Mechanical overstimulation causes acute injury and synapse loss followed by fast recovery in lateral-line neuromasts of larval zebrafish.

Excess noise damages sensory hair cells, resulting in loss of synaptic connections with auditory nerves and, in some cases, hair-cell death. The cellular mechanisms underlying mechanically induced hair-cell damage and subsequent repair are not completely understood. Hair cells in neuromasts of larval zebrafish are structurally and functionally comparable to mammalian hair cells but undergo robust regeneration following ototoxic damage. We therefore developed a model for mechanically induced hair-cell damage in this highly tractable system. Free swimming larvae exposed to strong water wave stimulus for 2 hr displayed mechanical injury to neuromasts, including afferent neurite retraction, damaged hair bundles, and reduced mechanotransduction. Synapse loss was observed in apparently intact exposed neuromasts, and this loss was exacerbated by inhibiting glutamate uptake. Mechanical damage also elicited an inflammatory response and macrophage recruitment. Remarkably, neuromast hair-cell morphology and mechanotransduction recovered within hours following exposure, suggesting severely damaged neuromasts undergo repair. Our results indicate functional changes and synapse loss in mechanically damaged lateral-line neuromasts that share key features of damage observed in noise-exposed mammalian ear. Yet, unlike the mammalian ear, mechanical damage to neuromasts is rapidly reversible.

Isoliquiritin promote angiogenesis by recruiting macrophages to improve the healing of zebrafish wounds.

Licorice is a widely used herbal medicine for the treatment of various diseases in southern Europe and parts of Asia. It has been reported that the isoliquiritin (ISL) from Glycyrrhiza root has the activity of promoting angiogenesis. The purpose of this study was to investigate the effect of ISL on the wound healing activity of zebrafish and its mechanism. 6-month-old zebrafish were injured in the skin (2 mm in diameter) and then treated with ISL. By measuring wound size and by histological examination, we found that ISL improved wound healing. In addition, 4-day-old zebrafish embryos of double transgenic line [Tg(fli-1:EGFP)]/[Tg(mpeg:mCherry)] were suffered from tissue traumas and then treated with ISL. Through fluorescent microscopy, we found that ISL promoted macrophage recruitment and angiogenesis in the wound area. Through qPCR analysis, we found that ISL up-regulated the expression of genes related to inflammation and angiogenesis in zebrafish embryos. These results showed that ISL could promote inflammatory response and angiogenesis, which played key roles in promoting wound healing. Therefore, ISL can be used as a promising candidate to promote wound healing.

Bacteria evoke alarm behaviour in zebrafish.

When injured, fish release an alarm substance (Schreckstoff) that elicits fear in members of their shoal. Although Schreckstoff has been proposed to be produced by club cells in the skin, several observations indicate that these giant cells function primarily in immunity. Previous data indicate that the alarm substance can be isolated from mucus. Here we show that mucus, as well as bacteria, are transported from the external surface into club cells, by cytoplasmic transfer or invasion of cells, including neutrophils. The presence of bacteria inside club cells raises the possibility that the alarm substance may contain a bacterial component. Indeed, lysate from a zebrafish Staphylococcus isolate is sufficient to elicit alarm behaviour, acting in concert with a substance from fish. These results suggest that Schreckstoff, which allows one individual to unwittingly change the emotional state of the surrounding population, derives from two kingdoms and is associated with processes that protect the host from bacteria.

A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals.

Spinal cord injury (SCI) is a complex condition, with limited therapeutic options, that results in sensory and motor disabilities. To boost discovery of novel therapeutics, we designed a simple and efficient drug screening platform. This innovative approach allows to determine locomotor rescue properties of small molecules in a zebrafish (Danio rerio) larval spinal cord transection model. We validated our screening platform by showing that Riluzole and Minocycline, two molecules that are in clinical trials for SCI, promote rescue of the locomotor function of the transected larvae. Further validation of the platform was obtained through the blind identification of D-Cycloserine, a molecule scheduled to enter phase IV clinical trials for SCI. Importantly, we identified Tranexamic acid and further showed that this molecule maintains its locomotor recovery properties in a rodent female contusion model. Our screening platform, combined with drug repurposing, promises to propel the rapid translation of novel therapeutics to improve SCI recovery in humans.

