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Tissue development and regeneration are dynamic processes involving complex cell migration and cell-cell interactions. We have developed a protocol for complementary time-lapse and three-dimensional (3D) imaging of tissue for developmental and regeneration studies which we apply here to the zebrafish cardiac vasculature. 3D imaging of fixed specimens is used to first define the subject at high resolution then live imaging captures how it changes dynamically. Hearts from adult and juvenile zebrafish are extracted and cleaned in preparation for the different imaging modalities. For whole-mount 3D confocal imaging, single or multiple hearts with native fluorescence or immuno-labeling are prepared for stabilization or clearing, and then imaged. For live imaging, hearts are placed in a prefabricated fluidic device and set on a temperature-controlled microscope for culture and imaging over several days. This protocol allows complete visualization of morphogenic processes in a 3D context and provides the ability to follow cell behaviors to complement in vivo and fixed tissue studies. This culture and imaging protocol can be applied to different cell and tissue types. Here, we have used it to observe zebrafish coronary vasculature and the migration of coronary endothelial cells during heart regeneration. [Display omitted] • Imaging approaches to investigate zebrafish juvenile heart development and adult regeneration. • Novel whole volume, medium throughput imaging optimized for zebrafish heart. • Reliable and convenient tissue clearing and antibody labeling. • Live time-lapse imaging to capture cardiac regenerative processes. • Methods optimized for coronary vessels but applicable to other models/organs. [ABSTRACT FROM AUTHOR]

R4269 --> 851.10 --> Background: Heart failure with preserved ejection fraction (HFpEF) is a common and serious condition that lacks evidence‐based therapies due to an incomplete understanding of its pathogenesis, although cardiac hypoperfusion is clearly implicated. Our aim was to characterize leukocyte interactions in the coronary microvasculature during HFpEF using the novel imaging technique of intravital cardiac multiphoton microscopy (MPM), with the goal of identifying cellular mechanisms of hypoperfusion. Methods: Male and female, 8–12‐week‐old C57BL/6 mice were fed a high fat diet and the nitric oxide synthase inhibitor, L‐NAME, in their drinking water (HFpEF) or a normal chow diet (Chow) for 15‐weeks. Mice were assessed for systolic and diastolic function (echocardiography and Doppler imaging), blood pressure (tail‐cuff), hypertrophy (wheat‐germ‐agglutin), hypoxia (Hypoxyprobe™), exercise intolerance (treadmill exhaustion), and heart RNA sequencing. Intravital cardiac MPM was performed in the anesthetized mechanically ventilated mouse following a left thoracotomy and placement of a customized imaging window on the left ventricle. Transgenic‐reporter mice were used to visualize neutrophil (Rosa26‐Cre‐Ly6G+/TdTom) and macrophage/monocyte (Cx3Cr1+/GFP‐CCR2+/RFP) populations. Intravenous injection of fluorescent dyes labeled the vasculature. Results: HFpEF mice developed systemic and clinical features of HFpEF compared to Chow mice; obesity, hypertension, left ventricular hypertrophy, diastolic dysfunction, preserved systolic function, pulmonary remodeling, myocardial hypoxia, and exercise intolerance (p<0.05 for all). Intravital MPM visualized Ly6G+ neutrophil motion in capillaries, revealing an increased frequency of slowed and arrested neutrophils in capillaries in HFpEF hearts compared to Chow (p<0.0001; Kolmogorov‐Smirnov test). Immunohistology for tissue infiltrated (non‐vascular) neutrophils revealed no increase in HFpEF. The number and frequency of slowed and arrested Cx3Cr1+ and CCR2+ capillary monocytes were similar between HFpEF and Chow mice, while tissue resident Cx3Cr1+ macrophages increased in HFpEF (p<0.05). Gene sets associated with inflammatory signaling (TNF‐α and TGF‐β) and cell junctions (epithelial‐mesenchymal transition and apical junctions) were upregulated in HFpEF. Acute neutrophil depletion with anti‐Ly6G antibody (αLy6G) administration (4mg/kg body weight, 24h) reduced the incidence of slowed and arrested neutrophils (p<0.05) and improved exercise tolerance in HFpEF. Chronic αLy6G (2mg/kg every 3 days for 4 weeks) improved myocardial hypoxia and measures of diastolic function (mitral e/e') (p<0.05). Conclusion: We have used the novel imaging technique of intravital cardiac MPM to discover a new behavior of neutrophils within myocardial capillaries during HFpEF. We demonstrate that arrested capillary neutrophils impair myocardial perfusion to promote HFpEF. Targeting arrested neutrophil behavior offers potential therapeutic benefits. [ABSTRACT FROM AUTHOR]