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1.
Redox Biol ; 6: 516-523, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26454246

ABSTRACT

Autophagy is activated when the neonatal brain exposed to hypoxia ischemia (HI), but the mechanisms underlying its activation and its role in the neuronal cell death associated with HI is unclear. We have previously shown that reactive oxygen species (ROS) derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase play an important role in HI-mediated neuronal cell death. Thus, the aim of this study was to determine if ROS is involved in the activation of autophagy in HI-mediated neonatal brain injury and to determine if this is a protective or deleterious pathway. Initial electron microscopy data demonstrated that autophagosome formation is elevated in P7 hippocampal slice cultures exposed to oxygen-glucose deprivation (OGD). This corresponded with increased levels of LC3II mRNA and protein. The autophagy inhibitor, 3-methyladenine (3-MA) effectively reduced LC3II levels and autophagosome formation in hippocampal slice cultures exposed to OGD. Neuronal cell death was significantly attenuated. Finally, we found that the pharmacologic inhibition of NADPH oxidase using apocynin or gp91ds-tat decreased autophagy in hippocampal slice cultures and the rat brain respectively. Thus, our results suggest that an activation of autophagy contributes to neonatal HI brain injury this is oxidative stress dependent.


Subject(s)
Autophagy , Hypoxia-Ischemia, Brain/pathology , Oxidative Stress , Acetophenones/pharmacology , Animals , Animals, Newborn , Hippocampus/blood supply , Hippocampus/metabolism , Hippocampus/pathology , Hypoxia-Ischemia, Brain/metabolism , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/metabolism , Neurons/physiology , Rats, Sprague-Dawley , Tissue Culture Techniques
2.
Redox Biol ; 6: 112-121, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26209813

ABSTRACT

We have recently shown that increased hydrogen peroxide (H2O2) generation is involved in hypoxia-ischemia (HI)-mediated neonatal brain injury. H2O2 can react with free iron to form the hydroxyl radical, through Fenton Chemistry. Thus, the objective of this study was to determine if there was a role for the hydroxyl radical in neonatal HI brain injury and to elucidate the underlying mechanisms. Our data demonstrate that HI increases the deposition of free iron and hydroxyl radical formation, in both P7 hippocampal slice cultures exposed to oxygen-glucose deprivation (OGD), and the neonatal rat exposed to HI. Both these processes were found to be nitric oxide (NO) dependent. Further analysis demonstrated that the NO-dependent increase in iron deposition was mediated through increased transferrin receptor expression and a decrease in ferritin expression. This was correlated with a reduction in aconitase activity. Both NO inhibition and iron scavenging, using deferoxamine administration, reduced hydroxyl radical levels and neuronal cell death. In conclusion, our results suggest that increased NO generation leads to neuronal cell death during neonatal HI, at least in part, by altering iron homeostasis and hydroxyl radical generation.


Subject(s)
Hippocampus/metabolism , Hypoxia-Ischemia, Brain/metabolism , Iron/metabolism , Neurons/metabolism , Nitric Oxide/pharmacology , Aconitate Hydratase/antagonists & inhibitors , Aconitate Hydratase/genetics , Aconitate Hydratase/metabolism , Animals , Animals, Newborn , Cell Death/drug effects , Cell Hypoxia , Culture Media/chemistry , Deferoxamine/pharmacology , Ferritins/antagonists & inhibitors , Ferritins/genetics , Ferritins/metabolism , Gene Expression Regulation , Glucose/deficiency , Hippocampus/drug effects , Hippocampus/pathology , Hydroxyl Radical/metabolism , Hypoxia-Ischemia, Brain/chemically induced , Hypoxia-Ischemia, Brain/genetics , Hypoxia-Ischemia, Brain/prevention & control , Microtomy , NG-Nitroarginine Methyl Ester/pharmacology , Neurons/drug effects , Neurons/pathology , Rats , Rats, Sprague-Dawley , Receptors, Transferrin/agonists , Receptors, Transferrin/genetics , Receptors, Transferrin/metabolism , Tissue Culture Techniques
3.
PLoS One ; 8(8): e70750, 2013.
Article in English | MEDLINE | ID: mdl-23976956

