NADH Fluorescence Research Articles

Enzymatic Activity of Renal H-K-ATPase in the Outer Medullary Collecting Duct of Transgenic Mice

The H-K-ATPase (HKA), a potassium-dependent proton transporter in the outer medullary collecting duct (OMCD) plays an important role in acid-base homeostasis. The OMCD contains two HKA isoforms; gastric (HKAα1), dominant under normal dietary conditions (ND), and colonic (HKAα2), induced under a K-free diet (KD). The enzymatic activity (EA) of HKA in the OMCD is incompletely understood. The focus of the present study is elucidating the EA of the HKA in HKAα1 and HKAα2 knockout (KO) mice under ND and KD. KO mice were subjected to ND or KD for 10 days. Ten OMCD tubules were extracted, half placed in potassium-free media (Solution 2), half in potassium-containing media (Solution 3). Fluorescence measurements are based on the hydrolysis of ATP to ADP, coupled with the oxidation of NADH. ADP is determined by a decrease in NADH fluorescence. In K presence, NADH fluorescence of HKAα1 KO mice read 13.5 ± 0.7 ppm for ND and 10.3 ± 0.2 ppm for KD, indicating stimulation of the colonic isoform. HKAα2 KO mice averaged 6.8 ± 0.3 ppm for ND and 5.4 ± 0.3 ppm for KD in solution 2 (p p α2 isoform. A significant difference in ATP production in HKAα2 KO mice is likely due to enhanced EA of H-ATPase under potassium depletion.

Metabolic characterization of hematopoietic stem cells.

An important feature of stem cells is their maintenance in their respective hypoxic niche. Survival in this low-oxygen microenvironment requires significant metabolic adaptation. We demonstrated that mouse HSCs utilize glycolysis instead of mitochondrial oxidative phosphorylation to meet their energy demands. We have adapted various tools for characterization of the metabolic properties of hematopoietic stem cells (HSCs). These techniques include flow cytometric profiling of HSCs based on mitochondrial potential and NADH fluorescence as well as measurement of ATP content, oxygen consumption rate, and glycolytic flux in purified HSCs.

NADH is an Endogenous Reporter for Alpha-Synuclein Aggregation in Live Cells

Alpha-synuclein aggregation is amply investigated for its involvement in Parkinson's disease etiopathogenesis. It has been shown that alpha-synuclein monomers, under pathological conditions, self-assembly to form oligomeric species that further aggregate into amyloid fibrils. Alpha-synuclein fibrils are the main constituent of Lewy Bodies, which are one of the characteristic hallmarks of Parkinson's disease.Alpha-synuclein aggregation is studied in vitro and in cellular models with the aim to correlate toxicity mechanisms to defined aggregation products. However, the characterization of the aggregation process in cells is a difficult task that typically needs cell lysis or fixation, or the use of exogenous dyes.Moreover, several different toxic mechanisms were ascribed to alpha-synuclein aggregates, i.e. clearance mechanisms impairment, mitochondrial dysfunctions, oxidative stress, neuroinflammation. In particular, mitochondria seem to be a target for alpha-synuclein to exert its toxicity. Several independent results suggested that alpha-synuclein overexpression and/or aggregation may cause impairment of cellular metabolism due to mitochondrial fragmentation and complex I dysfunction.On these premises, we report here the results obtained from the characterization of NADH fluorescence properties variation in vitro and in cell models during alpha-synuclein aggregation.The application of the phasor approach for the study of NADH fluorescence lifetime and spectra allowed the determination of specific variation in the NADH fluorescence properties correlated to alpha-synuclein oligomerization and amyloid fibrils formation in vitro and in live cells.The results presented here suggest that alpha-synuclein aggregation may be associated to impairment in cell metabolism due to damage to complex I in mitochondria and disruption of NADH and NAD+ equilibrium. Moreover, NADH can be used as an endogenous fluorescence reporter for alpha-synuclein aggregation in vitro and in cellular models.(Work supported in part by NIH grants P50 GM076516 and P41 GM103540, and by Aldo Gini Foundation.)

