An in vitro ischemic penumbral mimic perfusate increases NADPH oxidase-mediated superoxide production in cultured hippocampal neurons

Pamenter, Matthew E.; Ali, Sameh S.; Tang, Qingbo; Finley, J. Cameron; Gu, Xiang; Dugan, Laura L.; Haddad, Gabriel G.

doi:10.1016/j.brainres.2012.03.004

Cited by 21 publications

(32 citation statements)

References 39 publications

(60 reference statements)

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“…In hippocampal slice models and in cell cultures, mammalian neurons become electrically excited and rapidly depolarize within minutes of IS onset at 241C or 371C, and V m does not recover after normoxic reperfusion (Yao and Haddad, unpublished observations). Furthermore, after a 24-hour IS treatment B90% of neurons take up PI and > 90% of cellular lactate dehydrogenase is released (Yao et al, 2007), while [ATP] is depleted > 85%, and DNA exhibits extensive oligonucleosomal fragmentation and TUNEL (terminal deoxynucleotidyl transferase-mediated 2 0 -deoxyuridine 5 0 -triphosphatebiotin nick end labeling)-positive staining characteristic of late-stage apoptosis (Galluzzi et al, 2007;Pamenter et al, 2010Pamenter et al, , 2012. Similar results have been reported in a wide variety of in vivo and in vitro models of the ischemic mammalian penumbra, where in general ischemic stress induces: electrical hyperexcitation and extreme V m depolarization within minutes; ionic and neurotransmitter derangements, [ATP] depletion, and necrotic cell rupture or extensive activation of apoptotic mechanisms within < 1 to 4 hours; and extensive or total neuronal cell death within 6 to 24 hours (Broughton et al, 2009;Hansen and Nedergaard, 1988;Nedergaard and Hansen, 1993;Siesjo, 1992).…”

Section: Discussionmentioning

confidence: 99%

Painted Turtle Cortex is Resistant to anin VitroMimic of the Ischemic Mammalian Penumbra

Pamenter

Hogg

et al. 2012

J Cereb Blood Flow Metab

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Anoxia or ischemia causes hyperexcitability and cell death in mammalian neurons. Conversely, in painted turtle brain anoxia increases c-amino butyric acid (GABA)ergic suppression of spontaneous electrical activity, and cell death is prevented. To examine ischemia tolerance in turtle neurons, we treated cortical sheets with an in vitro mimic of the penumbral region of strokeafflicted mammalian brain (ischemic solution, IS). We found that during IS perfusion, neuronal membrane potential (V m ) and the GABA A receptor reversal potential depolarized to a similar steady state (À92 ± 2 to À28 ± 3 mV, and À75 ± 1 to À35 ± 3 mV, respectively), and whole-cell conductance (G w ) increased > 3-fold (from 4 ± 0.2 to 15 ± 1 nS). These neurons were electrically quiet and changes reversed after reperfusion. GABA receptor antagonism prevented the IS-mediated increase in G w and neurons exhibited enhanced electrical excitability and rapid and irreversible rundown of V m during reperfusion. These results suggest that inhibitory GABAergic mechanisms also suppress electrical activity in ischemic cortex. Indeed, after 4 hours of IS treatment neurons did not exhibit any apparent damage; while at 24 hours, only early indicators of apoptosis were present. We conclude that anoxia-tolerant turtle neurons are tolerant of exposure to a mammalian ischemic penumbral mimic solution.

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Section: Discussionmentioning

confidence: 99%

Painted Turtle Cortex is Resistant to anin VitroMimic of the Ischemic Mammalian Penumbra

Pamenter

Hogg

et al. 2012

J Cereb Blood Flow Metab

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“…Samples were treated for up to 48 hrs (as indicated) in four primary treatment categories: (1) cell death-negative control (“treatment media”, comprised of DMEM/F12 media (Invitrogen) supplemented with 1% bovine calf serum and 1% Pen/Strep), gassed with 21% O 2 , 5% CO 2 , balance N 2 , (2) an ischemic penumbral perfusate mimic (IS, in mM: K + 64, Na + 51, Cl − 77.5, Ca 2+ 0.13, Mg 2+ 1.5, glucose 3.0, glutamate 0.1, [315 mOsM, pH 6.5, 1.5% O 2 , 15% CO 2 , balance N 2 ]) [11], [17], [20], (3) an apoptosis-positive control (treatment media containing the pro-apoptotic agent STS (2.5 µM)), and (4) an autophagy-positive control (treatment media containing Oligomycin A (10 µM)). Many stages of apoptosis are ATP-dependent [26], therefore we chose to examine cellular viability and cell death pathway activation in the first 24–48 hours of treatment since in ATP-luciferase experiments cellular [ATP] was wholly depleted in all non-control experimental groups and cell types following 24–48 hours of treatment.…”

