scholarly journals Normobaric hyperoxia stimulates superoxide and nitric oxide production in the caudal solitary complex of rat brain slices

2016 ◽  
Vol 311 (6) ◽  
pp. C1014-C1026 ◽  
Author(s):  
Geoffrey E. Ciarlone ◽  
Jay B. Dean
Keyword(s):  
Nitric Oxide ◽  
Brain Slice ◽  
Neuronal Excitability ◽  
Nitrosative Stress ◽  
Brain Slices ◽  
Normobaric Hyperoxia ◽  
Fluorogenic Dyes ◽  
Cellular Excitability ◽  
Cellular Dysfunction ◽  
Solitary Complex

Central CO2-chemosensitive neurons in the caudal solitary complex (cSC) are stimulated not only by hypercapnic acidosis, but by hyperoxia as well. While a cellular mechanism for the CO2response has yet to be isolated, previous data show that a redox-sensitive mechanism underlies neuronal excitability to hyperoxia. However, it remains unknown how changes in Po2affect the production of reactive oxygen and nitrogen species (RONS) in the cSC that can lead to increased cellular excitability and, with larger doses, to cellular dysfunction and death. To this end, we used fluorescence microscopy in real time to determine how normobaric hyperoxia increases the production of key RONS in the cSC. Because neurons in the region are CO2sensitive, we also examined the potential effects of CO2narcosis, used during euthanasia before brain slice harvesting, on RONS production. Our findings show that normobaric hyperoxia (0.4 → 0.95 atmospheres absolute O2) increases the fluorescence rates of fluorogenic dyes specific to both superoxide and nitric oxide. Interestingly, different results were seen for superoxide fluorescence when CO2narcosis was used during euthanasia, suggesting long-lasting changes in superoxide production and/or antioxidant activity subsequent to CO2narcosis before brain slicing. Further research needs to distinguish whether the increased levels of RONS reported here are merely increases in oxidative and nitrosative signaling or, alternatively, evidence of redox and nitrosative stress.

scholarly journals Oxygen measurements in brain stem slices exposed to normobaric hyperoxia and hyperbaric oxygen

2001 ◽  
Vol 90 (5) ◽  
pp. 1887-1899 ◽  
Author(s):  
Daniel K. Mulkey ◽  
Richard A. Henderson ◽  
James E. Olson ◽  
Robert W. Putnam ◽  
Jay B. Dean
Keyword(s):  
Hyperbaric Oxygen ◽  
Brain Slice ◽  
Barometric Pressure ◽  
Normobaric Hyperoxia ◽  
Carbon Fiber Microelectrodes ◽  
Solitary Complex ◽  
Oxygen Measurements ◽  
Brain Stem Slices ◽  
Stem Slices ◽  
Tissue Oxygen Consumption

We previously reported ( J Appl Physiol 89: 807–822, 2000) that ≤10 min of hyperbaric oxygen (HBO2; ≤2,468 Torr) stimulates solitary complex neurons. To better define the hyperoxic stimulus, we measured Po 2 in the solitary complex of 300-μm-thick rat medullary slices, using polarographic carbon fiber microelectrodes, during perfusion with media having Po 2 values ranging from 156 to 2,468 Torr. Under control conditions, slices equilibrated with 95% O2at barometric pressure of 1 atmospheres absolute had minimum Po 2 values at their centers (291 ± 20 Torr) that were ∼10-fold greater than Po 2values measured in the intact central nervous system (10–34 Torr). During HBO2, Po 2 increased at the center of the slice from 616 ± 16 to 1,517 ± 15 Torr. Tissue oxygen consumption tended to decrease at medium Po 2 ≥ 1,675 Torr to levels not different from values measured at Po 2 found in all media in metabolically poisoned slices (2-deoxy-d-glucose and antimycin A). We conclude that control medium used in most brain slice studies is hyperoxic at normobaric pressure. During HBO2, slice Po 2 increases to levels that appear to reduce metabolism.

scholarly journals Propofol Restores Brain Microvascular Function Impaired by High Glucose via  the Decrease in Oxidative Stress

