhibition mitigated brain injury, brain edema, cell death, neuronal degeneration, neuroinflammation, and neurobehavioral deficits, whereas its activation exacerbated these outcomes. EP1R inhibition also was protective in middle-aged female mice and aged male mice after collagenase-induced ICH and in middle-aged male mice after blood- or thrombin-induced ICH. EP1R inhibition also reduced oxidative stress, white matter injury, and brain atrophy and improved functional outcomes. Histologic results were confirmed by MRI. Src kinase phosphorylation and matrix metalloproteinase-9 activity were increased by EP1R activation and decreased by EP1R inhibition. EP1R regulated matrix metalloproteinase-9 activity through Src kinase signaling, which mediated EP1R toxicity after collagenase-induced ICH. We conclude that prostaglandin E₂ EP1R activation plays a toxic role after ICH through mechanisms that involve the Src kinases and the matrix metalloproteinase-9 signaling pathway. EP1R inhibition could be a novel therapeutic strategy to improve outcomes after ICH.     

Excitotoxic swelling occurs in oxygen and glucose deprived human cortical slices

The experimental evidence linking glutamate to ischemic neuronal injury is derived from in vitro or in vivo animal stroke models. We, therefore, developed an in vitro preparation to determine whether glutamate contributes to early neuronal swelling in oxygen and glucose deprived (OGD) human neocortical slices. In order to monitor neuronal swelling, we measured extracellular tissue resistance in brain slices by passing constant current pulses through two electrodes and recording the voltage drop between them. We verified that NMDA (30 μM) or OGD induced a rise in tissue resistance in rat neocortical slices. We then examined human neocortical slices from 11 patients undergoing resections for intractable epilepsy. Both the rodent and human neocortical slices swelled within 10 min of OGD. In both, the glutamate antagonist dizocilpine (MK-801) reduced the swelling. In the rats, MK-801 (5 μM) prolonged the latency to onset of neuronal swelling following OGD from 7.6±0.6 min (mean±S.E.M., n=16) to 17.4±2.6 min (n=6; p<0.01). Other putative neuroprotective agents were much less effective in this paradigm. In the human slices, MK-801 again prolonged the latency to resistance increase from 8.6±0.4 min (n=8) to 17.2±1.7 min (n=9, p<0.01). This is the direct demonstration that glutamate receptor activation leads to neuronal swelling in substrate deficient human brain. These results, which are similar to those obtained in the rodent brain slices, help validate the animal slices as appropriate models for the study of OGD in human brain.     

1,3-Dipropyl-8-cyclopentylxanthine attenuates the NMDA response to hypoxia in the rat hippocampus

Excitatory amino acids may cause neuronal damage and death in cerebral hypoxia and ischemia, through the activation of different subtypes of glutamate receptors, in particular of the (NMDA) receptor. In the present work, the effect of hypoxia on the component of the field excitatory postsynaptic potential (fepsp) mediated by the NMDA receptor was studied in the hippocampal CA1 area of the rat. A period of 15 min of hypoxia induced virtual abolition of the NMDA receptor-mediated fepsp and a 94.8 ± 0.7% maximal decrease in the fepsp. A period of 3 min of hypoxia induced a 89.3 ± 12.3% maximal decrease in the NMDA receptor-mediated component of the fepsp and only a 50.8 ± 11.5% maximal decrease in the fepsp. Both periods of hypoxia thus induced a more pronounced depression of the NMDA receptor-mediated component of the fepsp than of the fepsp. We found that 48.5 ± 9.1% decrease (about half of the total decrease) in the NMDA receptor-mediated fepsp, and 51.6 ± 19.6% decrease (approximately all decrease) in the fepsp induced by hypoxia (3 min) were reversed in the presence of the selective adenosine A₁ receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (50 nM), and thus likely to be mediated by endogenous adenosine, through the activation of adenosine A₁ receptors. On the other hand, under the conditions we assumed to be normoxic in our slices, DPCPX (50 nM) induced a much larger increase in the amplitude of the NMDA receptor-mediated fepsp compared to the increase in the fepsp, which suggest that endogenous adenosine is inhibiting predominantly the NMDA receptor-mediated fepsp under these conditions. Hypoxia markedly decreases the NMDA receptor-mediated fepsp in the hippocampal CA₁ area. The contribution of endogenous adenosine to the inhibition of the NMDA receptor-mediated fepsp may be fundamental for its neuroprotective effects.     

