The zebrafish embryo as a model to quantify early inflammatory cell responses to biomaterials

Abstract

To rapidly assess early inflammatory cell responses provoked by biomaterials in the full complexity of the living organism, we developed a zebrafish embryo model which allows real time analysis of these responses to biomaterial microspheres. Fluorescently labeled microspheres with different properties were injected into embryos of selected transgenic zebrafish lines expressing distinct fluorescent proteins in their neutrophils and macrophages. Recruitment of leukocytes and their interactions with microspheres were monitored using fluorescence microscopy. We developed a novel method using ImageJ and the plugin ObjectJ project file "Zebrafish-Immunotest" for rapid and semi-automated fluorescence quantification of the cellular responses. In the embryo model we observed an ordered inflammatory cell response to polystyrene and poly (e-caprolactone) microspheres, similar to that described for mammalian animal models. The responses were characterized by an early infiltration of neutrophils followed by macrophages, and subsequent differentially timed migration of these cells away from the microspheres. The size of microspheres (10 and 15 mm) did not influence the cellular responses. Poly (e-caprolactone) microspheres provoked a stronger infiltration of neutrophils and macrophages than polystyrene microspheres did. Our study shows the potential usefulness of zebrafish embryos for in vivo evaluation of biomaterial-associated inflammatory cell responses.

Monitoring local delivery of vancomycin from gelatin nanospheres in zebrafish larvae

“…The husbandry and maintenance of adult zebrafish followed the welfare regulations of animals approved by the local animal welfare committee (Dierexperimentencommissie, DEC) and were described in detail before. 40,43 After harvesting, zebrafish larvae were maintained in E3 medium as described. 40,43 Microinjection of gelatin nanospheres into zebrafish larvae At 3 days post-fertilization, zebrafish larvae were anesthetized by using 0.03% tricaine (buffered 3 aminobenzoic acid ethyl ester; Sigma-Aldrich), placed in agarose plates (with U-shaped grooves, home-made) containing E3 medium.…”

“…40,43 After harvesting, zebrafish larvae were maintained in E3 medium as described. 40,43 Microinjection of gelatin nanospheres into zebrafish larvae At 3 days post-fertilization, zebrafish larvae were anesthetized by using 0.03% tricaine (buffered 3 aminobenzoic acid ethyl ester; Sigma-Aldrich), placed in agarose plates (with U-shaped grooves, home-made) containing E3 medium. Gelatin nanosphere suspension was vortexed for 1 minute and subsequently loaded into a glass capillary (Harvard Apparatus, Holliston, MA, USA) which was connected to an Eppendorf FemtoJet microinjector (Nijmegen, the Netherlands).…”

“…This allowed injection of a volume of ~3 nL. To study the distribution of gelatin nanospheres and macrophagegelatin nanospheres interactions, the suspension of gelatin nanospheres was injected either into the blood circulation of zebrafish larvae via the Duct of Cuvier 46 or into the tail muscle tissue, 40,46 under a light microscope.…”

“…32,39 Moreover, the optical transparency of zebrafish larvae enables real-time visualization and intravital analysis of cellular responses to pathogens or materials. 34,37,40 To this end, a variety of transgenic zebrafish lines expressing different fluorescent proteins in different types of cells (eg, macrophages and neutrophils) has been developed. 37 The aim of the present study was to evaluate the cellular response to gelatin nanospheres in vivo in zebrafish larvae and assess the potential of gelatin nanospheres as carriers to allow for internalization of vancomycin by macrophages, as studied in live larvae using fluorescence microscopy.…”

Photochemical internalization enhances cytosolic release of antibiotic and increases its efficacy against staphylococcal infection

“…The zebrafish embryo experiments were performed according to the EU Animal Protection Directive 2010/63/EU. Adult wild type (WT) or transgenic (Tg) zebrafish and embryos were maintained as described [33] and handled in compliance with animal welfare regulations, as approved by the local animal welfare committee (DEC).…”