ABSTRACT

We have recently shown that p38MAP kinase (p38MAPK) stimulates ROS generation via the activation of NADPH oxidase during neonatal hypoxia-ischemia (HI) brain injury. However, how p38MAPK is activated during HI remains unresolved and was the focus of this study. Ca²âº/calmodulin-dependent protein kinase II (CaMKII) plays a key role in brain synapse development, neural transduction and synaptic plasticity. Here we show that CaMKII activity is stimulated in rat hippocampal slice culture exposed to oxygen glucose deprivation (OGD) to mimic the condition of HI. Further, the elevation of CaMKII activity, correlated with enhanced p38MAPK activity, increased superoxide generation from NADPH oxidase as well as necrotic and apoptotic cell death. All of these events were prevented when CaMKII activity was inhibited with KN93. In a neonatal rat model of HI, KN93 also reduced brain injury. Our results suggest that CaMKII activation contributes to the oxidative stress associated with neural cell death after HI.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Hippocampus/enzymology , Hypoxia-Ischemia, Brain/enzymology , Neurons/enzymology , Animals , Animals, Newborn , Benzylamines/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Cell Death/drug effects , Cell Hypoxia , Enzyme Activation , Gene Expression Regulation , Glucose/deficiency , Hippocampus/drug effects , Hippocampus/pathology , Hypoxia-Ischemia, Brain/genetics , Hypoxia-Ischemia, Brain/pathology , Hypoxia-Ischemia, Brain/prevention & control , NADP/genetics , NADP/metabolism , Neurons/drug effects , Neurons/pathology , Oxidative Stress , Protein Kinase Inhibitors/pharmacology , Rats , Rats, Sprague-Dawley , Signal Transduction , Sulfonamides/pharmacology , Superoxides/antagonists & inhibitors , Superoxides/metabolism , Tissue Culture Techniques , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism
4.
Free Radic Biol Med ; 53(5): 1139-51, 2012 Sep 01.
Article in English | MEDLINE | ID: mdl-22728269

ABSTRACT

Neonatal brain hypoxia-ischemia (HI) results in neuronal cell death. Previous studies indicate that reactive oxygen species, such as superoxide, play a key role in this process. However, the cellular sources have not been established. In this study we examine the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in neonatal HI brain injury and elucidate its mechanism of activation. Rat hippocampal slices were exposed to oxygen glucose deprivation (OGD) to mimic the conditions seen in HI. Initial studies confirmed an important role for NADPH oxidase-derived superoxide in the oxidative stress associated with OGD. Further, the OGD-mediated increase in apoptotic cell death was inhibited by the NADPH oxidase inhibitor apocynin. The activation of NADPH oxidase was found to be dependent on the p38 mitogen-activated protein kinase-mediated phosphorylation and activation of the p47(phox) subunit. Using an adeno-associated virus antisense construct to selectively decrease p47(phox) expression in neurons showed that this led to inhibition of both the increase in superoxide and the neuronal cell death associated with OGD. We also found that NADPH oxidase inhibition in a neonatal rat model of HI or scavenging hydrogen peroxide reduced brain injury. Thus, we conclude that activation of the NADPH oxidase complex contributes to the oxidative stress during HI and that therapies targeted against this complex could provide neuroprotection against the brain injury associated with neonatal HI.


Subject(s)
Hippocampus/metabolism , Hypoxia-Ischemia, Brain/metabolism , NADPH Oxidases/metabolism , Neurons/metabolism , Neurons/pathology , Organ Culture Techniques , Superoxides/metabolism , Acetophenones/pharmacology , Animals , Animals, Newborn , Cell Death/drug effects , Glucose/deficiency , Glucose/metabolism , Microtomy , Neurons/drug effects , Neurons/enzymology , Oxygen/metabolism , Rats , Rats, Sprague-Dawley
5.
J Neurosurg ; 112(3): 631-9, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20192670

