Propentdyopents as Heme Degradation Intermediates Constrict Mouse Cerebral Arterioles and Are Present in the Cerebrospinal Fluid of Patients With Subarachnoid Hemorrhage

Rationale: Delayed ischemic neurological deficit is the most common cause of neurological impairment and unfavorable prognosis in patients with subarachnoid hemorrhage (SAH). Despite the existence of neuroimaging modalities that depict the onset of the accompanying cerebral vasospasm, preventive and therapeutic options are limited and fail to improve outcome owing to an insufficient pathomechanistic understanding of the delayed perfusion deficit. Previous studies have suggested that BOXes (bilirubin oxidation end products), originating from released heme surrounding ruptured blood vessels, are involved in arterial vasoconstriction. Recently, isolated intermediates of oxidative bilirubin degradation, known as PDPs (propentdyopents), have been considered as potential additional effectors in the development of arterial vasoconstriction. Objective: To investigate whether PDPs and BOXes are present in hemorrhagic cerebrospinal fluid and involved in the vasoconstriction of cerebral arterioles. Methods and Results: Via liquid chromatography/mass spectrometry, we measured increased PDP and BOX concentrations in cerebrospinal fluid of SAH patients compared with control subjects. Using differential interference contrast microscopy, we analyzed the vasoactivity of PDP isomers in vitro by monitoring the arteriolar diameter in mouse acute brain slices. We found an arteriolar constriction on application of PDPs in the concentration range that occurs in the cerebrospinal fluid of patients with SAH. By imaging arteriolar diameter changes using 2-photon microscopy in vivo, we demonstrated a short-onset vasoconstriction after intrathecal injection of either PDPs or BOXes. Using magnetic resonance imaging, we observed a long-term PDP-induced delay in cerebral perfusion. For all conditions, the arteriolar narrowing was dependent on functional big conductance potassium channels and was absent in big conductance potassium channels knockout mice. Conclusions: For the first time, we have quantified significantly higher concentrations of PDP and BOX isomers in the cerebrospinal fluid of patients with SAH compared to controls. The vasoconstrictive effect caused by PDPs in vitro and in vivo suggests a hitherto unrecognized pathway contributing to the pathogenesis of delayed ischemic deficit in patients with SAH.

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Acute changes in neurovascular reactivity after subarachnoid hemorrhage in vivo

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x15621253 ◽

2016 ◽

Vol 37 (1) ◽

pp. 178-187 ◽

Cited By ~ 9

Author(s):

Matilde Balbi ◽

Masayo Koide ◽

Susanne M Schwarzmaier ◽

George C Wellman ◽

Nikolaus Plesnila

Keyword(s):

Subarachnoid Hemorrhage ◽

Neuronal Damage ◽

Brain Slices ◽

Neurovascular Coupling ◽

Differential Interference Contrast Microscopy ◽

Two Photon Microscopy ◽

Two Photon ◽

Normal Increase ◽

Acute Changes

Subarachnoid hemorrhage causes acute and long-lasting constrictions of pial arterioles. Whether these vessels dilate normally to neuronal activity is of great interest since a mismatch between delivery and consumption of glucose and oxygen may cause additional neuronal damage. Therefore, we investigated neurovascular reactivity of pial and parenchymal arterioles after experimental subarachnoid hemorrhage. C57BL/6 mice were subjected to subarachnoid hemorrhage by filament perforation or sham surgery. Neurovascular reactivity was assessed 3 h later by forepaw stimulation or inhalation of 10% CO2. Diameters of cerebral arterioles were assessed using two-photon microscopy. Neurovascular coupling and astrocytic endfoot Ca2+ were measured in brain slices using two-photon and infrared-differential interference contrast microscopy. Vessels of sham-operated mice dilated normally to CO2 and forepaw stimulation. Three hours after subarachnoid hemorrhage, CO2 reactivity was completely lost in both pial and parenchymal arterioles, while neurovascular coupling was not affected. Brain slices studies also showed normal neurovascular coupling and a normal increase in astrocytic endfoot Ca2+ acutely after subarachnoid hemorrhage. These findings suggest that communication between neurons, astrocytes, and parenchymal arterioles is not affected in the first few hours after subarachnoid hemorrhage, while CO2 reactivity, which is dependent on NO signaling, is completely lost.