Molecular Cloning and Characterization of a Candidate ENOX Protein of <i>Saccharomyces cerevisiae</i> with a 25 Min Period Insensitive to Simalikalactone D Inhibition and Melatonin

A yeast deletion library was screened based on NADH fluorescence using a 384-well plate assay to identify a yeast isolate lacking a previously identified cell surface oxidase exhibiting an oscillatory pattern with a period length of 25 min and resistant to the ENOX1-specific inhibitor simalikalactone D (YNOX for yeast-specific ENOX = ENOX4). The cDNA was cloned from a yeast over expression library using NADH fluorescence analyzed by Fast Fourier transform and decomposition fits. The objective was to identify and sequence an ENOX homologue in Saccharomyces cerevisiae with a 25 min rather than a 24 min period length (YNOX). The finding identified YDR005C as the yeast ENOX protein with a temperature-independent 25 min period length and insensitive to inhibition by simalikalactone D. The encoded protein was expressed in bacteria and characterized. Gel slices corresponding to 55 kDa and 39 kDa His-tagged proteins exhibited 25 min oscillatory patterns not inhibited by 1 μM simalikalactone D for both NADH oxidation and reduced coenzyme Q10 oxidation as well as a protein disulfide-thiol interchange activity which alternated with the oxidative activities. Activities were phased by low-frequency electromagnetic fields but, in contrast, to yeast ENOX1, not by addition of melatonin. The assay in the presence of D2O shifted the length of the oscillatory period from 25 min to 32 min. The YDR005C deletion mutant cells lacked the ENOX4 clock output present in the wild type yeast.

Two-Photon Fluorescence Lifetime Imaging for Metabolic Profiling of Cochlear Dysfunction

More than 120,000 individuals treated with lifesaving antibiotics develop hearing or balance disorders annually. Although research has shown that of the two cochlear cell types, sensory and supporting cells, sensory cells are readily damaged due to age-related hearing loss, acoustic trauma and ototoxins, the reason for this remains unknown. Furthermore, cochlear sensory hair cells can be divided into two types, inner and outer hair cells (IHCs, OHCs). OHCs in the high-frequency region of the cochlea exhibit the greatest sensitivity to the above conditions. To determine if variations in sensory and supporting mitochondrial metabolism account for these differences, two-photon fluorescence lifetime microscopy (FLIM) was used to measure changes in the metabolic reporter molecule NADH in sensory and supporting cells from explanted murine cochleae. Mitochondrial uncouplers, inhibitors and an ototoxic antibiotic, gentamicin (GM), were used to assess high- and low-frequency IHC, OHC and supporting cell mitochondrial metabolism. Chemically induced changes in metabolic state resulted in a reorganization of specific NADH lifetimes into altered subcellular fluorescence lifetime pools. Variations in NADH intensity and average NADH lifetime were greatest in high-frequency OHCs. Pretreatment with GM significantly increased NADH intensity in high-frequency sensory cells but not supporting cells. Treatment with GM significantly increased the average NADH fluorescence lifetime within IHCs but not OHCs. GM also caused a significant increase of NADH concentration in OHCs, not IHCs. These results demonstrate: differences between sensory and supporting cell metabolism; GM alters mitochondrial metabolism; and IHCs and OHCs display differing metabolic effects when exposed to GM. Such fundamental differences between sensory and supporting cell mitochondrial metabolism indicate differing metabolic changes during antibiotic exposure. Understanding these antibiotic-induced metabolic changes may explain the ototoxic effects of these drugs which is crucial for preventing and treating numerous auditory deficits and diseases.

Subcellular Level Optical Metabolic Imaging of Induced Pluoripotent Stem Cells using Endogenous Fluorophore