ABSTRACT

OBJECT: Delayed vasospasm is a significant cause of morbidity and mortality after subarachnoid hemorrhage (SAH). Proteomic therapeutics offers a new modality in which biologically active proteins or peptides are transduced into cells via covalent linkage to cell permeant peptides (CPPs). The hypothesis of this study was that either intrathecal or intravenous delivery of a phosphopeptide mimetic of the small heat shock-related protein, HSP20, linked to a CPP, would inhibit delayed decreases in cerebral perfusion after experimental SAH in a rat model. METHODS: This study was conducted in 3 parts: 1) prevention and 2) reversal of delayed decreases in cerebral perfusion via either intrathecal or intravenous administration of a CPP linked to phosphopeptide mimetics of HSP20 (AZX100) and 3) determining the effect of intravenous administration of AZX100 on blood pressure and heart rate. Subarachnoid hemorrhage was induced in rats by endovascular perforation. Subsequently, AZX100 was administered intrathecally via a cisternal catheter or intravenously. Cerebral perfusion was determined by laser Doppler monitoring. Blood pressure was monitored by telemetry in a separate group of naïve animals treated with AZX100 for 24 hours. RESULTS: The maximal decrease in cerebral perfusion occurred 3 days after SAH. Cisternal administration of AZX100 (0.14-0.57 mg/kg) 24 hours after hemorrhage prevented decreases in cerebral perfusion after SAH. Animals receiving lower doses of AZX100 (0.068 mg/kg) or a scrambled sequence of the active HSP20 peptide linked to CPP developed decreases in cerebral perfusion similar to those seen in control animals. Intravenous administration of AZX100 (1.22 mg/kg) 24 hours after hemorrhage prevented the decreases in cerebral perfusion seen in the controls. Intravenous administration (0.175 mg/kg and 1.22 mg/kg) of AZX100 on Days 2 and 3 after SAH reversed decreases in cerebral perfusion as early as Day 3. There was no impact of AZX100 on blood pressure or heart rate at doses up to 2.73 mg/kg. CONCLUSIONS: Cisternal administration of AZX100 24 hours after hemorrhage prevented decreases in cerebral perfusion. Intravenous administration of AZX100 also prevented and reversed decreases in cerebral perfusion at doses that did not induce hypotension. Transduction of biologically active motifs of downstream regulators like HSP20 represents a potential novel treatment for SAH.


Subject(s)
Cerebrovascular Circulation/drug effects , Heat-Shock Proteins, Small/therapeutic use , Neuroprotective Agents/therapeutic use , Phosphoproteins/therapeutic use , Subarachnoid Hemorrhage/drug therapy , Animals , Biomimetics , Blood Pressure/drug effects , Disease Models, Animal , HSP20 Heat-Shock Proteins , Heart Rate/drug effects , Heat-Shock Proteins, Small/administration & dosage , Male , Neuroprotective Agents/administration & dosage , Phosphoproteins/administration & dosage , Rats , Rats, Wistar , Subarachnoid Hemorrhage/mortality , Time Factors
6.
Neurosurgery ; 51(1): 204-10; discussion 210-1, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12182419

ABSTRACT

OBJECTIVE: The mechanisms of cerebral vasospasm after subarachnoid hemorrhage (SAH) remain controversial. Recent data have implicated two small heat shock proteins (HSPs), namely HSP20 and HSP27, in the regulation of vascular tone. Increases in the phosphorylation of HSP20 are associated with vasorelaxation, and increases in the phosphorylation of HSP27 are associated with impaired vasorelaxation. Therefore, we hypothesized that alterations in the expression and/or phosphorylation of these two small HSPs might play a role in cerebral vasospasm after SAH. METHODS: A rat model of endovascular perforation was used to induce SAH. Middle cerebral arteries were harvested from control animals, sham-treated animals, and animals with SAH, 48 hours after SAH induction. Dose-response curves for endothelium-independent (sodium nitroprusside, 10(-8) to 10(-4) mol/L) and endothelium-dependent (bradykinin, 10(-10) to 10(-5) mol/L) relaxing agents were recorded ex vivo. Physiological responses were correlated with the expression and phosphorylation of HSP20 and HSP27 by using one- and two-dimensional immunoblots. RESULTS: There was impaired endothelium-independent and endothelium-dependent relaxation in cerebral vessels after SAH. These changes were associated with decreased expression of both total and phosphorylated HSP20 and increases in the amount of phosphorylated HSP27. CONCLUSION: In this model, impaired relaxation of cerebral vessels after SAH was associated with increases in the amount of phosphorylated HSP27 and decreases in the expression and phosphorylation of HSP20. These data are consistent with alterations in the expression and phosphorylation of these small HSPs in other models of vasospasm.


Subject(s)
Endothelium, Vascular/physiopathology , Heat-Shock Proteins/physiology , Phosphopeptides/physiology , Phosphoproteins/physiology , Subarachnoid Hemorrhage/physiopathology , Vasospasm, Intracranial/physiopathology , Animals , Cerebral Arteries/pathology , Cerebral Arteries/physiopathology , Disease Models, Animal , Endothelium, Vascular/pathology , HSP20 Heat-Shock Proteins , Male , Phosphorylation , Rats , Rats, Wistar , Subarachnoid Hemorrhage/pathology , Vasospasm, Intracranial/pathology
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