Development of an ImageJ-based method for analysing the developing zebrafish vasculature

Zebrafish with fluorescently labelled blood vessels provide an excellent model for studying angiogenesis. Most commonly the growth of the intersegmental blood vessels is investigated in response to compounds or manipulation of gene expression and analysed using manual methods, typically scoring the connectivity of these blood vessels to the dorsal longitudinal anastomotic vessel. Such methods are laborious and best suited to time points after the connectivity of these vessels has been established. By contrast, reported image processing-based methods are difficult to implement and often depend on specialist software. This study aimed to develop and evaluate a computational method using the freely available ImageJ software to quantify the development of intersegmental blood vessel formation. This methodology enabled rapid analysis of vascular development. The outputs of total vessel length and number of junctions best documented defective vascular development at differing levels of severity and gave comparable results to the frequently used manual approach of calculating the percentage connectivity of the intersegmental vessels. This ImageJ-based analysis method allowed objective quantitation of vascular network formation in zebrafish enabling a free, straightforward and rapid approach to determine the effect of novel compounds or genetic manipulation of the angiogenic process.