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

René R Sevag Packard,Tyler Beebe,Yonghe Ding,Yichen Ding,Peng Fei,Kyung In Baek,Jonathan Y Tang,Michael Chen,Feng Shi,Po-Heng Chen,Bong Jin Kang,Jonathan Gau,Debiao Li,Yu-Huan Shih,Nelson Jen,Xiaolei Xu,Tzung K Hsiai

doi:10.1038/s41598-017-09152-x

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.

Highlights

To characterize the ultra-structural changes in doxorubicin-induced cardiomyopathy, we developed an automated segmentation approach based on histogram analysis for cardiac Light-Sheet Fluorescent Imaging (LSFI), leading to rapid image processing and 3-D reconstruction with high axial resolution and depth penetration
We developed an automated segmentation process based on histogram analysis of cardiac light-sheet fluorescent imaging (LSFI) studies to standardize the volumetric quantitation of dynamic 3-D architectural changes occurring in experimental anthracycline-induced cardiac toxicity and repair
Our results demonstrate that doxorubicin-induced cardiac injury develops ventricular remodeling, followed by activation of Notch signaling to promote cardiac regeneration and restoration of contractile function; providing a high-throughput model with translational implications to drug discovery and genetic modifiers of chemotherapy-induced cardiomyopathy

Summary

Introduction

To characterize the ultra-structural changes in doxorubicin-induced cardiomyopathy, we developed an automated segmentation approach based on histogram analysis for cardiac Light-Sheet Fluorescent Imaging (LSFI), leading to rapid image processing and 3-D reconstruction with high axial resolution and depth penetration. Current efforts are underway to include the use of machine learning processes to facilitate automated interpretation and improve quality control measures[15] In this context, we developed an automated segmentation process based on histogram analysis of cardiac LSFI studies to standardize the volumetric quantitation of dynamic 3-D architectural changes occurring in experimental anthracycline-induced cardiac toxicity and repair. The PW Doppler signals further enabled us to establish the myocardial performance index by determining ventricular inflow tract isovolumic contraction and relaxation times as well as ejection times[19] In this context, the paralleled advances of light-sheet fluorescent imaging with automated histogram analysis and high-frequency ultrasonic transducers enabled us to unravel the 3-D architecture and electromechanical coupling of doxorubicin-induced cardiac injury and regeneration. Our results demonstrate that doxorubicin-induced cardiac injury develops ventricular remodeling, followed by activation of Notch signaling to promote cardiac regeneration and restoration of contractile function; providing a high-throughput model with translational implications to drug discovery and genetic modifiers of chemotherapy-induced cardiomyopathy

Methods

Results

Conclusion