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Dynamic Inositol Trisphosphate-mediated Calcium Signals within Astrocytic Endfeet Underlie Vasodilation of Cerebral Arterioles

The Journal of General Physiology ◽

10.1085/jgp.200609650 ◽

2006 ◽

Vol 128 (6) ◽

pp. 659-669 ◽

Cited By ~ 56

Author(s):

Stephen V. Straub ◽

Adrian D. Bonev ◽

M. Keith Wilkerson ◽

Mark T. Nelson

Keyword(s):

Flash Photolysis ◽

Ryanodine Receptors ◽

Brain Slices ◽

Spatiotemporal Resolution ◽

Temporal Properties ◽

Cerebral Arterioles ◽

High Degree ◽

Astrocytic Endfeet ◽

Arteriolar Diameter

Active neurons communicate to intracerebral arterioles in part through an elevation of cytosolic Ca2+ concentration ([Ca2+]i) in astrocytes, leading to the generation of vasoactive signals involved in neurovascular coupling. In particular, [Ca2+]i increases in astrocytic processes (“endfeet”), which encase cerebral arterioles, have been shown to result in vasodilation of arterioles in vivo. However, the spatial and temporal properties of endfoot [Ca2+]i signals have not been characterized, and information regarding the mechanism by which these signals arise is lacking. [Ca2+]i signaling in astrocytic endfeet was measured with high spatiotemporal resolution in cortical brain slices, using a fluorescent Ca2+ indicator and confocal microscopy. Increases in endfoot [Ca2+]i preceded vasodilation of arterioles within cortical slices, as detected by simultaneous measurement of endfoot [Ca2+]i and vascular diameter. Neuronal activity–evoked elevation of endfoot [Ca2+]i was reduced by inhibition of inositol 1,4,5-trisphosphate (InsP3) receptor Ca2+ release channels and almost completely abolished by inhibition of endoplasmic reticulum Ca2+ uptake. To probe the Ca2+ release mechanisms present within endfeet, spatially restricted flash photolysis of caged InsP3 was utilized to liberate InsP3 directly within endfeet. This maneuver generated large amplitude [Ca2+]i increases within endfeet that were spatially restricted to this region of the astrocyte. These InsP3-induced [Ca2+]i increases were sensitive to depletion of the intracellular Ca2+ store, but not to ryanodine, suggesting that Ca2+-induced Ca2+ release from ryanodine receptors does not contribute to the generation of endfoot [Ca2+]i signals. Neuronally evoked increases in astrocytic [Ca2+]i propagated through perivascular astrocytic processes and endfeet as multiple, distinct [Ca2+]i waves and exhibited a high degree of spatial heterogeneity. Regenerative Ca2+ release processes within the endfeet were evident, as were localized regions of Ca2+ release, and treatment of slices with the vasoactive neuropeptides somatostatin and vasoactive intestinal peptide was capable of inducing endfoot [Ca2+]i increases, suggesting the potential for signaling between local interneurons and astrocytic endfeet in the cortex. Furthermore, photorelease of InsP3 within individual endfeet resulted in a local vasodilation of adjacent arterioles, supporting the concept that astrocytic endfeet function as local “vasoregulatory units” by translating information from active neurons into complex InsP3-mediated Ca2+ release signals that modulate arteriolar diameter.

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Functional analysis of Pro-inflammatory properties within the cerebrospinal fluid after subarachnoid hemorrhage in vivo and in vitro

Journal of Neuroinflammation ◽

10.1186/1742-2094-9-28 ◽

2012 ◽

Vol 9 (1) ◽

Cited By ~ 20

Author(s):

Ulf C Schneider ◽

Jennifer Schiffler ◽

Nahid Hakiy ◽

Peter Horn ◽

Peter Vajkoczy

Keyword(s):

Functional Analysis ◽

Cerebrospinal Fluid ◽

Subarachnoid Hemorrhage

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Inversion of neurovascular coupling after subarachnoid hemorrhage in vivo

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x16686595 ◽

2017 ◽

Vol 37 (11) ◽

pp. 3625-3634 ◽

Cited By ~ 23

Author(s):

Matilde Balbi ◽

Masayo Koide ◽

George C Wellman ◽

Nikolaus Plesnila

Keyword(s):

Blood Flow ◽

Cerebral Blood Flow ◽

Subarachnoid Hemorrhage ◽

Neuronal Activity ◽

Ex Vivo ◽

Brain Slices ◽

Neurovascular Coupling ◽

Vessel Diameter ◽

Cerebral Arterioles

Subarachnoid hemorrhage (SAH) induces acute changes in the cerebral microcirculation. Recent findings ex vivo suggest neurovascular coupling (NVC), the process that increases cerebral blood flow upon neuronal activity, is also impaired after SAH. The aim of the current study was to investigate whether this occurs also in vivo. C57BL/6 mice were subjected to either sham surgery or SAH by filament perforation. Twenty-four hours later NVC was tested by forepaw stimulation and CO2 reactivity by inhalation of 10% CO2. Vessel diameter was assessed in vivo by two-photon microscopy. NVC was also investigated ex vivo using brain slices. Cerebral arterioles of sham-operated mice dilated to 130% of baseline upon CO2 inhalation or forepaw stimulation and cerebral blood flow (CBF) increased. Following SAH, however, CO2 reactivity was completely lost and the majority of cerebral arterioles showed paradoxical constriction in vivo and ex vivo resulting in a reduced CBF response. As previous results showed intact NVC 3 h after SAH, the current findings indicate that impairment of NVC after cerebral hemorrhage occurs secondarily and is progressive. Since neuronal activity-induced vasoconstriction (inverse NVC) is likely to further aggravate SAH-induced cerebral ischemia and subsequent brain damage, inverse NVC may represent a novel therapeutic target after SAH.

