Automated Segmentation of Light-Sheet Fluorescent Imaging to Characterize Experimental Doxorubicin-Induced Cardiac Injury and Repair

Abstract

This study sought to develop an automated segmentation approach based on histogram analysis of raw axial images acquired by light-sheet fluorescent imaging (LSFI) to establish rapid reconstruction of the 3-D zebrafish cardiac architecture in response to doxorubicin-induced injury and repair. Input images underwent a 4-step automated image segmentation process consisting of stationary noise removal, histogram equalization, adaptive thresholding, and image fusion followed by 3-D reconstruction. We applied this method to 3-month old zebrafish injected intraperitoneally with doxorubicin followed by LSFI at 3, 30, and 60 days post-injection. We observed an initial decrease in myocardial and endocardial cavity volumes at day 3, followed by ventricular remodeling at day 30, and recovery at day 60 (P < 0.05, n = 7–19). Doxorubicin-injected fish developed ventricular diastolic dysfunction and worsening global cardiac function evidenced by elevated E/A ratios and myocardial performance indexes quantified by pulsed-wave Doppler ultrasound at day 30, followed by normalization at day 60 (P < 0.05, n = 9–20). Treatment with the γ-secretase inhibitor, DAPT, to inhibit cleavage and release of Notch Intracellular Domain (NICD) blocked cardiac architectural regeneration and restoration of ventricular function at day 60 (P < 0.05, n = 6–14). Our approach provides a high-throughput model with translational implications for drug discovery and genetic modifiers of chemotherapy-induced cardiomyopathy.

Saak Transform-Based Machine Learning for Light-Sheet Imaging of Cardiac Trabeculation

“…In response to chemo-induced cardiac injury, the SVR was significantly reduced (doxorubicin: SVR= 0.10 ± 0.02, n=5; control: SVR= 0.14 ± 0.03, n=5; p = 0.03). This finding was consistent with the previously reported reduction in cardiac trabeculation in response to chemo-induced myocardial injury [13,23].…”

“…Our custom-built LSFM system was designed to image the changes in trabecular network in the zebrafish model of cardiac injury and regeneration. Our LSFM system adapting from [23,47] generated a Gaussian light-sheet at ~4.5 µm in thickness ( Figure 2), and the detection unit was perpendicularly positioned to the illumination plane. Fluorescence was orthogonally captured through an objective lens (Ob1) and a tube lens (TL1) to minimize photobleaching while providing high axial resolution (Inset in Figure 2A).…”

“…LSFM systems have the capacity to investigate cardiac ultra-structure and function [5][6][7][8][9][10][11][12][13][14], providing the moving Baek, Zhaoqiang Wang, Mehrdad Roustaei, Dengfeng Kuang, C.-C. Jay Kuo †, and Tzung K. Hsiai † boundary conditions for computational fluid dynamics [15,16] and the specific labeling of trabecular network for interactive virtual reality [17,18]. However, efficient and robust structural segmentation of cardiac trabeculation remains a post-imaging challenge [19][20][21][22][23]. Manual segmentation of cardiac images in zebrafish and mice remains as the gold standard for ground truth despite being a labor-intensive and error-prone method.…”

Organ‐on‐a‐Chip for Cancer and Immune Organs Modeling

“…Many characterization techniques, such as immunohistochemical staining and transcriptome/proteome profiling, have been implemented to ensure that organoids generated in vitro can faithfully represent their counterparts in vivo . With advanced sectioning‐based imaging techniques such as confocal and light‐sheet microscopy, more information can be obtained at the cellular, tissue, and organ levels to validate similar behavior between in vitro models and in vivo systems.…”

Integration of multiple imaging platforms to uncover cardiac defects in adult zebrafish

“…In a clinical setting, diagnosing the onset and progression of cardiovascular diseases often requires a combination of multiple imaging analyses including echocardiography, magnetic resonance imaging (MRI) and computed tomography (CT) (Marwick et al, 2013;Fuchs et al, 2016;Petersen et al, 2017;Plana et al, 2018). Recently, researchers have adapted light-sheet microscopy (Packard et al, 2017), MRI (Koth et al, 2017;Merrifield et al, 2017) and microcomputed tomography (µ-CT) (Descamps et al, 2014;Weinhardt et al, 2018;Ding et al, 2019) to define the morphology of zebrafish organs. Others have used echocardiography to characterize the functional impact of cardiac disease and regeneration in adult zebrafish (Ho et al, 2002;Sun et al, 2008;Parente et al, 2013;González-Rosa et al, 2014;Hein et al, 2015;Huang et al, 2015;Wilson et al, 2015;Ernens et al, 2016;Lee et al, 2016;Wang et al, 2017;Benslimane et al, 2019).…”