2008 ◽  
Vol 108 (2) ◽  
pp. 269-275 ◽  
Author(s):  
Katsutoshi Nakahata ◽  
Hiroyuki Kinoshita ◽  
Toshiharu Azma ◽  
Naoyuki Matsuda ◽  
Keiko Hama-Tomioka ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
High Glucose ◽  
Brain Slice ◽  
Brain Slices ◽  
Neuronal Nitric Oxide Synthase ◽  
Superoxide Production ◽  
Oxide Synthase ◽  
The Brain

Background Vascular dysfunction induced by hyperglycemia has not been studied in cerebral parenchymal circulation. The current study was designed to examine whether high glucose impairs dilation of cerebral parenchymal arterioles via nitric oxide synthase, and whether propofol recovers this vasodilation by reducing superoxide levels in the brain. Methods Cerebral parenchymal arterioles in the rat brain slices were monitored using computer-assisted videomicroscopy. Vasodilation induced by acetylcholine (10 to 10 m) was obtained after the incubation of brain slices for 60 min with any addition of l-glucose (20 mm), d-glucose (20 mm), or propofol (3 x 10 or 10 m) in combination with d-glucose (20 mm). Superoxide production in the brain slice was determined by dihydroethidium (2 x 10 m) fluorescence. Results Addition of d-glucose, but not l-glucose, reduced arteriolar dilation by acetylcholine, whereas the dilation was abolished by the neuronal nitric oxide synthase inhibitor S-methyl-l-thiocitrulline (10 m). Both propofol and the superoxide dismutase mimetic Tempol (10 m) restored the arteriolar dilation in response to acetylcholine in the brain slice treated with d-glucose. Addition of d-glucose increased superoxide production in the brain slice, whereas propofol, Tempol, and the nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase inhibitor apocynin (1 mm) similarly inhibited it. Conclusions Clinically relevant concentrations of propofol ameliorate neuronal nitric oxide synthase-dependent dilation impaired by high glucose in the cerebral parenchymal arterioles via the effect on superoxide levels. Propofol may be protective against cerebral microvascular malfunction resulting from oxidative stress by acute hyperglycemia.

scholarly journals Acute hypercapnic hyperoxia stimulates reactive species production in the caudal solitary complex of rat brain slices but does not induce oxidative stress

2016 ◽  
Vol 311 (6) ◽  
pp. C1027-C1039 ◽  
Author(s):  
Geoffrey E. Ciarlone ◽  
Jay B. Dean
Keyword(s):  
Oxidative Stress ◽  
Rat Brain ◽  
Nitrosative Stress ◽  
Brain Slices ◽  
Radical Mechanism ◽  
Oxidative And Nitrosative Stress ◽  
Fenton Chemistry ◽  
Nitrated Lipids ◽  
Species Production ◽  
Solitary Complex

Central CO2 chemoreceptive neurons in the caudal solitary complex (cSC) are stimulated by hyperoxia via a free radical mechanism. Hyperoxia has been shown to increase superoxide and nitric oxide in the cSC, but it remains unknown how changes in Pco2 during hyperoxia affect the production of O2-dependent reactive oxygen and nitrogen species (RONS) downstream that can lead to increased levels of oxidative and nitrosative stress, cellular excitability, and, potentially, dysfunction. We used real-time fluorescence microscopy in rat brain slices to determine how hyperoxia and hypercapnic acidosis (HA) modulate one another in the production of key RONS, as well as colorimetric assays to measure levels of oxidized and nitrated lipids and proteins. We also examined the effects of CO2 narcosis and hypoxia before euthanasia and brain slice harvesting, as these neurons are CO2 sensitive and hypothesized to employ CO2/H+ mechanisms that exacerbate RONS production and potentially oxidative stress. Our findings show that hyperoxia ± HA increases the production of peroxynitrite and its derivatives, whereas increases in Fenton chemistry are most prominent during hyperoxia + HA. Using CO2 narcosis before euthanasia modulates cellular sensitivity to HA postmortem and enhances the magnitude of the peroxynitrite pathway, but blunts the activity of Fenton chemistry. Overall, hyperoxia and HA do not result in increased production of markers of oxidative and nitrosative stress as expected. We postulate this is due to antioxidant and proteosomal removal of damaged lipids and proteins to maintain cell viability and avoid death during protracted hyperoxia.