Neuroprotective effect of the new thiadiazolidinone NP00111 against oxygen-glucose deprivation in rat hippocampal slices: implication of ERK1/2 and PPARgamma receptors

Thiadiazolidinones (TDZDs) are small molecules that inhibit glycogen synthase kinase 3-β (GSK3-β) activity in a non competitive manner to ATP. NP00111, a new TDZD, besides causing inhibition of GSK-3β, has also shown to be an agonist of PPARγ . Since phosphorylation and consequent inhibition of GSK-3β by PI-3K/Akt and agonism of PPARγ have shown to afford neuroprotection in several in vitro and in vivo models, we have studied the potential neuroprotective effect of NP00111 in an “in vitro” model of ischemia-reperfusion. NP00111, at the concentration of 10 μM, significantly protected adult rat hippocampal slices subjected to oxygen and glucose deprivation (OGD) for 1 h followed by 3 h re-oxygenation, measured as lactic dehydrogenase (LDH) released to the extracellular media. The protective effects of NP00111 were more pronounced during the re-oxygenation period in comparison to the OGD period. Other GSK-3β inhibitors like lithium or AR-A014418 did not afford protection in this model. However, the PPARγ agonist rosiglitazone was protective at 3 μM. Protection afforded by NP00111 and rosiglitazone were prevented by the PPARγ antagonist GW9662, suggesting that both NP00111 and rosiglitazone were preventing cell death caused by oxygen-glucose deprivation via activation of PPARγ. NP00111 increased by two fold phosphorylation of ERK1/2 and its protective effects were lost when the hippocampal slices were co-incubated with the mitogen-activated protein kinase (MAPK) inhibitor PD98059. In conclusion, the novel TDZD NP00111 was protective against OGD in rat hippocampal slices by a mechanism related to phosphorylation of ERK1/2 via activation of PPARγ.     

Effect of induced tolerance on biochemical disturbances in hippocampal slices from the gerbil during and after oxygen/glucose deprivation.

To evaluate whether the state of tolerance is stable enough to be studied under in vitro conditions after induction by ischemic preconditioning in vivo, metabolic disturbances of hippocampal slices prepared from control and preconditioned gerbils were evaluated during and after oxygen/glucose deprivation (OGD). Slices were subjected to 5, 10 or 15 min OGD with or without 2h recovery. During the state of metabolic stress, changes in energy metabolism were identical in slices taken from control and preconditioned gerbils. Following OGD, however, recovery of protein synthesis was significantly improved in hippocampal slices of preconditioned animals, indicating that the effect of preconditioning on metabolic disturbances induced by transient OGD in vitro or transient ischemia in vivo is similar. It is suggested that the hippocampal slice preparation is an in vitro model suitable for the study of basic mechanisms underlying the induction of tolerance in vivo.     

P2 receptor antagonists prevent synaptic failure and extracellular signal-regulated kinase 1/2 activation induced by oxygen and glucose deprivation in rat CA1 hippocampus in vitro