Circadian genes period1b and period2 differentially regulate inflammatory responses in zebrafish.

The circadian clock has been shown to regulate various immune processes in different animals. Our previous report demonstrated that the innate immune responses in zebrafish show significant rhythmicity that could be regulated by melatonin. Here, we used diurnal zebrafish to determine the role of circadian genes in the inflammatory responses. Our results indicate that circadian genes exhibit rhythmic oscillations in zebrafish leukocytes, and mutations of the clock genes period1b (per1b) and period2 (per2) considerably affect these oscillations. Using a wounded zebrafish inflammation model, we found that under constant dark conditions (DD), the expression of pro-inflammatory cytokines is significantly downregulated in per1b gene mutant zebrafish and significantly upregulated in the per2 gene mutant zebrafish. Furthermore, using real-time imaging technology, we found that the per1b gene markedly disturbs the rhythmic recruitment of neutrophils toward the injury, whereas the per2 gene does not show a significant effect. Taken together, our results reveal differential functions of the circadian genes per1b and per2 in the inflammatory responses, serving as evidence that circadian rhythms play a vital role in immune processes.

Foxj1a is expressed in ependymal precursors, controls central canal position and is activated in new ependymal cells during regeneration in zebrafish.

Zebrafish are able to regenerate the spinal cord and recover motor and sensory functions upon severe injury, through the activation of cells located at the ependymal canal. Here, we show that cells surrounding the ependymal canal in the adult zebrafish spinal cord express Foxj1a. We demonstrate that ependymal cells express Foxj1a from their birth in the embryonic neural tube and that Foxj1a activity is required for the final positioning of the ependymal canal. We also show that in response to spinal cord injury, Foxj1a ependymal cells actively proliferate and contribute to the restoration of the spinal cord structure. Finally, this study reveals that Foxj1a expression in the injured spinal cord is regulated by regulatory elements activated during regeneration. These data establish Foxj1a as a pan-ependymal marker in development, homeostasis and regeneration and may help identify the signals that enable this progenitor population to replace lost cells after spinal cord injury.

Silver nanoparticles enhance wound healing in zebrafish (Danio rerio).

Silver nanoparticles (AgNPs) were successfully synthesized by a chemical reduction method, physico-chemically characterized and their effect on wound-healing activity in zebrafish was investigated. The prepared AgNPs were circular-shaped, water soluble with average diameter and zeta potential of 72.66 nm and -0.45 mv, respectively. Following the creation of a laser skin wound on zebrafish, the effect of AgNPs on wound-healing activity was tested by two methods, direct skin application (2 µg/wound) and immersion in a solution of AgNPs and water (50 µg/L). The zebrafish were followed for 20 days post-wounding (dpw) by visual observation of wound size, calculating wound healing percentage (WHP), and histological examination. Visually, both direct skin application and immersion AgNPs treatments displayed clear and faster wound closure at 5, 10 and 20 dpw compared to the controls, which was confirmed by 5 dpw histology data. At 5 dpw, WHP was highest in the AgNPs immersion group (36.6%) > AgNPs direct application group (23.7%) > controls (18.2%), showing that WHP was most effective in fish immersed in AgNPs solution. In general, exposure to AgNPs induced gene expression of selected wound-healing-related genes, namely, transforming growth factor (TGF-ß), matrix metalloproteinase (MMP) -9 and -13, pro-inflammatory cytokines (IL-1ß and TNF-α) and antioxidant enzymes (superoxide dismutase and catalase), which observed differentiation at 12 and 24 h against the control; but the results were not consistently significant, and many either reached basal levels or were down regulated at 5 dpw in the wounded muscle. These results suggest that AgNPs are effective in acceleration of wound healing and altered the expression of some wound-healing-related genes. However, the detailed mechanism of enhanced wound healing remains to be investigated in fish.

Image-Based Measurement of H2O2 Reaction-Diffusion in Wounded Zebrafish Larvae.

