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In JoVE (1)
Other Publications (18)
- Chemical Communications (Cambridge, England)
- Molecular and Cellular Biology
- Chemical Communications (Cambridge, England)
- Oncogene
- Molecular and Cellular Biology
- Langmuir : the ACS Journal of Surfaces and Colloids
- Journal of Colloid and Interface Science
- NeuroImage
- Optics Letters
- Optics Express
- Optics Express
- NeuroImage
- Optics Letters
- Brain Research
- Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism
- Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism
- Journal of Biomedical Optics
- Cell Research
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Articles by Weicheng Wu in JoVE
JoVE Neuroscience
الأكسجة الدماغية قياس الأوكسجين في الدم واستنادا إلى الرواية التي تعتمد على التفسفر
1Optics Division, Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 2Department of Biochemistry and Biophysics, University of Pennsylvania, 3Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, 4Departments of Neurosciences and Radiology, University of California
نقدم إجراء التجارب لقياس الضغط الجزئي للأوكسجين (PO2) في الأوعية الدموية الدماغية استنادا الأوكسجين التي تعتمد على التبريد من تفسفر. وقد حددت إعداد الحيوان وإجراءات التصوير لكلا ميدانية واسعة من التصوير CCD القائم على ضوء PO2 في الفئران و2 - الفوتون التصوير القائم على الإثارة PO2 في الفئران.
Other articles by Weicheng Wu on PubMed
A Novel Catalyst for Hydrazine Decomposition: Molybdenum Carbide Support on Gamma-Al2O3
An alumina-supported Mo2C catalyst is found to be as active as a conventionally used Ir/gamma-Al2O3 catalyst for catalytic decomposition of hydrazine tested in a monopropellant thruster.
Sphingosine Kinase Mediates Vascular Endothelial Growth Factor-induced Activation of Ras and Mitogen-activated Protein Kinases
Vascular endothelial growth factor (VEGF) signaling is critical to the processes of angiogenesis and tumor growth. Here, evidence is presented for VEGF stimulation of sphingosine kinase (SPK) that affects not only endothelial cell signaling but also tumor cells expressing VEGF receptors. VEGF or phorbol 12-myristate 13-acetate treatment of the T24 bladder tumor cell line resulted in a time- and dose-dependent stimulation of SPK activity. In T24 cells, VEGF treatment reduced cellular sphingosine levels while raising that of sphingosine-1-phosphate. VEGF stimulation of T24 cells caused a slow and sustained accumulation of Ras-GTP and phosphorylated extracellular signal-regulated kinase (phospho-ERK) compared with that after EGF treatment. Small interfering RNA (siRNA) that targets SPK1, but not SPK2, blocks VEGF-induced accumulation of Ras-GTP and phospho-ERK in T24 cells. In contrast to EGF stimulation, VEGF stimulation of ERK1/2 phosphorylation was unaffected by dominant-negative Ras-N17. Raf kinase inhibition blocked both VEGF- and EGF-stimulated accumulation of phospho-ERK1/2. Inhibition of SPK by pharmacological inhibitors, a dominant-negative SPK mutant, or siRNA that targets SPK blocked VEGF, but not EGF, induction of phospho-ERK1/2. We conclude that VEGF induces DNA synthesis in a pathway which sequentially involves protein kinase C (PKC), SPK, Ras, Raf, and ERK1/2. These data highlight a novel mechanism by which SPK mediates signaling from PKC to Ras in a manner independent of Ras-guanine nucleotide exchange factor.
Cyclodehydrogenation of Di- and Tetra(benzimidazol-2-yl)benzenes to Give Model Heteroaromatic Discotic Systems
Di-and tetra(benzimidazol-2-yl)benzenes upon oxidation undergo cyclodehydrogenation with formation of N-N bonds to form planarized polycyclic compounds which are models for the cores of heteroatom-containing discotic materials, and which can be readily reduced back to the original compounds, thus demonstrating a molecular redox switch.