To investigate the role of purinergic P2 receptors under ischemia, we studied the effect of P2 receptor antagonists on synaptic transmission and mitogen‐activated protein kinase (MAPK) activation under oxygen and glucose deprivation (OGD) in rat hippocampal slices. The effect of the P2 antagonists pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonate (PPADS, unselective, 30 μm ), N ⁶‐methyl‐2′‐deoxyadenosine‐3′,5′‐bisphosphate (MRS2179, selective for P2Y₁ receptor, 10 μm ), Brilliant Blue G (BBG, selective for P2X₇ receptor, 1 μm ), and 5‐[[[(3‐phenoxyphenyl)methyl][(1S)‐1,2,3,4‐tetrahydro‐1‐naphthalenyl]amino]carbonyl]‐1,2,4‐benzenetricarboxylic acid (A‐317491, selective for P2X₃ receptor, 10 μm ), and of the newly synthesized P2X₃ receptor antagonists 2‐amino‐9‐(5‐iodo‐2‐isopropyl‐4‐methoxybenzyl)adenine (PX21, 1 μm ) and 2‐amino‐9‐(5‐iodo‐2‐isopropyl‐4‐methoxybenzyl)‐N ⁶‐methyladenine (PX24, 1 μm ), on the depression of field excitatory postsynaptic potentials (fEPSPs) and anoxic depolarization (AD) elicited by 7 min of OGD were evaluated. All antagonists significantly prevented these effects. The extent of CA1 cell injury was assessed 3 h after the end of 7 min of OGD by propidium iodide staining. Substantial CA1 pyramidal neuronal damage, detected in untreated slices exposed to OGD injury, was significantly prevented by PPADS (30 μm ), MRS2179 (10 μm ), and BBG (1 μm ). Western blot analysis showed that, 10 min after the end of the 7 min of OGD, extracellular signal‐regulated kinase (ERK)1/2 MAPK activation was significantly increased. MRS2179, BBG, PPADS and A‐317491 significantly counteracted ERK1/2 activation. Hippocampal slices incubated with the ERK1/2 inhibitors 1,4‐diamino‐2,3‐dicyano‐1,4‐bis(2‐aminophenylthio)butadiene (U0126, 10 μm ) and α‐[amino[(4‐aminophenyl)thio]methylene]‐2‐(trifluoromethyl) benzeneacetonitrile (SL327, 10 μm ) showed significant fEPSP recovery after OGD and delayed AD, supporting the involvement of ERK1/2 in neuronal damage induced by OGD. These results indicate that subtypes of hippocampal P2 purinergic receptors have a harmful effect on neurotransmission in the CA1 hippocampus by participating in AD appearance and activation of ERK1/2.     

Delayed neuronal injury induced by sub-lethal NMDA exposure in the hippocampal slice

Stroke produces neuronal death by two general processes which differ in their temporal course. Acute neuronal death occurs within minutes, while delayed neuronal death evolves within 24 h. To better examine mechanisms of delayed death, we developed a new in vitro model of delayed neuronal injury using extended electrophysiological recordings in paired hippocampal slices. We exposed one hippocampal slice of each pair to 10 μMN-methyl-d-aspartate (NMDA) until the orthodromic CA1 PS disappeared. Thereafter, NMDA-treated slices regained near full recovery of PS amplitude within one hour. However, 10 h later, NMDA-treated slices demonstrated a rapid decline in PS amplitude of 82% ± 15. CA1 orthodromic evoked PS was lost completely at an average 12.4 ± 1.6 h after NMDA exposure. This sudden loss of response contrasted with paired, untreated slices, where CA1 PS could be elicited for 22.6 ± 4.0 h (P < 0.05). Treatment with 10 mM MgCl₂ begun after NMDA exposure and continued for 35 min, prevented delayed loss of CA1 orthodromic PS, which then could be elicited for 20.3 ± 2.1 h. These results indicate that delayed injury can be evaluated using the hippocampal slice. They also suggest that activation of NMDA receptors can induce delayed neuronal injury in CA1 neurons, and that magnesium treatment after NMDA can prevent this injury.     