Epithelial injury induces rapid recruitment of antimicrobial leukocytes to the wound site. In zebrafish larvae, activation of the epithelial NADPH oxidase Duox at the wound margin is required early during this response. Before injury, leukocytes are near the vascular region, that is, ∼100-300 µm away from the injury site. How Duox establishes long-range signaling to leukocytes is unclear. We conceived that extracellular hydrogen peroxide (H2O2) generated by Duox diffuses through the tissue to directly regulate chemotactic signaling in these cells. But before it can oxidize cellular proteins, H2O2 must get past the antioxidant barriers that protect the cellular proteome. To test whether, or on which length scales this occurs during physiological wound signaling, we developed a computational method based on reaction-diffusion principles that infers H2O2 degradation rates from intravital H2O2-biosensor imaging data. Our results indicate that at high tissue H2O2 levels the peroxiredoxin-thioredoxin antioxidant chain becomes overwhelmed, and H2O2 degradation stalls or ceases. Although the wound H2O2 gradient reaches deep into the tissue, it likely overcomes antioxidant barriers only within ∼30 µm of the wound margin. Thus, Duox-mediated long-range signaling may require other spatial relay mechanisms besides extracellular H2O2 diffusion.

Assessing toxic effects of [Omim]Cl and [Omim]BF4 in zebrafish adults using a biomarker approach.

In the present study, the toxic effects of 1-octyl-3-methylimidazolium chloride ([Omim]Cl) and 1-octyl-3-methylimidazolium tetrafluoroborate ([Omim]BF4) on the zebrafish livers were studied at 0, 5, 10, 20, and 40 mg L(-1) on the 7th and 14th days. In addition, the concentrations of [Omim]Cl and [Omim]BF4 in the test water, the acute toxicity of the two ionic liquids (ILs), and the influence of anions on the toxicity of the ILs were evaluated. The acute toxicity test results showed 50 % lethal concentration (LC50) values of 152.3 ± 12.1 mg L(-1) for [Omim]Cl and 144.0 ± 11.4 mg L(-1) for [Omim]BF4. At the lowest concentration investigated (5 mg L(-1)), [Omim]Cl and [Omim]BF4 did not significantly affect zebrafish during the exposure period. However, the toxic effects of these substances were enhanced as dosing concentrations and exposure times were increased. Levels of reactive oxygen species (ROS) were significantly enhanced on the 7th day after 20 mg L(-1) and on the 14th day after 10 mg L(-1) of either substance was applied, resulting in oxidative damage, such as lipid peroxidation and DNA damage. The experimental results also indicated little effect of the anions on the toxicity of ILs and consistent toxic effects of [Omim]Cl and [Omim]BF4. Graphical Abstract The graphical abstract for the present study after exposure to [Omim]Cl and [Omim]BF4. The letter R represents the anions Cl(-) and BF4 (.)

H2O2: a chemoattractant?

H2O2 is a relatively stable, rapidly diffusing reactive oxygen species that has been recently implicated as a mediator of leukocyte recruitment to epithelial wounds and transformed cells in zebrafish. Whether H2O2 activates the innate immune response by acting as a bona fide chemoattractant, enhancing chemoattractant sensing, or triggering production of other chemoattractive ligands remains largely unclear. Here, we describe the basic experimental procedures required to study these questions. We present a detailed protocol of the zebrafish tail fin wounding assay and explain how to use it for analyzing leukocyte chemotaxis in vivo. We further outline a method for H2O2 measurement in live zebrafish larvae using the genetically encoded sensor HyPer on a wide-field and a spinning disk confocal microscope. These methods provide a basis for dissecting the role of H2O2 in leukocyte chemotaxis in a vertebrate animal.

In vivo monitoring of cardiomyocyte proliferation to identify chemical modifiers of heart regeneration.