VEGF Receptor Expression and Signaling in Human Bladder Tumors
Overexpression of vascular endothelial growth factor receptors (VEGFRs) has been reported in a variety of tumor types. Here we find that 11 out of the 14 bladder tumor cell lines examined express one or more VEGF receptors. Analysis of the T24 bladder tumor cell line reveals a functional autocrine loop involving VEGF and the Flk-1 receptor. Blocking VEGF expression in T24 cells results in a decrease in DNA synthesis. The Flk-1 receptor in T24 cells is phosphorylated in response to VEGF-121 or VEGF-165, and an Flk-1 inhibitor blocks VEGF to ERK signaling. We report that VEGF stimulation of T24 cells results in activation of H- and N-Ras and this is dependent on cellular sphingosine kinase 1 (SPK1) activity. Previously, we found VEGF-induced activation of Ras appears to be independent of a Ras-guanine nucleotide exchange factors (GEFs). Here we report that sphingosine can stimulate Ras-GTPase activating protein (GAP) activity in vitro, and sphingosine-1-phosphate (SPP) can block the stimulatory effects of sphingosine. We present a model where the balance between sphingosine and SPP regulates Ras-GAP activity such that stimulation of SPK1 favors downregulation of Ras-GAP and thereby the activation of Ras proteins. These data highlight a VEGF pathway that may be involved in the survival and proliferation of bladder tumor cells as well as other tumor cell types.
Sphingosine Kinase Protects Lipopolysaccharide-activated Macrophages from Apoptosis
Lipopolysaccharide (LPS) signaling is critical for the innate immune response to gram-negative bacteria. Here, evidence is presented for LPS stimulation of sphingosine kinase (SPK) in the RAW 264.7 murine macrophage cell line and rat primary hepatic macrophages (HMs). LPS treatment of RAW 264.7 cells resulted in a time- and dose-dependent activation of SPK and membrane translocation of SPK1. Further, LPS-induced SPK activation was blocked by SPK1-specific small interfering RNA (siRNA). Overexpression of Toll-like receptor 4 and MD2, the receptor and coreceptor of LPS, in HEK 293 cells activated SPK activity in the absence of LPS treatment. Inhibition of SPK by the pharmacological inhibitor N,N-dimethylsphingosine (DMS) or SPK1-specific siRNA blocked LPS stimulation of extracellular signal-regulated kinase 1/2 and p38 but enhanced LPS-induced c-Jun N-terminal kinase activation. The SPK inhibitor DMS and dominant-negative SPK1 also blocked LPS activation of Elk-1 and NF-kappaB reporters in RAW 264.7 cells. Inhibition of SPK sensitized RAW 264.7 cells and HMs to LPS-induced apoptosis. These data demonstrate the critical role of SPK1 in LPS signaling in macrophages and suggest that SPK1 is a potential therapeutic target to block hyperimmune responses induced by gram-negative bacteria.
Dibenzodioxin Adsorption on Inorganic Materials
Dibenzodioxin adsorption/desorption on solid surfaces is an important issue associated with the formation, adsorption, and emission of dioxins. Dibenzodioxin adsorption/desorption behaviors on inorganic materials (amorphous/mesoporous silica, metal oxides, and zeolites) were investigated using in situ FT-IR spectroscopy and thermogravimetric (TG) analysis. Desorption temperatures of adsorbed dibenzodioxin are very different for different kinds of inorganic materials: approximately 200 degrees C for amorphous/mesoporous silica, approximately 230 degrees C for metal oxides, and approximately 450 degrees C for NaY and mordenite zeolites. The adsorption of dibenzodioxin can be grouped into three categories according to the red shifts of the IR band at 1496 cm(-1) of the aromatic ring for the adsorbed dibenzodioxin: a shift of 6 cm(-1) for amorphous/mesoporous silica, a shift of 10 cm(-1) for metal oxides, and a shift of 14 cm(-1) for NaY and mordenite, suggesting that the IR shifts are proposed to associated with the strength of the interaction between adsorbed dibenzodioxin and the inorganic materials. It is proposed that the dibenzodioxin adsorption is mainly via the following three interactions: hydrogen bonding with the surface hydroxyl groups on amorphous/mesoporous silica, complexation with Lewis acid sites on metal oxides, and confinement effect of pores of mordenite and NaY with pore size close to the molecular size of dibenzodioxin.