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Targeting Potassium Channels to Reprogram Glioblastoma Microenvironment: in Vitro and in Vivo Studies

Case Medical Research ◽

10.31525/ct1-nct03954691 ◽

2019 ◽

Author(s):

Keyword(s):

Potassium Channels ◽

In Vivo Studies

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TRAF3 mediates neuronal apoptosis in early brain injury following subarachnoid hemorrhage via targeting TAK1-dependent MAPKs and NF-κB pathways

Cell Death and Disease ◽

10.1038/s41419-020-03278-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yan Zhou ◽

Tao Tao ◽

Guangjie Liu ◽

Xuan Gao ◽

Yongyue Gao ◽

...

Keyword(s):

Subarachnoid Hemorrhage ◽

Brain Injury ◽

Therapeutic Target ◽

Cortical Neurons ◽

Neuronal Apoptosis ◽

Early Brain Injury ◽

Neurological Deficits ◽

Human Brain Tissue

AbstractNeuronal apoptosis has an important role in early brain injury (EBI) following subarachnoid hemorrhage (SAH). TRAF3 was reported as a promising therapeutic target for stroke management, which covered several neuronal apoptosis signaling cascades. Hence, the present study is aimed to determine whether downregulation of TRAF3 could be neuroprotective in SAH-induced EBI. An in vivo SAH model in mice was established by endovascular perforation. Meanwhile, primary cultured cortical neurons of mice treated with oxygen hemoglobin were applied to mimic SAH in vitro. Our results demonstrated that TRAF3 protein expression increased and expressed in neurons both in vivo and in vitro SAH models. TRAF3 siRNA reversed neuronal loss and improved neurological deficits in SAH mice, and reduced cell death in SAH primary neurons. Mechanistically, we found that TRAF3 directly binds to TAK1 and potentiates phosphorylation and activation of TAK1, which further enhances the activation of NF-κB and MAPKs pathways to induce neuronal apoptosis. Importantly, TRAF3 expression was elevated following SAH in human brain tissue and was mainly expressed in neurons. Taken together, our study demonstrates that TRAF3 is an upstream regulator of MAPKs and NF-κB pathways in SAH-induced EBI via its interaction with and activation of TAK1. Furthermore, the TRAF3 may serve as a novel therapeutic target in SAH-induced EBI.

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KCNN4 promotes the progression of lung adenocarcinoma by activating the AKT and ERK signaling pathways

Cancer Biomarkers ◽

10.3233/cbm-201045 ◽

2021 ◽

pp. 1-15

Author(s):

Ping Xu ◽

Xiao Mo ◽

Ruixue Xia ◽

Long Jiang ◽

Chengfei Zhang ◽

...

Keyword(s):

Potassium Channels ◽

Lung Adenocarcinoma ◽

Potassium Channel ◽

Signaling Pathways ◽

Epithelial To Mesenchymal Transition ◽

Tumor Biology ◽

Erk Signaling ◽

Mesenchymal Transition

BACKGROUND: Potassium channels, encoded by more than seventy genes, are cell excitability transmembrane proteins and become evident to play essential roles in tumor biology. OBJECTIVE: The deregulation of potassium channel genes has been related to cancer development and patient prognosis. The objective of this study is to understand the role of potassium channels in lung cancer. METHODS: We examined all potassium channel genes and identified that KCNN4 is the most significantly overexpressed one in lung adenocarcinoma. The role and mechanism of KCNN4 in lung adenocarcinoma were further investigated by in vitro cell and molecular assay and in vivo mouse xenograft models. RESULTS: We revealed that the silencing of KCNN4 significantly inhibits cell proliferation, migration, invasion, and tumorigenicity of lung adenocarcinoma. Further studies showed that knockdown of KCNN4 promotes cell apoptosis, induces cell cycle arrested in the S phase, and is associated with the epithelial to mesenchymal transition (EMT) process. Most importantly, we demonstrated that KCNN4 regulates the progression of lung adenocarcinoma through P13K/AKT and MEK/ERK signaling pathways. The use of inhibitors that targeted AKT and ERK also significantly inhibit the proliferation and metastasis of lung adenocarcinoma cells. CONCLUSIONS: This study investigated the function and mechanism of KCNN4 in lung adenocarcinoma. On this basis, this means that KCNN4 can be used as a tumor marker for lung adenocarcinoma and is expected to become an important target for a potential drug.