In this work we show label-free metabolic imaging of induced pluoripotent stem (iPS) cell delineating regions of different metabolic activity at subcellular level. iPS cells, which are derived from adult somatic cells (skin fibroblast in this case) by nuclear reprogramming, possess pluoripotency. Hence, these cells offer a unique platform for stem cell research, drug discovery and testing, disease treatment development and toxicological screening. The phasor approach was applied to endogenous NADH fluorescence lifetime imaging microscopy (FLIM) to map free to bound NADH ratio distribution within single cells. The FLIM phasor distribution shows a clear difference between metabolic states of the nucleus and cytoplasmic regions. The nuclei had a larger fraction of free NADH indicated by a distribution shifted more toward the shorter lifetime as compared to the phasor distribution of the cytoplasm. Also within the nucleus, two distinct lifetime distributions were observed with the perinuclear region having more bound fraction of NADH and an abundance of free NADH in the central nucleolus region. A similar observation was reported in myoblasts [Microscopy research and technique,75(12),1717-22,(2012)]. Furthermore, within the cytoplasm there are some regions with more bound NADH. This could be due to presence of more protein bound NADH within the mitochondria. The phasor analysis also revealed a unique long lifetime distribution which arose from small granular like structures within the iPS cells. These areas also had higher intensities compared to rest of the cells. This could be liquid droplet-associated granules (LDAGs) which were previously observed by Stringari et al [Journal of biomedical optics 17(4),046012,(2012)] in embryonic stem cell. Hence, we show mapping of metabolic activity of iPS cells at subcellular resolution employing a non-invasive, label-free, optical microscopy technique.Work supported in part by NIH grants P50GM076516, P41GM103540 and UH2TR000481-0.

A Micro-Perfusion System for the Fluorescence-Based Monitoring of Physiological Responses to High Hydrostatic Pressures

Hydrostatic pressures of 102 to 103 atm affect a range of cellular processes, including motility, cell division, nutrient uptake, fermentation, translation and transcription, protein synthesis, and ultimately viability. In order to perform the real time monitoring of pressure effects, we present a micro-perfusion system designed for spectroscopic measurements on cellular systems under high pressure. The system consists of an optically-compatible pressure chamber and interchangeable fluid reservoirs. Perfusion is achieved using a dual pressure-generator configuration, where one positive-displacement generator is compressed while the other is retracted, thus maintaining a pressurized volume while achieving fluid flow. Control over perfusion rates (typically in the 10 μl/s range) and the ability to change between fluid reservoirs while under pressure (up to 600 atm) are demonstrated. Next, the system is used for the time-gated, spectral monitoring of endogenous NADH fluorescence under pressure. Spectrofluorimetric measurements utilize a nitrogen-discharge laser for sub-nanosecond pulse width, 337-nm wavelength excitation, and an intensified CCD coupled to a spectrograph for nanosecond-gated spectral detection. Because emission from NADH is routinely used at ambient pressure for monitoring mitochondrial function, we validate the system by observing the pressurized response of Saccharomyces cerevisiae (baker's yeast) to mitochondrial functional modifiers (e.g., cyanide). Because the system is compatible with both spectroscopy and sub-cellular resolution microscopy imaging, the system represents a robust tool for investigating the biophysical effects of pressure on cellular systems.

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD(+)]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD(+)]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD(+)]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD(+)]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD(+)]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.

NADH changes during hypoxia, ischemia, and increased work differ between isolated heart preparations

Langendorff-perfused hearts and working hearts are established isolated heart preparation techniques that are advantageous for studying cardiac physiology and function, especially when fluorescence imaging is a key component. However, oxygen and energy requirements vary widely between isolated heart preparations. When energy supply and demand are not in harmony, such as when oxygen is not adequately available, the imbalance is reflected in NADH fluctuations. As such, NADH imaging can provide insight into the metabolic state of tissue. Hearts from New Zealand white rabbits were prepared as mechanically silenced Langendorff-perfused hearts, Langendorff-perfused hearts, or biventricular working hearts and subjected to sudden changes in workload, instantaneous global ischemia, and gradual hypoxia while heart rate, aortic pressure, and epicardial NADH fluorescence were monitored. Fast pacing resulted in a dip in NADH upon initiation and a spike in NADH when pacing was terminated in biventricular working hearts only, with the magnitude of the changes greatest at the fastest pacing rate. Working hearts were also most susceptible to changes in oxygen supply; NADH was at half-maximum value when perfusate oxygen was at 67.8 ± 13.7%. Langendorff-perfused and mechanically arrested hearts were the least affected by low oxygen supply, with half-maximum NADH occurring at 42.5 ± 5.0% and 23.7 ± 4.6% perfusate oxygen, respectively. Although the biventricular working heart preparation can provide a useful representation of mechanical in vivo heart function, it is not without limitations. Understanding the limitations of isolated heart preparations is crucial when studying cardiac function in the context of energy supply and demand.