TMOD-31. AN ORGANOTYPIC TISSUE PLATFORM TO BRIDGE IN VITRO AND IN VIVO ASSAYS FOR BRAIN CANCER TREATMENT

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi222-vi222
Author(s):  
Breanna Mann ◽  
Noah Bell ◽  
Denise Dunn ◽  
Scott Floyd ◽  
Shawn Hingtgen ◽  
...  
Keyword(s):  
Cell Lines ◽  
Brain Cancer ◽  
Brain Slice ◽  
Brain Slices ◽  
In Vitro Assays ◽  
Preclinical Model ◽  
Short Period ◽  
The Brain

Abstract Brain cancers remain one of the greatest medical challenges. The lack of experimentally tractable models that recapitulate brain structure/function represents a major impediment. Platforms that enable functional testing in high-fidelity models are urgently needed to accelerate the identification and translation of therapies to improve outcomes for patients suffering from brain cancer. In vitro assays are often too simple and artificial while in vivo studies can be time-intensive and complicated. Our live, organotypic brain slice platform can be used to seed and grow brain cancer cell lines, allowing us to bridge the existing gap in models. These tumors can rapidly establish within the brain slice microenvironment, and morphologic features of the tumor can be seen within a short period of time. The growth, migration, and treatment dynamics of tumors seen on the slices recapitulate what is observed in vivo yet is missed by in vitro models. Additionally, the brain slice platform allows for the dual seeding of different cell lines to simulate characteristics of heterogeneous tumors. Furthermore, live brain slices with embedded tumor can be generated from tumor-bearing mice. This method allows us to quantify tumor burden more effectively and allows for treatment and retreatment of the slices to understand treatment response and resistance that may occur in vivo. This brain slice platform lays the groundwork for a new clinically relevant preclinical model which provides physiologically relevant answers in a short amount of time leading to an acceleration of therapeutic translation.

Renal and cardiac oxidative/nitrosative stress in salt-loaded pregnant rat

2007 ◽  
Vol 293 (4) ◽  
pp. R1657-R1665 ◽  
Author(s):  
Annie Beauséjour ◽  
Véronique Houde ◽  
Karine Bibeau ◽  
Rébecca Gaudet ◽  
Jean St-Louis ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Nitrosative Stress ◽  
Gestational Hypertension ◽  
Sodium Intake ◽  
Pregnant Rats ◽  
High Sodium ◽  
High Sodium Intake ◽  
Arterial Blood ◽  
Cardiac Ventricle

Sodium supplementation given for 1 wk to nonpregnant rats induces changes that are adequate to maintain renal and circulatory homeostasis as well as arterial blood pressure. However, in pregnant rats, proteinuria, fetal growth restriction, and placental oxidative stress are observed. Moreover, the decrease in blood pressure and expansion of circulatory volume, normally associated with pregnancy, are prevented by high-sodium intake. We hypothesized that, in these pregnant rats, a loss of the balance between prooxidation and antioxidation, particularly in kidneys and heart, disturbs the normal course of pregnancy and leads to manifestations such as gestational hypertension. We thus investigated the presence of oxidative/nitrosative stress in heart and kidneys following high-sodium intake in pregnant rats. Markers of this stress [8-isoprostaglandin F2α (8-iso-PGF2α) and nitrotyrosine], producer of nitric oxide [nitric oxide synthases (NOSs)], and antioxidants [superoxide dismutase (SOD) and catalase] were measured. Then, molecules (Na+-K+-ATPase and aconitase) or process [apoptosis (Bax and Bcl-2), inflammation (monocyte chemoattractant protein-1, connective tissue growth factor, and TNF-α)] susceptible to free radicals was determined. In kidneys from pregnant rats on 1.8% NaCl-water, NOSs, apoptotic index, and nitrotyrosine expression were increased, whereas Na+-K+-ATPase mRNA and activity were decreased. In the left cardiac ventricle of these rats, heightened nitrotyrosine, 8-iso-PGF2α, and catalase activity together with reduced endothelial NOS protein expression and SOD and aconitase activities were observed. These findings suggest that oxidative/nitrosative stress in kidney and left cardiac ventricle destabilizes the normal course of pregnancy and could lead to gestational hypertension.

