High Resolution Imaging of Vascular Function in Zebrafish

Rationale: The role of the endothelium in the pathogenesis of cardiovascular disease is an emerging field of study, necessitating the development of appropriate model systems and methodologies to investigate the multifaceted nature of endothelial dysfunction including disturbed barrier function and impaired vascular reactivity. Objective: We aimed to develop and test an optimized high-speed imaging platform to obtain quantitative real-time measures of blood flow, vessel diameter and endothelial barrier function in order to assess vascular function in live vertebrate models. Methods and Results: We used a combination of cutting-edge optical imaging techniques, including high-speed, camera-based imaging (up to 1000 frames/second), and 3D confocal methods to collect real time metrics of vascular performance and assess the dynamic response to the thromboxane A2 (TXA2) analogue, U-46619 (1 μM), in transgenic zebrafish larvae. Data obtained in 3 and 5 day post-fertilization larvae show that these methods are capable of imaging blood flow in a large (1 mm) segment of the vessel of interest over many cardiac cycles, with sufficient speed and sensitivity such that the trajectories of individual erythrocytes can be resolved in real time. Further, we are able to map changes in the three dimensional sizes of vessels and assess barrier function by visualizing the continuity of the endothelial layer combined with measurements of extravasation of fluorescent microspheres. Conclusions: We propose that this system-based microscopic approach can be used to combine measures of physiologic function with molecular behavior in zebrafish models of human vascular disease. © 2012 Watkins et al

Spontaneous DNA damage to the nuclear genome promotes senescence,redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline

Spontaneous DNA damage to the nuclear genome promotes senescence, T redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline

Time-lapse imaging of endothelial barrier dysfunction in response to the serine protease, thrombin.

<p>Extended depth of focus projection of the trunk vessels from a 5 dpf <i>Tg(kdrl:GFP)^la116</i> zebrafish larva (<b>A</b>). The EGFP-labeled endothelium appears green while the 0.1 µm FluoSpheres are pseudocolored red. Data were collected at baseline and at 10 minute intervals following exposure to thrombin (0.01 units/µL) for a total collection period of 3 hours. Localized increases in permeability, as evidenced by accumulation of microspheres outside the vessel wall (white arrows) were apparent by 1 h (panel <b>2</b>) and increased over time (panels 3–6). The zoomed images (<b>B</b>) from boxed region of panel A1 show a clear break in the endothelial layer at 1 hour (panel 4). A total of 8 larvae were examined with equivalent results. These data were confirmed in a subset of larvae (n = 3) using high magnification (60X, 1.4 NA) point scanning confocal imaging and showed extensive accumulation of Fluospheres outside the vessel wall (<b>C</b>). In contrast, 5 dpf zebrafish larvae (n = 8) exposed to the thromboxane mimetic, U-46619 showed no evidence for disrupted barrier function (<b>D</b>).</p

Quantitation of aortic blood flow velocity in zebrafish embryos using intravital imaging.

<p>Image processing was performed using Imaris software (Bitplane, Saint Paul, MN). Red blood cells shown in a representative single time point (<b>A</b>) were segmented by size, shape, and fluorescence intensity (<b>B</b>) and tracked using an auto-regressive tracking algorithm (<b>C</b>). The frequency of image collection was set such that erythrocyte displacement is less than one cell diameter/frame and therefore individual cells can be tracked with confidence. Only cells that could be tracked for >250 µm were included in the analysis. Quantitation revealed pulsatility of blood flow in both the 3 (<b>D</b>) and 5 (<b>E</b>) dpf embryos, and confirmed our ability to measure flow velocities greater than 2 mm/s in response to the thromboxane analogue U46619 (example shown in 5 dpf embryos, panel <b>E,</b> open circles).</p

The thromboxane analogue, U-46619 decreased vessel diameter.

<p>3D imaging of the dorsal aorta in a 5 dpf <i>Tg(kdrl:GFP)^la116</i> zebrafish larva with dorsal downwards and anterior right (<b>A-D</b>). Changes in aortic flow velocity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044018#pone-0044018-g005" target="_blank">Figure 5</a>) in response to U-46619 in the 5 dpf embryos, were accompanied by decreases in vessel diameter (<b>E</b>, n = 5, <i>P</i><0.05). χ denotes statistical differences from baseline (time 0).</p

Time sequence of red blood cell flow through a segment of dorsal aorta.

<p>This montage shows individual images from a time sequence (collected every 2 ms, with a 2 ms exposure). The images shown are semi-sequential (4 ms intervals). Erythrocytes are readily visible, and the tracking of the cells from right to left can be readily seen. (Scale Bar  = 100 microns).</p

Intravital imaging of vascular dynamics in a 5 dpf zebrafish larva.

<p>An extended depth of focus projection (dorsal upwards, anterior left) of the vasculature of a <i>Tg(kdrl:GFP)^la116;Tg(gata1:dsRed)^sd2 embryo with the fluorescence defining the endothelium (</i><b><i>A</i></b><i>)</i>. The outlined box shows the area of the aorta used for the time-based imaging of blood flow velocity and vessel diameter. The lower panel (<b>B</b>) shows a representative image from the same fish of the DsRed labeled erythrocytes within the dorsal aorta. This image was collected with a 2 ms exposure. With these settings it was readily possible to visualize the red blood cells flowing through the dorsal aorta over time. (Scale Bar  = 100 microns).</p

Comparison of aortic blood flow velocities in 3 dpf and 5 dpf zebrafish embryos.

<p>Plots show the mean data (± SE) for peak (<b>a</b>), minimum (<b>b</b>) and mean (<b>c</b>) aortic blood flow velocities and mean heart rate (<b>d</b>) in 3 dpf (open circles, n = 15) and 5 dpf (closed circles, n = 10) zebrafish embryos; and the changes in these parameters in response to the thromboxane A₂ (TXA₂) analogue, U-46619. α denotes statistical differences (ANOVA, <i>P</i><0.05) between age groups; β denotes statistical differences from baseline (time 0).</p