Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Ellett, Felix; Irimia, Daniel

doi:10.3791/56498

Cited by 5 publications

(4 citation statements)

References 20 publications

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“…Irradiated spores still stained well with calcofluor. For intravascular delivery experiments, microinjection of conidia utilised polydimethylsiloxane (PDMS) microstructured surface arrays [ 93 ], while imaging was performed following mounting in PDMS imaging devices, as previously described [ 94 ].…”

Section: Methodsmentioning

confidence: 99%

Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo

et al. 2018

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Neutrophils and macrophages provide the first line of cellular defence against pathogens once physical barriers are breached, but can play very different roles for each specific pathogen. This is particularly so for fungal pathogens, which can occupy several niches in the host. We developed an infection model of talaromycosis in zebrafish embryos with the thermally-dimorphic intracellular fungal pathogen Talaromyces marneffei and used it to define different roles of neutrophils and macrophages in infection establishment. This system models opportunistic human infection prevalent in HIV-infected patients, as zebrafish embryos have intact innate immunity but, like HIV-infected talaromycosis patients, lack a functional adaptive immune system. Importantly, this new talaromycosis model permits thermal shifts not possible in mammalian models, which we show does not significantly impact on leukocyte migration, phagocytosis and function in an established Aspergillus fumigatus model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions in vivo. Following parenteral inoculation, T. marneffei conidia were phagocytosed by both neutrophils and macrophages. Within these different leukocytes, intracellular fungal form varied, indicating that triggers in the intracellular milieu can override thermal morphological determinants. As in human talaromycosis, conidia were predominantly phagocytosed by macrophages rather than neutrophils. Macrophages provided an intracellular niche that supported yeast morphology. Despite their minor role in T. marneffei conidial phagocytosis, neutrophil numbers increased during infection from a protective CSF3-dependent granulopoietic response. By perturbing the relative abundance of neutrophils and macrophages during conidial inoculation, we demonstrate that the macrophage intracellular niche favours infection establishment by protecting conidia from a myeloperoxidase-dependent neutrophil fungicidal activity. These studies provide a new in vivo model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting T. marneffei’s opportunity for macrophage parasitism and thereby enhancing this pathogen’s exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.

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Section: Methodsmentioning

confidence: 99%

Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo

et al. 2018

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“…FPR1 sensitivity has been shown to vary widely between mammalian species, with mouse and rat neutrophils exhibiting poor recruitment in response to fMLP compared to human cells (29). There is evidence that zebrafish neutrophils do respond to formylated peptides (30, 31), although experiments in this model have been complicated by inability to distinguish direct responses to chemoattractant from recruitment to injured tissue at the site of microinjection. To avoid this complication in our experiments, we delivered pre-treated conidia at one site (the duct of Cuvier) and analyzed neutrophil responses at a spatially distant site (the caudal venous plexus) (Figure 5Ai).…”

Section: Resultsmentioning

confidence: 99%

“…To enable better measurement of neutrophil-specific responses, primitive macrophage differentiation was restricted by blocking translation of spi1 or irf8 using anti-sense morpholino oligonucleotides ( spi1 -MO) as previously described (34, 38). The morphant larvae were raised to 2 days post-fertilization, and then microinjected into the vasculature with a solution of A. fumigatus conidia pre-stained with Calcofluor together with C-001 (10 nM), C-016 (100 nM), or DMSO, using microstructured surface arrays developed for this purpose (31, 45). Imaging was performed on a fully automated Nikon TiE microscope.…”

Section: Methodsmentioning

confidence: 99%

Bifunctional Small Molecules Enhance Neutrophil Activities Against Aspergillus fumigatus in vivo and in vitro

Ellett

et al. 2019

Front. Immunol.

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Aspergillosis is difficult to treat and carries a high mortality rate in immunocompromised patients. Neutrophils play a critical role in control of infection but may be diminished in number and function during immunosuppressive therapies. Here, we measure the effect of three bifunctional small molecules that target Aspergillus fumigatus and prime neutrophils to generate a more effective response against the pathogen. The molecules combine two moieties joined by a chemical linker: a targeting moiety (TM) that binds to the surface of the microbial target, and an effector moiety (EM) that interacts with chemoattractant receptors on human neutrophils. We report that the bifunctional compounds enhance the interactions between primary human neutrophils and A. fumigatus in vitro , using three microfluidic assay platforms. The bifunctional compounds significantly enhance the recruitment of neutrophils, increase hyphae killing by neutrophils in a uniform concentration of drug, and decrease hyphal tip growth velocity in the presence of neutrophils compared to the antifungal targeting moiety alone. We validated that the bifunctional compounds are also effective in vivo , using a zebrafish infection model with neutrophils expressing the appropriate EM receptor. We measured significantly increased phagocytosis of A. fumigatus conidia by neutrophils expressing the EM receptor in the presence of the compounds compared to receptor-negative cells. Finally, we demonstrate that treatment with our lead compound significantly improved the antifungal activity of neutrophils from immunosuppressed patients ex vivo . This type of bifunctional compounds strategy may be utilized to redirect the immune system to destroy fungal, bacterial, and viral pathogens.

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“…The microfluidic device breaks through the limitations of conventional methods such as significantly reducing the damage to fish and no need for anesthetics while observing the zebrafish. This significantly reduces the cost of the inspection, and easy for drug administration to inspect the development of organs in zebrafish with a higher survival rate 17 – 19 . In this context microfluidic technologies enact a promising direction that can support the development of the next-generation systems for phenotype-based physiological analysis 20 .…”

Section: Introductionmentioning

confidence: 99%

Edible additive effects on zebrafish cardiovascular functionality with hydrodynamic assessment

Wang

Chen

Subendran

et al. 2020

Sci Rep

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Food coloring is often used as a coloring agent in foods, medicines and cosmetics, and it was reported to have certain carcinogenic and mutagenic effects in living organisms. Investigation of physiological parameters using zebrafish is a promising methodology to understand disease biology and drug toxicity for various drug discovery on humans. Zebrafish (Danio rerio) is a well-acknowledged model organism with combining assets such as body transparency, small size, low cost of cultivation, and high genetic homology with humans and is used as a specimen tool for the in-vivo throughput screening approach. In addition, recent advances in microfluidics show a promising alternative for zebrafish manipulation in terms of drug administration and extensive imaging capability. This pilot work highlighted the design and development of a microfluidic detection platform for zebrafish larvae through investigating the effects of food coloring on cardiovascular functionality and pectoral fin swing ability. The zebrafish embryos were exposed to the Cochineal Red and Brilliant Blue FCF pigment solution in a concentration of (0.02‰, 0.2‰) cultured in the laboratory from the embryo stage to hatching and development until 9 days post fertilization (d.p.f.). In addition, zebrafish swimming behaviors in terms of pectoral fin beating towards the toxicity screening were further studied by visualizing the induced flow field. It was evidenced that Cochineal Red pigment at a concentration of 0.2‰ not only significantly affected the zebrafish pectoral fin swing behavior, but also significantly increased the heart rate of juvenile fish. The higher concentration of Brilliant Blue FCF pigment (0.2%) increased heart rate during early embryonic stages of zebrafish. However, zebrafish exposed to food coloring did not show any significant changes in cardiac output. The applications of this proposed platform can be further extended towards observing the neurobiological/hydrodynamic behaviors of zebrafish larvae for practical applications in drug tests.

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Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Cited by 5 publications

References 20 publications

Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo

Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo

Bifunctional Small Molecules Enhance Neutrophil Activities Against Aspergillus fumigatus in vivo and in vitro

Edible additive effects on zebrafish cardiovascular functionality with hydrodynamic assessment

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