The Study of Thiophene Adsorption Onto La(III)-exchanged Zeolite NaY by FT-IR Spectroscopy
Zeolites NaY and LaNaY (ion-exchanged with aqueous lanthanum nitrate solution) were used as adsorbents for removing organic sulfur compounds from model gasoline solutions (without and with toluene) and fluid catalytic cracked gasoline in fixed-bed adsorption equipment at room temperature and atmosphere pressure. The adsorptive selectivity for organic sulfur compounds was significantly increased when Na(+) ions in zeolite NaY were exchanged with lanthanum ions. IR spectra of thiophene adsorption indicate that thiophene is adsorbed onto La(3+) ions via direct S-La(3+) interaction and Na(+) ions via pi-electronic interaction for La(3+)-exchanged zeolite NaY, but only via pi-electronic interaction with Na(+) ions for NaY. The amount of adsorbed thiophene on La(3+)-exchanged zeolite Y was slightly decreased by coadsorption of benzene, but greatly reduced on NaY. The adsorption of thiophene via interaction with La(3+) on La(3+)-exchanged zeolite Y is hardly replaced by benzene coadsorption. The direct S-La(3+) interaction might be the essential reason for the evidently improved adsorptive selectivity of LaNaY for removing organic sulfur compounds from solutions containing large amount of aromatics.
Coupling Between Somatosensory Evoked Potentials and Hemodynamic Response in the Rat
We studied the relationship between somatosensory evoked potentials (SEP) recorded with scalp electroencephalography (EEG) and hemoglobin responses recorded non-invasively with diffuse optical imaging (DOI) during parametrically varied electrical forepaw stimulation in rats. Using these macroscopic techniques we verified that the hemodynamic response is not linearly coupled to the somatosensory evoked potentials, and that a power or threshold law best describes the coupling between SEP and the hemoglobin response, in agreement with the results of most invasive studies. We decompose the SEP response in three components (P1, N1, and P2) to determine which best predicts the hemoglobin response. We found that N1 and P2 predict the hemoglobin response significantly better than P1 and the input stimuli (S). Previous electrophysiology studies reported in the literature show that P1 originates in layer IV directly from thalamocortical afferents, while N1 and P2 originate in layers I and II and reflect the majority of local cortico-cortical interactions. Our results suggest that the evoked hemoglobin response is driven by the cortical synaptic activity and not by direct thalamic input. The N1 and P2 components, and not P1, need to be considered to correctly interpret neurovascular coupling.
Depth-resolved Microscopy of Cortical Hemodynamics with Optical Coherence Tomography
We describe depth-resolved microscopy of cortical hemodynamics with high-speed spectral/Fourier domain optical coherence tomography (OCT). Stimulus-evoked changes in blood vessel diameter, flow, and total hemoglobin were measured in the rat somatosensory cortex. The results show OCT measurements of hemodynamic changes during functional activation and represent an important step toward understanding functional hyperemia at the microscopic level.
Optical Monitoring of Oxygen Tension in Cortical Microvessels with Confocal Microscopy
Evaluating cerebral oxygenation is of critical importance for the understanding of brain function and several neuropathologies. Although several techniques exist for measuring cerebral oxygenation in vivo, the most widely accepted techniques offer limited spatial resolution. We have developed a confocal imaging system for minimally invasive measurement of oxygen tension (pO(2)) in cerebral microvessels with high spatial and temporal resolution. The system relies on the phosphorescence quenching method using exogenous porphyrin-based dendritic oxygen probes. Here we present high-resolution phosphorescence images of cortical microvasculature and temporal pO(2) profiles from multiple locations in response to varied fraction of inspired oxygen and functional activation.
Quantitative Cerebral Blood Flow with Optical Coherence Tomography
Absolute measurements of cerebral blood flow (CBF) are an important endpoint in studies of cerebral pathophysiology. Currently no accepted method exists for in vivo longitudinal monitoring of CBF with high resolution in rats and mice. Using three-dimensional Doppler Optical Coherence Tomography and cranial window preparations, we present methods and algorithms for regional CBF measurements in the rat cortex. Towards this end, we develop and validate a quantitative statistical model to describe the effect of static tissue on velocity sensitivity. This model is used to design scanning protocols and algorithms for sensitive 3D flow measurements and angiography of the cortex. We also introduce a method of absolute flow calculation that does not require explicit knowledge of vessel angles. We show that OCT estimates of absolute CBF values in rats agree with prior measures by autoradiography, suggesting that Doppler OCT can perform absolute flow measurements in animal models.