Membrane‐tethered AKT kinase regulates basal synaptic transmission and early phase LTP expression by modulation of post‐synaptic AMPA receptor level

The serine/threonine kinase AKT/PKB plays a fundamental role in a wide variety of neuronal functions, including neuronal cell development, axonal growth, and synaptic plasticity. Multiple evidence link AKT signaling pathways to regulation of late phase long‐term synaptic plasticity, synaptogenesis, and spinogenesis, as well as long‐term memory formation. Nevertheless, the downstream effectors mediating the effects of AKT on early phase long‐term potentiation (eLTP) are currently unknown. Here we report that using different regimes of pharmacological activation and inhibition of AKT activity in acute hippocampal slices, we found that AKT regulates the post‐synaptic expression of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPA) receptors affecting solely the expression of eLTP, with no effect on its induction and maintenance. We further show that both maintenance of basal synaptic activity and expression of eLTP require plasma membrane tethering by activated AKT and that basal synaptic activity may be regulated via the direct effects of AKT1 on the expression level of post‐synaptic AMPA receptors bypassing the canonical AKT signaling. Finally, we establish that eLTP expression requires the involvement of both the canonical AKT signaling pathways and the direct effect of AKT1 on AMPA receptor activity/expression in the post‐synaptic membrane. © 2016 Wiley Periodicals, Inc.     

Neuroprotection Promoted by Guanosine Depends on Glutamine Synthetase and Glutamate Transporters Activity in Hippocampal Slices Subjected to Oxygen/Glucose Deprivation

Guanosine (GUO) has been shown to act as a neuroprotective agent against glutamatergic excitotoxicity by increasing glutamate uptake and decreasing its release. In this study, a putative effect of GUO action on glutamate transporters activity modulation was assessed in hippocampal slices subjected to oxygen and glucose deprivation (OGD), an in vitro model of brain ischemia. Slices subjected to OGD showed increased excitatory amino acids release (measured by d-[³H]aspartate release) that was prevented in the presence of GUO (100 µM). The glutamate transporter blockers, DL-TBOA (10 µM), DHK (100 µM, selective inhibitor of GLT-1), and sulfasalazine (SAS, 250 µM, Xc^? system inhibitor) decreased OGD-induced d-aspartate release. Interestingly, DHK or DL-TBOA blocked the decrease in glutamate release induced by GUO, whereas SAS did not modify the GUO effect. GUO protected hippocampal slices from cellular damage by modulation of glutamate transporters, however selective blockade of GLT-1 or Xc- system only did not affect this protective action of GUO. OGD decreased hippocampal glutamine synthetase (GS) activity and GUO recovered GS activity to control levels without altering the kinetic parameters of GS activity, thus suggesting GUO does not directly interact with GS. Additionally, the pharmacological inhibition of GS activity with methionine sulfoximine abolished the effect of GUO in reducing d-aspartate release and cellular damage evoked by OGD. Altogether, results in hippocampal slices subjected to OGD show that GUO counteracts the release of excitatory amino acids, stimulates the activity of GS, and decreases the cellular damage by modulation of glutamate transporters activity.     

Kainate-induced status epilepticus leads to a delayed increases in various specific glutamate metabotropic receptor responses in the hippocampus

Neuronal loss and gliosis were detected in the rat hippocampus soon after unilateral intra-amygdala injection of kainate (KA) (2.5 nmol) while solid mossy fiber sprouting could be seen only fourteen days after this injection. Using this experimental model, we examined the metabotropic glutamate receptor (mGluR)-induced inositol phosphate (IP) formation in hippocampal synaptoneurosomes and slices. In synaptoneurosomes prepared from ipsilateral hippocampi fourteen days following injection, there were no significant changes in mGluR- and carbachol(CARB)-stimulated IPs syntheses when sham-operated and KA-injected animals were compared. In the corresponding hippocampal slices, significant increases of the mGluR responses mediated by ibotenate (IBO) and aminocyclopentane-trans-1,3-dicar☐ylate (t-ACPD) were noted after KA application. The net stimulation values respectively expressed in a pair-wise fashion for buffer-injected control and KA-treated animals were IBO: 1,947 ± 457 and 10,553 ± 1,242; t-ACPD: 1,557 ± 662 and 9,449 ± 2,251 dpm/mg protein respectively. Significantly augmented mGluR responses in hippocampal slices were also measured at 7,42 and 92 days after KA injection. There were, however, no significant increases in CARB-stimulated phosphoinositide hydrolysis in the hippocampal slices at all time-intervals after KA administration. These findings show that there are differences between the mGluR responses in hippocampal synaptoneurosome and slice preparations, suggesting the presence of two distinct populations of mGluR in each of these two models. The large specific increases in certain mGluR activities after KA-induced status epilepticus in hippocampal slices could represent one of the molecular mechanisms which underlie the profound morphological changes, in particular gliosis or mossy fiber sprouting, which follow the KA-induced status epilepticus.     