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Development of 18F-Labeled Radiotracers for PET Imaging of the Adenosine A2A Receptor: Synthesis, Radiolabeling and Preliminary Biological Evaluation

International Journal of Molecular Sciences ◽

10.3390/ijms22052285 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2285

Author(s):

Thu Hang Lai ◽

Susann Schröder ◽

Magali Toussaint ◽

Sladjana Dukić-Stefanović ◽

Mathias Kranz ◽

...

Keyword(s):

Specific Binding ◽

Brain Slices ◽

Total Activity ◽

Biological Evaluation ◽

Adenosine A2a Receptor ◽

Positron Emission ◽

A2a Receptor ◽

Adenosine A2a

The adenosine A2A receptor (A2AR) represents a potential therapeutic target for neurodegenerative diseases. Aiming at the development of a positron emission tomography (PET) radiotracer to monitor changes of receptor density and/or occupancy during the A2AR-tailored therapy, we designed a library of fluorinated analogs based on a recently published lead compound (PPY). Among those, the highly affine 4-fluorobenzyl derivate (PPY1; Ki(hA2AR) = 5.3 nM) and the 2-fluorobenzyl derivate (PPY2; Ki(hA2AR) = 2.1 nM) were chosen for 18F-labeling via an alcohol-enhanced copper-mediated procedure starting from the corresponding boronic acid pinacol ester precursors. Investigations of the metabolic stability of [18F]PPY1 and [18F]PPY2 in CD-1 mice by radio-HPLC analysis revealed parent fractions of more than 76% of total activity in the brain. Specific binding of [18F]PPY2 on mice brain slices was demonstrated by in vitro autoradiography. In vivo PET/magnetic resonance imaging (MRI) studies in CD-1 mice revealed a reasonable high initial brain uptake for both radiotracers, followed by a fast clearance.

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Physiology, Pharmacology, and Topography of Cholinergic Neocortical Oscillations In Vitro

Journal of Neurophysiology ◽

10.1152/jn.1997.77.5.2427 ◽

1997 ◽

Vol 77 (5) ◽

pp. 2427-2445 ◽

Cited By ~ 61

Author(s):

Heath S. Lukatch ◽

M. Bruce Maciver

Keyword(s):

Current Source ◽

Brain Slices ◽

Receptor Activation ◽

Brain Regions ◽

Oscillatory Activity ◽

Cortical Layers ◽

Eeg Activity ◽

Layer 2

Lukatch, Heath S. and M. Bruce MacIver. Physiology, pharmacology, and topography of cholinergic neocortical oscillations in vitro. J. Neurophysiol. 77: 2427–2445, 1997. Rat neocortical brain slices generated rhythmic extracellular field [microelectroencephalogram (micro-EEG)] oscillations at theta frequencies (3–12 Hz) when exposed to pharmacological conditions that mimicked endogenous ascending cholinergic and GABAergic inputs. Use of the specific receptor agonist and antagonist carbachol and bicuculline revealed that simultaneous muscarinic receptor activation and γ-aminobutyric acid-A (GABAA)-mediated disinhibition werenecessary to elicit neocortical oscillations. Rhythmic activity was independent of GABAB receptor activation, but required intact glutamatergic transmission, evidenced by blockade or disruption of oscillations by 6-cyano-7-nitroquinoxaline-2,3-dione and (±)-2-amino-5-phosphonovaleric acid, respectively. Multisite mapping studies showed that oscillations were localized to areas 29d and 18b (Oc2MM) and parts of areas 18a and 17. Peak oscillation amplitudes occurred in layer 2/3, and phase reversals were observed in layers 1 and 5. Current source density analysis revealed large-amplitude current sinks and sources in layers 2/3 and 5, respectively. An initial shift in peak inward current density from layer 1 to layer 2/3 indicated that two processes underlie an initial depolarization followed by oscillatory activity. Laminar transections localized oscillation-generating circuitry to superficial cortical layers and sharp-spike-generating circuitry to deep cortical layers. Whole cell recordings identified three distinct cell types based on response properties during rhythmic micro-EEG activity: oscillation-on (theta-on) and -off (theta-off) neurons, and transiently depolarizing glial cells. Theta-on neurons displayed membrane potential oscillations that increased in amplitude with hyperpolarization (from −30 to −90 mV). This, taken together with a glutamate antagonist-induced depression of rhythmic micro-EEG activity, indicated that cholinergically driven neocortical oscillations require excitatory synaptic transmission. We conclude that under the appropriate pharmacological conditions, neocortical brain slices were capable of producing localized theta frequency oscillations. Experiments examining oscillation physiology, pharmacology, and topography demonstrated that neocortical brain slice oscillations share many similarities with the in vivo and in vitro theta EEG activity recorded in other brain regions.

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