scholarly journals Protective effect of 20-HETE inhibition in a model of oxygen-glucose deprivation in hippocampal slice cultures

2012 ◽  
Vol 302 (6) ◽  
pp. H1285-H1293 ◽  
Author(s):  
Marija Renic ◽  
Suresh N. Kumar ◽  
Debebe Gebremedhin ◽  
Matthew A. Florence ◽  
Nashaat Z. Gerges ◽  
...  
Keyword(s):  
Cell Death ◽  
Protective Effect ◽  
Caspase 3 ◽  
Hippocampal Slice ◽  
Hippocampal Slices ◽  
Glucose Deprivation ◽  
Dienoic Acid ◽  
Oxygen Glucose Deprivation ◽  
Iodide Uptake ◽  
Hippocampal Slice Cultures

Recent studies have indicated that inhibitors of the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) may have direct neuroprotective actions since they reduce infarct volume after ischemia reperfusion in the brain without altering blood flow. To explore this possibility, the present study used organotypic hippocampal slice cultures subjected to oxygen-glucose deprivation (OGD) and reoxygenation to examine whether 20-HETE is released by organotypic hippocampal slices after OGD and whether it contributes to neuronal death through the generation of ROS and activation of caspase-3. The production of 20-HETE increased twofold after OGD and reoxygenation. Blockade of the synthesis of 20-HETE with N-hydroxy- N′-(4-butyl-2-methylphenol)formamidine (HET0016) or its actions with a 20-HETE antagonist, 20-hydroxyeicosa-6( Z),15( Z)-dienoic acid, reduced cell death, as measured by the release of lactate dehydrogenase and propidium iodide uptake. Administration of a 20-HETE mimetic, 20-hydroxyeicosa-5( Z),14( Z)-dienoic acid (5,14-20-HEDE), had the opposite effect and increased injury after OGD. The death of neurons after OGD was associated with an increase in the production of ROS and activation of caspase-3. These effects were attenuated by HET0016 and potentiated after the administration of 5,14-20-HEDE. These findings indicate that the production of 20-HETE by hippocampal slices is increased after OGD and that inhibitors of the synthesis or actions of 20-HETE protect neurons from ischemic cell death. The protective effect of 20-HETE inhibitors is associated with a decrease in superoxide production and activation of caspase-3.

Protective effect of resveratrol against oxygen–glucose deprivation in organotypic hippocampal slice cultures: Involvement of PI3-K pathway

2006 ◽  
Vol 24 (1) ◽  
pp. 170-182 ◽  
Author(s):  
Lauren L. Zamin ◽  
Patrícia Dillenburg-Pilla ◽  
Ricardo Argenta-Comiran ◽  
Ana Paula Horn ◽  
Fabrício Simão ◽  
...  
Keyword(s):  
Protective Effect ◽  
Hippocampal Slice ◽  
Glucose Deprivation ◽  
Oxygen Glucose Deprivation ◽  
Hippocampal Slice Cultures ◽  
Organotypic Hippocampal Slice Cultures

scholarly journals Isoflurane Neurotoxicity Is Mediated by p75NTR-RhoA Activation and Actin Depolymerization

2011 ◽  
Vol 114 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Brian P. Lemkuil ◽  
Brian P. Head ◽  
Matthew L. Pearn ◽  
Hemal H. Patel ◽  
John C. Drummond ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Hippocampal Slice ◽  
Hippocampal Slices ◽  
Neurotrophin Receptor ◽  
P75 Neurotrophin Receptor ◽  
Actin Depolymerization ◽  
Rhoa Activation ◽  
Hippocampal Slice Cultures ◽  
Developing Neurons

Background The mechanisms by which isoflurane injured the developing brain are not clear. Recent work has demonstrated that it is mediated in part by activation of p75 neurotrophin receptor. This receptor activates RhoA, a small guanosine triphosphatase that can depolymerize actin. It is therefore conceivable that inhibition of RhoA or prevention of cytoskeletal depolymerization might attenuate isoflurane neurotoxicity. This study was conducted to test these hypotheses using primary cultured neurons and hippocampal slice cultures from neonatal mouse pups. Methods Primary neuron cultures (days in vitro, 4-7) and hippocampal slice cultures from postnatal day 4-7 mice were exposed to 1.4% isoflurane (4 h). Neurons were pretreated with TAT-Pep5, an intracellular inhibitor of p75 neurotrophin receptor, the cytoskeletal stabilizer jasplakinolide, or their corresponding vehicles. Hippocampal slice cultures were pretreated with TAT-Pep5 before isoflurane exposure. RhoA activation was evaluated by immunoblot. Cytoskeletal depolymerization and apoptosis were evaluated with immunofluorescence microscopy using drebrin and cleaved caspase-3 staining, respectively. Results RhoA activation was increased after 30 and 120 min of isoflurane exposure in neurons; TAT-Pep5 (10 μm) decreased isoflurane-mediated RhoA activation at both time intervals. Isoflurane decreased drebrin immunofluorescence and enhanced cleaved caspase-3 in neurons, effects that were attenuated by pretreatment with either jasplakinolide (1 μm) or TAT-Pep5. TAT-Pep5 attenuated the isoflurane-mediated decrease in phalloidin immunofluorescence. TAT-Pep5 significantly attenuated isoflurane-mediated loss of drebrin immunofluorescence in hippocampal slices. Conclusions Isoflurane results in RhoA activation, cytoskeletal depolymerization, and apoptosis. Inhibition of RhoA activation or prevention of downstream actin depolymerization significantly attenuated isoflurane-mediated neurotoxicity in developing neurons.