AMPA receptor-mediated cell death is reduced by docosahexaenoic acid but not by eicosapentaenoic acid in area CA1 of hippocampal slice cultures

Over the last decade, several studies have shown that high levels of polyunsaturated fatty acids (PUFAs) in the brain might limit neuronal damage in various pathological conditions. For example, in animal models of Alzheimer's disease, omega-3 type PUFAs such as docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids have been proposed to decrease both the cognitive and the cellular manifestations of premature ageing. The mechanisms by which they promote brain integrity remain to be established, and the experiments on cultured hippocampal slices described here examine the possibility that omega-3 fatty acids can modulate brain cell viability by interacting with glutamate receptors. We observed, by lactate dehydrogenase release and propidium iodide (PI) uptake, that excitotoxicity triggered by an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor agonist was markedly reduced in hippocampal slices treated with DHA. PI uptake experiments also revealed that neuroprotection by DHA was restricted to the hippocampal CA1 region and could not be reproduced by EPA or arachidonic acid, an omega-6 PUFA. Moreover, the beneficial effect of DHA was specific to AMPA receptor stimulation, insofar as the toxicity induced by N-methyl-D-aspartate or kainate receptor agonists was not diminished by DHA preincubation. Biotinylation experiments finally indicated that the neuroprotective actions of DHA could result from down-regulation of AMPA receptors in hippocampal membranes. This investigation thus provides the first indication that a beneficial outcome of DHA on the brain could derive from specific modulation of AMPA-mediated toxicity, reinforcing the notion that dietary DHA uptake might be useful in preventing neurodegenerative diseases.     

Neuroprotection induced in vitro by ischemic preconditioning and postconditioning: modulation of apoptosis and PI3K-Akt pathways

Preconditioning and postconditioning are mild ischemic exposures before or after severe injurious ischemia, respectively, that elicit endogenous neuroprotective responses. Molecular mechanisms of neuroprotection through preconditioning and postconditioning are not completely understood. Here we optimized the in vitro oxygen and glucose deprivation (OGD) models of preconditioning and postconditioning in primary cortical neuron cultures that allow the studies of the corresponding molecular mechanisms of neuroprotection. We found that the cortical cells preconditioned with a single 45-min OGD treatment administered 24 h prior to injurious 2 h OGD were robustly protected after both 3 h and 16 h of reperfusion. For the postconditioning treatment, we found that three cycles of 15 min OGD followed by 15 min reperfusion, applied immediately after injurious 2 h OGD and prior to complete reperfusion, resulted in effective neuroprotection at both 3 h and 16 h of reperfusion. Using real-time RT–PCR arrays focused on genes of the apoptosis and PI3K–Akt pathways, we found that injurious OGD mainly induced apoptosis-related and repressed PI3K–Akt pathway-related genes after either 3 h or 16 h of reperfusion. Preconditioning treatment resulted in the activation of both pro-survival and anti-apoptotic pathways after 3 h of reperfusion and mainly anti-apoptotic pathway after 16 h of reperfusion. In contrast, the activation of PI3K–Akt pathway mainly contributed to the neuroprotective effect by the postconditioning treatment after 3 h of reperfusion, but differential gene expression likely contributed minimally, if at all, to the neuroprotection observed after 16 h of reperfusion. Among the novel markers of neuroprotection, Nol3 gene upregulation was observed after 3 h of reperfusion following either preconditioning or postconditioning treatments and after 16 h of reperfusion following preconditioning treatment.     