scholarly journals Cooperative Roles of Nitric Oxide-Metabolizing Enzymes To Counteract Nitrosative Stress in Enterohemorrhagic Escherichia coli

2019 ◽  
Vol 87 (9) ◽  
Author(s):  
Takeshi Shimizu ◽  
Akio Matsumoto ◽  
Masatoshi Noda
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Stationary Phase ◽  
Nitrosative Stress ◽  
Enterohemorrhagic Escherichia Coli ◽  
The Other ◽  
Anaerobic Growth ◽  
Bacterial Cells ◽  
Metabolizing Enzymes ◽  
Microaerobic Conditions

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) has at least three enzymes, NorV, Hmp, and Hcp, that act independently to lower the toxicity of nitric oxide (NO), a potent antimicrobial molecule. This study aimed to reveal the cooperative roles of these defensive enzymes in EHEC against nitrosative stress. Under anaerobic conditions, combined deletion of all three enzymes significantly increased the NO sensitivity of EHEC determined by the growth at late stationary phase; however, the expression of norV restored the NO resistance of EHEC. On the other hand, the growth of Δhmp mutant EHEC was inhibited after early stationary phase, indicating that NorV and Hmp play a cooperative role in anaerobic growth. Under microaerobic conditions, the growth of Δhmp mutant EHEC was inhibited by NO, indicating that Hmp is the enzyme that protects cells from NO stress under microaerobic conditions. When EHEC cells were exposed to a lower concentration of NO, the NO level in bacterial cells of Δhcp mutant EHEC was higher than those of the other EHEC mutants, suggesting that Hcp is effective at regulating NO levels only at a low concentration. These findings of a low level of NO in bacterial cells with hcp indicate that the NO consumption activity of Hcp was suppressed by Hmp at a low range of NO concentrations. Taken together, these results show that the cooperative effects of NO-metabolizing enzymes are regulated by the range of NO concentrations to which the EHEC cells are exposed.

Major Role For Tonic GABAA Conductances in Anesthetic Suppression of Intrinsic Neuronal Excitability

2004 ◽  
Vol 92 (3) ◽  
pp. 1658-1667 ◽  
Author(s):  
Mark C. Bieda ◽  
M. Bruce MacIver
Keyword(s):  
Synaptic Transmission ◽  
Neuronal Excitability ◽  
Brain Slices ◽  
Pyramidal Cells ◽  
Intrinsic Excitability ◽  
High Potassium ◽  
Ca1 Neurons ◽  
Adult Rat ◽  
Adult Rat Brain ◽  
Sites Of Action

Anesthetics appear to produce neurodepression by altering synaptic transmission and/or intrinsic neuronal excitability. Propofol, a widely used anesthetic, has proposed effects on many targets, ranging from sodium channels to GABAA inhibition. We examined effects of propofol on the intrinsic excitability of hippocampal CA1 neurons (primarily interneurons) recorded from adult rat brain slices. Propofol strongly depressed action potential production induced by DC injection, synaptic stimulation, or high-potassium solutions. Propofol-induced depression of intrinsic excitability was completely reversed by bicuculline and picrotoxin but was strychnine-insensitive, implicating GABAA but not glycine receptors. Propofol strongly enhanced inhibitory postsynaptic currents (IPSCs) and induced a tonic GABAA-mediated current. We pharmacologically differentiated tonic and phasic (synaptic) GABAA-mediated inhibition using the GABAA receptor antagonist SR95531 (gabazine). Gabazine (20 μM) completely blocked both evoked and spontaneous IPSCs but failed to block the propofol-induced depression of intrinsic excitability, implicating tonic, but not phasic, GABAA inhibition. Glutamatergic synaptic responses were not altered by propofol (≤30 μM). Similar results were found in both interneurons and pyramidal cells and with the chemically unrelated anesthetic thiopental. These results suggest that suppression of CA1 neuron intrinsic excitability, by these anesthetics, is largely due to activation of tonic GABAA conductances; although other sites of action may play important roles in affecting synaptic transmission, which also can produce strong neurodepression. We propose that for some anesthetics, suppression of intrinsic excitability, mediated by tonic GABAA conductances, operates in conjunction with effects on synaptic transmission, mediated by other mechanisms, to depress hippocampal function during anesthesia.