Nicotinamide pre-treatment ameliorates NAD(H) hyperoxidation and improves neuronal function after severe hypoxia

Prolonged hypoxia leads to irreversible loss of neuronal function and metabolic impairment of nicotinamide adenine dinucleotide recycling (between NAD+ and NADH) immediately after reoxygenation, resulting in NADH hyperoxidation. We test whether the addition of nicotinamide (to enhance NAD+ levels) or PARP-1 inhibition (to prevent consumption of NAD+) can be effective in improving either loss of neuronal function or hyperoxidation following severe hypoxic injury in hippocampal slices. After severe, prolonged hypoxia (maintained for 3min after spreading depression) there was hyperoxidation of NADH following reoxygenation, an increased soluble NAD+/NADH ratio, loss of neuronal field excitatory post-synaptic potential (fEPSP) and decreased ATP content. Nicotinamide incubation (5mM) 2h prior to hypoxia significantly increased total NAD(H) content, improved neuronal recovery, enhanced ATP content, and prevented NADH hyperoxidation. The nicotinamide-induced increase in total soluble NAD(H) was more significant in the cytosolic compartment than within mitochondria. Prolonged incubation with PJ-34 (>1h) led to enhanced baseline NADH fluorescence prior to hypoxia, as well as improved neuronal recovery, NADH hyperoxidation and ATP content on recovery from severe hypoxia and reoxygenation. In this acute model of severe neuronal dysfunction prolonged incubation with either nicotinamide or PJ-34 prior to hypoxia improved recovery of neuronal function, enhanced NADH reduction and ATP content, but neither treatment restored function when administered during or after prolonged hypoxia and reoxygenation.

Non-invasive monitoring of cell metabolism and lipid production in 3D engineered human adipose tissues using label-free multiphoton microscopy

Non-linear optical microscopy methods can characterize over time multiple functional properties of engineered tissues during development. Here, we demonstrate how the combined use of third-harmonic generation (THG) and two-photon excited fluorescence (2PEF) imaging can provide direct quantitative biomarkers of adipogenic stem cell differentiation and metabolic state, respectively. Specifically, we imaged over nine weeks silk scaffolds embedded with human mesenchymal stem cells and exposed to either propagation (PM) or adipogenic differentiation media (AM). THG was employed to visualize the formation of lipid droplets. 2PEF was used to assess the metabolic state of the cells through the redox ratio defined based on the endogenous FAD and NADH fluorescence. The redox ratio of cells in the AM scaffold was significantly lower than that in the PM scaffold during week 5 and 9, and correlated with significant increases in lipid-to-cell volume ratio, and number and size of lipid droplets in the AM scaffold. These findings indicate that the decrease in redox ratio during adipogenic differentiation is associated with fatty acid synthesis and lipid accumulation. Our methods therefore enabled us to identify and measure dynamic correlations between lipid droplet formation and cell metabolic state, while providing insight on the spatial heterogeneity of the observed signals.

NADH-Fluorometric Biochemical Gas Sensor (Bio-Sniffer) for Evaluation of Indoor Air Quality

A fiber-optic biochemical gas sensor (bio-sniffer) for assessment of indoor formaldehyde (FA) is fabricated and tested in monitoring of FA degradation by TiO <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sub> . The biosniffer measures FA vapor as fluorescence of reduced nicotinamide adenine dinucleotide (NADH), which is the product of enzymatic reaction of FA dehydrogenase (FALDH). Usually, an enzyme loses its specific activity in the gas phase. This makes biochemical gas monitoring difficult. We employ a microflow-cell with a FALDHimmobilized membrane to prevent the FALDH from deactivation at the optode. An ultraviolet light-emitting diode (UV-LED) with peak emission of 335 nm is employed as an excitation light source. Emission of the UV-LED is introduced to the optode through an optical fiber and fluorescence of NADH is picked up coaxially at the optode. To improve the sensitivity, a multi-LED light source is introduced instead of the previously reported system. A photomultiplier tube is utilized as a photodetector. Continuous FA monitoring with biochemical method is successfully conducted with high selectivity at subppb level. Owing to the improved excitation source, the detection limit is improved to 750 ppt. A real-sample test is also carried out using a photocatalytic titania. A standard gas is flowed into a cell, in which photocatalyst glasses are located, and the FA level is measured using the biosniffer. Therefore, degradation of FA by the catalytic reaction of titania is monitored as reduction of FA in real time. According to the results, it is expected to be useful in fast and convenient monitoring of indoor FA.