The Effect of Different Anesthetics on Neurovascular Coupling
To date, the majority of neurovascular coupling studies focused on the thalamic afferents' activity in layer IV and the corresponding large spiking activity as responsible for functional hyperemia. This paper highlights the role of the secondary and late cortico-cortical transmission in neurovascular coupling. Simultaneous scalp electroencephalography (EEG) and diffuse optical imaging (DOI) measurements were obtained during multiple conditions of event-related electrical forepaw stimulation in 33 male Sprague-Dawley rats divided into 6 groups depending on the maintaining anesthetic - alpha-chloralose, pentobarbital, ketamine-xylazine, fentanyl-droperidol, isoflurane, or propofol. The somatosensory evoked potentials (SEP) were decomposed into four components and the question of which best predicts the hemodynamic responses was investigated. Results of the linear regression analysis show that the hemodynamic response is best correlated with the secondary and late cortico-cortical transmissions and not with the initial thalamic input activity in layer IV. Baseline cerebral blood flow (CBF) interacts with neural activity and influences the evoked hemodynamic responses. Finally, neurovascular coupling appears to be the same across all anesthetics used.
Rapid Volumetric Angiography of Cortical Microvasculature with Optical Coherence Tomography
We describe methods and algorithms for rapid volumetric imaging of cortical vasculature with optical coherence tomography (OCT). By optimizing system design, scanning protocols, and algorithms for visualization of capillary flow, comprehensive imaging of the surface pial vasculature and capillary bed is performed in approximately 12 s. By imaging during hypercapnia and comparing with simultaneous CCD imaging, the sources of contrast of OCT angiography are investigated.
Study of Neurovascular Coupling by Modulating Neuronal Activity with GABA
Fundamental to the interpretation of neurovascular coupling is determining the neuronal activity that accounts for functional hyperemia. Recently, synaptic and not spiking activity has been found to be responsible for the hemodynamic response. Using pharmacological manipulation in rats, we want to further determine whether the cortical synaptic activity generated by the thalamic input or the subsequent synaptic activity related to secondary cortical processing is driving the hemodynamic response. In this study, we topically applied γ-aminobutyric acid (GABA) in the somatosensory cortex and used electrical forepaw stimulation to evoke neural and vascular activity. In a group of 8 animals, using laminar electrophysiology, we verified that topical application of GABA for 20min does not affect layer IV synaptic activity but reduces subsequent activity in the supragranular and infragranular layers. In another group of 8 animals, we simultaneously measured the electrical and vascular responses with scalp electroencephalography (EEG) and diffuse optical imaging (DOI), respectively. We decomposed somatosensory evoked potentials (SEP) into three major components: P1, N1, and P2, where P1 represents the thalamic input activity originating in layer IV and N1 and P2 represent the subsequent cortical transmissions. We verified that GABA infusion in the somatosensory cortex does not significantly reduce the P1 SEP component but strongly reduces the N1 and P2 components. We found that GABA also elicits a large reduction in the hemodynamic responses, which correlate with the reduction in N1 and P2 components. These results suggest that the hemodynamic response is predominantly driven by cortico-cortical interactions and not by the initial thalamocortical activity in layer IV.
Microvascular Oxygen Tension and Flow Measurements in Rodent Cerebral Cortex During Baseline Conditions and Functional Activation
Measuring cerebral oxygen delivery and metabolism microscopically is important for interpreting macroscopic functional magnetic resonance imaging (fMRI) data and identifying pathological changes associated with stroke, Alzheimer's disease, and brain injury. Here, we present simultaneous, microscopic measurements of cerebral blood flow (CBF) and oxygen partial pressure (pO(2)) in cortical microvessels of anesthetized rats under baseline conditions and during somatosensory stimulation. Using a custom-built imaging system, we measured CBF with Fourier-domain optical coherence tomography (OCT), and vascular pO(2) with confocal phosphorescence lifetime microscopy. Cerebral blood flow and pO(2) measurements displayed heterogeneity over distances irresolvable with fMRI and positron emission tomography. Baseline measurements indicate O(2) extraction from pial arterioles and homogeneity of ascending venule pO(2) despite large variation in microvessel flows. Oxygen extraction is linearly related to flow in ascending venules, suggesting that flow in ascending venules closely matches oxygen demand of the drained territory. Oxygen partial pressure and relative CBF transients during somatosensory stimulation further indicate arteriolar O(2) extraction and suggest that arterioles contribute to the fMRI blood oxygen level dependent response. Understanding O(2) supply on a microscopic level will yield better insight into brain function and the underlying mechanisms of various neuropathologies.