scholarly journals The yjeB (nsrR) Gene of Escherichia coli Encodes a Nitric Oxide-Sensitive Transcriptional Regulator

2006 ◽  
Vol 188 (3) ◽  
pp. 874-881 ◽  
Author(s):  
Diane M. Bodenmiller ◽  
Stephen Spiro
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Inverted Repeat ◽  
Nitrosative Stress ◽  
Protein A ◽  
Repeat Sequence ◽  
Negative Regulator ◽  
Inverted Repeat Sequence ◽  
Multiple Copies ◽  
Regulatory Model

ABSTRACT Microarray studies of the Escherichia coli response to nitric oxide and nitrosative stress have suggested that additional transcriptional regulators of this response remain to be characterized. We identify here the product of the yjeB gene as a negative regulator of the transcription of the ytfE, hmpA and ygbA genes, all of which are known to be upregulated by nitrosative stress. Transcriptional fusions to the promoters of these genes were expressed constitutively in a yjeB mutant, indicating that all three are targets for repression by YjeB. An inverted repeat sequence that overlaps the −10 element of all three promoters is proposed to be a binding site for the YjeB protein. A similar inverted repeat sequence was identified in the tehA promoter, which is also known to be sensitive to nitrosative stress. The ytfE, hmpA, ygbA, and tehA promoters all caused derepression of a ytfE-lacZ transcriptional fusion when present in the cell in multiple copies, presumably by a repressor titration effect, suggesting the presence of functional YjeB binding sites in these promoters. However, YjeB regulation of tehA was weak, as judged by the activity of a tehA-lacZ fusion, perhaps because YjeB repression of tehA is masked by other regulatory mechanisms. Promoters regulated by YjeB could be derepressed by iron limitation, which is consistent with an iron requirement for YjeB activity. The YjeB protein is a member of the Rrf2 family of transcriptional repressors and shares three conserved cysteine residues with its closest relatives. We propose a regulatory model in which the YjeB repressor is directly sensitive to nitrosative stress. On the basis of similarity to the nitrite-responsive repressor NsrR from Nitrosomonas europaea, we propose that the yjeB gene of E. coli be renamed nsrR.

scholarly journals Formin Activity and mDia1 Contribute to Maintain Axon Initial Segment Composition and Structure

2021 ◽  
Author(s):  
Wei Zhang ◽  
María Ciorraga ◽  
Pablo Mendez ◽  
Diana Retana ◽  
Norah Boumedine-Guignon ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Initial Segment ◽  
Protein Transport ◽  
Neuronal Excitability ◽  
Brain Slices ◽  
Axon Initial Segment ◽  
Composition And Structure ◽  
The Stability ◽  
Protein Density ◽  
Voltage Gated Ion Channels

AbstractThe axon initial segment (AIS) is essential for maintaining neuronal polarity, modulating protein transport into the axon, and action potential generation. These functions are supported by a distinctive actin and microtubule cytoskeleton that controls axonal trafficking and maintains a high density of voltage-gated ion channels linked by scaffold proteins to the AIS cytoskeleton. However, our knowledge of the mechanisms and proteins involved in AIS cytoskeleton regulation to maintain or modulate AIS structure is limited. In this context, formins play a significant role in the modulation of actin and microtubules. We show that pharmacological inhibition of formins modifies AIS actin and microtubule characteristics in cultured hippocampal neurons, reducing F-actin density and decreasing microtubule acetylation. Moreover, formin inhibition diminishes sodium channels, ankyrinG and βIV-spectrin AIS density, and AIS length, in cultured neurons and brain slices, accompanied by decreased neuronal excitability. We show that genetic downregulation of the mDia1 formin by interference RNAs also decreases AIS protein density and shortens AIS length. The ankyrinG decrease and AIS shortening observed in pharmacologically inhibited neurons and neuron-expressing mDia1 shRNAs were impaired by HDAC6 downregulation or EB1-GFP expression, known to increase microtubule acetylation or stability. However, actin stabilization only partially prevented AIS shortening without affecting AIS protein density loss. These results suggest that mDia1 maintain AIS composition and length contributing to the stability of AIS microtubules.

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