Selective and Absolute Quantification of Endogenous Hypochlorous Acid with Quantum-Dot Conjugated Microbeads

Endogenous hypochlorous acid (HOCl) secreted by leukocytes plays a critical role in both the immune defense of mammalians and the pathogenesis of various diseases intimately related to inflammation. We report the first selective and absolute quantification of endogenous HOCl produced by leukocytes in vitro and in vivo with a novel quantum dot-based sensor. An activated human neutrophil secreted 6.5 ± 0.9 × 10⁸ HOCl molecules into its phagosome, and kinetic measurement for the secretions showed that the extracellular generation of HOCl was temporally retarded, but the quantity eventually attained a level comparable with its intraphagosomal counterpart with a delay of about 1.5 h. The quantity of HOCl secreted from the hepatic leukocytes of rats with or without stimulation of lipopolysaccharide was also determined. These results indicate a possibility to extend our approach to not only clinical settings for quantitative assessment of the bactericidal capability of isolated leukocytes of patients but also fundamental biomedical research that requires critical evaluation of the inflammatory response of animals

Toward Functional Screening of Cardioactive and Cardiotoxic Drugs with Zebrafish <i>in Vivo</i> Using Pseudodynamic Three-Dimensional Imaging

Given the high mortality in patients with cardiovascular diseases and the life-threatening consequences of drugs with unforeseen adverse effects on hearts, a critical evaluation of the pharmacological response of cardiovascular function on model animals is important especially in the early stages of drug development. We report a proof-of-principle study to demonstrate the utility of zebrafish as an analytical platform to predict the cardiac response of new drugs or chemicals on human beings. With pseudodynamic 3D imaging, we derive individual parameters that are central to the cardiac function of zebrafish, including the ventricular stroke volume, ejection fraction, cardiac output, heart rate, diastolic filling function, and ventricular mass. We evaluate both inotropic and chronotropic responses of the heart of zebrafish treated with drugs that are commonly prescribed and possess varied known cardiac activities. We reveal deranged cardiac function of a zebrafish model of cardiomyopathy induced with a cardiotoxic drug. The cardiac function of zebrafish exhibits a pharmacological response similar to that of human beings. We compare also cardiac parameters obtained in this work with those derived with conventional 2D approximation and show that the latter tends to overestimate the cardiac parameters and produces results of greater variation. In view of the growing interest of using zebrafish in both fundamental and translational biomedical research, we envisage that our approach should benefit not only contemporary pharmaceutical development but also exploratory research such as gene, stem cell, or regenerative therapies targeting congenital or acquired heart diseases

Spatiotemporal Characterization of Phagocytic NADPH Oxidase and Oxidative Destruction of Intraphagosomal Organisms <i>in Vivo</i> Using Autofluorescence Imaging and Raman Microspectroscopy

Spatiotemporal Characterization of Phagocytic NADPH Oxidase and Oxidative Destruction of Intraphagosomal Organisms in Vivo Using Autofluorescence Imaging and Raman Microspectroscop

Dependence of the sarcomeric length on the dosage and the duration of treatment.

<p>The zebrafish was treated with concentrations 0.05, 0.5 and 50 µM at 72 hpf for either 12 h (red) or 120 h (black). The images were obtained at the end of the treatments on regions between somites 5∼8. The statistics were calculated based on 21 images obtained from three zebrafish.</p

Shortened sarcomeres caused by demolition of the myosin filament.

<p>(A) A sarcomere subject to normal contraction. (B) A sarcomere subject to demolition of the myosin filament (B). (C, D, E) Comparison of sarcomeric microstructures of zebrafish subject to no treatment (the control), a treatment of statin (50 µm) at 72 hpf for 120 h, and that of caffeine (8 mM) at 72 hpf for 3 h. All images were obtained at the end of treatments on regions between somites 5∼8. (C) Sarcomere length. The statistics were calculated based on 21 SHG images obtained from three zebrafish. (D) Full width at half maximum (FWHM) of the single band. The statistics were calculated based on 15 SHG images obtained from three zebrafish. (E) Bare length of the double band. The statistics were calculated based on six images obtained from three zebrafish. The FWHM and the bare length were determined on analysis of cross-sectional plots of the single and double bands, respectively, as illustrated in the insets of (D) and (E).</p

Rescuing effect of mevalonate on the shortened sarcomere induced with statin.