Isoflurane Preconditions Hippocampal Neurons against Oxygen–Glucose Deprivation

2005 ◽  
Vol 103 (3) ◽  
pp. 532-539 ◽  
Author(s):  
Philip E. Bickler ◽  
Xinhua Zhan ◽  
Christian S. Fahlman
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Hippocampal Slice ◽  
Binding Protein ◽  
Glucose Deprivation ◽  
Mitogen Activated Protein Kinase ◽  
Oxygen Glucose Deprivation ◽  
Hippocampal Slice Cultures ◽  
Mitogen Activated Protein ◽  
Associated Proteins

Background Isoflurane preconditions neurons to improve tolerance of subsequent ischemia in both intact animal models and in in vitro preparations. The mechanisms for this protection remain largely undefined. Because isoflurane increases intracellular Ca2+ concentrations and Ca2+ is involved in many processes related to preconditioning, the authors hypothesized that isoflurane preconditions neurons via Ca2+-dependent processes involving the Ca2+- binding protein calmodulin and the mitogen-activated protein kinase-ERK pathway. Methods The authors used a preconditioning model in which organotypic cultures of rat hippocampus were exposed to 0.5-1.5% isoflurane for a 2-h period 24 h before an ischemia-like injury of oxygen-glucose deprivation. Survival of CA1, CA3, and dentate neurons was assessed 48 later, along with interval measurements of intracellular Ca2+ concentration (fura-2 fluorescence microscopy in CA1 neurons), mitogen-activated protein kinase p42/44, and the survival associated proteins Akt and GSK-3beta (in situ immunostaining and Western blots). Results Preconditioning with 0.5-1.5% isoflurane decreased neuron death in CA1 and CA3 regions of hippocampal slice cultures after oxygen-glucose deprivation. The preconditioning period was associated with an increase in basal intracellular Ca2+ concentration of 7-15%, which involved Ca2+ release from inositol triphosphate-sensitive stores in the endoplasmic reticulum, and transient phosphorylation of mitogen-activated protein kinase p42/44 and the survival-associated proteins Akt and GSK-3beta. Preconditioning protection was eliminated by the mitogen-activated extracellular kinase inhibitor U0126, which prevented phosphorylation of p44 during preconditioning, and by calmidazolium, which antagonizes the effects of Ca2+-bound calmodulin. Conclusions Isoflurane, at clinical concentrations, preconditions neurons in hippocampal slice cultures by mechanisms that apparently involve release of Ca2+ from the endoplasmic reticulum, transient increases in intracellular Ca2+ concentration, the Ca2+ binding protein calmodulin, and phosphorylation of the mitogen-activated protein kinase p42/44.

scholarly journals Concurrent Assessment of Calpain and Caspase-3 Activation after Oxygen–Glucose Deprivation in Primary Septo-Hippocampal Cultures

2001 ◽  
Vol 21 (11) ◽  
pp. 1281-1294 ◽  
Author(s):  
Jennifer K. Newcomb-Fernandez ◽  
Xiurong Zhao ◽  
Brian R. Pike ◽  
Kevin K. W. Wang ◽  
Andreas Kampfl ◽  
...  
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Glucose Deprivation ◽  
Apoptotic Cell Death ◽  
Oxygen Glucose Deprivation ◽  
Necrotic Cell Death ◽  
Necrotic Cell ◽  
Lactate Dehydrogenase Release ◽  
Hippocampal Cultures

The contributions of calpain and caspase-3 to apoptosis and necrosis after central nervous system (CNS) trauma are relatively unexplored. No study has examined concurrent activation of calpain and caspase-3 in necrotic or apoptotic cell death after any CNS insult. Experiments used a model of oxygen–glucose deprivation (OGD) in primary septo-hippocampal cultures and assessed cell viability, occurrence of apoptotic and necrotic cell death phenotypes, and protease activation. Immunoblots using an antibody detecting calpain and caspase-3 proteolysis of α-spectrin showed greater accumulation of calpain-mediated breakdown products (BDPs) compared with caspase-3–mediated BDPs. Administration of calpain and caspase-3 inhibitors confirmed that activation of these proteases contributed to cell death, as inferred by lactate dehydrogenase release. Oxygen–glucose deprivation resulted in expression of apoptotic and necrotic cell death phenotypes, especially in neurons. Immunocytochemical studies of calpain and caspase-3 activation in apoptotic cells indicated that these proteases are almost always concurrently activated during apoptosis. These data demonstrate that calpain and caspase-3 activation is associated with expression of apoptotic cell death phenotypes after OGD, and that calpain activation, in combination with caspase-3 activation, could contribute to the expression of apoptotic cell death by assisting in the degradation of important cellular proteins.

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