Label-free high-throughput assays to screen and characterize novel lactate dehydrogenase inhibitors

Catalytic turnover of pyruvate to lactate by lactate dehydrogenase (LDH) is critical in maintaining an intracellular nicotinamide adenine dinucleotide (NAD+) pool for continuous fueling of the glycolytic pathway. In this article, we describe two label-free high-throughput assays (a kinetic assay detecting the intrinsic reduced nicotinamide adenine dinucleotide (NADH) fluorescence and a mass spectrometric assay monitoring the conversion of pyruvate to lactate) that were designed to effectively identify LDH inhibitors, characterize their different mechanisms of action, and minimize potential false positives from a small molecule compound library screen. Using a fluorescence kinetic image-based reader capable of detecting NADH fluorescence in the ultra-high-throughput screening (uHTS) work flow, the enzyme activity was measured as the rate of NADH conversion to NAD+. Interference with NADH fluorescence by library compounds was readily identified during the primary screen. The mass spectrometric assay quantitated the lactate and pyruvate levels simultaneously. The multiple reaction monitoring mass spectrometric method accurately detected each of the two small organic acid molecules in the reaction mixture. With robust Z′ scores of more than 0.7, these two high-throughput assays for LDH are both label free and complementary to each other in the HTS workflow by monitoring the activities of the compounds on each half of the LDH redox reaction.

Multiphoton morpho-functional imaging of healthy colon mucosa, adenomatous polyp and adenocarcinoma.

Two-photon spectral resolved imaging was used to image fresh human biopsies of colon tissue and to characterize healthy colon mucosa, adenomatous polyp and adenocarcinoma by means of a morpho-functional analysis. Morphological examination, performed using endogenous tissue fluorescence, discriminated adenomatous and adenocarcinoma tissues from normal mucosa in terms of cellular asymmetry and nucleus-to-cytoplasm ratio. Good agreement was found between multiphoton images and histological examination performed on the same samples. Further characterization, performed by means of spectral-resolved analysis of NADH and FAD fluorescence, demonstrated an altered metabolic activity in both adenomatous and adenocarcinoma tissues compared to healthy mucosa. This morpho-functional approach may represent a powerful method to be used in combination with endoscopy for in vivo optical diagnosis of colon cancer and may be extended to other tissues.

Acute metabolic amplification of insulin secretion in mouse islets is mediated by mitochondrial export of metabolites, but not by mitochondrial energy generation

ObjectiveThe β-cell metabolism of glucose and of some other fuels (e.g. α-ketoisocaproate) generates signals triggering and acutely amplifying insulin secretion. As the pathway coupling metabolism with amplification is largely unknown, we aimed to narrow down the putative amplifying signals. Materials/MethodsAn experimental design was used which previously prevented glucose-induced, but not α-ketoisocaproate-induced insulin secretion. Isolated mouse islets were pretreated for one hour with medium devoid of fuels and containing the sulfonylurea glipizide in high concentration which closed all ATP-sensitive K+ channels. This concentration was also applied during the subsequent examination of fuel-induced effects. In perifused or incubated islets, insulin secretion and metabolic parameters were measured. ResultsThe pretreatment decreased the islet ATP/ADP ratio. Whereas glucose and α-ketoisovalerate were ineffective or weakly effective, respectively, when tested separately, their combination strongly enhanced the insulin secretion. Compared with glucose, the strong amplifier α-ketoisocaproate caused less increase in NAD(P)H-fluorescence and less mitochondrial hyperpolarization. Compared with α-ketoisovalerate, α-ketoisocaproate caused greater increase in NAD(P)H-fluorescence and greater mitochondrial hyperpolarization. Neither α-ketoacid anion enhanced the islet ATP/ADP ratio during onset of the insulin secretion. α-Ketoisocaproate induced a higher pyruvate content than glucose, slowly elevated the citrate content which was not changed by glucose and generated a much higher acetoacetate content than other fuels. α-Ketoisovalerate alone or in combination with glucose did not increase the citrate content. ConclusionsIn β-cells, mitochondrial energy generation does not mediate acute metabolic amplification, but mitochondrial production of acetyl-CoA and supplemental acetoacetate supplies cytosolic metabolites which induce the generation of specific amplifying signals.