<p>Left: the control, untreated zebrafish; middle: zebrafish subject to a treatment of statin (0.5 µM) at 24 hpf for 12 h; right: zebrafish subject to a co-treatment of statin (0.5 µM) and mevalonate (100 µM) at 24 hpf for 12 h. All SHG images used to determine the sarcomere length were measured at 36 hpf on somites near the head (black, somites 5∼8) and the tail (red, somites 21–24). The two images displayed as insets are representative results of co-treatment measured near the head (left) and the tail (right), respectively. Image size: 50×50 µm. The statistics were calculated based on 21 images obtained from three zebrafish.</p

Structural modification of zebrafish muscles induced on treatment with statin.

<p>(A) A representative SHG image of the control (an untreated zebrafish larva, 72 hpf). (B) A representative SHG image of a zebrafish larva (72 hpf) subject to treatment with statin (50 µM) for 12 h. Image size: 140 µm (w)×100 µm (h). (C) Effect of statin treatment on zebrafish with underdeveloped sarcomeres. The zebrafish was subject to a treatment of statin (0.5 µM) at 24 hpf for 12 h, and imaged at 36 hpf (black: somites 5∼8; red: somites 21–24). (D) Effect of statin treatment on zebrafish with fully developed sarcomeres. The zebrafish was subject to a treatment of the same dosage at 72 hpf for 12 h, and the image was taken at 84 hpf (head, somites 5∼8).</p

Sarcomeric length determined with SHG imaging.

<p>(A) A cartoon illustration of a sarcomere and the architectural arrangement of its major constituent filaments. The length of a sarcomere is defined as the separation between two <i>z</i>-disks. (B) A high-resolution SHG image of zebrafish muscles. The image exhibits two characteristic patterns that are denoted as double (gray arrow) and single bands (white arrow), respectively. Scale bar: 5 µm. (C, D) Representative cross-sectional plots of a double band (C) and a single band (D). The length of the sarcomere was determined from the distance between the two dashed lines as shown in the two cross-sectional plots. (E) Comparison of the averaged length of the sarcomere determined from the two sarcomeric patterns. The statistics were calculated based on 12 images obtained from somites near the head (fifth to eighth somite) of three 72-hpf zebrafish.</p

Sarcomeric length of living and fixed zebrafish, and developing larva at different stages.

<p>(A) Comparison of length of sarcomeres determined from zebrafish living and fixed with paraformaldehyde. The SHG images used to measure the lengths were measured on somites near the head (black, somites 5–8) and the tail (red, somites 21–24). The statistics were calculated based on 21 images obtained from three 72-hpf zebrafish. (B) Representative SHG images of zebrafish measured at three developmental stages (24, 36 and 72 hpf). The images were measured on regions near the head (somites 5∼8) and the tail (somites 21–24). (C) Growth of the sarcomere from 1 to 12 dpf. The SHG images used to evaluate the length were measured on somites near the head (black, somites 5∼8) and the tail (red, somites 21–24). The statistics were calculated based on 21 images obtained from three 72-hpf zebrafish.</p

Molecular Imaging of Ischemia and Reperfusion in Vivo with Mitochondrial Autofluorescence

Ischemia and reperfusion (IR) injury constitutes a pivotal mechanism of tissue damage in pathological conditions such as stroke, myocardial infarction, vascular surgery, and organ transplant. Imaging or monitoring of the change of an organ at a molecular level in real time during IR is essential to improve our understanding of the underlying pathophysiology and to guide therapeutic strategies. Herein, we report molecular imaging of a rat model of hepatic IR with the autofluorescence of mitochondrial flavins. We demonstrate a revelation of the histological characteristics of a liver in vivo with no exogenous stain and show that intravital autofluorescent images exhibited a distinctive spatiotemporal variation during IR. The autofluorescence decayed rapidly from the baseline immediately after 20-min ischemia (approximately 30% decrease in 5 min) but recovered gradually during reperfusion (to approximately 99% of the baseline 9 min after the onset of reperfusion). The autofluorescent images acquired during reperfusion correlated strongly with the reperfused blood flow. We show further that the autofluorescence was produced predominantly from mitochondria, and the distinctive autofluorescent variation during IR was mechanically linked to the altered balance between the flavins in the oxidized and reduced forms residing in the mitochondrial electron-transport chain. Our approach opens an unprecedented route to interrogate the deoxygenation and reoxygenation of mitochondria, the machinery central to the pathophysiology of IR injury, with great molecular specificity and spatiotemporal resolution and can be prospectively translated into a medical device capable of molecular imaging. We envisage that the realization thereof should shed new light on clinical diagnostics and therapeutic interventions targeting IR injuries of not only the liver but also other vital organs including the brain and heart