Optical Coherence Tomography for the Quantitative Study of Cerebrovascular Physiology
Doppler optical coherence tomography (DOCT) and OCT angiography are novel methods to investigate cerebrovascular physiology. In the rodent cortex, DOCT flow displays features characteristic of cerebral blood flow, including conservation along nonbranching vascular segments and at branch points. Moreover, DOCT flow values correlate with hydrogen clearance flow values when both are measured simultaneously. These data validate DOCT as a noninvasive quantitative method to measure tissue perfusion over a physiologic range.
Two-photon Microscopy of Cortical NADH Fluorescence Intensity Changes: Correcting Contamination from the Hemodynamic Response
Quantification of nicotinamide adenine dinucleotide (NADH) changes during functional brain activation and pathological conditions provides critical insight into brain metabolism. Of the different imaging modalities, two-photon laser scanning microscopy (TPLSM) is becoming an important tool for cellular-resolution measurements of NADH changes associated with cellular metabolic changes. However, NADH fluorescence emission is strongly absorbed by hemoglobin. As a result, in vivo measurements are significantly affected by the hemodynamics associated with physiological and pathophysiological manipulations. We model NADH fluorescence excitation and emission in TPLSM imaging based on precise maps of cerebral microvasculature. The effects of hemoglobin optical absorption and optical scattering from red blood cells, changes in blood volume and hemoglobin oxygen saturation, vessel size, and location with respect to imaging location are explored. A simple technique for correcting the measured NADH fluorescence intensity changes is provided, with the utilization of a parallel measurement of a physiologically inert fluorophore. The model is applied to TPLSM measurements of NADH fluorescence intensity changes in rat somatosensory cortex during mild hypoxia and hyperoxia. The general approach of the correction algorithm can be extended to other TPLSM measurements, where changes in the optical properties of the tissue confound physiological measurements, such as the detection of calcium dynamics.
S Phase-dependent Interaction with DNMT1 Dictates the Role of UHRF1 but Not UHRF2 in DNA Methylation Maintenance
Recent studies demonstrate that UHRF1 is required for DNA methylation maintenance by targeting DNMT1 to DNA replication foci, presumably through its unique hemi-methylated DNA-binding activity and interaction with DNMT1. UHRF2, another member of the UHRF family proteins, is highly similar to UHRF1 in both sequence and structure, raising questions about its role in DNA methylation. In this study, we demonstrate that, like UHRF1, UHRF2 also binds preferentially to methylated histone H3 lysine 9 (H3K9) through its conserved tudor domain and hemi-methylated DNA through the SET and Ring associated domain. Like UHRF1, UHRF2 is enriched in pericentric heterochromatin. The heterochromatin localization depends to large extent on its methylated H3K9-binding activity and to less extent on its methylated DNA-binding activity. Coimmunoprecipitation experiments demonstrate that both UHRF1 and UHRF2 interact with DNMT1, DNMT3a, DNMT3b and G9a. Despite all these conserved functions, we find that UHRF2 is not able to rescue the DNA methylation defect in Uhrf1 null mouse embryonic stem cells. This can be attributed to the inability for UHRF2 to recruit DNMT1 to replication foci during S phase of the cell cycle. Indeed, we find that while UHRF1 interacts with DNMT1 in an S phase-dependent manner in cells, UHRF2 does not. Thus, our study demonstrates that UHRF2 and UHRF1 are not functionally redundant in DNA methylation maintenance and reveals the cell-cycle-dependent interaction between UHRF1 and DNMT1 as a key regulatory mechanism targeting DNMT1 for DNA methylation.