Section: Methodsmentioning

confidence: 99%

“…To address this need, ‘ischemic solution’ (IS) mimics the key ionic, pH, O 2 %, glucose, and neurotransmitter changes previously discovered in the ischemic penumbra in vivo [11], [17]. Preliminary investigations indicate that IS induces deleterious reactive oxygen species generation and up-regulation of innate immune pathways in primary neurons and cell lines, similar to changes observed in the ischemic penumbra in vivo [18], [19], [20], [21]. Nonetheless, the cell death and stress pathways activated by this penumbral mimic model remain poorly understood.…”

Section: Introductionmentioning

confidence: 98%

Autophagy and Apoptosis Are Differentially Induced in Neurons and Astrocytes Treated with an In Vitro Mimic of the Ischemic Penumbra

et al. 2012

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The development of clinical stroke therapies remains elusive. The neuroprotective efficacies of thousands of molecules and compounds have not yet been determined; however, screening large volumes of potential targets in vivo is severely rate limiting. High throughput screens (HTS) may be used to discover promising candidates, but this approach has been hindered by the lack of a simple in vitro model of the ischemic penumbra, a clinically relevant region of stroke-afflicted brain. Recently, our laboratory developed such a mimic (ischemic solution: IS) suitable for HTS, but the etiology of stress pathways activated by this model are poorly understood. The aim of the present study was to determine if the cell death phenotype induced by IS accurately mimics the in vivo penumbra and thus whether our model system is suitable for use in HTS. We treated cultured neuron and astrocyte cell lines with IS for up to 48 hrs and examined cellular energy state ([ATP]), cell and organelle morphology, and gene and molecular profiles related to stress pathways. We found that IS-treated cells exhibited a phenotype of mixed apoptosis/autophagy characteristic of the in vivo penumbra, including: (1) short-term elevation of [ATP] followed by progressive ATP depletion and Poly ADP Ribose Polymerase cleavage, (2) increased vacuole number in the cytoplasm, (3) mitochondrial rupture, decreased mitochondrial and cristae density, release of cytochrome C and apoptosis inducing factor, (4) chromatin condensation, nuclear lamin A and DNA cleavage, fragmentation of the nuclear envelope, and (5) altered expression of mRNA and proteins consistent with autophagy and apoptosis. We conclude that our in vitro model of the ischemic penumbra induces autophagy and apoptosis in cultured neuron and astrocyte cell lines and that this mimic solution is suitable for use in HTS to elucidate neuroprotective candidates against ischemic penumbral cell death.

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“…First, inhibition of NOX2 with either apocynin (139,169,172), diphenylene iodonium (7), or a tatconjugated peptide inhibitor of NOX2 assembly (16,17,150) all block superoxide formation and neuronal death that otherwise result from NMDA receptor activation. Second, p47…”

Section: Fig 1 Nadph Oxidase (Nox)mentioning

confidence: 99%

NADPH Oxidase-2: Linking Glucose, Acidosis, and Excitotoxicity in Stroke

Brennan-Minnella

Won

Swanson

2015

Antioxidants & Redox Signaling

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Significance: Neuronal superoxide production contributes to cell death in both glutamate excitotoxicity and brain ischemia (stroke). NADPH oxidase-2 (NOX2) is the major source of neuronal superoxide production in these settings, and regulation of NOX2 activity can thereby influence outcome in stroke. Recent Advances: Reduced NOX2 activity can rescue cells from oxidative stress and cell death that otherwise occur in excitotoxicity and ischemia. NOX2 activity is regulated by several factors previously shown to affect outcome in stroke, including glucose availability, intracellular pH, protein kinase f/d, casein kinase 2, phosphoinositide-3-kinase, Rac1/2, and phospholipase A2. The newly identified functions of these factors as regulators of NOX2 activity suggest alternative mechanisms for their effects on ischemic brain injury. Critical Issues: Key aspects of these regulatory influences remain unresolved, including the mechanisms by which rac1 and phospholipase activities are coupled to N-methyl-D-aspartate (NMDA) receptors, and whether superoxide production by NOX2 triggers subsequent superoxide production by mitochondria. Future Directions: It will be important to establish whether interventions targeting the signaling pathways linking NMDA receptors to NOX2 in brain ischemia can provide a greater neuroprotective efficacy or a longer time window to treatment than provided by NMDA receptor blockade alone. It will likewise be important to determine whether dissociating superoxide production from the other signaling events initiated by NMDA receptors can mitigate the deleterious effects of NMDA receptor blockade. Antioxid. Redox Signal. 22,[161][162][163][164][165][166][167][168][169][170][171][172][173][174]

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An in vitro ischemic penumbral mimic perfusate increases NADPH oxidase-mediated superoxide production in cultured hippocampal neurons

Cited by 21 publications

References 39 publications

Painted Turtle Cortex is Resistant to anin VitroMimic of the Ischemic Mammalian Penumbra

Painted Turtle Cortex is Resistant to anin VitroMimic of the Ischemic Mammalian Penumbra

Autophagy and Apoptosis Are Differentially Induced in Neurons and Astrocytes Treated with an In Vitro Mimic of the Ischemic Penumbra

NADPH Oxidase-2: Linking Glucose, Acidosis, and Excitotoxicity in Stroke

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