Developmental modulation of mouse hypoglossal nerve inspiratory output in vitro by noradrenergic receptor agonists

The ontogeny of the noradrenergic receptor subtypes modulating hypoglossal (XII) nerve inspiratory output was characterized. Noradrenergic agents were locally applied over the XII nucleus of rhythmically active medullary slice preparations isolated from mice between zero and 13 days of age (P0–P13) and the effects on XII inspiratory burst amplitude quantified. The α₁ receptor agonist phenylephrine (PE, 0.1–10 μM) produced a dose-dependent, prazosin-sensitive (0.1–10 μM) increase in XII nerve inspiratory burst amplitude. The magnitude of this potentiation increased steadily from a maximum of 15±8% in P0 mice to 134±4% in P12–P13 mice. The β receptor agonist isoproterenol (0.01–1.0 mM) produced a prazosin-insensitive, propranolol-sensitive potentiation of XII nerve burst amplitude. The isoproterenol-mediated potentiation increased with development from 27±5% in P0–P1 slices, to 37±3% in P3 slices and 45±4% in P9–P10 slices. The α₂ receptor agonist clonidine (1 mM) reduced XII nerve inspiratory burst amplitude in P0–P3 slices by 29±5%, but had no effect on output from P12–P13 slices. An α₂ receptor-mediated inhibition of inspiratory activity in neonates (P0–P3) was further supported by a 19±3% reduction in XII nerve burst amplitude when norepinephrine (NE, 100 μM) was applied in the presence of prazosin (10 μM) and propranolol (100 μM). Results indicate that developmental increases in potentiating α₁ and, to a lesser extent, β receptor mechanisms combine with a developmentally decreasing inhibitory mechanism, most likely mediated by α₂ receptors, to determine the ontogenetic time course by which NE modulates XII MN inspiratory activity.     

Resveratrol attenuates early pyramidal neuron excitability impairment and death in acute rat hippocampal slices caused by oxygen-glucose deprivation

Accumulating evidence indicates that the polyphenol resveratrol (trans-3, 5, 4”-trihydroxystibene, RVT) potently protects against cerebral ischemia neuronal damage due to its oxygen free radicals scavenging and antioxidant properties. However, it is unknown whether RVT can attenuate ischemia-induced early impairment of neuronal excitability. To address this question, we simulated ischemic conditions by applying oxygen-glucose deprivation (OGD) to acute rat hippocampal slices and examined the effect of RVT on OGD-induced pyramidal neuron excitability impairment using whole-cell patch clamp recording. 100 µM RVT largely inhibited the 15 min OGD-induced progressive membrane potential (Vm) depolarization and the reduction in evoked action potential frequency and amplitude in pyramidal neurons. In a parallel neuronal viability study using TO-PRO-3 iodide staining, 20 min OGD induced irreversible CA1 pyramidal neuronal death which was significantly reduced by 100 µM RVT. No similar effects were found with PQQ treatment, an antioxidant also showing potent neuroprotection in the rat rMCAO ischemia model. This suggests that antioxidant action per se, is unlikely accounting for the observed early effects of RVT. RVT also markedly reduced the frequency and amplitude of AMPA mediated spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons, which is also an early consequence of OGD. RVT effects on neuronal excitability were inhibited by the large-conductance potassium channel (BK channel) inhibitor paxilline. Together, these studies demonstrate that RVT attenuates OGD-induced neuronal impairment occurring early in the simulated ischemia slice model by enhancing the activation of BK channel and reducing the OGD-enhanced AMPA/NMDA receptor mediated neuronal EPSCs.     

