Zebrafish Caudal Fin Angiogenesis Assay—Advanced Quantitative Assessment Including 3-Way Correlative Microscopy

Abstract

BackgroundResearchers evaluating angiomodulating compounds as a part of scientific projects or pre-clinical studies are often confronted with limitations of applied animal models. The rough and insufficient early-stage compound assessment without reliable quantification of the vascular response counts, at least partially, to the low transition rate to clinics.ObjectiveTo establish an advanced, rapid and cost-effective angiogenesis assay for the precise and sensitive assessment of angiomodulating compounds using zebrafish caudal fin regeneration. It should provide information regarding the angiogenic mechanisms involved and should include qualitative and quantitative data of drug effects in a non-biased and time-efficient way.Approach & ResultsBasic vascular parameters (total regenerated area, vascular projection area, contour length, vessel area density) were extracted from in vivo fluorescence microscopy images using a stereological approach. Skeletonization of the vasculature by our custom-made software Skelios provided additional parameters including “graph energy” and “distance to farthest node”. The latter gave important insights into the complexity, connectivity and maturation status of the regenerating vascular network. The employment of a reference point (vascular parameters prior amputation) is unique for the model and crucial for a proper assessment. Additionally, the assay provides exceptional possibilities for correlative microscopy by combining in vivo-imaging and morphological investigation of the area of interest. The 3-way correlative microscopy links the dynamic changes in vivo with their structural substrate at the subcellular level.ConclusionsThe improved zebrafish fin regeneration model with advanced quantitative analysis and optional 3-way correlative morphology is a promising in vivo angiogenesis assay, well-suitable for basic research and preclinical investigations.

Splitting of circulating red blood cells asin vivo-mechanism of erythrocyte maturation in developing zebrafish, chick and mouse embryos

“…Aside from murine models, the zebrafish is another lab species that is increasingly utilized in hematopoietic research because of its amenability to genetic manipulations and regenerative capacity (Hlushchuk et al, 2016;Poss et al, 2003). Primitive erythropoiesis in the zebrafish takes place in various regions, most prominently in the inner cell mass (Orkin and Zon, 2008).…”

Novel Heparin Receptor Transmembrane Protein 184a Regulates Angiogenesis in the Adult Zebrafish Caudal Fin

“…Zebrafish also express several HSPGs including syndecans which have been shown to promote angiogenesis by signaling through the Vegfr2 complex (Chen et al, 2004 , 2005 ; Gorsi et al, 2014 ; Venero Galanternik et al, 2015 ). The ease of visualizing and quantitating vascular regeneration in the caudal fin has been demonstrated (Huang et al, 2003 ; Santoro, 2014 ; Hlushchuk et al, 2016 ) in part due to the availability of various transgenic reporter lines, such as Tg(Fli1:EGFP) which expresses EGFP specifically in ECs. Upon amputation of the caudal fin, a plexus is formed and new vessels develop over several days.…”

Ex vivo microangioCT: Advances in microvascular imaging

“…The p anels a`-c` display at higher magnification the regions indicated in a -c. The glomeruli are indicated with circles in a`-c`. Reproduced from Hlushchuk R. et al [4].…”

“…Unfortunately, the often-frustrating results from late-stage clinical studies indicate an urgent need for improved assessment of the pro-and antiangiogenic compounds in preclinical models [3,4].…”