In VivoNADH Fluorescence Imaging Indicates Effect of Aquaporin-4 Deletion on Oxygen Microdistribution in Cortical Spreading Depression

Using in vivo two-photon imaging, we show that mice deficient in aquaporin-4 (AQP4) display increased fluorescence of nicotinamide adenine dinucleotide (NADH) when subjected to cortical spreading depression. The increased NADH signal, a proxy of tissue hypoxia, was restricted to microwatershed areas remote from the vasculature. Aqp4 deletion had no effects on the hyperemia response, but slowed [K(+)]o recovery. These observations suggest that K(+) uptake is suppressed in Aqp4(-/-) mice as a consequence of decreased oxygen delivery to tissue located furthest away from the vascular source of oxygen, although increased oxygen consumption may also contribute to our observations.

Abstract 384: NADH oxidase, Enox1 activity, is required for vasculogenesis in Zebrafish.

Abstract Establishment of an adequate vascular network is a crucial event for tumor development. Emerging research has implicated the endothelial redox state as a significant regulator of vascular growth and remodeling. ENOX1 encodes a protein that is expressed in mammalian endothelial cells and exhibits NADH oxidase activity. Recent research suggests that ENOX1 functions as a positive regulator of angiogenesis. However, direct genetic evidence supporting this hypothesis is lacking. We used a zebrafish model to directly examine the role of Enox1 during embryonic vascular development. Whole mount in situ hybridization coupled with immunocytochemistry performed on zebrafish cryosections show that enox1 message and the translated protein are expressed in heart and blood vessels during embryonic development. Furthermore, use of immunocytochemistry demonstrates that Enox1 co-localizes with vascular expression of the ETS-domain transcription factor fli in heart and intersegmental vessels. Using in vivo multiphoton microscopy, we show that morpholino-mediated knockdown of Enox1 in Zebrafish embryos increases NADH fluorescence, an expected consequence of loss of Enox1 oxidase activity. Phenotypic manifestations resulting from loss of Enox1 function in embryos include vascular defects, most notably loss of fli-eGFP and flk-eGFP (receptor kdr, VEGFR2 in mammals) expression in intersegmental vessels and a loss of blood circulation in the intersegmental vessels. Pharmacological inhibition of Enox1 activity using a novel small molecule phenocopies the defects observed following morpholino-mediated functional knockdown. Together, these results show that Enox1-mediated redox regulation is essential for vasculogenesis in vertebrates in vivo. These results, in conjunction with previous work that demonstrated that inhibition of ENOX1 radiosensitized tumor vasculature, support the hypothesis that ENOX1 represents a novel target for impairing tumor angiogenesis. Citation Format: Amudhan Venkateswaran, Daniel S. Levic, David B. Melville, Konjeti R. Sekhar, Alexandra J. Walsh, Melissa C. Skala, Peter A. Crooks, Ela W. Knapik, Michael L. Freeman. NADH oxidase, Enox1 activity, is required for vasculogenesis in Zebrafish. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 384. doi:10.1158/1538-7445.AM2013-384

Label-freein vivoimaging of human leukocytes using two-photon excited endogenous fluorescence