Adult mammalian cardiomyocytes have little capacity to proliferate in response to injury, a deficiency that underlies the poor regenerative ability of human hearts after myocardial infarction. By contrast, zebrafish regenerate heart muscle after trauma by inducing proliferation of spared cardiomyocytes, providing a model for identifying manipulations that block or enhance these events. Although direct genetic or chemical screens of heart regeneration in adult zebrafish present several challenges, zebrafish embryos are ideal for high-throughput screening. Here, to visualize cardiomyocyte proliferation events in live zebrafish embryos, we generated transgenic zebrafish lines that employ fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. We then performed a chemical screen and identified several small molecules that increase or reduce cardiomyocyte proliferation during heart development. These compounds act via Hedgehog, Insulin-like growth factor or Transforming growth factor ß signaling pathways. Direct examination of heart regeneration after mechanical or genetic ablation injuries indicated that these pathways are activated in regenerating cardiomyocytes and that they can be pharmacologically manipulated to inhibit or enhance cardiomyocyte proliferation during adult heart regeneration. Our findings describe a new screening system that identifies molecules and pathways with the potential to modify heart regeneration.

Neutrophil-delivered myeloperoxidase dampens the hydrogen peroxide burst after tissue wounding in zebrafish.

Prompt neutrophil arrival is critical for host defense immediately after injury [1-3]. Following wounding, a hydrogen peroxide (H(2)O(2)) burst generated in injured tissues is the earliest known leukocyte chemoattractant [4]. Generating this tissue-scale H(2)O(2) gradient uses dual oxidase [4] and neutrophils sense H(2)O(2) by a mechanism involving the LYN Src-family kinase [5], but the molecular mechanisms responsible for H(2)O(2) clearance are unknown [6]. Neutrophils carry abundant amounts of myeloperoxidase, an enzyme catalyzing an H(2)O(2)-consuming reaction [7, 8]. We hypothesized that this neutrophil-delivered myeloperoxidase downregulates the high tissue H(2)O(2) concentrations that follow wounding. This was tested in zebrafish using simultaneous fluorophore-based imaging of H(2)O(2) concentrations and leukocytes [4, 9-11] and a new neutrophil-replete but myeloperoxidase-deficient mutant (durif). Leukocyte-depleted zebrafish had an abnormally sustained wound H(2)O(2) burst, indicating that leukocytes themselves were required for H(2)O(2) downregulation. Myeloperoxidase-deficient zebrafish also had abnormally sustained high wound H(2)O(2) concentrations despite similar numbers of arriving neutrophils. A local H(2)O(2)/myeloperoxidase interaction within wound-recruited neutrophils was demonstrated. These data demonstrate that leukocyte-delivered myeloperoxidase cell-autonomously downregulates tissue-generated wound H(2)O(2) gradients in vivo, defining a new requirement for myeloperoxidase during inflammation. Durif provides a new animal model of myeloperoxidase deficiency closely phenocopying the prevalent human disorder [7, 12, 13], offering unique possibilities for investigating its clinical consequences.

Cellular proliferation and neurogenesis in the injured retina of adult zebrafish.

The retinas of adult teleost fish can regenerate neurons following a chemical or mechanical injury. Previous studies have demonstrated that mechanical excision of fish retina induces a hyperplasia within the retinal sheet, including the formation of a proliferative blastema from whence new retinal cells are produced to fill the excision site. The current study was designed to address two issues regarding injury-induced retinal hyperplasia: (1) Retinas of adult zebrafish can regenerate following a surgical excision, but compared to other fish they contain very few proliferative cells: Might retinal injury in adult zebrafish therefore induce minimal, or perhaps no, hyperplasia? (2) The fate of injury-induced, proliferative retinal cells outside surgical excision sites has yet to be determined. Do such cells produce retinal neurons? Evidence is presented that mechanical injury to the adult zebrafish retina induces a dramatic increase in the number of proliferative cells both within and external to the lesion site, and some of these cells apparently migrate within the radial dimension of the retina. Evidence is also presented that injury-induced proliferative cells outside a lesion site can produce retinal neurons--including cone photoreceptors, interplexiform cells, and amacrine cells--that are incorporated into the extant retina. The results suggest that the adult zebrafish retina contains a latent population of cells that is induced to proliferate following retinal injury, and that these cells might represent a novel avenue for pluripotent neurogenesis within the intact adult teleost retina.