Antioxidant and pro-oxidant properties of boldine on hippocampal slices exposed to oxygen-glucose deprivation in vitro

Boldine is one of the most potent natural antioxidants and displays some important pharmacological activities, such as cytoprotective and anti-inflammatory activities, which may arise from its free radical scavenging properties. Given that the pathogenesis of brain ischemia/reperfusion has been associated with an excessive generation of oxygen free radicals, the aim of this study was to evaluate the neuroproperties of boldine using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD), followed by reoxygenation, to mimic an ischemic condition. The OGD ischemic condition significantly impaired cellular viability, increased lactate dehydrogenase (LDH) leakage and increased free radical generation. In non-OGD slices, incubation with 100 μM boldine significantly increased LDH released into incubation media and decreased mitochondrial activity, suggesting an increase of tissue damage caused by boldine. However, slices incubated with 10 μM boldine during and after OGD exposure had significantly increased cellular viability with no effect on cell damage. Total reactive antioxidant potential (TRAP) levels measured for this alkaloid showed an antioxidant potential three times higher than Trolox, which acts as a peroxyl radical scavenger. Moreover, boldine prevented the increase in lipoperoxidation levels induced by ischemia, but higher concentrations potentiated this parameter. These results confirm the potent antioxidant properties of this alkaloid, and add evidence to support the need for further investigations in order to confirm the potential pro-oxidant effects of boldine at higher doses.     

Protection of adult mouse progenitor cells and human glioma cells by de novo decorin expression in an oxygen- and glucose-deprived cell culture model system.

We employed an in vitro hypoxia cell culture model system and gene transfer technology to examine the effect of the decorin gene on cell survival against oxygen and glucose deprivation (OGD). Ectopic expression of decorin in subventricular zone (SVZ) cells from adult male mouse brain and human glioblastoma U-87 cells kept the cells viable against 24 h of OGD. Fewer than 1% of decorin-synthesizing cells were apoptotic after 12 h of OGD. In contrast, 100% of the control cells were apoptotic even after 4 h of OGD. De novo decorin synthesis in SVZ and U-87 cells induced expression of p21, p27 and Ras, AKT (acutely transforming retrovirus AKT8 in rodent T-cell lymphoma), and phosphorylated AKT. Blocking of phosphoinositide 3-kinase (PI-3K), Ras, and the epidermal growth factor receptor with specific inhibitors had no effect on induction of Ras, p21, and p27 at the messenger RNA level in decorin-synthesizing SVZ and U-87 cells. PI-3K inhibitors significantly increased apoptosis in decorin-expressing cells. Our data indicate that induction of p21, p27, Ras, AKT, and phosphorylated AKT by decorin inhibits apoptosis and protects U-87 and SVZ cells against OGD. Therefore, our data suggest that decorin is a potent trophic factor that protects neuronal progenitor cells and glioma cells from OGD.     

Effect of metabotropic glutamate receptor activation on receptor-mediated cyclic AMP responses in primary cultures of rat striatal neurones

Co-activation of group I metabotropic glutamate (mGlu) receptors and adenosine receptors resulted in an augmented cyclic AMP response in primary cultures of rat striatal neurones. -glutamate and the selective group I agonist, (S)-dihydroxyphenylglycine (S-DHPG) evoked concentration-dependent potentiations of cyclic AMP accumulation stimulated by the adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA), with EC₅₀ values of 3.41±0.39 and 5.69±1.64 μM, respectively, and maximal augmentations of approximately 350% at concentrations of 100 μM. The S-DHPG potentiation was inhibited by group I mGlu receptor antagonists and a protein kinase C inhibitor, Ro 31-8220, implicating products of PI hydrolysis in this effect. Furthermore, -glutamate and S-DHPG stimulated PI hydrolysis in striatal neuronal cultures with similar EC₅₀ values to those observed for the augmentation of NECA cyclic AMP responses (5.19±1.18 and 3.78±1.42 μM, respectively). In situ hybridization and immunofluorescence techniques indicate that group I mGlu receptor-evoked potentiations are likely to be mediated via mGlu₅ receptors, which are expressed at high levels in these cultures. In contrast to cross-chopped slices of neonatal rat striatum, of equivalent age, the group II mGlu receptor agonist, (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) was without effect on NECA- or forskolin-stimulated cyclic AMP responses in primary striatal neuronal cultures. This lack of effect might be due to a low level of expression of group II mGlu receptors in cultured striatal neurones.