We demonstrate that two-photon excited endogenous fluorescence enables label-free morphological and functional imaging of various human blood cells. Specifically, we achieved distinctive morphological contrast to visualize morphology of important leukocytes, such as polymorphonuclear structure of granulocyte and mononuclear feature of agranulocyte, through the employment of the reduced nicotinamide adenine dinucleotide (NADH) fluorescence signals. In addition, NADH fluorescence images clearly reveal the morphological transformation process of neutrophils during disease-causing bacterial infection. Our findings also show that time-resolved NADH fluorescence can be potentially used for functional imaging of the phagocytosis of pathogens by leukocytes (neutrophils) in vivo. In particular, we found that free-to-bound NADH ratios measured in infected neutrophils increased significantly, which is consistent with a previous study that the energy consumed in the phagocytosis of neutrophils is mainly generated through the glycolysis pathway that leads to the accumulation of free NADH. Future work will focus on further developing and applying label-free imaging technology to investigate leukocyte-related diseases and disorders.

Spectral profiles of acute and healed myocardial radiofrequency ablation lesions

A spectral approach to differentiate between acute and healed myocardial lesions was explored. The long‐term goal of these studies is improved treatment of atrial fibrillation using percutaneous RF catheters. The epicardial surface of excised rat hearts was illuminated by continuous wavelength spectrum via an optical fiber coupled to a spectrofluorometer. Emitted, scattered and reflected light was acquired by a high sensitivity CCD camera. Spectra were separated into three groups: unablated tissue, acute and healed RF lesions. Structure of the lesions was confirmed by cytopathology. Spectra between all three groups differed significantly throughout the 300–800nm range. The largest difference was observed in the 300–400nm illumination range accounting for the loss of NADH fluorescence in acute lesions, but not in healed tissue. In the 400–620nm illumination range both acute and healed epicardial lesions exhibited significantly higher intensity compared to control tissue. In the 620–800nm range difference between three groups diminished. The results point to a possibility of in situ visualization of acute and chronic lesions based on the spectral differences in reflected and emitted light. The sensitivity and specificity of lesion identification can be further increased by ratiometry of images acquired at different illumination wavelengths (Supported by the NIH HL095828 &amp; NSF CBET1231549)

Hydrogen sulfide modulates sinusoidal constriction and contributes to hepatic micorcirculatory dysfunction during endotoxemia

Hydrogen sulfide (H₂S) affects vascular resistance; however, its effect on the hepatic microcirculation has not been investigated. Hepatic sinusoidal perfusion is dysregulated during sepsis, contributing to liver injury. Therefore, the present study determined the effect of H₂S on the hepatic microcirculation and the contribution of endogenous H₂S to hepatic microcirculatory dysfunction in an endotoxin model of sepsis. Portal infusion of H₂S increased portal pressure in vivo (6.8 ± 0.2 mmHg before H₂S vs. 8.6 ± 0.8 mmHg peak during H₂S infusion, P < 0.05). Using intravital microscopy, we observed decreased sinusoidal diameter (6.2 ± 0.27 μm before H₂S vs. 5.7 ± 0.3 μm after H₂S, P < 0.05) and increased sinusoidal heterogeneity during H₂S infusion (P < 0.05) and net constriction. Since hepatic H₂S levels are elevated during sepsis, we used the cystathionine γ lyase inhibitor DL-propargylglycine (PAG) to determine the contribution of H₂S to the hypersensitization of the sinusoid to the vasoconstrictor effect of endothelin-1 (ET-1). PAG treatment significantly attenuated the sinusoidal sensitization to ET-1 in endotoxin-treated animals. ET-1 infusion increased portal pressure to 175% of baseline in endotoxemic animals, which was reduced to 143% following PAG treatment (P < 0.05). PAG abrogated the increase in sinusoidal constriction after ET-1 infusion in LPS-treated rats (30.9% reduction in LPS rats vs. 11.6% in PAG/LPS rats, P < 0.05). Moreover, PAG treatment significantly attenuated the increase in NADH fluorescence following ET-1 exposure during endotoxemia (61 grayscale units LPS vs. 21 units in PAG/LPS, P < 0.05), suggesting an improvement in hepatic oxygen availability. This study is the first to demonstrate a vasoconstrictor action of H₂S on the hepatic sinusoid and provides a possible mechanism for the protective effect of PAG treatment during sepsis.